key: cord-281410-y558a5jf authors: akashi, h.; inaba, y.; miura, y.; sato, k.; tokuhisa, s.; asagi, m.; hayashi, y. title: propagation of the kakegawa strain of bovine coronavirus in suckling mice, rats and hamsters date: 1981 journal: arch virol doi: 10.1007/bf01314841 sha: doc_id: 281410 cord_uid: y558a5jf the kakegawa strain of bovine coronavirus was easily propagated in suckling mice. infected animals died with nervous symptoms, and serial passage was readily accomplished by intracerebral inoculation with brain emulsions. the 3rd passage viral material from infected mice evoked the same disease in suckling mice, rats and hamsters inoculated by the intracerebral or by the subcutaneous route. viruses recovered from mice, rats and hamsters could be clearly differentiated from mouse hepatitis virus strain 2 by the neutralization test. w'ith 1 figure accepted november 5, 1980 summary the kakegawa strain of bovine coronavirus was easily propagated in suckling mice. infected animals died with nervous symptoms, and serial passage was readily accomplished by intraeerebral inoculation with brain emulsions. the 3rd passage viral material from infected mice evoked the same disease in suckling mice, rats and hamsters inoculated by the intracerebral or by the subcutaneous route. viruses recovered from mice, rats and hamsters could be clearly differentiated from mouse hepatitis virus strain 2 by the neutralization test. a bovine coronavirus (bcv) has been recognized as one of the causative agents of diarrhea in calves (8) . l~ecently the kakagawa strain isolated from the feces of a cow with cpizootic diarrhea has been identified as bcv (1, 9) . however, the study of bovine diarrheal disease produced by coronavirus has been greatly hampered by difficulty in isolating virus in cell cultures and the lack of experimental hosts other than cattle. recently many members of the eorona~drus family" have been found to grow in suckling mice (5--7). the present paper describes briefly our recent observation that the kakegawa strain of bcv readily propagates in suckling mice, rats and hamsters. the kakegawa strain of bcv used for mouse inoculation was at, the 10th passage level in primary bovine kidney cell cultures. conventionally reared oneday-old mice (strain ddy), rats and syrian hamsters were each inoculated with 0.01-ml of the viral materials by the intracerebral (ic) route and with 0.02-ml by the subcutaneous (s.c.) route. virus recovery and infectivity assay were carried out. by inoculation into cell cultures of bek-1 ecll, derived from bovine embryonic kidney (3). the a:nti-kakegawa strain immune serum having a homologous neutralizing antibody titer of 4096 was prepared in specific germ-free rabbits with virus grown in bek-1 cells as described previously (1), and the anti-mouse hepatitis virus (mhv) strain 2 rabbit immune serum having a neutralizing antibody titer of about 10,000 by 50 per cent plaque reduction neutralization test was kindly supplied by dr. k. fujiwara, university of tokyo, japan. neutralization (nt) test was carried out by the method described previously (1) . viruses recovered from the brains of affected animals containing 200 tcids0 were mixed with an equal volume of two-fold serum dilutions. each mixture was assayed for infectivity by using two tubes of bek-1 cell cultures per dilution. the antibody titer was expressed as the reciprocal of the highest serum dilution that showed no cytopathic changes in at least, one of the two tubes. fig. 1 . four-day-old mouse 3 days after intraeerebral inoculation with the 3rd mouse brain passaged kakegawa strain (left) and an uninoeulated 4-day-old mouse (right) four litters (10 to 12/litter) of suckling mice were inoculated intracerebrally with infected tissue culture fluid containing 106-5 tcid50/ml. at daily intervals, four animals were sacrificed and their brains taken. the inoculated animals developed an illness 4 days post-inoculation (p. i.) and began to die at 5 days p. i. the affected animals became anemic and showed mild neural symptoms such as weak limbs and sluggish staggering gait. the virus titers of infected brains rose at 2 days p. i. with a subsequent gradual rise to a maximum of 10 s.~ tcids0/g at 5 days p. i. serial passage in suckling mice was readily accomplished by the i. c. route with 10 per cent, (w/v) brain emulsions (fig. 1) . the incubation period fell to 2 to 3 days by the 3rd passage. uninoculated mice and mice inoculated with normal brain emulsions remained healthy. virus at the 3rd passage level in suckling mice containing 105.2 tcids0/ml also produced a clinical illness with the same symptoms described above. it caused death in all animals when ineoulated into one litter of suckling mice (10/litter) by the s.c. route, and into rats (9 or 12/litter) and hamsters (7 or 8/litter) by the i.e. or s.c. route. table 1 neutralizing antibody titer the results of one way cross nt test are summarized in table 1 . the homologous nt titers were much higher than the heterologous liters. these findings showed that the viruses recovered from the brains of affected animals were antigenieally the same as the cell culture passaged virus and could be clearly differentiated from the mttv strain 2. a histological study of affected animals showed inflammation in the brains and vacuolar degeneration of the intestinal villi only in the case of s.c. inoculation. however, there was no evidence of inflammation in the liver and other organs (dr. 3/[. kubo, personal communiea, tion). kaye et al. (4) reported that they had adapted the american strain of calf diarrhea coronavirus to suckling mouse brain. on ~he other hand, dea et al. (2) reported that the american strain and their new isolates of calf diarrhea coronavirus were not virulent for suckling mice, hamsters and guinea pigs. our present results support kaye's findings. nt tests and histological studies have ruled out contamination with mhv. further studies, however, wil] be necessary to investigate the immunological and pa~thological differences between bcv and mi-iv. properties of a coronavirus isolated from a cow with epizootic diarrhea physicochemical and biological properties of neonatal calf diarrhea eoronaviruses isolated in quebec and comparison with the nebraska calf eoronavirus replication of bovine coronavirus in cell line ben-i culture calf diarrhoea coronavirus antigenic relationship between human eoronavirus strain oc 43 and hemagglutinating encephalomyelitis virus strain 67n of swine: antibody responses in human and animal sera coronaviruses: a comparative review feline infectious peritonitis virus. ii. propagation in suckling mouse brain neonatal calf diarrhea: purification and electron microscopy of a coronavirus-iike agent epizootic diarrhoea of adult cattle associated with a coronavirus-like agent authors' address: dr. h. akas~i, national institute o~' animal ttealth a-1011 wien. fiir den text~eil verantwortlieh: dr. wilhelm schwabl, m61kerbastei 5, a-1011 wish. fiir den anzeigenteil verantwortlieh: niag. bruno schweder, m61kerbastei 5, a-1011 wien key: cord-000249-hkc4vbmj authors: schughart, klaus title: sysgenet: a meeting report from a new european network for systems genetics date: 2010-07-11 journal: mamm genome doi: 10.1007/s00335-010-9273-7 sha: doc_id: 249 cord_uid: hkc4vbmj the first scientific meeting of the newly established european sysgenet network took place at the helmholtz centre for infection research (hzi) in braunschweig, april 7-9, 2010. about 50 researchers working in the field of systems genetics using mouse genetic reference populations (grp) participated in the meeting and exchanged their results, phenotyping approaches, and data analysis tools for studying systems genetics. in addition, the future of grp resources and phenotyping in europe was discussed. sysgenet is funded through the cost framework (http://www.cost.eu/about_cost). cost is an intergovernmental framework for european cooperation in science and technology that promotes and coordinates nationally funded research in europe, through funding of research networks. sysgenet is coordinated by klaus schughart (helmholtz center for infection research, braunschweig, germany) . more detailed information about sysgenet can be obtained from the website http://www.helmholtzhzi.de/sysgenet/. infectious diseases continue to represent a threat to human health. due to global warming and travel, newly emerging diseases are spreading at an unprecedented rate around the world. examples are the dissemination of antibioticsresistant mycobacteria, the new swine influenza virus variant, sars, and west nile virus (wnv). several research groups are using mouse grps to identify complex genetic influences on the host susceptibility to infections. grps have been and will continue to be an important basis for understanding infectious diseases in humans. a very good example for translational research was presented by pascal rihet, who identified genomic susceptibility regions to malaria in human populations in africa (delahaye et al. 2007 ) and then continued to compare these results with studies in mouse grps. in this way, a region on human chromosome 5 and its homologous regions on mouse chromosomes 11 and 18 were identified. subsequent expression studies in mice will now help to determine the molecular networks and genes involved. paul denny described the mapping of genetic susceptibility to streptococcus pneumonia infections in mouse inbred strains to chromosomes 7 (denny et al. 2003) and 4 (unpublished) . infection susceptibility to influenza was described by klaus schughart, who also pointed out that high susceptibility includes a hyperreactive immune response in the host (srivastava et al. 2009 ). xavier montagutelli generated a unique resource of mus spretus 9 c57bl/6 j interspecific recombinant congenic strains that carry different genomic fragments of mus spretus on a c57bl/6 j background (burgio et al. 2007 ). this grp was used to identify resistance and susceptibility regions to various pathogens, including rift valley fever, west nile virus, yersinia pestis, and influenza. the first lines of the collaborative cross strains have been screened by fuad iraqi for a number of susceptibility loci to various pathogens (unpublished). it was remarkable to see that several quantitative trait loci (qtl) showed high significance and that the genomic intervals for several loci were very narrow, which should make it possible to identify quickly the underlying quantitative trait genes. metabolic diseases in humans are dramatically on the rise; obesity and related diseases in particular represent a serious challenge to future health systems. several groups addressed the complex genetics of metabolic functions and disorders using different mouse grps. gudrun brockmann reported on the mapping of qtls for obesity in a specific mouse strain isolated in berlin and the bxd congenic strain set (neuschl et al. 2010) . the future goal is to relate these qtls to genetic polymorphisms that influence the immune system. joan campbell-tofte presented the use of herbal extracts for the treatment of type 2 diabetes in humans. she nicely illustrated the use of mouse models: from human to mouse to humans and back to the mouse. pénélope andreux reported on the setting up of a mouse clinic in lausanne for a systematic analysis of mouse grps for a large number of metabolic phenotypes, including mitochondrial functions (koutnikova et al. 2009 ). juan m. falcon-perez described the genomic, proteomic, and metabolic phenotyping capabilities of their technological platform and introduced extracellular microvesicles and metabolomic profiles as two new biological sources for identifying biomarkers for the detection and monitoring of hepatic diseases (hackenberg et al. 2009 ). abnormalities and diseases of the liver are also the subject of studies presented by karl kashofer (kashofer et al. 2009 ). several loci for (nonalcoholic) steatohepatitis have been mapped in chromosome substitution strains, and a more detailed mapping in subcongenic strains is ongoing. although rats in general were the species of choice for use by experimental psychologists to study behavior, mice have been the preferred animal for behavior geneticists since at least the 1940 s. in addition, the adaptation of behavioral assays and the development of new methods have confirmed the mouse as one of the most preferred experimental systems to learn more about the genetic underpinnings of behavior and associated phenotypes. martien kas described the currently underlying scientific rationale. precise measurement of a well-described behavioral trait across a grp will lead to the identification of associated genes and genomic regions. in the next step these genes can be used to find homologous genes and pathways that contribute to the development of neuropsychiatric disorders in humans. the translational value of this interspecies genetic approach was nicely exemplified in a study in which a qtl for avoidance behavior in mice was related to bipolar disorders in humans (de mooij-van malsen et al. 2009 ). in a similar approach, iris hovatta used a cross-species neurogenomics comparison to correlate brain region-specific gene expression patterns and anxiety-like behavior in mouse grps to polymorphisms in the finnish population for anxiety disorders (hovatta et al. 2005) . the mouse genes allowed identification of potential candidate genes in humans who predispose to anxiety disorders. paul franken presented studies on the identification of genetic traits that influence homeostatic and circadian aspects of sleep, and the electroencephalogram in the bxd grp and in inter-and backcross panels of mice (shaw and franken 2003) . several genes that play a decisive role were identified, and further phenotyping of the extended bxd grp is planned. eero vasar and sulev kõks described the role of the wfs1 gene in knockout mice for anxiety behavior, an altered response to morphine and the release of striatal dopamine (koks et al. 2009; luuk et al. 2009 ). ewelina knapska reported the use of a highly sophisticated cage system, intellicage, to automatically record a number of different complex behavioral traits in mice (jaholkowski et al. 2009 ). mice can be housed in social groups but nevertheless tested individually. ryszard przewlocki reported on a systematic study on various inbred mouse strains to identify genetic determinants of alcohol and drug addiction (piechota et al. 2010) . combining these studies with comprehensive gene expression analyses revealed that glucocorticoid receptor-activated gene expression pathways play an important role. wim crusio studied behavioral traits in learning and related them to brain anatomy (crusio and schwegler 2005) . thereby, the extent of neuron projections in the hippocampus could be correlated to more efficient learning capabilities and these two phenotypes are very strongly correlated genetically. guus smit gave an overview on a collaborative effort in the netherlands in which several research groups have determined various behavioral phenotypic traits and qtls in a commonly used bxd population (loos et al. 2009 ). also, they established a mouse facility in which they are using automated screening cages with sophisticated video recording and analysis. cancer is still one of the most frequent causes of death in western countries, and understanding its molecular causes as well as establishing appropriate animal model systems for the development of new treatment strategies is very important. fragiskos kolisis reported on the setting up of an infrastructure for a systems biology approach to carcinogenesis and aging (chatziioannou et al. 2009 ). understanding proteasome function and dysfunction as well as studying the alterations of the genome and proteome that account for different cancer phenotypes in a mouse skin carcinogenesis model are among the research goals. javier santos used grps to identify genetic traits for the susceptibility to radiation-induced thymic lymphomas in interspecific recombinant congenic and consomic mouse strains (santos et al. 2009 ). frank lammert developed assay systems to determine genetic causes of liver fibrosis and inflammatory liver carcinogens in the bxd mouse recombinant congenic grp (weber et al. 2008) . leonard schalkwyk studied allele-specific methylation in humans (schalkwyk et al. 2010) . he estimated that potentially more than 35,000 sites in the genome exhibit allele-specific modifications, and of these 10% are not in cis, a number that largely exceeds the number of known imprinted loci. these findings suggest that individual genetic heterogeneity may be much larger than estimated thus far and may contribute to individual phenotypic differences. jiri forejt used inter-subspecific consomic strains (gregorova et al. 2008) to investigate male sterility and its consequences for interspecies hybrid sterility (mihola et al. 2009 ). furthermore, in the livers of inter-subspecific hybrid strains he discovered new patterns of gene expression that were absent from both parental strains. the capture, storage, handling, and analysis of large data sets will present a specific challenge for future systems genetics projects. ritsert jansen and pjotr prins presented their approaches to integrate data from various phenotypic studies, encompassing gene expression, metabolome, and classical traits, and to develop new tools for advanced and improved mapping of qtls (jansen et al. 2009; li et al. 2008; swertz and jansen 2007) . these tools will be provided to the scientific community. andreas beyer gave a report on how to integrate data obtained at the post-transcriptional level with rna expression data. several loci that influence the post-transcriptional regulation of gene products could be identified in yeast. steffen möller presented his suite for the analysis of expression qtl (http://eqtl.berlios.de), which is being applied to the analysis of experimental autoimmune encephalomyelitis in mouse and rat. anastasios bezerianos presented a platform and developments for the identification of gene regulatory networks integrating protein-protein interactions and microarray data (bezerianos and maraziotis 2008). they started with yeast data and will soon expand to mouse, concentrating on time-varying gene regulatory networks. morris swertz presented xgap, a software platform developed for data management and integration of large data sets from phenotyping and genotyping studies (swertz et al. 2010) . grant morahan described the development of an extended tool for webqtl that allows a genome-centric analysis of qtl interactions (unpublished). the collaborative cross (cc) is currently being generated as a community resource for more sensitive and refined mapping of qtls. the goal is to breed a large population of recombinant inbred strains starting from eight founder strains. the eight founder strains were selected to capture a large portion of the genetic variation in the mouse genome. in fact, the genetic variation represented in the cc will be twice the genetic variation present in the human population (reviewed in valdar et al. 2006) . the three sites where the resource is being generated reported the present status of their breeding colonies; the final goal is to generate a total of 700 lines (chesler et al. 2008; iraqi et al. 2008; morahan et al. 2008a) . grant morahan gave an update on the ''southern cross'' being established in perth, australia. an inbreeding depression was observed at generations 7-9. at present, about 200 strains have been bred beyond generation 10. the first 40 strains are expected to be inbred by the end of the year. david threadgill reported the status of the breeding colony at the university of north carolina, chapel hill, nc, usa. about 300 lines are currently breeding at unc. the first 50 recombinant inbred lines will be available by the end of this year and 200 lines by the end of 2011. to speed up the inbreeding process, markerassisted breeding will be used to create homozygous lines beyond generation 12. genome analysis demonstrated that all parental genomes are well represented in the advanced generations. the first phenotyping analysis showed a large variation in body weight, exercise propensity, and susceptibility to pathogens. richard mott described the genome structure of a smaller cc colony, funded by the wellcome trust, which was developed by fuad iraqi and is presently housed in tel aviv, israel. a first phenotyping analysis for the qtls that affect recombination frequencies was performed. a full-genome sequencing project to complete the parental strains with high coverage is underway at the sanger institute. the two-day meeting in braunschweig has clearly demonstrated the great value of mouse grps in identifying genetic determinants of complex genetic traits for various phenotypic traits related to diseases in humans. the partners of the network collectively have great expertise in disease phenotyping and analysis of genetic reference populations. several examples that illustrated the translation of the knowledge gained in the mouse experimental systems to humans were presented. links to clinical researchers already exist at several places but will have to be further expanded in the future. furthermore, mouse grps can be ideally combined with mouse mutant lines carrying a gene-knockout mutation to determine the effect of a strong genetic defect in combination with modifier genes. it also became clear that a strong and sustained financial investment in mouse breeding and phenotyping facilities as well as in bioinformatic infrastructure is urgently needed to further advance a systems genetics approach in europe. open access this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. greece; sulev kõks, university of tartu, estonia; frank wfs1-deficient mice display impaired behavioural adaptation in stressful environment detection and interpretation of expression quantitative trait loci (eqtl) a mouse speciation gene encodes a meiotic histone h3 methyltransferase establishment of ''the gene mine'': a resource for rapid identification of complex trait genes systems genetics can provide new insights into immune regulation and autoimmunity a unique genetic defect on chromosome 3 is responsible for juvenile obesity in the berlin fat mouse a new set of bxd recombinant inbred lines from advanced intercross populations in mice the mouse as a model for human biology: a resource guide for complex trait analysis the dissection of transcriptional modules regulated by various drugs of abuse in the mouse striatum the polymorphism architecture of mouse genetic resources elucidated using genome-wide resequencing data: implications for qtl discovery and systems genetics a role for stroma-derived annexin a1 as mediator in the control of genetic susceptibility to t-cell lymphoblastic malignancies through prostaglandin e2 secretion allelic skewing of dna methylation is widespread across the genome perchance to dream: solving the mystery of sleep through genetic analysis host genetic background strongly influences the response to influenza a virus infections beyond standardization: dynamic software infrastructures for systems biology xgap: a uniform and extensible data model and software platform for genotype and phenotype experiments simulating the collaborative cross: power of quantitative trait loci detection and mapping resolution in large sets of recombinant inbred strains of mice genetic determinants in hepatic fibrosis: from experimental models to fibrogenic gene signatures in humans bao l, wei l, peirce jl, homayouni r, li h et al (2006) combining gene expression qtl mapping and phenotypic spectrum analysis to uncover gene regulatory relationships. key: cord-288133-h3wmo0xj authors: hickman, debra l title: evaluation of the neutrophil:lymphocyte ratio as an indicator of chronic distress in the laboratory mouse date: 2017-06-23 journal: lab anim (ny) doi: 10.1038/laban.1298 sha: doc_id: 288133 cord_uid: h3wmo0xj when evaluating the effect of husbandry and biomethodologies on the well-being of laboratory mice, it is critical to utilize measurements that allow the distinguishing of acute stress from chronic stress. one of the most common measurements of stress in laboratory animals is the corticosterone assessment. however, while this measurement provides a highly accurate reflection of the animal's response to acute stressors, its interpretation is more prone to error when evaluating the effect of chronic stress. this study evaluated the use of the neutrophil:lymphocyte (ne:ly) ratio as an assessment of chronic stress in male and female c57bl/6n mice as compared to serum corticosterone. one group of mice was exposed to mild daily stressors for 7 days, while the control group was handled with normal husbandry. the ne:ly ratio and serum corticosterone levels were significantly elevated in the chronically stressed mice, though a significant increase in corticosterone was only significant in males when compared by sex. the chronically stressed mice also demonstrated significantly fewer entries into the open arms and less time spent in the open arms of the elevated plus maze, suggesting that the mild daily stressors had induced a state of distress. the findings of this study confirm that the ne:ly ratio is a valid measurement for chronic stress in the laboratory mouse. however, these assays do not distinguish between distress or eustress, so behavioral and physiological assessments should always be included to determine a complete assessment of the well-being of the mouse. supplementary information: the online version of this article (doi:10.1038/laban.1298) contains supplementary material, which is available to authorized users. when designing studies that use animal models, it has been recognized that the results are more translatable to human disease when the subjects are healthy with minimal stressors [1] [2] [3] [4] [5] [6] . but, not all stress is bad, so it is critical that evaluations of animal well-being differentiate between distress (that which results in decreases in animal well-being as the animal is unable to cope or adapt) and eustress (stressors which enhance the functioning of the animal) 7, 8 . appropriate selection of assessments of animal well-being can assist laboratory animal professionals as they seek to evaluate and improve current practices 9 , especially with mice. generally, the most common method to assess laboratory mouse well-being has been focused on assessment of the response to acute stressors (i.e. an injection, brief restraint, a cage change) 10 . these stress events have consistently been associated with elevations in mouse serum and fecal corticosterone 6, 11, 12 and neuropeptides 13 . mice that have been exposed to acute stressors also have shown increased anxiety when assessed by the elevated plus maze and other behavioral assessments [14] [15] [16] [17] , in addition to elevations of heart rate and blood pressure [18] [19] [20] . it has been more challenging to measure states of chronic stress, where an animal was exposed to the stressors for hours or days instead of seconds or minutes. corticosteroids are commonly used in these assessments, but interpretation is complicated because corticosterone levels are influenced by factors that include the method of sample collection, sex, immune status, circadian rhythm, and nutritional status 10 . they also increase when an animal experiences extreme fear (distress) or extreme pleasure (eustress) 21 , requiring the addition of behavioral assessment to accurately determine the state of the animal. changes in body weight and organ weights relative to body weight have been among the more reliable measures of chronic stress as these measure change more slowly, making them a stronger reflection of the effects of exposure to chronic stressors 22-24 . work by this laboratory and others have noted that chronic stressors that induce increase glucocorticoid responses also induce when evaluating the effect of husbandry and biomethodologies on the well-being of laboratory mice, it is critical to utilize measurements that allow the distinguishing of acute stress from chronic stress. one of the most common measurements of stress in laboratory animals is the corticosterone assessment. however, while this measurement provides a highly accurate reflection of the animal's response to acute stressors, its interpretation is more prone to error when evaluating the effect of chronic stress. this study evaluated the use of the neutrophil:lymphocyte (ne:ly) ratio as an assessment of chronic stress in male and female c57bl/6n mice as compared to serum corticosterone. one group of mice was exposed to mild daily stressors for 7 days, while the control group was handled with normal husbandry. the ne:ly ratio and serum corticosterone levels were significantly elevated in the chronically stressed mice, though a significant increase in corticosterone was only significant in males when compared by sex. the chronically stressed mice also demonstrated significantly fewer entries into the open arms and less time spent in the open arms of the elevated plus maze, suggesting that the mild daily stressors had induced a state of distress. the findings of this study confirm that the ne:ly ratio is a valid measurement for chronic stress in the laboratory mouse. however, these assays do not distinguish between distress or eustress, so behavioral and physiological assessments should always be included to determine a complete assessment of the well-being of the mouse. an apparent immunosuppression that presents within a couple of hours of elevated corticosterone exposure as a neutrophilia that may include a concurrent lymphopenia [25] [26] [27] [28] . these immunologic changes are also seen in disorders which are associated with elevated glucocorticoids 29, 30 and other chronic illnesses, supporting the role of glucocorticoids in the apparent immunosuppression. the high correlation coupled with the relatively low cost to obtain a complete blood count has resulted in use of the neutrophil:lymphocyte (ne:ly) ratio to assess stress in multiple species 25, 27 . the objective of this study was to determine if the ne:ly ratio was consistent with serum corticosterone as a measurement of chronic stress in the laboratory mouse. additionally, we included the elevated plus maze to determine if the ne:ly ratio accurately measured distress in the chronically stressed mice, as compared to the behavior of the non-manipulated, non-stressed mice. the indiana university school of medicine (iusm) animal care and use program was in compliance with federal regulations and accredited by aaalac, international. all procedures were approved by the iusm iacuc prior to initiation of the study. the study was structured with a between-subjects design where subjects were randomly assigned to the independent variable of "stress exposure" which had two groups. the first group ("chronically stressed") was exposed to random, mild daily stressors for 7 consecutive days. the second group ("non-stressed") was housed and handled as per usual in the animal facility. each group consisted of 6 males and 6 females which were randomly assigned to their treatment group upon arrival at the animal facility. random assignment was accomplished by assigning each animal a number, then using a random number generator to assign animals to treatment groups. twelve male and twelve female c657bl/6ncrl mice (charles river laboratories, wilmington, ma) were used for this study. the mice were approximately 10 weeks of age. as social housing in mice has been demonstrated to prevent stress-associated responses 31, 32 , the mice in the chronically stressed group were housed individually, while the non-stressed animals were group housed. they were allowed to acclimate for 7 days prior to initiating the experimental manipulations. all animals were housed in shoebox caging with filter tops (alternative design, siloam springs, ar) on corncob bedding (bed o' cob, andersons, maumee, oh) under static conditions. all mice were provided with tissue material for nesting. both males and females were housed in the same room, but the mice in each treatment group were housed in different rooms as described below. the cages measured 18.5 cm w x 30 cm d x 12 cm h. feed (labdiet 2018, st. louis, mo) and water were provided ad libitium. standard operating procedures for the animal facilities required that all cages be changed at least once weekly. soiled cages were sanitized in a mechanical cage washer with a final rinse temperature of 180° f (82° c) and autoclaved prior to reuse. the macroenvironment was kept on a 12:12-h light:dark cycle (lights on at 0:700, off at 19:00) with temperature and humidity maintained at 72°f (22°c) and at least 30%, respectively. indirect sentinels were utilized to assess colony health. at the time of this study, the colony was free of mouse parvovirus, mouse coronavirus, mouse theliovirus, pneumonia virus of mice, sendai virus, reovirus 3, mouse rotavirus, lymphocytic choriomeningitis virus, mycoplasma pulmois, and ectoparasite and endoparasites. half of the mice were exposed to a daily stressor for 7 consecutive days. these stressors were presented in a random order and included: (1) forced swim, (2) heat stress, (3) shaker stress, and (4) restraint stress modified from previously published work 33 . for the forced swim, the mouse was gently placed in a 13 cm diameter x 30 cm tall clear plexiglass cylinder filled with water at 70° to 75° f (21° to 24° c). they remained in the water until they began floating with positional adjustments or 2 minutes, whichever was longer. they were removed, dried and allowed to warm. after 10 minutes of rest, the swim stress was repeated for a maximum of 4 sessions per mouse. for the heat stress, the mouse was placed in a clean cage with no bedding and a wire mesh to prevent escape. an incandescent light was positioned above the cage to create heat in the cage. the temperature was monitored and the target temperature was 85° f (29° c). the mouse was removed if the intracage temperature exceeded 90° f (32° c). they stayed in the cage for 2 hours and were continuously monitored during this stressor. for the shaker stress, the mouse remained in its home cage which was placed on a plate shaker that provided gentle, continuous shaking at no more than 160 rpm for 2 hours. mice were assessed every 30 minutes during this stressor. for the restraint stress, mice were restrained in a 50ml centrifuge tube with air-holes for a maximum of 3 hours. mice were continuously monitored through this stressor. the actual times and stressor assignments are provided as supplemental data. these mice were individually housed and held in a room isolated from other mice and outside of the main animal facility to minimize the potential effect of pheromones on other ongoing studies, including the non-stressed controls for this study. the cages for mice in this group were situated so that they were at average human eye level. these mice were tested and euthanized on day 8 of the study. the non-stressed mice were housed in groups of 3 mice of the same sex per cage. they were handled normally, with no additional interactions with the research staff. the cages for mice in this group were situated so that they were at average human thigh to knee level. the non-stressed mice were tested and euthanized on day 7 of the study to avoid release of pheromones from the stressed mice influencing the response of the non-stressed mice. all mice were assessed on an elevated plus maze at the end of the 7 consecutive days of daily stressors for the chronically stressed mice. the maze measured 25 cm l x 5 cm w open arms, 25 cm l x 5 cm w x 16 cm h closed arms, center platform of 5 cm x 5 cm. the closed arms were opaque. mice were brought to the testing room and allowed a minimum of 30 minutes to acclimate to the training room before being tested. each mouse was placed on the center platform to start the epm and digitally recorded for 10 minutes. at the end of 10 minutes, the mouse was removed from the maze and the maze was cleaned with 70% isopropyl alcohol before the next mouse. the proportion of entries into the open arms relative to the total number of arm entries was compared between treatment groups. the proportion of time each mouse spent in the open or closed arms relative to the total time in the maze was also compared between treatment groups. each mouse was tested once. all trials were between 8:00 and 12:00. all non-stressed mice were tested separately from the chronically stressed mice. data from two non-stressed and one chronically stressed mouse were not collected due to complications with the digital recording. after being tested on the elevated plus maze, each mouse was anesthetized with pentobarbital (390 mg/ml, fatal plus, vortech pharmaceuticals, dearborn, mi) administered ip. once anesthetized, a terminal blood collection was collected from the heart. half of the sample was placed in an edta treated tube and the remainder was placed in a serum separator tube and subsequently centrifuged to allow collection of the serum. the serum samples were stored at −80° f (−62° c) until processed. all samples were collected between 8:00 and 12:00 to control for alterations due to circadian rhythm. the edta sample was placed in a hemavet (drew scientific, waterbury, ct) and a complete blood count was performed. the mean of the white blood count, total number of neutrophils, total number of lymphocytes, and ne:ly ratio were compared between treatment groups. the serum corticosterone levels were assessed using a commercially available elisa kit (corticosterone rat/mouse, mp biomedicals, llp, solon, oh). the mean serum corticosterone levels were compared between treatment groups. we were unable to collect enough blood for serum from 2 of the non-stressed mice. to determine the appropriate animal numbers to be used prior to the initiation of the study, we performed a power analysis using our previous work with the neutrophil:lymphocyte ratio in our laboratory as the basis for the analysis. an online calculator was utilized 34 with µ a set at 0.4, µ b set at 0.25, σ set at 0.09, a sampling ratio (ê) of 1, power at 0.80 and α at 5%. this calculated a sample size of 6 animals per sex per group. data analyses were performed using a generalized linear model (jmp 10, cary, nc). the animal was the experimental unit. a single observation was made for each animal and this data was averaged per animal for analysis using a simple model of treatment, sex, and their interaction. if interactions were found to be non-significant, the models were updated by removing the interactions terms to retest the main effects and these values were reported. a full-factorial glm was completed for each observational unit. if interactions were found to be significant, these results were reported. all summary data are presented as box-and-whisker plots with whiskers representing min and max values, boxes first and third quartiles, and middle line the median value. there were no significant differences between the treatment groups in the total white blood cell count (f[1,23] = 2.0245, p=0.1682) ( fig. 2a; see supplementary figs. 6-9 for raw data plots), the total number of neutrophils (f[1,23] = 0.5778, p=0.4549) (fig. 2b; see supplementary figs. 6-9 for raw data plots), or the total number of lymphocytes (f[1,23] = 3.4353, p=0.0767) ( fig. 2c ; see supplementary figs. 6-9 for raw data plots). the ne:ly ratio of the chronically stressed mice was significantly increased as compared to the non-stressed mice (f[1,23] = 7.4200, p=0.0121) ( fig. 2d ; see supplementary figs. 6-9 for raw data plots). there was no interaction of sex with treatment for the total white blood cell count (p=0.8018), total number of neutrophils (p=0.6877), total number of lymphocytes (p=0.8665), and the ne:ly ratio (p=0.5542). the chronically stressed mice exhibited a significant increase in their levels of serum corticosterone as compared to the non-stressed mice (f[1,21 = 5.3431, p=0.0310). there was a significant interaction of sex and treatment (p=0.0389). chronically stressed male mice expressed significantly higher levels of serum corticosterone as compared to the non-stressed male mice (f[1,8] = 9.0825, p=0.0167), but females expressed no significant differences between non-stressed and chronically stressed mice (f [1, 11] = 0.1282, p=0.7270) ( fig. 3 ; see supplementary figs. 10 and 11 for raw data plots). the mice that had been exposed to 7 days of mild stressors mice demonstrated a significant increase in their serum corticosterone levels, suggesting that these mice were more stressed as compared to the mice that were handled routinely. not unexpectedly, the study also documented a sex difference in serum corticosterone levels, with the chronically stressed male mice exhibiting a significantly higher corticosterone level as compared to the chronically stressed female mice. additionally, the behavioral assessment of the chronically stressed mice demonstrated that they were less likely to spend time on the open arms and less likely to enter the open arms of the elevated plus maze. as demonstrated by previous studies, these findings suggest that the mice were more anxious and distressed 35 as compared to the mice that were handled routinely. these behavioral findings suggest that the daily stressors were adequately disturbing to create a situation of distress for these mice. the finding that the ne:ly ratio was also increased in the chronically stressed mice suggests that this assessment can be a reliable tool for the measurement of stress in the laboratory mouse. there was also a non-significant neutrophilia and lymphopenia in the chronically stressed mice, but all values were within published normal for c57bl/6ncrl mice 36 . these findings are consistent with other published studies that chronic exposure to elevated corticosterone causes a shift of neutrophilia with or without a concurrent lymphopenia 10, 25, 27 , and can be measured successfully by evaluating the ne:ly ratio of experimental treatment groups as compared to control groups 25, 27 . because the differences were robust and reflective of the behavioral changes, this suggests that the ne:ly ratio is an appropriate measurement to use when assessing chronic distress in a clinically healthy mouse. the measurement of the ne:ly ratio also has significant advantages over the measurement of serum corticosterone. although this study utilized an automated hemocytology machine, the ne:ly ratio can be calculated from a blood smear by manually performing a differential 25, 27 . measurement of serum corticosterone requires the use of specialized kits and additional machinery, such as elisa readers, for accurate calculation of the concentrations. as previously reported in the laboratory rat, the ne:ly ratio also is not influenced by the response to acute stressors, such as handling to obtain the blood collection 27 . it is also less sensitive to potentially confounding variables such as time of day, sex (which was seen in the serum corticosterone results of this study) and appetence, though it can be affected by chronic pathogen exposure 25, 27 . in this study, we conclude that the ne:ly ratio is an acceptable immunological measurement of exposure to chronic stress in the clinically healthy laboratory mouse. it can be run inexpensively without specialized equipment and is reliably robust in the face of individual and environmental factors. however, as should be considered for all assessments of stress in animals, this method should not be used alone, but as part of a multi-faceted panel of assessments that include physiologic and behavioral assessments to determine if changes are due to distress, eustress, or disease status 9,10 . note: any supplementary information and source data files are available in the online version of the paper. reducing mouse anxiety during handling: effect of experience with handling tunnels aspen shaving versus chip bedding: effects on breeding and behavior the effect of handling method on the mouse grimace scale in two strains of laboratory mice cage enrichment with paper tissue, but not plastic tunnels, increases variability in mouse model of asthma happy animals make good science effects of cage-change frequency and bedding volume on mice and their microenvironment stress and distress corticotropin-releasing factor in the hippocampus: eustress or distress? defining, measuring, and interpreting stress in laboratory animals domestic animal behaviour and welfare a robust and reliable non-invasive test for stress responsivity in mice glucocorticoids in the nonobese diabetic (nod) mouse: basal serum levels, effect of endocrine manipulation and immobilization stress stress and welfare: two complimentary concepts that are intrinsically related to the animal's point of view antidepressant action of agomelatine (s 20098) in a transgenic mouse model unaltered hormonal response to stress in a mouse model of fragile x syndrome marble burying as a test of the delayed anxiogenic effects of acute immobilisation stress in mice feeding, exploratory, anxiety-and depression-related behaviors are not altered in interleukin-6-deficient male mice hypertensive response to acute stress is attenuated in interleukin-6 knockout mice sympathetic and angiotensindependent hypertension during cage-switch stress in mice mouse strain differences in autonomic responses to stress causes and consequences of stress body condition scoring: a rapid and accurate method for assessing health status in mice effects of chronic social stress on neuroendocrine responsiveness to challenge with ethanol, dexamethasone and corticotropin-releasing hormone the context specificity of anxiety responses induced by chronic psychosocial stress in rats: a shift from anxiety to social phobia? the use of leukocyte profiles to measure stress in vertebrates: a review for ecologists corticosteroid-induced modulation of immunoglobulin secretion by human b lymphocytes: potentiation of background mitogenic signals evaluation of the neutrophil-lymphocyte ratio as a measure of distress in rats endogenous corticosteroids mediate the neutrophilia caused by platelet-activating factor in the mouse diseases of the adrenal cortex of dogs and cats pergolide treatment for cushing's syndrome in a horse activation of the mouse odorant receptor 37 subsystem coincides with a reduction of novel environment-induced activity within the paraventricular nucleus of the hypothalamus social modeling of conditioned fear in mice by nonfearful conspecifics dynamics and correlation of serum cortisol and corticosterone under different physiological or stressful conditions in mice a review of the validity and variability of the elevated plus-maze as an animal model of anxiety c57bl/6 mouse clinical pathology data this study was funded from internal sources. the author thanks melissa swan, jessica peveler, and brittany baker for their assistance with the data collection for this study. the author declares no competing financial interests. key: cord-306516-5t3ix35e authors: li, minghui; su, yangang; yu, yong; yu, ying; wang, xinggang; zou, yunzeng; ge, junbo; chen, ruizhen title: dual roles of calpain in facilitating coxsackievirus b3 replication and prompting inflammation in acute myocarditis date: 2016-10-15 journal: int j cardiol doi: 10.1016/j.ijcard.2016.07.121 sha: doc_id: 306516 cord_uid: 5t3ix35e background: viral myocarditis (vmc) treatment has long been lacking of effective methods. our former studies indicated roles of calpain in vmc pathogenesis. this study aimed at verifying the potential of calpain in coxsackievirus b3 (cvb3)-induced myocarditis treatment. methods: a transgenic mouse overexpressing the endogenous calpain inhibitor, calpastatin, was introduced in the study. vmc mouse model was established via intraperitoneal injection of cvb3 in transgenic and wild mouse respectively. myocardial injury was assayed histologically (he staining and pathology grading) and serologically (myocardial damage markers of ck-mb and ctni). cvb3 replication was observed in vivo and in vitro via the capsid protein vp1 detection or virus titration. inflammation/fibrotic factors of mpo, perforin, ifnγ, il17, smad3 and mmp2 were evaluated using western blot or immunohistology stain. role of calpain in regulating fibroblast migration was studied in scratch assays. results: calpastatin overexpression ameliorated myocardial injury induced by cvb3 infection significantly in transgenic mouse indicated by reduced peripheral ck-mb and ctni levels and improved histology injury. comparing with cvb3-infected wild type mouse, the transgenic mouse heart tissue carried lower virus load. the inflammation factors of mpo, perforin, ifnγ and il17 were down-regulated accompanied with fibrotic agents of smad3 and mmp2 inhibition. and calpain participated in the migration of fibroblasts in vitro, which further proves its role in regulating fibrosis. conclusion: calpain plays dual roles of facilitating cvb3 replication and inflammation promotion. calpain inhibition in cvb3-induced myocarditis showed significant treatment effect. calpain might be a novel target for vmc treatment in clinical practices. viral myocarditis (vmc) is a common cardiovascular disease in clinical practice [1] . while many vmc cases showed a self-limited regression, there are still approximately 30% cases progressing into dilated cardiomyopathy (dcm) and heart failure that might require mechanical circulatory support or heart transplant at the end stage despite of the optimal pharmacotherapy [1, 2] . so an effective treatment of vmc in the early stage is meaningful. vmc treatment has always been a challenge, especially with regard to the immunosuppressive treatment [2] . many researchers considered vmc an autoimmune disease [3] , which made immunosuppression treatment reasonable. however, virus persistence in the myocardium [1, 4] holds back the intended host immune suppression because this might enhance virus replication. clinical observations of myocarditis immunosuppression therapy also returned controversial results [1] . as such, guidelines also weight the words on this issue [2] . this ambiguity might be attributed to several reasons. 1) the core concern might be that it is a risk to prescribe immunosuppressant agents considering the role of host immune responses in virus clearance. 2) besides, we haven't completely clarified the life cycle of the intruded-viruses. so we don't know the timing to interfere the disease course. 3) the patterns that viruses interact with hosts were basically vague. so we don't know the appropriate methods to interfere. 4) last but not least, myocarditis-causing viruses' spectrum has always been shifting temporally and regionally [5] , which adds another variable for clinical specialists when facing a unique patient suffering from vmc. thus, logically speaking, a candidate compromising these two factors, virus replication and host inflammation response, might be an optimal choice. we found in our former study that calpain, a family of neutral cysteine proteases, could facilitate coxsackievirus b3 (cvb3, a common causing virus of vmc) replication in vitro [6] . besides, many studies demonstrated that calpain mediates systemic inflammation response in vivo [1] . so, calpain might participate both in the virus life cycle and the host inflammation response simultaneously. if it is the case, calpain would be an optical choice of myocarditis treatment. calpain activity is regulated by the endogenous inhibitor, calpastatin. calpastatin is an intracellular 110-kda protein that inhibits calpain activity specifically. binding of calpastatin across the calpain active site would block its access to substrates in vivo. however, calpastatin could not penetrate across the cell membrane for the large molecular weight and cannot be used as an inhibitor in vitro. thus, we introduced here a transgenic mouse overexpressing calpastatin in vivo to discuss this hypothesis in the mouse model of cvb3induced viral myocarditis. coxsackievirus b3 (nancy strain) was preserved in key laboratory of viral heart diseases, zhongshan hospital, fudan university. calpain inhibitor i (n-acetyl-leu-leu-norleucinal, alln), an inhibitor of neutral cysteine proteases, was purchased commercially (biomol research laboratories inc. usa, catalog no.: p-120) and dissolved in dimethyl sulfoxide (dmso, amresco llc., usa, product code: n182) with a final concentration of less than 0.1% in cell culture studies. alln was further diluted using culture medium into target concentrations during the research. the calpastatin transgenic mouse strain (tg-cast) was introduced from the laboratory of tianqing peng (lawson health research institute, canada) and was bred in the department of laboratory animal science, fudan university in a standard specific pathogen free (spf) environment. male inbred c57bl/6 mice aging3-6 weeks were used for the experiment and were grouped as: wild type control group (con. n = 10), transgenic mouse control group (tg-cast, n = 10), cvb3-infected group (virus n = 15) and cvb3-infencted transgenic mouse group (virus + tg-cast, n = 15). all littermates were genotyped following the protocol of peng's lab. we give assurance that all animals received humane care and that study protocols was approved by the ethical committee of fudan university. the cvb3-induced viral myocarditis model was established following the protocol sd previous [7] . in short, 100,000 tcid 50 /ml × 0.3 ml cvb3 was injected intraperitoneally with the same amount of pbs for the controls. then, all mice were kept in spf environment for 7 days before sacrifice. occasional dead mouse were excluded from the study. blood samples were collected via picking the eye balls and serum were separated via centrifuge (3000 r/min for 5 min). methods and grading standards were prescribed elsewhere [7] . level of peripheral ctni and ck-mb were measured by elisa assays following the protocol in former study [6] . heart tissue were grinned using homogenizer in lysis buffer (50 mm tris-hcl, ph 7.2, 250 mm nacl, 0.1% np-40, 2 mm edta, 10% glycerol) containing protease inhibitor cocktail on ice. tissue homogenate were centrifuged at 12,000 g for 10 min at 4°c. the protein concentration was determined by the bradford assay (bio-rad). detailed western blot protocol was described elsewhere in our former study [6] . in brief, equal amounts of protein were loaded for sds gel electrophoresis and then transferred to nitrocellulose membranes. after block, the blots were incubated overnight at 4°c with the primary antibody of α-fodrin no.: sc-101154, 1:1000) or mmp2 (bioworld technology, inc., usa, catalog no.: bs1236, 1:500) respectively followed by incubation for another hour with the corresponding secondary antibody (1:4000). bands were visualized by ecl (super signal west pico kit; pierce) and quantified by densitometry using quantity one software (bio-rad). gapdh was used as internal reference. virus titer was measured using tcid 50 assay in hela cells. heart tissue homogenates were prepared at 4°c using ice cold homogenizer followed by centrifugation to harvest virus-containing supernatant. decimal serial dilutions of cell culture supernatant or tissue homogenate supernatant were added to the cultured hela cells and incubated for 1 h at 37°c. then the infected hela cells were cultured at 37°c. cytopathic effect was observed daily for 7 days. and tcid 50 were calculated using reed-muench method. mmp activity was detected according to the instruction of the commercial assay kit (anaspec, inc., ca, catalog no.: 71151). tissues homogenate in assay buffer were centrifuged for 15 min at 10,000 g at 4°c followed by incubation with apma at a final concentration of 1 mm in the assay buffer for 1 h at 37°c. activate mmp immediately before the experiment. dilute mmp-2 substrate 1:100 in assay buffer. add 50 μl per well of sample and mmp2 substrate solution in 96-well plate. mix the reagents by shaking the plate gently for 30 s. incubate the reaction at 37°c for 50 min. keep plate away from direct light. add 50 μl pe well of stop solution. mix the reagents and measure fluorescence intensity at ex/em = 490/520 nm. plot data as rfu fold change comparing with the control. immunohistochemistry staining of vp1 (leica biosystems newcastle ltd., united kingdom, catalog no.: ncl-entero, 1:200), ifnγ (bio legend inc. usa, catalog no.: 505811, 1:500) and il17 (abcam inc. usa, catalog no.: ab79056, 1:500) were carried out according to the method prescribed elsewhere [7] . mpo activity was assayed using commercial kit (sigma-aldrich co. llc, usa, catalog no.: mak069). standard curve was prepared according to the instruction. then tissue samples (10 mg heart tissue + 5 volume mpo assay buffer) were prepared using mpo assay buffer followed by making master reaction mix. at room temperature, the fluorescence intensity was read and mpo activity was calculated according to the instruction. primary cardiomyocytes were separated from neonatal rat heart. in short, neonatal rat (1-3 days old, supplied by department of laboratory animal science, fudan university) were fertilized and hearts were taken out quickly, sliced into pieces and digested in 0.1% trypsin at 37°c for 10 min and centrifuged at 3000 r/min to collect the digested cells and suspended the cells in dmem. repeat the digest-centrifuge cycles until the left tissue become apparent. the collected cells were suspended in dmem containing 10% fbs and cultured in 37°c for 2 h. the adherent cells are fibroblast and the still suspending cells are primary cardiomyocytes. then the cardiomyocytes were counted and seeded for research. the fibroblasts were passaged as routine and the third generation of cells was used for the study. prepared cardiomyocytes were fasted in dmem overnight followed by inoculation with cvb3 for 1 h in 37°c. then the supernatant were changed into dmem containing 10% fbs and cultured in 37°c for the designed time course. fibroblasts were seeded into 6-well plate at the density of 1 * 10 5 cells/well and cultured for 48 h. then the cells were synchronized by fasting in dmem without fbs for 24 h. scratch injury were made using the tips of 200 μl pipets. then dmem containing 10% fbs were changed with or without 5 μg/ml of calpain inhibitor alln (biomol research laboratories inc., usa, catalog no.: p-12) and cultured for another 24 h. then pictures of the scratch trace were taken and the distance were measured and analyzed. data were specified as mean values ± sd and analyzed using spss software (version 16.0; spss, inc., chicago, il, usa). 2-group comparison was performed using student t test. p b 0.05 was considered statistically significant. transgenic mouse had lower mortality post cvb3 challenge (fig. 1a) . calpain inhibition decreased the hw/bw ratio significantly (fig. 1b) . and myocardial damage markers of ck-mb and ctni were also decreased (fig. 1c, d) in the peripheral blood indicating the protective effect of calpastatin overexpression. to make certain the calpain activity in the transgenic mouse, the degradation product of calpain-specific substrate, α-fodrin, was blotted and as expected, the calpain activity was down-regulated in the transgenic mouse heart tissue (fig. 1e ). to observe the role of calpain activity in cvb3 replication, cvb3 capsid protein vp1 was blotted. it showed that comparing with wild type mouse cvb3-infected transgenic mouse had a lower level of vp1 expression in the myocardium (fig. 2a) . vp1 detection with immune histology method also showed that virus capsid protein decreased significantly (fig. 2b) . virus load in heart tissue were titrated and it demonstrated that virus titer were lower in the heart of transgenic mouse (fig. 2c) . in further, we verify this finding in vitro. neonate rat cardiomyocytes were separated accordingly. post virus infection, the cells were inoculated with culture medium containing calpain inhibitor alln of various dilutions (5 μg/ml, 2.5 μg/ml and 1 μg/ml) respectively. cardiomyocytes inoculated with culture medium without alln were set as normal control. and 12 h later, virus load in the supernatant were titrated. we found that comparing with the normal control 2.5 and 5 μg/ml of alln hindered virus replication significantly (fig. 2d ). to observe the effect of calpastatin over-expression on cvb3-induced myocardial inflammation, tissue sections were he stained. comparing with wild littermates, inflammation infiltration in transgenic mouse heart tissues was ameliorated significantly (fig. 3a) . mpo, the marker of neutrophils infiltration, was also decreased in transgenic mouse hearts (fig. 3b) . perforin is the effective protein of cytotoxic lymphocytes and natural killers. in the transgenic mouse's heart, calpain inhibition was accompanied with significant perforin down-regulation post virus infection (fig. 3b) . this was also the case for the inflammation factors of ifnγ (fig. 4ab) and il17 (fig. 4cd) , which demonstrated the role of calpain in mediating inflammation infiltration in virus-induced myocarditis. fibrosis is the key pathophysiology course in myocarditis progression. thus, we observed here whether calpain participated in fibrosis in cvb3-induced myocarditis. the classic fibrotic agents of smad3 (fig. 5a) and mmp2 (fig. 5b) were blotted and it demonstrated that calpain inhibition resulted in the downregulation of the two factors as well as the total mmps activity (fig. 5c) . to observe if calpain could regulate myocardial fibroblasts function, we proceed scratch assays with fibroblast. cultured fibroblasts were incubated with the calpain inhibitor alln (5 μg/ml) for 24 h with a this study, utilizing a calpastatin-overexpression transgenic mouse model of viral myocarditis, demonstrated that endogenous calpain inhibition ameliorated myocardial injury significantly. simultaneously, calpain inhibition was associated with decreased cvb3 replication and ameliorated inflammation/fibrosis formation. these data indicated the dual involvements of calpain in both virus replication and host inflammation. this might be an answer for the ambiguous problem of immunosuppressive therapy in viral myocarditis treatment [1] . calpain is a family of proteinases that cleave substrates into peptide segments. its activity was regulated by the endogenous inhibitor of calpastatin [8] . as we have demonstrated, transgenic mouse 5 . calpain was involved in myocardium fibrosis. a, b: western blot of smad3 and mmp2 respectively. n = 6 for each group. *p b 0.05 vs. con. group; # p b 0.05 vs. virus group. c: heart tissue total mmp activity assay. con. group mmp activity was set 1 arbitrarily. *p b 0.05 vs. con. group; # p b 0.05 vs. virus group. n = 4 for each group. d: represented scratch assay picture of cultured fibroblasts with or without calpain inhibitor alln. longer distance indicated weaker migration ability of the cells. *p b 0.05 vs. con. group. the experiment was repeated for 3 times. overexpressing calpastatin have a lower level of calpain activity in vivo (fig. 1d ). many researchers have reported the involvement of calpain in viruses' life cycles. for examples, calpain participated in the replication of influenza a virus [9] , severe acute respiratory syndrome coronavirus [10] , echovirus 1 [11] and human immunodeficiency virus [12] . in our former study, we found that cvb3-induced calpain activation facilitates the progeny virus replication in the early phase of infection in vitro [6] . bozym et al. [14, 13] reported the roles of calpain in mediating cvb both the entry into and the release from endothelial cells. yoon et al. [15] demonstrated that cvb4 uses autophagy for replication after calpain activation in rat primary neurons. as we presented in this paper, calpain inhibition reduced cvb3 loads both in vivo and in vitro (fig. 2) , which further proves the calpain involvement in facilitating cvb3 replication. role of calpain in regulating inflammation has been observed in different animal models, such as allergic encephalomyelitis [16] , sepsis [17] , abdominal aortic atherosclerosis [18] , ventilatorinduced lung injury [19] and hypercholesterolemic nephropathy [20] . in the realm of viral myocarditis, debiasi et al. [21] found in a mice model of reovirus strain 8b-induced myocarditis that calpain inhibition protects against virus-induced apoptotic myocardial injury, which presented the role of calpain in treating myocarditis. but few evidence of inflammation was mentioned as we presented here in a transgenic mouse. mechanisms of calpain involvement in inflammation might be associated with its regulation on inflammation cell behaviors. this point is supported by evidences from several studies focusing on different inflammation cells. in neutrophil, calpain participated in cell migration, adhesion, arrest and oxidative burst [22] . calpain1 also contributes to ige-mediated mast cell activation [23] . calpastatin overexpression can impair post infarct scar healing in mice by compromising reparative immune cell recruitment and activation [24] . and in leukocytes, calpain deficiency reduces angiotensin ii-induced inflammation and atherosclerosis [25] . we presented here in cvb3-induced myocarditis model that myocardium inflammation infiltration was significantly ameliorated in transgenic mouse, as well as the inflammation factors of mpo activity, il17, perforin and ifnγ. although there might be some background levels of expression of the above factors, it is reasonable considering the wide spread of inflammatory cells even in physiology conditions without virus infection. actually, a variety of inflammation factors whose production or secretion were regulated by calpain have been reported elsewhere including il5 [26] , il6 [27] , il16 [28] and il33 [29] . combined what we have reported in this paper, we could conclude that calpain participates in the regulation of a wide spectrum of inflammation factor production. researches from peng's lab [30, 31] in the animal models of sepsis and myocardial infarction have demonstrated respectively that calpain induces myocardial nf-κb activation, which might explain the mechanism that calpain regulates inflammation factors production. data from another group of our lab also found the role of calpain in regulating nf-κb activity in cvb3-induced myocarditis mice (unpublished data). inflammation factors are always fibrotic agents. the most solid evidence of calpain involvement of tissue fibrosis might be from cystic fibrosis patients [32] . and jiang et al. [33] reported that calpain1 regulates mmp2 activity in vascular smooth muscle cells to facilitate ageassociated aortic wall fibrosis. so we further observed the effect of calpain on myocardium fibrosis. as we have mentioned here that smad3/mmp2 pathway was inhibited in the transgenic mouse indicating role of calpain in regulating this classic fibrosis pathway. this was coincided with what peng's lab have demonstrates that calpain inhibition ameliorates myocardial remodeling after myocardial infarction [31] and myocardial fibrosis in diabetic cardiomyopathy [34] . what's more, we also observed the role of calpain in regulating fibroblasts migration in vitro and found calpain inhibition hinders fibroblast migration in scratch assays. this might explain in a novel angle the mechanisms of calpain in mediating fibrosis. this study also has some drawbacks.1) this is an animal study, and the results should be interpreted in caution in clinical practices. data from vmc patients should be collected in future clinical studies. 2) the data were mainly observational. mechanisms studies including roles of calpain in cvb3 replication and mediating inflammation in vivo should be performed in further studies respectively. in summary, in cvb3-induced myocarditis model, we presented here that calpain plays a dual role of both facilitating cvb3 replication and mediating inflammation/fibrosis. endogenous inhibition of calpain activity showed significant treatment effect for cvb3induced myocarditis. this might provide a novel target for viral myocarditis treatment in the future. we declare no conflicts of interests. viral myocarditis-diagnosis, treatment options, and current controversies current state of knowledge on aetiology, diagnosis, management, and therapy of myocarditis: a position statement of the european society of cardiology working group on myocardial and pericardial diseases autoimmunity in viral myocarditis viral myocarditis: from experimental models to molecular diagnosis in patients parvovirus b19 is a bystander in adult myocarditis coxsackievirus b3-induced calpain activation facilitates the progeny virus replication via a likely mechanism related with both autophagy enhancement and apoptosis inhibition in the early phase of infection: an in vitro study in h9c2 cells initial weight and virus dose: two factors affecting the onset of acute coxsackievirus b3 myocarditis in c57bl/6 mouse-a histopathologybased study the calpain system and diabetes targeting host calpain proteases decreases influenza a virus infection severe acute respiratory syndrome coronavirus replication is severely impaired by mg132 due to proteasomeindependent inhibition of m-calpain calpain 1 and 2 are required for rna replication of echovirus 1 human immunodeficiency virus-1 tat activates calpain proteases via the ryanodine receptor to enhance surface dopamine transporter levels and increase transporter-specific uptake and vmax calcium signals and calpain-dependent necrosis are essential for release of coxsackievirus b from polarized intestinal epithelial cells release of intracellular calcium stores facilitates coxsackievirus entry into polarized endothelial cells coxsackievirus b4 uses autophagy for replication after calpain activation in rat primary neurons calpain inhibition attenuated morphological and molecular changes in skeletal muscle of experimental allergic encephalomyelitis rats muscle-specific calpastatin overexpression prevents diaphragm weakness in cecal ligation puncture-induced sepsis calpain inhibition attenuates angiotensin ii-induced abdominal aortic aneurysms and atherosclerosis in low-density lipoprotein receptor-deficient mice activation of calpains mediates early lung neutrophilic inflammation in ventilator-induced lung injury nicotinic acetylcholine receptor α1 promotes calpain-1 activation and macrophage inflammation in hypercholesterolemic nephropathy calpain inhibition protects against virus-induced apoptotic myocardial injury calpain inhibition impairs tnf-alpha-mediated neutrophil adhesion, arrest and oxidative burst calpain-1 contributes to ige-mediated mast cell activation calpastatin overexpression impairs postinfarct scar healing in mice by compromising reparative immune cell recruitment and activation leukocyte calpain deficiency reduces angiotensin ii-induced inflammation and atherosclerosis but not abdominal aortic aneurysms in mice parp-1 deficiency blocks il-5 expression through calpain-dependent degradation of stat-6 in a murine asthma model overexpression of a minimal domain of calpastatin suppresses il-6 production and th17 development via reduced nf-κb and increased stat5 signals secretion of il-16 through tnfr1 and calpain-caspase signaling contributes to mrsa pneumonia caspase-1, caspase-8, and calpain are dispensable for il-33 release by macrophages cleavage of iκbα by calpain induces myocardial nf-κb activation, tnf-α expression, and cardiac dysfunction in septic mice deficiency of capn4 gene inhibits nuclear factor-κb (nf-κb) protein signaling/inflammation and reduces remodeling after myocardial infarction evidence for alteration of calpain/calpastatin system in pbmc of cystic fibrosis patients calpain-1 regulation of matrix metalloproteinase 2 activity in vascular smooth muscle cells facilitates age-associated aortic wall calcification and fibrosis targeted inhibition of calpain reduces myocardial hypertrophy and fibrosis in mouse models of type 1 diabetes funding for this study were provided by the national science key: cord-303662-ro9879dl authors: wang, fun-in; stohlman, stephen a.; fleming, john o. title: demyelination induced by murine hepatitis virus jhm strain (mhv-4) is immunologically mediated date: 1990-11-30 journal: journal of neuroimmunology doi: 10.1016/0165-5728(90)90050-w sha: doc_id: 303662 cord_uid: ro9879dl abstract the neurotropic mouse hepatitis viruses (mhv), in particular strain jhm (jhmv or mhv-4), cause experimental central nervous system demyelination that pathologically resembles multiple sclerosis, an important human demyelinating disease. the mechanism of jhmv-induced demyelination remains unclear, though its tropism for oligodendrocytes had led to the belief that jhmv causes demyelination by direct lysis of these myelin-producing cells. however, several studies have also implicated the involvement of immune responses in the demyelinating process. in this communication, we present evidence that generalized immunosuppression with gamma irradiation prevents jhmv-induced demyelination, a finding that was not limited to a particular strain of jhmv or to one strain of mouse. in addition, significant paralytic-demyelinating disease was restored to infected, irradiated mice after the adoptive transfer of nylon wool nonadherent splenic cells and appeared to be restricted by the major histocompatibility complex (mhc). these observations indicate that the principal mechanisms of jhmv-induced demyelination are most likely immunopathological. has been used to study demyelination experimentally. these models include experimental allergic the murine coronavirus jhmv, also desig-encephalomyelitis (eae) and infection by theiler's hated mhv-4, was originally isolated from mice virus, canine distemper virus, semliki forest virus, which spontaneously developed hindlimb paralysis a59 coronavirus, visna virus, and others (reviewed and demyelination (bailey et al, 1949; cheever et by raine, 1984; martin and nathanson, 1979; dal al., 1949) . subsequently, jhmv infection of ro-canto and rabinowitz, 1982; stohlman and dents has been one of several model diseases which kyuwa, 1990) . for the viral models of experimental demyelination, rodriguez (1988) has outlined a use-gory of direct viral cytopathic effect on oligo-representative mice bled and tested by enzymedendrocytes, the cells which produce and maintain linked immunosorbent assay (elisa; fleming and myelin; in this mechanism, the immune system pen, 1988) for antibodies to murine coronaviruses plays no role or merely has a scavenging function, were seronegative. this hypothesis is supported by findings that the isolation and characterization of jhmv jhmv replicates and causes acute cytopathology antigenic variant 2.2-v-1 in oligodendrocytes (lampert et al., 1973; and a small plaque variant jhmv-ds (stohlman 1973; powell and lampert, 1975; fleury et al., et al., 1982) have been described previously. 1980), as well as by several reports that animals viruses were propagated under serum-free condiwhich are either immunosuppressed or im-tions and quantitated by plaque assay on dbt munodeficient nonetheless develop jhmv-in-cells (stohlman and weiner, 1978) . prior to inocduced demyelination to a variable degree (soren-ulation, viruses were diluted in dulbecco's minimal sen et al., 1982, 1987) . on the other hand, studies essential medium. mice were injected either i.c. have shown that mhv may elicit a variety of with 30 /xl containing 103 plaque-forming units potentially immunopathological responses, includ-(pfu) of virus or i.p. with 0.5 ml containing 106 ing alteration in major histocompatibility complex pfu of virus. infectious virus titers in brain ho-(mhc) antigen expression (massa et al., 1986; mogenates were measured on l2 cells and by suzumura et al., 1986) , reactivity to myelin basic infectious center assays as previously described protein (watanabe et al., 1983) , anti-viral antibod(stohlman and weiner, 1978) . ies (fleming et al., 1983 ) and anti-viral t cells (sussman et al., 1989) . despite the evidence show-gammdirradiation ing that jhmv is capable of causing (1) direct mice were irradiated with a ~3vcs gamma verticytopathology and (2) potentially immunopatho-cal beam source at 150 rad/min (gamma cell 40, logical responses, controversy remains about the atomic energy of canada) under the experimenextent to which either of these processes actually tal conditions noted below. in experiments conpredominates in vivo. ducted to compare the effects of irradiation of the two approaches were taken to directly study central nervous system (cns) with those of systhe role of the immune system in the jhmv-in-temic compartments, mice were anesthetized using duced demyelination. first, infected animals were pentobarbital (75 mg/kg; i.p.), placed in a plastic immunosuppressed by gamma irradiation early in restrainer, and protected by 25 mm thick lead the disease course. second, immunosuppressed shields introduced above and below the mice mice were reconstituted by the adoptive transfer longitudinally to either cover the cns (systemic of spleen cells from immune donors. these experi-exposure; 4 mm dorsal shield) or the non-cns, ments revealed that jhmv-induced demyelination systemic areas (cns exposure; ventral shield to is prevented by gamma irradiation and partially within 4 mm of the back). mock experiments and restored by the transfer of immune splenocytes, dissections confirmed that differential irradiation providing direct evidence for a central role of the of the cns (brain and spinal cord) or systemic immune system during jhmv-induced demyelina-compartments (including spleen, lymph nodes, and tion. bone marrow) was achieved under these shielding conditions. donor mice were either not immunized (naive donors) or immunized i.p. with approximately 106 six-week-old male c57bl/6j and balb/cj pfu of virus (immune donors) at 6 days prior to mice were obtained from jackson laboratories adoptive transfer. single-cell suspensions were (bar harbor, me, u.s.a.). mice were held for prepared from spleens of donor mice, and 5 x 10 v 48-72 h before intracerebral (i.c.) infection or cells were injected intravenously (i.v.) into recipiintraperitoneal (i.p.) immunization with virus. all ent mice which had received 850 rad of irradiation immediately prior to transfer. in some experi-statistical analyses ments, donor cells were fractionated into nylon both clinical and histological scores were comwool adherent (nwa) and nylon wool non-adher-pared for statistical significance using the mannent (nwna) populations (sussman et al., 1989) whitney test for nonparametric samples (statsoft prior to transfer, statistical programs, tulsa, ok, u.s.a.). a probability (p) < 0.05 by this test was considered sig-clinical evaluation nificant. in instances where observers disagreed, mice were evaluated for clinical signs of de-the histological score assigned was~either the lower myelination using a scale modified from brown et grade (one-step disagreement, n = 31/90 observaal. (1982) . numerical values were assigned as fol-tions) or intermediate grade (two-step disagreelows: 0, normal; 1, minimal gait abnormality; 2; ment, n = 2/90 observations). moderate paraparesis; 3, severe paraparesis; and 4, paraplegic. evaluations were scored at day 12 postinfection (p.i.), as almost all mice that will results develop subacute or chronic disease after 2.2-v-1 infection show some abnormal clinical signs at or whole body irradiation before day 12 p.i. . to study the immunosuppressive effects of irradiation, different amounts of whole body histological evaluation gamma irradiation were administered daily mice were sacrificed at day 12 p.i. and tissues throughout the disease course in c57bl/6j mice were fixed in clarke's solution (75% absolute al-after i.c. infection with jhmv variant 2.2-v-1. cohol and 25% glacial acetic acid), embedded in irradiation dramatically reduced paralytic-deparaffin, and stained with hematoxylin and eosin myelinating disease if 850 rad were administered (h&e) or luxol fast blue (lfb) counterstained at day 6 p.i. or earlier. at day 7 p.i. or later, with eosin . for quantitative however, disease was unaffected by as much as assessments, a single longitudinal section of h& 1250 rad. based on these data, we chose to admin-e-stained spinal cord was reviewed independently ister 850 rad on day 6 p.i. in subsequent experiby two observers without knowledge of the ments in which clinical and histological diseases animal's experimental group. evaluation focused were monitored quantitatively. on the degree of inflammation, edema, and dis-severe paralysis and demyelination were eviruption of tissue architecture in the white matter, dent by day 12 p.i. in infected, untreated mice pathology was graded as follows: 0, normal; i, (table 1 , group 1) as reported previously (fleming slight (mild, focal) ; 2, moderate (mild, diffuse); 3, et al. , 1986) . fig. 1 shows several histological marked (intense, focal) and 4, severe (intense, features of the demyelinating lesions observed in confluent). grade 3 and 4 lesions correspond to group 1, including inflammatory hypercellularity the fully developed plaques of acute, primary (a, b, c, e), scanty viral antigen and the presence jhmv-induced demyelination first described by of naked axons (d). in contrast, infected mice bailey et al. (1949) and weiner (1973) . using irradiated at day 6 p.i. had a marked reduction in myelin staining and electron microscopy, we have both paralysis and demyelination (table 1 , group previously shown that jhmv antigenic variant 2; fig. 2b ). irradiation given to uninfected, naive 2.2-v-1 produces lesions identical to those initially mice had no demonstrable clinical or histological described, in which the principal changes are effect (data not shown). to determine if the dimyelin loss and axonal preservation (fleming et minution in disease could be attributed to the al., 1986, 1987) . viral antigen was detected by inhibition of virus replication by irradiation, the immunoperoxidase staining , virus titer in brains of irradiated (850 rad) and using monoclonal antibody (designated j.3.3) control mice were compared. fig. 3 shows that the specific for the jhmv nucleocapsid protein as virus titer in the irradiated mice exceeded that of primary antibody, and counterstained with hema-the unirradiated controls. these data indicate that toxylin, the absence of disease was not due to an inhibisix-week-old male c57bl/6j mice were given 103 pfu of jhmv 2.2-v-1 i.c. on day 0, as indicated by ' +' sign. 850 tad of whole body irradiation were given at day 6 p.i., as indicated by ' +' sign. c clinical observations and blinded histological evaluations were performed as indicated in materials and methods (0-4 scales, with grade 4 being maximal disease). the mean, standard deviation, and number (n) of mice tested are shown. underlined values indicate a statistically significant difference (p < 0.05) between groups 1 and 2 as determined by the mann-whitney test for nonparametric samples. tion of virus replication. in addition, immunohis-infected with 103 pfu of 2.2-v-1 also developed tochemical studies showed that following 2.2-v-1 severe paralytic-demyelinating disease by day 12 challenge, viral antigen was scanty or absent in p.i. disease was also prevented in these mice by unirradiated mice (fig. 1d ) but was very abun-850 rad of whole body irradiation at day 6 p.i. dant in irradiated mice ( fig. 2a) . many of the (data not shown). these findings are essentially antigen-positive cells in irradiated, infected mice identical to those obtained in c57bl/6j mice appeared to be oligodendrocytes ( fig. 2a) ; infected with 2.2-v-1, suggesting that abrogation surprisingly, these cells showed few or no morpho-of demyelination by irradiation is not dependent logical abnormalities, on an unusual characteristic of a particular jhmv in control experiments paralleling those shown strain or mouse strain. in table 1 , mice were infected with a second jhmv strain, jhmv-ds (stohlman et al., 1982) , and were either irradiated or not irradiated. un-in view of the finding that whole body irradiairradiated mice demonstrated intense disease at tion at day 6 p.i. prevents jhmv-induced paraday 9 p.i. by contrast, mice given 850 rad at day 6 lytic-demyelinating disease, differential irradiation p.i. had few histological changes at day 9 p.i. in studies were conducted to determine whether critiaddition, a second mouse strain, balb/cj mice cal radiosensitive targets reside in the systemic or a immune donor mice were 6-week-old c57bl/6j males given 106 pfu of jhmv 2.2-v-1 i.p. 6 days prior to transfer. naive donors were identical mice not given virus. in each group, 5 x 10 v spleen cells were transferred i.v. into recipient mice on day 6 p.i. b recipient mice were 6-week-old c57bl/6j male inoculated (virus) or not (naive) with 103 pfu of jhmv 2.2-v-1 i.c. on day 0. all recipient mice were given 850 rad prior to adoptive transfer on day 6 p.i. scoring was performed as indicated in table 1 . underlined values are clinical or histological scores which are significantly greater than respective scores of jhmv-infected, irradiated mice not given splenocytes ( cns compartments. when 850 rad were delivered at day 6 p.i. to systemic regions (including spleen, to the cns only (brain and spinal cord) at day 6 lymph nodes, and bone marrow) but not to the after 2.2-v-1 infection, marked white matter pa-cns, white matter appeared normal (fig. 4c, d) . thology was observed (fig. 4a, b) . in the con-these findings are essentially identical to those verse experiment in which 850 rad were delivered found in eae (hickey and kimura, 1988) and im m k ,,. ~'l_m suggest that radiosensitive cells residing in the formed, using balb/cj (h-2 d) donor and systemic compartment at day 6 p.i. are essential c57bl/6j (h-2 h) recipient mice. as shown in for subsequent lesion development. table 3 (groups 6 and 7), allogeneic transfers did not restore disease, suggesting that mhc restriction is, in fact, required. to further define the characteristics of cells to jhmv-induced disease, spleen cells from donor which are active in the adoptive transfer of dismice were transferred into irradiated recipient mice ease, syngeneic immune spleen cells were sep-( table 2 ). when 2.2-v-l-infected, irradiated mice arated by nylon wool to yield t cell-enriched received immune spleen cells (group 3, table 2 ), (nylon wool nonadherent) and t cell-depleted significant disease was restored (clinical score, p (nylon wool adherent) fractions. as shown in ta-= 0.0023; histological score, p = 0.0587). while this effect was often quite dramatic in individual 60 mice (fig. 5a) , the mean clinical and histological scores in these animals were not as high as those of virally infected, unirradiated mice (group 1, oe ,0 table 1) . surprisingly, adoptive transfer of naive ~ spleen cells into infected, irradiated recipients ~ /,~/~p je3 (group 4, table 2 ; fig. 5b ) also produced mod-~o ,0 ~ .... crate disease. on the other hand, following trans-g / fer of immune splenocytes to naive, uninfected j 2° / mice (group 5, table 2 ) recipients were completely // normal (fig. 5c) the ability of spleen cells from naive mice to fig. 3 . viral replication in the brains of c57bl/6j mice induce disease in infected, irradiated mice (group inoculated with jhmv 2.2-v-1 on day 0 and sacrificed at the indicated time points, each of which represents the mean titer 4, table 2) raised the possibility that a nonfrom a group of 4-6 mice. mice were either not irradiated (i~), mhc-restricted cell, such as a macrophage, might or subjected to 850 rad of whole body irradiation at either day be responsible for mediating this effect. to test -1 (o) or at day + 6 (o) relative to the day of viral inoculathis hypothesis, allogeneic transfers were per-tion. a allogeneic (balb/cj, h-2 d) or syngeneic (c57bl/6j, h-2 b) donor mice were given 106 pfu of jhmv 2.2-v-1 i.p. 6 days prior to transfer (immune) or not (naive), as noted in table 2 . in groups 6 and 7, 5 x 107 spleen cells were transferred i.v. into each recipient at day 6 p.i. nwna indicates nylon wool nonadherent ceils, and nwa indicates nylon wool adherent cells (selected from a total of 5 x 107 spleen cells) transferred i.v. to recipients at day 6 p.i. b in all groups, recipient mice were 6-week-old c57bl/6j males given 103 pfu of jhmv 2.2-v-1 i.c. on day 0 and irradiated on day 6 p.i. prior to transfer. c scoring was also performed as in table 1 . underlined values are those which are significantly greater than respective scores of jhmv-infected, irradiated mice not given splenocytes ( ble 3, the ability to transfer clinical disease was vestigation cannot exclude some contribution of contained in the t cell-enriched population (group direct viral cytolysis of oligodendrocytes (lampert 8) . although histological scores were elevated in et al., 1973) to the jhmv pathogenesis. clearly, these mice relative to irradiated, infected mice not however, the role of this mechanism, if present, given spleen cells (group 2, table 1 ), this value did must be minor, since it should be unaffected or not achieve statistical significance, possibly due to even enhanced by an immunosuppressive dose of the relatively small number of animals in the irradiation. in fact, infected irradiated mice show experiment. taken together, the characteristics of little or no evidence of demyelination or cell de-mhc restriction and nylon wool nonadherent struction (fig. 2) , despite marked increases in suggest that spleen cells which are most active in both viral antigen-positive oligodendrocytes (fig. adoptive transfers are likely to be t lymphocytes. 2a) and brain viral titers (fig. 3) . prior studies in athymic (sorensen et al., 1982 ), immunosuppressed (sorensen et al., 1982 zimmer and dales, discussion 1989) , or lethally challenged sussman et al., 1989) rodents have established a the major finding of this study is that ira-protective role for cellular immunity early in the munosuppression of jhmv-infected mice by jhmv pathogenesis by limiting virus infection in means of gamma irradiation abrogates viral-in-susceptible cells, such as oligodendrocytes and duced demyelination. this result strongly argues neurons. again, these studies have limited relethat jhmv causes demyelination through ira-vance to the study of jhmv-induced demyelinamunopathological mechanisms. however, our in-tion, since early challenge of animals with severe immunodeficiency or with large amounts of viru-the two jhmv strains, 2.2-v-1 and jhmv-ds, lent virus primarily results in an acute, fulminant used in the present study are essentially identical panencephalitis, with little or no demyelination, to most jhmv strains in all respects except neu-further support for an immunopathological rovirulence (weiner, 1973; stohlman et al., 1982 ; mechanism of jhmv-induced demyelination . in terms of neurovirulence, comes from the adoptive transfer studies. these they resemble the original jhmv isolates, which experiments indicate that populations of murine in early passages primarily caused a nonfatal paradonor spleen cells, which are enriched for t lytic disease (bailey et al., 1949; cheever et al., lymphocytes and appear to be mhc-restricted, 1949) ; only after many i.c. passages did the virus restore demyelination to infected, irradiated re-acquire marked neurovirulence. most importantly, cipient mice (tables 2 and 3 ). two features of the the minimal neurovirulence of jhmv 2.2-v-1, adoptive transfers were unexpected, however, and jhmv-ds, and similar jhmv strains (hirano et indicate that these experiments must be interpret-al., 1981; knobler et al., 1982; dalziel et al., 1986 ) ed cautiously. first, cells from naive donors (ta-allows virus-induced demyelination to be studied ble 2, group 4) were nearly as effective as cells directly, by minimizing the confounding fatal enfrom immune donors ( the only previous study of irradiation during p.i. this result may reflect the fact that in an jhmv infection was that of love et al. (1987), established cellular immune response within the who applied regional irradiation to the spinal cords cns, the majority of lymphocytes present locally of mice which had been infected with jhmv, may not be antigen-specific (ceredig et al., 1987; strain ts8 (knobler et al., 1982 ) 2 months previ-fallis et al., 1987 . second, although the degree to ously. under these circumstances, irradiation had which adoptive transfers reconstituted disease was no effect on the clinical or histological course of clearly significant in the aggregate (table 2) and jhmv pathogenesis. we have confirmed the reoften dramatic in individual animals (fig. 5a, b) , suit of love et al. (1987) , that irradiation applied the mean clinical and histological scores of recon-to the cns alone or applied after disease has stituted mice were considerably lower than those become firmly established has no effect on of the infected unirradiated mice (table 1, group jhmv-induced demyelination. the suggestion of 1). thus, the disease was, on average, only parlove et al. (1987) , that irradiation administered at tially restored by adoptive transfer of spleen cells, an earlier time in the course of disease might have this result may reflect technical factors, such as an effect on demyelination was shown to be corthe quantity or quality of transferred cells or the rect in the present study (table 1) . ability of transferred cells to reach specific targets. in conclusion, we have shown that jhmv-in-alternatively, it is possible that irradiation at day duced paralytic-demyelinating disease may be pre-6 p.i. irreversibly damages critical elements in the vented by immunosuppressive dose of gamma cascade leading to demyelination. in order to ad-irradiation and partially restored by the adoptive dress these questions, further experiments in which transfer of spleen cells, which are most likely t adoptive transfers performed during the early, in-lymphocytes. taken together, these findings indiductive phase of disease are in progress, cate that the primary mechanism of jhmv-in-an immunopathological mechanism has not duced demyelination is immunopathological, been established previously for jhmv-induced rather than being due to direct viral lysis of demyelination, possibly due to the use of a rela-oligodendrocytes. tively virulent virus strain coupled with cyclophosphamide-mediated immunosuppression applied simultaneously with virus infection (lampert et a murine virus (jhm) causing disseminated 279-280. encephalomyelitis with extensive destruction of myelin myelinating disease caused by theiler's virus, mouse (1987) phenotypic analysis of the inflammatory exudate in hepatitis virus or experimental allergic encephalomyelitis. j. murine lymphocytic choriomeningitis animal models of new encephalomyelitis with extensive destruction of myelin viral med~ 90, 181-194. particles induce la antigen expression on astrocytes analysis of autoimmune demyelination: its 4 peplomer glycoprotein e2 results in reduced neuroviruimpact upon multiple sclerosis mechanisms of virus-induced demyelina-fallis in vivo and in bridoma supernatants and ascites in absolute units by vitro models of demyelinating disease. xvii. the infectious sensitive and reliable enzyme-linked immunosorbent assays process in athymic rats inoculated with jhm virus. microb. (elisa) pathogenesis of a murine (1983) improvements in obtaining and characterizing mouse coronavirus, strain jhm in the central nervous system of cerebrospinal fluid. application to mouse hepatitis virus-inmice 53-61. variant of murine coronavirus jhm selected with mono-stohlman in vivo effects of coronavirus-specific t cell pathog. 3, 9-20. clones: dth inducer cells prevent a lethal infection but do fleury further ultrastructural observations of virus t-cell-mediated clearance of mouse hepatitis virus duced demyelinating encephalomyelitis pathogenesis of demyelination induced by suzumura, a in vivo and in vitro models 993. of demyelinating diseases. xxiv. the infectious process in adoptive cyclosporin a treated wistar lewis rats inoculated with transfer of eae-like lesions from rats with coronavirus-in-jhm virus acknowledgements al., 1973; weiner, 1973) . in this setting, the majority of mice died of fulminant acute encephalitiswe are very grateful to thomas bohlmann, and did not manifest typical jhmv-induced sub-cindy fabricius-segal, wen-quiang wei and acute or chronic paralysis and demyelination.ligaya key: cord-329061-1xut73dq authors: bhatt, pravin n.; percy, dean h.; jonas, albert m. title: characterization of the virus of sialodacryoadenitis of rats: a member of the coronavirus group date: 1972-08-17 journal: j infect dis doi: 10.1093/infdis/126.2.123 sha: doc_id: 329061 cord_uid: 1xut73dq the virus that causes sialodacryoadenitis in rats has been isolated in mice and in primary cultures of rat-kidney cells and has been characterized as a heat-labile rna virus that is sensitive to lipid solvents and is relatively stable at ph 3.0. this virus is antigenically related to the virus of hepatitis in mice and to coronavirus of rats. the range of hosts of this agent appears to be narrow. on the basis of available biologic characteristics, it has been placed in the coronavirus group. cated that neither source had detectable serum antibodies to sda virus and that both were susceptible to infection with this agent. rats were inoculated intranasally with 0.1 ml of virus-infected salivary-gland suspension, observed daily for evidence of overt illness, and sacrificed at various intervals. all rats were maintained in rigid plastic isolators with high-efficiency air filters. tissue culture. three cell lines, baby-hamster kidney (bhk-21), vera, and hep-2, and primary monolayer cultures of rat embryo, rabbit kidney, rhesus-monkey kidney, guinea-pig-embryo skin, muscle, and kidney were used as previously described [l ] . monolayers obtained from explant cultures of submaxillary, parotid, harderian, and exorbital glands of germfree rats, monolayers of trypsin-dispersed brain cells of infant mice, and a line of polyoma-transformed mouse cells (py-al/n) [6j were also tested. at a later stage in the study, primary rat-kidney (prk) cultures prepared from kidneys of weanling charles river cd germfree or conventional inbred dark agouti fda) rats were used. inoculated tubes were kept in a roller drum at 37 c and observed for cytopathic effect (cpe) at intervals of two to four days for at least 21 days. the fluid medium was changed when necessary. in the absence of cpe, a blind passage was made between the eighth and 16th day after inoculation. cultures for passage were observed for one to two weeks for development of cpe, and, in the absence of cpe, vera, bhk-21, and pmk cultures were challenged on the 12th day after inoculation with chandipura [8j virus, an arbovirus of the vesicular stomatitis viral group, for determination of interference. in addition, the fluid from each of the second-passage cultures was inoculated ic into infant mice, and the mice were observed for 21 days. in some instances tissueculture fluid from inoculated tubes was passaged into infant mice without further passages in tissue culture. monolayer cultures of prk, infant-mouse brain, and py-al/n cells were also examined by indirect immunofluorescence for the presence of viral antigen [5] . characterization of the virus. for determination of the effect of 5-bromodeoxyuridine (5-budr), lipid solvent, low ph, and various temperatures, methods described by bhatt et al. [9j were used. the hemagglutination method will be bhatt, percy, and jonas described under results. staining with acridine orange was done according to the method of hsiung [10] . preparation of immune sera. hyperimmune sera were prepared in rats and mice by repeated inoculation of a suspension of salivary glands from infected rats and of brains from infected mice, respectively. complement-fixation test. complement-fixing antigen was prepared by sucrose-acetone extraction from infected brains of two-to four-day-old mice. polyvalent mouse-hepatitis cf antigen prepared in tissue culture was obtained from microbiological associates. the cf test was performed by the micromethod [l l ] using two units of complement and four to eight units of antigen. serum of mice immune to sda strain 681 was tested against 118 viral antigens by the cf test. neutralization test. the neutralization (n) test with sera immune to murine viruses was performed in infant mice, and these animals were observed for 14 days after inoculation. prk cultures were used for cross-n tests, using sda strain 681 and parker's rcv. sera were inactivated at 56 c for 30 min. cultures were examined on the third and fifth days after inoculation. the titers of antibody and virus were calculated by the method of reed and muench [12j. fluorescent-antibody method. pieces of mouse brain 2-4 mm thick were quick-frozen in a dry ice-alcohol bath and stored at -83 c. sections 6-8f!m thick were cut in a cryostat, two sections were mounted per slide, and then the slides were fixed in acetone at 25 c for 15-20 min and dried at 37 c for 15 min. sections were stained immediately or stored at -25 c for 1-30 days before use. tissue-culture cover slips were similarly prepared but at times were kept in chilled acetone at -25 c for 18 hr. the section and cover-slip preparations were reacted with sera immune to virus for 20 min at 25 c and then exposed to mouse or rat antiglobulin conjugate for 20 min. phosphate-buffered saline (pbs) was used for washing. preparations were examined with a carl zeiss microscope fitted with an hbo 200 w/4 supermercury lamp, a ug-5 exciter filter, and a 47/65 barrier filter. histopathology. histologic examination of tissue from inoculated rats included harderian, exorbital, parotid, and submaxillary glands. in suckling mice, coronal sections of brain and serial transverse sections of the thoracic and abdominal regions were examined. attempts to induce disease in weanling mice. sixty female mice, three-to four-weeks old, were given 2.5 mg of cortisone im twice a week beginning a week before inoculation and continuing until the end of the experiment. twenty mice each were inoculated ic and ip with 0.03 ml and 0.1 ml of viral suspension containing 2 x 1()3·9 and 6.3 x 10 3 . 9 infant mouse ld50 (imld50), respectively. twenty control mice were inoculated with diluent, 10 by the ic route and 10 by the ip route. another group of controls was neither inoculated nor given cortisone. six mice from each group were killed for histologic studies on the seventh day after inoculation and two were killed on the 14th day after inoculation. remaining mice were observed until the 21st day after inoculation, when the experiment was terminated. a complete necropsy was done on each mouse. induction of sialodacryoadenitis in susceptible rats by mouse-brain-adapted virus. eleven rats, weighing 250 g and from a colony known to be susceptible to sda virus, were inoculated by the intranasal route with fourth passage, infectedmouse-brain material. the inoculum contained approximately 6.6 x 10 3 . 8 imld 50 of virus. rats were sacrificed on the fifth, sixth, and eighth days after inoculation. harderian and submaxillary glands were processed for isolation of virus and histologic examination, whereas parotid gland was collected for histologic examination only. adaptation to mice and related observations. a 10% suspension of infected salivary glands was inoculated ic into one-day-old mice. one mouse was sick on the fifth day after inoculation, eight more were sick on the seventh day after inoculation, and six on the eighth day after inoculation. some of these animals were killed, and tissues were harvested for passages and histologic study, but mice that were sick but not killed died on the 10th day after inoculation. one mouse was unaffected and survived until the 21st day, when it was discarded. the disease was characterized by ataxia and uncoordination, followed by paresis, paralysis, and death. the same pattern of illness was observed on further passages. by the fifth mouse-brain passage, the incubation period was 125 shortened to two to three days. there was usually a random pattern of illness and death from two to eight days and occasionally up to 10 days after inoculation. the pattern has remained unchanged for 29 passages with this strain of virus. one other observation made during the first passage in mice and amply confirmed during subsequent work was emaciation of sick mice as compared to uninoculated control mice of the same age. these differences were more marked in mice that were two to four days old or older when inoculated. other significant observations can be summarized as follows: (l) the agent of sda does not cause detectable illness in weanling (three-to four-week-old) mice when inoculated ic or ip or in infant mice inoculated ip. (2) a comparative titration was done in mice two days old, 13 days old, and 22 days old that were inoculated ic with viral stock passaged 12 times in mouse brain. titers were 10 4 . 00, 10 4 . 25 , and <10 2 . 0 imld50/0.015 mi, respectively. (3) virus has undergone 29 serial ic passages in zero-to six-day-old mice, the cumulative dilution of which exceeds 10-100. (4) the titer of virus between the fifth and 29th passage in mouse brain has remained relatively stable at 10 3 . 5_105.0 imld50/0.015 ml (usually around 1q3·7 imld50/0.015 ml). (5) the original salivary-gland suspension was titrated in one-day-old mice and had a titer of 1()3·6 imld 50/o.015 ml. (6) when inoculated intranasally into susceptible rats, mouse-brain-adapted virus produced sialodacryoadenitis. (7) brains from two uninoculated mice (two days old) were harvested as controls; seven serial ic passages of this material were made at intervals of six to seven days in mice three to four days old. no agent pathogenic for mice was isolated from these control animals. histopathologic and immunofluorescent observations in inoculated mice. in general, histologic changes observed in the central nervous system of inoculated mice were characterized by diffuse and focal neuronal degeneration with minimal inflammatory cell response. regions of brain most frequently involved were the cortices of the occipital and parietal lobes. other foci of neuronal destruction were scattered elsewhere in the central nervous system; there was relatively little destruction 0 some important observations are summarized as follows. (l) cultures were most sensitive when used within a week after seeding; then sensitivity decreased. the cpe was delayed and less extensive in older cultures. (2) development of virus in prk cells was monitored by cpe, detection of viral antigen by indirect immunofluorescence, and quantitation of infectious virus in prk tubes. results are presented in table 1. significantly, detectable viral antigen developed by 12 hr and was followed by release of infectious virus into the medium. cpe was detected at 24 hr. beyond 24 hr, quantitation of viral antigen was difficult due to lysis of cell sheets, and after 36 hr, titer of infectious virus decreased. (3) the sensitivity of inoculation of mice ic with strain 681 virus was compared with that of inoculation of prk cultures. titers obtained with a mouse-brain-adapted virus were 2.5 x 10 4 imld50 in mice and lox 10 4 . 3 tcid50 in prk cultures. similar differences were also noted in other experiments. characterization of the agent. the effect of 5-budr on viral multiplication was determined by the method of bhatt et al. [9] . chandipura virus was used as rna control (p. n. bhatt, unpub-hour in the cerebellum. affected neurons were pyknotic and densely eosinophilic. in addition, there was a scattering of shrunken, densely staining astrocytes in these areas. occasionally there was hypertrophy and hyperplasia of capillary endothelial cells and minimal perivascular cuffing with mononuclear cells. sometimes a few polymorphonuclear leukocytes were scattered in areas of destruction. spinal cord, salivary glands, lung, heart, liver, kidney, spleen, and intestine were histologically normal. immunofluorescence procedures detected viral antigen in regions where frank cellular necrosis was seen by standard histologic techniques. in addition, intense staining was observed in neuronal cytoplasm of scattered cells that were intact and not associated with frank necrosis. serial coronal sections had immunofluorescence staining in dorsal cortical areas, the ventral portion of ammon's horn, the hypothalamus, and the brain stem, but fluorescence was rarely found in cerebellar folia and white matter. a ttempts to adapt the agent to monolayer cell cultures. the original salivary-gland suspension inoculated onto various monolayer cell cultures produced no detectable cpe up to 21 days after inoculation. when blind passages were made and cultures were challenged with chandipura virus [8] , interference was not observed. fluid from the second passage in tissue culture was inoculated ic into infant mice; the results were negative. attempts were made to propagate mouse-brainadapted virus to cell-culture systems, such as monolayers obtained from explant cultures of parotid, harderian, exorbital, and submaxillary glands of germfree rats and monolayers of trypsindispersed infant-mouse-brain cultures. there was no detectable cpe. similar results were obtained with the py-al/n cell line. infectious virus or viral antigen was not detected when tissue-culture fluids from infected-mouse-brain and py-al/n cultures were inoculated ic into infant mice or when monolayers were examined by indirect immunofluorescence. however, prk cultures showed cpe characterized by formation of multinucleated giant cells, which were seen as highly reflective masses. these cells fell off the glass wall a few hours later and were seen floating in medium. tissue-culture fluids of these cultures contained virus as detected in infant mice, and cultures were positive for viral antigen by indirect immunofluorescence. sensitivity of sda virus to a lipid solvent was also tested. the titer of virus was 10 3 . 8 and < 10 2 . 0 imld50/0.015 ml for controls and chloroformtreated samples, respectively. the test was repeated with similar results, and it was concluded that the agent is sensitive to lipid solvents. effect of low ph on infectivity. the test was performed as described by leibhaber [13] . tenfold serial dilutions of infected-mouse-brain suspension kept at different ph values were made in eagle's minimal essential medium in earle's base with 3% fetal bovine serum (fbs). the ph of each dilution was adjusted to approximately 7.0 by addition of tris, and this solution was inoculated into mice. end points of infectivity were calculated by the method of reed and muench [12] . the titers of infectious virus detected in pbs after incubation for 3.0 hr at 25 c was 103. 6 imld50/0.015 ml, whereas at ph 7.0 and ph 3.0 it was 10 3 . 9 imld50/0.015 m1 and lq2·8 1mld50/ 0.015 ml, respectively. thus infectious virus was relatively stable at low ph. effect of temperature on infectivity. the effect of a temperature of 37 c on infectious virus was determined as outlined by bhatt et al. [9] . to determine the effect of a temperature of 56 c, infectivity was determined at intervals of 0, 5, and 10 min. an aliquot of viral stock was kept at 4 c, and infectivity was determined on days 0, 7, and 28. infectivity of viral strain 681 was stable in pbs plus 3% fbs at 37 c for 3 hr; the titer then decreased by 1.1 log., by 5 hr. at 56 c the infectivity decreased from 10 3 . 8 1mld50 at zero time to trace levels by 5 the size of infectious viral particles. the approximate size of infectious viral particles was determined by the method of atoynatan and hsiung [14] and casals [15] as modified by bhatt et al. [9] . a fresh, 10% suspension of mouse brain was made in pbs plus fbs, clarified by centrifugation at 1,000 g for 20 min, and filtered through miliipore filters (millipore corp., bedford, mass.) of various pore sizes. viral titers obtained were 103. 5, 104.2, 103. 8 , 10 3 . 0, and < lq2 1mld50/0.015 m1 for unfiltered virus and after filtration through pore sizes 1,200 urn, 450/lm, 220/lm, and 100 urn, respectively. results indicate that the size of the virus is less than 220 urn but greater than 100 urn. the particles without membranes measured by electron microscopy were previously reported to be 6<f--70 /lm [1] . detection of hemagglutination. two sources of antigen were used. (1) a 10% suspension of infected salivary gland was tested for its capacity to hemagglutinate red blood cells (rbcs) of rabbits, guinea pigs, and geese by the method of ashe [16] . (2) either a 10% suspension of infected mouse brain in pbs or infected mouse brain extracted with sucrose and acetone was tested for hem agglutinability with rbcs of rats, mice, guinea pigs, and geese. the microtiter method [11] was used for all tests, and incubation was at 4 c, 25 c, and 37 c. rbcs were suspended (0.5%) in pbs. hemagglutination was not detected at 4 c, 25 c, or 37 c using a suspension of infected salivary gland and rabbit, guinea pig, or goose cells. a 10% suspension of infected brain in pbs or sucrose-acetone-extracted antigen from mouse brain gave unsatisfactory results with rbcs of rats, guinea pigs, and mice at 25 c and 37 c, and there was no ha activity after incubation overnight at 4 c. neither was ha detected for goose rbcs at 4 c, 25 c, or 37 c. acridine-orange staining. the cytoplasm of infected cells stained orange-red with acridine orange, indicating that virus replicates in the cytoplasm. antigenic relationship to other viruses. results of these experiments are summarized as follows. (1) serum-neutralization tests were conducted with strain 681 sda virus with immune sera to * names of the individual arboviruses will be furnished upon request. theiler encephalomyelitis virus (strain gd vii), sendai, mvm, and mouse adenovirus were negative. immune sera to toolan h-l virus, kilham rat virus, and simian myxovirus sv 5 (at dilutions of 1: 5) did not neutralize sda virus. (2) sera of mice immune to strain 681 were tested by cf at dilutions of 1: 4, 1: 8, and 1: 16 for reactivity to 118 viral antigens. these agents are listed in table 3. there was no reaction, indicating absence of antigenic relationship between our agent and these 118 agents under the conditions of the cf test. (3) a checkerboard cross-cf test was performed, using two different sera from mice immune to sda agent and commercial mhv antigen and corresponding immune serum. on the table 5 . results of cross-neutralization tests with sialodacryoadenitis virus (sda) and rat coronavirus (rev) and respective immune sera. (4) cross-neutralization tests were performed with parker's rat coronavirus and strain 681, using both homologous and heterologous immune sera. as shown in table 5, there is cross-neutralization, but there are also antigenic differences between these viruses. induction of disease in weanling mice. neither overt illness nor any gross or histologic evidence of lesions was associated with this agent. more specifically, there were no lesions compatible with infection by mhv either in the group treated with cortisone or in the untreated group. production of sda in susceptible rats by mousebrain-adapted strain 681. there was no overt illness in any rat, and at necropsy all organs, including the lacrimal and salivary glands, were grossly normal. histopathologic examination of the harderian, exorbital, parotid, and submaxillary salivary glands showed evidence of sda. there was considerable variation in the severity of the reaction of affected glands. in general, lesions were most numerous and most severe in the parotid and exorbital glands. two important features of this study were the relative severity of lesions in the parotid salivary glands and the failure to isolate an agent pathogenic for mice from these animals. these findings suggest that the mouse-brainadapted virus caused mild but definite lesions compatible with sda. it has been approximately 10 years since the first recognized outbreak of sda in rats. on the basis of the information presented in this report, it is concluded that a viral agent causing this disease in rats has been isolated and adapted to grow in brains of infant mice and in primary rat-kidney cultures. the agent probably has rna as its nucleic acid and is sensitive to lipid solvents. it is antigenically related to the rat coronavirus of parker and to mouse-hepatitis virus. it apparently multiplies in the cytoplasm and forms multinucleated giant cells in tissue culture. some coronaviruses are acid labile, but the sda agent is relatively stable at ph 3.0. the virus of transmissible gastroenteritis is also stable at ph 3.0, yet is considered a coronavirus [17] . thus ph stability may be a variable feature of this group. on the basis of this information, we conclude that this virus belongs to the corona group, even though information on the morphology of the negatively stained viral particle and its mode of replication in cells (as determined by electron microscopy) has yet to be obtained. in the affected tissues, the particles are frequently found in cytoplasmic vesicles that eventually contain lysosomal activity (a. m. jonas, unpublished observation). the titer of infectious virus in mouse brain was not increased by serial passage. this may be a reflection of the facts that only selected cells are involved in viral multiplication and that there may be a low yield of infectious virus per infected cell. (the former supposition has been confirmed by detailed histopathologic study supplemented by fluorescent-antibody tracing of infected cells.) when working with mouse-adapted viruses of rats, it is essential to prove that one has not picked up agents from mice. evidence against this possibility includes the following. (l) sickness in mice is produced only when the animals are inoculated with a suspension of infected salivary gland and not when normal salivary gland is used. (2) agents pathogenic for mice were not recovered when brains from normal mice were passaged consecutively several times by the intracerebral 129 route in mice. (3) dr. r. e. shope tested 118 viral antigens prepared from infected mouse brains with antiserum to sda virus. these mice were from the same colony used for our work. he did not find any reaction in cf tests with immune serum to sda virus. (4) immune sera prepared in rats with virus passaged in salivary glands reacted with antigen from murine brain, while sera taken from the same rats before immunization did not. (5) sda was reproduced in rats by inoculation of mouse-brain-adapted virus. failure to adapt sda virus to tissue-culture systems other than primary rat-kidney cultures confirms the observation that coronaviruses are fastidious in their cultural requirements [18] . absence of viral multiplication in mono1ayers of ratsalivary-gland and mouse-brain tissue may be due to absence in culture of cells that normally support growth of virus in the intact host. complement-fixing and neutralizing antibodies to mhv were detected in the sera of men and rats by hartley et al. [19] and were interpreted as indication of infection by antigenically-related, species-specific viruses. this hypothesis has been confirmed in part by isolation of coronaviruses from man [18] . sda agent and parker's rat corona agent may be responsible for antibodies to mhv found in rats. biologically, sda virus behaves differently from known strains of mhv, since the former does not multiply in the py-al/ n cell line, which is highly susceptible to mhv [6] . in addition, sda virus does not induce lesions compatible with those due to mhv if inoculated ic into infant mice or if inoculated ic or ip into cortisone-treated weanling mice. therefore, our failure to detect antigenic differences between these agents by the complement-fixation test should be interpreted with caution. more information may be obtained for serologic differentiation by use of immune sera prepared by different schedules of immunization and then tested by neutralization, complement-fixation, fluorescent-antibody, and gel-diffusion methods. since there is more than one serotype of human and mouse coronavirus [18, 20] , it is not surprising that, although there is much crossneutralization, there seem to be antigenic differences between our virus and the one isolated by parker et al. [4] . several serotypes of rat coronaviruses may therefore exist. it is necessary to delineate further the serotypes, the disease poten-tials, and the epidemiology of both agents. for example, our agent is known to cause sda, while parker's rat coronavirus causes pulmonary lesions. the ability of mouse-brain-adapted virus to produce the disease in rats indicates that we are dealing with the same agent. an evaluation of the glands involved and the extent of involvement may indicate a shift in tissue tropism and also an apparent reduction in virulence of virus. sda virus was not recovered from submaxillary salivary glands when mouse-adapted-virus was inoculated into rats, but a change in tissue tropism may have accounted for this difficulty. attempts were not made to isolate virus from parotid and exorbital glands, but histopathologically they demonstrated classical lesions. these tissues were only minimally involved in previous studies with the strain passaged in rats. the apparent shift in tissue tropism needs confirmation in both germfree and conventional, susceptible rats of the same strain, particularly since the transmission experiments were performed in susceptible, specific-pathogenfree wistar rats. sialodacryoadenitis in the rat. a light and electron microscopic study studies on the agent of sialodacryoadenitis of rats proc. p rat coronavirus (rcv): a prevalent, naturally occurring pneumotropic virus of rats. arch. gesamte virusforsch fluorescent antibody methods enhanced growth of murine corona viruses in cells transformed by polyoma virus proc. p tissue culture technics for diagnostic virology chandipura, a new arbovirus isolated in india from patients with febrile illness isolation and characterization of a herpeslike (hsiung-kaplow) virus from guinea pigs herpes and pox viruses: laboratory procedures application of a microtechnique to viral serological investigations a simple method of estimating fifty per cent endpoints studies of a myxovirus recovered from patients with infectious hepatitis. i. isolation and characterization. 1 ultrafiltration of simian viruses casals, 1. filtration of arboviruses through "millipore" membranes previously unrecognized virus from submaxillary glands of gnotobiotic and conventional rats diseases of swine corona viruses of man antibodies to mouse hepatitis viruses in human sera viruses of laboratory rodents. national cancer institute monograph no key: cord-000539-uh3q65we authors: zhang, yi; sun, honglei; fan, lihong; ma, yuan; sun, yipeng; pu, juan; yang, jun; qiao, jian; ma, guangpeng; liu, jinhua title: acute respiratory distress syndrome induced by a swine 2009 h1n1 variant in mice date: 2012-01-03 journal: plos one doi: 10.1371/journal.pone.0029347 sha: doc_id: 539 cord_uid: uh3q65we background: acute respiratory distress syndrome (ards) induced by pandemic 2009 h1n1 influenza virus has been widely reported and was considered the main cause of death in critically ill patients with 2009 h1n1 infection. however, no animal model has been developed for ards caused by infection with 2009 h1n1 virus. here, we present a mouse model of ards induced by 2009 h1n1 virus. methodology principal findings: mice were inoculated with a/swine/shandong/731/2009 (sd/09), which was a 2009 h1n1 influenza variant with a g222d mutation in the hemagglutinin. clinical symptoms were recorded every day. lung injury was assessed by lung water content and histopathological observation. arterial blood gas, leukocyte count in the bronchial alveolar lavage fluid and blood, virus titers, and cytokine levels in the lung were measured at various times post-inoculation. mice infected with sd/09 virus showed typical ards symptoms characterized by 60% lethality on days 8–10 post-inoculation, highly edematous lungs, inflammatory cellular infiltration, alveolar and interstitial edema, lung hemorrhage, progressive and severe hypoxemia, and elevated levels of proinflammatory cytokines and chemokines. conclusions/significance: these results suggested that we successfully established an ards mouse model induced by a virulent 2009 h1n1 variant without previous adaptation, which may be of benefit for evaluating the pathogenesis or therapy of human ards caused by 2009 h1n1 virus. a novel influenza a (h1n1) virus of swine origin emerged among humans in mexico during the spring of 2009 and rapidly spread worldwide [1] . the pandemic prompted the world health organization (who) to raise the alert level to the highest rating of six, the pandemic phase, within 2 months [2] . in august 2010, who officially declared that the disease was in the post-pandemic period [3] ; however, it is still circulating among humans, together with seasonal viruses. although most influenza cases caused by 2009 h1n1 virus infection typically display mild upper respiratory tract syndrome, some cases progress to severe pneumonia and acute respiratory distress syndrome (ards) [4, 5] . many studies have shown that ards caused by 2009 h1n1 virus results in 17.3-56% mortality [4, 6, 7, 8] , which was regarded as the major cause of death by 2009 h1n1 virus infection [9] . ards is the result of acute injury to lung tissue, commonly resulting from sepsis, trauma, and severe pulmonary infections [10] . infectious factors, most of which are viruses, have become one of the most important causes of ards in humans [11, 12, 13] . clinical cases and established animal models have revealed that the pathogenesis and pathological features of ards induced by different viral pathogens are distinct [14, 15] . however, knowledge of the pathogenesis of 2009 h1n1 virus, especially ards induced by 2009 h1n1 virus, is still limited and hinders therapeutic strategies. therefore, it is necessary to evaluate the pathogenesis of ards caused by 2009 h1n1 virus infection in an appropriate animal model to assess potential therapies. mice are a good model for evaluating the pathogenesis and antiviral therapy of influenza pneumonia, due to the general fidelity of the illness in mice to the human disease [16] . moreover, a mouse model of ards caused by highly pathogenic h5n1 avian influenza virus infection has been well established [13] . the typical 2009 h1n1 virus, such as a/california/04/2009 (ca/04), can efficiently replicate in mouse lungs without prior host adaptation. however, it only causes moderate lung lesions and no mortality, even when inoculated at a high dose of 10 6 pfu [17, 18] . thus, such typical 2009 h1n1 viruses may not be able to induce ards in a mouse model. in the present study, we used a virulent variant 2009 h1n1 virus, which was isolated from a pig and possessed a virulenceassociated ha-d222g mutation, to establish an ards mouse model. the model established here provides a useful tool to explore the mechanism of ards, as well as screening and therapeutic options. six-week-old female mice were infected intranasally (i.n.) with 10 2.5 pfu sd/09 virus. some of the infected mice showed signs of illness, such as altered gait, inactivity, ruffled fur, and anorexia on day 2 post-infection (p.i.). from day 2 p.i., the body weight of most mice significantly decreased ( figure s1 ). by day 6 p.i., most mice presented with more severe clinical signs of respiratory disease, including labored respiration and respiratory distress, and most mice lost almost 20% of their initial body weight. on day 8 p.i., most mice were nearly unable to respond to exterior stimuli, and acute respiratory rates and labored respiration were observed (video s1, and video s2 for control). approximately 60% of mice died between days 8 and 10 p.i. gross observation of infected mice showed that the lungs were highly edematous, with profuse areas of hemorrhage and consolidation. no obvious gross lesions were observed in the kidneys, liver, spleen or brain of infected mice. mice were infected i.n. with 10 2.5 pfu sd/09 virus, and three mice were euthanized on days 2, 4, 6, 8, 10 and 14 p.i., and the virus titers in viscera were determined. as shown in figure 1a , the virus titer in the lung gradually increased between days 2 and 6 p.i., and reached a peak on day 6 p.i. the virus titers in the lung gradually decreased from day 6 p.i., and only one of three mice possessed detectable virus in the lungs on day 10 p.i. no viruses were detected in other organs, including heart, spleen, liver, kidneys, blood and brain, at the indicated time. these results indicate that sd/09 virus could replicate efficiently in mouse lung but did not cause systemic infection. as shown in figure 1b , the effect of sd/09 viral infection on lung wet:dry weight ratio did not change significantly within 2 days p.i. however, a dramatic increase was observed from day 4 p.i., and reached a peak on day 8 p.i., which was nearly twice that observed in control group lungs (p,0.01). the change in lung wet weight:body weight ratio was similar to the change in lung wet:dry weight ratio ( figure 1c ). the results indicated that the sd/09 virus could induce acute lung edema in mice. kinetic observation of lung lesions of sd/09-virus-infected mice is shown in figure 2 . on day 4 p.i., lung lesions were characterized by dropout of mucous epithelium and inflammatory cells adhering to the bronchiolar surface ( figure 2c , d). on day 6 p.i., severe edema could be seen around blood vessels ( figure 2e ); interstitial pneumonia was also observed that showed interstitial edema and thickening of the alveolar walls; and the alveolar lumen was flooded with detached alveolar cells, erythrocytes, and inflammatory cells ( figure 2e , f). on day 8 p.i., the virus caused more severe interstitial pneumonia and peribronchiolitis, characterized by edema and extensive of lymphocytes, neutrophils and plasma cells around the area of bronchiolitis ( figure 2g , h). lesions in the lungs of infected mice were still severe on day 14 p.i., with extensive alveolar collapse, and remaining alveoli were filled with fibrin, desquamated alveolar cells, and inflammatory cells. lymphocytes and alveolar macrophages were the predominant inflammatory cells observed at high magnification ( figure 2j ). masson's stain revealed that alveolar walls and spaces were filled with collagen fibers ( figure 3a , b), indicating that proliferative fibroblastic lesions may develop. in comparison, lungs from mockinfected control mice had no apparent histological changes ( mice were inoculated i.n. with 10 2.5 pfu sd/09 viruses; tissues were collected at indicated times p.i. and viruses were titrated in mdck cells. body weight, lung wet and dry weight were determined and recorded. the lung wet weight:body weight ratio and lung wet:dry weight ratio were calculated and used as an indicator of lung edema. *p,0.05, **p,0.01, ***p,0.001, comparison between ratios obtained from the virusinfected and control groups. bars represent means 6 sd of data from three mice. doi:10.1371/journal.pone.0029347.g001 immunohistochemistry revealed viral antigens in the epithelial cells of the bronchioles ( figure 3d ), terminal bronchioles, and alveolar epithelial cells ( figure 3e ). these data indicated that sd/ 09 virus could infect the epithelia of the lower airway and cause viral pneumonia in mice. as shown in table 1 , virus-infected mice showed a slightly decreased partial pressure of arterial oxygen (pa o2 ), saturation of arterial oxygen (sa o2 ), and slightly increased partial pressure of arterial carbon dioxide (pa co2 ) from days 4 to 6 p.i. most infected mice presented with severe clinical signs of respiratory distress on day 8 p.i., and blood gas analysis also showed that pa o2 and sa o2 dramatically decreased compared with the controls (p,0.05). these results suggested acute respiratory dysfunction and severe hypoxemia in virus-infected mice. the number of leukocytes in balf from sd/09-infected mice showed an increase from day 4 p.i. ( table 2 ). the balf of virusinfected mice on day 6 p.i contained 1.1610 6 cells/ml and was significantly different from the 1.6610 5 cells/ml observed for pbsinoculated mice (p,0.001). these data indicate a dramatic increase in inflammatory cells in the lungs of sd/09-infected mice. to quantify the immune cell subpopulations responding to viral infection, we next determined cell differential counts in the infected lungs by wright staining. compared with pbs-inoculated animals, mice infected with sd/09 virus exhibited an increase of neutrophils from 4 days p.i., and the peak was 18-fold greater than that of the control group on day 8 p.i. leukopenia was detected on day 4 p.i., and was statistically significant on day 6 p.i. (p,0.05); the lowest value appeared on day 8 p.i. (p,0.001). furthermore, differential blood counts revealed that the number of lymphocytes sharply decreased in infected mice. the lowest number of lymphocytes observed occurred on day 8 p.i. (figure 4b ), which dropped to ,20% of the control group number (p,0.001). to determine the cytokine responses that occur after sd/09 virus infection, we measured the levels of five cytokines and chemokines in lungs of infected mice on days 2, 4, 6, 8 and 10 p.i. as shown in figure 5 , all five were significantly different between virus-infected and control mice. interleukin (il)-6 and il-10 in the virus-infected mice reached peak levels as early as day 2 p.i. and were significantly higher than those of the control group (p,0.001). interferon (ifn)-c, monocyte chemotactic protein (mcp)-1, and tumor necrosis factor (tnf) dramatically increased in mouse lungs on days 6-8 p.i. (p,0.001), consistent with the appearance of pulmonary lesions. these results showed that infection with sd/09 viruses resulted in elevated amounts of proinflammatory chemokines and cytokines in the lungs of mice. in the spring of 2009, a novel influenza a(h1n1) virus rapidly spread worldwide, resulting in the first influenza pandemic of the 21st century [1] . critically ill cases caused by 2009 h1n1 virus retrospectively showed that most had progressed or died due to ards [4, 6] . however, the pathogenesis and therapeutic intervention of ards caused by 2009 h1n1 infection have still not been elucidated. animal models of disease are important for characterizing pathogenesis and developing the preclinical evidence for revised approaches to ventilating patients with ards [19] . here, we present a mouse model for the study of ards induced by sd/09 virus, a virulent 2009 h1n1 variant. previous studies have indicated that typical 2009 h1n1 viruses such as ca/04 bind only to a-2,6-linked sialic acid (sa) receptor [17] , but only the a-2,3-linked sa receptor is found in the mouse respiratory tract [20] . therefore, such a typical 2009 h1n1 virus may not be able to induce ards in mice. in fact, we used ca/04 virus to induce ards in mice; however, animals inoculated with a high dose of ca/04 virus (10 6.5 pfu) only showed moderate respiratory symptoms, and no lethality was observed (unpublished data). it has been shown that 2009 h1n1 virus possessing a d222g mutation in hemagglutinin (ha) could increase the pathogenicity in mice [21, 22] and binding to the a-2,3 sa receptor [23] . moreover, clinical data indicate that such variants are only associated with severe h1n1 human infection [24] . therefore, we suggest that the variant possessing the d222g mutation in ha can induce ards in a mouse model. the virus used in the present study was isolated from swine in 2009, and sequence analysis revealed that all the eight genes of the isolate had a close relationship with the 2009 h1n1 influenza virus circulating in humans. notably, the swine isolate, sd/09, had a d222g mutation in ha. compared with ca/04 virus (ld 50 .10 6 pfu), sd/09 showed significantly increased virulence in mice, with an ld 50 of 10 2.25 pfu, which was nearly identical to that of the mouse-adapted strain a/hong kong/415742md/09 (ld 50 = 10 2.2 pfu) [17, 21] . mice infected i.n. with 10 2.5 pfu sd/ 09 virus showed obvious respiratory symptoms, including visually prominent signs of respiratory distress and abdominal respiration, with approximately 60% mortality between days 8 and 10 p.i. the lungs of virus-infected mice were highly edematous, which was also demonstrated by dramatically increased lung wet:dry weight ratio. pathological changes presented a progressive pattern, typically diffuse alveolar damage, interstitial and alveolar edema, neutrophil and macrophage-dominant inflammatory cellular infiltration, and areas of hemorrhage and necrotizing bronchiolitis. arterial blood gas saturation is a key parameter of ards in humans [25] . in the present mouse model, pao 2 and sao 2 of infected mice were significantly lower than in the control group from day 2 p.i., especially on day 8 p.i., where these parameters sharply decreased, and most virus-infected mice began to die. these changes in arterial blood gas demonstrated that most infected mice developed severe hypoxemia consistent with of the appearance of clinical signs and lung lesions of ards. previous studies showed that mice infected with typical 2009 h1n1 virus only exhibited mild interstitial inflammatory infiltration and limited alveolitis [18, 21] , whereas severe lung damage was found in the sd/09-infected mice, including severe edema around the blood vessels and bronchiolitis, and extensive inflammatory accumulation from 4 to 8 days p.i. at 10 days p.i., the surviving mice developed an irreversible fibrosis involving collagen deposition in alveolar walls and spaces, which was similar to that observed in human ards patients with 2009 h1n1 infection [26] . our histopathological results were consistent with ards induced by other influenza viruses. mice infected with mouse-adapted virus of the a/puerto rico/8/34 (h1n1), or high pathogenic h5n1 virus also showed a progressive series of pathological changes from interstitial pneumonia to diffuse alveolar damage [13, 27] . however, in contrast to highly pathogenic h5n1 virus, mice infected with sd/09 virus did not show viral spread to extrapulmonary organs. immunohistochemical examination revealed the presence of viral antigens in the bronchioles, terminal bronchiolar epithelium, and alveolar epithelial cells. perhaps sd/09 virus infection of the alveoli, particularly type ii pneumocytes, rather than bronchioles, is a key to the development of ards. type ii pneumocytes are responsible for the production and secretion of surfactant to lower the surface tension of water and allow membrane separation, and insufficient pulmonary surfactant in the alveoli may result in alveolar collapse [28, 29] . the 1918 pandemic h1n1 and high pathogenic h5n1 viruses preferentially infect type ii pneumocytes and alveolar macrophage in mice [15, 30] . alveolar macrophages may play a critical role in disease pathogenesis, not through production of infectious virus but rather through the upregulation of proinflammatory cytokines that may further damage alveolar pneumocytes [31] . these phenomena suggest that viral cell tropism may determine the processes of ards. pulmonary aberrant immune response is considered a significant feature of ards induced by 2009 h1n1 virus [19, 32] . in the present mouse model, the number of leukocytes observed in the balf of virus-infected mice significantly increased compared with the control mice on day 8 p.i. different counts in balf showed that the proportion of neutrophils dramatically increased. these innate immune cells were capable of reducing the virus load in the lung [33] ; however, they could cause lung injury through direct or indirect mechanisms. neutrophil oxidants and proteases can cause direct injury of cells in the alveolar-capillary membrane [34] . neutrophils and macrophages can secrete copious amounts of chemokines and cytokines that can recruit more immune cells into lung tissues, and produce a ''cytokine storm'', one of the most important factors in the production of ards [35] . a retrospective cohort study of 74 2009 h1n1 patients found that higher levels of proinflammatory cytokines and chemokines in plasma were observed in the ards-death group compared with the survived-without-ards or the mild-disease groups [5] . another study in critically ill patients with ards caused by 2009 h1n1 virus infection has shown that the hallmarks of disease severity were elevated levels of il-6, il-15, il-8 and tnf-a [36] . we examined the levels of five cytokines and chemokines in infected mouse lungs and found significant differences between the virus-infected and mock groups. it has proved that high levels of il-6 were able to mediate acute lung injury [37] , and had a negative correlation with the pa o2 :fi o2 ratio in severely affected patients with 2009 h1n1 virus infection [36] . our data showed sd/09 viral infection induced high levels of il-6 in mouse lung, which may also play an important role in the course of ards. hagau etc. found the levels of tnf-a increased significantly in the 2009 h1n1-related ards patients [36] . in present study, tnf levels also dramatically increased in the lungs of virus-infected mice, and were consistent with the clinical symptoms and reached peak levels when mice began to die. in addition, high levels of il-10, ifn-c and mcp-1 were also present in the virus-infected mouse lungs, similar to observations found in severely affected humans with 2009 h1n1 infection [38] . in summary, we successfully established an ards mouse model induced by a virulent 2009 h1n1 variant, which demonstrated key human ards clinical and pathological features, such as respiratory distress, low pa o2 , exudative, proliferative and fibrotic lung, and high levels of inflammatory cells and cytokines. the mouse model may contribute to the study of the pathogenesis and therapy of ards induced by 2009 h1n1 virus. to determine ld 50 of sd/09 virus, eight 6-week-old female balb/c mice per group were inoculated i.n. with 10 1 -10 6 pfu (50 ml) viruses and monitored for 14 days. the value of mld50 was calculated using the spearman-karber method and expressed by pfu per mld 50 [39] . we evaluated the pathogenicity of the virus in mice and found that it could efficiently replicate in the lungs of mice with high lethality (10 2.25 pfu per mld 50 ). to determine the optimal dose of inoculation, 10 mice in each group were infected i.n. with 10 1.5 , 10 2.5 or 10 3.5 pfu viruses, and the signs, body weight, and mortality were monitored daily for each group for 14 days. pilot experiments indicated that a dose of 10 2.5 pfu was optimal, because the course of the disease was prolonged and the mice presented with obvious signs of respiratory illness. balb/c mice were lightly anesthetized and inoculated i.n. with 50 ml 10 2.5 pfu sd/09 virus in pbs. mock-infected animals were inoculated i.n. with 50 ml pbs. at the indicated time, infected mice were sacrificed, and the parameters that present the course of the disease were determined. twenty mice (10 infected with sd/09 virus and 10 inoculated with pbs) were used to investigate clinical signs and mortality for 14 days. three mice were euthanized on days 2, 4, 6, 8, 10 and 14 p.i. and their organs were collected. the collected tissues were weighed, and 10% homogenates were prepared in cold pbs. the homogenates were centrifuged at 3000 rpm for 10 min to remove cell debris, and then the supernatants were 10-fold serially diluted for viral titer determination by plaque assay in mdck cells. virus titers were expressed as mean log pfu/g 6 standard deviation (sd). three mice were euthanized on days 2, 4, 6, 8, 10 and 14 p.i., and the lungs were removed and weighed and then desiccated in an oven at 60uc for 72 h. the lung wet weight:body weight ratio and lung wet:dry weight ratio were calculated and used as an indicator of lung edema, as previously described [40] . three mice were euthanized on days 4, 6, 8 and 14 p.i. the lungs were fixed in 10% buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. lungs on day 14 p.i. were also stained with masson's trichrome. lung tissue sections taken on day 6 p.i. were stained for influenza a virus antigens. an anti-influenza nucleoprotein monoclonal antibody (aa5h; abcam, hong kong) was used to identify influenza a virus nucleoprotein in sections. secondary antibody (millipore, billerica, ma, usa) against the primary antibody was labeled with horseradish peroxidase, and the color reaction was developed with a horseradish peroxidase reaction kit (diaminobenzidine-tetrahydrochloride; sigma, st. louis, mo, usa). blood gas analysis was performed as previously described [13, 41] . three mice were anesthetized with zoletil (tiletamine-zolazepam; virbac; 20 mg/g) on days 4, 6, 8, 10 and 14 p.i. arterial blood samples were withdrawn into a heparinized syringe by percutaneous left ventricular sampling of lightly anesthetized mice that were spontaneously breathing room air. blood gas analysis was immediately performed using a vetstat electrolyte and blood gas analyzer (idexx laboratories, westbrook, ma, usa). leukocyte counts in balf were performed as previously described [42, 43] . briefly, three mice were euthanized on days 4, 6, 8 and 10 p.i., and the lungs were lavaged twice in situ with the chest cavity opened by midline incision with a total volume of 1.0 ml saline (4uc) inserted through an endotracheal tube. the rate of recovery of balf was not less than 90% for all animals tested. after the amount of fluid recovered was recorded, an aliquot of balf was diluted 1:1 with 0.01% crystal violet and 2.7% acetic acid for leukocyte staining and erythrocyte hemolysis. the number of leukocytes in the balf was counted with a hemacytometer under a microscope. for differential counts, the balf samples from each mouse were stained with wright stain, and the numbers of monocytes, neutrophil and lymphocytes were determined, on the basis of morphologic criteria, under a light microscope, with evaluation of at least 200 cells per slide. all slides were counted twice by different observers blinded to the status of the animal. heparinized blood samples were collected on days 4, 6, 8 and 10 p.i. the total numbers of leukocytes and differential blood counts for three individual mice were analyzed using an automated hematology analyzer. il-6, il-10, tnf, ifn-c and mcp-1 levels were determined in lung homogenates using a cytometric bead array technique (bd cytometric bead array mouse inflammation kit; bd bioscience, san diego, ca, usa) according to the manufacturer's instructions. briefly, 50 ml mouse inflammation capture bead suspension and 50 ml pe detection reagent were added to an equal amount of sample standard dilution and incubated for 2 h at room temperature in the dark. subsequently, samples were washed by adding 1 ml wash buffer and centrifugation at 2006 g at room temperature for 5 min. supernatants were discarded and 300 ml wash buffer was added. samples were analyzed on a bd facsarray bioanalyzer (bd bioscience) according to the manufacturer's instructions. standard curves were prepared similar to the method above. data were analyzed using bd cba software (bd bioscience). finally, the chemokine or cytokine levels were recorded as pg/ml homogenate. data were analyzed by two-way analysis of variance using graphpad prism version 5.00 (graphpad software, san diego, ca, usa). when a significant effect was observed, pairwise comparisons were performed using the bonferroni post-hoc test. all 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enos-null mice is hyperresponsive to mild hypoxia respiratory reovirus 1/l induction of intraluminal fibrosis, a model of bronchiolitis obliterans organizing pneumonia, is dependent on t lymphocytes role of p38 mitogen-activated protein kinase in a murine model of pulmonary inflammation the authors thank dr. yanxin hu and dr. deping han for technical assistance in histopathologic observation. key: cord-001675-9717nzr7 authors: sugiyama, michael g.; armstrong, susan m.; wang, changsen; hwang, david; leong-poi, howard; advani, andrew; advani, suzanne; zhang, haibo; szaszi, katalin; tabuchi, arata; kuebler, wolfgang m.; van slyke, paul; dumont, dan j.; lee, warren l. title: the tie2-agonist vasculotide rescues mice from influenza virus infection date: 2015-06-05 journal: sci rep doi: 10.1038/srep11030 sha: doc_id: 1675 cord_uid: 9717nzr7 seasonal influenza virus infections cause hundreds of thousands of deaths annually while viral mutation raises the threat of a novel pandemic strain. antiviral drugs exhibit limited efficacy unless administered early and may induce viral resistance. thus, targeting the host response directly has been proposed as a novel therapeutic strategy with the added potential benefit of not eliciting viral resistance. severe influenza virus infections are complicated by respiratory failure due to the development of lung microvascular leak and acute lung injury. we hypothesized that enhancing lung endothelial barrier integrity could improve the outcome. here we demonstrate that the tie2-agonist tetrameric peptide vasculotide improves survival in murine models of severe influenza, even if administered as late as 72 hours after infection; the benefit was observed using three strains of the virus and two strains of mice. the effect required tie2, was independent of viral replication and did not impair lung neutrophil recruitment. administration of the drug decreased lung edema, arterial hypoxemia and lung endothelial apoptosis; importantly, vasculotide is inexpensive to produce, is chemically stable and is unrelated to any tie2 ligands. thus, vasculotide may represent a novel and practical therapy for severe infections with influenza. whether as an agent of pandemics or as a seasonal pathogen, the influenza virus exacts a heavy toll on global public health. despite vaccination programs and antiviral drugs, seasonal influenza alone causes millions of cases of severe illness and hundreds of thousands of deaths annually 1, 2 . there are concerns that ongoing mutation will lead to a novel strain of the virus that is both highly transmissible and highly virulent, as occurred in the 1918 pandemic leading to the deaths of 50 million people 3 . existing treatments for the virus are inadequate: antiviral drugs are not completely effective at reducing mortality 4, 5 and exhibit declining efficacy unless given at the time of infection, a problematic limitation since the time of infection is usually unknown 6, 7 . there is also the problem of antiviral resistance; of the two classes of drugs approved for use against influenza, one is largely ineffective due to widespread resistance, while sporadic cases of resistance to the other have been reported 8, 9 . thus, there is a need for novel therapies; those that target the host rather than the pathogen may be ideal as they should be less susceptible to viral resistance. most deaths from influenza virus infection occur due to pulmonary complications, in particular the development of acute respiratory distress syndrome (ards) 10, 11 , a potentially fatal syndrome of pulmonary edema that occurs due to increased permeability of the lung microvasculature 12, 13 . blood vessels in the lung are lined by a continuous layer of endothelium; thus, loss of endothelial barrier integrity is a prerequisite for ards. the fact that mortality persists despite antiviral therapy suggests that elements of the host response may be maladaptive 14, 15 . given the prominence of ards in severe infections, we hypothesized that enhancement of lung endothelial barrier function might improve survival. we tested the novel tie2-agonist tetrameric peptide, vasculotide; this peptide has no sequence homology with any endogenous ligands, is easy to produce and is chemically stable 16 . here we report that administration of vasculotide significantly improves survival from influenza virus infection, even when started several days post-infection. we first infected c57bl/6 mice with a lethal dose of influenza virus (hkx31;h3n2) followed by daily intraperitoneal injection of vasculotide (500 ng, see schematic, fig. 1a ). while no infected mice survived longer than 8 days, treatment with vasculotide markedly improved survival even if started as late as 72 hours post-infection (80% survival if started at 24 hours after infection; 70% survival if at 72 hours, p < 0.001); treatment starting 96 hours after infection improved survival although this did not achieve protocol used to test vt in mice infected with influenza virus. arrows indicate the time after infection (hours) at which daily administration of vt was begun. c57bl/6j mice were infected with mouseadapted influenza, strain x31 and received placebo or 500 ng vt per day starting at the indicated times. (b) survival was monitored daily; numbers in parentheses indicate the number of mice per group. ***p < 0.001 vs. flu-alone mice. (c) arterial oxygen saturation on day 5 after infection, ***p < 0.001. (d) lung edema was measured by wet-to-dry ratio in mice sacrificed 5 days after infection. (e) left ventricular ejection fraction on day 5 after infection was measured by echocardiography. (f) c57bl/6j mice were infected with mouseadapted influenza, strain pr8 and received placebo or 500 ng vt daily starting at 48 hours after infection, *p < 0.05. scientific reports | 5:11030 | doi: 10.1038/srep11030 statistical significance (p = 0.07; fig. 1b ); treatment with a lower dose of vasculotide (100 ng) did not appear to be as effective (supplemental figure 1 ). prior to receiving vasculotide, mice displayed typical signs of progressive illness such as arterial hypoxemia, hypothermia, and declining body weight; these features are apparent as early as 48-72 hours after infection. vasculotide treatment started at 72 hours post-infection is able to rescue a significant subset of infected mice (supplemental figures 2,3) . furthermore, virus-induced hypoxemia and lung edema were greatly ameliorated with the drug (fig. 1c-d) . echocardiography revealed that lung edema was not due to impairment of left ventricular systolic function by the virus, consistent with an increase in vascular leak from lung injury 13 ; analogously, the reduction in lung edema by vasculotide was not from an improvement in left ventricular systolic function (fig. 1e) . of note, the drug was similarly effective against a lethal dose of pr8 (h1n1), a second strain of influenza virus that is highly virulent in mice 17 (fig. 1f) . next, we infected mice with the 2009 swine-origin pandemic h1n1 influenza virus. although the mortality rate in these mice was only 25%, all of the vasculotide-treated mice survived (supplemental figure 4) . thus, vasculotide appears to be effective against lung injury caused by multiple strains of influenza. in a clinical setting, an agent like vasculotide is most likely to be administered in combination with an antiviral drug. we therefore treated influenza virus-infected mice with amantadine 18 alone or in combination with vasculotide. treatment with amantadine modestly but significantly improved survival (from 0 to 14%, fig. 2a ). adjuvant vasculotide markedly increased survival even when started as long as 72 hours after infection (83% survival) ( fig. 2a and supplemental figure 5 ). this was in association with significant improvements in arterial hypoxemia (fig. 2b ), hypothermia and weight loss ( fig. 2c-d) . these findings are especially important because mortality from influenza virus persists despite antiviral therapy and because the efficacy of such therapy declines rapidly over time 4, 6, 7 . to prove that the benefit of vasculotide is mediated through the tie2 receptor, and because homozygous deletion of tie2 is embryonically lethal 19 , we obtained haploinsufficient tie2 +/− mice bred onto a cd1 background. these mice express significantly less tie2 on the lung endothelium 20 . infection of cd1 wild-type mice with influenza virus (hkx31;h3n2) resulted in significant lethality (only 22% survival) that was greatly improved by vasculotide (78% survival), even if started 48 hours after infection (fig. 3a) . conversely, infection of haploinsufficient tie2 +/− mice resulted in a similar degree of mortality (25% survival) that was not significantly changed by the drug (50% survival), although there was a trend towards an intermediate phenotype (fig. 3b) . furthermore, in contrast to wild-type mice, vasculotide had no effect on influenza virus-induced hypoxemia or hypothermia in the haploinsufficient mice ( fig. 3c-d) . thus, the benefit of vasculotide is dependent on tie2 expression. while tie2 is widely thought to be almost exclusively expressed on endothelial cells 21 , we considered the possibility that its benefit might reflect unanticipated activity on non-endothelial tissues or on the virus itself. we first established that vasculotide has no antiviral activity in vitro and in vivo (fig. 4a) . second, vasculotide did not affect chemotaxis of human and murine monocytes and neutrophils in vitro (fig. 4b-c and data not shown). to our surprise, despite significantly improved arterial oxygen saturation and body weight ( fig. 4d -e), vasculotide-treated mice exhibited no difference in the severity of lung injury determined by blinded quantification of lung histology at 5 days post-infection ( fig. 4g ) (this timepoint was chosen as infected mice die soon afterwards). concordantly, while infection induced a marked increase in alveolar neutrophil recruitment, this was unaffected by treatment with vasculotide ( fig. 4f ); alveolar and serum cytokine levels on day 5 post-infection were also unaffected (supplemental figure 6 ). thus, the mechanism of benefit of vasculotide appears downstream of innate immune responses and instead involves the lung endothelial barrier. as expected, vasculotide induced tie2 phosphorylation and significantly attenuated thrombin-induced lung endothelial leak, as measured by electrical resistance (supplemental figure 7a -b). while the drug had no effect on human lung endothelial proliferation (supplemental figure 8) , it significantly attenuated lung endothelial apoptosis in vitro in response to influenza virus, as assessed by cleavage of caspase-3 (supplemental figure 7c ); we observed a similar reduction in cleaved caspase-3 in lungs from infected mice who received vasculotide (supplemental figure 7d ). although the apoptotic cells in the lungs of infected mice undoubtedly reflect contributions from epithelium and leukocytes, we observed colocalization of cleaved caspase-3 with the endothelial-specific marker ve-cadherin, consistent with our in vitro data. finally, while influenza infection caused loss of tie2 from the lungs of infected mice, this was significantly attenuated by treatment with vasculotide. vasculotide treatment also markedly increased levels of phospho-akt in the lung, an event that is known to be downstream of tie2 activation 22 and that acts as an endothelial pro-survival signal (supplemental figure 9 ). our findings have a number of important implications. first, these data strongly implicate failure of the lung endothelial barrier as the cause of death in murine models of severe influenza, as vasculotide conferred a significant survival benefit against multiple strains of the virus in two strains of mice. the effect of the drug is tie2-dependent, appears to be downstream of innate immune activation and does not require inhibition of viral replication. second, the enhancement of lung endothelial barrier integrity did not impair leukocyte recruitment to the lung, supporting the notion that vascular leak and leukocyte transmigration may be regulated independently 23 . accordingly, administration of vasculotide at the time of infection (i.e. even before leukocyte infiltration could occur) was not harmful and still conferred great benefit (fig. 1b) . lastly, administration of vasculotide even 3-4 days after infection significantly improved survival, an observation with practical implications given that the moment of infection in patients is usually uncertain. while other ligands for tie2 have been studied as therapies for vascular leak (as reviewed in 24 ), none have been reported in severe influenza virus infection and almost all required administration to be prophylactic or simultaneous with the time of infection 25, 26 , limiting their clinical utility. the potential clinical use of recombinant angiopoietin 1, the endogenous ligand for tie2, is limited by its tendency to bind to the extracellular matrix and to form undesirable aggregates 27 ; furthermore, modified forms of the protein are reportedly difficult to synthesize and are unstable 27 . in contrast, vasculotide was invented based on phage display experiments in which over 1 billion unique peptides were screened to identify those capable of binding with high affinity to tie2 28, 29 . vasculotide bears no sequence homology to angiopoietin-1 and exhibits no activity against or binding to some 500 cellular receptors (unpublished data); its specificity for tie2 is also strongly supported by our data using tie2 haploinsufficient mice. given the existing literature on ang1 and other putative barrier-enhancing agents 14, 26, 30 , the ability of delayed administration of vasculotide to rescue mice dying of influenza (supplemental figure 2 ) is unexpected and novel. this finding, combined with its relatively low cost of production and its chemical stability, suggests that the drug might constitute a practical therapy. our data indicate that vasculotide increases endothelial barrier integrity and reduces lung endothelial apoptosis, consistent with its agonism of tie2 31 . further work will determine whether it affects other mediators of vascular instability including angiopoietin-2 32 and ve-ptp 33 . as its mechanism of action is independent of viral replication and instead involves modulation of the host response, it is possible that vasculotide may not engender viral resistance, but this hypothesis will require direct testing. since influenza virus infections are often complicated by secondary bacterial pneumonia 34 , novel agents that impair the innate immune response may not be without risk. the fact that vasculotide does not impede neutrophil recruitment to the lung is therefore reassuring. finally, whether the impressive benefit of vasculotide in the therapy of the influenza virus carries implications for other infectious agents 35 or non-infectious causes of ards will require further investigation. experimental design. fourteen-week old c57bl/6j mice were purchased from jackson laboratories for all experiments, mice were sedated with 5% isoflurane and infected intranasally with influenza virus (see virus section, below) diluted in pbs to a final volume of 80 μ l. after infection, mice were separated into weight-matched control and treatment groups; unless otherwise indicated, we used ten mice per group and mice were infected with 64 hau of influenza virus, which caused greater than 90% mortality by day 7. in the first experiment, we included the following groups (see fig. 1b ): mice that received the influenza virus alone (flu) and infected mice that also received vasculotide (500 ng in 0.1 ml pbs by intraperitoneal injection, the dose based on previous work 16 ); of the latter, mice that received vasculotide were divided a priori into those who received it at the time of infection (vt0) and then once daily; those that in other experiments to test vt as an adjuvant therapy ( fig. 2 and supplemental figure 5 ), we divided mice into the following six groups: influenza-virus infected mice (flu); infected mice that received a dose of amantadine by oral gavage twice daily at a concentration of 138 mg/kg/day 18 (flu/amant); and infected mice that received amantadine and a daily intraperitoneal injection of vt starting 0, 24, 48 to test the requirement for tie2 (fig. 3) , cd-1 wildtype and tie2 +/− mice bred onto the cd-1 background were obtained from sunnybrook research institute (toronto, ontario); tie2 +/− mice were backcrossed onto the cd-1 background for a minimum of 6 generations. these mice were divided into the following four groups, depicted in fig. 3 : cd-1 wildtype mice infected with influenza virus (flu, fig. 3a) ; cd-1 wildtype mice that were infected and given vt daily starting 48 hours after infection (flu/vt48, fig. 3a) ; tie2 +/− mice infected with influenza virus (flu, fig. 3b) ; tie2 +/− mice that were infected and given vt daily starting 48 hours after infection (flu/vt48, fig. 3b) . in all experiments, mice were monitored 3 times daily for the duration of the experiment (12-14 days after infection) and were scored for weight loss, hypothermia, hypoxemia, spontaneous activity (scored from 1 (moribund) to 5 (normal), as described in 36 ) , and other clinical features of influenza infection. mice were euthanized if two or more of the following occurred: weight loss exceeded 30% of initial weight; temperature fell below 31 °c; the animal appeared moribund. in some experiments, mice were sacrificed on the indicated day for collection of blood and tissue samples. 37 . in vitro measurement of viral replication was also measured by qpcr as previously described 39 . pulse oximetry measurements. arterial oxygen saturation was measured using the mouse ox plus device and software (starr life sciences, oakmont, pa) on awake (non-anesthetized) mice using either the small or medium collar clips for c57bl/6j and cd-1 and tie2 +/− mice, respectively. for c57bl/6j mice, a chemical depilatory cream was used to remove hair around the base of the neck several days before infection. for so 2 measurements, mice were allowed to acclimate to the collar clip for several minutes in their home cage before recording the maximal so 2 measurement. mice treated with or without vt starting 48 hours after infection were sedated and sacrificed 5 days after infection by cardiac puncture. this timepoint was chosen as infected mice die soon afterwards. after sacrifice, the trachea was exposed and the lungs were inflated with 0.8 ml cold pbs with 0.6 mm edta. after 30 s, 0.7 ml balf was recovered and centrifuged at 300x g to pellet the cells. supernatant was collected and immediately frozen for analysis of balf cytokines. the balf pellet was resuspended in 300 μ l pbs. neutrophils were stained with 0. measurement of edema. c57bl/6j mice were infected with hkx31and vt (500 ng) or pbs was injected by ip injection once daily for 5 days. on day 5, mice were sacrificed by cardiac puncture of the right ventricle. the circulatory system was flushed with 10 ml cold pbs by instillation through the left ventricle. the entire right lung was removed, patted dry, and then weighed for determination of wet weight. lung tissues were dried for 24 hours and then weighed for the dry weight. degree of lung edema is expressed as the wet-to-dry ratio of lung weights. echocardiography. c57bl/6j mice were infected with hkx31 influenza and vt (500 ng daily) or pbs treatment was started 48 hours after infection. on day 4, mice were lightly sedated and a depilatory cream was used to remove hair from the ventral surface of the thorax and abdomen. on day 5, mice were lightly sedated and left ventricular function was measured by echocardiography as previously reported 40 . histology and immunohistochemistry. hkx31-infected c57bl/6j mice (n = 5 per group and scientific reports | 5:11030 | doi: 10.1038/srep11030 and eosin; a lung pathologist (d.h.) blinded to the experimental design scored acute lung injury based on consensus criteria, yielding a composite score between 0 (no injury) to 1 (highest degree of lung injury) 41 . degree of lung injury was determined by scoring 20 randomly selected high power fields (40x magnification) per mouse. for detection of cleaved caspase-3, immunofluorescence microscopy was performed on formalin-fixed paraffin embedded lung tissue as previously described 42 . sections were dual-stained with a goat polyclonal antibody directed against ve-cadherin (santa cruz biotechnology) and a rabbit polyclonal antibody directed against cleaved caspase-3 (cell signaling technology). slides were washed and subsequently incubated with alexa fluor 555 donkey anti-goat and alexa fluor 488 donkey anti-rabbit secondary antibodies, both from life technologies inc. (burlington, on, canada) . blinded quantification of cleaved caspase-3 was performed on 10 randomly selected lung fields per mouse acquired by spinning disc confocal microscopy (zeiss axiovert 200 m microscope, settings kept constant between conditions) and analyzed using imagej (nih). cell culture and reagents. primary human lung microvascular endothelial cells (hpmecs) obtained from promocell (heidelberg, germany) were cultured in ebm-2 media (lonza, basel, switzerland) with the recommended supplements and used in passages 6-7. the thp-1 human monocytic cell line was a gift from amira klip (toronto). human neutrophils were obtained from healthy volunteers and were isolated from whole blood by density gradient separation 43 . vasculotide, a pegylated tetrameric peptide agonist of the tie2 receptor, was synthesized as previously described 16 and prepared to a concentration of 50 ng/μ l in sterile phosphate-buffered saline (pbs) without calcium and magnesium. a working solution of vt was prepared by diluting the stock ten-fold in pbs. immunoblot. hpmec-l were grown to 90% confluency on 6-well plates and treated with x31 influenza or x31 influenza plus 20 ng/nl vt for 24 hours. lysates were prepared with lysis buffer (62.5 mm tris-hcl ph 6.8, 2% sds, 10% glycerol, 10 mm dtt). sds page was performed on a 12% polyacrylamide gel. immunoblotting was performed as previously described 44 with the exception of the blocking step, which used 5% bovine serum albumin in tbs instead of 5% milk. in some experiments, lung homogenates were generated from infected mice as follows: after euthanasia, the circulation was flushed with 10 ml ice-cold pbs. the lungs were homogenized in buffer containing 137 mm nacl, 2 mm edta, 10% glycerol, 1% triton x-100, 20 mm tris-hcl, 5 mm sodium orthovanadate, and complete protease inhibitor cocktail tablets and separated using 8% polyacrylamide gels. proteins were transferred to nitrocellulose membranes, blocked for 1 hour in 5% milk in tbs, and probed overnight with primary antibody at 4 °c. after washing, blots were incubated with hrp-conjugated secondary antibodies for 1 hour, washed, and then visualized by chemiluminescence (thermoscientific). band intensity was quantified using imagej (nih) and normalized to the loading control after background correction. the anti-phospho-akt (ser473) antibody was from cell signaling; the anti-tie2 (c20) antibody was from santa cruz; all other antibodies were from santa cruz biotechnology. chemotaxis assay. 2 × 10 5 neutrophils (isolated from healthy human volunteers) in 100 μ l of dmem with low-glucose or 2 × 10 5 thp-1 cells in 100 μ l of rpmi 1640 with 10% fbs and 1% penicillin/streptomycin containing 20 ng/ml or 100 ng/ml vt or pbs were seeded into the upper part of a transwell chamber (8-μ m pore size, 6.5 mm in diameter; corning life sciences). the lower chamber of the transwells was filled with 300 μ l of dmem containing vehicle (dmso) or 1 μ m fmlp for neutrophil chemotaxis or with 300 μ l of rpmi 1640, 10% fbs, 1% penicillin/streptomycin containing 10 μ m atp for thp-1 chemotaxis. after 4 h of incubation at 37 °c, the migrated neutrophils or thp-1 cells were recovered from the lower chamber and counted using a hemocytometer. electric cell substrate impedance sensing (ecis). an ecis ztheta system (applied biophysics, troy, ny) was used to follow barrier function. hpmecs were seeded on applied biophysics ecis culture arrays pretreated with 10 mm cysteine at 4.0 × 10 4 /well and grown for 48 h to allow the establishment of a confluent layer. next, the arrays were placed into the ecis system and impedance and capacitance (c) data were collected (and r values calculated by the software) continuously using the frequency scan mode. after obtaining a baseline, the measurement was paused and thrombin was added to the arrays in 20 μ l medium to induce endothelial permeability, at a final concentration of 1 unit/ml. some wells received vt alone or in combination with thrombin at a concentration of 20 ng/ml. controls received 20 μ l medium without thrombin. for each condition measurements were performed in duplicate. curves were normalized to the resistance level prior to thrombin administration using the ecis software. the ecis trace represents the data collected at 500 hz and is representative of 5 independent experiments. mtt and scratch assay. hpmecs were seeded on 96 well tissue culture plates at a density of 3.0 × 10 4 cells/ml (8 wells per condition). cells were incubated for four hours to allow cells to attach. after 4 hours (time 0), the media was changed to egm-2 (control) or egm-2 with 20 ng/ml vt. for baseline and endpoint measurements, mtt (sigma) was added to the cells at time 0 and 24 hours after treatment followed by a 2-hour incubation. mtt and media were aspirated and the cells were solubilized in 100 ul dmso and shaken for 5 minutes. absorbance was measured at 538 nm. for the scratch assay, hpmecs were grown to confluency on 12-well tissue culture plates marked on the bottom with an open square to define the scratch area. a single scratch was made through the scratch area using a sterile p1000 pipette tip. the initial scratch area was imaged by phase-contrast microscopy. cells were washed once with media and then treated with egm-2 or egm-2 with 20 ng/ ml vt for 24 hours. after 24 hours the cells were imaged again to assess the degree of wound healing. phase-contrast images were exported to imagej for quantification of baseline and endpoint wound areas. wound healing is expressed as a fraction of the endpoint area occupied by cells over the baseline scratch area. quantification of wound healing was performed in a blinded fashion. statistics. all statistics were performed using graphpad prism (la jolla, ca) software. comparisons between two groups were made using unpaired, two-tailed student's t-tests with statistical significance set at p < 0.05. comparisons of more than 2 groups were made using one-way anova with post-hoc analyses applying bonferroni's adjustment for comparisons between selected groups. for comparing kaplan-meier survival curves, the log-rank test was used with p < 0.05. statistics for ecis experiments were conducted with repeated measures anova and sidak's test for multiple comparisons. the tlr4 antagonist eritoran protects mice from lethal influenza infection mortality associated with influenza and respiratory syncytial virus in the united states the pathology of influenza virus infections antiviral therapy and outcomes of influenza requiring hospitalization in ontario neuraminidase inhibitors for preventing and treating influenza in healthy adults and children the neuraminidase inhibitor oseltamivir is effective against a/anhui/1/2013 (h7n9) influenza virus in a mouse model of acute respiratory distress syndrome effects of oseltamivir treatment on duration of clinical illness and viral shedding and household transmission of influenza virus antiviral susceptibility 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isolation of angiopoietin-1, a ligand for the tie2 receptor, by secretion-trap expression cloning the angiopietin-1-tie2 pathway prevents rather than promotes pulmonary arterial hypertension in transgenic mice tie2 expression and phosphorylation in angiogenic and quiescent adult tissues akt is a major angiogenic mediator downstream of the ang1/tie2 signaling pathway endothelial lsp1 is involved in endothelial dome formation, minimizing vascular permeability changes during neutrophil transmigration in vivo mending leaky blood vessels: the angiopoietin-tie2 pathway in sepsis angiopoietin-1 protects the adult vasculature against plasma leakage protective role of angiopoietin-1 in endotoxic shock orchestral actions of angiopoietin-1 in vascular regeneration acceleration of diabetic wound healing by an angiopoietin peptide mimetic a short synthetic peptide inhibits signal transduction, migration and angiogenesis mediated by tie2 receptor signaling and functions of angiopoietin-1 in vascular protection angiopoietin-1 inhibits endothelial cell apoptosis via the akt/survivin pathway excess circulating angiopoietin-2 may contribute to pulmonary vascular leak in sepsis in humans ve-ptp regulates vegfr2 activity in stalk cells to establish endothelial cell polarity and lumen formation predominant role of bacterial pneumonia as a cause of death in pandemic influenza: implications for pandemic influenza preparedness do viral infections mimic bacterial sepsis? the role of microvascular permeability: a review of mechanisms and methods a mouse model for studying the interaction of bisdioxopiperazines with topoisomerase iialpha in vivo influenza: propagation, quantification, and storage mesenchymal stromal (stem) cell therapy fails to improve outcomes in experimental severe influenza influenza infects lung microvascular endothelium leading to microvascular leak: role of apoptosis and claudin-5 conditional cardiac overexpression of s100a6 attenuates myocyte hypertrophy and apoptosis following myocardial infarction an official american thoracic society workshop report: features and measurements of experimental acute lung injury in animals the (pro)renin receptor: site-specific and functional linkage to the vacuolar h+ −atpase in the kidney neutrophil isolation protocol co-regulation of transcellular and paracellular leak across microvascular endothelium by dynamin and rac this work was supported by grants from the canadian institutes of health research (ocn126577 and mop130564), the physicians' services incorporated foundation (10) (11) (12) (13) (14) and an early researcher award (government of ontario), all to wll. the authors thank chris spring, pamela plant, xiaofeng lu, caterina di ciano-oliveira and judy trogadis from the research core facility at the keenan research centre for technical assistance; johnny su for assistance with densitometry, julie khang for assistance with luminex and qinghong dan for assistance with ecis. key: cord-312305-ll29frwc authors: sun, shihui; gu, hongjing; cao, lei; chen, qi; yang, guan; li, rui-ting; fan, hang; ye, qing; deng, yong-qiang; song, xiaopeng; qi, yini; li, min; lan, jun; feng, rui; guo, yan; qin, si; wang, lei; zhang, yi-fei; zhou, chao; zhao, lingna; chen, yuehong; shen, meng; cui, yujun; yang, xiao; wang, xinquan; wang, hui; wang, xiangxi; qin, cheng-feng title: characterization and structural basis of a lethal mouse-adapted sars-cov-2 date: 2020-11-11 journal: biorxiv doi: 10.1101/2020.11.10.377333 sha: doc_id: 312305 cord_uid: ll29frwc the ongoing sars-cov-2 pandemic has brought an urgent need for animal models to study the pathogenicity of the virus. herein, we generated and characterized a novel mouse-adapted sars-cov-2 strain named mascp36 that causes acute respiratory symptoms and mortality in standard laboratory mice. particularly, this model exhibits age and gender related skewed distribution of mortality akin to severe covid-19, and the 50% lethal dose (ld50) of mascp36 was ∼100 pfu in aged, male balb/c mice. deep sequencing identified three amino acid mutations, n501y, q493h, and k417n, subsequently emerged at the receptor binding domain (rbd) of mascp36, which significantly enhanced the binding affinity to its endogenous receptor, mouse ace2 (mace2). cryo-electron microscopy (cryo-em) analysis of mace2 in complex with the rbd of mascp36 at 3.7-angstrom resolution elucidates molecular basis for the receptor-binding switch driven by amino acid substitutions. our study not only provides a robust platform for studying the pathogenesis of severe covid-19 and rapid evaluation of coutermeasures against sars-cov-2, but also unveils the molecular mechanism for the rapid adaption and evolution of sars-cov-2 in mice. one sentence summary a mouse adapted sars-cov-2 strain that harbored three amino acid substitutions in the rbd of s protein showed 100% mortality in aged, male balb/c mice. coronavirus disease 2019 caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), has resulted in a public health crisis (1) . the symptoms of covid-19 are similar to those of sars-cov and mers-cov infections, ranging from fever, fatigue, dry cough and dyspnea, and mild pneumonia to acute lung injury (ali) and the acute respiratory distress syndrome (ards) in severe cases. in fatal cases, multi-organ failures accompanied by a dysregulated immune response have been observed (2) (3) (4) . numerous studies have highlighted age and gender related discrepancies in the distribution of covid-19 cases where the elderly and men tend to have a higher case-fatality ratio when compared to the young and females, suggesting that aged man are more likely to succumb to covid-19 (5, 6) . coronaviridae family, and is an enveloped, single stranded positive-sense rna virus. human angiotensin-converting enzyme 2 (ace2), has been demonstrated as the functional receptor for sars-cov-2 (7, 8) . sars-cov-2 cannot infect wild-type laboratory mice due to inefficient interactions between its s protein and the ace2 receptor of mouse (mace2). (9) . so, several hace2 expressing mouse models such as hace2 transgenic mice (10) , aav-hace2 transduced mice (11) and ad5-hace2 transduced mice (12) have been developed. furthermore, mouse adapted strains of sars-cov-2 have also been developed via either in vivo passaging or reverse genetics (13) (14) (15) . however, all these models cause only mild to moderate lung damage in mice. a small animal model capable of recapitulating the severe respiratory symptoms and high case fatality ratio of covid-19 remains to be established. in this study, we have developed and characterized a new lethal strain of sars-cov-2 named mascp36 by using the in vivo passaging method. previously, we reported the development of mascp6, a mouse-adapted sars-cov-2 strain using a similar strategy (16) . remarkably, intranasal injection of mascp36 caused 100% fatality in aged balb/c mice. prior to death, all infected animals developed severe malfunctions of the respiratory system, including acute respiratory distress syndrome in our previous study, we generated a mouse-adapted strain of sars-cov-2 (mascp6) by 6 serial passages of a sars-cov-2 in the lung of aged balb/c mice, which caused moderate lung damage and no fatality in mice. herein, we further serially passaged for additional 30 times to generate a more virulent sars-cov-2, and the resulting virus at passage 36 ( named as mascp36) was used for stock preparation and titration. to characterize the pathogenicity of mascp36, groups of balb/c were subjected to intranasal injection of varying doses of mascp36. strikingly, survival curve analysis showed that high doses of mascp36 caused almost 100% mortality within ten days in all aged mice, while young mice were resistant to mascp36 challenge and no animals developed disease and died in this group (fig. 1a-d) . aged mice challenged with high dose of mascp36 developed typical respiratory symptoms and exhibited features like ruffled fur, hunched back, and reduced activity. of particular note, tachypnea was common in all moribund animals (supplemental video). for the aged mice, male animals were more susceptible to mascp36 in comparison to female ones, and the ld50 was calculated to 58 pfu and 690 pfu, respectively (fig. 1a, b) . additionally, this unique gender-dependent mortality was also recorded in aged c57bl/6 mice challenged with mascp36 ( fig. s1 ) where 80% of the males challenged with the virus died from respiratory symptoms. thus, serial passaging of mascp6 generated a more virulent mascp36, which was sufficient to cause 100% mortality in aged balb/c mice at low dose (~1200 pfu). we further characterized the in vivo replication dynamics of mascp6 in both young and aged mice, and the results from qrt-pcr showed that high levels of sars-cov-2 subgenomic rnas were persistent in the lung and tracheas till 4 day post infection (dpi) in aged mice (fig. 1e) . marginal viral rnas were also detected in the intestine, heart, liver, spleen, brain, and kidney. the young mice had a similar tissue distribution as the aged ones upon mascp36 challenge, and lung and tracheas represented the major tissues supporting viral replication (fig. 1f ). multiplex immuno staining showed that mascp36 predominantly targeted the airway cc10+ club cells and spc+ at2 cells, while foxj1+ ciliated cells and pdpn+ alveolar type 1 (at1) cells detected few positive signals (fig. 1g ). more sars-cov-2-infected cells were detected in the lung from aged mice than those from young animals at 1 dpi (fig. 1h) , which was in agreement to the high ratio of spc+ at2 cells that co-express ace2 in aged mice ( fig. s2 ). consequently, a striking loss of spc+ at2 cells with apoptosis were observed in the lung from aged mice ( fig. s3 ). collectively, aged male mice that developed severe respiratory symptoms and 100% fatality serve as the most suitable animal host for mascp36 and were chosen for subsequent analysis. to further characterize the pathogical outcome in mascp36 infected aged male mice, the lung as well as other organs were collected at 4 dpi and subjected to histopathological and immunostaining analysis. when observed by the naked eye, lung tissue samples from mascp36 infected animals showed visible lung injury characterized with biolateral cardinal red appearance. furthermore, a lot of sticky secretion was seen at the lung surfaces when compared with the control animals ( fig. 2a ). according to the metrics of acute lung injury (ali) laid out by the american thoracic society (ats), mascp36 infection induced necrotising pneumonia and extensive diffuse alveolar damages (dad) and even ards on day 4 (17) . the microscopic observation showed large quantities of desquamative epithelial cells in bronchiole tubes (yellow arrow) and a large area of necrotic alveoli epithelial cells, fused alveoli walls with inflammatory cells infiltration especially neutrophils in alveolar septae or alveolar airspace, serious edema around vessels (cyan arrow) and scattered hemorrhage (blue arrow) ( fig. 2a ). in addition, foamy cells, polykaryocytes, fibrin cluster deposition, and hyaline membrane formation were common in the mascp36 infected animals (fig. 2b) , indicative of acute respiratory distress syndrome (ards), which is well characterized in severe covid-19 patients (4). interestingly, typical viral inclusion bodies were also observed occasionally in lungs of infected mice (fig. 2b) . comparatively, lung damage in young mice infected with mascp36 were much milder than those of the aged mice; less visual lung damage were seen in gross necropsy, and only thickened alveolar septa and activated inflammatory cell infiltration (19, 20) . these pathological damages caused by mascp36 in mice recapitulated most spectrums of seriously ill covid-19 patients caused by sars-cov-2 infection. additionally, immunostaining of lung sections showed a significant infiltration of cd68 + macrophages and ly-6g + neutrophils in mascp36 infected mice ( fig. s7 ). interestingly, more cd68 + macrophages and ly-6g + neutrophils were detected in young mice than that in aged mice 1 dpi, and reversed on 4 dpi, which indicated rapid and short-lived immune response to limite viral replication in young mice. to test the utility of mascp36 infected mouse model for evaluation of antiviral candidates, h014, a known human monoclonal antibody targeting the rbd of sars-cov-2 (21), was examined for its ability to confer benefit during the infection. to deduce the genetic basis for the lethal phenotype of mascp36, deep sequencing was performed to identify the mutations emerged during the in vivo passaging history. and fig. s8b ). to clarify the potential role of these mutations, the rbd of these different adaptive strains were expressed to assay their binding affinities to mace2 ( fig. s9 ). as expected, the wt rbd presented no detectable binding, but rbds from mouse-adapted strains (rbdmascp6, rbdmascp25 and rbdmascp36) gain gradually enhanced binding abilities to mace2 with affinities ranging from ~500 μm to 2 μm (fig. 4b ). the increased affinity between mace2 and the rbd of mouse-adapted strains probably contribute to the enhanced virulence in mice. to further elucidate the molecular basis for the gradual change in specificity of mascp36, structural investigations of the mace2 in complex with rbdmascp25 or rbdmascp36 were carried out. two non-competing fab fragments that recognize the rbd beyond the mace2 binding sites were used to increase the molecular weight of this complex for pursuing an atomic resolution by cryo-em reconstruction (fig. s9-s12). interestingly, cryo-em characterization of the mace2 in complex with rbdmascp25 revealed that the complex adopts three distinct conformational states, corresponding to tight binding (state 1), loose binding (state 2) and no binding modes (state 3) (fig. s13), indicative of a quick association and quick dissociation interaction manner between the mace2 and rbdmascp25. however, only the tight binding conformation was observed in the mace2-rbdmascp36 complex structure, reflecting a more stable/mature binding mode for the rbdmascp36 to mace2, akin to that of the rbdwt and hace2. we determined asymmetric cryo-em reconstructions of the mace2-rbdmascp36 complex at 3.7 å and three states of the mace2-rbdmascp25 complex at 4.4 to 8.2 å (figs. s11-s12 and table s1). the map quality around the mace2-rbdmascp36 interface was of sufficient quality for a reliable analysis of the interactions ( fig. 4c and fig. s9 ). the overall structure of the mace2-rbdmascp36 complex resembles that of the rbdwt-hace2 complex with a root mean square deviation of 1.0 å (fig. 4c ). the rbdmascp36 recognizes the helices (α1 and α2) located at the apical region of the mace2 via its receptor binding motif (rbm) (fig 4c-4e ). the interaction area on the mace2 could be primarily divided into three patches (pi, pii and piii), involving extensive hydrophilic and hydrophobic interactions with three regions separately clustered by three adaptation-mediated mutated residues (k417n, corresponding to clus1; q493h, corresponding to clus2; and n501y, corresponding clus3) in the rbm ( fig 4c-4e ). coincidentally, a number of amino acid substitutions, such as q493k, q498y and p499t, in the rbm identified in other reported mouse-adapted sars-cov-2 isolates (15, 22, 23) were included either in the clus2 or clus3, underlining the putative determinants for cross-transmission (fig 4f-4g ). an extra clus1 is further accumulated in the mascp36 to gain utmost binding activity and infection efficacy ( fig 4f-4g ). the extensive hydrophobic interactions in clus3 constructed by y501 (or y498 or h498 in other mouse-adapted sars-cov-2 isolates), y505 in the rbdmascp36 and y41, h353 in the mace2, hydrogen bonds in clus2 formed h493 (k493 in other mouse-adapted strain) in the rbdmascp36 and n31, e35 in the mace2 and hydrophilic contacts constituted by n417 in the rbdmascp36 and n30, q34 in the mace2 contribute to the tight binding of the mascp36 to mace2. contrarily, structural superimposition of the rbdwt over the mace2-rbdmascp36 complex reveals the loss of these interactions, leading to the inability of the rbdwt to bind mace2 (fig 4g) . these analysis pinpoints key structure-infectivity correlates, unveiling the molecular basis for adaptation-mediated evolution and cross-transmission of sars-cov-2. clinically, the severe covid-19 disease onset might result in death due to massive alveolar damage and progressive respiratory failure (24) (25) (26) . distinct from all currently reported animal models which mimic the mild to moderate clinical symptoms of covid-19, the mascp36 infected mouse model could manifest many of the severe clinical syndromes associated with covid-19 disease such as pulmonary oedema, fibrin plugs in alveolar, hyaline membrane, and scattered hemorrhage (25, 27) . the as the functional receptor of sars-cov and sars-cov-2, ace2, is highly expressed on vascular endothelial cells and smooth muscle cells in multiple organs, which probably leads to the observed viral tropism contributing to cellular injury. in this model, closely correlated with the higher ratio of ace2-positive cells in type ii pneumocytes in aged mice when compared to those in young mice, massive injury of (at2 cells) type ii pneumocytes was observed in aged mice. therefore, age-related ace2 expression pattern in lungs might contribute to the severe phenotype observed in aged mice. although sars-cov-2 viral antigen has been detected in kidney of postmortem specimens (28) , no viral antigen or viral rna were detected in our model. so in this mascp36 infected mouse model, the kidney injury may arise due to secondary endothelial injury leading to proteinuria. in addition, although sars-cov-2 has also been implicated to have neurotropic potential in covid-19 (29), we did not find typical characteristics of viral encephalitis in this model. importantly, the imbalanced immune response with high-levels of proinflammatory cytokines, increased neutrophils and decreased lymphocytes, which were in line with sars and mers infections (30), playing a major role in the pathogenesis of covid-19 (31) , were also observed in this model. the skewed age distribution of covid-19 disease was reproduced in the mascp36 infected mouse model where more severe symptoms were observed in aged mice when compared to young mice. different from h1n1 pandemic(32), covid-19 appears to have a mild effect on populations under 30 years, and the elderly are more likely to progress to severe disease and are admitted to intensive care unit (icu) worldwide (33) . ace2, the functional receptor of sars-cov-2, expressed increasingly in the lungs with age, which might provide an explanation to the higher disease severity observed in older patients with covid-19 (34) . more importantly, the host immune response may determine the outcome of the disease. our immune system is composed of innate immunity and adaptive immunity. the innate immunity comprises of the first line of defense against pathogens and is acute as well as short lived. however, aging is linked with insufficient, prolonged and chronic activation of innate immunity associated with low-grade and systemic increases in inflammation (inflamm-aging) which can be detrimental for the body (35) . the delicate co-operation and balance are interrupted by the chronic activation of innate immunity and declined adaptive immune responses with increasing age in covid-19 (36) . in the mascp36 infected mouse model, the young mice presented acute inflammatory response with more innate immune cells infiltration on day 1, while lagged and sustained immune response in aged mice. the different immune response in mice model may be vital in limiting virus replication at early times and contribute to different outcome on day 4 in young or aged mice. in addition to the age-related skewed distribution of covid-19, gender-related differences in distribution of covid-19 disease is also recapitulated in this mascp36 infected mouse model with increased susceptibility and enhanced pathogenicity observed in male mice when compared to their female counterparts. biological sex is an important determinant of covid-19 disease severity (37) . in china, the death rate among confirmed cases is 2.8% for women and 4.7% for men (34) . in italy, half of the confirmed covid-19 cases are men which account for 65% of all deaths (38) . this pattern is generally consistent around the world. the skewed distribution of covid-19 suggests that physiological differences between male and female may cause differential response to infection. so the hypothesis that females display reduced susceptibility to viral infections may be due to the stronger immune responses they mount than males (39) . it has been studied that androgens may lower and estrogens may enhance several aspects of host immunity. in addition, androgens facilitate and estrogens suppress lymphocyte apoptosis. furthermore, genes on the x chromosome important for regulating immune functions, and androgens may suppress the expression of disease resistance genes such as the immunoglobulin superfamily (40) . in the mascp36 infected mouse model, we found out that it presented higher mortality of the male than the female infected with the same dose of virus, indicating the successful recapitulation of covid-19 and also its potential application in the study of the pathogenesis of the disease. learning from sars-cov ma15 with increased virulence in mice, multiple gene products may contribute independently to the virulence. unlike the sars-cov ma15, three subsequent emerged mutations (n501y, k417n, and q493h) in the mascp36 were located in rbd, which breaks a barrier for cross-species transmissions of sars-cov-2, enabling gradually adapted recognition of sars-cov-2 to mace2 during the in vivo passages in mice. the serially increased affinities between mace2 and the rbd of mouse-adapted strains confer to enhanced infections in mice. cryo-em structures of the mace2 in complex with rbdmascp25 and rbdmascp36 define preciously the atomic determinants of the receptor-binding switch, providing novel insights into adaptationmediated evolution and cross-transmission of sars-cov-2. in addition, there are also 9 amino acid substitutions outside the s protein of mascp36 (fig. s8) . at present, we cannot rule out the contribution of these mutations, and further validation with reverse genetic tools will help understand the biological function of each single mutation (41) . figs. s1 to s13 center, beijing institute of microbiology and epidemiology (approval number: iacuc-dwzx-2020-002). mouse adapted strain of sars-cov-2 (mascp6) was developed in our previous study (16) . additional serial passage of 30 times was performed as previously described (16) . multiplex immunofluorescent assay. the multiplex immunofluorescence assay was conducted as previously described (16) . briefly, the retrieved sections were incubated with primary antibody for 2 h followed by detection using the hrp-conjugated secondary antibody and tsa-dendronfluorophores (neon 7-color allround discovery kit for ffpe, histova biotechnology, nefp750). afterwards, the primary and secondary antibodies were thoroughly eliminated by heating the slides in retrieval/elution buffer (abcracker®, histova biotechnology, abcfr5l) for 10 sec at 95°c lung homogenates from mascp36-infected mice or mock treated mice were processed as previously described (16) and subjected to rna-seq. total rna from lung tissue were extracted using trizol (invitrogen, usa) and treated with dnase i (neb, usa). sequencing libraries were generated using nebnext® ultratm rna library prep kit for illumina® (neb, usa) following the manufacturer's recommendations and index codes were added to attribute sequences to each sample. the clustering of the index-coded samples was performed on a cbot cluster generation system using hiseq pe cluster kit v4-cbot-hs (illumina) according to the manufacturer's instructions. after cluster generation, the libraries were sequenced on illumina novaseq6000 platform and 150 bp paired-end reads were generated. after sequencing, perl script was used to filter the original data (raw data) to clean reads by removing contaminated reads for adapters and low-quality reads. clean reads were aligned to the mouse genome (mus_musculus grcm38.99) using hisat2 v2.1.0. the number of reads mapped to each gene in each sample was counted by htseq v0.6.0 and tpm (transcripts per kilobase of exon model per million mapped reads) was then calculated to estimate the expression level of genes in each sample. deseq2 v1.6.3 was used for differential gene expression analysis. genes with padj≤0.05 and |log2fc| > 1 were identified as differentially expressed genes (degs). degs were used as query to search for enriched biological processes (gene ontology bp) using metascape. heatmaps of gene expression levels were constructed using heatmap package in r (https://cran.rstudio.com/web/packages/pheatmap/index.html). dot plots and volcano plots were constructed using ggplot2 (https://ggplot2.tidyverse.org/) package in r. production of fab fragment. the b8 and d14 fab fragments (43) were generated using a pierce fab preparation kit (thermo scientific). briefly, the antibody was mixed with immobilized-papain and then digested at 37 ˚c for 3-4 h. the fab was separated from the fc fragment and undigested iggs by protein a affinity column and then concentrated for analysis. surface plasmon resonance. mace2 was immobilized onto a cm5 sensor chip surface using the nhs/edc method to a level of ~600 response units (rus) using biacore® 3000 (ge healthcare) and pbs as running buffer (supplemented with 0.05% tween-20). wtrbd, rbdmascp25 and rbdmascp36, which were purified and diluted, were injected in concentration from high to low. the binding responses were measured, and chip surfaces were regenerated with 10 mm glycine, ph 1.5 (ge healthcare). the apparent binding affinity (kd) for individual antibody was calculated using biacore® 3000 evaluation software (ge healthcare). for the competitive binding assays, the first sample flew over the chip at a rate of 20 ul/min for 120 s, after which the second sample was injected at the same rate for another 120s. all antibodies were evaluated at a saturation concentration of 500 nm, while mace2 was applied at 1000 nm concentration. all surfaces of chips were regenerated with 10 mm glycine, ph 1.5 (ge healthcare). the response units were recorded at room temperature and analyzed using the same software as mentioned above. for cryo-em sample preparation, the quaternary complex (rbdmascp25/rbdmascp36-fabb8-fabd14-mace2) was diluted to 0.8 mg/ml. holy-carbon gold grid (quantifoil r0.6/1.0 mesh 300) were freshly glow-discharged with a solarus 950 plasma cleaner (gatan) for 60s. a 3 μl aliquot of the mixture complex was transferred onto the grids, blotted with filter paper at 22℃ and 100% humidity, and plunged into the ethane using a vitrobot mark iv (fei). for rbdmascp25/rbdmascp36-fabb8-fabd14-mace2 complex, micrographs were collected at 300 kv using a titan krios microscope (thermo fisher), equipped with a k2 detector (gatan, pleasanton, ca), using serialem automated data collection software (43) movies (32 frames, each 0.2 s, total dose 60 e − å −2 ) were recorded at final pixel size of 1.04 å with a defocus of between -1.25 and -2.7 μm. image processing. for rbdmascp25-fabb8-fabd14-mace2 complex, a total of 2,109 micrographs were recorded. for rbdmascp36-fabb8-fabd14-mace2 complex, a total of 2,982 micrographs were recorded. both sets of the data were processed in the same way. firstly, the raw data were processed by motioncor2, which were aligned and averaged into motion-corrected summed images. then, the defocus value for each micrograph was determined using gctf. next, particles were picked and extracted for two-dimensional alignment. the partial well-defined particles were selected for initial model reconstruction in relion. the initial model was used as a reference for three-dimensional classification. after the refinement and postprocessing, the overall resolution of rbdmascp36-fabb8-fabd14-mace2 complex was up to 3.69å, on the basis of the gold-standard fourier shell correlation (threshold = 0.143) (44) . for rbdmascp25-fabb8-fabd14-mace2 complex, the class i complex the resolution achieved was 7.89 å, classii complex had a resolution of 8.17 å, while class iii complex was reconstructed to a resolution of 4.4 å. the quality of the local resolution was evaluated by resmap (45) . model building and refinement. the wtrbd-hace2 (pdb id: 6m0j) structures were manually docked into the refined maps of rbdmascp36-fabb8-fabd14-mace2 complex using ucsf chimera (46) and further corrected manually by real-space refinement in coot (47) . the atomic models were further refined by positional and b-factor refinement in real space using phenix (48) . validation of the final model was performed with molprobity (49) . the data sets and refinement statistics are shown in table s1. statistical analyses were carried out using prism software (graphpad). all data are presented as means ± standard error of the means (sem). statistical significance among different groups was calculated using the student's t test, fisher's exact test, two-way anova or mann-whitney test *, **, and *** indicate p < 0.05, p < 0.01, and p < 0.001, respectively. dying with sars-cov-2 infection-an autopsy study of the first consecutive autopsy in suspected covid-19 cases the pathological autopsy of coronavirus disease 2019 (covid-2019) in china: a review. pathogens and disease considering how biological sex impacts immune responses and covid-19 outcomes clinical features of covid-19 in elderly patients: a comparison with young and middle-aged patients structure of the 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(sars-cov-2) directly decimates human spleens and lymph nodes. medrxiv human kidney is a target for novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection. medrxiv structural basis for neutralization of sars-cov-2 and sars-cov by a potent therapeutic antibody mouse-adapted sars-cov-2 replicates efficiently in the upper and lower respiratory tract of balb/c and c57bl/6j mice a mouse-adapted model of sars-cov-2 to test covid-19 countermeasures histopathology and ultrastructural findings of fatal covid-19 infections in washington state: a case series pathological study of the 2019 novel coronavirus disease (covid-19) through postmortem core biopsies. modern pathology : an official journal of the united states and canadian academy of pathology clinical features of covid-19 and factors associated with severe clinical course: a systematic review and meta-analysis. ssrn pathological findings of covid-19 associated with acute respiratory distress syndrome. the lancet identification of a potential mechanism of acute kidney injury during the covid-19 outbreak: a study based on singlecell transcriptome analysis neurological associations of covid-19 lung pathology of fatal severe acute respiratory syndrome a new coronavirus associated with human respiratory disease in china pandemic preparedness and response--lessons from the h1n1 influenza of report of the who-china joint mission on coronavirus disease 2019 (covid-19) sars-cov-2: virus dynamics and host response chronic inflammation in the etiology of disease across the life span disparities in age-specific morbidity and mortality from sars-cov-2 in china and the republic of korea characteristics of covid-19 patients dying in italy report based on available data on the lethal sex gap: covid-19 sex differences in immune responses spike mutation d614g alters sars-cov-2 fitness acknowledgements: this work was in memory of prof this work was supported by the national key plan for scientific research and development of china quantifying the local resolution of cryo-em density maps visualization system for exploratory research and analysis tools for macromolecular model building and refinement into electron cryo-microscopy reconstructions towards automated crystallographic structure refinement with phenix.refine molprobity: all-atom structure validation for macromolecular crystallography relion: implementation of a bayesian approach to cryo-em structure determination automated electron microscope tomography using robust prediction of specimen movements chadox1 ncov-19 vaccine prevents sars-cov-2 pneumonia in rhesus macaques data and materials availability: all requests for resources and reagents should be directed to c.-f.q. (qincf@bmi.ac.cn and qinlab313@163.com) and will be fulfilled after completion of a materials transfer agreement. key: cord-000261-ip32y0j5 authors: becker, pablo d.; legrand, nicolas; van geelen, caroline m. m.; noerder, miriam; huntington, nicholas d.; lim, annick; yasuda, etsuko; diehl, sean a.; scheeren, ferenc a.; ott, michael; weijer, kees; wedemeyer, heiner; di santo, james p.; beaumont, tim; guzman, carlos a.; spits, hergen title: generation of human antigen-specific monoclonal igm antibodies using vaccinated “human immune system” mice date: 2010-10-04 journal: plos one doi: 10.1371/journal.pone.0013137 sha: doc_id: 261 cord_uid: ip32y0j5 background: passive transfer of antibodies not only provides immediate short-term protection against disease, but also can be exploited as a therapeutic tool. however, the ‘humanization’ of murine monoclonal antibodies (mabs) is a time-consuming and expensive process that has the inherent drawback of potentially altering antigenic specificity and/or affinity. the immortalization of human b cells represents an alternative for obtaining human mabs, but relies on the availability of biological samples from vaccinated individuals or convalescent patients. in this work we describe a novel approach to generate fully human mabs by combining a humanized mouse model with a new b cell immortalization technique. methodology/principal findings: after transplantation with cd34(+)cd38(−) human hematopoietic progenitor cells, balb/c rag2(−/−)il-2rγc(−/−) mice acquire a human immune system and harbor b cells with a diverse igm repertoire. “human immune system” mice were then immunized with two commercial vaccine antigens, tetanus toxoid and hepatitis b surface antigen. sorted human cd19(+)cd27(+) b cells were retrovirally transduced with the human b cell lymphoma (bcl)-6 and bcl-xl genes, and subsequently cultured in the presence of cd40-ligand and il-21. this procedure allows generating stable b cell receptor-positive b cells that secrete immunoglobulins. we recovered stable b cell clones that produced igm specific for tetanus toxoid and the hepatitis b surface antigen, respectively. conclusion/significance: this work provides the proof-of-concept for the usefulness of this novel method based on the immunization of humanized mice for the rapid generation of human mabs against a wide range of antigens. hyper-immune sera containing polyclonal immunoglobulins (igs) have been widely used in both therapeutic and prophylactic clinical settings [1] . however, the use of polyclonal sera was associated with several problems, such as the stimulation of allergic reactions, low reproducibility between clinical batches and high off-label use, which finally caused a decline in their use [2] . the advent of technologies to make monoclonal antibodies (mabs) derived from animals, especially mice, has overcome many of the problems associated with the use of polyclonal sera. the technology to make monoclonal cell lines of antibody-producing cells by fusing antibody producing plasma cells with myeloma cells was described for the first time in 1975 by milstein and kohler [3] . the therapeutic potential of mabs was immediately recognized and in 1980 the first mab, okt3, was approved for therapeutic applications. this antibody inactivates t cells, thereby preventing rejections of organ transplants [4] . however, because of the animal origin of the first generation of mabs that were used in clinical trials, human subjects treated with these antibodies developed vigorous immune reactions against the animal proteins, which were thereby eliminated preventing their therapeutic actions [5] . to overcome these problems technologies were developed to diminish the immunogenicity of mouse antibodies by replacing part or the complete mouse antibody backbone by its human equivalent, first generating chimeric, and subsequently fully humanized antibodies [6] . in a parallel approach transgenic mice bearing the human ig region were created to obtain fully human antibodies following immunization. the use of these mice obviates the elaborate molecular engineering of antibodies that is needed to humanize antibodies generated in wild-type mice, however, the maturation process of the mouse b cells expressing human igs is different from that of fully human b cells [7] . immortalization of b cells from immune humans seems to be the logical strategy to avoid these problems. however, the methods to achieve this goal have showed low efficiencies, although some progress has recently been reported [8, 9] . nevertheless, the major disadvantage of human b cells immortalization is the need for cells from either vaccinated individuals or patients who had recovered from an infection. thus, to fully exploit the ig repertoire of human b cells in an in vivo setting, we explored the possibility to raise mabs following de novo induction of human b cell responses in mice carrying elements of the human immune system (his). his mice are generated by engrafting immunodeficient mice with human hematopoietic stem cells (hsc) with or without human lymphoid tissues from fetal origin [10, 11, 12] . in particular, mice deficient for the recombinase activating gene-2 (rag2) and the common gamma chain of the il-2 receptor (il2rg) on a balb/c or a non-obese diabetic (nod) background are permissive for human hsc xenografts. inoculation of newborn mice from these strains with human hsc of fetal or umbilical cord blood origin gives rise to robust engraftment of a number of immune cells, including t, b, nk and dendritic cells. in this work, we describe a convenient approach to generate fully human mabs based on the immunization of balb/c rag2 2/2 il-2rcc 2/2 engrafted with human cd34 + cd38 2 hsc [13, 14] . to this end, his mice were immunized with commercial vaccines against hepatitis b virus (hbv) and tetanus. following immunization, human cd19 + b cells were sorted based on surface cd27 expression, as a marker of memory phenotype, and the isotype of surface igs. the sorted b cell populations were immortalized in vitro by retroviral transduction with human b cell lymphoma (bcl)-6 and bcl-xl genes and antigen-specific b cell clones were established and characterized. the obtained results provided the proof-of-concept for the usefulness of this generic approach based on his mice combined with immortalization of human b cells for the rapid and inexpensive development of human mabs against a wide range of antigens. the use of fetal liver tissue obtained from elective abortions with gestational age ranging from 14 to 20 weeks was approved by the medical ethical committee of the amc-uva and was contingent on informed written consent. balb/c rag2 2/2 il-2rcc 2/2 mice were bred and maintained in individual ventilated cages, and fed with autoclaved food and water. his mice were generated as previously described [13, 14, 15, 16] , with the approval of the animal ethical committee of the amc-uva (permit number dhl-100970). in brief, human fetal livers were obtained from elective abortions with gestational age ranging from 14 to 20 weeks. magnetic enrichment of cd34 + cells (.98% pure) was performed by using the cd34 progenitor cell isolation kit (miltenyi biotech), after preparation of single cell suspensions and isolation of mononuclear cells by density gradient centrifugation over lymphoprep (axis shield). finally, newborn (,5 days old) sub-lethally irradiated (3.5 gy) balb/c rag2 2/2 il-2rcc 2/2 mice were injected via intra-hepatic route with 5-10610 4 sorted cd34 + cd38 2 human fetal liver hematopoietic stem cells in 30 ml. all manipulations of his mice were performed under laminar flow. cell suspensions were prepared in rpmi medium supplemented with 2% fetal calf serum (fcs). twelve to sixteen weeks after cd34 + cd38 2 hsc engraftment, his mice were killed and single cell suspensions of splenocytes were prepared. red cells lysis was performed in 1 ml of red cell lysis buffer (sigma) for 10 min. splenocytes were washed, resuspended in 600 ml of rlt lysis buffer (qiagen) and homogenized by passing through a 21-gauge needle several times using rnase free syringes. rna was prepared using rneasy mini kits (qiagen) according to manufactures instructions. bcr v h immunoscope was performed as previously described [17] . briefly, cdna was prepared and real-time pcr performed by combining primers for the different v h chains (v h 1-7) and specific fluorochrome-labeled probes against the different constant regions (c h m, c h a and c h c). an additional four pcr cycles 'run-off reactions' were then performed on the pcr products using fluorescent primers specific for the constant regions (fcm, fca and fcc). products were gel separated to determine cdr3 lengths. analysis of six individual his-mice containing greater than 30% human chimerism in the spleen was performed. the number of human cd19 + b cells in chimeric spleens ranged from 5-12610 6 . eight weeks after hsc transplantation, blood was taken from his mice to verify the level of engraftment by flow cytometry, as described elsewhere [18] . his mice with a good level of human reconstitution (.20% hcd45 + cells) were immunized by intramuscular route (biceps femoris) using a 29g needle, three times on weeks 14, 16 and 18 with either 100 ml of the hbv vaccine (engerix-b, glaxosmithkline) or 50 ml of tetanus toxoid (tt) containing vaccine (tetanus vaccine, the netherlands vaccine institute). these amounts correspond to 1/10 of the normal human dose. negative controls received the same volume of pbs buffer. two weeks after the last immunization, his mice were exsanguinated under isofluran/oxygen narcosis. spleens and mln were removed aseptically and cellular suspensions were prepared. the bm cells were isolated from the femur and tibia. from beckman coulter; cd3 (sk7), cd4 (sk3), cd8 (sk1), cd19 (hib19), cd38 (hit2), cd45 (2d1 and hi30), cd45ra (hi100), cd138 (mi15), igm (g20-127), igd (ia6-2), igg (g18-145) and ccr7 (3d12) from bd biosciences; cd27 (lt27) from abd-serotec; cd27 (lg.7f9) from ebioscience. tt-specific b cells were also occasionally stained with pe-coupled tt, kindly provided by dr. andreas radbruch (german rheumatism research center, berlin, germany). dead cells were excluded based on dapi incorporation. all washings and reagent dilutions were done with pbs containing 2% fcs and 0.02% nan 3 . stained cells were analyzed with an lsr-ii interfaced to a facs-diva software system (bd biosciences). cell sorting of b cell subsets were performed on his mouse bm and spleens using a facs-aria cell sorter interfaced to a facs-diva software system (bd biosciences). for these experiments, all washings and reagent dilutions were done with 2% fcs supplemented pbs without nan 3 . the human bcl6 [19, 20] and bcl-xl [21] encoding cdnas were further cloned in a lzrs retroviral expression vector, around a t2a cleavage-promoting peptide sequence and upstream a cassette containing an internal ribosome entry site (ires) and the gene encoding gfp. we therefore obtained a lzrs vector in the following configuration: bcl6-t2a-bclxl-ires-gfp [9] . transfection of phoenix-galv packaging cells and virus production were performed as previously described [22] . before retroviral transduction, memory b cells were activated on c-irradiated (50 gy) mouse l cell fibroblasts stably expressing cd40l (cd40l-l cells) in the presence of 25-50 ng/ml recombinant mouse interleukin-21 (rmil-21, r&d systems) for 36 h [19] . the b cells were washed, mixed with retroviral supernatants in retronectin-coated plates (takara), centrifuged at room temperature for 60 min at 360 g, and subsequently incubated with the retroviruses at 37uc, 5% co 2 for 6-8 h. transduced b cells were maintained in co-cultures using cd40l-l cells (10 5 cells/ml) and in standard imdm (gibco) culture medium supplemented with 8% fetal bovine serum (fbs; hyclone), penicillin/streptomycin (roche) and 25 ng/ml rmil-21. the analysis of human ig-v h sequences was performed as follows. total rna was isolated from approximately 5610 5 monoclonal b cells with trizol (invitrogen). the cdna was generated and subjected to pcr with primers specific to the different v h family members. pcr products were sequenced to determine the cdr3 region of the different clones. sequence analysis was performed using bigdye terminator chemistry (applied biosystems inc.) and codoncode aligner software. the plasma harvested from his mice (7 days after the first and second immunization; 10 days after the third immunization) and b cell clone culture supernatants were screened by elisa for the presence of total human igm, total human igg and antigenspecific antibodies. measurement of total igm and igg was performed by coating 96-well plates either with affinipure f(ab') 2 fragment goat anti-human igm (fc5m-specific, jackson immu-noresearch) or affinipure goat anti-human igg (fcc fragmentspecific; jackson immunoresearch). control human serum protein calibrator (dako) with known igm (0.8 mg/ml) and igg (10.4 mg/ml) concentrations was used as a standard to be compared to the samples. for the detection of antigen-specific antibodies, 96-well plates were coated either with tetanus vaccine (nederlands vaccin instituut) or engerix b (glaxosmithkline) (106 diluted in pbs) for 1 h at 37uc or overnight at 4uc. alternatively, ridascreen tetanus igg elisa plates (biopharm) were also used to screen for tt-specific antibodies. after coating, the plates were washed in elisa wash buffer (pbs, 0.5% tween-20). a pbs solution containing 4% of milk was used as a blocking agent, before adding serial dilution of his mouse plasma (starting at a dilution of 1:5) or cell culture supernatants (starting at a dilution of 1:2). enzyme-conjugated detection antibodies were added at a dilution of 1:2500 for hrp-conjugated anti-igg and a dilution of 1:5000 for hrp-conjugated anti-igm (both from jackson immunoresearch). then, tmb substrate/stop solution (biosource) was used for the development of the elisa assay. statistical analyses were performed using graphpad prism version 5.02 for windows (graphpad software). data were subjected to two-tailed unpaired student t test analysis. the obtained p values were considered significant when p,0.05. we have generated his mice by transplanting human hsc into alymphoid balb/c rag2 2/2 il-2rcc 2/2 newborn mice ( figure 1a ). as reported previously, multilineage human hematopoietic reconstitution is observed in his mice, which demonstrate human thymopoiesis, b cell differentiation, nk cell and plasmacytoid dendritic cell development, and myelopoiesis [10, 13, 14, 15, 16] . human immune cells accumulate in lymphoid tissues, and several b cell subsets are observed in his mice ( figure 1b) . we analyzed the human b cell repertoire present in naive his mice by using b cell receptor (bcr) immunoscope analysis based on quantitative pcr of ig variable (v h ) and constant (c h ) region gene segments [17] . due to the lack of human spleen samples, the cells isolated from his mouse spleens, which contained sufficient numbers of human b cells to perform the immunoscope analysis, were compared to control human peripheral blood mononuclear cells (pbmc) samples, which were considered acceptable for the purpose of the performed comparison. we observed that igm-expressing b cells as well as ig isotype-switched b cells are found in naive his mice ( figure 1c) . the vast majority of b cells of his mice expressed an igm (97.961.0%), whereas igg (1.861.0%) and iga (0.0760.04%) expressing b cells represented minor populations. only the frequency of iga-expressing b cells was found significantly higher in control human pbmc samples (p,0.0001). at 10-14 weeks post-transplantation (i.e. in steady state conditions), the human ig concentrations in the blood were 12268 mg/ml (igm) and 143612 mg/ml (igg) ( figure 1d ), as previously reported [8, 16] . in comparison, the normal range for ig concentration in healthy humans is 400-3100 mg igm/ml and 7200-14700 mg igg/ml. in brief, despite a low frequency of igg-expressing cells, both human igm and igg accumulated in the plasma of ,3 month-old his mice to levels representing around 10% and 1% of adult human igm and igg concentrations, respectively. we further examined the antigen receptor repertoire diversity in his mice, by determining the length of cdr3 hypervariable regions for each ig-v h gene family. the analysis of cdr3 length distribution of individual his mouse splenocytes showed that igm repertoires are undistinguishable from normal human pbmc igm repertoires, as measured by the bcr immunoscope analysis ( figure 1e ). this observation suggests that his mice contain a broad variety of naive igm + b cell clones. the v h -family usage was large and similar to control human pbmc ( table 1) . the bcr immunoscope analysis was also performed for igg and iga repertoires and we observed more restricted repertoires, as expected from b cells undergoing clonal selection and ig class switch recombination ( figure s1 ). immunization of his mice with hbv and tetanus vaccines results in the generation of antigen-specific antibody responses since his mice contained broad naïve b cell repertoires, we analyzed the induction of human antigen-specific b cell responses after immunization with commercially available human vaccines. we designed a vaccination protocol based on repeated intramuscular immunizations (3 injections with 2-week intervals) of 10-14-week old his mice with vaccines containing hepatitis b surface antigen (hbsag) or tt. seven days after the last immunization mice were sacrificed, the blood and the lymphoid organs were harvested, and the phenotype and function of human cells was analyzed. all his mice showed human reconstitution (.20% hcd45 + cells) in the blood before starting the immunization protocol, which correlated with human engraftment in lymphoid organs. overall, 42% of hbsag-vaccinated (8 out of 19 vaccinated animals) and 40% of tt-vaccinated (6 out of 15) his mice showed significant production of antigen-specific igm antibodies, as detected by elisa (figure 2a) . we performed a kinetic monitoring of antigen-specific plasma ig levels in individual hbsag-vaccinated responder his mice and we observed that after the first immunization antigen-specific igs were rarely detected. in contrast, after the second immunization antigen-specific igm was detected, which steadily increased after the third immunization with approximately 25-40% of responder mice also showing an antigen-specific igg response (figure 2a) . this suggests that repeated vaccination leads to enhanced antigen-specific antibody production. the responder mice exhibited higher total igm (173641 mg/ml) and total igg (4596140 mg/ml) concentrations in their plasma, as compared to pbs-injected (igm: 37612 mg/ml; igg: 191658 mg/ml) and non-responder vaccinated (igm: 44611 mg/ml; igg: 192667 mg/ml) animals ( figure 2b) . at the end of the immunization protocol, vaccinated animals showed significantly higher numbers of hcd45 + cells in all organs (i.e. spleen, bone marrow (bm) and mesenteric lymph nodes (mln)) in comparison to mock-injected control mice. responder his mice exhibited higher numbers of human t and b cells in the spleen, as well as t cells in the bm ( table 2; table s1 ), suggesting that the vaccination protocol had a positive impact on the accumulation of human b and t cells. moreover, the mln isolated from vaccinated his mice contained 4 to 5-fold more hcd45 + cells than those of control animals ( figure 2c ), suggesting that the mln structure might play a role in eliciting an immune response in the his mice. in humans, the cd27 + memory b cell population contains the majority of antigen-experienced b cells [23, 24] , and we reasoned that the same should be true in vaccinated his mice. we therefore cell sorted several different cd19 + cd27 + b cell subsets from individual his mice. we used two strategies to isolate the following human b cell (cd45 + cd19 + ) subsets from bm and spleens of vaccinated his mice: (i) cd27 hi cd38 hi , (ii) cd27 + cd38 lo/int igd + , and (iii) cd27 + cd38 lo/int igd 2 on the one hand ( figure 3 , although the number of cells was increased for each of these subpopulations in the vaccinated animals, as expected from the enhanced number of total b cells ( table 2) . we only observed a significant increase in the frequency of igg + b cells within the cd27 hi cd38 hi plasmablast population of vaccinated responder his mice, as compared to pbs-injected animals (13.262.7% and 4.361.8% of cd27 hi cd38 hi b cells, respectively; p = 0.0311). in order to identify, isolate and immortalize the antigen-specific antibody-producing b cells, the aforementioned b cell subsets were transduced immediately after cell sorting with a retroviral vector encoding both human bcl6 and bcl-xl [9, 19] . by ectopically expressing bcl6 and bcl-xl in splenic or peripheral blood memory b cells and culturing them with factors produced by follicular helper t cells (cd40l and il-21), we generated highly proliferative, bcr positive b cell lines that secrete igs. since these cells express bcl6, the differentiation of memory b cells to terminal plasma cells is blocked [28, 29, 30] . therefore, the resulting b cells can expand extensively in vitro for long periods of time in presence of cd40l and il-21, and provide a tool to generate antigen-specific human bcr-positive, antibody-secreting b cell lines. the number of isolated cells from spleen and antigen-specific b cell clones that were generated with the bcl6/bcl-xl transduction approach is provided in the table s2 . since the frequency of antigen-specific b cell clones was unknown, we started with microcell cultures ranging from 0.6 to 640 cells per well. the wells containing antigen-specific b cells -as determined by hbsagspecific or tt-specific elisa -were subsequently cultured by limiting dilution to obtain monoclonal b cell lines. overall, we generated 15 anti-hbsag igm + b cell clones from 3 his mice vaccinated with hbsag, and 18 anti-tt igm + b cell clones from 3 his mice vaccinated with tt ( table s2 ). the estimated frequency of hbsag-specific b cells (clones) in the his mice after vaccination was 1/350. the igm secretion level of the b cell clones were in the range of 1 mg per 10 5 cells over 3 days in culture, which was in a similar range of secretion (0.6-5 mg/10 5 cells/3 days) to what was previously reported for b cell clones generated from human blood [9] . the igm v h regions of the bcr of the antigen-specific igm + b cell clones were sequenced. overall, the bcr of hbsag-specific and tt-specific b cell clones exhibited a v h sequence close to the germ-line sequence, although limited frequencies of somatic hyper-mutations were observed (table s3 and table s4 ). somatic hyper-mutations were occasionally detected in all framework regions (fr) and complementary determining regions (cdr), and most of the bcr diversity was the result of nadditions in the cdr3 region. based on the bcr sequence, we observed that 12 out the 15 anti-hbsag igm + b cell clones were unique, as well as 5 out the 18 anti-tt igm + b cell clones ( table s2, table s3 and table s4 ). the supernatants of tt-specific b cell clones were further tested for their capacity to recognize different antigens by elisa. we observed that igm mabs did not cross-react with unrelated antigens (i.e., hbsag and respiratory syncytial virus (rsv) antigens) ( figure 4a) . the tt-specific b cell clones were also screened by flow cytometry for direct binding of the tt antigen labeled with a fluorochrome ( figure 4b) . interestingly, three types of clones that produced antibodies that gave a similar signal in elisa were detected, with high, intermediate and low binding of the fluorescent tt antigen. in the present work we established a new approach to generate fully human mabs. we immortalized b cells from vaccinated his mice by transduction with bcl6 and bcl-xl followed by expansion in presence of il-21 and cd40l. antigen-specific b cell clones were obtained that expressed the bcr on their cell surface and secreted antigen-specific antibodies. similarly to methods based on the immortalization of human memory b cells from individuals that were either vaccinated or exposed to pathogens, our strategy exploits the antibody repertoire of human b cells which is likely to be different from that of b cells of mice expressing human ig gene segments. naïve his mice display an extensive human igm-expressing b cell repertoire. based on the analysis of the length of the cdr3 regions, this igm b cell repertoire is similar to the repertoire of healthy individuals. thus, his mice have no obvious limitations for the generation of human igm mabs against any possible antigen. upon intramuscular vaccinations with either tt or hbsag, approximately 40% of the his mice were able to mount an antigen-specific antibody response. human igm-producing b cell lines against both antigens were obtained after isolation of memory b cells followed by ex vivo differentiation into plasmablastlike cells. it is important to highlight that the selection of the antigen-specific human b cell clones relied on relevant bioassays (e.g., elisa or neutralization test). in contrast to ebv-based approaches, human b cell immortalization using transduction with bcl-6 and bcl-xl preserves the expression of the bcr at the surface and antigen-specific b cell clones can also be selected by binding of labeled antigen to the bcr of immortalized memory b cells (e.g. by using a labeled antigen). even when igg was used as a selection criterion, we were unable to establish antigen-specific igg + human b cell clones. the reason for this might be that t cell help in this system is suboptimal as indicated by the absence of antigen-specific t cell responses after vaccination (not shown). we also observed that the bcr of the b cell clones had a close to germ-line sequence, suggesting that also the induction of somatic hyper-mutation is sub-optimal in his mice. in our hands the great majority of the vaccinated his mice showed a defective formation of germinal centers [14, 16] , which further explains the absence of antigenspecific ig-class switched b cells. so far, humanized mouse models based on the transplantation of human hsc only -i.e. without additional human tissues -share these limitations, and immunization strategies result in the limited generation of class-switched antigen-specific b cell responses [14, 31, 32] . similar patterns are observed in human hsc-transplanted immunodeficient mice infected with lymphotropic pathogens, such as hiv [33] or ebv [34] , although dengue virus infection in his mice was reported to induce an igg response in a majority of the responder animals [35] . it is not clear why igg antigen-specific responses are limited while serum igg can accumulate efficiently, considering the low frequency of igg + b cells in his mice. it remains to be determined whether this apparent discrepancy might be explained by the conjunction of particularly effective igg production on a cell basis by igg + b cells (which might occur in a t cell independent manner, such as in the case of the igg3 subclass), long-term stability of human igg in the his mouse serum as compared to human igm, and/or defective survival of igg + b cells under specific conditions (e.g. after antigen-specific triggering of the bcr). although igm mabs might already be useful for some specific applications or could be modified by ig class swapping to obtain igg mabs [36] , optimized humanized mouse models with improved b cell function are highly desirable. one reason for the suboptimal interaction of t and b cells may be the poor survival resulting in a high turnover of human t cells (discussed in [10, 37] ), making it very likely that procedures leading to improved accumulation of human t cells may promote b cell responses and isotype switching. it was already shown that human b cells undergoing isotype switching can be obtained in humanized mice, provided that a human environment supporting this process is present, e.g. in scid mice transplanted with human fetal bones, thymus and lymph nodes [38] . consistent with this notion, enhanced human peripheral t cell accumulation was observed in nod/scid mice transplanted with human bone marrow hsc, fetal liver and fetal thymus tissues (referred to as blt mice), as compared to conventional humanized mouse systems [39] . interestingly, blt mice consistently generated an antigen-specific igg response after hiv-infection [40] . although it is yet unknown whether the isotype switch observed in blt mice is truly t cell dependent, those data might support the idea that improved t cell homeostasis has a positive impact on b cell responses. to obtain humanized mouse models with improved b and t cell homeostasis, alternative strategies not relying on the transplantation of human fetal tissues -which are not necessarily easy to access to, for ethical, legal or practical reasons -will likely be favored in the future. the replacement of mouse genes involved in the hematopoietic system by their human equivalent is a valuable strategy to improve development, maintenance and/or function of several hematopoietic cell subsets in humanized mouse models, as shown with cytokines, such as il-7 and il-15 [15, 21, 41, 42] , or mhc molecules (n.d.h and j.p.d., manuscript submitted) [43, 44] . the fact that the human cd47 was shown to be unable to properly interact with the mouse sirpa indicates that reintroducing a functional phagocyte inhibition mechanism via the cd47/sirpa signaling axis is another strategy of potential interest [45] . in conclusion, our results show using two standard vaccine antigens the general applicability of an innovative b cell immortalization method in combination with the his mouse model to generate human mabs. similarly to methods based on the immortalization of human memory b cells from vaccinated or convalescent individuals [9] , our approach exploits the broad antibody repertoire of human b cells, overcoming the potential limitations of conventional humanized murine mabs such as laboriousness or impaired biological properties, synthetic antibody libraries that require a known target antigen, and transgenic mice bearing the human ig locus that have limited b cell repertoires. in addition, our method enables to exploit experimental infection models and immunization regimes that would be unethical or untenable in humans. considering the upcoming advances in his mice models [37] , this new approach will provide a powerful tool to generate human mabs for either diagnostic or therapeutic purposes. figure s1 igg/iga b cell repertoire in naïve his mice. similarly to figure 1e , the naive igg (a) and iga (b) b cell repertoires of his (balb-rag/c) mice were evaluated on splenocytes by performing a bcr immunoscope for each v h family. the profiles obtained with control human pbmc are also shown. found at: doi:10.1371/journal.pone.0013137.s001 (1.61 mb tif) figure 3 . limited dilutions of b cells transduced with bcl-6 and bcl-xl were performed with 6.4 and 0.64 cells/well. after sub-cloning of the positive wells, we generated 15 igm + anti-hbsag mabs, of which 13 are unique (as determined by ig-v h sequence, see table s3 ), and 18 igm + anti-tt mabs, of which 5 are unique (see table s4 ). in the case of hbsag vaccination, the number of screened b cells was ((192*6.4)+(96*0.64))*3 = 3870, which eventually suggests that the frequency of hbsag-specific b cells is at least 1/350 b cells. found at: doi:10.1371/journal.pone.0013137.s003 (0.13 mb doc) igm v h amino-acid sequence of generated hbsagspecific b cell clones. the germ-line sequence is given for each v h family, with indication of framework regions (fr) and complementary determining regions (cdr). highlighted amino-acids correspond to n-additions (in the cdr3 region) and somatic hyper-mutation events, whether it results in a silent mutation (green) or not (red). clones with identical bcr sequences are grouped together. found at: doi:10.1371/journal.pone.0013137.s004 (0.02 mb pdf) the mechanism of diphtheria immunity and tetanus immunity in animals immunogenicity of therapeutic monoclonal antibodies continuous cultures of fused cells secreting antibody of predefined specificity upping the ante on antibodies mice with a human touch molecular engineering and design of therapeutic antibodies from xenomouse technology to panitumumab, the first fully human antibody product from transgenic mice an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus generation of stable monoclonal antibody-producing b cell receptorpositive human memory b cells by genetic programming experimental models to study development and function of the human immune system in vivo human-hemato-lymphoid-system mice: opportunities and challenges humanized mice in translational biomedical research monitoring the effect of gene silencing by rna interference in human cd34+ cells injected into newborn rag2-/-gammac-/-mice: functional inactivation of p53 in developing t cells development of a human adaptive immune system in cord blood celltransplanted mice il-15 trans-presentation promotes human nk cell development and differentiation in vivo t cellindependent development and induction of somatic hypermutation in human igm+ igd+ cd27+ b cells many human peripheral vh5-expressing igm+ b cells display a unique heavy-chain rearrangement experimental model for the study of the human immune system: production and monitoring of ''human immune system'' rag2-/-gamma c-/-mice stat3-mediated up-regulation of blimp1 is coordinated with bcl6 down-regulation to control human plasma cell differentiation a senescence rescue screen identifies bcl6 as an inhibitor of anti-proliferative p19(arf)-p53 signaling il-7 enhances thymic human t cell development in ''human immune system'' rag22/2il-2rgammac2/2 mice without affecting peripheral t cell homeostasis stat5 regulates the self-renewal capacity and differentiation of human memory b cells and controls bcl-6 expression cd27: a memory bcell marker human b cell subsets rapid cloning of high-affinity human monoclonal antibodies against influenza virus human immunoglobulin m memory b cells controlling streptococcus pneumoniae infections are generated in the spleen human blood igm ''memory'' b cells are circulating splenic marginal zone b cells harboring a prediversified immunoglobulin repertoire control of inflammation, cytokine expression, and germinal center formation by bcl-6 bcl-6 represses genes that function in lymphocyte differentiation, inflammation, and cell cycle control the bcl-6 protooncogene controls germinal-centre formation and th2-type inflammation antigen-specific antibody production of human b cells in nog mice reconstituted with the human immune system antigen-specific human t-cell responses and t cell-dependent production of human antibodies in a humanized mouse model disseminated and sustained hiv infection in cd34+ cord blood cell-transplanted rag2-/-gamma c-/-mice a new humanized mouse model of epstein-barr virus infection that reproduces persistent infection, lymphoproliferative disorder, and cell-mediated and humoral immune responses dengue virus infection and immune response in humanized rag2(2/2)gamma(c)(2/2) (rag-hu) mice potent antibody therapeutics by design humanized mice for modeling human infectious disease: challenges, progress, and outlook generation of primary antigen-specific human t-and b-cell responses in immunocompetent scid-hu mice humanized mice mount specific adaptive and innate immune responses to ebv and tsst-1 intrarectal transmission, systemic infection, and cd4+ t cell depletion in humanized mice infected with hiv-1 lentiviral vector delivery of human interleukin-7 (hil-7) to human immune system (his) mice expands t lymphocyte populations human lymphoid and myeloid cell development in nod/ltsz-scid il2r gamma null mice engrafted with mobilized human hemopoietic stem cells generation of functional human t-cell subsets with hla-restricted immune responses in hla class i expressing nod/scid/il2r gamma(null) humanized mice priming of protective t cell responses against virus-induced tumors in mice with human immune system components polymorphism in sirpa modulates engraftment of human hematopoietic stem cells we thank berend hooibrink for expert cell sorting and maintenance of the amc-uva flow cytometry facility. we acknowledge the bloemenhove clinic (heemstede, the netherlands) for providing fetal tissues and the staff of the animal research institute amsterdam for animal care. access to technologies available at the centre d'immunologie humaine of the institut pasteur was greatly appreciated. key: cord-292157-hrm69640 authors: stull-lane, annica r.; lokken-toyli, kristen l.; diaz-ochoa, vladimir e.; walker, gregory t.; cevallos, stephanie a.; winter, andromeda l. n.; muñoz, ariel del hoyo; yang, guiyan g.; velazquez, eric m.; wu, chun-yi; tsolis, renée m. title: vitamin a supplementation boosts control of antibiotic-resistant salmonella infection in malnourished mice date: 2020-10-02 journal: plos negl trop dis doi: 10.1371/journal.pntd.0008737 sha: doc_id: 292157 cord_uid: hrm69640 disseminated disease from non-typhoidal salmonella enterica strains results in >20% mortality globally. barriers to effective treatment include emerging multidrug resistance, antibiotic treatment failure, and risk factors such as malnutrition and related micronutrient deficiencies. individuals in sub-saharan africa are disproportionately affected by non-typhoidal s. enterica bloodstream infections. to inform a clinical trial in people, we investigated vitamin a as a treatment in the context of antibiotic treatment failure in a mouse model of vitamin a deficiency. vitamin a-deficient (vad) mice exhibited higher systemic bacterial levels with a multidrug-resistant clinical isolate in comparison to mice on a control diet. sex-specific differences in vitamin a deficiency and disseminated infection with s. enterica serotype typhimurium (s. typhimurium) were observed. vad male mice had decreased weight gain compared to control male mice. further, infected vad male mice had significant weight loss and decreased survival during the course of infection. these differences were not apparent in female mice. in a model of disseminated s. typhimurium infection and antibiotic treatment failure, we assessed the potential of two consecutive doses of vitamin a in alleviating infection in male and female mice on a vad or control diet. we found that subtherapeutic antibiotic treatment synergized with vitamin a treatment in infected vad male mice, significantly decreasing systemic bacterial levels, mitigating weight loss and improving survival. these results suggest that assessing vitamin a as a therapy during bacteremia in malnourished patients may lead to improved health outcomes in a subset of patients, especially in the context of antibiotic treatment failure. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 in susceptible populations globally, there is a vicious cycle of infection and malnutrition [1] . malnutrition overlaps with deficiencies in micronutrients, such as iron, iodine, zinc and fatsoluble vitamins like vitamin a [2] . severe or recurrent infections can lead to vitamin a deficiency due to impaired nutrient absorption and decreased food intake, as well as direct nutrient loss through urine [3, 4] . in turn, vitamin a deficiency compromises the gastrointestinal mucosal epithelial barrier, increasing susceptibility to enteroinvasive bacteria and sepsis [4] . one of the most common enteric pathogens is salmonella enterica [5] . non-typhoidal serotypes of s. enterica cause significant morbidity and mortality worldwide, resulting in 93.8 million cases of gastroenteritis and 155,000 associated deaths annually [6] . invasive non-typhoidal salmonella (ints) infection, a severe febrile illness with bacteremia, disproportionately affects infants, older adults and immunosuppressed individuals. an estimated 3.4 million cases of invasive disease occur globally per year, resulting in 680,000 deaths [7] . malnutrition and concurrent malaria are key risk factors for systemic disease, and populations in sub-saharan africa are disproportionately affected [8] . barriers to effective treatment of ints illness include appropriate diagnostic tools, antibiotic treatment failure and antimicrobial resistance [9] . notably, the mortality rate due to ints in children reaches 20-25% [10] , indicating that more effective treatment is vital. vitamin a administration has been studied as treatment of infectious disease, such as for measles in children [11] . in addition, the who recommends vitamin a supplementation in the treatment of children that present with severe acute malnutrition [12] . given the importance of vitamin a in immune function [4, 13, 14] , these treatment recommendations suggest a role for vitamin a as a host-directed therapy [15] . however, vitamin a has not been studied as a treatment for nonspecific febrile illnesses like ints disease, especially in the context of antibiotic treatment failure. this study aimed to explore the therapeutic potential of vitamin a administered after onset of infection, simulating care of a sick patient presenting to clinic. our hypothesis was that vitamin a therapy could boost the effect of subtherapeutic antibiotic treatment and improve treatment outcomes for a drug-resistant ints infection. to address this question, we used mice to model antibiotic treatment failure of s. enterica serotype typhimurium in the setting of vitamin a deficiency. mouse experiments were carried out in compliance with the national institutes of health policies on animal welfare, the animal welfare act, and all other applicable federal, state and local laws. mice were cared for by research staff and university of california (uc) davis teaching and research animal care services (tracs) under a program accredited by the association for assessment and accreditation of laboratory animal care international (aaalac). tracs maintains a health monitoring program administered through the uc davis comparative pathology laboratory. when needed, campus veterinary services is available to provide interventional and supportive care. all mouse experiments were approved by the uc davis institutional animal care and use committee (iacuc) under protocol 19900. inbred c57bl/6j-slc11a1 +/+ mice are a c57bl/6j congenic strain containing a genomic segment with slc11a1 from s29s1 mice that was introgressed into chromosome 1 [16] . this mouse line was maintained at uc davis under specific pathogen-free conditions in cages with sterile alpha-dri bedding manufactured by shepherd specialty papers (watertown, tn). to generate vad and control mice, c57bl/6j-slc11a1 +/+ dams were given a vad diet at 2 weeks of gestation. pups were placed on either vad or control diets at weaning and were maintained as groups throughout each experiment. mouse weights were monitored weekly during growth. custom vad and control diets were obtained from envigo teklad diets (madison, wi). when the model was established, liver retinol concentration was confirmed by hplc. liver retinol concentration of control mice ranged from 120-300 nmol/g, whereas liver retinol concentration of vad mice ranged from 0-7 nmol/g. inbred c57bl/6j mice were purchased from the jackson laboratory (bar harbor, me) and were maintained on standard chow. d23580, a multidrug-resistant salmonella enterica serotype typhimurium clinical bloodstream isolate, was received from robert heyderman at the malawi-liverpool-wellcome trust clinical research centre [17] . inocula were cultured aerobically in luria-bertani (lb) media with 30 μg/ml chloramphenicol shaking for 16-18 hours at 37˚c, subcultured to a calculated optical density (od) of 0.001, and grown 16-18 hours rocking at 37˚c in a 96-well plate containing lb with serial dilutions of enrofloxacin ranging from 0 μg/ml to 2.56 μg/ml. enrofloxacin (baytril 100 by bayer) was obtained from the pharmaceutical service at the uc davis veterinary medical teaching hospital. the od was read at 595 nm on a biorad model 680 microplate reader (hercules, ca). the experiment was repeated six times, and the average minimum inhibitory concentration (mic) was recorded. a determination of susceptible (s), intermediate (i) or resistant (r) was made according to clsi guidelines [18] . inocula of s. typhimurium strains d23580, a multilocus sequence type (st) 313 strain isolated from blood, sl1344, an st19 isolate from a calf with salmonellosis [19] and sara16, an st19 human isolate and reference strain [20, 21] , were cultured in lb with shaking for 16-18 hours at 37˚c. the identity of each strain was confirmed by antibiotic resistance profiling. mice received 100 μl of sterile pbs or 100 μl of 1000 colony-forming units (cfu) diluted in sterile pbs by intraperitoneal (i.p.) injection. mouse weights were monitored daily during infection. mice were euthanized if they showed signs of lethal morbidity. systemic bacterial levels were characterized at day 1, 4 or 5 post-infection by determining tissue loads of s. typhimurium (cfu). liver and spleen were collected in pbs, weighed and homogenized using an ultra turrax t25 basic mixer from ika (staufen, germany). blood was collected in k2 edta microtainer tubes (becton, dickinson and company, franklin lakes, nj), kept on ice for one hour, and then incubated for 10 minutes with 100-200 μl of 1% triton x-100. liver, spleen and blood samples were serially diluted and plated on lb agar plates containing appropriate antibiotic selection. cfu/gram tissue or cfu/ml was calculated after overnight growth at 37˚c. mice were infected with s. typhimurium d23580 as previously described. for preliminary antibiotic treatment experiments, s. typhimurium systemic bacterial levels (cfu) were assessed for male (n = 5) and female (n = 5) mice on a standard diet 5 days post-infection for the following treatment groups: 0 mg/ml, 0.01 mg/ml, 0.05 mg/ml, and 0.10 mg/ml enrofloxacin delivered in the drinking water. water and mouse weights were collected daily, and the average antibiotic consumed (μg/kg/day) was determined. for co-treatment experiments with mice on special diets, 4-6 mice were assessed for control and vad male and female mice in the following groups: mock-treated, mock-treated and enrofloxacin, vitamin a only, and vitamin a and enrofloxacin co-treatment. mock-treated mice received 100 μl of sterile pbs administered by oral gavage on day 1 and day 2 post-infection. vitamin a-treated mice received 600 iu of retinyl palmitate (interplexus, inc., kent, wa) in 100 μl of sterile pbs administered by oral gavage on day 1 and day 2 post-infection and were changed to control diet at day 1 post-infection. enrofloxacin (0.05 mg/ml or 0.01 mg/ml) was administered at day 2 post-infection in the drinking water. enrofloxacin (baytril 100 by bayer) was obtained from the pharmaceutical service at the uc davis veterinary medical teaching hospital. water weights were monitored daily to determine average antibiotic consumption per mouse. cfu/ gram tissue or cfu/ml was calculated for liver, spleen and blood. for measurement of plasma enrofloxacin concentrations, blood was first centrifuged for 10 minutes at 1000 x g. plasma was stored at -80˚c for further analysis by liquid chromatography with tandem mass spectrometry (lc-ms/ms). the remaining blood sample was incubated at room temperature for 10 minutes with 100-200 μl of 1% triton x-100 and plated for cfu as previously described. lc-ms/ms was used to quantify enrofloxacin and ciprofloxacin concentrations in the mouse plasma. first, a mixed master stock solution of 10 μg/ml of enrofloxacin (milliporesigma, st. louis, mo) and ciprofloxacin (milliporesigma, st. louis, mo) was made in control cd-1 mouse plasma (bioreclamation ivt, westbury, ny) with k2 edta. this stock solution was then further diluted with the same control mouse plasma to establish a mouse plasma calibration curve. the following calibrator concentrations were used: 0, 1, 2.5, 5, 10, 50, 100, 500, 1000 and 5000 ng/ml. calibrators 10, 100 and 1000 ng/ml also served as quality control (qc) samples. moxifloxacin (milliporesigma, st. louis, mo) in acetonitrile (acn) (fisher scientific, hampton, nh) was used as an internal standard such that 160 μl of 200 ng/ml moxifloxacin in acn was added to 40 μl of the mouse plasma calibrators, qc samples and the experimental mouse plasma samples collected as previously described. all samples were vortexed for 10 seconds. samples were then centrifuged at room temperature for 5 minutes at 17,000 x g, and 25 μl of resulting supernatant was diluted with 175 μl of water. next, 2 μl of the final solution was injected into the acquity ultra performance liquid chromatography (uplc) system with a beh c18 column, 1.7 μm, 2.1 mm x 50 mm (waters corporation, milford, ma). the following mobile phases were used: a (h 2 o with 0.1% formic acid), and b (acn with 0.1% formic acid) (fisher scientific, hampton, nh). mobile phase a was also used for purging, and acn/h 2 o at 50/50 (v/v) was used for the needle washing between each injection. the flow rate was 0.2 ml/min, the column temperature was set at 50˚c and the autosampler temperature was set at 10˚c. the following uplc gradient program was used for the separation: 0-1 min, 10% b; 2.5 min, 72.5% b; 2.51-4.75 min, 95% b; 4.76-7 min, 10% b. the output of the uplc was fed to a xevo tq-s triple quadrupole mass spectrometry (ms/ms) system (waters corporation, milford, ma), which was used to ionize target molecules with electrospray ionization (esi+) and monitor the ion m/z fragmentation transitions from 360.1-245.2 for enrofloxacin quantification, 332.2-245.2 for ciprofloxacin quantification and 402.2-261.1 for moxifloxacin quantification in multiple reaction monitoring (mrm) mode. the retention times were 2.45, 2.38 and 2.58 minutes for enrofloxacin, ciprofloxacin and moxifloxacin, respectively. the calibration curve was fitted with a weighted (1/x 2 ) least-squares linear regression algorithm. the detection range was from 1 ng/ml to 5000 ng/ml for enrofloxacin and from 2.5 ng/ml to 5000 ng/ml for ciprofloxacin. the extraction yield was about 80-100% for all three antibiotics and the matrix effect enhanced both enrofloxacin and ciprofloxacin ms signals by 10% and 4%, respectively. the matrix effect enhanced moxifloxacin ms signals by 48%. both inter-and intra-batch accuracy were lower than 10% (% deviation) except that ciprofloxacin had 11% of inter-batch deviation at 2.5 ng/ml. both intra-and inter-batch precision were also lower than 10% coefficient of variation (cv), except that enrofloxacin and ciprofloxacin had 20-40% cv at 1 ng/ml and 2.5 ng/ml, respectively. the lower limit of quantification (lloq) for enrofloxacin was determined to be 1 ng/ml and the lloq for ciprofloxacin was determined to be 2.5 ng/ml. to assess survival in untreated mice, control and vad male and female mice were infected with s. typhimurium d23580 as previously described, and weights were monitored daily for up to 4 days post-infection. percent survival for each group (n = 8-9) was reported with a kaplan-meier survival curve. to assess male-specific survival with treatment conditions, control and vad male mice were infected with s. typhimurium as previously described. control mice and one group of vad mice were mock-treated with pbs. other vad groups received either enrofloxacin treatment alone (0.01 mg/ml) or co-treatment of enrofloxacin and vitamin a as previously described. percent survival of 6-16 mice/group at 8 days post-infection was assessed with a kaplan-meier survival curve. to assess treatment of vitamin a alone, control and vad male and female mice were infected with s. typhimurium as previously described. control mice were mock-treated with pbs, one group of vad mice was treated with vitamin a as previously described and a second group of vad mice received mock-treatment. percent survival of 12 mice/group at 13 days post-infection was assessed with a kaplan-meier survival curve. for all survival curve experiments, mice were weighed daily and humanely euthanized when they approached 20% weight loss or displayed signs of lethal morbidity, according to our institution's humane endpoints policy. statistical analyses were performed with graphpad prism 8 (graphpad, la jolla, ca). since data were found not to have a normal distribution, nonparametric analyses were utilized. significance of differences between two groups was determined with a mann-whitney test. significance of differences between multiple groups from a control group was determined with a kruskal-wallis test and a post-hoc dunn's multiple comparisons test. for the survival curves, statistical significance was determined using a log-rank (mantel-cox) test. data for weights, systemic bacterial levels, antibiotic consumed and plasma enrofloxacin concentration were reported as mean ± sem. a p<0.05 was considered significant. in order to model how malnutrition and associated vitamin a deficiency are risk factors for developing systemic s. typhimurium infection in people, we established a mouse model of vitamin a deficiency and disseminated disease (fig 1a) . mice were maintained on either a vad diet or a control diet containing adequate vitamin a. to model systemic infection, mice were infected with s. typhimurium d23580 via the intraperitoneal (i.p.) route. at 4d postinfection, systemic levels of s. typhimurium in liver, spleen and blood were significantly higher in vad mice compared to control (fig 1b) , and there was no sex difference observed in bacterial colonization (s1a fig). in a similar experiment with a necropsy at 5d post-infection, no sex differences in systemic bacterial burden was observed in control mice (s1b to generate vitamin a-deficient (vad) mice, c57bl/6 slc11a1 +/+ pregnant mice were put on a vad diet 2 weeks into gestation (a). at 3 weeks, pups were weaned onto either a vad or control diet. male (n = 7) and female (n = 7-8) mice on each diet were infected with s. typhimurium (stm) d23580 via the intraperitoneal (i.p.) route at 9 weeks of age and systemic bacterial burden was characterized by colony-forming units (cfu) in liver, spleen and blood collected at necropsy (nx) 4 days after infection (b). data represent mean ± sem. significance between control and vad mice was determined with a mann-whitney test of log-transformed values. a p<0.05 was considered significant. data demonstrated that our model of vitamin a deficiency could be utilized to study its effect on susceptibility to systemic s. typhimurium infection. it has been known for decades that vitamin a deficiency affects males and females differently [22] . to assess if this held true for our mouse model, both male and female mice were included. during maintenance on vad and control diets, mice were weighed weekly. while diet had no effect on weight gain of female mice, vad male mice gained significantly less weight than control mice starting at postnatal week 5 (fig 2a) . during the course of infection with s. typhimurium d23580, vad male mice had significant weight loss from day 0 to day 4 of infection, and 50% of mice in the vad male group exhibited lethal morbidity by day 4 ( fig 2b and 2c ). in contrast, vad females and control mice of both sexes maintained their weight over the course of the experiment (fig 2c) . while vitamin a deficiency had sex-specific effects on infection-induced morbidity ( fig 2b and 2c ), both male and female mice were similar in exhibiting significantly higher systemic burdens of s. typhimurium in vad compared to control mice (s1c fig). these results indicated that while vitamin a deficiency compromised control of systemic s. typhimurium infection in both sexes, male mice were affected more strongly, both by reduced growth while on the vad diet and by increased morbidity during infection. since there was no sex difference in systemic bacterial burden by day 4 post-infection (fig 1b) but vad males lost weight and had decreased survival compared to female mice (fig 2) , we investigated whether an earlier time point would yield sex differences in salmonella pathogenesis. further, we assessed if this trend would hold true for multiple strains of s. typhimurium, including d23580 (an st313 strain), and the st19 strains sl1344 and sara16. there was no sex difference in systemic bacterial burden for any of the s. typhimurium strains in mice fed a control diet (fig 3a) . however, at day 1 post-infection, male mice on a vad diet had significantly more or a trend towards a higher systemic burden of all three s. typhimurium strains than vad female mice (fig 3b) . these results suggest that vitamin a deficiency has a more marked effect in male mice on responses that control systemic replication of s. typhimurium in the early stages of infection. emerging multidrug-resistant s. typhimurium strains like d23580 are resistant to the firstline drugs for ints: ampicillin, chloramphenicol and trimethoprim/sulfamethoxazole. for this reason, the fluoroquinolone ciprofloxacin is one of the currently recommended treatments for ints [23] . however, emergence of fluoroquinolone resistance in salmonella strains and host factors like malnutrition can contribute to treatment failure [24, 25] . we chose to model antibiotic treatment failure with the fluoroquinolone enrofloxacin since it can be administered orally through the drinking water to mice and because we could measure it in the bloodstream. we first confirmed susceptibility of d23580 to enrofloxacin in vitro. the average mic was 0.04 μg/ml, which by clsi guidelines is susceptible [18] . we next sought to determine an in vivo mic. in order to assess the subtherapeutic concentration of enrofloxacin administered in the drinking water, systemic bacterial levels were assessed for male and female mice at a range of concentrations at day 5 post-infection. to model antibiotic treatment failure, enrofloxacin administration was delayed to day 2 post-infection. we used c57bl/6j mice fed a standard diet for titration experiments. no sex difference was observed in systemic bacterial levels (s2 fig) . a significant decrease in bacteria in liver, spleen and blood was seen at an enrofloxacin concentration of 0.10 mg/ml (fig 4a) . the concentration 0.01 mg/ml was subtherapeutic, as it failed to significantly decrease systemic s. typhimurium levels. the concentration 0.05 mg/ml was partly subtherapeutic, as it did not significantly decrease s. typhimurium levels in the liver. water weights and mouse weights were monitored daily during infection, and the vitamin a and antibiotics synergize against antibiotic resistant salmonella average amount of enrofloxacin consumed was calculated in μg/kg/day for days 2-3, 3-4 and 4-5. enrofloxacin consumption tracked with dosage, although 0.01 mg/ml and 0.05 mg/ml were not statistically significant from the untreated group (fig 4b) . taken together, these data demonstrated that 0.01 mg/ml and 0.05 mg/ml could model antibiotic treatment failure and thus were used for subsequent experiments with mice on special diets. to assess the therapeutic potential of vitamin a in the context of antibiotic treatment failure, systemic levels of s. typhimurium d23580 at day 4 post-infection were assessed for male and female mice on either control or vad diets with the following treatment groups: mocktreated, vitamin a only, enrofloxacin (0.05 mg/ml) only, and vitamin a and enrofloxacin cotreatment (fig 5a) . in vad male mice, co-treatment significantly decreased bacterial levels at all three systemic sites in comparison to mock treatment; however, individual treatments did not (fig 5b) . systemic colonization of vad male mice in all groups combined was significantly higher if the mouse had >10% weight loss than if the mouse had <10% weight loss (s3 fig). mock-treated vad male mice lost significant weight from day 0 to day 4, but none of the vitamin a and antibiotics synergize against antibiotic resistant salmonella treatment groups were significant (s4 fig). in contrast, vad female mice either showed no significant improvement with any treatment (spleen), or equal effectiveness with enrofloxacin alone and co-treatment (liver, blood) (fig 5c) . in male and female mice on a control diet, both enrofloxacin (0.05 mg/ml) and co-treatment yielded significant or trending decreases in systemic s. typhimurium (s5a and s5b fig). since 0.05 mg/ml enrofloxacin was successful, independent of vitamin a, at decreasing s. typhimurium in liver and blood in vad female mice and control mice for both sexes, a concentration of 0.01 mg/ml was also assessed for these groups. however, co-treatment of 0.01 mg/ml enrofloxacin and vitamin a yielded no benefit in vad female mice compared to mock-treated animals (s6 fig). for males on a control diet, co-treatment of 0.01 mg/ml enrofloxacin and vitamin a showed a slight decrease in splenic bacterial levels, but there was no difference in the liver and blood (s7a fig). there was no benefit of any therapy for females on a control diet (s7b fig). enrofloxacin is a common antibiotic used in veterinary medicine, and it is converted to ciprofloxacin by the liver [26] . no significant differences in plasma enrofloxacin or plasma ciprofloxacin levels were detected between enrofloxacin only and co-treated male (s8a fig) and as 0.01 mg/ml was subtherapeutic such that there was still lethal morbidity in vad male mice, these conditions were used to assess survival of vad male mice with treatments. mice were monitored for 8 days post-infection and euthanized upon showing signs of lethal morbidity, and a kaplan-meier survival curve was generated (fig 5d) . of the infected mice fed control diets, 100% of males survived. the percent survival of mock-treated vad males was only 25%, but this was improved with enrofloxacin only (41.7%) or co-treatment of vitamin a and enrofloxacin (66.7%). significance between each treatment group and survival of mice on a control diet was assessed with a log-rank (mantel-cox) test. further, for the 44 male mice 6-16 ). an enrofloxacin concentration of 0.01 mg/ml was used in drinking water treatment, and oral gavage treatments were given as previously described. significance was determined with pairwise comparisons between the control group and each other treatment group using a log-rank (mantel-cox) test ( � , p<0.05; ns, not significant). data from untreated controls are also included in fig 1. https://doi.org/10.1371/journal.pntd.0008737.g005 represented in the survival curve (fig 5d) , weight loss correlated with survival. over the course of the experiment, if a mouse lost less than 10% of its weight, it was more likely to have survived (16/21; 76% survival) than if a mouse had lost more than 10% of its weight (7/23; 30% survival). in a separate experiment with a longer time course grouping sexes, vitamin a treatment alone improved survival by 66.7% in vad mice (s9 fig). taken together, these data indicated that vitamin a synergized with subtherapeutic (0.05 mg/ml) enrofloxacin treatment to decrease systemic s. typhimurium levels and improve survival in vad male mice. epidemiologic studies have shown that vitamin a deficiency and disseminated non-typhoidal salmonella infections are co-endemic, disproportionately affecting sub-saharan africa [10, 27] . the high mortality rate of disseminated infection has been attributed to antimicrobial resistance of st313 strains that circulate regionally in sub-saharan africa, to genomic features of these strains associated with increased systemic dissemination, and to the high prevalence of co-morbidities that predispose to systemic disease [17, 23, 24] . the current study was designed as a preclinical trial in mice to assess the potential utility of vitamin a as an adjunct to antibiotic therapy. our results suggest a benefit of combined antibiotic treatment and vitamin a supplementation, with the effect being most apparent in male mice. it is well-appreciated that vitamin a supplementation can be a useful tool for disease prevention and treatment. in populations with a �1% prevalence of night blindness or a �20% prevalence of vitamin a deficiency, the who recommends vitamin a supplementation for children 12-59 months of age [28] . vitamin a supplementation was found to significantly reduce the risk of all-cause morbidity and mortality due to diarrhea [29] . in children 2 and under presenting with measles, the who-recommended treatment of two consecutive daily megadoses of vitamin a [30] reduces overall and pneumonia-specific mortality [11] and decreases duration of diarrhea and fever. however, the utility of vitamin a for bacterial bloodstream infections has not been assessed. s. typhimurium bloodstream infections typically present as a febrile illness; diarrhea is often absent or not a prominent symptom [10, 31, 32] . our results suggest that co-treatment with vitamin a and antibiotics may improve outcomes during bloodstream infections with drug-resistant s. typhimurium. despite who's recommendation for vitamin a supplementation, full coverage is still lacking in many countries. therefore, supplementing with vitamin a during episodes of febrile illness is a potential strategy to increase coverage and improve infection outcomes. a notable finding of our study was the sex-specific effect of vitamin a supplementation with antibiotics for treatment of s. typhimurium infection in mice. the picture emerging from recent studies of innate immunity is that multiple sex-specific differences impact resistance of males and females to infection. human males are more susceptible to many infectious diseases, whereas females are more at risk of developing an autoimmune condition [33] . in current events, males are at a higher risk of severe covid-19 infection [34] . males also have a higher risk of developing sepsis [35, 36] . a study on sepsis in children found that vitamin a deficiency was more common in the sepsis group as compared to control, and vitamin a deficiency rates were~80% in the subgroups of severe sepsis and septic shock [37] , indicating how a vad status can predispose to severe disease. the majority of patients with sepsis in this study were male. in addition, some genes encoding for innate immune molecules are located on the x chromosome [33] , suggesting a genetic basis for some of the differences observed. it is therefore possible that malnutrition and vitamin a deficiency may compromise the immune response to systemic salmonella infection in a sex-specific manner. we speculate that vitamin a may be playing the role of a host-directed therapy by boosting the immune response of vad male mice, synergizing with an initially subtherapeutic dose of antibiotic, and leading to better immunological control of infection. while the current study did not investigate the mechanisms underlying sex differences in the response to vitamin a supplementation, possible explanations include sex differences in innate immune cell function [33] , intestinal microbiota composition [38] , and hormone levels [39] . antibiotic treatment failure is the persistence of symptoms despite initiation of antibiotics. common causes include wrong diagnosis, infection with a resistant organism, or host failure such as malnutrition [25] . we modeled antibiotic treatment failure with a subtherapeutic enrofloxacin concentration that yielded no difference in systemic bacterial burden, but circulating antibiotic was still present in the mouse. it can be the joint action of antibiotics and host defense mechanisms that leads to clearance of bacteria and resolution of disease. while antibiotics are typically studied for their bactericidal or bacteriostatic activity against susceptible bacteria, they can also exist at subtherapeutic or subinhibitory concentrations, i.e. at concentrations below the mic. subinhibitory antibiotic concentrations can still affect both the host and the bacterium [40] . it has been proposed that some antibiotics may influence antiphagocytic cell-surface structures and lead to enhanced phagocytosis, modifying immune cell function [41] . for example, neutrophil phagocytosis and intracellular killing activity have been enhanced by pre-incubation with subinhibitory antimicrobial concentrations in some studies, although it depends on the antibiotic used and organism studied [42] . one study reported that subinhibitory levels of fluoroquinolone antibiotics like ciprofloxacin lead to enhanced binding of anti-lps antibodies to enterobacteriaceae like escherichia coli [43] . taken together, these findings suggest that vitamin a treatment may synergize with subtherapeutic antibiotics to enhance immune function in male mice. our study has limitations. we studied the effect of vitamin a co-treatment with only a single antibiotic, enrofloxacin, and a single clinical isolate, d23580. common treatments for ints have included first-line antibiotics of ampicillin, chloramphenicol and trimethoprim/sulfamethoxazole [23] . d23580 is resistant to all of these first-line drugs. it is sensitive to fluoroquinolones at certain concentrations in vitro and in vivo. however, future studies should incorporate s. typhimurium strains also resistant to fluoroquinolones, as this is an emerging issue [9] . in addition, it would be important to determine whether vitamin a supplementation can prevent treatment failure with other commonly used antibiotics. there are also limitations to using a mouse model of infection, including the low genetic variation of inbred mice used and the short infection time course permitted by the vad male mouse model. future studies could incorporate additional mouse lines, routes of infection and challenge doses. however, given the track record of using vitamin a as prevention and treatment for other infections, this information on whether vitamin a supplementation could be effective against disseminated infections with antibiotic-resistant salmonella might be best obtained in a clinical study. 1-7) . the same data is represented in two different ways. initially there were more vad male mice in the study but they reached lethal endpoint prior to day 5 and were likely to have a higher cfu than in the figure. c. levels of s. typhimurium in liver, spleen and blood at day 4 and 5 post-infection combined (n = 8-15). data shown in this figure are a re-analysis of data presented in s1a and s1b. data represent mean ± sem. significance between groups was determined with a mann-whitney test of logadditionally, vad mice treated with retinyl palmitate were placed on a control diet replete with vitamin a one-day after s. typhimurium infection. data represent percent survival of 12 mice per group from three independent experiments. pairwise comparisons between the control diet group and each other group was determined with a survival analysis log-rank (mantel-cox) test ( � , p<0.05). (tif) burden of infection on growth failure malnutrition and health in developing countries increased urinary retinol loss in children with severe infections vitamin a, infection, and immune function antimicrobial susceptibility testing of bacteria that cause gastroenteritis the global burden of nontyphoidal salmonella gastroenteritis global burden of invasive nontyphoidal salmonella disease the global burden and epidemiology of invasive non-typhoidal salmonella infections fluoroquinolone-resistant enteric bacteria in sub-saharan africa: clones, implications and research needs invasive non-typhoidal salmonella disease: an emerging and neglected tropical disease in africa vitamin a for treating measles in children guideline: updates on the management of severe acute malnutrition in infants and children. who guidelines approved by the guidelines review committee. geneva: world health organization vitamin a supplementation in infants and children vitamin a deficiency alters rat neutrophil function host-directed therapies for bacterial and viral infections tlr signaling is required for salmonella typhimurium virulence loss of multicellular behavior in epidemic african nontyphoidal salmonella enterica serovar typhimurium st313 strain d23580 m100: performance standards for antimicrobial susceptibility testing the estimation of doses of salmonella typhimurium suitable for the experimental production of disease in calves reference collection of strains of the salmonella typhimurium complex from natural populations defining the core genome of salmonella enterica serovar typhimurium for genomic surveillance and epidemiological typing the sex difference in vitamin-a metabolism fluoroquinolone resistance in salmonella: insights by whole-genome sequencing epidemic multiple drug resistant salmonella typhimurium causing invasive disease in sub-saharan africa have a distinct genotype recommendations for treatment of childhood non-severe pneumonia pharmacology of the fluoroquinolones: a perspective for the use in domestic animals global prevalence of vitamin a deficiency in populations at risk 1995-2005: who global database on vitamin a deficiency. geneva: world health organization guideline: vitamin a supplementation in infants and children 6-59 months of age. who guidelines approved by the guidelines review committee. geneva: world health organization vitamin a supplementation for preventing morbidity and mortality in children from six months to five years of age treating measles in children. department of immunization, vaccines and biologicals. geneva: world health organization non-typhoidal salmonella bacteraemia among hiv-infected malawian adults: high mortality and frequent recrudescence clinical presentation of non-typhoidal salmonella bacteraemia in malawian children sexual dimorphism in innate immunity sex differences in mortality from covid-19 pandemic: are men vulnerable and women protected? jacc case rep predictors of sepsis in moderately severely injured patients: an analysis of the national trauma data bank gender differences in human sepsis vitamin a deficiency in critically ill children with sepsis neonatal vitamin a supplementation and vitamin a status are associated with gut microbiome composition in bangladeshi infants in early infancy and at 2 years of age the early postnatal period, mini-puberty, provides a window on the role of testosterone in human neurobehavioural development role of antibodies and antibiotics in aerobic gram-negative septicemia-possible synergism between antimicrobial treatment and immunotherapy. reviews of infectious diseases subinhibitory antimicrobial concentrations: a review of in vitro and in vivo data influence of subinhibitory levels of antibiotics on expression of escherichia-coli lipopolysaccharide and binding of antilipopolysaccharide monoclonal-antibodies the authors thank greg barton for providing inbred c57bl/6-slc11a1 +/+ mice, robert hey key: cord-292596-ulu5y140 authors: lee, su hae; jee, seung wan; hwang, dae youn; kang, jong koo title: characterization of changes in global gene expression in the hearts and kidneys of transgenic mice overexpressing human angiotensin-converting enzyme 2 date: 2020-07-29 journal: lab anim res doi: 10.1186/s42826-020-00056-y sha: doc_id: 292596 cord_uid: ulu5y140 human angiotensin-converting enzyme 2 (hace2) has recently received a great attention due to it play a critical role as sars-cov receptor in the infection of human body. however, no further analysis for gene regulation has been performed in target tissues of model mice during hace2 overproduction. to characterize changes in global gene expression in the hearts and kidneys of rtta/hace2 double transgenic (dtg) mice in response to hace2 overexpression, total rna extracted from these tissues from dtg mice after doxycycline (dox) treatment was hybridized to oligonucleotide microarrays. briefly, dtg mice were generated by cross-mating pα-mhc/rtta tg mice with ptre/hace2 tg mice. the expression level of hace2 protein was determined to be high in hearts, kidneys, and brains of dtg mice, whereas lung, liver, and testis tissues expressed low levels. the level of hace2 was significantly enhanced in hearts and kidneys of the dox+dtg group compared to that in vehicle+dtg mice although consistent levels of mouse ace2 (mace2) remained in the same tissues. based on the microarray analysis of heart tissue, 385 genes were differentially expressed, including 168 upregulated and 217 downregulated, when comparing non-tg and vehicle+dtg mice, whereas 216 genes were differentially expressed, including 136 upregulated and 80 downregulated, between vehicle+dtg and dox+dtg mice. in the kidneys, 402 genes were differentially expressed, including 159 upregulated and 243 downregulated, between non-tg and vehicle+dtg mice. dox-treated dtg mice exhibited the differential expression of 4735 genes including 1636 upregulated and 3109 downregulated. taken together, these findings suggested that several functional groups and individual genes can be considered biomarkers that respond to hace2 overexpression in dtg mice. moreover, our results provided a lot of useful information to predict physiological responses when these dtg mice are applied as a susceptible model for novel coronavirus (sars-cov, covid-19) in both vaccine and drug development. angiotensin-converting enzyme 2 (ace2) is the first known human homolog of ace and was cloned from a human heart failure and human lymphoma cdna library [1, 2] . ace2 might play a pivotal role as a new element of the renin angiotensin system (ras) by reducing ang ii and increasing levels of ang1-7 [3] [4] [5] [6] and is distributed in a wide variety of tissues, including the brain, lung, heart, liver, kidney, and testis, as well as most cardiovascular-relevant tissues [7] [8] [9] [10] [11] . in the heart, myocardial infarction increases ace2 expression, which is localized to the vascular endothelium, smooth muscle, and cardiomyocytes of both rats and humans [12] . ace2 participates in the regulation of blood pressure and cardiac and renal functions and is associated with major cardiac and renal pathophysiological processes. especially, it has been reported that ace2 gene expression is upregulated in humans with heart failure [13] . it is also predominantly expressed in the proximal tubular brush border, distal tubules, and glomerular epithelial cells in the kidneys of humans, rats, and mice [14] [15] [16] [17] . the colocalization of ace2 with ang1-7 in renal tubules reveals the functional ability of ang1-7 to counteract the action of ang ii, and these vasodilator peptides might be a critical link, mediating regulatory feedback between ace and ace2 [18] [19] [20] . although the function of ace2 in the brain is poorly understood, there is considerable evidence of a role for ang1-7. previous studies have shown that ang1-7 is an important neuromodulator of cardiac baroreflex mechanisms [21] . the ace1/2 and neutral endopeptidase or neprilysin (nep) are zinc metallopeptidases [22] . however, more recently, ace2 has aroused considerable attention as a receptor for the coronavirus that causes severe acute respiratory syndrome and a protector against severe lung failure [23, 24] . in the past decade, functional studies on hace2 have continued using transgenic animals to clarify the mechanism related to heart and renal failures, as well as other pathophysiological conditions. first, in knockout mice, the genetic disruption of ace2 leads to severe cardiac contractile dysfunction, increases in ang ii levels, the upregulation of hypoxia-induced genes, and decreases in ace2 transcript and protein levels in the heart [25] . however, it is necessary to additionally study the effects related to hace2 expression levels to clarify whether increased levels can have a beneficial effect on cardiac and renal functions. myosin heavy chain (mhc), which is found in the contractile apparatus and is a major protein, is composed of two heavy chains and four light chains. in the cardiac muscle, two distinct mhc genes, which encode αand β-mhc isoforms, have been identified [26] . previously, the α-mhc gene promoter was used to direct tissue-and developmental-specific expression of the transgene in transgenic (tg) animals [27] . further, there are many transcriptional regulation systems, and tetracycline regulatory systems have been widely used for conditional gene expression. the tetracycline-controlled transactivator (tta) is generated by fusing the dnabinding domain of the tetracycline-resistance operon (tetr) encoded by tn10 of e. coli with the transcriptional activation domain of vp16 of herpes simplex virus [28] . there are two basic variants of this, tta and reverse tta (rtta) systems. to compensate for the tta system, which requires long-term administration of doxycycline (dox) for induction of the transgene, the other inducible system, namely rtta (tet-on system), has been developed. this system requires two dna constructs, a transcriptional regulatory unit and the responsive element teto sequences linked to a p cmv -derived target gene. in the presence of dox, rtta binds teto sequences and p cmv , which activates the target gene. in contrast, rtta does not bind teto and the target gene is not transcribed in the absence of dox [29, 30] . the doxinducible gene regulatory system allows for the tight and adjustable control of a transgene of interest to study organ development and disease pathogenesis. previously, tg rats expressing human ace under the control of the rat cardiac myosin light chain 2 (mlc2) promoter and tg mice expressing human ace2 under the control of the mouse cardiac α-mhc promoter have been generated [31, 32] . these tg mice had a high incidence of sudden death and rhythmic disturbances with sustained ventricular tachycardia including terminal ventricular fibrillation and heart block. however, in surviving older mice, spontaneous downregulation of the ace2 transgene was observed, which was associated with the restoration of nearly normal conduction, rhythm, and connexin expression. because sudden death occurs earlier in higher-expressing transgenic lines, a promoter that can be regulated, such as the tet-on or off system, might be useful to examine cardiac and renal pathophysiology at the basal level. the aim of this study was to characterize changes in global gene expression in the hearts and kidneys of dtg mice, created with pα-mhc/ rtta and ptre/hace2 vectors, in response to the overproduction of hace2 protein. first, pα-mhc/rtta was constructed by fusing the rtta-m2 gene with the α-mhc promoter (gene bank accession no. u71441). the puhrt62-1 was a gift from dr. wolfgang hillen at the university of heidelberg, germany [33] . this plasmid contains a mutagenized rtta-m2 fragment harboring the s12g, e19g, and a56p mutations. the α-mhc sequence was amplified by pcr, with the genomic dna as a template, which was isolated from the tail of bdf2 mice. the following primers were used for the amplification: the sense primer, 5′-ctcct tcctt gttgc atctt cc-3′ (corresponding to nucleotides 2,278-2,299 of α-mhc), and the antisense primer, 5′-cagga ggaag atgga gaaga cag-3′ (corresponding to nucleotides 4,578-4,600 of α-mhc). the amplified α-mhc product was cloned into pgem-t (pα-mhc-t). the α-mhc fragment obtained by the digestion of pα-mhc-t with saci and sacii was cloned into puhdrtta2s-m2-splice, in which the human cmv promoter has been eliminated via digestion with xhoi and sacii (pa-mhc/rtta). second, ptre/hace2 was constructed by inserting the hace2 gene into the noti site within the multiple cloning site of ptre2hyp (clontech laboratories inc., mountain view, ca, usa). the ptre2hyp gene contains a tet response element (tre), which consists of seven copies of the 19-bp tetracycline operator sequence (teto) and hygromycin resistance genes. the hace2 cdna was amplified by pcr, using a sense primer (5′-gacga tgtca agctc ttcct g-3′) with nucleotides 100-120 and an antisense primer (5′-gccta cagat cttct tcaga aataa gtttt tgttc aaagg tctga acatc atcag tg-3′; lowercase letter, c-myc tag) with nucleotides 2,494-2,521 based on hace2 (gene bank accession no. nm_021804). full-length rna was used as the template, which was isolated from hek-293 cells (atcc crl-1573). the amplified hace2 product was inserted into pgem-t (phace2-t; fig. 1a and b). the experimental protocol for dtg mice was carefully reviewed based on ethical and scientific care guidelines and approved by the national institute of food and drug safety evaluation-institutional animal care and use committee (nifds-iacuc; approval no. nitr0631). all c57bl/6nkorl and dba/2korl mice at 6 weeks of age were provided by the department of laboratory animals resources in nifds (cheongju, korea). all mice used in this study were provided with ad libitum access to water and an irradiated standard chow diet (purina mills inc., pyeongtaek, korea). mice were housed in cages under specified pathogen free (spf) condition under a strict light cycle (light on at 06: 00 h and off at 18:00 h). all mice were handled in an accredited nifds animal facility in accordance with the aaalac international animal care policies (accredited unit-the ministry of food and drug safety: unit number-001492). for the first lineage of tg mice, a linear 5.8-kb α-mhc/ rtta fragment was microinjected into the pronucleus of fertilized eggs of bdf1 mice, which had been obtained by mating c57bl/6nkorl (males) and dba2korl (females) mice. for the second lineage of tg mice, the linear 7.9-kb fragment of tre/hace2 was microinjected into the pronucleus of a fertilized embryo after dilution to a concentration of 4 ng/μl. microinjection was performed using microscopy (nikon, tokyo, japan) and a micromanipulator (narishge, tokyo, japan). each tg line was established by back crossing the founder mice with a parental strain of c57bl/6nkorl mice. the dtg mice were obtained by crossing the first lineage of α-mhc/rtta tg mice with the second lineage of tre/ hace2 tg mice. the single and double tg founder mice were back crossed onto the parental strain of the c57bl/6nkorl background to establish homogenous tg lines. the transgene was identified by dna-pcr analysis of the genomic dna isolated from the tail of 4-week-old founder mice. the precipitated dna was separated by centrifugation at 15,000 rpm for 10 min. after washing in 700 μl 70% et-oh, dna was dried for 15 min and dissolved in 200 μl of distilled water. the α-mhc/rtta gene was amplified as a template using the sense primer (5′-ctgtc ttctc catct tcctc ctg-3′) with a complementary α-mhc promoter ranging from 4578 to 4600 nucleotides and an antisense primer (5′-caggg taggc tgctc aactc-3′) with a complementary rtta gene ranging from 888 to 908 nucleotides. the tre/hace2 gene was also synthesized as a template using a sense primer (5′-gacga tgtca agctc ttcct g-3′) and antisense primer (5′-catat aatgg cctca gctgc-3′) with a complementary hace2 gene ranging from 100 to 120 and from 625 to 644 nucleotides, respectively. pcr amplification was carried out in a thermal cycler (perkin elmer, norwalk, ct, usa) using the following cycling conditions: one cycle: 94°c, 4 min; 27 cycles: 30 s at 94°c, 1 min at 62°c, and 45 s at 72°c; one elongation step of 7 min at 72°c. the dtg mice were distributed into two groups (3-5 mice per group), namely vehicle and dox treatment. drinking water containing 2 mg/ml dox (sigma-aldrich co., st. louis, mo, usa) was administered to mice of the dox-treated group for 4 weeks, whereas tap water was administrated to mice of the vehicle group for the same period. the brain, heart, lung, liver, kidney, and testis tissues were collected from mice of subset groups and homogenized with 1% nnonidet p-40 in 15 mm nacl, 10 mm tris hcl, and 1 mm edta supplemented with a protein inhibitor mixture (roche, basel, switzerland), which was followed by centrifugation at 4°c for 15 min. for western blot analysis, the proteins were separated by electrophoresis using a 4-20% gradient sds-polyacrylamide gel for 2 h and then transferred to a nitrocellulose membrane with transfer buffer containing 25 mm tris-base, 192 mm glycine, and 20% methanol at 45 v for 2 h. membranes were blocked with 3% (w/v) non-fat dried milk in pbs (137 mm nacl, 2.7 mm kcl, 10 mm na 2 hpo and 2 mm kh 2 po 4 ) solution containing 0.05% tween-20 (sigma-aldrich co.). the membrane was washed with pbs and incubated overnight 4°c with primary antibodies as follows: anti-human ace2 (santa cruz fig. 1 reverse tet promoter-controlled transactivator (rtta) and hace2 vectors and identification of α-mhc/rtta and tre/hace2 transgenes. a construction of the pα-mhc/rtta and ptre/hace2 expression vectors. rtta and hace2 (human ace2) were placed under the control of the α-mhc and tre promoter, respectively. two independent lineages of transgenic (tg) mice were produced and mated together to obtain the double tg (dtg) mice. in the dtg mice, the expression of the hace2 is induced by rtta in the presence of doxycycline (dox). the arrow (--->) indicates transcription. b features of the tet response element (tre) promoter sequence. the tre promoter is contained with seven copies of the teto sequence, a tata box, and an hcmv promoter. c the genomic dna was isolated from the tail of the founder mouse, and the 174-bp and 545bp products were shown in the dual transgenic mice carrying the α-mhc/rtta and tre/hace2 transgenes, respectively biotechnology inc., santa cruz, ca, usa), anti-human ace (santa cruz biotechnology inc.), and anti-α-tubulin (sigma-aldrich co.). after washing, each antigen-antibody complex was visualized with biotinylated secondary antibodies as follows: goat anti-mouse igg (h+l) hrpconjugated antibody, rabbit anti-goat igg (h+l) hrpconjugated antibody, and goat anti-rabbit igg (h+l) hrp-conjugated antibody (zymed laboratories inc., san francisco, ca, usa) at a 1:1000 dilution in pbs buffer containing 3% non-fat dried milk/pbs buffer at room temperature for 1 h. immunoreactive proteins were detected by an enhanced chemiluminescent substrate (ecl, amersham pharmacia biotech, inc., amersham, england) reaction, which was followed by exposing the membranes to hyperfilm ecl. the hearts and kidneys from non-tg, vehicle+dtg, and dox+dtg mice were used for the isolation of total rna using rnazol (tel-test inc., friendswood, texas, usa) according to the manufacturer's instructions. the frozen tissues were minced with scissors and homogenized in rnazol b solution using a teflon glass homogenizer. the rna pellet was suspended in depc-treated dh 2 o and purified using a qiaquick purification kit (qiagen inc., chatsworth, ca, usa). the integrity of the 18s/ 28s rrna was analyzed using a bioanalyzer 2100 (quality agilent technology inc., santa clara, ca, usa), with the rna quality checked based on the ratio of absorbance at 260 nm to that at 280 nm using a biophotometer (hamburg-eppendorf, hamburg, germany). the integrity of each rna sample was confirmed by 1% agarose gel electrophoresis, which showed the presence of intact 18s and 28s ribosomal bands. crna synthesis and labeling were performed using a chemiluminescent rt-ivt labeling kit (applied biosystems, foster city, ca, usa) according to the manufacturer's instruction. individual samples were submitted in randomly assigned pairs representing tissues from non-tg and tg mice. three micrograms of total rna was used to synthesize cdna. double stranded cdna was synthesized for 2 h at 16°c in a reaction mixture containing nuclease free water, 5× 2 nd strand buffer, and 2 nd strand enzyme and then purified using a purification column. finally, the biotinylated crna was generated from the cdna using a bioarray crna high yield rna transcript kit containing purified double stranded cdna, 5× ivt buffer mix, dig-utp and ivt enzyme mix. changes in global gene expression in the hearts and kidneys of mice overexpressing human ace2 were analyzed using the mouse genome survey microarray (applied biosystems) containing oligonucleotide probes for 44, 000 genes. the crna was fragmented in fragmentation buffer for 30 min at 60°c prior to chip hybridization. fifteen micrograms of fragmented crna was then added to the hybridization cocktail. each sample was hybridized to a separate oligonucleotide array for 16 h at 55°c in the genechip hybridization oven 640. after hybridization, the solution was removed and the slides were washed twice with 2× ssc containing 0.1% sds for 5 min at 42°c. the slides were incubated for 20 min with anti-dig-ap in cl blocking buffer and then developed using the chemiluminescence substrate. thereafter, the hybridized arrays were scanned using an abi700 analyzer. scanning and basic analyses were performed using geneplex software release 1.0 (istech inc., seoul, korea). logged gene expression ratios from the fluorescent intensity of each spot were normalized based on regression. tests for significance were performed using a one-way anova test of variance (spss for windows, release 10.01, standard version, and chicago, il, usa). all values are reported as the mean ± standard deviation (sd). microarray data and the statistical significance of differential expression were assessed by hypergeometric distribution analysis. p values less than 0.05 were considered significant. first, we produced one male founder mouse carrying the pα-mhc/rtta construct and one female founder mouse carrying the tre/hace2 construct using microinjection techniques. these showed different rates of transgene insertion into the chromosome at 18 and 7%, respectively. moreover, α-mhc/rtta and tre/hace2 single tg mice with a bdf2 background were backcrossed with c57bl/6nkorl mice to change their background. during this process, α-mhc/rtta and tre/hace2 constructs were transmitted into the genomes of their offspring of both sexes at a rate of approximately 50% hemizygous animals based on mendelian inheritance. furthermore, both construct genes were transmitted into the genomes of dtg mice at a 16.3% efficiency. to test whether the hace2 transgene was expressed under the control of rtta in a tissue-specific manner, its expression level was detected in various tissues including the brain, heart, lung, liver, kidney, and testis of dtg mice treated with 2 mg/ml dox for 4 weeks. the expression level of hace2 was higher in the heart, kidney, and brain than in other organs, although the highest level was detected in the heart. a low level of hace2 protein expression was observed in the lung, liver, and testis (fig. 2) . however, any significant expression of hace2 protein was not detected in heart and kidney of non-tg and c57bl/6 mice (data not shown). therefore, these results indicate that the hace2 protein might be successfully expressed in various tissues of dtg mice through regulation of the dox-controlled rtta system. to compare the expression level of mace and hace2 proteins in the heart and kidney with or without dox treatment, both proteins were detected with specific antibodies in these tissues of non-tg, ve-hicle+dtg, and dox+dtg mice at the age of 10 weeks. a similar expression pattern for both proteins was observed in the heart and kidney. the hace2 gene was highly expressed in only dox+dtg mice, whereas in non-tg and vehicle+dtg groups, expression was maintained at low level. however, the expression level of mace protein remained constant regardless of hace2 expression and dox treatment ( fig. 3a and b) . thus, these results suggest that hace2 protein can be successfully overexpressed in the hearts and kidneys of dtg mice after dox treatment. further, it was suggested that these organs would have great potential for microarray analysis to characterize the changes in global genes during overexpression of the hace2 protein. to analyze the differential expression of global genes during hace2 overproduction in the heart and kidney, microarray data were normalized based on the lowest values obtained from the experiments repeated in triplicate. the normalized log ratios of various genes in each experiment were used to compare relative expression between two independent experiments. based on these arbitrary differences, a substantial number of genes were expressed at elevated or reduced levels in the tg group before and after dox treatment. in the heart, 385 transcripts were selected as differentially expressed genes between non-tg and vehicle+dtg groups based on a 2-fold change in expression, whereas 216 differential transcripts were identified between vehicle+dtg and dox+dtg groups. among the former 385 genes, 168 were upregulated and 217 were downregulated in the heart tissue of the dtg group as compared to levels in the non-tg group before dox treatment. moreover, 216 genes, including 136 upregulated and 80 downregulated, were differentially expressed in the dtg group after dox treatment (table 1 ). in the kidney, a total of 402 genes, including 159 upregulated and 243 downregulated genes, were changed in the vehicle+dtg mice compared to levels in non-tg mice. furthermore, after dox treatment for 4 weeks, 4735 genes, comprising 1636 upregulated and 3109 downregulated, were altered in the dtg group based on a 3-fold change in (table 1) . overall, these results suggest that hace2 overproduction is more closely related to differentially expressed genes in the kidney than in the heart. to analyze the kegg pathways associated with hace2regulated gene expression in the heart and kidney of non-tg and dtg mice based on dox treatment, genplex software release 1.0 was used. the results are presented in tables 2 and 3 . genplex identified 15 pathways that were associated with significant changes in the heart ( table 2 ). as presented in table 2 , the largest numbers of genes in the heart were mainly associated with cytokine-cytokine receptor interaction, the calcium signaling pathway, apoptosis, and ecm-receptor interaction. in the kidney, 17 pathways were identified (table 3) . especially, genes involved in wnt signaling were highly changed, followed by those related to oxidative phosphorylation, prostate and colorectal cancer, the tgf-beta signaling pathway, and apoptosis. furthermore, we characterized the genes that were downregulated and upregulated by hace2 overproduction in the hearts and kidneys of dox+dtg mice. of the 136 upregulated genes in the heart, the highest difference was detected for cap1, followed by pik3c2g, syt16, hecw1, and tmod2, whereas downregulated genes included calgranulin a, calgranulin b, paip1, ptx3, and hspa1a. in the kidney, gabrg1 was associated with the highest increase, followed by wdr66, ppp1r14c, bin1, and mef2c among 1636 upregulated genes, but slc16a4, fgfr3, slc16a10, stam2, and mrps2 were significantly decreased after hace2 overproduction. taken together, this showed that hace2 overexpression is mainly associated with cellular pathways related to cytokine-cytokine receptor interactions, the calcium signaling pathway, apoptosis, ecm-receptor interactions, wnt and tgfbeta signaling, cancer, and oxidative phosphorylation in the hearts and kidneys of dtg mice. recently, advances in molecular biology have enabled functional analyses of interesting genes by using transgenic animals and stable cell lines with constant gene expression. however, if the transgene is related to the embryogenesis, the animal might be genetically predisposed to tolerate the effects of the transgene products [34] . to overcome this problem, it is necessary to develop transgenic animals in which transgene expression can be induced at selected time points but kept silent for an extended period. conditional gene expression has been achieved using a variety of model systems [35, 36] . non-regulatable promoters in tg animals cannot direct the genetic switches to upregulate or downregulate expression from the promoter-linked target gene, and therefore, it is impossible to know how the target gene interacts and regulates the pathophysiological processes at both the basal and inducible level. the tet-on and tet-off expression systems are the most widely used inducible regulatory systems. in the tet-on system, the reverse tetracycline-controlled transactivator (rtta) acts as an activator of gene transcription [29, 37] . dox binds the dox-binding site of the rtta protein, which represents random mutagenesis of the tta fusion protein between the tet repressor dna-binding domain (207 amino acids) and the vp-16 transcriptional activation domain (130 amino acids) of the herpes simplex virus. the dox-rtta complex then binds the tet sequence, which brings the vp-16 activation domain in close proximity to the minimal human cmv promoter, thereby activating the target gene in the presence of dox. indeed, rtta under the control of the cmv promoter was previously shown to activate the expression of a target gene in various organs [38, 39] . in this study, the inducible tg mice expressing α-mhc-controlled, rtta-regulated hace2 were generated to address the hypothesis that unregulated expression of the hace2 transgene leads to the generation of defects including cardiac contractile dysfunction, rhythmic disorders, and sudden death. first, single tg mice expressing α-mhc-controlled rtta and hace2 were successfully developed by directly introducing each gene into fertilized eggs. tg mice were mated to induce rtta-regulated hace2 expression, which generated founder mice in which hace2 expression could be increased by dox. in the established tg line, neither the location of the transgenes in the genome nor the locus was impacted by gestation or neonatal imprinting. because of this, all offspring in this experiment exhibited the transmission of α-mhc/rtta and tre/hace2 genes into their genomes in approximately 50% of hemizygotes. ace is considered the central enzyme in the ras, converting ang i to ang ii. however, the identification of novel ras components such as ace2 and collectrin, a homologue of ace, capable of degrading ang ii and forming ang1-7, has emphasized the increasing complexity and multiplicity of biochemical pathways forming the ras. in a previous study, non-regulatable promoters have been used to create transgenic rats or mice expressing ace or ace2 genes under control of the rat cardiac mlc2 or mouse cardiac α-mhc promoter [7, 32] . unexpectedly, the loss of ace2 in mice results in profound contractile dysfunction. however, the complete rescue of the heart phenotype in ace/ace2double mutant mice indicates that ace expression has a causative role in the onset of heart dysfunction [25] . because ace2 is expressed in the vascular endothelium and not in cardiac myocytes, local increases in ang ii might lead to vasoconstriction, resulting in hyperperfusion and hypoxia in the myocardium. it has been established that ang ii can induce oxidative stress in endothelial cells, and thus, its increase could result in dysfunction of the vascular endothelium via the induction of oxidative stress in the heart [25, 40, 41] . in this study, a heart-specific promoter system was employed to induce rtta-regulated hace2 expression at a physiologically relevant site. hace2 protein was abundantly expressed in heart and kidney tissue of dox+dtg mice, although this protein was also detected in several other tissues. these results suggest that the binding of dox to the rtta protein might successfully induce hace2 expression in the heart, kidney, and other organs of dtg mice. however, any significant histopathological changes was not observed in heart and kidney of dox+dtg mice after induction of hace2 expression for only 4 weeks (supplement fig. 1) . interestingly, the ras can be seen as a dual function system in which vasoconstrictor or vasodilator actions are primarily driven by the ace/ace2 balance [42] [43] [44] . elevated ace2 activity concomitant with reduced ace activity leads to a decrease in ang ii levels by converting it into ang1-7, which in turn promotes vasodilatation [45] . according to this concept, dox-driven hace2 expression resulted in a decrease in ace levels in both hearts and kidneys of dox-inducible tg mice compared to that in non-tg mice. these results imply that the effects of hace2 expression, via the formation of ang1-7, have a counter regulatory role in ace activity in heart and kidney functions. the present study also investigated gene profiles to offer critical insight into complexity of the ras, as well as heart and renal failure related to hace2 overexpression. inducible tg mice overexpressing hace2 and non-tg mice were used to address the hypothesis that genes many genes involved in cardiovascular and renal disorders are modulated as compared to levels in dox+dtg and non-tg mice. the results identified 136 genes from the hearts of dox+dtg mice that were significantly upregulated and 80 genes that were downregulated compared to levels in the hearts of vehicle+dtg mice. a total of 216 genes associated with cellular pathways comprising 15 categories mainly related to ecmreceptor interaction, cytokine-cytokine receptor interaction, apoptosis, the calcium signaling pathway, and the tgf-beta signaling pathway were identified. of these genes, s100a8 and s100a9, encoding s100 calciumbinding proteins that bind several types of proinflammatory cytokines, such as tnf-alpha, il-6, and il1beta, to form the proinflammatory cytokine complex in acute inflammation, play a role in calcium-mediated signaling [46, 47] . ptx3, a pentraxin-related gene, is induced in vascular smooth muscle cells via atherogenic modified low density [48] and acts as a nonredundant regulator of tissue damage in acute myocardial ischemia and reperfusion [49] . thus, these results suggest that the overexpression of hace2 might be involved in acute myocardial infarction and ischemia in the hearts of inducible rtta-regulated tg mice. in the kidney, 1636 genes in dox-treated tg mice were upregulated and 3109 genes were downregulated. these genes were also associated with cellular pathways comprising 17 categories related to apoptosis, the calcium and wnt signaling pathway, oxidative phosphorylation, cancer, and the tgf-beta signaling pathway. especially, hace2 expression was mainly associated with the cellular pathway related to cytokine-cytokine receptor interactions, the calcium signaling pathway, apoptosis, ecm-receptor interactions, wnt and tgf-beta signaling, cancer, and oxidative phosphorylation, and rtta-controlled hace2 expression regulates the expression of genes related to binding, receptor, transferase, oxidoreductase, and hydrolase activities in the hearts and kidneys of dtg mice. however, our study provides limited information since only mrna level were analyzed to characterize global gene expression in response to hace2 overexpression. furthermore, protein expression analyses and molecular mechanism studies are necessary to conform the effects of identified genes in future study. taken together, we produced rtta/hace2 dtg mice that overexpress the hace2 gene based on a doxinducible system and analyzed the changes in global gene expression in heart and kidney tissue using a microarray. the results of the present study suggest that hace2 protein was successfully expressed in the heart, kidney, and brain of dtg mice after dox treatment. moreover, our microarray analysis was able to identify several functional groups of genes and individual genes that respond to hace2 overexpression in the hearts and kidneys of tg mice. however, additional work should address the extent to which these changes are correlated with hace2 expression levels to determine whether beneficial effects can be obtained by reducing expression levels or if the increased expression of ace2 at any level is deleterious with respect to cardiac and renal disease. furthermore, it will be necessary to study the functions of differentially expressed genes, which could represent targets for the development of novel drugs, using pharmacoproteomics. supplementary information accompanies this paper at https://doi.org/10. 1186/s42826-020-00056-y. additional file 1. additional file 3. abbreviations ace2: angiotensin-converting enzyme 2; mhc: myosin heavy chain; rtta: reverse tetracycline-controlled transactivator; tetr: tetracyclineresistance operon; tre: tet response element a novel angiotensin-converting enzyme-related carboxypeptidase (ace2) converts angiotensin i to angiotensin 1-9 a human homolog of angiotensin-converting enzyme. cloning and functional expression as a captopril-insensitive carboxypeptidase counter regulatory actions of angiotensin-(1-7) angiotension-(1-7) and antihypertensive mechanisms the renin-angiotensin system and diabetes: an update. vasc health risk manag prevention of angiotensin ii-induced cardiac remodeling by angiotensin differential expression of neuronal ace2 in transgenic mice with overexpression of the brain renin-angiotensin system angiotensin ii at1 receptors regulate ace2 and angiotensin-(1-7) expression in the aorta of spontaneously hypertensive rats tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis distribution of angiotensin-(1-7) and ace2 in human placentas of normal and pathological pregnancies age-and gender-related difference of ace2 expression in rat lung myocardial infarction increases ace2 expression in rat and humans ace2 gene expression is upregulated in the human failing heart organ-specific distribution of ace2 mrna and correlating peptidase activity in rodents characterization of renal angiotensin-converting enzyme 2 in diabetic nephropathy glomerular localization and expression of angiotensin-converting enzyme 2 and angiotensinconverting enzyme: implications for albuminuria in diabetes angiotensin metabolism in renal proximal tubules, urine, and serum of sheep: evidence for ace2-dependent processing of angiotensin ii ace and ace2: their role to balance the expression of angiotensin ii and angiotensin temporal-spatial expression of ang-(1-7) and angiotensin-converting enzyme 2 in the kidney of normal and hypertensive pregnant rats the role of ace2 in pulmonary diseases--relevance for the nephrologist pressor and reflex sensitivity is altered in spontaneously hypertensive rats treated with angiotensin exploring the structure and function of zinc metallopeptidases: old enzymes and new discoveries a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung injury angiotensinconverting enzyme 2 is a functional receptor for the sars coronavirus angiotensin-converting enzyme 2 is an essential regulator of heart function molecular characterization of two myosin heavy chain genes expressed in the adult heart tissuespecific regulation of the alpha-myosin heavy chain gene promoter in transgenic mice tight control of gene expression in mammalian cells by tetracycline-responsive promoters transcriptional activation by tetracyclines in mammalian cells tetracycline-controlled transcriptional regulation systems: advances and application in transgenic animal modeling over-expression of angiotensin converting enzyme-1 augments cardiac hypertrophy in transgenic rats heart block, ventricular tachycardia, and sudden death in ace2 transgenic mice with downregulated connexins exploring the sequence space for tetracycline-dependent transcriptional activators: novel mutations yield expanded range and sensitivity conditional control of gene expression in the mouse tools for targeted manipulation of the mouse genome tetracycline-inducible expression systems: new strategies and practices in the transgenic mouse modeling generation of cells expressing improved doxycycline-regulated reverse transcriptional transactivator rtta2s-m2 doxycycline-mediated quantitative and tissue-specific control of gene expression in transgenic mice generation of the regulatory protein rtta transgenic mice vascular protective effects of angiotensin converting enzyme inhibitors and their relation to clinical events endothelial dysfunction in cardiovascular diseases: the role of oxidant stress liver fibrosis: a balance of aces? emerging evidence for a functional angiotensin-converting enzyme 2-angiotensin-(1-7)-mas receptor axis: more than regulation of blood pressure? angiotensin-(1-7) and the renin-angiotensin system the therapeutic potential of angiotensin-(1-7) as a novel renin-angiotensin system mediator possibility of formation of the s100a8/a9-proinflammatory cytokine complexes in vivo in acute inflammation and their functional roles molecular basis of the complex formation between the two calcium-binding proteins s100a8 (mrp8) and s100a9 (mrp14) modified atherogenic lipoproteins induce expression of pentraxin-3 by human vascular smooth muscle cells cardioprotective function of the long pentraxin ptx3 in acute myocardial infarction publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank the animal technician, seon m. choi, and mee k. jang for directing the animal care and use at the division of laboratory animal resources, njfds at korea. availability of data and materials available. the authors declare that there are no financial conflicts of interest with respect to the publication of these results. key: cord-003315-r1wkx0ml authors: jacobs, sophie; wavreil, fanny; schepens, bert; gad, hans henrik; hartmann, rune; rocha-pereira, joana; neyts, johan; saelens, xavier; michiels, thomas title: species specificity of type iii interferon activity and development of a sensitive luciferase-based bioassay for quantitation of mouse interferon-λ date: 2018-11-01 journal: j interferon cytokine res doi: 10.1089/jir.2018.0066 sha: doc_id: 3315 cord_uid: r1wkx0ml the type iii interferon (ifn-λ) family includes 4 ifn-λ subtypes in man. in the mouse, only the genes coding for ifn-λ2 and -λ3 are present. unlike mouse and human type i ifns (ifn-α/β), which exhibit strong species specificity, type iii ifns were reported to act in a cross-specific manner. we reexamined the cross-specificity and observed that mouse and human ifn-λ exhibit some species specificity, although much less than type i ifns. mouse ifn-λ3 displayed clear species specificity, being 25-fold less active in human cells than the closely related mouse ifn-λ2. this specificity likely depends on amino acids in α helices a and f that diverged from other ifn-λ sequences. human ifn-λ4, in contrast, retained high activity in mouse cells. we next developed a firefly luciferase-based reporter cell line, named fawa-λ-luc, to detect ifn-λ in biological fluids with high specificity and sensitivity. fawa-λ-luc cells, derived from mouse epithelial cells that are responsive to ifn-λ, were made nonresponsive to type i ifns by inactivation of the ifnar2 gene and strongly responsive to ifn-λ by overexpression of the mouse ifnlr1. this bioassay was as sensitive as a commercially available enzyme-linked immunosorbent assay in detecting mouse ifn-λ in cell culture supernatant, as well as in serum and bronchoalveolar lavage samples of virus-infected mice. the assay also enabled the sensitive detection of human ifn-λ activity, including that of the divergent ifn-λ4 with a bias, however, due to variable activity of ifn-λ subtypes. t ype i and iii interferons (ifns) are typically produced in response to viral infection, and these cytokines induce an antiviral state in target cells (isaacs and lindenmann 1957; kotenko and others 2003; sheppard and others 2003) . type i ifns consist of 13 ifn-a subtypes and ifn-b, -o (human) , and -z (mouse) -e and -k. the type iii family of ifns in humans comprises 3 functional genes that express ifn-l1 (il-29), ifn-l2 (il-28a), and ifn-l3 (il-28b) (kotenko and others 2003; sheppard and others 2003) . a fourth ifn-l subtype (ifn-l4) is present in a part of the human population, depending on a dinucleotide frameshift polymorphism upstream of the ifnl3 gene, which creates or disrupts the open reading frame (orf) encoding ifn-l4 (prokunina-olsson and others 2013) . in the mouse, ifnl1 is a pseudogene, and only ifn-l2 and ifn-l3 are expressed. unlike mouse and human type i ifns that exhibit strongly species-specific activity (veomett and veomett 1979) , type iii ifns were reported to act on cell types from both origins (lasfar and others 2006; hermant and others 2014) . the type i ifn family members signal through a unique heterodimeric receptor (ifnar), composed of the ifnar1 and ifnar2 subunits. type iii ifns engage a distinct receptor (ifnlr), composed of 2 chains: the ifn-l-specific ifnlr1, and il10rb which is shared by other il10-related cytokines (kotenko and others 2003; sheppard and others 2003) . while ifnar is ubiquitously expressed, ifnlr is expressed by a restricted range of cell types and mostly acts at mucosal surfaces. epithelial cells are well-established targets of ifn-l in vivo (sommereyns and others 2008) , although some immune cells such as neutrophils and dendritic cells have been characterized as ifn-l responders (koltsida and others 2011; blazek and others 2015; broggi and others 2017; espinosa and others 2017) . although type i and type iii ifns act on distinct receptors, they activate a similar jak-stat transduction pathway, leading to the phosphorylation of stat1 and stat2 that associate with irf9 to form the isgf3 complex. type i and type iii ifn signaling leads to the transcription of an overlapping set of ifn-stimulated genes (isgs) (dumoutier and others 2004; ank and others 2006) . type i ifn in biological samples can be measured by a variety of bioassays, but efficient techniques for type iii ifn quantification are largely lacking. enzyme-linked immunosorbent assay (elisa) for ifn-l detection is time and cost intensive and fails to detect ifn-l4. conventional cytopathic effect reduction bioassays to quantify antiviral activity as a proxy for the presence of ifn require the manipulation of infectious virus. luciferase reporter cells that have previously been used for recombinant human ifn-l detection were still responsive to type i ifn and would not allow specific ifn-l detection from biological samples (uze and monneron 2007) . in this work, we reexamined the species specificity of ifn-l activity, and we developed a very sensitive luciferase-based bioassay specific for type iii ifn detection. the lkr10 cell line (kind gift from guido bommer, de duve institute, brussels, belgium) is derived from lung adenocarcinoma tissues from a k-rasla1 mouse ( johnson and others 2001) . a549 cells (atcc) were kindly provided by pierre coulie, balb/3t3 fibroblasts (aaronson and todaro 1968) those cells, and derivatives, were maintained in dulbecco's modified eagle's medium (dmem) (lonza, vervier, belgium) containing 4.5 g/l glucose, supplemented with 10% fetal calf serum (fcs) (sigma-aldrich, overijse, belgium). african green monkey kidney (vero) cells (atcc) were cultured in dmem supplemented with 10% fcs, nonessential amino-acid, l-glutamine, and sodium pyruvate. bhk-21 cells (atcc) were cultured in glasgow's minimum essential medium (gibco; thermo fisher scientific, asse, belgium) supplemented with 10% newborn calf serum and 2.95 g/l tryptose phosphate broth. all media were supplemented with 50 u/ml penicillin and 50 mg/ml streptomycin (lonza). expression vectors used in this study are listed in table 1 . plasmids psj1 and psj7 are pcdna3 derivatives (invitrogen; thermo fisher scientific) used for mouse ifn-l2 and ifn-l3 expression in 293t cells. these plasmids were constructed by cloning the orf encoding ifn-l2 (psj1) and between the bamhi and xbai sites of the vector. coding sequences were polymerase chain reaction (pcr)-amplified from liver cdna samples of a c57bl/6 mouse infected with influenza turh (h7/n1) strain (hermant and others 2014) , using the following primers: forward (fw): bamhi-agei-kozak-mifn-l2/3, 5¢-aaa agg atc cac cgg tgc cac cat gct cct cct gct gtt gcc tct g-3¢; reverse (rev): mifn-l2/3-stop-xbai, 5¢-aaa atc tag att atc aga cac act ggt ctc cay tgg c-3¢. the sequence of psj7 matches the genomic ifnl3 sequence but diverges from a few nucleotides from that of the previously constructed pcs59 plasmid (sommereyns and others 2008) . both plasmids encode functional ifn-l3. pph23 was obtained by pcr-cloning the ifna2 orf from hela-m cell cdna, between the ecori and noti sites of pcdna3 (fw: ecori-kozak-ifna2, 5¢-aaa aga att cac cat ggc ctt gac ctt tgc ttt-3¢; rev: ifna2noti, 5¢-aaa agc ggc cgc tca ttc ctt act tct taa act tt-3¢). pmk03 is a lentiviral vector, derived from ptm897, that carries the mouse mx1 gene promoter driving the expression of the firefly luciferase gene. it is derived from pcclsin. cppt.hpgk.gfp.pre (follenzi and others 2000) in which a cloning polysite was first inserted to replace the coding sequences, yielding ptm897. the firefly luciferase gene from pgl4.16 (promega) was inserted between the xhoi and xbai sites of the vector, and the mx1 promoter was then cloned from pbsk-mx1, a gift from peter staeheli (freiburg university, freiburg, germany), as a bamhi/bsabi fragment. the lentiviral vector psj12, used for expression of the mouse ifn-l receptor, was obtained by cloning the ifnlr1 orf between the bamhi and xbai sites of ptm945, a lentiviral vector allowing the coexpression of the cloned gene and of mcherry (hermant and others 2013) . the ifnlr1 orf sequence was subcloned in this plasmid from pcr2.1-licr2, kindly provided by laure dumoutier (de duve institute, brussels, belgium). gene inactivation was done with px461 plasmid (pspcas9n-2a-gfp) coding for the cas9 nickase and green fluorescent protein (gfp) (ran and others 2013a). the 2 single guide rnas (sgrnas) were designed using the mit clustered regularly interspaced short palindromic repeats (crispr) design tool website (http://crispr.mit.edu). the selected sgrna pair (sgrna1-fw: 5¢-cac cgt caa att ctg gcg gct caa g-3¢; rev: 5¢-aaa cct tga gcc gcc aga att tga c-3¢; sgrna2-fw: 5¢-cac cga gac cac ata aac gtg acg a-3¢; rev: 5¢-aaa ctc gtc acg ttt atg tgg tct c-3¢) was targeting exon 6 of the mouse ifnar2 gene (genbank: y09865.1) and exhibited no expected off-target cleavage site. the sgrnas were cloned into px461 to form the plasmids psj37 and psj38. these constructs were cotransfected in lkr10 cells, using transit ò -lt1 transfection reagent (mirus bio llc, madison), according to the manufacturer's instructions. after 48 h, gfp-positive cells were sorted by fluorescence activated cell sorting (facs aria iii; bd biosciences) and cloned in 96-well plates. clones were screened for loss of type i ifn response with an antiviral assay. genome editing of the targeted exon was further confirmed by sequencing (genewiz). isg expression was measured by reverse transcriptionquantitative pcr. ifnar2-knockout (ko) and wild-type (wt) lkr10 cells were treated with 100 u/ml mouse ifn-aa, 700 pg/ml mouse ifn-l3, or control supernatant (mock) for 8 h before rna extraction. total rna extraction, reverse transcription, and sybr green quantitative pcr for mrna encoding mouse b-actin, oasl2, and usp18 were performed as previously described (paul and michiels 2006) . for infection analysis, cells were dissociated with trypsin-edta and suspended in phosphate-buffered saline (pbs) containing 5% of filtered fcs and 0.5% of paraformalde-hyde. data acquisition was done with an lsrfortessa flow cytometer (bd biosciences) using facsdiva software. data were analyzed using flowjo 9.6.4. the rate of infection was defined as the percentage of mcherry-positive cells 24 h postinfection with 0.5 pfu/cell tm967. for cell sorting, transduced cells were suspended in pbs containing 1% fcs and 1 mm edta. mcherry-or gfppositive cells were cloned at 1 cell per well in 96-well plates using the facs aria iii (bd biosciences). immunoblotting stat1, p-stat1, and b-actin were detected by western blot using sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels containing 10% acrylamide and run in a tris-glycine buffer. blots were probed with anti-stat1 polyclonal (sc-346; santa cruz biotechnology, heidelberg, germany), p-stat1 monoclonal (#9167; cell signaling technology, leiden, the netherlands), and anti-b-actin monoclonal (a5441; sigma-aldrich) antibodies. mouse ifn-aa, ifn-b, ifn-l2, ifn-l3, and human ifn-a2 were produced as described previously from 293t cells transfected with pcdna3-ifn-aa, pcdna3-ifn-b (van pesch and others 2004), pcdna3-ifn-l2 (psj1), and pcdna3-ifn-l3 (psj7). human ifn-a2 was produced similarly, using pcdna3-ifn-a2 (pph23). supernatant collected from 293t cells transfected in parallel with the empty pcdna3 vector was used for control treatment (mock) of cells and diluted as for ifns. recombinant mouse ifn-l3 (1789-ml-025) and human ifn-l1 (1598-il-025), ifn-l2 (1587-il-025), and ifn-l3 (5259-il-025) were purchased from r&d (r&d systems, minneapolis). recombinant human ifn-l4 was produced as described (hamming and others 2013) . mouse (ref 315-05) and human (ref 300-02) ifn-g were purchased from pe-protech (london, united kingdom). genbank accession numbers for mouse and human ifn-l are listed in table 2 . ifns were diluted in culture medium for cell treatment or in reagent diluent for elisa. mouse ifn-l2 and ifn-l3 were quantified by elisa. mouse ifn-aa and human ifn-a2 antiviral activities were quantified, as described previously, by a cytopathic effect reduction assay in mouse balb-3t3 and human hela-m cells, respectively, using mengovirus (van pesch and michiels 2003) . similar assays were conducted in human a549 cells and mouse lkr10 cells for quantification of ifn-l cross-species activity. ifnl4 was quantified by a cytopathic effect reduction assay in a549 cells, using recombinant human ifn-l3 as a standard, which was reported to have a similar specific activity (hamming and others 2013). mouse ifn-l2/3 measurement by elisa was performed using the mouse ifn-l2/3 duoset (r&d systems), according to the manufacturer's protocol. mouse sera were diluted 5-to 10-fold, and bronchoalveolar lavage fluids (balfs) were diluted 5-fold. when required (in case of samples derived from infected cells or mice), infectious virus present in biological samples was ultraviolet light (uv)-inactivated. for uv inactivation, 2.5 mm thick fluid samples were exposed at fixed uv dose (0.25-0.5-1-2 j/cm 2 ) with the uv irradiation system biolink 254 (vilber lourmat, eberhardzell, germany), either in 24-(470 ml) or in 96-well (80 ml) plate. uv-exposed samples were titrated on bhk-21 cells by a standard plaque assay to confirm virus inactivation. to confirm that the uv irradiation procedure that was established for picornaviruses effectively inactivates respiratory syncytial virus (rsv), 3 samples of 470 ml containing 1.5 · 10 6 pfu of rsv were exposed to 2 j/cm 2 with the uv irradiation system bio-link 254 at room temperature in a 24-well plate (uv irradiated samples). as controls 3 similar samples of 470 ml containing 1.5 · 10 6 pfu of rsv were incubated at room temperature in a 24-well plate (untreated samples). the titers of replicating rsv in the uv irradiated and untreated samples were determined by plaque assay using vero cells. cells were seeded at 160,000 cells per well in 24-well plates for cell supernatant and recombinant ifn analysis or at 32,000 cells per well in 96-well plates for mouse serum and bal analysis. forty-eight hours after seeding, cells were treated with ifn-l2/3 or mock-treated or incubated with uv-treated mouse serum (50-and 100-fold dilutions) or bal (5-fold dilutions). samples were diluted in culture medium. recombinant mouse ifn-l3 provided in the elisa kit was used as a standard. firefly luciferase activity was measured 6 h after treatment using the luciferase assay system (promega). twenty microliters and 100-ml lysis buffer were used in 96-and 24-well plates, respectively; 10 ml of the lysate was mixed with 25 ml of substrate for detection. luminescence was measured with a glomax 20/20 luminometer (promega). the limit of detection (lod) for each test was established based on the mean of mock-treated samples, plus 3 standard deviations, in all assays. tm967 is a theiler's murine encephalomyelitis virus (tmev) (strain da) derivative that carries a capsid adapted to infect l929 cells and the orf encoding the mcherry fluorescent protein cloned as a xbai/bsiwi fragment to replace codons 5-67 of the leader protein coding region, in the pkj6 vector ( jnaoui and michiels 1998) . mengovirus (a strain of encephalomyocarditis virus) used for ifn bio-assay is an attenuated variant that has been described previously (hermant and others 2013) . those viruses were quantified by plaque assay on bhk21 cells. rsv propagation and enrichment were performed as described in schepens and others (schepens and others 2014) . mouse experiments were conducted according to the national (belgian law 14/08/1986 and 22/12/2003, belgian royal decree 06/04/2010) and european (eu directives 2010/ 63/eu, 86/609/eeg) animal regulations. animal protocols were approved by the ethics committee of ghent university (permit no. la1400091, approval id 2013-030). all efforts were made to avoid or ameliorate suffering of animals. specific pathogen-free female balb/c mice at the age of 7-8 weeks were purchased from charles river. the mice were housed in a specific-pathogen-free temperature controlled environment with 12-h light/12-h dark cycles and given water and food ad libitum. mice were used at 9 weeks of age after adaptation in the animal room for 1 week. for challenge, the mice were sedated with isoflurane and infected intranasally with 4 · 10 6 pfu of human rsv. mock infection of the control group was performed with hank's balanced salt solution. five days postinfection, bal was isolated under anesthesia with an intraperitoneal injection of avertin (2.5% in pbs). a 23gauge cannula was inserted into the trachea, and cells were collected by washing the airway lumen twice with 0.5 ml pbs containing 0.05 mm edta. after removing the cells by centrifugation (5 min at 400g), the balf was stored at -80°c. serum samples from norovirus-infected and plasmidelectroinjected mice were reused from a previously published experiment (rocha-pereira and others 2018) to minimize animal experimentation. statistical analysis was performed using prism 7 software (graphpad software, san diego, ca). [***p < 0.001, **p < 0.01, *p < 0.05, and ns no statistically significant difference (p ‡ 0.05)]. unlike mouse and human type i ifns (ifn-a/b) that exhibit strong species specificity, type iii ifns were reported to act in a cross-species manner. to confirm whether a mouse cellbased bioassay would permit the detection of ifn-l subtypes from 2 different mammalian species, the species specificity of mouse and human type iii ifns was reexamined. crossreactive antiviral activity was systematically compared in a cytopathic effect reduction assay, using epithelial cell lines from mouse (lkr10) and human (a549) origin. as expected, type i human (ifn-a2) and mouse (ifn-aa) ifns were extremely species-specific in the antiviral assay, with an activity difference higher than 10,000-fold when applied to mouse and human cells. ifn-l exhibited some level of species specificity although much less than type i ifns (fig. 1a) . mouse ifn-l3 displayed the most pronounced species specificity. for a quantity that yielded the same antiviral activity in mouse cells, ifn-l3 was 25 times less active in human cells than the closely related mouse ifn-l2. human ifn-l1, ifnl-2 and, to a lesser extent, ifnl-3 exhibited significant antiviral effect on mouse epithelial cells. as ifn-l4 has been shown to have a specific activity similar to that of human ifn-l3 in human cells, it was quantified accordingly using the antiviral assay (hamming and others 2013) . remarkably, human ifn-l4 displayed much more antiviral activity on mouse cells than equivalent amounts of human ifn-l3. accordingly, treatment of lkr10 cells with concentrations of human ifn-l3 and ifn-l4 that yielded equivalent antiviral activities in human cells resulted in clear stat1 phosphorylation after ifn-l4 but not after ifn-l3 treatment (fig. 1b) . in line with their epithelial origin, mouse lkr10 cells respond to ifn-l. to ensure a selective detection of type iii ifns, the gene coding for the ifnar2 subunit of the type i ifn receptor was inactivated using the double nickase crispr/crispr-associated (cas) 9 technology (ran and others 2013b). an lkr10-ifnar2-ko clone was selected for the absence of response to type i ifn and intact response to type iii ifn. sequencing of the targeted exon revealed frameshift mutations in both ifnar2 alleles, resulting in premature stop codon appearance. in contrast to ifn-l treatment, ifn-a treatment of this clone failed to induce the expression of the ifn-stimulated genes, oasl2 ( fig. 2a) and usp18 (fig. 2b) , and to protect against infection with tm967, a tmev derivative expressing mcherry (fig. 2c) . to develop ifn-l reporter cells, the lkr10-ifnar2-ko clone was transduced with a lentiviral vector encoding the firefly luciferase gene under the control of the mx1 pro-moter. among the clones tested for luciferase activity induction after ifn-l treatment, the best one displayed not more than 10-fold luciferase activity induction. to try to boost the reporter gene responsiveness to ifn-l, this clone was transduced with sj12, a lentiviral vector that coexpresses the mouse ifnlr1 and mcherry. transduced cells were sorted, based on mcherry expression, and cloned. a selected clone, named ''fawa-l-luc,'' responded vigorously to ifn-l3. this clone displayed increased constitutive stat1 expression and strong stat1 phosphorylation after ifn-l3 but not after ifn-a treatment (fig. 2d) . a significant luciferase activity increase was measured in fawa-lluc cells as soon as 1 h after treatment with 700 pg/ml (quantified by elisa) of mouse ifn-l3. maximal induction was reached within 6 h and lasted up to 16 h posttreatment (fig. 3a) . the sensitivity of the fawa-l-luc assay was compared to that of an available elisa test for mouse ifn-l. mouse ifn-l2 and -3 produced by transfected 293t cells and recombinant mouse ifn-l3 were quantified in parallel by elisa and the fawa-l-luc assay. in the elisa, responses were linear between 1,000 and 16.25 pg/ml, the experimental lod of this test (fig. 3b ). in the fawa-l-luc assay, linearity was reached in a narrower concentration range (1.95-62.5 pg/ml for mouse ifn-l2; 3.9-125 pg/ml for mouse ifn-l3), but a higher sensitivity was observed with the detection limit being below 1.95 pg/ml and 3.9 pg/ml, for ifn-l2 and -3, respectively (fig. 3c, d) . based on elisa quantification, mouse ifn-l2 bioactivity in this assay was 2-fold higher than that of mouse ifn-l3. importantly, after up to 29 passages, the intensity of the luciferase signal in the fawa-l-luc cells tended to decrease, but the sensitivity of the bioassay was preserved because table showing the relative antiviral activity of mouse and human ifn-l on mouse lkr10 and human a549 cells. species specificity index was calculated as the ratio between the relative antiviral activity on cells of homologous to nonhomologous species (eg, for mouse ifn: relative activity in mouse cells/relative activity in human cells). relative activities in a given cell line were calculated as the ifn dilutions (starting from 1 ng/ml) that yielded similar antiviral activities. *due to the low amount of ifn-l4 available, the initial concentration of this ifn was estimated by comparison with human ifn-l3 antiviral activity that was reported to have a similar specific activity. (b) western blot showing stat1 phosphorylation in mouse lkr10 cells 30 min after treatment with control medium (mock) or ifn-l. concentrations of human (hu) ifn-l3 (615 pg/ml) and ifn-l4 that yielded the same antiviral activity on human a549 cells were used. as a control, mouse ifn-l3 was used at a concentration (2.5 ng/ml) showing equivalent antiviral activity as human ifn-l4 on mouse cells. results are representative of 3 experiments. the background activity also decreased with high passage numbers. to confirm the specificity of fawa-l-luc cells for ifn-l, we also evaluated any possible luciferase induction after treatment with type i and type ii (ie, ifn-g) ifns. as expected, no signal was observed after treatment with up to 2,500 iu/ml of either mouse ifn-aa or ifn-b. human ifn-g also failed to induce a detectable luminescent signal in the fawa-l-luc cells in the tested concentration range (0-25 ng/ml). the lod for mouse ifn-g was 12.55 ng/ml, a concentration at least 10-fold higher than what is observed in mouse fluids in infectious contexts (pomeroy and others 1998; claser and others 2017) . ifn-g is thus not expected to interfere with ifn-l detection in biological samples. we tested the sensitivity of our bioassay for human ifn-l detection. responses were analyzed individually for all 4 human ifn-l subtypes (fig. 4a-d) . as their cross-species activity differed, different detection sensitivities were reached with our mouse cell-based reporter assay: 15.125 pg/ml for ifn-l1, 500 pg/ml for ifn-l2, 31.25 pg/ml for ifn-l3, and 1.95 pg/ml for ifn-l4. the bioassay turned out to be extremely sensitive for human ifn-l4 detection. it is, however, worth noting that the recombinant ifn-l4 stock concentration was determined by comparison of its activity with that of a human ifn-l3 standard. when testing biological samples, potential occurrence of infectious virus may interfere with the viability of reporter cells. it is therefore important to inactivate infectious viruses in such samples. as ifn-l had previously been shown to be acid labile (kugel and others 2011) , uv inactivation of the virus was preferred. we thus tested whether virucidal uv doses would affect ifn-l activity. to this end, tm967 virus, mouse ifn-l2, and mouse ifn-l3 were irradiated with increasing uv doses. mock-and uv-exposed samples were then analyzed with the bioassay for mouse ifn-l2 and ifn-l3 detection and by plaque assay to determine viral titers. a 2 j/cm 2 uv exposure was sufficient to ensure a virus titer reduction of more than 100,000-fold (5.5 · 10 5 pfu to <1 pfu). at this uv dose, mouse ifn-l2 activity was not significantly reduced and mouse ifn-l3 showed a bioactivity reduction of less than 25% (fig. 5a) . similarly, ifn-l1 and ifn-l4 bioactivities were reduced by 25%, while human ifn-l2 and ifnl-3 were slightly more sensitive to irradiation, with about 2-fold activity decrease (fig. 5b) . we then compared the sensitivities of the fawa-l-luc and of the elisa assays to detect ifn-l in mouse serum and bal samples. ifn-l was first quantified in the serum of mice that were either injected intramuscularly with an ifn-l3 expressing plasmid (pcs59), thus expected to produce circulating ifn-l, or infected with mouse norovirus (rocha-pereira and others 2018). a preliminary test revealed that the minimal serum dilution that did not interfere with spiked-in ifn-l detection was 16fold for the bioassay and 5-fold for the elisa (fig. 5c, d) with both the fawa-l-luc and elisa assays. ifn-l was clearly detected at day 2 and 4 in the serum of mice that were electroinjected with the ifn-l3 expressing plasmid, as well as at day 3 postnorovirus infection (fig. 6a, b) . at day 2 after a single ifn-l3 injection, the cytokine was detectable in 1 out of 4 mice by elisa and in 3 out of 4 mice by the bioassay. the detection limit was higher for the elisa (194 pg/ml) than for the bioassay (9.29 pg/ml), showing the higher sensitivity of the luciferase-based bioassay (fig. 6a, b) . next, we compared ifn-l detection by the fawa-l-luc assay and by elisa in balf of balb/c mice that were mock infected or infected with rsv. balf and lungs were collected 5 days postinfection. plaque assay revealed that balf of all infected mice contained between 1.8 · 10 2 and 7.33 · 10 2 pfu/ ml of live replicating virus. the used uv irradiation procedure proved to readily inactivate rsv (rsv titer reduction of more than 46,000-fold; 4.6 · 10 4 pfu to <1 pfu). five-fold diluted control balf did not modify ifn-l2/3 detection in any of the 2 assays. using 5-fold dilutions of the balf as in the bioassay, we failed to detect any ifn-l by elisa (fig. 6c ). in contrast (fig. 6d) , ifn-l was detected by fawal-luc assay at a very low concentration in the balf of the rsv infected mice (5/6) and not detected in the control group. percentage of ifn-l activity (mean and sd) remaining after uv treatment (n = 3) at 2 j/cm 2 . ifn activity was measured in fawa-l-luc cells for ifn concentrations that yielded equivalent luciferase activities (20,000 rlu) before uv treatment (250 pg/ml mifn-l2 and mifn-l3, 125 pg/ml human ifn-l1, 10 ng/ml huifn-l2, 500 pg/ml huifn-l3, and 62.5 pg/ml huifn-l4). (c, d) influence of serum dilution on ifn-l detection by fawa-l-luc cells (c) and elisa (d). (c) fawa-l-luc cells were treated in triplicate with fixed doses of 15 and 250 pg/ml mifn-l2 supernatant and with 2-fold serial dilutions (2-128-fold) of control mouse serum. (d) 125 pg/ml recombinant mifn-l3 was mixed with 2-fold serial dilutions of control mouse serum (2.5-10-fold) before detection by elisa. (a-c) reporter cells were exposed to ifn for 6 h before luciferase assay. student's t-test: */**/***denotes a significant difference in signal compared to no uv exposure (a) or the absence of serum (c). uv, ultraviolet light. we conclude that the fawa-l-luc-based assay described in this study allows to quantify ifn-l from biological samples in a highly sensitive way. we reexamined the species specificity of ifn-l and highlighted a previously overlooked difference in bioactivity between mouse and human type iii ifns. mouse ifn-l3 displayed a surprising species specificity, with a 50-fold difference in relative antiviral activity when applied to lkr10 (mouse) and a549 (human) cells. this contrasted strongly with the closely related mouse ifn-l2 (93% amino acid sequence identity), which displayed little species specificity and was 25 times more active in human cells than mouse ifn-l3. in contrast, human ifn-l4 displayed a strikingly strong activity on mouse cells compared to human ifn-l3. although ifn-l4 is not expressed in mouse and rat, it is highly conserved in many mammalian species (key and others 2014) where it was shown to be cross-reactive. the nonhuman ifn-l4 orthologs were reported to activate ifnlr1 in human cells, sometimes with a higher efficacy than human ifn-l4 (eg, dog ifn-l4) (paquin and others 2016). the mouse cell response to human ifn-l4 is thus in line with the ability of ifn-l4 to be cross-reactive among mammalian species, although we do not have any physiological explanation for this phenomenon. ifn-l proteins follow the typical class ii cytokine structure, consisting of 6 secondary structure elements (a-f) (pestka and others 2004) . helix a is involved in both ifnlr1 and il10rb binding, while helix d binds il10rb and helix f binds ifnlr1 (gad and others 2010). among amino acid residues that have been characterized as crucial for ifnlr1 binding and activation (gad and others 2010), a few divergences are observed between sequences from mouse and human ifn-l (fig. 7) . notably, the more species-specific mouse ifn-l3 has 4 amino acid residues in a helices a and f that diverge from the 5 other ifn-l sequences, including the related mouse ifn-l2. in helix a, gly37 uniquely replaces the asp residue present in the other sequences. in helix f, 3 residues are unique to mouse ifn-l3: asp 150 (ala / asp), gln158 and bioassay (b) in the serum of ag129 mice 2 or 4 days after electroinjection of mouse ifn-l3 (mifn-l3) expressing (pcs59) or empty (pcdna3) plasmids, 2 days after injection of pcs59, or 3 days after infection with mouse norovirus. ifnl detection in the serum by elisa was performed without uv exposure to keep maximal sensitivity. (c, d) ifn-l2/3 detection by elisa (c) and bioassay (d) in the bronchoalveolar lavage of balb/c mice, 5 days postinfection with rsv, compared to control mice (mock). balf were uv-exposed before testing. (a-d) the horizontal dotted line represents the lod. mann-whitney: */**indicates a significant difference compared to pcdna3 group at days 2 or 4 (a, b) or compared to mock (d). balf, bronchoalveolar lavage fluid; rsv, respiratory syncytial virus. (arg / gln), and leu161 (thr / leu). future site-directed mutagenesis studies could help defining the key residues involved in the species specificity. we designed a highly sensitive bioassay named ''fawa-lluc'' based on luciferase reporter cells, specific for ifn-l detection and quantification. the bioassay is based on a cell line that is naturally responsive to ifn-l in which the type i ifn receptor gene was inactivated. overexpression of the mouse ifnlr1 receptor in those cells led to increased induction of the reporter gene after ifn-l treatment. unlike previously described bioassays, such as hl-116 fibroblasts stably transfected with the human ifnlr1 (uze and monneron 2007), fawa-l-luc cells permit the selective detection of type iii ifn because they lack the type i ifn receptor. they also fail to respond to ifn-g concentrations that are reached in infected mouse serum. this novel bioassay is efficient in measuring ifn-l from mouse bal and serum. ifn-l activity was hardly affected by uv exposure at virucidal doses, thus allowing detection in infected biological fluids. the high sensitivity of the assay permits sparing biological material as 10-fold more diluted serum samples could be used for bioassay detection (50-100-fold dilution) compared to the elisa (5-10-fold). when cell toxicity of the tested samples is suspected, cell viability may independently be confirmed with nonlytic viability assays such as resazurin-based tests, which are compatible with luciferase detection. importantly, our bioassay offers a physiologic analysis, attesting of the functionality of the ifn-l present in the samples, irrespective of specific activity. the bioassay also allows to detect human ifn-l. given the divergent cross-species activity and the higher uv lability of the human type iii ifns, the bioassay might be used for individual subtype analysis, but would not fit for their detection in complex biological samples. finally, it offers a sensitive detection of the divergent ifn-l4, for which no efficient commercial test is available. development of 3t3-like lines from balb-c mouse embryo cultures: transformation susceptibility to sv40 lambda interferon (ifn-lambda), a type iii ifn, is induced by viruses and ifns and displays potent antiviral activity against select virus infections in vivo ifn-lambda resolves inflammation via suppression of neutrophil infiltration and il-1beta production ifn-lambda suppresses intestinal inflammation by non-translational regulation of neutrophil function host resistance to plasmodiuminduced acute immune pathology is regulated by interleukin-10 receptor signaling basis for regulated rna cleavage by functional analysis of rnase l and ire1p role of the interleukin (il)-28 receptor tyrosine residues for antiviral and antiproliferative activity of il-29/interferon-lambda 1: similarities with type i interferon signaling type iii interferon is a critical regulator of innate antifungal immunity gene transfer by lentiviral vectors is limited by nuclear translocation and rescued by hiv-1 pol sequences the structure of human interferon lambda and what it has taught us interferon lambda 4 signals via the ifnlambda receptor to regulate antiviral activity against hcv and coronaviruses ifn-l sequence alignments. sequences of human and mouse ifn-l regions implicated in receptor binding were aligned. key amino acid residues involved in receptor activation that differs between mouse and human ifn-l2 and ifn-l3 sequences are indicated in bold in mouse sequences. residues unique to mouse ifn-l3 in helices a and f are indicated in bold red. *indicates identical amino acids between human ifn-l3 and ifn-l4 human but not mouse hepatocytes respond to interferon-lambda in vivo ifnepsilon is constitutively expressed by cells of the reproductive tract and is inefficiently secreted by fibroblasts and cell lines virus interference. i. the interferon adaptation of theiler's virus to l929 cells: mutations in the putative receptor binding site on the capsid map to neutralization sites and modulate viral persistence somatic activation of the k-ras oncogene causes early onset lung cancer in mice selection on a variant associated with improved viral clearance drives local, adaptive pseudogenization of interferon lambda 4 (ifnl4) il-28a (ifn-lambda2) modulates lung dc function to promote th1 immune skewing and suppress allergic airway disease ifn-lambdas mediate antiviral protection through a distinct class ii cytokine receptor complex novel nonviral bioassays for mouse type i and type iii interferon characterization of the mouse ifn-lambda ligand-receptor system: ifn-lambdas exhibit antitumor activity against b16 melanoma comparative functional analysis of 12 mammalian ifn-lambda4 orthologs cardiovirus leader proteins are functionally interchangeable and have evolved to adapt to virus replication fitness interferons, interferonlike cytokines, and their receptors role of interferon-gamma in murine cytomegalovirus infection a variant upstream of ifnl3 (il28b) creating a new interferon gene ifnl4 is associated with impaired clearance of hepatitis c virus double nicking by rna-guided crispr cas9 for enhanced genome editing specificity genome engineering using the crispr-cas9 system interferon lambda (ifn-lambda) efficiently blocks norovirus transmission in a mouse model protection and mechanism of action of a novel human respiratory syncytial virus vaccine candidate based on the extracellular domain of small hydrophobic protein il-28, il-29 and their class ii cytokine receptor il-28r ifnlambda (ifn-lambda) is expressed in a tissue-dependent fashion and primarily acts on epithelial cells in vivo il-28 and il-29: newcomers to the interferon family characterization of the murine alpha interferon gene family characterization of interferonalpha 13, a novel constitutive murine interferon-alpha subtype species specificity of interferon action: maintenance and establishment of the antiviral state in the presence of a heterospecific nucleus the authors are grateful to nicolas dauguet for his help in flow cytometry (ludwig institute for cancer research). work was supported by the eos joint programme of fonds de la recherche scientifique-fnrs and fonds wetenschapellijk onderzoek-vlaanderen-fwo (eos id: 30981113), by interuniversitary attraction poles program initiated by the belgian science policy office (iap-p7/45 belvir), by actions de recherche concertées (arc), and by a pdr grant (t.0185.14) of fnrs to t.m., and b.s. is a doctoral assistant at the department of biomedical molecular biology of ghent university. no competing financial interests exist. key: cord-299605-j1ewxk4q authors: lin, jing-wen; sodenkamp, jan; cunningham, deirdre; deroost, katrien; tshitenge, tshibuayi christine; mclaughlin, sarah; lamb, tracey j.; spencer-dene, bradley; hosking, caroline; ramesar, jai; janse, chris j.; graham, christine; o’garra, anne; langhorne, jean title: signatures of malaria-associated pathology revealed by high-resolution whole-blood transcriptomics in a rodent model of malaria date: 2017-02-03 journal: sci rep doi: 10.1038/srep41722 sha: doc_id: 299605 cord_uid: j1ewxk4q the influence of parasite genetic factors on immune responses and development of severe pathology of malaria is largely unknown. in this study, we performed genome-wide transcriptomic profiling of mouse whole blood during blood-stage infections of two strains of the rodent malaria parasite plasmodium chabaudi that differ in virulence. we identified several transcriptomic signatures associated with the virulent infection, including signatures for platelet aggregation, stronger and prolonged anemia and lung inflammation. the first two signatures were detected prior to pathology. the anemia signature indicated deregulation of host erythropoiesis, and the lung inflammation signature was linked to increased neutrophil infiltration, more cell death and greater parasite sequestration in the lungs. this comparative whole-blood transcriptomics profiling of virulent and avirulent malaria shows the validity of this approach to inform severity of the infection and provide insight into pathogenic mechanisms. scientific reports | 7:41722 | doi: 10.1038/srep41722 offers a feasible alternative as it is one of the "highways" of the immune system via which naïve, and activated or primed immune cells travel between lymphoid organs and the tissues affected by the infection. by profiling global transcriptomes of whole blood, insights can be obtained into the complex changes in systemic or even local host responses brought about by an infection, and thus inform more targeted mechanistic studies. to investigate the use of genome-wide transcriptomic profiling of the whole blood in identifying pathology signatures in malarial infection, and to gain insights into the mechanisms underlying pathology, we used the well-establised p. chabaudi chabaudi rodent malaria model to study malarial immunology and pathology 2,3 . using two strains of p. c. chabaudi, as and cb, that differ in virulence in c57bl/6 mice, we performed high-resolution comparative whole-blood transcriptomic analysis throughout the acute phase of the blood-stage infection, and identified several transcriptomic signatures associated with severe malarial pathology before the onset of pathology or disease. the virulent cb strain of p. c. chabaudi induces more severe pathology in the acute phase of a blood-stage infection compared with the avirulent as strain. infection of c57bl/6 mice was initiated by intraperitoneal inoculation of 10 5 infected red blood cells (irbc) of p. c. chabaudi as or cb. infection with the cb strain gave rise to a more severe infection, resulting in 40% (range 20-60%) of mice reaching the humane end points (more than 25% weight loss, persistent laboured breathing and severe hypothermia), while all as infected mice survived the infection without showing severe pathologies (fig. 1a) . as and cb infected mice showed comparable irbc loads (parasitemia multiplied by total rbc numbers), despite the fact that higher peak the total numbers of infected red blood cells (irbc) per ml of blood (b) and the parasitemia (percentage of infected irbc) (c) in the mice infected with as or cb parasites. (d,e) the change in rbc numbers during the infection in as or cb infected mice (d) and the hemoglobin (hgb) concentration in the blood of the infected mice (e). (f,g) the percentage change in temperature (f) and weight (g) during the infection in as or cb infected mice. data were pooled from 21 mice in 3 independent experiments. graphs show mean with sem, mann-whitney u test was performed (* p < 0.05, **p < 0.005, ***p < 0.0005). scientific reports | 7:41722 | doi: 10 .1038/srep41722 parasitemias were observed in the acute cb infection (fig. 1b,c) . a more severe rbc loss with a significantly lower hemoglobin concentration was observed in cb infection at 10 days post infection (dpi) compared to that in as infected mice, agreeing with previous observations, which showed more severe anemia in cb infected balb/c mice 4 . moreover, the rbc loss in cb infected mice is longer lasting, even after the peak of infection at 12 dpi (fig. 1d,e) . in addition, at 10 dpi, cb infected mice showed greater temperature and weight loss (fig. 1f,g) . virulent cb and avirulent as strains of p. c. chabaudi induce distinct responses in host whole blood transcriptome. to investigate whether as and cb parasites induce different host responses that might contribute to the differences in severity of the blood-stage infection we carried out genome-wide transcriptomic analyses of whole blood during the acute phase of infection. peripheral blood was collected into tempus tubes via cardiac puncture from c57bl/6 mice infected with as or cb at 2, 4, 6, 8, 10, and 12 dpi. blood samples collected from age-matched uninfected animals at day 0 and day 12 were used as naïve controls to exclude transcriptional changes due to time. the total rna was extracted, depleted of globin mrna and analysed using illumina mouse wg-6 v2.0 beadarrays. spearman's rank correlation coefficient (r s ) analysis of unfiltered transcripts normalised across the median of all samples, revealed high levels of similarity amongst naïve and 2, 4 dpi samples in both as and cb infections (fig. 2ai ,ii, r s ranging from 0.73 to 0.88), while from 6 dpi onwards the whole blood transcriptomes diverge significantly from the earlier time points (r s ranging from 0.08 to − 0.59 compared to naïve controls). when comparing as and cb infections at each of the time points, lower correlation values were observed between 6-10 dpi (fig. 2aiii , r s = 0.37, 0.67, 0.44, respectively). this indicates that as and cb infections induce different host responses at these times of infection. as infected mice and those cb infected mice that survived this phase of infection showed similar profiles at 12 dpi (fig. 2aiii , r s = 0.82). differential expression analysis (anova unequal variance with post-hoc hsd test, fdr < 0.01) was performed on transcripts normalised to the median of their respective naïve controls (supplementary table 1 ). at any of the post-infection time points examined, there were 6226 differentially expressed transcripts with a greater than 2-fold change in the infected mice compared to the naïve mice (fig. 2b, supplementary fig. 1a) . consistent with correlation analyses of unfiltered transcripts (fig. 2a) , only a few transcripts were differentially expressed at 2 and 4 dpi (fig. 2b) . strikingly, at 6 dpi, a large majority of transcripts were down-regulated compared to naïve controls in both as and cb infections. however, the number was greater in cb infected mice compared to as (as 1856 out of 2265 transcripts, cb 4306 out of 4471 transcripts) (fig. 2b) . the number of down-regulated transcripts increased steadily from 6 to 12 dpi in as infection (fig. 2b) . while down-regulation of transcripts was also observed at 6-8 dpi in the cb infection, the level of down-regulation was significantly lower at 10 dpi and there was a sudden up-regulation of transcripts occurred only in cb infected mice at this time point (fig. 2b , supplementary fig. 1a ), a critical time point when infected animals were either recovering from acute infection or suffering from severe pathologies leading to death (fig. 1) , indicating that these up-regulated transcripts may relate to the manifestation of severe pathology in the cb infection. indeed this higher level of transcriptional up-regulation was further confirmed in 4 cb infected mice that had reached humane end points at 9 dpi ( supplementary fig. 1b ). this up-or down-regulation of gene expression in whole blood are due to both the leukocyte population changes and transcript regulation during the infection ( supplementary fig. 1c ). in the blood associated with severe pathology. a hierarchical clustering of the 6226 differentially expressed transcripts of whole blood revealed 2 clusters of transcripts comprised of 22 genes that were significantly more up-regulated in cb infected mice between 8 and 10 dpi than in the blood of as infected mice ( supplementary fig. 2 , 22 genes in fig. 3a) , during which severe clinical signs were manifested (severe hypothermia, weight loss and host death) (fig. 1 ). ingenuity pathway analysis (ipa) diseases and function analysis showed that some of these genes were significantly enriched in 'functions' of inflammation and myeloid cell movement (fig. 3b) . interestingly, 5 genes (fig. 3a , indicated in red) were enriched in 'disease' of severe acute respiratory syndrome (sars) (fig. 3b) . these 5 genes have been shown to be amongst the top 10 up-regulated genes in transcriptomic analysis of pbmcs (peripheral blood mononuclear cells) isolated from sars patients suffering from severe lung inflammation 5 (fig. 3c) . importantly, in 4 of the cb infected mice that had reached humane end points at 9 dpi, these 5 genes showed an even higher level of up-regulation compared to naïve controls (fig. 3c) , indicating possible lung pathology in these 4 cb infected mice. to identify further pathology-related blood transcriptomic signatures, we included the microarray data of blood isolated from the 4 cb-infected mice that had reached the humane end points, and followed the same differential expression analysis as described above, yielding a total of 6321 differentially expressed transcripts (supplementary table 2 ). a self-organising map (som) clustering method was used to generate clusters of co-expressing transcripts ( supplementary fig. 3 , supplementary table 3) 6 . the cluster expression level was defined as the average fold-change of all transcripts within each module compared to that of naïve controls (fig. 3d) . the clusters were annotated with ipa diseases and function analysis and manually curated. several clusters, c8, c10, c13, c15, c16 and c20 showed greater up-regulation in cb infection during 9-10 dpi, indicating their association with lethality/severe pathology in cb infection (fig. 3d , indicated in red). modular analysis identified an early platelet aggregation signature only associated with the virulent p. c. chabaudi cb infection. clusters c15 and c20, were exclusively up-regulated in the cb infection, especially in the mice that had reached humane end points at 9 dpi (fig. 3d ). c15 showed a maximum average of 2.3-fold up-regulation and c20 showed a maximum average of 1.4-fold up-regulation in cb infected mice that were dying from the infection. twenty-seven genes within these 2 clusters are associated with platelet aggregation/pro-coagulation, a common feature of severe malaria infection 7 (fig. 4a) . they are significantly enriched in canonical pathways integrin signalling (−log(p value) 4.3, z score 2) and actin cytoskeleton signalling (−log (p value) 4.2, z score 2). importantly, this set of genes was significantly up-regulated in all cb infected mice as early as 2 dpi (fig. 4b) , at the time of which very few genes were differentially expressed compared to naïve controls (fig. 2b) . modular analysis identified a more pronounced and longer-lasting anemia signature in the virulent p. c. chabaudi cb infection. two clusters, c16 and c17 contained 21 genes related to anemia (fig. 5a ). for most of these genes, their down-regulation is associated with anemia, and up-regulation is associated with alleviation of anemia ( supplementary fig. 4 ). the anemia signature was already present at 6 dpi in both infections ( fig. 5a ), prior to clinical observation of rbc and hemoglobin loss at 8 dpi (fig. 1 ). the mean normalised intensity of these 21 genes was significantly down-regulated compared to naïve controls, and cb infected mice had even significantly lower values (fig. 5b) . the peak activation z scores calculated by ipa for this anemia signature of cb infection was 1.6-fold higher compared with that of as (fig. 5c , 3.6 vs 2.2, 6 dpi), agreeing with the more severe rbc loss (area under the curve analysis) in cb than as infection (1.5-fold, 36.0 vs 23.5) (fig. 1d) . interestingly, at 8 dpi, 13 out of the 21 genes within this signature were already up-regulated in as infections compared to naïve controls; by contrast, the majority (15) of these genes were still down-regulated in the blood of cb infected mice (fig. 5a, supplementary fig. 4 ). the activation z score was − 2.0 in as infected mice indicating anemia alleviation at this time point, compared to that of 2.0 in cb infected mice (fig. 5c ), which indicates a longer-lasting anemia. this is consistent with the more severe rbc and hemoglobin loss in cb infection at 10 dpi compared to as (fig. 1d,e) . moreover, in support of this data, ipa canonical pathway analysis of heme biosynthesis ii pathway also showed that genes in this pathway were more down-regulated or less up-regulated in cb than in as infected mice at 8 dpi compared to naïve controls (fig. 5d ). together, these data suggest a deregulated or stressed erythropoietic response in cb infected mice, leading to a stronger and prolonged severe anemia (fig. 1d ,e). the lungs of p. c. chabaudi cb infected mice. in addition to the 5 genes we identified from hierarchical clustering, the som analysis revealed a further 18 genes in clusters c8 and c20 related to sars, and the majority of these 18 genes were up-regulated only in cb infected mice that reached human end point at 9 dpi ( supplementary fig. 5a ). we therefore investigated whether this lung inflammation signature was reflected by more severe lung pathology in cb infected animals. lungs isolated from systemically perfused cb infected animals were of a darker coloration than lungs of uninfected or as infected mice (fig. 6a) . examination of bronchoalveolar lavage fluid showed significantly higher levels of igm in the lungs of mice infected with as or cb parasites compared to naïve mice (fig. 6b) . the hematoxylin and eosin (h&e) stained sections of perfused lungs a) were even more highly up-regulated compared to naïve controls in the blood extracted from 4 cb infected mice that had reached humane end points at 9 dpi compared to randomly selected mice infected with as or cb parasites at 10 dpi. fold changes in sars patients were from ref. 13 . (d) the twenty-four modules identified by using self-organising map (som) method were presented with the average fold change of all differentially expressed transcripts within each module compared to naïve controls. red represents positive mean fold change above zero (white) and blue indicates negative mean fold change. the clusters were annotated with ipa diseases and function analysis with manual curation. clusters indicated in red showed greater up-regulation in cb infection during 9-10 dpi. from both as and cb infected mice showed signs of leukocyte infiltration. in the lungs of some cb infected mice, patches of dense leukocyte accumulation between epithelial walls were observed in close proximity to hemozoin (hz) crystals ( supplementary fig. 5b) . we also observed more cell death in the lung tissues of cb infected mice (greater than two fold) compared to as infected mice by tunel staining (fig. 6c) . flow cytometry analysis confirmed leukocyte infiltration in the lungs of both as and cb infected mice compared to that in naïve mice (fig. 6d , gating strategy in supplementary fig. 6 ). we further characterised the cell populations within the infiltrating leukocytes in the lungs of infected animals. although cd3 + t cells, cd19 + b cells and cd3 − cd19 − innate cell numbers all increased in both infections, they did not significantly differ between as and cb infections; however, there was a trend towards higher t cell numbers in the as infection, and more innate cells in the cb infection ( supplementary fig. 6b,c) . within the innate cell populations, both the percentage and the cell numbers of ly6g + cd11b + neutrophils were significantly increased in lungs of cb infected mice compared to as infected mice (fig. 6e) , whereas other myeloid cell populations did not significantly differ between the two infections ( supplementary fig. 6d ). this observation of neutrophil infiltration is consistent with the ipa 'disease and function' analysis on the sars signature, which was up-regulated more in cb infection compared to as infection, indicating myeloid cell movement (fig. 3b) , especially neutrophils (z score 2.182). one of the top up-regulated genes, s100a9 (mrp14, myeloid-related protein 14) , indicates that neutrophils may be involved in the lung pathology of the cb infection. we therefore investigated whether the up-regulation of mrp14 transcript in the blood is associated with higher protein level and neutrophil infiltration in the lungs. we analysed the concentration of mrp14 protein in the serum at 9 dpi. while high level of mrp14 protein was also detected in sera of all (12) cb infected mice, it was below detection limit by elisa in the sera from 5 out of 12 as infected mice and all (12) from naïve mice . 6f) . we next measured the amount of mrp14 in the whole lung lysates, and found that cb infected mice contained more than twice the amount of mrp14 protein compared to that of lungs of as infected mice at 9 dpi; moreover, this upregulation was observed as early as 6 dpi (fig. 6g) . ifng, il6, kc (cxcl1) and lix (cxcl5) were higher in the lungs of cb infected mice, indicating a heightened proinflammatory response in the cb infection ( supplementary fig. 7a ). immunohistochemical staining of mrp14 on lung sections showed more mrp14 + cells present in the lungs of cb infected mice compared to naïve and as infected mice ( supplementary fig. 7b) , and flow cytometry analysis confirmed a greater than two fold increase of mrp14 hi ly6g + neutrophils in cb compared to as infected mice (fig. 6h) . together, these data show that the blood transcriptomic signature of lung inflammation is linked to an mrp14-associated neutrophil response in the lungs of mice infected with the more virulent cb strain of p. c. chabaudi compared with the avirulent as strain. amount of hz accumulated in the lungs of cb infected mice compared to as infected mice (fig. 7a) . this observation suggested a higher level of sequestration/accumulation of irbc in the lungs of cb infected mice. to investigate the level of sequestration of irbcs in p. c. chabaudi as and cb infected mice, we generated transgenic parasites, pccasluc 230p and pcccbluc 230p , expressing luciferase constitutively throughout the plasmodium life cycle under the control of eef1a promoter (supplementary fig. 8 ). at day 4, 6 and 9 dpi, the total parasite load was determined by measuring luciferase activity from 2 μ l tail blood when the parasites were at late trophozoite stage 8 , and the level of sequestration in different organs was investigated during schizogony. consistent with the peripheral load of irbc (fig. 1b) , there were no significant differences in luciferase activity between pccasluc 230p and pcccbluc 230p infected mice at 6 dpi either in peripheral blood or by whole body imaging (fig. 7b) . after intensive systemic perfusion, the luciferase activities in isolated organs were measured and relative ratio of sequestration was calculated as the level of luciferase activity per organ (total flux per second) relative to the total parasite load measured in peripheral blood before schizogony (relative light unit, rlu) (fig. 7c) . consistent with previous findings 8 , both as and cb irbc, sequester/accumulate mainly in the spleen, liver and lungs, with no significant signals observed in the kidney or brain. the relative levels of sequestration/accumulation in the spleen and liver were similar between as and cb infections. by contrast, a significantly higher level of sequestration in the lungs occurred in the cb infection at 6 dpi, and the trend was still maintained at 9 dpi (fig. 7c) . the higher level of sequestration/accumulation of schizonts in the lungs is consistent with the observation of greater amounts of hz in the lungs of cb infected mice compared to as infected mice (fig. 7a ). host genetics and immune status play important parts in the outcome of an infection with plasmodium 9,10 . however, there is an increasing amount of evidence showing that genetic diversity of the parasite also contributes to the varying severity of malarial disease. in this study we used a top-down systems analysis of peripheral blood to investigate whether transcriptomic signatures could be identified that would indicate or predict severity of acute blood-stage malaria caused by 2 strains of p. c. chabaudi of differing virulence. using high-resolution (c) bar charts showing the relative ratio of sequestration in different organs, which was quantified as the level of luciferase activities in the perfused ex vivo organs relative to the total parasite load measured in peripheral blood at late trophozoite stage (b, left). all data in (b,c) were pooled from 2 independent experiments (n = 9-12 in total). in all bar charts, median values are shown and each dot represents an individual mouse. mann-whitney u test was performed, p values are provided when significant difference was observed. profiling of the whole blood transcriptomics over multiple time points during the acute phase of infection, and data-driven modular analysis, we investigated the involvement of biological processes rather than specific genes, and uncovered several transcriptomic signatures related to severe pathologies in the virulent cb infection. these include distinct signatures for platelet aggregation, anemia and lung inflammation, which can be seen at different time points and distinguished the two infections. this analysis also revealed several signatures common between avirulent as and virulent cb infections, but they occurred at different time points or were of different magnitude. this highlights the value of studying pathological factors in the host induced by parasites over the course of the infection and not at a single time point. the platelet aggregation signature was highly up-regulated in all cb infected mice that had reached humane end points. this was the earliest pathology signature identified in this study and similar to the anemia signature was detected before the onset of severe disease. this set of genes was up-regulated as early as 2 days post infection in all cb infected mice regardless of eventual survival. it has been shown that in severe p. falciparum infections, platelets mediate irbc clumping and adhesion 11, 12 . these observations of association between infection severity and platelets aggregation suggest that similar mechanisms underlie pathology in both the p. c. chabaudi model of malaria and in human infections, and the experimental model may be useful to explore the underlying mechanisms. it would be of great interest to analyse the platelet aggregation signature in human malarial infections and investigate whether this transcriptomic signature could be used as an early marker to predict development of severe pathology. the anemia signature identified was present in the whole blood transcriptome ahead of the clinical onset of rbc loss in both avirulent as and virulent cb infections, but it was stronger and lasted longer in the cb infection. this transcriptomic signature predicted the more severe and longer-lasting anemia we have observed in cb infections 4 . both the anemia signature and the heme biosynthesis ii pathway analysis indicate a deregulated or stressed host erythropoietic response in the more severe cb infection. in addition to the platelet aggregation and anemia signatures, we identified a lung inflammatory signature in cb infected mice. although sequestration of p. c. chabaudi as parasites in lungs has been documented 8 , lung damage has not been previously reported for this experimental model. we confirmed that this sars-related lung inflammation signature in the blood was indeed associated with a more severe pulmonary neutrophilic infiltration and more cell death in the lungs in cb infections. furthermore, it was linked to a higher level of sequestration of cb irbc in this organ. both p. falciparum and p. vivax can sequester within the pulmonary microvasculature and cause lethal malaria-associated acute respiratory distress syndrome (ma-ards) 13 . members of the pfemp1 family (p. falciparum erythocyte membrane protein-1) of variant surface-expressed parasite proteins have been shown as parasite ligands mediating parasite cytoadherence 14 . although pfemp1 is lacking in other plasmodium parasites, another multigene family pir (plasmodium interspersed repeat) is present in most, if not all, species of plasmodium; and there is evidence that some pirs in p. vivax bind to icam-1 endothelial receptor in vitro 15 . it is possible that differential pirs expression between p. c. chabaudi as and cb is responsible for this differential pulmonary sequestration ability. however, it is also possible that as parasite is removed more effectively from the lung than cb due to the higher inflammation caused by cb infection. the higher level of cb pulmonary sequestration leaves greater amounts of hemozoin compared with the as parasite. there is evidence that hz can directly induce pulmonary proinflammatory responses 16 . in addition it has been shown that parasite-derived microparticles can induce macrophage activation in a tlr4 (toll-like receptor 4)-myd88 dependent manner 17 . in our study, cb infection induced higher level of inflammation (ifng, il-6 and mrp14) in the lungs of infected mice. mrp14 (s100a9) is one of the top up-regulated genes identified in the lung inflammation signagure. together with s100a8 (mrp8), mrp8/14 forms a heterodimer complex that has previously been shown to be a potent chemotactic factor for myeloid cells, especially neutrophils 18 . mrp8/14 are tlr4 ligands and are recognized as damage-associated molecular pattern molecules (damp) involved in many inflammatory diseases and infections 19 . for example, in tuberculosis and influenza infection, mrp8/14 is shown to exacerbate pro-inflammatory responses, cell-death and pathogenesis 20, 21 . of relevance here, mrp14 protein is significantly increased in p. falciparum and p. vivax infected patients 22, 23 . interestingly, in our p. c. chabaudi mouse model, mrp14 was detectable in all mice infected with the virulent cb strain; by contrast, it was detectable in only 40% of mice infected with the avirulent as strain. moreover, when mrp14 was detected in as infected mice, it was present at significantly lower level than that in cb infected mice. this coincided with a significantly higher number of mrp14 hi ly6g + neutrophils in the lungs of cb infected mice. it is possible that mrp14 + cells respond to the microparticles upon rupture of sequestered cb schizonts, leading to proinflammatory response and recruiting more mrp14 + neutrophils. our analysis offers evidence that different parasite strains, exhibiting different sequestration tendencies, can lead to different levels of lung inflammation and damage. deciphering the complex host immune responses during acute malaria is extremely challenging. here we demonstrate that whole blood transcriptomic signatures can help to reveal severe malaria-associated pathologies, often preceding clinical observations. our data demonstrate the potential in searching further transcriptomic signatures in human malaria for severity diagnosis and prognosis. furthermore, these blood signatures can also provide crucial information about the pathogenic processes taking place in organs or tissues during infection, as demonstrated here with the neutrophil-related lung inflammation signature. this unbiased modular analysis of blood transcriptomic data also offers a promising method to search for protective mechanisms in mouse and human malarial infections. this is particularly important for p. vivax infections of humans, because of its greater genetic diversity 24 , and the recent surge in reports of severe and fatal p. vivax malaria 25, 26 . mice. female c57bl/6 aged 6-8 weeks from the spf unit at the francis crick institute mill hill laboratory were housed under reverse light conditions (light 19.00-07.00, dark 07.00-19.00 gmt) at 20-22 °c, and had continuous access to mouse breeder diet and water. core body temperature was measured with an infrared surface thermometer (fluke); body weight was calculated relative to a baseline measurement taken before infection; and erythrocyte density was determined on a vetscan hm5 haematology system (abaxis). this study was carried out in accordance with the uk animals (scientific procedures) act 1986 (home office licence 80/2538 and 70/8326), and was approved by the francis crick institute ethical committee. walliker, university of edinburgh, uk and subsequently passaged through mice by injection of infected red blood cells (irbc) at the mrc national institute for medical research, uk and cryopreserved as described 27 . for experimental work, infections were initiated by intraperitoneal (i.p.) injection of 10 5 irbc derived from cryopreserved stocks. the course of infection was monitored on giemsa-stained thin blood films by enumerating the percentage of rbc infected with asexual parasites (parasitemia). the limit of detection for patent parasitemia was 0.01% infected erythrocytes. mice were culled upon reaching humane end points by showing the following signs: emaciation (more than 25% weight loss), persistent labored breathing, severe hypothermia (body temperature below 28 °c), inability to remain upright when conscious or lack of natural functions, or continuous convulsions lasting more than 5 min. p. c. chabaudi as and cb expressing luciferase under the control of the constitutive promoter eef1a were generated by transfection with the construct ppc-luc230p, targeting the neutral p230p locus (pchas_0308200 or pchcb_0308200). transfection and cloning of transgenic p. chabaudi parasites were performed as described previously 28 , and integration was verified by southern blot analysis of chromosomes separated by pulsed field gel (pfg) as described 29 . the construct ppc-luc230p was modified from ppc-luccam 8 by replacing the p. chabaudi ssu targeting region with 230p targeting region, chab03 277001-278950, generated by gene synthesis (genewiz llc, nj, usa). female c57bl/6 mice aged between 6-8 weeks were intraperitoneally infected with 10 5 irbc of p. c. chabaudi as or cb. at 2, 4, 6, 8, 10 and 12 days post infection, 0.5 ml of blood was collected via cardiac puncture into 1 ml tempus rna stabilising solution (applied biosystems). naïve samples were also collected at day 0 (the day of infection) and day 12 (the end of the experiment) and used as controls. samples were snap frozen on dry ice and stored at − 80 °c until rna isolation. total blood rna was extracted using perfectpure rna blood kit (5 prime) and globinclear kit (ambion) was used to remove globin mrna according to the manufacturer's instructions. crna samples were prepared from 300 μ g globin reduced blood rna using illumina totalprep rna amplification kit (ambion) and hybridized to illumina mouse wg-6 v2.0 beadarrays according to the manufacturer's protocols. at each step, the quantity and quality of the rna samples was verified using nanodrop 1000 spectrophotometer (thermo fisher scientific) and caliper labchip gx (caliper life sciences). microarray data analysis. illumina beadstudio/genomestudio software was used to subtract background and scale average samples' signal intensity and genespring gx 13.1 software (aigent technologies) was used to perform further normalization and analyses as described previously 30 . first, all signal intensity values less than ten fluorescent units were set to equal ten, log 2 transformed and per chip normalised using 75 th percentile shift algorithm. transcripts were further normalized to the median across all samples or to the median of control samples. transcripts were first selected if they were present (cut off 0.99) in ≥ 10% of all samples, and further filtered with a minimum of 2-fold expression up-or down-change compared with the median intensity across all samples. all microarray data are deposited in geo under accession number gse93631. genespring software was used to perform statistical tests, anova unequal variance with post-hoc tukey's hsd (honest significant difference) test, followed by benjamini-hochberg multiple test correction (fdr < 0.01); fold change was further performed on the combined list of transcripts differentially expressed either in as or cb infection, with 2-fold cut off compared to naïve controls. the set of transcripts was defined as differentially expressed transcripts and was used for further analyses. hierarchical clustering of samples at different infected time points compared to naïve were performed using pearson uncentred correlation with an average-linkage-clustering algorithm that organizes all transcripts according to their trend of expression across all samples. the hierarchical clustering on the 6226 transcripts differentiall expressed in as or cb (supplementray table 1 ) across the acute phase of infection was performed using euclidean distance metric with ward's linkage rule. for the pathological modules (in fig. 4) , prediction was performed on the 6321 transcripts differentially expressed in as or cb infection including the samples collected at 9 dpi (supplementray table 2 ) using the self-organising map algorithm in genespring. euclidean distance was used for similarity measurement and the maximum number of iterations was set at 10e5. the initial learning rate was set at 0.03 and initial neighbourhood radius 5, the number of grids was tested from 10 × 10, till less than 15% of clusters have similarities above 90%, at a final grid of 4 × 6 (24 clusters). ingenuity pathways analysis (ipa) (qiagen) was used to identify enriched disease and functions and networks. the significance of the association between the dataset and each analysis was measured using fisher's exact test, z score cut-off 2 and/or p value cut-off 0.01. this program was also used to map the canonical pathways and overlay it with expression data from the dataset. module annotation was determined using disease and function analysis in ipa. to obtain bronchoalveolar lavage fluid (balf), mice were terminally anaethetised, and lungs were cannulated and inflated with 500 μ l pbs. the liquid was retrieved and spun at 300 g for 10 min at 4 °c. supernatants were obtained and kept at − 80 °c till further analysis. igm levels in balf were quantified using a sandwich elisa. (southern biotech). detection of mrp14 and cytokines in sera and lung lysate. mouse serum was collected via cardiac puncture, clotted in room temperature for 30 min and collected by centrifuging twice at 10,000 g for 10 min at 4 °c. lung proteins were extracted in ripa lysis buffer with protease inhibitor cocktails (sigma) and homogenized with polytron homogenizer (kinematica) on ice. protein levels were quantified by pierce bca protein assay (thermo scientific) as per the manufacturer's instructions. all plates were read on a safire ii plate reader (tecan). mouse s100a9 elisa kit (r&d systems) was used to determine the level of s100a9/mrp14 in serum and lung lysate samples following manufacturer's instructions. cytokine concentrations were determined using cytometric bead array (biolegend) following the manufacturer's manual. histology and immunohistochemical analyses. the lungs were extensively perfused with 10 ml pbs and then inflated by injection of 3 ml of 10% neutral buffered formalin (nbf) through the tracheal cannula. tissue was then fixed overnight in 10% nbf, and transferred into 70% ethanol until embedded in paraffin and sectioned. each lung specimen was stained with haematoxylin and eosin (h&e). for each mouse, the number of hemozoin crystals were quantified from 10 randomly selected fields on h&e stained slides under leica light microscopy (400× ). immunohistochemical staining was performed to examine the expression of mrp14 on paraffin-embedded lung sections with anti-mrp14 antibody (clone 2b10). tunel staining was performed using apoptag ® fluorescein in situ apoptosis detection kit (merck millipore) following the manufacturer's protocol. imaging of slides was performed on a vs120 slide scanner (olympus) with a vc50 camera, a uplsapo lens, at a magnification of 20× or 40× . images were processed and analysed using olyvia image viewer 2.6 (olympus) and fiji 31 . and tunel positive cell numbers were quantified in an area of 1nm 2 using olyvia image viewer 2.6. in vivo imaging and luciferase assay. mice were infected intraperitoneally with 10 5 rbc infected with pccasluc 230p or pcccbluc 230p parasites; and at each time point 2 μ l of heparinized tail blood was collected before sequestration 8 . bioluminescence was assessed with the luciferase assay system (promega) according to the manufacturer's protocol and quantified with the tecan safire2 plate reader and magellan software (tecan). under these conditions, bioluminescence intensity is proportional to the amount of parasites in this blood volume 8 , which reflects the total parasite load before sequestration. at the time of maximum sequestration (12.00-14.00 h gmt, reverse light) 8 , d-luciferin (150 mg/kg, caliper life sciences) was injected subcutaneously 5 min before whole-body and organ imaging. mice were terminally anaesthetized and systemically perfused by intracardiac injection of 10 ml pbs 8 . the brain, lungs, liver, spleen, left kidney and gut were removed immediately and luciferase assessed using in vivo imaging system ivis lumina (xenogen), with a 10 cm field of view, a binning factor of 4, and an exposure time of 10 s. bioluminescence (total flux per second) was quantified with the software living image (xenogen) by adjusting a region of interest to the shape of each organ. to account for the influence of total parasite load on the number of parasites sequestered in the organs, bioluminescence in the organs was normalized to total parasite load. luciferase activities measured in the organs were divided by parasite load in 2 μ l blood (see above), allowing comparison between mice with different parasite burdens. quantifying genetic and nongenetic contributions to malarial infection in a sri lankan population malaria infection changes the ability of splenic dendritic cell populations to stimulate antigen-specific t cells disruption of il-21 signaling affects t cell-b cell interactions and abrogates protective humoral immunity to malaria the 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antituberculosis therapy fiji: an open-source platform for biological-image analysis were made in graphpad prism, each dot represents an individual biological replicate and p-values were derived from mann whitney u test or multiple t-test. we would like to thank thibaut brugat, audrey vandomme and barbara capuccini for critical reading of the manuscript. we are grateful to the brf at mill hill for animal husbandry, to the high-throughput screening (hts), flow cytometry, experimental histopathology and microscopy facilities at mill hill for excellent technical support. this work is supported by the francis crick institute, which receives its funding from the uk medical research council (grant u117584248), cancer research uk, and the wellcome trust (grant wt104777ma). key: cord-292402-u3sfc1yz authors: watanabe, rihito; kakizaki, masatoshi; ikehara, yuzuru; togayachi, akira title: formation of fibroblastic reticular network in the brain after infection with neurovirulent murine coronavirus date: 2016-04-28 journal: neuropathology doi: 10.1111/neup.12302 sha: doc_id: 292402 cord_uid: u3sfc1yz cl‐2 virus is an extremely neurovirulent murine coronavirus. however, during the initial phase of infection between 12 and 24 h post‐inoculation (hpi), the viral antigens are detected only in the meninges, followed by viral spread into the ventricular wall before invasion into the brain parenchyma, indicating that the viruses employ a passage between the meninges and ventricular wall as an entry route into the brain parenchyma. at 48 hpi, the passage was found to be constructed by er‐tr7 antigen (erag)‐positive fibers (erfibs) associated with laminin and collagen iii between the fourth ventricle and meninges at the cerebellopontine angle. the construct of the fibers mimics the reticular fibers of the fibroblastic reticular network, which comprises a conduit system in the lymphoid organs. in the meninges, erfibs together with collagen fibers, lining in a striped pattern, made up a pile of thin sheets. in the brain parenchyma, mature erfibs associated with laminin were found around blood vessels. besides mature erfibs, immature erfibs without associations with other extracellular matrix components like laminin and collagen appeared after infection, suggesting that the cns creates a unique conduit system for immune communication triggered by viral invasion. coronaviruses have relatively high mutation and rna recombination rates and rapidly undergo cross-species transmission events in vitro and in vivo. [1] [2] [3] in humans, viruses newly emerging out of members of coronaviridae cause serious diseases, such as severe acute respiratory syndrome (sars) 4 and middle east respiratory syndrome (mers). 5 interest in the pathogenicity of murine coronaviruses, particularly, mouse hepatitis virus (mhv), has grown because of the high incidences of mutation rates with consequences of different pathogeneses 6-10 as well as their ability, especially in jhm strains of mhv (jhmv), to cause both acute and chronic cns diseases , 10, 11 because neuropathogenic viruses can induce significant neuronal dysfunction and degeneration of specific neuronal populations, sometimes leading to devastating, life-threatening consequences in infected humans. 12, 13 among jhmvs, cl-2 exhibits extremely high neurovirulence. 10, 14, 15 a less virulent viral clone, srr7, has been isolated from cl-2, as a soluble receptor-resistant mutant. 16, 15 all mice infected with cl-2 die within 3 days post-inoculation (dpi), whereas mice infected with srr7 exhibit no clinical sign before 5 dpi and some of them survive after 10 dpi. however, there is no difference in the viral growth rate during the initial phase of infection between these two strains. 15, [17] [18] [19] the different virulence of these viruses is attributed to their capability to infect neurons, 15, 20 or their infectivity being independent or dependent upon a major mhv-receptor, carcinoembryonic cell adhesion molecule 1. 21, 22 in agreement with their similar viral proliferation rates during the early phase of infection, the two viruses exhibit almost the same patterns of viral spread in the brain during the initial phase of infection. the viral antigens are first detected in the infiltrating cells, namely cd11b-positive (cd11b + ) cells of monocyte lineage, in the meninges at 12 h post-inoculation (hpi), and not at the site of the inoculation in the frontal lobe. 17, 19, 23 during 24 to 48 hpi, infected cd11b + cells appear in the ventricular cavity followed by infection of cell components of the ventricular wall, before viral spread into the brain parenchymal cells. 17, 18 the ventricular wall is also the target site during th einitial phase of infection with other species of viruses, including herpes simplex virus 24 and lymphocytic choriomeningitis virus. 25 the manner of the viral spread after infection with cl-2 or srr7 indicated that there could be some other routes of viral entry into the brain parenchyma than crossing over the blood brain barrier (bbb), which has been almost exclusively studied or hypothesized as the mechanism of neuropathogenic viral entry into the brain parenchyma, [26] [27] [28] [29] [30] including a pioneering study of poliovirus using genemanipulated mice introduced by the human-type polioviral receptor-gene. 31 however, the mechanism of viral distribution of a neurovirulent strain of polioviruses, the mahoney strain, infecting motor neurons in the cns, was not elucidated through studies of its ability to circumvent the bbb in comparison with that of an attenuated viral strain, sabin strain. 32 our investigation to identify the viral entry route focused on the area between the fourth ventricle (ivv) and meninges in the cerebellopontine angle, because viral antigens are often found in the area during the early phase of infection between 24 to 48 hpi, 17, 18 and there is a conduit, the foramen of luschka, for csf to flow from the ventricle into the subarachnoid space. another intriguing feature of viral antigen localization after infection with cl-2 or srr7 is that the viral antigens were found in fibrous structures, which appeared to be the extracellular matrix (ecm), in the brain and spleen. 17, 18 viruses proliferate and produce viral antigens in living cells, and it is unusual to find them in the ecm area through a light-microscopical observation. however, such colocalization of viral antigen or viral particles with the ecm has been reported to occur in the lymphoid organs. after infection with lymphocytic choriomeningitis virus (lcmv), viral antigens are colocalized with the components of reticular fibers, including collagen iii and laminin, in the spleen. 33 similar phenomena have been reported in infection with extremely virulent viruses, such as ebola, 34 marburg and lassa viruses. 35 these viruses infect fibroblastic reticular cells (frcs) in the lymphoid organs, followed by immune dysfunction leading to viral persistence 33 or tissue destruction. 35 frcs are considered to maintain reticular fibers, which compose fibroblastic reticular network (frn) in the lymph nodes [36] [37] [38] and spleen. 39, 40 erasmus university rotterdam thymic reticulum antibody 7 (er-tr7) has been used to define frcs, 41, 42, 33 although the antigen of er-tr7 has yet to be determined. furthermore, there have been no reports on the size or determinant of the antigen using immune reactions, including western blotting or immunoprecipitation. each reticular fiber comprising the frn is around 1 μm in diameter, and contains collagen fibers as a core surrounded by an er-tr7 antigen-positive (erag + ) microfibrillar layer, and is further enclosed with a basement lamina at the outer surface. the entire fiber is largely ensheathed by frcs. 43 the frn functions as a conduit system for immunocompetent cells and inflammation-associated molecules such as cytokines and ligands or foreign antigens to reach appropriate sites in order to cause immune reactions in the lymph nodes 44 and spleen, 39 or to guide homing of the cells. 42, 40 subsequently, frcs, which are podoplanin (gp38)-positive (pod + ) and cd31-negative (cd31 à ), have been reported to co-operate with other stromal cells in the lymph nodes, such as pod + cd31 + lymphatic endothelial cells, pod à cd31 + blood endothelial cells, and pod à cd31 à myofibroblastic pericytes, to play a key role in the frn function, 45, 46 indicating that there should be organ-specific specialization of the fibroblastic stroma. in this report, we show the virus antigens detected in the brain as fibrous structures colocalized with erag and laminin as the same images, as previously reported in the spleen infected with lcmv. 33 this finding encouraged us to prove the expression of erag along the reticular system in the brain, which could function as a scaffold for viral entry and inflammatory cell invasion into the brain. the highly neurotropic mhv strain mhv-jhm cl-2 (cl-2) 14 was used. this virus was propagated and titrated using dbt cells maintained in dulbecco's modified eagle's minimal essential medium (dmem) (gibco, grand island, ny) supplemented with 5% fetal bovine serum (fbs) (sigma, tokyo japan), as previously described. 14 specific pathogen-free inbred balb/c mice purchased from charles river (tokyo, japan) were maintained according to the guidelines set by the committee of our university. for the experiment with infectious agents, mice were transferred to a biosafety level 3 (bsl-3) laboratory after permission from the committee. at 7-8 weeks old, each mouse was inoculated with 1 × 10 2 of cl-2 virus into the right frontal lobe under deep anesthesia. as described previously, 17 the scar caused by the needle tip was found in the right frontal lobe after post mortem examination. between 12 and 72 hpi, organs including the brains and spleens were removed from treated or untreated mice after exsanguination under deep anesthesia. parts of the organs were embedded in tissue tek oct (sakura, tokyo, japan) and frozen to prepare frozen sections. remaining portions were processed for viral titration, or fixed in 4% paraformaldehyde buffered with 0.12 mol/l phosphate (pfa) for pathological examination using paraffin-embedded sections to check that each experiment had been appropriately performed as we previously reported using cl-2 for infection, 17, 15 and no contradiction was found (data not shown). some mice were perfused with pfa from the aorta via the left ventricle under deep anesthesia using diethyl ether and 0.2 mg of chlorar hydrate (mylan seiyaku, tokyo, japan) per mouse to obtain sections for the detection of dexran labeled with texas red, for the three-dimensional construct, or for double or triple immunostaining that includes podoplanin detection. ten-micrometer cryostat sections prepared from unfixed frozen tissues were fixed in ice-cold acetone for 10 min. pfa-fixed tissues were rinsed with graded concentrations of sucrose overnight, and processed to prepare cryosections as previously described, 18 which were used without further fixation. chamber slides with mixed primary culture were fixed either in cold ethanol for 1 min followed by fixation in cold acetone for 5 min, or in pfa for 5 min to perform double or triple immunostaining that includes podoplaninstaining. immunostaining was carried out using the antibodies and reagents listed in table 1 , as previously described. 18 fluorescence was visualized with a confocal laser scanning microscope (leica microsystems, heidelberg, germany) to obtain images of double or triple immunostaining and three-dimensional constructs. a fluorescence microscope (keyence, osaka, japan) equipped with a bz analyzer (keyence) was used for statistical study. one mg of 10 kilodalton lysine fixable dextran labeled with texas red (invitrogen corp., carlsbad, ca, usa) was injected intravenously in 400 μl of pbs through the tail vein. ten minutes after the injection, mice were perfused with pfa, the brains and spleens were immersion-fixed overnight in pfa, and then processed for cryosections as described earlier. the pfa-fixed brains were processed either for 40-μm cryosections, or thick sections of 90-150 μm thick, which were prepared by slicing the brain with sharp stainless blades under a dissecting microscope. the cryosections were stained in the same way as the other cryosections described earlier. all procedures to stain the thick sections were performed in a floating state. after rinsing in 0.12 mol/l tris-hcl (ph 7.4) for 1 h, thick sections were incubated with blocking solutions for 1 h, 18 followed by incubation with primary antibodies overnight. each of the second and third steps of staining were conducted for 30 min. three-dimensional images were generated with leica software (leica microsystems, heidelberg, germany). twenty-four-bit color images with a 1360 × 1024 pixel resolution were acquired with a fluorescence microscope (keyence). three independent approaches were performed to quantify the observed immunofluorescence signal using the bz analyzer. the background fluorescence intensity (fi) in areas of the field of view was subtracted from total measurements of fi. the average fluorescence intensity (afi) within the unit area was obtained by dividing its integrated fi by its area. an increased level of erfibs in the restricted area of the brain was already detectable at 12 hpi with cl-2 ( fig. 1a -c), with an increased and intense staining for erfib compared with that of an uninfected mouse (fig. 1b ) or sham infected mouse ( fig. 1c ) in the root of the trigeminal nerve, especially in the area where gfap-positive activated astrocytes were observed near the border with the peripheral nervous system (fig. 1a3) . a more extended distribution of erfibs was observed during the later phase of infection (fig. 1d , e). furthermore, virus antigens in fibrous structures reported in our previous study 17, 18 were found to colocalize with laminin and erag ( fig. 1f ) with almost the same image observed in the spleen of mice infected with lcmv, 33 which indicated that viruses are concentrated in the narrow space of reticular fibers 33, 43 and are recognized by immunofluorescence, and they use the reticular conduit system as a scaffold. 33, 35 in order to quantify the increase and association of erfibs with laminin, the average fluorescence intensity (afi) of divided areas (0.9-2.4 × 10 4 μm 2 ) was measured in sections which included the pons ( fig. 2a) . there was a high positive correlation of the distribution and intensity between laminin and erag expression in an untreated mouse (r 2 = 0.82) and virally infected mouse at 48 hpi (r 2 = 0.78, fig. 2a) , as well as an increase of the expression at 48 hpi with cl-2 compared with that at 12 hpi ( table 2 ). the laminin expression of the mouse at 12 hpi with cl-2 was more rapid compared with the erag expression, and was more extensive at 48 hpi than those of medium or untreated mice (table 2) . next, a detailed study of afi in the area around the foramen of luschka, which provides a conduit for csf between the fourth ventricle and subarachnoid space at the cerebellopontine angle, was performed, because the viral antigens are initially detected in inflammatory cells that have infiltrated the meninges, followed by inflammatory cells in the ventricular cavity and cell components of the ventricular wall. 17, 18 afis in the dotted areas in figure 3e were compared among those of cl-2-, sham-infected and untreated mice (fig. 2b) . because of variable afi levels in individual mice, especially in infected mice showing high sds, a significant increase of afi in the area of cl-2-infected mice compared with that of sham-infected mice at 48 hpi was obtained in the deeper area of the fourth ventricle, 300-500 μm inside from the outermost portion of the lateral recess of the fourth ventricle (ov) (fig. 2b) . at 72 hpi, the expression of erfib remained at high levels in cl-2-infected mice, whereas the relatively high levels in the outer areas of the lateral recess of sham-infected mice at 48 hpi decreased at 72 hpi to the levels of untreated mice (fig. 2b ). the expression of erag was either colocalized with that of components of the extracellular matrix (ecm) such as laminin (fig. 1d ) and collagen (fig. 1e ) in the same way as reticular fibers reported in lymphoid organs, 33 or found without such colocalization as shown in the brain parenchyma ( fig. 1d2 and d3) and trigeminal root (supplemental figure s1a ). in the ventricle, we also found erag without an association with other ecm (figs 1e and 4d ), which indicated that there could be immature erfibs formed during initial events of the host reaction, compared with the mature form of erag + reticular fibers associated with several kinds of ecms observed in lymphoid organs. 33, 45, 47 therefore, we compared the length of erfibs colocalized with laminin in the ventricle between those of infected and sham infected mice, and found a greater difference between those in the two groups (fig. 2c ) than on comparison of erfibs alone. erag + structure as a conduit in the brain in order to examine whether the expression of erfib plays a role as a conduit system in the ventricle as reported in the spleen and lymph node, 39,44 fluorescence-labeled dextran was intravenously injected. mice infected with the virus showed the infiltration of many particles of dextran along with erfibs into a deep area in the ventricle ( fig. 3a and supplemental figure s1b ). a few dextran particles were detected in the hilum of the ventricle near the border with the meninx of sham-infected mice (fig. 3b) , and no trace of the particles was seen in the ventricles of mice with no treatment (fig. 3c) . the dextran particles were appropriately introduced into the recipients, because a high level of the particles was seen in the spleen of mice with no treatment along with erfbs, especially in the marginal zone around the follicle (fig. 3d) , and partially in the follicle (insert in fig. 3d ), as previously reported. 39 the viral antigens were detected in the ventricle of cl-2-infected mice in a similar way as dextran particles. in the deeper area of the ventricle at the positions corresponding to b and c in figure 3e , viral antigen-positive cells closely associated with erfibs were observed ( fig. 3g and h, respectively) . however, in the area near the junction between the meninges and ventricle at position a in figure 3e , the viral antigens did not always distribute along with erfibs (fig. 3f) , possibly because viruses brought into the ventricle earlier than in the deeper area had time to induce the antigens in the infiltrated and domestic cells after infection. because viral antigens are already detectable in cells of the monocyte lineage (mocs) that have infiltrated the meninges during the initial phase of infection at 12 hpi, 17 cd11b-positive (cd11b + ) cells as a carrier to transport viruses into the ventricle were examined. double staining for cd11b and erag revealed a close association of mocs and erfibs in the ventricle (fig. 3i) . as expected, many of the cd11b + cells in the ventricle were infected (fig. 3j ). in order to study stereostructures of erfbs, we threedimensionally reconstructed consecutive confocal images with prominent astrocytic activation (a3)), the expression of er-tr7 antigen (erag) was detected. the dotted area in a1 is shown at higher magnifications (a2 and a3). the orientation of this area is shown at a lower magnification in supplemental figure s1a2 . b: a low level of erag was detected (b1) especially in the area of the peripheral nerve (gfap-negative lower area). the area of the peripheral nerve is shown as a laminin-positive area in the serial section (b2). c: moderately more intensive staining of erag was observed in the sham-infected mouse than in the uninfected mouse. the boxed area is shown at a higher magnification (c2 and c3). in the area of the peripheral nerve (lower area), erag was not colocalized with laminin (supplemental fig. s1a ), although, in the peripheral nervous system, there were abundant amounts of laminin (b2) comprising the basement lamina around schwann cells. d: at 48 hpi with cl-2, erag colocalized with laminin around the blood vessels (arrows) and meninx (triangles) became prominent. away from the blood vessels and meninx, erag + areas without such colocalization were distributed. higher magnifications (d2 and d3) of boxed areas in d1 show that some areas of the erag + laminin arrangement exhibit cellular structures, and some fibrous configurations (arrowhead). e: erag and collagen iii, in the fourth ventricle. f: colocalization of laminin, collagen iii and the viral antigens in the meninx around the pons is shown in white. single and double bars indicate 50 and 20 μm, respectively. of 40 μm-thick sections of the brains. the integrated intensity of immunofluorescence of 35 slices of a 1.5 μm thickness showed robustly stained fibrous structures with a more than 5 μm thickness in the meninx (fig. 4a) and ventricle (fig. 4e) , which were double-stained for erag and collagen or laminin, respectively. in the meninx, most of the erfbs seemed to localize together with collagen based on the view of the integrated image (fig. 4a) . however, rotation of the image revealed a sheet composed of erfbs and collagen fibers in a fine striped pattern ( fig. 4c and supplemental movie s1). at the meninx with inflammation, many cells were observed, after triple staining for the nucleus, erfibs and collagen, in the narrow space between the sheets composed of an association of erfibs and collagen (fig. 4b) . another finding obtained from three-dimensional analysis was that a hammock-like structure composed of fine erfbs table 2 . b: the area in the fourth ventricle (ivv) including the space and ependymal cell layers was divided into five areas between the outermost portion of the lateral recess of the ivv (ov) and 500 μm distant from ov. afi of erag is shown as fi of each area per pixel obtained from three independently treated mice for each group. vertical lines indicate sd. c: the average length per μm 2 of erag-positive fibers associated with laminin expression in ivv obtained from three independently treated mice for each group. vertical lines indicate sd. * and ** indicate the p-value (p) examined by student's t-test <0.05 and <0.005, respectively. hanging on bold lines of laminin, which were partially colocalized with erfibs, was formed in the ventricle (fig. 4e and supplemental movie s2), which could have been ignored on non-specific staining based on typical confocal images of two dimensions because of their overly faint staining. in the meninges, arachnoid cells with the expression of cytokeratin or zic2 showed colocalization with erag (fig. 5c, d) . arachnoid cells are reportedly podoplaninpositive (pod + ), 48 just as with frcs in the lymphoid organs. [45] [46] [47] however, in our analysis, only part of zic2 + cells in the meninges was pod + (supplemental fig. s1c) . a thick pod + layer in the meninges was colocalized with erag ( fig. 5b) , most of which could be produced by fibroblasts, which are located in the meninges. 49 some podoplaninnegative (pod à ) cells (arrowheads in fig. 5b ) as well as structures making up the basal area (arrows in fig. 5b ) of meninges were also found to be erag + . in the ventricle, some of the ependymal cells with cytokeratin expression were found to produce erag. in the brain parenchyma, many gfap-positive astrocytes, especially those comprising glia limitans at the brain surface (fig. 5f) , or near the border with the peripheral nerve (fig. 1c) , were found to be erag + . deep in the parenchyma, erag + astrocytes were observed around the blood vessel (fig. 5g) . however, in the brain parenchyma, many erag + cells without colocalization with laminin (fig. 1d3) , which were not located around the blood vessels, were seen. furthermore, astrocytes situated away from blood vessels deep in the brain parenchyma, even though they had contact with erag + cells, did not produce erag (supplemental fig. s1d) . therefore, we examined other cell types which produce erag in the brain parenchyma, and found erag + neurons and oligodendrocytes (fig. 5h, i) , in addition to endothelial cells (fig. 5j) and pericytes (fig. 5k ) of blood vessels (bv) in the brain parenchymal area (pabv). however, brain parenchymal cd11b low cells (arrowheads in supplemental fig. s1e ), possibly microglia, 50 as well as infiltrating cd11b high cells in the meninges (arrows in supplemental fig. s1e ) and ventricle ( fig 3i) were erag à . in addition, erag + cells distributed in the area away from the viral antigen-positive site (fig. 5l) indicated that the signal of viral entry reached these erag + cells via an undetermined pathway. a classical neuropathology states that, in the brain in the presence of infectious diseases, there is an increase in reticular fibers, detected by silver staining, and these fibers are associated with inflammatory cells. 51 a recent study also detected an increase of reticular fibers as second harmonic generation structures in toxoplasmic encephalitis (te). 52 although the modern sophisticated technique revealed a three-dimensional structure of the reticular network, which may be used as a scaffold for antigen-specific t cells to fig. 2a, respectively) , where 2% of the population measured in (v-) mouse were found. when afis are higher than the highest afi measured in the (v-) mouse, which corresponds to an afi of >15.4 or 6.8 in laminin or erag staining, respectively, that was defined as highly positive (high). hps, hours post-infection; n, no matched areas. figures in the parentheses are mean ± sd, and the figures without ± show that one area was counted under the term. the significance of the difference was analyzed by student's t-test, with p-value shown. migrate into the infected sites in the brain, questions that were raised half a century ago 51 (what are the components of the reticular fibers, and which cells in the cns produce the components?), still remain to be answered. 52 in this report, we showed erfibs as one of such components, which increase during infection and comprise a reticular network unique to the brain (brrn) but in part analogous to frn in the lymphoid organs, detected as erag + fibrous structures and forming a conduit system . 39, 44, 45 erag + structures have been detected in the brains after infection 52, 53 or physical treatment, 49 and in the retina of gene-manipulated mice, 54 as well as in normal mice at low basal levels in the meninges 49, 52 and basement membrane of blood vessels. 52 in these reports, erag was used as a marker of reticular fibroblasts, although the figures in the papers show the co-localization of erag and gfap , 49, 54 and the contribution of erfibs to immune-mediating communication or a scaffold is not mentioned. one of the reasons that such an important role of erfibs to operate a conduit system in other organs was not described could be that the authors might have been obliged to limit the character of erag + structures, because an increase of these structures in injured regions, which were examined at around 1 week or more after the treatment of the mice, might not be large enough to trace and prove that they function as a conduit system. for example, erag + structures in the meninges have been reported to be found only in the sulcus 52 where the arachnoid membrane does not exist. in contrast, our results showed high expression of erag + structures in the arachnoid membrane. the reason why we could trace the erfibs could be that the virus cl-2 is an extremely neurovirulent strain, leading infected mice to a morbid state in 48 hpi. 8, 9, 14 the more vigorous the insult is, the more extended the host reaction would be. actually, the increased thickness of the arachnoid membrane formed during concentrated time periods after the infection enabled us to observe the architecture of a striped pattern consisting of erfibs and collagen fibers, forming the sheet of a thin membrane. each of the sheets was approximately 0.7-1.0 μm thick, which corresponds to the diameter of each reticular fiber serving as a unit of the conduit system in the lymphoid organs, 43, 45 and the sheets had been considered to be meningeal fibers on the two-dimensional observation of thin sections. furthermore, between the sheets, many cells were detected, many of which should be infected cd11b + cells. 17, 18 a classic neuropathological understanding of the sites of infiltrating cells observed in meningitis has theoretically assumed a subarachnoid space between the arachnoid membrane and pia mater. the erfib-embedded sheets may guide inflammatory cells after extravasation to migrate into the meningeal region, but on the other hand, the sheets might restrict the inflammatory lesion in the meninx blocking the direct spread of inflammatory cells and viral particles from the meninx into the brain parenchyma. at least at 48 hpi, the prevention of direct invasion of the inflammatory cells and viruses from the meninx to the brain parenchyma seemed to be successful, as has also been observed in meningitis induced by other infectious agents. [55] [56] [57] before our findings, such prevention was thought to be operated by the pia mater and astrocytes comprising glia limitans at the brain surface, 58 because inflammatory cells and infectious agents have been considered to be able to freely move around the presumed subarachnoid space. the increase in erfibs after infection had already occurred at 12 hpi. however, the possibility that all of the newly formed erfibs function analogous to frn in the lymph nodes and spleen is not conceivable, because invasions of cd11b + cells and viruses into erag-upregulated areas were not prominent at 12 hpi. in addition, although erfibs in the fourth ventricle, especially near the area of fig. 4 three-dimensional images were constructed after staining er-tr7 antigen (erag) (er) and collagen iii (col) in the meninges of the cerebellopontine angle (a d), or laminin (la) in the fourth ventricle (e) of a mouse infected with cl-2 at 48 hours post-infection (hpi). nuclear counter-staining was performed using hoechst 33342 (hoe). the letters in parenthesis in b,c indicate the areas of images corresponding to the boxed areas in a. between the sheets, many cells were found to be packed (b1). the thickness of the sheet was estimated at around 0.5 1.0 μm after viewing the sheet from a different angle (b2 and supplemental figure s2 ). the rotation of images around the y axis disclosed sheet structures (supplemental movie s1) composed of erag-positive fibers (erfibs) and collagen fibers (c and d at a higher magnification of the dotted area in c3). the ventricle contained a fine erag + structure without colocalization with laminin (arrows in e) after the rotation of images (supplemental movie s2). the double bar in a indicates 20 μm. single bars indicate 2 μm (b2 d) and 10 μm (b1 and e). the junction of the ventricle and meninx, were increased in sham-infected mice compared with non-treated mice, fluorescence-labeled dextran particles, which have been used as tracers to show the function of frns in the spleen, 39 were not detected in the fourth ventricle. in contrast, in cl-2infected mice, labeled dextran particles were found in the ventricle showing a close association with erfibs. this and subsequent findings indicated that erfibs need to be allied with some other substances including ecm components detected in frn to function as a scaffold. the erfibs in the ventricle of infected mice were co-localized with laminin at a higher level compared with those of sham-infected mice. in addition, erfibs that appeared at 12 hpi were not colocalized with laminin, although in the area of peripheral nerves there were high levels of pre-existing laminin as a component of the basement membrane around schwann cells. another immature form of erfibs appeared after the three-dimensional construction of images. in the fourth ventricle, an erag + net-like structure was found hanging on two pole-like structures of laminin, without forming firm fibers. the findings were supported by a study using a primary brain mix culture, where several stages of erag + expression with various types of association with ecm were observed (data not shown). besides collagen and components of the basement lamina including laminin, 43 several kinds of molecules, such as decorin, biglycan and fibromodulin, which are surrounded by the erag + matrix and localized to the conduit core, 45 are supposed to be engaged in the maturation , oligodendrocyte lineage transcription factor 2 (oli), cd31 and cd13 antigens, and nuclear counter-staining (nuc) by immunofluorescence. a e: arachnoid cells shown as ck-positive (ck + ) and zic-2 + and ependymal cells shown as ck + cells produced erag. pod is reportedly expressed in arachnoid cells, 48 but only a part of zic2 + cells were pod + (supplemental fig. s1c, respectively) . f and g: astrocytes producing erag at the brain surface making up glia limitans (f), and those deep in the brain parenchyma, where astrocytes around the blood vessels (pabvs) produce erag (g), but not those distant from pabvs, even in the area with many erag-producing parenchymal cells (supplemental fig. s1d ). the dotted area in f1 is shown at a higher magnification in f2 and f3. h k: cns components other than astrocytes produced erag, such as neurons (h), oligodendrocytes (i), endothelial cells (j), and pericytes (k). however, neither cd11b high nor cd11b low cells produced erag (supplemental fig. s1e ). triangles and ivv indicate the meninges and fourth ventricle, respectively. single and double bars indicate 50 and 20 μm, respectively. of frn with the erag + matrix to function as a conduit in the lymph nodes. in addition, frcs, a core cell population to maintain the ecm and conduit system in the lymph nodes, produce large amounts of other molecules involved in the matrix and elastic fiber assembly, including lysyl oxidase (lox), lox-like 1, microfibril-associated glycoprotein 2 (mfap-2), mfap-5 and fibulin-1. 45 future studies should elucidate the roles of these factors in regulating the reticular network in the brain. frcs are thought to be highly specialized to the lymphoid microenvironment. 45, 59 in order to study the corresponding cell population in the brain, we investigated cells that produce erag, which is one of the whole markers of frcs and frn . 33, 40, 41 besides expected cell populations such as pod + erag + cells in the meninges, various types of cells in the brain showed erag expression, including arachnoid cells, ependymal cells, endothelial cells and pericytes, which were judged through the expression of representative cell marker proteins, their localization in the tissue and their shape. in addition, to our surprise, all kinds of the brain parenchymal cells except for cd11b low cells, provbably microglia, showed erag expression. in spite of these findings, it is not plausible that the erag produced by brain parenchymal cells was directly employed in the conduit system to guide the invasions of the virus or inflammatory cells, as indicated in the meninges and ventricle, because the viral antigens and inflammatory cell invasions were not observed at 48 hpi in the large area where they were produced by brain parenchymal cells. furthermore, most of them were not colocalized with laminin fibers, except for the close association of gfap + and laminin + fibers around the blood vessels, where the basement membrane is formed facing astrocytes at the side of blood vessels even under normal conditions. 26 however, the colocalization of erag and laminin + fibers around the blood vessels was detected. the results indicate that a conduit system could be created around the pabv, because information that virus-bearing cd11b + cells had invaded the meninges and ventricle must reach to the area with erag expression in the brain parenchyma. the conduits in the lymphoid organs are able to transfer such information through micro-and macromolecules, including cytokines and ligands. 36, 39 in the brain, foot processes of astrocytes, neurons and microglia adhere to the basement lamina around the blood vessels. 26, 60 when the information reaches the cells around the blood vessels, cell-to-cell-mediated immune cross-talk through an immune synapse reported in lymphoid cells, 61 and /or the use of not only immune-related molecules but also several neurotransmitters as a tool, 62 could possibly trigger an immune reaction in the brain parenchymal cells distant from blood vessels. astrocytes might play such a role in cellular communication because of their ability to produce high levels of erag and their localization around the blood vessels, ventricles and brain surface. therefore, local immune reactions relevant to brain pathology should be further examined, especially on cross-talk between brain parenchymal cells, 63 possibly after receiving information from the conduit around the pabv. cns region in the trigeminal root (trigr), fourth ventricle (ivv), meninx (mx), inferior cerebellar peduncle (icp), and pons (po). figure (fig.) numbers in parenthesis indicate the figures where the legend of each image is described. long arrows and arrowheads in e indicate cd11b high and cd11b low cells, respectively. j: the area of the trigeminal root in figure 1a , which is indicated by the boxed area, is indicated by closed lines. the border between the cns area (upper right) and peripheral nerve (lower left) is indicated by a dotted line. single and double bars indicate 50 and 20 μm, respectively. supplemental figure s2 . infiltrating cells indicated by nuclear staining (blue) between thin sheets with collagen iii (red) and erag (green), illustrated in figure 4b , are viewed from different angles. bar indicates 10 μm. supplemental movie s1. supporting info item supplemental movie s2. supporting info item episodic evolution mediates interspecies transfer of a murine coronavirus persistent infection promotes cross-species transmissibility of mouse hepatitis virus feline aminopeptidase n 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tissue survival the immunological synapse: a focal point for endocytosis and exocytosis chemokines: a new class of neuromodulator? neuronal 'on' and 'off' signals control microglia this work was supported in part by grants from the ministry of education, culture, sports, science and technology grant number: 22 590 368). additional supporting information may be found in the online version of this article at the publisher's web site: supplemental figure s1 . immunofluorescence to detect erag (er), laminin (la), jhmv (v), zic2, podoplanin (pod), gfap (gf), cd11b, neun and nucleus (nuc). images were taken in areas of the peripheral nerve (pn) or key: cord-327568-5vo4nmei authors: tosini, fabio; ludovisi, alessandra; tonanzi, daniele; amati, marco; cherchi, simona; pozio, edoardo; gómez-morales, maria angeles title: delivery of sa35 and sa40 peptides in mice enhances humoral and cellular immune responses and confers protection against cryptosporidium parvum infection date: 2019-05-15 journal: parasit vectors doi: 10.1186/s13071-019-3486-8 sha: doc_id: 327568 cord_uid: 5vo4nmei background: cryptosporidium parvum is a major cause of diarrhea in children and ruminants at the earliest stages of life. maternal antibodies represent the main shield of neonate mammals for most of the infections. two recombinant antigens (sa35 and sa40), portions of two c. parvum proteins, were tested for their ability to induce immune responses in adult mice and for protection on neonate balb/c mice born from females immunised by mucosal delivery of both peptides. methods: adult balb/c mice were intraperitoneally immunised with sa35 and sa40, separately or mixed, and their immune response was characterised. furthermore, balb/c pregnant mice were immunised by mucosal delivery with an sa35/40 mix, before and during pregnancy. soon after birth, their offspring were infected with two doses (1 × 10(5) and 5 × 10(3)) of c. parvum oocysts and the parasitic burden was determined at 5 and 9 days post-infection. results: intraperitoneal immunisation with sa35 and sa40 induced specific igg and igg1 in serum, specific iga in the intestinal mucosa, increase of cd3+/cd4+ and cd30+ cells in splenocytes, which produced ifn-γ. neonates born from immunised mice and infected with 1 × 10(5) oocysts showed a significant reduction of oocysts and intestinal forms (23 and 42%, respectively). a reduction of all parasitic forms (96%; p < 0.05) was observed when neonates were infected with 5 × 10(3) oocysts. conclusions: sa35 and sa40 peptides induce specific humoral and cell-mediated immune responses to c. parvum in adult mice. moreover, mucosal administration of the sa35/40 mix in pregnant mice reduces c. parvum burden in their litters. the cryptosporidium genus includes 38 species that infect a wide range of vertebrates, including many mammals. humans are also susceptible to these parasites, and domesticated and wild ruminants, particularly livestock, represent important sources of zoonotic transmission [1] . in mammals, cryptosporidium spp. mainly affect the gut, causing gastrointestinal symptoms and diarrhoea. approximately 90% of human infections are caused by cryptosporidium parvum and cryptosporidium hominis, although over 20 species can infect humans [2] . in immunocompetent patients, cryptosporidiosis is a self-limiting infection of the small intestine that causes watery diarrhoea and can last up to ten days, whereas the infection can become a chronic and life-threatening disease in immunocompromised individuals such as aids subjects [3] . parasites & vectors *correspondence: fabio.tosini@iss.it; mariaangeles.gomezmorales@iss.it european union reference laboratory for parasites, istituto superiore di sanità, rome, italy transmission occurs via the faecal-oral route through the ingestion of oocysts contaminating food or water. oocysts are highly resistant to environmental conditions, chlorination and other sterilisation treatments, and contamination of water plants can therefore cause massive outbreaks [4] . it follows that the food and agriculture organisation and the world health organisation consider protozoa of the genus cryptosporidium to be one of the most significant food-borne parasites [5] . in livestock husbandry, c. parvum is a major cause of severe diarrhoea among neonate calves and lambs, resulting in substantial costs for farmers. a recent study on diarrhoea conducted on calves younger than one month reported that c. parvum is responsible, as the sole agent, for 37% of diarrhoeal infections and for 20% of coinfections with other intestinal pathogens [6] . in recent years, infections by c. parvum and c. hominis have emerged as significant causes of infantile diarrhoea. an extended case-control study, referred to as the global enteric multicenter study (gems) [7] , has shown a high prevalence of cryptosporidium spp. among children in developing countries, ranking these protozoa among the four pathogens responsible for the majority of diarrhoea cases in children younger than five years [8] . the prevalence of these parasites is higher in infants aged less than 11 months and, in this age range, cryptosporidium spp. is the second most common cause of death associated with diarrhoea, after rotavirus [9] . the susceptibility of neonates and children to cryptosporidium spp. is not completely understood. in the nursing period, the innate immune response of enterocytes, which is usually triggered by the stimulation of toll-like receptor 4 (tlr4), is almost completely inhibited so as to favour the establishment of commensal flora in the gut [10] . given that the immune response to cryptosporidium spp. begins with tlr4 stimulation and the consequent activation of the nf-kappa b pathway [11] , the temporary inhibition of the innate enterocyte response could promote the proliferation and dissemination of cryptosporidium spp. in the neonatal intestine. at present, nitazoxanide is the only drug approved for cryptosporidiosis by the us food and drug administration (us-fda), but this drug cannot be used in children younger than one year and is ineffective in immunodeficient patients [12] . a vaccine for cryptosporidiosis is not yet available, and immune protection of neonates is difficult to achieve because of their early-age immune status. however, various cryptosporidium spp. proteins, particularly those considered to be virulence factors, have been proposed as possible vaccine candidates [13, 14] . in this context, the passive immunisation of neonates might be an alternative to a conventional vaccine. indeed, passive immunity provided through hyperimmune bovine colostrum has been shown to establish an appreciable level of protection in human patients with aidsassociated cryptosporidiosis [15, 16] . more recently, the protective role of maternal anti-cryptosporidium antibodies has been demonstrated in two natural contexts. a survey of a child cohort in bangladesh reported that the presence of anti-cryptosporidium iga in breast milk protects neonates from cryptosporidiosis [17] . a second study, conducted in an endemic area of tanzania on a cohort of breastfeeding mothers and their infants (0 to 6 months), has shown that the infection rate increases with a reduction in maternal milk ingestion [18] . this study investigates the feasibility of an immunological preventive treatment to impede or reduce cryptosporidiosis in newborn mammals. as a preliminary step, two recombinant antigens, sa35 and sa40 [19] , were inoculated in adult mice to achieve the onset of a specific immune response. a group of female mice were then immunised by mucosal delivery of the same antigens prior to and during their pregnancies in parallel with a control group treated only with the adjuvant. the offspring of these mice were then infected with two different doses of c. parvum oocysts (1 × 10 5 and 5 × 10 3 ) and the parasite burden was measured by flow cytometry, microscopic count and a quantitative pcr assay. our results indicated that neonates born from immunised mothers had a reduced parasitic load compared to the neonates born from the control mice. cryptosporidium parvum oocysts (isolate code issc6) were collected from faeces of experimentally infected calves and purified by centrifuging with sucrose and percoll ® (sigma-aldrich, saint louis, mo, usa) densitygradients [20] . cryptosporidium parvum-crude extract (cce) was obtained as previously described [21] . heatlabile enterotoxin (lt) was obtained from chiron spa (siena, italy), and ketavet and xylazine solutions were provided by the animal care unit of the istituto superiore di sanità in rome, italy. a rabbit anti-c. parvum igg polyclonal antibody, which recognises all stages of c. parvum [22] , was used for ifa assays. the recombinant peptides sa35 (mw, 35 kda) and sa40 (mw, 40 kda) represent antigenic portions of two microneme proteins of c. parvum named cpa135 (cgd7_1730) and gp900 (cgd7_4020), respectively [23, 24] . protein id codes refer to sequences in the cryptodb database, release 37 [25] . the two peptides were purified as previously described [26] , dialysed in phosphate buffered saline (pbs) plus 50% glycerol and stored in aliquots at −20 °c. both peptides were used as a sole immunising agent or as a 1:1 mixture of the two antigens (referred to hereafter as the sa35/40 mix). eight-to ten-week-old balb/c female mice (charles river laboratories, milan, italy) were housed in the animal care unit of the istituto superiore di sanità in accordance with european directive 63/2010. the in vivo protocol was approved by the italian ministry of health (approval no. 8/2014-b, 15 january 2014). three groups of five female mice each were immunised intraperitoneally: one group with 5 μg of sa35, a second group with 5 μg of sa40, and a third group with the sa35/40 mix (5 μg plus 5 μg of sa35 and sa40, respectively). a control group (n = 5) was injected with an equal volume of pbs instead of antigens, according to the same immunisation schedule, which was as follows: day 0, peptides or pbs were administered in freund's complete adjuvant (sigma-aldrich); day 14, the same amount of peptides or pbs were inoculated using freund's incomplete adjuvant (sigma-aldrich); day 28, the last booster was provided using the same quantity of peptides or pbs without adjuvant. the mice were bled on days 0, 7, 14, 21, 28 and 35 following initial immunisation. individual sera were stored at −20 °c. faecal samples for an iga assay were collected weekly from each mouse separately, weighed and dissolved in 1.0 ml of pbs with 0.1% sodium azide per 100 mg of faecal material, and a cocktail of protease inhibitors (sigma-aldrich) was added at a ratio of 1:20 to each sample. the samples were then vortexed for 5-10 min and centrifuged to remove debris; supernatants were collected and stored at −70 °c. levels of igg, igg1 and iga in serum samples and iga in faecal samples specific to c. parvum were determined by elisa. levels of igg and iga specific to sa35 and sa40 peptides were also determined by elisa. microtiter plates (nunc-immuno plate polysorp ™ , sigma-aldrich, roskilde, denmark) were blocked using 200 μl per well of 1% w/v bsa in pbs, at room temperature (rt) for 90 min and coated with 1 µg/ml of recombinant peptides (sa35 or sa40) or 5 µg/ml of cce in a 50 mm carbonate/bicarbonate buffer with a ph of 9.6, and incubated at 37 °c for 90 min. serum samples were diluted in pbs with 0.5% w/v bsa and 0.05% v/v tween-20. supernatants of faecal samples were diluted as described above. plates were washed three times with 0.05%-tween-20 in pbs after incubation with sera, and washes were repeated after incubation using conjugated antibodies. dilutions of 1:50 for sera and 1:10 for faecal supernatants were established to be the optimal working conditions. these dilutions were then added in duplicate to wells and incubated overnight at 4 °c. as negative controls, the corresponding pre-immune faecal and serum samples from each mouse were used at the same dilution in elisa. bound antibodies were detected by incubation with 1:500 dilutions of biotin-conjugated rat monoclonal anti-mouse igg, igg1, and iga antibodies (bd pharmingen tm , san diego, ca, usa) at rt for 5 h followed by 2.5 mg/ml avidin-peroxidase (sigma-aldrich) for 30 min. the peroxidase substrate tmb (3,3′,5,5′-tetramethylbenzidine, kierkegaard and perry laboratories, gaithersburg, md, usa) was added to each well and the optical density was measured by a multiskan spectrum elisa reader (thermo scientific, vantaa, finland) at 450 nm. elisa was performed for each isotype using the same protocol. at the end of the immunisation schedule, the mice were sacrificed and their spleens were harvested under sterile conditions and disrupted using a syringe. the erythrocytes were lysed, and the splenocytes were then suspended in complete medium [rpmi-1640 containing 10% foetal bovine serum, 25 mm hepes, 2 mm l-glutamine, 100 u/ml penicillin, 100 µg/ml streptomycin, 1 mm sodium pyruvate, 5.5 × 10 −5 m 2-mercaptoethanol and 0.1 mm non-essential amino acids, all from hyclone laboratories (logan, ut, usa)]. the final cell concentration for proliferation analysis was 1 × 10 6 cells/ml and cells were settled in flat-bottom 96-well plates (costar corporation, cambridge, ma, usa). for cytokine and phenotypic analysis, cells were suspended at 2 × 10 6 cell/ ml in 5 ml tubes (becton dickinson, franklin lakes, nj, usa). cell cultures were stimulated with 5 μg/ml of cce, 1 μg/ml of sa35 or 2.5 μg/ml of sa40 and incubated in 5% co 2 at 37 °c for five days. cell cultures for negative controls were grown without any stimulants, whereas positive control cells were stimulated with 1.0 μg/ml of concanavalin a. lymphocyte proliferation was measured after 12 h of culture by 3 h-thymidine incorporation in the presence of 0.5 µci/well [ 3 h]-thymidine (amersham life science, buckinghamshire, uk) sampled in triplicate. proliferation was expressed as a stimulation index (si) (i.e. counts per minute of cce-stimulated cells divided by counts per minute of unstimulated cells). an si above 3.0 was considered positive. for the cytokine analysis, culture supernatants were harvested after five days of culture and stored at −70 °c until assayed. cytokine levels (ifn-γ, il-4, il12) were measured in culture supernatants by elisa. essentially, 96-well plates (nunc-immuno tm plate maxisorp tm surface, sigma-aldrich, roskilde, denmark) were coated with a solution of 1 μg/ml (in 0.1 m na 2 hpo 4 , ph 9.0) of monoclonal rat anti-mouse cytokine antibody (bd pharmingen). after overnight incubation at 4 °c, the plates were washed with pbs-t and blocked with 1% bsa (sigma-aldrich) in pbs for 2 h at 37 °c. after washing, serial dilutions of culture supernatants and standards, i.e. recombinant mouse cytokines, were added to the wells and incubated overnight at 4 °c. the plates were then washed and the appropriate biotin-conjugated rat antimouse cytokine antibody (bd pharmingen) was added to the well and incubated for 2 h at 37 °c. after washing and the addition of hrp-conjugated streptavidin (bd pharmingen) for 2 h at 37 °c, the tmb (kirkegaard & perry laboratories) substrate was added to the wells and absorbance was measured at 450 nm after 10-20 min. standard curves were generated for each cytokine using the corresponding murine recombinant standard. the minimum detection level for each cytokine by elisa was < 15 pg/ml for ifn-γ, < 4 pg/ml for il-4, and < 15 pg/ ml for il-12. to analyse the lymphocyte subsets after incubation with cce, the splenocyte suspension from each mouse was washed with pbs containing 2% bsa, and further re-suspended in 100 µl of the same buffer. the cells were then incubated with 5 µl of the following fluorescein isothiocyanate (fitc) or phycoerythrin (pe) conjugated monoclonal antibodies to murine leukocyte differentiation molecules: anti-cd3, anti-cd4, anti-cd8, anti-cd25, and cd30 (bd pharmingen). the cells were analysed for single and dual fluorescence assay in a fluorescence-activated cell-sorter (facscalibur; becton dickinson, franklin lakes, nj, usa) and data were acquired by cell-quest software (becton dickinson). the instrument was set to measure the fluorescence intensities of forward-angle light scatter (fsc), side-angle light scatter (ssc-h), fitc (fl1), pe (fl2) and fitc (fl1). cells incubated with fitc-and pe-conjugated mouse igg1/igg2a served as isotype control. the proportion (%) of cells expressing a given molecule was determined as the average of three replicas. two groups (experimental and control groups) of five mice each were anaesthetised with ketavet (100 mg/ml) and xylazine (20 mg/ml) at 50 mg/kg and 3 mg/kg, respectively. the mice from the experimental group were then immunised intranasally with 15-20 μl of pbs containing 1 μg of lt with 5 μg of sa35/40 mix, whereas the mice from the control group were inoculated intranasally with 15-20 μl of pbs containing 1 μg of lt. after seven days, the same immunisation protocol was repeated. then, bedding from cages housing male mice was transferred to the cages housing females for 48 h to induce oestrus. subsequently, one male and two females were kept in the same cage for 72 h and the females were examined daily for the presence of copulatory plugs. the day of plug detection was assumed to be day 0 of pregnancy [27] . immunisations were repeated at days 7, 14, and 21 of pregnancy ( fig. 1 ). blood samples were taken by tail bleeding on days 0, 7, 14, 21, 28, 35 and 230 after initial immunisation. individual sera were stored at −20 °c until analysis. faecal samples were collected at the same times and treated and stored as described above. specific igg and iga to c. parvum were measured in serum and faecal samples by elisa. proliferation assays and phenotypic analysis of spleen cells were performed as described above. to study the protection induced by mucosal immunisation, six litters of three-day-old balb/c mice (39 mice) were used. two litters, which were born from mothers immunised intranasally with sa35/40 with lt, were infected with 10 5 oocysts and then sacrificed five [28] and nine days post-infection (p.i.), respectively [29] . another litter born from an intranasally immunised mother was infected with 5 × 10 3 oocysts and sacrificed at nine days p.i. the remaining three litters were the respective control groups. all suckling mice were experimentally infected by the oral route using oocysts suspended in 20 µl of pbs with a 24-gauge gavage needle and kept with their mothers. suckling mice were anaesthetised before euthanasia and the whole intestine was collected. lymphocytic phenotypic analysis was performed on spleens from mice born from intranasally immunised mothers, infected with 10 5 oocysts and sacrificed five days later. the entire intestinal content was collected after washing of the intestinal tract with pbs. samples were vortexed for 30 s with a 1-min pause. the supernatant was centrifuged at 2500×g for 10 min at 4 °c. the pellet containing oocysts and free forms of the parasite was then suspended in 1 ml of pbs and vortexed for 30 s. each sample was divided in two aliquots: one aliquot was tested by an immunofluorescence assay, i.e. flow cytometry or indirect immunofluorescence assay (ifa), whereas the second aliquot was used to extract dna for qpcr (see below). using flow cytometry, the number of oocysts in the faecal sample was evaluated using an acquisition gate based on the forward-angle light scatter (fsc-h) characteristic and the fluorescence intensities of fitc (on fl1 detector) of a positive control [30, 31] . the positive control consisted of an oocyst suspension in pbs incubated with the anti-c. parvum igg. flow cytometry analysis was performed three times for each mouse and the number of oocysts was expressed as the mean ± sd for each mouse group. intracellular parasites were counted in the ileum homogenate from each mouse [28, 32] . homogenisation was carried out individually in 1 ml of pbs for 1 min at 20×g using a potter homogeniser and further diluted 1:5 in pbs. the suspension was then filtered using a 30-µm filcon syringe (beckton dickinson) and incubated with the anti-c. parvum igg and an anti-rabbit fitc antibody for 30 min at rt. intracellular parasites were counted using flow cytometry as above. the number of intracellular stages was expressed as the mean ± sd for each mouse group. when significant differences between the experimental and control groups were not found, the number of oocysts was also evaluated using ifa (see below). for ifa, the oocyst suspension was mixed with 20 µl of a solution of 0.5 µg/ml anti-c. parvum igg in pbs. the mixture was kept in the dark at 4 °c for 30 min and then centrifuged for ten min at 2500×g. the pellet was resuspended in 1 ml of pbs, and 20 µl of an anti-rabbit fitc antibody was added. the samples were kept in the dark at 4 °c for 30 min and then centrifuged at 2500×g for 10 min. the pellet was suspended in 1 ml of pbs and transferred to a polystyrene tube and kept at 4 °c. all samples were analysed on the day of oocyst collection. the number of oocysts was evaluated by fluorescence microscopy (zeiss axioplan 2; carl zeiss, jena, germany) at 400× magnification [31] . after washing (see above), the intestines from mice infected with 5 × 10 3 oocysts were fixed in 4% formaldehyde in pbs for 4 h for histological examination, which was performed using the following procedure. the ileum portion was cut and washed in tap water overnight, dehydrated in increasing concentrations of ethanol from 50 to 100%, placed in 100% xylol and finally included in paraffin. the paraffinated tissue was cut using a leica rm2125 rts microtome (leica biosystems, nussloch, germany) into microscopic slices of 4-6 μm, distributed on microscopic slides and dried overnight at 37 °c. the slices were then deparaffinated by immersion in 100% xylol for 5 min and re-hydrated with decreasing concentrations of ethanol from 100 to 50% and a final wash in distilled water for 2 min. antigen retrieval from fixed tissue was performed using the following "pressure cooking protocol": the microscopic slides were placed in a stainless steel slide rack in the boiling citrate buffer (10 mm sodium citrate, 0.05% tween 20, ph 6.0) in a pressure cooker heated by a hot plate; the lid was then closed for steaming at high pressure for 90 s, and the pan was then rapidly cooled using running tap water on the lid. immediately after the slides were prepared for indirect immunofluorescence assay (ifa), blocked using 2% foetal calf serum (fcs) in pbs for 1 h at room temperature, incubated for 1 h with anti-c. parvum rabbit serum [26] diluted 1:500 in 2% fcs in pbs, and washed three times (5-min wash) with pbs to remove unbound antibodies. secondary incubation was conducted for 1 h at room temperature using goat anti-rabbit antibody conjugate with fluorescein (fitc) (biorad, hercules, ca, usa) diluted 1:1000 in 2% fcs in pbs, and the slides were then washed as above and sealed using prolong antifade (thermo fisher scientific, waltham, ma, usa). microscopic inspection of the tissue slices was conducted using a fluorescence microscope (zeiss axioplan 2) at 1000× magnification and digital images were obtained using axiovision software (carl zeiss). for qpcr, aliquots of whole intestinal content from mice infected with 5 × 10 3 oocysts (see above) were centrifuged, suspended in 1 ml of 50% ethanol in pbs and stored at −20 °c until dna extraction. each aliquot was centrifuged, washed once with pbs and resuspended in 100 µl of lysis buffer (10 mm dtt, 1.8 mg/ml proteinase k in pbs), transferred to a 96-well plate and incubated for 1 h at 55 °c. for automated extraction, the plates were transferred to a biosprint 96 apparatus (qiagen, hilden, germany) using the protocol for blood samples and one-for-all vet kit reagents (qiagen), and dna was eluted in a final volume of 50 µl. dna was also extracted in the same way from referenced dilutions of purified oocysts in triplicate to obtain samples with 10 5 , 10 4 , 10 3 , 100, 10, 1 and a theoretical 0.1 oocysts, and used for a standard curve based on the median crossing threshold (ct) in qpcr experiments. dna samples from infected mice and referenced dilutions were analysed using real-time pcr assay (qpcr) based on the cowp gene [33] . this assay was tested for sensitivity using reference samples, obtaining positive curves with a single oocyst (ct = 36 in three out of three replicas) and up to a theoretical 0.1 oocysts (ct = 39 in one out of three replicas), and samples producing less than 40 ct were considered positive. cowp primers were synthesised by primm (milan, italy), and taqman probe and cowp probe labelled with 5′-hexachlorofluorescein (hex; λ em = 553 nm) were from tib molbiol (berlin, germany). each pcr reaction was completed in a final volume of 25 μl, comprising 12.5 μl of lightcycler 480 probes master mix containing fast-start taq dna polymerase, a proprietary buffer including deoxynucleotide triphosphates (dntps), mgcl 2 at a final concentration of 3.2 mm, 300 nm of each primer, 500 nm of taqman probe, and 5 µl (1/20 of whole intestinal content) as dna sample. all reactions were performed on a 96-well plate in a lightcycler 480 pcr system (roche diagnostics, mannheim, germany) in triplicate and three negative controls were included in each plate. the pcr conditions were: 10 min of incubation at 95 °c followed by 40 cycles of 95 °c for 15 s and 60 °c for 1 min. fluorescence data (three data real-time points) were collected at the end of each cycle. to compare each experimental group with the relevant control group, the mann-whitney u-test was used. a p-value < 0.05 was considered significant. the ip immunisation of adult balb/c mice to a single antigen (sa35 or sa40) or to a mixture of the two antigens (sa35/40 mix) induced specific anti-cryptosporidium igg in serum after day 14 following initial administration. later, the level of specific igg progressively increased over time, reaching the highest response 35 days after initial immunisation (sa35, u ( fig. 2c ). as expected, sera from mice immunised with sa35, sa40 or sa35/40 mix reacted with their respective homologous antigens by elisa (data not shown). specific anti-cryptosporidium iga were detected in faeces from day 7 after initial immunisation until the last sampling (day 35) with all the three antigen formulations (u (0) = 2, z = −2.50672, p = 0.0079 for sa35, sa40 and sa35/40 mix; fig. 2d ). no specific iga response was detected in faeces at day 0 in the control mice. cce induced proliferation in splenocytes from sa35, sa40 and sa35/40 mix in ip immunised mice. the highest responses to cce were obtained in splenocytes from mice immunised with sa35 alone or the sa35/40 mix. splenocytes from immunised and control mice responded to concanavalin a. single peptides also induced proliferation in splenocytes from mice immunised with homologous peptides (fig. 3) . cce induced ifn-γ production in the splenocytes from all groups of immunised mice. splenocytes from mice immunised with sa35 or sa40 produced higher levels of ifn-γ after stimulation with homologous peptides than mice immunised with the sa35/40 mix and stimulated with the same mix. il-12 production was observed in all mouse groups; the highest level of il-12 production was observed in splenocytes from mice immunised with sa35. minimal basal production of il-4 was detected in spleen cells from some groups of immunised mice (table 1) . induced cell populations in splenocytes were analysed at day 35 after initial immunisation. the percentages of cd3+ and cd4+ t cells were similar in mice immunised with sa35, sa40 and the sa35/40 mix and were significantly higher than in non-immunised control mice. however, the percentage of cd8+ t cells was similar among all groups, including the controls. moreover, the percentage of activated cd4+ lymphocytes co-expressing the high affinity interleukin-2 receptor (cd25) was decreased in all immunised groups, particularly in the sa35 immunised group (u (0) = 5, z =, 2.80224, p = 0.0022; fig. 4 ). the mucosal delivery of sa35/40 mix in female balb/c mice induced specific anti-cryptosporidium igg (mainly igg1) in serum 21 days after initial immunisation. the igg level increased over time from day 28 until day 35. high igg levels were detected until the last observation on day 230 (versus control group u (0) = 2, z = −2.50672, p = 0.01208 for igg and igg1; fig. 5a , b). a low specific iga level was detectable in serum until the end of the experiment (230 days after initial immunisation; fig. 5c ). in faeces, specific anti-cryptosporidium igg and iga began to increase on the day 28 following the first delivery and then over time (versus control group u (0) = 3, z = 2.64733, p = 0.0022 and u (0) = 5, z = −2.80224, p = 0.00512 for igg and iga, respectively). no specific igg or iga responses were detected in faeces at day 0 or in the control mice (fig. 5d, e) . cce and cona induced proliferation in splenocytes from mice immunised intranasally with the sa35/40 mix. splenocytes from control mice responded only to concanavalin a (fig. 6) . the cytometric analysis was performed in cce-stimulated splenocytes from both adult mice immunised intranasally and their litters. spleens were harvested from adult mice at day 236 following initial immunisation. the percentages of cd3+/cd4+ and cd30+ t cells were increased in adult mice treated with the sa35/40 mix. the same results were obtained with the cce-stimulated splenocytes from passively immunised litters infected with 10 5 oocysts (table 2) . when infected with 10 5 oocysts, passively immunised suckling mice showed similar levels of reduction at day 5 and day 9 following infection. in particular, a significant reduction of 23% of excreted oocysts (u (20) = 38, z = −2.76956, p = 0.0028) and 42% of intracellular forms (u (14) = 23, z = −2.43588, p = 0.00734) in the ileum was observed using flow cytometry (fig. 7a) . infection of neonates was also undertaken with a lower dose of oocysts in order to get closer to a natural infective dose using 5 × 10 3 oocysts. the histological examination of the ileums of infected mice did not show any evidence of the infection (i.e. parasitophorous vacuoles) in the analysed sections from neonates born from immunised females (fig. 8c, d) . by contrast, newborns from non-immunised females had numerous parasitophorous vacuoles disseminated along the epithelial lining of the intestinal lumen (fig. 8c, d) . indeed, a quantitative analysis of the entire intestinal content at day 9 p.i. showed a striking 96% reduction in parasites in newborns from immunised females (u (4) = 12, z = 2.65165, p = 0.00402), as evaluated by microscopic counting after ifa (fig. 7b) . to confirm the overall reduction in parasitic forms in passively immunised newborn mice infected with the lower infective dose (i.e. 5 × 10 3 oocysts), the intestinal contents of these newborns were evaluated using a realtime qpcr assay. as shown in fig. 9 , ct values in faecal samples from suckling mice born from immunised mothers ranged between 34 and 40, i.e. equivalent to 5-0.1 oocysts. four out of 6 samples showed a ct value higher than 37, thus with less than one oocyst equivalent in their content. ct values for the control mice ranged between 27 and 33, i.e. equivalent to 10-100 oocysts. therefore, qpcr analysis confirmed that the parasitic load in the intestinal contents of newborns from immunised mothers was extremely low when newborns were infected with the lower dose of 4.5 × 10 3 oocysts. control of neonatal cryptosporidiosis is a challenging task because of the lack of appropriate drugs and the difficulties in providing effective immunisation in the early phase of life. here, we explored the protective effect of anti-c. parvum maternal antibodies transferred to newborns through the placenta during gestation and through the colostrum in the first days of life. to this end, two recombinant peptides (sa35 and sa40) were first immunologically characterised by ip injection in adult mice to evaluate their potential as immunogens. both peptides, separately and in a mixed formulation, induced specific igg and iga responses. sa35 and sa40 peptides have been previously described as immunodominant antigens involved in the maintenance of t-cell response in healthy c. parvum-sensitised individuals [20] . it is important to note that the amino acid sequences of sa35 and sa40 peptides do not show similarities with mammalian proteins, but, by contrast, are also highly conserved in other cryptosporidium species and particularly in other human pathogens (data not shown). therefore, it is most likely that antibodies for sa35 and sa40 also react with the strictly related proteins of c. hominis and c. ubiquitum. in this study, it has been demonstrated that ip immunisation with these recombinant peptides is able to support an effective cell-mediated immunity in response to specific stimuli: cce-, sa35-and sa40-induced specific proliferation in splenocytes from all immunised adult mice. however, maximum proliferative responses to cce were obtained in splenocytes from mice following ip immunisation with sa35 or the sa35/40 mix (fig. 3) . the induced cell populations showed a higher percentage of cells expressing cd3+ and cd4+. moreover, splenocytes from mice immunised with sa35 showed a significant reduction in the percentage of regulatory cells (cd4+/cd25+) compared to the control group, whereas the others did not (fig. 4) . overall, the three antigen formulations (sa35, sa40 or sa35/40 mix) induced ifnγ, il-12 and, to a lesser extent, il-4 in immunised mice (table 1) . what is noteworthy is that stimulation with the homologous antigen resulted in the highest level of production of these cytokines even when the splenocytes also responded to stimulation with cce (table 1) . experimental studies and clinical cases have demonstrated that activation of cd4+ lymphocytes and production of ifn-γ are crucial for parasite clearance [34] [35] [36] [37] . indeed, ifn-γ production is one of the essential functions of cd4+ cells in response to cryptosporidium antigens [38, 39] . with regard to humoral response, all three antigenic formulations (sa35, sa40 or sa35/40 mix) induced specific igg, igg1 and secretory iga (fig. 2) . the production of these transferable antibodies is a fundamental requirement in conferring protective immunity for neonates. therefore, the antigenic sa35/40 mix was used for mucosal immunisation of female mice in order to evaluate the protective effect on their offspring. the notes: adult female balb/c mice were mucosally immunised with sa35/40 mix. cells were stimulated with c. parvum crude extract and were gated for 90-95% cd45 + . analysis of splenocytes from adult mice was carried out on the last day of the experiments. the number of mice is shown in fig. 1 . newborn mice born from intranasally immunised mothers were infected with 10 5 oocysts and sacrificed five days later the comparison between c. parvum-infected littermates born from immunised mothers and their respective controls, i.e. c. parvum-infected littermates born from mothers immunised only using a mucosal adjuvant, showed that maternal protection is highly effective with the lower infective dose (5 × 10 3 ), since there was a 96% reduction in parasitic burden (fig. 7) . in adult females, mucosal immunisation with the sa35/40 mix leads to a long-lasting (33 weeks) humoral response at systemic and intestinal level (fig. 5) . moreover, splenocytes from balb/c mothers specifically proliferated in response to cce and the resulting population showed an increase in the percentages of cd3+/cd4+ and cd30+ cells ( table 2) . the experimental infection of neonate mice born from immunised mothers showed that when neonates were infected with a massive dose of oocysts (1 × 10 5 ), the protective effect on the progeny is measurable but ineffective (−23% of excreted oocysts). conversely, a drastic reduction (96%) in oocyst emission was observed when neonates were infected with a lower dose (5 × 10 3 oocysts), which still represents a huge amount given the small weight (about 1 g) of newborn mice (fig. 7) ; in fact, this dose is comparable to a dose of 30 × 10 7 oocysts in a 60 kg human. these oocyst numbers were tested in the first instance to allow a quantitative analysis of excreted oocysts and of all parasitic forms using flow cytometry, although the 5 × 10 3 dose yielded parasite amounts below the sensitivity threshold for the instrument. to overcome this technical limitation, ifa/microscopic counting and qpcr were used to evaluate the minimal quantity of parasites in infected mice. moreover, dna samples for qpcr were prepared using an ad-hoc automated method in order to reduce handling errors. cell-mediated immunity showed unexpected signs of activity in splenocytes harvested from litters from immunised mothers, as there was an increase in the percentage of cd3+-cd4+ cells as well as cd30+ cells (table 2) . similarly, t-cell responses were identified after maternal vaccination in newborn mice of a plasmodium yoelii model, although there was no parallel increase in antibody response [40] . recent studies indicate that during pregnancy, maternal molecules such as inflammatory cytokines, as well as microbial products, are transferred in utero to the foetus and this influences the foetal immune system [41] . thus, neonatal hosts may compensate for an insufficiency in adaptive immune responses by having a heightened capacity to mount certain types of innate immune responses [42] . attempts at passive immunisation through administration of hyperimmune colostrum have been performed in humans and in other mammals. indeed, hyperimmune bovine colostrum was given to aids patients with prolonged cryptosporidiosis, resulting in a reduction in diarrhoea and an improvement in symptoms [15, 16] . in livestock, passive immunisation with recombinant antigens has shown a certain efficacy in protecting passively immunised calves and kids [43, 44] . more recently, heterologous protection was also successfully tested through administration of hyperimmune ovine colostrum in neonate mice [45] . unlike previous studies, this study combined the use of a mixture of two defined antigens extensively characterised as immunogens. a further peculiarity of this study is the mucosal immunisation of females to confer protection for their progeny. maternal immunisation protocols for other pathogens have been applied successfully to prevent neonatal infectious diseases in various contexts. with regard to livestock, a vaccine for rotavirus, coronavirus and escherichia coli (rotavec-corona ® , msd) is commonly used to immunise heifers and preparturient cows. this treatment induces a higher titre of specific antibodies for these pathogens in colostrum and milk [46] and has significant efficacy in reducing diarrhoea in neonate calves [47] . in humans, maternal immunisation with tetanus toxoid has fig. 9 quantification of cowp gene dna copies by qpcr in the intestinal content of neonate mice infected with 5 × 10 3 cryptosporidium parvum oocysts. values are expressed as the mean of cycles for crossing thresholds (ct), and each sample was tested in triplicate. standard curve (black dots) was obtained from dna extracted from the following oocysts dilutions: 1 × 10 5 (1), 1 × 10 4 (2), 1 × 10 3 (3), 100 (4), 10 (5), 1 (6) and 0.1 (7) . blue dots represent ct values from control mice. red dots represent ct values from immunised mice. values from control mice are between 33 and 29 cycles, equivalent to dna amounts from 6 to 100 oocysts, whereas values from immunised mice are between 39 to 35 cycles, equivalent to dna amounts from 0.1 to 5 oocysts been the first example of effective prevention for neonates [48] and it has been extensively applied as part of the who maternal and neonatal tetanus elimination (mnte) program. successful immunisations have been also achieved with influenza virus [49] and with the acellular pertussis antigen vaccine that protects infants from clinical pertussis [50] . therefore, maternal immunisation can be an effective strategy to limit neonatal cryptosporidiosis before the development of a mature immune system. the sa35 and sa40 peptides enhance humoral and cellmediated immune responses to c. parvum in adult mice. mucosal immunisation of pregnant mice with a mixture of sa35 and sa40 antigens (sa35/40 mix) resulted in a remarkable reduction in parasite load in the gut of their infected offspring. overall, this result represents the proof of concept that maternal immunisation can effectively prevent neonatal cryptosporidiosis. genetic diversity and population structure of 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recombinant protein protection of kids against cryptosporidium parvum infection after immunization of dams with cp15-dna oral administration of hyperimmune anti-cryptosporidium parvum ovine colostral whey confers a high level of protection against cryptosporidiosis in newborn nmri mice serological, colostral and milk responses of cows vaccinated with a single dose of a combined vaccine against rotavirus, coronavirus and escherichia coli f5 (k99) evaluation of a protocol to reduce the incidence of neonatal calf diarrhoea on dairy herds neonatal tetanus in new guinea. effect of active immunization in pregnancy maternal immunization with inactivated influenza vaccine: rationale and experience effect of a prepregnancy pertussis booster dose on maternal antibody titers in young infants publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we are very grateful to dr silvia vendetti for her helpful suggestions on the mucosal delivery mouse model. we would like to thank simone m. cacciò for the critical reading of the manuscript. all data presented in this manuscript are available at the istituto superiore di sanità. not applicable. the authors declare that they have no competing interests.received: 28 november 2018 accepted: 6 may 2019 key: cord-002341-v4r5d26a authors: chan, jasper fuk-woo; zhang, anna jinxia; chan, chris chung-sing; yip, cyril chik-yan; mak, winger wing-nga; zhu, houshun; poon, vincent kwok-man; tee, kah-meng; zhu, zheng; cai, jian-piao; tsang, jessica oi-ling; chik, kenn ka-heng; yin, feifei; chan, kwok-hung; kok, kin-hang; jin, dong-yan; au-yeung, rex kwok-him; yuen, kwok-yung title: zika virus infection in dexamethasone-immunosuppressed mice demonstrating disseminated infection with multi-organ involvement including orchitis effectively treated by recombinant type i interferons date: 2016-11-12 journal: ebiomedicine doi: 10.1016/j.ebiom.2016.11.017 sha: doc_id: 2341 cord_uid: v4r5d26a background: disseminated or fatal zika virus (zikv) infections were reported in immunosuppressed patients. existing interferon-signaling/receptor-deficient mouse models may not be suitable for evaluating treatment effects of recombinant interferons. methods: we developed a novel mouse model for zikv infection by immunosuppressing balb/c mice with dexamethasone. results: dexamethasone-immunosuppressed male mice (6–8 weeks) developed disseminated infection as evidenced by the detection of zikv-ns1 protein expression and high viral loads in multiple organs. they had ≥ 10% weight loss and high clinical scores soon after dexamethasone withdrawal (10 dpi), which warranted euthanasia at 12 dpi. viral loads in blood and most tissues at 5 dpi were significantly higher than those at 12 dpi (p < 0.05). histological examination revealed prominent inflammatory infiltrates in multiple organs, and cd45 + and cd8 + inflammatory cells were seen in the testis. these findings suggested that clinical deterioration occurred during viral clearance by host immune response. type i interferon treatments improved clinical outcome of mice (100% vs 0% survival). conclusions: besides virus dissemination, inflammation of various tissues, especially orchitis, may be potential complications of zikv infection with significant implications on disease transmission and male fertility. interferon treatment should be considered in patients at high risks for zikv-associated complications when the potential benefits outweigh the side effects of treatment. zika virus (zikv) is an emerging flavivirus that has been largely neglected for n 60 years after its discovery due to its restricted geographical distribution and its presumed low clinical significance (chan et al., 2016a) . since 2007, large-scale outbreaks of zikv infection have occurred in the pacific islands, latin america, and most recently, usa and southeast asia (duffy et al., 2009; musso and gubler, 2016; zhu et al., 2016) . as of 27 october 2016, n 70 countries/territories have ebiomedicine 14 (2016) 112-122 reported continuing mosquito-borne transmission of zikv (world health organization. zika situation report. october 27, 2016) . in addition to mosquito-borne transmission, sexual and transplacental transmissions of zikv have also been reported (chan et al., 2016a; musso et al., 2015a; foy et al., 2011; calvet et al., 2016) . these non-vectorborne transmission routes render the control of the continuing epidemic more complicated. zikv was not considered as an important human pathogen in the past as most infected adult patients were asymptomatic or developed a self-limiting acute febrile illness which resolved within 1-2 weeks (chan et al., 2016a; duffy et al., 2009 ). however, it has been recently recognized that infected mothers may transmit the virus transplacentally to developing fetuses, leading to congenital malformations, including microcephaly, cerebral malformations, ophthalmological and hearing defects, and arthrogryposis (chan et al., 2016a; mlakar et al., 2016; de paula et al., 2016; leal et al., 2016) . some infected adults may also develop severe neurological complications, such as guillain-barré syndrome, meningoencephalitis, and myelitis (cao-lormeau et al., 2016; carteaux et al., 2016; mecharles et al., 2016) . moreover, zikv-related fatalities have been increasingly recognized. most of the patients with fatal infection had underlying medical conditions and some were markedly immunosuppressed, including a patient with systemic lupus erythematosus and rheumatoid arthritis who was on corticosteroid therapy and died of disseminated infection with detectable zikv rna in blood, brain, spleen, liver, kidney, lung, and heart obtained at postmortem examination (pan american health organization/world health oganization (paho/who), 2015; sarmiento-ospina et al., 2016; arzuza-ortega et al., 2016) . a number of animal models have been developed for studying the pathogenesis and evaluating countermeasures for zikv infection. rhesus macaques with subcutaneous zikv inoculation develop mild clinical signs that resemble the self-limiting illness in most infected immunocompetent adults (dudley et al., 2016) . this non-human primate model provides a robust platform for the evaluation of vaccines and host immune response (abbink et al., 2016) . however, the mild clinical disease in these primates is suboptimal for antiviral treatment evaluation. moreover, expertise and facilities for working with non-human primates are not available in most research laboratories. wild-type adult balb/c mice are not susceptible to intraperitoneal zikv inoculation (dick, 1952) . suckling and young mice with intracerebral zikv inoculation develop disease that is localized to the central nervous system (dick, 1952; way et al., 1976; weinbren and williams, 1958; bell et al., 1971) . pregnant mice and fetal mice with partially intact type i interferon signaling response (fetuses of female mice deficienct in type i interferon signaling response crossed to wild-type male mice) were used to study pathogenesis in pregnancy and maternalfetal transmission, but these models are technically more demanding (miner et al., 2016; cugola et al., 2016) . type i/ii interferon-signaling-/ receptor-deficient mice with intraperitoneal or subcutaneous zikv inoculation develop fatal, disseminated infection (lazear et al., 2016; dowall et al., 2016; aliota et al., 2016; rossi et al., 2016) . these models are useful for the evaluation of countermeasures for zikv infection as the protective effects of antivirals drugs and vaccines are more easily observed in treated mice. however, such models have complete/nearcomplete deficiency in interferon response and do not resemble the real clinical situation in immunosuppressed humans. moreover, these mice are suboptimal for the study of host immune response and may be too expensive for laboratories in resource-limited areas. because of these limitations and knowledge gaps, we developed and characterized a more readily available mouse model which resembles immunosuppressed hosts with disseminated infection. we showed that these mice developed inflammation in multiple organs, including the testes, which may have important implications on zikv's longterm outcome and effects on fertility. we also utilized this novel animal model to show that early treatment with clinically approved recombinant type i interferons improved the clinical outcome of these mice. a clinical isolate of zikv (puerto rico strain prvabc59) was kindly provided by brandy russell and barbara johnson, centers for disease control and prevention, usa. the virus was amplified by three additional passages in vero cells (atcc) in minimum essential medium (mem) supplemented with 1% fetal calf serum and 100 units/ml penicillin plus 100 μg/ml streptomycin to make working stocks of the virus. for virus titration, aliquots of zikv were applied on confluent vero cells in 96well plates for 50% tissue culture infectious dose (tcid 50 ) assay as we previously described with slight modifications (zhou et al., 2014) . briefly, serial 10-fold dilutions of zikv were inoculated in a vero cell monolayer in quadruplicate and cultured in penicillin/streptomycinsupplemented mem. the plates were observed for cytopathic effect for 5 days. viral titer was calculated with the reed and münch endpoint method. one tcid 50 was interpreted as the amount of virus that causes cytopathic effect in 50% of inoculated wells. approval was obtained from the committee on the use of live animals in teaching and research of the university of hong kong. male and female balb/c mice, 6-8 weeks old, were obtained from the laboratory animal unit of the university of hong kong. the mice were kept in biosafety level-2 housing and given access to standard pellet feed and water ad libitum. virus inoculation experiments were performed in a biosafety level-2 animal facility according to the standard operating procedures approved by the committee on the use of live animals in teaching and research of the university of hong kong as we described previously (zhang et al., 2014) . the mice were randomly divided into 11 groups and given different regimens of virus inoculation, dexamethasone, and recombinant interferon treatment (table 1) . phosphate-buffered saline (pbs) was used to dilute the virus stocks to the desired concentration, and inocula were back-titrated to verify the dose given. on the day of virus inoculation, a dose of the virus equivalent to 6 × 10 6 tcid 50 (3.24 × 10 6 plaque forming units) in 200 μl of pbs was inoculated via the intraperitoneal route into mice under ketamine (100 mg/kg) and xylazine (10 mg/kg) anesthesia. mice in the negative-control groups (groups 5 to 8) were injected with the same volume of pbs. mice were monitored three times each day for clinical signs of disease and a numerical score was assigned at each observation as previously described (dowall et al., 2016; graham et al., 2015) . their body weight and survival were monitored for 14 days post-inoculation (dpi) or until euthanasia. three mice in each group (except groups 7 and 8 which included mock-infected control mice without dexamethasone immunosuppression and group 9 which included zikv-inoculated, dexamethasone-immunosuppressed mice without dexamethasone withdrawal) were sacrificed at 5 dpi for virological, histological, and immunohistochemistry analyses. the remaining mice were sacrificed at 14 dpi or euthanized when there was a 20% weight loss or 10% weight loss with ≥1 clinical sign (dowall et al., 2016) . samples of brain, testis/ epididymis (male), prostate (male), ovary/uterus (female), kidney, urinary bladder, spleen, liver, pancreas, intestine, heart, lung, and salivary gland were collected at necropsy. the specimens were separated into two parts, one immediately fixed in 10% pbs-buffered formalin, the other immediately frozen at −80°c until further experiments. blood samples were also collected for rna extraction and real-time pcr analysis. paraffin-embedded tissues were cut into 4-6 μm sections, mounted on slides, and stained with hematoxylin and eosin (h&e) for light microscopy examination as we previously described (zheng et al., 2008) . for immunohistochemical staining of zikv-ns1 antigen, mouse antiserum against zikv-ns1 protein prepared as we previously described was used as primary antibody (chan et al., 2016b) . de-paraffinized and rehydrated tissue sections were treated with antigen unmasking solution according to manufacturer's instructions (vector laboratories inc., burlingame, ca, usa) and then stained with mouse on mouse polymer ihc kit (abcam, cambridge, united kingdom). the primary antibody mouse anti-zikv-ns1 antiserum (1:1000 dilution with 1% bsa/pbs) was incubated at 4°c overnight. this was followed by mouse on mouse hrp polymer kit (abcam) with horseradish peroxidase-conjugated secondary antibody for 15 min. color development was performed using 3,3′-diaminobenzidine (dab) (vector laboratories, burlingame, ca, usa). for immunohistochemical staining of cd45 and cd8, the sections were incubated at 4°c for overnight with primary antibody (rabbit anti-mouse cd45, or rat anti-mouse cd8α (abcam) after antigen unmasking and blocking. this was then followed by incubation with biotin-conjugated goat anti-rabbit igg or goat anti-rat igg (calbiochem, darmstadt, germany) for 30 min at room temperature. streptavidin/peroxidase complex reagent (vector laboratories) was then added and incubated at room temperature for 30 min. color development was done with dab (vector laboratories). all tissue sections were examined microscopically by two pathologists in an operatorblinded manner. images were captured with nikon80i imaging system equipped with spot-advance computer software. total nucleic acid (tna) was extracted from the blood and necropsied tissues using ez1 virus mini kit v2.0 and qiasymphony dsp virus/pathogen mini kit (qiagen, hilden, germany), respectively, as we previously described (zheng et al., 2008; chan et al., 2016b; chan et al., 2015a) . zikv envelope gene was measured by using quantinova probe rt-pcr kit (qiagen) in lightcycler 96 real-time pcr system (roche diagnostics, basel, switzerland). 5 μl of purified tna was amplified in a 20 μl-reaction containing 10 μl of 2× quantinova probe rt-pcr master mix, 0.2 μl qn probe rt-mix, 0.8 μm forward primer, 0.8 μm reverse primer, and 200 nm probe. forward primer (5′-cgytgcccaacacaagg-3′), reverse primer (5′-ccacyaaygttcttttgcabaca-3′), and probe (5′-hex-agcctaccttgayaagcartcagacactc-iabkfq-3′) targeting the zikv envelope gene as we previously described were used (chan et al., 2016b) . reactions were incubated at 45°c for 10 min, followed by 95°c for 5 min, and then thermal cycled for 50 cycles (95°c for 5 s, 55°c for 30 s). internal control β-actin gene was measured by using quantinova sybr green rt-pcr kit (qiagen) in lightcycler 96 real-time pcr system. 5 μl of purified tna was amplified in a 20 μl-reaction containing 10 μl of 2× quantinova sybr green rt-pcr master mix, 0.2 μl qn sybr green rt-mix, 0.5 μm forward primer (5′-acggccaggtcatcactattg-3′) and 0.5 μm reverse primer (5′-caagaaggaaggctggaaaag-3′) for the β-actin gene. reactions were incubated at 50°c for 10 min, followed by 95°c for 2 min, and then thermal cycled for 50 cycles (95°c for 5 s, 60°c for 10 s). a series of 10-fold dilutions equivalent to 1 × 10 2 to 1 × 10 6 copies/reaction mixture were prepared to generate standard curves and run in parallel with the test samples. all data were analyzed with graphpad prism software (graphpad software, inc). kaplan-meier survival curves were analyzed by the log rank test, and weight losses were compared using two-way anova. student's t-test was used to determine significant differences in virus titers, and tukey-kramer post hoc tests were used to discern differences among individual treatment groups as previously reported rossi et al., 2016) . p-values b0.05 were considered statistically significant. to establish a novel mouse model for zikv infection, we compared the clinical, histological, and virological findings of male (group 1) and female (group 2) mice with dexamethasone immunosuppression and zikv inoculation with those of the appropriate controls (groups 3 to 8) (table 1 ). in terms of the clinical parameters, the dexamethasone-immunosuppressed mice developed mild (~5%) weight loss (fig. 1a) and no mortality at 5 dpi ( fig. 1b and c) . the weight losses of the dexamethasone-immunosuppressed mice with zikv inoculation (groups 1 and 2) table 1 eleven groups of mice receiving different regimens of virus inoculation, dexamethasone, and antiviral treatment in this study a . group gender routes and inoculum of zikv (0 dpi) dexamethasone antiviral treatment b date of sacrifice/euthanasia 1 m ip 6 × 10 6 tcid 50 50 mg/kg q24h ip, from 3 days before to 9 dpi inclusively no 5 (n = 3) and 12 (n = 6) dpi c 2 f ip 6 × 10 6 tcid 50 50 mg/kg q24h ip, from 3 days before to 9 dpi inclusively no 5 (n = 3) and 14 (n = 6) dpi 3 m ip 6 × 10 6 tcid 50 no no 5 (n = 3) and 14 (n = 6) dpi 4 f ip 6 × 10 6 tcid 50 no no 5 (n = 3) and 14 (n = 6) dpi 5 m no 50 mg/kg q24h ip, from 3 days before to 9 dpi inclusively no 5 (n = 3) and 14 (n = 6) dpi 6 f no 50 mg/kg q24h ip, from 3 days before to 9dpi inclusively no 5 (n = 3) and 14 (n = 6) dpi 7 no 14 (n = 6) dpi 9 m ip 6 × 10 6 tcid 50 50 mg/kg q24h ip, from 3 days before to 13 dpi inclusively no 14 (n = 6) dpi 10 m ip 6 × 10 6 tcid 50 50 mg/kg q24h ip, from 3 days before to 9 dpi inclusively pegylated ifn-α2b 1920 iu/dose q96h sc at 1, 5, and 9 dpi 5 (n = 3) and 14 (n = 8) dpi 11 m ip 6 × 10 6 tcid 50 50 mg/kg q24h ip, from 3 days before to 9 dpi inclusively ifn-β1b 160,000 iu/dose q48h ip at 1, 3, 5, 7, and 9 dpi 5 (n = 3) and 14 (n = 8) dpi abbreviations: dpi, days post-inoculation; f, female; ifn, interferon; ip, intraperitoneal; m, male; sc, subcutaneous. a 6-8 week-old balb/c mice were used in all the groups. b the preparations of pegylated ifn-α2b and ifn-β1b used in this study were pegintron (merck & co., inc., whitehouse station, nj, usa) and betaferon (bayer schering pharma ag, berlin, germany), respectively. c the mice in group 1 were euthanized at 12 dpi as they had ≥10% weight loss and ≥1 clinical symptom. were consistently more significant than those of their comparators, including the zikv-inoculated mice without dexamethasone immunosuppression (groups 3 and 4) and mock-infected mice without dexamethasone immunosuppression (groups 7 and 8) starting at 1 dpi (p b 0.05). minimal histological changes and inflammatory infiltrates were seen in the tissues of the male and female mice with dexamethasone immunosuppression and zikv inoculation (groups 1 and 2). on the other hand, zikv-ns1 protein expression was detected by immunohistochemical staining in most tissues of these mice, but not in dexamethasone-immunosuppressed mice with mock infection (groups 5 and 6), suggesting that the viral protein expression was specific and not related to dexamethasone effects (fig. 2) . the dexamethasone-immunosuppressed mice with zikv inoculation (groups 1 and 2) also had high mean viral loads in blood and most tissues at 5 dpi, especially in the testis/epididymis, ovary/uterus, prostate, spleen, and pancreas (figs. 3a and b and s1a and b). these findings at 5 dpi were suggestive of disseminated but non-lethal zikv infection involving different organs with minimal inflammatory response due to dexamethasone immunosuppression. to investigate the possible effects of immune reconstitution in the dexamethasone-immunosuppressed mice, dexamethasone was stopped after 9 dpi. this led to prominent weight loss and increased symptoms in the dexamethasone-immunosuppressed mice (groups 1 and 2). the most prominent body weight loss was observed in the male mice with dexamethasone immunosuppression and zikv inoculation (group 1), with all 6 mice having weight loss of ≥ 10% at 12 dpi (fig. 1a) . all of the female mice with dexamethasone immunosuppression and zikv inoculation (group 2) also had progressive weight loss and 4/6 (66.7%) of them had ≥ 10% weight loss at 14 dpi. in contrast, none of the mice with dexamethasone immunosuppression from 3 days before to 13 days post-infection (group 9) developed abrupt weight loss between 10 dpi and 14 dpi (fig. 1a) . the weight loss of mice in groups 1 and 2 became consistently more than those of their comparators in the other control groups (groups 3 to 8), including those in the dexamethasone-immunosuppressed mice with mock infection (groups 5 and 6) since 10 dpi (p b 0.05). together, these findings suggested that the combination of immune reconstitution after dexamethasone withdrawal and disseminated virus infection were responsible for the abrupt clinical deterioration. all of the mice in groups 1 and 2 developed rapid breathing, lethargy, and/or ruffled fur since 11 dpi (group 1) or 12 dpi (group 2), shortly after dexamethasone was stopped (fig. 1b) . the reasons for the earlier onset of weight loss and symptoms in the male mice were not fully understood, but might be related to the higher cumulative dose of dexamethasone because of their higher baseline body weights and/or possible effects of androgen on virus replication (tian et al., 2012) . based on the predefined criteria, all 6 (100%) male and 4/6 (66.7%) female mice were euthanized at 12 dpi and 14 dpi, respectively (fig. 1c) . comparatively, all the mice in the other control groups (groups 3 to 9) had either gained weight or had b 5% weight loss with spontaneous recovery at 14 dpi (fig. 1a) , remained asymptomatic (fig. 1b) , and survived through the study period (fig. 1c) . at euthanasia (12-14 dpi), h&e staining of the necropsied tissues of these mice showed prominent acute inflammatory reactions with predominantly lymphocytic infiltrates. the most prominent inflammatory changes were seen in the brain (cortical parenchymal and perivascular lymphocytic infiltrates) (fig. 4a to c), kidney (acute tubulitis and interstitial inflammation) (fig. 4d to f), and testis (necrotic and hemorrhagic seminiferous tubules with marked lymphocytic infiltration in the perimeter of the tubules and the interstitium) (fig. 5a to d) . zikv-ns1 protein expression was still visible, but to a lesser degree, in the immunohistochemical staining of the testis/epididymis, ovary/uterus, kidney, spleen, small intestine, pancreas, and salivary gland of the dexamethasone-immunosuppressed mice with zikv inoculation at 12-14 dpi compared with 5 dpi. in contrast, no inflammatory reaction and viral protein expression were seen in any organ of the control mice with zikv inoculation alone (groups 3 and 4) or dexamethasone immunosuppression alone (groups 5 and 6) ( fig. 5c and d) . these findings confirmed that mice with zikv inoculation but no dexamethasone immunosuppression were not susceptible to infection as previously reported, and that the histological changes in the model mice (groups 1 and 2) were unrelated to dexamethasone-induced effects such as drug-induced testicular toxicity (lazear et al., 2016; dowall et al., 2016; aliota et al., 2016; rossi et al., 2016; khorsandi et al., 2013) . the absence of inflammatory infiltrates in zikv-inoculated, dexamethasone-immunosuppressed mice without dexamethasone withdrawal (group 9) supported the role of the host immune response in eliciting the clinical and histological changes in the zikv-inoculated mice with dexamethasone withdrawal (groups 1 and 2). to further confirm the presence of inflammatory infiltrates and characterize the cell types involved in the host immune response, we stained the necropsied testis of the dexamethasone-immunosuppressed mice with zikv inoculation and those of the dexamethasoneimmunosuppressed mock-infected control mice with cd45 (pan-leukocyte) and cd8 (cytotoxic t lymphocyte) antibodies. corroborative to the histological findings, only the testis of the dexamethasone-immunosuppressed mice with zikv inoculation, but not those of the control mice, stained positive for cd45 ( fig. 5e and f) and cd8 antibodies ( fig. 5g and h). these findings confirmed the presence of inflammatory infiltrates and especially cd8+ t lymphocytes in the testis of the zikv-infected mice. the dexamethasone-immunosuppressed mice with zikv inoculation (groups 1 and 2) also had significantly lower mean viral loads in blood and most tissues at euthanasia at 12-14 dpi as compared with those collected at 5 dpi (↓ 1-4 log 10 copies/10 6 β-actin at 12-14 dpi) (figs. 3a and b and s1a and b). at euthanasia (12-14 dpi), viral rna was still detectable in most tissues of the male mice (up to 3 log 10 copies/10 6 β-actin), but viremia was absent. the control mice with zikv inoculation but no dexamethasone immunosuppression had undetectable viral rna in blood and most tissues, which was consistent with previous reports (lazear et al., 2016; dowall et al., 2016; aliota et al., 2016; rossi et al., 2016) . overall, these findings were suggestive of multi-organ inflammation upon immune reconstitution with partial viral clearance in the mice after withdrawal of dexamethasone immunosuppression. we next evaluated the effects of recombinant type i interferon treatment in our mouse model. we used male mice as they had earlier onset of weight loss and clinical symptoms requiring necropsy at 12 dpi. the mice were treated with pegylated interferon-α2b (pegintron®, merck & co., inc., whitehouse station, nj, usa) 1920 iu/dose every 96 h subcutaneously at 1 dpi, 5 dpi, and 9 dpi (group 10) or interferon-β1b (betaferon®, bayer schering pharma ag, berlin, germany) 160,000 iu/dose every 48 h intraperitoneally at 1 dpi, 3 dpi, 5 dpi, 7 dpi, and 9 dpi (group 11). as shown in fig. 6a , the mice treated with pegylated interferon-α2b (group 10) or interferon-β1b (group 11) had b10% weight loss with spontaneous recovery at 14 dpi. the weight loss of the untreated group became significantly more than those of the mice treated with either interferon-α2 or interferon-β1b starting at 10 dpi (p b 0.05). all of these mice remained asymptomatic and survived through the study period ( fig. 6b and c) . none of their tissues showed prominent inflammatory reactions in h&e staining at 5 dpi or 14 dpi. zikv-ns1 protein expression was only rarely seen in the immunohistochemical staining of the testis/epididymis, kidney, spleen, small intestine, lung, and pancreas collected at 5 dpi, and testis, epididymis, kidney, and spleen at 14 dpi. they had reduced mean viral loads in blood and all the tissues (↓2-4 log 10 copies/10 6 β-actin) as compared with those of the untreated mice at 5 dpi and 14 dpi ( fig. 7a and b) . the reductions were most significant in the tissues with high viral loads, such as the spleen, testis, pancreas, and prostate (p b 0.05). overall, these findings suggested that early use of systemic recombinant type i interferons improved the clinical, histological, and virological parameters of mice with disseminated zikv infection. the full spectrum of clinical manifestations and complications of zikv infection remains incompletely understood as of today. the previous assumption that zikv infection is an entirely self-limiting disease without severe or long-lasting sequelae has been overturned by the increasing recognition of congenital malformations, neurological complications, immune-mediated thrombocytopenia, and even fatality in some immunosuppressed patients (pan american health organization/ a b fig. 3 . viral loads in the blood and major organs of dexamethasone-immunosuppressed balb/c male and female mice with zikv inoculation. mice ((a) male (5 dpi, n = 3 from two independent experiments; 14 dpi, n = 6 from two independent experiments) and (b) female (5 dpi, n = 3 from two independent experiments; 14 dpi, n = 6 from two independent experiments)) with dexamethasone immunosuppression had higher blood and tissue viral loads at 5 dpi while they were on dexamethasone than at euthanasia (12 dpi for male mice and 14 dpi for female mice) after dexamethasone was stopped (10 dpi). zikv rna copies in the blood and tissues of the mice were determined by real-time rt-pcr and normalized by β-actin as described in the text. * denotes p-values of b0.05. *** denotes p-values b0.001. error bars represent standard error of the mean. world health oganization (paho/who), 2015; sarmiento-ospina et al., 2016; arzuza-ortega et al., 2016; duijster et al., 2016) . notably, patients with severe non-pregnancy-related complications of zikv often deteriorated suddenly after an initially mild disease phase as the viral load began to decrease (cao-lormeau et al., 2016; mecharles et al., 2016; sarmiento-ospina et al., 2016) . this has led us to hypothesize that, like many other flavivirus infections, including yellow fever, dengue, and west nile virus infection, the host immune response may also play a role in these zikv-associated complications, especially during the viral clearance phase by the host immune system (quaresma et al., 2013; screaton et al., 2015; wang et al., 2003) . in this study, we characterized a novel and readily available mouse model for severe zikv infection which attempts to provide an alternative venue for studying the host immune response of and evaluating countermeasures for zikv infection. the findings in our study have important implications on the pathogenesis, potential complications, and treatment of zikv infection. the dexamethasone-immunosuppressed mice with zikv inoculation in our study developed disseminated infection with viremia and multi-organ involvement, including the brain, urogenital tract, intestine, liver, spleen, pancreas, heart, lung, and salivary gland as evident by zikv-ns1 protein expression on immunohistochemical staining and/or detectable viral load in these tissues. immunohistochemistry staining of the testis confirmed the presence of inflammatory cell infiltrate (pan-leukocyte marker cd45 +) with predominantly cd8 + t lymphocytes. clinically, the male mice developed earlier onset of disease than the female mice, with ≥10% weight loss and ≥1 clinical sign, which warranted euthanasia at 12 dpi. their weight loss, clinical scores, and histological evidence of inflammatory reactions were most severe soon after dexamethasone withdrawal, when viral loads had already decreased by about 2-5 log 10 copies/10 6 β-actin. overall, these findings suggested that, like the other related flaviviruses, the host immune response might have led to marked clinical deterioration in the face of disseminated zikv infection at the time when immune-mediated clearance of the virus began. our findings provided an additional explanation for the pathogenesis of fatal zikv infection, which has been proposed to be related to uncontrolled virus dissemination in previously described mouse models utilizing types i/ii interferon-signaling-/receptor-deficient mice that were unable to mount a robust host innate immune response. our mouse model is also useful for studying zikv's tissue tropism and potential complications of severe zikv infection. in addition to the reported findings of detectable virus particles and/or rna in the brain, spinal cord, kidney, spleen, liver, testis, ovary, heart, lung, muscle, and blood of types i/ii interferon-signaling-/receptor-deficient mice with zikv infection, our study identified intestine, pancreas, and salivary gland as other possible tissues and anatomical sites for virus infection (dick, 1952; lazear et al., 2016; dowall et al., 2016; aliota et al., 2016; rossi et al., 2016) . this tissue tropism of zikv in our mouse model concurs with the in-vitro observation that zikv efficiently replicates in diverse cell types of neuronal, testicular, prostatic, renal, intestinal, hepatic, and placental origin (chan et al., 2016b; brault et al., 2016; hughes et al., 2016) . such degree of virus dissemination and multiorgan involvement is also compatible with the clinical findings in patients with severe and/or fatal zikv infection, in whom viral particles and/or rna were detected in multiple organs at post-mortem examination (pan american health organization/world health oganization (paho/who), 2015; sarmiento-ospina et al., 2016; arzuza-ortega et al., 2016) . while inflammatory neurological complications, such as guillain-barré syndrome, meningoencephalitis, and myelitis, have been recently reported in patients with zikv infection, inflammatory disorders of the other non-neuronal tissues were not well recognized (cao-lormeau et al., 2016; carteaux et al., 2016; mecharles et al., 2016) . our findings showed that inflammation could be observed in multiple organs including the testis, kidney, spleen, liver, intestine, pancreas, lung, and salivary gland outside the nervous system. among these non-neuronal tissues, the inflammatory reactions were most prominent in the testis of our model mice. some patients with zikv infection reported hematospermia, pelvic pain, and dysuria with detectable viral particles and/or rna in their semen (chan et al., 2016a; musso et al., 2015a; foy et al., 2011) . histological evidence of orchitis has not been reported due to the difficulty in obtaining the patients' testicular tissues for histological examination. previous mouse fig. 4 . representative histological findings in the brain and kidney of dexamethasone-immunosuppressed balb/c mice with zikv inoculation and dexamethasone-immunosuppressed mock-infected control mice. the brain and kidneys of all sacrificed mice were examined. each organ was entirely embedded in one paraffin block, and one full-face paraffin section at the maximum diameter of each organ was examined per block. sections of the brain (12 dpi) of a dexamethasone-immunosuppressed mouse with zikv inoculation showing (a) moderate degree of perivascular lymphocytic infiltrate and (b) marked lymphocytic infiltration in the cortical parenchyma (h&e, original magnification ×400). section of the brain (12 dpi) of dexamethasone-immunosuppressed mock-infected mouse showing normal architecture in the parenchyma (c) (h&e, original magnification ×400). sections of the kidney (12 dpi) showing (d) acute tubulitis with a large amount of inflammatory exudation in tubular lumens and (e) moderate degree of interstitial inflammation (h&e, original magnification ×400). (f) section of the kidney (12 dpi) of a dexamethasone-immunosuppressed mock-infected mouse showing normal architecture (h&e, original magnification ×400). models for zikv infection utilizing types i/ii interferon-signaling-/receptor-deficient mice have also showed that viral particles and rna could be detected in the mice's testes, but histological analysis were not reported (lazear et al., 2016) . the markedly necrotic and hemorrhagic seminiferous tubules observed in our mice are highly alarming as orchitis may have long-term effects on fertility. these changes were not accountable by dexamethasone-induced testicular toxicity, as they were morphologically different from the latter, and were not present in any of the testes of the control mice with dexamethasone treatment and mock infection (khorsandi et al., 2013) . during revision of this work, similar findings were reported in the testes of c57bl/6 mice treated with anti-ifna1 blocking monoclonal antibody and inoculated with zikv . clinical studies to confirm the presence of orchitis and to assess the fertility of convalescent male patients should be conducted to ascertain the long-term consequences of zikv infection regarding reproductive and hormonal derangements. inflammation of other organs, such as acute tubulitis, interstitial nephritis, sialadenitis, hepatitis, enteritis, and acute pancreatitis have been reported in patients with zikv or other flavivirus infections (chan et al., 2016a; sarmiento-ospina et al., 2016; arzuza-ortega et al., 2016; duijster et al., 2016; bonaldo et al., 2016; gourinat et al., 2015; musso et al., 2015b; mercado et al., 2016; bhagat et al., 2012; torres et al., 2000; macnamara, 1954; chatterjee et al., 2014) . these potential complications may be increasingly recognized as the zikv epidemic continues to expand into developed countries with a large ageing and immunosuppressed population. finally, our mouse model also provided a novel avenue for the evaluation of anti-zikv treatment. type i interferons have broad-spectrum antiviral activities including those against zikv, but type i interferonsignaling-/receptor-deficient mice were not suitable for evaluation of the effects of recombinant type i interferons. we therefore evaluated the antiviral effects of two commercially available preparations of recombinant type i interferons in this new mouse model (hamel et al., 2015; zumla et al., 2016; chan et al., 2013; chan et al., 2015b) . we showed that the early use of either drug was associated with improved clinical outcome with no fatality (100% fatality in untreated mice), markedly decreased inflammatory response after dexamethasone withdrawal, and reduced viral loads in various tissues of the mice as compared to those of the untreated mice. the viral load reductions were especially significant in the early phase of the disease (5 dpi), when the mice were on dexamethasone. these findings suggested that the early use of recombinant interferons might help to control viral replication during the initial phase of infection, and prevent the subsequent development of severe complications related to an exaggerated immune response in the presence of high viral loads as seen in the untreated mice. it is important to further confirm these results in our mouse model using different zikv strains and in clinical trials because zikv antagonizes mouse stat2 less efficiently than human stat2, and thus may be more susceptible to type i interferons in mice (grant et al., 2016) . while most patients with mild zikv infection may not require systemic interferon treatment, clinical trials should be considered to evaluate the benefits of the early use of interferon treatment in patients at risk of developing severe zikv-associated complications, such as those with underlying comorbidities (sarmiento-ospina et al., 2016) . the increased risk of fetal loss and low birth weight associated with interferon therapy in the first trimester of pregnancy may be outweighed by the risk of congenital malformations due to zikv infection. the optimal timing of treatment commencement should be further investigated as late commencement of interferon treatment may be useless or deleterious and should be avoided (solomon et al., 2003) . in summary, this novel mouse model is useful for investigating host immune response-associated damage of and evaluating countermeasures for zikv infection. inflammation of different visceral organs may be important complications of zikv that should be further studied in infected humans. long-term monitoring of the testicular function of zikvinfected male patients should be considered. clinical trials should be , inc., whitehouse station, nj, usa) 1920 iu/dose every 96 h subcutaneously at 1 dpi, 5 dpi, and 9 dpi or interferon-β1b (betaferon®, bayer schering pharma ag, berlin, germany) 160,000 iu/dose every 48 h intraperitoneally at 1 dpi, 3 dpi, 5 dpi, 7 dpi, and 9 dpi. no interferon treatment group (n = 6), interferon-α2b treatment group (n = 8), and interferon-β1b treatment group (n = 8). results were combined from two independent experiments. p-values b0.05 are indicated by (no interferon treatment versus interferon-α2b treatment) and * (no interferon treatment versus interferon-β1b treatment). abbreviation: ifn, interferon. clinical scores: normal = 0; ruffled fur = 2; lethargy, pinched, hunched, wasp waisted = 3; labored breathing, rapid breathing, inactive, neurological = 5; and immobile = 10. considered for evaluating the effects of recombinant interferon treatments in patients at high risk for zikv-associated complications when the potential benefits may outweight the side effects of treatment. supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.ebiom.2016.11.017. jfwc, ajz, ccsc, and kyy designed the study. ajz, ccsc, hz, and vkmp performed infections. ccyy, jolt, kkhc, and khc prepared virus stocks and viral titrations. ajz, wwnm, vkmp, and rkhay prepared histology and immunohistochemistry slides. ccyy, kmt, zz, and jpc performed viral load studies. jfwc, ajz, rkhay, vkmp, and kyy acquired images at the microscope, analyzed, and quantified the data. fy, khk, dyj provided technical assistance and edited the manuscript. jfw, ajz, and kyy wrote the manuscript. jasper f.w. chan has received travel grants from pfizer corporation hong kong and astellas pharma hong kong corporation limited, and was an invited speaker for gilead sciences hong kong limited. the other authors declared no conflict of interests. the funding sources had no role in study design, data collection, analysis or interpretation or writing of the report. the corresponding authors had full access to all the data in the study and had final responsibility for the decision to submit for publication. we are grateful to can li, andrew chak-yiu lee, and shuofeng yuan for their facilitation of the study. this work was partly supported by the donations of larry 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agonist imiquimod in combination with influenza vaccine expedites and augments humoral immune responses against influenza a(h1n1)pdm09 virus infection in balb/c mice delayed antiviral plus immunomodulator treatment still reduces mortality in mice infected by high inoculum of influenza a/ h5n1 virus active replication of middle east respiratory syndrome coronavirus and aberrant induction of inflammatory cytokines and chemokines in human macrophages: implications for pathogenesis comparative genomic analysis of pre-epidemic and epidemic zika virus strains for virological factors potentially associated with the rapidly expanding epidemic coronaviruses -drug discovery and therapeutic options key: cord-001958-2gt3fwpy authors: meseda, clement a.; atukorale, vajini; kuhn, jordan; schmeisser, falko; weir, jerry p. title: percutaneous vaccination as an effective method of delivery of mva and mva-vectored vaccines date: 2016-02-19 journal: plos one doi: 10.1371/journal.pone.0149364 sha: doc_id: 1958 cord_uid: 2gt3fwpy the robustness of immune responses to an antigen could be dictated by the route of vaccine inoculation. traditional smallpox vaccines, essentially vaccinia virus strains, that were used in the eradication of smallpox were administered by percutaneous inoculation (skin scarification). the modified vaccinia virus ankara is licensed as a smallpox vaccine in europe and canada and currently undergoing clinical development in the united states. mva is also being investigated as a vector for the delivery of heterologous genes for prophylactic or therapeutic immunization. since mva is replication-deficient, mva and mva-vectored vaccines are often inoculated through the intramuscular, intradermal or subcutaneous routes. vaccine inoculation via the intramuscular, intradermal or subcutaneous routes requires the use of injection needles, and an estimated 10 to 20% of the population of the united states has needle phobia. following an observation in our laboratory that a replication-deficient recombinant vaccinia virus derived from the new york city board of health strain elicited protective immune responses in a mouse model upon inoculation by tail scarification, we investigated whether mva and mva recombinants can elicit protective responses following percutaneous administration in mouse models. our data suggest that mva administered by percutaneous inoculation, elicited vaccinia-specific antibody responses, and protected mice from lethal vaccinia virus challenge, at levels comparable to or better than subcutaneous or intramuscular inoculation. high titers of specific neutralizing antibodies were elicited in mice inoculated with a recombinant mva expressing the herpes simplex type 2 glycoprotein d after scarification. similarly, a recombinant mva expressing the hemagglutinin of attenuated influenza virus rga/viet nam/1203/2004 (h5n1) elicited protective immune responses when administered at low doses by scarification. taken together, our data suggest that mva and mva-vectored vaccines inoculated by scarification can elicit protective immune responses that are comparable to subcutaneous vaccination, and may allow for antigen sparing when vaccine supply is limited. the eradication of smallpox, a disease that caused the death of hundreds of millions of people over many centuries, was accomplished primarily by the use of replication-competent vaccinia virus strains as vaccines. traditional (first-generation) smallpox vaccines, as well as more recently developed cell culture-derived second-generation smallpox vaccines such as acam2000 [1, 2] , the currently licensed smallpox vaccine in the united states, are inoculated into vaccine recipients by scarification of the skin surface, also known as percutaneous, skin or cutaneous vaccination [3] . rare but severe adverse reactions caused by these vaccines, including generalized vaccinia, eczema vaccinatum, and the more recently recognized cases of myopericarditis [4, 5, 6, 7] , limit the use of these vaccines for routine preventative vaccination of the general populace in the absence of any immediate risk of exposure to variola virus (the etiologic agent for smallpox) or other pathogenic orthopoxviruses such as monkeypox virus. thus, as early as the 1930s, efforts were made to develop safer smallpox vaccines by attenuating existing strains of vaccinia virus [8, 9] . as part of this effort, the modified vaccinia virus ankara (mva) was developed in the early 1970s. mva was derived from the chorioallantois vaccinia virus ankara (cva) strain of vaccinia virus, by more than 570 passages in chick embryo fibroblast (cef) cells [10] . during the course of passage of cva in cef cells, several genes (mainly host-range and immunomodulatory genes) were lost, resulting in the severely attenuated mva. about 15% of the viral genome was lost during passage in cef cells, and mva does not replicate productively in most mammalian cells [11, 12, 13] . mva has been extensively evaluated in different animal models [14, 15, 16, 17] and in clinical trials, and found to be less reactogenic when compared with replication-competent first and second generation smallpox vaccines [18, 19] . mva is licensed as a smallpox vaccine in europe and canada, and currently undergoing clinical development in the united states. the severe attenuation of mva and its consequent loss of the capacity to replicate efficiently in mammalian cells is evident in its inability to produce a "vaccine take", a pustular lesion that develops at the inoculation site, when vaccinia virus is inoculated on the skin surface. apart from its potential use as a smallpox vaccine in immunocompromised individuals, mva has the capacity to accommodate heterologous dna, and express encoded proteins, thus serving as a useful viral vector in vaccine development against different types of pathogens. several recombinant mva vectors expressing heterologous proteins of different human pathogens are at various phases of clinical development [20, 21] some of the mva-vectored vaccines in clinical trials include those expressing human immunodeficiency virus antigens [22, 23, 24] , mycobacterium tuberculosis 85a antigen [25, 26, 27] , malaria antigens [28, 29, 30] , human papilloma virus antigen [31] , hepatitis c antigens [32, 33] , respiratory syncytial virus antigens [34] , influenza virus antigens [35, 36, 37] , epstein-barr virus antigen [38, 39] and more recently, ebola virus antigens [40] . several other mva-vectored vaccines have also been evaluated in preclinical studies [41, 42, 43] . in most preclinical and clinical studies, mva or recombinant mva vectors, unlike replication-competent vaccinia virus strains, are inoculated into subjects via the intramuscular, intradermal or subcutaneous routes. although mva has been demonstrated to have a better safety profile than replication-competent vaccinia virus, a relatively large inoculum volume of 0.05 to 0.10ml and 0.5 to 1ml of mva or recombinant mva is typically used in small animal models and in non-human primates/humans, respectively. this often results in local reactions at the site of inoculation, including muscle ache, pain, and tenderness [44, 36] . in addition, the inoculation of prophylactic or therapeutic regimens with needles and syringes can be problematic for some people, a global problem commonly called needle phobia or fear of the needle in common phraseology. although the use of needle-free injection devices such as the jet injector [45] has become increasingly popular, hypodermic syringes and needles remain in wide use for the delivery of prophylactic and therapeutic remedies. previously, we [46] described a recombinant vaccinia virus, a33r/b5rko, that was severely attenuated for plaque formation in permissive cell lines due to deletions of the a33r and b5r genes, encoding the a33 and b5 proteins, respectively, of the extracellular virion form of vaccinia virus. the severe attenuation and growth characteristics of the a33/b5rko virus is reminiscent of the properties of mva. a33r/b5rko elicited vaccinia-specific igg and neutralizing antibodies when balb/c mice were inoculated by scarification at the base of the tail. in an intranasal challenge model, using the western reserve strain of vaccinia virus, the a33r/ b5rko virus conferred comparable levels of protection on mice as those vaccinated with a clonal isolate of dryvax, dv-3. during the course of the work above [46] , antibody [47] and robust cell-mediated immune responses after the inoculation of recombinant mva vectors through the skin, were also reported [48, 49, 50] , suggesting that percutaneous inoculation of mva may elicit equivalent or higher immune responses than subcutaneous injection. in the work described here, we demonstrate in mouse models that percutaneous inoculation of mva elicited protective immune responses against lethal intranasal challenge with the western reserve (wr) strain of vaccinia virus, and at low doses of mva, lower morbidity was recorded in mice that were vaccinated via the percutaneous route than in those immunized via the intramuscular or subcutaneous routes. in addition, we show in two models that percutaneous inoculation of recombinant mva expressing heterologous antigens elicited specific immune responses, including neutralizing antibodies, at levels that are comparable to subcutaneous inoculation. a recombinant mva expressing herpes simplex virus 2 (hsv-2) glycoprotein d elicited gd2-specific igg and hsv-2 neutralizing antibodies, and a recombinant mva expressing the hemagglutinin of influenza rga/vietnam/1203/04, h5n1, elicited protective immune responses when inoculated by the percutaneous route. our data suggest that vaccination via the percutaneous route is efficient in stimulating protective immune responses, and may find clinical relevance in immunizations with mva and mva-vectored vaccines. the modified vaccinia virus ankara (mva; atcc # vr-1508) was provided by drs. linda wyatt and bernard moss (niaid/nih) and vaccinia virus strain western reserve (vv-wr; atcc # vr-1354) was provided by dr. bernard moss. we have previously described the construction of a recombinant mva expressing the herpes simplex virus type 2 glycoprotein d (mva-gd2), in which the us6 gene of hsv-2 (strain ms) was inserted into the deletion ii region in mva by homologous recombination [51] . expression of glycoprotein d is driven by the synthetic vaccinia early/late promoter [52] . recombinant mva expressing the hemagglutinin (h5) of influenza virus rga/viet nam/1203/2004 (h5n1) (mva-ha) was constructed by homologous recombination of the h5 gene into the deletion iii region of mva [53] . expression of the influenza h5 is driven by the vaccinia h5r promoter. mva and recombinant mva vectors were prepared from primary chick embryo fibroblast cells (cef) or df-1 cells (atcc # crl-12203) that had been infected with the appropriate virus as previously described [54] , and partially purified viruses were obtained by passing infected cell lysates through a 36% sucrose cushion. mva and mva recombinants were titered on df-1 cell monolayers. vv-wr was prepared from infected bsc-1 cells (atcc # ccl26) , and was also partially purified through a 36% sucrose cushion, and titered on bsc-40 cell monolayers (atcc # crl-2761). the attenuated influenza virus rga/viet nam/1203/2004 (h5n1) was grown in the allantoic cavity of embryonated eggs and titrated on mdck cells. cells were cultivated and regularly maintained in dmem containing 10% fetal bovine serum (5% for mdck cells) and 5μg/ml gentamicin. balb/c mice (4-5 weeks old) were obtained from the jackson laboratories, bar harbor, maine. mice received feed and water freely. the animal study protocol for the work described in this manuscript was approved by the institutional animal care and use committee (iacuc) of the center for biologics evaluation and research, usfda (cber/fda). intramuscular inoculation into the muscles of the hind legs and subcutaneous inoculation at the base of the tail using 1ml tuberculin syringes affixed with 25-gauge needles, were performed as previously described [17] . tail scarification of anesthetized animals was also performed as previously described [17] . briefly, the inoculum was diluted in sterile endotoxin-free phosphate-buffered saline (pbs) such that the desired unit dose is contained per 2μl. mice were anesthetized by intraperitoneal injection of 1x avertin (2,2,2,-tribromoethanol in tert-amyl alcohol (sigma-aldrich, st. louis, missouri)) diluted in pbs at 20 μl per gram body weight. using a 25-gauge needle, 15 to 20 needle scratches/puncture were made at the base of the tail and 2μl of the inoculum was applied to the scarified surface. vaccinia virus (strain wr) and attenuated influenza a virus (rga/viet nam/1203/2004 (h5n1)), a 6+2 reassortant vaccine strain bases on influenza a/puerto rico/8/34, were used in challenge experiments. for intranasal mouse challenge, a dose of ten 50% lethal dose (10 ld 50 ) (equivalent to 1.5 x 10 5 pfu) of vv-wr was used. for challenge with rga/viet nam/1203/2004 (h5n1)), a dose of 10 6 pfu was used. we previously determined this dose to be lethal in our influenza virus challenge model. mice were anesthetized by intraperitoneal injection of 1x avertin (20 μl per gram body weight), followed by inoculation of the challenge virus in a total of 20 μl suspension into the nares (10μl per naris) as previously described [17] . mice were monitored and weighed daily for two weeks post challenge. mice that lost 25% of their initial body weight were considered to have reached the study endpoint, and were humanely euthanized per the iacuc-approved animal study protocol. testing of antisera for antigen-specific antibodies (igg) was performed by standard enzymelinked immunosorbent assay (elisa). elisa for vaccinia-specific antibodies was performed using psoralen/uv-inactivated vaccinia virus (dryvax) as the antigen, as previously described [17] . hsv-2 gd-specific igg elisa using partially-purified hsv-2 glycoproteins (50 ng/well) as the antigen, and hsv-2 neutralization assay were performed in 96-well immulon 2 hb plates (fisher scientific) and 96-well tissue culture plates (corning), respectively, as previously described [51] . influenza virus h5-specific igg was quantified using sanofi pasteur's inactivated rga/viet nam/1203/2004 vaccine (cber reference antigen #50) as the coating antigen. immulon 2 hb plates were coated with cber reference antigen at 1 μg/well, and plates were stored at 4°c overnight. plates were washed with phosphate-buffered saline containing 0.5% tween-20 (pbst), and then blocked with pbst containing 10% fetal bovine serum for 2 hours. subsequent incubation of the antigen with the test serum samples and completion of the assay followed standard elisa protocol as previously described [51] . testing of antisera for the neutralization of influenza a virus rga/viet nam/1203/2004 (h5n1), an attenuated vaccine strain generated by reverse genetics, was performed using a microneutralization assay as previously described [55] . the presence of virus was detected using biotin-conjugated antibody to influenza np (clone a; milipore, billerica, ma, usa) followed by hrp-labeled streptavidin (kpl, gaithersburg, md, usa). mice were vaccinated with 10 7 pfu of mva-gd2 via the subcutaneous or percutaneous route. a control group was vaccinated with 10 7 pfu of mva. seven (7) days after vaccination, mice were euthanized, spleens were collected from dissected mice, and lymphocytes were isolated from spleen homogenates as previously described [51] . spleen cells were cultured in 24-well tissue culture plates and re-stimulated with live hsv-2 (strain ms) at a multiplicity of infection of 1.0. culture supernatants were harvested after 48 hours, clarified of cells by centrifugation, and tested for il-2 and ifn-γ by capture elisa as described [51] , and reagents obtained from bd pharmingen. unpaired, two-tailed student's t-test for statistical comparison of antibody titers was performed using graphpad prism 6.02 software (graphpad software, inc.). the kaplan-meier survival logrank test was performed using the sigmaplot 13.0 software (systat software, inc.), and was used to compare differences in the number of surviving animals in the various treatment groups after virus challenge. in all cases, a p value <0.05 indicates statistically significant differences between treatment groups. in a preliminary experiment to investigate the utility of the percutaneous route for the delivery of mva, we observed that mva delivered by tail scarification, while statistically insignificant (p = 0.298), elicited a higher vaccinia-specific igg response and protection in mice than the same dose (10 6 pfu) delivered by the intramuscular route (s1 fig) . the experiment using 10 6 pfu of mva did not allow us to observe any differences in antibody response, disease progression and protection between the two immunization routes. thus in subsequent experiments, lower doses of mva were evaluated. groups of mice were vaccinated with 10 5 pfu of mva via the intramuscular, subcutaneous, or percutaneous routes. for comparison, a set of three groups of mice were similarly vaccinated with 10 5 pfu of the licensed acam2000 smallpox vaccine, via the same three routes. an untreated group was included as a control. three weeks after vaccination, serum samples were obtained from all mice and tested for vaccinia-specific antibodies by elisa, using inactivated dryvax as the antigen. among mice in the mva treatment cohort, an igg response was detectable in 2/5 mice in the subcutaneous group, and 1/5 in the percutaneous group. the untreated mice and all mice in the mva intramuscular group had no detectable igg at this time point. by contrast, all but three mice (2/5 and 1/5 in the intramuscular and subcutaneous groups, respectively) in the acam2000 cohort had detectable levels of igg (fig 1a) . at 4 weeks postimmunization, all mice were challenged intranasally, with a lethal dose (10 ld 50 ) of vv-wr. all mice in the untreated group showed severe symptoms of infection, reaching the study endpoint of 25% weight loss by day 7 post-challenge, and had to be euthanized (fig 1b & 1c) . except for a mouse in the mva-intramuscular group, all other mice vaccinated with mva or acam2000, survived. among the mva vaccinated animals, mice in the intramuscular group lost the most weight (fig 1b) , with a mean peak loss of about 16% on day 9 post-challenge. weight loss among the mva subcutaneous and percutaneous groups were similar, with peak losses of 9.6% (day 6) and 8.9% (day 7), respectively. however, mice in the mva percutaneous group recovered more quickly (98.4% of original mean body weight on day 14) than the subcutaneous group (94.4% mean weight on day-14). among the acam2000 treatment groups ( fig 1c) , the average peak weight loss was 8.8% (day-5), 7.7% (day-6), and 6.2% (day-6), for the intramuscular, subcutaneous, and percutaneous groups, respectively. in another experiment, mice in groups of five were vaccinated with 10 3 pfu or 10 5 pfu of mva by scarification. two other groups were similarly vaccinated with 10 3 pfu or 10 5 pfu of acam2000, and a control group was scarified with pbs. antisera were collected after three weeks and mice were challenged with 10 ld 50 of vv-wr. none of the mice in the pbs group had detectable igg and all succumbed to vv-wr infection ( table 1 ). all mice in the 10 3 pfu mva group had no detectable igg and 2/5 in the 10 5 mva group had detectable igg. however, 1/5 and 5/5 of mice survived in the 10 3 pfu and 10 5 pfu mva, respectively. in the acam2000 cohort, 2/5 and 5/5 of mice in the 10 3 pfu and 10 5 pfu groups, respectively, were seropositive. three of five (3/5) and . morbidity, as measured by mean weight changes post-challenge, is shown for the mva treatment groups (b), and for the acam2000 treatment groups (c). the "untreated" control group was the same for both the mva and acam2000 groups. a "+" sign represents a mouse that succumbed to infection. 5/5 of mice in the acam2000 cohort survived vv-wr challenge (table 1) . these sets of data suggest that in this mouse model, mva inoculation by the percutaneous route elicits equivalent or greater protective immune responses than inoculation via the intramuscular or subcutaneous routes. percutaneous vaccination with recombinant mva-gd2 elicits hsv-2-specific immune responses mva is an attractive vector being used for the expression of transgenes and has been used in the expression of antigens of a variety of pathogens. similar to studies investigating mva as a smallpox vaccine, preclinical and clinical evaluation of mva-vectored vaccines in development has relied predominantly on the use of intramuscular, intradermal or subcutaneous routes of mva delivery. in order to determine whether the percutaneous route will be useful for the evaluation of mva-vectored recombinant vaccines, a recombinant mva (mva-gd2) expressing the glycoprotein d of herpes simplex virus type-2 (hsv-2, strain ms) was evaluated for immunogenicity in the balb/c mouse model. in this recombinant mva, the hsv-2 us6 gene encoding glycoprotein d was inserted into the deletion ii site in mva by homologous recombination [51] . groups of mice were vaccinated with mva-gd2 at three dose levels: 10 5 pfu, 10 6 pfu, and 10 7 pfu; with each dose level administered subcutaneously or percutaneously. the control group received 10 7 pfu of mva subcutaneously. all treatment groups were vaccinated using a prime/boost immunization strategy as previously described [51] . antisera were collected 3 weeks after the priming vaccination, as well as at 3 weeks after the boost, and were tested for hsv-2-specific igg at both time points, using partially-purified hsv-2 glycoproteins as the antigen (fig 2a) . antisera collected at the 6-week time point were also tested for hsv-2 neutralizing antibodies (fig 2b) . antisera obtained from mice vaccinated with the mva vector had no detectable hsv-2 specific antibodies at both time points. hsv-2 specific antibodies were detected in antisera obtained from mice vaccinated with mva-gd2 at both time points (fig 2a) . the mean igg titers (log 10 the serum samples obtained at the 6-week time point were tested for the ability to neutralize hsv-2 infectivity in vero cells. similar to the igg elisa result, antisera from the mva control group did not neutralize hsv-2 (mean serum neutralization (sn) titer was below the level of quantitation of 0.30 log 10 ). low to modest neutralizing antibody titers were detected in mva-gd2 vaccinated mice (table 2 ). in the subcutaneous vaccination sub-groups, mean sn titers (log 10 ) of 0.96 (±0.13), 1.08 (±0.46), and 2.41 (±0.43), were obtained for the 10 5 pfu, 10 6 pfu, and 10 7 pfu groups, in another experiment, cellular immune response to mva-gd2 was evaluated. mice were vaccinated with 10 7 pfu of mva subcutaneously, or with 10 7 pfu of mva-gd2 either subcutaneously or percutaneously. spleen cells were harvested from mice 7 days after vaccination and tested for cytokine (il-2 and ifn-γ) secretion as previously described (meseda et al., 2002) . spleen cells were re-stimulated in vitro by live infection with hsv-2 (strain ms) at a multiplicity of infection of 1.0. supernatants were collected from the cultured spleen cells and tested for levels of il-2 and ifn-γ. whereas the levels of il-2 and ifn-γ in the supernatants from the spleen of mva-infected mice were below detection, all mice in both mva-gd2 groups had detectable levels of il-2 and ifn-γ, ( table 2 ). mean il-2 levels of 40.3 ± 21 pg/ml and 52.3 ± 40.1 pg/ml were obtained for the mva-gd2 subcutaneous and percutaneous groups, respectively. similarly, ifn-γ levels were 28.5 ± 17.4 pg/ml, and 82.3 ± 114.3 pg/ml, for the subcutaneous and percutaneous mva-gd2 groups, respectively. taken together, this set of data suggests that the inoculation of recombinant mva-gd2 by scarification is capable of eliciting antigen-specific immune responses that are comparable or higher than delivery by subcutaneous inoculation. mva is used as a vector for the expression of heterologous antigens. the deletion sites in mva, including the deletion ii and deletion iii sites, are commonly used for the insertion of transgenes. in order to broaden our understanding of the utility of the percutaneous route for the delivery of mva-vectored vaccines, we further evaluated a recombinant mva, mva-ha, in which the hemagglutinin gene of influenza a virus rga/viet nam/1203/2004 (h5n1), was inserted in the deletion iii site of mva. in a series of experiments, the antibody response following vaccination via the subcutaneous or percutaneous routes was characterized, and the protective effectiveness of vaccination via these routes was evaluated in a mouse intranasal challenge model. in the first experiment, groups of mice (five per group) were vaccinated with 10 5 , 10 6 , or 10 7 pfu of mva-ha on a prime-boost schedule at an interval of 3 weeks between vaccinations. each dose of mva-ha was administered via the subcutaneous or percutaneous routes. a control group was vaccinated with 10 7 pfu of mva via the subcutaneous route. three weeks after vaccination, serum samples were obtained from mice and tested for h5-specific igg, and all mice were challenged with 10 6 pfu of attenuated influenza rga/viet nam/1203/2004 (h5n1) via the intranasal route. except for a mouse in the mva control group that had a low level of non-specific igg, all other mice in the control group had no h5-specific igg, and 4/5 (including the one with detectable igg) succumbed to influenza virus challenge. all mice that were vaccinated with mva-ha, irrespective of the route, had high levels of h5-specific igg titers in the second experiment, mice in groups of mice (five per group) were vaccinated with 10 4 , 10 5 , or 10 6 pfu of mva-ha via the subcutaneous or percutaneous route on a prime-boost schedule at an interval of 3 weeks between vaccinations. a control group was vaccinated with 10 6 pfu of mva, subcutaneously. antisera were obtained from mice three weeks after the booster vaccination, and all mice were challenged with 10 6 pfu of influenza rga/viet nam/ 1203/2004. all mice in the mva group had no detectable h5-specific igg. by contrast, all mice inoculated with mva-ha, irrespective of the dose, had high titers of h5-specific igg (fig 3a) . the differences in igg levels between the subcutaneous and percutaneous cohorts at each dose level of mva-ha were not statistically significant, although mean igg titers were slightly higher in the subcutaneous cohort. following intranasal challenge with influenza rga/viet nam/1203/2004, all mice in the mva group succumbed to infection, and all mice vaccinated with mva-ha, irrespective of the dose, survived with varying degrees of morbidity. among mva-ha vaccinated mice, the 10 4 pfu/subcutaneous group recorded the severest weight loss (fig 3b) , and the difference in weight changes between the subcutaneous and percutaneous groups vaccinated with 10 4 pfu was statistically significant (two-tailed p-value = <0.001). a summary of the neutralizing antibody titers of pooled antisera for each treatment group from the two mva-ha experiments described above is presented in table 3 . the mva control group had no detectable neutralizing antibody in the serum samples obtained at any time point. among the groups vaccinated with mva-ha, neutralizing antibody was below detection in post-prime antisera, except in the 10 7 pfu subcutaneous group where a titer of 10 was obtained. however, following the administration of booster inoculations, all mva-ha treatment groups had detectable levels of neutralizing antibody that increased with increase in vaccine dose. neutralizing antibody titers in mice in the subcutaneous cohort were 15, 40, and 80, for the 10 4 , 10 5 , and 10 6 pfu, respectively. similarly, neutralizing antibody titers among the percutaneous cohort were 15, 60, and 80, for the 10 4 , 10 5 , and 10 6 pfu, respectively. interestingly, the levels of neutralizing antibody for the percutaneous treatment groups were similar to the subcutaneous group at the same mva-ha dosage, in spite of slightly higher total h5-specific igg levels in the subcutaneous cohort (difference is not statistically significant). this set of data suggests that the antibody response, both total igg and neutralizing antibodies, elicited by mva-ha was comparable between mice that were inoculated via the subcutaneous route and those inoculated via the percutaneous route. further, we observed that all mice inoculated with 10 4 pfu of mva-ha by prime-boost, were protected from influenza virus challenge. in the experiments with mva-ha described above, all mice that were vaccinated on a primeboost schedule with 10 4 pfu of mva-ha elicited antibody responses and protection that were indistinguishable between subcutaneous and percutaneous treatment cohorts. in order to further scrutinize the differences between the two inoculation routes, lower doses of mva-ha were used in experiments. in one experiment, groups of mice (five per group) were vaccinated with mva-ha at doses of 10 2 , 10 3 or 10 4 pfu, each dose administered via the subcutaneous or percutaneous route. a control group was inoculated subcutaneously with 10 4 pfu of mva. booster inoculations in the same amount of mva-ha or mva were administered three weeks after mice were primed. at three weeks after the booster doses, mice were challenged with attenuated rga/viet nam/1203/2004, at 10 6 pfu per mouse, and were evaluated daily for two weeks as described above. none of the animals vaccinated with mva survived. all mice vaccinated with 10 3 pfu mva-ha in this prime/boost schedule survived. in the 10 2 pfu mva-ha group, 2/5 and 1/5 survived in the subcutaneous and percutaneous groups, respectively. influenza virus pathogenesis in these mice, as measured by weight loss (fig 4) shows a doseresponse with respect to mva-ha, with weight loss being inversely proportional to the dose of mva-ha. there were no major differences in survival rates between mice in the subcutaneous and percutaneous groups inoculated with the same dose of mva-ha. at 10 2 pfu of mva-ha, the mean weight loss was higher in the percutaneous group than the subcutaneous group, but the difference was not statistically significant (two-tailed p-value = 0.084). finally, in two independent experiments, mice in groups of five in each experiment were vaccinated with low doses of mva-ha that were administered once, and were challenged three weeks after vaccination. groups of mice were inoculated at doses of 10 2 , 10 3 , or 10 4 pfu of mva-ha subcutaneously or percutaneously. control groups received 10 4 pfu of mva subcutaneously. a summary of the number of surviving mice is presented in table 4 . none of the mice vaccinated with mva survived. among mice in the mva-ha subcutaneous vaccination cohort, 3/10, 5/10, and 8/10 survived in the 10 2 pfu, 10 3 pfu, and 10 4 pfu vaccination groups, respectively. similarly, among mice in the percutaneous vaccination cohort, 5/10, 6/10, and 8/ 10 survived viral challenge. the mean weight loss among mice in these experiments are shown in fig 5. statistical comparisons of the number of surviving mice show that differences in survival between mice that were vaccinated with 10 4 pfu of mva-ha (irrespective of vaccination route) and the mva control group, were statistically significant (table 4 ). however, a comparison of the observed differences in survival between the percutaneous and subcutaneous groups at the 10 2 pfu dose level indicates the difference is not statistically significant. this set of data suggests that even at low vaccination doses, differences between mouse groups vaccinated via the subcutaneous and percutaneous routes are not apparent in this challenge model. the modified vaccinia virus ankara (mva) is licensed in europe and canada as a third generation smallpox vaccine, and currently in clinical development for licensure in the united states. the relatively better safety record of mva compared to first and second generation smallpox vaccines is well documented. this, in addition to its large capacity to accommodate heterologous genes, express encoded proteins, and elicit both humoral and cell-mediated immune responses also makes mva an attractive vector for the delivery of several candidate vaccines for a variety of infectious and non-infectious human and veterinary diseases [56, 57, 58, 59, 60] . evidence for the delivery of antigens through the skin in asia dates back to the 1500s with the practice of variolation and continued with the advent of the smallpox vaccine in the late 18 th century [3] . thus replication-competent smallpox vaccines, including those that were used in the successful eradication of smallpox, such as dryvax, lister, livp, temple of heaven, and em-63, were mostly administered by skin scarification [61] . the current us-licensed secondgeneration smallpox vaccine, acam2000, is also administered by skin scarification, a procedure that is believed to be partly responsible for the success of the global eradication of smallpox by provoking robust innate and adaptive immune responses [62] . due to the severe attenuation of mva, as epitomized by its inability to replicate productively in many mammalian cells [11, 12, 13] , mva and mva-vectored vaccines are usually administered via routes other than percutaneous in preclinical studies. clinical investigations of mva-vectored vaccines have mostly used intramuscular [23, 34, 35, 36] and intradermal routes [38, 39] , and to a lesser extent, the subcutaneous injection route [63] . local reactogenicity following vaccination with mva or mva-vectored vaccines is believed to be more severe with subcutaneous and intradermal inoculations than via intramuscular route [57, 63, 64] . in a comparison of the safety and immunogenicity of an mva-vectored hiv vaccine, individuals vaccinated with mva.hiva by the subcutaneous and intradermal routes were found to develop more severe local reactions than those vaccinated via the intramuscular route [63] . however, intramuscular and subcutaneous tissues have relatively fewer antigen presenting cells than the skin tissue and may not be adequate for optimal immune responses [65] . administering vaccines against infectious diseases through the skin has generated significant interest in recent years, including its use in the delivery of the bcg tuberculosis vaccine [66] , and has been further boosted by the development of the microneedle patch technology, which delivers vaccines intradermally. microneedle inoculation of vaccines has been used in preclinical evaluation of several vaccines, including inactivated polio vaccine [67] , influenza vaccine [68, 69, 70] , and measles vaccine [71] . clinical application of vaccines to the skin has also been documented for a number of vaccines, including influenza vaccine [72, 73] , and rabies vaccine [74] . recent studies have suggested that vaccine delivery through the skin takes advantage of the abundant presence of skin-resident antigen-presenting cells, including different subsets of dendritic cells and langerhans cells, as well as infiltrating antigen presenting cells, to provoke robust immune responses that include both humoral and cell-mediated immune responses [65] , and induce long-lived cd8 + t cell memory [75, 76] . moreover, data on the delivery of different types of vaccines through the skin suggest that both live vaccines and subunit vaccines can be administered through the skin, with successful immunization outcomes [67-71, 73,74] . earlier data from our laboratory [17] as well as from clinical trials [57, 77] suggest that delivery of the modified vaccinia virus ankara into the intradermal layer of the skin elicited robust immune responses that were higher than intramuscular or subcutaneous inoculations, and protected mice from intranasal challenge with vaccinia virus [17] . we further showed that a severely attenuated recombinant vaccinia virus that fails to form visible plaques in several mammalian cell lines, reminiscent of mva, elicited protective immune responses when used to vaccinate mice by scarification [46] . in the work described here, we expanded our investigation on the delivery of mva as well as mva-vectored antigens through the skin. in a preliminary experiment, we observed that igg titers were higher in mice that received 10 6 pfu of mva by skin scarification than in mice that received the same dose of mva by intramuscular inoculation. in subsequent experiments, antibody responses and protection of mice that were vaccinated with mva subcutaneously or by tail scarification were higher than in those vaccinated via the intramuscular route. melamed et al. [47] compared the antibody response elicited in response to mva and two recombinant mvas that had been genetically modified to replicate in vero and bsc-1 cells, after inoculating mice by intramuscular injection or tail scarification. their data indicate that tail scarification was efficient at inducing an antibody response, although the intramuscular route elicited higher geometric mean titers of antibody and conferred higher survival rates. the difference between their observation and the one reported here may be due to differences in experimental procedures and/or assay methods. for instance, in our work, mva for tail scarification is typically in 2 μl volume per dose, making it easier to handle than the 10μl used by melamed et al [47] . in subsequent experiments, the route comparison was limited to subcutaneous versus percutaneous routes, since the subcutaneous route is more commonly used in vaccination studies of mva vectors. as the utility of mva as a viral vector for the expression of heterologous antigens is expanding [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] 78] , we also compared the antibody responses and protection conferred by vaccination with two mva recombinants, one expressing the hsv-2 glycoprotein d, and the other expressing the h5 hemagglutinin of influenza virus rg/a viet nam/1203/2004 (h5n1). mice that were vaccinated with mva-gd2 by tail scarification, elicited higher than or similar titers of hsv-2 gd2-specific igg and neutralizing antibodies to those vaccinated by subcutaneous inoculation. the observed differences in igg titers were not statistically significant between the two routes at 10 6 or 10 7 pfu, but were statistically significant at 10 5 pfu, suggesting that the percutaneous delivery of mva-gd2 may be more effective than subcutaneous inoculation in eliciting hsv-2 neutralizing antibodies at lower vaccine doses. consistent with previous reports [48, 49] , percutaneous inoculation of mva-gd2 also elicited cell-mediated immune responses, as evident in the secretion of ifn-γ and il-2 by re-stimulated immune splenocytes. mva vectors expressing influenza antigens have been shown to elicit protective immune responses in animal models [79] , including mice [80] [81] [82] [83] , ferrets [84] and macaques [85, 86] . in the second recombinant mva vaccine model, mva-ha, expressing influenza virus h5, was used to vaccinate mice by subcutaneous injection or by tail scarification, and the h5-specific antibody response and protective effectiveness against intranasal challenge with the homologous attenuated influenza virus rga/viet nam/1203/2004, were assessed. the data indicate that comparable levels of antibody titers and protection were conferred by the two immunization routes. interestingly, higher survival rates among mice vaccinated by tail scarification were recorded at low vaccine doses. among the advantages attributed to skin delivery of vaccines is the possibility of antigen dose sparing [70, 72] . the data described in this manuscript support these earlier reports as lower doses of mva or mva-gd2 or mva-ha were found to elicits full or partial protection of mice. in summary, we showed that mva, and recombinant mva vectors expressing hsv-2 gd2 or influenza virus h5 elicited protective immune responses in the mouse model. taken together, the data presented in this work suggest that mva and mva-vectored vaccines can be effective when delivered through the skin. with the advantage that antigen delivery to the skin requires a volume that is at least 25 times (in murine models) to 200 times (in humans) less than the volume used in subcutaneous vaccination, a more comprehensive investigation of the clinical benefit of delivering mva and mva-vectored vaccines through the skin is necessary. apart from its efficiency in provoking robust immune responses, it may also help to ameliorate or obliterate some of the commonly reported volume-related local reactions associated with subcutaneous or intramuscular vaccine delivery, such as pain at the site of injection, and may be more acceptable in people with needle phobia, thus enhancing compliance with scheduled immunization programs. although the preclinical evaluation of vaccines by skin scarification, as described in our study, involves the use of improvised needles for the scarification process prior to the application of vaccines, advances in the development of the microneedle patch [87, 88, 89] should facilitate painless application of vaccines to the skin without the use of hypodermic injection needles or the bifurcated needle used in administering smallpox vaccines. in preclinical studies, microneedle delivery of mva-vectored vaccines has been shown to be effective in eliciting robust immune responses against malaria [90] that are comparable to the levels attained by intradermal vaccination. thus with further refinement, the use of microneedle delivery appears to hold a promising future for the application of viral-vectored vaccines through the skin. were vaccinated subcutaneously or percutaneously with 10 5 , 10 6 , or 10 7 pfu of mva-ha by prime-boost at an interval of 3 weeks between vaccinations. a control group received 10 7 pfu of mva prime-boost, subcutaneously. serum samples obtained 3 weeks after priming (week-3) and 3 weeks after boosting (week-6) were tested for h5-specific igg (a). error bars represent standard deviation. mice were subsequently challenged with 10 6 pfu of influenza rga/viet nam/1203/2004, and weighed daily for two weeks (b). a "+" sign represents a mouse that succumbed to infection. (tif) acam2000 clonal vero cell culture vaccinia virus (new york city board of health strain)-a second-generation smallpox vaccine for biological defense acam2000: a newly licensed cell culture-based live vaccinia smallpox vaccine cutaneous vaccination: antigen delivery into or onto the skin cardiac adverse events following smallpox vaccination-united states myopericarditis following smallpox vaccination among vaccinia-naive us military personnel incidence and followup of inflammatory cardiac complications after smallpox vaccination smallpox vaccination-associated myopericarditis is more common with the newest smallpox vaccine jennerian prophylaxis by means of intradermal injections of culture vaccine virus smallpox vaccination and its consequences: first experiences with the highly attenuated smallpox vaccine mva the smallpox vaccination strain mva: marker, genetic structure, experience gained with the parenteral vaccination and behavior in organisms with a debili modified vaccinia 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recombinant modified vaccinia ankara vaccine encoding epstein-barr virus (ebv) target antigens: a phase i trial in uk patients with ebv-positive cancer phase i trial of recombinant modified vaccinia ankara encoding epstein-barr viral tumor antigens in nasopharyngeal carcinoma patients use of chad3-ebo-z ebola virus vaccine in malian and us adults, and boosting of malian adults with mva-bn-filo: a phase 1, single-blind, randomised trial, a phase 1b, open-label and double-blind, dose-escalation trial, and a nested, randomised, double-blind, placebo-controlled trial high, broad, polyfunctional, and durable t cell immune responses induced in mice by a novel hepatitis c virus (hcv) vaccine candidate (mva-hcv) based on modified vaccinia virus ankara expressing the nearly full-length hcv genome a novel poxvirus-based vaccine, mva-chikv, is highly immunogenic and protects mice against chikungunya infection comparative assessment of vaccine vectors encoding ten malaria antigens identifies two 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glycoprotein d elicits greater specific antibody and cytokine responses than dna vaccine alone compact, synthetic, vaccinia virus early/late promoter for protein expression production and characterization of mammalian virus-like particles from modified vaccinia virus ankara vectors expressing influenza h5n1 hemagglutinin and neuraminidase preparation of cell cultures and vaccinia virus stocks detection of antibody to avian influenza a (h5n1) virus in human serum by using a combination of serologic assays protective efficacy of recombinant modified vaccinia virus ankara delivering middle east respiratory syndrome coronavirus spike glycoprotein safety and immunogenicity of modified vaccinia ankara (acam3000): effect of dose and route of administration cardiac safety of modified vaccinia ankara for vaccination against smallpox in a young, healthy study population efficacy assessment of an mva vectored rift valley fever vaccine in lambs evaluation of mva-5t4 as a novel immunotherapeutic vaccine in colorectal, renal and prostate cancer smallpox vaccines for biodefense protective properties of vaccinia virus-based vaccines: skin scarification promotes a nonspecific immune response that protects against orthopoxvirus disease studies of a prophylactic hiv-1 vaccine candidate based on modified vaccinia virus ankara (mva) with and without dna priming: effects of dosage and route on safety and immunogenicity comparing the safety and immunogenicity of a candidate tb vaccine mva85a administered by intramuscular and intradermal delivery microneedle-mediated vaccine delivery: harnessing cutaneous immunobiology to improve efficacy efficacy of percutaneous versus intradermal bcg in the prevention of tuberculosis in south african infants: randomised trial inactivated polio vaccination using a microneedle patch is immunogenic in the rhesus macaque enhanced immune responses by skin vaccination with influenza subunit vaccine in young hosts delivery of subunit influenza vaccine to skin with microneedles improves immunogenicity and long-lived protection dose sparing enabled by skin immunization with influenza virus-like particle vaccine using microneedles measles vaccination using a microneedle patch dose sparing with intradermal injection of influenza vaccine comparative immunogenicity of trivalent influenza vaccine administered by intradermal or intramuscular route in healthy adults long-term anti-rabies antibody persistence following intramuscular or lowdose intradermal vaccination of young vietnamese children skin infection generates non-migratory memory cd8+ t(rm) cells providing global skin immunity skin vaccination with live virus vectored microneedle arrays induce long lived cd8(+) t cell memory comparison of lyophilized versus liquid modified vaccinia ankara (mva) formulations and subcutaneous versus intradermal routes of administration in healthy vaccinia-naive subjects modified vaccinia virus ankara as antigen delivery system: how can we best use its potential? candidate influenza vaccines based on recombinant modified vaccinia virus ankara recombinant modified vaccinia virus ankara-based vaccine induces protective immunity in mice against infection with influenza virus h5n1 vaccinia virus-based multivalent h5n1 avian influenza vaccines adjuvanted with il-15 confer sterile cross-clade protection in mice vectors based on modified vaccinia ankara expressing influenza h5n1 hemagglutinin induce substantial cross-clade protective immunity broad protection against avian influenza virus by using a modified vaccinia ankara virus expressing a mosaic hemagglutinin gene evaluation of a modified vaccinia virus ankara (mva)-based candidate pandemic influenza a/h1n1 vaccine in the ferret model preclinical evaluation of a modified vaccinia virus ankara (mva)-based vaccine against influenza a/h5n1 viruses modified vaccinia virus ankara encoding influenza virus hemagglutinin induces heterosubtypic immunity in macaques coated microneedles for transdermal delivery microneedle-based vaccines coated microneedle arrays for transcutaneous delivery of live virus vaccines microneedle array design determines the induction of protective memory cd8+ t cell responses induced by a recombinant live malaria vaccine in mice the authors wish to thank drs. bernard moss and linda wyatt, niaid/nih, for the mva and vv-wr viruses. we also thank dr. maryna eichelberger and dr. alonzo garcia, cber/ fda, for reviewing this manuscript. this work was supported by intramural research funds key: cord-304855-7v0cncid authors: raaben, matthijs; prins, henk‐jan; martens, anton c.; rottier, peter j. m.; de haan, cornelis a. m. title: non‐invasive imaging of mouse hepatitis coronavirus infection reveals determinants of viral replication and spread in vivo date: 2009-02-10 journal: cell microbiol doi: 10.1111/j.1462-5822.2009.01298.x sha: doc_id: 304855 cord_uid: 7v0cncid bioluminescence imaging (bli) is a powerful new method to study virus dissemination in the live animal. here we used this method to monitor the spatial and temporal progression of mouse hepatitis coronavirus (mhv) infection in mice using luciferase‐expressing viruses. upon intranasal inoculation, virus replication could initially be observed in the nasal cavity and the cervical lymph nodes, after which the infection spread to the brain and frequently to the eyes. the kinetics of virus spread to and clearance from the brain appeared to depend on the inoculation dose. after intraperitoneal inoculation, virus replication was predominantly observed in the liver and occasionally in the intestines, but interestingly also in the tail and paws. bli thus elucidated new anatomic locations of virus replication. furthermore, mhv dissemination was shown to be critically depended on the viral spike protein, but also on the mouse strain used. widespread dissemination was observed in mice lacking a functional type i interferon response. the importance of the type i interferon system in limiting viral spread was also demonstrated by the administration of type i interferons to mice. our results provide new insights in coronavirus pathogenesis and demonstrate the potential of bli to study coronavirus–host interactions in vivo. current insights into infection processes of pathogens in their hosts and into the dynamics of their spread are largely based on studies using conventional methodologies. these methods have, however, many limitations. they require the experimental animals to be sacrificed in order to identify the sites of infection and to quantify the replication of the pathogens. they hence require large numbers of animals and are costly. they do not allow the real-time monitoring of spatial and temporal progression of infection in the same animal. important variations in host-pathogen interactions might therefore be overlooked, while dissemination of a pathogen to unexpected anatomical locations might be missed, simply because the infected tissue is not harvested and analysed (hutchens and luker, 2007; luker and luker, 2008a) . as of recently many of these limitations of the conventional techniques can be overcome by powerful new methods that involve non-invasive imaging of pathogen replication and spread in infected live animals. as a member of the coronavirus (cov) family, the mouse hepatitis virus (mhv) provides a practical model system for studying cov-induced pathogenesis in mice. depending on the inoculation route, the virus strain and the genetic background of the host, mhv infection can result in a variety of pathological disorders (compton et al., 1993) . the most commonly used laboratory strains primarily infect the liver and the brain, thereby providing animal models for studying encephalitis and hepatitis as well as for immune-mediated demyelinating disease that sometimes develop during a later stage of infection (perlman, 1998) . inoculation of susceptible mice, either intracranially or intranasally, with neurotropic mhv strains can result in a number of different outcomes, ranging from acute encephalomyelitis to chronic demyelinating disease (houtman and fleming, 1996) . besides a mild encephalitis, mhv strain a59 also causes enteric disease and moderate hepatitis (lavi et al., 1986) . the differences in pathogenesis between various mhv strains have been linked mainly to the spike protein which mediates viruscell attachment and subsequent membrane fusion (phillips et al., 1999) . however, other viral genes have been shown to also significantly contribute to pathogenesis (de haan et al., 2002; sperry et al., 2005; iacono et al., 2006) . the role of the immune system in response to mhv infection has been extensively studied. both humoral and cellular immune reactions are essential to guard against mhv infections marten et al., 2001; morales et al., 2001) . clearance of virus during acute infection is predominantly regulated by a typical expression pattern of proinflammatory chemokines that attract cd8 + and cd4 + t lymphocytes to sites of infection (glass et al., 2002; lane et al., 2006; stiles et al., 2006) . also interferon (ifn)-and perforin-mediated mechanisms are involved in the clearance of mhv from different cell types (weiss and navas-martin, 2005) . the kinetics and the extent of the host immune response, which aims to limit the production of infectious virus and viral spread without inducing extensive deleterious effects, is important in determining survival of the host. infectious virus is usually cleared from mhv-infected mice within 2 weeks. most animals, however, do not appear to obtain complete sterile immunity, since viral rna is often found to persist within the central nervous system (cns). recent biotechnological advances now allow the realtime imaging of pathogen replication and spread in living animals by making use of bioluminescence imaging (bli). to this end, light emitted by luciferase reporter proteins is detected by a cooled charge-coupled device (ccd) camera (contag et al., 1997; wu et al., 2001) . important advantages of bli are an intrinsically low background combined with a very high sensitivity for monitoring light emission in vivo (contag and bachmann, 2002; hutchens and luker, 2007) . furthermore, the substrate for firefly luciferase (fl), d-luciferin, is able to cross cellular membranes as well as the intact blood-brain barrier, thereby allowing the imaging of any anatomic location. in addition, d-luciferin is non-toxic, allowing the monitoring of individual mice over time through consecutive imaging. thus, fewer animals are typically needed to acquire statistically meaningful data at multiple time points as compared with conventional approaches (hutchens and luker, 2007) . initially, bli was used to identify sites of bacterial replication in intact animals (contag et al., 1995) . nowadays, this technique is widely used to study the formation and spread of cancer metastases and to visualize the effect of chemotherapy (shah et al., 2004) . bli has also been used to study the dissemination of herpes simplex virus, sindbis virus, vaccinia virus, and infectious hematopoietic necrosis virus (rodriguez et al., 1988; luker et al., 2002; cook and griffin, 2003; harmache et al., 2006) . all previous studies describing mhv infections and pathogenesis in mouse models have been relying on the sacrifice of infected mice in order to determine virus distribution and titres in various organs over time. although these conventional approaches have defined important factors that dictate replication and virulence of mhv in vivo as described above, non-invasive whole-body imaging of mhv infection in living mice is likely to offer new insights into virus replication, dissemination and pathogenesis. here, we have taken advantage of bli to study the replication and spread of fl-expressing mhv (de haan et al., 2003; 2005a) in mice in real-time. we were also able to demonstrate differences in virus replication and spread, resulting either from differences in virus doses, mutations in the spike gene, differences in host susceptibility or from the administration of antiviral compounds. in addition, we identified new anatomic sites of virus replication. our results provide new insights in cov pathogenesis and demonstrate the potential of bli to study cov-host interactions in vivo. in our previous studies we extensively characterized various recombinant mhvs expressing luciferase reporter genes. the insertion of an fl expression cassette at different positions in the viral rna genome was shown not to appreciably affect virus multiplication in vitro, while the fl expression levels were demonstrated to be a reliable measure for virus replication (de haan et al., 2003; verheije et al., 2008) . to verify the feasibility of detecting mhv infection in vivo with bli, we compared two recombinant mhvs containing the luciferase gene at different positions in the viral genome with a recombinant wildtype mhv-a59 with respect to virus replication. therefore, we prepared new stocks of mhv-eflm (containing the fl gene as an additional expression cassette between the e and m gene), mhv-2afls (containing the fl expression cassette at the position of the haemagglutinin esterase pseudo-gene) and wild-type mhv-a59 (fig. 1a ). in agreement with the previous studies we observed that the growth characteristics of the luciferaseexpressing viruses in tissue culture were highly similar to that of the parental mhv-a59 (fig. 1b) , with the fl reporter gene being expressed at high levels for both mhv-eflm and mhv-2afls. for the characterization of the fl-expressing viruses in vivo we used balb/c mice, which have been shown to be susceptible to mhv infection (ohtsuka and taguchi, 1997) . as the inoculation route largely determines the dissemination of virus infection in an animal, we made use of both intranasal and intraperitoneal injection. the 6-to 8-week-old mice were inoculated with 10 6 tissue culture 50% infectious dose (tcid50) of virus or with phosphate-buffered saline (pbs) (control) and sacrificed at 5 days post inoculation, at which day virus titres peak (macnamara et al., 2005) . as mhv-a59 mainly targets the liver and brain (lavi et al., 1984; haring and perlman, 2001) , these organs were taken for analyses. tissue homogenates were prepared and subsequently tested for virus titres, rna levels and fl activity. in intranasally inoculated animals, we detected high virus titres in the brains of all infected mice, with no disparity between the wild-type and fl-expressing viruses ( fig. 2a) . infectious virus could not be recovered from the liver homogenates. also the viral rna levels in the brains of the animals, as determined by quantitative reverse transcriptase polymerase chain reaction (rt-pcr), were indistinguishable between groups. however, viral rna in the liver could only be detected in wild-type mhv-infected mice (fig. 2b ). all mice infected with the fl-expressing recombinants showed high fl activity in the brain, as well as some fl activity in the liver (fig. 2c ). although not statistically significant, the fl expression levels of mhv-2afls were somewhat lower than those of mhv-eflm, which is consistent with the relative expression levels observed in vitro (de haan et al., 2003) . after intraperitoneal inoculation, infectious virus could only be recovered from the livers of wild-type mhv-a59-infected animals ( fig. 2a) . although viral rna was detected in the livers of all groups, the levels were approximately a 1000-fold lower for the fl-expressing mhv recombinants (fig. 2b) . furthermore, only in some livers of mhv-eflm-and mhv-2afls-infected mice could fl activity above background level be detected. overall, these data show that the introduction of the fl expression cassette into the genome of mhv-a59 affected virus multiplication in the liver but, importantly, not in the brain of 6-to 8-week-old balb/c mice. high levels of fl activity were measured in the brains of mhv-eflm-and mhv-2afls-infected mice at 5 days post infection, indicating that these viruses are attractive candidates to be used for bli after intranasal inoculation. in vivo imaging of mhv infection 827 thus, we applied whole-body bli to study the replication and spread of the two fl-expressing mhvs. to this end, balb/c mice were inoculated intranasally with 10 6 tcid50 of virus after which virus replication was followed over time in individual mice. in general, very similar patterns of virus dissemination were observed for both viruses. at 2 days post inoculation, a strong signal was observed, apparently emanating from the nasal cavity, when the mice were imaged at their dorsal side (fig. 3a ). when imaged from the ventral side, replication additionally could be observed in two distinct spots, probably representing the cervical lymph nodes of the animal. at this time point, no signal coming from the brain could yet be detected. three days later, dissemination of virus replication to the brain was apparent, while replication in the cervical lymph nodes was no longer observed. at 7 days post inoculation, the signal coming from the brain appeared more dispersed, while its intensity was clearly declining. at 9 days post inoculation, signal from the brain was the 6-to 8-week-old female balb/c mice were inoculated either intranasally or intraperitoneally with pbs or with 10 6 tcid50 of wild-type mhv-a59, mhv-eflm or mhv-2afls. at 5 days post inoculation, all mice were sacrificed and brains and livers were collected. homogenates were prepared as described in the experimental procedures. a. viral infectivity in the homogenates was determined by performing a plaque assay on lr7 cells. the viral titres are expressed as plaque-forming units per gram (pfu g -1 ) tissue. b. the amounts of viral genomic rna relative to total rna, isolated from the liver and brain homogenates, were determined by quantitative taqman rt-pcr. c. luciferase activities (in rlu) in each of the homogenates were determined by using a luminometer as described above. standard deviations (n = 4) are indicated in all graphs. scarcely detectable in most mice, while it could no longer be detected at day 12 (data not shown). with bli, it is important to note that the measurable photon flux decreases with increasing depth of the target tissue (luker and luker, 2008b) . thus, the signal coming from the brain is significantly attenuated when compared with the signal from the olfactory epithelium, because photons emitted from brain cells must penetrate considerably more tissue to be detected (fig. 3b) . as a consequence, signal intensities should only be compared when photons emanate from the same tissue. photon quantification of the head regions demonstrated that mice infected with mhv-eflm showed significantly higher signal intensities than mice infected with mhv-2afls at 2 days post inoculation (fig. 3b ). in conclusion, with bli the temporal and spatial spread of mhv replication could be readily visualized in living mice. while mice infected with different luciferase-expressing viruses displayed very similar patterns of virus dissemination, bli allowed the quantitative detection of relatively small differences in fl-expression levels between mhv-eflm and mhv-2afls, which is consistent with our earlier observations (de haan et al., 2003) . we next analysed whether we could visualize virus inoculation dose-dependent effects. to this end, balb/c mice were infected with two different doses of mhv-eflm, a high dose of 5 ¥ 10 6 and a low dose of 2 ¥ 10 3 tcid50, and progression of infection was subsequently monitored by bli at different time points post inoculation (fig. 4a ). as expected, photon quantification of the head regions at 2 days post inoculation demonstrated a clear correlation between the virus inoculation dose and the total signal produced from the olfactory epithelium ( fig. 4b ). in addition, spread of virus replication from the nasal cavity into the cns was more rapidly observed for the mice inoculated with the higher dose. this effect was quantified by dividing the head region into two sections (i.e. anterior versus posterior) after which the relative amount of photons emitted from these sections was determined (fig. 4c ). with a high virus inoculation dose, approximately 50% of the total amount of photons was coming from the posterior part at day 5, while with the low virus dose this degree of dissemination was delayed until around day 7. consistently, while mice that received the high dose showed virus replication in the cervical lymph nodes after 2 days, this was apparent in the mice inoculated with the low dose only after 5 days (table s1a) . interestingly, mice infected with the higher dose appeared to clear the virus infection faster than animals infected with the low dose. mhv replication was easily detectable at day 9 in mice inoculated with the low dose, whereas the luciferase signal of the high dose-inoculated group was almost undetectable at this time point. in some mice infected with a high virus dose, we could additionally visualize infection of the liver and lungs after 2 days, whereas at later time points (9 days), strong signals from the eyes were occasionally detectable. for a summary of the results see table s1a . in conclusion, virus dissemination was shown to differ in mice infected with a low or a high dose. in mice that received the low dose virus replication was initially restricted to the nasal cavity, while much faster dissemination of virus infection was observed after inoculation with a high dose. strikingly, and counter intuitively, this rapid dissemination appeared to come at a cost, as these mice were able to clear the infection faster than the mice that received the low dose. we next analysed the essential contribution of the s protein to virus dissemination by using bli. the s protein of mhv is a major determinant of pathogenesis and tropism (phillips et al., 1999) . it mediates virus-cell attachment and fusion via binding of the mhv receptor ceacam1a both in vitro and in vivo hemmila et al., 2004) . schickli et al. (1997) have described a limited set of mutations in the s protein of an mhv-a59 variant that had been acquired after extensive passaging in cell culture. we have recently shown that fig. 4 . the inoculation dose affects virus dissemination. balb/c mice were inoculated intranasally with 5 ¥ 10 6 or 2 ¥ 10 3 tcid50 of mhv-eflm. at the indicated times post inoculation (days p.i.), mice were processed for bli. a. dorsal images of a representative mouse are portrayed. the emitted photons were measured at the indicated time points and are displayed as a heat map in an overlay representation. scaling [in arbitrary units (au)] is similar in all depicted images. the vertical line in all panels represents the arbitrary border between the anterior (nose) and posterior (brain) regions of the head, which were selected for photon quantification. b. the total amounts of emitted photons from the complete head regions at the different time points were quantified and are expressed as integrated intensities on a log 2 scale with standard deviations (n = 4). c. the relative amounts of emitted photons from the anterior and posterior regions of the head (i.e. brain versus nasal epithelium) at the different time points were quantified and are expressed as percentage of the total signal, with standard deviations for both virus doses (n = 4). d. interesting phenotypes of mice at early (i.e. 2 days; ventral images portrayed) or late (i.e. 9 days; dorsal images portrayed) times post inoculation with 5 ¥ 10 6 tcid50 of mhv-eflm are shown: (a) liver, (b) paw, (c) lung, (d) eye and (e) cervical lymph nodes. note that the scaling [in arbitrary units (au)] differs between the ventral and dorsal images. these mutations enable the virus to use heparan sulfate as an additional attachment/entry factor. as a result, viruses carrying these mutations appeared to have acquired an extended host range as they were capable of entering cells also in a ceacam1a-independent manner (schickli et al., 1997; de haan et al., 2005b) . the physiological consequences of this adaptation in vivo, however, remain to be elucidated. here, we examined the infection of mice by an mhv with such an extended host range. therefore, we made use of a previously described recombinant virus (de haan et al., 2005b) , which contains the mutant spike gene (srec) and the fl reporter gene. thus, this recombinant virus mhv-2aflsrec only differs from its control virus mhv-2afls in its spike protein. both viruses replicated to comparable titres in murine lr7 cells as demonstrated by the one-step growth curve shown in fig. 1b and displayed similar fl expression kinetics ( fig. 1c) . next, balb/c mice were inoculated via the intranasal route with these viruses. clearly, mhv-2aflsrec replicated to a lower extent than the parental virus mhv-2afls at all time points measured ( fig. 5a and b) . although the luciferase signal exhibited by the virus with the extended host range increased until 5 days post infection, the infection did not progress towards the cns as was observed for the control virus. apparently, the acquisition of a heparan sulfate-dependent tropism significantly attenuated the ability of the virus to replicate and spread in vivo. replication of viruses in vivo is not only dependent on the genetic make-up of the virus, but also that of its host. it has been observed that the susceptibility of inbred mouse strains to different virus infections can vary significantly (parker et al., 1978; jubelt et al., 1991; thach et al., 2000) . in order to comparatively evaluate the mhv infection process in mice, we infected three commonly used mouse strains with mhv-eflm. to this end, balb/c, c57bl/6 and 129svev mice were inoculated in parallel each with 10 6 tcid50 via the intranasal route after which virus replication was monitored over time. clearly, at 5 days post inoculation balb/c mice displayed the highest signal in the brain, followed by c57bl/6 mice ( fig. 6a and b) . interestingly, 129svev mice were significantly less susceptible to mhv-eflm infection. in contrast to balb/c and c57bl/6 mice, virus replication was no longer detectable from 7 days post inoculation in these animals (data not shown). to confirm these observations, we inoculated mice also with mhv-a59 wild-type virus and determined the viral rna load in the brain by quantitative rt-pcr at 5 days post inoculation. in agreement with the bli results, balb/c mice showed the highest viral rna levels, while the brains from c57bl/6 and 129svev mice contained considerably less viral rna (fig. 6b ). next we studied virus replication and spread of the fl-expressing virus after intraperitoneal inoculation of balb/c and c57bl/6 mice. to this end 4-week-old mice were inoculated intraperitoneally with 10 6 tcid50 of mhv-eflm (fig. 5c ). at 2 days post inoculation we could visualize virus replication of the liver, with no apparent fig. 6 . replication of mhv-eflm in mice of different genetic background. the 6-to 8-week-old female balb/c, c57bl/6 and 129svev mice were inoculated intranasally with 10 6 tcid50 of mhv-eflm. a. bli was performed at 5 days post inoculation as described in the experimental procedures. here, the biospace photon imager was used as detector for reporter gene expression. the emitted photon counts were measured and are displayed as a heat map in an overlay image. a representative dorsal image is shown for the different mouse strains. b. the emitted photons from the head regions of each individual mouse were quantified and are expressed as the total number of counts measured within the 10 min imaging period. data are corrected for the background values (signal from uninfected mice). c. mice infected with wild-type mhv-a59 were sacrificed at day 5 post infection, after which the individual brains were collected. total rna was isolated and taqman rt-pcr was performed targeting mhv genomic rna sequences. the relative viral rna (vrna) levels and standard deviations are depicted (n = 4). d. the 4-week-old balb/c and c57bl/6 mice were inoculated intraperitoneally with 10 6 tcid50 of mhv-eflm. bli was performed at 2 days post inoculation. also here, the biospace photon imager was used. the emitted photons were measured and are displayed as a heat map in an overlay image. two representative ventral images are shown for the two different mouse strains. the anatomic locations displaying virus replication are indicated and include: (a) liver and (b) intestine. the arrows point to sites of virus replication in the tail and paws of the mice. difference between the two mouse strains. in some mice, infection of the intestine was also observed. interestingly, in all mice we could observe infection of the tail and paws, hitherto unidentified sites of infection. the signal rapidly decreased in time, with infection no longer being visible at 5-7 days post inoculation (data not shown). these results show that although replication of fl gene-containing viruses is decreased in 6-to 8-week-old mice (fig. 2) , which hardly enabled the detection of mhv replication by bli (data not shown), the infection could easily be monitored in younger mice, which are known to be more susceptible to infection. in addition, the technology allowed the identification of new anatomic sites of mhv replication that had been missed with the conventional techniques (table s1b) . next, bli was used to investigate the importance of the antiviral type i ifn system in controlling acute mhv infection. to this end, the effect of ifn alpha receptor knock-out (ifnar-/-) was studied using 129svev mice. wild-type and knock-out mice were inoculated via the intranasal route with 10 6 tcid50 of mhv-eflm and the infection process was monitored as before. already at 2 days post infection a significant difference between the ifnar-/-and wild-type mice was apparent when the animals were examined from the ventral side (fig. 7a) . a much higher signal was emitted from the nasal cavity in the ifnar-/-mice compared with the wild-type mice while, in addition, infection of the cervical lymph nodes was only detected in the knock-out mice. at later time points, infection was rapidly cleared from the wild-type mice, whereas dissemination to other organs, including liver and intestine, was manifest in the ifnar-/-mice. virus dissemination was accompanied by severe clinical signs, which included a significant decrease in body weight (fig. 7b) . mice had to be euthanized at 7 days post infection. interestingly, virus replication in the tail and paws could again be detected in the ifnar-/-mice, similar to what we had observed after inoculation of balb/c and c57bl/6 mice via the intraperitoneal route ( fig. 5c) . ex vivo imaging of the abdominal region of the ifnar-/-mice clearly identified the liver as the major site of mhv-eflm replication (fig. 7c) , the organ showing a focal pattern of luciferase expression. this observation was confirmed by the histological analysis of liver sections, as focal lesions were only observed in ifnar-/mice, but not in the control mice (fig. 7d) . overall, these results indicate that mice lacking a functional type i ifn response exhibit disseminated infection after intranasal inoculation with mhv-a59, which could be readily visualized using bli. in view of the increasing availability of all kinds of mutant mice, bli is an attractive approach to investigate the role of a particular host protein or pathway in virus replication and dissemination in vivo. as apparently uncontrolled virus dissemination was observed in mice lacking a functional type i ifn response, we next evaluated the anti-coronaviral effect of exogenous administration of type i ifn. thus, a cocktail of ifna/b was applied to balb/c mice intranasally. it has been established before that ifn can bypass the bloodbrain barrier after intranasal delivery (ross et al., 2004) . following subsequent intranasal inoculation with 2 ¥ 10 3 tcid50 of mhv-eflm, mice were processed for bli at the indicated time points as described above (fig. 8a) . at day 2, replication of mhv was not significantly affected by the application of ifn. however, while virus replication spread to the brains of the mock-treated animals, this was not observed in the ifn-treated mice. rather, the luciferase signal was lost by day 7. these observations were confirmed by the photon quantification of the head regions of the individual mice (fig. 8b) . thus, exogenous delivery of type i ifn did not prevent the initial replication in the nose, but prevented dissemination of virus infection to the cns. the results also indicate that bli is a promising technique for the non-invasive screening of antiviral compounds, the major advantage being that this system allows the detection of virus replication and spread after application of an antiviral compound over time within the same animal. b. the average body weight of both groups of mice is shown as the percentage relative to the initial weight at the beginning of the experiment. standard deviations are indicated (n = 4). c. in vivo versus ex vivo imaging of an ifnar-/-mouse infected with mhv-eflm at 7 days post inoculation. after the standard bli procedure, this mouse was sacrificed and immediately processed for imaging of the internal organs. note that the scaling [in arbitrary units (au)] in both images is different in order to visualize the focal infection pattern in the liver after ex vivo imaging. d. the liver of the ifnar-/-mouse in c was subsequently processed for histology as described in the experimental procedures. as a control, a liver section of a mhv-eflm-infected 129svev wild-type mouse is shown. typical focal lesions resulting from infection with mhv are indicated by the black arrows. mouse models are essential for defining factors that regulate replication and virulence of viruses. however, the conventional approaches for studying viral infections in mouse models are frequently limited by the need to sacrifice large numbers of animals to quantify viral titres, and establish the complete pattern of virus dissemination. the in vivo imaging of mhv infection 835 application of non-invasive imaging methods for the monitoring of virus infections in living animals can significantly facilitate studies on determinants of viral spread and pathogenesis (hutchens and luker, 2007) . we used bli for the real-time monitoring of mhv infection by using recombinant mhv viruses that express fl reporter proteins. we confirm and extend previous observations by demonstrating that mhv dissemination is critically dependent on the virus inoculation route and dose, the viral spike protein, the genetic background of the host, and the type i ifn system. in addition, the technology provided insights into the kinetics and dynamics of the infection process under these different conditions. furthermore, bli revealed an animal-to-animal variation in viral spread and elucidated new anatomic locations of virus replication. despite its many attractive features for studying mhv replication and spread, the bli technology has also some inherent limitations. first of all, photon transmission is affected by hair and organ pigmentation. thus, photon flux from the surface of an animal will be superior to that from an internal organ, because light is attenuated about 10-fold for every centimetre of tissue through which it has to pass (contag and bachmann, 2002) . as a consequence it is impossible to quantitatively compare bioluminescent signals derived from different anatomic locations. this phenomenon also explains why the photon flux from the head region at 2 days post infection is much higher than at later time points as the signal initially originates from the nasal cavity but thereafter increasingly derives from the cns, where it is much more shielded by surrounding tissue. to partly overcome this limitation, we routinely imaged the mice from both the ventral and the dorsal side. second, the introduction of a foreign gene into the viral genome may affect virus replication in vivo. interestingly, no significant differences could be observed between the replication of wild-type and fl-expressing viruses in the brain of intranasally infected mice, indicating that introduction of the foreign gene did not affect replication in vivo per se. however, after intraperitoneal application the replication of reporter gene expressing viruses in the liver was clearly much lower. the cause of this apparent discrepancy is currently unknown. importantly, however, fl expression was found at all the known sites of mhv replication (robbins et al., 1990; holmes and lai, 1996; komurasaki et al., 1996; mcintosh, 1996; perlman et al., 1999; macnamara et al., 2008) . the mhv spike protein is an important determinant of pathogenesis (phillips et al., 1999) . using bli, we monitored virus replication and spread of a recombinant virus that carried mutations in the spike gene shown to result in an extended host range in vitro due to the virus' ability to enter cells in a ceacam1a-independent but heparan sulfate-dependent manner (schickli et al., 1997; de haan et al., 2005b; . although it has been suggested that host range variants might arise in vivo during persistent infection of tissues that express low levels of the native mhv receptor (schickli et al., 1997; , this particular host range extension was obtained after serial passaging in vitro. clearly, the mutant virus replicated to a much lower extent in vivo and was not able to spread to the brain. many viruses adapt to heparan sulfate as an entry receptor upon propagation of the virus in cell culture (olmsted et al., 1984; mandl et al., 2001; lee et al., 2004) . often, but not always (liu and thorp, 2002; de haan et al., 2008) , this adaptation results in a reduction of virulence in living animals, consistent with our results. inbred mouse strains are known to differ in their susceptibility to many virus infections (parker et al., 1978; jubelt et al., 1991; compton et al., 1993; thach et al., 2000) . in our experiments we monitored the replication of mhv in three different mouse strains. c57bl/6 and 129svev mice appeared to be less susceptible to infection compared with balb/c mice after intranasal inoculation. similar results were obtained when mice were infected with wild-type mhv-a59 and viral rna load monitored using quantitative rt-pcr. strikingly, mhv replicated to approximately the same extent in balb/c and c57bl/6 mice after intraperitoneal inoculation. previously, mouse susceptibility to mhv infection was found to be linked to the viral receptor genotype (ohtsuka and taguchi, 1997) . however, quantitative rt-pcr analysis of the ceacam1a expression levels in brain and liver revealed only relatively small differences in the mhv receptor levels between the different mice strains, which did not correlate with the observed differences in viral replication (data not shown). apparently, other yet unknown host factors contribute to or limit the dissemination of mhv. the resistance of 129svev mice to infection is not restricted to mhv, because also vesicular stomatitis virus replicated much more efficiently in balb/c than in 129svev mice after intranasal inoculation (durbin et al., 2002) . the difference in mhv replication between balb/c and c57bl/6 mice might be associated with the mouse strain variation in immune responses which have been described for other virus infections (scalzo et al., 1992; brenner et al., 1994; thach et al., 2000; leipner et al., 2004; weinberg et al., 2004) . in general, c57bl/6 mice have a propensity to elicit a predominant t helper cell 1 response, while balb/c mice have a tendency to elicit a predominant t helper cell 2 response. our results confirm and extend previous observations that dissemination of mhv infection is determined both by the route and by the dose of inoculation. interestingly, clear differences in the spatio-temporal dissemination of the infection could be observed after intranasal inoculation with different virus doses. as expected, the virus inoculation dose correlated with the luminescent signal at 2 days post infection. however, inoculation with a low virus dose resulted in delayed spreading of the infection to the cns and subsequent delayed clearance. this result suggests that a certain level of replication in the nasal epithelium is required for subsequent dissemination. strikingly, at 9 days post infection higher levels of virus replication could be observed after inoculation with the low dose compared with the high dose. thus after inoculation with the high dose, virus was cleared faster from the brain. apparently, clearance of the virus is triggered by the extent of virus replication in the brain. probably high levels of virus replication more effectively induce an antiviral innate immune response. these results are corroborated by the observation that the expression levels of type i ifn and other cytokines positively correlate with the viral load in the brain of mhvinfected mice (m. raaben, unpublished results). type i ifn is a fundamental component of the innate immune response, which upon induction elicits an important antiviral signalling cascade that controls and orchestrates the outcome of numerous virus infections (zuniga et al., 2007) . here, we analysed the full extent of mhv dissemination in mice lacking a functional type i ifn response. these mice appeared to be highly susceptible to mhv and showed severe spread of the infection throughout the body when analysed with bli after intranasal inoculation. in contrast, the parental 129svev animals only showed some low levels of viral replication in the nasal cavity. these results confirm and extend recent studies in which mhv-a59-infected ifnar-/-mice were also shown to be highly susceptible to infection, both after intraperitoneal (cervantes-barragan et al., 2007) and after intracranial inoculation (roth-cross et al., 2008) . we additionally demonstrated the importance of the type i ifn system in controlling mhv dissemination by administration of type i ifns. intranasal delivery of recombinant type in vivo imaging of mhv infection 837 i ifns inhibited the spread of the infection to the brain, consistent with a previous study using the mhv strain jhm (minagawa et al., 1987) . it was interesting to observe that, while the spread to the brain was affected, the initial replication in the nasal epithelium was not decreased by the intranasally administered ifns. the experiments demonstrate the power of the bli technology for studying the effects of antiviral agents on virus replication and dissemination in animal models as was also illustrated earlier (luker et al., 2002; . our observations show that virus dissemination in an organism is a multifactorial process in which the genetic make-up of pathogen and host, the inoculation dose and route, and the status of the immune system play important roles. in addition, through the application of bli it has now become evident that, besides the typical, reproducible dissemination characteristics of mhv in mice, clear animal-to-animal variation also occurs. thus, while the infection invariably targets organs like brain, cervical lymph nodes and liver, depending on the inoculation route/dose, other anatomical sites of replication are less frequently detected (see table s1 for an overview). occasionally, mhv replication was measured in the lungs, in the intestine or in the eyes. quite frequently infection was also detected in the tail and paws, body parts not described earlier as sites of mhv replication. these results suggest that mhv can spread to and replicate in bone marrow in vivo, consistent with earlier in vitro studies demonstrating infection of bone marrow-derived macrophages and dendritic cells (zhou and perlman, 2006; roth-cross et al., 2008) . strikingly, mhv replication in tail and paws was never observed throughout the entire length of these extremities, but occurred only in distinct sites that differed between animals. in this respect, the dissemination of mhv is reminiscent of cancer metastases, which often also occur at distinct anatomical locations (molloy and van't veer, 2008) . similar to the importance of micro-environmental host factors in determining the non-random pattern of tumour localization, replication of mhv may also be enabled by local conditions in a specific micro-environment. lr7 mouse fibroblast cells (kuo et al., 2000) were maintained in dulbecco's modified eagle's medium (cambrex bio science) containing 10% (v/v) fetal calf serum (bodinco b.v), 100 u ml -1 penicillin, and 100 mg ml -1 streptomycin, supplemented with geneticin g418 (250 mg ml -1 ). mhv strain a59 and the derivates expressing the fl reporter gene (mhv-eflm, mhv-2afls and mhv-2aflsrec) have been described previously (de haan et al., 2003; 2005b) . all viruses were grown in lr7 cells. virus stocks were concentrated by pelleting through sucrose-cushion centrifugation, and subsequently resuspended in pbs. balb/c and c57bl/6 mice at different ages were obtained from charles river, while type i ifn receptor knock-out mice (ifnar-/-) (muller et al., 1994) and the parental 129svev mice were obtained from b&k universal ltd. mice were inoculated either intraperitoneally or intranasally with various doses of the different viruses. infected mice were either processed for bli, or sacrificed at the indicated time points for organ dissection. when indicated, mice were (pre-)treated with 1000 u of a cocktail of recombinant mouse ifn alpha/beta (ifn-a/b; sigma-aldrich). the cytokines were applied intranasally on each of days -2, -1, 0, 1 and 2 relative to the inoculation with mhv-eflm. control animals were treated with pbs. whole brains and livers were dissected from the mhv-infected and control mice. the tissues were added to lysing matrix d tubes (mp biomedical), containing 1 ml of pbs, and processed using a fastprep instrument (mp biomedical). the tissues were homogenized at 6000 r.p.m. for 40 s and immediately placed on ice. subsequently, the homogenates were centrifuged at 14 000 r.p.m. for 10 min at 4°c and supernatants were harvested, aliquoted and stored at -80°c. the aliquots were processed for the different assays as described below. when indicated, whole livers from mhv-infected mice were harvested, fixed in phosphate-buffered formalin, embedded in paraffin, sectioned and stained with haematoxylin. total rna was isolated from brain and liver homogenates using the trizol reagent (invitrogen) according to the manufacturer's protocol. rna was further purified using the rneasy mini-kit with subsequent dnasei treatment on the column (qiagen). the relative amounts of viral genomic rna were determined by quantitative taqman rt-pcr as described before (de haan et al., 2004) . titration of virus stocks was performed by determination of the tcid50 in lr7 cells (verheije et al., 2006) . the viral titres in tissue homogenates were determined by plaque assays. briefly, lr7 cells in 6 well microplates were inoculated with fourfold serial dilutions of the homogenates. at 1 h post inoculation, medium was replaced with an agar overlay. at approximately 48 h post inoculation, the lr7 cell monolayers were fixed and stained with crystal violet, after which the total amount of plaques was counted. the viral titres are expressed as plaque-forming units per gram (pfu g -1 ) tissue. monolayers of mhv-infected lr7 cells were lysed with 1¥ passive lysis buffer (promega). luciferase expression was measured according to the manufacturer's instructions, and relative light units (rlu) were determined in a turner designs td-20/20 luminometer. in case of the tissue homogenates, 40 ml was mixed with 40 ml 2¥ passive lysis buffer and incubated for 5 min at room temperature after which the luciferase expression was measured as described above. when indicated, mhv replication in mice was assessed by in vivo bli with a highly sensitive, ccd camera (versarray 1300b, roper scientific inc.) mounted in a light-tight imaging chamber (roper scientific inc.). imaging and quantification of signals was controlled by the acquisition software metavue (universal imaging corporation). prior to imaging, mice were anaesthetized by intraperitoneal injection of kxa: ketamine hydrochloride (100 mg kg -1 ; vétoquinol bv) plus xylazine (10 mg kg -1 ; eurovet animal health bv) and atropine (0.05 mg kg -1 ; pharmachemie bv). the substrate d-luciferin sodium salt (synchem laborgemeinschaft ohg) dissolved in pbs was injected intraperitoneal at a dose of~125 mg kg -1 . mice were positioned in a specially designed box and placed onto the stage inside the light-tight camera box. four mice were imaged simultaneously exactly 5 min after the injection of d-luciferin. the integrated light intensity of a stack of sequential 1 min exposures (10 from the dorsal side and 10 from the ventral side) was used to calculate the amount of emitted light from the animals. a low-intensity visible light image was made and used to create overlay images. images were further analysed with metamorph imaging software (universal imaging corporation). in some bli experiments, the detection of emitted photons was performed by the equally sensitive photon imager from biospace laboratory. bli images obtained with the biospace ccd camera were analysed by photovision software (biospace laboratory). table s1a . anatomical sites of mhv-eflm infection in balb/c mice inoculated with different doses (i.e. 2 ¥ 10 3 and 5 ¥ 10 6 tcid50). the numbers of mice from a group of four which showed infection at the indicated anatomical sites are indicated at the different time points post infection (days p.i.). table s1b . anatomical sites of infection in mhv-eflm infected mice of different genetic background (i.e. balb/c, c57bl/6, 129svev and ifnar-/-). note that mice were inoculated intranasally (i.n.) or intraperitoneally (i.p.) with 10 6 tcid50 of mhv-eflm and subsequently imaged at different time points post inoculation. the numbers of mice from a group of eight or four animals which showed infection at the indicated anatomical sites at any time point post inoculation are indicated. please note: wiley-blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. any queries (other than missing material) should be directed to the corresponding author for the article. in vivo imaging of mhv infection 841 cd8 t cell mediated immunity to neurotropic mhv infection targeted disruption of the ceacam1 (mhvr) gene leads to reduced susceptibility of mice to mouse hepatitis virus infection similar immune response to nonlethal infection with herpes simplex virus-1 in sensitive (balb/c) and resistant (c57bl/6) strains of mice control of coronavirus infection through plasmacytoid dendritic-cell-derived type i interferon the cellular and molecular pathogenesis of 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sclerosis murine coronavirus mouse hepatitis virus is recognized by mda5 and induces type i interferon in brain macrophages/ microglia the effect of the cmv-1 resistance gene, which is linked to the natural killer cell gene complex, is mediated by natural killer cells the murine coronavirus mouse hepatitis virus strain a59 from persistently infected murine cells exhibits an extended host range the n-terminal region of the murine coronavirus spike glycoprotein is associated with the extended host range of viruses from persistently infected murine cells molecular imaging of gene therapy for cancer single-amino-acid substitutions in open reading frame (orf) 1b-nsp14 and orf 2a proteins of the coronavirus mouse hepatitis virus are attenuating in mice t cell antiviral effector function is not dependent on cxcl10 following murine coronavirus infection differences between c57bl/6 and balb/cby mice in mortality and virus replication after intranasal infection with neuroadapted sindbis virus redirecting coronavirus to a nonnative receptor through a virus-encoded targeting adapter mouse hepatitis coronavirus rna replication depends on gbf1-mediated arf1 activation mouse strain differences in the chemokine response to acute lung infection with a murine gammaherpesvirus coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus noninvasive optical imaging of firefly luciferase reporter gene expression in skeletal muscles of living mice preferential infection of mature dendritic cells by mouse hepatitis virus strain jhm type i interferon during viral infections: multiple triggers for a multifunctional mediator additional supporting information may be found in the online version of this article this work was supported by grants from the m.w. beijerinck virology fund, royal netherlands academy of arts and sciences, and the netherlands organization for scientific research (nwo-vidi-700.54.421) to c.a.m. de haan. we thank monique oostra, marne hagemeijer and mijke vogels for stimulating discussions. key: cord-324326-q014b5ym authors: murakami, makoto title: lipoquality control by phospholipase a(2) enzymes date: 2017-11-10 journal: proc jpn acad ser b phys biol sci doi: 10.2183/pjab.93.043 sha: doc_id: 324326 cord_uid: q014b5ym the phospholipase a(2) (pla(2)) family comprises a group of lipolytic enzymes that typically hydrolyze the sn-2 position of glycerophospholipids to give rise to fatty acids and lysophospholipids. the mammalian genome encodes more than 50 pla(2)s or related enzymes, which are classified into several subfamilies on the basis of their structures and functions. from a general viewpoint, the pla(2) family has mainly been implicated in signal transduction, producing bioactive lipid mediators derived from fatty acids and lysophospholipids. recent evidence indicates that pla(2)s also contribute to phospholipid remodeling for membrane homeostasis or energy production for fatty acid β-oxidation. accordingly, pla(2) enzymes can be regarded as one of the key regulators of the quality of lipids, which i herein refer to as lipoquality. disturbance of pla(2)-regulated lipoquality hampers tissue and cellular homeostasis and can be linked to various diseases. here i overview the current state of understanding of the classification, enzymatic properties, and physiological functions of the pla(2) family. in terms of signal transduction, the phospholipase a 2 (pla 2 ) reaction, which hydrolyzes the sn-2 position of phospholipids to yield fatty acids and lysophospholipids, has been considered to be of particular importance, since arachidonic acid (aa, c20:4), one of the polyunsaturated fatty acids (pufas) released from membrane phospholipids by pla 2 , is metabolized by cyclooxygenases (coxs) and lipoxygenases (loxs) to lipid mediators including prostaglandins (pgs) and leukotrienes (lts), which are often referred to as eicosanoids (fig. 1) . lysophospholipids or their metabolites, such as lysophosphatidic acid (lpa) and platelet-activating factor (paf), are categorized into another class of pla 2 -driven lipid mediators ( fig. 2a, b) . more recently, a novel class of anti-inflammatory lipid mediators derived from b3 pufas, such as eicosapentaenoic acid (epa, c20:5) and docosahexaenoic acid (dha, c22:6), has also been attracting much attention (fig. 2c ). these lipid mediators exert numerous biological actions on target cells mainly by acting on their cognate g protein-coupled receptors. the pathophysiological roles of individual lipid mediators have been summarized in recent reviews. 1)-4) however, this principal concept appears to be insufficient to fully explain the biological aspects and physiological roles of the pla 2 family. phospholipids comprise numerous molecular species that contain various combinations of fatty acids esterified at the sn-1 and sn-2 positions and several polar head groups at the sn-3 position. many, if not all, pla 2 enzymes recognize such differences in the fatty acyl and/or head group moieties in their substrate phospholipids. moreover, several enzymes in the pla 2 family also catalyze the phospholipase a 1 (pla 1 ), lysophospholipase, neutral lipid lipase, or even transacylase/ acyltransferase reaction rather than or in addition to the genuine pla 2 reaction. therefore, the fatty acids and lysophospholipids released by different pla 2 s are not always identical; rather, in many situations, specific fatty acids and lysophosholipids can be released by a particular pla 2 in the presence of a given microenvironmental cue. in this context, pla 2 enzymes act as one of the critical regulators of spatiotemporal lipid profiles, namely the quality of lipids (lipoquality). to comprehensively understand the lipoquality regulation by individual pla 2 s in various pathophysiological contexts, their precise enzymatic, biochemical and cell biological properties, tissue and cellular distributions, and availability of phospholipid substrates in various pathophysiological settings should be taken into consideration. herein, i overview current understanding of the biological aspects of various pla 2 enzymes in the context of lipoquality. obviously, the substrate specificity of individual pla 2 s is the critical determinant of lipoquality. the in vitro enzymatic activity of pla 2 s may be influenced by the assay conditions employed, such as the composition of the substrate phospholipids, concentrations of pla 2 s and substrates, presence of detergents, and ph. hence, the enzymatic properties of individual pla 2 s determined in different studies may not be entirely identical. since natural membranes contain numerous phospholipid molecular species, the results obtained using artificial phospholipid vesicles comprising only one or a few phospholipid species may not always reflect the true enzymatic properties of a given pla 2 . addition of an excess amount of recombinant or purified pla 2 to an enzyme assay often results in hydrolysis of bulk phospholipids, which makes precise evaluation of its substrate specificity difficult. the results obtained using a commercially available pla 2 assay kit, in which a synthetic, chromophoric phospholipid is used as a substrate, should be interpreted carefully, since some pla 2 s are unable to hydrolyze it efficiently. in this regard, mass spectrometric examination of the in vitro hydrolysis of natural membrane phospholipids extracted from the affected tissues or cells by pla 2 , particularly at a low (physiologically relevant) concentration of the enzyme, could provide a valuable clue to the in vivo substrates and products of this enzyme. 5)-7) the overall tendency in this in vitro assay using natural membranes is recapitulated in several in vivo systems, often with even more selective patterns of hydrolysis that are relevant to the results of studies using pla 2 knockout and/or transgenic mice (see below). importantly, the mobilization of distinct lipids by pla 2 s in vivo relies not only on their intrinsic enzymatic properties, but also on tissue-or disease-specific contexts such as the lipid composition of target membranes, the spatiotemporal availability of downstream lipid-metabolizing enzymes, or the presence of cofactor(s) that can modulate the enzymatic function, which may account for why distinct pla 2 enzymes even in the same subfamily exert specific functions with different lipid profiles in distinct settings. hereafter, i describe the current understanding of various pla 2 s in the context of lipoquality. the classification, distributions, properties and functions of individual pla 2 s, whose pathophysiological functions have currently been studied using their gene-manipulated mice, are summarized in table 1 enzymes whose in vivo functions have been analyzed using knockout mice are summarized. the cpla 2 family. the cytosolic pla 2 (cpla 2 ) family comprises 6 isoforms (,-1), among which cpla 2 o, /, c and 1 map to the same chromosomal locus (fig. 3a) . 8) cpla 2 , (also known as group iva pla 2 ) is undoubtedly the best known pla 2 and its biological roles in association with lipoquality have been well documented. 9) cpla 2 , is the only pla 2 that shows a striking substrate specificity for aa-containing phospholipids. strictly speaking, cpla 2 , can also hydrolyze phospholipids containing epa, yet the low abundance of this b3 pufa relative to other fatty acids including b6 aa in cell membranes allows cpla 2 , to release aa rather specifically in most situations. upon cell activation, cpla 2 , translocates from the cytosol to the phosphatidylcholine (pc)-rich perinuclear, endoplasmic reticulum (er) and golgi membranes (particularly golgi) in response to an increase in the µm range of cytosolic ca 2d concentration, and is maximally activated by phosphorylation through mitogen-activated protein kinases (mapks) and other kinases. 10),11) in addition, the phosphoinositide pip 2 and ceramide-1-phosphate modulate the subcellular localization and activation of cpla 2 ,. 12),13) the aa released by cpla 2 , is converted by the sequential action of constitutive cox-1 or inducible cox-2 and terminal pg synthases to pgs or by the sequential action of 5-lox and terminal lt synthases to lts (fig. 3b ). mice deficient in cpla 2 , display a number of phenotypes that can be explained by reductions of pgs and/or lts. under physiological conditions, cpla 2 ,-deficient mice display a hemorrhagic tendency, impaired female reproduction, gastrointestinal ulcer, and renal malfunction, among others. 14)18) under pathological conditions, cpla 2 ,-deficient mice are protected against bronchial asthma, pulmonary fibrosis, cerebral infarction, alzheimer's disease, experimental autoimmune encephalomyelitis, collagen-induced arthritis, metabolic diseases, intestinal cancer and so on, whereas they suffer from more severe colitis and spinal cord injury. 15),19)-24) most of these phenotypes are recapitulated in mice lacking one or more of the biosynthetic enzymes or receptors for pgs and lts, lending strong support to the notion that cpla 2 , lies upstream of eicosanoid biosynthesis in many situations. for instance, as is the case for cpla 2 ,-deficient mice, mice lacking ltc 4 synthase (ltc 4 s), ltd 4 receptor (cyslt1), ltb 4 receptor (blt1), or pgd 2 receptor (dp1) are protected from asthma, 25)-27) revealing the critical role of the cpla 2 ,-ltb 4 /ltc 4 /pgd 2 axis in this allergic disease. likewise, the decrease of pge 2 in cpla 2 ,-deficient mice can account largely, even if not solely, for the mitigation of arthritis, autoimmune encephalomyelitis, cancer and neurodegeneration as well as the exacerbation of colitis, since these phenotypes are mimicked by mice lacking pge 2 synthase (mpges-1) or either of the four pge 2 receptors (ep194). 28)-32) furthermore, cpla 2 ,-triggered release of aa by platelets is coupled not only with biosynthesis of the pro-thrombotic eicosanoid thromboxane a 2 (txa 2 ), but also with o-oxidationmediated bioenergetics for blood clotting. 33) importantly, inherited human cpla 2 , mutations are associated with reduced eicosanoid biosynthesis, platelet dysfunction, and intestinal ulceration, 34) , 35) thus mimicking cpla 2 , deletion in mice. on the other hand, the enzymatic activities and biological functions of cpla 2 isoforms other than cpla 2 , have remained largely unknown. reportedly, cpla 2 o (group ivb pla 2 ), which has a unique jimc domain in the n-terminal region, display pla 1 , pla 2 and lysophospholipase activities. 36) cpla 2 . (group ivc pla 2 ), which uniquely lacks the c2 domain characteristic of the cpla 2 family, is cterminally farnesylated and possesses lysophospholipase and transacylase activities in addition to pla 2 activity. 37) cpla 2 / (group ivd pla 2 ), whose expression is elevated in human psoriatic skin, 38) shows pla 1 activity in preference to pla 2 activity. 36) cpla 2 c (group ive pla 2 ) exhibits a unique transacylase activity that transfers sn-1 fatty acid of pc to an amino residue of phosphatidylethanolamine (pe) to form n-acyl-pe, a precursor of the endocannabinoid lipid mediator n-acylethanolamine. 39) cpla 2 1 (group ivf pla 2 ) displays both pla 1 and pla 2 activities without fatty acid selectivity. 40) however, these enzymatic properties of cpla 2 o-1 vary according to the in vitro assays employed, implying that analyses using gene-manipulated mice for these enzymes will be necessary for clarifying their biological roles in the context of lipoquality. the ipla 2 /pnpla family. the human genome encodes 9 ca 2d -independent pla 2 (ipla 2 ) enzymes (fig. 4) . these enzymes are now more generally referred to as patatin-like phospholipase domain-containing lipases (pnpla199), as all members in this family share a patatin domain, which was initially discovered in patatin (ipla 2 ,), a potato protein. 41 ),42) mammalian ipla 2 /pnpla isoforms include lipid hydrolases or transacylases with specificities for diverse lipids such as phospholipids, neutral lipids, sphingolipids, and retinol esters. generally speaking, enzymes bearing a large and unique n-terminal region (pnpla699) act mainly on phospholipids (phospholipase type), whereas those lacking the n-terminal domain (pnpla195) act on neutral lipids (lipase type). analysis of mutant mouse models and clinical symptoms of patients with mutations for these enzymes have provided valuable insights into the physiological roles of the ipla 2 / pnpla family in various forms of homeostatic lipid metabolism that are fundamental for life. among the ipla 2 /pnpla family, pnpla9 (ipla 2 o, also known as group via pla 2 ) is the only isoform that acts primarily as a pla 2 with poor fatty acid selectivity. 43),44) although pnpla8 (ipla 2 . or group vib pla 2 ) displays pla 2 activity, it acts as a pla 1 toward phospholipids bearing sn-2 pufa. 45), 46) accordingly, hydrolysis of pufa-bearing phospholipids by pnpla8/ipla 2 . typically gives rise to 2-lysophospholipids (having a pufa at the sn-2 position) rather than 1-lysophospholipids (having a saturated or monounsaturated fatty acid at the sn-1 position). pnpla6 (ipla 2 /) and its closest paralog pnpla7 (ipla 2 3) have lysophospholipase activity that cleaves lysophosphatidylcholine to yield fatty acid and glycerophosphocholine. 47),48) genetic mutations or deletions of these phospholipid-targeting pnplas cause various forms of metabolic dysfunction and neurodegeneration. 49)-53) in particular, pnpla9/ipla 2 o is also referred to as the parkinsonism-associated protein park14, whose mutations impair ca 2d signaling in dopaminergic neurons. 54) apart from the metabolic and neurodegenerative phenotypes, the lack of pnpla9/ipla 2 o leads to male infertility through an unknown mechanism. 55) pnpla2 (ipla 2 1), more generally known as adipose triglyceride lipase (atgl), is a major lipase that hydrolyzes triglycerides in lipid droplets to release fatty acids as a fuel for o-oxidation-coupled energy production, a process known as lipolysis. 56) genetic deletion or mutation of pnpla2 leads to massive accumulation of triglycerides in multiple tissues leading to multi-organ failures, 57) while protecting from cancer-associated cachexia by preventing fat loss. 58) the activity of pnpla2 is regulated positively by abhd5 (see below) and negatively by perilipin and g0s2, which modulate the accessibility of pnpla2 to lipid droplets. 59) the fatty acids released from lipid droplets by pnpla2 act as endogenous ligands for the nuclear receptor ppar, or ppar/, which accelerates energy consumption. 59), 60) the regulatory mechanisms and metabolic roles of pnpla2 have been detailed in other elegant reviews. 61),62) mutations of pnpla3 (ipla 2 c) are highly associated with non-alcoholic fatty liver disease. 63) although the catalytic activity of pnpla3 is controversial, it may serve as a triglyceride lipase, since its loss-of function mutation increases cellular triglyceride levels. 64) furthermore, recent evidence suggests that pnpla3 acts as a retinyl-palmitate lipase in hepatic stellate cells to fine-tune the plasma levels of retinoids. the expressions of pnpla2 and pnpla3 are nutritionally regulated in a reciprocal way; pnpla2 is upregulated, while pnpla3 is downregulated, upon starvation, and vice versa upon feeding. 65) biochemical and cell biological studies have suggested that pnpla4 (ipla 2 2, which is absent in mice) might be involved in retinol ester metabolism 66) and that pnpla5 might participate in triglyceride lipolysis coupled with autophagosome formation, 67) although the in vivo relevance of these in vitro observations is unclear. unlike most pnpla isoforms that are ubiquitously expressed in many tissues, pnpla1 is localized predominantly in the upper layer of the epidermis. pnpla1 acts as a unique transacylase, catalyzing the transfer of linoleic acid (la; c18:2) in triglyceride to the b-hydroxy residue of ultra-longchain fatty acid in ceramide to form b-o-acylceramide, a lipid component essential for skin barrier function. 68),69) accordingly, genetic deletion or mutation of pnpla1 hampers epidermal b-o-acylceramide formation, thereby severely impairing skin barrier function and causing ichthyosis. the unique role of pnpla1 in the acylceramide-metabolic pathway in the epidermis is depicted in fig. 5 . the pafah family. the paf-acetylhydrolase (pafah) family comprises one extracellular and three intracellular pla 2 s that were originally found to have the capacity to deacetylate and thereby inactivate the lysophospholipid-derived lipid mediator paf. 70),71) type-i pafah is a heterotrimer composed of two catalytic subunits, group xiiia and xiiib pla 2 s, and a regulatory subunit lis-1, the causative gene for a type of miller diecker syndrome. 72) deficiency of type-i pafah leads to male infertility through an unknown mechanism. 73) type-ii pafah (group viib pla 2 ) preferentially hydrolyzes oxidized phospholipids (i.e., phospholipids having an oxygenated fatty acid at the sn-2 position) in cellular membranes, thereby protecting cells from oxidative damage. 74) although plasma-type pafah (group viia pla 2 ) is a secreted protein, it is described here as its structure is close to type-ii pafah. plasma-type pafah is now more generally called lipoprotein-associated pla 2 (lp-pla 2 ), existing as a low-density lipoprotein (ldl)-bound form in human plasma. 75) a series of studies have revealed the correlation of lp-pla 2 with atherosclerosis, likely because this enzyme liberates toxic oxidized fatty acids from modified ldl with pro-atherogenic potential. 76),77) furthermore, deficiency of lp-pla 2 decreases intestinal polyposis and colon tumorigenesis in apc min/d mice, 78) suggesting an anti-tumorigenic role for paf in this setting. lysosomal pla 2 . lysosomal pla 2 (lpla 2 ), also known as group xv pla 2 , is homologous with lecithin cholesterol acyltransferase (lcat) and catalytically active under mildly acidic conditions. 79) lpla 2 hydrolyzes both sn-1 and sn-2 fatty acids in phospholipids and contributes to phospholipid degradation in lysosomes. genetic deletion of lpla 2 results in unusual accumulation of non-degraded lung surfactant phospholipids in lysosomes of alveolar macrophages, leading to phospholipidosis, 80) perturbed presentation of endogenous lysophospholipid antigens to cd1d by invariant natural killer t (inkt) cells, 81) and impairment of adaptive t cell immunity against mycobacterium. 82) the plaat family. the pla-acyltransferase (plaat) family (3 enzymes in humans and 5 enzymes in mice) is structurally similar to lecithin retinol acyltransferase (lrat). members of this family, including group xvi pla 2 (pla2g16), display pla 1 and pla 2 activities, as well as acyltransferase activity that synthesizes n-acyl-pe, to various degrees. 83) pla2g16 is highly expressed in adipocytes, and pla2g16-deficient mice are resistant to diet-induced obesity. 84) pla2g16 and its paralogs in this family have also been implicated in tumor invasion and metastasis, 85) vitamin a metabolism, 86) peroxisome biogenesis, 87) and cellular entry and clearance of picornaviruses. 88) the abhd family. the ,/o hydrolase (abhd) family is a newly recognized group of lipolytic enzymes, comprising at least 19 enzymes in humans. 89) enzymes in this family typically possess both hydrolase and acyltransferase motifs. although the functions of many of the abhd isoforms still remain uncertain, some of them have been demonstrated to act on neutral lipids or phospholipids as lipid hydrolases. abhd3 selectively hydrolyzes phospholipids with medium-chain fatty acids. 90 lipoquality control by phospholipase a 2 enzymes no. 9] abhd4 releases fatty acids from multiple classes of n-acyl-phospholipids to produce n-acyl-lysophospholipids. 91) abhd6 acts as lysophospholipase or monoacylglycerol lipase, the latter being possibly related to the regulation of 2-arachidonoyl glycerol (2-ag) signaling. 92),93) 2-ag is an endocannabinoid lipid mediator that plays a role in the retrograde neurotransmission and is considered to be produced mainly by diacylglycerol lipase ,. 94) interestingly, in the brain, the aa released from 2-ag by monoacylglycerol lipase, rather than that released from phospholipids by cpla 2 , (see above), is linked to the production of a pool of pge 2 that promotes fever. 2),95) abhd12 hydrolyzes lysophosphatidylserine (lysops), and is therefore referred to as lysops lipase. 96) mutations in the human abhd12 gene result in accumulation of lysops in the brain and cause a disease called pharc, which is characterized by polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract. 97) abhd16a acts as a phosphatidylserine (ps)-selective pla 2 (referred to as ps lipase), being located upstream of abhd12 in the ps-catabolic pathway. 96) although abhd5 (also called cgi-58) does not have a catalytic activity because of the absence of a serine residue in the catalytic center, it greatly enhances pnpla2directed hydrolysis of triglycerides in lipid droplets by acting as an essential lipolytic cofactor. 98) 4. lipoquality control by secreted pla 2 s general aspects. the secreted pla 2 (spla 2 ) family contains 10 catalytically active isoforms and one inactive isoform in mammals. 42),99) based on the structural and evolutional relationships, these enzymes are categorized into classical (ib, iia, iic, iid, iie, iif, v and x) and atypical (iii and xii) classes (fig. 6) . the spla 2 family strictly hydrolyzes the sn-2 position of phospholipids, a feature that differs from intracellular pla 2 s that often display pla 1 , lysophospholipase, lipase, or transacylase/acyltransferase activity (see above). individual spla 2 s exhibit unique tissue and cellular distributions, suggesting their distinct biological roles. as spla 2 s are secreted and require ca 2d in the mm range for their catalytic action, their principal targets are phospholipids in the extracellular space, such as microparticles, surfactant, lipoproteins, and foreign phospholipids in microbe membranes or dietary components. the biochemical properties and pathophysiological functions of spla 2 s have been detailed in several recent reviews. 5), 100) here, i describe several key features of lipoquality regulation by the spla 2 family. in terms of the lipoquality, spla 2 s have long been considered to display no apparent selectivity for sn-2 fatty acid species in the substrate phospholipids. this view was based on the fact that spla 2 -ib and -iia, two prototypic spla 2 s that were initially identified through classical protein purification from the pancreas and sites of inflammation, respectively, 101),102) as well as a number of snake venom pla 2 s that belong to group i and ii spla 2 s, are capable of releasing fatty acids non-selectively. however, recent lipidomics-based evaluation of the substrate specificity of spla 2 s toward natural membranes (see above) has revealed that several spla 2 s can distinguish sn-2 fatty acyl moieties in phospholipids under physiologically relevant conditions. in general terms, spla 2 -ib, -iia and -iie do not discriminate fatty acid species, spla 2 -v tends to prefer those with a lower degree of unsaturation such as oleic acid (oa; c18:1), and spla 2 -iid, -iif, -iii and -x tend to prefer pufas including aa and dha. several spla 2 s can also distinguish differences in the polar head groups of phospholipids. for instance, spla 2 -x is very active on pc, while spla 2 -iia has much higher affinity for pe than for pc, and this substrate selectivity has been partly ascribed to their crystal structures. 103),104) therefore, in order to comprehensively understand the specific biological roles of this enzyme family, it is important to consider when and where different spla 2 s are expressed, which isoforms are involved in what types of pathophysiology, why they are needed, and how they exhibit their unique functions by driving specific types of lipid metabolism. classical spla 2 s. spla 2 -ib, also known as "pancreatic spla 2 ", is synthesized as an inactive zymogen in the pancreas, and its n-terminal propeptide is cleaved by trypsin to yield an active enzyme in the duodenum. 101) the main role of spla 2 -ib is to digest dietary and biliary phospholipids in the intestinal lumen. perturbation of this process by gene disruption or pharmacological inhibition of spla 2 -ib leads to resistance to diet-induced obesity, insulin resistance, and atherosclerosis due to decreased phospholipid digestion and absorption in the gastrointestinal tract. 105)-108) the human pla2g1b gene maps to an obesity-susceptible locus. 109) spla 2 -iia is often referred to as "inflammatory spla 2 ", since its expression is induced by proinflammatory cytokines such as tnf, and il-1o or by bacterial products such as lipopolysaccharide. 110) in mice, however, spla 2 -iia in mice is distributed only in intestinal paneth cells (in balb/c, c3h, nzb and dba, etc.) or not expressed at all due to a natural frameshift mutation (in c57bl/6, a/j, c58/ j, p/j, 129/sv and b10.riii, etc.). 111),112) the bestknown physiological function of spla 2 -iia is the degradation of bacterial membranes, thereby providing the first line of antimicrobial defense in the host. 113),114) consistent with this, spla 2 -iia preferentially hydrolyzes pe and phosphatidylglycerol, which are enriched in bacterial membranes. under sterile conditions, spla 2 -iia attacks phospholipids in microparticles, particularly those in extracellular mitochondria (an organelle that evolutionally originated from bacteria), which are released from activated platelets or leukocytes at inflamed sites. 115) hydrolysis of microparticular phospholipids by spla 2 -iia results in production of pro-inflammatory eicosanoids and lysophospholipids as well as in release of mitochondrial dna as a danger-associated molecular pattern (damp). thus, spla 2 -iia is primarily involved in host defense by killing bacteria and triggering innate immunity, while over-amplification of the response leads to exacerbation of inflammation. spla 2 -iia, -iic, -iid, -iie and -iif are often classified into the group ii subfamily (spla 2 -iic is a pseudogene in human), since they share structural characteristics and map to the same chromosome locus. spla 2 -iid is constitutively expressed in dendritic cells (dcs) in lymphoid organs. spla 2 -iid is an "immunosuppressive spla 2 " that attenuates dc-mediated adaptive immunity by hydrolyzing pe probably in microparticles to mobilize antiinflammatory b3 pufas and their metabolites such as resolvin d1 (rvd1). 7) as such, spla 2 -iid-null mice exhibit more severe contact hypersensitivity and psoriasis, whereas they are protected against infection and cancer because of enhanced anti-viral and anti-tumor immunity. 7),116),117) unlike spla 2 -iia, which is stimulus-inducible (see above), spla 2 -iid is downregulated by pro-inflammatory stimuli, consistent with its anti-inflammatory role. in mice, spla 2 -iie instead of spla 2 -iia is upregulated in several tissues under inflammatory or other conditions. spla 2 -iie is expressed in hair follicles in association with the growth phase of the hair cycle 118) and induced in adipose tissue in association with obesity in mice. 119) spla 2 -iie hydrolyzes pe without apparent fatty acid selectivity in hair follicles and lipoproteins, and accordingly, spla 2 -iie-deficient mice display subtle abnormalities in hair follicles 118) and are modestly protected from diet-induced obesity and hyperlipidemia. 119) spla 2 -iif has a long c-terminal extension containing a free cysteine, which might contribute to formation of a homodimer, and is more hydrophobic than other spla 2 s. 120) physiologically, spla 2 -iif is an "epidermal spla 2 " that is expressed predominantly in the upper epidermis and induced by il-22, a th17 cytokine, in psoriatic skin. 6) spla 2 -iif preferentially hydrolyzes pufa-containing plasmalogen-type pe in keratinocyte-secreted phospholipids to produce plasmalogen-type lysophosphatidylethanolamine (p-lpe; lysoplasmalogen), which in turn promotes epidermal hyperplasia (fig. 7a-c) . accordingly, spla 2 -iif-null mice are protected against epidermal-hyperplasic diseases such as psoriasis and skin cancer, while spla 2 -iif-transgenic mice spontaneously develop psoriasis-like skin. 6) although spla 2 -v was previously thought to be a regulator of aa metabolism, 121), 122) it is now becoming obvious that this spla 2 has a preference for phospholipids having fatty acids with a lower degree of unsaturation. spla 2 -v is markedly induced in adipocytes during obesity as a "metabolic spla 2 " and hydrolyzes pc in hyperlipidemic ldl to release oa and to a lesser extent la, which counteract adipose tissue inflammation and thereby ameliorates obesity-associated metabolic disorders. 119) transgenic overexpression of spla 2 -v, but not other spla 2 s, results in neonatal death due to a respiratory defect, which is attributable to the ability of spla 2 -v to potently hydrolyze pc with palmitic acid (pa, c16:0), a major component of lung surfactant. 123) this unique substrate preference of spla 2 -v has also been supported by a recent lipidomics analysis of the spleen (a tissue where spla 2 -v is abundantly expressed), in which the levels of fatty acids with a lower degree of unsaturation (e.g. pa, oa and la), rather than pufas (aa, epa and dha), are significantly reduced in spla 2 -v-deficient mice relative to wild-type mice (fig. 8) . this is in contrast to the spleen of spla 2 -iid-deficient mice, in which b3 pufas and their metabolites are selectively diminished, 7) revealing distinct lipoquality regulation 0.0e+00 2.0e+08 4.0e+08 6.0e+08 8.0e+08 , were significantly reduced in spla 2 -v-deficient mice relative to control mice. accordingly, la metabolites, including 9-and 13-hydroxyoctadecadienoic acids (hodes) among others, were substantially decreased in mutant mice relative to control mice, whereas none of the aa, epa and dha metabolites differed significantly between the genotypes. these results are consistent with the view that spla 2 -v has a propensity to preferentially hydrolyze phospholipids having sn-2 fatty acids with a lower degree of unsaturation, as illustrated at right bottom. by different spla 2 s. another intriguing feature of spla 2 -v is that it is the only "th2-prone spla 2 " induced in m2 macrophages by the th2 cytokines il-4 and il-13 and promotes th2-driven pathology such as asthma. gene ablation of spla 2 -v perturbs proper polarization and function of m2 macrophages in association with decreased th2 immunity, 124) although the underlying lipid metabolism responsible for this event remains obscure. probably because of this alteration in the macrophage phenotype, spla 2 -v-null macrophages have a reduced ability to phagocytose extracellular materials. accordingly, spla 2 -v-null mice are more susceptible to fungal infection and arthritis due to defective clearance of hazardous fungi and immune complexes, respectively. 125),126) likewise, spla 2 -v-null mice suffer from more severe lung inflammation caused by bacterial or viral infection, 127) which could also be explained by poor clearance of these microbes by alveolar macrophages. among the mammalian spla 2 s, spla 2 -x has the highest affinity for pc leading to release of fatty acids, with an apparent tendency for pufa preference. spla 2 -x is activated by cleavage of the nterminal propeptide by furin-type convertases. 128) spla 2 -x is expressed abundantly in colorectal epithelial and goblet cells and has a protective role in colitis by mobilizing anti-inflammatory b3 pufas. 24) consistently, spla 2 -x-transgenic mice exhibit global anti-inflammatory phenotypes in association with elevation of systemic b3 pufa levels. 24) in the process of reproduction, spla 2 -x secreted from the acrosomes of activated spermatozoa hydrolyzes sperm membrane phospholipids to release dha and docosapentaenioc acid (dpa, c22:5), the latter facilitating fertilization. 24),129) additionally, spla 2 -x-null mice are protected from asthma, accompanied by decreased levels of pulmonary b6 aa-derived eicosanoids. 130) unlike the situation in spla 2 -v-null mice (see above), however, the th2 response per se is not affected in the asthma model 131) and the lung damage is milder following influenza infection 132) in spla 2 -x-null mice, illustrating the distinct actions of different spla 2 s in the same tissue. atypical spla 2 s. spla 2 -iii is unusual in that it consists of three domains, in which the central spla 2 domain similar to bee venom group iii spla 2 is flanked by large and unique n-and cterminal domains. 133) the enzyme is processed to the spla 2 domain-only form that retains full enzymatic activity. 134) although spla 2 -iii does not discriminate the polar head groups, it tends to prefer sn-2 pufas in the substrate phospholipids. spla 2 -iii is expressed in the epididymal epithelium and acts on immature sperm cells passing through the epididymal duct in a paracrine manner to allow sperm membrane phospholipid remodeling, a process that is prerequisite for sperm motility. 135) spla 2 -iii is also secreted from mast cells and acts on microenvironmental fibroblasts to produce pgd 2 , which in turn promotes proper maturation of mast cells. 136) accordingly, mice lacking spla 2 -iii exhibit male hypofertility and reduced anaphylactic responses. spla 2 -xiia is evolutionally far distant from other spla 2 s. 137) spla 2 -xiia is expressed in many tissues at relatively high levels, yet its enzymatic activity is weaker than that of other spla 2 s. the properties and physiological roles of spla 2 -xiia are currently unclear and await future studies using spla 2 -xiia-deficient mice. apart from lipoquality regulation, spla 2 -xiib is a catalytically inactive protein due to substitution of the catalytic center histidine by leucine. 138) spla 2 -xiib deficiency impairs hepatic lipoprotein secretion, 139) although the mechanism is unclear. beyond the lipoquality control by spla 2 s, several spla 2 s binds to spla 2 receptor (pla2r1, also known as the c-type lectin clec13c) with different affinities. 140) in mice, pla2r1 binds to spla 2 -ib, -iia, -iie, -iif and -x with high affinity, spla 2 -v with moderate affinity, and spla 2 -iid, -iii and -xiia with low or no affinity. 138) pla2r1 is homologous to spla 2 -inhibitory proteins present in snake plasma and exists as an integral membrane protein or as a soluble protein resulting from shedding or alternative splicing. pla2r1 may act as a clearance receptor or endogenous inhibitor that inactivates spla 2 s, as a signaling receptor that transduces spla 2 -dependent signals in a catalytic activity-independent manner, or as a pleiotropic receptor that binds to non-spla 2 ligands. in support of its clearance role, pla2r1 !/! mice show more severe asthma, likely due to defective clearance of pro-asthmatic spla 2 -x. 141) in support of its signaling role, pla2r1, probably through binding to myocardial spla 2 s or other ways, promotes the migration and growth of myofibroblasts and thereby protects against cardiac rupture in a model of myocardial infarction. 142) pla2r1 has recently attracted attention as a major autoantigen in membranous nephropathy, a severe autoimmune disease leading to podocyte injury and proteinuria, 143) although it is not clear whether this role of pla2r1 is spla 2dependent or -independent. by applying lipidomics approaches to knockout or transgenic mice for various pla 2 s, it has become evident that individual enzymes regulate specific forms of lipid metabolism, perturbation of which can be eventually linked to distinct pathophysiological outcomes. knowledge of lipoquality control by individual pla 2 s acquired from studies using animal models should be translated to humans. current knowledges on the relationship between pla 2 gene mutations and human diseases are summarized in table 2 . nonetheless, future development of more comprehensive and highly sensitive lipidomics techniques will contribute to the discovery of novel pla 2driven lipid pathways that could be biomarkers or druggable targets for particular diseases. nakanishi, h., ikeda, k., taguchi, r., kabashima deletion of cytosolic phospholipase a 2 promotes striated muscle growth lipid signaling in cytosolic phospholipase a 2 ,-cyclooxygenase-2 cascade mediates cerebellar longterm depression and motor learning acute lung injury by sepsis and acid aspiration: a key role for cytosolic phospholipase a 2 cytosolic phospholipase a 2 ,-deficient mice are resistant to collagen-induced arthritis an essential role of cytosolic phospholipase a 2 , in prostaglandin e 2 -mediated bone resorption associated with inflammation cytosolic phospholipase a 2 ,-deficient mice are resistant to experimental autoimmune encephalomyelitis phospholipase a 2 reduction ameliorates cognitive deficits in a mouse model of alzheimer's disease group x secreted phospholipase a 2 releases b3 polyunsaturated fatty acids, suppresses colitis, and promotes sperm fertility cysteinyl leukotrienes regulate th2 celldependent pulmonary inflammation leukotriene b 4 receptor blt1 mediates early effector t cell recruitment prostaglandin d 2 as a mediator of allergic asthma dual roles of pge 2 -ep4 prostaglandin e 2 -ep4 signaling promotes immune inflammation through th1 cell differentiation and th17 cell expansion acceleration of intestinal polyposis through prostaglandin receptor ep2 in apc "716 knockout mice involvement of prostaglandin e 2 in production of amyloid-o peptides both in vitro and in vivo the prostaglandin receptor ep4 suppresses colitis, mucosal damage and cd4 cell activation in the gut mapping the human platelet lipidome reveals cytosolic phospholipase a 2 as a regulator of mitochondrial bioenergetics during activation the enteropathy of prostaglandin deficiency inherited human cpla 2 , deficiency is associated with impaired eicosanoid biosynthesis, small intestinal ulceration, and platelet dysfunction interfacial kinetic and binding properties of mammalian group ivb phospholipase a 2 (cpla 2 o) and comparison with the other cpla 2 isoforms a novel calcium-independent phospholipase a 2 , cpla 2 ., that is prenylated and contains homology to cpla 2 cloning of a gene for a novel epithelium-specific cytosolic phospholipase a 2 , cpla 2 /, induced in psoriatic skin a calcium-dependent acyltransferase that produces n-acyl phosphatidylethanolamines function, activity, and membrane targeting of cytosolic phospholipase a 2 1 in mouse lung fibroblasts mammalian patatin domain containing proteins: a family with diverse lipolytic activities involved in multiple biological functions recent progress in phospholipase a 2 research: from cells to animals to humans a novel cytosolic calcium-independent phospholipase a 2 contains eight ankyrin motifs multiple splice variants of the human calcium-independent phospholipase a 2 and their effect on enzyme activity cyclooxygenase-2 mediated oxidation of 2-arachidonoyl-lysophospholipids identifies unknown lipid signaling pathways activation of mitochondrial calcium-independent phospholipase a 2 . (ipla 2 .) by divalent cations mediating arachidonate release and production of downstream eicosanoids identification of an insulin-regulated lysophospholipase with homology to neuropathy target esterase evidence that mouse brain neuropathy target esterase is a lysophospholipase genetic ablation of calcium-independent phospholipase a 2 . prevents obesity and insulin resistance during high fat feeding by mitochondrial uncoupling and increased adipocyte fatty acid oxidation loss of function variants in human pnpla8 encoding calcium-independent phospholipase a 2 . recapitulate the mitochondriopathy of the homologous null mouse loss-offunction mutations in pnpla6 encoding neuropathy target esterase underlie pubertal failure and neurological deficits in gordon holmes syndrome mutations in pnpla6 are linked to photoreceptor degeneration and various forms of childhood blindness impairment of park14-dependent ca 2d signalling is a novel determinant of parkinson's disease male mice that do not express group via phospholipase a 2 produce spermatozoa with impaired motility and have greatly reduced fertility fat mobilization in adipose tissue is promoted by adipose triglyceride lipase defective lipolysis and altered energy metabolism in mice lacking adipose triglyceride lipase adipose triglyceride lipase contributes to cancer-associated cachexia the g 0 /g 1 switch gene 2 regulates adipose lipolysis through association with adipose triglyceride lipase atgl-mediated fat catabolism regulates cardiac mitochondrial function via ppar-, and pgc-1 lipolysis -a highly regulated multienzyme complex mediates the catabolism of cellular fat stores biochemistry and pathophysiology of intravascular and intracellular lipolysis genetic variation in pnpla3 confers susceptibility to nonalcoholic fatty liver disease chronic overexpression of pnpla3 i148m in mouse liver causes hepatic steatosis a feedforward loop amplifies nutritional regulation of pnpla3 identification of a novel keratinocyte retinyl ester hydrolase as a transacylase and lipase neutral lipid stores and lipase pnpla5 contribute to autophagosome biogenesis has a crucial role in skin barrier function by directing acylceramide biosynthesis pnpla1 is a transacylase essential for the generation of the skin barrier lipid b-o-acylceramide intracellular pafacetylhydrolase type i intracellular plateletactivating factor acetylhydrolase, type ii: a unique cellular phospholipase a 2 that hydrolyzes oxidatively modified phospholipids brain acetylhydrolase that inactivates plateletactivating factor is a g-protein-like trimer targeted disruption of intracellular type i platelet activating factor-acetylhydrolase catalytic subunits causes severe impairment in spermatogenesis protection against oxidative stress-induced hepatic injury by intracellular type ii platelet-activating factor acetylhydrolase by metabolism of oxidized phospholipids in vivo antiinflammatory properties of a platelet-activating factor acetylhydrolase inhibition of lipoprotein-associated phospholipase a 2 reduces complex coronary atherosclerotic plaque development phospholipase a 2 inhibitors in atherosclerosis: the race is on deficiency of phospholipase a 2 group 7 decreases intestinal polyposis and colon tumorigenesis in apc min/d mice lysosomal phospholipase a 2 is selectively expressed in alveolar macrophages lysosomal phospholipase a 2 and phospholipidosis role for lysosomal phospholipase a 2 in inkt cell-mediated cd1d recognition lysosomal phospholipase a 2 : a novel player in host immunity to mycobacterium tuberculosis generation of n-acylphosphatidylethanolamine by members of the phospholipase a/acyltransferase adpla ablation increases lipolysis and prevents obesity induced by high-fat feeding or leptin deficiency pla2g16 phospholipase mediates gain-of-function activities of mutant p53 lrat-specific domain facilitates vitamin a metabolism by domain swapping in hrasls3 interaction of phospholipase a/ acyltransferase-3 with pex19p: a possible involvement in the down-regulation of peroxisomes represents a switch between entry and clearance of picornaviridae in vivo metabolite profiling as a means to identify uncharacterized lipase function: recent success stories within the alpha beta hydrolase domain (abhd) enzyme family metabolomics annotates abhd3 as a physiologic regulator of mediumchain phospholipids abhd4 regulates multiple classes of n-acyl phospholipids in the mammalian central nervous system the serine hydrolase abhd6 controls the accumulation and efficacy of 2-ag at cannabinoid receptors the serine hydrolase abhd6 is a critical regulator of the metabolic syndrome the endocannabinoid 2-arachidonoylglycerol produced by diacylglycerol lipase , mediates retrograde suppression of synaptic transmission fever is mediated by conversion of endocannabinoid 2-arachidonoylglycerol to prostaglandin e 2 immunomodulatory lysophosphatidylserines are regulated by abhd16a and abhd12 interplay abhd12 controls brain lysophosphatidylserine pathways that are deregulated in a murine model of the neurodegenerative disease pharc adipose triglyceride lipase-mediated lipolysis of cellular fat stores is activated by cgi-58 and defective in chanarin-dorfman syndrome a new era of secreted phospholipase a 2 the roles of the secreted phospholipase a 2 gene family in immunology pancreatic phospholipase a 2 : isolation of the human gene and cdnas from porcine pancreas and human lung cloning and recombinant expression of phospholipase a 2 present in rheumatoid arthritic synovial fluid crystal structure of human group x secreted phospholipase a 2 . electrostatically neutral interfacial surface targets zwitterionic membranes structures of free and inhibited human secretory phospholipase a 2 from inflammatory exudate protection against diet-induced obesity and obesity-related insulin resistance in group 1b pla 2 -deficient mice group 1b phospholipase a 2 -mediated lysophospholipid absorption directly contributes to postprandial hyperglycemia the phospholipase a 2 inhibitor methyl indoxam suppresses diet-induced obesity and glucose intolerance in mice group 1b phospholipase a 2 inactivation suppresses atherosclerosis and metabolic diseases in ldl receptor-deficient mice linkage and potential association of obesity-related phenotypes with two genes on chromosome 12q24 in a female dizygous twin cohort phospholipase a 2 -a mediator between proximal and distal effectors of inflammation a natural disruption of the secretory group ii phospholipase a 2 gene in inbred mouse strains the secretory phospholipase a 2 gene is a candidate for the mom1 locus, a major modifier of apc min -induced intestinal neoplasia mobilization of potent plasma bactericidal activity during systemic bacterial challenge. role of group iia phospholipase a 2 staphylococcus aureus adenosine inhibits spla 2 -iia-mediated host killing in the airways platelets release mitochondria serving as substrate for bactericidal group iia-secreted phospholipase a 2 to promote inflammation dual roles of group iid phospholipase a 2 in inflammation and cancer critical role of phospholipase a 2 group iid in age-related susceptibility to severe acute respiratory syndrome-cov infection expression and function of group iie phospholipase a 2 in mouse skin the adipocyte-inducible secreted phospholipases pla2g5 and pla2g2e play distinct roles in obesity on the diversity of secreted phospholipases a 2 . cloning, tissue distribution, and functional expression of two novel mouse group ii enzymes the functions of five distinct mammalian phospholipase a 2 s in regulating arachidonic acid release. type iia and type v secretory phospholipase a 2 s are functionally redundant and act in concert with cytosolic phospholipase a 2 regulation of delayed prostaglandin production in activated p388d1 macrophages by group iv cytosolic and group v secretory phospholipase a 2 s transgenic expression of group v, but not group x, secreted phospholipase a 2 in mice leads to neonatal lethality because of lung dysfunction group v secretory phospholipase a 2 is involved in macrophage activation and is sufficient for macrophage effector functions in allergic pulmonary inflammation group v secretory phospholipase a 2 modulates phagosome maturation and regulates the innate immune response against candida albicans a novel anti-inflammatory role for secretory phospholipase a 2 in immune complexmediated arthritis group v phospholipase a 2 in bone marrowderived myeloid cells and bronchial epithelial cells promotes bacterial clearance after escherichia coli pneumonia group x secreted phospholipase a 2 proenzyme is matured by a furin-like proprotein convertase and releases arachidonic acid inside of human hek293 cells group x phospholipase a 2 is released during sperm acrosome reaction and controls fertility outcome in mice importance of group x-secreted phospholipase a 2 in allergen-induced airway inflammation and remodeling in a mouse asthma model key role of group v secreted phospholipase a 2 in th2 cytokine and dendritic celldriven airway hyperresponsiveness and remodeling lack of group x secreted phospholipase a 2 increases survival following pandemic h1n1 influenza infection novel human secreted phospholipase a 2 with homology to the group iii bee venom enzyme cellular arachidonate-releasing function of novel classes of secretory phospholipase a 2 s (groups iii and xii) group iii secreted phospholipase a 2 regulates epididymal sperm maturation and fertility in mice mast cell maturation is driven via a group cloning and recombinant expression of a structurally novel human secreted phospholipase a 2 novel mammalian group xii secreted phospholipase a 2 lacking enzymatic activity hepatocyte nuclear factor-4, regulates liver triglyceride metabolism in part through secreted phospholipase a 2 gxiib biochemistry and physiology of mammalian secreted phospholipases a 2 deficiency of phospholipase a 2 receptor exacerbates ovalbumin-induced lung inflammation circulating levels of secretory type ii phospholipase a 2 predict coronary events in patients with coronary artery disease thrombospondin type-1 domain-containing 7a in idiopathic membranous nephropathy efficient phagocytosis requires triacylglycerol hydrolysis by adipose triglyceride lipase desnutrin/ atgl activates ppar/ to promote mitochondrial function for insulin secretion in islet o cells patatin-like phospholipase domain-containing protein 3 is involved in hepatic fatty acid and triglyceride metabolism through x-box binding protein 1 and modulation of endoplasmic reticulum stress in mice patatin-like phospholipase domain-containing 3/ adiponutrin deficiency in mice is not associated with fatty liver disease brainspecific deletion of neuropathy target esterase/ swisscheese results in neurodegeneration group vib calcium-independent phospholipase a 2 (ipla 2 .) regulates platelet activation, hemostasis and thrombosis in mice mitochondrial dysfunction and reduced prostaglandin synthesis in skeletal muscle of group vib ca 2d -independent phospholipase a 2 .-deficient mice mice deficient in group vib phospholipase a 2 (ipla 2 .) exhibit relative resistance to obesity and metabolic abnormalities induced by a western diet genetic ablation of calcium-independent phospholipase a 2 . leads to alterations in hippocampal cardiolipin content and molecular species distribution, mitochondrial degeneration, autophagy, and cognitive dysfunction genetic ablation of calcium-independent phospholipase a 2 . leads to alterations in mitochondrial lipid metabolism and function resulting in a deficient mitochondrial bioenergetic phenotype smooth muscle cell arachidonic acid release, migration, and proliferation are markedly attenuated in mice null for calcium-independent phospholipase a 2 o age-related changes in bone morphology are accelerated in group via phospholipase a 2 (ipla 2 o)-null mice neuroaxonal dystrophy caused by group via phospholipase a 2 deficiency in mice: a model of human neurodegenerative disease group via phospholipase a 2 in both host and tumor cells is involved in ovarian cancer development platelet-activating factor and metastasis: calciumindependent phospholipase a 2 o deficiency protects against breast cancer metastasis to the lung loss of pafah1b2 reduces amyloid-o generation by promoting the degradation of amyloid precursor protein c-terminal fragments paf-ah catalytic subunits modulate the wnt pathway in developing gabaergic neurons increase of smooth muscle cell migration and of intimal hyperplasia in mice lacking the ,/o hydrolase domain containing 2 gene age-related pulmonary emphysema in mice lacking ,/o hydrolase domain containing 2 gene skin barrier development depends on cgi-58 protein expression during late-stage keratinocyte differentiation growth retardation, impaired triacylglycerol catabolism, hepatic steatosis, and lethal skin barrier defect in mice lacking comparative gene identification-58 (cgi-58) /o-hydrolase domain 6 deletion induces adipose browning and prevents obesity and type 2 diabetes platelet microparticles are internalized in neutrophils via the concerted activity of 12-lipoxygenase and secreted phospholipase a 2 -iia secreted phospholipases a 2 are intestinal stem cell niche factors with distinct roles in homeostasis, inflammation, and cancer secretory group v phospholipase a 2 regulates acute lung injury and neutrophilic inflammation caused by lps in mice group v secretory phospholipase a 2 promotes atherosclerosis: evidence from genetically altered mice group v secretory phospholipase a 2 plays a pathogenic role in myocardial ischaemia-reperfusion injury group v secretory phospholipase a 2 enhances the progression of angiotensin ii-induced abdominal aortic aneurysms but confers protection against angiotensin ii-induced cardiac fibrosis in apoe-deficient mice group x secretory phospholipase a 2 regulates insulin secretion through a cyclooxygenase-2-dependent mechanism group x secretory phospholipase a 2 enhances tlr4 signaling in macrophages group x secreted phospholipase a 2 limits the development of atherosclerosis in ldl receptor-null mice group x secretory phospholipase a 2 augments angiotensin ii-induced inflammatory responses and abdominal aortic aneurysm formation in apoe-deficient mice group x secretory pla 2 in neutrophils plays a pathogenic role in abdominal aortic aneurysms in mice mutations cause autosomal recessive congenital ichthyosis in golden retriever dogs and humans the gene encoding adipose triglyceride lipase (pnpla2) is mutated in neutral lipid storage disease with myopathy neuropathy target esterase gene mutations cause motor neuron disease genetic associations of nonsynonymous exonic variants with psychophysiological endophenotypes genome-wide association study identifies variants at 9p21 and 22q13 associated with development of cutaneous nevi lipoprotein-associated phospholipase a 2 adds to risk prediction of incident coronary events by c-reactive protein in apparently healthy middle-aged men from the general population: results from the 14-year follow-up of a large cohort from southern germany tagging-snp haplotype analysis of the secretory pla 2 iia gene pla2g2a shows strong association with serum levels of spla 2 iia: results from the udacs study phospholipase a 2 group iia expression in gastric adenocarcinoma is associated with prolonged survival and less frequent metastasis a novel polymorphism in secretory phospholipase a 2 -iid is associated with body weight loss in chronic obstructive pulmonary disease phospholipase a 2 group iii and group x have opposing associations with prognosis in colorectal cancer a gene involved in oxidative stress induced death, is associated with alzheimer's disease tagging snp haplotype analysis of the secretory pla 2 -v gene, pla2g5, shows strong association with ldl and oxldl levels, suggesting functional distinction from spla 2 -iia: results from the udacs study biallelic mutations in pla2g5, encoding group v phospholipase a 2 , cause benign fleck retina key: cord-287670-z6ckhkgg authors: magrini, elena; mantovani, alberto; garlanda, cecilia title: the dual complexity of ptx3 in health and disease: a balancing act? date: 2016-06-30 journal: trends in molecular medicine doi: 10.1016/j.molmed.2016.04.007 sha: doc_id: 287670 cord_uid: z6ckhkgg the humoral arm of innate immunity is complex and includes various molecules that serve as markers of inflammation with complementary characteristics, such as the short pentraxins c-reactive protein (crp) and serum amyloid p (sap) and the long pentraxin ptx3. there is a growing amount of evidence – including mouse and human genetics – that suggests that ptx3 is essential in conferring host resistance against selected pathogens and, moreover, that it plays a dual antagonistic role in the regulation of inflammation. dissection of such a yin-and-yang role of pentraxins in immunity and inflammation is timely and significant as it may pave the way for better clinical exploitation against various diseases. the dual complexity of ptx3 in health and disease: a balancing act? elena magrini, 1 alberto mantovani, 1,2, * and cecilia garlanda 1 the humoral arm of innate immunity is complex and includes various molecules that serve as markers of inflammation with complementary characteristics, such as the short pentraxins c-reactive protein (crp) and serum amyloid p (sap) and the long pentraxin ptx3. there is a growing amount of evidenceincluding mouse and human geneticsthat suggests that ptx3 is essential in conferring host resistance against selected pathogens and, moreover, that it plays a dual antagonistic role in the regulation of inflammation. dissection of such a yin-andyang role of pentraxins in immunity and inflammation is timely and significant as it may pave the way for better clinical exploitation against various diseases. the innate immune response is the first line of defense against invading microbes and tissue damage and comprises both a cellular and a humoral arm. sensing of microbes and tissue injury through pattern recognition molecules (prms) (see glossary) triggers a complex response in the organism that includes the production of inflammatory cytokines, the activation of the acute phase response, and leukocyte recruitment and polarization [1, 2] . this response has general significance in defense and orchestration of tissue repair; however, it is potentially part of the pathogenic mechanisms associated with the original cause of a particular diseasefor instance, in sepsis or in chronic inflammatory diseases such as arthritis. the cellular arm of innate immunity comprises cell-associated prms located in different cellular compartments (plasma membrane, endosomes, cytoplasm) belonging to different molecular classes, such as the toll-like receptors (tlrs), the nucleotide-binding oligomerization domain (nod), and rig-like receptors, as well as the scavenger receptors. the humoral arm of innate immunity includes biochemically heterogeneous molecules such as the classic short pentraxins crp) and sap, the long pentraxin ptx3, complement recognition molecules such as c1q and ficolins, and the collectins. the activation of such molecules represents an important component of the host response against invading microbes or tissue injury [3] . these fluid-phase prms harbor antibody-like properties, recognizing microbial moieties, exhibiting opsonic activity, and activating and regulating the complement cascade [4] , as well as interacting with extracellular matrix (ecm) components [5] . the expression of humoral prms is induced following infection or injury in various cell types and with different kinetics, thus providing a continuous presence of these molecules both in the circulation and in tissues [3] . the liver supports the expression and production of short pentraxins systemically. other cell types, particularly macrophages, dendritic cells (dcs), and endothelial cells (ecs), produce ptx3 in a gene expression-dependent fashion [4] . finally, neutrophils act as a reservoir of ready-made ptx3, rapidly released within minutes to sites where tissue damage or microbial stimulation are occurring, thus representing a primary source of soluble prms [6] . the long pentraxin ptx3 is an essential component of humoral innate immunity and plays a role in the regulation of inflammation. ptx3 has complex effects on the vasculature, including an interaction with the angiogenic growth factor fgf2 and the regulation of vessel wall tone. by modulating complement-driven inflammation, ptx3 acts as an oncosuppressor gene in mice and selected human tumors. by interacting with provisional matrix components, ptx3 contributes to the orchestration of wound healing and tissue repair/remodeling. ptx3 and the related pentraxins creactive protein (crp) and serum amyloid p (sap) can exert dual roles in inflammation and antimicrobial resistance, by either exerting a protective function or amplifying tissue damage. dissection of the yin-yang role of pentraxins in immunopathology may pave the way towards better exploitation of these molecules as envisaged disease markers and candidate therapeutic agents. the rapid production of pentraxins at the systemic level or within tissues has been shown to correlate with the severity of various clinical conditions, such as cardiovascular diseases [7] [8] [9] . this in turn appears to sustain their high levels of expression and consequently has often raised the question of their precise role in a given disease: are they simple markers, innocent bystanders, or players in disease pathogenesis [7] [8] [9] ? in particular, crp is a widely used biomarker of inflammation in humans; however, lack of strict evolutionary conservation between mouse and human has precluded the use of straightforward genetic approaches to explore its functions in vivo [10, 11] . by contrast, gene-targeted mice have allowed us to define the role of ptx3 in innate immunity and inflammation as a predecessor to antibody functions and an active player in tissue remodeling [5, [12] [13] [14] . even if most animal studies on the long pentraxin ptx3 are supported by human genetic findings suggesting that ptx3 is important in conferring host resistance to infection [15, 16] , a prevalent concept has surfaced: depending on the disease context, the cellular source, or the levels of protein released, ptx3 may contribute to disease pathogenesis [12, [17] [18] [19] . consequently, this review focuses on the multifaceted and yin-yang role of ptx3 in humoral innate immunity, microbial defense, and regulation of inflammation as well as in tissue remodeling and repair. this concept is presented at an exciting moment, when key players in innate immunity, the pentraxins, have emerged as being capable of exerting potential contradictory roles in health and disease. this is an opportune time to begin making a greater effort to elucidate the exact roles of ptx3 in disease pathogenesis and to better dissect its precise mechanisms of action in a context-specific manner. pentraxins are conserved multimeric proteins characterized by the presence of a conserved eight-amino-acid sequence, the 'pentraxin domain', in their carboxy terminus [4] . based on the primary structure of the protomer, pentraxins have been divided into short pentraxins, including crp and sap, and long pentraxins, such as the prototype long pentraxin ptx3 [4] . crp and sap are approximately 25-kda proteins organized in five identical subunits arranged with pentameric radial symmetry [3, 4] . crp and sap are the main acute phase proteins produced by human and murine liver, respectively [3] . they act as players in the innate immune response by regulating the complement system, recognizing pathogens, and interacting with fcg receptors (fcgrs), thus favoring cytokine secretion and phagocytosis of microorganisms by immune cells [20] . ptx3 was the first long pentraxin identified [4] , followed by other long pentraxins including guinea pig apexin, neuronal pentraxin (np) 1, np2, neuronal pentraxin receptor (npr), and ptx4 [21] . the gene encoding ptx3 is localized to chromosome 3 in humans and mice and comprises three exons encoding the leader signal peptide, the n-terminal domain, and the c-terminal pentraxin domain [4] . the expression of ptx3 is mainly induced by inflammatory stimuli such as inflammatory cytokines [tumor necrosis factor alpha (tnf/) and il-1b] and damage-associated molecular patterns (damps) or microbial moieties. in particular, il-1 is a major inducer of local ptx3 production in sterile tissue damage, such as in mouse models of acute myocardial infarction and in 3-methylcholanthrene (3-mca)-induced carcinogenesis [12, 22] . in skin-wound healing, tlr sensing and il-1 amplification are involved in the expression and production of ptx3 [5] . in urinary tract infections (utis) mediated by uropathogenic escherichia coli (upec), ptx3 production by human and murine uroepithelial cells has been shown to be under the control of the tlr4/myd88 signaling pathway [14] . accordingly, the human and murine ptx3 gene promoters have potential binding sites for many inflammatory transcription factors, including pu.1, ap-1, nf-kb, sp-1, and nf-il-6 [23, 24] . the pi3k/akt axis and jnk have also been shown to activate ptx3 transcription [14] . in addition, epigenetic mechanisms have been implicated in the regulation of human ptx3 expression, since methylation of a ptx3 glossary arthritogenic alphaviruses: include viruses such as chikv and rrv. they are enveloped, positive-sense, single-stranded rna viruses that are usually transmitted by mosquitoes and cause rheumatic disease. c1q: subcomponent of complement c1. c1q has binding sites for antibodies and initiates the complement cascade via the classical pathway. collectins: oligomeric proteins containing a c-terminal carbohydrate recognition domain linked to a collagen-like region through an alphahelical hydrophobic neck region and an n-terminal region. they have the capacity to interact with carbohydrates and lipids exposed on pathogen surfaces. they also bear opsonic activity and can activate the lectin pathway of the complement system. complement cascade: a multimolecular biologic process sustained by more than 20 proteins. these molecules act in concert and in a specific sequence called the complement cascade. three different pathways (i.e., the classical pathway, alternative pathway, and lectin pathway) can be activated. the terminal pathway of the complement cascade leads to the formation of a membrane-attack complex on target surfaces, which induces cell lysis. conidia: also called chlamidospores or mitospores; asexual, nonmotile spores of a fungus. they develop at the tip of specialized hyphae in fungi called conidiophores and are formed by the abstriction at the top of a hyphal branch. this process induces the separation and release of a mature spore by the formation of a septum. cumulus cells: a cluster of cells forming the cumulus oophorus, which is a follicular mass surrounding the oocyte in the ovarian follicle. damage-associated molecular patterns (damps): also known as danger-associated molecular patterns or endogenous alarmins; host cellderived molecules released by stressed cells and necrotic cells (e.g., hmgb1, il-1/). damps can engage receptors (e.g., tlrs) to induce and sustain the inflammatory response. fcg receptors (fcgrs): receptors for the fc (tail) region of igg. based on their structure, they comprise fcgri (cd64), fcgrii (cd32), and fcgriii (cd16). fcgrs participate in the regulation of the immune response. besides their capacity to recognize igg, fcgrs can act as receptors for opsonins (e.g., pentraxins). m2-like polarization: in response to external signals, macrophages can undergo m1 activation (also called classical activation) on stimulation by tlr ligands and ifng or m2 activation (also called alternative activation) on stimulation by il-4/il-13. the m2 or m2-like polarization state has been associated with high production of il-10, low production of il-12, and resolution or smoldering of chronic inflammation. neutrophil extracellular traps (nets): extracellular fibrillary networks formed and released by activated neutrophils. nets comprise dna and histones and contain a set of proteins from neutrophil granules (e.g., mpo, neutrophil elastase, ptx3). nets have the capacity to trap microbes and favor their elimination. nucleotide-binding oligomerization domain (nod) and rig-like receptors: intracellular sensors of microbial moieties and damps associated with cell stress or viruses, respectively. opsonic activity: opsonization is a process by which a molecule (i.e., opsonin) interacts with microbes leading to facilitated microbial recognition and elimination by phagocytosis. pattern recognition molecules (prms): a set of germline-encoded receptors used to discriminate selfversus non-self and modified-self antigens. based on their localization, prms have been divided into cellassociated receptors (e.g., tlrs) and soluble molecules (e.g., collectins, pentraxins). restenosis: common adverse event in endovascular procedures; involves the recurrence of narrowing of a blood vessel, usually an artery. toll-like receptors (tlrs): a family of prms characterized by an extracellular leucine-rich domain and a cytoplasmic domain sharing homology with the receptor for il-1 and the toll protein of drosophila. tlrs are cell-associated molecules located in cytoplasmic membranes or in the membranes of endosomal vesicles. they sense molecules in extracellular compartments and in the lumen of endosomal vesicles. enhancer and a ptx3 promoter has been deemed responsible of ptx3 gene silencing in human colorectal and esophageal cancer cell lines [12] . the protein is a multimer with a complex quaternary structure comprising two tetramers linked by interchain bridges to form an octamer of 340 kda. the protomer comprises 381 amino acids including a 17-amino-acid signal peptide, an n-terminal domain unrelated to any known protein, and a c-terminal pentraxin domain homologous to the short pentraxins crp and sap [25] . a single n-glycosylation site localized in the c-terminal domain at asn220 is occupied by corefucosylated and sialylated complex-type oligosaccharides [26] , which have been shown to modulate the interaction of human ptx3 with complement components such as human c1q [26] , factor h [27] , and ficolin-1 [28] and to be required for influenza virus recognition [29] and binding to the human and murine adhesion molecule p-selectin in vitro and in vivo [30] . various cell types, including dcs, monocytes, macrophages, epithelial cells, ecs, fibroblasts, and adipocytes, produce ptx3 on stimulation with inflammatory cytokines (e.g., tnf/, il-1b), tlr agonists and microbial moieties [e.g., lipopolysaccharide (lps), klebsiella pneumoniae outer membrane protein a (kpompa), other pathogens such as upec and fungal aspergillus fumigatus] [14] ( figure 1 ). neutrophils store ptx3 in specific granules and rapidly release it on encounter with microorganisms or on cell stimulation due to infection, such as in aspergillosis, or with sterile tissue damage, as in the case of human acute myocardial infarction [6, 31] . ptx3 ɵssue damage tlr agonists, inflammatory cytokines (il-1, tnf) ... mrna is transcribed only in myeloid precursors (promyelocytes and myelocytes/metamyelocytes) and not in resting mature cells in mice and humans [6] . the binding of ptx3 to select microorganisms, including bacteria, fungi, and viruses, leads to the activation of various antimicrobial effector mechanisms [3] (figures 1 and 2 ). in addition, ptx3 behaves as an immunoregulatory molecule; it can interact with p-selectin, which results in the modulation of neutrophil recruitment, but it can also interact with components of the complement cascade and tune complement activation [30, 32] . during infection, such as in the case of human sepsis, ptx3 blood levels rapidly increase from approximately 2 ng/ml to 200-800 ng/ml, correlating with both infection and disease severity and predicting patient survival [16, [33] [34] [35] [36] ( figure 1 ). importantly, although ptx3 has long been demonstrated to exert a protective role during microbial infections, it has now emerged as a potential player in contributing to immunopathology under specific circumstances. for instance, in vitro specific binding of ptx3 to fungal components has been observed for a. fumigatus [37] , paracoccidioides brasiliensis, and zymosan [38] . in particular, in vivo studies have revealed that global ptx3-deficient mice are more susceptible to a. fumigatus infection due to defective recognition of conidia by alveolar macrophages and dcs and to the induction of an impaired t helper (th) 1 lymphocyte response [37] . intravenous (i.v.) or intraperitoneal (i.p.) treatment with recombinant ptx3 alone or in combination with antifungal agents such as amphotericin b or voriconazole has resulted in therapeutic outcomes such as the reduction of fungal burden in murine and rat models of pulmonary aspergillosis [6, 37, 39, 40] and by favoring phagocytosis. ptx3 is stored in neutrophil granules and in response to microbial recognition has been found in human neutrophil extracellular traps (nets), suggesting that ptx3 is involved in the antimicrobial activity of nets [6] . in this same study, the recognition and elimination of a. fumigatus conidia by ptx3-deficient neutrophils was poorly efficient, but opsonization of conidia by ptx3 reversed this phenotype [6, 39] . furthermore, neutrophil adoptive transfer protected ptx3-deficient mice from aspergillosis and reduced fungal burden, revealing that neutrophil-associated ptx3 exerted major control of a. fumigatus infection [6] . ptx3 has also been reported to increase recognition and phagocytosis of microbes in an fcgriiand complement-dependent manner, as evidenced from studies with c1q-, c3-, and fcgrdeficient mice that displayed impaired phagocytosis of conidia [39] . in particular, the binding of ptx3-opsonized conidia to fcgrii (which acts as a pentraxin receptor) [20] induced inside-out activation of cd11b, resulting in facilitated phagocytosis of c3b-opsonized conidia by human neutrophils [39] . in addition, other in vitro studies have also shown that the interaction and heterocomplex formation of ptx3 with ficolin-2, and, of ptx3 with mannose-binding lectin (mbl) on fungal surfaces, could increase complement deposition on the same surfaces of a. fumigatus and candida albicans, respectively [41, 42] . zymosan has also been shown to induce the expression of ptx3 in mouse peritoneal macrophages in vitro [38] . ptx3, in turn, by binding to zymosan particles as well as to the yeast form of p. brasiliensis, was found to induce their aggregation and phagocytosis by macrophages (in high numbers) through a dectin-1-dependent mechanism, as demonstrated using blocking anti-dectin-1 antibodies [38] . moreover, snps and haplotypes in the human ptx3 gene have been associated with high susceptibility to invasive aspergillosis following allogeneic hematopoietic stem-cell transplantation [43] . since then, a link between ptx3 genetic variants and susceptibility to mold infections has been confirmed for more than 1000 patients in the swiss organ transplantation cohort [44] as well as in a small cohort of lung transplantation patients [45] . this indicates that the results obtained with ptx3 in mouse models of fungal infections can be translated to human scenarios. ptx3 can also bind various bacteria, including pseudomonas aeruginosa, klebsiella pneumoniae, neisseria meningitidis, and upec [13, 14, 37, 39, 46] . in particular, ptx3 has been reported to behave as an opsonin of p. aeruginosa and upec, facilitating their recognition and ingestion by human and mouse phagocytes [14, 39] (figure 2 ). accordingly, in acute and chronic models of p. aeruginosa lung infection in mice ptx3 has exhibited therapeutic outcomes such as reduced bacterial burden and inflammation [47, 48] . in addition, in utis, uroepithelial cells have been found to rapidly express ptx3 in response to upec infection in mice and humans, with ptx3 augmenting phagocytosis by, and phagosome maturation in, peripheral blood neutrophils [14] . in agreement with these findings, global ptx3-deficient mice displayed a defective capacity to clear bacteria concomitant with an exacerbated inflammatory response, as evidenced by increased production of inflammatory mediators and leukocyte recruitment in the urinary tract [14] . ptx3 binds to outer membrane vesicles (omvs) from n. meningitidis and to three selected meningococcal molecules (gna0667, gna1030, and gna2091) [13] . moreover, global ptx3deficient mice have shown a defective antibody response in vaccination protocols using n. meningitidis omv, while coadministration of ptx3 was found to increase antibody responses relative to controls. importantly, administration of ptx3 reversed the defective humoral responses to vaccination with omv of ptx3-deficient mice but also protected infant rats from infection when they were challenged with n. meningitidis [13] . these results indicate that even if a direct effect of ptx3 in adaptive immune responses has not been described so far, the ptx3dependent facilitated recognition of microbial components by antigen-presenting cells results in improved adaptive immune responses. the relevance in humans of the results obtained in bacterial infection animal models has been confirmed by several genetic studies showing a clear association between specific ptx3 genetic variants and augmented susceptibility to mycobacterium tuberculosis pulmonary infection [49] , acute pyelonephritis and cystitis [14] , or p. aeruginosa lung infection in cystic fibrosis patients [50] . regarding viral infections, ptx3 has been proposed to play a protective role in defense against viruses such as human and murine cytomegalovirus (cmv) and influenza virus type a (iva) [29, 51] (figure 2 ). increased susceptibility to cmv infection and to specific strains of influenza virus infections have been observed in global ptx3 à/à mice [29, 51] . in these cases, ptx3 was found to exert a protective role in the host by binding human and murine cmv, which reduced viral entry into permissive cells and dcs and induced interferon regulatory factor 3 (irf3) activation in in vitro experiments [51] . accordingly, intraperitoneal injection of ptx3 resulted in therapeutic efficacy against primary cmv infection and cmv reactivation in hematopoietic stem-cell transplantation experiments in mice, as indicated by the observed reduced viral load and tissue damage and increased animal survival [51] . ptx3 also recognizes specific strains of h3n2-subtype iav by interacting with viral envelope hemagglutinin and neuraminidase glycoproteins through a sialic acid residue on its glycosidic moiety [29] . in this manner, human and murine ptx3 were shown to act as 'receptor decoys' for the virus, preventing viral spread and infection by specific iav strains [29] . in line with these data, ptx3 à/à animals were more susceptible to h3n2 infection than wild-type mice and administration of ptx3 resulted in a protective effect, as evidenced by increased survival and reduced viral burden [29] . by contrast, ptx3 did not result in antiviral effects against seasonal or pandemic h1n1 and other h3n2 iav strains because specific amino acid residues of individual viral ha sequences led to a lack of interacting ability with the sialylated residue of ptx3 [52,53]. hence, these data suggested selective pressures on ha leading to viral escape from the neutralizing activity of ptx3. another study has reported that ptx3 exerts a protective role in defense against coronavirus murine hepatitis virus strain 1 (mhv-1) in vitro and in vivo [54] . ptx3 was shown to bind to mhv-1 with resulting reduced infectivity of cells in culture. in the same study, global ptx3-deficient mice were more susceptible than their wild-type counterparts to mhv-1 infection [54]. administration of ptx3 led to protection against mhv-1, because reduced lung injury and inflammation and accelerated viral clearance were observed [54]. although collectively these results have demonstrated a protective role of ptx3 during microbial invasion, there are other studies where ptx3 has been found to promote immunopathology in a context-specific manner (figure 2 ). in a model of k. pneumoniae infection in mice, ptx3 overexpression was reported to play antagonistic roles depending on the bacterial concentrations used to infect animals [17] . with high bacterial loads, transgenic mice expressing multiple copies of ptx3 under the control of its own promoter displayed faster lethality, reduced lung infiltration of neutrophils, and increased bacterial dissemination in blood compared with wild-type animals [17] . by contrast, with lower k. pneumonia pulmonary inocula ptx3 overexpression conferred protection on mice by increasing the expression of proinflammatory cytokines and the recruitment of neutrophils into lung tissues and by promoting bacterial phagocytosis [17] . kpompa, a conserved major component of the outer membrane of enterobacteriaceae, is one of the few characterized microbial ptx3 ligands [46] . in the mouse air pouch and footpad swelling model, kpompa has been found to induce ptx3 production and ptx3, in turn, can amplify tlr2-dependent inflammatory responses such as leukocyte recruitment induced following kpompa recognition of the scavenger receptors lectin-like oxidized lowdensity lipoprotein receptor-1 (lox-1) and scavenger receptor expressed by ecs i (srec-i) [46] . these results suggest that k. pneumoniae-induced, amplified ptx3-dependent inflammatory responses either play protective roles for the host or might contribute to immunopathology, depending on microbial load. this underlies the relevance of balanced and finely tuned inflammatory responses and innate resistance to this type of bacterial infection (figure 2 ). although future research might further clarify whether these type of findings could be influenced by particular experimental conditions, or whether the results hold true for different pathogen infections, the fact remains that ptx3 plays a complex role in immunoregulation. furthermore, a pathogenic role of ptx3 has been demonstrated in arthritogenic alphavirus infections induced by chikungunya virus (chikv) and ross river virus (rrv) [55] (figure 2 ). for instance, the expression of ptx3 mrna in peripheral blood leukocytes and ptx3 levels in serum have been found to be increased during the acute phase of alphavirus infection both in patients and in animal models [55] . increased ptx3 expression was associated not only with enhanced viral load but also with disease severity based on temperature, pulse rate, and platelet counts [55] . a murine model of acute rrv infection revealed a delay in disease progression, as shown by increased animal strength and hind-leg paralysis as well as fast recovery in global ptx3 à/à mice compared with wild-type controls. in addition, reduced leukocyte recruitment, expression of inflammatory mediators, and viral replication were reported with ptx3 deficiency. it was proposed that this phenotype could be explained by early rrv and chikv viral entry and replication, which were promoted by the binding of ptx3 to alphavirus, through still undefined mechanisms [55] . interestingly, this suggested that ptx3 might potentially be exploited by viruses to facilitate an increase in viral entrance and replication in host cells [55] (figure 2 ). in addition to its involvement in immunoregulation, ptx3 has been implicated in various other biological processes. for instance, it has been postulated that ptx3 also contributes to tissue remodeling under physiological and inflammatory conditions ( figure 1 ). the involvement of ptx3 in physiological tissue remodeling was first identified in ptx3-deficient infertile female mice [56]. this phenotype was reported to be due to defective assembly of the hyaluronan (ha)-rich matrix that forms around oocytes in preovulatory follicles, a process that is absolutely required for fertilization in vivo [56]. ptx3 produced by cumulus cells during the preovulatory phase was found to interact through its n-terminal domain with two ha-binding proteins, tnf/-induced protein 6 (tnfaip6 or tsg-6) and the serum proteoglycan inter-/-trypsin inhibitor (i/i), which are major functional proteins of the cumulus ecm, thus providing structural integrity to the cumulus matrix [56, 57] . ptx3 is thus required to facilitate murine female fertility. ptx3 has also been found to bind various fibroblast growth factors (fgfs) via its n-terminal extension, including fgf2, fgf6, fgf8b, fgf10, and fgf17 [58] [59] [60] , inhibiting fgf-dependent ec proliferation in vitro and angiogenesis in vivo in an animal model using chick embryo chorioallantoic membranes [61] . in addition, ptx3 has been demonstrated to inhibit fgf2dependent smooth muscle cell (smc) proliferation and suppressed the mitogenic and chemotactic activity exerted by fgf2 on these cells [62] . it has been therefore suggested that ptx3 produced by ecs and inflammatory cells, which are a major source of ptx3, may affect the autocrine and paracrine activity of fgfs on endothelium and smcs, providing, in turn, a mechanism for finely tuning the neovascularization processes and restenosis of carotid arteries in rodent models of arterial balloon injury [63, 64] . additional evidence has indicated that ptx3 is involved in modulating inflammation and tissue damage in animal models of sterile injury [22, 65] . for example, in a murine model of cardiac ischemia/reperfusion injury, global ptx3 deficiency was associated with increased tissue damage and neutrophil infiltration in the myocardium [22] . since higher deposition of complement c3 was observed in the infarct area of ptx3-deficient mice relative to controls [22] , it was hypothesized that this phenotype could be attributed to the potential defective regulation of complement activation via factor h [27] . surface-bound ptx3 was found to enhance the recruitment of factor h, which retained its cofactor activity leading to c3b cleavage [27] . this indicated that ptx3 participates in the localization of functionally active factor h in sites of tissue damage [27] . moreover, ptx3-deficiency in apolipoprotein e knockout mice (apoe mice) was associated with increased atherosclerosis and macrophage accumulation within atherosclerotic plaques as well as with more pronounced inflammatory profiles in vascular walls, as revealed by the increased expression of inflammatory mediators and macrophage recruitment within plaques [65] . in addition to regulating complement, ptx3 has been reported to selectively bind p-selectin via its n-linked glycosidic moiety, thus inhibiting leukocyte rolling on endothelium and providing a negative feedback loop that prevents excessive p-selectin-dependent recruitment of neutrophils and tissue damage in murine models of acute lung injury, pleurisy, and mesenteric inflammation [30] as well as in post-ischemic acute kidney injury [66] . recently ptx3 was demonstrated to play a nonredundant role in tissue repair, through a novel mechanism [5] . in various murine models of tissue damage, including skin-wound healing, chemically induced sterile liver and lung injury and arterial thrombosis, global ptx3 deficiency was associated with increased clot formation as well as with fibrin and collagen deposition/ persistence in sites of tissue damage. under these conditions, macrophages and mesenchymal cells produced ptx3 in response to tlr activation and amplification by il-1, localizing to the pericellular matrix of macrophages and mesenchymal remodeling cells [5] . moreover, ptx3deficient mesenchymal remodeling cells exhibited defective pericellular fibrinolysis in vitro and impaired directional collective migration in the provisional fibrin-rich inflammatory matrix in mice in vivo. it was proposed that this phenotype resulted from the interaction of the ptx3 n-terminal domain with fibrin and plasminogen proteins at an acidic ph and that this occurred in vivo as a consequence of cell metabolic adaptation under conditions of tissue damage-associated hypoperfusion and hypoxia [5] . furthermore, this work argued that the tripartite interaction between ptx3, fibrin, and plasminogen at sites of tissue repair facilitated plasminogen-dependent fibrinolysis [5] . deposited fibrin in damaged tissues acts as a provisional matrix component and its timely remodeling is essential for normal tissue repair [67] . consequently, the phenotype of ptx3 à/à mice described by doni et al. [5] demonstrates that the ptx3-dependent promotion of fibrin-rich inflammatory matrix remodeling contributes to tissue repair. along the same lines, in another murine study ptx3-deficient mesenchymal stromal cells were found to be impaired in their ability to promote skin-wound healing and were also associated with defective pericellular fibrinolysis and cell migration through fibrin [68] . these studies provide further evidence of the interaction between humoral pattern recognition molecules and ecm components and underscore the evolutionary link that exists between microbial and ecm recognition in the humoral arm of innate immunity [3, 69] . regarding the involvement of ptx3 in inflammatory processes, other studies have provided interesting data. for example, in a model of mouse cardiomyocyte death resulting from coxsackievirus b3 (cvb3) viral infection, global ptx3 deficiency increased heart injury, as evidenced by increased serum creatinine kinase activity and cardiomyocyte apoptosis, albeit without affecting viral titers [70] and through a poorly defined mechanism. in this context, it was proposed that the catalytic activity of the immunoproteasome that prevents exacerbation of cvb3-induced myocardial destruction regulated the timely availability of certain factors (erk1/2 and p38) for controlling ptx3 mrna expression and protein production during the infection [70] . this suggested that the cardioprotective function of immunoproteasome-dependent ptx3 expression was a crucial mechanism of stress-induced damage response in myocardial inflammation during cvb3 viral infection [70] . from a different perspective, ptx3 has also been shown to play a protective role in various brain disorders. for example, in a murine model of limbic seizure ptx3 synthesis was induced in the brain, exerting a protective role in seizure-induced neurodegeneration as evidenced by a lower number of degenerating neurons in wild-type mice compared with ptx3-deficient mice [71] . in cerebral ischemia mouse models, ptx3 production was induced in neurons and glia, and although ptx3 deficiency did not affect acute ischemic brain injury it did, however, compromise blood-brain barrier integrity and resolution of brain edema during recovery as well as impairing glial scar formation and neurogenesis [72, 73] . thus, ptx3 has been proposed to support bloodbrain barrier integrity [74] . ptx3 induction in association with tissue damage has also been identified in other pathological contexts [70] (figure 3 , key figure) . for instance, in a mouse model of superior mesenteric artery ischemia and reperfusion, ptx3 deficiency was associated with inhibition of local and remote inflammation and tissue injury, whereas genetic ptx3 overexpression or i.v. ptx3 administration worsened tissue injury and lethality [75, 76] . accordingly, in another murine study, ptx3 overexpression resulted in increased inflammatory responses and a faster decline in a model of ventilator-induced lung injury [77] . ptx3 overexpression also led to increased hypertrophic responses and ventricular dysfunction following increased pressure overload in a murine cardiac injury model [78] . more recently, ptx3 has been found to induce mouse ec dysfunction by inhibiting acetylcholine-evoked vasorelaxation in an in vitro model of the vascular reactivity of resistance vessels and by inducing morphological changes in ecs [79] . these effects were the yin-yang of ptx3 in innate immunity, inflammation, tissue damage, and cancer reported to be mediated by a p-selectin/matrix metalloproteinase-1-dependent pathway leading to impaired phosphorylation of enos and nitric oxide production in vitro [79] . in agreement, i.v. ptx3 administration caused hypertension in wild-type animals but not in p-selectin-deficient mice [79] . these results suggest that ptx3 may have a possible direct role on blood pressure homeostasis and endothelial function. inflammation is an essential component of tumor microenvironments in cancer, sustaining tumor development and growth [80] . however, the role of the humoral arm of the innate immune system in cancer is poorly understood. various reports have associated increased plasma levels of crp with cancer risk [81] [82] [83] . furthermore, elevated local or systemic ptx3 levels have been observed for several cancers, including glioma, liposarcoma, lung cancer, prostate carcinoma, pancreatic carcinoma, and breast cancer bone metastases, that correlate with either grade of malignancy or a poor prognosis [18, [84] [85] [86] [87] [88] . gene expression profiling has identified ptx3 as one of the expressed genes associated with the stromal response/ecm signature and poor prognosis in human ovarian cancer [89] . in addition, ptx3 genetic variants have been associated with increased ptx3 plasma levels and a risk of developing hepatocellular carcinoma in hepatitis c virus-infected subjects [90] . to investigate the role of ptx3 as a marker of cancer-related inflammation and malignant transformation and/or an active player in pathogenesis, our group addressed the susceptibility of ptx3-deficient mice to mesenchymal and epithelial carcinogenesis in models of 3-mca-induced carcinogenesis and 7,12-dimethylbenz [/] anthracene/terephthalic acid (dmba/tpa)-induced skin carcinogenesis [12] . in these models global ptx3 deficiency was associated with more pronounced cancer-related inflammation relative to controls, displayed a higher number of tumor-infiltrating macrophages that showed upregulated expression of genes associated with macrophage m2-like polarization, as well as increased angiogenesis, secretion of proinflammatory cytokines, and complement activation (with increased c3 and lower factor h localization in tumor tissues in addition to higher c5a levels in tumor homogenates) [12] (figure 4) . moreover, ptx3-deficient tumors were also characterized by enhanced genetic damage as indicated by increased trp53 mutations, oxidative dna damage, and expression of dna damage markers, in line with the hypothesis that inflammation contributes to genetic events that can lead to cancer and to the genetic instability observed in tumors [91] . importantly, c3 deficiency in mice or inhibition through a blocking antibody of the chemokine ccl2 led to a reduction in tumor macrophages, an outcome that was sufficient to revert the increased susceptibility to chemically induced carcinogenesis of ptx3-deficient mice. these results indicate that, at least in mice, ptx3 exerts a protective role in carcinogenesis and that this effect is based on the regulation of complement activation [12] (figure 3 ). the potential protumoral role of the complement system has also been suggested by a recent study showing that deficiency in key components of the complement pathway (e.g., c3, c5, c5a receptor) can be linked to colitis-associated colon cancer resistance in mice [92] . however, in specific mouse models, cancer-related inflammation was found to be complement independent [93, 94] , indicating that complement activation may contribute to cancer-related inflammation depending on the tissue context and driving molecular pathways. the functional relevance of ptx3 in human cancer has been further strengthened by results showing that the ptx3 promoter and regulatory regions of the locus are highly methylated in human mesenchymal and epithelial tumors, in contrast to normal healthy tissue, and, furthermore, that such ptx3 methylation can result in silencing of ptx3 protein expression [12] . ptx3 promoter hypermethylation and reduced ptx3 expression have also been reported in human esophageal squamous cell carcinomas [95] . taken together, these studies indicate that ptx3, which is an essential component of humoral innate immunity and a regulator of complement activation, also acts as an extrinsic oncosuppressor gene in mouse and man. moreover, its oncosuppressive role appears to act at the level of complement-mediated, macrophagesustained, tumor-promoting inflammation inhibition [95] . of note, since ptx3 binds certain fgfs (i.e., fgf2, fgf8), impairing the interaction with their cognate receptors [60, 61] , ptx3 plays a protective role in inhibiting tumor growth by regulating fgf-dependent effects as well [60, 96] (figure 4 ). ptx3 overexpression, under the control of the endothelium-specific tie2/tek transcription regulatory sequences, has been associated with reduced malignant growth of fgf2-dependent mouse tramp-c2 prostate cancer and b16 melanoma as well as reduced melanoma metastasis and cancer-associated angiogenesis [96] . furthermore, ptx3 has also been shown to impair cell proliferation and epithelial-mesenchymal transition (emt) in human melanoma cells in vitro [60] . however, contrary to the reports describing a protective role of ptx3 against cancer, other studies have suggested a protumorigenic effect (figure 3 ). ptx3 has been reported to promote in vitro migration and invasion of human pancreatic tumor cells, breast carcinoma, and head and neck squamous cell carcinoma, as well as macrophage chemotaxis, by still poorly defined molecular mechanisms [82, 84, 97, 98] . in another study, ptx3 silencing was found to promote the gastric cancer cell migratory potential, the recruitment of macrophages, and their subsequent binding to gastric cancer cells [98] . in addition, the transcription factor ccaat/enhancerbinding protein delta (cebpd) was shown to activate ptx3 transcription by directly binding to its promoter region in response to cisplatin or 5-fluorouracil treatment in human and murine m2 figure 4 . ptx3 as an extrinsic oncosuppressor in cancer. in cancer, ptx3 gene and protein expression is controlled by the presence of inflammatory mediators (e.g., il-1) and/or by genetic and epigenetic mechanisms. ptx3 regulates complement-dependent tumor-promoting inflammation in a host, including macrophage infiltration and cytokine production, as well as fibroblast growth factor (fgf)-dependent angiogenesis, which can lead to genetic instability and tumor promotion. macrophages and cancer-associated fibroblasts. as a result, ptx3 was found to promote the growth, metastasis, and invasion of drug-resistant human breast cancer cells in immunodeficient mice [19] . these murine studies and the reports showing a positive correlation between ptx3 expression and poor prognosis in specific human cancers suggest that the tumor-promoting or tumorsuppressing role of ptx3 might very likely depend on tissue, cancer type, and cellular source (e.g., cancer cells versus tumor-associated macrophages or fibroblasts) and contradict the assumption that ptx3 plays a unique role in cancer (figures 3 and 4) . however, it should be emphasized that hard genetic data in mice and humans demonstrate that this molecule is a bona fide cancer gene acting as an extrinsic oncosuppressor. further research should help clarify these collective context-dependent findings. pentraxins are a phylogenetically conserved superfamily of humoral prms exerting common essential functions in innate responses to pathogens and in tissue repair. crp is a widely used biomarker of inflammation in humans. however, the lack of strict evolutionary conservation between mouse and human has precluded the use of straightforward genetic approaches to explore its functions in vivo. by contrast, gene-targeting mouse studies have allowed better definition of the functional role of ptx3 in innate immunity, inflammation, and tissue remodeling. in addition, genetic and epigenetic data are consistent with the hypothesis that ptx3 exerts similar functions in humans. from the results described here it has been inferred that, on selective binding of conserved microbial molecules, ptx3 generally exhibits protective antibodylike functions such as complement activation, opsonization of microbes, and glycosylationdependent regulation of inflammation. furthermore, studies in tissue repair models have shown that ptx3-dependent promotion of fibrin-rich inflammatory matrix remodeling contributes to tissue repair and underline the evolutionary link that exists between microbial and ecm recognition in the humoral arm of innate immunity. thus, genetic evidence in mouse and human indicates that ptx3 is essential for resistance against selected pathogens and that it also promotes tissue repair. the described findings strongly support the promising potential of ptx3 in prophylaxis and therapy against infectious diseases in specific clinical settings. of note, the regulation of complement activation also appears to be an essential mechanism of action for the oncosuppressive role of ptx3 that has been recently described, suggesting that novel therapeutic approaches could be envisioned. however, several issues remain (see outstanding questions). for example, it is important to take into account that ptx3 could potentially lead to harmful consequences in an organism under certain circumstances. consequently, when considering the exploitation of ptx3 by viruses (such as alphaviruses) to facilitate viral entry into host cells [55] (figures 2 and 3) , the interaction of ptx3 with collectins and ficolins, which can lead to potent synergic complement activation [32] , as well as its potential to induce endothelial dysfunction [79] and inflammation [19, 76] ( figure 3 ), future clinical trials involving ptx3 administration (or blockade) should carefully assess the possibility of generating unbalanced inflammatory responses and other adverse clinical outcomes. finally, a call is made to strive to achieve a better understanding of the multiple mechanistic pathways in which ptx3 is involved. deciphering more clearly the multifaceted functional roles of ptx3 in physiology may facilitate the development of targeted therapeutic approaches in various clinical conditions. the . what is the impact of ptx3 as a genetic or circulating protein marker on disease management for 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cardioprotective function of the long pentraxin ptx3 in acute myocardial infarction promoter structure and transcriptional activation of the murine tsg-14 gene encoding a tumor necrosis factor/interleukin-1-inducible pentraxin protein characterization of the promoter for the human long pentraxin ptx3. role of nf-kb in tumor necrosis factor-/ and interleukin-1b regulation structural characterization of ptx3 disulfide bond network and its multimeric status in cumulus matrix organization structure and function of the long pentraxin ptx3 glycosidic moiety: fine-tuning of the interaction with c1q and complement activation binding of the long pentraxin ptx3 to factor h: interacting domains and function in the regulation of complement activation m-ficolin interacts with the long pentraxin ptx3: a novel case of cross-talk between soluble pattern-recognition molecules antiviral activity of the long chain pentraxin ptx3 against influenza viruses regulation of leukocyte recruitment by the long 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synergy between ficolin-2 and pentraxin 3 boosts innate immune recognition and complement deposition ficolin-1-ptx3 complex formation promotes clearance of altered self-cells and modulates il-8 production genetic ptx3 deficiency and aspergillosis in stem-cell transplantation polymorphisms and invasive mold infections after solid organ transplant ptx3-based genetic testing for risk of aspergillosis after lung transplant complexity and complementarity of outer membrane protein a recognition by cellular and humoral innate immunity receptors the therapeutic potential of the humoral pattern recognition molecule ptx3 in chronic lung infection caused by pseudomonas aeruginosa prototypic long pentraxin ptx3 is present in breast milk, spreads in tissues, and protects neonate mice from pseudomonas aeruginosa lung infection dc-sign (cd209), pentraxin 3 and vitamin d receptor gene variants associate with pulmonary tuberculosis risk in west africans ptx3 interacts with inter-/-trypsin inhibitor: implications for hyaluronan organization and cumulus oophorus expansion identification of an antiangiogenic fgf2-binding site in the n terminus of the soluble pattern recognition receptor ptx3 role of the soluble pattern recognition receptor ptx3 in vascular biology long pentraxin-3 as an epithelial-stromal fibroblast growth factor-targeting inhibitor in prostate cancer selective recognition of fibroblast growth factor-2 by the long pentraxin ptx3 inhibits angiogenesis pentraxin 3 inhibits fibroblast growth factor 2-dependent activation of smooth muscle cells in vitro and neointima formation in vivo fibroblast growth factor-2 antagonist and antiangiogenic activity of long-pentraxin 3-derived synthetic peptides anti-fgf2 approaches as a strategy to compensate resistance to anti-vegf therapy: long-pentraxin 3 as a novel antiangiogenic fgf2-antagonist deficiency of the long pentraxin ptx3 promotes vascular inflammation and atherosclerosis endogenous and exogenous pentraxin-3 limits postischemic acute and chronic kidney injury loss of fibrinogen rescues mice from the pleiotropic effects of plasminogen deficiency mesenchymal stromal cell-derived ptx3 promotes wound healing via fibrin remodeling the primary role of fibrinogen-related proteins in invertebrates is defense, not coagulation the immunoproteasome controls the availability of the cardioprotective pattern recognition molecule pentraxin3 dynamic induction of the long pentraxin ptx3 in the cns after limbic seizures: evidence for a protective role in seizure-induced neurodegeneration the acute-phase protein ptx3 is an essential mediator of glial scar formation and resolution of brain edema after ischemic injury pentraxin 3 mediates neurogenesis and angiogenesis after cerebral ischaemia astrocyte-derived pentraxin 3 supports blood-brain barrier integrity under acute phase of stroke the long pentraxin ptx3 is crucial for tissue inflammation after intestinal ischemia and reperfusion in mice increased mortality and inflammation in tumor necrosis factor-stimulated gene-14 transgenic mice after ischemia and reperfusion injury pentraxin 3 accelerates lung injury in high tidal volume ventilation in mice long pentraxin ptx3 exacerbates pressure overload-induced left ventricular dysfunction pentraxin 3 induces vascular endothelial dysfunction through a p-selectin/matrix metalloproteinase-1 pathway cancer-related inflammation c-reactive protein and risk of breast cancer: a systematic review and meta-analysis clinical impact of pentraxin family expression on prognosis of pancreatic carcinoma construction and validation of a prognostic index for patients with metastatic pancreatic adenocarcinoma elevated pentraxin 3 in bone metastatic breast cancer is correlated with osteolytic function pentraxin-3 is a novel biomarker of lung carcinoma antitumor and anti-inflammatory effects of trabectedin on human myxoid liposarcoma cells the long pentraxin ptx3 as a correlate of cancer-related inflammation and prognosis of malignancy in gliomas pentraxin 3: a novel biomarker for predicting progression from prostatic inflammation to prostate cancer novel molecular subtypes of serous and endometrioid ovarian cancer linked to clinical outcome genetic variation in ptx3 and plasma levels associated with hepatocellular carcinoma in patients with hcv cancer-related inflammation, the seventh hallmark of cancer: links to genetic instability complement activation promotes colitisassociated carcinogenesis through activating intestinal il-1b/il-17a axis fcrg activation regulates inflammationassociated squamous carcinogenesis early neoplastic progression is complement independent aberrant methylation of the 3q25 tumor suppressor gene ptx3 in human esophageal squamous cell carcinoma long-pentraxin 3 derivative as a smallmolecule fgf trap for cancer therapy ptx3 gene activation in egf-induced head and neck cancer cell metastasis pentraxin-3 silencing suppresses gastric cancer-related inflammation by inhibiting chemotactic migration of macrophages key: cord-001569-jd028cyg authors: dos santos, gimena; rogel, micah r.; baker, margaret a.; troken, james r.; urich, daniela; morales-nebreda, luisa; sennello, joseph a.; kutuzov, mikhail a.; sitikov, albert; davis, jennifer m.; lam, anna p.; cheresh, paul; kamp, david; shumaker, dale k.; budinger, g. r. scott; ridge, karen m. title: vimentin regulates activation of the nlrp3 inflammasome date: 2015-03-12 journal: nat commun doi: 10.1038/ncomms7574 sha: doc_id: 1569 cord_uid: jd028cyg activation of the nlrp3 inflammasome and subsequent maturation of il-1β have been implicated in acute lung injury (ali), resulting in inflammation and fibrosis. we investigated the role of vimentin, a type iii intermediate filament, in this process using three well-characterized murine models of ali known to require nlrp3 inflammasome activation. we demonstrate that central pathophysiologic events in ali (inflammation, il-1β levels, endothelial and alveolar epithelial barrier permeability, remodelling and fibrosis) are attenuated in the lungs of vim(−/−) mice challenged with lps, bleomycin and asbestos. bone marrow chimeric mice lacking vimentin have reduced il-1β levels and attenuated lung injury and fibrosis following bleomycin exposure. furthermore, decreased active caspase-1 and il-1β levels are observed in vitro in vim(−/−) and vimentin-knockdown macrophages. importantly, we show direct protein–protein interaction between nlrp3 and vimentin. this study provides insights into lung inflammation and fibrosis and suggests that vimentin may be a key regulator of the nlrp3 inflammasome. supplementary information: the online version of this article (doi:10.1038/ncomms7574) contains supplementary material, which is available to authorized users. t he cytoskeleton comprises microfilaments, microtubules and intermediate filaments (ifs) 1 . vimentin is the most abundant if protein and has a critical role in stabilizing intracellular architecture 2 . until recently, the importance of vimentin remained elusive because vimentin knockout (vim à / à ) mice displayed a relatively normal phenotype 3 . however, a thorough characterization of vim à / à mice under stress conditions revealed prominent abnormalities [4] [5] [6] , complementing the metabolic, signalling and regulatory abnormalities displayed by patients with if-related diseases and mice expressing functional mutations in ifs 2, [7] [8] [9] [10] . recently, evidence to support a role for ifs as scaffolds for macromolecular complexes involved in critical cell signalling functions was reported 11, 12 . in this study, we demonstrate that vimentin is required for the activation of the nlrp3 (nacht, lrr and pyd domains-containing protein 3) inflammasome, a macromolecular complex that orchestrates early inflammatory responses of the innate immune system. the nlrp3 inflammasome is activated in response to a broad spectrum of infectious agents known to cause acute lung injury (ali), including the bacterial pathogens staphylococcus aureus, pseudomonas aeruginosa and mycobacterim tuberculosis [13] [14] [15] and viral pathogens such as influenza a virus 16 . the wide range of pathogens known to activate the nlrp3 inflammasome suggests that nlrp3 indirectly senses microbes by probing for hostderived danger-associated molecular patterns that are produced or released after cellular or tissue injury 17, 18 . for example, uric acid released from cells exposed to bleomycin triggers the nlrp3 inflammasome, leading to maturation of interleukin 1b (il-1b) and inflammation 19 . in both human patients and animals treated with bleomycin, il-1b released from injured cells results in production of the pro-fibrotic cytokine transforming growth factor b (tgf-b), which contributes to the development of pulmonary fibrosis 20 . in addition, exposure to crystalline particles such as asbestos and silica also cause pulmonary fibrosis 21 that is regulated by the nlrp3 inflammasome 22, 23 . the nlrp3 inflammasome must be tightly regulated to avoid deleterious effects from the wide array of pathogens, pathogenassociated molecular patterns, danger-associated molecular patterns and environmental irritants, which are known to trigger its activation. unlike other inflammasome-associated nod-like receptor (nlr) proteins, nlrp3 is expressed at very low levels in naive macrophages. activation of the nlrp3 inflammasome requires two distinct pro-inflammatory stimuli. in the first step, toll-like receptor (tlr) signalling activates nf-kb and upregulates nlrp3 and pro-il-1b 24 . the second step involves the assembly of the multiprotein complex, which induces selfcleavage of caspase-1, which in turn mediates the cleavage of pro-il-1b into its biologically active form 25 . the mechanism by which the inflammasome is ultimately activated is still a subject of intensive research. we postulate that vimentin is required for nlrp3 inflammasome assembly and activation. the experiments described below use three well-established rodent models of nlrp3 inflammasome activation and result in increased levels of biologically active il-1b that contribute to lung injury. our data suggest that the if protein vimentin may act as an additional checkpoint to control the nlrp3 inflammasome. vimentin deficiency suppresses inflammation and injury. lipopolysaccharide (lps) is a potent inducer of inflammation and a known nlrp3 inflammasome activator [26] [27] [28] . to determine whether vimentin is important in the inflammatory response, wild-type (wt) and vimentin knockout (vim à / à ) mice were subjected to a lethal dose of lps, which induces both proinflammatory cytokine expression and a systemic inflammatory response that is associated with significant mortality. wild-type mice displayed about a threefold increase in mortality compared with vim à / à mice. vim à / à mice had a median survival of 138 h, whereas wt animals had a median survival of b40 h (fig. 1a) . surviving vim à / à mice exhibited mild lethargy, coat ruffling, febrile shaking and eye watering but recovered by day 7. to study whether vimentin affects lps-induced inflammation and ali, we used a sublethal dose of lps, as previously described 29, 30 . histopathological examination showed that lungs of lps-treated wt mice had more severe alveolar damage characterized by interstitial oedema and more fluid and debris in the airspace than in lps-treated vim à / à mice (fig. 1b) . wild-type mice exposed to lps exhibited an increase in wet-todry lung weight ratio and total protein concentration in the bronchoalveolar lavage fluid (balf), whereas vim à / à mice failed to exhibit such an increase (fig. 1c,d) . exposure to lps is followed by large increases in immune cells in the airspace 29, 30 . therefore, to further characterize the nature of immune cell recruitment into the lungs, we performed a systematic flow cytometric analysis of whole-lung lysates 31 . no differences were observed in the myeloid cell population between wt and vim à / à mice treated with saline ( supplementary fig. 1a ). as expected, both wt and vim à / à mice challenged with lps had significant increases in neutrophils and moderate increases in macrophages compared with saline-treated animals ( supplementary fig. 1a ); no differences were observed between wt and vim à / à mice challenged with lps ( supplementary fig. 1a) . these data suggest that the protection from the lpsinduced increase in alveolar capillary barrier permeability observed in vim à / à mice was not associated with impaired recruitment of inflammatory cells to the lung. lps induces rapid production and release of pro-inflammatory cytokines and chemokines, which are sufficient to induce lung inflammation and ali in mice and humans 32 . when we exposed wt and vim à / à mice to lps for 24 h, significantly more caspase-1 and mature il-1b were found in balf from wt mice than in vim à / à mice (fig. 1e,f) . furthermore, whole-lung homogenates prepared from wt and vim à / à mice exposed to lps had increased levels of il-1b messenger rna (mrna, supplementary fig. 1b) . these data suggest that vimentin is not involved in the tlr-mediated nf-kb activation and upregulation of pro-il-1b and nlrp3 (refs 26-28) . rather, vimentin must be required for the second step in lps-induced caspase-1 activation and il-1b maturation via the nlrp3 inflammasome. vimentin required for asbestos-induced injury and fibrosis. the nlrp3 inflammasome has been implicated in the pathological increase of il-1b production in a variety of pulmonary inflammatory diseases that lead to fibrosis, including asbestosis [21] [22] [23] . in a model of asbestosis, we sought to determine whether vim à / à mice showed diminished cytokine production and whether these animals were protected from lung injury and pulmonary fibrosis. wild-type and vim à / à mice were exposed to crocidolite asbestos or an inert particle, titanium dioxide (tio 2 ); markers of injury, inflammation, cytokine production and fibrosis were assessed. confirming previous reports 22, 23 , asbestosexposed mice had increased total cell numbers in balf compared with tio 2 -exposed animals. however, significantly fewer cells were recruited to the lungs of vim à / à mice after asbestos exposure (fig. 2a) . additional analysis of immune cell recruitment into the lungs of wt and vim à / à mice was performed by fluorescence-activated cell sorting analysis of whole-lung lysates ( supplementary fig. 2a) . wild-type mice had profound inflammatory cell infiltrates following exposure to asbestos as observed by haematoxylin and eosin (h&e) staining; in contrast vim à / à mice showed reduced inflammation ( supplementary fig. 2b ). wild-type mice exhibited an increase in total protein concentration in balf, whereas vim à / à mice failed to exhibit such an increase (fig. 2b) . the reduction in inflammation in vim à / à mice exposed to asbestos was associated with reduced levels of caspase-1 and il-1b in balf compared with wt mice (fig. 2c,d) . these data suggest that vimentin is required for asbestos-mediated activation of the nlrp3 inflammasome (step 2), but not for nf-kb activation and upregulation of pro-il-1b and nlrp3 (step 1). furthermore, we observed that wt mice exposed to asbestos develop pulmonary fibrosis, as assessed by masson trichrome and picrosirius red stain (a dye that binds specifically to collagen fibrils, fig. 2e ) and sircol assay (fig. 2f) . vimentin deficiency prevented pulmonary fibrosis, as no significant increase in collagen deposition was observed in vim à / à mice (fig. 2e,f) . collectively, these results demonstrate that loss of vimentin prevents asbestos-induced collagen deposition and fibrosis. vimentin required for bleomycin-induced injury and fibrosis. bleomycin is one of the best characterized rodent models of pulmonary fibrosis 33 ; intratracheal delivery of the drug causes severe ali, which is followed by clearance of alveolar inflammatory cells, fibroblast proliferation and increased collagen deposition 34 . moreover, bleomycin-induced fibrosis has been shown to be dependent on the nlrp3 inflammasome 35 . wt and vim à / à mice were exposed to bleomycin for 5 days. wt mice displayed intense lung inflammation as assessed by h&e staining (fig. 3a) , increased wet-to-dry lung weight ratio (fig. 3b) , and increased protein concentration in balf (fig. 3c) . all of these end points were significantly attenuated in vim à / à mice (fig. 3a-c) . exposure to bleomycin resulted in a large increase in immune cells in the airspace of both wt and vim à / à mice as assessed by flow cytometric analysis of whole-lung lysates ( supplementary fig. 3a ). vim à / à mice failed to exhibit an increase in caspase-1 (fig. 3d ) and il-1b (fig. 3e) in balf following the exposure to bleomycin; in contrast, wt mice showed a robust activation of caspase-1 and production of mature il-1b (fig. 3d,e) . as a proinflammatory cytokine, macrophage-produced il-1b is known to induce production of interleukin 6 (il-6). we observed that wt mice, but not vim à / à mice, had increased levels of il-6 following the exposure to bleomycin (fig. 3f ). tgf-b1 is a critical mediator of remodelling and fibrotic responses in the lung. total tgf-b1 was detected after activation in balf from wt mice 5 days following the exposure to bleomycin, but remained at the baseline level in balf from vim à / à mice (fig. 3g ). no significant difference was observed between wt and vim à / à mice in nlrp3 inflammasome-independent cytokines tnf-a and mcp-1 (fig. 3h,i) . interestingly, il-1b mrna levels were increased in the lungs of both wt and vim à / à mice following the exposure to bleomycin ( supplementary fig. 3b ). bleomycin exposure results in chronic inflammation and progressive pulmonary fibrosis 19, 35 . the role of vimentin in pulmonary fibrosis was examined 21 days following the administration of bleomycin. the lung architecture was similar for saline-treated wt and vim à / à mice, but in wt mice bleomycin induced extensive fibrotic areas with abundant collagen production, as assessed by masson trichrome stain ( fig. 4a and supplementary fig. 4a ). cellular infiltrates, alveolar wall destruction and collagen deposition were significantly reduced in vim à / à mice. similarly, we observed increases in collagen deposition in bleomycin-treated wt mice by staining with picrosirius red (fig. 4b) . in contrast, there was virtually no collagen deposition in the lungs of vim à / à mice (fig. 4b) . lung collagen concentrations, as assessed by sircol assay, were (a) survival of wt and vim à / à mice subjected to a lethal dose of lps (80 mg kg à 1 , intraperitoneally) was measured and compared. the survival curves were analysed using the log rank test, which calculates the chi-square (w 2 ) for each event time for each group and sums the results. the summed results for each group were added to derive the ultimate chi-square to compare the full curves of each group. the log rank test for the entire data set was p ¼ 0.001. (b-f) wt and vim à / à mice were challenged with a sublethal dose of lps and markers of ali were measured after 48 h, as assessed by h&e staining (scale bar, 200 mm) (b); by wet-to-dry lung weight ratio (c); and by protein content in the balf (d). inflammasome activation was measured by elisa for caspase-1 (e) or il-1b (f) in balf from vim à / à and wt mice. data in b-e are from three independent experiments of n ¼ 6-8 animals per group and presented as mean±s.d. **po0.001, ***po0.0001 relative to wt versus vim à / à , by one-way analysis of variance with a correction provided by the bonferroni multiple comparisons test. nature communications | doi: 10.1038/ncomms7574 article 0.57 ± 0.09 and 0.31 ± 0.03 mg per lung in bleomycin-treated wt and vim à / à mice, respectively (fig. 4d ). in patients with pulmonary fibrosis, pulmonary function tests typically reveal a parenchymal-restrictive ventilatory defect due to decreased lung compliance 36 . lung compliance is influenced by the underlying connective tissue, which is rich in extracellular matrix components such as collagen 37 . because vim à / à mice exhibited less collagen deposition following treatment with either bleomycin or asbestos, we reasoned that lungs of these mice would maintain normal compliance. to test this hypothesis, we measured the lung mechanics of wt and vim à / à mice using a scireq ventilator 21 days following the bleomycin administration. in comparison with saline treatments, lungs of wt mice exhibited a 2.5-fold decrease in quasi-static compliance following the bleomycin administration, whereas the compliance of lungs from vim à / à mice decreased only 1.3-fold (fig. 4e) . bleomycin administration creates heterogeneous regions of lung fibrosis in mice (fig. 4a) , which is a pathologic feature of the human disease as well 38 . to assess focal changes in lung tissue stiffness, we performed regional nano-indentations on lung sections (200-400 mm thick) using atomic force microscopy (afm). the micro-indentations provide a measure of the stiffness or elastic modulus (e) of lung tissue, which is inversely related to lung compliance. elasticity maps from afm micro-indentation from bleomycin-treated wt lungs clearly displayed the wide ranges of tissue stiffness (fig. 4c) , which corresponded to the heterogeneity of collagen deposition (fig. 4a) . the median elastic modulus of lung parenchymal tissue from wt mice increased 2.4-fold in response to bleomycin compared with control lung tissue (27 to 7.5 kpa, respectively), whereas the elastic modulus of lung tissue from bleomycin-treated vim à / à mice was significantly decreased (po0.0001) compared with wt bleomycin-treated lung tissue (fig. 4f) . the wide range of e-values is reflected in the frequency of occurrence and in the median and interquartile ranges for each condition ( supplementary fig. 4b ). in addition, there was a strong correlation (pearson r ¼ à 0.99) between the mean compliance values for each condition and the mean e-values for each condition (for example, wt-bleomycin-afm versus wtbleomycin-scireq). thus, the mechanical properties of the lung were preserved in vim à / à mice following the bleomycin treatment, which is evident at both the global tissue scale and in focal regions of lung. injury and fibrosis reduced in vim à / à bone marrow chimeras. because multiple cell types contribute to the inflammatory and fibrotic responses, we next addressed the respective roles of bone marrow-derived cells in bleomycin-induced lung injury and fibrosis by generating bone marrow chimeras (fig. 5a) . reconstitution of bone marrow from wt donor mice into irradiated recipient mice (wt) resulted in protein accumulation in balf in response to bleomycin, whereas in recipient mice reconstituted with bone marrow from vim à / à donor mice (vim à / à ), the balf protein was unchanged from saline controls (fig. 5b) . secretion of il-1b (fig. 5c ), il-6 ( fig. 5d ) and tgf-b (fig. 5e ) in the balf of wt mice significantly increased in response to bleomycin; the response in vim à / à mice was significantly attenuated. finally, wt mice displayed a significant degree of collagen deposition and fibrosis, as shown by masson trichrome wt and vim à / à mice were treated with pbs containing either 200 mg of asbestos crocidolite or the control particle titanium dioxide (tio 2 ), intratracheally. markers of inflammation and fibrosis were assessed 3 and 6 weeks after instillation, respectively. (a) total cell count (macrophages, neutrophils, eosinophils, erythrocytes and lymphocytes ) and (b) protein levels were assessed in balf collected from vim à / à mice 3 weeks after asbestos administration. inflammasome activation in wt and vim à / à mice was measured by elisa for caspase-1 (c) and il-1b (d) levels in balf at the same time point. collagen deposition was evaluated in asbestos-treated vim à / à and wt mice by masson's trichrome and picrosirius red staining of lung slices (e; scale bar, 200 mm) and by measuring total collagen content by sircol assay (f). data are from two independent experiments n ¼ 6-8 animals per group, and presented as mean ± s.d. *po0.05, **po0.001, relative to wt versus vim à / à , by one-way analysis of variance with a correction provided by the bonferroni multiple comparisons test. sircol assay ( fig. 5f ), whereas this response was severely attenuated in vim à / à mice ( fig. 5f ,g and supplementary fig. 5c ). all together, these results suggest that vimentin-expressing bone marrow-derived cells are important for bleomycin-induced activation of the nlrp3 inflammasome and pulmonary fibrosis. loss of vimentin prevents nlrp3 activation. as stated above, nlrp3 inflammasome activation requires two distinct stimuli. an inflammatory stimulus, such as lps, primes cells to transcribe and synthesize pro-il-1b (signal 1) and then another stimulus such as extracellular adenosine triphosphate (atp), monosodium urate (msu) or asbestos (signal 2) is necessary for inflammasome complex formation, caspase-1 activation and il-1b maturation 19, 22, 39 . to further investigate the possibility that nlrp3 inflammasome activation requires vimentin, we isolated primary alveolar macrophages from wt and vim à / à mice. macrophages were primed with lps and stimulated with atp as previously described 39 . levels of mature caspase-1 and il-1b were significantly increased in the supernatants collected from wt macrophages following lsp and atp treatment compared with saline treatment, but in the supernatant collected from vim à / à macrophages there was only a slight increase (fig. 6a,b) . there was no difference in pro-il-1b levels in total cell lysates between activated wt and vim à / à alveolar macrophages ( fig. 6c ; full blots are presented in supplementary fig. 7) . as an independent approach, we expressed a small hairpin rna (shrna) targeted to vimentin (vim kd ) or a scrambled shrna (ctrl) in j774a.1 macrophages and treated the cells with atp. caspase-1 is required to cleave pro-il-1b into its biologically active form and like other caspases is proteolytically activated from a pro-enzyme to produce a tetramer of its two active subunits, p20 and p10 (ref. 28 ). the levels of mature caspase-1 p20, but not pro-caspase-1, were considerably reduced in vim kd cells (fig. 6d ,e; full blots are presented in supplementary fig. 7 ) compared with ctrl cells treated with atp, further supporting the hypothesis that vimentin deficiency prevents nlrp3 inflammasome activation. uric acid released from cells exposed to bleomycin triggers nlrp3 inflammasome assembly leading to il-1b maturation and inflammation in the lungs 19, 40 . in an in vitro model, we examined whether silencing vimentin would impair nlrp3 inflammasome activation in response to msu. a human macrophage-like cell line was treated with silencing rna (sirna) targeted to vimentin (vim kd ) or a scrambled sirna (ct; fig. 7a ; full blots are presented in supplementary fig. 8 ) and subsequently activated with msu. no differences were observed in the levels of pro-il-1b between ct and vim kd cells (fig. 7a) , suggesting that vimentin deficiency does not impair signal 1. nlrp3 inflammasome activation was repressed in vim kd cells as assessed by il-1b and caspase-1 levels in cell supernatants (fig. 7b,c) . similar results were obtained when ct and vim kd cells were activated with asbestos (fig. 7b,c) . in addition, bone marrow-derived macrophages (bmdms) were isolated from wt and vim à / à mice, primed with lps and activated with msu. caspase-1 activation was assessed using a specific fluorescent probe, fam-yvad-fmk 41 . wt bmdms showed robust caspase-1 activation following treatment with msu, with caspase-1 forming aggregates throughout the cytoplasm. however, caspase-1 activation was severely reduced in bmdms isolated from vim à / à mice (fig. 7d) . no caspase-1 activation was observed in untreated bmdms isolated from either wt or vim à / à mice. vimentin interacts with inflammasome components. inflammasome activation involves the physical interaction of nlrp3, asc (apoptosis-associated speck-like protein containing a card) and caspase-1 (ref. 28 ), but the mechanism by which the inflammasome assembles is not clear. if the vimentin network serves as a protein scaffold upon which the nlrp3 inflammasome components are assembled, then vimentin should interact with inflammasome proteins. immunoprecipitation of caspase-1 and nlrp3 from human macrophage lysates resulted in a robust co-precipitation of vimentin, which was enhanced following activation of cells with atp ( fig. 8a ; full blots are presented in supplementary fig. 9 ). these interactions were specific for vimentin, as no actin was detectable in immunoprecipitates. we also used solution-binding assays to demonstrate a direct protein-protein interaction between vimentin and nlrp3. bacterially expressed and purified s-tag his-vimentin was incubated with extracts prepared from either control or msutreated human macrophage cells. the s-tag vimentin was precipitated using s-protein agarose. the precipitated proteins (c) representative elastographs from afm micro-indentation of lung tissue from wt and vim à / à animals. the colour bar indicates elastic modulus, e. the darkest red corresponds to elastic modulus values of 50 kpa and above. the data represent 256 indentations per region, with at least two regions per tissue section from at least three mice per group. (d) collagen content in lungs from wt and vim à / à mice assessed by sircol assay. (e) mice were ventilated 21 days after intratracheal instillation of bleomycin or pbs. shown are quasistatic compliance measurements of wt and vim à / à lungs. (f) elastic modulus frequency plot obtained from live, unfixed lung tissue of saline and bleomycin-treated wt and vim à / à animals. microindentation data were fit using the hertz model to acquire the elastic modulus of regions of lung tissue. images in a-b represent data obtained from at least three animals per group; data shown in d-e are mean ± s.d. of at least five animals. **po0.001, ***po0.0001 relative to wt versus vim à / à , by one-way analysis of variance with a correction provided by the bonferroni multiple comparisons test. were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page), transferred to nitrocellulose and then immunoprobed with antibodies directed against nlrp3. we observed a 3.0 ± 0.8-fold increase in binding between vimentin and nlrp3 in extracts prepared from msu-treated cells, as compared with untreated control cells (supplementary fig. 6 ). article a complementary method, bio-layer interferometry (bli), was also used to investigate the interaction between nlrp3 and vimentin. bli is an optical technique that analyses the interference pattern of white light reflected from two surfaces: a layer of immobilized protein on the biosensor tip and an internal reference layer 42 . any change in the number of molecules bound to the biosensor tip causes a shift in the interference pattern that can be measured in real time, providing detailed information on the kinetics of association and dissociation between the two molecules (k a , k d and k d ). commercially available nlrp3 was bound to the biosensor tip, then various concentrations (55 mm, 18 mm, 6 mm, 1.8 mm, 180 nm, 18 nm and 1.8 nm) of full-length vimentin were added to determine the association kinetics with nlrp3. in agreement with other methods (fig. 8a ,b,d and supplementary fig. 6 ), the assay demonstrated that nlrp3 binds to vimentin with a measureable affinity; a global dissociation constant (k d ) of 640 nm was determined using blitz pro. consistent with the co-immunoprecipitation and solutionbinding results, we observed co-localization of nlrp3 and vimentin in stimulated alveolar macrophages by confocal immunofluorescence microscopy (fig. 8d) . following activation, the wt cells, but not the vim kd cells, were spread and displayed lamellipodia. furthermore, the vimentin network spanned the cytoplasm in the wt activated cells, whereas in unstimulated cells, the network appeared to be collapsed around the nucleus and lamellipodia were not observed (fig. 8d) . vimentin filaments (red) strongly co-localized with nlrp3 (green) in both perinuclear and distal regions of the cells. co-localization studies were performed using fiji 1.48r (64-bit, win) and the coloc 2 test 43 . co-localization analyses were calculated on rois drawn around the lamellipodia, determined by the vimentin image. the thresholded manders co-localization scores were 0.89 ± 0.15 for vimentin and 0.86 ± 0.18 for nlrp3. nlrp3 and asc also colocalized in stimulated macrophages ( supplementary fig. 5 ); the score for nlrp3 co-localization with asc in untreated cells was 0.20 ± 0.11, indicating weak co-localization. in contrast, the score for nlrp3 co-localization with asc in cells treated with atp was 0.85 ± 0.21, indicating strong co-localization. however, knockdown of vimentin resulted in a lack of co-localization between nlrp3 and asc, even in activated macrophages ( supplementary fig. 5 ). taken together, these results demonstrate that vimentin associates with components of the inflammasome and is required for inflammasome activation. inflammation, the process aimed at restoring homeostasis after an insult, can be more damaging than the insult itself if uncontrolled, excessive or prolonged. growing evidence indicates that il-1b is a critical mediator of acute inflammation, remodelling and fibrosis in the lung. using three well-characterized murine models of ali and pulmonary fibrosis, which were previously shown to be dependent on the nlrp3 inflammasome 19, 22, 23, 35 , we show that nlrp3 inflammasome activation, which leads to il-1b maturation, is dependent on the type iii if protein vimentin. our data obtained in vimentin knockout mice and vimentin-null cells suggest that vimentin is required for nlrp3 inflammasome activation. dysregulated inflammation, excessive leukocyte accumulation and increased permeability of endothelial and alveolar epithelial barriers are the central pathophysiologic events in ali and its most severe form, the acute respiratory distress syndrome (ards) 44 . all these end points were observed in wt mice challenged with lps, bleomycin and asbestos. in contrast, vim à / à mice exposed to the same treatments experienced mild increases in protein levels in balf and limited alterations in lung histopathology, suggesting that they were protected from lps-, bleomycin-and asbestos-induced ali. inflammasome-regulated cytokines have a key role in the initiation and propagation of the aforementioned features of ali 45 . in fact, alveolar macrophages from patients with ards produce excessive amounts of il-1b 46 , and il-18 was elevated in the plasma of patients with ards, which correlated with increased morbidity and mortality 45 . since vimentin-deficient mice were protected from ali, we speculated that failure to produce inflammasome-mediated cytokines (for example, il-1b) caused this protection. in fact, vimentin-deficient mice failed to increase mature il-1b levels following exposure to lps, asbestos or bleomycin. moreover, we demonstrated that caspase-1 activation and mature il-1b production in vitro requires vimentin, as no increase was observed in activated macrophages either isolated from vim à / à mice or in which vimentin was silenced using rna interference. resolution of ali/ards depends on a precise balance of inflammatory and molecular signalling 44 ; an imbalance in that process caused by il-1b overproduction might prevent cellular restoration and lead to pulmonary fibrosis. in fact, il-1b produced by the inflammasome has been recently identified as an initiator of fibrosis in response to bleomycin 35 , and administration of il-1b alone recapitulates much of the lung pathology caused by bleomycin 47 . because vimentin-deficient mice did not produce the same amount of il-1b as wt mice, we reasoned that vim à / à mice would be protected from bleomycin-and asbestos-induced pulmonary fibrosis. indeed, lung pathology was significantly reduced in vimentin-deficient mice, pointing to the essential role of vimentin in pulmonary fibrosis. we found substantially less collagen deposition in vim à / à mice compared with wt mice. the excessive collagen deposition in lungs of wt mice led to loss of mechanical compliance, whereas the lungs of vim à / à mice maintained compliance similar to that of saline-treated mice. these results are consistent with previous reports demonstrating decreases in the compliance of fibrotic lungs 33 . the elastic modulus measurements strongly suggest that increased tissue stiffness at the microscale corresponds to areas of increased collagen deposition. importantly, macroscale measurements of lung compliance were highly correlated with the microscale elastic modulus measurements obtained by afm. a number of studies have demonstrated that il-1b can induce ali and contribute to the progression of pulmonary fibrosis 19, 23, 35, 48 . il-1b-induced fibrosis is associated with an increase in tgf-1b, demonstrating that ali initiated by proinflammatory cytokines can result in progressive fibrosis 35, 48 . vim à / à mice were treated with recombinant mouse il-1b (rmil-1b 0.05 mg kg à 1 body weight, intranasal (i.n.)), but we were unable to detect latent tgf-b1 in balf from vim à / à mice after i.n. rmil-1b at days 1, 7 and 14 (data not shown), whereas latent tgf-b1 was detected 5 days after bleomycin administration in wt mice (fig. 3) . gasse et al. 19 reported that collagen deposition was increased by rmil-1b in wild-type mice; vim à / à mice treated with rmil-1b had no increase in collagen deposition. we reason that the incomplete suppression of mil-1b in vim à / à mice is a reflection of incomplete chimera, with only b90% engraftment. studies with il-1r1 à / à , myd88 à / à , asc à / à , caspase-1 à / à and nlrp3 à / à mice have suggested that uric acid (induced by bleomycin), asbestos and silica are detected by the nlrp3 inflammasome in macrophages, likely leading to il-1r1/myd88 signalling in pulmonary epithelial cells, then to inflammation, neutrophil and lymphocyte recruitment and fibroblast activation 35 . lung fibroblast activation results in collagen deposition and pulmonary fibrosis. importantly, it was reported that vimentin is required for the stabilization of collagen mrnas 49 . we reason that the vim à / à mice treated with either rmil-1b or wt bone marrow failed to increase collagen deposition because vimentin knockout fibroblasts fail to produce type i collagen due to decreased stability of collagen a1(i) and a2(i) mrnas 49 . protection from injury, inflammation and fibrosis has been observed in mice lacking any components of the nlrp3 inflammasome, similar to our observations of vim à / à mice following injury 19, 22, 23, 35, 50 . these phenotypes led us to investigate whether vimentin might serve as a scaffold for the inflammasome by binding to one or more proteins in the inflammasome complex. several studies have suggested that ifs, including vimentin, act as scaffolds for signalling molecules 2,11,12 . in fact, vimentin interacts with and is involved in the translocation of perk1/2 to the nucleus 51 , limits the ability of 14-3-3 to interact with raf and cdk1, and regulates the release and translocation of roka following rhoa activation. these findings exemplify how vimentin ifs can act as scaffolds for signalling molecules. recently, vimentin was reported to bind to nod2 (nucleotide-binding oligomerization domain-containing protein 2), a member of the nlr family, via the leucin-richrepeat (lrr) 52 . the lrr is a protein-binding domain shared by 19 other nlr family members, including nlrp3. therefore, we sought to determine whether vimentin interacts with nlrp3. in our experiments, immunoprecipitation of both nlrp3 and caspase-1 in lysates from wt alveolar macrophages resulted in a robust co-precipitation of vimentin, which was enhanced following activation of the nlrp3 inflammasome. these data were confirmed by immunofluorescent confocal microscopy of human and murine macrophages. the co-localization score for vimentin with nlrp3 was 0.89±0.15, indicating strong colocalization 43 . moreover, we were able to demonstrate a direct protein-protein interaction between vimentin and nlrp3 by bli assays, further establishing a role for vimentin in assembly of the nlrp3 inflammasome. intermediate filaments, including vimentin, have been shown to provide a scaffold whereby active subunits of caspase-9 can activate caspase-3, which, in turn, can activate more caspase-9 resulting in an amplification loop 53, 54 . caspase-3, caspase-6 and caspase-7 have also been shown to interact with vimentin 54 ; to our knowledge, we are the first to report an interaction between caspase-1 and vimentin. we can only speculate about where and how vimentin associates with nlrp3 and caspase-1 during inflammasome assembly, since the crystal structures of the vimentin dimer and all components of the inflammasome except for pyd are unknown [13] [14] [15] . nlrp3 proteins have been shown to form inactive preassembled complexes in unstimulated cells [13] [14] [15] . under stimulation, these proteins undergo conformational changes whereby the inflammasome is activated. the vimentin if network could potentially associate with the nlrp3 inflammasome through one or more domains of nlrp3 and mediate the conformation change or stabilization of nlrp3 that leads to activation. we reason that vimentin may interact with nlrp3 in two distinct regions: the nacht domain and the lrr; vimentin has been previously shown to interact with nod2 in the lrr domain 52 . because asc is localized to the nucleus in unactivated cells, no interaction with vimentin is anticipated in nonactivated cells. furthermore, on the basis of the structure of asc, a pyd and card domain with a short linker, we do not anticipate a direct interaction with vimentin as vimentin lacks either of the asc domains, therefore, any asc signal following cell activation is anticipated to be due to vimentin interaction with other proteins in the inflammasome protein complex. an unresolved issue is how vimentin mediates inflammasome activation. mitochondrial-derived reactive oxygen species (ros) have been linked to inflammasome activation 55 . vimentin if are known to modulate mitochondrial motility 56 , and lack of vimentin results in increased ros production 57 . this suggests that vimentin-deficient cells should have more robust inflammasome signalling, the exact opposite of what we have demonstrated. in a very provocative paper, however, bauernfeind et al. 24 demonstrated that mitochondrial-derived ros is required for priming, not activation, of the inflammasome, as ros inhibitors blocked the priming step required for nlrp3 expression, but activation was not affected. this correlates with our findings that signal 1 is unaffected by the lack of vimentin. further studies on the relationship between vimentin, mitochondria and ros will likely provide insights into the regulation behind both signals 1 and 2 during inflammation, but currently they are beyond the scope of this paper. in summary, this study demonstrates a novel function for the type iii if, vimentin, in innate immunity and inflammation leading to ali and pulmonary fibrosis. in our experiments, vimentin deficiency resulted in a blunted inflammatory response in the early stages of injury, which was consistent among the different animal models of lung injury used: lps, bleomycin and asbestos. we further demonstrate that vimentin is required for il-1b maturation through its interaction with the nlrp3 inflammasome. vimentin may act as a scaffold for assembly of the inflammasome protein complex and thus regulate its function. overall, this study provides new insights into lung inflammation and fibrosis, and importantly, suggests that vimentin may be a key regulator of the nlrp3 inflammasome. mice and induction of acute lung injury. age-and sex-matched 8-to 10-weekold mice were used in all the experiments. the 129/sv6 vimentin-deficient (vim à / à ) mice were a gift from albee messing (madison, wi). bleomycin (0.07 units in 50 ml of pbs; hospira) and asbestos (200 mg in pbs; us epa) were administered intratracheally via two 25-ml aliquots. lps (80 mg kg à 1 , lethal dose; 24 mg kg à 1 , sublethal dose; sigma-aldrich) was administered via intraperitoneal injection. wt and vim à / à mice were treated with a single intratracheal injection of 50 ml of phosphate-buffered saline (pbs) or bleomycin (0.07 units in 50 ml of pbs; hospira). all mice were bred in our animal facility and the experiments were approved by the northwestern university animal care and use committee. histology. a 20-gauge angiocath was sutured into the trachea, heart and lungs were removed en bloc and inflated with 0.8 ml of 4% paraformaldehyde. the heart and lungs were fixed and embedded in paraffin; 5-mm sections were stained with h&e, picrosirius red or masson trichrome 58 by the mouse histology phenotyping laboratory (northwestern university, chicago, il). sections were visualized using the tissuegnostics tissue/cell high throughput imaging analysis system (vienna, austria) and captured using tissuefaxs software (tissuegnostics, los angeles, ca) at the northwestern university cell imaging facility. specific regions of interest were visualized using a zeiss axioskop microscope and captured using the cri nuance spectral camera software suite. wet-to-dry weight ratios. mice were anaesthetised and lungs were surgically removed en bloc. the left lung was ligated, excised and weighed in a tared container. the left lung was then dried at 70°c in a speed-vac sc100 evaporator (thermo scientific, waltham, ma) until a constant weight was obtained, and the wet-to-dry weight ratio was calculated. bronchoalveolar lavage. to perform bronchoalveolar lavage (bal), a 20-gauge angiocath ligated into the trachea and 1 ml of sterile pbs was instilled, then removed through the angiocath; the process was repeated three times. the samples were pooled and a 200 ml aliquot of the bal fluid was placed in a cytospin and centrifuged at 500 g for 10 min. the pellet was re-suspended in 1 ml of pbs, plated on glass slides, stained with crystal violet and subjected to a blinded manual cell count and differential. cytokine and chemokine levels in the supernatant were measured using the bd cytometric bead array (bd biosciences). samples were analysed in triplicate using the mouse inflammation kit (bd biosciences) according to the instructions provided. this kit detects interleukin (il)-6, mcp-1 and tumour necrosis factor (tnf)-a. total transforming growth factor (tgf)-b1 was measured using the tgf-b1 rii duoset enzyme-linked immunosorbent assay (elisa, r&d systems, minneapolis, mn) according to the manufacturer's instructions. total interleukin (il)-1b was measured using the il-b1 ready-set-go elisa (ebioscience, san diego, ca) according to the manufacturer's instructions. caspase-1 activity was measured using the caspase-1 fluorometric assay (r&d systems), according to the manufacturer's instructions. sircol assay. total collagen content was determined by harvesting lungs from mice 21 d after treatment with bleomycin or pbs, or 12 weeks after treatment with asbestos or tio 2 . exsanguination was accomplished by a left nephrectomy, followed by perfusion of the right ventricle with 2 ml of cold pbs. both lungs were removed and homogenized (1 min with a tissue homogenizer followed by 12 strokes in a dounce homogenizer) in 1 ml of 0.5 n acetic acid supplemented with roche edta protease inhibitor. aliquots of lung homogenate were then assayed for total lung collagen levels and compared with a standard curve prepared from rat tail collagen by using the sircol collagen dye binding assay (biocolor ltd, newtownabbey, uk), according to the manufacturer's directions. compliance measurements. wt and vim à / à mice were anaesthetised with ketamine and xylazine (45 mg per 100 g of body weight) and a tracheotomy was performed. pancuronium was administered intraperitoneally (0.08 mg kg à 1 of body weight). the mouse was mechanically ventilated on a computer-controlled piston ventilator (flexivent; scireq, montréal, québec, canada) with a tidal volume of 10 ml kg à 1 at a frequency of 150 breaths per minute against 2-3 cm h 2 o positive end-expiratory pressure. following two total lung capacity manoeuvres to standardize volume history, pressure and flow data (reflective of airway and tissue dynamics) were collected during a series of standardized volume perturbation manoeuvres. these data were used to calculate both total lung resistance (r) and elastance (e) using the single-compartment model and used to derive further parameters of respiratory function. the flexivent calibration procedure removes cannula impedance from the reported data. residual impedence (i) is therefore negligible and is not reported herein. atomic force microscopy measurements. lungs were used at 17-21 days following bleomycin challenge. following instillation of 2.5% low-gel point agar (50 ml kg à 1 ), lungs were sectioned using a vibratome (leica vt1000 s) into 200mm or 400-mm thick slices. these slices were used within 24 h of harvest; microindentations were performed using a bruker bioscope catalyst located at the northwestern university nuance facility. modified probes with 5-mm spheres (novascan, ames, ia) were used to indent 80 mm â 80 mm areas on the lung slices. force and indentation depth were converted to elastic modulus using the hertz spherical indentation model 21, 59 and a custom matlab program (generated by dr esra roan, university of memphis). a poisson ratio of m ¼ 0.4 was assumed for the tissue 60-62 . cell culture. to isolate bone marrow-derived macrophages (bmdm), bone marrow was extracted from mice femurs and tibia then purified through a ficoll-paque gradient. upon purification, cells were differentiated on charge-free plates in dmem containing 20% endotoxin-reduced fetal bovine serum and 30% l929 cell supernatant for 5 days 63 . peritoneal macrophages were obtained from untreated mice. briefly, the abdomen of anaesthetised mice was exposed and 5 ml of sterile pbs was instilled intraperitoneally using a 20-gauge syringe. following agitation, the fluid was removed and centrifuged. the cells were plated in dulbecco modified eagle medium containing 10% fetal bovine serum and used for western blotting. alveolar macrophages were obtained from balf of untreated mice. briefly, 1 ml of sterile pbs was instilled through a 20-gauge angiocath ligated into the trachea and the fluid then removed; this process was repeated three times. the fluid was centrifuged and cells were used for elisa. the second-generation lentiviral pgipz expression vectors encoding shrnas against mouse vimentin (clones v2lmm_14765, v2lhs_171946 and v2lhs_171949) and scrambled shrna control (catalogue no. rhs4346) originated from the thermo fisher scientific shrna library (waltham, ma) and were provided by rnai/throughput robotics core of northwestern university (evanston, il). lentiviral stocks (with titres of 10 7 -10 8 tu ml à 1 ) encoding shrnas against mouse vimentin were produced by the dna/rna delivery core (skin disease research center, northwestern university, chicago, il). western blotting. primary murine macrophages were cultured overnight in 1% serum and treated or not with lps (100 ng ml à 1 ) for 7 h. atp (50 mm) was added to lps-stimulated cells during the last 45 min. cells were lysed in sample buffer and equal volumes loaded in 10% sds-page gels transferred to nitrocellulose membranes. the presence of indicated proteins in cell lysates and medium was assessed by western blotting using the following antibodies: mouse monoclonal anti-vimentin (clone v9; 1:500; sigma-aldrich no. treated according to manufacturer's instructions. bound proteins were prepared for western blotting. protein-protein interaction. the interaction between nlrp3 and vimentin was analysed using a bli, blitz (fortebio, menlo park, ca). antibodies directed against nlrp3 (adipogen) were diluted 1:4 then attached to a protein a biosensor. next, commercially available, purified nlrp3 (abnova) was attached to the biosensor for 3 min. finally, each labelled biosensor was placed into a specific concentration of purified vimentin 66 and binding was measured for 120 s followed by dissociation in pbs for 120 s. at the start of each binding experiment, vimentin was diluted into pbs to assemble filaments 67 real-time reverse transcriptase polymerase chain reaction. total rna extraction and quantitative pcr were performed as previously described (). briefly, total rna was isolated using the aurum total rna mini kit (qiagen). cdna was synthesized using the qscript cdna supermix (quanta biosciences) and amplified using 40-cycle two-step pcr with sequence-specific primer pairs (idt). sybr green fluorescence was quantified with a cfx96 real-time system thermal cycler (bio-rad) using ddct analysis. relative il-1b mrna expression was determined via normalizing to b-actin mrna expression. statistical analysis. data are expressed as means ± s.d. or means ± s.e.m. differences between two groups were assessed by using a student's t-test. differences between three or more groups were assessed using one-way analysis of variance with a bonferroni multiple comparisons test. values of po0.05 were considered to be significant. the log rank test, which calculates the chi-square (w 2 ) for each event time for each group and sums the results, was used in the analysis of the kaplan-meier curve was used. all analyses were performed using graphpad prism software version 4.00 for windows (graphpad software, san diego, ca). wild-type j774.1 cells and j774.1 cells expressing shrna against vimentin were fixed in methanol ( à 20°c) for 7 min and processed for indirect immunofluorescence as described previously 64,65 using the following antibodies: mouse monoclonal anti-nlrp3 (adipogen international no. ag-20b-0014 images of fixed, stained preparations were taken with either a zeiss lsm 510 microscope or a nikon a1r microscope immunoprecipitated caspase-1 in the right panel could not be detected due to masking by the signal from the heavy chain of the antibody. protein a/g agarose was incubated with cell lysates in the absence of an antibody to control for non-specific binding. to enable caspase-1 detection in immunoprecipitates, caspase-1 antibodies were crosslinked to protein a/g agarose using crosslink ip kit (thermo) cell mechanics and the cytoskeleton introducing intermediate filaments: from discovery to disease mice lacking vimentin develop and reproduce without an obvious phenotype impaired wound healing in embryonic and adult mice lacking vimentin a role for vimentin in crohn disease functions of the intermediate filament cytoskeleton in the eye lens if-pathies": a broad spectrum of intermediate filamentassociated diseases intermediate filaments: primary determinants of cell architecture and plasticity dysfunctions of neuronal and glial intermediate filaments in disease novel functions of vimentin in cell adhesion, migration, and signaling intermediate filaments as signaling platforms inflammasome-mediated production of il-1beta is required for neutrophil recruitment against staphylococcus aureus in vivo activation of inflammasome signaling mediates pathology of acute p. aeruginosa pneumonia mycobacterium 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syndrome inflammasome-regulated cytokines are critical mediators of acute lung injury elevated interleukin-1 release by human alveolar macrophages during the adult respiratory distress syndrome bleomycin and il-1 beta-mediated pulmonary fibrosis is il-17a dependent transient expression of il-1b induces acute lung injury and chronic repair leading to pulmonary fibrosis a novel role of vimentin filaments: binding and stabilization of collagen mrnas the highly conserved nuclear lamin ig-fold binds to pcna: its role in dna replication vimentin binding to phosphorylated erk sterically hinders enzymatic dephosphorylation of the kinase the intermediate filament protein, vimentin, is a regulator of nod2 activity intermediate filaments control the intracellular distribution of capases during apopptosis caspase cleavage of vimentin disrupts intermediate filaments and promotes apoptosis a role for mitochondria in nlrp3 inflammasome activation vimentin intermediate filaments modulate the motility of mitochondria vimentin is secreted by activated macrophages proapoptotic bid is required for pulmonary fibrosis norepinephrine increases alveolar fluid reabsorption and na,k-atpase activity poissons' ratio of lung parenchyma and parenchymal interaction with bronchi feedback amplification of fibrosis through matrix stiffening and cox-2 suppression oxidative stress, plasminogen activator inhibitor 1, and lung fibrosis a nuclear receptor atlas: macrophage activation shear stress induced reorganization of the keratin intermediate filament network requires phosphorylation by protein kinase c zeta vimentin is sufficient and required for wound repair and remodeling in alveolar epithelial cells structure and assembly properties of the intermediate filament protein vimentin: the role of its head, rod and tail domains vimentin intermediate filament formation: in vitro measurement and mathematical modeling of the filament length distribution during assembly dr esra roan (university of memphis, tn) generously assisted with the afm microindentation matlab code and analysis of results. we thank mr luke skertich for assistance with immunofluorescence confocal microscopy. histological staining was performed by the mouse histology phenotyping laboratory. imaging work was performed at the northwestern university cell imaging facility generously supported by nci ccsg p30 ca060553 awarded to the robert h. lurie comprehensive cancer center. afm measurements were produced in the nifti facility (nuance center, northwestern university), which has received support from the nsf-nsec, nsf-mrsec, keck foundation, the state of illinois and northwestern university. key: cord-312692-jv3425w1 authors: iwata-yoshikawa, naoko; okamura, tadashi; shimizu, yukiko; kotani, osamu; sato, hironori; sekimukai, hanako; fukushi, shuetsu; suzuki, tadaki; sato, yuko; takeda, makoto; tashiro, masato; hasegawa, hideki; nagata, noriyo title: acute respiratory infection in human dipeptidyl peptidase 4-transgenic mice infected with middle east respiratory syndrome coronavirus date: 2019-01-09 journal: journal of virology doi: 10.1128/jvi.01818-18 sha: doc_id: 312692 cord_uid: jv3425w1 middle east respiratory syndrome coronavirus (mers-cov) infection can manifest as a mild illness, acute respiratory distress, organ failure, or death. several animal models have been established to study disease pathogenesis and to develop vaccines and therapeutic agents. here, we developed transgenic (tg) mice on a c57bl/6 background; these mice expressed human cd26/dipeptidyl peptidase 4 (hdpp4), a functional receptor for mers-cov, under the control of an endogenous hdpp4 promoter. we then characterized this mouse model of mers-cov. the expression profile of hdpp4 in these mice was almost equivalent to that in human tissues, including kidney and lung; however, hdpp4 was overexpressed in murine cd3-positive cells within peripheral blood and lymphoid tissues. intranasal inoculation of young and adult tg mice with mers-cov led to infection of the lower respiratory tract and pathological evidence of acute multifocal interstitial pneumonia within 7 days, with only transient loss of body weight. however, the immunopathology in young and adult tg mice was different. on day 5 or 7 postinoculation, lungs of adult tg mice contained higher levels of proinflammatory cytokines and chemokines associated with migration of macrophages. these results suggest that the immunopathology of mers-cov infection in the tg mouse is age dependent. the mouse model described here will increase our understanding of disease pathogenesis and host mediators that protect against mers-cov infection. importance middle east respiratory syndrome coronavirus (mers-cov) infections are endemic in the middle east and a threat to public health worldwide. rodents are not susceptible to the virus because they do not express functional receptors; therefore, we generated a new animal model of mers-cov infection based on transgenic mice expressing human dpp4 (hdpp4). the pattern of hdpp4 expression in this model was similar to that in human tissues (except lymphoid tissue). in addition, mers-cov was limited to the respiratory tract. here, we focused on host factors involved in immunopathology in mers-cov infection and clarified differences in antiviral immune responses between young and adult transgenic mice. this new small-animal model could contribute to more in-depth study of the pathology of mers-cov infection and aid development of suitable treatments. m iddle east respiratory syndrome coronavirus (mers-cov) was originally isolated as a novel coronavirus from a fatal case of acute respiratory distress syndrome and renal failure in 2012 (1). a human receptor for the virus, called human cd26/dipeptidyl peptidase 4 (hdpp4), was identified subsequently (2) . many epidemiological and virological investigations have been undertaken since then; however, information about the pathogenesis of mers-cov is limited. in addition, because mers-cov is endemic in the middle east, the development of effective prophylactic and therapeutic treatment strategies remains a high priority. therefore, appropriate animal models are needed to better understand the pathogenesis of mers-cov and facilitate development of effective vaccines and drugs. some research groups experimentally infected nonhuman primates and small experimental animals with mers-cov (3) (4) (5) (6) (7) . rhesus macaques appear to develop a transient lower respiratory tract infection after a combination of intratracheal, ocular, oral, and intranasal inoculation with mers-cov (3), whereas the common marmoset develops progressive and severe pneumonia, which can be lethal (4) . however, animal models based on nonhuman primates present both ethical and economic problems. thus, establishing a small-animal model of mers-cov infection is desirable. unfortunately, mers-cov does not infect or replicate in small rodents such as syrian hamsters (8) , mice (9) , or rats (10) because they lack a functional mers-cov receptor. zhao et al. described lung infection in a mouse model transduced with an adenovirus expressing hdpp4 (11) ; thus, a transgenic (tg) mouse carrying hdpp4 should be suitable for mers-cov studies (5) . some research groups developed tg mice overexpressing the hdpp4 receptor under the control of cag or cytokeratin 18 promoters (5) (6) (7) . these mice developed severe lung disease, along with infection of the brain. autopsy data are available from only one mers patient; therefore, it is unclear whether mers-cov causes a systemic infection although there is no evidence that mers-cov infects the human brain. other studies describe development of an hdpp4 knock-in mouse (12) (13) (14) . although the tissue distribution and expression levels of hdpp4 in these models are largely equivalent to those of dpp4 in wild-type mice, the phenotype that determines mers-cov susceptibility varies from model to model. the hdpp4 knock-in mouse model described by coleman et al. (12) succumbed to infection with wild-type mers-cov. in contrast, model mice described by cockrell et al. (14) and li et al. (13) are susceptible to infection by serially passaged mers-cov, which induces severe lung pathology and diffuse alveolar damage (dad). these mice would be good models for studying pathogenesis of mers. here, we developed a new tg mouse model expressing hdpp4 under the control of its endogenous promoter to better mimic physiological expression of hdpp4. these tg mice were then backcrossed onto th1prone c57bl/6 mice. after evaluating susceptibility to mers-cov infection, we investigated age-dependent differences in disease pathogenesis because older age is one of the common factors related to mers severity and mortality (15) (16) (17) (18) (19) (20) . both young and adult tg mice infected with mers-cov showed transient weight loss along with moderate pneumonia and mers-cov replication in the lung; however, they did not recapitulate the severe disease and lethal infection seen in humans. young and adult tg mice infected with mers-cov did, however, show different immunopathologies. adult tg mice showed higher levels of proinflammatory cytokine-and chemokinemediated macrophage infiltration of the lungs than young tg mice. taken together, these results suggest that age affects the immunopathology of mers-cov infection in tg mice. the data suggest that other factors are required to recapitulate severe human disease in these tg mice; however, this mouse model will be useful for identifying host mediators that protect against mers-cov infection. this animal model will provide new insight into factors that cause severe mers-cov infection. expression of hdpp4 in tg mouse tissues. to generate tg mice showing tissueor cell-type-specific hdpp4 expression mimicking that in humans, we first looked at research involving tg mice harboring human enterovirus receptors (such as the human poliovirus receptor) and scarb2 receptor-driven endogenous promoters (21, 22) . promoter sequences, which normally include a transcriptional start site, are usually isolated from the upstream regions of endogenous mammalian genes (23) . therefore, we used a bacterial artificial chromosome (bac) clone (rp11-345j9) containing the complete hdpp4 gene and an endogenous promoter to generate tg mice harboring hdpp4 (fig. 1a) . to screen the tg mice generated, we confirmed presence of the transgene by pcr genotyping using two primers sets specific for hdpp4 (table 1, exons 3 and 10) . hdpp4 is a protease expressed on the surface of cells in various organs, including t cells (24, 25) . the enzyme is expressed by approximately 60% of resting t cells isolated from blood (26) . since handling of peripheral blood in a laboratory is relatively simple, we conducted flow cytometry analysis using a fluorescein isothiocyanate (fitc)-conjugated anti-human cd26/hdpp4 monoclonal antibody that does no react with murine dpp4 to detect expression of hdpp4 in mice. cd3-positive lymphocytes from 2/15 tested pups were positive for hdpp4. these mice were then crossed with c57bl/6 mice to establish two independent tg lines (tg1 and tg2), which were maintained as hemizygotes carrying the hdpp4 gene. the tg animals were born at the the open and filled circles denote the centromere (cen) and telomere (tel) of human chromosome 2, respectively. the gray arrows indicate the genes located downstream of the hdpp4 gene (white arrow). the cloned region in the bac construct is denoted by a black line. gcg, glucagon gene; fap, fibroblast activation protein; ifih1, interferon induced with helicase c domain 1. (b) expression of hdpp4 on peripheral blood cd3-positive t lymphocytes from the transgenic (tg) mice. tg1 and tg2, hdpp4 ϩ/ϫ transgenic mouse lines 1 and 2; non-tg, hdpp4 ϫ/ϫ mouse. (c) genomic dna was extracted from tg2 mice, and human dpp4 exons 1 to 26 were subjected to pcr using specific primers. expected mendelian ratio and were outwardly indistinguishable from control littermates. because cd3-positive lymphocytes from peripheral blood of line tg2 showed higher hdpp4 expression than those from line tg1 (fig. 1b) , tg2 was used for further analyses. pcr genotyping using primer sets specific for hdpp4 revealed that the complete hdpp4 gene had integrated into the genome of tg2 mice ( fig. 1c and table 1) . to examine hdpp4 expression in human and tg2 tissues, we first performed western blot analysis with a goat anti-cd26/hdpp4 polyclonal antibody (af1180; r&d systems), which detected hdpp4 but cross-reacted weakly with mouse dpp4. bands of about 110 kda (hdpp4) were detected in all tested human tissues (liver, spleen, kidney, heart, lung, stomach, small intestine, large intestine, pancreas, brain, spinal cord, and skeletal muscle), except brain ( fig. 2a) . all of the tissues from hdpp4 tg mice expressed hdpp4, including liver, spleen, kidney, heart, lung, stomach, small intestine, large intestine, pancreas, brain, spinal cord, and skeletal muscle ( fig. 2a) . these results suggest that the human transgene was expressed in the majority of organs/tissues in tg2 mice. to further determine hdpp4 distribution in tissues, immunohistochemistry (ihc) was performed (fig. 2b ). ihc using a goat anti-cd26/hdpp4 antibody detected hdpp4 antigens in pneumocytes in the lung, in bile capillaries in the liver, in renal tubular epithelium, on the surface of epithelial cells lining the small intestine, in pancreatic islets, in lymphocytes in the lymph nodes, and in several types of endothelial cell and serous membranes (fig. 2b , left column). while hdpp4 expression was undetectable in brain tissue by western blot analysis, ihc revealed that endothelial cells lining blood vessels and leptomeninges of the human brain were positive for hdpp4 although neurons and glia were negative. in tg2 mice, pneumocytes and bronchial epithelial cells in the lungs, bile capillaries in the liver, the renal tubular epithelium, and the surface of epithelial cells in the small intestine were positive for hdpp4 (fig. 2b ). in addition, several types of endothelial cells and serous membranes in all tested tissues, including the central nervous system, from tg2 mice were positive for hdpp4. notably, most lymphocytes in the t cell zones of the spleen and lymph nodes from tg2 mice were positive for hdpp4. staining of tissues from non-tg mice was very weak or absent (except for the small intestine) (fig. 2b) . these data suggest that the pattern of hdpp4 expression in tg2 mice is similar to that in humans (except for pancreas and lymphoid tissues). expression of hdpp4 was higher in lymphocytes from tg2 mice than in those from humans ( fig. 1c and fig. 2b ). therefore, we investigated the immune response profile in tg2 mice. to assess innate immune responses in the lungs of tg2, non-tg, and c57bl/6 mice, all animals received intranasal administration of pbs with or without (b) immunohistochemical analysis of hdpp4 expression in human, tg2, and non-tg mouse tissues stained with an anti-hdpp4 polyclonal antibody. sections were counterstained with hematoxylin. scale bars, 50 m (large images of liver, kidney, small intestine, pancreas, spleen, and lymph node), 20 m (large images of lung and brain), and 25 m (insets). poly(i·c), a synthetic analog of double-stranded rna (fig. 3) . there was no statistically significant difference in cytokine expression levels between tg2, non-tg, and c57bl/6 mice at 24 h after inoculation with poly(i·c) or phosphate-buffered saline (pbs) (fig. 3) . however, when we set expression levels after pbs treatment as 1, we noted that expression of macrophage inflammatory protein 1␣ (mip-1␣), granulocyte-macrophage colony-stimulating factor (gm-csf), interleukin-1␤ (il-1␤), il-12, and il-2 in poly(i·c)treated tg2 mice was 0.8-to 2-fold higher than in the other two strains. these results suggest that hdpp4 expression in mice does not have a marked effect on basal innate immune responses in the three mouse strains; however, tg2 mice show slightly stronger or earlier innate immune responses than c57bl/6 mice and non-tg mice. thus, when we investigated immune responses in this animal model, we made comparisons between mers-cov-infected and noninfected tg mice. susceptibility of hdpp4-tg mice to mers-cov infection. in this experiment, c57bl/6 mice were used instead of non-tg mice because we were unable to prepare a sufficient number of non-tg mice from littermate mice for this experiment. after intranasal inoculation of 10-week-old tg2 and c57bl/6 mice with 10 5 50% tissue culture infectious doses (tcid 50 ) of mers-cov, neither group was lethargic; however, tg2 mice showed mild but transient weight loss from days 6 to 7 postinoculation (p.i.) (tg2 mice, n ϭ 8 [four females and four males]; c57bl/6 mice, n ϭ 10 [five females and five males]) (fig. 4a ). tg2 mice showed seroconversion at 35 days p.i., whereas c57bl/6 mice did not (tg2 mice, n ϭ 5 [three females and two males]; c57bl/6 mice, n ϭ 6 [three females and three males]) (fig. 4b ), suggesting that tg2 mice are susceptible to infection by mers-cov. we next examined viral replication kinetics and sites of viral replication in 10-weekold tg2 and c57bl/6 mice (n ϭ 3-4 [2 females and 1 to 2 males per time point]). tg2 mice and c57bl/6 mice were inoculated intranasally with 10 5 tcid 50 of mers-cov, and tissue specimens (the maxilla [including the nostril], nasal wash fluid, lung, and lung wash fluid) were collected at 6 h p.i. and on days 1, 3, 5, and 7 p.i. the nasal wash fluid from three out of four tg2 mice contained barely detectable levels of infectious virus at days 1 and 5 p.i. (fig. 4c ). supernatants from maxilla tissue homogenates (20%) from tg2 mice contained 10 2.8 tcid 50 /g at day 1 p.i. although the titer fell by 5 days p.i. the viral titers in lung wash fluid and supernatants of lung tissue homogenates (20%) from tg2 mice contained 10 2.3 tcid 50 /ml and 10 4.6 tcid 50 /g, respectively, at 1 day p.i.; these values were significantly higher than those at 6 h p.i. (p ͻ 0.05 and p ͻ 0.01, respectively; one-way analysis of variance [anova]). the virus titers in the lungs were detectable up until 5 days p.i. virus was undetectable in the respiratory tract at 7 days p.i. although ihc revealed that various organs from tg2 mice were positive for the hdpp4 antigen (fig. 2b ), no infectious virus was detected in the liver, spleen, kidney, heart, intestines, and brain up to 7 days p.i. (table 2 ). in contrast, virus was detected in the respiratory tract of c57bl/6 mice at 6 h p.i. only. these observations suggest that mers-cov infects and replicates mainly in the lower respiratory tract of tg2 mice and is eliminated within 7 days of infection. several research groups have developed tg mouse models of mers-cov infection; however, in these models, viral replication and mers-cov rna were detected in the brain (5-7, 27). thus, we next measured the amount of viral rna in the brain of tg2 mice at 6 h and at 1, 3, 5, and 7 days p.i. by real-time reverse transcription-pcr (rt-pcr) using primers specific for mers-cov; however, no mers-cov rna was detected in the brain of tg2 mice. furthermore, another study showed that experimental infection of common marmosets with mers-cov resulted in viremia (4) . quantitative examination of viral rna levels in tg2 mice revealed very low copy numbers of viral rna in the blood at 3 and 5 days p.i., while sera from tg2 mice were negative for the virus ( table 2 ). these results suggest that although intranasal inoculation of tg2 mice with mers-cov causes no neuroinvasion, it may induce viremia. several animal studies have identified mers-cov rna in the lymphoid organs of infected animals (3, 4, 28) , but no infectious virus was detected in the spleen from tg2 mice up to 7 days p.i. (table 2 ). to investigate whether lymphoid organs in tg2 mice are susceptible to mers-cov infection, we harvested splenocytes from tg2 and c57bl/6 mice and estimated infectivity and mers-cov rna levels (fig. 4d) . although the amount of infectious mers-cov in splenocytes was below the detection limit, viral rna was detected in splenocytes from tg2 mice (peaking at 2 days p.i.). thus, lymphoid organs of tg2 mice were as susceptible to mers-cov infection as those reported in other animal studies although lymphoid organs were not a major site of mers-cov replication in tg2 mice after intranasal inoculation. acute inflammatory changes in the lungs of hdpp4-tg mice after mers-cov inoculation. mers-cov infection of the nasal cavity, lungs, brain, spinal cord, liver, spleen, kidney, heart, and gastrointestinal tract of tg2 mice was examined histopathologically on days 1, 3, 5, 7, 14, and 35 p.i (n ϭ 3; one or two females and one or two males per time point). histopathological investigations revealed that tg2 mice showed progressive pulmonary inflammation associated with acute virus infection, from which they recovered (fig. 5 ). on day 1 p.i., there was no obvious infiltration of the lungs in tg2 mice; however, mild cellular degeneration and viral antigen-positive cells were seen in the bronchioles and a few alveolar areas ( fig. 5a to c). double-immunofluorescence staining revealed that mers-cov antigen colocalized with hdpp4-positive bronchioles and alveolar cells on day 1 p.i. (fig. 6 ). on days 3 and 5 p.i., inflammatory reactions, including partial and/or mild perivascular and peribronchiolar infiltration by mononuclear cells and eosinophils in response to viral antigens, were observed in alveolar areas of lung tissue from tg2 mice (fig. 5d to i). from day 3 p.i. onwards, the alveolar area was the main site of inflammatory responses to viral replication (fig. 5f , i, and l). on day 7 p.i., the point at which tg2 mice showed weight loss, there was evidence of severe lung inflammation, including perivascular and alveolar septal thickening, caused by infiltrating mononuclear cells; some alveoli were positive for viral antigens (fig. 5k ). at day 14 p.i., focal cellular infiltration was still evident in the peribronchioles and alveolar septa although viral antigens and inflammatory responses had cleared from the lungs by day 35 p.i. (fig. 5m to p). there was no evidence of diffuse alveolar damage in the lungs up to day 35 p.i. these findings suggest that progressive immunopathology occurred uniformly throughout the lungs but resolved within 14 days after infection. neither histopathology nor ihc detected inflammatory infiltration or viral antigens in the nasal cavity through 35 days p.i. in addition, there was no inflammation or viral antigen expression in the brain through 35 days p.i. i a g i a g i a g i a g i a g i a g i a (fig. 7) . in contrast, c57bl/6 mice showed no histopathological changes or viral antigen in any organ, including the lung. these results indicate that tg2 mice suffer acute pneumonia after infection of the lungs with mers-cov, which is related to expression of hdpp4 in the bronchiolar epithelium and pneumocytes. splenocytes from tg2 mice (fig. 4d) ; however, the spleen and other lymphoid tissues did not harbor viral antigens. furthermore, mers-cov induced t cell apoptosis upon infection in vitro (28) , whereas immunohistochemical staining detected no evidence of apoptosis in lymphoid tissues, including spleen and lymph nodes, of mers-cov-infected tg2 mice. similar to findings in the other mouse models in which mouse dpp4 was replaced with hdpp4 (12), mers-cov replication and pathology were localized in the lungs in tg2 mice. differences in the immunopathology of mers-cov infection between young and adult hdpp4-tg mice. according to an epidemiological study (29) , age (ͼ45 years) is considered to be one of the risk factors for mers-cov infection in humans. therefore, we infected 25-week-old mice with mers-cov. tg2 mice showed significant weight loss from days 7 and 8 p.i. before recovering by day 14 p.i. (tg2 mice, n ϭ 6 [one female and five males]; non-tg mice, n ϭ 7 [three females and four males]) (fig. 8a) . however, 25-week-old tg2 mice showed no obvious clinical signs (such as respiratory illness and mortality). the 25-week-old tg2 mice had seroconverted by 35 days p.i., whereas the c57bl/6 mice had not (fig. 8b) . next, we examined viral replication kinetics and sites of viral replication in 25-week-old tg2 and non-tg mice (n ϭ 4; two females and two males per time point). the nasal wash fluid from one out of four tg2 mice contained barely detectable levels of infectious virus at 1, 3, and 5 days p.i. (fig. 8c) . supernatants from maxilla tissue homogenates (20%) from tg2 mice contained 10 1.7 and 10 2.0 tcid 50 /g at 1 and 3 days p.i., respectively, although the titer was undetectable at 5 days p.i. the viral titers in lung wash fluid from tg2 mice were detectable up until 3 days p.i., while the supernatants from lung tissue homogenates (20%) from tg2 mice showed a high viral load from 1 to 5 days p.i.; infectious virus was detectable up until 7 days p.i. viral loads in the respiratory tract peaked at 3 days p.i. although no virus was detectable in the respiratory tract of 10-week-old tg mice at 7 days p.i., infectious virus was detected in the lungs of 25-week-old tg mice up until 5 days p.i. in addition, infectious virus was detected in the lungs of one of four 25-week-old tg mice (10 2.5 tcid 50 /g) even at 7 days p.i. we also found that viral titers in the nasal wash fluid, maxilla (including nostril), lung wash fluid, and lungs of 25-week-old tg2 mice on day 3 p.i. (10 2.6 tcid 50 /ml, 10 3.3 tcid 50 /ml, 10 2.3 tcid 50 /ml, and 10 4.9 tcid 50 /ml, respectively) were slightly higher than those in 10-week-old tg2 mice (10 1.6 tcid 50 /ml, 10 2.9 tcid 50 / ml, 10 1.8 tcid 50 /ml, and 10 4.5 tcid 50 /ml, respectively) (p ϭ 0.04, student's t test with welch's correction). histopathological analysis revealed that 25-week-old tg2 mice showed delayed and prolonged inflammatory responses in the lung compared with those in 10-week-old tg2 mice (fig. 9) . interestingly, viral antigen-positive cells were seen in the alveolar area on day 1 p.i. and then in the bronchi on day 3 p.i., along with a sparse cellular infiltrate (fig. 9a to f) . cellular infiltration (which included mononuclear cells and polynuclear cells) was observed in the alveoli from 5 days p.i.; this expanded on day 7 p.i. (fig. 9g to l). focal cell infiltration was seen in the alveoli on day 14 p.i., and lymphoid cell aggregates were seen around bronchioles and blood vessels on day 35 p.i. (fig. 9m to r). next, we compared inflammation of the alveoli in 10-week-old tg mice and 25week-old tg mice on day 7 p.i. ionized calcium binding adaptor molecule 1 (iba-1) is expressed specifically by monocytes/macrophages and is upregulated when these cells are activated. the predominant inflammatory infiltrate within the lungs of both 10week-old and 25-week-old tg mice comprised iba-1-positive large cells and cd3positive mononuclear cells (fig. 10) . phagocytic vacuoles were prominent in large iba-1-positive cells from 25-week-old tg mice. (30) . therefore, we measured the levels of 20 cytokines and chemokines in lung samples from both 10-week-old and 25-week-old tg2 mice inoculated with either mers-cov or minimal essential medium (mem). measurements were made at 6 h and at 1, 3, 5, and 7 days p.i. (n ϭ 3 to 4 [2 females and 1 to 2 males per time point] and n ϭ 4 [2 females and 2 males per time point]) ( fig. 11a and b, for 10-week-old and 25-week-old mice, respectively). on day 3 p.i., we observed early expression of gamma interferon (ifn-␥)-induced protein 10 (ip-10) in the lungs of both 10-and 25-week-old tg2 mice, a level which was significantly higher than that in control mice; this increase lasted through day 7. this was followed by a transient increase in expression of interleukin-6 (il-6), il-13, and monocyte chemotactic protein-1 (mcp-1) in lungs from both 10-and 25-week-old tg2 mice at day 5 p.i. high levels of macrophage inflammatory protein 1␣ (mip-1␣) and monokine induced by ifn-␥ (mig) were detected in the lungs of both groups of tg2 mice from days 3 or 5 to 7 p.i., whereas il-12 levels increased at day 7 p.i. ifn-␥ production in the lungs of 10-week-old tg2 mice peaked significantly on day 5 p.i. while high values were seen in both infected and noninfected 25-week-old mice during the observation period. in contrast, expression of ip-10, il-12, and il-1␤ in 25-week-old tg mice was higher than that in 10-week-old tg2 mice. interestingly, il-1␣ and il-17 were detected only in 25-week-old tg2 mice infected with mers-cov. il-1␣, a potent proinflammatory cytokine associated with inflammation and fever, was detected from 3 days p.i. and remained significantly elevated until 7 days p.i.; il-17 (a proinflammatory cytokine that recruits monocytes and neutrophils) was detected from 3 days p.i. and peaked at 5 days p.i. before falling at 7 days p.i. these results indicate that mers-cov infection induces production of acute inflammatory chemokines and cytokines in the lungs. in addition, aging causes more severe immunopathology; this means that young and adult tg mice show different pathologies in the lung after mers-cov infection. furthermore, we measured expression of mrna encoding ifn-␣4 and ifn-␤ (type i ifn with antiviral activity) in the lungs of 10-and 25-week-old mice at 6 h and at 1, 3, 5, and 7 days p.i. by real-time reverse transcription-pcr (rt-pcr) (31). we did not find any evidence of ifn-␣4 or ifn-␤ in the lungs from 10-week-old tg2 mice at early times postinfection; however, we observed a transient increase in ifn-␣4 expression in the lungs of two out of four tg2 mice at 5 days p.i.; this level fell by 7 days p.i. (fig. 11c) . day 5 p.i. was the time point at which the amount of virus in the lungs of tg2 mice began to fall. no ifn-␤ mrna was detectable in lung samples from either group. on the other hand, the lungs of 25-week-old tg2 mice showed a transient increase in ifn-␣4 and ifn-␤ mrna expression at 3 days p.i. (fig. 11c ). in addition, ifn-␣4 and ifn-␤ mrna expression was higher than that in 10-week-old tg2 mice. taken together, these results indicate that both type i and type ii ifn contribute to the immunopathology of the lungs of 25-week-old tg2 mice infected with mers-cov. here, we describe a new hdpp4-tg mouse expressing the human gene under the control of an endogenous human promoter. this mouse model shows a pattern of hdpp4 expression that closely mimics that in human tissues and is similar to that in other recent models (5) (6) (7) . this mouse model also shows susceptibility to infection by mers-cov; this mimics the nonlethal observations in other mouse models (13, 14) . after intranasal inoculation with a human isolate of mers-cov, the tg mice developed acute and mild interstitial pneumonia; however, the infection was nonlethal and so did not mimic severe cases seen in human mers-cov patients. mers-cov infection can cause severe illness, resulting in acute respiratory distress syndrome, although a large number of mers-cov infections follow a mild or asymptomatic course in healthy individuals (32) (33) (34) (35) . thus, this tg mouse model reflects the natural course of a mild mers-cov infection. the majority of severe mers-cov cases occur in middle-aged and older males (36, 37) . therefore, we infected tg2 mice aged 25 weeks with mers-cov. the mice showed prominent proinflammatory responses and prolonged pulmonary inflammation compared with responses in tg2 mice aged 10 weeks. however, none of the infected tg2 mice had a severe outcome, such as respiratory distress, that led to death. epidemiologically, patients with diabetes, kidney failure, or chronic lung disease, all of which might weaken the immune system, tend to have a poor outcome after infection by mers-cov (http://www.who.int/csr/don/23-september-2015-mers-kuwait/en/). thus, it is presumed that a combination of older age and underlying disease might also increase mortality in this animal model. this hdpp4-tg mouse model, which lacks the clinical signs and mortality associated with severe mers-cov infection, is likely to be less advantageous than other lethal mouse models with respect to development of novel vaccines or antiviral agents (12) (13) (14) . when we asked why the tg2 mice showed nonlethal responses to infection, we could not ignore the fact that virus levels in lungs were lower than those reported for other mers mouse models (5, 12, 13, 38, 39) . one reason for this is that the transgene used in this study is a hemizygote, meaning that the copy number or expression level may be lower than that in mice homozygous for hdpp4. in addition, the dpp4 protein is active as a dimer (40) , but the tg2 mice harbor both murine and hdpp4. we presume that the viral yield in the lungs of tg2 mice was low because of heterodimers formed by hdpp4 and murine dpp4. to address this, we constructed structural models of homo-and heterodimers comprising human and mouse dpp4. notably, the estimated interaction energy of the dpp4 heterodimers was greater than that of murine and hdpp4 homodimers (ϫ358.2, ϫ347.6, and ϫ344.6 kcal/mol, respectively). these results suggest that dpp4 heterodimers are as stable as dpp4 homodimers. cockrell et al. reported that mouse dpp4 does not support mers-cov entry (38) . thus, the presence of stable dpp4 heterodimers may be a reason for the lower levels of infection in our mouse model. further study is necessary to clarify this. some research groups generated a mouse-adapted mers-cov for use in severe/ lethal mers-cov infection mouse models (13, 14) . this may be one way to establish severe mers infection in our tg2 mice. while the tg2 mice expressed hdpp4 protein in the liver, spleen, kidney, heart, lung, gastrointestinal tract, pancreas, and brain, viral infection and replication were limited (mainly) to the lower respiratory tract, with little upper respiratory tract involvement, after intranasal inoculation of mers-cov. tg2 mice developed interstitial pneumonia, and mers-cov antigens were detected in the lungs. virus yields in the lung were up to 100-fold higher than those in the upper respiratory tract. most mers patients exhibit a severe lower respiratory tract infection, with little involvement of the upper respiratory tract (36) . this suggests that mers-cov infection in tg2 mice mimics mild infection in humans. in vitro analysis of mers-cov suggests that the virus also infects human t cells and macrophages (28, 41, 42) . we detected mers-cov rna in serum and spleen cells from tg2 mice. these data are similar to those generated from another tg mouse model in which mouse dpp4 was replaced with hdpp4 under the control of the endogenous mouse dpp4 promoter (12) . mers-cov infection of t cells might affect immunopathology or induction of apoptosis in tg mice, but we found no clear evidence of this. although tg2 mice showed systemic viremia, infection of organs (except lung) did not lead to secondary complications. the disease phenotype (including clinical symptoms, viral titer in the lung, and acute pneumonia) appeared to be driven by infiltration by macrophages and lymphocytes; this is similar to the phenotype observed in another tg mouse model harboring hdpp4 under the control of the endogenous mouse dpp4 promoter (12) . the tg2 mice described herein did not show any brain or renal lesions after mers-cov infection. other tg mouse models in which hdpp4 is expressed under a strong ubiquitous promoter show high levels of viral rna and inflammation in the lungs, which are accompanied by brain lesions (5, 7, 11) . a fatal case of human mers-cov infection published by ng et al. showed no sign of mers-cov infection in the brain (43) . to date, no reports suggest that mers-cov shows tropism for brain tissue. the primary target of mers-cov is the lower respiratory tract; however, patients with mers often show signs of acute kidney failure (1, 43). in addition, mers-cov was identified in the urine of mers patients (44, 45) . data from the first autopsy case did find pathological signs in the patient's kidneys although ihc revealed no evidence of mers-cov replication in the kidneys (43) . in addition, acute renal failure in this patient was not caused by mers-cov directly; rather, it was caused by hypotension (43) and/or acute respiratory distress syndrome (46) . histopathological analysis identified cd3-positive t cells and iba-1-positive macrophages in the lungs of tg2 mice on day 7 p.i., which correlated with expression of inflammatory cytokines and inflammatory infiltrates in the lung. tg2 mice aged 10 and 25 weeks showed increased expression of cytokines and chemokines associated with migration of t cells and activation of macrophages, including ip-10, il-6, il-13, mcp-1, ifn-␥, mip-1␣, mig, and il-12, in the lungs at day 5 and/or 7 p.i. this result is the same as that observed in a hdpp4 knock-in mouse model reported by coleman et al. (12) . in this hdpp4 knock-in mouse model, cd8 ϩ t cells and macrophages affected the course of mers-cov-induced disease (12) . in addition, tg2 mice expressed mrna encoding the type i ifn, ifn-␣4, during the early phase of the mers-cov infection. importantly, the pathogenic and immune response data from the knock-in mouse model and our own model are similar. thus, an acute inflammatory reaction (including production of type i and type ii ifns) and infiltration by macrophages might clear the virus from the lung, thereby preventing progression to mers. interestingly, we detected il-1␣ and il-17 in the lungs of 25-week-old tg2 mice, but not those of 10-week-old tg2 mice, after mers-cov infection. both il-1␣ and il-17 are proinflammatory cytokines that attract monocytes and neutrophils; therefore, they may exacerbate immunopathology after infection. these findings support the notion that the severity of mers-cov infection is age dependent. age is one of the most common factors related to severity and mortality of mers infection; however, the underlying pathology is unclear (47) . many studies have examined immune responses of mers patients (30, (48) (49) (50) (51) (52) . indeed, elevated serum levels of il-6, il-12, ip-10, and ifn-␥ are observed in patients during the early period after severe infection (48) (49) (50) (51) . in addition, a prominent proinflammatory th1 and th17 response, including production of ifn-␥, tumor necrosis factor alpha (tnf-␣), il-15, and il-17, is seen in patients during the acute phase of mers-cov infection (52) . in contrast, we found that administration of poly(i·c) to tg2 mice induced a mild increase (or earlier induction) in innate immune responses compared with those in c57bl/6 mice and non-tg mice. this suggests that overexpression of hdpp4 might influence immune responses in tg2 mice. thus, we must exercise caution when assessing the relationship between immunopathology and outcome in patients and animal models of mers-cov; however, proinflammatory responses seem to contribute to immunopathology during the acute phase of mers-cov infection in both patients and mouse models. in summary, we generated an hdpp4-tg mouse model showing mild respiratory infection by mers-cov. while this tg mouse has limitations as a model for human mers (i.e., lower virus titer in the lungs and mild disease), the immunopathology seems to resemble a mild and early stage of infection in humans. even though it has limitations, this tg mouse model will increase our understanding of the mechanisms underlying mers-cov infection. indeed, we recently used this mouse model to confirm a role for transmembrane protease serine type 2 (tmprss2) during mers-cov infection (53) . this animal model may provide new insight into disease pathogenesis and guide development of therapeutic interventions that mitigate mers. ethics statements. experiments using recombinant dna and pathogens were approved by the committee for experiments using recombinant dna and pathogens at the national institute of infectious diseases, tokyo, japan. all animal experiments were approved by the animal care and use committee of the national institute of infectious diseases and the national center for global health and medicine (ncgm) research institute and were conducted in accordance with institutional guidelines for the care and use of animals. all animals were housed in a japan health sciences foundation-certified facility. all human samples used in this study were obtained from us biomax, inc., genetex, inc., alpha diagnostics international, or protein biotechnologies. the protocols were approved by the health insurance portability and accountability act (hipaa) or institutional review board (irb). cells and viruses. mers-cov, hcov-emc 2012 strain, was kindly provided by bart haagmans and ron fochier (erasmus medical center, rotterdam, the netherlands) and was used throughout the study. vero e6 cells, purchased from the american type cell collection (manassas, va), were cultured in eagle's mem containing 5% fetal bovine serum (fbs), 50 iu/ml penicillin g, and 50 g/ml streptomycin (5% fbs-mem). stocks of mers-cov were propagated and titrated on vero e6 cells and cryopreserved at ϫ80°c. viral infectivity titers are expressed as the tcid 50 /milliliter on vero e6 cells and were calculated according to the behrens-kärber method. work with infectious mers-cov was performed under biosafety level 3 conditions. virus isolation and titration. lung wash fluids and liver, kidney, heart, spleen, intestine, and brain tissue samples from tg2, non-tg, and c57bl/6 mice were collected at the time of postmortem examination and stored at ϫ80°c. tissue homogenates (20%, wt/vol) were prepared in 2% fbs-mem, and samples were inoculated onto vero e6 cell cultures, which were then examined for cytopathic effects (cpe) for 5 days. blind passage was performed after freezing and thawing cells from the first-or second-round passages. if mers-cov-specific cpe were not observed in the first-, second-, or third-round cultures, the samples were deemed negative for infectious virus. viral infectivity titers were determined in vero e6 cell cultures using the microtitration assay described above. mers-cov neutralizing assay. blood was obtained from each mouse and allowed to clot. sera were then obtained by centrifugation and inactivated by incubation at 56°c for 30 min. one hundred tcid 50 aliquots of mers-cov were incubated for 1 h in the presence or absence of mouse serum (serially diluted 2-fold) and then added to confluent vero e6 cell cultures in 96-well microtiter plates. the presence of viral cpe was determined on day 5, and the titers of neutralizing antibody were determined as the reciprocal of the highest dilution at which no cpe were observed. the lowest and highest serum dilutions tested were 1:2 and 1:512, respectively. generation of hdpp4-tg mice. to generate tg mice expressing hdpp4, a bac vector carrying the hdpp4 gene (clone rp11-345j9) was purchased from advanced genotechs co., japan. the bac dna was purified using a large-construct kit (qiagen) according to the manufacturer's instructions and suspended in te buffer (10 mm tris-hcl and 0.1 mm edta, ph 7.5) at a concentration of 4 ng/l. tg mice were generated using standard procedures (23). the purified bac clones were microinjected into the pronuclei of fertilized eggs from bdf1ϫc57bl/6ncr mice (slc, inc., hamamatsu, japan) and then transplanted into pseudopregnant icr mice (slc inc.). expression of the transgene was assessed by fluorescence-activated cell sorter (facs) analysis as described below. the tg mice were then backcrossed onto c57bl/6ncr for five generations. after weaning, the mice were tested for tg integration by pcr and facs analysis. briefly, genomic dna isolated from ear punch tissues was subjected to pcr using hdpp4-specific primers ( table 2) , and lymphocytes were isolated from blood taken from the tail vein. lymphocytes were screened for hdpp4 protein expression by flow cytometry analysis. tg mice and their non-tg littermates were used for the mers-cov infection study. flow cytometry analysis. blood was collected from the retro-orbital venous plexus under isoflurane anesthesia using heparinized capillary tubes. the samples were then treated with red blood cell lysis buffer (sigma-aldrich, st. louis, mo) to remove erythroid cells. for immunofluorescence staining, cells kit (thermo fisher scientific). a panel of inflammatory cytokines and chemokines (basic fibroblast growth factor [bfgf], granulocyte-macrophage colony-stimulating factor [gm-csf], ifn-␥, il-1␣, il-1␤, il-2, il-4, il-5, il-6, il-10, il-12p40/p70, il-13, il-17, ip-10, keratinocyte chemoattractant [kc] , mcp-1, mig, mip-1␣, tnf-␣, and vascular endothelial growth factor) was measured according to the manufacturer's protocols. isolation of splenocytes and infection with mers-cov. spleens were removed aseptically from tg2 and c57bl/6 mice (n ϭ 3 each), dissociated in rpmi 1640 medium, and pressed gently through a 40-m-pore-size nylon mesh filter. the cell suspension was centrifuged at 400 ϫ g for 10 min, and red blood cells were lysed with blood cell lysis buffer (final concentrations of 155 mm nh 4 cl, 10 mm khco 3 , and 0.1 mm edta, ph 7.3) at room temperature for 5 min. the cells were washed twice with rpmi 1640 medium and centrifuged at 1,000 ϫ g for 10 min. hdpp4-expressing cd3 ϩ t cells within the splenocyte population were detected by flow cytometry analysis. the percentage of cd3 ϩ t cells was 39.11% ϯ 3.8%, and that of hdpp4-expressing cd3 ϩ t cells was 26.46% ϯ 1.9%. the cells were resuspended in the medium and infected with mers-cov (multiplicity of infection [moi] of 1). viral replication was determined after 1 and 2 days of culture. viral infectivity titers were measured in vero e6 cell cultures using a microtitration assay. to detect the mers-cov genome in splenocytes, rna from splenocytes infected with mers-cov was extracted at 1 and 2 days p.i. and subjected to quantitative real-time rt-pcr (10) . molecular modeling of dpp4 homo-and heterodimers. dpp4 dimer models were constructed using the molecular operating environment (moe) (chemical computing group, inc., montreal, qc, canada) based on the crystal structure of hdpp4 at a resolution of 2.55 å (pdb accession number 2onc). stereochemical quality was assessed using the ramachandran plot and atom clashes applications in moe. interaction energy, which is an indicator of the affinity of the dimer, was calculated using the potential energy application in moe. statistical analysis. data are expressed as the means and standard errors of the means. statistical analyses were performed using graphpad prism, version 5, software (graphpad software, inc., la jolla, ca). intergroup comparisons were performed using one-way and two-way anova or student's t test with welch's correction. a p value of ͻ0.05 was considered statistically significant. 05% nan 3 ), and fc receptors were blocked by incubation for 20 min on ice with an unlabeled anti-mouse cd16/cd32 monoclonal antibody (clone 2.4g2; bay bioscience co., ltd ca) and an allophycocyanin (apc)-labeled anti-human cd3 antibody human skeletal muscle lysates were purchased from protein biotechnologies (ramona, ca) and used under irb-approved protocols. to prepare protein samples from the tg and non-tg mouse organs, tissues were homogenized in 0.5 ml of radioimmunoprecipitation assay (ripa) buffer (50 mm tris/hcl and the protein concentrations were measured using a pierce bicinchoninic acid (bca) protein assay kit after being blocked, the membranes were incubated for 1 h with a goat anti-hdpp4 antibody (0.1 g/ml) (af1180 followed by incubation with a donkey anti-goat horseradish peroxidase (hrp)-conjugated antibody (abcam) and an anti-rabbit hrp-conjugated antibody (abcam). the bands were detected by an immobilon western chemiluminescent hrp substrate (millipore) and an las-3000 apparatus inflammatory cytokine profiles in 20% (wt/vol) lung homogenates were detected using a commercial mouse cytokine 20-plex antibody bead kit (thermo fisher scientific), as described by the manufacturer. inoculation of mice with mers-cov. tg2 and non-tg mice (9 to 10 weeks or 25 weeks old) and tg2-balb mice (12 to 22 weeks old) were anesthetized and inoculated intranasally with 1 ϫ 10 5 tcid 50 (30 l) of mers-cov. body weight was measured daily for 14 days (n ϭ 4 to 8 per group), and animals were sacrificed at 6 h and at 1, 3, 5, 7, 14, and 35 days p.i. to analyze virus replication, hematological parameters, cytokine expression, and disease pathology (n ϭ 3 to 6 per group) for double staining of cd3 (t cells) and iba-1 (macrophages) antigen, we used a rabbit anti-human cd3 antibody tucson, az) and a rabbit anti-human iba-1 antibody diaminobenzidine (dab 0) for 10 min at 121°c. the second staining was performed for iba-1 with vina green. nuclei were counterstained with hematoxylin for 10 s. 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respiratory syndrome comparative and kinetic analysis of viral shedding and immunological responses in mers patients representing a broad spectrum of disease severity immune responses to mers coronavirus during the acute and convalescent phases of human infection mers-cov infection in humans is associated with a pro-inflammatory th1 and th17 cytokine profile tmprss2 contributes to virus spread and immunopathology in the airways of murine models after coronavirus infection synthetic double-stranded rna poly(i:c) combined with mucosal vaccine protects against influenza virus infection we thank bart haagmans and ron fouchier for providing mers-cov (isolate hcov-emc/2012) and satoshi koike, ken fujii (tokyo metropolitan institute of medical science), and shutoku matsuyama (national institute of infectious diseases) for helpful discussions. we also thank ayako harashima and midori ozaki for technical assistance.this work was supported by the following: a grant-in-aid for research (h25-shinko-wakate-004) from the ministry of health, labor, and welfare, japan; the research program on emerging and reemerging infectious diseases (grants jp17fk0108313, jp18fk0108058, and jp18fk0108072) from the japan agency for medical research and development; grants-in-aid for scientific research from the ministry of education, culture, sports, science and technology, japan (16k09951 and 18h02665), and a grant from the national center for global health and medicine (27a1102). key: cord-267482-afqfymbq authors: ryu, seungjin; shchukina, irina; youm, yun-hee; qing, hua; hilliard, brandon k.; dlugos, tamara; zhang, xinbo; yasumoto, yuki; booth, carmen j.; fernández-hernando, carlos; suárez, yajaira; khanna, kamal m.; horvath, tamas l.; dietrich, marcelo o.; artyomov, maxim n.; wang, andrew; dixit, vishwa deep title: ketogenesis restrains aging-induced exacerbation of covid in a mouse model date: 2020-09-12 journal: biorxiv doi: 10.1101/2020.09.11.294363 sha: doc_id: 267482 cord_uid: afqfymbq increasing age is the strongest predictor of risk of covid-19 severity. unregulated cytokine storm together with impaired immunometabolic response leads to highest mortality in elderly infected with sars-cov-2. to investigate how aging compromises defense against covid-19, we developed a model of natural murine beta coronavirus (mcov) infection with mouse hepatitis virus strain mhv-a59 (mcov-a59) that recapitulated majority of clinical hallmarks of covid-19. aged mcov-a59-infected mice have increased mortality and higher systemic inflammation in the heart, adipose tissue and hypothalamus, including neutrophilia and loss of γδ t cells in lungs. ketogenic diet increases beta-hydroxybutyrate, expands tissue protective γδ t cells, deactivates the inflammasome and decreases pathogenic monocytes in lungs of infected aged mice. these data underscore the value of mcov-a59 model to test mechanism and establishes harnessing of the ketogenic immunometabolic checkpoint as a potential treatment against covid-19 in the elderly. highlights natural mhv-a59 mouse coronavirus infection mimics covid-19 in elderly. aged infected mice have systemic inflammation and inflammasome activation murine beta coronavirus (mcov) infection results in loss of pulmonary γδ t cells. ketones protect aged mice from infection by reducing inflammation. etoc blurb elderly have the greatest risk of death from covid-19. here, ryu et al report an aging mouse model of coronavirus infection that recapitulates clinical hallmarks of covid-19 seen in elderly. the increased severity of infection in aged animals involved increased inflammasome activation and loss of γδ t cells that was corrected by ketogenic diet. aging-driven reduced resilience to infections is dependent in part on the restricted t cell repertoire diversity together with impaired t and b cell activation as well as inflammasomedriven low-grade systemic inflammation that compromises innate immune function (akbar and gilroy, 2020; camell et al., 2017; youm et al., 2013) . consequently, 80 percent of deaths due to in us are in adults > 65 years old (https://www.cdc.gov/) and aging is the strongest factor to increase infection fatality (pastor-barriuso et al., 2020; perez-saez et al., 2020; ward et al., 2020) . lack of an aging animal model that mimics sars-cov-2 immunopathology has been a major limitation in the effort to determine the mechanism of disease and to develop effective therapeutics for the elderly. inability of mouse ace2 to bind sars-cov-2 is a significant hurdle in understanding the basic mechanism of covid-19. accordingly, several approaches have been employed to develop models including introduction of human-ace2 in mice and transient induction of hace2 through adenoviral-associated vectors. these models have begun to yield important information on the mechanism of disease development. for example, epithelial cell specific induction of hace2 (k18-hace2) as a model of sars-cov-2 infection demonstrated that post intranasal inoculation, animals develop lung inflammation and pneumonia driven by infiltration of monocytes, neutrophils and t cells . also, initial studies that employ lung ciliated epithelial cell-specific hfh4/foxj1 promoter driven hace2 transgenic mice show sars-cov-2 infection induces weight loss, lung inflammation and approximately 50% mortality rate, suggesting the usefulness of this model to understand the mechanism of immune dysregulation (jiang et al., 2020) . however, significant hurdles remain to understand the mechanism and test therapeutic interventions that are relevant to disease severity in elderly, as complicated breeding and specific mutations need to be introduced in hace2 transgenic strains in addition to the time required to age these models. the mouse model of sars-cov-2 based on adeno-associated virus (aav)-mediated expression of hace2 may allow circumvention of the above constrains. the delivery of hace2 into the respiratory tract of c57bl/6 mice with aav9 causes a productive infection as revealed by >200 fold increase in sars-cov-2 rna and show similar interferon gene expression signatures as covid-19 patients . however, in young wild-type mice, this model induces mild acute respiratory distress syndrome (ards) and does not cause neutrophilia, weight loss or lethality . other studies using replication deficient adenovirus-mediated transduction of hace in mice and infection with sars-cov-2 produced 20% weight-loss including lung inflammation (hassan et al., 2020; sun et al., 2020) . furthermore, genetic remodeling of the sars-cov-2 spike receptor binding domain that allow interaction with mace demonstrated peribronchiolar lymphocytic inflammatory infiltrates and epithelial damage but no weight-loss in infected mice (dinnon et al., 2020) . moreover, middle aged female mice (one year old, analogous to approx. 43 year old human), display greater lung pathology and loss of function post infection with 10% weight-loss followed by spontaneous recovery 7 days post infection (dinnon et al., 2020) . however, it remains unclear if this model replicates the clinical, systemic inflammation and immunological response and mortality observed in covid-19. the mouse hepatitis virus (mhv) and sars-cov-2 are both ards-related beta coronaviruses with a high degree of homology (gorbalenya et al., 2020) . until the emergence of sars-cov-2, the natural infection with mhv mouse coronavirus-a59 (mcov-a59) has traditionally been thought to be of minor relevance to human disease and largely been a primary veterinary concern to maintain the specific-pathogen free status of mouse research facilities (hickman and thompson, 2004) . however, lack of an aging mouse model of necessitates re-evaluation of mcov-a59 to investigate the mechanism of multi-organ inflammation, morbidity and mortality caused by the disease. importantly, the mcov-a59 utilizes the entry receptor ceacam1, which is expressed on respiratory epithelium, but also on enterocytes, endothelial cells, and neurons, much like ace2 (godfraind et al., 1995) thus allowing the study of wide-ranging systemic impacts of infection. moreover, natural infection with mcov-a59 causes ards in c57bl/6j animals, while all other mhv strains require the a/j or type-i interferon-deficient background, for the development of severe disease (de albuquerque et al., 2006; khanolkar et al., 2009; yang et al., 2014) limiting their use. lastly, another major practical advantage of natural infection with mcov-a59 mouse model is that it does not require limited bsl3 facilities, thus allowing the allocation of precious world-wide bsl3 specialized laboratory space to be prioritized for human sars-cov-2 virus studies in primates and other models, which cannot be achieved by using mouse-adapted sars-cov-2 models. aging-induced chronic inflammation in the absence of overt infections is predominantly driven by the nlrp3 inflammasome (bauernfeind et al., 2016; camell et al., 2017; youm et al., 2013) , a myeloid cell-expressed multiprotein complex that senses pathogen associated molecular patterns (pamps) and danger associated molecular patterns (damps) to cause the processing and secretion of il-1β and il-18. there is increasing evidence that sars-cov-2 infection activates the nlrp3 inflammasome (siu et al., 2019) with increased levels of il-18 and lactate dehydrogenase (ldh) levels due to inflammasome mediated pyroptotic cell death (lucas et al., 2020; zhou et al., 2020) . it is now known that increased glycolysis, which activates inflammasome is associated with worsened covid-19 outcome (codo et al., 2020) . this raises the question whether the substrate switch from glycolysis-to-ketogenesis, can be employed to stave off covid-19 in high risk elderly population. here, we establish that intranasal infection with mcov-a59 recapitulates clinical features of covid-19 seen in elderly and demonstrate that ketone metabolites protect against disease through inhibition of nlrp3 inflammasome and expansion of protective γδ t cells in lungs. to determine the underlying deficits in immune and inflammatory response in aging, we investigated the impact of mcov-a59 intranasal inoculation on adult (2-6 month) and old male mice (20-24 month) ( figure 1a ). the ld-0 infectious dose of mcov-a59 in adult (pfu 7e3) caused 100 percent lethality in aged mice ( figure 1b ). compared to adults, the old infected mice displayed greater weight-loss ( figure 1c ), hypoxemia ( figure 1d ), and anorexia ( figure 1e ) without a significant difference in viral load in lungs ( figure s1a ). interestingly, aging led to a significant reduction in cd4, cd4:cd8 ratio ( figure 1f and figure s1b ) and ϒδ t cells ( figure 1g and figure s1c ) in lungs and spleen ( figure s1d ) with increased neutrophils ( figure 1h ), ly6c hi monocytes ( figure 1i and s1e) and no change in eosinophils ( figure s1f ). in addition, the lungs of old infected mice displayed increased frequency of cd64 + mertk + cells ( figure 1j and s1e) with no significant differences in the total population of alveolar or interstitial macrophages ( figure s1g and s1h). transmission electron microscopy confirmed the dissemination of the viral particles in pneumocytes in lungs ( figure 2a ). following mcov-a59 inoculation, ihc analyses by he and msb staining, in both 6 month and 20-24-month old mice, there is perivascular inflammation (arrows, arrowhead) as well as perivascular edema (*) and increased perivascular collagen/fibrosis (msb, blue) that is more severe in the 20-24-month mcov-a59 infected mice ( figure 2b ). further, 20-24 month old mice inoculated with mcov-a59 have dense foci visible at low power (box) and amphophilic material (fibrosis) with few scattered brightly eosinophilic erythrocytes (grey arrowhead) admixed with lymphocytes and plasma cells. by msb stain, at higher power this same focus (***) in the 20-24 month infected mice revealed that the end of a small blood vessel (bv) terminates in to a mass of collapsed alveoli, without obvious septa admixed with inflammatory cells, disorganized fibrin/collagen fibers (blue) suggestive of ante mortem pulmonary thrombosis in contrast to post mortem blood clots where erythrocytes are yellow (** msb, yellow) ( figure 2b ). taken together, consistent with ards, the lungs of aged mice infected with mcov-a59 had increased foci of inflammation, immune cell infiltration, perivascular edema, hyaline membrane formation and type ii pneumocyte hyperplasia, organizing pneumonia, interstitial pneumonitis, and occasional hemorrhage and microthrombi, affecting approximately 75% of the lungs ( figure 2b ). we next investigated whether mcov-a59 infection in aged mice mimics the hyperinflammatory systemic response seen in elderly patients infected with covid-19. compared to young animals, old mice infected with equivalent doses of mcov-a59 displayed significant increase in circulating il-1β, tnfα, il-6 ( figure 3a -c) and mcp-1 without affecting mip-1β ( figure s2a and s2b). similar to covid-19, the infection with mcov-a59 caused increased cardiac inflammation in old mice as evaluated by greater number of infiltrating cd68 + myeloid cells ( figure 3d ). given that increased visceral adiposity is a risk factor for covid-19 severity and expression of ace2 is upregulated in adipocytes of obese and diabetic patients infected with sars-cov-2 (kruglikov and scherer, 2020) , we next studied whether mcov-a59 infection affects adipose tissue. given the prevalence of obesity is 10% among younger adults aged 20-39, 45% among adults aged 40-59 years and 43% among older adults aged 60 and over (hales et al., 2020) we investigated adipose tissue inflammation as a potential mechanism that contributes to infection severity in the aged. interestingly, consistent with the prior findings that adipose tissue can harbor several viruses (damouche et al., 2015) , the mcov-a59 rna was detectable in vat ( figure 3e ). despite similar viral loads, vat of aged infected mice had significantly higher levels of the pro-inflammatory cytokines il-1β, tnfα and il-6 ( figure 3f -h). moreover, compared to young mice, old animals infected with mcov-a59 had increased caspase-1 cleavage (p20 active heterodimer), a marker of inflammasome activation ( figure 3i and s2c). in addition, similar to sars-cov-2 invasiveness in cns, the mcov-a59 was detectable in the hypothalamus ( figure 3j ). compared to adults, the hypothalamus of aged infected mice showed increased expression of tnfα and caspase-1 ( figure 3k and l) with no significant differences in il-1β, il-6 ( figure 3m and n) and nlrp3 ( figure s2d ). infection in both young and aged mice caused significant increases in markers of astrogliosis and microglia activation ( figure s2e and f). interestingly, mcov-a59 reduced the mrna expression of orexigenic neuropeptide-y (npy) ( figure s2e and f), consistent with the fact that infected mice display anorexia ( figure 1c and e). however, mcov-a59 infection completely abolished the expression of preopiomelanocortin (pomc) in the hypothalamus, a transcript expressed by pomc neurons, which is involved in the control of the autonomic nervous system and integrative physiology. therefore, further investigation will be necessary to test the involvement of the hypothalamus in the pathogenesis of covid-19 and organ failure due to alterations in autonomic nervous system. given the switch from glycolysis to fatty acid oxidation reprograms the myeloid cell from proinflammatory to tissue reparative phenotype during infections (ayres, 2020; buck et al., 2017; galván-peña and o'neill, 2014) , we next investigated whether mcov-a59-driven hyperinflammatory response in aging can be targeted through immunometabolic approaches. hepatic ketogenesis, a process downstream of lipolysis that converts long-chain fatty acids into short chain β-hydroxybutyrate (bhb) as a preferential fatty acid fuel during starvation or glucoprivic states, inhibits the nlrp3 inflammasome activation (youm et al., 2015) and protects against influenza infection induced mortality in mice (goldberg et al., 2019) . moreover, given our recent findings that ketogenesis inhibits inflammation and expands tissue resident ϒδ t cells (goldberg et al., 2019) while sars-cov-2 infection in patients is associated with depletion of ϒδ t cells (lei et al., 2020; rijkers et al., 2020) , we next tested whether elevating bhb by feeding a ketogenic diet (kd) protects against mcov-a59-driven inflammatory damage in aged mice. we infected bone marrow derived macrophages (bmdms) with mcov-a59 in vitro in tlr4 ( figure 4a and s3a) and tlr1/2 primed cells ( figure 4b and s3b). infection with mcov-a59 caused robust activation of inflammasome as measured by cleavage of active il-1β (p17) in bmdm supernatants ( figure 4a and b) as well as in cell lysates ( figure s3a and b). given our prior findings that ketone metabolites specifically inhibits the nlrp3 inflammasome in response to sterile damps such as atp, ceramides, silica and urate crystals (youm et al., 2015) , we next tested whether bhb impacts inflammasome activation caused by mcov-a59. interestingly, bhb treatment reduced pro and active cleaved il-1β (p17) in both conditions when protein level was measured in the supernatant ( figure 4a and b) and cell lysate ( figure s3a and b). mechanistically, post mcov-a59 infection, the bhb reduced the oligomerization of asc, which is an adaptor protein required for the assembly of the inflammasome complex ( figure 4c and d). this data provides evidence that the ketone metabolite bhb can lower inflammation in response to coronavirus infection and deactivate the inflammasome. however, inflammasome activation is also required for mounting adequate immune response against pathogens including certain viruses. therefore, we next investigated if induction of ketogenesis and ketolysis in vivo by feeding a diet rich in fat and low in carbohydrates that elevates bhb level impacts inflammasome and host defense against mcov-a59 infection in aged mice. ketogenesis is dependent on hydrolysis of triglycerides and conversion of long chain fatty acids in liver into short chain fatty acid bhb that serves as primary source of atp for heart and brain when glucose is limiting. aging is associated with impaired lipid metabolism which includes reduced lipolysis that generates free fatty acids that are essential substrates for bhb production. thus, it is unclear whether in context of severe infection and aging, if sufficient ketogenesis can be induced. to test this, the aged male mice (20-21 months old) were fed a kd or control diet for 5 days and then intranasally infected with mcov-a59 ( figure 4e ). despite mcov-a59's known effects in causing hepatic inflammation (navas et al., 2001) , we observed that compared to chow fed animals, old kd fed mice achieved mild physiological ketosis between 0.6 to 1mm over the course of infection for one week ( figure s3c ). compared to chow fed animals, the mcov-a59 infected mice fed kd displayed similar levels of food intake, blood glucose, core-body temperature, heart rate, and respiration ( figure s3d -i). interestingly, mcov-a59 infected kd fed mice were protected from infection-induced weight-loss and hypoxemia ( figure 4f and g). importantly, consistent with in vitro data, kd feeding caused significant reduction in inflammasome activation in the vat ( figure 4h ), a major source of inflammation in aging. together, these data show that kd lowers exuberant inflammasome activation in old mice and can potentially be therapeutically employed. the elderly covid-19 patients exhibit multi-organ failure with systemic viremia and inflammation. therefore, we next investigated the impact of kd on the inflammatory response in lungs, adipose tissue and hypothalamus in old mice post mcov-a59 infection. consistent with the improved clinical outcome and protection afforded by ketone bodies in infected mice, we found that kd-fed mice inoculated with mcov-a59 had significantly reduced expression of pro-inflammatory cytokines il-1β, tnfα and il-6 in lung, vat and hypothalamus ( figure 5a -c). aging and mcov-a59 increases inflammasome activation which is increasingly implicated in the pathogenesis of covid-19 (vijay et al., 2017; youm et al., 2013) . the sars-cov open reading frame 3a (orf3a) and orf8b activates the nlrp3 inflammasome (siu et al., 2019) by inducing er stress and lysosomal damage (shi et al., 2019) . moreover, ability of bats to harbor multiple viruses including coronaviruses is due to splicevariants in the lrr domain of nlrp3 which prevents inflammasome mediated inflammatory damage (ahn et al., 2019) . interestingly, in the aging mouse model of mcov-a59 infection, kd significantly lowered nlrp3 and caspase-1 mrna in lung, vat and hypothalamus ( figure 5d and e) and decreased myeloid cell infiltration in heart ( figure 5f ). the ketogenesis in infected old mice did not affect the frequency of cd4, cd8 effector memory or macrophage subsets in lungs suggesting that reduction in pro-inflammatory cytokines was not a reflection of reduced infiltration of these cell types ( figure s4 ). interestingly, we found that kd feeding rescued mcov-a59-induced depletion of ϒδ t cell in lungs of aged mice ( figure 5g and s4a). to determine the mechanism of ketogenesis-induced protection from mcov-a59 driven inflammatory damage in aging, we next investigated the transcriptional changes in lung at the single-cell level. the scrna sequencing of whole lung tissues ( figure 6a ) found that kd feeding in old infected mice caused significant increase in goblet cells ( figure 6b ), expansion of ϒδ t cells ( figure 6b ) and significant decrease in proliferative cell subsets and monocyte populations ( figure 6b ). comparison with scrna-seq of the lungs from young and old noninfected animals highlighted that only loss of proliferative myeloid cells was associated with the baseline aging process ( figure s5 ), while other age-specific changes in cellular subpopulations emerged as a result of interaction between virus and host. moreover, the old mice showed reduced interferon responses, suggesting increased vulnerability to the viral infection ( figure s5 ). interestingly, some of the most striking changes occurred in t cells, where ketogenesis led to a substantial increase in ϒδ but not αβ t cells ( figure 6c and figure s6b ). to understand whether expansion of ϒδ t cells was also accompanied by the changes in their regulatory programs, we sorted the lung ϒδ t cells from aged mice fed chow diet and kd and conducted bulk rna sequencing to determine the mechanism of potential tissue protective effects of these cells in mcov-a59 infection. we found that kd in aging significantly increased the genes associated with reduced inflammation ( figure 6d ), increased lipoprotein remodeling and downregulation of tlr signaling, plk1 and aurora b signaling pathways in ϒδ t cells ( figure 6e ). furthermore, rna sequencing revealed that lung ϒδ t cells from ketogenic mcov-a59 infected old mice displayed elevated respiratory electron transport and complex i biogenesis ( figure 6e ). in addition, golgi to er retrograde transport and cell cycle are downregulated, suggesting the reduced activation status of ϒδ t cell ( figure 6e ). this may indicate that ϒδ t cells expanded with kd are functionally more homeostatic and immune protective against mcov-a59 infection. zooming in into monocyte subpopulation we observed three distinct monocyte clusters ( figure 6f ), characterized by ifi44, lmna, and cd300e respectively ( figure 6g ). strikingly, ketogenesis-induced change in the monocyte compartment was driven by a loss of cluster 1 (characterized by high levels of chil3, lmna, il1r2, lcn2, cd33, cd24a, figure 6h and figure s6d and e). in addition, the loss of monocyte subpopulation was observed in cells with low interferon-response further suggesting the immune protective response induction post ketogenesis in infected mice. this is an intriguing finding that is consistent with recent observations that dietary interventions can impact plasticity of the monocyte pool in both mouse and human (collins et al., 2019; jordan et al., 2019) . immune-senescence exemplified by inflammasome-mediated basal activation of myeloid cells, expansion of pro-inflammatory aged b cells, impaired germinal center and antibody responses together with thymic demise and restriction of t cell repertoire diversity all contribute to increased risk of infections and vaccination failures in elderly (akbar and gilroy, 2020; frasca et al., 2020; goldberg and dixit, 2015; goronzy and weyand, 2019) . it is likely that multiple mechanisms partake in aging-induced mortality and morbidity to sars-cov-2. however, study of immunometabolic mechanisms that control aberrant inflammatory response in elderly covid-19 patients are hindered due to lack of availability of an aging mouse model of disease that recapitulates the key features of sars-cov-2 immunopathology and multi-organ inflammation. despite the severity of this viral infection, it is currently unclear what underlies the symptom diversity and the mortality of this pandemic. epidemiological data strongly support that elderly and aged individuals with late-onset chronic diseases-including diabetes, obesity, heart conditions, pulmonary dysfunctions and cancer-present a much higher disease severity compared to young healthy adults (cai et al., 2020; chen et al., 2020) . these observations suggest that it is the vulnerability of the various tissues that occur in these chronic conditions that predispose elderly to develop severe forms of covid-19. rodent covs are natural, highly contagious pathogens of mice and rats (compton et al., 2003; compton et al., 1993) . they are efficient and safe platforms for recapitulating and examining factors and interventions that impact disease. these models enable basic and translational covid-19 studies by minimizing studies requiring sars-cov-2 infection, thereby conserving and reserving limited bsl-3 space for studies using the most promising candidates in a homologous sars-cov-2 model. among the diverse rodent covs, mouse hepatitis virus (mhv) is a collection of mouse cov strains which have clinical diseases ranging from clinically silent (enterotropic) to mortality (polytropic/respiratory tropic). of particular interest for covid-19 are the strains of mhv that are respiratory tropic (yang et al., 2014) . given, these advantages, mhv mcov-a59 infection in c57bl/6 mice can be a powerful tool to rapidly study the disease as well as test therapeutic interventions. we demonstrate that compared to all reported models (dinnon et al., 2020; hassan et al., 2020; israelow et al., 2020; jiang et al., 2020; sun et al., 2020; winkler et al., 2020) , mhv mcov-a59 infection recapitulates severe features of covid-19 that includes, up to 30% weight-loss, sickness behavior exemplified by anorexia, loss of oxygen saturation, lung pathology including neutrophilia, monocytosis, loss of γδ t cells, lymphopenia, increase in circulating pro-inflammatory cytokines, hypothalamic, adipose and cardiac inflammation and inflammasome activation. importantly, ld0 dose of mhv mcov-a59 induces 100% lethality in 2 year old male mice, suggesting that this model allows investigation of covid-19 relevant immunometabolic mechanisms that control disease development and severity with aging. mechanistically, nlrp3 inflammasome has been demonstrated to be an important driver of aging-induced chronic inflammation and organ damage (bauernfeind et al., 2016; camell et al., 2017; youm et al., 2013) . covid-19 patients have inflammasome dependent pyroptosis and increase in il-18 (lucas et al., 2020; zhou et al., 2020) . consistent with the hypothesis that aging may exacerbate inflammasome activation in sars-cov-2 infection, our data demonstrates that in vivo, mcov infection increases nlrp3 inflammasome mediated inflammation. emerging evidence demonstrates that ability of bats to harbor multiple viruses including coronaviruses is due to splice-variants in the lrr domain of nlrp3 which prevents inflammasome mediated inflammatory damage (ahn et al., 2019) . furthermore, recent study shows that mhv-a59 also activates the nlrp3 inflammasome in vitro bone marrow derived macrophages . in addition severe cases of covid-19 are accompanied with dyregulation of monocyte populations with increased level of s100a8/a9 or calprotectin (schulte-schrepping et al., 2020; silvin et al., 2020) , which can prime and induce the inflammasome activation . these data underscore that enhanced innate immune tolerance mediated by inflammasome deactivation maybe an important strategy against covid-19. the integrated immunometabolic response (iimr) is critical in regulating the setpoint of protective versus pathogenic inflammatory response (lee and dixit, 2020). the iimr involves sensing of nutrient balance by neuronal (sympathetic and sensory innervation) and humoral signals (e.g. hormones and cytokines) between the cns and peripheral tissues that allow the host to prioritize storage and/or utilize substrates for tissue growth, maintenance and protective inflammatory responses. peripheral immune cells, both in circulation and those residing within tissues, are subject to regulation by the metabolic status of the host. ketone bodies, bhb and acetoacetate are produced during starvation to support the survival of host by serving as an alternative energy substrate when glycogen reserves are depleted (newman and verdin, 2017) . classically, ketone bodies are considered essential metabolic fuels for key tissues such as the brain and heart (puchalska and crawford, 2017; veech et al., 2017) . however, there is increasing evidence that immune cells can also be profoundly regulated by ketone bodies (youm et al, 2015 , goldberg et al 2020 . for example, stable isotope tracing revealed that macrophage oxidation of liver-derived acac was essential for protection against liver fibrosis (puchalska et al., 2019) . given our past findings that ketone bodies inhibit nlrp3 inflammasome activation induced by sterile damps, we next hypothesized that coronavirus mediated inflammasome activation and disease severity in aging could be improved by bhb driven improved metabolic efficiency and nlrp3 deactivation. in support of this hypothesis, we found that bhb inhibits the mcov-a59 induced nlrp3 inflammasome assembly and kd reduces caspase-1 cleavage as well as decreases gene expression of inflammasome components. we next investigated the mechanism of protection elicited by kd that is relevant to aging. interestingly, scrna sequencing analyses of lung homogenates of old mice fed kd revealed robust expansion of immunoprotective γδ t cells, which are reported to decline in covid-19 patients (lei et al., 2020; rijkers et al., 2020) . the kd activated the mitochondrial function as evidenced by enhanced complex-1 biogenesis and upregulation of etc in immunoprotective γδ t cells. moreover, the kd feeding blocked infiltration of pathogenic monocyte subset in lungs that has high s100a8/9 and low interferon expression. our data shows that the mcov-a59 murine model offers an efficient and biosafety level-2 platform for recapitulating covid-19 to test mechanism of age-related immune decline and can thereby fast-track testing of interventions that impact disease outcome. our findings assumes strong clinical significance as recent studies, demonstrate that γδ t cells were severely depleted in covid-19 patients in two highly variable cohorts and disease progression was correlated with near ablation of vγ9vδ2 cells that are dominant subtype of circulating γδ t cells (laing et al., 2020) . taken together these data demonstrate that a ketogenic immunometabolic switch protects against mcov-a59 driven covid in mice and this anti-inflammatory response in lung is coupled with reduction of inflammasome activation, restoration of protective ϒδ t cells and remodeling of the pool of the inflammatory monocytes. finally, our results suggest that acutely switching infected or at-risk elderly patients to a kd may ameliorate covid-19 and, therefore, is a relatively accessible and affordable intervention that can be promptly applied in most clinical settings. mouse remains imperfect to model human biology and disease. instead of following the current approaches to make human sars-cov-2 amenable to infecting an unnatural murine host with unknown biological compatibility, we focused on natural mouse hepatitis virus (mhv)-a59 because like sars-cov-2, it belongs to the family of ards-related beta coronaviruses that are highly homologous. however, the obvious limitation of the model is that mhv-a59 is not sars-cov-2 virus. although, both these beta coronaviruses display high degree of homology, mhv-a59 uses ceacam1 instead of ace2 for binding and infectivity. however, cellular expression of ceacam1 is similar to ace2 in humans and similarity of viral orfs offer significant advantages in studying tissue responses. like most models of disease, this study shows that not all features of sars-cov-2 infection seen in humans are seen in mice. this includes lack of development of fever instead mice become hypothermic. also, despite monocyte infiltration in heart, the aged mice did not die due to cardiac failure and displayed normal heart rate. mcov-a59 virus induced hypothalamic inflammation and led to anorexia that included reduction in orexigenic npy but almost complete loss of pomc gene expression. it remains unclear, if this alters pomc derived peptides including melanocortins and endogenous opioids peptides, in addition these data suggest potential dysregulation of autonomic nervous system which can play a role in organ failure post infection. in terms of mechanism, our data shows that the inflammasome is activated in infection and kd-induced protection is associated with nlrp3 inflammasome deactivation. future studies are required to test if aged nlrp3 deficient mice are protected from mcov-a59 or if absence of γδ t cell increases mortality. aw and vdd conceived the project and helped with data interpretation and manuscript preparation. the authors declare they have no competing interests. fed chow (old-chow, n=6) or ketogenic diet (old-kd, n=5). the mice were provided with diet from 5 days before infection. after infection, the phenotype was evaluated until 7 days post infection. weight change (%) (f), and % o2 saturation (g) in old mice fed chow or kd. (g) western blot analysis of caspase-1 inflammasome activation in vat of infected old-chow and old-kd mice. error bars represent the mean ± s.e.m. two-tailed unpaired t-tests were performed for statistical analysis. * p < 0.05; ** p < 0.01. further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, vishwa deep dixit (vishwa.dixit@yale.edu). this study did not generate new unique reagents. the single cell rna-sequencing and bulk rna-sequencing data has been uploaded to gene expression omnibus (gse155346 and gse155347) respectively. all mice used in this study were c57bl/6 mice. old mice (20-24 month old) were received from nia, maintained in our laboratory. young mice (2-6 month old) were from nia or purchased from jackson laboratories or bred in our laboratory. the mice were housed in specific pathogenfree facilities with free access to sterile water through yale animal resources center. mice were fed a standard vivarium chow (harlan 2018s) or a ketogenic diet (envigo, td.190049) for indicated time. the mice were housed under 12 h light/dark cycles. all experiments and animal use were conducted in compliance with the national institute of health guide for the care and use of laboratory animals and were approved by the institutional animal care and use committee (iacuc) at yale university. mhv-a59 was purchased from bei resources (nr-43000) and grown in bv2 cells. mice were anesthetized by intraperitoneal injection of ketamine/xylazine. 700 or 7000 pfu of mhv-a59 was delivered in 40ul pbs via intranasal inoculation. vital signs were measured before and after infection. arterial oxygen saturation, breath rate, heart rate, and pulse distention were measured in conscious, unrestrained mice via pulse oximetry using the mouseox plus (starr life sciences corp.). lungs were fixed in 10% formaldehyde, osmicated in 1% osmium tetroxide, and dehydrated in ethanol. during dehydration, 1% uranyl acetate was added to the 70% ethanol to enhance ultrastructural membrane contrast. after dehydration, the lungs were embedded in durcupan and ultrathin sections were cut on a leica ultra-microtome, collected on formvar-coated single-slot grids, and analyzed with a tecnai 12 biotwin electron microscope (fei). h&e and msb staining of lung tissues were performed on sections of formalin-fixed paraffinembedded at the comparative pathology research core at yale school of medicine. for immunohistochemistry, the hearts were harvested from mhv-a59 infected mice, fixed in 4% pfa overnight and embedded in oct after dehydration with 30% sucrose and serial sections of aortic root were cut at 6 μm thickness using a cryostat. sections were incubated at 4°c overnight with cd68 (serotec; #mca1957) and alexa fluor™ 594 phalloidin (thermofisher, a12381) after blocking with blocker buffer (5% donkey serum, 0.5% bsa, 0.3% triton x-100 in pbs) for 1 hour at rt, followed by incubation with alexa fluor secondary antibody (invitrogen, carlsbad, ca) for 1 hour at rt. the stained sections were captured using a carl zeiss scanning microscope axiovert 200m imaging system and images were digitized under constant exposure time, gain, and offset. results are expressed as the percent of the total plaque area stained measured with the image j software (imagej version 1.51). plaque assay l2 cell (1.5 ml of 6x10 5 cells/ml) were seeded on 6 well plates (corning, 3516) in supplemented dmem and allowed to adhere overnight. tissue samples were homogenized in unsupplemented dmem, and spun down at 2000 rpm for 5 min. supernatant was serially diluted and 200 μl of each sample was added to aspirated l2 cells in 6 well plates. plates were agitated regularly for 1 hour before adding overlay media consisting of 1 part 1.2% avicel and 1 part 2x dmem (thermo fisher, 12800) supplemented with 4% fbs (thermo fisher, a3840301), penicillin-streptomycin (gibco, 15140122), mem non-essential amino acids solution (gibco, 11140050), and hepes (gibco, 15630080). after a four-day incubation, cells were fixed in 10% formaldehyde (sigma aldrich, 8187081000) diluted with pbs for 1 hour. cells were then stained in 1% (w/v) crystal violet (sigma aldrich, c0775) for 1 hour, washed once in distilled water, and then quantified for plaque formations. serum cytokine and chemokine level was measured by procartaplex multiplex assay (thermo fisher scientific). assay was prepared following manufacture's instruction. 25 µl of collected serum from each mice in this study was used. customized assay including il-1β, tnf , il-6, mcp-1, and mip-1β was used. luminex xponent system was used to perform the assay. to extract and purify rna from tissues, rneasy plus micro kit (qiagen) and direct-zol™ rna miniprep plus kit (zymo research) were used according to manufacturer's instructions. cdna was synthesized with isolated rna using iscript cdna synthesis kit (bio-rad). to quantify amount of mrna, real time quantitative pcr (qpcr) was done with the synthesized cdna, gene specific primers, and power sybr green detection reagent (thermo fischer scientific) using the lightcycler 480 ii (roche). analysis was done by ddct method with measured values from specific genes, the values were normalized with gaphd gene as an endogenous control. bone marrow derived macrophage was cultured by collecting mouse femurs and tibias in complete collecting media containing rpmi (thermo fischer scientific), 10% fbs (omega scientific), and 1% antibiotics/antimycotic (thermo fischer scientific). using needle and syringe, bone marrow was flushed into new complete media, followed by red blood cells lysis by ack lyses buffer (quality biological). in 6 well plate, the collected cells were seeded to be differentiated into macrophages incubated with 10 ng/ml m-csf (r&d) and l929 (atcc) conditioned media. cells were harvested on day 7, and seeded as 1x10 6 cell/well in 24 well plate for experiments. to infect bmdm, mhv-a59 was incubated with bmdm as a moi 1 (1:1) for 24 hour. for inflammasome activation, lps (1ug/ml) or pam3csk4 (1ug/ml) were pre-treated with or without bhb (1, 5, 10 mm) for 4 hour before mhv-a59 infection for 24 hour or atp (5mm) treatment for 1 hour. to prepare samples for western blotting, tissues were snap frozen in liquid nitrogen. ripa buffer with protease inhibitors were used to homogenize the tissues. after cell supernatant was collected, cells were harvested by directly adding ripa buffer on cell culture plate. after quantification of protein amount by the dc protein assay (bio-rad), same amount of protein was run on sds-page gel followed by transferring to nitrocellulose membrane. specific primary antibodies and appropriate secondary antibodies (thermo fisher scientific) were used to probe blots and bands were detected by ecl western blotting substrate (pierce). the following primary antibodies were used for experiments. antibodies to caspase-1 (1:250, genentech), βactin (1:1,000, 4967l; cell signaling), il-1β (1:1000, gtx74034, genetex), and asc (1:1000, ag-25b-0006, adipogen) were used. to detect asc oligomers, cells were harvested in np-40 lysis buffer which contains 20mm hepes-koh (ph 7.5), 150 mm kcl, 1% np-40, 0.1 mm pmsf, and protease inhibitors. the cells in lysis buffer were incubated on ice for 15 min, and centrifuged at 6,000 rpm at 4°c for 10 min. supernatant was collected and kept for cell lysate western blotting. the pellet was vortexed with 1 ml of np-40 lysis buffer, and centrifuged at 6,000 rpm at 4°c for 10 min. the pellet was incubated with 50ul of np-40 lysis buffer and 1 ul of 200 mm dss (disuccinimidyl suberate) for 30 min at room temperature, then centrifuged at 6,000 rpm at 4°c for 10 min. the pellet with sds sample buffer and reducing reagent was loaded for western blotting. lung was digested in rpmi (thermo fisher) with 0.5mg/ml collagenase i (worthington) and 0.2mg/ml dnase i (roche) for 1 hour. digested lung tissues were minced through 100 μm strainer. spleen was directly minced through 100 μm strainer. minced tissues were additionally filtered with 40 μm strainer after red blood cell lysis by ack lysing buffer (quality biological). after incubation with fc block cd16/32 antibodies (thermo fisher scientific), the cells from lung and spleen were further incubated with surface antibodies for 30 min on ice in the dark. washed cells were stained with live/dead™ fixable aqua dead cell stain kit (thermo fisher scientific). bd lsrii was used for flow cytometry and results were analyzed by flowjo software. the following antibodies were used for flow cytometry analysis to detect cd4 t cell, cd8 t cell, γδ t cell, neutrophil, eosinophil, and macrophage: cd45-bv711, mertk-fitc, cd64-bv605, f4/80-efluor450, ly6c-percp-cy5.5, cd11c-apc, cd169-pe, cd86-pe-cy7, cd3-bv605, cd4-pe-cy7, cd8-efluor450, tcr γ/δ-pe, ly6g-apc, siglecf-percp-cy5.5, cd62l-percp-cy5.5, cd44-apc-cy7. lung cells were prepared as mentioned above for flow cytometry and equal amount of cells were pooled as indicted in the experiments. single-cell rna sequencing libraries were prepared at yale center for genome analysis following manufacturer's instruction (10x genomics). novaseq6000 was used for sequencing library read. the cell ranger single-cell software suite (v3.0.2) (available at https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-iscell-ranger) was used to perform sample demultiplexing, barcode processing, and single-cell 3' counting. cellranger mkfastq was used to demultiplex raw base call files from the novaseq6000 sequencer into sample-specific fastq files. subsequently, fastq files for each sample were processed with cellranger counts to align reads to the mouse reference (version mm10-3.0.0) with default parameters. for the analysis, the r (v3.5.0) package seurat (v3.1.1) (butler et al., 2018) was used. cell ranger filtered genes by barcode expression matrices were used as analysis inputs. samples were pooled together using the merge function. the fraction of mitochondrial genes was calculated for every cell, and cells with high (>5%) mitochondrial fraction were filtered out. expression measurements for each cell were normalized by total expression and then scaled to 10,000, after that log normalization was performed (normalizedata function). two sources of unwanted variation: umi counts and fraction of mitochondrial reads -were removed with scaledata function. for both datasets platelet clusters as well as a cluster of degraded cells (no specific signature and low umi count) were removed and data was re-normalized without them. in case of old-keto and old-chow dataset we additionally removed neutrophils, doublets, and red blood cells. the most variable genes were detected using the findvariablegenes function. pca was run only using these genes. cells are represented with umap (uniform manifold approximation and projection) plots. we applied runumap function to normalized data, using first 15 pca components. for clustering, we used functions findneighbors and findclusters that implement snn (shared nearest neighbor) modularity optimization-based clustering algorithm on top 20 pca components using resolution of 0.3 for both datasets. to identify marker genes, findallmarkers function was used with likelihood-ratio test for single cell gene expression. for each cluster, only genes that were expressed in more than 10% of cells with at least 0.1-fold difference (log-scale) were considered. for heatmap representation, mean expression of markers inside each cluster calculated by averageexpression function was used. single cell rna-seq differential expression to obtain differential expression between clusters, mast test was performed via findmarkers function on genes expressed in at least 1% of cells in both sets of cells, and p-value adjustment was done using a bonferroni correction (finak et al., 2015) . pathway analysis was performed using clusterprofiler package (v3.12.0) (yu et al., 2012) with hallmark gene sets from msigdb. significantly different genes were used (padj < 0.05) if percent difference between conditions (|pct.1 -pct.2|) was over 1%. to visualize pathway expression for each cell z-scores of all pathway genes were averaged. to separate ab t cells and gs t cells we subset raw values of t cell (cluster 2), normalized and clustered corresponding data separately as described above with clustering resolution 0.2. obtained subclusters were projected on the original umap, splitting cluster 2 in 2_1 (ab t cells) and 2_2 (gs t cells). monocyte clusters 0 and 1 were subset and re-analyzed in the same manner with clustering resolution 0.3. umap was recalculated for monocytes only as shown in figure 6f . bulk rna sequencing of sorted gδ t cells cells from lung were prepared as mentioned above for flow cytometry and γδ t cell was sorted by flow cytometry (live cd45+ cd3+ cd4-cd8-tcr γ/δ+). rna was isolated from sorted cells using rneasy plus micro kit (qiagen). quality checked rna was used for rna sequencing library preparation at yale center for genome analysis following manufacturer's instruction (illumina). novaseq6000 was used for sequencing library read. fastq files for each sample were aligned to the mm10 genome (gencode, release m25) using star (v2.7.3a) with the following parameters: star --genomedir $genome_dir --readfilesin $work_dir/$file_1 $work_dir/$file_2 --runthreadn 12 --readfilescommand zcat --outfiltermultimapnmax 15 --outfiltermismatchnmax 6 --outreadsunmapped fastx --outsamstrandfield intronmotif --outsamtype bam sortedbycoordinate --outfilenameprefix ./$ (dobin et al., 2013) . quality control was performed by fastqc (v0.11.8), multiqc (v1.9) (ewels et al., 2016) , and picard tools (v2.21.6). quantification was done using htseq-count function from htseq framework (v0.11.2): htseqcount -f bam -r pos -s no -t exon $bam $annotation > $output . differential expression analysis was done using deseq function from deseq2 package (love et al., 2014) (v1.24 .0) with default settings. significance threshold was set to adjusted p-value < 0.05. gene set enrichment analysis via fgsea r package (sergushichev, 2016) (v1.10 .0) was used to identify enriched pathways and plot enrichment curves. to calculate statistical significance, two-tailed student's t test was used. level of significance was indicated as follow. *p < 0.05; **p < 0.005; ***p < 0.001; ****p < 0.0001 respectively. all statistical tests used 95% confidence interval and normal distribution of data was assumed. biological replication numbers for each experiment were indicated in each figure and figure legend. data were shown as mean ± s.e.m. graphpad prism software was used for all statistical tests to analyze experimental results. expression analysis in hypothalamus of young uninfected and young infected mice (e), and old uninfected and old infected mice (f). error bars represent the mean ± s.e.m. two-tailed unpaired t-tests were performed for statistical analysis. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. figure s5a split by sample. color represents expression of mki67. (e) summary of cluster-by-cluster differential expression comparison of young and old samples. each dot represents a gene, significant genes are shown in red. (f) gene set enrichment analysis of significantly up-and down-regulated genes described in figure s5e. (g, h) umap as in figure s5a split by sample. color shows average z-scores of genes in selected pathways. figure 6c . color represents expression of trac. (c) summary of cluster-by-cluster differential expression comparison of old-kd and old-chow. each dot represents a gene, significant genes are shown in red. (d) percentage of each monocyte subset as identified in figure 6f relative to total number of monocytes in the corresponding sample. 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sepsis-like traits associated with poor prognosis. medrxiv the phenotypic changes of γδ t cells in covid-19 patients. medrxiv moderated estimation of fold change and dispersion for rna-seq data with deseq2 longitudinal analyses reveal immunological misfiring in severe covid-19 murine coronavirus spike protein determines the ability of the virus to replicate in the liver and cause hepatitis hydroxybutyrate: a signaling metabolite. annual review of nutrition sars-cov-2 infection fatality risk in a nationwide seroepidemiological study. medrxiv serology-informed estimates of sars-cov-2 infection fatality risk in multi-dimensional roles of ketone bodies in fuel metabolism, signaling, and therapeutics hepatocyte-macrophage acetoacetate shuttle protects against tissue fibrosis more bricks in the wall against sars-cov-2 infection: involvement of γ9δ2 t cells severe covid-19 is marked by a dysregulated myeloid cell compartment an algorithm for fast preranked gene set enrichment analysis using cumulative statistic calculation. biorxiv sars-coronavirus open reading frame-8b triggers intracellular stress pathways and activates nlrp3 inflammasomes elevated calprotectin and abnormal myeloid cell severe acute respiratory syndrome coronavirus orf3a protein activates the nlrp3 inflammasome by promoting traf3-dependent ubiquitination of asc generation of a broadly useful model for covid-19 pathogenesis, vaccination, and treatment virus-induced inflammasome activation is suppressed by prostaglandin d<sub>2</sub>/dp1 signaling antibody prevalence for sars-cov-2 in england following first peak of the pandemic: react2 study in 100,000 adults. medrxiv sars-cov-2 infection of human ace2-transgenic mice causes severe lung inflammation and impaired function coronavirus mhv-a59 infects the lung and causes severe pneumonia in c57bl/6 mice the ketone metabolite β-hydroxybutyrate blocks nlrp3 inflammasome-mediated inflammatory disease canonical nlrp3 inflammasome links systemic low-grade inflammation to functional decline in aging clusterprofiler: an r package for comparing biological themes among gene clusters impaired nlrp3 inflammasome activation/pyroptosis leads to robust inflammatory cell death via caspase-8/ripk3 during coronavirus infection clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study key: cord-298117-9ycl7mn6 authors: monk, caroline title: ocular surface disease in rodents (guinea pigs, mice, rats, chinchillas) date: 2018-11-17 journal: vet clin north am exot anim pract doi: 10.1016/j.cvex.2018.08.001 sha: doc_id: 298117 cord_uid: 9ycl7mn6 this article discusses the clinical appearance, differential diagnoses, and treatment considerations of corneal disease in the most common domesticated species of rodent: mouse, rat, chinchilla, and guinea pig. many corneal diseases are related to inbred strains of either research or pet rodents. diseases are complicated by husbandry and treatment-related challenges in this small, social species. this article is broken down by species, first discussing normal anatomy, then discussing commonly encountered diseases, and concluding with treatment considerations. attending veterinarian. mice, rats, and chinchillas are thought to be predominantly nocturnal to crepuscular, while guinea pigs are diurnal. their eye anatomy reflects their origins. all rodents have a cornea divided into stratified epithelium, a bowman layer (epithelial basement membrane), stroma, descemet membrane, and the endothelium. guinea pigs are the largest of the rodents covered in this section. they live an average of 4 to 5 years, but can live as long as 8 years. they are not found in the wild, but are a domesticated relative of other species of cavies. once used frequently for research studies, they are now primarily a household pet. fine superficial corneal neovascularization (usually extending from limbus axially to the first or second third of the cornea) has been reported as a normal finding in fetal, newborn, and adult guinea pigs. 1 however, a later confocal microscopy study did not confirm this. 2 small palpebral conjunctival outpouchings were noted in the upper and lower fornices in almost all examined animals according to dwyer and colleagues (1983) , and were determined via histology to be masses of lymphoid tissue. their prevalence across individuals resulted in researchers concluding these foci to be a normal finding. guinea pigs have a remarkably low corneal sensitivity (or a high pressure threshold is needed to elicit a blink), replicated in 3 studies. 1, 3, 4 in these studies, the guinea pig was observed to have limited to no reflex tearing, which may be a component of the lack of corneal sensitivity ( fig. 1, table 1 ). this plays an important clinical role. staphylococcus epidermidis, a-hemolytic streptococcus, and corynebacterium were the top 3 aerobic commensal isolates from normal healthy guinea pigs. 4 trichiasis, or hair from the skin contacting the cornea, occurs in a wire-haired breed of guinea pigs, texel cavies; 0.8% of surveyed animals had this congenital abnormality. 11 after birth the wiry hair can curl inwards into the eye, causing corneal ulcers and epiphora. lubricating the eyes and hair around frequently immediately after birth until the hair grows in a more controlled manner can help. entropion, either primary or cicatrial after an eyelid trauma, is reported in guinea pigs. entropion is typically treated via a variety of blepharoplasties. to the author's knowledge, blepharoplasty in a guinea pig has not been reported. because of their small adnexal structures, they may be more suited to a hyaluronate injection that can evert the lids semipermanently or to frequent lubrication of the cornea to mitigate pathology. to the author's knowledge, guinea pigs are the only species discussed in this article formally reported to exhibit periocular dermoids. 12 large corneal dermoids can compromise vision by their opacification of the visual axis. haired dermoids may contaminate the conjunctival sac by harboring foreign material and debris. the treatment of choice for dermoids is surgical removal via keratectomy, but given the thinness of the guinea pig cornea ( table 2) , this treatment should be undertaken with great care. corneal lipidosis (bilateral stromal lipid dystrophy) is also reported. 11 this has been described as a bilateral paracentral lipid deposition with varying degrees of density and coverage. this appears to be less common than the corneal dystrophy reported in mice and rats. as previously discussed, guinea pigs have been found to have a low corneal sensitivity and a negligible ability to produce reflex tears, thereby placing them at an unusual risk for ocular foreign bodies. they have also been noted to have an unusually low blink rate. 2, 5 this is compounded by their typical housing, in a bedded enclosure with straw and hay at eye level. in a survey of 1000 guinea pigs, 4.7% had conjunctivitis, which was frequently secondary to a traumatic injury from a foreign body. 11 this had also been seen in a previous survey of guinea pigs. 1 it has been theorized that the corneal vessels and lymphoid tissue unique to guinea pigs eyes were an evolved strategy over increased blinking and discomfort seen in other animals. that being said, foreign bodies should still be taken seriously and treated promptly. per williams and sullivan (2010), these foreign bodies, along with congenital trichiasis, seemed to be the most irritating for the animals. guinea pigs have lost their ability to endogenously form ascorbic acid, like people and capybaras, and are therefore at greater risk for scurvy relative to other species of rodent. scurvy often starts with mucous membrane disease including petechiation and ulceration. dry eye has also been reported as a sequela. vitamin c is used in other species as an anticollagenase. 18 therefore, supplementation during times of ulceration, especially a melting ulcer (fig. 2) , is likely to be beneficial regardless of nutritional status. many foods are rich in vitamin c, and there are also nutritional supplements available. because guinea pigs are the largest of the species discussed here, topical treatment is more feasible in terms of administration. systemic absorption is still a risk, and so prolonged treatment, especially with topical steroids and nonsteroidal antiinflammatory drugs, should be approached cautiously. guinea pigs are typically docile and amenable to handling. given their common place as a household pet, individualized treatment including frequent topical medication is more achievable. mice are the smallest rodent discussed in this section. they are one of the most successful mammals on earth today and are viewed as pets, research subjects, vermin, and vectors. they are the most common experimental laboratory animal in recent decades because of their homology with people, small size, and rapid reproduction. breeding onset is about 50 days, and the life span is typically 1 to 2 years when kept as pet. a detailed comparative study was performed using confocal microscopy of normal 4-month-old swiss mice. 15 bowman layer was observed between basal epithelial cells and the anterior stroma. similar to rats, the anterior and posterior stroma had numerous anuclear reflective stellate structures. endothelial cell density was also determined (see table 2 ). just like guinea pigs, corneal vascularization has been reported to be a normal finding in 2 mouse strains -athymic and euthymic nude mice. 19 schirmer tear test with traditional whatman filter paper is not possible in the species based on size, but normal tear volume can be assessed via phenol red thread testing (see table 1 ). 6 no article on mice would be complete without touching on their significance as a research model. mice are models for certain ocular surface diseases, most notably sjö gren syndrome (ss), a common autoimmune dry eye syndrome in people. mice exhibiting ss-like characteristics have a mononuclear cell infiltration into exocrine glands, loss of acinar tissue, and secretory dysfunction. corneal pathology includes corneal vascularization, keratinization, and the propensity for bacterial corneal ulcers (fig. 3) . numerous murine strains have been used for their ss-like disease manifestations, and an excellent review article summarizing the various models has been published. 20 initially these characteristics were spontaneously arising, but later the alterations were associated with gene knockout, resulting in transgenic mice that model aspects of the disease. mice have helped make a breakthrough in the link between primary open-angle glaucoma and the thickness of the central cornea. mice were recently used to identify a transcription factor, pou6f2, that is associated with central corneal thickness and susceptibility of retinal ganglion cells to injury. 21 corneal deposits are a common finding in laboratory mice (fig. 4) . deposition of calcium in bowman layer with or without accompanying vascularization has been reported in both normal and ss-model mice (mrl/mp strain). 22 it is speculated that these deposits are genetically linked, but excess ammonia in cage bedding may also contribute. 23 after investigating husbandry, treatment of the deposits is not performed. peter anomaly, a form of anterior segment dysgenesis, has been documented spontaneously mice and is being used as a model for the disease. 24, 25 the keratolenticular adhesion results in the presence of a leukoma (corneal opacity) of varying size. corneal transplant is the treatment in people, but even then overall visual prognosis is poor. 26 this treatment has not been reported in veterinary patients. additionally, intraocular pressure should be assessed, as aggressive glaucoma often accompanies these changes. 24 fig. 4 . severe example of corneal dystrophy in a laboratory mouse. this individual was considered comfortable (no blepharospasm) and clinically acceptable as a research subject. the primary limiting factor in treatment of mice is their small size. corneal transplants have been performed successfully, but the success rate of these surgeries has not been published, as most studies focus on graft rejection, excluding those leaking or infected grafts from statistical analysis. 27, 28 in veterinary medicine, conjunctival grafts are more commonly performed relative to clear corneal transplants, but to the author's knowledge, this surgical technique has not been reported in any the species covered in this article. the small size of the mouse also means many topical treatments have the potential for significant systemic absorption. even the application of a topical sodium channel blocker to facilitate examination should be applied with judiciousness given its potential for systemic toxicity. 29 rats and mice are closely related and only differentiated by their size, not specific taxonomic criteria. this section focuses on the most common rat species, rattus norvegicus (brown rat and laboratory rat), rattus rattus (black rat), and fancy rat (rattus norvegicus). life span is typically 2 years when kept as a pet, but they can live up to 3 years. rats and mice have 3 tear-producing glands, the intraorbital, the extraorbital, and the harderian. the harderian is a well-studied exocrine gland associated with the third eyelid. this is what produces the porphyrin and lipid-laden tears in rats and is of clinical significance because of the vivid, visible tears it produces in several diseases. 30 tear staining or chromodacryorrhea refers to a dark stain below the inner corner of the eye, caused by porphyrin-pigmented secretion from the harderian gland. it indicates stress in rats but can have other indirect causes. chromodacryorrhea was produced within 15 minutes in young rats following intravenous injection of acetylcholine or acute stress induced by limb restraint. 31 by a similar mechanism, the presence of chromodacryorrhea was even identified as a welfare indicator on commercial pig farms. 32 this finding may gain more attention in the laboratory animal sphere, where welfare is paramount. the presence of chromodacryorrhea should not be ignored, as it is not a normal finding. as discussed, it may be caused by environmental stress, physical illness, or underlying disease. furthermore, even when not hypersecreted, porphyrins are labile until photic energy. exposure to high-intensity light induced necrosis of the glandular cells in a study on a research population of wistar rats. 33 the injury appeared to be caused by the creation of free radicals within glandular cells, probably as a result of photodynamic action on the porphyrins in the gland. proper husbandry with a day-night cycle is essential for rat health and welfare. certain strains of rat are reported to have a high rate of subepithelial mineralization (corneal dystrophy), just like mice. clinically, these opacities are subepithelial (associated with abnormal epithelial basement membrane, bowman layer). they vary from a few punctate opacities only visible with a slit lamp to marked dense opacities covering a majority of the corneal surface (fig. 5) . a thorough ophthalmologic and histopathological study was performed on fischer-344 (f344) rats that demonstrated a high incidence of corneal basement membrane dystrophy. 34 in the most severely affected ocular surface disease in rodents strains, this correlated to a systemic basement membrane disorder. it was speculated that these opacities are relatively under-reported for several reasons, and in this study anywhere from 50% to 100% of rats examined from various breeders had corneal dystrophy. thankfully, these opacifications did not appear to cause the animals discomfort or have an adverse effect on normal physiologic function. however, in people, epithelial basement membrane dystrophy is associated with recurrent corneal erosions. 35 f344 rat strains are also unusually prone to intraocular tumor, which may manifest as a pigmented opacity in the cornea. orbital malignant schwannomas and amelanotic melanoma are reported. 36 sialodacryoadenitis (sda) is a highly contagious common viral infection in rats. sda is caused by rat coronavirus and can spread rapidly, especially through laboratory colonies. anorexia occurs during these viral infections. the virus has a tropism for epithelial cells and can infect the harderian and extraorbital glands, causing ocular disease. 37 often, lacrimal gland involvement leads to reduced tear production. young rats are especially susceptible to sda, and the infection can occur in the lower respiratory tract, resulting in pneumonia. thankfully, the primary disease is usually selflimiting and resolves within a week; however, secondary signs may take up to a month to fully resolve. diagnosis is confirmed by detecting coronavirus antigen with reverse transcriptase polymerase chain reaction (rt-pcr) 38 or serologic testing. 39 treatment limitations are similar between rats and mice. their small size and colony habitat often preclude topical treatment. of the species covered, chinchillas are the most recently domesticated and the least studied. they are also the longest lived in this group of rodents, living on average 10 years in captivity, although chinchillas living into their 20s have been reported. 10 chinchillas were originally bred for fur but since have become pets and are used in scientific research. of the species discussed here, chinchillas have the most recent literature regarding presentation as a pet for ocular examination, and less research-based publications relating to their ocular biology. in a recent retrospective study over the course of 10 years, 7.8% of chinchillas presenting to a tertiary clinic had primary ophthalmic complaints. 10 chinchillas are characterized as having a shallow orbit, and proptosis can easily be induced with pressure on the eyelids; therefore, care must be taken when examining them. 40 chinchilla endothelial cell density has been determined via specular microscopy (see table 2 ). 17 like in other species, cell density decreases and pleomorphism increases with age. of the available publications and consensus among zoo veterinarians, ocular discharge appears to be the most common condition. the discharge/epiphora is suspected to be secondary to dental disease, as in all rodents, chinchillas are hypsodonts with continually growing teeth. this continual growth is compounded by their longevity and potential for inappropriate husbandry. 41 root extension into the nasolacrimal duct and subsequent epiphora is the most common clinical sign. chinchillas with only clear epiphora are typically considered primary dental disease patients. 10 bacteria normally populate the conjunctival fornix. in chinchillas, 9 lima and colleagues (2010) found that streptococcus species (27.45%) was most commonly isolated, followed by staphylococcus aureus (23.52%), and finally coagulase-negative staphylococcus (19.60%). this is in comparison to 42 ozawa and colleagues (2017), who studied chinchillas affected by bacterial conjunctivitis. in diseased individuals, 61.5% yielded a gram-negative isolate (50% being pseudomonas aeruginosa). the remainder yielded gram-positive isolates, staphylococcus species being most common (26.9%). chinchillas with acute conjunctivitis (1-3 days) were much more commonly affected by gram-negative organisms, and most cases were unilateral; 36.7% had concurrent dental disease. as chinchillas often experience conjunctivitis secondary to other disease, a thorough dental examination addressing the underlying cause is essential. however, despite this clinical paradigm, most chinchillas with bacterial conjunctivitis are reported to fully resolved with topical with or without oral antimicrobial therapy within 3 weeks. 42 being larger in size than rats and mice and more typically housed in a pet environment, chinchillas may be more amenable to topical medication, similar to guinea pigs. most diseases specific to rodents are either congenital or related to husbandry. unfortunately, their small size limits many of the typical surgical treatments routinely performed in other veterinary species. once husbandry is addressed, treatment may be limited to mitigation of the disease process but not complete resolution. unusual features in the conjunctiva and cornea of the normal guinea-pig: clinical and histological studies the cornea of guinea pig: structural and functional studies correlation between corneal sensitivity and quantity of reflex tearing in cows, horses, goats, sheep, dogs, cats, rabbits, and guinea pigs results of diagnostic ophthalmic testing in healthy guinea pigs schirmer tear test, phenol red thread tear test, eye blink frequency and corneal sensitivity in the guinea pig a mouse dry eye model induced by topical administration of benzalkonium chloride degeneration and regeneration of corneal nerves in response to hsv-1 infection effect of topical anesthesia on evaluation of corneal sensitivity and intraocular pressure in rats and dogs the chinchilla eye: morphologic observations, echobiometric findings and reference values for selected ophthalmic diagnostic tests reference values for selected ophthalmic diagnostic tests and clinical characteristics of chinchilla eyes (chinchilla lanigera) ocular disease in the guinea pig (cavia porcellus): a survey of 1000 animals conjunctival dermoid in two guinea pigs: a case report central corneal thickness does not correlate with tonolab-measured iop in several mouse strains with single transgenic mutations of matricellular proteins in vivo pachymetry in normal eyes of rats, mice and rabbits with the optical low coherence reflectometer comparative anatomy of laboratory animal corneas with a new-generation high-resolution in vivo confocal microscope regeneration of corneal endothelial cells following keratoplasty in rats with bullous keratopathy specular microscopy to determine corneal endothelial cell morphology and morphometry in chinchillas (chinchilla lanigera) in vivo efficacy of systemic vitamin c supplementation in reducing corneal opacity resulting from infectious keratitis vascularization of corneas of hairless mutant mice sjö gren's syndrome: studying the disease in mice genomic locus modulating corneal thickness in the mouse identifies pou6f2 as a potential risk of developing glaucoma spontaneous development of corneal crystalline deposits in mrl/mp mice effects of cage-change frequency and bedding volume on mice and their microenvironment genetic dissection of anterior segment dysgenesis caused by a col4a1 mutation in mouse a novel mouse model of anterior segment dysgenesis (asd): conditional deletion of tsc1 disrupts ciliary body and iris development outcome of penetrating keratoplasty for peters anomaly dynamics of donor cell persistence and recipient cell replacement in orthotopic corneal allografts in mice the balance of th1/th2 and lap1tregs/th17 cells is crucial for graft survival in allogeneic corneal transplantation systemic toxicity and resuscitation in bupivacaine-, levobupivacaine-, or ropivacaine-infused rats the eye and harderian gland chromodacryorrhea in laboratory rats (rattus norvegicus): etiologic considerations tear staining in pigs: a potential tool for welfare assessment on commercial farms sequential changes in the harderian gland of rats exposed to high intensity light spontaneous corneal dystrophy and generalized basement membrane changes in fischer-344 rats recurrent corneal erosions and epithelial basement membrane dystrophy intraocular and orbital malignant schwannomas in f344 rats comparison of the pathogenicity in rats of rat coronaviruses of different neutralization groups reverse transcriptase polymerase chain reaction-based diagnosis and molecular characterization of a new rat coronavirus strain coronavirus infection in the laboratory rat: immunization trials using attenuated virus replicated in l-2 cells clinical ocular findings in a colony of chinchillas (chinchilla laniger) skull size and cheek-tooth length in wild-caught and captive-bred chinchillas epidemiology of bacterial conjunctivitis in chinchillas key: cord-267965-84sotgds authors: noll, kelsey e.; ferris, martin t.; heise, mark t. title: the collaborative cross: a systems genetics resource for studying host-pathogen interactions date: 2019-04-10 journal: cell host & microbe doi: 10.1016/j.chom.2019.03.009 sha: doc_id: 267965 cord_uid: 84sotgds host genetic variation has a major impact on infectious disease susceptibility. the study of pathogen resistance genes, largely aided by mouse models, has significantly advanced our understanding of infectious disease pathogenesis. the collaborative cross (cc), a newly developed multi-parental mouse genetic reference population, serves as a tractable model system to study how pathogens interact with genetically diverse populations. in this review, we summarize progress utilizing the cc as a platform to develop improved models of pathogen-induced disease and to map polymorphic host response loci associated with variation in susceptibility to pathogens. pathogens often cause variable disease outcomes across infected individuals, ranging from asymptomatic infection to severe or fatal disease. multiple factors influence an individual's susceptibility to a pathogen, including variation in pathogen dose or virulence as well as host age, prior immune experience, microbiome/coinfection, and genetics. a role for host genetics in pathogen susceptibility is supported both by twin studies (kallmann and reisner, 1943) and by evidence of pathogen-driven selective pressure on the evolution of the human immune system (cagliani and sironi, 2013) . human genetic studies have demonstrated a role for host genetics in pathogen susceptibility and identified polymorphic genetic loci and genes associated with variation in susceptibility to specific pathogens (e.g., hiv) (mclaren et al., 2015) , classes of pathogens (e.g., mycobacteria) (bustamante et al., 2014) , and more generalized primary immunodeficiencies (pids) that result in severe immune defects (chapman and hill, 2012) . this information has enhanced our understanding of how host genetic variation affects pathogen susceptibility and how specific genes regulate human immunity and host-pathogen interactions. while advances in human genetic analysis have led to the identification of several host genes that regulate pathogen susceptibility in humans (newport and finan, 2011) , much of our understanding of how specific genes affect infectious disease pathogenesis in mammals has come from studies using mouse models (masopust et al., 2017) . this is in large part due to the existence of inbred mouse strains as well as the vast amount of immunological and molecular genetic tools available for the mouse, including the early generation of the mouse reference genome. inbred strains, in which each mouse is essentially genetically identical, allow control of host genetics as well as other factors that can confound human studies, such as pathogen dose, nutrition, and prior immune exposure. additionally, targeted knockout technology has been used in the mouse genome for almost 30 years (bouabe and okkenhaug, 2013) . while advances in gene editing techniques (e.g., crispr) have recently extended these approaches to other species, genetically modified mice have been an unparalleled resource for studying the role of specific genes in the response to a variety of pathogens (masopust et al., 2017) . furthermore, the use of classical genetic crossing approaches (e.g., intercrosses), as well as reproducible mouse genetic reference populations (grps), has facilitated the discovery of key regulators of host immunity and pathogen susceptibility such as the innate immune receptor tlr4, oligoadenylate-synthetase oas1b, and divalent metal transporter nramp1 (perelygin et al., 2002; qureshi et al., 1999; vidal et al., 1993) . the recent development of multiparental grps, such as the collaborative cross (cc), promises to further extend genetic mapping capabilities, while also leading to the development of mouse models that more accurately reproduce the genetic diversity and phenotypic range seen in human populations (churchill et al., 2004) . this review will summarize recent advances in the use of mouse grps, with an emphasis on the use of the cc for studying infectious diseases and immunity. the impact of host genetics on pathogen susceptibility can be illustrated by genes exhibiting large effects such as the chemokine receptor ccr5 and fucosyltransferase 2 (fut2), for which specific variants have a major impact on resistance to viral infection (hiv and norovirus, respectively) (quillent et al., 1998; thorven et al., 2005) . likewise, deleterious mutations in immune genes can result in pids, which cause enhanced susceptibility to specific or entire classes of pathogens (carneiro-sampaio and coutinho, 2007) . to date, over 350 genetically driven pids have been identified in humans (picard et al., 2018) . the identification of these monogenic resistance and susceptibility genes has enhanced our understanding of human immunity and hostpathogen interactions while contributing to the development of antiviral therapies, such as hiv entry inhibitors, as well as gene therapy and immune replacement therapies (collins and thrasher, 2015; lenardo et al., 2016) . though genes of large effect can impact human health, inheritance patterns suggest that susceptibility to infectious disease is largely polygenic (driven by tens to thousands of variants of smaller effect) (hill, 2012) . this is supported by results from genome-wide association studies (gwas) designed to map loci associated with variation in pathogen susceptibility and vaccine response. for example, gwas of hiv viral load have shown that mutations in the hiv co-receptor ccr5 and variants in human leukocyte antigen (hla) explain approximately 15% of the total variance, with an additional 5% of heritable variance explained by additive effects of other genes (mclaren et al., 2015) . gwas on malaria susceptibility have identified variants in multiple genes including the calcium atpase atp2b4, the glucose phosphate dehydrogenase gp6d, and hlas, as well as confirming previously identified variants in hemoglobin genes (sickle cell and thalassemia) (hedrick, 2011; timmann et al., 2012) . multiple loci have been associated with clearance of hepatitis c virus, including the cytokine il-28b and hla genes (duggal et al., 2013) , and over 20 loci have been associated with leprosy susceptibility (wang et al., 2016) . while gwas have been applied to and enhanced our understanding of infectious diseases, these studies explain only a fraction of the heritable variation in pathogen susceptibility (hill, 2012) . this is partly attributable to factors such as small cohort sizes, ethnic striations, and lack of replicates, which limit detection and interpretation of results (du et al., 2012) . rare genetic variants are unlikely to be detected by gwas, due to both lack of power and lack of tagging on conventional genotyping arrays (auer and lettre, 2015) . a specific concern for gwas in infectious diseases is that studies can be confounded by pathogen genetics, pathogen dose, and other environmental factors. furthermore, validation and mechanistic analysis can be difficult in humans due to factors such as lack of replicates and inability to routinely access many affected tissue types. for these reasons, the use of appropriate animal models has been a critical resource for identifying and studying pathogen susceptibility genes. while a variety of model genetic systems have been used to analyze host-pathogen interactions (e.g., caenorhabditis elegans, danio rerio, and drosophila melanogaster) (allen and neely, 2010; dionne and schneider, 2008; marsh and may, 2012) , the laboratory mouse (mus musculus) provides the most robust mammalian system for dissecting genetic regulation of immune responses in human-relevant diseases. mice are relatively inexpensive, can be easily controlled in diet and environment, and reproduce quickly. a wide variety of well-characterized mouse immunological reagents are available, as well as genetic tools such as reproducible inbred strains, gene-specific knockouts, genome-wide mutagenesis strategies (e.g., n-ethyl-n-nitrosourea [enu] and crispr/cas9), and transgenic technologies. these systems facilitate the identification and mechanistic characterization of specific host genes that affect disease pathogenesis and/or immunity (pelletier et al., 2015; takaki et al., 2017) . though over 450 commercially available inbred mouse strains and outbred stocks are available (beck et al., 2000) , the majority of research in infectious disease and immunology is conducted in a limited set of strains. one of the major reasons for the focus on specific strains is the need to control for genetic background when analyzing gene-specific knockouts or performing largescale mutagenesis screens. large-scale initiatives such as the international mouse phenotyping consortium (impc) depend upon the ability to control background strain to analyze the impact of specific gene deletions or mutations on a wide range of developmental and immune phenotypes (muñ oz-fuentes et al., 2018) , and have thus generated these mutants on common backgrounds using largely identical procedures. c57bl/6j is the most commonly used inbred mouse strain; it is the source of the mouse reference genome and the genetic background for the majority of knockout mice. other strains, such as the balb/c substrains, also have a rich history of use in immunology and infectious disease research (watanabe et al., 2004) . while the ongoing use of such strains is driven by the need to compare results across studies, it is important to note that every inbred strain has a unique set of genetic features, and thus no one strain is representative of all mice, let alone of a genetically complex population such as humans. although genetically modified mice have been an essential resource for gene discovery and mechanistic analysis, there has been a growing acknowledgment in the field that knockout approaches have limitations. approximately 15% of genes cannot be knocked out because they are developmentally essential (nih, 2015) . similarly, other knockouts have no observable phenotype, due to genetic redundancy and/or lack of robustness in the phenotyping pipeline (barbaric et al., 2007) . in other cases, knockouts have disparate phenotypes on different genetic backgrounds due to gene-gene interactions (doetschman, 2009) . furthermore, gene knockouts result in null alleles (complete loss of gene function), whereas naturally occurring genetic variants are most likely to be hypomorphic or hypermorphic alleles (partial loss or gain of gene function; alterations in timing and magnitude of expression). lastly, while single-gene approaches are straightforward, they are isolated systems that do not model the simultaneous contribution of variants in multiple pathways, as would most likely be observed in a natural system. thus, to better model and understand the role of genes in complex phenotypes such as immunity and infectious disease, it is pertinent to consider complex genetic interactions across diverse genetic backgrounds. in contrast to the approaches discussed above, researchers have also studied the role of natural genetic variants by leveraging differential phenotypes across inbred strains, using classical genetic breeding strategies to identify pathogen susceptibility genes such as oas1b, immune cell activating receptor ly49h, and large interferon-induced gtpase mx1 (casanova et al., 2002) . in contrast to mapping crosses between stocks (e.g., f2 crosses), where each mouse is unique and therefore ephemeral, grps serve as reproducible models to study the role of genetic diversity. grps consist of anywhere from a few to hundreds of inbred lines derived and split from a common ancestral population, and each line has a fixed and known genome that can be replicated indefinitely. the existence of reproducible yet genetically diverse individual mouse strains facilitates study designs that involve case versus control, genotype by environment interactions (gxe), and phenotypic penetrance or threshold traits. these populations further facilitate the integration of phenotypic, genotypic, and molecular data for systems-level data analysis while also allowing retrospective integration of new layers of data. the first recombinant inbred panel, the cxb (balb/cj 3 c57bl/6j), was created by donald bailey to facilitate mapping of the mhc locus (bailey, 1971) . subsequent biparental recombinant inbred panels include the axb/bxa (a/j 3 c57bl/6j, c57bl/6j 3 a/j), the bxh (c57bl/6j 3 c3h/hej), and the bxd (c57bl/6j 3 dba/2j), which is the largest and most extensively used of these panels (peirce et al., 2004) . though originally developed to study monogenic traits, use of these panels has been extended to study genetically complex traits such as susceptibility to murine plasmodia (hoffmann et al., 1984) , murine leukemia virus (panoutsakopoulou et al., 1998) , group a streptococcus (chella krishnan et al., 2016) , and influenza a virus (iav) (nedelko et al., 2012) . as results from these panels accumulated, there was a community-wide recognition that larger and more genetically diverse grps would improve the ability to genetically dissect more complex traits (threadgill et al., 2002) . based on results from the early grps as well as genetic mapping studies, the complex trait consortium proposed to create a second-generation, multi-parent advanced intercross grp to promote complex trait genetic mapping and systems genetics research (threadgill et al., 2002) . in contrast to single-gene approaches, systems genetics considers phenotypes in the context of global genetic variation as well as intermediate molecular factors such as gene expression, protein abundance, and environmental interactions. the resource ultimately derived from this proposal was the cc, a large panel of recombinant inbred strains derived from eight genetically diverse founder strains. the cc was designed to facilitate systems genetics approaches with improved resolution and higher statistical power compared to traditional grps, to expand the pool of genetic variation segregating in the population, and to bridge the analysis of genetic and molecular networks across diverse phenotypes (churchill et al., 2004) . of the eight cc founder strains, five are classical laboratory strains chosen for their rich history of use in mouse genetics (c57bl/6j and 129s1/svimj) or their relevance as models of common diseases including cancer, type 1 diabetes, metabolic syndrome, and obesity (a/j, nod/shiltj, and nzo/hiltj). the other three cc founders are wild-derived inbred strains, which represent the three major subspecies of mus musculus: casteneus (cast/eij), musculus (pwk/phj), and domesticus (wsb/eij), and introduce much of the genetic diversity into the cc. these eight founder strains were interbred in a funnel breeding scheme to produce recombinant mice with genomic contributions from each founder, which were then bred to homozygosity to produce recombinant inbred (ri) cc strains (figure 1 ). although the initial goal was to produce 1,000 cc strains, only around 100 cc strains survived the inbreeding process due to high rates of infertility and breeding issues, largely caused by genomic incompatibility introduced by the wild-derived strains. extinction of cc strains during the inbreeding process inspired the creation of the diversity outbred (do) panel, an outbred population derived from a set of incipient cc strains (churchill et al., 2012) . the do now serves as its own important genetic resource that has proven useful for high-resolution genetic mapping as well as exploration of phenotypic diversity ( while the do has only been used in a limited number of infectious disease studies (mchugh et al., 2013; niazi et al., 2015) , the do and cc should be viewed as complementary resources for studying how genetic variation affects pathogen susceptibility and immunity. while classical mouse grps remain actively used, and in some ways provide a more straightforward analytical path than the cc due to reduced complexity, several aspects of the cc make it more attractive for discovery-based research. the cc captures approximately 90% of common genetic variants described within laboratory mice, with up to eight distinct haplotypes at any region of the genome (roberts et al., 2007) . due to higher genetic diversity with novel allele combinations and epistatic interactions, the cc yields more phenotypic variation and extreme phenotypes than classical grps. the selection of founder strains and the breeding design, as well as higher levels of recombination, result in lower levels of long-range disequilibrium and population structure than classical grps, allowing for higher resolution quantitative trait locus (qtl) mapping (iraqi et al., 2012; threadgill et al., 2002) . genetic variation is also more uniformly distributed across the genome, removing several genomic ''blind spots'' that interfere with mapping in classical inbred strains and grps (roberts et al., 2007) . although the cc has only been available for a relatively short period of time, it has been utilized across a number of disciplines, including response to environmental toxins (venkatratnam et al., 2017) , nutrition (schoenrock et al., 2018) , body composition (mathes et al., 2011) , and immunity and pathogenesis (discussed further here). importantly, these studies have highlighted previously uncharacterized phenotypic diversity, including expansion of the phenotypic range, disassociation of traits previously thought to be connected, and identification of completely novel phenotypes (srivastava et al., 2017) . the cc is an especially useful resource for studying the role of host genetics in hostpathogen interactions because it allows for control of many of the factors described above that confound infectious disease studies in humans. over the past decade, a variety of studies utilizing the cc, including non-fully inbred incipient cc lines (commonly termed the pre-cc) and derived cc populations (such as cc-f1s), have been used to probe the role of host genetics in the response to infectious disease. here we will discuss the current work in this field, including studies focusing on phenotypic characterization (e.g., model development and molecular signatures of disease response) and genetic mapping of disease trait-associated qtl. there is an ongoing debate over the validity of mouse models in recapitulating human disease (seok et al., 2013; takao and miyakawa, 2015) . while the utility of the mouse is undeniable in the infectious disease field, it is equally true that individual classical inbred strains do not fully recapitulate human disease and most models recapitulate only a portion of the disease phenotypes observed in humans. in an effort to improve model fidelity to human disease, a number of researchers have turned to the cc to develop new mouse models. genetically diverse cc strains show a range of responses to pathogens, from variation in disease severity to completely novel disease phenotypes. this phenotypic variability better mimics the diversity of disease presentation across the human population, thus providing a more comprehensive platform to develop newer, more representative mouse models. ebola virus ebola virus (ebov) causes severe, and often lethal, hemorrhagic fever in humans. while classical inbred strains are susceptible to mouse-adapted ebov (ma-ebov), they do not display many of the characteristic symptoms observed in severe human cases such as coagulopathy, hemorrhagic manifestations, and rash (st claire et al., 2017) . rasmussen et al. infected a panel of f1 crosses between cc strains (cc-f1s) following an initial assessment in founder strains (rasmussen et al., 2014) . they observed significant phenotypic variation following ma-ebov infection, from high resistance to complete lethality, and covering a spectrum of different pathologies similar to the range of clinical disease observed in humans. importantly, some strains presented with severe hemorrhagic disease and liver damage, consistent with human ebov disease. follow-up studies with representative susceptible and resistant strains found differential transcriptional responses, highlighting the central transcriptional regulatory gene tek, for which haplotypes across these cc-f1s correlated with weight loss and mortality following ma-ebov infection. west nile virus (wnv), a neuroinvasive flavivirus, induces a diverse spectrum of immune responses and clinical outcomes in humans that classical mouse models do not fully recapitulate (graham et al., 2015) . graham et al. assessed wnv susceptibility in a panel of cc-f1 crosses that were heterozygous for the h-2b mhc haplotype, which allowed quantification of wnv-specific t cells with the same mhc tetramer. wnv outcome ranged from highly resistant to highly susceptible, including a novel outcome group in which mice were outwardly asymptomatic despite higher viral titers and immunopathology in the brain. graham et al. followed up on one unique cc-f1 ((cc032/ geniunc 3 cc013/geniunc)-f1), in which half of the mice that survived infection displayed sustained weight loss and persistent viral loads in the cns. they found that these mice exhibited a rapid early innate inflammatory response characterized by increased early expression of ifn-b and the interferonstimulated gene ifn1, as well as high early viral titers and the ability to control, but not clear, viral replication in the cns figure 1 . representative cc funnels cc founders were bred in funnel breeding schemes to produce progeny with genetic contributions from each of the eight founders, at which point they were inbred for generations until reaching near homozygosity. many different funnels were set up, each to produce a unique cc ri line. (graham et al., 2016) . additionally, (cc032/geniunc 3 cc013/ geniunc)-f1 mice had a unique immunoregulatory signature, with a high number of t regulatory cells in both infected and uninfected animals and a distinctive post-infection gene expression profile that indicated reduced cytolytic ability. the initial graham et al. study (graham et al., 2015) found that wnv response largely tracked with the haplotype of oas1b, a known flavivirus resistance gene, although there was still considerable disease variation within oas1b haplotype groups. green et al. continued to dissect the impact of oas1b on the innate immune transcriptional landscape following wnv infection (green et al., 2017) by performing genome-wide micro-array analysis across multiple time points post-infection in seven cc-f1s carrying different combinations of functional (wild-derived) or nonfunctional (classical inbred strain-derived) oas1b haplotypes. transcriptional correlation analysis of differentially expressed immune genes across cc-f1s yielded three gene clusters, and pathway analysis led to the construction of innate immune regulatory networks associated with wnv infection between oas1b haplotype groups. mycobacterium tuberculosis (mtb) infection varies broadly in disease presentation and pathology in humans. likewise, the efficacy of the standard tb vaccine, the live-attenuated bcg, is variable across individuals (mangtani et al., 2014) . smith et al. studied mtb pathogenesis and bcg vaccine efficacy in the cc founders and three ri strains that were selected based on a pilot study assessing mtb susceptibility (smith et al., 2016) . the authors observed a wide range of susceptibility to mtb and dissociation of previously associated phenotypes such as bacterial burden, immune cell recruitment, and tissue damage. when the bcg vaccine was administered prior to mtb challenge, vaccine efficacy was low overall and not correlated with susceptibility to primary infection. while bcg vaccination reduced mtb burden in four strains, there was an increased bacterial burden in nzo/hlltj mice, a model for obesity and type 2 diabetes. in addition to highlighting the importance of host genetics on mtb susceptibility and bcg-induced vaccine responses, the study illustrates the potential impact of comorbidities, such as type 2 diabetes in nzo/hlltj mice, on vaccine responses in genetically diverse populations. maurizio et al. analyzed the heritability of iav-induced disease using cc-f1s as well as f1 crosses between founder strains (maurizio et al., 2018) . these studies determined that iavinduced weight loss was 57% heritable at day 4 post-infection, and that this heritability was mostly composed of additive effects largely attributable to the haplotype of mx1, a polymorphic largeeffect anti-iav gene. the genetic dominance of the protective mx1 haplotype varied depending on subspecies origin, with the m. musculus musculus allele acting dominantly and the cast/eij allele, identified by ferris et al. (2013) , acting additively. consistent with ferris et al. (discussed below) , these authors determined that when controlling for mx1, non-mx1 heritable effects still accounted for 34% of the phenotypic variation, and this effect was consistent across founder diallel, pre-cc, and cc-f1 populations. human studies have identified blood transcriptomic and proteomic signatures associated with iav infection that could be used to predict patient outcome. elbahesh and schughart observed that gene signatures from iav-infected humans were reproduced in the peripheral blood of cc founders (elbahesh and schughart, 2016) . in a follow-up study, kollmus et al. characterized the transcriptional response to iav in the peripheral blood of eleven cc strains (kollmus et al., 2018) . though both human and mouse datasets were globally heterogeneous, the cc dataset showed that genetic background strongly influenced gene expression, highlighting the importance of genetics in driving the immune response. for the most differentially expressed genes, the transcriptional responses in mice and humans were largely similar, demonstrating the utility of the cc in recapitulating iav-induced host response in humans. xiong et al. characterized transcriptomic variation in response to severe acute respiratory syndrome coronavirus (sars-cov) and iav across the eight cc founder strains (xiong et al., 2014) . differential gene expression analysis at days 2 and 4 post sars-cov or iav infection showed significant differences driven by mouse strain, infection status, and time point. genes with strain-specific differential expression patterns were largely enriched for immune pathways, while more generic differential expression patterns were enriched for basic biological functions. the study also noted the presence of strain-specific isoforms and novel transcripts not present in the c57bl/6j reference annotation. pseudomonas aeruginosa is an opportunistic bacterial pathogen with variable clinical outcomes across susceptible individuals. human studies, including twin studies as well as association studies, have identified multiple genes associated with susceptibility to p. aeruginosa. lorè et al. studied p. aeruginosa infection in 17 pre-cc lines, focusing on survival time and early body weight change, and observed a large amount of variation in these phenotypes (lorè et al., 2015) . genetic mapping of disease response in the cc as described above, the cc was initially envisioned as a genetic mapping population with approximately 1,000 strains (threadgill et al., 2002) . while the number of available cc ri strains is closer to 100, this population has proven to be sufficient for successful qtl mapping. mapping studies have identified qtls spanning the genome that are driven by variants from all eight cc founders and show very little overlap across pathogens (figure 2 ; table 1 ; discussed below). overall, infectious disease phenotypes mapped in the cc show a broad range in overall heritability (the proportion of population-wide variation explained by differences between strains) as well as effect sizes (the proportion of population-wide variation explained by specific loci). across the studies described in table 1 , reported estimates for heritability range from 12% to over 80%, while reported estimates for effect size range from 5% (for a qtl of small effect influencing sars-cov viral titer) to over 40% (for a large effect qtl containing the gene mx1 on influenza-induced weight loss). authors have narrowed many of these qtls to lists of candidate genes, and a small number of these candidates have been subsequently validated. to date, most publications have highlighted qtls mapped by phenotyping large panels of cc strains; however, this is not the only successful strategy. as mentioned above, some cc strains present with unique disease phenotypes, which may be driven by complex genetic regulatory networks involving multiple loci with epistatic interactions. when one or a few cc strains possess the combination of genetic variants needed to manifest the novel phenotype, population-wide studies may not possess sufficient mapping power. in these cases, researchers have successfully used more traditional f2 or backcross approaches to identify causal genes (rogala et al., 2014) . in this section, we will highlight results of both population-wide and intercrossmediated cc studies of relevance to infectious disease research. the first host-pathogen study in the cc examined susceptibility to the fungus aspergillus fumigatus, which is pathogenic in immunocompromised individuals. durrant et al. found that survival time varied between 4 days and over 28 days post-infection in 66 pre-cc lines (durrant et al., 2011) . the authors mapped multiple genome-wide significant qtls for survival time that were largely driven by wild-derived founder haplotypes. candidate genes were identified using merge analysis, a procedure that compares the pattern of sequence variants (e.g., snps or indels) to the pattern of haplotype effects (impact of each founder strain haplotype on the phenotype) observed under the qtl (yalcin et al., 2005) . the application of merge analysis to the cc enables more effective gene refinement compared to studies in classic biparental grps, where the presence of only two haplotypes means that every genetic variant in a locus is a putative candidate causal variant. merge analysis supported the candidate gene irf2, an interferon regulatory transcriptional factor gene, under the most significant qtl; however, for the remaining qtls there was no strong concordance between a priori and merge-supported candidates. klebsiella pneumoniae vered et al. challenged 73 pre-cc lines with k. pneuomonia, which causes severe pneumonia and sepsis in immunocompromised individuals (vered et al., 2014) . the pre-cc mice displayed broad and heritable variation in survival time exceeding that observed in classic inbred strains, as well as dissociation of survival time from temperature or body weight changes. the study identified one qtl at day 2 post-infection and two at day 8 post-infection. the authors used merge analysis to refine candidate genes; however, they found that the best merge candidates showed simpler allele effect patterns than the more complex haplotype effects observed under the qtl. the strongest candidate genes identified were ikbkap, a transcriptional elongation factor complex component, and actl7a, actl7b, and ctnnal1, which are involved in cell adhesion and cytoskeletal structure. bottomly et al. analyzed a set of 99 pre-cc lines infected with mouse-adapted influenza a/pr/8/34 and assessed weight loss, clinical score, and mortality through 4 days post-infection (bottomly et al., 2012) . a subset of pre-cc lines, classified as high and low responders based on a composite metric of weight loss and histopathological scoring, were selected for transcriptomic profiling. over 2,000 transcripts were differentially expressed between susceptible and resistant classes, and mapping identified 21 significant expression qtls (eqtls). twelve of the eqtls were validated in cc founder strains, and structural equation modeling was applied to these candidates to infer reactive expression networks underlying the transcriptional differences. in a companion study, ferris et al. further characterized variation in response to influenza a/pr/8/34 in 155 pre-cc lines (ferris et al., 2013) . while disease-associated phenotypes (weight loss, viral titer, and inflammation) were largely correlated, subsets of these pre-cc mice showed breakdowns in these relationships, resulting in novel phenotypic combinations not observed in classical mouse strains (e.g., high weight loss with minimal inflammation). this study identified four significant qtls, including one strong qtl that explained approximately 42% of iav-induced weight loss, over the iav resistance gene mx1. importantly, while mx1 is well studied, the authors identified a novel allelic variant derived from cast/eij that protects from weight loss but less efficiently inhibits viral replication compared to the functional m. musculus musculus-derived allele carried by nzo/hiltj and pwk/phj. consistent with ferris et al.'s finding of a uniquely functional cast/eij mx1 variant, leist et al. found that cast/eij mice have a unique response to an h3n2 strain of iav (leist et al., 2016) . while the mx1 locus had a dominating effect in the ferris et al. study, large phenotypic variation occurred within mx1 classes, suggesting the presence of modifier alleles. three additional qtls were mapped when controlling for mx1 haplotype, corresponding to variation in weight loss, pulmonary edema, and airway neutrophils. notably, aside from the mx1 locus, none of the qtls identified by ferris et al. matched influenza susceptibility loci found in mapping studies conducted in the bxd population. (boon et al., 2009; nedelko et al., 2012) . these differences may reflect the more complex genetics of the cc compared to the bxd and/or differences in the strain of influenza or other experimental variables across studies. salmonella enterica salmonella enterica serovar typhimurium (s. typhimurium) causes typhoid fever, and previous mouse mapping studies have identified multiple loci associated with susceptibility (roy significance threshold levels: *<0.1, **0.1, ***0.05. genome coordinates marked with 0 refer to genome assembly gscv38/mm10; otherwise, coordinates refer to mgscv37/mm9. et al., 2006; sebastiani et al., 1998) . to identify novel loci associated with s. typhimurium susceptibility, zhang et al. infected 35 cc ri strains and measured bacterial load in the liver and spleen at 4 days post-infection (zhang et al., 2018) . qtl scans for bacterial load mapped multiple significant or suggestive qtl (figure 2 ; table 1 ), which differed from those identified in studies conducted in other mapping populations (roy et al., 2006; sebastiani et al., 1998) . zhang et al. used merge analysis in combination with immune cell gene expression, gene ontology, and analysis of known protein functions to narrow the list of candidate genes under the two significant loci. they identified candidate genes cul4a (ubiquitin ligase), lamp1 (lysosomal membrane protein), mcf2l (guanine nucleotide exchange factor), pcid2 (trex-2 complex component involved in mrna nuclear export), and a high-priority candidate slc35f1, which has lactate dehydrogenase activity that may be important in the s. typhimurium pyruvate metabolism pathway. the study also noted that one strain, cc042/geniunc, had extremely high bacterial loads, suggesting that it may be uniquely susceptible to s. typhimurium. sars-cov causes severe acute respiratory syndrome in humans, but the genes regulating these processes are poorly understood. gralinski et al. infected the cc founder strains and 147 pre-cc lines with mouse-adapted sars-cov and studied disease through day 4 post-infection (gralinski et al., 2015) . variation in weight loss and viral titer was significant in the cc founders and even broader in the pre-cc mice. similar to iav, phenotypic dissociation was observed in some pre-cc lines, with viral titer not correlated with weight loss. qtl mapping identified a significant main effect qtl, as well as a modifier qtl for vascular cuffing in the lungs, explaining 26% and 21% of the variation, respectively. suggestive qtls were identified for eosinophilia (26% of variation) and viral titer (22% of variation) (figure 2 ; table 1 ). the candidate region for the main effect vascular cuffing qtl was narrowed to a small 450 kb region of shared ancestry between the high-response haplotypes. this region contained only one functional gene, trim55, a ring zinc-finger-containing protein expressed in smooth muscle, which had not previously been implicated in any immune phenotypes. follow-up validation studies using trim55 knockout mice, which showed altered chemokine and tight junction gene expression as well as altered inflammatory cell recruitment, confirmed a role for this gene in sars-covinduced vascular cuffing. the authors also noted a high-priority candidate under the modifier qtl, the cadherin family member cdhr2, which may be involved in migration of inflammatory cell from the blood into the lung. in a subsequent study, gralinksi et al. used an f2 cross between two cc-ri strains with highly divergent sars-cov susceptibilities to map five significant loci affecting weight loss, viral titer, and other disease phenotypes, including one main effect qtl that explained between 6% and 12% of the variation in every phenotype (gralinski et al., 2017) . ticam2, a tlr4 adaptor protein, was identified as a high-priority candidate gene under the main effect qtl given the known importance of tlr4 in sars-cov pathogenesis (totura et al., 2015) . ticam2 knockout mice had increased sars-cov-induced weight loss, early viral titers, and pulmonary hemorrhage. in addition to exhibiting high levels of variation in pathogeninduced responses, the cc also exhibits variation in baseline (homeostatic) immunity. variation in immune homeostasis has been associated with variation in vaccine responses in humans (tsang et al., 2014) , and the cc provides a novel resource to study how variation in pre-existing immunity affects the host response to infection or vaccination. phillippi et al. analyzed subsets of lymphocytes and antigen-presenting cells in the cc founders, cc founder f1s, and 66 pre-cc strains (phillippi et al., 2014) . they observed variation in the pre-cc exceeding that in the founders, as well as novel extreme phenotypes such as lymphopenia and inverted cd4/cd8 ratios. while some immune phenotypes were highly correlated, others showed little or no correlation to other immune populations. mapping identified 10 significant qtls across 8 phenotypes, including b/t cell ratio and mean fluorescence intensity for cd23, also known as fc epsilon rii. the cd23 qtl contains the gene fcer2a, which codes for cd23 itself, and a combination of merge analysis, conditional association, and residue conservation was used to identify specific coding polymorphisms within fcer2a driving the qtl. graham et al. also performed an extensive examination of homeostatic immunity in over 100 cc-f1 crosses, with a focus on t cell populations, expression of activation markers, and production of inflammatory cytokines (graham et al., 2017) . cc strains exhibited high levels of phenotypic variation that extended well beyond the variation observed between c57bl/6j and balb/c, the most common laboratory strains in immunological studies. mapping identified two highly significant qtls driving the frequency of specific t regulatory subsets, and candidates were chosen based on concordance of haplotype effects with founder polymorphisms that were coding or splice variants. similar to the cis-qtl for cd23 expression identified by phillippi et al., the qtl mapped by graham et al. for cxcr3+ t regulatory cells contains the cxcr3 gene itself. kri sti c et al. studied variation in igg glycosylation, which is important for antibody structure and function, in 95 cc strains and observed nearly double the variation observed in humans (kri sti c et al., 2018) . glycosylation patterns were up to 80% heritable, and multiple qtls associated with variation in different glycans were identified. variation in 5 different glycans all mapped to the immunoglobulin heavy chain locus. variation in 2 other glycans mapped to a locus containing the glycosyltransferaseencoding gene mgat3, which was identified as a strong candidate. commensals and the microbiome in the cc while the focus of this review is on genetic control of immune populations and response to infectious agents, there is a growing appreciation for the role commensals play in shaping health and modulating immune responses. two studies have addressed these responses in the cc (snijders et al., 2016) and do (carmody et al., 2015) populations. both found that there were significant contributions of host genetic backgrounds. further, the cc study found strong genomic signals for many bacterial groups (otus) around the genome. given the increasing appreciation and awareness of the microbiome in modulating a variety of biomedically relevant traits, as well as the impact of commensals and the virome (virgin, 2014) on directly modulating immune responses (masopust et al., 2017) , future work within the cc will allow for the disentangling of direct roles on host genetic variants on disease responses, as well as indirect roles mediated through effects on commensals or prior immune exposure. the cc is a powerful resource for model development, extreme phenotype discovery, population-based qtl mapping, intercross mapping, validation of candidate genes across genetic backgrounds, and a variety of other approaches. here, we provide an overview of different study designs that can be implemented to study the impact of host genetic variation on pathogen outcomes or immunity using the cc (figure 3) . many of the genetic mapping studies described above utilized large panels of cc strains and were able to identify loci of moderate to large effects on pathogen outcomes. however, these types of studies are extremely resource intensive and may not always be the optimal approach. rather, we suggest a tiered approach to study design within the cc, starting with a small-scale analysis in a set of strains (e.g., 12-16) that sample most of the haplotypes present across the cc genomes, and expanding as necessary. the efficacy of this type of approach is demonstrated by the success of small cc screens that have identified strains with unique phenotypes and proceeded with further targeted studies such as f2 crosses to reveal complex polygenic networks driving the outlier phenotype (gralinski et al., 2017; rogala et al., 2014) . furthermore, even when investigators opt for large mapping studies, they may still identify strains with unique outlier phenotypes that cannot be explained by qtls identified in the initial screen. this missing heritability may be driven by complex genetic interactions such as epistasis, which are difficult to study in complex populations and are more effectively studied in targeted crosses (rogala et al., 2014) . initially screening small sets of cc strains (e.g., 12-16) allows the investigator to obtain insight into the distribution of trait variation (e.g., continuous, bimodal), while testing whether any strains show unique or strong outlier phenotypes (gralinski et al., 2015; rasmussen et al., 2014; rogala et al., 2014) . these small screens also facilitate estimation of the proportion of phenotypic variation explained by genetics within the test population (heritability), identification of potential confounding factors (e.g., mouse size and activity levels), and performance of power calculations before embarking on a large-scale screen. in contrast to conducting the initial screen in the cc founders, cc strains exhibit more phenotypic diversity due to re-assortment of allelic variants throughout the genome. furthermore, a specific set of cc strains can be chosen to avoid the presence of large effect resistance genes such as mx1 or oas1b that may dominate the response to some pathogens. likewise, strains can be selected that carry specific gene haplotypes to facilitate specific assays (e.g., mhc haplotypes for antigen-specific t cell analysis) (graham et al., 2015 (graham et al., , 2017 . following an initial screen, researchers interested in developing new models can pursue the strains with the most relevant phenotypes, while those interested in genetic mapping may either perform a more extensive population-wide study (e.g., across the entire cc) or conduct a focused analysis of one or more strains with extreme or novel phenotypes via classical intercrosses. unlike cc mice, which are fully inbred and genotyped, each mouse in an f2 or other intercross mapping population will need to be genotyped. in both cases, genetic mapping should identify a subset of the variants impacting the phenotype of interest (e.g., disease). while an in-depth discussion of mapping methods is beyond the scope of this review, several robust mapping strategies have been successfully applied to the cc, including doqtl, bagpipe, and r/qtl (arends et al., 2010; gatti et al., 2014; valdar et al., 2009 ). once qtls have been mapped, investigators can confirm the underlying haplotype effects by screening additional cc strains with high or low responder haplotypes under the qtls that were not included in the initial screen, or by screening a set of mice from the related do population. while not essential, this step confirms the initial mapping studies and provides confidence in the qtl effects prior to embarking on candidate gene investigation. the identification and validation of candidate genes can be a challenging and intensive process. qtls are often large, containing tens to hundreds of genes. in contrast to mapping in biparental grps or intercross populations, where there are only two haplotypes and every variant is a putative candidate, in the cc, merge analysis (yalcin et al., 2005) can be used to narrow candidates based on how the pattern of variants (e.g., snps or indels) for each founder matches the distribution of phenotypic responses associated with each founder haplotype. as described above, merge analysis has been a useful tool in the cc; however, a drawback of merge analysis is that it assumes a single snp variant driving the locus. in multi-parental populations, different snps or other mutations within the same gene may phenocopy one another (e.g., different mx1 null alleles; ferris et al., 2013) . furthermore, other genetic variants (e.g., copy number variants and transposable elements) are often missed by traditional merge databases. other in silico tools, including baseline tissue and/or haplotype-specific gene expression (e.g., immgen and gecco) (shay and kang, 2013 ; http://csbio.unc.edu/gecco) and protein functional consequence prediction (e.g., sift; sim et al., 2012) , can help narrow candidates to a single high-priority candidate gene or small set of candidates (ferris et al., 2013; gralinski et al., 2015) . alternatively, if causal variants are suspected of altering transcript levels, de novo gene expression analysis can be used to further refine candidate genes under the locus. following candidate gene identification, there are several options for testing whether the candidate is actually causal. when the phenotype is driven by an individual cellular factor, such as an antiviral molecule or receptor, gene function can be validated in vitro using techniques such as crispr or ectopic expression of different variants. when the phenotype has a more systemic cause, in vivo validation is generally necessary. this can be facilitated by the wide availability of knockout mouse lines (gralinski et al., 2015 (gralinski et al., , 2017 . however, in vivo validation studies should take into account factors such as genetic background of the knockout, the effect of the variant, and the distribution of haplotypes (e.g., a knockout on a c57bl/6j background may not provide an interpretable phenotype if, in the cc, the c57bl/6j haplotype is an extreme response haplotype, or if the c57bl/6j allele is hypomorphic or amorphic). more recent advances in crispr technology open up the possibility of knocking out or performing allele swaps in genes directly in cc founders or ri strains, enhancing the utility of the cc as a resource for both identifying and studying polymorphic genes affecting pathogen susceptibility or other phenotypes of interest. in less than two decades since the cc was conceptualized (churchill et al., 2004) , the cc has proven to be a fruitful resource across many fields, including infectious disease and immunology. expanding use of the cc has highlighted certain challenges and considerations that should be recognized to drive future development in both experimental design and resource expansion. (1) small screen in a subset of cc strains (12-16), selected based on availability or a genotype of interest (e.g., mhc haplotype). (2) assess variation across strains, identify outliers, measure heritability, perform power calculations, and identify confounding variables and reassess experimental design if necessary. (3) perform a larger screen either in the full cc population or a targeted intercross. following mapping, this can be repeated to follow-up on modifier alleles or other unexplained loci. (4) reassess variation, outliers, and heritability across mapping population. (5) map qtls driving phenotype of interest; analyze founder haplotype effects if mapping is done in the cc. (6) rationally select candidate genes using different tools as applicable. (7) perform validation studies in vitro and/or in vivo to confirm effect of candidate gene on phenotype. genetic mapping studies in the cc have successfully identified loci underlying infectious disease and immune phenotypes; however, relatively few have subsequently validated the initially identified candidate genes. even more challenging than candidate gene validation is the identification of specific causal variants within a gene. while strategies such as merge analysis can help refine candidates and variants, these tools are not always informative. there is a need for more refinement methodologies that can be applied in combination with experimental validation to narrow and identify causal variants. the wide array of genomic, molecular, and immunological reagents available is a valuable asset for mouse research; however, these do not necessarily uniformly work across the cc and should be validated before use. existing cc-specific resources, such as gecco, are not necessarily informative for infectious disease research since lymphoid organs, such as the spleen, thymus, and bone marrow, are not profiled. furthermore, the development of new cc-specific tools, such as cc-derived cell lines (e.g., mefs and ipscs), will expand the utility of the cc across fields, including the infectious diseases and immunology fields. one of the largest challenges across all of model organism research is connecting back to human relevancy. the cc has already proven useful in providing improved models of humanlike disease, as highlighted above. a number of genes that have been highlighted in the cc, such as mx1 and oas1b, have human paralogs relevant in infectious disease susceptibility (lin and brass, 2013; simon-loriere et al., 2015) , demonstrating the overlap between important genetically diverse infectious disease pathways in mouse and humans. however, the majority of studies have not yet made direct connections back to human datasets. as cc research develops, it will be increasingly important to continue to extend analysis from the cc to human genetics and vice versa. this will require more crosstalk between mouse and human geneticists to bridge gaps in research and communication. host immunity and susceptibility to infectious disease is a complex response driven by a multitude of factors, from environment and immune experience to the genetics of both the pathogen and the host. studying the role of host genetics in infectious disease directly in humans is challenging because of this complexity, and therefore much of the research in the field leans heavily on the mouse as a model organism. while the role of traditional laboratory strains and genetically modified mice in driving advances in the field cannot be understated, these models do not capture the genetic diversity and phenotypic complexity observed in the natural population. building on the success of classical intercrosses and biparental grps, the cc is a highly diverse and reproducible resource for studying and mapping complex traits, including infectious disease susceptibility. the reproducibility of cc strains will allow the community to cross-compare genes and genetic networks that regulate response across pathogens (e.g., iav and sars-cov; xiong et al., 2014) , while also exploring the impact of factors such as the microbiome, co-infections, and genetic variation of the pathogen in the context of a controlled, genetically diverse model population. the cc also holds promise for the development of new models and testing platforms that will better reproduce specific human disease outcomes or host responses to pathogens and vaccines. trolling for the ideal model host: zebrafish take the bait r/qtl: highthroughput multiple qtl mapping rare variant association studies: considerations, challenges and opportunities recombinant-inbred strains. an aid to finding identity, linkage, and function of histocompatibility and other genes appearances can be deceiving: phenotypes 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project and systems immunology quantitative trait mapping in diversity outbred mice identifies two genomic regions associated with heart size sift web server: predicting effects of amino acid substitutions on proteins high anti-dengue virus activity of the oas gene family is associated with increased severity of dengue high-resolution genetic mapping in the diversity outbred mouse population identifies apobec1 as a candidate gene for atherosclerosis tuberculosis susceptibility and vaccine protection are independently controlled by host genotype influence of early life exposure, host genetics and diet on the mouse gut microbiome and metabolome genomes of the mouse collaborative cross animal models of ebolavirus infection development of mouse models for analysis of human virus infections genomic responses in mouse models greatly mimic human inflammatory diseases a homozygous nonsense mutation (428g->a) in the human secretor (fut2) gene provides resistance to symptomatic norovirus (ggii) infections genetic dissection of complex and quantitative traits: from fantasy to reality via a community effort genome-wide association study indicates two novel resistance loci for severe malaria toll-like receptor 3 signaling via trif contributes to a protective innate immune response to severe acute respiratory syndrome coronavirus infection global analyses of human immune variation reveal baseline predictors of postvaccination responses mapping in structured populations by resample model averaging editor's highlight: collaborative cross mouse population enables refinements to characterization of the variability in toxicokinetics of trichloroethylene and provides genetic evidence for the role of ppar pathway in its oxidative metabolism susceptibility to klebsiella pneumonaie infection in collaborative cross mice is a complex trait controlled by at least three loci acting at different time points natural resistance to infection with intracellular parasites: isolation of a candidate for bcg the virome in mammalian physiology and disease a large-scale genome-wide association and metaanalysis identified four novel susceptibility loci for leprosy innate immune response in th1-and th2-dominant mouse strains genomic profiling of collaborative cross founder mice infected with respiratory viruses reveals novel transcripts and infectionrelated strain-specific gene and isoform expression using progenitor strain information to identify quantitative trait nucleotides in outbred mice identification of new loci involved in the host susceptibility to salmonella typhimurium in collaborative cross mice we would like to thank sharon taft-benz, sanjay sarkar, emily madden, and brea hampton for critical reading of the manuscript. chromosome ideograms shown in figure 2 were generated using the karyoploter package for r (gel and serra, 2017) . this work was supported by u19 ai100625 and p01ai132130. k.e.n. received support from t32ai007419. key: cord-335424-h84jtx94 authors: kirkland, j. l.; tchkonia, t. title: senolytic drugs: from discovery to translation date: 2020-08-04 journal: j intern med doi: 10.1111/joim.13141 sha: doc_id: 335424 cord_uid: h84jtx94 senolytics are a class of drugs that selectively clear senescent cells (sc). the first senolytic drugs dasatinib, quercetin, fisetin and navitoclax were discovered using a hypothesis‐driven approach. sc accumulate with ageing and at causal sites of multiple chronic disorders, including diseases accounting for the bulk of morbidity, mortality and health expenditures. the most deleterious sc are resistant to apoptosis and have up‐regulation of anti‐apoptotic pathways which defend sc against their own inflammatory senescence‐associated secretory phenotype (sasp), allowing them to survive, despite killing neighbouring cells. senolytics transiently disable these scaps, causing apoptosis of those sc with a tissue‐destructive sasp. because sc take weeks to reaccumulate, senolytics can be administered intermittently – a ‘hit‐and‐run’ approach. in preclinical models, senolytics delay, prevent or alleviate frailty, cancers and cardiovascular, neuropsychiatric, liver, kidney, musculoskeletal, lung, eye, haematological, metabolic and skin disorders as well as complications of organ transplantation, radiation and cancer treatment. as anticipated for agents targeting the fundamental ageing mechanisms that are ‘root cause’ contributors to multiple disorders, potential uses of senolytics are protean, potentially alleviating over 40 conditions in preclinical studies, opening a new route for treating age‐related dysfunction and diseases. early pilot trials of senolytics suggest they decrease senescent cells, reduce inflammation and alleviate frailty in humans. clinical trials for diabetes, idiopathic pulmonary fibrosis, alzheimer’s disease, covid‐19, osteoarthritis, osteoporosis, eye diseases and bone marrow transplant and childhood cancer survivors are underway or beginning. until such studies are done, it is too early for senolytics to be used outside of clinical trials. ageing is associated with increasing risk for developing multiple chronic diseases, the geriatric syndromes, impaired physical resilience and mortality [1] [2] [3] [4] . these risks increase exponentially in the last 25% of the lifespan [5] . amongst the diseases for which chronological age is a leading risk factor are congestive heart failure, myocardial infarction, dementias, strokes, most cancers, diabetes and metabolic diseases, renal dysfunction, chronic lung diseases, osteoporosis, arthritis, blindness and many others [6] [7] [8] . the geriatric syndromes include frailty, sarcopenia, falls, incontinence and mild cognitive impairment amongst others. decreased resilience can be manifested as delayed recovery following myocardial infarction, strokes, injuries or surgery, increased severity of infections such as influenza and coronaviruses and impaired development of antibodies after immunization [3] . combinations of several of these diseases, geriatric syndromes and resiliency impairments frequently occur in older people. indeed, the incidence of multi-morbidity, with 3 or more conditions being present simultaneously, increases exponentially beginning in adulthood [5] . analogous increases in disease burden occur with increasing age across multiple species. the geroscience hypothesis posits that fundamental ageing mechanisms are 'root cause' contributors to the increasing burden of disorders and diseases with advancing age that are responsible for the bulk of morbidity, mortality and health costs in the developed and developing worlds [9] . these fundamental ageing processes include: (1) chronic low grade 'sterile' (absence of bacteria, fungi, etc.) inflammation often accompanied by fibrosis, 2) macromolecular dysfunction (e.g. dna damage, telomere uncapping, protein misfolding and aggregation, decreased autophagy, increased advanced glycation end-products [ages], lipotoxicity and accumulation of bioactive lipids) and organelle dysfunction (altered nuclear membranes related to deficient lamin b, mitochondrial dysfunction leading to reduced fatty acid metabolism, higher glucose utilization, depletion of nad + and increased reactive oxygen species [ros] generation, etc.), 3) stem, progenitor and immune cell dysfunction (including altered proliferative capacity and dysdifferentiation with failure to develop into functional mature cells, declines in 'geroprotective' factors [e.g. a-klotho], contributing to stem and progenitor cell dysfunction) and 4) cellular senescence. our unitary theory of fundamental aging processes hypothesizes that these fundamental ageing processes may be interlinked. if this unitary theory is correct, then accelerating or targeting any one fundamental ageing process (e.g. cellular senescence) genetically or with drugs should affect many or perhaps all of the rest. indeed, consistent with the unitary theory, senescent cells contribute to inflammation, fibrosis, dna damage, development of protein aggregates, failed autophagy, lipotoxicity, mitochondrial dysfunction, depletion of nad + , ros generation and stem, progenitor and immune cell dysfunction [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] . cellular senescence, first reported in 1961 by hayflick and moorehead, is a cell fate that entails essentially irreversible replicative arrest, sustained viability with resistance to apoptosis, and frequently, increased metabolic activity [6, 8, [25] [26] [27] (fig. 1 ). intra and extracellular signals that can contribute to cells' entering the senescent cell fate mainly include signals related to tissue or cellular damage and/or cancer development. these include dna damage, telomeric uncapping or dysfunction, exposure to extracellular dna, oncogene activation, replicative stress or inducers of proliferation (such as growth hormone/igf-1), protein aggregates, misfolded proteins, failed protein removal fig. 1 cellular senescence: causes, mechanisms and consequences. a number of factors can combine to cause a cell to enter the senescent cell fate, as opposed to apoptosis or necrosis, through transcription factor cascades that can involve p16 ink4a , rb, p53, p21 cip1 or others. note that not every senescent cell exhibits increased p16 ink4a , rb, p53 or p21 cip1 . it can take up to 6 weeks for a cell to become a fully senescent, nondividing cell. this indicates that senescent cells can be removed intermittently, rather than having to treat with agents continuously to remove these cells. some, but not all senescent cells can develop a sasp. this sasp is a 'root cause contributor' to the stem cell, progenitor, tissue and systemic dysfunction that predispose to multiple disorders, which account for the bulk of morbidity, mortality and health costs. through decreased autophagy, presence of ages due to the reaction of reducing sugars with amino groups in proteins (e.g. haemoglobin a1c is an age), saturated lipids and other bioactive lipids (bradykines, certain prostaglandins, etc.), reactive metabolites (e.g. ros, hypoxia or hyperoxia), mechanical stress (e.g. bone-on-bone stress in osteoarthritis or shear stress such as occurs on the venous side of av fistulae for haemodialysis or around atherosclerotic plaques), inflammatory cytokines (e.g. tnfa), damage-associated molecular patterns (damps, e.g. released intracellular contents signalling breakage of neighbouring cells), and pathogen-associated molecular patterns (pamps, e.g. bacterial endotoxins). these inducers active one or more senescence-promoting transcription factor cascades, in some cases involving p16 ink4a -retinoblastoma protein (rb), in others, p53 and p21 cip1 , both of these pathways, or other pathways. these transcription factor cascades enforce replicative arrest and cause altered expression of hundreds of genes as well as epigenetic changes in dna. cellular senescence takes longer to become established than other cell fates, such as replication, differentiation, apoptosis or necrosis. from initiation to the attainment of a completed state of cellular senescence takes from 10 days to 6 weeks, at least in cell culture, depending on the cell type and the inducers driving the cell into the senescent fate. some, but not all senescent cells can acquire a senescence-associated secretory phenotype (sasp) [28, 29] , which we term here as deleterious (d-) senescent cells (fig. 2) . the sasp can include: 1) inflammatory, pro-apoptotic, insulin resistanceinducing cytokines, such as tnfa [30] , interleukin-(il-) 6, il-8 and others, 2) chemokines that attract, activate and anchor immune cells, 3) matrix metalloproteinases (mmps), such as mmp-3, à9 and à12 that cause tissue destruction, 4) tgfb family members that can contribute to fibrosis and stem cell and progenitor dysfunction, 5) activins and inhibins that also induce stem cell and progenitor dysfunction and dysdifferentiation [12] , 6) factors such as the serpines (e.g. plasminogen activator inhibitor [pai] à1 and à2) that can cause blood clotting and fibrosis [31, 32] , 7) growth factors that can exacerbate tumour spread [33] , 8) bioactive lipids that also contribute to inflammation and tissue dysfunction (e.g. bradykines, ceramides or prostaglandins) [34] , 9) micro-rnas (mirnas) that contribute to stem and progenitor cell dysfunction, inflammation and insulin resistance [35] and 10) exosomes that can carry cytotoxic and senescenceinducing cargos locally and systemically [36] . d-senescent cells can comprise 30 to 70 % of the senescent cell population. sasp components produced by d-senescent cells depend on the cell type that became senescent and the cause of senescence (e.g. mechanical stress, repeated replication, radiation, chemotherapy, pamps, etc.). the sasp can be modulated by hormones, drugs, pathogens and other factors. for example, endogenous or pharmacological glucocorticoids [37] can attenuate release of a subset of sasp factors, as can rapamycin and metformin [38, 39] . bacterial toxins such as lipopolysaccharide can exacerbate the sasp, as can tnfa [40] (unpublished observations). the sasp can vary over time due to factors inside or outside of senescent cells. for example, after a few months, transpositional events can begin to occur in senescent cells involving line-1 or other elements [41, 42] . this can lead to rearrangement of chromosomes and activation of cgas/sting, cellular pathways that recognize dna damage and exacerbate production of inflammatory sasp factors through interferon-1. thus, the sasp varies considerably depending on the tissue where d-senescent cells are located, the originating cell type, senescence inducers, hormonal milieu, drugs, pathogens and passage of time. other senescent cells, termed here helper-(h-) senescent cells, do not appear to release inflammatory and pro-apoptotic factors substantially and may be involved in promoting stem and progenitor cell determination into appropriate lineages and functions [43, 44] (unpublished observations). hsenescent cells might account for 30 to 70% of senescent cells. we speculate there could be mechanisms through which d-and h-senescent cells can interconvert, but this remains to be fully explored. d-and h-senescent cells may both have beneficial functions, including remodelling of tissues during development, release of factors by placental senescent cells during childbirth that promote passage of the infant through the birth canal, removal of damaged tissue during initial phases of wound repair, and cancer defence [44] [45] [46] [47] . cellular senescence can protect against cancer: senescence is induced by oncogenic insults, leading to replicative arrest of cancerous cells, as well as a pro-apoptotic sasp that can eliminate surrounding tumour cells. additionally, interfering with mechanisms involved in ability of cells to become senescent by decreasing capacity to express p16 ink4a , rb, p53 or p21 cip1 promotes cancer development [48, 49] . however, clearing already formed senescent cells, many of which harbour oncogenic mutations or cause cancer-promoting inflammation due to their sasp, prevents or delays cancer development [46] . indeed, senolytic drugs, agents that selectively eliminate senescent cells [50] , delay development of multiple forms of cancer in old mice as well as cancer-prone, dna repair-deficient ercc1 -/d mice [51, 52] . furthermore, although capacity for cells to become senescent is a cancer defence mechanism, already formed, chronically persisting senescent cells can accelerate cancer growth because of suppression of immune responses, growth factors in the sasp, production of proteases that can interfere with encapsulation of tumours and therefore can enhance spread of cancer, and promotion of mesenchymal transition of cells [10, 53] . thus, targeting transcription factor cascades that allow cells to become senescent can exacerbate cancer development, whilst clearing already formed senescent cells with senolytics can prevent cancer development, growth and spread. there are no completely sensitive or specific markers of senescent cells. most senescent cells are larger than nonsenescent cells. they generally have multiple dna damage foci identifiable by immunostaining for c-histone-2ax (ch2ax), but cancer cells and other cell types, including normal cells, can also have ch2ax-positive foci [54] . in senescent cells, frequently 2 or more ch2ax foci can be located within telomeric dna (telomereassociated foci [tafs]), one of the more sensitive and specific indicators of senescent cells [55] . senescent cells can develop peri-centromeric distended satellite dna (senescence-associated distension of satellites [sads]), which can be detected with antibodies to cenp-b, another marker for detecting senescent cells [56] . senescent cells can have increased lysosomal b-galactosidase activity (senescence-associated b-galactosidase [sa-b-gal]), but so can macrophages and other cell types [57, 58] . some, but not all senescent cells may have high expression of p16 ink4a , but p16 ink4a expression can be high in activated macrophages and many other cell types [59] . like p16 ink4a , p21 cip1 is not fully sensitive or specific for detecting senescent cells [60] . a subset of senescent cells can express sasp factors or damage signals, such as cytosolic hmgb1 [61] , but again, these indicators are not fully sensitive or specific. because of the less than perfect specificity and sensitivity of markers for cellular senescence, generally several need to be tested. this will especially be the case for the initial clinical trials of senolytics for different diseases that are already underway. some senescent cells have enlarged, fused mitochondria and changes in mitochondrial function, including a shift from utilizing fatty acids for energy generation towards utilizing glucose, much like the 'warburg shift' in cancer cells [62, 63] . this shift to dependence on glycolysis can be associated with lipid accumulation, resulting in lipid droplet accumulation and lipotoxicity, contributing to an adipocyte-like appearance of senescent mesenchymal cells or 'mad cells' (mesenchymal adipocytelike default cells) that are insulin-insensitive [15] . an example is the accumulation of pro-inflammatory, perilipin-expressing, lipid-laden senescent ependymal mad cells around the third ventricle in obesity [24] . these cells contribute to neuroinflammation, failed brain microcirculation, impaired neurogenesis and neuropsychiatric dysfunction in obese, insulin-resistant mice. although senescent cells can appear at any point during life in vertebrates, even in 32 cell stage human blastocysts [45] , senescent cells accumulate in multiple tissues during the latter part of the lifespan. for example, skin senescent cells increase sharply in otherwise healthy humans at a point usually between ages 60 and 80 (although there has been debate about this) [64] [65] [66] [67] [68] . the same is true for human adipose tissue biopsied from healthy subjects donating a kidney as well as adipose depots of ageing mice [22, 69] . senescent cells can accumulate at aetiological sites of multiple diseases throughout the lifespan. for example, in humans, senescent cells accumulate in adipose tissue in diabetes and obesity, in the hippocampi and frontal cortex in alzheimer's disease (ad), the substantia nigra in parkinson's disease (pd), bone and marrow in age-related osteoporosis, lungs in idiopathic pulmonary fibrosis, liver in cirrhosis, retinae in macular degeneration, plaques in psoriasis, kidneys in diabetic kidney disease, endothelium in pre-eclampsia, and the heart and major arteries in cardiovascular disease, amongst many other conditions [6, 7, 13, 19, 23, [70] [71] [72] [73] [74] [75] [76] [77] [78] [79] [80] . consistent with a causal contribution of senescent cells to chronic diseases, transplanting small numbers of autologous or syngeneic senescent cells near the knee joints of younger mice causes an osteoarthritis-like state, with radiographic findings similar to those of human spontaneous osteoarthritis, as well as knee pain and impaired mobility, whilst transplanting nonsenescent cells does not [81] . transplanting 10 6 syngeneic adipose cell progenitors or autologous ear fibroblasts made senescent by irradiation, doxorubicin exposure or repeated replication is sufficient to cause frailty and accelerate death from all causes, not just one or a few, in middle-aged mice, whilst transplanting nonsenescent cells does not [51] . transplanting 10 6 senescent human cells into immunodeficient mice also causes frailty. additionally, transplanting senescent porcine renal scattered tubular-like cells into the aorta of young mice causes kidney fibrosis and inflammation in the recipients, whilst transplanting nonsenescent cells does not [82] . there is a lag between senescent cell accumulation and development of functional effects. frailty was evident a month after transplanting senescent cells into middle-aged mice and increased mortality became apparent 3 months later [51] . this study demonstrated that if only 1/10,000 cells in recipient mice is a transplanted senescent cell, an accelerated ageing-like state ensues. transplanting 500 000 senescent cells did not do this, suggesting a senescent cell 'threshold effect'. consistent with this, transplanting 500 000 senescent cells into old mice, as opposed to middle-aged recipients, did cause frailty. furthermore, transplanting 500 000 senescent cells into middle-aged but diet-induced obese (dio) mice caused frailty, whilst 10 6 senescent cells had to be transplanted into age-matched, lean mice for frailty to occur. thus, pre-existing senescent cell burden, together with acute acquisition of yet more senescent cells, can surpass a threshold and cause clinical effects. a potential reason for this 'threshold effect' is that d-senescent cells can spread senescence to normal cells, even at a distance [51] . if senescent cells are labelled with luciferase, so transplanted senescent cells can be distinguished from the recipients' own cells, and the labelled senescent cells are transplanted into the abdominal cavity where they remain, senescent cells originating from the recipient's own cells appear throughout the recipient, including in the limbs. since senescent cells resist cell death and are normally removed by the immune system [10] , the threshold effect may be a result of the rate of spread of senescence exceeding the capacity of the immune system to clear them. this'threshold theory of cellular senescence' may explain the rapid increase in senescent cell abundance in skin and adipose tissue of otherwise healthy people at a point after age 60 [22, [64] [65] [66] [67] , as well as the lag between the first histological evidence for increased senescent cell abundance and subsequent development of symptoms. an article by the sharpless group in 2004 led us to start searching for interventions that remove senescent cells [83] . they reported delayed accumulation of p16 ink4a -, sa-b-gal-positive cells in mice in which healthspan had been extended by caloric restriction or a mutation that decreases growth hormone signalling. this indicates there is an inverse association between senescent cell burden and healthspan. beginning in 2005, we attempted to develop fusion proteins with a senescent cell recognition site and a toxin to kill senescent cells. we then attempted to find chemicals that would kill senescent but not nonsenescent cells using high-throughput screens of human senescent vs. nonsenescent cells. neither approach was successful. the high-throughput screening approach suffered from difficulties in selecting the right controls. parallel nondividing, nonsenescent cells had to be serum-starved or confluent in order to prevent replication. however, serum starvation or confluence both cause artefactual effects. alternatively, dividing nonsenescent cells could be used as controls. however, these dividing control cells have to be compared to senescent cells that, by definition, cannot divide, introducing a confounder that interferes with interpretation of the results. thus, rather than random high-throughput drug library screening or other traditional drug development strategies, we turned to a hypothesisdriven, mechanism-based drug discovery paradigm to develop the first senolytic drugs. our strategy was based on the 1995 observation by e. wang that senescent cells resist apoptosis [84] . this apoptosis resistance occurs despite senescent cell production of pro-apoptotic sasp factors. the existence of the sasp had been discussed within the field for a couple of years before it was published [28, 29, 85] . our hypotheses upon which senolytic drug discovery was based were: 1) senescent cells can resist apoptotic stimuli, implying increased pro-survivalanti-apoptotic defences and 2) in some respects, senescent cells are like cancer cells that do not divide, including apoptosis resistance and metabolic shifts. we speculated that senescent cells might prevent their own removal through protective senescent cell anti-apoptotic pathway (scap) networks [86] . from proteomic and transcriptomic databases, we found that scap pathways were more highly expressed by senescent than nonsenescent cells [86] . we interrogated the scap network using rna interference and demonstrated that senescent cells with a pro-apoptotic sasp do indeed depend on this network to evade self-destruction. the small interfering rna's (sirna's) against antiapoptotic regulators that were effective in selectively decreasing senescent cell viability depended on the senescent cell type. for example, we found that sirna's that decrease bcl-xl or other bcl-2 family members were effective in selectively causing apoptosis of senescent vs. nonsenescent human umbilical vein endothelial primary cells (huvec's) [86] , as was confirmed by others [87] . however, the same sirna's were ineffective in selectively inducing apoptosis of senescent vs. nonsenescent human primary adipocyte progenitor cells. furthermore, more than one scap pathway had to be targeted to kill some types of senescent cells, indicating that senescent cell defences against apoptosis are in some cases redundant. next, we used bioinformatics approaches to find compounds whose mechanisms of action involve targeting the scap network nodes that we had identified as being critical for protecting senescent cells from apoptosis. we identified 46 potentially senolytic compounds. to accelerate translation into clinical application, the compounds that we focused on initially were drugs and natural products already in use for other indications in humans, many as anti-cancer agents. these included the tyrosine kinase inhibitor, dasatinib (d), which has been approved for clinical use in the united states since 2006, and quercetin (q), the naturally occurring flavonoid that makes apple peels taste bitter. unlike other tyrosine kinase inhibitors such as imatinib, which are not senolytic, d promotes apoptosis caused by dependence receptors, such as the ephrins, in part by inhibiting src kinase [88, 89] . fisetin (f) is another naturally occurring flavonoid that is senolytic [52, 90] . the senolytic flavonoids act in part by inhibiting bcl-2 family members such as bcl-xl as well as hif-1a and other scap network components. based on their known targets, we predicted that d would target senescent human cultured primary adipocyte progenitor cells, whilst q would target senescent cultured huvec's. these predictions proved to be correct. the combination of d + q caused apoptosis of both senescent human primary adipocyte progenitor cells and senescent huvec's, but not nonsenescent adipocyte progenitor cells or huvec's. furthermore, likely because of redundancy of scap pathways, in some cell types, for example, mouse embryonic fibroblasts, neither d nor q are senolytic on their own, whilst the combination of d + q is senolytic [86] . the target of true senolytics is senescent cells, not a single receptor, enzyme or biochemical pathway. efforts to discover senolytics by finding agents that act on multiple scap network nodes effectively increases specificity for senescent cells, reduces off-target effects on nonsenescent cells compared with candidates that act on a single molecule, such as a receptor or an enzyme, and likely flatten sideeffect profiles. consistent with this prediction, candidates that do act upon single or limited targets, for example, navitoclax (n) [91, 92] or nutlin3a [93] , have substantial off-target apoptotic effects on nonsenescent cell types, such as platelets and immune cells [94, 95] , making them potentially 'panolytic'. also, nutlins can actually cause senescence [96] and drugs like n, which act on a single or few scap nodes, eliminate only a restricted range of senescent cells. for example, n and other bcl-2 family member inhibitors such as a1331852 or a1155463, are not senolytic against human adipocyte progenitors, one of the most abundant senescent cell types in the elderly as well as patients with diabetes and obesity [90, 91] . thus, candidates that act on only one or a limited range of scap-related molecules are generally less specifically senolytic and are more 'panolytic', causing apoptosis or dysfunction of multiple cell types other than senescent cells, than agents or combinations of agents that act on multiple nodes across the scap network. the first senolytics were discovered using bioinformatics approaches to identify agents that transiently disable the scap networks that allow dsenescent cells to survive the hostile microenvironment they create that kills the nonsenescent cells around them (table 1 ). in this respect, the development of senolytics has been distinct from the conventional approaches for developing drugs usually followed in other fields. approaches for developing senolytics has held more in common with those used to develop antibiotics than the traditional one target-one drug-one disease approach. a second generation of senolytic agents is now being identified using more traditional drug discovery methods, including random highthroughput drug library screens, such as one in which both senescent and nonsenescent cells are within the same wells, overcoming the control cell problem [97] . several other strategies have recently been used to develop potentially senolytic interventions. these include the development of galacto-oligosaccharide-coated nanoparticles with toxic cargos that are engulfed by senescent cells and opened by sa-b-gal, vaccines and immunomodulators [10, [98] [99] [100] . perhaps some of these will make it through preclinical studies and into clinical trials within the next few years. d + q and f have been shown to reduce senescent cell abundance in vivo whilst not destroying nonsenescent cells [51, 52] . after intraperitoneal transplantation of 10 5 radiation-induced syngeneic senescent adipocyte progenitors, which could be detected due to constitutive sffv promoter-driven luciferase expression, into middle-aged mice, treatment with oral d + q for 3 days substantially decreased luminescence 2 days after the last dose [51] . no decrease in luminescence was evident after treating senescent cell-transplanted mice with vehicle, nor was decreased luminescence found after treating mice transplanted with nonsenescent luciferase-expressing cells with either d + q or vehicle. oral treatment with f of progeroid ercc -/d mice expressing luciferase driven by a p16 ink4a promoter element also resulted in decreased luminescence, whilst treating these mice with vehicle had no effect. thus, d + q and f are senolytic in mice, each eliminating 30 to 70% of transplanted or endogenous senescent cells, respectively. to test if d + q is senolytic in human tissue, abdominal subcutaneous adipose tissue fragments that were freshly isolated during surgery from patients with diabetes and obesity, who have extensive adipose tissue senescent cell accumulation [108] [109] [110] [111] [112] , were immediately placed in organ culture and exposed to d + q or vehicle [51] . within 2 days, abundance of senescent cells detected by tafs had decreased by 70%. this was accompanied by increases in cleaved caspase-3, confirming that the decrease was caused by apoptosis. furthermore, the sasp factors, il-6, il-8, mcp-1, pai-1 and gm-csf, decreased journal of internal medicine significantly in conditioned medium from the organ cultures treated with d + q compared to vehicle. also, f reduced senescent cell number in adipose tissue organ cultures [52] . thus, d + q and f cause decreased senescent cell abundance by apoptosis in human tissue within 2 days. senolytic drugs alleviate a number of disorders in mouse disease models [6] [7] [8] 108] (table 2) . intermittent oral administration of d + q enhances cardiac and vascular function in 24-month-old mice (equivalent to roughly 80 years in humans), with partial reversal of age-related declines in cardiac ejection fraction, reduction in conduit vessel calcification and restoration of conduit vessel reactivity to nitroprusside and acetylcholine [113] . d + q decreases cardiac senescent cell burden, as manifested by lowering of p16 ink4a , and partly reverses age-related cardiomyocyte hypertrophy and left ventricular fibrosis [13] . senescent cells can develop in the venous arm of arterialvenous (av) fistulae of the type surgically fashioned for haemodialysis, potentially contributing to clotting and failure of the av fistulae [114] . in mice, d + q decreases senescent cell abundance in av fistulae. furthermore, d + q reduces senescent cell burden and calcification in the aortic media of high fat-fed apoe -/mice, a mouse model of atherosclerosis [113] . n is also senolytic in the hearts of old mice [18] . following 1 month of n, there was decreased cardiac fibrosis and a 40% decrease in taf + cardiomyocytes (it appears nondividing cells such as cardiomyocytes can develop a senescent-like state). thus, senolytics alleviate age-and lipid-related cardiovascular dysfunction in mice. intermittent oral d + q administration enhances insulin sensitivity in dio as well as genetically obese mice, as manifested by improvements in glucose and insulin tolerance [111] . d + q does so mainly by reducing peripheral insulin resistance. this is in part due to restoring adipose tissue akt responses to insulin and in part to restoration of the capacity of adipocyte progenitors to differentiate into insulin-responsive adipocytes. furthermore, intermittent d + q reduces the adipose tissue inflammation that is a causal contributor to the pathogenesis of type 2 diabetes. d + q does so in part by reducing homing of monocytes to adipose tissue: there is decreased trafficking of luciferase-expressing monocytes injected into the tail vain into adipose tissue of genetically obese mice treated with d + q, but not in mice treated with vehicle. d + q does not kill the activated macrophages that can accumulate in adipose tissue in diabetes [51] . these macrophages have increased p16 ink4a expression but are not senescent [59] . consistent with this, senolytic drugs also do not target atherosclerotic foam cells, which are essentially activated macrophages, as opposed to the truly senescent cells that are deeper within plaques or the media of atherosclerotic blood vessels that are ablated by d + q [113] . n, which is not effective in targeting senescent adipocyte progenitors [91] , does decrease senescent high-throughput compound library screens [98] vaccines [100, 108] toxin-loaded nanoparticles preferentially lysed by senescent cells [99] immunomodulators [101] pancreatic b cells, enhancing pancreatic function in dio mice [115] . thus, senolytics can alleviate diabetes by decreasing sasp-expressing adipose tissue senescent cells, leading to increased peripheral insulin sensitivity, reducing chemo-attraction and activation of pro-inflammatory macrophages that further contribute to insulin resistance, and/ or enhancing pancreatic insulin production. senolytics decrease several complications of diabetes in mouse models. liver fat accumulation and consequent cirrhosis and liver failure are increasingly common complications of diabetes and obesity [108, 109] . d + q reduces liver senescent cell accumulation related to hepatic steatosis in genetically obese mice [136] . additionally, proteinuria and renal dysfunction are frequent complications of diabetes. dio mice develop increased kidney senescent cell burden manifested by p16 ink4a and p53 expression and sasp factors in renal tubular cells along with glomerulomegaly, decreased renal function and impaired cortical oxygenation compared to lean mice [128] . proteinuria is reduced in dio mice treated with d + q [110] . dio mice with renal dysfunction that were treated with oral q for 5 days every 2 weeks for 10 weeks had decreased kidney senescent cells, decreased sasp factors, decreased renal fibrosis, increased renal cortical oxygenation and decreased plasma creatinine levels compared to mice treated with vehicle [128] . another complication of diabetes/obesity is neuropsychiatric dysfunction, particularly anxiety and its complications [24] . in genetically obese mice, senescent adipocyte-like ependymal mad cells can accumulate near the third ventricles near the limbic system, which regulates emotions. d + q reduces the burden of these senescent adipocytelike periventricular ependymal cells. administering d + q also increases neuronal markers, indicating improved neurogenesis and reduces neuro-inflammation in obese mice. importantly, intermittent table 2 . conditions with emerging evidence for a causal contribution of cellular senescence or benefits of senolytics diabetes/ obesity [109, 110, 112, [116] [117] [118] cardiac dysfunction [13, 18, 114] vascular hyporeactivity/ calcification [114] av fistulae [115] frailty [21, 51, 52, 87] age-related muscle loss (sarcopenia) [119] chemotherapy complications [21, 51, 93, 120, 121] radiation complications [122] cancers [51] bone marrow transplant complications [120] organ transplantation complications [123, 124] myeloma/ mgus [125] age-related cognitive dysfunction [126] alzheimer's disease [23, 127] parkinson's disease [76] amyotrophic lateral sclerosis [128] ataxia [87] obesity-related neuropsychiatric dysfunction [24] renal dysfunction [111, 129] urinary incontinence [87] osteoporosis [14, 71, 130] osteoarthritis [131] age-related intervertebral disc disease [87, 132] idiopathic pulmonary fibrosis [21, 133] hyperoxic lung damage [134] chronic obstructive pulmonary disease [135] tobacco [136] hepatic steatosis [137] cirrhosis [78] primary biliary cirrhosis [138] progerias [52, 87] pre-eclampsia [72] macular degeneration [75, 139] glaucoma [140] [141] [142] cataracts [143] prostatic hypertrophy [144] [145] [146] psoriasis [81] healthspan [51, 52, 87] lifespan [51, 52, 87] d + q alleviates anxiety in these obese mice, as manifest by reduced fear in open-field testing. thus, senolytics not only restore peripheral insulin responsiveness in diabetic/obese mice, they alleviate complications of diabetes for which no effective mechanism-based treatments currently exist. in addition to diabetic/obesity-related neuropsychiatric dysfunction, senolytics alleviate alzheimer's-like changes in tau + as well as b-amyloid + (ab) mouse models of alzheimer's disease [23, 126] . accumulation of tau protein aggregates is a common pathology across multiple degenerative brain diseases, including ad, progressive supranuclear palsy, traumatic brain injury and over twenty others. furthermore, tau-containing neurofibrillary tangles correlate with cognitive decline and brain cell loss. neurofibrillary tangle-containing neurons from postmortem human ad brains have expression profiles that are consistent with cellular senescence. also, in both mice and humans, p16 ink4a expression correlates directly with brain atrophy and neurofibrillary tangles. in 23-monthold tau nft -mapt 0/0 mice that overexpress tau, d + q every 2 weeks for 3 months reduced total neurofibrillary tangle density, neuronal loss, neuro-inflammation, gliosis, small vessel hypoperfusion and ventricular enlargement and increased cortical volume and neurogenesis, despite the advanced age of these mice. administering d + q to ab + mice was also effective, with selective removal of senescent cells from the ad-associated plaque environment, reduced neuro-inflammation, decreased ab load and alleviation of deficits in cognitive function, with improved memory and executive function [126] . thus, senolytic drugs may potentially prove to be effective, mechanismbased interventions for alleviating ad and other chronic brain diseases, depending on results of the clinical trials that are underway. senolytics alleviate dysfunction in mouse models of chronic lung diseases. idiopathic pulmonary fibrosis (ipf) is a progressive, fatal, senescence-driven lung disease for which there are no highly effective interventions apart from lung transplantation [21] . it affects older patients, leading to respiratory failure, pronounced frailty and weight loss. targeting senescent cells with d + q was effective in alleviating frailty as shown by improved treadmill endurance, reducing extent of weight loss, and enhancing lung compliance in a mouse bleomycin inhalation-induced pulmonary fibrosis model of ipf. another lung condition that might be alleviated by senolytics is hyperoxia-induced damage to airway structure and function [133] . supplemental o 2 can be a necessary intervention in premature infants, but appears to contribute to development of neonatal and paediatric asthma. at least in cell culture, d + q shows promise for alleviating complications of supplemental o 2 . foetal airway smooth muscle (asm) exposed for 7 days to 40% o 2 developed markers of senescence, including sa-bgal and increased p16 ink4a , p21 cip1 , p-p53, ch2a.x and pro-fibrotic and inflammatory sasp factors. treating na€ ıve asm cells with media from hyperoxia-exposed senescent asm cells increased collagen, fibronectin and contractility. d + q selectively reduced the number of hyperoxia-induced senescent asm cells. hence, senolytic drugs may become interventions for multiple lung diseases. indeed, in the first clinical trial of senolytics, d + q alleviated physical dysfunction in a preliminary study in patients with ipf [70] (see below). additionally, the senolytic, f, reduces mortality in mice infected with mouse bcoronavirus and covid-19 viral antigens exacerbate the sasp in human senescent cells (unpublished observations). based on these discoveries, a clinical trial of f for older hospitalized covid-19 patients to prevent progression to cytokine storm and acute respiratory distress syndrome has been approved by the us food and drug administration (fda) and is about to start. age-related, as opposed to hormone-deficiencyrelated, osteoporosis appears to be associated with senescent cell accumulation, particularly senescent-like osteocytes and bone marrow cells [71, 129] . osteoclasts cause bone resorption. conditioned medium from senescent cells increases early monocyte osteoclast progenitors due to enhanced survival of these cells [14] . the sasp factors, il-6, il-8 and pai-1, contribute to this increased survival of osteoclast progenitors. in old (20 months) mice with osteoporosis, d + q treatment once monthly for 4 months led to significant reductions in bone p16 ink4a mrna and sads + senescent osteocytes [14] . in d + q-treated mice, trabecular bone histomorphometry demonstrated reduced osteoclasts vs. in vehicle-treated mice. d + q treatment increased femoral cortical thickness and bone strength. this was associated with reduced endocortical surface osteoclast numbers and increases in endocortical numbers of osteoblasts, the cells that form new bone, mineral apposition rate and bone formation rate. these beneficial effects of targeting senescent cells resulted in suppression of bone resorption accompanied by either maintenance (trabecular) or increased (cortical) bone formation. thus, unlike the coupled decreases in bone resorption and formation that results from currently available antiresorptive treatments for osteoporosis, d + q causes decreased bone resorption whilst not causing a coupled decrease in bone formation. these beneficial effects result from a decrease in development of bone-resorbing osteoclasts and an increase in development of bone-forming osteoblasts from their respective stem cells/progenitors. furthermore, intermittent d + q was as effective as continuous d + q, consistent with our contention that a 'hit-and-run' approach of senolytic administration is effective and could decrease adverse effects. a clinical trial of d + q for agerelated osteoporosis is currently underway (see below). n also decreases bone senescent cells in old mice but, unlike d + q, does not alleviate agerelated osteoporosis because, consistent with its 'panolytic' side-effect profile, n is directly cytotoxic to bone-forming osteoblasts [146] . in addition to age-related osteoporosis [129] and osteoarthritis [130] , senescent cells contribute to another skeletal disorder, age-related intervertebral disc disease. glycosaminoglycan content of the nucleus pulposus of intervertebral discs declines with ageing in progeroid mice [86] . d + q increases spinal glycosaminoglycan levels in progeroid mice compared to animals receiving vehicle only. since senolytics increase vertebral proteoglycans in preclinical models, they might prove to be an effective treatment for intervertebral disc disease. d + q or f alleviate physical dysfunction across a range of mouse models in which senescent cells are linked to frailty: naturally aged mice, progeroid ercc -/d mice, senescent cell-transplanted younger mice and mice with bleomycin-induced pulmonary fibrosis [21, 51, 52, 86] . in these mouse models, senolytics prevented frailty or restored physical function as assessed by daily activity (movement detected by laser measurement), distance run on a treadmill, treadmill speed, grip strength, time able to hang on a suspended wire, and/or ability to remain on a rotating spindle (rotarod test). d + q as well as f alone both extended remaining lifespan in old mice by up to 35% [52, 86] . consistent with the geroscience hypothesis, d + q delayed death in old mice not due to any single age-related disease, but from multiple disorders that cause death in naturally aged, untreated mice [86] . as would be expected from their use as anticancer drugs, the senolytics d + q and f delay death from cancers in mice [51, 52] . over 50% of deaths in most mouse strains are caused by cancers, and d + q and f delay deaths of old mice by up to 35 and 17%, respectively. furthermore, even though f might cause aneuploidy in cultured nonsenescent cells [147] , f delays cancers in cancer-prone erc-c1 -/d mice that have a dna repair enzyme mutation [52] . in these preclinical studies, it would be desirable to prove that a candidate agent alleviates a phenotype because it is actually a senolytic drug. an approach for proving senolytic effects is to use a modified set of koch's postulates. if an agent is truly senolytic, then: 1) senescent cells should be present in association with the phenotype, 2) individuals without senescent cells should not have the phenotype, 3) inducing accumulation of senescent cells should cause the phenotype, 4) clearing these induced senescent cells should alleviate the phenotype, 5) clearing naturally occurring senescent cells should alleviate the phenotype, 6) the drug should induce few or no effects related to the phenotype in young individuals without senescent cells, 7) administering the senolytic candidate intermittently should be effective and 8) the senolytic candidate should alleviate multiple agerelated conditions. in the case of d + q, these modified koch's postulates have been met in mice for age-and cellular senescence-related physical dysfunction, insulin resistance, cognitive dysfunction, osteoporosis, osteoarthritis and cancers in the published studies cited above and in unpublished studies. evidence consistent for a true senolytic effect of f is mounting. to our knowledge, there is not yet comprehensive evidence for a true senolytic effect of n or nutlin3a that meets this modified set of koch's postulates. since clearing senescent cells with senolytic drugs is a completely new therapeutic paradigm, a novel strategy for translating senolytics into interventions for humans is needed (fig. 3) . in particular, ethical risk/benefit concerns have to be addressed: situations in which senolytic drugs enter the first clinical trials should ideally be serious and be those for which currently available treatments are ineffective (fig. 3) , such as ipf, complications of advanced diabetes, diastolic heart failure, advanced osteoporosis, dementias or covid-19 infections in hospitalized elderly patients. to accelerate progress, our approach has been to start multiple small, parallel trials for such conditions instead of conducting trials one after the other in series. if these trials in serious disorders show acceptable side-effect profiles, if the agents are well tolerated, if target engagement (i.e. killing senescent cells) can be demonstrated, and if the candidate senolytic drugs are effective in treating serious diseases, then use of senolytics could go on to be tested for less serious conditions related to cellular senescence. depending on results of such trials, the step after that would be to test effectiveness of the candidate senolytic drugs in delaying or preventing clinically manifest disease in subjects in whom presence of senescent cells can be demonstrated by blood, imaging, saliva or urine tests or biopsies. this could be feasible, since we discovered that there is a lag between accumulation of senescent cells and appearance of clinically apparent disease: after transplanting senescent cells into middle-aged mice, it took time for frailty to develop [51] . as this progression from: 1) administration of senolytics in clinical trials for serious diseases, 2) to less serious conditions, 3) to prevention in at-risk individuals proceeds, clinical trials will need to become progressively larger, longer and more expensive. ideally, the first clinical trials should be with agents that have, insofar as possible, an established clinical safety profile, with strong preclinical data available about the drugs and indications to be tested. intermittent administration, as opposed to continuous dosing, could reduce off-target sideeffects. the clinical trials should be carefully monitored for safety, tolerability, target engagement (removal of senescent cells) and effectiveness. to speed translation, ideally several of these trials would be conducted in parallel across several diseases, rather than conducting one trial after the other in series. if safe and effective in initial trials for serious diseases, moving a senolytic drug towards less serious conditions and, eventually, to prevention in humans with an increased senescent cell burden and who are at risk for subsequently developing cell senescence-associated diseases, could be a translational strategy. it is currently too early for practicing physicians to prescribe senolytic agents outside the context of carefully monitored clinical trials. a consideration is whether systemic or local administration of candidate senolytic drugs is the best approach for initial clinical trials. for agents with an unknown or poor safety profile, local administration for senescence-related conditions could be an option, but the following points run counter to this: 1) senescence spreads from cell to cell [51] , meaning that apparently local accumulations of senescent cells could be the 'tip of the iceberg', with an increased systemic senescent cell burden being a possible consequence. this could be the case even if effects of this systemic senescent cell burden are not yet apparentas discussed above, there is a lag between senescent cell accrual and appearance of clinically apparent symptoms. 2) also consistent with the concern that local injection of senolytics would be less beneficial than systemic administration is the geroscience hypothesis: multiple age-and figure 3 early clinical studies of senolytics: balancing benefit and risk. since senolytic drugs are at the forefront of a completely new potential therapeutic paradigmtargeting fundamental ageing processes to delay, prevent or alleviate age-related phenotypes, multiple diseases, geriatric syndromes and reductions in physical resiliencea careful risk-benefit balance must be struck within firstin-human senolytic drug clinical trials, since potential short-and long-term side-effects from clearing senescent cells are not yet fully known. senescence-related diseases cluster within individuals, reducing the possible benefits from local injection. 3) locally injected drugs generally do not stay local, particularly if the injection is into, for example, an osteoarthritic knee joint in which the synovial cavity is damaged. in the absence of an intact synovial membrane, local injection into the damaged knee joint could be effectively systemic, more like an intramuscular injection than a truly intra-articular injection into a patient with an intact knee joint. 4) the previous points notwithstanding, local skin application, particularly of poorly absorbed senolytics, might be a viable strategy for treating senescence-related skin disorders, such a melanotic naevi or psoriasis. therefore, in general, systemic senolytic administration in initial clinical trials appears to be a reasonable approach, especially in the cases of candidates with an established safety profile, such as d, which has been approved for clinical prescription in the united states since 2006, or the natural product, q. this would be less true for n or a1331852, which are not yet approved for general use as prescription drugs in the united states. in a preliminary report from an early, open-label clinical trial of 3 days of d + q administration orally in patients with diabetes complicated by renal dysfunction, evidence for target engagement was found. compared to biopsies before d + q administration, 11 days after the last dose of d + q (which has an 11 hour elimination half-life), p16 ink4a+ and sa-bgal + senescent cells had decreased in the diabetic subjects' adipose tissue [116, 117] . furthermore, d + q decreased adipose tissue inflammation as evident by reduced macrophages and immune infiltration-related crown-like structures. a composite score of blood sasp factors was also decreased by d + q in these patients. thus, as in mice, d + q can successfully decease senescent cell burden in humans. the first-in-human trial of senolytics was of d + q for patients with ipf [70] (n = 14 patients with stable ipf) in an open-label study of intermittent d + q (3 days/week over 3 weeks). physical function was evaluated by testing 6-minute walk distance, 4-metre gait speed and chair-stands time. these assessments were significantly and clinically meaningfully improved by d + q (p < 0.05). furthermore, correlations were found between change in function and change in sasp-related pro-inflammatory cytokines, matrix-remodelling proteases and micro-rnas (23/48 markers; r ≥ 0.50). this pilot study supports the feasibility of further clinical trials using senolytics. it provided initial evidence that senolytics can alleviate physical dysfunction in ipf, supporting evaluation of d + q in larger randomized, controlled trials for senescence-associated diseases. several clinical trials of senolytics are currently underway or planned ( it should be understood that the target of senolytics is senescent cells, not a single molecule or pathway, since targeting networks might yield more truly senolytic, less panolytic, drugs. additionally, consistent with true senolytic activity, intermittent treatment with d + q or f appears to be as effective as continuous administration, despite their short elimination half-lives. d + q already satisfies a modified set of koch's postulates for certain disorders and mounting evidence indicates that these postulates could also be satisfied for a number of other conditions in the cases of d + q and f. true senolytic effects remain to be established for n or nutlin3a. consistent with the unitary theory, senolytics appear to restore progenitor dysfunction, attenuate tissue inflammation and alleviate age-and disease-related metabolic dysfunction across cell types and tissues. senolytics appear to delay, prevent or alleviate multiple age-related conditions and chronic diseases and enhance healthspan and lifespan in experimental animals. therefore, these agents could lead to interventions for humans that delay, prevent or treat senescence-and age-related conditionsif clinical trials continue to demonstrate effectiveness and low toxicity. however, unless and until such clinical trials are completed and demonstrate safety, tolerability, target engagement and effectiveness, candidate senolytics should not be prescribed or used by general patient populations. they should only be administered in the course of carefully monitored clinical trials. translating the science of aging into therapeu-ticiinterventions translating advances from the basic biology of aging into clinical application resilience in aging mice life expectancy and education: the author replies risk of developing multimorbidity across all ages in an 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of transcription factor brn3b in an ocular hypertension rat model of glaucoma glaucoma: a disease of early cellular senescence effects of senescent lens epithelial cells on the severity of age-related cortical cataract in humans: a case-control study cellular senescence in the pathogenesis of benign prostatic hyperplasia tchkonia 18 ª 2020 the authors expression of senescence-associated beta-galactosidase in enlarged prostates from men with benign prostatic hyperplasia the senescenceassociated secretory phenotype promotes benign prostatic hyperplasia the senolytic drug navitoclax (abt-263) causes trabecular bone loss and impaired osteoprogenitor function in aged mice a comparative study of the aneugenic and polyploidy-inducing effects of fisetin and two model aurora kinase inhibitors dr. yi zhu worked with the authors in 2013 to discover the first senolytic drugs as reported in march, 2015 [87] . the authors acknowledge support from the journal of internal medicine, us nih grants r37 ag013925, p01 ag062413 and r33 ag061456 (translational geroscience network), the connor fund, robert j. and theresa w. ryan and the noaber foundation. the authors have a financial interest related to this research. patents on senolytic drugs are held by mayo clinic. this research has been reviewed by key: cord-300372-h5g4z8ts authors: kelvin, alyson a.; degousee, norbert; banner, david; stefanski, eva; leon, alberto j.; angoulvant, denis; paquette, stéphane g.; huang, stephen s. h.; danesh, ali; robbins, clinton s.; noyan, hossein; husain, mansoor; lambeau, gerard; gelb, michael h.; kelvin, david j.; rubin, barry b. title: lack of group x secreted phospholipase a(2) increases survival following pandemic h1n1 influenza infection date: 2014-04-01 journal: virology doi: 10.1016/j.virol.2014.01.030 sha: doc_id: 300372 cord_uid: h5g4z8ts the role of group x secreted phospholipase a(2) (gx-spla(2)) during influenza infection has not been previously investigated. we examined the role of (reviewer 2 minor comment 2) gx-spla(2) during h1n1 pandemic influenza infection in a gx-spla(2) gene targeted mouse (gx(−/−)) model and found that survival after infection was significantly greater in gx(−/−) mice than in gx(+/+) mice. downstream products of gx-spla(2) activity, pgd(2), pge(2), ltb(4), cysteinyl leukotrienes and lipoxin a(4) were significantly lower in gx(−/−) mice bal fluid. lung microarray analysis identified an earlier and more robust induction of t and b cell associated genes in gx(−/−) mice. based on the central role of spla(2) enzymes as key initiators of inflammatory processes, we propose that activation of gx-spla(2) during h1n1pdm infection is an early step of pulmonary inflammation and its (reviewer 2 minor comment 2) inhibition increases adaptive immunity and improves survival. our findings suggest that gx-spla(2) may be a potential therapeutic target during influenza. influenza is a leading source of morbidity and mortality worldwide that is caused by ever changing and newly emerging influenza viruses including the introduction of the 2009 a/h1n1/2009 (h1n1pdm) and novel avian h7n9 viruses (fisher et al., 2005; groom and luster, 2011; widegren et al., 2011) . while effective vaccines and antiviral drugs have been developed for circulating strains of human influenza (santone et al., 2008) , continued antigenic drift and shift generate novel virus strains that pose a threat to immunologically naïve populations. the emergence of pandemic influenza h1n1pdm in the spring of 2009 led to hundreds of thousands of hospitalizations with significant numbers of fatalities in north america (update: influenza activity -united states, 2009-10). severe cases were characterized by viral pneumonia and uncontrollable pulmonary inflammation, and were similar to the inflammation observed in severe cases of sars, h5n1 and spanish influenza patients (baillie and digard, 2013; bermejo-martin et al., 2010; cameron et al., 2012; curfs et al., 2008) importantly there is little understood regarding the pathways driving the pulmonary inflammatory process for these diseases. host-defenses against influenza include anatomic barriers, mucociliary clearance, anti-microbial secretions and innate and adaptive immune responses. early host responses are characterized by the mobilization of leukocytes, such as alveolar and circulating macrophages, polymorphonuclear leukocytes (pmn) (world health organization, 2012) and nk cells which continues into the activation of adaptive immune cells such as t-cells, b-cells and dendritic cells. importantly, factors that lead to the activation of these cells and cell networks are increased after h1n1pdm infection (influenza activity -united states and worldwide, 2010; ohtsuki et al., 2006; ohtsuki et al., 2006; paquette et al., 2014) . these include inflammatory mediators such as chemokines, cytokines and lipid mediators like eicosanoids. the first step in the generation of eicosanoids, inflammatory mediators that participate in the regulation of the inflammatory response, is catalyzed by pla 2 enzymes, which release arachidonic acid (aa) from phospholipids (del et al., 2007) . to date, 11 secreted pla 2 enzymes (spla 2 ; ib, iia, iic, iid, iie, iif, iii, v, x, xiia and xiib) , six cytosolic pla 2 enzymes (cpla 2 s; α, β, γ, δ, ε, and ξ) (kim et al., 2007) , nine ca 2 þ -independent pla 2 enzymes (ipla 2 s; α, β, γ, δ 2 , δ, ε, ε2 ξ, θ, and η) , and two lysosomal pla 2 enzymes (femling et al., 2005) have been described. the spla 2 enzymes are structurally related, ca 2 þ -dependent proteins with unique biological properties, enzymatic activities against membrane phospholipids and tissue and cellular locations, suggest distinct roles for these enzymes in various pathophysiological events. spla 2 enzymes are implicated in lipid mediator release, degranulation, cellular proliferation, destruction of invading bacteria , viruses (kennedy et al., 1995; mazur et al., 2007) and activation of intracellular signaling cascades (kim et al., 2007) . gx-spla 2 is expressed in alveolar macrophages and epithelial cells in the lungs of patients with pneumonia (marshall et al., 2000) , neuronal cells (gaudreault and gosselin, 2008) , male reproductive organs (dennis, 1994) and atherosclerotic plaques , and is cleaved to its active form in inflamed tissues (lu et al., 2006) . arachidonic acid (aa) is the precursor of prostaglandins, thromboxanes, leukotrienes and lipoxins, eicosanoids that regulate pulmonary vascular and bronchial responses, leukocyte activation, adhesion and emigration (guan et al., 2013; henderson et al., 1995) . eicosanoids also regulate antigen presenting cell [apc] function (degousee et al., 2001; murakami et al., 2011; truchetet et al., 2012) , t cell maturation (myers et al., 2012) and th17 expansion (stephenson et al., 1988; zhao et al., 2011) . we found that il-17 and th17 cells are dysregulated during human h1n1pdm infection (baillie and digard, 2013; bermejo-martin et al., 2010; ohtsuki et al., 2006) . therefore, spla 2 enzymes and eicosanoids may play a central role in determining the outcome of pulmonary viral infection. the role of spla 2 enzymes in the immune responses to h1n1pdm infection in vivo has not been evaluated. gx-spla 2 has been highly implicated in the inflammatory response including pattern recognition receptor function, and displays the highest activity among all mammalian spla 2 s on phosphatidylcholine-rich liposomes in vitro (crooks and stockley, 1998; henderson et al., 1995) . recently, gx-spla 2 has been suggested as a signal amplifier in tlr4 stimulation which further suggests a role for gx-spla 2 in the regulation of the inflammatory response (schultz-cherry and jones, 2010) . considering the potential of gx-spla 2 in the inflammatory response, spla 2 enzymes may play a central role in determining the outcome of pulmonary viral infections, which cause uncontrolled inflammatory destruction of the respiratory tract (henderson et al., 1995) . we have developed a robust lethal mouse model of h1n1pdm infection to study innate host defense mechanisms and antiviral compound activity . we have shown that h1n1pdm infection in this mouse model leads to pulmonary inflammation, a histopathological picture similar to what is observed in fatal human cases, and over 90% lethality within 5-8 days . in this study, we document a marked increase in gx-spla 2 expression in lung following infection in gx þ / þ mice. to specifically evaluate the pathophysiological role of gx-spla 2 in our lethal influenza mouse model, we subjected gx þ / þ and gx à / à mice (henderson et al., 1995) to h1n1pdm infection in vivo. our results showed that, in two distinct mouse strains, targeted deletion of gx significantly increased survival following h1n1pdm infection in comparison with gx þ / þ mice. in addition, eicosanoid accumulation in bal fluid was attenuated and induction of t cell and b cell associated genes was higher in gx à / à than gx þ / þ mice after h1n1pdm infection. taken together, our data suggests a negative role for gx-spla 2 in the immune response to pulmonary infection with h1n1pdm influenza in vivo. furthermore, these findings implicate gx-spla 2 as a potential therapeutic target during severe influenza infection. airway epithelial cells and myeloid cells can both express gx-spla 2 (marshall et al., 2000) . previously, we have investigated the host immune responses to pulmonary viral infections, including infection with the influenza viruses h5n1 and h1n1pdm (baillie and digard, 2013; bermejo-martin et al., 2010; bhavsar et al., 2010; cameron et al., 2008; cameron et al., 2012; escoffier et al., 2010; huang et al., 2009; huang et al., 2012; kudo and murakami, 1999) . furthermore, we have also delineated the biology and molecular regulation of many of the enzymes that catalyze eicosanoid biosynthesis in vitro and in vivo degousee et al., 2006; degousee et al., 2008; degousee et al., 2002; degousee et al., 2003; leon et al., 2012; lu et al., 2006; saez de et al., 2011) . to begin to investigate the role of gx-spla 2 during h1n1pdm infection, we evaluated the pulmonary expression of gx-spla 2 in our h1n1 pandemic influenza mouse model. gx þ / þ mice were infected intranasally with a/mexico/4108/ 2009 (h1n1pdm) and lung tissues were harvested at baseline and on day 3, 6 and 14 post infection (pi). real-time pcr was performed on the extracted rna and identified a significant increase in the ratio of gx-spla 2 to gapdh mrna on day 3 and day 14, but not day 6 pi (fig. 1ai) . gx-spla 2 /gapdh mrna increased approximately four fold on day 3 and three fold on day 14 compared to baseline. furthermore, we also determined the regulation of cytosolic pla 2 (cpla 2 ) (fig. 1aii ) and the spla 2 family member gv-pla 2 (fig. 1aiii ) in both gx þ / þ and gx à / à mice. there was negligible total protein upregulation of cpla 2 and gv-pla 2 was not regulated throughout the infection time course. the absence of the change of total protein of cpla 2 was also confirmed by immunohistochemistry (data not shown). no statistical differences in mrna transcripts for cpla 2 and gv-pla 2 were noted between the gx þ / þ or gx à / à mice. these results demonstrated that h1n1pdm influenza infection stimulated a bimodal increase in pulmonary gx-spla 2 mrna expression which was /4108/2009) and the lungs were assessed for the mrna and protein expression and localization of plas during a 14 day time course. gx-spla 2 mrna (ai), cpla 2 mrna (aii) and gv-spla 2 mrna (aiii) expression quantified by real-time rt-pcr was normalized to gapdh, gx þ / þ (open bars) and gx à / à (filled bars) mice (c3h/hen background mice). gx þ / þ and gx à / à mouse lungs were perfusion fixed in situ with 4% paraformaldehyde, sectioned and subject to immunohistochemical analysis with the igg fraction of rabbit anti-mouse gx-spla 2 antiserum (1/100 dilution) (b). giia and gx-spla 2 protein expression determined by immunoblot analysis of lung tissue homogenates of wild type gx þ / þ (lane 1) and knockout gx à / à (lane 2) mice (c). for each blot, the corresponding recombinant spla 2 enzyme (rec spla 2 ) was run alone (lane 3) as a control. representative results for five separate experiments are shown. all the mice used in these experiments were genotyped littermates and grouped and analyzed by their genotype. a, p o 0.05 gx þ / þ or gx à / à vs. base; b, p o 0.05 gx þ / þ vs. gx à / à at any time point, anova followed by paired t-test, two tailed, assuming unequal variance. nz 8 per group; 400 â ; scale bar: 50 μm for immunohistochemistry. specific for this spla 2 since neither cpla 2 nor gv-spla 2 was upregulated. since gx-spla 2 mrna levels increased in response to h1n1pdm infection, we investigated the spatial and temporal expression of gx-spla 2 protein in mouse lungs after influenza infection. lungs from gx þ / þ and gx à / à mice infected with h1n1pdm were harvested at baseline, 3, 6 and 14 days pi and subjected to immunohistochemical analysis with anti-mouse gx-spla 2 antiserum. visualization by light microscopy revealed gx-spla 2 protein accumulation in the lungs of infected mice compared to baseline (fig. 1b) . gx-spla 2 protein was identified in inflammatory cells that had infiltrated in the alveolar space on day 3 and 6 in gx þ / þ mice infected with h1n1pdm (shown by arrows). gx-spla 2 protein was also clearly identified in epithelial cells lining the bronchioles on day 3, 6 and 14 in gx þ / þ mice infected with h1n1pdm (fig. 1b , upper right panels). no staining for gx-spla 2 protein was observed in gx à / à mice at baseline or at any time point after infection with h1n1pdm (fig. 1b , lower panel rows). similarly, no proteins cross reacting with the secondary antibody alone was identified in gx þ / þ or gx à / à mice (fig. 1b , left hand panels). we confirmed the loss of gx-spla 2 in the gx à / à mice by immunoblot analysis. we indeed observed a specific depletion of gx-spla 2 but no change in the expression of giia-spla 2 in the gx à / à mice compared to gx þ / þ mice (fig. 1c ). taken together, these results show that intranasal infection with h1n1pdm increases gx-spla 2 rna and protein expression in the lung that corresponds to the increase in lung inflammation associated with influenza infection. this suggests a possible role for gx-spla 2 in the pathogenesis of pulmonary h1n1pdm influenza infection. since gx-spla 2 was upregulated in the lung during h1n1pdm infection, we explored its role in the host response to pulmonary infection with h1n1pdm influenza. we first examined the clinical outcome of gx-spla 2 deletion by assessing weight loss and survival of g þ / þ and gx-spla 2 gene targeted mice à / à mice on two different genetic backgrounds following infection and assessed weight loss and survival. in the first series of infections, gx þ / þ (n¼25), gx þ /-(n¼32), and gx à /à (n¼24) mice on a c57bl/6j background were infected intranasally with h1n1pdm influenza a/mexico/4108/2009 ( fig. 2a ). mice on this background lack the giia-spla 2 gene (karabina et al., 2006) . animals were euthanized if their body weight decreased to less than 80% of baseline weight, or if the 14-day duration of the study was completed. survival 14 days after h1n1pdm influenza infection was 70% in gx à /à mice (blue line), 48% in gx þ /mice (green line) and 15% in gx þ / þ mice (red line). the difference in survival between gx à /à and gx þ /mice, and between gx à /à and gx þ / þ mice after h1n1pdm infection was statistically significant, pr0.01. to independently confirm these findings, we evaluated the survival of gx þ / þ (n¼ 71) and gx à / à (n ¼ 57) mice on a c3h/hen background (fig. 2b) which have a functional giia-spla 2 gene (karabina et al., 2006) . as with the studies with the c57bl/6j mice, animals were infected intranasally with a/mexico/4108/2009 and euthanized if their body weight decreased to less than 80% of baseline weight, or at the end of the study. survival of gx à / à mice on the c3h/hen background was again significantly higher (62%, blue line) following h1n1pdm infection than survival of gx þ / þ mice on a c3h/hen background (36%, red line). together, these studies showed that targeted deletion of gx-spla 2 in two different mouse models led to increased survival following h1n1pdm infection in vivo. furthermore, since the c3h/hen mice expressed endogenous giia-spla 2 , these results demonstrate that the ability to express giia-spla 2 does not compensate for the loss of gx-spla 2 during host immune responses to pulmonary h1n1pdm influenza infection. depletion of gx-spla 2 during h1n1pdm infection leads to a decrease in downstream phospholipid catalysis (aa) products but no difference in innate cell recruitment gx þ / þ and gx-spla 2 gene targeted mice à / à on a c3h/hen background were infected with a/mexico/4108/2009, and bal fluid was harvested 3 or 6 days post h1n1pdm infection. to assess the general inflammatory response and lung tissue destruction that typically occurs during h1n1pdm infection (ohtsuki et al., 2006; paquette et al., 2012) , we investigated the histopathology by h&e staining of lungs isolated from both gx à / à and gx þ / þ mice at baseline, day 3, 6 and 14 pi (fig. 3a) . the pulmonary pathology peaked quickly by day 3 pi in infected gx þ / þ and gx à / à animals. bronchiolitis and alveolitis with mononuclear cell and neutrophil infiltration were observed in several loci of the infected lungs of both groups. hemorrhage, edema, and necrotizing respiratory epithelia were also observed with similar severity among both groups. pathology persisted until day 7 pi where mononuclear cell and neutrophil infiltration were still profound and caused patches of consolidation in the lung tissue in both groups. it seemed by day 14 pi that the pulmonary pathology was slightly more minimal in the gx à / à mice with reduced level of leukocyte infiltration. in contrast, multi foci cell infiltration and tissue consolidation was still prominent in the gx þ / þ lungs by day 14 pi. to further examine the inflammatory cell types that may be recruited to the lung during h1n1pdm infection we analyzed lung homogenates from day 0, 3, 6 and 14 pi from both gx þ / þ and gx à / à mice by immunoblot for neutrophil and leukocyte cell markers, mpo and cd45 respectively. mpo was induced on day 3 and day 6 pi in both the mouse genotypes and returned to baseline on day 14 and cd45 was induced from baseline for all time points measured. neither mpo nor cd45 showed any variation in the lungs between gx à / à or gx þ / þ throughout the infection time course (fig. 3bi) ; densitometry did not reveal any statistical differences (fig. 3bii) . furthermore, we also analyzed gx à / à and gx þ / þ lungs for the presence and activation of macrophages by immunohistochemistry and real-time rt-pcr ( fig. 3ci , cii and d). here we found that the macrophage marker mac-3 was significantly increased and peaked at day 6 following infection as determined by immunohistochemistry staining which was further confirmed by quantifying the staining and quantification of the signal ( fig. 3ci and cii). furthermore the mrna for the inflammatory chemokine ccl2 was also significantly increased following infection (days 3 and 6) and decreased by day 14 (fig. 3d) . no difference was determined for mac-3 or ccl2 expression between gx à / à or gx þ / þ mice. taken together, these results suggest a similar inflammatory and innate response in both the gx þ / þ and gx à / à mice. to assess the role of gx-spla 2 in leukocyte infiltration into the bronchoalveolar space after h1n1pdm infection, we measured total leukocyte cell counts and the levels of different leukocyte cell types in the bal fluid of h1n1pdm infected in gx þ / þ and gx à / à mice (fig. 4a ). no significant difference in total cell counts ( fig. 4ai ) or the percentage of cd4 þ , cd8 þ , b or natural killer cells, or granulocytes were identified in the bal fluid of gx þ / þ and gx à / à mice 6 days after h1n1pdm infection (fig. 4aii ). in addition, targeted deletion of gx-spla 2 had no effect on lung viral titers 3 or 6 days pi (fig. 4b ). we next determined by elisa the levels of diferent aa metabolites including pgd 2 , ltb 4 , cysteinyl leukotrienes, pge 2 , a stable pge metabolite, and lipoxin a 4 which are known to regulate bronchiolar reactivity and inflammatory cell adhesion, migration and activation (henderson et al., 1995) were determined by elisa. levels of pgd 2 ( 5g ) were all significantly lower in the bal fluid from gx à / à mice (solid bars) on day 3 pi compared to gx þ / þ mice. conversely, on day 6 pi, the levels of these metabolites were similar in gx þ / þ and gx à / à mice ( fig. 5a-f) . in summary, these results show that deletion of gx-spla 2 in mice led to a transient but significant decrease in the levels of a panel of aa metabolites when mice were infected with a lethal h1n1pdm influenza virus that was not associated with alterations in inflammatory cell infiltration or viral clearance. increased expression of immunoglobulin chain, lymphocyte differentiation, antigen processing genes and presence of cd3 þ t cells in the lungs of mice lacking gx-spla 2 after h1n1pdm infection to increase our understanding of the molecular events leading to increased survival following h1n1pdm infection in gx à / à mice, we conducted microarray analysis of rna extracted from the lungs of gx þ / þ and gx à / à influenza infected animals. as previously reported by our group paquette et al., 2014; , influenza infection caused a progressive increase in the total number of upregulated genes in the lung tissue of gx þ / þ mice (1246 genes at 3 days pi and 2469 genes at 6 days pi). genes that belonged to different functional groups, such as immune response, inflammatory response and prostaglandin signaling pathways (figs. 6 and 7) showed a progressive increase that was parallel to the global evolution of gene expression. conversely, the expression of cytokine-related genes reached maximal levels 3 days pi and were maintained thereafter (fig. 6a) . at first sight, lack of gx-spla 2 did not modify the global evolution of gene expression in the lungs. similarly to gx þ / þ mice, gx à / à mice showed a progressive increase in the number of upregulated genes (1578 at 3 days pi and 2469 at 6 days pi). further analysis demonstrated that on day 3 pi, gx à / à mice showed significantly higher levels of the cytokines lta and ltb, the chemokines ccl19, cxcl9 and cxcl13 and the chemokine receptors cxcr3 and cxcr5 ( fig. 6b and c). in contrast, the expression pattern of cytokines and chemokines showed no differences between gx þ / þ and gx à / à mice 6 days pi (data not shown). interestingly, expression of 21 immunoglobulin chains, including heavy and light chains, was identified in gx à / à mice 3 days pi, while no expression of immunoglobulin chain genes was identified in gx þ / þ mice at this time point. in addition, the number of immunoglobulin chain related genes was higher in gx à / à than gx þ / þ mice 6 days after h1n1pdm infection (fig. 6b ). no differences were observed in the patterns of interferon regulated genes between gx þ / þ and gx à / à mice after h1n1pdm infection (data not shown). to determine which functional pathways are differentially enriched between gx þ / þ and gx à / à mice after h1n1pdm infection, we performed intersect analysis of the respective sets of upregulated genes (fig. 7) . at 3 days pi, expression of interferon regulated, inflammatory response and innate immune response genes were common to both gx þ / þ and gx à / à mice. a number of genes related with eicosanoid synthesis and their receptors were found to be regulated during influenza infection; however, gx-spla 2 deficiency did not cause any major alterations in their expression profiles (fig. s1) . the set of genes specifically enriched in the gx à / à mice at 3 day pi were those related to adaptive immune responses, such as immunoglobulin chains, lymphocyte differentiation and antigen processing and presentation. on the other hand, the set of genes more enriched in the gx þ / þ mice were genes involved in the tissue development category at 3 days pi (fig. 7a ). at 6 days pi (fig. 7b) , the enrichment profiles of upregulated genes in gx þ / þ and gx à / à mice were nearly identical. while immunoglobulin chain gene expression was identified in both gx þ / þ and gx à / à mice, expression of immunoglobulin chain genes remained elevated only in the gx à / à set of genes 6 days pi, while the gx þ / þ set of genes continued to show enrichment in the tissue development category at day 6 pi. to further evaluate the adaptive immune system of the gx à / à mice infected with h1n1pdm we investigated the t and b cell responses within the lung during infection. here we stained lung sections with anti-cd3 to assess infiltration of t cells using immunocytochemistry ( fig. 8a and b) . we found a significant increase of cd3 positive t cells in the lung on day 3 pi in the gx à / à mice compared to gx þ / þ mice (fig. 8a , upper right panels) by approximately 2 fold (fig. 8b) . interestingly, cd3 staining of the gx þ / þ animals had increased to similar levels seen in the gx à / à mice by day 6 and both genotypes had sustained levels of cd3 on day 14. moreover, we also investigated cd8a and igg (ighg) mrna levels in the lungs of the gx à / à mice throughout the time course and there was a slight trend for increase cd8a levels. taken together, the results from the cd3 and igg analysis supported the microarray studies where the adaptive immune system of the gx à / à had a faster and more robust initiation. gx-spla 2 has been highly implicated in various inflammatory diseases of the respiratory tract, including th2 cytokine-driven asthma (de jong et al., 2006; henderson et al., 2007) and lung injury (napolitani et al., 2009 ), but its role during influenza infection has not been previously investigated. here we evaluated the pathophysiological role of gx-spla 2 during severe influenza a h1n1pdm infection in the mouse. we found that gx-spla 2 expression was increased following infection, and that targeted deletion of gx-spla 2 led to increased survival in mice. lack of gx-spla 2 resulted in decreased levels of pgd 2 , ltb 4 , cysteinyl leukotrienes, pge 2 and lipoxin a 4 and increased adaptive immune responses at 3 but not 6 days following h1n1pdm infection. this demonstrates that gx-spla 2 plays an important role in the production of several biologically active inflammatory lipid mediators during the early phase of the inflammatory response that follows h1n1pdm influenza infection. human patients with a severe respiratory disease caused by influenza infection have a dysregulated inflammatory response that leads to lung pathogenesis associated with hypercytokinemia in most cases (bermejo-martin et al., 2010; curfs et al., 2008) . taken together with the previous findings showing a role of gx-spla 2 in inflammatory lung diseases, our work supports the further investigation of the therapeutic potential of attenuating gx-spla 2 during severe influenza infection as well as the interplay between eicosanoids and adaptive immunity. spla 2 has previously been implicated in pulmonary disease onset and progression putting it forth as a potential biomarker for severe respiratory diseases (henderson et al., 1995; henderson et al., 2007) . we show that gx-spla 2 protein and mrna expression increased in the lungs of gx þ / þ mice following h1n1pdm infection, suggesting that gx-spla 2 may be used as a possible biomarker of severe influenza infection. this is the first report of increased gx-spla 2 expression following influenza virus infection. both epithelial cells and leukocytes were found to be sources of gx-spla 2 during infection, and gx-spla 2 expression was detected in epithelial cells 3 days prior to the infiltration of leukocytes. in will be interesting to determine in future experiments whether the specific deletion of gx-spla 2 expression in epithelial cells vs. infiltrating leukocytes or both is responsible for the increased survival. here we observed a bimodal expression pattern of gx-spla 2 during the 14 day time course of infection. it is possible that this occurred due to the protein stability as it is used to regulate bioactive lipid mediator synthesis. if the protein does not remain stable throughout the course of infection and recovery, it may be important to have a second increase in gx-spla 2 in the later stages of infection to compensate for the loss of protein. it is in fact possible that the protein exerts distinct roles in the clearance of the virus and tissue remodeling in addition to the regulation of immune cells. such a scenario would require inductions at specific time points during infection. it will be important to further explore the local expression in the virus niche and the stability of protein gx-spla 2 during influenza infection to better understand how gx-spla 2 stability may influence influenza severity in the initiation of the innate immune response, adaptive maintenance, and recovery. furthermore, it would also be of value to investigate the source of gx-spla 2 by expression analysis of each cell type and also by investigating the role of hematopoietic gx-spla 2 compared to epithelial gx-spla 2 . the latter could be studied by employing bone marrow transplantation experiments from gx à / à mice into gx þ / þ and the reverse. while the association between gx-spla 2 and influenza related complications has not previously been investigated, ltb 4 , a downstream product of gx-spla 2 has been suggested to be a biomarker for pulmonary disease and respiratory complications following trauma (influenza activity -united states and worldwide, 2010; henderson et al., 2011; shridas et al., 2011) . multiple studies have implicated gx-spla 2 in the pathophysiology of pulmonary diseases onset and progression, suggesting gx-spla 2 might be a suitable therapeutic target in lung (henderson et al., 1995; morita et al., 2013) . deletion of gx-spla 2 in a th2 cytokine-driven mouse asthma model significantly impairs development of asthma (henderson et al., 1995) and accordingly, administration of a human gx-spla 2 selective inhibitor in a human gx-spla 2 knock-in mouse model led to a significant reduction in airway inflammation, mucus hypersecretion and airway hyperresponsiveness (henderson et al., 2007) . furthermore, although not specific for human gx-spla 2 , the indole-based spla 2 inhibitor varespladib has been shown to significantly inhibit spla 2 activity in the bal fluid of infants with post-neonatal ards (de jong et al., 2006) during induced asthma, suggesting the involvement of spla 2 among other spla 2 s. our results showing increased survival of the gx à / à mice after infection with h1n1pdm further support the notion that gx-spla 2 is a therapeutic target in pulmonary diseases due to viral infection and that infection with h1n1 might be better controlled by inhibiting this spla 2 . one of the main functions of gx-spla 2 is likely the generation of bioactive lipid mediators which play important roles in lung inflammatory diseases (gao et al., 2013; gaudreault and gosselin, 2007; van elssen et al., 2011) . although we did not see any major differences in the mrna analysis of the eicosanoid pathways between the gx à / à and gx þ / þ mice, measuring the mrna levels of these genes may have limited value to determine the level of activation of their signaling pathways. conversely, we observed decreased levels of pgd 2 , ltb 4 , cysteinyl leukotrienes, pge 2 and lipoxin a 4 in bal fluid 3 day pi in the gx à / à mice, indicating that gx-spla 2 acts upstream of these bioactive lipid mediators during influenza infection and thereby suggests a possible role of these bioactive mediators in pulmonary pathogenesis after influenza infection. in agreement with our findings, pgd 2 has been implicated during influenza a infection as pgd 2 expression in the lungs of older animals inhibits regulatory dendritic cells activity and t cell responses (zhang et al., 2000) . other eicosanoids have been implicated in different lung diseases, and the dysregulation of leukotrienes and lipoxins have been reported as contributing factors to the pathogenesis and severity of other respiratory diseases (bermejo-martin et al., 2009) . ltb 4 has been suggested to play a destructive inflammatory role in the lung by priming neutrophils for adhesion, chemotaxis and stimulation of granule release (cameron et al., 2007) . as well pgd 2 , pgd receptor, lipocalin-type pgd synthase and ltb 4 have been implicated in asthma pathogenesis (arima and fukuda, 2011; masuda et al., 2005; rusinova et al., 2012) . although asthma and pulmonary disease due to influenza infection differ in derivation, both are characterized by hyper-inflammation of the respiratory tract. taken together, our data supports a role of gx-spla 2 signaling and bioactive mediator production in the regulation of the pulmonary response to h1n1pdm infection. in the future it would be important to specifically determine whether pgd 2 , ltb 4 , cysteinly leukotrienes, pge 2 , lipoxin a 4 or another aa metabolite specifically modulates the response to h1n1pdm infection. the inflammatory response may be simultaneously beneficial and destructive during lung infection (baillie and digard, 2013; fig. 2 . increased survival of gx à / à vs. gx þ / à or gx þ / þ mice following a/mexico/ 4108/2009 infection. gx þ / þ (n¼ 25), gx þ / à (n ¼32), and gx à / à (n¼24) mice (c57bl/6j background, lacks giia-spla 2 ) were infected intranasally with a/mexico/ 4108/2009 and survival was assessed for a 14 day period (a). gx þ / þ (n¼71) and gx à / à (n¼ 57) mice (c3h/hen background, expresses giia-spla 2 ) were infected intranasally with a/mexico/4108/2009 and survival was assessed for a 14 day period (b). animals were sacrificed if their body weight decreased to less than 80% of baseline weight, or if the 14-day duration of the study was completed. log rank test, p o0.05 gx à / à vs. gx þ / þ and gx þ / à mice or po 0.05, gx à / à vs. gx þ / þ mice. all the mice used in these experiments were genotyped littermates and grouped and analyzed by their genotype. bermejo-martin et al., 2010) . although destructive killing of foreign pathogens is imperative for eradication and microbe clearing, the over production of inflammatory mediators leading to an overt inflammatory response may accentuate disease pathology, as is the case during severe influenza h5n1 and h1n1 infection (bermejo-martin et al., 2010; curfs et al., 2008; huang et al., 2009) . this illustrates the dual role of proinflammatory mediators, which has also been suggested for some gx-spla 2 downstream lipid mediators. for instance, ltb 4 has been shown to increase the activity of nasal neutrophil killing of human coronavirus, rsv, and influenza b virus (van elssen et al., 2011) and to induce the release of antimicrobial peptides in vivo in the lungs of fig. 3 . infection with h1n1pdm influenza induces similar pulmonary inflammation and recruitment of inflammatory cells in gx þ / þ and gx à / à mice. gx þ / þ and gx à / à mice (c3h/hen background mice) were infected with h1n1pdm (a/mexico/4108/2009) influenza and the lungs were perfusion fixed in situ with 4% paraformaldehyde on specific time points following infection, sectioned and subject to hematoxylin and eosin staining (a). mpo protein (neutrophil marker), cd45 protein (leukocyte marker) and gapdh protein (loading control) expression levels were determined by immunoblot analysis from lung tissue homogenates of gx þ / þ and gx à / à mice over a 14 day time course of h1n1pdm influenza infection (bi). densitometric analysis of mpo (bii) and cd45 (biii) protein levels normalized to gapdh levels in lung tissue of gx þ / þ (open bars) and gx à / à (filled bars) mice after h1n1pdm influenza infection, are presented. immunohistochemical analysis with specific rabbit primary antibody against mouse mac-3 antigen (marker for macrophages) is shown (ci). assessment of mac-3 positive cells (indicated by -) per high power field before, 3, 6 or 14 days after infection with h1n1pdm influenza is shown (cii). ccl2 mrna expression normalized to gapdh was determined by quantitative real-time pcr in lung tissue of gx þ / þ and gx à / à mice after h1n1pdm influenza infection (d). representative images ( â 200) from five independent experiments are shown. scale bar: 100 μm (a) or 50 μm (c). a, p o 0.05 gx þ / þ or gx à / à vs. base; b, p o0.05 gx þ / þ vs. gx à / à at any time point, anova followed by paired t-test, two tailed, assuming unequal variance. nz 8 per group. mice infected with viruses (gao et al., 2013; gaudreault and gosselin, 2007) . the lipid product protectin d1 has been implicated in influenza therapeutics (arima and fukuda, 2011; mitsuishi et al., 2006) . although these previous reports seem to suggest a conflicting role for gx-spla 2 in consideration of our data, it may be possible that ltb 4 and gx-spla 2 promote antiviral activity and are significant during a viral response but only at moderate levels. alternatively, it is possible that gx-spla 2 prevents h1n1 infection but also triggers excessive inflammation that is associated with lipid surfactant destruction. more work is needed to understand how the function of gx-spla 2 mediates both beneficial and deleterious roles during influenza infection. our survival data from gx gene targeted mice indicated that the loss of gx-spla 2 was beneficial to the host during influenza infection. the microarray mrna data from lungs of gx þ / þ infected mice were in agreement with our previously published data on pandemic h1n1 2009 virus infection, in mice including the progressive increase of immune and inflammatory responses and of the prostaglandin signaling pathway (paquette et al., 2014) . together, the results of microarray analysis, gene expression by real-time rt pcr and immunocytochemistry of the lungs suggested that gx à / à mice exhibited a more robust adaptive immune response than gx þ / þ mice. indeed, we observed significant differences in lymphocyte gene profiles at day 3 pi, associated with differences in the levels of lymphotoxin alpha and beta, b cell chemokines, t cell chemokine receptors and b cell immunoglobulin chains as measured which by immunofluorescence and rt-pcr. expression of b cell immunoglobulin chain genes were substantially increased on day 3 pi in the gx à / à mice but not in the gx þ / þ . b cell immunoglobulin gene expression was significantly greater on day 6 pi. and the expression of the t cell, b cell and dendritic cell chemokines and chemokine receptors, i.e., ccl19, cxcr3, etc., were significantly higher in the gx à / à samples than gx þ / þ . these results suggest that the downstream products of gx-spla 2 , such as pgd 2 , pge 2 , ltb 4 may inhibit the early adaptive immune responses of t and b cells during viral infection and this fits with the fact that aspirin, which attenuates eicosanoid production, can be an effective therapy for patients with influenza infection (matsuoka et al., 2000) . it would be of value in future studies to further investigate the effect of gx-spla 2 on the proliferation, activation and differentiation of t and b lymphocytes. consistent with this notion, the chemokines and chemokine receptors found to be upregulated in the h1n1pdm infected gx à /à mice are known to play significant roles in t and b cell migration and localization to the lymph nodes (goracci et al., 2010; muthuswamy et al., 2010) . cxcl13/cxcr5 signaling has been shown to activate b cells (sadik and luster, 2012) , which may explain the increased immunoglobulin chain gene expression observed in gx à /à mice. our data supports previous findings implicating pgd 2 in the inhibition of cell migration to lymph nodes (zhang et al., 2000) , and pge 2 in the inhibition of adaptive immune cellular events such as chemokine production by dcs and the attraction of naïve t cells (murakami et al., 2011; truchetet et al., 2012) . in conclusion, our findings provide new insights into the molecular pathophysiology of lethal influenza infection, highlighting a new role for gx-spla 2 during h1n1pdm infection. overall, the spla 2 appears as a negative effector but it may act at several steps during infection. we found that gx-spla 2 and its downstream products may have a role in the inhibition of adaptive immunity during viral infection in mice thereby contributing to pathogenesis. within this mechanism, it is in fact possible that t and b cell maturation and activation are initiated in mice lacking gx-spla 2 prior to virus infection, and that a more robust and earlier adaptive fig. 4 . lung viral titers and cell counts in bal fluid show similar cell numbers and cell population distributions following infection in gx þ / þ and gx à / à mice. gx à / à and gx þ / þ mice infected with a/mexico/4108/2009 were investigated for lung cell numbers, populations and viral load. bal fluid was harvested from infected gx þ / þ (open bars) and gx à / à (filled bars) mice (c3h/hen background mice) on day 0 and 6 and the cell numbers (ai) and cell population distributions (aii) were assessed by facs. viral load was determined on day 0, 3 and 6 pi of gx þ / þ (open bars) and gx à / à (filled bars) mice by real-time rt-pcr vrna quantification (b). all the mice used in these experiments were genotyped littermates and grouped and analyzed by their genotype. a, p o0.05 gx þ / þ vs. base, anova followed by paired t-test, two tailed, assuming unequal variance. nz 7 per group. immune response increased the survival of gx à / à mice after h1n1pdm infection. since gx-spla 2 may contribute to inflammatory response dysregulation during influenza infection and contribute to the morbidity and mortality associated with hospitalized influenza patients, this work may shed important insight into the molecular mechanisms of severe influenza infection. our findings further support the notion that gx-spla 2 is an interesting therapeutic target in lung inflammatory diseases. whether inhibition or attenuation of gx-spla 2 activity during severe influenza infection has a therapeutic effect remains to be demonstrated. to dissect the role of gx-spla 2 in the molecular regulation of pulmonary infection with h1n1pdm influenza, mice that lack gx-spla 2 (gx à / à mice) on the c57bl/6j background previously described were used (matsuoka et al., 2000) . this mixed background strain has a naturally occurring mutation in the gene encoding giia-spla 2 (karabina et al., 2006) , which has been fig. 5 . decreased eicosanoid levels in the bal fluid 3 but not 6 days after infection with a/mexico/4108/2009 in gx à / à vs. gx þ / þ mice. the eicosanoid levels in the bal fluid of gx à / à and gx þ / þ mice (c3h/hen background) were investigated at baseline and following infection with a/mexico/4108/2009. gx þ / þ (open bars) and gx à / à (filled bars) mice (c3h/hen background mice) bal fluid was harvested by instilling ice cold nacl (1 ml) five times and pooled. levels of pgd 2 mox (a), ltb 4 (b), cysteinyl leukotrienes (c), pge 2 (d), stable pge metabolite (e), pge 2 plus pge metabolite (f) and lipoxin a 4 (g) were assessed by elisa. these results are the mean of 5 independent studies. all the mice used in these experiments were genotyped littermates and grouped and analyzed by their genotype. gx þ / þ (open bars) and gx à / à (filled bars). results are expressed in pg/ml. a, p o0.05 gx þ / þ or gx à / à vs. base; b, p o0.05 gx þ / þ vs. gx à / à at any time point, anova followed by paired t-test, two tailed, assuming unequal variance. n z7 per group. implicated in bacterial phospholipid hydrolysis . furthermore, we generated gx à / à mice on the c3h/hen background (fig. 1c ) by backcrossing the c57bl/6j gx à / à mice for 10 generations which had functional giia-spla 2 . mice were maintained on standard animal feed and water ad libitum in the conventional environmental conditions and controlled temperature and humidity with a 12 h light and dark cycle. for infection studies, animals were housed in hepa-filtered cage racks adherent to absl2 þ conditions (toronto general hospital, animal resource centre, toronto, canada). all animal procedures were performed in a certified class ii biosafety cabinet (baker company, sanford, nc, usa). housing and experimental procedures were approved by the animal care committee of the university health network, and were in accordance with the guide for the care and use of laboratory animals research statutes, ontario (1980) . all infection experiments were conducted with h1n1pdm strain, a/mexico/4108/2009 (h1n1pdm), provided by the centers for disease control and prevention (atlanta, ga, usa). virus was propagated and titrated in embryonated eggs and titrated prior to animal challenge. viral stocks were stored in liquid nitrogen and thawed prior to use. mice were weighed and randomly assigned for sample collection, and were infected through intranasal instillation with 50 ml phosphate-buffered saline (mock infection) or 50 ml a/mexico/4108/2009 (h1n1pdm) at 1 â 10 5 or 1 â 10 4 50% egg infectious dose (eid) 50 . virus dosage were 1 â 10 4 eid 50 and â 10 5 eid 50 for host response profiling in c57bl/6j mice and 1 â 10 4 eid 50 for comparing disease severity between gx þ / þ and gx à / à mice. throughout infection experiments, animal survival, clinical signs, and weights were recorded daily. in accordance with animal care committee recommendation, mice were euthanized when recorded body weight fell below 80% of original body weight. at day 0, 3 and 6 pi, 3 gx þ / þ and 3 gx à / à mice were euthanized and lung homogenates collected for viral load determination by either madin-darby canin kidney (mdck) cell growth determination or real-time rt-pcr (rna analysis methods and table s1 ). for mdck determination lungs were homogenized (10% w/v) in high glucose (4.5 g/l) dulbecco's modified eagle medium (dmem), supplemented with 1% bovine serum albumin, 50 mg/ml gentamycin, 100 u/ml penicillin, 100 mg/ml streptomycin, and 1 mg/ml tpck-trypsin (vdmem). homogenates were then serially diluted (0.5 log 10 ) in quadruplicate over madin-darby canine kidney cells, cultured at 2.0 â 10 4 cells/well in 96-well plates. cells were incubated for 2 h at 37 1c and 5% co 2 . homogenates were then removed and replaced with fresh vdmem. cells infected were incubated for 6 days at 37 1c and 5% fig. 6 . effect of gx-spla 2 deficiency in the mrna expression levels of cytokines, chemokines and their receptors and immunoglobulin chains in the lung tissue of mice during influenza infection. gx þ / þ and gx à / à mice (c3h/hen background mice) were infected with influenza a/mexico/4108/2009 and the gene expression profiles were analyzed in the lung tissue at day 0, 3, and 6 days after infection by microarray analysis (n¼ 4 per group). evolution of gene enrichment (fisher's exact test) of the kegg category "cytokine-cytokine receptor interaction" (a). differences in the expression levels of cytokines (b) and chemokines (c) and their receptors at 3 days pi. the heatmaps show the genes that are significantly upregulated with respect to the control group and the blue boxes indicate that the expression levels of those genes are significantly higher in the gx à / à than in the wild type mice at the same time-point. evolution in the expression levels of immunoglobulin genes: total number of regulated genes (d) and overview of different experimental groups and time-points (e). all the mice used in these experiments were genotyped littermates and grouped and analyzed by their genotype. fig. 7 . intersect analysis of the genes up-regulated in the lung tissue of gx à / à and gx þ / þ mice during influenza infection and functional classification of the resulting gene subsets. venn diagrams are representative of the total number of genes that are significantly up-regulated with respected to the uninfected mice. david annotation tool was used to classify the genes of each subset, and the fold enrichment is shown for each category. all the mice used in these experiments were genotyped littermates and grouped and analyzed by their genotype. * the "immunoglobulin chains" category was manually curated and contains 84 genes. nn the "interferon responses category". fig. 8 . gx-spla 2 deficiency increases t cell recruitment and immunoglobulin heavy chain mrna expression in the lung tissue of mice during influenza infection. day 0 and 3 pi with h1n1pdm (a/mexico/4108/2009) influenza the lungs from gx þ / þ and gx à / à mice (c3h/hen background mice) were perfusion fixed in situ with 4% paraformaldehyde, sectioned and subject to immunofluorescence analysis with specific rabbit primary antibody against mouse cd3 antigen (marker for t-cell) (ai). representative images (at â 400 with 3.4 zoom factor) from five independent experiments are shown. assessment of cd3 positive cells per high power field for day 0, 3, 6 and 14 days following infection with h1n1pdm influenza is shown (aii). igg (b) and cd8a (c) mrna expression normalized to gapdh were determined by quantitative real-time pcr in lung tissue of gx þ / þ and gx à / à mice after h1n1pdm influenza infection. scale bar: 10 μm. a, po 0.05 gx þ / þ or gx à / à vs. base; b, p o 0.05 gx þ / þ vs. gx à / à at any time point, anova followed by paired t-test, two tailed, assuming unequal variance. nz 8 per group. all the mice used in these experiments were genotyped littermates and grouped and analyzed by their genotype. co 2 , after which cell culture supernatants were tested for the presence of virus by hemagglutination assay using 0.5% (v/v) turkey red blood cells (lampire biological laboratories, pipersville, pa, usa). viral loads were determined as the reciprocal of the dilution at which 50% of wells were positive for viral infection. viral loads were reported as tcid 50 per gram of lung tissue. limit of detection of 10 1 tcid 50 /g. lung tissues from both gx à / à and gx þ / þ mice were collected at 3 and 6 days post infection (pi) and from uninfected controls (four mice per group). rna was purified from lung tissue using tripure (roche, indianapolis, in, usa). purified rna was then reverse transcribed using improm-ii reverse transcription system (promega, madison, wi, usa). real time rt-pcr was performed using the abi-prism 7900ht sequence detection systems (applied biosystems, foster city, ca, usa). data was collected with applied biosystems sequence detection systems version 2.3 software. each reaction well contained 4 μl of 0.625 ng/μl cdna, 0.5 μl each of forward and reverse primers (final concentration of 200 nm), and 5 μl sybr green master mix, for a total reaction volume of 10 μl and run in quadruplicate. host gene expression was normalized to the glyceraldehyde-3-phosphate dehydrogenase (gapdh) housekeeping gene, and quantified by relative standard curve method. viral load was quantified by the absolute standard curve method, normalized to gapdh housekeeping. primer sequences are listed in table s1 . histology, immunohistochemistry, immunofluorescence and immunoblotting gx þ / þ and gx à / à mice were euthanized at baseline, day 3, day 6 and day 14 pi (n 45 mice) and the mouse whole body was vascular perfused by cardiac puncture in situ with a fixative solution of 10% buffered formalin by a continuous release pump under pressure and volume-controlled conditions. fixed lung tissues were paraffin wax embedded for histology and immunohistochemistry. for h&e, tissue slides were then stained with hematoxylin-eosin for histopathology assessment. rabbit antimurine gx-spla 2 (degousee et al., 2008) was used to assess the tissue distribution of the gx-spla 2 protein. sections were counterstained with hematoxylin and eosin and observed under light microscope (accu-scope s , commack, ny, usa). images were captured using a digital camera and se premium software (micrometrics tm , londonderry, nh, usa) . for mac-3 tissue expression, rat anti-mouse mac-3 was used (bd biosciences, mississauga, on). for cd3 immunofluorescence, following heat-induced antigen retrieval, lung tissue sections were blocked with donkey serum and stained with primary antibodies rabbit anti-cd3 (dako, burlington, on). donkey anti-rabbit cy3 was used as secondary antibodies (millipore, billerica, ma) and dapi (sigma) for nuclear counterstain. images were recorded with an olympus fluo view 1000 confocal laser scanning microscope (olympus, tokoyo, japan). immunoblots for gx-spla 2 were carried out as described by our group (degousee et al., 2008) . for immunoblotting detection antibodies against mpo (upstate, lake placid), cd45 (bd biosciences, mississauga, on), and gapdh (santa cruz biotechnology, dallas, tx). lungs were lavaged at 0 and 6 days post h1n1 infection with 5 ml of normal saline. the bal fluid was centrifuged at 250g for 10 min and the supernatant was used for estimation of pgd 2 , pge 2 , ltb 4 , cysteinyl leukotriens and lipoxin a 4 content. analysis of pgd 2 in bal fluid 0.5 ml of bal fluid was mixed with 0.5 ml of ice-cold acetone, incubated on ice for 5 min and centrifuged for 10 min at 3000g at 4 1c. after supernatant aspiration, the pellet was extracted with 1 ml of ice-cold acetone and centrifuged again. the acetone extracts were combined and the acetone evaporated under nitrogen. all the samples were then methoximated (pgd 2 -mox eia kit (cayman chemical), and purified on oasis hlb columns (waters corporation) equilibrated with methanol/0.2% formic acid. methanol eluants were evaporated in a savant speed vac concentrator and samples dissolved in cayman eia buffer before eia analysis, according to the manufacturer's instructions. analysis of pge 2 , ltb 4 and cysteinyl leukotriens in bal fluid 1.2 ml of bal fluid was mixed with 2.4 ml of methanol containing 0.2% formic acid, incubated on ice for 5 min and centrifuged at 3000g for 10 min at 4 1c. after adjustment of methanol to 15%, supernatants were loaded on oasis hlb column equilibrated with methanol/0.2% formic acid (waters corporation). columns were processed in a vacuum manifold (waters corporation). after wash with water/0.03% formic acid, the samples were eluted with methanol/0.2% formic acid, methanol eluants were evaporated in a savant speed vac concentrator and samples dissolved in cayman eia buffer before eia analysis for pge 2 , ltb 4 and cysteinyl leukotrienes (eia kits, cayman chemical) according to the manufacturer's instructions. 0.6 ml of bal fluid was extracted with 1.2 ml of ice-cold methanol, incubated on ice for 5 min and centrifuged at 3000g for 10 min at 4 1c. the supernatants were diluted with water to achieve 11% methanol concentration and adjusted to ph 3.5 with 1n hcl. samples were purified on c18 sep-pak columns (waters corporation) preconditioned with methanol. after column wash with water followed by hexane, samples were eluted with methyl formate. the eluants were evaporated under nitrogen and the samples reconstituted in eia buffer and assayed for lipoxin a 4 content (neogen corporation) according to the manufacturer's protocol. lung tissues from both gx à / à and gx þ / þ mice were collected at 3 and 6 days pi and from uninfected controls (four mice per group) as with the real time rt-pcr. rna was purified from lung tissue using tripure (roche, indianapolis, in, usa) and amplified with illumina totalprep rna amplification kit (ambion, austin, tx, usa). 1.5 mg of crna was labeled and hybridized to mouse wg-6 v2.0 expression beadchip (illumina, san diego, ca, usa) and scanned on illumina beadstation 500gx. raw data was processed with illumina genomestudio v2010.3 software. the data sets were subjected to quantile normalization, variance stabilization and log 2 transformation. genes were considered significantly regulated if the expression levels with respect to the uninfected controls were z1.5fold different and the student t-test's p value was o0.05. david bioinformatics resource v6.7 (http://david.abcc.ncifcrf.gov/home.jsp) (hicks et al., 2007) was used to perform functional classification of differentially expressed genes. additionally, interferon regulated genes were selected by using the interferome (v2) database (http://interfer ome.its.monash.edu.au/interferome/home.jspx) (rubin et al., 2005) . immunoglobulin chains and prostaglandin-related gene categories were defined by searching for relevant keywords in the annotated microarray datasets. multiexperiment viewer v4.7.2 (http://www.tm4. org/mev/) was used to perform complete hierarchical clustering and generate heatmap representations of selected genes. 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stabilization improves cardiac contractile function following hemorrhagic shock and resuscitation influenza vaccines: the good, the bad, and the eggs group x secretory phospholipase a 2 enhances tlr4 signaling in macrophages1 increased concentrations of leukotrienes in bronchoalveolar lavage fluid of patients with ards or at risk for ards prostaglandin i(2) analogues enhance already exuberant th17 cell responses in systemic sclerosis3 update: influenza activity -united states, 2009-10 season inflammation-restraining effects of prostaglandin e2 on natural killer-dendritic cell (nk-dc) interaction are imprinted during dc maturation1 ltb4 increases nasal neutrophil activity and conditions neutrophils to exert antiviral effects2 global influenza programme. (ref type: online source) innate immunity and pulmonary host defense age-related increases in pgd (2) expression impair respiratory dc migration, resulting in diminished t cell responses upon respiratory virus infection in mice1 supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.virol.2014.01.030. key: cord-326223-q6e60nf8 authors: gembardt, florian; sterner-kock, anja; imboden, hans; spalteholz, matthias; reibitz, franziska; schultheiss, heinz-peter; siems, wolf-eberhard; walther, thomas title: organ-specific distribution of ace2 mrna and correlating peptidase activity in rodents date: 2005-02-16 journal: peptides doi: 10.1016/j.peptides.2005.01.009 sha: doc_id: 326223 cord_uid: q6e60nf8 biochemical analysis revealed that angiotensin-converting enzyme related carboxy-peptidase (ace2) cleaves angiotensin (ang) ii to ang-(1–7), a heptapeptide identified as an endogenous ligand for the g protein-coupled receptor mas. no data are currently available that systematically describe ace2 distribution and activity in rodents. therefore, we analyzed the ace2 expression in different tissues of mice and rats on mrna (rnase protection assay) and protein levels (immunohistochemistry, ace2 activity, western blot). although ace2 mrna in both investigated species showed the highest expression in the ileum, the mouse organ exceeded rat ace2, as also demonstrated in the kidney and colon. corresponding to mrna, ace2 activity was highest in the ileum and mouse kidney but weak in the rat kidney, which was also confirmed by immunohistochemistry. contrary to mrna, we found weak activity in the lung of both species. our data demonstrate a tissueand species-specific pattern for ace2 under physiological conditions. in the regulation of heart function and blood pressure, different peptide systems are involved, e.g. the renin-angiotensin system (ras), the kallikrein-kinin system, and the natriuretic peptide system. in these systems, proteases like angiotensin-converting enzyme (ace) or neutral endopeptidase (nep) have the distinction of generating or catabolizing biologically active peptides [10, 39, 42] . the newly discovered angiotensin-converting enzyme-related carboxypeptidase (ace2) has considerable sequence homology to ace (40% identity and 61% similarity), contains a hexxh zinc-binding domain, and conserves other critical residues typical of the ace family [12, 37] . the first step in generating angiotensin peptides is the cleavage of angiotensinogen to angiotensin (ang) i by renin. ang i is hydrolyzed by either ace or chymase to ang ii, which mediates its biological actions via the at1 and at2 receptors [15, 21] . ang i is also metabolized by nep to ang-(1-7) [15] , which mediates distinct effects through its receptor mas [35] . importantly, ang-(1-7) can also be directly metabolized from ang ii by ace2, whereas aminopeptidase a converts ang ii to ang iii [18] . ace2 also hydrolyzes ang i to ang-(1-9), although there is no hydrolysis of ang-(1-9), ang-(1-7), and ang(1) (2) (3) (4) (5) . moreover, ace2 hydrolysis is also specific for des-arg [9] bradykinin and its shorter fragments, although it cleaves neither bradykinin nor bradykinin-(1-7) [40] . ace2 mrna is expressed in many tissues but shows a less ubiquitous profile than ace. first studies in mice detected the highest expression in the ileum by quantitative reverse transcriptase polymerase chain reaction (qrt-pcr) [23] . ace2 is an important part of the ras, which counteracts the function of ace. it was also shown that ace2 expression can be upregulated by blockade of at 1 -receptors [27] . the importance of ace2 in cardiovascular regulation was confirmed by targeted disruption of ace2 in mice. the absence of ace2 in mice leads to a severe cardiac contractility defect, increased ang ii levels, and upregulation of hypoxia-induced genes in the heart [11] . in addition to its peptidolytic function, recent investigations have discovered that ace2 is a functional receptor for the coronavirus, which causes the severe acute respiratory syndrome (sars) [30] . in this investigation, we (i) measured the mrna distribution of ace2 through different tissues in both species. moreover, we (ii) quantified ace2 protein by western blot using a commercial polyclonal antibody to ace2. we (iii) measured ace2 activity in different tissues of mice and rats. we (iv) established a monoclonal antibody against ace2 to complete the investigation of tissue distribution by immunohistochemistry. finally, we compared (v) the distribution of ace2 in both species on the mrna and protein level. all experiments were done according to the guidelines of the federal law on the use of experimental animals in germany and were approved by the local authorities. for this investigation we used c57bl/6 mice and sprague-dawley (sd) rats in an age of 3-5 months. animals were killed by cervical dislocation. for rnase protection assay (rpa), ace2 activity assay and western blot, the tissues were snap frozen in liquid nitrogen. the samples were stored at −80 • c until further processing (all organs in total, heart divided into atria and ventricles). the tissues for immunohistochemistry were put in 4% formalin. after 24 h they were embedded and processed to paraffin sections. the polymerase chain reaction (pcr) amplified a 358 bp fragment (probe: mmace2) from mouse kidney cdna using the 5 -primer ctc agt gga tgg gat ctt gg (mmace25) and the 3 -primer tgt agc cat ctg ctc cct ct (mmace23), respectively a 342 bp fragment (probe: rnace2) from rat lung cdna using the 5 -primer cgg gga aag atg tca agc tcc tgc (rnace25) and the 3 -primer ctt gtc tgg tga cag cgc (rnace23), which were subcloned in a t-vector (promega gmbh, mannheim, germany). a sp6 polymerase transcribed a radioactive probe complementary to mmace2 (resp. rnace2) mrna, and a rna complementary to 127 nucleotides of the rl32 mrna was used as positive control [2] . ace2-specific mrna for mouse and rat were identified by rnase protection assay (rpa) using the ambion rpa ii kit (ambion (europe) ltd., huntingdon, uk). total rna was isolated from tissues using the trizol reagent (invitrogen gmbh, karlsruhe, germany) with subsequent chloroform-isopropanol extraction according to the manufacturer's instructions. a 15 g total rna fraction of each sample was hybridized with approximately 50 000 cpm for ace2 and 50 000 cpm for rl32 of the radiolabeled antisense probes in the same assay. equal loading has been insured by mrna measurements and mrna gel electrophoresis using 1 g of each sample (not shown). the hybridized fragments protected from rnase a + t1 digestion were separated by electrophoresis on a denaturing gel (5%, w/v polyacrylamide, 8 m urea) and analyzed using a fujix bas 2000 phospho-imager system (raytest gmbh, straubenhardt, germany) to perform quantitative analysis by measuring the intensity of the ace2 bands. the blots of each species were calculated to ace2 mrna expression in kidney, which was present on both blots of each species. the expression level in the lung was set to 100%. ace2 activity was measured similar to the method by vickers et al. [40] . tissue was homogenized in assay buffer (50 mm 2-morpholinoethanesulfonic acid, 300 mm nacl, 10 m zncl 2 , 0.01% brij-35, ph 6.5). protein concentration was determined using roti-quant (carl roth gmbh and co. kg, karlsruhe, germany) by the manufacturers instruction. we used mca-apk(dnp) (biosynthan gmbh, berlin, germany) dissolved in dmso (50 m, final concentration) as the ace2 substrate. the assay was performed in assay buffer and was started by adding 10 l of tissue homogenate. after 2 h incubation at ambient temperature (24 • c), the reaction was suppressed by adding 100 m o-phenanthrolin (final concentration). parallel control tests were performed in the presence of 1 m dx 600 (data not shown) [25] . after centrifugation (10 min, 10 000 × g) the fluorescence was measured at 320 nm (excitation) and 405 nm (emission) with the perkin-elmer fluorescence reader lambda 5 (perkin-elmer las gmbh, rodgau, germany). the molecular standardization was performed with mca-ap (biosynthan gmbh, berlin, germany) and calculated per mg protein. the functionality of the assay was proven by a standardized solution with defined, recombinant ace2 activity (r&d systems gmbh, wiesbaden, germany). tissue was homogenized in phosphate-buffered solution (pbs) containing protease inhibitor mixture (complete, roche diagnostics gmbh, mannheim, germany). protein concentration was determined with bca protein assay kit (perbio science gmbh, bonn, germany). sample proteins (10 g/lane) and a prestained protein-weight marker (amersham biosciences gmbh, freiburg, germany) were size fractionated by sds-polyacrylamide gels (10%) and transferred to pvdf membranes with a pegasus semidry-blotter (phase gmbh, lübeck, germany). equal loading has been insured by staining control gels with simply-blue safe stain (invitrogen gmbh, karlsruhe, germany) using 10 g of each sample (not shown). the membranes were blocked at room temperature in 5% dry milk powder (blotting grade, non-fat dry milk, bio-rad laboratories gmbh, munich, germany) prepared with tris-buffered saline containing 0.1% tween 20 (ttbs) for 1 h, incubated with goat polyclonal antibody against ace2 (santa cruz biotechnology inc., heidelberg, germany, 1:250 diluted in 5% dry milk powder ttbs, 1 h), and then washed three times with ttbs (15 min each). subsequently, the membranes were incubated with horseradish peroxidase-conjugated antigoat igg (dakocytomation a/s, glostrup, denmark, 1:1000, 1 h) and washed three times. specific immunoreactive proteins were detected by enhanced chemiluminescence (amersham biosciences gmbh, freiburg, germany). the bands on the x-ray film were quantified by densitometry scanning and expressed as percentage of the kidney protein signal. monoclonal antibodies against the synthetic peptide avgeimslsaat (aa 403-414 of murine ace2) have been raised. for immunization of the mice peptide was cross-linked with glutaraldehyde to albumin fraction v from bovine serum. balb/cj female mice were injected with the conjugate. following four booster injections the spleen lymphocytes were fused with fo myeloma cells by using polyethylene glycol 1500 (roche diagnostics gmbh, mannheim, germany) following the manufacturers instructions. the different hybridoma supernatants were screened for specific antibodies by using the synthetic peptide in the nctest [26] . for production of monoclonal antibodies, positive hybridoma cells were grown in celline incubators (integra biosciences gmbh, fernwald, germany). the mouse monoclonal antibodies were affinity purified on a mabtrap g ii column (amersham-pharmacia gmbh, otelfingen, switzerland) from cell culture supernatants. immunoglobulin class and subclasses were determined with the immuno type kit (sigma-aldrich chemie gmbh, taufkirchen, germany). paraffin sections of mouse tissues were prepared and stained using standard histology procedures. for immunostainings, deparaffinized and rehydrated tissue slides were first treated for 30 min with 30% h 2 o 2 to block the endogenous peroxidase. after rinsing in ddh 2 o and soaking in pbs for 5-10 min, slides were treated with 10% (w/v) bsa in pbs to eliminate non-specific protein binding sites. the slides were then exposed (overnight, 4 • c) to the monoclonal ace 2 antibodies (clone 7e7, 1d3) at concentrations of 1 and 4 g/ml, respectively. after removing excess antibody, slides were treated with biotin-labeled anti-mouse (dianova gmbh, hamburg, germany) antibody for 30 min at 37 • c and finally with horse-radish peroxidase (hrp) labeled streptavidine (zymed laboratories inc., san francisco, usa) for 20 min at 37 • c. after washing, slides were incubated in aminoethylcarbazol (sigma-aldrich co., st. louis, usa) for 10 min at room temperature. slides were counterstained with hematoxylin, and cover slipped according to conventional procedures. slides were examined under a conventional microscope after removing the excess substrate in ddh 2 o. negative controls were performed without the primary antibody, just applying dilution buffer of the primary antibody. data were analyzed by t-test using spss11 software (spss benelux bv, gorinchem, the netherlands). each value was expressed as the mean ± s.e.m., and statistical significance was accepted for p < 0.05. ace2 mrna could be detected in all investigated organs, but with profound distinction between different organs. in both species, only a low amount was found in ventricle, liver, testis, forebrain, and spleen ( figs. 1 and 2) , whereas in the lungs a moderate and comparable expression of ace2 mrna was found and set to 100%. the highest levels were found in the ileum of both species (fig. 3) . between the species several differences in tissue specific expression of ace2 mrna were found. the expression in mouse was most pronounced higher than in rat in kidney (∼31.9-fold), colon (∼18.6-fold), and ileum (∼12.0-fold) (fig. 3) , whereas in bladder (∼2.5-fold) and ventricle (∼2.1-fold) ace2 expression in rat exceeded the mouse. in accordance with the rna expression data, highest activity for ace2 was found in the ileum of mouse and rat (table 1) , whereas the activity in the mouse was 3.2fold higher. lowest ace2 activity was found for both species in spleen. low activity was also found for liver of mice and thymus of rats. corresponding to the differences on mrna levels in the kidney the ace2 activity fig. 3 . quantification of the rpas of mice (white columns) and rats (black columns). the mrna amount of the lungs is set to 100% (n ≤ 4) . the values are shown as mean + s.e.m. 1. ventricle, 2. kidney, 3. lung, 4. liver, 5. testis, 6. bladder, 7. forebrain, 8. spleen, 9. thymus, 10. stomach, 11. ileum, 12. colon, 13. brainstem, 14. atrium, 15. adipose tissue. * p < 0.05, ** p < 0.01, *** p < 0.0001 compared mouse vs. rat. was much higher in mice than in rats (∼13.9-fold). the activity of ace2 in the lung was different to mrna and 2.6-fold higher in rats than in mice. in contrast to rpa data the activity in colon was comparable between both species. using a commercial polyclonal antibody in western blot for the quantification of protein levels in mouse and rat tissues (fig. 4) a pattern completely different from rna expression and ace2 activity was found. a moderate and comparable expression could be detected in the kidney of both species and was set to 100%. thus, the highest amount of protein could be detected in atrium of both species (mouse: 124.5%; rat: 131.5%) and ventricle (mouse: 131.7%; rat: 143.3%). for the mouse less ace2 protein was found in lung (19.7%) and testis (28.7%), whereas no protein was detectable in these two tissues in rat. in thymus (mouse: 44.4%; rat: 50.6%) and forebrain (mouse: 87.9%; rat: 80.7%) of both species a moderate expression was detectable, whereas no ace2 protein was found in spleen of mouse and rat. to further clarify the discrepancy between rpa and activity on one side and western blot on the other, immunohistochemistry was performed in lung, kidney (fig. 5) , and testis (data not shown) of mice and rat with new monoclonal ace2 antibodies (clones 7e7 and 1d3), we generated. the antibodies were determined to belong to the igg1 subclass. in the lungs of both species alveolar macrophages and type 2 cells (fig. 5 , upper row) were stained with both monoclonal ace2 antibodies (data for clone 1d3 not shown). the epithelium of the renal tubuli was strongly stained (fig. 5 , lower row, left) in the kidney of mice. in rats only a weak signal, but the same pattern as in mouse, was detected, what aligned with mrna and ace2 activity (fig. 5, lower row, right) . in recent investigations it was shown that peptidases like ace and nep are important regulators of cardiovascular and endothelial function as well as myocardial remodelling [1, 7, 36, 41] . consequently, after its discovery in 2000, ace2 became an enzyme of interest for scientific investigation of its impact in cardiovascular physiology and pathophysiology [11, 12, 37] . to elucidate some of its physiological functions we investigated the tissue distribution of mrna and protein in a variety of tissues of c57bl/6 mice and sprague-dawley rats. while we could see correlating patterns of mrna and ace2 activity in most of the examined tissues, we also found significant divergences between the investigated species. the huge difference between mrna and protein levels in the lung may be due to shedding as demonstrated for ace [4, 12, 32] . this shedding leads to an increased secretion of ace2 and lowered its protein content in the lung by even high mrna expression. the significant differences that we found between the species on ace2 protein and mrna levels in kidney could be explained by the varying interspecies regulation and expression of peptidases, as shown in the literature for nep activity in rat and rabbit kidneys [14] . comparing our mrna and activity data with the western blot pattern, we have to conclude that the commercial polyclonal antibody is not detecting ace2 protein in organ homogenates and is not suitable for ace2 staining. in contrast, using immunohistochemistry our new monoclonal ace2 antibodies produce staining patterns comparable to our mrna and activity data. we have shown that ace2 expression in rodents is highest in ileum among the examined organs. it was shown for other peptidases of ras like ace and nep that they are also present at high levels in the intestine [29] . however, the distinct function of these peptidases in the ileum is not yet known. further investigations have to clarify the physiological and pathophysiological functions of the peptidases in the gastrointestinal tract. beside its physiological function as a peptidase, ace2 is used by coronavirus as a co-receptor in severe acute respiratory syndrome (sars) [30] . it was shown that the sars coronavirus only can enter cells which express ace2 [24] . ace2 distribution in the small intestine, lung and vascular endothelium may offer a point of entry for the sars coronavirus, but does not reflect its basic function [22, 30, 38] . interestingly, the distribution patterns we found for mrna and ace2 activity contradict investigations using a commercial northern blot for detecting mrna [12, 37] but have been confirmed by recent papers using rt-pcr [23] . this discrepancy may be a species-specific alteration of tissue distribution, since they used human tissue for northern blot, or it may be due to technique differences (commercial northern versus rpa and activity assay). the first possibility is at least supported by our finding that significant differences in ace2 expression patterns exist between the close relatives mouse and rat. recent investigations revealed biological activity for angiotensin peptides other than angii, like ang-(1-7) [16, 33, 34] . ace2 can generate ang-(1-7) by cleaving the cterminal amino acid from angii [40] . ace2 is also involved in another pathway leading to the generation of ang-(1-7). it cleaves angi to ang-(1-9) [12] . ang-(1-9) is then hydrolyzed by ace to ang-(1-7) . we demonstrated that ang-(1-7) is an endogenous ligand for the g protein-coupled receptor (gpcr) mas [35] . mrna of the gpcr mas was found at high levels in testis and certain brain regions and at fig. 5 . immunohistochemical visualization of ace2 positive cells. sections of lungs (upper row) and kidneys (lower row) from mouse (left panel) and rat (right panel). in the lungs of both species alveolar macrophages and type 2 cells were stained positive. the tubulus epithelium in mouse kidney was stained positive, whereas in the rat kidney only weak staining was seen. moderate levels in kidney and heart [2, 3, 31] . it was shown that high concentrations of ang-(1-7) were present in heart, kidney, and brain [5, 6, 8, 28] . in recent investigations, it was demonstrated that ace2, mas, and its endogenous ligand ang-(1-7) are present in the same cells of the kidney [9] . as we recently postulated, this indicates a relevant impact of the ace2/ang-(1-7)/mas axis on blood pressure regulation and cardioprotection. actual investigations indicate an upregulation of ace2 in heart failure, pointing to the relevance of ace2 in cardiac function [20, 32, 43] . however, there was a high incidence of sudden death in animals overexpressing ace2. electrophysiology revealed severe, progressive conduction and rhythm disturbances with sustained ventricular tachycardia that progressed to fibrillation and death [13] . while anti-arrhythmic actions were demonstrated for ang-(1-7) in low concentra-tions (0.22 nm) by stimulating its own receptor, 100-fold higher concentrations of ang-(1-7) lead to arrhythmias by stimulating the at1 receptor [17, 19] . therefore, the overexpression of ace2 may lead to a high increase in the production of ang-(1-7), turning its cardioprotective actions into effects causing arrhythmias by unspecific at1 stimulation. in future studies, the actions of ang-(1-7) and its concentrationdependency on ace2 expression on heart rhythm have to be proven in in vivo experiments with at1-and mas-deficient animals. our data on tissue and species-specific ace2 expression point to the fact that the ras becomes increasingly complex. since we identified an expression pattern markedly different from ace, we conclude that the expression levels of the involved peptidases like ace, ace2, and nep that generate and/or degrade the bioactive peptides of the ras are predic-tive of either the occurrence of vasoconstriction or dilatation or the dominance of pathophysiological stimuli over beneficial conditions. the acute infarction ramipril efficacy (aire) study investigators, effect of ramipril on mortality and morbidity of survivors of acute myocardial infarction with clinical evidence of heart failure imprinting of the murine mas protooncogene is restricted to its antisense rna cell type-specific expression of the mas proto-oncogene in testis a point mutation in the juxtamembrane stalk of human 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angiotensin-converting enzyme 2 after myocardial infarction by blockade of angiotensin ii receptors angiotensin-(1-7) immunoreactivity in the hypothalamus of the (mren-2d)27 transgenic rat burrell lm. differential tissue and enzyme inhibitory effects of the vasopeptidase inhibitor omapatrilat in the rat angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus expression of the mouse and rat mas proto-oncogene in the brain and peripheral tissues the role of ace2 in cardiovascular physiology vasodilator action of angiotensin-(1-7) on isolated rabbit afferent arterioles angiotensin-(1-7): an update angiotensin-(1-7) is an endogenous ligand for the g protein-coupled receptor mas remodeling of myocardium and arteries by chronic angiotensin converting enzyme inhibition in hypertensive patients a human homolog of angiotensin-converting enzyme. cloning and functional expression as a captopril-insensitive carboxypeptidase exploring the pathogenesis of severe acute respiratory syndrome (sars): the tissue distribution of the coronavirus (sars-cov) and its putative receptor, angiotensin-converting enzyme 2 (ace2) the angiotensin-converting enzyme gene family: genomics and pharmacology hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase at1 receptor blockade increases cardiac bradykinin via neutral endopeptidase after induction of myocardial infarction in rats vasopeptidase inhibitors: will they have a role in clinical practice? increased angiotensin-(1-7)-forming activity in failing human heart ventricles: evidence for upregulation of the angiotensin-converting enzyme homologue ace2 florian gembardt is paid by a grant from the "deutsche forschungsgemeinschaft" (german research foundation)[grk865]. this study was also supported by the "stiftung zur förderung der wissenschaftlichen forschung an der universität bern". we thank helmut würdemann and susanne gygax for their technical assistance. key: cord-333043-fe24ezt6 authors: traavik, t.; mehl, r.; kjeldsberg, elisabeth title: “runde“ virus, a coronavirus-like agent associated with seabirds and ticks date: 1977 journal: arch virol doi: 10.1007/bf01314476 sha: doc_id: 333043 cord_uid: fe24ezt6 from 206i. uriae collected in the seabird colonies at runde, norway, two identical virus strains demonstrating no antigenic relationships to major arbovirus groups were isolated. the new strains demonstrated a corona-virus like morphology, haemagglutinated chicken red cells and were sensitive to sodium desoxycholate. multiplication with cpe was demonstrated in bhk 21/c13 and bsc-1 cells, and without cpe in vero and gmk cell cultures. the mouse pathogenicity was relatively low. in gel precipitation three to five specific lines were seen. precipitating antibodies have been found in seabird species commonly infested byi. uriae. the ecological circumstances of the isolates indicate an earlier unrecognized arbovirus circulating between seabirds andi. uriae. this corona-like virus has been tentatively termed runde virus. in 1973 investigations were undertaken to evaluate the extent, distribution and ecological circumstances of arbovirus loci in norway. until then., no arbovirus isolates had been reported from this country, although ecological and cli-nicai/epidemiological considerations (3, 24, 26) and a limited serological survey on bovine sera (28) indicated the existence of central-european tick-borne encephalitis virus fool. the early phase of the project concerns the study of the importance of ticks as vectors; later, mosquito-borne agents will be investigated. in norway five ixodes species are abundant: _[. ricinus (23) , i. tria.nguticeps (24) , i. hexagonus (17) , i. tividus (16) and 1. uriae (15, 23) . the latter species is present at great numbers in the vast norwegian seabird colonies. its ability to attack man and mammms has been questionable, but this has been recently documented (18) . this paper describes studies with ixodes uriae as a possible vector. these studies were undertaken since: a) the tick is part of rather confined ecosystem which might be suitable for studies on virus-vector-host interrelationships. b) virus-transmission by this tick might have mainly zoonoticm but also some public health importance. c) reports of arbovirus isolates from seabird colonies have been presented earlier (9, 10, 12, 32, 33) . the seabird colonies at runde were chosen from a great number of possibilities due to relative ease of access, and because an unusually high and unexplainable chick mortality had recently been reported. from 206 i. uriae ticks collected at runde in late september 1973, three virus strains have been isolated. one of these isolates belongs to the uukuniemi group (30, 31) . the additional two strains are serologically closely related or identical. some characteristics and ecological circumstances of the latter viruses are reported in the present article. description o/ the biotope the island runde is situated at 62°25'n ' 5°38'e, approx. 30 kin from the city alesund (fig. 1) . the total area is about 6.4 km 2 and the circumference 20 kin. there are two small settlements on the island, l~unde in southeast and goksoyr in northeast : most of the island consists of an uneven mountain platea.u, and has altitudes varying between 100 and 330 metres above sea level. in the southwest part, the plateau is terminated by cliffs bordering the sea. most seabird colonies are situated in this area. approaching sea-level there is naked stone with niches and clefts inhabited mainly by rissa tridactyla, sula bassana, alca torda and uria aalge. further up towards the plateau there are rockfalls covered by thin layers of soil supporting various gramnivor plants. these parts are inhabited by the common puffin (fratercula arctica). we visited the island between september 25--27, 1973 , and collected ixodes uriae in the puffin rockfalls. according to local ornithologists, the puffins had migrated south about 3 weeks earlier. we found the ticks under and between the rocks. signs of engorgement were never seen, and they seemed to have entered into diapause. most of the ticks were to varying degrees surrounded by desiccated skinflaps indicating that they had newly emerged to their present stage. some had not completed their development from nymph to adult, and were totally surrounded by nymph cutis. the ticks were kept on dram-vials with moist plaster of paris, and transported alive to the laboratory. we collected a totm of 257 i. uriae, 206 of which were processed for virus-isolation (table 1) . isolation procedures the ticks were divided according to stage and sex, pools of imagos consisting of 5 and nymph pools of 20 individuals. after rinsing in saline, the ticks were ground in a mortar in 0.4 per cent bovine albumin in pbs pi-i 7.4 (apbs) with mycostatin, streptomycin and penicillin. suspensions were clarified by centrifugation at 1500 × g and inoculated i.c. into baby mouse litters, born: nmri (spe), aged one to three days. suspensions not inoculated the same day as processed were kept at --70°c (l~evco ultrmow). mice were observed during 3 weeks for signs of illness. diseased mice were killed, brains removed aseptically and homogenated in apbs with antibiotics, centrifuged and passed into new baby mice. litters which remained healthy were killed after 3 weeks, brains processed and passed as described. two blind passages were performed routinely. calculations of 50 per cent end point, titers, lds0 baby mice (bmlda0) were performed according to ir,~d and mu~nci4 (20) , based on ten-fold titrations in mice. virus-multiplication has been tested in bhk21/c t3, itela bristol, bsc-1, rk-13 and vero celt lines and also in primary gmk cells. bi-ik21/c i3 cells were grown in the medium described by maephe~asos" and sto~e~ (it). all other cultures were grown in eagle's medium with 2 per cent inactivated calf serum. tubes were seeded with 105 cells in 1 ml medium. cultures were used when confluent monolayers were present. cells were washed with saline, virus was diluted ~enfold from 10 -1 to t0 -6 in the medium, a volume of 0.2 ml of each dilution was inoculated into three tubes and allowed to adsorb for 1 hour at room temperature before washing with saline and addition, of new medium, culture tubes were incubated for 8 days at 37 ° c and inspected daily for a cytopathie effect (cpe). after 8 days culture fluids were inoculated i.c. in baby mice litters, 0.0t ml per mouse. cell controls were treated in parallel. bhk21/e 13 cultures in roux bottles were established by seeding 6 × 10a cells/ml. efforts to plaque the isolates were performed in bitk 21/c 13 cells under carboxymethylcellulose overlay by a method recently described (22) . one to three hundred times concentration of cell culture fluids were performed by precipitation with polyethylene glycol (peg) 6000 and nac1 as described by mcs~arry and benzingee (14) . in some instances the precipitates were sonicated at 100w for 3 × 15 seconds in a branson b 12 sonifier, sensitivity to sodium-desoxycholate (si)c) was determined in baby mouse litters by the method of t~ei~a (25) . for haemagglutination (ha), immunoelectroosmophoresis (ieop) and closed hexagon immundiffusion (chi) experiments, the following antigen preparations have been employed : a) crude suckling mouse brain (smb) preparations: a 20 per cent infected suspension in apbs which has been centrifuged for 15 minutes at 10,000 rpm. b) sucrose-aceton (sa) extracted infected mouse brains: prepared according to c~ar~:e and casals (4), but omitting the final lyophilization step. c) cell culture antigens: culture fluids from infected bhk21/c13 roux bottle cultures were precipitated with 6 per cent peg 6000 (macrogolum, norsk medisinaldepot) and 2.2 per cent naci (14) . the 100--300 times concentrated virus-precipitates were sonieated. antibodies were produced in adult, white mice inoculated with infectious suckling mouse brain preparations. initially each mouse received a virus dose of approximately 2.5 x 104-5 bmld~0 in 0.25 ml brain suspension mixed thoroughly with 0.25 mi freund's complete adjuvant (difco). subsequently 3 weekly injections with approximately 5× 104-5 bmld~0 in 0.5 ml suspension were given. the last injection, identical to the initial one, was performed one week thereafter. another 7--8 days later paraeentesis and bleeding from the retro-orbital sinus were performed. arltibody preparations for tahyna virus were produced in the same way. a fowl antiserum to avian infectious bronchitis virus (aib) with a ni of 6.1, was kindly provided by the institute of veterinary medicine, oslo. reference mouse antibody preparations to tbe, tribec, eee, wee and uukuniemi (s 23) were supplied by the yale arbovirus research urlit. an ornithological expedition to tternyken, rost (approx. 67 ° 28°n, 12°e (fig. 1) , collected 19 seabird sera in early may 1975. the composition of this material is cited in table 6 . serological methods tiaemagglutination (ha) and haemagglutination inhibition (hai) tests were performed according to clarke and casals (4), modified for microtitration equipment (cooke eng. co.). ha activity was tested within the ph range 5.6--7.2 at 4 °, 22 ° and 37 ° c. erythrocytes from a variety of species have been investigated. these results will be reported separately, and all ha and hai titers in the present paper refer to the employment of 0.5 per cent chicken erythrocytes. (ieop) twelve ml gel was poured onto precoated 8 × 8 cm lantern slides. for antigen detection i per cent agarose (l'industr. biol. franc.) and for antibody detection a mixture of 0.6 per cent agarose and 0.4~ per cent difco baeto agar was used (29) . three rows of paired wells with 3 mm diameter were punched out. the well interdistance was 3 ram. in the i-lepascreen eleetrophoresis apparatus (spectra biologicals, oxnard, calif., 9303) 2 slides were run simultaneously for i--2 hours. in screenings, this allows testing of 58 unknown samples in one run. gel precipitation this was performed by a sensitive modification of the micro ouehterlony technique termed closed hexagon immunodiffusion (chi) (27) , and also with varying patterns on lantern slides. to obtain the maximum number and intensity of the precipitation lines, the antigens were applieated several times (2--5) during the 16 24 hours prior to antiserum application. due to the lower diffusion rate of antigens, an interval of 2--4 hours between the last antigen and the antiserum application was adopted. the first lines then could be seen 4--5 hours after antiserum application. the gels were incubated at room temperature overnight, and then several days at -~4 ° c before staining and final reading. ieop and chi gels were stained by 4 per cent tannic acid as previously described (8) . negative contrast eiv[ infected culture fluids from bgk21/cl3 cells grown in roux bottles were concentrated 300 times by peg6000/nac1 precipitation (14) , resuspended in borate saline pit 9 or pbs pti 7.4 and sonicated. then 0.3 ml suspension was diluted 1/10 in pbs and centrifuged for 1 hour in a sorvm1 rc 2-b with rotor 55--34 at 20,000 rpm. the pellet was suspended in a few drops of distilled water and negatively stained with 2 per cent phosphotungstie acid pi-i 7.0 or 0.5 per cent uranyloxalate pi-i 6.0. one drop virus-stain mixture was placed on a formvar-earbon coated grid and excess fluid was withdrawn with filterpaper. the grid was examined in a jem 100b electron miscroseope at a magnification of 50,000 ×. thin section em infected and control bhk 21/c 13 cultures were harvested 4 days p.i. by means of a rubber policeman. the ceils were fixed in 3 per cent glutaraldehyde for 1 hour at -~ 4 ° c, washed three times in eacodylate buffer, postfixed in 1 per cent osmiumtetroxyde for 1 hour at room temperature and centrifuged for 3 minutes at 2500 rpm. the cell pellet was resuspended in a few drops of caeodylate buffer and centrifuged in micro capillary tubes in a hematocrit eentrifuge for 3 minutes at 12,500 rpm. the pellets were removed from the capillary tubes, dehydrated in acetone and embedded in spurt by a rapid procedure (6) . ultrathin sections (silver) were cut on a u 2-reichert ultramierotome and doubly stained by saturated uranylacetate in 50 per cent ethanol and reynolds lead citrate (21) . electron microscopy was carried out in a jem 100b at a magnification of 50,000 ×. two pools, both processed from 5 unengorged female i. urine, termed e81 and e85 were inoculated into mouse litters 2 and 3 days old respectively. the mice seemed unaffected during the 3 weeks observation period. however, in the first passage (m1) the mice were moribund after 1.4 days for e81 and 13 days for e85. in the second passage (m2), incubation periods were reduced to i0 and 9 days respectively and in the subsequent passages to 5--7 days for both strains. the viruses were reisolated twice from the original tick pools during the following year. these results are summarized in table 2 . the original tick pools were diluted 1/10 in bhk-medimn and inoculated on five bhk21/c 13 tube cultures. the tubes were harvested after five days. no cpe was recorded. the culture fluids were incubated into three baby mouse litters each. the mice were moribund after 8--10 days. the viruses were shown to be serologically identical to the strains isolated in mice by hai, chi and ieop. the ru e8t tick pool was diluted 1/100 in bhk-medium, and three bhk cultures in roux bottles were infected. tile cultures were harvested after four days. infected culture fluid was inoculated i.c. into 5 baby mouse litters, which were paralysed at day 10 p.i. the rest of the culture fluids were concentrated 100 times by peg 6000/naci, and used as antigen. the isolates were shown to be serologiealty identical, and also identical to the mouse-passaged strains by hai, chi and ieop. a 20 per cent brain suspension from the third suckling mouse brain passage (m3) titered 105-s bmlds0/ml in 1 day old mice. the virus suspension was titrated in parallel in 7 and 1.4 days old mice. as shown in table 3, 1 week old mice were nearly as sensitive as the newborn, while there was a drop in infectivity of 1.2 logs and a prolongation of average survival time from 6.3 to 10.5 days for 2 week old mice. during production of antibody preparations it was demon-strafed that adult mice were refractary to intraperitoneally injections with both e81 and e85. in hela bristol and rk-13 cultures no signs of virus multiplication were detected during primary inoculation and 3 consecutive blind passages. no cpe was present, and culture media concentrated 1--300 times by peg 6000/nac1 precipitation did not affect 1--3 days old suckling mice. as mentioned before, virus multiplication but no cpe was recorded after infection of bhk 21/c13 cultures with the original tick pools. plaquing in bhk 21/e 13 cells was not successful by the method used. both i~unde es1 and e85 virus demonstrated a very marked sensitivity to treatment with sodiumdesoxycholate. the titers were reduced from 5.5 to 3.4 in the case of ru e81 and 5.8 to 3.6 in the case of l~u e36. l~unde virus produces antigens in suckling mouse brains as well as in bhk 21 l c 13. the antigens demonstrate specific reactions with mouse antisera and immune aseitie fluids in hai, chi and ieop. the antigenic preparations have been kept at, --20 ° c for 11 months without loss in reactivity, itaemagglutinating (ha) antigens can be produced ~om infected smb by the sa extraction method of cl~r~:e and casals (4), hut mso suspensions of crude infected smb and peg 6000/nac1 precipitated cell culture fluids contain haemagglutinating activity. the ability to agglutinate chicken erythrocytes is relatively independent of pit and temperature. the highest titers and most clearcut endpoints have however been attained by pit 6.4--6.8 at 4 ° c. in hai no serologic differences between rue81 and e85 could be demonstrated. the mouse antisera and immune ascitie fluids were inhibitory to 4i-ia units of antigen to dilutions of 1/80--640. normal mouse sera were not inhibitory, neither was a fowl antiserum to aib virus or antisera to tbe, tribec, eee, wee and tahyna. precipitating antigens, as revealed by chi and i e o p , also were present in the same preparations as cited above. i n citi 3 --5 specific lines have been noted (fig. 2) , while in i e o p double lines were seen. no antigenic difference between the two strains was detected. virus from mouse brains and cell culture demonstrated total i d e n t i t y b y these methods. no reaction was observed with a n antiserum aga, inst avian infectious bronchitis virus. the 19 seabird sera were screened by i e o p , and the specificity secured by chi. foul" out of 19 birds had precipitating antibodies to r u e 8 t . the composition of the material and the results are sho~nt in table 4 . negative contrast em a series of electron micrographs of negatively stained preparations are presented in figs. 3, 4. fig. 3 shows corona-virus-like structures in a preparation from l~u e 81 infected cell culture supernate. similar structures were never seen in uninfected controls. most of the particles have an approximate total diameter of 170 nm, but in other preparations particles with diameters varying from l l 0 to 220 n m have been seen. fig. 4 a demonstrates an intuct particle at a greater magnification. i t seems to contain an inner structure 80 nm in diameter. fig. 4 b and 4c demonstrate partly and completely disrupted particles with loss of knobs and release of inner helical material. staining with 0.5 per cent uranyloxalate (fig. 4d ) produces a sort of 3-dimensional picture of the virus-like particle. thin section em thin sections of r u es1 infected b h k 21/e13 cultures 4 days p.i., demonstrate virus-like particles in the cytoplasm and extraeellularly between broken and intact cells (fig. 5) . the particles have a diameter of 100--110 nm. some of them look empty, while others are penetrated by stain and expose inner structures. the particles seem to have a double outer membrane, but no knobs are visible by thin sections and positive staining. when the runde virus strairts were first isolated, it had to be kept in mind that they might originate from the staff handling the tick pools, or from the baby mice used for isolation. the reisolations from the original tick suspensions (table 2) , a~d the properties differing from known human, rodent and also avian eoronaviruses (2, 19) , could not entirely rule out these possibilities. but the fact that antibodies to the virus were demonstrated irt seabirds and the isolation of virus also in cell cultures seem to settle this question. circumstances of tickcollection strongly support that runde virus is an arbovirus in the ecological sense, and not a mechanical pick-up : 1. i t is highly unlike that a relatively labile virus should survive for 3 weeks in i. uriae without active multiplication. 2. the lack of engorgement or pregnancy of the female i. uriae, and the fact that they were in diapause and surrounded by desiccated skinlaps, strongly indicate that t~unde virus m a y have passed interstadially in the virus-carrying ticks. still, the final inclusion of t~unde virus amortg the arboviruses must await the demonstratiorl of true biological transmission by .i. uriae. although the morphology of gunde virus strikingly resembles that of the eoronavirus group, some characteristics of the virus differ considerably from 3* those of known coronaviruses. this holds true for the pathogenicity to mice (2, 13) , the ability to grow in cell cultures (1, 2, 5) and the haemagglutinating properties (2, 7) . besides, the size of the virion seems larger than for other members of the coronavirus group (19) . the discrepancy in size between negative contrast and thin sections may be partly due to shrinkage during fixation and dehydration prior to embedding and thin sectioning, and partly to collapse of the particles by negative staining adding to their actual size. the most reliable staining procedure for size calculations probably is negative staining by uranyloxalate which also stabilizes the structure of the particles. detailed taxonomic considerations for runde virus must await further characterization of biological, serological and molecular properties. but runde virus is probably a "new" coronavirus on the grounds of: its morphology, the lack of cross reaction with avian infectious bronchitis virus and the lack of serological reactions with control mouse sera probably eliminating the possibility of mi-iv isolates. also, our department of experimental animals have no indications of mouse hepatitis in the mouse colony which have been used in these studies. the demonstration of this hitherto unrecognized virus associated with seabirds and ticks also necessitates further efforts to clarify-the ecological and possible epizootologieal implications. as alre~ody mentioned, an unusually high chick mortality ill the seabird colonies at runde has been reported during the ias~ few years. bacteriological and toxocological research has not been able to reveal any definite etiological factor, although pesticide residues have been demonstrated to some extent. this must of course bring viruses into consideration. a synergistic action between pesticides and virus seems one possible working hypothesis in this connection. since norwegian seabird colonies in general are rather frequently visited by scientists, students, holiday travellers and the local public, the recent documentation of the ability of i. uriae to attack man (18) raises the question of public health implications. isolations from i. urine of uukuniemi viruses (30, 31, 33) orbiviruses (12, 32) and flaviviruses (9, 33) ill addition to runde virus further stresses this point. at present nothing is known concerning the distribution and general infection rates of runde virus. some indications that it might be widespread in norwegian seabird eolonies can be given, however. firstly, the isolation of two virus strains from only 206 i. urine collected at runde indicates a rather high infection rate of the vectors in this location. secondly, the demonstration of a high rate of seropositive birds at tlernyken, rest, proves that virus-circulation is not restricted to runde. further field work and experiments which may throw more light upon th(~ ecological interrelationships of runde virus as well as upon the characteristics of the virion are in progress. we are indebted to eva mood petterson, einar brunvold and hatlgrim sog£rd for their skilfui technical assistance, and to tone lea for her valuable help with the preparation of figures. the propagation of "eoronaviruses" in tissue-culture coronaviruses of mart meningoeneephalomyeloradieulitis in sunnhordland. tidsskr. for den norske l~egeforening 11 techniques for haemagglutination inhibition with arthropod-borne viruses replication of avian infectious bronchitis virus in african green monkey kidney cell line veto two-hour embedding procedure for intracellular detection of viruses by electron microscopy some characteristics of haemagglutination of certain strains of "ibv-like" virus a serological tool for the diagnostics of virus b hepatitis. aeta path. microbiol, scand tuleniy virus--a new group b arbovirus isolated from ixodes (ceratixodes) putus pick.--camb. 1878 collected on tuleniy island sakhalin v i r u s --a new arbovirus isolated from ixodes (ceratixodes) put~ts p i c k . --c a m b . 1878 collected on tuleniy island, sea of 0khotsk polyoma transformation of hamster cell c l o n e s --a n investigation of genetic factors affecting cell competence great island and bauline : two new kemorovo group orbiviruses from ixodes uriae in eastern canada growth in suckling mouse brain of "ibv-like" viruses from patients with upper respiratory tract disease concentration and purification of vesicular stomatitis virus by polyethylen glycol "precipitation lopper og lundelus p£ sjofugl p& t~ost 1968. fauna (oslo) 21 records of eetoparasitic insects and mites on birds and mammals in norway lopper, f~tt og midd p~ piggsvin i norge. fauna (oslo) 25 ultrastructure of animm viruses and bacteriophages: an atlas a simple method of estimating fifty percent endpoints the use of lead citrate at high p i t as an electron opaque stain in electron:microscopy arboviruses in finland. ii. isolation and characterization of uukuniemi virus, a virus associated with ticks and birds notes on norwegian ticks ixodes ricinus som mulig vektor for sykdommer hos mennesker i norge action of sodium desoxycholate on arthropod-borne viruses arbovirusencephalitt overfort av ixodes ricinus a sensitive modification of the ouehterlony technique. detection of hepatitis associated antigen by immunodiffusion in a closed hexagonal system. acta path. mierobiol, seand serological investigations indicating the existence of tick-borne encephalitis virus loci along the norwegian coast. aeta path. microbiol, seand development of a modified immunoelecgroosmophoresis method for uukmfiemi and i~unde virus serology tickborne viruses in norway uukuniemi group viruses isolated ill norway tickborne viruses in western north america. ii. yaquina head, a new arbovirus of the kemorovo group isolated from ixoedes uriae tick-borne viruses from seabird nesting sites in oregon and alaska authors' address: dr. t. t~aavik, institute of medical biology, university of tromso, 9001 tromso, norway.received august 24, 1976 key: cord-332233-01rdlf8l authors: tully, thomas n. title: chapter 12 mice and rats date: 2009-12-31 journal: manual of exotic pet practice doi: 10.1016/b978-141600119-5.50015-9 sha: doc_id: 332233 cord_uid: 01rdlf8l publisher summary this chapter focuses on mice and rats, and provides detailed information that may be useful for veterinarians treating these animals. mice are continuous, polyestrous rodents that should be bred in polygamous or monogamous setups because of the males' aggressive territoriality behavior. when breeding mice that have been housed in a polygamous ratio, there may be one male with two to six females. females are removed from a polygamous cage before parturition, whereas the monogamous pair is maintained together with the young until weaning. mice are maintained in environments that are similar to other small rodents but require a thorough cleaning of their cage more often because of their malodorous urine. ventilation is essential for small rodent housing to prevent irritation of the respiratory tract from ammonia vapors generated by urine. quarantining is important when a new animal is being introduced into a setting in which there is an established group. as with other animals, a 30-day quarantine period is recommended, along with a physical examination and fecal parasite check. to maintain oversight of breeding animals' health and reduce the exposure of young animals to infectious disease and parasites, routine screening of representative animals within the colony is recommended. in very large colonies, special caging, food, and water may be necessary to prevent exposure to disease organisms. c h a p t e r 1 2 thomas n. tully, jr. this chapter, on mice and rats, contains information on the companion species that is useful for veterinarians treating the animals or providing information to the owners. although similarities exist for mice and rats-in care, husbandry, diagnostic testing, and treatment-emphasis will be on the differences between the species. mice may be maintained as pets because of their size and playful nature. although playful, mice can be aggressive to cagemates and owners. the aggressive nature often manifests as barbering and/or fi ghting with cagemates and biting their owners. any potential owner should be educated on the aggressive nature of these small dynamos. domestic mice (mus musculus) and the african pygmy mouse (baiomys spp.) are commonly sold for pets and are available in several varieties. the mice varieties sold in pet stores include white, black, or tan colored, satin hair coat (shiny), pied or spotted, and longhaired 1,2 (figure 12-1) . the size and ability of mice to escape quickly from the grasp of a human handler make these animals, as pets, better suited for older individuals. the timid nature of mice predispose these animals to biting if handled roughly, which commonly occurs with a young owner. mice are nocturnal animals and may be a disturbance at night if maintained in a bedroom. female mice produce less odor than males and therefore may be more desirable as pets. as mentioned earlier, male mice are territorial, and if placed with other male mice, may fi ght. advantages of having mice as pets include their size, ability to be a good companion animal for the educated (regarding mouse behavior) owner, and adorable appearance. mice also rarely become infected with bacterial diseases, and their life span is approximately 2 years (box 12-1). the integument of mice is commonly associated with a number of disease presentations. a hair coat that has not been adequately maintained is often the fi rst clinical sign associated with disease. mice are fastidious with their grooming and, when healthy, maintain a very tidy hair coat. common problems associated with a hair coat that is not maintained include general illness, parasitism (internal and external), aggression by cagemates as a result of psychological trauma, and infectious dermatitis. neoplasia is a disease condition that affects mice. there are some mouse strains in which mouse mammary tumor viruses have been identifi ed that have a neoplasia incidence as high as 70%. 3 tumor viruses, as well as a very short life span (up to 2 years), predispose these animals to cancer. mammary adenocarcinomas and fi brosarcomas are the most common tumors that affect mouse mammary tissue. 4 unfortunately, by the time many of these tumors are diagnosed, the malignant masses are large and ulcerated. 4 fibrosarcomas may be hormonally induced, and the incidence of this disease process can be reduced through an ovariectomy at an early age. the small size of the female mouse often makes the ovariectomy surgery challenging for the veterinary surgeon. although relatively uncommon, urethral obstruction can occur in male mice as a result of preputial and/or bulbourethral gland infections. 4 male mice suffering from an urethral obstruction may have a mutilated penis, a sign often associated with the disease process. mice are continuous, polyestrous rodents that should be bred in polygamous or monogamous setups because of the males' aggressive territoriality behavior. 2 when breeding mice that have been housed in a polygamous ratio, there may be one male with two to six females. 2 females are removed from a polygamous cage before parturition, whereas the monogamous pair is maintained together with the young until weaning. 2 mice can chew out of enclosures; therefore, it is important that the housing be "mouse proof." if an animal escapes, the best way to capture the pet is to place food in the center of the room. once it has been determined in which room the animal is staying, then it should be sealed and measures taken to look for the pet. because mice are nocturnal animals, capture at night in a dark room with a fl ashlight may work best. this technique will work for capture of other small rodents and pocket pets too. mice are maintained in environments that are similar to other small rodents but require a thorough cleaning of their cage more often because of their malodorous urine. ventilation is essential for small rodent housing to prevent irritation of the respiratory tract from ammonia vapors generated by urine. the recommended housing unit for mice is 12-15″ × 12-15″ × 6″ (length × width × height) for each adult mouse; females and young require 2 to 3 times the space listed. 2 the major considerations for selecting an enclosure for mice is that it be resistant to their escape and easy to clean. an open screen top of the enclosure is recommended for proper ventilation. available rodent cages that fi t this criterion are wire or metal mesh, plastic, and plexiglas. if the plastic tube housing systems are used, there should be routine cleaning of the sections using hot water and a mild detergent. owners should be informed of the small distance required for a mouse to escape and their ability to chew through plastic. an owner must always monitor the cage for possible escape avenues, especially if the animal has a predilection for modifying the cage opening through chewing. it is very important to provide cage toys and exercise opportunities for mice. the enclosure should be large enough to house not only food and water containers but also cage toys and an exercise wheel (figure 12-2 ). although not a cage toy, a hide box, in some form, should also be included for the psychological well-being of the animal. this hide box can be manufactured or a small cardboard container that has one end cut out. mice like to hide and also sleep during the day. this piece of cage furniture will enable the mouse to sleep undisturbed during the daylight hours. mice, especially males, have odiferous urine. to prevent the buildup of urine and fecal material in the cage, commercially available paper rodent bedding or hardwood shavings should be used as a cage substrate. although softwood (pine) and cedar shavings are available, the volatile oils that radiate from these substrates are irritating to small animals. these irritating compounds can cause dermal and respiratory infl ammation, often leading to secondary bacterial infections. cardboard tissue tubes can be placed in a mouse enclosure; mice like to chew and run through these items. because many fabrics have strong thread and elastic, it is not recommended to put clothes items in enclosures with animals that chew. often mice will shred the clothing items, exposing thread and elastic that can become entangled around an extremity; this can lead to necrosis distal to the stricture. it is recommended to change the substrate within the enclosure at least twice a week-more often if there is a problem with odor or excessive excreta. temperature, humidity, and lighting within the enclosure should follow what is commonly maintained with the ambient conditions within the house. if the animals are housed outside or in an outbuilding, the temperature range should be 65° f to 85° f and humidity 30% to 70%, although the recommended humidity is on the higher end of the range provided. 2 mice, especially males, are extremely territorial animals. if mice are housed alone, it is better to keep them separate, as introduction of new animals will often lead to aggression and fi ghting. to prevent aggression, fi ghting, and psychologically induced adverse behavior (e.g., barbering), a single mouse is recommended as a companion animal. for those who intend to breed the animals, enclosures with a single pair is necessary for reproductive success. because mice are commonly used as laboratory animals in investigations of human disease processes, much research literature is available on the recommended nutritional requirements of these animals. the benefi t of these dietary studies is the availability of commercially produced diets that provide the recommended daily nutritional requirements of the mouse. the problem that most owners face is the multitude of dietary products available for mice and the lack of knowledge regarding which food to buy. most new mouse owners think that rodents eat seed-based diets. although mice will happily eat seed, seed-based diets are lacking in a number of the nutritional requirements needed to maintain long-term health. commercial rodent biscuits or pellets with more than 14% protein are the recommended diets for mice. 2 the commercial rodent biscuits or pellets are the only food mice need to obtain their required nutrients (figure 12-3) . again, seed-based diets are not recommended, nor is a signifi cant supplementation of fruits, nuts, vegetables, cheese, or other human foods (e.g., peanut butter). if a treat is to be given, yogurt or dried fruit treats manufactured specifi cally for rodents and/or mice should be provided 2 to 3 times a week. for younger mice, less than 3 weeks old, softer pellets are needed because babies start eating pellets and drinking water at 2 to 3 weeks of age. 2 a sipper bottle, placed on the outside of the cage, is easy to maintain and does not take any space within the enclosure. if the cage is plastic or plexiglas, modifi cations will be needed to attach the sipper bottle on the outside of the cage. most sipper bottles come with attachment hardware to attach to wire cages ( figure 12 -4). fresh bottled water is recommended for mice, although chlorinated tap water is acceptable. the water should be checked on a daily basis and clean water supplied at least every 2 days, if not every day. quarantining an animal is important when a new animal is being introduced into a setting in which there is an established group. as with other animals, a 30-day quarantine period is recommended, along with a physical examination and fecal parasite check. unfortunately, there is no time period that will screen 100% against potential infectious agents, and with mice, time is critical because their life span and reproductive time frame are so short. the animals should be quarantined in conditions similar to those in which they will be permanently maintained. providing adequate food and water will make for a smooth transition. it is imperative that the owner screen the new animal for diseases and watch for any signs of illness. the most common sign of clinical illness is a rough hair coat. the lack of grooming is most often related to the animal feeling depressed or sick, thereby not having the energy to perform routine behaviors. if a number of animals are being quarantined, sacrifi cing an apparently diseased animal is the most effi cient way to determine a rapid defi nitive diagnosis. if it is a single or an expensive animal, routine diagnostic testing will be required. to maintain oversight of breeding animals' health and reduce the exposure of young animals to infectious disease and parasites, routine screening of representative animals within the colony is recommended. in very large colonies, special caging (e.g., fi lter), food, and water may be necessary to prevent exposure to disease organisms. 2 before a veterinarian examines a mouse, the examination table should be disinfected with either a dilute sodium hypochlorite or chlorhexidine solution. the examiner should always wash his or her hands and, if necessary, wear examination gloves to capture and restrain the patient. the diffi culty with using latex gloves when examining a mouse is the small size of the patient and its ability to twist and turn. the only way to restrain some mouse patients is without gloves. if gloves are not worn, the examiner's hands must be washed thoroughly after the examination is completed. to catch a mouse, the tail should be grabbed with the thumb and forefi nger, allowing the mouse to hold on to an object with their front feet (figure 12-5 ). when the mouse securely attaches itself to an object, the opposite hand then grabs the dorsal skin in the cervical region while keeping the tail in a fi rm grasp. if one is unable to adequately restrain a mouse while the animal is conscious, inhalant anesthesia may be used for sedation purposes. the animal should be placed in a small induction chamber and isofl urane gas permeated into the closed space. once the animal has stopped moving, the enclosure top is removed and a nose cone placed on the anesthesia tube and placed on the patient's face. a syringe case can be modifi ed as a nose cone for small rodents, including mice. once the physical examination is complete, the nose cone is removed, allowing the patient to breath oxygen from the anesthesia unit. obtaining a detailed history of the mouse patient is very important, as it is with other animal patients that are brought to a veterinarian's offi ce. it is important to get as much information from the owner as possible, even if it is a routine health examination. typical background information required includes how long the mouse been owned, where it was acquired, how often it is handled, and what the character of the feces and urine is. husbandry questions should focus on the animal's housing and whether it is allowed to roam unobserved; cage location; type, size, and material of the cage; cage substrate, furniture, and toys; and the frequency with which the cage is cleaned and what disinfectant is used. when investigating the diet, the veterinarian should ask not only if pellets are fed and in what quantity, but also what the animal is eating and what the primary diet is. supplemental offerings and frequency of feeding are important data for the case work-up. the veterinarian should fi nd out about the water supply, how often the water is changed, and how much the animal drinks on a daily basis. because there are transmissible diseases among animals, the fi nal questions should center on the other pets in the household, if new animals have been added to the family, and if the animals are housed together. a description of any previous problems and a complete chronological description of the presenting problem are needed to complete the history form. before the animal is restrained, an observation should be made on the attitude, activity, and posture of the animal. the next step is to weigh the mouse in a basket on a digital gram scale. if possible, temperature, respiration, and pulse should be measured and any abnormalities in rate and/or character noted. the veterinarian should start the physical examination at the head, looking for any abnormalities. eyes, ears, and nares are observed, looking for discharge or infl ammation. the oral cavity is diffi cult to examine in mice because of the small opening and tendency for the buccal mucosa to encroach toward the middle of the mouth. a small speculum (e.g., modifi ed paper clip) or an otoscope may be used to examine the oral cavity and cheek teeth. mucous membranes help determine hydration status using capillary refi ll time and moisture. body condition and abdominal palpation are important information that should be obtained. lymph nodes and limbs are palpated before checking the nails and plantar surface of each foot. the patient should have a normal posture, be aware of its surroundings, and move properly. any problems should be noted in the record. finally, a dermatologic exam considers hair coat quality, alopecia, external parasites, and any skin abnormalities. all abnormal fi ndings are written in the record for case review, differential diagnoses determination, diagnostic testing, and treatment considerations. most of the common problems noted during the physical examination of mice involve the skin and hair coat. as mentioned previously, an unkempt hair coat may point toward generalized illness or external parasitism. abrasive lesions or pustules on the skin can be signs of external parasites or infectious dermatitis. if external parasites are a problem, the mouse may be uncontrollably scratching. hair loss may be associated with barbering, either cagemate or self-induced, or with infection (e.g., ringworm). although not common, trichophyton mentagrophytes will cause hair loss in mice, from the face, head, and neck. 4 mice will commonly present with tumors and abscesses. the entire body of the mouse should be palpated for any evidence of masses. if a mass is identifi ed, a fi ne needle aspirate is recommended for basic diagnostic evaluation. with most fi ne needle aspirate samples, the clinical pathology results are not very rewarding. if a diagnosis cannot be obtained through cytologic examination, a biopsy of the lesion is required, and if possible, an excisional biopsy is the procedure of choice. as with many rodents, respiratory disease is commonly identifi ed in mice. mice may have presenting symptoms of dyspnea, sneezing, coughing, chattering, and sniffl ing if they have a respiratory infection. in cases where respiratory signs are observed, anesthetizing the animal to perform the physical examination is not recommended, and handling should be kept to a minimum. the two most common respiratory disease conditions in mice are sendai virus and mycoplasma pulmonis. 4 these two organisms are very diffi cult to identify through routine diagnostic measures, and obtaining samples significantly stresses the patient. if the animal is being examined for placement into a colony or breeding environment, sacrifi ce to identify the disease condition is recommended. if the animal is an individual companion animal, doxycycline treatment should be initiated. although treatment with doxycycline will not clear the organism from the animal, it will reduce clinical disease and improve the quality of life. the treated animal should always be considered a carrier of these respiratory diseases if a defi nitive diagnosis cannot be obtained. male mice that are licking and/or mutilating their penis may be suffering from a urethral obstruction. infection of accessory sex glands, young males with aggressive breeding activity, urolithiasis, and trauma may initiate this self-induced trauma to the penis. 4 pasteurella pneumotropica is often isolated from accessory sex gland infections as well as subdermal abscesses. 4 treatment for this organism may aid in the recovery of a mouse affected by urethral obstruction. diarrhea in mice is rare, but endoparasite examinations should be performed if the animal has diarrhea. although pinworms are considered nonpathogenic in mice, syphacia obvelata and aspiculuris tetraptera may cause rectal prolapse due to straining in immunosuppressed individuals. 5 as with other animals, a tape test is the best way to identify these parasites. a tape test follows a procedure of pressing the sticky side of the tape against the rectum and perianal area and then examining the tape under a microscope for eggs and parasites. 5 as with other pocket pets, blood collection from mice can be quite diffi cult. approximately 0.5 to 0.7 ml/100 g body weight can be safely removed from a nonanemic healthy mouse. 2 the mouse patient should always be anesthetized for blood collection procedures. usually inducing the animals in a closed chamber and maintaining the patients in a mask will allow the technician plenty of time to collect the blood sample. a small, 25-, 26-, or 30-gauge needle, usually placed on a 1-ml syringe, is recommended for collecting the blood. recommended blood collection sites in a mouse are a warmed ventral tail vein or the tip of the tail. 2 box 12-2 describes the technique used for retroorbital bleeding in mice. the retroorbital bleeding technique is commonly used in laboratory animal settings and is easily performed by an experienced veterinarian or veterinary technician. 5 on smaller rodents, the saphenous vein or lateral vein of the tarsus may be used for multiple blood collections without the use of anesthesia. the patient must be properly immobilized in a restraint tube (35-ml syringe) with the leg extended and the skin held tight on the medial aspect of the thigh using the thumb and forefi nger. 2 the taut skin on the lateral aspect of the thigh allows exposure of the saphenous vein. a 23-gauge needle is used to puncture the vein, and blood is collected in a microhematocrit tube as it fl ows from the vessel. 5 blood samples obtained from nail and ear clips are not considered appropriate diagnostic samples. cardiac puncture is recommended only in terminal cases and when the animal is maintained under general anesthesia because of possible complications involving the lungs and heart vessels. reference ranges for complete blood counts and serum biochemistry panels for mice are listed in boxes 12-3 and 12-4. 1,2 marrow samples may be obtained from the ilium, tibia, sternum, femur, or the bones of the proximal one third of the tail. 5 preparation of the samples is consistent with that of other mammalian patients. standard rodent cages can be used without substrate to collect urine and feces when the patient is hospitalized. after placing a rodent in a zip-lock bag, urination frequently occurs and the sample can be collected. 4 in a similar manner, urine can be collected over a disinfected stainless steel examination table. the urine is voided while the animal is restrained and can be collected in a capillary tube for evaluation on urine reagent strips. 1 urinalysis reference values for mice are listed in box 12-5. under most practice settings, the size of a mouse patient limits imaging capabilities to radiographs. restraint is essential for quality diagnostic images and is even more important when patients weigh as little as 40 g. to obtain adequate restraint and prevent movement of the mouse patient during radiographic evaluation, sedation is required. as with all diagnostic box 12-2 retroorbital blood collection for mice from procedures involving avian and exotic animals, an evaluation of the patient is required to determine its ability to withstand the stress associated with the assessment. if it is determined that the patient cannot withstand sedation, then it should be stabilized until it is deemed healthy enough for diagnostic imaging evaluation. sedating the mouse using an induction chamber and isofl urane anesthetic, as described for restraint, is necessary to obtain diagnostic fi lms. high-speed, 300-ma machines with fi ne or detail-intensifying screens should be adequate for most small exotic mammal images. with mice, dental x-ray units that can focus at short distances may be advantageous for both isolation of focal anatomy and full body radiographs in extremely small patients. for extreme detail in small exotic mammal medicine, using a traditional radiography unit, the min r mammography system (eastman kodak) with min r single-intensifying screen cassettes, in conjunction with min r single-emulsion fi lm, has been recommended. 1 digital radiography is bringing a new dimension to radiograph evaluations and detail. the secret to success in either traditional or digital radiographic imaging is to restrict patient movement during exposure. two views, ventrodorsal and lateral, are recommend for mice patients. again, because of a mouse patient's size, whole body radiographs are commonly obtained during the radiographic evaluation, even using dental radiographic equipment. for the mouse patient in which microbiological testing is recommended, standard collection techniques used for other animals is appropriate. the diffi culty when culturing mice is acquiring a representative sample of the suspect area that will provide diagnostic information. sick mice often are extremely stressed when being examined or when diagnostic samples are being collected. it is also important to know what the common disease conditions are and the common etiologies associated with those clinical signs. with the most common respiratory conditions in mice, one is sendai virus and the other a mycoplasma sp. both sendai virus and mycoplasma spp. will not be isolated using standard aerobic or anaerobic culture techniques. if in doubt on collecting, preserving, and/or shipping a microbiological sample, the diagnostic laboratory must be contacted for full instructions as they relate to mouse submissions. routine fecal parasite evaluations should be performed on small rodents when they are brought to the veterinary clinic for a health examination or an abnormal stool. common protozoal organisms can be detected using a direct fecal examination, and mouse pinworms are commonly diagnosed using the sticky side of clear cellophane tape to make an impression of the perianal area. the anal tape test will aid in diagnosing syphacia spp. the sticky surface will pick up any of the banana-shaped pinworm eggs that can be observed under a microscope. diagnosis of ectoparasites in small rodents is similar to that in other species listed in this text. the pelage tape test is performed by pressing clear cellophane tape against the pelt of a mouse, dorsally and ventrally, from the nose to the base of the tail. myobia spp. and myocoptes spp. can be diagnosed using the pelage tape test. a skin scraping of the affected area may also help in identifying ectoparasites. diagnosis of intestinal parasites can be made by fi nding individual eggs during fecal examination or whole worms within the lumen of the small intestine during necropsy. 6 even though most of the diagnostic testing is performed through pathologic examination for mice, there are serologic tests available that can be run on a minimum of 50 μl of undiluted serum. sound diagnostics, inc. (woodinville, washington) provides serologic testing on a number of common mouse diseases, including ectromelia virus, mouse hepatitis virus, mouse parvovirus, mouse minute virus, rotavirus, theiler's murine encephalomyelitis virus, pneumonia virus of mice, reovirus 3, sendai virus, mycoplasma pulmonis, lymphocytic choriomeningitis virus, and mouse adenovirus. the serologic tests are done using enzyme-linked immunosorbent assay (elisa) and, when indicated, are confi rmed by indirect fl uorescent antibody testing. many of the diseases for which there are available serologic tests are benefi cial in maintaining disease-free animals in laboratory settings or large breeding facilities. in cases of unknown disease conditions for the individual pet mouse, there are panels available that test for multiple diseases from a single serum sample. it is very important for the veterinary clinician to understand both the signifi cance of any test that is being promoted for mice and how the results are interpreted. bacterial infections associated with dermatitis lesions and subcutaneous abscesses in mice are commonly caused by staphylococcus aureus, pasteurella pneumotropica, and streptococcus pyogens. 4 acute and chronic respiratory infections may be caused by sendai virus or mycoplasma pulmonis. 4 acute respiratory infections usually are associated with sendai virus; adults live while neonates often die. chronic respiratory infection, with clinical signs being pneumonia, suppurative rhinitis, and occasionally otitis media, may be the result of a mycoplasma pulmonis infection. 4 both infections should be treated using supportive care, whereas mycoplasmosis can be treated with enrofl oxacin in combination with doxycycline hyclate for 7 days. 7 p. pneumotropica has been associated with urethral obstruction caused by infl ammation and swelling related to infection of accessory sex glands. 4 although uncommon, trichophyton mentagrophytes (ringworm) can cause alopecia of the face, head, and neck of mice. 4 in addition to the disease organisms mentioned in this chapter (which are the most common disease organisms identifi ed in pet mice), there are other bacterial, fungal, and viral organisms that can cause disease in mice. proper diagnostic protocol should always be followed to identify the causal agent. once the cause is identifi ed, the proper treatment can be initiated and control measures implemented to prevent other animals' exposure to the disease. numerous parasites have been identifi ed in mice. hymenolepis nana (dwarf tapeworm) and cysticercus fasciolaris (the larval stage of the cat tapeworm) can be isolated from these small rodents. 5 the dwarf tapeworm is often found in young mice that are dead or have severe gastroenteritis resulting in diarrhea. diagnosis of the dwarf tapeworm can be made following either a fecal fl otation exam or necropsy, when the adults may be found in the small intestine. treatment for the dwarf tapeworm is with praziquantel. the mouse pinworm (syphacia obvelata) is a commensal oxyurid nematode that feeds on bacteria that inhabit the intestinal tract of mice. 6 although these nematodes are nonpathogenic in most cases, an overwhelming number of them can cause severe irritation of the terminal gastrointestinal tract. these nematode parasites can be diagnosed using transparent tape and applying it to the rectal area. after removing the tape from the affected rectal area, it is placed on a slide and the ova may be viewed under a microscope. ivermectin and fenbendazole have been used effectively in treating this parasite in mice. 6 giardia muris is a common protozoal parasite affecting mice. 5 this organism can be seen using a direct fecal examination of an affected patient's fecal material. metronidazole is the treatment of choice for giardia spp. infections in small rodents. myobia musculi, myocoptes musculinis, radfordia affi nis, and psorergates simplex are fur mites that can cause severe selfmutilation and hair loss in mice. 6 polyplax serrata (house mouse louse) can cause anemia, pruritus, dermatitis, and death in severely infested mice. 6 diagnosis of ectoparasites in mice is similar to that in other companion animal species (e.g., skin scraping, examining hair samples for nits). treatment of ectoparasites can be accomplished with ivermectin and a topical miticide. as with other rodent species, there has been an increasing ectoparasite resistance to ivermectin treatment. because most rodent diets are manufactured in the form of pellets or small biscuits that provide all recommended nutrients, there are few nutritional disease problems diagnosed in pet mice. if mice are fed an all-seed diet, there could be nutritionally related consequences. a lack of vitamin a could result in roughened hair coat or skin lesions. a breakdown of the protective epithelial lining of the gastrointestinal and/or respiratory tract could predispose the mouse to secondary bacterial infections affecting those body systems. nutritional defi ciencies may also result in poor reproductive activity. it is important to provide an adequate supply of fresh water and appropriate rodent food on a daily basis. following these basic nutritional instructions should provide a nutritional basis for mice to live healthy lives. the average life span of a mouse is 2 years old. this short life span and aging process increase the prevalence of tumors in these small rodents. if an animal is over 18 months of age, neoplastic disease should be considered as a differential diagnosis, especially if an asymmetrical mass is present. the most common tumors diagnosed in mice are mammary adenocarcinomas and fi broadenomas. 4 other tumor types are identifi ed in mice, and if possible, the suspect tissue should be completely excised. representative tissue sections should be submitted for histopathological evaluation. although there are treatment options available for mice that have been diagnosed with neoplasia (e.g., radiation, chemotherapy), its small size does not allow it to withstand the stress and negative side effects of these healing modalities. in mice, the recommended treatment for neoplasia is removal of the affected tissue, if possible, to increase its quality of life. the most common disease in mice colonies is barbering. barbering is defi ned as the dominant mouse nibbling off the whiskers and hair around the face of the subservient cagemates. 5 the hair clipping is very close to the skin without causing skin lesions. this behavior is noted often among female mice, whereas male mice are more likely to fi ght. mice may injure themselves on cage furniture or poorly designed cages and sustain abrasions on the face or nose. individual animals may show stereotypic behavior that results in abrasions and/or alopecia. enrichment furniture and toys may help alleviate this initial cause of dermatologic abnormality. the size of mice contributes to the diffi culty in properly administering therapeutic medications. the scruff of the neck or caudal fl ank are the subcutaneous sites that may be utilized to administer medication, fl uids, or both. 2 the semitendinosus, triceps, and epaxial muscles are commonly used for intramuscular injections, whereas intraperitoneal injections are used in extremely small animal species, including mice. 2 intraosseous catheters are much easier to place in mice than are intravenous catheters. the tibial plateau and the greater trochanter are the sites of choice for intraosseous catheter placement in mice. 5 although there are many methods that may be used to administer medication in small mammals, oral treatment using a dropper or tuberculin syringe is the easiest to administer and the least stressful for the patient. medication can be added to the food or water, but often the patient is anorexic or the medicated food or water is not palatable, in which case, the patient does not receive the treatment. although a relatively diffi cult procedure in mice, gastric gavage can be performed using a bulbed stainless steel feeding needle or fl exible red rubber feeding tube. the length of the tube is premeasured, externally from the nares to the last rib, and marked. a water-based lubricant is applied to the feeding tube before it is inserted through the intradermal space toward the back of the oral cavity. 2 the tube should advance without diffi culty until the premeasured mark is reached. restraint of mice for this procedure is accomplished by pinching the dorsal neck skin fold and holding the body with the remaining free fi ngers. both oral medication and nutritional supplementation can be administered through an oral gastric feeding tube. medication dosages for mice can be found in a number of formularies. before administration of any therapeutic agent, the dosage should be checked twice and calculations thoroughly evaluated to make sure they are correct. because mice are very small patients, any overdose of medication can have adverse consequences on the patient's recovery. inducing and maintaining mouse patients under anesthesia can be very challenging. with the widespread use of isofl urane anesthesia in small animal practices, the previous problems associated with methoxyfl urane and halothane anesthetic agents has been overcome. there are still concerns and differences in anesthetic protocol because of these animals' small size, but in general, isofl urane is a safe agent when used on a relatively healthy surgical candidate. although there are doses of injectable agents for sedation, the small size of the mouse makes it diffi cult to respond to anesthetic crises that may appear during a diagnostic or surgical procedure. therefore, inhalant anesthetic agents (e.g., isofl urane, sevofl urane) are recommended for anesthetizing mouse patients. an induction chamber is used to induce the patients, and then they are maintained under gas anesthesia using a face mask. it may be possible to intubate a larger rodent, but it is very diffi cult in mice; under most conditions, a modifi ed syringe case face mask is used. 5 to guard against aspiration of stomach contents in a patient that is not intubated, the veterinary surgeon should place the head and neck in a position slightly higher than the body. box 12-6 provides basic guidelines for proper techniques used in surgical preparation and surgical assistance in small mammals, including mice. 9 premedication and sedation doses for mice and rats are listed in surgical procedures that are performed on mouse patients require similar techniques to those used with larger animals. veterinarians should follow the same protocol but remember that the surgical fi eld is smaller and patient manipulation more delicate with patients that weigh less than 50 g. zoonotic diseases associated with mice maintained as pets are rare. possibly the most commonly reported zoonotic condition associated with mice is an allergic reaction to their dander and urine. as with all animals, it is important that a handler wash his or her hands after interacting with a mouse. salmonella spp. may colonize the intestinal tract of small mammals, including mice. 7 contact with the feces or anus of a mouse may expose the handler to this enteric pathogen. washing hands after handling a mouse will help prevent exposure to possible pathogenic organisms a mouse may carry. one can monitor the individual mouse and mice colonies for subclinical carriers of zoonotic organisms by culturing the terminal intestinal tract. mice are carriers of two potentially deadly viruses: the hantaan disease virus and an arenavirus that causes lymphocytic choriomeningitis (lcm) in humans. 7 mice that are propagated for sale in the pet trade are rarely if ever exposed to the hantaan disease virus. 7 lymphocytic choriomeningitisinfected mice generally show no clinical signs, although weight loss, photophobia, tremors, and convulsions may occur. 7 humans are exposed to lcm through contaminated feces, urine, or a bite wound. disease signs in humans infected with lcm are fl u-like (e.g., malaise, headaches, fever, myalgia, arthritis). 7 fatal aseptic meningitis or meningoencephalitis in humans infected with lcm is rare. hymenolepis spp. are cestodes found in mice that can infect humans if contaminated feces are ingested. 7 rarely identifi ed in pet mice, giardia spp. are protozoan parasites that can cause severe gastroenteritis in humans. because feces have to be ingested for someone to be exposed, it is more common for children to become infected with zoonotic intestinal parasites than for adults. using proper sanitary practices after handling mice helps reduce exposure to zoonotic intestinal parasites and other potentially transmissible diseases. although there are diseases listed in this zoonotic section that can be transmitted from mice to humans, the occurrence of this happening is extremely rare when commercially bred mice are purchased at reputable pet stores. rats, like mice, are common laboratory animals and are propagated for commercial sale in the pet trade and for use as reptile food. the life expectancy of a rat is 2 to 3 years. unlike mice, rats have excellent pet characteristics that include a charming personable disposition and extreme intelligence (figure 12-6 ). the common rat species maintained as a companion animal is rattus norvegicus, with the white rat and hooded rat being the most common variations. although rats have an excellent temperament for companionship, they can infl ict a serious bite if provoked. also, as with other animal species, humans can be allergic to their hair, skin dander, and urine and salivary proteins. 2 rats are not as territorial as other rodent species and are very social. the integument of rats is commonly associated with a number of presenting symptoms of disease. as with mice, a rat's hair coat that has not been adequately maintained is often the fi rst clinical sign associated with disease. rats are fastidious with their grooming and, when healthy, maintain a very tidy hair coat. common problems associated with a hair coat that is not maintained include general illness, parasitism (internal and external), aggression by cagemates as a result of psychological trauma, and infectious dermatitis. staphylococcus aureus infections initiated by self-trauma due to a fur mite infestation will manifest as ulcerative dermatitis. 4 the most common tumor in rats is a mammary-associated fi broadenoma. 4 fibroadenomas rarely metastasize but are locally invasive and can grow to a large size. a coronavirus is the etiologic agent behind sialodacryoadenitis which results in infl ammation and swelling of the cervical salivary glands. 4 the most common disease presentations in rats involve the respiratory system. the three major pathogens that infect rat respiratory systems are mycoplasma pulmonis, streptococcus pneumoniae, and corynebacterium kutcheri. 5 respiratory disease in rats often produces "red tears" and a red nasal discharge. this red staining around the eyes and nose is not blood but porphyrins produced by an irritated harderian gland located behind each eye. the red staining at the nasal opening is due to the drainage of the porphyrins via the nasolacrimal duct. renal disease may present in older males in the form of chronic progressive nephrosis. 4 on necropsy, kidneys in affected animals are enlarged, appear pale, and have a pitted mottled surface. 4 rats are continuous, polyestrous rodents that should be bred in polygamous or monogamous systems. 2 when breeding rats that are housed in a polygamous ratio, it is recommended to have one male with two to six females. 2 females are removed from a polygamous cage on day 16 of gestation, whereas the monogamous pair is maintained together with the young until weaning. 2 baseline rat physical information is listed in box 12-7. rats, like mice, can chew out of enclosures; therefore, it is important that the housing be "rat proof" regarding the animal's ability to chew through the substance and escape into the house. rats are maintained in environments that are similar to those of other small rodents, but rats do not produce the quantities of odiferous urine associated with other rodents. for rats, large wire cages with easy-to-remove plastic bottoms are the optimal enclosure (figure 12-7) . the recommended housing unit size for rats is 15-20″ × 15-20″ × 7-10″ (length × width × height) for each adult rat, with females and young requiring 2 to 3 times the space listed. 2 rats like to climb ramps and rope for exercise, so a cage that is taller than the listed requirements would be better suited for most singly housed animals. the major considerations for selecting an enclosure for rats, as with mice, are that it is resistant to their escape and easy to clean. an open screen top on the enclosure is recommended for proper ventilation. available rodent cages that fi t this criterion are wire or metal mesh, plastic, and plexiglas. if the plastic tube housing systems are used, there should be routine cleaning of the sections using hot water and a mild detergent. an owner must always monitor the cage for possible escape avenues, especially if the animal adult male 450-520 g adult female 250-320 g birth weight 5-6 g rectal body temperature 99°-101° f normal heart rate 250-450 beats/min normal respiratory rate 70-115 breaths/min daily food consumption 10 g/100 g body weight daily water consumption 10-12 ml/100 g body weight has a predilection for modifying the cage opening through chewing. it is very important to provide cage toys and exercise opportunities for rats; therefore, the enclosure should be large enough to house not only food and water containers but also cage toys and an exercise wheel. although not a cage toy, a hide box, in some form, should also be included, for the psychological well-being of the animal. this hide box can be a manufactured one or a small cardboard container that has one end cut out. rats like to hide and also sleep during the day. by providing this piece of cage furniture or deep enough substrate for the rat to burrow, it will be able to sleep undisturbed during the daylight hours. to prevent the buildup of urine and fecal material in the cage, commercially available paper rodent bedding or hardwood shavings should be used as a cage substrate (figure 12-8) . although softwood (pine) and cedar shavings are available, the volatile oils that radiate from these substrates are irritating to small animals, especially because they are in very close contact with the material. these irritating compounds can cause dermal and respiratory infl ammation, often leading to secondary bacterial infections. cardboard tissue tubes can be placed in a rat enclosure for chewing and play. clothes items are not recommended in cages with animals that chew. rats will shred the clothing items, exposing thread and elastic material that can become entangled around a rat's extremity, leading to necrosis distal to the stricture. it is recommended to change the substrate within the enclosure at least twice a week and more often if there is a problem with odor or excessive excreta. temperature, humidity, and lighting within the enclosure should follow what is commonly maintained with the ambient conditions within the house. for animals housed outside or in an outbuilding, the temperature range should be 65° f to 85° f. 2 the preferred light cycle for rats is 12 hours of light alternating with 12 hours of darkness. 2 there have been a number of studies investigating the recommended nutritional requirements of rats. these studies have taken place because of the fact that rats are one of the most commonly used laboratory animals when investigating human disease processes. the benefi t of these dietary studies is the availability of commercially produced diets that provide the recommended daily nutritional requirements of the rat. the problem that most owners face is the multitude of dietary products available for rats, and owners often buy seed-based diets. like mice, rats will happily eat seed although seed-based diets are lacking in a number of the nutritional requirements needed to maintain long-term health. commercial rodent biscuits or pellets with 20% to 27% protein are the recommended diets for rats. the commercial rodent biscuits or pellets are the only food rats need to obtain their required nutrients. again, seed-based diets are not recommended, nor is a signifi cant supplementation of fruits, nuts, vegetables, cheese, or other human foods (e.g., peanut butter). if a treat is to be given, yogurt or dried fruit treats manufactured specifi cally for rodents and/or rats should be provided 2 to 3 times a week (figure 12-9) . for younger rats, less than 3 weeks old, softer pellets are needed because babies start eating pellets and drinking water at 2 to 3 weeks of age. 2 a sipper bottle, placed on the outside of the cage is easy to maintain and does not take up space within the enclosure. if the cage is plastic or plexiglas, modifi cations will be needed to attach the sipper bottle on the outside of the cage. most sipper bottles come with attachment hardware to attach to wire cages. fresh bottled water is recommended for rats, although chlorinated tap water can also be used. the water should be checked on a daily basis and clean water supplied at least every 2 days, if not every day. quarantining an animal is important for the pet owner who is introducing a new animal into a setting in which there is an established group. as with other animals a 30-day quarantine period is recommended, along with a physical examination and fecal parasite check. unfortunately, there is no time period that will screen 100% against potential infectious agents, and with rats, time is critical because their life span and repro ductive time frame is so short. quarantining the introduced animals in conditions similar to those in which they will be maintained permanently, reduction of handling, and providing adequate food and water will make for a smooth transition. it is imperative that the owner screen the new animal for diseases and watch for any signs of illness. all rats that are captive, either as pets or in a breeding setup, should have no access to insects, wild rodents, or other animals. the most common sign of clinical illness is an unkempt hair coat. the lack of grooming is most often related to the animal feeling depressed or sick and thereby not having the energy to perform routine behaviors. if a number of animals are being quarantined, sacrifi cing an apparently diseased animal is the most effi cient way to determine a rapid defi nitive diagnosis. if it is a single or an expensive animal, routine diagnostic testing will be required. to maintain oversight of a breeding animal's health and reduce the exposure of young animals to infectious disease and parasites, routine screening of representative animals within the colony is recommended. in very large colonies, special caging (e.g., fi lter), food, and water may be necessary to maintain disease control. 1 before examining a rat, the examination table should be disinfected with either a dilute sodium hypochlorite or chlorhexidine solution. the examiner should always wash his or her hands and, if necessary, put on gloves before the capture and restraint of the patient. the diffi culty with using latex gloves when examining a rat is the small size of the patient and its ability to twist and turn. some rat patients can be restrained only if the examiner is not wearing gloves. if gloves are not worn, the examiner must thoroughly wash his or her hands, as with all cases, after the examination is completed. to restrain a rat, the animal should be picked up with one hand being placed over the back and rib cage, restraining the head with the thumb and forefi nger directly behind the jaws. the other hand is grasping the tail, stabilizing the animal (figure 12-10) . the skin on the dorsal cervical region may also be used, as with other rodents, to pick up the rat. a plastic sandwich bag can be modifi ed as a restraint device for rats and small rodents. the rat's head is placed toward a bottom corner and the sides folded over the animal's back to limit movement. if one is unable to adequately restrain a rat while the animal is conscious, an inhalant anesthetic may be used for sedation purposes. the animal should be placed in a small induction chamber and isofl urane gas permeated into the closed space (figure 12-11) . once the animal has stopped moving, the enclosure top is removed and a nose cone placed on the anesthesia tube and placed on the patient's face. a syringe case can be modifi ed as a nose cone for small rodents. after the physical examination is completed, the nose cone is removed and the patient is allowed to recover, breathing oxygen from the anesthesia unit. injectable agents are another form of sedation used with rats for examination and diagnostic sample collection purposes (see table 12 -2). a thorough assessment of the patient is necessary before using injectable drugs for sedation and anesthesia because of the inability of veterinarians or technicians to rapidly manipulate the effects of these treatments. obtaining a detailed history for the rat patient is very important. it is imperative to get as much information from the owner as possible as it relates to the patient's presenting problem, even if it is routine health examination. typical background information required for the rat patient includes how long the owner has had the animal, where it was acquired, how often it is handled, and the appearance of the feces and urine. husbandry questions should focus on where the animal is housed, whether it is allowed to roam unobserved, where its cage is located; type, size, and material of the cage; cage substrate, furniture, and toys; how often the cage is cleaned and what disinfectant is used. when investigating the diet it is important to ask not only if pellets are fed and in what quantity, but also what the animal is eating and what its primary diet is. supplemental offerings and frequency of feeding are very important data for the case work-up. the technician should fi nd out about the water supply, how often the water is changed, and how much the animal drinks on a daily basis. because there are transmissible diseases among animals, the fi nal questions should center on other pets in the household, if new animals have been added to the family, or if the animals are housed together. a description of any previous problems and a complete chronological description of the presenting problem are needed to complete the history form. before restraining the animal, an observation should be made on its attitude, activity, and posture. the next step is to weigh the rat in a basket on a digital gram scale. if possible, temperature, respiration, and pulse should be measured and any abnormalities in rate and/or character noted. the physical examination starts at the head where the veterinarian should look for any abnormalities. eyes, ears, and nares are observed for signs of discharge or infl ammation. the oral cavity is diffi cult to examine in rats because of the small opening and tendency for the buccal mucosa to encroach toward the middle of the mouth. rat incisors have a tendency to overgrow, so examination of the front teeth for normal occlusion and length is essential. a small speculum (e.g., modifi ed paper clip) or an otoscope may be used to examine the oral cavity and cheek teeth. mucous membranes help determine hydration status using capillary refi ll time and moisture. body condition should be examined and abdominal palpation performed. lymph nodes and limbs are palpated before checking the nails and plantar surface of each foot. the patient should have a normal posture, be aware of its surroundings, and move properly. any problems should be noted in the record. finally, a dermatologic exam considers hair coat quality, alopecia, external parasites, or any skin abnormalities. all abnormal fi ndings are written in the record for case review, differential diagnoses determination, diagnostic testing, and treatment considerations. respiratory disease is often the primary complaint offered by owners when presenting a sick rat to the veterinary hospital. bacterial and viral pathogens can infect rats and are easily spread within breeding colonies because most of the organisms are transmitted by subclinical carriers. ocular and nasal discharge is often observed in combination with dark red pigment. this pigment may be mistaken for blood by owners but actually is porphyrins produced by the harderian glands located behind the eyes. the irritation of the harderian glands causes the excessive production of porphyrins, which are secreted around the eye and down the nasolacrimal duct to the nose. rats have incisors that grow continuously. the continuous growth is not a problem for animals with normal occlusion. if the incisors do not line up, then there is a condition that leads to overgrowth of these teeth. the upper incisors will curve around, and if they are not cut, they will lodge in the dorsal aspect of the oral cavity. the ventral incisors will fl are out from the oral cavity. a common tumor found in older intact female rats is the mammary tumor. mammary tumors can be large and extensive. rat mammary tissue extends, on their ventrum, from the inguinal area to the thoracic inlet and up to their dorsal lateral body wall. the most common subcutaneous mammary tumor in the rat is a fi broadenoma (figure 12-12) . 4 removing the ovaries at an early age reduces the incidence of the hormonally infl uenced tumor later in a female rat's life. swelling of the cervical salivary gland is caused by a virus. the etiologic agent of this condition is a coronavirus, sialodacryoadenitis virus, resulting in upper respiratory infl ammation and the previously mentioned swelling of the cervical salivary and lacrimal gland. 4 there is no treatment for this highly contagious viral disease. external and internal parasites may affect rats in the form of a rough hair coat or dermal lesions. rats that are pruritic may cause abrasions or scratches that become infected with staphylococcus aureus bacteria, leading to secondary ulcerative dermatitis. 4 routine parasite examinations are necessary so that the veterinarian can identify parasitic infestations and administer treatment. rats can also present with neurologic, gastrointestinal, and urinary disease signs. a detailed history and examination will help direct the clinician toward a differential diagnosis list in which the appropriate diagnostic tests can be submitted to obtain a defi nitive diagnosis. as with other pocket pets, blood collection from rats can be quite diffi cult. approximately 0.5 to 0.7 ml/100 g body weight can be safely removed from a nonanemic healthy rat. 2 a rat patient should always be anesthetized for blood collection procedures, to reduce patient stress and movement. usually inducing the animals in a closed chamber and maintaining the patients in a mask will allow the technician plenty of time to collect the blood sample. using a warmed ventral tail vein, 0.5 ml can be collected through a 23-gauge butterfl y needle. 2 to collect from the tail vein, warm the distal two-thirds of the ventral aspect of the tail in a container of warm water for approximately 15 minutes. 2 the tail vein can be observed more clearly in the distal aspect of the tail. a 22-to 25-gauge butterfl y needle is used, and free fl owing blood can be collected directly into the microtainer tubes. 2 a tape bandage should be placed on the injection site to aid in hemostasis. the easiest way to collect blood from many small mammals, including rats, is from the anterior vena cava. with the rat under general anesthesia, a 25-gauge, 1-inch needle attached to a 3-ml syringe is directed toward the umbilicus from the right side of the manubrium. 2 box 12-8 describes the technique used for retroorbital bleeding in rats and is not recommended for companion animals. the retroorbital bleeding technique is commonly used in laboratory animal settings and is easily performed by an experienced veterinarian or veterinary technician. as with smaller rodents, the saphenous vein or lateral vein of the tarsus may be used for multiple blood collections without the use of an anesthetic. the patient must be properly immobilized in a restraint tube (35-ml syringe) with the leg extended and the skin on the medial aspect of the thigh held tightly between the handler's thumb and forefi nger. the taut skin on the lateral aspect of the thigh allows exposure of the saphenous vein. a 23-gauge needle is used to puncture the vein, and blood is collected in a microhematocrit tube as it fl ows from the vessel. 2 blood samples obtained from nail and ear clips are not considered appropriate diagnostic samples. cardiac puncture is recommended only in terminal cases and when the animal is maintained under general anesthesia because of possible complications involving the lungs and heart vessels. the collection of samples for diagnostic testing in rats is similar to that in other rodents. the reference ranges for complete blood counts and serum biochemistry panels are listed in boxes 12-9 and 12-10. marrow samples may be obtained from the ilium, tibia, sternum, femur, or the bones of the proximal one third of the tail. 5 preparation of the samples is consistent with that of other mammalian patients. standard rodent cages can be used without substrate to collect urine and feces when the patient is hospitalized. by placing a rodent in a ziplock bag, urination frequently occurs and the sample can be collected. in a similar manner the urine in a rat can be collected over a disinfected stainless steel examination table. 1 the urine is voided while the animal is restrained and can be collected in a capillary tube for evaluation on urine reagent strips. 1 urinalysis reference values for rats are listed in box 12-11. under most practice settings, the size of a rat patient limits imaging capabilities to radiographs. restraint is essential for quality diagnostic images. to obtain adequate restraint and prevent movement of the patient during radiographic evaluation, sedation is required. as with all diagnostic procedures involving avian and exotic animals, an evaluation of the patient is required to determine its ability to withstand the stress associated with the assessment. if it is determined that the patient cannot withstand sedation, then it should be stabilized until it is deemed healthy enough for diagnostic imaging evaluation. a rat can be sedated in an induction chamber following a technique similar to that described for mice. because rats are larger than mice, they are easier to intubate. using a special otoscope adapter, exposure is enhanced for endotracheal tube placement. high-speed, 300-ma machines with fi ne or detail-intensifying screens should be adequate for most small exotic mammal images. dental x-ray units that can focus at short distances may be advantageous for both isolation of focal anatomy and full body radiographs in extremely small patients, including rats. for extreme detail in small exotic mammal medicine, using a traditional radiography unit, the min r mammography system (eastman kodak) with min r singleintensifying screen cassettes in conjunction with min r singleemulsion fi lm has been recommended. 1 digital radiography is bringing a new dimension to radiograph evaluations and detail. the secret to success with either traditional or digital radiographic imaging is to restrict patient movement during exposure. 2 two views, ventrodorsal and lateral, are recommend for rat patients. if there are rotational problems with the rat patient, a dorsoventral view may provide better imaging quality. 2 the patient should be taped to the cassette or positioning board with white cloth tape or masking tape. taping the rat to the cassette or positioning board will aid in image consistency with that particular patient and among similar animals. in the lateral views, the dependent legs should be positioned cranial to the contralateral limbs. 1 again, as with mice, whole body radiographs are commonly obtained for rat patients during the radiographic evaluation. normal radiographic appearance of the rat patient is described in box 12-12. for the rat patient in which microbiological testing is recommended, standard collection techniques used for other animals are appropriate. with the most common respiratory conditions in rats being viral and bacterial organisms, it is important to try and aid the diagnostic laboratory in organism isolation through listing the potential pathogens. as always, if in doubt on collecting, preserving, and/or shipping a microbiological sample, the diagnostic laboratory should be contacted for full instructions regarding rat submissions. parasitology routine fecal parasite evaluations should be performed on small rodents when they are brought to the veterinary clinic for a health examination or an abnormal stool. common protozoal organisms can be detected using a direct fecal examination. an anal tape test is useful for identifying syphacia spp. in rats. diagnosis of ectoparasites in small rodents is similar to that in other species listed in this text. a skin scraping of the affected area or a pelage tape test (to identify radfordia spp.) will usually yield the parasite. diagnosis of intestinal parasites can be made by fi nding individual eggs in a fecal fl otation of a macerated fecal pellet and in the lumen of the small intestine during necropsy. staphylococcus aureus is the most common cause of ulcerative dermatitis in rats. 4 this organism is ubiquitous in most rat colonies and survives on the animals' skin. unless there is a break in the skin, there is no infection or disease caused by the organism. in cases where there is a fur mite (radfordia ensifera) infestation, rats become pruritic, developing lesions that seed the s. aureus organism. 4 the ulcerative dermatitis can be treated with an appropriate antibiotic through both systemic and topical application, cleaning the affected areas, and clipping the toe nails of the rear feet. mycoplasma pulmonis, streptococcus pneumoniae, corynebacterium kutscheri, sendai virus, and cilia-associated respiratory bacillus have been isolated and identifi ed as infectious agents causing rat respiratory disease. 4 with the respiratory pathogens, clinical signs can vary from mild dyspnea to severe pneumonia and death. rats with mycoplasmosis are treated in much the same way as infected mice. sialodacryoadenitis virus is a highly contagious disease that causes rhinitis and infl ammation of the cervical salivary and lacrimal glands. 4 there is no treatment for this viral disease, and supportive care is recommended for the affected patient. there are other infectious diseases that can infect rats, as evidenced by the available serologic tests (e.g., kilham's rat virus, pneumonia virus of mice, rat parvovirus, rat coronavirus, toolan's h-1 virus, theiler's murine encephalomyelitis virus, mouse adenovirus, reovirus 3). these infectious diseases are diagnosed less commonly than those previously identifi ed. even so, veterinarians should be aware of the infectious disease organisms to which rat patients may be exposed and aware that tests are available for antemortem diagnosis. specifi c intestinal parasites identifi ed in rats include the dwarf tapeworm (hymenolepis nana), pinworms (syphacia muris), nematodes (specifi cally trichosomoides crassicauda), and giardia muris. 5 diagnosis of intestinal parasites is important for the implementation of the appropriate treatment plan. pinworms are diagnosed through the anal tape test, protozoan parasites (e.g., giardia muris) through a direct fecal exam, and cestodes and nematodes from fecal fl otation on macerated fecal pellets. 5 once the parasite has been identifi ed, proper treatment can be initiated and environmental recommendations applied to prevent exposure of other animals and reexposure of the patient being treated. rats do not appear as susceptible to ectoparasites as mice, but the following have been identifi ed: ornithonyssus bacoti (tropical rat mite), radfordia ensifera (rat fur mite), demodex nanus (demodex), polyplax spinulosa (the spined rat louse), and fl eas. 5 diagnosis and treatments are similar to those recommended for mice although the dosages and duration differ. treatment of ectoparasites can be accomplished with ivermectin and a topical miticide. as with other rodent species, there has been an increasing ectoparasite resistance to ivermectin treatment. because most rodent diets are manufactured in the form of pellets or small biscuits that provide all recommended nutrients, there are few nutritional disease problems diagnosed in pet rats. it is also important to provide an adequate supply of fresh water and appropriate rodent food on a daily basis. if rats are fed an all-seed diet, there could be nutrition-related consequences. a lack of vitamin a could result in roughened hair coat or skin lesions. a breakdown of the protective epithelial lining of the gastrointestinal and/or respiratory tract could predispose the rat to secondary bacterial infections affecting those body systems. 2 nutritional defi ciencies may also result in poor reproductive activity. vitamins b complex, c, and e plus selenium are recommended for treatment of female rats diagnosed with pregnancy toxemia. 2 rats, if fed a high quantity of human food, will develop dental plaque on the cheek teeth. 8 proper diet, primarily rodent biscuits or pellets, will reduce the formation and buildup of dental plaque. 8 if left unattended, the excess plaque may lead to gum disease and dental caries. if caries develop within cheek teeth, the recommended treatment is removal of the affected teeth. 8 rats are susceptible to tumors, most likely because of their short life span and physiologic predisposition. the most common subcutaneous tumor in rats is the fi broadenoma of the mammary tissue. 4 the mammary tumors can reach very large sizes and affect both males and females. to reduce the incidence of fi broadenomas, ovariohysterectomies are advocated at an early age in female animals. whereas mammary tumors in mice are almost always malignant, rat mammary tumors are usually localized and respond to surgical resection. 4 rats have continuously growing incisors. under normal anatomic conditions, the incisors maintain their length by grinding on their occlusive surface. if the position of the incisors is compromised, malocclusion will occur, causing overgrowth of their front teeth. the teeth, if left untrimmed, can cause trauma to the dorsal surface of the oral cavity (upper incisors) and the lower teeth extending outside of the mouth. in severe cases the teeth may adversely affect the animal's ability to eat. the teeth can be trimmed with a high-speed motor tool (e.g., dremel ® ) with a cutting disk attachment. a common disease in older male rats is chronic progressive nephrosis; the disease can also affect females. 4 the protein level in the urine commonly exceeds 10 mg/day and progressively increases as the animal ages. 4 the pathophysiology of chronic progressive nephrosis involves glomerulosclerosis with tubulointerstitial disease primarily affecting the convoluted proximal tubule. 4 the progression of the disease process may be slowed through a low-protein diet and anabolic steroid therapy. 4 therapeutics fluid therapy can be given to rats through intramuscular, subcutaneous, intraperitoneal, and intravenous routes. using a 25-gauge needle, a volume of up to 0.2 ml of fl uid can be injected into the quadriceps muscle. 2 up to 5 ml of fl uid can be injected under the loose skin covering the neck or lateral body wall. 2 intraperitoneal fl uids should be administered while the rat is properly restrained to restrict movement. with one of the rat's hindlimbs extended, a 25-gauge needle is inserted into the center of the caudal quadrant of the abdomen, following the line of the extended leg. 2 up to 5 ml of fl uids can be given via the intraperitoneal route. 2 the most diffi cult method of providing fl uids is intravenously to the lateral tail vein. to access the tail vein, warming the tail to dilate the vessel in warm water for approximately 15 minutes is required to aid in visualization. 2 the older the animal is, the more diffi cult it is to see the vessel through the tail skin. although there are many methods that may be used to administer medication in small mammals, oral treatment with a dropper or tuberculin syringe is the easiest for the veterinarian and least stressful to the patient. medication can be added to the food or water, but often the patient is anorexic or the medicated food/water is not palatable, in which case, the patient does not receive treatment. both oral medication and nutritional supplementation can be administered through an oral gastric feeding tube to rat patients. the techniques described earlier for mice can also be applied to rats. medication dosages for rats can be found in a number of formularies. before administration of any therapeutic agent, the dosage should be checked twice and calculations thoroughly evaluated to make sure they are correct. the same surgical and anesthetic techniques described for mice can be used for rats (see box 12-6). dosages for rat injectable anesthetic and sedative agents and analgesic treatment can be found in tables 12-1 and 12-2. rats are larger than mice and can be intubated using a modifi ed otoscope head (figure 12-13) . when using inhalant anesthesia, an intubated animal can be ventilated and there is less risk of aspiration from fl uid that enters the oral cavity. surgical procedures that are performed on rat patients use similar techniques utilized on larger animals. veterinary surgeons should follow the same protocols but remember that the surgical fi eld is small and patient manipulation is delicate. the most common surgical procedures performed on rats are castration, ovariohysterectomy, and tumor (especially mammary tumor) removal. occurrences of humans becoming infected with zoonotic diseases associated with pet rats are uncommon. as with mice, an basic anatomy, physiology, husbandry, and clinical techniques small rodents: exotic companion medicine handbook for veterinarians box 12-12 normal radiographic appearance of the rat from johnson-delaney ca, harrison lr: small rodents: exotic companion medicine handbook for veterinarians basic anatomy, physiology, husbandry, and clinical techniques small rodents: exotic companion medicine handbook for veterinarians tumours of the mammary gland disease problems of small rodents a technician's guide to exotic animal care parasites of ferrets, rabbits, and rodents zoonotic diseases small mammal dentistry allergic reaction to rat dander and urine may be the most commonly reported zoonotic condition. as with all animals it is important that a handler always wash his or her hands after interacting with a rat. salmonella spp. may colonize the intestinal tract of rats. 7 there has been a human case of chorioamnionitis in which corynebacterium kutscheri has been isolated. 7 this grampositive bacillus is routinely isolated from the nasopharynx of rats and therefore can be considered a zoonotic agent.streptobacillus moniliformis and spirillum minus are organisms that initiate the disease called rat-bite fever. 7 a rat bite is required to seed the bacteria in a human, and after a 6-to 10day incubation cycle, the human begins to show clinical signs associated with the disease (e.g., relapsing fever, chills, vomiting, myalgia, regional lymphadenopathy). 7 rats appear to be one of the primary rodent carriers of streptococcus pneumoniae. the rodent develops respiratory disease through which the organism is shed and humans are exposed. infected humans have respiratory and meningeal disease signs associated with s. pneumoniae illness. 7 it should be mentioned that the rodent fl ea (xenopsylla cheopis) transmits the plague organism yersinia pestis. 7 it is wild rodents in which the plague organism is of most concern, but the disease is found worldwide, especially in the american southwest. this is a very good reason why rats under the care of humans should have no access to wild animals.leptospira interrogans is transmitted from the infected urine of rats. 7 again, restricting contact with wild rodents will reduce the risk of pet exposure. humans infected with l. interrogans have chills and fever and often develop septicemic conditions that can lead to organ dysfunction. 7 hymenolepis sp. is a cestode found in rats, which can infect humans if contaminated feces are ingested. 7 giardia sp. is a protozoan parasite, rarely identifi ed in pet rats, that can cause severe gastroenteritis in humans. 7 because feces have to be ingested for exposure, it is more common for children to become infected with zoonotic intestinal parasites than for adults. proper sanitary practices after handling rats will help reduce humans' exposure to zoonotic intestinal parasites and other potentially transmissible diseases. all potential rat owners and current rat owners should know that the occurrence of zoonotic disease transmission is extremely rare when commercially bred rats are purchased from reputable pet stores. key: cord-266745-jit1xeqc authors: liou, jenn-fa; chang, chih-wei; tailiu, jui-jane; yu, chun-keung; lei, huan-yao; chen, lih-ren; tai, chein title: passive protection effect of chicken egg yolk immunoglobulins on enterovirus 71 infected mice date: 2010-11-29 journal: vaccine doi: 10.1016/j.vaccine.2010.09.089 sha: doc_id: 266745 cord_uid: jit1xeqc the objective of this study is to evaluate the passive protective efficiency of immunoglobulin in yolk (igy) specific against human enterovirus type 71 (ev71). the antibody was raised by intramuscular immunization to 10 white leghorn hens, with inactivated human ev71 serving as the antigen. the titer and specificity of the antibody were analyzed from purified igy in the egg yolks of immunized hens. results indicate that the titer of igy specific against ev71 increased from the third week after the first immunization. the content of total igy was 190 ± 26 mg/yolk, with an average concentration of specific igy of 6.34 ± 3.38 mg/yolk in the eggs from 3 to 18 wk after immunization. the results of the neutralization effect of specific igy in ev71-challenged mice demonstrate that the ev71-specific igy, either by intraperitoneal injection or oral administration, was able to significantly reduce the morbidity and mortality in ev71 infected mice pups. researchers have extensively studied passive immunization in both humans and animals using specific antibodies to protect against pathogens. an increase in antibiotic-resistant bacteria and the desire to treat pathogens that do not respond to antibiotics, such as viral pathogens, has prompted many researches to use antibodies as alternative to antibiotics [1] . igy (immunoglobulin in yolk) antibodies are the predominant serum immunoglobulin in birds, reptiles, and amphibians, and are transferred from serum to egg yolk in the females to confer passive immunity to their embryos and neonates [2] . the potential of orally administered igy for the prevention and treatment of many pathogens has been studied for many years, for escherichia coli [3] , helicobacter pylori [4] , salmonella [5] , rotavirus [6] , staphylococcus [7] , streptococcus mutans [8] , yersinia [9] , coronavirus [10] , and the porcine epidemic diarrhea virus [11] . this paper evaluates the efficiency of igy against enterovirus 71 (ev71). enterovirus 71 belongs to the human enterovirus a family of picornaviridae. these virions consist of a non-enveloped capsid surrounding a core of single-stranded, positive-polarity rna approximately 7.5 kb in size [12] . since the initial description of ev71 in 1974 [13] , outbreaks of this virus have been identi-fied periodically in countries throughout the world, including the usa, australia, sweden, japan, bulgaria, hungary, hong kong, and malaysia [14] . in 1998, an outbreak of ev71 infections occurred in taiwan, in which 405 children were hospitalized and 78 died [15] . ev71 infections are generally mild, like hand-foot-and-mouth disease (hfmd) and herpangina, but occasionally lead to severe diseases such as aseptic meningitis, poliomyelitis-like paralysis, and even fatal encephalitis in neonates [16] [17] [18] . in recent years, some efforts have been made to control ev71 infections. the most promising antiviral agents for ev71 are win-group compounds, which have undergone clinical trials [19, 20] . in addition, a research group from taiwan has developed two candidate ev71 vaccines, including a formalin-inactivated whole virus vaccine and a vp1 expressing dna vaccine [21] . the efficacy of both vaccine constructs is currently being tested in animal models. several approaches have been attempted in the treatment of viral diseases. researchers tried to block the replication of virus through binding the capsid of virus with some chemicals, such as pyridazinamines [22] , phenoxyl imidazoles [23] , or pleconaril [24, 25] . however, the treatment of ev71 with pleconaril was not significant, the binding of pleconaril with capsid was limited under higher viral concentration in vitro [26] . a specific antibody which could bind with the virus and hence reduce the contact of virus with host cells is an alternative choice. human's immunoglobulin has been applied in the treatment of ev71 infections in babies whose immune system are not well-developed [27, 28] . there is no routine therapy for the treatment of ev71 infection with human's immunoglobulin so far. however, some animal model of ev71 challenged mouse has been developed, and provided some information about the protective effects of the neutralizing antibody on ev71 infection [29] . compared to traditional antibody production, chicken as bio-factory can produce higher yield of igy antibodies than mammals' igg. a chicken can lay 280 eggs in a year and an egg yolk contains 100-200 mg of igy antibodies. this study was subjected to produce igy against enterovirus 71 (anti-ev71 igy) and evaluated the inhibition effects of specific igy on ev71, including in vitro virus neutralization test and in vivo icr mice model. this study also provided an animal model for the application of igy in the cure or prevention of ev71. the ev71 strains 4643 and mp4. strain 4643 was originally derived from a patient with ev71 encephalitis [30] , while strain mp4 was a mouse-adapted enterovirus 71 with increased virulence in mice, and it was generated after four serial passages of the 4643 strain [31] . ev71 stock virus of strains 4643 and mp4 were grown in rd (rhabdomyosarcoma) cells, which were maintained in dulbecco's modified eagle's medium (dmem, invitrogen, u.s.a.) containing 10% fetal bovine serum (fbs, invitrogen, u.s.a.), 2 mm of l-glutamine (invitrogen, u.s.a.), 100 iu/ml of penicillin (invitrogen, u.s.a.), and 100 g/ml of streptomycin (invitrogen, u.s.a.). all animals received humane care as outlined in the guide for the care and use of experimental animals and viral challenge (institutional animal care and use committee; iacuc approval no. 940020). four 6-month-old white leghorn laying hens, obtained from livestock research institute, council of agriculture, executive yuan, taiwan, were utilized for the production of anti-ev71 igy. a formalin-inactivated ev71 of strain 4643 was utilized as the antigen. one milliliter of ev71 antigen (400 g/ml, 5.4 × 10 6 pfu) was emulsified with an equal volume of freund complete adjuvant and immunized intramuscularly to chickens at 4 sites in the breast muscle. four booster injections with freund's incomplete adjuvant were given at weeks 2, 4, 6, and 13 after the first immunization. the eggs were collected daily from the first day to 3 weeks after the last immunization, and stored at 4 • c. the egg yolk was separated, pooled, and kept at −20 • c prior to igy purification, and all egg yolks from each chicken for each week were pooled into one analysis sample. the isolation of igy was carried out as described by akita and nakai [32, 33] , with some modifications. the egg yolk was first mixed with one of nine volumes of cold distilled water (acidified with 0.1 n hcl to ph 5.0) and stored overnight at 4 • c. the mixture was then centrifuged at 3125 × g at 4 • c for 40 min to obtain the water soluble fraction (wsf). the wsf was collected and filtered to remove solids. the resulting igy-containing wsf was further purified by ultra filtration using amicon ultra-15 filter (pl-100, millipore), condensing the sample to 1/30 to 1/40 of its original volume. these wsf concentrates were then subjected to the virus neutralization test and mice challenge experiments. the antibody activity of anti-ev71 igy was determined using the elisa method described by lee et al. [34] , with some modifications. briefly, microtiter plates were coated with 100 l of inactivated ev71 4643 antigen (10 g/well), while control wells were coated with rabbit anti-chicken igy antibody (10 g/ml, sigma c2288). the plate was then incubated overnight at 4 • c. after washing with pbs-tween 20 buffer, 2% bsa blocking was conducted overnight at 4 • c. the wells were then washed with pbs-tween 20 buffer. next, 100-fold diluted wsf was added to the sample wells (100 l/well) for testing. wsf from the same chicken before immunization was used as a control. to generate the standard curve, wells were filled with 100 l serial-diluted pure chicken igy at a concentration from 0.015 g/ml to 1 g/ml (promega, g116a) and incubated at 4 • c for overnight. after washing with pbs-tween 20 buffer, 100 l of alkaline phosphate-conjugated goat anti-chicken igy (promega, g115a) was added to the wells and incubated at 37 • c for 2 h. after washing with pbs-tween 20 buffer, 100 l of disodium p-nitrophenyl phosphate was added to each well as a substrate (sigma, n9389) and allowed to react at 37 • c for 10 min. the absorbance was then measured at 405 nm using a microplate reader (multiskan ms: thermo labsystems). the resulting absorbance of standard curves provided a relative measurement of anti-ev71 igy concentration. for the total igy determination, each well on the microtiter plate was first coated with 100 l of rabbit anti-chicken igy antibody (10 g/ml, sigma c2288), to which 100 l of 10,000-fold diluted wsf was then added. the following experiments were performed following the same protocol described above. the ev71 was run on sds-polyacrylamide gel electrophoresis (sds-page) and transferred electrically onto a nitrocellulose membrane. the membrane was divided into 3 parts and soaked in blocking buffer (5% nonfat dry milk in tbst) for 30 min at room temperature, then incubated with anti-ev71 or non-specific igy for 1 h. after washing 4 times with tbs-t, the membranes were incubated with peroxidase-conjugate goat anti-chicken igy (1000fold dilution in blocking buffer for each) at room temperature for 1 h. immunoreactivity was detected by incubating the membranes with 0.02% (w/v) 4-methoxy-1-naphthol in tbs and 0.02% (v/v) h 2 o 2 . the tcid 50 of mp4 virus and the neutralization titer of anti-ev71 igy were determined using the method described by yu et al. [35] . in the tcid 50 tests, each 96-well 8 × 10 3 rd cell was added to a microplate well, followed by the 10-fold serial diluted virus. the 50% tissue culture infective dose was determined after incubation under 5% co 2 at 37 • c for 7 days. in the neutralization tests, each 50 l sample of serial diluted anti-ev71 igy concentrate solution was mixed with 50 l of 100 tcid 50 ev71 in a microplate well. two hours later, the rd cell suspensions (8 × 10 3 cell/well) were added and further incubated under 37 • c and 5% co 2 for 7 days. neutralization antibody titers were then identified as the highest dilution of purified igy solution that completely inhibited virus growth. the challenge test of ev71 of the mouse model in this study was modified from wang et al. [30] . trials 1-4 were designed to test the effect of igy with different neutralization titers on mortality in suckling mice. each one-day-old icr strain mouse was intraperitoneally (ip) inoculated with 1 × 10 5 pfu of mp4 virus, which is a mouse-adapted 4643 strain of ev71. thereafter, igy with different neutralization titers (64, 128, 256, and 512) was injected ip into the challenged mice 1-3 days after inoculation (dpi). the dosage of igy week after immunization mg anti-ev71 igy was 100 l per mouse. in trials 5 and 6, the igy treatment dates were 2-4 dpi and 4-6 dpi, respectively. trials 7-8 were used to evaluate the protection effect of anti-ev71 igy by oral treatment. specifically, trials 7 and 8 used a 24-gauge feeding tube to orally inoculate mice of 4-5 d (2.5-3.0 g) and 5-6 d old (3.0-3.5 g), respectively. the mice were given 100 l of mp4 virus (3 × 10 6 pfu/mouse) after 12-h of fasting. for each pup, 100 l of anti-ev71 igy with a titer of 512 was orally administered either 1 h before or 1 h after viral challenge. all mice were checked daily for their body weight and the syndromes of ev71 infection, including limb paralysis and abnormal hair coat until 2 weeks after viral infection. morbidity and mortality data was collected for two weeks after the viral challenge. the concentration of total igy and specific anti-ev71 igy was analyzed by elisa. the content of igy in the wsf of egg yolk was investigated for a period of 18 weeks, from the first week of immunization until 6 weeks after the last booster injection. the average content of total igy in each yolk was 190 ± 26 mg (table 1) , which was 32% of the egg yolk's total protein. after condensation by ultrafiltration, the total protein per egg was 231 ± 25 mg on average, and the purity of total igy increased to 65%. the content of specific anti-ev71 igy increased gradually from the third week after the first immunization, as fig. 1 indicates. a variation in immune response among individual hens was noticed from the large standard deviation. the average anti-ev71 igy concentration reached 6.34 ± 3.38 mg per yolk between the third to eighteenth weeks, equivalent to 3.33% of the total igy. the neutralization titer of specific anti-ev71 igy was determined by the cytopathic effect (cpe) of ev71 on rd cells (fig. 2) . the resulting neutralization titers of specific igy obtained from eggs of all immunized chickens ranged from 0 to 2048 (data not shown). comparing the neutralization titers to the contents of specific igy in the respective yolks showed no significant corb the igy-containing wsf was further purified by ultra filtration using amicon ultra-15 filter (pl-100, millipore), condensing the sample to 1/40 of its original volume. these wsf concentrates were then subjected to the virus neutralization test. relation between these two sets of data (r = −0.396, data not shown). western blotting analysis of the immunoreactivity of anti-ev71 igy. the ev71 was run sds-page and transferred onto nitrocellulose filter and probe with ev71 immunized and non-immunized igy. the ev71 was detected of ∼52 kda molecular mass (fig. 3a) and anti-ev71 igy was specifically bound to the proteins witch molecular weight corresponding of ev71 virus (fig. 3b ). in the first part of the animal tests, icr mice showed both limb paralysis and abnormal hair growth after ev71 challenge in the ip model (fig. 4) . in trials 1-6 was conducted to investigate the effect of ip injected specific igy on curing ip ev71-challenged mice (table 3) . when one-day-old icr mice were challenged with ev71 of strain mp4 with a dose of 10 5 pfu per mouse, the average morbidity was 96.5 ± 6.1% in the control group. the resulting mortality was 88.0 ± 8.4% based on the challenged mice, or 91.2 ± 7.6% when based on illness in mice injected with only non-immunized igy (table 3) . we ip administrated the purified specific igy derived from condensation of the immunized yolk wsf to ev71-challenged mice. this administrated was applied continuously for 3 days, from the day after viral infection (1 dpi) to the third day post infection (3 dpi). the therapeutic effects of igy varied according to the neutralization titers of the specific igy administered. the infection rate was 100% when the titer of specific igy was 64, which is same infection rate as in the control group (table 3 ). the treatment of igy with a titer of 64 did not significantly reduce mortality in trial 1. however, when the igy titers were 128 or higher, the morbidities reduced to 20%, 7%, and 0%, respectively for igy treated groups with titers of 128, 256 or 512. only the group of titer 128 (trial 2) had some deaths after viral infection and igy injections, with a mortality rate of 10%. without the treatment of specific igy, the challenged mice showed high morbidities and mortality. in trials 5 and 6, if the treatment between 64 and 512) . c non-immunized igy. # differences in proportions morbidity and mortality were tested with the use of the x 2 statistic (p < 0.05). time of specific igy began at 2 or 3 dpi, the morbidities increased to 43% and 89%, respectively. this reduction in cure effects increased mortalities by 21% and 63%, respectively. the mortalities of mice showing illness were 50% and 71% in trials 5 and 6, respectively, indicating a decrease in cure effects of specific igy following the delay of igy administration for one or 2 days. the second part of the animal tests involved oral administration of specific igy to ev71 orally challenged mice. in trial 7, a dosage of 3 × 10 6 pfu mp4 strain ev71 was orally fed to pups 4 or 5 days old with a body weight of 2.5-3.0 g. the results indicated that if 100 l of specific igy was orally fed 1 h before the viral challenge, the morbidity and mortality of challenged mice was 24% and 20%, respectively. both morbidity and mortality were significantly reduced compared to those of the control group, at 69% and 62%, respectively (table 4 ). when the specific igy was given to the pups 1 h after the viral challenge, the infection rate was 38% with a final mortality rate of 31%. the morbidity and mortality rates of the specific igy treatment groups were lower than those of the control group. regardless of whether the igy was fed 1 h before or after the viral challenge, virus neutralization by specific igy was effective. comparing the mortality of mice exhibiting infection symptoms, the igy treated groups showed a similar rate to the control groups, which all exceeded 80%. the igy application in trial 8 was similar to that in trial 7, changing only the age of challenged pups from 5-to 6-days old, with their body weight ranging from 3.0 to 3.5 g. under this situation, the average infection rate decreased to 13% in the igy treated group and 29% in the control group. also, the mortality in the igy treated group among ill pups was lower than that in the control groups. although the average mortality of igy treated groups was as low as 8%, the control groups also exhibited a lower mortality (26.5%) than the control groups in trial 7. in recent years, pathogen-specific igy has also been demonstrated to be effective in the passive protection of human's diseases, such as staphylococcus for holotoxin [7] , rotavirus for diarrhea [36] , dental caries caused by s. mutans [37] , and h. pylori for gastric ulcers [4] . in this study, we tried to produce the specific igy against human ev71. this enterovirus was first reported in the united states in 1974 [13] and has subsequently been reported worldwide [38, 39] . in taiwan, some severe cases of human ev71 are reported every year after the devastating ev71 outbreak of 1998 [15] . the seriousness of ev71 infection lies in its complications, which include hand-foot-and-mouth disease, herpangina, aseptic meningitis, poliomyelitis-like paralysis, and fatal encephalitis [40] . most fatal cases occur in children under 3 years old, and high mortality is correlated with brainstem encephalitis [41] . the specific igy against ev71 in the egg yolk increased after the third week after the first immunization, though there were obvious differences among individual hens. this variation in antibody pro-duction, based on the specific igy production, might be the result of differences in individual immune responses [3] . the origin of antigen also plays an important role in the immune response, as different outer membrane proteins or fimbrial adhesions from the bacteria affected the rate of igy production [5, 9] . when the protein antigen contained whole bacteria, or when the virus achieved better specific antibody production, the in vivo neutralization effect of the resultant antibody was also better [7] . this study used the whole virus as the antigen source to obtain a better igy titer. the total protein obtained from an egg yolk by the water dilution method is around 595 mg per egg. this protein contains few lipids and lipoproteins, and accounts for one-sixth of the total protein of a yolk [42] . in this study, the purity of igy in wsf was approximately 30-40% (table 1 ). this demonstrates that the water dilution method can easily separate the igy from other egg yolk components, as described previously [4, 34] . for the production of igy in trials 1-8, the immunization dosage of antigen used was doubled and the immunization schedule was modified. a longer interval of 7 weeks was applied after the fourth immunization, and the last boost was given on week thirteen. thereafter, the average specific-igy against ev71 between 3 and 18 weeks increased to 3.33% of the total igy, which is significantly higher than that of the pre-test which is only 1% of the total igy (p < 0.5). however, whether or not this modified protocol is beneficial to the production of specific igy remains to be clarified. an analysis of the relationship between the concentration of specific igy with the neutralization titer of the same sample revealed no significant correlation (table 2 ). this indicated that the amount of specific igy estimated by the elisa method was not a good indicator of the neutralization titer of ev71 virus in this study. previous studies on the effect of antibodies against bacillus spores, botulinum pentavalent (abcde) toxoid and anthrax vaccine also suggest that elisa is not a reliable indicator for the neutralization titers [43, 44] . the neutralization titer itself is a better indicator of antibody functionality. we determined the concentration of specific igy by elisa method using the cell lysate from the whole virus particle as the coating antigen. the binding between all viral proteins and specific igy would be counted as part of the igy concentration, whether or not the specific antibody was effective in virus neutralization. kovacs-nolan et al. [45] studied the epitopes of human rotavirus (hrv) by igy and found that only three out of the five sequential neutralization epitopes on the wa strain subunit protein vp8 were directly involved in hrv neutralization [46, 47] . our results indicate that the therapeutic function was significant when neutralization titer of the anti-ev71 igy was 128 or higher (table 2) . however, further research is necessary to identify the antibodies directly involved in the neutralization of ev71. this study includes two panels of animal tests for the ev71 challenge and igy treatment. in trial 1, we challenged 1-day-old mice with a mouse-adapted ev71 strain mp4 by intraperitoneally administering a dosage of 10 5 pfu per mouse, and treated with specific igy of neutralization titer 64. the results revealed no therapeutic effect. on the contrary, if the neutralization titer value of specific igy was 128 or higher, the protective effects were significant (table 2 ). previous studies also show that this level of neutralization titer is effective for the serum igg of mice [31, 35] . compared to the control group, the mice in specific igy treated group not only had a lower mortality, but also had normal weight gains. most of the mice in control group showed a weight loss 5 days after the viral challenge, and continued to lose body weight until they died. less than 10% of the sick mice in the control group survived the artificial infection of ev71 at a dose of 10 5 pfu per mouse. the surviving mice gained some weight despite showing signs of illness. after reaching a body weight of 4.5-5.0 g, they recovered from the infection and returned to healthy status. however, their subsequent weight gains were only 60-70% of the mice in the specific igy treated group (data not shown). some surviving mice also exhibited persistent paralysis of the hind limb and remained paralyzed for the rest of their lives, just like some children infected by ev71 [47] . in trials 1-4, the ip injection time of specific igy began from the second day after viral infection and lasted for three consecutive days, before the ev71 syndrome appeared. in practical situations, children infected by ev71 will probably only receive the igg treatment after they exhibit the typical symptoms. to determine the critical timing of effective treatment, trials 5 and 6 applied specific igy treatment one and two days later, respectively. clearly, the starting time of specific igy injection was essential in reducing the mortality of infected mice. if igy was given on 4-6 dpi, at which point most mice exhibited the symptoms of hind limb paralysis, only 37% of infected mice survived. it has been reported that on the first day of ev71 infection, the virus is kept in the small intestine, and then spreads to other organs on 2 dpi. the virus attacks most organs on 3 dpi, including the brain stem, lung, spinal cord, and muscle [31, 48] . if we started the specific igy treatment on 2 dpi, which corresponds to the onset of clinical symptoms in children, then the therapeutic outcome was improved. in this case, the survival rate of the ev71-infected mice could be increased to 79%, highlighting the importance of the timing of specific igy treatment. before the on-set of clinical symptoms, specific igy treatment has a better chance of achieving effective therapy. some animal studies use oral feeding of egg yolk powder directly, without any purification of specific antibodies from the yolk [10, 49] . these studies prove the possibility of passive protection from the de novo form of yolk powder. in this study, 4 to 5 day-old mice were orally challenged with a viral dosage of 3 × 10 6 pfu per mouse. challenged mice in the specific igy-treated group showed a lower morbidity rate of 29% (12/41) than the control group, which showed a morbidity rate of 79% (38/48) . this indicates that the orally fed specific igy effectively neutralized the viral attack in the gastroenteric duct, thereby blocking the infection of virus in challenged mice. the neutralization function appeared to be effective no matter if it was given 1 h before or after the viral challenge. the mortality among specific igy treated mice was 24% (10/41), which was significantly lower than that of the control group, at 69% (33/48). however, the effect of specific igy declined for pups aged 5 to 6 days old with a body weight ranging from 3.0 to 3.5 g. previous research also shows that the age of mice affects the infection results [50] . mice more than 6 days old would not be infected by ev71 after artificial challenge, indicating that the immune system of mice at this age could provide suitable protection. when mice are challenged orally or received the igy through an oral passage, the virus and igy pass through gastroenteric duct and be digested by gastric juice, reducing the viral infectivity of the igy function. this was noticed by the lower morbidity after oral challenge with the same strain of ev71 and a less protective function of igy among infected mice, compared to the results in trials 1-6, where an infection rate near 100% and a curing function more than 90% was obtained. the igy specific to human rotavirus exhibits a passive protection against diarrhea in mice [51] . although gastric juice had some influence on igy, was still protective when a sufficient amount of igy was provided. oral administration of spray dried yolk antibodies specific against salmonella typhimurium or salmonella dublin is effective in preventing salmonella infections in calves [5] . igy raised against etec antigen has also been proven effective in controlling the diarrhea of piglets by oral administration [49] . nevertheless, because the 4 to 6 day-old mice pups in this study could not consume egg yolk, we applied a purified antibody from igy instead. this purified igy, derived from the water soluble fraction of egg yolk, was more accessible to the gastric juice and easily exposed to digestive enzymes. the activity of might be influenced by the change of structure and loss of bioactivity [5, 7] . based on the limitations of oral administration in suckling pups, a higher titer of igy or a continuous treatment of igy after viral challenge might achieve greater protection. this possibility is worthy of further research. the results of this study, however, indicate the potential of igy in the prevention of ev71 infection in young children. several studies have been conducted to evaluate the stability of these antibodies. the igy is not degraded during pasteurization at 60 • c [52] igy was stable after 30 min in 60-65 • c, but was no longer active after 20 min in 80 • c [53] . so igy technology being new potential market applications in medicine, public health, veterinary medicine and food safety. a broader use of igy technology could be applied as biological or diagnostic tool, nutraceutical or functional food development, oral-supplementation for prophylaxis, and as pathogen-specific antimicrobial agents for infectious disease control. in conclusion, the icr mice ip challenged with mouse-adapted strain mp4 of ev71 at a dose of 10 5 pfu per mouse induced an ev71 infection resulting in a high mortality rate. however, a survival rate of 98.3% (60/61) was achieved if the challenged mice were ip injected, 1-3 dpi for 3 consecutive days, with a purified igy antibody with a neutralization titer of 128 or more. when the challenge was carried out orally with a dose of 3 × 10 6 pfu per mouse, the lower morbidity in igy treated group proved that the protection could be achieved by the neutralization of virus in the gastroenteric duct, thereby reducing the mortality of challenged mice. this is the first study to evaluate the effect of igy treatment on ev71 infection, and our positive results indicate that further application in the prevention of ev71 is possible. in the form of an egg-yolk-added drink, yolk powder tablet, or capsule, it is can potentially be used to prevent the early 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enterovirus 71 infections and neurologic disease-united states, 1977-1991 monoplegia caused by enterovirus 71: an outbreak in hong kong enterovirus type 71 infections: a varied clinical pattern sometimes mimicking paralytic poliomyelitis clinical spectrum of enterovirus 71 infection in children in southern taiwan, with an emphasis on neurological complications general chemical composition of hen eggs search for correlates of protective immunity conferred by anthrax vaccine immunological correlates for protection against intranasal challenge of bacillus anthracis spores conferred by a protective antigen-based vaccine in rabbits fine mapping of sequential neutralization epitopes on the subunit protein vp8 of human rotavirus identification of immunodominant sites on the spike protein of severe acute respiratory syndrome (sars) coronavirus: implication for developing sars diagnostics and vaccines outbreak of poliomyelitis-like paralysis associated with enterovirus 71 a murine oral enterovirus 71 infection model with central nervous system involvement passive protective effect of egg-yolk antibodies against enterotoxigenic escherichia coli k88+ infection in neonatal and early-weaned piglets outbreaks of hand, foot and mouth diease by enterovirus 71. high incidence of complication disorders of central nervous system antibodies to rotaviruses in chickens' eggs: a potential source of antiviral immunoglobulins suitable for human consumption productivity and some properties of egg yolk antibody (igy) against human rotavirus compared withr abbit igg suppressive effect of functional drinking yogurt containing specific egg yolk immunoglobulin on helicobacter pylori in humans key: cord-337464-otwps68u authors: parray, hilal ahmed; shukla, shivangi; samal, sweety; shrivastava, tripti; ahmed, shubbir; sharma, chandresh; kumar, rajesh title: hybridoma technology a versatile method for isolation of monoclonal antibodies, its applicability across species, limitations, advancement and future perspectives date: 2020-05-27 journal: int immunopharmacol doi: 10.1016/j.intimp.2020.106639 sha: doc_id: 337464 cord_uid: otwps68u the advancements in technology and manufacturing processes have allowed the development of new derivatives, biosimilar or advanced improved versions for approved antibodies each year for treatment regimen. there are more than 700 antibody-based molecules that are in different stages of phase i/ii/ iii clinical trials targeting new unique targets. to date, approximately more than 80 monoclonal antibodies (mabs) have been approved. a total of 7 novel antibody therapeutics had been granted the first approval either in the united states or european union in the year 2019, representing approximately 20% of the total number of approved drugs. most of these licenced mabs or their derivatives are either of hybridoma origin or their improvised engineered versions. even with the recent development of high throughput mab generation technologies, hybridoma is the most favoured method due to its indigenous nature to preserve natural cognate antibody pairing information and preserves innate functions of immune cells. the recent advent of antibody engineering technology has superseded the species level barriers and has shown success in isolation of hybridoma across phylogenetically distinct species. this has led to the isolation of monoclonal antibodies against human targets that are conserved and non-immunogenic in the rodent. in this review, we have discussed in detail about hybridoma technology, its expansion towards different animal species, the importance of antibodies isolated from different animal sources that are useful in biological applications, advantages, and limitations. this review also summarizes the challenges and recent progress associated with hybridoma development, and how it has been overcome in these years to provide new insights for the isolation of mabs. antibodies are the glycoproteins produced by the b-cells also known as immunoglobulins, which are present in higher eukaryotes. immunoglobulins are present in either as a soluble form (blood or plasma) or as membrane-bound form (b cell receptors). antibodies are the major component of the humoral immune system that provides protection against the invading pathogens i.e. viruses and bacteria [1] . an antibody is made up of two structural unit's i.e. heavy and light chain. generally, each heavy chain has one variable and three constant regions whereas the light chain has one variable and one constant region. the variable region of antibodies is mainly responsible for its interactions with the invading pathogen and antigen recognition. the antigen-antibody recognition mechanism works like a lock and key fashion. each antibody has a particular paratope (i.e. lock) that binds to a particular antigen (i.e. key). one type of b cell produces one type of antibody against a particular antigen. there are five different types of heavy chains based on the structure of crystallizable fragments (fc) that is attached to the antigen-binding fragments. on the basis of different fc region, antibodies are grouped into five different isotypes i.e; igm, igg, iga, igd, and ige. among all the isotypes igg is the smallest and the most common isotype with the highest therapeutic potential. it makes 70-80% of the total antibodies. iggs have a longer half-life and are permeable to extravascular spaces [2, 3] . https://doi.org/10.1016/j.intimp.2020.106639 received 11 april 2020; received in revised form 6 may 2020; accepted 22 may 2020 antibodies are potentially used for various applications as extraordinary tools in biomedical research for many years. high specificity and selective binding have expanded the scope of antibodies to various applications such as flow cytometry, magnetic cell sorting, immunoassays, therapeutic approaches etc. [4] . antibodies have developed about 40 years ago and have expanded the scope of antibodies to various applications due to their specificity and selective binding ability. these antibodies are classified into two primary subtypes, monoclonal and polyclonal on the means they are basis of their origin from the lymphocytes [5, 6] . both polyclonal and mabs have their advantages and limitations which make them equally suitable for different applications. polyclonal antibodies (pabs) are a pool of immunoglobulin molecules that are secreted by different b cell lineages and react against multiple epitopes of a specific antigen. the pabs are generated by injecting an immunogen into an animal using a prime-boost immunization strategy to produce high titres of antibodies against the particular antigen. after immunization, pabs can be used directly or in the purified form (through affinity column chromatography to remove other serum protein components). polyclonal sera display multiple epitope binding properties which make them an attractive reagent for various purposes, like its use as research or therapeutic reagent either directly or in purified form. the polyclonal serum is widely used for several decades for the treatment of toxin-mediated bacterial and viral diseases [7] . emil adolf von behring was awarded nobel prize in physiology and medicine in 1901 for his work on serum therapy, especially its application against diphtheria, through which he opened doors for new ways of treatment in medical sciences [8, 9] . animal serum-derived therapy has been successfully applied for different medical aspects like overdosing of medication, viral disease (like rabies) and as antitoxins in snakebite envenoming [10] . the beneficial effects of pabs come from its polyclonal nature and biophysical diversity. the poly clonality nature allows targeting multiple sites in a single window of the application and biophysical diversity provides greater stability in environmental changes [11, 12] . despite having beneficial effects of serumderived pabs therapy has several limitations, which need to be evaluated before introducing new interventions. as blood-derived products, intravenous polyclonal immunoglobulins (ivig) have limited availability, batch-to-batch variability; carry the risk of blood-borne disease transmission and only a small fraction of antibodies from the pool of antibodies binds to the target of interest to exert the desired effect. this sometimes results in low specific activity and relatively needs high doses to observe a desired beneficial clinical effect [13, 14] . the other limitation of polyclonal serum is that it cannot be used for the treatment of chronic diseases [15] . due to some of these potential drawbacks of pabs, the way for mab isolation, need, and urgency comes into the light. however, mabs are most suitable and frequently used because of their high sensitivity, specificity, affinity and homogenous nature [16] . monoclonal antibodies are monospecific in nature and produced by identical b cells having high affinity and specificity towards a single epitope of an antigen. in 1975 hybridoma based technology was used to generate the mabs showing very minimal and acceptable batch to batch variation, produced in an indefinite amount continuously. the mabs can be produced against any given epitope present on an antigen or immunogen. moreover, they can be used to detect, purify and characterize the substance of interest. since the development of mab, the scope of antibodies has expanded to various further applications due to their target specificity [17] . this has made mabs a powerful tool in the fields of biochemistry, molecular biology, and medicine. antibody-based biologics are one of the best-selling classes of biomolecules in today's market. advancement in mab generation technologies in recent years has made ease in identification of new target antigens to be explored in diagnostic and therapeutic approaches [18, 19] . several mab generation technologies had been developed over the years, to isolate mabs from immune and non-immune sources using hybridoma, display methods, and more advanced novel mab development technologies like single b cell amplification and culturing methods. unlike the hybridoma method, other methods rely mostly on recombinant production of mabs. each of these technology platforms has their respective advantage, limitations, and applicability [18, 20, 21] . hybridoma technology is the primitive, most fundamental and successful methodology in the field of mab isolation [19] . this technology is quite robust and useful in discovering thousands of antibodies for different applications [22] . the basic practical advantage associated with hybridoma technology is, once the hybridoma clones are established, the production of mab becomes simple and efficient. the antibodies isolated through hybridoma methodology preserves the native pairing of variable and constant regions gene combination, which further supports studies on both direct and indirect functions of a mab. hybridoma technology relies on b cells that are matured in secondary lymphatic organs in response to an antigen. these b cells undergo natural antibody maturation process where the variable region of antibodies diversified by accumulating somatic hypermutations which further results in the selection of high-affinity tight binders [18, 20] . resulting antibodies possess the natural pairing of variable heavy and light chain genes with naturally class-switched matured constant region gene through class switch recombination (csr). such freedom of natural csr is not possible in other mab isolation method that makes hybridoma a unique way to produce naturally matured in vivo antibodies in the laboratory [19] . nevertheless, hybridoma technology is the most preferred technology for mab discovery for in vivo applications. presently there are more than 90% of the antibodies approved by the united states food and drug administration (us fda) are generated by traditional hybridoma technology and are used either directly or in chimeric or humanized versions [19, 23, 24] . a list of these fda approved antibodies has been listed in table 1 . the dominance of this technology has been continued with the recent development of transgenic humanized animals that has opened new avenues for more effective generation of high-quality human antibodies in the modern biotherapeutic era. in this review, we have discussed in detail about hybridoma technology, how it has been expanded to different animal species and their associated biological applications which determine their usage for certain intends. we have also discussed the advancement and challenges associated with hybridoma development, and how it has been overcome in years. georges kohler and cesar milstein in 1975 invented the hybridoma technology, for which they received the nobel prize in 1984 in physiology and medicine [25] . during the same period, herve bazin in louvain-la-nerve belgium created a rat myeloma cell line ir983, allowing the generation of first rat mabs (https://www.synabs.be/2019/ 11/25/hybridoma-vs-display-the-fight-of-the-century/). hybridoma cells are generated via fusion between a short-lived antibody-producing b cell and an immortal myeloma cell. each hybridoma cell constitutively expresses a large amount of one purely specific mab, and favoured hybridoma clones can be cryopreserved for continuous mab production for a long period. hybridoma generation process takes advantage of a host animal's natural ability to generate functional, highly specific and high-affinity mabs [19] . to date, several mabs are developed using this technology and are presently used for diagnosis, prevention, and treatment of different diseases tables 1, 2 and 3. initially, hybridoma technology was limited to murine antigens but with advancement in this field, it is a well-established technology to develop mabs against a vast range of antigens and from different species like rabbits [26] , humans [27, 28] , chickens [29] , goats, sheep [30] , cows [31] , mice [32] , guinea pigs, and rats [33] . choice of animal species especially for mab isolation depends on several factors such as the presence of a homologous protein in the immunized species, table 3 a list of fda approved rabbit mabs that are used in diagnostics. device name availability of suitable fusion partner, the amount of protein or antigen available for immunization, the time required to obtain an antibody response and finally, a purpose for which these mabs are needed. most commonly used hosts for mab production are the mice followed by rabbits. the inbred balb/c strain of mice is usually the right choice and preferably suited for mab isolation [34] . chickens are also considered as a preferred host of choice due to its distinct phylogenetic relationship between the antigen donor and the antibody producer [16, 35] . hybridoma technology has not gained much attention because its success mainly depends on the availability of a suitable fusion partner. in the initial years of hybridoma discovery, technology was limited to mice but in progressive years researchers used this platform for human and rabbit hybridoma development. however major limitation associated with the production of mabs from other species is the instability of hybridoma clones produced with fusion partners from heterologousspecies [18] . this instability resulted in the hybridoma clones are due to the fusion of two cells from different species which leads to chromosomal instability. to overcome this, in the past few years many different strategies have been used to increase the fusion efficiency. generally, there are two types of hybridomas one is homo-hybridomas and second is hetero-hybridomas. in homo-hybridomas both, the igg secreting b cells and fusion partners are from the same species. in hetero-hybridomas the antibody-secreting b cells and fusion partners are from two different species. homo hybridomas are genetically more stable and secrete stable igg as compared to hetero-hybridomas as it gradually lost the chromosomal recombinants during the clonal selection step due to their genetic instability. 3. animal species used for hybridoma development over the years: mouse polyclonal and mabs held the largest market in 2019 as they are more specific and easier to produce in nature. the structural similarities between human and mice antibodies are the prime reason for their high acceptability rate. upgradation and simplicity of mice hybridoma process have made it a more prime reason for their high adoption rate in research and therapeutics [36] . the mice hybridoma technology is a multi-step process that takes advantage of a host animal's natural ability to produce highly specific, high-affinity and fully functional mabs. it involves the development and optimization of specific immunogenic antigen (ag). following the optimization, a host animal is immunized with the ag along with adjuvant for several weeks. the sera from immunized animals are tested for their reactivity and specificity to the immunizing antigen while the animals with high titres of binding antibodies are selected further for splenocytes isolation [32] . the spleen cells are fused with the immortalised myeloma cells in the presence of fusogenic agents like viruses, chemicals and electric pulses. the most common myeloma fusion cell lines are x63-ag 8.6539 [37] and sp2/0-ag 1410 [38] , with the origin from balb/c mouse. the fused cells are then selected on hypoxanthine-aminopterin-thymidine (hat) medium. the myeloma cells are sensitive to hat medium as they lack hypoxanthine-guanine phosphoribosyltransferase (hgprt) gene required for nucleotide synthesis by the de novo or salvage pathways while the unfused b cells die as of short life span. in this process, only the hybrid (b cell-myeloma) survives, as they harbour the functional hgprt gene from the b cells. however, hybrid cells retain the dual properties, antibody secreting property of b cells and continuously growing property (immortality) from myeloma cells. fused or hybrid cells are then screened by "limited dilution cloning" method or with semi solid selective medium to select only those hybridoma that produce antibodies of appropriate specificity. a detailed schematic representation of steps involved in hybridoma production is shown in fig. 1 . production of antibodies by mouse hybridoma technology is quite robust and has been useful to discover thousands of antibodies for different applications. academic and industrial research groups with expertise in the field of antibody isolation, hybridoma methodology continues being the methodology of the first choice, particularly if the goal is to obtain antibodies for analytical purposes. the main associated advantage is that, once the hybridoma clone is isolated, mab production in mouse ascites is simple, efficient and reproducible. these hybridomas can be stored in liquid nitrogen for several years making them virtually immortal [39] . the mouse mabs can be potentially used for diagnostic, therapeutic as well as for other research applications. the first therapeutic antibody that was approved by the food and drug administration (fda) in 1985, developed by murine hybridoma technology was potentially used to reduce the graft rejection in transplant patients [18] . okt3, the first mab to be used in organ transplantation and during the past one decade there has been an extensive experience of its use both for prevention and treatment of organ transplantation rejection. okt3 blocks t cell function by modulating cd3 and the t cell receptor from the t cell surface, functions as an immunosuppressant [40] . since its discovery, okt3 was further improvised in progressive years from chimeric to humanized version, to further reduce adverse effects and to increase immunologic efficiency [41] . the use of murine derived mabs has less therapeutic value as they are entirely from mouse origin and can cause immunogenic reactions in the target host. such limitations have been addressed by chimerization and humanization of antibodies, potentially removing the mouse immunogenic content [42] . several murine mabs has been approved by the fda for use in diagnostic and therapeutic purposes over the years as listed in table 2 . since the discovery of mouse mabs, rabbit hybridoma has been potentially used as a dominant tool in the field of research, diagnostics and therapeutics from several decades [4] . however, a new technology for generating mabs with improved affinity, specificity and having the ability to recognize non-immunogenic rodent's epitopes, are in need as an alternative for the scientific community. the rabbit immune system has been documented as a vehicle for developing antibodies with higher affinity and more diverse recognition of many molecules including phospho-peptides, carbohydrates and immunogens that are not otherwise immunogenic in mouse [24] . antibodies produced in rabbits usually have about 10 to 100 fold greater affinity than those produced by mice. rabbits generate more diverse and complex immune response towards target antigen as compared to human and mice because of gene conversion and somatic hypermutations phenomenon leads towards more mutations in rabbit antibody repertoire [43, 44] . the gene conversion is responsible for introducing mutations and affinity maturation of variable antibody fragments which takes place in double-stranded rearranged v(d)j dna segment of antibody gene via homologous recombination [19, 45] . the rabbit iggs are somewhat simpler than the mouse and human antibodies. rabbit igg has only one subclass i.e. cî³ gene and the majority (90-95%) of light chains are derived from isotype cîº1. only 5% to 10% of the total igg light chains are isotype l. fig. 2 . several efforts were made to generate rabbit mabs after the development of mouse hybridoma technology in the 1970s. due to the favourable properties of rabbit antibodies, many scientific groups tried to develop methods for the generation of rabbit hybridomas. this endeavour was significantly complicated by the absence of rabbit myeloma cell lines. viral transformation of rabbit b cells to generate myeloma-like cell lines also proved to be difficult and rather inefficient [46] . for these reasons, substantial efforts are focused on generating rabbit-mouse hetero-hybridomas. unfortunately, all hetero-hybridomas generated in the early days of hybridoma technology revealed poor fusion efficiency, genetic instability and impaired functional rabbit heavy-and light-chain pairings. in 1988, raybould et al. generated the first stable rabbit-mouse hetero-hybridoma by polyethylene glycol-mediated fusion of rabbit spleen b cells with the mouse myeloma cell line sp2/0-ag14. even though they observed stable rabbit igg expression for several months, other groups observed genetic instability and concomitant decrease of mab secretion [47] . these shortcomings could be partially addressed by extensive efforts to regularly subclone the rabbit-mouse hetero-hybridoma in 1996, weimin zhu and robert pytela, at the university of california, developed an improved rabbit hybridoma fusion partner by repeatedly subcloning. after multiple rounds of subcloning, they selected high fusion efficiency clones based on characteristics like robust growth, morphological characteristics and named it as a new cell line 240e-w, with better fusion efficiency and stability. since then this cell line 240e-w has been further developed and optimized to eliminate endogenous igg and has been used for the production of rabbit mabs for research and commercial applications [48] . the cell line 240e-w was further modified to a superior version 240e-w2. abcam patented this technology to develop highly specific mabs, under the name of ranmab, which has been potentially explored for the production of diagnostic and research antibodies. a large number of rabbit mabs are used in basic laboratory research. rabbit mabs are preferred over the mouse and human mabs in diagnostics and pharmaceuticals applications because of their high specificity and affinity. various rabbit mabs have been approved by the fda to use as in vitro tumour diagnostic tool [49] . a list of fda approved rabbit mabs for diagnostics are listed in table 3 . recently, wei et al. developed an ultrasensitive test for ebola virus diagnostics using carbon nanoparticle-labelled pad with rabbit anti-ebola virus (ebov)-vp40 igg for rapid detection lateral flow test strip for ebola virus [50] . limited success has been gained in the development of rabbit mabs as therapeutic agents but the potential use of rabbit pabs for the prophylaxis and treatment of acute rejection against t cells in organ transplant opened venture to explore the use of humanized rabbit mabs as a therapeutic agent [46] . a company name epitomics has developed a humanized rabbit monoclonal drug candidate and demonstrated in vitro and in vivo efficacies [51] . recent fda approval of humanized rabbit monoclonal single-chain antibody fragment, brolucizumab in 2019 for the treatment of wet age-related macular degeneration (amd) has increased hope and venture of other humanized rabbit monoclonal for therapeutics in near future [52] . a detailed description of these antibodies has been reviewed by mage et al. 2018 [53] . human hybridoma technology which allows the direct generation of human antibodies in a native form, is the most direct effective approach for the production of natural therapeutic and diagnostic antibodies with no additional modifications require [54] . it is believed to be the most promising and convenient technological platform for the isolation of therapeutic mabs. however, success of human hybridoma technology for the therapeutic purposes has been limited since years due to several technical challenges like unavailability of human fusion partners, as most of the fusion partners available are from rodent origin or heteromyelomas. a fusion of human b cells with different fusion partners limits the use of these mabs for therapeutic applications. several hetero-myelomas fusion has been successfully employed for the generation of mabs of human origin for different diseases like hiv [28, 55] , chikungunya [56, 57] , dengue [58] etc. however, such hybridomas are unstable, leads to a loss in the ability of antibody secretion hence there is a challenge in achieving desired pharmacokinetic characteristics of natural human antibodies in terms of distribution, metabolism and excretion [54] . several scientific groups have attempted to develop natural human fusion partner cell lines but limited success stories are reported using these fully human fusion partners. one such example of fusion cell is human karyochi cells which were successfully used to develop complete human stable hybridomas with igg secreting properties for several months. the fusion efficiency of these human karyochi cells was in the range of 10 -5 to 10 -3 with no reports on the endogenous generation of immunoglobulin or chains that can interfere with subsequent synthesis, assembly and purification of mabs. the other major limiting step in the development of human hybridomas is low fusion efficiency of human b cells with the myeloma partner (0.001%) and secondly, the low percentage of circulating antigen-specific b cells in the peripheral blood (0.01%) also limits the use of this technology [59] . frequency of antigen-specific b cells is very rare in circulation therefore, selection of a proper blood donor is critical for the success of this method. acutely infected patients have a higher number of circulating b cells as compared to the convalescent patients. donors showing a high titre of serum binding/neutralizing antibodies may have a higher frequency of peripheral b cells, and an indication of the greater chance of successful production of hybridomas. to overcome these challenges different groups have tried ebv(epstein-barr virus) transformation approach to enrich the population of b cells [60] . the most commonly used cell line b95-8 is a continuous cell line releases high titres of transforming ebv in supernatants [61] . the b95-8 cell line was initiated by exposing marmoset blood leukocytes to ebv fig. 2 . schematic drawing of natural rabbit, mouse, chicken and human igg. generally 150-kda igg comprises of two identical îº or î» light chains paired with two identical heavy chains. the light chain consists of an n-terminal variable domain (vl), followed by one constant domain (cl). the heavy chain consists of an nterminal variable domain (vh), followed by three constant domains (ch1, ch2 and ch3) generally, however, the heavy chain of avian igy contains four constant regions (ch1, ch2, ch3 and ch4). schematic drawing of natural rabbit antibodies in igg format. the~150-kda rabbit igg molecule contains two identical îº (white) or î» (light grey) light chains paired with two identical heavy chains (dark grey). the light chain consists of an n-terminal variable domain (vl), shown with its three cdrs, followed by one constant domain (cl). the heavy chain consists of an n-terminal variable domain (vh), also shown with its three cdrs, followed by three constant domains (ch1, ch2 and ch3). ch1 and ch2 are linked through a flexible hinge region that has the amino-acid sequence apstcskptcp (or apstcskpmcp in an allotypic variant) and anchors three disulphide bridges (orange) of the igg molecule, one for each of the two light-and heavy-chain pairs, and one for the heavychain pair. notably, rabbits have two îº light chains, k1 and k2. the more frequent îº light chain, k1, contains an additional disulfide bridge that links vl and cl. rabbits of the commonly used new zealand white strain have~90% igg-îº (k1),~10% igg-îº (k2) and < 1% igg-î» antibodies. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) extracted from a human leukocyte cell line. b95-8 provides a source for ebv to establish continuous lymphocytic cell lines from human donors. ebv mainly binds with the cells in peripheral blood that contains cd21 receptor and b cells in peripheral blood express these antigens on their surface and activates latent membrane protein (lmp) 2a and lmp1 [62, 63] . due to low antibody-secreting and chromosomal instability characteristics transformed b cells cannot be cultured for a long period. the transformation efficiency of ebv to b cells is low, ranges from 0.1 to 1%, however, the transformation efficiency of b cells in presence of ebv can be increased by the addition of cpg, which acts as toll-like receptor (tlr) 9 agonist [27] . cpg oligo-deoxynucleotides (or cpg odn) are defined as short single-stranded synthetic dna molecules where c refers for a cytosine triphosphate deoxynucleotide ("c") followed by a guanine triphosphate deoxynucleotide ("g"). the "p" denotes to the phosphodiester bond between consecutive nucleotides [64] . in unmethylated form, these cpg motifs act as immune stimulants and are recognized by the pattern recognition receptor (prr) toll-like receptor 9 (tlr9), which is constitutively expressed on immune system cells like b cells. these b cells undergo polyclonal response to cpg dna stimulation by proliferation and differentiation to antibody-producing cells [65] . these transformed b cells are grown for a specific time for the emergence of immortalized cells, which further fused with the fusion partner to establish hybridomas. this cpg activation method has been successfully used to produce mabs against severe acute respiratory syndrome coronavirus (sars-cov) [66] and hiv [27] . the main concern of developing hybridoma for therapeutic purpose is that the final hybrid cells should be free of ebv and other human viruses [67] . the two most common cell lines used for fusion are shm-d3327 and hmma 2.5. the shm-d33; produced by fusing the human myeloma cell line fu-266, clone e-1 (hat sensitive, 8-azaguanine resistant and resistant to g-418 -an antibiotic similar to gentamicin) with the murine myeloma p3x63ag8.653 [68] . this cell line has been used as a fusion partner to stabilize the lymphoblastoid cell lines (lcl's) secreting immunoglobulins to produce mabs against envelope proteins of hiv-1 and parvovirus b19 [55, 69] . hmma 2.5 is a human ã� mouse cell line that was generated by fusing mouse myeloma cell line p3x6ag8.653 with bone marrow mononuclear cells of a patient with iga myeloma [70] . several mabs have been produced using this cell line as a myeloma fusion partner [71] . the other limiting steps in human hybridoma development are fusion efficiency. fusion is mainly performed by three methods (i) chemical agents like polyethylene glycol (peg) (ii) viral agent mediated methods and (iii) electric fusion methods. peg mediated fusion is the most common and traditional method of fusion due to its simplicity and most commonly convenient fusing agent of choice for hybridoma production. peg fuses the plasma membranes of two adjacent mammalian cells by dehydrating the lipid head groups, leading to the asymmetry of the membrane bilayer, favouring fusion of two cells leading to a single cell with two or more nuclei. one major drawback of peg mediated fusion is a generation of non-specific fusion between different kinds of cells [72] . over the year's viral agents have also been used as a successful agent to perform fusion among two different cells. sendai and vesicular stomatitis virus are the two most commonly viruses used as fusion partners. the most efficient and innovative method for cell fusion is electrical cytofusion. this mainly works on the principle of fusion of cells in the presence of high-intensity electric field pulse that causes transient membrane permeabilization. this method has higher fusion efficiency over chemical or viral-based fusion methods. however, electrofusion yields in low fusion efficiency when fusion partner cells are different in size [73] . hmma 2.5 myeloma cell line is found to be the most preferred cell line for electrofusion showing maximum fusion efficiency as compared to other myeloma cells [71] . rems et al., developed a numerical calculation method to fuse cells with shorter, nanosecond (ns) pulses. the performance of this method works on the principle of contact areas between two fusion cells, regardless of their cell size [73] . use of cephalin as fugenic reagent along with emetine and actinomycin d, golestani as selection method dramatically increased the fusion efficiency and recovery (19-34%) [74] . the major advantage of human hybridoma technology is that antibodies produced by this technology are derived from human origin have more therapeutic applications than rodent derived counterparts due to attributable differences between the human and rodent immune systems. due to evolutionary differences between mammalian (e.g. human, mice) and avian species (chickens), the avian/chicken immune system recognizes more epitopes on mammalian proteins as foreign and generates a more vigorous and diverse immune antibody repertoire [35] . phytogenic differences among different species have been illustrated in fig. 3 . as the phylogenetic difference between the immunizing antigen and the immunized animal increases, the immune response increases accordingly. it is, therefore, possible to produce antibodies in chicken that are difficult or impossible to produce in mammals such as g-protein-coupled receptors (gpcrs) against which producing antibodies in rodents is a challenge as of high sequence conservation (> 70%) at the protein level. chickens are emerging as valuable immunization hosts specifically for therapeutic antibody discovery for difficult targets having sequence conservation in mammals. the use of chicken egg yolk for antibody production represents a reduction in animal use (ethical issues) as chicken produces a larger amount of antibodies than laboratory rodents [75] . the advantages of chicken (gallus domesticus) antibodies as diagnostic and therapeutic biomolecules are less characterized than their mammalian counterparts [16] . initially, in 1989 a successful attempt was made to generate chicken hybridomas against newcastle disease virus (ndv) by fusing the peripheral blood lymphocytes (pbl) and thymidine-kinase deficient (tk-) chicken myeloma cells [76] . surprisingly, the secreted antibody hybridomas were initially obtained, but they lose the ability to produce antibody in the culture immediately [77] . to overcome this issue nishinaka s et al., in 1991 developed a new improved fusion cell line r27h4, for the production of chicken mabs [29] . the new cell line was efficient in the development of antibody-producing hybridomas with highly reactive igg secretion ability of 6 months. these cell lines were further improved and several chicken hybridomas were successfully developed [29, 78] . however, chicken hybridoma technology has been explored in limits and most of the investigators prefer to use phage display method over hybridoma for the development of chicken mabs [79] . the main avian antibody isotype igy shares structural and functional homology to mammalian igg and ige isotype counterparts but the difference in a constant region of antibodies, igy has 4 constant regions whereas igg has 3 constant regions [80] [81] [82] fig. 2 . igy present in chicken sera gets passed to the embryo through the egg yolk [83] . egg igy antibodies have been used previously against bacterial and viral infections [84, 85] . humanization of these antibodies can have great potential for biopharmaceutical development [86, 87] . the recent development of transgenic chicken with human immunoglobulin loci has expedited the use of transgenic chicken derived mabs directly for human therapeutic use [88, 89] . these engineered transgenic chickens express antibodies from immunoglobulin heavy and light chain loci containing human variable regions and exhibit normal b cell development raising immune responses to conserved human targets that are non-immunogenic in mice [90] . these transgenic chickens can be potentially used for the development of hybridoma secreting antibodies of human origin. however, like others, the unavailability of robust hybridoma fusion partner limits its potential utility [88] . in recent years different research groups have extensively explored the display method to overcome the limitations of chicken hybridoma. antibodies isolated through hybridoma methodologies have the advantage of being used directly and could be cryopreserved for future uses till an indefinite time, as the fusion partners are myelomas possessing remodelled transcriptional machinery to secrete a continuously large amount of antibodies [91] . advancement in recombinant technology has overcome the challenges by cloning of variable heavy (vh) and variable light (vl) from unstable hybridoma; cloning in transient transfection vectors to produce antibodies in mammalian expression system [92] . currently the development of stable cell lines has advanced the antibody production system by developing stable cell lines from unstable hybridomas to produce consistent antibody production. chinese hamster ovary cell line (cho) is the most preferred cells used for large scale production of mabs. however, the development of stable cell lines is a tedious process that sometimes takes a month to year time where the success of this process depends on random genome integration of transgenes [93] . development of crispr-cas9 in recent years has overcome by immunogenomics reprogramming using plug and play technology, where crispr-cas9 was used to engineer immunogenomics by homology-directed repair to replace endogenous immunoglobulin region by exogenous donor counterpart with the help of guide rna. this technology platform has enabled the rapid generation of full-length antibody-secreting cell lines [94] . a major limitation of hybridoma technology is the lack of suitable fusion partners which limits the use of this technology and limits its applicability to other species. to overcome the problem of suitable fusion partners, the transgenic mice model h-2k b -tsa58 has been developed. h-2k b -tsa58 transgenic mice express the simian virus 40 (sv40) antigens (tag) under the control of mouse major histocompatibility complex h-2k b promotor. this promotor allows differential expression of sv40 antigen in different tissues at various levels. expression of this antigen can be increased by simply increasing the levels of interferons (ifns) [95] . the higher expression level of this antigen is responsible for tumorigenesis and aberrant development. in this approach transgenic mice h-2k b -tsa58 were specifically used to isolate monoclonal against the filamentous phage. transgenic mice were immunized with filamentous phage suspension. splenocytes were recovered from the immunized animals and spleen cells were limited diluted from 6, 24-, 96-well plates. cells from the positive wells were selected to develop monoclonal lines. the main advantage of this technology is that it eliminates the use of fusion partners and can parallelly be used for the development of monoclonal and polyclonal based therapies [96] . the other major challenge associated with hybridoma development is the requirement of purified antigen to generate a specific immune response. in some of the cases, it's a challenging task to purify the antigen. the lack of specific immune reagents for characterization and monitoring of these numerous proteins limits the overall time process for the production of hybridoma. expression and purification of recombinant protein are time-consuming and sometimes not cost-effective. additionally, immunization of animals with these purified recombinant proteins in formulation with adjuvants sometimes leads to alteration of native conformation of these proteins which finally leads to an undesired immune response in animals. in recent years, different approaches have been successfully implemented to overcome these challenges; a novel strategy has been developed to isolate mabs against the native proteins [55] . in this strategy, animals were directly immunized with transiently transfected hek cells that express desire protein on the surface of these cells in a proper folded and glycosylated form. this modified technology is mainly useful for the isolation of mabs against native antigens. the advantage of this technology is that expressed protein on transfected cells closely mimics as it expresses in naturally infected cells. a similar type of strategy was developed by hazen m et al; where they had attempted to isolate monoclonal against the native conformation of extracellular loops & multi transmembrane proteins using dna based immunization strategy in combination with immunomodulatory agents [97] . use of immunomodulators in dna immunization has positively enhanced the immune response and proved to be a successful approach to isolate desired specificity binding mabs to native conformations. kato et al., in 2019 developed a cell-based immunization and screening (cbis) method for isolation of mabs for podoplanin (pdpn) [98] . in this strategy, they immunized the mice with transfected cells overexpressing pdpn and screened the fused hybridoma cells using flow cytometry. these cell-based immunization methods are useful in the isolation of mabs against various membrane proteins which is still difficult to achieve otherwise [56, 57] . the other challenge in hybridoma technology is animal immunization. the overall efficiency of hybridoma depends upon the efficiency of immunization. some factors that affect the efficiency of immunization are; route of administration of antigen, dose, choice of adjuvant, number of boosts and immunization protocol. dna based immunizations are generally more preferred where it is difficult to express fulllength proteins and immune response are mainly targeted towards native or conformational epitopes because the structural integrity of protein is critical for induction of functional mabs. the major routes of dna based delivery are intramuscular, intravenous and intrasplenic. intrasplenic routes are considered to be the most efficient route. a single dose of dna delivery is sufficient to induce antibody responses. antibodies against different proteins can be produced at the same time by immunizing with several nucleic acids encoding for different proteins or single plasmid encoding different subunits of the protein. in such cases multiple booster doses are usually avoided, to reduce immune-dominance amongst antigens. another major challenge in the field of human hybridomas is the requirement of lymphocytes from actively infected patients or who have been exposed to antigen. if the active immune response is absent or not sufficient then the probability of circulating b cells in such cases is very poor or negligible. due to ethical issues and considerations, it is generally not possible to immunize humans. to overcome these limitations li et al., in 2006 have developed a novel and rapid combined ex-vivo immunization strategy with morphogenics platform process for the isolation of therapeutic human mabs. in this strategy, the group has purified b cells (cd19+) and cd4 positive t cells from healthy volunteer blood samples using magnetic bead-based sorting. these b/t cells were cultured in the presence of growth factor and antigen to activate b cells. the pool of these b and t cells were then fused with myeloma fusion partner using electric cyto plus. the fused cells were screened for antigen-specific antibody secreting properties by limited dilution method. the isolated mabs had shown high specificity, biological activity and high affinity. this method avoids the collection and screening of a large number of patient samples which are normally the basic requirement for the generation of therapeutic human hybridomas. it also avoids the risk of potential viral transmission associated with conventional methods where pbmcs are sometimes used from viral infected patients. screening of volunteers and blood cells can be performed before ex vivo immunization and after hybridoma development. this platform technology offers a rapid and cost-effective way for therapeutic human mabs having natural pairing of immunoglobulin genes [99] . after cell fusions between b cells and myeloma cells, protocols of hybridoma technology include multistep screening and cloning processes to identify antigen-specific hybridomas, which is labour-intensive and time-consuming. however, recent advances in robotic screening methods have alleviated this to some extent [88] . the screening process on semi-solid selective medium has made it easy and reduces the overall time in hybridoma production by repeated selection and cloning steps. this screening technology has been used by companies to sale ready to use kit based systems for the development of murine hybridoma. it has also facilitated the use of murine hybridoma technology in a less cumbersome and user friendly. methylcellulosebased semi-solid selective medium is preferentially used in hybridoma selection [56] . technically it avoids the loss of rare clones from an overgrowth of faster-growing cells, which can occur during selection in a liquid medium. the selected clones are further dispersed into a liquid growth medium for screening and expansion. similarly, paul et al recently developed microarray-based screening technology for direct identification of high-affinity clones which avoids the loss of slowgrowing clones. additionally, this approach eliminates the enrichment, isolation, and purification of igg for the characterization process. the crude culture supernatants can be directly used and thus avoids expensive and lengthy screening steps [100] . the screening process has advanced through flow cytometry-based methodology where single cells could be sorted from a bulk mixture of fused hybridoma cells. it has advanced by saving time and labour instead of the traditional multimicro well plate seeding and limiting dilution sub-cloning [101] . in recent years tedious hybridoma screening and cloning processes are replaced with flow cytometry-based sorting methods. these methods avoid effort and time of tedious repeated screening processes. this method could also differentiate igm and igg secreting hybridoma. facs based screening methodology can be applied in any laboratory easily setup as it doesn't require any special reagents [102] [103] [104] . the other alternative approach that some groups had used is the screening of hybridoma supernatant directly by bio-layer interferometry based on disassociation rates to select clones containing high-affinity antibodies for further expansion and subsequent characterization. the main drawback of the elisa based screening method is that clones that express high levels of a low-affinity antibody can give an equivalent signal to clones that express low levels of a high-affinity antibody. as a consequence, superior clones can be overshadowed by inferior clones because elisa method score antibodies based on the binding signal strength and do not provide accurate affinities or dissociation rate constants [105, 106] . current drug approval rates underline the revolutionary effect of fully human mab therapeutics on drug development. antibodies isolated for therapeutic applications from different species excluding human needs a multistep process of humanization and developability through rational sequence optimization [107] . mouse is the most common and preferred progenitor used in mab isolation. to avoid the multistep process of humanization, the concept of transgenic mice harbouring the human antibody repertoire has gain attention, where large human immunoglobulin loci are transferred into the mice germline using yeast artificial chromosome approach [108] . the xenomouse and humab mouse are the first engineered transgenic mice that carry the majority of human vh & vl antibody repertoire [109] . xenomouse transgenic technology was developed by cell-genesya a biotech company (now a part of bristol myers squibb, new york, usa). the first therapeutic antibody developed by this transgenic technology was approved by the fda in 2006 for the treatment of advanced colorectal cancer. the strength of transgenic technology can be evaluated by the recent data on approved mabs. more than 18 fully human mabs developed by transgenic animal-based technology are used for human therapy. a list of fda approved mabs developed by transgenic technology is listed in table 4 . initially, this technology was limited to mice but over the years this technology has been established for other animal models like rabbits, rats, and cows. the success of this technology table 4 a list of fda approved mabs derived from transgenic technology. treatment of colorectal cancer mainly depends on the proper representation of the target antigen to the immune system. in addition, designing a proper immunogen is very critical for success. to avoid the ambiguity of immunogen design in soluble form, genetic immunizations are more preferred over the traditional methods. in contrast to human, mouse possesses very less antibody diversity represents the main limiting step in xenomouse transgenic technology. human antibody repertoire shows diversity more than 10 11 , however, the number of b cells in a mouse is~10 8 only. a single mouse can harbour the only fraction of the antibody repertoire from human antibody [110, 111] . moreover, immunization of a large cohort of mice to increase the diverse response against antigens could be used to overcome the limited antibody diversity. the other transgenic mice technologies, where kymouse & trianni mouse models were developed to represent a more diverse human antibody repertoire that allows the selection of diverse human antibodies and overcomes the limitations of xenomouse [112] . fig. 4 represents an illustration of transgenic antibody technology showing the antibody production route. besides, the other limitation of this technology platform is immune tolerance when attempting to raise an immune response against human targets. a large number of human targets possesses a very high degree of sequence and structural homology, because of this homology the transgenic immune system recognizes these antigens as self-antigen. different groups have tried to overcome the immune tolerance mechanism by adding t cell epitopes to the antigen [89, 113] . the other similar approach tried by different groups to abolish the expression of murine orthologues gene [114] . but the major limitation with these defected mice models is that sometimes these mice suffer from health issues and some of these knockdown genes are necessary for the development of a foetus. recent developments of transgenic rat and fig. 4 . illustration of transgenic antibody technology shows the antibody production route: mouse immunoglobulin gene loci were functionally inactivated in embryonic stem (es) cells by targeted gene deletion used to generate mice homozygous for the necessary deletions. crossbreeding between the transgenic mice (containing both human and mouse antibodies) with mice incapable of producing mouse immunoglobin, resulting in the xenomouse strain which expresses human antibodies but not the mouse antibodies. b cells, isolated from immunized xenomouse, are fused with myeloma cells to produce hybridomas producing human mabs. chicken (omnichicken) models have partially overcome these limitations. the transgenic model-based technology harbours some advantages over the phage display derived antibodies. the antibody developed by transgenic technology requires less development or optimization and thus require a shorter time to reach the product development stage. production of stereo-specific mabs is still a very big challenge. there are hardly any practical technologies available for the generation of stereospecific antibodies, because of hurdles like how to immunize a mouse maintaining the antigen structure intact in the presence of adjuvant. in addition, adjuvants allow more effective sensitization, disrupting the native structure of proteins. however, if an adjuvant is not used, immunization efficiency could be very low. another difficulty is strict selection of stereospecific mab producing sensitized b lymphocytes. although, if the immunization is successful with a native intact antigen, the number of desired sensitized b lymphocytes is extremely small, accounting for very less population of total spleen cells after repeated immunization [72] . most of the established therapeutic mabs have specificity for the antigen targets having primary structures. generally mabs can recognize two types of antigen epitopes which includes linear in the primary structures of proteins and the conformational, dependent on secondary and tertiary structures [115] . stereo-specific mabs recognizing conformational structures of target antigens may thus offer a markedly more versatile approach. besides primary, secondary and tertiary structures, proteins may also exhibit quarterly structures. which are formed by hetero or homo-subunits, providing unique interfacial geometric structures on their complexes. native or conformation-specific mabs are quite reasonable and attractive for future therapeutic purpose. there is need to replace the conventional mabs with stereospecific mabs in the near future for therapeutic medicine as they recognize the 3 dimensional conformation, which intrinsically determine their fundamental biological functions [116] . stereospecific mabs that recognize 3d molecular configuration has advantages over linear epitope-specific mabs that consider only 2d configuration. in general, mabs against primary structures, in target therapeutic antigens with the dominant secondary and tertiary folding are having affinity only with full antigens in restricted areas, as their linear epitope may be masked due to conformational folding. alternatively, target antigens in the immune system can be identified by assuring indigenous structures via immunizing dna of the target antigens expressed on the surface of the cell, allowing healthy and intact structural conformation. this can be linked to membranes, which mimics membrane protein, with a soluble protein harbouring sufficient signal peptides and membrane-penetrating areas that are genetically linked to the 5â�² and/or 3â�² terminals of genes of the desired soluble proteins as nucleic acid sequences to express as a fusion protein [117] . in the emerging diseases like human immunodeficiency viruses-1 (hiv-1), coronavirus -19 (sars-cov2), dengue, and chikungunya, the immunogenic proteins of pathogens harbour complex structural glycoproteins, needs an urgent high-quality stereospecific mabs to control their spread. development of therapeutic antibodies against these pathogenic diseases is aimed to the structural antigens. the complex native structures of envelope proteins on viral surface facilitate the attachment of virus to the host cell, and subsequently entry inside the cell [118] . these native structural proteins induce predominantly cross reactive neutralizing antibodies. the neutralizing antibodies have shown promising results in the protection against pathogens however nonneutralizing antibodies help the virus in evading immune system [119] . in hiv-1, researchers are working to develop broadly neutralizing antibodies targeting conformational epitopes [120] . similar approaches have been taken for other viral antigenic targets [121] . in many of these viral infections "conventional abs" are generated in response to virus infection, but the virus adopts numerous evasion strategies like conformational masking of antigenic targets by glycosylation, high mutation rate etc. [122] . generation of stereospecific mabs are required to display impressive breadth and potency against the conformational proteins. these stereospecific mab productions with promising therapeutic potential can be achieved by inclusion of critical steps like i. dna or soluble protein immunization with native like targets or structural proteins ii. selection of antigen specific myeloma cells and iii. selective fusion of myeloma and b cells to generate hybridoma cells secreting stereospecific mabs. different novel approaches and attempts are being taken to produce the soluble trimers which can display native-like conformational stable structure mimicking with virus surface. these conformational protein structures are promising targets for protein or dna immunization and could subsequently require for the production of stereospecific mabs. one major challenge associated with the use of native like antigens for the development of stereospecific antibodies is use of adjuvants in immunization process. most of the adjuvants usually disrupts the original protein native structure and hence impede its ability to present relevant epitopes or occludes the trimer conformational epitope [115] . in recent years' studies using iscom class of adjuvants in animal preceded by in vitro analyses showed that it has no adverse effect on native trimer conformation or antigenicity [123] . the other way to generate stereospecific antibodies is the direct immunization of mammalian cells expressing cell surface antigens in its native conformation [124] . a number of stereospecific antibodies has been generated against several targets like receptors [125] , ligands [126] , antagonist and chemical compounds [127] . a detailed schematic representation of different approaches used for isolation of stereospecific mab is shown in fig. 5 . the production of stereo-specific mabs could be achieved by tweaking the conventional hybridoma fusion through stereo-specific targeting (sst) technique invented for the first time by tsumoto et al [115] . crucially, it involves a strict selection of the required sensitized b lymphocytes by intact antigens, expressed on myeloma cells, through b-cell receptors (bcrs) and their selective electrofusion (only attached cells can be fused among themselves) to generate a specific hybridoma [128] . sst technology offers selective production, against different protein types, of monoclonal stereospecific antibodies not only for membranous but also for soluble non-membranous antigens. morshed et al recently showed efficiency in activation of the g-protein coupled receptor which holds seven-transmembrane domains by a stereo-specific mab [117] . furthermore, the new promising class of therapeutic mabs, catalytic antibodies are capable of identifying and degrading antigens, has fundamentally demonstrated. hifumi et al. have developed a catalytic antibody to degrade the active site for urease o helicobacter pylori and eliminates the bacterial infection in the mouse [129] . moreover, the catalytic antibodies have proven their utility in suppressing infection of the rabies virus [130] and the influenza virus [131] in vitro and in vivo using human antibody light chains. in addition, they have recently been noted in their ability to effectively reduce the accumulated ã�-amyloid in the mice's brain [132, 133] . medi9447 is a mab that inhibits cd73 (ecto-5â�²nucleotidase) activity on a non-competitive basis and is considered a promising immuno-oncology target [134] . this mab is antagonistic to cd73 employing dual inter-cd73 dimer cross-linking and/or steric blockage mechanisms that prevent the adoption of the cd73 catalytic active conformation [135] . bispecific mabs with stereo-detection may be especially effective for cancer cell detection of membranous antigens which have not been easily detected by conventional linear mabs. in a nutshell, as proteins retain their native conformation in nature, development of stereospecific, alternate forms of bispecific and catalytic mabs, for selective therapy is the founding factor in therapeutic future drugs. the mab has come a long way since the days when unmodified murine mab was explored against the cancer-causing agents. from the last two decades, mabs have been a standard element of cancer therapy, however, with still much room for further improvement in future [64] . the mabs are always preferred over the chemical compound based therapies because of their high specific reactivity and affinity towards the target antigen recognition. they show minimal side effects with favourable pharmacotoxicity and pharmacokinetics properties. the high specificity of mabs towards their targets presents an attractive and successful option for the development of medical treatment and molecular drug targets. [136] . early clinical attempts exploring mab-based therapeutics were very primitive and disappointing 20 years ago, some clinical experts considered the antibody-based therapy treatment for cancer as a failed hypothesis [137] . the first mab which was clinically evaluated against cancer was the murine mab. although there were some fascinating hints that mab therapy could be successful, however, the problems related to the administration of murine mab to humans limited their clinical utility and applications [138] . rise in immune response against the therapeutic mab, very rapid clearance of the mab from the system and suboptimal ability of the murine mab to interact with the human immune system in a manner that led to immune destruction were the challenging tasks. however, some investigators tried continuously to explore the use of mab as a possible cancer treatment. they also evaluated other strategies such as using; igg to target cancer directly, alter the host immune response to cancer, provide cytotoxic substances to cancer, and retarget the cellular immune response towards cancer [64] . over the last twenty years, the effectiveness of antibodies in the treatment of patients with cancer and other deadly diseases has been increasingly recognized, as mentioned in tables 1 and 4. many of these antibodies are specific for antigens expressed by the disease-causing agents itself [139] . in the case of viral targets, mab-based therapeutics have shown limited success. however neutralizing antibodies play an essential part in antiviral immunity and human protection against viral diseases is primarily mediated by the humoral immune response [120, 122] . it is well documented that early administration of mabs in the treatment regimen reduces mortality rate significantly up to 95% [140] . to date, only two mab is licensed for viral infection i.e. respiratory syncytial virus (rsv) and the other one is ibalizumab, which has been recently approved in 2018 for the treatment of hiv positive people with multidrug resistance towards anti-retroviral therapy (art) [18, 141] . several other candidates are at the different stages of clinical trials e.g. leronlimab, an anti-ccr5 igg4 for hiv infection and regn-eb3: a mixture of 3 igg1 mabs for ebola virus infection (https://www. antibodysociety.org/antibodies-to-watch-in-2020-at-pegs-europe/). lack of vaccines against various deadly viral diseases necessitates the development of antibody therapeutics to save the loss of lives and control deadly diseases [142] . however, it is always easy to generate monoclonal to protect the population at the time of the outbreak in a much shorter time as compared to vaccine production. though vaccines are one of the most cost-effective ways to manage infections, vaccines also require time to elicit protective immunity and depend on the host's ability to mount an immune response. a number of a prophylactic vaccine against pathogens such as; herpes simplex virus type-1 (hsv-1) and human immunodeficiency type-1 (hiv-1) have shown protection in animal immunization studies, but, so far, no effective human vaccine against these diseases are available [58, 59] . antigenic drift and high diversity among the emerging pathogens; such as influenza virus and hiv have been reported [60, 61] which further add to the complexity and may lead to vaccine mismatch drop in vaccine effectiveness against circulating serotypes and strains. in developing countries, it is not economically feasible to make a vaccine of every disease because of a lack of awareness of disease burden [62] . the mabs are widely used in the fields of diagnostic, therapeutic and biological applications due to their high specificity and affinity. at present, the majority of mabs approved for therapeutics are humanized or the chimeric versions of mouse mabs and were generated using hybridoma technology. in recent years, these engineered humanize and chimeric antibodies are potentially used to generate different forms of antibody fragments such as scfvs [143, 144] , diabodies [145] , tandom abs [146] , and domain antibodies [147] , pegylated fabs [148] to target novel antigenic sites. technical advancement in the applicability of hybridoma technology to other animal species (other than mice) of different phytogenic origin has led to the development of novel mabs to conserve human antigens. it has opened a new path for therapeutic and diagnostic mabs with high specificity and affinity to poorly immunogenic targets. with the recent development of high throughput mab generation technologies, hybridoma technology is the most favoured method due to its indigenous nature to preserve natural cognate pairing information of antibodies that is lost in other methodologies, reduces the specific diversity of antibodies [149] . advancement in recombinant dna technology methods like chimerization and humanization has increased the potential of hybridoma technology to a great extent. an antibody that undergoes the process of humanization preserves the natural specificity and limits risk of cdrs causing an immune response. the unnatural pairing of antibodies in terms of affinity maturation and recombination pairing in display methods sometimes results in high immunogenic response [19] . all these features have make hybridoma platform as first and most preferred mab isolation technology. recent advances in development of hybridoma cells secreting stereo-specific mabs have opened new avenues of future therapeutics. in comparison with other anti-viral drug treatments, a stereospecific antibody-based therapy could offer potent anti-viral actions by more comprehensive target and potent neutralizing effect. the mab market has shown tremendous increase in the last five years as a diagnostic and therapeutic reagent. the commercial development of therapeutic mabs commenced in the early 1980s, and by 1986 the first therapeutic mab was fda approved for the prevention of kidney transplant rejection. over the years mab market has changed rapidly as a major class of therapeutic agents for the treatment of many human diseases, in terms of global sales revenue for all mab products was~$115.2 billion in 2018. a graphical representation of the global antibody-based therapeutic market trend is represented in figs. 6 and 7 . the global mab therapeutics market is expected to grow at a compound annual growth rate (cagr) of 12.80% and is expected to reach market revenue of around usd 218.97 billion by the end of 2023 [150] . humanized mab accounts for the largest revenue-generating share among antibody-based therapeutics, showing to its widespread acceptance for numerous diseases including cancer, autoimmune diseases, inflammatory diseases, infectious diseases, haematological diseases, and others. the other potential area where mab has shown great success is diagnostics. the mab -based diagnostic reagents potentially identify abnormal cell targets, infectious agents, or elements 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antigen-binding proteins protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain fv analogue produced in escherichia coli diabodies: small bispecific antibody fragments bispecific tandem diabody for tumor therapy with improved antigen binding and pharmacokinetics binding activities of a repertoire of single immunoglobulin variable domains secreted from escherichia coli efficacy of a novel pegylated humanized anti-tnf fragment (cdp870) in patients with rheumatoid arthritis: a phase ii doubleblinded, randomized, dose-escalating trial veterinary sources of nonrodent monoclonal antibodies: interspecific and intraspecific hybridomas storyid=1096715878&title= global-monoclonal-antibody-therapeutics-market-to-be-worth-usd-21897-billion-by-2023: global monoclonal antibody therapeutics market to be worth usd 218.97 billion by 2023 supplementary data to this article can be found online at https:// doi.org/10.1016/j.intimp.2020.106639. key: cord-295194-xbla6tu7 authors: stripecke, renata; münz, christian; schuringa, jan jacob; bissig, karl‐dimiter; soper, brian; meeham, terrence; yao, li‐chin; di santo, james p; brehm, michael; rodriguez, estefania; wege, anja kathrin; bonnet, dominique; guionaud, silvia; howard, kristina e; kitchen, scott; klein, florian; saeb‐parsy, kourosh; sam, johannes; sharma, amar deep; trumpp, andreas; trusolino, livio; bult, carol; shultz, leonard title: innovations, challenges, and minimal information for standardization of humanized mice date: 2020-06-24 journal: embo mol med doi: 10.15252/emmm.201708662 sha: doc_id: 295194 cord_uid: xbla6tu7 mice xenotransplanted with human cells and/or expressing human gene products (also known as “humanized mice”) recapitulate the human evolutionary specialization and diversity of genotypic and phenotypic traits. these models can provide a relevant in vivo context for understanding of human‐specific physiology and pathologies. humanized mice have advanced toward mainstream preclinical models and are now at the forefront of biomedical research. here, we considered innovations and challenges regarding the reconstitution of human immunity and human tissues, modeling of human infections and cancer, and the use of humanized mice for testing drugs or regenerative therapy products. as the number of publications exploring different facets of humanized mouse models has steadily increased in past years, it is becoming evident that standardized reporting is needed in the field. therefore, an international community‐driven resource called “minimal information for standardization of humanized mice” (mishum) has been created for the purpose of enhancing rigor and reproducibility of studies in the field. within mishum, we propose comprehensive guidelines for reporting critical information generated using humanized mice. antibody-dependent cellular cytotoxicity, is an immune defense mechanism whereby effector cells such as nk cells lyses target cells that have been bound by specific antibodies aml acute myeloid leukemia art anti-retroviral therapy bdbv bundibugyo ebolavirus bite bispecific t-cell engagers is a registered trademark for a class of recombinant bispecific monoclonal antibodies which bind to the cd3 receptor and to a tumor-specific antigen blt bone marrow-liver-thymus bm bone marrow bnabs broadly neutralizing antibodies are antibodies capable of neutralizing different types of viral strains brgf balb/c rag2 à/à il2rg à/à flt3 à/à mouse strain expressing macrophage colonystimulating factors (m-csf), il-3, il-6, gm-csf, and thrombopoietin (tpo) mscs mesenchymal stromal cells myelo-ablated mice are mice treated with irradiation or chemotherapy in order to decrease the bone marrow activity in order to improve the engraftment of transplanted stem cells myelodysplasia is an abnormal accumulation of immature blood cells in the bone marrow myelofibrosis is the replacement of the bone marrow with scar tissue due to proliferation of immature blood cells nash non-alcoholic steatohepatitis nih national institutes of health nk natural killer nod non-obese diabetic nog nod.cg-prkdc scid il2rg tm1sug /jic nrgf nod-rag1 à/à il2rg à/à flk2 à/à nrg nod-rag1 tm1mom il2rg tm1wjl /szj nsg nod.cg-prkdc scid il2rg tm1wjl /szj pbmcs peripheral blood mononuclear cells pd-1 programmed death receptor 1 pd-l1 pd-1 ligand 1 pdx-mi pdx model minimal information standard pdx patient-derived xenograft pirf por à/à /il2rg à/à /rag2 à/à /fah à/à rag1 recombination activating gene studies of human stem cell engraftment, hematopoiesis, and immunity studies using immunocompetent mice have provided critical insights into the development and regulation of hematopoiesis and immunity. however, such studies do not always reflect responses in humans because of multiple species-specific differences. therefore, mice developing components of the human immune system (his) mice were created. these models have provided tools for the understanding of human hematopoiesis and immunity in vivo and to test new therapies or vaccines without incurring risks to patients. the simplest engraftment method is the adoptive administration of human peripheral blood mononuclear cells (pbmcs) into severely immunodeficient mice ( fig 1a, table 1 ). since the adoptive human t cells react forcefully against the xenogeneic major histocompatibility complex (mhc) class i and ii expressed by mouse tissues, this so-called "hupbl" model faces the hardship of fulminant xenograft graft-versus-host disease (gvhd) occurring 2-4 weeks after pbmc transfer. these models have limited applicability to follow specific antigenic responses, but can be used to test human immunosuppressive agents. improvement of the hupbl model has been described with novel mouse strains lacking mouse mhc class i and ii, resulting in lower occurrences of gvhd (yaguchi et al, 2018; brehm et al, 2019) . a more complex approach covered here in detail is the hematopoietic stem cell transplantation (hct) of preconditioned immunodeficient mice with human hematopoietic stem cells (hscs). despite the full mismatch between the human leukocyte antigens (hla) expressed on the human hematopoietic cells and the mouse mhc expressed on tissues, hct leads to "fully" humanized his models ( fig 1a, table 1 ). human hscs can differentiate into multiple human hematopoietic lineages, giving rise to mature leukocytes, including several lineages of the human immune system. robust engraftment with human hematopoietic and lymphoid cells was pioneered back in 1988 with the description of the cb17-prkdc scid severely compromised immunodeficient (scid) strain engrafted with human fetal liver hematopoietic cells and autologous thymic tissues (mccune et al, 1988) . this scid-humanized (scidhu) system showed initially only a transient presence of human t cells and human immunoglobulin g (igg) in the circulation. the critical relevance of the strain background for engraftment success of human cells was later appreciated when it was observed that non-obese diabetic (nod)-scid mice had a much higher capacity to support human hsc engraftment. this was elucidated to be due to the expression of a human-like signal regulatory protein alpha (sirpa) allele in the nod strain, popularly known as the "don't eat me signal", bypassing phagocytosis of human cells by mouse macrophages (takenaka et al, 2007; shultz et al, 2012) . targeting the interleukin 2 (il-2) receptor common gamma chain (il2rg) resulted in the absence of mouse natural killer (nk) cell activity as well as ablation of t and b lymphocyte lineages. in addition, the development of mice lacking the expression of recombination activating gene 1 (rag1) à/à and rag2 à/à provided radioresistant mouse models lacking mature host t cells as well as b cells (shultz et al, 2007) . currently, there are approximately 50 diverse humanized mouse models available from biorepositories. most of these models are homozygous for the scid, il2rg, rag1, or rag2 mutations and express the nod or human sirpa allele. the nod-scid il2rg (à/à) (nsg), the nod-rag1 à/à il2rg à/à (nrg), and the nod/shi-scid il2rg (à/à) (nog) are broadly used strains for xenografting a large variety of human cells, but several other strains are prospering (for recent reviews, see shultz et al, 2019; allen et al, 2019) . it is important to be thoughtful also about the nature of the human hscs. although humans and mice differ greatly in their biological characteristics, human hscs can essentially engraft in myelo-ablated or irradiated mice and reside in the mouse bone marrow (bm) niche. this hct approach opened several doors for the understanding of the basic properties for long-term durable repopulation of human hscs. as sources of human hscs, cord blood (cb) or fetal liver are mostly used, as they have high frequencies of hscs. generally, a range of 1 × 10 4 -10 5 isolated hscs is administered per mouse in order to enable efficient human hematopoietic engraftment and long-term reconstitution. several laboratories have opted to use fetal tissues due to the higher abundance in the numbers of hscs, which can be explored to generate larger cohorts of humanized mice (n = 30-40) compared with cord blood (n = 10-20). some groups have tried to overcome this limitation by pooling hscs from several donors, but upon development of immune systems that are not hla-matched, once the t cells develop, allograft reactions among donors can complicate the analyses of the immune responses. additionally, it is important to take into consideration that hscs in fetal and neonatal tissues may be intrinsically different regarding the stage of the hematopoietic development. further, it is important to consider ethical constraints and difficulties in procurement of human fetal tissues. in fact, the us national institutes of health (nih) is currently supporting investigators to seek and develop humanized mouse models that do not rely on human fetal tissues (allen et al, 2019) . human hsc cell surface markers have been used to allow their identification, purification, and analyses, in order to define the hsc populations with highest engraftment and/or repopulation capacity. xenotransplantation of human cd34 + hscs into preconditioned immunodeficient mice is the most broadly used procedure to generate his mice, and this approach is corroborated by the clinical evidence that transplantation with human-enriched cd34 + hematopoietic/stem/progenitor cells (hspcs) is a salvage procedure when the hla is not optimally matched between patients and donors. remarkably, a defined cd93 hi sub-fraction within the lineage negative (lin à ) cd34 à cd38 à cell present in cb has high repopulating capacity in nod-scid mice (danet et al, 2002) . cd49f is an adhesion molecule serving as a hsc marker and intra-femoral injection of single cd49f + cells into female nsg mice can generate long-term (20 weeks) multilineage grafts (notta et al, 2011) . thus, the quest for the archetypical human hsc population and whether other defined cd34 à hscs subpopulations should also be considered for the generation of his mice and how to eventually expand these cells ex vivo without compromising their self-renewal potential remains to be clarified. another aspect to be taken in account is that the ability of human hspcs to engraft and differentiate into different hematopoietic lineages may largely depend on their interactions with the mouse bm microenvironment constituents. as some human factors may be absent in the mouse bm niche, sponge scaffolds seeded with human bm-derived mesenchymal stromal cells (mscs) have been implanted subcutaneously into nsg mice to allow the formation of niches for human hscs to differentiate (antonelli et al, 2016; reinisch et al, 2016; abarrategi et al, 2017) . using two-photon microscopy for high-resolution non-invasive in vivo analyses, these implants are currently enabling the clarification of the human bm microenvironment requirements in regulating human normal and malignant hematopoiesis in vivo . another limitation in his models is the lack or low levels of human factors and cytokines in mouse tissues or circulating in the plasma and needed for human hsc self-renewal or differentiation. transgenic expression of human interleukin 3 (il-3)/granulocyte macrophage-colony-stimulating factors (gm-csf)/stem cell factor (scf) in nsg mice resulted in enhanced levels of human myeloid cells and regulatory t cells (t reg ) (billerbeck et al, 2011) . very promising models are his mice generated with a mouse strain expressing several human cytokines such as macrophage colonystimulating factors (m-csf), il-3, il-6, gm-csf, and thrombopoietin (tpo), the "mistrg-6", and showing improved human t, b, and nk cell development (das et al, 2016; yu et al, 2017) . this is a valid approach, and expression of several different human growth factors and cytokines to support differentiation of early or mature lymphoid or myeloid cells has been performed (rongvaux et al, 2014; bryce et al, 2016; jangalwe et al, 2016) . some recent development was also exemplified by transgenic expression of human thymic-stromalcell-derived lymphopoietin (tslp) that supported lymph node development in immunodeficient mice (li et al, 2018) . dendritic cells (dcs) are main orchestrators of the adaptive immune system presenting processed peptide antigens to t cells through mhc classes i/ii and expressing key costimulatory molecules such as cd40 ligand (cd40l) required for b-cell activation and class switching (steinman, 2012) . different types of dcs exist in mice but they are not homologous to human dcs. further, in his mice, the human dc development and maturation are not optimal. novel his models based on the balb/c rag2 (à/à) il2rg (à/à) flt3 (à/à) (brgf) and nod.cg-rag1 tm1mom il2rg tm1wjl/szj flk2/flt3 à/à (nrgf) mice with a mutated receptor tyrosine kinase flk2/flt3 were created. human dc development is increased in brgf and nrgf mice with exogenous administration of human flt3 ligand (flt3l) after hct, leading to a major increase also in the numbers of human nk and t cells (li et al, 2016; douam et al, 2018) . a factor to be taken into account for the generation of his is the gender of the mice. females show better hsc engraftment and faster human t-cell immune development and maturation (volk et al, 2017) . when setting up these models, it is important to keep in mind that the kinetics of human immune reconstitution is not linear and the time of analyses after hct has to be longitudinally established for different strains and methods. for example, human t cells show maturation, activation, and functionality at 15-20 weeks after cb-hct in nrg-his mice (volk et al, 2017; theobald et al, 2018) , but this varies considerably for other his models. future improvements are seeking a better development of human t cells in his mice so that they will be equipped with functional t-cell receptors (tcrs) able to interact with the matched hla complexes on antigen-presenting cells. this critical advance relies essentially on the substitution of the mouse mhc class i and ii by different hla haplotypes. to solve this mismatch problem, a transgenic nrg mouse strain called "drag" was developed that expresses hla-dr4 . drag mice transplanted with hla-dr4 + hscs developed more cd4 + t cells and higher levels of human immunoglobulins g and m (igm and igg; kim et al, 2017) . hsc-humanized mice expressing class ii hla-dr4 and class i hla-a2 transgenes ("draga" mice) generated cd8 + -specific t cells and influenza-specific antibody responses (mendoza et al, 2018a) . similarly humanized brgs mice expressing human hla-a2 and dr2 transgenes (brgsa2dr2) showed faster development of cd4 + and cd8 + t cells and higher concentration of iggs in plasma (masse-ranson et al, 2019). the practical limitation of these hla-transgenic strains is that it is difficult to find hscs that express a particular combination of hlas. another method to improve and accelerate the regeneration of human t and b cells in his mice is the adoptive transfer of geneengineered human dcs from the hsc donor that are long-lived in vivo (salguero et al, 2014; daenthanasanmak et al, 2015; volk et al, 2017) . this approach significantly enhanced the regeneration of lymph nodes in his-nrg mice promoting maturation of functional human t cells, b cell class switching, and development of antigenspecific iggs (salguero et al, 2014; daenthanasanmak et al, 2015) . as a take-home message, development of human immunity in his mice depends on several variables ( fig 1a, table 1 ). different approaches are being taken concurrently to accelerate and optimize human immune responses in mice. a structured approach to converge the reporting in scientific publications of the materials and methods (such as specific mouse strains, sex of the mice, methods used for hct, time-points of analyses) will facilitate the interactions in the community to boost these promising preclinical models (table 1 ). the liver is a vital organ responsible for key metabolic functions of the body and the site for several human-specific viral infections. for efficient generation of mice xenografted with human liver tissues, a combination of a growth disadvantage of the murine liver and a regeneration stimulus for the human cells is required. several approaches resulted in high human liver chimerism in mice (dandri et al, 2001; mercer et al, 2001; bissig et al, 2007; hasegawa et al, 2011) and the resulting models have pros and cons (reviewed in ref. bissig et al, 2018) . for example, the transgenic upa (urokinase-type plasminogen activator expressed under the albumin promoter) mouse has a profound dysfunction and triggers apoptosis of murine hepatocytes (heckel et al, 1990; dandri et al, 2001; mercer et al, 2001) . therefore, salvage human hepatocyte transplantation is required within 2-4 weeks after birth. nevertheless, humanized upa mice maintain considerable health problems. transgenic upa mice are difficult to breed, which is also a limitation of another utilized mouse strain based on nog mice expressing transgenic herpes simplex virus type 1 thymidine kinase (hsvtk) under the albumin promoter (tk-nog; hasegawa et al, 2011) . conversely, the metabolic dysfunction of the il2rg à/à rag2 à/à mice with a knock-out for the fumarylacetoacetate hydrolase gene (fah à/à ) can be regulated by a small drug and bred efficiently but the mice frequently develop murine hepatocellular cancer (azuma et al, 2007; bissig et al, 2007) . all these models show some remaining mouse liver tissue that can blur human-specific liver metabolism ( fig 1b, table 1 ). therefore, next-generation models seek to eliminate the interfering mouse metabolism. one such model is the por à/à il2rg à/à rag2 à/à fah à/à (pirf) mouse (barzi et al, 2017) , lacking murine p450 cytochrome function and allowing a human-only cytochrome metabolism in mice. human liver chimeric mice have also been used to model metabolic disorders. the first xenograft model for metabolic liver disease was established using human hepatocytes from a patient with familial hypercholesterolemia with a low-density lipoprotein (ldl) receptor deficiency; bissig-choisat et al, 2015) . it would be desirable to extend metabolic disease models also to more prevalent disorders such as non-alcoholic steatohepatitis (nash). a nash model would also require a functional immune system in addition to the liver chimerism. such dual humanizations have been achieved previously (gutti et al, 2014; strick-marchand et al, 2015; billerbeck et al, 2016; dagur et al, 2018) . the combined human liver and immune system models can show formation of fibrosis upon hepatitis c virus (hcv) or hepatitis b virus (hbv) infections (washburn et al, 2011; bility et al, 2014) . another promising approach to study human liver function is the combination of organoid technology with humanized mouse models to examine the immune response to regenerative cellular therapies and cancer. organoid technology allows the generation of unlimited numbers of non-malignant (sampaziotis et al, 2017) or cancer cells (broutier et al, 2017 ; fig 1b, table 1 ). if derived from the same hsc donor used to humanize the mice, this approach can potentially be used to compare the immunogenicity of autologous and allogeneic cellular therapies or investigation of safety and efficacy of autologous cancer immunotherapies. studies of type 1 diabetes (t1d) are also prospering with the use of humanized mice. backcrossing the insulin 2 (akita) mutation into nrg mice (nrg-akita) followed by human hct into newborn mice resulted in > 50% of the nrg-akita mice rejecting human islet allografts (brehm et al, 2010 ; fig 1b, table 1 ). the akita model was also used to demonstrate the efficacy of stem cell-derived human beta cells (sc-beta) to regulate blood glucose levels in vivo. the effect of viral infections was also established for studies of t1d, showing that coxsackievirus b accelerated the destruction of insulinproducing beta cells of pancreatic islets (gallagher et al, 2015) . his models are currently being developed to recapitulate the course of disease in human t1d, including the interactions between human immune system and beta cells (tan et al, 2017; walsh et al, 2017) . recent studies have included engraftment of mice with diverse hematopoietic and non-hematopoietic human tissues and cell populations, human-induced pluripotent (ips) stem cells and embryonic stem (es) cell-derived tissues (shultz et al, 2014) . exciting advances on the development and use of emerging humanized mouse models in multiple disciplines are ongoing (fig 1b, table 1 ). technical and analytical annotation and standardization strategies to harmonize the use of human tissues implanted into humanized mice will be needed (table 1) . infections with human-specific pathogens his mice offer a unique possibility to study infectious disease agents with a tropism toward human leukocytes, hepatocytes, and lung ª 2020 the authors embo molecular medicine 12: e8662 | 2020 epithelia, to characterize the induced immune responses and to develop therapeutic interventions against associated pathologies ( fig 1c, table 1 ). one such pathogen is the epstein-barr virus (ebv), a common c-herpesvirus that persistently infects more than 95% of the human adult population and was the first oncogenic virus identified in man (mü nz, 2019). accordingly, it is associated with around 2% of all malignancies in humans (cohen et al, 2011) . despite the threat of primarily b and epithelial cell transformation in infected individuals, ebv remains asymptomatic in most carriers, presumably due to a near perfect immune control of the virus by cytotoxic lymphocytes (taylor et al, 2015) . his mice can model this cell-mediated immune control by primarily cd8 + t lymphocytes (mchugh et al, 2019) . ebv infection resulted in dramatic cd8 + t-cell expansion in humanized mice with a peculiar phenotype (chatterjee et al, 2019; danisch et al, 2019) . the expanding cd8 + t cells carried the programmed death receptor (pd)-1 and t-cell immunoglobulin and mucin domain-containing protein 3 (tim-3) but retained cytokine production and were even superior in cytotoxicity to pd-1 negative cd8 + t-cell populations. nonetheless, pd-1 blockade with antibodies did not improve ebvspecific immune control in his mice (chatterjee et al, 2019) . in contrast, pd-1 inhibition led to elevated ebv titers, increased il-10 production, and associated lymphomagenesis. long-term infection of his mice with another herpes virus, the human cytomegalovirus (hcmv) is also possible as human cd34 + cell serves as a latent reservoir, whereas lytic reactivation in monocytes and macrophages can be stimulated with granulocyte-colony-stimulating factor (g-csf) (for a review, see koenig et al, 2020) . hcmv infection and reactivation result in different immunological responses and reactivation is associated with a higher pd-1 expression on t cells (theobald et al, 2018) . other pathogens that challenge immune compromised humans, especially pediatric patients after hct, are human adenoviruses. human adenovirus 2 (hadv2) infection of his mice resulted in liver pathology in one-third of mice, while two-thirds of infected mice remained asymptomatic (rodriguez et al, 2017) . his mice with asymptomatic hadv2 infection developed virus-specific igm and interferon (ifn)-c-producing t-cell responses. in blood and bm of mice not showing pathology, only early viral rna transcripts could be detected, which suggested the establishment of a persistent infection. in contrast, severely affected mice showed both early and late transcripts in many tissues as well as virus production in the liver (rodriguez et al, 2017) , all signs of disseminated disease, similar to what is observed in hct patients that suffer severe hadv infections (lion, 2014) . viruses for which dichotomous outcomes of infection can be modeled in his mice are the filoviruses of the genus ebolavirus his mice also offer platforms to explore new therapeutic avenues. this has primarily been investigated for human immunodeficiency virus (hiv; marsden & zack, 2017) . for example, treatment of hiv infection with broadly neutralizing antibodies (bnabs) was established in his mice (klein et al, 2012) . these studies primarily explored antibodies against four regions of the hiv envelope protein that consists of three heterodimers of glycoprotein (gp)41 and gp120 (caskey et al, 2019) . these four regions are the cd4 binding site, the v3 loop, the membrane proximal region, and the v1/v2 region. in these bnab treatment studies, mutational escape from single bnab treatment of viremic his mice was observed, while mixtures of several bnabs were able to suppress hiv viral titers for several weeks (klein et al, 2012) . based on these successful treatments in his mice and control of hybrid simian and human immunodeficiency virus (shiv) in macaques (nishimura et al, 2017) , hiv-specific bnabs were also tested in patients with and without prior anti-retroviral therapy (art; caskey et al, 2015 caskey et al, , 2017 . similar to his mice, individual bnab treatment suppressed hiv viremia only transiently with escape mutation development. even transfer of two bnabs only achieved suppression in hivinfected individuals after prior art treatment (bar-on et al, 2018; mendoza et al, 2018b) . thus, multiple bnabs probably need to be maintained at sufficient plasma levels to suppress hiv long term in his mice and patients. improving t cell-mediated immune control of hiv through gene therapy is another avenue that is explored in his mice. studies fall into two main categories, either improving t-cell reactivity by hivspecific tcr and chimeric antigen receptor (car) expression or rendering t cells resistant to hiv infection . for improving t-cell function, hiv-specific tcrs were introduced in hscs and could suppress hiv infection (kitchen et al, 2012) . as another strategy, in order to target cells replicating hiv and with gp120 surface expression, an extracellular domain of cd4 fused to the cd3f signaling domain was expressed in t cells (zhen et al, 2015 . downregulation of the hiv co-receptor, a chemokine receptor targeted by r5 tropic hiv strains (ccr5), has primarily been explored. hiv infection in his mice could be significantly compromised by ccr5 deletion or downregulation by zinc finger nuclease-mediated gene editing or rna silencing, respectively (holt et al, 2010; myburgh et al, 2015; shimizu et al, 2015) . the longterm survival of such engineered t cells might potentiate a functional cure of hiv, but also raises concerns with respect to toxicities and adverse effects of the respective cellular products, which can be assessed in his mice. engraftment of additional human tissues, like liver, bone, lung, and thymus, has been reported, but mainly hepatocytes and human lung tissue have been explored for infections with human pathogens. for example, humanized liver mouse models have been used to study infections by different hepatitis viruses (dandri et al, 2001; mercer et al, 2001; bissig et al, 2010; lutgehetmann et al, 2012; allweiss et al, 2016) . however, simultaneous reconstitution of human tissues with autologous human immune cells remains a challenge. nevertheless, hbv and hcv infections have been explored in his mice with allogeneic or autologous hepatocyte engraftment (washburn et al, 2011; billerbeck et al, 2016; dusseaux et al, 2017) . more recently, bone marrow, liver, and thymus (blt) engrafted mice have also been combined with ectopic human lung transplants (wahl et al, 2019) . intra-organ infection of these 8 of 16 embo molecular medicine 12: e8662 | 2020 ª 2020 the authors animals with different human-specific viruses (middle east respiratory syndrome-related coronavirus (mers), zika virus, respiratory syncytial virus (rsv), and hcmv) showed virus replication within lung implants, as well as antigen-specific humoral and t-cell responses. further studies in this direction are necessary to widen the application of humanized mice to additional human pathogens and immune responses against them. these examples of the use of humanized mice to recapitulate human infections and associated immune responses illustrate the vigor and translational value of these models. standardization of the reporting will improve the interpretation of results for single infections and for cross-reference among the different pathogens studied (table 1) . over the past decades, mouse xenograft models have significantly contributed to a better understanding of human malignancies. cancer-derived immortalized cell lines can adapt to in vitro growth and do not replicate the original malignant physiology seen in patients, potentially leading to artifacts in oncology studies. thus, patient-derived xenograft (pdx) models are currently the state-ofthe-art approach. development of liquid and solid pdx models relies on the availability of material obtained from patients with defined types of cancer, which after minimal manipulation is transferred by several routes into immunodeficient mice (fig 1d, table 1 ). nevertheless, whereas cell line-based xenografts allow an easier standardization of models, pdx samples are highly variable, can adapt to the murine environment and the human tumor stroma can be eventually replaced by murine cells. for leukemia research, while the generation of immunodeficient mouse strains like nsg has enabled functional in vivo studies on human hematopoiesis, engraftment of (in particular) myeloid malignant cells has remained challenging. the absence of a human bone marrow niche and species-specific growth factors underlies these challenges, and most notably, it has been difficult to maintain selfrenewal properties of malignant stem cells. since these populations are thought to be the therapy-resistant cells that frequently cause relapse of disease, it is of critical importance to use xenograft models in which specifically these cells can be propagated and stemness maintained. in order to further humanize xenograft models, transgenic and knock-in strains have been generated that (over)express growth factors like il-3, gm-csf, scf, tpo, and/or m-csf, as well as others (wunderlich et al, 2010; rongvaux et al, 2014 ). an alternative approach has been to develop a human microenvironment in the mouse initiated by mesenchymal stem cells coated on 3d scaffolds (antonelli et al, 2016; abarrategi et al, 2017; carretta et al, 2017) or embedded in matrigel (reinisch et al, 2016) . these models have allowed the engraftment of various hematological malignancies, including those that are notoriously difficult to engraft in regular nsg models such as low-risk acute myeloid leukemia (aml), myelodysplastic syndrome (mds), and myelofibrosis. importantly, self-renewal was better maintained in these models as shown by serial transplantation experiments and transcriptome studies. as such, these models more faithfully capture the disease phenotypes as seen in human patients and therefore are likely to produce more clinically relevant and translatable results when used in drug screens. a challenge that remains is that myeloid malignancies, in particular aml, display a complex clonal heterogeneity. multiple genetically distinct subclones can co-exist within an individual patient, each driven by a similar founder mutation but with different secondary driver mutations. these clones are not only genetically distinct; they also differ remarkably at the transcriptome, epigenome, and cell biological level (de boer et al, 2018) . to develop curative therapies, this clonal heterogeneity needs to be taken into account. it has become clear that not all clones of an individual patient might engraft equally efficiently in mice, and also the level of humanization of the model used might impact on whether the true clonal heterogeneity is preserved in vivo (klco et al, 2014; antonelli et al, 2016; carretta et al, 2017; wang et al, 2017; de boer et al, 2018) . both (deep) sequencing technologies and flow cytometry-based approaches are useful tools to dissect clonal heterogeneity, in vitro as well as in mouse models. for instance, an "infinicyt"based approach, which combines expression profiles of multiple aberrant aml-specific plasma membrane proteins, can provide subclone-specific insights into the clonal complexity of the malignancy under study (de boer et al, 2018) . implementation of such technologies is warranted in any conducted in vivo xenograft experiment to link drug responses to specific genetic features of malignant clones. in the case of solid tumors, the use of pdxs for preclinical drug development holds potential to improve our knowledge of the principles underlying responsiveness to individualized treatment regimens (hidalgo et al, 2014; byrne et al, 2017 ). yet, many questions are still open, in particular concerning the ability of the pdx approach to directly influence clinical decision making (aparicio et al, 2015) . not all cells that compose the parental tumor successfully engraft in the mouse, which introduces a selective pressure for genetic variants conferring better survival fitness (ben-david et al, 2017) . the subsequent propagation steps may also affect clonal dynamics, with further deviation of serially passaged samples from the primary tumor from which they were derived (eirew et al, 2015) . the lack of a fully functional immune system in the host and the fact that human stromal components -such as cancer-associated fibroblasts, endothelial cells, and inflammatory cells-are replaced by murine counterparts add extra layers of divergence over native tumors (hidalgo et al, 2014; aparicio et al, 2015; byrne et al, 2017) . these limitations notwithstanding pdx models of solid tumors offer considerable opportunities for biomarker and target nomination. first, although serially passaged pdxs are likely to be genetically different from the matched tumor of their donor patients, they are expected to display genomic makeups and polyclonality patterns that, on a probabilistic basis, may be similar to those of tumors that spontaneously develop in unrelated individuals (eirew et al, 2015; byrne et al, 2017) . these factors make pdxs critical tools in the translational oncology domain, whereby predictive biomarkers discovered in pdxs may be leveraged for the prospective identification of patients with tumors exhibiting the same biomarker repertoire. second, responses to therapies that target driver oncoproteins are thought to be only partly influenced by microenvironmental parameters and more directly dependent on cancer cell-intrinsic features, which affords results in pdxs with adequate predictive power for cancer cell-directed treatments (byrne et al, 2017) . finally, vast pdx collections are poised to capture inter-patient tumor diversity on a population scale, thus representing powerful platforms for large-scale genotype-response associations (gao et al, 2015) . the above examples illustrated the importance of pdx models in understanding the evolution of tumor growth, investigating the mechanisms of drug resistance, and developing personalized treatments. critical to these studies is ensuring researchers have access to high-quality pdx models and molecular datasets that give sufficient power to perform informative analyses. however, the complex nature of pdx models and the heterogeneous resources that generate them often lead to crucial information about tumors, host strains, transplant, and quality assurance processes being inconsistently presented. to address this challenge, the pdx research community developed the pdx model minimal information standard (pdx-mi) that defines the critical metadata needed to exchange knowledge about pdx models (meehan et al, 2017) . pdx-mi describes the clinical attributes of a patient's tumor, the processes of implantation and passaging of tumors in a host mouse strain, quality assurance methods, and the use of pdx models in cancer research. since its inception, pdx-mi has been adopted by producers of pdx models including the international pdxnet and europdx consortia as well as the pdx finder catalog that captures, harmonizes, and disseminates data about pdx models and associated omic datasets (conte et al, 2019) . pdx-mi promotes reuse of models and data, maximizing the impact of these models on oncology research and facilitating the development of new treatments. therapeutic modulation of the human immune system to improve recognition and response to tumors is a clinically accepted revolution in oncology treatment for multiple tumor types (pardoll, 2012) . immuno-oncology (io) is considered a breakthrough due to significant and durable tumor regression coupled with increased long-term survival. however, these clinical responses only occur in a subset of patients. therefore, considerable investment in preclinical research is still necessary to identify new and improved approaches to cancer cell-specific immune response as well as testing of combinatorial strategies. these and other approaches are typically developed in syngeneic mouse models of oncology to work out mechanisms of action. nonetheless, human-specific immune modulators require in vivo models with human-specific targets on both human immune cells and human tumors to validate preclinical responses, accelerate development, and improve translation to the clinic. ideally, io in vivo studies will rely on the combination of his and pdx models. as described in the following sections, his mice co-engrafted with human tumors are proving to be a valuable tool in the development of new strategies for human-specific immuno-oncology therapies. nonetheless, as these models are per se complex and the matching of the tumor and immune system from the same patient is currently a difficult task, several studies explore cell line derived xenograft (cdx) implanted after humanization in his mice or administration of human peripheral blood lymphocytes (pbl; fig 1e, table 1 ). these io studies have been used, for example, to test immune modulation caused by engineered agonistic or antagonistic monoclonal antibodies (mab; scott et al, 2012) , bispecific t-cell engagers (bite; baeuerle & reinhardt, 2009) , or t-cell bispecific antibodies (tcb; bacac et al, 2018) . a very important and commonly asked question regarding human tumor cell engraftment in his mice is whether the cdx or pdx tumor and hematopoietic donor must be 100% hla-matched to allow co-engraftment. neonatal irradiated nsg mice co-injected into the liver with cd34 + cord blood-derived hsc and hlamismatched human breast cdx, resulted in the development of a human immune system together with human tumor growth, including metastases in the lung and brain (wege et al, 2011 (wege et al, , 2014 . tumors were partially infiltrated with t cells, b cells, and myeloid cells. more detailed analyses of the spleen revealed not only a t-cell specific activation pattern but also b-cell maturation and the production of tumor-specific antibodies (wege et al, 2014) . moreover, the tumor engrafted his mice were used for a preclinical trial to test the potential of il-15 in combination with trastuzumab (anti-her2 mab) therapy with the intention to enhance antibody-dependent cellular cytotoxicity (adcc; wege et al, 2017) . il-15 treatment triggered immune activation and promoted tumor depletion but also induced systemic inflammation, resulting in death of the treated mice (wege et al, 2017) . in another study, 3-week-old nsg mice were engrafted with cord blood-derived cd34 + hsc first to establish mature multilineage immune engraftment and then injected subcutaneously 12-16 weeks later with partially hla-matched pdx tumors (wang et al, 2017) . despite the presence of a wide range of functional immune cells, the his mice were capable of engrafting the tumors and in many cases the growth kinetics of these tumors did not vary significantly from immunodeficient controls not engrafted with hsc. however, not all partially hla-matched tumor/hsc donor combinations escape immune-mediated changes in growth kinetics and some tumors are rejected, highlighting the importance of empirically testing tumor growth against multiple hsc donors. subcutaneously engrafted tumors were infiltrated with a wide range of human innate and adaptive immune cells and both the frequency and distribution of immune cell types varied across different tumor types (wang et al, 2017) . one mechanism known to prevent t cells from responding to tumors is the pd-1 and its ligand (pd-l1) checkpoint pathway. the clinically approved checkpoint inhibitor pembrolizumab (anti-pd-1 mab) has been tested in tumor-bearing his mice and suppression of tumor growth was observed using both cdx and pdx tumors (wang et al, 2017) . suppression of tumor growth with pembrolizumab only occurred in mice co-engrafted with human immune cells and the response was abrogated when mice were pretreated with anti-human cd8 mab to deplete human cd8 + t cells, demonstrating human cd8 + t cells mediated the effector response following release from checkpoint inhibition. efficacy studies with pembrolizumab were run with multiple hsc donors distributed among both control and treatment arms of each tumor tested for response. multiple hsc donors allowed the observation that not all tumor/hsc combinations show a response to pembrolizumab, and the frequency of donor-related response (~25-30%) is similar to what is observed in the clinic (topalian et al, 2012) . regulatory t cells (tregs) infiltrate a wide range of tumor types and mechanism of action studies performed in syngeneic mouse tumor models revealed that depletion of these cells from the tumor could release t effector cells from treg suppression (smyth et al, 2014) . preclinical efficacy for this approach was demonstrated when his mice were engrafted with sk-mel-5 human melanoma cdx and treated with an anti-human mab targeting the glucocorticoidinduced tnfr family-related (gitr) protein, highly expressed on tregs. tumor growth was significantly suppressed, the percentage of tregs was reduced in tumor and spleen, and tumor-infiltrating lymphocytes showed increased secretion of the effector cytokines il-2 and ifn-c (mahne et al, 2017) . as more io treatments move through clinical trials, clinicians are seeing an association between strong immune-mediated tumor killing responses and cytokine release syndrome (crs). given these observations, preclinical studies able to recapitulate crs in his mice are becoming highly relevant. in a recent report, his mice were co-engrafted with a diffuse large b-cell lymphoma (wsu-dlcl2) and treated with either obinutuzumab (anti-cd20 mab) or a novel cd20-t-cell bispecific antibody (tcb) containing two cd20 binding domains and one cd3e domain in a head-to-tail orientation to one of the cd20 regions (bacac et al, 2018) . cd20-tcb promoted a more extensive killing response than obinutuzumab. further, cd20-tcb administration was associated with increased expression of multiple human inflammatory cytokines indicating a crs response that was not observed with obinutuzumab treatment. an alternate strategy was tested where treatment was initiated with a single dose of obinutuzumab followed by multiple high doses of cd20-tcb. the pretreatment with obinutuzumab strategy enabled rapid and extensive tumor killing with minimal crs response. these types of preclinical experiments with his mice demonstrate their value in working out protocols designed to maximize both efficacy and safety. the question of immuno-therapy-mediated toxicity, particularly in the context of crs, is a key component of preclinical evaluation and a reliable assay is needed. neither in vitro assays nor nonhuman primates have proven reliable for assessment of crs (stebbings et al, 2007) . a team at the us food and drug administration recently published two reports testing crs using mab therapies known to have a strong cytotoxic response in the blt-his mice (yan et al, 2019a,b) . blt mice were injected with adalimumab (anti-tnf-a mab) as a negative control because it is used clinically without evidence of crs. the test article was tgn1412 (anti-cd28 mab), a reagent known to be associated with clinical crs. tgn1412-treated blt mice released multiple cytokines into peripheral blood within 2-4 hours of treatment, indicating a strong crs response that was not observed in the adalimumab-treated group (yan et al, 2019a) . the mice also showed a decrease in human cd45 + , cd3 + , cd4 + , cd8 + , and cd19 + cells in peripheral blood similar to human patients and showed an increase in murine serum amyloid a, indicating severe liver inflammation. a second study compared muromonab (anti-cd3 mab, okt3) to adalimumab in blt mice. the muromonab-treated mice released multiple proinflammatory cytokines associated with crs into peripheral blood within 2-4 h, and pretreatment of mice with methylprednisolone prior to muromonab blunted or delayed development of crs (yan et al, 2019b) . together, these studies show that blt-his mice are capable of recapitulating multiple aspects of a strong crs response when dosed with mab therapies designed to stimulate strong t-cell activation. to our knowledge, no direct comparisons between pbmc-his and blt-his model mice have been made to date. there are several published studies using pbmc-his mice demonstrating crs (brady et al, 2014; weissmuller et al, 2016) with the mice being used between 6 and 16 days of pbmc injection. in both reports, the authors state there were no signs of gvhd present when used. in yan et al (2019a) , a limited comparison of cd34-his mice and blt-his mice was undertaken and blt-his mice showed clear evidence of crs and no gvhd, whereas cd34-his mice did not show any difference as compared to control treatment, suggesting that the cd34-his mouse does not show a clear signal for crs and may not be an appropriate model. as previously published for the blt-his mouse (weaver et al, 2019) , when present, gvhd is clearly evident and can be differentiated from other processes, such as crs. gvhd occurs rapidly and for all pbmc-his mice, but does not occur with high frequency in blt-his mice. with respect to crs, pbmc-his mice would potentially demonstrate crs for drugs specifically impacting t cells, but not other tissues or cell types. blt-his mice have much broader engraftment in terms of cell types and presence in non-lymphoid organs, suggesting that a wider range of targets could demonstrate crs if present. for preclinical use, the stebbings in vitro assay (stebbings et al, 2007) should be initially undertaken. studies with his mice would be adjunctive and informed by both the target of the therapeutic and what organ system(s) were targeted. additional circumstances in which in vivo testing could be helpful include higher risk drug targets and non-t-cell targets. in conclusion, his mice are a powerful tool for io research. his mice do not recapitulate every aspect of human immunity, but they are capable of answering a wide range of important scientific questions that form a critical guide for preclinical io discovery. future challenges will include the understanding of the donor-to-donor immune variability observed in some of these treatment strategies. this will provide opportunities to identify predictive markers and clinical diagnostics assays helpful in assisting patient enrollment for improved treatment outcomes. tumor populations that escape response in subsets of mice can be further analyzed for understanding mechanism of resistance. humanized mouse models result from the sum of several components: choice and availability of human donors, human cells or tissues, mouse recipient, types of manipulations, human infections, and human tumor types. furthermore, the materials available for analyses and methods of analyses provide another level of complexity. it is quite clear that humanized mouse models are customized by the different laboratories around the globe and it would be unrealistic to standardize how the models should be built. nevertheless, reporting of minimal information provided by specific guidelines can facilitate independent validation of published data, which is a fundamental cornerstone for scientific advancements. the list of variables provided in table 1 is an initial attempt brought up by the faculty participating in the 2017 and 2019 embo practical courses created for training young investigators on the development of humanized mouse models. this initiative called "minimal information for standardization of humanized mouse models" (mishum) is built on the experience of the authors of this manuscript in developing similar reporting standards for non-humanized pdx models (meehan et al, 2017) . the main aim of this workgroup is, similar to pdx-mi, to promote material and protocol exchange, transparency in reporting of assays and analyses performed. the standards described here represent a starting point. the longer term intention is to extend this initiative beyond the embo courses to include input from multiple stakeholders from both academia and industry. further, in order to enable a standardized interpretation of already published results, the collection of technical information and quantifiable data can in the future be procured within mishum for the creation of a digitalized database. once these standards and database are evolving, a parallel data mining activity will provide the opportunity to explore and discover convergent signatures and patterns of human immunology, infections, and oncology in vivo in humanized mice. our long-term goal is that, in the future, these models will become largely reproducible and predictive models for the understanding of human physiology, immunology, and oncology, which require a living experimental system. beyond the gain of scientific information, we hope to make humanized mouse models more environmentally sustainable by optimizing the methods and reducing the number of humanized mice used for experimentation. ultimately, we seek to support the "3r" principles: (i) replace the use of humanized mice if alternative in vitro techniques (such by the use of organoids or chips), metadata or data mining eventually prove to be as solid as the in vivo results; (ii) reduce the number of humanized mice to a minimum within each experimental cohort; and (iii) refine the experimental setup using the best and ethically available human material and analyses, also making sure that invasive approaches can be minimized to mitigate the suffering of the animals. only with a consensus checklist and with a coherent reporting policy, we will be able to identify the best material, methods, and analyses that will ultimately lead to optimized humanized mouse models. below is a summary of main topics identified by our community that remain to be solved on a case-by-case basis depending on the use of the humanized mouse model: human hematopoiesis and immunity: methods to expand human hscs will enable larger experimental cohorts. novel methods for matching the hla between human hematopoietic cells and mouse epithelial cells will improve human t-cell development in a hlarestricted manner. better development of lymph nodes and germinal centers within the spleen that would improve innate and adaptive immune responses. novel mouse strains or methods allowing regeneration of lymph nodes will allow b cell class switch and production of human high-affinity igg and iga antibodies. human metabolism: mice and human display different rates and pathways of metabolism, and particularly for liver metabolism, it is essential to address this limitation of humanized mice. since many metabolites are diffusible, new models are needed that can eliminate or temporarily block murine metabolism while using chimeric mice. human infections: engraftment of human peripheral tissues (i.e., liver, lung, skin, and brain) will allow infections with human pathogens targeting other tissues than immune cells, and the possibility of combining them with hlas matched human hematopoietic stem cells will create a more complete model of human infection. better engraftment of the human erythrocyte lineage will allow further studies of erythrocyte-infecting pathogens (i.e., plasmodium). human oncology: further humanization of xenograft mouse models such as implantation of human msc-coated 3d scaffolds or nsg mice (over) expressing human cytokines has improved engraftment rates of primary tumor cells. however, for each individual cancer patient case, it will have to be established which (sub)clones preferably grow out. also, it will need to be carefully evaluated how the transcriptome and epigenome of the original patient samples are preserved in the pdx models. human immuno-oncology: the hla matching of the human immune system and tumor will be required. a functional enhancement of the cd4 + and cd8 + t cells will be needed to allow higher tumor infiltration and anti-tumor responses. an improved and faster development of "avatar" pdx models will allow decisions about personalized therapeutic options. (iii) https://www.mdpi.com/journal/vaccines/special_issues/humanized_mice in this special issue "humanized mice in vaccinology: opportunities and challenges", aspects related to the use of humanized mice in vaccinology, opportunities, and the challenges ahead are discussed. (iv) http://www.pdxfinder.org/pdx-standard/ the pdx minimal information document represents the results of a broad community effort to develop a standard regarding the essential information needed to describe a pdx model. versatile humanized niche model enables study of normal and malignant human hematopoiesis humanized immune system mouse models: progress, challenges and opportunities human liver chimeric mice as a new model of chronic hepatitis e virus infection and preclinical drug evaluation establishing human leukemia xenograft mouse models by implanting human bone marrow-like scaffold-based niches examining the utility of patientderived xenograft mouse models robust expansion of human hepatocytes in fah à/à /rag2 à/à /il2rg à/à mice cd20-tcb with obinutuzumab pretreatment as next-generation treatment of hematologic malignancies bispecific t-cell engaging antibodies for cancer therapy safety and antiviral activity of combination hiv-1 broadly neutralizing antibodies in viremic individuals a novel humanized mouse lacking murine p450 oxidoreductase for studying human drug metabolism patient-derived xenografts undergo mouse-specific tumor evolution hepatitis b virus infection and immunopathogenesis in a humanized mouse model: induction of human-specific liver fibrosis and m2-like macrophages development of human cd4+ foxp3+ regulatory t cells in human stem cell factor-, granulocyte-macrophage colony-stimulating factor-, and interleukin-3-expressing nod-scid il2rgamma(null) humanized mice humanized mice efficiently engrafted with fetal hepatoblasts and syngeneic immune cells develop human monocytes and nk cells repopulation of adult and neonatal mice with human hepatocytes: a chimeric animal model human liver chimeric mice provide a model for hepatitis b and c virus infection and treatment p450-humanized and human liver chimeric mouse models for studying xenobiotic metabolism and toxicity development and rescue of human familial hypercholesterolaemia in a xenograft mouse model prospective isolation and characterization of genetically and functionally distinct aml subclones preclinical screening for acute toxicity of therapeutic monoclonal antibodies in a hu-scid model human immune system development and rejection of human islet allografts in spontaneously diabetic nod-rag1null il2rgammanull ins2akita mice lack of acute xenogeneic graft-versus-host disease, but retention of t-cell function following engraftment of human peripheral blood mononuclear cells in nsg mice deficient in mhc class i and ii expression human primary liver cancer-derived organoid cultures for disease modeling and drug screening humanized mouse model of mast cell-mediated passive cutaneous anaphylaxis and passive systemic anaphylaxis interrogating open issues in cancer precision medicine with patient-derived xenografts genetically engineered mesenchymal stromal cells produce il-3 and tpo to further improve human scaffold-based xenograft models new approaches for the enhancement of chimeric antigen receptors for the treatment of hiv viraemia suppressed in hiv-1-infected humans by broadly neutralizing antibody 3bnc117 antibody 10-1074 suppresses viremia in hiv-1-infected individuals broadly neutralizing anti-hiv-1 monoclonal antibodies in the clinic cd8 + t cells retain protective functions despite sustained inhibitory receptor expression during epstein-barr virus infection in vivo epstein-barr virus: an important vaccine target for cancer prevention pdx finder: a portal for patient-derived tumor xenograft model discovery engineered dendritic cells from cord blood and adult blood accelerate effector t cell immune reconstitution against hcmv human hepatocyte depletion in the presence of hiv-1 infection in dual reconstituted humanized mice repopulation of mouse liver with human hepatocytes and in vivo infection with hepatitis b virus c1qrp defines a new human stem cell population with hematopoietic and hepatic potential spatiotemporally skewed activation of programmed cell death receptor 1-positive t cells after epstein-barr virus infection and tumor development in long-term fully humanized mice microenvironment-dependent growth of preneoplastic and malignant plasma cells in humanized mice selective expansion of myeloid and nk cells in humanized mice yields human-like vaccine responses viral load affects the immune response to hbv in mice with humanized immune system and liver dynamics of genomic clones in breast cancer patient xenografts at single-cell resolution comparative pathogenesis of ebola virus and reston virus infection in humanized mice viral infection of engrafted human islets leads to diabetes high-throughput screening using patientderived tumor xenografts to predict clinical trial drug response human hepatocytes and hematolymphoid dual reconstitution in treosulfan-conditioned upa-nog mice the reconstituted 'humanized liver' in tk-nog mice is mature and functional neonatal bleeding in transgenic mice expressing urokinase-type plasminogen activator patient-derived xenograft models: an emerging platform for translational cancer research human hematopoietic stem/ progenitor cells modified by zinc-finger nucleases targeted to ccr5 control hiv-1 in vivo improved b cell development in humanized nod-scid il2rgamma(null) mice transgenically expressing human stem cell factor, granulocytemacrophage colony-stimulating factor and interleukin-3 tracking human immunodeficiency virus-1 infection in the humanized drag mouse model in vivo suppression of hiv by antigen specific t cells derived from engineered hematopoietic stem cells functional heterogeneity of genetically defined subclones in acute myeloid leukemia hiv therapy by a combination of broadly neutralizing antibodies in humanized mice modeling human cytomegalovirus in humanized mice for vaccine testing a novel flt3-deficient his mouse model with selective enhancement of human dc development a human immune system mouse model with robust lymph node development adenovirus infections in immunocompetent and immunocompromised patients ebola virus disease in mice with transplanted human hematopoietic stem cells humanized chimeric upa mouse model for the study of hepatitis b and d virus interactions and preclinical drug evaluation dual roles for regulatory t-cell depletion and costimulatory signaling in agonistic gitr targeting for tumor immunotherapy humanized mouse models for human immunodeficiency virus infection accelerated thymopoiesis and improved t-cell responses in hla-a2/-dr2 transgenic brgs-based human immune system mice the scid-hu mouse: murine model for the analysis of human hematolymphoid differentiation and function infection and immune control of human oncogenic gamma-herpesviruses in humanized mice pdx-mi: minimal information for patient-derived tumor xenograft models generation and testing anti-influenza human monoclonal antibodies in a new humanized mouse model combination therapy with anti-hiv-1 antibodies maintains viral suppression hepatitis c virus replication in mice with chimeric human livers latency and lytic replication in the oncogenesis of the epstein barr virus lentivector knockdown of ccr5 in hematopoietic stem and progenitor cells confers functional and persistent hiv-1 resistance in humanized mice early antibody therapy can induce long-lasting immunity to shiv isolation of single human hematopoietic stem cells capable of long-term multilineage engraftment the blockade of immune checkpoints in cancer immunotherapy bioengineering of humanized bone marrow microenvironments in mouse and their visualization by live imaging a humanized bone marrow ossicle xenotransplantation model enables improved engraftment of healthy and leukemic human hematopoietic cells humanized mice reproduce acute and persistent human adenovirus infection development and function of human innate immune cells in a humanized mouse model dendritic cellmediated immune humanization of mice: implications for allogeneic and xenogeneic stem cell transplantation reconstruction of the mouse extrahepatic biliary tree using primary human extrahepatic cholangiocyte organoids antibody therapy of cancer rnai-mediated ccr5 knockdown provides hiv-1 resistance to memory t cells in humanized blt mice humanized mice in translational biomedical research humanized mice for immune system investigation: progress, promise and challenges human cancer growth and therapy in immunodeficient mouse models humanized mouse models of immunological diseases and precision medicine targeting regulatory t cells in tumor immunotherapy cytokine storm" in the phase i trial of monoclonal antibody tgn1412: better understanding the causes to improve preclinical testing of immunotherapeutics decisions about dendritic cells: past, present, and future e8662 | 2020 human hepatocytes polymorphism in sirpa modulates engraftment of human hematopoietic stem cells type 1 diabetes induction in humanized mice the immunology of epstein-barr virus-induced disease signatures of t and b cell development, functional responses and pd-1 upregulation after hcmv latent infections and reactivations in nod.rag.gamma mice humanized with cord blood cd34(+) cells safety, activity, and immune correlates of anti-pd-1 antibody in cancer multidimensional analysis integrating human t-cell signatures in lymphatic tissues with sex of humanized mice for prediction of responses after dendritic cell immunization precision mouse models with expanded tropism for human pathogens humanized mouse models of clinical disease patient-derived xenotransplants can recapitulate the genetic driver landscape of acute leukemias a humanized mouse model to study hepatitis c virus infection, immune response, and liver disease blt-immune humanized mice as a model for nivolumab-induced immune-mediated adverse events: comparison of the nog and nog-exl strains humanized tumor mice-a new model to study and manipulate the immune response in advanced cancer therapy co-transplantation of human hematopoietic stem cells and human breast cancer cells in nsg mice: a novel approach to generate tumor cell specific human antibodies il-15 enhances the anti-tumor activity of trastuzumab against breast cancer cells but causes fatal side effects in humanized tumor mice (htm) tgn1412 induces lymphopenia and human cytokine release in a humanized mouse model aml xenograft efficiency is significantly improved in nod/ scid-il2rg mice constitutively expressing human scf, gm-csf and il-3 human pbmc-transferred murine mhc class i/ii-deficient nog mice enable long-term evaluation of human immune responses evaluation of a tgn1412 analogue using in vitro assays and two immune humanized mouse models bone marrow-liverthymus (blt) immune humanized mice as a model to predict cytokine release syndrome a novel humanized mouse model with significant improvement of class-switched, antigen-specific antibody production hiv-specific immunity derived from chimeric antigen receptor-engineered stem cells long-term persistence and function of hematopoietic stem cell-derived chimeric antigen receptor t cells in a nonhuman primate model of hiv/aids license, which permits use, distribution and reproduction in any medium, provided the original work is properly cited we thank the european molecular biology organization (embo) and the jackson laboratory (jax) for funding, and we thank the staff of the euro key: cord-308461-4lhh3du0 authors: ueki, hiroshi; wang, i-hsuan; zhao, dongming; gunzer, matthias; kawaoka, yoshihiro title: multicolor two-photon imaging of in vivo cellular pathophysiology upon influenza virus infection using the two-photon impress date: 2020-01-29 journal: nat protoc doi: 10.1038/s41596-019-0275-y sha: doc_id: 308461 cord_uid: 4lhh3du0 in vivo two-photon imaging is a valuable technique for studies of viral pathogenesis and host responses to infection in vivo. in this protocol, we describe a methodology for analyzing influenza virus–infected lung in vivo by two-photon imaging microscopy. we describe the surgical procedure, how to stabilize the lung, and an approach to analyzing the data. further, we provide a database of fluorescent dyes, antibodies, and reporter mouse lines that can be used in combination with a reporter influenza virus (color-flu) for multicolor analysis. setup of this model typically takes ~30 min and enables the observation of influenza virus–infected lungs for >4 h during the acute phase of the inflammation and at least 1 h in the lethal phase. this imaging system, which we termed two-photon impress (imaging pathophysiology research system), is broadly applicable to analyses of other respiratory pathogens and reveals disease progression at the cellular level in vivo. in vivo two-photon imaging is an analytical approach that can be used to visualize cell dynamics and hemodynamics in organs or tissues of live animals. information in real time obtained by using this approach, such as changes in cell behavior and morphology, tissue localization, and blood flow, has revealed highly sophisticated and dynamic systems of living organisms. during in vivo imaging, the blood circulation in the tissue being observed is maintained; therefore, this technique is also effective for analyzing the migration and invasion of immune cells in the inflammatory environment. observations in physiological environments deepen our understanding of host response mechanisms under both steady-state and disease conditions. computed tomography, x-ray, and ivis spectrum (an in vivo imaging system) imaging methods have been used as non-invasive approaches; however, these techniques have low spatiotemporal resolution and have been able to estimate only the site of inflammation in an organ 1, 2 . therefore, it is impossible to observe cellular responses of the immune system using these approaches. by contrast, a two-photon excitation laser microscope, the light source of which is a near-infrared laser that produces low damage to cells but has long-reaching depth in tissue, enables us to capture the movement of cells in living animals at high resolution. two-photon imaging has been in use in biological science since the 1990s; it has progressed at a remarkable rate, and observation methods for various organs, including brain, liver, and lymph nodes, have been reported 3, 4 . in this protocol, we describe how to use it to image virus-infected lungs. we have previously demonstrated that this protocol works by using mice infected with mouse-adapted seasonal influenza virus (h1n1) or highly pathogenic avian influenza virus (h5n1) 5 . the lung, which is a respiratory organ, has contact with the outside environment and is an important organ for research on immunity to infectious diseases. in the seventeenth century, marcello malpighi discovered pulmonary capillaries and alveoli in the frog lung by using optical microscopy 6 ; now fluorescent reporter mice facilitate the study of disease models in conjunction with two-photon excitation microscopy (table 1) . however, a challenge encountered when imaging the lung is that it is constantly moving during respiration. the lung has been stabilized in several ways during in vivo observation by microscopy, including bronchus clamping, prolonged apnea, gluing, and suction 7, 8 ; however, it is difficult to reduce motion artifacts due to lung respiratory movement under physiological conditions and hence to obtain high-quality images. bronchus clamping can suppress respiratory motion artifacts of the lung lobe 9, 10 ; however, it is not suitable for long-term observation because it causes severe hypoxia. although prolonging apnea is less invasive [11] [12] [13] , it does not allow researchers sufficient time to observe the lung for image acquisition by two-photon excitation microscopy, and the quality of the images tends to deteriorate over time. gluing addresses the above limitations 14, 15 ; however, it can induce shear force injury and inflammation, which affect the interpretation of results. a suction window, which is currently the most commonly used stabilizing system during lung imaging, achieves moderate immobilization of the lung and high-quality images [16] [17] [18] [19] ; however, the observation period is limited to ≤12 h. ex vivo imaging of lungs and in vivo imaging of trachea have also been performed as complementary methods 8 . each of these methods has its advantages and disadvantages, and it is important to select and optimize the method best suited to the goal of the experiments and disease model. in vivo observation of lungs has been performed using various lung disease and experimental models, including bacterial infection, allergen inoculation, tumor metastasis, and lipopolysaccharide (lps)-induced sepsis (table 1) . however, for viral respiratory diseases, such as influenza, other than an observation in a methodology report 20 , only analyses of the trachea in vivo [21] [22] [23] and isolated lungs had been performed 24 , with no analysis of the lung in vivo, until our recent publication 5 (table 1) . unlike ex vivo methods, which involve isolated or sliced lungs, in vivo imaging using two-photon excitation microscopy of live animals enables researchers to observe hemodynamics, migration and extravasation of immune cells, as well as interactions among immune cells during influenza virus infection. however, it is technically demanding to perform two-photon excitation microscopy of live influenza virus-infected lung, which exhibits severe inflammation, requiring the development of highly sophisticated, less invasive instruments and surgical techniques. in addition, when observing animals infected with pathogenic viruses, specialized facilities and instruments are frequently required to avoid the spread of the virus. furthermore, because many types of immune cells infiltrate the infected lung in an inflammatory environment, it is necessary to distinguish the target immune cells from the infected cells by using fluorescent labels in the infected microenvironment. to detect multiple fluorescent signals excited simultaneously by a two-photon excitation laser, fluorochromes with different spectra and equal brightness must be selected; however, there is currently no comprehensive database of fluorescent reagents, fluorescent reporter viruses, and reporter mouse lines available for lung in vivo imaging. we therefore also provide a database of fluorescent dyes, antibodies, and reporter mouse lines that can be used in combination with a reporter influenza virus (color-flu) [25] [26] [27] for multicolor analysis under pathological conditions in this protocol. our system uses suction-based lung stabilization 16, 28 to improve an existing in vivo two-photon imaging system for influenza virus-infected lung as a model of an acute inflammatory respiratory disease 5 . we have successfully used c57bl/6 mice and transgenic mice of the c57bl/6 background (6-to 10-week-old males and females). by using our method, described in detail here, it is possible to visualize and analyze the behavior of immune cells and their interactions with infected cells during an influenza virus infection, which creates an acute inflammatory environment. a limitation of two-photon excitation microscopy is that the observation depth that can be achieved is a maximum of~70 μm. therefore, we cannot observe the bronchial region. this limitation is linked to the wavelength of the infrared laser and detector capability of the microscope. however, as laser technology develops, the observation depth achievable using this method will improve. in this protocol, we describe the application of this methodology to influenza virus infection of the lungs because this is what we have used it for previously. this protocol could be applied not only to studies of the early stages of inflammation due to infection or other causes, but also to analyses of tissue regeneration mechanisms in lungs that are in the process of recovering from infection or other protocol nature protocols injuries. the information provided will also be useful to those using two-photon imaging analysis for the evaluation of the effects of drugs and vaccines, as well as biological events in the lungs and other organs (e.g., liver, spleen) 5 . moreover, with minor modifications, the approach could be applied to analyses of other respiratory diseases, including other infectious models (e.g., severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers)), pulmonary fibrosis, and tumor metastasis. . dna fingerprinting showed that this cell line has the same origin as one obtained from atcc (cat. no. ccl-34, rrid:cvcl_0422) ! caution all viruses and infected animals should be handled in accordance with your institution's biosafety regulations. all work on highly pathogenic avian influenza viruses must be performed under biosafety level 3 (bsl3) regulations. accordingly, all our in vivo imaging studies were performed in the bsl3 facility at the university of tokyo (tokyo, japan), which is approved for such use by the ministry of agriculture, forestry, and fisheries of japan c critical the cells should be regularly checked to ensure that they are not contaminated with mycoplasma. • isoflurane (msd animal health) ! caution isoflurane is an anesthetic gas associated with adverse health outcomes. it should be used in a well-ventilated room or with another appropriate removal system. store it in a locked drawer at room temperature (18-25°c). • sevoflurane (maruishi pharmaceutical) ! caution sevoflurane is an anesthetic gas associated with adverse health outcomes. it should be used in a well-ventilated room or with another appropriate removal system. store it in a locked drawer at room temperature. c critical all reagents should be prepared under sterile conditions. fluorescent reagents should be protecting from light during the setup procedure because they are light sensitive. to prepare 10 mg/ml of tamoxifen solution in sunflower seed oil, dissolve 100 mg of tamoxifen in 1 ml of ethanol (99.5%) and add 9 ml of sunflower seed oil. after adding the ethanol and sunflower seed oil, mix well by vortexing and sonication. this solution can be stored in a refrigerator (2-8°c) for a week. ! caution tamoxifen powder should be handled in a hood. to avoid inhalation and contact with skin, wear rubber gloves and a surgical mask. prepare a solution at a concentration of 2 mg/ml in sterile 1× pbs or saline, make aliquots in 1.5-ml microtubes, and store them in a refrigerator (2-8°c) for up to 2 weeks. inject 50 μl (100 μg) of fluorescent dextran i.v. per mouse. qtracker 655 vascular labels immediately before use, add 5 μl of the stock solution to 95 μl of sterile 1× pbs or saline to make 100 μl total and inject 50 μl i.v. at a concentration of 0.1 μm. prepare a solution at a concentration of 1 × 10 8 beads/ml in sterile 1× pbs or saline, make aliquots of the solution in dark 1.5-ml microtubes, and store them in a refrigerator (2-8°c) for long periods (~3 months). immediately before use, mix well and inject 50 μl i.v. per mouse. qdot 655 wga immediately before use, add 5 μl of the stock solution to 95 μl of sterile 1× pbs or saline to make 100 μl total and i.v. inject 50 μl. prepare a solution at a concentration of 100 μm in sterile 1× pbs or saline, dispense the solution into dark 1.5-ml microtubes, and store them in a refrigerator (2-8°c) for up to 2 weeks. inject 50 μl of fluorescent dextran i.v. per mouse. divide the 5 mm dmso stock solution into dark 1.5-ml microtubes and store them at −20°c for up to 3 months. immediately before use, prepare a solution at a concentration of 50 μm in sterile 1× pbs or saline and i.v. inject 50 μl per mouse. prepare a solution at a concentration of 100 mm in sterile 1× pbs or saline, dispense the solution in dark 1.5-ml microtubes, and store them at −20°c for up to 3 months. immediately before use, prepare a solution at a concentration of 1 mm in sterile 1× pbs or saline and inject 50 μl i.v. per mouse. prepare a solution at a concentration of 10 mm in sterile 1× pbs or saline, make aliquots of the solution in dark 1.5-ml microtubes, and store them in a refrigerator (2-8°c) for up to 2 weeks. inject 50 μl of the solution i.v. per mouse. prepare a working solution according to the vendor's manual, dissolve pan caspase in vivo probe in 5 μl of dmso, and add 55 μl of 1× injection buffer (from the kit). inject 60 μl of the solution i.v. per mouse within 1 h of preparation. prepare a working solution according to the vendor's manual, dissolve 100 μl of pkh26pcl in 900 μl of ethanol and store at room temperature for up to 3 months. immediately before use, prepare a solution at a concentration of 10 μm in sterile dilution buffer (from the kit) and inject 50 μl intranasally per mouse. cellrox green, orange, and deep red immediately before use, add 50 μl of the stock solution to 450 μl of sterile 1× pbs or saline to make 500 μl total and inject 50 μl i.v. at a concentration of 250 μm. lysotracker blue, green, red, and deep red immediately before use, add 50 μl of the stock solution to 450 μl of sterile 1× pbs or saline to make 500 μl total and inject 50 μl i.v. at a concentration of 100 μm. mitotracker orange cmtmros, red cm-h2xros, and red fm immediately before use, dilute 50 μg of mitotracker in 1 ml of dmso and inject 50 μl i.v. at a concentration of 100 μm. c critical the mitotracker solution should be prepared fresh each time immediately before use. prepare the solution at a concentration of 10 mm in sterile 1× pbs or saline, make aliquots in dark 1.5-ml microtubes, and store them in a refrigerator (2-8°c) for up to 2 weeks. immediately before use, prepare a solution at a concentration of 10 μm in sterile 1× pbs or saline and inject 50 μl i.v. per mouse. prepare the solution at a concentration of 10 mm in dmso, make aliquots in dark 1.5-ml microtubes, and store them in a refrigerator (2-8°c) for up to 2 weeks. immediately before use, prepare a working solution at a concentration of 1 mm in sterile 1× pbs or saline and inject 50 μl i.v. per mouse. prepare each solution at a concentration of 1 mm in dmso, make aliquots in dark 1.5-ml microtubes, and store them in a refrigerator (2-8°c) for up to 1 week. immediately before use, prepare solutions at a concentration of 100 μm in sterile 1× pbs or saline and inject 50 μl i.v. per mouse. dilute fluorescent antibodies to a concentration of 1 µg per 10 μl with sterile 1× pbs or saline and inject 50 μl i.v. per mouse. ! caution it should be noted that antibody staining may affect the target cell behavior; for example, at a high dose (~200 μg), antibodies may neutralize cell activities and/or cause antibody-dependent cytotoxic activity [35] [36] [37] . in our studies, we use 5 µg of antibody for brightness screening because inoculation of fluorochrome-conjugated anti-ly-6g antibody at low doses (1-40 µg) into mice does not affect neutrophil recruitment 38 . the contribution of ly-6g, which is expressed predominantly on murine neutrophils, to recruitment during inflammation remains a matter of debate. it has been reported that low-dose antibody treatment inhibited ly-6g ligation and the recruitment of neutrophils to the site of inflammation 39 ; however, a more recent study indicated that ly-6g knockout did not affect either neutrophil differentiation or recruitment to the site of inflammation in catchup mice 32 . therefore, a low dose of anti-ly-6g antibody is used in our protocol. laser path adjustment system an overview of the laser path adjustment system is shown in fig. 1 . our two-photon excitation laser (chameleon vision ii) unit is placed on an anti-vibration table outside the bsl3 facility. the laser beam enters the bsl3 room, where the two-photon excitation scanning microscope is located, through a window (composed of a small glass window (wg12012-b) and a planar window (rs seal)) connecting the inside and the outside of the bsl3 facility (fig. 1c,d) . the laser path connecting the laser source unit and the two-photon excitation microscope is adjusted by automated laser beam alignment and the aligna 4d stabilization system is adjusted with two active mirrors. ! caution this system adjusts the laser path passing from the outside to the inside of the bsl3 facility for maintenance purposes, so there is no need for this setup unless you are using pathogens that require bsl3 containment. heat is generated when the laser source unit is running, so keep the temperature and humidity constant by using air conditioning equipment. ! caution the system should be operated only by users trained to deal with unenclosed high-power invisible beams and should be placed in an appropriate enclosure with interlocking doors. two-photon excitation laser scanning microscopy system for in vivo imaging of virus-infected mouse lungs in a bsl3 facility a schematic of the arrangement of the in vivo lung imaging system for virus-infected mouse is shown in fig. 2a, and layout examples are shown in fig. 2b -g. this in vivo lung imaging system is based on the upright microscope lsm 780 nlo system, which is equipped with four different lasers (excitation at 405, 488, 543, and 633 nm) for confocal imaging and a two-photon excitation laser (excitation at 630-1,050 nm). to be able to perform the surgical procedure on the mouse, we replaced the sample stage with a large, flat one (microscope stage for in vivo experiment) as shown in fig. 2b ,c. to efficiently excite multiple fluorescent proteins and fluorescent dyes simultaneously, the wavelength of the infrared laser should be set at 910 nm. all fluorescent spectra between the 410-and 695-nm wavelengths can be detected using a 20× water immersion lens, and we record signals in lambda image stacks (0.13 frames per s, 1,024 × 1,024 pixels) and acquire z-stack images with z-depths of 5 μm (total of 10-μm z-depth). we perform spectral separation of the acquired lambda stacks by using the linear unmixing function of the zen software. although the lsm 780 microscope system is controlled by a primary personal computer, we recommended adding >64 gb of ram for appropriate imaging analysis. we keep the mice on a heated stage on the sample stage and record their vital signs using a labox-1 pulse oximeter. to observe the lungs of the mice with a thoracotomy, we place the ventilator with an airway pressure monitor and anesthesia machine for rodents in appropriate positions on the stage. we installed high-efficiency particulate air (hepa) filters in the exhalation duct of the ventilation system (fig. 2b,d) , and the operator wore a positive pressure mask (versaflo faceshields) and a tyvek suit (fig. 2e-g) to avoid exposure to the viruses. ! caution the wavelength and power of the excitation laser should be adjusted appropriately according to the experimental conditions. increasing the laser power enhances target signals and enables detection of second-harmonic generation (shg), in which structures with repeating patterns lead to the formation of a signal. shg is a useful phenomenon for visualizing collagen fibers in the lung without staining; however, it should be noted that the autofluorescence of lung tissue is also enhanced under excessive excitation conditions ( supplementary fig. 1 ). when using this protocol, we did not perform experiments under which shg occurs, in order to minimize autofluorescence; it is better to adjust the laser power according to the experimental purpose. when the wavelength of the excitation laser is too short, the autofluorescence signal becomes very strong and it is difficult to observe properly. by contrast, when the laser wavelength is too long, it becomes difficult to obtain a signal because of the short excitation energy (supplementary fig. 2 ). ! caution although color separation of emission using a conventional optical band-pass filter is also available for this protocol, multispectral imaging is a useful approach for simultaneously analyzing multiple targets by eliminating tissue autofluorescence and identifying fluorescent labels with overlapping spectra 40, 41 . in vivo two-photon imaging is performed under conditions of single stimulation with a two-photon excitation laser; limitations exist regarding available fluorescent reagents/proteins for multiple labeling of target cells and lung architecture. therefore, we recommend using a multispectral approach to produce crosstalk-free images of fluorescence with overlapping spectra that cannot be separated by using band-pass filters. before starting experiments, it is necessary to collect spectral signatures of the emission signal of each fluorescent reagent and protein as reference spectra under the same excitation condition as will be used in the experiment. to observe the mouse lung using an upright microscope, it is necessary to prepare a thoracic suction window to immobilize the lung. in the bsl3 facility, animal experiments must be performed while wearing two or three layers of latex gloves; therefore, the thoracic suction window was designed for easy handling, even in the bsl3 facility, and to be minimally invasive for the infected animals (fig. 3a-c and supplementary fig. 3 ). to position a cover glass for each observation, flatten the upper surface of the thoracic suction window so that a commercially available cover glass will fit. this device is also designed to reduce concavity and convexity as much as possible so that blood containing virus cannot accumulate. connect the thoracic suction window to an aspirator through a waste tank and a suction regulator. to prevent the spread of virus-containing aerosols, install hepa filters between the waste tank and the suction regulator as shown in fig. 3d . starting up the imaging system equipment • timing 20-30 min 2 on the day of analysis, turn on the two-photon excitation laser and the aligna 4d control unit placed outside the bsl3 facility, and verify that they are working. c critical the aligna 4d control unit needs to be kept on. 3 wearing a tyvek suit, positive pressure mask, and gloves according to the guidelines for the bsl3 facility, enter the bsl3 facility where the imaging system is housed. ? troubleshooting 4 turn on the microscope controllers, confocal lasers, and the computer for the two-photon excitation microscope and the aligna 4d system. 5 launch the microscope control software zen and turn on the lasers, including the two-photon excitation laser. 6 launch the aligna 4d control software kangoo and adjust the laser path connecting the laser source unit and the microscope (supplementary fig. 4) . ? troubleshooting 7 wrap the hot plate with aluminum foil, turn it on, and keep it at 35°c. sterilize the surgical area and tools with 70% ethanol and place all instruments within easy reach. animal anesthesia • timing 2-3 min 8 turn on the gas anesthesia vaporizer and supply 5% isoflurane to a mouse anesthesia induction chamber. 9 anesthetize the influenza virus-infected mouse with 5% isoflurane in a mouse anesthesia induction chamber. subsequently, transfer the mouse to the hot plate while supplying 2% isoflurane via an anesthetic mask. ? troubleshooting 10 inject the chosen fluorescent dyes and antibodies via the retro-orbital plexus (as shown in supplementary video 1) using an insulin syringe. tables 4 and 5 show the brightness levels of antibodies and fluorescence of dyes, respectively, in vivo. ! caution when working with viruses in a bsl3 containment, it is not safe to use needles, so we avoid them as much as possible, which is a standard precaution in high-containment laboratories. in addition, in the bsl3 facility, animal experiments must be performed wearing two or three layers of latex gloves. tail-vein administration is a common method; however, it is not easy to perform these procedures with so many layers of gloves. use tweezers to hold down the mouse to make the administration route. when an infected animal is not used, an administration route can be created via the tail vein or the jugular vein. ? troubleshooting surgical procedure • timing 10-15 min c critical before experimenting with infected animals, practice the surgical procedures with euthanized animals. 11 place the mouse on its back and tape the anterior limbs with adhesive tape (fig. 4a) . 12 using straight scissors, cut the skin beneath the chin in the middle and expose the trachea (fig. 4b) . insert a tracheal cannula and intubate the mouse to facilitate mechanical ventilation with a ventilator (fig. 4c) . turn on the ventilator, ventilate the mouse at a respiratory rate of 120 breaths per min, and apply positive-end expiratory pressure (peep;~6 cm h 2 o) and a tidal volume of 0.5 ml. deliver isoflurane continuously at 2% to maintain anesthesia. ! caution perform the surgery with care so as not to cut the blood vessels. if bleeding occurs, stop the bleeding with fine bulldog forceps for microsurgery. 13 place the mouse in the right lateral decubitus position and re-fix its anterior limbs with the tape (fig. 4d) . make an incision in the skin at the left axilla using straight scissors, straight iris scissors, and hooked forceps (fig. 4e) . ! caution carefully change the mouse's position in order to avoid cannula drop off. 14 expose the left lung lobe by surgical intercostal incision between ribs 3 and 4, and keep it exposed by using retractors (fig. 4f) . the brightness of each fluorescent protein during in vivo lung imaging was scored as relative fluorescence intensity compared with fluospheres fluorescent microspheres as an internal standard. for relative intensities of 0-0.2, 0.2-0.6, 0.6-0.9, and >0.9, the brightness scores are represented as +, ++, +++, and ++++, respectively. the brightness of each fluorochrome during in vivo lung imaging was scored as relative fluorescence intensity compared with fluospheres fluorescent microspheres as an internal standard. for relative intensities of 0-0.2, 0.2-0.6, 0.6-0.9, and >0.9, the brightness scores are represented as +, ++, +++, and ++++, respectively. af, alexa fluor; nd, not detected. ! caution perform the surgery with care so as not to cut the blood vessels. if bleeding occurs, stop the bleeding with fine bulldog forceps for microsurgery. c critical because lungs infected with viruses often shrink, secure a large field of surgical view so that the suction window can reach it. 15 place the mouse beneath the objective lens and connect a device to monitor the heart rate of the mouse (we use a labox-1 pulse oximeter). starting up the thoracic vacuum window system • timing 2-3 min 16 turn on the aspirator connected to the thoracic suction window. 17 fix the thoracic suction window to the holding block at a 90°angle and put a round cover glass on the tip of the suction device. ? troubleshooting 18 turn on the suction pressure regulator and adjust the suction pressure to 25-30 mmhg. 19 lower the thoracic suction window gently to immobilize the mouse lungs (fig. 4g,h) . the thoracic suction window should cause the lung to stick to the cover glass because of negative pressure. ! caution carefully move the suction window so as not to scratch the objective. 20 position the objective lens above the thoracic window. 21 put water drops on the cover glass by using a pasteur pipette and lower the objective lens to the thoracic suction window (fig. 4i) . unmixing of spectrum data and analyzing the images • timing 1-2 h per sample 24 to unmix the spectrum data, prepare a reference image of each spectrum in advance. to make a reference image, acquire each fluorescent dye or protein separately without any co-staining and analyze the single fluorescent spectrum. we use the linear unmixing module of the zen software for separating spectrum data; however, other commercial or open-source software is available (table 6 ). 25 subject unmixed time-series stacks to image registration to correct for tissue drifts and respiratory artifacts. this step is critical to certain analyses, such as long-term tracking of individual cells or subcellular structures. in some cases, a reference channel is required for determining the shift and distortion of the objects. in our studies, we use time-series stacks of blood vessels or collagens for such use, because their localizations are constant over time without substantial changes in shape or structure during the observation. ! caution some image registration algorithms may cause spatial distortion. choose algorithms that generate corrected data suitable for your subsequent analyses, especially when examination of the shape and structure of cells and tissues is required. 26 analyze the movies as required for your experiment. troubleshooting advice can be found in table 7 . step 1, infection: 10-20 min anticipated results the imaging system described in this protocol enables the observation of the behavior of virusinfected cells and immune cells in infected lungs in real time. typical images of influenza virus-infected lung are shown in fig. 5a and supplementary video 2. when observing while using a multicolor fluorescent label, it is easier to analyze the detected images if the brightness level of each fluorophore is adjusted to make them similar. it is better to choose fluorescent dyes or proteins that possess high fluorescence stability for long-term observations (tables 2, 4 and 5). we have found that use of ma-cerulean-viruses or ma-venus-viruses for infection produces influenza virus-infected cells with sufficient brightness (table 3) . for labeling immune cells and alveolar cells, we have achieved good results by using the fluorochrome phycoerythrin (pe) for antibody staining and rosa-tdtomato 42 or -mtfp1 33 mice that were crossed with cell-specific cre-expressing mice. if using reporter mice expressing a fluorescent protein such as gfp, which is regulated by an endogenous promoter, the expression level of the fluorescent protein should be confirmed. to visualize the lung structure, we use texas-red dextran or qtracker 655 vascular labels for the red to far-infrared channel. mice die during anesthesia the level of anesthesia is too high decrease the concentration of anesthesia as soon as the mouse shows loss of righting reflex 10 mice regain consciousness during anesthesia the level of anesthesia is too low confirm the concentration of anesthesia; administer the reagents again after a brief pause 15 no heart rate is measured the monitoring probe is mispositioned make sure that the monitoring probe is in the appropriate place 17 the cover glass falls off the cover glass does not hold on the suction device put water droplets on the tip of the suction device and then place the cover glass on it influenza virus-infected lungs are infiltrated by numerous immune cells, including neutrophils and monocytes [43] [44] [45] . an immune cell-specific reporter mouse line can be used to visualize cells infiltrating the alveoli and cells in blood vessels, whereas it is preferable to label intravascular cells by intravenous administration of fluorochrome-conjugated antibodies 5, 46, 47 . consistent with previous reports, intravenously injected antibodies will label only the cells in contact with the blood flow and not those in extravascular regions under our experimental conditions 5 . by administering a fluorescently labeled antibody against neutrophils into neutrophil reporter mice, we can observe the behavior of both the neutrophils infiltrating the influenza-infected lungs and the neutrophils in blood vessels separately (fig. 5b) . to observe the interaction between different kinds of infiltrating immune cells, such as neutrophils and monocytes, double-reporter mice expressing fluorescent proteins with different spectra but similar brightness have a major advantage (fig. 5c and supplementary video 3) . co-infection of the host with different strains of influenza virus can lead to the emergence of reassortant viruses. by infecting mice with color-flu viruses that produce different fluorescence spectra, we detected alveolar epithelial cells that simultaneously expressed two fluorescent proteins in vivo (fig. 6) . visualization of co-infected cells might enable us to better understand the reassortment process of influenza viruses in vivo. in summary, the use of this in vivo imaging system for infected animal and multicolor imaging enables us to analyze pathology and immune cell dynamics at the cellular level, which would not be possible by using conventional histopathology methods. this imaging system thus provides a novel and useful approach for investigating viral pathogenicity. further information on research design is available in the nature research reporting summary linked to this article. the data that support this study are available from the corresponding author upon reasonable request. the matlab scripts are available at https://github.com/kawaokalab/ueki_pnas_2018. for all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or methods section. the exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement a statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly the statistical test(s) used and whether they are one-or two-sided only common tests should be described solely by name; describe more complex techniques in the methods section. a description of all covariates tested a description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons a full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) and variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) for null hypothesis testing, the test statistic (e.g. f, t, r) with confidence intervals, effect sizes, degrees of freedom and p value noted give p values as exact values whenever suitable. for hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes estimates of effect sizes (e.g. cohen's d, pearson's r), indicating how they were calculated our web collection on statistics for biologists contains articles on many of the points above. policy information about availability of computer code to efficiently excite multiple fluorescent proteins and fluorescent dyes simultaneously, the wavelength of the infrared laser was set at 910 nm. all fluorescent spectra between 410 and 695 nm wavelengths were detected using a 20x water immersion lens (carl zeiss ag, germany) and the signals were recorded in lambda image stacks. we use the linear unmixing module of zen software for separating spectrum data. unmixed time-series stacks are subjected to image registration to correct for tissue drifts and respiratory artefacts. a reference channel is required for determining the shift and distortion of the objects. in our studies, we employ time-series stacks of blood vessels or collagens for such use, as their localizations are constant over time without significant changes in shapes or structures during the observation. for manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers. we strongly encourage code deposition in a community repository (e.g. github). see the nature research guidelines for submitting code & software for further information. policy information about availability of data all manuscripts must include a data availability statement. this statement should provide the following information, where applicable: -accession codes, unique identifiers, or web links for publicly available datasets -a list of figures that have associated raw data -a description of any restrictions on data availability the data that support this study are available from the corresponding author upon reasonable request. october 2018 field-specific reporting please select the one below that is the best fit for your research. if you are not sure, read the appropriate sections before making your selection. for a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf all studies must disclose on these points even when the disclosure is negative. only one sample was shown as a representative example that can be obtained by using the imaging protocol. data exclusions no data was excluded since one representative image was shown. no repeated measurements were performed in this paper since one image has been shown as a representative image by using the imaging protocol. • microsurgery straight scissors (13.5 cm • microsurgery bulldog forceps (brc, cat. no. 70052-30cii/r) • insulin syringes (0.5 ml, 100 u, 30 gauge × 10 mm; nipro, cat. no. 08277) • pasteur pipettes (bd falcon, cat. no. 357575) • customized surgical retractor • thoracic suction window (sakura seiki, custom made) stage for mounting a thoracic suction window (sakura seiki, custom made) • suction regulator (iwaki, cat. no. 1450050) • cover glass (matsunami glass, cat. no. c013001) • hot plate (hipet, cat • confocal microscope system (zeiss, model no. lsm 780 nlo) • infrared laser (coherent, model no. chameleon vision ii) • 20× water immersion lens (zeiss, plan-apochromat model) • beam-pointing stabilizer (tem messtechnik, model no. aligna 4d system high-efficiency particulate air (hepa) filters (vacushield; pall, cat. no. 4402) • artificial ventilator (shinano, cat • airway pressure monitor (shinano) • gas anesthesia vaporizer • mouse anesthesia induction chamber • mouse anesthesia mask • positive pressure mask (versaflo faceshields 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environmental cues drive group 2 innate lymphoid cell dynamics in mice and humans long-term high-resolution intravital microscopy in the lung with a vacuum stabilized imaging window cancer cells induce metastasis-supporting neutrophil extracellular dna traps a permanent window for the murine lung enables high-resolution imaging of cancer metastasis embryonic stem cells and mice expressing different gfp variants for multiple non-invasive reporter usage within a single animal cre reporter strains produced by targeted insertion of eyfp and ecfp into the rosa26 locus a global double-fluorescent cre reporter mouse in vivo depletion of cd11c + dendritic cells abrogates priming of cd8 + t cells by exogenous cell-associated antigens notch-rbp-j signaling controls the homeostasis of cd8 -dendritic cells in the spleen zbtb46 expression distinguishes classical dendritic cells and their committed progenitors from other immune lineages absence of mhc class ii on cdcs results in microbial-dependent intestinal inflammation identification of a dendritic cell receptor that couples sensing of necrosis to immunity a macrophage colony-stimulating factor receptor-green fluorescent protein transgene is expressed throughout the mononuclear phagocyte system of the mouse analysis of fractalkine receptor cx(3)cr1 function by targeted deletion and green fluorescent protein reporter gene insertion fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis generation of cd4creer(t(2)) transgenic mice to study development of peripheral cd4-t-cells notch2 integrates signaling by the transcription factors rbp-j and creb1 to promote t cell cytotoxicity b lymphocyte-specific, cre-mediated mutagenesis in mice genetic analysis of basophil function in vivo lethal influenza infection in the absence of the natural killer cell receptor gene ncr1 high-efficiency type ii cell-enhanced green fluorescent protein expression facilitates cellular identification, tracking, and isolation hyperspectral phasor analysis enables multiplexed 5d in vivo imaging the orfeo toolbox remote sensing image processing software optimizing imaging parameters for the separation of multiple labels in a fluorescence image spectral imaging and linear un-mixing enables improved fret efficiency with a novel gfp2-yfp fret pair blind source separation techniques for the decomposition of multiply labeled fluorescence images image matching as a diffusion process: an analogy with maxwell's demons diffeomorphic demons: efficient non-parametric image registration automated motion artifact removal for intravital microscopy, without a priori information nonrigid registration using free-form deformations: application to breast mr images imart software for correction of motion artifacts in images collected in intravital microscopy automated filtering of intrinsic movement artifacts during twophoton intravital microscopy removing physiological motion from intravital and clinical functional imaging data software tools for single-cell tracking and quantification of cellular and molecular properties cellprofiler: image analysis software for identifying and quantifying cell phenotypes icy: an open bioimage informatics platform for extended reproducible research additional information supplementary information is available for this peer review information nature protocols thanks megan macleod and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. reprints and permissions information is available at www.nature.com/reprints. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank s. watson for editing the manuscript. we thank k. iwatsuki-horimoto, l. wu, s. fukuyama, y. matsuzawa, and k. miyake randomization no randomization is included in this paper since one image has been shown as a representative image by using the imaging protocol. blinding was not relevant to this study which is describing a imaging protocol and anticipated results. we require information from authors about some types of materials, experimental systems and methods used in many studies. here, indicate whether each material, system or method listed is relevant to your study. if you are not sure if a list item applies to your research, read the appropriate section before selecting a response. all antibodies used are commercialized and the fluorescence has been tested in this study. the information is included in table 4 . animals and other organisms policy information about studies involving animals; arrive guidelines recommended for reporting animal research laboratory animals six-ten-week-old c57bl/6 mice (japan slc, inc.) and transgenic mouse lines were used in this study. all animal care and experiments conformed to the guidelines for animal experiments of the university of tokyo, and were approved by the animal research committee of the university of tokyo (pa17-31 and pa17-17). all in vivo imaging studies were performed in the biosafety level 3 facility at the university of tokyo (tokyo, japan), which is approved for such use by the ministry of agriculture, forestry, and fisheries of japan. field-collected samples not applicable. all experiments with mice were performed in accordance with the university of tokyo's regulations for animal care and use and were approved by the animal experiment committee of the institute of medical science, the university of tokyo.note that full information on the approval of the study protocol must also be provided in the manuscript. key: cord-324617-yok7mh70 authors: andreata-santos, robert; alves, rúbens prince dos santos; pereira, sara araujo; pereira, lennon ramos; de freitas, carla longo; pereira, samuel santos; venceslau-carvalho, alexia adrianne; castro-amarante, maria fernanda; favaro, marianna teixeira pinho; mathias-santos, camila; amorim, jaime henrique; ferreira, luís carlos de souza title: transcutaneous administration of dengue vaccines date: 2020-05-06 journal: viruses doi: 10.3390/v12050514 sha: doc_id: 324617 cord_uid: yok7mh70 in the present study, we evaluated the immunological responses induced by dengue vaccines under experimental conditions after delivery via a transcutaneous (tc) route. vaccines against type 2 dengue virus particles (denv2 new guinea c (ngc) strain) combined with enterotoxigenic escherichia coli (etec) heat-labile toxin (lt) were administered to balb/c mice in a three-dose immunization regimen via the tc route. as a control for the parenteral administration route, other mouse groups were immunized with the same vaccine formulation via the intradermic (id) route. our results showed that mice vaccinated either via the tc or id routes developed similar protective immunity, as measured after lethal challenges with the denv2 ngc strain. notably, the vaccine delivered through the tc route induced lower serum antibody (igg) responses with regard to id-immunized mice, particularly after the third dose. the protective immunity elicited in tc-immunized mice was attributed to different antigen-specific antibody properties, such as epitope specificity and igg subclass responses, and cellular immune responses, as determined by cytokine secretion profiles. altogether, the results of the present study demonstrate the immunogenicity and protective properties of a dengue vaccine delivered through the tc route and offer perspectives for future clinical applications. infection with one of the four dengue virus serotypes (denv1-4) may cause a spectrum of diseases ranging from an acute, self-limiting febrile illness (df) characterized mainly by fever, retro-orbital headache, rash, arthralgia, and to more severe, life-threatening, conditions that may include hemorrhagic manifestations, increased vascular permeability, thrombocytopenia, and shock [1] [2] [3] . in fact, it is estimated that 3.9 billion people in 128 countries are at risk of infection [2, 4] . denv causes approximately 390 million infections, of which 500,000 cases develop into severe forms, making denv infection one of the most economically and epidemiologically relevant arthropod-borne diseases the murine model elected for this study was based on the balb/c mouse line. male mice from 6 to 8 weeks old were supplied by the parasitology department animal house at the university of são paulo. the animals were considered free of pathogens and routinely subjected to standard monitoring. aedes albopictus cells of the c6/36 strain were used for viral propagation, while cercopithecus aethiops kidney epithelial cells (vero) were used for plaque assays. briefly, vero cells (1 × 10 5 /well) were plated in 24-well culture plates and incubated at 37 • c in a co 2 incubator overnight. denv supernatant aliquots (100 µl) were 10-fold serially diluted in medium, added to the vero cells, and incubated at 37 • c for 1 h. the viral supernatant was aspirated, and a prewarmed solution of 1% carboxymethyl cellulose medium (synth, diadema, brazil) was added to each well. after 7 days incubation, the cells were fixed with 4% paraformaldehyde solution for 15 min at room temperature (rt), washed with water and stained with a 1% crystal violet solution for 10 min. the staining solution was removed and the plates washed to remove residual staining. finally, after drying on the bench, plaque forming units (pfu) were counted to obtain the virus titers, which were expressed in pfu/ml. the denv2 new guinea c (ngc) strain was previously isolated and adapted to infect and kill immunocompetent mice after intracranial (i.c.) administration [20] . the lt was purified from a recombinant e. coli k12 strain transformed with a bspks (-) vector carrying the sequence encoding the toxin originally expressed by enterotoxigenic e. coli [21] . the recombinant lt1 was purified by affinity chromatography with immobilized d-galactose, as previously described [22] . c6/36 cells were grown in leibovitz-15 (l-15) medium (vitrocell, campinas, brazil) supplemented with 10% fetal bovine serum (fbs) (vitrocell, campinas, brazil) until they reached approximately 50% confluence in plastic culture bottles (corning, new york, ny, usa). then, the cells were washed and infected with the denv2 ngc at a standard moi equal to 1, and when the cells displayed syncytial characteristics, the supernatant was harvested. the cells were centrifuged (eppendorf, hamburg, ger) at 3200× g for 15 min and then lysed by freezing at −80 • c for 5 min and melting through the liquid state three times. after being lysed, the cells were centrifuged one more time, and the supernatant containing the released virus was added to the cell culture supernatant. the whole supernatant was mixed with polyethylene glycol (peg-6000) (synth, diadema, brazil) in a proportion of 2.5 ml of peg to 10 ml of supernatant and incubated overnight at −4 • c. after incubation, the supernatants were centrifuged at 3200× g for 30 min, the supernatant was discarded, and the precipitate viruses were suspended in mem supplemented with hepes buffer. the concentrated viruses (up to 100-fold) were titrated, and the content of total protein was also determined using an epoch take3 spectrophotometer (biotek, winooski, vt, usa) and maintained at −80 • c until being used for immunization assays. the purification of denv2 virus particles was performed by ion exchange chromatography using the akta fplc chromatograph (amersham pharmacia biotech, little chalfont, uk) associated with a 1 ml hitraptm anx (high sub) (gelifesciences, boston, ma, usa) anion exchange column previously loaded with 60 mm sodium phosphate buffer (ph 7.2). a total of 10 ml of infected cell culture supernatant (vero cell previously infected for 7 days with denv2, moi of 0.1) was used for purification. the elution procedure was carried out by applying a linear gradient from 60 to 600 mm of sodium phosphate (ph 7.2) in 15 column volumes (cv). the virus-containing fractions were combined, added with 5% sterile glycerol and stored at −80 • c for later use. for the id immunizations, balb/c mice (n = 5) were immunized with different vaccine formulations containing 40 µg/ml of polymyxin b, 10 or 50 µg of denv2 ngc virus particles administered alone or with 10 µg of purified lt1. the vaccination regimen consisted of three doses given at two-week intervals (14 days). serum samples were collected through the submandibular plexus before each immunization (days −1, 13, 27, 41). the administration was performed in the dorsal region on the back of the mice with a 25 mm gauge needle loaded with 25 µl of the vaccine formulation. the same vaccine formulations were used in the tc immunizations. the dorsum of balb/c mice (n = 5/group) was shaved with an electric clipper and depilatory cream. before the administration of the vaccine patches, a slight abrasion with fine-grained sandpaper was applied to the shaved skin to disrupt the stratum corneum (sc). the patches were prepared with a double-sided waterproof adhesive film (smith&nephew, london, uk) cut in squares of approximately 2.5 cm 2 . an absorbent sterile gauze piece (approximately 1 cm 2 ) was placed in the center of the adhesive film to form a pad that received the vaccine. the vaccines were placed on the back of the animals for 24 h, and bandages were applied to prevent their removal by the animals ( figure s1 ). mouse sera were individually tested for the presence of the virus-specific abs by elisas. maxisorp plates (nunc, roskilde, dk) were coated with pbs containing 200 ng of denv2 ngc virus particles and incubated overnight at 4 • c. the plates were blocked with 3% gelatin in pbs at 37 • c for 2 h. serum samples were then serially diluted in 1% gelatin diluted in 0.05% pbs-tween (pbst) and incubated at 37 • c for 1.5 h. after three washes with pbst, the plates were treated with goat anti-mouse igg (1:6000), igg1 (1:3000) or igg2a (1:2000) conjugated with horseradish peroxidase (sigma-aldrich, san luis, mo, usa) at 37 • c for 1.5 h. after a new wash cycle with pbst, a solution containing orthophenylenediamine dihydrochloride (opd) (sigma-aldrich, san luis, mo, usa) and h 2 o 2 was added for a final volume of 100 µl per well. the plates containing the developer solution were maintained in the dark for 15 min, at which time, the reaction was stopped by the addition of 50 µl of 2 n h 2 so 4 . the od 492nm was measured by an epoch take3 spectrophotometer (biotek, winooski, vt, usa), and the ab concentration values were calculated by comparison with a reference igg preparation at defined concentrations that had been added to each plate. antigen avidity with serum abs was determined with serum pools collected after the last vaccine dose was administered and carried out as previously reported [23] . after incubation with normalized concentrations of antivirus sera, plates were washed and exposed to phosphate-buffered saline (pbs)-diluted ammonium thiocyanate sigma-aldrich, san luis, mo, usa ranging from 0 to 8 m. the plates were allowed to stand for 15 min at room temperature before being washed. the concentrations of the ammonium thiocyanate required to dissociate 50% of the bound abs were determined. the percentage of binding was calculated as follows: od 492nm in the presence of ammonium thiocyanate x 100/od 492nm in the absence of ammonium thiocyanate. the values are expressed as percentages compared with those of the sample not subjected to the ammonium thiocyanate treatment. mice were sacrificed, and the spleens were aseptically harvested. splenocytes were counted after red blood cell lysis and suspended in 10% fbs/rpmi medium. splenocytes were plated (2.5 × 10 5 cells/well) and stimulated with 2 µg of thermally inactivated denv2 for 96 h. the levels of secreted il-10, tnf-α, ifn-γ, il-4, and il-2 were determined with a cytometric bead array (cba) mouse th1/th2/th17 cytokine kit (bd, franklin lakes, nj, usa) according to the manufacturer's protocol. the measurements were performed using bd lsrfortessa (bd, franklin lakes, nj, usa), and the results were processed with system software. three technical repeats were carried out for each animal, and the results were statistically evaluated using one-way anova with bonferroni's post-test. two weeks after receiving the last vaccine dose, the immunized mice were first anesthetized with a mixture of ketamine and xylazine and subsequently challenged with 1 × 10 6 plaque forming units (pfus) of the ngc strain to a final volume of 50 µl administered through the intracranial (i.c.) route, as previously described [24] . these inoculated mice were monitored for 21 days for survival. all statistical analyses were calculated using prism 5 and 6 software (graphpad software inc, la jolla, ca, usa), and differences with p ≤ 0.05 were considered significant. to compare results generated in several groups, a two-way anova test was applied in association with bonferroni's post-test. a comparison of the results generated in three groups was performed using one-way anova in association with bonferroni's post-test. statistical significance based on the mortality curves was determined by mantel-cox tests. male balb/c mice received three vaccine doses via the tc or id route with either 10 or 50 µg of concentrated denv2 particles (ngc strain) with or without purified lt1 ( figure s1e ). as shown in figure 1 , the mice inoculated with denv2 mounted a low antigen-specific igg response following the tc and id immunizations ( figure 1a ,b). the addition of lt1 to the vaccines significantly enhanced the denv2-specific serum igg responses elicited in the mice immunized via the tc or id route, with the maximal values reached two weeks after the third vaccine dose ( figure 1a ,b). similar serum antibody responses were also measured in mice immunized virus particles purified from cell culture supernatants by anionic chromatography ( figure s2 ). male balb/c mice received three vaccine doses via the tc or id route with either 10 or 50 μg of concentrated denv2 particles (ngc strain) with or without purified lt1 ( figure s1e ). as shown in figure 1 , the mice inoculated with denv2 mounted a low antigen-specific igg response following the tc and id immunizations ( figure 1a ,b). the addition of lt1 to the vaccines significantly enhanced the denv2-specific serum igg responses elicited in the mice immunized via the tc or id route, with the maximal values reached two weeks after the third vaccine dose ( figure 1a ,b). similar serum antibody responses were also measured in mice immunized virus particles purified from cell culture supernatants by anionic chromatography ( figure s2 ). the results also demonstrated that the serum denv2-specific igg responses increased according to the amount of inoculated antigen in the id-immunized mice but not in the mice immunized via the tc route ( figure 1a ,b). the tc-immunized mice that received the higher antigen dose (50 μg) combined with lt1 showed an increased serum igg response two weeks after the third dose ( figure 1c ). the id-immunized mice showed significant differences in denv2-specific serum igg responses in the presence of lt1, particularly in the group immunized with 50 μg of the tested antigen ( figure 1d ). in conclusion, tc administration of the anti-denv vaccine induced serum antigen-specific antibody responses but at lower levels than those elicited by the id administered vaccine. the results also demonstrated that the serum denv2-specific igg responses increased according to the amount of inoculated antigen in the id-immunized mice but not in the mice immunized via the tc route ( figure 1a,b) . the tc-immunized mice that received the higher antigen dose (50 µg) combined with lt1 showed an increased serum igg response two weeks after the third dose ( figure 1c ). the id-immunized mice showed significant differences in denv2-specific serum igg responses in the presence of lt1, particularly in the group immunized with 50 µg of the tested antigen ( figure 1d ). in conclusion, tc administration of the anti-denv vaccine induced serum antigen-specific antibody responses but at lower levels than those elicited by the id administered vaccine. the vaccinated mice were also monitored for different physiological aspects in a search of possible vaccine-induced adverse effects ( figure s3 ). in general, there was no indication of significant hematological alterations or tissue damage, such as in a tissue damage marker like ldh, in mice immunized via the tc or id route, with the exception of decreased hematocrit values and increased platelet numbers in the id-immunized mice receiving lt1 and 50 µg of the vaccine antigen ( figure s3b ). no other adverse reactions, such as weight loss or altered animal behavior, were observed in the mice in which the vaccine regimens were tested [25] . collectively, these results indicate that the anti-denv vaccines were safe under the experimental test conditions. we first measured the igg-specific subclass responses in the mice subjected to different immunization regimens. the serum igg1 subclass response prevailed in the mice immunized without adjuvant two weeks after they received the third dose. a higher igg1/igg2a ratio was observed among the mice immunized via the tc route compared with that of the mice immunized via the id route (figure 2a,b) . the mice immunized with lt1 also developed a predominant igg1 response, but those immunized with the higher antigen dose developed a more balanced igg1/igg2a ratio, particularly the animals immunized via the id route (figure 2a,b) . aiming for an initial characterization of the antibodies capable of binding conformational epitopes in the mice immunized with the tested vaccine formulations, we measured the specificity of denv2-specific antibodies against intact viral epitopes. for this experiment, we used heat-denatured and intact virus particles in the solid phase. as shown in figure 2c ,d, igg was increased in the mice immunized via the tc or id route, but significantly fewer igg molecules were bound to the denv particles after heat-denaturation treatment. the reduction in the anti-denv titers for groups inoculated with lt1 adjuvant and was greater in the tc-immunized mice (55% to 58%) than it was in the id-immunized mice (25% to 34%) ( figure s4a ,b). these results suggest that a significant fraction of antibodies generated in mice immunized with intact virus particles target conformational epitopes on the virus surface. in addition, the results indicate that the proportion of antibodies targeting conformational epitopes was higher (p = 0.0321) in the mice immunized via the tc route than it was in the mice immunized via the parenteral route. finally, we measured the avidity of the denv-specific antibodies in the mice subjected to different vaccine regimens tested. for this experiment, we followed a previously described elisa protocol with an additional dissociation step performed with ammonium thiocyanate. taken together, the results presented in figure 2e ,f demonstrate that the addition of lt1 steadily promoted an increase in the avidity of the antibodies to virus particles administered through both routes. however, in accordance with the measurements of the antibody titers, the amount of antigen had an impact only in the id-immunized mice, which presented enhanced avidity after the mice received higher antigen doses ( figure 2e ,f and figure s4c ,d). finally, we measured the avidity of the denv-specific antibodies in the mice subjected to different vaccine regimens tested. for this experiment, we followed a previously described elisa protocol with an additional dissociation step performed with ammonium thiocyanate. taken together, the results presented in figure 2e ,f demonstrate that the addition of lt1 steadily promoted an increase in the avidity of the antibodies to virus particles administered through both routes. however, in accordance with the measurements of the antibody titers, the amount of antigen had an impact only in the id-immunized mice, which presented enhanced avidity after the mice received higher antigen doses ( figure 2e ,f and figure s4c ,d). to analyze the immune responses elicited in the vaccinated mice, we assessed the cytokine secretion profile of spleen cells collected two weeks following the administration of the last vaccine dose. as indicated in figure 3 , different cytokine secretion patterns were observed according to the tested administration route. mice immunized via the tc route showed higher tumor necrosis factor alpha (tnf-α) and interleukin 4 (il-4) levels than shown by those immunized via the id route ( figure 3b,d) . in contrast, id-immunized mice mounted higher il-10 and il-2 responses than shown by those immunized via the tc route ( figure 3c,e) . nonetheless, an increase in interferon gamma (ifn-γ) was found following immunization via both routes. flow-cytometry assays were also performed with spleen cells after the immunization protocols prior to challenge, but no significant difference among immunized versus nonimmunized mice was observed [26] . alpha (tnf-α) and interleukin 4 (il-4) levels than shown by those immunized via the id route ( figure 3b,d) . in contrast, id-immunized mice mounted higher il-10 and il-2 responses than shown by those immunized via the tc route ( figure 3c,e) . nonetheless, an increase in interferon gamma (ifn-γ) was found following immunization via both routes. flow-cytometry assays were also performed with spleen cells after the immunization protocols prior to challenge, but no significant difference among immunized versus nonimmunized mice was observed [26] . mice immunized via the tc or id route were subjected to a lethal i.c. challenge with the denv2 ngc strain. as shown in figure 4 , the immunized mice developed partial or complete protective immunity to a lethal challenge with the denv2 strain. in the tc-immunized mice, the conferred protection was dependent on the addition of lt1 and the antigen amount, with maximal survival values observed in the mice immunized with 50 μg of denv2 combined with lt1 (100% survival in comparison with 20% survival in the sham-treated group) ( figure 4a ). among the id-immunized mice, the group immunized with the higher antigen dose and lt1 were fully protected against the lethal challenge ( figure 4b ). we also looked for signs of morbidity in the mice challenged with the denv2 ngc strain ( figure 4c,d) . among the immunized mice, only those immunized with the higher antigen dose combined with lt1 displayed the maximal protection compared to that shown by the sham-treated animals ( figure 4d ). at the end of the observation period, all the mice immunized via the id route and 80% of the mice immunized via the tc route showed no signs of mice immunized via the tc or id route were subjected to a lethal i.c. challenge with the denv2 ngc strain. as shown in figure 4 , the immunized mice developed partial or complete protective immunity to a lethal challenge with the denv2 strain. in the tc-immunized mice, the conferred protection was dependent on the addition of lt1 and the antigen amount, with maximal survival values observed in the mice immunized with 50 µg of denv2 combined with lt1 (100% survival in comparison with 20% survival in the sham-treated group) ( figure 4a ). among the id-immunized mice, the group immunized with the higher antigen dose and lt1 were fully protected against the lethal challenge ( figure 4b ). we also looked for signs of morbidity in the mice challenged with the denv2 ngc strain ( figure 4c,d) . among the immunized mice, only those immunized with the higher antigen dose combined with lt1 displayed the maximal protection compared to that shown by the sham-treated animals ( figure 4d ). at the end of the observation period, all the mice immunized via the id route and 80% of the mice immunized via the tc route showed no signs of morbidity. altogether, these results indicated that the administration of the tested vaccine formulation administered via either the tc or id route induced protective immunity against denv2. viruses 2020, 12, 514 9 of 16 viruses 2020, 12, x for peer review 9 of 15 morbidity. altogether, these results indicated that the administration of the tested vaccine formulation administered via either the tc or id route induced protective immunity against denv2. despite a number of reports describing the use of tc for the delivery of different antibacterial and antivirus vaccines [5] [6] [7] [8] [14] [15] [16] [17] [18] [19] [27] [28] [29] [30] , thus far, only two studies addressed the performance of denv vaccines, delivered with microneedle pads, that were tested under experimental conditions [31, 32] . here, we showed mice immunized with whole denv2 particles through the use of adhesive patches applied on the surface of the skin and compared the results with regard to mice immunized via a parenteral route (id). the results demonstrated that induction of dose-dependent serum virus-specific antibody responses required the incorporation of an adjuvant (lt1). in addition, although mice immunized via the tc route developed lower serum igg responses, the protective immunity elicited in both mouse groups was similar. such results indicate that differences in the properties of antigen-specific antibodies or activation of differential cellular immune responses contribute to the protective immunity induced in tc-immunized animals. there are several ways to perform tc immunizations that include the use of different technologies to break the skin barrier. microneedle devices represent an alternative to deliver the antigens right below the sc [32] . on the other hand, tc immunization may be performed with adhesive patches soaked with the antigen and applied directly on the surface skin after a gentle superficial abrasion [33] . the use of adhesive patches has several inherent advantages over parenteral routes, such as the lack of needles and pain and reduced risks of contamination, but precise determination of the amount of antigen actually delivered into the host is not possible. a precise definition of the efficiency of the tc administration route would, therefore, require comparison of immune responses and protective immunity induced after immunization with the same vaccine delivered via a parenteral route. in the present study, we performed, for the first time, such a comparison with denv antigens and demonstrated that antigen delivered via the tc route induced similar immune responses and, more relevantly, protective immunity to denv, thus representing an alternative for the administration of denv vaccines. the recent report of microneedle-based tc immunization with a live-attenuated denv vaccine further supported the use of this delivery route for the administration of live virus vaccines [32] . previous reports demonstrated that the tc route is a safe and efficient immunization method [15] [16] [17] [18] [27] [28] [29] 34, 35] . in particular, the tc administration route prevents the toxicity of certain adjuvants, such as lt1, which may induce local and systemic side effects when delivered via parenteral routes [23, 36] . indeed, our results demonstrate that administration of denv2 particles admixed with lt1 and delivered through the tc route led to the generation of specific antibody and cellular responses without any significant deleterious effects or local inflammatory reactions. studies describing the use of viral antigens administered via the tc route usually exhibit a wide range of antigen doses [7, 14, 19, 27, 37] . the antigen loads tested in the present study were designed to permit comparisons with mice immunized under the same conditions via a parenteral (id) route. interestingly, similar amounts of denv envelope (e) protein were shown to be immunogenic and protect against challenge after skin application of the microneedle device [31] . immunization of mice with the same vaccine via two different administration routes allowed for comparisons of the impact on the antigen-specific immune responses and the induction of protective immunity. under the test conditions, the induction of anti-denv serum igg responses was lower in the mice subjected to tc immunization than it was in the mice immunized via the id route. this difference was clear after the third immunization dose and may be ascribed to the natural limitations of the naked ablated skin to efficiently deliver antigens compared with that delivered via parenteral inoculations. despite such differences, mice immunized via the tc or id route developed similar protective immunity after challenge with the denv2 ngc strain. the immune responses elicited in the mice immunized via the tc route, therefore, confer a more efficient protective immunity against denv. antibodies increased in the mice immunized via the tc route displayed a different reactivity toward conformation-dependent epitopes presented on the virus particles. heat denaturation of the virus particles is expected to disrupt conformational epitopes present on structural proteins, while linear epitopes would not be affected to the same extent after similar exposure to the protein denaturation step. indeed, results from previous studies based on human monoclonal abs (hmabs) generated from patients infected with denv demonstrated that protective antibodies react preferentially to tertiary and quaternary conformational epitopes of the e protein [38] . thus, the lower reactivity of the antibodies to the heat-denatured viruses in the tc-immunized mice may reflect differences in epitope specificity. antigen avidity has been reported to affect the capacity of antibodies to neutralize viruses [39] . although mice immunized via the id route developed antibodies with higher antigen avidity than those immunized via the tc route, particularly the mice immunized with the higher antigen concentration (50 µg/dose), the protective immunity to denv2 was similar in both groups. thus, antigen avidity did not seem to play a relevant role in the immune protection against denv observed under the test conditions. the classic protection correlate for denv infection consists of the induction of neutralizing antibodies, but several results generated under experimental and clinical conditions indicated that the cellular arm of the immune system also plays a relevant role in the development of antiviral immunity [40, 41] . modulation of immune responses, particularly the production of th1-related cytokines, such as ifn-γ and tnf-α, enables the activation of immune cells and control of the intracellular replication of viruses through both direct destruction of infected host cells and noncytotoxic pathways [42] . the abundant presence of intraepithelial antigen-present cells (apcs), such as langerhans cells (lcs), capable of processing exogenous antigens and priming naïve t cells, is expected to improve the performance of vaccines delivered via this route [13] . furthermore, a previous report showed that, in contrast to other antigen-presenting cells, few lcs are infected by denv [43] , indicating the more efficient activation of b and t lymphocytes. in this study, the mice immunized via the tc route secreted high amounts of ifn-γ and, particularly, tnf-α. on the other hand, spleen cells collected from the id-immunized mice secreted ifn-γ and il-2. the production of ifn-γ and tnf as associated with the expression of cd107a was previously correlated with protection mediated by cd8 + t cell responses [44, 45] . moreover, ifn-γ, tnf-α and il-2 production was correlated with subclinical denv secondary infections [46] and may contribute to protection through multifunctional cd4 + t cells after vaccination [47] . the same pattern was observed in hiv patients with lower viral loads, indicating that proinflammatory responses are relevant for viral control [48] . spleen cells collected from the mice immunized via the tc route also secreted higher amounts of il-4 and had lower production of il-10 in comparison with the mice immunized via the id route. both il-4 and il-10 were previously correlated with severity increases in denv diseases [49] [50] [51] . il-4 is associated with the upregulation of denv-targeted receptors, leading to an increase in denv infectivity in dermal apcs [52] . the production of il-4 is modulated by denv during infection, which explains the crescent levels after the increase in viral particle administration or adjuvant-mediated inflammation. il-4 also induces the transformation of b cells in plasma cells [52] , increasing the amount of specific antibodies produced, as observed at a level of significance in the group that received 50 µg of denv2. il-10 is usually associated with the suppression of pathogen-mediated inflammation and is found in high amounts in the sera of denv-infected mice and humans [53, 54] . the presence of il-10 + monocytes after yellow fever (yfv) vaccination was associated with a reduction in immune responses and immune system adverse reactions [55, 56] . accordingly, higher levels of il-10 were expected to be observed after id inoculation with lt1. furthermore, il-10 + cd8 + t cells showed higher cytolytic activity than did the il-10 -cd8 + t cells during the control of coronavirus-induced acute encephalitis [57] , suggesting possible enhancement of the survival and activation of cd8 + t cells during acute inflammation. collectively, these results demonstrated that the same vaccine formulation delivered by either the id or tc routes triggered different cellular immune responses, as indicated by the cytokine secretion pattern of total spleen cells collected from the vaccinated animals. the experimental virus challenge model used in the present study depended on the i.c. inoculation of a mouse-adapted denv2 strain (ngc). although this infection route did not correspond to the infection process observed under natural conditions, the mouse-adapted virus strain represents a classic tool for the determination of protective immunity elicited in immunocompetent mice [58] [59] [60] . it has been demonstrated that serum antibody responses are only partial contributions to the encephalitis developed in i.c. challenge with denv viruses [43] . furthermore, the depletion of cd4 + or cd8 + t lymphocytes drastically reduced the vaccine-based protection observed in the encephalitis model [43, 61] . thus, the high protective status induced by the tested vaccine administered via both inoculation routes suggests the relevant role of cellular immune responses. this study adds further experimental evidence that, similar to other virus vaccines, the tc route represents a promising route for vaccine administration capable of inducing protective immunity without the concerns associated with needle-delivered vaccines. despite difficulties in standardizing the amount of antigen effectively delivered into the host tissues, this vaccine administration route is safer and a similarly effective alternative to parenterally administered denv vaccines. further studies based on preclinical models should contribute to the translation of the present results into an alternative for the clinical testing of denv vaccines in humans. the following are available online at http://www.mdpi.com/1999-4915/12/5/514/s1, figure s1 : antigens and transcutaneous immunization procedures adopted in the study. figure s2 : serum igg responses in mice immunized with purified denv2 particles. figure s3 : hematological analyses of mice submitted to tc or id immunizations with concentrated denv2 particles. figure s4 : binding of anti-denv antibodies to viral particles following antigen heat denaturation or treatment with ammonium thiocyanate. cost of dengue outbreaks: literature review and country case studies the global distribution 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natural polymorphic variant of a heat-labile toxin (lt-i) produced by enterotoxigenic escherichia coli (etec): research article a genetic and pathologic study of a denv2 clinical isolate capable of inducing encephalitis and hematological disturbances in immunocompetent mice targeting the non-structural protein 1 from dengue virus to a dendritic cell population confers protective immunity to lethal virus challenge other adverse reactions, such as weight loss or altered animal behavior, were observed in the mice in which the vaccine regimens were tested preferential amplification of cd8 effector-t cells after transcutaneous application of an inactivated influenza vaccine: a randomized phase i trial transcutaneous yellow fever vaccination of subjects with or without atopic dermatitis coated microneedle arrays for transcutaneous delivery of live virus vaccines transcutaneous anti-influenza vaccination promotes both cd4 and cd8 t cell immune responses in humans efficient delivery of dengue 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dengue virus infection protective role of cross-reactive cd8 t cells against dengue virus infection cells can mediate short-term protection against heterotypic dengue virus reinfection in mice gamma interferon can prevent herpes simplex virus type 1 reactivation from latency in sensory neurons antibodies are not required to a protective immune response against dengue virus elicited in a mouse encephalitis model a protective role for dengue virus-specific cd8 + t cells cells are not required for the induction of dengue virus-specific cd8 + t cell or antibody responses but contribute to protection after vaccination intracellular cytokine production by dengue virus-specific t cells correlates with subclinical secondary infection primary vaccination with low dose live dengue 1 virus generates a proinflammatory, multifunctional t cell response in humans presence of hiv-1 gag-specific ifn-γ + il-2 + and cd28 + il-2 + cd4 t cell responses is associated with nonprogression in hiv-1 infection vascular endothelium: the battlefield of dengue viruses differing influences of virus burden and immune activation on disease severity in secondary dengue-3 virus infections thrombocytopenia in dengue fever dermal cd14 + dendritic cell and macrophage infection by dengue virus is stimulated by interleukin-4 il-10 levels in dengue patients: some findings from the exceptional epidemiological conditions in cuba vascular endothelial growth factor kdr receptor signaling potentiates tumor necrosis factor-induced tissue factor expression in endothelial cells innate immunity phenotypic features point toward simultaneous raise of activation and modulation events following 17dd live attenuated yellow fever first-time vaccination characterization of main cytokine sources from the innate and adaptive immune responses following primary 17dd yellow fever vaccination in adults highly activated cytotoxic cd8 t cells express protective il-10 at the peak of coronavirus-induced encephalitis protection against dengue type 2 virus induced in mice immunized with a dna plasmid encoding the non-structural 1 (ns1) gene fused to the tissue plasminogen activator signal sequence production of a recombinant dengue virus 2 ns5 protein and potential use as a vaccine antigen immunogenic and protective response in mice immunized with a purified, inactivated, dengue-2 virus vaccine prototype made in fetal rhesus lung cells flow-cytometry assays were also performed with spleen cells after the immunization protocols prior to challenge we thank mariana cintra and pietro pisani for technical support in tc immunizations and eduardo gimenes martins for the administrative and technical support. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord-353600-5wo74ms4 authors: tyrrell, daniel j.; goldstein, daniel r. title: ageing and atherosclerosis: vascular intrinsic and extrinsic factors and potential role of il-6 date: 2020-09-11 journal: nat rev cardiol doi: 10.1038/s41569-020-0431-7 sha: doc_id: 353600 cord_uid: 5wo74ms4 the number of old people is rising worldwide, and advancing age is a major risk factor for atherosclerotic cardiovascular disease. however, the mechanisms underlying this phenomenon remain unclear. in this review, we discuss vascular intrinsic and extrinsic mechanisms of how ageing influences the pathology of atherosclerosis. first, we focus on factors that are extrinsic to the vasculature. we discuss how ageing affects the development of myeloid cells leading to the expansion of certain myeloid cell clones and induces changes in myeloid cell functions that promote atherosclerosis via inflammation, including a potential role for il-6. next, we describe vascular intrinsic factors by which ageing promotes atherogenesis — in particular, the effects on mitochondrial function. studies in mice and humans have shown that ageing leads to a decline in vascular mitochondrial function and impaired mitophagy. in mice, ageing is associated with an elevation in the levels of the inflammatory cytokine il-6 in the aorta, which participates in a positive feedback loop with the impaired vascular mitochondrial function to accelerate atherogenesis. we speculate that vascular and myeloid cell ageing synergize, via il-6 signalling, to accelerate atherosclerosis. finally, we propose future avenues of clinical investigation and potential therapeutic approaches to reduce the burden of atherosclerosis in old people. the number of old people (aged >65 years) is rising worldwide, and cardiovascular diseases are the largest contributor to morbidity and mortality in this popu lation 1, 2 . changes in diet and lifestyle contribute to the high cardiovascular morbidity and mortality in old indi viduals, but many biological processes that are altered with ageing also contribute to this increased cardio vascular risk. as a result, therapies for cardiovascular disease that are effective in young and middle aged peo ple might be less effective in older people. additionally, novel therapies might be required to improve disease management specifically in old people. deciphering the mechanisms by which ageing promotes atheroscle rotic cardiovascular disease will be fundamental for the development of novel therapies to reduce the burden of atherosclerosis with ageing. the development of new therapies is especially relevant with the coronavirus dis ease 2019 (covid19) pandemic, because old people and particularly those with cardiovascular diseases are at a substantially higher risk of morbidity and death 3, 4 . in this review, we describe two major areas by which ageing promotes atherosclerosis. first, we dis cuss age related factors that are extrinsic to the vascula ture, focusing on the effects of ageing on myeloid cells of the immune system. age related effects in the bone marrow skew the differentiation of haematopoietic cells towards the myeloid cell lineage. ageing also promotes the generation of clones of haematopoietic cells with out clear development of haematopoietic malignancy or other known clonal disorder, a phenomenon known as clonal haematopoiesis of indeterminate potential (chip) [5] [6] [7] [8] . clinical studies from the past decade have revealed that the presence of chip increases the risk of cardiovascular diseases 6, 7 . intriguingly, the increased risk of cardiovas cular disease associated with the presence of chip is abrogated in patients with a loss of function mutation in il6 (ref. 9 ). however, the precise mechanisms by which chip promotes the development of cardiovascular diseases are yet to be fully clarified. we next address vascular intrinsic factors, discussing how ageing impairs vascular bioenergetics by compro mising mitochondrial function and how this alteration connects with inflammatory pathways within the vascu lature to promote atherosclerosis 10, 11 . we describe studies in mice and humans showing that, in the aorta, ageing impairs both mitochondrial function and the removal of damaged mitochondria (mitophagy) 10, 11 . we describe experimental evidence reported in 2020 demonstrating that the age mediated increase in the levels of the plei otropic cytokine il6 in the aorta occurs in a positive clonal haematopoiesis of indeterminate potential (chip) . clonal expansion of haematopoietic stem cells that carry certain somatic mutations that confer a cell proliferation advantage. feedback loop with vascular mitochondrial dysfunction and that these alterations promote atherosclerosis 11 . the cantos study 12 demonstrated that il1β block ade reduces the risk of recurrent cardiovascular events in patients aged >60 years. importantly, the greatest bene fit of il1β blockade was seen in patients who had low plasma il6 levels 13 . this pivotal clinical study indicates that chronic inflammation, potentially via il6 signal ling, is a major contributor to age related atherosclerosis. given this observation, we speculate that increased ath erosclerosis with ageing could result from a synergy between myeloid cells of the immune system and the vasculature via il6 signalling (fig. 1 ). this mechanism is especially important because clinically approved agents targeting this pathway (such as anti il6 therapies) are already available and could reduce the risk of cardio vascular disease in old people. finally, we propose that future experimental and clinical investigation will be required to determine the contribution of this inflamma tory pathway in age related atherosclerosis. we acknowl edge that other inflammatory pathways and cytokines could contribute to age related atherosclerosis, and the source of these cytokines (including il6) could be senescent adipocytes. a detailed discussion of the con tribution of ageing to senescence and atherosclerosis has been published previously 14 . ageing affects the immune system in complex ways (as reviewed previously [15] [16] [17] , and various components of the immune system contribute to atherosclerosis 18, 19 . this review focuses on clones of myeloid cells that increase with ageing and how these clones contribute to athero sclerosis. we do not describe how ageing affects other cells of the immune system, which has been reviewed previously (for example, b cells 20 , t cells 21 , eosinophils or dendritic cells 22 ). in addition, we focus on vascular mito chondrial function and how mitochondrial dysfunction could influence inflammatory pathways within the vasculature. however, given that most of the available evidence indicates that oxidative stress is not a major driver of biological ageing [23] [24] [25] , and given the complex roles that oxidative stress has in atherosclerosis 26 , we do not describe in detail the contributions of oxidative stress in age related atherosclerosis. neither do we describe in detail how ageing affects other processes within the arte rial wall, such as extracellular matrix remodelling or pro duction of pro fibrotic and pro calcific factors, which can promote atherosclerosis indirectly via increasing arterial stiffness and hypertension 27, 28 , and which have been previously reviewed 29, 30 . effects of ageing on myeloid cell production. numerous subpopulations of immune cells of various lineages have been implicated in atherosclerosis, including macrophages 31 , dendritic cells 32 , t helper 1 (t h 1) cells 33 and b cells 34 (reviewed previously 18, 35 ), all of which are affected by ageing. immune cells are generated in the bone marrow via haematopoiesis from regenerative haematopoietic stem cells (hscs) 36, 37 . monocytes, mac rophages and neutrophils are derived from myeloid biased hscs. with ageing, although the absolute number of hscs increases 38 , hscs lose their regener ative capacity 39, 40 . this loss of regenerative potential is accompanied by an expansion of the number of hscs that are committed to the platelet (megakaryocytes) and myeloid lineages 38, 41 . competitive bone marrow transplantation studies in mice have demonstrated that aged hscs have a reduced repopulation capacity, with an imbalance towards myeloid cell differentiation, compared with young hscs 38, 42 . several major biological pathways contribute to ageing, including dna damage, mitochondrial dysfunction, cell senescence, impaired autophagy, epigenetic alterations and gene transcrip tion dysregulation 25 . transcriptomic studies in mice have shown that with ageing, hscs upregulate stress responses and inflammatory pathways and downregu late the expression of genes related to genetic stability 43 . with ageing, hscs exhibit an increase in epigenetic dysregulation, specifically downregulation in chroma tin remodelling and transcriptional silencing 43 , and increases in dna methylation (as reviewed previously 44 ). these epigenetic alterations are accompanied by func tional defects in hscs, including a reduction in hsc homing to the bone marrow and hsc proliferation 43, 45 . importantly, mutations in genes such as idh2, which alter epigenetic regulation, lead to impairments in hae matopoietic progenitors in mice 46 and are associated with t cell lymphomas in humans 47 , a malignancy that increases with ageing. although whether hscs, or stem cells in general, undergo senescence is questionable 48 , the clearance of senescent cells improves hsc engraft ment in bone marrow transplantation mouse models and reduces myeloid skewing 49 . autophagy deficient young mice have increased mitochondrial content and metabolism that lead to mitochondrial stress in hscs compared with wild type young mice 50 . these features are also observed in aged wild type mice and are asso ciated with a skewing towards the myeloid lineage and a reduced proliferative capacity of hscs 50, 51 . loss of microrna146a (mir146a) in hscs with ageing also promotes a myeloid bias 52 . furthermore, myeloid cells derived from mir146a deficient hscs have elevated levels of both il6 and tumour necrosis factor (tnf) 52 , which connects altered regulation of transcription in hscs to inflammation. overall, these studies indicate that ageing has effects on hscs via several complex pathways that lead to reduced hsc function. ageing also alters haematopoiesis by influencing the bone marrow niche independently of the direct effects • ageing-related alterations in the bone marrow increase the phenomenon of clonal haematopoiesis of indeterminate potential (chip) and promote a skewing towards myeloid cell differentiation, both of which can accelerate atherosclerosis. • the increased risk of atherosclerotic cardiovascular diseases associated with the presence of chip might be mediated by il-6 signalling and/or inflammasome activation. • ageing is associated with a decline in mitochondrial function and an increase in il-6 levels in the vasculature, and both effects probably accelerate atherosclerosis independently of chronic hyperlipidaemia. • the role of the vasculature and myeloid cells of the immune system in promoting age-related atherosclerosis might be mediated by shared inflammatory pathways, in particular il-6 signalling. senescence a state of permanent replicative arrest in normally proliferative cells. www.nature.com/nrcardio on hscs. the bone marrow niche provides a support ing environment for hsc function and includes mesen chymal cells and endothelial cells 53 . how ageing affects the bone marrow niche is not clear, but the presence of chronic systemic inflammation might contribute. ageing leads to a chronic systemic low grade inflammatory state 54, 55 , which might be mediated by cellular senescence that leads to the production of inflammatory mediators (termed the senescence-associated secretory phenotype (sasp)) 56,57 . one source of senescent inflammatory cells is the adipose tissue, which typically increases in size with ageing 58, 59 . the number of adipocytes also increases in the bone marrow with ageing, accompanied by an elevation in the levels of pro inflammatory cytokines, including il6 (refs 60,61 ). these cytokines promote a skewing towards myeloid cell differentiation and an increase in platelet production, the latter of which could contribute to thrombosis 60, 61 . importantly, adipocytes arising from leptin receptor positive progenitors in the bone marrow, but not within other fat depots, synthesize stem cell factor, which promotes hsc regeneration 62, 63 . senescent stromal cells in the bone marrow are another potential source of inflammation 64 . these cells can dif ferentiate into adipocytes in the ageing bone marrow 65 and further promote an inflammatory environment. the function of bone marrow endothelial cells also declines with ageing 66 . furthermore, the number of vascular niches in the bone marrow that support hsc regenera tion decreases with ageing, but can be restored in aged mice by activating notch signalling in endothelial cells 53 . activation of the innate immune receptor toll like receptor 4 (tlr4) induces myeloid differentiation in hscs in mice 67 . ageing is associated with alterations in gut microbiota 68 , which could act as a microbial source for tlr4 stimulation (for example, lipopolysaccharide (lps) from gram negative bacteria activates tlr4). tlr4 activation could then increase the imbalance of hsc differentiation towards the myeloid lineage. in a 2019 study in mice, β 2 adrenergic receptor signalling in the bone marrow niche was found to increase with ageing in association with increased generation of myeloid cells and platelets through an il6 dependent mechanism 60 . this study also demonstrated that the bone marrow niche switches from an endosteal to a non endosteal mutation show an increased production of il-6 and il-1β, which can contribute to accelerated atherosclerosis. ageing can also have pro-atherogenic effects directly on the vasculature. ageing is associated with an increase in the levels of il-6, possibly mediated by increased production by vascular smooth muscle cells (vsmcs), and mitochondrial genomic instability and with a decline in mitochondrial function in the vasculature. the reduced mitochondrial function alters mitophagy and increases il-6 levels, creating a positive feedback loop that accelerates atherogenesis. vascular ageing also leads to the production of chemoattractants that increase myeloid cell recruitment into the arterial wall, further promoting atherosclerosis. impaired mitochondrial function combined with reduced mitophagy might lead to increased levels of reactive oxygen species (ros). senescence-associated secretory phenotype (sasp). secretion of cytokines, chemokines, growth factors and proteases by senescent cells. nature reviews | cardiology niche with ageing, indicating that ageing shifts myeloid cell production away from the bone tissue to further within the bone marrow 60 . this study also found that a mouse model of hutchinson-gilford progeria syn drome, which is associated with accelerated ageing 69 , had an imbalance favouring myeloid cells over lymphoid cells in the peripheral blood 60 . this effect was mitigated by administration of a β 3 adrenergic receptor agonist 60 . overall, clear evidence indicates that ageing alters the bone marrow niche via multiple mechanisms to impair hsc function and promote myeloid cell differentiation. clonal haematopoiesis and cardiovascular disease: clinical correlation. the positive selection and expansion of clones of hscs carrying certain somatic mutations, known as clonal haematopoiesis, occurs commonly with ageing. approximately 10% of individuals aged >70 years carry mutations associated with clonal haemato poiesis, whereas these mutations are rare in individu als aged <40 years 7, 70, 71 . these clones of haematopoietic cells harbour single somatic mutations most commonly in genes associated with haematological malignan cies, such as dnmt3a, tet2 and asxl1. individuals with mutations in these genes have an increased risk of developing haematological malignancies (hr 11-12, depending on the study) 7, 70, 71 . all cause mortality is increased in individuals with any somatic mutation asso ciated with clonal haematopoiesis (hr 1-2) compared with those with no mutations 7 . interestingly, the cause of the increased mortality in these individuals is not only the higher rate of haematological malignancies but also a higher rate of adverse cardiovascular events 7,70-72 . the association between clonal haematopoiesis and the risk of adverse cardiovascular events remained even after adjustment for traditional cardiovascular risk fac tors, such as diabetes mellitus, hypertension, smoking and bmi, in multivariate analyses 6 . as a result of these studies, the term chip was coined to distinguish the phenomenon of clonal haematopoiesis without clear development of haematopoietic malignancy or other known clonal disorder from the pre malignant clonal haematopoiesis of clinical importance 73 . a follow up clinical study provided further evidence of the association between cardiovascular disease and chip 6 . in particular, old individuals (aged 60-70 years) with chip had an approximately twofold higher risk of incident coronary artery disease, a fourfold higher risk of early onset myocardial infarction and a three fold higher coronary artery calcium score than similarly aged individuals without chip 6 . importantly, the size of the chip clone, defined as the variant allele frequency (vaf), correlates with the risk of cardiovascular dis ease. specifically, individuals with a chip clone with a vaf of >10% have a 12 fold increased risk of cardi ovascular disease compared with individuals with no mutations, whereas the risk of cardiovascular disease is not significantly increased in chip carriers with a vaf of <10% 6 . this study has established that chip is associated with the risk of cardiovascular diseases and has developed a potential new paradigm that certain clones of haematopoietic cells accelerate atherogenesis 6 . however, to date, the presence of chip can be used only as a biomarker of atherosclerosis and is not therapeutically actionable. mutations in tet2 are the second most prevalent somatic mutations associated with chip after those in dnmt3a. mouse models have been used to elucidate the mechanistic contributions of tet2 mutations to ath erogenesis. in irradiated, atheroprone ldlr −/− mice, those reconstituted with either tet2 −/− or tet2 +/− bone marrow had increased atherosclerotic lesion size compared with mice receiving wild type bone marrow 6, 74 . these data imply that deletion of one copy of the tet2 gene is sufficient to increase atherosclerosis in mice. further studies in mice showed that myeloid cell specific tet2 deficiency increases atherosclerotic plaque size 74 . interestingly, tet2 deficiency in bone marrow derived macrophages leads to an elevated secretion of il6 and il1β (a signature cytokine produced by inflammasome activation) 75 in response to various stimuli (such as ldl, lps and ifnγ) in vitro 74 . furthermore, the increased atherogenic potential of tet2 −/− bone marrow cells is reduced when bone marrow transplantation recipients are treated with a small molecule inhibitor of the nlrp3 inflammasome 74 . the effect of tet2 deficiency might not be limited to vascular diseases, because experimen tal studies have demonstrated that transfer of tet2 −/− bone marrow cells into non irradiated mice accelerates the development of age related cardiac hypertrophy and fibrosis 76 , and tet2 deficiency in myeloid cells worsens the development of heart failure in mice after acute injury 77 . the contribution of il6 to the cardiovascular risk in individuals with large chip clones (vaf >10%) was evaluated in 35,416 individuals without prevalent cardi ovascular disease enrolled in the uk biobank registry 9 . the investigators examined whether an il6r coding mutation that leads to reduced il6 signalling alters the association between chip and the risk of adverse cardiovascular events (myocardial infarction, coronary artery disease revascularization, stroke or death). the study revealed that the presence of the il6r mutation mitigated the increased risk of adverse cardiovascular events in individuals with large chip clones but not in individuals without chip 9 . these data indicate that il6 signalling is causally linked to the increased risk of cardiovascular disease associated with chip. il6 is released following inflammasome activation 78 ; therefore, the observed link between il6 and chip suggests that inflammasome activation is a mecha nism by which chip promotes the development of cardiovascular diseases. this concept is compatible with the study in mice discussed above, showing that the nlrp3 inflammasome contributes to the increased atherosclerotic burden induced by tet2 −/− bone marrow transplantation 74 . the contribution of il1β to cardio vascular disease in humans was demonstrated in the cantos study 12, 13 , which showed that a monoclonal antibody against il1β reduces the risk of recurrent car diovascular events in patients with previous myocardial infarction. the effects of il1β blockade in the cantos trial were greater in patients who had lower circulating variant allele frequency (vaf) . the proportion of sequences that match a gene mutation divided by the overall coverage at that gene locus. www.nature.com/nrcardio il6 levels after il1β blockade than in those with higher circulating il6 levels 13 . however, the role of il1β and il6 in experimental models of atherosclerosis is not completely clear because data indicating that each of these cytokines has atheroprotective effects have been reported 79, 80 . however, these studies were performed in young mice, so these cytokines might have increasingly pathogenic roles with ageing. monocytes and macrophages contribute to both the initiation of the chronic inflammatory process of ath erosclerosis and the resolution of the chronic vascular inflammation 81 . ageing directly influences the function of monocytes and macrophages 16 . human monocytes have lower levels of tlrs and a reduction in tlr dependent pro inflammatory cytokine production with ageing 16 . a study comparing bone marrow derived monocytes from young and aged atheroprone ldlr −/− mice showed that ageing leads to a downregulation in the expression of tnf and il1b but monocyte chemotaxis is preserved 82 . aged (6 month old) atherosclerotic apoe −/− mice have a reduction in the number of vascular progenitor cells in the bone marrow compared with 1 month old atheroscle rotic apoe −/− mice 83 . furthermore, administration of bone marrow derived hscs from young non atherosclerotic mice to non irradiated 6 month old apoe −/− mice reduced atherogenesis after feeding a high fat diet 83 . this finding suggests that ageing is accompanied by a reduc tion in the number of atheroprotective progenitor cells in the bone marrow. aged (18-21 month old) mice with chronic or induced acute hyperlipidaemia have more macrophage infiltration into atherosclerotic lesions than young mice 11, 82 . furthermore, the aortas of aged athero sclerotic mice (12 months old) and rats (30 months old) have higher levels of macrophage attracting chemokines and il6 than the aortas of young atherosclerotic mice (2 months old) and rats(10 months old) 82, 84 . although macrophages and monocytes can have an increased basal secretion of inflammatory cytokines, such as il1β, il6 and il8, with ageing 85 (possibly owing to senescence) 57 , whether these cells are the major contributors to the increased vascular production of il6 with ageing during atherogenesis is unclear. vascular cells such as vascular smooth muscle cells (vsmcs) have been shown in animal models to have an elevated il6 production with ageing before any signs of atherosclerosis development 86, 87 . efferocytosis is a crucial mechanism for resolving plaque inflammation and reducing atherosclerosis progression 88 . in vivo and in vitro assays have indi cated that the phagocytic function of tissue alveolar macrophages to take up apoptotic neutrophils declines with ageing 89 and is associated with reduced expres sion of scavenger receptor cd204 (ref. 90 ). in a mouse model of peritonitis, ageing led to reduced resolution of acute inflammation and was associated with reduced levels of pro resolution lipid mediators, specifically resolvins 91 . resolution of inflammation was also delayed with ageing in a human model of skin blistering 92 . this phenotype is related to reduced expression of the effe rocytotic receptor tim4 in macrophages. reduced tim4 expression with ageing was caused by elevations in p38 mitogen activated protein kinase activity in macrophages, and treatment with an oral p38 inhibitor increased the resolution of blister inflammation in old individuals 92 . overall, macrophages show impaired inflammation resolution properties with ageing; how ever, whether this impaired macrophage function contributes to increased atherosclerosis is not yet clear. vascular mitochondrial dysfunction with ageing before atherogenesis initiation. ageing affects the vasculature before the development of atherosclerosis. generally, ageing is associated with remodelling of the arterial wall, with evidence of reduced endothelial cell function, increased collagen deposition, fibrosis and functionally stiffer vessels 28, 93, 94 . in addition, vsmcs acquire a more proliferative and synthetic function with ageing 86 . vsmcs also show an increased generation of reactive oxygen spe cies (ros) and high oxidative damage 95 . endothelial cells also have a dysregulated antioxidant capacity with ageing (mediated by the disruption of nuclear factor erythroid 2 related factor 2 signalling), thereby contributing to vas cular ageing 96, 97 . all these effects of ageing can contribute to the development of hypertension, a major risk factor for cardiovascular disease. most studies on vascular ageing in rodent models have been performed in normolipidaemic animals. these studies provide evidence that mitochondrial dysfunction, a known hallmark of ageing 25 , contrib utes to vascular ageing before the initiation of athero genesis. disease free, normolipidaemic mice develop mitochondrial dysfunction in the aorta as they age, first detected at 11 months of age (measured as a decline in oxygen consumption rate (ocr)) and becoming more evident as the mice reach 18 months of age 98 . the reduction in ocr is accompanied by an increase in mitochondrial dna (mtdna) damage 98 , a sign of mito chondrial genomic instability, which is another hallmark of ageing 25 . furthermore, reduced vascular mitochon drial function with ageing is accompanied by a decrease in the expression of the mtdna helicase twinkle 98 , an enzyme involved in preserving mtdna integrity. aged transgenic mice expressing high levels of twinkle show delayed vascular ageing; in particular, the decrease in aortic compliance and the increase in aortic stiffness are delayed in these mice compared with aged wild type mice 98 . overall, experimental evidence indicates that mitochondrial dysfunction and mitochondrial genomic instability contribute to vascular ageing. in humans, atherosclerotic plaques show evidence of damage to mtdna, which is associated with reduced mitochondrial function, specifically lower ocr in the fibrous cap and core regions of the atherosclerotic plaque than in the shoulder region of the plaque or in non overtly diseased regions of the aorta 10 . these findings are compatible with those of previous experimental work indicating that apoe −/− mice fed a low fat, standard chow diet have increased vascular mtdna damage but not nuclear dna damage as the mice age 99, 100 . furthermore, human atherosclerotic plaques have lower levels of mitochondrial complex i and complex ii than non diseased aortic regions 10 . similar findings are noted in efferocytosis phagocytosis of apoptotic cells by phagocytic cells. atherosclerotic apoe −/− mice fed a high fat diet 10 . apoe −/− mice overexpressing twinkle have a reduced necrotic core area in atherosclerotic plaques compared with con trol apoe −/− mice 10 . mitochondrial dysfunction probably has a central role in ros generation but the interaction between these two factors is complex. for example, low levels of ros might improve cell fitness and pro mote survival, a concept known as mitohormesis 25, 101 . however, higher levels of ros might contribute to age related chronic vascular diseases. the complex inter action between mitochondrial dysfunction and ros might explain why disruption of some mitochondrial enzymatic pathways (such as nadph oxidase 1 (nox1) and nox2 signalling) in atherosclerotic mice has no effect on age related atherosclerosis 102 , whereas partial deficiency of ros scavenging enzymes (such as super oxide dismutase) 103 in atherosclerotic mice contributes to atherosclerosis 99 . however, one study found that mtdna damage occurs in both vsmcs and monocytes and cor relates with atherosclerotic burden in humans but with out evidence of alterations in ros levels 100 . furthermore, clinical trials on antioxidants have yet to reveal a benefi cial effect in patients with atherosclerotic cardiovascular disease 104, 105 . overall, mitochondrial dysfunction occurs during chronic hyperlipidaemia and atherogenesis, and this mitochondrial dysfunction promotes atherosclero sis. however, the precise role of ros in this context is complex and requires further investigation. part of the challenge of using stand ard mouse models of atherosclerosis (such as ldlr −/− or apoe −/− mice) to understand the role of ageing on atherogenesis is that even when fed a standard low fat diet, these mice age with chronic hyperlipidaemia. therefore, the effects of ageing cannot be dissected from the effects of chronic hyperlipidaemia. a study in mice published in 2020 circumvented this issue by first examining mitochondrial function in the aortas of young and aged wild type mice without hyperlipidaemia or vascular diseases 11 . consistent with previous studies, aged mice had evidence of reduced ocr in the aortas compared with young mice 11 . this ocr reduction in the vasculature from aged mice was accompanied by increased expression of the mitophagy protein parkin and increased basal mitophagy (box 1), a macroau tophagy process to remove damaged mitochondria. altered mitochondrial quality control in the ageing vas culature without hyperlipidaemia is linked to arterial stiffening in mice 106 . the mitochondrial dysfunction and elevated parkin levels with ageing in the mouse aorta are accompanied by an increase in tlr9, myd88 and il6 levels 11 . importantly, blocking il6 in aged mouse aortas in vitro increased the ocr and reduced parkin levels. this study identified a positive feedback loop in which mitochondrial dysfunction and elevated il6 levels coexist and positively influence each other 11 . however, the exact identity of the il6 producing and il6 responsive cell(s) has yet to be identified, although evidence suggests that vsmcs secrete more il6 with ageing 87 . to study the link between the changes occurring with normolipidaemia in the aged aorta and atherogen esis, young and aged wild type mice were made acutely hyperlipidaemic by inducing a decrease in ldl recep tor levels with adeno associated virus vector mediated during homeostasis, damaged mitochondria are recycled via mitophagy, which is a specialized subset of macroautophagy (see the figure) . mitophagy reduces the production of mitochondrial damage-associated molecular patterns (mtdamps) and limits inflammation. mitochondrial depolarization results in the accumulation of the serine/threonine protein kinase pink1 at the outer mitochondrial membrane, leading to the recruitment of parkin, an e3 ubiquitin ligase that ubiquitylates mitochondrial membrane proteins including mitofusin 1 (mfn1), mfn2 and voltage-dependent anion-selective channel protein 1 (vdac1). this ubiquitylation primes the mitochondria for targeting by the autophagy machinery, including sequestosome 1 (p62) and microtubule-associated protein 1 light chain 3 (lc3), to package mitochondria in autophagosomes and deliver them to lysosomes for degradation. other mitophagy mechanisms involve the apoptotic bcl-2 family proteins bcl-2/ adenovirus e1b 19 kda protein-interacting protein 3 (bnip3) and nip3-like protein (nix; also known as bnip3l), which dimerize and bind directly to lc3 and function as adaptors between mitochondria and autophagosomes. bnip3 and nix can also facilitate apoptosis and cell death by participating in the release of mitochondrial cytochrome c and opening of the mitochondrial permeability transition pore. pgc1α, proliferator-activated receptorγ co-activator 1α; tf, transcription factor. release of mtdamps www.nature.com/nrcardio delivery of pcsk9 and by feeding the mice a high fat diet for 10 weeks 11 , which is an established technique 107 . with this protocol, young and aged mice had similar and durable levels of hyperlipidaemia; however, aged hyperlipidaemic mice had larger atherosclerotic lesions with larger necrotic cores than young hyperlipidaemic mice 11 . importantly, administering spermidine, an agent that increases macroautophagy and mitophagy, to aged hyperlipidaemic mice reduced the levels of both il6 and parkin in the aorta and reduced the size of ather osclerotic plaques 11 . this finding is consistent with pre vious studies showing that treatment with spermidine or trehalose, an agent that increases mitophagy, reduces stiffness in the aged vasculature in normolipidaemic, non atherosclerotic aged mice 106, 108 . the cantos study 12 showed that in old patients (aged >60 years) with cardiovascular disease, il1β blockade reduces the risk of recurrent cardiovascular events, indi cating that chronic inflammation is a major contribu tor to age related atherosclerosis. as described above, mitochondrial dysfunction might coexist in a positive feedback loop with il6 signalling 11 to increase chronic inflammation in vascular ageing. furthermore, mito chondrial components that are released to the cyto sol after mitochondrial damage can stimulate innate immune responses 109 . mitochondrial injury in turn can be induced by tlr stimulation leading to the activa tion of caspase 4 and caspase 5 in humans or caspase 11 in mice 110 . these inflammatory caspases cleave gasder min d, which enables gasdermin d to form pores in the outer mitochondrial membrane, leading to impaired mitochondrial membrane potential and further increas ing mitochondrial injury 110 . whether this pathway is activated during ageing and in particular vascular age ing is unclear. nevertheless, the involvement of such a pathway could explain why chronic tlr activation, via either microbial products or sterile inflammatory medi ators, could lead to a chronic basal inflammatory state and mitochondrial dysfunction in the vasculature with ageing. mitochondrial injury leads to the release of mito chondrial components, known a s m it oc ho nd rial damageassociated molecular patterns (mtdamps), including mtdna, that when in the cytosol, can activate intracel lular innate immune signalling pathways, such as the dna sensing receptor cyclic gmp-amp synthase and the inflammasome 75, [110] [111] [112] . transfer of mitochondrial components into endosomes also activates the tlr9 inflammatory pathway 111 , but the detailed mechanisms are not fully elucidated. mitochondria also contain n formylated peptides that induce inflammation via engaging the n formyl peptide receptor 1 to increase neutrophil chemoattraction 113 , arterial injury and ros release 114 . cardiolipin, a component of mitochondrial membranes, can directly bind to nlrp3 and activate the nlrp3 inflammasome 115 . if chronically activated, all these pathways could promote vascular ageing and also diminish mitochondrial function, although ascertain ing the definitive contributions of each pathway requires future investigation. age related atherosclerosis might be mediated by alterations in the vasculature and mye loid cells via a shared inflammatory pathway. a potential candidate pathway is il6 signalling because available evidence indicates that the level of il6 is elevated with ageing in both the immune system and the vasculature. in the bone marrow niche in mice, il6 levels increase with ageing, which is probably mediated by increased β 2 adrenergic receptor signalling and increased num bers of adipocytes 60 (fig. 1) . il6 directly acts on hscs to promote a bias towards myeloid cell differentiation 60 . in mouse macrophages, tet2 deficiency, which is one of the most common genetic alterations found in the age related condition chip, increases il6 secretion in vitro 74 . importantly, the atherosclerosis promoting effects of chip seem to be abrogated in individuals with a loss of function il6 genetic polymorphism 9 . in the vasculature, the level of il6 increases with ageing, which is at least in part mediated by il6 production by vsmcs 87 . il6 is associated with ageing in general and is part of the 'inflammageing' phenotype 15, 16, 116 . why ageing leads to elevated basal secretion of inflammatory cytokines (not solely il6 but also other inflammatory mediators such as tnf) is not clear but might be caused by alter ations in the microbiota 117 , increased adiposity 118 , and changes in the immune system 17 and the vasculature 29, 30 . elevated cytokine levels with ageing could also be a manifestation of chronic, latent infections such as with herpesviruses 119 , of cellular senescence 57, 86, 87 or, potentially, of mitochondrial dysfunction. the role of il6 in young animal models of ath erosclerosis remains unclear and might relate to the complexities of il6 signalling (box 2). specifically, signalling via the classic il6 pathway occurs in a restricted number of cells (such as hepatocytes and some immune cells) and involves il6 binding to the membrane bound il6 receptor (il6r), with subse quent association with the signal transducing il6r subunit β (also known as gp130). evidence indicates that classic il6 signalling is important for tissue home ostasis, regeneration and host defence (as reviewed previously 120 ). soluble il6r can also engage il6 in the circulation and activate a broader range of cells than the classic pathway, via membrane activation of gp130. this pathway is termed il6 trans signalling (box 2) and can result in chronic inflammation 120 . these different il6 signalling pathways might explain the pleiotropic effects of il6 in different tissues and cellular compartments and also the divergent role of il6 in experimental atherosclerotic models. for instance, one study in apoe −/− mice showed that admin istration of exogenous il6 worsens atherosclerosis 121 . by contrast, another study in apoe +/− mice showed that il6 deficiency worsens atherosclerosis 80 nature reviews | cardiology atherosclerotic ldlr −/− mice 122 . the study found that inhib iting il6 trans signalling reduced atherosclerosis 122 , indicating that il6 trans signalling might have a path ogenic role in atherosclerosis. therefore, clinical ther apeutics to reduce atherosclerosis should focus on this il6 pathway. whether il6 has a causal role in age related ather osclerosis is not known yet. the contribution of il6 to age related atherosclerosis should be investigated in the future and should determine the main il6 producing and il6 responding cells. furthermore, the iden tification of the major il6 producing cells (fig. 2) and whether il6 activation occurs via the classic or trans signalling pathway with ageing could lead to more targeted therapeutics for atherosclerosis, especially given the availability of clinically approved agents to target il6 (refs 123,124 ). importantly, the risk-benefit balance of targeting il6 in atherosclerosis will need to be deter mined, given that anti il6 therapies in human studies increased the risk of infections 120 , similar to other bio logical agents (such as anti il1β antibodies) that have been used to reduce atherosclerosis 12, 13 . however, other biological agents such as tnf inhibitors 125 might be ben eficial for the treatment of atherosclerotic cardiovascular disease and should be investigated in age related ather osclerosis. finally, other inflammatory cytokines (such as tnf, c c motif chemokine 2 and il18, which are all part of the sasp) 56 might have a pathogenic role in age related atherosclerosis and should be assessed in future studies. therapies that can mitigate some of the detrimental biological effects of ageing, such as removing senescent cells (including senescent adipocytes) 126, 127 , improving mitochondrial function (for example, with metformin therapy) 128 , or augmenting macroautophagy (for exam ple, with rapamycin therapy) 129 or mitophagy, might reduce the burden of atherosclerosis in old people and should be investigated in future clinical studies. agents that increase mitophagy, such as spermidine, have been shown in experimental studies to reduce atherosclero sis in both young 130 and aged 11 mice. some or all these agents might have pleiotropic effects, which could reduce inflammation. furthermore, these agents might synergize with specific anti inflammatory therapies to reduce atherosclerosis with ageing, which will require future clinical investigation. ageing influences atherogenesis via multiple complex pathways, and one sole factor is unlikely to be a domi nant pathophysiological mechanism. in this review, we provide an overview of how ageing affects two systems, myeloid cell haematopoiesis and the vasculature, to pro mote atherosclerosis. we lay a framework of a poten tial shared inflammatory pathway, mediated by il6 signalling, that connects the role of the two systems in box 2 | il-6 signalling il-6 can signal via a classic signalling pathway and a trans-signalling pathway (see the figure) . in the classic il-6 signalling pathway, il-6 engages the membrane-bound il-6 receptor (il-6r) and subsequently interacts with the il-6r subunitβ (also known as gp130). intracellular signalling mainly involves activation of the janus kinase (jak) and the signal transducer and activator of transcription 3 (stat3). the classic pathway is generally restricted to hepatocytes and immune cells such as myeloid cells and lymphocytes. the trans-signalling pathway is activated by il-6 binding to soluble il-6r (sil-6r) in the circulation and then binding of the il-6-sil-6r complex to membrane-bound gp130 on a broad range of cells. sil-6r is released by enzymatic cleavage of membrane-bound il-6r by disintegrin and metalloproteinase domain-containing protein 17 (adam17). activation of the il-6 trans-signalling pathway generally leads to chronic inflammation, whereas the il-6 classic signalling pathway is involved in cell growth, regeneration and host defence. age related atherosclerosis and propose future avenues of investigation to determine whether il6 and/or other inflammatory pathways are feasible and effective ther apeutic targets to reduce the burden of atherosclerosis in old people. anti inflammatory strategies should be considered in the context of other therapies that aim to reduce many of the detrimental biological effects of ageing. overall, we hope that with the pursuit of further clinical investigation and trials, therapeutic options will be available in the future to reduce the burden of ath erosclerosis in the increasing number of old people in our society. fig. 2 | il-6 as a potential therapeutic target in age-related atherosclerosis. il-6 is upregulated in multiple tissues that have important roles in the increase in atherogenesis with ageing. therefore, blockade of il-6 might be an effective therapeutic strategy to reduce atherosclerosis development and progression during ageing. blocking il-6 might interfere with the increased il-6 signalling in bone marrow adipocytes that occurs with ageing (which promotes a skewing towards myeloid cell differentiation), thereby reducing the risk of clonal haematopoiesis of indeterminate potential (chip). il-6 blockade might also reduce the inflammatory potential of clones of myeloid cells associated with chip. il-6 blockade might reduce atherosclerosis burden, although direct comparisons of efficacy and safety with il-1β inhibition requires future investigation. il-6 blockade might increase mitochondrial function and reduce the expression of parkin, a mitochondrial stress protein, which might also contribute to reducing atherogenesis during ageing. hsc, haematopoietic stem cell; vsmc, vascular smooth muscle cell. prevalence and determinants of carotid plaque in the cross-sectional refine-reykjavik study heart disease and stroke 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multidatabase cohort study senolytics improve physical function and increase lifespan in old age chronic senolytic treatment alleviates established vasomotor dysfunction in aged or atherosclerotic mice metformin as antiaging therapy: is it for everyone? rapamycin, but not resveratrol or simvastatin, extends life span of genetically heterogeneous mice spermidine reduces lipid accumulation and necrotic core formation in atherosclerotic plaques via induction of autophagy acknowledgements d.j.t. is supported by nih award f32-hl1400728, and d.r.g. is supported by nih awards r01-hl127687, r01-ai138347 and k07-ag050096. both authors researched data for the article, discussed its content, wrote the manuscript, and reviewed and edited it before submission. the authors declare no competing interests. nature reviews cardiology thanks c. leeuwenburgh, h. oliveira, a. tedgui and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-004774-fvf671jn authors: kjeldsberg, elisabeth; hem, annelise title: detection of astroviruses in gut contents of nude and normal mice date: 1985 journal: arch virol doi: 10.1007/bf01310560 sha: doc_id: 4774 cord_uid: fvf671jn gut contents of nude and normal mice were examined by electron microscopy in association with an outbreak of diarrhea in a colony of nude mice. virus-like particles with a morphology consistent with previous descriptions of astroviruses of other species were demonstrated in a high percentage of the animals. gut contents of nude and normal mice were examined by electron microscopy in association with an outbreak of diarrhea in a colony of nude mice. virus-like particles with a morphology consistent with previous descriptions of astroviruses of other species were demonstrated in a high percentage of the animals. various viruses have been detected in recent years by electron microscopy (em) of stools and contents of the intestinal tract from both man and animals (15) . the relationship of some of these viruses to gastroenteritis has been established or postulated. different viruses are causative agents of diarrhea in mice. the epizootic diarrhea of infant mice (edi~[) virus was demonstrated in 1947 by cheeve~ and muell~ (3) . the morphology of this virus was described in 1972 by muck and zajac (12) and it was later classified as a rotavirus. lethal intestinal virus of infant mice (livim) was first described by k~aft in 1962 (8) . this virus could be distinguished from edim by its pathology and clinical signs, and by serological tests (9) and it was later shown to belong to the coronavirus group (1, 2). recently another coronavirus was isolated from an infant mouse in association with diarrhea and designated as diarrhea virus of infant mice (dvim) (14) . astroviruses were first detected in human faeces in association wit, h gastroenteritis (10) , and morphologically indistinguishable particles have been reported in diarrheal faeces of lambs (13) , calves (16) , birds (11) and cats (6) . astrovirus has not been demonstrated in mice so far. in this note we report the detection of astrovirus-like partieles in gut contents from nude mice, with and without clinical signs of illness, and from normal symptomless mice in association with an outbreak of diarrhea. gut contents of mice from different sources and of different strains and stocks were examined: 1. han:nmri nu/nu bred in isolators in the animal unit. 2. balb/e/nu/nu bom. 3. nih:bg/nu/nu bred in isolators in the animal unit. 4. nih:nihs/nu/nu bred in isolators in the animal unit. 5. bom:nml~i (normal controls). all the animals were caesarean derived and barrier maintained and of specified pathogen free quality when delivered from the breeder. the nude and thymus deficient mice were kept in a barrier unit. they were given irradiated altromin 1410 pelleted diet ad libitum. the water was acidified to ph 2.5 with hydrochloric acid. autoclaved woodshavings (hahnflock 3) were used as bedding. the diarrhea episode started during a heat wave in the summer when the temperature in the anim m room varied between 22--32 ° c and the relative humidity between 25--70 per cent. the duration of the disease in the individual animal was generally protracted. when it was obvious that 'the animal was ill with diarrhea or wasting it was killed. the mice with normal haircoat were kept in a conventional animal room for 2--3 days after arrival from the breeder before they were submitted to examination. only animals received from the same breeder were kept in this room. the age of the animals varied between 4 weeks and 9 months. both sexes were represented about evenly in nude and normal animals. the gut contents of the mice were scraped off and suspended to 10 per cent v/v in phosphate buffered saline (pbs) ph 7. the suspension was shaken with a vortex mixer for one minute to disperse the material, left on the bench for 30 minutes at room temperature and shaken by hand from time to time. the extract was centrifuged at 640 g for five minutes and the supernatant was kept at --20 ° c until required for use. in a few experiments gut contents were suspended to 20 per cent v/v in pbs containing 1 per cent w/v triton x-100 (pbs/tx-100) and disrupted with ten strokes of a glass homogenizer. the mixture was cleared at i040 × g for 10 minutes, the pellet washed with a small volume of pbs/tx-i00 and the supernatants pooled. for electron microscopy the extracts from gut contents were treated as earlier described (7). 2.5 ml extract was centrifuged for 30 minutes at 6000 r.p.m, and the supernatant reeentrifuged for 90 minutes at 20,000 r.p.m. in a sorvall rc2-b centrifuge ss-34 rotor. the deposit was suspended in a few drops of distilled water and treated in a branson 220 ultrasonic bath for 3 minutes to disperse the virus particles. negative staining was performed by mixing equal volumes of virus suspension and 2 per cent potassium phosphotungstate pit 7 and layering the mixture on a formvar carbon coated 400 mesh copper grid. after one minute excess fluid was removed with filtering paper, and the grid was air-dried. the specimen was examined in a jem 100b electron microscope at a magnification of 50,000 x using 80 kv accelerating voltage. the magnification had been calibrated with a diffraction grating specimen. a sample was considered negative if no virus particles were observed within 15 minutes examination. intestinal scrapings from 72 nude mice with and without clinical signs of illnes and from 10 normal symptomless mice were treated and examined symptomless or with symptoms other than diarrhea in the electron microscope as described above. particles resembling astrovirus were demonstrated in 50 of the nude mice. the particles were roughly spherical in outline and had a diameter of about 30 nm. the 5--6 pointed star configuration, characteristic of astroviruses, was seen on some of the particles, which mostly appeared in aggregates (fig. 1 a--c) . on some of the micrographs the virus particles showed a smooth outer edge (fig. 1 a) . often, however, fiber-like structures which seemed to form bridges between the virus particles were seen (fig. 1 b) . electron microscopy of pbs/tx-100 extracts of gut contents occasionally showed astrovirus particles penetrated with stain revealing an inner core structure 13--14 nm in diameter (fig. 1 c) . virus particles were demonstrated in both apparently healthy and diseased animals. the results of the examination of 72 nude and 10 normal mice are summarized in table 1 . seventeen of the nude mice examined suffered from diarrhea while 55 animals were killed for various reasons; i.e. abcesses, bite wounds, termination of experiments. astrovirus was demonstrated in 16 (94 per cent) of the animals with diarrhea and in 34 (62 per cent) from the control group. no attempt was made to quantify the amount of virus in the samples, but there appeared to be a higher number of virus particles in the samples from animals with diarrhea than from those without. small amounts of astrovirns-like particles were also demonstrated in 4 of l0 normal mice showing no sign of illness. the morphology of the virus-like particles detected in gut contents of nude and normal mice corresponds to the previous description of astroviruses. the occurrence of the virus in association with diarrhea is consistent with the demonstration of astrovirnses in humans and other animal species in association with gastroenteritis. the failure to demonstrate virus in one of the animals with diarrhea may be due to low sensitivity of the technique. as the majority of astrovirns particles are usually aggregated (5) a great deal is probably lost in the low speed centrifugation. demonstration of aggregates of virus-like structures in deposits after low speed centrifugation confirms this assumption. the presence of large aggregates of virus-like particles in the intestinal scraping suggests a multiplication of the astrovirus in epithelial cells of the intestinm tract, which would be in agreement with earlier findings in infected lambs (4). demonstration of astrovirus in a high percentage of nude and normal mice without diarrheal symptoms might suggest that the animals are symptomless carriers of the virus and that the pathogenicity in the nude mice is enhanced by break-down of the climatic control or heavy experimental stress. this hypothesis needs further support. studies on the pathogenicity and characteristics of the mouse astrovirus and the relationship of the virus to diarrhea in these animals are in progress. lethal enteritis in infant mice caused by mouse hepatitis virus lethal intestinal virus of infant mice is mouse hepatitis virus epidemic diarrheal disease of suckling mice. i. manifestations, epidemiology and attempts to transmit the disease ultrastructure of the small intestine in astrovirusdnfeeted lambs purification and characterization of ovine astrovirus detection of astroviruses in feces of a eat with diarrhea comparison of solid-phase (immune electron microscopy, direct electron microscopy and enzyme-linked immunosorbent assay for detection of rotavirus in faecal samples an apparently new lethal virus disease of infant, mice epizootic diarrhea of infant mice and lethal intestinal virus infection of infant mice viruses in infantile gastroenteritis detection of astroviruses in turkey faeces by direct electron microcopy purification and characterization of epizootic diarrhea of infant mice virus detection and transmission of 30 nm virus particles (astroviruses) in faeces of lambs with diarrhea morphnogicat and biological properties of a new eoronavirus associated with diarrhea in infant miee virus infections of the gastrointestinal tract isolation of small viruses resembling astroviruses and caliciviruses from acute enteritis of calves authors' address : dr. elisabeth kjeldsberg, national institute of public health key: cord-352480-1ay8y7li authors: wang, ting; yin, huiquan; li, yan; zhao, lingxiao; sun, xiahui; cong, hua title: vaccination with recombinant adenovirus expressing multi-stage antigens of toxoplasma gondii by the mucosal route induces higher systemic cellular and local mucosal immune responses than with other vaccination routes date: 2017-04-03 journal: parasite doi: 10.1051/parasite/2017013 sha: doc_id: 352480 cord_uid: 1ay8y7li toxoplasmosis caused by toxoplasma gondii, an obligate intracellular protozoan, is a cause of congenital disease and abortion in humans and animals. various vaccination strategies against toxoplasmosis in rodent models have been used in the past few decades; however, effective vaccines remain a challenge. a recombinant adenovirus vaccine expressing ubiquitin-conjugated multi-stage antigen segments (ad-umas) derived from different life-cycle stages of t. gondii was constructed previously. here, we compared the immune responses and protection effects in vaccination of mice with ad-umas by five vaccination routes including intramuscular (i.m.), intravenous (i.v.), subcutaneous (s.c.), intraoral (i.o.), and intranasal (i.n.). much higher levels of t. gondii-specific igg and iga antibodies were detected in the sera of the intraoral and intranasal vaccination groups on day 49 compared with controls (p < 0.05). the percentages of cd8(+) t-cells in mice immunized intranasally and intraorally were larger than in mice immunized intramuscularly (p < 0.05). the highest level of il-2 and ifn-γ was detected in the group with nasal immunization, and splenocyte proliferation activity was significantly enhanced in mice immunized via the oral and nasal routes. furthermore, the higher survival rate (50%) and lower cyst numbers observed in the intraoral and intranasal groups all indicate that ad-umas is far more effective in protecting mice against t. gondii infection via the mucosal route. ad-umas could be an effective and safe mucosal candidate vaccine to protect animals and humans against t. gondii infection. toxoplasma gondii is a single-cell obligate intracellular protozoan with an infection rate of approximately 25%-30% in the worldwide population [6, 13, 29] . in pregnant women, this parasite may threaten health or even be fatal concerning congenitally infected fetuses [30] . it is also a major opportunistic infection in immunodeficient individuals, causing toxoplasmic encephalitis and retinochoroiditis [7, 17, 33] . as a result, an effective vaccine against t. gondii is urgently needed to prevent this disease. various vaccination strategies against toxoplasmosis in rodent models have been used in the last few decades. however, effective vaccines remain a challenge as tested vaccines have not been able to confer sterile immunity against infection [19] . current research on t. gondii vaccines only focuses on antigens expressed in the tachyzoite stage, which only induce partial protective immunity [20, 28, 38, 44] . the development of a variety of epitope combinations from different stages of the life cycle, including tachyzoites, bradyzoites (in tissue cysts), and sporozoites (in oocysts), are likely to induce full protection [2, 3, 4, 43] . with the development of bioinformatics, epitope vaccines are considered a novel immunization approach to prevent t. gondii infection. in addition, an appropriate delivery system of vaccines could increase the transfection efficiency of immune cells. adenovirus, which can penetrate host cells delivering vaccine antigen to antigen-presenting cells (apcs) and elicit vigorous and sustained t-cell responses, is a promising t. gondii vaccine vector and can be used effectively to transport immunogens [27] . recent studies have also found that recombinant canine adenovirus type-2 expressing tgrop16 and tgrop18 provides partial protection against acute t. gondii infection in mice, and indicate that adenovirus vectors may be potentially useful in the development of an effective vaccine against t. gondii infection [21, 22] . the mucosal immune system consisting of specialized epithelial cells can trigger both humoral and cell immune responses when antigens are administered with appropriate adjuvants or attenuated live vaccines via mucosal routes (oral, nasal, sublingual, ocular, genital, or rectal) [18, 23] . furthermore, local mucosal immunization can lead to antigen-specific t-and b-cell responses not only in mucosal sites but also systemically [5, 24] . hence, there is a greater need for effective vaccines that exert protective effects at mucosal surfaces, especially for intracellular parasites such as t. gondii. in this research, we utilize a recombinant adenovirus vaccine expressing ubiquitin-conjugated multi-stage antigen segments (ad-umas) derived from different life-cycle stages of t. gondii to evaluate the immune responses obtained with the different intramuscular vaccination routes described in our earlier study [41] . to further explore the optimal immune pathway for this recombinant adenovirus t. gondii vaccine, we plan to determine the immune responses and protection efficacy by vaccinating balb/c mice via five routes (intramuscular (i.m.), intravenous (i.v.), subcutaneous (s.c.), intraoral (i.o.), and intranasal (i.n.)). all the experimental procedures with animals used in the present study were granted prior approval by the institutional animal care and use committee of shandong university under contract ll201602044. humane endpoints are chosen to terminate the pain or distress of the experimental animals via euthanasia. mice were monitored daily over 11 weeks for signs of toxoplasmosis including food and water intake difficulties, fatigue, severe ascites, and any test animals that showed signs of illness were euthanized immediately with co 2 gas. tissue cysts of the low-virulence prugniaud (pru) strain (type ii) and tachyzoites of the high-virulence rh strain (type i) of t. gondii, which were a kindly provided by professor xingquan zhu at lanzhou veterinary research institute, were propagated and harvested as described in our previous studies [41] , and then used for the in vivo challenge of mice. ad-umas vaccine was constructed as previously described [40] . briefly, ad-umas was constructed using the admax system (hanbio, shanghai, china). the ubiquitinconjugated multi-stage antigen segments (umas) were cloned into a shuttle plasmid, phbad-mcmv-gfp. ad-umas was generated by homologous recombination of phbad-mcmv-gfp-umas with phbad-bhg in hek-293 cells. ad-umas particles, with a titer of 10 11 plaque-forming units (pfu)/ml, were purified by cesium chloride gradient centrifugation and then stored in storage buffer (10 mm tris, 2 mm mgcl 2 , and 5% sucrose, ph 8.0) at à80°c. specific-pathogen-free female balb/c mice aged 6-8 weeks were purchased from shandong university laboratory animal centre (jinan, china). the animals for vaccination included intramuscular, intravenous, subcutaneous, intraoral, and intranasal immunization groups (15 mice per group). for the intramuscular group, the mice were injected 100 ll (3 · 10 8 pfu) ad-umas in the quadriceps muscle. for the intravenous group, the mice were injected 50 ll (3 · 10 8 pfu) ad-umas in the caudal vein. the subcutaneous and oral groups were administered 200 ll (3 · 10 8 pfu) ad-umas by subcutaneous injection and intragastric administration. for intranasal vaccinations, mice were anesthetized with 3% isoflurane in oxygen and were given a nasal drip of 10 ll (3 · 10 8 pfu) ad-umas with the head canted 45°for 10 min. each group also had 15 mice that were treated with phosphate-buffered saline (pbs) as the control. mice were vaccinated twice at 3-week intervals. table 1 summarizes the treatments given to the mice. figure 1 shows the study flowchart for vaccinated mice. serum samples were collected by retro-orbital bleeding on days 0, 14, 35, and 49. standard elisas were used to determine the levels of t. gondii-specific antibodies, igg, igg1, igg2a, and iga, in the serum samples from the inoculated mice. this was done with some changes to the method previously described [3, 5] . briefly, a flat-bottom 96-well plate was pre-coated with the umas peptide pool at a concentration of 10 lg/ml in a 50 mm carbonate-bicarbonate buffer (ph 9.6) overnight at 4°c. the mouse sera were diluted in pbs (1:100) and incubated at 37°c for 1 h. after washing the plates, bound antibodies were then reacted with horseradish peroxidase (hrp)-conjugated goat anti-mouse igg, igg1, igg2a, or iga (sigma-aldrich, usa) at 37°c for 1 h. peroxidase activity was revealed by 3, 3 0 , 5, 5 0 -tetramethylbenzidine (tmb, 10 mg/ml) and stopped by adding 50 ll of 2 m h 2 so 4 . the optical density (od) at 450 nm was measured by a thermo scientific multiskan fc microplate photometer (thermo scientific, usa). spleens were removed from three immunized mice per group four weeks after the last immunization. single-cell splenocytes were harvested following the method described in a previous study [5, 42] . isolated splenocytes were plated in 96-well plates, at a density of 1 · 10 6 per well, in 100 ll rpmi-1640 medium (sigma-aldrich, usa) supplemented with 10% fetal calf serum and cultured with concanavalin a (cona, 2 lg/ml, sigma-aldrich) or the umas peptide pool (10 lg/ml). cell proliferative activity was measured according to the manufacturer's instructions on a dojindo cell counting kit-8 (dojindo, japan). the results were expressed as absorbance at 450 nm. the level of cytokine production was determined using splenocytes from three mice per group 4 weeks after the last immunization. commercial elisa kits (r&d systems, usa) were used following the manufacturer's instructions to assay il-2 at 24 h, il-10 at 72 h, and ifn-c at 96 h in culture supernatants obtained as previously described [40] . this was done by collecting the cell supernatant of three wells from the 96-well plates and centrifuging to discard cell debris. the supernatant was tested for cytokines. the detection was replicated three times for each spleen. cells were stained with fluorescein isothiocyanate (fitc)labeled anti-mouse cd8 + monoclonal antibody and phycoerythrin (pe)-labeled anti-mouse cd4 + monoclonal antibody; tlymphocyte subsets were measured by a beckman coulter fc500 flow cytometer (beckman coulter, usa). four weeks after the last immunization, mice were challenged with the type 1 or type 2 parasites to evaluate the protective effect. six mice per group were infected intraperitoneally with 1 · 10 3 tachyzoites of t. gondii rh strain (type 1 parasite) after the last immunization, and the survival time of the mice was observed and recorded. another six immunized mice per group were infected via the gastric route with 20 cysts of the t. gondii pru (type 2 parasite) strain. the challenged mice were sacrificed 4 weeks later and the brains were removed and homogenized in 1 ml pbs. the mean number of cysts per brain was determined by counting three samples of 10 ll aliquots from each homogenized brain under an optical microscope [20] . statistical significances between the groups were calculated by one-way analysis of variance (anova) using spss 19.0 software. the survival rate was compared by the kaplan-meier method. a p-value of less than 0.05 (p < 0.05) was considered to be significant. to assess the systemic humoral immune responses obtained with the five immunization routes of ad-umas, we collected sera at different times after the immunization, and anti-t. gondii igg and igg subtypes were detected by elisa. as shown in figure 2a , the levels of antibody titers all increased in the five ad-umas immunization routes. compared to the control, levels of t. gondii-specific igg antibodies increased dramatically in the sera of all vaccination mice from day 14 to day 35 and achieved the highest on day 49 (p < 0.05). among different immunization routes, the intramuscular group achieved the highest titer of igg antibodies, then the subcutaneous, nasal, and oral groups, in decreasing order (fig. 2b) . in the intravenous vaccination group, the titer of igg increased rapidly at 2 weeks and gradually more slowly thereafter, making the final titer relatively low. further evaluation of serum igg subtype antibody revealed that t. gondii-specific igg1 and igg2a antibody levels were both significantly increased in five ad-umas vaccination groups as compared with the control (p < 0.05), while their differences were substantial in igg2a but not in igg1. the values of igg2a antibody were relatively higher in the intramuscular and subcutaneous vaccination groups than with other vaccination routes (figs. 2c and 2d ). to evaluate the ability of ad-umas to induce local mucosal immune responses via the five pathways, mucosal iga antibody response was detected in the collected mouse sera. as shown in figure 2e , vaccination in the intraoral and intranasal groups elicited strong t. gondii-specific iga antibody levels that were significantly higher than other vaccination routes (p < 0.05) (fig. 2e) . however, there were no statistically significant differences of iga titer in the intramuscular, intravenous, and subcutaneous vaccination groups compared to control groups (p > 0.05). cellular immune responses were first evaluated by analysis of the t-cell subset. the cd4 + and cd8 + t lymphocyte subsets in immunized mice were assayed by flow cytometry. table 2 shows the percentage of cd4 + and cd8 + t-cells, in comparison with control groups; the percentage of cd8 + t-cells in all vaccination groups with ad-umas increased significantly (p < 0.05). the percentages of cd8 + t-cells in mice immunized intranasally (36.5 ± 1.8%), intraorally (29.7 ± 0.2%), and intramuscularly (27.6 ± 1.3%) were higher than in mice immunized by the subcutaneous and intranasal routes (p < 0.05). the cell-mediated immunity induced in the immunized mice was further evaluated by measuring the amount of cytokines il-2, il-10, and ifn-c. table 3 shows the values of il-2, ifn-c, and il-10 in all vaccination groups. the highest levels of il-2 and ifn-c were detected in the nasal immunization group at 638.7 ± 17.6 pg/ml and 1429.8 ± 37.6 pg/ml, respectively. furthermore, the lymphocyte proliferation ability was also higher in the mice inoculated nasally with ad-umas than the controls (p < 0.05). however, there were no statistically significant differences in the amount of il-10 between all the immunization group and control groups (p > 0.05) ( table 3) . to evaluate the protective immunity, six mice of each group were given an intraperitoneal injection of 1 · 10 3 tachyzoites of t. gondii rh strain at 4 weeks after vaccination. the survival rates of mice immunized via different routes are shown in figure 3 . the mice immunized with ad-umas via i.m., i.o., and i.n. showed an almost 50% survival rate and lower survival rates of 40% were found in the i.v. and s.c. immunization groups 28 days after challenge. all the control mice died within 8 days. the level of protective effect against chronic infection was evaluated by counting cysts in the brains of mice challenged with 20 cysts of the pru strain of t. gondii (fig. 3 ). an obvious reduction in the burden of brain cyst was detected in all the immunized mice compared with the control mice (p < 0.05). the brain cyst numbers in the oral and nasal immunization groups were significantly reduced to 459 ± 34 and 398 ± 61, respectively, which is much lower than the other immunization groups with injection in muscle (569 ± 23), via the venous route (892 ± 51), and via the subcutaneous route (675 ± 65) (p < 0.05). the antigen delivery pathway is a key parameter for the induction of protective immune responses by vaccines against intracellular pathogens [10] . as a novel inoculation, the splenocyte culture supernatants taken from mice (n = 3, each group) 2 weeks after the last immunization were stained with fitclabeled anti-mouse cd8 + monoclonal antibody and pe-labeled anti-mouse cd4 + monoclonal antibody; t lymphocyte subsets were measured using flow cytometry. asterisks mark the significant difference: * p < 0.05; ** p < 0.01. each bar represents the mean od (± sd). there is general agreement that effective mucosal vaccines (oral, nasal, sublingual, and genital tract vaccines) could dramatically stimulate protective immune responses not only against mucosal infections but also against hiv, mycobacterium tuberculosis, and many other pathogens [8, 37] . in this study, we compared the capability of ad-umas to induce systemic humoral and cellular immune responses, and local mucosal immune responses via five vaccination pathways (i.m., i.v., s.c., i.o., and i.n.). mucosal vaccines are advantageous compared with systemic vaccines from a production and regulatory perspective [18, 23] . previous studies have revealed that a middle east respiratory syndrome (mers) vaccine greatly enhances local mucosal immune responses in immunized balb/c mice with the intranasal inoculation method [24] . ideal attenuated vaccines could be engineered to have a limited capacity for replication and to deliver an effective antigen, while avoiding unwanted inflammatory responses [35, 39, 41] . recombinant adenoviruses are some of the most intensively investigated vaccine carriers in many pathogenic infections, such as ebola virus, hiv, and the malaria parasite [1, 11, 16, 32] . several clinical trials have shown that vaccines based on adenoviruses could elicit vigorous and sustained t-cell responses, with the advantage of safety and high immunogenicity [25] . our results showed that the ad-umas vaccine could effectively improve the humoral response intramuscular pathway. systemic humoral igg antibody responses induced by the intraoral and intranasal vaccination pathway were comparable to those induced by the other pathway in the collected mouse sera. interestingly, we found that igg antibody in the intravenous vaccination group increased very sharply at first but slowly later on, even after the boost immunization. it should be considered that antigens that are transmitted to the bloodstream directly can induce strong immune responses in the early stage, but can also cause immune tolerance, explaining why antibody levels increase slowly in the following time [25] . moreover, evaluation of serum igg subtype antibody responses revealed that all vaccination pathways induced a relatively higher level of th1-associated igg2a than th2associated igg1 in this study. it should be noted that ad-umas induced a slightly biased th1-associated antibody (a) (b) figure 3 . evaluation of the protective ability against t. gondii infection. (a) six immunized mice per group were infected intraperitoneally with 1 · 10 3 tachyzoites of t. gondii rh strain 4 weeks after the last immunization. survival was monitored daily for 28 days after challenge. (b) another six immunized mice per group were infected intragastrically with 20 cysts of t. gondii pru strain and the cyst burden in the brain of vaccinated mice was counted 4 weeks later. the mean number of cysts in every mouse group was based on each mouse brain cyst in the group. asterisks mark the significant difference: *p < 0.05; **p < 0.05. each bar represents the mean od (± sd). response. it is known that production of igg subtype is driven by cytokines secreted during cellular immune responses. therefore, the presence of igg1 and igg2a indirectly suggests that the vaccination protocol also promoted activation of a cellmediated response [12, 31] . these data suggest that ad-umas immunization by the mucosal vaccination route was capable of eliciting similar systemic humoral antibody responses compared with those elicited by the intramuscular pathway in vaccinated mice. mucosal vaccines against toxoplasma infection require the induction of protective responses at mucosal surfaces and also the induction of systemic protective immune responses in the systemic compartment [18] . as the first line of defense in protecting mucous membranes, specific siga plays a protective role against many pathogens. therefore, vaccines capable of inducing strong local mucosal immunity represented by secretory iga would be helpful in preventing t. gondii infection [9] . our data indicated that t. gondii-specific iga antibody responses in vaccinated mouse sera were elicited by i.n. and i.o vaccination of ad-umas, which were significantly higher than those induced by other vaccination routes, confirming the ability of ad-umas to induce strong mucosal immunity via the mucosal route. the memory t-cell consists of cd4 + and cd8 + t-cells, which play a vital role in the establishment of protective immunity in hosts [31] . cd8 + t-cells can control the spreading and development of t. gondii infection in synergy with cd4 + t-cells [12] . in our study, cd4 + and cd8 + t lymphocyte subsets from immunized mice were assayed by flow cytometry. there was a marked increase in the percentage of cd8 + t-cells in mice immunized with the ad-umas vaccine via mucosal routes. cytokines play a critical role in the activities of th cells. as we know, th1 cells are responsible for limiting tissue extension of the parasite through the production of il-2 and ifn-c [15] . our results show that in contrast with the blank control, high levels of il-2 and ifn-c were induced in mice immunization with ad-umas especially treated intraorally and intranasally. however, there is no difference in il-10 production between vaccinated and control groups. additionally, splenocyte proliferation activity was significantly enhanced in mice immunized via oral and nasal routes. these results clearly suggest that mucosal vaccination with ad-umas can significantly augment th1-mediated cellular immune responses in which cd8 + t lymphocytes are considered as major effectors responsible for controlling parasite infection and secreting ifn-c during the cellular response against toxoplasmosis. in our study, a highly virulent rh strain and a mild virulent pru strain of t. gondii were used for the challenge study. with the rh strain, it is easy to induce acute infection in mice through intraperitoneal injection with tachyzoites. the pru strain is more likely to induce chronic infection in mice following intragastric administration with cysts [36] . in order to simulate acute and chronic infection in the host, mice were infected with the rh strain and pru strain by different challenge routes. when challenged with lethal doses of t. gondii (1 · 10 3 ), all five groups immunized with ad-umas had a prolonged survival time as compared with control mice. in particular, mice immunized intranasally and intraorally showed higher survival rates than with the other immunization routes. since protection via vaccination was not 100% because the strain of the parasite has high mortality and the dose of rh parasites for immunized mice was larger, the optimal challenge dose should be determined in the future. furthermore, the higher mucosal immune responses that were induced by intraoral and intranasal vaccination of ad-umas may explain the significant reduction in the cyst burden in the mice immunized with ad-umas intraorally and intranasally, which exhibited reductions of 75.3% and 78.6% of brain cysts, respectively. earlier studies [14, 26, 34] have determined mortality, cytokine production, and parasite burden after challenge infection in susceptible and resistant strains of mice with the same or similar doses of t. gondii parasites. these studies found that different inbred strains of mice have markedly different susceptibility to t. gondii infection. these susceptibility differences may be due to differences in the virulence of the parasite itself or differences in the genetic make-up of the mice in terms of their immune response. in our study, only one inbred strain of mice was used for the experiment. it is therefore necessary to assess the different immunizations in different strains of mice in future research. in summary, mucosal immunization with recombinant adenovirus vaccine expressing ubiquitin-conjugated multistage antigen segments (ad-umas) is a potential vaccination route to elicit protective immune responses. compared to other immunization routes, oral and nasal immunizations with ad-umas elicit both robust systemic and mucosal immune responses, accompanied by a significant increase in survival rates after challenge with highly virulent parasites and a dramatic reduction in brain cyst burdens in vaccinated mice after challenge with mild strains of t. gondii. therefore, the results of this study should contribute to the development of an effective and safe mucosal vaccine for preventing t. gondii infection. the authors declare that they have no competing interests. a single dose respiratory recombinant adenovirus-based vaccine provides long-term 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immune responses than with other vaccination routes reviews, articles and short notes may be submitted. fields include, but are not limited to: general, medical and veterinary parasitology including entomology, acarology, helminthology and protistology, and molecular analyses; molecular biology and biochemistry; immunology of parasitic diseases; host-parasite relationships; ecology and life history of parasites all papers in parasite are published in english. manuscripts should have a broad interest and must not have been published or submitted elsewhere. no limit is imposed on the length of manuscripts parasite (open-access) continues parasite (print and online editions, 1994-2012) and annales de parasitologie humaine et comparée (1923-1993) and is the official journal of the société franc¸aise de parasitologie. editor-in-chief acknowledgements. this study was supported by grants from the national natural science foundation project of china (grant no. 81171604) and the development project of shandong province (grant no. 2016gsf201201). key: cord-351011-v4zmksio authors: golden, joseph w.; cline, curtis r.; zeng, xiankun; garrison, aura r.; carey, brian d.; mucker, eric m.; white, lauren e.; shamblin, joshua d.; brocato, rebecca l.; liu, jun; babka, april m.; rauch, hypaitia b.; smith, jeffrey m.; hollidge, bradley s.; fitzpatrick, collin; badger, catherine v.; hooper, jay w. title: human angiotensin-converting enzyme 2 transgenic mice infected with sars-cov-2 develop severe and fatal respiratory disease date: 2020-07-09 journal: biorxiv doi: 10.1101/2020.07.09.195230 sha: doc_id: 351011 cord_uid: v4zmksio the emergence of sars-cov-2 has created an international health crisis. small animal models mirroring sars-cov-2 human disease are essential for medical countermeasure (mcm) development. mice are refractory to sars-cov-2 infection due to low affinity binding to the murine angiotensin-converting enzyme 2 (ace2) protein. here we evaluated the pathogenesis of sars-cov-2 in male and female mice expressing the human ace2 gene under the control of the keratin 18 promotor. in contrast to non-transgenic mice, intranasal exposure of k18-hace2 animals to two different doses of sars-cov-2 resulted in acute disease including weight loss, lung injury, brain infection and lethality. vasculitis was the most prominent finding in the lungs of infected mice. transcriptomic analysis from lungs of infected animals revealed increases in transcripts involved in lung injury and inflammatory cytokines. in the lower dose challenge groups, there was a survival advantage in the female mice with 60% surviving infection whereas all male mice succumbed to disease. male mice that succumbed to disease had higher levels of inflammatory transcripts compared to female mice. this is the first highly lethal murine infection model for sars-cov-2. the k18-hace2 murine model will be valuable for the study of sars-cov-2 pathogenesis and the assessment of mcms. sars-cov-2 is a betacoronavirus and the causative agent of covid-19, a febrile 55 respiratory human disease that emerged in late 2019 in china and subsequently spread throughout the world (1, 2). covid-19 is primarily a respiratory disease with a wide spectrum of severity ranging from a mild cough, to the development of hypoxia, and in some cases resulting in a lifethreatening acute respiratory distress syndrome (ards) requiring mechanical ventilation (3, 4). the most severe cases are generally skewed towards the aged population (>50) and those with 60 underlying health conditions such as hypertension or cardiovascular disorders (5, 6) . sars-cov-2 human infections can also cause vasculature damage and coagulopathies, leading to infarction (7-9). acute disease often presents with elevated levels of inflammatory cytokines, including il-6, and these host molecules may play a role in the pathogenic process (10, 11) . additionally, ~30% of cases include signs of neurological disease such as headache, anosmia (loss of smell), ataxia, 65 meningitis, seizures and impaired consciousness (12) (13) (14) . to-date, sars-cov-2 has infected over eight million people world-wide and resulted in the death of more than 400,000. there is an urgent need for medical countermeasures to prevent this disease or limit disease severity in a post exposure setting. similar to sars-cov (15) , sars-cov-2 binds to target cells via an interaction between the 70 139 kda viral spike protein and the host angiotensin-converting enzyme 2 (ace2) protein (16) (17) (18) (19) ). an important factor in host tropism of the virus is this receptor interaction and reduced affinity between these two molecules greatly impacts host susceptibility to infection. both sars-cov and possibly to a greater extent sars-cov-2 bind to murine ace2 (mace) poorly compared human ace2 (hace2) (20) . accordingly, mice are inherently refractory to infection by ace2 utilizing 75 golden jw, et al sars-cov-2 pathogenesis in k18-hace2 mice human coronaviruses (21) (22) (23) . in these animals, infection by sars-cov (22) and sars-cov-2 (23) is rapidly controlled, although older mice are more permissive to lung replication, but nevertheless lung injury is limited and mortality is generally low. indeed, even infection in mice lacking adaptive immunity due to rag deficiencies (no t-cells or b-cells) are protected against severe sars-cov disease, whereas mice genetically devoid of stat-1, an important molecule 80 involved in innate immunity, are sensitive to infection by sars-cov(24-26). however, disease in stat-1 mice is protracted and not highly representative of human infections (25, 26) . in response to the need for small animal models to study sars-cov, several laboratories 32) . k18 limits expression to airway epithelial cells, colon and to a lesser extent kidney, liver, spleen and small intestine. a minor level 90 of hace2 expression was also detected in the brain. these mice are susceptible to sars-cov strain urbani and develop severe respiratory disease subsequent to intranasal exposure characterized by lung inflammation, serum cytokine and chemokine production as well as high lethality (30). as with several mouse strains transgenic for hace2, sars-cov infects the brains of k18-hace2 mice (30, 32) . cns localization is speculated to play a major role in host mortality 95 due to neuronal cell death, particular cell loss in cardiorespiratory control center (32) . some of these previous hace2 transgenic mice are being used for sars-cov-2 research and newer golden jw, et al sars-cov-2 pathogenesis in k18-hace2 mice systems have been developed using crsipr-cas9 (23, 33, 34). however, none were shown to produce a consistent lethal disease and in models where lung injury occurred, it was most pronounced in aged animals (23, 34). here, we evaluated susceptibility of k18-hace2 mice to 100 sars-cov-2. we found that mice developed severe disease that included respiratory distress with weight loss and mortality, as well as brain infection. sars-cov-2 produces severe and fatal infection of k18-hace2 mice. two groups of 9-week old k18-hace2 mice (n=14 per group) were intranasally infected with 2x10 4 or 2x10 3 pfu of 105 sars-cov-2. these groups equally divided by sex (n=7 per group/sex). on day 3, two mice per group were euthanized to assess disease severity. the remaining 5 mice per group were monitored up to 28 days. we also infected c57bl/6, balb/c and rag2 deficient mice with a challenge dose of 2x10 4 pfu. on day 4, all groups of infected k18-hace2 mice began to lose weight, which was more pronounced in the female mice compared to the male mice in either challenge dose group 110 ( fig. 1a & fig. s1a ). non-transgenic mice did not lose any weight and no animal succumbed to disease. starting on day 5, several k18-hace2 animals began to show signs of respiratory disease, included labored breathing and conjunctivitis. on day 5 through day 7 the majority of mice (15/20 mice) began to meet euthanasia criteria (n=13) or died (n=2). an additional animal in the low dose male group succumbed to disease on day 11, after a period of weight loss. the highest mean weight 115 loss was in the female groups (>20%), although male mice lost >12% weight (fig s1a,b) . of the k18-hace2 mice in the high dose challenge groups, all females and 80% of the male mice succumbed to disease. most female mice survived in the low dose group with 40% mortality, but all male mice succumbed to disease. the difference in survival between low dose male and female golden jw, et al sars-cov-2 pathogenesis in k18-hace2 mice mice was significant (log-rank; p=0.040), as was mortality between the high and low dose female 120 groups (log-rank; p=0.037). there was no statistical significance in survival between the other groups. lung homogenates taken on day 3 showed high levels of virus in k18-hace2, in contrast to c57bl/6 or rag2-deficient mice which had low levels of virus (fig. 1b,c) . the virus rna levels were nearly identical between all the infected k18-hace2 groups and remained high in most of the euthanized mice, with the exception of the mouse that died on day 11. that animal had 125 markedly lower levels of detectable genomic rna (fig. 1c) . compared to non-transgenic mice and uninfected k18-hace2 mice, infected k18-hace2 mice had comparatively higher serum levels of tnf-α, il-6 and il-10 as well as the monocyte chemoattractants mcp-1 (ccl2) and mcp-3 (ccl7) (fig. 1d) . levels of cytokines observed in mice that succumbed to disease were generally higher compared to those sampled on day 3. overall, these findings indicated that 130 sars-cov-2 causes a severe disease in k18-hace2 mice following intranasal exposure. lungs of k18-hace2 mice exposed to sars-cov-2 show signs of acute disease. lungs were collected from k18-hace2 on day 3 or at the time of euthanasia due to disease severity. in k18-hace2, viral rna was detected by in situ hybridization (ish) in all mice on day 3 and in most 135 mice succumbing to disease on days 5-7, but these levels diminished as disease progressed (fig 2a, fig s2, table s1 ). additionally, ish labeling was patchy (fig. s2) and most severe at the day 3 time point. ish labeling was present in both inflamed and normal appearing alveolar septa. positive ish labeling for sars-cov-2 was identified multifocally in alveolar septa in the lung, suggesting infection of pneumocytes and macrophages. this was confirmed by the detection of 140 golden jw, et al sars-cov-2 pathogenesis in k18-hace2 mice sars-cov-2 protein in e-cadherin positive cells (pneumocytes) and cd68 positive (alveolar and infiltrating macrophages) using immunofluorescence assay (ifa, fig. 2b ,c). in comparison with the normal lung architecture in uninfected control animals, infected mice necropsied on day 3, and those succumbing to disease on days 5-11, had varying levels of lung injury including area of lung consolidation characterized by inflammation/expansion of 145 alveolar septa with fibrin, edema and mononuclear leukocytes and infiltration of vessel walls by numerous mononuclear leukocytes (fig 3a, fig s4, and table s1 ). type ii pneumocyte hyperplasia was identified in less than half of infected animals. this lesion had a relatively patchy distribution except in the most severely affected animals where it is more abundant. in areas of septal inflammation, exudation of fibrin and edema into alveolar lumina from damaged septal 150 capillaries was observed in most animals. vasculitis was the most common finding and was present in ~95% percent of all mice (fig. 3a,b) . the lesion encompassed small and intermediate caliber vessels, and was often characterized by near circumferential infiltration of the vessel wall by numerous mononuclear inflammatory cells. this lesion also contained small amounts of fibrin and occasional necrotic debris, affecting all tunics and obscuring the vessel wall architecture. however, 155 the endothelial cells surrounding lung vasculature were largely free of viral rna (fig. 3b) . in one low dose male mouse that died on day 11, evidence of numerous fibrin thrombi were identified in the small and intermediate vessels, suggestive of a hypercoagulable state within the lung. this same animal had marginally detectable virus in the lung (fig. 1c) , and fibrin thrombi were not identified in other organs including liver and kidney. infected k18-hace2 mice had elevated 160 numbers of tunel positive cells, suggesting increased cell death (fig. 3c) . we also detected increased expression of ki-67, a marker for cellular proliferation, likely expressed by proliferating golden jw, et al sars-cov-2 pathogenesis in k18-hace2 mice pneumocytes and potentially by replicating alveolar macrophages (fig. 3d) . neutrophilia was detected by h&e staining, consistent with an increase in myeloperoxidase (mpo)-positive cells (neutrophils, basophil and eosinophil) detected by ifa (fig. s3a) . however, the mpo positive 165 cells were devoid of viral antigen (fig. s3a) . there was also an increase in the presence of cd68 positive macrophages (fig. s3b) and a pronounced increase in cd45 and cd3 positive cells in infected lungs, indicative of infiltrating leukocytes including t-cells, which is consistent with histologic findings (fig. s3c) . these data indicate that k18-hace2 mice develop a pronounced lung injury upon exposure to sars-cov-2. transcriptional profiles of host immunological and inflammatory genes in lung homogenates from sars-cov-2 infected k18-hace2 and c57bl/6 mice were examined by nanostring-based gene barcoding on day 3 (k18-hace2 and c57bl/6) or in k18-hace2 mice at the time euthanasia. 478 transcripts were increased in k18-hace2 mice and 430 decreased at a log2 fold cutoff of 1 and a p value <0.05. many of the increased transcripts in the k18-hace2 175 mice were inflammatory genes including il-6, interferon gamma and chemokines (ccl2, ccl5, ccl9 and cxcl10) (fig. 4a) . the type i interferon transcripts ifna1 and ifnb1 along with the cytokines il-9 and il-2 were decreased. consistent with the increased presence of cd68 macrophages, cd68 transcripts were also significantly increased in k18-hace2 mice. viral sensing pathways were elevated in infected mice with severe disease, indicated by high transcript was observed in the nasal turbinates in the majority of mice and rarely within the eyes of infected mice (fig. 5a, fig. s5 and table s1 ). viral rna in the eye was localized to the retina, suggesting 195 viral infection of neurons in the inner nuclear layer and ganglion cell layer (fig. s5) . despite infection, evidence of inflammation or other damage in the retina or elsewhere in the eye was not present. viral rna and spike protein were also detected to some degree in the nasal turbinate epithelium (predominantly olfactory epithelium) as early as day 3 post-infection (fig. 5b) , as indicated by co-staining with cytokeratin ( fig 5c) . pathology was minimal, predominantly 200 isolated to few areas of olfactory epithelium atrophy, with degeneration or erosion present in the epithelium lining the dorsal and lateral nasal meatuses (fig. 5d) . some cellular sloughing was also detected and these sloughed cells contained viral rna (fig. s5) . in mice succumbing to disease on days 5-7, virus was present in the olfactory bulb and most animals showed asymmetrical staining, with one bulb more positive than the other. viral rna was detected throughout the 205 olfactory bulb, including in the olfactory nerve layer (onl), glomerular layer (gl), external golden jw, et al sars-cov-2 pathogenesis in k18-hace2 mice plexiform layer (epl), and mitral cell layer (mcl) of olfactory bulbs in most of animals. viral protein co-localized with the neuronal marker neun, suggesting virus was present within neurons in the olfactory bulb (fig. s6) . virus was not detected in the olfactory bulb of animals taken on day 3, suggesting that virus trafficked to this region on day 4 or 5. these data indicated that sars-210 cov-2 infects cells within the nasal turbinates, eyes and olfactory system and that infection was observed in epithelial cells and neurons. brain infection was not observed in the majority of animals examined on day 3, but was prevalent in mice necropsied on days 5-11 (table 215 s1 ). evidence of sars-cov-2 was found throughout the brain including strong but variably diffuse signal in regions of the thalamus, hypothalamus, amygdala, cerebral cortex, medulla, pons and midbrain (fig. 6a, table s1 ). similar intense but less diffuse signal was present within the hippocampus. ish positive cells included neurons of thalamic nuclei. in contrast, cells within the vessel walls and perivascular spaces infiltrated by mononuclear inflammatory cells were negative 220 for viral genomic rna (fig. 6a) . histopathological changes were detected in the brains of several infected k18-hace2 mice euthanized on day 5-11, but not in most mice taken on day 3 (fig. 6b , table s1 and fig. s7 ). in the thalamus/hypothalamus, vasculitis was the most common lesion characterized by endothelial hypertrophy and increases in mononuclear leukocytes within the vessel wall and/or filling the perivascular space. small amounts of necrotic debris were also 225 identified. in the majority of these cases, the vascular lesion was characterized by the presence of increased numbers of microglia within the adjacent neuropil. occlusive fibrin thrombi were also detected within the thalamus in a few mice. meningitis was observed in a subset of animals and golden jw, et al sars-cov-2 pathogenesis in k18-hace2 mice was associated with infiltration of mononuclear leukocytes (majority lymphocytes) and is most prominent adjacent to vessels. in the mouse that died late on day 11 with massive pulmonary 230 clotting, the rostral cerebral cortex brain lesions included small to intermediate size vessel walls multifocally expanded by mononuclear inflammatory cells and perivascular hemorrhage extending into the adjacent neuropil (fig. 6b) . increased numbers of microglia were readily detected on h&e stained sections, expanding outward from the perivascular neuropil. an increase in numbers of microglia were found in the neuropil surrounding affected vessels. additionally, brains also 235 showed signs of neuroinflammation indicated by increased staining of iba-1 and gfap indicating microgliosis and astrocytosis, respectively (fig. 6c) . necrosis was identified in at least five animals, and was most prominent within the periventricular region of the hypothalamus as well as in the amygdala. the lesion was characterized by moderate numbers of shrunken, angular cells with hypereosinophilic cytoplasm, pyknotic nuclei and surrounded by a clear halo (fig. s7) . the 240 morphology and location of individual cells was suggestive of neuronal necrosis, but further investigation will be required to confirm this finding. viral spike protein was detected in neun positive cells, indicating viral infection of neurons (fig. 6d) . viral antigen was absent in gfap positive cells suggesting virus does not productively infect astrocytes. these data indicate that similar to sars-cov, sars-cov-2 also targets the brain of k18-hace2 mice, causing brain 245 injury. as indicated by duplex ish labeling of brain, neurons are positive for both hace2 transgene expression and viral genomic rna (fig. s8) . no animal showed outward signs of neurological deficient, such as hind limb paralysis, head tilting or tremors. 250 golden jw, et al sars-cov-2 pathogenesis in k18-hace2 mice other murine infection models for sars-cov-2 involving transgene expression of the human ace2 protein have been reported (23, 33, 34) . however in these models, sars-cov-2 only produces a transient weight loss with some lung injury, but the animals generally recover. additionally, several of these models required mice aged >30 weeks for the most severe disease to occur, diminishing the practicality of these systems given the urgent need for medical 255 countermeasures (mcms) (23, 34). one system tested sars-cov-2 infection in mice in which hace2 was expressed under the control of the hfh4 promoter (33) . infection in these mice was only ~40% lethal (2/5 mice) and lung injury (assessed by plethysmography) and weight loss were absent. lethality in this system was exclusive to animals where virus was detected in the brain. other recently reported sars-cov-2 murine models involved transduction of mouse lungs with 260 a replication-incompetent adenovirus virus or an adeno associated virus (aav) encoding the hace2 gene (35, 36) . transduced lung cells expressing hace2 supported sars-cov-2 replication and lung pathology ensued along with weight loss. however, disease was generally mild with no lethality. blockade of the type i interferon system using pharmacological intervention was needed to produce the most severe disease 26 . murine systems faithfully producing the major 265 elements of severe disease observed in humans will be more useful for identifying the most promising mcms. the k18-hace2 mice lost considerable weight >12% in males and >20% in females and lethality in the high dose exceeded 90%. acute lung injury was detected in all animals succumbing to disease, with vascular damage the most common lesion. similar to findings with sars-cov, sars-cov-2 infected the brains of k18-hace2 mice. brain infection resulted in 270 vasculitis and inflammation, with sars-cov-2 antigen detected in neurons. it is possible virus enters the brain via the olfactory bulb, as has been reported for sars-cov, although more studies golden jw, et al sars-cov-2 pathogenesis in k18-hace2 mice will be required as virus may also have entered the brain via inflamed vessels. whether mortality results directly from brain infection is not clear, but this has been speculated as the major cause of mortality in sars-cov infected mice (32) . infection in the brain was delayed by at least four 275 days, as it was an uncommon finding in day 3 animals. thus, early during infection lung appears to be the primary target. we have not yet evaluated the protective efficacy of mcms in this model, but it has been reported that antibodies protect against sars-cov, demonstrating the k18-hace2 system is useful for evaluating countermeasures against ace2-targeting human coronaviruses. importantly, in our study some animals at the lower dose survived infection despite significant infection of k18-hace2 mice by sars-cov-2 produces a disease similar to that observed in acute human cases, with development of an acute lung injury associated with edema, production 285 of inflammatory cytokines and the accumulation of mononuclear cells in the lung. impacted lungs had elevated levels of transcripts consistent with respiratory damage such as increased expression of himf, which is involved in activation of lung endothelial cells in response to lung inflammation (37) . we also found increased levels of sgpl1, a molecule associated with lung injury (38) and known to be increased in mechanically ventilated mice (39) . a prominent finding in infected k18-290 hace2 mice system was vasculitis. endotheliitis/vasculitis has been observed in human covid-19 patients and a role for the endothelium in acute disease is becoming more apparent (7, 8, 40) . direct viral infection in human endothelial cells has also been reported (7). curiously in the mice, virus was absent in these areas suggesting vasculitis was due to host inflammatory processes, but golden jw, et al sars-cov-2 pathogenesis in k18-hace2 mice further study will be required to determine if vascular damage is incurred by direct or indirect viral 295 effects. it has been speculated that during human acute disease, inflammatory cytokines (il-6 in particular) are major drivers of tissue damage and this is supported by data from humans showing il-6 receptor targeting with pharmacological antagonists (tocilizumab) can reduce morbidity (41). we found il-6 transcripts, as well chemokines, are elevated in mouse lungs. during human disease, pulmonary inflammation is associated with increases in lung granulocytes and an increase 300 in macrophages (4, 42, 43) . some have speculated that these macrophages play an important role in host injury (44) . it is still unclear if human macrophages are directly infected by sars-cov-2, though we found virus antigen in cd68 macrophages and others report infection in murine mac2-positive macrophages (23). further study will be required to determine the role macrophages play in sars-cov-2 lung injury and given the regents available, murine systems 305 may be highly suited for these studies. in addition to vascular issues, coagulopathies are a common findings in humans (9), with pulmonary embolism having been reported, along with clotting abnormities leading to loss of limbs (45) . at least some mice produced evidence of these clotting issues, with one mouse presenting with fibrin thrombi in the lungs. during human infections, males have been reported to have more severe outcomes despite 310 a similar infection rate (46). in our experiments, we observed a statistically significant difference in survival of female and male mice infected at the lower dose of virus, with 60% of females but no males surviving infection. despite surviving, the female mice challenged with the low dose still lost a significant amount of weight (>20%). transcriptomic profiling in mouse lungs indicated that female mice that succumbed to disease had modestly lower levels of il-6, cxcl-2 and il-1ra 315 suggesting a less intense inflammatory response. our work only involved a small number of golden jw, et al sars-cov-2 pathogenesis in k18-hace2 mice animals, and more work will be required to determine if the k18-hace2 system recapitulates this sex difference in disease severity. the neuroinvasive aspects of covid-19 are becoming more appreciated (12) . indeed, sars-cov-2 causes neurological sequela in at least a third of human cases including headache, infection of these cells may help explain the loss of smell associated with some covid-19 cases. however, the virus may also gain access via disruption of the blood brain barrier, as these were inflamed in most animals and neurovasculitis has been found in humans (48). overall, the k18were then heated in kit-provided antigen retrieval buffer and digested by kit-provided proteinase. 405 sections were exposed to ish target probe pairs and incubated at 40°c in a hybridization oven for 2 h. after rinsing, ish signal was amplified using kit-provided pre-amplifier and amplifier conjugated to alkaline phosphatase and incubated with a fast red substrate solution for 10 min at room temperature. sections were then stained with hematoxylin, air-dried, and coverslipped. epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study a pneumonia outbreak associated with a 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focus on severity and mortality 465 this project was funded by a grant awarded to j.w.g. and j.w.h. from the military infectious disease program. we thank the usamriid histology lab and comparative medicine division for their assistance. we also express gratitude to the jackson laboratory for providing early access to duplex in situ hybridization. duplex in situ hybridization was performed using the rnascope 2.5 hd duplex assay kit (advanced cell diagnostics) according to the manufacturer's instructions with minor modifications. in addition to sars-cov-2 genomic rna probe mentioned above (#854841, green), another probe with c2 channel (#848031-c2, red) specifically targeting human ace2 (nm_021804.3) was designed and synthesized by advanced cell diagnostics. ish signal was amplified using kit-provided pre-amplifiers and amplifiers conjugated to either alkaline phosphatase or horseradish peroxidase, and incubated sequentially with a fast red and green chromogenic substrate solution for 10 min at room temperature. sections were then stained with hematoxylin, air-dried, and coverslipped. key: cord-314333-hkyiy1gm authors: nagata, noriyo; iwata, naoko; hasegawa, hideki; fukushi, shuetsu; harashima, ayako; sato, yuko; saijo, masayuki; taguchi, fumihiro; morikawa, shigeru; sata, tetsutaro title: mouse-passaged severe acute respiratory syndrome-associated coronavirus leads to lethal pulmonary edema and diffuse alveolar damage in adult but not young mice date: 2008-06-30 journal: the american journal of pathology doi: 10.2353/ajpath.2008.071060 sha: doc_id: 314333 cord_uid: hkyiy1gm advanced age is a risk factor of severe acute respiratory syndrome (sars) in humans. to understand its pathogenesis, we developed an animal model using balb/c mice and the mouse-passaged frankfurt 1 isolate of sars coronavirus (sars-cov). we examined the immune responses to sars-cov in both young and adult mice. sars-cov induced severe respiratory illness in all adult, but not young, mice on day 2 after inoculation with a mortality rate of 30 to 50%. moribund adult mice showed severe pulmonary edema and diffuse alveolar damage accompanied by virus replication. adult murine lungs, which had significantly higher interleukin (il)-4 and lower il-10 and il-13 levels before infection than young murine lungs, rapidly produced high levels of proinflammatory chemokines and cytokines known to induce macrophage and neutrophil infiltration and activation (eg, tumor necrosis factor-α). on day 2 after inoculation, young murine lungs produced not only proinflammatory cytokines but also il-2, interferon-γ, il-10, and il-13. adult mice showed early and acute excessive proinflammatory responses (ie, cytokine storm) in the lungs after sars-cov infection, which led to severe pulmonary edema and diffuse alveolar damage. intravenous injection with anti-tumor necrosis factor-α antibody 3 hours after infection had no effect on sars-cov infection. however, intraperitoneal interferon-γ injection protected adult mice from the lethal respiratory illness. the experimental model described here may be useful for elucidating the pathophysiology of sars and for evaluating therapies to treat sars-cov infection. in the severe acute respiratory syndrome-associated coronavirus (sars-cov) epidemic of winter 2002 to 2003, ϳ800 people (10% of the ͼ8000 sars patients) suffered progressive respiratory failure and died. [1] [2] [3] [4] [5] common symptoms of sars include fever, nonproductive cough, myalgia, and dyspnea. an age of 60 years or older, co-morbid disease, male sex, high neutrophil counts, and several biochemical abnormalities are associated with poor outcomes. 6 -10 the sars-cov spike (s) protein mediates the infection of cells bearing an appropriate receptor. 11 one such receptor is angiotensin-converting enzyme 2 (ace2), which binds sars-cov s protein with high affinity. [11] [12] [13] [14] that the binding of sars-cov to ace2 may contribute to sars-cov-associated pathology is suggested by several reports showing that angiotensin ii expression promotes severe lung failure on acute lung injury whereas ace2 expression protects from lung injury. 15, 16 however, it is likely that the acute lung injury caused by sars-cov infection is also attributable to a complex pathophysiological process in which inflammatory cytokines released by activated alveoli macrophages induce immune system dysregulation. [17] [18] [19] [20] to understand the pathogenesis of sars-cov, the sars-cov susceptibility of experimental animals such as monkeys, cats, ferrets, mice, pigs, guinea pigs, ham-sters, chickens, and rats has been investigated. 2, 4, [21] [22] [23] [24] [25] [26] [27] [28] all of these animals are susceptible to sars-cov after intrarespiratory inoculation and exhibit virus excretion in pharyngeal or nasal swabs, histopathological pulmonary lesions, and seroconversion. however, the course of infection in these animals is shorter than that in humans. as in humans, an advanced age correlates positively and independently with adverse outcomes and is a predictor of mortality in animal models. 6 -10 moreover, sars-cov isolates replicate better in aged balb/c mice than in younger mice. 29 it is likely that the correlation between poor outcome and advanced age reflects the weakened immune responses of the elderly, in particular their impaired cytokine responses. this is significant because cytokines regulate the immune response to infection. indeed, analysis of the cytokine responses of elderly individuals to respiratory infections that lead to severe pulmonary diseases (eg, listeria monocytogenes, respiratory syncytial virus, influenza virus) 30 -33 have revealed unbalanced th1-type and th2-type responses. we recently succeeded in establishing a rat model of sars using rat-passaged sars-cov. 34 although the ratpassaged sars-cov was not lethal, it induced more severe pathological lesions in adult f344 rats than in young rats. we found that the severe inflammation in the adult rats was associated with high levels of inflammatory cytokines in the serum and lung homogenates, especially interleukin (il)-6, along with low levels of the immunosuppressive cytokine il-10. il-6 is an inflammatory cytokine that is produced by monocytes, leukocytes, endothelial cells, fibroblasts, and alveolar epithelial cells. sars patients have significantly elevated serum il-6 levels. 19 il-10 is produced by macrophages, th2 lymphocytes, and b cells and inhibits tumor necrosis factor (tnf)-␣ production and neutrophil activation in lipopolysaccharide-induced acute lung injury, thereby suppressing lung tissue injury. 35 it has been reported that serum il-10 levels increase in sars patients during the convalescence phase. 19 in this study, we established a new and more useful experimental small animal model for sars by using balb/c mice and mouse-passaged sars-cov. this model allows us to better characterize the virus-host relationship and determine which immune responses are antiviral and which are pathogenic. here, we sought to determine why sars-cov infection is more frequently lethal in elderly patients by comparing sars-cov-infected adult and young mice in terms of their pulmonary pathology and immune responses. the frankfurt 1 isolate of sars-cov used in this study was kindly supplied by dr. john ziebuhr, institute of virology and immunology, university of wü rzburg, wü rzburg, germany. the virus was propagated twice in vero e6 cells purchased from american type cell collection (manassas, va) that were cultured in eagle's minimal essential medium (mem) containing 5% fetal bovine serum, 50 iu of penicillin g, and 50 g of streptomycin per ml. titers of this stock virus were expressed as 50% of the tissue culture infectious dose (tcid 50 )/ml on vero e6 cells, which was calculated according to the behrens-kä rber method. work with infectious sars-cov was performed under biosafety level 3 conditions. compared to the original virus, the frankfurt 1 isolate used in our laboratory has one amino acid change at position 641 (his to tyr) in the s protein and another in open reading frame (orf) 1a 429 (ala to ser). these changes presumably arose during the passage through vero e6 cells. female 4-week-old or 6-month-old balb/c mice were purchased from japan slc (shizuoka, japan) and maintained in specific pathogen-free facilities. on experimental infection, these animals were housed in biosafety level 3 animal facilities. these animal experiments were approved by the animal care and use committee of the national institute of infectious diseases, tokyo, japan. the frankfurt 1 isolate of sars-cov was serially passaged 10 times in 4-week-old female balb/c mice, as follows. after intranasal inoculation, three mice were sacrificed on day 3 after inoculation and their bronchoalveolar wash fluids were collected. these bronchoalveolar fluids were then used to inoculate three additional balb/c mice, whose bronchoalveolar fluids on day 3 after inoculation were used to inoculate fresh mice. after 10 such passages in mice, the lungs were removed under sterile conditions, washed three times, and homogenized in 1 ml of phosphate buffer containing 0.1% bovine serum albumin, 20 iu of penicillin g, 20 l of streptomycin, and 1 g of amphotericin b per ml. the lung homogenates were centrifuged at 1000 ϫ g for 20 minutes, and 1 ml of the supernatants in 10 ml of mem containing 2% fetal bovine serum were used to infect vero e6 cells. after 1 hour of adsorption, the inoculum was removed and mem containing 2% fetal bovine serum was added. the cell cultures were incubated at 37°c with 5% co 2 for 2 days and then treated once with freezethawing. after centrifugation at 1000 ϫ g for 20 minutes, the supernatants (referred to here as f-musx-veroe6) were used as the virus inoculum. compared to the original virus, f-musx-veroe6 has amino acid mutations in the s protein at positions 480 (asp to glu) and 641 (his to tyr); the latter change is identical to one of the mutations found in the frankfurt 1 isolate. in the completely sequenced genome, f-musx-veroe6 also has two additional mutations in orf1a 3534 (phe to leu) and orf1ab 5172 (thr to ile). the mutation in orf1a 429 found in the frankfurt 1 isolate was not present. mice were anesthetized by intraperitoneal injection with a 0.1 ml/10 g body weight mixture of 1.0 mg ketamine and 0.02 mg xylazine. the animals were then inoculated intranasally in the left nostril with the frankfurt 1 isolate or f-musx-veroe6 (2 ϫ 10 6 tcid 50 in 20 l) and observed for clinical signs. body weight was measured daily for 10 or 21 days. infected animals were also sacrificed at various time points after inoculation to analyze virus replication, hematology, cytokine expression, and pathology (n ϭ 3 in each group). twenty percent (w/v) tissue homogenates of the lung, maxilla (including the nasal cavity), cervical lymph node, spleen, liver, and kidney were prepared in mem containing 2% fetal bovine serum, 50 iu penicillin g, 50 g streptomycin, and 2.5 g amphotericin b per ml (mem-2fbs). bronchoalveolar and nasal wash fluids were also collected for analysis of virus replication. viral infectivity titers of respiratory tract and wash fluids were determined as described above. virus isolation from other tissues was performed by blind passage after freezing and thawing the first-round passage using vero e6 cells. total blood cell counts in peripheral blood collected in sodium-heparinized tubes were measured by an autoanalyzer (cell tuck; nihon koden, tokyo, japan). neutrophil, lymphocyte, monocyte, eosinophil, and basophil counts were determined by microscopic analysis. antibodies used for flow cytometry were anti-cd4-phycoerythrin-cy5 (ebioscience, san diego, ca), anti-cd8␤phycoerythrin (santa cruz biotechnology, santa cruz, ca), and anti-pan-nk cells-fluorescein isothiocyanate (ebioscience). cells incubated with these surface-binding antibodies were fixed in 2% paraformaldehyde in phosphate-buffered saline (pbs) and subjected to flow cytometry (epics elite; beckman coulter, fullerton, ca) using expo cytometer software (beckman coulter). homogenized lung tissue samples were diluted 1:1 with cell extraction buffer [10 mmol/l tris, ph 7.4, 100 mmol/l nacl, 1 mmol/l edta, 1 mmol/l egta, 1 mmol/l naf, 20 mmol/l na 4 p 2 o 7 , 2 mmol/l na 3 vo 4 , 1% triton x-100, 10% glycerol, 0.1% sodium dodecyl sulfate, and 0.5% deoxycholate (biosource international, inc., camarillo, ca)], incubated for 30 minutes on ice with vortexing at 10 minute intervals, and then centrifuged at 15,000 ϫ g for 10 minutes at 4°c. supernatants were diluted 1:5 in assay diluent of the mouse cytokine 20-plex antibody bead kit (biosource international). sera and the 20% lung homogenate supernatants were subjected to ultraviolet irradiation for 10 minutes to inactivated virus infectivity and stored at ϫ80°c until they were used to determine the presence of mouse cytokines, namely, basic fibroblast growth factor, gm-csf, interferon (ifn)-␥, il-1␣, il-1␤, il-2, il-4, il-5, il-6, il-10, il-12p40/p70, il-13, il-17, ip-10, keratinocyte chemoattractant (kc), monocyte chemoattractant protein 1 (mcp-1), mig, mip-1␣, tnf-␣, and vascular endothelial growth factor (vegf), by using the mouse cytokine 20-plex antibody bead kit and luminex 100tm (luminex co., austin, tx). animals (n ϭ 3 in each group) were anesthetized and perfused with 2 ml of 10% phosphate-buffered formalin. fixed lung, heart, kidney, liver, spleen, small and large intestine, brain, spinal cord, and maxilla (including nasal cavity) tissues were routinely embedded in paraffin, sectioned, and stained with hematoxylin and eosin. maxilla samples were decalcified in phosphate-buffered saline (ph 7.4) plus 10% edta before being embedded. immunohistochemical detection of the sars-cov antigens was performed on paraffin-embedded sections, as follows. after deparaffinizing with xylene, sections were rehydrated in ethanol and immersed in pbs. antigens were retrieved by hydrolytic autoclaving for 20 minutes at 121°c in 10 mmol/l sodium citrate-sodium chloride buffer (ph 6.0). after cooling, the sections were immersed in pbs. endogenous peroxidase was blocked by incubation in 1% hydrogen peroxide in methanol for 30 minutes. after washing in pbs, the sections were incubated with normal rabbit serum for 5 minutes, and then with rabbit antibody against sars-cov 32,36 overnight at 4°c. after three washes in pbs, the sections were incubated with biotin-conjugated anti-rabbit igg for 30 minutes at 37°c, followed by reaction with streptavidin-peroxidase for 30 minutes at room temperature. peroxidase activity was detected by development with diaminobenzidine containing hydrogen peroxide. nuclei were counterstained by hematoxylin. sars-cov-and mock-infected adult and young mice were euthanized 1, 3, and 5 days after inoculation by exsanguination under excess ether anesthesia, after which the lungs were harvested for pathological examination (three mice per group). mock infection was performed by using mem containing 2% fetal bovine serum. for staining with anti-mac-3 and anti-surfactant d (sp-d) antibodies and to detect sars-cov antigens, the lungs were fixed with 4% paraformaldehyde in pbs at 4°c for 15 to 18 hours and embedded in paraffin according to the manufacturer's instructions (bd biosciences pharmingen, san diego, ca). the paraffin-embedded sections were then subjected to a double-immunofluorescence staining method 37 using a polyclonal rabbit antibody against sars-cov 36 and the skot9 monoclonal mouse antibody against nucleocapsid protein 38 or a monoclonal rat anti-mac-3 antibody against mouse mononuclear phagocytes (bd biosciences pharmingen), and a poly-clonal rabbit anti-sp-d antibody (chemicon international, inc., billerica, ma). briefly, after deparaffinization with xylene, the sections were rehydrated in ethanol and immersed in pbs. antigens were retrieved by hydrolytic autoclaving for 10 minutes at 121°c in 10 mmol/l sodium citrate-sodium chloride buffer (ph 6.0). after cooling, the sections were immersed in pbs, and then incubated with primary antibodies overnight at 4°c. to block background staining, normal donkey serum or the m.o.m. immunodetection kit for primary mouse monoclonal antibody (vector laboratories, burlingame, ca) were used according to the manufacturer's instructions. after three washes in pbs, the sections were incubated for 30 minutes at 37°c with biotin-conjugated secondary antibodies, ie, a donkey anti-rat serum (jackson immunoresearch, west grove, pa) to detect the mac-3 antibody or a goat anti-rabbit serum (jackson immunoresearch) to detect the sp-d antibody. after three washes in pbs, the sections were incubated with streptavidin-alexa fluor 488 (molecular probes, eugene, or) for 60 minutes at room temperature. after three washes in pbs, to detect the sars-cov antibodies (the skot9 or the rabbit antibody), the sections were incubated with anti-rabbit or anti-mouse alexa fluor 568 (molecular probes) for 60 minutes at room temperature. the sections were counterstained with to-pro-3 nucleic acid staining (molecular probes) and images were captured and analyzed by confocal laser microscopy (fluoview, fv1000; olympus, tokyo, japan). three hours after intranasal inoculation of f-musx-veroe6 (2 ϫ 10 6 tcid 50 in 20 l), adult (6-month-old) balb/c females mice were injected intravenously with 100 l of anti-mouse tnf-␣ rat monoclonal antibody (1 g/l, biosource), or isotype-matched control rat antibody (1 g/l; mp biomedicals, solon, oh) in pbs, or injected intraperitoneally with 100 l of recombinant mouse ifn-␥ (0.05 g/l; r&d systems, minneapolis, mn) or intraperitoneal injection with 100 l of pbs/0.1% bovine serum albumin was used as control. at least two independent experiments were performed (n ϭ 5 or 8 per group). sars-cov-and mock-infected mice were injected intravenously with 100 l of 1% evans blue dye (tokyo kasei, tokyo, japan) 1 hour before sacrifice (n ϭ 3 in each group). mock infection was performed by using mem containing 2% fetal bovine serum. after perfusion with isotonic saline, the whole lung was removed and immersed in 10% phosphate-buffered formalin. the fixed lungs were immediately frozen in cold acetone with dry ice in 100% o.c.t. compound (sakura finetechnical co. ltd., tokyo, japan). cryosections (5 m) (cm1900; leica, wetzlar, germany) were mounted on mas-coated slides (matsunami, osaka, japan), air-dried, and examined with a fluorescence microscope. statistical significance was determined by student's ttest. p values ͻ0.05 were considered significant. the frankfurt 1 isolate was passaged twice on veroe6 cells and then serially passaged 10 times in young balb/c mice (4-week-old females) by intranasal inoculation of bronchoalveolar fluids from infected mice. the f-musx-veroe6 strain showed higher replication and pathogenicity in the respiratory tract of young balb/c mice than the original frankfurt 1 isolate, as follows (figure 1 , a-e). on day 3 after inoculation, f-musx-veroe6 replication in the lung washes was higher than that of the original frankfurt 1 isolate (p ϭ 0.055) but lower in the nasal washes (p ͻ 0.01) ( figure 1a ). compared to frankfurt 1 isolate-inoculated young mice, the f-musx-veroe6inoculated young mice also evinced more lung inflammation, as shown by neutrophil, macrophage, and lymphocyte infiltration and virus antigen-positive cells in the alveolar spaces ( figure 1 , b-e). however, the f-musx-veroe6-inoculated young mice did not develop any obvious respiratory illnesses, although they did show transient weight loss for a few days after inoculation (data not shown). because advanced age is associated with higher mortality in human sars patients and sars-cov replicates better in aged mice, 6 -10,29 we experimentally infected 6-month-old (adult) female balb/c mice with f-musx-veroe6 or the frankfurt 1 isolate. although none of the mice showed clinical signs of illness after intranasal inoculation with frankfurt 1 isolate, all f-musx-veroe6-inoculated mice became severely ill, as revealed by significant weight loss (ϳ20% of their initial body weight), hunching, ruffled fur, and dyspnea on day 2 after inoculation ( figure 1f ). three of the ten mice became moribund and died of severe respiratory illness on days 3, 6, and 10 after inoculation (30% mortality rate). the surviving animals recovered their body weight during days 4 to 6 after inoculation. these results demonstrated that serial in vivo passage of sars-cov in mice increased the virulence of the virus, especially in adult mice. thus, we characterized the clinical and pathological features of f-musx-veroe6-infected young and adult mice up until day 5 after inoculation in more detail. the young mice again showed transient weight loss of up to 8.2% (sd ϭ 3.7%) during days 2 to 4 after inoculation but had recovered their weight by day 5 after inoculation ( figure 2a ). in contrast, the adult mice showed continuous weight loss of up to 23.0% (sd ϭ 4.5%) of their initial body weight that continued until day 5 after inoculation. all adult mice showed virtually identical clinical manifestations during days 1 to 2 after inoc-ulation (such as hunching and ruffled fur) that were not observed in the young mice. severe respiratory symptoms such as dyspnea were also observed in the adult mice from 2 days after inoculation onwards. in this experiment, 50% of the adult mice had died by day 5 after inoculation. the lungs of infected young and adult mice were weighed on days 0 to 5 after inoculation. the progressive increase in lung weight of the adult mice suggested the development of pulmonary edema (figure 2 , b and c). by day 5 after inoculation, the adults showed significantly greater lung weight changes than the young mice (p ͻ 0.01). the lungs of infected young and adult mice were also subjected to histopathological analysis on days 1 to 5 after inoculation (figure 3, a-h) . on day 1 after inoculation, both young and adult mice had antigen-positive epithelial cells in the bronchi and alveoli. the antigenpositive cells in the alveoli were considered on the bases of morphology and immunohistochemistry to be mainly type ii pneumocytes (figure 3 , a and e; see supplemental figure s1 at http://ajp.amjpathol.org). on day 2 after inoculation, antigen-positive atrophic and necrotic cells were seen in the alveolar area of both mice ( figure 3b ). in addition, antigen-positive activated alveolar macrophages associated with inflammatory infiltrations were seen in the alveolar area of adult mice ( figure 3f ). no antigen-positive cells were seen in the bronchi on day 2 after inoculation or afterward in either young or adult mice. on day 3 after inoculation, the young mice had diffuse inflammatory infiltrates consisting mainly of mononuclear cells (figure 3 , c and d), and virus antigenpositive cells were seen in the alveolar area. activated macrophages, lymphocytes, and neutrophils were seen in the alveoli on days 4 and 5 after inoculation. in contrast, the adult mice evinced severe pulmonary edema, and congestion on day 3 after inoculation ( figure 3 , g and h). in these mice, the main inflammatory cells around the adult blood vessels and alveolar area on days 3 to 5 after inoculation were neutrophils and activated macrophages. fibrin deposition and hyaline membrane formation in the alveolar duct and alveoli were also observed ( figure 3h ), and microhemorrhages was seen in the alveolar area. the adult mice also had high virus titers in the lung and maxilla (including nasal cavity) and their fluid (figure 2, d and e) . after the infection, virus continued to be isolated from the cervical lymph nodes, spleen, liver, and kidneys of adult mice after day 2 after inoculation whereas virus could no longer be isolated from any young mouse tissue (apart from the lung) after this time point (table 1) . to analyze the immune responses of young and adult mice after infection with f-musx-veroe6, we examined their peripheral blood white blood cell counts (figure 4) , and measured the levels of 20 different chemokines and cytokine levels in their plasma and lung homogenates (figures 5 and 6 ). before infection (day 0), the adult mice had significantly lower white blood cells counts, especially with regard to lymphocytes (including cd4 ϩ and cd8␤ ϩ t cells), than the , lung wash fluid, and lung homogenates of young mice on days 3, 5, and 7 after inoculation (n ϭ 3 per group). the detection limit was 10 1.5 tcid 50 /g of tissue. asterisks indicate statistically significant differences between f-musx-veroe6 and the frankfurt isolate (p ͻ 0.05). b-e: histopathological features of the lungs of young mice on day 3 after inoculation. b: after infection with the frankfurt 1 isolate, inflammatory infiltrates in the lung were not detected. moreover, very few alveolar pneumocytes and alveolar duct and alveolus epithelial cells were sars-cov antigen-positive (c, arrowheads). in contrast, extensive cellular infiltration (d) and many virus antigen-positive cells (e) were seen in the alveolar area after f-musx-veroe6 infection. f: clinical illness in individual 6-month-old adult balb/c mice after frankfurt isolate or f-musx-veroe6 infection (n ϭ 10 per group). shown are the changes in body weight (expressed as percentages of the body weight on day 0). the mean initial body weight of the two mouse groups (on day 0) were 24.72 ϯ 1.04 g and 25.44 ϯ 1.55 g, respectively. significant differences in body weight change were detected on days 2 to 8 after inoculation. for example, the average body weight f-musx-veroe6-infected adult mice on day 5 after inoculation was 83.4 ϯ 9.88% of the mean day 0 body weight. this was significantly lower than the average body weight change of frankfurt 1 isolate-infected adult mice on day 5 after inoculation (102.4 ϯ 2.99%). three f-musx-veroe6-infected adult mice died (crosses) of severe pulmonary edema on days 3, 6, and 10 after inoculation. young mice (figure 4 ). after infection, the neutrophil counts in the adult mice increased; however, this change was not observed in the young mice. in young mice, relative to counts on day 0, lymphocyte counts decreased signifi-cantly (p ͻ 0.05) on days 2, 3, and 4 after inoculation but then recovered, cd8␤ ϩ t-cell counts decreased significantly on day 2 and then recovered, and cd4 ϩ t-cell counts decreased slightly and then showed a significant to assess the lungs for pulmonary edema, the lungs were weighed after mice were sacrificed on days 1 to 5 after inoculation by exsanguination under anesthesia (n ϭ 3 per group). c: lungs from virus-and mock-infected young and adult mice obtained at the indicated time points after inoculation. arrowheads indicate focal congestion. on day increase on day 5. in contrast, although the lymphocyte counts of adult mice also dropped and were significantly lower than day 0 counts on days 3 and 4, they did not evidence an improvement on day 5 after inoculation. moreover, the cd8␤ ϩ t-and cd4 ϩ t-cell counts of the adult mice also showed significant drops on days 1, 3, and 4 after inoculation (p ͻ 0.05) but had recovered poorly on day 5 after inoculation, unlike the counts in young mice. with regard to the pannk ϩ cells counts, both the young and adult mice showed a marked drop on day 1 after inoculation that was followed by a brief recovery and then another loss on day 4 after inoculation cell count loss at day 1 and 5 days after inoculation compared with 0 days after inoculation in adult mice (p ͻ 0.05). with regard to the cytokine responses of the mice, the lung homogenates of adult mice on day 1 after inoculation had significantly higher levels of monocyterelated chemokines [ie, mcp-1, macrophage inflammatory protein 1 (mip-1), and ifn-␥-inducible protein 10 (ip-10)] than those from young mice ( figure 5 ). in contrast, on day 2 after inoculation, the lung homogenates of young mice exhibited elevated levels of these three cytokines as well as kc, monokine induced by ifn-␥ (mig), and vascular endothelial growth factor (vegf) ( figure 5 ). compared to young mice, the lung homogenates of adult mice on day 1 after inoculation also had higher levels of il-1␣, il-1␤, and tnf-␣, and on day 3 after inoculation, higher levels of il-6 were 0 0 0 0 0 0 0 0 0 1d a y 3 3 3 1 0 3 3 3 3 0 2 days 3 3 2 2 0 3 3 2 2 1 3 days 3 0 0 0 0 3 2 1 1 1 4 observed ( figure 6 ). in contrast, the lung homogenates of young mice had significantly higher levels of ifn-␥ (on day 2 after inoculation), il-2 (on days 2 to 5 after inoculation), il-10 (on days 0, and 2 to 5 after inoculation), and il-13 (on days 0 to 2, and 4 and 5 after inoculation). notably, the lung homogenates of preinfected adult mice (day 0) had higher il-4 and lower il-10 and il-13 levels than young murine lungs. these observations indicate that the patterns of cytokine/chemokines responses are different between young and adult mice after sars-cov infection. adult mice showed early and acutely excessive proinflammatory responses in the lung after sars-cov infection. to determine whether the tnf-␣ response of the adult mice and the ifn-␥ produced by the young but not adult mice played significant roles in the development of sars-like illness by the f-musx-veroe6-infected adult mice, we treated adult mice with an anti-tnf-␣ antibody or ifn-␥ 3 hours after infection (figure 7 , a and b). although the intravenous injection with anti-tnf-␣ antibody delayed the onset of this weight loss in the infected adult mice, as well as the onset of respiratory illness, both the anti-tnf-␣ antibody-treated and control adult mice showed significant body weight loss up until 6 days after inoculation and there were no significant differences in mortality rates between treated and control adult mice (treated adult mice: 62.5%, 50% mortality rate; control adult mice: 37.5%, 37.5% mortality rate in two separate experiments) ( figure 7a ). in contrast, the ifn-␥-treated mice rapidly recovered from the illness as evidenced by their body weight loss and severe acute respiratory symptoms and all animals survived after onset 3 days after inoculation ( figure 7b ). in contrast, 50% of the control adult mice died. . asterisks indicate statistically significant higher or lower chemokine levels in adult mice (p ͻ 0.05) compared to young balb/c mice. adult mice showed earlier induction of mcp-1, mip-1, and ip-10 in the lungs than young mice but these three chemokines and mig and vegf were at significantly higher levels in the lungs of young mice on day 2 after inoculation. to determine whether the protective effect of ifn-␥ treatment is attributable to suppression of viral replication, the virus titers on the day 3 after inoculation of nasal washes, maxilla homogenates, lung washes, and lung homogenates of ifn-␥-treated and pbs-treated adult mice after the infection were compared. however, the two groups did not differ significantly in terms of virus titers in the respiratory tracts ( figure 7c ). the ifn-␥-treated mice showed much milder histopathological changes than the untreated mice because only very mild edema with slight mononuclear cell infiltration was observed around the blood vessels after the infection ( figure 7d ). in contrast, the pbs-treated mice exhibited severe edema and infiltration of inflammatory cells, mainly neutrophils, around blood vessels ( figure 7e ). by examining evans blue dye extravasation, we found the ifn-␥treated mice also had lower blood vessel permeability than the pbs-treated mice (figure 8, a-g) . together, these results suggest that ifn-␥ treatment 3 hours after inoculation protected the mice from severe sars-covinduced pulmonary edema that was responsible for the death of the untreated adult mice. to understand better the pathogenesis of sars after sars-cov infection, we developed a useful experimental mouse model of sars. when the frankfurt 1 isolate of sars-cov was serially passaged in vivo in young balb/c mice, the passaged virus (f-musx-veroe6) exhibited in-creased infectivity in the murine lung. f-musx-veroe6 was also able to induce severe sars-like illness in adult (6-month-old) balb/c mice and several animals died of severe pulmonary edema and acute alveolar damage. however, young (4-week-old) mice were relatively resistant to f-musx-veroe6 and did not evince any obvious respiratory illness. when the immune responses of infected young and adult mice were compared, the adult animals had significantly lower pre-existing lymphocyte and cd4 ϩ and cd8 ϩ t-cell counts, their lungs expressed significantly higher il-4 and lower il-10 and il-13 levels before infection, and their lungs did not show the strong up-regulation of il-2 (a t-cell cytokine), il-10, il-13, and ifn-␥ that was exhibited by the young murine lungs after infection. during the infection, the lungs of adult mice also produced inflammatory chemokine-/cytokine-related macrophages (ie, mcp-1, mip-1 and ip-10, il-1␣, il-1␤, and tnf-␣) earlier than young mice (day 1 after inoculation) and at higher levels. they also had higher il-6 levels on day 3 after inoculation. in contrast, the young mice produced high levels of inflammatory chemokines and cytokines (ie, mcp-1, mip-1, ip-10, kc, mig, vegf, and il-1␣) on day 2 after inoculation and produced very little il-6 at any time point. these observations suggest that advanced-age balb/c mice have an early and acutely excessive proinflammatory cytokine reactions (ie, a cytokine storm) in response to f-musx-veroe6. this cytokine storm results in severe pulmonary edema with macrophage and neutrophil infiltration, which causes severe acute lung injury and is likely to be the cause of death in infected adult mice. supporting this scenario are reports of age-related differences in pulmonary cytokine and chemokine responses to other pathogens, especially th1 and th2 cytokine imbalances. [31] [32] [33] moreover, recent reports showed that unregulated ifn responses during acute sars prevented sars-cov-infected patients from switching from innate to adaptive immunity, 39, 40 and some reports described that ifn-␥ may be responsible for lung immunopathology in sars patients. 41, 42 interestingly, in our model, injection of ifn-␥ 3 hours after inoculation significantly reduced the acute pulmonary edema induced by infection. notably, these protective effects of ifn-␥ injection were not associated with reductions in viral titers in the lung. this animal model and human results seem not to be consistent. however, these observations together suggest that the failure of the adult animals to produce ifn-␥, which is a prominent immunomodulator that plays a key role in host defense against intracellular pathogens, 43,44 was responsible for their inability to control the excessive and pathogenic innate immune response to the virus in the lung. supporting this is our previous study on the rat sars model, which showed that although adult rats developed severe inflammatory reactions that led to pulmonary edema, all of the infected animals survived; significantly, the infected adult rats had similar cytokine profiles as infected adult mice except that they also produced ifn-␥. 34 thus, a strong and timely ifn-␥ response may be needed to prevent the immunopathology induced by sars-cov infection. with regard to the inflammatory reactions in the f-musx-veroe6-infected adult lung, our histopathological and chemokine/cytokine analyses suggested that on day 1 after inoculation, the affected respiratory epithelial cells and pulmonary macrophages released acute inflammatory chemokines (mcp-1, mip-1, ip-10) and cytokines (il-1, tnf-␣, il-12). thus, pulmonary macrophage activation associated with the release of tnf-␣ and il-1 appears to predominate in the early phase of the inflammatory reaction in adults. tnf-␣ and il-1 are classic acute inflammatory cytokines that recruit neutrophils and monocytes to the area of infection along with increasing vascular permeability, where they activate these cells so that they can eradicate the pathogens. however, when tnf-␣ is overexpressed, it can induce systemic clinical and pathological abnormalities such as depressing cardiac dysfunction and cardiomyocyte death. 45, 46 this double-edged aspect of tnf-␣ function may be why there were no effects of neutralizing tnf-␣ antibodies on sars-cov infection in this model. supporting the key role of tnf-␣ in f-musx-veroe6-induced immunopathology is that the lungs of the adult mice on days 3 and 5 after inoculation were infiltrated with predominantly neutrophils; these mice also exhibited neutrophilia. young mice did not evince these changes. this suggested that infected adult mice produce tnf-␣, which then induces neutrophil-mediated inflammation in their lungs. although the significance of this is not clear, high neutrophil infiltration in the lung is known to be the cause or the result of lung injury characterized by hyaline membrane formation and pulmonary edema. 47 high neutrophil counts in human sars cases have also been associated with poor outcomes. 8, 10 we observed in the anti-tnf-␣ antibody and ifn-␥injection experiments that the control groups, which were injected 3 hours after infection with rat igg serum intravenously or pbs intraperitoneally, showed body weight loss starting on day 1 after inoculation. in contrast, untreated mice (such as those shown in figures 1f and 2a) only started to lose weight on day 2 after inoculation. it may be that the injection with serum or pbs after the infection enhanced the pulmonary edema by hyperpermeability in the lung. we previously reported that serial in vivo passage of sars-cov in f344 rats increased the infectivity of the virus in rats, and that this correlated with a single amino acid change in the virus receptor-binding domain of the s protein. 34 we detected the similar change in f-musx-veroe6 along with another amino acid change in the receptor-binding domain of the s protein in f-musx-veroe6. the amino acid change may be responsible, at least in part, for the increased replication of f-musx-veroe6 in the pulmonary tissue of balb/c mice. [12] [13] [14] [15] 34, 48 using another sars-cov isolate (urbani), roberts and colleagues 48 have described the development and characterization of the mouse-adapted strain ma15, which is lethal for young (6-to 8-week-old) female balb/c mice after intranasal inoculation. compared with the original urbani isolate, the ma15 strain had six coding mutations that may have been responsible for the mouse adaptation and increased virulence of this strain. the fewer amino acid mutations in our mouse-adapted f-musx-veroe6 strain may be responsible for the fact that it is less virulent than ma15. in conclusion, we developed an experimental mouse animal model of sars by using in vivo-passaged sars-cov and balb/c mice. we found that the virus was lethal in adult mice because they generated an excessive innate immune response that induced lethal pulmonary edema and diffuse alveolar damage with infiltration of macrophages and neutrophils. ifn-␥ appears to play an important role in modulating the 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disease and mortality in balb/c mice key: cord-004663-a47pkh8q authors: tardieu, m.; goffinet, a.; harmant-van rijckevorsel, g.; lyon, g. title: ependymitis, leukoencephalitis, hydrocephalus, and thrombotic vasculitis following chronic infection by mouse hepatitis virus 3 (mhv 3) date: 1982 journal: acta neuropathol doi: 10.1007/bf00690797 sha: doc_id: 4663 cord_uid: a47pkh8q mouse hepatitis virus 3 (mhv 3) is either avirulent (resistant mice), hepatotropic (susceptible mice). or neurotropic (semisusceptible mice), depending on the strain of mice infected. in semisusceptible mice, infection led first to a transient meningitis, ependymitis, and leukoencephalitis, followed by a permanent communicating hydrocephalus and, later on, to a chronic thrombotic vasculitis affecting meningeal and parenchymal vessels at the brain stem level. small foci of ischemic necrosis related to vascular occlusions were seen in the dorsal brain stem. cyclophosphamide treatment of semisusceptible mice significantly reduced the meningeal infiltrates but did not prevent the development of hydrocephalus and other neuropathologic changes. identical lesions occurred in fully susceptible mice infected with a low dose of virus, but no neurologic disorder could be induced in genetically resistant mice even following immunosuppression or intracranial inoculation. the leukoencephalitis differed from the demyelinating lesions observed with mhv4. vascular lesions were of particular interest. more attention should be given to the possibifity of virus induced chronic cerebral vasculitis in man. differences in susceptibility to virus infection depend on characteristics of injected virus (weiner et al. 1977 wolinsky and stroop 1978) , the route of infection (johnson 1964) , and on host genetic factors and immune function (allison 1965; hirsch et al. 1970; levy-leblond et al. 1979 ). mouse hepatitis virus 3 (mhv 3), a member of the corona virus group, is either avirulent, hepatotropic, or neurotropic, depending on the strain of mice infected. adult a/j mice are fully resistant to the virus. susceptible strains (e.g., dba/2 or balb/c) develop an acute hepatic necrosis leading to death within a few days. some mouse strains (e.g., c3h/he) as well as f 1 hybrids between resistant and susceptible strains exhibit a "semisusceptibility" resulting either in early death or, in surviving animals, in the development of a chronic disease with neurologic manifestations and virus persistence (dupuy et al. 1975; le prevost et al. 1975a, b) . ependymitis and vasculitis have been reported . susceptibility or resistance to mhv 3 infection has been shown to be under the influence of at least two major gene complexes, one for the acute disease and the other, h-2 linked, for the chronic disease (levy-leblond et al. 1979 ). in addition to genetic factors, one of us has observed that host defense against mhv 3 infection requires at least three types of immunocompetent cells: t-lymphocytes, splenic macrophages, and nk-like lymphoid cells (tardieu et al. 1980) . the aim of the present neuropathologic study was to better delineate the interaction of mhv 3 with the different cells of the central nervous system (cns), as influenced by the genetic background and immunologic status of the host. of particular interest were an early transient leukoencephalitis and a chronic thrombotic vasculitis in the later stage of the disease. x a/j) fi hybrids (thereafter designed as baf 1 mice) were bred in our mouse colony as previously described (le prevost et al. 1975 a) . passage, assay, and recovery of mhv 3 were performed in susceptible dba/2 mice as reported previously (le prevost et al. 1975 a) . virus titer was expressed in ld 50/g of tissue. the virus was injected intraperitoneally (i.p.) at a dose of 103 ld 50 in 0.1 ml of saline buffer unless otherwise specified. in a group of a/j strain mice, 103 ld 50 of viral suspension were injected intracerebrally through a burhole in the parietal bone (total injected volume: i gl). a total of 158 mice were injected. ninety-one survived and were available for study. at various times after infection [on days 2, 4, 7, 9, 14, 21, 28, 35, 43, 60, 78, 90, 100, and 130 post infection (p.i.) ], mice were anesthetized with ether and perfused through the left ventricle with either formaldehyde (4 % w/v in phosphate buffer) for opto-microscopic studies, or formaldehyde (2 % w/v in phosphate buffer) plus glutaraldehyde (2.5% v/v) for electron-microscopic studies. the whole cranium was fixed overnight. the brain was then removed, embedded in paraffin, and sliced coronally on a serial basis for the preparation of paraffin sections which were processed by conventional techniques. in a series of mice prepared for electron-microscopic studies, selected fragments of ependyma, white matter, cortex and brain stem were obtained, postfixed in 1% oso 4, and embedded in epoxy resin. semithin sections were stained with toluidine blue or with the pas method. thin sections were stained with uranyl acetate and lead citrate and examined using a philips em 300 electron microscope. cyclophosphamide treatment. cyclophosphamide was reconstituted with saline buffer and a freshly prepared solution was injected i.p. at a dose of 150 mg/kg. cyclophosphamide treatment, in semisusceptible baf 1 mice, given either before or just after mhv 3 infection, leads to 100 % mortality (willenborg et al. 1973) . to avoid acute mortality, cyclophosphamide was injected on days 7, 10, 14 p.i. or 10 and 14 p.i. in some experiments, a/j strain mice were injected with cyclophosphamide 9, 13, and/or 17 days before infection. a9strontium treatment. 100 cu of 89strontium (89sr) were injected i.p. in baf 1 mice on two separate occasions, 5 weeks apart. six weeks after the last injection, animals were considered "898rtreated", as described previously (tardieu et al. 1980) . mhv 3 was injected 5 weeks after the second injection. after infection, 54 (62 %) of the semisusceptible mice (baf 1, ch 3) survived and developed a progressive neurologic disease characterized by incoordination, paresis of the hind limbs, enlargement of the head, and progressive neurologic deterioration leading to death within 3-5 months. in this group of animals, no neuropathologic changes could be detected when examined on days 2 and 4 p.i. the first abnormalities, at 7 days p.i., consisted of an important meningeal in-filtration of polymorphonuclear and mononuclear cells. at 14days p.i., the infiltrate consisted of small lymphocytes, plasmocytes, and macrophages. after 21 days p.i., the diffuse meningeal infiltrate diminished progressively and was minimal after 60 days p.i. during the same period inflammatory cuffing of meningeal vessels became progressively more marked. perivascular inflammatory cells consisted of small lymphocytes, plasmocytes, and macrophages. between 7 and 14 days p.i. perivascular infiltrations of mononuclear cells and microglial nodules appeared in the hemispheric white matter (fig. l a) . in rare instances, a microglial nodule was observed in the adjacent cortex. at 21 days the inflammatory lesions in the white matter had disappeared, leaving widespread destruction and cavitation (fig. i b) . at 10 days p.i., the first signs of an ependymitis appeared. between 21 and 35 days p.i. the granular ependymitis was particularly marked. inflammatory lesions predominated in the fourth ventricle and aqueduct ( fig. 2a) . focal aggregates of inflammatory cells consisting of mononuclear cells were formed beneath the ependyma and the ependymal cell line was focally disrupted. some of these granulomas bulged into the ventricular lumen and became even detached to form rosettes which were found free in the csf or were attached to the cilia of the ependymal cells. under the electron microscope, these granulomatous buds were seen to consist of lymphocytes and histio-monocytes together with ependymocytes. no viral particles could be seen within the affected ependymal cells. the cells of the subependymal aggregates did not stain with an antiserum against gfap, suggesting that they did not contain proliferating astrocytes. no changes were detected in the chroid plexus. after 35 days p.i., the ependymal lesions became progressively less active and demonstrated residual scarring. the ependymal cell line was focally disrupted, and in the posterior part of the fourth ventricle it was completely destroyed. proliferation of subependymal atrocytes was not conspicuous (fig. 2b) . the first evidence of ventricle enlargement was noted at 21days p.i. hydrocephalus progressed thoughout the life span of the animals. on serial sectioning of the aqueduct no stenosis was observed at 100 and 130days p.i. in mice killed after 45 days p.i. vascular lesions were observed. numerous meningeal arterioles showed severe alterations : hyaline necrosis of the media, fragmentation of the elastic lamina and intimal proliferation (fig. 3) . some arterioles and capillaries were occluded by an inflammatory thrombus (figs. 3, 4) consisting of plasmocytes, a few polymorphonuclear leukocytes, and necrotic cells of uncertain origin (fig. 5) . between 60-130 days p.i. vasculitis and thrombosis were seen to affect intraparenchymal yesfig. 1a , b. inflammatory nodules in hemispheric white matter. neocortex is situated below and subiculum above the central white matter. fragments of ependyma are seen amidst the inflammatory cells. a9 days p.i. nissl, x 108. b widespread destruction of cerebral white matter, no inflammation; 21 days p.i. he, x 94 sels beneath the iv ventricle and in the dorsolateral part of the brain stem (fig. 6) . a t some points, the nervous tissue surrounding these vessels showed distinct signs of ischemic necrosis, characterized by the presence of macrophages and proliferating astrocytes. aggregates of inflammatory cells were seen around vessels and in the surrounding parenchyma. n o microglial nodules and no intranuclear inclusions were observed. the infected semisusceptible mice which died acutely during week 1 showed lesions similar to those observed in fully susceptible mice, i.e., acute hepatitis with a normal appearance of the cns. cyclophosphamide treatment. a group of 14 baf 1 mice were injected i.p. with mhv 3 (103 ld 50). two died acutely and the 12 others were treated with cyclophosphamide as described in material and methods. among the 12 infected and cyclophosphamide-treated mice, 10 survived and two died at 23 and 27 days p.i. pathologically, the ependymal lesions and ventricular dilatation were identical to non-treated mice. hyaline necrosis of the walls of meningeal arteries and thrombosis existed as in controls. in contrast, there was a marked diminution of the meningeal and perivascular inflammatory cell infiltration as compared to control mice which were infected by mhv 3 but were not treated with cyclophosphamide. there was no evidence of leukoencephalitis. 89 strontium treatment. treatment with 89 sr resulted in acute liver necrosis with death between day 5 and 9 p.i. in the five semisusceptible mice tested. these mice showed a pattern of lesions similar to that observed in susceptible mice. when six susceptible balb/c mice were injected i.p. with mhv 3 (103ld50), they died of an acute hepatic necrosis 5 -8 d a y s after m h v 3 infection, and no neuropatholigic lesion was observed except a slight degree of meningeal infiltration. a group of 10 balb/c mice was then infected with a low dose of virus (10 ld50). eight of these animals died acutely with in 5 -8 days, the other two survived. the neuropathologic lesions were identical to those observed in semisusceptible mice, i.e., meningitis, leukoencephalitis, ependymitis, ventricular dilatation, and vasculitis. if the dose of inoculated virus was further lowered (1 ld 50), neither acute nor chronic lesions developed in five injected animals. to determine whether resistant mice could develop a chronic disease, three experiments were performed. first, three resistant a/j mice were injected in the same way as the semisusceptible animals. no clinical manifestations or neuropathologic abnormalities were observed. secondly, 5 a/j strain mice were immunosuppressed with cyclophosphamide. after infection with mhv 3, the most profoundly immunosuppressed animals (two or three cyclophosphamide injections before mhv 3 infection) died acutely as previously shown (willenborg et al. 1973) ; in the two surviving animals a slight meningeal infiltration and a perivascular infiltrate were observed on day21 p.i. without any ependymal damage, in a third experimental group mhv 3 was injected directly into the brain of nine mice. only minimal neuropathologic lesions were observed (a light microglial infiltration at the point of injection) without any ependymal or vascular change. systemic infection of semisusceptible strains of mice with mhv 3 leads to a remarkable sequence of brain lesions, such as meningitis and leukoencephalitis to mention those appearing first. the diffuse meningeal infiltrate tends to disappear progressively within a few weeks without fibrotic changes. however, perivascular infiltrates remain, especially at the level of the brain stem. the acute leukoencephalitis (fig. 1 a) , which is more marked in c3h, is restricted to the central white matter of the cerebral hemisphere and is visible only during a limited period of time between days 7 and 14 p.i., leaving behind severe destructive lesions (fig. 1 b) . this type of leukoencephalitis is different from white matter lesions produced by mhv 4, which consist of noninflammatory patchy demyelination (waksman and adams 1962; lampert et al. 1973; weiner 1973; herndon et al. 1975; haspel et al. 1978; weiner and stohlman 1978; stohlman and weiner 1981) . the presence of glial nodules in the white matter and occasionally in the adjacent cortex, may be considered as an indication of a direct action of the virus on glial cells. inflammatory changes in the ependyma appeared approximately at day 14 and persisted for about 1 month (fig. 2a) . they resulted in widespread destruction of the ependymal lining (fig. 2b) . viral particles were not detected with the electron microscope in the ependymal cells, in contrast with the findings in other viral ependymitis (nielsen and baringer 1972; wolinsky 1977) . progressive hydrocephalus starting at about 3 weeks p.i. was probably a major factor in the death of the animals. virus-induced experimental hydrocephalus has been attributed either to stenosis of the aqueduct produced by an ependymitis (johnson et al. 1967; johnson and johnson 1968; johnson 1975) or to a post infectious fibrosis of the meninges (masters et al. 1977) . in our animals aqueductal stenosis was not observed (at least until the 130th day p.i.), and there was no meningeal fibrosis (arachnoid villi were not examined). immunosuppression of semisusceptible animals with cyclophosphamide did not prevent the appearance of hydrocephalus; its most remarkable effect was the nearly complete suppression of the meningeal infiltrate. fibrosis of the meninges can, therefore, probably be discounted as the cause of intraventricutar hypertension and the mechanism of hydrocephalus remains unsettled. however, the parallelism between the intensity of ependymitis and hydrocephalus should be noted: both abnormalities were more important in baf 1 mice than in c3h mice. in contrast to the early, self-limiting, inflammatory lesions observed in the cerebral white matter and ependyma, vasculitis appeared late, after ependymitis had subsided, and increased progressively thoughout the life of the animals. these remarkable lesions consisted in hyaline necrosis of vessel walls and thromboses (figs. 3, 4) . plasmocytes were remarkably numerous in the inflammatory thrombi (fig. 5 ). perivascular cuffings with lymphocytes and plasmocytes were even more abundant than in the early phases of the disease. arterioles, capillaries, and venules were affected, essentially in the meninges surrounding the pons and medulla, deep in the subependymal region of the ivth ventricle, and within the dorsolateral parts of the brain stem, where small loci of ischemic necrosis related to vascular occlusions were observed (fig. 6) . it is not impossible that some of the parenchymal lesions could be ascribed to an encephalitic process; however, the presence of vascular occlusions and ischemic necrosis in these areas is unquestionable. in man, particularly in the fetus, the possibility for a chronic viral infection to induce ischemic brain lesions after a long delay, through a slowly progressive thrombotic vasculitis, has not been sufficiently considered. such a sequence of events is known, however, in congenital rubella, where degeneration of vessel walls leads to multifocal ischemic necrosis of the brain (rorke and spiro 1967; singer et al. 1967; rorke 1973 ) and similar lesions have been shown in experimental animals (rorke et al. 1968) . a rare condition, granulomatous angiitis has been related to varicella-zoster infection (kolodny et al. 1968; rosenblum and hodfield 1972; linnemann and alvira 1980) and to other viral infections (reyes et al. 1976 ) in man. also, it is known that inflammatory perivascular infiltrates perist for many months in human poliomyelitis (esiri 1980) . at the time vascular lesions develop, a low titer of virus can still be detected in the brain, as late as 6 months p.i. (le prevost et al. 1975b), and dupuy et al. (1973) have demonstrated at that stage a severe non specific immunodepression (dupuy et al. 1973) . to investigate possible immune mediation in the generation of vascular lesions, we studied the effects of two immunomodulating regimen in semisusceptible mice: cyclophosphamide, which depresses t-and b-cell activities (stockman et al. 1973) and s9sr, which abolishes selectively nk cell activity (kumar et al. 1976; tardieu et al. 1980 ). cyclophosphamide did not prevent necrosis of vessel walls and thrombosis. 89 sr treatment changed the pattern from semisusceptible to susceptible mice, dying of acute hepatic necrosis. to study further the effect of 898r, preliminary experiments were performed in which mice were injected with mhv 3 1 day after the second injection of 89sr, to avoid acute hepatic necrosis. the intensity of vasculitis in these mice and in controls was identical. these experiments suggest that t, b, and nk-cells do not play a prominent role in the induction of vascular lesions. however, there is as yet no clear evidence in favor of a direct action of the virus on the vessels, and further research is needed into the pathogenesis of the thrombotic vasculitis following mhv 3 infection. the same neurologic disease can occur in fully susceptible mice infected with a low dose of virus, but attempts to induce this disease in genetically resistant mice have been unsuccessful, even following immunosuppression or intracranial inoculation. these findings suggest that host cells in the brain from fully susceptible and semisusceptible strains are equally able to support viral replication and are equally susceptible to give rise to destructive lesions. the absence of disease in resistant mice may be related to an inability of host cells to either bind or replicate mhv 3. it is also possible that other means of immunosuppression may alter this genetic resistance. genetic factors in resistance against virus infection persistent virus infection with neurological 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mechanism of genetic resistance to friend leukemia virus in mice. i. role of 89sr-sensitive effector cells responsible for rejection of bone marrow allografts mechanism of demyelination in jhm virus encephalomyelitis. electronmicroscopic studies immunopathology of mouse hepatitis virus type 3 infection role of humoral and cell-mediated immunity in resistance mechanisms b) immunopathology of mouse hepatitis virus type 3 infection. iii. clinical and virologic observations of a !aeristent viral infection genetic study of mouse sensitivity to hmv 3 infection: influence of the h-2 complex pathogenesis of varicella-zoster angiitis in the cns pathogenesis ofreovirus type i hydrocephalus in mice. significance of aqueductal change reovirus-induced aqueductal stenosis in hamsters. phase contrast and electron-microscopic studies virus-like particles in granulomatous angiitis of the central nervous system cerebral lesions in congenital rubella syndrome experimental cerebrovascular lesions in congenital and neonatal rubella-virus infections of ferrets nervous system lesions in the congenital rubella syndrome granulomatous angiitis of the nervous system in cases of herpes zoster and lymphosarcoma pathology of the congenital rubella syndrome differential effects of cyclophosphamide on the b-and t-cell compartments of adult mice chronic central nervous system demyelination in mice after jhm virus infection neonatal susceptibility to mhv 3 infection in mice. ii. role of natural effector marrow cells in transfer of resistance neuropathological effects of persistent infection of mice by mouse hepatitis virus infectious leukoencephalitis. a critical comparison of certain experimental and naturallyoccuring viral leukoencephalitis with experimental allergic encephalomyelitis pathogenesis of demyelination induced by a mouse hepatitis virus (jhm virus) molecular basis of reovirus virulence: role of the s 1 gene viral models of demyelination effect of cyclophosphamide on the genetic resistance of c3h mice to mouse hepatitis virus mumps virus-induced hydrocephalus in hamsters. ultrastructure of the chronic infection virulence and persistence of three prototype strains of mumps virus in newborn hamster key: cord-288253-wqrhiq08 authors: park, jung-eun; park, eui-soon; yu, jung-eun; rho, jaerang; paudel, sarita; hyun, bang-hun; yang, dong-kun; shin, hyun-jin title: development of transgenic mouse model expressing porcine aminopeptidase n and its susceptibility to porcine epidemic diarrhea virus date: 2015-02-02 journal: virus res doi: 10.1016/j.virusres.2014.12.024 sha: doc_id: 288253 cord_uid: wqrhiq08 porcine coronavirus infections have known as they are specific to pigs with predominantly enteric or respiratory diseases. no laboratory animal model is yet been developed in porcine coronaviruses study. here, we report that development of a transgenic mouse model expressing porcine apn which is susceptible to porcine coronavirus infection. the porcine apn transgene was constructed by fusing with mouse proximal apn promoter at 5′ terminus and bovine growth hormone polyadenylation site at its 3′ terminus. after screen on pubs from the microinjected mice, we confirmed two transgenic lines expressing porcine apn in various organs. we confirmed the susceptibility to porcine epidemic diarrhea virus, one of the porcine coronaviruses. these transgenic mice will be an important tool for research into the porcine coronaviruses. the coronaviruses belong to the family coronaviridae within the order nidovirales. they are classified into four genera, alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus, based on genetic similarities (adams et al., 2014) . the primary replication of the coronaviruses is often confined to respiratory-or gastrointestinal-tract epithelial cells, so they usually induce respiratory or enteric diseases, but also hepatic, renal and neuronal infections (lednicky et al., 2013; masters, 2006; weiss and navas-martin, 2005) . the pathogenesis of several porcine coronaviruses, including transmissible gastroenteritis virus (tgev) and porcine respiratory coronavirus (prcov), has been broadly studied (enjuanes et al., 1995; saif, 2004a,b; saif, 2004a,b; weiss and navas-martin, 2005) . tgev is a major cause of viral enteritis and fetal diarrhea in swine, most severely in neonates and with a high mortality rate, which causes significant economic losses worldwide (enjuanes et al., 1995) . prcov is reported to be an attenuated variant of tgev. prcov infects lung epithelial cells, and prcov antigen has been found in type i and type ii pneumocytes and alveolar macrophages, and infection is followed by interstitial pneumonia (halbur et al., 1993; saif, 2004a,b) . porcine epidemic diarrhea virus (pedv) is the causative agent of porcine epidemic diarrhea, which is characterized by watery diarrhea, as is tgev infection (chasey and cartwright, 1978; pensaert and de bouck, 1978; turgeon et al., 1980) . pedv-infected piglets usually show typical enteric signs, including profuse watery diarrhea, weight loss, and loss of milk uptake, entailing high mortality. pedv has been reported in europe and asia, and also recently in the united states (hess et al., 1980; huang et al., 2013; pan et al., 2012; park et al., 2013; wang et al., 2014) . all these porcine coronaviruses belong to the genus alphacoronavirus. a hemagglutinating enteric coronavirus, a member of the genus betacoronavirus, is antigenically unrelated to the other porcine coronaviruses and uses a 5-nacetyl-9-o-acetylneuraminic-acid-containing moiety as its cellular receptor. coronavirus infections are mediated by the spike (s) glycoprotein, a large surface glycoprotein on the viral envelope (delmas and laude, 1990; masters, 2006) . coronavirus s glycoproteins recognize cellular receptors and mediate virus-cell fusion (masters, 2006; peng et al., 2011) . most alphacoronavirus, including tgev, pedv, and prcov, use aminopeptidase n (apn/cd13) as their cellular receptor (delmas et al., 1992; li et al., 2007; ren et al., 2010; tresnan and holmes, 1998; yeager et al., 1992) . apn/cd13 is a 150-kda, zinc-dependent metalloprotease consisting of 967 amino acids (rawlings and barrett, 1995) . mammalian apn is ubiquitously expressed as a glycosylated homodimer on the surfaces of epithelial cells in the liver, intestine, kidney, and respiratory tract, and in fibroblasts and leukocytes, and plays multiple roles in many physiological processes, including coronavirus entry (barnes et al., 1994; luan and xu, 2007; mina-osorio et al., 2008; miura et al., 1983) . the natural hosts of porcine coronaviruses are young piglets, and clinical illness has only been observed in sucking piglets. however, in vivo studies using suckling piglets have many disadvantages, including their high cost, difficulty in handling the piglets, limited reagents, etc. because the use of laboratory animal models (e.g., mouse models) can circumvent these limitations, several transgenic animal models have been generated to study porcine viruses (benbacer et al., 1998; ono et al., 2006) . here, we generated transgenic mice expressing porcine apn under the control of the mouse proximal apn promoter. we tested their susceptibility to pedv with reverse transcription-polymerase chain reaction (rt-pcr) and immunochemical analyses, and found that the porcine apn transgenic mice were susceptible to pedv infection. vero monkey kidney cells were maintained in minimal essential medium containing 10% fetal bovine serum (fbs), 100 u/ml penicillin g, 100 g/ml streptomycin, and 250 g/ml amphotericin b in a 5% co 2 atmosphere at 37 • c. 293t human embryonic kidney cells were maintained in dulbecco's modified eagle's medium containing 10% fbs, 100 u/ml penicillin g, 100 g/ml streptomycin, and 250 g/ml amphotericin b. all tissue culture reagents were purchased from gibco (carlsbad, ca, usa). kpedv-9, a cell-adapted vaccine strain of pedv, was grown and titrated in the vero cells, as described previously, and stored at −80 • c until use (cruz and shin, 2007; hofmann and wyler, 1988) . the polyclonal antibodies specific for pedv and porcine apn were generated in balb/c mice immunized with kpedv-9 and purified porcine apn, respectively, as described previously (cruz et al., 2008) . the antibody specificities were confirmed with enzyme-linked immunosorbent assays. the anti-flag m2 monoclonal antibody (anti-flag) and anti-flag m2 affinity gels (gel beads) were purchased from sigma-aldrich (st. louis, mo, usa). fluorescein isothiocyanate (fitc)-conjugated anti-mouse igg antibody was purchased from santa cruz biotechnology (santa cruz, ca, usa). the mouse proximal apn promoter region (starting at nucleotide-1044) was amplified from c57bl/6j genomic dna by pcr with primers 5 -cccgcggccgcaagatttgaaacagtgga-3 and 5 -cccaagcttgatgccggtggacaggga-3 , containing flanking noti and hindiii restriction endonuclease sites, respectively. the pcr product was cloned into the pbluescript ks (+) vector and the pgl3-basic vector (promega, madison, wi, usa), which contains a promoterless luciferase reporter gene. the porcine apn gene was amplified from the total rna isolated from porcine enterocytes with rt-pcr using specific primers and cloned into the pbluescript ks (+) vector. a sequence encoding the flag epitope (dykddddk) was fused to the 3 terminus of porcine apn with pcr with primers 5 -cccaagcttaccatggccaagggattctac-3 and 5 -cccctcgagtcacttgtcgtcatcgtctttgtagtcgctgtgctctat-gaacca-3 , which contain flanking hindiii and xhoi restriction endonuclease sites, respectively. the bgh-polya sequence was amplified from the pcdna3.1 vector (invitrogen, carlsbad, ca, usa) with pcr using primers 5 -cccctcgagcgactgtgccttctagtt-3 and 5 -cccggtaccccatagagcccaccgcat-3 , which contain flanking xhoi and kpni restriction endonuclease sites, respectively. the pcr product was cloned into the pbluescript ks (+) vector. all pcr products were confirmed with automated sequencing. to generate the porcine apn transgene, porcine apn-flag and the mouse proximal apn promoter were ligated into pbluescript ks-bgh-polya after they were digested with hindiii/xhoi and noti/hindiii, respectively. 293t cells (2.5 × 10 5 cells/ml) were plated in 24-well plates. after 24 h, the cells were transfected with pgl3-basic-mouse proximal apn promoter (mapn-luc) (0.2 g or 1 g). the cells were transfected in triplicate using lipofectamine 2000 (invitrogen), according to the manufacturer's instructions. after 24 h, the cells were lysed for assay with the luciferase assay system (promega), according to the manufacture's protocol. all luciferase activities were normalized to ␤-galactosidase activity. the porcine apn transgene was linearized by restriction with noti and purified with gel extraction (qiagen, valencia, ca, usa). gain-of-function gene transfer was performed by microinjecting the purified dna into the pronuclei of icr mouse zygotes, which were then transferred into the oviducts of female recipient mice. the transgenic mice were identified with pcr analysis of tail genomic dna with primers: pcr1-f: 5 -cccaagcttaccatggccaagggattctac-3 and pcr1-r: 5 -gaagttggagagcatcct-3 ; and pcr2-f: 5 -ggcgtcctacttgcatgc-3 and pcr2-r: 5 -cccctc-gagtcacttgtcgtcatcgtctttgtagtcgctgtgctctatgaacca-3 . the founder mice were backcrossed to the c57bl/6j background for five generations. all the mice used in this study were maintained in a specific-pathogen-free facility at the biomedical research center at the korea advanced institute of science and technology, daejeon, korea. all animals were cared for and the experiments were performed at the animal facility at chungnam national university (cnu), korea, with the permission of and according to protocols approved by the institutional animal care and ethics committee of cnu (permission number 20110825). the porcine apn transgenic and nontransgenic wild-type mice were orally inoculated with 5 × 10 6 tcid 50 of kpedv-9 or phosphate-buffered saline (pbs, ph 7.2) as the negative control. their clinical signs were monitored and their feces collected for 5 days. two mice from each group were killed on the indicated days after viral infection. the tissues were aseptically collected and prepared for rt-pcr and immunohistopathological analysis. total rnas were extracted from the feces and tissue samples using trizol ® reagent (invitrogen) and transcribed to cdna using power cdna synthesis kit (intron biotechnology, korea), according to the manufacturer's protocol. the porcine apn-flag cdna was pcr amplified with the specific primers used for the genotyping pcr. as a control, specific primers were used to amplify ␤-actin (mouse ␤-actin-f: 5 -cggttccgatgccctgaggctctt-3 and mouse ␤-actin-r: 5 -cgtcacacttcatgatggaattga-3 ). the cycling parameters were initial denaturation at 94 • c for 2 min, followed by 30 cycles of denaturation at 94 • c for 15 s, annealing at 60 • c for 40 s, and extension at 72 • c for 90 s, with a final extension step at 72 • c for 5 min. to detect the viral genome, viral rna was extracted from tissue homogenates using the viral gene-spin viral dna/rna extraction kit (intron biotechnology), according to the manufacturer's instructions. m-mlv reverse transcriptase (intron biotechnology) was used for first-strand cdna synthesis, together with 10 l of extracted viral rna in a 50 l randomly primed reaction. the specific primers used for the detection of the n gene were pedv-n forward (5 -ggtaccatggcatctgtcagcttt-3 ) and pedv-n reverse (5 -ggatccttaatttcctgt tcgaa-3 ). rt-pcr was performed at 94 • c for 2 min, followed by 30 cycles of 94 • c for 20 s, 54 • c for 10 s, and 72 • c for 2 min, with a final extension at 72 • c for 10 min. the pcr products were analyzed by gel electrophoresis and visualized with ethidium bromide staining and uv transillumination. 293t cells were lysed with cell lysis buffer (25 mm tris-cl [ph 7.5], 150 mm nacl, 1 mm edta, 1 mm naf, 1 mm sodium orthovanadate, 1 mm pmsf, 5% glycerol, 0.5% triton x-100, and protease inhibitors [roche, indianapolis, in, usa]). the small intestines were rinsed with pbs and homogenized in lysis buffer (1% np40, 150 mm tris-cl, 50 mm nacl, 1 mm edta). the cell or tissue lysates were separated with 8-15% gradient sds-page and transferred into pvdf membrane (amersham-pharmacia biotech, piscataway, nj, usa). the proteins were probed with an anti-flag monoclonal antibody diluted 1:4000 and an anti-␤-actin antibody diluted 1:10,000. the immune complexes were detected with peroxidase-conjugated goat anti-mouse igg antibody (santa cruz biotechnology) and visualized with amersham ecl western blotting detection reagent (amersham-pharmacia biotech). 293t cells were transfected with the porcine apn transgene. after 24 h, the cells were rinsed with pbs and detached with non-enzymatic cell dissociation solution (sigma-aldrich) for 2 min. the detached cells were harvested and resuspended in fluorescenceactivated cell sorting (facs) staining buffer (pbs containing 2% fbs). the cells were stained with the anti-flag m2 monoclonal antibody (1:200) for 15 min at 4 • c and then with fitc-conjugated anti-mouse igg secondary antibody (1:500) for 15 min at 4 • c. the antibody-stained cells were analyzed with flow cytometry (fac-scalibur, becton-dickinson biosciences, franklin lakes, nj, usa). kidney and liver tissues collected from mice were rinsed with pbs, and the renal or hepatic cells were isolated from the tissues with a 70 m mesh filter (becton-dickinson biosciences), resuspended in facs staining buffer, and analyzed as described above. small intestine samples were fixed in 10% buffered formalin and embedded in paraffin. serial 5 m sections were mounted on silane-coated slides. the sections were deparaffinized for immunohistochemical analysis, and the endogenous peroxidases were blocked with 0.6% h 2 o 2 in methanol for 30 min. antigens were retrieved with microwave heating in citrate buffer for 10 min, and the slides were incubated with 1.5% horse serum for 30 min at room temperature. polyclonal anti-porcine apn antiserum, monoclonal anti-flag antibody, or polyclonal anti-pedv antiserum were applied overnight at 4 • c. the biotinylated anti-mouse igg secondary antibody was detected with an avidin-biotin-peroxidase kit (vectastain abc kit, vector laboratories, burlingame, ca, usa). antibody binding was detected with the chromogen diaminobenzidine (vector laboratories), and the cells were counterstained with hematoxylin and eosin. 2.11. pedv replication in the small intestines of porcine apn transgenic mice both wild type and porcine apn transgenic mice were infected with pedv orally on day 0. viral titer in inoculum was 5x tcid 50 10 6 . two mice from each group were killed daily up to 5 days, and small intestine samples from all mice were collected in mem and homogenized. samples were centrifuged and supernatants were kept in −80 • c until processed for titration. titration was performed using vero cells. the pedv titer was measured and described in log value. the transcription of mouse apn is regulated by two different promoters, as shown in fig. 1a (bhagwat et al., 2001) . the distal promoter is located 8 kb upstream from the transcription -1 (3 and 4) , and cnupedm-3 mice (5 and 6). porcine apn rnas were amplified using specific primers targeting porcine apn, as described in section 2. ␤-actin was detected as the loading control. (b) expression of porcine apn protein in the small intestines. small intestine samples were collected from cnupedm-1 and cnupedm-3 mice. porcine apn protein was detected in small intestine homogenates with immunoblotting using an anti-flag monoclonal antibody (2 and 4). no recombinant protein was detected in the small intestine samples derived from nontransgenic wild-type mice (1 and 3). ␤-actin was used as the loading control. (c) immunohistochemical analysis of porcine apn transgenic mice. small intestine sections were prepared from porcine apn transgenic mice (a-c: cnupedm-1; d-f: cnupedm-3) and incubated with anti-flag monoclonal antibody (anti-flag) (a and d), mouse anti-porcine apn polyclonal antiserum (anti-papn) (b and e), or normal mouse serum (c and f). the immune complexes were visualized with avidin-biotin-peroxidase and the cells were counterstained with hematoxylin and eosin. magnification, ×40. start site and controls apn expression in myeloid and fibroblast cells, whereas the proximal promoter regulates apn expression in epithelial cells (olsen et al., 1991; shapiro et al., 1991) . following references and preliminary studies, we choose the mouse proximal apn promoter to express porcine apn in mouse epithelial cells. to test the promoter's activity, the sequence encoding the promoter region was amplified and cloned into the pgl3-basic promoterless vector (fig. 1b) , and the promoter activity was measured by luciferase assay. as shown in fig. 1b , the luciferase activity was about eight-fold higher in 293t human embryonic kidney cells transfected with the vector containing the mouse proximal apn promoter (mapn-luc) than in cells transfected with the empty vector. we constructed a vector encoding porcine apn, which was regulated by the mouse proximal apn promoter (porcine apn transgene) (fig. 2a) . the porcine apn cdna was amplified from porcine enterocytes and tagged at the c-terminus with the flag epitope to distinguish exogenously expressed porcine apn from endogenously expressed murine apn. the 2.9-kb flag-epitopetagged porcine apn cdna was cloned into the pbluescript ks (+) vector under the control of a 1.1-kb genomic sequence containing the mouse proximal apn promoter. the splice sites and polyadenylation signal for the cdna were provided by a 0.2-kb bovine growth hormone polyadenylation signal (bgh-polya). the expression of the porcine apn protein was detected with immunoprecipitation and immunoblotting in 293t cells transfected with the porcine apn transgene. the flag-tagged recombinant protein, with a molecularweight of about 150 kda (the expected molecular mass for porcine apn), was confirmed with immunoprecipitation (fig. 2b) . we also measured the surface expression of porcine apn with flow cytometry using an anti-flag antibody, and found that 21.3% of cells were flag-positive (fig. 2c ). our data demonstrate that the mouse proximal apn promoter efficiently induced porcine apn expression from our construct. the porcine apn transgene was linearized with noti restriction and the dna microinjected into mouse zygotes. to screen for porcine apn expression, we designed two genomic pcr primers, as indicated in fig. 3a (arrowheads) . the presence of the porcine apn transgene in mouse litters was monitored with pcr analysis on tail genomic dna (fig. 3b) . microinjection of the porcine apn transgene produced two transgenic founders, designated cnupedm-1 and cnupedm-3. the transgenic mice were healthy and showed no negative effects of transgene expression. because the major pathological changes of the porcine coronaviruses (e.g., tgev and pedv) involves enteric diseases, we measured porcine apn expression in the small intestine by rt-pcr, immunoblotting, and ihc. all the tested litters descended from cnupedm-1 or cnupedm-3 expressed porcine apn mrna in their small intestines (fig. 4a ). the small intestines expressed a recombinant protein with the molecular mass expected for porcine apn (fig. 4b ). an immunohistochemical analysis, with both anti-flag and anti-porcine apn antibodies, clearly confirmed porcine apn expression in the brush borders of the absorptive cells in the small intestines of the mouse model (fig. 4c) . these results demonstrate that the porcine apn transgenic mice expressed porcine apn in their small intestines. we also screened for porcine apn expression in various other tissues with rt-pcr. porcine apn mrna was strongly expressed in the lung, kidney, and the intestine, and was slightly expressed in the liver (fig. 5a) . the extracellular expression of porcine apn was examined in the kidney and liver by facs analysis (fig. 5b) . consistent with the rt-pcr results, the number of flag-positive renal cells was significantly higher (2-4.3-fold) in the porcine apn transgenic mice than in the nontransgenic wild-type mice, whereas the number of flag-positive hepatic cells was also slightly higher (1.6-3-fold) in summary, we confirmed the expression of porcine apn in the small intestines, lungs, livers, and kidneys of the porcine apn transgenic mice. we examined the susceptibility of the porcine apn transgenic mice to pedv, one of the enteropathogenic porcine coronaviruses. porcine apn transgenic or nontransgenic wild-type mice were inoculated orally with kpedv-9, a vero-cell-adapted korean strain of pedv. in infected transgenic mice, other than slightly watery feces observed at 3 days after inoculation (fig. 6a) , whereas no clinical signs (e.g., watery diarrhea, vomiting, fever, weight loss, or death) were observed for 5 days. viral replication was detected in various tissue extracts with rt-pcr. viral rna was confirmed in the small intestines (for at least 5 days), kidneys (for at least 2 days), and spleen (for at least 3 days) only in the porcine apn transgenic mice (table 1) , and not in the nontransgenic mice. an immunohistochemical analysis detected pedv antigen in the brush borders of the small intestines, at the same location at which porcine apn is expressed (fig. 6b) , which has been demonstrated in pedvinfected piglets (debouck and pensaert, 1980; pensaert et al., 1981) . however, no histopathological changes were observed in the small intestines that shown in pedv infected piglets. overall, these data indicate that porcine apn transgenic mice are susceptible to pedv infection, although they showed none of the enteric disease symptoms typical of pedv-infected piglets (fig. 7) . 3.6. pedv replication in the small intestines of porcine apn transgenic mice pedv replication in both wild type and porcine apn transgenic mice were confirmed and compared by viral load in the small intestines. there were no detectable pedv in any of wild type mice samples. in porcine apn transgenic mice, the mean value of pedv was tcid 50-10 3.1 on day 1. it increased to tcid 50-10 3.8 , tcid 50-10 4.8 , tcid 50-10 5.0 , tcid 50-10 5.1 , on day 2, 3, 4, and 5, respectively. there was no big change in titer between 3 and 5 days pi. we could confirm clear pedv replication in the small intestines of porcine apn transgenic mice. laboratory animal models are crucial tools for the study of viral pathogenesis in vivo, especially for highly pathogenic human viruses or viruses with restricted host ranges (calvert et al., 2014; chiu et al., 2014; deruaz and luster, 2013) . unlike in vitro cell culture systems, laboratory animal models offer researchers invaluable opportunities to study the biological, pathological, and histological characteristics of human and animal diseases. for these purposes, many transgenic mouse models have been developed to study viral pathogenesis, immune responses, and vaccines (darling et both wild type and porcine apn transgenic mice were infected with pedv (5x tcid5010 6 ) orally on day 0. two mice from each group were killed daily up to 5 days, and intestine samples were collected and prepared for titration. titration was done using vero cells. y value is pedv titer in tcid50 in log value. reynaud et al., 2014) . in the field of coronavirus research, studies of severe acute respiratory syndrome coronavirus (sars-cov) and human coronavirus 229e (hcov-229e) have been conducted with transgenic mouse models (lassnig et al., 2005; tseng et al., 2007) . the host range of the porcine coronaviruses is strongly limited to pigs, so pigs are the only animals available in which to study viral pathogenesis. however, in vivo experiments using pigs are relatively challenging because they require special treatments, reagents, and facilities. because most porcine coronaviruses use porcine apn as their receptor, we generated porcine apn transgenic mice, which are susceptible to porcine coronaviruses. we constructed the porcine apn transgene by attaching the mouse proximal apn promoter to the 5 terminus of the porcine apn gene and the bgh-polya signal at its 3 terminus. we thus generated porcine apn transgenic mice expressing porcine apn in the brush borders of their small intestines and various tissues (lungs and kidneys). previous attempts to generate apn transgenic mice susceptible to tgev or hcov-229e have been documented (benbacer et al., 1998; lassnig et al., 2005) . benbacer et al. generated an rt-pcrpositive lineage, but failed to confirm either the expression of the porcine apn protein in the intestine or tgev replication in their porcine apn transgenic mouse model (benbacer et al., 1998) . a susceptible mouse model for hcov-229e infection was successfully developed using comprehensive apn regulatory elements to generate human apn +/+ stat1 −/− double transgenic mice, and a virus was adapted to grow in primary embryonic fibroblasts from these mice (lassnig et al., 2005) . a transgenic mouse model of sars was generated by expressing human angiotensin-converting enzyme 2, a functional receptor for the virus, under the regulation of a global promoter (tseng et al., 2007) . the significance of our study is that we successfully generated transgenic mice expressing porcine apn with minimal modifications, by using the mouse apn proximal promoter and the bgh-polya signal to ensure the strong expression and correct splicing of the gene (pfarr et al., 1986) . therefore, we could express porcine apn in the brush borders of the mouse small intestines and on the surfaces of their renal cells (figs. 4 and 5) . among the porcine coronaviruses, pedv is relatively poorly studied, and many questions and controversies remain regarding its outbreaks and pathogenesis. outbreaks of porcine epidemic diarrhea have been limited to few countries in east asia in the cold season, whereas it has spread to most swine farms in the asian region (mole, 2013; pan et al., 2012; park et al., 2013; song and park, 2012; stevenson et al., 2013) . recently, pedv has also spread rapidly among swine farms in the united states, causing high piglet mortality in more than 17 states (huang et al., 2013; mole, 2013; stevenson et al., 2013; wang et al., 2014) . more importantly, although vaccination is practiced on most swine farms in korea, reported outbreaks are still increasing. moreover, a method for isolating wild-type pedv is not well established. for these reasons, we tested the susceptibility of porcine apn transgenic mice to pedv. although significant clinical illness was not observed when the transgenic mice were infected with pedv, their susceptibility to the virus was confirmed by the detection of viral rna in various organs with rt-pcr and viral proteins in the small intestines with ihc. more clearly, pedv replication was confirmed by increase in viral titer up to 5 days pi compared to those in wild type mice. we could found clear increase in viral titer in the small intestines of papn transgenic mice but not from wild type mice. these results also confirmed that porcine apn plays a role as the cellular receptor for pedv. however, similar experiments using other porcine coronaviruses are required. although we have presented evidence of pedv infection in the small intestines of porcine apn transgenic mice, no clinical signs were observed, apart from moderately soft feces. it has previously been shown that pedv can infect cells lacking extracellular trypsin, although trypsin is essential for the generation of a cytopathic effect (cpe) in infected cells (park et al., 2011) . therefore, we assume that cellular cofactors (e.g., trypsin) other than the primary receptor might be involved in and required for pedv pathogenesis in vivo. however, these factors are probably present at insufficient levels in mice. proteases are generally required for efficient viral entry and the cpes of coronaviruses (matsuyama et al., 2005; simmons et al., 2004 simmons et al., , 2013 . although the viral receptors are usually expressed in various organs, the diseases caused by coronavirus infections are limited to the small intestines and/or lungs, which are enriched cellular proteases. this suggests that proteases play a role in the pathogenesis of the coronaviruses in vivo, as is already well established for influenza virus infections (hatesuer et al., 2013; sakai et al., 2014; tarnow et al., 2014) . the porcine apn transgenic mouse model should extend our understanding of the pathogenesis of and other studies to porcine coronavirus infections. this laboratory animal model should also circumvent all the disadvantages and difficulties related to the study of porcine 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work was supported by a grant from animal and plant quarantine agency of korea (grant no. z-ad-14-2009-10-02), and a grant from the technology development program for agriculture and forestry, ministry for food, agriculture, forestry and fisheries, the korean research foundation grants (grants no. 20120008358, 2011grants (grants no. 20120008358, -0023942, 211-2006. key: cord-345359-okmkgsbr authors: ohno, marumi; sekiya, toshiki; nomura, naoki; daito, taku ji; shingai, masashi; kida, hiroshi title: influenza virus infection affects insulin signaling, fatty acid-metabolizing enzyme expressions, and the tricarboxylic acid cycle in mice date: 2020-07-02 journal: sci rep doi: 10.1038/s41598-020-67879-6 sha: doc_id: 345359 cord_uid: okmkgsbr although the severity of influenza virus infections has been associated with host energy metabolism, the related mechanisms have not yet been clarified. here we examined the effects of influenza virus infection on host energy metabolism in mice. after infecting mice with intranasal applications of 500 plaque-forming units of a/puerto rico/8/34 (h1n1; pr8) virus, the serum levels of most intermediates in the tricarboxylic acid (tca) cycle and related metabolic pathways were significantly reduced. these data suggest that substrate supply to the tca cycle is reduced under these conditions, rather than specific metabolic reactions being inhibited. then, we focused on glucose and fatty acid metabolism that supply substrates to the tca cycle. akt phosphorylation following insulin injections was attenuated in the livers of pr8 virus-infected mice. furthermore, glucose tolerance tests revealed that the pr8 virus-infected mice showed higher blood glucose levels than the vehicle-inoculated control mice. these results suggest that influenza virus infection impairs insulin signaling, which regulates glucose uptake. however, increases in the hepatic expressions of fatty acid-metabolizing enzymes suggest that fatty acids accumulate in liver cells of infected mice. collectively, our data indicate that influenza virus infection dysregulates host energy metabolism. this line of investigation provides novel insights into the pathogenesis of influenza. www.nature.com/scientificreports/ investigated metabolic changes by determining the serum levels of metabolites, insulin sensitivity in the liver, glucose availability, and hepatic gene expressions in the early stages of symptom onset as well as the lethal phase of influenza in a mouse model. the results of this study indicate that influenza virus infection dysregulates glucose and fatty acid metabolism and decreases tricarboxylic acid (tca) cycle activity, leading to enhanced degradation of adenosine triphosphate (atp) and guanosine triphosphate (gtp). we believe that this research provides novel insights into the pathogenesis of influenza and contributes to the development of novel influenza drugs. metabolome analysis indicated reduced tca cycle activity and enhanced purine degradation in mice with severe influenza. upon infection of 500 plaque-forming units of pr8 virus, the mice showed significant body weight loss starting at 3 day post infection (1 dpi, 98.3% ± 2.1% in control mice, 99.5% ± 0.9% in pr8 virus-infected mice; 3 dpi, 100.2% ± 2.2% in control, 85.9% ± 0.7% in pr8 virus-infected mice; 6 dpi, 100.2% ± 2.4% in control mice, 75.5% ± 2.2% in pr8 virus-infected mice). mice were sacrificed when weight loss reached a humane endpoint (25% at 6 dpi). to investigate the systemic effect of influenza virus infection on the host metabolic system, metabolome analyses were performed with serum samples collected at 1, 3, and 6 dpi for samples at very early-stage, onset of symptoms, and lethal phase during influenza, respectively. metabolome analyses identified 74 metabolites in the serum samples of the control and pr8 virus-infected mice at all time points. peak areas and relative ratios of all metabolites are provided in supplemental tables s1 and s2. samples collected at 6 dpi showed the greatest effect of pr8 virus infection on serum metabolomes, as indicated by principle component analyses with high contribution rates of principle component 1 (pc1; 51.4%; supplemental fig. s1 ). moreover, pc1 scores for the control and pr8 virus-infected mice were negative and positive, respectively, suggesting that the pc1 score was positively related to the effects of pr8 virus infection. accordingly, metabolites with high positive and negative factor loading in pc1 tended to increase and decrease with pr8 virus infection, respectively (table 1) . relative increases in the serum levels of hypoxanthine, xanthine, and inosine were 4.56-, 2.90-, and 2.68-fold, respectively, in the pr8 virus-infected mice. these molecules are intermediates of purine degradation, and their increased serum levels are considered indicative of enhanced adenosine triphosphate (atp) and guanosine triphosphate (gtp) catabolism. in agreement, the relative serum levels of the upstream purine metabolites adenosine monophosphate (amp), adenosine, guanosine monophosphate (gmp), and guanosine, were decreased by 0.033-, 0.010-, 0.0075-, and 0.25-fold, respectively, at 6 dpi. among them, the serum levels of amp and adenosine did not differ significantly between the control and infected groups at any time point owing to large individual differences in the control groups (fig. 1) . thus, the lethal phase of influenza is considered to be characterized by increased degradation of atp and gtp. metabolites that were present at reduced levels in the sera of pr8 virus-infected mice were mainly related to the tca cycle, urea cycle, and amino acid metabolism, as indicated by the serum levels of metabolite in these pathways at 1, 3, and 6 dpi (fig. 2) . the effects of 6-day pr8 virus infections on the serum levels of metabolite are summarized in fig. 3 . the levels of tca cycle metabolites pyruvic acid, 2-ketoglutaric acid, fumaric acid, and malic acid were significantly reduced in pr8 virus-infected mice at 6 dpi (0.60-, 0.54-, 0.24-, and 0.27-fold, respectively). succinic acid levels were significantly increased by 1.62-fold at 1 dpi but decreased by 0.26-fold at 6 dpi in pr8 virus-infected mice. in addition, 4-aminobutyric acid, which is synthesized from glutamic acid and converted to succinic acid, was present at reduced in pr8 virus-infected mice at 3 and 6 dpi (0.61-and 0.55-fold, respectively). the levels of urea cycle metabolites argininosuccinic acid, ornithine, and citrulline were decreased by 0.35-, 0.57-, and 0.61-fold, respectively, in pr8 virus-infected mice at 6 dpi, whereas the level of arginine was not altered significantly at any time point. given that the levels of most metabolites were significantly reduced in the tca cycle and related pathways, we suggest that pr8 virus infection reduces flux through these pathways, particularly through the tca cycle. as for amino acids, while the serum levels of ketogenic amino acids did not show any apparent decreases, those of glucogenic amino acids were significantly reduced in pr8 virus-infected mice at 6 dpi. specifically, the serum levels of asparagine, glycine, proline, serine, and tyrosine were significantly decreased by 0.49-, 0.50-, 0.62-, 0.52-, and 0.58-fold, respectively. furthermore, to estimate the effect of pr8 virus infection on glutaminolysis in which glutamine is converted to 2-ketoglutaric acid via glutamic acid and enters the tca cycle, we compared the ratio of the peak areas of glutamic acid to those of glutamine and the ratio of the peak areas of ketoglutaric acid to those of glutamic acid. while the ratio of glutamic acid to glutamine was not changed by the infection, the ratio of 2-ketoglutaric acid to glutamic acid was significantly decreased by 0.48-and 0.65-fold, respectively, at 3 and 6 dpi (supplemental table s3 ). therefore, it was indicated that glutaminolysis contribution to the tca cycle was attenuated by influenza virus infection. pathway analysis using metaboanalyst further confirmed that the tca cycle and related pathways were significantly affected by pr8 virus infection at 6 dpi. the top 10 pathways are listed in table 2 . given that the tca cycle generates gtp and contributes electrons to mitochondrial oxidative phosphorylation to generate a substantial amount of atp, suppression of the tca cycle in pr8 virus-infected mice is considered to cause imbalanced synthesis and degradation of atp and gtp. moreover, these data suggest that suppression of the tca cycle was due to reduced substrate supply to the pathway due to pr8 virus infection, rather than inhibition of specific metabolic reactions. tca cycle flux is associated with glucose and fatty acid metabolism because both these energy sources are eventually converted to acetyl-coa, which enters the tca cycle. because the liver plays a predominant role in whole-body energy metabolism, we further investigated the effect of pr8 virus infection on liver function in terms of glucose and fatty acid metabolism. www.nature.com/scientificreports/ insulin regulates cellular glucose uptake from the blood and intracellular glycolysis 9,10 , which supplies substrates to the tca cycle. upon insulin binding to the insulin receptor on the cell surface, the signal is transduced to downstream molecules and phosphorylates akt. phosphorylated akt activates glucose transporters to increase glucose uptake. therefore, phosphorylation of akt is a good indicator of activation of insulin signaling. here we examined the effect of influenza virus infection on insulin sensitivity in the livers according to ratios of insulin-induced phosphorylatable 1 . increased or decreased serum levels of metabolites in pr8 virus-infected mice at 6 dpi. metabolites with high factor loading (> 0.7) and significant differences (p < 0.05, t-test) were selected as significant metabolites and are listed here. the relative serum levels of each metabolite from pr8 virus-infected mice are presented as fold changes relative to those from control mice. factor loading was defined for each metabolite as correlation coefficients between pc1 scores and metabolite levels that were normalized by autoscaling in each sample. www.nature.com/scientificreports/ tion of akt. western blotting analyses ( fig. 4a ) of phosphorylated and total akt in whole liver lysates of control and pr8 virus-infected mice at 6 dpi revealed that the ratios of phosphorylated akt to total akt were increased by 13.0-fold after insulin treatments in control mice, but this ratio was increased by only 4.6-fold in pr8 virusinfected mice (fig. 4b) . similar experiments were performed with the livers collected at other time points. at 3 dpi, akt phosphorylation was clearly inhibited by pr8 virus infection, but no clear differences were observed in samples collected at 1 dpi (supplemental fig. s2 ). taken together, these results demonstrated that influenza virus infection impairs insulin actions in the liver. we also performed glucose tolerance tests (gtt) in control and pr8 virus-infected mice at 6 dpi to investigate whether pr8 virus infection affected glucose uptake in response to high doses of glucose (fig. 4c) . we found no significant differences in fasting blood glucose levels between the groups. moreover, at 30 min after glucose injections, the control and pr8 virus-infected mice showed similar blood glucose levels (394.4 ± 18.2 mg/dl vs. 363.2 ± 29.7 mg/dl). subsequently, blood glucose levels were rapidly decreased in control mice (60 min, 274.2 ± 13.7 mg/dl; 90 min, 193.4 ± 8.5 mg/dl; 120 min, 165.4 ± 9.0 mg/dl). conversely, decreases in blood glucose levels were delayed and higher blood glucose levels were observed in pr8 virus-infected mice, compared with the control mice, at all time points (60 min, 387.0 ± 35.4 mg/dl; 90 min, 258.6 ± 11.1 mg/dl; 120 min, 184.2 ± 4.4 mg/dl). significant differences in blood glucose levels were identified at 60 and 90 min between the groups (p < 0.05, two-way anova). these results indicate that pr8 virus infection impairs insulin signaling in the liver and induces a tendency toward glucose intolerance, potentially reflecting reduced glucose uptake. fatty acid accumulation in the liver of infected mice was suggested by gene expression assays. we also investigated the effects of pr8 virus infection on the hepatic expressions of genes that are transcriptionally regulated by insulin signaling (fig. 5) . pck2, which encodes a gluconeogenic enzyme, was expressed dpi. mice were intranasally inoculated with pbs alone or pbs comprising pr8 virus, and serum samples were collected for metabolome analysis at 1, 3, and 6 dpi. the serum levels of purine metabolites in pr8 virusinfected mice were expressed relative to those in control mice at each time point. bars represent mean ± sem of 3 or 4 animals. in each panel, white, gray, and black bars indicate data from pr8 virus-infecetd mice at 1, 3, and 6 dpi, respectively; *p < 0.05, unpaired t-test using the holm-sidak correction method with an alpha value of 0.05, control vs. pr8 virus-infected mice at each time point. the right panel shows a schematic of the effects of pr8 virus infection on purine metabolism at 6 dpi. upward and downward black arrows indicate significant increases and decreases, respectively. downward gray arrows indicate observed but not significant decreases. www.nature.com/scientificreports/ at slightly but significantly increased (1.3-fold) levels in the liver of infected mice at 3 and 6 dpi (fig. 5a ). this gene is negatively regulated by insulin signaling through inactivation of a transcriptional factor forkhead boxcontaining protein o sub-family 1 (foxo1) which reduces glycolysis and activates gluconeogenesis 11 . therefore, the increase in pck2 expression in the present study also suggests reduced insulin activity or sensitivity in pr8 virus-infected mice. in addition to glucose, fatty acids are important sources of acetyl-coa for the tca cycle. fatty acid metabolism was previously reported to be inhibited by influenza virus infection 12, 13 . therefore, we investigated whether pr8 virus infection altered the hepatic expressions of genes encoding fatty acid-metabolizing enzymes (fig. 5b) . cd36 and pnpla2 play important roles in fatty acid transport and lipolysis, respectively, and their gene expressions are downregulated by insulin signaling 14, 15 . here, cd36 was expressed at significantly increased levels in the livers of pr8 virus-infected mice, with 2.44-and 2.38-fold increases at 3 and 6 dpi, respectively. in addition, pnpla was significantly induced by 2.06-and 2.57-fold at 3 and 6 dpi, respectively. significant increases in the expressions of figure 2 . relative serum levels of intermediates of the tca cycle and related pathways in control vs. pr8 virusinfected mice at 1, 3, and 6 dpi. mice were intranasally inoculated with pbs alone or pbs comprising pr8 virus, and serum samples were collected for metabolome analysis at 1, 3, and 6 dpi. the serum levels of tca cycle metabolites in pr8 virus-infected mice are presented relative to those in control mice at each time point. each panels show metabolites of the tca cycle, urea cycle, and amino acid metabolism, respectively. bars represent mean ± sem of 3 or 4 animals. in each panel, white, gray, and black bars indicate data from pr8 virus-infected mice at 1, 3, and 6 dpi, respectively; *p < 0.05, unpaired t test using the holm-sidak correction method with an alpha value of 0.05, control vs. pr8 virus-infected mice at each time point. www.nature.com/scientificreports/ cd36 and pnpla2 as well as of pck2 suggested the attenuation of insulin signaling in the liver of pr8 virus-infected mice. conversely, we observed no changes in the expressions of acox1 and cpt1β, which encode key enzymes of fatty acid β-oxidation. in addition, the serum levels of carnitine were significantly decreased by 0.52-fold at 6 dpi (fig. 2) . because carnitine is involved in fatty acid transport into mitochondria, the observed decrease would possibly reduce fatty acid β-oxidation in mitochondria. these changes suggest fatty acid accumulation in the liver cells of pr8 virus-infected mice, although further analyses on protein expressions and enzymatic activities are warranted in the future. in the present study, metabolome analyses demonstrated that pr8 virus infection decreases the serum levels of most tca cycle intermediates and metabolites in the related metabolic pathways, suggesting reduced flux through the tca cycle. because the tca cycle greatly contributes electrons for mitochondrial oxidative phosphorylation, suppression of the tca cycle leads to reduced atp synthesis. hence, the rates of atp and gtp degradation were possibly greater than the respective rates of their synthesis in pr8 virus-infected mice, leading to significant increases in the serum levels of metabolites, such as xanthine, hypoxanthine, and inosine. energy depletion indicated by the reduced levels of atp and gtp has been described in several studies on infectious diseases and endotoxemia [16] [17] [18] . reduced atp levels in blood and various organs, including the liver, have been previously associated with influenza virus infection in a mouse model 16 . decreased gtp levels have been shown www.nature.com/scientificreports/ in the lung tissues of rabbits treated with bacterial endotoxin lipopolysaccharide 17 . increases in the serum levels of hypoxanthine and inosine have also been reported in patients with primary dengue virus infection at the febrile stage, suggesting an imbalance of atp and/or gtp synthesis and degradation during the acute stage of the dengue fever 18 . these studies and ours indicate that regulatory energy depletion is a common symptom of infectious diseases. tca cycle flux is associated with glucose and fatty acid metabolism because these energy sources are eventually converted to acetyl-coa for entry into the tca cycle. if either pathway is inhibited, the other pathway becomes activated to compensate for the reduced substrate supply to the tca cycle. in the case of hfd-induced obese mice with insulin resistance, fatty acid oxidation is increased in a leptin-dependent manner, and tca cycle activity is enhanced 19, 20 . conversely, db/db mice, a well-known mouse model of type 2 diabetes, reportedly developed insulin resistance without activation of fatty acid oxidation due to lack of leptin receptor and showed reduced serum levels of the tca cycle intermediates citric acid, 2-ketoglutaric acid, malic acid, and fumaric acid with disease progression 21 . in the present study, no changes in the expression of cpt1 or acox1 in the liver suggest that fatty acid oxidation was not enhanced in pr8 virus-infected mice despite insulin resistance. although the effect of pr8 virus infection on fatty acid oxidation was not evaluated in the present study, reduced fatty acid oxidation during influenza has been demonstrated previously 12, 13 . if both insulin signaling and fatty acid oxidation are inhibited during influenza, it might result in suppression of the tca cycle due to reduced substrate supply. given significant body weight loss in pr8 virus-infected mice, however, decrease in food intake during influenza could affect the serum levels of the tca cycle and related pathways. further investigations, such as studies with pair-fed control and lower titer of virus, will provide helpful information to confirm the underlying association. here we demonstrated for the first time that influenza virus infection impairs insulin signaling in liver tissues, which critically regulates glucose metabolism 9,10 , and induces a tendency toward glucose intolerance. importantly, nagao et al., reported a high blood glucose level (over 150 mg/dl) as one of the worst prognostic factors in pediatric patients with influenza-associated encephalopathy 22 . in addition to the elevated glucocorticoid levels, as speculated by nagao et al., our study suggests the impairment of insulin signaling as a mechanism of elevated blood glucose levels in children with severe influenza. conversely, another study reported that glucose uptake was activated in the lungs of patients with respiratory viral infections, including influenza virus infection 23 . this discrepancy may be associated with tissue-specific metabolic changes in response to influenza virus infection. however, given the direct activating effect of influenza virus proliferation on glucose metabolism in cultured mice were intranasally inoculated with pbs alone or pbs comprising pr8 virus. at 6 dpi, the mice were intraperitoneally injected with pbs or insulin after overnight fasting, and liver samples were collected after 15 min for whole lysate preparation. western blotting was performed to quantitate phosphorylated and total akt protein levels in lysates. (a) a representative western blotting analysis shows total akt and akt phosphorylated at ser473 on the same membrane that was sequentially immunoblotted with corresponding antibodies. (b) relative akt phosphorylation levels were calculated from band densities and expressed relative to data from pbs-treated control mice. bars represent mean ± sem of 3 animals. white and black bars indicate data from pbs-and insulin-treated mice, respectively; p < 0.05, two-way anova, pbs vs. insulin (*), control vs. pr8 virus-infected mice (#). (c) mice were intranasally inoculated with pbs alone or pbs comprising pr8 virus. at 6 dpi, gtt was performed after overnight fasting. to this end, mice were intraperitoneally injected with glucose, and their blood glucose levels were sequentially measured at 0, 30, 60, and 90 min post injection. dots represents mean ± sem of 5 animals; *p < 0.05, unpaired t test using the holm-sidak correction method with an alpha value of 0.05, control vs. pr8 virus-infected mice at each time point. www.nature.com/scientificreports/ madin-darby canine kidney cells 24 , the hepatic insulin resistance demonstrated here could have been induced by host factors and not the virus itself. upon intranasal infection of pr8 virus, proliferation of the virus occurs mainly in the lung and not in the liver of mice 25 . hence, the hepatic insulin resistance observed herein is possibly induced by host factors, such as systemically secreted cytokines. the direct roles of cytokines on insulin signaling have been demonstrated in a study showing that the proinflammatory cytokine interleukin-6 (il-6) inhibited insulin-dependent phosphorylation of akt in hepg2 cells and human primary hepatocytes 26 . furthermore, il-6 treatments reportedly impaired insulin signaling and inhibited insulin-induced decreases in blood glucose levels in mice 27 ; in this previous experiment, the serum levels of il-6 reached approximately 125 pg/ml, which was comparable to that observed herein in pr8 virus-infected mice at 3 and 6 dpi when reduced insulin-induced phosphorylation of akt was observed (supplemental fig. s3, 109.3 and 137.1 pg/ml at 3 and 6 dpi, respectively). thus, we suggest that this cytokine is one of host factor candidates that affect insulin resistance, particularly in the liver. in addition to il-6, interferon-gamma (ifn-γ) was reportedly induced by murine cytomegalovirus infections and was recently shown to induce insulin resistance in skeletal muscle 28 . besides il-6, the serum levels of ifn-γ were elevated in pr8 virus-infected mice at 6 dpi but were very low at 3 dpi in our study (supplemental fig. s3; 1 .391 and 313.4 pg/ml at 3 and 6 dpi, respectively). therefore, we propose that influenza virus infection induces hepatic insulin resistance through systemic cytokine secretion. as stated above, in addition to glucose, fatty acids are important sources of the tca cycle substrate acetyl-coa. reduced fatty acid metabolism during influenza has been reported in previous reports showing mitochondrial abnormalities, decreases in the expressions of relevant enzymes, and hepatic steatosis in mice 12, 13 . consistently, our gene expression data indicate altered fatty acid metabolism in the livers of pr8 virus-infected mice. in particular, the transcription levels of the fatty acid transporter cd36 and the lipolytic enzyme pnpla2 were upregulated at 3 and 6 dpi, possibly elevating extracellular fatty acid delivery and stored triglyceride lysis in liver cells. however, no changes were observed in the mrna expressions of acox1 and cpt1b. these encoded enzymes are rate limiting factors for fatty acid β-oxidation in peroxisomes and mitochondria, respectively. taken together, our data suggest that fatty acids are accumulated in the cytoplasm of liver cells in influenza virus-infected mice. although anorexia due to influenza virus infection may induce similar alterations in gene expressions and subsequent hepatic steatosis, the findings of pair-fed studies on influenza eliminated the possibility that starvation www.nature.com/scientificreports/ is solely responsible for these changes 12, 13 . moreover, fasting generally increases the mrna expressions of acox1 and cpt1b, thereby discriminating the effects of influenza virus infection on fatty acid metabolism from those of starvation 29, 30 . fatty acid accumulation can induce mitochondrial dysfunction, as reported previously in cultured hepatocytes 31 . mitochondrial dysfunction increases oxidative stress via reactive oxygen species production. reactive oxygen species and diacylglycerol, converted from triacylglycerol by adipose triglyceride lipase (encoded by pnpla2), inhibit insulin signaling 32 . it is suggested that accumulation of intracellular fatty acids triggers and complicates insulin resistance in the liver of infected mice. given the higher influenza-associated mortality and morbidity rates in patients and mice with energy metabolism disorders 2-6 , energy metabolism dysregulation induced by influenza virus infection is possibly associated with pathogenicity. increased mortality rates with influenza have been reported in diet-induced obese mice which probably had insulin resistance as well as in long-chain acyl-coa dehydrogenase-knockout mice, which had reduced mitochondrial fatty acid oxidation 2,5 . interestingly, both these mouse models presented with subdued immune responses to virus infection and similar or lower lung virus titers compared with the control mice. hence, increased influenza severity in animals and patients with energy metabolism disorders can be explained neither by the increased levels of inflammatory cytokines nor by the reduced clearance of pathogens 2,5 . as described above, dysregulation of energy metabolism appears to be a downstream response to cytokine and inflammatory signaling and thus could be more directly associated with the pathogenesis and severity of influenza. previous cohort studies have demonstrated that antiinflammatory corticosteroids have no beneficial effects and that these corticosteroids can exacerbate outcomes in patients with severe influenza 33 . given that glucocorticoids inhibit insulin signaling as well as cytokine-dependent pathogen clearance 34, 35 , corticosteroid treatments could confound pathogenicity. thus, improvements in host energy metabolism would be a novel therapeutic target for severe influenza rather than immune suppression. moreover, this concept could be expanded to other infectious diseases because energy metabolism dysregulation is possibly induced by host factors, such as proinflammatory cytokines, and not by the viruses themselves. given the recent emerging infectious diseases such as the pandemic influenza in 2009 and severe pneumonia by the novel corona virus in 2019, the importance of developing novel therapeutic drugs that target host factors against common symptoms of various diseases is important. infants and children younger than 5 years of age are generally at high risks of mortality and morbidity due to influenza compared with middle-aged adults 36 . previously, a negative correlation of nasal lavage and plasma inflammatory cytokine levels with age has been reported and thought to be a reason for the high mortality and severe illness caused by influenza virus infection in young children 37, 38 . in addition to cytokine responses, age-specific features of energy metabolism could provide insights into the pathogenesis of influenza at different ages. in pediatric patients with type i diabetes, the youngest population (0-5 years of age) reportedly showed the highest prevalence of diabetic ketoacidosis, which has an impact on morbidity and mortality 39 , indicating that attenuation of insulin signaling by virus infection could result in more severe symptoms in such populations. the present study provides novel insights into host responses during influenza. based on the findings of the present and previous studies, we conclude that influenza virus infection induces dysregulation of both insulinregulating glucose metabolism and fatty acid oxidation, leading to decreased tca activity. in future studies, we aim to investigate the effect of influenza virus infection at lethal and sublethal doses of various virus strains for further understanding of influenza pathogenesis and examine the therapeutic effects of interventions that improve host energy metabolism. materials. phosphate-buffered saline (pbs) was purchased from gibco/life technologies (carlsbad, ca). tris-buffered saline (tbs) tablets were purchased from takara bio (otsu, japan). urea, tween 20, and glucose were purchased from sigma-aldrich (st louis, mo). sodium dodecyl sulfate (sds) was purchased from wako (tokyo, japan). mouse. male c57bl/6 mice were purchased from hokudo (sapporo, japan) and were kept at a bsl-2 laboratory at the research center for zoonosis control, hokkaido university, under standard laboratory conditions (room temperature 22 °c ± 2 °c, relative humidity 50% ± 10%) and a 12/12-h light/dark cycle. the mice were administered a standard ce-2 chow diet purchased from clea japan (sapporo, japan) with water ad libtum. experiments were performed on 9-14 week-old mice. virus infection and sample collection. pr8 virus particles at 500 plaque-forming units in 50 µl of pbs or pbs only (control) were intranasally inoculated into the mice under inhalation anesthesia with isoflurane. at 1, 3, or 6 dpi, the mice were euthanized, and their liver and blood samples were collected. blood samples were incubated at room temperature for 1 h to clot and were then centrifuged at 1,000g for 20 min. supernatants were collected as serum and were stored at − 20 °c until further analysis. tissue samples were stored at − 80 °c until further analysis. to evaluate insulin sensitivity, the mice were administered insulin (humulin r, eli lilly, indianapolis, in) at 2 u/kg body weight intraperitoneally at 6 dpi after overnight fasting. after 15 min, the mice were euthanized, and their liver samples were collected and frozen immediately in liquid nitrogen. liver samples were stored at − 80 °c until further analysis. research (kyoto, japan) using liquid chromatography-tandem mass spectrometry (lc-ms/ms). briefly, after the addition of an internal standard (2-isopropylmalic acid) to the serum samples, sample preparation was conducted using a series of hydrochloride and acetonitrile extractions. after centrifugation at 10,000 rpm for 5 min at room temperature, the supernatants were divided into two tubes: one was used for analyses of amp, gmp, aconitic acid, citric acid, and fumaric acid, whereas in the other one, the supernatant was mixed with hydrochloride and used for analysis of other compounds. chromatographic separations were performed on a 2 discovery hs f5-3 column (2.1 mm × 150 mm, 3 μm, sigma-aldrich). the oven temperature for the column was maintained at 40 °c. mobile phases a and b were 0.1% formic acid-water solution and 0.1% formic acid-acetonitrile, respectively. separation was performed using gradient elution at a flow rate of 0.25 ml/min with a nexera uhplc system (shimadzu). compounds were eluted by changing proportions of mobile phase b as follows: 0% (0-2 min), 0%-25% b (2-5 min), 25%-35% b (5-11 min), 35%-95% (11-15 min), 95% (15-20 min), 95%-0% (20-20.1 min), and 0% (20.1-25 min). compounds were detected using an lcms-8050 (shimadzu) instrument with electrospray ionization. the peak areas of each compound were measured using labsolutions (shimadzu) and were normalized to that of an internal standard. the metabolome analyses identified 74 molecules in our sample sets. among them, cysteine, cytosine, and methionine sulfoxide were not detected in some samples from pr8 virus-infected mice at 3 and 6 dpi (supplemental table s1 ). to examine the effects of pr8 virus infection on the serum levels of various compounds, the relative peak areas for pr8 virus-infected mice were expressed as fold changes relative to the average peak areas for the control mice at corresponding time points. the peak areas and relative ratios are provided in supplemental table s1 and s2. serum metabolome data analysis using metaboanalyst. utilizing peak areas of 74 molecules, principal component analysis and pathway analysis were performed with metaboanalyst (https ://www.metab oanal yst.ca). the values were normalized by autoscaling function. missing values in cysteine, cytosine, and methionine sulfoxide were replaced by small values (a half of the minimum positive values in the original data). factor loading was defined for each metabolite as the correlation coefficient between the pc1 score and the level of the metabolite after normalizing by autoscaling for each sample. metabolites with high factor loading (> 0.7) and significant differences (p < 0.05, t-test) were selected as significant metabolites and are listed in table 1 . pathway analyses were performed based on the kyoto encyclopedia of genes and genomes (https ://www.genom e.jp/ kegg/), and the most significantly altered metabolic pathways in pr8 virus-infected mice were identified. the top 10 pathways identified are listed in table 2 . evaluation of phosphorylated akt by western blotting. liver samples were homogenized in tbs comprising 8 m urea and 1% sds using an ultrasonic homogenizer (q125 sonicator, qsonica, newtown, ct) and centrifuged at 15,000g for 10 min at 10 °c to obtain supernatants as whole liver lysates. protein levels were measured using pierce bca protein assay kits (thermo fisher scientific, waltham, ma). samples for western blotting were prepared by mixing whole liver lysates with the nupage lds sample buffer (thermo fisher scientific) and heating at 70 °c for 10 min. subsequently, 10 μg aliquots of total protein were separated using 10% sds-polyacrylamide gel electrophoresis (sds-page) and were transferred to polyvinylidene difluoride membranes. after blocking with 5% nonfat dry milk in tbs buffer comprising 0.1% tween 20 (tbst), the membranes were probed with an anti-akt phosphorylated at ser473 antibody (1:1,000, #9271, cell signaling technology, beverly, ma) or an anti-akt antibody (1:1,000, cell signaling technology) in tbst buffer comprising 5% bovine serum albumin overnight at 4 °c. the membranes were then treated with secondary horseradish peroxidase-conjugated antirabbit antibody (1:5,000, sc-2357, santa cruz) for 1 h at room temperature. protein bands on membranes were detected using supersignal west femto maximum sensitivity substrate (thermo fisher scientific) and an imagequant las4000 system (ge, buckinghamshire, uk). the bands were quantified using the imagequant tl analysis system (ge). full-length images are provided in supplemental fig. s4 . pbs at 2 g/kg body weight after overnight fasting. blood was collected from tail veins at 0, 30, 60, and 90 min after injections, and plasma glucose concentrations were measured using an accu-chek glucose meter (roche diagnostic, mannheim, germany). www.nature.com/scientificreports/ amount of assay buffer for serum samples or serum matrix for standards and controls. magnetic beads coated with antibodies against the target cytokines were added to each well, and the plates were incubated on a plate shaker overnight at 4 °c. after washing with washing buffer in the kit, the samples were reacted with biotinylated detection antibodies for 1 h and then with streptavidin-phycoerythrin for 30 min. after washing and addition of loading buffer from the kit, the samples were analyzed by the magpix system (luminex, austin, tx, usa). the results are presented in supplemental fig. s3 . statistical analysis. statistical analyses were performed using prism 7 (graphpad software, san diego, ca, usa). differences were identified using unpaired t-tests or two-way anova and were considered significant when p < 0.05. data are presented as mean ± standard error of the mean (sem). all experiments were performed at least twice to confirm the reproducibility. ethical statement. all mouse experiments were performed with approval from the animal care and use committee of hokkaido university following the fundamental guidelines for proper conduct of animal experiment and related activities in academic research institutions under the jurisdiction of the ministry of education, culture, sports, science and technology in japan. body weight losses were monitored daily after infection, and mice were humanely euthanized when weight loss reached 25%. 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2009 pandemic influenza a (h1n1) virus infection ketoacidosis at diagnosis of type 1 diabetes in french children and adolescents we thank the national institute of infectious disease in japan for kindly providing influenza virus a/puerto rico/8/34 (h1n1). this work was supported by japan science and technology agency basic research programs, jsps kakenhi (grant number 17k15367), and northern advancement center for science and technology, hokkaido japan (grant number 786k001018). the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/10.1038/s4159 8-020-67879 -6.correspondence and requests for materials should be addressed to h.k. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creat iveco mmons .org/licen ses/by/4.0/. key: cord-350593-bvmg7f15 authors: mcdonald, r.s.; sambol, a.r.; heimbuch, b.k.; brown, t.l.; hinrichs, s.h.; wander, j.d. title: proportional mouse model for aerosol infection by influenza date: 2012-08-21 journal: j appl microbiol doi: 10.1111/j.1365-2672.2012.05402.x sha: doc_id: 350593 cord_uid: bvmg7f15 aims: the aim of this study was to demonstrate a prototype tool for measuring infectivity of an aerosolized human pathogen – influenza a/pr/8/34 (h1n1) virus – using a small‐animal model in the controlled aerosol test system (cats). methods and results: intranasal inoculation of nonadapted h1n1 virus into c57bl, balb/c and cd‐1 mice caused infection in all three species. respiratory exposure of cd‐1 mice to the aerosolized virus at graduated doses was accomplished in a modified rodent exposure apparatus. weight change was recorded for 7 days postexposure, and viral populations in lung tissue homogenates were measured post mortem by dna amplification (qrt‐pcr), direct fluorescence and microscopic evaluation of cytopathic effect. plots of weight change and of pcr cycle threshold vs delivered dose were linear to threshold doses of ~40 tcid (50) and ~12 tcid (50), respectively. conclusions: mid (50) for inspired h1n1 aerosols in cd‐1 mice is between 12 and 40 tcid (50); proportionality to dose of weight loss and viral populations makes the cd‐1 mouse a useful model for measuring infectivity by inhalation. significance and impact of the study: in the cats, this mouse–virus model provides the first quantitative method to evaluate the ability of respiratory protective technologies to attenuate the infectivity of an inspired pathogenic aerosol. whereas aerosols and contact are accepted as modes of transmitting many disease-causing organisms, including legionella pneumophila, smallpox, severe acute respiratory syndrome (sars), coronaviruses, rhinovirus (fiegel et al. 2006 ) and tuberculosis (wells 1934; riley et al. 1959; mcclement and christianson 1980) , the role of bioaerosols as a transmission mechanism for influenza is less clearly understood (tellier 2006; tellier 2007a,b; lemieux and brankston, 2007; brankston et al. 2007; tang and li, 2007; lee 2007) . although a few publications have documented the transmissibility of influenza a through inhalation routes (tellier 2006 (tellier , 2009 , few studies to date have utilized a mouse model to investigate susceptibility to and pathogenicity of measured aerosol exposures. the lack of aerobiology studies results from several factors, including the need for specialized equipment to generate and monitor bioaerosols, the technical difficulty involved, inconsistency among reported techniques (lore et al. 2011 ) and the considerable cost of conducting this research (sherwood et al. 1988) . therefore, the most commonly described method of infecting mice with influenza virus is through the installation of fluid into the nasal passages (lu et al. 1999; govorkova et al. 2007; gillim-ross et al. 2008; chen et al. 2011) . for more than 75 years, laboratory mice have served as models for susceptibility to and pathogenesis of influenza disease (andrewes et al. 1934) . their low cost, small size, relative susceptibility to the virus and ease of handling make mice a favourable platform for studying influenza virus infections. the mouse is currently considered the primary model for the evaluation of influenza antiviral agents because it is a predictive indicator of the efficacy of such treatments in humans (sidwell and smee 2004) . the use of a well-characterized mouse model is especially important in studying the infectious pathways of new pandemic (pdm) subtypes of influenza a. indeed, the latest emergence of influenza has reignited interest in the use of mouse models (beigel and bray 2008) . although mathematical models have been used for decades (findeisen 1935; yeh et al. 1976 ; international commission on radiological protection (icrp) 1994; asgharian and anjilvel 1998; heyder 2004) to calculate particle deposition within the respiratory tract, such calculations of particle placement are able to rationalize but not to predict the resulting clinical effect. animal models allow closer approximation to a human response (schulman 1968; lowen et al. 2006; gustin et al. 2011) , and therefore, it is important to continue to further develop these models kawaoka 2012) . experimental inhalation exposure systems are an established tool and the subject of several reviews (drew and laskin 1973; macfarland 1983; cheng and moss 1995; jaeger et al. 2006; wong 2007) . the purpose of this study was to identify and evaluate a mouse model as a complement to a measured-dose bioaerosol delivery apparatus termed cats (controlled aerosol test system) for testing the clinical effectiveness of media used in respiratory protective equipment (rpe). this validation, which includes complementary data measured postmortem, renders the mice available to serve as a detector to evaluate the clinical significance of articles of rpe by directly measuring the change in infectivity the protective article causes. exposure to influenza virus often leads to a disease presenting as an acute and temporarily incapacitating infection of the upper respiratory tract that can be fatal. influenza illness is often associated with occurrences of annual or near-annual epidemics in temperate climate zones. within the last 100 years, influenza pandemics have occurred four times [1918 (h1n1), 1957 (h2n2), 1968 (h3n2) and 2009 (h1n1)] (oxford 2000) . pandemics are infrequent but often severe events because of the emergence of novel, unpredictable strains of influenza a virus caused by recombination of genetic material from two or more circulating virus subtypes. this antigenic shift can often lead to a new virus subtype with the ability to jump from one species into another, potentially naive species (e.g. avian influenza), and cause a large proportion of influenza-related deaths. a number of animal models have been studied to evaluate new vaccines and other approaches for preventing influenza-related disease (gubareva et al. 1998; ng et al. 2010) . green and kass (1964) conducted studies on the clearance of inhaled microbial aerosols from the murine respiratory tract. schulman and kilbourne (1963) studied mouse-to-mouse transmissibility of influenza virus. a factor complicating viral research in animal models is that a virus may be present in a host without causing disease. this may be due to restrictions such as the absence of appropriate receptors on certain cell types (e.g. tissue tropism) and the lack of intracellular processes required to generate infectious progeny viruses or induce cytolytic effects. differences in viral receptors have been documented for respiratory epithelial cells based on location in either the pharynx or peripheral lung (van riel et al. 2007 (van riel et al. , 2010 . in addition, either the organism or host cell may mount an immune response or generate intracellular molecules that disrupt the viral effects. therefore, differences may appear at either the cellular or tissue level or among susceptible hosts depending upon the method of infection, especially in regard to aerosol exposures (phalen et al. 2008) . this study examined these parameters related to efficient (near threshold) infection versus overwhelming infection of mice by exposure to aerosolized influenza virus. the cats is an apparatus that was designed, constructed and validated (stone 2010; stone et al., 2012) to deliver a precisely measured aerosol concentration, either directly or after passage through a filter medium, through a nose-only directed-flow inhalation exposure system (noies; ch technologies, westwood, nj, usa, jaeger et al. 2006) to individual mice (figs 1 and 2) . this low-flow, singlepass design consists of an aerosol generator, diffusion drier, charge neutralizer, filter holder, sampling points and noies unit (stone 2010; stone et al., 2012) . the cats generates a biological aerosol over a range of constant concentrations andafter conditioning and treatment, if any is applieddelivers the particles to the nose of a mouse constrained in a polycarbonate tube (cht-247; ch technologies) as a pure respiratory exposure. the main system components were connected using 0á5-inch (12á7-mm) stainless steel tubing with a minimum number of gradual bends. in operation, a single-jet collison nebulizer (bgi inc., waltham, ma, usa), regulated to 25-30 psi was used to atomize the viral suspensions. the aerosol, which acquires surface charges during atomization, passes through a 9á5-inch (23-cm) diffusion drier and then through a 2-mci 85 kr charge neutralizer (tsi inc., shoreview, mn, usa) to restore the 'normal' boltzmann equilibrium charge distribution. after passage through the filter holder and any filter medium mounted in it, the aerosol enters the 12-port noies, from which it exits the test system through the hepa-filtered exhaust. total flow rate through the system was regulated at the nebulizer to deliver 2á0 ± 0á1 l min à1 measured on exit by a mass flow metre (tsi model 4043e). the entire system was designed to fit inside a biological safety cabinet (baker company, sanford, me, usa; sg603-ats) for additional protection from generated aerosols. the unique feature of the system is an optional filter holder (triosyn corp, williston, vt, usa), which is capable of holding filter media samples 47 mm in diameter. smaller discs of filter media can be accommodated with the use of reducers. correlation of sampling ports stone et al. (2012 , stone 2010 ) demonstrated uniform distribution of aerosol to several ports of the cats. following transport and installation of the cats in an animal biosafety level 3 (abl3) facility at the university of nebraska medical center, the exposure system was retested to verify consistency of particle counts among all 12 ports, using tap water to generate test aerosol particles. from each of the 12 exposure ports, samples of particles delivered by a single-jet collison nebulizer were measured in triplicate using a scanning mobility particle sizer (smps) system (tsi inc., minneapolis, mn, usa). the aerosol particle size and concentration were determined using 6á0 l min à1 sheath air and 0á6 l min à1 sample air. data outputs from the smps were collected by the aerosol instrument manager ® software (ver. 8.1.0.0; tsi inc., minneapolis, mn, usa). influenza a/pr/8/34 (h1n1) virus was obtained from american type culture collection (rockville, md, usa) as frozen stock (atcc vr-1469). virus was propagated using cdc unit 15g.1 protocol (szretter et al. 2006) . titres were performed and calculated using the spearman-kärber method (armitage and allen 1950; finney 1964) . to assess the psd of aerosols containing influenza virus, samples were taken in triplicate from the sampling port located downstream from the cats, diluted with filtered air and routed to the smps. results indicated a single-mode, polydisperse aerosol in the size range 10-400 nm. three strains of female mice (c57bl, balb/c and cd-1) were purchased from charles river laboratories (portage facility, mi, usa). the mice were 6-8 weeks old and ranged in weight from 18 to 30 g. mice were randomly divided into groups assigned to specific exposure time points, and no more than five (all in a given exposure group) were housed per cage. animals were provided rodent chow (harlan teklad, usa) and water ad libitum and maintained on a 12-h light/dark cycle. all animal work was carried out in an abl3 facility following institutional and regulatory procedures. to minimize artefacts caused by stress during respiratory exposure sessions, mice were preconditioned daily during the week preceding their exposure sessions (nrc 2003 (nrc , 2011 ) by insertion into a mouse restraint device (ch247; ch technologies) for a period that did not exceed the maximum exposure time for that experiment. to select a suitable mouse strain for infection with the influenza a/pr/8/34 (h1n1) (not mouse-adapted) strain used in this study, two inbred (balb/c and c57bl) and one semi-outbred strain (cd-1) of 20-25 g female mice were tested for susceptibility to infection by the virus. individual base weights were determined prior to exposure, and all mice were weighed daily at a uniform, scheduled time throughout the study. the average weights from surviving exposed mice at day 7 were compared to the averages of control mice. all mice were euthanized by day 7 postinoculation. the inoculum, consisting of 30 ll of virus at a concentration of 4á74 9 10 7 median tissue culture infectious dose (tcid 50 ) ml à1 , was placed intranasally into each mouse. the dose was divided equally and placed dropletby-droplet by pipette into each naris of the anesthetized (ketamine/xylazine) mouse. following the same procedure, a 1 : 10 dilution of virus stock in 19 phosphatebuffered saline (pbs) medium was used to inoculate a second set of mice of the same three strains. in all, five mice per strain per dilution were used to determine susceptibility to the virus. three mice per strain were used as controls. each control mouse was intranasally inoculated with 30 ll total 1 9 pbs medium as previously described (jerrells et al. 2007) . in a preliminary study conducted to establish a baseline dose of virus capable of causing infection following aerosolization, the working stock of influenza virus was diluted 1 : 30 in endotoxin-free water (sigma, st. louis, mo, usa) and delivered into the collison nebulizer at 1á58 9 10 6 tcid 50 ml à1 . four sets of three cd-1 mice were emplaced in polycarbonate restraints, installed into the noies with the filter holder empty, and exposed to aerosolized virus at exposure times of 2, 6, 20 and 60 min. aliquots of the influenza working stock (4á74 9 10 7 tcid 50 ml à1 titre) were subsequently diluted to 1 : 300 and 1 : 1000 (v/v) in endotoxin-free water to prepare concentrations aerosolized during three successive mouse exposure series described below. the single-jet collison nebulizer was charged and allowed to run for 5 min to stabilize the system. the bypass valve directly upstream of the cats directed the aerosol through two hepa filters connected in series until exposure was initiated. nonanesthetized mice were carefully immobilized in the polycarbonate tubes so that the tip of the nose projected out of an opening in the front of the holder. the tubes were then inserted securely into a port on the cats. once the animals were emplaced, the test aerosol was directed through the system. vents inside the cavity of the cats directed an airstream containing the filtered aerosol at the nares of the mouse as her only source of breathing air. excess aerosol flow and exhaled air were continuously swept away to preclude inhalation of previously exhaled air. a spread of delivered doses (proportional to concentration, c, 9 time, t) around each dilution was achieved by varying the time of exposure. the bioaerosol dose received is calculated as follows: where vsf is the ratio of viable airborne counts, in tcid 50 ml à1 , to viable counts, in tcid 50 ml à1 , in the collison reservoir, and r a is the respiration rate per minute, and v a is the tidal volume in ml a (ml of air), respectively, of the cd-1 mouse. r a and v a are reported (fairchild 1972) to be 261 respirations/min and 0á16 ml a , respectively. thus, a mouse exposed for 2 min to a 300 : 1 dilution of a suspension containing 4á74 9 10 7 tcid 50 ml à1 of virus inhales a dose of d p ¼ 4á74 â 10 7 tcid 50 ml à1 â 1=300 â 9 â 10 à7 ml ml à1 a â 261 resp min à1 â 0á16 ml a resp à1 â 2 min ¼ 12 tcid 50 mice were exposed in groups for each preselected time (tables 2-4 ). at the end of the exposure period, the polycarbonate tubes holding the mice were removed, and the next group was inserted, until all mice for that series of experiments were exposed. when time points allowed, the mice were inserted in overlapping groups. unused ports were sealed with the supplier's standard plugs. all exposures were carried out within a biological safety cabinet. control mice for 1 : 30 (1á58 9 10 6 tcid 50 ml à1 ) and 1 : 300 (1á58 9 10 5 tcid 50 ml à1 ) exposure groups were placed in polycarbonate tubes during the testing equal to the maximum exposure time and exposed to aerosols generated from endotoxin-free water (sigma) containing no virus. for the 1 : 1000 (4á74 9 10 4 tcid 50 ) exposure group, two sets of controls were used. one mouse group was exposed as earlier, while a second group was exposed to uninfected allantoic fluid processed in the same manner as from influenza-infected eggs. mice were observed and weighed each of 7 days postexposure. severely distressed mice were euthanized after the day's weighing and, following the final weighing, all surviving mice were euthanized by administration of an overdose of ketamine/xylazine by intraperitoneal injection. a necropsy was conducted and selected portions of the lungs were selected for molecular, histological or virus culture assessment. lung tissues aseptically placed into 2á7 ml of cold bd universal virus transport medium (becton, dickinson and co., franklin lakes, nj, usa) were homogenized by hand using a closed ultra tissue grinder system (fisher scientific, pittsburgh, pa, usa) and then stored at à80°c. cell culture and molecular assays tcid 50 /cpe and dfa assays starting viral titres were quantified by cell culture endpoint-dilution assays performed using madin-darby canine kidney (mdck) cells and calculated using the spearman-kärber method in units of log 10 tcid 50 ml à1 . cell culture plates containing mdck cells were grown and maintained using standard cell culture techniques. presence of viable virus in homogenates of murine lung tissue was qualitatively assessed by two-concentration cell culture endpoint assays performed using mdck cells. cell culture plates containing mdck cells were grown and maintained using standard cell culture techniques. aliquots (1á0 ml) of lung homogenates were plated in serial 1 : 10 dilutions (in serum-free eagle's minimal essential medium (emem)) from 10 à1 to 10 à4 in quadruplicate on confluent cell monolayers. the samples remained in contact with the monolayer for a 1-h incubation before 1% bsa-serumfree emem with trypsin was added (bovine serum albumin). the plates were incubated for 5-6 days under 5% co 2 at 37°c prior to visualization under the microscope for cytopathic effect (cpe) or fluorescent-labelled antibody evaluation. test plates were read using a +/à system, in which + showed disruption of the monolayer and à showed that the monolayer remained confluent. direct fluorescent antibody assay (dfa) was used to qualitatively determine influenza infection of the mdck cell line using the d 3 ultra dfa respiratory virus screening and id kit (diagnostic hybrids inc., athens, oh, usa) per manufacturer's instructions. rna extraction and qrt-pcr ribonucleic acid (rna) was extracted from samples using the qiaamp ® minelute ® virus spin kit following the manufacturer's protocol (qiagen, valencia, ca, usa). rna amplification was performed using invitrogen's superscript iii platinum one-step quantitative real-time polymerase chain reaction (qrt-pcr) kit (invitrogen, grand island, ny, usa). the qrt-pcr assay was run on the roche lightcycler ® 480 real-time pcr system (roche diagnostics, indianapolis, in). assay conditions and reaction volumes were used from protocols previously described by the cdc (who 2009 ). the cycle threshold (ct) values from replicate runs were averaged for each time point and rounded to two decimal places. the recommended cut-off ct value of 30 was used as the criterion for infection. following fixation and routine processing, tissue sections were stained with hematoxylin and eosin and reviewed under standard light microscopy. compared to normal tissue, influenza-infected lungs showed lobular pneumonia with interbronchial inflammation. infected lungs also showed focal chronic inflammatory cell infiltration with a few neutrophils and some interstitial thickening. figure 3 shows the infiltrates in the infected tissue compared to uninfected tissue. after installation in the abl3 cabinet, a reverification of cats performance was conducted with water. the par-ticle counts at each port were averaged and again seen to be uniform within 10% (data not shown). a subsequent delivery of 100 mg l à1 sodium chloride in water showed number mean diameter (d 50,n ) = 74 nm and mass mean diameter (d 50,m ) = 208 nm over the range of particle diameters from 10 to 407 nm. figure 4 plots the coefficient of variation (cov) as a function of particle size at the 12 ports for the nacl aerosol measurements. groups of five mice were inoculated intranasally with one of two doses of virus and weighed daily for a week. per cent changes in average weights of exposed and control groups are indicated in table 1 . the nonmouse-adapted influenza virus produced obvious infection in all three strains of mice used. as no difference in gross infectivity was indicated by weight loss (table 1) , the less-expensive cd-1 mice were selected for further study. aerosol exposure (1á58 9 10 6 tcid 50 ml à1 ) two mice were used as unexposed controls. all of the mice survived to day 7, when they were euthanized and necropsied, and their lung tissue was examined by three different assays. at all four exposure time points, mouse lung tissues gave positive results from the qrt-pcr assay, for which a positive value was defined to be 31 ct. dfa and cpe assays on the lung tissue were also all positive ( table 2 ). there was a direct correlation between dose received (for convenience reckoned as ct values (product of concentration and exposure time)), with lower pcr ct values resulting from prolonged exposure. the mean ct value for control mice was 37, which was defined to be a negative response. values of 37 > ct > 31 were considered indeterminate. weight losses again showed proportionality to dose delivered. the results demonstrated that the influenza virus remained viable and capable of causing infection in cd-1 mice when aerosolized under test conditions. aerosol exposure (1á58 9 10 5 tcid 50 ml à1 ) because exposure to the 1 : 30 dilution of aerosolized virus resulted in massive but graduated infection of all tested mice, a greater dilution of the working stock was delivered in an effort to identify a threshold infective dose. the dilution was increased tenfold (to 1 : 300, resulting in delivery of a 1á58 9 10 5 tcid 50 ml à1 dispersion from the nebulizer and an overlap (as ct products) of the two smaller doses from the 1 : 30 dilutions), and the initial aerosol exposure sequence was repeated. three mice per time point were assayed by qrt-pcr. variation in ct values was observed in the 2-min exposure group, one mouse being clearly positive as reflected by ct values, and the other two mice falling within the indeterminate range (table 3 ). all mice in the 6-, 20-and 60-min time points were positive (<31 ct) with minimal variation in ct values. all control mice were negative (ct values ! 37). tcid 50 assays were performed on mice from group 1 at each time point. one mouse lung homogenate in group 1 that was indeterminate by qrt-pcr (36 ct) was negative by tcid 50 assay. all other lung homogenates tested positive by both methods. aerosol exposure (4á7 9 10 4 tcid 50 ml à1 ) although weight loss by the mice appeared to have reached baseline at the dose delivered in 6 min at 1 : 300 dilution, ct results from the aerosol challenge at 1 : 300 dilution show that all the mice exposed for 6 min or more received an infectious dosethat is, weight loss is an indicator of dose but ct measurements provide better sensitivity to detect an endpoint. in an effort to better define the threshold at which viral infection occurs, the stock suspension was further diluted to 4á7 9 10 4 tcid 50 ml à1 (1 : 1000), the exposure times were reduced, the number of mice per time group was increased to five, and two additional time points were added to increase the range and dose of aerosol exposure. results of this test are shown in table 4 . 4á74 9 10 7 tcid 50 ml à1 4á74 9 10 6 tcid 50 ml à1 controls sd, standard deviation. *one mouse died before day 7. table 2 results of three assays [pcr, direct fluorescent antibody assay (dfa) and cpe] from the homogenates of cd-1 murine lung tissue exposed to an aerosol generated from 1á58 9 10 6 tcid 50 ml à1 at the 3-min exposure time, no mice were positive for influenza virus as determined by ct value. in all four of the longer-exposure groups, a single mouse displayed a positive ct value. a trend may be suggested by the pattern of indeterminate values, but the quantitative ct values show an equally unconvincing opposite trend. all of the lung homogenates were tested by virus cell culture assay for tcid 50 and dfa. for homogenates whose ct value is <31, both tcid 50 and dfa were positive. it is accepted that influenza may be contracted through a variety of methods including large droplet and contact transmission. the only route of infection examined during this study was inhalation of droplet nuclei through nasal passageways. results should be expected to be different if other mucosal surfaces had been dosed with the same viral aerosols. this study sought only to determine the existence and scale of a measurable threshold aerosol infective dose in this animal model and to set parameters such as the mouse restraints in the experimentthat limit other exposure. although this study demonstrated infectivity of the aerosol, its residence time as dispersed fine particles was shorthundreds of milliseconds from nozzle to noseand viruses are known to spontaneously lose viability, so the importance of such bioaerosols as an environmental component remains uncertain. however, the experiment accurately simulates direct exposure to droplet nuclei generated by a cough, which can accordingly be concluded to be a mechanism for immediate transmission of this virus. the range of states resulting from influenza virus spans from asymptomatic infection to mild symptoms to pneumonia, which is often fatal. factors such as the strain of influenza virus that caused the illness, immune status of the host and/or age of the affected individual play an important role in recovery or progression of disease. this was evident in the 2009 outbreak of influenza with the influenza a virus h1n1 pandemic (pdm) strain, during which a disproportionately high percentage of morbidity occurred in children and young adults as well as in individuals with underlying conditions such as obesity and or diabetes (jhung et al. 2011) . influenza a (h1n1) virus selected for this study was chosen based on reports of its infectivity in mice (smee et al. 2008 )although it had not been mouse adaptedand its known hardiness in culture systems as a starting point for development of a mouse model for aerosolized influenza. our results showed a significant variability in morbidity and mortality among the mice exposed to aerosolized influenza virus. this appears to have been owing to individual susceptibility of the mice, because variability in the uniformity of the aerosol delivered was found not to be significant when each of the ports was analysed. in addition, significant differences were noted when mice were exposed over times ranging from 6 to 18 min with minimal or no morbidity when a low quantity of virus was sprayed over an extended period of time. our preferred explanation is that some of the mice were able to process and clear virus, while in others, it caused clinical infection and disease. additionally, the hardiness of immune response to influenza varies among the mice, resulting in different levels of susceptibility. for example, in table 3 at the lowest exposure time, one of the three mice was positive for infection, whereas in the lowest dose experiment (table 4) , one mouse was positive following only 6 min of exposure. as with many viruses, influenza produces a significant number of defective particles incapable of causing infection (huang 1973; pathak and nagy 2009) . this is further demonstrated by the wide variationranging from hundreds to thousandsin reported gene copy (total virions)-to-tcid 50 (infectious virion fraction) ratio (yang et al. 2011 ). sidorenko and reichl (2004) table 4 pcr ct values from homogenates of cd-1 murine lung tissue exposed to aerosol generated from a 4á74 9 10 4 tcid 50 ml à1 dilution of influenza virus over five different exposure times are indicated in parentheses: x = mouse death; pos = positive, ct < 31; ind = indeterminate, 37 > ct ! 31; and neg = negative, ct ! 37 level for influenza a (h1n1) to be 1 tcid 50 for using qrt-pcr and 0á1 tcid 50 using nested qrt-pcr. recognizing that a direct correlation may not always exist between a method that detects viable organisms and one based on viral genomes, our system showed excellent correlation between classical virology methods, morphology based on histological examination, clinical features and molecular quantification by qrt-pcr. influenza infection in mice has been monitored by several different parameters including mean time to death, lung weight and change in body weight (sidwell and smee 2000) . however, these indicators are difficult to interpret when the infectivity and challenge dose of the virus does not clinically manifest an illness (morbidity or mortality). therefore, we utilized qrt-pcr in comparison with tcid 50 to minimize the variability. virus replication in lung tissue is considered the most informative endpoint for efficacy studies because even modest changes in virus load can have a large impact on survivability (haga and horimoto 2010) . assays of postsacrifice tissue samples from the mice were uniformly positive in the 1 : 30 dilution series. in contrast with the data shown in fig. 4 , in the 1 : 300 dilution series only, the 2-min exposure (12 tcid 50 dose) group contained subjects that were not unequivocally positive for infection, both by ct (2 of 3) and by cpe (1 of 3). one-way anova and a two-tailed t-test did not find any statistical differences between the change in the mouse's weight and the pcr data; however, a more precise threshold value and statistical significance of this difference can be expected when the number of mice (n) in each exposure group is increased. likewise, the two sets of subjects that shared ct products (2 min 9 1 : 30 and 20 min 9 1 : 300, and 6 min 9 1 : 30 and 60 min 9 1 : 300) appear to show increased sensitivity with increasing aerosol concentration, which could be taken to imply that an acute exposure leads to greater infectivity than the same dose experienced more gradually. although this interpretation is intuitively reasonable, the volume of data supporting it is too limited to justify such a conclusion. owing to logistical constraints in the abl-3 facility, the psd was not measured during the exposures. however, stone et al. (2012; stone 2010 ) measured bioaerosol particles in the range 100~500 nm for a slightly smaller virus (ms2 coli phage) in the same apparatus. the absorbed dose was likely slightly smaller than the presented dose because deposition of inspired particles in this size range is incomplete and size dependent (stuart 1973; clay and clarke 1987; heyder 2004) . a plot of weight loss vs calculated inhaled dose (fig. 5 ) was fitted to a straight line, which intersects the mean weight change of the control group at approximately 40 tcid 50 . a third series of exposures was performed to a 1 : 1000 dilution, intended to improve definition of the threshold dose for weight loss; however, the results were equivocal, likely because the delivery was gradual enough that the mice developed an immune response that was able to manage the challenge, and/or the n of five was too small to average out what we presume to have been idiosyncrasies among the subjects. our results showed that qrt-pcr was more sensitive or that an excess of genome was present in comparison with the number of infectious virions as determined by tcid 50 and dfa assays. as the gold standard (schrauwen et al. 2011) for determining the moi has been tcid 50 and quantification of virus in mice exposed to influenza aerosols by qrt-pcr has not been previously reported, additional confirmatory studies were needed. we chose seven days as the terminal point for our study based on symptomatology in humans where virus production peaks approximately 48 h postinfection, and few virus particles are shed after day 6 (taubenberger and morens 2008). our results showed the delivered aerosol mid 50 to be at least 12 tcid 50 as determined by qrt-pcr ct value and significantly <40 tcid 50 as determined by obvious clinical response. however, the sample size must be expanded in the future to achieve greater resolution and statistical significance. in addition, future studies will utilize the influenza virus a (h1n1) pdm strain to determine variation in mid 50 between the two strains. the data and methods presented here contribute to a fundamental basis for refining studies of aerosol delivery of particles into animal models for study of a variety of clinical subjects, such as infectious doses and vaccine delivery. further work will be needed to more precisely define the median infective dose (mid 50 ) of the current influenza strain in the cd-1 mouse, and to better understand the effect of bioaerosol ageing and dose rate on infectivity. work presented herein validates aerosolization of one organism and delivery by a pure respiratory pathway into one murine host as a technique for assaying infectivity in the challenging bioaerosol. this work can serve as a starting point for a continuation of work using other microbial organisms and other animal hosts. the intended application of the aerosol influenza animal model described here is the assessment of the clinical effect of respiratory protection devices incorporating antimicrobial treatments. various approaches have been proposed to increase the effectiveness of respiratory filtering media including the addition of bioactive media. although materials such as silver nanoparticles (lala et al. 2007) , copper oxide (borkow et al. 2010) , iodinated compounds (heimbuch and wander 2006) and others (cecchini et al. 2004) have shown biocidal potential, only the iodine vector has been proposed (lee et al. 2009 ) to operate by a noncontact mechanism. additional studies will focus on evaluating such new technologies and, after replacement of the filter holder with a larger enclosure able to collect aerosols behind a filtering facepiece respirator (ffr) worn by an articulated headform, on quantifying the effect on protectivity of seal leakage and on optimizing the particle removal efficiency of ffrs and other rpe to maximize net protectivity. the susceptibility of mice to the viruses of human and swine influenza methods of estimating the ld 50 in 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mice deposition of inhaled aerosols current protocols in microbiology; unit 15g.1 influenza: propagation, quantification, and storage reflection and reactiontransmission of influenza a in human beings review of aerosol transmission of influenza a virus questioning aerosol transmission of influenza: in response reflection and reaction-transmission of influenza a in human beings aerosol transmission of influenza a virus: a review of new studies on air-borne infection. study ii. droplets and droplet nuclei cdc protocol of realtime rtpcr for influenza a(h1n1) revision 2 inhalation exposure systems: design, methods, and operation concentrations and size distributions of airborne influenza a viruses measured indoors at a health centre, a day-care centre and on aeroplanes factors influencing the deposition of inhaled particles pilgrim's pride farms (live oak, fl) donated 100 embryonic eggs from stock-breeding hens, of which 80 were used for virus propagation and 20 served as controls. this work was supported in part by contract no. fa8650-07-c-5911, funded by the defense threat reduction agency. protocols for animal use were approved by the university of nebraska medical center institutional animal care and use committee and all active participants received all required training prior to the acquisition of mice. disclaimerpublication of this work does not indicate endorsement or approval of this work by the department of defence. key: cord-347039-eap592i7 authors: lee, seung-hwan; dimock, ken; gray, douglas a; beauchemin, nicole; holmes, kathryn v.; belouchi, majid; realson, john; vidal, silvia m. title: maneuvering for advantage: the genetics of mouse susceptibility to virus infection date: 2003-08-31 journal: trends in genetics doi: 10.1016/s0168-9525(03)00172-0 sha: doc_id: 347039 cord_uid: eap592i7 abstract genetic studies of host susceptibility to infection contribute to our understanding of an organism's response to pathogens at the immunological, cellular, and molecular levels. in this review we describe how the study of host genetics in mouse models has helped our understanding of host defense mechanisms against viral infection, and how this knowledge can be extended to human infections. we focus especially on the innate mechanisms that function as the host's first line of defense against infection. we also discuss the main issues that confront this field, as well as its future. genetic studies of host susceptibility to infection contribute to our understanding of an organism's response to pathogens at the immunological, cellular, and molecular levels. in this review we describe how the study of host genetics in mouse models has helped our understanding of host defense mechanisms against viral infection, and how this knowledge can be extended to human infections. we focus especially on the innate mechanisms that function as the host's first line of defense against infection. we also discuss the main issues that confront this field, as well as its future. viral infections in humans are notable for the diversity of host responses, rates of progression, and disease outcomes. a large body of evidence indicates that these differential responses depend not only on viral factors but also on inherited components affecting host susceptibility. significant advances in our understanding of the host response to infection in humans and other animal species have recently been achieved through the use of mouse models of infection. in laboratory mice, years of inbreeding have fixed deleterious alleles, which can be detected as susceptibility phenotypes. two major breakthroughs advanced this field. one was our ability to manipulate the mouse genome using transgenic and gene knockout technologies. this 'reverse genetics' approach allows direct testing of specific genes for their role in host resistance or susceptibility to infection. the other was our ability to clone genes responsible for natural variation in susceptibility to infection among inbred strains of mice, through molecular genetics. this 'forward genetics' approach can uncover novel mechanisms of host defense that are crucial to effective and protective responses to infection. to date more than 30 mouse loci (table 1 ) and many fewer human genes ( table 2 ) have been associated with the outcome of virus infection [1, 2] . each locus controls infection by a single virus family or strain. this is probably related to the vast diversity of virus life cycles and the likelihood that the product of each host resistance gene interacts with unique molecules encoded by individual viruses (fig. 1) . in this review we illustrate the contribution of mouse genetics to our understanding of mechanisms of host resistance to virus infection. these mechanisms might manifest themselves as: (1) barriers to infection at the host cell membrane; (2) intracellular host responses to infection; or (3) recognition or destruction of infected cells. viruses used as examples are described in table 3 . barriers to infection at the host cell membrane the first step in the life cycle of a virus is attachment to a receptor on the cell surface and delivery of the viral genome into the interior. usually the virus takes advantage of a host molecule with an unrelated function, such as an adhesion molecule or complement regulator, for its own benefit. receptors are recognized as important determinants of virus host range and tissue tropism; and some host resistance/susceptibility loci encode molecules that are expressed on the cell surface. for example, sjl mice are 10 000 times more resistant to a lethal dose of mouse hepatitis virus (mhv) -a murine coronavirus -than c57bl/6 or balb/c mice [3] . resistance is due to allelic variation in the gene encoding carcinoembryonic antigen-related cell adhesion molecule 1 (ceacam1) [4] . susceptible strains carry the ceacam1a allele, which encodes the principal mhv receptor, cea-cam1a. resistant strains, such as sjl, are homozygous for the ceacam1b allele, which encodes a 27-amino-acid substitution in the n-terminal virus-binding domain of ceacam1a [4] . an immunoreceptor tyrosine-based inhibitory motif (itim) (see glossary) is present in the cytoplasmic domain of ceacam1, also suggesting a potential immunomodulatory function for this molecule during mhv infection. although no role for human ceacam1a has been described during viral infection of humans [5] , a possible immunomodulatory function is consistent with the observation of a ceacam1-mediated suppression of cd4 þ t-cell activation during infection by the pathogenic bacteria neisseria gonorrhoeae, neisseria meningitidis and haemophilus influenzae. these human pathogens bind to domain 1 of ceacam1, very close to the site recognized by mhv [6] . another example of natural host resistance is the restriction of ecotropic murine leukemia virus (mulv) infection by the mouse fv4 gene. binding of retroviral env glycoproteins to cellular receptors triggers fusion of viral and cellular membranes, and allows the virus nucleocapsid to enter the cell. the fv4 (or akvr) locus is a defective endogenous ecotropic provirus that encodes an env protein, which also has a fusion defect [7, 8] . it has been proposed that the env protein encoded by fv4 blocks mulv infection by interacting with the cellular receptor and restricting the amount of receptor available for exogenous retrovirus attachment (box 1) [9] . in humans, resistance to human immunodeficiency virus (hiv) is also mediated by a barrier at the cell surface. cd4 is the primary or 'attachment' receptor for hiv, but cd4 is necessary but not sufficient for the productive entry of hiv into target cells. the identification of ccr5 as a coreceptor for hiv prompted genetic screening of individuals that escape hiv disease despite high-risk behavior [10] . individuals carrying a homozygous 32 bp deletion in the coding sequence of ccr5 (ccr5d32) are extremely resistant to the 'r5'strain of hiv because the deletion results in a frame shift and generates a nonfunctional receptor [10] . the ccr5d32 mutation is found predominantly in the caucasian population and is absent in africans, american indians and east asians [11] . it has been speculated that this distribution is consistent with resistance to an agent that predates hivand caused enormous mortality. one candidate is yersinia pestis, the causative agent of bubonic plague, although other pathogens targeting the macrophage/monocyte lineage cannot be excluded [12] . delivery of a viral genome to the interior of an infected host cell does not guarantee that an infection will be glossary advanced intercross lines : mouse lines generated by producing an f2 generation between two inbred strains and then intercrossing in each subsequent generation, but avoiding sibling matings. this provides increasing probability of tightly linked genes recombining. congenic strain : a mouse strain that has been bred to be identical to an inbred strain, except for a selected differential chromosomal segment. congenic strains are derived by backcrossing to a parental inbred strain for at least ten generations while selecting for heterozygosity at a particular locus. consomic strain : a variation on a congenic strain in which recombinants between two inbred strains are backcrossed to produce a strain that carries a single chromosome from one strain on the genetic background of the other. epistasis : the interaction between alleles at different loci that allows an allele at one locus to alter the effects of alleles at a different locus. immunoreceptor tyrosine-based activation motif (itam) : a cytoplasmic tyrosine-containing motif that is the site of tyrosine phosphorylation. these motifs are associated with tyrosine kinases and other phosphotyrosinebinding proteins involved in cellular activation. immunoreceptor tyrosine-based inhibitory motif (itim) : a cytoplasmic tyrosine-containing motif. in contrast to itams, phosphorylation of the tyrosine residues within itims recruits the src-homology-2-domain-containing tyrosine phosphatase shp-1 and/or shp-2, transducing a negative signal that inhibits cellular activation. interferons (ifns) : a group of immunoregulatory proteins that are produced by cells infected with a virus and have the ability to inhibit viral growth. ifns are classified as type i (ifn-a/b), which have antiviral properties, and as type ii (ifn-g), which is known as immune interferon. despite the use of different receptors, certain ifn-a/b and ifn-g functions are shared because the signal transduction pathways activated through these receptors partly overlap. murine leukemia virus (mulv) : there are four subgroups of naturally occurring mulvs based on receptor usage on mouse cells: ecotropic mulvs, which are able to infect only mouse cells utilizing the cationic amino acid transporter as a receptor; amphotropic mulvs, which are able to infect mouse cells as well as those of other species (including human) by binding to an inorganic phosphate transporter protein; polytropic mulvs, which can infect cells from mouse as well as nonmurine species using yet another cellular receptor protein, thought to be a g-protein-coupled receptor; and xenotropic mulvs, which use receptors from cells of most species except mice. quantitative trait loci (qtls) : the locations of genes that affect a trait that is measured on a quantitative (linear) scale, such as body weight or blood pressure in animals, as identified by statistical analysis. these traits are typically affected by more than one gene, and also by the environment. recombinant congenic strain : a variation on recombinant inbred strains in which the initial outcross is followed by several generations of backcrossing before inbreeding. retroviral interference : the phenomenon by which prior infection of cells with a retrovirus confers strong resistance to infection of the same cells by a retrovirus that utilizes the same receptor; however, cells remain susceptible to viruses that use a different receptor. this process results from masking or downregulation of the receptor due to interaction with the glycoprotein of the established virus. an important element of resistance to retroviral infection is provided by endogenous retroviral elements. some retroviral elements in the mammalian genome are transcriptionally active but carry deletions and point mutations that render them unable to replicate. some of their gene products, such as the fv4 and fv1 proteins, interfere with infection by their exogenous relatives thus providing a selective advantage to their hosts. for example, fv4 encodes an env protein that interferes with ecotropic murine leukemia virus (mulv) infection (see main text) at the level of viral entry. examples of this phenomenon can be found in mice, chickens and cats, suggesting that env-mediated retroviral interference is commonly used for limiting virus spread [76] . gene therapy based on the principle of receptor interference has demonstrated that introduction of fv4-envtransduced bone marrow cells into a thymectomized host confers resistance to friend leukemia virus-induced leukemogenesis [77] . these findings suggest that a similar approach could be used as a therapeutic strategy to inhibit infections by other retroviruses in vivo, including immunodeficiency virus. for example, transfer of a gene encoding a normally processed but fusion-defective retroviral env protein into susceptible cells would interfere with viral entry and potentially reduce the infectiousness of viruses emerging from the cell. the fv1 locus [78, 79] encodes a retrovirus capsid-like protein [80] that restricts mulv infection at a post-entry stage, before integration of the provirus [81] . because all retroviruses replicate in very similar ways, it is conceivable that similar restriction mechanisms could be found in humans. interestingly, although an fv1 ortholog was not detected in nonmurine species, a mechanism of preintegration interference, resistance factor 1 (ref1), has been demonstrated in human cell lines [82] . at the time of writing, the ref1 gene has not been cloned, but there is speculation that, like fv1, ref1 might consist of endogenous retroviral sequences. a recent study suggests the existence of a ref1-like restriction in humans and nonhuman primates that determines lentivirus tropism [83] . consistent with this proposal, the target for this restriction is within the capsid protein of hiv [83] . an example of an alternative mechanism of resistance to retroviral infection is provided by fv2, which determines the progression of friend virus complex-induced erythroleukemia. fv2 is identical to ron, which encodes stk, a member of the met family of receptor tyrosine kinases. susceptible mice express a positive-acting truncated version of stk lacking most of the extracellular domain, which is associated with proliferation of sffv-infected erythroblasts [84] . successful, because potent defense mechanisms act at the intracellular level. one of the initial cellular responses to viral infection is the production of type i interferons (ifns). ifns, in turn, stimulate the expression of several gene products, including the double-stranded rna-activated protein kinase (pkr) and rnase l, which leads to the establishment of an antiviral state in cells, characterized by a general inhibition of protein synthesis. the ifn-induced mx1 protein is one of the best-studied determinants of innate immune responses to viral infection. the inbred mouse strain a2g is resistant to doses of mouse-adapted influenza virus (an orthomyxovirus) that are lethal for other inbred strains [13] . resistance in these mice is determined by a single dominant locus, mx1, whose gene product, mx1, rapidly accumulates in the nuclei of cells following influenza virus infection, and blocks virus spread [14] . susceptible mice produce no functional mx protein due to either a nonsense mutation (cba/j) or gene deletion (balb/c) [15] . the mx1 product belongs to the dynamin superfamily of large gtpases conserved in all vertebrates. in mice there are actually two mx gene products, mx1 and mx2. mx1 protein blocks transcription of influenza virus, probably via interaction with the pb2 subunit of the influenza virus polymerase [16] . by contrast, the mx2 protein is cytoplasmic and has been shown to inhibit viruses that replicate in the cytoplasm, such as vesicular stomatitis virus (vsv) and members of the family bunyaviridae [14] . mx proteins inhibit virus replication by interfering with the transport of viral nucleocapsids, and by sorting them to locations where they become unavailable for the generation of new virus particles [14] , possibly to promyelocytic leukemia protein nuclear bodies (pml nbs) [17] , which are known to be a site of proteasome-mediated degradation. humans also possess mx genes, mxa and mxb, but only the mxa gene product has antiviral properties. however, in contrast to its mouse mx1 ortholog, the mxa gtpase accumulates in the cytoplasm of ifn-treated cells, where it inhibits not only the replication of orthomyxoviruses, vsv and bunyaviruses but also the replication of other rna viruses such as measles virus, coxsackievirus b4 and semliki forest virus [14] . a mechanism of resistance to flaviviruses, which also appears to be ifn-mediated, was recently revealed by the cloning of flv, which confers resistance (flv r ) or susceptibility (flv s ) to flavivirus infection. virus titers in the brains of resistant mice infected with murray valley encephalitis virus, yellow fever virus or west nile virus are orders of magnitude lower than in susceptible animals. viral yields in tissue cultured from resistant or susceptible animals are also dramatically different, indicating that the flv gene product acts intracellularly on flavivirus replication [18] . genetic mapping localized flv1 to chromosome 5 [19] and, more recently, it was demonstrated that flv susceptibility is associated with a nonsense mutation in a member of the 2 0 ,5 0 -oligoadenylate synthetase (oas) family, oas1b [20, 21] . oas proteins are induced by ifns and play a central role in producing the antiviral state by binding double-stranded rna and catalyzing the synthesis of 2 0 ,5 0 -oligoadenylates (2-5a), which, in turn, interact with and activate rnase l to degrade singlestranded viral and cellular rnas. whereas oas1b genes from resistant mice encode full-length proteins, those from susceptible mice encode proteins truncated at the c-terminus that might not form active synthetases. in contrast to the single oas1 gene found in humans, there are eight closely linked oas1 genes in the murine genome [20, 22] , but only oas1b is associated with resistance to flaviviruses. in addition to resistance imparted by barriers to virus entry or by intracellular antiviral defense mechanisms, cytotoxic cells can control the level of viral replication in an animal (box 2). host recognition of virus-infected cells is mediated by two players: natural killer (nk) cells and cytotoxic t cells. herpesviruses avoid recognition and activation of the adaptive immune system by downregulating major [24] . strains of the c57bl background carry a dominant resistance allele, cmv1 r , that restricts viral replication in target organs, whereas other mouse strains carry a recessive susceptibility allele, cmv1 s , that allows rapid proliferation of the virus [25] . cmv1 r encodes an activating nk-cell receptor, ly49h [26 -28] , which is absent in susceptible strains. mouse strains express different repertoires of ly49 molecules, which are part of a large family of polymorphic receptors expressed on overlapping populations of nk cells. upon binding of mhc class i ligands, different ly49 molecules deliver either activating or inhibitory signals that modulate nk-cell function [29] . recent studies have revealed that ly49h recognizes mcmv-infected cells via direct interaction with the viral antigen, m157, which shares structural homology with mhc class i molecules and is expressed on the surface of infected cells [30, 31] . m157 also binds to an inhibitory receptor, ly49i, expressed in susceptible strain 129 mice, suggesting that m157 could have evolved as a mechanism to escape nk-cell killing by targeting inhibitory receptors [30] . in resistant mice, ly49h might have evolved as a countermeasure against virus-encoded mhc class i homologs, providing an overriding activating signal to the nk cell, and promoting the elimination of mcmv-infected cells (fig. 2a) . considering the prevalence of human cytomegalovirus (hcmv) in the human population, we expect that there are likely to be hcmv-specific activating receptors present on human leukocytes that might function in a manner analogous to ly49h. the mhc (box 2) is a set of genes, present in all vertebrates, with immunological and nonimmunological functions. mhc class i genes play a crucial role in in vertebrates, host defense against virus infection is mediated by innate and adaptive immunity. innate immunity constitutes the first line of defense, providing a rapid response through germ-line encoded proteins that pre-exist or are induced within hours of infection. adaptive immunity is a slower, yet highly specific response mediated by b cells and t cells that confers effective and long-lasting protection against infection, and is characterized by immunological memory. major histocompatibility complex (mhc) molecules are highly polymorphic glycoproteins encoded by mhc class i and ii genes, which are mainly involved in the presentation of peptide antigens to t cells. mhc class i molecules bind peptides derived from proteins synthesized in the cytosol, such as viral proteins, and present them to cytotoxic cd8 þ t cells. by contrast, mhc class ii molecules are loaded with peptides derived from exogenous antigens engulfed within intracellular vesicles, for presentation to cd4 þ t cells. mhc class i molecules also play an important role in modulating the cytotoxic activity of natural killer (nk) cells. nk cells are a component of the innate system, so named because of their propensity to kill certain neoplastic and virus-infected cells without prior sensitization. the cytotoxic activity of nk cells is regulated by signals elicited by inhibitory receptors containing immunoreceptor tyrosine-based inhibitory motifs (itims), or stimulatory receptors associated with immunoreceptor tyrosine-based activation motifs (itam)-bearing adaptor molecules. inhibitory receptors interact with mhc class i molecules and prevent cell killing of healthy cells by autologous nk cells. in situations where mhc class i is downregulated, such as during tumorigenesis or viral infection, reduced inhibitory signals result in nk-cell-mediated lysis [85] . activating nk-cell receptors recognize stress-induced or pathogenencoded mhc class i-like proteins and stimulate killing of cells expressing these molecules [85] . review trends in genetics vol. 19 no.8 august 2003 combating viral infection in mice (fig. 3a) . congenic mice with intra-mhc recombinations provide an important tool for dissecting the contribution of mhc class i subregions to virus infection. for example, comparisons of t-cell responses in h2 congenic mice that differ in their recovery from friend leukemia virus infection were used to localize friend leukemia virus resistance loci rfv1 and rfv2. rfv1 mapped to the h2d subregion and determined t-cell activation. rfv2 mapped to the ia subregion and determined unresponsiveness of t cells in a proliferation assay [35] . resistance to acute lethal infection of mcmv is also controlled by genes linked to h2, with the k haplotype being more resistant than b or d. resistance was associated with genes of subregions k/ia and d. interestingly in this model, transfection of macrophages with sequences encoding mhc molecules such as h2-k d , d d or k b renders these cells, which are major reservoirs for mcmv, sensitive to mcmv infection [37] , consistent with a role for mhc molecules as mcmv receptors. in humans, association analysis between mhc and specific viral infections has also suggested a differential role of specific alleles in susceptibility to hiv, hepatitis b virus (hbv) and hepatitis c virus (hcv) (fig. 3b) . for example, hla-drb1*1302 is associated with resistance to chronic hbv infection in both african and european populations [42, 43] whereas hla-b*35-cw*04 is associated with the rapid progression of aids in caucasian populations [38] . although polymorphisms in the classical mhc class i and class ii genes are known to be related to antigen presentation, it is important to note that there are other genes within the mhc class iii region, such as components of the complement cascade (c2, c4), cytokines -tumor necrosis factor (tnf)-a and -b -and proteins involved in antigen processing (lmp2, tap1), which also have an important function in immunity [51] . therefore, disease resistance or susceptibility that maps to the mhc must be interpreted with caution, to differentiate the role of antigen presentation from the other roles of mhcassociated genes. although advances in genomic analysis have paralleled the discovery of host susceptibility traits that are determined by single alleles, most differences in host susceptibility to viral infection are the result of interactions among different genes with multiple alleles. the study of infectious disease under complex genetic control in humans is complicated by a variety of factors including genetic heterogeneity, low penetrance and environmental factors. the tools currently available to the mouse geneticist make the identification and isolation of disease susceptibility genes much more attainable. an example is the response to theiler's murine encephalomyelitis virus (tmev), a rodent model for multiple sclerosis [52] . in genetically susceptible mice, certain tmev strains cause persistent infection, inflammation, and a chronic demyelinating disease. one of the loci determining theiler's murine encephalomyelitis virus persistence (tmevp1), maps to the mhc region and corresponds to the h2d gene [52] . mice transgenic for the h2d b allele acquired resistance to persistent virus infection, suggesting that the gene effect was at the level of peptide presentation by h2d b [53] . using the amount of viral rna in the spinal cords of persistently infected mice as a phenotype, and a genetic dissection strategy based on genome scans, a second locus, tmevp2, was localized near the type ii interferon (ifn-g) gene on distal chromosome 10 [54] . analysis of a panel of congenic mice that inherited different portions of mouse chromosome 10 revealed that tmevp2 was actually two distinct loci, now termed tmevp2 and tmevp3, and excluded the ifn-g gene from the candidate region [55] . recently, a strong candidate for tmevp3, named tmevpg1, has been identified as a noncoding rna expressed in immune cells infiltrating the central nervous system of resistant mice, where an inverse relationship with ifn-g mrna levels has been observed [56] , suggesting a role for this rna in regulating ifn-g synthesis. consistent with this observation, the same reciprocal pattern of rna expression was observed for tmevpg1, the human counterpart, and ifn-g in human nk cells and t cells. several complementary avenues promise significant acceleration in the identification of genes that determine resistance or susceptibility in complex models of virus infection. the well-defined sets of recombinant congenic strains (rcs) [57] , consomic (chromosome substitution) strains [58] and advanced intercrossed lines [59] that are now available can be used to map a locus of interest and to characterize the specific phenotypic component of disease susceptibility under genetic control. also, traditional genetic mapping techniques are now complemented by high-throughput methods for studying gene function and regulation. for example, high-density oligonucleotide arrays [60] or cdna arrays [61] allow for gene expression monitoring on a genome-wide scale and offer an opportunity to establish functional links between genotype and phenotype. a strategy that combines congenic mapping with microarray expression profiling would aid in the identification of novel susceptibility genes and biochemical pathways not previously known to be involved in disease etiology. furthermore, the availability of the human and mouse genome sequences represents the ultimate breakthrough in quantitative trait loci (qtl) studies by placing the identification of candidate genes within a defined genetic interval only a (computer) mouse click away. one of the current limitations of using inbred strains of mice to study susceptibility to viral infection is the limited extent of genetic variation, mainly due to a small number of original progenitor strains [62] . therefore, although these resources are valuable, they do not permit the identification of all possible resistance or susceptibility loci. wild-derived inbred strains of mice are an important source of additional resistance alleles [63] that is just beginning to be exploited, as demonstrated by the characterization of flv. chemical mutagenesis, a method that has been successfully applied to the study of other model organisms, from bacteria to drosophila, is another promising strategy for creating novel susceptibility alleles in the mouse. the alkylating agent n-ethyl-n-nitrosourea (enu) introduces point mutations and creates random variation throughout the genome [64] . this is highly advantageous for defining host susceptibility phenotypes because it represents an unbiased method for identifying previously unrecognized physiological pathways because no assumptions are made about the relevant genes. enu mutagenesis yields models of simple inheritance that are readily accessible to analysis by positional cloning [64] . the effort to understand the genetic basis of susceptibility to viral disease is driven by three considerations: (1) the increased public awareness of the toll imposed by viruses on the host; (2) the increase in susceptible human populations because of longer life expectancy, frequently accompanied by chronic illness, and the consequences of advances in medical technology, including immunosuppressive therapies for organ transplantation or treatment of malignancy; and (3) the need to develop new therapies for infections caused by multidrug-resistant human killer-cell immunoglobulin-type receptor (kir) is considered to be a functional homolog of mouse ly49. an epistatic interaction between kir3ds1 and hla-b delays progression to aids, suggesting that hla-b behaves as a ligand for kir3ds1 [32] . the peptide presented on hla-b that is responsible for this interaction remains to be identified. because kir3ds1 receptor is also associated with the adaptor molecule dap12, the intracellular signaling cascade leading to cellular activation and killing of virus-infected cells seems to be similar to that triggered by mouse ly49h. by using inhibitory small molecules, represent promising approaches to antiviral therapy. therapies based on the principle of receptor interference for viruses other than retroviruses (box 1) could be explored. type i ifn induces an intracellular antiviral response against many different rna and dna viruses. mouse genetics has provided a starting point for dissecting intracellular mechanisms of host defense, and has revealed that several ifn-inducible gene products have inhibitory effects on a much more restricted number of viruses than expected. the precise nature (sequence or structural) of determinants recognized by effector proteins such as mx or oas1b remain to be characterized. the identification of additional effector molecules and viral targets must continue to be an important area of active research, particularly in view of the existence of orthologous human genes. mouse genetics has also demonstrated that recognition and destruction of virus-infected cells by nk cells is mediated by specific interactions between activating nkcell receptors and viral target molecules. viruses pose a particular problem for specific recognition because all the components of the virus are synthesized and assembled in the host cell. important questions to be answered include: (1) do other activating nk-cell receptors have specialized functions in virus recognition? (2) is the mhc class i fold a recognition-associated structure? (3) what is the extent of the repertoire of target molecules for nk-cell-activating receptors? in addition, studies of mouse nk-cell receptors, such as the ly49 family, have provided a conceptual framework for understanding human nk-cell biology. because no functional human ly49 counterpart has been identified, prime candidates for the recognition of viral pathogens by human nk cells are members of the killercell immunoglobulin-like receptor (kir) family [65] . although the ligands of activating kir receptors remain to be identified, it has been proposed that activating kir molecules have evolved to recognize infectious agents (fig. 2b) [32] . it is suspected that several common and/or chronic human diseases under complex genetic control are triggered by a viral infection. this idea is supported by experimental evidence derived from mouse models for initiation and exacerbation of atherosclerosis following mcmv infection [66] , for diabetes [67] or dilated cardiomyopathy [68] following coxsackievirus infection, and for multiple sclerosis-like disease following tmev [33] or mhv [69] infection. identifying genes that control susceptibility to acute and chronic viral infection in these models is a crucial step in understanding the development of these complex pathologies. comparisons between mouse and human mechanisms of host resistance or susceptibility to viral infection have increased our awareness not only of their differences but also, more importantly, of their similarities. in the future, the use of comparative genomic approaches in animal model systems will provide more comprehensive knowledge of the impact of viruses on human health. forward genetics of infectious diseases: immunological impact genetics of susceptibility to human infectious disease resistance to fatal central nervous system disease by mouse hepatitis virus strain jhm. i. genetic analysis specificity of coronavirus/ receptor interactions human aminopeptidase n is a receptor for human coronavirus 229e crystal structure of murine sceacam1a[1,4]: a coronavirus receptor in the cea family molecular characterization of the akvr-1 restriction gene: a defective endogenous retrovirus-borne gene identical to fv-4r fv-4 resistance gene: a truncated endogenous murine leukemia virus with ecotropic interference properties fv-4: identification of the defect in env and the mechanism of resistance to ecotropic murine leukemia virus chemokine receptors as hiv-1 coreceptors: roles in viral entry, tropism, and disease global distribution of the ccr5 gene 32-basepair deletion dating the origin of the ccr5-delta32 aids-resistance allele by the coalescence of haplotypes resistance of mice to mouse adapted influenza a virus interferon-induced mx proteins: dynamin-like gtpases with antiviral activity influenza virus-susceptible mice carry mx genes with a large deletion or a nonsense mutation function of the mouse mx1 protein is inhibited by overexpression of the pb2 protein of influenza virus interferon-induced antiviral mx1 gtpase is associated with components of the sumo-1 system and promyelocytic leukemia protein nuclear bodies genetically determined resistance to infection with group b arboviruses. ii. increased production of interfering particles in cell cultures from resistant mice development and characterization of new flavivirus-resistant mouse strains bearing flv(r)-like and flv(mr) alleles from wild or wild-derived mice positional cloning of the murine flavivirus resistance gene a nonsense mutation in the gene encoding 2 0 -5 0 -oligoadenylate synthetase/l1 isoform is associated with west nile virus susceptibility in laboratory mice gene structure of the murine 2 0 -5 0 -oligoadenylate synthetase family selective rejection of h-2-deficient lymphoma variants suggests alternative immune defence strategy inhibition of natural killer cells by a cytomegalovirus mhc class i homologue in vivo cmv-1, a genetic locus that controls murine cytomegalovirus replication in the spleen susceptibility to mouse cytomegalovirus is associated with deletion of an activating natural killer cell receptor of the c-type lectin superfamily vital involvement of a natural killer cell activation receptor in resistance to viral infection murine cytomegalovirus is regulated by a discrete subset of natural killer cells reactive with monoclonal antibody to ly49h the ever-expanding ly49 gene family: repertoire and signaling direct recognition of cytomegalovirus by activating and inhibitory nk cell receptors recognition of a virus-encoded ligand by a natural killer cell activation receptor epistatic interaction between kir3ds1 and hla-b delays the progression to aids genetics of susceptibility to theiler's virus infection serial backcross analysis of genetic resistance to mousepox, using marker loci for rmp-2 and rmp-3 h-2d control of recovery from friend virus leukemia: h-2d region influences the kinetics of the t lymphocyte response to friend virus genetic control of sensitivity to moloney leukemia virus in mice. iii. the three h-2 linked rmv genes are immune response genes controlling the antiviral antibody response are mhc proteins cellular receptors for cmv? hla and hiv-1: heterozygote advantage and b*35-cw*04 disadvantage influence of combinations of human major histocompatibility complex genes on the course of hiv-1 infection hla alleles determine human t-lymphotropic virus-i (htlv-i) proviral load and the risk of htlv-i-associated myelopathy a tumor necrosis factor-alpha (tnf-alpha) promoter polymorphism is associated with chronic hepatitis b infection hla-drb1*1301 and *1302 protect against chronic hepatitis b association between an mhc class ii allele and clearance of hepatitis b virus in the gambia class ii hla alleles and hepatitis b virus persistence in african americans influence of mhc class ii genotype on outcome of infection with hepatitis c virus. the hencore group. hepatitis c european network for cooperative research association between mhc class ii alleles and clearance of circulating hepatitis c virus. members of the trent hepatitis c virus study group genes of the major histocompatibility complex class ii influence the outcome of hepatitis c virus infection association between hla class ii genotype and spontaneous clearance of hepatitis c viraemia influence of hla haplotypes on the clinical courses of individuals infected with hepatitis c virus hla drb1 and dqb1 alleles and haplotypes influencing the progression of hepatitis c complete sequencing and gene map of a human major histocompatibility complex (mhc) the infection of mouse by theiler's virus: from genetics to immunology fvb mice transgenic for the h-2db gene become resistant to persistent infection by theiler's virus mapping loci influencing the persistence of theiler's virus in the murine central nervous system two loci, tmevp2 and tmevp3, located on the telomeric region of chromosome 10, control the persistence of theiler's virus in the central nervous system of mice tmevpg1, a candidate gene for the control of theiler's virus persistence, could be implicated in the regulation of gamma interferon recombinant congenic strains derived from a/j and c57bl/6j: a tool for genetic dissection of complex traits analysing complex genetic traits with chromosome substitution strains simultaneous detection and fine mapping of quantitative trait loci in mice using heterogeneous stocks high density synthetic oligonucleotide arrays quantitative monitoring of gene expression patterns with a complementary dna microarray the mosaic structure of variation in the laboratory mouse genome wild mice: an ever-increasing contribution to a popular mammalian model enu mutagenesis: analyzing gene function in mice divergent and convergent evolution of nk-cell receptors does cytomegalovirus play a role in atherosclerosis coxsackieviruses and diabetes the transition from viral to autoimmune myocarditis mouse hepatitis virus infection of the central nervous system: chemokine-mediated regulation of host defense and disease susceptibility to hiv infection and progression of aids in relation to variant alleles of mannose-binding lectin genetic restriction of hiv-1 pathogenesis to aids by promoter alleles of il10 mutation of gene of mannose-binding protein associated with chronic hepatitis b viral infection tuberculosis and chronic hepatitis b virus infection in africans and variation in the vitamin d receptor gene host response to ebv infection in x-linked lymphoproliferative disease results from mutations in an sh2-domain encoding gene the x-linked lymphoproliferative-disease gene product sap regulates signals induced through the co-receptor slam endogenous retroviruses and the evolution of resistance to retroviral infection a gene therapy model for retrovirus-induced disease with a viral env gene: expression-dependent resistance in immunosuppressed hosts susceptibility to two strains of friend leukemia virus in mice inheritance of susceptibility to friend mouse leukemia virus. 11. spleen foci method applied to test the susceptibility of crossbred progeny between a sensitive and a resistant strain positional cloning of the mouse retrovirus restriction gene fv1 physical mapping of the fv-1 tropism host range determinant of balb/c murine leukemia viruses a conserved mechanism of retrovirus restriction in mammals cellular inhibitors with fv1-like activity restrict human and simian immunodeficiency virus tropism fv2 encodes a truncated form of the stk receptor tyrosine kinase natural killer cells, viruses and cancer we are grateful to christine di donato for critical reading of the manuscript. our laboratories are supported by grants from the canadian institutes of health research (cihr) and natural sciences and engineering research council of canada (nserc). seung-hwan lee is supported by a cihr doctoral scholarship and silvia m. vidal is a junior scientist of cihr. key: cord-003389-0yh5k6jk authors: patton, john b.; bennuru, sasisekhar; eberhard, mark l.; hess, jessica a.; torigian, april; lustigman, sara; nutman, thomas b.; abraham, david title: development of onchocerca volvulus in humanized nsg mice and detection of parasite biomarkers in urine and serum date: 2018-12-12 journal: plos negl trop dis doi: 10.1371/journal.pntd.0006977 sha: doc_id: 3389 cord_uid: 0yh5k6jk background: the study of onchocerca volvulus has been limited by its host range, with only humans and non-human primates shown to be susceptible to the full life cycle infection. small animal models that support the development of adult parasites have not been identified. methodology/principal findings: we hypothesized that highly immunodeficient nsg mice would support the survival and maturation of o. volvulus and alteration of the host microenvironment through the addition of various human cells and tissues would further enhance the level of parasite maturation. nsg mice were humanized with: (1) umbilical cord derived cd34(+) stem cells, (2) fetal derived liver, thymus and cd34(+) stem cells or (3) primary human skeletal muscle cells. nsg and humanized nsg mice were infected with 100 o. volvulus infective larvae (l3) for 4 to 12 weeks. when necropsies of infected animals were performed, it was observed that parasites survived and developed throughout the infection time course. in each of the different humanized mouse models, worms matured from l3 to advanced fourth stage larvae, with both male and female organ development. in addition, worms increased in length by up to 4-fold. serum and urine, collected from humanized mice for identification of potential biomarkers of infection, allowed for the identification of 10 o. volvulus-derived proteins found specifically in either the urine or the serum of the humanized o. volvulus-infected nsg mice. conclusions/significance: the newly identified mouse models for onchocerciasis will enable the development of o. volvulus specific biomarkers, screening for new therapeutic approaches and potentially studying the human immune response to infection with o. volvulus. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 onchocerciasis, caused by the parasitic filarial nematode onchocerca volvulus, remains a significant source of morbidity throughout sub-saharan africa [1] . o. volvulus infection is commonly diagnosed through the presence of microfilariae in skin snips, but skin samples can be further analyzed by qpcr for enhanced sensitivity [2] . antibody tests (e.g. ov16 [3, 4] ) are available but do not have the ability to differentiate between past and present infections which is problematic in areas where the infection is endemic [5] . recently, a limited number of biomarkers have been identified in the urine that can distinguish between o. volvulus-infected and non-infected individuals [6] [7] [8] . a significant obstacle for studying the biology of o. volvulus and for the development of new therapeutics and diagnostics has been the absence of small animal models. the only susceptible animal hosts for o. volvulus are chimpanzees [9, 10] and mangabey monkeys [11] . chimpanzees infected with o. volvulus had patent infections that lasted between 6 to 9 years with adult-worm bundles located in deep tissues with microfilariae in skin snips being detected 12-18 months post infection [12, 13] . while immunologically intact mice are resistant to infection with the infective larvae (l3) of o. volvulus [14] , adult worms within nodules have been successfully transplanted into scid mice (nod.cb17-prkdc scid /j) with the worms surviving greater than 20 weeks [15] . this observation was confirmed by the successful transplantation of adult onchocerca ochengi into scid mice [16] . as an alternative approach, o. volvulus l3 were implanted in primates and rodents within diffusion chambers that consist of a lucite ring enclosed with permeable membranes, allowing for migration of cells and other humoral factors into the diffusion chamber with simultaneous containment of the parasites. o. volvulus in diffusion chambers implanted in multiple species of rodents and primates showed limited growth of larvae in all host species [17] . several strategies have been employed to overcome murine resistance to infection with various human pathogens. the collaborative cross (cc) is a large group of inbred mouse strains that were developed to address many of the different shortcomings found within the existing experimental mouse populations, including small numbers of homozygous strains, limited genetic diversity, and non-ideal population structures. based on the hypothesis that there is a genetic basis for mouse susceptibility and resistance to infection, novel strains of cc mice have been identified that are susceptible to specific bacteria, viruses, and parasites of humans [18] [19] [20] [21] [22] [23] . as an alternative approach to overcome murine resistance to infection, mice have been developed with greatly diminished immune responses. scid mice were shown to be susceptible to infection with brugia malayi while immunocompetent mice were resistant to the infection [24] . nod.cg-prkdc scid il2rg tm1wjl /szj (nsg) mice are a highly immune-compromised strain of mice that have profound defects in the adaptive and innate immune responses [25] . the most notable defects include those in: 1) macrophages, dendritic cells and in the complement cascade [26] [27] [28] ; 2) maturation of t and b cells [29] ; 3) nk cells; 4) signaling of 6 different cytokines [30] and 5) the presence of eosinophils in the peripheral circulation and in tissue [31] . nod-rag1 tm1mom il2rg tm1wjl mice (nrg) are phenotypically similar to nsg mice with disruption in the b and t-cell production [32] . interestingly, nsg mice can support the complete lifecycle of the human nematode strongyloides stercoralis, whereas immunologically intact mice cannot [31] . a third strategy to enhance pathogen survival in mice has been to add human-derived cells required for survival and growth [33] . nsg mice have the unique ability to support several different xenografts including human hematopoietic stem cells (cd34 + stem cells) that allow for the development of an immature partially-functional human immune system [25, 27, 30, 34] . nod.cg-prkdc scid il2rg tm1wjl tg (cmv-il3, csf2, kitlg)1eav/mloyszj (sgm) mice have an additional three human genes il3, csf2, and kitlg under the cmv promoter to enhance the overall microenvironment for the development of human xenografts. this results in increased numbers of cd33 + myeloid cells, b-cells, t-cells, and hematopoietic stem cells [35] . humanized blt mice are nsg mice that received a xenograft of human cd34 + stem cells and a transplant of human fetal thymus and liver implanted under the kidney capsule, which results in the formation of a "human immune organ" [36] . control and potential elimination of onchocerciasis has been significantly impeded by the limited number of available drugs and the development of resistance to those therapies [37, 38] . in addition, biomarkers to assess the infection status of treated individuals or macrofilaricidal activity are sorely lacking [6] [7] [8] . one of the critical barriers blocking drug and biomarker development has been the absence of suitable small animal hosts for experimentation. hence, this study was focused on the development of a small animal model that would support the growth and maturation of o. volvulus and could serve to identify parasite specific biomarkers. to this end we tested multiple genetically defined mouse strains and xenografted (with human cells) immunodeficient mice to identify those microenvironments suitable for o. volvulus development. in so doing, we were able to develop convenient and tractable murine models that support the development of o. volvulus l3 into advanced larval stages. moreover, we were able to use these o. volvulus-infected animals to identify parasite-derived biomarkers measurable in both urine and serum. the parasite material was collected during the years 1994-1999 in the research facility at the tropical medicine research station, kumba, cameroon. the procedures used for the production of o. volvulus forest strain third-stage-larvae (l3) were approved by an nih accredited institutional review board of the medical research council kumba, cameroon (protocol 001). the protocol was reviewed and approved annually. l3 were collected from black flies (simulium damnosum) that were fed on consenting infected donors. after seven days the flies were dissected and the developed l3 were collected, cleaned and cryopreserved. the cryopreserved l3 were shipped to the new york blood center in liquid nitrogen and upon arrival in new york were stored in liquid nitrogen. all protocols using the l3 cryopreserved samples in this study were approved by the new york blood center's irb (protocol 321 and protocol 603-09). all l3 samples were anonymized. all experimental procedures in mice were performed in compliance with the ethical and regulatory standards set by the nih for animal experimentation. the animal use protocol (01469) was approved by the thomas jefferson university institutional animal care and use committee. the animal care and use protocol adhered to the "guide for the care and use of laboratory animals" published by the national research council, usa. cryopreserved l3 were prepared as previously described [39] [40] [41] . briefly, black flies (simulium damnosum) were fed on consenting donors infected with o. volvulus, and after 7 days the developed l3 were collected from dissected flies, cleaned, and cryopreserved in dimethyl sulfoxide and sucrose using biocool ii computerized freezing equipment (fts systems inc., stone ridge, ny) [42] . cryopreserved l3 were removed from liquid nitrogen storage and placed on dry ice for 15 minutes followed immediately by a 37˚water bath. the l3 were then washed 5 times in a 1:1 mixture of nctc-135 and iscove's modified dulbecco's medium (sigma, st. louis mo) supplemented with 100 u penicillin, 100 μg streptomycin (corning, tewksbury ma) 100 μg gentamicin and 30 μg chloramphenicol per ml (sigma). l3 isolated from different collection days were tested first for viability in diffusion chambers implanted in balb/byj for 21 days, as previously described [17] . batches of l3 with viabilities greater than 50% at 21 days post implantation were used in these studies. one hundred worms (except where noted) were then counted and loaded into 1 ml tuberculin syringes with 21 g needle for subcutaneous injection of the larvae into the nape of the neck. all mice were housed in micro-isolator boxes in a pathogen-free room at the laboratory animal science facility at thomas jefferson university (philadelphia, pa). collaborative cross (cc) mouse strains, cast/eij, il16680 (cc055/tauunc), au8052 (cc052/geniunc), au8049 (cc038/geniunc) and or13067 (cc003/unc), were purchased and imported from the systems genetics core facility of university of north carolina (unc-chapel hill). information about the cc strains can be found on the unc systems genetics website at http:// csbio.unc.edu/ccstatus/index.py. the mice were kept under temperature, humidity and light cycle-controlled conditions and fed autoclavable rodent chow and given water ad libitum. nod-scid il2rg null (nsg), nod-rag1 null il2rg null (nrg), and nod-scid il2rg null -3/ gm/sf (sgm) mice were obtained from the jackson laboratories (bar harbor, me). an nsg, nrg, and sgm mouse breeding colony was maintained in the laboratory animal science facility, at thomas jefferson university with breeding trios given acidified water and low fat 5k52 animal chow (labdiet, st. louis, mo). the following human cell types were individually transferred into nsg mice: (1) human keratinocytes (hacat) (atcc, manassas, va), (2) bovine embryo skeletal muscle cells (besm) in the initial experiments, mice were injected with 5×10 6 besm, hacat, lec, or huskmc cells subcutaneously weekly throughout the experiment. the frequency of injection was subsequently determined by in vivo imaging experiments. genes encoding green fluorescent protein (gfp) and luciferase were inserted into huskmc cells using lentiviral vectors following the manufacturer's recommendations (cell biolabs, inc, san diego, ca). cells expressing gfp were isolated using the gfp marker by fluorescence-activated cell sorting using a bd facs aria (bd biosciences, franklin lakes, nj). the isolated cells were grown in huskmc media as described above and 5×10 6 cells were injected subcutaneously into nsg mice. mice were injected with vivoglo (promega, madison, wi) and imaged following manufacturer's recommendations on an ivis lumina xr (promega). two approaches were used to create humanized mice containing multiple human-cell types [25, 36] . human umbilical cord blood was obtained through collaboration with thomas jefferson university hospital department of obstetrics and gynecology from full term natural deliveries. cd34 + stem cells were isolated from cord blood using magnetic assisted cell sorting (macs) and cryopreserved until use. sgm, nrg and nsg mice were humanized with cd34 + umbilical cord derived stem cells by intrahepatic injection of 5x10 5 cd34 + stem cells into 48-hour old pups that were irradiated with 1.5 gray. six weeks following injection of the stem cells, peripheral blood from the mice was screened for the presence of human cells and mice with counts greater than 600 human cd45 + hematopoietic cells per μl of whole blood were used for experimentation, following previously published protocols [31] . blt mice were purchased from the jackson laboratories or were prepared following previously established protocols [27] . briefly, nsg mice (4-to 6-week old) were implanted with 1 mm 3 sections of fetal thymus and liver (advanced biomedical resources, alameda, ca) under the kidney capsule. two weeks post-implantation the mice were treated with busulfan (sigma) (20 mg/kg ip) and were injected retro-orbitally with 5x10 5 cd34 + stem cells, isolated from the donor fetal liver using macs, (miltenyi biotec inc. auburn, ca). eight weeks following the stem cell xenograft the peripheral blood from the blt mice was screened for the presence of human cells. blt mice were screened and selected using the same protocol described above for the cd34 + cord blood mice. pcr screening for o. volvulus dna was performed on all the infected mouse tissues to identify the presence of current or past o. volvulus larvae in that location. mice were anesthetized and exsanguinated, and the internal organs were removed, and the skin was removed from the muscle. the muscle and skin were then divided into 100 different sections and individually frozen in 1.7 ml eppendorf tubes. dna was extracted from the tissue sections using the promega genomic dna kit a1125 following the manufacturer's directions. realtime pcr was performed using custom taqman probes (integrated dna technologies, coralville, ia) against the ov-150 [44] [45] [46] repeats and an abi onestep-plus (thermofisher). mice were necropsied following previously established protocols for the isolation of filarial worms from tissues [47, 48] . briefly, mice were anesthetized using isoflurane gas and exsanguinated. the head was removed from the body of the mouse and discarded. the remaining internal organs and skin were removed from the muscles, and the muscle was divided into upper and lower sections at the bottom of the rib cage. all portions of the mouse (muscle, skin, and all internal organs with the exception of the head) were soaked overnight in rpmi containing 10% fbs and with 100 u penicillin, 100 μg streptomycin (corning), and emerging parasites were then collected and enumerated. infected mice were evaluated using two criteria: 1) percent established, measured the proportion of mice in a group of infected animals from which live parasites were recovered; and 2) the geometric mean number of live worms recovered per mouse within the group. recovered worms were placed in boiling fixative consisting of 95% ethanol (deacon labs, king of prussia, pa) and 5% glycerol (fisher, fair lawn, nj). after allowing the alcohol to evaporate, glycerol was added, and the worms were transferred to glycerin jelly (gelatin 10 g, ddh 2 o 60.0 ml, glycerine 70.0 ml, phenol 1.0 ml). fixed worms were measured using an olympus szx16 dissecting scope connected to a dp26 camera (olympus, center valley, pa). cellsens dimensions software (olympus) was used to measure the length of the recovered worms. serum and urine were collected and frozen as terminal procedures during necropsy. serum was thawed on ice and 25 μl removed for processing from each mouse. sera from 4 mice from each group/strain were pooled for maximizing the protein identifications. abundant proteins were depleted using an affinity chromatography (mars-ms-3, agilent) according to the manufacturer's directions. urine was thawed on ice, centrifuged and then filtered through a 0.22 μm filter (corning). serum and urine samples were prepared for mass spectrometry by digestion using the filter-assisted sample preparation (fasp) method [49] . briefly, the samples brought to 1% sodium deoxycholate (sdc), 50 mm tris-hcl, ph 7.6, 3 mm dithiothreitol, sonicated briefly, and incubated in a thermo-mixer at 90 o c, 1,000 rpm for 20 min. samples were centrifuged to clarify and the supernatant was transferred to a passivated 30 kd mwco device (millipore, merck kgaa, darmstadt, germany) and centrifuged at 13,000g for 30 min. the remaining sample was buffer exchanged with 1% sdc, 100 mm tris-hcl, ph 7.6, then alkylated with 15 mm iodoacetamide. the sdc concentration was reduced to 0.1%. samples were digested using trypsin at an enzyme to substrate ratio of 1:100, overnight, at 37 o c in a thermo-mixer at 1,000 rpm. digested peptides were collected by centrifugation and the filter washed with 0.5 nacl to elute electrostatically bound peptides. digested peptides were desalted using reversed phase stop-and-go extraction tips [50] . peptides were eluted with 80% acetonitrile, 0.5% formic acid and lyophilized in a speedvac (thermo savant, holbrook, ny) to near dryness, approximately 1 h. each digestion mixture was analyzed by ultra-high performance liquid chromatography tandem mass spectrometry (uhplc-ms/ms). lc was performed using an easy-nlc 1000 uhplc system (thermo fisher scientific, waltham, ma). mobile phase a was 97.5% milliq water, 2% acetonitrile, 0.5% formic acid. mobile phase b was 99.5% acetonitrile, 0.5% formic acid. the 240 min lc gradient ran from 0% b to 35% b over 210 min, then to 80% b for the remaining 30 min. samples were loaded directly to the column. the column was 50 cm x 75 um i.d. and packed with 2 μm c18 media (thermo easy spray pepmap). the lc was interfaced to a quadrupole-orbitrap mass spectrometer (q-exactive, thermo fisher scientific, waltham, ma) via nano-electrospray ionization using a source with an integrated column heater (thermo easy spray, thermo fisher scientific, waltham, ma). the column was heated to 50˚c. an electrospray voltage of 2.2 kv was applied. the mass spectrometer was programmed to acquire, by data-dependent acquisition, tandem mass spectra from the top 10 ions in the full scan from 400-1200 m/z. dynamic exclusion was set to 15 s, singly-charged ions were excluded, isolation width was set to 1.6 da, full ms resolution to 70,000 and ms/ms resolution to 17,500. normalized collision energy was set to 25, automatic gain control to 2e5, max fill ms to 20 ms, max fill ms/ms to 60 ms and the underfill ratio to 0.1%. mass spectrometer raw data files were converted to mzml format using msconvert [51] . mgf files were generated from mzml using the peak picker hires tool, part of the openms framework [52] . all searches were performed on amazon web services-based cluster compute instances using the proteome cluster interface. detailed search parameters are printed in the search output xml files. briefly, all searches required 10 ppm precursor mass tolerance, 0.02 da fragment mass tolerance, strict tryptic cleavage, up to 2 missed cleavages, fixed modification of cysteine alkylation, variable modification of methionine oxidation and protein-level expectation value scores of 0.0001 or lower. proteome cluster builds species-and genus-specific protein sequence libraries from the most current uniprotkb distribution [53] . mgf files were searched using the most recent protein sequence libraries available from uniprotkb using x!tandem [54] and omssa [55] . xml output files were parsed and non-redundant protein sets determined using proteome cluster based on previously published rules [56] . ms1-based isotopoic features were detected and peptide peak areas were calculated using the featurefindercentroid tool, part of the openms framework [52] . proteins were required to have 1 or more unique peptides across the analyzed samples with e-value scores of 0.0001 or less. geometric means (gm) were used as measures of central tendency. data were analyzed for larval growth by multifactorial analysis of variance anova with post-hoc fisher's least significant difference (lsd) testing in systat v.11 (systat inc., evanstown, il, usa). probability values less than 0.05 were considered statistically significant. all experiments were performed a minimum of 2 times. to determine if there was an underlying genetic basis for the resistance of mice to infection with o. volvulus [14] , 5 mouse strains having a wide range of genetic diversity from the cc (cast/eij, il16680, au8052, au8049 and or13067) [57] were screened for their susceptibility to o. volvulus. five mice from each of the different strains were infected with 100 o. volvulus l3 and necropsied at 4-weeks post infection. none of the 5 strains of cc mice tested was susceptible to infection with o. volvulus l3. to assess the role of the mouse immune system in mediating resistance to o. volvulus, immunodeficient mouse strains were assessed for their susceptibility to infection with o. volvulus. in a preliminary set of studies, 250 o. volvulus l3 were injected into 2 nsg mice. after 4-weeks the skin and muscle from the mice was divided into 100 anatomically distinct sections and qpcr for o-150 was performed on extracted dna from each section. twenty-three of the sections were positive from, spatially widespread regions of the body. it was concluded that parasites survived in nsg mice and had the ability to migrate extensively. this indicated that all regions of the mice had to be inspected for the presence of parasites following infection. nsg mice were infected with 100 o. volvulus l3 and necropsied at 4-and 8-weeks following infection. infected mice had an established infection rate of 63% with a gm worm recovery of 2.0 (range 1 to 4) worms recovered at 4-weeks following infection. at 8-weeks, 75% of mice had an established infection, with a gm recovery of 1.4 (range 1 to 3) worms per mouse ( fig 1a) . the recovered worms were measured and found to be significantly increased in size (p<0.0001; min: 677 μm, max 843 μm, gm: 717 μm) at 4-weeks compared to l3. they also significantly increased in size between 4 and 8 weeks (p<0.0001) reaching a maximum of 1,085 μm (min: 700 μm, gm: 1034 μm) (fig 2) . table) . mice engrafted with lec had 20% established infection and gm of 4 worms recovered per mouse with a range of 1 to 4 worms per mouse (s1 table) . hacat cell engrafted mice had a 60% established infection and gm recovery 1.7 worms recovered per mouse with between 1 and 2 worms per mouse (s1 table) . huskmc cell engrafted mice had an 81% estab(fig 1b) . at each time point tested the parasites in huskmc-engrafted mice demonstrated continued parasite growth. at 4-weeks recovered parasites had gm lengths of 713 μm (min: 592 μm, max: 788 μm), at 8-weeks lengths of 751 μm (min: 642 μm, max: 1,081 μm), and at 12-weeks the gm length was 1,121 μm with a maximum length of 2,086 μm observed (min: 748 μm), representing a 4-fold increase in length over l3 (fig 2) . these growth rates represented significant changes between l3 and 4-week worms (p = 0.0010) and between 8-and 12-week worms (p<0.0001). sgm, nrg and nsg (hunsg) mice humanized with cd34 + umbilical cord derived stem cells were infected with 100 o. volvulus l3. each of these humanized mice had human hematopoietic lineage cells at a concentration greater than 600 cells per μl of blood. while a complete picture of the cell populations in these specific mice was not determined, based upon flow analysis during the screening process all humanized mice had both human b and t-cells present in their blood. at 4-weeks post-infection humanized sgm mice contained a gm of 2.4 (range 1-7) worms/mouse, humanized nrg mice had a gm of 1.7 (range 1 to 4) worms/ table) . hunsg at 4-weeks post-infection had a 69% established infection rate with a gm of 3.4 (range 1-14) worms/mouse, and at 8-weeks post infection hunsg mice had a 46% established infection rate and a gm of 2.6 (range 1-18) worms/mouse (fig 1c) . larval growth in hunsg mice was comparable to that seen in nsg and in nsg mice engrafted with huskmc (fig 2) . a significant increase in length was seen between the l3 and 4-week recovery 12 ). after 4-, 8-or 12-weeks the percent established (the proportion of mice in a group of infected animals from which live parasites were recovered) (fig 1a-1d ) and the geometric mean number of live worms recovered per mouse within the group was determined (fig 1e-1h) . https://doi.org/10.1371/journal.pntd.0006977.g001 humanized nsg (hunsg) mice: nsg mice that had received a human cd34 + stem cell transfer, (4) blt: nsg mice that had been engrafted with human fetal liver derived cd34 + stem cells and engrafted with fetal thymus and liver tissues. after 4, 8 or 12-weeks, animals were necropsied and worms were recovered and measured. solid colored bar is the geometric mean of the lengths of larvae recovered. solid black line is the geometric mean of the length of l3 recovered from black flies and dotted line is the 95 th confidence interval. � asterisk represents statistical difference, p value � 0.05, in length of larvae recovered from mice. complete statistical analyses for all groups are included on s2 blt mice humanized with fetal thymus, liver and cd34 + stem cells, which display an enhanced repertoire of the developing human cells [58] , were infected with 100 o. volvulus l3 and then necropsied at 4-and 8-weeks post infection. at 4-weeks post infection blt mice had an established infection rate of 77% with a gm of 4.8 (range 2-10) worms/mouse. at 8-weeks post infection blt mice had an established infection rate of 60% with a gm of 2.3 (range 2-3) worms/mouse (fig 1d) . growth of the parasites was equivalent to that seen in the nsg mice and the other humanized models with the maximum length reaching 1,080 μm at 4-weeks (min: 391 μm, gm: 623 μm) and 1,448 μm at 8-weeks (min: 730 μm, gm: 949 μm) (fig 2) . the overall growth was significant between the l3 and worms 4-weeks post infection (p<0.0001) and between worms recovered at the 4-and 8-week time points (p<0.0001). during necropsy, mice were sectioned into 4 groupings: upper muscles, lower muscles, skin, and the complete set of internal organs. no nodules were found in any of these tissues upon necropsy. worms were recovered from all four of the different tissue groupings with no apparent preference for any region of the animal in all of the mice tested. detailed morphological analyses focused on worms recovered from blt mice infected for 8-weeks and huskmc humanized mice infected for 12-weeks. although there were clear differences in lengths of these worms, their morphological characteristics were similar. no differences in the ratio of males and female worms recovered from the mice was observed, however it was noted that most of the longer worms were females. both the anterior and posterior ends in both sexes were bluntly rounded and only slightly tapered (fig 3a, 3b and 3f ). other than growth in length, the major change from l3 was development of the reproductive systems. in the l3, both the female and male systems are rudimentary genital primordia consisting of only several cells. in the 8-12-week old worms, the female ovejector had formed and had attached to the body wall. the ovejector was ovoid in shape, relatively large and filled the body cavity, and had a distinct lumen (fig 3c and 3d) . rudimentary cellular growth of the reproductive tubes was also evident (fig 3d) . in males, the testis, located at approximately mid-body, had become elongate in shape and had looped posteriorly to form a classic shepherd's crook (fig 3e) . in addition, the spicule pads were well developed and demarcated (fig 3f) but were still oval in shape and had not yet started to take on the shape of the spicules nor was there any evidence of cuticularization. these observations are consistent with parasite development into advanced fourth stage larvae (l4). global proteomic analyses were performed with serum and urine collected from blt mice infected for 8-weeks and huskmc humanized mice infected for 12-weeks. a total of 7,430 proteins were identified based on the spectral matching to a combined protein database of human, mouse, o. volvulus and its wolbachia (wov) endosymbiont. because of the ambiguity in distinguishing certain spectral matches for proteins commonly found in both humans and mice, these proteins were grouped as non-o. volvulus proteins (4,743 in serum, 2,836 in urine, s1 development of onchocerca volvulus in nsg mice serum and urine of the infected mice, the blt and huskmc mice had 5 proteins (ovoc115 56, ovoc835, ovoc10244, ovoc4009 and ovoc9087) in common in the serum and 5 (ovoc7220, ovoc4139, ovoc224, ovoc8249 and ovoc9267) in the urine (fig 4b) . almost all the proteins identified have been shown through rnaseq to be transcribed by various stages of the o. volvulus parasite (fig 4b) . the objective of this project was to identify small animal models that would support the development of black-fly derived o. volvulus l3 into advanced mammalian-adapted stages of the parasite. these small animal models are critically needed for identifying biomarkers released from the early stages of the infection and for screening potential new anthelmintics. published findings on the susceptibility of mice to infection with the l3 of o. volvulus suggest that mice are resistant to infection when the larvae were injected subcutaneously [14] . however, o. volvulus l3 have been shown to survive and develop in mice when implanted within diffusion chambers at rates comparable to those seen in susceptible primates [17] . genetic traits can play a significant role in the susceptibility of animals to infection as has been clearly demonstrated by the diversity of susceptibility of different mouse strains to infection with litomosoides sigmodontis [59] . the cc mouse project was developed to produce an extremely diverse set of inbred animals that could be used for mapping different genetic traits [57] . five different cc mouse strains, selected based on their diverse genetic backgrounds, were tested for susceptibility to o. volvulus and were completely resistant to the infection. this observation suggests that either mice are missing some integral factors required for parasite growth or the immune responses in mice are effective at eliminating the infection. the question remains as to why parasites are recovered live from diffusion chambers implanted in mice but not when injected into the tissues. there are several possible explanations including: (1) the diffusion chamber acts as a barrier from the immune response creating an immune privileged site, (2) the larvae within the diffusion chamber are blocked from migrating through the tissue releasing excretory and secretory products and thereby eliciting an immune reaction, or (3) the diffusion chamber attracts host components to the parasite microenvironment that are beneficial for parasite development. to test the hypothesis that mouse-intrinsic immune responses control o. volvulus infections, nsg mice that lack both functional innate and adaptive immune systems were infected with o. volvulus l3. advanced stages of the parasites were consistently recovered from the infected nsg mice, and parasites survived and developed over the 8-week time course into advanced l4. these findings demonstrate that the mouse immune response was capable of controlling infection with o. volvulus, with elements of the mouse immune response eliminating the infection in immunologically intact mice. many mechanisms have been described for innate immune control of nematode infections in mice [39, [60] [61] [62] [63] [64] [65] [66] [67] all or some of which may be effective against o. volvulus. interestingly, tissues and cells in nsg mice provide required factors for parasite development and based on pcr analyses, the larvae actively migrated far from the infection site. the present study did not identify the point at which o. volvulus ceased surviving and developing in nsg mice. studies with the human parasite s. stercoralis have demonstrated that the entire parasite life cycle will develop in nsg mice within 4-weeks [31] . given the significant difference in time that it takes for o. volvulus adults to develop (12-15 months in chimpanzees [9, 10] ) and their size as mature adults (females are 50 cm [68] ) it is unlikely that the entire o. volvulus life cycle, including mating and development of microfilariae, will occur in nsg mice. in vitro studies on the development of filarial worms including o. volvulus [69, 70] and brugia malayi [71] have demonstrated that host cells are needed in the culture wells to optimize parasite growth and development. furthermore, optimal development and survival of larval t. spiralis in mice requires the presence of mouse eosinophils [33] . it was thus hypothesized that adding human cells to the nsg mice, from the tissues that the parasites are normally found juxtaposed in humans, might provide additional required nutritional or developmental elements found in humans required for parasite development and survival. four different single cell xenografts were screened: huskmc, lec, hacat and besm. of these four cell lines, huskmc was found to support the highest average percent survival of the implanted worms and consistent infection rates over a 12-week time period (figs 1 and 2) as an alternative to adding single cell populations to the nsg mice, multipotential umbilical cord stem cells were transferred to the immunodeficient mice. nsg, nsg-sgm, and nrg mice, all of which lack functional immune responses, were humanized with cd34 + umbilical cord stem cells. the humanization of the various immunocompromised mouse strains resulted in the development of an immature human immune system. the developing immune system in these mice displays a t-independent response, limited antigen-specific igm responses, and the presence of multiple innate immune cells has been noted [25] . when humanized nsg, nsg-sgm, and nrg mice were infected with o. volvulus l3, higher gm parasite recoveries were observed when compared to nsg mice without any human cells but these enhancements were not significant. although nsg-sgm did have consistent infection rates with o. volvulus we had significant difficulties establishing reliable engraftment of human cells in this strain of mice. extending this concept further, blt mice which contain cd34 + stem cells in addition to fetal tissue were used. blt mice are known to develop a more mature version of a human immune system. limited t-dependent recognition is seen within these mice and better overall functionality of both the b and t-cells has been documented [36] . blt mice supported the highest average parasite recoveries of any mouse model tested at 4-weeks, although the average fell to be in line with the hunsg and huskmc models by 8-weeks post infection. the mean lengths of the parasites recovered from 4-and 8-week time points from the nsg mice were comparable to those recovered from blt mice. this suggests a link between the enhanced survival, may be related to the presence of the human immune cells, but the growth of the parasites that survive may not be related to the human cell byproducts. although cellular engraftments levels in blt and hunsg mice were not rechecked at the end of the experiment previously published data have shown that these animals reliably hold their engraftments for extended periods of time [72, 73] . the mouse models developed in this study offer a number of advantages over the models that currently exist. l3 implanted within diffusion chambers in both non-human primates and mice successfully molt into l4 but the lengths of the recovered parasites were significantly shorter than those recovered at 8 and 12 weeks post infection from the mice tested in this study [17] . while non-human primates can support the development of the parasites from l3 to microfilariae producing adults, cost and ethical considerations limit the utility of primates for drug discovery and antigen identification [10, 14] . nodules containing adult worms recovered from humans have been implanted into scid mice and the female worms survived and released microfilariae [15] . this method allows for identification of adult antigens and biomarkers, but lacks the intermediate l3 and l4 stages of development. finally, an alternative model using adult male o. ochengi implanted into scid mice has been developed for the identifying filaricides [16] , which may also be effective against o. volvulus. the nsg models developed in this study have the critical advantage of working with o. volvulus, thereby allowing species-specific screening of filaricides and identification of biomarkers. it was of interest to note that the immune response found in normal mice was highly efficient at eliminating the l3 of o. volvulus, but the human immune cells in nsg mice were supportive rather than destructive of the parasites. it is possible that the mouse immune cells were evolutionally adapted to eliminate the parasites, whereas the human cells were evolutionally adapted to support the parasites as observed in nature. a comparison between mouse and human immune reactions to the worms might yield important new insights into the etiology of resistance and susceptibility to infection with o. volvulus. the source of the l3 used in these studies was from parasites cryopreserved in liquid nitrogen. after defrosting, 100 individual parasites were selected, attempting to identify only the viable/undamaged l3. it is reasonable to predict that a percentage of larvae exiting from the mouth parts of a black fly have the potential to resume development in the human host and that the cryopreservation process damaged some of those worms. approximately 50 percent of the larvae recovered after cryopreservation survived in diffusion chambers implanted in mice, which may explain the overall number of worms recovered from the different mouse models in this study. while minor differences in the overall larval recovery levels were observed between individual batches of cryopreserved larvae, multiple batches were combined before implantation to help ensure a consistent viability level going into the animal hosts. even with the combination of multiple batches of larvae it cannot be ruled out that the overall viability of the injected larvae played a role in the observed recovery rates. the recovery rates of o. volvulus larvae after developing in nsg mice with or without human cells was in the same order of magnitude as that reported for the recovery of adult filarial worms, where infections were initiated by larvae recovered directly from the insect vector. these infections included brugia pahangi in cats [74] , brugia malayi in leaf-monkey [75] , and onchocerca ochengi in cattle [76] . in the final analysis, optimal parasite recovery was observed in mice humanized with huskmc (maximum of 10 worms), hunsg (maximum of 18 worms) and blt mice (maximum of 10 worms). it was clear that worms increased in size during the infection period with individual worms achieving up to 4 times the size of the original l3. growth of worms within a single mouse was not consistent and suggests that there is significant variability within the infecting larval population. it does verify, however, that humanized mice have the potential to support extended development of o. volvulus. both male and female worms grew in length and resumed their sexual development. although no cast cuticles were observed, it was evident from organ development that the parasites had molted into fourth-stage larvae. urine and serum was collected from humanized mice infected for 8-12-weeks with o. volvulus for the identification of biomarkers. infected huskmc mice or blt mice were selected for this analysis so the biomarkers identified would develop in the presence of human cells thereby potentially enhancing their specificity. several o. volvulus-specific peptides were identified in the serum and urine of blt and huskmc mice (s3 table) , however, no o. volvulusspecific proteins were found in both urine and serum from either mouse source. the most likely useful biomarkers were the proteins listed in fig 4b. though most of the proteins were identified by one or more unique peptide(s), among the proteins with unknown function (ovoc9087, ovoc835, ovoc224), ovoc9087 does not have orthologues in other filarial species and hence would likely be able to distinguish o. volvulus from other filarial infections. because of the expected low number and abundance of o. volvulus-specific protein identification in the serum and urine from any given mouse in the current system, mass spectrometry was carried out with pooled serum and urine samples from each group of mice. in conclusion, novel small-animal hosts, nsg mice, have been identified that support the survival and development of o. volvulus l3 into advanced l4 mammalian stages. humanized mice have also been shown to be effective at identifying biomarkers for early o. volvulus infections. it is anticipated that these small-animal hosts for o. volvulus will also be useful as part of the effort to identify new anthelminthic drugs. finally, the fact that o. volvulus survives and develops in nsg mice humanized with human immune cells may provide the opportunity to study the human immune response to early infection with o. volvulus in a small animal model. global, regional, and national comparative risk assessment of 79 behavioural, environmental and occupational, and metabolic risks or clusters of risks in 188 countries, 1990-2013: a systematic analysis for the global burden of disease study detection of onchocerca volvulus 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proteome analysis protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using stagetips a cross-platform toolkit for mass spectrometry and proteomics openms-an open-source software framework for mass spectrometry uniprot: a hub for protein information tandem: matching proteins with tandem mass spectra open mass spectrometry search algorithm masssieve: panning ms/ms peptide data for proteins the collaborative cross, a community resource for the genetic analysis of complex traits the utility of the new generation of humanized mice to study hiv-1 infection: transmission, prevention, pathogenesis, and treatment resistance and susceptibility to filarial infection with litomosoides sigmodontis are associated with early differences in parasite development and in localized immune reactions complement component c3 is required for protective innate and adaptive immunity to larval strongyloides stercoralis in mice role of eosinophils and neutrophils in innate and adaptive protective immunity to larval strongyloides stercoralis in mice strongyloides infection in rodents: immune response and immune regulation extracellular traps are associated with human and mouse neutrophil and macrophage mediated killing of larval strongyloides stercoralis human and mouse macrophages collaborate with neutrophils to kill larval strongyloides stercoralis innate and adaptive immunity to the nematode strongyloides stercoralis in a mouse model strongyloides stercoralis: protective immunity to third-stage larvae inbalb/cbyj mice immunity to onchocerca spp. in animal hosts morphology of onchocerca volvulus the use of cell-conditioned medium for the in vitro culture of onchocerca spp. larvae (nematoda: filarioidea) onchocerca volvulus: biochemical and morphological characteristics of the surface of third-and fourth-stage larvae in vitro cultivation of third-stage larvae of brugia malayi to the young adult stage sustained engraftment of cryopreserved human bone marrow cd34(+) cells in young adult nsg mice long-term human cd34+ stem cell-engrafted nonobese diabetic/scid/il-2r gamma(null) mice show impaired cd8+ t cell maintenance and a functional arrest of immature nk cells repeated infection of cats with brugia pahangi: parasitological observations experimental infection of the leaf-monkeys, presbytis cristata and presbytis melalophos with subperiodic brugia malayi onchocerca ochengi infections in cattle as a model for human onchocerciasis: recent developments we thank the consenting infected donors from the villages around kumba, cameroon (marumba i, marumba ii, boa bakundu, bombanda, and bombele) who participated in the study. we also thank dr. peter enyong and the staff at the tropical medicine research station for their technical support in the production and cryopreservation of the o. volvulus l3s.we thank dr. timothy manser, thomas jefferson university, for his support and encouragement for using human-stem cell engrafted humanized mice; dr. judy sakanari, university of california san francisco for her gift of bovine embryo skeletal muscle cells and dr. fernando pardo manuel de villena, ms. darla r. miller, and dr. linda d. siracusa for guidance with collaborative cross strain import and husbandry. key: cord-006588-aavpj5r3 authors: schwarte, l.a.; zuurbier, c.j.; ince, c. title: mechanical ventilation of mice date: 2000 journal: basic res cardiol doi: 10.1007/s003950070029 sha: doc_id: 6588 cord_uid: aavpj5r3 due to growing interest in murine functional genomics research, there is an increasing need for physiological stable in vivo murine models. of special importance is support and control of ventilation by artificial respiration, which is difficult to execute as a consequence of the small size of the animal and the technically demanding breathing pattern. in addition, numerous genetically altered mice show depressed spontaneous ventilation or impaired respiratory responses. after an introduction in murine respiratory physiology we describe options for ventilatory support, its monitoring and the potential side effects. this review will provide an overview on current possibilities in the field of airway support in mouse research. rate (rr) of 79 min ð1 , a ventilatory tidal volume (vt) of 25µl, an inspiratory fraction (inspiratory to total time of the respiratory cycle, ti/tt) of 0.44 and a resulting ventilatory minute volume (ve) of 1.98 mlámin ð1 was found. similar values are found for wild type and endothelin-1 knock-out mice (et (ð/ð) ) when birth was achieved by cesarean section (30) . rather low respiratory values were found in a study comparing wild type neonates with littermates deþcient for the ret-protooncogene: rr ranged from 28ð40 min ð1 , vt from 7ð10 µl and ve from 0.26ð0.32 mlámin ð1 , with no significant differences between the groups. the authors suggest that a reduced body temperature in their setting contributed to their þndings (4) . with respect to control of breathing, newborn wild type mice (1 g bodyweight) show similar ventilatory responses to hypercapnia compared to other newborn mammals (34) . neonates deþcient in endothelin-1 (129sv/j x icr, et-1 (ð/ð) ) have an attenuated ventilatory response to hypoxia and hypercapnia, compared to their wild type littermates (30) . a signiþcantly reduced ventilatory response to hypercapnia was also found in neonate mice deþcient in the ret-protooncogene, compared to wild type littermates, whereas no consistent differences were obtained during hypoxic challenge (4) . parameters for the spontaneous ventilation of adult mice are given in table 1 , where the following characteristics of mice ventilation are given: rr = 180ð270 mlámin ð1 , vt = 0.1ð0.2 ml and ti/tt = 0.30ð0. 35 . little information is currently available on the lung mechanics of mice (37) . murine lung-thorax compliance was measured in pancuronium-relaxed, mechanically ventilated mice (balb/c, no weight stated) in the range of 31ð38 µl (cmh 2 o) ð1 under control conditions, and 12 µl (cmh 2 o) ð1 after experimental surfactant inactivation (50) . respiratory system resistance was measured in age matched a/j-and c3h/hejmice, revealing signiþcant strain differences under baseline conditions (1 vs. 2 cmh 2 oám/ ð1 ás ð1 ) and especially after i.v. acetylcholine treatment (3 vs. 15 cmh 2 oám/ ð1 ás ð1 ) (11) . in the same study, signiþcant differences were also found for the respiratory system elastance under both conditions (25 vs. 30 cm h 2 oáml ð1 at baseline and 28 vs. 95 cm h 2 oáml ð1 after i.v. acetylcholine). again, one has to realize that marked differences exist between mouse strains in respect to respiratory physiology and pathophysiology. divergent data is available on physiologic values for murine arterial blood gasses, regarded as the gold standard for the evaluation of adequacy of ventilation. information on tissue pco 2 of the mouse indicates that it þts within the normal mammalian range (53) . later it was suggested that mice have considerably lower alveolar and arterial pco 2 than other mammals, as low as 20 mmhg (at ph 7.4) (31) . indeed, recent data support these lower pco 2 (24 mmhg in chronically instrumented 129sv/j x icr mice (30) , 34 mmhg in chronically instrumented c57bl/6 mice (39)). although effects of (30, 39) or restrainment cannot completely be excluded in these studies, the data does indicate a lower paco 2 for the mouse, e.g., compared to mammals ranging from rats to humans (paco 2 33ð41 and 35ð45 mmhg respectively). mechanical mouse ventilation is indicated unter the following circumstances. first, the òanesthesiologicaló indication: one of the major goals of anesthesia is achievement of more (39, 52) , mice lacking the brain derived neurotropic factor bdnf (1), ret-protooncogene deþcient mice with depressed ventilatory response (4), and endothelin-1 deficient mice with altered blood gas-values (e.g., signiþcantly lower po 2 than wild type littermates) and impaired respiratory response to hypoxia and hypercapnia (30) . however, it is often unknown when during development and under which conditions alterations occur in the respiratory system or the control of breathing (27) . also among inbred wild type strains the control of respiration differs substantially, e.g., the c57bl/6-strain was classiþed as highly responsive to hypercapnia, whereas the dba/2-strain was highly responsive to hypoxia (51) . therefore, in these animals mechanical ventilation offers the possibility to study effect of differences in genetic background independent of changes in ventilation. second, the òsurgicaló indication: certain surgical procedures have an improved outcome or can only be performed with support of mechanical ventilation. examples are the bilateral ligation of the carotid artery (36) , where non-ventilated mice often die from respiratory disturbances, or the growing þeld of cardiac research that needs an open-chest murine model (15, 18, 29, 44) . hereby opening of the thorax and the adherent interpleural space induces lung collapse with respiratory insufficiency, whereas with support of a mechanical respirator the lungs can be inßated and ventilated. there are several possibilities of accessing and maintaining the airway in mice. in early studies using a mechanical ventilator with active in-and expiration for mice (28) , the airway was accessed by advancing a tube through the mouth into the pharynx, but not further into the trachea. although technically less demanding than endotracheal intubation, this attempt risked gastric gas-insufflation (including aspiration of stomach content) and did not provide peep capability. a different, nontraumatizing mode to access the airways is performed with the brady-newsom apparatus (3): it consists of a gas-chamber around the murine head, sealed at the thorax by a tight neoprenecollar. elevating the pressure in this chamber leads to increased airway pressures, with respect to ambient air. thus this concept provides continuous positive airway pressure (cpap) if the mouse ventilates spontaneously, or mechanical ventilation, if periodic pressure changes in the chamber are generated. although this apparatus has advantages, e.g., the technically less demanding airway access, it is also hampered with the disadvantage of gastic gas insufflation and aspiration of gastric content. to circumvent this problem, mechanical ventilation of mice today is usually performed after securing the airways by orotracheal intubation or via tracheotomy and consecutive endotracheal intubation. tracheotomy has been performed in preterm neonate mice (1.15 g bodyweight) with insertion of polyethylene tubes (sp8, natsume, tokyo, japan; tapered 0.4 mm outer diameter (od)) (30), whereas for adult mice the od of the endotracheal tube ranges from 0.9 mm (25ð38 g mice) (29) to 1.1 mm (20ð35 g mice) (15) . for experiments requiring recovery after surgery, temporary orotracheal intubation is performed, ideally via direct laryngoscopy with a purpose-made laryngoscope (13) . the visualization of relevant airway structures requires a strong small-focus light-source, allowing transillumination of the laryngeal cavity (with vocal cords and tracheal oriþce), when put on the ventral cervical region from the outside (2). orotracheal intubation is performed with or without visual conþrmation of the tubeõs tip-position (tube position judged by respiratory excursions). visualization of the trachea (after surgical access to the deeper cervical structures) and controlled advancement of the endotracheal tube allows more accurate positioning of the tube, but is more invasive and has therefore an increased morbidity and mortality. for animals sacriþced at the end of the experiment, direct surgical access to the airways is an alternative option. since most of our mouse surgeries require preparation of deeper cervical structures (e.g., catherization of a carotid artery and jugular vein) performing tracheotomies is convenient, with the additional advantage of minimized dead space (vd). a reliable surgical procedure for this airway access is brießy described: after induction of anesthesia the mouse is put into the supine position and the head is reclined (silk or rubber band around the upper incision teeth and attached to the operating surface) to allow easy access to the ventral neck. a median cerival skin incision (upper thorax aperture to lower jaw) is performed and layerwise skin, fascia, fat and connective tissue covering the thyroid gland are removed. the thyroid lobes are divided bluntly at their isthmus, pulled aside and kept retracted by bulldogclamps. the pretracheal muscles are spread bluntly and pulled aside to allow access to larynx and trachea. tracheotomy is performed by a transversal cut between two tracheal rings, usually in the lower third of the trachea. for the endotracheal intubation vascular pe-catheters (abbocath-t¨, abbott-venisystems) are used, cut to a length of about 5 mm with an angled tip (45¡) to allow smooth introduction. the correct position of the endotracheal tube is conþrmed by judging chest excursions, e.g., if respiration is still visible and symmetric. the tube is secured by ligation (silk 6-0, davis & geck¨) around the trachea. if maintenance of spontaneous ventilation is desired, the endotracheal tube is shortened to minimize vd and airway resistance. for mechanical ventilation, initially applied rr and/or vt should be sufficiently higher than spontaneous rr and vt of the anesthetized mouse to decrease respiratory drive (e.g., òbreathing against the ven-tilatoró) by moderate hyperventilation. after connecting the endotracheal tube, anesthesia can be safely deepened for an additional depression of ventilatory drive and facilitation of mechanical ventilation. alternatively, this goal can be achieved (avoiding additional cardiocirculatory depression) by a non-depolarizing muscle relaxant (vecuronium-bromid 0.25 mgákg ð1 bodyweight i.p., norcuron¨, organon). using this option it should be realized however that anesthesia depth cannot be judged from parameters like spontaneous movement or limb-withdrawal to paw pinch, but instead on changes in hemodynamics. a side aspect of murine airway access is the preparation of a ventilated (and perfused) isolated lung. this model has been described for the investigation of cytokines released from a hyperventilated mouse lung (57) . the physiological lung variables obtained from this murine model (female balb/c, 22ð30 g body weight) after 60 min of npv (negative pressure ventilation) are a vt = 187 µl, dynamic compliance (cdyn) of 0.022 ml/cmh 2 o and an airway resistance of 0.45 cm h 2 oás/ml. there are two basic modes of respiration, spontaneous ventilation and controlled mechanical ventilation (cmv), where work of breathing is taken over by the ventilator. between these there are a variety of mixed forms, like respiratory assistance, augmenting inspiration if shallow or labored (rsp1002, kent-scientiþc¨, us) or supported spontaneous ventilation with continuous positive airway pressure (cpap), adopted for mice by brady and colleagues (3) . however, these mixed modes are not largely established for mice yet, and will therefore not discussed in detail. although mechanical ventilation of mice has major advantages, it is confounded by difficulties: obviously, the small size of the animals makes airway access more challenging than in other laboratory animals. for example, in 25ð30 g mice, the trachea has an accessible length of only 3ð5 mm and a circumference of about 2.5 mm (49) . appropriate endotracheal tubes have an outher diameter ranging from 0.4 mm for neonate mice (30) to 1.0 mm for adult mice (13) . the actual mechanical ventilation of the mouse is complicated by the technically demanding respiratory pattern of this species, e.g., the high rr, the low ti/tt ratio and the small vt, with the latter also demanding a minimized deadspace (vd) to prevent rebreathing. in commercially available rodent ventilators, the relatively large system-vd allows substantial gas compression, thereby minimizing the vt delivered to the mouse. ewart et al. (11) reported that 2 ml of added vd (compliancẽ 0.002 ml/cmh 2 o) and a peak inspiratory pressure of 40 cm h 2 o reduce the delivered vt by about 50 %. thus, mouse ventilators (see table 2 ) should have a range of technical modalities to enable proper murine ventilation. since some authors did not report on the type of ventilator used (see table 3 ) the list might be incomplete. the most obvious difference between spontaneous respiration and usual modes of mechanical ventilation (mv) are inverted pressure relations, with respect to ambient pressure, during the respiratory cycle: during spontaneous inspiration the expansion of the intrathoracic volume (mainly caused by contraction of the diaphragm and extension of the rib cage) generates a negative intrathoracic pressure and allows gas ßow into the lungs. during expiration mainly passive elastic forces of lung and rib cage generate a positive intrathoracic pressure, leading to exhalation of air. applying usual mv, the air is pushed during inspiration into the miceõs lungs, leading to a positive intrathoracic pressure. during expiration intrathoracic pressure equilibrates with ambient pressure (intermittent positive pressure ventilation, ippv) or remains positive (continuous positive pressure ventilation, cppv). therefore it is important to notice that mean airway pressure during mechanical ventilation is higher than during spontaneous ventilation. these higher pressures contribute to side effects induced by mechanical ventilation. mechanical ventilators are classified according to their method of cycling between the inspiratory and expiratory phases. small animal ventilators are usually time-cycled: the ventilator switches between the respiratory phases after a certain time has elapsed, depending on the preset respiratory rate. although other methods of cycling might be favorable under certain experimental conditions (e.g., volume-cycled to prevent pulmonary volutrauma in murine open chest preparations), they are not established yet. however, although the method of cycling is classiþed according to a single parameter (e.g., time), some mouse respirators allow a more sophisticated control of the ventilation by limiting relevant parameters: to prevent high inspiratory peak airway pressures, for example, most mouse ventilators allow the limitation of the ventilatory system pressure. most commercial mouse ventilators are time cycled and pressure limited. the primary goal of mechanical ventilation is delivery of an adequate respiratory minute-volume (ve) to the murine 514 basic research in cardiology, vol. 95, no. 6 (2000) © steinkopff verlag 2000 lung. to achieve this, the rr and vt have to be adjusted to supply the actual ventilatory requirements. rr in most mouse ventilators is the preset cycling parameter, but vt-lung may vary, depending on the compliance of lungs and tubing and whether respiration is pressure limited. a decrease in compliance of the respiratory system, for example in murine models of interstitial pulmonary diseases (ards, pneumonia), increases the airway pressure for a given vt ventilator . if ventilation is pressure limited, then vt lung will be considerably less than vt ventilator . in contrast, if higher peak pressures are accepted to maintain vt lung , the risk to traumatize the delicate murine lung will increase (17) . to prevent the occurrence of these high pressures, inspiration time relative to expiration can be increased for a given vt (up to inverse ratio ventilation (irv), with inspiration longer than expiration). disadvantages of a higher inspiratory fraction during mechanical ventilation are possibly insufficient times for complete exhalation (òair trappingó) and the prolonged phases of elevated intrathoracic pressure, which may cause a decreased cardiac preload and consequently lowered blood pressures (7). a feature provided by some mouse-ventilators is the possible application of external positive end-expiratory airway pressure (peep): during expiration, airway pressure is not allowed to equilibrate with ambient pressure (0 cmh 2 o), but kept at a higher level (usually 2ð10 cmh 2 o). mechanical ventilation with peep prevents the endexpiratory collapse of small airways and possibly recruits atelectatic pulmonary areas for gas exchange by increase of functional residual capacity (frc). since pulmonary compliance increases (up to a certain degree) with increased frc, ventilation with peep supports pulmonary inßation. whether this holds true for mice has not been shown yet. additionally, ventilation with peep might mimic the physiologic end-expiratory tonus of inspiratory muscles and glottis of spontaneous ventilating mice (56) . typical side effects, demonstrated in a murine model of peep, are discussed below. a list of ventilator settings is given in table 3 . most experiments have mechanical ventilation set at rr = 100ð 150ámin ð1 , vt = 0.2ð0.7 ml, ti/tt 0.2ð0.4. when these values are compared to values of spontaneous ventilating mice (table 1) , it becomes obvious that lower rr and higher vt are commonly used to mechanically ventilate mice. a more rare feature provided by some mouse ventilators is the intermittent sigh-ventilation. here an inspiration with higher tidal volume (hyperinßation-or sigh-cycle) is administered after a certain number of respiratory cycles (for example every 50 ventilatory cycles) or triggered manually (rsp-1002, kent-scientific¨corporation, litchfield, ct, usa or 60ð 1100/1099, harvard instruments¨, us). although not proven, this imitation of physiological sighing is thought to result in recruitment of atelectatic pulmonary areas, without the need for a continuous peep, circumventing constant elevation of airway pressures. intermittent positive pressure ventilation (ippv) and continuous positive pressure ventilation (cppv) are the two common modes in murine mechanical ventilation, as provided by commercial ventilators. however, for the ventilation of newborn mice (1.0ð1.5 g bodyweight, vt 5ð20 µl) the concept of the òiron lungó, based on the principle of body surface negative pressure ventilation has been adopted (27) to prolong survival time of nmda-receptor mutant mice (32) , usually dying within 20 h after birth. only a few studies using mechanical ventilation in mice report on additional ventilatory parameters, like inspiratory gas ßow or pressures in the ventilatory system. using a rr of 140ámin ð1 , a vt of 0.7 ml and an inspiratory time of 0.2 s (resulting in a inspiratory fraction (ti/tt) of 0.41, table 3 data are also available on ventilation regimen for murine open-chest preparations: guo et al. (18) compared respiratory rates of 95, 105 and 121ámin ð1 (harvard rodent ventilator, room air with oxygen (21ámin ð1 ), vt = 2.2 ml with endotracheal tube loosely connected) in male icr mice (8ð12 weeks old, mean body weight 33.3 g) with respect to the resulting arterial blood gases. adequate ventilation was obtained with a rr of 105ámin ð1 , resulting in an arterial pao 2 of 327 mmhg (177 and 321 mmhg for 95ámin ð1 and 121ámin ð1 respectively), an arterial paco 2 of 31 mmhg (43 and 18 mmhg for 95ámin ð1 and 121ámin ð1 respectively) and an arterial ph of 7.4 (7.3 and 7.5 for 95ámin ð1 and 121ámin ð1 respectively). other regimens for the ventilation of open chest mice are listed in table 3 . during controlled mechanical ventilation (cmv), the most frequent applied mode of artiþcial respiration in mice, the work of breathing is taken over by the respirator. to facilitate this controlled mechanical ventilation (e.g., prevention of òbreathing against the ventilatoró) it is usually performed under deep anesthesia. this ensures that relevant compensatory mechanisms (e.g., onset of spontaneous ventilation induced by hypercapnia or hypoxia) are impaired and underline the importance of monitoring mechanical ventilation. attempts to monitor mechanical mouse ventilation are complicated by the fact that even standard variables for ventilation are not established yet or show conßicting results (see table 1 ). several possibilities are applicable to monitor adequacy of ventilation. a highly subjective way, requiring experience, is to observe the thorax-excursions with respect to rate, amplitude, symmetry and regularity as indicators of ventilatory rate, tidal volume, endotracheal tube position and attempt to spontaneous ventilatory effort. dalkara et al. (7) suggest for a physiological mouse ventilation (mean paco 2 = 35 mmhg) keeping òthe volume at a level at which thoracic movements þrst become noticeably observedó. information on the oxygenation can be obtained from the ear and tailskin color, e.g., with cyanosis indicating poor oxygenation (desoxy-hemoglobin 5 gá100 ml ð1 ). these measures might serve as a þrst orientation in the evaluation of the adequacy of mechanical ventilation. more objective and in-depth measures are gas ßows, airway pressures and partial pressures (see below). the measurement of respiratory gas ßow in mice has been a technical challenge, mainly because pneumotachographs attached to the endotracheal tube increase the ventilatory dead space (vd). recently pneumotachographs have been developed for mice that allow both direct airway gas ßow measurement and plethysmography (kent-scientiþc¨small animal pneumotachs, trn3100, kent scientiþc corporation). but even modern devices in this setting are suspected to increase dead space to a crucial extent, leading to partial rebreathing and consequently to hypercapnia and eventually hypoxia. the direct, non-invasive measurement of murine airway pressures is technically difficult; therefore, substitutes have been established to obtain estimates. in commercially available mouse ventilators, the airway pressures are measured inside the ventilator. the advantage of this construction is that it circumvents the need for an additional pressure transducing line, ideally originating from the endotracheal tube itself, to a pressure transducer. a major disadvantage however is that pressures measured in the ventilator need to be extrapolated to airway pressures within the endotracheal tube. this extrapolation is determined by the compliance of the respiratory gases and the tubing system (òsignal dampingó). it is therefore desirable to measure airway pressures as close as possible to the endotracheal tube. with respect to this, we have developed an integrated endotracheal tube within a small aluminum frame (10 x 5 x 2 mm, 5 g), consisting of temperature controlled adapters for in-and expiratory tubing, an inline-capnography cell (vd = 25 µl) and a liquid-filled airway-pressure transducer. although airway pressures are usually monitored to maintain safe values for mean-and peak-airway pressure, this measure can also serve to titrate damage in murine models of respirator-induced lung impairment, such as baro-or volutrauma (17) , where tidal volumes (vt) have been adjusted to achieve traumatizing target airway pressures. fresh-gas inßow composition to the mechanical ventilator can be monitored with respect to the concentrations of the different respiratory gases, e.g., oxygen (o 2 ), nitrogen (n 2 ), nitrous oxide (n 2 o) and volatile anesthetics. when rebreathing of exhaled air is excluded in the ventilatory circuit, analysis of fresh gas streaming to the respirator should accurately reßect the composition of inspiratory gases. this attempt is advantageous, compared to sample lines attached to the inspiratory tubing-limb of the system, because it circumvents the need for gas extraction from the inspiratory tubing with the consequence of decreased inspiratory airways-pressures and ventilatory tidal volumes. the actual measurement can be performed online with commercially available multi-gas analyzers (datex¨, hp¨). it has been argued that ventilation with room air may not be ideal in mice (24) , based on observed high murine p 50 values (po 2 corresponding to 50 % hemoglobin saturation) of 65 mmhg (46) or even 71 mmhg (48) . however, most studies have found a p 50~ 40 mmhg for the mouse, when measured under standard conditions (ph = 7.4, pco 2 = 40 mmhg, 37 ¡c) (25, 31, 35, 42, 47) . a p 50 of ~ 40 mmhg is still rightshifted, when compared to humans (p 50 = 28 mmhg). nevertheless, although even with a high p 50~ 65 mmhg blood is virtually 100 % saturated at room air (46) , increased fio 2 may be helpful in the mouse when uncertainty exists whether mechanical ventilation optimally expands the lungs. if volatile anesthetics (halothane, enßurane, isoßurane or more recent ones such as sevoßurane and desßurane) are used, additional considerations have to be taken into account: clinically used vaporizers for volatile anesthetics (draeger¨, series 19) are usually precalibrated for gas ßows higher than that required for mice and will therefore give unreliable results when used with low gas ßows. although recalibration of the vaporizer for low air-ßow is the best option, a more common approach is to use an appropriate high gasßow (several hundred mlámin ð1 ) with the abundant air released via a t-type adapter or valve. disadvantages of the latter approach are higher costs and the generation of relatively large amounts of waste gas giving environmental contamination and increased toxicity for exposed personnel. in contrast to maintenance of sufficient arterial oxygenation, which even under conditions of poor ventilatory support can be achieved by an increased fraction of inspiratory oxygen (fio 2 ), adequate pulmonary elimination of co 2 is the more challenging aspect of mechanical ventilation. although the arterial blood gas analysis is regarded as the gold standard for evaluation of proper ventilation, the drawbacks are that it is a discontinuous and invasive method requiring a substantial vol-ume of arterial blood (0.1ð0.2 ml for a single measurement, 5ð10 % of the total blood volume of a 25 g mouse (58)), the latter aspect adding new sources of instability to the model. though often neglected, measurement of respiratory carbon dioxide concentration (capnography) is therefore recommended in mouse research. especially the determination of the end-tidal co 2 (etco 2 ), as a measure for alveolar co 2 -concentration, is described as essential to maintain stable physiological parameters during mechanical mouse ventilation (7) . however, capnography has rarely been performed in mice due to technical difficulties, like gas dilution artifacts occurring in side stream systems, which prevent accurate detection of endtidal co 2 -peaks or-plateaus. therefore, to use the potential of capnography to its full extent, fast responding mainstreamcapnography is required. to this end we have developed an integrated mouse endotracheal tube with an implemented capnography-cell with minimal gas-mixing artifacts. since this cell is located in the endotracheal tube it allows real-time in-and expiratory mainstream breath-by-breath capnography. a typical capnography curve using this device is shown in fig. 1 , showing several single breath capnography curves (inner section with high chart speed, rr ~ 100 min ð1 ). capnography is not only a measure for the adequacy of mechanical ventilation, but can also serve to detect changes in hemodynamics. an example for this is given in fig. 2 : during steady-state anesthesia 0.2 ml blood was withdrawn from an arterial line (black arrow). the end-tidal carbon dioxide concentration drops immediately, likely due to a decrease in pulmonary perfusion (decreased cardiac output). resuscitation is performed by rapid infusion of 0.2 ml albumin solution (white arrow), which increased capnography values back to baseline, probably by restoring pulmonary perfusion. since time resolution is enhanced by our breath-by-breath capnography, the relation between expiratory co 2 and speciþc events can be described precisely. additionally, the capnography curves can be analyzed with respect to irregularity (e.g., due to intermittent spontaneous breathing) and shape alterations (e.g., airway obstruction). a few mouse capnographs are commercially available, mostly using side stream sampling (e.g., kent scientiþc corporation sc-210 respiratory co 2 monitor). the only main stream capnograph commercially available for mice to our knowledge is the sc-300 (kent-scientiþc corporation) with an external co 2 -probe to be placed in the ventilation tubing. however, we were unable to þnd studies reporting on this device. a disadvantage of this device, as reported by the manufacturer, is that rr is limited to 150 min ð1 , which might not meet the demands of all experimental designs (table 3) . the enhanced stability of the murine homeostasis is a major goal of mechanical ventilation, but typical side effects have to be considered, possibly adding new sources of instability to the murine model. these side effects can be classiþed as regional (airways and lungs affected) or systemic (distant organs affected). a typical example for a ventilator-induced regional side effect is the mechanical hyperinßation of the murine lung, e.g., when large tidal volumes or high airway pressures are applied. this induces histological damage of the delicate murine pulmonary structures. this volutrauma has been shown in mice to trigger the onset of inßammation, including expression of cytokines (57) . the systemic side effects of mechanical ventilation are (at least partly) due to a compromised hemodynamic situation. mechanical ventilation per se results in larger intrapulmonary pressures as compared to spontaneous ventilation, possibly inhibiting venous return to the heart. in addition, prolonged phases of positive airway pressures, e.g., when the inspiratory fraction (ti/tt) is increased or when external peep is applied, depresses the circulation by several mechanisms, also via an impaired venous return to the heart. as an example for a global hemodynamic variable, arterial blood pressure decreases immediately after application of higher levels of peep: fig. 3 shows a typical blood pressure trace of a deeply anesthetized mouse (c57bl/6, 25 g bodyweight, anesthesia with ketamine (35 mgákg ð1 áh ð1 ) and medetomidine (35 µgákg ð1 áh ð1 ) indicating the immediate hypotensive reaction after application of 10 cmh 2 o peep. the extent of hypotension induced by mechanical ventilation is further related to the respiratory phase, as demonstrated in fig. 1 , a simultaneous recording of capnography and arterial blood pressure in a c57bl/6mouse (anesthetized with fentanyl (0.8 mgákgá ð1 áh ð1 ), ßuanison (25 mgákgá ð1 áh ð1 ) and midazlam (3 mgákgá ð1 áh ð1 ), a more hypotensive anesthesia than used in fig. 3 , explaining the different baseline blood pressures). at end-expiration (white arrow), where intrathoracic pressure is minimal, arterial blood pressure reaches its peak value and vice versa (black arrow). a second example for side effects of the mechanical ventilation with peep distant to the lung is the impairment of the intestinal microcirculation. in anesthetized c57bl/6-mice (terminal ileum exposed) we were able to visualize a marked reduction of perfused capillaries and a decrease in capillary blood flow velocity, using orthogonal polarized spectral (ops-) imaging (16) . figure 4 a/b shows a typical example for peep-induced microcirculatory impairment. both images show the same intestinal region, before (4a) and 15 minutes after (4b) onset of 5 cmh 2 o external peep, with white arrowheads marking corresponding capillaries (black) in both images. in contrast to fig. 4a the capillaries in 4b are markedly less numerous and appear dashed, indicating erythrocyte aggregations formed at low capillary blood flow velocities. respiratory control of mice is a field of growing interest. although spontaneous ventilation is the more physiological mode of breathing, only mechanical ventilation maintains physiological values (blood gases, acid-base status) in situations with impaired spontaneous breathing, as occurring during anesthesia, surgery or constitutively in murine strains with impaired spontaneous ventilation. there are currently several commercially available small rodent ventilators feasible for use in mice. if mechanical ventilation is performed, then monitoring of this intervention is required, ideally by means of capnography. research still has to be performed on how the most physiological ventilation can be achieved in mice, which ventilatory patterns are appropriate and how ventilation related side effects can be limited. 518 basic research in cardiology, vol. 95, no. 6 (2000) © steinkopff verlag 2000 brain derived neurotropic factor is required for normal development of the central respiratory rhythm in mice in vivo electrophysiologic studies in mice effect of positive pressure breathing on the vibration tolerance of mice ret-protooncogene is important for the development of respiratory co2 sensitivity physiological characteristics of the chest and lungs and the work of breathing in different mammalian species breathing and pulmonary surfactant function in mice 24 h after ozone exposure cerebrovascular responses under controlled and monitored physiological conditions in the anesthetized mouse the effect of restraint on ventilatory response to hypercapnia and hypoxia in adult mice contribution of type i nos to expired gas no and bronchial responsiveness in mice whole body 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better simulate human peripheral artery disease date: 2020-02-26 journal: sci rep doi: 10.1038/s41598-020-60352-4 sha: doc_id: 4416 cord_uid: qw6tusd2 peripheral arterial disease (pad) develops due to the narrowing or blockage of arteries supplying blood to the lower limbs. surgical and endovascular interventions are the main treatments for advanced pad but alternative and adjunctive medical therapies are needed. currently the main preclinical experimental model employed in pad research is based on induction of acute hind limb ischemia (hli) by a 1-stage procedure. since there are concerns regarding the ability to translate findings from this animal model to patients, we aimed to develop a novel clinically relevant animal model of pad. hli was induced in male apolipoprotein e (apoe(−/−)) deficient mice by a 2-stage procedure of initial gradual femoral artery occlusion by ameroid constrictors for 14 days and subsequent excision of the femoral artery. this 2-stage hli model was compared to the classical 1-stage hli model and sham controls. ischemia severity was assessed using laser doppler perfusion imaging (ldpi). ambulatory ability was assessed using an open field test, a treadmill test and using established scoring scales. molecular markers of angiogenesis and shear stress were assessed within gastrocnemius muscle tissue samples using quantitative polymerase chain reaction. hli was more severe in mice receiving the 2-stage compared to the 1-stage ischemia induction procedure as assessed by ldpi (p = 0.014), and reflected in a higher ischemic score (p = 0.004) and lower average distance travelled on a treadmill test (p = 0.045). mice undergoing the 2-stage hli also had lower expression of angiogenesis markers (vascular endothelial growth factor, p = 0.004; vascular endothelial growth factorreceptor 2, p = 0.008) and shear stress response mechano-transducer transient receptor potential vanilloid 4 (p = 0.041) within gastrocnemius muscle samples, compared to animals having the 1-stage hli procedure. mice subjected to the 2-stage hli receiving an exercise program showed significantly greater improvement in their ambulatory ability on a treadmill test than a sedentary control group. this study describes a novel model of hli which leads to more severe and sustained ischemia than the conventionally used model. exercise therapy, which has established efficacy in pad patients, was also effective in this new model. this new model maybe useful in the evaluation of potential novel pad therapies. major amputation, renal failure and death) and poor long-term durability [5] [6] [7] [8] . there is great interest in developing novel medical therapies for the leg symptoms of pad. recent efforts have focused on stimulating development of new blood vessels within the leg through angiogenesis or by encouraging the remodelling of existing small vessels into improved collateral channels (arteriogenesis). promising results for novel treatments, such as viral vectors carrying angiogenesis promoting agents and stem cells, in pre-clinical models of pad have not been consistently replicated in large clinical trials 9-12 . in patients that have pad, atherosclerosis-associated arterial narrowing develops gradually over many years allowing the legs to adjust to the gradual decrease in blood flow through compensatory mechanisms within the blood vessels and muscle fibres 13 . in contrast, the most commonly used animal model for initial testing of novel therapies for pad is a model of acute blood supply interruption through ligation or excision of the femoral artery (referred to here as the 1-stage hind limb ischemia (hli) model) 14, 15 . previous studies report that the ligation and excision of the femoral artery in the 1-stage model leads to increased fluid shear stress within the limb collateral arteries resulting in altered gene expression patterns through shear stress responsive elements which promote arterio-and angio-genesis [16] [17] [18] . hind limb blood supply in this 1-stage model therefore usually naturally recovers over a period of approximately 4 weeks 15 . this model does not therefore simulate the clinical presentation of pad. patients typically present with a history of acute exacerbation of chronic symptoms of leg pain on walking and have ongoing ischemic symptoms. the 1-stage hli model may therefore not be an ideal model to study therapeutic angiogenesis and arteriogenesis 14, 19 . another approach to inducing hli is the placement of an ameroid constrictor around the femoral artery to induce gradual occlusion 16, 20, 21 . previous studies suggest that this approach leads to mild ischemia 20 and that blood flow recovery occurs within 2-3 weeks 16, 21 . furthermore, previous pre-clinical pad research has mainly focused on assessing hind limb blood supply with limited assessment of ambulatory ability 14, 15 . on the other hand, the assessment of novel treatments in pad patients usually involves measures of walking ability using treadmill or corridor walking tests 22, 23 . there is therefore a need for an increased focus on functional tests of the limb within clinically-relevant rodent models. we hypothesised that the limb ischemia produced by the current 1-stage hli model would be more severe and sustained if the model was modified to include an initial more slowly progressive arterial narrowing over 14 days prior to the induction of acute ischemia (i.e. a 2-stage model). our overall aim was to develop a more clinically relevant rodent model that could incorporates stable on-going limb ischemia in order to test therapeutic interventions. mice. male apolipoprotein e deficient (apoe −/− ) mice (n = 118, obtained from animal resources centre, western australia) were used for the experiments. mice were housed in a temperature-controlled room (21 ± 1 °c) with an automatic 12:12-h light/dark cycle (07:00 to 19:00 hours). mice were singly housed in a clear individually-ventilated, temperature and humidity-controlled cage system (aero ivc green line; tecniplast) with enrichment. all experiments were performed during the light phase (10:00-18:00 hours) and mice were fed with standard rodent chow and water ad libitum during the course of these experiments. approval for the animal studies was obtained from the institutional ethics committee (animal ethics committee, james cook university) and experimental work performed in accordance with the institutional and ethical guidelines of james cook university, australia, and conforming to the guide for the care and use of laboratory animals (national institutes of health, usa). hli models. the first phase of the study utilised two hli models: the most commonly used unilateral acute hli model (1-stage hli) 16, 24, 25 and the newly developed 2-stage hli model. male apoe −/− mice aged 20 months were randomly divided into 4 groups as follows: group 1 = 1-stage hli model (n = 25), group 2 = 1-stage sham (n = 19), group 3 = 2-stage hli model (n = 25) and group 4 = 2-stage sham (n = 19). body weight and primary outcome measures were recorded at regular intervals as illustrated in fig. 1a . all functional assessments were performed in a subset of mice randomly selected from each experimental group (n = 5-7). the creation of the 1-stage hli model involved exposure of the left femoral artery through a vertical 0.5-1 cm skin incision under a stereotactic microscope (leica). the femoral artery and its side branches were then ligated with 6-0 silk sutures (ethicon) immediately distal to the inguinal ligament and proximal to the popliteal bifurcation before being excised ( supplementary fig. s1a,b) . femoral nerves were carefully preserved. the wound was irrigated with sterile saline and then the overlying skin was closed using 4-0 vicryl sutures (ethicon). post-operative pain was reduced using lignocaine (troy laboratories). a similar surgery without ligation or excision of the femoral artery was performed on the 1-stage sham controls. the 2-stage hli model was performed using a 2-stage surgical procedure. the left femoral artery was exposed as described above and 2 custom made miniature ameroid constrictors of 0.25 mm internal diameter (research instruments sw) were positioned on the artery. one was placed on the femoral artery immediately distal to the inguinal ligament and one was positioned proximal to the sapheno-popliteal bifurcation ( supplementary fig. s1c ). after 14 days, a new incision was made and the femoral artery ligated and excised, as described for the 1-stage hli model. a similar two-stage surgery was performed without placement of ameroids, nor ligation and excision of the femoral artery for the 2-stage sham controls. assessment of the effect of exercise training in the 2-stage hli model. during the second phase of the study, the effect of an exercise program on male apoe −/− mice aged 6 months undergoing the 2-stage hli model (male apoe −/− mice, n = 30) was tested. mice subjected to 2-stage hli were randomly allocated to an exercise training or control group (n = 15 per group). mice in the exercise group received 180 to 200 m (between 30-45 mins of running wheel access) of exercise each day on a running wheel (8 station home cage running the movement of animals were monitored and functional scores of the various groups were assessed according to the scoring criteria detailed in the materials and methods section. the 2-stage hli model showed reduced function throughout the experimental period compared to the 1-stage hli model. data shown as mean ± sem and analysed by repeated measures 2-way anova, and p value significant at ≤0.05. (e) graph showing modified ischemia scores. the animals were monitored and scored for signs of ischemia according to previously published criteria detailed in the materials and methods. the 2-stage hli model showed a higher level of ischemia compared to the 1-stage hli depicted by a lower scoring throughout the study period. data shown as median ±sem and analysed by repeated measures 2-way anova, and p value significance set at ≤0.05. suggested that the hli typically resolved over the course of 28 days, with hind limb blood supply reaching a plateau between 21 and 28 days after surgery 28 . the ldpi measurements were therefore performed at the following time points for 1-stage hli model: day 0 prior to surgery, immediately after surgery, days 3, 7, 14, 21 and 28 after surgery. for the 2-stage hli model, the ldpi measurements were performed at the following time points: day 0 prior to the first operation (ameroid placement), immediately after the first operation, days 3, 7, and 14 after the first operation, immediately after the second operation (femoral artery excision), and 7, 14, 21 and 28 days after the second operation (i.e. 14, 21, 28, 35 and 42 days after the first operation). a schematic illustration of the experimental design is shown in fig. 1a . body mass was also measured on the same day as the ldpi measurements. in clinical practice pad patients are treated to improve pain free walking capacity and resolve rest pain and tissue loss (critical limb ischemia, cli). in clinical trials, these are usually investigated by walking tests and assessment of pain. similar to clinical trials, in the current study ambulatory ability was assessed with a treadmill test, voluntary physical activity examined through an open field test and foot pain estimated through a mechanical allodynia test. all outcomes were assessed by an assessor blinded to mice group. semi-quantitative assessments of limb function and ischemia were performed at the same time points as the blood flow measurements ( fig. 1a ; supplementary tables s1, s2). limb function was assessed using the clinical use score (tarlov scale) as: 0 = no movement; 1 = barely perceptible movement, no weight bearing; 2 = frequent and vigorous movement, no weight bearing; 3 = supports weight, may take 1 or 2 steps; 4 = walks with only mild deficit; 5 = normal but slow walking and 6 = full and fast walking 29, 30 . limb ischemia was scored using the ischemia scoring scale as previously reported: 0 = auto-amputation of leg; 1 = leg necrosis; 2 = foot necrosis; 3 = two or more toe discoloration; 4 = one toe discoloration; 5 = two or more nail discolorations; 6 = one nail discoloration and 7 = no necrosis 30 . all scoring was performed by two independent observers and found to be identical. treadmill test. mice were run on a six lane excer3/6 treadmill (columbus instruments) without incline. mice were acclimatised to the treadmill by ambulating on it at 5 m/min for 5 min once daily on three consecutive days prior to any testing. before each treadmill test, mice were fasted for 1 h. the speed of the treadmill was controlled using the software and calibrated using an inbuilt speedometer mounted on the treadmill platform. a treadmill test involved an initial warm up at 5 m/min for 5 min followed by a progressive speed increase from 5 to 10 m/min, accelerated at 1 m/min. following this the treadmill speed remained at 10 m/min for up to a total running time of 20 min. during the test a stimulus grid of 3 hz was kept on until mouse exhaustion as previously reported 31 . exhaustion of the mouse was defined if the mouse returned to the stimulus grid 10 times despite a 3 hz electrical stimulus to encourage walking on the belt. the treadmill software recorded the total distance walked by a mouse until exhaustion. a blinded observer supervised the experiment to assess outcomes. the treadmill belt and lanes were cleaned with water and 70% alcohol and dried with paper towel after each test to remove any body scent. treadmill testing was carried out before ameroid placement, 10 days after ameroid placement, and 1 and 3 weeks after completion of the 2-stage hli (fig. 1a) . for the 1-stage hli model, treadmill testing was performed before ligation and excision of the femoral artery and 1 and 3 weeks after ischemia induction. voluntary physical activity test. the open field test is a common measure of voluntary physical activity in rodents suggested to be similar to a 6-min walk test used in humans 32 . to ensure consistency prior to the test, mice were brought to the testing room in their home cages at least 1 hr prior to the start of behavioural testing. the mice were fasted during the acclimatisation period and given free access to water under normal lighting. the open field box was made of opaque plastic (40 × 40 × 40 cm), divided into an outer field (periphery) and a central field (20 × 20 cm) which was primarily used for analysis. mice were individually placed in the centre of the arena and movements of the mice were recorded using a video camera (logitech) supported with acquisition software (capture star ver. 1; cleversys inc) and analysed by the topscan lite software (high throughput version 2.0; cleversys inc). the test protocol used was identical for each mouse assessed. after each test the open field box was cleaned with water and 70% alcohol and dried with a paper towel to remove the body scent, which could be a cue to movement of the mice. room lighting, temperature, and noise levels were kept consistent for all tests. the mouse movements were recorded for 20 min, to mimic the short timed nature of the 6-min walk test. rest time was recorded as motion measure score <0.05 in the software and average speed was calculated only for motion measure score >0.05. total distance travelled (m), frequency of movement, time spent in the arena (s) and average velocity in the arena (mm/s) were measured. mechanical allodynia test. the paw pressure transducer and the pressure application measurement device (pam; ugo basile) is a non-invasive tool for measuring mechanical allodynia threshold and hypersensitivity in rodents. the pam device allows an accurate measurement of primary mechanical hypersensitivity in rodents 33, 34 . a gradually increasing squeeze force is applied across the joint at a rate of approximately 300 gms/sec until a behavioural response (paw withdrawal, freezing of whisker movement, wriggling or vocalization) is observed with a cut-off of 5 sec. the peak gram force (gf) applied immediately prior to limb withdrawal was recorded by the base unit, and this value was designated the limb withdrawal threshold (lwt). lwt was measured twice in both the ipsilateral and contralateral limbs by two independent observers. the measurements were averaged and presented as a ratio of operated left limb to the un-operated right limb. blood tests. blood was collected by cardiac puncture at the completion of the experiments. platelet poor plasma was separated as described previously 35, 36 . the plasma concentrations of interleukin (il)-6, interferon (ifn)-γ, monocyte chemoattractant protein-1 (mcp-1) and tumour necrosis factor (tnf)-α were determined scientific reports | (2020) 10:3449 | https://doi.org/10.1038/s41598-020-60352-4 www.nature.com/scientificreports www.nature.com/scientificreports/ using a cytometric bead array kit (cba, bd biosciences). the inflammatory markers were assessed in samples (n = 10/group) selected from each group using a random number generator. briefly, 50 μl of mixed capture beads and 50 μl of serially diluted standard or plasma sample and 50 μl of phycoerythrin (pe) detection reagent, were incubated in the dark for 2 h in sample assay tubes. samples were then washed twice with 1 ml of the wash buffer, resuspended, and acquired on the cyan adp flow cytometer (beckman coulter). results were analysed and quantified by fcap array ™ software (v3, bd biosciences). we previously reported this method to have good reproducibility with an inter-assay coefficient of variation of 6-9% (n = 8-10) 36 . total nitrate was measured in plasma samples by a nitrate/nitrite colorimetric assay kit following the manufacturer's protocol (inter-assay coefficient of variation 2.7%; cayman chemicals) as reported previously 37 . briefly, nitrate was converted to nitrite using nitrate reductase. subsequently, addition of the griess reagents converted nitrite into a deep purple azo compound and the absorbance was measured at 540 nm using an omega plate reader. histological assessments. low capillary density has been reported in the gastrocnemius muscle of pad patients and animal hli models and associated with functional impairment 38, 39 . hence at the end of experiments gastrocnemius muscle samples were collected from the mice and stored in optimal cutting compound (oct, proscitech) which was progressively frozen in isopentane (sigma) suspended in liquid nitrogen. sections (5 µm-thick) were obtained on poly l-lysine coated slides (proscitech) from each sample with muscle fibres oriented in the transverse direction. all histological assessments were performed on sections that were examined in a blinded fashion at 40x magnification. muscle fibre structure. fixed cryostat sections were stained with hematoxylin and eosin (h&e, proscitech), examined at magnifications of 100x or 400x to assess the integrity of the tissues. degenerating muscle fibres were identified in the h&e stained sections by morphological assessment. assessors looked for the presence of mature skeletal muscle fibres (small peripheral nuclei) versus immature skeletal muscle myoblasts (large lobulated central nuclei) 40 . muscle fibre number and size were examined in 5 separate fields in 4 distinct areas in each specimen. muscle fibrosis. the extent of skeletal muscle fibrosis was assessed by staining the cryostat sections (5µm-thick) with picrosirius (proscitech). briefly, tissue sections were stained and examined under 40x power light microscope and skeletal muscle fibrosis was analysed using the image analysis software (zeiss axio imager z1). quantification of fibrosis was expressed as the percentage of fibrotic tissue present within the section (1 mm 2 tissue area) using a previously published protocol 40 . immunohistochemistry and morphometric analysis of capillary and arteriolar density. these were performed as previously reported 41, 42 . the gastrocnemius muscles from ischaemic and non-ischaemic hind-limbs were collected and embedded in oct compound (proscitech), frozen, and cut into 5 µm-thick sections. the slides were fixed at −20 °c in 95% ethanol for 1 hr. slides were washed three times in cold pbs with 1% horse serum (5 min/ wash) and blocked overnight with 5% horse serum in pbs at 4 °c. immunohistochemistry was performed using primary antibodies against cd31 (1:100 dilution; abcam) and smooth muscle α-actin (α-sma, 1:200 dilution; abcam). bound primary antibodies were detected by using appropriate secondary antibodies (biotinylated anti-goat igg and biotinylated anti-rat igg, all at 1:100 dilutions, vector labs) using avidin-biotin-peroxidase (vector labs) as described previously 43 . pictures from four random areas of each section and three sections per mouse were taken by using a digital camera (nikon eclipse sci epifluorescence microscope, nikon corporation) at 40× magnification. capillary density were quantified by measuring the percentage of cd31 and α-sma staining out of the total area as previously described. western blotting. gastrocnemius muscles were mainly harvested at the end of studies (i.e. 4 weeks after full ischemia induction). gastrocnemius muscles were also harvested from a subset of mice (n = 15) subjected to the 2-stage hli (n = 5/time-point) prior to and days 3 and 7 after ameroid placement. samples were frozen in liquid nitrogen, and stored in oct compound (proscitech) at −80 °c. tissues were pulverised in ripa buffer (150 mm sodium chloride, 1.0% np-40 or triton x-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mm tris, ph 8.0) with protease inhibitors (roche diagnostics, australia) and phostop (roche diagnostics, australia) to extract proteins and quantitated using the bradford protein assay kit (biorad, usa). samples (25 μg of protein/lane) were loaded onto a 10% sds-polyacrylamide electrophoresis gel. after electrophoresis (110 v, 90 min), the separated proteins were transferred (15 ma, 60 min) to a polyvinylidene difluoride membrane (biorad, usa). non-specific sites were blocked with 5% non-fat dry milk for 60 min, and the blots were then incubated with following antibodies: anti-vascular endothelial growth factor (anti-vegf www.nature.com/scientificreports www.nature.com/scientificreports/ mrna analysis by quantitative real-time pcr. at the end of the study gastrocnemius muscle samples were harvested, placed in rna later (qiagen) and stored at −80 °c. samples (n = 10/group) were selected from each group using a random number generator and were processed for gene expression analysis. total rna was isolated using an rneasy mini kit (qiagen) according to manufacturer's instructions and quantified spectrophotometrically using nanodrop 2000. rna samples (20 ng) were subjected to quantitative real time pcr (qrt-pcr) analysis of genes of interest using the quantitect sybr green one-step rt-pcr assay (qiagen). qrt-pcr was performed using primers for mouse vegf-r1 (ppm03066f), vegf-r2 (ppm03057a), trpv4 (ppm36070a), klf4 (ppm25088b) and gapdh (qt01658692). the relative expression of these genes were calculated by using the concentration-ct-standard curve method and normalized using the average expression of mouse gapdh for each sample using the rotor-gene q operating software (version 2.0.24) as previously reported 44,45 . statistical analyses. all data were tested for normality using the d' agostino-pearson normality test. data with normal distribution were expressed as mean ± standard error of mean (sem) and analysed using parametric tests. non-normally distributed data were expressed as median and interquartile ranges (iqr) and analysed using non-parametric tests. statistical significance was determined using the unpaired student t test for comparison between two groups or analysis of variance followed by student-newman-keuls post-hoc analysis for comparison between multiple groups. comparison of the time course of ldpi indices, clinical scores, open field tests and treadmill exercise tests were done by 2-way anova for repeated measures, followed by bonferroni post hoc analysis or by linear mixed effect method using r studio software. difference in the clinical ischemia score were determined by fisher's exact test. analyses were performed using prism (graphpad software, san diego, ca) or r software. a p value of ≤0.05 was considered to be statistically significant. mice undergoing 2-stage hli had more severe ischemia than those undergoing 1-stage hli. immediately after femoral artery excision, limb perfusion assessed by ldpi was similarly reduced in both hli models by approximately 70%. mice subjected to the 1-stage hli had more rapid recovery of hind limb perfusion than those subjected to the 2-stage procedure (p = 0.014, fig. 1b,c, supplementary fig. s2 ). by 28 days after ischemia induction limb perfusion was similar in mice subjected to the 1-stage procedure and sham controls (p = 0.510) but still reduced in mice subjected to the 2-stage procedure by comparison to sham controls (p < 0.001). there was no change in overall body mass after ischemia induction (supplementary fig. s3 ). mice subjected to 2-stage hli showed more severely impaired hind limb use than those undergoing 1-stage hli. after ischemia induction, mice subjected to both methods of hli developed limb oedema, paleness of skin and occasional muscle necrosis. mice in all experimental groups exhibited a severe functional deficit after surgery (fig. 1d) . functional score was significantly worse in mice subjected to the 2-stage hli than those subjected to the 1-stage hli (p = 0.014). both the hli models showed increased ischemia compared to the respective shams. there were no cases of auto-amputation or foot or limb necrosis (supplementary fig. s4 ). mice subjected to the 2-stage hli showed a significantly worse ischemic score compared to those subjected to the 1-stage hli (repeated measures 2 way anova, p = 0.004, fig. 1e ). mice subjected to 2-stage hli had reduced treadmill performance. mice subjected to the 1-stage hli showed no significant difference in total distance travelled during the study period on a treadmill test when compared to shams (repeated measures 2 way anova, p = 0.818; fig. 2a ). after the first procedure of the 2-stage model (ameroid placement) the treadmill ambulatory performance of mice was not significantly affected ( supplementary fig. s5a ). in contrast mice subjected to 2-stage hli had a significant reduction in total distance travelled on the treadmill compared to their sham controls (p = 0.050) and mice subjected to 1-stage hli (p = 0.045; fig. 2a ). supplementary fig. s5b-d) . this reduction in physical activity was maintained after completing the 2-stage hli by comparison to sham controls and also mice subjected to 1-stage hli (fig. 2b) . the reduction in physical activity of mice subjected to 2-stage hli was reflected in less distance travelled, less total time in motion and lower velocity of the movement compared to sham controls (fig. 2c-e) . when compared to the 1-stage hli, the 2 stage-hli model showed a reduction in the total distance travelled in the open field arena (linear mixed effect model, p = 0.036). mice subjected to hli had enhanced mechanical allodynia. mice subjected to both 1-stage and 2-stage hli showed significantly increased sensitivity to pressure compared their respective sham controls and there was no significant difference in pressure sensitivity between the two models (fig. 2f ). hli induces systemic inflammation. the plasma concentrations of the cytokines assessed were below the detectable ranges in both the sham control groups (table 1) . mice subjected to hli, irrespective of model, had plasma cytokine concentration significantly higher than the sham controls although levels were not significantly different between models ( table 1 ). the plasma concentrations of nitric oxide (no) metabolites were higher in mice undergoing 1-stage hli than the sham control group (p < 0.001) and mice undergoing 2-stage hli (p = 0.031, table 1 ). myofibers which are healthy and functionally active are characterised by peripheral nuclei, while myofibrils with central nuclei are immature and do not show optimal contraction 46 . in gastrocnemius muscle samples removed from sham controls, myocytes were angular with peripheral nucleus (fig. 3a) . at day 28 after ischemia induction, mice subjected to 2-stage hli showed microscopic changes such as cellular swelling, focal necrosis and interstitial oedema. there were also numerous infiltrating inflammatory cells. gastrocnemius muscle samples from mice subjected to 1-stage hli showed more homogenous appearance with all myocytes showing peripheral nuclei and limited inflammatory cell infiltration (fig. 3a) . histological evaluation revealed that tissues of mice subjected to 1-stage hli had fewer immature myofibers cells than the tissues from 2-stage hli model. furthermore, muscle samples from mice subjected to 2-stage had prominent oedema, myofibre separation and multifocal neutrophilic infiltration. neutrophils were observed throughout the tissue sections. necrotic muscle fibres were prominent and formed confluent areas (fig. 3a) . muscle fibrosis was assessed by picrosirius red staining which suggested that 2-stage hli led to increased skeletal muscle fibrosis compared to 1-stage hli (p = 0.007) or sham controls (p = 0.001; fig. 3b ,e). mice subjected to 2-stage hli had fewer hind limb collaterals. consistent with the reduced perfusion as assessed by ldpi, both arteriogenesis and angiogenesis was inhibited in the ischemic gastrocnemius muscles of the 2-stage hli model (fig. 3c,d) . measurement of angiogenesis by cd31 immunostaining showed that the presence of arterioles was significantly reduced in samples from the 2-stage hli compared to the 1-stage hli (p = 0.002) or sham controls (p = 0.004; fig. 3 ). the arteriolar density was also significantly less in samples from the 2-stage hli model compared to the 1-stage hli (p = 0.001) or sham control (p = 0.004; fig. 3 ). protein concentrations of angiogenesis and shear stress response markers were downregulated in the gastrocnemius muscles of mice undergoing 2-stage hli. the relative total vegf and vegfr-2 (but not vegfr-1, p-enos/enos and hif-α) protein levels in gastrocnemius tissue collected from mice 4 weeks after 2-stage hli were significantly less than in sham controls and mice undergoing 1-stage hli (fig. 4a-d, supplementary fig. s6 ). analysis of tissues from the 2-stage hli model prior to ameroid placement and day 3 and day 7 after ameroid placement suggested no significant changes in concentrations of trpv4 or vegf in response to ameroid constriction ( supplementary fig. s7 ). at the end of the experiment, protein concentrations of trpv4 and klf4 were significantly less in the gastrocnemius muscle samples from mice undergoing 2-stage hli compared to sham controls and mice undergoing 1-stage hli (fig. 4e,f) . qrt-pcr showed that the relative expressions of vegf-r2, trpv4 and klf4 in gastrocnemius muscle of mice subjected to 2-stage hli were significantly lower than within the gastrocnemius muscle of mice undergoing 1-stage hli or in sham controls (fig. 4g-j) . exercise training improved functional capacity but not limb perfusion in mice subjected to 2-stage hli. supervised exercise training is an established method to improve functional capacity in pad patients 47, 48 and previous studies show that mice respond positively to exercise training 49, 50 . in order to examine whether an established clinically effective therapy was effective in the novel animal model, the effect of exercise training (using a running wheel) on functional capacity of mice subjected to 2-stage hli was assessed. exercise training was commenced 5 days after ischemia induction. exercise training did not affect limb perfusion as shows the data from the ischemic limbs from the 1-stage and 2-stage hli models and the respective sham controls (n = 8/group, scale bars in all images = 25 µm). (e-g) quantitative bar graphs showing the effect of hli on (e) skeletal muscle fibrosis by picrosirius red staining, (f) angiogenesis by immunohistochemical staining against cd31 and (g) arteriogenesis by immunohistochemical staining against α-sma. all values are median and interquartile ranges (n = 8/group) and p value significance set at ≤0.05. (2020) 10:3449 | https://doi.org/10.1038/s41598-020-60352-4 www.nature.com/scientificreports www.nature.com/scientificreports/ www.nature.com/scientificreports www.nature.com/scientificreports/ assessed by ldpi (linear mixed effect test, p = 0.700; fig. 5b ,c). mice subjected to exercise training showed a significant increase in average treadmill walking capacity compared to sedentary controls (p = 0.003; fig. 5d ). exercise training upregulated gastrocnemius muscle vegf and trpv4 levels. since improvement in ambulatory performance as a result of exercise training could be due to enhanced angiogenesis, the expression of angiogenesis and shear stress responsive proteins vegf, vegf-r1, vegf-r2, trpv4 and klf4 were assessed in the gastrocnemius muscles (fig. 6). vegf (p = 0.001, fig. 6b ) and trpv4 (p = 0.027, fig. 6e ) but not vegf-r1, vegf-r2 and klf4, were highly upregulated following exercise training (fig. 6c,d,f ). this report describes the development of a novel model of hli which results in more severe and prolonged ischemia than the traditional model. mice subjected to the 2-stage hli had functional and ambulatory impairment and a positive response to exercise training as has been reported for pad patients 51 . previous rodent studies suggest that placing of ameroid constrictors alone without manipulating the femoral artery results in mild ischemia 20 and blood flow recovers within 2 weeks 16 . hence, the novel hli model was based on placement of two ameroid constrictors on the femoral artery to promote gradual occlusion followed by excision of intervening segment after 14 days to induce severe ischemia. a previous report suggests that recovery of hind limb blood flow is reduced in apoe −/− compared to c57bl/6 j mice due to their limited collateral arteries and hence apoe −/− mice were used in the current study 24 . pad patients are generally older and exhibit metabolic derangements that limit angio-and arterio-genesis and previous studies suggest that apoe −/− mice have delayed skeletal muscle healing 52 , reduced angiogenesis responses and impaired functional recovery after hli further supporting the rationale for choosing this mice species 26, 28, 53 . mice subjected to 1-stage hli had rapid recovery of limb blood flow and reached a perfusion level similar to the sham controls within 28 days as has been reported by other investigators 28, 54, 55 . ligation and sudden excision of the femoral artery is believed to generate a pressure gradient between the proximal and distal ends of the occluded vessel, resulting in increased shear stress and a redirection of blood flow towards the collaterals and through numerous branches arising from the internal iliac artery, resulting in rapid improvement in blood flow 14, 56 . ameroid constrictors have been shown to cause luminal occlusion within 14 days, however, the blood flow slowly increases in the next 2-3 weeks 16, 21 . hence, in the new model, we superimposed a secondary acute event by excising the intervening femoral artery segment along with the ameroid constrictors. in contrast to the 1-stage hli, mice subjected to 2-stage hli had on-going limb ischemia and a prolonged functional deficit on both forced and voluntary ambulation tests. these findings support the value of the novel model for the testing of interventions aimed at achieving clinical improvements in pad patients. since angiogenesis is an inflammation-driven process 57 , the concentrations of circulating cytokines were measured. these markers of systemic inflammation increased in response to hli in both models examined. twenty eight days after hli induction, the concentrations of circulating cytokines were similar in the two models studied. gastrocnemius muscle samples obtained from the 2-stage hli model had marked neutrophilic infiltration. it has been previously reported that inflammatory cells accumulate in hypoxic tissues and promote angiogenesis 58 . it is possible that the systemic concentrations of cytokines were not reflective of the level of inflammation within the hind limb. markers of angiogenesis and arteriogenesis in gastrocnemius tissue, such as cd31 and α-smc, were found to be less evident in the 2-stage than the 1-stage hli model. furthermore, gastrocnemius vegf and vegf-r2 protein levels and amount of total plasma no metabolites were significantly lower in the 2-stage than 1-stage hli model. vegf promotes angiogenesis 59 through binding to vegf-r2 60 expressed on endothelial cells 61 . vegf induces the release of no 62 thereby promoting microvascular perfusion and endothelial progenitor cell mobilization [63] [64] [65] . endothelial cell derived microrna, such as mir-16, have also been implicated in controlling angiogenesis through inhibiting rho gdp dissociation inhibitor (rhogdi)-α, an important regulator of enos phosphorylation 66 . it appears likely that the low levels of pro-angiogenic markers in the 2-stage hli model reflect less activation of endothelium-dependent pro-angiogenesis signalling pathways which were stimulated by collateral flow within the 1-stage model. shear stress promotes arteriogenesis by stimulating remodelling of collaterals 67, 68 . endothelial cells transduce changes in shear stress into intracellular signals which promote expression of a distinct set of genes which can control the response to ischemia [69] [70] [71] . previous studies suggest that increased shear stress promotes phosphorylation and upregulation of mechano-sensors, such as trpv4 [72] [73] [74] [75] . it was postulated that the distinct ways of inducing hli in the two models studied might be reflected in different trpv4 expression. mice subjected to 2-stage hli had lower expression of trpv4 compared to those undergoing 1-stage hli. furthermore, in the 2-stage hli model, there was no change in trpv4 protein levels after ameroid placement, suggesting that ameroid constriction is a gradual process that does not lead to shear stress changes capable of stimulating mechano-sensors like trpv4. these findings also suggest that 2-stage hli results in more limited collateral flow than the 1-stage approach. the acute reduction in arterial pressure gradient following femoral artery excision in the 1-stage hli model it thought to be registered by endothelial shear stress response elements resulting in upregulation of angiogenesis and arteriogenesis promoting genes 21 . simulation of trpv4, for example by 4α-phorbol 12, of vegf-r1 (g), vegf-r2 (h), trpv4 (i) and klf4 (j) assessed in the gastrocnemius muscles of mice undergoing hli. quantitative real time pcr (qrt pcr) was performed on extracted total mrna using specific primers and normalised to glyceraldehyde 3 phosphate dehydrogenase (gapdh) expression. data analysed by mann-whitney u test (n = 10 samples/group). (2020) 10:3449 | https://doi.org/10.1038/s41598-020-60352-4 www.nature.com/scientificreports www.nature.com/scientificreports/ 13-didecanoate, has been reported to promote increase no release and increased hind limb blood flow 73 . trpv4 deficient rodents have impaired vasodilatation [76] [77] [78] [79] . this supports the important role of trpv4 in promoting adaptation to acute limb ischemia. four weeks of exercise training led to an approximate 2-fold increase in treadmill ambulatory distance in the mice subjected to 2-stage hli, paralleling findings from pad patients [80] [81] [82] [83] . these findings suggest the novel 2-stage hli mouse model simulates the walking impairment experienced by patients. in support of the relevance of this model to patients, we found that exercise therapy improved treadmill performance without improving limb perfusion, a finding similar to that described in pad patients 47, 48, 84 . since exercise training increases shear stress in pre-existing collaterals we examined the expression of trpv4 and angiogenesis markers 85 . compared to sedentary controls, mice undergoing exercise training showed increased total protein levels of vegf and trpv4, which was in accordance with previous reports showing enhanced expression of pro-angiogenesis markers after exercise training [86] [87] [88] . overall these findings suggest flow-mediated upregulation of shear stress responsive genes is important in stimulating angiogenesis responses in the new model. this study have several strengths and weaknesses. the 1-stage hli model which is usually utilised for pad research has many limitations including its disparate pathophysiological mechanisms compared to patient presentations, the temporary nature of the ischemia and its relative responsiveness to a variety of therapies which are not effective in patients 14 . this study suggests the novel 2-stage model has clear advantages over the 1-stage model since ischemia is more severe and prolonged and 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hind-limb collaterals this research was funded by a faculty administered grant and a research infrastructure block grant from james cook university and funding from the queensland government. jg holds a practitioner fellowship from the national health and medical research council, australia (1117061) and a senior clinical research fellowship from the queensland government. smo was supported by funding from the graduate research school and college of medicine, james cook university. we would like to acknowledge with thanks the help of prof. zoltan sarnyai (james cook university) who provided access to open field test assessment facility, dr. joseph moxon (james cook university) who assisted with the lme used to analyse data from the exercise study and dr. pacific huynh (james cook university) who provided the mouse images in figs. 1a and 5a. s.m.k. lead the design of the research, undertaking of experiments, and interpretation of data and writing of the manuscript. s.m.o., j.l., s.m. and r.j.j. performed parts of the experiments, contributed to interpretation of the data and gave critical comments on the manuscript. j.g. contributed rationales for the studies, led funding applications, co-wrote the manuscript and contributed to project supervision and data interpretation. the authors declare no competing interests. supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-60352-4.correspondence and requests for materials should be addressed to j.g.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. key: cord-010278-loey5xq9 authors: huh, changgoo; nagle, james w.; kozak, christine a.; abrahamson, magnus; karlsson, stefan title: structural organization, expression and chromosomal mapping of the mouse cystatin-c-encoding gene (cst3) date: 1995-01-23 journal: gene doi: 10.1016/0378-1119(94)00728-b sha: doc_id: 10278 cord_uid: loey5xq9 cystatin c (cstc) is a potent cysteine-proteinase inhibitor. the structure of the mouse cstc-encoding gene (cst3) was examined by sequencing a 6.1-kb genomic dna containing the entire gene, as well as 0.9 kb of 5′ flanking and 1.7 kb of its 3′ flanking region. the sequence revealed that the overall organization of the gene is very similar to those of the genes encoding human cstc and other type-2 cst, with two introns at positions identical to those in the human gene. the promoter area does not contain typical tata or caat ☐es. two copies of a spl-binding motif, gggcgg, are present in the 5′ flanking region within 300 bp upstream from the initiation codon. a hexa-nucleotide, tgttct, which is a core sequence of the androgen-responsive element (are), is found in the promoter region. this region also contains a 21-nucleotide sequence, 5′-agactagcagctgactgaagc, which contains two potential binding sites for the transcription factor, ap-1. the mouse cst3 mrna was detected in all of thirteen tissues examined by northern blot analysis. cst3 was mapped in the mouse to a position on distal chromosome 2. the cystatins (cst) are a group of potent cysteineproteinase inhibitors. there are at least five distinct types in the cst superfamily, each type consisting of several proteins (rawlings and barrett, 1990; devos et al., 1993) . cstc belongs to the family of type-2 cst and consists of 120 aa, with two intrachain disulfide bonds (barrett et al., 1986) . although the proteinase-inhibiting function of the cstc has been thoroughly investigated, less is known about its broader biological role. recent reports indicate that cstc may play a role in cancer progression (sloane, 1990) , bone resorption (lerner and grubb, 1992) , modulation of neutrophil chemotactic activity and inflammation (leung-tack et al., 1990a,b) , and resistance to viral infection (collins and grubb, 1991) . furthermore, a point mutation in the cst3 gene, resulting in a leu~gln substitution, is the primary cause of autosomal dominant hereditary disorder, hereditary cstc amyloid angiopathy (hccaa) (grubb et al., 1984) . as young adults, carriers of this mutation suffer from repeated and massive brain hemorrhages due to deposition of the mutant protein in the walls of the cerebral arteries. the gene structures for several type-2 cst have been determined. the human cst1 and cst2 genes (saitoh et al., 1987) , and the human cst3 (abrahamson et al., 1990) and cst4 (freije et al., 1991) genes show very similar structural organization with respect to the number and position of the introns. structural analysis of the genes for these proteins will be necessary to understand the function and evolution of the members in the cystatin multigene superfamily. in this paper, we report the structural organization and expression of the mouse cst3 gene, compare its regulatory elements with that of other cst genes and map the cst3 gene in the mouse. using two primers mcyc3: (5'-atg gcc agc ccg ctg cgc tcc ttg-3') and mcyc4: (5'-ggc att ttt gca gct gaa ttt tgt cag-3'), a 420-bp dna fragment within the coding region was generated from mouse cst3 cdna, labeled with 32p by random primer extension, and used as a probe to screen a ~,fixli genomic dna library from 129/sv mice (purchased from stratagene, la jolla, ca, usa). among several hybridizing clones, one clone, ~,cygl3, was chosen for characterization. southern blot and pcr analysis indicated the presence of the mouse cst3 gene in a 15-kb dna insert. southern blot analysis of the genomic dna did not show any evidence for the presence of a cst3 pseudogene. a 6.1-kb genomic dna fragment containing the entire gene, as well as 0.9 kb of 5' flanking and 1.7 kb of 3' flanking region, was subjected to nt sequencing. the 6125-nt sequence covering the entire mouse cst3 gene is shown in fig. 1 . comparison of the mouse cst3 gene sequence with that of the corresponding cdna (solem et al., 1990) revealed that the gene contains two intron sequences located between the nt triplets encoding aa 55-56 and 93-94 of the proposed mature polypeptide chain, exactly as in the human cst3 gene. the presence of two introns, at homologous positions, has also been reported in the other type-2 cst genes fully characterized to date: the human csti, cst2 and cst4 genes. the intron-exon junctions in the mouse cst3 gene are all close matches to the consensus sequences for the donor and acceptor splice sites of introns (mount, 1982) . some differences were observed between the exons of the genomic dna sequence and the published cdna, the five differences are summarized in table 1 . two of these positions, 994 and 3421, are in the coding region. the nt 994 in exon 1 results in a gcc codon (coding for ala) towards the c-terminal part of the leader sequence. however, a ggc codon (coding for gly) was reported at this position in the published cdna. this gcc codon found in the genomic dna exists in the corresponding site of the rat and human cst3 cdnas. the nt 342~ in exon 2 forms a ttg codon for leu (ttt coding for phe in cdna). the differences between genomic and cdna sequence may be due to an error during cdna synthesis or due to polymorphism between the mouse strains 129/sv and balb/c. the sequence of the 0.9-kb segment flanking exon 1 of the mouse cst3 gene at the 5' end, did not reveal a typical tata or caat box in the suggested promoter area. however, a tata-box-like taaaa sequence is present at 78-82 nt upstream from the start codon. a similar slightly atypical tata-box is found at the homologous position in the human cst3 gene (ataaaa), the human cst4 gene (ataaat), the human csti and cst2 genes (ataaa). the tata-box is preceded by a spl-binding gc-box sequence (pugh and tjian, 1990) with the core consensus sequence, gggcgg, ending 23 nt upstream from the at-sequence. a corresponding sequence in the human cst3 gene is located slightly closer to the tatabox (distance 16 nt). in the human cst4 gene, a gc-box is also found in the immediate 5'-flanking region (upstream distance from the at-sequence 41 nt). by contrast, in the human csti and cst2 genes, a segment similar to the caat consensus is found instead of the gc box. a core sequence of the are, tgttct, is found 22 nt downstream from the taaaa sequence. this hexanucleotide is located in a partial palindromic setting. some naturally occurring sequences and synthetic constructs containing this core sequence in a partial palindromic structure were shown to be inducible with androgens (ham et al., 1988) . this are is not found in the promoters of any other type-2 cst genes published. however, it is present in cst-related protein-encoding genes whose expression is regulated by androgen in the ventral prostate and lachrymal gland (chamberlain et al., 1983 ). an exact match of nine nt with the pituitary transcription factor (pit-l) recognition element is centered around nt -795 from the start codon, but is probably of low significance for the expression of the gene because multiple recognition elements have been shown to be needed for markedly increased expression of the rat prolactin gene by pit-1 (ingraham et al., 1988 recognized by the leader binding protein (lbp-1), 5'-wctgg-3' or its inverse, that is present in several copies in the hiv-1 promoter and contribute to its basal function (jones et al., 1988) , is strikingly abundant in the 5'-flanking region of the mouse cst3 gene. another five lbp-i motifs are found within a 300-bp segment in the first part of the first intron. transcription factor ap-l-binding sites that bind the jun-fos protooncogene complexes contain the consensus sequence 5'-tgactcagc. the mouse cst3 promoter contains two ap-l-like binding sequences within the sequence 5'-agactagcagctgactgaagc, immediately upstream from the first spl-binding site. this 21-mer sequence contains direct repeats of two adjacent potential ap-l-binding sites, each slightly deviating from the consensus sequence. it has been shown that two adjacent ap-l-like binding sites act synergistically to confer inducibility beyond that observed for a single ap-1 consensus sequence (friling et al., 1992) . the presence of the two ap-l-like binding sites in the promoter indicates that differences between the mouse cst3 gene sequence and that of the published edna (solem et al., 1990) position" genomic dna b cdna c aa a (genomic/cdna) 994 c (11) g ala/gly 3421 g(5) t leu/phe 4393 c 15) t 4564-5 gc (3) at a the nt positions refer to fig. 1 . b the nt refers to the genomic dna sequence. sequence was determined on both strands (number of independent sequencing runs in parentheses). c the nt refers to the cdna sequence. d the aa encoded by nt in columns genomic dna and cdna. transcription factor ap-1 may play a role in the cst3 gene expression. there is evidence that induction of gene expression by tgf-13 is mediated by transcription factor ap-1. autoregulation of tgf-131 expression is mediated by the binding of ap-1 to a loose consensus binding site, tgagaca, in the tgf-131 promoter (kim et al., 1990) . a strong positive regulation of the cst3 gene by tgf-13 in serum free mouse embryo cells has also been reported (solem et al., 1990) . the presence of ap-l-like binding sites in the mouse cst3 promoter suggests that cystatin c induction by tgf-[3 may be mediated by the ap-i complex, the y-flanking region of the human cst3 gene has a notably high g+c content, with >70% g+c in the 400 bp sequence upstream from the start codon. the g+c-rich region also includes the coding part of exon 1 and the 5' part of the first intron, which together represents a 900-bp segment with a g+c content of 73%, and contains cpg/gpc dinucleotides in a ratio close to unity (abrahamson et al., 1990) . the immediate 5'-flanking region of the mouse cst3 gene does not have such a strikingly high g + c content, but is more similar to the human cst1, cst2 and cst4 genes in having a gc content of approx. 60%. however, the cpg/gpc ratio is 1/1.3 in the 300 bp region upstream from the start codon (as compared to 1/4.1 over the entire 6. l-kb sequenced region), differing markedly from ratios of 1/6, 1/9 and 1/16 for the human cst4, csti and cst2 genes, respectively. thus, the mouse cst3 gene y-flanking region is not a typical housekeeping gene promoter having extremely high g + c content. rather it is similar to these promoters and the human cst3 promoter because it displays several spl-binding sites and contains a high number of cpg dinucleotides. this may indicate a low degree of methylation due to constant transcription of the gene (bird, 1986) . proposed promoter regions of several type-2 cst are compared in fig. 2 (saitoh et al., 1987 : abrahamson et al., 1990 : freije et al., 1991 . sequence determination of the mouse cst3 gene 1.7-kb 3'-flanking segment revealed no alternative polyadenylation signals in addition to the one present in the corresponding edna, 213bp downstream from the stop codon. analysis of short tandem repeats within the entire gene sequence revealed the presence of(gt)zl and (ga)26 in the region immediately 3'-flanking the polyadenylation signal and three stretches of perfect ct repeats, (ct)13, (ct)14 and (ct)25, 1300 bp further downstream. analysis of two multilocus crosses .was used to define the chr location for the mouse cst3 gene: (nfs/n or c58/j × m. m. musculus) × m. m. musculus ) and (nfs/n ×m. spretus)×m, spretus or c58/j (adamson et al., 1991) . dnas extracted from parental mice and progeny of the crosses were typed by southern blotting analysis for rflps of cst3 using the mouse cst3 cdna as probe. ssti digestion produced fragments of 8.6 and 6.0 kb in m. spretus and 6.0 and 2.8 kb in nfs/n and c58/j mice, and bamhi fragments of 22.0 and 18.8 kb were detected in m. m. musculus and nfs/n, respectively. inheritance of the parental fragments was followed in both crosses and compared with inheritance of almost 650 markers previously typed and mapped to all 19 autosomes and the x chromosome. as shown in fig. 3 the numbers given between adjacent loci represent percent recombination±the standard error calculated according to green (1981) . (b) chr 2 linkage maps. the map on the right was generated from the two crosses described here and indicates the position of cst3 relative to the other markers typed in this cross. distances between adjacent markers (in centimorgans) are indicated to the immediate left of the map. the map on the left is an abbreviated version of the composite genetic map (siracusa and abbott, 1993) . numbers to the left of the map are centimorgan distances from the centromere. human map locations for homologs of the underlined mouse genes are indicated to the far left of this map. and distal to snap (encoding synaptosomal associated protein), markers which were typed in these crosses as previously described (joseph et al., 1990; grimaldi et al., 1992) . it has recently been shown that the human homolog of this gene maps to 20pll (schnittger et al., 1993) . this is consistent with our results, since the distal end of mouse chr 2 contains a substantial region of linkage conservation with human chr 20 (siracusa and abbot, 1993; fig. 3b ). the human cst3 is part of a cluster which includes up to eight members of the cst gene family (schnittger et al., 1993) further suggesting that the mouse homologs of these genes are likely to map to the same site on chr 2. we examined the expression of the mouse cst3 gene in 13 different tissues by northern blot analysis using the mouse cst3 cdna probe. as expected, cst3 mrna was detected in all tissues examined, including stomach, brain, intestines, liver, muscle, spleen, heart, kidney, lung, pancreas, testis, uterus and ovary (data not shown). the pattern of the mouse cst3 gene expression is similar to that of its human counterpart. both species show expression of the gene in all tissues examined with high level of cst3 messenger rna in brain and testis, and lowest level in pancreas. this overall similarity between the two species indicates that mouse may be suitable for generating an animal model for the human genetic disease hccaa. structure and expression of the human cystatin c gene the mouse homolog of the gibbon ape leukemia virus receptor: genetic mapping and a possible receptor function in rodents nomenclature and classification of the proteins homologous with the cysteine-proteinase inhibitor chicken cystatin cpg-rich islands and the function of dna methylation isolation, properties, and androgen regulation of a 20-kilodalton protein from rat ventral prostate inhibitory effects of recombinant human cystafin c on human coronaviruses structure of rat genes encoding androgen-regulated cystatin-related proteins (crps): a new member of the cystatin superfamily structure and expression of the gene encoding cystatin d, a novel human cysteine proteinase inhibitor two adjacent ap-l-like binding sites form the electrophile-responsive element of the murine glutathione s-transferase ya subunit gene genetics and probability in animal breeding experiments genomic structure and chromosomal mapping of the murine cd40 gene abnormal metabolism of 7-trace alkaline microprotein characterization of response elements for androgens, glucocorticolds and progestins in mouse mammary tumour virus a tissue-specific transcription factor containing a homeodomain specifies a pituitary phenotype structural arrangements of transcription control domains within the 5'-untranslated leader regions of the hiv-i and hiv-2 promoters characterization and expression of the complementary dna encoding rat histidine decarboxylase autoinduction of transforming growth factor 131 is mediated by the ap-1 complex molecular genetic markers spanning mouse chromosome 10 neutrophil chemotactic activity is modulated by human cystatin c, an inhibitor of cysteine proteases modulation of phagocytosis-associated respiratory burst by human cystatin c: role of the n-terminal tetrapeptide lys-pro-pro-arg human cystatin c, a cysteine proteinase inhibitor, inhibits bone resorption in vitro stimulated by parathyroid hormone and parathyroid hormone-related peptide of malignancy a catalogue of splice junction sequences mechanism of transcriptional activation by sp 1; evidence for coactivators evolution of proteins of the cystatin superfamily human cysteineproteinase inhibitors: nucleotide sequence analysis of three members of the cystatin gene family cystatin c (cst3), the candidate gene for heretary cystatin c amyloid angiopathy (hccaa), and other members of the cystatin gene family are clustered on chromosome 20pl 1.2 mouse chromosome 2. mammal cathepsin b and cystatins: evidence for a role in cancer progression transforming growth factor beta regulates cystatin c in serum-free mouse embryo (sfme) cells we thank dr. jakob reiser for critical reading of the manuscript. key: cord-010187-ymhcfyxx authors: gromeier, matthias; lu, hui-hua; wimmer, eckard title: mouse neuropathogenic poliovirus strains cause damage in the central nervous system distinct from poliomyelitis date: 2005-03-25 journal: microb pathog doi: 10.1016/s0882-4010(05)80002-6 sha: doc_id: 10187 cord_uid: ymhcfyxx poliomyelitis as a consequence of poliovirus infection is observed only in primates. despitea host range restricted to primates, experimental infection of rodents with certain genetically well defined poliovirus strains produces neurological disease. the outcome of infection of mice with mouse-adapted poliovirus strains has been described previously mainly in terms of paralysis and death, and it was generally assumed that these strains produce the same disease syndromes in normal mice and in mice transgenic for the human poliovirus receptor (hpvr-tg mice). we report a comparison of the clinical course and the histopathological features of neurological disease resulting from intracerebral virus inoculation in normal micewith those of murine poliomyelitis in hpvr-tg mice. the consistent pattern of clinical deficits in poliomyelitic transgenic mice contrasted with highly variable neurologic disease that developed in mice infected with different mouse-adapted polioviruses. histopathological analysis showed a diffuse encephalomyelitis induced by specific poliovirus serotype 2 isolates in normal mice, that affected neuronal cell populations without discrimination, whereas in hpvr-tg animals, damage was restricted to spinal motor neurons. mouse neurovirulent strains of poliovirus type 2 differed from mouse neurovirulent poliovirus type 1 derivatives in their ability to induce cns lesions. our findings indicate that the characteristic clinical appearance and highly specific histopathological features of poliomyelitis are mediated by the hpvr. our data lead us to conclude that the tissue tropism of mouse-adapted poliovirus strains in normal mice is fundamentally different from that of poliovirus in hpvr-tg mice and primates, and that this is indicative of an as yet unknown mechanism of adsorption and uptake of the virus into cells of the murine cns. poliomyelitis is a rare neurological complication of primate infection with poliovirus (pv), a member of the picornavirus family, genus enterovirus. ingestion of virulent particles results in intestinal uptake, presumably through m-cells, 1 initial viral replication in subjacent lymphatic tissue, spread to deeper cervical and mesenteric *author to whom correspondence should be addressed. 0882-4010/95/040253+15 $08.00/0 o 1995 academic press limited lymph nodes (lymphatic phase), and ultimately entry of the virus into the bloodstream (viremic phase). 2 the latter is a prerequisite for passage of the blood brain barrier 3 and subsequent lytic infection of motor neurons. only a small proportion of pv infections lead to a distinctive neurological syndrome that ranges in severity from transient flaccid monoparesis to progressive flaccid paraplegia with respirato.ry impairment and sometimes bulbar involvement. 4 the hallmark of poliomyelitis histopathology is selective damage to anterior horn motor neurons along the entire spinal cord. spinal neurons outside the motor neuron system are characteristically spared, 5 in spite of their close anatomical relationship to the target of polioviral attack. the predominant molecular determinant of pv tropism is the hpvry the nucleotide sequence of hpvr identified it to be a member of the immunoglobulin superfamily, whose four mrna isoforms are the result of alternative splicing events, and give rise to different receptor molecules. 6'8 hpvrc~ and hpvr& are integral membrane proteins with divergent cytoplasmatic domains, whereas hpvr/~ and hpvr7 are secreted molecules lacking the putative transmembrane domain. 6 the hpvr is a highly glycosylated protein with an apparent molecular weight of 80kda2 the animal model for poliomyelitis in hpvr-tg mice showed pv-induced damage of comparable anatomical distribution as in primates, 1°'11 an observation confirming views of the hpvr as the critical determinant conferring pv susceptibility. unexpectedly, hpvr mrna and hpvr-related proteins were shown to be present in a wide variety of tissue homogenates not known to be sites of pv replication, ~'1° an observation suggesting additional limiting factors of pv susceptibility, mrnas specifying the simian 12 and murine ~3 homologues to hpvr have been isolated, but only the monkey-specific pvr can promote pv infection. ~2 attempts to use rodents as possible models of pv-induced disease resulted in the isolation of mouse-neurovirulent (ran) strains. a 1937 pv serotype 2 field isolate from lansing, michigan, [pv2(l)], which caused a syndrome described as 'polioencephalitis' upon intracerebral injection in the cotton rat, tm provided the first system of pv encephalitogenesis in the wild type (wt) mouse. ~s histopathological analyses of murine infection with the rodent-passaged pv2(l) isolate described a pattern of damage in accordance with concepts of primate poliomyelitis. ~6 a structural element conferring mouse neurovirulence to pv2(l) was determined to map in the capsid region. ~7 sufficient in causing this effect was a segment within the bc-ioop of vp1 since mouse avirulent pv type 1 (mahoney) [pvi(m)], after transposition of the lansing bc-ioop, caused neurological disease in mice. 18' 19 surprisingly, point mutations in distant regions of vp1 and vp2 could be shown to exert a mn phenotype. 2°.2~ the histopathological features of neurological disease caused by these genetically well defined mn pv strains have not been reported thus far. in addition to the selection of wt pv strains expressing a mn phenotype in mice, attenuated pv strains with a mouse host range phenotype but reduced neurovirulence have also been described [pv type 2, strain w222]. the attenuated (att) phenotype of pv2(w2) is reminiscent of the sabin strains of pv that express an att phenotype for primates. 23 the genetic determinants of viral neurotropism have been studied in several viral systems. most frequently, these determinants mapped to the envelope gene; examples are the alphaviruses sindbis virus 24 we have infected swiss-webster-, and icr-mice with a variety of mn virus isolates of different serotypical origin. type 2 strains pv2(l), pv2(mef-1), a recent pv2 isolate from india (ind), and pv2(w2) caused a fatal diffuse encephalomyelitis in these mice with considerable differences in intensity between individual viral strains. pvl(lsa), a mouse-adapted derivative of pvl(m), 37 caused a characteristic non-progressive panmyelitic syndrome. we have also studied the histopathology of murine infection with pvi(m), carrying a single mutation in position 54 of capsid protein vp1 that we constructed according to a published report. 2~ infections with each of these strains did not follow the stereotypic course of predictable progression seen in pv-induced poliomyelitis in hpvr-tg icr-mice. syndromes with distinct and consistent features followed intracerebral infection with each group of strains. these syndromes could be separately characterized and distinguished from hpvr-mediated poliomyelitis, on both clinical and histopathological grounds. poliovirus isolates and experimental animals. pv transgenic icr-mice expressing the hpvr ~° were a generous gift from a. nomoto (the university of tokyo, japan). swiss-webster-and normal icr-mice were obtained from taconic (germantown, ny, u.s.a). ultraviolet (uv) irradiation conditions. uv irradiation was performed in a uv-stratalinker (stratagene, lajolla, ca, u.s.a) a suspension of pv was irradiated at a distance of ca. 10 cm with an intensity of ca. 3.5 j/m z. virus was exposed to the irradiation source in ice-cooled plastic dishes at a solution depth of ca. 1ram. the loss of infectivity was confirmed in a plaque assay. poliovirus intracerebral infections. virus preparations were used to infect tg or normal mice with the desired input titer by intracerebral inoculation. mice were anesthetized, and a 25 gauge hypodermic needle was used to inject maximally 30 pl of virus suspension in dulbecco's minimal essential medium (dmem). the point of injection was the middle on the median between the ear pinnacle and the eye. infected mice were regularly observed for symptoms. none of the treated animals showed any external signs of damage or neurological disturbances in 24 h following injection. tissue processing, sectioning, and staining. affected animals in the final stage of their disease were sacrificed according to approved protocols, and their bodies were immediately perfused with 15 ml of phosphate buffered saline (pbs), followed by 15 ml of 4% neutral buffered paraformaldehyde. the brain and spinal cord were removed and fixed for 2 h at room temperature in the fixative used for perfusion. tissue specimens were rinsed for 30 rain in icecold pbs, then placed in 70% ethanol over night. dehydration was achieved through a scheme of gradually increasing ethanol concentration, followed by clearing in toluene for 2 h and infiltration with paraffin at 57.5°c for 2 h. neural tissues were embedded and cut on a rotary microtome at a thickness of 10/~m. tissue sections were placed on microscopic slides treated transgenic mice expressing the pv receptor tm were infected intracerebrally with pvi(m) with an amount of virus ranging from 10 4 to 5x 10 6 plaque forming units (pfu). all infected mice developed a characteristic neurological syndrome displaying stereotypic clinical features irrespective of input titer of virus and differing only with regard to onset of symptoms (table 1) . once visible signs of functional impairment were apparent, the progression of disease followed a predictable course. two to five days after intracerebral injection, initial signs were invariably a flaccid paralysis of the lower extremities and tail (fig. 1a) . the condition was rapidly progressive leading to complete immobilization and respiratory difficulty within 48 h. respiratory distress caused visible signs of bobbing of the head, strenuous respiratory effort, insufficient thoracic excursions, and nasal flaring. animals in the final stage of the disease were killed and their cns tissues processed according to standard procedures. histopathological analysis of the cns of pvl(m)-infected hpvr-tg mice revealed the spinal pathology of poliomyelitis described in earlier reports. 1°' 11 selective loss of anterior horn motor neurons along the entire spinal cord was invariably present (fig. 2b) . foci of virally-induced damage in the higher cervical cord extended into the brain stem and were accompanied by minor signs of inflammation in a predominantly perivascular distribution. characteristically, apart from a clearly defined lesion in the pyramidal cell layer of the hippocampal formation, lesions above the brain stem were absent in all cases analyzed. the appearance of initial lesions in the lumbar spine, distant from the injection site without evidence of damage to cortical motor neurons or descending tracts, indicated that virus had been disseminated via the hematogenous or cerebrospinal fluid route. twenty eight day old swiss-webster-and icr-mice were injected intracerebrally with pvi(m), pv2(l), pv2(mef-1), pv2(ind), or pv2(w2), with amounts of virus ranging from 104 to 5x 106. three groups of four animals each with different genetic backgrounds, swiss-webster and icr, were injected with the same viral strain. it is important to note that we were unableto detect clinical or histological evidence for differences in susceptibility towards pv infection between both outbred mouse strains used. therefore, in the following text, the term 'normal mouse' will refer to swiss-webster mice. all mn pv strains assayed caused poliomyelitis when injected into hpvr-tg mice (clinical and histopathological details are described later). none of the normal mice injected with pvi(m) showed clinical signs of neurological damage, whereas inoculation of type 2 pv strains produced signs of cns infection ( table 2) . mice infected with pv2(mef-1) and pv2(ind) were most severely affected. initial symptoms appeared 2-5 days post infection (p.i.) in all animals but they were inconsistent regarding the sites of manifestation and quality of functional impairment. motor symptoms consisted predominantly of spastic weakness involving the lower or upper extremities in a random fashion ( table 2) . paraplegic animals had a characteristic posture marked by kyphoscoliotic deformity (fig. 1b) . the progression of motor symptoms did not follow any apparent topographical scheme. pareses frequently were accompanied by gait ataxia or motor incoordination. clinical signs of respiratory involvement as described above usually did not appear during the course of the disease ( table 2 ). the clinical course proceeded to a terminal stage within 4 days after onset of symptoms. preterminal animals were severely emaciated once functional impairment supervened, and refused intake of fluid and food offered within their reach. compared to pv2(mef-1) and pv2(ind) a proportionally lower number of pv2(l)infected animals died (table 1) . their clinical syndrome was less variable, but also diverged from the regular pattern of disease localization and progression seen in poliomyelitic hpvr-tg mice. motor symptoms similar to those in pv2(mef-1) infected littermates dominated the clinical picture. progression of motor involvement did not follow any recognizable pattern and it only rarely included respiratory function ( table 2 ). eventually immobilization led to quick deterioration of the starving animals. pv2(w2) was previously reported to be of attenuated neuropathogenicity with respect to pv2(l) in mice. 44 accordingly, the proportion of infected animals that developed disease, and the severity and pace of progression of symptoms, were less than in animals infected with other pv type 2 isolates (table 1 ). this observation confirms the attenuated nature of the strain." the spectrum of functional deficits observed was identical to pv2(l)-induced disease. likewise, there was considerable variability in the sites involved in production of symptoms, course of progression, the clinical discrepancies between wt pv-induced poliomyelitis in hpvr-tg mice, and mouse-adapted pv2 infections in normal mice, were confirmed by histopathological differences. the severity and variability of clinical signs secondary to infection with the pv2 strains in normal mice was consistent with the histopathological findings. extent, morphology and localization of viral lesions in swiss-webster-and icr-mice were comparable. the range of vitally-induced damage encompassed a variety of lesions indiscriminately affecting structures within the cerebral hemispheres and the entire spinal cord. characteristically, spinal pathology was patchy and irregular, and it did not follow the consistent pattern of damage to the entire cord seen in pvinfected hpvr-tg mice. unexpectedly, anterior horn motor neurons remained frequently unaffected in normal mice infected with mn pv2, although severe pathological changes within the spinal cord were apparent (fig. 2c,d) . in contrast, routine poliomyelitis in hpvr-tg mice invariably led to complete and exclusive elimination of the motor neuron population in the spinal cord with only minor infiltrative changes ( fig. 2b) . unlike the limitation of vitally-induced lesions in pv infections of hpvr-tg mice to the spinal cord, pv2-induced disease involved the entire cns. microglial nodules accompanied by mixed lymphocytic and neutrophilic infiltrates were scattered throughout the brain and were found within the insular, piriform and temporal cortex (fig. 2f) , and the basal ganglia and thalamus (fig. 2h) . these structures were never affected in infected hpvr-tg mice (fig. 2e,g) . perivascular cuffing and dense infiltration of perivascular and periventricular parenchyma were distributed ubiquitously in the cns. the cerebral white matter was a frequent site of viral lesions. lymphocytic infiltrates within the cerebral or cerebellar peduncles, the internal capsule, or the long descending tracts and posterior columns within the upper cervical cord, occasionally caused rarefaction necrosis with secondary demyelination (fig. 21) . immunohistochemical staining for viral proteins with a polyclonal anti-pv2 antiserum revealed positive signals in those structures frequently involved by virallyfig. 3 . signs of widespread lesions and diffuse encephalomyelitis were most commonly associated with the isolates pv2(mef-1) and pv2(ind), whereas in pv2(l) cases a myelitic pattern of disease predominated over encephalitis. parallel to the clinical findings, overall cns damage induced by pv2(w2) was moderate in comparison with the other serotype 2 isolates. neurological damage focused on the spinal cord with rare cerebral involvement. spinal pathology qualitatively resembled the pv2 panmyelitis described above but the extent and number of lesions were reduced (data not shown). to analyze the clinical course of murine infection with pv2 in mice expressing the hpvr, tg mice were inoculated intracerebrally with pv2(mef-1) or pv2(l). the resulting clinical picture featured signs typically seen in murine poliomyelitis. the onset of disease was marked by a flaccid paraparesis, followed by ascending progressive symptoms as in poliomyelitis. all affected animals developed a preterminal dyspneic stage before they were killed. associated histopathology showed elements characteristic of hpvr-mediated poliomyelitis, as well as pv2-induced encephalomyelitis. anterior horn motor neurons had changes typical for poliomyelitis. in addition, there was almost always severe hemispheric involvement, never associated with hpvr-tg murine poliomyelitis resulting from infection with pvi(m). lesions equivalent to those seen in pv2-induced encephalomyelitis were distributed in the same widespread indiscriminate manner throughout the cns. gray matter structures were affected as well as cerebral white matter (data not shown). two groups of ten 28-day-old swiss-webster-and icr-mice were each infected intracerebrally with pvi(ls-a) at a range of 105 to 5 x 106pfu. as in previous assays, there was no notable difference in clinical signs and histopathological features in mice with different genetic background. all infected animals developed a specific neurological syndrome with stereotypical onset, clinical course, functional deficits, and pattern of progression. four days p.i., mice showed the first signs of an insidious spastic paraparesis (fig. 1c) . no ascending motor deficits occurred, respiratory function remained unaffected, and there were no fatalities. intracerebral inoculation of pvl(ls-a) was required to produce symptoms, since intravenous injection of virus with concomitant intracerebral needle puncture produced no neurological impairment. intracerebral inoculation of equal particle numbers of pvi(ls-a) virus, inactivated by uv-irradiation, also caused no neurological signs of cns damage. this finding indicated viral replication to be a prerequisite for pvi(ls-a) neuropathogenicity. histopathology of the neurological syndrome caused by this variant centered exclusively on the spinal cord. extensive infiltrates, originating from spinal blood vessels, invaded both the gray and white matter. pathologic changes involved the entire spinal cord, but did increase in extent of damage caudally (fig. 4a,b) . there was no apparent predilection for any cell subtype. motor neuron damage was seen within areas of destructive necrotic tissue change secondary to the inflammation. at no time during the infection could damage to motor neurons within the thoracic or cervical cord be distinguished (fig. 4b) , despite prominent inflammation affecting these regions. spinal sections taken from animals which underwent partial reversal of neurological defects, 4 weeks p.i., revealed complete regression of infiltrative changes and preservation of motor neuron populations (fig. 4c ). a mutant pvi(m) virus, which had previously been reported to exert neuropathogenic potential in normal mice 21 was tested in a similar manner as the above mentioned strains. pvl(vp1-54) carried a point mutation at position 54 (p1054s) within capsid protein vp1. intracerebral inoculation of each four swiss-webster-and icr-mice with at least i x 109pfu resulted in visible signs of neurological damage in 50% of affected animals in both groups. with the genetic variant constructed in this laboratory, this large inoculum was required to induce clinical signs. the nature of the neurological syndrome was partly obfuscated by the vigorous host response to the application of excess viral antigen, and the resulting clinical deterioration. sick mice showed prominent systemic signs of disease such as ruffled fur, reduced activity, and emaciation. symptoms of minor motor impairment were present in half of the diseased mice, but the limb pareses were difficult to assess in quality due to the general weakened state of the animal. mild limb weakness did not progress with any apparent disease pattern. severely weakened, emaciated animals succumbed to the effects of infection, without the clear causal relationship of neurological disease and fatal outcome seen in poliomyelitis, hpvr-tg mice injected with pv1(vp1-54) developed a neurological condition indistinguishable from pvi(m) related poliomyelitis (data not shown). similar to clinical findings, the histopathologic features induced by pvl(vp1-54) were less defined than in the other described infections. there was no supraspinal involvement, and spinal cord sections at all levels displayed widespread parenchymal invasion and reactive migroglial proliferation. no targeted attack against specific cell populations was apparent (fig. 4d) . histopathologic evidence for a primary thoracic and cervical cord affection was not present. with the rare exception of certain serotype 2 pv strains, e.g. pv2(mef-1) and pv2(l), naturally occurring pv strains can only infect primates, and even pv2(mef-1) and pv2(l) are mouse neurovirulent only when artificially inoculated by intracerebral injection. the narrow host range of pv results from its stringent dependence on the express exactly the same cell tropism in the murine cns as they do in the primate cns unlikely and, hence, that mn pvs would cause the same specific disease syndrome (poliomyelitis) in normal mice as they do in primates. to shed some light on these complex problems we have compared the disease syndromes caused by a variety of different pv strains in hpvr-tg and normal mice. we have come to the conclusion that mn pv strains do not produce poliomyelitis in normal mice. the occurrence of poliomyelitis in hpvr-tg mice corroborates the function of the hpvr as a major determining factor in the pathogenesis of the peculiar histopathological and clinical features of this disease. 1°'11 poliomyelitis in tg mice followed a stereotypic course: initial flaccid pareses of the lower limbs, followed by cranial progression, involving respiratory function, and fatal outcome. in contrast, the clinical symptoms resulting from mn pv infections in normal swiss-webster-and icr-mice could not be explained by classical terms of the syndrome poliomyelitis. infection of normal mice with mn pv strains caused a diffuse encephalomyelitis with highly variable neurological symptoms appearing in random order, at topographically unrelated sites. mouse neuropathogenicity was shared by several serotype 2 field isolates, the mouse adapted attenuated strain pv2(w2), the pvi(m) derivative pvi(ls-a), and pvi(m) with a mutation in residue 54 of capsid protein vpi. interestingly, the type 2 pv field isolates pv2(mef-1) and pv2(ind), have been reported to be mn without a lengthy process of adaptation, whereas other type 2 [pv2(l)] and type 1 strains [pvi(ls-a)] were passaged in the rodent cns before they acquired the mn phenotype. mn type 1 or type 3 pv field isolates have not been described. the molecular basis underlying this peculiar property of type 2 pv strains is not understood. earlier reports stressed the similarity between primate poliomyelitis and encephalomyelitis caused by pv2(l) in normal mice. 16 comparative studies at that time were impeded by the lack of a murine model of hpvr-mediated poliomyelitis. viral antigen was shown to be expressed in spinal anterior horn motor neurons, ~8 but in situ hybridization revealed the presence of viral rna in cells in posterior portions of the spinal cord as well as the white matter. "s previous analyses of pv neurovirulence using mn strains were based on the assumption of a similar pathogenesis of mn pv infections in wt mice and in primates. one parameter to assess the potential of viral neurovirulence is the ldso value, and in earlier studies, animals showing evidence of neurological functional impairment were generally killed without further histopathological analysis (see for example, ref. 17) . our results suggest that the expression of the mn phenotype of pv strains in mice should not be compared to the expression of pv neurovirulence in primates, the normal host. indeed, it appears that the expression of a mn phenotype in wt mice can result from very different genetic changes in pv strains, and that each genetic change can produce different syndromes. this is apparent not only from various type 2 mn strains, but also from the type 1 mn strain pvi(ls-a) [a derivative of pvi(m) whose genotype has been recently elucidated38]. histopathological changes within the cns should therefore be monitored when murine infections with mn pv strains are studied. a major determinant for the mn phenotype of pv2(l) is the bc-ioop of vp1 ~7 a surface protrusion of about 10 amino acids located at the apex of the poliovirion 46 that functions as a neutralization antigenic site. 47 exchange of the vp1 bc-ioop of the mouse-inert pvi(m) with the bc-ioop of pv2(l) produces the mn phenotype, ~8' 19 an observation that prompted speculation that the vp1 bc-ioop is involved in the recognition of a putative mouse receptor. no evidence for this hypothesis has been obtained as yet. strain pvi(ls-a), a mn derivative of pvi(m), accumulated 54 point mutations during passage in non-human tissue, of which 10 mutations led to amino acid exchanges in the capsid region28 these capsid mutations, however, were insufficient to produce the mn phenotype. instead, the mutations in capsid protein vp1 plus, surprisingly, five mutations in the coding region of the non-structural protein 2a "r°, a proteinase, produced mouse neurovirulence. the syndrome pv1 (ls-a) ir~duced in normal mice, however is distinct from the encephalomyelitis caused by type 2 pv strains. a pathological entity different from those observed with all other mn poliovirus strains, wes seen with pv1(vp1-54), a virus that we constructed according to a previous report. 21 pv1(vp1-54) is a mn derivative of pvi(m) that carries only a single amino acid exchange in capsid protein vp1 (p1054s). the mutation, located at an interpentameric interface inside the capsid, was suggested to destabilize the virion in the mouse cns, but the neurological syndrome caused by infection with pv1(vp1-54) was not histopathologically characterized. 21 infection of mice with pv1(vp1-54) led to neurological damage only after intracerebral inoculation of excess viral antigen. the poorly defined histopathological features of cns involvement lacked characteristics of specificity observed in hpvr-mediated poliomyelitis. the genotypic differences between mn pv strains and the clinical syndromes they cause, which differ from hpvr-mediated poliomyelitis, challenge the hypothesis of common determinants of a mn phenotype. rather, it appears that a multitude of non-related genetic elements affect this phenotypic marker in various ways. as indicated before, the nature of the receptor(s) used by the mn pv strains in the mouse cns remains to be determined. available data suggest that the mouse homologue to the hpvr does not serve as surrogate for the mn pv strains. 13 it is more likely that the mn pv strains enter neuronal tissue via cell-surface protein(s) unrelated to hpvr. if so, it is not surprising that the syndromes produced by these viral strains are distinct from poliomyelitis. in recent years picornaviruses other than poliovirus have been implicated as causative agents of neurological disease. these include enterovirus (ev) 70, 48 the etiologic agent of acute hemorrhagic conjunctivitis, or ev 71. 49 however, it has also been reported that the encephalomyelitis caused by the newly emerging human pathogen ev 71 did not always resemble poliomyelitis. s° the evolution of enterovirus strains with new neuropathogenic properties distinct from previously non-neuropathogenic ancestors might be due to adaptation to new receptor entities. the mouse-neurotropic pv variants with altered cell tropism constitute a precedent for the emergence of non-poliomyelitic picornaviral cns disease. we thank a. nomoto for the generous gift of the hpvr-tg mouse strain used in this study and b. jubelt and o. kew for kindly providing pv type 2 strains. we are grateful to p. coyle for critical review of this manuscript. we thank n. peress for helpful discussion and d. colflesh for help with microscopic imaging. this work was supported in part by nci grant ca28146, and nih grants ai15122 and ai32100. m.g. is a recipient of a grant from the stipendienprogramm infektionsforschung, heidelberg, germany. poliovirus 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hybrids simultaneously expressing antigenic determinants from all three serotypes poliovirus infection of cyclophosphamide-treated mice results in persistence and late paralysis: i. clinical, pathologic, and immunologic studies molecular pathogenesis of type 2 poliovirus in mice the three-dimensional structure of poliovirus at 2.9 ,a, resolution antigenic structure of picornaviruses radiculomyelitis following acute haemorrhagic conjunctivitis an apparently new enterovirus isolated from patients with disease of the central nervous system enterovirus type 71 infections: a varied clinical pattern sometimes mimicking paralytic poliomyelitis key: cord-007094-ur9sz21s authors: mahabir, esther; bauer, beth; schmidt, jörg title: rodent and germplasm trafficking: risks of microbial contamination in a high-tech biomedical world date: 2008-01-01 journal: ilar j doi: 10.1093/ilar.49.3.347 sha: doc_id: 7094 cord_uid: ur9sz21s high-tech biomedical advances have led to increases both in the number of mice used for research and in exchanges of mice and/or their tissues between institutions. the latter are associated with the risk of dissemination of infectious agents. because of the lack of international standardization of health surveillance programs, health certificates for imported rodents may be informative but may not address the needs of the importing facility. preservation of mouse germ-plasm is achieved by cryopreservation of spermatozoa, embryos, or ovaries, and embryonic stem cells are used for the production of genetically engineered mice. after embryo transfer, recipients and rederived pups that test negative in microbiological screening for relevant microorganisms are released into full barrier holding areas. however, current research shows that embryos may also transmit microorganisms, especially viruses, to the recipient mice. in this article, we discuss regulations and practical issues in the shipping of live mice and mouse tissues, including spermatozoa, embryos, ovaries, and embryonic stem cells, and review work on microbial contamination of these biological materials. in addition, we present ways to reduce the risk of transmission of pathogens to mice under routine conditions. k nowledge of the prevalence, replication, and persistence of pathogens in a mouse facility aids both in assessments of the risk of transmission to other mice and mouse facilities and in the development of suitable management strategies to eliminate such risks. but there is a lack of current, comprehensive information on the prevalence of bacteria and parasites in mice in europe (for detailed information on us prevalence of these and murine viruses see carty 2008) . with respect to murine viruses, a summary of data from the past decade shows that the most prevalent in the united states (carty 2008; livingston and riley 2003) and europe (schoondermark-van de ven et al. 2006 ) are mouse hepatitis virus (mhv 1 ), parvoviruses, mouse rotavirus, theiler's murine encephalomyelitis virus (tmev 1 ), reovirus type 3 (reo 3), sendai virus, and mouse adenovirus (madv) (fl + k87). the report on european prevalence (schoondermark-van de ven et al. 2006) showed that the main viruses of concern there are mhv (12%), parvoviruses (8.8%), mouse rotavirus (3.7%), tmev (2.2%), reo 3 (0.6%), pneumonia virus of mice (pvm, 0.2%), sendai virus (0.2%), and polyoma (0.1%). the same report noted that viruses such as lymphocytic choriomeningitis virus (lcmv), mouse k virus, ectromelia virus, madv, and mouse thymic virus (mtv) have not been detected in europe in the last decade. these data are based on serological analysis of 80% of samples obtained between 2000 and 2003 from more than 100 different institutions including universities, research centers, breeding companies, and industry in the netherlands (45%), france (35%), belgium (7%), and other european countries (13%, mainly germany and switzerland). since the discovery of murine norovirus (mnv 1 ) in 2003 (karst et al. 2003) , its prevalence is reported to be 22.1% from 12,639 mouse serum samples collected in the united states and canada in 2003 (hsu et al. 2005) . perdue and colleagues (2007) reported a prevalence ranging from 2% to 83% in sentinel mice from five us mouse facilities. in some german mouse facilities, mnv had a prevalence of 60% (nicklas et al. 2006) , 64.3% (müller et al. 2007) , and 0% to 69%, depending on the type of mouse holding area (mahabir et al. 2007b) . some viruses, such as mhv, mouse rotavirus, and sendai virus, are shed for only a short period of time, whereas ectromelia virus and pvm are shed for a moderate length of time, and other viruses such as lcmv, lactate dehydroge-nase-elevating virus (ldhv), murine cytomegalovirus (mcmv), minute virus of mice (mvm 1 ), mouse parvovirus (mpv 1 ), tmev, and mtv persist for longer periods of time (compton and riley 2001) . a comprehensive list of viruses and their target tissues is available in compton and riley (2001) . transportation of live mice and mouse cells and tissues from one institution to another is crucial to biomedical research in the international community. for the benefit of the mice, it is important to thoroughly plan such transportation before making shipping decisions. major proactive points of consideration include the observance of national legislation of all aspects of genetic technology and biosafety, international shipping regulations, packaging requirements, and consultations with and requirements of the exporting and importing institutions. us guidelines have recently been published for the transportation of laboratory animals (nrc 2006) , and european guidelines are summarized in appendix a of the european convention for the protection of vertebrate animals used for experimental and other scientific purposes (ets no. 123 ; http://conventions.coe.int). the uk laboratory animal science association (lasa) also has published guidance on the transport of laboratory animals (swallow et al. 2005) . for shipments of live mice, specialized courier companies provide professional and comprehensive services. they are experienced in global logistics as well as country-and species-specific requirements and regulatory documents and concerns. in addition to importing/exporting country requirements, the international air transport association (iata) regulations should be followed when packing and preparing live animals for air transport (iata 2007) . packages for any kind of animal transport should be designed to prevent the animals' escape, exclude the entry of microorganisms, allow visual inspection of the animals without compromising their microbiological status, and allow external disinfection of the package on arrival at the receiving facility (lee et al. 2007 ). in addition, the following measures are indispensable to ensure minimal transit time and facilitate the animals' arrival in good health at their final destination: • a recent health report by a veterinarian of the exporting institution's animal colony including a 1 1 ⁄2-year history of the colony's health status, • packaging in adequate transport boxes, with bedding, food, and water, • safety and stress reduction measures for the animals, and • early contact with the shipping corporation or carrier. for regional shipping, ground transport may pose the least stress as it entails minimal handling of the shipping container and a single environment during transportation. ground transportation should be carried out by licensed personnel in climate-controlled vehicles using the shortest routes. regional shipments by ground transportation normally involve one licensed carrier, whereas long distance or transcontinental shipping generally requires coordination of both ground transport and air freight and may be performed by multiple subcontractors, resulting in longer transit times and more handling, both of which may be more stressful for the mice (nrc 2006) . for the animals' comfort, it is particularly important to ensure suitable environmental conditions throughout the shipping. further to the stress involved in any shipment, longer journeys (particularly international or transcontinental journeys) also affect their diurnal rhythm. mice that experience shifts in light/dark cycles require up to 2 weeks to normalize (weinert et al. 1994 ) and thus require a longer period of overall adaptation and restoration, ranging from 1 to 7 days to several weeks or even months, depending on the stress to which they were exposed during shipment (obernier and baldwin 2006) . the us department of agriculture (usda) guidelines for the importation of live laboratory animals set forth regulations on international transport of laboratory mice and their tissue to the united states (http://www.aphis.usda.gov/ vs/ncie/ilive-mam.html). other us regulatory agencies that may have oversight of research animal importation or shipment are the centers for disease control and prevention (cdc), the department of transportation (dot), and the us fish and wildlife service. in general, research mice are exempt from these regulations provided that they are not carrying infectious diseases and are from the genus mus musculus. imports to countries of the european union (eu) require the permission of the local government. to ensure us customs clearance, the following documentation should accompany the shipment: a pro forma invoice stating that the shipment contains "live laboratory mice" and listing the species (mus musculus), number, gender, age, type of package, and names and addresses of the exporting and importing institutions. the invoice should also include a statement that the animals do not meet the criteria for an endangered species nor pose a risk to human health. in addition, a health certificate is required with, if appropriate, a veterinarian's statement that "animals are healthy and have not been exposed to or inoculated with any livestock or poultry disease agents exotic to the united states" and that "the animals have not originated from a facility where work with exotic disease agents affecting livestock or poultry is conducted." some countries may have additional exportation documents that must be completed before the animals leave the country of origin, and commercial airlines also may ask for additional documents, which should be applied for and prepared well ahead of the time of shipment. for live mice and the cells and tissues derived from them some institutions require material transfer agreements (mtas) or, if the material is transferred on a collaborative basis, a short plan of the research project before shipment. exportation of live mice from the united states or the european union is not regulated but the shipment must conform to iata regulations and the destination country's regulations. because each country has its own regulations and requirements it is highly advisable to use a customs broker and/or a courier company knowledgeable in live animal shipping for international shipments of live mice. cryopreservation of embryos (glenister et al. 1990; shaw and nagakata 2002) and spermatozoa has become a routine mechanism to preserve mouse models and to transfer single mice or colonies without the welfare concerns that arise in the shipping of live animals. additional advantages to shipping cryopreserved germplasm (spermatozoa, oocytes, and resulting embryos) are the suitability of cryopreserved materials for storage and use when needed and for preliminary testing of sample aliquots to determine microbiological status before use. many institutions (including both of ours) have established transgenic animal cores that can assist in the recovery of cryopreserved materials. but if this expertise is not available at an investigator's institution there are established regional repositories in countries around the world that can assist in the recovery of cryopreserved materials; a number of these repositories have joined together to form the federation of international mouse resources (fimre; table 1 ). many members are working together to assist the transfer of models across continents from one repository to another to facilitate their use by individual investigators (davisson 2006 ). these cooperative agreements will enable investigators to obtain recovered live mouse models from their local regional repository even if the model is cryopreserved and held in another country. cryopreserved oocytes, embryos, spermatozoa, and ovaries are typically shipped in a "dry shipper" in which the liquid nitrogen is absorbed in the shipper liner. these containers usually maintain an ultralow temperature for 7 to 21 days, depending on the model of shipper. in compliance with iata regulations, documentation of an inversion test should be included to demonstrate that no free liquid nitrogen is present in the shipper. cell lines, including murine embryonic stem (es 1 ) cells, and tissues are usually shipped on dry ice, which is considered a hazardous material and has special packaging and labeling requirements (set forth in the iata regulations). alternatively, for short-term (less than 24 hours) shipping, embryos can be transported in holding medium in cryopreservation straws at room temperature. likewise, murine es cells can be sent directly in the culture flask at room temperature. the importation paperwork for cryopreserved laboratory mouse tissues and cell lines is similar to that required for live animal importation to the united states (i.e., a pro forma invoice and declaration statements). in addition, any cells or tissues grown in vitro before shipment must be declared free of animal cell culture-derived products of livestock origin, particularly fetal or adult bovine serum. shipments of cell lines or tissues that contain a material of livestock origin such as fetal bovine serum require a usda importation permit. above all, proactive communication between the exporting institution, carrier, veterinary and legal authorities, regulatory agencies, and importing institution is of paramount importance for safe and successful deliveries. the oocyte and preimplantation embryo are surrounded by a zona pellucida (zp 1 ), essentially a sulphated glycoprotein gel of the order of 2% to 6% (weight/volume) (green 1997) that protects the embryo from its environment (epifano and dean 1994; wasserman et al. 1996) . after removal of the surrounding cumulus cells, the zp is composed of a complex fibrous network interspersed with numerous pores that are largest at the outer surface and decrease in size centripetally. in viremic mice the reproductive tract may be susceptible to infectious pathogens either locally or systemically and viruses can spread from there to various tissues and organs. in mouse germplasm cells, pathogens may (1) be present in the oocyte at fertilization and replicate in the embryo, fetus, or pups; (2) be present in or attached to the spermatozoa and carried into the oocyte at fertilization; (3) traverse, become embedded in, or adhere to the zp; or (4) be present in the embryo after damage to the zp during handling or manipulation such as assisted reproductive technologies (arts). oocytes and embryos with an intact zp subjected to vigorous pipetting may rupture, and are further at risk when the zp is partially disrupted from arts such as intracytoplasmic sperm injection (icsi), subzonal injection (suzi), microinjection of oocytes, and blastocyst injection. there are typically four main experimental designs to determine the risk of pathogen transmission by in vivoderived embryos (hare 1990 ): • in vitro-in vitro: embryos from clean donors are exposed to the pathogen in vitro and assayed in vitro • in vitro-in vivo: embryos from clean donors are exposed to the pathogen in vitro and transferred to seronegative recipients, which are screened for the development of antibodies to the pathogen • in vivo-in vitro: embryos are collected from infected and/or seropositive donors and assayed in vitro • in vivo-in vivo: embryos are collected from infected and/or seropositive donors and transferred to seronegative recipients, which are screened for the development of antibodies to the pathogen. in efforts to gain more knowledge about the epidemiological potential of current and emerging arts, the main difficulty of working with an in vivo system is the selection of mice that are carrying the virus as they may be seropositive but not necessarily virus positive. many of the wellknown murine pathogens are shed for a short period of time and diagnosis of virus-positive mice depends heavily on environmental monitoring. in some cases, fecal samples are adequate for antemortem determination of the microbiological status (e.g., for parvoviruses, mnv, mhv, mouse rotavirus, reo 3, and tmev). in order to gain as much information as possible about the biological interaction of the germplasm with pathogens it is generally speedier to perform experimental work that more or less simulates in vivo conditions. however, experimental designs often include infections with high viral doses to demonstrate the "worst case" scenario. in the in vitro system, pathogens may be present either in the collecting media or in or on the gametes or embryos themselves. in vitro fertilization (ivf 1 ) with infected spermatozoa and/or oocytes is an effective method to determine the risks posed by gametes or in vitro-derived embryos. the resulting embryos are analyzed in vitro by polymerase chain reaction (pcr) and/or are transferred to seronegative recipients that are monitored for seroconversion. it is also useful to screen their pups for the presence of the virus by pcr or serological analysis. a further step in determining the epidemiological potential of such germplasm is to monitor the collecting and washing fluids for the presence of the infectious agent, usually by pcr and virus isolation in cell culture. embryos are usually kept in short-term culture (e.g., for 1 day) to prevent low levels of viruses from replicating to such an extent that they pose a real threat. ivf with fresh samples of mouse spermatozoa resulted in live offspring as early as 1969 (mukherjee and cohen 1970) , and cryopreservation of sperm with subsequent ivf and recovery of live mice started in the early 1990s (yokoyama et al. 1990 ). advances in cryopreservation of spermatozoa, ivf, icsi, and embryo culture techniques make sperm cryopreservation a reliable means for preserving mouse strains and lines. few studies have examined the possibility of transmission of murine pathogens by spermatozoa. dutko and oldstone (1979) found that murine cytomegalovirus (mcmv) replicated in germ cells of the testes. baskar and colleagues (1986) confirmed this observation and found viral particles in spermatids; however, they did not detect any decreased fertility in these male mice and embryos developed normally to the blastocyst stage. tebourbi and colleagues (2002) examined whether mcmv microinjected directly into zygote-stage embryos (simulating icsi conditions) would result in infected live pups; they were able to infect the embryos, and the virus was present up until the blastocyst stage but disappeared in fetuses and pups. retroviruses have been demonstrated to replicate in epididymal sperm epithelium and associate with spermatozoa as they move through the epididymis, suggesting that venereal transmission is possible (kiessling et al. 1989) . recent studies have focused on murine pathogens that are prevalent in mouse colonies. raspa (2004, 2006) examined testes and epididymis for the presence of helicobacter typhlonius and mhv, both of which were present transiently. in these two studies, spermatozoa collected from males with testes that tested positive for either h. typhlonius or mhv failed to produce positive live pups after ivf and embryo transfer (et 1 ). peters and colleagues (2006) demonstrated that standard washing procedures in the ivf and embryo culture system are sufficient to eliminate mhv transmission when spermatozoa and oocytes are incubated with high levels of virus. mpv was recently found in spermatozoal samples (agca et al. 2007 ), but it remains to be determined if embryos generated using mpvcontaminated sperm result in infected offspring. in a recent study (mahabir et al. 2007a) we demonstrated that mvmexposed in vivo-derived embryos washed ten times are still capable of transmitting the virus to recipient female mice. however, we also demonstrated that the ivf-et procedure with mvm-contaminated spermatozoa resulted in the production of virus-free seronegative pups (mahabir et al. 2008) . additional studies are needed to determine whether other prevalent viruses such as mnv, rotavirus, or tmev can be transmitted by spermatozoa. methods to remove pathogens from mouse spermatozoa have not been widely explored but one study did demonstrate that percoll separation of spermatozoa was not sufficient to remove mpv (agca et al. 2007 ). pathogens can be transmitted by et to recipients by contaminated personnel, instruments, or equipment, and transport or wash medium, as well as in or on the embryo itself, although an intact zp is usually regarded as a mechanism for preventing pathogen transmission during et. agents that infect embryos could be viruses, bacteria, fungi, mycoplasmas, or parasites, but because of their size viruses are the most likely pathogens to be transmitted during et. oocytes are typically collected and immediately used in icsi or ivf with cryopreserved sperm, although cryopreservation of oocytes is possible and becoming more common (endoh et al. 2007) . females are superovulated and the cumulus oocyte complexes (cocs) can either be used as intact clutches for ivf or treated with hyaluronidase to remove the cumulus cells for icsi. wild-type oocytes are usually collected from female donors held in known pathogen-free colonies and are coincubated with spermatozoa containing the mutation or gene of interest. however, in rare instances, oocytes or ovarian tissue (gunasena et al. 1997; hani et al. 2006 ) may be used as the primary means to recover mutant or transgenic mouse lines and the risk posed by pathogens may be higher in these cases. ovarian tissue cryopreservation is particularly useful for mouse models in which male infertility occurs (such as the x-autosome translocation mouse lines) or for rescue of lines in which only female animals are available (disteche et al. 1979; takahashi et al. 2001) . ovarian tissue transplantation carries an increased risk compared to gametes and embryos because it is a complex organ with multiple cell types, and tissue from viremic donors is likely to transfer infectious organisms. retroviral contamination of donor tissue by a recipient female has occurred with transmission to recovered offspring (lock et al. 1988) , demonstrating that genetic alteration via retroviruses can occur in offspring as a consequence of either the donor or recipient female. ovarian tissue from mice naturally infected with mhv has been shown to transmit mhv to the recipient females and offspring (scavizzi and raspa 2004) . in contrast, h. typhlonius-infected ovarian tissue transplantation did not result in infection of the recipient female or offspring (scavizzi and raspa 2006) . researchers have found mpv in both ovarian tissue and oocytes (agca et al. 2007 ), but it is unknown whether transplantation of infected ovarian tissue results in infection of recipient females and their offspring. very small viruses belonging to the picornaviridae family-for example, mengo virus, which is 27 to 28 nm (gwatkin 1963 (gwatkin , 1966 (gwatkin , 1967 , and the coxsackie b-4 virus, which is 30 nm (heggie and gaddis 1979)-have been shown to traverse the zp of murine embryos. investigators have documented lcmv in mouse oocytes and embryos, providing evidence of transmission via the oocyte (mims 1966) , and sendai virus (100 to 200 nm) in the murine zp (lavilla-apelo et al. 1991 , 1992 tuffrey et al. 1972) . one study reported infection with polyoma in the trophectoderm but not in the inner cell mass of murine embryos (abramczuck et al. 1978 ). we include es cells in this overview as they are fundamental to the production of genetically engineered mice. during blastocyst injection, besides feeder cells and the blastocysts themselves, the es cells may harbor infectious pathogens. the exchange of es cells between laboratories worldwide may increase the risk of transmitting mouse infectious agents as es cells are often not screened for viruses, and viral contamination and infection are not readily detectable by cell morphology. although a survey of 46 es cell lines did not show the presence of murine infectious agents (nicklas and weiss 2000) , two studies showed that murine es cells infected with mhv-2 (kyuwa 1997; okumura et al. 1996) and mhv-a59 (kyuwa 1997) grew in vitro without showing either cytopathic effects or signs of differentiation okumura et al. 1996) . in addition to mhv, parvovirus and mycoplasma contamination has been found in es cells submitted to diagnostic laboratories for pcr testing (bb personal communication with lela riley, university of kansas, august 2007). the risk of es cell contamination by feeder cells and embryos can be reduced by obtaining them from mice that are free of relevant pathogens. washing protocols for embryos remove the viruses from the germplasm to a level below an infectious and/or immunogenic dose. although there are well-established washing protocols for the embryos of some domestic animals such as cattle and pigs (stringfellow and seidel 1998), none exist for murine embryos. some major contributing factors to reducing the risk of pathogen transmission during washing of the embryos include working under sterile conditions, repeated testing and use of pathogen-free reagents, use of a new pipette between washing drops, dilution of the washing fluids in a 1:100 range, and removal of zp-less or damaged embryos before transfer to suitable recipient mice. because of the zp's porous nature and depending on the virus size, even after washing there may be a risk of viruses nesting in the zp micropores. for example, reports show that trysin treatment of embryos has not been successful in removing sendai virus even after 12 washings (lavilla-apelo et al. 1991) . experimental infection with mvm and washing of embryos 10 times with medium revealed transmission of the virus by in vivo-derived embryos (mahabir et al. 2007a) but not by embryos produced by ivf of oocytes with mvm-coincubated spermatozoa (mahabir et al. 2008) . this was an unexpected result that may be due to differences in the characteristics of the embryos themselves. with respect to in vitro-derived embryos, the cumulus cells surrounding the oocytes may have adsorbed some of the virus thereby reducing the quantity left for entrapment in the micropores. furthermore, the cumulus cells block entry of the virus as micropores are present only after removal of these cells. removal of the cumulus cells and washing of oocytes have been shown to eliminate mpv contamination in mouse oocytes (agca et al. 2007) , and this approach should be sufficient to remove the majority of viral contaminants when they are not present in the oocyte itself. embryo transfer recipients in rederivation programs should be held in individually ventilated cages (ivcs 1 ) until testing shows that they are free of all unwanted microorganisms, including those listed in appendix 3 of the federation of laboratory animal science associations (felasa) recommendations (nicklas et al. 2002) . there should be a minimum of 6 weeks between the et and microbiological examination of the recipients, coinciding with weaning of the offspring at 3 or 4 weeks of age (wild-type littermate pups should not undergo health monitoring until they are at least 6 weeks old). some mouse facilities screen recipients before releasing pups into full barrier areas and, for subsequent screening of the pups themselves, use sentinel mice or random samples from the colony. other mouse facilities screen the recipient mother and at least one pup from each litter (usually a wild-type pup) before releasing rederived pups into full barrier areas. if wild-type pups are not available for screening, it may be preferable to use cohoused wild-type sentinels placed at the time of weaning rather than relying on test results of genetically altered mice whose immune status is usually unknown. a number of reports have shown that in vitro-derived 2-cell embryos (peters et al. 2006 ) and in vivo-derived 1-cell (van keuren and saunders 2004) and 2-cell embryos (carthew et al. 1983 (carthew et al. , 1985 mahabir et al. 2007a; reetz et al. 1988; suzuki et al. 1996) did not pose a risk of transferring mhv during embryo transfer. in addition, embryos from mice from holding areas that were positive for madv, mouse rotavirus, mpv, or tmev led to rederivation of the mouse lines (van keuren and saunders 2004) . the efficacy of embryo transfer for rederiving mice is reported to be high. due to short periods of virus shedding, donor mice may be seropositive but may not be virus carriers, thus leading to the production of virus-free seronegative pups. laboratories that deal with frequent imports of mice and biological materials should establish and implement effective measures to reduce the risk of transmitting infectious agents to staff or existing animal colonies at the importing institution. even collection of germplasm under sterile conditions does not prevent dissemination of infectious agents from colonies where they are prevalent. although health certificates that precede or accompany an import colony provide information on the animals' health status and a 1-to 1 1 ⁄2-year history of health data, because of the interval between collection and analysis of the samples and preparation of the health report, both mice and biological materials should be considered carriers of microorganisms unless analyses prove the absence of contaminants. imported mice should therefore be kept in quarantine pending reevaluation of their health status and biological materials should be carefully screened before use. to enable facility management to obtain sound information on imported animals' current health status at any given time, each facility should design and implement health monitoring programs based on its needs. these programs use randomly sampled mice, contact sentinels, or sentinels exposed to soiled bedding. some protocols also include monitoring of exhaust air for pathogens not transferred by soiled bedding (brielmeier et al. 2006; compton et al. 2004) . in recent years, there has been an increase in the use of ivc rack systems in laboratory rodent facilities. recent data (brielmeier et al. 2006) show that ivcs provide complete biocontainment when infected and noninfected mice are kept in separate cages in the same ivc rack provided that cage bedding is changed in class ii laminar flow hoods or cage changing cabinets (cccs) and appropriate standard operating procedures (sops) are strictly observed. in a typical ivc rack, each cage receives high-efficiency particulate air (hepa)-filtered air. mice are kept under positive pressure to protect them from airborne infectious or other noxious particulate agents in the environment (clough et al. 1995; cunliffe-beamer and les 1983; lipman et al. 1993; lipman 1999) . similarly, the exhaust air from the cages is hepa-filtered before being returned to the room environment or to the heating, ventilation, and air conditioning (hvac) system. maintaining mice under negative pressure prevents dissemination of pathogens into the environment and minimizes (gordon et al. 2001; lipman 1999) or prevents transfer of infectious agents from cage to cage in an ivc rack or room, depending on the ivc rack model. for biocontainment purposes, ivcs are optimal for holding imported mouse colonies under quarantine pending the completion of adequate microbiological analyses. irrespective of the health report from the exporting institution, some facilities conduct routine prophylactic treatments of all imported and quarantined mice for pinworm and ectoparasites. others refuse import applications for mice infected with parasites and authorize such imports only if the mice have been successfully treated at the exporting institution before shipment. once the health status of an imported mouse colony is found compatible with that of the importing institution mice may be transferred from the quarantine area to an experimental unit and released for investigators' use. although ivcs meet a number of requirements for breeding and holding mice under specific pathogen-free (spf) conditions in experimental units, filter-top cages are an economical and suitable alternative. for core breeding units and long-term holding of laboratory mice under spf conditions, full barrier areas with a wet entry system or air shower and the use of ivcs have become an international standard. in general, these units do not allow access to investigators and mice are imported into the barrier only by et. trafficking of biological materials, including germplasm and es cells, should meet the same hygienic standards as those required for importing laboratory mice. care should be taken to ensure that collection and/or processing of cells and tissues of the germplasm are performed under sterile conditions. to determine microbiological status-for example, if investigators suspect the presence of a particular agent or wish to screen for a limited number of agents-it may be useful to test aliquots of biological materials, collection media, or washing drops by molecular biological analysis. well-established sensitive pcr techniques are available for determining the microbiological status of murine biological materials in-house or by commercial laboratories. in addition, the mouse antibody production (map) test blank et al. 2004; bootz et al. 2003; livingston et al. 2004; mahabir et al. 2004 ) is also performed as antibodies to several viruses are detectable by standard serological analyses. for such tests, mice should be free of all felasa-listed microorganisms (as well as mnv) and held under conditions that prevent dissemination of infectious agents. taken together, health monitoring, evaluation of the risk of pathogen transmission by mouse and germplasm trafficking, and appropriate managerial strategies are fundamental components of a facility's quality assurance program. last but not least, the establishment and implementation of suitable quality assurance programs are based on the expertise of the facility's supervisory staff, who must have sufficient knowledge of and experience in laboratory animal science (nevalainen et al. 1999) . infection of mouse preimplantation embryos with simian virus 40 and polyoma virus detection of mouse parvovirus in mus musculus gametes, embryos, and ovarian tissues by polymerase chain reaction assay murine cytomegalovirus infection of mouse testes comparison of the mouse antibody production (map) assay and polymerase chain reaction (pcr) assays for the detection of viral contaminants virus pcr assay panels: an alternative to the mouse antibody production test comparison of the sensitivity of in vivo antibody production tests with in vitro pcr-based methods to detect infectious contamination of biological materials microbiological monitoring of laboratory mice and biocontainment in individually ventilated cages: a field study elimination of sendai (parainfluenza type 1) virus infection from mice by embryo transfer pathogenicity of mouse hepatitis virus for preimplantation mouse embryos opportunistic infections of mice and rats: jacoby and lindsey revisited a positive, individually ventilated caging system: a local barrier system to protect both animals and personnel detection of infectious agents in laboratory rodents: traditional and molecular techniques efficacy of three microbiological monitoring methods in a ventilated cage rack effectiveness of pressurized individually ventilated (piv) cages in reducing transmission of pneumonia virus of mice (pvm) (abstr) federation of international mouse resources: global networking of resource centers late replication in an xautosome translocation in the mouse: correlation with genetic inactivation and evidence for selective effects during embryogenesis murine cytomegalovirus infects spermatogenic cells the developmental ability of vitrified oocytes from different mouse strains assessed by parthenogenetic activation and intracytoplasmic sperm injection biology and structure of the zona pellucida: a target for immunocontraception genome cryopreservation: a valuable contribution to mammalian genetic research elimination of mouse allergens in the working environment: assessment of individually ventilated cage systems and ventilated cabinets in the containment of mouse allergens three-dimensional structure of the zona pellucida live births after autologous transplant of cryopreserved mouse ovaries effect of viruses on early mammalian development. i. action of mengo encephalitis virus on mouse ova cultivated in vitro effect of viruses on early mammalian development. iii. further studies concerning the interaction of mengo encephalitis virus with mouse ova passage of mengovirus through the zona pellucida of the mouse morula fertility of mice receiving vitrified adult mouse ovaries design and analysis of research on infectious disease transmission by embryos effects of viral exposure of the two-cell mouse embryo on cleavage and blastocyst formation in vitro development of a microsphere-based serologic multiplexed fluorescent immunoassay and a reverse transcriptase pcr assay to detect murine norovirus 1 infection in mice live animal regulations stat1-dependent innate immunity to a norwalk-like virus epididymis is a principal site of retrovirus expression in the mouse replication of murine coronaviruses in mouse embryonic stem cell lines in vitro characterization of embryonic stem-like cell lines derived from embryoid bodies the effectiveness of trypsin treatment to remove sendai virus adhering to the zona pellucida of mouse preimplantation embryos the effect of experimental infection of mouse preimplantation embryos with paramyxovirus sendai efficacy of disinfectants against mvm-and mnv-contaminated surfaces isolator rodent caging systems (state of the art): a critical view evaluation of isolator caging systems for protection of mice against challenge with mouse hepatitis virus diagnostic testing of mouse and rat colonies for infectious agents polymerase chain reaction (pcr) testing of biological materials for rodent pathogens as an alternative to the mouse antibody production (map) test. (abstract) 9th felasa symposium studies of the mechanism of spontaneous germline ecotropic provirus acquisition in mice mouse antibody production test: can we do without it? transmission of mouse minute virus (mvm) but not mouse hepatitis virus (mhv) following embryo transfer with experimentally exposed in vivo-derived embryos prevalence of mouse norovirus (mnv) in a large breeding and experimental mouse facility. (abstract) 10th felasa symposium production of virus-free seronegative pups from murine embryos arising from in vitro fertilization with mice minute virus-exposed spermatozoa immunofluorescence study of the carrier state and mechanism of vertical transmission in lymphocytic choriomeningitis virus infection in mice development of normal mice by in vitro fertilization genetic diversity and recombination of murine noroviruses in immunocompromised mice felasa guidelines for education of specialists in laboratory animal science (category d): report of the federation of laboratory animal science association's working group on education of specialists (category d) accepted by the felasa board of management survey of embryonic stem cells for murine infective agents recommendations for the health monitoring of rodent and rabbit colonies in breeding and experimental units mouse norovirus (mnv): erste untersuchungsergebnisse (abstract) 44. gv-solas annual meeting guidelines for the humane transportation of research animals establishing an appropriate period of acclimatization following transportation of laboratory animals maintenance of pluripotency in mouse embryonic stem cells persistently infected with murine coronavirus naturally occurring murine norovirus infection in a large research institution risk assessment of mouse hepatitis virus infection via in vitro fertilization and embryo transfer by the use of zona-intact and laser-microdissected oocytes rederivation of inbred strains of mice by means of embryo transfer tissue distribution and duration of mouse hepatitis virus in naturally infected immunocompetent icr (cd-1) and immunodeficient athymic nude-nu mouse strains used for ovarian transplantation and in vitro fertilization helicobacter typhlonius was detected in the sex organs of three mouse strains but did not transmit vertically prevalence of naturally occurring viral infections, mycoplasma pulmonis and clostridium piliforme in laboratory rodents in western europe screened from cryopreservation of transgenic mouse lines manual of the international embryo transfer society rederivation of mice by means of in vitro fertilization and embryo transfer guidance on the transport of laboratory animals rescue of a transgenic mouse line by transplantation of a frozen-thawed ovary obtained postmortem failure to infect embryos after virus injection in mouse zygotes sendai (parainfluenza 1) infection of mouse eggs rederivation of transgenic and genetargeted mice by embryo transfer constructing the mammalian egg zona pellucida: some new pieces of an old puzzle resynchronization of the circadian corticosterone rhythm after a light/dark shift in juvenile and adult mice production of normal young following transfer of mouse embryos obtained by in vitro fertilization using cryopreserved spermatozoa key: cord-354325-r73datur authors: berger, mitchell; shankar, vidya; vafai, abbas title: therapeutic applications of monoclonal antibodies date: 2002-07-31 journal: the american journal of the medical sciences doi: 10.1097/00000441-200207000-00004 sha: doc_id: 354325 cord_uid: r73datur abstract researchers have sought therapeutic applications for monoclonal antibodies since their development in 1975. however, murine-derived monoclonal antibodies may cause an immunogenic response in human patients, reducing their therapeutic efficacy. chimeric and humanized antibodies have been developed that are less likely to provoke an immune reaction in human patients than are murine-derived antibodies. antibody fragments, bispecific antibodies, and antibodies produced through the use of phage display systems and genetically modified plants and animals may aid researchers in developing new uses for monoclonal antibodies in the treatment of disease. monoclonal antibodies may have a number of promising potential therapeutic applications in the treatment of asthma, autoimmune diseases, cancer, poisoning, septicemia, substance abuse, viral infections, and other diseases. i n 1975, kohler and milstein revolutionized the field of immunology by developing monoclonal antibodies (mabs). since that time, many mabs have been developed for use in diagnostic procedures and in immunotherapy. ever since it was observed that the therapeutic use of heterologous mabs elicited immunogenic responses in humans, significant research efforts have been devoted toward creating chimeric and humanized antibodies for use in human patients. major achievements have been in the production of mabs in transgenic plants and animals. the use of phage display libraries has created customized antibodies with defined affinity and specificity. this review describes how rodent, chimeric, and humanized antibodies have each been used, with varying degrees of success to treat cancer, septicemia, autoimmune disorders, and infectious diseases. we also describe here recent applications of antibody engineering, such as the use of bispecific antibodies and antibody fragments in immunotherapy. von behring and kitasato discovered in 1890 that the serum of vaccinated persons contained certain substances, which they termed "antibodies." in 1895, they treated diphtheria with an antiserum raised against the toxin. on the basis of research on tetanus toxin and trypanosome parasites, ehrlich proposed in 1900 the "side-chain theory" of antibody formation, which hypothesized that physiologically active substances, including toxins, attach to cell surface receptors that are produced in response to toxin-cell interactions and then ejected from the cells into the bloodstream, leading to circulating antibodies. 1, 2 not until the 1950s, however, did scientists' understanding of antibodies become sufficient to lay the foundation for the development of mabs. jerne 3 postulated in 1955 a theory of natural selection for antibody formation. animals vaccinated with an antigen were expected to produce several distinct antibodies against several epitopes of the antigen. frank macfarlane burnet subsequently refined and expanded jerne's theory. 4 burnet's "clonal selection theory," as it is generally known, postulates that cells specific for synthesizing 1 type of antibody are spontaneously generated due to random somatic mutations during the maturation of the immune system and that these cells proliferate when exposed to an antigen. at about the same time, porter isolated fragment antigen binding (fab) and fragment crystalline (fc) from proteolytically cleaved rabbit ␥-globulin. 5 until the 1960s, antibody-producing cells were difficult to maintain in culture, because they died after a few days. in addition, only polyclonal antibodies could be obtained. in 1964, littlefield 6 developed a way to isolate hybrid cells from 2 parent cell lines using the hypoxanthine-aminopterin-thymi-dine (hat) selection media. in 1975, khler and milstein 7 provided the most outstanding proof of the clonal selection theory from results of heterokaryons-cell hybrids formed by the fusion of normal and malignant cells. twenty-five years after kohler and milstein produced the first monoclonal antibodies, dramatic progress has been made in using antibodies for diagnostic purposes, but the uses of mabs to treat disease have-until recently-remained somewhat limited. 8, 9 however, as this review indicates, the potential of mabs to aid in the treatment of a wide range of diseases is now beginning to be realized. antibodies are y-shaped proteins composed of peptides called heavy and light chains, the ends of which vary from antibody to antibody. each combination of heavy and light chains binds to a particular antigenic site. these glycoprotein chains are folded into domains of about 110 amino acids that become twisted into the immunoglobulin (ig) fold and are stabilized by disulfide bonds. structurally, each ig molecule consists of 2 50-kd heavy chains and 2 25-kd light chains, linked by disulfide bonds. human immunoglobulins are divided into 5 classes or isotypes based on the amino acid composition of their heavy chains: ␣, ␦, ⑀, ␥, and , denoted iga, igd, ige, igg, and igm, respectively. there are 2 kinds of light chains, (k) and (l), which are common to all 5 classes. four subclasses of igg (igg1, igg2, igg3, and igg4) and 2 subclasses of iga (iga1 and iga2) exist, each with a distinct function. secretory iga exists in dimeric form held together by a j chain and is associated with a secretory component that helps it pass through the cell membrane. 10, 11 each chain has a constant domain to bind host effector molecule and a variable domain to bind to the target antigen. light chains have 1 variable (v l ) and 1 constant domain (c l ), whereas heavy chains have 1 variable (v h ) and either 3 (␣, ␦, and ␥ chains) or 4 constant domains (⑀ and heavy chains) depending upon the isotype class. each variable domain contains 3 regions known as "hypervariable loops," also known as complementarity-determining regions (cdrs), that identify the antigen. the other amino acids in the variable (fv) domain are known as framework residues and act as a scaffold to support the loops. the v l and the v h , the c h 1 and the c l , and the 2 c h 3 domains are paired; the 2 c h 2 domains have carbohydrate side chains attached to them and are not paired. the folded constant domains may be homologous among different species, allowing hybrid domains (eg, mouse-human) to be produced. the variable domain confers specificity and affinity. the cdr amino acid sequences are extremely variable and play a large role in interac-tion with the targeted antigen. the chain type, region, and distance from the amino terminus characterize the domains. thus, c h 2 domain refers to the second constant domain of the heavy chain. upon digestion with papain, the antibody molecule is cleaved on the amino-terminal side of the disulfide bridges into 2 identical fabs and an fc fragment, whereas pepsin cleaves the antibody on the carboxy-terminal side of the disulfide bridges into 1 f(ab') 2 fragment containing both the arms of the antibody, and many small pieces of the fc fragment. currently, the following are available: whole antibodies, enzymatically produced 50-kd fab fragments, engineered 25-kd single-chain fv (scfv) antibodies consisting of the v h and v l connected by a flexible peptide linker, diabodies (noncovalent dimers of scfv), minibodies (scfv-c h 3 dimers), and heavy chain iggs found in species of the camelidae family, which are devoid of light chains and are referred to as v hh . the first mab described by kohler and milstein was created by the fusion of murine myeloma cells with murine-antibody-secreting lymphocytes. 7, 8 myeloma cells are immortalized b lymphocytes capable of secreting homogeneous antibodies. the immortal myeloma cell lacks the enzyme hypoxanthine guanosine phosphoribosyl transferase (hgprt) and is sensitive to the hat media. however, a hybrid cell known as a hybridoma, generated by the fusion of myeloma cell and an antibody-producing b cell, can survive in the hat media. the spleen b lymphocytes contribute the hgprt gene to the hybrid cell and, hence, unfused myeloma cells and spleen cells die in the hat media. the conventional method of generating mabs is the hybridoma technology in which spleen cells from immunized mice are fused with murine myeloma cells. whereas the myeloma cell imparts immortality to the hybridoma allowing cells to be cultivated indefinitely, the immune spleen b-cell confers antigen specificity. because each hybridoma is derived from a single cell, the cells within a hybridoma cell line are identical and make the same antibody molecule with same antigen-binding site and isotype, hence it is called a mab. among several excellent reviews that detail mab production with hybridomas is a recent review by dean and shepherd. 12 initial attempts to bypass the mouse to make human mabs involved fusion of human immune spleen lymphocytes with nonsecreting human myeloma partners to obtain hybrid cells that continually secrete a specific antibody. however, poor fusion of human myelomas, unsatisfactory performance of the hybrid cells, and the difficulty in accessing immune lymphocytes have prevented success. although heteromyelomas-which are fusions of hu-man and mouse myelomas-work better, these hybrids are usually unstable. attempts to use mouse myeloma cells to create hybrids and derive human mabs led to the loss of human chromosomes and the inability to make human igs. 13 unfortunately, in vitro immunization is limited by its inability to produce a secondary response and by the absence of the affinity maturation process that occurs in vivo. 13 affinity maturation process is a complex phenomenon, a consequence of intense bcell proliferation, somatic hypermutation of ig variable domain genes, and selection for b cells with high-affinity antigen binding, all occurring in the specialized microenvironment of the germinal center within the lymphoid tissue. thus, the search for an ideal fusion partner for generating human mabs has been difficult, which is why the epstein-barr virus (ebv) technique for immortalization of b lymphocytes is preferred. but immortalized b cells do not always replicate exactly the in vivo antibody response, because the tissues from which these cells were selected and the manipulations to which they are subjected to in the laboratory may alter the antibody specificity. 14, 15 protocols for the preparation of ebv virus, b cells, and cell fusion have been described elsewhere. 14, 16, 17 methods used for large-scale production of mabs may include the generation of ascites tumors in mice or in vitro mammalian cell culture fermentation by using bioreactors and continuous perfusion culture systems. 18, 19 the key issues in scale-up productions are the growth media, fermenter size, fermentation time, and purification procedures. 19 purification or downstream processing is accomplished by chromatography, fragmentation, conjugation with chelating agents, ultrafiltration, and controlled precipitation. 18 a more recent technique for producing antibodylike molecules uses what is known as the phage display library. it involves the construction of v h and v l gene libraries and their expression on the surface of a filamentous bacteriophage. developed in the 1990s, the phage display method requires repeated "panning" or screening of different antibodies based on their affinity for a specific antigen. antibody genes are linked to bacteriophage coat protein genes and the bacteriophages with the fusion genes are used to infect bacteria to create the phage display library. the resulting bacteriophages express the fusion proteins and display them on their surface, and the phage display library comprises recombinant phages, each displaying a different antigenbinding site on its surface. the phage expressing an antigen-binding domain specific for a particular antigen can be detected and isolated by binding to the surface coated with that antigen. libraries of v h and v l genes may be generated from nonimmunized donors, immunized donors who have an immune response against a particular antigen or from a synthetic library consisting of antibody fragments. 20, 21 one promising way to increase antibody yield or develop new antibodies may be by using genetically altered animals and plants. abgenix, a company in fremont, ca, has developed the transgenic "xeno-mouse," in which the mouse antibody-producing genes have been inactivated and functionally replaced by approximately 90% of the human ig gene loci in germline configuration, coding for the heavy and light chains. 22, 23 upon immunization with any specific human or nonhuman antigens, the "xeno-mouse" generates mabs, which are fully human igs, with high affinities and antigen-binding specificities. "xenomouse" strains producing specifically igg1, igg2, or igg4 isotypes have also been created to generate panels of diverse and highly specific mabs. kirin brewery company, japan, has developed another transgenic mouse known as the "trans-chromo" mouse. the endogenous igh and igg loci of the "trans-chromo" mouse were inactivated, but it harbors 2 individual human chromosome fragments, derived from human chromosomes 2 and 14, that contain whole human ig light-and heavy-chain loci, respectively. 24 these mice are capable of producing every subtype of fully human ig, including iga and igm. in these transgenic mouse models, human antibodies with high affinity to an immunized antigen are naturally selected by the murine immune system via an affinity maturation process, and thereby show increased diversity of the mabs. transgenic mice may be a suitable alternative to chimeric or humanized antibody production or the use of phage display systems to create less immunogenic or novel antibodies. 25 for instance, transgenic mice that express mabs to coronavirus in their milk have been developed. 26, 27 because ruminant animals, such as cows, goats, and sheep, produce relatively large amounts of milk, genetically-modified members of these species could also be used to produce large quantities of therapeutic proteins, including mabs. 28 plants may be a potential source of recombinant proteins, including mabs. 29 -31 plant virus vectors, such as the tobacco mosaic virus, may be used to make mabs. transgenic tobacco plants may also be used for large-scale production of recombinant iga, which is used in passive mucosal immunotherapy. 30, 31 this mab could be added to toothpaste to effectively protect against bacteria that cause tooth decay. 31, 32 recombinant antigens obtained from plants may also have therapeutic applications. for instance, attempts to immunize mice with escherichia coli heat labile enterotoxin b produced in transgenic tobacco and potato plants have proved promising. 33 hepatitis b viral surface protein produced in transgenic tobacco plants has been shown to be immunogenic in mice. 32 the genes coding for murine malignant b-cell specific markers are inserted into tobacco mosaic virus to cultivate an immunogenic protein in tobacco plants that may eventually be used in developing a vaccine against non-hodgkin lymphoma. 34, 35 other immunotherapeutic proteins under development include norwalk virus capsid proteins produced in tobacco and potatoes, cholera toxin and ct-b produced in potatoes, hepatitis b antigen produced in tobacco, anti-human igg used to detect nonagglutinating antibodies produced in alfalfa, and humanized anti-herpes simplex virus (hsv)-2 grown in soybeans. 36 -39 the food and drug administration (fda) considers antibodies to be "biopharmaceuticals"; as such, mab applications are regulated by the agency's center for biologics evaluation and research (cber) and the center for drug evaluation and research. 40, 41 the fda has created a "points to consider" document advising manufacturers of factors to consider in the production and testing of mabs intended for human use and identified information that should appear in investigational new drug or biologics license applications. 40 the "points to consider" document serves to "indicate the agency's current thinking on mab products for human use." 40 the agency's recommendations are aimed at protecting human health because viruses and cellular dna from antibody-producing cells with malignant phenotypes may be integrated into host cells after transformation. accordingly, among the points to consider are several steps-such as taking care to ensure purity of immunoconjugates and demonstrating the ability of any purification scheme to remove adventitious agents-designed to prevent contamination of the final product by human pathogens. 40 manufacturers must also adhere to animal care standards and detail steps to prevent contamination of cell culture. the points to consider document includes a list of normal human tissues used in cross-reactivity testing, tests for murine viruses, and organs to be considered in dosimetry estimates. the fda recognizes that because of species differences, animal models expressing the antigen of interest or cross-reactive epitopes are not always available. the agency has also published a guidance entitled s6 preclinical safety evaluation of biotechnology derived pharmaceuticals based on international conference of harmonization technical requirements. 41 before beginning phase 1 clinical studiesconducted to assess the safety of the drug and its mechanism of action-the fda recommends that researchers conduct in vivo and in vitro testing of the mab to assess cross-reactivity with human tissues or non-target-tissue binding. preclinical safety testing aims to evaluate the immunogenicity, crossreactivity, stability and effector functions of mabs. the metabolism, carcinogenicity, and genotoxicity of the product should also be evaluated. 41 guidance has also been published for mabs used as reagents in drug manufacturing. 42 the guidance emphasizes biological safety, performance characteristics of the reagent, and potential presence of residual amounts of the reagent in the final product. the fda general principles for testing and manufacturing are broadly applicable to many classes of antibodies, including those produced by phage display systems or transgenic plants and animals. depending on the expression system being used, other fda guidance documents, such as cber's points to produce biologicals should also be referenced. [43] [44] [45] humanizing monoclonal antibodies rodent mabs with excellent affinities and specificities have been generated using conventional hybridoma technology, but their use in clinical medicine is limited due to the immune responses they elicit in humans. for instance, the human antimouse antibody (hama) response can compromise the clinical effectiveness of murine mabs. although hama responses are directed against the murine constant regions, which represent the major antigenic features of the mouse ig, significant responses are also directed toward the murine variable regions. as a result, patients may mount an immune response against the injected murine antibodies, leading to allergic or immune complex hypersensitivities, rapid clearance of the antibody, and reduced clinical efficacy. 13,46 -51 initially, morrison and colleagues 48,52 introduced chimeric mabs in 1984, which showed several advantages over unmodified rodent antibodies. generally, chimeras combine the human constant regions with the intact rodent variable regions, replicating the rodent antibody variable regions by pcr and then cloning them into eukaryotic expression vectors containing human constant regions. 49 ideally, this allows better interaction with human effector cells and the complement system. because the fc region has little influence on the structure of the fv region, the chimeric constructs' affinity and specificity are virtually unchanged, and both rodent and chimeric antibodies cause apoptosis at a similar rate and intensity against target cells in vitro. 50, 51, 55 although chimeric antibodies have helped solve some of the problems associated with the use of rodent mabs, they still show significant immunogenicity in humans; because of their approximately 30% mouse sequence, they cause an human-antichimeric antibody response. humanized antibodies containing only the cdrs of the rodent variable region grafted onto the human variable region framework have been introduced to overcome these deficiencies. 53 the early work on recombinant, chimeric, and rodent/human antibodies happened during the mid 1980s and, by the late 1980s, greg winters and his colleagues demonstrated that a functional human-like antibody could be created by grafting the antigen-binding cdrs from variable domains of rodent antibodies onto human variable domains. numerous humanized antibodies have now been designed and constructed, and many are currently being evaluated in clinical trials. 13, 14, 39, 47, 54 efficient procedures for constructing humanized antibodies have been developed. 13, 14, 47, 55 the first step is to clone and sequence the complementary dnas (cdnas), coding for the variable domains of the mouse antibody to be humanized. the mouse hybridoma cell line is grown in an appropriate culture medium, and cells are harvested for rna isolation. polymerase chain reaction (pcr) primers that hybridize to the 5' ends of the mouse leader sequences and to the 5' ends of the mouse constant regions are designed for cloning light chain variable regions and heavy chain variable regions. cdna is synthesized from total rna, followed by pcr amplification with light and heavy chain specific primers. positive bacterial colonies containing mouse variable regions are then screened. construction of a chimeric antibody involves modifying the cloned mouse leader-variable regions at the 5'-and 3'-ends, using pcr primers to create restriction enzyme sites for convenient insertion into expression vectors, to incorporate sequences for efficient eukaryotic translation, and to incorporate splice-donor sites for rna splicing of the variable and constant regions. the adapted mouse light and heavy chain leader-variable regions are inserted into vectors containing, for example, human cytomegalovirus enhancer and promoter for transcription, a human light or heavy chain constant region, a neomycin gene for selection of transformed cells, and the simian virus 40 origin of replication in cos cells. these vectors are designed to express chimeric or reshaped human light and heavy chains in mammalian cells. the design and construction of an engineered human antibody require an analysis of the primary amino acid sequences of the mouse variable regions to identify the residues most critical in forming the antigen-binding site. a structural model of the mouse variable region is built on the basis of homology to known antibody variable regions. the framework regions (frs) of the new variable regions are modeled on frs from structurally similar immunoglobulin variable regions. the design process involves selecting human light and heavy chain variable regions that will serve as templates for the construction of a reshaped human antibody. the mouse cdrs are then joined to the frs from selected human variable regions. the primary amino acid sequences are then carefully analyzed to ascertain whether they would recreate an antigen-binding site that mimics the original mouse antibody. within the frs, the amino acid differences between the mouse and the human sequences are examined, and the relative importance of each amino acid in the formation of antigen-binding site is evaluated. minimum changes in the frs are desirable and should closely match the sequences from natural human antibodies. any potential glycosylation site in the frs of either mouse or human sequence needs to be identified and its influence on antigen binding considered. the dna sequences coding for the reshaped human variable regions, either made synthetically or based on an existing sequence that is very similar to the newly designed reshaped human variable region, are modified by pcr with specially designed oligonucleotide primers. the human variable regions together with their leader sequences are then cloned into a mammalian expression vector that already contains human constant regions. each human variable region is linked to the desired constant region via an intron. preliminary expression and analysis of the reshaped human antibodies are done by cotransfection of mammalian cell-expression vectors, 1 coding for human light chain and 1 coding for human heavy chain. the vectors will replicate in the cos cells and transiently express and secrete reshaped human antibodies. the concentration of the antibody produced can be analyzed by using an enzyme-linked immunosorbent assay. specific changes in the amino acids of the framework region may also be required to preserve the orientation and structure of the rodent cdr required for binding. computer modeling using databases containing human variable genes will identify sequences homologous to the rodent v regions. 13, 47 a computer model of the rodent fv can identify the non-cdr residues that interact with the cdr sequences, and choices can be made regarding which residues need to be included in the variable region. antibodies humanized in this way may have binding affinities up to one-third greater than the corresponding murine antibodies. 49 allergenicity is also significantly reduced. about 20 to 40% of patients exhibit a hama reaction to murine antibodies, whereas only about 7% have a similar reaction to humanized antibodies. 47,53,56 -58 humanization of mabs still has several practical difficulties. first, a detailed knowledge of the antibody structure and function is required. second, methods for efficient construction of humanized mabs are limited. third, unpredictable immunogenicity may result when a new amino acid sequence is introduced to balance affinity retention. fourth, the antibody repertoire is limited to the animal in which the progenitor mab originated. fifth, the rodent mab producing hybridoma must be isolated and thoroughly characterized. however, obstacles to humanization are gradually being surmounted. karpas et al 15 reported creating a hat-sensitive and ouabain-resistant human myeloma cell line that can fuse with human lymphoblast cells. a hat-sensitive subline of the myeloma cells that secreted only light chains was fused with ebv-transformed white blood cells that produced igg mabs to hiv-1 gp41. eventually, a clone was isolated that was polyethylene glycol-resistant and would not revert when placed in hat medium. standard polyethylene glycol fusion protocol was followed to fuse the myeloma cells with both ebvtransformed white cells and fresh white cells obtained from the peripheral blood of adults and tonsil cells from 2 children. the authors reported promising rates of hybridoma formation, stable ig production, and high yield of secreted antibodies compared with antibody-producing cell lines from mouse myeloma cells. the authors have now developed 40 hybridomas that have been secreting cells for more than 5 months. a human myeloma cell line may eventually prove useful in creating mabs against certain autoimmune diseases and cancers. this technique may also make it easier for other researchers to generate human mabs for use in therapy. antibodies may act directly when binding to a target molecule by inducing apoptosis, inhibiting cell growth, mimicking or blocking a ligand, or interfering with a key function. 53, 59 in addition, antibodies may modulate or potentiate drugs or other therapies. the antibody may itself act as an effector-as in antibody-dependent cellular cytotoxicity (adcc) or antibody-dependent complement-mediated cascade-or it may involve effector elements such as cytotoxins, enzymes, radioactive isotopes, signals for other parts of the immune system, and/or cytotoxic drugs. 49,50,60 -62 adcc occurs if fc regions on the antibodies are recognized by receptors present on cytotoxic cells, such as natural killer cells, macrophages, granulocytes, and monocytes. complement-mediated cytotoxicity ensues if the antibody binding prompts a complement cascade to occur. active immunotherapy can be accomplished if the antigen can provoke a long-lasting t-cell response, which may be achieved by administering whole cancer cell extracts or by using small antigenic peptides isolated from tumors in experimental patients or animals. 58 for instance, mabs mimicking breast cancer-specific antigens elicit anti-idiotype antibodies; this is another way of creating active immunity, which can lead to a humoral auto antibody-like immune response. 58 the use of bispecific molecules that recognize antigens on both the target cell and effector cell can increase adcc. the most effective mechanisms are blockade of a crucial ligand or growth factor or adcc in which tumor cells are killed by fc receptorbearing cytotoxic effectors. cell killing by adcc is proportional to the amount of antibody bound to a cell, whereas the blockade of an essential growth factor may not show effects until most of its receptor is saturated. a higher antigen expression by the target cell will increase antibody binding and subsequent adcc. but high receptor expression will also make it difficult to prevent the binding of a cytokine or ligand at minimum threshold. 53, 59, 63 with a bispecific antibody, the specificity of mab is combined with the cytotoxicity of immune effector cells, for instance, to neutralize a tumor. 9, 45, 63, 64 bispecific antibodies link the tumor cell directly to the killer cell via cytotoxic trigger molecules, such as t-cell receptors or fc receptors, leading to lysis and/or phagocytosis by the effector cells. although bispecific antibodies can enrich effectors at the tumor site and activate tumor bound effector cells by enabling cross-linking between effector and target cells, they can cause system-wide immune activation because of t-cell receptor cross-linking. bispecific antibodies can also mediate cellular cytotoxicity via various effector cells, including phagocytes, natural killer cells, and t-lymphocytes. another way to use the binding properties of antibodies is by conjugating antibodies to cytotoxic drugs, radioisotopes, or toxins. 49, 58, 63, 65, 66 techniques for conjugation have been described in several recent reviews and articles. 49, 58, 67, 70 mabs can be conjugated to chemotherapeutic drugs such as doxorubicin, mitomycin, and methotrexate. chemotherapeutic agents constitute cytotoxic or cytostatic drugs that can be conjugated to antibodies; thus far, however, these drugs have shown poor specificity for target cells and frequently lead to toxicity. 55, 62, 65 often, antibodies may lose reactivity upon conjugation with such agents. immunotoxins used in cancer therapy are conjugated antibodies that combine plant (ricin, gelonin, saponin, abrin, pokeweed antiviral protein) or bacterial (diphtheria toxin and pseudomonas toxin a) toxins with antibody specificity. 52, 59, 62, 63, 65, 66, 68, 69 toxins may inhibit protein synthesis even at picomolar concentrations. natural toxins, such as diphtheria toxin, can act at low concentrations, enter cells, and disrupt key cellular processes. 69 enzymes may also be conjugated to antibodies in antibody-directed enzyme-prodrug therapy. 55, 61, 62, 66, 71 an enzyme that can convert a prodrug to an active form may be conjugated to an antibody and targeted to a desired location using the antibody-binding region. they may more effectively localize tumors than chemical conjugates, and a prodrug is converted to an active drug by the enzyme at the target cells. another therapeutic technique relies on using antibody fragments, which may be produced by the traditional method (proteolytic digestion with papain) or with newer methods that may reduce damage to the binding sites. 66, 71 some newer methods attempt to construct mimetics that combine multiple cdrs from several antibodies into single molecules with molecular weights substantially lower than single chain fragments. because of their smaller size, antibody fragments may be able to penetrate tissues and tumors more readily and be less immunogenic than whole mabs. however, although antibody fragments may have better penetration than whole antibodies, their shorter half-lives may compromise clinical usefulness. 68 heteropolymerized antibodies have recently been developed by chemically linking a mouse igg mab, specific to a complement receptor site (cr1) on the human or nonhuman primate erythrocyte, to a second mouse igg mab that is specific to the targeted antigen. 72 the heteropolymer technique is based on immune adherence, in which antibody-antigen immune complexes bind to a complement receptor on the red blood cell, facilitating the phagocytosis of these complexes. 72 once introduced into the patient, the heteropolymers will bind to both the red blood cells and the targeted antigen. the antigen-antibody heteropolymer complexes are then transported to the liver and spleen, where the complexes are destroyed by macrophages, whereas the red blood cells return to the circulation unharmed. [72] [73] [74] [75] a wide variety of conditions, of both autoimmune and foreign origin, may potentially be treated with this method, including hiv infection, systemic lupus erythematosus, marburg virus infection, and myasthenia gravis. 76 -78 although mouse antibodies are currently being used with this technique, chimeric mousehuman antibodies could also be constructed. 72 it may also be possible to use the heteropolymers as "sentinels" by injecting them before antigen exposure. in 1 recent study, multiple infusions of heteropolymers provided nonimmunized monkeys with protection against an antigen (⌽174) for as long as 2 weeks. 79 when milstein and kohler announced their isolation of a mab in 1975, many thought mabs would provide an effective cancer treatment. however, early clinical trials were disappointing. 80 -81 producing mabs to tumor antigens is a complex process. first, proteins peculiar to human cancer cells are identified; then, mice are injected with human tumor cells (antigen) to stimulate an immune response. after about 30 days, the mouse lymphocytes are removed and fused with myeloma cells to isolate and reproduce hybrid cells specific to a predetermined antigen. rodent antibodies alone have been used in several trials, but the clinical effects are often minimal because of poor activation of human effector or complement cells by the fc region of the murine antibody and the accompanying hama responses. 55, 57 substituting the rodent fc region with a human fc fragment may help overcome these effects. 81 with the discovery of proto-oncogenes, secondgeneration trials in the 1990s turned to more specific tumor antigens that could be potential targets. 58 in 1994, mab 17-1a, an antibody to epithelial cell surface antigen expressed on human colorectal carcinomas, was approved for the identification of adenocarcinomas; mab17-1a may reduce the mortality and occurrence rate of colorectal cancer. 82, 83 in 1997, rituximab, a mouse-human chimeric anti-cd20 antibody, was approved for the treatment of non-hodgkin b cell lymphoma. 53, 83 rituximab binds to cd20 antigen on b cells and b cell tumors and then elicits a natural immune response that can kill malignant cells. recent animal trials have shown the potential of mab therapy in cancer. 52 increases in epidermal growth factor (egf) receptor expression have been found on many human cancers, and a fully human anti-egf receptor igg2 mab has been shown to inhibit human cancer growth in vitro and in vivo. 22 other efforts are focusing on antibody to her2 antigen in breast cancer. patients with cancer often mount an immunologic response to tumor-associated antigens with increases in cytotoxic lymphocytes and antibodies. 14 several tumor-reactive antibodies have been cloned against melanomas, colon carcinomas, ovarian, breast, and lung tumors. often, these antibodies cross-react with other malignant tissues or cell lines. 14 recently, trastuzumab (herceptin), a humanized mab that binds directly to the c-erb2 protein (her2), has been approved by the fda for the treatment of breast cancer. 52,84 -89 herceptin can be used to treat metastatic breast cancer in patients whose tumors overexpress the her2 protein (about 30% of breast cancer patients) and may be used in conjunction with other therapies such as paclitaxel (taxol). 89 antibody was developed by humanizing murine mab 4d5 by inserting antigen-binding regions of mab 4d5 into the framework of a consensus human igg1, resulting in rhumab-her2 or trastuzumab. 85 cancer cells have antigens that are specific to the tumor (tumor-specific antigens) or are present in greater concentration than normal (tumor-associated antigens). antibodies may eliminate target cells by complement action or through adcc. the variable region of the antibody recognizes and attaches to a specific antigen and the constant region, then joins with an effector cell capable of killing the targeted cancer cell. when the antigen and antibody bind, precipitation and agglutination may isolate the complex. anti-idiotype mabs that mimic-tumor associated antigens also may be used in cancer therapy. 82 the idiotype network hypothesis, formulated by lindemann and jerne, suggests that because each mature b cell secretes an antibody with unique antigenbinding specificity in the variable domain (referred to as the idiotype), anti-idiotype mabs can be generated and used as surrogate antigens or vaccines for immunization against the tumor. 82, 90 a murine anti-idiotype mab, aca125, which mimics the tumor-associated antigen ca125, was recently found to induce a specific anti-anti-idiotypic immune response in 28 of 42 patients with platinum-pretreated recurrent ovarian cancer enrolled in a phase i/ii clinical trial. 90,91 a positive immune response was associated with statistically significant (p ͻ 0.0001) prolonged survival times (19.9 ϯ 3.1 months in patients shown to have an anti-antiidiotypic response compared with 5.3 ϯ 4.3 months in patients who were anti-anti-idiotypic negative). 90, 91 in addition, peripheral blood lymphocyte mediated lysis of ca-125 expressing tumor cells increased in 9 of 18 patients after vaccination with the mab. 91 despite the use of a murine antibody, both this study and a previous phase i study involving patients with ovarian cancer found minimal side effects. 90, 91 promising early results have also been demonstrated in patients with advanced colorectal carcinoma who received a murine anti-idiotype mab that mimics an epitope of carcinoembryonic antigen (ceavac) 82, 90 and in patients with malignant melanoma who received an anti-idiotype mab (trigem) that mimics disialoganglioside gd2 92 despite promising developments, however, there are still several obstacles to effective cancer therapy with mabs. problems with the tumor, the mab, or conjugate characteristics all continue to challenge researchers. 52, 63, 84 thus far, chemotherapeutic mab therapies have faced obstacles due to the poor ability of agents to affect tumor cells preferentially over healthy cells, the intrinsic insensitivity of many tumors to these drugs, and the rapid development of resistance in tumor cells. 65, 66, 93, 94 mabs often decrease the size of tumors, but rarely lead to complete remission of solid tumors. 59 at the cellular level, mabs at low doses ideally should bind with excellent affinities. because no antigens have been identified that are expressed exclusively on tumor cells, cross-reactivity of mabs must be examined with histochemical tests on tissue sections or in animal models. careful testing must also be done to ensure that the effector molecules such as drugs, toxins, or isotopes in the conjugates do not inadvertently target healthy cells. although humanized antibodies have greater affinity than murine antibodies, this potential has yet to be translated into improved clinical outcomes against cancer. 52 however, recent success using the xenomouse technology to develop a fully human igg2 mab specific to epidermal growth factor receptor indicates the potential for future advances in this area. 22 recombinant immunotoxins, fusion proteins containing the fv of a mab and a bacterial toxin, are under development for cancer therapy. because immunotoxins exploit only the variable region-binding function of the mab, in theory just a fragment of the binding region, such as the bivalent f(ab')2 fragment, monovalent fab, scfv, or disulfide-stabilized fv fragments may be used as opposed to the larger and probably more immunogenic whole antibody. disulfide-stabilized fragments may be more stable or have higher affinity for the antigen than scfv, in which the linker may interfere with binding or fail to stabilize the fragment. 66 recombinant immunotoxins derived from pseudomonas enterotoxin have been shown to be active against lymphomas, solid tumors, and leukemias. 66 because binding specificity and affinity are the key elements, it is not known which form of the immunoconjugate is usually more effective in treating human tumors; some animal tests suggest that the whole antibody, with its longer half-life and bivalent binding, is more effective than the antibody fragments with decreased binding affinity. bispecific antibodies and single chain fvs are being developed that may be more effective because they clear faster from nontumor tissues and more deeply penetrate tumors than whole antibodies. 49, 64, 70 bispecific antibodies may be used to target tumor vascular endothelial cells, thereby limiting the tumor's blood supply and possibly inhibiting its spread. 70 advances in radiolabeling have allowed immunoconjugates to be delivered to cells and showed promise in clinical trials. 14, 63, 80, 95, 96 radioimmunotherapy uses a radiolabeled mab to deliver radioactive isotopes to targeted cells. radioisotopes such as iodine-131 and yttrium-90, which are ␤ emitters, can cause damage not only to the bound cell but also to cells adjacent to tumor cells that antibodies may not be able to reach within the tumors. lack of knowledge about the appropriate dose, biodistribution, and shedding of antigen hinders use of radioisotopes. radiolabeled mabs may also affect normal cells, depending on the extent to which reticuloendothelial cells expressing fc receptors bind to the constant regions of intact antibody molecules. using antibody fragments or constructs may modify this nonspecific uptake. 63, 68, 82, 93 radiotherapy exerts most of its effect by emitting a low dose, which exponentially decreases continuous radiation, but the antibody per se may have a cytotoxic effect. the duration of radiotherapy is determined by the half-lives of the antibody and the isotope used. the success of radioimmunotherapy is affected by the specificity, affinity, dose, and immunoreactivity of the antibody, heterogeneity of antigen expression, diffusion rate, tumor volume, blood supply and tumor location, dose rate effects, and variability in dose deposition. the choice of radionuclide, selection of the chelate used to link the mab to the radionuclide, and the mab selected are critical in development of radiolabeled mabs. 80 suitable radio nuclides include yttrium-90, iodine-131, and copper-67. iodine-131 was the first radioisotope to be used in treatment of hodgkin disease and other lymphomas. 68 yttrium-90 is potentially useful for lymphoma therapy because it decays with ␤, but not ␥, emissions that may kill other tumor cells in a cross-fire effect. 68 the energy released by yttrium is 5 times higher than that of iodine-131, which degrades rapidly after uptake into a tumor cell, causing toxicity. 80 in addition to their uses in radiotherapy, radiolabeled mabs can also be used to diagnose cancer. 18, 56, 57, 82, 94 radioactive isotopes linked with mabs may help localize tumors in a form of diagnostic imaging called immunoscintigraphy. for instance, oncoscint, a mab coupled to indium-111, may be used to detect an antigen (tag-72) found on colorectal adenocarcinomas. 56 mabs may potentially be used to suppress the immune system after transplant or to induce tolerance to transplanted organs or tissues. thus far, however, only antibodies to cd3 and cd25 are licensed for clinical use. 97 okt3, a murine igg2a antibody to human cd3, and antibodies to cd25 (il-2 receptor) have been used to reduce allograft rejection. 97 graft-versus-host disease (gvhd) is a complication of allogeneic stem cell transplant that occurs despite histocompatibility testing and use of cyclosporine and its analogs. 98 gvhd is a frequent cause of illness or death in allogeneic transplant patients and occurs when alloselective donor t cells recognize and interact with major and minor histocompatibility antiigens in the host, leading to cytokine release. mab therapy against gvhd is most effective when it is administered after a bone marrow transplant but before gvhd development by targeting t-cells before their activation. 99 for instance, 1 target is the interleukin-2 receptor ␣-chain (il-2r␣ tac protein or cd25), whose expression is a crucial step in the activation of alloreactive t cells. 97, 100 recently, it was reported that daclizumab, a humanized anti-il-2a antibody, was an effective complement to dual immunosuppression therapy in renal transplant patients. 101 because only activated cells express il-2, antibodies to this cytokine might inhibit t cells during allograft rejection and prevent generation of cytotoxic t cells. the development of hama response in patients and the decreased effectiveness of murine mab relative to human mab have thus far compromised the effectiveness of this approach. however, humanized anti-tac mabs, with better human effector functions, may survive longer in vivo and may prove less immunogenic than its murine counterpart. short-term cd4 antibody therapy may also contribute to long-term acceptance of skin and islet allografts, even after gaining immunocompetence. 102 more generally, the ability of mabs to induce tolerance to transplanted tissues and self-antigens may hold great therapeutic potential. 97, 102 mabs to cd4 and cd8 have been studied, although thus far these have not resulted in clinical success. 97 depletion or blockade of t cells by antibodies may facilitate tolerance by preventing t cells from attacking the graft, and recent t cell depletion studies in primates have proven promising. 97 mab treatments for other complications after transplants are also under study, including potential treatment of posttransplant lymphoproliferative disorder with the anti-cd20 mab rituximab and the use of anti-lfa-1 mab (odulimomab) to protect against ischemia-reperfusion injury after kidney transplants. [103] [104] [105] daclizumab, a humanized antibody that targets the anti il-2 receptor, may reduce the risk of acute rejection of a renal transplant and also lower cytomegalovirus infection rates among transplant recipients. 106 the inflammatory bowel disorder known as crohn disease has also been treated with mab therapy. 107 a chimeric igg1k antibody, infliximab (remicade), acts by binding to soluble and transmembrane tumor necrosis factor ␣ (tnf␣), preventing it from binding to its receptors on activated macrophages. the anti-tnf␣ antibody may provide relief to patients with moderately to severely active crohn's disease. in addition, the fda recently approved infliximab for the treatment of rheumatoid arthritis in combination with methotrexate. 108 -111 high levels of ige may cause bronchial hyperresponsiveness, a risk factor for asthma. [112] [113] [114] im-therapeutic applications of monoclonal antibodies mune responses mediated by ige are important in the pathogenesis of allergic asthma. in 1 recent study with subjects reporting moderate to severe allergic asthma, twice-weekly injections of recombinant humanized anti-ige antibody (rhumab-e25), which forms complexes with free ige and blocks its interaction with mast cells and basophils led to a fall in serum ige levels and slightly decreased asthma symptom scores relative to the placebo group. 112 patients receiving anti-ige were able to reduce reliance on corticosteroids. although the study must be interpreted cautiously, it is nonetheless a promising step in finding more effective asthma therapies. 112, 114, 116 approximately 400,000 cases of sepsis in the us and about 25,000 cases in the uk are reported each year. 116, 117 the mortality rate could be as high as 40 to 70%, depending on the population studied. 117, 118 mabs have been targeted to tnf␣ and tnf␣ receptors, which are key elements of the inflammatory response. unfortunately, this may inhibit many cytokines, which can impair the patients' ability to fight infection and increase the risk of secondary sepsis; also inhibiting a few cytokines may not be sufficient. although mabs targeted toward components of the host immune system, such as individual receptors or mediator cells, help in the treatment of septic shock, targeting the bacterial endotoxin or lipid a of gram-negative bacteria with mabs may be more efficacious. lipopolysaccharide endotoxin, a component of the outer cell membrane of gramnegative bacteria, consisting of a highly variable o-linked polysaccharide chain, an r core region, and lipid a, which in turn is composed of a glucosamine disaccharide backbone substituted with amide-and ester-linked long-chain fatty acids, is believed to be important in many cases of septic shock and septicemia, and can trigger an inflammatory cascade that can cause serious injury and even death. 119, 120 lipid a is linked by the core region to the o-linked side chain, and variations in the o-linked side chains result in an enormous diversity among gram-negative bacteria, making adoptive immunotherapy against the o-linked antigenic determinants difficult. 114 directing antibodies to the core region or lipid a, which tend to be more conserved, would allow for therapy against a diverse array of gram-negative bacteria. favorable results with polyclonal j5 antibody have spurred attempts to develop a mab. 116, 117 a murine igm mab, e5 (xoma, berkeley, ca) binds to an epitope on lipid a of e coli j5. a human igm, ha-1a (centoxin) is derived from a heterohybridoma fused from spleen cells of a patient vaccinated with e coli j5 before splenectomy. 116, 117, 119, 121 both the antibodies have thus far shown mixed success in patients with sepsis and neither is currently cleared by the fda for therapeutic use. because patients with bacteremia often have endotoxemia, ha-1a, an igm antibody to endotoxin may be effective against endotoxin in the bloodstream as well. unfortunately, the high cost of antibody ($3500/dose in europe) combined with the uncertainty that the sepsis is caused by gram-negative bacteria has hindered the widespread use of ha-1a. 112, 115, 117 although it is less efficacious, the only other option may be to target tnf␣, but 1 recent study reported a high (54.1%) rate of adverse reactions in patients receiving an anti-tnf␣ antibody. 118 the antibody was no more effective than the placebo given to control subjects. however, research on a mab for the treatment of septicemia continues. cytomegalovirus (cmv) causes serious illness affecting the immunocompromised, such as patients with aids and those undergoing organ transplants. infection rates may be up to 75% in those negative for cmv who receive kidneys from seropositive patients. 122 cmv infection can result in retinitis and gastroenteritis in hiv-infected patients and may also cause chronic intrauterine infection. 122, 123 up to 40,000 cases of congenital cmv infection are reported each year; mental retardation and hearing loss may occur in about 25% of these cases. 122 currently, there is no vaccine against cmv. ganciclovir, foscarnet, and (s)-1-[3-hydroxy-(2 phosphonylmethoxy)propyl]cytosine are some of the potential treatments for cmv infection. 122 another mode of treatment is via administration of anti-cmv hyperimmunoglobulin, derived from pooled sera of cmvseropositive persons. passive immunization has been shown to reduce the severity of cmv and prevent mother-to-infant transmissions. 122 moreover, antibodies may be able to clear the virus from infected tissues, a function previously thought to be exclusive to cytotoxic t lymphocytes. many physicians use a combination of antiviral agents and immunoglobulins in patients at risk for cmv infection. mabs may also decrease the amount of antiviral agents required for treatment. mabs against murine cmv polypeptides have been shown to be protective in animal models. 124 although mabs may act synergistically with foscarnet or ganciclovir in vitro, it is unclear whether this advantage could be extrapolated to in vivo conditions. experiments in which murine cmv is used as a model system suggest that high concentrations of antibody, along with ganciclovir or (s)-1-[3-hydroxy-(2 phosphonylmethoxy)propyl]cytosine, synergistically inhibit the growth of murine cmv in cell culture, whereas lower antibody concentrations produce only an additive effect. however, antibody ad-ministered along with ganciclovir in severe combined immunodeficient mice also produced only an additive effect. 123 the extent to which a mab can protect the host from cmv infection cannot reliably be predicted from its immunoreactivity, neutralizing titers in vitro, or from its antigen-binding data, because the mechanism of action of a mab in vitro and in vivo may be entirely different. 124 if an antibody is used along with drugs, not only is the treatment efficacy improved but also the side effects of a high drug concentration may be avoided. for instance, 25 mg of ganciclovir/kg of body weight, along with an antibody, was almost as effective as 50 mg/kg ganciclovir administered alone in cmv-infected severe combined immunodeficient mice. 124 the msl109 antibody, a human igg1 directed against human cmv glycoprotein, is marginally synergistic in inhibiting cmv in vitro when conjugated to ganciclovir or foscarnet. in a recent trial involving 209 aids patients with active cmv retinitis, the rate of retinitis progression was similar in the group receiving the antibody (60 mg, intravenous) for 2 weeks and the placebo control group. 125 surprisingly, mortality increased in the msl-109 group, and the poor performance of msl-109 in this trial may have been due to the inability of the antibody to neutralize the established cmv infection in aids patients or its failure to cross the blood-ocular barrier in sufficient quantities. liver transplant patients are also at risk of cmv infection and may require treatment with the murine mab okt3, an anti-t cell antibody that is directed to cd3 antigen, along with ganciclovir to mitigate transplant rejection. 126 human mabs neutralizing cmv were tested for their safety and pharmacokinetics over a period of 3 to 73 days in bone marrow transplant recipients and were found to be safe and efficacious, as evidenced by a steady increase in the neutralizing activity. 127 also, a human anti-cmv mab designated c23 that was purified from hybridomas generated by the fusion of human lymphocytes with mouse myeloma cells has virus neutralization titers about 1000-fold higher than those produced after conventional human ␥ globulin used in humans. 128 a humanized mab has been developed that binds to the 86-kd glycoprotein, gpul75 (gh), of cmv, recognizes a variety of virus strains, and neutralizes clinical isolates of cmv; it could be used as a potential agent for the prevention or treatment of cmv infections in humans. 129 respiratory syncytial virus (rsv) causes serious lower respiratory tract disease requiring considerable supportive care, administration of humidified oxygen, and respiratory assistance. the fda approved the use of a humanized mab against rsv, which afflicts mostly infants and children younger than 24 months of age. 130, 131 known as palivizumab, the antibody (produced by synagis and medimmune, inc.) treatment showed a 55% reduction in rsv infection in hospitalized patients. 130, 131 the american academy of pediatrics' committee on infectious diseases has published recommendations on the use of this antibody in children at risk for rsv infection. 130, 131 mabs have also been used in the therapy of hsv infections. the incidence of neonatal herpes is 30 to 50% in babies born vaginally to mothers with primary infection but only 1 to 3% in babies born to mothers with recurrent infections. 132 this difference is attributed to the passive transfer of protective antibodies to the fetus. antibodies to hsv-1 may also confer partial protection against type 2 strains. based on this observation, the potential of mab therapy against herpes has been studied for more than a decade. experiments in mice, for instance, indicate that microgram quantities of antibodies may promote healing of corneal opacity and blepharitis caused by herpes simplex. 133 however, attempts to confer immunity in humans and other higher animals by passive immunization alone have been disappointing, because antibodies to hsv are restricted to only a few specific epitopes. 133 among the 11 glycoproteins expressed by the herpes virus on its surface, 5 glycoproteins (gb, gd, gh, gk, and gl) are thought to be crucial to infection. the initial step in hsv infection seems to be the binding of gc to heparan sulfate proteoglycans followed by the binding of gb, and the binding of gb and gc is stabilized by gd. gh is involved in initiation of viral fusion and other glycoproteins may help in expanding the fusion process. evidence that neutralizing antibodies to gh, gd, and gb prevent membrane fusion but not viral attachment is consistent with this model. 133 most human antibodies are directed against gd and gb epitopes. mabs can target at least 7 different antigenic sites on hsv glycoprotein d in the murine model. 133, 134 glycoprotein d is necessary for virus replication in tissue culture. yamamoto et al 135 reported the antibody-dependent cellular cytotoxicity effect of hs1, a neutralizing mab to glycoprotein b of hsv, in athymic nude mice inoculated with hsv intracutaneously. when hs1 is administered, the skin lesions healed in 50% of the mice given the antibody. also, latent infection in the ganglia was prevented in mice that survived, as evidenced by the failure to detect hsv upon co-cultivation with vero cells. administration of hs1 after development of zosteriform lesions (5 to 9 days after infection) reduced the virus in the ganglia and prolonged survival time; however, the disease was not totally avoided and eventually the mice succumbed to the disease. the antibody therapy could prevent or decrease the severity of herpetic keratitis, iritis, and blepharitis after corneal infection by hsv. 136, 137 thus, mabs could potentially be used to prevent congenital herpes and sexual transmission of the herpes virus. 47, 133, 138, 139 currently, no treatments exist for the hemorrhagic fever caused by the filovirus ebola. recently, a team of researchers at the us army's medical research institute identified protective antibodies directed against epitopes on ebola virus membraneanchored glycoprotein. some of the antibodies protected mice as long as 2 days after exposure; doses were equivalent to acceptable (3 to 5 mg/kg) human levels. antibody specificity is important, because some mabs studied bound to ebola zaire but not to the ivory coast or sudan serotypes. much research remains to be done before the potential of antibodies in the treatment of ebola will become realized. 140 toxins are poisonous proteinaceous substances. antibodies were first clinically used in the 1970s for protection against the toxin digitalis. 48, 102 today, antidigitalis immunotoxicotherapy is a standard therapy. digoxin is produced from sheep immunized with digoxin-serum albumin conjugate. igg antibody specific for digoxin is purified and cleaved into fabs, because the smaller molecular mass of the fragments allows faster renal clearance and more rapid action than the whole antibody. the fab fragments bind to circulating digitalis molecules and generate a complex that is unable to bind to receptors. adverse effects are rare, and the immunotherapy is generally effective against digoxin and digitoxin within 1 hour. 120, 141 however, because polyclonal antibodies may be difficult to produce and may cause hypersensitive reactions, researchers have attempted to develop humanized mabs against digoxin and other toxins. 141 for example, 1 group has used a transgenic mouse to produce hybridomasecreted human mabs against digoxin. 141 more recently, the effect of goat anti-colchicine igg antibodies has been studied in mice exposed to a lethal intraperitoneal dose of anti-inflammatory colchicines, and preliminary studies in a 25-yearold woman with colchicine poisoning showed promise. 102, 141 attempts have also been made to use drug-specific goat antibody fragments to treat anti-tricyclic antidepressant (tca) overdose. 120, 141 antidepressant overdose is the most common cause of intentional drug overdose in the us. however, lethal doses of tricyclics are 10-to 100-fold higher than those of colchicine, digoxin, and snake venom, which require higher antibody doses (up to several g/kg) to reverse the toxicity. 120, 142 high-affinity tricyclic antidepressant-specific mabs have been shown to reverse the cardiovascular toxicity of antidepressant desipramine in rats and prolong their survival, and tricyclic antidepressant-specific fab fragments, along with sodium bicarbonate, a standard treatment for tricyclic overdose, also minimizes the required dose of antibody. 142 the use of smaller antibody fragments, such as single chain fv fragments, half the size of a fab, may also be a promising approach because the single chain fragment retains affinity and has shorter half-life in the body. monoclonal antibodies may also help to alleviate poisoning due to environmental contamination. hexachlorobiphenyl, paraquat, atrazine, 3,5,6-trichloro-2-pyridinol, the chief degradation byproduct of the insecticides chloropyrifos, triclopyr, and chloroprifosmethyl, and other chemicals may soon be detected and remediated with the help of antibodies. 139, [143] [144] [145] antibodies may also be used in the treatment of poisoning due to paraquat, hexachlorobiphenyl, domoic acid [amnesic shellfish poisoning], and other contaminants that are otherwise difficult to remove from the body or surrounding environment. [145] [146] [147] [148] the primary target of many abused drugs is the central nervous system (cns), and immunotherapy against such chemicals must be able to penetrate the cns. 149 pcp or phencyclidine (angel dust) is a type of arylcyclohexamine that affects multiple sites in the brain. 150, 151 it is linked to violent psychotic episodes that are similar to schizophrenia. 149 treatment is difficult because there is no known antagonist identified, pcp has a high volume of distribution, and about 95% of it is cleared after being metabolized. 151 recently, mabs have been described that could bind to cocaine or pcp and act like sponges in the bloodstream to prevent them from reaching the brain. 152 it has been shown that a single dose of antibody reduced pcp effects for up to 2 weeks in animals, which might be equivalent to a several months in humans. high-affinity antibody 6b5 fragments against pcp function better in that the fragments with bound pcp are more quickly cleared from the body than the whole antibodybound pcp. 153 the crystal structure of the antibody fragment complexed with pcp has been studied in detail. 154 antibody fragments are also preferable to whole antibodies in the treatment of pcp addiction because of their lower antigenicity and improved pharmacokinetics. 149, 151 anti-idiotype antibodies could potentially be produced against the drug-mab binding site and mimic drug structural features, although these antibodies do not cross the bloodbrain barrier. 151 by increasing protein binding in the vascular compartment and lowering the drug's volume of distribution, the antibody acts like a pharmacokinetic antagonist. 150 evidence that the antibody can also reverse effects of other potent arylcyclohexylamine drugs suggests that antibody medications can be used to treat different classes of drugs. 150, 151 an anti-pcp igg has been produced from a hybridoma cell line using both ascites and bioreactor methods, and the pharmacological and immune specificity of the antibody has been confirmed by administering the anti-pcp fragment to rats in three different studies without producing observable effects in behavior. 155 active immunotherapy against methamphetamine addiction is also being contemplated. 156 antibody therapy for cocaine addiction is also a possibility, especially with the development of catalytic antibodies having enzyme-like characteristics that can be used to inactivate drugs. 151, [157] [158] [159] antibodies with esterase activity have been successfully mimicked, and because cocaine is believed to be metabolized by in vivo esterases, such antibodies would be useful in the treatment of cocaine addiction. 151 catalytic antibodies probably would not be as useful as high-affinity anti-cocaine antibodies, but could be used in medical emergencies or as part of a withdrawal therapy. 151 potential antibody applications against cocaine addiction have been tested in rats with a vaccine approach by (1) attaching cocaine molecules to a carrier protein for immunizing rats to produce antibodies against cocaine and (2) humanizing anticocaine antibodies derived from rats and producing them in bacteria. 152, 160 a catalytic mab (15a10) has recently been reported to attenuate cocaine's cardiovascular effects in mice. 155, 159 conclusion about 200 years have elapsed since edward jenner vaccinated a young child against smallpox. since that time, the field of immunology has evolved at a rapid pace and has yielded many critical developments. although vaccination has thus far proven to be the most cost-effective method of preventing diseases worldwide, the development of mabs that use the specificity of immunological responses is one of the most successful applications of immunology to date. chimeric and humanized antibodies have reduced the risk of allergenicity from exposure to nonself antibodies and heightened the clinical effectiveness of mab treatments. developments in radiology and pharmacology have allowed radiolabeled and immunoconjugated antibodies to be produced. antibody fragments, heteropolymers, and bispecific antibodies are now available in addition to whole mabs. these promising developments may soon allow mabs to be used to treat afflictions as varied as substance abuse, cancer, asthma, viral infection, septicemia, and poisoning. as heddy zola observed in 1995, "the therapeutic application of mabs, whilst still limited in scope, promises to break its substantial shackles and realize the potential forecast by its proponents." 46 on immunity: with special reference to cell life ehrlich's passion: the origins of his receptor immunology the natural selection theory of antibody formation a modification of jerne's theory of antibody production using the concept of clonal selection the hydrolysis of rabbit gamma globulin and antibodies with crystalline papain selection of hybrids from matings of fibroblasts in vitro and their presumed recombinants continuous cultures of fused cells secreting antibody of predefined specificity with the benefit of hindsight antibodies in diagnostics-from immunoassays to protein chips igg subclasses-a review biological activities of immunoglobulins of different classes and subclasses preparation of rodent monoclonal antibodies by in vitro somatic hybridization monoclonal antibodies in clinical applications monoclonal antibodies: the second generation. herndon (va): bios scientific a human myeloma cell line suitable for the generation of human monoclonal antibodies the optimal technological approach to the development of human hybridomas continuous production of monoclonal rheumatoid factor by ebv-transformed lymphocytes clinical use of monoclonal antibodies production of clinical grade monoclonal antibodies. presentation at international business communications fifth annual antibody production & downstream processing conference introduction to antibody engineering and phage display natural and designer binding sites made by phage display technology development of abx-egf, a fully human anti-egf receptor monoclonal antibody, for cancer therapy antibody engineering via genetic engineering of the mouse: xenomouse strains are a vehicle for the facile generation of therapeutic human monoclonal antibodies double trans-chromosomic mice: maintenance of two individual human chromosome fragments containing ig heavy and loci and expression of fully human antibodies the trimera mouse: generating human monoclonal antibodies and an animal model for human diseases lactogenic immunity in transgenic mice producing recombinant antibodies neutralizing coronavirus transgenic mice secreting coronavirus neutralizing antibodies into the milk the future of transgenics. regulatory affairs focus expression and assembly of a full-length monoclonal antibody in plants using a plant virus vector characterization of a recombinant plant monoclonal secretory antibody and preventive immunotherapy in humans generation and assembly of secretory antibodies in plants exploring transgenic plants as a new vaccine source oral immunization with a recombinant bacterial antigen produced in transgenic plants tobacco can be good for you. the new york times rapid production of specific vaccines for lymphoma by expression of the tumor-derived single-chain fv epitopes in tobacco plants production of antibodies in transgenic plants a humanized monoclonal antibody produced in transgenic plants for immunoprotection of the vagina against genital herpes production of a diagnostic monoclonal antibody in perennial alfalfa plants using monoclonal antibodies to prevent mucosal transmission of epidemic infectious diseases points to consider in the manufacture and testing of monoclonal antibody products for human use guidance for industry. s6 preclinical safety evaluation of biotechnology-derived pharmaceuticals center for biologics evaluation and research and center for drug evaluation and research, us food & drug administration points to consider in the production and testing of therapeutic products for human use derived from transgenic animals points to consider in the production and testing of new drugs and biologicals produced by recombinant dna technology points to consider in the characterization of cell lines used to produce biologicals. center for biologics evaluation and research, us food & drug administration monoclonal antibodies: the second generation human antibodies by design genetically engineered antibody molecules engineering antibodies for therapy recombinant antibodies: alternative strategies for developing and manipulating murine-derived monoclonal antibodies monoclonal antibody-based therapy for solid tumors: an overview monoclonal antibodies: innovations in diagnosis and therapy unconjugated monoclonal antibody therapy of lymphoma monoclonal antibodies: the second generation. herndon (va): bios scientific preparation of recombinant antibodies from immune rodent spleens and the design of their humanization by cdr grafting the promise and pitfalls of monoclonal antibody therapeutics radiolabeled monoclonal antibodies monoclonal antibody-based therapy of breast cancer monoclonal antibodies in cancer therapy antibody-based therapies for hodgkin's disease principles of antibody therapy reconstruction of monoclonal antibodies by genetic engineering genetically engineered monoclonal antibodies for direct anti-neoplastic treatment and cancer cell specific delivery of chemotherapeutic agents immunotherapeutic perspective for bispecific antibodies monoclonal antibodybased therapy of cancer recombinant fv immunotoxins and fv fragments as novel agents for cancer therapy and diagnosis photoreduction of monoclonal antibodies for conjugation and fragmentation monoclonal antibody-based therapies of leukemia and lymphoma immunotoxin therapy of lymphoma: studies with anti-b4-blocked ricin the use of bispecific antibodies in tumor cell and tumor vasculature directed immunotherapy production of fab fragments of monoclonal antibodies using polyelectrolyte complexes use of heteropolymeric monoclonal antibodies to attach antigens to the c3b receptor of human erythrocytes: a potential therapeutic treatment bispecific monoclonal antibody complexes bound to primate erythrocyte complement receptor 1 facilitate virus clearance in a monkey model bispecific monoclonal antibody complexes facilitate erythrocyte binding and liver clearance of a prototype particulate pathogen in a monkey model the primate erythrocyte complement receptor (cr1) as a privileged site: binding of immunoglobulin g to erythrocyte cr1 does not target erythrocytes for phagocytosis antigenbased heteropolymers facilitate, via primate erythrocyte complement receptor type 1, rapid erythrocyte binding of an autoantibody and its clearance from the circulation in rhesus monkeys quantitative studies of heteropolymer-mediated binding of inactivated marburg virus to the complement receptor on primate erythrocytes antigen-based heteropolymers, a potential therapy for binding and clearing autoantibodies via erythrocyte cr1 infusion of bispecific monoclonal antibody complexes into monkeys provides immunologic protection against later challenge with a model pathogen radioimmunotherapy of interleukin-2r alpha-expressing adult t-cell leukemia with yttrium-90-labeled anti-tac monoclonal antibody therapy of cancer colorectal cancer as a model for immunotherapy advances in cancer immunotherapy antibody-targeted immunotherapy for treatment of malignancy first mab approved for treatment of metastatic breast cancer breast cancer biology blossoms in the clinic response of metastatic breast cancer to trastuzumab anti-her2 antibody and heregulin suppress growth of her2-overexpressing human breast cancer cells through different mechanisms tyrosine kinase inhibitors targeted to the epidermal growth factor receptor subfamily: role as anticancer agents are solid tumor anti-idiotype vaccines ready for prime time? immunological consolidation of ovarian carcinoma recurrences with monoclonal anti-idiotype antibody aca125: immune responses and survival in palliative treatment clinical and immune responses in advanced melanoma patients immunized with an anti-idiotype antibody mimicking disialoganglioside gd2 delivery of monoclonal antibodies to the tumor cells monoclonal antibody-based therapy of melanoma radioimmunoconjugate therapy of non-hodgkin's lymphoma tumor markers as targets for selective diagnostic and therapeutic procedures monoclonal antibody therapy in organ transplantation immunotoxin therapy of graft-versus-host disease immunotoxins: an update anti-tac-h, a humanized antibody to the interleukin 2 receptor, prolongs primate cardiac allograft survival reduction of acute renal allograft rejection by daclizumab. daclizumab double therapy study group how do monoclonal antibodies induce tolerance? a role for infectious tolerance? treatment of posttransplant lymphoproliferative disorder with the anti-cd20 monoclonal antibody rituximab alone in an adult after liver transplantation: a new drug in therapy of patients with posttransplant lymphoproliferative disorder after solid organ transplantation rituximab (anti-cd20 monoclonal antibody) for the treatment of patients with clonal lymphoproliferative disorders after orthotopic liver transplantation: a report of three cases protective effect of an anti-lfa 1 monoclonal antibody (odulimomab) on renal damage due to ischemia and kidney autotransplantation cytomegalovirus infections after treatment with daclizumab, an anti il-2 receptor antibody, for prevention of renal allograft rejection infliximab for the treatment of fistulas in patients with crohn's disease therapeutic efficacy of multiple intravenous infusions of anti-tumor necrosis factor alpha monoclonal antibody combined with lowdose weekly methotrexate in rheumatoid arthritis antibodies to tumour necrosis factor alpha as treatment for crohn's disease tumor necrosis factor blockers in rheumatoid arthritis monoclonal antibody therapy in rheumatoid arthritis treatment of allergic asthma with monoclonal anti-ige antibody. rhu mab-e25 study group treatment of allergic asthma with monoclonal anti-ige antibody anti-ige therapy for asthma treatment of allergic asthma with monoclonal anti-ige antibody monoclonal antibodies in sepsis and septic shock treatment of gram-negative bacteremia and septic shock with ha-1a human monoclonal antibody against endotoxin. a randomized, double-blind, placebo-controlled trial double-blind randomised controlled trial of monoclonal antibody to human tumour necrosis factor in treatment of septic shock. norasept ii study group adoptive immunotherapy of gram-negative sepsis: use of monoclonal antibodies to lipopolysaccharide antibodies proposed as therapeutic agents anti-endotoxin monoclonal antibodies human monoclonal antibodies against a plethora of viral pathogens from single combinatorial libraries effects of monoclonal antibody combined with ganciclovir or (s)-1-[3-hydroxy-(2-phosphonylmethoxy)-propyl]cytosine against murine cytomegalovirus infections in cell culture and in severe combined immunodeficient mice protection against murine cytomegalovirus infection by passive transfer of neutralizing and non-neutralizing monoclonal antibodies msl-109 adjuvant therapy for cytomegalovirus retinitis in patients with acquired immunodeficiency syndrome: the monoclonal antibody cytomegalovirus retinitis trial. the studies of ocular complications of aids research group ganciclovir prophylaxis decreases frequency and severity of cytomegalovirus disease in seropositive liver transplant recipients treated with okt3 monoclonal antibodies human monoclonal antibodies neutralizing cytomegalovirus (cmv) for prophylaxis of cmv disease: report of a phase i trial in bone marrow recipients characterization of human anti-cytomegalovirus monoclonal antibody as biologics a humanized antibody against human cytomegalovirus (cmv) gp ul75 (gh) for prophylaxis or treatment of cmv infections american academy of pediatrics committee on infectious diseases and committee of fetus and newborn. prevention of respiratory syncytial virus infections: indications for the use of palivizumab and update on the use of rsv-igiv respiratory syncytial virus (rsv) immune globulin and palivizumab for prevention of rsv infection directed selection of recombinant human monoclonal antibodies to herpes simplex virus glycoproteins from phage display libraries effective antibody therapy in herpes simplex virus ocular infection: characterization of recipient immune response use of monoclonal antibody directed against herpes simplex virus glycoproteins to protect mice against acute virus-induced neurological disease ability of monoclonal antibody to herpes simplex virus glycoprotein gb to promote healing of herpetic skin lesions in nude mice protection against herpetic ocular disease by immunotherapy with monoclonal antibodies to herpes simplex virus glycoprotein passive immunization with monoclonal antibodies against herpes simplex virus glycoproteins protects mice against herpetic ocular disease characterization of a type-common human recombinant monoclonal antibody to herpes simplex virus with high therapeutic potential topically applied human recombinant monoclonal igg 1 antibody and its fab and f(ab')2 fragments protect mice from vaginal transmission of hsv-2 epitopes involved in antibody-mediated protection from ebola virus isolation and characterization of human monoclonal antibodies to digoxin drug-specific antibodies as antidotes for tricyclic antidepressant overdose automated immunosensing system for 3,5,6-trichloro-2-pyridinol application to surface water samples exploiting antibody-based technologies to manage environmental pollution pharmacokinetic mechanisms for obtaining high renal coelimination of phencyclidine and a monoclonal antiphencyclidine antigen-binding fragment of immunoglobulin g in the rat prevention of paraquat toxicity in suspensions of alveolar type ii cells by paraquatspecific antibodies production and characterization of a monoclonal antibody against domoic acid and its application to enzyme immunoassay antiphencyclidine monoclonal antibody therapy significantly changes phencyclidine concentrations in brain and other tissues in rats pharmacodynamics of a monoclonal antiphencyclidine fab with broad selectivity for phencyclidine-like drugs antibodies as pharmacokinetic and metabolic modifiers of neurotoxicity antibodies may treat overdoses, addiction phencyclidine-specific fab fragments alter phencyclidine disposition in dogs crystal structure of monoclonal 6b5 fab complexed with phencyclidine disposition of a monoclonal anti-phencyclidine fab fragment of immunoglobulin g in rats drug-fighting drugs reactive immunization a catalytic antibody against cocaine attenuates cocaine's cardiovascular effects in mice: a dose and time course analysis natural and artificial enzymes against cocaine. i. monoclonal antibody 15a10 and the reinforcing effects of cocaine in rats evaluation of anti-cocaine antibodies and a cocaine vaccine in a rat selfadministration model we thank carolyn m. black and the national center for infectious diseases editorial office for the critical review of this manuscript. key: cord-007726-bqlf72fe authors: rydell-törmänen, kristina; johnson, jill r. title: the applicability of mouse models to the study of human disease date: 2018-11-09 journal: mouse cell culture doi: 10.1007/978-1-4939-9086-3_1 sha: doc_id: 7726 cord_uid: bqlf72fe the laboratory mouse mus musculus has long been used as a model organism to test hypotheses and treatments related to understanding the mechanisms of disease in humans; however, for these experiments to be relevant, it is important to know the complex ways in which mice are similar to humans and, crucially, the ways in which they differ. in this chapter, an in-depth analysis of these similarities and differences is provided to allow researchers to use mouse models of human disease and primary cells derived from these animal models under the most appropriate and meaningful conditions. although there are considerable differences between mice and humans, particularly regarding genetics, physiology, and immunology, a more thorough understanding of these differences and their effects on the function of the whole organism will provide deeper insights into relevant disease mechanisms and potential drug targets for further clinical investigation. using specific examples of mouse models of human lung disease, i.e., asthma, chronic obstructive pulmonary disease, and pulmonary fibrosis, this chapter explores the most salient features of mouse models of human disease and provides a full assessment of the advantages and limitations of these models, focusing on the relevance of disease induction and their ability to replicate critical features of human disease pathophysiology and response to treatment. the chapter concludes with a discussion on the future of using mice in medical research with regard to ethical and technological considerations. although the genetic lineages of mice and humans diverged around 75 million years ago, these two species have evolved to live together, particularly since the development of agriculture. for millennia, mice (mus musculus) were considered to be pests due to their propensity to ravenously consume stored foodstuff (mush in ancient sanskrit means "to steal" [1] ) and their ability to adapt to a wide range of environmental conditions. since the 1700s, domesticated mice have been bred and kept as companion animals, and in victorian england, "fancy" mice were prized for their variations in coat color and comportment; these mouse strains were the forerunners to the strains used in the laboratory today. robert hooke performed the first recorded inquiry-driven experiments on mice in 1664, when he investigated the effects of changes in air pressure on respiratory function [2] . more recently, with data from the human genome project and sequencing of the mus musculus genome showing remarkable genetic homology between these species, as well as the advent of biotechnology and the development of myriad knockout and transgenic mouse strains, it is clear why the mouse has become the most ubiquitous model organism used to study human disease. in addition, their small size, rapid breeding, and ease of handling are all important advantages to scientists for practical and financial reasons. however, keeping in mind that mice are fellow vertebrates and mammals, there are ethical issues inherent to using these animals in medical research. this chapter will provide an overview of the important similarities and differences between mus musculus and homo sapiens and their relevance to the use of the mouse as a model organism and provide specific examples of the quality of mouse models used to investigate the mechanisms, pathology, and treatment of human lung diseases. we will then conclude with an assessment of the future of mice in medical research considering ethical and technological advances. as a model organism used to test hypotheses and treatments related to human disease, it is important to understand the complex ways in which mice are similar to humans, and crucially, the ways in which they differ. a clear understanding of these aspects will allow researchers to use mouse models of human disease and primary cells derived from mice under the most appropriate and meaningful conditions. in 2014, the encyclopedia of dna elements (encode) program published a comparative analysis of the genomes of homo sapiens and mus musculus [3] , as well as an in-depth analysis of the differences in the regulatory landscape of the genomes of these species [4] . encode, a follow-up to the human genome project, was implemented by the national human genome research institute (nhgri) at the national institutes of health in order to develop a comprehensive catalog of protein-encoding and nonproteincoding genes and the regulatory elements that control gene expression in a number of species. this was achieved using a number of genomic approaches (e.g., rna-seq, dnase-seq, and chip-seq) to assess gene expression in over 100 mouse cell types and tissues; the data were then compared with the human genome. overall, these studies showed that although gene expression is fairly similar between mice and humans, considerable differences were observed in the regulatory networks controlling the activity of the immune system, metabolic functions, and responses to stress, all of which have important implications when using mice to model human disease. in essence, mice and humans demonstrate genetic similarity with regulatory divergence. specifically, there is a high degree of similarity in transcription factor networks but a great deal of divergence in the cis-regulatory elements that control gene transcription in the mouse and human genomes. moreover, the chromatin landscape in cell types of similar lineages in mouse and human is both developmentally stable and evolutionarily conserved [3] . of particular relevance regarding modeling human diseases involving the immune system, in its assessment of transcription factor networks, the mouse encode consortium revealed potentially important differences in the activity of ets1 in the mouse and human genome. although conserved between the two species, divergence in ets1 regulation may be responsible for discrepancies in the function of the immune system in mouse and human [4] . certainly, the biological consequences of these differences in gene expression and regulation between human and mouse invite further investigation. the anatomical and physiological differences between model organisms and humans can have profound impacts on interpreting experimental results. virtually every biological process under investigation in experimental studies involves at least one anatomical structure. to aid in interpretation, many anatomy compendia have been developed for model organisms; the most useful organize anatomical entities into hierarchies representing the structure of the human body, e.g., the foundational model of anatomy developed by the structural informatics group at the university of washington [5] . although an analysis of the myriad differences between mouse and human anatomy is beyond the scope of this chapter, a few of the most critical issues that have an impact on the interpretation of data from mouse experiments should be mentioned. the most obvious difference between mice and humans is size; the human body is about 2500 times larger than that of the mouse. size influences many aspects of biology, particularly the metabolic rate, which is correlated to body size in placental mammals through the relationship bmr ¼ 70 â mass (0.75), where bmr is the basal metabolic rate (in kcal/day). thus, the mouse bmr is roughly seven times faster than that of an average-sized human [6] . this higher bmr has effects on thermoregulation, nutrient demand, and nutrient supply. as such, mice have greater amounts of metabolically active tissues (e.g., liver and kidney) and more extensive deposits of brown fat [6] . furthermore, mice more readily produce reactive oxygen species than do humans, which is an important consideration when modeling human diseases involving the induction of oxidative stress (i.e., aging, inflammation, and neurodegeneration) [6] . the lung provides an excellent example of the similarities and differences between human and mouse anatomy. similar to the human organ, the mouse lung is subdivided into lobes of lung parenchyma containing a branching bronchial tree and is vascularized by the pulmonary circulation originating from the right ventricle. there are a number of subtle variations in this general structure between species, i.e., the number of lobes on the right and left, the branching pattern, and the distribution of cartilage rings around the large airways, but the most important differences between the mouse and human lung are related to the organism's size (airway diameter and alveolar size are naturally much smaller in the mouse) and respiratory rate. moreover, there are important differences in the blood supply of the large airways in humans versus mice [7] . specifically, the bronchial circulation (a branch of the high-pressure systemic circulation that arises from the aorta and intercostal arteries) supplies a miniscule proportion of the pulmonary tissue in mice (the trachea and bronchi) compared to humans; the majority of the lung parenchyma is supplied by the low-pressure, high-flow pulmonary circulation. in the mouse, these systemic blood vessels do not penetrate into the intraparenchymal airways, as they do in larger species [8] . this difference, although subtle, has important ramifications regarding the vascular supply of lung tumors which, in humans, is primarily derived from the systemic circulation [9] . these differences may also have profound consequences when modeling human diseases involving the lung vasculature. the adaptive immune system evolved in jawed fish about 500 million years ago, well before the evolution of mammals and the divergence of mouse and human ancestral species [10] . many features of the adaptive immune system, including antigen recognition, clonal selection, antibody production, and immunological tolerance, have been maintained since they first arose in early vertebrates. however, the finer details of the mouse and human immune systems differ considerably, which is not surprising since these species diverged 75 million years ago [6] . while some have claimed that these differences mean that research into immunological phenomena in mice is not transferable to humans, as long as these differences are understood and acknowledged, the study of mouse immune responses can continue to be relevant. research on mice has been vital to the discovery of key features of both innate and adaptive immune responses; for example, the first descriptions of the major histocompatibility complex, the t cell receptor, and antibody synthesis were derived from experiments performed on mice [6] . the general structure of the immune system is similar in mice and humans, with similar mediators and cell types involved in rapid, innate immune responses (complement, macrophages, neutrophils, and natural killer cells) as well as adaptive immune responses informed by antigen-presenting dendritic cells and executed by b and t cells. however, due to the anatomical and physiological differences between these species as described above, divergence in key features of the immune system, such as the maintenance of memory t cells (related to the life span of the organism) and the commensal microbiota (related to the lifestyle of the organism), has arisen [11] . similar to what has been discovered regarding the genetics of mice and humans, i.e., broad similarities in structure but considerable differences in regulation, there are a number of known discrepancies in the regulation of innate and adaptive immunity in mouse models of human disease mice versus humans, including the balance of leukocyte subsets, t cell activation and costimulation, antibody subtypes and cellular responses to antibody, th1/th2 differentiation, and responses to pathogens (described in detail in table 1 ). in addition to these differences in immune cell functions, the expression of specific genes involved in immune responses also differs, particularly those for toll-like receptors, defensins, nk inhibitory receptors, thy-1, and many components of chemokine and cytokine signaling; additionally, differences between mouse strains are known to exist for many of these mediators [12] . another important consideration when using mice to perform immunological research (with a view to translating these findings to human medicine) is the availability of hundreds of strains of genetically modified mice that have enabled exquisitely detailed studies on immune cell function, regulation, and trafficking. many of these strains involve the expression of inducible cre or cas9 that allow for targeted knockdown or overexpression of key immune function-related genes in specific cell types at specific moments in time. however, it is important to note that drift between mouse colonies has long been known to occur. in fact, a recent report described the fortuitous discovery of a point mutation in the natural cytotoxicity receptor 1 (ncr1) gene in the c57/bl6 cd45.1 mouse strain, resulting in absent ncr1 expression. this mutation was found to have profound effects on the response of mice to viral infection, i.e., the mice were resistant to cytomegalovirus infection but more susceptible to influenza virus [23] . this cautionary tale highlights the importance of understanding the genetic evolution of laboratory strains of mice, the effect of these genetic and immunological changes on mouse biology, and the impact on the translation of these results to human medicine. in addition to the differences between mouse and human genetics, physiology, and immunology highlighted above, several factors must also be taken into account when performing in vitro assays using isolated mouse cells and applying these findings to our understanding of human disease. particularly with regard to stem cell research, it should be noted that the telomeres of mouse cells are five-to tenfold longer than human telomeres, resulting in greater replicative capacity [24] . there are also important differences in the regulation of pluripotency and stem cell differentiation pathways in humans and mice [25] . moreover, there are considerable species differences in the longevity of cultured cells; for example, mouse fibroblasts are capable of spontaneous immortalization in vitro, whereas human fibroblasts become senescent and ultimately fail to thrive in culture [26] . in summary, although there are considerable differences between mice and humans, constant improvement in the analytical techniques used to delineate these differences and their effects on whole organism and cell function have provided vital information and contributed to our understanding of both murine and human biology. experimentation employing mouse models of human disease will continue to provide key insights into relevant disease mechanisms and potential drug targets for further clinical investigation. however, several important considerations must be taken into account when selecting a mouse model of human disease, as described in the following section, using mouse models of human lung disease to illustrate this point. the two most salient features of a mouse model of human disease are the accuracy of its etiology (it employs a physiologically relevant method of disease induction) and its presentation (its ability to recapitulate the features of human disease). the relevance of any given mouse model can be judged on the basis of these two criteria, and there is considerable variation within mouse models of human disease in this regard. as a full assessment of the advantages and limitations of all currently available mouse models of human disease would be prohibitively long and complex, here we have elected to assess the accuracy of currently available models of human lung diseases, i.e., asthma, chronic obstructive pulmonary disease, and pulmonary fibrosis, focusing on the relevance of disease induction in these models and their ability to replicate critical features of human disease pathophysiology and response to treatment. the first and foremost notion when modeling human disease in mice is to acknowledge the species differences, which are significant [27] . as described above, genetics, anatomy, physiology, and immunology differ between mice and humans, but despite these differences, mouse models of human disease are useful and necessary, as long as data interpretation is performed appropriately. an elegant example of differences between mice and humans that must be considered when designing a mouse model of human inflammatory lung disease is the key effector cell type in human asthma, i.e., mast cells. these leukocytes differ in granule composition as well as localization in the mouse and human airways [28] . mice mostly lack mast cells in the peripheral lung [29] , whereas humans have numerous mast cells of multiple subpopulations in the alveolar parenchyma [30] . another example is anatomy: in contrast to humans, mice lack an extensive pulmonary circulation, which may have significant effects on leukocyte adhesion and migration, and subsequently inflammation [31] . still, as long as these differences are taken into consideration, mouse models can be powerful tools in the discovery and exploitation of new targets for the treatment of human disease. the world health organization (who) defines asthma as a chronic disease characterized by recurrent attacks of breathlessness and wheezing, which may vary in severity and frequency from person to person. the disease is characterized by airway hyperresponsiveness, airway smooth muscle thickening, increased mucus secretion and collagen deposition, as well as prominent inflammation affecting both large and small airways [32] . nowadays, it is recognized that asthma is not a single homogenous disease but rather several different phenotypes united by similar clinical symptoms [32, 33] . only a few animal species develop asthma naturally, including cats and horses [34, 35] , whereas mice do not [31] . however, mice can be manipulated to develop a type of allergic airway inflammation, which is similar in many ways to the human disease, in response to different aeroallergens [36] . importantly, these models are capable of recapitulating only the allergic type of human asthma and have less relevance for other types of asthma (i.e., endotypes induced by medication, obesity, and air pollution). as with many human diseases, asthma has a complex and multifaceted etiology, where environmental factors, genetic susceptibility, and microbial colonization all contribute; thus, it is important to take strain differences into consideration. generations of inbreeding have created mouse strains that differ not only in coat color and disposition but also from a physiological, immunological, and genetic perspective. different strains may be more susceptible to allergic airway inflammation or pulmonary fibrosis, whereas others are more or less resistant. choosing the right strain to model a specific disease or pathologic event is thus essential. the most widely used strains for models of allergic airway inflammation are balb/c and c57bl/6. these strains differ regarding the type of immune response mounted to an inhaled allergen: c57bl/6 is generally considered a t h 1-skewed strain, whereas balb/c is regarded as a t h 2-skewed strain [36] . due to their strong t h 2response, and subsequent development of robust asthmatic responses, balb/c has been commonly used to model asthma [37] . however, most humans do not express such a strongly t h 2-skewed immune system, suggesting this strain may not be the best model of human disease; instead, c57bl/6 may be more suitable as immune responses in this strain are more similar to those of atopic human subjects [37] . furthermore, as c57bl/6 is the most commonly used strain for the development of genetically manipulated mice, using these mice allows for very specific investigations into disease pathology; thus, this strain is increasingly used in models of human lung disease. besides the genetic differences in the mouse strains used in these models, the etiology (the method of disease induction) of commonly used models of asthma is highly variable. in humans with allergic asthma, environmental allergen exposure occurs at the airway mucosa; the immune response is coordinated in the bronchopulmonary lymph nodes, and the t cells, macrophages, and eosinophils recruited as part of this response travel to the lung where they mediate the cardinal features of asthma: airway inflammation, structural remodeling of the airway wall, and airway hyperreactivity [38] . ideally, these features should be found in a physiologically relevant mouse model of asthma. however, for the sake of cost and convenience, early mouse models of asthma used the surrogate protein ovalbumin (ova) [31] rather than an environmental allergen to induce an immune response, which also requires the use of a powerful t h 2-polarizing adjuvant such as alum delivered via the intraperitoneal route, followed by ova nebulization-a clear divergence from the etiology of human asthma [36] . in terms of disease presentation, mice develop some hallmarks of asthma, including airway eosinophilic inflammation, goblet cell metaplasia, and increased airway smooth muscle density [31] . after the cessation of ova exposure, most of the remodeling resolves, although some structural alterations remain up to 1 month after the last challenge [39] . based on these attributes, the ova model is primarily a model to investigate the initiation of inflammation, rather than the chronic progression and maintenance of inflammation [31] . a clear advantage with the ova model is the number of studies where it is used; both the pros and cons are familiar. it is easy to find a suitable protocol, and the model is readily accessible and flexible regarding the number of sensitizations and allergen doses. the model is relatively easy to reproduce, as ova and different adjuvants are easily obtained. however, the resolution of remodeling following the cessation of allergen provocations is a disadvantage, as is the practical problem with the nebulization of an allergen-it ends up in the mouse's coat and is ingested during grooming, potentially resulting in systemic exposure (this is particularly relevant in models employing systemic, intraperitoneal sensitization). in addition, concerns have been raised against the use of adjuvants to induce the immunological response, as well as the clinical relevance of ova as an allergen, which have driven the development of more clinically relevant allergens and models [31] . the common environmental aeroallergen house dust mite (hdm) extract is increasingly used to initiate disease in mouse models of allergic airway inflammation, as it is a common human allergen (around 50% of asthmatics are sensitized to hdm [40] ) that evokes asthma attacks and other allergic responses in susceptible individuals. in addition, hdm has inherent allergenic properties, likely due to components with protease activity [40] , so there is no need to use an adjuvant, thus improving the etiological similarity of these models with the clinical situation [41] . in contrast to ova, prolonged exposure of hdm (up to 7 weeks) induces asthma-like severe airway inflammation with prominent eosinophilia, severe hyperreactivity to methacholine, and robust remodeling of the airway wall [41] , i.e., the presentation of chronic respiratory hdm exposure in mice effectively recapitulates the key features of human allergic asthma. importantly, the airway structural changes induced by chronic hdm exposure, such as increased collagen deposition, airway smooth muscle thickening, and microvascular alterations, persist for at least 4 weeks after the cessation of hdm exposure [42] , another commonality with human asthma in which airway remodeling is currently considered to be irreversible. thus, the advantages of using hdm as the allergen in mouse models of asthma are the clinical relevance of the allergen [43] and the route of delivery via the respiratory tract. moreover, studies have shown that the type of inflammation and characteristics of tissue remodeling are relatively similar to those seen in human asthmatics [35, 41, 43] . one disadvantage is the complexity of hdm extract; as a consequence of this complexity, variations exist in some components between batches, particularly regarding the content of lipopolysaccharide, so reproducibility in these studies may be problematic. with similarity to hdm, these models were developed to be as clinically relevant as possible, as many patients suffer from allergy toward cockroach allergen, molds, and other environmental irritants. a common feature of these allergens is their complex nature, as they commonly consist of a mix of different allergic epitopes and fragments. this complexity is most likely why the immunological reaction in mice is relatively similar to that seen in asthmatics [44] . cockroach allergen (cra) is a common allergen, known to induce asthma in susceptible individuals; thus, it shares with hdm the advantage of being highly clinically relevant [45] . cra induces peribronchial inflammation with significant eosinophilic inflammation and transient airway hyperresponsiveness, both of which can be increased by repeated administrations of the allergen [45] . colonization of the airways with aspergillus fumigatus is the cause of allergic bronchopulmonary aspergillosis (abpa), a disease where the lungs are colonized by the fungus, but allergens from aspergillus fumigatus can also induce asthma similar to other allergens [46] . the reaction to aspergillus allergens is robust, and often no adjuvants are needed to elicit inflammation [46] . in addition to aspergillus, other fungi such as penicillium and alternaria can also induce asthma in humans and have been used to model disease in mice [47] . a common difficulty with these allergens is the method of administration, as the physiological route is believed to be the inhalation of dry allergens; mimicking this route with a nebulizer introduces the risk of the animals ingesting the allergen and thus causing systemic responses [47] . exacerbations of asthma are defined as the worsening of symptoms, prompting an adjustment in treatment, and are believed to be associated with increased inflammation in the distal airways. clinically, exacerbations are believed to be induced by infections (most common), allergen exposure, or pollutants, which can be modeled in different ways [48, 49] : 1. infections with viruses and bacteria or exposure to proteins/ dna/rna derived from these microbes. 2. administration of a high dose of allergen in a previously sensitized animal. 3. exposure to environmental pollutants, such as diesel exhaust or ozone. modeling exacerbations adds a layer of complexity, as robust ongoing allergic airway inflammation needs to be established first, before challenge with the exacerbating agent. both the ova and hdm models are used in this respect, and in both cases chronic protocols extending for several weeks before triggering an exacerbation have been used [48] . chronic obstructive pulmonary disease (copd) is characterized by chronic airway obstruction, in contrast to asthma where the obstruction is reversible (particularly in response to bronchodilator treatment). clinically, in copd, chronic bronchitis and emphysema can occur either separately or in combination. copd is almost always associated with either first-or secondhand tobacco smoking or in rare cases with a deficiency in the production of α1antitrypsin (a serpin that prevents elastin breakdown as a result of neutrophil degranulation) [50] . the etiology of copd is highly complex and is believed to develop after many years of smoking in combination with other known factors such as genetic susceptibility or environmental factors [51] . in similarity to asthma, inflammation is a major component in copd, but the leukocyte profile is very different: the most prominent players in copd-related inflammation are neutrophils and, to some degree, macrophages [51] . due to the complex etiology of copd, it is difficult to recapitulate all aspects of this disease in a single model, so in most cases, the aim is to induce copd-like lesions by exposing mice to tissue-damaging substances (usually cigarette smoke) or to mimic emphysema by the administration of tissue-degrading enzymes [27, 51] . clearly, mice do not smoke cigarettes on their own, so to model copd by cigarette smoke (cs) inhalation, the mice need to be exposed to unfiltered cs in an induction chamber; moreover, in an attempt to better model the chronic aspects of copd, this needs to be performed for a prolonged period of time. mice are very tolerant to cs, but eventually (over a period of several weeks), cs induces pulmonary neutrophilic inflammation that is associated with some degree of tissue degradation and destruction [51] . an important advantage of this model is the fact that cs is the actual irritant responsible for disease in humans, and mice develop several features similar to the clinical disease, making this model highly clinically relevant [27] . a significant drawback is the self-limitation of the model-the pathological changes do not progress after the cessation of cs exposure [51] . furthermore, the exposure time needed for mice to develop copd-like pathology is extensive, i.e., studies have shown that an exposure protocol of 5 days per week for a minimum of 3 months is needed to generate robust structural changes to the lung [52]. the pathological image in copd is complex and varies greatly between patients, commonly encompassing chronic bronchitis and bronchiolitis, emphysema, fibrosis, and airway obstruction. although mice develop some of these symptoms when exposed to cs, they do not develop all the symptoms of human disease; thus, cs has advantages as a model but fails to mimic the complexity of the clinical situation and disease presentation [27] . other models of copd rely on the administration of proteases (protein-degrading enzymes) that are believed to be involved in the pathology of this disease in a subset of patients, such as elastindegrading elastase. this approach mimics the emphysematous changes seen in copd, but the pathological process underlying tissue destruction is likely very different compared to the clinical situation [51] , as very few patients show evidence of elastase dysregulation [27] . however, if the aim of the study is to investigate the general effect of protease-induced tissue destruction and regeneration, then this is a highly relevant method [51] . some studies on copd have also used genetically modified animals, such as mice overexpressing collagenase, which results in tissue destruction without inflammation or fibrosis with an end result fairly similar to the type of emphysema observed in copd [53] . pulmonary fibrosis, the accumulation of fibrotic tissue within the alveolar parenchyma, is merely a symptom of disease, and the etiology of this pathology in humans varies greatly [54] . the most enigmatic class is perhaps the idiopathic interstitial pneumonias, especially idiopathic pulmonary fibrosis (ipf). ipf is a debilitating and progressive disease with a grave prognosis, characterized by progressive fibrosis believed to reflect aberrant tissue regeneration [55] . as the reason behind this defective repair is unknown, although a combination of immunological, genetic, and environmental factors are suspected, it is very difficult to model disease in a clinically relevant fashion [56] . the most common method used to model pulmonary fibrosis in mice is administration of the chemotherapeutic agent bleomycin; this agent is known to cause pulmonary fibrosis in humans as well, but this may not accurately reflect the true etiology of most cases of human disease. the strain of choice is c57bl/6, as it is prone to developing pulmonary fibrosis, whereas balb/c is relatively resistant, a feature believed to reflect the cytokine response following cellular stress and damage [57] . bleomycin administration can be performed locally or systemically, producing very different results. the most common model of pulmonary fibrosis is a single intranasal or intratracheal administration of bleomycin, with analysis 3 to 4 weeks later. during this time, the drug causes acute tissue damage in a restricted area of the lung (where the solution ends up during administration), followed by intense inflammation in this area and subsequent fibrosis, which gradually resolves within weeks. however, if older mice are used, the fibrosis will persist longer than in younger mice, which is in accordance with clinical ipf, where the majority of the patients are 65 years of age or older [56, 58] . a great advantage of this model is how well-characterized it is. in addition, local administration is labor-effective, as only one administration is required and the result is highly reproducible. the fibrosis is robust, only affects the lungs, and the accumulation of extracellular matrix can be easily measured using standard techniques [58] . furthermore, as it is used throughout the world, studies performed in different labs and by different groups can be compared relatively easily. unfortunately, the intense pulmonary inflammation may be lethal, and fatalities are to be expected with this model [59] , representing an important ethical limitation. furthermore, fibrosis is heterogeneous-it develops where the bleomycin solution is deposited. the solution usually deposits within the central lung, a localization that is not in agreement with the clinical situation where fibrosis is located in the more distal regions of the lung parenchyma. in addition, the fibrosis that develops as a result of severe tissue damage is self-limiting and reversible, unlike what is observed clinically [58] . the severe degree of tissue damage induced by bleomycin may in fact be more relevant for modeling acute lung injury (ali) or acute respiratory distress syndrome (ards). bleomycin can also be administered systemically, through intravenous or subcutaneous injection. in contrast to local administration, this route requires multiple administrations and is thus more laborintensive [58] . some studies have described the usage of osmotic mini-pumps, where bleomycin is slowly administered over a short period of time, and then fibrosis continues to develop over subsequent weeks [60] . irrespective of the route of delivery, systemic administration results in more homogenous fibrosis, affecting the entire lung through the pulmonary endothelium and persisting much longer than following local administration [61] . the main advantages of systemic administration are that inflammation is limited, while the fibrosis is more apparent and displays a more distal pattern, all of which mimics the clinical situation relatively well. the multiple administrations allow for lower doses with each injection; this is less stressful to the animals and results in little to no mortality [61] and is thus more ethically acceptable. a major disadvantage with this model is that it takes time for fibrosis to develop [58] , which may be the reason it is used relatively scarcely, and thus the pathological development is less well-understood. in addition, as ipf is a local disease, local administration of the etiologic agent may better mimic the clinical reality [56] . the administration of fluorescein isothiocyanate (fitc) induces focal inflammation, primarily involving mononuclear cells and neutrophils, and localizes in areas where the fitc solution is deposited [58] . antibodies against fitc can be detected after 1 week, and the fibrosis persists for up to 5 months after instillation [58] . the benefits of this model are mainly related to the persistent fibrosis that does not appear to be self-limiting, thus reflecting the clinical situation, and it is also very easy to determine which part of the lung has been exposed to fitc, as the molecule is fluorescent [58] . it is also an advantage that both c57bl/6 and balb/c mice are susceptible and develop fibrosis following fitc administration [56] . the disadvantages of this model include profound variability due to differences between batches of fitc, as well as in the method used to prepare the solution before instillation. importantly, given the characteristics of the etiologic agent used to induce this model of ipf, this model is considered a very artificial system with limited clinical relevance [56] . adenovirus vectors have been used to overexpress the pro-fibrotic cytokine transforming growth factor (tgf)-β, which results in pulmonary fibrosis. as tgf-β overexpression in the lungs is known to be crucial in the development of fibrosis in humans [62] , this model mimics an important feature of disease etiology. however, the delivery system has some drawbacks, as the virus itself initiates an immune response. moreover, adenoviruses display significant tropism for epithelial cells and rarely infect other cell types such as fibroblasts [58] , which are the cells meant to be targeted in this model. as tgf-β has major effects on fibroblast biology, the main feature of this model is the effect of epitheliumderived tgf-β on fibroblasts and myofibroblasts, resulting in the deposition of ecm proteins and areas of dense fibrosis [63] . an advantage of this model is the relatively low degree of inflammation, as well as what appears to be a direct effect on fibroblasts/ myofibroblasts [63] , which is in accordance with the clinical situation (as we understand it today). silica administration induces a similar pathology in mouse lungs as in humans exposed to silica, and as is also observed in human silicainduced fibrosis, structural remodeling persists when administration is halted [56] . following the administration of silica particles, fibrotic nodules develop in mouse lungs, with considerable resemblance to the human lesions that develop after exposure to mineral fibers [56] . the fibrotic response is accompanied by a limited inflammatory response, and different pro-fibrotic cytokines such as tgf-β, platelet-derived growth factor, and il-10 are involved in disease development, which is in accordance with the clinical situation [56] . another advantage is that nodules develop around silica fibers, and these fibers are easy to identify by light microscopy. the response in this model is strain-dependent, with c57bl/6 mice being the most susceptible. the main drawbacks are the time required to establish disease, i.e., 30-60 days, and the need for special equipment to aerosolize the silica particles. however, since the route of administration, the driving etiologic agent, and the resulting pathobiology are all similar to the characteristics of this subtype of pulmonary fibrosis [56, 58] , the silica exposure model can be considered to have very good clinical relevance. 7 what does the future hold for mouse models of human disease? medical research using experimental animals (not only mice but other animals including rats, guinea pigs, zebrafish, and fruit flies) has greatly contributed to many important scientific and medical advances in the past century and will continue to do so into the near future. these advances have contributed to the development of new medicines and treatments for human disease and have therefore played a vital role in increasing the human life span and improving quality of life. despite the acknowledged benefits of performing research using experimental animals, a number of considerations must be made before embarking on this type of research. of course, the financial aspects of conducting this type of work are an important limitation, as the costs of purchasing and housing mice can be prohibitive, especially when genetically modified mice and colony maintenance are required for the study. the practicalities of working with animals such as mice may also be an issue, as this type of work requires specialized facilities, equipment, and staff to ensure studies are carried out in a manner that is safe for both the researchers and the animals. moreover, as discussed in detail in this chapter, the relevance of the selected animal model to human disease must be carefully evaluated to ensure that these experiments provide robust results that are translatable to human health and disease. another important and demanding aspect of biomedical research using animals is the ethics of imposing pain and suffering on live animals. although there has been a considerable reduction in the numbers of animals used in research in the last 30 years, animal research remains a vital part of biomedical research. however, no responsible scientist wants to cause unnecessary suffering in experimental animals if it can be avoided, so scientists have accepted controls on the use of animals for medical research. in the uk, this ethical framework has been enshrined in law, i.e., the animals (scientific procedures) act 1986. this legislation requires that applications for a project license to perform research involving the use of "protected" animals (including all vertebrates and cephalopods) must be fully assessed with regard to any harm imposed on the animals. this involves a detailed examination of the proposed procedures and experiments, and the numbers and types of animal used, with robust statistical calculations to support these numbers. the planned studies are then considered in light of the potential benefits of the project. both within and outside the uk, approval for a study involving protected animals also requires an internal ethical review process, usually conducted by the research institution where the work is taking place, with the aim of promoting animal welfare by ensuring the work will be carried out in an ethical manner and that the use of animals is justified. additionally, the uk has a national animal use reduction strategy supported by the national centre for the replacement, refinement and reduction of animals in research (nc3rs; london, uk). this consortium was established in 2004 to promote and develop high-quality research that takes the principles of replacement, refinement, and reduction (the 3rs) into account. replacement strategies often involve the use of alternative, non-protected species (e.g., zebrafish, fruit flies, flatworms) and in vitro correlates (two-dimensional cell culture or threedimensional organoids containing multiple cell types) to test hypotheses and assess the effects of therapeutic interventions. the main obstacle with studies on non-protected animals is the difficulty of accurately mimicking the complex physiological systems involved in human health and disease, as described in detail above. for example, the fruit fly drosophila melanogaster is an excellent model organism for studies on genetic diseases, aging, and pathogen-borne illnesses but may be less relevant for studies on complex lung diseases. importantly, model organisms such as fruit flies, zebrafish, and flatworms do not possess lungs, which somewhat limits the translatability of research on these animals in the field of respiratory disease. as such, it is likely that rodents will remain the model organism of choice for studies into lung disease for some time to come. there has been considerable progress recently in imitating single organs such as the liver, lung, and brain in vitro using multiple cell types and a physical scaffold. as an important advantage, these in vitro tests have replaced a large number of rodents in initial drug discovery experiments, while also speeding up the process [64] . these studies still require further refinement and validation to establish them as suitable models for an entire organ; importantly, these in vitro organoids cannot take into account interactions between organ systems in complex, multisystem diseases such as copd. refinement involves selecting the most clinically relevant model for the disease available, informed by the discussion above on closely recapitulating the etiologic agent and disease pathobiology associated with clinical cases. another important factor is refining the management of pain. an assessment of the procedures used and the effects of the substance on the animal, as well as the degree of handling, restraint, and analgesia, are other important aspects of refinement. this standard of animal care is achieved through strict regulations and controls on how personnel are trained to carry out experiments on live animals. adequate training is an important aspect of refinement and should be reviewed and improved on an ongoing basis. moreover, refinement can be achieved by improving animal housing by environmental enrichment, e.g., providing a place for mice to hide in the cage and housing social animals such as mice in appropriate-sized groups. these simple changes can improve the physiological and behavioral status of research animals; this not only increases animal well-being but also contributes to the quality of the experimental results by reducing stress levels. the 3rs aspect of reduction focuses on the statistical power of experiments and by following the animal research: reporting of in vivo experiments (arrive) guidelines, originally published in plos biology in 2010. these guidelines provide a framework to improve the reporting of research performed on live animals by maximizing the quality of the scientific data and by minimizing unnecessary studies. the arrive guidelines provide a checklist of aspects that must be considered in good quality research using live animals. the guidelines are most appropriate for comparative studies involving two or more groups of experimental animals with at least one control group, but they also apply to studies involving drug dosing in which a single animal is used as its own control (within-subject experiments). the guidelines provide recommendations on what should be considered when preparing to report on the results of experiments involving live animals, i.e., by providing a concise but thorough background on the scientific theory and why and how animals were used to test a hypothesis, a statement on ethical approvals and study design including power and sample size calculations, a clear description of the methods used to ensure repeatability, objective measurements of outcomes and adverse effects, and interpretation of the results in light of the available literature and the limitations of the study. in addition to the positive impact of the arrive guidelines on reducing the number of animals used in experiments, this checklist provides an easy-tofollow roadmap on what is required for good quality reporting of experimental results. in conclusion, the use of animals in research will continue to be an important aspect of medical research, and these procedures can be ethically justified provided the proper controls are in place. the benefits of animal research have been vital to the progress of medical science; abandoning these studies would have severe negative consequences on human health. by considering aspects such as the 3rs and the arrive guidelines 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signaling involved in pulmonary fibrosis and emphysema adenovector-mediated gene transfer of active transforming growth factor-beta1 induces prolonged severe fibrosis in rat lung the ethics of animal research. talking point on the use of animals in scientific research key: cord-003634-iq0e1qp1 authors: otxoa-de-amezaga, amaia; miró-mur, francesc; pedragosa, jordi; gallizioli, mattia; justicia, carles; gaja-capdevila, núria; ruíz-jaen, francisca; salas-perdomo, angélica; bosch, anna; calvo, maria; márquez-kisinousky, leonardo; denes, adam; gunzer, matthias; planas, anna m. title: microglial cell loss after ischemic stroke favors brain neutrophil accumulation date: 2018-12-22 journal: acta neuropathol doi: 10.1007/s00401-018-1954-4 sha: doc_id: 3634 cord_uid: iq0e1qp1 stroke attracts neutrophils to the injured brain tissue where they can damage the integrity of the blood–brain barrier and exacerbate the lesion. however, the mechanisms involved in neutrophil transmigration, location and accumulation in the ischemic brain are not fully elucidated. neutrophils can reach the perivascular spaces of brain vessels after crossing the endothelial cell layer and endothelial basal lamina of post-capillary venules, or migrating from the leptomeninges following pial vessel extravasation and/or a suggested translocation from the skull bone marrow. based on previous observations of microglia phagocytosing neutrophils recruited to the ischemic brain lesion, we hypothesized that microglial cells might control neutrophil accumulation in the injured brain. we studied a model of permanent occlusion of the middle cerebral artery in mice, including microgliaand neutrophil-reporter mice. using various in vitro and in vivo strategies to impair microglial function or to eliminate microglia by targeting colony stimulating factor 1 receptor (csf1r), this study demonstrates that microglial phagocytosis of neutrophils has fundamental consequences for the ischemic tissue. we found that reactive microglia engulf neutrophils at the periphery of the ischemic lesion, whereas local microglial cell loss and dystrophy occurring in the ischemic core are associated with the accumulation of neutrophils first in perivascular spaces and later in the parenchyma. accordingly, microglia depletion by long-term treatment with a csf1r inhibitor increased the numbers of neutrophils and enlarged the ischemic lesion. hence, microglial phagocytic function sets a critical line of defense against the vascular and tissue damaging capacity of neutrophils in brain ischemia. electronic supplementary material: the online version of this article (10.1007/s00401-018-1954-4) contains supplementary material, which is available to authorized users. neutrophil infiltration under conditions of sterile inflammation can contribute to tissue injury. neutrophils are transiently detected in the brain after stroke since they are rapidly attracted to the injured brain peaking between 1 and 3 days post-ischemia [10, 23, 24, 51] . compelling evidence suggests that neutrophils are contributors to tissue damage after ischemic stroke [35, 44, 51, 61, 64] , in spite of the fact that diverse experimental strategies inhibiting neutrophil activation or depleting neutrophils provided conflicting results [16, 61] . likely, the differences between experimental studies depend on the efficacy and potential side effects of the diverse neutrophil depleting or inhibiting strategies, status of capillary reperfusion, lesion severity, and integrity of the blood-brain barrier (bbb). moreover, several aspects of neutrophil infiltration after acute ischemic brain damage remain controversial. neutrophils accumulate in perivascular spaces in murine and human strokes [17, 55] . the presence of neutrophils in the brain parenchyma has been reported in rodent models of permanent ischemia [23, 51, 55] , but it is more controversial in experimental models of transient ischemia [17, 64] . several studies reported the presence of neutrophils in the brain parenchyma in postmortem samples of patients deceased between day 1 and 5 [55] , or 3 days after stroke onset but not at other time points [56, 76] . in other studies, neutrophils were not detected in the brain parenchyma of stroke patients [17] . therefore, the molecular determinants underlying perivascular neutrophil accumulation and the conditions facilitating the potential access of neutrophils to the brain parenchyma need further clarification. the observation that microglia phagocytose neutrophils in the ischemic brain [50] [51] [52] led us to hypothesize that microglia function may be critical to explain neutrophil accumulation in the injured brain tissue. microglial cells react to brain ischemia in different ways depending on the regional location and temporal course of the lesion. microglial cells are vulnerable to ischemia and previous reports showed death of microglia after oxygen and glucose deprivation in tissue slices [18] and cell cultures [41, 73] . in addition, microglial reduction has been reported after transient mcao [43] , and microglial dysfunction and loss was detected in classical neuropathological studies of brain ischemia in rodents and primates [3, 4] . classical histopathological studies have shown long-lasting microgliosis surrounding the infarction several days after ischemic stroke onset. however, the progression of this reaction from the very acute phase of stroke is less precisely determined mainly due to the fact that microglia and infiltrating macrophages show many common features and markers leading to the frequent terminology of microglia/macrophages to describe the mononuclear myeloid cell reaction that follows stroke. microglia have a unique transcriptomic signature distinguishable from that of macrophages or monocytes [7, 32] . therefore, reactive microglia and infiltrating macrophages likely play different functions in the injured brain tissue. current developments allow the distinction between these cells with antibodies against more specific microglia markers [1, 7] , availability of fluorescent reporter mice [74] , or transfer of fluorescent reporter leukocytes [46] . by exploiting some of these novel experimental possibilities, we investigated the neutrophil-microglia crosstalk after brain ischemia. the results show that microglial cells effectively remove brain-infiltrating neutrophils, hence microglia dysfunction or death is associated with neutrophil accumulation into the injured brain tissue. we used adult male mice on the c57bl/6 background. mice expressing tamoxifen-inducible cre recombinase under the direction of the cx3cr1 promoter in the mononuclear phagocyte system (cx3cr1 cre/ert2 ) [74] (#020940 jax ® mice) were crossed with either ai 9 mice harboring a loxp-flanked stop cassette that prevents transcription of the red fluorescent protein tdtomato (tdt) (b6.cg-gt(rosa) 26sortm9 (cag-tdtomato)hze/j (#007909 jax ® mice) [42] , or colony stimulating factor 1 receptor (csf1r) +/flox mice (b6.cg-csf1r tm1jwp /j, #021212 jax ® mice). we used heterozygous catchup ivm mice expressing tdt in ly6g +/− neutrophils [26] . homozygous catchup ivm (ly6g −/− ) mice were crossed with cx3cr1 gfp/gfp mice to obtain double heterozygous mice with red fluorescent neutrophils and green fluorescent microglia [50, 75] . we also obtained cells from dsred mice constitutively expressing the red fluorescent protein dsred under the control of the actin promoter [8] . wildtype mice were obtained from a commercial source (janvier, france). mice were maintained in the animal house of the school of medicine of the university of barcelona under controlled spf conditions. animal work was conducted with the approval of the ethical committee of the university of barcelona (ceea) and the direcció general de polítiques ambientals i medi natural, departament de territori i sostenibilitat de la generalitat de catalunya. studies complied with the "principles of laboratory animal care" (nih publication no. 86-23, revised 1985), and the spanish national law (real decreto 53/2013). the brains of six patients suffering from acute ischemic stroke who died between 1 and 6 days after stroke onset at the stroke unit of the hospital clinic of barcelona were used after obtaining written consent from their relatives or legal representatives for tissue removal after death at the neurological tissue bank of the biobank-hospital clinic-institut d'investigacions biomèdiques august pi i sunyer (idibaps). the ethics committee of this hospital approved the study. online resource 1 shows a summary of patient characteristics. the elapsed time from death to autopsy was 2-8 h. an expert neuropathologist dissected the ischemic core, periphery, and a portion of non-ischemic tissue (control) obtained from a region distant to infarction, as described [55] . samples were embedded in oct and immediately frozen in liquid nitrogen for sectioning at 5 µm in a cryostat. the bone marrow of transgenic dsred mice [46] was used to generate chimeric mice, as reported [37] . in brief, recipient adult (2-month old) wild-type mice received three intraperitoneal injections of the chemotherapeutic agent busulfan (30 mg/g body weight) 7, 5 and 3 days prior to transfer via the tail vein of five million bone marrow cells from dsred donor mice. mice were used 8 weeks after grafting and reconstitution was assessed by flow cytometry analysis. to impair microglial function, mice received a daily oral administration by gavage of the csf1r inhibitor gw2580 [12] (75 mg/kg body weight in a volume of 0.2 ml) (#s8042, selleckchem) for 4 days, which is a dosing regimen that does not challenge microglial survival [54] . treatment controls received the same volume of the vehicle (0.5% hydroxypropylcellulose, 0.1% tween-80). treatment started 2 h prior to induction of ischemia, it was randomly allocated, and was administered in a blinded fashion. for microglia depletion, mice received the csf1r inhibitor plx5622 (plexxikon) following previously reported protocols [15, 33, 69] . the inhibitor was mixed into ain-76a standard chow at 1200 ppm (brogaarden, denmark). mice (8-week-old) received the diet ad libitum for 3 weeks prior to induction of ischemia and the diet was maintained until the mice were killed. treatment controls received ain-76a diet for the same period of time. both diets were given in parallel in groups of five animals per cage. surgery was carried out under isoflurane anaesthesia and mice received analgesia (buprenorphine, 140 µl of a 0.015 mg/ml solution, via s.c.). permanent occlusion of the middle cerebral artery (mcao) was induced by coagulation of the distal portion of the right mca together with ligation of the ipsilateral common carotid artery. this experimental model induces a focal cortical lesion in the ipsilateral hemisphere. a subset of mice receiving the above diets (control or plx5622) was used to study the volume of the lesion 1 day after induction of ischemia by t2w mri in a 7.0 t biospec 70/30 horizontal animal scanner (bruker biospin, ettlingen, germany), as reported [13] . sample size was calculated using g*power 3.1 software (university of dusseldorf) with an alpha level of 0.05, statistical power of 0.95, and estimating a size effect of 1.8 based on sd of previous results from our laboratory and published data on the effect of microglia depletion on infarct volume in other stroke models [65] . one mouse died (control diet), and one mouse was excluded (plx5622 diet) due to surgical problems. bromodeoxyuridine (brdu) (10 mg/ml) (#550891, bs pharmingen) was daily injected (150 μl) via i.p. into mice starting 1 day after mcao until day 4. one-hour after the last brdu administration mice were killed and processed for immunofluorescence. brdu was detected in brain tissue sections using a rat monoclonal fitc-anti-brdu antibody (1:50, #ab74545, abcam, cambridge, uk) [46] . mouse blood and brain tissue were processed for flow cytometry as described [46] . fc receptors were blocked by previous incubation for 10 min with cd16/cd32 (clone 2.4g2, bd pharmingen) in facs buffer (pbs, 2 mm edta, 2% fbs) at 4 °c. live/dead aqua cell stain (molecular probe, invitrogen) was used to determine the viability of cells. cells were incubated with the following mix of microglia cells from adult mice (9-14 weeks old) were isolated and cultured using immunomagnetic separation (miltenyi biotec, germany). mice were perfused via the left ventricle with 60 ml of cold saline and collected in hanks' balanced salt solution (hbss) buffer without calcium/magnesium (#14175-05; life technologies). the brain tissue was enzymatically dissociated using the neural tissue dissociation kit-p (#130-092-628; miltenyi biotec). the gentlemacs™ dissociator with heaters (#130-096-427; miltenyi biotec) was used for mechanical dissociation steps during 30 min at 37 °c. the digested tissue was filtered (70 µm) with hbss buffer with calcium and magnesium (#14025-050; life technologies) and prepared for myelin removal process (myelin removal beads ii, #130-096-733; miltenyi biotec). then, cells were magnetically labeled with cd11b microbeads (#130-093-634; miltenyi biotec) diluted in pbs supplemented with 0.5% bsa for 15 min in the dark in the refrigerator (2-8 °c) . cd11b + cells were collected using magnetic field columns (miltenyi biotec). cell suspensions (35 μl) were then plated in complete medium consisting of dmem medium (#10569010; gibco-brl) supplemented with 10% fetal bovine serum (fbs; gibco-brl) containing 40 u/ml penicillin and 40 μg/ml streptomycin (#15140122; gibco-brl) added as a drop in the middle of each well of a poly-l-lysine (#p4832; sigma) pre-coated 8-well plate (µ-slide 8 well, ibidi #80826). cells were incubated for 30 min at 37 °c and then 250 µl of complete medium were carefully added to each well. twenty-four hours later, we replaced 50% of complete medium, and we did a full medium change at day 5. the cells were maintained at 37 °c in a humidified atmosphere of 5% co 2 for 7 div. we obtained human microglial cells from the ischemic tissue of one patient deceased 5 days after fatal stroke. fresh brain tissue (about 500 mg) was harvested at autopsy (8 h after death) and was placed in a falcon tube with sterile cold rpmi 1640 medium (#21875-034, gibco). visible meninges were removed, the tissue was cut in small pieces using a scalpel and incubated in a 0.25% trypsin-edta solution in pbs at rt for 30 min. then, dmem/f12 (#11330032; gibco-brl) with 20% fbs and dnase i (200 units/ml) was added (1:1), the tissue was disaggregated, centrifuged for 7 min at 250×g and the pellet was re-suspended in 30 ml dmem/f12 supplemented with 10% fbs, 10% l-cell conditioned medium obtained from the l929 cell line, and 100 u/ml penicillin/100 μg/ml streptomycin (#15140122; gibco-brl). cells were seeded in poly-l-lysine coated t25 flasks, incubated in 5% co 2 at 37 °c and allowed to adhere. culture medium was changed twice a week and at 7div the cells were scrapped and seeded in a 8-well plate (µ-slide 8 well, ibidi #80826) previously coated o/n with poly-l-lysine. a time-lapse microscopy study was initiated 6 h later after addition of fresh bone marrow neutrophils. afterwards, we fixed the cells for an immunofluorescence study with antibodies against the purinergic receptor p2y, g-protein coupled, 12 (p2ry12) (1:200, #as55042a, anaspec). neutrophils were obtained from the bone marrow of adult (10-14 weeks old) mice. the bone marrow was flushed using a 25-gauge needle with rpmi 1640 (#21875-034, gibco) supplemented with 10% fbs onto a 50 ml falcon tube through a 70-μm cell strainer. cells were centrifuged at 300×g for 5 min. the supernatant was discarded and cells were then incubated for 2 min with an erythrocyte lysis solution (150 mm nh 4 cl, 1 mm khco 3 , 0.1 mm edta). after washing with cold pbs supplemented with 2% fbs, cells were incubated at 4 °c for 15 min with a mix of fcblock (1/200; clone 2.4g2; bd pharmingen; bd bioscience), and the antibody ly6g (clone 1a8, fitc; bd pharmingen) with 10 µl/10 7 cells. cells were washed with pbs-0.5% bsa, and were then incubated with anti-fitc microbeads (#130-048-701, miltenyi biotec) for 15 min at 4 °c with 10 µl microbeads/10 7 cells. after washing, the fraction of positive ly6g cells was magnetically collected and prepared for immediate use or cells were frozen in fbs serum with 10% of dmso until the day of the experiment. human neutrophils were isolated from the blood by density gradient centrifugation. human and mouse neutrophils were stained with celltracker™ green cmfda (#c2925; ther-mofisher scientific). fig. 1 localization of microglia and infiltrating leukocytes. we generated chimeric mice by administering dsred fluorescent bone marrow cells to 2-month old wild-type receptor mice (n = 10). after 2 months, we induced ischemia and 4 days later we studied the brain by immunofluorescence (n = 5) (a, b) and flow cytometry (n = 5) (c, e). a cd45 hi cd11b hi cells infiltrating the ipsilateral hemisphere are mostly dsred + whereas cd45 dim cd11b dim microglial cells are dsred − . b flow cytometry shows an increase in infiltrating dsred leukocytes (cd11b hi cd45 hi ) (mann-whitney test, **p = 0.008). c immunostaining of astrocytes (gfap, green) showed the presence of dsred cells at the border and core of the lesion separated from the peripheral area that shows a prominent astroglial reaction. d microglial cells were stained with an antibody against p2ry12 (green), which did not co-localize with dsred + leukocytes. the morphology of the microglia in the different regions is illustrated with representative images. cell nuclei were stained with to-pro3 (blue). e p2ry12 + microglial cell at the border of the lesion nearby dsred + infiltrating leukocytes. f schematic representation of the distribution of microglia (green) and infiltrating leukocytes (red) in the different regions of the ischemic hemisphere. scale bar c 50 µm; d, e 10 µm . c-f analysis of microglial morphology in the core and periphery of the ipsilateral cortex and the contralateral cortex (contral.) (n = 25-49 cells per region of 5 different mice represented by the different colors) using imagej tools. g, h imaris analysis of 3d-reconstructions of the above cells (b). i counting the number of microglial cells per area showed an increased microglial density in the periphery and a decrease in the core (n = 17-27 fields in 5 different mice). statistical analyses in c-i were carried out with the kruskal-wallis test followed by the dunn's test. **p < 0.01 and ***p < 0.001 vs. contralateral microglia; && p < 0.01 and &&& p < 0.001 vs. peripheral microglia. j flow cytometry after dissecting out the core and periphery regions of the ipsilateral hemisphere and mirror regions of the contralateral hemisphere 1 and 4 days after mcao in an independent group of mice (n = 5 mice per time point). for comparative purposes the number of microglial cells (cd45 low cd11b + ) is expressed by mg of brain tissue. data analysis was conducted with two-way anova by region (p < 0.0001) and time point (p = 0.039) followed by the bonferroni test. the number of microglia was higher in the periphery than the core of infarction 1 day ( && p < 0.01) and 4 days ( &&& p < 0.001) after mcao. furthermore, the number of microglia decreased in the core of infarction versus the corresponding contralateral region 1 day (**p < 0.01) and 4 days (*p < 0.05) postischemia. in the periphery, the number of microglia cells increased in the ipsilateral versus the corresponding contralateral hemisphere at day 4 ( # p < 0.05). scale bar 10 μm isolated and stained neutrophils (75,000 cells/ml) were added to the adult microglia cultures at 7div. automated multiposition live cell imaging was carried out using a leica tcs sp5 confocal microscope (leica microsystems, heidelberg, germany) equipped with adaptive focus control to keep the specimen in focus and an incubation system with temperature and co 2 control. cells were subjected to a timelapse study while maintained at 37 °c in a humidified atmosphere of 5% co 2 . all images (3-4 z sections) were acquired using a apo 63 × (numerical aperture 1.3) glycerol immersion objective lens, pinhole set at 1.5 airy units. images of cmfda and dsred were acquired sequentially line by line using 488 and 561 laser lines and detection ranges at 500-550 and 570-650, respectively. simultaneously, bright field images were acquired. multiposition confocal images were acquired every 4 min during 12-14 h, with an image matrix of 512 × 512 pixel; 600 hz; 2 × line average and autofocus control. manual analysis was performed using fiji software (version 2.0.0-rc-67/1.52d). we recorded 3-4 timelapse videos per well and analysed 180-210 frames in each video. in every frame, manual tracking of neutrophils was performed using the mtrackj plugin [45] to identify phagocytosis of neutrophils by microglial cells. we studied in parallel four wells per genotype (csf1r +/+ or csf1r +/− microglial cells) in each independent experiment and conducted five independent experiments. the analysis was performed in a blinded fashion by assigning a code to each video that did not reveal the identity of the genotype. we used green fluorescent zymosan a bioparticles (#z-23373; thermo fisher scientific) in the phagocytosis assay. at 7 div, microglial cells were exposed to zymosan fluorescent beads (75,000 particles/ml) for 1 h. following 3-4 washes to remove all the non-phagocyted particles, cells were fixed with cold 4% paraformaldehyde for 20 min, permeabilized with 0.2% triton x-100 (sigma) in pbs 0.1 m for 15 min, blocked with 3% goat serum in pbs for 1 h, and incubated overnight at 4 °c with the primary rabbit antibody against the p2ry12 receptor (1:200, #as55042a, anaspec). the next day, cells were washed and incubated with red fluorescence alexa fluor ® 546 dye-labelled goat anti-rabbit igg antibody (#a10036, life technologies) for 1 h at room temperature. dapi (#d3571, life technologies) stained was performed to visualize the cell nuclei. cells were then covered using fluoromount-g ® (southern biotech, birmingham, al, usa). images were obtained with a fluorescence inverted microscope (leica ctr 40000). mice were perfused via the left heart ventricle with 40 ml of cold saline (0.9%) followed by 20 ml of cold 4% paraformaldehyde (pfa) diluted in phosphate buffer (pb) ph 7.4. the brain was removed, fixed overnight with the same fixative, and immersed in 30% sucrose in pb for cryoprotection for at least 48 h until the brains were completely sunk to the bottom of the tube. after that, brains were frozen in isopentane at − 40 °c. cryostat brain sections (14-μm thick) were fixed in ethanol 70%, blocked with 3% normal serum, and incubated overnight at 4 °c with primary antibodies: rat monoclonal antibodies against ly6g cell nuclei were stained with dapi or to-pro3 (invitrogen). cryostat sections from human brain tissue were processed for immunofluorescence as described above with a rabbit polyclonal antibody against p2ry12 (1:200, #5042a, anaspec) and a mouse monoclonal antibody against ki67 (1:400, #9449, cell signaling tech). consecutive sections were stained with thionine for examination of the lesion at the light microscope. confocal images were obtained (tcs-spe-ii or sp5 microscopes from leica microsystems; or a zeiss lsm880 microscope) and were not further processed except for enhancing global signal intensity in the entire images for image presentation purposes using las software (leica), imagej, or adobe photoshop. for estimation of the density of p2ry12 + cells and ki67 + cells in human brain sections, images were obtained (40 × objective), the number of immunostained cells and cell nuclei per image were counted in ten different fields per brain region of each subject, and average values per region and time group were calculated. for cell counting in mouse brain sections, we obtained 5-6 confocal images of the immunostaining (63 × objective) in three different brain sections per mouse. microglia morphology was assessed using fiji software (version 2.0.0-rc-67/1.52d) and imaris software (ima-ris bitplane v.9.0). basic shape descriptors such 1 3 as the circularity index (ci) or the area were performed with the plugin shape descriptors [68] ; other parameters, such us the ramification index (ri), were obtained using the sholl analysis plugin [21] . parameters such as volume or sphericity index were measured using imaris software after creating a 3d surface in the maximum intensity projection image. then, microglial cells were thresholded by the huang method [34] to generate a binary mask (with a 1.5 mean filter). the ci parameter was calculated by the shape descriptors plugin (4p[area]/[perimeter] 2 ). the highest count of intersections (max inters) reflects the highest number of processes in the cell. two-group comparisons were carried out with the mann-whitney u test. for multiple group comparisons we used the kruskal-wallis test followed by the dunn's test. comparisons were two-sided. comparisons of groups by brain region and time were carried out with two-way anova followed by the bonferroni post-hoc analysis. twoway anova by genotype and experiment, with an experiment-matched design, was used to analyze quantification of in vitro studies. statistical analyses were performed with graphpad software. the specific test used in each experiment and n values are reported in the figure legends. to unequivocally distinguish microglia from infiltrating leukocytes we generated chimeric mice with the hematopoietic system derived from fluorescent (dsred) donor mice where microglia remained non-fluorescent, whereas a high proportion of peripheral myeloid cells were dsred + (fig. 1a; online resource 2). most myeloid cells infiltrating the ipsilateral brain hemisphere were dsred + 4 days after mcao, as assessed by flow cytometry (fig. 1a, b) , and they were preferentially located in the lesion core where the expression of gfap is lost (fig. 1c) . we stained microglia with anti-p2ry12 antibodies [7, 28, 59] and verified that the infiltrating dsred + cells were not p2ry12 + . microglia and infiltrating dsred + leukocytes co-existed at the border of infarction, whereas microglia were abundant at the infarct periphery but scarce in the core of the lesion (fig. 1d-f ). microglia acquired a reactive phenotype at the periphery and border of infarction with thicker ramifications compared to microglia of the contralateral hemisphere. in contrast, microglial cells in the infarcted core showed a dystrophic morphology since the cell body became smaller and there were only a few long ramifications showing an appearance of discontinuity as if they were broken or beaded, and the density appeared to be reduced (fig. 1d) . to ensure that in wild-type mice we did not miss microglial cells in the infarcted core due to downregulation of the markers used to label microglia, i.e. iba1 and p2ry12, we studied the cx3cr1 cre/ert2 mice [74] crossed with floxed rosa26:tdt reporter mice [42] , which express the red fluorescent protein in microglia (fig. 2a, b; online resource 3) obtaining the same findings as in wild-type mice. next, we analysed microglia morphology (online resource 4) in the contralateral hemisphere, and in different zones of the ipsilateral hemisphere, i.e. periphery and infarcted core, 4 days after mcao (fig. 2c-h) . shape descriptors showed increased circularity and reduced area of microglia in the ipsilateral hemisphere that was more marked in cells located in the core region (fig. 2c, d) . a sholl analysis showed that ischemia reduced the number of ramifications, and maximal intersections per microglial cell, and again the changes were greater in the core (fig. 2e, f) . analysis of 3d-reconstructions of the cells showed a reduced volume and higher sphericity index after ischemia, particularly in the core (fig. 2g, h) . furthermore, the quantification of cells per area showed that while microglial cell density increased in the periphery, it was reduced in the core of the lesion (fig. 2i) . this result was confirmed by flow cytometry after excising the core and periphery of the ipsilateral hemisphere and mirror regions of the contralateral hemisphere 1 and 4 days after mcao. microglia cell number was severely reduced in the core of the lesion at 1 and 4 days post-ischemia (fig. 2j) . altogether, these findings show that microglial cells are sensitive to persistent ischemic conditions and are lost in the infarcted core. fig. 3 microglial proliferation after ischemic stroke in mouse and human brain. immunofluorescence in mouse (a-c) and human (d-g) brain tissue. a-c brdu incorporation (green) in dsred − ramified microglial cells expressing p2ry12 (blue). d human tissue of stroke patients deceased at different time points after stroke onset. the brains were grouped according to the day of death after stroke in '1-3 days' (n = 3) and '4-6 days' (n = 3) groups. the number of p2ry12 + cells per area tended to decrease in the core of the lesion in both groups and to increase at the periphery of infarction in the '4-6 days' group. **p < 0.01 vs. periphery. the percentage of ki67 + cells among the p2ry12 + cell population increased at the periphery of infarction in the '4-6 days' group (***p < 0.001 vs. control and vs. core). e-g representative images of double-immunopositive p2ry12 (green) and ki67 (red) microglial cells (arrowheads) at the periphery of infarction from different stroke patients deceased 5 (e-f) or 6 (g) days after stroke. nuclei are stained with to-pro3 (blue). scale bar 10 µm in contrast to the lesion core, the microglial cell number increased at the periphery of infarction 4 days post-ischemia (fig. 2i, j) . this effect was attributable to microglial proliferation as shown in dsred chimeric mice that received injections of the cell proliferation marker brdu after mcao (fig. 3a-c) . by cell counting, we calculated that 26.8 ± 8.8% (mean ± sd, n = 3 mice) of the microglial cells at the periphery of infarction incorporated brdu suggesting microglial proliferation, in agreement with previous reports [14, 40] . in contrast, microglial cells were brdu − in the contralateral non-ischemic hemisphere. notably, brdu was found in ramified microglial cells suggesting that microglia could undergo cell division without regression of their differentiation status. we extended the results to the human brain by showing an increased number of microglial cells and proliferating ki67 + microglia at the periphery of infarction in post-mortem human brain tissue of stroke patients (fig. 3d-g) . we also found reduced microglial cell numbers in the lesion core of stroked human brains (fig. 3d) thus supporting that the findings in mice might be relevant to human stroke. our data identified the edge of the lesion as a site for possible interactions between microglia and leukocytes since both cell types were abundant in this zone. indeed, we observed numerous microglial cell processes surrounding the blood vessels and getting into contact with infiltrating dsred + leukocytes by sampling the dsred + cells adjacent to the vascular endothelium (online resource 5). furthermore, we detected microglial processes surrounding dsred + cells suggestive of engulfment of leukocytes (fig. 4a-c; online resource 6). 3d-reconstructions of confocal images showed phagosomes completely wrapping dsred + leukocytes (fig. 4b, c) . the csf1r signaling pathway is critical for survival of microglia and maintenance of their functions [2] , including endocytic processes [60] . consequently, by interfering with microglial function after oral administration of the csf1r inhibitor gw2580 [12] for 4 days, we found more dsred + cells at the periphery of infarction not surrounded by microglia or in apparent contact with microglia, suggesting that microglia dysfunction impaired the process of phagocytosis of leukocytes (fig. 4d) . neutrophils showed bright dsred fluorescence in the dsred chimeric mice, as assessed by flow cytometry and immunofluorescence 4 days post-ischemia (fig. 5a) . we detected ly6g + dsred + neutrophils shrouded by microglia at the periphery of infarction (fig. 5b, c) . furthermore, processes of microglial cells located nearby the surface of the cortex seemed to traverse the external cortical basement membrane and surround neutrophils located in the subpial space (fig. 5d) . catchup ivm mice crossed with transgenic cx3cr1 gfp/gfp mice allowed obtaining heterozygous double reporter mice with red fluorescent neutrophils and green fluorescent microglia [50, 75] . in these mice, we observed microglia (green) adjacent to the basal lamina of blood vessels and surrounding extravasated neutrophils (red) after mcao (fig. 5e to further investigate the phagocytosis of neutrophils by microglia, we carried out an in vitro study using time-lapse confocal microscopy. we isolated microglia from adult mouse brains, cultured the cells in vitro for 7 days, and exposed them to freshly isolated bone marrow neutrophils (stained with cmfda) to study phagocytosis. microglial cells phagocytosed neutrophils in vitro (fig. 6a, b , and timelapse microscopy movie in online resource 8). by following neutrophil trajectories along time (time-lapse microscopy movie in online resource 9), we counted the number of neutrophils phagocytosed by microglia. we validated that allogenicity in the phagocytosis experimental design was not interfering with the assay by comparing phagocytosis of neutrophils obtained from different mice with the phagocytosis of neutrophils from the same mouse. we found no differences, ruling out prime effects of allogenicity in the fig. 4 microglia cells phagocytose infiltrating dsred leukocytes. a-c chimeric mice were generated by bone marrow transfer from donor dsred mice to recipient wild type mice and were subjected to ischemia (n = 5). immunofluorescence showing iba1 + dsred − microglial cells engulfing dsred leukocytes at the periphery of infarction at day 4 after mcao. a confocal images of an iba1 + (green) dsred − microglial cell engulfing a dsred leukocyte. the images correspond to different z planes. b 3d-reconstructions of iba1 + dsred − microglial cells (blue) engulfing dsred + leukocytes (red). c microglial cell (iba1 + , blue) with engulfed red cells in several prolongations. nuclei are stained with dapi (white). magnified details of engulfed dsred cells are shown on the right hand side. f dsred chimeric mice received oral administration of either the csf1r inhibitor gw2580 or the vehicle (n = 5 per group) starting 2 h prior to mcao and then daily for 3 days. the brain was studied 4 days post-ischemia by immunofluorescence using anti-p2ry12 antibody to label microglia (green), dsred for infiltrating leukocytes, and to-pro-3 for staining the cell nuclei (blue). the image shows a microglial cell at the periphery of infarction engulfing dsred leukocytes. schematic representation of this cell illustrating: (a) a dsred cell completely surrounded by a microglial process; (b) a microglial process making apparent contact with a dsred leukocyte (touching); and (c) a dsred leukocyte separated from microglia (free). counting the number of dsred cells in the above status (a-c) in relation to microglia showed that the csf1r inhibitor increased (***p < 0.001) the number of free dsred cells. two-way anova by treatment and condition followed by the bonferroni test. scale bar corresponds to a, b 5 µm; c 10 µm for left image and 5 µm for the magnifications on the right; d 10 µm ◂ phagocytosis of neutrophils by microglia (online resource 10). given that we observed signs of impaired phagocytosis of neutrophils in vivo after ischemia in mice after shortterm treatment with a csf1r inhibitor (fig. 4d) , we investigated in vitro the role of csf1r in this process. to this end, we obtained microglia from adult mice with heterozygous csf1r +/− microglia (obtained by crossing cx3cr1 cre/ ert2 with csf1r flox/+ mice). less neutrophils were phagocytosed by csf1r +/− microglia compared to csf1r +/+ microglia, as assessed by time-lapse microscopy where we counted the neutrophils phagocytosed by microglia during the 14-h duration of the experiment (fig. 6c ). in addition, csf1r +/− microglia showed reduced phagocytic activity in a phagocytosis assay with fluorescent beads (fig. 6d-f ). we then studied the potential relevance of these findings for human cells by obtaining human microglia from the post-mortem brain of an ischemic stroke patient deceased 5 days after stroke onset. after maintaining the human cells in culture for 7 days, we exposed the cells to neutrophils obtained from blood of a donor control subject. we observed very active phagocytosis of neutrophils (fig. 6g, h and timelapse microscopy movie in online resource 11). by immunofluorescence after the ex vivo assay, we detected p2ry12 expression in the cells and observed that they contained material from neutrophils (fig. 6i) . neutrophil numbers were higher in the core of infarction than the periphery 1 and 4 days after mcao, as assessed by flow cytometry after dissection of these brain regions (fig. 7a) . neutrophils were seen in perivascular spaces of venules (fig. 7b) but also in the parenchyma outside the basement membrane in the core of the lesion (fig. 7c, d) . strikingly, neutrophils were found in the brain parenchyma in zones of the core where microglia was absent or showed signs of dystrophy, as assessed in immunostained brain sections of wild-type mice and reporter mice, including the cx3cr1 cre/ert2 -rosa26:tdt mice (fig. 7e ) and the catchup ivm -cx3cr1 +/− double reporter mice (fig. 7f-h) . at the periphery of infarction neutrophils were surrounded by reactive microglia, suggesting that microglia phagocytosed neutrophils thereby preventing their accumulation in this region. in contrast, in the core of infarction, dysfunction or loss of microglia might facilitate the accumulation of neutrophils. to obtain further evidence that microglia remove neutrophils after ischemia, we depleted microglia by feeding the mice for 3 weeks with a diet containing a csf1r antagonist (plx5622) [15, 33, 69] . plx5622 diet caused a strong reduction (90%) of the microglia population (fig. 8a ) but did not affect blood leukocyte counts (online resource 12 shows the gating strategy and cell quantifications are shown in online resource 13), in agreement with previous reports [20, 70] . however, we detected a reduction of a minor subset of blood ly6c − monocytes (online resource 13), which is dependent on csf1r [39] . in brain tissue, plx5622 diet reduced the numbers of infiltrating monocytes (40%) and f4/80 + macrophages (60%) versus the control diet 4 days post-ischemia (online resource 14), in agreement with previous findings suggesting a role for microglia in the recruitment of monocytes into the brain [20] . in contrast, the plx5622 diet increased the numbers of neutrophils in the brain tissue after ischemia, as assessed by flow cytometry at day 4 (fig. 8b) . immunofluorescence in cx3cr1 cre/ert2 :r26-tdt mice showed extravasated neutrophils that seemed more abundant in the absence of microglia (fig. 8c, d) . by counting the number of neutrophils located either in the parenchyma or associated with blood vessels (hence not crossing the parenchymal basal lamina) we found an increase in the percentage of parenchymal neutrophils in the absence of microglia 1 and 4 days post-ischemia (fig. 8e) . of note, neutrophils were often observed on the fig. 5 microglia engulf neutrophils. a-c chimeric mice generated by transfer of dsred bone marrow cells to wild type recipients. a flow cytometry shows cd11b + ly6g + dsred neutrophils in the ipsilateral but not the contralateral hemisphere (mann-whitney test, **p = 0.008, n = 5) 4 days post-ischemia. infiltrating neutrophils (nimp-r14 + , ly6g + ) are dsred + . b, c p2ry12 + microglial cells (blue) engulf neutrophils at the periphery of infarction. images obtained 4 days after mcao representative of n = 5 chimeric mice. d image of a cx3cr1 cre/ert2 :rosa26-tdt mouse 4 days after mcao showing a microglial cell (red) sending a process across the external basal lamina of the cortex (α4-laminin, blue) to trap a neutrophil (ly6g + , green) in the subpial space. e-i images obtained from cx3cr1 gfp/+ /catchup mice, which have gfp + microglia (green) and tdt + neutrophils (red) 1 day after mcao. sequence of confocal z-images showing the basal lamina (pan-laminin, blue) of a capillary with a neutrophil in the lumen and an extravasated neutrophil, surrounded by a microglial cell (e). z projections of one plane and confocal projection illustrating interaction of microglia with basal lamina and neutrophils (f-g). 3d-reconstructions showing the microglial cell (green) attached to the capillary (blue) and intraluminal (h) and extravasated (i) neutrophils (red). nuclei are stained with dapi (white). images were obtained from superficial cortical layers (a-c, e-i) and the brain surface (d) in zones corresponding to the core of the lesion (a), border of the lesion (d-i), and periphery (b, c). scale bar 10 μm ◂ 1 3 basement membrane of capillaries (fig. 8d) and venules (fig. 8f) suggesting a possible interaction of these cells with basal lamina components. in line with this, microglia depletion increased the size of the ischemic lesion (fig. 8g) , further supporting a beneficial effect of microglia at the periphery of the infarction. this study supports the concept that microglia phagocytose and remove neutrophils after brain ischemia [14, [50] [51] [52] and demonstrates that neutrophil accumulation in the brain parenchyma is associated with reduced microglial phagocytic activity, attributable to ischemia-induced microglial cell dysfunction due to loss or dystrophy. morphometric analysis of microglia showed changes in the periphery of the lesion compatible with microglial reactivity and similar to those reported [27] . overall, morphological changes of microglia within the lesion core were larger than in the periphery, for instance regarding the notable loss of ramifications and reduced cell size. such profound morphological changes of microglia in the lesion core might indicate a further process of transformation from reactive microglia to dystrophic microglia, potentially associated with cell dysfunction. furthermore, we found reduced microglial cell numbers in the core of infarction. while our results support microglial degeneration in the infarcted core, microglial cells proliferated and accumulated at the periphery of infarction in mouse and human brain, in agreement with previous findings in the mouse brain [14, 40] . these reactive microglial cells at the periphery of infarction phagocytosed neutrophils, suggesting that the phagocytic activity of microglia prevented neutrophil accumulation in this region. accordingly, the numbers of neutrophils were higher in the core than the periphery of the lesion. microglial activity and survival are critically dependent on csf1r [12] . consequently, drug-induced inhibition of csf1r or genetic reduction of csf1r expression in microglia impaired their phagocytic capacity in vivo and in vitro. previous studies reported that csf-1 promotes phagocytosis of ab1-42 peptide by primary human microglia in vitro [62] , and it regulates cell motility in macrophages [58] . csf1r is a tyrosine kinase that upon activation shows phosphorylation of several intracellular tyrosine residues [77] . upon activation, csf1r associates with several signaling molecules, notably phosphoinositide 3-kinase (pi3k) [58] . csf1r also activates akt [11] , and it induces erk1/2-mediated signaling in microglia [9] . akt [22, 67] and erk1/2 [19] are involved in the phagocytic process. however, the specific signaling molecules downstream of csf1r participating in phagocytosis in microglia after brain ischemia, and the precise step(s) of the phagocytic process affected by csfr1 remain to be identified. microglia depletion induced by long-term inhibition of csf1r in vivo [15, 33, 65, 69] increased the numbers of neutrophils in the ischemic brain tissue, further supporting the view that microglial cells contribute to neutrophil removal. neutrophils are attracted to the injured brain after acute stroke [10, 23, 30, 31, 50, 51] . thereby, neutrophils adhere to venules and migrate through the vessel wall to reach perivascular spaces [17] . in addition, neutrophils access perivascular spaces of penetrating cortical vessels from the leptomeninges [55] . accumulation of neutrophils in the leptomeninges might be due to extravasation from pial vessels. in addition, neutrophil migration from the skull bone marrow through direct anatomic connections [29] might explain the presence of neutrophils in the subarachnoid space, although migration of neutrophils from there to perivascular spaces of cortical vessels still needs further investigation. subpial neutrophils are separated from the brain parenchyma by the basement membrane and glia limitans. likewise, the parenchymal basal lamina and surrounding astrocyte end-feet separate perivascular cells from the brain parenchyma. interestingly, we observed ramifications of microglial cells apparently crossing the basal lamina suggesting the possibility that reactive microglia might sample the perivascular space and also the subpial space after brain ischemia. using intravital fig. 6 microglia phagocytose neutrophils in vitro under the control of microglial csf1r expression. microglial cells were obtained from adult mice (a-f) or a human stroke patient deceased 5 days after ischemic stroke (g-i). after 7 days in culture, the cells were exposed to corresponding control mouse or human neutrophils and studied by time-lapse confocal microscopy for 14 h. a, b microglia were obtained from dsred mice (red) and neutrophils were stained with cmfda (green). 3d-reconstructions of image sequences (1) (2) (3) (4) (5) of the time-lapse video (see supplementary video 3 and 4) illustrating the phagocytosis of neutrophils by mouse microglia (a). the time point of each image is indicated (hours:minutes). an original confocal image is shown b for illustrative purposes. c microglia cultures were obtained from csf1r +/+ (wt) and csf1r +/− littermate mice (n = 5 mice per genotype) and the cell cultures were exposed to neutrophils and studied by time-lapse microscopy, where microglial cells were seen by phase contrast and neutrophils were stained with cmfda and detected by green fluorescence. we studied four wells per mouse in each independent experiment (n = 5), recorded 3-4 videos of different fields per well, and analysed 180-210 frames per time-lapse video. quantification of the number of neutrophils phagocytosed by microglia (normalized by the number of microglia in each well) shows that heterozygous csf1r +/− microglia phagocytose less neutrophils than wt microglia. two-way anova by genotype and experiment, with an experiment-matched design, show a genotype effect ***p < 0.0001. d-f cultures of wt and csf1r +/− microglia were exposed to green fluorescent zymosan beads (n = 3 independent experiments). at the end of the experiment cells were fixed and immunostained with anti-p2ry12 antibodies (red) and the number of cells containing fluorescent beads was counted. compared to wt microglia (d), csf1r +/− microglia (e) also shows a reduced capacity to phagocytose zymosan beads (green) (f). two-way anova by genotype and experiment, as above, shows a significant genotype effect **p < 0.01. g sequential snapshot images (1-6) of human microglia obtained during the 12 h time-lapse microscopy study in which 720 frames were studied. the images illustrate neutrophil phagocytosis by human microglia at the indicated times (hours:minutes). h magnification of the indicated part of sequence 6 in g is shown to illustrate the engulfment of a neutrophil by a human microglial cell. i after the time lapse-experiment, human cells were fixed and stained with anti-p2ry12 antibodies (red). nuclei are shown in blue (dapi). the microglial cells contain material (green fluorescence) derived from digested neutrophils. scale bar 20 μm ◂ 1 3 microscopy, we previously found evidence that microglia phagocytosed neutrophils before they extravasated to the brain parenchyma [50, 51] . however, further studies are required to demonstrate whether microglia can really cross the external cortical basement membrane after brain ischemia. although we detected engulfment of complete cells by microglia, some of the images suggest that microglia may take portions of the neutrophils while they are located in the perivascular or subpial spaces, potentially through a process of trogocytosis [71] . the blood vessel glycocalyx and basement membrane composition varies between organs and inflammatory conditions suggesting that leukocytes may have to use diverse strategies to access different inflamed tissues [53] . in the brain, neutrophils cross the endothelial cell layer and the endothelial basal lamina of venules to reach the perivascular spaces after ischemia [17] . then, they accumulate in the perivascular spaces because they do not seem to readily cross the parenchymal basal lamina [17] , at least not at the same pace as they transmigrate through the former layers. however, the precise molecular determinants of this process remain to be identified. the different molecular composition of the two layers of basal lamina surrounding the perivascular spaces, local molecular diversity, and the finding that certain basal lamina components inhibit leukocyte transmigration [63] , might explain why neutrophils have more difficulty to cross the parenchymal than the endothelial basal lamina after brain ischemia. in a model of transient ischemia, there is evidence suggesting that neutrophils are kept in the perivascular spaces without infiltrating the brain parenchyma [17] , whereas other studies suggested that neutrophils reach the brain parenchyma [64] . it is plausible that stroke severity, status of microglia function, and time point of the study are critical determinants of the presence of neutrophils in the brain parenchyma. neutrophils located in the perivascular spaces might damage the basement membrane by releasing proteolytic enzymes and/or undergoing netosis [55] . however, at this stage we cannot exclude the possibility that neutrophils gained access to the brain parenchyma in a passive fashion after loss of vessel integrity in the ischemic core. several lines of evidence support that after brain ischemia neutrophils release proteolytic enzymes, promote matrix metalloproteinase (mmp) activation, and cause bbb breakdown [25, 35, 36, 57, 66, 72] . accordingly, blocking neutrophils or neutrophil-derived mmp-9 is markedly protective in models of systemic inflammation and stroke, e.g. [35, 44] . furthermore, pharmacological inhibition of neutrophil elastase or genetic deficiency of this enzyme reduced bbb disruption and vasogenic edema after transient mcao [64] suggesting that neutrophils contributed to vascular damage following stroke. in this study we showed that, after permanent ischemia, neutrophils gained access to the brain parenchyma of the lesion core when it was already severely damaged and microglia was lost due to persistent ischemia. under these conditions, parenchymal neutrophils might be bystanders of severe tissue damage. therefore, it is likely that preventing the access of neutrophils to the brain parenchyma in this model would not have a major impact on the size of the brain lesion since the damage is already established by the time the cells reach the parenchyma and the core of infarction will not recover. this possibility agrees with the finding that inhibition or deficiency of neutrophil elastase was not protective in models of permanent mcao [64] . in contrast, inhibiting microglial phagocytic activity in this model might bear negative effects by favoring neutrophil accumulation in the ischemic periphery. accordingly, detrimental effects of neutrophils became apparent in our study after microglia depletion causing an abnormal increase in neutrophils and larger ischemic lesions. a limitation of our study is that we did not assess stroke outcome in the long term. future work should investigate how microglia depletion affects the progression of the ischemic brain lesion and the neurological deficits. the results highlight an aspect of microglia phagocytic function that may be beneficial for the ischemic tissue. nonetheless, several mechanisms can contribute to the detrimental effect of microglia depletion and csf1r deficiency. for instance, pioneer studies demonstrated increased ischemic lesions related to reduced production of neurotrophic factors after depleting proliferating microglia [38] , and neuroprotective functions mediated by csf1r [47] . moreover, we previously identified that absence of microglia significantly augmented infarct size in a model of transient ischemia, in part mediated by dysregulation of fig. 7 the presence of neutrophils in the brain parenchyma is associated with dystrophy and loss of microglia. a flow cytometry analysis of cd11b + ly6g + neutrophils in the core and periphery of infarction and in mirror regions of the contralateral hemisphere 1 and 4 days after mcao (n = 5 mice per time point) shows that ischemia increases the number of neutrophils in the core of infarction more than the periphery (two-way anova, bonferroni test, ***p < 0.001). b-d brain confocal images 1 day after mcao (n = 8) show ly6g + neutrophils (green) in the perivascular space of a venule (b) and the parenchymal side of capillaries (c, d). the basal lamina is stained with pan-laminin (red) and nuclei are stained with to-pro3 (blue). e neutrophil (ly6g + , green) accumulation is higher at the border (dotted line) of the infarcted core than the periphery, whereas microglia (red cells, cx3cr1 cre/ert2 :rosa26-tdt mice) accumulate in the infarct periphery and are scarce in the core. the vascular basal lamina is shown in blue (pan-laminin). f-h images obtained from catchup mice crossed with cx3cr1 gfp/gfp mice at day 1 (f, g) and day 4 (h) postischemia. extravasated neutrophils (red) away from the vascular basal lamina (pan-laminin, blue) are seen in the core of the lesion where microglia (green) is absent or dystrophic, whereas neutrophils are surrounded by reactive microglial cells in the periphery (f). g examples (1-5) of microglial cells sending prolongations towards neutrophils 1 day after pmcao. neutrophils located in perivascular spaces extend protuberances crossing the basal lamina (1). extravasated neutrophils are seen near vessels with discontinuous basal lamina (2). they are surrounded by reactive microglia at the border of the lesion (2-4), but dystrophic microglia seem to be unable to fully reach the extravasated neutrophils (5) . h at day 4, microglial cells sending prolongations towards neutrophils are seen at the border of the lesion (1). extravasated neutrophils are seen in zones where microglia is dystrophic (1-2) or absent (3) (4) (5) . the vascular basal lamina is hardly detected at places where the neutrophils are located (3) . arrowheads indicate neutrophils, whereas arrows indicate microglia. the cell nuclei (dapi, white) are shown in g (4) (5) and h (1-3). scale bar 10 μm ◂ neuronal activity [65] . the latter model caused moderate leukocyte infiltration and we failed to observe a significant impact of microglia depletion on bbb injury and leukocyte recruitment, at least at the times examined [65] . in contrast to the findings suggesting beneficial effects of microglia in brain ischemia, several lines of evidence support that the phagocytic activity of microglia could exert negative effects by removing viable neurons through phagoptosis [5, 6, 48, 49] . it is possible that any negative consequences of phagoptosis of neurons might predominate under mild ischemic conditions where the inflammatory response is low, vascular integrity is preserved, and neutrophil attraction to the brain is negligible. collectively, our results support a model where microglia removes neutrophils from the parenchyma and perivascular and subpial spaces after brain ischemia. severe ischemic conditions induce local microglia loss/dystrophy facilitating the presence of neutrophils in perivascular spaces first and in the brain parenchyma later. overall, this study shows that reactive microglial cells phagocytose and remove neutrophils, whereas microglial loss or dysfunction enhances neutrophil accumulation in the ischemic lesion. our results, hence, suggest that microglia function is critical to prevent neutrophil infiltration to the brain parenchyma and to minimize the negative impact of neutrophils in the vascular bed after ischemic stroke. fig. 8 microglia depletion increases the number of neutrophils in the brain parenchyma. a schematic representation of the experimental design. eight-week old mice received a control or plx5622-containing diet for 21 days. then mcao was induced and the animals continued with the corresponding diet one or four more days until the end of the study. b flow cytometry (n = 7 mice per group) showed the microglia (cd45 low cd11b + cells) depleting effect of the plx5622 diet, and an increased number of neutrophils (cd11b + ly6g + ) in the ipsilateral hemisphere of mice with depleted microglia. two-way anova by treatment and brain hemisphere, and bonferroni posthoc test, **p < 0.01. c control or plx5622-containing diet was given to cx3cr1 cre/ert2 :rosa26-tdt mice with the same dosing regimen. microglia is shown in red, neutrophils in green (ly6g + ), the vascular basal lamina is stained with pan-laminin (blue), and nuclei (dapi) are shown in white. the absence of microglia is associated with an increased presence of extravasated neutrophils (arrowheads). d is a magnified image showing extravasated neutrophils after a plx5622containing diet. e, f distribution of neutrophils in parenchymal versus perivascular or intravascular locations, as assessed by cell counting in brain sections immunostained with ly6g, pan-laminin and to-pro3. mice treated with the plx5622 diet show a higher (mann-whitney test) percentage of parenchymal neutrophils 1 day (**p = 0.004) and 4 days (**p = 0.006) post-ischemia (n = 5-7 mice per group). f illustrates the previous staining after plx5622 diet. g brain lesion volume was measured with t2w mri 24 h post-ischemia in both diet groups (n = 9 per group). the brain lesion was larger in mice depleted of microglia (mann-whitney test, *p = 0.031). scale bar 20 μm (c); 10 μm (e, f) ◂ new tools for studying microglia in the mouse and human cns diverse requirements for microglial survival, specification, and function revealed by defined-medium cultures the origin of lipid phagocytes in the central nervous system i. the intrinsic microglia the 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axon regeneration after acute injury image thresholding by minimizing the measure of fuzziness targeting neutrophils in ischemic stroke: translational insights from experimental studies neutrophil infiltration increases matrix metalloproteinase-9 in the ischemic brain after occlusion/reperfusion of the middle cerebral artery in rats bone marrow cell recruitment to the brain in the absence of irradiation or parabiosis bias selective ablation of proliferating microglial cells exacerbates ischemic injury in the brain specific contributions of csf-1 and gm-csf to the dynamics of the mononuclear phagocyte system proliferation of parenchymal microglia is the main source of microgliosis after ischaemic stroke oligodendrocytes and microglia are selectively vulnerable to combined hypoxia and hypoglycemia injury in vitro a robust and high-throughput cre reporting and characterization system for the whole mouse brain antibodies to cd11b, cd68, and lectin label neutrophils rather than microglia in traumatic and ischemic brain lesions systemic inflammatory stimulus potentiates the acute phase and cxc chemokine responses to experimental stroke and exacerbates brain damage via interleukin-1-and neutrophil-dependent mechanisms methods for cell and particle tracking immature monocytes recruited to the ischemic mouse brain differentiate into macrophages with features of alternative activation microglia overexpressing the macrophage colony-stimulating factor receptor are neuroprotective in a microglial-hippocampal organotypic coculture system phagocytosis executes delayed neuronal death after focal brain ischemia inhibition of microglial phagocytosis is sufficient to prevent inflammatory neuronal death beware the intruder: real time observation of infiltrated neutrophils and neutrophil-microglia interaction during stroke in vivo very-late-antigen-4 (vla-4)-mediated brain invasion by neutrophils leads to interactions with microglia, increased ischemic injury and impaired behavior in experimental stroke microglia cells protect neurons by direct engulfment of invading neutrophil granulocytes: a new mechanism of cns immune privilege leukocyte migration into inflamed tissues pharmacological targeting of csf1r inhibits microglial proliferation and prevents the progression of alzheimer's-like pathology neutrophil recruitment to the brain in mouse and human ischemic stroke cerebral neutrophil recruitment, histology, and outcome in acute ischemic stroke: an imaging-based study mmp-9-positive neutrophil infiltration is associated to blood-brain barrier breakdown and basal lamina type iv collagen degradation during hemorrhagic transformation after human ischemic stroke phosphorylation of csf-1r y721 mediates its association with pi3k to regulate macrophage motility and enhancement of tumor cell invasion selective expression of gi/o-coupled atp receptor p2y12 in microglia in rat brain a comprehensive profile of chip-seq-based pu.1/spi1 target genes in microglia the paradox of the neutrophil's role in tissue injury m-csf increases proliferation and phagocytosis while modulating receptor and transcription factor expression in adult human microglia endothelial basement membrane laminin 511 contributes to endothelial junctional tightness and thereby inhibits leukocyte transmigration neutrophil elastase and neurovascular injury following focal stroke and reperfusion microglia protect against brain injury and their selective elimination dysregulates neuronal network activity after stroke implications of mmp9 for blood brain barrier disruption and hemorrhagic transformation following ischemic stroke akt signaling pathway in macrophage activation and m1/ m2 polarization ijblob: an imagej library for connected component analysis and shape analysis homeostasis of microglia in the adult brain: review of novel microglia depletion systems microglia are required for protection against lethal coronavirus encephalitis in mice microglia remodel synapses by presynaptic trogocytosis and spine head filopodia induction blood-brain barrier breakdown in acute and chronic cerebrovascular disease ischemic vulnerability of primary murine microglial cultures fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis neutrophil migration into the infected uroepithelium is regulated by the crosstalk between resident and helper macrophages dominant role of microglial and macrophage innate immune responses in human ischemic infarcts csf-1 receptor structure/function in maccsf1r −/− macrophages: regulation of proliferation, differentiation, and morphology acknowledgements aoda had a predoctoral fellowship from the mineco-fpi program and fmm had a peris award by the health department of generalitat de catalunya. part of this work was performed at the centre de recerca biomèdica cellex, barcelona. the cerca programme of generalitat de catalunya supports the institut d'investigacions biomèdiques august pi i sunyer (idibaps). plx5622 was provided by plexxikon under materials transfer agreement. we acknowledge the cytomics and image platforms of idibaps for access to equipment. we would like to thank elisenda coll (advanced optical microscopy-ccitub) for excellent technical assistance. we thank the neurological tissue bank of the biobank-hospital clinic-idibaps for sample and data procurement, and to patient's relatives for giving consent to sample use for research purposes. key: cord-009388-k3exf8a4 authors: agarwal, yash; beatty, cole; biradar, shivkumar; castronova, isabella; ho, sara; melody, kevin; bility, moses turkle title: moving beyond the mousetrap: current and emerging humanized mouse and rat models for investigating prevention and cure strategies against hiv infection and associated pathologies date: 2020-04-10 journal: retrovirology doi: 10.1186/s12977-020-00515-3 sha: doc_id: 9388 cord_uid: k3exf8a4 the development of safe and effective combination antiretroviral therapies for human immunodeficiency virus (hiv) infection over the past several decades has significantly reduced hiv-associated morbidity and mortality. additionally, antiretroviral drugs have provided an effective means of protection against hiv transmission. despite these advances, significant limitations exist; namely, the inability to eliminate hiv reservoirs, the inability to reverse lymphoid tissues damage, and the lack of an effective vaccine for preventing hiv transmission. evaluation of the safety and efficacy of therapeutics and vaccines for eliminating hiv reservoirs and preventing hiv transmission requires robust in vivo models. since hiv is a human-specific pathogen, that targets hematopoietic lineage cells and lymphoid tissues, in vivo animal models for hiv-host interactions require incorporation of human hematopoietic lineage cells and lymphoid tissues. in this review, we will discuss the construction of mouse models with human lymphoid tissues and/or hematopoietic lineage cells, termed, human immune system (his)-humanized mice. these his-humanized mouse models can support the development of functional human innate and adaptive immune cells, along with primary (thymus) and secondary (spleen) lymphoid tissues. we will discuss applications of his-humanized mouse models in evaluating the safety and efficacy of therapeutics against hiv reservoirs and associated immunopathology, and delineate the human immune response elicited by candidate hiv vaccines. in addition to focusing on how these his-humanized mouse models have already furthered our understanding of hiv and contributed to hiv therapeutics development, we discuss how emerging his-humanized rat models could address the limitations of his-mouse models. despite combination antiretroviral therapy (cart)mediated suppression of human immunodeficiency virus (hiv) replication and promotion of immune reconstitution in patients, hiv-associated morbidity persists and is associated with the latent reservoir, unresolved immune abnormalities, and fibrosis in lymphoid organs [1] . additionally, hiv transmission remains endemic across the globe, and development of a functional cure and/or an effective vaccine will be required to end this epidemic [2] . hiv is a human specific pathogen; thus, animal models for evaluating the safety and efficacy of therapeutics and vaccines directly against hiv requires the incorporation of human lymphoid tissues and/or hematopoietic lineage cells. such mouse models exist, and are termed, human immune system (his)-humanized mice [3, 4] . to construct his-humanized mice, immunodeficient mice are myoablated to eradicate residual mouse bone marrow stem cells, and then engrafted with human peripheral blood mononuclear cells (pbmcs) or human hematopoietic stem cells (hsc) with or without transplantation of lymphoid tissues, such as thymus and/or spleen [5] . over a period of a few weeks to months, transplanted mice develop human immune cells, and reconstitution is confirmed by flow cytometry [5] (fig. 1) . his-humanized mice can then be employed in studies investigating hiv prevention or cure strategies [5] . in this review, we will discuss the myriad of approaches for developing hishumanized mouse models, and their applications in hiv therapeutics and vaccine development studies, along with their limitations. finally, we will discuss the potential of an emerging his-humanized rat model, which has a longer lifespan and greater physiological similarity to humans compared to mice, designed to enable longitudinal studies in evaluating therapeutics against hiv reservoirs and vaccine-induced immunity [6, 7] . human cd4 + t cells are the major target for hiv infection; thus, a mouse model with human cd4 + t cells provides a platform for modeling hiv/aids. various immunodeficient mouse models lacking mature t, b, [8] and nk cells, along with defects in macrophage phagocytic function [9] support robust reconstitution of human cd4+ t cells and other lymphocytes (e.g. cd8 + t cells) following transplantation of human pbmcs or cd4+ t cells. such cells can be transplanted via intravenous (iv) or intraperitoneal (ip) injection into myoablated, immunodeficient juvenile mice (6-8 weeks old) at a dose of 5-10 × 10^6 cell per mouse, to generate peripheral blood lymphocyte (pbl)-humanized mice. human cd4 + t cells are readily detectable in the blood fig. 1 construction of the human immune system-humanized mouse models. (i) immunodeficient mice are myoablated via irradiation or busulfan, followed by the administration of antibiotics and analgesics. general anesthesia is induced prior to surgery. (ii) to generate human lymphoid tissue xenografts along with autologous immune cell reconstitution, human fetal lymphoid tissue(s) and liver are processed into 1 mm 2 pieces, and autologous cd34 + hscs are isolated from the fetal liver via immunomagnetic selection. cd34 + hscs are then transplanted via retro-orbital injection following renal capsule transplantation of the lymphoid tissues. alternatively, to generate human immune cell only, pbmcs, cd4+ t cells, or cd34 + hscs are transplanted via iv or ip injection. (iii) transplanted mice are maintained under specific pathogen-free conditions and the human lymphoid tissue(s), and/or immune cell reconstitution in the peripheral blood and murine lymphoid tissues (humanized-murine tissues) are allowed to develop over a period of 2-10 weeks (or more), resulting in the his-humanized mouse model at 4 weeks post-transplantation [9, 10] , hence providing a humanized mouse model that can be generated in a relatively short period. the pbl-humanized mouse model supports hiv replication and provides a means of evaluating the efficacy of direct-acting therapeutics (e.g. antivirals drugs, antibodies) geared towards preventing hiv transmission [11] and controlling hiv replication [9] . additionally, pbl-humanized mouse models constructed using pbmcs from hiv-infected individuals with undetectable viral load can be employed as an in vivo assay (mouse-quantitative viral outgrowth assay) for evaluating the eradication of the hiv reservoir in said individuals [12, 13] . a major limitation of this model is the rapid development of graft-versus-host (gvhd) disease within 6-7 weeks following transplantation of lymphocytes, thus significantly restricting the experimental window [14] . additionally, the pbl-humanized mouse model does not incorporate human macrophages, which are a major hiv reservoir in various organs, including the brain [9, 15] . however, modification of the pbl-humanized mouse model via transplantation of hiv-infected monocyte-derived macrophages in the brain supports hiv infection and pathogenesis in the brain [15] . in order to generate a de novo human immune system and reconstitute a broader spectrum of human immune cells in his-humanized mice, myoablated, immunodeficient mice are transplanted with human cd34 + hscs via intrahepatic or intracardiac injection in neonatal mice [10, 16] or iv injection in juvenile/adult mice [16, 17] . these hscs can be obtained from a myriad of sources, including fetal liver tissue [17, 18] and neonatal cord blood cells [4, 16, 19] . human immune reconstitution in the hsc-humanized mouse model requires 10-12 weeks to develop [20] . various hematopoietic lineages, including t cells, monocytes/macrophages, b cells and dendritic cells are developed in the blood and other tissues (e.g., spleen, liver, brain) [20] . moreover, the hsc-humanized mouse model generates a naïve human immune system, which negates confounding factors associated with prior pathogen exposure [16, 21] . the hsc-humanized mouse model supports hiv infection, cd4+ t cell depletion, chronic immune activation and limited anti-hiv t and b cell immune responses [4] . a major advantage of the hsc-humanized mouse model over the pbl-humanized mouse model is the delayed and reduced incidence of gvhd, which provides the opportunity for long-term modeling of hiv infection and replication [9] . the hsc-humanized mouse model provides a means of evaluating the efficacy and safety of directacting therapeutics (e.g. antivirals drugs, antibodies) and immune-modulatory agents (e.g. pdc modulators [22] ) geared towards preventing hiv transmission, controlling hiv replication, and ameliorating cd4+ t cell depletion and chronic immune activation [4] . the hschumanized mouse model supports hiv transmission via the iv (along with ip) route [18] ; however, conflicting reports exist for the mucosal route of transmission [23, 24] . additionally, the reconstituted human t cells are educated in the mouse thymic epithelium, thus limiting antigen-specific responses [25] . this limitation of t cell education in the murine thymic epithelium has been partially overcome by the construction human leukocyte antigen (hla) class i transgenic-immunodeficient mice to support robust t cell development of hla-matched hsc transplants [26] . moreover, lymph nodes and spleen are poorly reconstituted, including a limited development of human b and myeloid cells in the white and red pulps of the spleen [27] . several modifications have been made to the hsc model to address these limitations. li et al. constructed an immunodeficient mouse model that incorporated a lymphoid tissue-stromal cytokine transgene (i.e. thymic-stromal-cell-derived lymphopoietin) and demonstrated improved lymph node development in hsc-humanized mice [27] . additionally, studies have demonstrated enhanced human b and myeloid cell development in murine secondary lymphoid tissues via transgenic expression of critical cytokines (i.e. il6; il3, gm-csf and scf) for b and myeloid cell maturation [28] [29] [30] . although incorporation of requisite human transgenes in his-humanized mice has been successful in demonstrating improved development of immune cells, often the resultant lineage is skewed, as the transgene expression is not synchronized for physiological expression and supporting stromal cells and other essential cytokines are absent [28] [29] [30] . another strategy for improving human immune cell development in his-humanized mouse models is to implant human lymphoid tissues containing the requisite microenvironment for supporting robust immune cell development. to facilitate human t cell education and associated function, human thymic tissues are incorporated in his-humanized mice, and termed, bone marrow-liver-thymus (blt)-humanized mice [31, 32] (fig. 2) . blt-humanized mice have served as a key animal model for hiv research for over a decade and are a cost-effective alternative to the surrogate, simian immunodeficiency virus (siv)-non-human primate (nhp) models. the blt-humanized mouse model is generated by surgically transplanting myoablated, immunodeficient mice with fetal human liver and thymus tissues, followed by iv injection of autologous cd34 + hscs [31, 33, 34] . transplanted mice require 10-12 weeks for systemic reconstitution of human cells post-transplantation [31, 33, 34] . the most widely utilized strain for constructing blt mice is the nod-prkdc scid il2rg tm1wjl (nsg) [21, 32] , which is readily available from jackson laboratory. blt-humanized mice can also be constructed using comparable immunodeficient mouse strains, such as, c57bl/6 rag2−/−γc−/−cd47−/− (tko) [21, 35] . the key benefit of the blt-humanized mouse model over pbl-and hsc-humanized mouse models is the presence of human thymic microenvironment, which facilitates t cell education in an autologous human tissue that contains the requisite stromal cells (as well as cytokines and factors, presumably at physiological levels) [21] . blt-humanized mice have systemic tissue reconstitution with human immune cells, including in mucosal tissues, which enables mucosal transmission [36] [37] [38] [39] [40] [41] [42] [43] and recapitulates the main route of hiv transmission in humans [36] [37] [38] [39] [40] [41] [42] [43] [44] . other hallmarks of hiv infection and replication in blt-humanized mice include robust t cell depletion [36, 42] , central nervous system infiltration [45, 46] , immune response [35, [47] [48] [49] [50] , and latency [51] [52] [53] . the blt-humanized mouse model is a robust platform for evaluating hiv prevention and cure strategies, including antiretroviral therapy, pre-exposure prophylaxis (prep), latency reversing agents (lra), vaccination, proviral excision, and t cell engineering (table 1) . despite significant advances gained from the blthumanized mouse model, the system does have some disadvantages. construction of blt-humanized mice requires advanced surgical expertise and extensive experience; therefore, these animals are constructed predominantly by specialized core facilities. additionally, blt-humanized mice are prone to gvhd, which limits the experimental window these animals can be utilized to approximately 6 months post-engraftment [54, 55] . however, blt-humanized mice constructed with a c57bl/6 immunodeficient background are resistant to gvhd [35, 53] . another disadvantage involves the use of human fetal tissues in constructing the model; these tissues are not readily available. furthermore, a typical human fetal thymus and autologous fetal liver-derived hscs can only support the construction of 15-25 blt-humanized mice. the limited availability of said human fetal tissues creates logistical and operational constraints. recently, a novel blt-like humanized mouse model has been developed using non-autologous human cord blood-derived hscs and human neonatal/pediatric thymus, which enable investigators to construct > 1000 blt-like humanized mice using cryopreserved thymus tissues and readily available cord blood-derived hscs [96] . recent studies demonstrate that these blt-like humanized mice develop human immune cells, support hiv infection and replication, and exhibit anti-hiv immune response (unpublished data from elie haddad, chloé colas, et al., at the cancure 5th annual general meeting-2019, poster session, in montreal, canada). despite systemic immune cell reconstitution and hiv-specific immune responses, both neonatal/pediatric tissue-and fetal tissue-derived blt-humanized mouse models blt mice do not develop a complete human immune system. the current widely used immunodeficient mouse models possess an il-2 receptor γ chain deletion [97] [98] [99] [100] . as a result, mouse lymphoid organs do not fully develop in such models, [21] and the loss of lymphoid tissue microenvironment impairs the ability of blt-humanized mice to develop a robust humoral immune response, as immunoglobulins are skewed towards igm or weak igg response [35, 49, 99, [101] [102] [103] . constructing blt-humanized mice using immunodeficient mouse models with requisite human transgenic factors/cytokines, may optimize human b cell development and overcome the limitations of humoral immune response in the model [28, 104, 105 ]. an alternative strategy, which is consistent with the blt-model to construct his-humanized mice and rats, immunodeficient mice and rats are myoablated, followed by engraftment of human lymphoid tissues (thymus with or without human spleen) under the kidney capsule, along with injection of autologous human cd34+ hematopoietic stem cells. in representative images, we show the engrafted human lymphoid tissues (human thymus xenograft-thymus, white tissue; human spleen xenograft-spleen, dark brown tissue and the reconstituted rodent-spleen (humanized spleen-hspleen). note: mouse and rat organs are not at the same scale pre-exposure prophylaxis (prep) c5a peptide [66] cc-griffithsin [67] cd4 asics [68] cd4-expressing lactobacillus acidophilus [69] cd4mc p-iii-48 [70] dtg-ultra la [71] efda [72] g2-s16 pcd [73] iga [74] mvc [75] ral-la [76] rpv-la [39, 77] siccr5 lfa-1 i-tsnp [78] tnv gel [79] [80] [81] vrc01 bnab [82] ftc, taf [83] ftc, tdf [36, 43, 84, 85] taf, evg [86] b12, vrc01, vrc07 g54w bnabs [87] latency-reversing agents (lras) azd5582 [88] panobinostat [89] suw133 (bryostatin analog) [90] vaccines plga-gag microparticles [49, 91] recombinant gp140∆683 [49, 91] proviral excision sacas9/sgrna [92] t cell engineering ccr5 shrna [93, 94] cd4 car [95] 3tc, lamivudine; asics, aptamer-sirna chimeras; azt, zidovudine; bnab, broadly neutralizing antibody; ccr5, c-c chemokine receptor type 5; car, chimeric antigen receptor; cd4, cluster of differentiation 4; cd4mc, cd4 mimetic compound; cc, caulobacter crescentus recombinant expressing; dca, didehydro-cortistatin a; ddi, didanosine; dtg, dolutegravir; dtg-ultra la, long acting dolutegravir; efda, 4′-ethynyl-2-fluoro-2′-deoxyadenosine; evg, elvitegravir; ftc, emtricitabine; idv, indinavir; ifnα14, interferon α suptype 14; iga, immunoglobulin a; lfa-1 i-tsnp, lymphocyte function-associated antigen-1 integrin-targeted and stabilized nanoparticle; mab, monoclonal antibody; mvc, maraviroc; pcd, polyanionic carbosilane dendrimers; pd-1, programmed cell death protein 1; plga, poly(lactic-co-glycolic) acid; ral, raltegravir; ral-la, long-acting raltegravir; rpv, rilpivirine; rpv-la, long acting rilpivirine; sacas9/sgrna, staphylococcus aureus crispr-associated protein 9/single-guide rna; shrna, short hairpin rna; siccr5, small interfering rna ccr5; taf, tenofovir alafenamide; tdf, tenofovir disoproxil fumarate; tnv; tenofovir strategy, is to incorporate the requisite human secondary lymphoid tissue (i.e. spleen) microenvironment for robust human immune cell (e.g. b cells, macrophages) development and response [5] . to address the limitations of the blt-humanized mouse model, namely, poor development of secondary lymphoid tissue and modest macrophage reconstitution, we incorporated human spleen into the blt-humanized mouse model, and termed these animals, bone marrow-liverthymus-spleen (blts)-humanized mice [5] (fig. 2) . blts-humanized mice exhibit significant improvement over the blt-humanized mice by addressing several limitations [5] . successful spleen growth dramatically lowers the incidence of gvhd in blts-humanized mice, thus allowing for experimental studies that extend up to 9 months post-transplantation [5] . we speculate that the decreased gvhd in blts-humanized mice results from appropriate modulation of t cell activation by the human antigen-presenting cells in the human spleen xenograft. the human spleen in the blts-humanized mouse model recapitulates human adult spleen architecture and facilitates better reconstitution of immune cells, including human red pulp macrophages, which are poorly reconstituted in the blt-humanized mouse model [5] . it is well established that macrophages can serve as a reservoir for hiv [106] [107] [108] ; thus, the blts-humanized mouse model provides a system for investigating human splenic macrophage-hiv interactions [5] . additionally, the spleen is a major lymphoid tissue reservoir, with b cell follicles in the white-pulp serving as an immune privilege site for anti-hiv t-cells [109] . the human spleen in bltshumanized mice provides a model for investigating anti-hiv immune response within the white-pulp and the role of b cell follicle in mediating hiv persistence. the blts-humanized mouse model supports cart-mediated hiv load suppression, and replication competent hiv reservoirs can be detected in human spleen tissues [5] . lymphoid tissue fibrosis is an immuno-pathogenic feature associated with hiv infection and plays a major role in mediating chronic inflammation and abrogating the development of a robust immune response [110] . a major advantage of the blts-humanized mouse model is that hiv infection results in lymphoid tissue fibrosis; this disease manifestation is absent in hiv-infected blt-humanized mice [5] . although the blts-humanized mouse model exhibits more robust immune reconstitution compared to its blt counterpart, the two models share some limitations. the transplantation of the human tissues under the renal capsule requires an individual with advanced surgical skills. the blts-humanized mouse model uses human fetal tissues, which introduces logistical and operational constraints. the use of frozen fetal tissues and hscs can alleviate some of those constraints (unpublished data). demonstrating robust anti-hiv immunity in his-humanized mouse models has been a long-term goal in the field because said system would allow robust evaluation of hiv vaccine candidates against circulating viral strains. the incorporation of human primary and secondary lymphoid tissues in hishumanized mice brings us closer to this goal. at present, we are actively investigating the anti-hiv human immunity in the blts-humanized mouse model to determine if this system provides a means for evaluating hiv vaccines. prior to the development of genetic engineering technologies for creating transgenic and knockout mice, rats were the predominant specie of rodents employed in biomedical research [6] . advantages of using rats include their longer lifespans (~ 3.5 years) and larger size (~ 350 grams), which facilitates longer experimental window and larger sampling volumes compared to the short lifespan (< 1 year) and small size (< 25 g) of mice [6] . rats provide a more ideal platform for in vivo imaging of diseases, as the larger size of rats provides better resolution [111] . additionally, rat models exhibit advanced cognitive skills and critical physiological parameters (e.g. heart rate, drug metabolism) that more closely mimic humans [112] [113] [114] [115] . recent advances in genetic engineering, such as the crispr/cas 9 technology has enabled the development of several immunodeficient rat models for transplanting and regenerating human tissues and cells [7, [116] [117] [118] . similar to currently used immunodeficient mouse models, immunodeficient rat models carry mutations in rag1/2 and il2rγ genes, with or without sirp1α transgene [7, [116] [117] [118] . a recent study demonstrated that these immunodeficient rats can be reconstituted with a myriad of human immune cells following transplantation with human-fetal liver-derived hscs and autologous thymus tissues [7] (fig. 2) . these his-humanized rat models could provide a means for robust longitudinal studies on the safety and efficacy of therapeutic agents targeting the hiv reservoir. additionally, his-humanized rats could provide a means for modeling hiv-associated end organ diseases, such as cardiovascular, neurocognitive and lung diseases. despite successful prevention of hiv transmission with antiviral drugs, it is likely that an effective vaccine provides the only means of ending the hiv epidemic [2] . over the past decades, several promising vaccine candidates have failed in large-scale safety and efficacy clinical trials [2] . the failure of those clinical trials suggests that improved "gate keeper" animal modeling systems are needed for better prediction of vaccine candidate outcomes in human clinical trials [119] . currently, the surrogate siv-nhp model is the sole "gate keeper" animal model for determining potential of vaccine candidates [119] . candidate hiv vaccines selected using this gatekeeper system have been unsuccessful in human clinical trials [119] ; suggesting, major improvements in animal modeling are needed. although significant advancements are still needed in improving his-humanized models, several recent advances, such as improved human-secondary lymphoid tissue development, along with the previously developed, robust primary lymphoid tissue development has made it possible to evaluate human immune responses to vaccines [5, 27] . ideally, these "improved" his-humanized mouse models will complement nhp models in addressing critical gaps, such as vaccine-induced immune responses against circulating hiv strains, vaccine safety in the context of hiv transmission, and human-correlates of immunity. although cart has significantly reduced the morbidity and mortality associated with chronic hiv infection, the hiv reservoir persists in people living with hiv (plhiv) and is associated with chronic inflammation, lymphoid tissue damage, and a myriad of end-organ diseases [1] . therefore, eradicating the hiv reservoir and associated chronic inflammation and end organ diseases remains a major challenge. the mechanisms of hiv persistence in plhiv, despite robust cart-mediated suppression of the virus, are thought to be multifactorial. these factors include persistence in transcriptionally quiescent resting memory cd4+ t cells in the peripheral blood and lymphoid tissues, infection of long-lived resident tissue macrophages in lymphoid tissues and immune privilege organs (e.g. brain, testes, b cell follicle, etc.), and dysregulation in anti-hiv immunity. by virtue of the multitude of factors that play a role in hiv persistence, in vivo models that recapitulate human host-hiv interactions are necessary for determining the safety and efficacy of therapeutic agents for eradicating hiv reservoirs. improved his-humanized mouse models with systemic reconstitution of human immune cells and robust lymphoid tissues development provide a means of evaluating both directacting and immune-modulatory hiv-cure therapeutics. further advances in improving the human-immune system in his-humanized mouse and rat models will provide better in vivo systems for evaluating the safety and efficacy of therapeutics and vaccines for hiv prevention and cure. not applicable. moses turkle bility is the senior author authors' contributions mtb conceived this review and contributed to the preparation of the manuscript. ya, cb, sb, ic, sh and km contributed equally to the preparation of the manuscript. all authors read and approved the final manuscript. this work was supported by the following nih grants (r21od024789, (national institute of allergy and infectious diseases) r21ai135412). not applicable. the human fetal liver, spleen and thymus (gestational age of 18-20 weeks) used in the transplantations were obtained from medically or elective indicated termination of pregnancy through magee-women' ). the use of human hematopoietic stem cells was 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rat model for human stem cell transplantation research hu-pbl-scid mice: a model for human immune function, aids, and lymphomagenesis a simple mouse model for the study of human immunodeficiency virus chronic, acute, and reactivated hiv infection in humanized immunodeficient mouse models hu-pbl-scid mice can be protected from hiv-1 infection by passive transfer of monoclonal-antibody to the principal neutralizing determinant of envelope gp120 the mouse viral outgrowth assay: avatars for the detection of hiv-1 reservoirs high activation and skewed t cell differentiation are associated with low il-17a levels in a hu-pbl-nsg-sgm3 mouse model of hiv infection xenogeneic graft-versus-host-disease in nod-scid il-2rgammanull mice display a t-effector memory phenotype generation of cytotoxic t cells against virus-infected human brain macrophages in a murine model of hiv-1 encephalitis parameters for establishing humanized mouse models to study human immunity: analysis of human hematopoietic stem cell engraftment in three immunodeficient strains of mice bearing the il2rgamma(null) mutation hiv-1 infection and pathogenesis in a novel humanized mouse model humanized mice engrafted with human hsc only or hsc and thymus support comparable hiv-1 replication, immunopathology, and responses to art and immune therapy comparison of human fetal liver, umbilical cord blood, and adult blood hematopoietic stem cell engraftment in nod-scid/gammac-/-, balb/c-rag1-/-gammac-/-, and cb-17-scid/bg immunodeficient mice generation of immunodeficient mice bearing human immune systems by the engraftment of hematopoietic stem cells humanized mice for immune system investigation: progress, promise and challenges flt3l-mediated expansion of plasmacytoid dendritic cells suppresses hiv infection in humanized mice rag2(-/-)gamma(-/-)(c) mice transplanted with cd34(+) cells from human cord blood show low levels of intestinal engraftment and are resistant to rectal transmission of human immunodeficiency virus mucosal transmission of r5 and x4 tropic hiv-1 via vaginal and rectal routes in humanized rag2-/-gammac -/-(rag-hu) mice the analysis of the functions of human b and t cells in humanized nod/shi-scid/gammac(null) (nog) mice (hu-hsc nog mice) generation of functional human t-cell subsets with hla-restricted immune responses in hla class i expressing nod/scid/il2r gamma(null) humanized mice a human immune system mouse model with robust lymph node development a novel humanized mouse model with significant improvement of class-switched, antigen-specific antibody production development and function of human innate immune cells in a humanized mouse model development of human cd4 + foxp3 + regulatory t cells in human stem cell factor-, granulocyte-macrophage colony-stimulating factor-, and interleukin-3-expressing nod-scid il2rgamma(null) humanized mice reconstitution of a functional human immune system in immunodeficient mice through combined human fetal thymus/liver and cd34(+) cell transplantation antiretroviral pre-exposure prophylaxis prevents vaginal transmission of hiv-1 in humanized blt mice humanized mice mount specific adaptive and innate immune responses to ebv and tsst-1 immunodeficient mouse model for human hematopoietic stem cell engraftment and immune system development blt-humanized c57bl/6 rag2-/-gammac-/-cd47-/-mice are resistant to gvhd and develop b-and t-cell immunity to hiv infection antiretroviral pre-exposure prophylaxis prevents vaginal transmission of hiv-1 in humanized blt mice art influences hiv persistence in the female reproductive tract and cervicovaginal secretions animal models for hiv/aids research long-acting rilpivirine (rpv) preexposure prophylaxis does not inhibit vaginal transmission of rpvresistant hiv-1 or select for high-frequency drug resistance in humanized mice immune reconstitution of the female reproductive tract of humanized blt mice and their susceptibility to human immunodeficiency virus infection superior human leukocyte reconstitution and susceptibility to vaginal hiv transmission in humanized nod-scid il-2rgamma(-/-) (nsg) blt mice intrarectal transmission, systemic infection, and cd4 + t cell depletion in humanized mice infected with hiv-1 human breast milk and antiretrovirals dramatically reduce oral hiv-1 transmission in blt humanized mice setting the stage: host invasion by hiv reduced antiretroviral drug efficacy and concentration in hiv-infected microglia contributes to viral persistence in brain t cells establish and maintain cns viral infection in hiv-infected humanized mice induction of robust cellular and humoral virus-specific adaptive immune responses in human immunodeficiency virus-infected humanized blt mice alpha interferon and hiv infection cause activation of human t cells in nsg-blt mice humoral immune responses in humanized blt mice immunized with west nile virus and hiv-1 envelope proteins are largely mediated via human cd5 + b cells rapid evolution of hiv-1 to functional cd8 + t cell responses in humanized blt mice generation of hiv latency in humanized blt mice hiv latency in the humanized blt mouse an advanced blt-humanized mouse model for extended hiv-1 cure studies graft versus host disease in the bone marrow, liver and thymus humanized mouse model human immune system development and survival of nonobese diabetic (nod)-scid il2rgamma(null) (nsg) mice engrafted with human thymus and autologous haematopoietic stem cells in vivo suppression of hiv rebound by didehydro-cortistatin a, a "block-and-lock" strategy for hiv-1 treatment efficient inhibition of hiv replication in the gastrointestinal and female reproductive tracts of humanized blt mice by efda pd-1 blockade in chronically hiv-1-infected humanized mice suppresses viral loads efficacy of broadly neutralizing monoclonal antibody pg16 in hiv-infected humanized mice therapeutic efficacy of vectored pgt121 gene delivery in hiv-1-infected humanized mice early initiation of antiretroviral therapy can functionally control productive hiv-1 infection in humanized-blt mice long-acting parenteral combination antiretroviral loaded nano-drug delivery system to treat chronic hiv-1 infection: a humanized mouse model study elite suppressor and chronic progressor hiv-1 isolates replicate vigorously and cause cd4 + t cell depletion in humanized blt mice targeted cytotoxic therapy kills persisting hiv infected cells during art concurrent administration of ifnα14 and cart in tko-blt mice enhances suppression of hiv-1 viremia but does not eliminate the latent reservoir protection efficacy of c5a against vaginal and rectal hiv challenges in humanized mice a caulobacter crescentus microbicide protects from vaginal infection with hiv-1jr-csf in humanized bone marrow-liver-thymus mice inhibition of hiv transmission in human cervicovaginal explants and humanized mice using cd4 aptamer-sirna chimeras blocking hiv-1 infection by chromosomal integrative expression of human cd4 on the surface of lactobacillus acidophilus atcc 4356 a small-molecule cd4-mimetic compound protects bone marrow-liver-thymus humanized mice from hiv-1 infection ultra-longacting removable drug delivery system for hiv treatment and prevention hiv pre-exposure prophylaxis for women and infants prevents vaginal and oral hiv transmission in a preclinical model of hiv infection prevention vaginally of hiv-1 transmission in humanized blt mice and mode of antiviral action of polyanionic carbosilane dendrimer inhibitory effect of hiv-specific neutralizing iga on mucosal transmission of hiv in humanized mice role of semen on vaginal hiv-1 transmission and maraviroc protection a long-acting formulation of the integrase inhibitor raltegravir protects humanized blt mice from repeated high-dose vaginal hiv challenges nanoformulations of rilpivirine for topical pericoital and systemic coitus-independent administration efficiently prevent hiv transmission rnai-mediated ccr5 silencing by lfa-1-targeted nanoparticles prevents hiv infection in blt mice rectal transmission of transmitted/founder hiv-1 is efficiently prevented by topical 1% tenofovir in blt humanized mice one percent tenofovir applied topically to humanized blt mice and used according to the caprisa 004 experimental design demonstrates partial protection from vaginal hiv infection, validating the blt model for evaluation of new microbicide candidates topical tenofovir disoproxil fumarate nanoparticles prevent hiv-1 vaginal transmission in a humanized mouse model vrc01 antibody protects against vaginal and rectal transmission of human immunodeficiency virus 1 in hu-blt mice nanoencapsulation introduces long-acting phenomenon to tenofovir alafenamide and emtricitabine drug combination: a comparative preexposure prophylaxis efficacy study against hiv-1 vaginal transmission prevention of vaginal and rectal hiv transmission by antiretroviral combinations in humanized mice systemic administration of antiretrovirals prior to exposure prevents rectal and intravenous hiv-1 transmission in humanized blt mice tenofovir alafenamide and elvitegravir loaded nanoparticles for long-acting prevention of hiv-1 vaginal transmission vectored immunoprophylaxis protects humanized mice from mucosal hiv transmission systemic hiv and siv latency reversal via non-canonical nf-kappab signalling in vivo in vivo analysis of the effect of panobinostat on cell-associated hiv rna and dna levels and latent hiv infection in vivo activation of latent hiv with a synthetic bryostatin analog effects both latent cell "kick" and "kill" in strategy for virus eradication immunization of blt humanized mice redirects t cell responses to gag and reduces acute hiv-1 viremia in vivo excision of hiv-1 provirus by sacas9 and multiplex single-guide rnas in animal models a highly efficient short hairpin rna potently down-regulates ccr5 expression in systemic lymphoid organs in the hu-blt mouse model engineering hiv-1-resistant t-cells from short-hairpin rna-expressing hematopoietic stem/progenitor cells in humanized blt mice stem-cell based engineered immunity against hiv infection in the humanized mouse model a humanized mouse model generated using surplus neonatal tissue cryptopatches are essential for the development of human galt generation of improved humanized mouse models for human infectious diseases human immune system mice: current potential and limitations for translational research on human antibody responses defective lymphoid development in mice lacking expression of the common cytokine receptor γ chain th1 and th17 immunocompetence in humanized nod/scid/il2rγnull mice the analysis of the functions of human b and t cells in humanized nod/shi-scid/ cnull (nog) mice (hu-hsc nog mice) hypogammaglobulinemia in blt humanized mice-an animal model of primary antibody deficiency improved b cell development in humanized nod-scid il2rgamma(null) mice transgenically expressing human stem cell factor, granulocyte-macrophage colony-stimulating factor and interleukin-3 enhanced antibody responses in a novel nog transgenic mouse with restored lymph node organogenesis the hiv reservoir in monocytes and macrophages hiv-1 reservoirs in urethral macrophages of patients under suppressive antiretroviral therapy macrophages sustain hiv replication in vivo independently of t cells the b-cell follicle in hiv infection: barrier to a cure lymphoid tissue fibrosis is associated with impaired vaccine responses technical and conceptual considerations for performing and interpreting functional mri studies in awake rats rodent models in neuroscience research: is it a rat race? rat models of cardiovascular diseases autoimmunity to myelin oligodendrocyte glycoprotein in rats mimics the spectrum of multiple sclerosis pathology 2011:497841. research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over 100m website views per year • at bmc a novel immunodeficient rat model supports human lung cancer xenografts severe-combined immunodeficient rats can be used to generate a model of perinatal hypoxic-ischemic brain injury to facilitate studies of engrafted human neural stem cells sprague dawley rag2-null rats created from engineered spermatogonial stem cells are immunodeficient and permissive to human xenografts monkeying around with hiv vaccines: using rhesus macaques to define 'gatekeepers' for clinical trials publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-022324-tcltmhi7 authors: barthold, stephen w. title: mouse hepatitis virus biology and epizootiology date: 2012-12-02 journal: viral and mycoplasmal of laboratory rodents doi: 10.1016/b978-0-12-095785-9.50032-9 sha: doc_id: 22324 cord_uid: tcltmhi7 nan mouse hepatitis virus (mhv) is a highly contagious and ubiquitous coronavirus of laboratory mice. it is present among mice in many commercial breeding colonies and most biomédical research institutions and has been documented in laboratory mice throughout the world. recognition of the ubiquity of mhv is often not realized because of unfamiliarity with its biology, reliance on insensitive diagnostic methods and the subtle effects of the virus. its subtle effects should not be underestimated, however, since mhv has been associated with a panoply of effects on mice and research data derived from them. considering the large financial investment in biomédical research and that mice represent the majority of animals used in such research, the potential scientific and economic impact of this single virus is enormous. since the original isolation of mhv in 1949 (1), a body of literature representing nearly 350 citations has been developed on the subject of mhv. the voluminous literature on mhv would not have been generated if mhv were simply a pathogen of mice. interest in mhv has evolved from a number of different perspectives, including emphasis on mhv as a model of viral hepatitis, viral encephalitis and demyelinating disease, genetic mechanisms of host resistance to viral infection, and the molecular biology of coronaviruses, using mhv as a prototype. these have been the subjects of several reviews (2-11). on the one hand, the literature clearly illustrates the complexity of mhv pathogenesis. virus strain, passage history, dose, route of inoculation, host genotype, age, immune function and co-infection with other agents all interact to determine the ultimate expression of mhv disease. on the other hand, this complexity interferes with a clear view of the biology of mhv as a natural pathogen of mice. many of these studies have emphasized different aspects of mhv-host interactions, using artificial routes of inoculation and analysis of only one or a few target organs or effects. as a result, still relatively little is known about the natural pathobiology of mhv in mice. without such information, the true significance of this agent cannot be assessed and its effect under diverse experimental conditions cannot be predicted. this review will discuss recent updates in mhv pathobiology and incorporate past literature in that discussion wherever possible. coronaviruses, including mhv, are enveloped viruses containing single-stranded, non-segmented, infectious rna with positive (messenger) polarity. the molecular biology of mhv will be discussed in dr. holmes' chapter and has been reviewed elsewhere (3,10). physical resistance properties have also been reviewed (4). coronaviruses infect many species of birds and mammals, causing respiratory or enteric infections in their hosts (table 1 ). the relationship among viruses of this family is complex. they do not share a common group antigen, yet many are antigenically and genetically related (8,10,12). although "mhv" is a singular term, it refers to many named and unnamed strains of mouse coronavirus (table 2) . certain prototype strains have served as standards for study of the mhv group, including mhv-l,-2,-3,-a59,-jhm and -s. these strains have been used in most experimental studies and as antigenic or genetic standards for mhv serodiagnosis and comparison to new mhv all mhv strains or isolates share cross-reacting antigens, but each strain also possesses strain-specific antigenicity, with considerable overlap (6,12-15)· similarly, while there is rna and polypeptide homology between mhv strains, there is also strain-related heterogeneity (16,17). this suggests ongoing "drift" or mutation as a factor in the biology of mhv. relatedness of one mhv strain to another is not a useful means of predicting biological behavior. for example, mhv-s and mhv-jhm are relatively unrelated genetically, yet induce similar patterns of disease. related strains such as mhv-s and mhv-s/cdc produce different patterns of disease (14, 16-20). furthermore, the behavior of a defined or named mhv strain depends on its passage history and thus varies from laboratory to laboratory. mhv also cross-reacts antigenically with coronaviruses of the rat and some strains of coronavirus of other species, including man (12,21-23). the biological significance of these relationships has not been thoroughly explored. mhv produces disease in mice by lytic infection of cells, although viral replication and exocytosis can take place in the absence of cytolysis. in addition to cytolysis, cell fusion is a hallmark of mhv, both in vivo and in vitro, although it is not necessary for viral replication (3,7 in contrast to respiratory mhv strains, very little is known about host resistance factors to enteric mhv. most of the mhv strains listed in table 2 have not been characterized as to their primary pattern of infection, but it is clear that both respiratory and enteric strains exist in contempory mouse populations (19). selective organotropisra is often stressed as an mhv strain-related phenomenon, such as neurotropism of mhv-jhm (37), hepatotropism of mhv-a59 (38), lymphocytotropism and bone marrow tropism of mhv-3 (39,40). such differences are only relative, with considerable overlap between mhv strains (19). many of these differences are simply a reflection of the emphasis an author places on a particular target organ. nevertheless, recent work is suggesting that selective cell tropism can take place with specific mhv strains and that host cell type can influence the outcome of mhv infection. in primary central nervous system cell cultures, mhv strains exhibit different cell tropism, cytopathic effects and viral assembly, which may account for different patterns of disease (10,41-48). understanding the pathogenesis of mhv is best approached by catagorizing infections into the respiratory and enteric patterns, which are determined by virus strain. relative differences in órgano-and cell tropism exist between virus strains in each of these catagories. furthermore, host factors play a significant role in severity of infection within these basic themes. the following discussion will detail what is known about the pathogenesis of respiratory and enteric patterns of mhv infection with supporting references for each pattern. these mhv strains rely on upper respiratory mucosa as a site of primary replication and excretion. in weanling or adult outbred mice infected with low-virulence mhv (mhv-s), infection is largely restricted to upper respiratory mucosa (18,20 since brain is often infected with several strains of mhv in susceptible hosts, neurotropism is not a particularly unique feature of any single mhv strain. however, some mhv strains (s and jhm) seem to possess the relatively unique ability to extend directly from the nasal lining into the olfactory tracts of the brain, even in older, resistant mice (18-20,37,53). within 48 hours after intranasal inoculation, mhv-jhm causes meningitis and necrosis of mitral cells in the olfactory bulbs, with extension into the olfactory tracts, meninges, piriform cortex, septum pelucidum, anterior commissure, and hippocampus within 120 hours. during this phase, both neurons and glia are infected. at 144 hours, virus is present in the pons, medulla and spinal cord and by 168 hours, severe demyelination is present. at this time, titers are diminishing (53). a similar course of events probably occurs with mhv-s (18,20). the demyelinating component has been shown to be due to selective destruction of oligodendroglia (54-57) and reportedly does not seem to occur with other encephalitogenic strains of mhv, such as mhv-3 (41,47,58). virus strain tropism for different nervous system cell types in vitro correlates with disease phenotype (41-48). the pattern of nervous system disease that occurs is also dependent on host age, genotype as well as dose and route of virus inoculation (45,57-59). duration of mhv infection is a subject of considerable contention. the behavior of the virus under natural conditions has created the impression that infections are chronic and latent, since many experimental manipulations such as immunosuppression, tumor transplantation or co-infection with other agents will precipitate acute disease (3). it appears, however, that this impression is created by the generally subclinical (but not latent) nature of mhv infection and that experimental manipulations that exacerbate disease are coincidentally applied during active infection. an overwhelming body of literature with a number of mhv strains clearly supports the concept of short-term infection, from which the host fully recovers within 2-3 weeks of infection (18,19,40,48,57,60-66). aggressive immunosuppression after the acute phase of mhv infection will not reactivate the virus, even when residual mhv antigen and lesions remain in the brain (66,67). a smaller number of reports exist, however, that uphold the hypothesis of persistence. mice of the semisusceptible c3h genotype that survive intraperitoneal inoculation of virulent mhv-3 can have persistent infections, in which virus can be recovered in low titer from liver, brain and lymphoid organs for up to 42 days (68). chronic brain infections have also been established in mice following intracerebral inoculation of mhv-3, intracerebral or intranasal inoculation of brainpassaged mhv-jhm and selected temperature sensitive jhm mutants (58, 69-71). however, others have shown neurotropic mhv is eventually cleared from the brain (18,19,48,57,67). these studies suggest that mhv can potentially cause chronic persistent infections, particularly under artificial experimental conditions, but limited infections, without a chronic carrier state, are the norm. an obvious exception is the athymic nude mouse, which suffers from a wasting syndrome due to chronic, persistent mhv infections. in contrast to their heterozygous euthymic counterparts, which recover within 2-3 weeks, homozygous athymic mice develop increasingly high titers of virus in multiple organs, ultimately dying from hepatitis or encephalitis. during the early acute phase of infection, euthymic and athymic mice have parallel titers and distribution of virus (28,60, 72,73). persistent mhv infections incontestably take place in vitro in cultured cells in the absence of immune surveillance in the live mouse. this has been demonstrated repeatedly with a variety of mouse and rat cell types (47, [74] [75] [76] [77] [78] [79] . the addition of a low concentration of antiviral antibody to the culture medium was found to modulate infection, producing a carrier state with intracytoplasmic antigen, but no virus production or expression of viral antigens on the cell surface. the carrier state continued in the absence of antibody, and virus could not be rescued by co-cultivation with susceptible cells. virus could only be recovered following fusion of persistently infected cells to permissive cells with polyethylene glycol (78,79). the significance of these observations for the mouse remain to be determined. host-related factors in mhv pathogenesis have been elucidated using several respiratory prototype mhv strains. mice less than 2-3 weeks of age are susceptible, regardless of genotype (2,26,80-82). other workers have suggested that resistance to intracerebral jhm infection further evolves at 6-12 weeks of age (83,84 mhv-jhm productively infects both neuronal and non-neuronal cells in vitro, while laboratory-created temperature-sensitive mutants of jhm cause non-productive infection of non-neuronal cells only, with expression of antigen without virus replication. on the other hand, mhv-a59 selectively infects non-neuronal cells in a productive fashion (41). differences among various mhv strains have been observed in their ability to productively infect adherent cells (macrophages) from liver expiant cultures (88). intrinsic susceptibility of cells in vitro can be modified with lymphokines (90,91) and interferon (82,96,97). lymphoreticular function in the whole animal also influences the course of mhv infection. agents that interfere with or stimulate macrophage activity alter the course of mhv infections (65,90,95,98-100). interferon (if) plays a disputed role in mhv pathogenesis. serum if levels are low in mhv-infected immature mice and elevated in adults (82), but others have found that resistant mouse genotypes may produce lower levels of if than susceptible genotypes in response to mhv infection (88,101). others have found the same degree of if levels (93). tissue if levels have also been variably correlated with resistance (88,102). it is apparent that if plays a role in the initial response of mice to mhv (97), and becomes elevated in adult, susceptible genotypes because of permissive infections, also resulting in increased natural killer (nk) cell activity in some mice. in resistant adults, virus titers, if and nk cells may all remain low (101). immunosuppression by a variety of means, including x-irradiation, neonatal thymectomy, antilymphocyte serum, graft vs host reaction, cyclophosphamide and corticosteroid treatments, renders resistant mice susceptible to mhv. immunosuppression, however, does not modify intrinsic genetically determined resistance of cells cultured ±n_ vitro or abrogate a secondary immune response to virus challenge (62,64,94,103,104). cell-mediated immunity is crucial in recovery from mhv infections. this fact is most evident in t cell deficient, athymic nude mice, in which mhv titers initially parallel their heterozygous euthymic counterparts, but continue to rise as hétérozygotes recover (73) only a single study has demonstrated delayed-type hypersensitivity in mhv immunized mice (62). it becomes apparent from this overview that mhv strains interact at different levels with mice of different ages and genotypes. expression of susceptibility or resistance is mediated first through the intrinsic ability of the target cell to support or restrict mhv replication. this can be further modified by if and nk cells, among other factors. secondly, infection is restricted by a specific immune response to the virus, resulting in susceptibility, attenuated disease or recovery. this is influenced by immunosuppressive regimens or impaired immune responsiveness. both intrinsic resistance and development of an effective immune response are age and genotype dependent (29,62,64,84, 97,101,103,109). this is why susceptibility to mhv can be predicted in genotypes that allow high-titer virus replication early in infection, prior to mounting an immune response (62,64,97,109). it also explains the confusion over whether genetic susceptibility or resistance are inherited as one or two genes, recessive or dominant, h-2 haplotype linked or unlinked with different mhv strains, different routes of inoculation and different mouse genotypes (2,45, 46,59,80,109). the course of mhv infection is notoriously sensitive to modification of lymphoreticular function. normally resistant mice are rendered susceptible by x-irradiâtion, cyclophosphamide, cortisone, neonatal thymectomy, antilymphocyte serum and graft vs host reaction, among other manipulations (3). co-infection with other infectious agents also potentiates mhv disease. eperythrozoon coccoides and k virus are normally minimally pathogenic, but they exacerbate mhv in resistant hosts (104,110). leukoviruses and even schistosoma mansoni also enhance mhv disease. these diversely related infectious agents have been suggested to have a common mechanism by blocking if responsiveness of the host to mhv (111-113). several enterotropic strains of mhv have been identified. like other mhv strains, they can be differentiated antigenically, but also share antigenic and genetic homology with other members of the mhv group (13,14,17,34-36). the upper respiratory mucosa is often a target of enterotropic mhv (19), but intestinal mucosa is the major target for virus replication and excretion. unlike respiratory mhv strains that infect intestine only in highly susceptible hosts, enterotropic mhv universally infects intestine, regardless of host age. in neonatal mice, epithelial degeneration, necrosis and syncytia predominate, resulting in erosions, ulcération and villus attenuation. virusinduced syncytia, or "balloon cells," are diagnostic. intracytoplasmic inclusions have also been described. lesions can be fully developed within 24-48 hours (32), but are usually most severe at three to five days (14,19,33). in surviving mice or mice exposed at older ages, there tends to be less necrosis and more compensatory mucosal proliferation, or hyperplasia. adult mice are also susceptible, but usually have mild infections with only minor lesions (3,13, 14,31,32,34). lesions can be found anywhere from pylorus to anus, but stomach is not involved. distal small intestine, cecum and ascending colon are preferential sites (3,13,14, 19,31-33). dissemination beyond the intestine can also occur. some mhv strains are highly restricted to intestinal mucosa and mesenteric lymph nodes, while others spread to liver and other organs (3,13,14,19,31,32). mhv-d, like mhv-jhm and -s, appears to share the ability of infecting the olfactory bulbs of the brain (33). these differences in behavior have not been carefully examined and may be either virus strain or host related. the influence of host genotype and immune response have not been well studied with enterotropic mhv. however, clinical symptoms and lesions associated with intestinal protozoal infestations are exacerbated by enterotropic mhv (3,31) and x-irradiation exacerbates enterotropic mhv infection. duration of intestinal mhv infections is limited, with no known carrier state beyond two weeks after exposure (3,13,19,31,32 ). an exception is the athymic nude mouse, which develops persistent infections of the intestinal and nasal mucosa. enteric mucosa tends to be segmentally hyperplastic with syncytia. involvement of other organs is minimal or absent. these mice suffer from progressive emaciation, but lesions and distribution of lesions differ markedly from the typical wasting syndrome caused by respiratory mhv strains (3,31,114). clinical disease is usually absent or mild and lesions are often restricted to the primary target organ (nose or intestine), except in highly susceptible hosts. disease and lesions are most apt to be seen in naive mouse populations experiencing an initial epizootic. suckling, genetically susceptible or immunologically compromised mice are the best candidates for diagnostic evaluation. suckling mice naturally infected with respiratory mhv suffer moderate to high mortality between 4-10 days of age. they may have signs of encephalitis, but more often signs are nonspecific. gross necropsy findings include multiple white spots on the liver (30,51). flaccid paralysis was described in adult swiss mice naturally infected with mhv-jhm (1), but this is uncommon. a variety of experimental manipulations will exacerbate otherwise mild infections in resistant mice (3). enterotropic mhv strains tend to produce explosive epizootics of diarrhea and high mortality among infants but these signs may be absent in enzootically-infected colonies. necropsy findings include enteritis, usually in the absence of liver lesions. surviviors are often runted and pot-bellied (3,14,19,31-34) . histopathology is a useful means of confirming a diagnosis of mhv, but only in susceptible mice during the active phase of infection. in susceptible hosts, particularly neonates and athymic nude mice, mhv induces multinucleate syncytia in multiple tissues, which are pathognomonic for mhv. syncytia are especially obvious in enteric mucosa of mice infected with enterotropic mhv. histopathology details are given elsewhere (3,30,31) . a more definitive diagnosis can be made with the use of immunohistochemical techniques such as immunofluorescence and immunoperoxidase. formalin-fixed, paraffin-embedded tissues can be ultilized for this purpose if subjected to prior trypsinization. antigen is very stable in formalin-fixed tissue, allowing retrospective analysis of archival cases (13,18,19,115) . electron microscopy can also be utilized. mhv has characteristic coronavirus morphology and buds into cisternae of endoplasmic reticulum. it can also produce intracytoplasmic aggregates of viral products (3,13,31) . virus recovery from actively infected tissues is difficult but can be accomplished using a variety of effective cell lines, such as nctc 1469, dbt, 17c1-1 or l929 cells. these cell lines, however, may not be successful substrates for some enterotropic mhv strains. syncytium formation is the hallmark mhv cytopathic effect. identification of a suspect agent as an mhv strain can be accomplished by immunocytochemistry. reciprocal serum neutralization is used as a means of characterizing the antigenic relatedness of a new isolate to prototype mhv strains. however, because of extensive and complex antigenic inter-relationships, this method is not definitive for strain classification (13-15, 19,116) . several serological assays can be used for detection of antibody in mhv-recovered mice. the complement fixation test was used extensively at one time, but is usually too insensitive to detect seroconversion following natural exposure (13,117-120). as mentioned above, serum neutralization against prototype mhv strains grown ±n_ vitro can be used, but it tends to be highly strain-specific. the neutralizing profiles of sera from different mouse populations or from different mhv outbreaks within a population can be compared to determine if the same or unrelated mhv strains are involved (13-15). hemagglutination inhibition is used for detecting antibody to coronaviruses of other species, but only one mhv isolate (dvim) has been found to possess a hemagglutinin (35,36). two very sensitive and specific assays for mhv antibody are an indirect immunofluorescence assay (ifa), using mhv-infected cells and an enzyme-linked immunosorbent assay (elisa) (118-120). both offer the advantage of using multiple mhv strains simultaneously as antigens in order to cover a broad range of reactivity to mhv serotypes. under experimental conditions, these two tests are nearly equally sensitive and both are more sensitive than serum neutralization (120). nude mice are not good candidates for serology. although they can produce neutralizing antibody to mhv, titers are widely variable and sometimes absent (60,141). morphological, immunohistochemical, virus recovery and serological methods can be embellished with the use of susceptible mhv hosts. for example, if mhv is suspected but cannot be confirmed in a mouse population, athymic nude mice can be placed in the animal room as sentinels. when these mice become infected, lesions and antigen are easily confirmed and virus is easier to isolate. infant mouse brain inoculation will amplify virus titers from suspect tissues, allowing easier antigen detection or virus recovery. similarly, inoculation of cortisonized mice will serve the same purpose. the mouse antibody production test can also be effectively utilized. inoculation of adult, virus antibody free mice with suspect material will result in seroconversion if mhv is present. under natural conditions, mhv is highly contagious and is transmitted by the respiratory or oro-fecal routes. expression of overt disease requires immature, immunocompromised or genetically susceptible mice. under experimental conditions, mice seroconvert by ifa or elisa between 7 to 14 days after inoculation (120) . under natural conditions, seroconversion occurs in 100% of mice within two weeks after introduction to an mhv-infected population, underscoring the rapidity of exposure that takes place (unpublished observations). outbreaks of clinical disease among susceptible neonatal mice are also very rapid with high morbidity (13,14). as already discussed, infections last less than two weeks with seroconversion at the time of recovery (18,120). maintenance of infection in a mouse population (enzootic infection) requires continual exposure of new, susceptible mice, either through newly introduced mice or breeding populations. in the absence of susceptible mice, mhv will die out of a population. frequently, mhv-free mice are received into enzootically infected mouse rooms. if they are neonatal mice, they suffer high mortality (51). however, extramural mice are usually weanlings or older, developing only subclinical infections upon exposure, but perpetuating the infectious cycle. use of these newly acquired exposed mice within the first one to two weeks after arrival creates a high potential for exacerbation of overt disease from subclinical infection when experimentally immunosuppressed or otherwise stressed. in enzootically infected populations, disease or lesions are most apt to be encountered in weanlings, rather than neonates, because neonates are protected by passive immunity. colostral igg from immune dams is partially protective under experimental conditions and seems to occur naturally (14,122). therefore, when mhv initially infects a naive mouse population, neonatal mice suffer high mortality, particularly with enterotropic mhv strains (13,14). once infection is enzootic, adult mice confer protection to neonates, so the infection cycle is perpetuated among older mice, when their passive immunity wanes. vertical transmission of mhv is possible, but unlikely in the short time span of an acute infection relative to the breeding life of a female mouse. transplacental transmission to fetuses has been shown in mice inoculated with mhv-jhm intravenously, but depended upon stage of gestation at the time of inoculation (123) . neither transplacental transmission to fetuses nor vaginal transmission to newborns could be demonstrated in dams inoculated intraperitoneally with mhv-3 (124) . thus, the potential is there, but it is not a likely consideration. one aspect of mhv epizootiology that is not well understood is whether repeated infections can occur with the same mhv strain or different mhv strains. challenge resistence to mhv has been accomplished by vaccination of mice with denatured virus or surface peplomers, but not with virus membrane or ribonucleoprotein subcomponents, suggesting that the most immunogenic components of the virion are virus-strain specific (60,108). the host range of mhv has not been defined. under natural conditions, rats and mice develop serum antibody to coronaviruses of the heterologous species but this is not surprising because of the antigenic cross-activity of rat and mouse coronaviruses (21,23). mice are experimentally susceptible to rat sialodacryoadenitits virus (sdav) (21). suckling mice infected with sdav develop encephalitis, but not hepatitis or enteritis. weanling mice develop interstitial pneumonia, with antigen in alveolar lining cells (type 1 pneumocytes). in contrast, mhv infects alveolar septal cells, but not lining cells in the mouse (19). suckling rats are susceptible to mhv by intranasal inoculation. lesions are restricted to the nasal mucosa and antigen is present for only 2-3 days (125) . intracereberal inoculation of rats with mhv-jhm causes encephalitis and demyelination (9). ιτ± vitro growth characteristics of viruses from these two species also differ. sdav will grow in primary rat kidney but not mouse cells and mhv will grow in mouse but not rat cells (23,126). sdav passaged through infant mouse brain remains unable to grow in mouse cells in vitro (126) . it therefore appears that cross-species infections are laboratory, rather than natural, phenomena. the antigenic relatedness of mhv to coronaviruses of other species, including man, has unexplored biological significance. mhv has a long history of interference with research. most mhv strains were isolated and identified as inadvertant contaminants that were exacerbated by experimental protocol. subclinical mhv infections are potentiated by a variety of experimental manipulations that modify immune or observed (38,127,131-133) . the ubiquity of mhv among laboratory mice creates a high potential for contamination of other agents by mhv or confusion with mhv when passed in mice. examples are not hard to find. tettnang virus was an unclassified virus isolated from ticks and man in germany, egypt and czechoslovakia. the agent was unrelated to a variety of other tick-born arboviruses, and was eventually found to be mhv. retrospective study revealed that there was a relationship among other examples include isolation of mhv when human encephalitis brain biopsy material was passed in infant mice (142), confusion over the human or mouse origin of coronaviruses isolated from multiple sclerosis material passed in mice or mouse tissue (143) and confusion over avian or mouse origin of coronavirus isolated from manx shearwaters suffering from puffinosis (144). mhv has a number of effects on immunologie and nonspecific host responses to antigens. following intraperitoneal injection of mhv-3, mice had a modified immune response to sheep red blood cells. during acute infections, if mice were infected prior to antigen exposure, immunodepression occurred, while simultaneous injection with mhv and antigen resulted in immunostimulation. mice inoculated oronasally with mhv-jhm and an enterotropic mhv isolate display transient but significant functional disturbances in t and b lymphocyte populations, in mitogenesis assays with splenocytes from susceptible (balb/cbyj) but not resistant (sjl) genotypes (146). it remains to be determined if these effects are due to direct or indirect action of the virus on lymphoid tissue. mhv certainly has the potential of direct action on lymphoid organs (19,39) . pre-infection of mice with mhv, either naturally or experimentally, alters the ability of normally permissive respiratory tract tissues to support replication of pneumonia virus of mice and also reduces susceptibility of dba/2j mice to sendai virus (147). regardless of the mechanisms involved, these observations have broad implications for experimental virology and laboratory animal science, since mhv-infected mice respond abnormally to respiratory and possibly other viruses. mhv has been shown to activate nk cells and alter the if responsiveness of infected mice (101,145) . -recovered mice (100,117,148, 149) . athymic nude mice infected with mhv develop a number of immunological peculiarities. responses to sheep red blood cells, including a secondary immune response (121, 151) . in addition, they are able to reject tumor xenografts (131, 134) . since mhv causes acute infections and because its contagiousness results in almost simultaneous infection of an entire population, it can be controlled like coronaviruses of the rat by simply breaking the infectious cycle (3). cessation of breeding or quarantine without introduction of new mice will achieve this objective. selection of seropositive breeding stock for re-establishment of a breeding colony will ensure that mice are recovered from infection. judgement on the success of this approach must be made only under conditions that prevent re-infection of the population. prevention of infection is extremely difficult in facilities receiving mice from outside sources at various intervals. such conditions can allow introduction of subclinically infected animals that can infect resident stock or, conversely, perpetuation of the infectious cycle by introduction of naive mice following exposure to infected populations. furthermore, mhv is so highly contagious that it is very likely to re-contaminate mouse populations that have become rid of the agent by re-derivation, re-population or quarantine, particularly if identical husbandry practices prevail. the best means of mhv control is preventing its entry into a facility. this can be achieved by purchase of mice from mhv-free sources and shipment in filtered boxes. also, transplantable tumors and other mouse biological products must be mhv-free. since most institutions are unable to achieve this goal, mhv-free mice can be maintained in properly controlled barrier facilities, plastic film isolators or filtered cage systems. filter-top cages offer a relatively inexpensive means of preventing mhv infections, but handling and service of the cages must be strictly aseptic (13,18,19,120) . vaccination has been shown to effectively reduce severity of challenge mhv infection, but not totally prevent it (60,108). since vaccination immunity is produced against virus peplomeric antigens, it is likely to be strainspecific (108). the number of antigenic mhv biotypes therefore makes vaccination impractical. furthermore, mhv usually causes subclinical infections, particularly when the virus is enzootic in a colony. it is likely that vaccination will cause greater effects on the host than natural infection· if exacerbation of mhv disease is a problem in an experimental protocol, newly arrived mice can be allowed to become infected and recover prior to use, or virus-free mice under appropriate preventive husbandry conditions can be used. a number of aspects of mhv biology are worthy of future research pursuit. rather than generating a list of such topics, the author has attempted to indicate throughout the text where deficiencies in knowledge exist. since scientific knowledge evolves best from a multi-disciplinary approach, it is hoped that scientists in various fields will seek ways to better understand this agent from their own perspectives. effect of corticosteroids on mouse hepatitis virus infection chronic central nervous system demyelination in mice after jhm infection immunopathology of mouse hepatitis virus type 3 infection. iii. clinical and virologie observation of a persistent viral infection mouse hepatitis virus-induced recurrent demyelination. a preliminary report virus persistence and recurrent demyelination 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during viral hepatitis · hematological changes in viral (mhv-3) murine hepatitis ιηάμ^ίοη of monocyte procoagulant activity by murine hepatitis virus type 3 parallels disease susceptibility in mice western equine encephalitis mimicking herpes simplex encephalitis coronavirus isolates sk and sd from multiple sclerosis isolation of a latent murine hepatitis virus from cultured mouse liver cells. am. j. gastroenterol. 58: 259-74. 78. stohlman, s.a., sakaguchi, a.y., weiner, l.p. (1979) .characterization of the cold-sensitive murine hepatitis virus mutants rescued from latently infected cells by cell fusion. virol key: cord-017521-z9l9c83i authors: kubota, tetsuya; kubota, naoto title: cuff-induced neointimal formation in mouse models date: 2015-11-05 journal: mouse models of vascular diseases doi: 10.1007/978-4-431-55813-2_2 sha: doc_id: 17521 cord_uid: z9l9c83i ischemic heart failure caused by atherosclerosis is a major cause of death worldwide. although remarkable technological advances have been made in the treatment of coronary heart disease, there is as yet no treatment that can sufficiently suppress the progression of atherosclerosis, including neointimal thickening. therefore, a precise understanding of the mechanism of neointimal hyperplasia will provide the development of new technologies. both apoe-ko and ldlr-ko mice have been employed to generate other relevant mouse models of cardiovascular disease through breeding strategies. although these mice are effective tools for the investigation of atherosclerosis, development of a progressive atherosclerotic lesion takes a long time, resulting in increase of both the costs and the space needed for the research. thus, it is necessary to develop simpler tools that would allow easy evaluation of atherosclerosis in mouse models. in this review, we discuss our experience in generating mouse models of cuff-induced injury of the femoral artery and attempt to provide a better understanding of cuff-induced neointimal formation. epidemiological studies reveal that coronary heart diseases caused by atherosclerosis, including myocardial infarction and other forms of ischemic heart disease, are major causes of death worldwide [1] . if coronary blood flow is impaired by the development of atherosclerosis, interventions such as balloon angioplasty and endovascular stent placement are employed to overcome the vascular occlusion. these interventions can produce mechanical damage to the vasculature, including endothelial cells, smooth muscle cells (smcs), and the adventitia [2] [3] [4] . destruction of the endothelial cell layer is observed in the early phase after these interventions, with the formation of a thin thrombus layer covering the vascular surface [5] . within several weeks, the vascular endothelial cells completely cover the neointima. endothelial injury causes recruitment and adherence of circulating leucocytes, which results in the progression of neointimal formation [6] . the extent of neointimal formation has been reported to be correlated with the number of macrophages in the neointima [7] . macrophages and neutrophils enhance the inflammatory response through the release of growth factors such as fibroblast growth factor (fgf), transforming growth factor-beta (tgf-β), platelet-derived growth factor (pdgf), and vascular endothelial growth factor (vegf) [5] . human studies have shown the existence of a correlation between chronic inflammation after stent placement and intimal thickening [7] . the release of the aforementioned growth factors and cytokines by the injured endothelium and infiltrating inflammatory cells leads to smc migration and proliferation, which is preceded by transition of the smcs from a contractile to a synthetic phenotype with excessive extracellular matrix (ecm) deposition in the intima [8, 9] . although remarkable technological advances have been made in the treatment of coronary heart disease, none of the available treatments up to date can sufficiently suppress atherosclerosis or entirely prevent restenosis after angioplasty [10] [11] [12] . although in-stent restenosis can be alleviated by the use of drug-eluting stents, a number of cases treated with drug-eluting stents are still reported to develop restenosis [13, 14] . there also remains the question of the safety of drug-eluting stents, including in relation to the higher frequency of occurrence of thrombotic events observed with the use of drug-eluting stents as compared to bare-metal stents [15, 16] . moreover, cases with neointimal hyperplasia occurring after bypass surgery or allograft cardiac transplantation cannot be treated with drug-eluting stents [17, 18] . thus, a more precise understanding of the mechanism of neointimal hyperplasia will provide the development of new technologies. in this review, we discuss our experience in generating mouse models of cuff-induced injury of the femoral artery and attempt to provide a better understanding of cuff-induced neointimal formation. many previous studies have reported rabbit models of cuff-induced injury. two types of materials have been used for the generation of these rabbit models, a polyethylene tube and a silastic tube. in 1969, mizukawa et al. have reported neointimal formation induced by the insertion of a polyethylene tube in the rabbit carotid artery [19] . histological analysis revealed that the neointima was composed of smcs, but had no other characteristics of atherosclerosis, e.g., foam cells [20] [21] [22] . importantly, the polyethylene tube produced only mild, not severe, injury of the endothelial cells. hirosumi et al. carried out an in-depth investigation of the morphometric changes induced in an artery by the insertion of a polyethylene tube by scanning electron microscopy and light microscopy (1.5 cm long pe-280; inner diameter, 2.15 mm; outer diameter, 3.25 mm; becton, dickinson and company) [23] . at 30 min after cuff placement, small endothelial defects and adherence of a small number of platelets were observed in the subendothelium. at 2 h, a number of leukocytes were recruited to this cuff-injured area. the endothelial defects increased for 24 h, and leukocytic infiltration of the internal elastic lamina and endothelial cells was observed. after the 3rd day, regeneration of spindle-shaped endothelial cells occurred and covered the exposed subendothelium. however, the leukocytic infiltration persisted. the internal elastic laminae were irregular, swollen, and wavy. the smcs under these laminae were markedly deformed and edematous. although the endothelial cells became flat after 1 week, gaps were often observed at the cell junctions. light microscopy revealed regression of the edematous media and smc-like intimal cell proliferation. after 2 weeks, the endothelial cells completely covered the subendothelium, and the gaps between the cells acquired normal tightness. leukocytes were no longer seen at the endothelial surface. on the other hand, booth et al. were the first to use a biologically inert soft and flexible silastic cuff, 2.1 cm length with an internal volume of 0.3 cm 3 in the carotid artery of a new zealand white male rabbit [24] . neointimal formation consisted largely of smc-like cells, similar to the case in the polyethylene cuff-induced injury. no foam cells were detected in any of the sections obtained from the carotid arteries of the new zealand white rabbits at 7 days after the placement of the silastic cuff. moreover, an intact endothelial layer was observed after 7 days. the endothelial cells remained in contact with each other, and no denudation or exposure of the subendothelium was seen at 24 h after placement of the silastic cuff [25] . kockx et al. revealed that silicone cuff-induced neointimal formation can be distinguished into three phases [26] . the first phase begins within 2 h, with polymorphonuclear leukocyte (pmn) infiltration from the luminal surface toward the intima and the inner media. in the second phase, which starts within 12 h, the replication rate of the smcs in the media increases by about 20-fold as compared with that in unmanipulated arteries. the third phase, beginning from day 3, is characterized by the appearance of subendothelial smcs that are immunoreactive for α-sm actin (α-sma). although the neointima formed in response to both injury caused by a polyethylene and that caused by a silastic cuff consists of smcs, the injury caused by the polyethylene cuff was associated with injury of the endothelial cells, whereas that caused by the silastic cuff was devoid of injury to the endothelium. mouse models have been used popularly as experimental animal models due to their well-defined genetic background and the possibility of manipulating the mouse genome. mouse models are useful for evaluating the roles of specific molecules in vascular remodeling, such as in the case of post-percutaneous coronary intervention (pci) restenosis. apolipoprotein (apo) e-knockout (ko) mice have been used extensively because they develop spontaneous atherosclerosis [27, 28] . furthermore, the atherosclerotic lesions in this mouse model closely resemble the atherosclerotic lesions, both stable and unstable, in humans. low-density lipoprotein (ldl) r-ko mice also serve as good models to evaluate human atherosclerotic lesions [29] . however, unlike the apoe-ko mice, the ldlr-ko mice do not develop spontaneous atherosclerosis [28] . both apoe-ko and ldlr-ko mice have also been utilized to generate other relevant mouse models of cardiovascular disease through a variety of breeding strategies. although these mice are effective tools for the investigation of atherosclerosis, development of a progressive atherosclerotic lesion takes a long time, resulting in an increase of both the costs and the space for the research. mouse arteries, unlike the arteries of larger animals, are too small for transluminal injury to be induced with a balloon. thus, it is necessary to develop tools for easier evaluation of atherosclerosis in mouse models. three methods for the induction of neointimal formation in mice have been mainly reported [10] . in the arterial ligation model, blood flow through the common carotid artery is disrupted by ligation near the distal bifurcation [30] . the decrease in the blood flow resulting from the dramatic reduction of the vessel diameter results in the formation of an extensive smc-rich neointima within 2-4 weeks. this model has the advantage of reproducibility due to the ease of use of the method for inducing neointimal hyperplasia. lindner and colleagues were the first to report mechanically induced endothelial denudation model, which involves passage of a flexible guidewire three times in the carotid artery [31] . this procedure completely removes the endothelial cell layer, which results in the recruitment of platelets to the denuded surface and activation of the medial smcs. neointimal hyperplasia is typically observed within 2 weeks after the injury. this procedure is the most similar to angioplasty. however, this procedure is a relatively challenging procedure, which leads to difficulty in achieving reproducible results. subsequently, sata et al. modified the wire injury method to develop a more convenient and reproducible results [32] . since the wire injury model shows complete denudation of the endothelial cell layer, it may not be suitable for investigation of the involvement of endothelial cell-specific molecules in the development of atherosclerosis. use of a polyethylene cuff also causes damage to the endothelial cell layer as described above, but it produces milder injury as compared to wire injury. the endothelium itself is not directly injured by a polyethylene cuff. in fact, endothelial cells have reported to remain relatively intact after perivascular cuff placement [33] . moroi et al. first adapted an external vascular cuff model used in rabbits to mice deleted with endothelial cell-specific enos gene [34] . since the virus-mediated vector and drug can be injected easily between the cuff and the vessel, this cuff-induced injury model is also excellent for the observation of changes in the adventitia associated with arteriosclerosis [35] . thus, the use of mouse models of cuff-induced injury has increased in recent years. dr. moroi kindly taught us the procedure for the cuff placement [34] . before the operation, the cuff (intramedic polyethylene tubing (pe-50, inner diameter ¼ 0.58 mm), becton dickinson) needs to be prepared. polyethylene tube was cut for each length 2 mm and cut in longitudinal direction. we tied polyethylene tube directly to 8-0 nylon suture with needle (bear medic corporation) in two places on both side and made stringlike. the mice were anesthetized by an intraperitoneal (ip) injection of sodium pentobarbital at the dose of 40-50 mg/kg. then, the mice were fixed in the supine position and their skin was shaved from the inguinal region to the medial aspect of the thigh above the knee and disinfected with 70 % ethanol. the skin was next lifted with tweezers under stereomicroscopic observation, and an incision about 7-10 mm length was placed in the inguinal region. the subcutaneous connective tissue was carefully removed to expose both the femoral artery and the femoral vein. the femoral artery is separated from the femoral vein by blunt dissection with micro tweezers. a stringlike nylon suture with polyethylene tube is sent under a separating femoral artery. the break of the polyethylene tube is expanded by pulling stringlike nylon suture of polyethylene tube. the polyethylene tube is placed around the femoral artery. finally, the surrounding tissue is straightened and the skin is sutured ( fig. 2.1) . a sham operation is performed without polyethylene tube placement in the other femoral artery, as control. the mice are anesthetized by ip injection of pentobarbital 14 days after the operation and fix in the supine position. after laparotomy, the diaphragm is incised to expose the heart. ice-cold 4 % paraformaldehyde is injected into the systemic circulation via the cardiac apex at a pressure of 100 cm h 2 o, followed by incision of the right auricular appendage. both the cuffed-femoral artery and the sham-operated artery of the opposite sides are removed and immersed in oct compound. for the immunohistochemical staining, 5-to 6-μm-thick sections are prepared from each sample. paraffin sections are more suitable for the measurement of intimal thickness than frozen sections. femoral artery samples embedded in paraffin were cut from one edge to the other edge of the cuffed portion. the areas of the lumen, intima, and media were measured in 10 cross sections using the image analyzer. images are digitized and captured with a ccd camera connected to a personal computer ( fig. 2 the medial thickness for each vessel cross section, the linear distance between the internal elastic lamina and external elastic lamina is measured independently in 10 places, separated by 90 each and then averaged. the ratio of the intimal to the medial area and the percent luminal stenosis are calculated based on these measurements [10, 34] . since cuff-induced neointimal formation showed positive staining with an α-smc antibody (fig. 2.2b) , the neointima appears to be almost entirely composed of smcs. neointimal formation induced by cuff placement can be considered as a consequence of migration and/or proliferation of the smcs induced by some factors such as pdgf-bb or inflammatory cytokines ( fig. 2.3 ). although the exact mechanisms underlying the neointimal formation in cuff-induced injury models are still uncertain, i would like to refer to reports from the literature of studies carried out using genetically manipulated mouse models to discuss the mechanisms of neointimal formation ( cuff replacement around the femoral artery induced neointimal hyperplasia, which was exclusively composed of smcs, as mentioned above. platelet-derived growth factor (pdgf)-bb, which is mainly secreted from platelets, is one of the most the expression levels of pdgf-bb mrna in the cuffed artery were significantly increased as compared to those in the sham-operated artery (fig. 2.3 ). an antagonist of the pdgf-bb receptor was shown to suppress the proliferation of vascular smcs after balloon-induced injury in a rat model [36] . pdgf-bb induces vascular smc proliferation via, at least in part, regulation of the cell cycle. schwaiberger et al. demonstrated that local treatment with a cyclin-dependent kinase inhibitor inhibited cuff-induced neointimal formation, accompanied by reduction of the phosphorylation level of signal transducer and activator of transcription 3 (stat3), but not of akt, erk1/2, or p38mapk activation [35] . these data suggest that pdgf-bb-stat3 signaling is involved in cuffinduced neointimal formation. in addition to pdgf-bb-stat3 signaling, pdgf-bb has been reported to augment the migration of smcs through the lr11, a member of the ldl receptor family/urokinase-type plasminogen activator receptor (upar) pathway [37] . ohwaki et al. found that balloon-injured intimal smcs showed strong expression of lr11 in rat arteries. high-fat diet-fed lr11-ko mice showed a decrease of cuff-induced neointimal formation [38] . in in vitro experiments, the secreted soluble form of lr11 (soilr11) promoted the migration of thp-1 induced by pdgf-bb, an effect that was completely canceled by the anti-upar antibody [38] . these data suggest that pdgf-bb induced by cuff-induced injury is one of strongest regulators of neointimal formation. the ecm has been reported to be a key player in arterial remodeling after vascular injury [4] . hyaluronan 2 (has2), which is a primary component of the ecm, is expressed in neointimal lesions in humans with atherosclerosis and at sites of wire-induced injury of the arteries in mice. mice overexpressing the murine has2 gene specifically in the vascular smcs (chas2/cresm22a mice) showed markedly enhanced cuff-induced neointimal formation, with augmentation of smc migration and proliferation, and production of inflammatory cytokines and ros [39] . consistent with these data, in a wire-induced injury model, a ha synthesis inhibitor markedly suppressed neointimal formation. the ecm induced by arterial cuff-induced injury promotes migration of smcs into the intima, leading to neointimal formation. the roles of bone marrow (bm)-derived cells in the pathogenesis of atherosclerosis have been extensively studied [40] . fluorescence in situ hybridization (fish) analysis has revealed that female-derived bm cells transplanted into male mice were detected in the neointima of male mice (fig. 2.4) . following bmt from green fluorescent protein (gfp)-transgenic mice to apoe-ko mice, gfp-positive cells were confirmed in the vascular neointima (4-10 %) and media (5-32 %) in the latter mice. following bone marrow transplantation (bmt) from lacz mice to wild-type (wt) mice, a number of lacz-positive macrophages were found in the neointima (25.7 %), media (7.3 %), and adventitia (73.7 %) of the arteries of the wt mice at 4 weeks after cuff placement [33] . it was worthy of note that more marked infiltration of the adventitia by macrophages was observed after injury induced by cuff placement than after wire injury or ligation injury. xu et al. transplanted the bm of gfp-transgenic mice into ldlr-ko mice to identify the cell lineage in the lesion [41] . two weeks after cuff placement following a high-fat diet for 4 weeks, atherosclerotic lesions developing in the intima predominantly consisted of a massive accumulation of foam cells with a number of α-smaand gfp-positive cells. in addition to macrophages, adventitial small vessels also showed positive staining for both the endothelium-specific marker cd31 and gfp in mice transplanted with bm obtained from gfp-transgenic mice [41] . most of the macrophages are gfp-positive, and some of the smcs and ecs are also gfp-positive. in a cuff-induced vascular injury model, bm-derived cells are recruited to the adventitia and differentiate into macrophages, smcs, and endothelial cells. treatment with a blocker of m-csf, which guides differentiation into macrophages, caused a prominent decrease of macrophages, and inhibition of the pdgf receptor suppressed the recruitment of smcs to the adventitia after cuff placement [42] . these data suggest that the adventitia plays a pivotal role in neointimal formation induced by cuff placement. scott et al. demonstrated that the adventitia is important in the first wave of growth after angioplasty following occurrence of neointimal hyperplasia [43] . the importance of t and b lymphocytes in the process of restenosis after pci has been pointed out [44] . rag-1-ko mice, which lack mature b and t cells, have been demonstrated to show increased thickness of cuff-induced neointima in the carotid artery. reconstitution of the rag-1-ko mice with b cells from wt mice reduced the neointimal formation. moreover, both igg and igm were detected in the cuffinjured carotid arteries of reconstituted rag-1-ko mice with b cells [45] . these data suggest that an increase of immunoglobulin by activation of b cells exerts a protective action against intimal thickening. the neointimal area and intima/media ratio were significantly reduced in mice treated with immunoglobulin administered intraperitoneally for 5 consecutive days starting 1 day prior to cuff placement. on the other hand, immunoglobulin could not suppress cuff-induced neointimal formation, when the treatment was commenced 3 days after cuff placement [46] . when pooled mouse igg or igm was given to rag-1-ko mice by intravenous injection, a significant reduction of intimal thickening was observed as compared with that in the untreated rag-1-ko mice. immunoglobulin treatments modify the serum complement c3 profile, and the amount of complement c3 was decreased in the injured arteries. depletion of complement c3 in the rag-1-ko mice significantly decreased the degree of intimal thickening [47] . these data suggest that igg, igm, and complement c3 are involved in the modulation of the neointimal hyperplasia response to cuff-induced injury. moreover, treatment with immunoglobulin significantly enhanced the secretion of interleukin (il)-10, suggesting activation of t cells by immunoglobulin [46] . rag-1-ko mice reconstituted with t cells from wt mice showed a reduction of neointimal formation after cuff placement [48] . dimayuga et al. carried out a detailed examination of the t-cell fraction. splenic cd8 + cd25 + t cells and cd8 + cd28 + t cells, but not cd4 + cd25 + and cd4 + cd28 + t cells, were also significantly increased after arterial injury in the wt mice. rag-1-ko mice given cd8 + t cells showed a significant decrease of neointimal formation as compared to rag-1-ko mice not given the cells. on the other hand, transfer of cd4 + t cells was not associated with inhibition of the neointimal formation [49] . neointimal formation induced by cuff placement was significantly reduced in cd4-ko mice as compared with that in the wt mice, because of the higher percentage of cd8 + t cells. moreover, adoptive transfer of cd8 + cd28 hi t cells into recipient rag-1-ko mice significantly reduced neointimal formation as compared to that of cd8 + cd28 + t cells [50] . although both cd8 + t cells and cd4 + t cells are activated in response to arterial injury, cd8 + t cells, which constitute at least a fraction of the cd8 + cd28 hi , are mainly involved in the inhibition of cuff-induced neointimal formation. nitric oxide (no) is an important vascular regulatory factor that is generated by the enzyme nitric oxide synthase (nos) [51] . three different isoforms of nos are recognized: among these is endothelial nos (enos), which is constitutively expressed mainly in the vascular endothelial cells [52] . enos can be an important factor modulating vascular endothelial function and is activated by acetylcholine (ach) and insulin. enos-ko mice exhibit impaired ach-induced vascular relaxation [53] . the second isoform is inducible nos (inos), which cannot be detected in normal tissue, but is expressed in several cell types, including macrophages and vascular smcs, after cytokine stimulation [54] , and the third isoform is neuronal nos (nnos), which is constitutively expressed mainly in the nervous tissues and skeletal muscle type ii. enos-ko mice exhibit increased neointimal formation following cuff placement [34] . consistent with these data, the neointimal formation induced by ligation is also significantly more pronounced in the enos-ko mice than in the wt mice [55] . since antiplatelet and antihypertensive treatments cannot attenuate the progression of neointimal formation, the neointimal hyperplasia observed in enos-ko mice is produced by the direct action of enos and not mediated by thrombus formation or high blood pressure [33] . when adenovirus-mediated human endothelial constitutive nos cdna (adcmvcenos) was transduced into the rat carotid artery after balloon injury, the intima/media ratio decreased significantly because of inhibition of smc proliferation [56] . these data suggest that no mediated by enos inhibits neointimal hyperplasia induced by vascular injury. inos has been shown to be expressed in the smcs after cuff-induced vascular injury in rabbits [57, 58] , and inos-ko mice showed a significant reduction of neointimal thickening induced by cuff placement [59] . unlike enos-deficient mice, inos-ko mice showed no reduction of the neointimal hyperplasia associated with mechanically induced endothelial denudation. both the medial area and medial thickness were increased in the inos-ko mice after mechanically induced endothelial denudation [60] . consistent with these data, yogo et al. demonstrated that vascular remodeling, but not neointimal hyperplasia, after carotid artery ligation was increased in the inos-ko mice [55] . the differences in the procedure used to induce vascular injury may be related to the degree of neointimal hyperplasia and vascular remodeling in the inos-ko mice. cuff placement evokes significant participation of inflammatory cells, including macrophages, in the adventitia as compared to the ligation model, as mentioned above. increase of inflammation in the adventitia may be associated with increased inos expression levels, which may promote neointimal formation induced by cuff placement. mechanically induced endothelial denudation and ligation may mainly induce migration of the smcs from the media to the intima, resulting in increased neointimal hyperplasia. similar results were also observed in soluble epoxide hydrolase (seh)/apoe double-ko mice and inhibition of seh by 12-(3-adamantan-1-yl-ureido) dodecanoic acid, which suppress metabolism of epoxyeicosatrienoic acids (eets). seh/apoe double-ko mice or mice with inhibition of seh showed significant reduction of the neointimal formation in the femoral artery cuff model, but not in the carotid artery ligation model. the expressions of proinflammatory genes were significantly reduced in the femoral arteries of the seh/apoe double-ko mice [61] . inflammation is considered as an important factor in human atherogenesis [8] . an inflammatory response to vascular injury, mediated by proinflammatory cytokines, influences the progression of neointimal formation and development of atherosclerotic lesions. it has been demonstrated that the expressions of toll-like receptors (tlrs) 2 and 4 are markedly enhanced in human atherosclerotic plaques and vascular adventitia [62] . tlr4 serves as the receptor for bacterial lipopolysaccharides (lps) and also recognizes cellular fibronectin, heat shock protein 60, and endogenous peptides that are produced in response to tissue injury [63] . in the adventitia, not all tlr4-positive cells are colocalized with macrophages. although application of lps between the cuff and artery augmented the neointimal formation induced by cuff-induced injury in the wt mice, no such finding was observed in the tlr4-ko mice [64] . application of pam3cys-sk4, a synthetic tlr2 ligand, significantly enhanced the neointimal formation induced by cuff placement in the femoral arteries of the wt mice. no such increase of the neointimal formation was observed in the tlr2-ko mice. in apoe-ko mice, application of pam3cys-sk4 led to a significant increase in the formation of atherosclerotic plaques [65] . tlr2 stimulation produced significant induction of inflammatory cytokines in human adventitial fibroblasts in vitro. treatment with cathepsin k (catk), which is one of the most potent of mammalian collagenases, increased the mrna levels of inflammatory cytokines, including tlr2 and tlr4. catk-ko mice showed significantly reduced neointimal formation following cuff placement and ligation, accompanied by a decrease in the expression levels of tlr2 and tlr4 mrna [66] . these findings provide evidence for a link between inflammatory cytokines in the adventitia and intimal lesion formation. although tlr2/4 are expressed on the cell surface, tlr7/9 are expressed on the endosomes. tlr7/9 were detected at sites of post-interventional remodeling and accelerated atherosclerosis [67] . in hypercholesterolemic apolipoprotein e*3-leiden mice, femoral artery cuff placement led to a strong increase of the tlr7/9 expressions [68] . blockade of tlr7/9 with a dual antagonist reduced neointimal thickening and foam cell accumulation; the intima/media ratio was reduced by 64.5 % and luminal stenosis by 62.8 %. application of the tlr7/9 dual antagonist also reduced arterial wall inflammation, with reduced macrophage infiltration and altered serum il-10 levels. stimulation of cultured macrophages with tlr7/9 ligands enhanced tnfα expression, which was decreased by coadministration of the tlr7/9 antagonist. furthermore, the antagonist abolished the tlr7/9-enhanced ldl uptake. the antagonist also reduced oxidized ldl-induced foam cell formation, most likely not via decreased influx, but via increased efflux induced by increased il-10 levels. the renin-angiotensin-aldosterone system (raas) has various physiological actions such as vasoconstriction and is known to be involved in the development of hypertension and atherosclerosis [69, 70] . specifically, modulation of the local raas may play a key role, just like that of the systemic ras, in the development of cardiovascular diseases. angiotensin (ang) ii is a peptide that exerts potent vasoconstrictive action via the at1 receptor-mapk pathway. although human renin (hrn)/human angiotensinogen (hang)-transgenic (tg) mice showed increased blood pressure and medial thickening even in the absence of cuff placement, the hrn/hang-tg mice showed even more pronounced inflammatory vascular remodeling after cuff placement. treatment with an at1 blocker inhibited cuff-induced neointimal formation associated with reduced inflammation, but independently of the blood pressure change [71] . when the direct renin inhibitor aliskiren was administered to c57bl/6 mice via an osmotic pump, it inhibited cuff-induced vascular remodeling. the number of adherent leukocytes was increased in the cuff-injured mice not treated with aliskiren, where it was significantly reduced in the aliskiren-treated mice without any change of the blood pressure. aliskiren decreased the adhesion of thp-1 cells to tnfα-stimulated human umbilical vein endothelial cells [72] . these data indicate that ras activation augments neointimal hyperplasia induced by cuff placement via increased release of inflammatory cytokines. moreover, the ace expression level increased in a time-dependent manner after cuff placement and was observed in the medial and neointimal layers and the adventitia of the cuffed arteries in fvb/n mice. the intima/media ratio after cuff placement was significantly decreased by ace inhibitor treatment [73] . ang ii appears to be one of the factors exacerbating cuff-induced neointimal formation. in fact, at1 receptor-ko mice showed decreased neointimal formation following cuff placement, accompanied by an increase of apoptotic cells among the smcs [74] . consistent with these data, the at1-selective receptor blocker olmesartan suppressed cuff-induced neointimal formation via reducing erk phosphorylation [75] . on the other hand, neointimal formation induced by cuff placement was increased in at2 receptor-ko mice. the expressions of bcl-2 and bcl-xl mrna, which are regulators of apoptosis, were enhanced in the at2 receptor-ko mice showing enhanced neointimal formation [74] . recently, in addition to the ang ii-at1 receptor pathway, ang-(1-7), which is synthesized from ang i and ang ii mainly via ace2 activity, has been reported to play a crucial role in vascular remodeling via mas receptor activation [76, 77] . mas-ko mice showed markedly increased neointimal formation after cuff placement, independently of the at1 receptor. treatment with ang-(1-7) also suppressed neointimal formation, associated with suppression of vascular smc proliferation, release of inflammatory cytokines and superoxide anion production in the injured artery. on the other hand, these inhibitory effects of ang-(1-7) were less marked in the mas-ko [78] . interestingly, treatment with an at1 receptor blocker inhibited neointimal formation induced by cuff placement, accompanied by a decrease in the expression levels of ace2 and mas mrna and an increase in the expression of at2 receptor mrna. at2 receptor-ko mice showed no reduction of the neointimal formation by treatment with ang(1-7) . these results suggest that in addition to the activities of the ace2/ang-(1-7)/mas axis, blockade of the at1 receptor could enhance the activities of the ace2/ang-(1-7)/at2 receptor axis and thereby inhibit neointimal formation induced by cuff placement. atherosclerosis is associated with increased production of reactive oxygen species (ros) in the vessel [79] . the superoxide dismutases (sods) are enzymes that catalyze the dismutation of superoxide anions to hydrogen peroxide; three isoenzymes of the sods have been identified, namely, manganese sod (mnsod), which is localized in the mitochondria; copper/zinc sod (cu/znsod), which is localized in the cytosol; and extracellular sod (ec-sod). the antioxidant enzyme cu/znsod metabolizes superoxide anions (o2-) in the vascular endothelial cells [80] . although there was no difference in the degree of cuff-induced neointimal formation between the cu/znsod-ko and wt mice, the former showed a significant decrease in the intima/media ratio after cuff placement [81] . this increased medial smcs in the cu/znsod-ko mice showed positive staining for smemb/ mhc-b, which is a useful molecular marker of embryonic-type smcs. moreover, the expression levels of tnfα, icam1, vcam1, and inos in the media were higher in the cu/znsod-ko mice than in the wt mice, suggesting that cu/ znsod-ko mice showed enhanced inflammation, expression of adhesion molecules, and altered structure of the media post-injury. when an adenovirus vector expressing ec-sod (axcaec-sod) was injected between the cuff and the adventitia of the femoral arteries in rat models, neointimal formation was significantly reduced in the axcaec-sod-transfected arteries [82] . furthermore, proliferation of smcs in the neointima and media was inhibited by ec-sod treatment. augmented inos expression, apoptosis, collagen content, and ros generation in the vascular wall were also reduced by ec-sod treatment. the amount of generation of ros may have influence on the degree of medial thickening as well as neointimal formation induced by cuff placement. insulin resistance has been reported to be associated with atherosclerosis; however, the underlying mechanism is still unknown [83] . insulin resistance is caused by impaired insulin signaling induced by some factors. insulin activates insulin receptor substrates (irs) 1 and 2, which are expressed ubiquitously in various tissues, including the endothelial cells and smcs, through the insulin receptor and the downstream signaling [84] . both irs1-and irs2-ko mice show features of the metabolic syndrome, including insulin resistance, dyslipidemia, and hypertension. although both genotypes of mice show a similar degree of insulin resistance, the irs2-ko mice show more pronounced cuff-induced neointimal formation than the irs1-ko mice, which in turn show more pronounced cuff-induced neointimal formation than the wt mice. irs2 expression, but not irs1 expression, is detected in the blood vessels [85] . insulin resistance in the blood vessels is considered to exacerbate cuff-induced neointimal formation. on the other hand, mice with phosphatase and tensin homology deleted on chromosome 10 (pten), which is an antagonist of phosphatidylinositol 3-kinase (pi3k), exhibited significant reduction of neointimal formation when adenovirusmediated human pten (adpten) type 1 was injected between the cuff and the adventitia. adpten reduced smc proliferation and release of inflammatory cytokines [86] . since insulin activates pi3k via the irs, the overexpression of pten in response to cuff-induced injury appears to be a seemingly contradictory finding to that observed in the irs-ko mice. however, as pi3k has known to be activated by some factors independently of insulin signaling, activated pi3k may enhance neointimal hyperplasia induced by cuff placement under some conditions. cuff placement causes mild endothelial cell damage, smc proliferation and migration, and inflammation of the adventitia. this is a convenient and reproducible tool and is relatively less expensive and time-consuming. this tool is effective for analyzing the mechanism of vascular remodeling using mice, including genetically engineered mice. further experiments to identify the precise mechanisms of neointimal hyperplasia by using this tool will provide the development of new technologies in the future. global, regional, and national age-sex specific all-cause and cause-specific mortality for 240 causes of death, 1990-2013: a systematic analysis for the global burden of disease study in stent restenosis: bane of the stent era the importance of the endothelium in atherothrombosis and coronary stenting mechanisms of post-intervention arterial remodelling biological responses in stented arteries acute and chronic 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bimodal role of ifn-gamma in immune deficiency enhanced neointima formation following arterial injury in immune deficient rag-1à/à mice is attenuated by adoptive transfer of cd8 t cells reduced neointima formation after arterial injury in cd4à/à mice is mediated by cd8+cd28hi t cells cellular and molecular mechanisms of endothelial cell dysfunction primary endothelial dysfunction: atherosclerosis hypertension in mice lacking the gene for endothelial nitric oxide synthase isoforms of nitric oxide synthase. characterization and purification from different cell types different vasculoprotective roles of no synthase isoforms in vascular lesion formation in mice human endothelial nitric oxide synthase gene transfer inhibits vascular smooth muscle cell proliferation and neointima formation after balloon injury in rats arterial smooth muscle cells express nitric oxide synthase in response to endothelial injury induction of nitric oxide synthase in the neointima induced by a periarterial 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cell formation, and migration angiotensin ii promotes atherosclerotic lesions and aneurysms in apolipoprotein e-deficient mice angiotensin ii-induced hypertension accelerates the development of atherosclerosis in apoe-deficient mice temporary treatment with at1 receptor blocker, valsartan, from early stage of hypertension prevented vascular remodeling dynamic observation of mechanically-injured mouse femoral artery reveals an antiinflammatory effect of renin inhibitor angiotensin converting enzyme inhibitor restrains inflammation-induced vascular injury in mice role of angiotensin ii-regulated apoptosis through distinct at1 and at2 receptors in neointimal formation effect of estrogen and at1 receptor blocker on neointima formation angiotensin-(1-7): beyond the cardio-renal actions possible involvement of angiotensin-converting enzyme 2 and mas activation in inhibitory effects of angiotensin ii type 1 receptor blockade on vascular remodeling possible role of angiotensin-converting enzyme 2 and activation of angiotensin ii type 2 receptor by angiotensin-(1-7) in improvement of vascular remodeling by angiotensin ii type 1 receptor blockade role of oxidative modifications in atherosclerosis vascular expression of extracellular superoxide dismutase in atherosclerosis deficiency of cuzn superoxide dismutase promotes inflammation and alters medial structure following vascular injury extracellular superoxide dismutase overexpression reduces cuff-induced arterial neointimal formation insulin sensitivity and atherosclerosis. the insulin resistance atherosclerosis study (iras) investigators snapshot: physiology of insulin signaling lack of insulin receptor substrate-2 causes progressive neointima formation in response to vessel injury pten reduces cuffinduced neointima formation and proinflammatory cytokines key: cord-032975-7hugs419 authors: sun, j. d.; dahl, a. r.; gillett, n. a.; barr, e. b.; crews, m. l.; eidson, a. f.; bechtold, w. e.; burt, d. g.; dieter, m. p.; hobbs, c. h. title: two-week, repeated inhalation exposure of f344/n rats and b6c3f mice to ferrocene(1) date: 1991-07-17 journal: fundam appl toxicol doi: 10.1093/toxsci/17.1.150 sha: doc_id: 32975 cord_uid: 7hugs419 two-week, repeated inhalation exposure of f344/n rats and b6c3f(1) mice to ferrocene. sun, j. d., dahl, a. r., gillett, n. a., barr, e. b., crews, m. l., eidson, a. f., bechtold, w. e., burt, d. g., dieter, m. p., and hobbs, c. h. (1991). fundam. appltoxicol. 17, 150-158. ferrocene (dicyclopentadienyl iron; cas no. 102-54-5) is a relatively volatile, organometallic compound used as a chemical intermediate, a catalyst, and as an antiknock additive in gasoline. it is of particular interest because of its structural similarities to other metallocenes that have been shown to be carcinogenic. f344/n rats and b6c3f, mice were exposed to 0, 2.5, 5.0, 10, 20, and 40 mg ferrocene vapor/m3, 6 hr/day for 2 weeks. during these exposures, there were no mortality and no observable clinical signs of ferrocene-related toxicity in any of the animals. at the end of the exposures, male rats exposed to the highest level of ferrocene had decreased body-weight gains relative to the weight gained by filtered air-exposed control rats, while body-weight gains for all groups of both ferroceneand filtered air-exposed female rats were similar. male mice exposed to the highest level of ferrocene also had decreased body-weight gains, relative to controls, while female mice had relative decreases in body-weight gains at the three highest exposure levels. male rats had a slight decrease in relative liver weight at the highest level of exposure, whereas no relative differences in organ weights were seen in female rats. male mice had exposure-relative decreases in liver and spleen weights, and an increase in thymus weights, relative to controls. for female mice, relative decreases in organ weights were seen for brain, liver, and spleen. no exposure-related gross lesions were seen in any of the rats or mice at necropsy. histopathological examination was done only on the nasal turbinates, lungs, liver, and spleen. the only exposure-related finding was histopathologic lesions in the nasal turbinates of both species. these lesions were primarily centered in the olfactory epithelium and were morphologically diagnosed as subacute, necrotizing inflammation. nasal lesions were observed in all ferrocene-exposed animals and differed only in severity, which was dependent on the exposure concentration. in vitro metabolism studies of ferrocene showed that nasal tissue, particularly the olfactory epithelium, had 10 times higher “ferrocene hydroxylating” activity than did liver tissue from the same animals. these results suggest that the mechanism of ferrocene toxicity may be the intracellular release of ferrous ion through ferrocene metabolism, followed by iron-catalyzed lipid peroxidalion of cellular membranes. lizer, a smoke suppressant for polymers, and a polymerization catalyst, as well as in lubricant additives, and rocket propellants. the annual production of ferrocene is reported to be less than 1000 pounds per year (usepa, 1982) . ferrocene was selected for study by the national toxicology program because of its structural similarities to other metallocenes, some of which are carcinogenic, and because of the increasing potential for human exposure to ferrocene. the current tlv for ferrocene is 10 mg/m 3 (acgih, 1986) . at temperatures of 20°c or higher and concentrations in air of less than 40 mg/m 3 , ferrocene exists mostly as a vapor (jacobs et al, 1983) . therefore, ferrocene vapor, as opposed to droplets or particles, is the most likely chemical form to be inhaled by people. despite the increasing use of ferrocene and similar organometallic compounds, there is virtually no information available regarding the effects of long-term inhalation of vapors or aerosols of such compounds. inhalation exposures to inorganic forms of iron have been shown to be relatively nontoxic (committee on medical and biological effects of environmental pollutants, subcommittee on iron, 1979) . however, because of the lipophilicity imparted by its organometallic structure, ferrocene is able to deliver iron to lipophilic, intracellular sites in tissues which neither insoluble or water-soluble iron can reach (dahl and briner, 1980) . as a result, there may be ironinduced toxic effects that are not elicited by inorganic forms of iron, because of the differences in microdosimetry at specific sites. dahl and briner (1980) studied the toxicokinetics of inhaled ferrocene vapor. for these studies, they used ferrocene that was labeled with both iron-59 and tritium. inhalation exposure to ferrocene vapor resulted in an initial deposition of 37% of the inhaled vapor. of this amount, approximately 60% was deposited in the nasopharyngeal region and 25% in the lung. over 75% of the tritium label was excreted within the first day, but the 59 fe label remained, largely in the bronchopul-monary and nasopharyngeal regions, over the 117-day duration of the study. this suggested that the ferrocene was being metabolized in the respiratory tract, probably to hydroxyferrocene, which then decomposed in the lipidcontaining environment of the cellular endoplasmic reticulum. these investigators hypothesized that, because of the tendency of iron ions to catalyze the production of hydroxy radicals, inhaling ferrocene created the potential for cellular injury in the respiratory tract, particularly at the sites where ferrocene could deliver its iron moiety. by using an estimated half-time of 200 days for clearance of iron introduced into rat lungs by inhaled ferrocene, dahl and briner calculated that a human breathing ferrocene at the tlv concentration of 10 mg/m 3 would have acquired 1.7 g of ferrocene-derived iron in the pulmonary region at equilibrium. the purpose of our study was to characterize the toxic effects of inhaled ferrocene vapor after 2-weeks of inhalation exposure. we also investigated the in vitro metabolism of ferrocene in selected tissues to gain further insight into the importance of metabolism of this compound as a mechanism of ferrocene-induced toxicity. chemicals. the ferrocene used for this study was obtained from midwest research institute and was >99% pure, as determined by high-performance liquid chromatography (hplq. we studied the potential for ferrocene degradation that might be caused by the generation of exposure atmospheres. hplc analyses and graphite furnace atomic absorption of the exposure atmosphere showed that ferrocene was degraded by less than 1%. "fe-labeled ferrocene was prepared by treating a mixture of anhydrous ferrous chloride and w fe-labeled ferric chloride with sodium cydopentadienide. the labeled ferrocene was purified by sublimation and then was dissolved in ether. the concentration of ferrocene in the ethereal solution was determined by measuring the visible absorbance at 440 nm and calculating the concentration, using the molar absorptivity (0.95 m"'cm"'). the m fe activity was determined by scintillation counting. from the concentration and activity of the w fe, we were able to determine the specific activity of the synthesized material, which was then corrected for loss of activity due to radiodecay, the half-life of w fe is 45 days. exposure atmosphere generation and exposure system. wright dust feeders (wdf) (bgi, inc., waltham, ma) were used to generate the ferrocene aerosols. the aerosol generated by each wdf was heated to 200°c to vaporize the ferrocene particles. a separate wdf generator/vapor system was used for each of the five exposure chambers. six hazleton h-2000 whole-body exposure chambers were used to house animals being exposed to each of the five levels of ferrocene (2.5, 5.0, 10, 20, and 40 mg ferrocene vapor/m 3 ) or to filtered air (control). the highest ferrocene exposure level represented the highest concentration of ferrocene vapor that could be generated without producing condensation aerosols of the vapor in the chamber. the method used to determine aerosol concentrations in the chambers was a bubbler sampling train. the hexane solution from each bubbler system was analyzed on a hewlett-packard model hp589oa gas chromatograph to determine the chamber vapor concentration. a real-time aerosol monitor (ram-s; gca corp., bedford, ma) was used to determine whether significant levels of particles were present in the chambers during exposures. during the exposures, we measured the homogeneity of the ferrocene vapor within each exposure chamber by injecting heated line samples directly into the gas chromatograph. animals. thirty male and 30 female f344/n rats and 30 male and 30 female b6c3f, mice (4 weeks old) were received from the simonsen laboratories, inc. (gilroy, ca). the animals were quarantined in cage units within a hazleton h-2000 chamber prior to exposure, 2 per cage, for the first 4 days and then 1 per cage for 11 (rats) or 12 (mice) days. animals were exposed for 5 days/week, starting on a monday, for 2 weeks, with 2 or 3 additional exposure days before terminal euthanization, for a total of 12 or 13 exposure days for both rats and mice. this exposure schedule was necessitated to accomplish day-before-euthanization exposure and to facilitate timely necropsy of euthanized animals. most animals (67%) were exposed for 12 days. prior to the start of exposures, we used a computerbased randomization system (path/tox, xybion corp.) to randomly assign the rats and mice. individual animals assigned to the study were identified by tail tatoos for rats and tail tatoos and ear tags for mice. serum was obtained from two males and two females of each rodent species prior to the first day of exposure and was analyzed for antibody liters to specific rat viruses (kilham rat virus, toolan h-1 virus, pneumonia virus of mice, sendai virus, rat coronavirus/sialodacryoadenitis virus) and mouse viruses (k. virus, polyoma virus, mouse hepatitis virus, ectromelia, mouse encephalomyelitis (theiler's gd vii), mouse adenovirus, lymphocytic choriomeningitis virus, minute virus of mice, sendai virus, pneumonia virus of mice, epizootic diarrhea of infant mice, and reovirus 3). titers were negative for all of these agents. the animals were fed zeigler nih-07 open formula rat or mouse ration (zeigler brothers, inc., gardners, pa). food was withheld during the 6-hr-exposure period. water was available at all times, provided by an automatic watering system. temperature and relative humidity in the inhalation chambers were monitored once every 5 min and averaged 22.8 ± 1.1 °c and 55 ± 6% (mean + sd), respectively. the light cycle was automatically controlled to provide 12 hr of fluorescent light and 12 hr of darkness each 24 hr (on at 6 am and off at 6 pm). experimental design. rats and mice were weighed prior to starting the study, after 1 week of exposure, and at terminal euthanization. detailed clinical examinations were also made at these times. morbidity/mortality checks were made twice daily on all animals. rats and mice were killed at terminal euthanization by cardiac puncture exsanguination while under halothane anesthesia, and each animal was given a complete gross necropsy examination. the liver, thymus, right kidney, right testicle, brain, heart, and lungs were weighed and then fixed in 10% neutral-buffered formalin. tissues for microscopic examination (nasal turbinates, lungs, liver, and spleen) were embedded in paraffin, sectioned at 5 nm, and stained with hematoxylin and eosin. in vitro metabolism of ferrocene. microsomes for in vitro studies of ferrocene metabolism were obtained from the cells of 14-20 week old unexposed male f344/n rats. the rats were killed by co 2 asphyxiation. nasal ethmo-and maxilloturbinates and the liver were removed and homogenized. mictosomes were separated and stored as described previously (hadley and dahl, 1982) . suspensions of microsomes, appropriate cofactors (hadley and dahl, 1982) , and the radiolabeled ferrocene were incubated for 20 min at 37°c. metabolism was stopped by adding 1 m k.oh to raise the ph to ~ 11; adding the koh not only stopped the reaction, but also ionized phenolic metabolites to keep this species water soluble. after exhaustive hexane extractions of unmetabolized ferrocene, the aqueous portion of the reaction mixture was evaporated to dryness to remove, by sublimation, any small amounts of ferrocene that had not been extracted into the hexane. the residue was analyzed for radioactivity by liquid scintillation spectroscopy, and the protein content was determined with lowry protein assay (lowry et a!.. 1951) . statistics. the data from male and female animals were analyzed separately, using bartlett's test for data homogeneity and dunnett's test for the equality of mean values. the criterion for significance was set at p < 0.05. the overall mean ferrocene concentrations in each exposure chamber (2.5, 5, 10, 20, and 40 mg vapor/m 3 ) during the 2-week, repeated study were within ± 10% of the target concentrations (table 1 ). the relative standard deviation of the daily means was within 45%. the homogeneity (i.e., the variation among " mean ± sd (n =• 78). each ferrocene exposure concentration was shown to be statistically different from all other exposure concentrations at p < 0.05. six position samples within each chamber) of the ferrocene vapor concentrations ranged from 5.7 to 17.6% among all exposure chambers. no rats or mice died during the study, nor were any clinical signs of toxicity found during either the daily or the detailed observation times. group mean body weights at the end of the study (day 17) and mean weight gain over the exposure period are given in tables 2 and 3 for rats and mice, respectively. relative to control mice, male rats exposed to the highest concentration of ferrocene (40 mg/m 3 ) had statistically significant decreases in terminal body weight and rate of weight gain during the exposures. no significant body-weight differences were seen among the groups of exposed and control female rats at the end of the study. male mice exposed to the two highest concentrations of ferrocene (20 and 40 mg/m 3 ) had significant decreases in terminal body weight and/or in the rate of weight gain, while female mice had decreases in terminal body weight and rate of weight gain at the three highest exposure levels (10, 20, and 40 mg/m 3 ). no exposure-related gross lesions were found at necropsy in either rats or mice from any exposure group. the only measured organ weight affected by ferrocene exposure in rats was that of the liver. group mean liver weights for male and female rats are given in table 4 . male rats exposed to the highest concentration of ferrocene vapor had a statistically significant, although small, decrease in liver weights, compared to control animals. statistically significant decreases in liver weight were not seen in female rats at any exposure concentration. postexposure, group mean organ weights for male and female mice are given in table 5 . male mice had a statistically significant decrease in liver and spleen weights after exposures to the two highest concentrations of ferrocene vapor, and a statistically significant increase in thymus weight after exposure to the highest concentration of ferrocene, when compared to control animals. female mice had exposurerelated decreases in brain, liver, spleen, and right kidney weights, relative to control female mice. the measured weights for other organs were not affected by ferrocene exposure (data not shown). because of the absence of any gross lesions in other organs, only lung, nasal turbinates, and high level, ferrocene-exposed mice (b) (x135). note the marked attenuation, necrosis and accompanying inflammation of the nasal epithelium from the ferrocene-exposed animal (b). similar nasal lesions were found in rats exposed to ferrocene vapor. epithelium present at the entry to the nasal cavity. the respiratory epithelium was minimally affected; occasional necrosis of individual cells and occasional cells containing a granular, light-brown pigment within the cytoplasm were observed in this epithelium. these alterations were primarily centered in the nasal septum. the transitional epithelium that extends between the squamous and respiratory epithelial regions exhibited minimal necrosis and epithelial hyperplasia. brown, cytoplasmic pigment, as described above, was also present within individual cells of this region. the severity of the lesions in the nonolfactory epithelium was exposure concentration-related, and both sexes and species were equally affected. however, the lesions were minimal in degree, even in the group exposed to the highest concentration of ferrocene (40 mg/m 3 ). the primary histopathological lesion for both rats and mice for both sexes and at all exposure levels was located in the olfactory epithelium (fig. 1) and was most severe in the dorsal meatus. this lesion was classified as subacute, necrotizing inflammation and encompassed the features described below. the olfactory epithelium was characterized by patchy areas of degeneration and by necrosis of neuroepithelial and sustentacular cells. many areas were markedly atrophic. inflammatory cells, predominantly neutrophils, were present; however, they were not a prominent component of the reaction. the relative pau-city of inflammatory cells that we observed is a common finding in lesions of the olfactory epithelium. the majority of the remaining epithelium was characterized by the presence of mitotic figures, rosette formation, and loss of basement membrane orientation of epithelial cells, all of which are indicative of marked regenerative attempts by the olfactory epithelium. many epithelial cells contained cytoplasmic, light-brown, granular pigment. bowman's glands were also characterized by patchy necrosis and atrophy. lesions were graded based on severity and distribution on a 1 to 5 scale, with 1 being minimal and 5 being severe. none of the animals had lesions that were considered to be more than moderate (grade 3) in degree. "minimal" lesions were those having rare individual cell necrosis and degeneration with subtle atrophy of the olfactory epithelium. "mild" lesions were characterized by patchy areas of degeneration and necrosis that involved less than 10% of the epithelium. "moderate" lesions were more extensive, involving up to approximately 25% of the epithelium; an inflammatory response was more prominent in lesions of this severity. the severity of the lesion in the olfactory epithelium was directly related to exposure dose. lesions were most severe in the groups exposed to the highest ferrocene concentration (40 mg/m 3 ) and were scored as moderate in degree. lesions of a similar magnitude were also observed in the groups exposed to 20 mg/ m 3 ferrocene. lesions, minimal to mild in degree, were seen in the groups exposed to 5 and 10 mg/m 3 ferrocene. lesions, minimal in degree, were seen in the group exposed to the lowest concentration of ferrocene, 2.5 mg/m 3 . we did not observe sex-related differences, and the severity of the lesion occurring after exposure to a given ferrocene concentration was about the same in rats and mice. no exposurerelated microscopic lesions were found in either livers or spleens. the in vitro studies of ferrocene metabolism showed that microsomes from the ethmoturbinates metabolized ferrocene more rapidly " values for v^ and k m and estimated 95% confidence intervals were taken from a plot of rate and concentrations of ferrocene derived by using 10 data points representing a range of ferrocene concentrations of 2 to 300 ^m. protein concentration was 0.5-1 mg microsomal protein, which was then normalized to a per gram tissue basis. than did those from the maxilloturbinates. also, microsomes from both nasal tissues had up to 10 times higher activities towards the ferrocene than did the liver microsomes (table 6). the localization of lesions in the olfactory mucosa is probably related to the high retention of ferrocene-derived iron in the nose (dahl and briner, 1980) , which, in turn, is probably a result of the high metabolic capacity in the nose, particularly in the olfactory mucosa. metabolism of ferrocene by cytochrome p450 probably proceeds according to the reactions shown in fig. 2 ; ferrocene is metabolized to unstable hydroxyferrocene (hanzlikeza/., 1978) , which then decomposes, releasing free ferrous ion intracellularly. the ferrous ion can then catalyze lipid peroxidation (dumelin and tappel, 1977) . this scenario could account for the lesions we observed. in addition to the high metabolic capacity of the olfactory epithelium, the greater sensitivity of this tissue to ferrocene toxicity as opposed to respiratory tract epithelium may also be due to a higher tissue dose and a lower rate of chemical clearance than respiratory epithelium. the olfactory epithelium lacks the ciliary clearance mechanism that is present in the respiratory epithelium. the olfactory epithelium also has fewer mucus-secreting cells and thus lacks the protective mucus layer that covers the respiratory epithelium. other inhalants that are metabolized to toxic products in the olfactory mucosa include dimethylnitrosamine (reznik-schiiller, 1983) , hexamethylphosphoramide (lee and trochimowicz, 1982) , and 3-trifluoromethylpyridine (gaskell el ai., 1988) . the feature that is unique for ferrocene is the suggestion that metabolism leads to free intracellular ferrous ion, which produces toxicity by catalytic peroxidation. experimental evidence for such a mechanism of toxicity for ferrocene has been provided by eidson et al. (1989) . other metabolically active sites, including the lung bronchioles, in which clara cells are located, and the liver, were not affected by inhaled ferrocene. this may reflect either less rapid ferrocene metabolism (as is the case for rat liver) or less penetration of ferrocene into those regions (dahl and briner, 1980) . exposure-related decreases in organ weights were seen for liver in rats, and in mice, liver, spleen, kidney, and brain weights also decreased, while thymus weights increased. while these results suggest that the named tissues may be target organs for toxicity resulting from long-term exposures to ferrocene, it is more likely that these changes were secondary effects brought on by the nasal lesions. however, in the case of liver, which, like olfactory epithelium, is highly active in xenobiotic metabolism, it is possible that primary toxic effects may result if ferrocene reaches this organ. also, the differences between the two species in the effects of ferrocene exposure on body and organ weights indicate that mice may be more susceptible to ferrocene toxicity than are rats, and that in longer-term exposure, female mice may be more sensitive to the toxic effects of ferrocene than are male mice. the results from these studies were used to select the exposure concentrations for a 13-week subchronic inhalation exposure study. these 13week subchronic inhalation exposures, using concentrations of 3.0, 10, and 30 mg/m 3 ferrocene, are currently in progress to determine the cumulative toxicity of ferrocene. threshold limit values and biological exposure indices for 1986-1987 biological fate of a representative lipophilic metal compound (ferrocene) deposited by inhalation in the respiratory tract of rats hydrocarbon gases produced during in vitro peroxidation of polyunsaturated fatty acids and decomposition of preformed hydroperoxides toxicity of ferrocene and vinylferrocene to rat nasal epithelium olfactory and hepatic changes following inhalation of 3-trifluoromethylpyridine in rats cytochrome p450 dependent monooxygenase activity in rat nasal epithelial membranes biological activity and disposition of the amphetamine analog 2-aminopropylferrocene the vapor pressure and enthalpy of sublimation of ferrocene metaplastic changes of nasal respiratory epithelium in rats exposed to hexamethylphosphoramide (hmpa) by inhalation protein measurement with folin phenol reagent nitrosamine-induced nasal cavity carcinogenesis chemicals in commerce information system (cicis), office of pesticides and toxic substances. chemical information division the authors are indebted to a number of individuals on the technical staff for their assistance on this project. this research was conducted in facilities fully accredited by the american association for accreditation of laboratory animal care. this research was sponsored by the national institute of environmental health sciences/national toxicology program under interagcncy agreement y01 -es-70157 and the office of health and environmental research of the u.s. department of energy under contract de-aco4-76ev01013. key: cord-104251-cq8ojfit authors: nan title: in vitro macrophage manifestation of cortisone-induced decrease in resistance to mouse hepatitis virus date: 1981-03-01 journal: j exp med doi: nan sha: doc_id: 104251 cord_uid: cq8ojfit genetically resistant g3h mice routinely yielded macrophages that were resistant when grown in 90% horse serum. these mice also routinely yielded macrophages that were susceptible to the same virus, mhv (pri), in vitro after the mice had been treated with three intraperitoneal doses, of hydrocortisone. dexamethasone and prednisolone when similarly administered also increased the susceptibility of c3h macrophages taken from the treated animal, but progesterone and testosterone did not. in addition, spleen cells from mice treated with cortisone made the resistant c3h macrophages 100 times more susceptible in vitro. increased in vitro susceptibility induced in this way by hydrocortisone was reversed by exposure to supernatant fluid removed from cultures of concanavalin a-treated spleen cells. effect of adding spleen cells from cortisone-treated mice. previous work from our laboratory has suggested that susceptibility of macrophages may be regulated by various lymphokines. therefore, cells from cortisone-treated mice were combined in vitro with normally resistant call macrophages and the susceptibility of these was tested. c3h mice were given 2.5 mg cortisone on day 0, and the spleens were removed 3 d later. 0.5 × 106 spleen cells were then added to each culture of normal c3h macrophages. control macrophage cultures received equal numbers of untreated spleen cells from syngeneic mice. various dilutions of virus [mhv(pri)] were inoculated into both groups of cultures. all cultures were kept for 6 d at 37°c. the results are summarized in table i . genetically resistant macrophages were destroyed by day 3 at 100-fold greater dilutions when they were cocultivated with spleen cells from cortisone-treated spleen cells but not the normal spleen cells. spleen cells that had been killed by heating to 56°c for 30 min or by freezing and thawing did not alter the resistance of call macrophages. direct addition of hc to peritoneal adherent cell populations. 10 table ii , however, shows that macrophages from hc-treated resistant mice (which had been treated three times) were 400-1,000 times more susceptible to the virus than those from untreated mice. when the mice were given only one injection of hc, the macrophages were not susceptible. viral growth was demonstrated primarily by cell destruction, which occurred as early as 2 d, but giant-cell formation also occurred which eventually ended in loss of contact with the culture tube. virus yield was determined in critical cases. the 1,000-fold increase in susceptibility of the macrophages was routinely observed if three doses of hc ranging from 50 #g to 5 mg were given to the mice, but when 10 mg was used, the cells were only 10-fold more susceptible. shif and bang (3) demonstrated that when high multiplicities of mhv(pri) were each group of csh mice (six per group) received three intraperitoneal doses (1 mg each) of the steroid after thioglycollate stimulation, and pec were harvested 24 h after the last dose. * all macrophages in the experiment were seeded in 16-mm 24-well tissue culture cluster plates and incubated at 37°c with 5% cc.~z and humidified air, given to c3h macrophages, a mutant virus, mhv(c3h), emerged that grew to high titer in both resistant and susceptible macrophages. following the above findings, macrophage cultures from hc-treated mice receiving 104 lds0 of mhv(pri), and which were destroyed by the virus, were assayed for virus yield on czh and c3hss macrophages on days 5 and 7 postinfection. virus harvested from macrophages of hc-treated mice on day 5 postinfection titered 10,000 times higher in cahss macrophages than in cnh macrophages. by day 7 postinfection, the difference had decreased to 2 logs, presumably because of late growth of the mutant. because of this, most of the subsequent experiments were limited to observations within 5 d of inoculation. the effect of cortisone is apparently glucoeorticoid-specific. prednisolone and dexamethasone were administered in the same way as hc: three successive injections, intraperitoneally, in vivo. two steroids (progesterone and testosterone) were similarly administered. the results showed that the active glucocorticoids increased susceptibility of the c3h macrophages, whereas the other steroids did not (table iii) . growth curve of mhv(pri) in macrophages from csi-iss, call, and hc-treated mice. virus (1.5 × 104 tissue culture infective doses/2 × 106 cells) was adsorbed for 60 min at 37°c in a roller drum. the fluid was then replaced by culture medium. at various intervals, individual tubes were harvested, frozen, then titered for total cellular and supernatant virus. as can be seen from fig. 1 , there was a sharp increase in virus titer in czhss macrophages during the first 10 h. there was no rapid initial rise in virus titer in either normal or hc-treated c3h macrophages. however, after about 11 h, the growth rate in the macrophages from hc-treated mice increased rapidly. at 21 h postinfection, virus replication in czh resistant macrophages started to decelerate, whereas in macrophages from hc-treated czh mice, it continued to increase. although virus growth in the latter attained almost the same final yield as in c~hss, it required ~20 h longer for this to occur. in vivo, however, c3h mice challenged with virus 3 d after cortisone (2.5 mg s.c.) die in just as short, or shorter, time period than susceptible czhss mice (mean survival time: 1.9 d for c3h; 2.5 d for c3hss). earlier, willenborg (7) had found that c3h mice challenged with virus 1 h after cortisone administration take longer to die than genetically susceptible pri mice. evidently, the time of cortisone administration may be a factor in the induction of susceptibility to the virus. it has been previously shown in our laboratory that genetically susceptible mice, as well as their macrophages, gain resistance when the mice are treated with con a, or when the macrophages are treated with the supernate from con a-treated spleen cells (4) . it was therefore of interest to determine whether con a would be antagonistic to cortisone. therefore, 0.1 ml of supernate fluid from con a-treated spleen cells (4) and also from normal spleen cells was added to macrophages from hc-treated mice and incubated for 14 h before inoculation of virus. table iv shows the complete reversal of the hc effect by supernatant fluid from con a-treated spleen cells but only a partial reversal by supernate fluid from normal spleen ceils. the addition of normal spleen cells did not make the macrophages from cortisonetreated mice resistant again. 0.5 × 106 spleen cells/ml, harvested from normal call mice, were added to macrophages from hc-treated mice and incubated for 24 h before viral infection. there was no significant difference between macrophages that received the spleen cells and those that did not (table iv) . although it has been known almost from the start of research on cortisone that this powerful anti-inflammatory drug depresses various immunologic mechanisms (1), the way in which it alters susceptibility to specific agents is not clear. the correspondence between in vivo and in vitro susceptibility of macrophages to mouse hepatitis made this model an attractive one to study. cortisone-induced increase in susceptibility of mice to mouse hepatitis virus has also been observed by a number of other investigators as summarized by vella and starr (8) . administration of cortisone to the genetically resistant czi-i mice (9) , as well as to outbred mice, rendered them susceptible to viral [mhv(pri)] infection. the lds0 of mhv(pri) virus was 108.5 in pri mice, 106.5 in cortisone-treated call mice, and <101'° in untreated call mice (9) . the slight or equivocal change in macrophage resistance, which had been demonstrated in vitro (7, 9) , was in no way comparable to the marked effects on the intact animal. gallily et al. (9) , using a 1:100 dilution of stock virus (106 lds0), had found that 85-100% of taylor et al. brief definitive report cortisone-treated cultures were killed by virus as compared to -20% of cultures given virus alone. willenborg (7), using a single dose of 2.5 mg cortisone, had found that the lds0 of mhv(pri) virus in macrophage cultures from cortisone-treated call mice was 103.4 , and in cultures from untreated mice, it was 103.2 . in our studies, it was apparently crucial to administer three doses of hc to the mice to achieve sufficient suppression of viral resistance for in vitro expression of the effect. presumably, a 1,000fold increase of susceptibility of macrophages may explain most, if not all, of the 105.5 difference in titer in normal and cortisone-treated mice. the mechanism whereby hc or cortisone changes the resistance of the all-important macrophage remains unclear. addition of spleen cells from cortisone-treated mice enhanced the susceptibility of call macrophages 100-fold. furthermore, cultures from hc-treated mice remained susceptible up to 72 h in culture, but fully regained resistance by 1 wk. the implication is that resistance of c3h macrophages is altered by some product of lymphoid cell activity induced by the steroid. that two other steroids, testosterone and progesterone, failed to cause the development of susceptible macrophages, whereas dexamethasone and prednisolone did, again suggests a correspondence of the in vitro results with the susceptibility of the whole animal. the addition of whole spleen cells from normal resistant mice to macrophages from hc-treated mice for a period of 24 h before viral infection did not restore resistance. however, adding 0.1 ml ofsupernate fluid from con a-stimulated spleen cells reversed the hc effect. this suggests that resistance is dependent, in part at least, upon a lymphokine released from t cells. because con a stimulates a variety of lymphokines, such as migration-inhibitory factor, interferon, soluble immune response suppressor, and t cell growth factor, it will be necessary to identify which specific lymphokine is responsible for the effect. the growth-curve experiment reveals that during the first 12 h of observation, virus replication was equal in both sets of czh macrophages, but that after this period, virus growth started to decelerate in the call control macrophages, although it continued to increase in macrophages from hc-treated mice. this suggests that resistance might be a result of the capacity of the call macrophage to produce an anti-viral substance during early growth which restricts viral growth. the work of gillis et al. (10) on how cortisone is able to suppress immune responses begins to explain the well known ability of cortisone to suppress antibody and t cell response. suppression of the production of a t cell growth factor by this corticosteroid is then followed by a failure of the t cell compartment to expand after either specific antigenic stimulus or administration of con a. that con a and cortisone act antagonistically in their studies (10) is very relevant to the similar blocking effect of one by the other in our system. however, because our studies seem to demonstrate that the cortisone-treated cells cause a change in the macrophage rather than prevent the release of a factor from lymphocytes, the analogy needs to be explored further. genetically resistant call mice routinely yielded macrophages that were resistant when grown in 90% horse serum. these mice also routinely yielded macrophages that were susceptible to the same virus, mhv(pri), in vitro after the mice had been treated with three intraperitoneal doses of hydrocortisone. dexamethasone and prednisolone when similarly administered also increased the susceptibility of csh macrophages taken from the treated animal, hut progesterone and testosterone did not. in addition, spleen cells from mice treated with cortisone made the resistant call macrophages 100 times more susceptible in vitro. increased in vitro susceptibility induced in this way by hydrocortisone was reversed by exposure to supernatant fluid removed from cultures of concanavalin a-treated spleen cells. received for publication 10 november 1980. mechanisms of glucocorticoid action on immune processes congenic strains of mice susceptible and resistant to mouse hepatitis virus in vitro interaction of mouse hepatitis virus and macrophages from genetically resistant mice. ii. biological characterization of a variant virus mhv(c3h) isolated from stocks of mhv(pri) blocking of in vitro and in vivo susceptibility to mouse hepatitis virus biochemical actions of glucocorticoids on macrophages in culture. specific inhibition of elastase, collagenase, and plasminogen activator secretion and effects on other metabolic functions a simple method of estimating fifty percent end-points the effect of chemical and physical agents on the genetic resistance of mice to mouse hepatitis virus mhv(pri) effect of x-irradiation and cortisone on mouse hepatitis virus infection in germ-free mice effect of cortisone on genetic resistance to mouse hepatitis virus in vivo and in vitro glucocorticoid-induced inhibition of t cell growth factor production. i. the effect on mitogen-induced lymphocyte proliferation key: cord-306535-j26eqmxt authors: robertson, matthew j.; kent, katarzyna; tharp, nathan; nozawa, kaori; dean, laura; mathew, michelle; grimm, sandra l.; yu, zhifeng; légaré, christine; fujihara, yoshitaka; ikawa, masahito; sullivan, robert; coarfa, cristian; matzuk, martin m.; garcia, thomas x. title: large-scale discovery of male reproductive tract-specific genes through analysis of rna-seq datasets date: 2020-08-19 journal: bmc biol doi: 10.1186/s12915-020-00826-z sha: doc_id: 306535 cord_uid: j26eqmxt background: the development of a safe, effective, reversible, non-hormonal contraceptive method for men has been an ongoing effort for the past few decades. however, despite significant progress on elucidating the function of key proteins involved in reproduction, understanding male reproductive physiology is limited by incomplete information on the genes expressed in reproductive tissues, and no contraceptive targets have so far reached clinical trials. to advance product development, further identification of novel reproductive tract-specific genes leading to potentially druggable protein targets is imperative. results: in this study, we expand on previous single tissue, single species studies by integrating analysis of publicly available human and mouse rna-seq datasets whose initial published purpose was not focused on identifying male reproductive tract-specific targets. we also incorporate analysis of additional newly acquired human and mouse testis and epididymis samples to increase the number of targets identified. we detected a combined total of 1178 genes for which no previous evidence of male reproductive tract-specific expression was annotated, many of which are potentially druggable targets. through rt-pcr, we confirmed the reproductive tract-specific expression of 51 novel orthologous human and mouse genes without a reported mouse model. of these, we ablated four epididymis-specific genes (spint3, spint4, spint5, and ces5a) and two testis-specific genes (pp2d1 and saxo1) in individual or double knockout mice generated through the crispr/cas9 system. our results validate a functional requirement for spint4/5 and ces5a in male mouse fertility, while demonstrating that spint3, pp2d1, and saxo1 are each individually dispensable for male mouse fertility. conclusions: our work provides a plethora of novel testisand epididymis-specific genes and elucidates the functional requirement of several of these genes, which is essential towards understanding the etiology of male infertility and the development of male contraceptives. the world human population reached nearly eight billion people in august 2019. this number continues to rise and is predicted to reach nearly ten billion by the year 2050 [1] . the increasing need to promote family planning through the development of reliable contraceptive options available to both men and women is widely recognized. currently there are numerous contraceptive options available to women; however, identification of a safe, non-hormonal contraceptive option for men is still an ongoing challenge. although several different fertility control alternatives for men have been investigated, none are currently clinically approved for use. our understanding of the mechanisms underlying male reproductive physiology is still at an early stage as the identification and elucidation of the function of key reproductive proteins is still an ongoing effort. identifying druggable protein targets expressed in the male reproductive tract has been the focus of numerous studies dedicated to the development of male contraception. the mammalian epididymis is a segmented organ comprised of a single, highly coiled tubule with functionally and morphologically distinct regions that can be subdivided most simplistically into a proximal, central, and distal region, conventionally named the caput, corpus, and cauda regions, respectively [2] . as mammalian spermatozoa transit through the epididymis, they acquire the ability to recognize and fertilize an egg, properties that they did not possess upon exiting the testis [3] . considering its essential role, the epididymis-in addition to maturing germ cells of the testis and spermatozoa-is a prime target for the development of a male contraceptive. to advance progress towards the development of a non-hormonal male contraceptive, several previous high-throughput studies have been published that identified a number of human, mouse, and rat genes as testis-specific or epididymis-specific [2, [4] [5] [6] [7] [8] [9] . in 2003, schultz et al. conducted the first study to identify male reproductive tract-specific genes using microarrays. through affymetrix-based genome-wide geneexpression analysis of meiotic-and post-meiotic spermatogenic cells, together with parallel analysis of available data from the ncbi unigene database, the authors identified 271 mouse genes as testis-specific, which included genes with both known and unknown function at the time [4] . in the following 5 years, through two additional microarray-based studies of rat testes and purified rat testicular cells, johnston et al. identified 58 [5] and 398 [8] additional or overlapping genes as testisspecific. in 2014, as part of the continued effort to identify novel contraceptive targets, the newer rna-seqbased transcriptomics methodology was utilized identifying 364 human genes as testis-specific [9] . together with antibody-based protein profiling, many of these genes were characterized in terms of the spermatogenic cell populations showing expression [9] . the first high-throughput transcriptomics study to identify epididymis-specific genes was a 2005 mouse epididymal transcriptome study, in which rna isolated from each of the 10 epididymal segments was analyzed by microarray analysis, identifying 75 epididymis-specific genes with distinct patterns of segmental gene regulation [2] . later in 2007, additional transcriptome profiling utilizing whole genome microarrays resulted in identification of 77 previously unreported epididymis-specific transcripts in the mouse [6] and 110 epididymis-specific transcripts in the rat [7] . a significant number of the identified mouse and rat genes in these studies were not known at the time, and only the probe identification numbers were presented. when evaluating potential druggability in a targetbased drug discovery process, one must consider the protein properties that are required for safe and effective inhibition. among the most significant is tissue expression specificity to minimize potential adverse effects, protein function and whether protein activity or interaction with other proteins is potentially druggable, sequence similarity to closely related paralogs that may be ubiquitously expressed, and whether genetically manipulated animal models demonstrate a functional requirement for the target of interest [10] . several noteworthy review publications have mentioned numerous genes whose critical functions, high expression, and specificity to the testes or epididymides make them viable nonhormonal male contraceptive targets [11] [12] [13] [14] [15] [16] [17] [18] . however, among the identified genes, a significant number either (1) are required for fertility, but are expressed in nonreproductive tissues, or (2) are reproductive tractspecific, but, when disrupted, lead to subfertility [10] . in either case, both are ineffective and highly undesirable outcomes for a potential male contraceptive target. therefore, the identification of additional novel male reproductive tract-specific genes would allow for further advances to be made in the quest to develop an effective and safe non-hormonal male contraceptive. in this study, 21 newly acquired and 243 previously published human and mouse rna-seq datasets [9, [19] [20] [21] [22] [23] [24] [25] [26] were processed in parallel through a custom bioinformatics pipeline designed to identify novel reproductive tractspecific and reproductive tract-enriched transcripts. additional databases obtained from illuminating the druggable genome [27] , mouse genome informatics [28] , and ensembl biomart [29] were utilized to stratify the results into subgroups based on protein druggability and on the availability of a mouse model. numerous reproductive tract-specific and reproductive tract-enriched, potentially druggable targets for which no published mouse model exists, congruent in expression across both mouse and human datasets were identified through our analysis and verified through conventional polymerase chain reaction (pcr). we present the data in a manner that should be most relevant and of substantial interest to the male contraceptive development field since identification of new targets worthy of consideration for further functional analysis in a knockout animal model and potential drug targeting continues to be of vast importance. through our results, we identified four novel epididymis-specific genes (spint3, spint4, spint5, and ces5a) and two novel testis-specific genes (pp2d1 and saxo1) worthy of functional validation in an animal model. through the crispr/cas9 system, we generated four individual gene knockouts (spint3, ces5a, pp2d1, and saxo1) and one double knockout mouse model (spint4/5) revealing an essential requirement for spint4 and spint5 in male mouse fertility, and the potential utility of pursuing spint4 in humans as a non-hormonal contraceptive target. despite significant advances in our understanding of the human and rodent testis and epididymis transcriptome, mostly through microarray-based studies, no prior studies have utilized purified human testis cells for the identification of human testis-specific transcripts, no prior studies have utilized the more state-of-the-art rna-seqbased transcriptomics methodology for analysis of human epididymis-specific transcripts, and no prior studies have utilized rna-seq analysis of rodent reproductive tissues or cells to identify rodent reproductive tractspecific transcripts. to address these gaps in knowledge, and to increase the number of identified reproductive tract-specific genes in both species using the most relevant high-throughput transcriptomics methodology, we analyzed in parallel on a custom bioinformatics pipeline a large number of published and newly acquired human and mouse rna-seq datasets. one hundred and sixtytwo previously published human and 81 previously published mouse rna-seq datasets were retrieved from the sequence read archive (sra). the sra value for each sample is listed in additional file 1: table s1 and additional file 1: table s2 . we also generated 12 new human and 9 new mouse reproductive tissue rna-seq samples (geo accession gse150854). the final dataset is comprised of 3 new and 5 previously published human testis datasets [9] , 27 previously published purified human germ cell datasets [23, 24] , 6 previously published purified human sertoli cell datasets [23, 26] , 9 new and 6 previously published human epididymis segment datasets [21] , 6 previously published mouse testis datasets [19] , 9 new mouse epididymis datasets, 10 previously published purified mouse germ cell datasets [22, 25] , and 3 previously published purified mouse sertoli cell datasets [20] . an additional 118 previously published datasets contributed to the 26 non-reproductive human tissues [30] and 62 previously published datasets contributed to the 14 non-reproductive mouse tissues [19] . figure 1a , b summarizes all the samples acquired for the study. we performed a principal component analysis to visualize the variation in the samples after correcting for batch effects. human reproductive and non-reproductive tissues grouped according to sample type. the reproductive tissue samples clustered by tissue type whether or not they were newly generated or acquired from the sra (fig. 1c) . mouse data showed a similar variation in the samples based on the tissue type (fig. 1d) . for both human and mouse reproductive tissues, samples separated by whether or not the rna-seq was performed on isolated cells or the whole tissue. epididymal tissue was distinct from testis tissue in both human and mouse (fig. 1c, d) . to identify potential male reproductive tract-specific drug candidates, we analyzed the aggregated rna-seq data to find genes that were statistically significant in expression when compared to the non-reproductive tissue that had the maximum expression for that gene. this gene list was then further refined by filtering for genes that were lowly expressed in the non-reproductive tissue that had the maximum expression for that gene (tpm less than or equal to 1.0 for human; tpm less than or equal to 2.0 for mouse). finally, this tpm filtered list was then filtered for the genes that had a reproductive tissue or cell expression value greater than or equal to 10.0 tpm for human, or 8.0 tpm for mouse (fig. 2a) . across all the reproductive tissues, 720 candidate genes were identified in the human and 1304 candidate genes were identified in the mouse samples (fig. 2b , additional file 2: fig. s1 ). additional file 3: table s3 and additional file 4: table s4 summarize the differential fold change, identity of the non-reproductive tissue with maximal gene expression based on the differential gene analysis, fdr, average and standard deviation tpm expression values, and log2 cpm gene expression value for the human and mouse samples, respectively. the results from the fdr and tpm expression value filtering for the human and mouse samples are summarized in additional file 5: table s5 and additional file 6: table s6 , respectively. additional file 5: table s5 and additional file 6: table s6 report the log2 fold change for the reproductive tissue or cell of interest compared to the tissue with maximal gene expression. the genes identified in additional file 5: table s5 and additional file 6: table s6 pass the filters in at least one of the reproductive tissues or cells of interest. in additional file 5: table s5 and additional file 6: table s6 , a value of zero for a given gene and fold expression comparison indicates that for that comparison, the gene did not pass the filters. the majority of genes were downregulated in the reproductive tissue of interest compared to the maximal gene expressing nonreproductive tissue (additional file 7: fig. s2 ). from the analysis, the majority of the candidate genes that passed the fdr and tpm filters were identified in the testis-or sperm-related cells in both human and mouse samples (additional file 7: fig. s2 ). the majority of candidate genes identified in our screen that were testis-specific were already identified by the human protein atlas [9] and/or our reanalysis of (see figure on previous page.) fig. 1 summary of the human and mouse rna-seq samples used in the identification of novel male reproductive tract-specific drug targets. the rna-seq samples used in the human (a) and mouse (b) analyses are schematically shown. principal component analysis was performed on the human (c) and mouse (d) non-reproductive and reproductive samples separately. the colors of the circles next to the tissues listed in a and b correspond to the colors used in the circles for the pca in c and d. sample size (n) values in red and/or black denote the number of new (red) and previously published (black) samples included in our analysis. fig. 2 identification of candidate drug male reproductive gene targets. a diagrammatic representation of overall methodology used to identify reproductive tract-specific candidate genes in humans (720 genes) and in mice (1062 genes). the maximum gene expression was determined across all the non-reproductive tissue samples for each gene for a reproductive tissue or cell sample of interest. genes were then filtered for significance using a false discovery rate (fdr) of less than or equal to 0.05 based on the differential gene expression analysis for the nonreproductive tissue with maximum gene expression and reproductive tissue or cell sample of interest. genes that passed the fdr filter were filtered such that the average tpm expression value of the maximum expressing non-reproductive tissue was less than or equal to 1.0 tpm and the average tpm expression value of the reproductive tissue or cell of interest was greater than or equal to 10.0 tpm. b diagrammatic representation of the number of human and mouse candidate genes in terms of (1) the number of orthologs in the opposite species, (2) the number of genes previously or not previously identified in a prior transcriptomics-based drug target report, (3) the availability and phenotypic outcome of any reported mouse models, and (4) the number of novel genes without a reported mouse model congruent across both species. the main value in each bubble represents the total number of candidate genes identified regardless of tissue or cell identified in. the numbers in parentheses comprise the total number of candidate genes that are either epididymis-specific or specific to testis and epididymis, but not testis and/or testis cell-specific only. the hpa testis datasets (additional file 8: fig. s3 and additional file 9: table s7 ). thirty-six out of the 91 genes that were identified across all the human epididymis tissue were also identified by the human combined (newly acquired and previously published datasets) testis candidate gene list. finally, the majority of the candidate genes, 300, identified from the combined newly generated and previously published human testis datasets were shared with genes identified from the various testis cell datasets. we identified more candidate genes in the newly generated human epididymis tissues compared to previously published data: 19 out of 54 genes were unique to the newly generated caput samples compared to only 1 out of 36 genes which was unique to the previously published samples, 19 out of 75 genes were unique in the newly generated corpus samples compared to 12 out of 68 genes which were unique to the previously published corpus samples, and 33 genes were unique to the newly generated cauda samples compared to 2 genes in the previously published cauda data with no overlap between the two cauda gene lists (additional file 8: fig. s3 and additional file 9: table s7 ). there were 117 candidate genes that overlapped between the newly generated human testis samples and mouse testis sample gene lists, while there were 134 candidate genes that overlapped between the previously published human testis sample and mouse testis sample gene lists (additional file 8: fig. s3 and additional file 9: table s7 ). across all human epididymis tissue samples, including the newly generated and previously published samples, there were 16 genes in common with the combined list of candidate genes across all the mouse epididymis tissue samples. there was a small overlap between the human and mouse samples when the newly generated human caput, corpus, and cauda tissues were individually compared to the mouse caput, corpus, and cauda tissues; there was an overlap of 10, 12, and 4 for the caput, corpus, and cauda, respectively (additional file 8: fig. s3 and additional file 9: table s7 ). this trend was continued for the candidate gene lists derived from the previously published human caput, corpus, and cauda samples when compared to the candidate gene list from the mouse caput, corpus, and cauda, with 7, 10, and 4 genes in common for the caput, corpus, and cauda comparisons, respectively (additional file 8: fig. s3 and additional file 9: table s7 ). additional file 9: table s7 details the genes that are unique and in common for each of the comparisons. to assess the potential usefulness of the candidate genes identified in each human reproductive tissue as drug targets, we assigned the genes to a protein family (i.e., gpcr or ion channel). the majority of identified genes were not from a traditional drug target family like kinases or enzymes. the testis and germ cell datasets provided the most potential targets while the epididymis datasets provided the fewest (additional file 10: fig. s4a ). the protein family classification for each candidate gene identified in each reproductive tissue is detailed in additional file 11: table s8 . the majority of the candidate genes do not have a reported mouse model (additional file 10: fig. s4b ). additional file 12: table s9 summarizes mouse model availability for each candidate gene identified from human reproductive tissues or cells. figure 3 shows the complete list of novel human genes without a reported mouse model as identified in each of the respective cell and/or tissue datasets. digital pcrs (heatmap) and conventional pcrs demonstrating expression of a subset of the novel human reproductive tract-specific genes without a reported mouse model that we identified are shown in figs. 4 and 5, respectively. additional file 13: fig. s5 shows the complete list of previously identified human genes that remain without a reported mouse model as identified in each of the respective cell and/or tissue datasets. additional file 14: fig. s6 shows the complete list of male reproductive tract-specific human genes for which a previously generated mouse model shows male infertility phenotype, as identified in each of the respective cell and/or tissue datasets. through our bioinformatics analysis of previously published and newly acquired rna-seq datasets, we identified a total of 720 genes as reproductive tract-specific in humans (fig. 2 ). of these genes, 122 genes do not have a mouse gene ortholog, while 598 genes have a mouse gene ortholog (fig. 2) . of those with a mouse gene ortholog, 477 have a single gene ortholog (324 have the same symbol in mouse, while 153 have a different symbol in mouse), while 121 have two or more orthologous mouse genes. seventy-six human genes had 2-3 orthologous mouse symbols, 36 genes had 4-10 orthologous mouse symbols, and 9 genes (fam205a, krta p10-6, magea10, or2ag1, pramef11, pramef2, ssx2, ssx3, and ssx4b) had greater than 10 orthologous mouse symbols (11-93 symbols) (additional file 5: table s5 ). of the 720 human genes that we identified as male reproductive tract-specific, 435 have not been previously identified in a transcriptomics-based male reproductive tract-specific study [2, [4] [5] [6] [7] [8] [9] . the sum of our human data confirms the findings of 232 out of 364 genes from djureinovic et al. [9] . after re-identification of gene symbols from reported affymetrix ids and consideration of orthologous genes (mouse to human and rat to human), our human data confirm the findings of 19 out of 39 genes from johnston et al. [5] , 77 out of 176 genes from schultz et al. [4] , 5 out of 32 genes from johnston et al. [6] , 36 out of 253 genes from johnston et al. [2] , 4 out of 58 genes from johnston et al. [8] , and 3 out of 19 genes from jelinsky et al. [7] . of the 598 genes that have a mouse gene ortholog, 346 have not been previously identified as male reproductive tractspecific, and of these, 233 human genes currently lack mouse phenotype information based on data obtained from ensembl biomart, mgi, impc, and ncbi. three hundred and eighty-six genes were identified as testis-specific through either the reanalysis of djureinovic et al. testis datasets (377 genes identified), analysis of our de novo testis datasets (322 genes identified), or both (additional file 5: table s5 ). three hundred and thirteen genes were congruent across both datasets, while 64 genes were uniquely identified through our reanalysis of djureinovic et al.'s datasets and only 9 genes [ac136352.4, ankrd20a1, ankrd62, fam230a, ggtlc2, iqcm, potec, prnt, and utf1] were uniquely identified through our de novo datasets (additional file 5: table s5 ). interestingly, of the 377 genes we identified through djureinovic et al.'s reanalyzed datasets, 143 were not previously identified in their report [9] or any of the other previous reports [2, [4] [5] [6] [7] [8] . of these 143 genes, we randomly verified 21 of these genes as testis-specific in humans through conventional pcr (fig. 5) . we also verified through rt-pcr an additional 15 genes-such as allc, cdkl3, cox7b2, or2h1, and sppl2c-that had been identified through previous studies (additional file 15: fig. s7 ). of the 386 genes identified through either testis datasets, 150 have not been previously identified; of these, 117 genes have one or more mouse orthologs; and of these, 76 genes are lacking reported phenotype information. of the 76 novel genes lacking a reported mouse model, 7 genes encode enzymes (adam20, cpa5, dusp21, naa11, plscr2, prss38, and triml1), 6 encode transcription factors (bhmg1, foxr2, prdm9, tgif2ly, znf560, znf729), 2 encode transporters (slc22a14, slc25a52), and 61 encode proteins of unknown drug target type two hundred and thirty-three novel human reproductive tract-specific genes that each have mouse orthologous genes but with no reported knockout mouse models. the listed genes were identified in one or more datasets as indicated in the venn diagram. underlined genes were also identified in our studies as reproductive tract-specific in mouse (109 genes). genes written in blue encode either enzymes, kinases, gpcrs, ogpcrs, transporters, transcription factors, or proteins involved in epigenetic regulation (74 genes) . genes written in dark red were identified in both testis (testis and/or testis cell) and epididymis (10 genes). (such as etdb, smim36, bend2, btg4, cnbd1, dppa2, efcab5, erich6, fthl17, iqcm, mroh2b, ms4a5, oosp2, pnma6e, ppp4r3c, rbmxl3, rtl9, spdye4, spem2). all of these genes are listed in fig. 3 , and many of these genes are listed in figs. 4, 5, and/or 6. to the best of our knowledge, no prior studies have utilized purified human testis cells for the identification of human testis-specific transcripts. through our analysis, we identified 291 genes as human testis-specific through one or more of the human germ cell datasets, but not through either of the human testis datasets (additional file 5: table s5 ). seventy-six genes were identified exclusively through one or more of the five human spermatogonia datasets (genes such as anp32d, c13orf42, dscr4, or13g1, or2d2, or52e4, ssx2, tle7), while 18 genes were identified exclusively through the human spermatocyte datasets (genes such as h2bfm, mageb17, mageb18, or2b6, tcp10, and znf709) and 79 genes were identified exclusively through the human spermatid datasets (genes such as ac013269.1, clec20a, or7e24, pramef2, spat a31a3, tmem191c, znf679). thirty-four genes were identified through all three cell types' datasets (genes such as ccdc166, eloa2, fam47a, heat r9, and spata31a1). many of these genes are listed in figs. 3, 4, 5, and/or 6. of the 291 genes identified as human testis-specific through one or more of the human germ cell datasets, 252 genes have not been previously identified, 201 of which have one or more equivalent mouse orthologs with 139 of these genes having not been knocked out in mouse. of these 139 novel genes with no mouse model, 8 encode enzymes (glt6d1, prss48, satl1, sult6b1, tmprss7, tpte2, triml2, and ttll8), 1 encodes an epigenetic protein (taf1l), 6 encode gpcrs (gpr156, tas2r13, tas2r30, tas2r46, tas2r50, vn1r2), 1 encodes a kinase (cdkl4), 33 encode ogpcrs (such as or2d3, or3a2, or52e5, or8g5, or10j1, and ) and obp2b met candidate threshold through our analysis of human testes datasets but did not meet candidate threshold from any of the germ cell or sertoli cell datasets, indicating potential expression in peritubular myoid cells, leydig cells, or other cell outside of the seminiferous epithelium. fam36a has not been previously identified, and neither mouse orthologs (1700011m02rik, gm9112) have been knocked out. obp2b was previously identified through djureinovic et al. [9] and johnston et al. [6] ; however, of the equivalent mouse orthologs (lcn4, obp2a, obp2b), only obp2a has been knocked out revealing abnormal coat/hair pigmentation [31] . ism2 and magec2 were identified through both human sertoli cell datasets, while also identified through testis and/or germ cell datasets. both genes have been previously identified (ism2 [8] , magec2 [9] ). ism2 knockout mice display non-reproductive phenotypes [9] . consistent with this finding, our mouse data do not identify ism2 as reproductive tract-specific in mice. magec2 lacks a mouse ortholog for functional analysis in mice. human sertoli cell-specific krtap2-3, krtap4-12, lhx9, and psg5 were identified through one or both human sertoli cell datasets but were not identified through any of the testis or germ cell three hundred and two novel mouse genes with human orthologs without a reported mouse model. the listed genes were identified in one or more mouse datasets as indicated in the venn diagram. underlined genes were also identified through our studies as reproductive tractspecific in human (111 genes). genes written in blue encode either enzymes, kinases, gpcrs, ogpcrs, transporters, transcription factors, or proteins involved in epigenetic regulation (60 genes). genes written in dark red were identified in both testis (testis and/or testis cell) and epididymis (14 genes). datasets indicating sertoli cell-specific expression in the testes (additional file 5: table s5 ). none of these genes have been previously identified as reproductive tractspecific in humans although lhx9 and psg5 have mouse orthologs that have been knocked out [32] [33] [34] [35] [36] [37] . human krtap2-3 has mouse orthologs krtap5-2, gm4559, gm40460, and gm45618, and human krta p4-12 has mouse orthologs krtap4-7 and gm11555; none of these mouse orthologs have been knocked out ( fig. 3 and additional file 5: table s5 ). psg5 knockout mice display non-reproductive phenotypes [32] [33] [34] [35] [36] ; however, lhx9 knockout mice display absent testes and sterility due to an essential requirement for lhx9 during mouse gonad formation [37] . a lhx9-gfpcreer knock-in mouse line-generated by knocking-in gfpcreer at the endogenous lhx9 locuscrossed with the rosa26-tdtomato reporter mouse line revealed cre recombinase activity in retinal amacrine cells, developing limbs, testis, hippocampal neurons, thalamic neurons, and cerebellar neurons [38] . thus, lhx9 is not reproductive tract-specific in mice. our mouse data confirm this finding. to the best of our knowledge, no prior studies have utilized rna-seq for analysis of human epididymis-specific transcripts. through our studies, we identified 39 genes as human epididymis-specific through one or more of the human epididymis segment datasets that were not identified through any of the other human male reproductive tissue or cell datasets, indicating true epididymis specificity (additional file 5: table s5 ). of these 39 genes identified as human epididymis-specific, 29 genes have not been previously identified, 24 of which have equivalent mouse orthologs with 16 of these genes having not been knocked out in mouse. of these 16 novel human epididymis-specific genes with no mouse model, 1 encodes an enzyme-related gene (spint3) and the remaining 15 encode proteins of unknown drug target type (such as actbl2, bsph1, mslnl, spag11a, spag11b, wfdc10a, and wfdc9) (fig. 3) . seven genes were identified through our de novo sequenced human epididymis segment datasets that were not identified through our reanalysis of the human epididymis segment datasets by browne et al.; two of these genes are considered novel without mouse models: defb104a and defb104b (fig. 3 , additional file 5: table s5 , and additional file 9: table s7 ). meanwhile, five genes were identified through our reanalysis of the human epididymis segment datasets by browne et al. that were not identified through our de novo-sequenced human epididymis segment datasets; two of these genes are considered novel without mouse models: actbl2 and mslnl (fig. 3 , additional file 5: table s5 , and additional file 9: table s7 ). fifty-two genes met the criteria for identification as epididymis-specific through one or more of the human epididymis segment datasets, while also being identified as reproductive tract-specific through one or more of the testes, germ cell, and/or sertoli cell datasets (additional file 5: table s5 ). thus, these targets are not epididymis-specific per se, but may be desirable potential male contraceptive targets considering their broader target availability. of these 52 genes identified as human male reproductive tract-specific and epididymisexpressed, 20 genes have not been previously identified, 15 of which have one or more equivalent mouse orthologs with 11 of these genes having not been knocked out in mouse. all 11 of these novel genes with no mouse model encode proteins of unknown drug target type (al163195.3, ccdc168, ccnb3, defb121, defb134, eppin-wfdc6, krtap2-3, magea11, pnma6e, spem2, tex44) ( fig. 3 and additional file 5: table s5 ). since model organisms other than mice may be of interest for the future functional study of human genes-especially those for which no known mouse ortholog exists-we list novel reproductive tract-specific human genes without a mouse ortholog in additional file 16: fig. s8 , which may be of interest for generating null rat or marmoset models [39] . digital pcr (heatmap) demonstrating expression of a subset of these novel human reproductive tract-specific genes without mouse orthologs is shown in additional file 17: fig. s9 . through our bioinformatics analysis of previously published and newly acquired mouse rna-seq datasets, we identified a total of 1062 genes as reproductive tractspecific in mice. of these genes, 303 genes do not have a human gene ortholog, while 759 genes do (fig. 2) . of those with a human gene ortholog, 632 have a single ortholog (451 with the same gene symbol in human; 181 with a different symbol), while 127 mouse genes have two or more ortholog human genes (fig. 2 ). ninety-two mouse genes have 2-3 orthologous human symbols, 16 genes have 4-10 orthologous human symbols, and 19 genes (such as 1700080o16rik, ankrd36, fam90a1b, gm15319, magea10, pramel1, spdye4a, spdye4b, and zfy1) have greater than 10 orthologous human symbols ranging anywhere from twelve to twenty-six symbols (additional file 6: table s6 ). of the 1062 mouse genes that we identified as male reproductive tract-specific in mouse, 743 have not been identified in a previous transcriptomics-based study [2, [4] [5] [6] [7] [8] [9] . the sum of our mouse data confirms the findings of 150 out of 271 mouse genes from schultz et al. [4] , 7 out of 54 mouse genes from johnston et al. [2] , and 6 out of 23 mouse genes from johnston et al. [6] (additional file 18: table s10 ). of the 759 mouse genes that have a human ortholog equivalent, 482 have not been previously identified as male reproductive tract-specific, and of these, 302 genes currently lack mouse phenotype information based on data obtained from ensembl biomart, mgi, impc, and ncbi (fig. 6 ). digital pcr (heatmap) demonstrating expression of a subset of the novel mouse reproductive tract-specific genes with human orthologs and no reported mouse model, and with human reproductive tract enrichment, is shown in fig. 7 . seventeen novel genes without mouse models (8030474k03rik, fthl17b, fthl17c, fthl17d, fthl17e, fthl17f, gm15262, gm18336, magea3, magea4, magea5, magea6, magea8, mageb2, xlr5a, xlr5b, and xlr5c) were identified through the mouse id4+ spermatogonia datasets that were also identified as spermatogonia-specific through the human datasets (bend2, bx276092.9, fam9a, fthl17, magea3, magea4, magea6, mageb6, and pnma6e) and were not identified through either mouse or human testis datasets, indicating restricted expression in spermatogonia, spermatogonial stem cells, or both (additional file 5: table s5 and additional file 6: table s6 ). eight genes (1700080o16rik, ccnb3, gm21964, gm4779, pet2, prr23a1, prr23a2, and prr23a3) were identified through the mouse id4+ spermatogonia datasets, genes whose human orthologs-ccnb3, dcaf8l1, magea10, magea11, magea12, magea9, magea9b, prr23a, prr23b, prr23c, and tle7-were also identified through the human spermatogonia datasets. since these genes were also identified through mouse, human, or both species' respective testis datasets, this indicates either strong expression in the spermatogonia compartment or expression outside of and in addition to the spermatogonia compartment. twenty-two genes were identified as mouse sertoli cellspecific as they were not otherwise identified as reproductive tract-specific through our analysis of mouse testes or germ cell datasets (encode project consortium testes and helsel et al.'s id4+ germ cell datasets) (additional file 6: table s6 ). of these 22 genes, 18 have human orthologs; of these 18 genes, 17 are novel and not previously identified as male reproductive tractspecific; of these 17 genes, 7 have not been previously knocked out in the mouse (c1ql2, gm45015, mageb18, mycs, shc4, sowahd, and tmsb15b2); and of these 7 genes, 2 genes (gm45015 and mageb18) were identified with human orthologs (pnma6e and mageb18) that were also identified through our human analysis as human reproductive tract-specific (additional file 5: table s5 and additional file 6: table s6 ). unlike the limited number of reproductive tract-specific genes we identified through human sertoli cell-specific datasets, a considerable number of genes-202 mouse genes-were identified as reproductive tract-specific through analysis of zimmermann et al.'s mouse postnatal day 35 sertoli cell datasets that were also identified through either the mouse testis datasets, mouse id4+ germ cell datasets, or both (additional file 6: table s6 ). of these 202 genes, 160 have one or more human orthologs; of these 160 genes, 54 are novel and not previously identified as male reproductive tract-specific; of these 54 genes, 36 have not been previously knocked out in the mouse; and of these 36 genes, 16 (1700011l22rik, 1700011m02rik, 1700018b08rik, 1700042g07rik, ankrd36, ankrd60, etd, gm39566, gm6657, gm9112, magea10, spata31d1b, tcp10c, tex44, tex48, and wfdc9) were identified with human orthologs that were also identified through our analyses as human reproductive tractspecific (fig. 6 , additional file 5: table s5 , and additional file 6: table s6 ). to the best of our knowledge, published rna-seq data of mouse whole epididymis or epididymis segments does not exist, for the identification of epididymis-specific transcripts or otherwise. therefore, we isolated caput, corpus, and cauda segments from adult (postnatal day 60) b6/129 mice and subjected the rna to sequencing. sixty-six genes were identified as mouse epididymisspecific as they were not identified as mouse male reproductive tract-specific through our reanalysis of the entable s6 ). of these 66 genes, 48 have human orthologs; of these 48 genes, 34 are novel and not previously identified as male reproductive tract-specific; of these 34 genes, 17 have not been previously knocked out in the mouse (ascl4, bsph1, c1s2, clec18a, cyp2j13, cyp4a30b, defb42, gm45826, lce6a, lcn6, muc15, odam, spag11a, spag11b, spink13, svs1, tchhl1); and of these 17 genes, 5 genes (bsph1, defb42, gm45826, spag11a, and spag11b) were identified with human orthologs (bsph1, defb136, spag11a, and spag11b) that are also human epididymis-specific (fig. 6 , additional file 5: table s5 , and additional file 6: table s6 ). sixty-five genes were identified as reproductive tractspecific in mouse with expression in both epididymis and testis and/or testis cell. of these 65 genes, 48 have human orthologs; of these 48 genes, 24 are novel and not previously identified as male reproductive tract-specific; of these 24 genes, 14 have not been previously knocked out in the mouse (1700009n14rik, 4922502d21rik, 4930563d23rik, d330045a20rik, defb28, dgkk, fam90a1b, gm6871, hrasls5, nxf3, shc4, spint3, trpc5os, wfdc9); and of these 14 genes, 3 genes (spint3, trpc5os, and wfdc9) were identified with human orthologs (spint3, trpc5os, and wfdc9) that are also human epididymis-specific (fig. 6 , additional file 5: table s5 , and additional file 6: table s6 ). through the aforementioned studies, we identified spint3, spint4, ces5a, pp2d1, and saxo1 as congruent in expression across both mouse and human datasets with expression restricted to either the epididymis (spint3, spint4, ces5a) or the testis (pp2d1 and saxo1) (figs. 3, 4, 5, and 6; additional file 5: table s5 ; additional file 6: table s6 ). spint5 was also identified as epididymisspecific in mouse (fig. 5 , additional file 6: table s6 ); however, in humans, spint5p is a pseudogene that is not processed into protein. conventional rt-pcr of a panel of mouse and human tissue cdnas confirmed epididymis-or testis-restricted expression of spint3, spint4, ces5a, pp2d1, and saxo1 in both species and spint5 in mouse (fig. 5) . to glean insight into the onset of expression for the epididymis-specific genes, spint3, spint4, spint5, and ces5a; whole epididymides from postnatal days (p) 3, p6, p10, and p14; and epididymis segments (caput, corpus, and cauda) from p21, p28, p35, and p60 aged mice were collected and analyzed through rt-pcr (additional file 19: fig. s10 ). spint3 expression begins as early as p3 (low) and gradually increases through p10 and p14 reaching steady levels throughout p21 to p60 in all three segments of the epididymis (additional file 19: fig. s10 ). in contrast, spint4 and spint5 display near identical expression levels with no expression at p3, p6, or p10, and expression apparent at p14 and later time points, with expression restricted to corpus only at p21 and p28, and caput and corpus, but not cauda at p35 and p60 (additional file 19: fig. s10 ). rnascope-based fluorescence in situ hybridization revealed a distinct segment-specific pattern of expression for spint4 that was identical to spint5, with both showing expression in most of the epithelial cells restricted to a brief region of distal caput/proximal corpus (additional file 20: fig. s11) . spint3, on the other hand, displayed a pattern of expression in a majority of epithelial cells that begins just a bit further downstream along the corpus, but persisting for much further along the corpus, throughout the corpus and into the cauda (additional file 20: fig. s11 ). these results indicate that spint3 shares a role that is distinct from spint4 and spint5 and indicates a potential redundancy between spint4 and spint5 and how humans may have lost the evolutionary pressure to keep spint5p as a protein-coding gene. to glean insight into the potential spermatogenic cell population(s) expressing pp2d1 and saxo1, we performed rt-pcr of mouse testes isolated at postnatal day (p) 3, a time point enriched for gonocytes; p6 (onset of expression of type a spermatogonia); p10 (early spermatocytes); p14 (late spermatocytes); p21 (spermatids); and p35 and p60, which display complete spermatogenesis [40] (additional file 19: fig. s10 ). expression of pp2d1 and saxo1 is detected at similar levels at p28 and later, but not at p21 or before indicating expression during spermiogenesis and spermiation (additional file 19: fig. s10 ). to determine the male reproductive requirement and potential functional role of the identified novel male reproductive tract-specific genes, spint3, ces5a, pp2d1, and saxo1 were individually ablated by crispr/cas9mediated zygote approach. since in humans spint5p is a pseudogene, and in mice, spint5 protein is most similar in sequence to mouse spint4, we simultaneously ablated both mouse spint4 and spint5 genes, which on mouse chromosome 2 are only separated by 12.9 kilobases. the efficiency of generating each mutant is summarized in additional file 1: table s11 . each of the genes contained deletions of differing sizes and genomic targets. the genomic sequences flanking the deletion in each mutant are presented in additional file 1: table s12 , and representative sanger sequencing results for each mutant are presented in fig. 8 . using the forward and reverse primer pairs presented in fig. 8a -e and listed in additional file 1: table s13 , offspring carrying the mutant alleles were identified through routine genotyping (fig. 8k-o) . spint3, spint4/5, ces5a, pp2d1, and saxo1 knockout mouse lines were examined in parallel with littermate controls of equivalent age to determine the effect of gene ablation on spermatogenesis, sperm maturation, and fertility in male mice. none of the knockout strains generated in this study displayed any overtly abnormal appearance, difference in body weight (fig. 9 ) or composition, or difference in behavior when compared to the controls. to determine the male reproductive requirement of each of the genes of interest, spint3, spint4/5, ces5a, pp2d1, and saxo1 knockout and control adult male mice were housed continuously with two females for 3 months and the size and number of litters were recorded. although spint3, pp2d1, and saxo1 knockout males sired a number and size of litters during the test mating period that was not significantly different from controls (fig. 9a-c) , spint4/5 and ces5a knockout males sired significantly fewer litters and pups over the test mating period (fig. 9a-c) . spint4/5 null males displayed a statistically significant 95% reduction in the number of litters and pups sired per male and statistically significant 64% reduction in litter size, over the 3-month mating period (n = 9 controls, n = 9 kos) (fig. 9 ). seven out of 9 males displayed complete infertility, and the two remaining males, who sired pups, sired pups at a significantly reduced number of litters and pups per month with litters of reduced litter size (fig. 9a-c) . this fertility defect in spint4/5 double ko males was not associated with any significant changes in epididymis and testis histology (additional file 21: fig. s12 ) or sperm numbers, motility, and morphology ( fig. 9g-i) . ces5a null males displayed a variegated phenotype with an overall statistically significant 50% reduction in the number of litters and pups sired per male, but no significant difference in litter size, over a 3-month mating period (n = 9 controls, n = 9 kos). the fertility defect in ces5a ko males was associated with significant changes in epididymis histology (additional file 22: fig. s13 ) and significant reductions in sperm motility and progressive motility (fig. 9h, i) . ces5a null males displayed a 50% reduction in sperm motility and progressive cells, a 50% increase in static cells, and a 25% decrease in average path velocity and progressive velocity after hyperactivation. no changes in testis histology were found (additional file 22: fig. s13) , and despite the sperm motility defect, scanning electron microscopy failed to identify a morphological defect in ces5a null sperm in comparison to controls (additional file 23: fig. s14) . the epididymides and testes weights of spint3, spint4/5, ces5a, pp2d1, and saxo1 knockout mice were not significantly different from littermate control mice (fig. 9e, f) . histological analyses of testes from spint3, spint4/5, ces5a, pp2d1, and saxo1 knockout mice revealed all had seminiferous tubules with intact epithelia and the presence of all germ cell subtypes and all stages of spermatogenesis (additional file 21: fig. s12 and additional file 24: fig. s15 ). histological analyses of caput, corpus, and cauda from spint3, spint4/5, pp2d1, and saxo1 ko mice revealed spermatozoa in tubule lumens of all knockouts with no significant differences in epididymal histology in comparison to controls (additional file 21: fig. s12 and additional file 24: fig. s15 ). however, ces5a knockout mice displayed significant histological abnormalities including lumen dilation (possibly from occlusion), inflammation, and the appearance of abnormal epithelia (additional file 22: fig. s13 ). computer-assisted sperm analysis of spint3, spint4/5, pp2d1, and saxo1 knockouts 9 phenotype analysis of crispr/cas9 generated null mice for determining the contraceptive potential of the selected genes. spint4/5 and ces5a null mice show significant fertility defects; meanwhile, spint3, pp2d1, and saxo1 null mice appear normal. fertility (a-c), body and reproductive organ weights (d-f), and sperm parameters (g-i) were all measured between knockout (−/−) and littermate control [wild-type (+/+) and heterozygous (+/−)] mice as indicated. bars represent mean ± sem. *p < 0.05, **p < 0.01, ***p < 0.005. ns, not significant. showed no statistically significant differences across all measured parameters including sperm concentration, sperm motility, and progressive motility (fig. 9g-i) . ces5a knockouts displayed significant decreases in sperm number and sperm motility (fig. 9h, i) ; however, cauda epididymal sperm isolated from a variety of ces5a null animals looked morphologically indistinguishable to controls (additional file 23: fig. s14 ). to date, the etiology of idiopathic male infertility is not fully understood, and hormonal male contraceptives have not been effective. therefore, identification of novel reproductive tract-specific genes, and elucidating the functional requirement or lack thereof of these genes, is essential towards understanding the etiology of male infertility and the development of male contraceptives. despite significant advances in our understanding of the human and rodent testis and epididymis transcriptome, mostly through microarray-based studies, no prior studies have utilized purified human testis cells for the identification of human testis-specific transcripts, no prior studies have utilized the more state-of-the-art rna-seq-based transcriptomics methodology for analysis of human epididymis-specific transcripts, and no prior studies have utilized rna-seq analysis of rodent reproductive tissues or cells to identify rodent reproductive tract-specific transcripts. to address these gaps in knowledge, and to increase the number of identified reproductive tract-specific genes using the most relevant high-throughput transcriptomics methodology, we analyzed in parallel on a custom bioinformatics pipeline a large number of published and newly acquired human and mouse rna-seq datasets. through our studies, we identified and verified many novel male reproductive tract-specific transcripts in both species, and through the crispr/cas9 system, we interrogated the reproductive requirement of a subset of these genes. we found that spint4 (together with spint5) in mice is required for normal male mouse fertility, and although not required for male fertility, we identified ces5a as playing a major biological role in the epididymis. we report the remaining genes that we knocked out-spint3, pp2d1, and saxo1as dispensable for male reproductive function, which is essential information to disseminate to the scientific community. our study also verified the male reproductive tract-specific expression of many previously identified genes (additional file 13: fig. s5 , additional file 15: fig. s7) , and genes for which previously published mouse models display male infertility phenotypes (additional file 14: fig. s6 ). this later group of already functionally validated genes serves as potential male contraceptive targets worth underscoring to the research community. prior to massively parallel microarray-based and rnaseq-based transcriptomics analyses for the identification of reproductive tract-specific genes, the ncbi unigene database was a valuable resource for many in the male reproductive biology field for identifying testis-specific transcripts [4, [41] [42] [43] . although in our study we only considered prior microarray-based and rna-seq-based studies when considering the novelty of the genes that we identified, it is worth noting that several genes that we identified-not previously identified in microarray-based and rna-seq-based transcriptomics studies-were previously identified through studies that solely utilized the unigene database [41, 42] . nineteen human genes that we identified-c16orf82, ccdc27, cpa5, fam217a, fam46d, fam47c, fbxo24, fbxw10, fkbp6, galn tl5, kcnu1, mageb3, mroh2b, nutm1, prdm9, rbmxl3, spata31e1, triml1, and trpc5os-were previously identified by liu et al. [42] , and seven mouse genes that we identified-1700013d24rik, akap3, ankrd36, hrasls5, spesp1, tex22, and ubqlnl-were previously identified by choi et al. [41] and liu et al. [42] . thus, our results confirm the findings of these previous studies. since more than half of all human protein-coding genes are categorized as unknown in terms of drug target potential (additional file 11 table s8 ), and only 30% encode for classically druggable enzymes, gpcrs, ion channels, nuclear receptors, and transporters (additional file 10 fig. s4) , the potential to find new undiscovered drug targets that can be drugged using classical approaches is somewhat limited. indeed, in our study, we found one hundred and nine genes to be novel in terms of previously published high-throughput transcriptomics studies, without a current reported mouse model, and reproductive tract-specific in both humans and mice (figs. 2, 3, and 6 ). many of these genes (93 genes; 85%) fall into the category of unknown and may otherwise be considered "undruggable" due to various challenges with existing targeting approaches [10] . however, the contraceptive potential of these genes should not be overseen, but rather investigated for potential identification of a high-affinity small molecule that can either interfere with protein-protein interaction (ppi) or target the protein specifically for degradation using a new technology called proteolysis targeting chimeras (protacs). protein-protein interaction targets are not deemed undruggable, based on the discovery of small molecules capable of deeper and higher affinity binding within the contact surfaces of the target protein [44] . additionally, once a high-affinity small molecule against a specific target protein is identified, an engineered protac molecule can mark a target protein for proteasomal degradation by linking the target protein to the polypeptide co-factor, ubiquitin [45] [46] [47] . there are currently various combinations of protacs developed to overcome the limitations of cell permeability, stability, solubility, selectivity, and tissue distribution [48] [49] [50] [51] . therefore, disrupting ppis or utilizing protacs provides the potential to greatly promote the development of contraceptive drugs against the "undruggable" nonenzymatic target protein space. if gene knockouts for closely related and ubiquitously expressed paralogs display no abnormal phenotype, then unintended drug targeting of these proteins may result in no side effects in humans. however, the burden of safety for a male contraceptive is extremely high, and if it can be avoided, targeting non-reproductive tractexpressed proteins in humans should be avoided since the functional requirements for these proteins may not be fully understood. further, although mice are one of the best models for human disease, they are unable to communicate when they are unwell, a phenomenon that may occur independent of any measurable phenotypic traits. thus, potential reported side effects in humans during clinical trials may have been present, but missed, during animal studies, or in fact be present in humans and not in mice because of the vast biological differences across these two species. thus, with drug safety in mind, reproductive tract-specific candidates should be prioritized based on somatic cell-expressed protein sequence similarity, especially in the drug binding pocket. according to ensembl, several novel reproductive tract-specific genes without mouse models that we identified and verified-ac022167.5, al672043.1, bhmg1, c1orf105, c2orf92, c4orf51, ccdc196, spint3, tex44, tex48, tex51, and trpc5os (figs. 3, 4, and 5; additional file 5: table s5 )-have no known associated paralogs, indicating reproductive tract-specific drug targeting is highly likely. additionally, spag11a and spag11b are epididymis-specific paralogs (figs. 3, 4 , and 5; additional file 5: table s5 ) with no other known paralogs according to ensembl. efcab5 (figs. 3 and 5, additional file 5: table s5 ) has a ubiquitously expressed paralog, nsrp1, with only 7% amino acid sequence similarity, indicating specific drug targeting potential for this candidate. likewise, erich6 and mroh2b (figs. 3 and 5, additional file 5: table s5 ) have ubiquitously expressed paralogs erich6b and mroh2a, respectively, with 20% and 30% amino acid sequence similarity, indicating reasonable potential for specific drug targeting. prr23a, prr23b, and prr23c are testis-specific paralogs (figs. 3 and 5, additional file 5: table s5 ) with prr23d2 as the next closest paralog according to ensembl. since prr23d2 has less than 26% amino acid sequence similarity to prr23a, prr23b, and prr23c, but appears to be epididymis-specific according to the human protein atlas [52] , all four proteins of unknown function make suitable drug candidates. likewise, spat a31d1, spata31d3, and spata31d4 are testis-specific paralogs (figs. 3 and 5, additional file 5: table s5 ) with spata31a5 as the next closest paralog according to ensembl. since spata31a5 has less than 26% sequence similarity to spata31d1, spata31d3, and spat a31d4, but also appears to be reproductive tractspecific according to hpa [52] , all four proteins with unknown function also appear to be worthy of consideration for potential drug targeting. tpte and tpte2 are testis-specific paralogs (figs. 3 and 5, additional file 5: table s5 ) with ubiquitously expressed pten as the next closest paralog according to ensembl. since pten has less than 25% sequence similarity to tpte and tpte2, off-target effects appear to be unlikely. likewise, wfdc10a, wfdc10b, and wfdc13 are all epididymis-specific paralogs with the closest nonreproductive tract-expressed paralog, wfdc5, having less than 26% sequence similarity to any of the rts paralogs, also indicating high drug specificity potential. several additional novel reproductive tract-specific enzyme and gpcr genes without mouse models that we identified-gpr156, prss38, prss48, sult6b1, tmpr ss7, triml2, and ttll8 (fig. 3, 4 , and 5; additional file 5: table s5 )-have non-reproductive tract-expressed paralogs with less than 35% sequence similarity indicating good drug specificity potential. a novel testis-specific transporter gene without a reported mouse model that we identified, slc25a52, would make a poor drug candidate since its closest paralog, slc25a51, is 93% similar in amino acid sequence and ubiquitously expressed. cpa5, iqca1l, and ppp4r3c have ubiquitously expressed paralogs, cpa1, iqca1, and ppp4r3b, respectively, with 50-60% protein sequence similarity indicating careful consideration must be made for potential drug targeting without off-target effects. of the seventy-three genes that our study identifies as reproductive tract-specific in humans and for which a published mouse model shows male infertility phenotype (additional file 14: fig. s6) [28, 29, 31] , it is worth noting that 21 genes-cnbd2, defb110, fam170a, fbxo47, meig1, meiob, meioc, odf1, odf4, rec114, rnf17, spaca1, spata22, spem1, spo11, sycp1, terb1, tex19, tex38, tnp2, and topaz-do not have any associated paralogs and, thereby, may be considered most suitable for further drug development. however, it is also worth noting that it may be possible that genes required for male fertility in mice may not necessarily be required for male fertility in humans. of the seventy-three human reproductive tract-specific genes our study identified with male mouse infertility phenotypes, twenty-seven genes-actl7b [53] , akap4 [54] , boll [55] , brdt [55] [56] [57] [58] [59] [60] , catsper4 [54] , ccdc155 [61] , fkbp6 [55, [61] [62] [63] , meig1 [64] , meiob [55] [56] [57] 65] , nanos2 [55, 61] , odf1 [55] , prdm9 [61, 66, 67] , prss37 [55] , rad21l1 [68] , rbmxl2 [55, 62] , rnf17 [69] , sohlh2 [61, 70] , spaca1 [55] , spata16 [55, 56] , spem1 [55] , spo11 [55, 58, 61] , sun5 [55] [56] [57] , sycp1 [55] , tex38 [55] , tnp2 [55] , tssk1b [62] , and zpbp [55] -are currently associated with mutations underlying human male infertility, confirming a similar functional requirement for these genes in humans may exist. for the remaining 45 genes, however, either these genes are not required for human male fertility as they are required in mice, or associated mutations in male infertile patients have not yet been reported. although many reproductive tract-specific genes have been studied through functional genetics approaches, many remain to be solved. elucidating the function of these novel genes is necessary to build a better understanding of the factors underlying spermatogenesis and sperm maturation, which has implications in understanding the etiology of male infertility and the development of male contraceptives. in this study, four epididymis-specific genes (spint3, spint4, spint5, and ces5a) and two testis-specific genes (pp2d1 and saxo1) were deleted in mice to determine their functional requirement in male fertility and potential utility as male contraceptive target. we chose to study these genes because all but saxo1 encode enzymes or enzyme-related protein products and are thus considered druggable in the classical sense. saxo1, a cilia-related gene, was chosen because prior literature demonstrated expression in sperm [71] . although not druggable in the classical sense, if targeted through non-canonical approaches, one could obtain a fast-acting drug with greater reversibility potential and a decreased likelihood of affecting testicular function and size. the epididymis-specific genes we chose to target for functional analysis, by the very nature of their tissue's expression, also fit this potential drug profile of modulating only the latest stages of sperm development. analyses of testis and epididymis organ weights and histology, sperm parameters and morphology, and fertility revealed no significant differences in spint3, pp2d1, and saxo1 knockout mice in comparison to littermate controls demonstrating that, individually, spint3, pp2d1, and saxo1 are not required for male mouse fertility and are not suitable targets for the development of a male contraceptive. however, we found partial effects on male fertility in ces5a knockout mice and profound effects on male fertility in spint4/5 double knockout mice. ces5a is a member of a multigene family of mammalian carboxylesterases that can hydrolyze ester, thioester, amide, and carbamate linkages in a wide variety of endogenous and exogenous molecular substrates, including triglycerides, thus playing key roles in both metabolism and detoxification [72] [73] [74] [75] . ces5a shares roughly similar percent homology (~40% homology) to all four of its related paralogs, ces1, ces2, ces3, and ces4a. human carboxylesterase 1 (ces1) is predominantly expressed in the liver and has been shown to have triglyceride hydrolase activity as overexpression of human ces1 in cells leads to an increase in cholesteryl ester hydrolysis and free cholesterol efflux [76] . further, mouse ces1g-a protein expressed by one of a cluster of eight syntenic genes (ces1a through ces1h) orthologous to the human ces1 gene-has been shown to have triglyceride hydrolase activity as ces1g null mice display hyperlipidemia and abnormal lipid homeostasis including increased liver and circulating cholesterol and triglycerides, and altered saturated and unsaturated fatty acid levels [77, 78] . therefore, it is likely that ces5a exhibits similar carboxylesterase activity in the epididymis hydrolyzing cholesteryl ester and affecting free cholesterol efflux. indeed, recombinant ces5a protein has been previously shown to have carboxylesterase activity hydrolyzing cholesterol ester and choline ester [79] . since sperm cholesterol content is significantly decreased during epididymal maturation [80, 81] and a proper cholesterol/phospholipid (c/pl) ratio of the sperm plasma membrane is required for sperm capacitation [82, 83] , ces5a may be pivotal in regulating sperm membrane cholesterol and lipid levels to ensure the normal function of male gametes in the last steps of the fertilization process. the most closely related paralog to spint4 is eppin, which, as reviewed in o'rand et al., has at least three physiological functions [16] . eppin inhibits sperm motility when it binds the semen coagulation protein semenogelin 1 (semg1) on the sperm surface [84] ; it modulates the proteolytic activity of prostate-specific antigen (psa), a serine protease, against its seminal plasma substrate, semg1 [85] ; and it exhibits strong antibacterial activity [86] . these functions are postulated to prevent premature hyperactivation and capacitation of sperm in the female reproductive tract [16] , and to protect spermatozoa from proteolytic and bacterial attack during transit in the female reproductive tract [11, 16] . thus, it is possible that the physiological function of spin t4 is similar. however, unlike spint1 and spint2, which have been shown to act as protease inhibitors against a wide variety of prss and tmprss proteases [87] [88] [89] [90] , when tested against a panel of eight proteases (including psa, trypsin, chymotrypsin, plasmin, urokinase, thrombin, factor xa, and elastase), spint3 and spint4 were shown to lack protease inhibiting capability [91] . this indicates that either the protease inhibiting properties of spint3 and spint4 were lost in favor of yet unknown functions or their protease activity has a narrower spectrum of inhibition against unknown targets. since spint4/5 null male mice are severely subfertile, without an apparent difference in epididymis histology, sperm number, sperm morphology, and sperm motility parameters in comparison to the wild-type (wt) mice, this phenocopies the reproductive phenotype of several null mice of testis-, epididymis-, or prostate-specific genes (sof1, tmem95, and spaca6; pate8, and pate10), which reveal a requirement in regulating sperm migration through the oviduct and sperm-oocyte fusion in mice [92, 93] . a severe fertility defect associated with normal sperm number, morphology, and motility is also shared among mice lacking the sperm membrane protein adam3, thought to be crucial in sperm-zp binding and sperm migration through the uterotubular junction (utj) [94, 95] . more than 10 proteins including 2 proteases (ace, adam1a, adam2, calr3, clgn, cmtm2a/b, pdilt, pmis2, prss37, rnase10, tex101, and tpst2) have been described that affect the processing and/or localization of adam3 protein in spermatozoa [96, 97] . further studies with spint4/5 null mice are required to determine whether sperm behavior in the female reproductive tract, specifically the sperm migration through the utj, is adversely affected. since a large 16,797-bp genomic region-including the intergenic region between the spint4 and spint5 genes-was deleted ( fig. 8 ; additional file 1: table s12 ), based on the evidence presented in this manuscript, we cannot exclude the possibility that cis-acting elements and/or trans-acting factors affecting the expression of other genes may have contributed to the phenotype of these mice. lack of protein-coding ability of human spint5p does not necessarily indicate that this pseudogene is functionally obsolete. pseudogenes have been shown to play roles in gene expression and gene regulation [98] . for example, pseudogene transcripts can act as competitive endogenous rnas (cerna) through competitive binding of mirna, which results in regulation of gene expression [99] . to this end, studying the functional requirement of spint5 in male mice is necessary to further our knowledge of evolutionarily conserved genes between species. since humans are genetically diverse, a limitation to phenotype characterization of genetically manipulated mice is the reliance of a single mouse background to the examination of complex genetic outcomes, such as fertility, that is under the control of many genes with different levels of contribution to the phenotype [100] . it is possible that a gene that causes complete infertility in an inbred mouse background may only cause partial infertility or subfertility in a different inbred line or more robust outbred background. since the mice used in our study were a cross between c57bl/6 (b6) mice and dba/2 (d2) mice, and thus, these b6d2f1 mice are heterozygous for b6 and d2 alleles at all loci in their genome, we can eliminate infertility susceptibility of either the b6 or the d2 background as the cause for fertility defects in ces5a and spint4/5 mice. it does remain to be determined, however, whether the phenotype of the genes we knocked out would be more or less severe on a different mouse background, and if required for male fertility in humans, the level of contribution to male fertility of these genes across genetically diverse men. a limitation to this study is the reliance on mrna abundance positively correlating with protein abundance. future studies are necessary to elucidate the relationship between mrna and protein expression levels of the candidate genes identified in our study. furthermore, despite batch corrections that were made, technical differences in sample preparation and integrity across the various published rna-seq datasets can influence the results of our findings. one of the major advantages to our study design is the use of rna-seq datasets from purified human and mouse germ cells and sertoli cells to identify reproductive tract-specific targets since the use of whole testes for the identification of cell typespecific transcripts in past studies is subject to dilution effects. however, this advantage could also be considered a disadvantage since purified cells from nonreproductive tissues were not used for comparison but, if analyzed, purified cells from non-reproductive tissues may have revealed significant levels of expression in non-reproductive tissues. ultimately, functional studies in animals and humans will help to confirm whether genes identified in our study are essential for male fertility and not any other physiological process. through the integration of hundreds of published and newly acquired human and mouse reproductive and non-reproductive tissue and cell rna-seq datasets, we have generated a list of novel genes expressed predominantly or exclusively in the male reproductive tract that are worthy of consideration for functional validation in an animal model and potential targeting for a male contraceptive. our results further validate a functional requirement for spint4/5 and ces5a in male mouse fertility, while demonstrating that spint3, pp2d1, and saxo1 are each individually dispensable for male mouse fertility. identifying novel reproductive tract-specific genes congruent across species adds insight into organismal biology and valuable information that can be used to identify potential male contraceptive drug target candidates. furthermore, elucidating the individual functional requirement or lack thereof of these novel genes builds a better understanding of the factors underlying spermatogenesis and sperm maturation, which has implications in understanding the etiology of male infertility and further validation of the utility of a potential male contraceptive target. the de novo isolated human testes and epididymides included in this study were obtained from three donors through a local organ transplant program in quebec, canada, called transplant quebec. all procedures were approved by the local ethics committee, and written consent was obtained from each respective donor's family. the donors were of 40, 52, and 65 years of age with no preexisting medical condition that could affect reproductive function. donor testes and epididymides were removed under artificial circulation to preserve other organs that were assigned for transplantation. each testis and epididymis were dissected in the laboratory of robert sullivan at université laval. each epididymis was dissected into three segments corresponding to the caput, corpus, and cauda regions and minced into small tissue pieces. testes and epididymides tissue fragments were immediately snap frozen in liquid nitrogen, stored at − 80°c, and shipped frozen to baylor college of medicine for further processing. eight non-reproductive tissue types (kidney, liver, lung, skin, spleen, and stomach) and 2 female reproductive tissues (ovary and uterus) were obtained from the baylor college of medicine tissue acquisition and pathology core. thirteen nonreproductive tissue types (adipose, adrenal gland, brain, colon, heart, leukocytes, pancreas, prostate, salivary gland, skeletal muscle, small intestine, smooth muscle, thyroid) were obtained as purified rnas from takara bio (kusatsu, japan). human testes and epididymis segments were used for de novo rna-seq analysis; all human tissues and/or resulting rnas were used for rt-pcr verification. mouse tissues [testis, caput, corpus, cauda, ovary, uterus, and 17 non-reproductive tissue types (adipose, bladder, brain, colon, eye, heart, kidney, liver, lung, prostate, skeletal muscle, skin, small intestine, spleen, stomach)] were obtained from dissection of b6/129 mice; the remaining 2 non-reproductive tissues (smooth muscle and thyroid) were obtained as purified rnas from takara bio. mouse epididymis segments were used for de novo rna-seq analysis; all mouse tissues and/or resulting rnas were used for rt-pcr verification. rna for both rna-seq and/or rt-pcr verification was isolated from human and mouse tissues using trizol/ chloroform extraction method followed by rneasy mini kit from qiagen with on-column dnase (qiagen) treatment using the manufacturer's protocol. rnas used for rna-seq were assessed by bioanalyzer for rna integrity. for rt-pcr, rna was reverse-transcribed to cdna using superscript iii reverse transcriptase from thermo fisher according to the manufacturer's protocol. cdna was then pcr amplified using gene-specific primers designed using ncbi primer design tool. primer sequences are listed in additional file 1: table s14 . library generation for rna-seq rna-seq libraries were made using kapa stranded mrna-seq kit (kk8420). briefly, poly-a rna was purified from total rna using oligo-dt beads; subsequently, it was fragmented to small size; and first strand cdna was synthesized. second strand cdna was synthesized and marked with dutp. resultant cdna was used for end repair, a-tailing, and adaptor ligation. finally, the library was amplified for sequencing on an illumina novaseq 6000 platform. the strand marked with dutp was not amplified, allowing strand-specific sequencing. sequence alignment, quantification, and differential gene expression human testes, human epididymis segments, and mouse epididymis segments were sequenced by the department of molecular and human genetics functional genomics core at baylor college of medicine (additional file 1: table s1 and additional file 1: table s2 ). previously published reproductive and non-reproductive tissue and cell sequences were downloaded from the sequence read archive (sra) [101] (additional file 1: table s1 and additional file 1: table s2 ). all sequences were trimmed using trim galore! and aligned against the human genome (grch38) or mouse genome (grcm38) using hisat2 [102, 103] . gene expression in each tissue was quantified using featurecounts, filtered for only protein-coding genes, and batch corrected by removing unwanted variation using the ruvr method from ruvseq [104, 105] . differential gene expression was determined for each reproductive tissue against each nonreproductive tissue using the r package edger [106] . principal component analysis (pca) was performed in the r statistical environment using the log2 counts per million (cpm) for each gene in the corresponding tissue after using the ruvr method to correct for batch variation as described above. our procedure for identifying a reproductive-specific gene was repeated for each reproductive tissue or cell sample independently. the following selection criteria were applied to each reproductive tissue or cell sample. first, the non-reproductive tissue with the maximum expression, expressed as the log2 fold change between the non-reproductive tissue and the reproductive tissue or cell of interest, was identified for each gene using the results from the differential gene expression analysis. second, we identified reproductive-specific gene drug candidates using three filters: a false discovery rate (fdr) filter, a maximum transcript per million (tpm) expression value filter on the non-reproductive sample with the maximum expression identified as described above, and a minimum tpm expression value filter on the reproductive tissue or cell sample of interest. a gene was kept if the fdr from the differential gene expression analysis was less than or equal to 0.05 for the comparison of the reproductive tissue or cell sample of interest to the non-reproductive tissue with the maximum expression. a gene was considered to be a male reproductive tissue-specific drug target if the average tpm expression value in the non-reproductive tissue with the maximum expression from the differential analysis was less than or equal to 1.0 for human (2.0 for mice), and if the average tpm expression value for that gene was greater than or equal to 10.0 for human (8.0 for mice) in the reproductive tissue or cell sample of interest. the average and standard deviation of the tpm expression value for each gene was calculated from the ruvr batch corrected counts per million expression value for each tissue or cell sample. we consolidated data from ensembl biomart [29] and mouse genome informatics (mgi) [28] to create a comprehensive database of mouse gene symbols orthologous to human genes and vice versa. each respective species' stable ensemble gene id was used for each conversion, with gene symbol as the final output. as mentioned, several notable high-throughput gene expression studies using microarrays or rna-seq, focused on identifying male reproductive tract-specific genes, have been previously published [2, [4] [5] [6] [7] [8] [9] . tables and supplementary tables from these studies were gathered to collect the lists of genes previously identified. for microarray-based studies, affymetrix ids were used to confirm the identity of a listed gene, based on current sequence mappings, or in many cases to identify de novo the identity of a gene only known at the time of the study by its affymetrix id and not gene symbol. for mouse and rat studies, gene symbols were converted to orthologous human symbols, to systematically catalog both the rodent and corresponding human symbols as previously identified. for example, 399 affymetrix probe ids were listed as reproductive tract-specific in johnston et al. [8] . after re-identification of gene symbols based on current ensembl sequence mappings, 42 identified gene symbols remained the same, 160 received an updated/replacement gene symbol identification, 103 genes that were previously unidentified received a new gene symbol identification, 26 affymetrix ids lost mapping to any gene symbol, and 67 remain unidentified. out of the total of 305 affymetrix ids that mapped to 301 current rat gene symbols, 257 rat gene symbols converted to at least one human ortholog gene symbol that was either the same symbol or different. both rat and human symbols, based on new mappings, were considered previously identified. for the complete list of previously identified genes, see additional file 18: table s10 . genes were classified as encoding either enzymes, epigenetic-related proteins, g protein-coupled receptors (gpcrs), ion channels, kinases, nuclear receptors, orphan gpcrs (ogpcrs), transcription factors, transporters, or unknown proteins based on data obtained from illuminating the druggable genome [27] (additional file 11: table s8 ). we used data obtained from ensembl biomart [29] , mgi [28] , the international mouse phenotyping consortium (impc) [31] , and pubmed searches to generate a comprehensive database identifying the existence of a mouse model for all mouse genes (additional file 12: table s9 ). we then queried our identified candidate human and mouse genes against this list. for a given human gene, we queried the equivalent mouse ortholog gene symbol(s). spint3, spint4/5, ces5a, pp2d1, and saxo1 knockout mice were produced at baylor college of medicine. b6d2f1 (c57bl/6 × dba2) mice were used as embryo donors, and cd1 mice were used as foster mothers. mice were purchased from charles river (wilmington, ma). all mice were housed in a temperature-controlled environment with 12-h light cycles and free access to food and water. mice were housed in accordance with nih guidelines, and all animal experiments were approved by the institutional animal care and use committee (iacuc) at baylor college of medicine. generation of spint3, spint4/5, ces5a, pp2d1, and saxo1 knockout mice to generate spint3, spint4/5, ces5a, pp2d1, and saxo1 knockout mice, grna/cas9 ribonucleoprotein complex was electroporated into fertilized eggs and transplanted into surrogate mothers as previously described [107] . briefly, to harvest fertilized eggs, card hyperova (0.1 ml, cosmo bio) was injected into the abdominal cavity of b6d2f1 females (charles river), followed by human chorionic gonadotropin (hcg) (5 units, emd chemicals). forty-eight hours after card hyperova, b6d2f1 males were allowed to mate naturally. twenty hours after mating, fertilized eggs with 2 pronuclei were collected for electroporation. custom crrnas targeting each gene were purchased from millipore-sigma. the sequences for all guide rnas used for crispr/cas9mediated gene editing are listed in additional file 1: table s11 . crrna and tracrrna (millipore-sigma) were diluted with nuclease-free water. the mixture was denatured at 95°c for 5 min and allowed to anneal by cooling gradually to room temperature (1 h). each grna was mixed with cas9 protein solution (thermo fisher scientific) and opti-mem media (thermo fisher scientific), and then incubated at 37°c for 5 min to prepare the grna/cas9 rnps [final concentration, 300 ng/μl cas9 for 250 ng/μl of each grna]. the grna/cas9 rnp solution was placed between electrodes with a 1mm gap in the ecm 830 electroporation system (btx). fertilized eggs were arranged between the electrodes, and then, the electroporation was performed with the following conditions: 30 v, 1-ms pulse duration, and 2 pulses separated by 100-ms pulse interval. for egg transfer, electroporated embryos were transplanted into the oviduct of pseudo-pregnant icr recipients. after 19 days, offspring were obtained by natural birth or cesarean section. the f0 mice with sequence-predicted heterozygous mutations were used for the mating with additional b6d2f1 mice to generate homozygous mutants. the f2 or later generations were used for the phenotypic analyses. for sanger sequence analysis of mutant mice, genomic dna was isolated by incubating tail tips in lysis buffer [20 mm tris-hcl (ph 8.0), 5 mm edta, 400 mm nacl, 0.3% sds, and 200 μg/ml actinase e solution] at 60°c overnight. polymerase chain reactions (pcrs) amplifying the genomic region containing the insertion/deletion events were performed using kod xtreme enzyme (toyobo, osaka, japan); pcr products were purified using the qiaquick pcr purification kit (qiagen, carlsbad, ca, usa) and sent for sanger sequencing on an abi 3130xl genetic analyzer (thermo fisher scientific, waltham, ma, usa) using the forward primer. for routine genotyping of mutant mice, genomic dna was isolated by separately incubating ear snips and tail tips in 50 mm naoh solution at 95°c overnight and inactivating with 1 m tris ph = 8.0. pcrs amplifying wild-type and mutant-specific amplicons were performed using 2x amfisure pcr master mix (gendepot, barker, tx). primer sequences are listed in additional file 1: table s13 . upon sexual maturation (6-7 weeks of age), knockout and littermate control male mice (n = 6-9 mice per genotype) were continuously housed with two 7-8week-old wild-type b6d2f1/j female mice per male for 12 weeks. during the fertility test, the number of pups was counted shortly after birth. the total number of litters and pups per male over the mating trial was calculated and divided by the number of months to generate averages and statistics per genotype. the average number of pups per litter is based on the average litter size per male where a litter is considered one or more pups. knockout and littermate control male mice (n = 5-13 mice per genotype) that were 12 weeks of age were used to examine body and reproductive organ weights, and testicular and epididymal histology. testes and epididymides were fixed in bouin's fixative, embedded in paraffin, sectioned at 5 μm thickness, and stained with 1% periodic acid-schiff (pas) stain followed by counterstaining with hematoxylin 2 solution. histological images were acquired with an aperio at2 slide scanner (leica microsystems). knockout and littermate control male mice (n = 5-13 mice per genotype) that were 12 weeks of age were used to examine sperm numbers and motility parameters using computer-assisted sperm analysis (casa). cauda of both epididymides was isolated, transferred into human tubule fluid (htf) (irvine scientific, santa ana, ca) containing 5 mg/ml of bsa, minced, and placed in a humidified incubator for 15 min at 37°c with 5% co 2 . following incubation, the sperm were diluted 1:50 in htf, added to a pre-warmed slide, and analyzed using a hamilton-thorne bioscience's ceros ii instrument. several fields of view were illuminated and captured until at least 200 cells were counted. rnascope 2.5 hd reagent kit (red) (cat. 322350, advanced cell diagnostics, newark, ca, usa) was used to detect spint3, spint4, and spint5 mrna transcripts on pfa-fixed, paraffin-embedded sections from 3-monthold wild-type epididymis. the probes against mm-spint3, mm-spint4, and mm-spint5 were custom-made, and the standard positive control (mm-ppib, cat. 313911) and negative control (dapb, cat. 310043) probes were used. the assay was performed according to the manufacturer's instructions. slides were counterstained using dapi and mounted using prolong glass antifade mountant (thermo fisher scientific inc.). multi-channel fluorescent images were acquired with an aperio versa (leica microsystems). all measurements are expressed as mean ± standard error of the mean. statistical differences were determined using student's t test. differences were considered statistically significant if the p value was less than 0.05. supplementary information accompanies this paper at https://doi.org/10. 1186/s12915-020-00826-z. additional file 1: tables s1, s2, s11, s12, s13, and s14. table s1 . summary of human rna-seq datasets. this table contains the sra value for each previously published human rna-seq dataset that was reanalyzed as part of this study. the geo accession number for each new human rna-seq dataset generated and subsequently analyzed in this study is also included. table s2 . summary of mouse rna-seq datasets. this table contains the sra value for each previously published mouse rna-seq dataset that was reanalyzed as part of this study. the geo accession number for each new mouse sample generated and subsequently analyzed in this study is also included. table s11 . single-guide rnas targeting the genes' upstream (u) and downstream (d) regions used for generating knockout mice. efficiency of embryo transplantation was presented using the number of total pups delivered by pseudopregnant mice divided by the number of total embryos used for oviduct transplantation (total pups/embryos transplanted). efficiency of genome editing was determined by the number of pups carrying enzymatic mutations divided by the number of pups subjected to genotyping (gm pups/pups genotyped). table s12 . sanger sequencing of detailed genotype of mutant dna sequences in all the five mouse lines. table s13 . primers and pcr conditions used for genotyping the mutant alleles of the knockout mouse lines. table s14 . human and mouse rt-pcr primer sequences used for verification of reproductive tract-specificity. additional file 2: fig. s1 . genes that passed the tpm and fdr filters in at least one of the measured reproductive tissues or cells were visualized using a heatmap of the ruvr batch corrected log2 cpm gene expression values for the human (a) and mouse (b) samples. additional file 3: table s3 . human expression summary. contains differential fold change, identity of the non-reproductive tissue with maximal gene expression based on the differential gene analysis, false detection rate (fdr) value, average and standard deviation tpm expression values, and log2 cpm gene expression value for the human samples. all protein-coding genes (18,305 genes) that had expression in at least one reproductive tissue or cell is listed. additional file 4: table s4 . mouse expression summary. contains differential fold change, identity of the non-reproductive tissue with maximal gene expression based on the differential gene analysis, false detection rate (fdr) value, average and standard deviation tpm expression values, and log2 cpm gene expression value for the mouse samples. all protein-coding genes (16,891 genes) that had expression in at least one reproductive tissue or cell is listed. additional file 5: table s5 . all human male reproductive tract-specific genes that met the criteria of identification as reproductive tract-specific in at least one male reproductive tissue or purified cell type, with the level of fold change listed under the tissue or cell if all criteria were met. the criteria of selection are as follows: fdr < 0.05; tpm repro > 10; tpm non-repro , max < 1. a fold change value of 0 indicates the criteria were not met for that that tissue or cell. additional columns indicating 1.) the equivalent mouse ortholog gene symbols (single or multiple symbols) that exist, and 2.) if our studies identified any of these mouse orthologs as reproductive tract-specific in mouse, are included. additional file 6: table s6 . all mouse male reproductive tract-specific genes that met the criteria of identification as reproductive tract-specific in at least one male reproductive tissue or purified cell type, with the level of fold change listed under the tissue or cell if all criteria were met. the criteria of selection are as follows: fdr < 0.05; tpm repro > 8; tpm non-repro , max < 2. a fold change value of 0 indicates the criteria were not met for that that tissue or cell. additional columns indicating 1.) the equivalent human ortholog gene symbols (single or multiple symbols) that exist, and 2.) if our studies identified any of these human orthologs as reproductive tract-specific in human, are included. received: 17 april 2020 accepted: 8 july 2020 population division: un the mouse epididymal transcriptome: transcriptional profiling of segmental gene expression in the epididymis1 the human epididymis: its function in sperm maturation a multitude of genes expressed solely in meiotic or postmeiotic spermatogenic cells offers a myriad of contraceptive targets identification of testis-specific male contraceptive targets: insights from transcriptional profiling of the cycle of the rat seminiferous epithelium and purified testicular cells identification of epididymis-specific transcripts in the mouse and rat by transcriptional profiling the rat epididymal transcriptome: comparison of segmental gene expression in the rat and mouse epididymides1 stage-specific gene expression is a fundamental characteristic of rat spermatogenic cells and sertoli cells the human testis-specific proteome defined by transcriptomics and 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functional diversity of caput, corpus and cauda regions transcriptome analysis of highly purified mouse spermatogenic cell populations: gene expression signatures switch from meiotic-to postmeiotic-related processes at pachytene stage dynamics of the transcriptome during human spermatogenesis: predicting the potential key genes regulating male gametes generation chromatin and single-cell rna-seq profiling reveal dynamic signaling and metabolic transitions during human spermatogonial stem cell development id4 levels dictate the stem cell state in mouse spermatogonia human sertoli cells support high levels of zika virus replication and persistence pharos: collating protein information to shed light on the druggable genome 2018: knowledgebase for the laboratory mouse analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics high-throughput discovery of novel developmental phenotypes carcinoembryonic antigen-related cell adhesion molecule 2 controls energy balance and peripheral insulin action in mice shp1 phosphatase-dependent t cell inhibition by ceacam1 adhesion molecule isoforms deletion of the carcinoembryonic antigen-related cell adhesion molecule 1 (ceacam1) gene contributes to colon tumor progression in a murine model of carcinogenesis ceacam1a-/-mice are completely resistant to infection by murine coronavirus mouse hepatitis virus a59 carcinoembryonic antigen-related cell adhesion molecule 10 expressed specifically early in pregnancy in the decidua is dispensable for normal murine development the lim homeobox gene lhx9 is essential for mouse gonad formation generation and characterization oflhx9-gfpcreert2knock-in mouse line targeted germline modifications in rats using crispr/cas9 and spermatogonial stem cells spermatogenic cells of the prepuberal mouse. isolation and morphological characterization integrative characterization of germ cell-specific genes from mouse spermatocyte unigene library comparative and functional analysis of testis-specific genes genome engineering uncovers 54 evolutionarily conserved and testis-enriched genes that are not required for male fertility in mice reaching for high-hanging fruit in drug discovery at protein-protein interfaces pharmacological perturbation of cdk9 using selective cdk9 inhibition or degradation catalytic in vivo protein knockdown by small-molecule protacs hijacking the e3 ubiquitin ligase cereblon to efficiently target brd4 lessons in protac design from selective degradation with a promiscuous warhead assessing different e3 ligases for small molecule induced protein ubiquitination and degradation cell-penetrating peptides: 20 years later, where do we stand? tat peptide-mediated cellular delivery: back to basics proteomics. tissue-based map of the human proteome single nucleotide polymorphisms: discovery of the genetic causes of male infertility a comprehensive gene mutation screen in men with asthenozoospermia malacards: an amalgamated human disease compendium with diverse clinical and genetic annotation and structured search genetic evaluation of patients with non-syndromic male infertility a systematic review on the genetics of male infertility in the era of nextgeneration sequencing genetics of male infertility: from research to clinic association study of single-nucleotide polymorphisms in faslg, jmjdia, loc203413, tex15, brdt, or2w3, insr, and tas2r38 genes with male infertility evaluation of 172 candidate polymorphisms for association with oligozoospermia or azoospermia in a large cohort of men of european descent point-of-care whole-exome sequencing of idiopathic male infertility the biology of infertility: research advances and clinical challenges mutation screening of the fkbp6 gene and its association study with spermatogenic impairment in idiopathic infertile men efficient typing of copy number variations in a segmental duplication-mediated rearrangement hotspot using multiplex competitive amplification genetic defects in human azoospermia single-nucleotide polymorphisms of the prdm9 (meisetz) gene in patients with nonobstructive azoospermia two single nucleotide polymorphisms in prdm9 (meisetz) gene may be a genetic risk factor for japanese patients with azoospermia by meiotic arrest single-nucleotide polymorphisms in the human rad21l gene may be a genetic risk factor for japanese patients with azoospermia caused by meiotic arrest and sertoli cell-only syndrome excess of rare variants in genes that are key epigenetic regulators of spermatogenesis in the patients with non-obstructive azoospermia mutations in sohlh1 gene associate with nonobstructive azoospermia human fam154a (saxo1) is a microtubule-stabilizing protein specific to cilia and related structures human carboxylesterases: a comprehensive review structure and catalytic properties of carboxylesterase isozymes involved in metabolic activation of prodrugs human carboxylesterases and their role in xenobiotic and endobiotic metabolism the mammalian carboxylesterases: from molecules to functions heterologous expression, purification, and characterization of human triacylglycerol hydrolase hepatic carboxylesterase 1 is essential for both normal and farnesoid x receptorcontrolled lipid homeostasis deficiency of carboxylesterase 1/esterase-x results in obesity, hepatic steatosis, and hyperlipidemia baculo-expression and enzymatic characterization of ces7 esterase lipid remodeling of murine epididymosomes and spermatozoa during epididymal maturation development changes occurring in the lipids of ram epididymal spermatozoa plasma membrane regionalization and redistribution of membrane phospholipids and cholesterol in mouse spermatozoa during in vitro capacitation variation in the cholesterol/phospholipid ratio in human spermatozoa and its relationship with capacitation analysis of recombinant human semenogelin as an inhibitor of human sperm motility characterization of an eppin protein complex from human semen and spermatozoa antimicrobial activity of human eppin, an androgen-regulated, sperm-bound protein with a whey acidic protein motif the role of type ii transmembrane serine proteasemediated signaling in cancer delineation of proteolytic and non-proteolytic functions of the membrane-anchored serine protease prostasin mechanisms of hepatocyte growth factor activation in cancer tissues regulation of cell surface protease matriptase by hai2 is essential for placental development, neural tube closure and embryonic survival in mice three genes expressing kunitz domains in the epididymis are related to genes of wfdc-type protease inhibitors and semen coagulum proteins in spite of lacking similarity between their protein products sperm proteins sof1, tmem95, and spaca6 are required for spermoocyte fusion in mice identification of multiple male reproductive tract-specific proteins that regulate sperm migration through the oviduct in mice disruption of adam3 impairs the migration of sperm into oviduct in mouse male mice deficient for germ-cell cyritestin are infertile co-expression of sperm membrane proteins cmtm2a and cmtm2b is essential for adam3 localization and male fertility in mice factors controlling sperm migration through the oviduct revealed by gene-modified mouse models pseudogenes regulate parental gene expression via cerna network interspecific recombinant congenic strains between c57bl/6 and mice of the mus spretus species: a powerful tool to dissect genetic control of complex traits the sequence read archive cutadapt removes adapter sequences from high-throughput sequencing reads transcript-level expression analysis of rna-seq experiments with hisat, stringtie and ballgown systematic selection of reference genes for the normalization of circulating rna transcripts in pregnant women based on rna-seq data featurecounts: an efficient general purpose program for assigning sequence reads to genomic features edger: a bioconductor package for differential expression analysis of digital gene expression data crispr/cas9 mediated genome editing in es cells and its application for chimeric analysis in mice publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank drs. yumei li all data generated or analyzed during this study are included in this published article, its supplementary information files, and publicly available repositories. the sra values for each of the 162 previously published reproductive and non-reproductive human rna-seq datasets [9, 21, 23, 24, 26, 30] and 81 previously published reproductive and non-reproductive mouse rna-seq datasets [19, 22, 25] are listed in additional file 1: table s1 and additional file 1: table s2 . all raw and processed data for the 12 new human and 9 new mouse samples generated in this study is deposited in ncbi geo (accession gse150854). all mice generated in this study, and any additional information about this study, are available from the corresponding authors upon request.ethics approval and consent to participate human tissue acquisition was approved by the ethics committee at université laval with written consent obtained from each respective donor's family. all animal experiments were approved by the institutional animal care and use committee (iacuc) at baylor college of medicine. additional file 7: fig. s2 . summary of number of statistically significant up and down-regulated genes, and quantification of candidate genes with respect to the individual reproductive tissue or cell of interest. the plots in panels (a) and (b) summarizes the number of statistically significant human or mouse genes respectively, that are up-regulated or downregulated in each reproductive tissue or cell of interest compared to the non-reproductive tissue with maximal gene expression. red columns depict the number genes that are up-regulated and blue columns depict the number genes that are down-regulated. changes in gene expression were considered statistically significant for an fdr of less than or equal to 0.05. the total number of candidate genes are designated by the black columns. candidate genes are genes that passed the fdr and tpm expression value filters.additional file 8: fig. s3 . venn diagrams comparing the overlap between the candidate male reproductive genes identified by the indicated reproductive tissues. the human testis combined gene list is the list of genes from both new samples we isolated and from previously published testis samples. the human epididymis combined gene list is the list of genes identified in either previously published samples or the newly generated samples across all sections of the epididymis. lastly, the mouse epididymis combined gene list is the list combined list of genes identified across all three sections of the mouse epididymis.additional file 9: table s7 . complete cross-sample comparison identifying human and mouse reproductive tract specific genes common to two or more samples and unique to each as identified through our studies.additional file 10: fig. s4 . classification of genes into different protein families and identification of the existence of an experimental mouse model. each candidate human gene was classified as an enzyme (enzyme), chromosome and histone modifiers (epigenetic), g-proteincoupled receptor (gpcr), orphan g-protein-couple receptor (ogpcr), kinase (kinase), transcription factor (tf), nuclear receptor (nr), ion channel (ic), chromosome and histone modifying transcript factor (tf; epigenetic), transporter (transporter) and unknown (a). the total number of candidate genes identified in our search for mouse models were plotted. orange columns designate the number of candidate genes where a model was identified while yellow designates candidate genes where a model was not identified (b).additional file 11: table s8 . drug target type classification for human genes. genes are listed according to the tissue and/or cell that they were identified as reproductive tract-specific in.additional file 12: table s9 . availability of a mouse model for human genes with a mouse ortholog. genes are listed according to the tissue and/or cell that they were identified as reproductive tract-specific in.additional file 13: fig. s5 . one-hundred and forty-two previously identified human male reproductive tract-specific genes that remain without a reported mouse model. the listed genes were identified in one or more datasets as indicated in the venn diagram. underlined genes were also identified in our studies as reproductive tract-specific in mouse. genes written in blue encode either enzymes, kinases, gpcrs, ogpcrs, transporters, transcription factors, or proteins involved in epigenetic regulation. genes written in dark red were identified in both testis (testis and/or testis cell) and in epididymis.additional file 14: fig. s6 . seventy-three human male reproductive tract-specific genes that each have a reported mouse model with male infertility phenotype. the listed genes were identified in one or more datasets as indicated in the venn diagram. underlined genes were also identified in our studies as reproductive tract-specific in mouse. genes written in blue encode either enzymes, kinases, gpcrs, ogpcrs, transporters, transcription factors, or proteins involved in epigenetic regulation. genes written in dark red were identified in both testis (testis and/or testis cell) and in epididymis.additional file 15: fig. s7 . rt-pcr confirmation of reproductive tractspecificity in both humans (a) and mice (b). the genes listed in this figure were identified through our studies and previous studies, but currently remain without a reported mouse model. gapdh and hprt are included as housekeeping genes.additional file 16: fig. s8 . eighty-nine novel human genes without a mouse ortholog. the listed genes were identified in one or more datasets as indicated in the venn diagram. genes written in blue encode either enzymes, kinases, gpcrs, ogpcrs, transporters, transcription factors, or proteins involved in epigenetic regulation. genes written in dark red were identified in both testis (testis and/or testis cell) and in epididymis.additional file 17: fig. s9 . novel reproductive tract-specific human genes that do not have any equivalent mouse orthologs. these genes may serve as potential contraceptive targets, however functional validation would need to be carried out in another model organism than mouse, such as rat or marmoset, which do have orthologs to these genes. the digital pcr (heatmap) depicts the average transcripts per million (tpm) value per tissue per gene from the indicated human rna-seq datasets as processed in parallel through our bioinformatics pipeline. white = 0 tpm, black ≥30 tpm. the expression profile of the human housekeeping gene, gapdh, is included as reference. for data obtained from published datasets, superscript values reference the dataset publication as previously mentioned.additional file 18: table s10 . 1064 previously identified genes. genes previously identified as male reproductive tract-specific through high throughput gene expression studies using either microarrays or rna-seq [2] [3] [4] [5] [6] [7] [8] . the human ortholog to genes identified in mouse and rat studies is included.additional file 19: fig. s10 . developmental expression pattern of spint3, spint4, spint5, pp2d1, and saxo1 in epididymis and testis of postnatal and adult mice. whole epididymides were used at postnatal days 3, 6, 10, and 14 and epididymis segments (caput, corpus, and cauda) were used at postnatal days 21, 28, 35, and 60. whole testes were used at all time points. the housekeeping gene, hprt, was used as reference.additional file 20: fig. s11 . multi-channel fluorescence images of bilateral epididymis serial sections stained with custom rnascope probes targeting either spint3, spint4, or spint5 mrna (red) and dapi (blue). the position of caput (cap), corpus (cor), and cauda (cau) is labeled in the overview image (left column). the position of the magnification over the epididymis is the same for all three sections (right column).additional file 21: fig. s12 . representative periodic acid-schiff staining of spint3 and spint4/5 knockout and littermate control (wild-type) testes and epididymis segments (caput, corpus, and cauda) at 3 months of age.additional file 22: fig. s13 . representative periodic acid-schiff staining of ces5a knockout and littermate control (wild-type) testes and epididymis segments (caput, corpus, and cauda) at 3 months of age.additional file 23: fig. s14 . representative scanning electron microscopy images of spint4/5 and ces5a ko and littermate control mouse sperm.additional file 24: fig. s15 . representative periodic acid-schiff staining of pp2d1 and saxo1 knockout and littermate control (wild-type) testes and epididymis segments (caput, corpus, and cauda) at 3 months of age. all authors have no competing interests. key: cord-104092-yau3r79c authors: tamming, renee j.; dumeaux, vanessa; langlois, luana; ellegood, jacob; qiu, lily r.; jiang, yan; lerch, jason p.; bérubé, nathalie g. title: atrx deletion in neurons leads to sexually-dimorphic dysregulation of mir-137 and spatial learning and memory deficits date: 2019-04-13 journal: biorxiv doi: 10.1101/606442 sha: doc_id: 104092 cord_uid: yau3r79c mutations in the atrx chromatin remodeler are associated with syndromic and non-syndromic intellectual disability. emerging evidence points to key roles for atrx in preserving neuroprogenitor cell genomic stability, whereas atrx function in differentiated neurons and memory processes are still unresolved. here, we show that atrx deletion in mouse forebrain glutamatergic neurons causes distinct hippocampal structural defects identified by magnetic resonance imaging. ultrastructural analysis revealed fewer presynaptic vesicles and an enlarged postsynaptic area at ca1 apical dendrite-axon junctions. these synaptic defects are associated with impaired long-term contextual memory in male, but not female mice. mechanistically, we identify atrx-dependent and sex-specific alterations in synaptic gene expression linked to mir137 levels, a known regulator of presynaptic processes and spatial memory. we conclude that ablation of atrx in excitatory forebrain neurons leads to sexually dimorphic outcomes on mir-137 and on spatial memory, identifying a promising therapeutic target for neurological disorders caused by atrx dysfunction. summary statement ablation of the atrx chromatin remodeler specifically in forebrain excitatory neurons of mice causes male-specific deficits in long-term spatial memory associated with mir-137 overexpression, transcriptional changes and structural alterations corresponding to preand post-synaptic abnormalities. alpha-thalassemia x-linked intellectual disability syndrome, or atr-x syndrome, is a rare congenital x-linked disorder resulting in moderate to severe intellectual disability (id), developmental delay, microcephaly, hypomyelination, and a mild form of alphathalassemia [omim: 301040] 1 . in a recent study of approximately 1000 individuals with id, atrx mutations were identified as one of the most frequent cause of non-syndromic id 2 , emphasizing a key requirement for this gene in cognitive processes. atrx-related id arises from hypomorphic mutations in the atrx gene, most commonly in the highly conserved atrx/dnmt3/dnmt3l (add) and switch/sucrose non-fermenting (swi/snf) domains 3, 4 . the former targets atrx to chromatin by means of a histone reader domain that recognizes specific histone tail modifications 5 , and the latter confers atpase activity and is critical for its chromatin remodeling activity 6,7 . atrx, in a complex with the histone chaperone daxx, promotes the deposition of the histone variant h3.3 at heterochromatic domains including telomeres and pericentromeres 8, 9 . however, atrx is also required for h3.3 deposition within the gene body of a subset of g-rich genes, presumably to reduce g-quadruplex formation and promote transcriptional elongation 10 . atrx is also required for the postnatal suppression of a network of imprinted genes in the neonatal brain by promoting long range chromatin interactions via ctcf and cohesin 11 . in mice, germline deletion of atrx results in embryonic lethality 12 while conditional deletion of atrx in neuroprogenitors leads to excessive dna damage caused by dna replication stress and subsequent tp53-dependent apoptosis 13, 14 . mice with deletion of exon 2 of atrx (atrx δe2 ) were generated that result in global reduction of atrx expression. these mice are viable and exhibit impaired novel object recognition memory, spatial memory in the barnes maze, and contextual fear memory 15 . some of the molecular defects identified in these mice included decreased activation of camkii and the ampa receptor in the hippocampus as well as decreased spine density in the medial prefrontal cortex, and altered dna methylation and increased expression of xlr3b in neurons 16 . our group also reported similar behavioural impairments in female mice exhibiting mosaic expression of atrx in the central nervous system 17 . however, the contribution of different cell types and sex difference to behavioural abnormalities have not yet been resolved. to start addressing these questions, we deleted atrx specifically in glutamatergic forebrain neurons in male and female mice. this approach bypasses deleterious effects of atrx loss of function that we previously observed during brain development caused by replication stress in proliferating neuroprogenitors 12, 13 . a comprehensive analysis of these mice reveals that atrx promotes long-term spatial learning and memory associated with morphological and synaptic ultrastructural changes in the hippocampus. we show that female mice lacking atrx in neurons are protected from spatial learning and memory defects and identify sex-specific effects of atrx loss on the expression of synaptic genes and mir-137. overall, we identify a novel sex-specific function for atrx in neurons in the regulation of long-term spatial memory associated with abnormal synapse ultrastructure. animal care and husbandry. mice were exposed to a 12-hour-light/12-hour-dark cycle and with water and chow ad libitum. the atrx loxp mice have been described previously 18 . atrx loxp mice were mated with c57bl/6 mice expressing cre recombinase under the control of the αcamkii gene promoter 19 . the progeny includes hemizygous male mice that produce no atrx protein in forebrain excitatory neurons (atrx-cko). the atrx-cko males were mated to atrx loxp females to yield homozygous deletion of atrx in female mice (atrx-cko fem ). male and female littermate floxed mice lacking the cre allele were used as controls (ctrl; ctrl fem ). genotyping of tail biopsies for the presence of the floxed and cre alleles was performed as described previously 18 arrive guidelines were followed: mouse groups were randomized, experimenters were blind to the genotypes, and software-based analysis was used to score mouse performance in all the tasks. all behavioural experiments were performed between 9:00 am and 4:00 pm. immunofluorescence staining. mice were perfused with 25ml phosphate buffered saline (pbs) followed by 25ml 4% paraformaldehyde (pfa) in pbs and the brain fixed for 24 hours in 4% pfa in pbs and cryopreserved in 30% sucrose/pbs. brains were flash frozen in cryomatrix (thermo scientific) and sectioned at 8µm thickness as described previously 13 . for immunostaining, antigen retrieval was performed by incubating slides in 10 mm sodium citrate at 95°c for 10 min. cooled sections were washed and blocked with 10% normal goat serum (sigma). the slides were incubated overnight in primary antibody sections were washed again three times for 5 min, counterstained with dapi and mounted with slowfade gold (invitrogen). all images were captured using an inverted microscope (leica dmi 6000b) with a digital camera (hamamatsu orca-er). openlab image software was used for manual image capture, and images were processed using the volocity software (demo version 6.0.1; perkinelmer) and adobe photoshop cs6 (version 13.0). cell counts of dapi, gfap, and iba1 were performed in adobe photoshop. dapi was counted per mm 2 and gfap and iba1 were counted as percentages of dapi + cells. one section from five pairs of ctrl/atrx-cko was counted. reverse transcriptase real-time pcr (qrt-pcr). total rna was isolated from control and atrx-cko frontal cortex and hippocampus using the mirvana total rna isolation kit (thermofisher) and reverse transcribed to cdna using 1 μg rna and superscript ii reverse transcriptase (invitrogen). real-time pcr was performed in duplicate using gene-specific primers under the following conditions: 95°c for 10 s, 58°c for 20 s, 72°c for 30 s for 35 cycles. all data were normalized against β-actin expression levels. primers used were as follows: β-actin (forward ctgtcgagtcgcgtccaccc, reverse acatgccggagccgttgtcg); atrx (forward agaaattgaggatgcttcacc, reverse tgaacctggggacttctttg). total rna was also used for reverse transcription of mirna using the taqman advanced microrna reverse transcription kit (thermofisher). qrt-pcr was performed using the short-term memory, mice were exposed to the original object (a) and a novel object (b; a blue plastic pyramid attached on top of a green prism base) 1.5 hours after training. to test long-term memory, mice were exposed to (a) and (b) 24 hours after training. novel object recognition was expressed as the percentage of time spent with the novel object as a fraction of the total time spent interacting. interaction was defined as sniffing or touching the object, but not leaning against or climbing on the object. morris water maze. the morris water maze was conducted as described previously 36 touchscreen assays. the paired associate learning (dpal) and visual paired discrimination (vpd) and reversal tasks were performed as previously described 37-39 . animals were food-restricted to 85% starting body weight. animals were separated into two counter-balanced subgroups to control for time of day of testing, and equipment variation. mice were tested in bussey-saksida mouse touch screen chambers (lafayette neuroscience) with strawberry milkshake given as a reward. for the dpal acquisition phase, animals were tested for their ability to associate objects with locations. mice were presented with two images in two of three windows; one image was in its correct location (s+) and one was in one of its two incorrect locations (s-). the third window was blank. a correct response triggered reward presentation and start of an inter-trial period. the pre-training was repeated until mice reached criterion (completion of 36 trials within 60 minutes). the dpal evaluation phase was performed for 45 sessions over 9 weeks. a correct response triggered reward presentation, whereas an incorrect response caused a 5 s time out and the house lights to turn on. an incorrect response also resulted in a correction trial, where the same s+/s-images were presented in the same two locations until the mouse responded correctly. the mouse was given 36 trials over 60 minutes per day. percent correct, number of correction trials, latency to a correct or incorrect response, and latency to retrieve reward were recorded for each week. vpd acquisition required the animal to touch the same image (s+) no matter which window it appeared in. the other screen had an incorrect image (s-). a correct response triggered reward presentation, whereas an incorrect response triggered the house lights to turn on, a time out of 5 s, and a correction trial to begin (previous trial repeated until a correct choice is made). this was repeated until mice reached criterion of 24/30 trials correct within 60 minutes over 2 consecutive days, after which baseline measurements were done for two sessions. parameters for baseline were identical to the acquisition steps. immediately following baseline measurements, the vpd task reversal began, where most parameters were the same as the acquisition, but the correct image associated with the reward was s-, and the incorrect response that triggers house lights was s+. this also determined total number of vesicles per synapse. vesicle cluster size was measured to calculate vesicle density. the area of the post-synaptic density was also quantified. statistics were calculated by two-way repeated measures anova with sidak's multiple comparison test or unpaired student's t-tests where applicable. statistical analyses. all data were analyzed using graphpad prism software with student's t test (unpaired, two-tailed) or one or two-way repeated measures anova with sidak's post-test where applicable. all results are depicted as mean +/-sem unless indicated otherwise. p values of less than 0.05 were considered to indicate significance. we generated mice lacking atrx in postnatal forebrain excitatory neurons by cre/loxp mediated recombination of the mouse atrx gene with the camkii-cre driver line of mice 49 . to confirm loss of atrx, we performed immunofluorescence staining of control and conditional knockout (atrx-cko) brain cryosections obtained from 3-month-old mice (figure 1a,b) . atrx is highly expressed in excitatory neurons of the hippocampus of control mice, including the cortex and hippocampal ca1, ca2/3, and dentate gyrus neurons, but is absent in these cells in the atrx-cko mice. additional validation of atrx inactivation in atrx-cko mice was achieved by qrt-pcr (figure 1c) , showing that atrx expression is decreased by 78% (+/-9.4%) and 81% (+/-1.7%) in the cortex and the hippocampus, respectively, which is expected from a neuron-specific deletion. the brain sub-region specificity of atrx loss was demonstrated by western blot analysis, showing reduced protein levels in the rostral and caudal cortices and hippocampus, but not in the cerebellum (figure 1d ). the mice survived to adulthood and had normal general appearance and behaviour. however, body weight measurements revealed a small but significant reduction in atrx-cko compared to control mice (figure 1e ). these findings demonstrate that we achieved specific deletion of atrx in excitatory neurons and while the mice were slightly smaller, they survived to adulthood, allowing further analyses in the adult brain. we first examined control and atrx-cko mouse brains for neuroanatomical anomalies by magnetic resonance imaging (mri). using a t2-weighted mri sequence, we were able to analyze and compare the entire brain as well as independent brain regions from 16 control and 13 atrx-cko male animals. the data obtained showed that the overall volume of the atrx-cko brain is significantly smaller compared to controls (92.8% of control volume, p<0.0001), as indicated by whole volume in mm 3 and cumulative serial slices of control and atrx-cko brains (figure 2a,b) , which correlates with the smaller body size of the mice. due to the reduction in body size and absolute total brain and hippocampal volumes of the atrx-cko mice, we next examined hippocampal neuroanatomy relative to total brain volume ( we postulated that the increase in relative volume of the ca1 sr/slm may be due to increased length or branching of ca1 apical dendrites. to investigate this possibility, golgi staining was used to sporadically label neurons (figure 3a ) and sholl analysis was performed on confocal microscopy images to evaluate apical dendrite branching of ca1 hippocampal neurons. however, no significant difference in dendritic branching or length was observed between control and atrx-cko mice, whether analyzed separately for apical or basal dendrites (figure 3b-g) . increased relative volume might also be caused by an increased number of cells, but immunofluorescence staining and quantification of astrocytes (gfap+) and microglia (iba1+) and total number of cells (inferred from dapi+ staining) revealed no differences in atrx-cko hippocampi (figure 3h-j) . overall, the increased relative volume of the ca1 sr/slm cannot be explained by increased length or complexity of dendritic trees or by an increased number of glial cells. based on the hippocampal structural alterations we detected by mri, we looked more closely at potential ultrastructural changes in the ca1 sr/slm area using transmission electron microscopy (tem) (figure 4a) . the presynaptic boutons were divided in 50nm bins from the active zone, and the number of vesicles in each bin was counted. the spatial distribution of vesicles in relation to the cleft was unchanged between the atrx-cko mice and controls (figure 4b) . however, we found that the total number of vesicles, the density of the vesicles, and the number of docked vesicles was significantly decreased at atrx-cko compared to control synapses (figure 4c-e) . we also analysed other structural aspects of synapses and found that the size of the postsynaptic density and the width of the synaptic cleft were both increased in atrx-cko compared to controls (figure 4f,g) . the length of the active zone, cluster size, or diameter of the vesicles did not vary significantly between control and atrx-cko samples ( figure 4h -j). these results suggest that atrx is required for structural integrity of the pre-and post-synapse, including maintenance of the synaptic vesicle pool at pre-synaptic termini and potential defects in postsynaptic protein clustering. we to investigate the effects of neuronal-specific atrx ablation on spatial learning and memory, we tested the mice in the morris water maze task. the atrx-cko mice showed a significant delay in latency to find the platform on day 3 of the learning portion of the task; however, by day 4 they were able to find the platform as quickly as the control mice (f=4.622, p=0.0404; figure 5a ). this finding was reflected in the distance traveled p=0.0251; figure 5f , g). these behavioural analyses suggest that atrx in required in excitatory neurons for long-term hippocampal-dependent spatial learning and memory. to determine whether loss of atrx in female mice would exhibit similar behavioural defects as seen in male mice, we generated atrx-cko female mice (atrx given the observed male-specific defects in spatial memory, we performed additional translational cognitive tasks on the atrx-cko male mice. the dpal touchscreen task in mice is analogous to cognitive testing done in humans by the cambridge neuropsychological test automated battery (cantab) 50,51 and normal performance in this task is thought to partly depend on the hippocampus 39,52 . control and atrx-cko mice were trained to identify the position of three images as depicted in figure 6a , undergoing 36 trials per day for 10 weeks. the results demonstrate that the atrx-cko mice exhibit a profound deficit in this task, indicated by both the percent correct (f=10.53, p=0.0031; figure 6b ) and the number of correction trials required (f=30.64, p<0.0001; figure 6c ) (supplementary video 1, 2) . these defects were not due to an inability to perform within the chamber or to attentional deficits, as latency to a correct answer (f=0.4802, p=0.4943), to an incorrect answer (f=0.1259, p=0.7255), and to retrieve the reward (f=0.9840, p=0.3300) was not significantly different between control and atrx-cko mice (figure 6d-f) . to determine if the impairment in the dpal task is caused by a vision problem rather than a learning defect, the mice were also tested in the visual paired discrimination (vpd) touchscreen task which requires the mice to discriminate between two images regardless of position on the screen. while the atrx-cko mice took significantly longer to reach the criterion pre-testing (t=2.945, p=0.0067; to identify the molecular mechanism(s) leading to spatial memory impairment, we performed rna-sequencing in both male and female hippocampi obtained from three pairs of littermate-matched ctrl/atrx-cko and ctrl fem /atrx-cko fem mice. there were 1520 transcripts differentially expressed in the atrx-cko males compared to control counterparts and 9068 transcripts in atrx-cko fem compared to the female controls (fdr < 0.20). to isolate transcripts that were likely to be causative to the impaired learning and memory phenotype in the male mice which was not found in the female mice, we focused on transcripts whose changes in expression with the atrx-cko were differential between male and female mice (n = 1054 transcripts, interaction term fdr < 0.05). the expression heat map of these transcripts illustrates that their expression levels are similar in control males and females but are differentially expressed when atrx is lost depending on sex (figure 7a) . we then utilized panther 53 , a tool for gene enrichment analysis based on functional annotations to examine gene ontology biological processes for which our list of transcripts was enriched (figure 7b) . the top five pathways included neurotransmitter receptor transport to postsynaptic membrane, protein localization to postsynaptic membrane, non-motile cilium assembly, and vesicle-mediated transport to the membrane. therefore, the rna sequencing revealed many transcripts related to synapses, supporting the tem data. certain mirna are enriched within presynaptic terminals and have been figure 5a ) as well as an enrichment for targets of mir-137 (supp. figure 5b) . we compared the list of genes downregulated in the atrx-cko male hippocampi to those predicted to be regulated by mir-137 through mirna.org (supp. table 1) . we found shank2, cadps2, glrb, and sgip1 expression to be inversely 64 . this data provides additional evidence that loss of atrx in the cortex and hippocampus of male mice leads to increased mir-137 expression and consequent downregulation of its target genes, starting at early stages of forebrain development. this study presents evidence that atrx is required in a sex-specific manner in excitatory forebrain neurons for normal spatial learning and memory (figure 8) [75] [76] [77] . in humans, neurological disorders such as autismspectrum disorders tend to preferentially affect males rather than females, possibly due to combinatorial contributions of hormonal and genetic factors in a phenomenon known as the female protective effect [78] [79] [80] , and this is regularly supported with mouse models [81] [82] [83] . the presence of estrogen and estrogen receptor in the female brain has been shown to be neuroprotective and leads to enhanced schaffer-collateral ltp 84 . in addition, certain x-linked genes involved in chromatin regulation (e.g. utx, a histone demethylase) are able to escape x-inactivation and so are expressed two-fold in females compared to males 85 in conclusion, our study presents strong evidence that atrx is required in forebrain excitatory neurons for spatial learning and long-term memory and regulation of genes required for efficient synaptic transmission. vpd touchscreen assays were performed at the robarts research institute neurobehavioural core facility, tem imaging at the biotron at western university and the rna sequencing at the london regional genomics centre. mutations in a putative global transcriptional regulator cause x-linked mental retardation with alpha-thalassemia (atr-x syndrome) targeted next-generation sequencing analysis of 1,000 individuals with intellectual disability mutations in transcriptional regulator atrx establish the functional significance of a phd-like domain microrna-34a regulates neurite outgrowth, spinal morphology, and function mir-27b shapes the presynaptic transcriptome and influences neurotransmission by silencing the polycomb group protein bmi1 the schizophrenia risk gene product mir-137 alters presynaptic plasticity the swi/snf protein atrx co-regulates pseudoautosomal genes that have translocated to autosomes in the mouse genome toppgene suite for gene list enrichment analysis and candidate gene prioritization the shank family of scaffold proteins the glycine receptor cloning and characterization of human cadps and cadps2, new members of the ca2+-dependent activator for secretion protein family intersectin 1 forms complexes with sgip1 and reps1 in clathrin-coated pits disruption of the direct perforant path input to the ca1 subregion of the dorsal hippocampus interferes with spatial working memory and novelty detection a macromolecular synthesis-dependent late phase of long-term potentiation requiring camp in the medial perforant pathway of rat hippocampal slices abnormal microglia and enhanced inflammation-related gene transcription in mice with conditional deletion of ctcf in camk2a-cre-expressing neurons lipopolysaccharide-induced microglial activation induces learning and memory deficits without neuronal cell death in rats learning, memory, and glial cell changes following recovery from chronic unpredictable stress disruption of the perineuronal net in the hippocampus or medial prefrontal cortex impairs fear conditioning modification of extracellular matrix by enzymatic removal of chondroitin sulfate and by lack of tenascin-r differentially affects several forms of synaptic plasticity in the hippocampus new translational assays for preclinical modelling of cognition in schizophrenia: the touchscreen testing method for mice and rats synaptic scaffold evolution generated components of vertebrate cognitive complexity x-linked alphathalassemia/mental retardation (atr-x) syndrome: localization to xq12-q21.31 by x inactivation and linkage analysis sexually dimorphic behavior, neuronal activity, and gene expression in chd8-mutant mice mecp2 organizes juvenile social behavior in a sex-specific manner mecp2 modulates sex differences in the postsynaptic development of the valproate animal model of autism epidemiology of pervasive developmental disorders a higher mutational burden in females supports a "female protective model" in neurodevelopmental disorders transcriptomic analysis of autistic brain reveals convergent molecular pathology human crmp4 mutation and disrupted crmp4 expression in mice are associated with asd characteristics and sexual dimorphism shank1 deletions in males with investigation of sex differences in the expression of rora and its transcriptional targets in the brain as a potential contributor to the sex bias in autism memory-related synaptic plasticity is sexually dimorphic in rodent hippocampus sex-specific differences in expression of histone demethylases utx and uty in mouse brain and neurons mirna expression profile after status epilepticus and hippocampal neuroprotection by targeting mir-132 microarray based analysis of microrna expression in rat cerebral cortex after traumatic brain injury the brain-specific microrna mir-128b regulates the formation of fear-extinction memory supplementary figure 5: transcriptional profiling reveals dysregulation of presynaptic genes in atrx-foxg1 mice. (a) differentially expressed genes between control and atrx-foxg1 p0.5 forebrain were used for gene ontology analysis and top 25 biological processes were listed by p-value. (b) top mirna predicted to regulate differentially expressed genes from control and atrx-foxg1 mice we are grateful to doug higgs and richard gibbons for the atrx floxed mice, vania prado and marco prado for the camkii-cre mice, michael miller for advice on statistical analyses, and tim bussey for discussions on the touchscreen assays. the dpal and the authors declare no competing or financial interests. 0 key: cord-103703-t03r6ny8 authors: nguyen-tu, marie-sophie; martinez-sanchez, aida; leclerc, isabelle; rutter, guy a.; da silva xavier, gabriela title: reduced expression of tcf7l2 in adipocyte impairs glucose tolerance associated with decreased insulin secretion, incretins levels and lipid metabolism dysregulation in male mice date: 2020-05-20 journal: biorxiv doi: 10.1101/2020.05.18.102384 sha: doc_id: 103703 cord_uid: t03r6ny8 transcription factor 7-like 2 (tcf7l2) is a downstream effector of the wnt/beta-catenin signalling pathway and its expression is critical for adipocyte development. the precise role of tcf7l2 in glucose and lipid metabolism in adult adipocytes remains to be defined. here, we aim to investigate how changes in tcf7l2 expression in mature adipocytes affect glucose homeostasis. tcf7l2 was selectively ablated from mature adipocytes in c57bl/6j mice using an adiponectin promoter-driven cre recombinase to recombine alleles floxed at exon 1 of the tcf7l2 gene. mice lacking tcf7l2 in mature adipocytes displayed normal body weight. male mice exhibited normal glucose homeostasis at eight weeks of age. male heterozygote knockout mice (atcf7l2het) exhibited impaired glucose tolerance (auc increased 1.14 ± 0.04 -fold, p=0.03), as assessed by intraperitoneal glucose tolerance test, and changes in fat mass at 16 weeks (increased by 1.4 ± 0.09-fold, p=0.007). homozygote knockout mice exhibited impaired oral glucose tolerance at 16 weeks of age (auc increased 2.15 ± 0.15-fold, p=0.0001). islets of langerhans exhibited impaired glucose-stimulated insulin secretion in vitro (decreased 0.54 ± 0.13-fold atcf7l2ko vs control, p=0.02), but no changes in in vivo glucose-stimulated insulin secretion. female mice in which one or two alleles of the tcf7l2 gene was knocked out in adipocytes displayed no changes in glucose tolerance, insulin sensitivity or insulin secretion. plasma levels of glucagon-like peptide-1 and gastric inhibitory polypeptide were lowered in knockout mice (decreased 0.57 ± 0.03-fold and 0.41 ± 0.12-fold, p=0.04 and p=0.002, respectively), whilst plasma free fatty acids and fatty acid binding protein 4 circulating levels were increased by 1.27 ± 0.07 and 1.78 ± 0.32-fold, respectively (p=0.05 and p=0.03). mice with biallelic tcf7l2 deletion exposed to high fat diet for 9 weeks exhibited impaired glucose tolerance (p=0.003 at 15 min after glucose injection) which was associated with reduced in vivo glucose-stimulated insulin secretion (decreased 0.51 ± 0.03-fold, p=0.02). thus, our data indicate that loss of tcf7l2 gene expression in adipocytes leads to impairments on metabolic responses which are dependent on gender, age and nutritional status. our findings further illuminate the role of tcf7l2 in the maintenance of glucose homeostasis. transcription factor 7-like 2 (tcf7l2) is a member of the high mobility group box family of transcription factors, a downstream effector of the wnt/beta-catenin signalling pathway, and a key regulator of development and cell growth [1] . tcf7l2 function is important for the proper function of tissues involved in the regulation of energy homeostasis. for example, tcf7l2 is required for the maintenance of functional pancreatic beta cell mass and insulin release from the endocrine pancreas [2, 3] . in the liver, ablation of tcf7l2 expression from hepatocytes has variously been shown to lead to reduced hepatic glucose production and improved glucose homeostasis [4] , or hyperglycemia [5] . studies by macdougald and colleagues [6] [7] [8] have demonstrated that tcf7l2 is involved in the regulation of the expression of proadipogenic genes during adipocyte development. additionally, insulin/insulin receptor substrate-1 and insulin growth factor 1 have been shown to cross-talk with the wnt signalling pathway to regulate insulin sensitivity in preadipocytes [9, 10] . the presence of tcf7l2 binding sites on the promoter of the insulin receptor gene also suggests a role of tcf7l2/wnt signalling pathway in regulating insulin action in adipocytes [11] . genome-wide association studies (gwas) have identified single nucleotide polymorphisms (snps; rs12255372 and rs7903146) in the tcf7l2 gene as being amongst the most strongly associated with an increased risk of type 2 diabetes [12] . humans carrying the t risk allele at rs7903146 have elevated proinsulin levels, lowered first-phase insulin secretion and impaired responses to the incretin hormone glucagon-like peptide 1 (glp-1) [13] [14] [15] [16] [17] . whilst tcf7l2 variants appear chiefly to affect pancreatic beta cell function in man, the mechanisms driving impaired insulin secretion are still poorly defined [18, 19] . although rs7903146 in tcf7l2 is not associated with changes in overall tcf7l2 transcription (i.e. of all isoforms), with conflicting data regarding the association of rs7903146 with specific tcf7l2 variant expression in subcutaneous fat [20] [21] [22] , tcf7l2 expression has been shown to be reduced in adipose tissue from type 2 diabetes subjects [23] and in obese mice [24] . additionally, surgery-induced weight loss has been shown to regulate alternative splicing of tcf7l2 in adipose tissue [25] . tcf7l2 splice variant expression is regulated by plasma triglycerides and free fatty acids [25, 26] , with evidence indicating that acute intake of fat leads to reduced expression of tcf7l2 in human adipocytes [27] . thus, overall these data suggest that changes in tcf7l2 expression may be linked to adaptation to changes in fuel intake. efforts to understand the causal relationship between tcf7l2 and diabetes led to studies on several metabolic tissues, with the data suggesting that the combined effects of loss of tcf7l2 in multiple tissues may account for the diabetes phenotype [28] . for example, previous reports have described the impact of loss of tcf7l2 expression on adipocyte development and insulin sensitivity [24, 29] . in the present study, we used a genetic model of ablation of tcf7l2 gene expression in mature adipocytes to determine whether tcf7l2 plays a role in adipocyte function, independent of its role in adipogenesis. we have focused on whether loss of tcf7l2 expression in the adipocyte impacts the release of hormones involved in glucose homeostasis, notably insulin and incretins. in this way, we sought to explore the possibility that altered tcf7l2 expression in the adipocyte may contribute to type 2 diabetes risk through downstream effects on multiple effector organs [28, 30] . to generate tissue-specific knockout of tcf7l2 alleles, we crossed mice in which exon 1 (encoding for the beta-catenin-binding domain) was flanked by loxp sites [2] with mice expressing cre recombinase under the control of the adiponectin promoter [31] (a kind gift from d. withers, imperial college london) to produce deletion of one tcf7l2 allele (atcf7l2het) or two alleles (atcf7l2ko). littermates used as controls did not express cre recombinase but were homozygous or heterozygous for the floxed tcf7l2 allele. adiponectin-cre [31] or mice with tcf7l2 gene flanked by loxp sites (tcf7l2-floxed) [2] did not display phenotypes that deviate from wild-type littermate control mice, consequently we used tcf7l2-floxed mice as controls in our test cohorts. mice were born at the expected mendelian ratios with no apparent abnormalities. animals were housed 2-5 per individuallyventilated cage in a pathogen-free facility with 12:12 light:dark cycle with free access to standard mouse chow (rm-1; special diet services) diet and water. high fat diet (hfd) cohort were put under a high sucrose high fat diet (d12331; research diets) for 12 weeks from 7-week-old. for the chow diet cohort, metabolic exploration was performed on each animal within a 2-week window at 2 stages (8-week-old and 16-week-old). all in vivo procedures described were performed at the imperial college central biomedical service and approved by the uk home office animals scientific procedures act, 1986 (ho licence ppl 70/7971 to gdsx). glucose tolerance was performed on 15 h-fasted mice after an oral gavage of glucose (ogtt, 2 g/kg of body weight) or intraperitoneal injection of glucose (ipgtt, 1g/kg body weight. ipgtt and ogtt were performed at two stages (at 8-week-old and at 16-week-old) for each individual mouse. insulin tolerance was performed after a 5 h-fast with an intraperitoneal injection of insulin (ipitt, 0.5 u/kg in females, 0.75 u/kg in males under chow diet, 1.5u/kg in males under hfd). in vivo glucosestimulated insulin secretion was assessed after oral or intraperitoneal administration of glucose and blood was collected at 0-and 15-minutes post-injection to assess plasma insulin levels using an ultrasensitive mouse insulin elisa kit (crystal chem, netherlands) or using a homogeneous time resolved fluorescence (htrf) insulin kit (cisbio, france) in a pherastar reader (bmg labtech, uk). to assess insulin sensitivity, male mice were starved for four hours and following an intraperitoneal injection of insulin (1ui/kg body weight) adipose tissues were collected and frozen in liquid nitrogen. adipose tissue proteins were extracted in lysis buffer (150 mmol/l nacl, 50 mmol/l tris-hcl ph 8.0, 1% np-40) supplemented with protease inhibitors (roche, germany) and phosphatase inhibitors (sigma-aldrich, uk) and analysed by western blotting using antibodies for tcf4/tcf7l2 (c48h11) (#2569, 1:500, cell signalling, neb, uk), phospho-akt (#9271, 1:1000, cell signalling, neb, uk), total-akt (#9272, 1:1000, cell signalling, neb, uk), gapdh (#2118, 1:10000, cell signalling, neb, uk), alpha-tubulin (t5168, 1:10 000, sigma-aldrich, uk). fiji software was used for densitometry quantification. uncut versions of all western blot images are presented in supplemental figure s1. islets were isolated by digestion with collagenase as described [32] . in brief, pancreata were inflated with a solution of collagenase from clostridium histolyticum (1 mg/ml; nordmark, germany) and placed in a water bath at 37 ⁰c for 12 min. islets were washed and purified on a histopaque gradient (sigma-aldrich, uk). isolated islets were cultured for 24 h in rpmi 1640 containing 11.1 mmol/l glucose, 10% foetal bovine serum and l-glutamine (sigma-aldrich, uk) and allowed to recover overnight. insulin secretion assays on isolated mouse islets were performed as previously described [32] . in measurement of intracellular calcium dynamics was performed as previously described [33] . in brief, whole isolated islets were incubated with fura-8am (invitrogen, uk) [34] for 45 min at 37⁰c in khb containing 3 mmol/l glucose. fluorescence imaging was performed using a nipkow spinning disk head, allowing rapid scanning of islet areas for prolonged periods of time with minimal phototoxicity. volocity software (perkinelmer life sciences, uk) provided interface while islets were kept at 37⁰c and constantly perifused with khb containing 3 mmol/l or 17 mmol/l glucose or 30 mmol/l kcl. epididymal adipose tissue were removed from euthanized mice, fixed overnight in 10% formalin and subsequently embedded in paraffin wax. adipose tissue slices (5 μm) were stained with hematoxylin and eosin (sigma-aldrich, uk) for morphological analysis. blood in the fed state was obtained by tail bleeding, and circulating factor concentrations were measured using the following kits according to the respective manufacturer's protocols: bio-plex protein array system (biorad, uk) with one multiplex panel was used to measure total glucagon-like peptide-1 (glp-1), glucose-dependent insulinotropic polypeptide (gip), leptin, adiponectin and plasminogen activator inhibitor-1 (pai-1); one multiplex panel was used to measure fatty acid binding protein 4 (fabp4) and resistin (r&d systems, uk); non-esterified fatty acid (nefa) serum levels were measured by colorimetric assay (randox, uk) and dipeptidyl peptidase 4 (dpp4; r&d systems, uk) levels were measured by elisa. rna was isolated from epididymal and subcutaneous adipose tissue, liver and pancreatic islets with trizol following manufacturer's instructions (invitrogen, uk). rna purity and concentration were measured by spectrophotometry (nanodrop, thermo scientific, uk). only rna with absorption ratios between 1.8-2.0 for 260/280 and 260/230nm were used. rna integrity was checked on an agarose gel. rna was reversed transcribed using high-capacity cdna reverse transcription kit (applied biosystems, uk). qpcr was performed with fast sybr green master mix (applied biosystems, uk). the comparative ct method (2 -ct ) was used to calculate relative gene expression levels using gapdh, βactin or ppia as an internal control. the primers sequences are listed in supplemental table s1. data are shown as means ± sem. graphpad prism 8.4 was used for statistical analysis. statistical significance was evaluated by the two-tailed unpaired student t-test and one-or two-way anova, with tukey or bonferroni multiple comparisons post-hoc test as indicated in the figure legends. p values of <0.05 were considered statistically significant. to determine the role of tcf7l2 in the mature adipocyte, we generated a mouse line in which tcf7l2 is deleted specifically in these cells through the expression of cre recombinase under the control of the adiponectin promoter [31] . cre recombinase mediated the excision of exon 1 of tcf7l2 generating one single tcf7l2 allele deletion (atcf7l2het) or a biallelic tcf7l2 deletion (atcf7l2ko). tcf7l2 mrna levels were decreased in inguinal adipose tissue (iwat) by 39.8 ± 13.3% (p=0.008) while in epididymal adipose tissue (ewat), a reduction by 46.3 ± 20.1% did not reach any statistical significance (p=0.09) in atcf7l2het mice compared to controls. in atcf7l2ko mice, expression was reduced by 77.3 ± 11.2 % and 57.8 ± 20.2%, respectively in ewat and iwat compared to controls. conversely, no changes in tcf7l2 expression were apparent in liver and pancreatic islets from atcf7l2het and atcf7l2ko mice vs islets from littermate controls (fig.1a) . correspondingly, the content of the two tcf7l2 protein isoforms (79kda and 58kda) in ewat was significantly reduced by 41 ± 11 % and 35 ± 7 % respectively in atcf7l2het mice and reduced by 77 ± 17 % and 80 ± 14 % in atcf7l2ko mice ( fig.1b and c) . body weight was unchanged in male ( likewise, we found no change in fat or lean mass in 8 weeks old male or female mice, as assessed by echomri ( fig.1g , i, k and m). however, fat mass was significantly increased, and lean mass was decreased, in male atcf7l2het mice on normal chow diet ( fig.1h and j) . female mice showed no change in body composition with age ( fig.1l and n) . to explore the effects of adipocyte-specific tcf7l2 ablation on whole body metabolism, we measured glucose tolerance, and found an age-dependent impairment in the response to glucose administration. glucose challenge was assessed in male and female mice at a young stage (8 weeks of age) and at an older stage (16 weeks of age). in younger mice, blood glucose levels after intraperitoneal injection of glucose were similar in atcf7l2het and atcf7l2ko compared to sex-matched littermate controls regardless of the gender ( fig.2a and fig.3a ). in older mice, glucose levels were impaired in male atcf7l2het mice rising to 17.7 ± 1.0 mmol/l at 15 minutes after intraperitoneal injection of glucose compared to littermate controls rising to 13.4 ± 0.8 mmol/l (fig.2b) . likewise, atcf7l2het and young atcf7l2ko mice had similar increases in glucose levels 15 minutes after oral administration of glucose (17.4 ± 1.1 mmol/l and 15.8 ± 0.7 mmol/l respectively) compared to littermate controls (16.7 ± 0.7 mmol/l; fig.2c) . surprisingly, glucose tolerance (as assessed by oral glucose tolerance test) was impaired in older atcf7l2ko mice (17 weeks) compared to littermate controls (fig.2d) . female mice exhibited no change in glucose tolerance regardless of age and genotype (fig.3a, fig.3b and fig.3c ). whole body insulin sensitivity was unaffected in both genders across all genotypes ( fig.2e and fig.3d ). in order to assess whether ablation of tcf7l2 in mature adipocyte affects organs involved in glycaemic control, beta cell secretory capacity was measured during glucose challenge. we sought first to examine whether impaired intraperitoneal and oral glucose challenge in older male atcf7l2het and atcf7l2ko mice was due to an impact on insulin secretion. fasting plasma insulin levels and in vivo glucose-stimulated insulin release were similar in atcf7l2het and atcf7l2ko mice vs littermate controls after intraperitoneal injection of glucose (fig.2f) or after oral administration of glucose (fig.2g) . glucose stimulated insulin secretion in isolated islets was decreased after high (17 mm) glucose incubation (0.32 ± 0.08% in atcf7l2ko vs 0.59 ± 0.13% in controls) while responses to kcl (30 mm) were not different in islets from atcf7l2ko compared to islets from littermate controls (fig.2h) . when exploring after tcf7l2 loss expression of key genes associated with normal beta cell function, we observed no significant changes in gene expression. indeed, no difference was observed for the expression of the insulin (ins1, ins2), or glucagon (gcg) genes in islets from all genotypes, however a reduction in the expression of the glucose transporter 2 (glut2/slc2a2) gene was observed in atcf7l2ko mice (0.73 ± 0.06 in atcf7l2ko vs 1.00 ± 0.03 in controls; fig.2i ). to further evaluate the origins of the secretory defects observed in isolated islets (fig. 2h) , we measured the changes in cytosolic calcium in response to incubation with varying concentrations of glucose (3 mm, 17 mm) in the presence or absence of kcl (30 mm) on isolated islets (fig.2j) . islets from atcf7l2ko male mice showed a diminished response to high glucose incubation compared to atcf7l2het animals, while differences compared to control mice did not reach any statistical significance. islets from atcf7l2het male mice showed an increase response to kcl compared to controls (fig.2j) . in vivo after intraperitoneal injection and in vitro glucose stimulated insulin secretion was unchanged in female atcf7l2kohet and atcf7l2ko mice ( fig.3e and f) . therefore, alterations in pancreatic beta cell function observed ex vivo in the absence of tcf7l2 in adipocyte have no impact on whole body glucose-stimulated insulin secretion. to investigate the causes of impaired oral glucose tolerance in male atcf7l2ko mice, we measured the circulating levels of other hormones regulating glucose metabolism. circulating glp-1 and gip levels in plasma were decreased in older male atcf7l2ko mice compared to age-and sex-matched littermate control mice (glp-1: 23.7 ± 6.8 vs 57.6 ± 11.6 ng/ml; gip: 335.1 ± 21.4 vs 583.8 ± 39.5 ng/ml respectively; fig.4a and fig.4b ). we observed a significant decrease in gip levels (421.8 ± 24.6 vs 583.8 ± 39.5 ng/ml in controls), but no robust or statistical differences in glp-1 levels in atcf7l2het mice ( fig.4a and b) . plasma dpp4 levels in male atcf7l2ko mice were not different compared to controls (fig.4c ). evidence suggests a role of wnt/tcf7l2 signalling in the control of lipid metabolism [24, 35] . we, therefore, sought to determine whether tcf7l2 could play a role as a regulator of fatty acid release from mature adipocyte. plasma levels of circulating nefa and the lipid carrier fabp4 were found to be increased respectively in atcf7l2ko mice compared to age-and sex-matched littermate controls (nefa: 0.79 ± 0.04 vs 0.62 ± 0.04 mmol/l, respectively; fabp4: 75.0 ± 13.5 ng/ml vs 42.1 ± 6.0 ng/ml, respectively; fig.3d and e) . finally, we investigated endocrine adipocyte function by measuring adipokines which are usually found to be affected in insulin resistance and metabolic diseases. plasma levels of adiponectin, leptin, resistin and pai-1 were found to be unchanged in male mice of all genotypes (fig.4f , g and supplemental fig.s2a and b) . we next explored whether loss of tcf7l2 expression may affect insulin signalling in adipocytes, since previous reports [24, 29] described hepatic insulin resistance in a mouse model of ablation of tcf7l2. pkb/akt ser473 phosphorylation was elevated at basal condition prior insulin stimulation in older (17 weeks) atcf7l2 male mice ( fig.4h and i) , but pkb/akt ser473 phosphorylation in response to insulin was similar adipocytes from atcf7l2ko mice and control littermates. however, relative expression of phosphorylated akt after insulin stimulation compared to basal appeared decreased in atcf7l2ko mice compared to controls but was not robust enough to reach statistical significance (p=0.07; fig.4j ). indicative of unaltered hepatic insulin sensitivity, liver levels of the gluconeogenic genes g6pase and pepck (pck1) did not differ between control and atcf7l2ko mice (fig.4j) . therefore, our results suggest that tcf7l2 could control lipolysis and lipid metabolism in adipocyte. in order to investigate whether nutritional status had an impact on glucose metabolism in the absence of tcf7l2 expression in adipocytes, we maintained atcf7l2ko male mice on high fat (60%) diet for 12 weeks. we focused on males as no change were observed in female mice on chow diet. body weight trajectory showed a similar increase in atcf7l2ko compared to control mice (fig.5a) . glucose tolerance was altered after intraperitoneal injection of a high concentration (2g/kg) of glucose by a delay of 15 minutes in the blood glucose peak after injection of glucose compared to controls (17.6 ± 1.5 vs 23.7 ± 2.2 mmol/l respectively; fig.5b and c) . no significant change in oral glucose tolerance ( fig.5d and e) , or insulin sensitivity (fig.5f) , was observed between mice of all genotypes. insulin secretion was impaired during in vivo oral glucose challenge in atcf7l2ko compared to littermate controls (at 15 minutes, 4.6 ± 0.2 vs 8.9 ± 1.0 ng/ml respectively; fig.5h ). we observed no robust changes following intraperitoneal injection of glucose to reach statistical significance (fig.5g) . ex vivo insulin release in response to high glucose (17 mm), glp-1 (20nm) and kcl (30 mm) was found to be no different between islets of langerhans isolated from atcf7l2ko mice and littermate control mice (fig.5i) . in the present study, we demonstrate that changes in the expression of tcf7l2 in murine adult adipose tissue may lead to alterations not only in adipocyte function but also in the function of other tissues involved in the regulation of energy homeostasis in a gender-, age-, and nutritional status-dependent manner. thus, we provide evidence that deletion of tcf7l2 in adipocytes leads to alterations in the function of adipocytes, pancreatic islet beta cells, and enteroendocrine cells, thereby highlighting a role for tcf7l2 in systemic glucose homeostasis. wnt signalling and its effectors beta-catenin and tcf7l2 are critical during adipogenesis [6, 8, 29] . the presence of this signalling module during adulthood suggests that it may also be important for the function of adult adipocytes. in the present study, we found that young mice presented no alteration of glucose tolerance or body composition, regardless of the gender, in the absence of tcf7l2 in adipocyte, whilst defects appear with age ( fig.1g and fig.2a) . recently, tian et al. revealed crosstalk between wnt signalling and females hormones through tcf7l2 to regulate lipid metabolism [35] . thus, gender differences observed when expressing a dominant-negative form of tcf7l2 in hepatocytes would suggest that sex hormones regulate glucose homeostasis via repressing hepatic gluconeogenesis and regulate lipid metabolism [35] . in our study, we also found that female mice were largely unaffected by the loss of tcf7l2 function in the adipocyte, suggesting a role for female hormones to maintain glucose homeostasis. the key effector of the wnt signalling pathway is the association of beta-catenin and a member of the tcf family which may include tcf7l2, tcf7, tcf7l1 and lef-1 [1] . the availability of free betacatenin entering the nucleus to bind tcf7l2 is crucial for activation of expression of downstream target genes. however, the regulation of tcf7l2 expression is also essential. high-fat feeding modulates tcf7l2 expression in pancreatic islets, in hepatocytes and in human adipocytes [27, 36, 37] . furthermore, studies have suggested that variation in tcf7l2 expression altered glucose metabolism and induces type 2 diabetes phenotype [38] . we found that deletion of a single tcf7l2 allele generates distinct features of obesity-induced glucose intolerance while biallelic tcf7l2 deletion reveals a disruption of endocrine signalling molecules. therefore, alterations induced by deletion of tcf7l2 in mature adipocytes could depend on age and on the dosage of tcf7l2 expression. we found that under chow diet fed mice lacking tcf7l2 selectively in adipocytes displayed an impaired response to oral glucose challenge (fig. 2d) but normal tolerance to intraperitoneal injection of the sugar (fig. 2b) . this suggests an impaired incretin effect, defined as the postprandial insulin response provoked by incretin hormones such as glp-1 and gip. however, insulin release in response to an oral glucose challenge was maintained (fig. 3d) , whilst glucose-stimulated insulin secretion ex vivo from isolated islets of langerhans is impaired (fig. 3e) . our data therefore suggest that a mechanism exists to maintain insulin release in vivo after deletion of tcf7l2 from adipocytes. however, when challenged with high fat feeding, impaired glucose tolerance is associated with impaired insulin secretion ( fig.5b and h) . future studies will need to assess further the effects of highfat diet feeding on beta cell function on a larger cohort, as exploration were suspended during the pandemic events of covid-19. a striking finding in the present study is that tcf7l2 is required in adipose tissue for normal incretin production and insulin secretion: we reveal that decreased tcf7l2 expression in mature adipocytes leads to lowered circulating levels of glp-1 and gip ( fig.4a and b) . this suggests that a compensatory effect is unmasked in atcf7l2ko mice to stimulate insulin secretion in pancreatic beta cells when the incretin effect is compromised. one possible explanation to reconcile our findings on in vivo and ex vivo glucose-stimulated insulin secretion is that elevated fatty levels compensate in part for the lowered levels of circulating incretins, acting to amplify insulin release through the action of fatty acid receptors. nefa and specifically long-chain fatty acids potentiate glucose-stimulated insulin secretion [39, 40] . moreover, a direct insulinotropic action of fabp4, a cytosolic lipid chaperone expressed and secreted by white and brown adipocytes may act directly on pancreatic beta cells as demonstrated in previous studies showing that recombinant fabp4 administration enhanced glucose-stimulated insulin secretion in vitro and in vivo [41, 42] . in a small type 2 diabetes cohort, increased serum fabp4 was correlated to enhanced insulin release [43] . taking these findings together, lowered circulating incretins may have provoked impaired glucose tolerance with no apparent change in beta cell function to release insulin in vivo-compensated by stimulation of glucose-stimulated insulin secretion by higher circulating fabp4 and nefa-in atcf7l2ko mice. nevertheless, there is a need to further investigate the mechanisms that may link impaired glucose tolerance with normal insulin secretion. these might include mechanisms such as reduced insulinindependent glucose disposal, or insulin resistance-induced altered glut2 trafficking in enterocytes [44] . by what mechanisms might depletion of tcf7l2 from adipocytes lead to a decrease in the circulating levels of glp-1 and gip? our data suggest that decreased incretin levels are unlikely to be due to an increase in the rate of degradation of these hormones in the bloodstream, as no change was found in circulating dpp4 in atcf7l2ko mice (fig.4c) . we observed elevated circulating plasma fatty acids and fabp4 levels ( fig.4d and e) , reflecting altered adipocyte metabolism in the absence of tcf7l2. wnt and tcf7l2 are regulators of lipid metabolism in hepatocytes [35] , consistent with elevated plasma triglycerides associated with tcf7l2 risk variant rs7903146 [45] . moreover, the genetic variant is localized near the regulatory region for acyl-coa synthetase long chain 5 (acsl5) which activates fatty acids to generate long chain fatty acyl coa [45] . consistent with our model, martchenko and colleagues [46] have recently reported fatty acid-induced lowering of circadian release of glp-1 from l-cells as a result of decreased bmal1 expression. similarly, filipello et al [47] reported decreased insulin-dependent glp-1 secretion from l-cell-derived glutag cells, and increased glucagon release, upon fatty acid treatment. similar findings on glp-1 secretion were reported by others [48, 49] , whist long chain saturated (palmitate) but not unsaturated (oleate) fatty acids lead to l-cell apoptosis [50] . on the other hand, activation of free fatty acid receptors with ffar1/gpr40 agonist tak-875 [51] or with short-chain fatty acids (ffar2/gpr43) [52] acutely increased glp-1 secretion from l-cells, indicating a balance between shorter term, and more chronic "lipotoxic" effects, is likely to govern overall glp-1 production, with the latter predominating after tcf7l2 deletion in adipocytes. to explore further the direct role of tcf7l2 in lipid metabolism, future studies will be necessary to assess lipolysis and insulin action in adipocyte lacking tcf7l2. in this study, high fat diet feeding altered glucose tolerance and insulin secretion. some discrepancies emerge from our report and the previous studies using the same genetic model. when examining the effects of conditional deletion tcf7l2 in mature adipocytes, chen et al. [29] found impairments in glucose tolerance after intraperitoneal injection of glucose in 3-month-old males and females under standard diet associated with hepatic insulin resistance. geoghegan et al. [24] also generated a conditional knockout of tcf7l2 in the adipocyte, reporting that knockout animals maintained on regular chow displayed no change in intraperitoneal glucose tolerance, whilst exaggerated insulin resistance and impaired glucose tolerance were apparent after high fat feeding [24] . the authors demonstrated a role for tcf7l2 in regulating lipid metabolism, finding a reduction in lipid accumulation and lipolysis during high fat feeding. we note that slightly different genetic strategies were used to create the mouse models in each case. thus, both our own and the earlier studies used a similar same loxp strategy with a cre recombinase under the control of the adiponectin promoter, but we have deleted exon 1, whereas chen et al. targeted exon 11 and geoghegan et al. targeted exon 5 [24, 29] . the tcf7l2 gene consists of 17 exons [53] and tissue-specific splicing variants could exert tissue-dependent distinct function and impact differently on specific cell type function such as the adipocyte or the beta cell [54] . alternative splicing of tcf7l2 potentially results in transcripts lacking exons 1 and 2 predicted to encode proteins lacking the β-catenin-binding domain [22] . might changes in tcf7l2 expression in adipose tissue contribute to the effects of type 2 diabetes-associated variants? whilst inspection of data in the gtex database [55] does not reveal any genotype-driven alteration in subcutaneous or breast adipose tissue tcf7l2 expression for rs7903146, we note that other intronic variants in this gene, such as rs6585195, reveal significant expression quantitative trait loci for tcf7l2, and may conceivably influence incretin and insulin levels through the mechanisms identified here. in summary, tcf7l2 could play a post-developmental role on metabolic tissues depending on its level of expression and the age of the mice. our findings provide an unexpected insight into the action of tcf7l2 and reveal a novel mechanism through which adipocytes may impact insulin and incretin secretion. in demonstrating an action on multiple tissues, we also reveal a new level of complexity in the effects of gene action at the systems level on disease risk. such findings may highlight the importance of early interventions with incretins when tcf7l2 expression is compromised and therefore the development of novel personalized therapies. m-sn-t co-designed the study, collected, analysed, interpreted the data and drafted the manuscript. gdsx conceived and co-designed the study, collected, interpreted the data and substantially critically revised the manuscript. gar conceived the study and co-wrote the manuscript. am-s contributed to the collection of data and critically revised the manuscript for important intellectual content. il contributed for resources. all authors gave final approval of the manuscript and gave consent to publication. gar is the guarantor of this work. awards, mrc programme grants (mr/r022259/1, mr/j0003042/1, mr/l020149/1) an mrc experimental challenge grant (diva, mr/l02036x/1), mrc (mr/n00275x/1), diabetes uk (bda/11/0004210 this project has received funding from the european association for the study of diabetes, and university of birmingham to gdsx, european union's horizon 2020 research and innovation programme via the innovative medicines initiative 2 joint undertaking under grant agreement no 115881 (rhapsody) to gar. this joint undertaking receives support from the european union's horizon 2020 research and innovation programme and efpia. duality of interest gar has received grant funding from the wnt signaling pathway effector tcf7l2 and type 2 diabetes mellitus abnormal glucose tolerance and insulin secretion in pancreas-specific tcf7l2-null mice selective disruption of tcf7l2 in the pancreatic beta cell impairs secretory function and lowers beta cell mass diabetes risk gene and wnt effector tcf7l2/tcf4 controls hepatic response to perinatal and adult metabolic demand tcf7l2 modulates glucose 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mechanisms by which common variants in the tcf7l2 gene increase risk of type 2 diabetes tcf7l2 variant rs7903146 affects the risk of type 2 diabetes by modulating incretin action the t allele of rs7903146 tcf7l2 is associated with impaired insulinotropic action of incretin hormones, reduced 24 h profiles of plasma insulin and glucagon, and increased hepatic glucose production in young healthy men tcf7l2 rs7903146 impairs islet function and morphology in non-diabetic individuals human pancreatic islet three-dimensional chromatin architecture provides insights into the genetics of type 2 diabetes transcription factor 7-like 2 polymorphisms and type 2 diabetes, glucose homeostasis traits and gene expression in us participants of european and african descent genotype and tissue-specific effects on alternative splicing of the transcription factor 7-like 2 gene in humans tissue-specific alternative splicing of tcf7l2 transcription factor tcf7l2 genetic study in the french population: expression in human beta-cells and adipose tissue and strong association with type 2 diabetes targeted deletion of tcf7l2 in adipocytes promotes adipocyte hypertrophy and impaired glucose metabolism adipose tissue tcf7l2 splicing is regulated by weight loss and associates with glucose and fatty acid metabolism tcf7l2 is associated with high serum triacylglycerol and differentially expressed in adipose tissue in families with familial combined hyperlipidaemia tcf7l2 expression is regulated by cell differentiation and overfeeding in human adipose tissue tcf7l2 and glucose metabolism: time to look beyond the pancreas the diabetes gene and wnt pathway effector tcf7l2 regulates adipocyte development and function tcf7l2 and diabetes: a tale of two tissues, and of two species transcriptional control of adipose lipid handling by irf4 isolation and culture of mouse pancreatic islets for ex vivo imaging studies with trappable or recombinant fluorescent probes the transcription factor pax6 is required for pancreatic beta cell identity, glucose-regulated atp synthesis, and ca(2+) dynamics in adult mice lipotoxicity disrupts incretinregulated human beta cell connectivity the developmental wnt signaling pathway effector beta-catenin/tcf mediates hepatic functions of the sex hormone estradiol in regulating lipid metabolism insulin treatment and high-fat diet feeding reduces the expression of three tcf genes in rodent pancreas the wnt signaling pathway effector tcf7l2 is upregulated by insulin and represses hepatic gluconeogenesis alterations in tcf7l2 expression define its role as a key regulator of glucose metabolism gpr40 is necessary but not sufficient for fatty acid stimulation of insulin secretion in vivo endogenous fatty acids are essential signaling factors of pancreatic beta-cells and insulin secretion identification of fatty acid binding protein 4 as an adipokine that regulates insulin secretion during obesity circulating adipocyte fatty acid-binding protein induces insulin resistance in mice in vivo serum fatty acid-binding protein 4 (fabp4) concentration is associated with insulin resistance in peripheral tissues, a clinical study insulin internalizes glut2 in the enterocytes of healthy but not insulin-resistant mice the type 2 diabetes presumed causal variant within tcf7l2 resides in an element that controls the expression of acsl5 suppression of circadian secretion of glucagon-like peptide-1 by the saturated fatty acid, palmitate chronic exposure to palmitate impairs insulin signaling in an intestinal l-cell line: a possible shift from glp-1 to glucagon production differential molecular and cellular responses of glp-1 secreting l-cells and pancreatic alpha cells to glucotoxicity and lipotoxicity glucagon-like peptide-1 production in the glutag cell line is impaired by free fatty acids via endoplasmic reticulum stress mechanistic insights into the detection of free fatty and bile acids by ileal glucagon-like peptide-1 secreting cells vascular, but not luminal, activation of ffar1 (gpr40) stimulates glp-1 secretion from isolated perfused rat small intestine short-chain fatty acids stimulate glucagon-like peptide-1 secretion via the g-protein-coupled receptor ffar2 the human t-cell factor-4 gene splicing isoforms, wnt signal pathway, and apoptosis in renal cell carcinoma tcf7l2 splice variants have distinct effects on beta-cell turnover and function the genotype-tissue expression (gtex) project blood glucose levels during ogtt in male mice after 12 weeks of hfd (n=4 control mice, n=3 atcf7l2ko mice). (f) blood glucose levels during ipitt in male mice after 12 weeks of hfd (n=3 mice/genotype). (g) insulin plasma levels after intraperitoneal injection of glucose (2g/kg) in male mice following 12 weeks of hfd (n=4 control mice, n=3 atcf7l2ko mice). (h) insulin plasma levels after oral administration of glucose (2g/kg) in male mice following 12 weeks of hfd (n=4 control mice, n=3 atcf7l2ko mice). data were analysed by unpaired student's t-test, p*<0.05 versus control. (i) insulin secretion on isolated islets from male mice after 12 weeks of hfd during static incubation of 3 mmol/l glucose (3g), 17 mmol/l glucose (17g), a combination of 17 mmol/l glucose and 20 nmol/l glp-1 (17g+glp-1) and 30 mol/l kcl, (n=3 mice/genotype). (j) proposed mechanism: decreased tcf7l2 expression in adipocyte provoked increased circulating levels of lipids and subsequently, decreased incretins and glucose-stimulated insulin secretion (gsis) were observed in vivo the authors would like to thank dominic withers (mrc london institute of medical sciences (lms) at imperial college london) for the adipoq-cre mouse, lorraine lawrence (research histology facility at imperial college london) and stephen rothery (facility for imaging by light microscopy film at imperial college london) for technical assistance. key: cord-022505-17khcmta authors: delaney, martha a.; treuting, piper m.; rothenburger, jamie l. title: rodentia date: 2018-10-26 journal: pathology of wildlife and zoo animals doi: 10.1016/b978-0-12-805306-5.00020-1 sha: doc_id: 22505 cord_uid: 17khcmta this chapter includes diseases of animals in the order rodentia, in which there are over 2000 species representing 40% of all mammals. this incredibly diverse order includes members inhabiting every continent, either naturally or in human-made environments. while rodents have been the cause or implicated in disease transmission that has lead to human pandemics, such as the black death, and the decimation of certain animal species, like island-dwelling birds; genetically modified rodents have contributed significantly to the advancement of biomedical research and human health. there are more than 50 species of endangered rats, mice, voles, squirrels, and marmots. the recent extinction of the bramble cay melomys represents the first human-induced rodent extinction linked to climate change. rodents are the reservoir host of several human and domestic pathogens of concern listed by oie. herein, we highlight those diseases of rodents that lead to clinically important gross and microscopic lesions. the order rodentia includes 29 families, 468 genera, and over 2052 species. two separate systems exist to classify rodents (carleton and musser, 2005) . the first classification scheme by j.f. brandt (1855) is based on morphology of the jaw and skull together with masticatory muscle position and has three suborders: sciuromorpha (squirrellike); myomorpha (mouse-like); and hystricomorpha (porcupine-like). the second separates rodents into two suborders, sciurognathi and hysticognathi, based on the position of incisors and the angle of the jaw (carleton and musser, 2005) . molecular studies yield seven clades containing three phylogenetic lineages: squirrel-related (sciuriodea and gliridae); mouse-related (anomaluromorpha, castoridae, geomyoidae, and myodonta); and ctenohystrica. with diverse ecologic strategies, rodents are found naturally in a variety of habitats globally except antarctica. the natural history and biology of members of this extensive order exhibit substantial variation. nevertheless, similarities exist among rodents including dentition, anatomy, and disease susceptibility. the unifying characteristic of all rodents is their dentition, which includes two pairs of continuously growing (elondont) incisors. brachydonts have elondont incisors with brachydont molars; in hystricomorphs, all of the teeth are elondont. dentition is important when considering the diet and husbandry needs of these groups. brachydonts, which include mice, rats, hamsters, and gerbils, need high calorie diets with low amounts of fiber, whereas the hystricomorphs (porcupine-like; guinea pigs, chinchillas, agoutis) are strictly herbivores and require ample abrasive roughage. dental formulae vary but in general, rodents have four incisors, no canines, 1-2 premolars, and numerous (up to 12) molars. as most rodents are omnivorous, their digestive system typically consists of a simple stomach (monogastric) and a variably adapted intestinal tract depending on the degree of hind gut fermentation (stevens and hume, 1998) . most practice coprophagy or cecotrophy to maintain normal microflora and absorb microbially derived amino acids and vitamins (e.g., b and k). as such, herbivorous, omnivorous, insectivorous, and carnivorous rodents have different gastric and large intestinal anatomy. some rodents lack gall bladders (e.g., rats and pocket gophers). rats and hamsters are unable to vomit due to a muscular sphincter at the esophageal-gastric junction. members of caviidae lack the enzyme l-gulonolactone oxidase, making vitamin c (ascorbic acid) a necessary dietary requirement for this group (see scurvy in section, nutritional diseases). rodents are typically small (as small as 10 g) and rotund with short legs, though the largest rodents are almost 70 kg (e.g., capybaras). rodents are nocturnal and though some species hibernate (e.g., woodchucks) but most of the species do not. there are numerous specializations among rodents including cheek pouches (food transport), extremity and claw development (e.g., burrowing, jumping, climbing, flying), sensory organ variations (e.g., vibrissae and large ears or eyes), special integument and pelage (e.g., porcupine quills and chinchilla fur), and tail adaptations (e.g., prehensile, fat storage). rodents have a strong sense of smell and a well-developed vomeronasal organ, which is important for reproduction and social behavior through the sensing of pheromones. since rodents lack sweat glands, they are susceptible to overheating. reproductive anatomy varies though most female rodents ovulate spontaneously and are polyestrous. like rabbits, chinchillas have two cervices that communicate with individual uterine horns. vaginas are imperforate prior to sexual maturing and during nonbreeding and/or postpartum intervals. placentation is discoid and hemochorial. mammary gland anatomy varies among species. the suborders 500 pathology of wildlife and zoo animals scuirognatha and stricognatha have altricial and precocial young, respectively. some male rodents have testes within a scrotum, though the testes of porcupines, agoutis, chinchillas, cavies, and capybaras are located within the inguinal canal, which remains open in most male rodents. male rodents also have well developed accessory sex glands and an os penis. prairie dogs have perianal sacs similar to canids and felids, which are important for sexual communication. male beavers have persistent paramesonephric ducts that resemble a uterus and are referred to as masculine uterus (doboszynska and zurowski, 1981; meier et al., 1998) . south american rodents including guinea pigs and related species have unique pulmonary anatomy as do burrow-dwelling, subterranean mole-rats that live in low oxygen tension environments, for example, large and dilated, primitive bronchi and bronchioles (ilgun et al., 2014; widmer et al., 1997) . lung morphology of 40 different rodent species representing 11 families is well described (wallau et al., 2000) . common microscopic findings in rodents that may be misinterpreted as lesions include: multinucleated, karyomegalic, and cytomegalic hepatocytes are common in several rodent species and can increase with age ( fig. 20 .1); hepatocellular intranuclear cytoplasmic invaginations (pseudoinclusions) (fig. 20 .1); eosinophilic cytoplasmic spherical inclusions in renal tubular epithelial cells and hepatocytes seen predominantly male mice, rats, and hamsters; splenic extramedullary hematopoiesis, which is very common in healthy rodents of all ages (fig. 20 .2); hemosiderin, lipofuscin, ceroid, and melanin (in dark or black coated animals) are commonly detected in various tissues, such as spleen, liver, kidney, and adrenal glands; cardiac muscle in the tunica of pulmonary veins in the lung is a normal finding in mice; male rodents may have refluxed seminal coagula in the urinary bladder and urethra that is thought to occur peri mortem; and adrenal x-zone vacuolation in female mice. furthermore, there are evident sexual dimorphisms of some organs and glands, such as the cuboidal parietal epithelium of the glomeruli in male rodents. hematological reference ranges have been established for some common domestic, zoo-housed, and ubiquitous free-ranging species. in general, the lifespan of rodent erythrocytes is shorter than other mammals, thus, higher levels of circulating reticulocytes (1%-6%), seen as polychromasia and anisocytosis, is considered normal. howell-jolly bodies may also be present. in all age groups, lymphocytes are more numerous than neutrophils. in fact, lymphocytes are the predominant white blood cells in guinea pigs (45%-80%). in addition, guinea pigs and capybaras have kurloff cells (fig. 20. 3), which are lymphocytes containing a single, large intracytoplasmic mucopolysaccharide inclusion body, known as a kurloff body (jara et al., 2005) . though the actual function of these cells is unknown, they are presumed to be natural killer cells. the number of kurloff cells in females fluctuate with the reproductive cycle stage, thus they are considered to be estrogen-dependent. pregnant females may have 2%-5% kurloff cells in circulation while males typically have fewer. guinea pigs have heterophils similar to rabbits. rats, mice, and hamsters commonly have neutrophils with ring-form nuclei. chinchillas and porcupines have welldefined and segmented neutrophil nuclei. band neutrophils may be a normal finding in some species including new world porcupines, coypu, and agoutis. serum biochemical parameters have been compiled for some rodent species (kurtz et al., 2017; . of note is that serum levels of alanine aminotransferase (alt) are not sensitive biomarkers of liver health in guinea pigs and figure 20.1 normal liver in an aged mouse. common findings inlcude anisocytosis, anisokaryosis, and eosinophilic intranuclear pseudoinclusions of invaginated cytoplasm. within the red pulp are hematopoietic elements including erythroid and myeloid lineages. note the large multinucleated megakaryocytes, which are readily visible at this magnification. some other rodents as they normally have low hepatic levels of this enzyme. in contrast, gamma glutamyl transferase is present in the liver of all rodents and can be evaluated to determine the hepatic injury or disease. hamsters and other desert species may have high levels of serum calcium. rodents like mice and rats, and specifically those evolved in arid regions, have a profound ability to concentrate their urine, thus it is often turbid and contain calcium crystals. the following list of diseases of rodents is not exhaustive but instead highlights the more common findings in species of which there is published literature or knowledge. a plethora of information exists regarding the anatomy, physiology, and diseases of rodents used as animal models in biomedical research (i.e., laboratory animals) and is a valuable resource when examining lesser-studied rodent species using a comparative approach greaves, 2011; suckow et al., 2012; treuting and dintzis, 2017) . as many rodents are important prey species, diseased or dead rodents are exceedingly difficult to detect in nature using the passive surveillance techniques of traditional wildlife disease monitoring. thus, less is known about the diseases and pathology of free-ranging rodents as compared to larger and charismatic mammals. one of the most well described nutritional diseases in rodents is hypovitaminosis c or scurvy. guinea pigs and a number of other species including capybaras and humans, lack the enzyme l-gulonolactone oxidase which is required for l-ascorbic acid (vitamin c) production therefore, these species require dietary vitamin c (cueto et al., 2000) . vitamin c is necessary for the formation of collagen via hydroxylation of proline/lysine for cross linking of fibrillar collagen. hypovitaminosis c results in decreased collagen fibril cross linking, leading to the increased fragility of blood vessels, cartilage, and osteoid. in affected individuals, gross and histologic lesions are characteristic and include capillary hemorrhage and lack of bone deposition and remodeling as a result of poorly formed and weak collagen. other gross lesions include evidence of anemia (e.g., pallor, reactive bone marrow), periarticular hemorrhage, and swollen joints, specifically at the costochondral junctions (scorbutic lattice). characteristic oral lesions include gingival swelling, erythema, hemorrhage, erosion, ulceration, and tooth loss. histological examination is necessary to confirm and define bone lesions, which include: dilation of metaphyseal blood vessels, irregular columnization and hypertrophy of physeal cartilage, calcification of metaphyseal cartilage with reduced or absent osteoid, microfractures with surrounding and subperiosteal hemorrhage, medullary fibrosis, and decreased osteoclastic activity and remodeling. tooth loss may result from a combination of abnormal odontoblasts, dentin resorption, pulp fibrosis, and a lack of/ abnormal alveolar bone remodeling. like lagomorphs, some hindgut-fermenting rodents are susceptible to gut stasis and related diseases if they are not able to obtain enough fiber from the diet. obesity can be common in rodent species if fed a high caloric diet and can predispose to diabetes mellitus, cardiac disease, pregnancy toxemia, and hepatic lipidosis in females. dystrophic mineralization occurs in guinea pigs, hamsters, and free-ranging rats (rothenburger et al., 2015a) . mineralization most often affects the myocardium but can also occur in other tissues, such as the diaphragm, tongue, liver, kidney, lungs, cornea, and aorta (see section congenital/ genetic). though the pathogenesis is not fully elucidated, mitochondria appear to be preferentially mineralized based on ultrastructural studies. gross lesions may include cardiomegaly with chalky to gritty white material and streaking within the myocardium of the atria and ventricles. histologically, affected cardiac myocytes are degenerative and/ or necrotic with rupture of the sarcolemma. damaged myocytes will have variable and progressive mineralization until the entire myofiber is densely mineralized. variably mature fibrosis surrounds individual and clusters of dead and mineralized myocytes. satellite cells are frequently hypertrophic. pregnancy toxemia occurs in overweight, pregnant, and lactating female guinea pigs and hamsters. there are two forms in guinea pigs, a fasting type that results from decreased carbohydrate intake and subsequent mobilization of fat stores and a circulatory type that occurs when the gravid uterus compresses the aorta and causes reduced blood flow to abdominal organs. characteristic gross and histologic lesions include hepatic lipidosis and fat deposition in renal tubular epithelial cells. similarly, hepatic lipidosis and associated metabolic effects occasionally affect overweight nongravid sows and boars. urolithiasis is common in guinea pigs, rats, and mice due to high urine ph and calcium concentrations. obstructive urolithiasis has also been reported in captive male agoutis resulting in bilateral hydronephrosis, urinary bladder rupture, and peritonitis (batista et al., 2010) . a number of other endocrine diseases have rarely been documented in various rodent species. they have typically been associated with hormone-producing neoplasms (adrenal, islet, thyroid, parathyroid, and pituitary tumors). fluorosis is well studied in rats as they are used as models in toxicology studies. guinea pigs and rabbits are also susceptible and fluorosis has occurred in free-ranging cotton rats in sites contaminated by petrochemical waste (rafferty et al., 2000) . pathognomonic lesions occur in teeth and bones. periosteal hyperostosis occurs first along the medial surface of the metatarsals, then progresses to affect the mandible, metacarpals, and ribs though all bones can be affected in severe, chronic disease. bones are brittle and chalkywhite. pathological fractures are common but joints are typically spared. although characteristic, enamel hypoplasia only occurs when fluorosis is present during tooth development. tooth lesions include dull, dry, and chalky hypomineralized or hypoplastic enamel with pitting, grooves, and discoloration (due to oxidation); excessive attrition affects the incisors first. irregular wear, malocclusion, and fractures may be present. histologically, ameloblasts are small while osteoblasts are vacuolated and disorganized. there is abundant globular dentin and hypomineralization of the outer enamel layer. a differential for enamel hypoplasia in young syrian hamsters is hamster parvovirus (hapv); concurrent testicular hypoplasia, and cerebral malacia is present with this viral infection. cholecalciferol (vitamin d) is used as a rodenticide. the deliberate poisoning of free-ranging rats and mice results in inadvertent toxicity of nontarget animals including other rodents, such as squirrels or carnivorous predators that consume affected carcasses. multisystemic mineralization occurs prior to death in affected individuals. unlike other rodents, naked mole-rats, beavers, and woodchucks do not require dietary vitamin d and toxicity can develop if they are fed rodent chow or diets supplemented with vitamin d. lesions consistent with calcinosis cutis and circumscripta can develop in naked mole-rats fed such diets. soft tis-sue calcification is not limited to pressure points and can be found throughout the body in severe cases. resolution occurs with correction of the diet (delaney et al., 2013) . anticoagulant rodenticides including warfarin and its derivatives are frequently used to control free-ranging mice and rat populations. these compounds antagonize vitamin k and thus inhibit production of vitamin k-dependent clotting factors (i, ii, vii, ix, x). gross lesions include multisystemic hemorrhage, particularly subcutaneous and periarticular hematomas. "bait," some of which are brightly colored (e.g., teal-green), may be present within the intestines. inadvertent consumption by nontarget wildlife may have fatal consequences. hindgut fermenting rodents are susceptible to similar antibiotic-related toxicities as rabbits (see chapter 19). nephrotoxicosis can be the result of exposure to numerous agents and has been extensively described in laboratory animals. for example, swainsonine toxicity occurs following the ingestion of plants from the genus swainsona and results in acquired alpha-mannosidosis in rats. histologically, renal tubular epithelial cells are severely and diffusely vacuolated. dystrophic mineralization, also known as dystrophic cardiac calcinosis is considered as a genetic disease in inbred mouse strains, particularly in aged individuals (eaton et al., 1978) . cardiac mineralization is detected grossly as pinpoint foci to larger irregular crusts composed of white, gritty material within and overlying the epicardium. histologic lesions vary depending on severity and chronicity with degeneration and necrosis of individual myocytes to extensive regions of myocyte loss and replacement by deeply basophilic granular material. in severe cases, mineralization extends into the myocardium resulting in scar formation and can also be seen in the kidneys and lungs. the prevalence of this disease in wild mice from which these strains originated is unknown. diabetes mellitus (dm) in chinese hamsters is a heritable, autosomal recessive disease (green et al., 1963) . symptomology and lesions are similar to those found in humans and include hydropic degeneration and degranulation of pancreatic islet î²-cells with secondary vascular lesions, such as arteriosclerosis and glomerulosclerosis. in addition, affected hamsters are predisposed to pancreatic adenocarcinomas. serum levels of î±-2 globulins are a useful indicator of risk for diabetes mellitus development in these populations. polycystic kidney disease has been diagnosed in captive populations of brazilian agoutis (mã¼ller et al., 2009) . most affected individuals have no clinical signs and are potentially related genetically. grossly, affected agoutis have a range of bilateral renal changes including atrophy, roughened granular cortical surfaces, and multiple cysts. histologically, there is cystic dilatation of renal tubules and bowman's capsules with mesangial and capsular thickening accompanied by mononuclear interstitial nephritis and fibrosis. many degenerative and age-related diseases are described in rodents used in toxicology studies pettan-brewer and treuting, 2011) . a significant disease confounding these studies is chronic progressive nephropathy, which is best described in rats though mice, naked mole-rats, and australian rodents, particularly the sticknest rat, are also affected (delaney et al., 2016a) . the exact pathogenesis is unknown; however genetic factors may play a role. grossly, kidneys are bilaterally pale and either enlarged or shrunken with numerous pinpoint to large, 1 mm cortical microcysts creating a nodular surface. histologically, renal tubules are markedly ectatic and tortuous and many contain brightly eosinophilic fluid (proteinosis) ( fig. 20.4 ). tubules have a range of degeneration, necrosis, and regeneration with occasional tubular hyperplasia and adenoma formation (predominantly in rats). glomeruli are variably affected by membranous and/or proliferative changes with thickened basement membranes, sclerosis, and obsolescence. synechiae, dilated bowman's capsules, and hypertrophied parietal epithelium are common. the interstitium is expanded by lymphoplasmacytic infiltrates with pigment-laden macrophages, mild hemorrhage, and edema. there is often substantial interstitial fibrosis and in severe cases, infarction. diagnosis is based on histologic evaluation though in some species, clinical pathology may be preferentially used to determine renal function. cardiomyopathy is common in aged mice, rats and hamsters; it is particularly common in syrian hamsters and is reported in woodchucks (chanut et al., 2013; rothenburger et al., 2015a; roth and king, 1986) . lesion severity varies though it appears to increase with age and is more common in males. though considered strain and thus possibly genetically related in laboratory rats, free-ranging rats develop similar disease (rothenburger et al., 2015a) . grossly, there may be cardiomegaly but macroscopic changes are not always apparent. histologically, myocardial lesions consist of multifocal and perivascular lymphoplasmacytic infiltrates accompanied by fibrosis, myocyte degeneration, and drop out ( fig. 20 .5a,b). trichrome, movat pentachrome, or other stains that highlight the fibrosis are useful to evaluate severity and chronicity. syrian hamsters develop cardiomyopathy associated spontaneous atrial thrombosis and subsequent coagulopathy that is often fatal (fig. 20.6 ). females appear to be affected earlier in the life than males. like other species, rodents are susceptible to skeletal degeneration and related diseases. guinea pigs, specifically the duncan-hartley breed and syrian hamsters, frequently develop degenerative joint disease. gross lesions include thickened joint capsules, variable osteophyte formation, roughened cartilage surfaces, eburnation, joint effusion, and synovial proliferation. depending on which joints are affected and chronicity, crepitus may be present when joints are manipulated. microscopic examination of joints reveals degeneration, necrosis, and ulceration of the articular cartilage with proliferation and fibrosis of the synovium and joint capsule, periarticular and periosteal bone formation, and remodeling. there may also be variable inflammation and hemorrhage within joint spaces. amyloidosis is typically seen in aged animals. amyloid is an extracellular accumulation of insoluble protein. excessive amyloid deposits can lead to tissue atrophy and dysfunction. amyloidosis is most common in myomorphs (mice, rats, hamsters, gerbils) and appears in the kidneys, spleen, adrenals, and liver as well as the gastrointestinal lamina propria (mouse) (fig. 20.7 ). there is a higher prevalence in females. of note is the eosinophilic substance within the nasal planum of aged mice that was once thought to be amyloid, through histochemical and ultrastructural studies, has instead been shown to consist of collagen and complex carbohydrates produced by the nasal gland epithelial cells (doi et al., 2007) . hemosiderosis is the accumulation of iron within various tissues, most commonly the liver. it is described in numerous species including primates, birds, rhinoceros, hyraces, and rodents. in one zoological institution, hemosiderosis was present in approximately 65% of adult naked mole rats (delaney et al., 2013) . grossly, livers appear bronze. iron accumulation appears histologically as zonal to diffuse hepatocellular accumulation of brown granular to figure 20.4 chronic progressive nephropathy in a naked molerat. renal tubules are variably ectatic, atrophied, degenerate, necrotic, and replaced by collagenous fibrous tissue. within the interstitium are accumulations of mononuclear cells (primarily lymphocytes and fewer macrophages). there is also sclerosis of glomeruli and periglomerular fibrosis. globular pigment. pigment is most intense in the periportal hepatocytes. the pigment stains positively with prussian blue and is not refractile with polarized light. aged mice can develop intrapulpal denticles, which are dysplastic tooth-like growths arising in the pulp of incisors. these may result in abnormal wear and or breakage of the incisors contributing to periodontitis and decreased ability to effectively gnaw (pettan-brewer and treuting, 2011). despite their protective quills, porcupines are particularly susceptible to cutaneous trauma and tears due to an exquisitely fragile integument. secondary infections resulting in pyoderma and persistent ulceration may occur. stress dermatosis (or dermatitis) is a condition of several south american rodents including, the agouti, acouchis, and capybaras. this syndrome is seen in captive and free-ranging individuals in densely populated groups. it presents as alopecic skin lesions and lacerations along the lumbosacral spine. stress is thought to increase skin fragility and overcrowding with intraspecific aggression contributes to lesion development. sun exposure, pruritus, and self-mutilation result in large patches of alopecia with erythema and excoriated skin with loss of the long hairs along the dorsum and tail. microscopic lesions correlate to severity and chronicity and include variable epidermal degeneration, necrosis, erosion/ulceration, and associated dermatitis with hemorrhage and edema. diagnosis is based on clinical history, demonstrated absence of a parasitic etiology in skin scrapings and histopathology. malocclusion is common especially in hysticomorphs, though it is also seen in brachydonts. it is a significant disease in captivity and may be related to genetics, nutrition and diet, and husbandry practices (losco, 1995) . ovarian cysts are common in rodents. cystic rete ovarii are prevalent in female guinea pigs (veiga-parga et al., 2016) and can be distinguished from other ovarian cysts as they arise from the rete and compress adjacent ovarian tissue ( fig. 20.8 ). other ovarian cystic structures reported in female rodents include: bursal cysts, epithelial cysts, follicular cysts, luteal cysts, and paraovarian cysts . the hemochorial placentation in rodents predisposes them to trophoblast emboli. this incidental lesion is best described in chinchillas and is not associated with clinical ramifications or gross lesions. histologically, intravascular trophoblasts are present in the lung, uterus, spleen, liver, and other sites, such as the adrenal glands with no associated inflammation. trophoblasts are large (>100 âµm) with abundant amphophilic granular cytoplasm and large nuclei with prominent nucleoli. many neoplasms of inbred mice and rats appear to be strain related. nevertheless, certain neoplasms occur more frequently among free-ranging and domesticated outbred rodents than in other mammalian orders. several of them are described below. a list of other common neoplasms in rodent species is listed in the supplemental materials (table e1) . naked mole-rats are a popular zoo species and are heralded as animal models for cancer and aging due to their extreme longevity and purported cancer resistance. rare cases of neoplasia have been reported in naked mole-rats and in the related damaraland mole-rat, which is also longlived and appears to be relatively resistant to cancer and agerelated diseases (delaney et al., 2016b; taylor et al., 2016,) . other subterranean rodents used as animal models for carcinogenesis research include some members of spalacidae (mole-rats, blind mole-rats, zokors, and bamboo rats). there are currently no reported spontaneous tumors in this group; however, subcutaneous tumors (fibromas and fibrosarcomas) may be induced with application of carcinogenic compounds. odontoma, or more aptly named odontogenic dysplasia, is described in several rodent species (rats, mice, guinea pig, prairie dogs, degus, chinchilla, chipmunk, squirrels; . most odontomas are not true neoplasms and instead represent dysplastic changes resulting from chronic inflammation or trauma and associated regional tissue response. as there is no demonstrated metastatic potential or criteria for malignancy, these lesions are increasingly identified as hamartomas versus neoplasms (and also referred to as pseudo-odontomas). nevertheless, these toothy masses are regionally destructive and almost exclusively affect the incisors. grossly, odontogenic dysplasia presents as nodular swellings around the maxillary or mandibular incisors. microscopically, these lesions are composed of disorganized and dysplastic proliferations of odontogenic epithelium, enamel matrix, and mineralized enamel, dentin, pulp, and cementum. in rodents, odontomas have been described as complex because they contain fully differentiated tooth elements but do not form the tooth-like structures grossly present in compound odontomas seen in other species (e.g., horse). histologically, rodent odontomas are composed of welldifferentiated odontogenic epithelium among an enamel organ with dental pulp mesenchyme and a variably mineralized enamel matrix. several important infectious and zoonotic diseases of rodents described are later with a focus on those that cause significant lesions in rodent species. additional pathogens, including zoonotic pathogens that occur in rodent species are listed in the supplemental materials (table e2 ). several references exist pertaining to rodent reservoirs of emerging and reemerging zoonotic pathogens ). poxviral diseases in rodents are caused by members of orthopoxviridae, which includes mousepox and cowpox (oie listed reportable zoonotic diseases). several species of rodents serve as the reservoir for cowpox virus, which results in cutaneous lesions in humans. in laboratory mice, mousepox, more commonly known as ectromelia, is an important disease. this infection can be acutely fatal with minimal lesions. in the chronic form, gross lesions include crusting lesions of face, legs, and tail, conjunctivitis, an enlarged, swollen and friable liver and spleen, and in cases of recovery, splenic fibrosis. necrosis of the kidney, thymus, and lymph nodes may also be grossly evident. the small intestine, urinary bladder, and vagina can have mucosal erosions and hemorrhage. histologically, skin and mucosal lesions include epithelial ballooning degeneration, hyperplasia, and erosions with characteristic intraepithelial, intracytoplasmic inclusion bodies. within the parenchymal organs, splenic and hepatocellular necrosis is typically coagulative and multifocal to coalescing. diagnosis is based on characteristic lesions with viral inclusions. electron microscopy, pcr, and virus isolation are useful for confirmation of the viral strain. in 2003, an outbreak of monkeypox (orthopoxviridae), a zoonotic virus, occurred in prairie dogs sold as companion animals in the united states (gaurner et al., 2004) . fatal infections in infected prairie dogs were characterized by necrotizing bronchopneumonia, conjunctivitis, and glossal ulcers. viruses and viral antigens were detected in multiple tissues and cell types via immunohistochemical staining, pcr, and electron microscopy. infected humans developed fever and vascular rashes. forty seven confirmed and probable cases of monkeypox were reported from six states. the prairie dogs contracted the virus following exposure to infected african rodents. other rodents that also developed disease and died included: rope squirrels, tree squirrels, gambian giant rats, brush-tailed porcupine, dormice, and striped mice. fibromas and fibromatosis of gray squirrels are potentially fatal conditions caused by squirrel fibroma virus (leporipoxviridae). disease is mainly reported in the eastern north america (terrell et al., 2002) but also occurs in red squirrels and has been reported in a fox squirrel (wilcoxen et al., 2015) . transmission is likely through biting arthropods (fleas, ticks, and mosquitoes). gross and histologic lesions are similar to shope fibromas in rabbits (both are leporipoxviruses). multifocal to coalescing tan, firm nodules may cover all parts of the body and begin as alopecic nodules with progression to plaque-like to pedunculated masses ( fig. 20.9a ). systemic disease manifests as renomegaly and multiple pulmonary nodules . histologically, the skin nodules exhibit marked epidermal hyperplasia with ballooning degeneration of keratinocytes, spongiosis, and intracytoplasmic eosinophilic viral inclusions (fig. 20.9b,c) . pulmonary nodules correlate histologically to adenomatous hyperplasia with similar viral inclusions. other microscopic lesions may include atypical mesencyhmal proliferation in the liver and seminal vesicles, and renal tubular epithelial hyperplasia with viral inclusions. diagnosis is based on characteristic lesions and detection of virus by pcr or isolation. parapoxvirus is an important cause of disease in red squirrels in the united kingdom and ireland where it is a suspected factor in severe population declines tompkins et al., 2002) . the causative agent is referred to as red squirrel parapoxvirus. grey squirrels are the likely maintenance hosts since a large percentage of healthy gray squirrels are positive for the virus and their range overlaps with that of red squirrels. lesions in red squirrels include exudative dermatitis with crusting that is similar to other poxviral diseases. viral inclusions can be detected histologically within the cytoplasm of infected cells. electron microscopy and pcr are used to detect the virus. woodchuck hepatitis virus (whv; hepadnaviridae) is a disease in captive and free-ranging animals . although not zoonotic, this disease is a valuable animal model for viral hepatitis and hepatocarcinogenicity associated with hepatitis b in humans. it affects both sexes and is typically found in animals greater than 4 years of age. activated t cells (cd8 +) cause cellular and dna damage leading to persistent hepatitis. simultaneously, viral dna integrates into the host genome at loci containing oncogenes, creating the potential for carcinogenic transformation. hepatocellular carcinomas are reported in 100% of experimentally infected woodchucks. grossly, most carcinomas are solitary and involve one hepatic lobe. histologically, neoplastic masses have differing growth patterns: trabecular, adenoid, or solid with and without cystic spaces, hemorrhage, and necrosis. an interesting feature of these tumors is the presence of atypical, bizarre, and giant neoplastic cells. adenomas may be present and are considered a precursor to carcinomas. mitotic index and cellular morphology are used to classify tumors as adenomas versus carcinomas. the remaining hepatic parenchyma often contains acute and/or chronic hepatitis with neutrophilic to mononuclear inflammation, variable hepatocellular degeneration, necrosis, fibrosis, and biliary changes. electron microscopic identification of the virus is definitive. beechey ground squirrels have a similar virus with variable hepatic lesions (cullen and marion, 1996) . hamster polyomavirus (hapyv), a papovavirus, causes cutaneous trichoepitheliomas in syrian hamsters . the virus is transmitted via urine and is highly contagious, with reported 50% morbidity in affected breeding colonies. early lesions consist of wartlike growths and alopecic nodules of the face and perineum with progression to severe, multifocal to coalescing nodules throughout the body. histologically, these characteristic skin tumors contain multiple islands of basilar to variably differentiated follicular epithelium and abrupt central keratinization with faded anuclear keratinocytes (ghost cells). rudimentary hairs may be present and follicular rupture may be associated with inflammation. other polyomaviruses-associated neoplasms include other cutaneous tumors, lymphomas and salivary gland tumors. in virally induced lymphomas, affected tissues are effaced by diffuse sheets of neoplastic lymphocytes among scant stroma. viral inclusions are not present. in a case of polyomavirus-induced, multicentric lymphoma in an 8-week old syrian hamster, the virus was detected in tumor cells, renal tubular epithelial cells, and enterocytes via in situ pcr indicating some degree of tissue tropism . diagnosis is based on clinical history, gross and histologic lesions, and detection of the virus via pcr and/or electron microscopy. mouse hepatitis caused by mhv (coronaviridae) is enzootic in some colonies of inbred laboratory mice . mouse hepatitis may also occur in nonlaboratory strains of mice in zoo settings. mhv causes polytropic and enterotropic disease syndromes, both of which occur following highly contagious viral infection by the oronasal route. grossly, there is hepatomegaly, splenomegaly, lymphadenomegaly, and lymphadenopathy with jaundice and ascites. there may be gross evidence of intestinal (ileal and cecal) disease with thickened and necrotic mucosa. within the liver, spleen, and lymph nodes, there are multiple coalescing and random white necrotic foci. histologically, polytropic disease includes necrotizing hepatitis with syncytial cells within necrotic foci, and demyelination in cns in immunodeficient mice. the enterotropic disease lesions are age-dependent and may include villous attenuation and syncytial cell formation with eosinophilic intracytoplasmic inclusion bodies in enterocytes, mesenteric lymph nodes, and endothelial cells; granulomatous serositis may also be present. diagnosis is based on histologic features and immunohistochemical demonstration of viral antigens within lesions. virus isolation, pcr, and serology are used to confirm infection and exposure, respectively. sendai virus infection (parainfluenza-1; paramyxoviridae) is common in wild and pet rodents and is actively excluded from laboratory populations of mice, rats, and gerbils (easterbrook et al., 2008) . disease manifestations occur quickly following the aerosol inhalation, direct contact, or in utero exposure. the virus targets respiratory epithelium and type ii pneumocytes, which may impair ciliary activity and predispose affected individuals to secondary mycoplasma pulmonis and other bacterial infections. gross lesions include dark purple discoloration, atelectasis, and consolidation of the cranioventral lung lobes and diffuse pulmonary edema. splenomegaly and lymphadenomegaly are common and if infection occurs in a gravid female, fetal death may occur. histologically, lesions include necrotizing rhinitis, laryngotracheitis, bronchitis, and bronchointerstitial pneumonia. in immunocompromised animals (e.g., athymic and scid mice), intracytoplasmic and intranuclear inclusion bodies may be present. diagnosis is based on supportive clinical history, histologic findings, and pcr detection of the virus. sialodacryoadenitis is a disease specific to rats and is caused by sialodacryoadenitis virus (sdav; coronaviridae). the virus may be enzootic in some research colonies and in pet and free-ranging rats (easterbrook et al., 2008) . virus is shed in nasal secretions and/or saliva. sdav infects and replicates in the respiratory tract and spreads to salivary and lacrimal glands resulting in secondary lesions of the nasopharynx, respiratory tract, and eyes. grossly, the parotid and submandibular salivary glands, harderian glands, exorbital glands, and cervical lymph nodes are enlarged, pale, and edematous. the harderian glands may exhibit brown pigmentation, which correlates to accumulations of porphyrin within acini. histologically, there is acute necrotizing adenitis. in chronic cases, there is fibrosis and extensive squamous metaplasia of glandular tissue. there may be rhinitis, tracheitis, bronchitis, and bronchiolitis in severe and chronic cases. histologic lesions are characteristic and supportive of the diagnosis. within this section are several zoonotic and notable nonzoonotic bacteria that infect rodents. several others that are notable but not associated with disease in their rodent host are listed in the supplemental materials (table e2 ). plague is an oie-listed and who-reportable zoonotic disease that is important to consider when working with rodents. fleas spread the causative agent, yersinia pestis, between hosts. cats and other rodent-eating predators can also contract infection through ingestion and rarely via inhalation. historically, plague has been implicated in the deaths of millions of humans, most notably in 14th century europe during the black death. there are two main host types: enzootic reservoirs (voles and deer mice) and epizootic amplifiers (prairie dogs, rats, and squirrels). with close to 100% mortality, plague outbreaks in prairie dog colonies are a devastating and significant threat not only this species but to endangered black-footed ferrets, who are susceptible to disease because prairie dogs are their primary food source (cully et al., 2010) . there are three disease forms: bubonic (lymphadenomegaly with abscessation); pneumonic ( necrotizing pneumonia with fibrinous pleuritis); and septicemic (multifocal necrosis of liver, lung, spleen, kidney, eye, brain). gross lesions include multifocal pulmonary hemorrhage (fig. 20.10) , hemorrhagic lymph nodes, erythema of axillary and inguinal skin, splenomegaly and localized hemorrhage and necrosis in the dermis and subutis (suspected site of flea bite). histologic lesions of bubonic disease include necrosuppurative lymphadenitis; in the pneumonic form, necrotizing pneumonia; and in the septicemic form, multifocal abscessation. all lesions contain gram and giemsa positive coccobacilli with characteristic bipolar morphology resembling a safety pin. bacterial culture must be completed at a certified, biosecure laboratory; identification using immunohistochemistry or immunofluorescence is a beneficial diagnostic aid. yersiniosis (pseudotuberculosis) is caused by y. pseudotuberculosis and y. entercolitica. rodents may serve as carriers of this zoonotic bacterial agent, though some species, including mice, voles, beavers, and muskrats may succumb to acutely fatal disease or develop more characteristic lesions and symptoms. gross and histologic lesions include multifocal necrotizing hepatitis and splenitis and/or fibrinonecrotizing and ulcerative enterocolitis (fig. 20.11 ). agoutis appear to be uniquely susceptible in zoo-settings (k. terio, personal communication). tularemia caused by francisella tularensis is an important zoonotic and oie listed reportable disease that is maintained by wild rodents and lagomorphs. disease occurs in many rodents including mice, beavers, and muskrats, while voles appear to be subclinical carriers and serve as a reservoir (nelson et al., 2014; rossow et al., 2014) . lesions of affected species are similar to those described in rabbits (see chapter 19). miliary to multifocal and coalescing hepatic necrosis with variable inflammation (depending on chronicity) characterize the disease (fig. 20.12) . unlike yersiniosis and similar to listeriosis, bacteria are not readily detected but can be better visualized with special stains. listeriosis is a common zoonotic bacterium in rodent species. it can cause variable, yet similar lesions as for rabbits (see chapter 19) . the systemic form is most common. during an outbreak in bushy-tailed jirds, death occurred without clinical signs (tappe et al., 1984) . salmonellosis is reported in guinea pigs, rats, and mice, though any rodent species can be infected and develop disease. salmonella enterica enteritidis or typhimurium are the most common zoonotic serovars. guinea pigs become septicemic and often die acutely. other rodents may develop gastrointestinal disease that includes diarrhea and abdominal pain prior to systemic spread. in hamsters, salmonellosis must be considered and ruled out in cases of "wet tail," which is typically associated with infection by lawsonia intracellularis. gross lesions of salmonellosis include cyanosis of mucous membranes and polyserositis with splenomegaly. hepatic, splenic, and lymph node necrosis are typical, as are multifocal necrosis and button ulcers in the colon with segmental intestinal infarction. histologic lesions correlate to gross findings and include small foci of hepatocellular necrosis containing necrotic cell debris and variable numbers of macrophages, lymphocytes, and fewer neutrophils (paratyphoid nodules) and kupffer cell hyperplasia; granulomatous hepatitis, splenitis, and lymphadenitis; and fibrinonecrotic ileotyphlocolitis. intestinal infarction can be attributed to vasculitis and thrombosis of the mesenteric or mesocolic vessels. culture of the causative agent and histologic lesions confirm the diagnosis. mycobacteriosis is a zoonotic disease that is rarely reported in rodents, though some species may serve as reservoirs. there are reports of mycobacteriosis (mycobacterium microti) in british voles and wood mice (cavanagh et al., 2002) , hamsters, and korean and richardson's ground squirrels in spain. chlamydophila caviae and less frequently c. psittaci cause disease in guinea pigs, primarily juveniles. conjunctivitis, bronchitis, and pneumonia may occur and result in debilitation and death. histologically, there is heterophilic keratoconjunctivitis and uveitis. pneumonia has a cranioventral pattern and is heterophilic. some animals may be asymptomatic. diagnosis is achieved through cytology of ocular discharge and/or pcr of affected tissues. immunohistochemistry of tissue sections is also available. bordetella bronchiseptica causes bordetellosis (epizootic pneumonia), a common respiratory disease in guinea pigs though is also reported in free-ranging squirrel populations . similar disease can occur in other rodents, particularly in the context of immunosuppression or coinfection. grossly, there is cranioventral pneumonia with multifocal to coalescing, discrete, reddish-gray consolidated regions affecting multiple lobes, and pleuritis. mucopurulent exudate may be present in the nares, nasal passages, trachea, and tympanic bullae. there is often crusting conjunctivitis. females may have metritis and pyosalpinx. histologically, lung lesions consist of necrotizing and heterophilic bronchopneumonia with obliteration of the airways. diagnosis is confirmed with bacterial culture or pcr. other predisposing pathogens (e.g., adenovirus, mycoplasma spp.) must be ruled out. ciliated associated respiratory (car) bacillus is an unclassified bacterium that colonizes the ciliated epithelium of the respiratory tract of rats, mice, rabbits, and a variety of other species. this agent acts synergistically with other respiratory pathogens including mycoplasma pulmonis, sendai virus, coronavirus, streptococcus pneumoniae, corynebacterium kutscheri, bordetella bronchiseptica, klebsiella pneumoniae, or pasteurella spp. lesions include suppurative to mucopurulent bronchopneumonia, perivascular and bronchiolar lymphoid hyperplasia, rhinitis and lymphoplasmacytic tracheitis in free-ranging, pet and laboratory rats rothenburger et al., 2015b) . coinfection of car bacillus and mycoplasmas has been reported in rats and spinifex hopping mice with pneumonia (mackie et al., 2001) . murine respiratory mycoplasmosis is caused by mycoplasma pulmonis . gross lesions include catarrhal exudate in the upper respiratory tract with cranioventral bronchopneumonia and mucopurulent exudate in airways with atelectasis. histologic lesions include peribronchial lymphoid cuffing with balt hyperplasia; neutrophilic bronchopneumonia; type ii pneumocyte hyperplasia of alveolar epithelium; and emphysema; bronchiectasis is a characteristic finding. histology alone cannot differentiate m. pulmonis and car bacillus respiratory infections (rothenburger et al., 2015b) . other lesions associated with m. pulmonis include endometritis, salpingitis, and perioophoritis. diagnosis is based on histologic features, culture or pcr detection, and elisa. pasteurellosis caused by p. multocida affects young and immunocompromised rodents, most frequently following direct transmission from the dam. strain virulence and species determine lesions, which in general, are similar to those reported in other species, including rabbits (see chapter 19) . prairie dogs develop upper respiratory disease and pneumonia, while conjunctivitis is more common in rats and mice. hamsters have variable susceptibility. cervical lymphadenitis, a disease classically described in guinea pigs, is caused by streptococcus equi subsp. zooepidemicus. infection is associated with coarse foods that cause traumatic lesions of the oral mucosa; bite wounds may serve as another route of entry. grossly, the cervical and submandibular lymph nodes are markedly enlarged and contain thick purulent yellow-white to red-gray exudate. lymph nodes in severely affected and chronic cases may rupture. histologically, affected lymph nodes are effaced by heterophilic lymphadenitis with central necrosis and peripheral fibrosis. thoracic lesions include fibrinous pleural adhesions, fibrinosuppurative bronchopneumonia, pleuritis, and pericarditis. chains of gram-positive cocci are present within lesions. diagnosis is based on gross and histologic lesions and culture of causative agent. tyzzer's disease, caused by clostridium piliforme, is a potentially fatal disease of many rodent species. it has been reported in captive gerbils, hamsters, and spinifex hopping mice (stannard et al., 2017) . infection results in a triad of gross lesions including icterus and hepatomegaly with military gray foci, congestion, and edema of the intestine, and lymphadenomegaly with edema and hemorrhage. sudden death without clinical disease or lesions is possible in peracute cases. histologically, multifocal random to coalescing hepatic necrosis is surrounded by hemorrhage, heterophils, and macrophages (fig. 20.13a ). at the necrotic margins, hepatocytes may accumulate characteristic criss-crossed bundles of faintly staining bacilli that are best visualized with silver stains (fig. 20.13b ). proliferative ileitis (wet tail) due to lawsonia intracellularis is most commonly reported in hamsters. similar to this infection in other species, there is segmental thickening of ileum with serosal nodules. histologically, there is marked crypt hyperplasia and herniation and villous elongation, hyperplasia, and fusion with variable necrosis and hemorrhage. silver stains and pcr are useful to confirm infection. a diverse array of pneumocystis spp. are carried without clinical signs by a variety of wild rodents; these organisms are thought to be highly host-specific. pneumocystis spp. are found within alveoli and alveolar macrophages and appear as small, eosinophilic round organisms with a small central body. in severe cases, organisms within alveolar spaces and alveolar macrophages can obliterate alveoli; in some cases they can be associated with interstitial pneumonia (fig. 20.14a,b) . this fungus can cause significant opportunistic infections in immunocomromised individuals. cryptococcosis (cryptococcus neoformans) can be a significant pathogen in some rodent species. for example, approximately 20% of slender-tailed cloud rats at one zoological institution died with or as a result of cryptococcal pneumonia over a 15-year period (berliner et al., 2007) . these large, arboreal rodents were likely infected by contaminated substrates and may be a highly susceptible species, similar to some species of nonhuman primates and felids. lesions included none or mild to severe, chronic granulomatous pneumonia with or without necrosis and regional lymphadenitis. disseminated disease can produce subcutaneous nodules ( cryptococcoma) and meningoencephalitis. microscopically, characteristic fungal yeasts with narrow-based budding are present in cytologic preparations and histologic sections. gms staining can enhance detection of fungal yeasts, which are 5-20 âµm diameter with a distinct, thick (5-10 âµm), nonstaining mucopolysaccharide capsule, the latter of which is best visualized using the pas reaction and mucin stains. culture can confirm infection ( fig. 20.15) adiaspiromycosis is a systemic fungal disease reported in several captive and free-ranging rodent species. it is caused by the saphrophytic fungi chrysosporium parum and c. crescens. once inhaled, these fungi cause pulmonary granulomas with fungal spherules and conidia. additional diagnostic tests may be useful, including immunodiffusion and complement fixation. these fungi are zoonotic. other systemic mycoses of rodents include infections caused by the endemic (and zoonotic) fungi blastomcyes dermatitides and histoplasma capsulatum. reported cases include juvenile mice and chinchillas. immunity to fungal pathogens appears limited in mice. dermatophytosis is a cutaneous fungal disease that can be found in any rodent species and is similar to that of other mammalian species. causative agents include trichophyton mentagrophytes, microsporum canis, m. gypseum, and epidermophyton. of note, these fungi are zoonotic and can cause skin lesions in other animals. additionally, sporothrix schneckii associated cutaneous disease is reported in several rodent species and can be zoonotic. dermatitis lesions vary from suppurative and necrotizing to granulomatous and fibrosing depending on chronicity. paspositive yeast are present within macrophages. character-istic organisms on cytologic preparations of skin scrapes prepared with potassium hydroxide support the diagnosis. capillaria hepatica (synonymn calodium hepaticum) affects a broad host range worldwide, including rodents and humans. it is a ubiquitous parasite of globally invasive norway and black rat populations . gross lesions consist of multifocal to coalescing, tortuous, white tracks in the liver parenchyma ( fig. 20.16a ). histologically, varying combinations of lymphoplasmocytic and granulomatous inflammatory infiltrates, fibrosis, and bioperculate eggs and occasionally adult parasites replace hepatic parenchyma (fig. 20.16b ). cross sections of viable or mineralized capillarid nematodes may be present. eggs are only released from the liver following carcass decomposition or ingestion by a predator/cannibalistic conspecific. therefore, diagnosis is based on the presence of the nematode and/or eggs in the liver and not fecal parasitology. many species of free-ranging rodents are susceptible to fatal visceral larval migrans associated with the raccoon nematode baylisascaris procyonis (fig. 20.17 ). significant clinical disease is often related to parasite migration in the brain, where they may be accompanied by low numbers of eosinophils, gliosis, spheroids, and rarefaction or necrosis of the neuropil. rodents, particularly squirrels are prone to notoedric mange cornish et al., 2001; . mites burrow into the stratum corneum inducing marked hypekeratosis, which results in patchy alopecia, and crusting and thickening of the skin (lichenification), which can appear yellow to gray. gross lesions are predominantly found on the ears, nose, tail, external genitalia, inguinal and perianal regions, and feet though they can cover the entire body in severe cases. peripheral lymphadenopathy may also be seen. skin scrapings and fecal floats are beneficial to best examine mite features and presence of eggs, respectively. histologically, there is marked epidermial hyperplasia with parakeratotic hyperkeratosis forming caps over tunnels containing mites with superficial, perivascular eosinophilic dermatitis ( fig. 20.18a,b) . secondary bacterial infections are common. additional notable parasites of rodents are listed in the supplemental materials (table e3) . chronic progressive nephropathy (cpn), naked mole-rat, kidney. in the kidney, there is marked ectasia of tubules and bowman's spaces forming numerous microcysts seen at low magnification. some ectatic tubules contain luminal proteinaceous material and there is multifocal tubular degeneration, necrosis, and regeneration. interstitium has areas of mixed inflammation, fibrosis and edema. glomeruli have segmental to global membranous change and multifocal sclerosis. (see fig. 20 .4). hepatic hemosiderosis is seen in sections of the liver. eslide: vm05054 20.e2 chronic progressive nephropathy (cpn), naked mole-rat, kidney. in the kidney, there is marked ectasia of tubules and bowman's spaces forming numerous microcysts seen at low magnification. some ectatic tubules contain luminal proteinaceous material and there is multifocal tubular degeneration, necrosis, and regeneration. interstitium has areas of mixed inflammation, fibrosis and edema. glomeruli have segmental to global membranous change and multifocal sclerosis. (see fig. 20 .4). hepatic hemosiderosis is seen in sections of the liver. eslide: vm05053 20.e3 chronic progressive nephropathy (cpn), naked mole-rat, kidney. there is mild to moderate ectasia of tubules and bowman's spaces forming numerous microcysts seen at low magnification. some ectatic tubules contain luminal proteinaceous material and there is multifocal tubular degeneration, necrosis, and regeneration. interstitium has areas of mixed inflammation, fibrosis and edema. glomeruli have segmental to global membranous change and multifocal sclerosis. (see fig. 20 .4). eslide: vm05057 20.e4 hepatic hemosiderosis, naked mole-rat, liver. prussian blue staining highlights bright blue granular intracytoplasmic (iron) pigments throughout the liver. the kidney has changes consistent with chronic progressive nephropathy. eslide: vm05058 20.e5 yersiniosis, beaver, liver and diaphragm. yersiniosis in the liver and diaphragm of a free-ranging north american beaver. bacterial infection causes multifocal random,necrosis throughout the hepatic parenchyma. large, central colonies of the causative agent, yersinia pseudotuberculosis are surrounded by mild neutrophilic inflammation. (see fig. 20 .11). eslide: vm04954 20.e6 tularemia, beaver, liver and spleen. tularemia in the liver and spleen of a free-ranging north american beaver. large, multifocal random areas of coagulative necrosis are present throughout the hepatic and splenic parenchyma. note the relative lack of inflammation. unlike yersiniosis, bacterial colonies are not readily detected with routine hematoxylin and eosin staining. (see fig. 20 .12). eslide: vm04952 20.e7 pneumocystis murina pneumonia, mouse, lung. multifocally, alveoli are partially to completely filled with large macrophages containing small round yeasts.(see fig. 20 .14). eslide: vm05178 20.e8 pneumocystis murina pneumonia, mouse, lung, gms. gomori methenamine silver (gms) staining highlights the intrahistiocytic yeasts within the alveoli. (see fig. 20 .14). eslide: vm05275 20.e9 pulmonary cryptococcosis, slender-tailed cloud rat, lung. the interstitium and air spaces are moderately to markedly expanded and distorted by myriad fungal yeasts that are associated with histiocytic to granulomatous inflammation. aggregates of lymphoplasmacytic inflammation are present but infrequent. (see fig. 20 .15). eslide: vm05089 20.e10 pulmonary cryptococcosis, slender-tailed cloud rat, lung. gms. intralesional yeasts are hhighlighed gray/ blue with silver staining. gms (see fig. 20 .15). eslide: vm05096 20.e11 pulmonary cryptococcosis, slender-tailed cloud rat, lung. mucicarmine. mucicarmine staining highlights the thick mucopolysaccharide capsule of the crytpococcal yeast and stains it bright pink. mucicarmine (see fig. 20 .15). eslide: vm05097 20.e12 capillaria hepatica (calodium hepaticum), norway rat, liver. capillaria hepatica in the liver of a freeranging norway rat. adult nematodes and eggs invade and efface the liver parenchyma. there are examples of viable and degenerative adults. the eggs are characteristically bioperculate with a thick shell. inflammation is lymphoplasmacytic to granulomatous and is associated with fibrosis indicating chronic infection. in some areas, there is multifocal mineralization adjacent to eggs. (see fig. 20 .16). eslide: vm04956 20.e13 baylisacaris visceral larval migrans, groundhog, brain. multifocally, multiple sections of larval nematodes with prominent lateral alae and lateral cords, a thick cuticle, and coelomyarian musculature, features consistent with larval ascarids, are present in the brain. inflammation, including eosinophils, gliosis, and spheroids and rarefaction of the neuropil due to parasite migration, are multifocal and occasionally associated with intralesional nematodes. (see fig. 20 .17). eslide: vm04961 (2012); krinke (1994) , krinke (1996) mice, rats mammary fibroadenoma rudman (2012); krinke (1994) , krinke (1996) gray squirrel mammary (mixed malignant) williams (1989) mice, rats zymbal gland adenoma/carcinoma rudman (2012); krinke (1994) , krinke (1996) mice, rats preputial/clitoral adenoma/carcinoma rudman (2012), , krinke (1994) , krinke (1996) female reproductive ovarian adenocarcinoma , krinke (1994) , krinke (1996) mice teratoma dixon (2014), mice, rats uterine decidual reaction/deciduoma dixon (2014) woodchuck, mice, rats uterine leiomyoma foley (1993) , mice, rats uterine leiomyosarcoma dixon (2014) woodchuck adenoma of the rete testis foley (1993) rat, woodchuck interstitial cells tumors foley (1993) , , creasy (2012) woodchuck sertoli cell tumors foley (1993) woodchuck seminoma foley (1993) woodchuck, mice teratoma foley (1993) , , creasy (2012) hepatobilliary foci of cellular alteration thoolen (2010) woodchuck, rat, mice hepatocellular adenoma roth (1991) , thoolen (2010) , krinke (1994) , krinke (1996) woodchuck, rat, mice hepatocellular carcinoma , thoolen (2010) , krinke (1994) , krinke (1996) hemolymphatic mice, rats histiocytic sarcoma krinke (1994) , krinke (1996) , kogan (2002) , guinea pig, rat lymphocytic leukemia yarto-jaramillo (2011), guinea pig, hamster, mice, woodchuck, rat lymphoma nagy (2002) , , yarto-jaramillo (2011), kogan (2002) , woodchuck, mice, rats myeloproliferative disease roth (1991) , kogan (2002) , (2008), , krinke (1994) , krinke (1996) ground squirrel integumentary gland adenocarcinoma carminato (2012) damaraland mole rat melanomas sura (2011) yellow cheeked vole sebacious gland adenoma williams (1989) capybara, tundra vole, arctic ground squirrel,woodchuck squamous cell carcinoma hamanno (2014) , takahisa (2014), anderson (1990) beaver, insular vole, porcupine squamous papilloma williams (1989) hamsters, red squirrels trichoepithelioma , guinea pig trichofolliculoma (2004) beaver rhabdomyoma williams (1989) red backed vole rhabdomyosarcoma williams (1989) tundra vole osteoma williams (1989) guinea pig alveologenic carcinoma yarto-jaramillo (2015) guinea pig bronchogenic carcinoma yarto-jaramillo (2015) guinea pig papillary adenoma yarto-jaramillo (2015) gray squirrels pulmonary adenomatosis red squirrels, mice pulmonary carcinoma , , renne mice, rats pulomnary adenoma , renne (2009) red and gray squirrels, mice, rats renal adenoma , williams (1989) , frazier spontaneous neoplastic and hyperplastic skin lesions of the woodchuck cutaneous and systemic poxviral disease in red (tamiasciurus hudsonicus) and gray (sciurus carolinensis) squirrels pathology of laboratory rodents and rabbits non-proliverative and proliferative lesions of the cardiovascular system of the rat and mouse endocrine system odontoma-like tumours of squirrel elodont incisors -elodontomas spontaneous hibernomas in sprague-dawley rats adenocarcinoma of the dorsal glands in 2 european ground squirrels (spermophilus citellus) synhimantus (nematoda) associated with gastric squamous tumors in muskrats proliferative and nonproliferative lesions of the rat and mouse male reproductive system nonproliferative and proliferative lesions of the rat and mouse female reproductive system neoplastic and nonneoplastic lesions of the reproductive tract of the woodchuck (marmota monax) polyomavirus infection in hamsters and trichoepitheliomas/ cutaneous adnexal tumors proliferative and nonproliferative lesions of the rat and mouse urinary system proliferative and non-proliferative lesions of the rat and mouse soft tissue, skeletal muscle and mesothelium squamous cell carcinoma in a capybara (hydrochoerus hydrochaeris) malignant pleural mesothelioma in a woodchuck (marmota monax) oral leiomyosarcoma in a woodchuck (marmota monax) changes in the major ocular glands nonneoplastic and neoplastic changes in the harderian and lacrimal glands pulmonary lesions produced by fibromas viruses in squirrels and rabbits bethesda proposal for classification of nonlymphoind hematopoietic neoplasms in mice lymphosarcoma in the laboratory woodchuck obstructive respiratory disease in prairie dogs with odontomas proliferative and nonproliferative lesions of the rat and mouse respiratory tract harderian gland neoplasms in captive, wild-caught beechey ground squirrels (spermophilus beecheyi) chronic hepatitis and hepatocellular carcinoma associated with persistent woodchuck hepatitis virus infection hepatic lesions in woodchucks (marmota monax) serongative for woodchuck hepatitis virus lesions associated with eucoleus sp. in the non-glandular stomach of wild urban rats (rattus norvegicus) proliferative and nonproliferative lesions of the rat and mouse mammary, zymbal's preputial and clitoral glands hamster polyomavirus infection in a pet syrian hamster (mesocricetus auratus) causes of mortality and pathological lesions observed post-mortem in red squirrels (sciurus vulgaris) in great 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the zoonotic pathogens carried by rodents that cause disease in wild animals and those deemed most relevent to wildlife and zoo animal specialists ear mange mites (notoedres muris) in black and norway rats (rattus rattus and rattus norvegicus) from inner-city vancouver ear mange mites (notoedres muris) in black and norway rats (rattus rattus and rattus norvegicus) from inner-city vancouver nathural pathogens of laboratory mice, rats, and rabbits and their effects on research toxoplasmosis in a woodchuck (marmota monax) and two american red squirrels (tamiasciurus hudsonicus) baylisascariosis-infections of animals and humans with 'unusual'roundworms echinococcus multilocularis detection in live eurasian beavers (castor fiber) using a combination of laparoscopy and abdominal ultrasound under field conditions synhimantus (nematoda) associated with gastric squamous tumors in muskrats besnoitia jellisoni (sporozoa: toxoplasmea) in rodents from utah and california a survey of hemoparasite infections in free-ranging mammals and reptiles in french guiana systemic toxoplasmosis in a five month old beaver, (castor canadensis) echinococcus vogeli rausch and bernstein, 1972, from the paca, cuniculus paca l.(rodentia: dasyproctidae), in the departamento de santa cruz pathomorphologic findings in short-tailed voles (microtus agrestis) experimentallyinfected with frenkelia microti pneumocystis carinii causes a distinctive interstitial pneumonia in immunocompetent laboratory rats that had been attributed to "rat respiratory virus more than a rabbit's tale-encephalitozoon spp. in wild mammals and birds studies on the nematode parasite, gongylonema neoplasticum (spiroptera neoplasticum) and avitaminosis a in the forestomach of rats: comparison with fibiger's results echinococcus multilocularis in a european beaver from switzerland frenkelia sp. from the brain of a porcupine (erethizon dorsatum) from alberta the role of wildlife rehabilitation as sentinels for one health issues at the wildlife and public health interface: reports of taenia crassiceps cysticercosis in woodchucks (marmota monax) and squirrels (sciurus carolinensis) in maryland and virginia cysticerci of taenia mustelae in the fox squirrel characteristics of natural infections of the stomach worm, obeliscoides cuniculi (graybill), in lagomorphs and woodchucks in canada first identification of echinococcus multilocularis in rodent intermediate hosts in sweden parasites of ferrets, rabbits, and rodents klossiella infection of the guinea pig the taxonomic status of echinococcus cruzi brumpt and joyeux, 1924 (cestoda: taeniidae) from an agouti (rodentia: dasyproctidae) in brazil capillaria hepatica in wild norway rats (rattus norvegicus) from vancouver lesions associated with eucoleus sp. in the non-glandular stomach of wild urban rats (rattus norvegicus) cutaneous leishmaniasis in the amazon: isolation of leishmania (v.) lainsoni rodent agouti paca (rodentia: dasyproctidae) in the state of para causes of mortality and pathological lesions observed post-mortem in red squirrels (sciurus vulgaris) in great britain pathologic findings in western gray squirrels (sciurus griseus) from a notoedric mange epidemic in san bernardino moutnains, california natural infection of the ground squirrel (spermophilus spp.) with echinococcus granulosus in china ear mange mites (notoedres muris) in black and norway rats (rattus rattus and rattus norvegicus) from inner-city vancouver. can nathural pathogens of laboratory mice, rats, and rabbits and their effects on research cutaneous and systemic poxviral disease in red (tamiasciurus hudsonicus) and gray (sciurus carolinensis) squirrels pathology of laboratory rodents and rabbits diseases of agouti (dasyprocta agouti) rained in captivity diagnosed by pathological examination cryptococcus neoformans pneumonia in slender tailed cloud rats (phloeomys pallidus): a review of seven cases odontoma-like tumors of squirrel elodont incisors -elondontomas order 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risks for public health persistent paramesonpehric ducts (masculine uterus) in the male north american beaver (castor candadensis) polycystic kidney disease in adult brazilian agoutis (dasyprocta leporina) francisella tularensis infection rodentia chapter | 20 515 without lesions in gray tree squirrels (sciurus griseus): a diagnostic challenge practical pathology of aging mice fluorosis risks to resident hispid cotton rats on land-treatment facilities for petrochemical wastes detection of francisella tularensis in voles in finland. vector borne zoonotic dis congestive cardiomyopathy in the woodchuck, marmota monax chronic hepatitis and hepatocellular carcinoma associated with persistent woodchuck hepatitis virus infection capillaria hepatica in wild norway rats (rattus norvegicus) from vancouver survey of cardiovascular pathology in wild urban rattus norvegicus and rattus rattus respiratory pathology and pathogens in wild urban rats (rattus norvegicus and rattus rattus) hamster polyomavirus infection in a pet syrian hamster (mesocricetus auratus) causes of mortality and pathological lesions observed postmortem in red squirrels (sciurus vulgaris) in great britain pathologic findings in western gray squirrels (sciurus griseus) from a notoedric mange epidemic in san bernardino moutnains, california contributions of microbes in vertebrate gastrointestinal tract to production and conservation of nutrients the laboratory rabbit, guinea pig, hamster, and other rodents neoplasia and granulomas surrounding microchip transponders in damaraland mole rats (cryptomys damarensis) four cases of spontaneous neoplasia in the naked mole-rat (heterocephalus glaber), a putative cancer-resistant species an epizootic of fibromatosis in gray squirrels (sciurus carolinensis) in florida parapoxvirus causes a deleterious disease in red squirrels associated with uk population declines listeriosis in seven bushy-tailed jirds spontaneous reproductive pathology in female guinea pigs lung morphology in rodents (mammalia rodentia) and its implication for systematics working underground: respiratory adaptations in the blind mole rat fibroma virus infection in an eastern fox squirrel (sciurus niger) from sangamon county illinois fowler's zoo and wild animal medicine chirodiscoides caviae skin none guinea pigs morrisey (1996) demodex skin alopecia, dermatitis, pruritis gerbil, hamster morrisey (1996) gliricola porcelli skin alopecia, crusting dermatitis, pruritis guinea pigs morrisey (1996) gyropus ovalis skin alopecia, crusting dermatitis, pruritis guinea pigs morrisey (1996) myobia musculi skin alopecia, pruritis, selfmutilation mice myocoptes musculinis skin alopecia, pruritis, selfmutilation mice morrisey (1996) notoedres sp. skin alopecia, crusting dermatitishamster, grey squirrel, ratsanholt (2014), , morrisey (1996) , ornithonyssus bacoti skin anemia rats morrisey (1996) polyplax serrata skin anemia, pruritis, dermatitis mice morrisey (1996) polyplax spinulosa skin anemia, pruritis rats morrisey (1996) psoregates simplex skin follicular nodules mice morrisey (1996) radfordia affinis skin alopecia, pruritis, selfmutilation mice morrisey (1996) trixacarus caviae skin alopecia, crusting dermatitis guinea pigs morrisey (1996) key: cord-254950-y6kayxie authors: morse, stephen s. title: mouse thymic virus (mtlv; murid herpesvirus 3) infection in athymic nude mice: evidence for a t lymphocyte requirement date: 1988-03-31 journal: virology doi: 10.1016/0042-6822(88)90262-0 sha: doc_id: 254950 cord_uid: y6kayxie abstract mouse thymic virus (mtlv; murid herpesvirus 3) is a lymphotropic herpesvirus that cytolytically infects developing t lineage lymphocytes in the thymus of neonatal mice. mtlv establishes a persistent infection and can be recovered indefinitely from infected mice, but nothing is known about requirements for this persistent infection. in order to determine whether t lineage lymphocytes are required for infection, young adult athymic nude (nulnu) mice and euthymic littermates were infected with mtlv and tested for virus shedding. although euthymic littermates regularly shed virus, in the nude mice only about 20% of isolation attempts up to 100 days postinfection were positive. blind passage yielded an additional three isolations out of 14 samples (21 %). in addition, unlike many other herpesviruses, the virus did not replicate in a number of epithelial and fibroblastic cell lines that were tested. these data confirm that the virus is preferentially t lymphotropic and suggest that infection may require t lineage lymphocytes. mouse iclymic virus (mtlv; murid herpesvirus 3) is a lymphotropic herpesvirus that infects and kills developing t lymphocytes in the thymus of neonatal mice (1) (2) (3) . the virus is also cytolytic for t lymphocytes in peripheral lymphoid tissues (4) . when adult mice are infected, the virus appears to bypass the thymus (2) but can be isolated indefinitely from salivary glands and on mouth swabs (1) (2) (3) . irrespective of virus dose and age at primary infection, regular lifetime virus shedding appears to be the rule (1, 2) . although thymus-derived lymphocytes appear to be the targets for primary infection in newborn mice, nothing is known about cells involved in persistent infection. the nude mouse is an ideal model for dissecting the effects of lymphotropic viruses. nude mice are athymic as a result of a genetic defect which prevents the development of the embryonic thymus (5) . because t lymphocytes mature in the thymus, this lymphocyte type is largely absent in nude mice (6). the nude gene is autosomal recessive; heterozygotes (+lnu) are phenotypically normal (5) and have essentially normal t lymphocyte function (6). in this study, experimentally infected nude mice and euthymic littermates were used to determine whether t lymphocytes are required for infection by mtlv. because there is no tissue culture system for assay of mtlv in vitro (2, 3) , the standard assay, and the most sensitive and reliable assay available at present is based on infectivity (thymic necrosis) in litters of newborn mice. the breeding stocks used to provide litters for our infectivity assay have been screened for thymic virus by infectivity testing of mouth swabs and salivary glands from retired breeders (i, 3) and are negative. in addition, the breeding stocks used were seronegative for all known murine pathogens. forvirus assay of mouth swabs, each swab was expressed into 1 ml dulbecco's modified eagle's medium supplemented with 10% fetal bovine serum and 50 pg gentamycin per milliliter (dme-1 0), and the liquid was inoculated intraperitoneally (0.05 ml per mouse; additional mice received up to 0.2 ml per newborn mouse) into litters of newborn (less than 24 hr old) balb/c mice (3). the pups were examined for thymic necrosis after 9-l 1 days (2, 3); any visible necrosis was considered positive. typically, these necrotic thymuses were ~20% of normal size. to determine whether mtlv infection requires thymus-derived lymphocytes, 4-week-old female icr swiss athymic nude (nulnu) and euthymic (+lnu) littermate controls (four each; memorial sloan-kettering cancer center nude mouse breeding colony) were inoculated intraperitoneally with either 40 or 200 ids0 of mtlv and virus shedding was tested by mouth swabs beginning 6 days after infection. all nude mice and littermate controls were negative for mtlv by virus isolation at the outset of the experiment (before inoculation). after inoculation, all the inoculated mice became infected with the virus. ability to recover virus was not correlated with input virus dose. virus could as readily be recovered from mice that had received 40 ids0 as from those that had received 200 idso. the infected mice remained healthy throughout the observation period (over 100 days), and no overt disease was attributable to mtlv infection in the nude mice. in the euthymic controls, all virus isolation attempts were positive through 100 days postinfection, with most of the inoculated pups developing macroscopic thymic necrosis (mean, 92%). in the athymic nude mice, by contrast, although virus was recovered at least once from each nude mouse (table l) , only 5 of 27 primary isolation attempts from day 6 through day 98 were positive, giving an isolation rate of 18.5%. in positive isolations, fewer inoculated pups showed thymic necrosis (mean, 14.2%) than pups that received swab fluids from controls (table 1) . for many viruses, virulence or tissue tropism can be altered by conditions of infection (7); introduction of mtlv into the nude mouse could therefore have altered virus infectivity. blind passage is a standard method for restoring virulence and increasing assay sensitivity (1, 2) . although limitations in the number of note. infected icr swiss athymic nude (nulnu) and euthymic (+/nu) littermate controls (total number of mice, eight) were tested forvirus shedding at the times shown. forvirus assay, mouth swabs were taken using cotton swabs moistened with dulbecco's modified eagle's medium + 10% fetal bovine serum and 50 pg gentamytin per milliliter (dme-1 0), and tested for mtlv by infectivity assay in newborn balb/c mice as described in the text. additional infectivity assays in normal newborn icr background swiss mice gave the same results. a values in parentheses represent number of nude mice yielding positive isolations on that day. infectivity assays from four nude mice through day 30, three on day 34, three on days 44/48 together, and two on day 98. litters available did not allow every negative sample to be tested, additional litters of normal newborn mice were inoculated with fresh homogenates (1 o-20%, w/v) of randomly selected negative thymuses and salivary glands, representing various test dates up to day 48, from 14 assay litters that had received swab fluids from nude mice. up to two blind passage cycles were performed. these passages yielded a total of three additional isolations (21%) ( table 2 ). in an additional biological passage, separate litters of normal neonatal mice were inoculated with swab fluids from each of the nude mice (day 50 postinfection) and these newborns were allowed to reach adulthood. homogenates of salivary glands and thymuses were prepared from these animals, and tested for mtlv by infectivity assay in fresh litters of newborn mice. one sample, from an animal that had received fluids from nude mouse 2, was positive on assay. this represented only the second isolation from this nude mouse, which was positive on primary isolation on day 6 and had been consistently negative thereafter. tissues from uninfected mice (as negative controls), or other negative samples, passaged at the same time as nude mouse samples were consistently negative. nude mice lack t lymphocyte function, but nonspecific immune functions, such as natural killer (nk) activity, are normal or even increased in the nude mouse (8). nk-deficient nude mice have recently been developed (9) . in order to determine whether reduced virus shedding in the nude mice could have been due to active suppression by nk cells or similar mechanisms, eight nk-deficient balb/c-c57 beige hybrid mice (life sciences, st. petersburg, fl) were infected with mtlv and tested at various times after infection. there were six positive or possibly positive isolations out of a total of 46 attempts (13%) from 1 to 14 weeks postinfection. virus was isolated consistently more than once from only one mouse, which was negative on day 6, positive on days 19, 26, and 41, positive with a lower level of virus shedding on day 33, and negative on day 48 and thereafter. one other nude mouse appeared to shed a low level of virus on day 19 and was possibly positive on day 34, but appeared negative on other isolation attempts including days 6, 41, and 48 postinfection. the other six nude mice were negative on all isolation attempts (e.g., days 6, 19, 26, 33-34, 41, 48 and thereafter through 14 weeks). thus, mtlv establishes a persistent infection in the nude mouse, but virus shedding may be reduced in comparison with euthymic mice. aside from thymusderived (t) lymphocytes, most other cell types appear to be normal in the nude mouse (5, 6) . therefore, if persistent mtlv infection were to involve mostly nonlymphoid cells, all isolations should have been positive after initial infection. that this did not appear to occur suggests that t lineage lymphocytes might well be the major target cells for persistent infection. this has been demonstrated in herpesvirus sylvilagus, a rabbit herpesvirus, which can be isolated from saliva and salivary glands of infected hosts, but the virus is associated with lymphocytes and not with epithelial cells (12) . mtlv similarly persists in salivary gland (1, 2), but electron microscopy of salivary gland epithelial cells of infected animals revealed no abnormalities (i), and no viral antigens could be detected in salivary gland epithelial cells (2) . there are two likely explanations for the apparently low level iof virus shedding in the nude mouse. although nude mice lack mature functional t lymphocytes, early t lineage precursors are present in the nude mouse (6, 8, 10); under certain conditions, these cells in the nude mouse can also differentiate extrathymically into mature t lymphocytes (7 1). in addition, several reports indicate that a small number of apparently matu"e (bearing the t lymphocyte marker thy 1) t lineage lymphocytes are present in nude mice throughout their lives (6, 10). a small population of this type could be persistently infected and responsible for the low level of virus shedding observed. this population reportedly increases with age or intercurrent infection (111. infection with the coronavirus mouse hepatitis virus (mhv), a common murine pathogen endemic in many nude mouse colonies, has also been reported to induce extrathymic t cell differentiation in nude mice (11) . some of our preliminary results (data not shown) suggest that nude mice older at primary infection, or nude mice that have been infected with mhv, appear more likely to shed mtlv. alternatively, there could be a secondary cell type (such as the macrophage) in which virus can persist at low levels even in the absence of t lymphocytes. macrophage infection by another t lymphocytolytic virus, human immunodeficiency virus (hiv-l), has been documented (13). our preliminary attempts to isolate mtlv from adherent peritoneal macrophages of persistently infected euthymic mice have been negative so far. however, low levels of infected cells might not be detected. specific molecular probes, such as genomic dna probes or monoclonal antibodies, are not presently available for this virus. we are in the process of producing specific high-titer antisera and other probes in order to test the presence of mtlv in various lymphoid and hematopoietic tissues and in nerve cells. as regards other susceptible cells, original attempts to grow the virus in mouse embryo cells or mouse embryo kidney cells, among others, were unsuccessful (1). our attempts to infect a variety of epithelial and fibroblastic cell cultures that support the replication of many other viruses have been uniformly negative. for infection, subconfluent monolayers of cells were incubated for 90 min with dme-10 containing 500-2000 ids0 mtlv and polybrene (30 pg/ml), then fed (dme supplemented with 5010 fetal bovine serum), incubated, and tested for mtlv by infectivity assay at 5-7 days and at 2 weeks. cells tested included ar42j rat glandular epithelium, balb cl.7 mouse fibroblast, balb 3t3 fibroblast, cl271 mouse mammary tumor, j774a.l mouse macrophage, nctc 1469 mouse liver, sc-l feral mouse embryo, tcmk-1 mouse kidney, 32 ill a rat hepatoma, a549 human lung carcinoma, hep-2 human epidermoid carcinoma, mdck canine kidney epithelium, vero african green monkey kidney, wish diploid human amniotic epithelium, 324k human kidney, and primary cultures of african green monkey kidney, cynomolgous monkey kidney, rabbit kidney, and rhesus monkey kidney. replicate cultures, observed up to a month for cytolysis or focus formation, remained negative throughout. additional cultures cocultivated with primary mtlv-infected thymocytes (5 x 105/ml, harvested day 5 postinfection) also remained uninfected. these results suggest that mtlv is unable to productively infect many cell lines that support the cytolytic replication of many viruses including other lymphotropic herpesviruses. taken together, these lines of evidence appear to implicate the t lineage lymphocyte as the primary target of both acute and persistent mtlv infection. co/d spring harbor conf. cell pro/if key: cord-013023-uanozm00 authors: crouse, richard b; kim, kristen; batchelor, hannah m; girardi, eric m; kamaletdinova, rufina; chan, justin; rajebhosale, prithviraj; pittenger, steven t; role, lorna w; talmage, david a; jing, miao; li, yulong; gao, xiao-bing; mineur, yann s; picciotto, marina r title: acetylcholine is released in the basolateral amygdala in response to predictors of reward and enhances the learning of cue-reward contingency date: 2020-09-18 journal: nan doi: 10.7554/elife.57335 sha: doc_id: 13023 cord_uid: uanozm00 the basolateral amygdala (bla) is critical for associating initially neutral cues with appetitive and aversive stimuli and receives dense neuromodulatory acetylcholine (ach) projections. we measured bla ach signaling and activity of neurons expressing camkiiα (a marker for glutamatergic principal cells) in mice during cue-reward learning using a fluorescent ach sensor and calcium indicators. we found that ach levels and nucleus basalis of meynert (nbm) cholinergic terminal activity in the bla (nbm-bla) increased sharply in response to reward-related events and shifted as mice learned the cue-reward contingency. bla camkiiα neuron activity followed reward retrieval and moved to the reward-predictive cue after task acquisition. optical stimulation of cholinergic nbm-bla terminal fibers led to a quicker acquisition of the cue-reward contingency. these results indicate bla ach signaling carries important information about salient events in cue-reward learning and provides a framework for understanding how ach signaling contributes to shaping bla responses to emotional stimuli. learning how environmental stimuli predict the availability of food and other natural rewards is critical for survival. the basolateral amygdala (bla) is a brain area necessary for associating cues with both positive and negative valence outcomes (baxter and murray, 2002; janak and tye, 2015; ledoux et al., 1990) . a recent study has shown that genetically distinct subsets of bla principal neurons encode the appetitive and aversive value of stimuli (kim et al., 2016b) . this encoding involves the interplay between principal neurons, interneurons, and incoming terminal fibers, all of which need to be tightly regulated to function efficiently. the neuromodulator acetylcholine (ach) is released throughout the brain and can control neuronal activity via a wide range of mechanisms. ach signals through two families of receptors (nicotinic, nachrs and muscarinic, machrs) that are differentially expressed on bla neurons as well as their afferents (picciotto et al., 2012) . ach signals through these receptors to increase signal-to-noise ratios and modify synaptic transmission and plasticity in circuits involved in learning new contingencies (picciotto et al., 2012) , especially in areas that receive dense cholinergic input, like the bla (woolf, 1991; zaborszky et al., 2012) . the effect of ach signaling can differ depending on the receptor, as metabotropic machrs work on a slower timescale than the rapid, ionotropic nachrs (gu and yakel, 2011; picciotto et al., 2012) . the overall impact of ach signaling on the bla is likely quite heterogeneous as machrs are coupled to both inhibitory and excitatory signaling cascades and nachrs are found on both glutamatergic and gabaergic bla neurons (picciotto et al., 2012) . the basal forebrain complex is a primary source of ach input to the bla. in particular, the nucleus basalis of meynert (nbm) sends dense cholinergic projections to the bla (woolf, 1991; zaborszky et al., 2012) . optical stimulation of bla-projecting cholinergic terminal fibers (nbm-bla) during fear conditioning is sufficient to strengthen fear memories (jiang et al., 2016) and may support appetitive behavior (aitta-aho et al., 2018) . cholinergic nbm neurons increase their firing in response to both rewarding and aversive unconditioned stimuli (hangya et al., 2015) . cholinergic signaling in the medial prefrontal cortex and visual cortex has been linked to cue detection (parikh et al., 2007) and reward timing (chubykin et al., 2013; liu et al., 2015) , respectively. a recent study has also demonstrated that nbm cells fire in response to a conditioned stimulus during trace fear conditioning, indicating that ach signaling may be involved in learning about cues that predict salient outcomes (guo et al., 2019) . we hypothesized that ach signaling in the bla is a critical neuromodulatory signal that responds to both unconditioned stimuli and cues that gain salience, thereby coordinating activity in circuits necessary for learning cue-reward contingencies. to test this hypothesis, we measured relative levels of bla ach (ach signaling), cholinergic nbm-bla terminal fiber activity (bla ach signal origin), and the activity of bla principal neurons (bla output) across all phases of learning in an appetitive operant learning task to evaluate how bla output and ach signaling are related to behavioral performance in this paradigm. we then optically stimulated cholinergic nbm fibers locally in the bla, while mice learned to nose poke in response to an auditory cue to receive a food reward to determine if accelerating the increase in ach signaling that occurs as mice learn the task would enhance performance. we also pharmacologically blocked different ach receptors during the learning task to determine the subtypes involved, and varied the timing of optical stimulation of cholinergic nbm-bla terminal fibers to determine whether ach release time-locked with the reward-predictive cue is necessary for the improvement of the task performance. these studies provide a novel framework for understanding how nbm ach signaling in the bla is recruited during the perception of novel stimuli and how it contributes to linking previously neutral cues to predictions about future salient outcomes. acetylcholine release in the bla occurs at salient points in the cuereward learning task and shifts as mice learn the cue-reward contingency the bla is critical for learning that previously neutral cues can predict future punishments or rewards and for assigning valence to those cues (baxter and murray, 2002; janak and tye, 2015) . the bla receives dense cholinergic input (woolf, 1991; zaborszky et al., 2012) and we speculated that, since ach signaling is involved in both attention and several types of learning (picciotto et al., 2012) , it could be essential for learning about cues that predict salient events, such as reward delivery. based on data showing that ach neurons fire in response to unexpected or salient events (hangya et al., 2015) , we also hypothesized that ach release might vary as mice learn a cue-reward contingency. therefore, we designed a cue-reward learning task in which food-restricted mice were trained to perform a nose poke when signaled by a cue (tone) to receive a palatable reward (ensure) on a 30 s variable intertrial interval (iti; figure 1a -d). we injected adeno-associated virus (aav) carrying an improved version of the fluorescent ach sensor grab ach3.0 (ach3.0; jing et al., 2018; jing et al., 2019) construct into the bla of mice and implanted an optical fiber above the bla to record ach signaling during the cue-reward learning task ( during the pre-training phase of the task, mice received reward and receptacle light presentation for performing a nose poke in the active port during tone presentation ( figure 1c , purple active nose poke coincident with tone) but there was no consequence for an incorrect nose poke ( figure 1c , red active nose poke not coincident with tone). animals quickly learned to make a high number of responses over the course of each pre-training session. in this paradigm, mice obtained most available rewards by day 5 of pre-training ( figure 2b , blue shaded region). however, this phase of training did not promote learning of the cue-reward contingency, (i.e. that they should only nose poke during tone presentation) seen by the high number of incorrect nose pokes (figure 2figure supplement 2a, blue shaded region). mice performed roughly eightfold more incorrect nose pokes than correct nose pokes, suggesting that mice were not attending to the task contingency. the training phase of the task was identical to pre-training except incorrect nose pokes resulted in a 5 s timeout, during which the house light was illuminated, that concluded with a restarting of the iti timer ( figure 1d , red active nose poke not coincident with tone). on day 1 of the training phase, all animals earned fewer rewards ( figure 2b , pink shading) and, while still high, incorrect nose pokes dropped (figure 2-figure supplement 2a, pink shading). animals that did not meet acquisition criterion by day 9 (defined as consistently earning 20 or more rewards per session, figure 2b , white horizontal line) were moved to a 20 s variable iti to promote responding ( figure 2b , pink shading day 10). following the change in iti, mice acquired the cue-reward behavior at different rates. after figure 1 . experimental timeline and cue-reward learning paradigm. (a) experimental timeline. mice began food restriction 7 d after surgery and were maintained at 85% free-feeding body weight for the duration of the experiment. after 7 d of handling, 5-6 d of operant familiarization prepared the mice for the cue-reward learning task (pre-training through extinction). (b) behavioral chamber setup. mice were placed in modular test chambers that included two nose poke ports on the left wall (active and inactive) and the reward receptacle on the right wall. a tone generator and timeout light were placed outside the modular test chamber. for fiber photometry (fp) and optical stimulation (laser) experiments, mice were tethered to a patch cord(s). (c-d) details of the cue-reward learning paradigm. (c) in pre-training, an auditory tone was presented on a variable interval 30 schedule (vi30), during which an active nose poke yielded ensure reward delivery but there was no consequence for incorrect nose pokes (active nose pokes not during tone). (d) training was identical to pre-training, except incorrect nose pokes resulted in a 5 s timeout, signaled by timeout light illumination, followed by a restarting of the intertrial interval (iti). during pre-training, when there were high numbers of both correct and incorrect nose pokes, there was a large increase in ach release following correct nose pokes, which were followed by reward delivery and receptacle light, but not incorrect nose pokes ( figure 2c + figure 2 -figure supplement 1b-c). we used bootstrapped confidence intervals (bcis) to determine when transients were statistically significant (bci did not contain the null of 0 [jeanrichard-dit-bressel et al., 2020] ). correct, but not incorrect, nose poke trials consistently showed a sustained, significant responses of mice expressing ach3.0 in bla. individual mice acquired the task at different rates as measured by rewards earned. horizontal white line: acquisition threshold, when a mouse began to earn 20 rewards consistently in training. incorrect nose pokes shown in figure 2 -figure supplement 2a. pre-training (pt): blue shaded area, training: pink shaded area, extinction (ext): orange shaded area. (c) fluorescence traces from bla of ach3.0expressing mouse. a significant increase in fluorescence representing bla ach release consistently coincided with correct (green line) but not incorrect (gray line) nose pokes on the last day of pt (data are shown from mouse 1). the mean z-scored precent df/f0 (z%df/f0) overlaid on bootstrapped 99% confidence intervals (99% bcis). shaded significance bars under traces represent time points where 99% bcis do not contain 0 for at least 0.5 s. correct: n = 24; downsampled incorrect: n = 24 of 58. traces of signal and reference channels (%df/f0) during nose pokes are shown in figure 2 figure supplement 1b-c. incorrect nose pokes on the last day of pt versus training day 1 shown in figure 2 -figure supplement 2b. (d) heatmap of bla ach signaling in mouse 1 across all training phases, aligned to tone onset (tone), correct nose poke (np), and receptacle entry (rec). each row is the average of rewarded trials across a training session. white dashed horizontal line: first training day earning 10 rewards. horizontal white line: acquisition threshold, when a mouse began to earn 20 rewards consistently in training. black horizontal lines: divisions between training phases. black vertical lines: divisions between breaks in time to allow for variable latencies in tone onset, correct nose poke, and receptacle entry (reward retrieval). the bci plot for mouse 1 is in figure 2 increase in fluorescence close to the time of nose poke onset ( figure 2c) . we also observed a significant decrease in fluorescence for most mice around 2-4 s after correct nose poke, which corresponds to the time of reward retrieval. ach release occurred in response to different events as mice learned the task (data for individual mice are shown in figure 2d + figure 2 -figure supplement 1d-g and averaged data across all mice at key time points in the task is shown in figure 2e + figure 2 -figure supplement 1h). during pre-training rewarded trials, the highest levels of ach release occurred close to the time of correct nose pokes (np), with a smaller peak at the time of reward retrieval (entry into the reward receptacle, rec). as training began, the ach release during reward trials shifted dramatically toward the time of reward retrieval, likely because the animals were learning that many nose pokes did not result in reward delivery. incorrect nose pokes that triggered a timeout were also followed by a modest but non-significant increase in bla ach levels ( ), ach level significantly increased at the time of the tone, suggesting that as animals learned the cue-reward contingency, the tone became a more salient event. at this time point, there was still a peak at the time of reward, but its magnitude was diminished. after task acquisition, the increase in ach following correct nose pokes remained but was diminished, and incorrect nose pokes did not elicit apparent ach release ( ) but met the acquisition criteria faster than initial mice because aspects of the behavioral setup were optimized (3d printed wall extensions) to allow the imaging apparatus to be used inside sound attenuating chambers (see materials and methods section). one difference observed in this group that learned the task more rapidly, was small magnitude, but significant, increases in bla ach release following tone onset late in pre-training (figure 2-figure supplement 3c-i). as behavioral performance during the training phase increased, ach release to tone onset became more pronounced, as in the initial cohort. in order to determine the source of the ach released in the bla during cue-reward learning, we recorded calcium dynamics as a measure of cell activity of chat + nbm terminal fibers in the bla (nbm-bla), since the nbm is a major source of cholinergic input to the bla (jiang et al., 2016; woolf, 1991; zaborszky et al., 2012) . we injected aav carrying a cre-recombinase-dependent, genetically-encoded calcium indicator (dio-gcamp7s) into the nbm of chat-ires-cre mice and implanted an optical fiber above the ipsilateral bla ( figure 2f . strikingly, nbm-bla cholinergic terminal activity most closely followed correct nose pokes in pre-training and shifted primarily to tone onset as mice learned the contingency during training. as in the replication cohort for the ach sensor, small magnitude, but significant, increases in terminal activity were observed following tone onset late in pre-training ( figure 2j in order to record nbm-bla cholinergic terminal activity and bla ach levels simultaneously in the same mouse, we injected aav carrying a construct for cre-recombinase dependent red-shifted genetically-encoded calcium indicator (dio-jrcamp1b) into the nbm of chat-ires-cre mice, ach3.0 sensor into the ipsilateral bla, and implanted a fiber above the bla (figure 2 -figure supplement 9a-e, mouse 1). dio-jrcamp1b was also injected into the nbm of a wild-type littermate so cre-mediated recombination would not occur to control for any crosstalk between the ach3.0 and jrcamp1b channels. while this was only a single animal and proof of principle for future studies, we found that nbm-bla cholinergic terminal activity coincided with ach levels (figure 2 -figure supplement 9f-g). importantly, this relationship between ach release and nbm-bla terminal fiber activity was not explained by signal crosstalk (figure 2 -figure supplement 9h-i), further indicating that the bla ach measured comes at least in part from the nbm. bla principal neurons respond to reward availability and follows cuereward learning glutamatergic principal cells are the primary output neurons of the bla (janak and tye, 2015) , and their firing is modulated by nbm-bla cholinergic signaling (jiang et al., 2016; unal et al., 2015) . bla principal neurons can increase their firing in response to cues as animals learn cue-reward contingencies (sanghera et al., 1979; schoenbaum et al., 1998; tye and janak, 2007) . calcium/calmodulin-dependent protein kinase (camkii) has been shown to be a marker for glutamatergic bla principal cells (butler et al., 2011; felix-ortiz and tye, 2014; mcdonald, 1992; tye et al., 2011) . to determine whether ach modulates principal neuron activity during cue-reward learning, we injected aav carrying a cre-recombinase dependent genetically encoded calcium indicator (dio-gcamp6s) into the bla of camkiia-cre mice to record bla principal cell activity during the learning task ( figure 3a + figure 3 -figure supplement 1a). as was seen for bla ach levels, there was a significant increase in bla camkiia cell activity following correct and a modest decrease in activity following incorrect nose pokes on the last day of pre-training ( figure 3b ). however, the activity peaked later after the correct nose poke response (~2.5 s) compared to the ach3.0 signal (~0.5 s) and appeared to align more tightly with reward retrieval ( during pre-training, the highest levels of bla camkiia cell activity followed reward retrieval. in addition, during the first few days of training, bla camkiia cell activity after reward retrieval was higher than it was during pre-training, and the magnitude of response decreased as mice learned the contingency and earned more rewards, ultimately reaching similar intensity to that observed during pre-training. concurrently, as mice approached acquisition of the task ( figure 3c , white horizontal line), bla camkiia cell activity significantly increased in response to tone onset ( figure 3d -e + figure 3 -figure supplement 1e-h, acq.), suggesting that the recruitment of bla camkiia cell activity likely reflects the association of the cue with a salient outcome (lutas et al., 2019; sengupta et al., 2018) . incorrect nose pokes that triggered a timeout did not elicit a different response in camkiia cell activity compared to before timeouts were incorporated (figure 3-figure supplement 2b-g). in an independent cohort of mice, those with more posterior fiber tip placements (mice 4 + 7) replicated the primary findings (figure 3-figure supplements 3-4). since ach released by nbm-bla terminals during training shifted to tone onset during acquisition of cue-reward learning ( figure 2e ,j), we hypothesized that ach may potentiate learning the cuereward contingency. we, therefore, tested whether increasing ach release in bla during learning could alter cue-reward learning by injecting aav carrying a cre-recombinase-dependent channelrhodopsin-eyfp (aav-dio-chr2-eyfp) construct bilaterally into the nbm of chat-ires-cre transgenic mice and placing fibers over the blas to optically stimulate cholinergic terminals originating from the nbm selectively ( figure 4a figure 4c ) and training ( figure 4d ). stimulation usually occurred during at least a portion of all three components of a during pre-training, auditory tones were presented on a variable interval 30 schedule (vi30), during which an active nose poke (correct) yielded ensure reward delivery and 2 s of optical stimulation but there was no consequence for incorrect nose pokes (active nose pokes not during tone). (d) training was identical to pre-training, except incorrect nose pokes resulted in a 5 s timeout, signaled by house light illumination, followed by a restarting of the iti. (e) behavioral performance in a cue-reward learning task improves with optical stimulation of chat + fibers in bla. eyfp-and chr2-expressing mice earn similar numbers of rewards during pt (blue shaded region). chr2-expressing mice more rapidly earn significantly more rewards than eyfpexpressing mice during training (pink shaded region). no significant differences were observed during extinction training (orange shaded region). horizontal white line: acquisition threshold, when a mouse began to earn~20 rewards consistently in training. mean ± sem, eyfp: n = 5, chr2: n = 6. figure 4 continued on next page rewarded trial: tone, correct nose poke, and reward retrieval, since these events were often separated by short latencies. as seen in previous experiments, during the pre-training phase animals made a high number of nose poke responses over the course of each session, obtained most available rewards by the last day ( figure 4e as the animals learned that a nose poke occurring outside of the cued period resulted in a timeout, both control eyfp and chr2 groups learned the contingency and improved their performance, resulting in acquisition of the cue-reward task (20 rewards earned). however, significant group differences emerged, such that chr2 mice earned significantly more rewards than eyfp controls ( figure 4e two-way repeated-measures anova, f (1, 9)=12.67, p=0.0061), suggesting that the chr2 group learned the tone-reward contingency more quickly than the eyfp group. eyfp mice were able to reach the same peak cue-reward performance as the chr2 group only after 4-6 additional days of training. once peak performance was achieved, there was no difference in extinction learning between the groups (main effect of group (eyfp versus chr2) in a two-way repeated-measures anova, f (1, 9)=2.293, p=0.1643). while sex differences in the behavior were not formally tested side by side, an independent cohort of male mice (eyfp n = 7, chr2 n = 7, figure 4 -figure supplement 4) was tested to determine whether both male and female mice would respond to ach stimulation, revealing similar trends during training for rewards earned ( in order to determine if optical stimulation of nbm-bla cholinergic terminals improved performance in the task by increasing the rewarding value of the outcome, rather than enhancing cuereward learning by some other means, we allowed mice to nose poke for optical stimulation rather than for ensure (figure 4-figure supplement 5a ). there were no differences between the eyfp control and chr2 groups (two-way repeated-measures anova, f (1, 9)=0.6653, p=0.4357). we also tested whether nbm-bla cholinergic terminal activation was reinforcing on its own by stimulating these terminals in a real-time place preference test. mice were allowed to explore two similar compartments to determine baseline preference, and nbm-bla cholinergic terminals were then stimulated in one of the two chambers to determine whether it increased time spent in the simulationpaired chamber. there was no difference between groups (figure 4-figure supplement 5b, main effect of group (eyfp versus chr2) in a two-way repeated-measures anova, f (1, 9)=0.1311, p=0.7257) in place preference, confirming that optical activation of nbm-bla cholinergic terminals is not innately rewarding. stimulation of nbm-bla cholinergic terminals also did not lead to changes in nose poke behavior in an uncued progressive ratio task ( muscarinic, but not nicotinic, receptors are required for acquisition of the cue-reward contingency ach signals through multiple receptor subtypes, with rapid, ionotropic signaling mediated through stimulation of nachrs, and metabotropic signaling mediated through stimulation of machrs (picciotto et al., 2012) . to determine which ach receptors were involved in this cue-reward learning task, mice were injected intraperitoneally with saline (n = 8), mecamylamine (non-competitive nicotinic antagonist, mec, n = 9), scopolamine (competitive muscarinic antagonist, scop, n = 8), or a combination of both antagonists (mec+scop, n = 9) 30 min prior to pre-training and training, during the same epochs of the task in which optical stimulation was administered ( figure 5a ). like optical stimulation, blockade of ach receptors during the pre-training phase of the task had no effect on rewards earned ( figure 5b we have shown that this dose of mecamylamine delivered i.p. has significant effects in tests of anxiety-like behavior and responses to inescapable stress. in addition, chronic treatment with mecamylamine at this dose is sufficient to decrease bla c-fos immunoreactivity (mineur et al., 2007) . consistent with the inability to acquire the cue-reward contingency, mice treated with scop or mec+scop also obtained very few rewards during extinction ( figure 5b ach-mediated accelerated cue-reward learning does not require contingent stimulation of chat + nbm terminals in the bla acetylcholine is often thought of as a neuromodulator (picciotto et al., 2012) , and the window for cholinergic effects on synaptic plasticity varies across ach receptor subtypes (gu and yakel, 2011) . it is therefore possible that ach signaling may result in intracellular signaling changes that outlast the cue presentation window. in order to determine if the effect of nbm-bla stimulation is there was no significant difference between saline controls and those receiving the nicotinic achr antagonist (mec) during training and mice extinguished responding at similar rates. figure 5 continued on next page dependent upon the timing of correct nose poke and laser stimulation contingency, we repeated the experiment in an independent cohort of mice with an additional non-contingent chr2 group that received the same number of stimulation trains as the contingent chr2 group, but in which light stimulation was explicitly unpaired with task events ( figure 6a + figure 6-figure supplement 1) . as in the previous experiment, there were no differences between the eyfp control (n = 6) and stimulation groups (contingent-chr2 n = 5 and non-contingent chr2 n = 5) during pre-training ( figure these results demonstrate that chr2-mediated ach release does not have to be time-locked to the cue, nose poke, or reward retrieval to improve performance of the task, suggesting that ach may alter the threshold for neuronal plasticity for cue-reward pairing over a much longer timescale than might be expected based on results from the ach3.0 recording and nbm-bla recordings, which could be consistent with the involvement of machr signaling in this effect. as in the previous experiment, once all groups reached criterion for acquisition of the cue-reward contingency, there were no differences between any of the groups during extinction ( figure it is increasingly recognized that the bla is involved in learning to predict both positive and negative outcomes from previously neutral cues (cador et al., 1989; janak and tye, 2015; ledoux et al., 1990) . cholinergic cells in the basal forebrain complex fire in response to both positive and negative reinforcement (hangya et al., 2015) . the results shown here indicate that ach signaling in the bla is intimately involved in cue-reward learning. endogenous ach is released in the bla in response to salient events in the task, and ach dynamics evolved as the subject formed associations between stimuli and reward. while the pattern of ach signaling in the bla may seem reminiscent of how dopamine neurons encode reward prediction errors as measured in other brain areas (schultz et al., 1997) , the current results suggest that ach release in the bla may instead be involved in signaling a combination of salience and novelty. ach release and nbm-bla activity increased following correct nose poke and, around the time that animals acquired the cue-reward task, following tone onset. however, earlier in training, incorrect nose pokes that resulted in a timeout were also followed by ach release, although this was smaller in magnitude. further, stimulating nbm-bla cholinergic terminals during learning enhanced behavioral performance, but was not intrinsically rewarding on its own and did not support responding for the tone alone. although ach was released in the bla at discrete points during the task, the effects of heightened bla ach signaling were relatively long lasting, since it was not necessary for stimulation to be time-locked to cue presentation or reward retrieval to enhance behavioral performance. thus, cholinergic inputs from the basal forebrain complex to the bla are a key component of the circuitry that links salient events to previously neutral stimuli in the environment and uses those neutral cues to predict future rewarded outcomes. we have shown that ach release in the bla is coincident with the stimulus that was most salient to the animal at each phase of the task. use of the fluorescent ach sensor was essential in determining these dynamics (jing et al., 2018; jing et al., 2019) . previous microdialysis studies have shown that ach is released in response to positive, negative, or surprising stimuli, but this technique is limited by relatively long timescales (min) and cannot be used to determine when cholinergic transients align to given events in an appetitive learning task and how they evolve over time (sarter and lustig, 2020) . in this cue-reward learning paradigm, when there was no consequence for incorrect nosepoking (pre-training phase), animals learned to perform a very high number of nose pokes and received a large number of rewards, and bla ach signaling peaked following correct nose pokes. both the behavioral response (nose poking that was not contingent with the tone) and the ach response (linked to the correct nose poke) suggest that the animals were not attending to the tone during most of the pre-training phase of the task, but rather were attending to the cues associated with reward delivery, such as the reward light or the sound of the pump that delivered the reward. consistent with this possibility, in the next phase of the task when mice received a timeout for responding if the tone was not presented, performance of all groups dropped dramatically. interestingly, in animals that had difficulty learning the cue-reward contingency, during early training sessions ach release shifted to reward retrieval, likely because this was the most salient aspect of the task when the majority of nose pokes performed did not result in reward. finally, as mice acquired the contingency between tone and reward availability, the tone also began to elicit ach release in the bla, suggesting that mice learned that the tone is a salient event predicting reward availability. since there are multiple sources of ach input to the bla, it was important to determine whether nbm cholinergic neurons were active during the periods when ach levels were high (woolf, 1991) . recordings from cholinergic nbm-bla terminal fibers showed similar dynamics to ach measurements, suggesting that the nbm is a primary source of ach across the phases of cue-reward learning. perhaps the most well-known example of dynamic responding related to learning cue-reward contingencies and encoding of reward prediction errors is the firing of dopaminergic neurons of the ventral tegmental area (vta; schultz, 1998) . after sufficient pairings, dopaminergic neurons will fire figure 6 . non-contingent stimulation of cholinergic nbm-bla terminals is sufficient to enhance cue-reward learning. (a) experimental details of laser stimulation in non-contingent-chr2 mice. non-contingent-chr2expressing mice received the same number of light stimulations as contingent-chr2-expressing mice, but stimulation was only given during the iti, when non-contingent mice had not made a response within 2 s. injection sites and fiber placements are shown in figure 6-figure supplement 1a-b. (b) non-contingent nbm-bla optical stimulation also improves behavioral performance in cue-reward learning task. there was no significant difference in the number of rewards earned between eyfp (n = 6), contingent-chr2 (n = 5), or non-contingent-chr2 (n = 5) mice during pre-training. contingent-and non-contingent-chr2-expressing mice more rapidly earned significantly more rewards during training than eyfp-expressing mice. no differences were observed between groups during extinction training. mean ± sem eyfp: n = 6, contingent-chr2: n = 5, non-contingent-chr2: n = 5. horizontal white line: acquisition threshold, when a mouse began to earn 20 rewards consistently in training. individual data are shown in figure 6 -figure supplement 2a. (c) incorrect nose pokes. there was no significant difference in the number of incorrect nose pokes between groups during pre-training. contingent-and non-contingent-chr2-expressing mice made significantly fewer incorrect nose pokes during training than eyfpexpressing mice. no differences between groups were observed during extinction training. mean ± sem eyfp: n = 6, contingent-chr2: n = 5, non-contingent: n = 5. individual data are shown in figure 6 in response to the cue that predicts the reward, and no longer to the rewarding outcome, which corresponds with behavioral changes that indicate an association has been formed between conditioned stimuli (cs) and unconditioned stimuli (us). it should be noted that dopamine signaling is not unique in this learning-related dynamic response profile. serotonergic neuronal responses also evolve during reward learning in a manner distinct from dopaminergic neurons (zhong et al., 2017) . plasticity related to learning has also been observed in cholinergic neurons in the basal forebrain complex during aversive trace conditioning, such that after several training days, neuronal activity spans the delay between cs and us (guo et al., 2019) . additionally, a recent study suggested that ach may signal a valence-free reinforcement prediction error (sturgill et al., 2020) . future studies on the selective inputs to nbm to bla cholinergic neurons would be of interest to identify the links between brain areas involved in prediction error coding. we found that bla camkiia cells were most reliably activated following reward retrieval before contingency acquisition (both when they were receiving several rewards but no timeouts in pre-training and few rewards early in training). similar to the recording of ach levels, after acquisition, the tone began to elicit an increase in bla camkiia cell population activity. however, activity of camkiia neurons differed from ach signaling in the bla in important ways. ach was released in response to the salient events in the task that were best able to predict reward delivery or availability. by contrast, the activity of bla camkiia neurons was not tightly time-locked to correct nose poking, and instead followed reward retrieval until acquisition, when activity increased in response to tone onset. the divergent dynamics of ach release and camkiia neuron activity underscores that ach's role in the bla is to modulate, rather than drive, the activity of camkiia neurons, and therefore may alter dynamics of the network through selective engagement of different populations of gaba interneurons (unal et al., 2015) . neuronal activity and plasticity in the bla is required for both acquisition of appetitive learning (conditioned reinforcement) and fear conditioning, however the inputs that increase activity in the structure during salient events likely come from many brain areas (mckernan and shinnick-gallagher, 1997; rogan et al., 1997; tye et al., 2008) . in particular, dopaminergic inputs to the bla are important for acquisition of conditioned reinforcement and for linking the rewarding properties of addictive drugs to cues that predict their availability (cador et al., 1989) . our results indicate that ach is a critical neuromodulator upstream of the bla that is responsive to salient events, such as reward availability, motor actions that elicit reward, and cues that predict reward. we show here that increasing endogenous ach signaling in the bla caused mice to perform significantly better than controls in an appetitive cued-learning task. heightened ach release during learning of a cueaction-reward contingency led to fewer incorrect responses and increased acquisition rate in both female and male mice. the optical stimulation was triggered by correct nose poke, thus the cholinergic nbm-bla terminal fiber stimulation overlapped with all three salient events: tone, nose poke, and reward retrieval, since the tone terminated 2 s after correct nose poke. we chose this stimulation pattern, as opposed to linking optical stimulation to tone onset, to ensure stimulation was dependent on behavioral responses. therefore, stimulation did not precisely recapitulate the ach release profile observed in mice that had already acquired the task (when ach increases following tone onset). this suggests that behaviorally-contingent increases in bla ach are sufficient to enhance task acquisition (but see below). it is also possible that optogenetic-mediated ach release may last longer than endogenous, tone-evoked release. a simultaneous stimulation and recording approach would be required to compare ach release under both conditions (pisansky et al., 2019) . it is important to note that basal forebrain neurons have been demonstrated to co-release ach and gaba (ma et al., 2018; saunders et al., 2015) , and cholinergic basal forebrain neurons that project to the bla have been shown to co-express a glutamate transporter (ma et al., 2018; nickerson poulin et al., 2006) . thus, it is possible that both fiber photometry and optogenetic results could be influenced, in part, by co-release of other neurotransmitters from chat-positive neurons. future studies employing additional fluorescent neurotransmitter sensors (marvin et al., 2013; marvin et al., 2018; marvin et al., 2019) could help understand the interaction between the different signals employed by basal forebrain neurons. it is possible that ach improved learning by increasing the intensity of the reward, potentiating the learned association, improving discrimination, or a combination of these phenomena. however, increasing ach release in the bla was not inherently rewarding, because it did not support self-stimulation or real-time place preference. this is at odds with a recent study that found stimulation of nbm-bla cholinergic terminals could induce a type of place-preference and modest self-stimulation (aitta-aho et al., 2018) . perhaps slight differences in targeting of chr2 infusion or differences in the behavioral paradigm could be responsible for the lack of direct rewarding effects of optical chat terminal stimulation in the current study. other recent work (jiang et al., 2016) has demonstrated that stimulating this nbm-bla cholinergic pathway is sufficient to strengthen cued aversive memory, suggesting that the effect of ach in the bla may not be inherently rewarding or punishing but instead potentiates plasticity in the bla, allowing learning of cue-outcome contingencies. similarly, it is possible that ach alters motor activity. however, there were no effects of optical stimulation on locomotion or responding in the inactive nose poke port. in addition, during the pre-training phase when there was no consequence for incorrect nose pokes, all groups earned the same number of rewards, regardless of optical stimulation or pharmacological blockade of ach receptors, suggesting that ach is not involved in the motor aspects of the task or the value of the reward. indeed, differences emerged only during the training phase, when attention to the tone was critical to earn rewards. further, incorrect nose poking remained high for mice administered scopolamine. this suggests that scopolamine-treated animals were seeking the reward, as in the operant familiarization and pre-training phases of training, but were unable to learn that they should only nose poke in response to the tone. cell-type-specific expression of achrs and activity-dependent effects place cholinergic signaling at a prime position to shape bla activity during learning. for instance, late-firing interneurons in the bla exhibit nachr-dependent epsp's when no effect is seen on fast-spiking interneurons, while principal neurons can be either excited or inhibited through machrs, depending on activity level of the neuron at the time of cholinergic stimulation (unal et al., 2015) . bla machrs can support persistent firing in principal neurons and can be important for the expression of conditioned place preference behavior, as well as trace fear conditioning (baysinger et al., 2012; egorov et al., 2006; mcintyre et al., 1998) . similar to studies of trace fear conditioning, in which activity of the network over a delay period must be maintained, we found that metabotropic (machrs) but not ionotropic (nachrs) ach receptors were required for learning the contingency of this cue-reward task. the timing of cholinergic signaling can be a critical factor in the induction of synaptic plasticity in other brain regions, so we hypothesized that the enhancement of cue-reward learning observed might be dependent upon when nbm-bla terminal fibers were stimulated with respect to tone presentation and/or behavioral responses (gu and yakel, 2011) . however, we found that heightened ach signaling in the bla improved behavioral performance even when stimulations were explicitly unpaired with the cue or correct nose poking. this suggests that the effect of increased cholinergic signaling in the bla is long lasting, and that stimulation during a learning session is sufficient to potentiate synaptic events linking the cue to a salient outcome-even if cs and/or reward delivery are presented tens of seconds later. given the findings from fiber photometry recordings, which showed endogenous ach release was time-locked to salient stimuli during the task and evolved with learning, it is surprising that time-locking of exogenous ach release was not necessary for enhancement of cue-reward learning. coupled with pharmacological evidence demonstrating that muscarinic signaling is necessary for reward learning in this task, these results suggest the involvement of metabotropic signaling downstream of muscarinic receptors that outlasts the initial cholinergic stimulation. to conclude, the abundant ach input to the bla results in ach release in response to stimuli that predict reward in a learned cue-reward task. increasing cholinergic signaling results in accelerated learning of the cue-reward contingency. these findings are consistent with the hypothesis that ach is a neuromodulator that is released in response to salient stimuli and suggests that ach signaling may enhance neuronal plasticity in the bla network, leading to accelerated cue-reward learning. key resources all procedures were approved by the yale university institutional animal care and use committee (protocol: 2019-07895) in compliance with the national institute of health's guide for the care and use of laboratory animals. experiments were performed in mice of both sexes, in keeping with the nih policy of including sex as a biological variable. sex of mice in behavioral graphs is indicated by circles for females and squares for males. female and male heterozygous mice with cre recombinase knocked into the choline acetyltransferase (chat) gene (chat-ires-cre, b6;129s6-chat tm2(cre)lowl/j , jackson laboratory, bar harbor, me) were bred in house by mating chat-ires-cre with c57bl6/j mice. camkiia-cre (tg(camk2acre)2gsc) mice obtained from ronald duman (casanova et al., 2001; wohleb et al., 2016) were bred in house as above. c57bl6/j mice were obtained from the jackson laboratory at 6-10 weeks of age, and tested at 5-7 months of age, following at least 1 week of acclimation. all mice were maintained in a temperature-controlled animal facility on a 12 hr light/dark cycle (lights on at 7:00 am). mice were group housed 3-5 per cage and provided with ad libitum food and water until undergoing behavioral testing. mice were single housed 1-3 weeks before surgery to facilitate food restriction and body weight maintenance. surgical procedures for behavior were performed in fully adult mice at 4-6 months of age, agematched across conditions. for viral infusion and fiber implantation, mice were anesthetized using isoflurane (induced at 4%, maintained at 1.5-2%) and secured in a stereotactic apparatus (david kopf instruments, tujunga, ca). the skull was exposed using a scalpel and bregma was determined using the syringe needle tip (2 ml hamilton neuros syringe, 30 gauge needle, flat tip; reno, nv). for fiber photometry surgeries, 0.4 ml of aav9 hsyn-ach3.0 (vigene biosciences inc) to measure bla ach levels ( figure 2a -e + figure 2 -figure supplements 1-2) was delivered unilaterally to the bla (a/p; à1.34 mm, m/l + or -2.65 mm, d/v à4.6 mm, relative to bregma) of chat-ires-cre or wild-type c57bl6/j mice at a rate of 0.1 ml/min. the needle was allowed to remain at the infusion site for 5 min before and 5 min after injection. a mono fiber-optic cannula (1.25 mm outer diameter metal ferrule; 6 mm long, 400 mm core diameter/430 mm outer diameter, 0.48 numerical aperture (na), hard polymer cladding outer layer cannula; doric lenses, quebec city, quebec, canada) was implanted above the bla (a/p; à1.34 mm, m/l + 2.65 mm, d/v à4.25 mm) and affixed to the skull using opaque dental cement (parkell inc, edgewood, ny). for bla camkiia cell calcium dynamic recordings (figure 3 + figure 3-figure supplements 1-2) , 0.5 ml of aav1 syn-flex-gcamp6s-wpre-sv40 (addgene, watertown, ma) was injected into the left bla using the same procedure and coordinates but was injected into camkiia-cre mice. cholinergic nbm-bla terminal fiber calcium dynamic recording ( figure dana et al., 2016; dana et al., 2019) . mice were allowed to recover in a cage without bedding with a microwavable heating pad underneath it until recovery before being returned to home cage. for 2 d following surgery, mice received 5 mg/kg rimadyl i.p (zoetis inc, kalamazoo, mi) as postoperative care. figure 6-figure supplements 1-2) , surgeries were performed as above except as follows: 0.5 ml of control vector (aav2 ef1a-dio-eyfp) or channelrhodopsin (aav2 ef1a-dio-hchr2(h134r)-eyfp; university of north carolina gene therapy center vector core, chapel hill, nc) was delivered bilaterally into the nbm (a/p: -0.7 mm, m/l ± 1.75 mm, d/v -4.5 mm) of chat-ires-cre mice. mono fiber-optic cannulas (1.25 mm outer diameter zirconia ferrule; 5 mm long, 200 mm core diameter/245 mm outer diameter, 0.37 na, polyimide buffer outer layer cannula; doric lenses) were inserted bilaterally above the basolateral amygdala (bla, a/p; à1.22 mm, m/l ± 2.75 mm, d/v à4.25 mm). mice were randomly assigned to eyfp or chr2 groups, controlling for average group age. for ex vivo local field potential electrophysiology experiments ( figure 4b) , the nbm was injected with dio-chr2-eyfp as described above, except mice were 8 weeks of age (see supplemental methods for current clamp recording methods). the coronal brain slices containing the nbm were prepared after 2-4 weeks of expression. briefly, mice were anesthetized with 1â fatal-plus (vortech pharmaceuticals, dearborn, mi) and were perfused through their circulatory systems to cool down the brain with an ice-cold (4˚c) and oxygenated cutting solution containing (mm): sucrose 220, kcl 2.5, nah 2 po 4 1.23, nahco 3 26, cacl 2 1, mgcl 2 6 and glucose 10 (ph 7.3 with naoh). mice were then decapitated with a guillotine immediately; the brain was removed and immersed in the ice-cold (4˚c) and oxygenated cutting solution to trim to a small tissue block containing the nbm. coronal slices (300 mm thick) were prepared with a leica vibratome (leica biosystems inc, buffalo grove, il) after the tissue block was glued on the vibratome stage with loctite 404 instant adhesive (henkel adhesive technologies, dü sseldorf, germany). after preparation, slices were maintained at room temperature (23-25˚c) in the storage chamber in the artificial cerebrospinal fluid (acsf; bubbled with 5% co 2 % and 95% o 2 ) containing (in mm): nacl 124, kcl 3, cacl 2 2, mgcl 2 2, nah 2 po 4 1.23, nahco 3 26, glucose 10 (ph 7.4 with naoh) for recovery and storage. slices were transferred to the recording chamber and constantly perfused with acsf with a perfusion rate of 2 ml/min at a temperature of 33˚c for electrophysiological experiments. cell-attached extracellular recording of action potentials was performed by attaching a glass micropipette filled with acsf on eyfp-expressing cholinergic neurons with an input resistance of 10-20 mw under current clamp. blue light (488 nm) pulse (60 ms) was applied to the recorded cells through an olympus bx51wi microscope (olympus, waltham, ma) under the control of the sutter filter wheel shutter controller (lambda 10-2, sutter instrument, novato, ca). all data were sampled at 3-10 khz, filtered at 3 khz and analyzed with an apple macintosh computer using axograph x (axograph). events of field action potentials were detected and analyzed with an algorithm in axograph x as reported previously (rao et al., 2008) . one week after surgery, mice were weighed daily and given sufficient food (2018s standard chow, envigo, madison, wi) to maintain 85% free-feeding body weight. all behavioral tests were performed during the light cycle. mice were allowed to acclimate to the behavioral room for 30 min before testing and were returned to the animal colony after behavioral sessions ended. two weeks after surgery, mice were handled 3 min per day for 7 d in the behavioral room. mice were given free access to the reward (ensureplus vanilla nutrition shake solution mixed with equal parts water (ensure); abbott laboratories, abbott park, il) in a 50 ml conical tube cap in their home cages on the last 3 d of handling to familiarize them to the novel solution. mice were also habituated to patch cord attachment during the last 3 d of handling for optical stimulation and fiber photometry experiments. immediately before training each day, a patch cord was connected to their optical fiber(s) via zirconia sleeve(s) (1.25 mm, doric lenses) before being placed in the behavioral chamber. all operant training was carried out using med associates modular test chambers and accessories (env-307a; med associates inc, georgia, vt). for optical stimulation experiments, test chambers were housed in sound attenuating chambers (env-022m). two nose poke ports (env-313-m) were placed on the left wall of the chamber and the reward receptacle (env-303lphd-rl3) was placed on the right wall. the receptacle cup spout was connected to a 5 ml syringe filled with ensure loaded in a single speed syringe pump (phm-100). nose pokes and receptacle entries were detected by infrared beam breaks. the tone generator (env-230) and speaker (env-224bm) were placed outside the test chamber, but within the sound attenuating chamber, to the left. the house light (used for timeout, env-315m) was placed on top of the tone generator to avoid snagging patch cords. each chamber had a fan (env-025f28) running throughout the session for ventilation and white noise. behavior chambers were connected to a computer running medpc iv to collect event frequency and timestamps. for optical stimulation experiments, a hole drilled in the top of the sound attenuating chambers allowed the patch cord to pass through. initial bla ach3.0 (figure 2a-e) and bla camkiia gcamp6s (figure 3 ) fiber photometry recordings occurred in a darkened behavioral room outside of sound attenuating chambers due to steric constraints with rigid fiber photometry patch cords. later behavioral chamber optimization (wall height was extended with 3d printed and laser cut acrylic panels to allow removing the test chamber lid while preventing escape) allowed all other fiber photometry cohorts to be tested inside sound attenuating chambers. for fiber photometry experiments, a custom receptacle was 3d printed that extended the cup beyond the chamber wall to allow mice to retrieve the reward with more rigid patch cords. each mouse was pseudo-randomly assigned to behavioral chamber when multiple chambers were used, counterbalancing for groups across boxes. three weeks after surgery, initial operant familiarization consisted of one 35 min session of free reward to demonstrate the location of reward delivery; all other sessions were 30 min. during free reward, only the reward receptacle was accessible. after 5 min of habituation, ensure (24 ml over 2 s) was delivered in the receptacle cup and a light was turned on above the receptacle. the receptacle light was turned off upon receptacle entry. the next phase of operant familiarization, mice learned to nose poke to receive reward on a fixed-ratio one (fr1) schedule of reinforcement. mice in experiments involving manipulations (optical stimulation and antagonist studies) were pseudo-randomly assigned to left or right active (reinforced) nose poke port. mice in fiber photometry experiments were all assigned to right active port to minimize potential across subject variability. the inactive (unreinforced) port served as a locomotor control. during fr1 operant familiarization, each nose poke response into the active port resulted in receptacle light and reward delivery. after the mice reached criterion on fr1 operant familiarization (group average of 30 rewards for 2 consecutive days, usually 4-5 d), mice were advanced to the pre-training phase. this phase incorporated an auditory tone (2.5-5 khz,~60 db) that lasted for at most 10 s and signaled when active nose pokes would be rewarded. only active nose pokes made during the 10 s auditory tone (correct nose pokes) resulted in reward and receptacle light delivery. the tone co-terminated with ensure delivery. during pre-training, there was no consequence for improper nose pokes, neither in the active port outside the tone (incorrect nose pokes) nor in the inactive port (inactive nose pokes). the number of inactive nose pokes were typically very low after operant familiarization and were not included in analysis. after reward retrieval (receptacle entry following reward delivery) the receptacle light was turned off and the tone was presented again on a variable intertrial interval schedule with an average interval of 30 s (vi 30), ranging from 10 to 50 s (ambroggi et al., 2008) . after 4-5 d of pre-training, mice progressed to the training phase, which had the same contingency as pre-training except incorrect nose pokes resulted in a 5 s timeout signaled by house light illumination, followed by a restarting of the previous intertrial interval. mice were considered to have acquired the task after earning 20 rewards during the training phase of the task. in order to promote task acquisition, mice that were not increasing number of rewards earned reliably were moved to a vi 20 schedule after 9 d of vi 30 training for bla ach3.0 or 6-7 d for bla camkiia cell mice. the vi 20 schedule was only needed for the two groups that were trained outside of the sound attenuating chambers. mice progressed to extinction after 12 d of training or, in the case of fiber photometry cohorts, once all mice met the acquisition criteria. extinction was identical to training except no ensure was delivered in response to correct nose pokes. the replicate cohorts of the bla camkiia gcamp6s and nbm-bla terminal fiber recording experiments were advanced to 1 d of extinction after only 7 d of training due to the covid-19 shutdown. between mice, excrement was removed from the chambers with a paper towel. at the end of the day chambers were cleaned with rescue disinfectant (virox animal health, oakville, ontario, canada) and ensure syringe lines were flushed with water then air. mice were excluded from analyses if a behavioral chamber malfunctioned (e.g. syringe pump failed) or they received the improper compound. fiber photometry mice were excluded from analyses if they did not meet the acquisition criterion by the last day of training. see supplementary file 1 for number of mice that acquired, were excluded, and further explanations for behavioral paradigm deviations. optical stimulation was generated by a 473 nm diode-pumped solid-state continuous wave laser (opto engine llc, midvale, ut) controlled by a ttl adapter (sg-231, med associates inc). the laser was connected to a fiber optic rotary joint (doric lenses) via a mono fiber optic patch cord (200 mm core, 220 mm cladding, 0.53 na, fc connectors; doric lenses). the rotary joint was suspended above the sound attenuating chamber with a connected branching fiber optic patch cord (200 mm core, 220 mm cladding, 0.53 na, fc connector with metal ferrule; doric lenses) fed into the behavioral box. laser power was adjusted to yield 10-12 mw of power at each fiber tip. the stimulation pattern was 25 ms pulses at 20 hz for 2 s modified from parameters in jiang et al., 2016. jiang et al. used a 20 hz pulse frequency, 5 ms pulses, and 10-12 mw power at the fiber tips. in this study, we used a 2 s stimulation duration because it matched the time of syringe pump activation for reward delivery and co-terminated with the pump and auditory tone. a 25 ms pulse width was used because our lasers were not able to generate sufficient power with 5 ms pulses. optical stimulation was only delivered during the pre-training and training phases of the operant task. both control (eyfp) and experimental (chr2) groups received identical light delivery, and stimulation was triggered by a correct nose poke and co-terminated with the auditory tone and ensure delivery. for the non-contingent experiment, the number of light stimulations was yoked to the concurrently running chr2 mouse. the timing of the non-contingent stimulation was explicitly unpaired with correct nose pokes or tones and was held in queue until the mouse had not made a response in the last 2 s, a tone was not going to be delivered within the next 2 s, or at least 5 s had passed since the mouse entered the receptacle after earning reward. fluorescent measurements of ach and calcium levels were recorded using two doric lenses 1-site fiber photometry systems: a standard 405/465 nm system and a 405/470/560 nm system. the standard 405/465 system was configured as follows: the fiber photometry console controlled the two connectorized leds (cleds, 405 nm modulated at 208.616 hz and 465 nm modulated at 572.205 hz) through the led module driver (cassidy et al., 2019) . each cled was connected via attenuating patch cord to the five-port fluorescence minicube (fmc5_ae(405)_af(420-450)_e1(460-490)_f1 (500-550)_s). a pigtailed fiber optic rotary joint was connected to the minicube and suspended above the behavioral chamber with a rotary joint holder in order to deliver and receive light through the implanted optical fiber. the other end of the rotary joint was connected to the mono fiber optic patch cord via m3 connector and attached with a zirconia sleeve to the implanted fiber optic as above. the f1 (500-550 nm) port of the minicube was connected to the photoreceiver (ac low mode, new focus 2151 visible femtowatt photoreceiver, new focus, san jose, ca) via a fiber optic adapter (doric lenses) that was finally connected back to the fiber photometry console through an analog port. the 405/470/560 nm system was set up similarly, except a 560 nm led was incorporated (modulated at 333.786 hz), a six-port minicube with two integrated photodetector heads was used (ifmc6_ie(400-410)_e1(460-490)_f1(500-540)_e2(555-570)_f2(580-680)_s), and doric fluorescence detector amplifiers were used (ac 1â or 10â mode, dfd_foa_fc). a ttl adapter (sg-231, med associates inc) was connected to the digital input/output port to allow for timestamping when events occurred in the behavioral chamber. signal was recorded using doric neuroscience studio (v 5.3.3.14) via the lock-in demodulation mode with a sampling rate of 12.0 ks/s. data were decimated by a factor of 100 and saved as a comma-separated file. pre-processing of raw data was performed using a modified version of a matlab (mathworks, natick, ma) script provided by doric. the baseline fluorescence (f0) was calculated using a firstorder least mean squares regression over the~30 min recording session. second-order least mean squares regressions were used when photobleaching of the sensor was more pronounced, as in the case of nbm-bla terminal fiber recordings. the change in fluorescence for a given time point (df) was calculated as the difference between it and f0, divided by f0, which was multiplied by 100 to yield %df/f0. the %df/f0 was calculated independently for both the signal (465 nm) and reference (405 nm) channels to assess the degree of movement artifact. since little movement artifact was observed in the recordings (figure 2-figure supplement 1b-c, figure 2 -figure supplement 5d-e, figure 3 -figure supplement 1c-d, tan lines), the signal %df/f0 was analyzed alone (the code provided allows for running the entire analysis pipeline with the reference channel %df/f0 if desired). the %df/f0 was z-scored to give the final z%df/f0 reported here. for the bla camkiia cell recordings (figure 3-figure supplement 1c-d) , the reference channel displayed some mirroring (moving in the opposite direction) compared to the signal. this is likely because 405 nm is not the 'true' isosbestic point for gcamp and we were instead measuring some changes in calciumunbound gcamp rather than calcium-insensitive gcamp signal alone (barnett et al., 2017; kim et al., 2016a; sych et al., 2019) . graphs and heatmaps for averaged traces aligned to actions were based on licking bout epoch filtering code from tdt (alachua, fl; link in code comments). combined action heatmaps were generated in matlab (2020a) by analyzing data 5 s preceding tone onset (rewarded trials only) to 5 s after receptacle entry. actions were aligned despite variable latencies by evenly splitting a maximum of 4 s post-tone onset/pre-correct nose poke and 1 s postcorrect nose poke/pre-receptacle entry for each trial within a day. the resulting aligned trials were averaged to generate daily averages that made up the rows of the individual animal heatmaps. blanks in the rows of heatmaps (black time bins) indicate time bins added for alignment, meaning that no trials for that day had a latency that stretched the entire window. only rewarded trials where the mouse entered the receptacle within 5 s after nose poke were analyzed. full or partial training days were excluded from analysis if there were acquisition issues such as the patch cord losing contact with the fiber or behavioral apparatus malfunction. lack of trials (fewer than three) for analysis or recording issues led to missing rows of fiber photometry data in the heatmap despite having behavioral data, in which case these rows were skipped rather than adding entire blank rows. due to individual differences in behavior, across-mouse average data was calculated by using a selection of days in which behavior was roughly similar or milestones such as the first and last day of pre-training, first day earning 10 rewards in training, first day crossing acquisition threshold (and maintaining afterward), last day of training, last day of extinction (with three or more rewarded trials that met analysis criteria). additional days were included in across-mouse average heatmaps when possible. incorrect nose poke heatmaps were generated by averaging signals for 5 s before and 5 s after incorrect nose pokes that were not preceded by an incorrect nose poke in the last 5 s. the incorrect nose poke heatmaps averaged across mice were generated using the same selection of days as the combined action heatmaps for a given experiment. bootstrapped confidence intervals (bcis) of the z-scored % df/f0 fiber photometry data within and across mice were generated using the methods described in jeanrichard-dit-bressel et al., 2020 for the following events: tone onset, correct nose poke, receptacle entry, and incorrect nose poke. for the within-mouse analysis by day, trials were aligned to event onset, and bcis were generated for events that had at least 3 trials from 5 s before to 10 s after each event. each series of data were bootstrapped 10,000 times and a two-sided 99% confidence interval was constructed. data were considered significantly different from baseline (z% df/f0 = 0) when their 99% bcis did not contain zero for an interval of time designated by a consecutive threshold of 0.5 s. to avoid comparing vastly different numbers of trials, in graphs where correct and incorrect nose pokes were plotted together, incorrect nose pokes were downsampled to match the number of correct nose poke trials. for incorrect nose pokes graphs where last pre-training day and training day 1 were plotted together, both days were downsampled to the number of correct nose pokes on the last pre-training day. for the combined action bci plots (tone onset, correct nose poke, and receptacle entry), the selection of days for each mouse matched that of the cohort-averaged combined action heatmaps. the three-event plots were combined by cropping to match the maximum latencies used in the combined action heatmaps. for the across-mouse averaged bci plots, analyses were carried out as above except the bootstrapping used mouse trial averages. the mean lines for across-mouse averaged bci plots were calculated by taking the mean of all individual trials together. the nbm-bla cholinergic terminal fiber experiment required combining the two independent cohorts to obtain n ! 3. for the incorrect nose poke bci plots, the number of trials used for each day was downsampled to 20 if a mouse performed more than 20. male wild-type c57bl/6j mice were injected i.p. 30 min before each pre-training and training session with a volume of 10 ml/kg with the following compounds: 1â dpbs (thermo fisher scientific, waltham, ma), 1 mg/kg mecamylamine hydrochloride (millipore sigma, st. louis, mo), 0.5 mg/kg (-) scopolamine hydrochloride (millipore sigma), or 1 mg/kg mecamylamine + 0.5 mg/kg scopolamine ( figure 5 + figure 5-figure supplement 1) . after completion of behavioral experiments, animals were anesthetized with 1â fatal-plus (vortech pharmaceuticals). once there was no response to toe-pinch, mice were transcardially perfused with 20 ml ice-cold 1â dpbs followed by 20 ml 4% paraformaldehyde (pfa, electron microscopy sciences, hatfield, pa). brains were extracted and post-fixed for at least 1 d in 4% pfa at 4˚c and transferred to 30% sucrose (millipore sigma) for at least 1 dy at 4˚c. brains were sliced 40 mm thick on a self-cooling microtome and stored in a 0.02% sodium azide (millipore sigma) pbs solution. brain slices were washed in pbs, blocked for 2-3 hr (0.3% triton x-100, american bioanalytical, canton, ma; 3% normal donkey serum, jackson immunoresearch, west grove, pa), then incubated overnight with primary antibodies (1:1000 + 1% normal donkey serum). slices were then washed in pbs and incubated with secondary antibodies (1:1000) for 2 hr, washed, stained with dapi for 5 min, washed, mounted, and coverslipped with fluoromount-g (electron microscopy sciences). all incubations were at room temperature. microscope slides were imaged using a fluoview fv10i confocal microscope (olympus). injection sites and fiber placements were designated on modified allen mouse brain atlas figures (lein et al., 2007) . mice were excluded from analyses if fluorescence was not observed at injection sites or if fiber tips were not identified at the intended site. operant behavioral data saved by medpc iv was transferred to excel using mpc2xl. data were organized in matlab and analyzed in prism (v8.3.0, graphpad software, san diego, ca). differences between groups and interactions across days for training were evaluated using two-way repeated measures anovas. we computed the required sample size for a 90% power level with an alpha of 0.05 by estimating the control (eyfp) group mean would be 10 rewards and the mean experimental (chr2) group would be 20 rewards with a standard deviation of 5. we utilized a power calculator for continuous outcomes of two independent samples, assuming a normal distribution. the result was six samples per group. each manipulation experiment started with at least six mice included in each group (sealed envelope, 2020) . in each experiment, each animal within a group served as a biological replicate. these studies did not include technical replicates. masking was not applied during data acquisition but data analyses were semi-automated in matlab and performed blind to condition. editing; miao jing, yulong li, resources, writing -review and editing; xiao-bing gao, data curation, formal analysis, investigation, writing -review and editing; yann s mineur, conceptualization, investigation, methodology, writing -review and editing; marina r picciotto, conceptualization, resources, supervision, funding acquisition, project administration, writing -review and editing supplementary files . supplementary file 1. number of mice that acquired the reward learning behavior, number that were excluded, and any training deviations. (a) mice in the initial bla ach3.0 group were trained outside of the sound attenuating chambers. these mice had 5 dof pre-training because they were trained concurrently with another cohort of mice (not shown) that required an extra day to reach two consecutive days of 20 rewards earned and were advanced to a vi 20 schedule of reinforcement during training after 9 d to promote responding. training was extended to allow all mice to acquire. due to time constraints during acquisition, mouse 3 in this cohort was moved to extinction after 20 d of training because it had acquired earlier, was earning the most rewards, and we wanted to record more extinction days. (b) mice in the bla ach3.0 and nbm-bla terminal fiber replicate experiments were advanced to 1 d of extinction after only 7 d of training due to the covid-19 shutdown. (c) bla ach3.0 and nbm-bla terminal fiber jrcamp1b mice were analyzed as dual channel mice just through pre-training and were instead used as replicates of the bla ach3.0 experiment. one of the mice had apparatus errors during training and had to be excluded. (d) mice in the initial bla camkiia gcamp6 were trained outside of the sound attenuating chambers. mouse 1 progressed from pre-training to training a day earlier than the rest of the group and was able to have an extra day of training before the 2 d of extinction. mice in this group were advanced to a vi 20 schedule of reinforcement during training after 6-7 d to promote responding. training was extended to allow more mice to acquire. . transparent reporting form data availability all data generated or analysed during this study are included in the manuscript and supporting files. source data files have been provided for all experiments on dryad digital repository: https://doi. org/10.5061/dryad.3xsj3txcf. the following dataset was generated: conceptualization, resources, data curation, software, formal analysis, validation, investigation, visualization, methodology, writing -original draft data curation, formal analysis, investigation, methodology, writing -review and editing 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differentially encode motivation and reinforcement impact of basal forebrain cholinergic inputs on basolateral amygdala neurons gaba interneurons mediate the rapid antidepressant-like effects of scopolamine cholinergic systems in mammalian brain and spinal cord the basal forebrain cholinergic projection system in mice. the mouse nervous system was connected to a computer running ethovision xt (version 10.1.856, noldus, wageningen, netherlands) to track the position of the mouse and deliver optical stimulation when the mouse was on the laserpaired side (via ttl pulse to otpg_4 laser controller (doric lenses) connected to the laser; 20 hz, 25 ms pulses). optical stimulation on a progressive ratio schedule (escalations given below) these studies were supported by grants da14241, da037566, mh077681. lw, dt, and pr were supported by ns022061, mh109104 from the national institutes of health, and by the intramural programs of ninds and nimh. x-bg was supported by da046160. rbc was supported by t32-ns007224. this work was funded in part by the state of connecticut, department of mental health and addiction services but this publication does not express the views of the department of mental health and addiction services or the state of connecticut. the views and opinions expressed are those of the authors. we thank samantha sheppard for the use of her mouse illustration and animal care assistance and nadia jordan-spasov for genotyping and laboratory help. li jiang performed the ex vivo current-clamp recordings. angela lee and wenliang zhou provided helpful input into experimental planning. colin bond, marcelo dietrich, usman farooq, onur iyilikci, sharif kronemer, matthew pettus, and zach saltzman provided insightful discussion and assistance with analysis and figure design. ralph dileone, stephanie groman, hyojung seo, and jane taylor offered helpful discussion about experimental design and analysis. the support teams at doric lenses (alex cô té and the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. olivier dupont-therrien) and tucker-davis technologies provided discussion, analysis support, and matlab code assistance. supplemental methods ex vivo electrophysiology slice preparation coronal brain slices were prepared from virus injected mice after 3 weeks from surgery. animals were anesthetized with a mixture of ketamine and xylazine (100 mg ketamine and 6 mg xylazine/kg body weight injected ip). then the mice were transcardially perfused with a sucrose-based solution (see below). after decapitation, the brain was rapidly transferred into a sucrose-based cutting solution bubbled with 95% o 2 and 5% co 2 and maintained at~3˚c. this solution contained (in mm): sucrose 230; kcl 2.5; mgso 4 10; cacl 2 0.5; nah 2 po 4 1.25; nahco 3 26; glucose 10 and pyruvate 1.5. coronal brain slices (300 mm) were prepared using a leica vt1000s vibratome (leica biosystems inc). slices were equilibrated with a mixture of oxygenated artificial cerebrospinal fluid (acsf) and sucrose-based cutting solution at room temperature (24-26˚c) for at least 1 hr before transfer to the recording chamber. pyruvate (0.15-0.75 mm) was added to reduce oxidative damage and enhance survival. with this protocol, slices are initially incubated in a mixture of 50% cutting solution with pyruvate and 50% acsf (in mm): sucrose 115; nacl 63; kcl 2.5; nah 2 po 4 1.25; mgso 4 5; cacl 2 1.25; mgcl 2 1; nahco 3 26; glucose 10; and sodium pyruvate 0.75 at 35˚c for 30 min and then transferred to a mixture of 10% cutting solution and 90% acsf (in mm): sucrose 23; nacl 113.4; kcl 2.5; nah 2 po 4 1.25; mgso 4 1; cacl 2 1.85; mgcl 2 1.8; nahco 3 26; glucose 10; and sodium pyruvate 0.15 at 35˚c for 1-4 hr before recording. the slices were continuously superfused with acsf at a rate of 2 ml/min containing (in mm); nacl 126, kcl 2.5, nah 2 po 4 1.25, nahco 3 26, cacl 2 2, mgcl 2 2 and glucose 10 bubbled with 95% o 2 and 5% co 2 at room temperature. brain slices were placed on the stage of an upright, infrared-differential interference contrast microscope (olympus bx51wi, olympus). nbm neurons were visualized with a 40 â water-immersion objective by infrared microscopy (cohu 4915 camera, cohu, inc, poway, ca). patch electrodes with a resistance of 4-6 mw were pulled with a laser-based micropipette puller (p-2000, sutter instrument company). signals were recorded with a multi clamp 700a amplifier and pclamp10 software (molecular devices, inc, san jose, ca). the pipette solution contained (in mm) 130 k-gluconate, 2 kcl, 2 mgcl 2 , 10 hepes, 0.5 egta, 1 atp and 0.2 gtp (ph = 7.3).to examine action potential firing frequency, nbm neurons were recorded in a current clamp configuration after forming a giga-ohm seal. membrane potentials were clamped at à60 mv by injecting 0-~50 pa current through the recording electrode as needed. cells that maintained steady membrane potentials for at least five mins were included in the analysis. channelrhodopsin was activated with a train of light flashes delivered through the 40â microscope objective. the light source was an olympus x-cite 120q lamp (olympus) gated with a ttl controlled shutter (lambda sc, sutter instrument). the filter cube contained an hq480/40x excitation filter, a q505lp bypass filter and an hq535/50 m emission filter (chroma technology corp., bellows falls, vt). the fluorescence illumination intensity delivered at the brain slices was adjusted to 1-3 mw/ mm 2 , measured with a pm100d optical power and energy meter (thorlabs inc, newton, nj). in the nbm, cholinergic neurons were identified by egfp fluorescence and light flashes were delivered at 1 hz, 5 hz, 10 hz, 15 hz, 20 hz, 25 hz, and 30 hz. after xtinction, responding was reinstated in training for 2 d. then mice underwent a modified training paradigm where correct nose pokes yielded only laser stimulation, without ensure delivery. locomotor data was collected using an accuscan instruments (columbus, ohio) behavior monitoring system and software. mice were individually tested in empty cages, with bedding and nesting material removed to prevent obstruction of infrared beams. mice were injected (i.p.) with saline, mecamylamine (1 mg/kg, sigma), scopolamine (0.5 mg/kg, sigma), or mecamylamine+scopolamine (1 mg/kg and 0.5 mg/kg, respectively) 30 min before locomotor testing. locomotion was monitored for 20 min using 13 photocells placed 4 cm apart to obtain an ambulatory activity count, consisting of the number of beam breaks recorded during a period of ambulatory activity (linear motion rather than quick, repetitive beam breaks associated with behaviors such as scratching and grooming). a rectangular box was divided evenly into a light (clear top, illuminated by an 8w tube light) and dark (black walls, black top) side with a black walled divider in the middle with a small door. the lid and divider were modified to allow the optical fiber and patch cord to pass through freely. mice were placed facing the corner on the light side furthest from the divider and the latency to crossing to the dark side was measured. the number of crosses and time spent on each side were measured for 6 min following the initial cross. key: cord-253459-tcn10pho authors: moreau, gregory brett; burgess, stacey l.; sturek, jeffrey m.; donlan, alexandra n.; petri, william a.; mann, barbara j. title: evaluation of k18-hace2 mice as a model of sars-cov-2 infection date: 2020-07-28 journal: am j trop med hyg doi: 10.4269/ajtmh.20-0762 sha: doc_id: 253459 cord_uid: tcn10pho murine models of sars-cov-2 infection are critical for elucidating the biological pathways underlying covid-19. because human angiotensin-converting enzyme 2 (ace2) is the receptor for sars-cov-2, mice expressing the human ace2 gene have shown promise as a potential model for covid-19. five mice from the transgenic mouse strain k18-hace2 were intranasally inoculated with sars-cov-2 hong kong/vm20001061/2020. mice were followed twice daily for 5 days and scored for weight loss and clinical symptoms. infected mice did not exhibit any signs of infection until day 4, when no other obvious clinical symptoms other than weight loss were observed. by day 5, all infected mice had lost around 10% of their original body weight but exhibited variable clinical symptoms. all infected mice showed high viral titers in the lungs as well as altered lung histology associated with proteinaceous debris in the alveolar space, interstitial inflammatory cell infiltration, and alveolar septal thickening. overall, these results show that the k18-hace2 transgenic background can be used to establish symptomatic sars-cov-2 infection and can be a useful mouse model for covid-19. an invaluable step in identifying effective vaccines and therapies to combat covid-19 is the availability of a mouse model of infection. the host receptor for sars-cov-2 is the human angiotensin-converting enzyme 2 (hace2), 1 which was previously identified as the receptor for the sars-cov-1 2 that causes sars, a disease that emerged from china in 2002. 3 the mouse ace2 ortholog, which has significant amino acid sequence variation in the viral receptor binding domain, cannot serve as an efficient receptor for either sars-cov-2 or cov-1. 4 a transgenic mouse model to study sars-cov-1 infection was developed that expresses the hace2 gene under the control of the human cytokeratin 18 promoter. 5 infection of these mice with sars-cov-1 results in a rapidly lethal infection. 5 four other hace2-expressing mouse lines have been created to date and tested for the ability to support sars-cov-2 infection. two lines express the hace2 gene under the control of the mouse ace2 promotor 6, 7 ; one was made using the crispr/cas9 technology. 7 the third strain uses the lung ciliated epithelial cell hepatocyte nuclear factor-3/ forkhead homologue 4 (hfh4) promoter. 8, 9 an additional approach was to transfect wild-type mice with an adenovirus carrying the hace2 gene. 10 overall, with the exception of the hfh4 mice, in which there was some lethality, infection of these three mouse strains with sars-cov-2 results in mild clinical symptoms and no lethality. here, we report the infection of k18-hace2 with sars-cov-2. although this infection resembled that of other strains, we observed variable clinical presentation, with some mice exhibiting more severe symptoms than reported using other models. overall, this work supports the usefulness of k18-hace2 transgenic mice as a model for human covid-19 infections. to investigate the potential of this transgenic mouse strain as a model for covid-19 infection, five k18-hace2 mice were intranasally inoculated with 8 × 10 4 median tissue culture infectious dose (tcid50) of sars-cov-2, and five mice were mock-infected with sterile dulbecco's modified eagle's medium (dmem). mice were followed twice daily for 5 days and scored for clinical symptoms (weight loss, eye closure, appearance of fur [piloerection] and posture, and respiration). the mock-infected mice did not exhibit any clinical symptoms or experience any weight loss throughout the experiment. infected mice did not exhibit any measurable clinical symptoms through day 3. on day 4, no other clinical symptoms other than weight loss were observed. on day 5, all the infected mice had lost around 10% of their original weight ( figure 1a ) and exhibited variability in other clinical signs of infection, with clinical scores ranging from 3 to 9 (maximal score 14) ( figure 1b ). although two of the infected k18-hace2 mice showed only mild symptoms at day 5 (weight loss and reduced activity), two mice exhibited piloerection. the most severe mouse had increased respiration, lethargy, and slight eye closure and met our criteria for euthanasia. because the study was ended on day 5, it is unclear whether the remaining four mice would have recovered if the study was carried past day 5. although the clinical severity was variable between infected k18-hace2 mice, our results suggest that these mice present with more symptomatic disease than other hace2 mouse models of sars-cov-2 infection. in the mouse model expressing hace2 under the mouse ace2 promoter, infected mice did not exhibit any clinical symptoms other than maximal weight loss on day 3 postinfection, and those mice recovered. 7 only mild ruffling of fur and up to 8% weight loss on day 5 were observed in the other model using the mouse ace2 promoter, and once again, all mice recovered. 6 in mice transfected with an adenovirus carrying the hace2 gene, mice exhibited about a 10% weight loss on day 4 postinfection but no lethality. 10 in contrast to these models, in which mice exhibited mild symptoms and recovered, only 60% of the mice survived past day 5 in the mouse strain expressing hace2 under the lung ciliated epithelial cell hfh4 promoter. 9 although this model had higher lethality, weight loss was only about 5% and these mice had no respiratory symptoms. the authors hypothesize that mortality may be due to neuroinvasion because virus was detected in the brain. in k18-hace2 mice infected with sars-cov-1, the course of infection is clearly different; the infection is uniformly fatal, beginning on day 4 postinfection, and mice were symptomatic with labored breathing and lethargy. 5 although the number of mice used in this study was small and we were not able to measure survival, our data support a difference in the disease progression between these two viruses. all mice were euthanized on day 5, and tissue was collected for dissection and enumeration of viral loads. no significant differences in histology of the spleen, small intestine, or liver were observed between infected and mock-infected mice, and these tissues were normal in size and appearance. dissection of the lungs of infected mice revealed a mottled or marbled appearance that was not observed in mock-infected mice (data not shown). lung sections were analyzed after staining with hematoxylin and eosin and scored based on tissue pathology. 11 sars-cov-2-infected mice exhibited significantly higher histopathology scores than mock-infected mice ( figure 2 ). the major histopathology findings in infected mice were proteinaceous debris in the alveolar space, neutrophils in the interstitial space, and alveolar septal thickening ( figure 2 ); these observations were consistent with other hace2 mouse models, which also detected signs of lung injury including interstitial pneumonia, inflammatory cell infiltrates, and alveolar septal thickening. 6, 7 consistent with the observed infiltrating neutrophils, granulocytes and inflammatory monocytes were also elevated in the bronchoalveolar lavage (bal) fluid from the infected mice ( figure 3 ). other hace2 mouse models of covid-19 infection have observed high viral titers in the lungs with limited viral load in organs such as the liver and spleen during intranasal infection. 6,7 although we did not investigate viral load in the liver or spleen, these organs appeared normal by histology, suggesting that there was limited viral titer in these tissues. virus was detected in the lungs of all infected mice, with titers generally in the range of 1 × 10 5 plaque forming units (pfu)/ml (table 1 ). viral titers in the lungs appeared somewhat associated with disease severity: mouse 1390, which had the highest lung titer, had the highest clinical score, histopathology score, and percent weight loss at day 5 (table 1 ) and the highest numbers of neutrophils, monocytes, and eosinophils in the bal (figure 3 ). in addition, mouse 1413, which had the lowest titer, had the lowest clinical score, second lowest percent weight loss at day 5 (table 1) , and lowest number of eosinophils and monocytes in the bal (figure 3 ). of note, mouse 1413 did not have the lowest histopathology score (table 1) . although there were trends toward higher viral titers in the lungs being associated with higher clinical and histopathology scores, these trends were not significant, and viral titer was not a strong predictor of percent weight loss. the power of this analysis is limited by the small sample size, but these results suggest that factors in addition to viral load, such as inflammatory responses, are driving the severity of disease. this would also potentially explain the sudden onset of clinical symptoms at 5 days post-inoculation. in this report, we have described the course of sars-cov-2 infection in k18-hace2 transgenic mice. our findings are consistent with other studies using hace2 mice, which observed successful infection with sars-cov-2 and a milder disease severity compared with sars-cov-1. 6, 7 the onset of symptoms was abrupt, manifesting on day 5. mice exhibited a similar degree of weight loss but a varying degree of symptoms and clinical/histopathological scores. the number of mice used in this study was too small to determine whether this was a result of experimental variability or natural variability in outcomes. the variance in clinical and histopathological scores may be partially explained by viral titer, but there are likely other factors, such as the host immune response, that contribute to the variance observed. the observation of more severe disease in a subset of the k18-hace2 mice is distinct from other hace2-expressing covid-19 models, which typically observed only mild clinical symptoms. 6, 7 this could be due to experimental differences such as strain differences or the challenge dose (table 2 ). to date, little is known about the possibility of virulence differences among isolates. hong kong/vm20001061/2020 and strain 2019n-cov/usa_wa1/ 2020 are closely related and have been classified as type ib. 12 the receptor-binding domains of these strains are 100% identical (data not shown). the phylogeny of hb-01 and wuhan/amms01/2020 has not been reported. the challenge dose used in each experiment is similar; our experiment used the lowest amount of inoculum. the resident microbiota in each mouse strain could also impact outcomes of infection. the other difference between these strains is in the level of hace2 receptor expression or tissue distribution. nonetheless, k18-hace2 transgenic mice may be a particularly useful , and respiratory rate (0-2). all mouse work was approved by the university's institutional animal care and use committee, and all procedures were performed in the university's certified animal biosafety level three laboratory. histology. tissues were fixed in formaldehyde. slides were scanned at ×20 magnification. histopathological scoring for lung tissues was performed according to the guidelines of the american thoracic society. 11 a two-tailed student's t test was used to determined statistical significance. viral titers. the left lobe of the lung was homogenized in 1 ml serum-free dmem with a disposable tissue grinder. plaque assays were performed as described. 13 in brief, vero c1008, clone e6 (atcc crl-1586) cells grown in dmem (gibco 11995-040) with fetal bovine serum (fbs) were seeded into at a concentration of 2 × 10 5 cells/well the night before the assay. serial dilutions were added to the wells. the plate was incubated at 37°c, 5% co 2 for 2 hours, shaking the plates every 15 minutes. after 2 hours, the media was replaced with a liquid overlay of dmem, 2.5% fbs containing 1.2% avicel ph-101 (sigma aldrich, st. louis, mo) and incubated at 37°c, 5% co 2 . after 3 days, the overlay was removed, wells were fixed with 10% formaldehyde, and stained with 0.1% crystal violet to visualize plaques. plaques were counted, and pfus were calculated according to the following equation: average # of plaques/dilution factor × volume diluted virus added to the well. 8, 9 not specified not specified ∼ 5% weight loss, no clinical symptoms, but only 60% survived adenovirus transfection 10 10 5 focus-forming units strain 2019n-cov/usa_wa1/2020 10% maximum weight loss, all recovered hace2 = human angiotensin-converting enzyme 2. this work was supported by nih grants r01 ai124214 to w. a. p., r01 ai146257 to s. l. b., and 5t32ai007496-23 to a. n. d., and the university of virginia's global infectious diseases institute. j. m. s. is a an ithriv scholar, a program supported in part by the national center for advancing translational sciences of the nih under award numbers ul1tr003015 and kl2tr003016 disclaimer: the content is solely the responsibility of the authors and does not necessarily represent the official views of the nih division of infectious diseases and international health receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus a cluster of cases of severe acute respiratory syndrome in hong kong structural basis of receptor recognition by sars-cov-2 lethal infection of k18-hace2 mice infected with severe acute respiratory syndrome coronavirus the pathogenicity of sars-cov-2 in hace2 transgenic mice. nature (epub ahead of print a mouse model of sars-cov-2 infection and pathogenesis sars-like wiv1-cov poised for human emergence pathogenesis of sars-cov-2 in transgenic mice expressing human angiotensin-converting enzyme 2 a sars-cov-2 infection model in mice demonstrates protection by neutralizing antibodies an official american thoracic society workshop report: features and measurements of experimental acute lung injury in animals genomic variations of sars-cov-2 suggest multiple outbreak sources of transmission viral concentration determination through plaque assays: using traditional and novel overlay systems acknowledgments: we would like to gratefully acknowledge the advice and assistance of angelina angelucci and young hahn at the university of virginia as well as caitlin woodson and kylene kehn-hall at george mason university. publication charges for this article were waived due to the ongoing pandemic of covid-19. this is an open-access article distributed under the terms of the creative commons attribution (cc-by) license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. key: cord-032982-xri24v40 authors: medinsky, m. a.; bechtold, w. e.; blrnbaum, l. s.; bond, j. a.; burt, d. g.; cheng, y. s.; glllett, n. a.; gulati, d. k.; hobbs, c. h.; plckrell, j. a. title: effect of inhaled azodicarbonamide on f344/n rats and b6c3f(1) mice with 2-week and 13-week inhalation exposures date: 1990-08-17 journal: fundam appl toxicol doi: 10.1093/toxsci/15.2.308 sha: doc_id: 32982 cord_uid: xri24v40 effect of inhaled azodicarbonamide on f344/n rats and b6c3f, mice with 2-week and 13-week inhalation exposures. medinsky, m. a., bechtold, w. e., birnbaum, l. s., bond, j. a., burt, d. g., cheng, y. s., gillftt, n. a., gulati, d. k., hobbs, c. h., and pick-rell, j. a. (1990). fundam. appl. toxicol 15, 308/319. azodicarbonamide (ada), a compound used in the baking and plastics industries, has been reported to cause pulmonary sensiti-zation and dermatitis in people. two-week repeated and 13-week subchronic inhalation exposures of f344/n rats and b6c3f, mice to ada were conducted to determine the toxicity of inhaled ada. the mean air concentrations of ada in the 2-week studies were 207, 102, 52, 9.4, or 2.0 mg/m3. no exposure-related mortality nor abnormal clinical signs were observed in rats or mice during or after exposure. the terminal body weights were slightly depressed in the highest exposure group. liver weights were lower in male rats exposed to 200 mg ada/m3. no significant lesions were noted on either gross or histologic evaluation of rats or mice. in the 13-week subchronic study, the mean air concentrations of ada were 204, 100, or 50 mg/m3. no mortality or clinical signs related to exposure were observed. the terminal body weights of exposed rats were not significantly different from those of control rats but were significantly depressed in mice exposed to 100 or 200 mg ada/m3. no histopathological lesions were noted in mice. lung weights were increased and enlarged mediastinal and/or tracheobronchial lymph nodes were noted in rats exposed to 50 mg ada/m3. no exposure-related lesions were observed microscopically in rats exposed to 100 or 200 mg ada/m3. all rats in the 50 mg ada/m3 exposure group only had lung lesions that consisted of perivascular cuffing with lymphocytes and a multifocal type ii cell hyperplasia, suggesting a possible immune reaction to an antigen in the lung. viral titers for rats exposed to 50 mg ada/m3 were negative for sendai virus and pneumonia virus of mice, which produce similar lesions. the possibility of an unknown viral antigen causing this lesion cannot be eliminated. lung tissue from male rats was analyzed for ada and biurea, the major metabolite of ada. no ada was detected. the amount of biurea in the lungs increased nonlinearly with increasing exposure concentration, suggesting that clearance was somewhat impaired with repeated exposures. however, even at the highest exposure concentration, this amount of biurea was less than 1 % of the estimated total ada deposited over the exposure period. in summary, ada is rapidly cleared from the lungs, even when inhaled at concentrations up to 200 mg/m3. exposure to ada for up to 13 weeks did not appear to be toxic to rodents ported by the fda, niosh, osha, and cpsc, because of (1) the high level of occupational exposure (~232,ooo workers) in the rubber, plastics, and baking industries; (2) levels of production as high as 10 million pounds per year, and (3) structural relationships to known carcinogens such as 3-aminotriazole, azoethane, and hydrazine. ada is an orange crystalline compound that is produced as a condensation product of urea and hydrazine. ada is an orange crystalline compound that is produced as a condensation product of urea and hydrazine. ada is manufactured predominantly as a fine yellowish powder milled to particle sizes in the range 2 to 10 mm (slovak, 1981) . principal worker exposure occurs in factories where it is ground and in bakeries where it is used as an additive in flour. because ada is used as a flour-maturing agent, toxicity testing using oral exposures was originally recommended. however, in making dough, ada is quantitatively reduced to biurea (1,2-hydrazinedicarboxylic acid diamide; h 2 nochn-nhconh 2 ). ada decomposes at temperatures > 180°c, releasing ammonia, nitrogen, and carbon monoxide and producing urazone, biurea, cyanuric acid, and cyanidine (herweh and fantazier, 1974) . subchronic toxicity testing of ada in rats and mice by gavage revealed only a nonspecific nephrotoxicity resulting from precipitation of ada (or biurea) in the kidneys of animals given 2.5 g/kg body wt or more. similarly, nonspecific nephrotoxicity was noted in dogs fed 5 or 10% biurea in their diet for up to 1 year (oser et al, 1965) . methemoglobin levels measured by oser et al. (1965) were below the limit of detection (0.2 mg/loog). ada has been reported to exhibit an antithyroid effect (gafford et al., \91\) and an inhibitory effect on cholinesterase activity in blood and liver (mel'nikova and selikhova, 1965) . anorexia, weight loss, and gross hematuria have also been observed in rats given ada intraperitoneally at a dose of 200 mg/ kg body wt daily for 1 week (gafford et al., 1971) . five out of eight rats died from this dose of ada. there were no deaths among rats given the same dose of ada orally. reports of airway constriction in workers exposed to ada suggest that ada might be an airway sensitizer or irritant. ferris et al. (1977) observed decreased forced vital capacity and forced expiratory volume in exposed workers. slovak (1981) reported the occurrence of asthma in almost one-fifth of workers in a factory manufacturing ada. whitehead et al. (1987) found symptoms similar to chronic bronchitis in workers in ada plants. one case of dermatitis, with a positive response to 1% ada in a patch test, was noted (bonsall, 1984) . studies of the effects of an acute, 1 -hr exposure of guinea pigs to ada (19, 5 8, or 97 mg/ m 3 ) demonstrated minor changes in tidal volume and respiratory frequency at the two higher concentrations (shopp et al., 1987) . no lesions were noted in respiratory tract tissue of animals euthanized immediately after exposure or 24 hr later. repeated exposure of guinea pigs to 51 or 200 mg ada/m 3 for 4 weeks did not result in either specific or nonspecific airway sensitization on inhalation challenge with ada or aerosolized histamine, respectively (gerlach et al., 1989) . the objective of the studies reported here was to describe the toxic effects in rats and mice of inhalation exposure for 2 or 13 weeks to airborne concentrations of ada as high as 200 mg/m 3 . azodicarbonamide, obtained from midwest research institute, was 98% pure, as determined by iodometric titration. the major impurity identified was biurea, which was from 0.4 to 0.7% of the bulk chemical. a compound stability study was also done to confirm that the chemical composition of ada did not change upon aerosolization. filter samples were analyzed by reverse-phase highperformance liquid chromatography (hplq, using the method of bechtold et al. (1988) . contamination of ada aerosols with biurea was less than 1 %. this was not different from the amount of contamination in the bulk samples. stainless-steel, multitiered, whole-body exposure chambers (h1000, hazleton systems, aberdeen, md), with a total internal volume of 1.0 m 3 , were used in both the 2-week and the 13-week studies. the flow rate through the chambers was 7 ± 1 ft 3 /min, corresponding to 12 ± 2 air changes per hour. chambers were maintained at a temperature between 21.2 and 25.4*c(mean 23.6"c). the average relative humidity ranged from 70 to 85%. to reduce the spatial variation of aerosol concentration and to increase the uniformity of mixing, the aerosol was diluted in a radial diluter before entering the chamber. animal cages were rotated on a regular basis to reduce any variation in the relative levels of ada inhaled by animals over the course of the study. rats and mice were housed in the same chambers, but in different cage units, for both studies. ada aerosol was generated by a jet-o-mizer/screw feed method (cheng el al., 1985) . ada powder was put into the hopper of the screw feeder (model 300, accu-rate, whitewater, wi) and delivered to the funnel of the jet-o-mizer (model 0101, fluid energy co., hatfield, pa) for dispersion. one generator was provided for each exposure chamber. the aerosol concentration in the exposure chamber was monitored by sampling at a flow rate of 0.5 liter/min for three, 2-hr periods during the 6-hr exposure. samples were collected on 25-mm fiberglass filters (type ae, gelman, ann arbor, mi). each exposure day, aerosol generation was started and sampling began after 12 min of rise time, when the chamber concentration had reached 90% of equilibrium concentration (t^). therefore, the total exposure was 6 hr plus a t^ of 12 min. the control chamber was also sampled daily. a ram-s continuous aerosol monitor (gca, bedford, ma) was used to monitor the stability of the aerosol concentration and to adjust the aerosol generator during exposure. it was operated on each chamber at the beginning, middle, and end of the filter sampling period as described previously (cheng el al., 1988) . aerosol size distribution was obtained once per study by using a lovelace multijet cascade impactor (newton et al.. 1977) , with a flow rate of 15 liters/min. thesanv pling period ranged from 1 to 6 hr, depending on the chamber concentration. two-week repeated study. four-week-old f344/n rats and b6c3f| mice were received from frederick cancer research facility (frederick, md). the animals were acclimated two per cage for the first week and then one per cage in cage units within two hazleton 1000 chambers prior to exposure [total of 21 days (males) or 22 days (females)]. exposures began when animals were 7 weeks old. animals were exposed 5 days per week for 2 weeks, plus an additional 2 (males) or 3 (females) consecutive exposure days before terminal euthanasia for a total of 12 exposure days for both sexes. exposures of females were initiated 1 day later than males. 13-week subchronic study. four-week-old f344/n rats and b6c3f, mice were received from simonsen laboratories, inc. (gilroy, ca). rats and mice were acclimated as described for the 2-week study, for a total of 18 days (males) or 22 days (females) prior to exposure. animals were exposed by inhalation, 5 days per week for a total of 13 weeks, with 2 or 3 (males) or 4 or 5 (females) consecutive dose days before terminal euthanasia. the total number of exposure days was 65 or 66 for both males and females. the feed used in both studies was zeigler nih-07 open formula rat ration (zeigler brothers, inc., gardners, pa). water was provided by an automatic watering system. the light cycle was automatically controlled to provide 12 hr of fluorescent light and 12 hr of darkness each 24 hr (lights on at 6 am and off at 6 pm). prior to study start, the rats and mice in both studies were randomly assigned by weight to treatment groups, using a computer-based system (path/tox, xybion corp.). animals within exposure groups were further randomized by use of computer-generated random numbers into groups for the basic studies or special studies and, for the 13-week study, into termination days (day 1 or day 2) for each group. in the 2-week study, serum (approximately 0.5 ml) was obtained from five male and five female rats and mice 3 days prior to the first day of exposure, and was analyzed for antibody titers to specific bacteria and viruses (mycoplasma pulmonis, m. arth, pneumonia virus of mice (pvm), sendai virus, rat cornavirus (rcv)/sda, and mycoplasma-rats; reo3, m. ad., m. pulmonis, m. arth, pvm, send, mhv, ectro, and gdvii-mice). titers were negative for these agents. in the 13-week study, serum was also obtained from five male and female rats and mice 3 days prior to study start. analyses for the agents described above showed positive titers for rcv/sda in the rats and negative titers for all agents in the mice. no gross lesions were noted in the rats on necropsy. tissue sections were taken from all lung lobes, liver, both kidneys, spleen, harderian gland, and mandibular salivary glands and processed for histology. lesions were only noted in the salivary gland or harderian gland tissue of 8 of the 10 animals. ductal squamous metaplasia was noted in both tissues and corresponded with those animals having positive titers to rcv/sda. these lesions are consistent with lesions induced by sialodacryoadenitis virus. at the end of the 13-week study, serum from 5 male and 5 female rats from all exposure groups and controls (40 rats total) were analyzed for titers to sendai virus, pvm, rcv/sda, and mycoplasma. all 40 sera were negative for titers to sendai, pvm, and mycoplasma. twenty-five of the 40 sera were positive for rcv/sda (>0.17 absorbance units by elisa assay). two-week repeated study. rats and mice in the basic study group (table 1) were weighed prior to study start, after 1 week of exposure, and at terminal euthanasia. detailed clinical observations were made at these times. morbidity/mortality checks were made twice daily on all animals. evaluations of gross and microscopic pathology and organ weight changes were made on the basic study group (table 1) . separate groups of rats and mice were used for evaluation of methemoglobin levels and cholinesterase levels in whole blood. thirteen-week subchronic study. rats and mice in the basic study group (table 1) were weighed weekly at the time of detailed clinical examinations. mortality/morbidity checks were performed twice daily on all animals. histopathology, hematology, organ weight, sperm morphology, and vaginal cytology evaluations were also made on this group of rats. each animal assigned to the special study groups (table 1) was used for the following determinations: levels of ada and biurea in lungs and kidneys, acetylcholinesterase activity in whole blood, and t3 and t4 levels in serum. necropsy and histopathology. all rats and mice in the basic study group, for both the 2-week and the 13-week studies, were given complete gross necropsy examinations. rats and mice were killed by cardiac puncture exsanguination while under halothane anesthesia and were necropsied immediately. weights of liver, thymus, right kidney, right testicle, brain, heart, and lungs (including trachea) were taken. tissues were fixed in 10% neutralbuffered formalin. tissues for microscopic examination were embedded in paraffin, sectioned at 5 nm, and stained with hematoxylin and eosin. the following tissues were trimmed and sectioned for histopathology: adrenals, bone (vertebra, with bone marrow and spinal cord; femur, rib), brain, epididymus or oviduct, esophagus, heart, small intestine (duodenum, jejunum, ileum), large intestine (cecum, colon, rectum), both kidneys, larynx, liver, lung (4 lobes), lymph nodes (bronchial, mandibular, mediastinal, mesenteric), mammary glands, nose (3 levels), pancrease (including islets), parathyroid gland, pituitary gland, prostate or uterus, salivary glands, seminal vesicles, skin, spleen, stomach (including forestomach and glandular portions), testes or ovaries, thymus, thyroid gland, trachea, and urinary bladder. all tissues from each rat and mouse exposed to filtered air or 200 mg ada/m 3 , for both the 2-week and the 13week studies, were examined microscopically. in addition, lungs of rats exposed to 50 or 100 mg ada/m 3 for 13 weeks were examined. hematology. for the 13-week study, blood was collected at necropsy from anesthetized rats and mice from the basic study group by cardiac puncture and placed in glass vials containing edta. a coulter electronics model s-550 was used for analysis of erythrocyte count, mean corpuscular volume, hemoglobin concentration, hematocrit (calculated), and leukocyte count. smears were made from the blood, stained with wright's stain, and examined under a light microscope to obtain differential leukocyte counts and counts of nucleated erythrocytes. additional blood smears were stained with new methylene blue and examined for the presence of reticulocytes. sperm morphology-vaginal cytology. in the 13-week study, vaginal cytology samples were taken on a daily basis from basic study females for 1 week before final termination. samples were obtained with sterile saline, placed on duplicate dakin slides, fixed with spray cyte (clay adams 7180), and stained with toluidine blue (0.5% in 20% ethanol). live sperm were obtained from the cauda of the right epididymis of male rats and mice at necropsy. sperm were incubated at 37*c in tyrode buffer, and viability was quantitated as the percentage of motile sperm in the sample. sperm density (number of sperm per gram of caudal tissue) was quantified on a hemocytometer. the number of sperm in the sample was divided by the weight of the cauda of the epididymis to obtain the value for sperm density. evaluations of sperm morphology were done using preparations of sperm fixed in ethanol and stained with eosin y. urinary enzymes. in the 13-week study, 1 week prior to termination, all rats in the basic study group were placed in metabolism cages for overnight urine collection. urine was collected on ice and analyzed for total amounts of lactate dehydrogenase (ldh), /3-galactosidase 03-g), a'-acetylglucosaminidase (nag), and alkaline phosphatase (ap) using methods described (medinsky el aj., 1989). lactate dehydrogenase was quantitated by using pyruvate as a substrate; 4-nitrophenyiphosphate was the substrate for alkaline phosphatase; /vnitrophenol was the substrate for both tv-acetyl-js-d-glucosaminidase and 0-galactosidase. determinations of methemoglobin in whole blood of rats and mice exposed to ada for 2 weeks were made to using the method of evelyn and malloy (1938) . acetylcholinesterase concentrations in whole blood of rats only in the 13-week study and both rats and mice in the 2week study were determined using the method of ellman el al. (1961) , in which acetylthiocholine is used as the substrate. serum thyroxine (t4 or tetraiodothyronine) and triiodothyronine (t3) levels for rats exposed to ada for 13 weeks were measured by radioimmunoassay (tietz, 1986) . 2 samples of lung and kidney from male rats exposed to ada for 13 weeks were analyzed for ada and biurea. this procedure involved derivatization of ada with triphenylphosphine and quantitation of the resulting derivative by hplc (bechtold el al, 1989) . biurea in the sample was oxidized to ada using potassium permanganate in acid solution. the ada produced was then quantitated by hplc (bechtold el al, 1989) . all data were analyzed separately for each sex. organ and terminal body weights on animals found dead or euthanized because of a moribund condition were not included in the statistical analysis. analysis of variance techniques were used for statistical evaluation. provided bartlett's test of homogeneity of variance was not significant, exposure groups were compared to controls by using dunnett's multiple range test. when bartlett's test was significant, comparisons with the control group were made by a modified student's i test, making allowance for unequal variance. all statistical tests were conducted at a 5%, two-sided risk level. the overall, mean ada concentration for each chamber was within 8% or less of target concentration ( table 2 ). the relative standard deviation of daily means was within 16%. the aerosol had an average mass median aerodynamic diameter (mm ad) of 2.13 jtm (range 1.89 to 2.45) with a mean geometric standard deviation {a%) of 1.9. there were no abnormal clinical observations during the 2-week exposures for either rats or mice. no rats died. one male (50 mg/ m 3 exposure group) and one female (200 mg/ m 3 exposure group) mouse died of undetermined causes. the mean terminal body weights for the basic study male rats exposed to 200 mg/m 3 ada were significantly less than those of controls (95% of control value). the mean terminal body weights for basic study male and female mice were significantly lower (89 or 94% of controls, respectively) for the 200 mg/m 3 exposure group. the mean liver weights of male rats and of male and female mice were significantly less at the 200 mg/m 3 exposure than those of controls (83, 79, and 79% of control values, respectively). other organ weights of either mice or rats were not influenced by ada exposure (data not shown). a complete set of tissues from rats and mice exposed to 200 and 0 mg ada/m 3 was examined histologically. the two exposure groups could not be distinguished on the basis of histologic findings. because no effect was seen at the highest exposure concentration, we did not examine tissues from rats exposed to lower concentrations. for mice, the only change noted that may have been related to the ada exposure was the degree of fine vesicular vacuolation of the hepatocyte cytoplasm, which is indicative of glycogen accumulation. all of the livers examined for both groups were considered to be within normal limits. however, the degree of cytoplasmic vacuolation varied among animals. the livers from the control and highdose groups were examined blindly and sorted on the basis of cytoplasmic vacuolation. seven of the high-dose animals and one control animal showed no cytoplasmic vacuolation, indicative of glycogen depletion. when the next lowest dose group (100 mg ada/m 3 ) was compared with the controls, four treated animals and one control showed no cytoplasmic vacuolation. it must be emphasized that none of the livers were regarded as being abnormal. however, there did appear to be a correlation between ada exposure and loss of hepatocyte vacuolation. possibly the ada-exposed animals were eating " values represent means (percentage standard deviation) of daily measurements over the 2-or 13-week exposure period. although rats and mice were housed in the same exposure chamber, the starts of exposure for each species were staggered by 1 week. * particle size was measured once during the study when both rats and mice were being exposed to ada. mmad, mass median aerodynamic diameter, <r,, geometric standard deviation. c no exposure at this target concentration. less, resulting in depletion of glycogen stored in the liver. the mice in the 200 mg/m 3 group had smaller body and liver weights than those of controls. there were no significant differences in methemoglobin levels or acetylcholinesterase activities in whole blood of male and female rats or mice exposed to any concentration of ada, when compared to controls. the overall mean ada concentration for each chamber was within 3% or less of target concentration ( table 2 ). the relative standard deviations of daily means were within 10%. the aerosol had an average mmad of 2.38 mm (range 2.33 to 2.45), with a mean <r g of 1.7. there were no abnormal clinical observations during the 13-week exposures for either rats or mice that could be attributed to exposure to ada. no deaths occurred that could be attributed to ada. one female rat (50 mg/ m 3 exposure group) was euthanized because of weight loss and dehydration. three male mice died of unknown cause, and one female mouse was accidentally killed. the mean terminal body weights for the basic study rats exposed to ada were not significantly different from those of the controls. the mean terminal body weights for basic study male mice were significantly depressed (93 and 91% of controls) at the 100 and 200 mg/ m 3 exposure levels, respectively. for female mice, terminal body weights were significantly depressed (94% of controls) only at the 200 mg/m 3 level. lung weights of male and female rats were increased (111% of control) at the 50 mg/m 3 exposure level. mean liver weights for male and female mice exposed to 200 ada mg/m 3 were lower than those of controls (85 or 86% of controls, respectively). lung weights of mice were not influenced by ada exposure (data not shown). no other effects of exposure on organ weights were noted. a complete set of tissues from rats and mice exposed to 200 and 0 mg ada/m 3 was examined histologicaljy. although several incidental lesions were present in both groups, lesions attributable to the ada exposure were not present. the decrease in hepatocyte vacuolation with increasing ada exposure described for the 2-week study was not observed in mice exposed to ada for 13 weeks. five male and six female rats exposed to 50 mg/m 3 ada were described as having enlarged bronchial or bronchial and mediastinal lymph nodes at gross necropsy. histological examination of these nodes showed a moderate-to-severe lymphoid hyperplasia (table 3) . subsequently, the lungs of all rats and mice in the 50 mg/m 3 exposure group were examined histologically. all male and female rats in the 50 mg/m 3 exposure group euthanized at the end of the 13-week exposure period had a spectrum of lung lesions that consisted of perivascular cuffing with lymphocytes and a multifocal type ii cell hyperplasia that was associated with a moderate number of mixed inflammatory cells (fig. 1) . the extent of lung involvement varied from mild to moderate among animals. in some animals, the perivascular cuffs were predomi-nant, while other animals showed both the perivascular cuffs and the epithelial hyperplasia (table 3 ). the lungs from all rats and mice exposed to 100 mg ada/m 3 , as well as the lungs of mice exposed to 50 mg/m 3 , were examined. there were no significant lesions on histologic examination. results obtained for hematology in rats and mice exposed to ada for 13 weeks indicated that there were no exposure-related alterations in blood parameters. there were no significant, exposure-related changes, in either male or female rats, in the amounts of the four urinary enzymes (ldh, ap, p-g, nag) excreted. there were no adverse effects from inhalation exposure to ada for 13 weeks, with respect to right caudal weight, right epididymal weight, right testicular weight, sperm motility, sperm count per gram caudal tissue, or incidence of abnormal sperm. however, there was a small, but significant, increase in sperm count in male rats exposed to 50 or 100 mg ada/m 3 . no abnormal effects were noted in male mice. there were no apparent adverse effects on estrual cyclicity or on estrous cycle length in any of the dose groups, except in 2 out of the 10 animals in the 200 mg/m 3 dose group. for these animals, estrous cycle length was >7 days or was not precisely determined. no abnormal effects were noted in female mice. there were no significant differences in acetylcholinesterase activities in whole blood of male and female rats exposed to any concentration of ada when compared to controls. t3 and t4 levels in serum from male rats increased with increasing ada exposure concentration in male rats. t3 and t4 levels in the highest exposure group were significantly elevated, relative to control. t3 was increased approximately 50%, while t4 was increased approximately 40%. t3 and t4 were unchanged in female rats exposed to ada. samples of lung and kidney of male rats exposed to ada for 13 weeks were analyzed for the presence of ada and biurea. these tissues were chosen, because they are the most likely tissues to contain significant quantities of one or both compounds, lung being the organ of exposure and kidney, because biurea had been detected in kidney (oser etal., \ 965) . the results of chemical analysis of ada and biurea in lung and kidney of male rats are summarized in table 4 . no ada was detected in either lung or kidney of male rats exposed to ada for 13 weeks. biurea, however, was detected in lungs, but not kidney, of rats exposed to 50, 100, and 200 mg/m 3 ada. the amount of biurea in the lungs increased nonlinearly with increasing exposure concentration. the percentage of biurea retained in lungs was calculated as a percentage of ada deposited on the last day of exposure (table 4 ). although 66% of the amount of ada expected to be deposited in rats exposed to 200 mg/m 3 on the last exposure day is retained in the lungs as biurea, this is a small percentage (~1%) of the total amount of ada deposited over the entire study. after inhalation of ada, the only apparently exposure-related lesions observed were those in lung and lymph nodes of rats exposed to 50 mg/m 3 for 13 weeks. the lung and lymph node lesions in rats exposed to 50 mg ada/m 3 for 13 weeks were striking and suggest an immune response to a virus or another antigen. several viral agents have been reported to produce similar pulmonary lesions in rats (jones el al, 1985; hamm, 1986) . one such agent is sendai virus. factors suggesting that sendai virus is not involved in this case are the following: (i) absence of bronchiolar epithelial involvement, including bronchiolar epithelial necrosis and hyperplasia; (ii) absence of similar lesions in mice housed in the same chamber as the rats (mice are even more susceptible to the virus than are rats); and (iii) negative viral titers for sendai virus from animals within the same chamber terminated at the same time. pvm has been reported to produce perivascular mononuclear cell infiltrates and a multifocal interstitial pneumonia in rats. however, as for sendai, negative viral titers for pvm were reported from animals within the same chamber as the rats having the lung lesions. for both sendai virus and pvm, serum antibody titers would be present at 8 to 14 days after exposure to the virus. it is unlikely that histologic lesions from such a viral infection would be present in the animals examined in the absence of positive antibody titers. rcv infection produces mild lung lesions in young adult rats, consisting of patchy interstitial pneumonia and lymphocytic perivascular infiltrates. mice are unaffected by the virus. rat coronavirus is closely related antigenically to sda, and serology cannot distinguish between exposure to either of these two coronaviruses. as previously indicated, rats in this study had positive titers to rcv/sda. we, therefore, cannot determine if the titers reflect only exposure to sda, or subsequent infection with rcv. because antibody titers for rcv/sda were equivalent across dose groups, any pulmonary lesions due to the viral infection would be expected to be present in all dose groups. 1. (a) lung section of a control rat exposed to filtered air. (b) lung section of a rat exposed to 50 mg ada/m 3 for 13 weeks. note prominent perivascular cuffs composed of lymphocytes. another possible etiology for the lung lesions we observed is that ada acts as a hapten, inducing an immune response within the lung. several reports in humans suggest that ada is capable of inducing an immune reaction and/or asthma-like responses (slovak, 1981; bonsall, 1984) . the absence of a response to the two higher exposure concentra" assuming a rat minute volume of 0.2 liter/min, a 6-hr (360 min) exposure, and a 10% deposition efficiency in the pulmonary region (lungs and bronchi), the amount of ada deposited in the lungs per day is estimated as (0.2 liter/min) x (200 mg/liter) x 0.1 x 360 min = 1440 mg/lungs and bronchi for the 200 mg/m 3 group, 720 mg for the 100 mg/m 3 group, and 360 mg for the 50 mg/m 3 group. * np, no peak was observed at the expected retention time. limit of quantitation is 100 mg per sample. ' nd, not determined. d mean mg/g tissue ± standard deviation; n = 5. tions used in the studies reported here (i.e., 100 and 200 mg/m 3 ) suggests that the lung lesions in rats are not related to ada exposure. in addition, two recent studies of acute and repeated inhalation exposure to ada in guinea pigs did not report any evidence of airway irritation (shopp, 1987) or a potential for sensitization to ada (gerlach et al., 1989) . pyelonephritis, renal tubular concretions, and intratubular renal crystals were noted after subchronic exposure of rats and mice to ada by gavage, at levels of 2.5 g/kg body wt or greater per day. in the present study, no renal lesions were observed in animals exposed to 200 mg ada/m 3 for up to 13 weeks. the lack of renal toxicity in the present study is most likely related to differences in the totaj amount of ada given to the animals in the two studies. for example, for the 2-to 3-ftm particle size used in this study, total deposition should be about 49% (raabe et al., 1988) . for animals exposed to 200 mg ada/ m 3 , with a 200 ml/min minute volume, the total amount of ada deposited should be about 20-40 mg/kg body wt per day for 338-g males or 190-g females (mean terminal body weights of 200 mg/m 3 group). this dose is 100-fold lower than that resulting in nephrotoxicity. the lack of an effect on whole blood cholinesterase activity in the inhalation studies, compared to those previously reported, is also most likely due to differences between the two studies in administered dose. mernikova and selikhova (1965) used oral exposures of up to 7000 mg/kg body wt for 5 days to achieve a 50% reduction in blood cholinesterase. the increased levels of t3 and t4 found in male rats after 13 weeks of exposure to ada differ from those observed by gafford et al. (1971) , who reported a decreased radioactive iodine uptake in rats 24 hr after being fed ada at 5% of their diet. no changes in iodine uptake were noted at lower levels of ada in the diet in studies of gafford et al. the reason for the difference between the results reported by gafford and those reported here is unclear, but may include differences in exposure route, exposure concentration, and duration ofexposure. the findings in male rats exposed to ada for 65 or 66 exposure days are generally consistent with the findings of mewhinney et al. (1987) , who showed that [ i4 c]ada deposited in the lungs of rats is rapidly absorbed into blood during the inhalation exposure and is cleared from tissues with a half-time of 1 day. the rats in the 13-week, ada subchronic study were euthanized 16 to 18 hr after exposure to ada was stopped. no ada or biurea was detected in kidney tissue. the detection limit was at least 100 mg/g tissue. thus, in the 13-week study, ada and biurea were cleared from the kidney at least as fast as for the rats in the previous studies. the nonlinearity of the amount of biurea in the lungs of rats exposed for 65 or 66 days to ada aerosols would not be expected, based on the results of the study by mewhinney et al., in which the rats were exposed once for a period of 6 hr. this may, of course, be due to the much greater amount of ada deposited in the lung over the 65-or 66-day period of exposure or to the higher exposure concentration in the 13-week study compared to that in the study involving 14 c. the amount of biurea present in the lungs of rats exposed to ada for 65 or 66 exposure days was small (~1%), compared to the total amount of ada inhaled and deposited over the 13-week period. in summary, ada is rapidly cleared from the lungs, even when inhaled at concentrations up to 200 mg/m 3 . exposure to ada for up to 13 weeks did not appear to be toxic to rodents. however, the question as to the nature of the chemical sensitization noted in workers exposed to ada still needs to be addressed. vironmental health sciences as part of the national toxicology program. the facilities used for this research were fully accredited by the american association for the accreditation of laboratory animal care. the authors gratefully acknowledge the technical and professional contributions of colleagues at the inhalation toxicology research institute and at the national institutes of environmental health sciences. the determination of biurea in the presence of azodicarbonamide by hplc azodicarbonamide: methods for the analysis in tissues of rats and inhalation disposition allergic contact dermatitis to azodicarbonamide use of a jet mill for dispersing dry power for inhalation studies evaluation of a real-time aerosol monitor (ram-s) for inhalation studies a new and rapid colorimetric determination of acetyl cholinesterase activity microdetermination of oxyhemoglobin, methemoglobin, and sulfhemoglobin in a single sample of blood apparent effect of an azodicarbonamide on the lungs effect of azodicarbonamide (i, l'-azobisformamidc) on thyroid function effect of four-week repeated inhalation exposure to azodicarbonamide on specific and nonspecific airway sensitivity of the guinea pig complications of viral and mycoplasmal infections in rodents to toxicology research and testing 1,1'-azobisformamide. ii. thermal decomposition. kinetics, products, and decomposition mechanism respiratory system correlation of urinary enzyme activity and renal lesions after injection of nickel chloride blood and liver cholinesterase in poisoning with porophor-505 and celogen the fate of inhaled azodicarbonamide in rats cascade impactor design and performance studies of the safety of azodicarbonamide as a flourmaturing agent regional deposition of inhaled monodisperse coarse and fine aerosol particles in small laboratory animals acute inhalation exposure of azodicarbonamide in the guinea pig occupational asthmas caused by a plastic blowing agent, azodicarbonamide use of doseresponse data to compare the skin sensitizing abilities of dicyclohexylmethane-4,4'-diisocyanate and picryl chloride in two animal species textbook of clinical chemistry respiratory symptoms associated with the use of azodicarbonamide foaming agent in a plastics injection molding facility this research was conducted under interagency agreement yo1-es-3o108 between the u.s. department of energy's office of health and environmental research (contract 76ev01013) and the national institute of enkey: cord-254155-860780z9 authors: wang, junyi; kaplan, nihal; wysocki, jan; yang, wending; lu, kurt; peng, han; batlle, daniel; lavker, robert m. title: the ace2‐deficient mouse: a model for a cytokine storm‐driven inflammation date: 2020-06-17 journal: faseb j doi: 10.1096/fj.202001020r sha: doc_id: 254155 cord_uid: 860780z9 angiotensin converting enzyme 2 (ace2) plays an important role in inflammation, which is attributable at least, in part, to the conversion of the pro‐inflammatory angiotensin (ang) ii peptide into angiotensin 1‐7 (ang 1‐7), a peptide which opposes the actions of angii. ace2 and angii are present in many tissues but information on the cornea is lacking. we observed that mice deficient in the ace2 gene (ace2(−/−)), developed a cloudy cornea phenotype as they aged. haze occupied the central cornea, accompanied by corneal edema and neovascularization. in severe cases with marked chronic inflammation, a cell‐fate switch from a transparent corneal epithelium to a keratinized, stratified squamous, psoriasiform‐like epidermis was observed. the stroma contained a large number of cd11c, cd68, and cd3 positive cells. corneal epithelial debridement experiments in young ace2‐deficient mice showed normal appearing corneas, devoid of haze. we hypothesized, however, that these mice are “primed” for a corneal inflammatory response, which once initiated, would persist. in vitro studies reveal that interleukins (il‐1a, il‐1b), chemokines (ccl2, cxcl8), and tnf‐α, are all significantly elevated, resulting in a cytokine storm‐like phenotype. this phenotype could be partially rescued by treatment with the angii type 1 receptor (at1r) antagonist, losartan, suggesting that the observed effect was mediated by angii acting on its main receptor. since the severe acute respiratory syndrome coronavirus 2 (sars‐cov‐2) utilizes human ace2 as the receptor for entry with subsequent downregulation of ace2, corneal inflammation in ace2(−/−) mice may have a similar mechanism with that in covid‐19 patients. thus the ace2(−/−) cornea, because of easy accessibility, may provide an attractive model to explore the molecular mechanisms, immunological changes, and treatment modalities in patients with covid‐19. angiotensin i converting enzyme 2 (ace2) is a critical component of the renin-angiotensin system (ras), due to its ability to hydrolyze angiotensin ii (angii). [1] [2] [3] angii is the major effector peptide of ras and regulates cell growth, and key events in the inflammatory process. 4 in its pro-inflammatory mode, angii directly stimulates pro-inflammatory mediators resulting in the infiltration of macrophages and moreover is profibrotic and may foster angiogenesis (4 and references therein). the expression of ace2 is most abundant in the kidney and intestine, followed by testis and the heart. [5] [6] [7] moreover, the surface expression of ace2 was found in lung epithelial cells. 8 several groups generated ace2-deficient mice [9] [10] [11] with conflicting responses on the contribution of ace2 to cardiac structure and function, and the control of blood pressure. 12, 13 due to its importance as an entry point for coronaviruses, the effects of ace2 depletion was tested in lung tissue and shown to be detrimental in the progression of lung injury following experimental perturbations. 14,15 ace2 depletion also created a "cytokine storm" like inflammation. 16, 17 a cytokine storm is a consequence of the secretion of a large number of cytokines and involves recruitment and activation of inflammatory cells such as macrophages. 18, 19 cytokine storms are known to occur in autoimmune diseases 20 and can be triggered by chemical insults such as corneal alkali burns 21 as well as infections, such as covid-19. 22 in covid-19 patients, ace2 is the target of the virus 23 and dramatic increases in plasma cytokines and chemokines such as il1b, ccl2 (mcp1), cxcl8 (il8), and tnfα have been observed. 22 ace2 is present in the retina 24 and recently, there has been a plethora of information regarding the expression in the cornea and conjunctiva due to the ongoing covid-19 pandemic. [25] [26] [27] [28] [29] [30] during our investigations using an ace2deficient mice, we noted that as the ace2-deficient mice aged, some developed cloudy corneas. in certain mice, cloudy corneas were bilateral, in others they were unilateral, whereas some adult aged mice had clear corneas. herein, we report that angii and ace2 are expressed in limbal and corneal epithelia in humans and mice. moreover, when challenged with corneal injury, ace2-deficient mice are "primed" for an increased corneal inflammatory response. once initiated, inflammation persists, which markedly alters the epithelial and stromal phenotypes. blockade of the angii type 1 receptor (at1r) partially restores the cytokine/chemokine balance due to ace2 deficiency. collectively, our findings establish a pivotal role of ace2 in the cornea and identifies angii blockade as a potential new target for corneal inflammation. moreover, the administration of soluble ace2 protein should have a therapeutic benefit by fostering the degradation of angii and the formation of ang 1-7 as it has proposed for other disease entities. 31 of note, ace2 is the main receptor for the coronavirus sars-cov-2 responsible for the current pandemic. 23 the associated organ injury, particularly acute lung injury, has been shown to involve a decrease in plasma bound ace2 and administration of such ace2 have the potential as an ideal treatment, and ace2 −/− mice a unique model to study inflammatory processes akin to those observed in severe covid-19. animal procedures were approved by the northwestern university animal care and use committee. we used an ace2-deficinet mouse (ace2 -/-) on a c57bl/6 background provided to us as a gift by susan gurley. 9 to generate debridement wounds in the central corneal epithelium, a rotating diamond burr was gently applied to the surface of the central cornea to remove the corneal epithelium while the peripheral corneal and limbal epithelia remained intact. gross images of mouse ocular globes were taken using a leica dissecting scope. corneal haze was graded on a scale of 0 through 4 32 as follows: 0, no corneal haze; 1, iris detail visible; 2, pupillary margin visible, iris detail obscured; 3, pupillary margin not visible; 4, cornea totally opaque. wound closure was examined by topically applying 20 μl of 0.5% fluorescein in pbs and imaging the wound using a leica dissecting scope under cobalt blue illumination. at the termination of the experiment, mouse whole eyes were fixed in 10% buffered formalin solution and paraffin embedded for histological analysis. h&e staining was conducted as described previously 33 and slides were imaged using an axiovision z1 fluorescence microscope system (carl zeiss, oberkochen, germany). normal human corneal tissues were obtained from the eversight eye banks (ann arbor, mi, usa) and embedded in paraffin blocks. immunohistochemical (ihc) staining of human and ace2 −/− mouse eyes were conducted as described previously. 34 antigen retrieval of the paraffin embedded sections was performed at 96°c in ph 6.0 citrate buffer for 30min. after blocking in 2.5% normal horse serum, sections were incubated overnight at 4°c with antibodies recognizing: angiotensinii (angii) rabbit polyclonal antibody (penlabs, san carlos, ca), ace2 goat polyclonal antibody(r&d systems, minneapolis, mn), cd68 rabbit polyclonal antibody (proteintech, chicago, il), cd3 rabbit polyclonal antibody (proteintech, chicago, il), and cd11c rabbit polyclonal antibody (proteintech, chicago, il) at 1:100 dilution. after rinsing with pbs containing 0.1% tween (pbst), the samples were incubated with immpress-ap (alkaline phosphatase) polymer anti-rabbit or anti-goat igg reagent (vector lab, burlingame, ca) at room temperature for 30 minutes. after rinsing with pbst, chromagen was detected using a vector red alkaline phosphatase substrate kit (vector lab, burlingame, ca) for 10-30 minutes. samples were counterstained with hematoxylin, and dehydrated with graded ethanol and xylene. images were taken using an axiovision z1 fluorescence microscope system (carl zeiss, oberkochen, germany). immortalized corneal epithelial cells, htcepi, were cultured in keratinocyte serum-free media (thermo fisher scientific, waltham, ma, usa) as described before. 35 for sirna knock-down experiments, cells were transfected with 10 nm sirna smartpools against ace2 and nontarget control (ge dharmacon, colorado, usa) as previously described. 33 two days after transfection, cells were treated with losartan (100μm) for 24 hours, and processed for total rna isolation. human monocytic thp-1-dual cells (invivogen, san diego, ca, usa) were cultured with rmpi 1640 with 10% heat inactivated fbs. about 10nm 12-o-tetradecanoylphorbol-l3acetate (pma) for 24 hours is used to induce thp-1-dual into macrophages. murine macrophagic raw-dual (invivogen, san diego, ca, usa) cells were cultured with dmem with 10% heat activated fbs. for the chemotaxis assay, the htcepi cells (5 × 10 4 cells/500 μl) transfected with sicontrol or siace2 were plated in the lower chamber of 24-well culture plates for 24 hours. the raw-dual cells (4 × 10 4 cells/200 μl) were resuspended in dmem with 10% heated-fbs media, and placed in transwell chambers (corning inc, corning, ny, usa) in 24 well culture plates. dmem with 10% heated-fbs was added to both the transwell and lower chamber. after 48 hours, transwell chambers were rinsed with pbs, and the cells on top were removed with a cotton tip applicator. the raw-dual cells that migrated to the bottom of the transwell were stained with crystal violet (sigma-aldrich, st. louis, mo, usa), and counted under an inverted zeiss microscope. total rnas were isolated from htcepi cells and purified with a mirneasy kit (qiagen, hilden, germany). real-time qpcr (rt-qpcr) was performed with a roche lightcycler 96 system using the roche faststart essential dna green master (roche, branchburg, nj, usa) according to the manufacturer's instructions. the primer pairs for rt-qpcr were designed using the idt primerquest primer design tool (idt, coralville, ia). primers (table s1) were used to recognize: human il1a, il1b, il6, ccl2, inos, cxcl8, tnfa. relative gene expression was calculated using the 2δδct. unpaired t test were performed to determine statistical significance. the data are shown as means ± standard deviation (sd). the differences were considered significant for p values of <.05. all experiments were replicated at least three times. immunohistochemical analysis revealed that the expression of angii was strong throughout the human limbal and corneal epithelia as well as the stromal vessels ( figure 1a ,b). in contrast, ace2 expression was prominent in the limbal basal cells and vasculature ( figure 1c ). angii and ace2 were detected throughout the wild-type (wt) mouse corneal epithelium (figure 2a ,c) but angii appeared to be more patchy in its distribution (figure 2a ). in the ace2-deficient mice (ace2 −/− ), no ace2 was detected in the corneal epithelium ( figure 2d ), whereas angii expression was markedly increased ( figure 2b ) compared with the wt (figure 2a ). aged ace2 −/− mice developed striking changes in corneal epithelial morphology as well as significant stromal angiogenesis and an inflammatory infiltrate ( figure 3 ). interestingly these changes appeared in approximately 70% of aged adult (60-80 weeks old) mice. in many instances, these changes only occurred in one eye. the contralateral eye appeared to have a normal corneal epithelium and a stroma containing highly organized collagen bundles with numerous keratocytes but devoid of inflammatory cells ( figure s1 ). those ace2 −/− mice that developed inflammation were readily recognized clinically by a cloudy, hazy cornea ( figure 3b-d) . a wide spectrum of corneal epithelial alterations were detected, ranging from a thin, disorganized epithelium ( figure 3f ) to a more stratified, thickened epidermoid tissue ( figure 3h ). in all eyes exhibiting haze or cloudiness the stroma consisted of a densely packed admixture of polymorphonuclear, mononuclear, and lymphocytic infiltrates along with numerous vascular profiles ( figure 3f -h). on higher magnification, the infiltrates were identified as neutrophils by h&e, and as macrophages, t cells, and dendritic cells identified by immunohistochemistry staining for cd68, cd3, and cd11c cells, respectively (figure 4 ). since in 30% (6/19 mice) of ace2 −/− mice, the cloudy corneas were not bilateral, this would suggest that the lesions are a direct sequelae of external perturbation rather than arising from a systemic deficiency. to test this idea, we made small 1mm circular debridement wounds in young ace2 −/− mice with clear corneas and compared the response to aged-matched wt littermates. in this well adopted procedure, the type of wound removes the corneal epithelium but does not result in a significant inflammatory infiltrate. 36 immediately following wounding, all mice received topical application of a 0.5% fluorescein stain, and the rate of epithelial healing was visualized. as expected, wt mice sealed wounds within 24 hours ( figure 5a ), whereas the ace2 −/− mice still had detectable fluorescein staining at this time ( figure 5a ). three days post wounding, wt mice had clear corneas devoid of fluorescein staining. similarly, the ace2 −/− mice had minimal fluorescein staining 3days post wound; however, there was development of haze prompting further clinical evaluation of corneal clarity 32 ( figure 5b ). day 7 post wounding, wt mice appeared normal, whereas the ace2 −/− mice still had cloudy corneas and patches of fluorescein staining detected on the surface, suggestive of a defective epithelial barrier. one day post wounding, the wt mice had completely re-epithelialized the surface as evidenced by a thin, 1-2 cell layered, well-organized epithelium ( figure 6a,b) . this was contrasted by the ace2 −/− mice, which were characterized by a single, thin layer of disorganized epithelial cells, lacking evidence of stratification ( figure 6c ,d). at day 1 post wounding, the stroma of the wt mice appeared normal consisting of a plywood-like organization of collagen bundles, interspersed with numerous keratocytes. few if any inflammatory cells were noted ( figure 6a,b) . the stroma of the ace2 −/− mice was filled with inflammatory cells (primarily polymorphonuclear cells) and numerous vascular profiles, characteristic of a brisk inflammation ( figure 6c,d) . seven days post wounding, wt mice had a multi-layered, stratified epithelium characteristic of an unwounded mouse ( figure 6e ,f) overlying a normal appearing stroma devoid of inflammatory cells. the corneal epithelium from ace2 −/− mice, 7 days post wounding was variable in appearance. for example, some mice had a markedly thickened multi-layered epithelium ( figure 6g ), whereas some mice had a thin, single-layered epithelium ( figure 6h ). in all cases the ace2 −/− mice, 7 days post wounding, had a stroma with variable amounts of inflammatory cells and vascular remnants ( figure 6g,h) . ace2 −/− mouse eyes (60-80 weeks old (b-d) were taken using a dissecting scope. clear and transparent corneas were observed in aged wt mice eyes (a), while increased central corneal haze with corneal neovascularization were noted in aged ace2 −/− mice eyes (b-d). (e-h) representative h&e stained histological images of wt mouse cornea with intact corneal epithelium and regular stroma (e) and ace2 −/− mouse corneas (f-h) with markedly thickened cornea and a large number of polymorphonuclear, mononuclear, and lymphocytic infiltrates along with numerous vascular profiles. twenty-six aged ace2 −/− mice (60-80 weeks old) were analyzed. about 19 mice developed corneal haze. among the 19 mice with corneal haze, 13 were bilateral and 6 were unilateral. total 32 eyes with cloudy cornea were observed 6 | wang et al. following a perturbation such as wounding, epithelial cells produce pro-inflammatory cytokines, which are known to initiate the inflammatory response. 37 to investigate the mechanism of this initial phase of inflammation in the context of ace2, we knocked-down ace2 in a corneal epithelial cell line (htcepi) and evaluated the expression of several cytokines. knockdown of ace2 in htcepi cells with an sirna smartpool increased the expression of il1a, il1b, f i g u r e 4 aged ace2-deficient mice show infiltration of immune cells. wt (a, c, e) and ace2 −/− (b, d, f) mouse corneas were stained for cd68 (marker for macrophage), cd3 (marker for t cells), and cd11c (marker for dendritic cells). n = 3 tnfa, ccl2, and cxcl8, compared to the sirna control ( figure 7) . to explore whether such an increase in cytokine expression in corneal epithelial cells enhanced recruitment of immune cells, we conducted trans-well chemotaxis assays using a modified boyden chamber co-cultured with the raw-dual macrophage cell line against htcepi cells on the bottom chamber. increased migration of macrophages was observed when ace2 was knocked-down in htcepi cells ( figure s2 ).this suggests that ace2 deficiency was at least in part responsible for the recruitment of macrophages into the inflammatory milieu. the increases observed in ccl2 and tnfa are associated with the recruitment and activation of macrophages. [38] [39] [40] [41] therefore, we explored the possibility that ace2-deficient epithelial cells could activate macrophages. we exposed a pma-stimulated thp-1-dual human monocytic cell line to conditioned media from htcepi cells lacking ace2. such treatment significantly increased the expression of il1a, il1b, il6, tnfa, ccl2, and cxcl8 compared with control htcepi conditioned media ( figure s3 ). these findings indicate that loss of ace2 in htcepi can activate macrophages. to examine if ace2 deficiency-induced cytokine production is due to an increase in angii activity, we investigated whether treatment of corneal epithelial cells with the at1r antagonist, losartan could rescue the phenotype. htcepi deficient in ace2 were treated with 100 µm losartan for 24 hours and the cytokine response was evaluated. losartan treatment decreased the expression of il1a, tnfa, and ccl2 (figure 7 ). this suggests that ace2 deficiency is responsible for the excessive expression of cytokines in the inflammatory milieu via the activation of angii. ras components are found in ocular tissues of various species, as well as humans. the major components including renin, angiotensinogen, and ace2 have been identified in the retina, ciliary body, vitreous fluid, iris, choroid, aqueous fluid, sclera, and conjunctiva 42 and references therein. until recently, little if any attention has been focused on defining ras in the limbus/cornea. [27] [28] [29] we now demonstrate that ace2 is present in both human and mouse limbal and corneal tissues and that the genetic deficiency of ace2 results in a marked inflammatory response in corneal epithelial and losartan treatment can partially reverse pro-inflammatory activity in ace2 depleted corneal epithelial cells. htcepi cells transfected with sicontrol or siace2 were treated with losartan for 24 hours. total rnas were isolated from these cells for rt-qpcr for inflammationrelated genes. n = 8. *p < .05 stromal tissues. moreover, the deficiency of ace2 resulted in marked upregulation of angii, the main peptide that is degraded normally by ace2. as ace2-deficient mice aged, more than 70% developed cloudy corneas in either one or both eyes. this led us to postulate that external stress (eg, scratching or wounding) to the corneal surface resulted in an inflammatory event that could not be resolved due to the inability to regulate angii. to test this concept, we induced gentle debridement of the corneal epithelium, which in wt mice does not result in a significant inflammatory response. 36 in the ace2 −/− , this procedure caused a significant inflammatory response in young mice with otherwise normal appearing corneas (figures 5, 6 ). this strongly supports the idea that ace2 −/− mice are "primed" for an inflammatory response and results in unresolvable inflammation after a mild perturbation. similarly, ace2 −/− mice lungs following an insult resulted in worsened inflammatory cell infiltration and lung damage compared with wt mice; while there were no differences in unperturbed lungs between ace2 −/− and wt mice. 14, 15, 43 in contrast, ace2 deficiency led to increases in inflammation in mouse kidney and aorta even without perturbation. 44, 45 although the lung and cornea are disparate tissues, the similarity in inflammatory responses raises the interesting question of what are the underlying mechanisms driving inflammation. as noted earlier in ace2-deficient mice, cloudy corneas were frequently observed in older (>60 weeks) mice and sometimes only in one eye. this most likely reflects an earlier external trauma. however, it is also possible that cloudy corneas seen in older mice are the result of an enhanced accumulation of reactive oxygen species due to alterations in ras, as it has been shown that ace2-deficient mice exhibit increased oxidative stress. 46 it is well established that with age, the well-developed corneal antioxidant defense systems diminish, leading to ros accumulation and oxidative stress (47 and references therein). this combination of ros and oxidative stress can lead to many age-related ocular diseases of the anterior segment (for reviews see 47, 48) . in other tissues, such as the brain, angii has been shown to induce nadph oxidase-dependent ros production in isolated cerebral microvessels, resulting in cerebrovascular dysregulation. 49 in the ace2-deficient mice, once an inflammatory response is initiated, inflammation persists becoming permanent, which remodels the stromal microenvironment. this microenvironmental change results in a wide range of epithelial phenotypes. the most dramatic is a cell fate switch from the transparent corneal epithelium to a keratinized, stratified squamous, psoriasiform-like epidermis. a similar cell-fate switch was reported in mice deficient in leucine-rich repeats and immunoglobulin-like domains 1 (lrig1). 50 in these mice, loss of lrig1 resulted in a pro-inflammatory state in the cornea through upregulation of stat3. the persistence of the inflammation eventually resulted in the generation of an epithelium that resembled human psoriasis. when ace2 is silenced in a corneal epithelial cell line, interleukins (il1a, il1b, il6), chemokines (ccl2, cxcl8), and tnfa, are all significantly elevated, resulting in a cytokine storm-like phenotype (51 and references therein). we observed that losartan treatment significantly blocked the induction of il1a, tnfa, and ccl2 by ace2 depletion in cell cultures but only slightly altered the expression of il1b and cxcl8, indicating a partial rescue. losartan is one of the angii receptor blockers (arbs). unlike other arbs, the effect of losartan can be overcome by high concentrations of angii, which means that losartan is surmountable. 52 in addition, losartan needs to be converted in the liver to form its active metabolite exp3174, which is 10-40 times more potent than losartan. 53 these two features of losartan may explain why we only see partial rescue in vitro. nonetheless, these findings clearly establish that the recruitment and activation of the macrophage component of the inflammatory response is related to angii acting via the at1 receptor. in the aggregate, our findings suggest that provision of ace2 may provide a new strategy for treating corneal inflammatory conditions, as well as other indications such as kidney disease. of potential clinical significance is the possibility that the ace2 −/− mouse cornea might be an excellent model for investigations into the etiology and treatment of a coronavirus-induced inflammation. 54 the severe acute respiratory system coronavirus (sars-cov), middle east respiratory syndrome coronavirus (mers-cov) and the novel human coronavirus (sars-cov-2) have all been demonstrated to utilize human ace2 as the receptor for entry. 23, 55, 56 in such a scenario, the coronavirus binds to and reduces the expression of ace2; such a reduction increases angii, leading to unregulated inflammation. recently, it was demonstrated that patients infected with sars-cov-2 had interstitial mononuclear inflammatory infiltrates, dominated by lymphocytes in both lungs. 57 as the condition worsened, a dramatic cytokine-storm-driven inflammation was noted. 57 this phenotype bears striking similarities to what we report in the ace2 −/− mice, suggestive that corneal inflammation in ace2 −/− mice may have a similar mechanism with that in sars-cov-2 infected patients. thus, the eye becomes extremely attractive as a model to explore the molecular mechanisms, immunological changes, and treatment modalities in patients with a sars-cov-2 infection for the following reasons: (i) mice cannot be infected with the sars-cov-2 making it difficult to find a direct experimental model; (ii) not having to use the live virus makes the ace2 −/− safer; and (iii) the relative ease of inducing and clinically detecting a corneal ace2-deficient inflammatory response (corneal haze) is extremely convenient for observation. angiotensin-converting enzyme-2 (ace2): comparative modeling of the active site, specificity requirements, and chloride dependence ang ii (angiotensin ii) conversion to angiotensin-(1-7) in the circulation is pop (prolyloligopeptidase)-dependent and ace2 (angiotensin-converting enzyme 2)-independent targeting the degradation of angiotensin ii with recombinant angiotensin-converting enzyme 2: prevention of angiotensin ii-dependent hypertension angiotensin ii: a double-edged sword in inflammation a novel angiotensin-converting enzyme-related carboxypeptidase (ace2) converts angiotensin i to angiotensin 1-9 a human homolog of angiotensin-converting enzyme. cloning and functional expression as a captopril-insensitive carboxypeptidase expression of the sars-cov-2 cell receptor gene ace2 in a wide variety of human tissues tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis altered blood pressure responses and normal cardiac phenotype in ace2-null mice deletion of angiotensin-converting enzyme 2 accelerates pressure overload-induced cardiac dysfunction by increasing local angiotensin ii angiotensin-converting enzyme 2 is an essential regulator of heart function the role of ace2 in cardiovascular physiology angiotensin-converting enzyme 2 gene targeting studies in mice: mixed messages a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung injury angiotensin-converting enzyme 2 protects from severe acute lung failure the anti-inflammatory potential of ace2/angiotensin-(1-7)/mas receptor axis: evidence from basic and clinical research differences in arterial and mixed venous il-6 levels: the lungs as a source of cytokine storm in sepsis cytokine storm of graftversus-host disease: a critical effector role for interleukin-1 current concepts in the diagnosis and management of cytokine release syndrome review: cytokine storm syndrome: looking toward the precision medicine era desiccating stress-induced mmp production and activity worsens wound healing in alkali-burned corneas clinical features of patients infected with 2019 novel coronavirus in wuhan a pneumonia outbreak associated with a new coronavirus of probable bat origin identification of angiotensin converting enzyme 2 in the rodent retina the ocular surface and the coronavirus disease 2019: does a dual "ocular route" exist? strengthening basic and clinical research on ocular infection caused by coronavirus risks posed to corneal transplant recipients by covid-19-affected donors replication competence, and innate immune responses of the coronavirus sars-cov-2 in human respiratory tract and conjunctiva: an analysis in ex-vivo and in-vitro cultures expression analysis of 2019-ncov related ace2 and tmprss2 in eye tissues coronavirus disease 2019 (sars-cov-2) and colonization of ocular tissues and secretions: a systematic review novel variants of angiotensin converting enzyme-2 of shorter molecular size to target the kidney renin angiotensin system comparison of prognostic value of roper hall and dua classification systems in acute ocular burns fih-1 engages novel binding partners to positively influence epithelial proliferation via p63 micrornas-103/107 coordinately regulate macropinocytosis and autophagy microrna-31/ factor-inhibiting hypoxia-inducible factor 1 nexus regulates keratinocyte differentiation wounding the cornea to learn how it heals the corneal wound healing response: cytokine-mediated interaction of the epithelium, stroma, and inflammatory cells the cytokine tnf promotes transcription factor srebp activity and binding to inflammatory genes to activate macrophages and limit tissue repair type i interferons and the cytokine tnf cooperatively reprogram the macrophage epigenome to promote inflammatory activation specific tumor-derived ccl2 mediated by pyruvate kinase m2 in colorectal cancer cells contributes to macrophage recruitment in tumor microenvironment lnmat1 promotes lymphatic metastasis of bladder cancer via ccl2 dependent macrophage recruitment four decdes of ocular renin-angiotensin and kallikrein-kinin systems instillation of particulate matter 2.5 induced acute lung injury and attenuated the injury recovery in ace2 knockout mice deletion of angiotensin-converting enzyme 2 exacerbates renal inflammation and injury in apolipoprotein e-deficient mice through modulation of the nephrin and tnf-alpha-tnfrsf1a signaling genetic ace2 deficiency accentuates vascular inflammation and atherosclerosis in the apoe knockout mouse ace2 deficiency increases nadph-mediated oxidative stress in the kidney the role of the reactive oxygen species and oxidative stress in the pathomechanism of the age-related ocular diseases and other pathologies of the anterior and posterior eye segments in adults mitochondrial dysfunction and oxidative stress in corneal disease angiotensin ii impairs neurovascular coupling in neocortex through nadph oxidase-derived radicals lrig1 inhibits stat3-dependent inflammation to maintain corneal homeostasis into the eye of the cytokine storm angiotensin ii type 1 receptor blockers clinical pharmacokinetics of angiotensin ii (at1) receptor blockers in hypertension is there an association between covid-19 mortality and the renin-angiotensin system-a call for epidemiologic investigations molecular pathology of emerging coronavirus infections soluble angiotensin-converting enzyme 2: a potential approach for coronavirus infection therapy pathological findings of covid-19 associated with acute respiratory distress syndrome we are thankful to amani a. fawzi for her initial clinical evaluation of ace2 ko eyes. the nu-sbdrc skin tissue engineering and morphology core facility assisted in morphologic analysis. the nu-sbdrc is supported by the national institute of arthritis and musculoskeletal and skin diseases grant ar075049. this research is supported by national institutes of health grants ey06769, ey017539, and ey019463 (to rml); a dermatology foundation research grant and career development award (to hp); an eversight research grant (to hp), ar064144 and ar071168 (to kql), an niddk grant r01dk104785 (to db), and the international postdoctoral exchange fellowship program key: cord-021413-1ht1xm88 authors: kraft, lisbeth m. title: viral diseases of the digestive system date: 2013-10-21 journal: diseases doi: 10.1016/b978-0-12-262502-2.50016-x sha: doc_id: 21413 cord_uid: 1ht1xm88 this chapter discusses three virus infections affecting the digestive system of mice and their properties: (1) epizootic diarrhea of infant mice (edim), (2) reovirus 3 infection, and (3) murine hepatitis virus infection (mhv). all three infections may cause serious, debilitating, and sometimes fatal diarrheal disease in nursling and weanling mice. mice of all ages can be infected by the edim virus but overt disease is restricted to animals up to about 12–13 days of age at the time of first exposure. the edim virus is worldwide in distribution. its prevalence is difficult to estimate because serologic tests have not been readily available, and it is not customary to sacrifice animals for the purpose of examining the appearance of their intestinal tract or for electron microscopic visualization of fecal contents. the acute disease of reovirus 3 infection affects mainly sucklings and weanlings, whereas the chronic disease is encountered in animals over 28 days of age. the mhv virus, on the other hand, has been found to affect cotton rats, rats, and hamsters. epidemic diarrhea of infant mice, it is common knowledge that virtually every colony of conventional mice suffered that infec tion to a greater or lesser extent. in recent years, with the advent of measures such as cesa rean derivation, barrier-sustained breeding and maintenance procedures, routine serologic surveillance, laminar flow hoods or rooms, and filter-top cages, the problems associated with these diseases have become somewhat less critical. neverthe less, they are at times and under certain circumstances still troublesome. all three infections may cause serious, debilitating, and sometimes fatal diarrheal disease in nursling and weanling mice. thus, the economic impact on commercial mouse col onies can be severe. furthermore, newborn mice, pooled from many dams and then redistributed to them at random, are used for the isolation and identification of certain viruses in diagnos tic and epidemiological studies, for example arboviruses (shope, 1980) , coronaviruses , and reoviruses (stanley, 1977) . clearly, indigenous infection with related or identical agents in only a few such infants could confound and compromise the validity of observations follow ing the inoculation of test materials. it should be pointed out, too, that collins and parker (1972) demonstrated both reovirus 3 and murine hepatitis virus as contaminants in some murine leukemia and transplantable tumor specimens. whereas it is mainly from these standpoints that the diseases in question derive importance, they should not be dismissed without considering their intrinsic value as models for elucidat ing the pathogenesis and control of related infections in man and other animals as well as their utility for the molecular biologist. cheever and mueller (1947, 1948) , pappenheimer and en ders (1947) , and pappenheimer and cheever (1948) were the first to describe the pathological changes and epidemiology of edim. runner and palm (1953) and cheever (1956) also con tributed to knowledge concerning the epidemiology and etiol ogy of the disease. thereafter kraft (1957 kraft ( , 1958 kraft ( ,1961 kraft ( ,1962b kraft ( , 1966 reported on studies regarding the etiology, mode of transmission, carrier state, immune response, pathogenesis, and control of the disease. serologic studies were also under taken by blackwell et al (1966) . kraft (1963, 1967) first demonstrated the agent in electron micrographs of infected infant mouse intestinal epithelium. banfield et al (1968) enlarged on those findings, comparing the virions to those of the reoviruses. particles of similar morphology were subsequently observed in the diarrheal feces of many species, primarily in the young: cattle (mebus et al, 1969) , man (flewett et al, 191 a) , horse (flewett et al, 1975) , pig (rodger et al, 1975) , sheep , rabbit , deer (tzipori et al, 1976) , goat (scott et al, 1978) , and dog (england and poston, 1980) . in addition, one virus, sa-11, was isolated from a nondiarrheal monkey, and another, oa (offal agent), was recovered from intestinal washings of sheep in an abattoir (els and lecatsas, 1972) . much and zajac (1972) purified edim virus from diarrheal infant mice and further characterized it. based on its moφhology and other known characters, edim virus has been placed into the genus rotavirus in the family reoviridae (matthews, 1979) . during the past decade, knowl edge of the rotaviruses as agents of diarrheal disease of young mammals has burgeoned, especially with regard to those af fecting children, calves, and piglets. reviews concerning the genus have been published by wyatt et al (1978 ), mcnulty (1978 , flewett and woode (1978) , andrewes et al (1978) , and holmes (1979) . additional comments may be found in the american veterinary medical association panel the young (anonymous, 1978) . the classification of edim virus in the genus rotavirus, family roeviridae, derives from its moφhology and mode of replication as seen in electron micrographs (adams and kraft, 1967; banfield et al, 1968) , which were later shown to re semble those observed in ultrathin sections in human intestinal material containing ''reovirus-like" particles (bishop et al, 1973) . the term rotavirus (l. rota, wheel) was proposed by flewett et al (1974) because of its moφhology as viewed in negative-contrast electron micrographs. it has become widely used in preference to duovirus, which is synonymous (david son et al, 1975) . there is no evidence that antigenic or pathogenetic variants of edim virus exist. analysis of rna segment and struc tural polypeptide variation, however, as has been accom plished for other rotaviruses derbyshire and woode, 1978; rodger and holmes, 1979) , may reveal differences among strains. further, several serotypes have been described a m o n g the h u m a n rotaviruses by m e a n s of the serum neutralization test (beards et al., 1980) . this technique may also prove useful in future studies of e d i m virus strains. a. morphology, size, and composition. kraft (1962b) , using millipore filters, determined the size of the infective e d i m virion to be between 33 and 100 n m . in electron micro graphs, a d a m s and kraft (1967) mined the m e a n diameter of 100 virions of purified e d i m virus to be 5 4 . 4 ± 2 n m . o n the other h a n d , melnick (1979) gives about 50% is lost at 4x for 24 hr or at 37t for 1 hr. although some infectivity (>0.05%) remains at either 56°c or 60°c for 30 min, it is abolished at 70t for 15 min (cheever and muel ler, 1947; kraft, 1957 kraft, , 1962b . much and zajac (1972) found that the purified virus is unstable at both 4° and -24''c for 2 weeks but found that at -70°c infectivity is retained for at least 4 weeks. c. ejfect of chemicals. in an intestinal filtrate, edim virus titer is not significantly reduced when held in ether or 0.1% sodium deoxycholate at 4°c for 24 hr (kraft, 1962b) . according to much and zajac (1972) , the purified virus is stable in 20% ether, 5% chloroform, or 0.1% sodium deoxycholate at 4°c for 1 hr and, as is true for other rotaviruses, is resistant to pancreatin. ward and ashley (1980a,b) examined the effects of the anionic detergent sodium dodecyl sulfate and of the chelating agent ethylenediaminetetraacetate on purified simian virus and determined that low concentrations and mild temper ature conditions readily inactivated the agent. both chemicals modified the viral capsid to prevent adsoφtion of the inacti vated virions to cells. indeed, this study was the outgrowth of a need to determine the survival of enteric viruses in wastewater. it had been determined that wastewater sludge reduced the heat necessary for simian rotavirus inactivation. ionic detergents in the sludge were identified as the active components. nonionic detergents did not destabilize the virus; further, these com pounds protected the virus from the destabilizing effect of sodium dodecyl sulfate. destabilization by both cationic and anionic detergents was found to be dependent on the ph of the medium. a systematic study of the stability of edim virus to extremes of ph has not been reported. much and zajac (1972) kraft (1966) replication of edim virus occurs in epithelial cells of the villi of the small intestine. in electron micrographs, kraft (1967), banfield etal. (1968) , and holmes etal. (1975) demonstrated replication by budding in distended cistemae of the endoplasmic reticulum. the significance of intracytoplas mic and intranuclear tubular structures encountered is not clear. using direct immunofluorescence, wilsnack et al. (1969) found that edim virus antigen in infant mice is limited to the cytoplasm of the villous epithelium from the duodenum to the colon and is detectable within 48 hr after peroral inoculation. stainable virus was also seen in the intestines of contact infant mice without necessarily causing clinical signs to appear. the virus could not be stained in the stomach or liver of mice infected either naturally or experimentally. kraft (1958) studied the distribution of infectious edim virus in 3-day-old mice following peroral inoculation. at 3 hr, virus was detected in the stomach and small and large intes tines (the cecum was not tested) and could not be recovered from the lungs, liver, spleen, kidney, or blood. at 22 hr, all those tissues were positive except for the kidney. the bladder and urine, as well as the brain, were also devoid of the agent, but at 30 hr, these too were positive. at 72 hr, the liver, spleen, kidney, and intestines yielded virus, but the brain was negative. the blood, stomach, lungs, bladder, and urine were not tested at that interval. at 6 days, blood, liver, and intestines, the only tissues examined, still contained virus. ingested virus from a diarrheal litter brings about an active infection in the nursing dam that previously had been nondiarrheal herself and had had only nondiarrheal litters (kraft, 1958) . blood, liver, spleen, and feces all contain infectious virus in the dam 1 week after her litter is given virus perorally as an intestinal filtrate. later, kraft (1961) determined that adult male mice can also be intestinal carriers for at least 17 days after a single peroral exposure. with regard to the mechanism of cell penetration. holmes et al. (1976) proposed that lactase of the villous brush border of the intestine may be the receptor that uncoats the rotavirus virion by attacking the glycolysated polypeptides of the outer capsid. in addition, pancreatic enzymes within the lumen may also be instrumental in the infectious process (see section ii,b,6). the question of cofactors that might enhance pathogenicity or of interfering substances, in addition to antibodies, that might inhibit edim virus replication is open. kraft (1958) found increased numbers of clostridium tertium in the intes tine of diarrheal animals, and pappenheimer and enders (1947) remarked on the persistent presence of ''coccoid bodies" in the intestines of diarrheal animals as seen by light microscopy. in both instances, these may be considered opportunistic or ganisms. their effect on the severity of the clinical disease is unknown, however. one report (labonnardiere and devaurieux, 1979) the use of standard methods to grow edim virus in tissue culture has resulted for the most part in failure. habermann (1959) (thiel et al., 1978) . further, babiuk et al. (1977) and al meida et al. (1978) state that calf rotavirus production is mark edly enhanced when passaged in the presence of trypsin. clark et al. (1979) produced high-titer calf rotavirus in a variety of continuous cell lines, also by utilizing trypsin, and matsuno et al. (1977) were successful in producing plaques with bovine rotavirus in monkey kidney cell monolayers when trypsin was incorporated in the overlay medium. estes and graham (1979) found that simian virus titers were enhanced by both trypsin and elastase in vero as well as ma 104 cells. ramia and sattar (1980) studied additional proteolytic enzymes utilizing plaque formation by simian virus sa-11 in ma 104 cells as the endpoint. elastase, a-chymotrypsin, subtilase, pronase, and pan creatin were as effective as trypsin, whereas pepsin, papain, and thermolysin were ineffective. another approach with human rotavirus has been to incoφorate the agent into cells by means of low-speed centrifugation of the two together (banatvala et al., 1975; bryden et al., 1977; schoub et al., 1979; wyatt et al., 1980) . it remains to be seen if similar treatments will lead to suc cessful cultivation of edim virus in tissue culture. chick embryos appeared unaffected when the chorioallantois was inoculated. the agent did survive up to 5 days, but an increase in infectious titer could not be demonstrated (kraft, 1958) . moon (1978) of each disease appears to be reflected not only in the particular group of cells but also in the numbers of host cells involved. lucid descriptions of the natural clinical disease are given by cheever and mueller(1947) , seamer (1967) , and mcclure et al. (1978) . the experimental disease does not differ signifi cantly (kraft, 1957 (kraft, , 1962b . overt illness is confined to pretypical appearance of infant mice infected with enterotropic mhv (pair of mice at left) or rotavirus (pair at right) compared with normal mice of the same age (center pair). note the evidence of inanition and dehydration in the mhv-infected animals, whereas those infected with rotavirus show mainly a somewhat prominent abdomen and only a slightly smaller size than the normal control mice. weanling mice. t h e first signs usually appear at 7 -8 days of age. in mild cases diarrhea is manifested by a minimal amount of pasty fecal material about the perineum. in severe cases the amount is copious, the entire infant becoming soiled. rectal impaction may occur at about 1 2 -1 6 days of a g e , and death can ensue if the impacted mass is not removed spontaneously or deliberately. death does not seem to be the result of the virus infection per se, but rather as a consequence of protracted obstipation. in pure e d i m virus infections, mice continue to nurse throughout their illness (in the absence of impaction). they may be slightly stunted (fig. 2 ), but those with mild cases soon attain the weight of their nondiarrheal peers. especially when diarrhea is mild, morbidity in the natural disease m a y be difficult to ascertain, but one could quite cor rectly assume that, if diarrheal signs are observed in a few litters, it is likely that all infants in a colony will sooner or later be affected. concerning mortality, cheever and mueller (1947) state that there was both 100% recovery and 100% lethality in their outbreaks. based on present k n o w l e d g e , the first is suggestive of e d i m virus infection and the latter of another disease, perhaps murine (mouse) hepatitis. at no time do a d u h mice exhibit signs of illness ascribable to e d i m virus infection. in the experimentally induced dis ease, the absence of external clinical signs cannot be relied upon for a negative diagnosis (kraft, 1957) . necropsy of each animal is essential. in this w a y , the appearance of the colonic contents can be observed, which in normal infant mice are burnt orange in color and semisolid but formed in consistency. t h e colon is not distended. in diarrheal m i c e , however, even in those with no external soiling, the colonic contents are fluid or m u c o i d , bright lemon yellow to amber, or gray-green, with no formed feces in evi dence. g a s is often seen in the colon and c e c u m , which are distended. t h e stomach, t o o , is distended with curdled milk (except in terminal cases with anal impaction). all other or gans appear normal. gross change is not seen in the intestines of adults exposed perorally. it is noteworthy that in germ-free infant m i c e , ex perimental e d i m virus infection appears in the gross as it does in conventional mice (kraft, 1966) . the histologic picture has been confirmed by others (adams and kraft, 1967; mcclure et al, 1978) . there is no inflam matory reaction in the intestines. inclusions may indeed be found in enterocytes near the tips of villi, especially in the jejunum, and infrequently in the sloughed cells seen in the lumen. epithelial cells near and at the tips of villi are fre quently vacuolated. acres and babiuk (1978) have pointed out that in all species studied, the rotaviruses cause diarrhea by attacking and de stroying the columnar epithelium of the small intestine. middleton (1978) considers that cell migration from the crypts is speeded in response to diarrhea and that the infected cells are relatively immature. enzyme levels, high thymidine kinase and low sucrase, are similar in such cells to those of normal crypt cells and thus support this view. the microscopic appearance belies the severity of the gross and clinical findings of edim. in this regard, d-xylose absorp tion has been studied in calves in which 60-90% reduction occurred during the acute illness with the calf rotavirus . aberrations in sodium transport have also been investigated in other animals and in humans (middleton, 1978) , but such studies have not been carried out in mice using the mouse agent. cheever and mueller (1947) and kraft (1957) demonstrated that the agent is transmissible perorally. kraft (1957) showed further that dissemination in a mouse colony is mediated prin cipally by the airborne route. kraft (1961) examined the neutralization test in edim and found that antibodies were not always formed following infec tion, and when they were present, they tended to be low in titer. sera from adult mice having had lifelong contact with the agent (in the form of consistently diarrheal litters) were more apt to neutralize the virus than those of mice exposed for the first time as adults. on the other hand, hyperimmunization of mice has resulted in the production of significant serum titers of both complement-fixing and neutralizing antibodies (blackwell etal, 1966) . antibody response measured by other serologic tests has not been investigated for edim, nor have immune substances in lacteal secretions been measured directly. suggestive of their presence, however, are data showing that infants of primiparae who themselves had been diarrheal in infancy resisted about 10 id50 more of edim virus than did offspring of previously nondiarrheal dams (kraft, 1961) . antirotaviral immunoglobu lins have been found in both colostrum and milk in man (yolken et al, 1978c; simhon and mata, 1978; thouless et al, 1977a) , bovines (acres and babiuk, 1978) , and lambs (snodgrass and wells, 1976) . indeed, local immunity afforded by lacteal immune substances is regarded as crucial for protection against any intestinal infection in the young (welliver and ogra, 1978; snodgrass and wells, 1976 as indicated, mice of all ages can be infected, but overt disease is restricted to animals up to about 12-13 days of age at the time of first exposure. there seems to be no predilection for a particular sex. the observation that first litters are more apt to show copious and protracted soiling than those of later parity has been reported by cheever and mueller (1947, 1948) and by runner and palm (1953 edim virus is probably worldwide in distribution. whether it occurs in wild mouse populations is unknown. its prevalence is difficult to estimate, since serologic tests have not been readily available, and it is not customary to sacrifice animals for the purpose of examining the appearance of their intestinal tract or for electron microscopic visualization of fecal contents. as has been shown experimentally, latent carriers can exist. the carrier rate in a diarrheal colony is not known, nor is the frequency of viral shedding under colony conditions. cheever and mueller (1948) examined seasonal variations in the weaning percentage in their mouse strains and found that there was a significant effect in only the cfw mice. they experienced the lowest weaning rate in the late fall and winter. runner and palm (1953) , studying c3h mice, indicated that there was a higher incidence of diarrhea in december/january (kraft, 1961; blackwell et al., 1966) , complement fixation (wilsnack et al., 1969; kapikian et al, 1976; thouless et al., 1977b) , direct immunofluorescent staining or precipitin (wilsnack et al., 1969; spence et al., 1975; foster α/., 1975; peterson α/., 1976) , immune electron microscopy (kapikian et al., 1974; bridger and woode, 1975) , immunoelectroosmophoresis (tufvesson and johnsson, 1976; middleton et al., 1976) , enzyme-linked im munosorbent assay (elisa) (scherrer and bernard, 1977; el lens etal., 1978; yolken etal., 1978a,b,c) , radioimmunoas say (acres and babiuk, 1978; kalica et al., 1977; middleton et al., 1977) , immunodiffusion (woode et al., 1976) , hemagglutination inhibition (fauvel et al., 1978) , enzymelinked fluorescence assay (elisa) (yolken and stopa, 1979) , an unlabeled soluble enzyme peroxidase-antiperoxidase method , plaque reduction test (estes and graham, 1980) , serologic trapping on antibody-coated electron microscope grids (nicolaieff et al., 1980) , a solid phase system (space, solid phase aggregation of coupled erythrocytes) for detection of rotaviruses in feces (bradbume et al., 1979) , and immune electron microscopy with serum in agar diffusion (lamontagne et al., 1980) . more recently, sheridan and aurelian (1981) have described an elisa test for edim virus which should prove beneficial for both practical (serologic) purposes and for investigations of the antigenic structure of the virion. in the absence of a reliable tissue culture system, edim virus isolation is generally impractical. bacteria-free filtrates of intestinal suspensions can, of course, be given to diarrheafree animals (gnotobiotes or axenics), but this is expensive and inefficient. on the other hand, such filtrates may be concen trated by ultracentrifugation and examined in the electron mi croscope for characteristic virus particles flewett, 1978) . the particles may also be identified by immune electron microscopy. a practical approach to a presumptive diagnosis would be to kill selected animals in order to examine the appearance of the colonic contents. the inclusion of sentinel dams with litters in a breeding colony should be considered. these might be sac rificed at intervals to determine the presence of edim in the colony. together with the clinical history of the mouse colony, this practice may provide a fairly reliable, although not pathognomonic, indication of the presence of edim. his topathologic examination could also be of value. based on experimental results, kraft et al. (1964) proposed the use of air-filter devices, essentially dust caps for each cage, for the practical control of airborne transmission of edim in a commercial mouse colony. it was subsequently shown (kraft, 1966 ) that 43% of first litters and 79% of all other litter parities were weaned from cages without filters, whereas in cages pro vided with filters, weaning percentages were 96 and 99%, respectively, during the same observation period. it should be pointed out that both edim and mouse hepatitis virus (livim) were present simultaneously in that colony. although the filter devices did not eliminate the disease(s), they probably reduced the pathogenic microbial load in the immediate environment of the susceptible animals, but the precise mechanism by which this control method succeeds to the extent that it does is obscure. since that time (1964), various devices based on a filter cage or filter-top design have been utilized. some of these are de scribed by simmons and brick (1970) . woods et al. (1974) evaluated them from the viewpoint of environmental factors within the cages. they found that dry bulb temperature dif ferentials, comparing environments inside and outside the cage, were not significantly different (about 2°c) between fil tered and unfiltered cages, but that dew point differentials were significantly greater in the filtered cages (about 5°c) than in unfiltered cages (about 3°c). however, they concluded that a suitable cage size for a particular species and number of ani mals could compensate for the higher wet bulb readings under filters to maintain acceptable conditions for the animals. cur rently, several types of filter covers or bonnets are available commercially. vaccination as a method of control has not been attempted. judging from the lack of reports to the contrary, caesarian derivation together with barrier maintenance apparently elimi nates and controls the infection. kunstyf (1962) attempted to control edim by decontamina tion of air by the use of triethylene glycol but was unable to do so. the use of antibiotics, too, is without value, although there seems to be amelioration of signs for a short period as secon dary organisms are temporarily reduced in number. as indicated by kraft (1966) , it may be relatively easy to establish a colony of mice free from edim virus infection. this may be accomplished by eliminating those breeding pairs whose first litter is diarrheal. filter devices are required for this, and they and the animals must be handled with the aid of a transfer or laminar flow hood using sterile techniques during observation and handling of the animals. the method is suita ble for small colonies, but it is impractical for commerce where caesarian derivation and barrier maintenance are the methods of choice. reovirus 3 was first isolated from the feces of an australian child manifesting a cough, fever, vomiting, hypertrophic tonsils, and bilateral bronchopneumonia. it was named hepatoencephalomyelitis virus by stanley et al. (1953) , who originally recovered the agent. sabin (1959) proposed the name reovirus for a group of agents associated with the respi ratory and enteric tracts of humans. they were found to be ether resistant, about 70 nm in size by membrane filtration but of unknown shape, and caused distinctive cytopathic effects in monkey kidney tissue cultures. one of the echo group of viruses, echo 10, now synonymous with reovirus, became a member of the new group on that basis. (echo is the acronym for enteric cytopathic /zuman 6>φhan, agents isolated in tissue culture from asymptomatic humans-so-called viruses in search of disease.) stanley (1961) then demonstrated that the hepatoencephalomyelitis virus was serologically identical to reovirus 3. additional strains have since been recovered from humans, other mammals, marsupials, birds, insects, and reptiles (for review, see stanley, 1974) , and from mouusks (meyers, 1979; meyers and hirai, 1980) . reoviruses have been divided into three serotypes on the basis of hemagglutination inhibition and neutralization tests (sabin, 1959; rosen, 1960) . reovirus 3 was established as an indigenous murine virus by hartley et al. (1961) and by cook (1963) . reviews concerning the biological and clinical aspects of disease caused by this agent have been prepared (stanley, 1974 (stanley, , 1977 . when gomatos et al. (1962) and discovered that reoviruses possess double-stranded rna, a unique characteristic among viruses, molecular biologists were inspired to study them in minute detail. currently, knowledge of reovirus replication on biochemical and biophysical planes is as extensive as that available for any other virus and is, for the most part, outside the scope of this chapter. interested readers are therefore referred to reviews by shatkin (1969) and joklik (1974) for extended literature coverage and detailed dis cussion and to andrewes et al. (1978) for a condensed over view. or reovirus, in the family reoviridae (melnick, 1979) . wild-type strains of reovirus 3 include: dearing, the pro totype strain (sabin, 1959) , isolated from a child with diarrhea; abney (rosen, 1960) , isolated from a child with a febrile upper respiratory infection; can 230, from a case of burkitt's lymphoma (bell et al., 1964) ; and several strains obtained from naturally infected cattle (rosen, 1960) . mutant, temperature-sensitive (ts) strains have been developed in the laboratory (fields and joklik, 1969) and have been used for studying the synthesis of viral rna and peptides (cross and fields, 1972; as well as for examining problems of pathogenesis. a neurotropic strain was also de-rived from the m o r e hepatotropic original isolate (stanley et al, 1954) . t h e reovirus 3 virion (fig. 3) has a m e a n diameter of about 6 0 -7 6 n m and is icosahedral in shape, with 5:3:2 symmetry. particles have a core, an inner layer or shell containing a n u m b e r of capsomers formalin at 56°c. ether treatment was ineffective. rozee and leers (1967) determined that although cholorform does not affect infectivity, it does destroy the hemagglutinin. wallis et al. (1964) found that mg^^ enhances the titer of reovirus at 50°c. the infective titer increased four to eight times by heat ing for 5-15 min in 2 μ mgcl2. other divalent cations and nacl were ineffective. hemagglutinin was not affected. it is thought that the high temperature and mg^+ caused activation of reovirus particles that were inactive in the original prepa rations. as with rotavirus. ward and ashley (1978) found that reovirus was sensitive to anionic detergents in wastewater sludge, i.e., these chemicals decreased the temperature needed to inactivate the virus. cationic detergents were more active than anionic, and nonionic detergents were inactive in decreas ing reovirus thermal stability. mutagens (nitrous acid, nitrosoguanidine, and proflavine) have been applied to reovirus 3 (fields and joklik, 1969) . the resulting mutants are of interest not only to the molecular biologist but to the clinical virologist as well, since some of them produce altered disease pictures (fields and raine, 1972) . for example, when inoculated into rats, wild-type virus produced a necrotizing encephalitis, whereas a mutant gave rise to a slowly progressive communicating hydrocephalus. the effects of enzymes are described below (see section iii,b,5). in phosphate-citrate buffers, stanley et al. (1953) ascertained that the virus is stable between ph 2.2 and 8.0. e. antigenic determinants. sabin (1959) demonstrated that the mammalian reoviruses known at that time could be divided into three serologic groups by neutralization tests. they could also be differentiated by hemagglutination inhibi tion (rosen, 1960 ), hull et al. (1956 having discovered that echo 10 virus possessed a hemagglutinin for human o eryth rocytes. later, reported that reovirus 3, but not reovirus 1 or 2, agglutinated ox erythro cytes. the reovirus 3 hemagglutinin was inhibited by nonspecific substances such as normal mouse, rabbit, or rat serum, and by vibrio cholerae filtrate. weiner et al. (1978) were able to show that the 57 rna segment, which is associated with type specificity, encodes the polypeptide that determines the hemagglutinating properties of the virion. complement-fixing antigens were prepared by stanley et al. (1953, 1954) and by j. c. parker et al. (1965, 1966) . these are group specific. leers et al. (1968) determined that reovirions display at least one type-specific and one to two group-specific antigens when studied by immunodiffusion. with the more sensitive im munoelectrophoresis technique, however, these authors en countered two type-specific and four group-specific precipitin lines. the agent is regarded as pantropic in mice. in neonates, stanley et al. (1953) (wolf et al., 1981) . papadimitriou (1965 papadimitriou ( , 1966 papadimitriou ( , 1968 also studied viral replica tion by electron microscopy in the mucosa of the common bile duct and in the liver. reovirus 3 has been reported to be oncolytic for a mouse ascites tumor by bennette (1960) , bennette et al. (1967,a,b) , and nelson and tamowski (1960) . most of the studies dealing with replication of the reoviruses have been accomplished in tissue culture, frequently in plaque assays in l-cell monolayers. other cells that have been suc cessfully employed are primary kidney monolayers of rhesus, patas, and capuchin monkeys, as well as those of pigs, cats, and dogs. continuous lines, such as fl human amnion, bs-c-1, and kb, have also been used (hsiung, 1958; cook, 1963; mcclain etal., 1967; rhim and melnick, 1961; harford et al., 1962) . harford et al. (1962) (1973a,b,c, 1974, 1979) . stanley et al. (1953, 1954) reported the development of pocks on the chorioallantois of 12-day-old chick embryos in oculated with infectious brain and liver. the embryos appeared unaffected, and with succeeding passages, the pocks could no longer be observed, although oral inoculation of suckling mice with chorioallantoic suspensions resulted in active disease. essentially the same results were found following amniotic inoculation. the experimental and natural diseases appear identical ex cept for variations in intensity of signs, perhaps due to dif ferences in infectious dose. up to 16 days after intraperitoneal inoculation, the mice appear emaciated and uncoordinated. the hair is oily and matted-the so-called oily hair effect (ohe)-an effect that can be demonstrated in contact animáis as well. however, in these mice it disappears as soon as the diseased animal is removed from the healthy ones. the effect was ultimately traced to a high proportion of fat in the intesti nal contents (steatorrhea): 12.9% in infected animals as com pared with 4.6% in normal mice. feces may contain as much as 29% fat in infected mice (stanley et al., 1953 (stanley et al., , 1954 . the thymus and other organs appeared normal. in the central nervous system, neuronal degeneration began about the ninth day and was most prominent in the brain stem and cerebral hemispheres. by the tenth day, perivascular cuf fing as well as neuronal satellitosis was evident. the meninges were infiltrated with round cells and netrophilic leukocytes. by the fourteenth day encephalitis was severe and widespread, with small hemorrhages occurring in necrotic regions. in suckling rats inoculated intracerebrally with reovirus types 1, 2, or 3, viral cytoplasmic inclusions and intranuclear bodies corresponding to cowdry type β inclusions have been ob served (margolis et al., 1975) . the latter were seen in cells that were free from intracytoplasmic inclusions; they were readily found in weanlings, were unassociated with inflam and their microvilli swollen. the common bile duct is dilated, and the ampullary region becomes obstructed with debris. it is this obstruction that leads to both hepatic and pancreatic dys function, for after studying the pancreatic lesions further, papadimitriou and walters (1967) concluded that, even though virions were seen in pancreatic acinar cells, the principal cause of acinar degeneration is ductal obstruction. based on the neutralization test, determined that the si genome segment is linked to type speci ficity, and finberg et al (1979) found that the same genome segment is also responsible for the production of cytolytic τ lymphocytes after reovirus infection. tytell et al (1967) , working with reovirus 3 rna, found that it was highly active in inducing interferon in rabbits and tissue culture, and lai and joklik (1973) showed that coreless virions as well as those lacking the outer capsid shell induce no interferon. the question of the role of interferon in protection of mice from either the acute or chronic infection remains an intriguing problem. in an effort to permit a precise definition of the host cellular immune response to viral antigens, weiner et al (1980b) and greene and weiner (1980) which also determined serotype-specific humoral (neutraliz ing) and cytolytic t-cell responses. it is clear that the agent can be transmitted by the oral route as well as by parenteral inoculation. mice respond to the natural infection with neutralizing, hemagglutination-inhibiting, and complement-fixing an tibodies. as indicated earlier (section iii,b,3,^), precipitating antibodies can also be demonstrated. there is no evi dence that any mouse strain is more or less susceptible to reovirus 3 infection than any other, provided the animals come from a colony that is free of the infection. the host range is broad. stanley (1974) cited at least 60 species that may be infected with reoviruses, and, as men tioned above, it is thought that the prevalence of antibodies in otherwise normal mammals is related to this fact and that mosquitoes or other insects may be operational in the spread of the infection. the acute disease affects mainly sucklings and weanlings, whereas the chronic disease is encountered in animals over 28 days of age. there is no indica tion that either sex is more or less susceptible than the other. in an epizootic described by cook (1963) , 130 of 800 first litters were affected. later-parity litters were involved hardly at all. in view of the absent or low complement-fixing antibody titers in the presence of significant hemagglutination-inhibiting titers that follow natural infection, prevalence estimation by immunologic means may be difficult to assess. the data cited by parker et al. (1966) , 82% of 34 colonies positive, and by descoteaux et al. (1977) , 100% of five colonies posifive, may be typical incidences for conventional mouse colonies. as already indicated, reovirus 3 infection is regarded as worldwide in distribution. this aspect of reovirus 3 infection has been covered above (section iii,b,l,2a,¿?). munoperoxidase method (enzyme-labeled antibody) has been employed for reovirus 1 (ubertini et al., 1971 ) and may find application for reovirus 3 diagnosis as well. although ohe may not be absolutely pathognomonic for reovirus 3 infection, it seems distinctive enough so that a provisional diagnosis may be made when it is seen. stronger evidence is afforded if the animals are also jaundiced and wasted. necropsy coupled with histopathologic examination is never a mistake and is to be encouraged. the placement of sentinel animals at strategic locations in an animal colony should be considered. such animals may be regarded as expendable for sacrific, necropsy, and virus isolation as well as for the acquisition of serum for antibody determinations. there is no evidence that epizootics occur preferentially at a particular time of the year. j. c. parker et al. (1965, 1966) discussed the serologic diagnosis of reovirus 3 infection, concluding that for these puφoses the hemagglutination inhibition test was the most reliable. in preparing type-specific antisera for standardizafion and controls, behbehani et al. (1966) found that the ubiquity of inhibitory substances (antibodies included) in most mamma lian species precluded accurate work. they therefore used domestic geese, in which virtually no hemagglutinafion inhibi tion or neutralizing antibodies could be detected prior to im munization. in any event, for routine surveillance, the hemagglutination inhibition test is currently utilized. comments similar to those expressed for edim virus recov ery and visualization apply. although it is possible to perform these procedures, they are inefficient for routine diagnostic puφoses. stanley (1977) has outlined methods for this pur pose. in brief, infectious material can be inoculated into tissue cultures (primary rhesus monkey or human kidney) or newbom mice (from reovirus 3 free colonies!), or the material may be subjected to immunofluorescent methods. an imcesarean derivation and barrier maintenance are believed to be suitable techniques for control and prevention of reovirus 3 infection. although no experimental evidence has been found, it is possible that the use of filter devices in conventional colonies might also be helpful in preventing the spread of infection. in the absence of information on the vertical trans mission of the agent, it is impossible to evaluate the influence of that route on successful control of the endemic disease. although therapy is impractical from the standpoint of con trolling epizootics of reovirus infection, it is nonetheless of considerable interest that willey and ushijima (1980) found that thymosin given intraperitoneally to 7-day-old mice that had been neonatally infected with reovirus 2 (2 ld50) signifi cantly increased their mean survival time, provided it was ad ministered at 2200 hr. when given at 0800 hr, significantly increased survival time was not observed, but when inoculated at 1200 hr, there was an apparent decrease in mean survival time. reviews on the subject include those by piazza (1969) , mcintosh (1977) , kapikian (1975) , andrewes et al (1978) , holmes (1979) , garwes (1979) , and robb and bond (1980) . based on their own electron microscopic studies and on those of david-ferreira and manaker (1965) , becker et al (1967) considered that mhv might be an 'tbv-like" (infecti ous bronchitis viruslike) agent. virions of similar moφhology seen in electron micrographs and also ether sensitive, as is ibv, were being isolated at that time from human cases of colds (almeida and tyrell, 1967) . the following year, the term coronavirus was proposed for the group (tyrell et al, 1968) , and in 1975 the coronaviridae became an official fam ily with a single genus, coronavirus (tyrell etal, 1975 additional agents may be added to this group, e.g., ''runde" virus (traavik et al, 1977) , a coronavirus causing car diomyopathy in rabbits (small et al, 1979) . as noted, many strains of mhv have been recovered from mice under various circumstances. in addition to jhm, these include: mhvl, from "white mice" (p or parkes strain), dur ing attempts to adapt human hepatitis virus to animals (the strain was originally isolated as a dual agent consisting of the virus and a protozoon parasite, eperythrozoon coccoides (gledhill and andrewes, 1951) ; mhv2, from mice used to propagate murine leukemia virus (nelson, 1952a,b) ; mhv3, also found during studies on adaptation of human hepatitis virus to mice (dick et al, 1956) ; mhv-b (ehf-120), from mice used for human epidemic hemorrhagic fever (hehf) adaptation attempts (buescher, 1952) ; an unnamed strain from mice undergoing murine leukemia chemotherapy trials (braunsteiner and friend, 1954) ; h747, following intracere bral inoculation of suckling mice with hehf materials (morris, 1959) ; mhv-a59, during transfer of moloney leukemia virus in mice (manaker et al, 1961) ; mhv-s, from cesareanderived mice that had been barrier-maintained before exposure to conventional mice ; mhv-c (mhv-balb/c), isolated during passage of spleens from leukemic mice (nelson, 1955) ; four addiiional strains from spleens of leukemic mice or from natural outbreaks (mhv-srl, -sr2, -sr3, -sr4) (nelson, 1963 (nelson, , 1965 ; lethal intestinal virus of infant mice (livim), from infant mice dying of a spontaneous infection (kraft, 1962a) , identified as an mhv strain by broderson et al (1976) and hierholzer et al (1979) ; and nuu, nua, nu66, from nude mice with hepatitis and wasting syndrome . other isolates have also been derived from nude mice (sebesteny and hill, 1974; ward et al, 1977) , and fox et al (1977) have described a strain that appeared during passage of an ascites myeloma cell line in balb/c mice. *hev may also denote the hepatoencephalomyelitis virus of stanley et al. (1954) , which is identical to reovirus 3. a s reviewed by mcintosh (1974) , coronaviruses are p l e o m o φ h i c , enveloped, and variable in size, measuring about 8 0 -1 5 0 n m in diameter. peplomers are 1 2 -2 4 n m in length (fig. 4 ) . using various techniques, a n u m b e r of workers have con firmed the diameter of m h v virions to fall within the range of the coronaviruses (gledhill et al., 1955; miyazaki et al., 1957; kraft, 1962a; starr etal., 1960) . svoboda etal. (1962) further described the virion as consisting of a nucleoid sepa rated from an outer m e m b r a n e by an electron-lucent space. david-ferreira and manaker (1965) , working with m h v -a 5 9 in tissue culture, found that the virions had a m e a n diameter of 75 nm with an electron-dense inner shell, 55 nm in diameter, separated from the outer double m e m b r a n e by an electronlucent space 8 nm wide. peplomers were 1 6 . 6 -2 3 . 4 n m long. not only was the virion significantly smaller than that of i b v , but the peplomers dif fered from those of both i b v and h c v 2 2 9 e , being conerather than club-shaped. mallucci (1965) in general, coronaviruses are inacti vated at 56°c in 10-15 min, at 37°c in several days, and at 4°c in several months (tyrell etal., 1968; kapikian, 1975) . wildtype mhv isolates also fall into this range of sensitivity gledhill and andrewes, 1951; gledhill et al., 1955; kraft, 1962a) . hirano et al. (1978) , however, indicated that mhv2 is not completely destroyed at 56°c for 30 min and is stable at 50t for 15 min in 1 μ mgclg or mgs04 but not in water. freezing and thawing or sonication at 20 kc for 3 min does not affect the virus titer. c. effect of chemicals. all coronaviruses are sensitive to ether when exposed overnight at 2°-4°c. chloroform also de stroys or reduces infectivity (mcintosh, 1974) . fifty percent glycerol inactivates mhvl after 6 weeks at 2°c (gledhill and andrewes (1951) . sodium deoxycholate reduces the titer of livim significantly (kraft, 1962a) , but calisher and rowe (1966) regard mhv virus as moderately resistant. according to hirano et al. (1978) , mhv2 is completely inactivated by ether, chloroform, sodium deoxycholate, and /3-propiolactone, but it is completely resistant to trypsin. mutagenesis has been reported by means of nmethyl-/v'-nitroguanidine or 5-fluorouridine and by 5-azacytidine or 5-fluorouracil (haspel et al., 1978) . the ph stability of all known murine hepatitis viruses has not been reported. for coronaviruses in general, acid sensitivity is regarded as variable (mcintosh, 1974) . hirano et al. (1978) found that mhv2 is stable be tween ph 3 and 9 at 37°c for 30 min. calisher and rowe (1966) although they have been sought, hemagglutinins have not been demonstrated for mhv strains (see, e.g., hirano et al., 1978; kraft, 1962a; bradbume, 1970; miyazaki etal., 1957) , but two human coronavirus strains do agglutinate human 0 erythrocytes (kaye and dowdle, 1969 cheever et al. (1949) found jhm virus to be unrelated to other neurotropic viruses, including gd vii, pseudorabies, lansing poliomyelitis, and mengo virus. kraft (1962b) found no relationship between livim, edim, and reovirus 3. with regard to other coronaviruses, the picture is somewhat different, for as a group, coronaviruses display complex serologic variability (bradburne, 1970; mcintosh et al., 1969) . mhv is serologically closely related to rcv and sdav in complement fixation tests and distantly related to rcv in cross-neutralization tests (parker et al., 1970; bhatt et al., 1972) . several strains of mhv are closely related to human coronaviruses oc 38 and oc 43 , and mhv3 is related to hcv-229e (bradbume, 1970) . antibody to mhv strains commonly found in human sera is probably present because of endemic human infection with related coronavimses (hartley et al., 1964) . electron micrographs and studies using fluorochrome stains indicate that coronaviruses develop exclusively in the cyto plasm of infected cells, that the virions collect in cytoplasmic vesicles of diverse size, that particles may also be seen in the matrix outside of the endoplasmic reticulum as well as in the golgi apparatus, and that they are not observed in the nucleus. in the main, replication involves budding into cytoplasmic cis temae, but tubular structures have also been seen within the cytoplasm during virus formation (ruebner et al., 1967; stan di α/., 1960; watanabe, 1969a,b) . wilsnack (1971) , confirming the work of boss and jones (1963) , elicited immunofluorescent antigen staining in sinusoidal lining cells in necrotic liver foci of weanling mice within 24 hr after intraperitoneal inoculation of the a59 strain. stainable antigen in intestinal impression smears of mice in fected by cage contact was also demonstrated. piazza et al. (1967) examined the fate of mhv3 after in travenous inoculation. the agent was not demonstrable be tween 40 min and 3.5 hr, when it appeared first in the spleen, then in the liver (4 hr) and blood (4.5 hr). at 5 hr it was recoverable from brain and kidney. high titers were then reached in all organs. barinsky and dementiev (1968) thereafter, the cells of the olfactory bulb and other brain re gions are affected. a number of cell systems have been successfully employed for in vitro growth of mouse hepatitis viruses: mhv-c in mouse embryo explants (mosley, 1961) ; mhvl in newborn mouse kidney explants (starr and pollard, 1959) ; mhv-s in mouse embryo explants (compels, 1953) and in liver (gallily et al, 1964) ; mhv-b in liver cell monolayers (paradisi and piccinino, 1968) ; mhv3 in liver explants (vainio, 1961) ; mhv-b in liver cells (miyazaki et al, 1957) ; mhv2 and mhv3 in dbt cells (hirano et al, 1978; takayama and kim, 1978) ; and various strains in nctc 1469 cells (david-ferreira and manaker, 1965; wilsnack etal, 1971; hartley and rowe, 1963) . mallucci (1965) , seamer (1965) , and lewis and starr (1972) described syncytium formation by mhv in mouse macrophage cultures, and a plaque assay for mhv2 in primary peritoneal macrophage cultures was described by shif and bang (1966) . laufs (1967) also described multinucleated giant cells with as many as 200 nuclei per cell in macrophage cultures infected with mhv3. using autoradiography, he ascertained that there was no dna synthesis in them and that they originated from cell fusion. macrophages derived from either liver or peritoneal wash ings are of enormous interest for the question of host cell-vims interactions. bang and warwick (1959, 1960) macrophages from the mice mirror these changes in resistance as the animals age. kantoch et al. (1963) determined that temporary susceptibility could be induced in resistant cells in culture if they were exposed to homogenates of susceptible cells, and gallily et al. (1964) showed that macrophages from genetically resistant mice treated with cortisone to enhance susceptibility behave in culture as if they were from susceptible animals. macrophages from mice susceptible to mhv2 virus can be converted to resistance by the intraperitoneal inoculation of concanavalin a in the donor mice. this enhanced resistance is also expressed in vivo (weiser and bang, 1977) . cheever etal. (1949) , nelson (1952b) , and kraft (1962a) in suckling mice, rapid wasting with or without neurologic signs may take place, accompanied in some cases by diarrhea, inanition, and dehydration (fig. 2) . mortality and morbidity are variable, ranging between al most 0 and 100% depending on factors like those affecting clinical signs. of importance to users and breeders of mice alike is the fact that a number of agents and procedures are known to modify the reactivity (and therefore the clinical signs) of mice to both spontaneous and experimental infection. examples of these, together with pertinent references, are presented in table i . leprévost et al. (1975a,b) have taken the view that there are three types of sensitivity to mhv3 infection in mice: resis tance, full susceptibility, and semisusceptibility. these are re flected in the susceptibility of their macrophages response of nuinu mice to sheep erythrocytes dba/2 mice, on the other hand, are regarded as fully suscep tible since deaths begin 4-6 days after infection, even when the mice are 90 days old, whereas the a/j strain is resistant, being susceptible to the acute disease only up to 3-4 weeks of age. although c3h mice are partially susceptible to the acute phase of the disease, they are fully susceptible to the chronic stage. in nude (nu/nu) mice, mhv takes on special significance. indeed, perhaps the best description of the clinical signs may be found in the original report describing this mutant mouse (flanagan, 1966) , published before runt disease, as the wast ing syndrome was called, came to be recognized as something other than a genetic effect. mhv-infected nude mice lose weight slowly or rapidly. they move stiffly with a stilted gait, and their faces assume a pointed, anxious appearance. partial paralysis may develop first in the hindlimbs and then in the forelimbs, resulting in almost total immobility. nu/+ heterozygotes are not affected in this way. flanagan (1966) found that at weaning, the nude animals were much smaller than controls (heterozygotes), that 55% died within 2 weeks of birth, and that 100% were dead by 25 weeks of age, whereas only 6% of controls died in the same period. it was not until sebesteny and hill (1974) uv-inactivated virus did not elicit the effect. the authors be lieve that fixed macrophages in athymic mice may be acti-vated, as was indicated by nickol and bonventre (1977) and by cheers and waller (1975) in certain bacterial infections in nude mice. t a m u r a et al (1979) (see, e . g . , r u e b n e r and bramhall, 1960; gledhill etal., 1952; nelson, 1953) . in liver regeneration m a y take place as early as 1 0 -1 4 days after infection (ruebner and bramhall, 1960) , ranging from complete healing to chronic scarring with intermediate gradations. kupffer cells were examined by r u e b n e r and miyai (1962) and r u e b n e r et al. (1967) . t h e y m a y undergo nuclear pyknosis and karyorrhexis 24 hr after intravenous inoculation of m h v 3 . in infection with neurotropic variants, such as j h m , the principal lesions appear in the central nervous system . meningitis is present but varies in degree and loca tion. in the brain, lesions m a y be found in all regions, but the hippocampus and its connections, the olfactory lobes, the periependymal tissues, and the brain stem seem to b e affected most often. necrotizing lesions predominate in the olfactory lobes and hippocampal regions, whereas demyelination is the major change in the brain stem. s o m e exudate m a y be found around blood vessels associated with lesions, and at about 5 days after infection, proliferating pericytes and scant lym phocytic cuffing can be seen. peripheral nerves show n o change. in sucklings, j h m virus produces extensive lesions in the brain and cord at 6 -8 days. meningitis is present, and large regions of necrosis with m a n y giant cells occur throughout the brain. in the cord, the lesions consist of spongy necrosis of the central gray matter. ganglion cells appear unaffected. powell and lampert (1975) resolved into small foci of fibrillary gliosis with an increased size and n u m b e r of astrocytic processes. t h e importance of this finding for investigations into the cause of multiple sclerosis and other demyelinating diseases in m a n is obvious and has been addressed b y , a m o n g others, lucas et al. (1977) and lampert (1978) . in sucklings infected with enterotropic strains (kraft, 1962a; broderson et al., 1976; ishida et al., 1978; ishida and fuji wara, 1979; r o w e et al., 1963; hierholzer et al., 1979) strains (dick et al., 1956; r u e b n e r and bramhall, 1960; hirano and ruebner, 1966) , and biggart and ruebner (1970) attribute the change to virus replication in lymphocytes. in other organs, minute superficial necrotic foci m a y be found in the stomach. n o changes are seen in the heart, lungs, pancreas, kidney, adrenals, voluntary m u s c l e , femoral o r ver tebral bone m a r r o w , or pituitary gland, although virus m a y be isolated from some of those organs. occasional giant cells are found in peripancreatic lymph nodes and in p e y e r ' s patches. flanagan (1966) islands of hepatocytes, markedly hypertrophied, remained. changes in other organs were not described. the liver lesions observed in other nude mice infected with mhv were similar. sebesteny and hill (1974) noted central nervous system lesions in their nude mice. ward et al. (1977) also encountered central nervous system lesions in addition to vascular changes, giant cell peritonitis, ascites, and giant cells in the villous epithelium of the intestines. since virus can be transmitted perorally and intranasally, it may be assumed that these are the principal routes of natural infection. transmission may be mediated by both the airborne and contact modes. feces, nasopharyngeal exudates, and perhaps urine would be sources of infection. evidence of vertical transmission is at hand, but reports are conflicting. piccinino et al. (1966) with or without booster, showed no antibody in the same colony. in a conventional colony of ddd mice, furthermore, seropositivity increased from 0 to 12% and in ddy mice from 6.3 to 45% as a result of the booster technique. in barrier-maintained animals, the booster did not elicit antibodies where there had been none before. presumably, these animals were free from mhv infection. from the foregoing, it is evident that the classic humoral immune response to mhv seems to be weak, and that a colony in contrast to the findings of bang and warwick (1960) , who concluded that one gene (or factor) was responsible for host susceptibility in mhv2 infection, stohlmann and freiinger (1978) showed that two genes are required for resistance of the central nervous system in sjl mice to fatal disease due to mhv-jhm. further, stohlmann et al. (1980) reported that there is an age-related change in resistant mice that protects them from acute central nervous system disease. they iden tified this change as due to a maturing adherent spleen or peritoneal exudate cell population. in extensive genetic studies, levy-leblond et al. (1979) found no correlation between the h-2 locus and either the acute or chronic disease in c57bl (susceptible) or a/j (resistant) animals. using congenie c3h lines, however, they were able to show that the h-2^ allele enables both heterozygous and homozygous animals to resist the development of the chronic disease. they believe, therefore, that mhv sensitivity appears to be influenced by at least two major genes: one for the acute disease, and the other, linked to the h-2 gene complex, for the chronic disease. mice also produce interferon as a result of mhv infection virelizier and gresser, 1978) , and taguchi et al (1979a) ascribe the greater susceptibility of suckling c3h/hejms mice to serum levels of interferon that are considerably lower than in weanling and aduh mice, as well as to greater macrophage sensitivity in the neonates. mhv-induced interferon may be regarded as the principal mechanism by which the virus modifies the immune respon siveness of mice to sheep red blood cells, for example. (see also (1949) infected cotton rats, rats, and hamsters intracerebrally, but rabbits and guinea pigs failed to respond. sebesteny and hill (1974) at tempted to infect infant wistar rats and hamsters using virus recovered from nude mice. all survived at least 21 days with out signs of illness. of considerable importance is a report by taguchi et al (1979c) concerning asymptomatic mhv-s infection in suckl ing rats following intranasal inoculation. the agent multiplied mainly in the nasal epithelium without any clinical signs. neu tralizing antibodies were produced, however, and could also be demonstrated in adult rats following infection. necrotic changes took place in the nasal mucosa, and cytoplasmic im munofluorescence was demonstrated in the nasal epithelium 2 days after intranasal inoculation of 10-day-old rats. age susceptibility has been amply addressed above in the consideration of virus growth in mice and tissue culture and in the discussion of the clinical picture. there appears to be no difference in susceptibility between the sexes (taguchi et al., 1976) . parker et al. (1966) found that the incidence of complement-fixing antibodies was greater in females than in males under colony conditions, as cribing this difference to continual exposure to virus-infected litters. there appears to be no evidence that first litters are significantly more susceptible than later ones. as noted above (section iv,β, 2 and c,l), the susceptibility of various mouse strains depends on the age of the host at the time of infection, the virus strain, and the host genotype. a completely resistant mouse strain has not been reported. prevalence of mhv infection in an animal colony is often difficult to ascertain. in addition to examples given earlier, descoteaux et al. (1977) found a low prevalence of hepatitis antibodies in three of five colonies studied in canada. complement-fixing antibodies ranged in titer from 8 to 32, 8 being considered positive. fewer than 20% of the animals, which were 6-9 months old at the time of testing, were posi tive. in distribution, mhv is regarded as occurring worldwide. these have been defined as occurring more than 2 months after intracerebral, intraperitoneal, intranasal, or intravenous inoculation (as reviewed in robb and bond, 1980) . the chronic clinical manifestations range from none to porenceph aly, paralysis, hepatitis, immunodeficiency manifestations, encephalitis, lymph node adenopathy, and vasculitis. virus may or may not be isolated. cells stainable by immunofluores cence may be found. demyelination with or without remyelination may occur. occasional scattered mononuclear cell infil trates may be evident. there is no evidence that seasonal changes influence the oc currence of epizootics of mhv infection. as indicated above, serologic testing is routinely carried out by means of the complement fixation test. cross-reactions with other coronaviruses must be taken into account when inteφreting the results, however. neutralization tests performed in tis sue culture systems are also possible. employing virus strain a59 as antigen in the elisa, peters et al. (1979) found a high prevalence of mhv antibodies in colonies with a low incidence of both complement-fixing and neutralizing antibodies. hierholzer and tannock (1977) have used the single radial hemolysis test for human coronavirus serodiagnosis. they then applied it to some mhv strains (hierholzer et al, 1979) . these techniques are helpful under certain circumstances, e.g., experimental investigations, but they are inefficient for field conditions. necropsy of dead or sick animals is always useful, although a definitive diagnosis in the absence of serologic evidence cannot be made. nevertheless, wherever possible, gross and microscopic pathologic examination should be undertaken. sentinel animals, especially gnotobiotes, could be ιηοοφοrated into a program of colony health surveillance. these ani mals can then be checked at predetermined intervals for clini cal, serologic, and histopathologic evidence of endemic dis ease in the colony. control of mhv is difficult in mouse colonies unless caesa rian derivation coupled with barrier maintenance is under taken. with the finding that vertical transmission is possible, however, barrier maintenance alone may not be adequate if, for example, such transmission is frequent. using small number of animals for experimental puφoses, kraft (1962a) tamura et al (1976) found that nu/nu mice could resist mhv infection when they previously received thymocytes from weanling nu/+ littermates. they were then not only able to produce antibody but survived a challenge infection as well. studies on rotaviral antibody in bovine serum and lacteal secretions, using radioimmunoassay epidemic diarrhea of infant mice. identification of the etiologic agent electron microscopic study of the intestinal epithelium of mice infected with the agent of epizootic diarrhea of infant mice (edim virus) the morphology of three pre viously uncharacterized human respiratory viruses that grow in organ culture the effect of trypsin on the growth of rotavirus viruses of verte brates panel report of the colloquium on selected diarrheal diseases of the young rotavirus isolation and cultivation in the presence of trypsin a murine virus (jhm) causing disseminated encephalomyelitis with extensive destruction of myelin. ii in vitro detection of human rotaviruses further observa tions on the virus of epizootic diarrhea of infant mice. an electron microscopic study genetics of resistance of animals to viruses: i. introduc tion and studies in mice macrophages and mouse hepatitis mouse macrophages as host cells for the mouse hepatitis virus and the genetic basis of their susceptibility virological and cytogenetic studies on the involvement of bone marrow of mice in some hepatoencephalotropic viral infections rotavirus serotypes by serum neutralisation moφhogenesis of avian infectious bronchitis virus and a related human virus (strain 229e) preparation of type-specific antisera to reoviruses isolation of a reovirus from a case of burkitt's lymphoma isolation of a non-pathogenic tumour-destroying virus from mouse ascites characteristics of a newborn runt disease induced by neonatal infection with an oncolytic strain of reovirus type 3 (reo3mh). ii. immunological aspects of the disease in mice characteristics of a newborn runt disease induced by neonatal infection with an oncolytic strain of reovirus type 3 (reo3mh). i. pathological investigations in rats and mice characterization of the virus of sialodacryoadenitis of rats: a member of the coronavirus group lymphoid necrosis in the mouse spleen produced by mouse hepatitis virus (mhv3): an electronmicroscopic study lethal intestinal virus infection of mice (livim). an important new model for study of the response of the intestinal mucosa to injury virus particles in epithelial cells of duodenal mucosa from children with acute non-bacterial gastroenteritis serological studies with an agent of epizootic diarrhea of infant mice pathogenic murine coronaviruses. ii. characterization of virus-specific proteins of murine coronaviruses jhmv and a59v specific monovalent cation effects on modification of reovirus infectivity by chymotrypsin digestion in vitro ex traordinary effects of specific monovalent cations on activation of reovirus transcriptase by chymotrypsin in vitro new intermediate subviral particles in the in vitro uncoating of reovirus virions by chymotrypsin reovirus transcriptase activation in vitro: involvement of an endogenous uncoating activity in the second stage of the process two modes of entry of reovirus particles into l cells hepatic localization of infectious agent in murine viral hepatitis antigenic relationships amongst coronaviruses a solid-phase system (space) for the detection and quantification of rotavirus in faeces small intestinal epithelial brush border enzymatic changes in suckling mice infected with reovirus type 3 reovirus type 3 infection in a suckling mouse: the effects on pancreatic structure and enzyme content viral hepatitis associated with trans plantable mouse leukemia. i. acute hepatic manifestations following treatment with urethane or methylformamide neonatal calf diarrhoea: identifica tion of reovirus-like (rotavirus) agent in faeces by immunofluorescence and immune electron microscopy lethal en teritis in infant mice caused by mouse hepatitis virus the laboratory diagnosis of epizoo tic diarrhoea of infant mice a rabbit rotavirus. vet. ree. 99 diagnosis of rotavirus infection by cell culture a hepatitis virus of mice two coronaviruses isolated from central nervous system tissue of two multi ple sclerosis patients mouse hepatitis, reo-3, and the theiler viruses activated macrophages in congenitally athymic "nude" mice and in lethally irradiated mice epidemic diarrheal disease of suckling mice epidemic diarrheal disease of suckling mice. i. manifestations, epidemiology, and attempts to trans mit the disease epidemic diarrheal disease of suckling mice. iii. the effect of strain, litter, and season upon the incidence of the disease a murine virus (jhm) causing disseminated encephalomyelitis with extensive destruction of myelin. i. isolation and biological prop erties of the virus serological inter relationships of murine hepatitis viruses production of high-titer bovine rotavirus with trypsin murine virus contaminants of leukemia viruses and transplantable tumors reovirus type 3 infection in laboratory mice temperature-sensitive mutants of reovirus type 3: studies on the synthesis of viral rna effect of corticosteroids on mouse hepatitis virus infection an electron microscope study of the development of a mouse hepatitis virus in tissue culture cells importance of a new virus in acute sporadic enteritis in children comparison of the morphology of three coronaviruses classification of rotaviruses: report from the worid health organization/food and agriculture or ganization comparative virology program serologic study on the prevalence of murine viruses in five canadian mouse colonies a virus related to that causing hepatitis in mice (mhv) immunopathology of mouse hepatitis virus type 3. ii. effect of im munosuppression in resistant mice the appearance of a hepatotrophic virus in mice thymectomized at birth comparison of five diagnostic methods for the detection of rotavirus antigens in calf faeces morphological studies on simian virus sa 11 and the "related" 0 agent electron microscopic identification and subsequent isolation of a rotavirus from a dog with fatal neonatal diarrhea enhancement of rotavirus infectivity by trypsin and elastase identification of rotaviruses of different origins by the plaque-reduction test hemagglutination and hemagglutination-inhibition studies with a strain of nebraska calf diarrhea virus (bovine rotavirus) isolation and preliminary genetic and biochemical characterization of temperature-sensitive mutants of reovirus altered disease in rats due to mutants of reovirus type 3 temperature-sensitive mutants of reovirus type 3: studies on the synthesis of viral peptides generation of cytolytic τ lymphocytes after reovirus infection: role of the si gene nude", a new hairless gene with pleiotropic effects in the mouse electron microscopy in the diagnosis of infectious diarrhea the rotaviruses relation between viruses from acute gastroen teritis of children and newborn calves virus diarrhoea in foals and other animals. vet. ree. 96 fluorescent virus precipitin test adverse effects of mouse hepatitis virus on ascites myeloma passage in the balb/cj mouse problems in checking inapparent infections in laboratory mouse colonies: an attempt at serological checking by anamnestic re sponse implication pancréatique chez la souris infectée avec le virus de l'hépatite murine immunisation de la souris "nude" contre le virus de l'hépatite murine par transfert de lymphocytes sensibilisés carrier state of anti body and viruses in a mouse breeding colony persistently infected with sendai and mouse hepatitis viruses effect of cortisone on genetic resistance to mouse hepatitis virus in vivo and in vitro ontogeny of macrophage resistance to mouse hepatitis in vivo and in vitro structure and physicochemical properties of coronaviruses comparison of an enzyme-linked immunosorbent assay for quantitation of rotavirus an tibodies with complement fixation in an epidemiological survey enhancement of the pathogenicity of mouse hepatitis virus (mhvl) by prior infection of mice with certain leukaemia agents a hepatitis virus of mice production of hepatitis in mice by the combined action of two filterable agents mouse hepatitis virus and its pathogenic action reactive sites of reovirus type 3 and their interaction with receptor substances reovirus type 3: physical characteristics and interaction with l cells the propagation of s virus of mouse hepatitis in tissue culture comparison of methods for immunocytochemical detection of rotavirus infections delayed hypersensitivity in mice infected with reovirus. ii. induction of tolerance and suppressor τ cells to viral specific gene products spontaneous diseases and their control in laboratory animals electron microscopic examination of cells infected with reovirus tissue culture cytopathic and plaque assays for mouse hepatitis viruses recovery of reoviruses from wild and laboratory mice antibodies to mouse hepatitis viruses in human sera temperature-sensitive mutants of mouse hepatitis virus produce a high incidence of demyelination mouse hepatitis virus-induced recurrent demyelination quantitation of antibody to non-hemagglutinating viruses by single radial hemolysis: serological test for human coronaviruses new strain of mouse hepatitis virus as the cause of lethal enteritis in infant mice isolation of low-virulent mouse hepatitis virus from nude mice with wasting syndrome and hepatitis studies on the mechanism of destruc tion of lymphoid tissue in murine hepatitis virus (mhv3) infection. i. selective prevention of lymphoid necrosis by cortisone and puromycin 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species measurement of rotavirus antibody by an enzyme-linked immunosorbent assay blocking assay secretory antibody directed against rotavirus in human milk-measurement by means of enzyme-linked immunosorbent assay key: cord-261036-zdhg4axx authors: shirato, kazuya; maejima, madoka; hirai, asuka; ami, yasushi; takeyama, natsumi; tsuchiya, kotaro; kusanagi, kouich; nunoya, tetsuo; taguchi, fumihiro title: enhanced cell fusion activity in porcine epidemic diarrhea virus adapted to suckling mice date: 2010-09-09 journal: arch virol doi: 10.1007/s00705-010-0790-1 sha: doc_id: 261036 cord_uid: zdhg4axx porcine epidemic diarrhea virus (pedv) is the major causative agent of fatal diarrhea in piglets. to study the pathogenic features of pedv using a mouse model, pedv with virulence in mice is required. in pursuit of this, we adapted a tissue-culture-passed pedv mk strain to suckling mouse brains. pedv obtained after ten passages through the brains (mk-p10) had increased virulence for mice, and its fusion activity in cultured cells exceeded that of the original strain. however, the replication kinetics of mk and mk-p10 did not differ from each other in the brain and in cultured cells. the spike (s) protein of mk-p10 had four amino acid substitutions relative to the original strain. one of these (an h-to-r substitution at residue 1,381) was first detected in pedv isolated after eight passages, and both this virus (mk-p8) and mk-p10 showed enhanced syncytium formation relative to the original mk strain and viruses isolated after two, four, and six passages, suggesting the possibility that the h-to-r mutation was responsible for this activity. this mutation could be also involved in the increased virulence of pedv observed for mk-p10. the group i coronavirus porcine epidemic diarrhea virus (pedv) is an enveloped rna virus with a single, positive-stranded genome about 30 kb in length [9] . pedv is the causative agent of pig diarrhea and induces a loss of appetite and weight in adult pigs, whereas it is lethal in piglets [16] . however, our understanding of the pathogenesis of diseases caused by pedv is limited due to a lack of appropriate small-animal models. coronaviruses cause various diseases in a species-specific manner, and they grow in cultured cells established from susceptible host species; human, feline, and porcine coronaviruses grow only in cells isolated from the respective species [24] . one of the major factors determining species specificity is the cellular receptor, which interacts with a given virus, but not with other viruses, when the virus initially encounters cells [5] . some of the group i coronaviruses, such as porcine transmissible gastroenteritis virus (tgev) and human coronavirus 229e (hcov-229e), utilize aminopeptidase n (apn) [1, 19, 22, 23] . however, the receptor of pedv has not yet been identified, although there was a controversial report on a pedv receptor protein [10] . expression of the receptor protein for mouse hepatitis virus (mhv) and severe acute respiratory syndrome (sars)-associated coronavirus (sars-cov) in cultured cells confers susceptibility to each of these viruses [2, 4, 25] . expression of the tgev receptor in mice from a transgene rendered otherwise resistant mice susceptible to tgev [1] . however, this approach is not available for pedv. an alternative approach is to isolate viruses that have adapted for growth within the animals and which exhibit virulence. such adaptations have been reported for two other coronaviruses, ibv and oc43 [3] . in the present study, we isolated a strain of pedv that was more virulent than the original tissue-culture-adapted virus after passage through mouse brain cells. the adapted virus showed enhanced cytopathology compared with the original virus. vero cells were obtained from the american type culture collection (atcc; manassass, va, usa). the cells were maintained in dulbecco's modified eagle's medium (dmem; sigma, st. louis, mo, usa) containing 5% fetal calf serum (fcs). the pedv strain used in this study was an mk strain isolated from the jejunum of a pig that had exhibited diarrhea in 1996 in japan, and which had been passed nine times in vero cell cultures. pedv was maintained in vero cells using a slightly modified version of a reported method [8] . briefly, vero cells were infected with the virus. after 1 h, the cells were washed with phosphate-buffered saline (pbs) and cultured in dmem containing 10% tryptose phosphate broth (tpb) and 2.5 lg/ml trypsin (sigma) for 15 h. next, the cells and supernatant were pooled, ultrasonicated, and stored at -80°c until use. the pedv infectious titer was determined by a plaque assay using vero cells. briefly, vero cell monolayers grown in 24-well plates coated with type i collagen (agc techno glass, chiba, japan) were inoculated with serially diluted virus samples. after 1 h to allow for adsorption of the virus, the inoculum was removed, the cells were washed with pbs, and dmem containing 10% tpb and 1.25 lg/ml trypsin was added. for titration of the virus, half of the concentration of trypsin was used to drive cell fusion in a procedure that is gentle compared with viral propagation. after a 15-h incubation, the cells were fixed and stained with pbs containing 0.1% crystal violet and 20% formalin under ultraviolet light. after staining for 2 h, the cells were washed with water and dried, and the plaques were counted under a light microscope as described for the infectious titration of human coronavirus 229e [6] . the viral titer in the brains of the mice is given as plaqueforming units (pfu)/50 ll of 10% homogenate, whereas the titer in the supernatant is given as pfu/ml of supernatant; the cell titer is given as pfu/well of a 24-well plate. isolation of a murine-adapted variant of pedv specific-pathogen-free (spf) pregnant icr mice were obtained from japan slc (shizuoka, japan). all animal experiments were approved by the committee on experimental animals, national institute of infectious diseases, japan, and all experimental animals were handled according to the guidelines of the same committee. all spf mice were confirmed by seromonitoring to be free from infection with mhv, lactate dehydrogenase virus, and other pathogenic microorganisms. newborn (within 24 h of birth; 0-day-old) mice were used in this study. pedv strain mk (2,500 pfu) was inoculated intracerebrally (i.c.) into the brains of 0-day-old mice (n = 12-16). five days post-infection (p.i.), the mice were sacrificed and their brains collected, pooled, and homogenized in pbs to yield a 10% suspension, which was centrifuged at 2,000 rpm for 10 min at 4°c. next, 10 ll of the supernatant from the 10% brain homogenate was inoculated i.c. into suckling mouse brains, and the brains were collected as described above. this cycle was repeated ten times, and the virus obtained was designated mk-p10. mk-p10 was amplified once by vero cell culture to match its condition to that of the original virus, then stored at -80°c until use. to investigate viral virulence and replication in mouse brains, 0-day-old mice were infected i.c. with 5,000 pfu of pedv and monitored daily for clinical features; they were also weighed daily. pbs was used in a mock infection. to examine viral replication in the brains, the mice were euthanized on the indicated day p.i., their brains were collected, and 10% homogenates were prepared as described above. to determine the replication kinetics in cultured cells, vero cells were infected with pedv at a multiplicity of infection (m.o.i.) of 0.1. after 1 h, the cells were washed with pbs and cultured in dmem containing 10% tpb and 2.5 lg/ml trypsin. at the indicated hour p.i., the supernatant and cells were collected separately, the cells were ultrasonicated, and the viral titers were determined using a plaque assay as described above. sequence analysis viral rna was extracted with trizol ls reagent (invitrogen, carlsbad, ca, usa) following the manufacturer's protocol. first-strand cdna was synthesized using m-mlv reverse transcriptase (takara-bio, shiga, japan) and oligod(t) 16 to determine the statistical significance of the survival curves, log-rank and generalized wilcoxon tests were performed. significance was determined in all other cases using unpaired t tests. p \ 0.05 was considered to be statistically significant. to obtain a murine-adapted variant of pedv, passage through suckling mice was performed. zero-day-old mice were inoculated i.c. with the mk strain of pedv, and the brains of the infected mice were collected four or 5 days later. the brains were pooled and used for the next round of mouse inoculation. surprisingly, mice infected with the non-adapted, original strain (mk) showed clinical signs and mortality; these observations differ from those reported for hcov oc43 and ibv [3] . however, in subsequent passages, clinical signs in the infected mice, such as trembling, tremors, and growth retardation, became more obvious. the process was stopped after ten passages, at which point the variant was designated mk-p10. the original mk strain and the mouse-adapted variant mk-p10 were inoculated onto vero cells in the presence of trypsin and cultured for 15 h. as shown in fig. 1 , more syncytium formation was seen in the cells infected with mk-p10 than in those infected with the original strain. the mean diameter of the mk-p10 syncytia was significantly greater than that of the mk syncytia (874 ± 177 vs. 348 ± 104 lm; n = 10, p \ 0.0001). the neurovirulence of mk-p10 in suckling mice was examined. zero-day-old mice were infected i.c. with 5,000 pfu of virus and monitored daily for clinical symptoms and mortality. all mice infected with mk and mk-p10 succumbed to infection; however, the mice infected with mk-p10 died more quickly than the mk-infected mice ( fig. 2a ; n = 25, p \ 0.01). the mice were also weighed daily ( fig. 2b; n = 12 ). the weights of the mice infected with mk or mk-p10 increased for 3-4 days after infection. subsequently, the weight of the mice decreased beginning 4 days p.i., whereas the mice infected with mk began to lose weight 8 days p.i.-4 days later than the mk-p10infected mice. the body weight of the mice infected with mk-p10 was determined 7 days p.i. because most of the mice had succumbed by 7-11 days p.i. the mock-infected mice did not exhibit weight loss or clinical symptoms. as described above, a mouse-adapted variant of pedv was successfully isolated. the growth kinetics of the original strain and mk-p10 were examined in suckling mouse brains. mk and mk-p10 exhibited similar growth kinetics in suckling mouse brain, peaking on day 4 p.i. (fig. 3a) . their growth kinetics in cultured vero cells was also determined. some coronaviruses tend to remain intracellular, and viral stocks are prepared by ultrasonication or repeated freeze-thawing cycles. to compare the amounts of budded and intracellular virus, the titers in the supernatant (fig. 3b) and cells (fig. 3c) were determined separately. similar to the growth kinetics in the brains of the mice, the growth pattern in vero cells did not differ significantly between mk and mk-p10 (fig. 3b, c) . these results suggest that the increased virulence in suckling mice and syncytium formation in cultured cells is not due to increased growth in the brain or cultured cells. because the s proteins of coronaviruses are reported to be associated with viral pathogenicity [20] , table 1) . among these, the amino acid substitutions at positions 804 and 1,381 resulted in a polarity change (hydrophobic to hydrophilic). to determine at which brain passage the amino acid changes occurred, we determined the primary sequences of s protein from the viruses isolated after two, four, six, and eight passages in mouse brain. as shown in table 2 , the amino acid at position 804 differed at the second passage, while those at positions 496 and 774 changed simultaneously at the fourth passage. the amino acid change at position 1,381 occurred at the eighth passage. as described above, the plaque size of mk-p10 was larger than that of the parental strain (fig. 1) . this larger plaque morphology was also detected in pedv after eight passages, whereas the viruses isolated after four and six passages showed syncytium formation similar to that in mk (fig. 4) . these results suggest that the amino acid change at position 1,381 (h to r) first detected at the eighth passage, which is located in the cytoplasmic tail (ct) region, contributed to the larger plaques of mk-p10, though other genes must be considered. two different coronaviruses, oc43 and ibv, have successfully adapted to grow in mice and show virulence after brain-to-brain passage in suckling mice [3, 12] . these viruses initially showed low virulence (i.e., clinical symptoms such as tremor and rigidity and lethargy in suckling mice), but the virulence increased with repeated passage in mouse brains. in this study, the passage of pedv in suckling mouse brains clearly strengthened its virulence, similar to the adaptation seen in oc43 and ibv. unexpectedly, the original mouse non-adapted pedv showed significant virulence for suckling mice. the primary sequences of s protein from the viruses isolated after repeated passages were determined. pedv passed in mouse brains showed mutations in the s protein, which had accumulated after ten passages. the h-to-r mutation at residue 1,381, which was first detected after eight passages, is likely responsible for the enhanced cell-cell fusion activity, as only those viruses with this mutation showed significantly larger syncytia compared with the original pedv; the s protein is a major determinant of the fusion activity of coronaviruses [5, 20] . it would be interesting to find out whether this substitution in the s protein is responsible for the enhanced fusion activity by expressing s protein alone. those mutations found in the mouseadapted viral s proteins could be responsible for the increased virulence; however, other genes have been reported to be involved in the virulence of such coronaviruses as mhv and sars-cov [7, 15, 17, 21] . to identify the genes responsible for the increase in virulence, we would need the entire genomic sequences of the original and mouse-adapted pdevs; additionally, reverse-genetic analyses would be essential. pedv differs from most coronaviruses in that it grows in cultures of cells derived from animal species not susceptible to pedv; the virus grows efficiently in vero cells derived from the kidneys of green monkey, which is not a natural host of the virus [8] . few cases are known in which coronaviruses multiply in cells derived from non-susceptible host animals. sars-cov can grow in a variety of cells established from several animal species [13, 14, 18, 24] , and it is somewhat capable of growing in the same animals; however, efficient infection is restricted to cells from animals that support sars-cov infection [24] . the growth potential of sars-cov in a variety of animal cells correlated with the utilization of its receptor molecule, ace2 [11] . most known coronaviruses become infective in cells when their functional receptor molecule is expressed by transfection with a plasmid encoding the receptor molecule [5] . this suggests that vero cells susceptible to pedv infection express a functional receptor. in our study, both mouse-adapted and non-adapted mk strains of pedv could infect and grow in suckling mouse brains, suggesting that a receptor molecule utilized by both viruses exists in suckling mouse brain. to determine which cell types in the brain express the receptor, we are attempting to identify the cell types that are infected by pedv in suckling mouse brain. this will provide valuable information related to the receptor molecule utilized by pedv. in summary, a highly neurovirulent variant of pedv was successfully isolated by passage following the i.c. infection of 0-day-old suckling mice. the s protein of the variant had four amino acid substitutions, which might be responsible for its enhanced cell-cell fusion activity. of these substitutions, the h-to-r substitution at position 1,381 in the ct region of the s protein is likely to be the determinant of the high fusion activity. to address this possibility, the expression of wild-type and mutant s proteins in cultured cells and an analysis of their fusion activity are necessary. reverse-genetic analyses of the entire pedv genome are also required to assess the effect of s protein mutations on the virulence and pathogenesis of pedv. p2 p4 p6 p8 p10 fig. 4 plaque morphology of pedv isolates after mouse brain passage pedv passed in mouse brains two (p2), four (p4), six (p6), eight (p8), or ten (p10) times as well as the original mk (p0) was inoculated onto vero cells. cell fusion assays were performed as described in the legend to fig. 1 aminopeptidase n is a major receptor for the entero-pathogenic coronavirus tgev cloning of the mouse hepatitis virus (mhv) receptor: expression in human and hamster cell lines confers susceptibility to mhv growth of infectious bronchitis virus in suckling mice brain susceptibility to sars coronavirus s protein-driven infection correlates with expression of angiotensin converting enzyme 2 and infection can be blocked by soluble receptor coronavirus receptors. in: siddell sg (ed) the coronaviridae proteasemediated entry via the endosome of human coronavirus 229e severe acute respiratory syndrome coronavirus open reading frame (orf) 3b, orf 6, and nucleocapsid proteins function as interferon antagonists isolation and serial propagation of porcine epidemic diarrhea virus in cell cultures and partial characterization of the isolate the molecular biology of coronaviruses porcine aminopeptidase n is a functional receptor for the pedv coronavirus angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus growth in suckling-mouse brain of ''ibv-like'' viruses from patients with upper respiratory tract disease pathology and virus dispersion in cynomolgus monkeys experimentally infected with severe acute respiratory syndrome coronavirus via different inoculation routes mouse-passaged severe acute respiratory syndrome-associated coronavirus leads to lethal pulmonary edema and diffuse alveolar damage in adult but not young mice inactivation of expression of gene 4 of mouse hepatitis virus strain jhm does not affect virulence in the murine cns a new coronavirus-like particle associated with diarrhea in swine reverse genetic characterization of the natural genomic deletion in sars-coronavirus strain frankfurt-1 open reading frame 7b reveals an attenuating function of the 7b protein in vitro and in vivo severe acute respiratory syndrome coronavirus infection of golden syrian hamsters coronavirus 229e susceptibility in man-mouse hybrids is located on human chromosome 15 coronaviruses: structure and genome expression single-amino-acid substitutions in open reading frame (orf) 1b-nsp14 and orf 2a proteins of the coronavirus mouse hepatitis virus are attenuating in mice feline aminopeptidase n serves as a receptor for feline, canine, porcine, and human coronaviruses in serogroup i feline aminopeptidase n is a receptor for all group i coronaviruses coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus receptor for mouse hepatitis virus is a member of the carcinoembryonic antigen family of glycoproteins acknowledgments we thank shutoku matsuyama, miyuki kawase, and makoto ujike for their valuable comments and encouragement. this work was supported by a grant-in-aid for scientific research (b; no. 19390135) from the ministry of education, culture, sports, science, and technology of japan. passage number 0 2 4 6 8 1 0 496 i i t t t t 774 t t m m m m 804 a d d d d d 1,381 h h h h r r fig. 3 the replication kinetics of mk (squares) and mk-p10 (circles). a viral replication in suckling mouse brain. zero-day-old mice were infected i.c. with 5,000 pfu of pedv. at the indicated days p.i., the brains were collected and 10% homogenates were prepared. the viral titer in the brains was determined using a plaque assay with vero cells and is expressed as pfu/50 ll of 10% homogenates (n = 3). b, c viral replication in cultured cells. vero cells were infected with pedv at an m.o.i. of 0.1. viral titers in the supernatants (b) and cells (c) were determined separately using plaque assays (n = 6). the viral titer in the supernatant is given as pfu/ml of supernatant, while that in the cells is given as pfu per well of a 24-well plate key: cord-026009-rdhuc2n2 authors: anderson, nancy l. title: pet rodents date: 2009-05-15 journal: saunders manual of small animal practice doi: 10.1016/b0-72-160422-6/50179-0 sha: doc_id: 26009 cord_uid: rdhuc2n2 nan many small rodents are commonly kept for companionship and enjoyment. this chapter provides information needed to diagnose and treat the most frequently encountered problems of mice, rats, gerbils, hamsters, guinea pigs, and chinchillas. • cages should be made of stainless steel, hard plastic, or glass. these materials are cleaned and sanitized easily and are resistant to gnawing or corrosion from urine and fecal matter. minimum floor space and height requirements are listed for each species in • guinea pigs can be housed in open-topped enclosures with walls higher than 10 inches. ensure that dogs, cats, wild animals, and small children do not have unsupervised access to these cages. • clean cages as needed, usually 1 to 3 times per week for most rodents. a scrub brush, dish soap, and water work well. if cages are not kept clean, ammonia, other irritants, moisture, and bacteria concentrations rise to harmful levels, predisposing animals to disease. • disinfect the cage twice a month with 1 part sodium hypochlorite (household bleach) mixed in 30 parts water. let the bleach solution stand for at least 15 minutes. rinse the cage well afterward. • all solid-floored cages need to be covered in bedding. shredded paper, non-resinous wood shavings, wood wool, and corn cobs are all acceptable. provide at least 2 inches of bedding. most rodents enjoy burrowing in deeper bedding when it is provided in one corner of a cage. do not, however, fill the entire cage with deeper bedding. this usually leads to poor sanitation as a result of owners' failure to recognize buildup of hidden wastes such as moisture from leaking water bottles, cached foods, urine, and feces. • wire mesh floors can be used successfully only if the dimension of the mesh is correct. size the openings to be just large enough for an adult to retract a tarsal joint back through the mesh. larger holes make it difficult for the animals to walk and cause pressure sores. smaller openings may cause injuries such as tibial fractures and self-mutilation while struggling to free trapped appendages. bedding above the wire keeps waste from dropping through the wire and therefore is not recommended. wire bottom cages do not work well for breeding animals because neonatal rodents must be surrounded by nesting material to maintain moisture in the nest and prevent dehydration. young rodents often cannot walk correctly on mesh sized for adult feet. • all pet rodents require visual security. tubes, jars, or cans made of nontoxic, nonabrasive substances work well for this purpose. also provide objects for gnawing. rodents possess open-rooted teeth, and constant wear is necessary to maintain normal dentition. mice, rats, gerbils, and hamsters enjoy and benefit from exercise wheels. • a good room temperature range for most pocket pets is 70°f to 75°f. keep rodents with disease at 85°f to 90°f unless hyperthermia is of concern (some chinchillas). • provide 10 to 12 hours of darkness to 12 to 14 hours of light. this light cycle is essential if breeding is desired. • hamsters, guinea pigs, and chinchillas that are exposed to temperatures below 65°f may hibernate for a few days or until the ambient temperature rises. heart rates may be less than 5 bpm during hibernation. m key point feed pet rodents laboratory animal chow appropriate for their species (table 177 -2). seed diets are deficient in protein and contain excessive fat. • seeds, as well as vegetables and other foods, may be fed as treats but not to provide more than 15% of calories. intermittent exposure to vegetables and seeds causes mild, transient diarrhea. • supplementation of vitamin c is recommended for all guinea pigs. • adult chinchillas that are not obese should be fed high quality, fresh grass hay ad libitum. obese animals may need to have the hay rationed. chinchillas require 1 / 8 to 1 / 4 -cup of fresh pellets per animal each day. feeding pellets free choice leads to obesity, and the high protein and calcium levels in these diets may predispose animals to urinary tract disease. most pellets also do not provide sufficient fiber to maintain normal gastrointestinal (gi) motility. • store food in tightly sealed containers at less than 60°f; keep food refrigerated if possible. • feed all diets within 90 days of milling to ensure the highest nutritional value. encourage owners to check dates on packages and ask pet store managers about providing dates on bulk items. • if possible, feed pet rodents except guinea pigs from overhead racks. these devices reduce wastage and eliminate fecal contamination of food. covered hoppers, heavy crocks, or stainless steel bowls that are attached to the side of the cage to eliminate spillage are acceptable and recommended for guinea pigs. • feed breeding females and their litters from the floor of their cages until the young are large enough to reach overhead feeders or crawl in and out of crocks. • cannibalism of neonates commonly occurs as the result of stress associated with cage cleaning. to minimize cannibalism, clean the cage and provide a 10to 13-day food supply 1 to 2 days before parturition. m key point provide fresh water in clean containers daily. • do not provide water in open crocks. these are contaminated or spilled easily and are a common cause of dehydration and poor sanitation. • sipper tubes and water bottles work well. clean with dish soap and water daily and disinfect them weekly. guinea pigs expel food from their mouths into their sipper tubes, so more frequent cleaning is needed. • some water bottles have special valves to minimize backflow. supplement guinea pigs' water daily with 200 mg vitamin c/l. if the water is not dechlorinated, it will inactivate the vitamin c. quarantine all newly acquired animals in a different room from current pets for a minimum of 30 days. feed and handle quarantined animals last. recommend that caretakers wash their hands and change clothes before handling current pets. avoid the introduction of adult animals because this frequently results in fighting. instead, place animals together while young and allow them to mature together. avoid keeping more than one male per cage because this also usually leads to fighting. a systematic history and physical examination are mandatory. many disease syndromes are caused by poor husbandry. pets that have been kept isolated from other rodents or acquired from a private breeder are less likely to harbor infectious disease than animals obtained from a pet store, laboratory, or wholesaler. see table 177 -3 for normal physiologic data. obtain the following information: • observe the pet in its cage for mentation, activity, locomotion, dyspnea, head posture, haircoat, and any grossly observable abnormalities. • note respiratory and heart rates before restraint when possible. observe the condition of cagemates. m key point if dyspnea or severe depression is detected, warn the owner that the animal is critically ill and could die of stress brought on by an examination. • handle such animals as little as possible. initially, treat severely ill animals symptomatically, then • note the type of diet and bedding as well as the level of sanitation and compare these with what was described in the history. • observe quantity and quality of feces and urine. diarrhea, soft stools, absence of stools, copious urine, and discolored urine all can be signs of illness. • coprophagy is a normal behavior in rodents. • check the diet and water supply for freshness, quantity, source, and accessibility. • evaluate the presence and suitability of cage furniture. adequate visual security and the ability to exercise and gnaw are extremely important. • an accurate weight in grams is extremely important for evaluating an animal's body condition, calculating drug dosages, and monitoring treatment. the easiest method of weighing a pet rodent is to place it in a box and then subtract the weight of the container. • restraint of pet rodents is easy with experience. pets that have been handled frequently and gently by the owners require only minimal restraint. gentle pressure directs the animal as needed. grasp less cooperative patients (except chinchillas and guinea pigs) by the scruff over the back of the neck with thumb and forefinger ( fig. 177-1) . take care to pinch enough skin to prevent the animal from turning around, yet leave enough slack for respiration. on smaller specimens, hold the base of the tail, if present, between the fourth and fifth fingers to provide additional restraint. • hold docile guinea pigs with the palm of one hand supporting the chest while the other hand supports the hind quarters ( fig. 177 -2). place the thumb and forefinger of the first hand in the axillas for additional control. • take care to minimize damage to the fur when handling chinchillas because they lose hair easily. grasp the animal by the tail and scoop it up into the palm of the same hand ( fig. 177-3 ). if necessary, grasp the thorax just behind the axillas. • calm uncooperative rodents by placing an appropriately sized towel over the head. complete the physical examination by wrapping the patient in a towel and exposing only needed areas. even oral, ophthalmic, and aural examinations can be performed with minimal effort if the animal is given the chance to relax in the towel "burrow." • remove particularly aggressive patients from their cages by scooping them up in a can or bucket; then slide them out onto a slick surface and pick them up or transfer them to a holding area or scale. m key point lift the hind quarters of mice and rats by the base of their tails to facilitate scruffing. never use the tip of the tail for restraint, or the skin of the tail may slough. once the animal is restrained properly, examine the head. assess the cranial nerves. check the nose for presence and character of discharge. examine the mouth for ptyalism, swelling, overgrown incisors, or discharges. to inspect the oral cavity, place an avian speculum across the mouth just caudal to the incisors. use a light source and a pair of hemostats as retractors to improve access. alternatively, use an otoscope with a pediatric head to examine the premolars and molars of guinea pigs and chinchillas for overgrowth. examine the cheek pouches of hamsters for swelling, impaction, or discharge. an ophthalmic examination, including a fundic examination, is important. • use a slit lamp to identify superficial pathology, especially corneal ulcers or foreign bodies. • if indicated, perform fluorescein stain and conjunctival scrapings or cultures. • note the presence of conditions such as discharge, asymmetry, and exophthalmos. m key point gerbils, rats, and mice produce red tears (chromodacryorrhea) with stress or disease. do not confuse them with hemorrhage. • guinea pigs suffering from hypovitaminosis c often produce dry, white tears. • check ears for discharge, foreign bodies, and mites. bluish discoloration of the ears is a sign of cyanosis. bright red injected coloration is associated with septicemia. sores behind the ears and on the neck are often a sequela of aural disease. • evaluate submandibular, axillary, inguinal, and popliteal lymph nodes for size and consistency. enlargements usually indicate infectious or neoplastic disease. • reevaluate respirations and heart rate after the stress of handling and compare them with the resting rate noted when the animal was in the cage. note dyspnea or respiratory sounds. auscultate animals weighing more than 200 g. counting every third or fourth beat and multiplying by the appropriate factor allows recording of heart rates up to 500 bpm. • palpate the abdomen. pay special attention to differentiating pregnancy from the bladder, kidneys, abdominal masses, enlarged cecum, and fecal balls in the colon. while palpating the abdomen, examine the mammary chain of all female rodents for signs of mastitis, lactation, or neoplasia. also check male mice and rats for mammary neoplasia. mammary tissue extends from the base of the neck to the base of the tail. gerbils typically have an elliptical sebaceous gland on their ventral midline. do not confuse this with neoplasia or infection. check the rectum and perineal area for signs of diarrhea, prolapse, irritation, parasites, and bite wounds. note that coprophagy is normal in rodents. • evaluate the urogenital tract for signs of inflammation, foreign bodies, urine scalding, and vaginal discharge. locate and palpate the testicles in males. the easiest method of determining sex in pet rodents is to compare the anogenital distance, which is twice as long in males as in females. other characteristics that allow the determination of sex are as follows: • visualization or palpation of testicles or extrusion of the penis from the prepuce indicates a male. • the presence of two external openings (i.e., anus and urethra) indicates a male. • the presence of three openings (i.e., anus, vagina, and urethra) indicates a female. • examine the skin and fur for conditions such as crusts, alopecia, masses, herniations, and wounds. • check tail and feet for swellings, coloration, sores, length of toenails, and condition of footpads. • evaluate the extremities for trauma or other abnormalities. apply cellophane tape to crusted areas of the skin and view under a microscope as an aid in diagnosing ectoparasites such as lice, mites, and fleas. skin scrapings are beneficial in detecting mites and dermatophytes. dermatophytes are diagnosed best through culture of broken hairs or crust on dermatophyte test medium. use small, cotton-tipped swabs to obtain ear swabs from animals weighing more than 25 g. mix debris with mineral oil and view under low magnification to test for ear mites, or roll onto a glass slide and gram stain to look for bacterial or yeast infections. collect urine by placing the rodent in a clean meshbottomed cage with a plastic liner. after enough urine has been produced, collect it off the bottom of the cage with hematocrit tubes or a syringe and a 25-gauge needle. perform cystocentesis on non-pregnant animals weighing more than 100 g with a 25-to 27-gauge needle. collect feces over several hours to provide a volume sufficient for fecal flotation. flotation allows the detection of nematodes and some trematodes and cestodes. cellophane tape applied to the perineal area and then viewed under a microscope often reveals oxyurid eggs. use a fresh saline smear or fecal sedimentation to diagnose protozoal parasites. fecal cultures are useful in diagnosing bacterial diarrhea. radiology is an extremely useful tool. machines capable of exposures as low as 40 kvp and 3 to 10 mas effectively image mice. most radiograph machines are capable of generating diagnostic radiographs of guinea pigs, chinchillas, and mature rats at settings used for kittens. positioning is accomplished with masking tape or velcro straps. sedate unruly animals. techniques used in cats for contrast studies of both urinary and gi systems are modified easily for use in pocket pets. • use lateral or medial saphenous veins to obtain samples in animals heavier than 100 g. liberally clip the area to allow exposure of the vessel before attempting venipuncture. place a 25-to 27-gauge needle in the vein and collect blood into microtainers or hematocrit tubes as it drips from the hub of the needle (fig. 177-4) . take extreme care not to col-lapse and lacerate the vein with overzealous aspiration if a syringe is attached to the needle. • it is also possible to use the cephalic vein in guinea pigs. • jugular veins are good alternatives in thin individuals under sedation. • an alternative technique that is useful in smaller animals is to coat the skin over the vein with a thin layer of petroleum jelly and then to puncture the vessel. blood is collected with a hematocrit tube as it exits the wound. samples up to 1% of the animal's weight are considered safe, even in stressed animals. m key point attempt tail bleeding only as a last resort in mice, rats, gerbils, and hamsters. these techniques often are not acceptable to owners. to bleed the tail, warm the tail with water or compresses to dilate the tail vessels. in large rats, perform venipuncture with a needle and obtain blood in the usual fashion. in smaller animals, lacerate the tip of the tail. blood from the wound is collected as described previously. see tables 177-5 and 177-6 for hematology and chemistry values. incorporate oral medications into a treat, or administer them in liquid form. if the medication is palatable, administer it by placing the tip of a dosing syringe into the diastema. m key point take care not to place the tip into the contralateral cheek pouch, or the patient may store the medication and expel it later. administer medication in small amounts. ensure that the animal swallows the medication in its mouth before more is administered. this technique is useful for force-feeding pellet gruels to anorexic pets if the caregiver is patient. medication or food that is administered too quickly will be spit out or aspirated. for rodents that are intractable or for administration of unpalatable substances, pass a stomach tube per os. • metal feeding needles, red rubber urinary catheters, or infant feeding tubes work well. selection is based on the size of the animal and individual preference. metal feeding needles with ball tips frequently are used in patients weighing less than 100 g . the metal provides the necessary stiffness to pass a tube of small diameter. the ball at the end of the needle makes it difficult (but not impossible) to pass the tube into the trachea. these tubes have the potential to create esophageal tears with improper restraint or when excessive force is applied. • measure the length from the tip of the nose to the last rib. ventroflex the head slightly, and place the tip of the tube through the diastema and over the tongue. if the tube does not pass easily down the esophagus to the premeasured distance, check the tube size and/or reposition the tube before attempting further advancement. the needle is easily palpable percutaneously if it is placed correctly. it is usually safe to administer up to 3 ml/100 g body weight. • a flexible catheter is ideal for use as a stomach tube in larger rodents ( fig. 177-6 ). use a speculum to prevent chewing on the tube. an otoscope head, avian speculum, or piece of wood or plastic with a hole drilled in the center works well. measure and mark the tube for the distance from the tip of the nose to the last rib. place the speculum in the mouth and over the tongue. pass the tube while holding the speculum in place and slightly ventroflexing the head. resistance is encountered if the tube is malpositioned or is an inappropriate size. the tube must pass over the tongue before it can be advanced down the esophagus. this is difficult in some animals. palpate the throat to confirm the presence of the feeding tube in the esophagus. m key point because the placement of a stomach tube is a blind procedure, administer a small volume of sterile saline into the tube before administering the medication to ensure that the tube is not in the trachea. misplaced medications are fatal. • this method is also useful for administration of nutrition to anorexic patients. place a pharyngostomy tube if repeated dosing is necessary, using the technique for cats. flush pharyngostomy tubes with water at least every 4 to 6 hours. nasogastric tubes are not recommended because they are difficult to place and maintain patency because of their small size. securely suture all tubes to the skin. place a tube collar made of radiographic film or use rear leg hobbles to prevent removal of tubes. • nutritional support is critical in rodents because of their high metabolic rate. provide supplements in animals that are anorexic for longer than 12 hours. if the gi tract is capable of digestion, use a slurry of pellets mixed with a high-calorie supplement. if the tube diameter is too small for this mixture, use avian hand-feeding formula or a mixture of vegetable and cereal baby foods in place of the pellets. if the ability of the gi tract to tolerate enteral feeding is questionable, first try isotonic electrolyte or dextrose solutions. parenteral nutrition is used successfully in research animals and may be feasible in select pet cases. administer sc injectable medications or fluids over the shoulder blades or in folds of skin on the flank. • avoid irritating substances in rats and mice because their mammary tissue extends into these areas. the resulting inflammatory response is thought to increase the occurrence of mammary neoplasia. m key point in general, avoid streptomycin and the carrier procaine in all pet rodents because of a high incidence of toxicity and hypersensitivity reaction. give im injections in the semimembranous and semitendinous muscles. inject only small volumes of nonirritating substances, or tissue damage with resulting self-mutilation may occur. use the epaxial or triceps muscles if repeated injections are necessary. m key point use intraperitoneal (ip) injections only as a last resort for large volumes of fluids or for irritating injections that cannot be administered via an iv or io route. express the bladder and aseptically prepare the abdomen. restrain the rodent with its head down to move the abdominal organs cranially. give the injection 0.5 to 2 cm lateral to the midline in the caudal abdomen. aspirate before injecting to ensure that the injection is not being given into the bladder or bowel. never use this technique in pregnant animals. give iv injections into any of the veins as previously described. in addition, the penile vein may be used in hamsters and guinea pigs. placement of iv catheters is possible in animals heavier than 100 g. for small rodents, give a bolus of fluids every 2-4 hours, followed by a diluted heparin flush. a pediatric iv pump is used for continuous infusion of fluids to mark distance from nose to last rib larger animals. maintenance of catheters in active animals is extremely difficult. for io injections, place a spinal needle into the proximal tibia or femur following the technique used for placing an intramedullary pin. once the needle is seated, remove the stylet. aspirate and check the hub of the needle for bone marrow. the tip of the needle should be in the bone marrow cavity that directly drains into the central venous system in normal bones (i.e., the cortex must be intact). administer drugs, blood, or fluids at a rate similar to that used for iv catheters. • in chinchillas and guinea pigs, withhold food for 6 hours before anesthesia. withhold food from smaller, mature rodents for 2 hours. withhold food from immature animals for up to 1 / 2 hour depending on age and condition. • use heat lamps and heating pads to prevent hypothermia. have a prewarmed incubator available for recovery. preoperative or intra-operative warmed sc or iv fluids are strongly recommended. place iv or io catheters whenever possible. • administer atropine preoperatively to reduce airway secretions. acepromazine, diazepam, or midazolam work well as premedications for other anesthetics. avoid acepromazine in gerbils because it potentiates seizures. see table 177 -7 for anesthetic drugs and dosages. surgical anesthesia is reached when toe, tail, and ear pinch fail to generate a withdrawal reaction. depth of anesthesia is best monitored by pulse and respiratory rate and character. pulses drop to within normal ranges after induction. further reduction, especially to less than 80% of the original stabilized value, is an indication to lighten the plane of anesthesia. monitor the electrocardiogram (ecg) of small patients by clamping the alligator clips onto the hubs of all-metal 27-gauge needles or steel sutures placed through the skin at the usual sites. tape cables to the table to maintain placement. doppler units taped over the chest also provide accurate heart rates. pulse oximeters are easier to use, more sensitive, and more expensive than the instruments mentioned previously. these instruments are easily taped to the patient's ear, foot, or tail and provide heart rates as well as information regarding oxygenation. respirations are often shallow and rapid during induction. they become deep and regular as a surgical plane of anesthesia is reached. the corneal reflex varies markedly between individuals and anesthetic agents. if the animal has a corneal reflex after induction and then loses it, reduce the anesthetic. induce gas anesthesia using small face masks purchased from laboratory supply houses or make them from syringe cases and latex gloves ( fig. 177-7) . induction in an anesthetic chamber is also possible. all rodents induced and maintained on gas anesthesia require some form of non-rebreathing system. usual induction is achieved between 2% and 3% for isoflurane and 2% and 4% for halothane. maintenance for isoflurane and halothane varies from 0.25% to 2%. there is marked individual variation in the amount of anesthetic required for induction and maintenance. use of 50% nitrous oxide in oxygen reduces anesthetic concentration requirements for other gases. m key point some chinchillas and guinea pigs hold their breath while being induced with gas anesthetics and then take deep rapid breaths. if the concentration of anesthetic gases is high enough, this behavior results in death. the risk of this behavior is reduced by premedication with tranquilizers, initial induction with nitrous oxide with later addition of primary anesthetic gas after relaxation, and low induction settings. changes in respirations, especially erratic or apneustic patterns and decreased respiratory rates, indicate deepening anesthesia. most pet rodents are not intubated for anesthesia because of their small size. when necessary, as in prolonged oral and other procedures, endotracheal intubation is accomplished with the animal in dorsal or ventral recumbency, depending on the clinician's preference. small non-cuffed or cole endotracheal tubes work well. a stylet usually is required to provide enough stiffness for the tube to pass the larynx. extend the animal's head and neck. grasp the tongue with forceps and use gentle traction. the tip of the tube then is advanced above the tongue and just past its base. the hard palate is used to deflect the tip of the tube ventrally into the glottis. this is a blind procedure that is difficult to master. use of a laryngoscope is helpful in larger rodents. another technique is to place an over-the-needle catheter in the trachea and move it up retrograde through the larynx to act as a guide. the catheter is removed after the endotracheal tube is in place. it is extremely important that the tube be checked for patency. rodents produce copious respiratory secretions, which frequently clog endotracheal tubes. the small diameter allows these tubes to collapse or kink, resulting in asphyxiation of the patient. check patency at least every 2 minutes by applying positive pressure ventilation at 10 to 15 cm water and watching for excursion of the chest wall. if extending the head and neck does not result in air flow, suction the tube. if this is either not successful or impossible, remove the tube and continue anesthesia with a mask or reintubate the animal with a new tube. because of the small diameter of the trachea, endotracheal tube-induced tracheitis and subsequent swelling of the trachea may become a life-threatening situation. doses and routes for injectable anesthetics are listed in table 177 -7. needed doses for injectable anesthetics are tremendously variable among species and individuals. most injectable anesthetics provide safe sedation for minor procedures, but very few induce a safe surgical plane of anesthesia on a consistent basis. • ketamine in combination with diazepam is easily obtainable, is given intramuscularly, and has a wide margin of safety, but it does not provide good analgesia. • intraperitoneal injections of barbiturates provide surgical anesthesia but have a low margin of safety and a significant mortality rate. barbiturate anesthesia can result in fatal ileus. euthanasia is performed easily by induction of inhalant anesthetic through a mask or chamber followed by an overdose of barbiturates given intraperitoneally, iv, or intracranially. euthanasia by ip injection of barbiturates alone causes pain in some animals. • surgical techniques for pet rodents are similar to those used in cats and birds. • hemostasis is critical because of small blood volumes. • electrosurgery for incisions and cautery is highly recommended. • if necessary, give fresh blood transfusions drawn from a donor of the same species and mixed with sodium heparin (1000 iu/ml) at a rate of 0.005 ml/1 ml of blood directly into an iv or io line. • the lack of a filter creates a potential for thrombosis. • transfusion reactions are possible. administer postoperative analgesics to all rodents undergoing surgical or dental procedures. common analgesics include buprenorphine, butorphanol, ketoprofen, carprofen and meloxicam. see table 177 -7 for dosages. the most common surgeries are laceration repair and removal of dermal or sc masses. • most rodents will not gnaw on skin sutures. • if this occurs, use steel sutures, subcuticular sutures, or tissue glue. • if an animal still chews at its suture line, physical restraint, such as a tube or an elizabethan collar, is required. castration is a common procedure in guinea pigs. this usually is performed when owners want to house more than one male together or do not wish to breed their female any longer. common abdominal surgeries include cystotomy for urolith removal in guinea pigs and rats, and cesarean section (c-section) in guinea pigs and chinchillas because of dystocia. use a technique similar to those described for dogs and cats. preplaced stay sutures are recommended to define incision edges for closure. use 4-0 polyglactin 910 or polydioxanone (pds) on a taper needle and suture in a simple continuous pattern to close the body wall. close the skin with a subcuticular suture (absorbable) or interrupted skin monofilament, nonabsorbable suture. fracture fixation is accomplished best with intramedullary pinning or kirschner apparatus. rodents gnaw on bandages until they remove them. if they are unable to remove a splint, self-mutilation often results in self-amputation. if a cast or splint is necessary, physical restraint often is required. healing usually takes 3 to 6 weeks. incisors can be trimmed with nail trimmers, but this technique often fractures the tooth, causing abscesses of the root. instead, use a high-speed dental burr or a flat cutting disk on a dremel hand tool. trim molars with a high-speed drill or pediatric rongeurs. a mouth speculum that deflects the tongue and other soft tissues is essential to prevent lacerations and provide working space ( fig. 177-8) . intubate the trachea to prevent aspiration pneumonia when working on molars. if a tooth is abscessed, extract both it and the occlusal tooth. • if necessary, approach cheek teeth via an incision through the cheek. • use a fine dental elevator to loosen the teeth. • patience and firm but gentle pressure are needed, or the root or surrounding bone may fracture. • the roots of the maxillary incisors curve dramatically back into the head. take care to follow the curve of the tooth. • packing an infected tooth socket with a calcium hydroxide paste may decrease the occurrence of persistent infection. remove the paste in 3 to 4 days. • administer meloxicam, carprofen or ketoprofen postoperatively to control pain. see table 177 -7 for dosages. in chinchillas with dental malocclusion, the roots of the molars can become impacted, causing swelling of the mandible or exophthalmos and epiphora. these teeth are extremely difficult to extract without causing extensive bony and soft-tissue damage. discourage breeding of animals with malocclusion, unless it was acquired as a result of trauma or infection, because this trait is hereditary. most pet and laboratory mice are derived from mus musculus, which is the common house mouse. mice sold in the pet trade are randomly bred and less likely to suffer from the genetic problems associated with inbred laboratory rodents. mice possess brown fat tissues between their scapulae that also are known as hibernating glands; these are thought to provide an energy store. the spleen in male mice is 50% larger than that of females. ectoparasites usually are found in new acquisitions. • alopecia and pruritus, especially on the back of the head and dorsal midline, usually are associated with lice (polyplax serrata), mites, or fleas. • mite infestation (e.g., mycoptes musculinus, myobia musculini, radfordia affinis) often causes a greasy haircoat and folliculitis. transmission of lice and mites occurs via direct contact. fleas are transmitted by other household pets, such as cats and dogs. • diagnosis is based on clinical signs, history, visualization of parasite, skin scrape, and cellophane tape test. • treatment of fleas and lice is with pyrethrin powder. ivermectin is recommended for treatment of mites (see table 177 -8). • change the bedding and thoroughly clean the cage between treatments to prevent reinfestation. occasionally, the surrounding environment needs to be treated with a premises spray used for killing fleas. • alopecia also may be the result of dermatophytes (see chapter 42). lesions are often hyperkeratotic. • diagnosis is made by skin scrape or isolation on culture. most dermatophytes found in pet rodents do not fluoresce under a wood's lamp. • treatment is with lime-sulfur dip or griseofulvin (table 177 -9). • ulcerative dermatitis is a common syndrome caused by staphylococcus aureus characterized by pododermatitis, mastitis, and abscesses in other areas. • administer antibiotics based on culture and sensitivity tests. chloramphenicol is recommended pending culture results (see table 177 -9). the application of hot packs, local drainage, and topical medications are also beneficial in selected cases. • mastitis also may be caused by escheria coli or pasteurella, klebsiella, pseudomonas, or streptococcus species. mastitis usually is caused by poor sanitation, abrasive bedding, or overly aggressive young. • preputial gland abscesses are fairly common in males and are usually caused by e. coli or s. aureus. local flushing and topical treatment are usually adequate. • sc abscesses can be the result of the aforementioned bacteria or actinobacillus spp. or corynebacterium kutscheri. corynebacteria is associated with widespread abscesses, septic arthritis, gangrene, and ulcerated draining tracts. diagnosis is based on finding grampositive pleomorphic rods on gram stain and isolation on culture. • the bacteria are usually sensitive to ampicillin, chloramphenicol, and tetracycline (see table 177 -9). • lymphoma and mammary neoplasia are common causes of sc masses. mammary neoplasia is usually malignant in mice, and metastasis to the lungs is common. (mice have five pairs of mammary glandsthree thoracic and two abdominal.) • obtain thoracic radiographs before surgery. • give a guarded prognosis for long-term survival. • other possibilities for sc masses are fungal granulomas, nodules from the psorergates simplex mite, hematoma, hernia, non-neoplastic lymphadenopathy, or emphysema. • otitis externa usually is caused by ear mites, although bacteria or fungi also may cause primary or secondary otitis. • clinical signs include erythema, pruritus, waxy debris, and excoriations behind the ears. • mites may be diagnosed by identification on otoscopic examination or microscopic examination of ear swabs (see "techniques"). • treatment requires cleaning debris from ears with a commercial ear cleanser followed by administration of three doses of ivermectin at 2-week intervals or topical acaricides used daily for 3 to 4 weeks (see table 177 -9). • bacterial and fungal otitis is diagnosed by identification of organisms or gram-stained specimens and isolation on culture. • treatment is similar to that used in cats. • otitis media/interna usually are caused by hematogenous spread or local invasion of bacteria from a primary abscess. • clinical signs include head tilt, circling, facial nerve paralysis, and otitis externa. • rule out mouse hepatitis virus as the cause of the head tilt (see "gastroenterology"). • if treatment of the primary disease is successful, the otitis media usually resolves, although a residual head tilt may persist. • if a cluster of pseudomonas infections occurs in a population, evaluate the water source and produce for contamination. use sodium hypochlorite in the drinking water at 10 ppm to control an outbreak while water quality is being restored. • damage to the pinnae can be associated with trauma, dermatitis, pox virus, hypersensitivity reactions, and vasculitis. dry gangrene is a common sequela and is usually self-limiting. i have observed a steroid-responsive pruritus in pet mice. the pruritus is severe enough to result in significant self-mutilation. this condition has been nonresponsive to treatment with ivermectin, lime-sulfur dips, griseofulvin, multiple antibiotics, oral prednisolone, and antihistamines. attempts at bacterial and fungal culture have failed to identify a pathogen. an inflammatory response is observed on histologic examination. mice with this condition respond to repository methylprednisolone injections every 2 to 4 weeks (1.0 mg/kg im). most owners have not elected to continue injections for longer than a few months. once the injections are stopped, the pruritus returns, often requiring euthanasia of the affected mouse. • bilateral alopecia found around the muzzle associated with no other abnormalities usually is caused by friction from overhead feeders. • alopecia occurring in smaller, weaker individuals is often the result of barbering. removal of the mice in best condition from the cage results in normal appearance of barbered mice in 2 to 3 weeks. • tailhead alopecia and scabbing are usually the result of aggression. separate affected animals, or additional trauma may occur. • other rare causes of alopecia are endocrinopathies, leprosy, and hereditary alopecia in nude mice. • epiphora is a common condition of pet mice. the most common causes in pets owned for longer than 2 months are ammonia fumes and overgrown incisors. • ammonia causing contact irritation is diagnosed by examining an uncleaned cage and checking for odor. • treatment is improved sanitation. • overgrown incisors are diagnosed easily by oral examination. treat by trimming the affected teeth and providing opportunities for gnawing. • foreign bodies and the resultant corneal ulcers can cause epiphora. an eye examination, including fluorescein stain, is indicated. treat by removing the foreign body and administering an ophthalmic antibiotic (gentamicin, tetracycline, or chloramphenicol in affected eye, q8h-q6h). • in newly acquired pets, epiphora is often the first clinical sign of an upper respiratory infection. pasteurella pneumotropica is the most common pathogen, although salmonella spp., mycoplasma, sendai virus, lymphocytic choriomeningitis, and mouse pox also may cause epiphoria. the ocular discharge later appears mucopurulent (see "respiratory"). retinal degeneration can be either hereditary or (in albino mice) may be caused by exposure to highintensity lighting. the resulting blindness often goes undetected because patients adapt well and behave normally in their cages. • clinical signs include dyspnea (often described as chattering), mucopurulent oculonasal discharge, hunched posture, and anorexia. animals with a chronic history of this disease are often cachexic. • radiology aids in determination of the extent and severity of the pneumonia and the absence or presence of distant foci of infection. • treatment with tylosin is successful in controlling the disease if it is not too advanced. tetracycline, enrofloxacin, and chloramphenicol also have been used (see table 177 -8). • many patients need nutritional support. • mice with pyometra or other abscesses require surgical debridement. • recovered animals are carriers and stress elicits clinical signs. strictly quarantine these animals. a common cause of pneumonia in newly acquired mice is sendai virus. acute fatalities are seen in suckling or weanling mice. • transmission is by aerosol or direct contact. • clinical signs in adults are caused by secondary bacterial infections and are similar to those in mrm. • diagnosis is based on clinical signs and serologic testing. • treat with antibiotics to control the secondary bacterial infection and provide supportive care as needed. • prohibit breeding for 4 to 6 weeks. • a killed vaccine is available. • recovered animals are resistant to new infection. common primary or secondary pathogens causing respiratory signs in mice are streptococcus pneumoniae, corynebacterium kutscheri, pasteurella pneumontropica, pseudomonas aeruginosa, and klebsiella pneumoniae. treatment is empiric or based on culture and sensitivity of a tracheal swab sample. dyspnea often is caused by metastasis to the lungs from mammary adenocarcinomas. primary lung tumors, especially pulmonary adenomas, also occur frequently. although not frequently diagnosed, cardiac disease can result in signs of respiratory disease. diagnosis is based on radiographic evidence of cardiomegaly and pulmonary edema. • tapeworms usually do not cause clinical signs. occasionally, heavy burdens may cause diarrhea or weight loss. the chief concern is the zoonotic potential of one species, hymenolepis nana, which is directly transmissible to humans. • diagnosis is made from visualization of eggs in the feces. treat with praziquantel or niclosamide (see table 177 -8). improve sanitation, and remove indirect hosts (e.g., fleas, beetles, roaches). • pinworms (syphacia obvelata, aspiculuris tetraptera) may cause anal pruritus and, in severe cases, rectal prolapse. • diagnosis is based on clinical signs and observation of eggs on cellophane tape after application to the perineal region. treat with piperazine or mebendazole every 7 to 10 days for three treatments or with fenbendazole once daily for 5 days (see table 177 -8) and provide improved sanitation. • the protozoal parasite spironucleus muris causes diarrhea and slow growth associated with a pot-bellied appearance in young mice. • diagnosis is by fecal wet mount, although falsenegative findings are common. • treat with oxytetracycline (see table 177 -9). supportive care to combat dehydration and hypothermia is extremely important. • control is achieved with improved sanitation. • giardia spp. and, rarely, eimeria falciformis show signs similar to spironucleus. giardiasis is zoonotic. treat with metronidazole. treat eimeria with sulfadiazine/trimethoprim (see table 177 -9). most other protozoa are considered nonpathogenic. • cysticercus fasciolarus causes nonpathologic cysts of the liver. these cysts are the infective form of taenia taeniaformis in carnivores. viral diseases • diagnosis is based on clinical presentation, serology, and presence of syncytial giant cells in the epithelium of the small intestine. • treat supportively, and quarantine affected individuals. the prognosis is grave. • although less commonly seen in pet mice, reovirus occurs in older suckling mice. it is characterized by an oily diarrhea, which results in a greasy haircoat. other signs are conjunctivitis, stunted growth, and tremors. transmission is by ingestion. • diagnosis is based on clinical signs, histology, and serology. • treat supportively. the long-term prognosis is grave. initial survivors are weak and jaundiced, suffer from alopecia, and eventually die. • transmissible murine colonic hyperplasia (mch) caused by citrobacter freundii is characterized by diarrhea followed by rectal prolapse and stunted growth. transmission is feco-oral. • diagnosis is made by clinical signs and fecal culture. • treat with neomycin, tetracycline, or sulfamethazine until sensitivity results are available (see table 177 -9). severe thickening of the distal half of the colon is observed at necropsy. • salmonellosis, also known as mouse typhoid, is transmitted by latent carriers or contaminated feed or bedding. incubation lasts for 3 to 6 days. • clinical signs are lethargy, anorexia, purulent conjunctivitis, arthritis, and diarrhea. • diagnosis is based on clinical signs and fecal culture. treat supportively. use of antibiotics is controversial. • quarantine survivors. • sanitation is extremely important because salmonella spp. are zoonotic. on gross postmortem examination, erythema of distal ileum and congestion of the spleen and liver are seen. with more chronic infections, necrotic foci are seen in the liver, spleen, and lymph nodes. prevent infection by feeding a fresh laboratory chow from a reputable source. thoroughly wash all produce and then dip it in a diluted bleach solution. rinse completely before feeding. • tyzzer disease is caused by bacillus piliformis. transmission is feco-oral. • clinical signs are precipitated by stress and consist of anorexia, diarrhea, and high mortality in weanlings. • diagnosis is made by clinical signs or isolation on culture. enteritis and multiple gray-yellow necrotic foci in the liver are seen on gross postmortem examination. • administer tetracycline for 4 to 5 days (see table 177 -9) and reduce stress to control the disease. breeding systems vary; from one to six females may be placed with one male. all animals are housed together, and the young are removed after weaning. females in proestrus have swollen vulvas. vaginal plugs are present post-copulation. female mice that have been bred within 4 days abort if a new male is placed in the cage. inappropriate light cycle, inappropriate age, crowding, and poor nutrition are the most common causes of infertility in pet mice. pyometra due to pasteurella pneumontropica, mycoplasma spp., or other bacteria is also common. desertion of litters is usually a result of stress, lack of nesting materials, agalactia, or mastitis. • urethral obstruction from proteinaceous plugs of inspissated ejaculum may develop in aged male mice. pseudomonas, e. coli, or proteus are the most frequently cultured pathogens. before complete obstruction, chronic hematuria may be noticed by the owner. • antibiotics, which are chosen based on the results of urine culture, are often curative with early presentation. complete obstruction requires surgical removal. • glomerulonephritis is very common in geriatric mice. it frequently is secondary to chronic viral infection. clinical signs are anorexia, lethargy, dehydration, and cachexia. urinalysis demonstrates proteinuria. as the disease progresses, the urine becomes isosthenuric, the blood urea nitrogen (bun) and creatinine levels rise, and other electrolyte abnormalities typical of chronic renal failure occur. treat supportively. prognosis for long-term survival is grave. • coccidia (e.g., klosseilla muris) occasionally is found in the urine. the clinical significance of its presence is unknown. • mice can be asymptomatic carriers of leptospirosis; however, this is rarely seen in pet mice. • diagnosis is based on darkfield microscopy of urine, serology, or histopathology. euthanasia of carriers is recommended. • infectious polyarthritis or mouse rheumatism is caused by streptobacillus moniliformis. in humans, it is known as rat bite or havernill fever. transmission is by direct contact. clinical signs are cachexia, keratitis, edema and ulceration of the appendages, and ankylosing arthritis. • diagnosis is based on the bacterial culture findings or the presence of caseous pericarditis and arthritis on necropsy. • treat with antibiotics chosen through the results of culture and sensitivity tests. use penicillin while awaiting results. supportive care is important. animals that recover remain arthritic. control is achieved through quarantine and sanitation. • the most frequently diagnosed neurologic disease in pet mice is head tilt resulting from bacterial otitis media (see "otitis"). the second most common cause of neurologic signs is trauma. • diagnosis is based on history and clinical signs. consider neoplasia in aged mice with slowly progressive signs. • lymphocytic choriomeningitis is a zoonotic arenavirus. transmission is airborne, transplacental, or by direct contact, fomites, or insect vectors. acute signs usually occur in mice that are 3 to 6 weeks old. approximately 20% of infected individuals show acute clinical signs, which include lethargy and photophobia followed by convulsions and paralysis. in animals that are latently infected, glomerulonephritis develops later. mice infected after weaning and before 1 year in age lose weight, appear arthritic, and show signs of conjunctivitis and photophobia. the virus runs its course in several weeks. animals that recover show no residual signs. • diagnosis is based on clinical signs and the presence of immunofluorescent antibody (ifa). pleural effusion, splenomegaly, and hepatic lipidosis are found on necropsy. treat supportively. house survivors separately. • prevent the disease by improving sanitation, providing pest control, and cleaning produce. consider euthanasia because of the zoonotic potential of the virus. • mouse poliomyelitis/encephalomyelitis, also known as theiler disease, causes clinical signs in 1 in 10,000 infected mice. two-thirds of healthy mice are carriers. transmission is by oral or respiratory routes. mice younger than 4 weeks of age show signs of encephalitis. animals that are 6 to 10 weeks old are weak in the rear legs and progress to paralysis. the tail may remain mobile. affected mice continue to eat and be alert. albino mice are predisposed to show clinical signs. • diagnosis is based on clinical signs, serology, or histopathology that shows necrosis of the ganglionic cells of the anterior horn of the spinal cord. • treat supportively. consider euthanasia because of poor prognosis. • seizures in mice commonly result from otitis media, trauma, liver or kidney failure, toxin, bacterial meningitis, neoplasia, or viral encephalitis. • leukemia in mice is usually viral in origin. transmission is trans-mammary or trans-placental. • clinical signs are anemia, dyspnea (with thymic involvement), and those signs that are compatible with chronic disease. • diagnosis is based on complete blood count (cbc), bone marrow aspirate, or histopathology. prognosis is grave. • eperythrozoon coccoides is a rickettsial red blood cell (rbc) parasite of mice. affected mice are usually asymptomatic. occasionally, fever, anemia, and splenomegaly develop in infected animals. transmission is through the louse polyplax serrata. control is by extermination of the louse. • treat with tetracyclines. pet rats are derived from the norwegian or brown rat (rattus norvegicus), which did not originate from norway, but from asia. breeds of rats are called strains when they are inbred extensively and stocks when strains are hybridized. rats have brown fat, as discussed in the section on mice. they do not possess a gallbladder. their mandibular symphysis is articulated normally. rats are neophobic; therefore, make gradual changes in food or environment when possible. • fleas, mites (e.g., radfordia ensifera, ornithonyssus bacoti), lice (i.e., polyplax spinulosa), ear mites (i.e., notoedres muris), and dermatophytes cause similar signs in both mice and rats. treatment also is similar (see "mouse"). • sc masses in rats are similar to mice. pasteurella pneumotropica is a very common pathogen in mastitis and sc abscesses. • treat with chloramphenicol until culture results are available (see table 177 -9). • mammary cancer develops in 50% to 90% of adult female rats and in approximately 15% of male rats. always submit biopsy specimens for histologic examination. most, but not all, of these tumors are fibroadenomas, which are benign. prognosis for longterm survival after surgical removal is good. other common neoplasms include interstitial cell tumors of the testes, which cause sc swellings in the inguinal region, and squamous cell carcinomas of the zymbals gland of the external ear canal. • ulcerative dermatitis occurs in rats as well as mice. staphylococcus aureus is the causative agent. c. kutscheri follows a similar course in rats and mice (see "mouse"). • ringtail is the formation of constrictive bands of fibrous tissue around the tail in nestling rats. these bands result in gangrene of the distal tail. this disease occurs when environmental humidity is less than 40%. • treat by making a longitudinal incision of the ring to release the stricture and apply topical dimethyl sulfoxide (dmso), steroid, and antibiotic solution (10 ml dmso, 6 ml 50 mg/ml amikacin, 4 ml 2 mg/ml dexamethasone) four times daily. • to prevent ringtail, keep humidity above 50%, use solid-bottom cages and provide ample nesting material. prognosis for life is excellent. prognosis for retention of the distal tail is guarded. • epiphora and blepharospasm are caused mostly by ammonia fumes, overgrown incisors, or foreign bodies (see "mouse"). • sialodacryoadenitis virus is a coronavirus that is endemic in many rat populations. • clinical signs vary from mild keratoconjunctivitis to blepharospasm, chromodacryorrhea, severe uveitis, hyphema, buphthalmos, periorbital swelling, and pneumonia. the clinical course of the disease lasts 10 to 14 days. rats maintain normal activity levels and appetite. • treatment is not necessary for mild infections. place rats showing marked ocular disease or discomfort on the appropriate ophthalmic ointments (e.g., atropine, antibiotic, steroid) based on presentation. administer parenteral antibiotics to animals that show signs of respiratory problems. recovery is usually complete unless the eye ruptures or selfmutilation occurs. • control is achieved by not introducing new animals for 4 weeks. • in contrast to mice, sendai virus rarely causes clinical signs in rats. • mucopurulent ocular discharge also may be caused by infection with mycoplasmosis, streptococcus pneumoniae, pseudomonas spp., and other less common bacterial or viral agents that cause pneumonia. • cataracts are primary hereditary defects or occur secondary to severe uveitis or diabetes mellitus. retinal dystrophy and colobomas are also inheritable traits in rats. retinal degeneration occurs in rats housed under intense lighting. • mrm is extremely common in pet rats. its presentation is similar to the disease in mice (see "mouse"). • streptococcus pneumoniae is normal flora for rats. however, during stressful situations, bacteremia may occur, resulting in pneumonia. clinical signs are similar to mrm. differentiation is based on culture and the presence of extensive fibrinopurulent pleural effusion on necropsy. • ampicillin controls clinical signs if treated early in the course of disease (see table 177 -9). prevent the condition by minimizing stress. • corynebacterium kutscheri and pasteurella pneumotropica cause signs similar to mrm (see "mouse"). there is a serologic test for c. kutscheri. see the mouse section for a discussion of pseudomonas aeruginosa. • pneumocystosis carinii is an uncommon protozoa that infects the lung. cysts and trophozoites live in the alveoli. clinical signs occur only in immunocompromised or geriatric individuals. signs are cachexia, cyanosis, and dyspnea. • diagnosis is based on clinical signs, tracheal wash, response to therapy, or histologic examination. • treat with sulfadiazine/pyrinrethamine (see table 177 -9). • myocardial degeneration and subsequent congestive heart failure are fairly common in geriatric rats. diagnosis is based on radiographs of the thorax and clinical signs. treat supportively, and use furosemide and digitalis at cat dosages to alleviate pulmonary edema. • polyarteritis nodosa is an idiopathic condition of geriatric rats that results in thickening and tortuosity of arteries, especially in the mesentery, pancreas, and testicles. affected areas are predisposed to clot formation and aneurysms. • nematode (syphacia muris), cestode, and intestinal protozoal parasite infestations are similar to those in mice. • capillaria hepatica has no clinical significance. yellow streaks on the liver are an incidental finding at necropsy. the causes and treatment of malocclusion are similar to those for mice. • epizootic diarrhea of suckling rats is a viral disorder found in rats 7 to 14 days old. the infection causes a mild diarrhea. most animals recover. occasionally, stunting occurs. treat supportively. • salmonellosis in rats is similar to that in mice. • if breeding is desired, take females showing signs of estrus (e.g., lordosis, hyperactivity, quivering ears, and swollen vulva) to a male rat's cage for 24 hours, or keep one male in a cage with up to six females. check females for a post-copulatory plug to confirm breeding. remove females just before parturition, and house females individually while raising the young. a vaginal discharge is seen 1.5 to 4 hours before labor. parturition is accompanied by stretching and extension of the rear legs. all neonates usually are delivered within 1 to 2 hours. • two extremely common conditions in geriatric rats are nephrocalcinosis and chronic progressive nephropathy. clinical signs are compatible with those of chronic renal failure. enlarged or small irregular kidneys may be found on physical or radiographic examination. isosthenuria and marked proteinuria are found in urinalysis. • definitive diagnosis is based on renal biopsy. • treat supportively. prognosis for long-term survival is grave. • trichasomoides crassicauda is an uncommon parasite of the urogenital tract. the adult worms usually reside in the kidney, but they occasionally may wander into the genital tract. the ova are passed in the urine. • clinical signs are hematuria and stranguria. proliferative mucosa of the bladder occasionally may be palpated as an abdominal mass. • treatment is somewhat successful with methyridene (see table 177 -8). sanitation is critical in control of this disease. • klossiella muris is an incidental coccidia of the urinary tract. • many geriatric pet rats have chronic progressive radiculoneuropathy. • clinical signs are compatible with cauda equina syndrome, including posterior paresis progressing to paralysis, urine retention, and incontinence. prognosis is grave. • treat supportively or euthanize. • streptobacillus moniliformis, a normal bacteria found in the oral, nasal, and pharyngeal cavities of rodents, is isolated from 43% of middle ear infections and 35% of chronic pneumonias in rats. the bacteria is nonpathogenic for gerbils and guinea pigs. • clinical signs vary with the site of infection. head tilt and circling, septic arthritis, and respiratory disease commonly are seen. • diagnosis is based on isolation on culture. the clinical signs mimic many other diseases, especially mrm and pseudomonas infection (see "mouse"). • head tilt in rats also may be the result of trauma or neoplasia, especially pituitary adenomas. • hemobartonella muris is an rbc parasite of rats that is nonpathogenic unless the rat is immunocompromised or splenectomized. transmission is through the louse polyplax spinulosa. • clinical signs result from hemolytic anemia and hemoglobinuria. • treat with tetracyclines (see table 177 -9). mesocricetus auratus, better known as the golden or syrian hamster, is a primarily nocturnal rodent that originated in the middle east. almost all hamsters in the united states are the offspring of three siblings imported in the 1930s. many color variations are available. long-haired hamsters are called "teddy bear" hamsters. the stomach has two compartments, a non-glandular forestomach, which functions like a rumen, and a glandular stomach. hamsters are very territorial. they possess flank glands, which are larger in males, that are rubbed against objects to mark their territory. females are larger than males. except during estrus, they use this size advantage to attack males. do not allow groups to estivate together or recently awakened animals may cannibalize sleeping hamsters. m key point hamsters are extremely sensitive to antibiotics. penicillins, clindamycin, lincomycin, streptomycin, tylosin, erythromycin, and cephalosporins eliminate the normal intestinal flora, allowing overgrowth of pathogenic bacteria, particularly clostridium difficile. diarrhea, which is almost always fatal within 3 to 7 days, subsequently occurs. even antibiotics considered to be safe can have this effect. treat by discontinuing antibiotics, providing a lactobacillus supplement, and giving supportive therapy. • hamsters are susceptible to demodex criceti and d. aurati mites. d. criceti is limited to skin folds. d. aurati causes hyperpigmentation, alopecia, and seborrhea sicca affecting the dorsal midline. demodex is carried by many normal-appearing hamsters. • clinical signs occur in immunosuppressed animals, as would occur with stress, chronic infection, pregnancy, or malnutrition. • diagnosis is based on clinical signs and deep-skin scrapings. • treat with amitraz every 2 weeks for two treatments past two consecutive negative skin scrapings. use the manufacturer's recommended dilution for dogs. • sarcoptes mites infrequently cause facial alopecia. diagnosis is based on skin scraping. treat with ivermectin (see table 177 -8). do not confuse this condition with alopecia caused by contact with feeders or barbering. • notoedres mites affect only the external ear canal in female hamsters but may affect the ears, feet, geni-talia, and tail in males. diagnosis is made by observation of mites on samples from ear swabs, skin scrapings, or both. treat with ivermectin (see table 177 -8). • other less common causes of alopecia in hamsters are dermatophytosis, endocrinopathies, and genetic defects. • dermal sc masses are usually abscesses caused by pasteurella pneumotropica, s. aureus, or streptococcus spp. treatment is based on results of culture and susceptibility testing. use chloramphenicol until culture results are available. other frequent causes of sc swellings are distended cheek pouches and testicles, mastitis, hernias, neoplasia, and lymphadenopathy. • epiphora and conjunctivitis are caused most frequently by increased environmental ammonia concentrations, incisor overgrowth, foreign body, or lymphocytic choriomeningitis (see "rat"; "mouse"). • mucopurulent discharge is caused by secondary infection by pasteurella or streptococcus spp. • hamsters are predisposed to rupture of the eye following trauma or infection. surgical enucleation is advised. electrosurgery is extremely helpful in controlling bleeding but do not apply heat to the stump of the optic nerve or vessels, or thermal injury to the brain may result. place gelfoam in the socket to enhance clot formation. • hamsters are susceptible to viral respiratory infections of humans. • clinical signs include nasal discharge, sneezing, otitis media, fever, and pneumonia. uncomplicated cases last 5 to 7 days. complications are usually the result of secondary bacterial infections. • treat supportively. use of antibiotics is indicated if copious nasal discharge, dyspnea, anorexia, or marked lethargy is observed. overuse of antibiotics may cause diarrhea-related death in hamsters that might have recovered uneventfully if left untreated. • most dyspnea in hamsters is caused by blunt thoracic trauma. hamsters often bite when startled. reflex actions on the part of humans, especially children, cause hamsters to be flung against hard objects. • diagnosis is by history and presence of fresh epistaxis. • treat supportively. emergency shock therapy, consisting of supplemental heat, oxygen administration, parenteral fluids, and glucocorticoids, frequently is required. • sendai virus can cause death in suckling hamsters housed with mice. adults show no clinical signs (see "mouse"). • primary bacterial pneumonia most frequently is caused by yersinia pseudotuberculosis, pasteurella pneumotropica, or streptococcus. clinical signs are compati-ble with those of pneumonia seen in other species, as well as weight loss and conjunctivitis. all three agents have a tendency to form distant abscesses, especially in the uterus. • diagnosis is based on clinical signs and isolation on culture. • treat with chloramphenicol until antibiotic susceptibility results are available. abscesses require surgical debridement; however, anesthesia in affected animals is very risky. recovered hamsters are carriers and must be quarantined from other rodents. prognosis is guarded. • cardiac thrombosis is seen in 73% of geriatric hamsters. most thromboses occur in the left atrium and are secondary to degenerative cardiomyopathy, cardiac amyloidosis, sepsis, or calcification of the great vessels. congenital myocardial necrosis also occurs. • clinical signs include cyanosis, dyspnea, and acute death. enlargement of the cardiac silhouette and pulmonary edema sometimes can be seen on thoracic radiographs. • furosemide and digitalis (using standard cat doses) may temporarily alleviate clinical signs. • hamsters can carry the zoonotic tapeworm h. nana (see "mouse"). • treat with niclosamide or praziquantel (see table 177 -8) and provide improved sanitation. • pinworms (aspicularis tetraptera, syphacia muris, s. obvelata) occur in hamsters as well as in mice. • treat with fenbendazole (see table 177 -8). • hamsters are predisposed to dental caries. a large percentage of affected teeth become abscessed, causing facial swelling, ptyalism, and anorexia. • diagnosis is based on clinical signs, oral examination, skull radiographs, and isolation on culture. • extract the tooth and administer antibiotic therapy based on results of susceptibility testing. prognosis is variable depending on the condition of the animal, tooth affected, and extent of the abscess (see "mouse"). • overgrown incisors also occur, as in mice. • the cheek pouches are very distensible. impaction of the pouches occurs on occasion. • clinical signs vary from ptyalism to swelling from abscess. in simple cases, removal of the material from the pouch with fine forceps is sufficient. sedation usually is not required. • if a fungal or bacterial infection of the pouch is present, remove the exudate, submit samples for gram staining and bacterial or fungal culture, and flush the pouch with diluted iodine solution. if cellulitis is present, administer systemic antibiotics as well. fistulas often heal spontaneously. • proliferative ileitis (i.e., wet-tail disease) is thought to be caused by a campylobacter-like organism with or without concurrent bacterial or viral infections. more than 90% of animals with clinical signs die. the highest morbidity and mortality rates occur in hamsters 3 to 8 weeks of age. teddy bear hamsters may be more susceptible to infection than shorter-haired varieties. transmission is feco-oral. • clinical signs include diarrhea, which mats on the ventrum and perineum, and results in anorexia, dehydration, and a hunched posture. the abdomen frequently seems painful on palpation. bowel loops often are distended on palpation because of ileal obstruction or intussusception. rectal prolapse usually occurs. • administer neomycin, gentamicin, metronidazole, or tetracycline (see table 177 -9). supportive care is critical. prognosis is grave, even with treatment. gross postmortem findings include gas and yellow diarrhea in the distal intestinal tract, mucosal thickening in the ileum and distal jejunum, peritonitis, and liver abscesses. • other common causes of bacterial diarrhea include e. coli, tyzzer disease, or salmonella spp. (see "mouse"). in hamsters older than 1 year of age, liver cysts that are derived from the biliary duct often develop. less frequently, similar cysts arise from the pancreas, epididymis, and seminal vesicles. this syndrome is called polycystic disease. no clinical signs are associated with cysts in these structures, which are an incidental finding on abdominal palpation. no treatment is recommended. • timing is critical to prevent injury to the male when breeding hamsters. transfer the female to the male's cage in the early evening 3 days after a creamy, viscous vaginal discharge is noticed. monitor the pair carefully. remove the male immediately if the female is aggressive. remove the male after mating or after 1 to 2 hours even if mating has not occurred. two days after successful copulation, a gray malodorous vaginal discharge is observed. pregnancy is highly likely if there is no translucent vaginal discharge 5 to 9 days post-breeding. pseudopregnancies last 8 to 12 days. normal gestation is 15 to 16 days. before par-turition, a hemorrhagic vaginal discharge appears, and the female may pant. hamsters rarely suffer from pregnancy toxemia (see "guinea pig"). • infertility may be caused by pyometra (see p. pneumotropica and lymphocytic choriomeningitis). • cannibalism is most frequently a result of stress or mastitis. • in almost 90% of geriatric hamsters, renal amyloidosis develops. the disease tends to develop more rapidly in females. • clinical signs include edema and ascites due to protein loss in the urine, as well as the typical signs of chronic renal failure. • treat supportively. prognosis for long-term survival is grave. • head tilt is usually secondary to otitis media. also consider lymphocytic choriomeningitis or neoplasia as differential diagnosis (see "rat"). • in hamsters fed all-seed diets and deprived of exercise, cage paralysis syndrome often develops. usually pets are presented for acute posterior paresis which, in reality, was slowly progressive. the distinction is important in ruling out trauma. in mild cases, the hamster is able to move its hind limbs but unable to support weight. vitamin d and e supplementation, along with nutritional improvement and providing exercise, is curative in 1 to 2 weeks. in severe cases, recovery is negligible or incomplete. lymphoma and lymphosarcoma may be viral in origin. diagnosis is made by biopsy or fine-needle aspiration of affected lymph nodes. rule out lymphadenopathy caused by lymphadenitis from infection with streptobacillus moniliformis (see "rat"). although many hamsters initially respond well to chemotherapy protocols established for cats and dogs, prognosis for long-term survival is grave. • rarely, demodex spp. mites cause alopecia in gerbils. • diagnosis is based on skin scraping. • treat with rotenone ointment or amitraz dips every 2 weeks for three to six treatments. use manufacturer's recommended dilution for dogs. • acute moist dermatitis usually is caused by s. aureus infection. infection on the face often begins with the harderian glands. the gland secretion is viscous and causes matting, with secondary staphylococcal infection occurring under the mats. attempts at grooming spread the infection to the feet and abdomen. • diagnosis is based on clinical signs and isolation on culture. • administer enrofloxacin, tetracycline, or chloramphenicol and apply warm, moist compresses to remove dried debris. remove possible irritants from cage (e.g., pine or cedar shavings, ammonia). occasionally, surgical removal of a chronically infected or inflamed gland is needed. • alopecia of the facial area, especially when it is symmetric, is usually the result of self-trauma from feeders, cage bars, or overzealous burrowing. • treat by changing cage construction or providing better visual security. • gerbils that catch their tails in crevices or are restrained inappropriately by their tails often are presented for avulsion of the skin from their tails. • treat initially by controlling hemorrhage and hypovolemic shock. • amputate the tail after patient stabilization to prevent ascending infection. in some animals, the infection is localized to the distal tail, which is sloughed in approximately 3 to 4 weeks. • generalized alopecia is normal in some weanling gerbils. the hair grows in as the animals mature. • melanomas are found most frequently on the ears, feet, or base of the tail. • diagnosis is based on biopsy. • treat by surgical removal. • sebaceous gland disease is usually the result of bacterial infections or neoplasia. • diagnosis is based on cytologic examination, culture, histologic examination, and response to antibiotic therapy. • treat bacterial infections with parenteral or topical antibiotics based on the severity of signs. • sebaceous gland adenomas, basal cell tumors, and squamous cell carcinomas are the most frequently encountered neoplasms. • take a radiograph of the thorax to diagnose metastases. prognosis for long-term survival is based on tumor type, stage, and character. • treat by surgical excision. chromodacryorrhea and epiphora occur as in mice. tapeworms (i.e., h. nana and h. diminuta) and pinworms (i.e., syphacia obvelata, dentostomella translucida, and aspicularis tetraptera) occur as in mice. incisor overgrowth occurs as in mice. • salmonella spp. cause transient diarrheas in gerbils. the source of infection is usually unwashed greens, contaminated feed, or carrier rodents of another species. most recover. animals that die have a fibrinosuppurative peritonitis. • treat supportively. use antibiotics in severe cases based on results of culture and susceptibility testing. • tyzzer disease, caused by bacillus piliformis, is seen most often in weanlings at 3 to 7 weeks of age and in post-partum females. • clinical signs include anorexia, lethargy, rough haircoat, and sometimes diarrhea. gross postmortem findings include yellow-gray nodules in the liver and hemorrhage at the ileocecal junction. • diagnosis is based on postmortem examination or response to therapy. • treat with oxytetracycline (see table 177 -9) and supportive care. hepatic lipidosis and gallstones are frequent sequela to lipemia in gerbils fed diets with excessive fat. • breeding is most successful if animals are paired at weaning and kept in these pairs. male gerbils aid in raising the young. pairing older animals causes fighting. an average of 20% of neonates fail to survive to weaning. this is usually the result of agalactia and crushing. • chronic hemorrhagic discharge from the vulva is usually the result of cystic ovaries or ovarian tumors. most tumors occur in animals older than 2 years of age and consist of granulosa cell tumors or theca cell tumors. leiomyomas of the uterus also cause similar clinical signs. • rule out urinary tract disease by performing a urinalysis via cystocentesis. large masses may be visualized on abdominal ultrasound. definitive diagnosis is based on vaginal cytology followed by exploratory laparotomy. • ovariohysterectomy is curative for cystic ovaries and tumors if they have not metastasized. • chronic renal failure develops in most gerbils older than 2.5 years of age. • clinical signs are polyuria, polydipsia, weight loss, and anorexia. urinalysis demonstrates proteinuria, hematuria, casts, and an increase in white and red blood cells. • treat supportively. prognosis for long-term survival is grave. • up to 50% of gerbils in certain family lines suffer spontaneous epileptiform seizures. the seizures are induced by stress and are self-limiting. seizures usually start as the gerbil reaches 2 months of age. • treatment is unnecessary. chinchilla laniger and c. brevicaudata are nocturnal rodents from the andes mountains in south america. most animals kept in the united states are the descendants of 11 animals. aside from pets, chinchillas are raised commercially for their pelts. the most common coat color is gray; the most valuable coat color is black. m key point chinchillas are sensitive to antibiotics (see "hamster"); therefore, avoid use of penicillins, lincomycin, erythromycin, and cephalosporins. house chinchillas in a cool environment because they are prone to overheating. if heat stroke occurs, treat with tepid water baths and supportive therapy. • chinchillas require dust baths to keep their skin in condition. use commercially available chinchilla dust only. sand substitutions do not condition the coat and occasionally cause conjunctivitis. offer dust at least once a week. • dermatophytosis occurs as in guinea pigs. • fur chewing is a serious problem in chinchillas that are farmed for pelts and often is seen in pet chinchillas that are recent culls from a ranch. the etiology of fur chewing is unknown. some cases seem to be related to chronic disease, malnutrition, poor caging, or stress. theories for undiagnosed cases include genetic abnormality; undiagnosed dermatophytosis; or adrenal, pituitary, or thyroid gland abnormalities. • diagnostics such as skin scraping, fungal culture, fecal, cbc, history profile, and biopsy are recommended. in general, if changes in diet and husbandry do not elicit a response or an underlying treatable disease condition is not discovered, prognosis for cure is grave. • one source advocates plucking all remaining underfur in chewed areas in an attempt to stimulate new hair growth. place collars after this procedure until the fur has grown in completely. • cystic sc masses may be caused by the intermediate stage of multiceps serialis. transmission is by ingestion of feed contaminated with canine feces. • diagnosis is made by histopathologic or cytologic examination of tissue samples. treat by surgical removal of the masses. • otitis caused by pseudomonas spp. occurs as in rats. • conjunctivitis occurs as in mice. • cataracts are congenital or developmental. • asteroid hyalosis occurs as a degenerative change. pneumonia occurs as in guinea pigs. parasites tapeworms (i.e., h. nana) occur as in mice. malocclusion of incisors and cheek teeth occurs as in guinea pigs. • diarrhea is caused most often by coccidia or giardia spp. or a bacterium. • clinical signs range from soft stools and weight loss to fluid diarrhea, dehydration, bloating, septicemia, and sudden death. • the protozoal parasites are best diagnosed on fresh saline smear or necropsy. • bacterial diarrhea is most often caused by contaminated feed and is diagnosed by isolation on culture. clostridium spp., pseudomonas aeruginosa, e. coli, salmonella enteritidis, and pasteurella spp. are the most common isolates. • treat supportively and use appropriate antiprotozoal or antibiotic drugs. • pasteurella pseudotuberculosis causes acute death from septicemia or a chronic weight loss with intermittent diarrhea. enlarged mesenteric lymph nodes are a hallmark of this disease. • diagnosis is based on clinical signs, histopathologic examination of tissue samples, and isolation on culture. • treat with sulfa drugs until sensitivity results are available. prognosis for recovery is poor. gross postmortem examination reveals yellow to white necrotic foci in the liver. • check male chinchillas four times per year for penile hair rings. roll back the prepuce and expose the penis. roll hair rings off the penis after application of a water-soluble lubricant. treat ulcerations topically or systemically as needed. • dystocia is fairly common in chinchillas (see "guinea pig"). • metritis is suspected when post-partum vaginal discharge, failure to return to a normal estrus cycle, anorexia, weight loss, polydipsia, polyuria, and chewing at flank and abdomen are present. • diagnosis is based on history, physical examination, abdominal radiographs, culture, ultrasound, and cbc. it usually is caused by bacteria introduced by the male or spread from an internal abscess. retained placentas, macerated fetuses, and dystocia are predisposing factors toward metritis. • treat with ovariohysterectomy after stabilization. females used only for breeding purposes may be treated with antibiotics alone, but the prognosis is poor. • female chinchillas are aggressive toward male chinchillas when not in estrus. breeding operations usually have separate cages for females and an interconnecting run for the male. females are kept out of the male's run by their larger size or collars. the young are precocious and do not need a nest. chinchillas only produce two litters per year. • clinical signs include hot, swollen mammary glands. suspect mastitis if previously healthy neonates become restless, then lethargic. • perform bacterial cultures on milk samples, and treat with antibiotics based on susceptibility testing. administer sulfa drugs until susceptibility results are available. local hot packing is also beneficial. occasionally, surgical drainage is required. foster neonates to another female if possible, or use puppy or kitten milk replacers to hand-raise babies. • chinchillas seem to be particularly sensitive to listeria monocytogenes. clinical signs can mimic p. pseudotuberculosis and include anorexia, lethargy, abortion, generalized central nervous system (cns) signs, hepatitis, mild enteritis, and mild emphysematous pneumonia. necropsy shows yellow foci in the liver. • diagnosis is based on isolation on culture. • treat with sulfa drugs (see table 177 -9) until sensitivity results are available. the prognosis is poor. • other less common causes of neurologic disease in chinchillas include lymphocytic choriomeningitis, streptococcus spp., balisascaris procyonis (i.e., aberrant migration of raccoon roundworm), lead poisoning, and thiamine deficiency. guinea pigs (caviae porcellus) are nocturnal rodents that originated in the andes mountains. they are known for their dietary need for vitamin c. they are used as a food source in their native lands. there are three basic types: english, which have short hair; abyssinian, which have short, cowlicked hair; and peruvian, which have long hair. male guinea pigs are known as boars and the females as sows. guinea pigs become neophobic as they mature. offer a variety of foods early in life and make changes in diet or environment gradually. guinea pigs stampede when excited. square cages and strategically placed barriers on external walls prevent the trampling of small or weak animals. the smooth muscle of the bronchial tree is quite developed in guinea pigs. this places them at high risk for asthmatic-type anaphylactic reactions. both male and female guinea pigs have one pair of inguinal mammary glands; however, only the female's are well developed. m key point antibiotic toxicity (see "hamster"): guinea pigs also may be sensitive to tetracyclines. • fleas occur as in mice. • lice (i.e., gliricola porcelli, gynopus ovalis) usually cause no clinical signs except occasional alopecia, seborrhea, and trauma secondary to pruritus. • diagnosis is made by observation of lice on skin scraping. • treat with ivermectin, 5% malathion dust, or pyrethrin shampoo (see table 177 -8). • the mite trixacarus caviae causes severe pruritus and is zoonotic. it mainly affects the dorsal midline and is difficult to find on skin scraping. it occurs most frequently in recently post-partum females, in which alopecia is the predominant clinical sign. treat with excellent sanitation and ivermectin (see table 177 -8). • chirodiscoides caviae lives on the hair shaft of the perineal regions. it does not cause clinical signs. • treat with 5% carbaryl or lime-sulfur dip (1:40) (see table 177 -8). sanitation is critical in preventing reinfestation. • about 6% to 13% of guinea pigs are carriers of trichophyton mentagrophytes. • clinical signs are alopecia and seborrhea sicca, usually starting on the face and spreading along the dorsum. • treat with lime-sulfur dips or griseofulvin (see table 177 -9) combined with topical povidone iodine or chlorhexadine shampoos. • other causes for alopecia are barbering, alopecia of the flanks in late-gestation females, and generalized alopecia of young at weaning. subclinical hypovitaminosis c causes a poor hair coat and seborrhea sicca, as well as anorexia and large, malodorous stools. • "lumps" is the lay terminology for cervical lymphadenitis, which is characterized by lymphadenopathy in the ventral neck region. • pododermatitis and sore hocks are very common in guinea pigs. predisposing factors are untrimmed toe nails, poor sanitation, and wire flooring. s. aureus is the most commonly cultured pathogen. • clinical signs range from small ulcers on the soles of the feet to abscesses and gangrene. radiography is essential in determining whether bony involvement is present. untreated pododermatitis usually develops into osteomyelitis, which is very difficult to cure. • treat mild cases by improving sanitation and grooming. place affected individuals in solidfloored cages with paper bedding. use sulfa drugs (see table 177 -9) until results of susceptibility testing are available. surgically remove or curette abscesses, and apply topical therapy and hot packing. amputation may be necessary when severe osteomyelitis exists. • conjunctivitis and epiphora occur as in mice. • inclusion body conjunctivitis is caused by chlamydia psittaci and is self-limiting in 3 to 4 weeks. • perform a conjunctival scraping to differentiate inflammatory conjunctivitis secondary to infection from allergy. i have observed an idiopathic, topical steroid-responsive lymphoplasmacytic conjunctivitis in guinea pigs. • white, dry ocular discharge is an early sign of hypovitaminosis c. • "pea-eyes" is the lay terminology for subscleral fatty deposits or protrusion of the lacrimal gland through the lower conjunctiva. the condition is thought to be hereditary. treatment is not required. • cataracts occur and are either congenital or developmental. • diabetes mellitus in guinea pigs also may cause cataracts. usually, no other clinical signs are present and urine glucose is greater than 100 mg/dl whereas blood glucose remains within normal limits. • corneal or scleral calcification is usually an incidental finding. a thorough workup, including serum chemistry profile and radiographs, is recommended to ensure that generalized metastatic calcification is not present. • pneumonia in guinea pigs usually is caused by infection with s. pneumoniae, s. zooepidemicus, or bordetella bronchiseptica. s. aureus, p. aeruginosa, klebsiella pneumoniae, and pasteurella multocida also are cultured frequently. transmission is by direct contact, fomites, or aerosol. hypovitaminosis c and stress often predisposes guinea pigs to bacterial respiratory infections. weanlings are particularly susceptible. clinical signs and diagnosis are similar to other small mammals (see "mouse"). • take radiographs to rule out abscesses, pleural effusion, or pericardial effusion in refractory cases. • treat with chloramphenicol, sulfa drugs, or enrofloxacin (see table 177 -9) and vitamin c (table 177 -10) until results of culture and susceptibility testing are available. cats, dogs, rabbits, and rats are reservoirs for bordetella spp. as in other rodents, respiratory infections may lead to otitis interna/media. bordetella spp. also cause pyometra and abortions. • nasal discharge is most frequently a sign of upper respiratory tract infection but also may be associated with allergies or volatile irritants. • the diagnosis of allergic rhinitis is made by exclusion and through response to antihistamines or environmental changes. • bronchogenic papillary adenoma develops in approximately 30% of guinea pigs older than 3 years of age. • diagnosis is often an incidental finding when thoracic radiography is performed for another problem. • occasionally, clinical signs are seen as a result of pressure on the heart or great vessels. • dyspnea most frequently is caused by heat stress or trauma. other causes are pregnancy toxemia, gastric bloat, volatile irritants, pleural effusion, pneumonia, or pulmonary edema. rhabdomyomatosis is a common necropsy finding. gross lesions appear as pale foci located on the endomyocardium and valves. histologic examination reveals myocardial cells that have stored excessive glycogen. do not confuse these areas with thrombi, abscesses, or neoplasia. their clinical significance is unknown. • paraspidodera ucinata is the cecal pinworm of guinea pigs. they are generally asymptomatic, but heavy infestations can cause diarrhea and weight loss. • diagnosis is based on fecal examination or cellophane tape test. • treat with piperazine or fenbendazole (see table 177 -8). • coccidiosis caused by eimeria caviae is a fairly common cause of diarrhea in guinea pigs recently purchased from pet stores. • clinical signs are tenesmus, diarrhea, dehydration, and death. • diagnosis is based on fecal examination. on gross postmortem examination, petechiation and thickening of the colon are seen. • treat supportively and administer sulfa drugs (see table 177 -9). • cryptosporidium wrairi and giardia spp. are found rarely. they cause a chronic enteritis. balantidium spp. are thought to be nonpathogenic. • malocclusion in guinea pigs is diagnosed on oral examination. • clinical signs are ptyalism and anorexia. the premolars are the most commonly affected teeth. • long-standing hypovitaminosis c predisposes guinea pigs to malocclusion. • treat malocclusion as in other rodents (see "dental procedures"). • hypovitaminosis c (i.e., scurvy) is associated with soft, malodorous feces. degeneration of the epithelium of the intestinal tract adversely affects digestion and absorption and allows secondary bacterial infections. • diagnosis of scurvy is based on clinical signs, the exclusion of other causes of diarrhea, and response to vitamin c therapy (see table 177 -10). • salmonellosis usually is contracted through contaminated feed. • clinical signs range from sudden death to diarrhea and anorexia. the diarrhea is frequently light colored. sepsis is common and may cause conjunctivitis, shock, pneumonia, abortion, and neurologic symptoms. • diagnosis is based on isolation on culture of feces or other appropriate tissue samples. • treatment is controversial because recovered individuals remain carriers. use sulfa antibiotics or enrofloxacin (see table 177 -9) until sensitivity testing results are available. supportive care is essential. • e. coli, arizona, and clostridium are other commonly cultured diarrhea-causing organisms. clostridium are diagnosed most easily by finding large numbers of spores on a gram stain fecal specimen. treat with metronidazole (see table 177 -9). • yersinia pseudotuberculosis either causes an acutely fatal diarrhea or localizes into regional lymph nodes. • diagnosis is based on culture. • treat by surgical removal or drainage of abscessed lymph nodes. mesenteric lymph node involvement necessitates abdominal surgery. treat with sulfa drugs or enrofloxacin until susceptibility testing results are available (see table 177 -9). • one male usually is housed with four to six females for breeding purposes. signs of estrus are vulvar swelling, lordosis, and opening of the vaginal closure membrane. fetuses are palpable at 4 to 5 weeks of gestation. parturition occurs within 48 hours after the pubic symphysis has reached 15 mm. neonates weighing less than 60 g have a grave prognosis for survival even with intensive care. neonates normally do not nurse for the first 12 to 24 hours. litters with five or more fetuses usually result in abortion. • dystocia commonly occurs in females bred after the age of 6 to 9 months. after this age, the symphysis fuses and is unable to open the 2 to 3 cm required to allow passage of a fetus. dystocia in younger guinea pigs may be caused by obesity, large fetal size, fetal malpresentation, subclinical ketosis, or uterine inertia. on presentation, check the pelvic symphysis. if active contractions are present and the symphyseal gap is less than 2 cm, perform a c-section. normal parturition is very rapid, with a rest of only 3 to 7 minutes between fetuses. • perform a c-section if active straining does not produce a fetus within 15 to 20 minutes. radiograph sows with a history of weak contractions to determine the stage of pregnancy and evaluate the size of the fetuses. if well-developed skeletons of appropriate size are seen and the pubis has not yet fused, give oxytocin and calcium (see table 177 -10). if no fetuses are produced within 15 to 20 minutes, perform a c-section. • if poorly developed fetuses are seen radiographically, consider fetal death, ketosis, or a nonreproductive disorder as possible causes of dystocia. m key point pregnancy toxemia usually is seen in obese sows with large litters in late pregnancy. other risk factors include systemic disease or diet change causing anorexia, genetics, stress, and first litter. • clinical signs are tachypnea, depression, malodorous breath, seizures, and icterus. a urine ph of less than 6 with marked proteinuria is compatible with pregnancy toxemia. a marked hyperkalemia and elevation of liver enzymes often occurs. thrombocytopenia may be present. • treat with iv or io saline, dextrose, glucocorticoids, and calcium. surgical abortion of the fetuses may be attempted, but the anesthesia risk is quite high. prognosis for survival is grave. do not rebreed affected females. do not breed sows heavier than 900 g. • large litters can cause a hemorrhagic syndrome. compression of the portal vein and liver causes hepatic dysfunction, which results in vitamin k and clotting factor deficiency. • treat with vitamin k supplementation (see table 177 -10). response is poor in severely compromised patients. affected individuals are at risk of ketosis developing. prognosis is guarded. • vaginitis in guinea pigs frequently is caused by foreign bodies, usually bedding. • diagnosis is made on vaginal examination. • treat by flushing the vagina to remove the foreign material. • vaginal discharge also can be caused by pyometra, uterine torsion, urinary tract infection, or urogenital neoplasia. • diagnosis is based on findings on abdominal palpation, vaginal cytology and culture, urinalysis, abdominal radiographs, ultrasound, and exploratory. • treatment varies with the condition and is similar to that used in cats. • ovarian teratomas and uterine tumors occasionally are diagnosed and usually resolve with ovariohysterectomy. • a symmetric alopecia with concurrent abdominal enlargement may be seen in female guinea pigs with cystic ovaries. • diagnosis is based on abdominal palpation, cytology, and ultrasound. • treat by performing an ovariohysterectomy. if the guinea pig is not a good candidate for surgery, human chorionic gonadotropin (hcg, 1000 usp units im, repeat in 1 week) may temporarily resolve clinical signs. • male guinea pigs are prone to preputial foreign bodies. a preputial discharge is the usual presenting complaint. • diagnosis is based on physical examination. • treat by removing foreign bodies and performing local flushing. chronic problems require a change in bedding. • male guinea pigs produce sebaceous secretions in the folds around their perineal area. clean these areas with soap and water semiannually to prevent localized pyoderma. • if pyoderma occurs, treat with topical therapy and oral antibiotics. bacterial cystitis and urolithiasis are relatively common in guinea pigs. diagnosis is based on a history of stranguria, hematuria, painful abdomen, and anorexia, in addition to abdominal palpation, urinalysis, urine culture, abdominal radiographs, and ultrasonography. treatment consists of antibiotics based on results of culture and susceptibility testing and surgical removal of calculi, if present. prevention of recurrence is difficult if the calculi are not caused by a bacterial infection. addition of vitamin c to the drinking water as well as changing the brand of diet are sometimes helpful in preventing recurrence of metabolic stones. klossiella cobayae is a coccidia that lives in the renal tubules. it has no clinical significance. the most common orthopedic problem seen in guinea pigs is overgrown toenails. this leads to pododermatitis and sore hocks as well as to degenerative joint disease and a predisposition to tibial fractures. tibial fractures are the most common fracture seen in guinea pigs. they most frequently occur after foot entanglement. internal fixation with an im pin or application of a kirschner apparatus is the repair of choice. m key point signs of hypovitaminosis c or scurvy start to develop in guinea pigs as early as 10-15 days if they are placed on diets 100% deficient in vitamin c. early signs are soft, malodorous stools, weight loss, poor hair coat, and anorexia. later, petechia, gingivitis, cutaneous and oral sores, swollen costochondral junctions, joint pain and hemorrhage resulting in lameness, and conjunctivitis become apparent. treat supportively and administer parenteral vitamin c (25 mg/day). • lymphocytic choriomeningitis occurs as in mice. • guinea pig paralysis syndrome starts with mild pyrexia and urinary incontinence, followed by weight loss and posterior paresis that progresses to paralysis. currently, the etiology is unknown, but it does not appear to be contagious. • treat with supportive care. prognosis for long-term survival is grave. • head tilt is usually the result of otitis or trauma (see "mouse"). cavian leukemia has a viral etiology. the liver, spleen, and lymph nodes are the primary organs involved. there is no current treatment. quarantine exposed individuals. death usually occurs within 5 days after discovery of lymphoblasts in the peripheral blood. neutrophils normally have red granules. kurloff bodies are normally occurring eosinophilic intracytoplasmic inclusion bodies that are found in mononuclear cells. they are seen most frequently in females and appear to correspond positively with estrogen levels. metastatic calcification occurs in most guinea pigs older than 1 year of age. it is more severe in females than in males. the stomach is one of the first organs affected. dysfunction in motility causes obstruction. the tendency appears to be exacerbated by high calcium and low phosphorus diets. exotic animal formulary the biology and medicine of rabbits and rodents veterinary clinics of north america key: cord-258129-c38q6xxs authors: russell, clark d; schwarze, jürgen title: the role of pro-resolution lipid mediators in infectious disease date: 2014-01-09 journal: immunology doi: 10.1111/imm.12206 sha: doc_id: 258129 cord_uid: c38q6xxs inflammation is an essential host defence against infection, but can be damaging when excessive. resolution of inflammation is an active process, and the pro-resolution effects of lipoxins, resolvins and protectins have received significant interest. here, we review emerging data on the role of these lipid mediators in infectious disease. lipoxins influence host control of mycobacterium tuberculosis, toxoplasma gondii, trypanosoma cruzi and plasmodium berghei cerebral malaria in mice. their effects are protective in toxoplasmosis, t. cruzi infection and cerebral malaria but detrimental in tuberculosis; related to the balance between pathogen-control and excessive immune response. topical lipoxin abrogates the tissue damage seen in a rabbit model of porphyromonas gingivalis periodontitis. the increased virulence of h5n1 influenza a virus in mice correlates with reduced expression of socs2, required to mediate the effects of lipoxin. mice unable to synthesize lipoxin suffer increased lung pathology during respiratory syncytial virus infection. protectin suppresses influenza a virus replication in vitro and increases survival in a mouse model of severe influenza infection. resolvins were investigated in a number of animal models of systemic bacterial infection, and were found to enhance phagocytic clearance of bacteria, reduce inflammation severity, promote neutrophil apoptosis, modulate neutrophil chemotaxis and importantly, reduce mortality. interestingly, resolvin also enhances the antibacterial effect of ciprofloxacin and vancomycin. topical resolvin application reduces the severity of herpes simplex virus ocular infection in mice. if the effects of these mediators translate from pre-clinical studies into successful clinical trials, they represent promising new strategies in managing infectious disease. inflammation is an essential host defence mechanism, required for protection against pathogenic micro-organisms and viruses. however, an excessive and non-resolving inflammatory response is damaging to the host. for example, in sepsis there is loss of homeostatic control over the inflammatory response mounted against an infectious agent, resulting in a pro-inflammatory, procoagulant state that can result in organ dysfunction and death. acute respiratory distress syndrome is another example, and describes the life-threatening hypoxic complication of excessive pulmonary inflammation that can be triggered by a range of insults, including pneumonia and sepsis. 1 chronic, non-resolving inflammation also underpins a number of other pulmonary diseases, including asthma, cystic fibrosis and bronchiectasis. inflamma critical events following the clearance of an infectious agent, to prevent the development of these 'complications' of excessive inflammation. this is now known to be an active process, termed catabasis, 2 driven by the pro-resolution lipid mediators resolvin, protectin and lipoxin. 3 lipoxins (trihydroxy-tetraene-containing eicosanoids) are derived from the omega-6 polyunsaturated fatty acid arachidonic acid through sequential reactions involving lipoxygenase enzymes (including 15-lipoxygenase type 1 and 5-lipoxygenase in human mucosal tissues). 4, 5 resolvins are derived from omega-3 polyunsaturated fatty acids and exist as two series (d and e). d-series resolvins are products of docosahexaenoic acid metabolism involving 15-lipoxygenase. e-series resolvins are synthesized from eicosapentaenoic acid involving aspirin-acetylated cyclo-oxygenase 2. 6 protectins are also omega-3 polyunsaturated fatty acid derivatives, generated from docosahexaenoic acid through a 15-lipoxygenase-mediated pathway. 7 the pro-resolution effects of these mediators are exemplified by their role in pulmonary inflammation, the setting in which they are best studied. resolvins, protectins and lipoxins all have a proresolution role in a mouse model of allergic airway inflammation. [8] [9] [10] [11] given that an excessive inflammatory response is implicated in the pathogenesis of a number of infectious diseases and their complications, including sepsis syndrome and acute respiratory distress syndrome, we conducted a review of the literature to determine what is currently known about the role of the pro-resolution lipid mediators lipoxin, resolvin and protectin in infectious disease. 13 in mice, h5n1 was more virulent (infection resulted in shorter survival time) than the h1n1 strain and disseminated to extra-pulmonary sites (brain and spleen) following intra-nasal inoculation. global transcriptional responses to infection were analysed and showed that the different strains of influenza a virus caused different early responses in gene expression, and that many of the differentially regulated genes were components of the inflammatory response. h5n1 infection was associated with up-regulation of inflammasome genes [including caspase 1 and interleukin-1 (il-1)], tumour necrosis factor-a (tnf-a), interferon-c (ifn-c) and protein kinase r, as well as other inflammation-related genes. using a bioinformatics approach to analyse gene expression, the authors found that extra-pulmonary dissemination was associated with down-regulation of genes involved in mediating the pro-resolution effects of lipoxin on leucocyte recruitment and counter-regulation of pro-inflammatory cytokine induction. these included the suppressor of cytokine signalling (socs) 2 gene, the product of which is activated by lipoxins and is an essential intracellular mediator of lipoxin's effects on inflammatory cell trafficking and cytokine induction. 14 therefore, loss of lipoxin's pro-resolution effects may be associated with greater influenza a virus virulence, suggesting a protective role for lipoxin in this infection, possibly related to the suppression of pro-inflammatory cytokines that are up-regulated in this model. the highly pathogenic avian h5n1 virus used in this study was less successful at spreading between human hosts compared with other influenza a virus strains, for example the 2009 h1n1 pandemic strain. a successful pathogen minimizes damage to its host to prolong the availability of its replicative niche and the high case-mortality seen with infection by this h5n1 strain indicates that it is not well adapted to humans. reduction of lipoxin-mediated pro-resolution effects may contribute to the virus's poor adaptation to humans. in a recent study, respiratory syncytial virus (rsv) infection of mice deficient in 5-lipoxygenase, an enzyme required for lipoxin production, failed to elicit alternative macrophage differentiation, which is known to be required for the resolution of rsv-induced lung injury. 15, 16 alternatively activated macrophages are thought to induce anti-inflammatory cytokine expression and drive lung tissue repair. 16 significantly, rsv infection of these 5-lipoxygenase-deficient mice resulted in greater lung pathology on histological analysis (increased peribronchiolitis, perivasculitis, interstitial pneumonitis and alveolitis) in comparison to infected wild-type mice. 15 treatment with lipoxin a4 and resolvin e1 partially restored the alternatively activated macrophage phenotype in the 5-lipoxygenase-deficient mice. these observations support a pro-resolution role for lipoxins in viral respiratory tract infection. alveolar macrophages are the major resident inflammatory cell of the respiratory tract and are able to produce lipoxins. alveolar macrophages isolated from rats that had previously been infected with a range of respiratory viruses (murine parainfluenza virus type 1, rat coronavirus, pneumonia virus of mice and mouse adenovirus) produced higher levels of lipoxin than macrophages from rats that had been kept in sterile conditions since birth. 17 these findings suggest that virus-induced pulmonary inflammation is a stimulus for the production of lipoxins. in a cell culture model of hiv central nervous system infection, following co-culture of hiv-infected monocytes with astroglia, tnf-a and il-1b were produced and this production correlated with synthesis of large amounts of leukotriene b4, leukotriene d4 and lipoxin a4. 18 this is the only study to demonstrate, albeit in vitro, that lipoxins are produced in direct response to viral infection. however, the role of lipoxin in this model of infection has not been investigated. following aerosol infection with mycobacterium tuberculosis, mice have been shown to produce high levels of lipoxin a4 during chronic infection. 19 pulmonary endothelial cells and macrophages were responsible for this. transgenic mice deficient in 5-lipoxygenase, an enzyme required for lipoxin production, were able to control m. tuberculosis infection better than wild-type mice, as demonstrated by lower pulmonary and splenic titres of viable m. tuberculosis (at 21 and 42 days). on histological examination of lung tissue, extensive inflammation and areas of necrosis were seen in wild-type mice whereas the transgenic mice had a lesser degree of inflammation and little evidence of necrosis. importantly, the transgenic mice unable to produce lipoxin enjoyed enhanced survival in this model of m. tuberculosis infection and had increased pulmonary levels of il-12, ifn-c and inducible nitric oxide synthase, which is known to have a protective role in host control of m. tuberculosis infection in mice. 20 oral administration of a lipoxin a4 analogue reversed the enhanced control of infection in the transgenic mice, and splenocytes from these mice showed reduced ifn-c production. in a mouse model of toxoplasma gondii infection (established through intraperitoneal inoculation of t. gondii cysts), serum levels of lipoxin a4 rise during infection and remain high once chronic infection has been established. 21 transgenic mice unable to produce lipoxin a4 suffered increased mortality following infection, compared with wild-type control animals. 14,21 interestingly, fewer t. gondii brain cysts were seen in the lipoxin-deficient mice and consistent with this, lower parasitic burden and increased serum levels of il-12 and ifn-c were seen when compared with wild-type mice. therefore, it seems likely that the increased mortality of the lipoxin-deficient mice is attributable to cytokine-mediated tissue damage, despite better parasite control. corroborating this, increased histological severity of meningitis and encephalitis was observed in the transgenic mice. administration of a lipoxin analogue rescued the lipoxin-deficient mice from this fatal phenotype and lowered il-12 and ifn-c levels (to wild-type levels in the case of ifn-c). interleukin-12, produced by antigen-presenting cells such as dendritic cells, has an important role in host control of intracellular pathogens such as t. gondii and viruses, but in excess can cause immunopathology. in mice, it has been shown that lipoxin a4 analogues suppress il-12 production by dendritic cells stimulated with t. gondii extract. 22 although this may be a host-driven response to curb excessive inflammation, induction of lipoxin production could be a strategy adopted by pathogens to modulate host immunity and perhaps facilitate chronic infection by reducing tissue damage. the contrasting roles of lipoxins in these two models of infection may relate to the dynamics of the specific pathogen-host interactions. toxoplasma gondii replicates more quickly than m. tuberculosis and the risk of excessive inflammation and ensuing immunopathology may therefore be greater. 19 by preventing this, lipoxins are beneficial to the host, and so enhance survival. mycobacterium tuberculosis induces a weaker t helper 1 cell response in mice than t. gondii and replicates more slowly, therefore the enhanced inflammatory response seen in the lipoxin-deficient mice may be advantageous, by 'topping-up' existing host control. the reduced tissue damage seen in lipoxin-deficient mice may be a result of better control of m. tuberculosis replication. it is also important to note that the local (lung versus peritoneum/ brain) biosynthesis and actions of lipoxin may differ between these two infection models and could provide an alternative explanation for the different outcomes observed. in a mouse model of trypanosoma cruzi infection, it was found that the administration of aspirin (acetylsalicylic acid) at doses of 25 or 50 mg/kg increased survival and decreased peak parasitaemia, whereas higher or lower doses had no effect. 23 the aspirin-triggered lipoxin, 15epi-lipoxin a4, was found to be increased by administration of aspirin in both in vitro and in vivo models of infection, with infection alone also increasing 15-epi-lipoxin a4 levels. exogenous administration of 15-epi-lipoxin a4 was found to decrease parasitaemia peaks and decrease cardiac inflammation and increase survival. the authors therefore hypothesized that the beneficial effect of aspirin on infected mice was a result of the production of 15-epi-lipoxin a4. this may represent a new therapeutic strategy in the acute phase of chagas disease. wild-type and 5-lipoxygenase-deficient mice were infected with plasmodium berghei-anka in a model of cerebral malaria. 24 wild-type mice enjoyed significantly increased survival in comparison to 5-lipoxygenase-deficient mice. levels of parasitaemia were similar between groups, but lipoxin-deficient mice had an increased cerebral parasite burden after 5 days despite reduced cerebral inflammation and less cd8 + ifn-c + t cells in brain tissue. the lipoxin-deficient mice were found to have higher serum levels of il-12 and ifn-c, both of which are associated with the pathogenesis of murine cerebral malaria. significantly, treatment with 15-epi-lipoxin a4 significantly prolonged survival in both wild-type and lipoxindeficient mice, as well as reducing expression of il-12 and ifn-c in the brain. the mortality rate did not differ significantly if lipoxin was administered on the day of infection or 3 days later. lipoxin therefore appears to increase host survival in a mouse model of cerebral malaria by limiting inflammation, not by enhancing control of parasite levels. serhan and co-workers used a rabbit model of periodontitis to investigate the effects of lipoxin in this infection. 25 the application of a dental ligature plus porphyromonas gingivalis resulted in soft tissue loss, histological (leucocyte infiltrates) and radiological (bone loss) features of periodontitis in wild-type rabbits. the authors then used a transgenic rabbit that over-expresses the enzyme 15-lipoxygenase, resulting in threefold to fourfold increased lipoxin production. in these transgenic animals, following the same procedure to establish periodontitis, none of the clinical, histological or radiographic features were found. in the wild-type animals, bone loss could be prevented by the administration of intravenous metronidazole, confirming that p. gingivalis infection was responsible for the features seen. wild-type rabbits subject to periodontitis induction were then administered a topical lipoxin analogue and this protected the animals from gross soft tissue destruction, leucocyte accumulation and bone loss. periodontitis is an infectious disease, though the tissue damage characteristic of the condition is thought to be driven by an aberrant host response to infection. 26 in this set of experiments, serhan et al. demonstrate that lipoxin has a role in curbing this excessive response and that this has a protective effect in vivo. by performing transcriptional profiling of model epithelial cells exposed to lipoxin, canny et al. 27 found that lipoxin up-regulated the expression of bactericidal/perme-ability-increasing protein (bpi). bpi is an innate immune defence molecule that is active against gram negative bacteria, through bactericidal effects on bacterial cell membranes, neutralization of lipopolysaccharide and as an opsonin. this was then confirmed in four other epithelial cell lines (okf6, t84, caco2 and pulmonary epithelial cells a549). building on the known bactericidal effect of epithelium-derived bpi on gram-negative bacteria, the authors sought to examine the effect of lipoxin on epithelial killing of salmonella typhimurium. epithelial cell culture was pre-exposed to lipoxin analogue and then incubated with s. typhimurium. this resulted in concentration-dependent increased bacterial killing compared with non-pre-treated cultures. this enhanced bactericidal activity could be significantly inhibited by the addition of anti-bpi, indicating that induction of bpi was largely responsible for this lipoxin-mediated enhanced killing of s. typhimurium. interestingly, in a mouse model of (noninfectious) dextran sodium-sulphate-induced colitis, oral administration of lipoxin analogue reduced weight loss, passage of blood per rectum and mortality, affirming its role in gastrointestinal mucosal inflammatory conditions. 28 caecal ligation and puncture in mice establishes microbial sepsis that is considered similar to the pathophysiology of sepsis in humans. 29 in this model, in comparison to a control treatment, intravenous administration of resolvin d2 reduced the viable bacterial load in blood and peritoneal exudates (in the absence of direct antibacterial effects) and reduced neutrophil migration into the peritoneal cavity. in addition, histological evidence was seen of enhanced phagocytosis of bacteria in inguinal lymph nodes and in vitro, incubation with resolvin d2 enhanced zymosan phagocytosis by macrophages obtained from the peritoneum of resolvin-naive mice. when incubated with resolvin d2, human neutrophils demonstrated enhanced phagocytosis of escherichia coli and also increased intracellular production of reactive oxygen species. furthermore, plasma levels of pro-inflammatory cytokines (il-6, il-1b, il-23 and tnf-a) were reduced in resolvin d2treated mice. importantly, the effect of these pro-resolution actions of resolvin was to increase survival from sepsis in this mouse model. chiang et al. 30 investigated the effects of resolvin using a mouse model of e. coli peritonitis, involving intraperitoneal inoculation of e. coli. they found that the intraperitoneal administration of resolvin d5 enhanced phagocytosis of bacteria in comparison to mice inoculated with e. coli without resolvin. resolvin d1 had a similar but smaller effect. administration of resolvin (d1 or d5) was also associated with a significantly lower titre of viable bacteria in the blood and peritoneal exudate, as well as a lesser degree of hypothermia. importantly, resolvin d1 increased survival in this model. as for the model of microbial sepsis discussed previously, plasma levels of pro-inflammatory cytokines (including tnf-a and il-1b) were reduced by resolvin administration. interestingly, it was found that resolvin d1 enhanced the antimicrobial effect of ciprofloxacin in resolving e. coli peritonitis. when given in combination, resolvin and ciprofloxacin had an additive effect on reducing the 'resolution interval' (time from maximum concentration of peritoneal leucocytes to 50%), greater than either agent given alone. in addition, the combined administration of ciprofloxacin and resolvin d1 resulted in lower titres of viable bacteria in blood and peritoneal exudate, and enhanced macrophage phagocytic activity (not seen with either agent alone). the effect of reducing bacterial titres was still seen even when ciprofloxacin was administered at sub-optimal concentrations with a cocktail of resolvin d1, d5 and protectin d1. in staphylococcus aureus skin infection, using mouse dorsal skin pouches, resolvins d1 and d5 plus protectin d1, when administered with vancomycin (at a sub-optimal concentration) into the pouch, enhanced the clearance of bacteria, resulting in lower viable bacterial titres in pouch exudate and reduced neutrophil infiltration. this effect was also seen when either resolvin or vancomycin was administered alone, but again, an enhanced effect was seen when both were given together. infection secondary to burns is an important clinical problem and burns-related sepsis is responsible for significant mortality in burns patients. using a rat model of burn injury, kurihara et al. 31 demonstrated that neutrophils isolated from burned rats were impaired in their ability to migrate towards a chemoattractant. this defect in neutrophil chemotaxis could result in an inability of neutrophils in burns patients to successfully reach and kill infectious agents. as a consequence, these 'lost' neutrophils activated by the burn injury may accumulate and initiate tissue damage inappropriately in healthy tissues. by administering resolvin d2 to the burned rats, kurihara et al. found that neutrophil chemotaxis was restored to almost normal. when burned rats received intravenous lipopolysaccharide 9 days after their burn injury, pretreatment with intravenous resolvin d2 improved survival significantly. similarly, resolvin d2 pre-treatment increased survival following caecal ligation after burns injury. a shorter duration of resolvin treatment that was insufficient to normalize neutrophil chemotaxis did not significantly improve survival in these two models of burns-related sepsis. therefore, this study suggests that administering resolvin d2 could improve outcomes in burns-related sepsis by modulating neutrophil chemotaxis. the role of resolvin e1 in bacterial pneumonia and acute lung injury was investigated using a mouse model of aspiration pneumonia. 32 mice were administered hydrochloric acid followed by e. coli into a lung. resolvin e1, when given intravenously before this insult, reduced pulmonary neutrophil infiltration, enhanced bacterial clearance and decreased levels of pro-inflammatory cytokines (including il-1b and il-6) present in lung tissue homogenates. importantly, mice administered resolvin e1 enjoyed increased survival (100% at 3 days, in comparison to 50% when only 0á9% saline was administered). neutrophil apoptosis is known to be a critical event in the resolution of pulmonary inflammation, and suppression of this process is thought to contribute to the pathology seen in acute respiratory distress syndrome and sepsis. in a case-control cohort study of 121 intensive therapy unit patients with or without sepsis, the level of neutrophil apoptosis in peripheral venous blood was found to inversely correlate with the severity of sepsis. 33 the role of resolvin e1 in this process has been investigated in mouse models of acute lung injury, including an e. coli pneumonia model and an e. coli peritonitis-associated acute lung injury model. 34 six hours after intra-tracheal instillation of e. coli (pneumonia model), intraperitoneal administration of resolvin e1 enhanced the resolution of e. coli-evoked pneumonia in this mouse model. in contrast to the control group, mice administered resolvin had lower bronchoalveolar lavage neutrophil counts, higher monocyte/macrophage counts, increased neutrophil apoptosis, reduced bronchoalveolar lavage il-6 levels and less severe pulmonary inflammation when assessed histologically. when a pan-caspase inhibitor was also added intraperitoneally, these pro-resolution effects of resolvin were abrogated, suggesting that neutrophil apoptosis plays a critical role here. in the e. coli peritonitis-associated acute lung injury model, intraperitoneal administration of resolvin e1 also reduced bronchoalveolar lavage neutrophil counts and il-6 levels, increased neutrophil apoptosis and reduced the histological severity of pneumonia as well as lung myeloperoxidase content (where myeloperoxidase is used as a marker of neutrophil activity). importantly, resolvin administration reduced 6 hr mortality from 70% to 30% in this model. in terms of a possible mechanism for increased neutrophil apoptosis, in vitro, resolvin e1 mitigates cellular survival signals generated in response to a number of stimuli relevant to the pathogenesis of acute lung injury in e. coli pneumonia and sepsis (neutrophil myeloperoxidase, serum amyloid a and bacterial cpg dna). similarly, 15epi-lipoxin a4 also has a pro-apoptotic effect on human neutrophils and in a mouse model of e. coli sepsisinduced lung injury, intravenous lipoxin reduced bronchoalveolar lavage neutrophil counts and increased caspase-mediated neutrophil apoptosis. 35 as discussed, although inflammation is an essential response to infection, an excessive response is detrimental and stromal keratitis demonstrates this. it is an inflammatory lesion of the cornea that is a sequela of ocular herpes simplex virus infection, usually appearing 1 week after primary infection. importantly, when stromal keratitis manifests, replicating virus cannot be detected; therefore the initiating virus has been cleared and the disease is a consequence of non-resolving excessive inflammation. in mice, ocular herpes simplex virus infection is a model of stromal keratitis in humans. topical administration of resolvin e1 was able to control the immunopathological manifestations of ocular herpes simplex virus and reduce the severity of stromal keratitis lesions. 36 the influx of cd4 + t cells and neutrophils was reduced, as were corneal levels of pro-inflammatory cytokines known to be involved in the pathogenesis of stromal keratitis. levels of the anti-inflammatory cytokine il-10 were increased. through a screen of the effect of a range of lipid mediators on the replication of h1n1 influenza a virus in human lung epithelial cells, protectin d1 was found to significantly reduce viral replication in vitro. 37 in a model of human severe influenza, virus was introduced through the intratracheal route in mice and protectin d1 levels were found to be reduced, suggesting that endogenous production was suppressed by the virus. when other influenza a virus strains were tested, the reduction in protectin levels was found to inversely correlate with the virulence of the virus strain used. in this same model of severe influenza, intravenous administration of protectin increased survival. administration of resolvin and lipoxin had no effect. significantly, the protective effect of protectin was still seen when administered 2 days after the start of infection. lipoxins, resolvins and protectins are endogenous lipid mediators with a pro-resolution effect in inflammation. much work has focused on their now well-recognized role in allergic airway inflammation. 38 however, an emerging theme is their importance in the resolution of infection and associated inflammation, and this article has reviewed emerging data relevant to this. table 1 summarizes what has been learnt from animal studies. the role of lipoxins may vary depending on the specific infection. in a mouse model of m. tuberculosis infection, transgenic mice unable to produce lipoxin were able to control the bacteria better with reduced mortality in comparison to wild-type, and expressed il-12, ifn-c and inducible nitric oxide synthase 2 at higher levels. in contrast, using the same transgenic mice in t. gondii infection, mortality was higher despite a lower parasitic burden and this was attributed to excessive cytokine-mediated tissue damage. beneficial effects were also seen in a mouse model of trypanosoma cruzi infection and cerebral malaria. topical lipoxin abrogated the tissue and bone damage seen in a rabbit model of p. gingivalis periodontitis. therefore, the proresolution effects of lipoxin may be advantageous in protecting the host from the damaging effect of excessive inflammation, but this benefit may be out-weighed in some situations by permitting unchecked pathogen replication (as seen in the model of m. tuberculosis). the finding that administration of lipoxin enhances survival in a mouse model of cerebral malaria suggests a potential new therapeutic strategy for human cerebral malaria. the effects of resolvin were investigated in a number of mouse models of systemic bacterial infection, and were found to enhance phagocytic clearance of bacteria, reduce neutrophil influx and inflammation severity, promote neutrophil apoptosis and clearance, modulate neutrophil chemotaxis and importantly, reduce mortality. interestingly, resolvin administration also enhanced the antibacterial effect of ciprofloxacin (in e. coli peritonitis) and vancomycin (in staphylococcus aureus skin infection) when the antimicrobial was administered at a sub-optimal concentration. topical resolvin application also reduced the severity of herpes simplex virus ocular infection in mice. finally, protectin was found to increase survival in a mouse model of severe influenza, even when administered 2 days after infection. given the well-recognized importance of these mediators in allergic airway inflammation, it is interesting to consider how their regulation may be disturbed in states of airway inflammation induced by infection, for examples acute respiratory distress syndrome. notably, pulmonary production of protectin was found to be suppressed by highly pathogenic influenza a strains. 37 evidence from cilloniz et al. 12 suggests that lipoxin-mediated activity is also altered during influenza a virus infection. disturbed lipoxin regulation could have a role in the predisposition towards asthma seen in infants following rsv bronchiolitis, 39, 40 especially when considering the finding that mice unable to synthesize lipoxin fail to elicit alternative macrophage differentiation, required for the resolution of rsv disease. 15, 16 if the beneficial effects of these mediators translate from pre-clinical studies into clinical trials, they represent promising new strategies in the management of infectious disease. the pro-resolution, anti-inflammatory and antimicrobial-enhancing effects of resolvins, protectin and possibly lipoxins make these appealing candidates for further study in humans. from a therapeutic perspective it is important to note that these pro-resolution mediators enjoy a substantial advantage over steroids for use in the treatment of infectious inflammation, or other systemic inflammatory states, as they are not immunosuppressive agents. 'aspirin-triggered' lipoxins and resolvins share the pro-resolution effects of lipoxin a4 and resolvin d1, respectively, and act by the same intracellular pathways. 11, 14, 41 this effect is unique to aspirin, and is not shared with non-steroidal anti-inflammatory drugs, which do not trigger the biosynthesis of these mediators. due to its ability to trigger the production of these pro-resolution mediators there may be a new role for aspirin in managing the inflammatory sequelae of infectious disease. the authors declare that they have no competing interests. the acute respiratory distress syndrome molecular circuits of resolution: formation and actions of resolvins and protectins resolvin e1 and protectin d1 activate inflammation-resolution programmes lipoxin biosynthesis and its impact in inflammatory and vascular events lipoxins and aspirin-triggered 15-epi-lipoxins are the first lipid mediators of endogenous anti-inflammation and resolution resolvins: a family of bioactive products of omega-3 fatty acid transformation circuits initiated by aspirin treatment that counter proinflammation signals anti-inflammatory actions of neuroprotectin d1/protectin d1 and its natural stereoisomers: assignments of dihydroxy-containing docosatrienes multi-pronged inhibition of airway hyper-responsiveness and inflammation by lipoxin a(4) resolvin e1 regulates interleukin 23, interferon-c and lipoxin a4 to promote the resolution of allergic airway inflammation protectin d1 is generated in asthma and dampens airway inflammation and hyperresponsiveness resolvin d1 and aspirin-triggered resolvin d1 promote resolution of allergic airways responses lethal dissemination of h5n1 influenza virus is associated with dysregulation of inflammation and lipoxin signaling in a mouse model of infection emergence and pandemic potential of swine-origin h1n1 influenza virus antiinflammatory actions of lipoxin a4 and aspirin-triggered lipoxin are socs-2 dependent role of the lipoxygenase pathway in rsv-induced alternatively activated macrophages leading to resolution of lung pathology control of rsv-induced lung injury by alternatively activated macrophages is il-4ra-, tlr4-, and ifn-b-dependent elevated formation of lipoxins in viral antibody-positive rat alveolar macrophages cytokines and arachidonic metabolites produced during human immunodeficiency virus (hiv)-infected macrophage-astroglia interactions: implications for the neuropathogenesis of hiv disease host control of mycobacterium tuberculosis is regulated by 5-lipoxygenase-dependent lipoxin production identification of nitric oxide synthase as a protective locus against tuberculosis parasite-induced lipoxin a4 is an endogenous regulator of il-12 production and immunopathology in toxoplasma gondii infection lipoxin-mediated inhibition of il-12 production by dcs: a mechanism for regulation of microbial immunity protective role of acetylsalicylic acid in experimental trypanosoma cruzi infection: evidence of a 15-epi-lipoxin a₄-mediated effect lipoxin a₄ and 15-epi-lipoxin a₄ protect against experimental cerebral malaria by inhibiting il-12/ ifn-c in the brain reduced inflammation and tissue damage in transgenic rabbits overexpressing 15-lipoxygenase and endogenous anti-inflammatory lipid mediators resolution of inflammation: a new paradigm for the pathogenesis of periodontal diseases lipid mediator-induced expression of bactericidal/permeability-increasing protein (bpi) in human mucosal epithelia lipoxin a4 analogs attenuate induction of intestinal epithelial proinflammatory gene expression and reduce the severity of dextran sodium sulfate-induced colitis resolvin d2 is a potent regulator of leukocytes and controls microbial sepsis infection regulates pro-resolving mediators that lower antibiotic requirements resolvin d2 restores neutrophil directionality and improves survival after burns the anti-inflammatory and proresolving mediator resolvin e1 protects mice from bacterial pneumonia and acute lung injury neutrophil apoptosis: a marker of disease severity in sepsis and sepsis-induced acute respiratory distress syndrome resolvin e1 promotes phagocytosis-induced neutrophil apoptosis and accelerates resolution of pulmonary inflammation 15-epi-lipoxin a4 inhibits myeloperoxidase signaling and enhances resolution of acute lung injury controlling herpes simplex virus-induced ocular inflammatory lesions with the lipid-derived mediator resolvin e1 the lipid mediator protectin d1 inhibits influenza virus replication and improves severe influenza resolvins: natural agonists for resolution of pulmonary inflammation severe respiratory syncytial virus bronchiolitis in infancy and asthma and allergy at age 13 respiratory syncytial virus in early life and risk of wheeze and allergy by age 13 years lipoxin (lx) a4 and aspirin-triggered 15-epi-lxa4 inhibit tumor necrosis factor 1a-initiated neutrophil responses and trafficking: regulators of a cytokine-chemokine axis key: cord-015569-vy49r1zd authors: nan title: abstracts from the 45(th) annual meeting of japanese association for the stusy of taste and smell (jasts 2011), kanazawa, japan, october 5-7(th), 2011 (the president of the meeting was dr. takaki miwa, kanazawa medical university) date: 2012-05-17 journal: chem senses doi: 10.1093/chemse/bjs052 sha: doc_id: 15569 cord_uid: vy49r1zd nan since umami taste receptors t1r1 and t1r3 have been identified, some reports revealed their structures and functions. umami taste receptors are member of g protein-coupled receptors, belonging to family c with long n-terminal extracellular domain. t1r1 and t1r3 percept umami taste as a heterodimer. we focused on leucine repeats of t1r1 and t1r3 in their transmembrane domains. we have reported the possibility of correlation between the food preference of animals and difference in the length of leucine repeats. poly leucine (polyl) is called homo polymeric amino acid, so called hpaa. it is reported that 1;2% of proteins in prokaryotic and eukaryotic organisms contain hpaas. it is also suggested that polyl could interact with each other more than with the other hpaas. some inherited diseases are caused by proteins in which hpaas such as polyq, polya are expanded to excessive length. activity of the protein containing hpaas is thought to be increased by expansion of hpaas, but with expansion of more than the threshold, activity of the protein is suddenly decreased. hpaas are one of the structures to be seen frequently. it is suggested that hpaas are important structures for some proteins to function. on the other hand normal function of hpaas remains unclear. from the above point of view, polyl might affect interaction of umami taste receptor t1r1 and t1r3. we also hypothesize polyl play an important role in the perception of umami. some odorant receptors also have the similar polyl structure, suggesting that under the hydrophobic condition some proteins are dimerized by interactions between leucine repeats. we named a leucine hpaas motif for protein-protein interaction as ''leucine glue.'' we are discussing the effect of polyl on protein-protein interaction by using split gfp assay. #3 comparative analysis of the umami receptor gene, t1r1 from cetaceans t1r1 and t1r3 are known to act as umami receptor. they are member of g protein-coupled receptors (gpcr) and have seven transmembrane domains. in 2010, it has been reported that the t1r1 gene of giant panda has frameshift mutation, resulting in inactivation of umami receptor. originally, they are used to be a carnivorous animal. it is suggested that the mutation of the t1r1 gene might be associated with their current food preference. we have already analyzed genomic sequences of the t1r1 genes from two different dolphins; lagenorhynchus obliquidens and tursiops truncatus. the results strongly suggested that umami receptor genes, t1r1 from both cetaceans are not functional, because frameshift mutations occurred in their coding sequences. nonsense codon caused by these framshift mutations appeared on their downstream region. in this study, we have also cloned two more t1r1 genes from cetaceans, balaenoptera acutorostrata and balaenoptera physalus. both the t1r1 genes had frameshift mutation and various mutations in their coding sequences. in addition, we have also cloned two more t1r1 genes from cetaceans, b. acutorostrata and b. physalus. both the t1r1 genes had frameshift mutations and various mutations in their coding sequences as well as dolphins described above. especially, in one allele from both b. acutorostrata and b. physalus, we found point mutiations at start codon atg which is a to g substitution, resulting in gtg. by this mutation, t1r1 could not be translated. cell-based expression studies have shown that mammalian t1r1 and t1r3 g-protein coupled receptors combine to form a broadly tuned l-amino-acid receptor. however, contradictory data in t1r3 knockout (ko) mouse models have been reported on taste nerve responsiveness. one study showed that taste nerve responses to amino acid stimuli in t1r3-ko mice were totally abolished. the other reported reduced but not abolished responses to glutamate in t1r3-ko mice. in the present study, we have tried to recharacterize the role of t1r3 for the taste nerve responses to amino acids using a wide variety of taste stimuli. we have recorded from whole-nerve chorda tympani nerve responses from t1r3-ko mice to lingual application of various amino acid stimuli including sweet-and umami-tasting amino acids. whole-nerve recordings of chorda tympani nerve in t1r3-ko mice showed robust responses to all the tested amino acids. comparing to those from c57bl/6 wild-type mice, only small reductions of responses to glycine and l-alanine, but no reduction in responses to l-proline, l-serine, l-glutamine and l-glutamate salts have been observed. these results suggest that t1r3 mediated receptor system may play limited role for detection of amino acids. type iii taste cells play to act to receive sour taste and also to act as ''taste integration cell'' to transmit taste information from type ii taste cells through atp. but, it is not known whether a type iii taste cell has both roles and whether two kinds of type iii taste cells share each role separately. using calcium imaging and immunocytochemistry, we revealed that atp activates type ii and iii taste cells and hcl activates type iii taste cells, and also that hcl and atp activate separate subpopulations of taste cells. we also showed that sour-responding taste cells do not have p2x 2 and p2y 1 as atp receptors in taste buds. moreover, we carried out double cell staining of mice circumvallate papillae using antibodies to atp receptor (p2x 2 , p2x 3 and p2y 1 ) and sour taste receptor (pkd2l1) using an antibody to type ii taste cell marker (plcb2) and type iii taste cell marker (pgp9.5). it was shown that p2x 2 and p2x 3 are expressed in taste nerves, p2y 1 is in type ii taste cells, and pkd2l1 is in type iii taste cells. no taste cell was stained by both atp receptor and sour taste receptor. although we could not cover all the atp or sour taste receptors, these results suggest that there are the two separate subpopulations of type iii taste cells: the one sensing sour taste and the other accepting the atp released from type ii taste cells. #6 development of gustatory papillae in the absence of six1 and six4 six family genes encode homeobox transcription factors, and a deficiency in them leads to abnormal structures of the sensory organs. in a previous paper, six1 was reported to be expressed in the taste bud-bearing lingual papillae of mice, and loss of six1 affected the development of these gustatory papillae. we show here that embryos lacking both six1 and six4 revealed more severe abnormalities than those lacking six1 alone during morphogenesis of their gustatory papillae. by in situ hybridization, six4 was shown to be broadly distributed in the epithelium of the lateral lingual swellings at embryonic day (e) 11.5, and in the tongue epithelium, mesenchyme, and muscles at e12.5. from e14, six4 was similar in expression pattern to six1, as previously reported; and in the fungiform papillae, six4 was expressed in the epithelium at e14-e16.5. in the circumvallate and foliate papillae, six4 expression observed in the trench wall of these papillae at e15.5-p0. although six4-deficient mice had no abnormalities, six1/six4-deficient mice showed distinct morphological changes: fusion of the lateral lingual swellings was delayed, and the tongue was poorly developed. the primordia of fungiform papillae appeared earlier than those in the wild-type or six1-deficient mice, and the papillae rapidly increased in size; thus fusion of each papilla was evident. the circumvallate papillae showed severe defects; e.g., invagination of the trenches started asymmetrically, which resulted in longer and shorter trenches. the foliate papillae elevated initially, and showed stunted trenches. therefore, six1 and six4 function synergistically to form gustatory papillae during development of the tongue. the life span of taste cells was previously estimated as 10-14 days by labeling newly proliferated taste cells with radioactive 3 hthymidine or bromo-d-uridine (brdu) incorporated during dna synthesis. to re-evaluate this observed lifetime of taste bud cells, we repeated the analysis of lingual tissues of mice after incorporation of ethynyl-d-uridine (edu) instead of brdu, followed by detection of edu by a click reaction. we injected edu into adult mice and sacrificed them 1, 3, 5, 6, 10, 15, 20, 25, 30, 35, or 40 days later. the vallate papillae were fixed, cryo-sectioned, immunostained for plcb2 (a marker for type ii cells) and examined with a confocal laser scanning microscope with optical sectioning at 2 lm. taste buds were identified in dic images captured in parallel. numerous cells with brightly fluorescent nuclei (edu+ cells) appeared at day 1 near and within taste buds. the number of those cells declined rapidly toward day 10. however, among type ii cells, very few appeared to be edu+ at day 1. the number of edu+ type ii cells increased at day 3, showed a broad peak around day 10, and then declined. occasional edu+ type ii cells were still detected at day 40. because labeling of dna with edu is much faster, brighter and more consistent than with brdu, this method will be useful to study proliferating cells in taste buds. by combining edu labeling and immunstaining for approapriate markers, the longevity of taste cell types can be examined. adiponectin is a white adipose protein that plays important roles in glucose homeostasis and lipid metabolism and is involved in cell proliferation and differentiation. the function of adiponectin is mediated by its receptors (adipor1 and adipor2), which have different expression patterns. in the gustatory system, adiponectin is contained in the saliva; however, the function of adiponectin signaling in gustatory tissues has not been elucidated yet. therefore, we have examined the expression patterns of adipor1 and adipor2 in mouse gustatory tissues. reverse transcription polymerase chain reaction assays have revealed that adipor1 and adipor2 mrnas were expressed in the circumvallate papillae. using immunohistochemistry, the anti-adipor1 antibody labeled at the taste hairs in the fungiform, foliate and circumvallate papillae. double-labeling experiments have demonstrated an expression pattern of adipor1-positive cells in a subset of basal cells within the taste buds and extragemmal epithelium cells to marker proteins for taste cell types (including types i, ii, iii, and basal cells). for the marker proteins analyzed, only shh was co-expressed with adipor1 in a subset of basal cells within the taste buds in the circumvallate papillae. these results show that adipor1 at taste hairs in gustatory tissues may detect saliva adiponectin and may play a role in taste sensing in the taste buds. in addition, adipor1 may play a role in cell proliferation and differentiation for the shh and adipor1 expressing basal cells within the taste buds. mash1 is expressed in subsets of neuronal precursors in both the central nervous system and the peripheral nervous system. however, involvement of mash1 in taste bud cell differentiation remained to be demonstrated. in the present study, to begin to understand the mechanisms that regulate taste bud cell differentiation, we have investigated the role of mash1 in regulating taste bud cell differentiation using mash1 ko mice and forced expression of mash1 in lingual epithelial cells. in mash1 ko mice, aadc-ir cells are missing both in the mash1 mutant circumvallate papilla epithelium and in the taste buds of soft palate. in mash1 ko/gad67-gfp mice, gfp-positive cells (gad67 expressing type iii cells) are also missing in the taste buds of soft palate. on the other hand, gustducin, a type ii cell marker of taste bud, is expressed in soft palate taste buds in mash1 mutant mice. these results suggest that mash1 plays an important role for expression of aadc and gad67 in type iii cells in taste buds. taste receptor cells are epithelial in sense that they have a limited life span and therefore must be replaced to maintain the structure of the epithelium. therefore gustatory nerves need to make synapse with appropriate taste receptor cells. however, mechanism of recognition of taste receptor cells which express appropriate taste receptor is still unknown. the cadherin superfamily of cell-cell adhesion molecules controls a series of interactions that regulates synapse formation. in this study, in order to test whether the cadherins are required for formation of synapse between gustatory nerve fibers and taste receptor cells, we have investigated expression patterns of cadherin superfamily in the taste buds. #10 induction of type iii-like taste cells in threedimensional co-culture and analysis of excitability of clonal cell lines derived from murine taste buds we have collected taste buds from a tongue of a p53-deficient mouse and established clonal cell lines (tbd cell lines). each of tbd cell lines expresses gustducin and neural cell adhesion molecule (ncam), suggesting that tbd cell lines have characteristics of both type ii and type iii taste cells. to reveal whether tbd cell lines have excitability or not, we analyzed the expression pattern of voltage-dependent sodium channels in one of tbd cell lines, tbd-a5 cells and performed patch clamp recording. scn2a, scn3a, scn5a and scn9a genes coding voltage-dependent sodium channels were detected in tbd-a5 cells by rt-pcr. patch clamp recording revealed that tbd-a5 cells had tonic outward currents, but lacked transient inward currents. these results suggest that functional voltage-dependent sodium channels are not expressed in tbd-a5 cells. next, we tried to establish a culture model mimicking the lingual taste cells of the mouse. tbd cell lines were co-cultured with a lingual epithelial cell line (20a cell line) and a lingual mesenchymederived cell line (tmd cell line). tbd, 20a and tmd cell lines were maintained in a triple co-culture, in which tbd cells were preseeded as aggregates or in suspension on the collagen gel containing tmd cells and 20a cells were laid over the tbd cells. tbd cells in the triple co-culture expressed ncam, however gustducin was not immunohistochemically detected in tbd cells. this result suggests that tbd cells in the co-culture exhibited a characteristic of type iii taste cells. this triple co-culture model would be useful to study morphogenesis and functions of the gustatory organ. immunohistochemistry of taste-signaling molecules is useful for the distinction of taste-bud cell types. however, the optimal immunohistochemical detection depends on specimen-preparing conditions. therefore, this study aimed to examine differences in immunoreactivities under various tissue-preparing conditions in rat vallate taste buds for some typical markers of gustatory cells as follows: gustducin, type iii inositol triphosphate receptor (ip 3 r3), synaptobrevin-2 (vamp2), protein gene product 9.5 (pgp9.5), and neural cell adhesion molecule (ncam). staining patterns and intensities of immunoreactivities for these molecules varied according to tissue-preparing conditions, especially the fixation. confirming our previous finding, gustducin immunoreactivities were localized to the upper part of the taste bud called taste hairs in case of short fixation, but to the cell body cytoplasm excluding the taste hairs in case of long fixation. the specific immunostaining for ip 3 r3 was strong in short-time fixation. on the other hand, those for vamp2, pgp9.5, and ncam were intense in long-time fixation. the present data suggest that different experimental protocols cause the discrepancies in immunoreactivities for these markers. further investigations based on this study should provide important information on the classification of the gustatory cell type. #12 immunohistochemical localization of nk1 and vpac1 receptors in the frog fungiform papilla hiroshi ando 1 , osamu tadokoro 2 , naokazu asanuma 1 , mihoko tomida 1 , takami nakamura 1 and eiji kondo 2 1 department of oral physiology and 2 department of oral anatomy, matsumoto dental university, shiojiri 399-0781, japan although the presence of substance p (sp) immunoreactive (ir) nerve fibers and vasoactive intestinal peptide (vip)-ir nerve fibers has been reported in the frog fungiform papilla, the functional roles of these fibers remain to be elucidated. to clarify whether these sp-ir and vip-ir fibers play any efferent roles in the taste disc in the fungiform papilla, we immunohistochemically examined the existence of neurokinin 1 receptor (nk1r), which is also a sp receptor, and vip and pituitary adenylate cyclase activating hormone 1 receptor (vpac1r), which is also a vip receptor, in the fungiform papilla of the frog, rana catesbeiana. many nk1r-ir structures were observed in the taste disc. these were conical in shape in the middle layer of the taste disc and had a rod-like thin apical process toward the upper layer of the taste disc. double-labeling immunohistochemistry showed that only a few nk1r-ir structures were located adjacent to the sp-ir nerve fibers in the taste disc. vpac1r immunoreactivity was observed in the taste disc cells and the glossopharyngeal nerve bundle beneath the taste disc. vpac1r-ir cells in the taste disc had either a thin or thick apical process. vip-immunonegative nerves beneath the taste disc showed vpac1r-ir. vip-ir neuronal cell bodies colocalized vapc1r in the grossopharyngeal nerve bundle within the tongue.the presence of the nk1r and vpac1r in the taste disc suggests that sp-ir and vip-ir nerve fibers play efferent roles in the taste disc in the frog fungiform papilla. #13 cesium-permeable potassium channel in the rod cells of frog taste disc in animal cells, the resting membrane potential is maintained by random open-shut gating of a leaky potassium channel. frog taste disc cells display the resting membrane potential of about -50 mv. the magnitude is not consistent with the equilibrium potential of potassium (-90 mv). when internal potassium was replaced with cesium in whole-cell configuration, the rod cells in frog taste disc increased a parabolic outward current gradually and displayed the resting membrane potential of -49 â± 2 mv (n=23). the wing cells did not possess the outward current and displayed the resting potential of 5 â± 1 mv (n=41) in the condition of internal cesium solution. the replacement of external normal saline solution with cesium saline solution turned the parabolic outward current to the inward current. the inward current reversed at 2 â± 1 mv (n=10) which is close to the equilibrium potential of cesium (4 mv). several experiments on cationic replacement showed the permeability ratio of p k :p cs :p na :p nmdg =1.67:1.00:0.29:0.15. carbenoxolone (cbx, a blocker of pannexin) inhibited the parabolic outward current moderately (ic 50 , 26 lm). the dialysis of 20 lm arachidonic acid in internal cesium solution changed the parabolic outward current to the exponential one and depolarized the membrane potential by 15 mv. the results suggest that the outward cesium current in the frog rod cells may flow through a novel channel different from mammalian cesium-permeable potassium channel. capsaicin, which is the main component of chili pepper and has many physiological functions such as increasing perspiration and energy metabolism, has negative effect of its strong pungency. capsiate was identified from ã�ch-19 sweetã� (capsicum annuun l.), a non-pungent cultivar of red pepper. like capsaicin, capsiate is thought to enhance energy metabolism by activating the sympathetic nervous system. human sensory evaluation tests demonstrated that capsiate and that analogs (capsinoids) had very weak pungency compared with capsaicin and capsaicin analogs (capsaicinoids), and the difference of threshold level between capsinoids and capsaicinoids was about 1000-fold. however, the in vivo studies focused on the reception of them in oral cavity are few because the reception mechanism of hot taste is unclear, and the reason of weak pungency of capsinoids is not well understood. therefore, this study focuses on the reception of capsinoids and capsaicinoids in the oral cavity. firstly, we performed two-bottle preference tests using rats and confirmed that they began to avoid capsinoids at about 1000 times higher concentrations than that of capsaicinoids. in addition, we recorded the lingual trigeminal nerve responses to capsinoids and capsaicinoids and found that capsinoids induced little responses while capsaicinoids evoked apparent intensive responses. such differences were also found in immunohistochemical detection of p-erk in the trigeminal ganglion after stimulation of capsinoids and capsaicinoids. recent studies using animal models of binge eating shows that the accumbal dopaminergic neurotransmission is elevated in bingeing. it suggests that the experience of bingeing modifies the activity of midbrain dopaminergic neurons in the ventral tegmental area (vta). however, few evidences directly clarify whether the activity of the vta is elevated anticipatorily, that is, before receiving the oral (taste) and/or post-oral (viscerosensory) cues of palatable food. to address the issue, we explored possible changes in the activity of the vta neurons in mice bingeing on sugar by immunohistochemical detection of c-fos expression as a marker of neuronal activation. under food restriction, mice were subjected to a binge-type eating training for 10 days (day 1-10), in which their intake of sucrose solution gradually increased day by day, resulting in its overconsumption. on day 11, mice were deeply anesthetized and transcardially perfused with 4% paraformaldehyde before receiving the sugar exposure. brain sections including the ventral midbrain were immunohistochemically processed to detect the c-fos gene expression product, fos protein. the number of fos immunopositive neurons in the vta of trained mice was larger than that of untrained naã¯ve mice. the finding suggests that the activity of the vta is elevated anticipatorily before sugar exposure and contributes to the stimulation of the mesolimbic dopamine system, resulting in sugar bingeing. #16 activation of output neurons in the basolateral nucleus of the amygdala by a learned aversive taste: a manganese-enhanced mri study tadashi inui 1 , chizuko inui-yamamoto 2 , izumi ohzawa 3 , yoshichika yoshioka 4 and tsuyoshi shimura 1 1 grad. sch. human sci., osaka univ., suita, 565-0871, japan, 2 dept. oral anatomy, osaka dental univ., hirakata 573-1121, japan, 3 grad. sch. frontier biosci., osaka univ., suita 565-0871, japan and 4 immunology frontier research center, osaka university, suita the basolateral nucleus of the amygdala (bla) is known to play an important role in conditioned taste aversion (cta). anatomical studies have shown that the bla has reciprocal neural projections with many brain regions. it is still unclear, however, which connections are involved in the retrieval of cta. to elucidate the neural circuits for the retrieval of cta, we examined the enhancement of activity of the efferents from the bla by the presentation of learned aversive taste, using a manganese-enhanced mri technique. rats received a pairing of 5 mm saccharin with 0.15 m lithium chloride or saline (cta or sham group, respectively). on the test day, thirty min after an injection of 40 mm mncl 2 into the bla, rats were intraorally infused with saccharin. sixty min after the mncl 2 injection, we acquired triple t1-weighed mr images at hourly intervals. the signal intensities of mr images, which reflect the movements of manganese by the activated bla neurons, were measured. the cta group showed significant time-dependent increase of signal intensity in the cea than did the sham group, indicating that the retrieval of cta activates efferents from the bla to the cea. furthermore, the analyses based on the injection site of mncl 2 (rostral or caudal) demonstrated the larger movements of manganese toward the cea and cortices (insular and piriform) in the rostrally-injected cta group. therefore, the outputs of efferent neurons in the rostral part of the bla may be separated in two directions, medial (cea) and lateral (cortices). this work was supported by kakenhi (22700748). the 13 c isotope-labelled acetic acid breath test is a common clinical method to evaluate gastric emptying and is a non-invasive and repeatable method in rodents. however, the sensitivity of this test to detect changes in gastric emptying of liquid diet in mice has not been studied thoroughly. the present study aimed to validate this method by using bethanechol chloride, atropine sulfate, clonidine hydrochloride, cck-8s and phenylthiocarbamide. male c57bl-6j mice after overnight fasting were placed in the metabolic chamber. commercial liquid diet containing 13 c-acetic acid was used as the test meal. after intragastric administration of the test meal, the expired air was collected at 5-min intervals for four hours by a fully automated system. the 13 co 2 / 12 co 2 levels were measured by the mass spectrometer. the 13 co 2 excretion data were analyzed for calculation of gastric half excretion time (t 1/2 ) as the gastric emptying parameter. t-test was used for statistical analysis (p<0.05). bethanechol chloride decreased t 1/2 of liquid diet and atropine sulfate and clonidine hydrochloride increased t 1/2 . cck-8s increased t 1/2 and cck1 receptor antagonist, devazepide, attenuated the delay of gastric emptying by cck-8s. bitter tastant, phenylthiocarbamide increased t 1/2 . our study indicated that the 13 c-breath test was sensitive to detect difference in gastric emptying of liquid diet induced by pharmacological agents, hormones and tastants. we concluded that this technique could be used to determine the effect of physiological, pharmacological and nutritional intervention on the motility of the gastrointestinal tract. in oral surgery treatments, acrinol hydrate is often used as a disinfectant for oral membranes; however, there are few reports about the effect on taste nerve response to this disinfectant. in this study, we tested if tongue treatment with acrinol hydrate affects taste nerve responses in mice. the chorda tympani nerve responses of c57bl/6 mice to 5 basic taste stimuli before and after treatment with acrinol hydrate were recorded and compared. the results of the treatment with acrinol hydrate indicated that the chorda tympani nerve response to 0.5m sucrose was suppressed by 60%; however, there was no change in the responses to 0.1m nacl, 0.01m hcl, 0.02m quinine hydrochloride and 0.1m monopotassium glutamate. the suppression of the sucrose response by this treatment recovered within 1 minute after washing. these results suggest that acrinol hydrate has an ability to suppress sweet taste. some mints, such as peppermint, are used as flavoring substances for foods and toothpaste widely. however, it is not clear whether mints affect the taste receptor mechanisms or not. in the present study, therefore, the chorda tympani nerve responses to 5 basic tastes were compared before and after mixing with 3 types of mints, such as 0.18% peppermint, 0.18% spearmint and 0.10% menthol, in c57bl/6 mice. when spearmint was mixed with 0.5 m sucrose, 20 mm denatonium benzoate, 0.1 m monopotassium glutamate (mpg), 5 mm 5#-inositol monophosphate (imp) and the mixture of 0.1 m mpg and 5 mm imp (m+i), the neural responses to these stimuli were suppressed. but mixing of spearmint did not change the responses to 0.1 m nacl, 20 mm quinine-hydrochloride and 10 mm hcl. mixing of peppermint or menthol did not change any responses to all tested stimuli in the present study. these results suggest that spearmint may work as a suppressant for some kind of receptors for sweet, umami and bitter. the majority of the experiments for investigating gustatory information processing in the nucleus of the solitary tract (nst) have been performed using rats as experimental animals. although the importance of mice in this kind of study is widely recognized from the view point of recent, rapid advance in gene manipulation techniques, there are very few studies using mice in this academic field. also, it is desirable for physiological studies on neural mechanisms of taste perception to use awake, or at least, weakly anesthetized animals. therefore, in the present study we tried to establish an experimental model for in vivo recording of nst neurons in slightly anesthetized or awake mice. for this, we surgically fixed a head restraining holder to the mouse scull, which made it possible to place the mouse head precisely in a stereotaxic position repeatedly without pain. after the recovery from this surgery, the gustatory area in the nst was localized electrophysiologically under anesthesia. then, single neuron activity was recorded in this area under slight anesthesia, and responsiveness to 4 standard taste solutions (nacl, sucrose, citric acid and quinine hcl) was tested. as a result, 9 gustatory neurons were recorded; of these, 4, 4 and 1 neurons were classified into nacl-best, sucrose-best and citric acid-best, respectively. furthermore, we found that the mouse could be easily trained to lick taste stimuli with the head restrained in the stereotaxic device. these results indicate that the present method is applicable to the investigation of gustatory information processing in the nst of mice under a physiological condition. #21 comparison of preference between sucrose and sugar alcohols in c57bl/6 mice yoshihisa katagawa 3 , toshiaki yasuo 1 , keika gen 3 , takashi yamamoto 2 and noritaka sako 1 1 department of oral physiology, asahi university school of dentistry, mizuho 501-0296, japan, 2 department of health and nutrition, faculty of health science, kio university, nara 635-0832, japan and although sorbitol tended to be less preferred compared to palatinit. the present study suggests that the sugar alcohols are less palatable than that to common sugars, such as sucrose, in mice. #22 novel peptides, dryamide-1, dryamide-2: comparison in regulatory effects on feeding behavior of the blowfly phormia regina because the drosophila orphan receptor cg5811 is similar to the vertebrate neuropeptide y receptor, we screened its putative ligands and purified two novel peptides: dryamide-1 and -2. to investigate their function, we injected these peptides into the thorax of the blow fly, phormia regina, and observed the subsequent changes in feeding behavior and taste sensitivity to sucrose. the blow fly was used for technical reasons, to facilitate injection. before and after peptide injection, we measured 1) the sucrose intake as an indicator of appetite, 2) the proboscis extension reflex (per) as an indicator of feeding motivation, and 3) the electrophysiological response of the labellar contact chemosensilla to measure the magnitude of the gustatory input inducing per. there was no significant change in sucrose intake after injection. injecting both peptides depressed per and reduced the percentage of flies that performed per. the electrophysiological response of the labellar taste sensillum to sucrose seemed to decrease after injecting dryamide-1, but not dryamide-2. therefore, once feeding starts, these peptides might not limit the food intake of the fly. nevertheless, the results of the per test suggest that these peptides contribute to feeding initiation. together with other peptides that restrict food intake, dryamide-1 and -2 might contribute to the overall regulation of feeding behavior in phormia regina. #23 stand off effect between the noble bioactive peptide and appetitive odor on feeding behavior in blowfly we compared the behavioral effects of feeding suppression by the two kinds of new peptides (dryamide-1, 2) in blowfly. both of gustatory and olfactory systems are required to elicit appropriate motor pattern in feeding behavior. feeding behavior was also controlled by internal condition based on many bioactive peptides. proboscis extension response (per) is indicator of feeding behavior that is generally controlled by the chemical cues from the maxillary palps. the behavioral experiment showed that they detect palatable chemical signals increasing their appetite in blowfly, phormia regina. phormia usually have detected the appetite odor like mushroom (1-octen-3-ol) by maxillary palps. however, some kinds of newly found peptide have an effect on feeding behavior. therefore, we compared the effect of two kinds of newly found peptides in drosophila. first, we test whether they affect feeding behavior by the amount of sucrose intake. after injection of one kind of peptide, food intake amount was obviously increased. in the next step, we investigated the odor of 1octen-3-ol by maxillary palps did not work as appetitive odor when they are starved sufficiently in behavioral experiments. the results showed that these peptides work as the appetitesuppressant in blowfly feeding behavior, and it did worked as counteractant to odor stimulation. sodium-deficient animals ingest sodium chloride at concentrations normally rejected, and this motivated behavioral state is known as sodium appetite. for neural mechanisms of sodium appetite, there is a line of electrophysiological evidence in rats, whereas there is less evidence in mice. in order to develop a model for sodium appetite in mice, we examined the effects of repeated sodium depletion on salt intakes in c57bl/6j mice. adult male c57bl/6j mice (n=5, 8 weeks, 21-26 g at the beginning of the experiment) were used; each animal was housed individually in a metabolism cage during the experiment. a combination of furosemide injection with sodium-deficient diet was used to induce acute sodium deficiency. in 3 of the 5 mice, furosemide was injected in the 1st week and saline was injected in the 2nd week. in the remaining 2 mice, the injection order was reversed. this pattern was repeated 4 times over 2 months. after 24 hours following the injection, intakes of 0.3 m nacl and distilled water were measured. the furosemide injection shortened the latency of, and increased the amount of, 0.3 m nacl intake. furthermore, the increase in 0.3 m nacl intake was tended to be enhanced at later furosemide injections. both urine volume and 24-h urinary sodium excretion were greater after the furosemide injection than after the saline injection, but there were no significant differences of these measures across repeated depletions. these results show that present procedure reliably elicits sodium appetite in c57bl/ 6j mice, suggesting that this is a suitable experimental model of sodium appetite in mice. the sensations of taste and smell are the important determinants guiding preference for foods and fluids. the dried-bonito dashi is a traditional japanese fish broth that enhances palatability of various dishes due to its specific flavor. however, the scientific evidence why dried-bonito dashi is important for japanese dishes is little known. in general, the main stimulus is considered as inosinate (an umami substance) but the dashi is a complex mixture of many taste substances such as lactate (sour), histidine (bitter), sodium (salty) and other minerals (generally bitter) as well as macromolecules (proteins and peptides) and over 400 odorants. here we investigated mechanisms of preference for dried-bonito dashi using 48-h two-bottle choice tests in adult male sprague-dawley rats. we found that preference for dashi was influenced by current nutrition, i.e., ingestion of high fat or high sucrose diet suppressed preference for dashi. in addition, past experience/learning of dashi ingestion was the most important determinant that could overcome the influences of current nutrition. for example, preference of the threshold concentrations (0.4%) of dashi increased gradually during repeated exposure to dashi solution. after the repeated ingestion of dashi solution, ingestion of high fat or high sucrose diet has no influence on dashi preference. in the descending concentration series, the concentration-preference function shifted 100-fold to the lower concentrations compared with the ascending one. these results suggested that postingestive consequences as well as oronasal sensations guide the preference for dashi solutions that are under the control of current nutrition and past experience/learning. food aversion learning is the long-term memory established after association of the properties of food, such as texture and taste (conditioned stimulus, cs), with visceral malaise (unconditioned stimulus, us). after the acquisition of the learning, the re-exposure to the cs induces the retrieval of the memory and the rejection of the cs occurs. previously, we showed that histamine release in the central nucleus of amygdala (cea) during the re-exposure to the cs was increased in soft pellets-conditioned rats, but not in sweet pellets-conditioned rats. in the present study, we studied the role of the histaminergic transmission in the cea in the retrieval of food aversion learning induced by two patterns of the cs. fos expression, a marker of neuronal activation, following the retrieval of food aversion learning induced by each cs (soft pellets or sweet pellets) and us pairing was examined. few fos-positive cells in the cea were observed by consuming both types of pellets in naã¯ve rats. in the conditioned rats, fos expression in the cea was increased in response to the re-exposure to soft pellets while it was not elevated by the re-exposure to sweet pellets. in addition, the microinjection of h 1 -antagonist to the bilateral cea just before the re-exposure to the cs impaired the aversive response to soft pellets but not to sweet pellets. these findings indicate that the neuronal activation through the histaminergic system via h 1 receptor is involved in the retrieval of texture aversion learning but not in that of taste aversion learning. #27 food entrained feeding behavior to highly sweet food in non-food deprived mice yasunobu yasoshima, megumi tanibuchi and tsuyoshi shimura division of behavioral physiology, department of behavioral sciences, graduate school of human sciences, osaka university, suita 565-0871, japan light-regulated circadian clock in each animal species affects its original feeding rhythm. nocturnal rodents mainly consume food during the dark phase under ad libitum food condition; however, their daily feeding rhythm is entrained to mealtime when they are restricted of food. food-entrainment is suggested to be mediated by increased arousal and food-oriented motivation derived from calorie deficiency, but recent studies indicate that taste palatability seems to be important in food-entrainment. however, the role of taste palatability (hedonic aspect of taste) in food-entrainment remains not fully understood. to study the issue, we developed a new, simple mouse model. ad libitum-fed c57bl/6j male mice received daily limited access to highly sweet chow (hsc) containing 65% sucrose for 2 hours in the light phase (9:00-11:00 a.m.). they received the regimen for 14 days as training. the intake of hsc gradually increased and was about 11-fold greater than their basal intake of normal chow at the corresponding period of time during pretraining phase. on the other hand, the intake of normal chow in another group during the corresponding timing did not increase. intake of normal chow during the dark phase in mice group receiving hsc, but not normal chow, significantly decreased during the training. the entrained increase of hsc intake was also observed in genetically hyperphagic strain, db/db (leptin receptor gene deficiency) mice. these results suggest that taste palatability and/or hedonics of sweeten food play a role in shifting of daily feeding rhythm. #28 sexual difference of gene expression of androgen receptor in the brain region related to extinction memory after conditioned taste aversion learning in immature mice mice acquire the conditioned taste aversion memory (ctam) by application of the conditioned stimulus (cs) such as a novel taste, sodium saccharin (sac), followed by the unconditioned stimulus (us) such as an malaise induced by an i.p. injection of licl. after conditioned taste aversion (cta) learning, the extinction of ctam is induced by repeated presentations of cs without us. recent studies have demonstrated that the extinction is a process of relearning, where mice acquire the extinction memory of cta. we have reported that the sexually mature male mice (c57bl/6) represented significantly higher retention of extinction memory (rem) than sexually immature male mice. in contrast, the significant difference was not observed in female mice. we have also reported that chronical administration of androgen into castrated mice during prematuration period results in an enhancement of rem in both sexes. here we investigated whether the sex difference of the gene expression of androgen receptor (ar) is observed in the ventral medial prefrontal cortex (vmpfc) and amygdala between sexually immature and mature mice by real-time pcr. the sexually immature male mice presented significantly higher level of ar gene in vmpfc and amygdala than the sexually mature male mice. however, there was no such difference in vmpfc and amygdala of the female mice. these results suggest that the sex difference in maturation process of rem is induced by androgen acting on vmpfc and amygdala during sexual prematuration period in mice. our previous study showed that weanling rats acquired conditioned flavor preference when the flavor was associated with low concentration of sucrose; while flavor aversion when associated with high concentration. the present study aims to confirm and extend this finding with various concentrations of sucrose. wister male 3-week-old rats were used. half of the rats received unsweetened grape-flavored water on odd-numbered days and sweetened (sucrose) cherry-flavored solution on even-numbered days. the remaining rats received sweetened grape-flavored solution on odd-numbered days and unsweetened cherry-flavored water on even-numbered days. during the acquisition session, the liquid was presented to each rat for 15 min daily across 6 consecutive days. in the following test session, each rat was presented with unsweetened cherry-and grape-flavored water simultaneously for 15 min daily across 4 consecutive days. the rats showed a significant preference for the flavor associated with 2% or 10% sucrose, but showed a significant aversion to the flavor associated with 30% sucrose, suggesting a hedonic shift from positive to negative with increasing the concentration. the association learning acquired at the age of 3 weeks was preserved when retested at the age of 20 weeks. short-term (5 min) drinking test showed that the volume of intake was significantly smaller for 30% than for 2% and 20% sucrose, indicating that 30% sucrose is aversive because of its oropharyngeal sensation rather than its post-ingestive effects. the present study suggests that weanling experience of food is important in the formation of feeding behavior in adulthood. tastes play important roles in detecting nutrients and irritant materials in food. preference for food tastes is critical for ingestive behavior because greater palatability is to evoke greater food intake. animals generally prefer sweetness but not bitterness. such taste preferences are known to be affected by aging which causes changes in the dietary and energy requirements. however, the mechanisms of shift in taste preference by aging still remain unclear. therefore, to elucidate differences in taste preference among the life stages, we measured the amount of intake of taste solutions by the two bottle test. we used juvenile (3-6 weeks), young-adult (8-11 weeks), adult (17-20 weeks), and middle-aged (34-37 weeks) sprague-dawley male rats. all rats were fed ad libitum during all tests. we used 0.1 m or 0.3 m nacl, 0.01 m or 0.1 m hcl, 0.3 m or 0.5 m sucrose, 5 mm saccharin-na, 3â·10 -4 m or 3â·10 -5 m quinine-hcl, and 0.1 m monosodium glutamate (msg). the preference ratio for 0.5 m sucrose and 0.1 m msg in middle-aged group was lower than that in juvenile and young-adult groups. on the other hand, the preference ratio for 3â·10 -5 m quinine-hcl in middle-aged group was higher than that in juvenile and young-adult groups. there were no significant differences in the preference for hcl and nacl tastes among groups. these results suggest that aging affects taste preferences for sucrose, msg and quinine-hcl which represent sweet, umami and bitter tastes, respectively, in humans. we investigated the effects of furanones as the maillard reaction products flavors on umami and sweet taste preferences in mice. 5-dimethyl-3-hydroxy-2,5-dihydrofuran-2-one; sotolon and 4-hydroxy-2,5-dimethyl-3(2h)-furanone; furaneol (food-grade certified products, safc supply solutions, usa) were chosen as the maillard-borne odorants. in the short time two-bottle tests, 0.001, 0.003, 0.03, 0.1 lg/kg sotolon flavored umami solutions containing 10 mm monosodium glutamate (msg) and sweet solutions containing 1 mm saccharin were significantly consumed more than distilled water. sotolon tended to enhance umami and sweet taste preference in a dose dependant manner. whereas there were no differences of intakes between sotolon flavored water and distilled water. in addition, furaneol and citral flavor agents did not enhance umami solution preferences. these results suggested that sotolon, one of the maillard reaction products, induced enhancement of umami and sweet taste preference in mice. supported by society for research on umami taste and kakenhi (22780127). zinc is one of the essential trace elements and its deficiency causes anorexia, growth retardation, and taste disorder. insulin in pancreatic beta-cells is known to be stored as microcrystals of the zinc-containing hexamer, and zinc is thought to be fundamentally important for assembly and release of insulin from beta-cells. however, the effect of dietary zinc on food intake or insulin secretion of model rats of diabetes has not been well elucidated. in this study, we followed the levels of food intake and insulin secretion in type 2 diabetes model gk (goto-kakizaki) rats fed on diets containing different levels of dietary zinc for 27 weeks. all the experimental rats were divided into four experimental groups of zinc-deficient (zn-def, rats fed on 2.2 mg/kg diet), zinc-sufficient (zn-suf, rats fed on 33.7 mg/kg diet), high-zinc (high-zn, rats fed on 67.4 mg/kg diet), and pair-fed (rats fed on zinc-sufficient diet with the same amount of diet consumed by zn-def one day later). food intake in zn-def gk rats followed typical cyclic-pattern seen with normal rats such as sd rats. plasma zinc concentrations and plasma insulin concentrations were found to have positive correlations in high-zn rats. orexins are neuropeptides synthesized mainly in the lateral hypothalamic area and are implicated in the regulation of food intake in addition to arousal. since swallowing is early step of feeding behavior, orexins might modify swallowing. in the present study, we examined the effects of orexins on the reflex swallowing using anaesthetized rats. orexin, which was dissolved in ringerã�s solution, was administered into the fourth ventricle. orexin-1 receptor antagonist (sb334867), which was dissolved in dimethyl sulfoxide, was administered into the fourth ventricle. three ll of drugs were administered using a microsyringe. swallowing was induced by the electrical stimulation (20 hz, 20 sec) of the central cut end of the superior laryngeal nerve (sln) and was monitored by recording the electromyogram of the mylohyoideus muscle. the frequency of swallowing during the electrical stimulation of the sln decreased after the administration of orexin-a in a dose-dependent manner. significant reduction in number of swallowing was observed after the administration of orexin-a at the dose of 1 or 3 nmol. the latency of the response tended to be longer in the presence of orexin-a. the administration of orexin-b did not affect the frequency of swallowing. pre-administration of sb334867 attenuated the magnitude of inhibition of swallowing frequency induced by the administration of orexin-a. these results suggest that orexin-a but not orexin-b inhibits reflex swallowing via type 1 receptors situated in the dorsal medulla where swallowing center is housed. this work was supported by a grant-in-aid for scientific research from the japan society for the promotion of science (no: 22592064 to mk). stimulation of water receptors in the laryngopharynx (lp) with water shortened the swallowing interval in voluntary swallowing in humans. we previously found that excitation of water receptors was due to absence or reduced concentration of clin fluids applied to the lp. unstimulated saliva contains a low concentration of cl -. thus, unstimulated saliva may excite water receptors in humans. the aim of the present study was to evaluate the role of unstimulated saliva in initiation of swallowing in humans. solutions were infused through a fine tube into the lp at a slow infusion rate (0.2 ml/min) that minimized the mechanical effect of infusion. facilitation of voluntary swallowing by infusion of unstimulated saliva was almost the same as that by infusion of water. topical anesthesia of the throat produced loss of facilitation of voluntary swallowing by both water infusion and unstimulated saliva infusion. the similar effects of water and unstimulated saliva on voluntary swallowing suggest that water receptors are responsible for facilitation of voluntary swallowing by unstimulated saliva. since facilitation by water infusion appeared without mechanical effect of infusion, it is likely that excitation of water receptors in the lp by a small amount of unstimulated saliva precedes excitation of mechanoreceptors caused by a certain amount of unstimulated saliva accumulated in the throat. excitation of water receptors by unstimulated saliva may initiate spontaneous swallowing and spontaneous swallowing probably plays an important role in clearance of the mouth of saliva. therefore, it appears that excitation of water receptors by unstimulated saliva may contribute to avoidance of aspiration. #35 peripheral chemical stimulation to trigger the swallowing reflex and its neuronal mechanisms in humans kensuke kawamoto 1 , takuya inoue 1 , kaya narimatsu 1 , yuki nakamura 2 , makoto inoue 2 , rika yahagi 2 and yasuyuki kitada 3 1 faculty of dentistry, niigata university, 2-5274 gakkocho-dori, chuo-ku, niigata 951-8514, japan, 2 division of dysphagia rehabilitation, niigata university graduate school of medical and dental sciences, 2-5274 gakkocho-dori, chuo-ku, niigata 951-8514, japan and 3 morioka taste and swallowing research institute, 1-15-12 higashi-kuroishino, morioka 020-0108, japan initiation of swallowing is mediated by the swallowing central pattern generator (cpg) in the brain stem. both peripheral and central inputs can activate the cpg. previous studies demonstrated that the ability to initiate repetitive voluntary swallowing varies even among healthy humans and proposed that appropriate chemosensory inputs, e.g., activation of water receptors in the pharynx and larynx, may compensate for difficulty in initiation of swallowing. however, whether variation also exists in reflexively evoked swallowing is unclear, and the nature of interindividual variation has not been clarified. the aim of this study was to investigate interindividual variation of swallowing initiated by central and peripheral inputs and to clarify the neuronal mechanisms that cause this variation. voluntary and reflexive swallowing activity was recorded during infusion of fluid (distilled water [dw] or 0.3 m nacl solution) into the pharynx and larynx at a very low rate in 20 healthy volunteers. dw and nacl were used to activate and inhibit the water receptors, the activation of which facilitates initiation of swallowing. swallowing intervals (sis) were measured between the fourth and ninth swallows, and facilitatory effects (fes) were calculated by subtraction of the si of dw from that of nacl. there was a significant positive correlation between sis of voluntary and reflexive swallowing with nacl. fes of both voluntary and reflexive swallowing appeared strongly in subjects with longer sis. the present results suggest that interindividual variation in the ability to initiate swallowing depends on the potency of the swallowing cpg. the effects of taste stimuli on the change in regional prefrontal cortex blood flow (rcbf) measured by multichannel near infrared spectroscopy (nirs) were evaluated in 10 healthy women volunteers. the test solutions were sucrose, nacl, citric acid and monosodium glutamate at three different concentrations each. six pairs of source and detector nirs probes were placed on the forehead with a 3 cm separation which allowed the measurement of 16 different channels every 0.65 sec. the changes in oxygenated hemoglobin (oxy-hb), which is the most sensitive indicator of changes in rcbf, were analyzed. subjects were positioned in a chair that supported their back and head while wearing a blindfold (eyemask) and headphones to minimize motion, visual and auditory disturbances. the entire oral area was exposed to 5 ml of solution for 15 sec applied using a syringe and rubber tube positioned above the head. another solution was applied after rinsing with deionized water, subjects were tested consecutively for 12 different solutions in random order. each subject was tested for the same solution three times, the data were averaged and the value for the water test data subtracted in order to determine subjectã�s value for a given solution. the taste hedonic tone with a five-rating scale was also measured for the preference test. oxy-hb in all channels increased during all taste stimuli. the increases in oxy-hb and preference scale values showed significant negative correlation. these findings indicate that the prefrontal cortex activation evaluated by nirs is possibly related to pleasant or unpleasant emotions associated with taste stimuli. we receive various different stimulus modalities including 5 tastants and other gustatory and / or somatosensory sensations in eating. sensory information from these modalities would be integrated and recognized as a ã�flavorã�. it is useful to understand their psychophysiological properties because it may help us to develop new functional foods. we investigated the effects of vanillyl butyl ether (vbe), l-menthol (lm) and carbonated water (cw) to the arousal level of 12 healthy subjects. these 3 stimuli were used as hot, cold, tingling stimulus respectively. the results of contingent negative variation showed that vbe, lm and cw increased subjectã�s arousal level just after ingested by aqueous solution whereas no significant change was observed after the water ingestion. the increase of the arousal level still remained for 20 min after ingesting vbe and lm. on the other hand, the arousal level of 20 min after ingesting cw decreased to the same level as before ingestion. apart of the subjects were asked to rate the general stimulus intensity by 9 point likert scale. all samples including water showed maximum intensity just after ingestion and decreased with time course. however, the significant increase of the stimulus intensity still remained for 20 min after ingesting 3 stimulus solutions. these discrepancies between the arousal level and subjective stimulus intensity of cw suggest that only stimulus intensity itself cannot explain the change of the arousal level. in order to understand the property of each stimulus, we need to look each stimulus into detail in the derived modality of the sensation, intensity, timing of appearance. we had reported that umami taste induced sustained saliva secretion. in this study, to investigate the effect of repetitive taste stimulation on saliva secretion, we compared the saliva weight secreted by single taste stimulation (single stimulation methods; ss) with repetitive taste stimulation (repetitive stimulation method; rs), using fourteen healthy adult subjects. in ss, the subjects sprayed the taste solution (approx. 0.25 ml) using a spray bottle into the mouth, and held for 30 seconds and spat the whole saliva into cups at every 30 second for 10 minutes. in rs, the subjects sprayed the taste solution into the mouth, and held for 30 seconds, then, spat the whole saliva into cup, and rested for 1 minute until the next stimulation. after this procedure was repeated ten times, the subject performed the same procedure as ss. the stimuli were 100 mm monosodium glutamate (msg) (umami), 3.8 mm citric acid (sour) and 440 mm xylitol (sweet), which had ã�moderateã� taste intensity on a labeled magnitude scale. during the repetitive stimulation in the rs of msg, the salivary flow was reduced gradually according to the decrease of umami taste intensity by adaptation. however, the msg stimulation caused a sustained salivary secretion after repetitive stimulation, and there was no significant difference in the 10 minutes saliva between ss and rs. thus, the repetitive umami stimulation did not reduce saliva secretion and could produce sustained salivary secretion. there was no adaptation in rs of citric acid and xylitol, and the saliva secretion pattern of rs was similar to that of ss, with less saliva secretion than msg. dried-bonito (db) dashi is a traditional japanese broth that enhances palatability of various cuisines due to its specific flavor. db dashi contains many components, such as histidine, lactate, inosinate, minerals, and proteins. ingestion of db dashi has been reported to have various physiological functions, e.g., effectiveness against physical and mental fatigues, enhancement of peripheral blood flow, and suppression of oxidative stress. in this study, we investigated the effects of db dashi on gastric electrical activity and hunger-satiety states in healthy japanese males. we performed a randomized crossover study for 19 healthy males (21.5 â± 0.5 yr). subjects were fasted for at least 10 h, and all measurements were performed in the morning. before and just after a single ingestion of 150 ml of db dashi (''hondzukuri ichiban-dashi katsuo'', ajinomoto co., inc., japan) or energy adjusted water as a control, gastric myoelectrical activity was measured by electrogastrography for 20 min. subjective motivation to eat (hunger and satiety) was rated by visual analogue scales before, immediately after, and 35 min after the ingestion. ingestion of db dashi significantly increased both electrogastrographic normogastric power and percentage in postprandial state. in addition, db dashi produced higher satiety sensations at 35 min after the ingestion, compared to the control diet. in conclusion, db dashi intake enhances the gastric motility and satiety in humans. we usually take sweet foods with drinking tea or coffee not only to increase nutrition but also to produce feelings of pleasantness and restfulness that belong category of emotion. when we take sweet foods, comfortable audio-visual environments, e.g. consonant interior decoration, good music and fine view may increase feelings of deliciousness. thus, it is hypothesize that environmental factors modify feelings of deliciousness. comprehensive delicious feelings during having sweet foods might be effectively produced in the comfortable environments by integration of various palatable sensory perceptions. electroencephalograms (eegs) were recorded from frontal region of the scalp of healthy participants under virtual scenes of tearoom and construction work, respectively. the participants were asked to rate deliciousness after the recordings. frequency analyses were performed from the eegs. during having the foods, occupancy rates of beta frequency band between tearoom scenes and construction work scenes were markedly different, but not in other frequency bands. during having no food, in contrast, there was no difference of occupancy rates in respective frequency bands between the two different scenes. with regard to deliciousness during having sweet foods, all participants rated high scores under the scenes of tearoom than those under the scenes of construction work. interestingly, there is a positive correlation between occupancy rates of beta frequency band and scores of deliciousness. these findings suggest that comfortable audio-visual environments play an important role for increasing delicious feelings during having sweet foods, in which beta frequency rhythms may be concerned with producing comprehensive feelings of deliciousness. though it has been reported that capsaicin possesses effects of stomach protection and appetite improvement, the influence of dietary habits of consuming pungent substance such as capsaicin on taste sensitivity has not been investigated. in order to elucidate the influence of dietary intake of capsaicin on taste and pungent sensitivities, we measured the thresholds for 5 basic tastes and capsaicin in thai and japanese, because thai have a dietary habit of much more use of pungent substances for meal than japanese. in addition, we conducted questionnaire study about dietary habits of both groups. regarding the recognition thresholds for 5 basic tastes, the threshold for umami tended to be higher in thai compared to japanese with no differences in other basic tastes. this result indicates that daily high intake of pungent substances hardly affect taste sensitivities. however, the threshold for capsaicin in thai was significantly higher than japanese, which was considered to result from much more use of hot pepper for meal in thai. when the thresholds for 5 basic tastes were measured immediately after capsaicin stimulation, the thresholds for acid and bitter tastes were elevated in thai whereas they did not change in japanese. this difference may be due to the concentration of pretreatment capsaicin because suprathreshold concentration was used in the experiment, showing that high concentration of capsaicin may cause transient impairment in some taste sensitivities. in conclusion, it was suggested that daily high consumption of pungent substances hardly affects taste sensitivities but high concentration of capsaicin causes transient hyposensitivity of some tastes. this study aimed to investigate what flavors show interaction with umami taste. twenty-one female university students participated in this study. solutions of 0.25 % msg were used as taste stimuli, and the 19 kinds of flavors were used as flavor stimuli. participants were asked to taste a pair of flavor and msg solution, and to evaluate the intensity of umami taste and the congruency of the flavor with the umami. when the participants taste msg solution with flavor of dried bonito, and of garlic, they evaluated the umami taste stronger than when they taste it without flavors (potentiation effect). when the participants taste msg solution with flavor of tomato, of garlic, and of onion, they evaluated those flavors more congruent with umami taste than when they taste it without flavors. they evaluated congruency of dried bonito and of matsutake less congruent with umami taste. there is a significant, but weak, interaction between the congruency with umami taste and the potentiation effect of umami taste (r=0.36, p<0.05). in general, the flavors that are experienced in our daily lives are evaluated more congruent with umami taste, and have potentiation effect on umami taste perception. preceding studies also suggest that the interaction between olfaction and taste, and perception of umami taste are based on learning. this study also supports the view of ''learned synesthesia'' of olfaction and taste. acknowledgment: this study was supported by society for research on umami taste. eating with someone makes the foods more palatable, and the family ties more strong. on the other hands, eating alone (ko-sho-ku, in japanese) has many problems, such as malnutrition, unbalanced meals, not good at communication etc. ko-sho-ku is very severe problems in modern japanese society. thus, we aimed to study the effect of ko-sho-ku on palatability of the foods. in the study 1, ratings for taste and palatability of confectioneries were compared between when they were eaten with friends and/or family members and when eaten alone. thirty six female university students participated in this study, and were asked to eat two kinds of chocolate and two kinds of cookies in a laboratory (alone) and in their home (with someone). palatability ratings were higher when they were eaten with someone in their home. in the study 2, ratings for taste and palatability of jellies were compared between when they were eaten with watching a child eating same jellies in a video and without watching it. fifteen female university students participated in this study, and were asked to eat three kinds of jellies in a laboratory and to rate a questionnaire about their ability of sympathy. students with high sympathy tend to rate the jellies more palatable. these results suggested that human perceptions of taste and food palatability were affected not only by foods but also by contextual and social variables. acknowledgement: part of this study was supported by a grant titled "effects of eating with someone on palatability of foods." from asahi breweries foundation. #44 taste and smell of the bread made using pre-germinated brown rice sake lees kana kogiso 1 , hiroko nakazawa 1 , yumi yoshioka 1 , akiko sato 1 and mitsuo okazaki 2 1 faculty of human life sciences, nagano prefectural collage, nagano, nagano 380-8525, japan and 2 okazaki brewer co, ltd., ueda, 386-0012, japan pre-germinated brown rice is prepared by soaking brown rice in water at 37 o c for 24-48 hours to initiate germination of sprouts not exceeding 0.5-1.0mm in length. in the traditional method of brewing sake, rice bran and embryo bud are separated from brewer's brown rice during sake brewing in order to remove the rough taste. this study tried to use sake lees in fermentation with pregerminated brown rice, since the sake made from pre-germinated brown rice has hardly appeared on the market. also, the use and benefit of pre-germinated brown rice-made sake and sake lees have not been reported. in this study, we made sensory evaluation to measure the difference in taste and smell between bread made using normal sake lees and bread made using pre-germinated brown rice sake lees. the result showed that the bread made with pre-germinated brown rice sake lees had significantly better smell (p = 0.0365) and stronger sake lees smell (p = 0.0026) as well as a stronger sake less taste (p < 0.0001) than the bread made with normal sake lees. the complex flavor (p = 0.0587) and saltiness (p = 0.0511) of the bread made with pre-germinated brown rice sake lees tended to be stronger than that of the made with normal sake lees. female college studentsã� preference to steamed and cured slices of sweet potato was evaluated to find their sensory characteristics. evaluation was carried out with the 10 sweet potato cultivars and lines, tamayutaka, izumi13, hoshikirari, kanto131, tamaotome, quick sweet, hitachi red, hamakomachi, purple sweet lord and sakukei20 in terms of appearance, fragrance, texture and taste by a rank-scoring method. the most preferable cultivars were hoshikirari, tamaotome, and kanto131; sweetness greatly contributed to the taste of the steamed and cured slices of these samples, while appearance and texture somehow influenced their taste scores. fish flakes are one of the traditional japanese ingredients for dashi. various methods are used to extract dashi depending on the thickness of flake. especially, the thick type flake that is often used for soba noodle sauce requires around 30 minutes of cooking time and extracting conditions vary as well. the energy saving has become recent tendency even in culinary field, and we focused on a cooking method using remaining heat. we analyzed the free amino acid and 5#-imp. sensory evaluation was carried out at the same time. as a result, we can suggest that there is a method for extracting the most effective use of remaining heat for 25 minutes after heating for 5 minutes. we were able to explain the taste improvement from the amino acid composition determined by accq-tag tm ultra method. this condition can be considered effective for both energy saving and flavor improvement of dashi. oolong tea is usually served with oil-rich chinese food. our group has been studying on the mechanism of oil rinsing in the mouth and, in particular, on the interactions between oolong tea components and oil. our previous studies showed that oolong tea had a higher affinity for oil than deionized water, mineral water and green tea did. it may thus be more effective in washing out remaining oil from the mouth. the objective of this study is to determine the effective oolong tea components contributing to removing the residual oily sensation. for the human sensory evaluation, sweetened whipped cream was used as an aliment, and mineral water or oolong tea was used as a washer, with the result that oolong tea more effectively reduced the sense of residual oiliness. an aqueous solution of epigallocatechin gallate (egcg) and epigallocatechin (egc) as tea constituents was used to measure its interfacial tension activity in the presence of soybean oil by a face surface tensiometer. also, we emulsified soybean oil with egcg or egc and examined the resulting average particle size with a laser diffraction particle size analyzer. it was found that egcg and egc did not influence the interfacial tension and average particle size. further studies are required to find some contributory factor. commercial granulated sugars are of 99.9% purity which is almost equivalent to the purity of a guaranteed reagent-grade sucrose. we observed that commercial sugars were different in melting point from one another and the tastes of caramel sauces prepared from these sugars were different. a white coarse bright sugar with larger grain size than that of regular granulated sugar used as an experimental sucrose crystal was milled into powder. in a differential scanning calorimetry of the crystals, a higher peak in the curve of endothermic heat flow shifted to higher temperature side after milling. the powder crystal became more stabilized on heating than the coarse crystal. using the coarse and the powder crystals, caramel sauces (''sauce c'' and ''sauce p'', respectively) were prepared. sensory tests showed that sauce c was more brownish in color and was stronger in sourness, bitterness and acridity. on the other hand, sauce p was better in aroma, stronger in sweetness and more preferable in taste and aftertaste. sauce c was lower in ph, giving stronger sourness. the use of a color difference meter showed a large difference between the two sauces (de=6.4). coarse crystal was less stable on heating and sauce c was more brownish in color, having stronger bitterness. in the sensory studies for flavor development, commercial products or trial products are often used as samples, because sensory evaluation is conducted considering usual behavior of eating habit. but in the case of odor-taste interaction studies, it is desired that odorants and tastants are completely separated and presented to subjects simultaneously. in this study, we established a simple and effective way of presenting odor and taste stimuli separately under two model systems: beverage and soup. experiment.1: evaluation of sweet/sour intensity in fruit flavor beverage firstly, we prepared simple equipment comprising clear plastic cup with scented papers attached on the upper inside edge. the use of non-flavored solution and flavor on the scented papers allow subjects to evaluate beverage. sugar-acid water solution was used as taste stimulus and, lemon and strawberry flavors were used as odor stimuli. untrained panel (n = 45) evaluated intensity of sweet and sour characters. the result showed that lemon flavor enhanced sour character and decreased sweet character when compared with non-flavored condition. experiment.2: evaluation for savory -citrus flavor interaction we prepared ordinary soup solution as taste stimulus, with two savory flavor (chicken and dried bonito) and two citrus flavor (lemon and yuzu) as odor stimuli. untrained panel (n=66) evaluated 6 characters (saltiness, umami, sourness, mild, fatty, animalic). the results showed flavor influenced taste intensity as is the case in experiment 1. in conclusion, we successfully showed the important effect of odor on taste perception by using this study. #50 added flavorings enhance salivary hemodynamic responses to tastes detected by near-infrared spectroscopy -flavor creation using optical imaging-dried-bonito (katsuobushi) broth, an important seasoning in japanese cuisine, has long been used to reinforce the flavor of appetitive foods. not only the taste but also the aroma components of the broth contribute well to the enhancement. to elucidate the effects of aroma from dried-bonito on broth tastes caused by the central integration of flavor, we performed optical imaging of salivary hemodynamic responses using near-infrared spectroscopy (nirs). when hemodynamic responses to a reconstituted dried-bonito flavored broth were compared to those of odorless broth taste solutions, the flavored broth produced a significantly larger response than the odorless broth. this was the case for five of the ten participants, who felt that the combination of the aroma with the tastes was congruent. in the remaining five who felt the combination incongruent, the flavored broth did not cause the enhancement of responses. moreover, when (z,z)-4,7-tridecadienal, one of the most important key aroma components of dried-bonito extracts, was added to the flavoring, the latter five as well as the former five participants came to feel congruency of the reconstructed aroma with broth tastes. the reconstruncted flavored broth produced significantly larger hemodynamic responses than the odorless broth in both participantsã� groups. accordingly, there is a positive correlation between the degree of congruency and increase of the salivary responses. these results indicate that nirs offers a sensitive method to detect the effect of flavoring on the taste-related salivary hemodynamic responses, dependent on the perceptual experience of the combination of aromas and tastes. minty flavors are utilized in foods, beverages, toothpastes and tobacco. minty flavors are considered to have a relaxing effect, which is necessary for us, living in modern and urban societies. the authors have reported that the participants evaluated themselves more relaxing after smelling minty odor, when they were tied out for solving hard puzzles. however, psychological and physiological effects of minty flavors are still unclear. this study was aimed to reveal brain responses to minty flavors and minty odors with nirs (near infrared spectroscopy). sixty female university students participated in this study. they received detailed explanation about the experiment and wrote consent for participating in this study. the three minty stimuli, menthol, spearmint and peppermint and the other three odor stimuli, rose, skatol and orange, were administered to the participants via the olfactometer. the participants were asked to smell the stimuli by an orthonasal or retronasal route or to taste them. brain responses were measured with nirs (bom-1, omegawave, co ltd) and recorded with ad converter (powerlab) connected to the imac. the participants were also asked to evaluate the intensity of the odor continuously with the apparatus which outputs 0;5 v being based on the participantsã� evaluations. the results showed that the stimuli were recognized faster and stronger when the stimuli were administrated via orthonasal and retronasal routes than via oral cavity. the rose odor and orange odor were recognized faster and stronger than the menthol odor. the right forebrain showed greater responses to the stimuli via an orthonasal route than via the other routes, and greater responses to the rose odor than the menthol and the peppermint odors. currently existing odorant sensors are mainly fabricated based on metal-oxide semiconductor devices, conducting polymers, quartz crystal microbalances, or surface acoustic wave detectors. these sensors have been studied for improving on various parameters, such as sensitivity, selectivity, response speed, and portability. however, it has been difficult to develop odorant sensor elements that incorporate a combination of desirable properties. in contrast, living organisms, especially insects, use numerous olfactory sensory cells, which express odorant receptors, to sensitively detect environmental odorants in real time. so far, we have developed a compact odorant sensor that consists of a multichannel fluidic device and xenopus laevis oocytes expressing insect odorant receptors. although this odorant sensor possessed fast responsiveness and good portability, it had some technical disadvantages in terms of the stability of response measurements in the oocytes. here, we report the development of new odorant sensor elements that enable us to acquire stable odorant response measurements. we introduced insect odorant receptors and the olfactory receptor co-receptor (orco) as well as a calcium indicator protein, gcamp3, into spodoptera frugiperda sf21 cells to construct sensor cell lines. when these cells were stimulated with a set of odorants in solution, intracellular calcium as monitored by fluorescence imaging showed sensitive responses in accordance to the ligand specificity of the expressed odorant receptors. these results show that our sensor cells can be applied as odorant sensor elements that detect various kinds of odorants with high sensitivity, selectivity, and stability. with promotion of globalization, our diet are changing in several directions (diversification, mass commercialization, or high quality). because of the change, objective evaluation techniques for taste or odor of foods and beverages are needed for effective r&d and strict quality control. in recent years, biomimetic sensors, such as taste sensor, have been more developed as practical objective evaluation tools for taste or odor. with development of these sensors, development of correlativity evaluation system between objective values (sensor outputs) and subjective values (sensory scores) is needed to realize objective quality evaluation of foods and beverages. mainly developing artificial-lipids-based taste sensors with global selectivity, our research group have studied for realization of ã�taste-odor fusion biosensor system,ã� which estimates quality (deliciousness and safety) of foods or beverages using several sensor outputs through analysis and evaluation of subjective-objective relation. in the studies, we have estimated relations between each taste sensory and each sensor output, but did not investigate about correlativity among quality of foods or beverages and sensor outputs. hence, in this study, we investigated correlativity among sensory of deliciousness and several sensorsã� outputs on a particular sample as the next step of realizing ã�taste-odor fusion biosensor system.ã� as a result, we found that sensory scores of deliciousness were able to be displayed on a superspace as a hyperplane and to be estimated by the hyperplane and sensor outputs. this means that a sensory score on the particular sample can be calculated using sensor outputs and an estimation equation indicating the hyperplane. it has been reported that taste disorder caused by systemic diseases is associated with zinc deficiency. this study included 101 patients with dysgeusia due to systemic diseases: 30 patients with diabetes mellitus (dm), 21 with renal failures, 19 with digestive diseases, 21 with digestive diseases post operation, and 8 with liver failure. taste function was tested by electrogustometry (egm) and filter paper disk method (fpd method) in the area controlled by the chorda tympani nerve. the severe grades of taste threshold before treatment were more than 60% for renal and liver failure patients. all patients were treated with zinc sulfate. the rates of serum zinc deficiency (< 80 lg/dl) were more than 80% for all systemic diseases. in patients with renal failure, the symptom improvement rate was the highest, and the curative period was shortest. however, in dm patients, the improvement rate was the lowest, and the curative period was longest. this study demonstrated that zinc deficiency is induced by systemic diseases, and might cause taste disorder. it is also necessary to consider the possibility of taste disorder caused by medication. this study analyzed 124 patients with bms. the threshold of the taste function was measured by electrogustometry (egm) and the filter-paper disk (fpd) method. both basal and stimulated salivary secretions were measured. in addition, the self-rating depression scale (sds) was assessed. serum zinc and copper levels were also measured. the egm threshold was more than 10 db in 80 (67.2%) of 119 patients, and the taste threshold measured by the fpd method was more than 4 points in 61 (51.3%) of 119 patients. basal and stimulated salivary secretions were less than 3 ml in 90 (78.3%) of 115 patients and less than 10 ml in 52 (45.2%) of 115 patients, respectively. based on the sds, 45 (56.2%) and 14 (17.5%) of 80 patients were diagnosed with neurosis and depression, respectively. blood tests showed that the serum levels of zinc were less than 70 or that of copper were more than 130 lg/dl, respectively, in 61 (54.5%) of 112 patients. the clinical outcome was cure in 38.1%, improvement in 13.6%, recurrence in 11.9%, unchanged in 11.0%, and dropped out in 16.9%. in this study, basal salivary secretions lower than the normal range compared with stimulated salivary secretions. this suggests that, even if the salivary secretory function is maintained to some extent, the sympathetic nerve activity is predominant due to tension. it is necessary to accumulate more clinical data, such as trigeminal sensitivity of the tongue in comparison with the electric taste threshold. postoperative oral tumor patients show a variety of conditions and often display taste dysfunction resulting from anti-cancer drugs, irradiation and defects of oral tissue. prosthetic rehabilitation should be required for the recovery of oral functional disorders. however, no reports have investigated taste disorder for patients wearing maxillofacial prostheses. the aim of this study was to clarify taste sensation in postoperative oral tumor patients using the filter-paper disc method, salivary secretions measured by gum chewing, serum zinc concentrations by blood tests. we investigated patients who had undergone prosthodontic treatment in the department of prothodontics and oral rehabilitation at iwate medical university dental center. results were as follows: 1) taste disorder was seen in 90% of cases; 2) within 2 years after radiation exposure, the degree of taste disorder was severe; 3) disease severity did not necessarily accord with awareness of taste disorder, and some patients with serious taste decline were unaware of their taste disorder; and 4) patients who were aware of their taste decline also showed declines in chewing score. objective inspection and liaison with other departments to include an otolaryngologist, doctor of psychosomatic medicine, and a dietitian is important. it is known that more than 70% of patients with taste dysfunction have zinc deficiency. carbonic anhydrase (ca) vi, also known as gustin, is a 37,000-da zinc metallo-protein found in human saliva and is involved in taste dysfunction. it can be detected by enzymelinked immunosorbent assay, but the test takes time. therefore, in this study, we developed a new quick and simple method of diagnosis using an immunochromatography assay to be used as an on-site test. first, reactivity with a serum antibody (prepared using a synthetic peptide designed from a 93-111 amino acid chain of human ca vi as a hapten conjugated to keyhole limpet hemocyanin and injected into two new zealand white rabbits) was evaluated and compared to a control (swine antirabbit igg) using a hi-flow plus120 membrane and detected with colloidal gold. a difference in color development was observed between samples of pbs (-) and the positive control (synthetic peptide). furthermore, it was density dependent, and in serially diluted parotid gland saliva, the color development was seen to be 640 times dilution density from 8 times dilution density. the results suggest that an immunochromatography assay using this novel antibody can be used to quantitatively measure ca vi, which may be useful in the diagnosis of taste dysfunction caused by zinc deficiency. #58 the epithelium of recessus olfactorius is a part of the accessory olfactory system in the japanese toad (bufo japonicus) hideo nakazawa 1 , kimiko hagino-yamagishi 2 , atsushi c. suzuki 1 and takatoshi nagai 1 the recessus olfactorius (ro) is a small pouch observed in the antero-lateral olfactory epithelium of anurans. ro is mostly covered with the epithelial cells with cilia, which is reminiscent of sensory function. however, if ro is a part of the main olfactory system has not been studied. we studied the epithelium of ro (ro epithelium) in japanese toads regarding central projection, ultrastructure and expression of proteins involved in olfactory transduction. fluorescent carbocyanine dye (dii) was placed on the ro epithelium to show a neuronal projection from ro. fluorescent signal was not detected in the main olfactory bulb, but in the accessory olfactory bulb. in contrast to the olfactory epithelium which does not contain microvillous receptor cells, the ro epithelium contained both ciliated cells and micro-villous cells. the expression of a-subunit of g-proteins (g aolf and g ao ) in the ro epithelium was examined by immunohistochemistry and in situ hybridization. regionally different expression of g aolf and g ao was detected in the ro epithelium, namely expression of g aolf in the apical cell layer and g ao in the basal cell layer. such a pattern of g-protein expression in the ro epithelium was similar to that in the vomeronasal epithelium. these results suggest that the ro epithelium belongs to the accessory olfactory system. however, the ro epithelium contained secretory cells with granules that were not contained in the vomeronasal epithelium. physiological experiments will further clarify if the ro epithelium is functionally similar to the vomeronasal epithelium. in mice, the female memorizes some urinary pheromones of the mating male, thereby preventing the pheromones from inducing pregnancy block. the memory depends on the neural changes in the accessory olfactory bulb (aob), the first relay in the vomeronasal system. microcircuits in the aob include the prominent reciprocal dendrodendritic synapse between mitral cell projection neurons and granule cell interneurons. a previous study has shown that in vivo infusion of a protein synthesis inhibitor, anisomycin, in the aob is able to prevent the pheromonal memory formation. other reports have indicated that antidromical stimulation of mitral cell axons induces long-term potentiation (ltp) at the mitral-to-granule cell synapse in slice preparations of the aob. using aob slices, we measured field epsps derived from granule cells to examine the effects of protein synthesis inhibition on ltp at the mitral-to-granule cell synapse. high frequency stimulation, consisting of a 100 hz, 100 pulse train applied four times at 3 min intervals, induced ltp lasting for 180 min. under bath application of anisomycin (20 lm), high frequency stimulation induced short-term potentiation and early-phase ltp, but failed to induce late-phase ltp. the results suggest that protein synthesis underlies late-phase ltp at the mitral-to-granule cell synapse in the aob. the goal of our research is to understand the mechanism of synaptic transmission in the aob that has been demonstrated to be critical to memory formation for male mouse pheromones. by measuring the reciprocal synaptic currents from mitral cells in the aob, we have demonstrated that an agonist for group ii metabotropic glutamate receptors (mglur2/mglur3), dcg-iv, suppressed dendrodendritic inhibition (ddi) in a reversible manner while the mglur2/mglur3 antagonist ly341495 enhanced it. the effects of these drugs were markedly impaired by genetic ablation of mglur2, indicating that dcg-iv-mediated suppression of ddi is mediated by mglur2. in these studies, glutamate release was triggered by an application of a voltage step from -70 to 0 mv. in the present study, to see whether mglur2 has similar effects on the ddi elicited by more physiological stimuli, ipsps triggered by spike trains in mitral cells in slice preparations were recorded using the patch-clamp technique in whole-cell configuration. aob slices were prepared from 23-to 36-day-old balb/c mice. ly341495 enhanced it. together with previous results, the present result suggests that mglur2/mglur3 can be activated by endogenous glutamate release from mitral cells, which results in the suppression of the synaptic transmission from mitral to granule cells in the aob. phenols are predominant in the total flavor compounds of dried bonito (katsuobushi). the influence of odors of 1,3,5-trimethoxybenzene (tm; m=168), 2,6-dimethoxyphenol (dt; m=154), and guaiacol (gu; m=124), which were the chemicals responsible for the katsuobushi aroma, on the production of b-endorphin (be) in the hypothalamus were examined in f344 male rats. rats (5 weeks of age) were exposed to 0.1% of odors dissolved in either ethyl alcohol (etoh: m=46) or triethyl citrate (tec: m=276) for 10h/ day for one or two weeks. be levels in water soluble extracts of hypothalamus were examined by elisa kits. among three phenols, tm is the most potent odor to increase be levels in rat hypothalamus, followed by dt and gu: the increasing rate as compared with the control was 170%, 160% and 140%, respectively. these phenols may activate glomeruli clusterd in the lateral domain of the dorsal surface of the mammalian olfactory bulb and probably suppress the activities of sympathetic nervous system. these results suggest that the long-term exposure to bonito bouillon-flavored diet enhanced ratã�s preference for bonito bouillon. neurogenesis occurs in the forebrain subventricular zone (svz) throughout life. these neurons migrate to the main olfactory bulb (mob) via the rostral migratory stream (rms), and upon reaching the mob they differentiate into granule cells and periglomerular cells. the information broadcast by general odorants is received by the olfactory sensory neurons and transmitted to the mob. newly generated neurons at the svz play important roles in odor discrimination and odor memory. recent studies have shown that reduction of mastication impairs neurogenesis at the hippocampus and brain functions. in the present study, bromodeoxyuridine-immunoreactive (brdu-ir) structures in the sagittal section of the svz, rms, mob and accessory olfactory bulb (aob) of female adult mice fed a soft diet were studied to explore the effects of reduction of mastication on newly generated neurons at the svz and mob. the numbers of brdu-ir cells in the svz of adult mice fed a soft diet for 1, 3, or 6 months were lower than those of mice fed a hard diet. the odor preferences of individual female mice to butyric acid were tested in a plexiglas y-maze preference apparatus. feeding the soft-diet affected the adverse responses of mice to butyric acid. the results suggest that feeding with a soft-diet reduces neurogenesis at the svz, which in turn reduces olfactory function at the mob. epigenetic mechanisms play an important role in memory formation and synaptic plasticity. specifically, histone-associated heterochromatin undergoes changes in structure during the early stages of long-term memory formation. before eye opening young rats depend on somatosensory and olfactory function for survival, as they can learn their damã�s odor and approach her without visual information. in order to establish olfactory learning, the pairing of odor and somatosensory stimulation is crucial. we have shown that synaptic plasticity in the ob underlies aversive olfactory learning. our behavioral pharmacological experiments have shown that long-term olfactory memory requires activation of creb. western blot analyses have revealed that expression of p-mapk/erk is increased for 1 hour after odor-shock training, followed by increase of p-creb lasting for 6 hours. therefore, we examined whether intrabulbar infusion of trichostatin a (tsa), a histone deacetylase (hdac) inhibitor, facilitates olfactory learning in young rats. tsa infusion during odor-shock training enhanced a conditioned odor aversion in a dose-dependent manner. we further tested whether odor-shock training leads to histone acetylation in the ob and defined the time course of the activation. the acetylation of histone h3 was significantly increased for 1 hour after odor-shock pairing compared with odor only or no stimulation. tsa infusion significantly increased histone acetylation levels as well. these results show that hdac inhibition is associated with aversive olfactory learning in young rats. the endopiriform nucleus (epn) is a large group of multipolar cells located in the depth of the pirifprm cortex (pc). although many studies have suggested that the epn plays a role in temporal lobe epilepsy, the normal function of the epn remains to be elucidated. by using optical imaging with voltage-sensitive dye in slice preparations, we previously found that paired-pulse stimulation to pc and/or gustatory cortex (gc) evoked the excitation propagation to the epn, agranular division (ai) of the insular cortex and further to the claustrum (paired-pulse facilitation). in the present study, we investigated the electrophysiological properties of neurons in the epn, claustrum and ai in anaesthetized rats. in response to electrical stimulation of the olfactory bulb, the evoked potentials with a latency of about 50 ms were elicited in these regions. paired-pulse facilitation of the evoked potential was also observed. of the 52 neurons in these regions tested with odor and gustatory stimulation, 8 (15%) showed excitatory responses to both odor and taste stimulation, 14 (27%) had excitatory responses to odor but not to taste stimulation, 2 (4%) had excitatory responses to taste but not to odor stimulation, and the remaining 28 (54%) had no response. further, we previously found significant increases in the numbers of c-fospositive cells in the pc, gc, epn, ai and claustrum following natural stimuli, such as feeding an apple. these results, together with previous results, suggest that the epn, claustrum and ai are the multimodal-chemosensory regions, where olfactory and gustatory information is centrally integrated. department of science education, faculty of education, chiba university, chiba 263-8522, japan we found that a simple and typical association learning of a flower odor with proboscis extension reflex to nectar in honeybees was inhibited by exposure to extremely low frequency (elf) alternating magnetic fields. we postulated that the elf magnetic fields could be detected by magneto-receptors. honeybees have two types of putative magneto-receptors which either contain magnetite crystals, abdominal dark short hairs or abdominal trophocystes. we previously reported that a cutting of the abdomen longitudinal connective nerve between 3rd ganglion (first ganglion in abdomen) and 4th ganglion had no effect on the inhibitory action of alternating magnetic fields. and we concluded that the trophocystes had no role in magnetoreception. in this work, we confirmed that the dark hairs had a role in magnetoreception and inhibitory action of olfactory learning by magnetic fields. an exposure to 100hz alternating magnetic fields of approximate 450lt inhibited the performance of conditioning learning in intact bees. when all dark hairs were pull out, an exposure to alternating magnetic fields had no inhibitory effects on the conditioning. so, it was shown that the dark hairs had a role in magnetoreception which impaired olfactory learning. #66 prediction of glomerular activity patterns using the graph structure of odorant molecules zu soh 1 , toshio tsuji 2 , yuichi kurita 2 , noboru takiguchi 3 and hisao ohtake 1 1 grad. school of engineering, osaka univ., suita 565-0871, 2 grad. school of engineering, hiroshima univ., higashihiroshima 739-8527, 3 grad. school of natural science and technology, kanazawa univ., quantitative evaluation of odor qualities is an important process especially in the fragrance, food and beverage industries. however, most of the conventional odor sensing systems are unable to assess odor qualities perceived by humans. this study therefore aims to develop an artificial sensory evaluation system that can be used to evaluate odors and support sensory testing. as the first step in the development of such a system, we proposed a neural network model for prediction of olfactory glomerular activity based on the results of previous studies, which reported that odors evoking similar glomerular activity patterns have similar odorant qualities. the proposed model consists of marginalized graph kernels and a multilayer neural network. the former is used to evaluate distances between odorant structures, and the latter converts these distances into glomerular activity patterns. however, a number of parameters included in the model required further discussion. we therefore explored the two parameters that most significantly influenced the prediction accuracy of the model. these were the number of the gaussian units and the number of neuron units in the neural network. the results indicated that optimal prediction ability was achieved when the number of the gaussian units was approximately sixty and the number of neuron units in the hidden layer was approximately double than in the input layer. based on these outcomes, future plans for further improvement of prediction ability were discussed. in food and beverage industries, quantitative assessment of odors is an important process in developing new products. however, conventional odor sensing systems focus on identifying or detecting specific odors, and are unable to assess odor qualities perceived by humans. against this background, our study aims to develop an odor assessment system that can be used to evaluate odors and support sensory testing. toward the development of a model for the evaluation of odor qualities, this study focuses on neural activity evoked by odorant stimuli in the olfactory system. this is because the results of previous studies suggested that odorants prompting common neural activity patterns in the olfactory system had similar odorant qualities. as a first step, we conducted a series of human sensory tests to investigate perceptual similarities between odorants, and then compared the results with activity patterns evoked on the glomerular layer of the olfactory bulb in rats. these animals were chosen because the structure of their olfactory system is similar to that of humans, and measurement data relating to their glomerular activity is available online. we defined three indices (correlation, overlap rate of activation, and sum of squares of difference between histograms) to describe similarities and differences between activity patterns, and also conducted sensory testing to determine similarities to human perception. comparison of the results revealed that each index showed a weak correlation to perceptual similarities. we therefore examined whether similarities between odorants could be predicted by combining the three indices using svm (support vector machine), and the results indicated a certain level of prediction ability. novel esters, alcohols, and ethers containing a tetrahydropyran ring were derived from (2,6,6-trimethyltetrahydropyran-2-yl)acetic acid (1) or 2-methyl-(2,6,6-trimethyltetra-hydropyran-2-yl)propanoic acid (2), and their odor properties were investigated. the relationships between the structures of the derived compounds and their odor properties were also examined. the methyl ester of carboxylic acid (1) had a refreshing, floral, and herbaceous odor, and the methyl ester of carboxylic acid (2) had a refreshing, woody odor. the propyl ester of carboxylic acid (1) had an adzuki-bean odor, and the propyl ester of carboxylic acid (2) had a vegetable and spicy odor. thus, the odor of methyl ester of carboxylic acid (1) was thus similar to that of methyl ester of carboxylic acid (2), but considerably different from that of the propyl ester. moreover, the odor of propyl ester of carboxylic acid (1) was considerably different from that of propyl ester of carboxylic acid (2). to determine the most stable structures of these esters, the molecular orbital calculation was carried out for each ester using mopac pm3. the bulkiness of the side chain of a tetrahydropyran ring of methyl ester of carboxylic acid (1) was comparable to that of methyl ester of carboxylic acid (2). however, the side chain of propyl ester of carboxylic acid (1) was bulkier than that of its methyl ester, and this increased bulkiness prevents the free rotation of the side chain of propyl ester. similarly, the bulkiness of the side chain of propyl ester of carboxylic acid (2) makes its free rotation more difficult than that of propyl ester of carboxylic acid (1). thus, the bulkiness and the degree of free rotation of the side chain of a tetrahydropyran ring influence the odor of compounds containing a tetrahydropyran ring. measured odor thresholds depend on both subject sensitivity and method. yet, threshold-methods have received relatively little systematic attention in olfaction. we measured psychometric, i.e., proportion correct detection vs. concentration, functions for acetic acid. a 2-outof-5, forced-choice procedure was used. stimuli were precisely controlled using an air-dilution olfactometer. the design had four factors, all randomized or counter-balanced: 1) practiced subjects vs. unpracticed subjects (between subjects); 2) 15-second inter-trial interval (iti) vs. 30-second iti (within subjects); 3) re-sampling allowed (i.e., subjects could smell each of the 5 stimuli presented during a trial as many times as they wished) vs. not allowed (within subjects); 4) concentrations presented in ascending order (lowest concentration first, moving up to the highest concentration, then starting again at the lowest concentration after a break) vs. random order (within subjects). a four-way anova (the above four factors) revealed a significant main effect of re-sample condition. these results highlight the importance of methods for measured thresholds, and have implications for laboratory practice. many researches have reported the influence of information to identify an odor. we tried to measure the prefrontal cortex activity during the smelling an odor using functional near-infrared spectroscopy (fnirs) to find out the effects of positive and negative information with a same odor. we used three kinds of odors which have been evaluated as moderately hedonic values by a preliminary experiment. eighteen subjects who had normal sense of odor were presented these odors with visual stimulation on a display. the visual stimulation showed the explanations of each odor with positive or negative information. during the experiment, subjects were measured for their prefrontal cortex activity with 42 channels (3x9 distributions) by fnirs (foire-3000, shimadzu corp). these odors were presented by odor-stimulation system through tubes and a mask. the experiment was conducted by the following protocol; pre-resting period (20s), task period (15s), evaluation period (25s) and post-resting period (25s). we had 24 trials (3 odors x positive/negative information x 4 trials) and 4 control tasks with odorless. during the evaluation period, subjects evaluated the intensity, pleasantness/unpleasantness and familiarity of each odor. the stimuli were presented by random order. as a result, we showed the different evaluation value on the same odor but different information condition. the positive information condition showed significantly higher pleasant value than negative information condition (p<.05). fnirs data also showed the significantly different prefrontal cortex activation between positive and negative information conditions. we showed the effects of positive and negative information on a same odor. first impression of a stranger is created by several perceptions, such as face, tone of voice, speed of words etc. there are many researches on development of first impressions, but there are few researches that study a role of olfaction on development of first impressions. we wear perfume not only because we like it, but also because we want someone to have good impressions. this study aimed to investigate whether the perfume that we wear affects othersã� first impression for us. sixty female university students participated in this study, and all of them were not acquaintance of the experimenters. ck one, citrus-floral perfume, and coco chanel, fresh oriental note perfume, were used as olfactory stimuli. the method to investigate for participantsã� impression for the female model (experimenter) was teg ii (tokyo university egogram ver. ii, published by kaneko shobo). when the model wore ck one, she was evaluated as having stronger sense of responsibility and the makings of leader. on the other hand, when the model wore coco, she was evaluated as having unique personality and strong self-assertion. these results show the perfume that we wear changes othersã� first impressions for us. the authors are continuing to study about this phenomenon, and confirm it with another models and with another perfumes. #72 a study of the image congruency between bottle appearance and fragrance of perfume appearance of bottles might influence on the evaluation of perfumes as visual information before sniffing fragrance. we investigated such congruencies/ incongruencies between visual and odor information in the market products of perfume through the two experiments. in the experiment1, we investigated broad constructs of image aroused from appearance of bottles and flagrance of perfumes, and reviewed congruencies between bottles and fragrances. we selected 10 products as samples, and used ''napping'' for experiment1. in napping, subject sort the samples on the space reflecting sensory similarities. 11 our subjects carried out napping under the two conditions. subjects were asked to sort samples according to similarities of scents. these scents were sniffed actually in the sniffing condition, but in the viewing condition, scents were imagined from appearance of bottles. distances matrixes between samples were measured from results of napping, and were used for multi-dimensional scaling (mds). the result of mds showed that dimension1 could represent major image of perfumes (for men or female) in the both conditions, and that diman-sion2 in the viewing condition could represent lightness of scents related to transparencies of bottles. however, we could not find systematic feature change of scents on dimension2 in sniffing condition. it may suggest complexity of scents sniffed actually. we then examined the qualitative aspects in congruencies/ incongruencies between bottles and flagrancies in the experiment2. we selected 5 among 10 perfumes used in experiment1, and used 10 descriptive terms to evaluate each of the samples. in the results of experiment 2, we could not find any relations between terms with perfumes. changes of the physiological parameters (saliva amylase, heart rate, and brain waves) of 10 subjects during the smelling of the bark oil of lindera umbellata were measured. the value of saliva amylase did not change during the smelling of the bark oil. the proportion of the parasympathetic nervous system increased significantly during the smelling of the oil, while that of the sympathetic nervous system decreased. on the other hand, the proportion of b-wave decreased significantly during the smelling of the oil. thus, the bark oil exhibited the relaxation effects for the subjects. the bark oils included monoterpene alcohols (73.19%) and ketones (6.99%), monoterpene oxides (5.59%), monoterpene hydrocarbons (5.18%), and others. among of these, linalool (63.70%) was the most abundant component. here we investigated relationship between the number of constituent odors and odor pleasantness. we hypothesized that the stability of the olfactory perception depends on the number of odor components and their molecular variability. we especially focused on odor pleasantness, which best explains molecular variability of odorants in the natural world. three different categories of odors, aliphatics, aromatics and terpenoids, were used in this study. we used 5 or 10 odors for preparing multi component odors. odor pleasantness for the multi component odors tended to be more stable against concentration changes than that of the single component odors. notably, multi component odors containing odorants of all categories showed higher stability than odors containing single odor category. future study for large number of components with different molecular variability will reveal rules for the prediction of the odor perception for odor mixtures. #75 fmri of brain responses to complex odor and visual stimuli one of the most important features of odor perception is the hedonic or emotional component. the purpose of this study was to measure the brain activity associated with human olfaction using functional magnetic resonance imaging (fmri) and to evaluate the influence of the pleasantness of odors by complex odor and visual stimuli. seventeen healthy right-handed subjects (9 men; 8 women) with normal olfaction participated in the study. an event-related mri method was used to analyze olfaction-induced responses on fmri. a puff of odorant was delivered to the nose of each subject through a nose mask with electrically controlled pneumatic valves. for the event-related task in the fmri experiment, two odorants (pleasant odor: amyl-acetate, unpleasant odor: iso-valeric acid) and two types of visual stimuli (pleasant image, unpleasant image) were randomly presented. two cross-modal sensual interactions were tested using spm-software for both matching/mismatching conditions and pleasant/unpleasant conditions, respectively. results revealed specific brain areas that were activated by cross-modal interactions with odor and visual stimuli. in advance of the fmri experiment, psychological odor experiments (cross-modal, complex stimuli with a visual and odor evaluation test) were conducted using a subjective judgment method or pleasantness/unpleasantness, respectively. based on the fmri and psychological experiments, we estimated the most suitable conditions for evaluating the pleasantness of odors. from analysis of the fmri experiments, two responses (1: emotional response to pleasantness/unpleasantness, and 2: response to matching/ mismatching) were obtained with the cross-modal, complex stimuli using odors and images. from the analysis of our experiments, activity associated with the response to pleasantness/unpleasantness was seen mainly in the right orbital frontal cerebrum and the supramarginal gyrus, middle frontal gyrus and amygdala. on the other hand, activity associated with the response to matching/mismatching was seen in areas of the brain corresponding to memory and cognition. thus, cross-modal, complex effects were suggested to be an emotional response to pleasantness/unpleasantness and cognitive and memory responses to matching/mismatching. as otorhinolaryngologists, it is important to grasp the grade of subjective symptoms and quality of life (qol) when we examine outpatients with olfactory dysfunction. however, the influence that the sense of smell exerts on the qol related to health is not clear. therefore, we investigated outpatientsã� qol using the ''health-related qol measure (sf-36),'' which is widely used around the world. concurrently, we applied a 1) visual analog scale (vas) to grade subjective symptoms (the smell of food and drink, perfumes, excreta and gases) and the deliciousness of meals (qol), 2) smell questionnaire for daily life, 3) t&t olfactometry, 4) intravenous olfaction test (alinamin test) and 5) card-type smell identification test (open essence). we then analyzed those tests and sf-36 for correlations. as the results, ''vitality (vt),'' which is a subscale of sf-36, correlated weakly with the subjective symptom vas for ''smell of perfume'' (r s =0.262) and the t&t recognition threshold (r s =0.203). ''role functioning emotional (re),'' which is another subscale of sf36, correlated weakly with the subjective symptom vas for each of ''smell of food and drink'' (r s =0.234), ''smell of perfume'' (r s =0.272) and ''smell of gas'' (r s =0.222). no other correlation was found for any of the olfactory tests. these results showed that, although olfactory dysfunction affected the vt and re of the patientsã� health-related qol, it was very slight. even if olfactory dysfunction develops, the effect on the overall qol of the patient is slight compared with other diseases. this likely explains why these patients are slow to consult a doctor. â½introduction olfactory dysfunction is most commonly caused by chronic rhinosinusitis (crs) in japan. within crs, eosinophilic chronic rhinosinusitis (ecrs) is said to cause severe olfactory dysfunction. however, there have been few reports that have compared ecrs with non eosinophilic chronic rhinosinusitis (necrs). â½patients and methods a prospective study was carried out at three institutions. a total of 621 patients underwent endoscopic sinus surgery between april 2007 and march 2008 and were pathologically diagnosed with crs. of those, 464 patients (318 males, 146 females; mean age: 48.2 years) were included in this study because they had undergone olfactory evaluation preoperatively. olfactory dysfunction was assessed using the standard olfactory threshold test (t&t) and subjective symptom score (0;6). data were analyzed using spss software ver. 11. â½results the 464 patients consisted of 228 necrs patients, 190 ecrs patients, 21 patients with allergic fungal rhinosinusitis (afrs), 13 patients with odontogenic maxillary sinusitis and 12 patients with fungal rhinosinusitis. t&t found that 54.9% of the total patients had olfactory dysfunction with a mean recognition threshold of â�¡4.1 (107 of 228 necrs patients; 46.9%, 119 of 190 ecrs patients; 62.6%), while 57.2% of the total patients had olfactory dysfunction with a subjective symptom score of â�¡ 3 (100 of necrs 228 patients; 43.9%, 135 of 190 ecrs patients; 71.1%). both the t&t result (or: 1.895) and the subjective symptom score (or: 3.309) were significantly worse in the ecrs patients than in the necrs patients. [introduction] we investigated the hypothesis that the prognosis of olfactory dysfunction (od) due to chronic rhinosinusitis (crs) becomes worse as the duration of od becomes longer. [patients and methods] 113 patients (70 males, 43 females; age range: 21-74 years; mean age: 50.9â±12.7 years) with od due to crs underwent appropriate surgery and postoperative treatment, and were followed up for at least 3 months. a visual analog scale (vas) was used to evaluate their pre-and post-treatment symptoms of od. [results and discussion] patients with an od duration of â�¡5 years showed significantly less improvement compared with an od duration of <5 years. the od duration and the post treatment vas showed a significant negative correlation (r s =ã�.399, n=113). it was particularly strong in male patients aged â�¡60 years (r s =ã�.632, n=27), and in the aspirin-intolerant asthma (r s =ã�.624, n=11), peripheral eosinophil count of â�¡800/microl (r s =ã�.528, n=17) and current smoking (r s =ã�.731, n=11) subgroups. multiple logistic regression analysis of 19 patients with severe od even after appropriate treatment identified the following as poor prognostic factors: 1) male gender (or=18.996), 2) od duration â�¡5 years (10.023), 3) age â�¡60 years (9.349) and 4) peripheral eosinophil count of â�¡800microl (8.234) (predictive rate: 89.4%). these results indicate that the prognosis of od due to crs becomes poorer as the duration of the disease becomes longer. in particular, patients who have these risk factors should be started on treatment as soon as possible and should be advised to quit smoking. motohiko suzuki, yoshihisa nakamura, hiroshi kawaguchi and shingo murakami causative viruses of postviral olfactory dysfunction have not yet been identified. we investigated causative viruses in patients with postviral olfactory dysfunction. we examined the presence of viruses in nasal discharge and examined the time course, with regard to changes in olfactory dysfunction and nasal obstruction in 24 patients with postviral olfactory dysfunction, using questionnaires, acoustic rhinometry, and olfactory tests. nasal discharge was collected from patients with postviral olfactory dysfunction. rhinoviruses were detected in 10 patients. viral serotypes were identified by nucleotide sequences to be human rhinovirus-40, human rhinovirus-75, human rhinovirus-78, and human rhinovirus-80. one of the four patients complained of dysosmia, whereas another complained of anosmia. results of acoustic rhinometry significantly improved in the four patients, although olfactory testing did not show significant improvement. two of the four patients complained of olfactory dysfunction even 6 months after the first visit. coronavirus and parainfluenza virus were detected in one patient each, and epstein-barr viruses were detected in three patients. the present study detected rhinovirus, coronavirus, parainfluenza virus, and epstein-barr virus in nasal discharge of patients with postviral olfactory dysfunction. furthermore, this study suggests that rhinoviruses cause olfactory dysfunction through mechanisms other than nasal obstruction. in this study, we evaluated 10 patients with olfactory dysfunction induced by central nervous damage. six of 10 patients developed hyposmia after head trauma, 2 patients by brain tumor, one patient after subarachnoid bleeding and one patient by encephalitis. five of 10 patients did not sense any smell by venous injection of prosultiamine. psychophysical evaluation of olfactory function was performed by t&t olfactometry, and the average threshold score of recognition was 5.2 (severe hyposmia). eight patients got mri brain scans and each of them had one or more intracranial lesions. frontal lobe abnormalities were seen in 7 of 8 patients. intracranial lesions were also observed in 3 temporal lobes and one parietal lobe and one occipital lobe. these mri findings suggested most of the olfactory disorders were come from damage of orbitofrontal cortex in frontal lobe, which is thought to be the olfactory center. on the other hand, 8 out of 10 patients developed taste disorder, despite no pathogenic lesion was found in medullary, thalamus and insula, which are thought to be the central pathway of taste and taste cortex. currently, we have yet to establish effective treatment of olfactory loss caused by central pathway damage. in clinical practice, some of these patients show improvements in olfactory function with several years, thus long-term follow-up of patients is needed to develop new clinical studies. the clinical study of patients with drug-induced olfactory disturbances cases (1.4 %) of drug-induced olfactory disturbance were investigated. anti-cancer drugs occupied 81 % in the frequency of causative drugs. tegafur was most common in the causes of olfactory disturbance by anti-cancer drugs. we showed the characteristics of drug-induced olfactory disturbance by tegafur. a total of 21 patients were identified. the averaged recognition threshold was 5.29 and the averaged detection threshold was 4.67 in t&t olfactometry. after treatment, re-examinations were performed in 15 patients. the averaged recognition key: cord-022082-1dq623oe authors: greaves, peter title: respiratory tract date: 2007-09-28 journal: histopathology of preclinical toxicity studies doi: 10.1016/b978-044452771-4/50007-9 sha: doc_id: 22082 cord_uid: 1dq623oe the chapter describes different aspects of the respiratory tract. in preclinical safety studies, pathologies of the respiratory system can be a result of an intercurrent disease or can be induced by systemically administered drugs. intranasal or inhalation modes of therapy pose particular challenges in terms of the formulations and technologies required to administer a drug. a complex technology is developed to support the assessment of adverse effects of inhaled substances in rodent and nonrodent species, and the extrapolation of experimental findings to humans. the nasal chambers are the structures that are first to be subjected to the effects of inhaled substances, whether microorganisms or chemical substances. in rodents, the relatively small size of the nose and nasal sinuses facilitates a histological examination. findings show that infectious agents cause inflammation in the nose and nasal sinuses, and this may be associated with inflammation in the conjunctiva, the middle ear, and the oral cavity. it has been observed that a particular response of the rodent nasal mucosa to some irritant substances, including pharmaceutical agents, is the formation of rounded eosinophilic inclusions in the cytoplasm of sustentacular cells of the olfactory epithelium, and to a lesser extent in respiratory and glandular epithelial cells. by far and away the most important pulmonary diseases in humans are related to the smoking of tobacco. however, occupational lung diseases caused by inhalation of industrial chemicals, particulate matter and antigens, are also important causes of morbidity and mortality. for this reason, considerable effort has been directed to the examination of airborne pollutants over recent years, including study of their effects in laboratory animals when administered by the inhalation route. extensive study has shown that a complex array of defensive mechanisms protects the lung against the adverse effects of airborne substances and pathogenic organisms. aerodynamic factors prevent access of particles larger than 10|im diameter for these are deposited on the walls of the nasal passages. particles measuring between 2 and 10 (im diameter tend to be trapped by the mucus-covered ciliated epithelium lining the bronchial tree and removed by mucociliary transport aided by the cough reflex. smaller particles may reach the alveoli where they are ingested and transported by pulmonary macrophages.^ considerations of airborne delivery to the lungs are also important in the development of therapies to be administered via the respiratory tract. whilst the inhalation route has been used for many years for volatile anaesthetic gases, the respiratory tract is increasingly being employed for delivery of therapy in not only for asthma and other lung diseases but also as a means of systemic delivery of polypeptides such as insulin. in contrast to the adverse pulmonary effects of cigarette smoke and industrial pollutants, therapeutic agents remain a relatively minor cause of pulmonary toxicity in humans although actual incidence is difficult to ascertain. however, drug-induced pulmonary disease appears to be an increasingly frequent clinical problem and the drugs associated with parenchymal pulmonary injury in humans continue to increase.^ although it is difficult to assess in the context of the underlying disease process, it has been suggested that about 10% of patients receiving wellestablished anticancer drugs develop various forms of pulmonary toxicity some novel antineoplastic therapies may have a similar liability.^ drug-induced toxicity usually occurs after exposure of lung tissue via the circulation to parent drug or metabolites, although increasingly the adverse effects of direct administration of drugs to the lungs needs consideration in preclinical studies. in patients a number of drugs have been associated with pulmonary toxicity which can occur through different mechanisms and take different forms.^"^ through their specific pharmacological action drugs can produce excessive effects on bronchial calibre or pulmonary function. drugs mediate allergic reactions in the bronchi or lungs. they may also produce a variety of obscure, diffuse pulmonary alveolar conditions including a pulmonary syndrome resembling systemic lupus er3^hematosis. as the respiratory tract is a major route by which microorganisms gain entry into the body, opportunistic pulmonary infections with bacteria, viruses, fungi or protozoa are consequences of immunosuppression or broad-spectrum antibacterial therapy. as in other organs, drugs that disturb coagulation may precipitate pulmonary thromboembolism or haemorrhage. localized lung lesions also result from accidental, diagnostic or therapeutic inhalation of xenobiotics. mucociliary clearance is also sensitive to therapeutic agents that affect the secretion of mucus and fluid, ciliary activity and transport.^ treatment with antacids or histamine h2 blockers can also increase the risk of pneumonia developing in patients in intensive care units through increasing gastric ph, which leads to an overgrowth of gram-negative bacteria in the stomach and retrograde pharyngeal colonization.^ in preclinical safety studies, pathology of the respiratory system can be the result of intercurrent disease or be induced by drugs administered systemically. intranasal or inhalation modes of therapy pose particular challenges in terms of the formulations and the technologies required to administer drug. the different anatomical and physiological characteristics of the airways also influence drug toxicity, disposition and metabolism. the development of drugs to be administered by inhalation or intranasal routes is particularly difficult because of the perceived risks of high local drug concentration in respiratory tissues and their use in potentially vulnerable patients with pulmonary disease.^ a complex technology has been developed to support the assessment of the adverse effects of inhaled substances in rodent and non-rodent species and the extrapolation of the experimental findings to humans.^'^ in order to administer drugs by inhalation, it is necessary to generate aerosols (suspensions of particles in a gas) with a well-defined composition, particle size and shape. they must be delivered to the respiratory tract of laboratory animals in a way that parallels the likely human exposure. in case of therapeutic agents, this should avoid non-respiratory pathways through the skin and food. when aerosols are inhaled, various fractions of the particles are deposited at different locations in the respiratory tract. site of deposition depends primarily on particle size, but variability in the sites of deposition occurs among different laboratory animal species and humans by virtue of the differences in the size and shape of the respiratory passages as well as breathing patterns. ^^ in addition, there are different types of inhalers used in human therapy to consider in the assessment, which can deliver different materials to the lungs, for example nebulizers, propellant-driven metered dose inhalers and dry powder inhalers for asthma treatment. ^^ the subsequent fate of inhaled particles depends not only on their size but also their shape, chemical nature, and solubility in body fluids. soluble substances are absorbed into the blood stream and are removed by the pulmonary circulation. they may also undergo metabolism by enzymes present in the cell populations of the respiratory tract and reactive metabolites may cause local pulmonary damage. insoluble, inert particles are removed primarily by the mucociliary transport system of the trachea and bronchi or through phagocytosis by macrophages. overload of the lung by even relatively inert, nonfibrous particles such as titanium dioxide or carbon black may impair alveolar macrophage-mediated particle clearance. ^^ this may lead in turn to accumulation of dusts over time with eventual fibrotic and tumorigenic responses, particularly in rats.^^ measurements of respiration rate, tidal volume, airway resistance, pulmonary gas exchange and the disposition of the inhaled substances have an important place in the evaluation of chemically induced lung damage in laboratory animals.^"^'^^ however, the key component of the evaluation of the adverse effects of inhaled substances is careful morphological assessment of the fixed tissues. even though there are novel and very sensitive physiological methods for the characterization of oedema following lung injury in rodents, light and electron microscopy of lung tissue provides vital qualitative evidence of the nature of injury. ^^ the nasal chambers are the structures which are first to be subjected to the effects of inhaled substances, whether microorganisms or chemical substances. although these chambers are not usually examined in great detail in conventional toxicity studies in which substances are administered orally or by parenteral routes, they are carefully examined histologically when drugs are administered by inhalation. study of nasopharyngeal silicone rubber casts has shown considerable species differences in the anatomy of this part of the airway. ^^"^^ relative to total nasal length, the nasopharynx is longest in rats and shortest in humans with the dog in an intermediate position. maxilloturbinates are relatively simple structures in man and non-human primates but highly complex in dogs and rodents. as a consequence, regional nasal airflow and disposition patterns vary considerably and this influences the distribution of lesions produced by inhaled xenobiotics in the nasal cavity.^^ however, comparison of the nasal cavity of rhesus monkey and humans using magnetic resonance imaging and nasal casts have shown that many similarities in structure exist in these species. ^^ the anterior nares are lined by stratified squamous epithelium. in other zones the sinuses are covered either by respiratory or olfactory epithelium with a zone of transitional epithelium at the junction between the two types. respiratory epithelium is similar to that found elsewhere in the respiratory passages being composed of ciliated cells, serous and mucous cells, brush cells, intermediate cells and progenitor basal cells. it represents a cellular system engaged in mucociliary clearance carrying surface secretions to the nasopharynx to be cleared by swallowing. although this epithelium is similar to that lining the other large airways, key differences are the particularly rich complement of secretory cells and the complex vasculature of the nose which can modulate capillary, arterial and venous blood flow through the mucosa.^^ mucins may be particularly important. it has been postulated that they not only have a physical protective function but also possess antioxidant properties by virtue of the scavenging behaviour of their high proportion of sugar groups. ^^ the proportion of the nose lined by olfactory mucosa is variable between species, being disposed over a much larger area in dogs and rodents than in primates. ^^ however, it is structurally similar in humans and rodents. it is located in more dorsal or posterior regions of the nasal passages out of the direct line of airflow during normal respiration. olfactory mucosa is a pseudostratified columnar epithelium composed of basal cells, sustentacular cells and sensory cells with mucus-secreting bowman's glands situated in the lamina propria. basal cells are composed of two distinct types, light and dark cells. the light type represents the primitive, stem cell population. sustentacular or supporting cells are non-ciliated, columnar cells possessing microvilli that extend into the overlying layer of mucus. cell bodies of olfactory sensory neurons are situated in the middle layer of the epithelium between sustentacular and basal cells. their dendritic processes extend above the epithelial surface to end in a ciliated expansion referred to as the olfactory vesicle that is believed to be the receptor of odour perception. olfactory axons extend from the cell body, penetrate the basement membrane in bundles to become surrounded by schwann cells and eventually join with the olfactory bulb. the olfactory system is of importance in toxicology for it can be selectively damaged by xenobiotics, including therapeutic agents, presumably as a result of its high metabolizing capacity. the superficial location of neural cells in the olfactory epithelium also provides a model system for the study of the effects of xenobiotics on neural cells. submucosal mucous glands have been well characterized in the rat, hamster and dog, where they are divided into lateral nasal glands and maxillary recess glands. these are both situated in the posterior parts of the nasal cavity and composed of mucus-secreting cells.^^"^^ immunocytochemical study using antisera raised against the major isoenzymes of rat hepatic microsomal cytochromes p450 induced by (3-napthoflavone, 3-methylcholanthrene, phenobarbitone and pregnenolone-16-a-carbonitrile as well as nadph-cytochrome p450 reductase, epoxide hydrolase and glutathione s-transferases b, c and e, has shown their presence in rat nasal mucosal cells.^^ cyp2a enzymes appear to be expressed at high levels in the respiratory tract mucosa.^^"^^ this suggests that the nasal mucosa not only has a capacity for metabolizing and activating xenobiotics by oxidation, but also for hydration and inactivation of potentially toxic epoxides and conjugating electrophilic, reactive metabolites with reduced glutathione. it has been shown that the distribution of immune-reactive enzymes is different in olfactory and respiratory mucosa.^^ xenobiotics can be metabolized within both olfactory and respiratory mucosa but the olfactory regions appear to possess greatest capability for oxidative metabolism. consequently, regional differences in nasal toxicity and tumour formation from inhaled materials may not only be a response to different water solubility and deposition patterns but also differences in the formation of reactive metabolites.^'* another feature of this metabolizing activity is that it can be induced by systemically administered xenobiotics and this can alter the distribution of enzyme activity in the nasal mucosa.^^ studies of the mouse olfactory mucosa have shown that whilst typical hepatic inducers of cyp2a5 do not significantly change its expression in the mucosa, olfactory toxicants can alter the pattern of enzyme distribution.^^'^^ like many other tissues exposed to external environmental agents, the nasal mucosa possesses aggregates of lymphoid tissue in the underlying lamina propria. in rats these areas, characterized by follicles containing both t and b cell areas, are located in the ventral aspects of the lateral walls of the nasal airways at the opening of the nasopharyngeal duct.^^'^^ like the gutassociated lymphoid tissue, these nasal follicles have been shown in the rat to be covered by specialized epithelium with islands of cells with microvilli, socalled m or membranous cells. little is known of any toxicity occurring in this tissue despite its strategic position in the respiratory tract.^^ in rodents, the relatively small size of the nose and nasal sinuses facilitates histological examination. usually this area is sectioned transversely into several standardized blocks following decalcification.^^ there have been a number of detailed publications describing the histological preparation and assessment and recording of pathology of the rodent nasal cavity.^^"^^ careful standardized histological sections, careful recording of lesions with the use of diagrams of the rodent nasal cavity are useful in the assessment of lesions in the nasal cavity found in inhalation studies."*^ in larger species, particularly dogs and primates, sectioning and blocking is more complex. although dissection is required, a similar procedure following decalcification can be adopted. careful examination of haematoxylin and eosin stained sections remains paramount in the assessment of the nasal cavity, although special stains may be helpful. examination of cytokeratin expression in the respiratory mucosa has been used as a marker of epithelial differentiation in the respiratory tract>^ a test system that relates to the innervation of the nasal mucosa is that proposed by alarie.^^ the trigeminal nerve endings in the nasal mucosa of mice mediate the response to sensory irritants and this can be measured by a decrease in respiratory rate. it has been shown that a good correlation exists between the decrease in respiration rate in mice exposed to airborne chemicals and the nasal irritancy potential of the chemicals in humans.^^ this enables the detection of airborne sensory irritants and the prediction acceptable levels of exposure to the upper respiratory tract in people. microbial pathogens infectious agents cause inflammation in the nose and nasal sinuses and this may be associated with inflammation in the conjunctiva, middle ear and oral cavity. murine pathogens may cause alterations in the respiratory tract that can confound the assessment of changes induced by xenobiotics.^^ in rats, microbiological agents implicated in the development of rhinitis and sinusitis include corynebacterium kutscheri (pseudotuberculosis), streptococcus pneumonia, pasteurella pneumotropica, klebsiella pneumoniae, mycoplasma pulmonis and the sialodacryoadenitis virus or rat corona virus.^^ rats infected with the sialodacryoadenitis virus show inflammation and necrosis of the upper respiratory epithelium as well as damage to salivary and lachrymal glands. the sendai virus, a paramyxovirus, also has marked tropism for the respiratory tract, including the nasal cavity, and is associated with systemic effects that can compromise studies in laboratory rodents. occasionally, fungal infections of the airways with. aspergillus fumigatus are reported.^^ a variable that has been shown to influence the severity of the rhinitis produced by mycoplasma pulmonis is the strain of rat. following housing of lewis and fischer 344 strains together to eliminate microbial and environmental differences it was shown that the lewis strain developed a more severe rhinitis following inoculation with mycoplasma pulmonis than fischer 344 rats, although the reason for the difference was unclear."^^ rats exposed to ammonia, a common pollutant of the air in laboratory animal cages, have also been shown to develop lesions of the dorsal meatus, dorsal nasal septum and prominence of the turbinates.^^ these lesions are characterized histologically by swelling or mild degeneration of the epithelium. it appeared that ammonia exposure potentiated the acute inflammatory response of the nasal cavity to microbiological pathogens. a microorganism reported in the nasal cavity of rhesus monkeys employed in inhalation studies is the nematode of the genus anatrichosoma.^^ sections of this nematode are found in the squamous epithelium of the nasal vestibule and are associated with acanthosis and hyperkeratosis of the epithehum and a multifocal or diffuse granulomatous inflammation in the submucosa. administration of toxic or irritant substances to laboratory animals by the inhalation route produces degenerative, inflammatory and reactive changes in the nasal mucosa. the range of histological features is similar to those found in other mucosal surfaces damaged by other exogenous agents. whilst therapeutic agents administered by the inhalation route do not usually produce a severe degenerative or inflammatory responses in the nasal mucosa, at least at therapeutic doses, the simple categories proposed by hardisty and colleagues in recording of degenerative and reactive lesions following exposure to volatile chemicals are useful.'^^ categories suggested are: degeneration, regeneration, atrophy (postdegenerative), respiratory metaplasia and basal cell hyperplasia and inflammation. degeneration is usually the earliest morphological change characterized by loss of sensory and sustentacular cells resulting in a thinner mucosa. bowman's glands and nerve bundles, individual cell necrosis may be seen in more severe cases. regeneration is characterized by proliferation of basal cells associated with an epithelium that loses its regular structural features. post-degenerative atrophy usually follows severe damage and is characterized by loss of sensory and sustentacular cells. respiratory metaplasia is a process whereby the normal olfactory mucosa is replaced by pseudostratified epithelium of respiratory type often with cilia. basal cell hyperplasia represents a longer term effect where the proliferating basal cells form a distinct layer of cells below the respiratory epithelium. an example of the type and distribution of the degenerative and inflammatory conditions which can be induced by inhaled irritants is provided by the study in which swiss-webster mice were given various irritants by inhalation for periods of 6 hours per day for 5 days at concentrations that produced a 50% decrease in respiratory rate (alarie test). although the degree of histological changes varied with different agents, the changes were broadly similar in type and distribution.^^ most agents examined produced little or no alteration in the squamous mucosa lining the anterior part of the nose apart from some mild increase in thickness of the squamous layers. principal sites of damage were shown to be the anterior respiratory epithelium adjacent to the vestibule and the olfactory epithelium of the dorsal meatus. there was a distinct decrease in severity in posterior regions. histologically, the lesions in respiratory epithelium ranged from mild loss of cilia and small areas of epithelial exfoliation to frank erosion, ulceration and necrosis of the epithelium and underlying tissues including bone. variable polymorphonuclear cell infiltration was also found. in some cases, early squamous metaplasia developed on the free margins or the naso-maxillo-turbinates. changes to the olfactory epithelium varied from focal to extensive loss of sensory cells associated with damage to sustentacular cells. in severe cases, complete loss of olfactory epithelium occurred. although the degree of histological change was shown to vary with different agents, lesions induced by the more water-soluble chemicals tended to remain localized in the anterior part of the nasal cavity whereas agents with relatively low water solubility produced lung lesions in addition. it was suggested that these findings demonstrated the powerful 'scrubbing' action of the nasal cavity for water soluble, airborne xenobiotics.^^ inflammatory alterations have been induced in the nasal cavity of rodents treated with therapeutic agents at high doses by inhalation. significantly irritant substances do not make viable therapies. however, the precise relevance of such changes for human therapy by the inhalation route are sometimes questionable when the nasal damage is limited to high doses and it is not associated with alterations in other parts of the respiratory tract. in the case of tulobuterol, a 32-adrenergic receptor agonist, it was argued that the nasal inflammation induced in rats in a one month inhalation toxicity study was the result of a particularly high exposure of the nasal epithelium to drug, not representative of the likely human exposure to tulobuterol by inhalation, where little or no nasal exposure would occur.^^ rp73401 [3cyclopentyloxy)-ar-(3,5-dichloro-4-pyridy)-4-methoxybenzamide], a novel type iv phosphodiesterase inhibitor which was being developed for the treatment of asthma and rheumatoid arthritis, was also reported to produce degeneration of the olfactory epithelium in rats but neither dogs nor mice after single and repeated oral doses and by inhalation.^^ histologically, the olfactory epithelium showed necrosis of the superflcial epithelial layers including the sustentacular and sensory cells, with sparing of the basal cell layer. there was also damage to bowman's glands. the development of proliferative lesions and ultimately tumours of principally neuroectodermal origin followed chronic treatment. as rp73401 was highly metabolized and the nasal lesions could be inhibited by treatment of rats with metyrapone, a non-speciflc inhibitor of cytochromes p450, it was postulated that the changes were the result of p450mediated activation in the olfactory tissues, not linked to its pharmacological phosphodiesterase activity.^^ nasal epithelial degeneration and necrosis has also been reported in both rats and dogs treated with another candidate anti-inflammatory drug ci-959 by the intranasal route. this agent affected olfactory epithelium more than respiratory mucosa, suggesting that metabolism was important in the generation of this toxicity^^ although the nasal cavity has not been often examined histologically in great detail in toxicity studies conducted on drugs administered orally or by parenteral routes, damage to the nasal mucosa can be induced by drugs administered by these routes. one example is methimazole, a thioureylene antithyroid drug used in clinical practice where oral doses of 0.2-2mg/kg/day are employed and abnormalities of taste and smell have been described.^^ administration of methimazole at relatively high doses to long-evans rats by single oral (50mg/kg) or intraperitoneal (25mg/kg) routes was shown to produce damage to the sustentacular and sensory cells with sparing of the basal cells and basement membrane.^^ bowman's glands were also involved. methimazole is metabolized by the flavin-containing monooxygenase system and it is employed as a model substrate for this enzyme in vitro. the presence of flavin-containing monooxygenase isoforms in olfactory mucosa of long-evans rats suggested that reactive intermediates may be responsible for the nasal toxicity ^^ similar changes have been reported in mice where depletion of glutathione in the olfactory mucosa has been demonstrated also suggesting formation of local reactive metabolites.^^ histological examination has also shown that intravenous administration of a single dose of vincristine to mice damages the olfactory epithelium.^^'^^ vincristine is a vinca alkaloid derivative used in cancer therapy which has antimitotic activity and binds to tubulin. cell death was noted in olfactory cells 2-5 days after dosing with a peak of cell proliferation at 5 days and repair after about 10 days. these features resemble those that can be seen in other proliferating tissues after single doses of antimitotic drugs. the risk of damage to human olfactory cells from agents with these effects in rat nasal mucosa often remains uncertain because an understanding of relative exposure and metabolism in different species and a better understanding of the metabolic potential of human olfactory mucosa is required. a particular response of the rodent nasal mucosa to some irritant substances, including pharmaceutical agents, is the formation of rounded eosinophilic inclusions in the cytoplasm of sustentacular cells of the olfactory epithelium and to a lesser extent in respiratory and glandular epithelial cells.^^'^^ these inclusions are pas-negative and ultrastructural examination shows that they are membrane-bound, ellipsoid bodies containing homogenous electron dense matrix. their significance remains uncertain. a consensus classification for the variety of proliferative, non-neoplastic changes and atypical epithelial lesions and neoplasms found in the rat nasal cavity has been defined by schwartz and colleagues.^^ the classification of the international agency for research on cancer provides a similar perspective for rats and mice.^^'^^ proliferative lesions may be occasionally seen in untreated rodents in carcinogenicity studies but are much more commonly induced by administration of xenobiotics in inhalation carcinogenicity studies. spontaneous nasal tumours are uncommon but most often squamous in type in rats whereas in mice spontaneous squamous tumours are extremely rare and haemangiomas and respiratory adenomas predominate.^^'^^ the generally agreed categories are described below: mucous (goblet) cell hypertrophy and hyperplasia affects the nasal respiratory epithelium and are characterized by the presence of enlarged mucus-filled goblet cells, some of which form clusters suggestive on intraepithial glands. squamous cell hyperplasia is seen in the stratified squamous epithelium of the nares and is characterized by a focal increase in the number of cell layers. cells may show atypia with irregular enlarged, pleomorphic nuclei and nucleoli. squamous metaplasia occurs to respiratory epithelium under conditions of chronic damage. it is characterized histologically by the presence of three or more layers of epithelial cells with eosinophilic cytoplasm and clear cell boundaries whereas advanced lesions show typical keratinization and formation of intercellular bridges. cellular atypia may also be seen and should be characterized when found. respiratory epithelial metaplasia (of the olfactory epithelium) represents atrophy and degeneration of the olfactory epithelium with loss of sensory cells and in advanced cases loss of sustentacular cells with replacement by ciliated and non-ciliated respiratory epithelium. it may be seen as a spontaneous focal lesion in aged rats. epithelial hyperplasia with cellular atypia (atypical hyperplasia, basal cell hyperplasia, dysplasia) is a term used to embrace proliferative lesions in the respiratory and olfactory mucosa in the nasal cavity in which there is varying degrees of altered differentiation and atypia. there is perturbation of the growth pattern of the epithelium such that the changes are not those found in the normal regenerative response to transient mucosal damage. adenomas (polypoid or villous adenoma, adenomatous or villous polyp) usually develop in the anterior part of the nasal cavity and are usually exophytic lesions that develop from respiratory epithelium or nasal glands. adenomas of respiratory epithelium may be papillary in form but are, by definition, well circumscribed with minimal cellular pleomorphism and atypia. they may very occasionally occur spontaneously in aged rats.^^ adenomas of nasal glands usually show an acinar pattern. squamous cell papillomas develop in the squamous epithelium of the nares or in areas of squamous metaplasia in respiratory or olfactory epithelium. they are exophytic lesions with limited connective tissue stroma. they may develop spontaneously in aged rats.^^ carcinomas of either squamous or glandular differentiation develop in the nasal mucosa. histologically, they have similar characteristics to those in other epithelial tissues. they are rare spontaneous lesions in aged laboratory rodents but may be induced by xenobiotics administered by inhalation, orally or by the parenteral route. squamous carcinomas have been reported to develop in a small number of untreated fischer 344 rats used in carcinogenicity studies in association with point mutations in the c-h-ras and c-k-ras gene.^^ olfactory neuroblastoma (ethesioneuroblastoma, olfactory neuroepithelioma, olfactory neuroepithelial carcinoma) show olfactory differentiation and arise from olfactory epithelium. they do not seem to occur as spontaneous lesions in rats or mice and only rarely induced.^^'^^ cells are arranged in lobules or in solid sheets with scanty stroma. cells are relatively uniform with scanty cytoplasm with round or oval hyperchromatic nuclei. true rosettes with lumens or pseudorosettes are also seen. poorly differentiated tumours of this type may require ultrastructural study for diagnosis. olfactory neuroblastomas typically show the presence of electron-dense neurosecretory granules, neurofilaments or axons. as there is no detailed understanding of the biological behaviour of these neoplasms in laboratory rodents, the generic term olfactory neuroblastoma is usually preferred. they are almost always invasive tumours.^^ olfactory carcinomas forming glands, follicles and rosettes have been occasionally reported in aged syrian hamsters.^^'^^ mesenchymal neoplasms may be seen in the nasal cavity, particularly after exposure to potent carcinogens. their histological features are similar to those in the soft tissues and bone elsewhere in the body (see chapter 2). the mucosa lining the larynx and trachea becomes involved as part of an upper or lower respiratory tract infection. for instance, in rats, an acute laryngitis or tracheitis has been shown to accompany experimental infection with mycoplasma pulmonis and the sialodycroadenitis virus."^^'^^ a spontaneous degenerative condition of tracheal and laryngeal cartilage of uncertain pathogenesis associated with granulomas has been reported in fischer 344 rats.^^ the condition increases in severity and incidence with advancing age although it is seen in rats as young as 6 weeks of age. tracheal cartilage rings may also show alterations in genetically engineered animals, such as the c57bl/6j-tgn(c3-l-tag)cjeg (tag) mice that have generalized defects in cartilage development.^^ the larynx of rodents is also susceptible to the effect of inhaled substances, notably tobacco smoke but also pharmaceutical agents and propellants.^^'^^ in view of the localized nature of induced lesions in the larynx, standardized histological sectioning techniques have been proposed for rats, mice and hamsters using anatomical landmarks.^^"^^ the target site is located on the ventral floor of the larynx near the base of the epiglottis cranial to the ventral laryngeal diverticulum. lesions tend to occur in the ventrolateral region, which is covered by respiratory epithelium and the inner aspect of the arytenoid processes which is lined by squamous mucosa. the larynx responds to inhaled irritants by inflammatory, degenerative and regenerative changes in a manner similar to other regions of the respiratory tract. these include disruption of the epithelial cells, inflammatory cell exudates and infiltration, goblet cell hyperplasia and squamous metaplasia.^^ however, these changes are not specific to inhaled irritants but also occur as a response to natural respiratory tract pathogens in conventionally housed rats.'^^ the pseudostratified ciliated and non-ciliated mucosa of the trachea may also show pathological alterations in inhalations studies, although sites at the bifurcation (carina) are those often first affected. consequently, the carina should be systematically included in examination of the respiratory tract for induced lesions.^^'^^ as in the nasal passages a range of proliferative lesions including squamous hyperplasia, mucous cell hyperplasia, as well as papilloma, carcinoma and mesenchymal tumours are occasionally reported in the airways in laboratory rodents. in humans and laboratory animals, the trachea terminates at the bifurcation giving rise to two main bronchi which serve left and right lungs. depending on species, the main bronchi subdivide into further branches that enter the different lobes. various forms of branching are recognized. bronchi may arise as side-branches from a parent or stem bronchus (monopodial). the parent bronchus can divide into two equal daughter bronchus (dichotomous) or several daughter bronchi ipolychotomous)j^ study of silicone rubber casts of the respiratory tract has shown that the bronchial trees of humans and non-human primates are essentially dichotomous, in contrast to the monopodial pattern of rodents.^^ the comparatively long trachea of the dog gives rise to dichotomous upper airways but monopodial branching develops peripherally within each lobe. the size of the lungs is generally dependent on size and weight of the different species. auometric studies have shown that lung volume, alveolar surface area and diffusing capacity increase proportionally with body weight across a broad range of mammalian species, although cell size and surface area appear to be more determined by cell function rather than species size.^^ dogs have comparatively smaller body mass and higher airway dimensions compared to humans.^^ the number of lobes is species-dependent. the human lung possesses an upper and lower left lobe and an upper, middle and lower right lobe. this contrasts with the upper, middle and lower left lobes and a fourth, azygos right lobe in rhesus monkeys and baboons.^^ the dog has three lobes on both right and left sides. rats, mice and hamsters show cranial, middle, caudal and postcaval right lobes with a single, left lobe in mice and rats and a superior and inferior lobe on the left side in hamsters. cell types lining the bronchi are generally similar between species.^ the majority of cells are the ciliated cells that are accompanied by variable but relatively smaller proportions of basal cells, intermediate cells, mucous or goblet cells, serous cells, neuroendocrine and brush cells. in addition, mucous cells line the adjacent bronchial glands.^^ unlike the tracheal mucosa, which is pseudostratified, the mucosa of intra-pulmonary bronchi is non-stratified. ciliated cells are tall, columnar cells attached to basal and intermediate cells by desmosomal junctions. tight junctions exist between adjacent specialized cells at the apex. each cell possesses 200 or more cilia that are engaged in mucociliary clearance.^^ the superficial cell surface also shows a pronounced glycocalyx. the cytoplasm of ciliated cells contains scattered profiles of rough endoplasmic reticulum, a supranuclear golgi and numerous mitochondria particularly near the apex where a prominent cytoskeleton is also found. mucous or goblet cells are typical mucus-secreting cells representing about 10% of the bronchial mucosa cell population in man but less than 1% in pathogenfree rats.^ the serous cell is a cylindrical or pyramidal cell containing small, round, closely packed serous granules.^^ basal cells are compact, pyramidal cells resting on the basement membrane. they are believed to be progenitor stem cells with the intermediate cells representing an intermediate stage of cell differentiation. the mucus-secreting and ciliated cells form the cellular basis for the mucociliary clearance mechanism of the main conducting airways. the epithelium is covered by a mucous blanket that is fairly complete in humans and rabbits but patchier in rats.^ the mucous layer is segregated into an upper layer or gel phase separated from epithelial cells by a serous layer or sol phase. the complex carbohydrates of the glycocalyx and secreted mucosubstances show species-related differences in their sugar residues, which can be demonstrated histochemically by the use of labelled-lectins.^^ mucociliary clearance mechanisms are sensitive to the effects of many therapeutic agents, particularly those that alter mucins, fluid or electrolyte balance and ciliary activity. anaesthetic gases, barbiturates, narcotics and alcohol depress clearance function. by contrast, topical, oral or parenteral administration of (3-adrenergic agonists, isoprenaline and adrenalin, produce a dose-dependent stimulation of mucociliary transport by an effect on ciliary beat frequency, probably mediated by increasing levels of cyclic adenosine monophosphate in ciuated cells rather than through vascular changes. although basal mucociliary function is dependent on normal vagal tone, parasympathomimetic agents can affect mucociliary transport. acetylcholine and cholinergic agents stimulate ciliary activity whereas anticholinergic drugs, atropine and hyoscine, inhibit ciliary activity and mucociliary transport. these substances may alter deposition of inhaled particles in the lung.^ clara cells or non-ciliated bronchiolar cells located in the bronchiolar epithelium, first described by clara in 1937, are small and cylindrical in shape with highly infolded nuclei, surface microvilli, well developed golgi, abundant endoplasmic reticulum and characteristic oval, homogeneous electron-dense granules in the apical cytoplasm. in rats, rabbits and humans the granules are pas-positive, although they are usually considered pas-negative in hamster and mouse.^^ clara cells have high metabolic activity. they contain cytochrome p450-dependent enzymes and secrete a variety of proteins.^^"^^ clara cell secretory protein is the major component of their cytoplasmic granules and they have been shown to produce mucin following antigen challenge.^^ in most laboratory rodents, the conducting airways terminate abruptly at the non-cartilaginous terminal bronchiole that opens directly into an alveolar type airway, the alveolar duct which in turn communicates with the alveoli.^^ squamous epithelial or type i cells form only about 10% of all lung cells but they line over 90% of the alveolar surface, by virtue of extremely long cytoplasmic extensions. the principal gas exchange takes place across this cell. in the rat, the typical thickness of this barrier is 20 nm for a cytoplasmic extension of a type i pneumocyte, 90 nm for basal lamina and 90 nm for an endothelial cell.^^ the type i cell contains juxtanuclear mitochondria and the long smooth cytoplasmic extensions contain many ribosomes and pinocytotic vesicles. the anatomical configuration and function of type i cells render them highly vulnerable to inhaled gases and particles. the other main alveolar lining cell is the granular pneumocyte or type ii cell which constitutes about 10% of all lung cells, but which covers only about 5% of the alveolar surface.^^ this cell does not possess the long cytoplasmic processes typical of type i cells and it shows many microvilli on its luminal surface. the cell cytoplasm contains rough endoplasmic reticulum, golgi apparatus, some mitochondria and characteristic oval, osmiophilic lamellar inclusions. surfactant, a microaggregate of phospholipid and protein which modifies alveolar surface tension at low inflation volumes, is secreted by type ii alveolar cells. ultrastructural immunocytochemistry has shown the presence of surfactant apoproteins in the synthetic organelles and in the lamellar bodies of these cells, in agreement with the concept that the surfactant apoproteins are synthesized in the rough endoplasmic reticulum, glycosylated in the golgi and are stored in lamellar bodies.^^ type ii cells are more resistant to the damaging effects of xenobiotics and unlike type i cells they retain the ability to undergo mitotic division. following damage to type i cells, increased numbers of mitoses are evident in type ii cells which results in the appearance of large undifferentiated epithelial cells which ultimately differentiate into type i and type ii cells.^^ the lung also contains a dense neural network and a population of endocrinelike cells believed to be important in lung function.^^ these neurosecretory cells (kultschitsky or apud cells) are scattered sparsely in the epithelial surface of the larynx, trachea bronchi, bronchioles and alveoli. these cells are oval or cuboidal with oval nuclei, and argyrophilic cytoplasm which electron microscopic examination shows to contain dense core granules. the role of neuroendocrine cells in the lung is uncertain but immunocytochemical study has shown them to contain a number of neuroendocrine substances including neurone-specific enolase, synaptophysin, chromogranin and a variety of other peptides similar to vasoactive intestinal peptide, bombesin, calcitonin, serotonin, leu-encephalin, p endorphin and acth.^^'^^ cells lining the bronchi, bronchioles and alveolar walls are capable of metabolizing xenobiotics. immunocytochemical study has shown the presence of immune-reactive cytochromes p450, nadph cytochrome p450 reductase, epoxide hydroxylase and glutathione s-transferase in bronchial epithelial cells, ciliated bronchiolar cells, clara cells, type ii and possibly type i pneumocytes in the rat lung.^^ different cell populations contain different amounts of enzymes, clara cells containing the greatest concentrations of the phenobarbitoneinducible isoenzyme of cytochrome p450, nadph-cytochrome p450 reductase and epoxide hydrolase. studies of microsomal enzyme activities suggest that lung tissue contains fewer p450 isoenzymes than liver, principally forms cyplal cyp2b1, cyp3a2 and cyp4bl^^ whereas p450 enzyme activity is highly concentrated in specific cell types, overall microsomal enzyme activity is low compared with liver on the basis of microsomal protein weight.^^ other important cells are the pulmonary alveolar macrophages and lymphocytes. lymphocytes are found in the epithelium of the airways, in the interstitium of alveoli and as part of follicles in bronchial walls. pulmonary macrophages form part of the specific immune defence system of the lung, involving, as elsewhere in the body, antigen presentation. in the rat and mouse, distinctive populations of pulmonary macrophages have been described based on enzyme activities and reactivity to monoclonal antibodies against monocyte and macrophages surface determinates.^^'^^ bronchus associated macrophages in rat and mouse have more acid phosphatase and less non-specific esterase activity than the populations found in the pulmonary alveoli and interstitial tissues. an important aspect of the immune system is the bronchus-associated lymphoid tissue or balt, which forms part of the mucosal lymphoid system found in other epithelia. the morphology of balt is a useful guide to the nature and degree of immune stimulus in the lung. balt is organized in a way that is characteristic of other peripheral lymphoid organs. it is structurally similar in the laboratory rat, mouse, rabbit, guinea pig as well as in man but its size and prominence is species and strain-dependent as well as a function of the degree of antigenic stimulus^"^"^^. in the rat, the balt is composed of lymphoid aggregates or fouicles located mostly between a bronchus and artery with a zone of lymphocytes situated immediately under the bronchial epithelium. as in other peripheral lymphoid tissue, balt is organized into b and t cell zones but in no predetermined manner. immunocytochemical staining has shown that b and t lymphocyte zones differ in location from one aggregate to another. there are about two t lymphoc3^es for every three b cells compared with a ratio of 2:5 in rat peyer's particles.^^ the ratios may be different in other species. quantitative observations of t cell subsets using monoclonal antibodies have also shown that rat balt normally contains twice as many t-helper as t-suppressor/cytotoxic lymphocytes.^^ the t cells are confined to one or two discrete zones with a light scattering of t cells within the b cell zones and immediately under the bronchial epithelium. in common with lymph nodes, interdigitating cells are also found. the epithelium overl3dng balt shows anatomical modifications. it is composed of ciliated and non-ciliated cells covered by microvilli. in conventional, untreated laboratory rats, balt shows little activity and germinal centres are usually absent, although balt may be more prominent in some rat colonies in association with non-specific inflammatory lesions in lungs.^^'^^ in one colony of young wistar rats germinal centres were not seen in balt in untreated animals but they developed following the administration of a single intratracheal dose of lipopolysaccharide, a t cell-independent antigen.^^^ single intratracheal doses of t cell dependent antigens such as horseradish peroxidase, bovine serum albumin and bcg have been shown to produce only minor morphological changes which include expansion of the zone of lymphocytes immediately under the epithelium and infiltration of the bronchial epithelium overlying balt by lymphocytes.^^^ in addition, perivascular, peribronchial or alveolar infiltrates of small and large lymphocytes and macrophages were observed in the lungs of rats given bcg. immunoc3^ochemical study of the rat balt following intratracheal challenge with horseradish peroxidase showed that the majority of cells that infiltrated the bronchial epithelium were t helper (cd4 positive) lymphocytes. ^^^ furthermore, la antigen expression of the epithelial cells overlying the balt was shown to increase, associated with an increase in the number and size of microvilli, a more pronounced glycocalyx and a decrease in number and size of cilia. immunocytochemical study of the balt tissue in c57b1/6 mice using monoclonal antibodies to lymphoid and macrophage populations has demonstrated quite similar arrangements of cells to those in the rat with the majority of t cells belonging to the t helper (cd4 positive) class.^^ the pulmonary lymphatic system drains into mediastinal or cervical lymph nodes. although among rat strains differences in the location of lymph nodes and their drainage occur, tracer studies in the fischer 344 rat using colloidal carbon have shown that the lung lymphatics drain mainly into posterior mediastinal lymph nodes and those in the tracheal wall drains primarily to the internal jugular and posterior cervical nodes.^^^ although a variety of fixation, embedding and staining procedures are available for light and electron microscopic examination of lung tissue, there is no substitute for initial, careful visual inspection of the lungs at autopsy. uneven collapse of lungs on opening the thoracic cavity, discoloration or alteration in texture of the pleural or cut surface, congestion and presence of fluid in the larger airways may indicate structural damage. in rodent lungs, small pulmonary adenomas may be detectable by inspection in good light. fresh lung weight is also a helpful measure in lung assessment, although passive vascular engorgement can significantly affect this value. nevertheless, studies in the normal fischer 344 rat have shown that after exsanguination, wet lung weights show a close relationship to body weight and that dry weight of lungs consistently represents about 20% of the wet weights regardless of age or body weight. ^^^ an increase in wet weight over dry weight appears to be a good index of pulmonary oedema. ^^ various methods of fixation have been employed although simple immersion fixation in formalin for conventional light microscopy has the virtue of simplicity and it avoids the risk of translocation or removal of exudates from airways and alveoli. mixtures of formaldehyde, paraformaldehyde and glutaraldehyde are used in initial fixation for electron microscopy.^^ the best overall appreciation of lung architecture is achieved by instillation of fixative via the trachea under an appropriate constant pressure or by perfusion fixation of the pulmonary arteries that is less liable to dislodge intra-alveolar exudate. in a review of methods employed routinely in rodent toxicity studies, instillation of fixative via the trachea was the preferred method in most laboratories because its advantages were seen to outweigh its disadvantages.^^ the sampling procedure is an important aspect of histological examination of the bronchi and lungs, particularly those of large laboratory animals. the extent of histological sectioning in conventional toxicity studies should be modulated to take account of lesions found by macroscopic examination, the type of study and the nature of the test substance. the bronchi should be carefully sampled to allow assessment of any alterations in bronchial epithelium. morphometric analysis represents a sensitive tool of value in the evaluation of drug-induced lung changes, but it requires particularly rigorous sampling and evaluation procedures.^^^'^^^ a tiered, multiple stage or cascade sampling technique is normally considered the most appropriate for morphometric studies.^^'^ this involves dividing the lung into a series of homogeneous compartments or strata from which randomly selected samples can be examined by appropriate light or electron microscopic techniques. conventional special stains for reticulin and collagen as well as pas and alcian blue for mucins are helpful in the characterisation of lung damage and changes to the respiratory epithelium. immunocytochemistry and enzyme cytochemistry are also useful in the study of the heterogeneous cell population of the lung. xenobiotic metabolizing activity can be studied both by enzyme cytochemical methods as well as by immunocytochemical techniques using antisera specific for pulmonary monooxygenases and related enzymes.^^ important structural components, particularly collagen and laminin can be studied both at light and ultrastructural level with immunocytochemical methods.^^^ cytokeratin immunocytochemistry can be used as a method for the characterization of changes to epithelial cells.^^ clara cells can be localized by the presence of clara cell secretory protein and ciliated cells by the presence of tubulin.^^ endocrine cells are visualized by immunocytochemistry using antibodies to general neuroendocrine markers such as chromogranin and synaptophysin or regulatory peptides.^^ other useful antigens, which can be demonstrated in the lung, include surfactants, lysozyme, immunoglobulins and those of microorganisms that infect the lung.^^^ electron microscopy is particularly useful for the detailed characterization of injury to the cells of the alveolar epithelium and endothelium ( figure 6 .1). pulmonary oedema is a component of many inflammatory conditions of the lung, including those induced by infectious agents. however, the term oedema is reserved for a poorly cellular exudate characterized by the presence of pale, homogenous eosinophilic material in the alveoli, sometimes associated with a similar exudate in the lung septae and perivascular connective tissue. it occurs in a number of spontaneous conditions such as in congestive cardiac failure, metastatic pulmonary neoplasms or as an agonal change in association with pulmonary congestion and haemorrhage. drugs may induce cardiogenic pulmonary oedema as a consequence of pulmonary hypertension or impaired ventricular contractility. cardiogenic oedema is often associated with vascular congestion and red blood cells and haemoglobin may leak into airspaces. this can give rise to the presence of haemoglobin crystals within the oedema fluid in formalin-fixed tissue sections. most importantly, pulmonary oedema may be a manifestation of acute lung injury. inhalation or systemic administration of toxic chemicals may produce acute pulmonary oedema (see figure 6 .1). some substances such as phenylthiourea and a-naphlythiourea produce massive pulmonary oedema in laboratory animals when administered orally, principally as a result of damage to the endothelium of pulmonary capillaries and venules. ^^^ over 30 drugs have been reported to produce non-cardiogenic pulmonary oedema in humans, either directly or through poorly understood immunogenic mechanisms.^ another form of pulmonary oedema involves the main airways. allergic reactions in sensitized airways of asthmatic individuals is believed to result from cross-linking of ige and activation of mast cells that degranulate and release inflammatory mediators. ^^^ this has been reproduced in the main airways of rats sensitized to ovalbumin and then challenged with ovalbumin by the intratracheal route. ^^^ this treatment leads to rapid accumulation of bronchial exudate, degranulation of mast cells and the development of mucosal oedema, most marked immediately below the respiratory epithelium. congestion and haemorrhage is a frequent finding in the lungs of laboratory animals, where it is usually related to certain modes of death. it can be associated with administration of drugs and chemicals that have adverse effects on cardiac function or on the coagulation system. administration of heparin to rats produces a characteristic extravasation of blood into the air spaces.^^^ lower respiratory tract infection is generally not a major health hazard among laboratory animals but it is nevertheless an ever-present threat that can cause overt respiratory disease within a colony or develop following administration of xenobiotics. subclinical pulmonary infections and infestations can also produce histological alterations in the bronchial airways or pulmonary parenchyma which mimic changes induced by inhaled irritants or systemically administered drugs.^^'^^ furthermore, some respiratory pathogens alter immune defences and exacerbate the effects of inhaled substances. ^^^ a range of bacterial and viral pathogens may produce inflammatory lung changes.^^ typically, bacterial pathogens such as steptococcus pneumoniae produce acute bronchitis associated with a variable degree of acute inflammation of the lung parenchyma (bronchopneumonia) or a confluent lobar pneumonia. viral agents are generally associated with histological features of bronchiolitis and interstitial pneumonia, characterized by an increase in mononuclear cells in the respiratory bronchioles and alveolar septa. the histological features are variable for they depend on the particular pathogen, species and strain, immune status, presence or absence of secondary infection and the particular stage at which the infection is examined. respiratory infections are frequently mixed. changes due to secondary bacterial infection are frequently superimposed on those induced by viruses. sequential histopathological examination of the lungs of laboratory animals following inoculation with respiratory tract pathogens has been able to characterize the evolution of pathological changes produced by individual organisms. for instance, following inoculation with mycoplasma pulmonis, one of the more important respiratory pathogens among laboratory rodents both lewis and fischer 344 rats were shown to develop upper and lower respiratory tract inflammation. in the lewis strain this was characterized after 28 days by a variable acute inflammatory exudate in bronchi and bronchioles with focal bronchiectasis, inflammation and hyperplasia of the epithelium with a predominantly macrophage infiltration of the alveoli and alveolar walls.^^'^^^ these changes were associated with marked hyperplasia of the bronchusassociated lymphoid tissue (balt), which extended down the airways and blood vessels towards the periphery of the lungs. although the lymphoid hyperplasia was also found in inoculated fischer 344 rats, it was less marked and accompanied by little or no mucopurulent exudate or active inflammation of the bronchial walls. this disparity in response suggested that differences were related to the degree of lymphocyte activation in the two strains, an imbalance in regulation of lymphocyte proliferation in lewis rats, or both.^^^ other studies have been conducted in both rats and mice infected with another important respiratory pathogen of laboratory rodents, the sendai virus (parainfluenza type 1). sequential studies showed that the initial damage to bronchial and bronchiolar epithelium is associated with polymorphonuclear and lymphocytic inflammation (bronchiolitis). immunocytochemical and ultrastructural studies revealed the presence of viral antigen in the mucosa.^^^ hyperplastic and multinucleated syncytial epithelial cells develop in the hyperplastic terminal bronchiolar epithelium and the inflammatory process extended to involve peribronchial or peribronchiolar parenchyma with infiltration of alveolar walls by mononuclear cells, macrophages and neutrophils. a similar cell population accompanied by cell debris and oedema fluid develops in air spaces. pulmonary arteries show only minor involvement with inflammatory cells and focal reactive hyperplasia of the endothelium. immunocytochemistry and ultrastructural examination suggested that virus replication takes place in alveolar type i and type ii epithelial cells and macrophages but not in endothelial or interstitial cells of the alveolar septae.^^^ it was shown that when repair occurs there may be residual distortion of bronchiolar and alveolar walls by collagen and hyperplastic cuboidal epithelium may line the thickened alveolar septa. air spaces may also contain enlarged macrophages with pale vacuolated cytoplasm.^^^ strain differences in susceptibility have also been demonstrated to this virus. there is differential pulmonary interleukin 12 (il-12) gene expression between virus-susceptible brown norway rats and resistant fischer 344 rats and il-12 treatment provides protection from virus-induced chronic airway inflammation and remodelling. moreover increased tumour necrosis factor a (tnfa) expression has been shown to be an important regulatory factor in the development of sendai virus-induced bronchiolar flbrosis in infected rats.^^^ virus-inoculated brown norway rats had increased tnfa pulmonary mrna levels and increased numbers of bronchiolar macrophages and fibroblasts expressing tnfa protein compared with virus-inoculated f344 rats.^^^ the corona virus, which causes sialodacryoadenitis in many rat colonies, also produces lower respiratory tract inflammation. this is characterized by acute bronchitis and bronchiolitis with focal extension into lung parenchyma. thickened oedematous, hypercellular alveolar walls infiltrated by monocytic cells are found.^^ immunocytochemistry has shown the presence of viral antigen in bronchial and bronchiolar epithelial cells. there is also peribronchial lymphocytic infiltration and increased prominence of balt. ultimately complete resolution occurs. viruses remain a potential source of spontaneous respiratory disease in laboratory dogs. canine adenovirus type 2, parainfiuenza sv5, canine herpes virus, coronavirus and parvovirus have all been isolated from laboratory dogs developing respiratory disease.^^^ the syndrome of visceral larva migrans also incites focal inflammation, granulomas and fibrosis in the lungs of species such as dog and primate in which parasites are prevalent. the syndrome of visceral larva migrans is usually defined as that which results from the migration of nematode larvae into the viscera. it has been well described in the beagle dog lung where it results from the larvae of toxocara species or metastrongyloid nematodes.^^^'^^^ the precise identification of parasites is not always possible in tissue sections. histological appearances of infested lungs are highly variable. nematodes surrounded by granulomas and granulomatous inflammation, mostly in a subpleural location, may be visible in sections. in affected lungs there may be perivasculitis and active arteriolitis, bronchiolitis and peribronchiolitis. pleural involvement by the inflammatory process can be marked, particularly in regions overlying granulomas. scarring develops and pleural and sub-pleural fibrosis is frequently associated with epithelial hyperplasia and squamous metaplasia of the associated airways ( figure 6 .2).^^^ the lesions may sufficiently severe to resemble those induced by high doses of anticancer drugs such as bleomycin (see below). pulmonary acariasis is a common infestation of many species of non-human primates caused by various species of the mite pneumonyssus. reproduction of the mites appears to take place in the terminal bronchioles. pneumonyssus simicola is the recognized form found in rhesus monkeys.^^^ although it is most prevalent in wild caught primates, the disease is not easily eliminated during breeding in captivity. ^^^ even when eliminated by ivemectin the lesions of chronic bronchiolitis, bronchiectasis and pigmentation may persist as an incidental finding. ^^^ as the mite can produce significant destructive pulmonary pathology and render animals susceptible to secondary pulmonary bacterial infections, it can disrupt or confound the interpretation of toxicity studies performed in primates. the lesions are located most frequently in cranial lobes and are characterized by the presence of bullae distending the pleural surface, parenchymal cysts, nodules and scar tissue.^^^'^^^ histologically, there is a wide range of inflammatory activity. fully developed lesions are characterized by granulomatous bronchiolitis and peribronchiolitis with involvement of immediately adjacent alveoli. cystic lesions involving the bronchiolar walls develop around the parasites giving rise to the appearance of walled-off cysts composed of highly cellular granulation tissue, associated with neutrophils, lymphocytes, macrophages, multinucleated giant cells and various pigments (see below). in less active lesions, dilated, cystic airways with walls composed of thick bands of smooth muscle and lined by squamous or cuboidal epithelium are found. pneumocystis carinii is an important cause of pneumonia in patients with the acquired immunodeficiency syndrome (aids) as well as in other immunocompromised patients, including those treated with immunosuppressive drugs.^^"^ the natural habitat of pneumocystis carinii is pulmonary alveoli and it is widely encountered in the human population without being associated with overt disease. both clinical and experimental evidence suggests that impaired cellular immunity is much more important as a predisposing factor than impaired humoral immunity. ^^^ as in humans, laboratory animals may have latent pneumocystis infection that becomes clinically evident following immunosuppression. it has been shown in the rat that chronic administration of various regimens of adenocorticosteroids, low protein diets, cyclophosphamide and other immunosuppressive drugs with concomitant antibiotic administration to prevent other infections gives rise to typical pneumocystis pneumonia. ^^^ rodents with genetically deficient cellular immunity also develop pneumocystis pneumonia. the importance of pneumocystis pneumonia in toxicology is that it can be considered as a sentinel of chronic immune depression. in haematoxylin and eosin stained sections, pneumocystis pneumonia is characterized in both humans and rodents by the presence of alveoli filled with foamy eosinophilic material containing a few macrophages and indistinct nuclei of pneumocystis (figure 6 .2fe). ovoid or crescent-shaped structures of the organisms become clearly visible with gomori methenamine silver or toluidine blue stains. ultrastructural study of rats with pneumocystis pneumonia shows that trophozoites attach themselves most frequently to type i pneumocytes by altering their morphology to the contours of the pneumocytes rather than by a process of invasion. ^^^ systemically administered therapeutic agents may produce histological changes within the lung parenchyma that mimic components of the normal response to respiratory pathogens. however, there is no sharp separation between agents that produce pulmonary oedema with those that are associated with acute inflammatory changes and histological features overlap because an acute inflammatory process is often accompanied by exudate within airspaces. an example of drug-induced pulmonary inflammation in laboratory animals and humans is reported following the administration of interleukin 2 (il-2). il-2 is a glycoprotein lymphokine, molecular weight 15kda, which is normally produced by activated t cells and mediates immunoregulatory responses. it has been produced in large quantities by recombinant dna technology for use in tumour immunotherapy. however, high doses have been associated with a number of adverse effects, notably the 'vascular leak' syndrome, characterized clinically by pulmonary oedema, pleural effusions and ascites. ^^^ the vascular leak syndrome has been reported in laboratory animals given high doses of this agent. histological examination of the lungs of b6d2f mice developing this syndrome following administration of il-2 showed infiltration of the alveolar walls with large lymphocytes and intra-alveolar proteinaceous exudate containing large lymphocytes, macrophages and red blood cells.^^^'^^^ pulmonary venules and arterioles showed the presence of lymphocytes attached to or lying beneath the endothelium, infiltrating vessel walls or in a perivascular location where they were accompanied by oedema fluid or red blood cells. similar, but less severe changes have been demonstrated in rats given il-2.^^^ in addition, treated rats showed an infiltration of pulmonary vasculature with eosinophils probably secondary to an eosinopoietic cytokine produced by il-2 stimulated lymphocytes. immunocytochemical evaluation of the lymphoid infiltrate in mice showed that most of the cells were thy 1.2positive (cd90) lymphocytes. furthermore, co-administration of asialo gml (ganglio-n-tetrosyl-ceremide) with il-2 not only abrogated the clinical signs but also reduced the number of asialo gml-positive lymphocytes in the tissue sections. as lymphoid cells expressing lyt-2 (cds, suppressor/cytotoxic t cells) were unaffected by asialo gml treatment, it was postulated that the vascular leak syndrome (but not antitumour efficacy) in these mice was mediated by an endogeneous subset of il-2 stimulated lymphocytes or lymphokine-activated killer cells. ^^^ corresponding changes were also observed in liver and lymphoid tissue. immunocytochemical and detailed electron microscopic studies in rats have supported the concept that il-2 induces cytotoxic vascular damage that is mediated both directly by lymphokine-activated killer cells and cytotoxic t lymphocytes with secondary release of inflammatory cytokines.^^^ as in humans, severe chronic pulmonary inflammatory disease in laboratory animals may compromise pulmonary function and lead to secondary alterations in other organs. although the mechanisms were not explored in detail, a diffuse interstitial pulmonary inflammatory process with lung haemorrhage was induced in rats treated for two years with prizidilol (skandf 92657-a2), an antihypertensive agent with both vasodilator and (3 adrenoceptor blocking properties.^^^ affected animals developed dyspnoea associated with reduction in lung volume, deformity of the thoracic spinal column and marked cardiac hypertrophy. inflammation with granulomas develops in the lungs of laboratory animals under a variety of different circumstances, which have been alluded to above. a common cause in rodents is granulomatous pulmonary inflammation resulting from aspiration of stomach contents or food particles (aspiration pneumonia). this is sporadically observed in aged rodent where it is associated with general ill health, particularly resulting from pressure effects of large pituitary adenomas and subsequent disturbance of pharyngeal or laryngeal reflex mechanisms. ^^^ histologically, the lungs show peribronchial and peribronchiolar granulomatous inflammation with macrophages and foreign body cells associated with fragments of refractive vegetable matter. the associated bronchial mucosa may also show reactive changes including goblet cell hyperplasia in long-standing cases. as dogs and primates are more liable to be infested by parasites, granulomatous inflammation in response to pulmonary larvae is more common in these species. pulmonary tuberculosis represents a potential problem among non-human primate colonies in view of its insidious onset and its liability for transmission from monkeys to humans. ^^^ pathological findings are similar to those so well known in the human disease. the disease is characterized by the presence of granulomas in lung parenchyma and lymph nodes. in florid cases there may be caseation surrounded by epithelioid and multinucleated giant cells and variable numbers of lymphocytes, plasma cells and flbroblasts. diffuse granulomatous pneumonia as a result of tuberculosis is also reported in non-human primates. granulomatous pneumonitis is also produced in laboratory animals by the intravenous injection of bcg. twenty-eight days following intravenous injection of bacille calmette-guerin (bcg), the lungs of c57b1/6 mice contained numerous granulomas composed of histiocytes and round cells which were surrounded by alveoli with thickened walls and associated with mild interstitial pneumonitis. ^^^ these histological changes were associated with an increase in the number of thy 1.2-positive (cd90) cells, especially lyt-1 (cds) positive lymphocytes. the histological changes were abrogated by treatment with cyclosporin a suggesting an important role for cd5-positive lymphocytes in the development of the granulomas. discrete granulomas occur in the lungs of experimental animals in response to intra-tracheal or intravenous injection of certain relatively insoluble substances (figure 6.3) . intra-tracheal administration of insoluble polymerized dextran and latex micro-particles to mice showed that the morphology and the systemic effects of granulomas depends on the nature of the injected substances. it has been shown that large granulomas develop rapidly in the pulmonary parenchyma around dextran particles that subsequently regress quickly, whereas latex particles produce small, discrete stable granulomas. ^^^ although both forms of granulomas are of foreign body or non-immunological in type, those produced by dextran but not latex beads, are associated with anergy-like immunosuppression, probably caused by release of soluble factors from the granulomas. it has been reported that granuloma formation after instillation of sephadex beads is associated with increases in the interleukin 1-(il-1) like activity in the lung.^^^ studies comparing the effects of inhaled crystalline silica and titanium dioxide have shown a correlation between the release of the macrophage derived cytokine il-1 and granuloma formation, suggesting that il-1 might be a useful biomarker for granuloma formation. ^^^ localized, angiocentric granulomas of foreign body type, clustered around pulmonary arteries and arterioles and occasionally alveolar capillaries and venules also develop following intravenous injection of relatively insoluble polysaccharides or other polymers. ^^^ characteristic epithelioid and large, foreign body type giant cells efface the smaller vessels although overt necrosis is not usually observed (figure 6.3) . haemosiderin-laden macrophages accumulate in the alveou of laboratory animals in association with chronic pulmonary congestion and haemorrhage. similar changes occur in patients in congestive cardiac failure where the haemosiderin-laden macrophages are termed 'heart failure' cells. the lungs of non-human primates are especially liable to contain alveolar, perivascular and peribronchial aggregates of macrophages laden with various brown pigments. iron-containing pigments have been associated with the inflammatory changes produced by simian lung mites (pneumonyssus simicola) which are prevalent in many non-human primates. in addition, lungs from some primate colonies may show perivascular and peribronchial collections of brown-grey macrophages containing highly refractive spicules and plates composed of high concentrations of silica.^^^'^^^ it has been shown that in old world primates including rhesus and cynomolgus monkeys, this pigment contains fossil diatomaceous material, compatible with the concept that the animals inhale dusts containing diatoms and other silicon fragments to which they are exposed in their semi-arid, natural habitats.^^^ chronic lung injury from a variety of different causes is frequently associated with the development of pulmonary fibrosis characterized by the replacement of the normal pulmonary structure by a thickened collagenous matrix with consequent reduction in the capacity for gas exchange. regardless of the inciting agent, the fibrogenic process appears to be generally characterized by disruption of the normal alveolar-capillary structure, leakage of exudate from the vascular compartment into the airspaces, subsequent invasion by infiammatory cells and fibroblasts associated with excess matrix formation. studies in laboratory animals with different fibrogenic agents as well as in humans have suggested that central to pulmonary fibrogenesis is increased production of tnfa by macrophages.^^'^^^'^^^"^^^ this cytokine is a not only a mitogen for fibroblasts but also a potent activator and chemo-attractant for macrophages, capable of stimulating release of other cytokines and inducing expression of adhesion molecule expression on endothelial cells. moreover, it has been shown that tnfa receptor knockout mice appear protected from the fibroproliferative effects of inhaled asbestos. ^^^ pulmonary fibrosis is a common sequel of chronic lower respiratory tract inflammation. it may be associated with, or preceded by interstitial pneumonitis, characterized by infiltration of lymphocytes, plasma cells and macrophages with scattered polymorphonuclear cells. ^^^ focal pulmonary fibrosis occurs spontaneously in laboratory animals, although this is usually most prevalent in dogs and non-human primates as a response to chronic infestation by parasites, which are not easily eliminated during breeding. in humans, conditions leading to pulmonary fibrosis vary widely. they include infections, shock lung syndrome, ionizing radiation, inhalation of irritant particulate matter, exposure to antigens or excessive amounts of oxygen as well as the results of the toxicity of paraquat and a range of both cytotoxic and noncytotoxic therapeutic agents which cause pulmonary parenchymal injury.^'^^^ the principal therapeutic agents that produce pulmonary fibrosis in both humans and laboratory animals are anticancer drugs. bleomycin, a glycopeptide preparation derived from streptomyces verticillus, is the best known example but pulmonary fibrosis is also associated with the clinical use of a number of other anticancer agents, including l,3-bis-(2-chloroethyl)-l-nitrosourea (bcnu or carmustine), cyclophosphamide, busulphan, mitomycin c and methotrexate.2 '108,147-150 the precise mechanisms involved in the induction of pulmonary fibrosis by antineoplastic drugs in humans are poorly understood. the true incidence for a particular drug is difficult to estimate because of confounding factors in cancer patients, such as concomitant administration of several drugs, radiation and oxygen therapy, diffuse pulmonary cancer and opportunistic infections. it is probable that drug-induced fibrosis is accentuated by concomitant administration of several antineoplastic agents, radiation therapy, hyperoxia, preexisting pulmonary damage and age of the patient. severity is often related to total dose of drug received.^^^ novel antineoplastic drugs may also produce lung toxicity.^ bleomycin is associated with the development of interstitial pneumonia and pulmonary fibrosis in clinical use and this can be reproduced in experimental animals. the histopathological appearances of bleomycin-induced pulmonary fibrosis in patients are in many instances different from those seen in laboratory animals because the lungs of patients treated with bleomycin are modified by the primary neoplastic disease, smoking, multiple drugs, radiation therapy and secondary pulmonary infections, interstitial pneumonitis and fibrosis.^^^ it has been postulated that tnfa is an important mediator in the development of bleomycin-induced fibrosis. ^'^ in the preclinical evaluation of bleomycin beagle dogs were given cycles of drug by the intravenous route for periods of up to 26 weeks. ^^^ dogs developed anorexia, weight loss, a variety of epithelial lesions as well as focal interstitial pneumonia and fibrosis. the focal lung lesions were characterized by increased elastic fibres, reticulin, collagen and acid mucosubstances. the lesions were situated predominantly in the pleural and subpleural zones, suggestive of a potentiating effect of friction between the pleural surfaces. histologically the lesions resembled those produced by larvae migrans in the dog (see figure 6 .2a). similar histological changes have also been described in both rats and mice treated with bleomycin by both the intravenous and intratracheal route.^^^'^^^ as fibrosis is such a consistent change, bleomycin-treated rodents have been extensively employed as a model for pulmonary fibrosis. early changes include mild, diffuse increases in interstitial lymphocytes, macrophages, polymorphonuclear cells and perivascular or interstitial oedema. after about a week, interstitial infiltrates also comprise fibroblasts with early collagen deposition, associated with proliferation of macrophages and type ii pneumocytes.^^^'^^^ subsequently, the amount of interstitial collagen increases, with eventual scarring and collapse of lung tissue in proportion to the cumulative dose given. ^^^ immunohistochemical and ultrastructural study of rats and mice treated with bleomycin shows a large accumulation of immune-reactive laminin and reduplication of the basement lamina within the thickened alveolar walls. ^^^ in bleomycin-treated rats three-dimensional scanning electron microscopy shows drug-induced capillary remodelling comprising irregular alveolar and pleural capillaries with increased diameter and decreased branching. ^^^ certain strains of mice have been shown to possess greater sensitivity to bleomycin fibrogenesis. the c57bl/6 strain produces a greater fibroblastic response than dba/2 and swiss mice and the balb/c strain demonstrates a particularly poor fibroblastic response. ^^^ therapeutic use of cyclophosphamide is also occasionally associated with the development of pulmonary interstitial fibrosis.^^^'^^^ it appears to be associated with two forms of pathology: an early-onset pneumonitis and a late onset progressive pulmonary fibrosis. ^^^ similar changes have been less easy to reproduce in laboratory animals. when mice were sequentially examined for periods of up to one year after a single intravenous dose of loomg/kg of cyclophosphamide, only slight pulmonary interstitial thickening and hypercellularity was observed in association with progressive multifocal accumulation of intra-alveolar macrophages. ^^^ however, these changes were also accompanied by a progressive increase in pulmonary hydroxyproline content and a decrease in pulmonary compliance with time in treated animals compared with controls. the changes were amplified by exposure to 70% ambient oxygen. the bronchiolitis, alveolar septal infiammation and fibrosis induced by gold therapy in patients with rheumatoid arthritis is probably immune-mediated. this condition is associated with peripheral eosinophilia and drug-induced alterations to the immune system.^^^ emphysema is characterized by abnormal, permanent enlargement of airspaces distal to terminal bronchioles, accompanied by destruction of their walls without obvious fibrosis. three principle types, centriacinar, panacinar and distal acinar emphysema, are recognized in humans. enlargement of air spaces as a result of congenital factors or fibrous scarring are grouped separately and not regarded as emphysema. ^^^ emphysema has been reported as an age-related spontaneous change in laboratory rats.^^^ however, several experimental rodent emphysema models have been developed, using intratracheal instillation of proteolytic enzymes papain, pancreatic and neutrophil elastase. this gives rise to histological appearances resembling panacinar emphysema in humans.^^^ irritant gases, notably oxides of nitrogen, are also capable of inducing changes in the lungs of laboratory rats and hamsters following long term exposure which resemble mild human, centrilobular emphysema.^^^'^^^ a variety of different names have been applied to membrane-bound, acid phosphatase-positive cytoplasmic inclusions with a lamella or crystalloid ultrastructural matrix. these include myeloid bodies, myelinoid bodies, myelin figures or myelinosomes. these lysosomal inclusions are seen in small numbers in a variety of normal cell types but they accumulate in various organs in laboratory animals following administration of a wide variety of drugs of diverse therapeutic classes.^'^^^"^^^ the generalized accumulation of these lysosomal cytoplasmic bodies is generally called phospholipidosis, a term coined to describe the tissue accumulation of phospholipids. ^^^ at the light microscopic level, phospholipids are characterized by the increase in the number of foam cells in the airspaces. examples of drug-induced phospholipidosis include the anorectic drug chlorphentermine, tricyclic antidepressants, inhibitors of cholesterol biosynthesis such as triparanol, the antihistamine chlorcyclizine and its analogues, the selective oestrogen receptor antagonist tamoxifen, chloroquine and the cardiovascular drugs amiodarone, 4,4'diethylamino-ethoxyhexestrol and perhexiline.^^^'^^^'^^^ many tissues and organs may develop the cytoplasmic inclusions, including lymphoid cells, liver, pancreas, endocrine tissue, nervous system, muscle cells, eyes and particularly lungs. aminoglycoside antibiotics may produce laminated phospholipid inclusions in the renal tubular cell and imidazol antifungals in hepatocytes (see under liver and kidney, chapters 9 and 10). many drugs that induce phospholipidosis usually share structural features, notably a hydrophilic cationic side chain, a primary, secondary or tertiary amine and a hydrophobic region that is usually an aromatic ring or ring system. as this structural pattern renders these molecules amphiphilic, these drugs probably bind with polar lipids by means of electrostatic and hydrophobic forces.^^"^ this leads to formation of drug-lipid complexes which are poorly degraded by lysosomal enzymes and which accumulate in the cell cytoplasm to form the inclusions described above. as the binding is not covalent, its reversibility depends on the dissociation rate constant under the particular intracellular conditions and drug concentration achieved. predictions of this activity based on molecular structure have shown reasonably good correlation with the ability of compounds to produce phospholipidosis in cultured rat peritoneal macrophages. more recently other cell based systems have been proposed for screening for phospholipidosis.^^^'^^^ however these perform less well in the prediction of in vivo potency, presumably because of differences in drug disposition in blood and tissues. it should be underlined that the accumulation of foamy macrophages in the alveolar spaces may also be a spontaneous change in laboratory animals. it has long been recognized as a spontaneous alteration in ageing rats.^^^ it may also be found in lung tissue distal to bronchial lesions that impede clearance mechanisms. in contrast to drug-induced changes, the spontaneous lipidosis characterized by accumulation of alveolar foam cells occurs sporadically in older rats and is observed in both controls and treated animals. drug-induced phospholipidosis occurs within a period of several months during which lungs of control animals remain fairly free of foam cell accumulation. the lungs appear especially vulnerable to drug-induced phospholipidosis, possibly because macrophages are in very close proximity to blood-borne agents. phospholipidosis is also more clearly visible microscopically in alveoli whereas it can be easily overlooked in other organs. the continuous uptake of phospholipid-rich surfactant material from the alveoli by macrophages leads to excessive accumulation of phospholipids when their catabolism is impaired.^^^'^^^ the fact that lungs are commonly affected is a potentially useful diagnostic feature because in many organs phospholipidosis can be extremely difficult to recognize in haematoxylin and eosin stained sections. although the changes in the lungs are not specific for drug-induced phospholipidosis, an increase in the number of lipid-containing lung macrophages in treated animals compared with controls is relatively easy to detect and provides a simple way for the pathologist to screen for this effect. in severe generalized phospholipidosis in rats, the lungs show irregular pale grey or yellowish patches of discoloration of the pleura and parenchyma. this is a result of patchy or confluent aggregates of large, pale, foamy macrophages. they may be free lying or packed in alveoli and accompanied by granular, extracellular material. their abundant cytoplasm shows a vacuolated appearance in which fine eosinophilic granules are sometimes visible. the nuclei are rounded and centrally located structures of variable size (figure 6.4) . multi-laminated cells are also occasionally seen, as are vacuolated cells firmly attached to alveolar walls, probably pneumocytes. these foamy cells stain typically for phospholipids (e.g. acid haematin), although neutral lipids may also be present and stain with oil red o. semi-thin plastic-embedded sections stained with toluidine blue allow better characterization of phospholipidosis in all organs, including the lungs. the macrophages in the air spaces contain unmistakable dense, dark round cytoplasmic inclusions of variable size, some over 5 mm diameter. ^^^ plasticembedded sections also show the inclusions in other pulmonary cells including pneumocytes attached to the alveolar walls, from which they can be seen discharging into the alveolar spaces. as in other organs affected by phospholipidosis, ultrastructural examination reveals dense, multi-lamellar membranes and numerous heterogeneous dense bodies of lysosomal origin (figure 6.4) . these bodies need to be distinguished from membranous bodies that form as a result of fixation for ultrastructural study. lipids tend to leach out and become hydrated to form myelinoid membranes during glutaraldehyde fixation. these structures are subsequently fixed by osmium to give rise to electron-dense membranous figures both outside and inside cells, particularly in mitochondria where they may be mistaken for pathological lesions.^^^ the lamella patterns seen in phospholipidosis may be simple alternating dense and clear lines spaced at 4-5 nm, or more complex arrangements of clear and dense lines. the other typical crystalloid inclusions of hexagonal aggregates of tubular subunits seen in other organs are not usually found in the lungs. the significance of these various forms is uncertain but they probably represent the various phases in which phospholipids exist and are influenced by proportions of lipids present. electron microscopic examination reveals that not only are pulmonary macrophages affected by these changes but that inclusions may be present in pneumocytes types i and ii, pulmonary capillary endothelial cells, smooth muscle cells, bronchiolar epithelium and occasionally neutrophils.^^^"^^^ the changes are typically still visible several weeks after withdrawal from treatment with the offending agent. although the extent of pulmonary phospholipidosis in the lungs varies between dosage regimen and animal species, studies with chlorphentermine, 4,4'diethylaminoethoxyhexestrol and amiodarone indicate that similar cytological and ultrastructural changes occur in most laboratory animal species studied including rats, mice, hamsters, guinea pigs, rabbits and dog.^^^'^^^'^^^'^^^ safety assessment -amphiphilic drugs what are the imphcations for humans of drugs that induce phosphohpidosis in laboratory animals? novel compounds continue to be found that possess the property of producing phosphohpidosis in laboratory animals with varying degrees of severity.^^^'^^^ although not all drugs that produce phosphohpidosis in animals have been studied in humans, only very few drugs that produce phosphohpidosis in animals have been shown capable of inducing significant phosphohpidosis in human clinical practice.^ agents such as chloroquine, 4,4'diethylamindethoyhexestrol and amiodarone, which have been shown to produce phosphohpidosis in patients, can also induce cellular damage in the same organs. however the phenomenon of phosphohpidosis where phospholipids are packaged behind lysosomal membranes may not be causally related to cellular damage in humans. indeed the weight of evidence suggests that druginduced phosphohpidosis per se is an adaptive phenomenon and does not in itself have functional or deleterious consequences unless excessive. ^^^ hence, the finding of phosphohpidosis in animal studies with a novel drug requires careful assessment on a case by case basis with respect to its implications for the safety of humans. an example of this issue is the iodinated benzofuran derivative amiodarone, a potent antiarrhythmic drug effective against ventricular arrhythmia. lung toxicity continues to be a problem in patients treated for cardiac arrhythmias with this drug.^^"^ not only does phosphohpidosis occur in a wide variety of organs in laboratory animals treated with amiodarone,^^^'^^^ but also in liver, peripheral nerve cells, skin, lymphoid cells and lungs in patients at therapeutic doses.^^^'^^^'^^^ although pulmonary interstitial fibrosis occurs in association with phosphohpidosis in patients, amiodarone-induced phosphohpidosis in rodents is not associated with pulmonary fibrosis or significant functional alterations. several theories have been proposed for the pulmonary alveolitis and interstitial fibrosis in humans. the weight of evidence to date suggests that the accumulation of lipid-laden histiocytes is not causally related to the alveolitis or pulmonary fibrosis. ^^^ indeed, overall there is little evidence that the mere presence of phosphohpidosis is deleterious to the organism.^^^ cytotoxicity, possibly through the metabolite desethylamiodarone, has been proposed and an immune-mediated mechanism has been postulated, possibly favoured by the binding of drug to components of pulmonary tissue.^^^ it might also involve free radical formation or indirect influences on inflammatory mechanisms.^^^ it is also possible that pulmonary disease results from an interaction of several mechanisms and metabolic factors unique to particular patients. ^^^ despite undoubted differences in tissue and species sensitivity to development of phosphohpidosis, dose, drug disposition, metabolism and elimination and the degree of tissue exposure to drug are important considerations in safety assessment of drugs that produce phosphohpidosis in laboratory animals. although phosphohpidosis is more likely to occur at high doses employed in toxicity studies than at lower therapeutic doses used in patients, it has been suggested that this may be offset by faster ehmination of the drug, characteristic of small laboratory animals. ^^^ the potential for drugs to accumulate in critical tissues such as eye and heart is especially important when drugs are administered for long periods of time, particularly as tissue/plasma ratios of some amphiphilic drug may exceed 100, following repeated administration.^^^ consequently, although phospholipidosis may not have functional consequences, any implications for humans of drugs that induce phospholipidosis in laboratory animals can only be assessed on a case by case basis, with due consideration of mechanism, drug disposition and clinical risk-benefit analysis. it is important to underline that similar morphological changes due to the increased presence of phospholipids in lysosomes can also result from treatment with compounds that are not cationic amphiphilic structures. mechanisms include direct or indirect inhibition of lysosomal enzyme activity. this reenforces the need to understand the mechanism of any chemically induced increase of phospholipids in the lungs of laboratory animals. for example, it has been shown that when glycosaminoglycans accumulate in inherited human lysosomal disorders they inhibit other lysosomal enzymes, thereby inducing lysosomal phospholipid inclusions. ^^^ this is reflected by administration of high doses of the trypanocidal drug suramin to rats which induces intracellular storage of glycosaminoglycans associated with phospholipid inclusions in diverse organs including lungs. ^^^ although at light microscopy clear vacuoles are typically seen, electron microscopic examination shows the presence of both clear vacuoles containing glycosaminoglycans and lamellar phospholipid inclusions. a similar effect seems to have been produced in rats by elmironâ®, a semi-synthetic heparin-like macromolecular carbohydrate derivative, chemically and structurally similar to glycosaminoglycans used clinically for anticoagulant effects and interstitial nephritis.^^^ another example is the induction of lysosomal inclusions in the lungs of rat and dogs by the macrolide antibiotic erythromycin.^^^ collections of foam cells were described in the lungs and lymphatic tissues of dogs and rats treated with high oral doses. foam cells in the lung showed a pattern of small whorls in vacuoles similar to that seen with other drugs that induce phospholipidosis. in vitro studies have suggested all macrolide antibiotics have the potential to cause phospholipidosis. biochemical studies suggest that drug binds to phosphatidylinositol-containing liposomes and inhibits activity of lysosomal phospholipase in close correlation with the number of cationic groups carried by each of the drugs. ^^^ hook reviewed other agents, such as oxidant gases and insoluble particles including silica, that can also increase phospholipid levels and histological appearances of phospholipidosis in the lungs.^^^ some of these agents inhibit phospholipid catabolism in the lungs giving rise to accumulation of surfactant protein a and surfactant lipoproteins and a clinico-pathological picture similar to pulmonary alveolar proteinosis in humans. studies from humans have shown that three chnically distinct forms of this condition occur: congenital, secondary or acquired. the congenital disease is caused by a diverse range of mutations in the genes encoding surfactant proteins or the (3c chain of the receptor of granulocyte-monocyte colony stimulating factor (gm-csf). the secondary form occurs in association with conditions where there is functional impairment or reduced numbers of alveolar macrophages, such as in haematological cancers, following immune suppression or inhalation of silica or toxic fumes. acquired or idiopathic alveolar proteinosis that accounts for over 90% of all cases (0.37 per 100000 persons) has been an enigma until recently. patients are at risk from infections, particularly nocardia, and the 5 year survival rate appears to be about 75%. studies from transgenic mouse models and in humans have shown that autoantibodies against gm-csf are important in the development of the acquired form of the disease as this antibody causes a defect in macrophages function which impairs the catabolism of surfactant lipids and proteins. ^^^ in this context it is of interest to note that treatment with imatinib, a tyrosine kinase inhibitor of the bcr-abl tyrosine kinase constitutively expressed in philadelphia chromosome positive myeloid leukemia, has been associated with the accumulation of lamellar inclusions in pulmonary macrophages in a leukaemia patient, although this might have been a result of the primary disease. ^^^ studies in mice have suggested that imatinib mesylate actually inhibits the fibrogenic activity of transforming growth factor (3 and prevents fibrosis induced by bleomycin.^^^ various forms of hyperplasia are found in the airways and lungs of laboratory animals. the mucosal surface of the bronchi may show hyperplasia of the goblet cells, squamous hyperplasia or metaplasia. the cells lining the terminal bronchiole and alveolus may also show hyperplasia and squamous metaplasia. standard classifications for the characterization of these changes in histological sections have been developed for use in rodent studies.^^"^^ goblet cell hyperplasia is a well-recognized response of the mucosa of conducting airways to chronic inflammation and inhalation of irritant substances such as cigarette smoke and sulphur dioxide.^^'^^'^^^'^^^ the degree of goblet cell hyperplasia is dictated by the severity and duration of the irritation or inflammatory process. florid cases of goblet cell hyperplasia are characterized by thickening and pseudostratification of the tracheal or bronchial mucosa by a population of tall, mucus secreting cells with abundant pale cytoplasm. in addition, goblet cells extend further down the airways than in normal animals and mucus may fill or distend the airways or impact in the alveoli. in less florid cases, a simple increase in the number of goblet cells may be found without other structural change.^^ goblet cell hyperplasia of the lining epithelium may be accompanied by an increase in size of the underlying submucosal glands. this has clearly been demonstrated in patients with chronic bronchitis and in rats where submucosal glands are normally quite prominent.^^^'^^^ species differences may exist because the airways of laboratory animals are variably endowed with goblet cells and submucosal mucous glands. the normal rat has more goblet cells lining the airways than either mouse or hamster. ^^^ the factors controlling these alterations are uncertain but is has been long suggested that increased mitotic activity as well as cell conversion, probably by metaplasia of serous or clara cells to mucous cells, is involved.^^^ it has more recently been shown in mice sensitized to ovalbumin and subject to a single antigen challenge by aerosol that clara cells in the proximal airways show great plasticity and become mucin-secreting cells.^^ pharmacological agents can induce goblet or mucous cell hyperplasia. rats given six or 12 daily injections of isoprenaline, a non-selective (3 receptor agonist showed a dose-and time-dependent increase in the number and size of alcian blue-positive goblet (mucous) cells as well as serous cells in the tracheal and bronchial mucosa. this was associated with an increase in length, width and depth of submucosal glands.^^^ similar changes were produced by pilocarpine, although both alcian blue-and pas-positive cells were increased in number following this agent, suggesting that pilocarpine induced both acid and neutral glycoprotein secretion. comparison of the distribution of these changes in the rat following isoprenaline, with those of salbutamol, pilocarpine and tobacco smoke, showed that there were regional differences in the distribution of these changes in the airways.^^'* isoprenaline produced a greater increase in secretory cells in peripheral airways than tobacco smoke, which itself produces a greater increase in mitotic activity. isoprenaline and pilocarpine produced a more diffuse change than the more selective (3 agonist, salbutamol. the changes induced by these therapeutic agents are presumably the result of their pharmacological activity.^^'^ sturgess and reid showed that the changes in the rat were accompanied by hypertrophy of the pancreas, submaxillary and parotid salivary glands^^^ (see digestive system, chapter 8). unlike the rat and mouse, the hamster appears predisposed to develop minor multifocal epithelial hyperplasia of the tracheal and bronchial mucosa spontaneously with advancing age. these changes are flat or polypoid in nature and are composed of clear cells and goblet cells.^^'^^ the epithelium of the bronchi shows squamous metaplasia in response to chronic irritation or injury. it is characterized by three or more layers of epithelial cells with abundant eosinophilic cytoplasm with prominent cell boundaries. it may be associated with degenerative alterations to the mucosa or goblet cell hyperplasia. squamous metaplasia can also develop in the alveolar parenchyma as a response to prolonged damage such as produced by large burden of inhaled irritant or insoluble dusts. the metaplasia is also characterized by the presence of several layers of flattened epithelial cells showing squamous differentiation. the term pulmonary keratinizing cyst has been recommended for pulmonary cystic lesions lined by non-neoplastic squamous epithelium without excessive proliferative change.^^^ hyperplasia, bronchiolo-alveolar (type ii cell hyperplasia) hyperplasia may involve the lining epithelium of the alveoli or bronchioli. this form of hyperplasia has been termed alveolar hyperplasia, adenomatosis, alveolar bronchiolization or epithelialization. it occurs spontaneously but can be induced by infections and administration of irritant xenobiotics in rats/^'^^^"^^^ j^j^g64,209 ^^^ hamsters.^^^ histologically, the lesions consist of localized but unencapsulated foci of hyperchromatic regular, cuboidal or columnar cells investing airspaces without appreciable distortion of alveolar walls. neuroendocrine hyperplasia is well described in hamsters. although small aggregates of neuroendocrine cells (neuroepithelial bodies) are found at various levels of the bronchi and bronchioli in normal hamsters, administration of nitrosamines and 4-nitroquinoline 1-oxide produces neuroendocrine hyperplasia.^^^"^^^ hyperplastic lesions are recognizable as groups of non-ciliated cuboidal, oval or columnar cells located in the bronchial or bronchiolar epithelium. they contain argyrophilic granules that show immunoreactivity for corticotrophin (acth) and neurone-specific enolase. ultrastructural examination reveals the presence of dense-core cytoplasmic granules of apud type. proliferative changes have also been reported in other species, including rats and humans in hypoxic conditions, although it has been suggested that these changes might be a result of increased peptide content rather than cell proliferation.^^'^^^ the most frequently diagnosed neoplasm world wide is lung cancer, where it is usually caused by smoking tobacco.^^^ bronchogenic squamous carcinoma is generally the most common subtype but in north america the incidence of adenocarcinoma now exceeds that of squamous cell tumours for reasons not fully understood. some of the most aggressive subtypes are small and large cell neuroendocrine lung cancers, defined as small or large tumour cells with greater than ten mitoses per 2mm^.^^^ these seem to be seen almost exclusively in heavy cigarette smokers. in contrast to findings in people, squamous cell lung tumours are only occasionally seen arising spontaneously in laboratory animals. even laboratory animals -rats, mice, hamster, monkeys and dogs -exposed to tobacco smoke for long periods and at high doses fail to develop an increase in lung tumours.^^^'^^^ moreover, there appears to be no good experimental model for neuroendocrine lung cancer. thus, particular caution is merited if using animal models for prediction of lung tumorigenic potential of inhaled substances. by far the most common primary pulmonary neoplasms found in laboratory rats, mice and hamsters are adenomas and adenocarcinomas. these appear to develop from the bronchiolar or alveolar epithelium, although their precise histogenesis is somewhat disputed. although spontaneous squamous neoplasms are uncommon in rodents cystic keratinizing lesions can be induced in rats by high burdens of particulate material in the lungs.^^^ pleural mesotheliomas and mesenchymal neoplasms also occur in these species but are uncommon. they can be induced in rodents by mineral fibres.^^^ mesenchymal tumours have similar histological features to those in soft tissues and mesotheliomas may show either epithelial or mesenchymal differentiation or both. in most rat strains alveolar or bronchiolar neoplasms occur spontaneously in relatively small numbers, but morphologically identical neoplasia can be induced by administration of chemical carcinogens.^^^ the most common are classified as bronchiolo-alveolar adenoma (pulmonary adenoma) and bronchioloalveolar carcinoma. the national toxicology program database on control fischer 344 rats used in carcinogenicity studies indicates an overall percentage of less than 3% of animals with bronchiolo-alveolar adenomas and less than 1% with bronchiolo-alveolar carcinomas.^^ however, the range of bronchioloalveolar adenomas in different studies was between 0 and 14% in this series. histologically, bronchiolo-alveolar tumours are mostly small, discrete, rounded nodules located in the lung parenchyma and composed of fairly uniform cells with moderately hyperchromatic nuclei arranged in solid (alveolar), tubular, papillary or mixed growth patterns. they usually compress surrounding tissues without infiltration or metastatic spread (adenoma), although loss of differentiation, infiltration and spread to adjacent tissues can occur (adenocarcinoma). ultrastructural study of bronchiolar-alveolar neoplasia in fischer 344 rats has shown the presence of osmiophilic, lamellated inclusion bodies similar to those found in alveolar type ii cells. therefore it has been suggested that the neoplasms are derived from this cell type.^^^ pulmonary squamous carcinoma occurs but is a very uncommon spontaneous neoplasm in the rat.^^ the large proliferative but benign cystic lesions found in the lungs of rats following accumulation of large amounts of particulate matter have been termed pulmonary cystic keratinizing epitheliomas for they have been regarded as benign neoplasms. when these lesions show evidence of tissue invasion they are regarded as pulmonary squamous cell carcinomas. similar lesions are very occasionally reported as spontaneous lesions.^^^ analogous neoplasms are found more commonly in most strains of laboratory mice used in carcinogenicity bioassays although considerable variation in incidence is reported. they are common in strain a mice where they are observed in low frequency at 3-4 months of age and incidences reach nearly 100% by 24 months of age.^^^ fewer, but significant numbers are found in b6c3fi mice, although there is considerable inter-laboratory variation.^^^ the national toxicology program database on control b6c3fi mice used in carcinogenicity studies indicates an overall percentage of about 16% of males and 6% of females with bronchiolo-alveolar adenomas but only about 5% and 2.5% respectively with bronchiolo-alveolar carcinomas.^^ however, the range of bronchioloalveolar tumour varied considerably between studies in this series. even in the same laboratory, mice housed under similar conditions show variation in incidence in these neoplasms with time. the incidence of lung adenomas and adenocarcinomas occurring in cd-i mice used as controls in 18-month carcinogenicity bioassays in the same laboratory under similar conditions for a period of 3 years varied from between 19 and 36% in males and 6 to 16% in females.^^^ by contrast, some strains of mice such as the c5781/10j strain show a very low predisposition to the development of lung adenomas.^^^ although these mouse pulmonary adenomas and adenocarcinomas do not resemble the common lung tumours in humans, strain differences have been exploited to study genetic susceptibility and resistance to pulmonary adenomas and carcinomas.^^^'^^^ histologically, pulmonary tumours of this type in mice are generally small, sharply circumscribed nodules composed of fairly uniform, closely packed columns of cuboidal or columnar cells arranged in tubular or papillary structures with scanty fibrovascular stroma ( figure 6 .5). they may be less well differentiated, with cellular pleomorphism, and show intrabronchial growth, invade lung parenchyma and produce metastatic spread. the histogenesis of mouse pulmonary adenomas and adenocarcinomas is disputed. on the basis of sequential light and electron microscopic study of pulmonary adenomas induced in bagg-webster swiss mice by transplacental exposure to ethylnitrosourea, it has been suggested that they develop from either alveolar type ii cells or clara cells.^^^'^^^ careful, stepwise analysis using light microscopic and electron microscopic examination has suggested that adenomas can be divided into three principal groups. some are composed of solid growths of uniform cuboidal cells with expanding margins limited to alveolar septae (alveolar pattern). these cells contained concentrically arranged cytoplasmic lamellar bodies and abundant, large mitochondria similar to mitochondria found in alveolar type ii cells. tubular or papillary patterns are composed of cuboidal cells showing histological and ultrastructural features of clara cell differentiation.^^^ however, immunocytochemical studies of chemically induced and spontaneous pulmonary neoplasia in b6c3f1, balb/c or a strain mice have shown that the majority of adenocarcinomas, including those showing papillary patterns, contain surfactant apoprotein, typical of type ii antigens, suggesting that most neoplasms show alveolar type ii differentiation.^^^ however, in view of the plasticity of clara cells, this does not exclude a clara cell origin of the tumours. immunocytochemistry of specific clara cell secretory protein expression in a transgenic mouse model of lung carcinomas developing from clara cells has shown that the protein is lost during tumour cell progression.^^^ it has also been shown in strain a mice that the proportion of tumours with papillary and solid/alveolar growth patterns varies with the inducing agent.^^^ this also suggests biological differences exist between histological subtypes. very few squamous carcinomas are reported in most series of mouse studies. a chemically induced mouse model of squamous cell carcinoma has been generated by administration of n-nitroso-tris-chloroethylurea. strain differences in susceptibility to squamous cancer development have been demonstrated in this model, with nih swiss, a/j and swr/j being highly susceptible, akr/j and c57bl/6j being resistant and fvb/j and balb/cj mice showing an intermediate response to carcinogen.^^^ the high incidence and the inherent variabihty of pulmonary adenomas and adenocarcinomas in conventional mouse carcinogenicity bioassays sometimes gives rise to statistically significant differences between control and treatment groups. there is considerable risk in over-interpretation of such group differences in conventional mouse bioassays. in the analysis of group differences, consideration needs to be given to tissue sampling procedure, age-standardization, historical control incidence, effects on food intake as well as the results of mutagenicity studies and carcinogenicity bioassays in other rodent species. indeed, a considerable number of widely employed therapeutic agents of different classes have produced an increase in benign or malignant pulmonary tumours in carcinogenicity studies performed in mice without this proving of any significance to humans. davies and monro counted at least 17 drugs of this type in the 1994 physicians' desk reference of the united states.^^^ for instance, in a carcinogenicity bioassay in which cfl mice were treated for 80 weeks with the synthetic analgesic tilidine fumarate, a statistically significant difference (p < 0.01) was reported in the incidence of lung adenocarcinomas between the top dose female group (24%) and concurrent controls (10%).^^^ it was argued that group differences did not indicate tumorigenic potential of tilidine fumarate on the basis that the incidence in the high dose group was within the historical control range (27%) and that there was no tumorigenic effect in a parallel 104 week rat carcinogenicity study. a more difficult evaluation concerned metronidazole, a nitroimidazole which is an important therapeutic agent active against anaerobic organisms and trichomonas species. administration of this compound led to an increased incidence of pulmonary adenomas and carcinomas in three separate mouse carcinogenicity bioassays.^^^'^^^ the analysis of these findings was somewhat complicated by evidence that metronidazole shows mutagenic activity in bacterial assays using some strains of salmonella typhimurium. it was argued that the risk to human patients was slight because the increase in prevalence in pulmonary tumours was likely to be a result of changes in nutritional status of the mice through the effect of metronidazole on gut fiora, as similar differences could occur between ad libitum fed mice and those fed the same but restricted diet.^^^ it was also postulated that the positive findings in bacterial mutagenesis assays were an inherent part of the antibacterial activity of metronidazole as a result of nitroreduction that does not occur in normal mammalian tissues. this conclusion was supported by negative effects in hamster carcinogenicity bioassays as well as lack of excess cancer risk in women followed up for 10 years or more.^^^ the common occurrence of lung adenomas in strain a mice has been utilized in the development of a quantitative bioassay for carcinogenic activity. this followed the demonstration that administration of carcinogens such as 3-methylcholanthrene to this strain could significantly increase the incidence of pulmonary adenomas within periods of up to six months.^^^ over many years the strain a mouse pulmonary tumour assay has been used to test a large number of chemicals of different classes, including polycyclic hydrocarbons, nitrosamines, food additives, alkyl halides, metals and chemotherapeutic agents.^^^'^^^ however, as with many test systems, correlation of results in the strain a test with 2 year carcinogenicity study data and genotoxicity results have been shown to be poor so prudence is needed in the use of this test.^^^ hamsters develop lung adenomas spontaneously in small numbers with advancing age. they are composed of uniform cylindrical cells similar to those found in bronchial epithelium or goblet cells showing distinct mucus production.^^'^^'^^^ an immunohistochemical study of similar pulmonary neoplasms induced in hamsters by n-nitrosodiethylamine showed the presence of clara cell antigen in 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mouse lung carcinogenesis strain a/j mouse lung adenoma patterns vary when induced by different carcinogens a chemically induced model for squamous cell carcinoma of the lung in mice: histopathology and strain susceptibility marketed human pharmaceuticals reported to be tumorigenic in rodents evaluation of chronic toxicity and carcinogenesis in rodents with the synthetic analgesic, tilidine fumarate induction of lung tumors and malignant lymphomas in mice by metronidazole toxicologic evaluation of metronidazole with particular reference to carcinogenic, mutagenic, and teratogenic potential induced pulmonary tumors in mice. ii: reaction of lungs of strain a mice to carcinogenic hydrocarbons. arc/iii;es of pathology strain a mouse pulmonary tumor test results for chemicals previously tested in the national cancer institute carcinogenicity tests animal model: spontaneous carcinoma of the lung in hamsters key: cord-353190-7qcoxl81 authors: nicklas, werner; bleich, andré; mähler, michael title: viral infections of laboratory mice date: 2012-05-17 journal: the laboratory mouse doi: 10.1016/b978-0-12-382008-2.00019-2 sha: doc_id: 353190 cord_uid: 7qcoxl81 viral infections of laboratory mice have considerable impact on research results, and prevention of such infections is therefore of crucial importance. this chapter covers infections of mice with the following viruses: herpesviruses, mousepox virus, murine adenoviruses, polyomaviruses, parvoviruses, lactate dehydrogenase-elevating virus, lymphocytic choriomeningitis virus, mammalian orthoreovirus serotype 3, murine hepatitis virus, murine norovirus, murine pneumonia virus, murine rotavirus, sendai virus, and theiler’s murine encephalomyelitis virus. for each virus, there is a description of the agent, epizootiology, clinical symptoms, pathology, methods of diagnosis and control, and its impact on research. in interpreting the microbiological status of laboratory animals, it must be understood that infection and disease are not synonymous. infection refers to the invasion and multiplication of microorganisms in body tissues and may occur with or without apparent disease. disease refers to interruption or deviation from normal structure and function of any tissue, organ or system. many of the infections with which we are concerned may not cause discernable disease in many strains of mice. however, they may cause inapparent or subclinical changes that can interfere with research. such interference often remains undetected, and therefore modified results may be obtained and published. the types of interference of an agent with experimental results may be diverse. there is no doubt that research complications due to overt infectious disease are significant and that animals with clinical signs of disease should not be used for scientific experiments. but clinically inapparent infections may also have severe effects on animal experiments. there are numerous examples of influences of microorganisms on host physiology and hence of the interference of inapparent infections with the results of animal experiments. many microorganisms have the potential to induce activation or suppression of the immune system, or both at the same time but on different parts of the the laboratory mouse ó 2012 elsevier ltd. all rights reserved. isbn 978-0-12-382008-2 doi: 10.1016/b978-0-12-382008-2.00019-2 immune system, regardless of the level of pathogenicity. all infections, apparent or inapparent, are likely to increase interindividual variability and hence result in increased numbers of animals necessary to obtain reliable results. microorganisms, in particular viruses, present in an animal may contaminate biological materials such as sera, cells or tumours [1, 2] . this may interfere with in vitro experiments conducted with such materials and may also lead to contamination of animals [3] . mouse antibody production (map) testing or polymerase chain reaction (pcr) testing of biologics to be inoculated into mice is an important component of a disease prevention programme. finally, latent infections may be activated by environmental factors, by experimental procedures, or by the combination and interaction between various microorganisms. for all these reasons, prevention of infection, not merely prevention of clinical disease, is essential. unfortunately, research complications due to infectious agents are usually considered artefacts and published only exceptionally. information on influences of microorganisms on experiments is scattered in diverse scientific journals, and many articles are difficult to find. to address this problem, several meetings have been held on viral complications on research. the knowledge available is summarized in conference proceedings [4, 5] and has later repeatedly been reviewed [6] [7] [8] . viral infections of mice have been studied in detail, and comprehensive information on their pathogenic potential, their impact on research, and the influence of host factors such as age, genotype, and immune status on the response to infection is available. the nomenclature and taxonomy of viruses is described based on recent nomenclature rules by the international union of microbiological societies [9] and the universal virus database of the international committee on the taxonomy of viruses (http://www. ictvdb.org). retroviruses are not covered in this chapter because they are not included in routine health surveillance programmes and cannot be eradicated with the methods presently available. this is because most of them are incorporated in the mouse genome as proviruses and thus are transmitted via the germline. the ability to accurately determine whether or not laboratory animals or animal populations have been infected with a virus depends on the specificity and sensitivity of the detection methods used. most viral infections in immunocompetent mice are acute or short term, and lesions are often subtle or subclinical. the absence of clinical disease and pathological changes has therefore only limited diagnostic value. however, clinical signs, altered behaviour or lesions may be the first indicator of an infection and often provide clues for further investigations. serology is the primary means of testing mouse colonies for exposure to viruses, largely because serological tests are sensitive and specific, are relatively inexpensive and allow screening for a multitude of agents with one serum sample. they are also employed to monitor biological materials for viral contamination using the map test. serological tests detect specific antibodies, usually immunoglobulin g (igg), produced by the host against the virus and do not actually test for the presence of the virus. an animal may have been infected, mounted an effective antibody response and cleared the virus, but remains seropositive for weeks or months or for ever, even though it is no longer infected or shedding the agent. active infection can only be detected by using direct detection methods such as virus isolation, electron microscopy or pcr. meanwhile, pcr assays have been established for the detection of almost every agent of interest. they are highly sensitive and, depending on the demands, they can be designed to broadly detect all members of a genus or only one species. however, good timing and selection of the appropriate specimen is critical for establishing the diagnosis. in practice, combinations of diagnostic tests are often necessary, including the use of sentinel animals or immunosuppression to get clear aetiological results or to avoid consequences from false-positive results. reports on the prevalence of viral infections in laboratory mice throughout the world have been published frequently. in general, the microbiological quality of laboratory mice has constantly improved during the last decades, and several agents (e.g. herpesviruses and polyomaviruses) have been essentially eliminated from contemporary colonies due to advances in diagnostic methodologies and modern husbandry and rederivation practices [10] [11] [12] [13] [14] [15] . they may, however, reappear, since most have been retained or are still being used experimentally. furthermore, the general trend towards better microbiological quality is challenged by the increasing reliance of biomedical research on genetically modified and immunodeficient mice, whose responses to infection and disease can be unpredictable. increasing numbers of scientists are creating genetically modified mice, with minimal or no awareness of infectious disease issues. as a consequence, these animals are more frequently infected than 'standard' strains of mice coming from commercial breeders, and available information on their health status is often insufficient. frequently they are exchanged between laboratories, which amplifies the risk of introducing infections from a range of animal facilities. breeding cessation strategies that have been reported to eliminate viruses from immunocompetent mouse colonies may prove to be costly and ineffective in genetically modified colonies of uncertain or incompetent immune status. it must also be expected that new agents will be detected, although only occasionally. infections therefore remain a threat to biomedical research, and users of laboratory mice must be cognizant of infectious agents and the complications they can cause. two members of the family herpesviridae can cause natural infections in mice (mus musculus). mouse cytomegalovirus 1 (mcmv-1) or murid herpesvirus 1 (muhv-1) belongs to the subfamily betaherpesvirinae, genus muromegalovirus. murid herpesvirus 3 (muhv-3) or mouse thymic virus (mtv) has not yet been assigned to a genus within the family herpesviridae. both are enveloped, double-stranded dna viruses that are highly host-specific and relatively unstable to environmental conditions such as heat and acidic ph. both agents are antigenically distinct and do not cross-react in serological tests, but their epidemiology is similar [16] . mcmv-1 is very uncommon in european and american colonies of laboratory mice and is found at a very low rate [11] or reported as not found [14, 15] . seropositivity has, however, been reported from asian countries [17, 18] . testing for mtv is not frequently reported, and no sample tested positive in recent studies [11] . the data available suggest that the prevalence of both viruses in contemporary colonies and thus their importance for laboratory mice is negligible. however, both mcmv-1 and mtv are frequently found in wild mice, which may be coinfected with both viruses [8, [19] [20] [21] . mouse cytomegalovirus 1 (mcmv-1) or murid herpesvirus 1 (muhv-1) natural infection with mcmv-1 causes subclinical salivary gland infection in mice. like other cytomegaloviruses, mcmv-1 is strictly hostspecific. it persists in the salivary glands (particularly in the submaxillary glands) and also in other organs [22] [23] [24] . the virus can be cultured in mouse fibroblast lines like 3t3 cells, but primary mouse embryo fibroblasts are more sensitive to infection and produce higher virus titres. however, passage in cell culture results in its attenuation. to maintain virulence, the virus is best propagated by salivary gland passages of sublethal virus doses in weanling mice of a susceptible strain (e.g. balb/c) [25] . most information concerning the pathogenesis of mcmv-1 infection is based on experimental infection studies. these results are very difficult to summarize because the outcome of experimental infection in laboratory mice depends on various factors such as mouse strain and age, virus strain and passage history [26] , virus dose and route of inoculation [24] . in general, newborn mice are most susceptible to clinical disease and to lethal infection and develop higher levels of resistance with increasing age. infection of neonates leads to abnormal brain development [27, 28] . virus replication is observed in newborn mice in many tissues and appears in the salivary glands towards the end of the first week of infection when virus concentrations in liver and spleen have already declined. resistance develops rapidly after weaning between days 21 and 28 of age. experimental infection of adult mice results in mortality only in susceptible strains and only if high doses are administered. not even intravenous or intraperitoneal injections of adult mice usually produce signs of illness in resistant strains [29] . mice of the h-2 b (e.g. c57bl/6) and h-2 d (e.g. balb/c) haplotype are more sensitive to experimental infection than are mice of the h-2 k haplotype (e.g. c3h), which are approximately 10-fold more resistant to mortality than those of the b or d haplotype [24] . subclinical or latent infections can be activated by immunosuppression (e.g. with cyclophosphamide or cortisone) or critical illness such as sepsis [30] . reactivation of mcmv-1 also occurs after implantation of latently infected salivary glands into prkdc scid mice [31] . immunodeficient mice lacking functional t cells or natural killer (nk) cells, such as foxn1 nu and lyst bg mice are more susceptible than are immunocompetent animals. experimental infection in prkdc scid mice causes severe disease or is lethal, with necrosis in spleen, liver and other organs, and multinucleate syncythia with inclusion bodies in the liver [32] . similarly to aids patients infected with human cytomegalovirus, athymic foxn1 nu mice experimentally infected with mcmv-1 also develop adrenal necrosis [33] . the virus also replicates in the lungs, leading to pneumonitis, whereas replication and disease are not seen in heterozygous (foxn1 nu/þ ) littermates [34] . the pathogenesis of mcmv-1 infection in immunocompetent and in immunocompromised mice, as well as the role of the immune system, have been reviewed by krmpotic et al. [35] . the most prominent histological finding of cytomegaloviruses is enlarged cells (cytomegaly) of salivary gland epithelium with eosinophilic nuclear and cytoplasmic inclusion bodies. the inclusion bodies contain viral material and are found also in other organs such as liver, spleen, ovary and pancreas [24] . depending on inoculation route, dose, strain, and age of mice, experimental infections may result in inflammation or cytomegaly with inclusion bodies in a variety of tissues, pneumonitis, myocarditis, meningoencephalitis or splenic necrosis in susceptible strains [8, 24, 36] . virus is transmitted by the oronasal route, by direct contact and is excreted in saliva, tears and urine for several months. the virus is ubiquitous in wild house mice worldwide. they serve as a natural reservoir for infection and can even be infected with different virus strains [37] . the virus is most frequently transmitted horizontally through mouse-to-mouse contact but does not easily spread between cages. sexual transmission and transmission with tissues or organs is also possible. the virus does not cross the placenta in immunocompetent mice, although infection of pregnant females results in fetal death or resorption and wasting of borne pups. however, fetal infection is possible by direct injection of mcmv-1 into the placenta [38] and also occurs by transplacental transmission in mice with severe immunodeficiency [39] . vertical transmission is also possible by milk during lactation [40] . it is generally assumed that mcmv-1 has a very low prevalence in contemporary colonies of laboratory mice. the risk of introduction into facilities housing laboratory mice is very low if wild mice are strictly excluded. monitoring is necessary if populations of laboratory mice may have been contaminated by contact with wild mice. as for other viruses, different serological tests, including multiplex fluorescent immunoassay (mfia) [41] , are used for health surveillance of rodent colonies. as the virus persists, direct demonstration of mcmv-1 in infected mice is possible by pcr [42] [43] [44] or by virus isolation using mouse embryo fibroblasts (3t3 cells). although mcmv-1 does not play a significant role as a natural pathogen of laboratory mice, it is frequently used as a model for human cytomegalovirus infection [45] . these aspects have been discussed in detail by shellam et al. [24] . mcmv-1 has also been used as a vaccine vector aiming at a disseminating mouse control agent by inducing immunocontraception in mice [46] . the virus is known to influence immune reactions in infected mice [47, 48] and may therefore have an impact on immunological research [6, 8] . mtv was detected during studies in which samples from mice were passaged in newborn mice. unlike other herpesviruses, mtv is difficult to culture in vitro and is usually propagated by intraperitoneal infection of newborn mice. the thymus is removed 7-10 days later, and thymus suspensions serve as virus material for further studies. the prevalence of mtv is believed to be low in laboratory mice, and for this reason, and also due to the difficulties in virus production for serological assays, it is not included in many standard diagnostic or surveillance testing protocols. limited data are available indicating that it is common in wild mice [8, 49] . further, mtv obviously represents a significant source of contamination of mcmv-1 (and vice versa) if virus is prepared from salivary glands, since both viruses cause chronic or persistent salivary gland infections and can coinfect the same host. all mouse strains are susceptible to infection, but natural or experimental infection of adult mice is subclinical. gross lesions appear only in the thymus and only if experimental infection occurs at an age of less than about 5 days. infection results in nuclear inclusions in thymocytes and their almost complete destruction within 2 weeks. virus is present in the thymus but may also be found in the blood and in salivary glands of surviving animals. salivary glands are the only site yielding positive virus isolations if animals are infected as adults. the virus persists here and is shed via saliva for months. mtv also establishes a persistent infection in athymic foxn1 nu mice, but virus shedding is reduced compared to euthymic mice, with virus recovery possible only in a lower percentage of mice [50] . pathological changes caused by mtv occur in the thymus, and reduced thymus mass due to necrosis in suckling mice is the most characteristic gross lesion [36] . lymphoid necrosis may also occur in lymph nodes and spleen [51] , with necrosis and recovery similar to that in the thymus. in mice infected during the first 3 days after birth, necrosis of thymus becomes evident within 3-5 days, and the animals' size and weight are markedly reduced at day 12-14. intranuclear inclusions may be present in thymocytes between days 10-14 after infection. the thymus and the affected peripheral tissues regenerate within 8 weeks after infection. regardless of the age of mice at infection, a persistent infection is established in the salivary glands, and infected animals shed virus for life. several alterations of immune responses are associated with neonatal mtv infection. there is transient immunosuppression, attributable to lytic infection of t lymphocytes, but activity (e.g. response of spleen cells to t-cell mitogens) returns to normal as the histological repair progresses [51] . selective depletion of cd4þ t cells by mtv results in autoimmune disease [52, 53] . information about additional influences on the immune system is given in textbooks [6, 8] . in experimentally infected newborn mice, oral and intraperitoneal infections similarly result in thymus necrosis, seroconversion and virus shedding, suggesting that the oral-nasal route is likely to be involved in natural transmission [54] . the virus spreads to cagemates after long periods of contact. it is transmitted between mice kept in close contact, and transmissibility from cage to cage seems to be low. mtv is not transmitted to fetuses by the transplacental route, and intravenous infection of pregnant mice does not lead to congenital damage, impairment in size or development, or abortion [55] . mtv and mcmv-1 do not cross-react serologically [16] . serological monitoring of mouse populations for antibodies to mtv is possible by indirect immunofluorescent assay (ifa) testing, which is commercially available; enzyme-linked immunosorbent assays (elisa) tests have also been established [41, 56] . elisa and complement fixation yield similar results [57] . serological tests based on recombinant proteins and direct detection of virus by pcr are currently not possible because the genome of the virus has not yet been sequenced. it must be noted that the immune response depends on the age at infection. antibody responses are not detectable in mice infected as newborns, whereas adult mice develop high titres that are detectable by serological testing. if neonatal infection is suspected, homogenates of salivary glands or other materials can be inoculated into pathogen-free newborn mice followed by gross and histological examination of thymus, lymph nodes and spleens for lymphoid necrosis [49] . alternatives to the in vivo infectivity assay for detecting mtv in infected tissues include a competition elisa [58] and map testing, although this is slightly less sensitive than infectivity assays [59] . there is very little experience of eradication methods for mtv because of its low prevalence in contemporary mouse colonies. methods that eliminate other herpesviruses will likely eliminate mtv. procurement of animals of known negative mtv status is an appropriate strategy to prevent infection. strict separation of laboratory mice from wild rodents is essential to avoid introduction of the virus into laboratory animal facilities. murid herpesvirus 2 (muhv-2) or rat cytomegalovirus infects rats and is also a member of the genus muromegalovirus. murid herpesvirus 4 (muhv-4) is a member of the genus rhadinovirus in the subfamily gammaherpesvirinae and is also known as mouse herpesvirus strain 68 (mhv-68). other murid herpesviruses are not yet assigned to a subfamily within the family herpesviridae. among these is murid herpesvirus 3 (mouse thymic herpesvirus), but also murid herpesvirus 5 (field mouse herpesvirus) which infects voles (microtus pennsylvanicus), murid herpesvirus 6 (sand rat nuclear inclusion agent), and murid herpesvirus 7 [60] . furthermore, a gammaherpesvirus of house mice (mus musculus) has been described recently which is clearly distinct from mhv-68 [61] . experimental infection of laboratory mice with mhv-68 is a frequently used model system for the study of human gammaherpesvirus pathogenesis, e.g. of kaposi's sarcoma-associated herpesvirus or epstein-barr virus (ebv) [62, 63] which are members of the same subfamily. they are also important models to study viral latency and immune mechanisms controlling latency [64] [65] [66] . mus musculus is not the natural host for this virus; it was first isolated in slovakia from bank voles (myodes glareolus). additional closely related strains (mhv-60, mhv-72) exist from the same host species, and similar strains (mhv-76, mhv-78) were isolated from wood mice (apodemus flavicollis and apodemus sylvaticus). apodemus sp. seem to be the major hosts for mhv-68 in great britain [67] . different virus strains exhibit different genetic and biological properties and also differ in their pathogenicity, e.g. for prkdc scid mice [68] . infections in laboratory mice take the same course as in their natural hosts [69] . there are, however, some differences as, e.g. higher virus levels are reached in the lungs of balb/c mice, and wood mice develop higher titres of neutralizing antibodies [70] . house mice develop an acute infection in the lungs after intranasal infection. a latent infection develops within 2 weeks and the virus persists lifelong in epithelial cells in the lungs and also spreads to the spleen and other organs (e.g. bone marrow, peritoneal cells) where it persists in different cells of the immune system. it behaves like a natural pathogen in inbred strains of mice and persists without causing disease. the mousepox (ectromelia) virus (ectv) is a member of the genus orthopoxvirus belonging to the family poxviridae. it is antigenically and morphologically very similar to vaccinia virus and other orthopoxviruses. poxviruses are the largest and most complex of all viruses, with a diameter of 200 nm and a length of 250-300 nm. mousepox (ectromelia) virus contains one molecule of double-stranded dna with a total genome length of nearly 210 000 nucleotides [71] . it is the causative agent of mousepox, a generalized disease in mice. experimental transmission to young rats (up to 30 days of age) is possible [72] . unlike various other orthopoxviruses, ectromelia virus does not infect humans [73] . the virus is resistant to desiccation, dry heat and many disinfectants. it is not consistently inactivated in serum heated for 30 min at 56 c [3, 74] and remains active for months when maintained at 4 c in fetal bovine serum [75] . effective disinfectants include vapour-phase formaldehyde, sodium hypochlorite and iodophores [8, 76] . historically, ectv has been an extremely important natural pathogen of laboratory mice. the virus was widespread in mouse colonies worldwide and can still be found in several countries. between 1950 and 1980 almost 40 individual ectromelia outbreaks were reported in the usa. the last major epizootic in the usa occurred in 1979-80 and has been described in great detail [77] . severe outbreaks were also described in various european countries [78] [79] [80] . a more recent outbreak in the usa, which resulted in the eradication of almost 5000 mice in one institution, was described by dick et al. [81] . another recent and well-documented case of mousepox was published by lipman et al. [3] . few additional but unpublished cases of ectromelia have been observed since then; the latest report of an outbreak was published in 2009 [74] . in general, positive serological reactions are occasionally reported from routine health surveillance studies [17] but the virus is extremely rare in european and american colonies of laboratory mice [13] [14] [15] . natural infections manifest differently, depending on many factors. mousepox may occur as a rapidly spreading outbreak with acute disease and deaths, or may be inconspicuous with slow spreading and mild clinical signs and may therefore be very difficult to diagnose [81] . the mortality rate can be very low in populations in which the virus has been present for long periods. the infection usually takes one of three clinical courses: acute asymptomatic infection, acute lethal infection (systemic form) or subacute to chronic infection (cutaneous form) [8, [81] [82] [83] . the systemic or visceral form is characterized clinically by facial oedema, conjunctivitis, multisystemic necrosis and usually high mortality. this form is less contagious than the cutaneous form because the animals die before there is virus shedding. the cutaneous form is characterized by typical dermal lesions and variable mortality. the outcome of infection depends on many factors including strain and dose of virus; route of viral entry; strain, age, and sex of mouse; husbandry methods and duration of infection in the colony. while all mouse strains seem to be susceptible to infection with ectv, clinical signs and mortality are strain-dependent [84] [85] [86] . acute lethal (systemic) infection occurs in highly susceptible inbred strains such as dba/1, dba/2, balb/c, a and c3h/hej. immunodeficient mice may also be very susceptible [87] . outbreaks among susceptible mice can be explosive, with variable morbidity and high mortality (>80%). clinical disease may not be evident in resistant strains such as c57bl/6 and akr, and the virus can be endemic in a population for long periods before being recognized. furthermore, females seem to be more resistant to disease than males, at least in certain strains of mice [84, 85] . killer cells are necessary to control mousepox infections [88] . mice that are resistant to mousepox may lose their resistance with increasing age, most likely due to the decreased number and activity of nk cells [89] . the mechanisms determining resistance versus susceptibility are not fully understood but appear to reflect the action of multiple genes. the genetic loci considered to be important include h2d b (termed rmp3, resistance to mousepox), on chromosome 17 [90] ; the c5 genes (rmp2, on chromosome 2); rmp1, localized to a region on chromosome 6 encoding the nk cell receptor nkr-p1 alloantigens [91] ; the nitric oxide synthase 2 locus on chromosome 11 [92] ; and the signal transducer and activator of transcription 6 locus on chromosome 10 [93] . mousepox infections are controlled for several days during the initial course of infection by the complement system until the adaptive immune system can react. loss of the complement system results in lethal infection [94] . clearance of the virus by the immune system is dependent upon the effector functions of cd8þ t cells while nk cells, cd4þ t cells and macrophages are necessary for the generation of an optimal response [95, 96] . t-and b-cell interactions and antibodies play a central role during recovery from a secondary infection [97] . mousepox (ectromelia) virus usually enters the host through the skin with local replication and extension to regional lymph nodes [8, 82, 86] . it escapes into the blood (primary viraemia) and infects splenic and hepatic macrophages, resulting in necrosis of these organs and a massive secondary viraemia. this sequence takes approximately 1 week. many animals die at the end of this stage without premonitory signs of illness; others develop varying clinical signs including ruffled fur, hunched posture, swelling of the face or extremities, conjunctivitis and skin lesions (papules, erosions or encrustations mainly on ears, feet and tail; figure 3 .2.1). necrotic amputation of limbs and tails can sometimes be seen in mice that survive the acute phase, hence the common gross lesions of acute mousepox include enlarged lymph nodes, peyer's patches, spleen and liver; multifocal to semiconfluent white foci of necrosis in the spleen and liver; and haemorrhage into the small intestinal lumen [36, 81, 86, 87] . in animals that survive, necrosis and scarring of the spleen can produce a mosaic pattern of white and red-brown areas that is a striking gross finding. the most consistent histological lesions of acute mousepox are necroses of the spleen (figure 3.2. 3), lymph nodes, peyer's patches, thymus and liver [3, 36, 81, 86, 87] . occasionally, necrosis may also be observed in other organs such as ovaries, uterus, vagina, intestine and lungs. the primary skin lesion, which occurs about a week after exposure at the site of inoculation (frequently on the head), is a localized swelling that enlarges from inflammatory oedema. necrosis of dermal epithelium provokes a surface scab and heals as a deep, hairless scar. secondary skin lesions (rash) develop 2-3 days later as the result of viraemia ( natural transmission of ectv mainly occurs by direct contact and fomites [8, 82, 98, 99] . the primary route of infection is through skin abrasions. faecal-oral and aerosol routes may also be involved [98] . in addition, the common practice of cannibalism by mice may contribute to the oral route of infection [99] . intrauterine transmission is possible at least under experimental conditions [100] . virus particles are shed from infected mice (mainly via scabs and/or faeces) for about 3-4 weeks, even though the virus can persist for months in the spleen of an occasional mouse [8, 99] . cage-to-cage transmission of ectv and transmission between rooms or units is usually low and largely depends on husbandry practices (e.g. mixing mice from different cages). importantly, the virus may not be transmitted effectively to sentinel mice exposed to dirty bedding [3] . various tests have been applied for the diagnosis of ectromelia. previous epidemics were difficult to deal with because of limited published data and information on the biology of the virus and the lack of specific and sensitive assays [101] . in the 1950s, diagnosis relied on clinical signs, histopathology and animal passages of tissues from moribund and dead animals. culture of the virus on the chorioallantoic membrane of embryonated eggs was also used. serology is currently the primary means of routine health surveillance for testing mouse colonies for exposure to ectv. the methods of choice are mfia, elisa and ifa; they are more sensitive and specific than the previously used haemagglutination inhibition (hi) assay [41, 102, 103] . serological tests based on virus particles detect antibodies to orthopoxviruses and do not distinguish between ectv and vaccinia virus or other orthopoxviruses, respectively. vaccinia virus is commonly used as an antigen for serological testing to avoid the risk of infection for mice. thus, false-positive serological reactions may be found after experimental administration of replication-competent vaccinia virus. it has been shown that even cage contact sentinels may develop antibodies, and vaccinia virus leading to seroconversion may even be transmitted by dirty bedding [104] . confirmation of positive serological results is important before action is taken because vaccinia virus is increasingly prevalent in animal facilities as a research tool (e.g. for vaccination or gene therapy). as observed in different outbreaks, serological testing is of little value in the initial stages of the disease. for example, in the outbreak described by dick et al. [81] depopulation was nearly completed before serological confirmation was possible. for this reason, negative serological results should be confirmed by direct detection methods (pcr, immunohistochemistry, virus isolation) or by histopathology, especially when clinical signs suggestive of mousepox are observed. pcr assays to detect different genes of poxviruses in infected tissues have been used [3, 81, 105] . other pcr tests which were developed to detect smallpox virus have also been shown to detect ectromelia virus and can be used as well [106, 107] . the key to prevention and control of mousepox is early detection of infected mice and contaminated biological materials. all institutions that must introduce mice from other than commercial barrier facilities should have a health surveillance programme and test incoming mice. perhaps even more important than living animals are samples from mice (tumours, sera, tissues). the virus replicates in lymphoma and hybridoma cell lines [108] , and such cells or material derived from them may therefore be a vehicle for inadvertent transfer between laboratories. the last three published outbreaks of ectromelia were introduced into the facilities by mouse serum [3, 74, 81] . lipman et al. [3] found that the contaminated serum originated from a pooled lot of 43 l that had been imported from china, but in both other cases, serum was obtained from animals in the usa. because mouse serum is commonly sold to the end user in small aliquots (a few millilitres), it has to be expected that aliquots of the contaminated lot may still be stored in freezers. these published cases of ectromelia outbreaks provide excellent examples of why testing should be performed on all biological materials to be inoculated into mice. in the case of ectromelia virus it was shown that pcr is more sensitive, and map testing failed to detect contamination [74] . eradication of mousepox has usually been accomplished by elimination of the affected colonies, disinfection of rooms and equipment, and disposal of all infected tissues and sera. while culling of entire mouse colonies is the safest method for eradication of mousepox, it is not a satisfactory method because of the uniqueness of numerous lines of genetically modified animals housed in many facilities. several studies indicate that mousepox is not highly contagious [75, 84, 99] and that it may be self-limiting when adequate husbandry methods are applied. therefore, strict quarantine procedures along with cessation of breeding (to permit resolution of infection) and frequent monitoring, with removal of clinically sick and seropositive animals, are a potential alternative. the period from the last births until the first matings after cessation of breeding should be at least 6 weeks [99] . sequential testing of immunocompetent contact sentinels for seroconversion should be employed with this option. in the past, immunization with live vaccinia virus was used to suppress clinical expression of mousepox. vaccination may substantially reduce the mortality rate, but it does not prevent virus transmission or eradicate the agent from a population [109, 110] . after vaccination, typical pocks develop at the vaccination site, and infectious vaccinia virus is detectable in spleen, liver, lungs and thymus [111] . vaccination also causes seroconversion so that serological tests are not applicable for health surveillance in vaccinated populations. it is therefore more prudent to control mousepox by quarantine and serological surveillance than by relying on vaccination. mortality and clinical disease are the major factors by which ectv interferes with research. severe disruption of research can also occur when drastic measures are taken to control the infection. the loss of time, animals and financial resources can be substantial. experimental mousepox infections are frequently used as a model to study various aspects of smallpox infections of humans [112] [113] [114] . mousepox shares many aspects of virus biology and pathology, and models the course of human smallpox. experimental mousepox infections are used to study vaccination procedures [115, 116] or anti-poxvirus therapies [117] . murine adenoviruses (madv) are non-enveloped, double-stranded dna viruses of the family adenoviridae. two distinct strains have been isolated from mice. the fl strain (madv-1) was first isolated in the usa as a contaminant of a friend leukaemia [118] and has been classified as a member of the genus mastadenovirus. the k87 strain (madv-2) was isolated in japan from the faeces of a healthy mouse [119] and has not yet been assigned to a genus. both strains are considered to represent different species [120] [121] [122] . they are host species specific and are not infectious for infant rats [123] . madv-1 can be cultured in vitro in mouse fibroblasts (e.g. 3t6 or l929 cells), madv-2 is usually cultured in vitro in a mouse rectum carcinoma cell line (cmt-93). in laboratory mice, seropositivity to adenoviruses was reported to be very low [11, 14, 15, 17, 18, 124] or negative [12, 13] . antibodies to madv are also found in wild mice [21, 125] and in rats [21, 126] . neither virus is known to cause clinical disease in naturally infected, immunocompetent mice. however, madv-1 can cause a fatal systemic disease in suckling mice after experimental inoculation [118, 127, 128] . disease is characterized by scruffiness, lethargy, stunted growth and often death within 10 days. experimental infection of adult mice with madv-1 is most often subclinical and persistent but can cause fatal haemorrhagic encephalomyelitis with neurological symptoms, including tremors, seizures, ataxia and paralysis in susceptible c57bl/6 and dba/2j mice [129] . balb/c mice are relatively resistant to this condition. athymic foxn1 nu mice experimentally infected with madv-1 develop a lethal wasting disease [130] . similarly, prkdc scid mice succumb to experimental infection with madv-1 [131] . gross lesions in response to natural madv infections are not detectable. occasional lesions observed after experimental infection with madv-1 include small surface haemorrhages in the brain and spinal cord of c57bl/6 and dba/2j mice [129] , duodenal haemorrhage in foxn1 nu mice [130] and pale yellow livers in prkdc scid mice [131] . histologically, experimental madv-1 infection of suckling mice is characterized by multifocal necrosis and large basophilic intranuclear inclusion bodies in liver, adrenal gland, heart, kidney, salivary glands, spleen, brain, pancreas and brown fat [8, 36, 127, 132] . in experimentally induced haemorrhagic encephalomyelitis, multifocal petechial haemorrhages occur throughout the brain and spinal cord, predominantly in the white matter, and are attributed to infection and damage to the vascular epithelium of the central nervous system (cns) [129] . histopathological manifestations in madv-1-infected prkdc scid mice are marked by microvesicular fatty degeneration of hepatocytes [131] . in contrast to madv-1, the tissue tropism of madv-2 is limited to the intestinal epithelium. naturally or experimentally infected mice develop intranuclear inclusions in enterocytes, especially in the ileum and caecum [8, 36, 133] . transmission of madv primarily occurs by ingestion. madv-1 is excreted in the urine and may be shed for up to 2 years [134] . madv-2 infects the intestinal tract and is shed in faeces for only a few weeks in immunocompetent mice [135] ; immunodeficient mice may shed the virus for longer periods [136] . murine adenovirus infections are routinely diagnosed by serological tests. however, there is a one-sided cross-reactivity of madv-1 with madv-2 [137] . serum from mice experimentally infected with madv-1 yielded positive reactions in serological tests with both viruses, while serum from mice infected with madv-2 reacted only with the homologous antigen [138] . smith et al. [126] reported that sera might react with madv-1 or madv-2 or both antigens. occasional reports of mice with lesions suggestive of adenovirus infections and negative serology (with madv-1) indicate that the infection may not be detected if only one virus is used as an antigen [139] . it is therefore usual to test sera for antibodies to both madv-1 and madv-2. the commonly used methods are ifa, elisa and mfia. the low prevalence in colonies of laboratory mice indicates that madv can easily be eliminated (e.g. by hysterectomy derivation or embryo transfer) and that barrier maintenance has been very effective in preventing infection. the low pathogenicity and the low prevalence in contemporary mouse populations are the main reasons why adenoviruses are considered to be of little importance, which is also indicated by the fact that recent publications about murine adenoviruses are very rare. however, the viruses might easily be spread by the exchange of genetically modified mice and therefore re-emerge. only a few influences on research attributable to madv have been published. for example, it has been shown that madv-1 significantly aggravates the clinical course of scrapie disease in mice [140] . natural infections with madv could also interfere with studies using adenovirus as a gene vector. a novel murine adenovirus classified as a mastadenovirus has recently been isolated from a striped field mouse (apodemus agrarius) [141] . it was cultured in vero e6 cells and named madv type 3 (madv-3). it revealed the highest similarity to madv-1 but it represents a separate serotype. however, there is some cross-reactivity between madv-3 and both other mouse viruses [142] . in addition to serological and antigenic differences it also shows a unique organotropism and infects predominantly the heart tissue of c57bl/6n mice after experimental infection. experimentally infected mice show no clinical signs. the virus is not easily transmitted from experimentally infected mice to contact sentinels [142] . polyomaviridae are enveloped, double-stranded dna viruses. two different agents of this family exclusively infect mice (mus musculus), and both belong to the genus polyomavirus. murine pneumotropic virus (mptv) was formerly known as 'newborn mouse pneumonitis virus' or 'k virus' (named after l. kilham who first described the virus). the second is murine polyomavirus (mpyv). both are related, but antigenically distinct, from each other [143] , and also viruslike particles from the major capsid protein (vp1) do not cross-react [144] . they are enzootic in many populations of wild mice but are very uncommon in laboratory mice. even older reports indicate that both have been eradicated from the vast majority of contemporary mouse colonies, and their importance is negligible [8] . seropositivity to these viruses was not reported in a recent survey conducted in the usa [13] , and other publications also indicate that these viruses do not presently play a significant role in laboratory mice [11, 14, 15] . because of their low prevalence, neither virus is included in the list of agents for which testing is recommended on a regular basis by felasa [145] . although polyomavirus genes, especially those of sv40, are widely used in gene constructs for insertional mutagenesis, very few reports have been published on spontaneous or experimental disease due to mpyv or mptv in the last 10-15 years. the reader is therefore referred to previous review articles for details [8, 146] . natural infections with mptv are subclinical. the prevalence of infection is usually low in an infected population. the virus may persist in infected animals for months and perhaps for life depending on the age at infection and is reactivated under conditions of immunosuppression. virus replicates primarily in endothelial cells, but renal tubular epithelial cells are the major site of viral persistence [147, 148] . clinical signs are observed only after infection of infant mice less than 6-8 days of age. infected pups suddenly develop respiratory symptoms after an incubation period of approximately 1 week, and many die within a few hours of onset of symptoms with an interstitial pneumonia caused by productive infection of and damage to pulmonary endothelium (figure 3.2.5). endothelial cells in other organs are also involved in virus replication [148, 149] (figure 3 .2.6). in older suckling mice, mptv produces a more protracted infection, and the virus or viral antigen can be detected for as long as 4 months. in adult animals, the virus produces a transient asymptomatic infection. even in immunodeficient foxn1 nu mice, experimental infection of adults is clinically asymptomatic, although virus is detectable for a period of several months [150] . in vitro cultivation of mptv is difficult. no susceptible permanent cell line is known to support growth. it can be cultured in primary mouse embryonic cells, but viral titres are not sufficient for use in serological assays [151] . for this reason, the hi test using homogenates of livers and lungs of infected newborn mice is still frequently used, but ifa and elisa tests are also available [152] . furthermore, a pcr test for demonstration of mptv in biological samples has also been published [153] . mpyv was first detected as a contaminant of murine leukaemia virus (mulv) when sarcomas developed in mice after experimental inoculation of contaminated samples. it has later been shown to be a frequent contaminant of transplantable tumours [1] . natural infection of mice is subclinical, and gross lesions including tumours are usually not found. tumour formation occurs when mice are experimentally infected at a young age or when inoculated with high virus doses. development of tumours may be preceded by multifocal necrosis and mortality during the viraemic stage [36] . parotid, salivary gland and mammary tumours are common, and sarcomas or carcinomas of kidney, subcutis, adrenal glands, bone, cartilage, teeth, blood vessels and thyroid also occur. virus strains vary with regard to the tumour types or lesions that they induce, and mouse strains vary in their susceptibility to different tumour types. those of c57bl and c57br/cd lineage are considered to be the most resistant strains; athymic foxn1 nu mice are considered to be most susceptible; c3h mice are particularly susceptible to adrenal tumours and a mice tend to develop bone tumours. immunosuppression or inoculation into immunodeficient strains (e.g. foxn1 nu ) also supports the growth of tumours. on the other hand, experimental infection of adult immunocompetent mice does not result in tumour formation because the immune response suppresses tumour growth, and newborn immunocompetent mice develop runting only if inoculated with high virus doses [154] . after experimental intranasal infection, mpyv initially infects the respiratory tract followed by a systemic phase in which liver, spleen, kidney and colon become infected [155] . the virus is shed in faeces and in all body fluids, and transmission occurs rapidly by direct contact between animals, but also between cages in a room. further, intrauterine transmission has been documented after experimental infection [156] . mpyv persists in all organs in prkdc scid mice while viral dna is detectable in immunocompetent mice after experimental infection for only a limited period of about 4 weeks [157] . however, virus may persist and can be reactivated by prolonged immunosuppression [158] or during pregnancy, at least in young mice [159] . it has been shown that interferon-gamma is an important factor of the host defence against tumour formation and mpyv infection [160] . biological materials of mouse origin are likely to be the most common source of contamination of laboratory mice, emphasizing the importance of map or pcr screening of biological materials to be inoculated into mice. the most frequently used tests for health surveillance of mouse colonies are elisa, mfia and ifa; in addition, the hi test is still used. latent infections can be detected by intracerebral inoculation of neonate mice or by map testing, but direct demonstration of virus in biological samples is also possible by pcr testing [153] . while mpyv infections are of low importance for laboratory animal medicine, the virus is used in models of persistent virus infection [161, 162] . virus-like particles from both murine polyomaviruses have been used as a vector for gene therapy or vaccines [163, 164] . parvoviruses are non-enveloped small viruses (approximately 20 nm in diameter) with a singlestranded dna genome of approximately 5000 nucleotides. murine parvoviruses are members of the family parvoviridae, genus parvovirus. they are remarkably resistant to environmental conditions like heat, desiccation, acidic and basic ph-values. up to date, two distinct species that infect laboratory mice are officially listed: the minute virus of mice (mvm), previously named mice minute virus (mmv), and the mouse parvovirus (mpv). non-structural proteins (ns-1 and ns-2) are highly conserved among both viruses whereas the capsid proteins (vp-1, vp-2, vp-3) are more divergent and determine the serogroup [165] . both viruses require mitotically active cells for replication. severe clinical signs are therefore not found in mature animals because of the lack of a sufficient number of susceptible cells in tissues. general aspects of rodent parvovirus infections and their potential effects on research results have been reviewed [6, 8, [166] [167] [168] [169] [170] . already in the mid-1980s mouse colonies were identified that gave positive reactions for mvm by ifa but not by hi tests. it was subsequently shown that these colonies were infected with a novel parvovirus, initially referred to as 'mouse orphan parvovirus'. the first isolate of mpv was detected as a contaminant of cultivated t-cell clones interfering with in vitro immune responses [171] and was named 'mouse parvovirus'. it does not replicate well in currently available cell cultures, and sufficient quantities of virus for serological tests are difficult to generate. hitherto, only very few isolates of mpv have been cultured and subsequently characterized on a molecular basis [165, 172] . on the basis of epidemiological analyses, further parvoviruses were recently identified in mice, sequenced, and tentatively named serially mpv-2 and mpv-3 [173] , mpv-4 (genbank fj440683) and mpv-5 (genbank fj441297). in addition, several variants are published for mpv-1 [172, 174, 175] . at present, mpv is among the most common viruses found in colonies of laboratory mice. the prevalence of sera positive for parvoviruses ranged from 1% to nearly 10% in western europe and north america, with the majority of sera being positive for mpv in studies differentiating between the two parvovirus species [12, 14, 15, 176] . these prevalence data are based on testing at commercial laboratories and do not reflect that, despite highly specific and sensitive test methods, enzootic parvovirus infections are difficult to detect due to virus-associated characteristics [169, 170] . a recent survey conducted in the usa showed that during a 24-36 month period mouse parvoviruses were detected at almost all facilities that responded to a questionnaire, with mpv being more often diagnosed than mvm [13] . clinical disease and gross or histological lesions have not been reported for mice naturally or experimentally infected with mpv. infections are subclinical even in newborn and immunocompromised animals [177, 178] . in contrast to many other viruses infecting mice, viral replication and excretion is not terminated by the onset of host immunity. tissue necrosis has not been observed at any stage of infection in infected infant or adult mice [177, 178] . humoral immunity to mpv does not protect against mvm infections, and vice versa [179] . serological surveys have indicated that mpv naturally infects only mice, with the exception that mpv-3 shows genetic similarity to hamster parvovirus, suggesting that a cross-species transmission has occurred, where the mouse probably served as the natural host [173, 180] . differences in mouse strain susceptibility to clinical mpv infection do not exist. however, seroconversion seems to be strain-dependent. after experimental infection with mpv-1b, seroconversion occurred in all c3h/hen mice, fewer balb/c, dba/2 and icr mice, and seroconversion could not be detected in c57bl/6 mice [181] . upon mpv-1f inoculation, antibody response was absent in balb/carc mice [182] . diagnosis of mpv infection by pcr testing of small intestine and mesenteric lymph nodes also depended on the mouse strain. mpv dna was detected in all mouse strains evaluated except dba/2 even though seroconversion was detected in these mice. after oral infection, the intestine is the primary site of viral entry and replication. the virus spreads to the mesenteric lymph nodes and other lymphoid tissues, where it persists for more than 2 months [178] , and seems to be excreted via the intestinal and the urinary tract. after experimental inoculation of weanling mice, mpv is transmitted to cagemates by direct contact for 2-6 weeks [177] , and transmission by dirty bedding is also possible. these results implicate a role for urinary, faecal, and perhaps respiratory excretion of virus. another study showed that naturally infected mice might not transmit the virus under similar experimental conditions [183] . serology is a useful tool to identify mpv infections in immunocompetent hosts, but reaching a diagnosis based on serological assays may be difficult and requires a good knowledge of the available techniques. neither the virion elisa nor hi is a practical screening test for mpv because they require large quantities of purified mpv, which is difficult to obtain. diagnosis of mpv infections has long been made on the basis of an mvm hi-negative result coupled with an mvm ifa-positive result. ifa provides the opportunity to detect both serogroup-specific vp proteins as well as ns proteins that are conserved among mouse parvoviruses. a generic rodent parvovirus elisa using a recombinant ns-1 protein as antigen has been developed [184] , but mpv ifa and mpv hi assays are more sensitive techniques than the ns-1 elisa and the mvm ifa [181] . in contrast, elisa tests that use recombinant vp-2 provide sensitive and serogroup-specific assays for the diagnosis of mpv infections in mice [176, 185] , although considerable cross-reactivity with heterologous capsid antigens exists [173] . nevertheless, when using the elisa technique, one needs to consider that mpv-2 may not consistently be detected by mpv-1 vp-2 elisa [168, 173] , especially when antibody titres are low (own observations). therefore, elisas using mpv-2 vp-2 and mpv-3 vp-2 antigens are also used for diagnostics. as parvovirus diagnostics using recombinant assays should be based on a combination of antigens, bead-based mulitplex assays are a convenient extension of traditional elisa, allowing the use of multiple antigens simultaneously. in immunodeficient mice that do not generate a humoral immune response, pcr assays can be used to detect mpv [186, 187] and other parvoviruses. mpv has been shown to persist for at least 9 weeks in the mesenteric lymph nodes [178] . this tissue is considered the best suited for pcr analysis, but spleen and small intestine can also be used with good success [181] . for antemortem detection, shedding of parvoviruses can also be detected by pcr of faecal samples [188] . the virus persists sufficiently long in mesenteric lymph nodes so that pcr assays may also be used as a primary screening tool for laboratories that do not have access to specific mpv antigen-based serological assays. the pcr is further a good confirmatory method for serological assays and has also been described for the detection of parvoviruses in cell lines and tumours [189] . in addition, the map test has been reported as a sensitive tool to detect mpv [183] . given the high environmental stability of the virus and the potential fomite transmission, together with the long virus persistence in infected animals, spontaneous disappearance from a mouse population (e.g. by cessation of breeding) is unlikely. eradication of infection is possible by elimination of infected animals and subsequent replacement with uninfected mice, and the agent can be eliminated from breeding populations by embryo transfer or by hysterectomy. it should be noted that recent studies suggest a risk of virus transmission by embryo transfer, though successful sanitation of immunodeficient mice was achieved despite antibody response in recipients and progeny after embryo transfer [190, 191] . although there are few published reports of confounding effects of mpv on research, it is lymphocytotropic and may perturb immune responses in vitro and in vivo. infections with mpv have been shown to influence rejection of skin and tumour grafts [192] . mvm is the type species of the genus parvovirus. the virus was intermediately named mice minute virus (mmv). it was originally isolated by crawford [193] from a stock of mouse adenovirus, and this prototype isolate was later designated mvmp. its allotropic variant was detected as a contaminant of a transplantable mouse lymphoma [194] and designated mvmi because it exhibits immunosuppressive properties in vitro. both variants have distinct cell tropisms in vivo and in vitro. mvmp infects fibroblast cell lines and does not cause clinical disease [195, 196] . both strains are apathogenic for adult mice, but the immunosuppressive variant is more pathogenic for neonatal mice than is mvmp. a third strain, the cutter strain mvmc, was isolated from bhk-21 cells [172] . in contrast to these three strains detected as cell culture contaminants, an isolate was obtained from naturally infected mice with a b-cell maturational defect maintained at the university of missouri and therefore denominated mvmm [173] . serological surveys show that the mouse is the primary natural host [19, 125, 197] , but the virus is also infective for rats, hamsters [168, 198] , and mastomys [199] during fetal development or after parenteral inoculation. natural infections are usually asymptomatic in adults and infants, and the most common sign of infection is seroconversion. kilham and margolis [200] observed mild growth retardation a few days after experimental infection of neonatal mice with mvmp. studies of transplacental infection yielded no pathological findings in mice [201] . the immunosuppressive variant, but not the prototype strain, is able to produce a runting syndrome after experimental infection of newborn mice [195] . depending on the host genotype, experimental infections of fetal and neonatal mice with mvmi produce various clinical presentations and lesions. infection in c57bl/6 mice is asymptomatic, but the virus causes lethal infections with intestinal haemorrhage in dba/2 mice. infection of strains such as balb/c, cba, c3h/he and sjl is also lethal and mice have renal papillary haemorrhage [196] . the mvmi also infects haematopoietic stem cells and mediates an acute myelosuppression [202, 203] . because of its dependence on mitotically active tissues, the fetus is at particular risk for damage by parvoviruses. mvm and other parvoviruses may have severe teratogenic effects and cause fetal and neonatal abnormalities by destroying rapidly dividing cell populations, often resulting in fetal death. adult prkdc scid mice develop an acute leukopenia 1 month after experimental infection with mvmi and die within 3 months. the virus persists lifelong in the bone marrow of these mice [204] . during a natural concurrent outbreak of mvmm and mpv, a runting syndrome with lymphohistiocytic renal inflammation and inclusion bodies in cells resembling splenic haematopoietic progenitor cells was reported in b-cell (ighm)deficient mice [205] . mmv is shed in faeces and urine. in faecal samples, mvm was detected for up to 4-6 weeks by pcr [206, 207] , although shorter periods (9-12 days) have been observed [208] . notably, shedding re-occurred after immunosuppression by irradiation [207] . contaminated food and bedding are important factors in viral transmission because the virus is very resistant to environmental conditions. direct contact is also important and the virus does not easily spread between cages. routine health surveillance is usually conducted by serological methods. unlike mpv, mvm can easily be cultured in cell lines so that antigen production for hi and elisa (using whole purified virions) is easy. hi is a highly specific diagnostic test whereas ifa always exhibits some degree of cross-reactivity with mpv and other closely related parvoviruses. elisa is probably the most frequently used test, but depending on the purity of the antigen preparation, cross-reactions with mpv may occur due to contamination with non-structural proteins that are common to both viruses. this problem can be avoided by the use of recombinant vp-2 antigen [176] . by using serological methods, one needs to consider that the mouse strain has a considerable effect on seroconversion so that an antibody response might not be detectable despite infection; while c57bl/6j mice showed good antibody response, seroconversion was observed only in some balb/c, akr/n, dba/2j, fvb/n and c3h/hen, but not in nmri and icr mice upon contact exposure to mvmi-inoculated mice [206] . viral detection is also possible by pcr in biological materials, organs (intestine, mesenteric lymph node, kidney, spleen) and faeces from infected animals [187, 189, 206, 207, 209] . although mvm was not thought to cause persistent infection in immunocompetent mice, recent data show that it can be detected in spleens for up to 16 weeks after exposure in some mouse strains [207] . therefore, pcr may be considered as a confirmatory method for serology. the virus can be eliminated from infected breeding populations by caesarean derivation or by embryo transfer. however, certain precautions such as careful washing and accompanying testing need to be minded, as mvm has been detected in reproductive organs and gametes and this virus firmly attaches to the zona pellucida or might even cross it [210, 211] . in experimental colonies, elimination of infected animals and subsequent replacement with uninfected mice is practical if careful environmental sanitation is conducted by appropriate disinfection procedures. it is important that reintroduction is avoided by exclusion of wild mice and by strict separation from other infected populations and potentially contaminated materials in the same facility. admission of biological materials must be restricted to samples that have been tested and found to be free from viral contamination. both allotropic variants of mvm have been used as models for molecular virology, and their small size and simple structure have facilitated examination of their molecular biology and expedited understanding of cell tropism, viral genetics and structure. the significance for laboratory mouse populations was considered low or uncertain because natural infections are inapparent. however, various effects on mouse-based research have been published [6, 7, 166, 167, 170] . because of their predilection for replicating in mitotically active cells, they are frequently associated with tumour cells and have a marked oncosuppressive effect [212] . special attention is also necessary for immunological research and other studies involving rapidly dividing cells (embryology, teratology). in addition, mvm is a common contaminant of transplantable tumours, murine leukaemias and other cell lines [1, 2, 213] . lactate dehydrogenase-elevating virus (ldv) is a single-stranded rna virus of the genus arterivirus belonging to the family arteriviridae. the genome organization and replication of ldv and other arteriviruses, their cell biology and other molecular aspects have been reviewed by snijder and meulenberg [214] . ldv has repeatedly been detected in wild mice (mus musculus), which are considered to be a virus reservoir [215, 216] . after infection of mice, virus titres of 10 10 -10 11 particles per ml serum are found within 12-14 h after infection. the virus titre drops to 10 5 particles per ml within 2-3 weeks and remains constant at this level for life. it persists in infected mice for the whole lifetime although it stimulates various immune mechanisms [216] [217] [218] [219] . the virus can be stored in undiluted mouse plasma at à70 c without loss of infectivity, but it is not stable at room temperature and is very sensitive to environmental conditions. only mice and primary mouse cells are susceptible to infection with ldv. it replicates in a subpopulation of macrophages in almost all tissues and persists in lymph nodes, spleen, liver, and testes tissues [220] . as suitable cell systems have not been available for virus production, routine serology has not been easily possible so that testing for ldv was not included in serological health monitoring programs. the prevalence of ldv in contemporary colonies of laboratory rodents is likely to be very low but detailed information about its prevalence comparable to most other agents is not available. ldv was first detected during a study of methods that could be used in the early diagnosis of tumours [221] . it produces a persistent infection with continuous virus production and a lifelong viraemia despite ldv-specific immune reactions of the host [217]. ldv has been found in numerous biological materials that are serially passaged in mice such as transplantable tumours including human tumours or matrigel prepared from such materials [1, 2, 222, 223] , monoclonal antibodies or ascitic fluids [224] , or infectious agents (e.g. haemoprotozoans, k virus, clostridium piliforme). these materials are contaminated after serial passage in an infected and viraemic mouse. contamination with ldv leads to the infection of each sequential host and to transmission of the virus by the next passage and remains associated with the specimen. it is therefore the most frequently detected contaminant in biological materials [1, 2] . infection with ldv is usually asymptomatic, and there are no gross lesions in immunocompetent as well as in immunodeficient mice. the only exception is poliomyelitis with flaccid paralysis of hindlimbs developing in c58 and akr mice when they are immunosuppressed either naturally with ageing or experimentally. it has been shown that only mice harbouring cells in the cns that express a specific endogenous mulv are susceptible to poliomyelitis [225] . the characteristic feature of ldv infection is the increased activity of lactate dehydrogenase (ldh) and other plasma enzymes [8, 226] , which is due to the continuous destruction of permissive macrophages that are responsible for the clearance of ldh from the circulation. as a consequence, the activity of plasma ldh begins to rise by only 24 h after infection and peaks 3-4 days after infection at 5-10-fold normal levels, or can even be up to 20-fold in sjl/j mice. the enzyme activity declines during the next 2 weeks but remains elevated throughout life. antigen-antibody complexes produced during infection circulate in the blood and are deposited in the glomeruli [226] . in contrast to other persistent virus infections (e.g. lymphocytic choriomeningitis virus), these complexes do not lead to immune complex disease and produce only a very mild glomerulopathy. the only gross finding associated with ldv infection is mild splenomegaly. microscopically, necrosis of lymphoid tissues is visible during the first days of infection. in mouse strains that are susceptible to poliomyelitis, ldv induces lesions in the grey matter of the spinal cord and the brainstem. ldv is not easily transmitted between mice, even in animals housed in the same cage. fighting and cannibalism increase transmission between cagemates, most likely via blood and saliva. infected females transmit the virus to their fetuses if they have been infected few days prior to birth and before igg anti-ldv antibodies are produced, but developmental and immunological factors (e.g. gestational age, timing of maternal infection with ldv, placental barrier) are important in the regulation of transplacental ldv infection [227, 228] . maternal immunity protects fetuses from intrauterine infection. immunodeficient prkdc scid mice also transmit virus to their offspring during chronic infection [229] . an important means of transmission is provided by experimental procedures such as mouse-to-mouse passage of contaminated biological materials or the use of the same needle for sequential inoculation of multiple mice. in principle, serological methods such as ifa may be used for detecting ldv infection [230] but they are not of practical importance. circulating virus-antibody complexes interfere with serological tests, and sufficient quantities of virus for serological tests are difficult to generate because ldv replicates only in specific subpopulations of primary cultures of murine macrophages and monocytes for one cell cycle [226] . however, it is meanwhile possible to use recombinant viral proteins of ldv as antigens [231] in elisa and mfia tests so that routine testing by serology is possible. in the past, diagnosis of ldv infection has primarily been based on increased ldh activity in serum or plasma of mice. ldv activity in serum or plasma can be measured directly, or samples (e.g. plasma, cell or organ homogenates) are inoculated into pathogen-free mice and the increase in ldh activity within 3-4 days is measured. an 8-10-fold increase is indicative of ldv infection. detection of infectivity of a plasma sample by the induction of increased ldh activity in the recipient animal is the most reliable means of identifying an infected animal. however, it is important to use clear non-haemolysed samples because haemolysis will (falsely) elevate activities of multiple serum or plasma enzymes, including ldh. this assay was usually included in a 'map test', but antibody detection similar to other viruses was not involved for reasons mentioned earlier. persistent infection makes ldv an ideal candidate for pcr detection in plasma or in organ homogenates [232, 233] . however, reports exist that pcr may produce false-negative results and should be used cautiously [234] . just as important as detecting ldv in animals is its detection in biological materials. this may be done by assay for increased ldh activity after inoculation of suspect material into pathogenfree mice [1, 2] or by pcr [232, 233, [235] [236] [237] . ldv spreads slowly in a population because direct contact is necessary. therefore, ldv-negative breeding populations can easily be established by selecting animals with normal plasma ldh activity. embryo transfer and hysterectomy derivation are also efficient. the presence of ldv in experimental populations may be indicative of contaminated biological materials. in such cases, it is essential that the virus is also eliminated from these samples. this is easily achieved by maintenance of cells by in vitro culture instead of by animal-to-animal passages [238] . due to the extreme host specificity of the virus, contaminated tumour samples can also be sanitized by passages in nude rats [223] or other animal species. another method to remove ldv from contaminated cells, which is based on cell sorting, has recently been described [239] . ldv is a potential confounder of any research using biological materials that are passaged in mice. once present in an animal, the virus persists lifelong. the most obvious signs are increased levels of plasma ldh and several other enzymes. ldv may also exhibit numerous effects on the immune system (thymus involution, depression of cellular immunity, enhanced or diminished humoral responses, nk cell activation, development of autoimmunity, and suppression of development of diabetes in nod mice); [218, 219, 224, [240] [241] [242] [243] [244] and enhance or suppress tumour growth [6, 7, 226] . interaction with other viruses has also been described [245] . lymphocytic choriomeningitis virus (lcmv) is an enveloped, segmented single-stranded rna virus of the genus arenavirus family, arenaviridae. it can easily be propagated in several commonly used cell lines like bhk-21 cells. however, cells are not lysed and a cytopathic effect (cpe) is not visible. the virus name refers to the condition that results from experimental intracerebral inoculation of the virus into adult mice and is not considered to be a feature of natural infections. mice (mus musculus) serve as the natural virus reservoir [246] , but syrian hamsters are also important hosts [247] . additional species such as rabbits, guinea-pigs, squirrels, monkeys and humans are susceptible to natural or experimental infection [248] . natural infection of callitrichid primates (marmosets and tamarins) leads to a progressive hepatic disease that is known as 'callitrichid hepatitis' [249, 250] . antibodies to lcmv have been found in wild mice in europe [251, 252] , africa [253] , asia [254] , australia [125] and america [255] . thus, it is the only arenavirus with worldwide distribution. infection with lcmv is rarely found in laboratory mice [248] . seropositivity to lcmv in laboratory mice was reported to be low during the last decade [11, 15, 17, 124] or negative [12] [13] [14] . in addition to laboratory mice and other vertebrate hosts, the virus has frequently been found in transplantable tumours and tissue culture cell lines from mice and hamsters [2, 256] . despite the low prevalence in laboratory mice, seropositivity to this zoonotic agent should raise serious concern for human health. lcmv is frequently transmitted to humans from wild mice and is also endemic to a varying degree in the human population [257] [258] [259] [260] [261] due to contact with wild mice. it has also been transmitted to humans by infected laboratory mice [262] and by pet and laboratory syrian hamsters [263] [264] [265] [266] . in addition, contaminated biological materials are important sources of infections for humans, and several outbreaks of lcm among laboratory personnel have been traced to transplantable tumours [267, 268] . transmission of lcmv to humans also occurred repeatedly by organ transplantation and was most likely transmitted to organ donors by close contact with infected pets [266, 269] . lcmv can cause mild-to-serious or fatal disease in humans [262, 270, 271] . congenital infection in humans may result in hydrocephalus, or fetal or neonatal death [272] . in mice, clinical signs of lcmv infection vary with strain and age of mouse, strain and dose of virus, and route of inoculation [8, 248, 251] . two forms of natural lcmv infection are generally recognized: a persistent tolerant and an (acute) non-tolerant form. the persistent form results from infection of mice that are immunotolerant. this is the case if mice are infected in utero or during the first days after birth. this form is characterized by lifelong viraemia and viral shedding. mice may show growth retardation, especially during the first 3-4 weeks, but they appear otherwise normal. infectious virus is bound to specific antibodies and complement, and these complexes accumulate in the renal glomeruli, the choroid plexus, and sometimes also in synovial membranes and blood vessel walls. at 7-10 months of age, immune complex nephritis develops with ruffled fur, hunched posture, ascites and occasional deaths. this immunopathologic phenomenon is called 'late onset disease' or 'chronic immune complex disease'. the incidence of this type of disease varies between mouse strains. gross lesions include enlarged spleen and lymph nodes due to lymphoid hyperplasia. kidneys affected with glomerulonephritis may be enlarged with a granular surface texture or may be shrunken in later stages of the disease process. microscopically, there is generalized lymphoid hyperplasia and immune complex deposition in glomeruli and vessel walls, resulting in glomerulonephritis and plasmacytic, lymphocytic perivascular cuffs in all visceral organs [36] . the non-tolerant acute form occurs when infection is acquired after the development of immunocompetence (in mice older than 1 week). these animals become viraemic but do not shed virus and may die within a few days or weeks. natural infections of adults are usually asymptomatic. surviving mice are seropositive and in most cases clear the virus to below detection levels of conventional methods. however, virus may persist at low levels in tissues (particularly spleen, lung and kidney) of mice for at least 12 weeks after infection as determined by sensitive assays such as nested reverse transcriptase (rt)-pcr or immunohistochemistry [273] . such non-lethal infection leads to protection against otherwise lethal intracerebral challenge. protection from lethal challenge is also achieved by maternally derived anti-lcmv antibodies through nursing or by the administration of anti-ldv monoclonal igg2a antibodies [274] . in experimentally infected mice, the route of inoculation (subcutaneous, intraperitoneal, intravenous, intracerebral) also influences the type and degree of disease [248] . intracerebral inoculation of adult immunocompetent mice typically results in tremors, convulsions and death due to meningoencephalitis and hepatitis. neurological signs usually appear on day 6 after inoculation, and animals die within 1-3 days after the onset of symptoms, or recover within several days. the classic histological picture is of dense perivascular accumulations of lymphocytes and plasma cells in meninges and choroid plexus. while infection following subcutaneous inoculation usually remains inapparent, reaction of mice to intraperitoneal or intravenous inoculation depends on the virus strain and on the mouse strain. infection by these routes primarily causes multifocal hepatic necrosis and necrosis of lymphoid cells. athymic foxn1 nu mice and other immunodeficient mice do not develop disease but become persistently viraemic and shed virus. as a general rule, all pathological alterations following lcmv infection are immune-mediated; and mice can be protected from lcmvinduced disease by immunosuppression [275] . lcmv disease is a prototype for virus-induced t-lymphocyte-mediated immune injury and for immune complex disease. for detailed information on the pathogenesis, clinical and pathological features of lcmv infection, the reader is referred to review articles [248, 276, 277] . in nature, carrier mice with persistent infection serve as the principal source of virus. intrauterine transmission is very efficient, and with few exceptions all pups born from carrier mice are infected. furthermore, persistently infected mice and hamsters can shed large numbers of infectious virions primarily in urine, but also in saliva and milk. the virus can replicate in the gastric mucosa after intragastric infection [278, 279] . gastric inoculation elicits antibody responses of comparable magnitudes as intravenous inoculation and leads to active infection with lcmv, indicating that oral infection is possible, e.g. by ingestion of contaminated food or by cannibalism. a self-limiting infection frequently results from infection of adult mice. the virus does not spread rapidly after introduction in populations of adult mice, and the infectious chain usually ends. however, if the virus infects a pregnant dam or a newborn mouse, a lifelong infection results, and soon a whole breeding colony of mice may become infected if the mice live in close proximity (which is the case under laboratory conditions). the virus is not easily transmitted to dirty-bedding sentinels, and it is important that colony animals or animals having had direct contact with a population are tested to exclude lcmv infection [280] . lcmv is most commonly diagnosed by serological methods such as mfia, ifa and elisa [281] . all strains show a broad cross-reactivity and are serologically uniform. however, subclinical persistent infections may be difficult to detect because they may be associated with minimal or undetectable levels of circulating antibody. it is important that bleeding of mice is done carefully because of a potential risk due to viraemic animals. historically, direct viral detection was performed by inoculating body fluids or tissue homogenates into the brain of lcmv-free mice or by subcutaneous injection into mice and subsequent serological testing (map test). more recently, pcr assays have been developed for the direct detection of viral rna in clinical samples or animals [282] [283] [284] . both map test and pcr can also be used to detect contamination of biological materials [235, 237] . specifically for exclusion of contamination by lcmv, it was requested by different authorities that virus is inoculated intracerebrally at a lethal dose 3-4 weeks after administration of the material to be tested. in case of contamination by lcmv and subsequent seroconversion, animals survive the challenge infection. vertical transmission of lcmv by transuterine infection is efficient so that this virus cannot reliably be eliminated by caesarean rederivation [280] . caesarean derivation may be effective if dams acquired infection after the development of immunocompetence (non-tolerant acute infection) and subsequently eliminated the virus, but such a strategy is difficult to justify in light of lcmv's zoonotic potential. in breeding colonies of great value, virus elimination might be possible soon after introduction into the colony by selecting non-viraemic breeders. this procedure is expensive and time consuming and requires special safety precautions. fortunately, infections of laboratory mice with lcmv are very uncommon. however, once lcmv has been detected in animals, or in biological materials, immediate destruction of all contaminated animals and materials is advisable to avoid risk of human infection. foxn1 nu and prkdc scid mice may pose a special risk because infections are silent and chronic [268] . cages and equipment should be autoclaved, and animal rooms should be fumigated with disinfectants such as formaldehyde, vaporized paraformaldehyde, hydrogen peroxide or other effective disinfectants. prevention of introduction into an animal facility requires that wild mice cannot get access to the facility. similarly important is screening of biological materials originating from mice and hamsters because these can be contaminated by lcmv. finally, it has been shown that the virus can also be introduced into a population by mice with an undetected infection [280] . appropriate precautions are necessary for experiments involving lcmv, or lcmv-infected animals or materials. biological safety level (bsl) 2 will be considered to be sufficient in most cases. bsl 3 practices may be considered when working with infected animals owing to the increased risk of virus transmission by bite wounds, scratching or aerosol formation from the bedding. animal biosafety level (absl) 3 practices and facilities are generally recommended for work with infected hamsters. appropriate precautions have been defined for different bsls or absls by cdc [285] . lcmv is frequently utilized as a model organism to study virus-host interactions, immunological tolerance, virus-induced immune complex disease, and a number of immunological mechanisms in vivo and in vitro [286] [287] [288] . accidental transmission may have a severe impact on various kinds of experiments [6, 7, 248, 251] and also affect infection with other agents [289] . mammalian orthoreoviruses (mrv) are nonenveloped, segmented double-stranded rna viruses of the family reoviridae, genus orthoreovirus. they have a wide host range and are ubiquitous throughout the world. the designation reo stands for respiratory enteric orphan and reflects the original isolation of these viruses from human respiratory and intestinal tract without apparent disease. the term 'orphan' virus refers to a virus in search of a disease. mammalian orthoreovirus can be grouped into three serotypes, numbered 1-3. mammalian orthoreovirus-3 (synonyms: hepatoencephalomyelitis virus; echo 10 virus) infection remains prevalent in contemporary mouse colonies and has been reported in wild mice [20, 125, 290] . a study in france reported antibodies to mrv-3 in 9% of mouse colonies examined [10] . in more recent studies in north america and western europe, such antibodies were detected in 0.01-0.2% of mice monitored [11, 14, 15] . schoondermark-van de ven et al. [12] found antibodies to mrv-3 in 0.6% of mouse samplings from western european institutions; and in a survey conducted by carty [13] , about 6% of responding institutions in the usa reported mrv-3 infection in their mouse colonies. in addition, contamination of mouse origin tumours and cell lines by mrv-3 has been reported many times [2, 8, 290] . experimentally, mrv-3 infection of infant mice has been used to model human hepatobiliary disease, pancreatitis, diabetes mellitus and lymphoma [8, 291] . the literature on mrv-3 infections in mice is dominated by studies on experimentally infected animals. the virus can cause severe pantropic infection in infant mice [290] [291] [292] . after parenteral inoculation, virus can be recovered from the liver, brain, heart, pancreas, spleen, lymph nodes and blood vessels. following oral inoculation, reoviruses gain entry by infecting specialized epithelial cells (m cells) that overlie peyer's patches. the virus then becomes accessible to leukocytes and spreads to other organs by way of the lymphatic system and the bloodstream. neural spread to the cns has also been well documented [293, 294] . the mechanisms of viral pathogenesis and their interactions with the host cell as well as the host's immune response are reviewed in detail by tyler et al. [295] , schiff et al. [296] and ward et al. [291] . natural infection by mrv-3 in a mouse colony is usually subclinical, although diarrhoea or steatorrhoea and oily hair effect in suckling mice may be noted [8, 36, [290] [291] [292] . the latter term has been used to describe the matted, unkempt appearance of the hair coat that results from steatorrhoea due to pancreatitis, maldigestion and biliary atresia. in addition, runting (attributed to immune-mediated destruction of cells in the pituitary gland that produce growth hormone), transient alopecia, jaundice (due to excessive bilirubin in the blood, which is attributed to the liver pathology, especially biliary atresia) and neurological signs such as incoordination, tremors or paralysis may develop. when present in natural infections, clinical signs and lesions are similar to but milder than in experimental neonatal infections. early descriptions of naturally occurring disease may have been complicated by concurrent infections such as mhv (murine hepatitis virus) or murine rotavirus a (murv-a)/epizootic diarrhoea of infant mice (edim) virus that contributed to the severity of the lesions especially in liver, pancreas, cns and intestine. the outcome of mrv-3 infection depends on age and immunological status of mouse, dose of virus and route of inoculation. adult immunocompetent mice typically show no clinical signs and have no discernible lesions even in experimental infections. mucosal and maternally conferred immunity are considered to be important in protection from or resolution of disease [297, 298] . experimental infection of adult prkdc scid mice is lethal [299] . depending on the route of inoculation, experimental infection of adult foxn1 nu mice is subclinical or results in liver disease [299, 300] . histological findings reported to occur after experimental mrv-3 infection of neonatal mice include inflammation and necrosis in liver, pancreas, heart, adrenal, brain, and spinal cord; lymphoid depletion in thymus, spleen, and lymph nodes; and hepatic fibrosis with biliary atresia [36, [290] [291] [292] 298] . transmission of reoviruses probably involves the aerosol as well as the faecal-oral route [8, 291] . fomites may play an important role as passive vectors because reoviruses resist environmental conditions moderately well. serological screening with mfia, elisa or ifa is in widespread use for detection of antibodies to mrv-3 in diagnostic and health surveillance programmes. both elisa and ifa detect cross-reacting antibodies to heterologous mrv serotypes that can infect mice [301] , although a recent report indicates that some ifa-positive mrv infections in mice may not be detected by commonly used elisas [302] . the hi test does not detect such cross-reacting antibodies but is prone to give false-positive results due to nonspecific inhibitors of haemagglutination [301, 303] . rt-pcr methods for the detection of mrv-3 rna [304, 305] or mrv rna [302, 306] are also available. reports on contamination of mouse origin tumours and cell lines by mrv-3 and its interference with transplantable tumour studies [307, 308] emphasize the importance of screening of biological materials to be inoculated into mice by map test or pcr. natural seroconversion to mrv-3 without clinical disease is also observed in laboratory rats, hamsters and guinea-pigs [8, 290] . caesarean derivation and barrier maintenance have proven effective in the control and prevention of mrv-3 infection [8, 291] . the virus may interfere with research involving transplantable tumours and cell lines of mouse origin. it has the potential to alter intestinal studies and multiple immune response functions in mice. in enzootically infected colonies, protection of neonates by maternal antibody could complicate or prevent experimental infections with reoviruses. it could further complicate experiments that require evaluation of liver, pancreas, cns, heart, lymphoid organs and other tissues affected by the virus. the term murine hepatitis virus (mhv; commonly referred to as 'mouse hepatitis virus') designates a large group of antigenically and genetically related, single-stranded rna viruses belonging to the family coronaviridae, genus coronavirus. they are surrounded by an envelope with a corona of surface projections (spikes). mhv is antigenically related to rat coronaviruses and other coronaviruses of pigs, cattle and humans. numerous different strains or isolates of mhv have been described. they can be distinguished by neutralization tests that detect strainspecific spike (s) antigens, by use of monoclonal antibodies, or by sequencing [309] . the beststudied strains are the prototype strains mhv-1, mhv-2, mhv-3, jhm (mhv-4), a59, and s, of which mhv-3 is regarded as the most virulent. like other coronaviruses mhv mutates rapidly, and strains readily form recombinants, so that new (sub)strains are constantly evolving. strains vary in their virulence, organotropism and cell tropism [310] . based on their primary organotropism, mhv strains can be grouped into two biotypes: respiratory (or polytropic) and enterotropic. however, intermediate forms (enterotropic strains with tropism to other organs) also exist. murine hepatitis virus is relatively resistant to repeated freezing and thawing, heating (56 c for 30 min) and acid ph but is sensitive to drying and disinfectants, especially those with detergent activity [8] . given the environmental conditions present in mouse rooms, mhv might remain infective for several days, at low humidity (20% relative humidity) or low temperatures (4 c) even for weeks on surfaces [311] . mus musculus is the natural host of mhv. it can be found in wild and laboratory mice throughout the world and is one of the most common viral pathogens in contemporary mouse colonies. while polytropic strains have historically been considered more common, this situation is thought to have reversed. monitoring results for research institutions across north america and europe indicate that the prevalence of mhv has decreased in the past, though it seems to have remained quite stable since the 1990s [11, 12] . recently 1.57% of north american laboratory mouse serum samples tested positive [15] . in europe, prevalence rates ranged from 3.25% to 12% [12, 14, 15] . a retrospective study in france covering the period from 1988 to 1997 reported antibodies to mhv in 67% of mouse colonies examined [10] , and a survey performed in 2006 revealed that almost half of north american research institutions detected mhv in their mouse populations [13] . suckling rats inoculated experimentally with mhv had transient virus replication in the nasal mucosa and seroconversion but no clinical disease [312] . similarly, deer mice seroconverted but showed no clinical disease after experimental infection [313] . mhv is also a common contaminant of transplantable tumours [1, 2] and cell lines [314, 315] . the pathogenesis and outcome of mhv infections depend on interactions between numerous factors related to the virus (e.g. virulence and organotropism) and the host (e.g. age, genotype, immune status, and microbiological status) [8, 36, 309, 310, 316, 317] . mhv strains appear to possess a primary tropism for the upper respiratory or enteric mucosa. those strains with respiratory tropism initiate infection in the nasal mucosa and then may disseminate via blood and lymphatics to a variety of other organs because of their polytropic nature. respiratory (polytropic) strains include mhv-1, mhv-2, mhv-3, a59, s and jhm. infection of mice with virulent polytropic mhv strains, infection of mice less than 2 weeks of age, infection of genetically susceptible strains of mice or infection of immunocompromised mice favour virus dissemination. virus then secondarily replicates in vascular endothelium and parenchymal tissues, causing disease of the brain, liver, lymphoid organs, bone marrow and other sites. infection of the brain by viraemic dissemination occurs primarily in immunocompromised or neonatal mice. additionally, infection of adult mouse brain can occur by extension of virus along olfactory neural pathways, even in the absence of dissemination to other organs. in contrast, enterotropic mhv strains (e.g. livim, mhv-d, mhv-y) tend to selectively infect intestinal mucosal epithelium, with no or minimal dissemination to other organs such as mesenteric lymph nodes or liver. all ages and strains are susceptible to active infection, but disease is largely age related. infection of neonatal mice results in severe necrotizing enterocolitis with high mortality within 48 h. mortality and lesion severity diminish rapidly with advancing age at infection. adult mice develop minimal lesions although replication of equal or higher titres of virus occurs compared with neonates. the age-dependent decrease in severity of enterotropic mhv disease is probably related to the higher mucosal epithelium turnover in older mice, allowing more rapid replacement of damaged mucosa. another factor that is of considerable importance to the outcome of mhv infections is host genotype. for example, balb/c mice are highly susceptible to enterotropic mhv disease while sjl mice, at the other end of the spectrum, are highly resistant [318] . unlike in polytropic mhv infection where resistance is correlated with reduced virus replication in target cells [319] , enterotropic mhv grows to comparable titres in sjl and balb/c mice at all ages [318] . therefore, the resistance of the sjl mouse to disease caused by enterotropic mhv seems to be mediated through an entirely different mechanism than resistance to polytropic mhv. furthermore, mouse genotypes that are susceptible to disease caused by one mhv strain may be resistant to disease caused by another strain [316] . it is therefore not possible to strictly categorize mouse strains as susceptible or resistant. the genetic factors determining susceptibility versus resistance in mhv infections are as yet poorly understood. both polytropic and enterotropic mhv infections are self-limiting in immunocompetent mice. immune-mediated clearance of virus usually begins about a week after infection, and most mice eliminate the virus within 3-4 weeks [316, 318, 320] . humoral and cellular immunity appear to participate in host defences to infection, and functional t cells are an absolute requirement [321] [322] [323] [324] . therefore, immunodeficient mice such as foxn1 nu and prkdc scid mice cannot clear the virus [317, 325] . similarly, some genetically modified strains of mice may have deficits in antiviral responses or other alterations that allow the development of persistent mhv infection [326] . recovered immune mice are resistant to reinfection with the same mhv strain but remain susceptible to repeated infections with different strains of mhv [327] [328] [329] . similarly, maternal immunity protects suckling mice against homologous mhv strains but not necessarily against other strains [329, 330] . however, maternal immunity, even to homologous strains, depends on the presence of maternally acquired antibody in the lumen of the intestine [330] . therefore, the susceptibility of young mice to infection significantly increases at weaning. most mhv infections are subclinical and follow one of two epidemiological patterns in immunocompetent mice [8, 310] . enzootic (subclinical) infection, commonly seen in breeding colonies, occurs when a population has been in contact with the virus for a longer period (e.g. several weeks). adults are immune (due to prior infection), sucklings are passively protected, and infection is perpetuated in weanlings. epizootic (clinical) infection occurs when the virus is introduced into a naive population (housed in open cages). the infection rapidly spreads through the entire colony. clinical signs depend upon the virus and mouse strains and are most evident in infant mice. typically, they include diarrhoea, poor growth, lassitude, and death. in infections due to virulent enterotropic strains, mortality can reach 100% in infant mice. some strains may also cause neurological signs such as flaccid paralysis of hindlimbs, convulsions and circling. adult infections are again usually asymptomatic. as the infection becomes established in the colony, the epizootic pattern is replaced by the enzootic pattern. in immunodeficient (e.g. foxn1 nu and prkdc scid ) mice, infection with virulent polytropic mhv strains is often rapidly fatal while less virulent strains cause chronic wasting disease [317] . in contrast, adult immunodeficient mice can tolerate chronic infection by enterotropic mhv, with slow emaciation and diarrhoea, or minimal clinical disease [316, 325] . subclinical mhv infections can be activated by a variety of experimental procedures (e.g. thymectomy, whole body irradiation, treatment with chemotherapeutic agents, halothane anaesthesia) or by coinfections with other pathogens (e.g. eperythrozoon coccoides, k virus; reviewed in [8, 309] ). in most natural infections, gross lesions are not present or are transient and not observed. gross findings in neonates with clinical signs include dehydration, emaciation, and in contrast to edim, an empty stomach [309, 331, 332] . the intestine is distended and filled with watery to mucoid yellowish, sometimes gaseous contents. haemorrhage or rupture of the intestine can occur. depending on the virus strain, necrotic foci on the liver [36, 309, 332] and thymus involution [331, 333] may also be seen in susceptible mice. liver involvement may be accompanied by jaundice and haemorrhagic peritoneal exudate. splenomegaly may occur as a result of compensatory haematopoiesis [334] . histopathological changes in susceptible mice infected with polytropic mhv strains include acute necrosis with syncytia in liver, spleen, lymph nodes, gut-associated lymphoid tissue, and bone marrow [8, 36, 309, 316] (figure 3.2.7) . recently, pulmonary inflammation has been observed in susceptible mouse strains (c3h/hej and a/j) after intranasal inoculation with polytropic mhv-1 [335, 336] . neonatally infected mice can have vascular-oriented necrotizing (meningo)encephalitis with demyelination in the brainstem and periependymal areas. lesions in peritoneum, bone marrow, thymus and other tissues can be variably present. mice can develop nasoencephalitis due to extension of infection from the nasal mucosa along olfactory pathways to the brain, with meningoencephalitis and demyelination, the latter of which is thought to be largely t-cell mediated [324] . this pattern of infection regularly occurs after intranasal inoculation of many mhv strains but is a relatively rare event after natural exposure. syncytium arising from endothelium, parenchyma or leukocytes is a hallmark of infection in many tissues including intestine, lung, liver, lymph nodes, spleen, thymus, brain and bone marrow. lesions are transient and seldom fully developed in adult immunocompetent mice, but they are manifest in immunocompromised mice. highly unusual presentations can occur in mice with specific gene defects. for example, granulomatous peritonitis and pleuritis were found in interferongamma-deficient mice infected with mhv [337] . histopathological changes caused by enterotropic strains of mhv are mainly confined to the intestinal tract and associated lymphoid tissues [8, 36, 309, 316] . the most common sites are terminal ileum, caecum and proximal colon. the severity of disease is primarily age-dependent, with neonatal mice being most severely affected. these mice show segmentally distributed areas of villus attenuation, enterocytic syncytia (balloon cells) and mucosal necrosis accompanied by leukocytic infiltration. intracytoplasmic inclusions are present in enterocytes. erosions, ulceration, and haemorrhage may be seen in more severe cases. lesions can be fully developed within 24-48 h, but are usually more severe at 3-5 days after infection. surviving mice may develop compensatory mucosal hyperplasia. mesenteric lymph nodes usually contain lymphocytic syncytia, and mesenteric vessels may contain endothelial syncytia. pathological changes in older mice are generally much more subtle and may only consist of transient syncytia. an occasional exception seems to occur in immunodeficient animals such as foxn1 nu mice, which can develop chronic hyperplastic typhlocolitis of varying severity [325] , but other agents such as helicobacter spp. may have been involved. in general, enterotropic mhv strains do not disseminate, but hepatitis and encephalitis can occur with some virus strains in certain mouse genotypes. in t-cell deficient mice, multisystemic lethal infection was observed after experimental infection with the enterotropic strain mhv-y [338] . mhv is highly contagious. it is shed in faeces and nasopharyngeal secretions and appears to be transmitted via direct contact, aerosol and fomites [8, 309] . vertical (in utero) transmission has been demonstrated in experimental infections [339] but does not seem to be of practical importance under natural conditions. mhv was transmitted by ovarian transplantation after reproductive organs became infected [340] . however, risk of mhv transmission by sperm or oocytes (ivf) or by embryo transfer seems to be low, though thorough washing of gametes and embryos is required [211, [340] [341] [342] . diagnosis during the acute stage of infection can be made by histological demonstration of characteristic lesions with syncytia in target tissues, but clinical signs and lesions can be highly variable and may not be prominent. suckling, genetically susceptible or immunocompromised mice are the best candidates for evaluation. active infection can be confirmed by immunohistochemistry [343] or by virus isolation. virus recovery from infected tissues is difficult but can be accomplished using primary macrophage cultures or a number of established cell lines such as nctc 1469 or dbt [301] . these cells, however, may not be successful substrates for some enterotropic mhv strains. virus in suspect tissue can also be confirmed by bioassays such as map testing or infant or foxn1 nu mouse inoculation [301, 344] . amplification by passage in these mice increases the likelihood of detection of lesions and antigen, or virus recovery. other direct diagnostic methods that have been successfully utilized to detect mhv in faeces or tissue of infected mice include monoclonal antibody solution hybridization assay [345] and a number of rt-pcr assays [346] [347] [348] [349] . because of the transient nature of mhv infection in immunocompetent mice, serology is the most appropriate diagnostic tool for routine monitoring. multiplex fluorescent immunoassay, elisa and ifa are well established and sensitive, and all known mhv strains cross-react in these tests [301, 350, 351] . the magnitude of antibody response depends on mhv strain and mouse genotype [319, 352] . dba/2 mice are poor antibody responders whereas c57bl/6 mice produce a high antibody titre and are therefore good sentinels. antibody titres remain high over a period of at least 6 months [327, 329] . infected mice may not develop detectable antibodies for up to 14 days after initial exposure [350] . in such cases, a direct diagnostic method, as discussed above, may be useful. another drawback of serology is that mice weaned from immune dams can have maternal antibodies until they are 10 weeks of age [353] . this may impact serological monitoring because the possibility must be considered that low positive results are due to maternally derived passive immunity. because the virus can be transmitted by transplantable tumours and other biological materials from mice, including hybridomas [354] and embryonic stem cells [355, 356] these materials should also be routinely screened for mhv contamination. mouse inoculation bioassay, map test and rt-pcr can be used for this purpose. therefore, surveillance programmes should combine careful evaluation of clinically ill animals, testing of biological materials and routine health monitoring. soiled-bedding sentinel mice, which are frequently used for routine monitoring, are likely well suited for detecting enterotropic strains of mhv, but might not indicate the presence of less contagious respiratory strains of mhv [309] equally well. the mouse strain used as sentinel should be considered as a critical factor. furthermore, duration of mhv shedding and stability of the virus, which seems to be lower in static microisolator cages than in ivc cages, might interfere with detection. the amount of bedding transferred seems not to be as critical as for, e.g. parvoviruses, at least for enterotropic strains [357] . use of contact and exhaust air sentinels and testing of exhaust filters by pcr was also shown to be effective at detecting mhv [358] . the best means of mhv control is to prevent its entry into a facility. this can be accomplished by purchasing mice from virus-free sources and maintenance under effective barrier conditions monitored by a well-designed quality assurance programme. control of wild mouse populations, proper husbandry and sanitation, and strict monitoring of biological materials that may harbour virus are also important measures to prevent infection. if infection occurs, the most effective elimination strategy is to cull the affected colony and obtain clean replacement stock. however, this is not always a feasible option when working with valuable mice (e.g. genetically modified lines, breeding stocks). caesarean derivation or embryo transfer can be used to produce virus-free offspring, and foster-nursing has also been reported to be effective [359] . quarantine of an affected colony with no breeding and no introduction of new animals for approximately 2 months has been effective in immunocompetent mice [360] . the infection is likely to be terminated because mhv requires a constant supply of susceptible animals. this method works best when working with small numbers of mice. large populations favour the development of new mhv strains that may result in repeated infections with slightly different strains [361] . it may be practical to select a few future breeders from the infected population and quarantine them for approximately 3 weeks [317] . this can be achieved in isolators, or in individually ventilated cages if proper handling is guaranteed. after this interval, breeding can resume. the 3-week interval should permit recovery from active infection, and the additional 3-week gestation period effectively extends the total quarantine to 6 weeks. it is advisable to select seropositive breeders because the possibility of active infection is lower in such animals. the breeding cessation strategy may not be successful if immunodeficient mice are used because they are susceptible to chronic infection and viral excretion [325] . genetically engineered mice of unclear, unknown or deficient immune status pose a special challenge because they may develop unusual manifestations of infection or may be unable to clear virus. rederivation is likely to be the most cost-effective strategy in such situations. along with the measures described, proper sanitation and disinfection of caging and animal quarters, as well as stringent personal sanitation, are essential to eliminate infection. careful testing with sentinel mice should be applied to evaluate the effectiveness of rederivation. if transplantable tumours are contaminated with mhv, virus elimination can be achieved by passage of tumours in athymic foxn1 rnu rats [362] . mhv is one of the most important viral pathogens of laboratory mice and has been intensively studied from a number of research perspectives (e.g. as a model organism for studying coronavirus molecular biology or the pathogenesis of viral-induced demyelinating disease). numerous reports document the effects of natural and experimental infections with mhv on host physiology and research, especially in the fields of immunology and tumour biology (reviewed in [6-8, 310, 316, 317] ). noroviruses are non-enveloped, single-stranded rna viruses with high environmental resistance and belong to the family caliciviridae, genus norovirus. they were first identified after an outbreak of acute gastroenteritis at a school in norwalk (ohio, usa) in 1968 and cause about 90% of non-bacterial epidemic gastroenteritis in humans. noroviruses found in animals include bovine, porcine and murine noroviruses. noroviruses are not known to cross species. murine norovirus (mnv) is endemic in many research mouse colonies and currently the most commonly detected viral agent in laboratory mice [14, 15, 363] . in the hitherto largest survey [15] , about 32% of mouse serum samples examined had antibodies against mnv. the first norovirus to infect mice was described in 2003 [364] . experimental inoculation studies with this murine norovirus (mnv-1) show that duration of infection and disease manifestation vary depending on the mouse strain [363] [364] [365] . in immunocompetent strains, mnv infection is variable in length (e.g. !7-14 days in 129s6 mice, !5 weeks in hsd:icr mice) and does not induce clinical signs. infection is associated with mild histopathological alterations in the small intestine (increase in inflammatory cells) and spleen (red pulp hypertrophy and white pulp activation) of 129s6 mice. in certain immunodeficient strains, however, infection can cause lethal systemic disease (encephalitis, vasculitis, meningitis, hepatitis and pneumonia in interferon-alpha-beta-gamma-receptor-deficient and stat1 tm1 mice) or persist without symptoms (!90 days in rag1 à/à and rag2 à/à mice). these findings indicate that components of the innate immune system are critical for resistance to mnv-1 induced disease. consistent with this hypothesis, it was demonstrated that mnv-1 replicates in macrophages and dendritic cells [366] . meanwhile, many additional strains of mnv with diverse biological properties were isolated [367, 368] . an analysis of 26 mnv isolates revealed 15 distinct mnv strains that comprise a single genogroup and serotype [368] . experimental inoculation studies show that several mnv strains are able to persist in various tissues (small intestine, caecum, mesenteric lymph node, spleen) of immunocompetent (c57bl/6j, hsd:icr, jcl:icr) and immunodeficient (cb17-prkdc scid ) mice with viral shedding in faeces for the duration of at least 35-60 days [367] [368] [369] . murine norovirus is transmitted via the faecaloral route and is efficiently transferred to sentinel mice by soiled bedding [370, 371] . mnv infection can be detected directly by rt-pcr on faecal pellets or tissue specimens (see above) and indirectly by serology (mfia, elisa, ifa) [363, 367, 369] . detection is facilitated by high stability of mnv rna in faeces (at least 2 weeks at room temperature) [371] and by broad serological cross-reactivity among different strains of mnv [367, 368] . embryo transfer [370] and hysterectomy [369] are most likely effective means of eliminating mnv from mouse colonies. since 1-to 3-day-old pups are resistant to infection, elimination of mnv may also be achieved by transferring neonates from infected dams to uninfected foster dams ('cross-fostering') [372] . this transfer should ideally be performed within 24 h after birth. mnv is used as a surrogate to evaluate resistance of human noroviruses to disinfectants. the impact of mnv on animal experiments remains to be evaluated. recent studies show that mnv is immunmodulatory and may alter disease phenotypes in mouse models of inflammatory bowel disease [373] [374] [375] and other experimental mouse models [376, 377] . murine pneumonia virus, commonly referred to as 'pneumonia virus of mice' (pvm), is an enveloped, single-stranded rna virus of the family paramyxoviridae, genus pneumovirus. it is closely related to human respiratory syncytial virus (hrsv). the virus name is officially abbreviated as 'mpv' according to the international union of microbiological societies [9] ; however, the former designation 'pvm' will be used in this chapter to avoid confusion with the official abbreviation of mouse parvovirus (mpv). pvm infection remains prevalent in contemporary colonies of mice and rats throughout the world. a serological survey in france demonstrated antibodies to pvm in 16% of mouse colonies examined [10] . in more recent studies in north america and western europe, the prevalence of pvm-specific antibodies in mice ranged between 0% and 0.1% [11, 14, 15] . schoondermark-van de ven et al. [12] found antibodies to pvm in 0.2% of mouse samplings from western european institutions. antibodies to pvm have also been detected in hamsters, gerbils, cotton rats, guinea-pigs and rabbits [8, 378, 379] . experimentally, pvm infection of mice is used as a model for hrsv infection and has therefore been extensively studied (reviewed by rosenberg and domachowske [380] ). in immunocompetent mice, natural infection with pvm is transient and usually not associated with clinical disease or pathological findings [8, 379, 381] . however, natural disease and persistent infection may occur in immunodeficient mice [382] [383] [384] . in particular, athymic foxn1 nu mice seem to be susceptible to pvm infection, which can result in dyspnoea, cyanosis, emaciation and death due to pneumonia [383, 384] . similar clinical signs have been reported for experimentally infected immunocompetent mice [385] . necropsy findings in naturally infected foxn1 nu mice include cachexia and diffuse pulmonary oedema or lobar consolidation [384] . pulmonary consolidation (dark red or grey in color) has also been found after experimental infection of immunocompetent mice [381] . histologically, natural infection of foxn1 nu mice with pvm presents as interstitial pneumonia [383, 384] . experimental intranasal inoculation of immunocompetent mice can result in rhinitis, erosive bronchiolitis and interstitial pneumonia with prominent early pulmonary eosinophilia and neutrophilia [381, 386] . hydrocephalus may result from intracerebral inoculation of neonatal mice [387] . susceptibility to infection is influenced by age and strain of mouse, dose of virus, and a variety of local and systemic stressors [8, 379, 386] . in terms of the extent of the alveolar inflammatory response, 129/sv and dba/2 mice are susceptible to pvm infection, while balb/c and c57bl/6 mice are relatively resistant [386] . in terms of the control of viral replication, mice of strains 129/sv, dba/2, balb/c and c57bl/6 are susceptible to pvm infection, while sjl mice are relatively resistant. pvm is labile in the environment and rapidly inactivated at room temperature [8, 379] . the virus is tropic for the respiratory epithelium [382, 385] , and transmission is exclusively horizontal via the respiratory tract, mainly by direct contact and aerosol [8, 379] . therefore, transmissibility in mouse colonies is low, and infections tend to be focal enzootics. serology (mfia, elisa, ifa or hi) is the primary means of testing mouse colonies for exposure to pvm. immunohistochemistry has been applied to detect viral antigen in lung sections [382, 384] ; however, proper sampling (see chapter 4.4, 'health management and monitoring') is critical for establishing the diagnosis due to the focal nature of the infection. an rt-pcr assay to detect viral rna in respiratory tract tissues has also been reported [388] . however, the use of direct methods requires good timing because the virus is present for only up to about 10 days in immunocompetent mice [381] . embryo transfer or caesarean derivation followed by barrier maintenance can be used to rear mice that are free of pvm. because active infection is present in the individual immunocompetent mouse for only a short period, strict isolation of a few (preferably seropositive) mice with the temporary cessation of breeding might also be successful in eliminating the virus [8, 378] . pvm could interfere with studies involving the respiratory tract or immunological measurements in mice. in addition, pvm can have devastating effects on research using immunodeficient mice because they are particularly prone to develop fatal disease [383, 384] or become more susceptible to the deleterious effects of other agents such as p. murina [389] . murv-a/edim (commonly referred to as 'mouse rotavirus' or 'epizootic diarrhoea of infant mice virus') is a non-enveloped, segmented double-stranded rna virus of the family reoviridae, genus rotavirus. it is antigenically classified as a group a rotavirus, similar to rotaviruses of many other species that cause neonatal and infantile gastroenteritis [291] . murv-a/edim infection remains prevalent in contemporary mouse colonies and appears to occur worldwide. large commercial laboratories found 0.6% to 9% of mouse sera from north american and european facilities to be positive for antibodies against murv-a/edim [11, 12, 14, 15] , and up to 30% of mouse colonies in the usa were identified as affected in a survey performed in 2006 [13] . experimentally, murv-a/edim infection in mice is used as a model for human rotavirus infection, especially in investigations on the mechanisms of rotavirus immunity and in the development of vaccination strategies [390] . clinical symptoms following murv-a/edim infection range from inapparent or mild to severe, sometimes fatal, diarrhoea. 'epizootic diarrhoea of infant mice' describes the clinical syndrome associated with natural or experimental infection by murv-a/edim during the first 2 weeks of life [8, 36, 291, 391, 392] . diarrhoea usually begins around 48 h after infection and persists for about 1 week. affected suckling mice have soft, yellow faeces that wet and stain the perianal region (figure 3.2.8) . in severe instances, the mice may be stunted, have dry scaly skin, or are virtually covered with faecal material. morbidity is very high but mortality is usually low. gross lesions in affected mice are confined to the intestinal tract. the caecum and colon may be distended with gas and watery to paste-like contents that are frequently bright yellow. the stomach of diarrhoeic mice is almost always filled with milk, and this feature has been reported to be a reliable means to differentiate diarrhoea caused by rotavirus from the diarrhoea caused by mhv infection. histopathological changes may be subtle even in animals with significant diarrhoea (figure 3.2.8 ). they are most prominent at the apices of villi, where rotaviruses infect and replicate within epithelial cells; the large intestinal surface mucosa may also be affected. though inflammation is minimal, the lamina propria may be oedematous, lymphatics may be dilated and mild leukocytic infiltration in the large intestinal mucosa and submucosa has been observed in a recent outbreak of disease [36, 393] . hydropic change of villous epithelial cells is the hallmark finding of acute disease. the villi become shortened, and the cells that initially replace the damaged cells are less differentiated, typically cuboidal instead of columnar, and lack a full complement of enzymes for digestion and absorption, resulting in diarrhoea due to maldigestion and malabsorption. undigested milk in the small intestine promotes bacterial growth and exerts an osmotic effect, exacerbating damage to the villi. intestinal fluid and electrolyte secretion is further enhanced by activation of the enteric nervous system [394] and through the effects of a viral enterotoxin called nsp4 (for non-structural protein 4) [395] . it is hypothesized that nsp4 is released from virus-infected cells and then triggers a signal transduction pathway that alters epithelial cell permeability and chloride secretion. susceptibility to edim depends on the age of the host and peaks between 4 and 14 days of age [8, 36, 291, 391, 392] . mice older than about 2 weeks can still be infected with murv-a/edim, but small numbers of enterocytes become infected, there is little replication of virus and diarrhoea does not occur. the exact reason for this agerelated resistance to disease is unknown. pups suckling from immune dams are protected against edim during their period of disease susceptibility [396] . in general, the infection is self-limiting and resolves within days. successful viral control and clearance is promoted by an intact immune response [396] [397] [398] [399] , and some immunodeficient mice (e.g. prkdc scid and rag2 tm1fwa mice) may shed virus for extended periods or become persistently infected [400, 401] . protection against murv-a/edim reinfection is primarily mediated by antibodies [396, 397] . murine rotavirus-a/edim is highly contagious and transmitted by the faecal-oral route [8, 291, 391] . dissemination of the virus occurs through direct contact or contaminated fomites and aerosols and is facilitated by the general property of rotaviruses that they remain infectious outside the body, show resistance to inactivation (e.g. low ph, non-ionic detergents, hydrophobic organic liquids, proteolytic enzymes), and are shed in high quantities (>10 11 particles/g faeces) [291] . murv-a/edim is stable at à70 c but otherwise tends to be susceptible to extreme environmental conditions, detergents and disinfectants containing phenols, chlorine or ethanol [291] . mfia, elisa and ifa are in widespread use for detection of serum antibodies to murv-a/ edim in diagnostic and health surveillance programmes; other assay systems such as those using latex agglutination are also used [402] . as murv-a/edim shares the vp6 protein determined group a antigen, for example, with human, simian or bovine rotavirus strains, commercially available elisa assays utilizing polyclonal or monoclonal antibodies have been used to detect rotavirus antigen in mice; however, great care must be taken in interpreting the results because some feeds have been reported to cause false-positive reactions with certain elisa kits [403] . electron microscopy of faeces of diarrhoeic pups should reveal typical wheel-shaped rotavirus particles, 60-80 nm in diameter. rt-pcr also can be used to detect rotavirus rna in faecal samples [404] . good timing is critical for establishing the diagnosis from faeces because virus is shed for only a few days in immunocompetent mice. embryo transfer or caesarean derivation followed by barrier maintenance is recommended for rederivation of breeding stocks [8] . in immunocompetent mice in which infection is effectively cleared, a breeding suspension strategy for 8-10 weeks combined with excellent sanitation, filter tops and conscientious serological testing of offspring and sentinel mice has also been reported to be effective, and prolongation of breeding cessation up to 12 weeks resolved infection even in immunocompromised mice [393] . murv-a/edim has the potential to interfere with any research using suckling mice. it may have a significant impact on studies where the intestinal tract of neonatal or infant mice is the target organ. the infection also poses a problem for infectious disease and immune response studies, particularly those involving enteropathogens in infant mice [405] . a disease-induced stress-related thymic necrosis may occur and alter immunology experiments [36] . in addition, runting could be interpreted erroneously as the effect of genetic manipulation or other experimental manipulation. sendai virus (sev) is an enveloped, singlestranded rna virus of the family paramyxoviridae, genus respirovirus. it is antigenically related to human parainfluenza virus 1. the virus was named after sendai, japan, where it was first isolated from mice. historically, infections were relatively common in mouse and rat colonies worldwide. in addition, there is evidence that hamsters, guinea-pigs and rabbits are susceptible to infection with sev [8, 301, 406, 407] ; however, some apparently seropositive guinea-pigs may in fact be seropositive to other parainfluenza viruses instead of sev. a study in france reported antibodies to sev in 17% of mouse colonies examined [10] . a low rate of seropositive mice (0.2%) was found in a survey in north america [11] . schoondermark-van de ven et al. [12] also found antibodies to sev in 0.2% of mouse samplings from western european institutions. in more recent surveys in north america and western europe, sev infection was not detected [13] [14] [15] , indicating that sev, like most viruses, has meanwhile been eliminated from the majority of mouse colonies. sev can contaminate biological materials [1] . sev is pneumotropic and can cause significant respiratory disease in mice. the pneumotropism is partially a consequence of the action of respiratory serine proteases such as tryptase clara, which activate viral infectivity by specific cleavage of the viral fusion glycoprotein [408] . in addition, the apical budding behaviour of sev may hinder the spread of virus into subepithelial tissues and subsequently to distant organs via the blood. two epidemiologic patterns of sev infection have been recognized, an enzootic (subclinical) and epizootic (clinically apparent) type [8, 379, 409] . enzootic infections commonly occur in breeding or open colonies, where the constant supply of susceptible animals perpetuates the infection. in breeding colonies, mice are infected shortly after weaning as maternal antibody levels wane. normally, the infection is subclinical, with virus persisting for approximately 2 weeks, accompanied by seroconversion that persists for a year or longer. epizootic infections occur upon first introduction of the virus to a colony and either die out (self-cure) after 2-7 months or become enzootic depending on colony conditions. the epizootic form is generally acute, and morbidity is very high, resulting in nearly all susceptible animals becoming infected within a short time. clinical signs vary and include rough hair coat, hunched posture, chattering, respiratory distress, prolonged gestation, death of neonates and sucklings and runting in young mice. breeding colonies may return to normal productivity within 2 months and thereafter maintain the enzootic pattern of infection. factors such as strain susceptibility, age, husbandry, transport and copathogens are important in precipitating overt disease. dba and 129 strains of mice are very susceptible to sev pneumonia, whereas sjl/j and c57bl/6/j and several outbred stocks are relatively resistant. resistance to sev infection is under multigenic control with epistatic involvement [410] . there is no evidence for persistent infection in immunocompetent mice, but persistent or prolonged infection may occur in immunodeficient mice and can result in wasting and death due to progressive pneumonia [411, 412] . clearance of a primary sev infection is mediated by cd8þ and cd4þ t-cell mechanisms [413, 414] . heavier than normal, consolidated, plumcolored or grey lungs are a characteristic gross finding in severe sev pneumonia [8, 36, 379, 409] . lymphadenopathy and splenomegaly reflect the vigorous immune response to infection. histologically, three phases of disease can be recognized in susceptible immunocompetent mice: acute, reparative and resolution phases [36, 409] . lesions of the acute phase, which lasts 8-12 days, are primarily attributed to the cellmediated immune response that destroys infected respiratory epithelial cells and include necrotizing rhinitis, tracheitis, bronch(iol)itis and alveolitis. epithelial syncytiae and cytoplasmic inclusion bodies in infected cells may be seen early in this phase. alveoli contain sloughed necrotic epithelium, fibrin, neutrophils and mononuclear cells. atelectasis, bronchiectasis and emphysema may occur as a result of damage and obstruction of airways. the reparative phase, which may overlap the acute phase but continues through about the third week after infection, is indicated by regeneration of airway lining epithelium. adenomatous hyperplasia and squamous metaplasia (with multilayered flat epithelial cells instead of normal columnar cells) in the terminal bronchioles and alveoli are considered to be a hallmark of sev pneumonia. mixed inflammatory cell infiltrates in this phase tend to be primarily interstitial, rather than alveolar, as they are in the acute phase. the resolution phase may be complete by the fourth week after infection and lesions may be difficult to subsequently identify. residual, persistent lesions that may occur include organizing alveolitis and bronchiolitis fibrosa obliterans. alveoli and bronchioles are replaced by collagen and fibroblasts, foamy macrophages and lymphoid infiltrates, often with foci of emphysema, cholesterol crystals and other debris, which represent attempts to organize and wall off residual necrotic debris and fibrin. lesions are more severe and variable when additional pathogens such as mycoplasma pulmonis are present [8] . otitis media has also been reported in natural infections with sev although some of these studies have been complicated by the presence of other pathogens [415] . sev has been detected in the inner ear after experimental intracerebral inoculation of neonatal mice [416] . sev is extremely contagious. infectious virus is shed during the first 2 weeks of infection and appears to be transmitted by direct contact, contaminated fomites and respiratory aerosol [8, 379] . serology (mfia, elisa, ifa, or hi) is the approach of choice for routine monitoring because serum antibodies to sev are detectable soon after infection and persist at high levels for many months, although active infection lasts only 1-2 weeks in immunocompetent mice. the short period of active infection limits the utility of direct methods such as immunohistochemistry [382] and rt-pcr [388, 417] . although sev is considered to be highly contagious, studies have shown that dirty bedding sentinel systems do not reliably detect the infection and that outbred stocks may not seroconvert consistently [418, 419] . map testing and rt-pcr can be used to detect sev in contaminated biological materials. sev infection in mouse colonies has proved to be one of the most difficult virus infections to control because the virus is highly infectious and easily disseminated. depopulation of infected colonies is probably the most appropriate means of eliminating the virus in most situations. embryo transfer, or caesarean derivation, followed by barrier maintenance, can also be used to eliminate the virus [8, 379] . a less effective alternative is to place the infected animals under strict quarantine, remove all young and pregnant mice, suspend all breeding and prevent addition of other susceptible animals for approximately 2 months until the infection is extinguished, and then breeding and other normal activities are resumed. vaccines against the virus have been developed [8, 379, 409] , but these probably do not represent a practical means to achieve or maintain the seronegative status of colonies that is in demand today. sev has the potential to interfere with a wide variety of research involving mice. reported effects include interference with early embryonic development and fetal growth; alterations of macrophage, nk-cell, and t-and b-cell function; altered responses to transplantable tumours and respiratory carcinogens; altered isograft rejection; and delayed wound healing (reviewed in [6] [7] [8] ). pulmonary changes during sev infection can compromise interpretation of experimentally induced lesions and may lead to opportunistic infections by other agents. they could also affect the response to anaesthetics. in addition, natural sev infection would interfere with studies using sev as a gene vector. theiler's murine encephalomyelitis virus (tmev), or murine poliovirus, is a member of the genus cardiovirus in the family picornaviridae. members of this genus are non-enveloped viruses with single-stranded rna. the virus is rapidly destroyed at temperatures above 50 c. it is considered to be a primary pathogen of the cns of mice and can cause clinical disease resembling that due to poliomyelitis virus infections in humans. antibodies to tmev have been identified in mouse colonies and feral populations worldwide, and mus musculus is considered to be the natural host of tmev [420] . the best-known and most frequently mentioned tmev strain is gdvii, which is virulent for mice. infant or young hamsters and laboratory rats are also susceptible to intracerebral infection. the original isolate is designated to (theiler's original) and represents a group of tmev strains with low virulence for mice. many additional virus strains have been isolated and studied, and they all fall in the broad grouping of to and gdvii. a similar virus strain has also been isolated from rats, but in contrast to mouse isolates, this virus is not pathogenic for rats and mice after intracerebral inoculation [421] . recently, another rat isolate has been characterized and shown to be most closely related to, but quite distinct from, other tmev viruses [422] . antibodies to tmev (strain gdvii) have been detected in guinea-pigs and are considered to indicate infection with another closely related cardiovirus [423] . seropositivity to tmev was reported in approximately 48% of french mouse colonies in a retrospective study [10] . in more recent studies, the prevalence of tmev infections was found to be lower. schoondermark-van de ven et al. [12] detected antibodies to tmev in 2.2% of mouse samplings from western european institutions. in a survey conducted by carty [13] , about 9% of responding institutions in the usa reported tmev infection in their mouse colonies. further surveys in north america and western europe revealed antibodies in 0.09-0.26% of mice monitored [11, 14, 15] . tmev is primarily an enteric pathogen, and virus strains are enterotropic. in natural infections, virus can be detected in intestinal mucosa and faecal matter, and in some cases it is also found in the mesenteric lymph nodes. however, histological lesions in the intestine are not discerned. virus may be shed via intestinal contents for up to 22 weeks, sometimes intermittently [424] , and transmission under natural conditions is via the faecal-oral route, by direct contact between mice, as well as by indirect contact (e.g. dirty bedding). the host immune response limits virus spread, but it does not immediately terminate virus replication in the intestines. virus is cleared from extraneural tissues, but persists in the cns for at least a year. clinical disease due to natural tmev infection is rare, with a rate of only 1 in 1000-10 000 infected immunocompetent animals [36] . in immunodeficient mice, especially in weanlings, clinical signs may be more common and mortality may be higher [425] . this group of viruses usually causes asymptomatic infections of the intestinal tract. they may spread to the cns as a rare event where they cause different neurological disease manifestations. the most typical clinical sign of tmev infection is flaccid paralysis of hindlimbs. the animals appear otherwise healthy, and there is no mortality. experimental infection in mice provides models of poliomyelitis-like infection and virusinduced demyelinating disease including multiple sclerosis [426] . after experimental infection, tmev causes a biphasic disease in susceptible strains of mice. the acute phase is characterized by early infection of neurons in the grey matter. encephalomyelitis may develop during this phase and may be fatal, but most animals survive and enter the second phase of the disease at 1-3 months after the acute phase. this phase is characterized by viral persistence in the spinal cord white matter, mainly in macrophages, and leads to white matter demyelination. persistence and demyelination occur only in genetically susceptible mouse strains, while resistant strains clear the infection after early grey matter encephalomyelitis through a cytotoxic t lymphocyte response. the severity and nature of disease depend on virus strain, route of inoculation, host genotype and age [8, 36, 427] . in general, virus isolates with low virulence produce persistent cns infection in mice whereas virulent strains are unable to cause persistent infection. intracerebral inoculation results in the most severe infections, but the intranasal route is also effective. experimental intracerebral infections with virulent fa and gdvii strains of tmev are more likely to cause acute encephalomyelitis and death in weanling mice 4-5 days after inoculation ('early disease'). death may be preceded by neurological manifestations of encephalitis such as hyperexcitability, convulsions, tremors, circling, rolling and weakness. animals may develop typical flaccid paralysis of hindlimbs, and locomotion is possible only by use of the forelimbs. interestingly, the tail is not paralyzed. experimental infections with low-virulence virus strains (e.g. to, da, ww) are more likely to cause persistent infection with development of mild encephalomyelitis followed by a chronic demyelinating disease after a few months ('late disease'). these virus strains infect neurons in the grey matter of the brain and spinal cord during the acute phase of viral growth, followed by virus persistence in macrophages and glial cells in the spinal cord white matter. sjl, swr and dba/2 strains are most susceptible to this chronic demyelinating disease. cba and c3h/he are less susceptible strains, and strains a, c57bl/6, c57bl/10 and dba/1 are relatively resistant [428] . differences in humoral immune responses play a role in resistance to tmev infection [429] , but genetic factors are also important. several genetic loci implicated in susceptibility to virus persistence, demyelination, or clinical disease have been identified, including the h-2d region of the major histocompatibility complex [430] . furthermore, the age at infection influences the severity of clinical disease. in infant mice, intracerebral infection with low-virulence virus strains (e.g. to) is often lethal. young mice develop paralysis after an incubation period of 1-4 weeks while adult mice often show no clinical signs of infection. the only gross lesions are secondary to the posterior paralysis and may include urine scald or dermatitis due to incontinence of urine and trauma to paralyzed limbs, or wasting or atrophy of the hindlimbs in long-term survivors. tmev infects neurons and glial cells, and histological changes in the cns include nonsuppurative meningitis, perivasculitis and poliomyelitis with neuronolysis, neuronophagia and microgliosis in the brainstem and ventral horns of the spinal cord [36] . demyelination in immunocompetent mice is considered to be immunemediated. susceptible strains develop a specific delayed-type hypersensitivity response which is the basis for inflammation and demyelination. this reaction is mediated by t cells that release cytokines leading to recruitment of monocytes and macrophages as a consequence of infection of macrophages and other cns-resident cells [431] [432] [433] . protection from chronic demyelinating disease is possible by vaccination with live virus given previously by subcutaneous or intraperitoneal inoculation [434, 435] . early immunosuppression at the time of infection, e.g. by treatment with cyclophosphamide or antithymocyte serum, inhibits or diminishes demyelination. immunosuppression in mice chronically infected with tmev leads to remyelination of oligodendrocytes [436] . further details related to the pathogenesis of tmev infections and the role of immune mechanisms have been reviewed by yamada et al. [437] , kim et al. [432] and lipton et al. [433] . experimental infection of foxn1 nu mice results in acute encephalitis and demyelination. demyelination associated with minimal inflammation and neurological signs, including the typical hindlimb paresis, develop 2 weeks after inoculation, and most animals die within 4 weeks. in foxn1 nu mice, demyelination is caused by a direct lytic effect of the virus on oligodendrocytes [438] . demyelination and lethality are reduced after administration of neutralizing antibodies [439] . histopathological changes in prkdc scid mice are very similar to those in foxn1 nu mice [440] . young mice born in infected populations usually acquire infection shortly after weaning and are almost all infected by 30 days of age. intrauterine transmission to fetuses is possible during the early gestation period, but a placental barrier develops during gestation and later prevents intrauterine infection [441] . all tmev isolates are closely related antigenically and form a single serogroup, as determined by complement fixation and hi [427] . hemelt et al. 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phenotype different roles for cd4þ and cd8þ t lymphocytes and macrophage subsets in the control of a generalized virus infection correlates of protective immunity in poxvirus infection: where does antibody stand? transmission of mouse-pox in colonies of mice mousepox in inbred mice innately resistant or susceptible to lethal infection with ectromelia virus. iii. experimental transmission of infection and derivation of virus-free progeny from previously infected dams intrauterine infection of mice with ectromelia virus mouse pox threat serological detection of ectromelia virus antibody evaluation of an enzyme-linked immunosorbent assay for the detection of ectromelia (mousepox) antibody administration of vaccinia virus to mice may cause contact or bedding sentinel mice to test positive for orthopoxvirus antibodies: case report and follow-up investigation specific detection of mousepox virus by polymerase chain reaction real-time pcr system for detection of orthopoxviruses and simultaneous identification of smallpox virus detection of human orthopoxvirus infections and differentiation of smallpox virus with real-time pcr observations on the replication of ectromelia virus in mouse-derived cell lines: implications for epidemiology of mousepox reexamination of the efficacy of vaccination against mousepox effect of vaccination on the clinical response, pathogenesis and transmission of mousepox pathogenesis of vaccinia (ihd-t) virus infection in balb/cann mice mouse models for studying orthopoxvirus respiratory infections animal models of orthopoxvirus infection ectromelia virus: the causative agent of mousepox a protein-based smallpox vaccine protects mice from vaccinia and ectromelia virus challenges when given as a prime and single boost postexposure immunization with modified vaccinia virus ankara or conventional lister vaccine provides solid protection in a murine model of human smallpox mousepox in the c57bl/6 strain provides an improved model for evaluating antipoxvirus therapies a new mouse virus apparently related to the adenovirus group an adenovirus isolated from the feces of mice i. isolation and identification genotypic differences between the mouse adenovirus strains fl and k87 molecular cloning, physical mapping and cross-hybridization of the murine adenovirus type 1 and type 2 genomes genetic relationship between mouse adenovirus-2 (strain k87) and human adenovirus-2 mouse adenoviruses microbiological contamination of laboratory mice and rats in korea from 1999 to a serologic survey for viruses and mycoplasma pulmonis among wild house mice (mus domesticus) in southeastern australia comparative biological characterization of mouse adenovirus strains fl and k 87 and seroprevalence in laboratory rodents pathogenesis of experimentally produced mouse adenovirus infection in mice age and susceptibility of swiss mice for mouse adenovirus, strain fl mouse adenovirus type 1 causes a fatal hemorrhagic encephalomyelitis in adult c57bl/6 but not balb/c mice duodenal lesions associated with adenovirus infection in athymic 'nude' mice murine adenovirus infection of scid mice induces hepatic lesions that resemble human reye syndrome experimental adenovirus infection of the mouse adrenal gland. i. light microscopic observations electron microscope study of experimental enteric adenovirus infection in mice experimental infection with mouse adenovirus in adult mice intestinal resistance in the experimental enteric infection of mice with a mouse adenovirus. i. growth of the virus and appearance of a neutralizing substance in the intestinal tract fluctuation of antiviral resistance in the intestinal tracts of nude mice infected with a mouse adenovirus biological and biophysical characteristics of mouse adenovirus, strain fl serological relationship between mouse adenovirus strains fl and k87 a naturally occurring intestinal mouse adenovirus infection associated with negative serologic findings acceleration of scrapie disease in mice by an adenovirus a novel cardiotropic murine adenovirus representing a distinct species of mastadenoviruses serological diagnosis of murine adenovirus 3 characterization of k virus and its comparison with polyoma virus murine pneumotropic virus vp1 virus-like particles (vlps) bind to several cell types independent of sialic acid residues and do not serologically cross react with murine polyomavirus vp1 vlps recommendations for the health monitoring of rodent and rabbit colonies in breeding and experimental units polyoma viruses the major site of murine k papovavirus persistence and reactivation is the renal tubular epithelium distribution of k-papovavirus in infected newborn mice morphological and immunohistochemical studies of the central nervous system involvement in papovavirus k infection in mice chronic infection of nude mice by murine k papovavirus serial passage of murine k-papovavirus in primary cultures of mouse embryo cells comparison of an enzymelinked immunosorbent assay, an immunofluorescence assay and a hemagglutination inhibition assay for detection of antibodies to k-papovavirus in mice diagnostic polymerase chain reaction assays for identification of murine polyomaviruses in biological samples a model for mixed virus disease: co-infection with moloney murine leukemia virus potentiates runting induced by polyomavirus detection of dna and rna virus genomes in organ systems of whole mice: patterns of mouse organ infection by polyomavirus transplacental transmission of polyoma virus in mice persistence of polyomavirus in adult scid c.b-17 mice immunosuppression and murine polyomavirus infection reactivation of polyoma virus in kidneys of persistently infected mice during pregnancy ifn-g controls mouse polyomavirus infection in vivo heterogeneity among viral antigen-specific cd4þ t cells and their de novo recruitment during persistent polyomavirus infection long-term infection of adult mice with murine polyomavirus following stereotaxic inoculation into the brain murine polyomavirus virus-like particles (vlps) as vectors for gene and immune therapy and vaccines against viral infections and cancer cd4þ, and cd8þ t cells can act separately in tumour rejection after immunization with murine pneumotropic virus chimeric her2/neu virus-like particles molecular characterization of a newly recognized mouse parvovirus the rodent parvoviruses viral and mycoplasmal infections of laboratory rodents: effects on biomedical research the mouse in biomedical research. diseases lurking in the shadows: emerging rodent infectious diseases coping with parvovirus infections in mice: health surveillance and control identification and propagation of a putative immunosuppressive orphan parvovirus in cloned t cells molecular characterization of newly recognized rodent parvoviruses identification of novel murine parvovirus strains by epidemiological analysis of naturally infected mice temporal transmission studies of mouse parvovirus 1 in balb/c and c.b-17/ icr-prkdc scid mice serologic prevalence of mpv1 in mouse strains in a commercial laboratory mouse colony determined by using vp1 antigen serodiagnosis of mice minute virus and mouse parvovirus infections in mice by enzyme-linked immunosorbent assay with baculovirus-expressed recombinant vp2 proteins in vivo studies with an 'orphan' parvovirus of mice characterization of mouse parvovirus infection by in situ hybridization humoral immunity and protection of mice challenged with homotypic or heterotypic parvovirus experimental infection of mice with hamster parvovirus: evidence for interspecies transmission of mouse parvovirus 3 effect of mouse strain and age on detection of mouse parvovirus 1 by use of serologic testing and polymerase chain reaction analysis strainand age-associated variation in viral persistence and antibody response to mouse parvovirus 1 in experimentally infected mice characterization of mouse parvovirus infection among balb/c mice from an enzootically infected colony expression of recombinant parvovirus ns1 protein by a baculovirus and application to serologic testing of rodents validation of an enzyme-linked immunosorbent assay for detection of mouse parvovirus infection in laboratory mice detection of newly recognized rodent parvoviruses by pcr detection of rodent parvoviruses by use of fluorogenic nuclease polymerase chain reaction assays antemortem detection of mouse parvovirus and mice minute virus by polymerase chain reaction (pcr) of faecal samples polymerase chain reaction for detection of rodent parvoviral contamination in cell lines and transplantable tumors embryo transfer rederivation of c.b-17/icr-prkdc scid mice experimentally infected with mouse parvovirus 1 detection of mouse parvovirus in mus musculus gametes, embryos, and ovarian tissues by polymerase chain reaction assay mouse parvovirus infection potentiates allogeneic skin graft rejection and induces syngeneic graft rejection a minute virus of mice immunosuppressive activity of a subline of the mouse el-4 lymphoma. evidence for minute virus of mice causing the inhibition pathogenicity of fibroblastand lymphocyte-specific variants of minute virus of mice pathogenesis of infection with a virulent allotropic variant of minute virus of mice and regulation by host genotype minute virus of mice. ii. prevalence, epidemiology, and occurrence as a contaminant of transplanted tumors electron microscopic localization of virions in developing teeth of young hamsters infected with minute virus of mice experimentally induced infection with autonomous parvoviruses, minute virus of mice and h-1, in the african multimammate mouse (mastomys coucha) pathogenicity of minute virus of mice (mvm) for rats, mice, and hamsters fetal infections of hamsters, rats, and mice induced with the minute virus of mice (mvm) in vitro myelosuppressive effects of the parvovirus minute virus of mice (mvmi) on hematopoietic stem and committed progenitor cells myeloid depression follows infection of susceptible newborn mice with the parvovirus minute virus of mice (strain i) severe leukopenia and dysregulated erythropoiesis in scid mice persistently infected with the parvovirus minute virus of mice reduced fecundity and death associated with parvovirus infection in b-lymphocyte deficient mice minute virus of mice: antibody response, viral shedding, and persistence of viral dna in multiple strains of mice presence of minute virus of mice in immunocompetent mice despite the onset of host immunity gender influences infectivity in c57bl/6 mice exposed to mouse minute virus a rapid and simple procedure to detect the presence of mvm in conditioned cell fluids or culture media risk assessment of minute virus of mice transmission during rederivation: detection in reproductive organs, gametes, and embryos of mice after in vivo infection transmission of mouse minute virus (mmv) but not mouse hepatitis virus (mhv) following embryo transfer with experimentally exposed in vivo-derived embryos antineoplastic activity of parvoviruses experience with viral contamination in cell culture the molecular biology of arteriviruses lactic dehydrogenase virus isolation of lactate dehydrogenase-elevating viruses from wild house mice and their biological and molecular characterization lactate dehydrogenaseelevating virus induces systemic lymphocyte activation via tlr7-dependent ifnalpha responses by plasmacytoid dendritic cells distinct gamma interferon-production pathways in mice infected with lactate dehydrogenase-elevating virus lactate dehydrogenase-elevating virus replication persists in liver, spleen, lymph node, and testis tissues and results in accumulation of viral rna in germinal centers, concomitant with polyclonal activation of b cells transmissible agent associated with 26 types of experimental mouse neoplasms from bench to cageside: risk assessment for rodent pathogen contamination of cells and biologics lactic dehydrogenase virus (ldhv) contamination in human tumor xenografts and its elimination contamination of a monoclonal antibody with ldh-virus causes interferon induction infection of central nervous system cells by ecotropic murine leukemia virus in c58 and akr mice and in in utero-infected ce/j mice predisposes mice to paralytic infection by lactate dehydrogenase-elevating virus lactate dehydrogenase-elevating virus regulation of transplacental virus infection by developmental and immunological factors: studies with lactate dehydrogenaseelevating virus transplacental lactate dehydrogenase-elevating virus (ldv) transmission: immune inhibition of umbilical cord infection, and correlation of fetal virus susceptibility with development of f4/80 antigen expression regulation of maternal-fetal virus transmission in immunologically reconstituted scid mice infected with lactate dehydrogenase-elevating virus immunofluorescent antibody response to lactic dehydrogenase virus in different strains of mice characterization of lactate dehydrogenase-elevating virus orf6 protein expressed by recombinant baculoviruses enzymatic amplification of lactate dehydrogenase-elevating virus detection of lactate dehydrogenase-elevating virus in transplantable mouse tumors by biological assay and rt-pcr assays and its removal from the tumor cell false negative results using rt-pcr for detection of lactate dehydrogenase-elevating virus in a tumor cell line comparison of the sensitivity of in vivo antibody production tests with in vitro pcr-based methods to detect infectious contamination of biological materials detection and typing of lactate dehydrogenase-elevating virus rna from transplantable tumors, mouse liver tissues, and cell lines, using polymerase chain reaction comparison of the mouse antibody production (map) assay and polymerase chain reaction (pcr) assays for the detection of viral contaminants relationship between the lactic dehydrogenase-elevating virus and transplantable murine tumors removal of lactate dehydrogenase-elevating virus from human-in-mouse breast tumor xenografts by cell-sorting infection of mice with lactate dehydrogenase-elevating virus leads to stimulation of autoantibodies suppression of development of diabetes in nod mice by lactate dehydrogenase virus infection natural killer cell activation after infection with lactate dehydrogenase-elevating virus effects of various adjuvants and a viral infection on the antibody specificity toward native or cryptic epitopes of a protein antigen lactate dehydrogenase-elevating virus infection at the sensitization and challenge phases reduces the development of delayed eosinophilic allergic rhinitis in balb/c mice suppression of acute anti-friend virus cd8þ t-cell responses by coinfection with lactate dehydrogenase-elevating virus mammalian reservoirs of arenaviruses risk to humans through contact with golden hamsters carrying lymphocytic choriomeningitis virus (author's transl) lymphocytic choriomeningitis virus first outbreak of callitrichid hepatitis in germany: genetic characterization of the causative lymphocytic choriomeningitis virus strains arenavirus-mediated liver pathology: acute lymphocytic choriomeningitis virus infection of rhesus macaques is characterized by high-level interleukin-6 expression and hepatocyte proliferation lymphocytic choriomeningitis virus spatial and temporal dynamics of lymphocytic choriomeningitis virus in wild rodents, northern italy antibodies to lymphocytic choriomeningitis virus in wild rodent sera in egypt seroepidemiological survey of lymphocytic choriomeningitis virus in wild house mice in china with particular reference to their subspecies lymphocytic choriomeningitis virus infection and house mouse (mus musculus) distribution in urban baltimore contamination of transplantable murine tumors with lymphocytic choriomeningitis virus human-rodent contact and infection with lymphocytic choriomeningitis and seoul viruses in an inner-city population seroprevalence of lymphocytic choriomeningitis virus in nova scotia lymphocytic choriomeningitis virus infection in a province of spain: analysis of sera from the general population and wild rodents mouse-tohuman transmission of variant lymphocytic choriomeningitis virus exposure to lymphocytic choriomeningitis virus lymphocytic choriomeningitis outbreak associated with nude mice in a research institute laboratory studies of a lymphocytic choriomeningitis virus outbreak in man and laboratory animals lymphocytic choriomeningitis virus in southern france: four case reports and a review of the literature lymphocytic choriomeningitis in laboratory personnel exposed to hamsters inadvertently infected with lcm virus pet rodents and fatal lymphocytic choriomeningitis in transplant patients outbreak of lymphocytic choriomeningitis virus infections in medical center personnel virus zoonoses and their potential for contamination of cell cultures transmission of lymphocytic choriomeningitis virus by organ transplantation lymphocytic choriomeningitis virus: an unrecognized teratogenic pathogen lymphocytic choriomeningitis virus: reemerging central nervous system pathogen lymphocytic choriomeningitis virus: emerging fetal teratogen persistence of lymphocytic choriomeningitis virus at very low levels in immune mice mechanisms of antibody-mediated protection against lymphocytic choriomeningitis virus infection: mother-to-baby transfer of humoral protection murine hepatitis caused by lymphocytic choriomeningitis virus. ii. cells involved in pathogenesis biology and pathogenesis of lymphocytic choriomeningitis virus infection lymphocytic choriomeningitis infection of the central nervous system murine infection with lymphocytic choriomeningitis virus following gastric inoculation timed appearance of lymphocytic choriomeningitis virus after gastric inoculation of mice lymphocytic choriomeningitis infection undetected by dirty-bedding sentinel monitoring and revealed after embryo transfer of an inbred strain derived from wild mice detection of the antibody to lymphocytic choriomeningitis virus in sera of laboratory rodents infected with viruses of laboratory and newly isolated strains by elisa using purified recombinant nucleoprotein development of a reverse transcription-polymerase chain reaction assay for diagnosis of lymphocytic choriomeningitis virus infection and its use in a prospective surveillance study detection of lymphocytic choriomeningitis virus by use of fluorogenic nuclease reverse transcriptase-polymerase chain reaction analysis quantitative pcr technique for detecting lymphocytic choriomeningitis virus in vivo centers for disease control and prevention and national institutes of health mechanisms of humoral immunity explored through studies of lcmv infection lymphocytic choriomeningitis virus and immunology viral persistence: parameters, mechanisms and future predictions stage of primary infection with lymphocytic choriomeningitis virus determines predisposition or resistance of mice to secondary bacterial infections reovirus type 3 infection, liver, mouse the mouse in biomedical research. diseases reovirus infection in laboratory rodents direct spread of reovirus from the intestinal lumen to the central nervous system through vagal autonomic nerve fibers type 3 reovirus neuroinvasion after intramuscular inoculation: direct invasion of nerve terminals and age-dependent pathogenesis reoviruses and the host cell orthoreoviruses and their replication passive immunity to fatal reovirus serotype 3-induced meningoencephalitis mediated by both secretory and transplacental factors in neonatal mice infectivity, disease patterns, and serologic profiles of reovirus serotypes 1, 2, and 3 in infant and weanling mice reovirus-induced liver disease in severe combined immunodeficient (scid) mice. a model for the study of viral infection, pathogenesis, and clearance histopathological characterization of the naturally occurring hepatotropic virus infections of nude mice detection methods for the identification of rodent viral and mycoplasmal infections reverse transcriptionpolymerase chain reaction detection and nucleic acid sequence confirmation of reovirus infection in laboratory mice with discordant serologic indirect immunofluorescence assay and enzyme-linked immunosorbent assay results diagnosis of murine infections in relation to test methods employed reovirus 3 not detected by reverse transcriptase-mediated polymerase chain reaction analysis of preserved tissue from infants with cholestatic liver disease detection of reovirus type 3 by use of fluorogenic nuclease reverse transcriptase polymerase chain reaction detection of reovirus by reverse transcription-polymerase chain reaction using primers corresponding to conserved regions of the viral l1 genome segment isolation of a non-pathogenic tumour-destroying virus from mouse ascites an oncolytic virus recovered from swiss mice during passage of an ascites tumour mouse hepatitis virus enterotropic mouse hepatitis virus effects of air temperature and relative humidity on coronavirus survival on surfaces asymptomatic infection of mouse hepatitis virus in the rat effects of experimental infection of the deer mouse (peromyscus maniculatus) with mouse hepatitis virus isolation of a latent murine hepatitis virus from cultured mouse liver cells induction of lytic plaques by murine leukemia virus in murine sarcoma virus-transformed nonproducer mouse cells persistently infected with mouse hepatitis virus mhv-s mouse hepatitis virus biology and epizootiology the cellular and molecular pathogenesis of coronaviruses enterotropic coronavirus (mouse hepatitis virus) in mice: influence of host age and strain on infection and disease response of genetically susceptible and resistant mice to intranasal inoculation with mouse hepatitis virus jhm duration of mouse hepatitis virus infection: studies in immunocompetent and chemically immunosuppressed mice effective clearance of mouse hepatitis virus from the central nervous system requires both cd4þ and cd8þ t cells role of cd4þ and cd8þ t cells in mouse hepatitis virus infection in mice antibody prevents virus reactivation within the central nervous system mouse hepatitis virus enterotropic mouse hepatitis virus infection in nude mice persistent transmission of mouse hepatitis virus by transgenic mice duration of challenge immunity to coronavirus jhm in mice virus strain specificity of challenge immunity to coronavirus duration and strain-specificity of immunity to enterotropic mouse hepatitis virus passively acquired challenge immunity to enterotropic coronavirus in mice epizootic coronaviral typhlocolitis in suckling mice isolation of mouse hepatitis virus from infant mice with fatal diarrhea thymus involution induced by mouse hepatitis virus a59 in balb/c mice adverse effects of mouse hepatitis virus on ascites myeloma passage in the balb/ej mouse murine hepatitis virus strain 1 produces a clinically relevant model of severe acute respiratory syndrome in a/j mice tolllike receptor 4 deficiency increases disease and mortality after mouse hepatitis virus type 1 infection of susceptible c3h mice granulomatous peritonitis and pleuritis in interferon-gamma gene knockout mice naturally infected with mouse hepatitis virus pathogenesis of enterotropic mouse hepatitis virus in immunocompetent and immunodeficient mice vertical transmission of mouse hepatitis virus infection in mice tissue distribution and duration of mouse hepatitis virus in naturally infected immunocompetent icr (cd-1) and immunodeficient athymic nudenu mouse strains used for ovarian transplantation and in vitro fertilization rederivation of inbred strains of mice by means of embryo transfer risk assessment of mouse hepatitis virus infection via in vitro fertilization and embryo transfer by the use of zona-intact and laser-microdissected oocytes mouse hepatitis virus immunofluorescence in formalin-or bouin's-fixed tissues using trypsin digestion comparison of isolation in cell culture with conventional and modified mouse antibody production tests for detection of murine viruses monoclonal antibody solution hybridization assay for detection of mouse hepatitis virus infection detection of rodent coronaviruses in tissues and cell cultures by using polymerase chain reaction sequence analysis and molecular detection of mouse hepatitis virus using the polymerase chain reaction detection of mouse hepatitis virus by the polymerase chain reaction and its application to the rapid diagnosis of infection detection of rodent coronaviruses by use of fluorogenic reverse transcriptase-polymerase chain reaction analysis an immunofluorescence test for detection of serum antibody to rodent coronaviruses simultaneous detection of antibodies to mouse hepatitis virus recombinant structural proteins by a microsphere-based multiplex fluorescence immunoassay differences in antibody production against mouse hepatitis virus (mhv) among mouse strains maternally-derived passive immunity to enterotropic mouse hepatitis virus mouse hepatitis virus: molecular biology and implications for pathogenesis maintenance of pluripotency in mouse embryonic stem cells persistently infected with murine coronavirus replication of murine coronaviruses in mouse embryonic stem cell lines. in vitro reliability of soiled bedding transfer for detection of mouse parvovirus and mouse hepatitis virus efficacy of three microbiological monitoring methods in a ventilated cage rack rederivation of mhv and mev antibody positive mice by cross-fostering and use of the microisolator caging system elimination of mouse hepatitis virus from a breeding colony by temporary cessation of breeding evolution of mouse hepatitis virus (mhv) during chronic infection: quasispecies nature of the persisting mhv rna the elimination of mouse hepatitis virus by temporary transplantation of human tumors from infected athymic nude mice into athymic nude rats (rnun/rnun) development of a microsphere-based serologic multiplexed fluorescent immunoassay and a reverse transcriptase pcr assay to detect murine norovirus 1 infection in mice stat1-dependent innate immunity to a norwalk-like virus murine norovirus 1 infection is associated with histopathological changes in immunocompetent hosts, but clinical disease is prevented by stat1-dependent interferon responses replication of norovirus in cell culture reveals a tropism for dendritic cells and macrophages persistent infection with and serologic cross-reactivity of three novel murine noroviruses murine noroviruses comprising a single genogroup exhibit biological diversity despite limited sequence divergence molecular detection of murine norovirus from experimentally and spontaneously infected mice naturally occurring murine norovirus infection in a large research institution soiled-bedding sentinel detection of murine norovirus 4 the use of cross-foster rederivation to eliminate murine norovirus, helicobacter spp., and murine hepatitis virus from a mouse colony impact of murine norovirus on a mouse model of ibd virus-plus-susceptibility gene interaction determines crohn's disease gene atg16l1 phenotypes in intestine murine norovirus: an intercurrent variable in a mouse model of bacteria-induced inflammatory bowel disease investigation of the impact of the common animal facility contaminant murine norovirus on experimental murine cytomegalovirus infection effects of murine norovirus infection on a mouse model of diet-induced obesity and insulin resistance mouse adenovirus, k virus, and pneumonia virus of mice sendai virus and pneumonia virus of mice (pvm) pneumonia virus of mice: severe respiratory infection in a natural host pneumonia virus of mice infection, lung, mouse, and rat persistence of pneumonia virus of mice and sendai virus in germ-free (nu/nu) mice fatal pneumonia with terminal emaciation in nude mice caused by pneumonia virus of mice respiratory disease and wasting in athymic mice infected with pneumonia virus of mice pathogenesis of pneumovirus infections in mice: detection of pneumonia virus of mice and human respiratory syncytial virus mrna in lungs of infected mice by in situ hybridization differential resistance/susceptibility patterns to pneumovirus infection among inbred mouse strains experimental pneumovirus infections: 1. hydrocephalus of mice due to infection with pneumonia virus of mice (pvm) detection of sendai virus and pneumonia virus of mice by use of fluorogenic nuclease reverse transcriptase polymerase chain reaction analysis lethal exacerbation of pneumocystis murina. pneumonia in severe combined immunodeficiency mice after infection by pneumonia virus of mice handbook of animal models of infection mouse rotavirus murine rotavirus infection, mouse successful sanitation of an edim-infected mouse colony by breeding cessation role of the enteric nervous system in the fluid and electrolyte secretion of rotavirus diarrhoea age-dependent diarrhoea induced by a rotaviral nonstructural glycoprotein the immunology of rotavirus infection in the mouse murine model of rotavirus infection evidence that resolution of rotavirus infection in mice is due to both cd4 and cd8 celldependent activities ifn-l determines the intestinal epithelial antiviral host defense persistent rotavirus infection in mice with severe combined immunodeficiency role of b cells and cytotoxic t lymphocytes in clearance of and immunity to rotavirus infection in mice comparison of methods for detection of serum antibody to murine rotavirus identification of nonspecific reactions in laboratory rodent specimens tested by rotazyme rotavirus elisa removal of inhibitory substances from human fecal specimens for detection of group a rotaviruses by reverse transcriptase and polymerase chain reactions synergistic rotavirus and escherichia coli diarrhoeal infection of mice infection of rabbits with sendai virus pathogenesis of sendai virus infection in the syrian hamster determinants of organ tropism of sendai virus sendai virus infection, lung, mouse, and rat multigenic control of resistance to sendai virus infection in mice naturally-occurring sendai virus infection of athymic nude mice signs and lesions of experimental sendai virus infection in two genetically distinct strains of scid/beige mice cooperation between cytotoxic and helper t lymphocytes in protection against lethal sendai virus infection. protection by t cells is mhc-restricted and mhc-regulated; a model for mhc-disease associations delayed clearance of sendai virus in mice lacking class i mhc-restricted cd8þ t cells naturally occurring sendai virus disease of mice affinity of sendai virus for the inner ear of mice detection of nucleoprotein gene of sendai virus in the lungs of rats by touchdown nested reverse transcription polymerase chain reaction the effectiveness of a microisolator cage system and sentinel mice for controlling and detecting mhv and sendai virus infections the efficacy of a dirty bedding sentinel system for detecting sendai virus infection in mice: a comparison of clinical signs and seroconversion serological evidence that mus musculus is the natural host of theiler's murine encephalomyelitis virus comparison of mhg virus with mouse encephalomyelitis viruses genetic analysis of a theiler-like virus isolated from rats a serological indication of the existence of a guineapig poliovirus duration and patterns of transmission of theiler's mouse encephalomyelitis virus infection a spontaneous outbreak of theiler's encephalomyelitis in a colony of severe combined immunodeficient mice in the uk axonal loss results in spinal cord atrophy, electrophysiological abnormalities and neurological deficits following demyelination in a chronic inflammatory model of multiple sclerosis the theiler's murine encephalomyelitis viruses susceptibility of inbred mice to chronic central nervous system infection by theiler's murine encephalomyelitis virus role of the humoral immune response in resistance to theiler's virus infection the genetics of the persistent infection and demyelinating disease caused by theiler's virus infection with theiler's murine encephalomyelitis virus directly induces proinflammatory cytokines in primary astrocytes via nf-kappab activation: potential role for the initiation of demyelinating disease innate immune response induced by theiler's murine encephalomyelitis virus infection cardioviruses: encephalomyocarditis virus and theiler's murine encephalomyelitis virus effect of immunization with theiler's virus on the course of demyelinating disease protection of sjl/j mice from demyelinating disease mediated by theiler's murine encephalomyelitis virus immunosuppression promotes cns remyelination in chronic virus-induced demyelinating disease pathogenesis of theiler's murine encephalomyelitis virus mechanism of theiler's virus-induced demyelination in nude mice survival of athymic (nu/nu) mice after theiler's murine encephalomyelitis virus infection by passive administration of neutralizing monoclonal antibody vacuolar neuronal degeneration in the ventral horns of scid mice in naturally occurring theiler's encephalomyelitis evolution of the placental barrier to fetal infection by murine enteroviruses enhanced detection of theiler's virus rna copy equivalents in the mouse central nervous system by real-time rt-pcr spontaneous demyelinating myelopathy in aging laboratory mice key: cord-319933-yp9ofhi8 authors: ruiz, sara i.; zumbrun, elizabeth e.; nalca, aysegul title: chapter 38 animal models of human viral diseases date: 2013-12-31 journal: animal models for the study of human disease doi: 10.1016/b978-0-12-415894-8.00038-5 sha: doc_id: 319933 cord_uid: yp9ofhi8 abstract as the threat of exposure to emerging and reemerging viruses within a naive population increases, it is vital that the basic mechanisms of pathogenesis and immune response be thoroughly investigated. by using animal models in this endeavor, the response to viruses can be studied in a more natural context to identify novel drug targets, and assess the efficacy and safety of new products. this is especially true in the advent of the food and drug administration's animal rule. although no one animal model is able to recapitulate all the aspects of human disease, understanding the current limitations allows for a more targeted experimental design. important facets to be considered before an animal study are the route of challenge, species of animals, biomarkers of disease, and a humane endpoint. this chapter covers the current animal models for medically important human viruses, and demonstrates where the gaps in knowledge exist. well-developed animal models are necessary to understand the disease progression, pathogenesis, and immunologic responses in humans. furthermore, to test vaccines and medical countermeasures, well-developed animal models are essential for preclinical studies. ideally, an animal model of human viral infection should mimic the host-pathogen interactions and the disease progression that is seen in the natural disease. a good animal model of viral infection should allow many parameters of infection to be assayed, including clinical signs, growth of virus, clinicopathological parameters, cellular and humoral immune responses, and virus-host interactions. furthermore, viral replication should be accompanied by measurable clinical manifestations, and pathology should resemble that of human cases such that a better understanding of the disease process in humans is attained. there is often more than one animal model that closely represents human disease for a given pathogen. small animal models are typically used for first-line screening, and for initially testing the efficacy of vaccines or therapeutics. in contrast, nonhuman primate (nhp) models are often used for the pivotal preclinical studies. this approach is also used for basic pathogenesis studies, with most experiments performed in small animal models when possible, and nhps only used to fill in remaining gaps in knowledge. the advantages of using mice to develop animal models are low cost, low genetic variability in inbred strains, and abundant molecular biological and immunological reagents. specific pathogen-free (spf), transgenic, and knockout mice are also available. a major pitfall of mouse models is that the pathogenesis and protection afforded by vaccines and therapeutics cannot always be extrapolated. additionally, blood volumes for sampling are limited in small animals, and viruses often need to be adapted through serial passage in the species to induce a productive infection. the ferret's airways are anatomically and histologically similar to that of humans, and their size enables larger or more frequent blood samples to be collected, making them an ideal model for certain respiratory pathogens. ferrets are outbred, with no standardized breeds or strains; thus, greater numbers are required in studies to achieve statistical significance and overcome the resulting variable responses. additionally, spf and transgenic animals are not available, and molecular biological reagents are lacking. other caveats making ferret models more difficult to work with are their requirement for more space than mice (rabbit-style cages), and the development of aggressive behavior with repeated procedures. nhps are genetically the closest species to humans; thus, disease progression and host-pathogen responses to viral infections are often the most similar to that of humans. however, ethical concerns of experimentation on nhps, along with the high cost and lack of spf nhps raise barriers for such studies. nhp studies should be carefully designed to ensure that the least amount of animals are used, and the studies should address the most critical questions regarding disease pathogenesis, host-pathogen responses, and protective efficacy of vaccines and therapeutics. well-designed experiments should carefully evaluate the choice of animal, including the strain, sex, and age. furthermore, route of exposure and the dose should be as close as possible to the route of exposure and dose of human disease. the endpoint for these studies is also an important criterion. depending on the desired outcome, the model system should emulate the host responses in humans when infected with the same pathogen. in summary, small animal models are helpful for the initial screening of vaccines and therapeutics, and are also often beneficial in obtaining a basic understanding of the disease. nhp models should be used for a more detailed characterization of pathogenesis and for pivotal preclinical testing studies. ultimately, an ideal animal model may not be available. in this case, a combination of different well-characterized animal models should be considered to understand the disease progression and to test medical countermeasures against the disease. in this chapter, we will be reviewing the animal models for representative members of numerous virus families causing human diseases. we will focus on the viruses for each family that are the greatest concern for public health worldwide. poliovirus (pv) is an enterovirus in the picornavirus family and causes poliomyelitis. 1 humans are the only natural host for the virus, but a number of nhp species are also susceptible. all three serotypes of pv cause paralytic disease, but it is relatively rare with only 1-2% of infected individuals ultimately developing paralysis. humans typically acquire and transmit the virus by the oral-fecal route, although transmission by aerosol droplets may also be possible. 2 the virus replicates in the oropharyngeal and intestinal mucosa, made possible by the resistance of pv to stomach acids. 3 cd155 expression in peyer's patches and m cells suggest that these cell types may be important during initiation of infection. 4 replication at extraneural sites 38 . in vivo models of viral diseases affecting humans precedes invasion into the central nervous systems (cnss), when it occurs. two effective vaccines, the salk killed polio vaccine delivered by the intramuscular route and the sabin live attenuated polio vaccine delivered orally, have been used very successfully to eliminate the disease from most parts of the world. 5 the world health organization has led a long and hard-fought global polio eradication campaign, with much success, but full eradication has not yet been achieved. since 2003, between 1000 and 2000 cases of pv infection are reported worldwide each year. 6 thus, animal models are also needed to test new vaccine approaches that could be used toward eradication of polio in the areas where it still persists. additionally, the recent focus of work with pv animal models has been fraught with urgency, as to gain understanding of pv pathogenesis before the eradication effort is complete and work with this virus ceases. animal models for the study of pv consist of nhp models and mouse models. mice are susceptible to certain adapted pv strains: p2/lansing, p1/lsb, and a variant of p3/leon. mice infected intracerebrally with p2/lansing develop disease with some clinical and histopathological features resembling that of humans. 7 wild-type mice are not susceptible to wildtype pv; however, the discovery of the pv receptor (cd155) in 1989 led to the use of hcd155 transgenic mice as a model of pv infection. 8 these mice are not susceptible to pv by the oral route and must be exposed intranasally or by intramuscular infection to induce paralytic disease. 9 interestingly, hcd155 mice that have a disruption in the interferon (ifn)-a/b receptor gene are susceptible to oral infection. 10 this finding has given rise to speculation that an intact ifn-a/b response may be responsible for limiting infection in the majority of individuals exposed to pv. thus, mouse models have proven to be very useful in gaining a better understanding of pv disease and pathogenesis. rhesus macaques are not susceptible to pv by the oral route, but they have been used extensively to study vaccine formulations for safety and immunogenicity, for monitoring neurovirulence of the live attenuated sabin vaccine, and in the past for typing pv strains. 11 bonnet monkeys are also susceptible to oral inoculation of pv, which results in the gastrointestinal shedding of virus for several weeks, with paralysis occurring in only a small proportion of animals. consistent paralytic disease can be induced in bonnet monkeys (macaca radiata) through exposure to pv by infection into the right ulnar nerve (at the elbow), resulting in limb paralysis that resembles human paralytic poliomyelitis both clinically and pathologically. 12 as such, bonnet monkeys can be used to study pv distribution and pathology and the induction of paralytic poliomyelitis or provocation paralysis. 13 hepatitis a virus causes jaundice, which is a public health problem worldwide. the incubation period lasts from 15 to 45 days with an average of 28 days. transmission between humans occurs by the oral-fecal route, person-to-person contact, or ingestion of contaminated food and water. 14 hepatitis a virus causes an acute and self-limited infection of the liver with a spectrum of signs and symptoms ranging from subclinical disease, to jaundice, fulminant hepatitis, and in some cases death. 15, 16 the disease can be divided into four clinical phases: (1) incubation period, during which the patient is asymptomatic but virus replicates and possibly transmits to others. (2) prodromal period, which might last from a few days to a week with patients generally experiencing anorexia, fever (<103 f), fatigue, malaise, myalgia, nausea, and vomiting. (3) icteric phase, in which increased bilirubin causes characteristic dark brownish colored urine. this sign is followed by pale stool and yellowish discoloration of the mucous membranes, conjunctiva, sclera, and skin. most patients develop an enlarged liver, and approximately 5-15% of the patients have splenomegaly. (4) convalescent period, with resolution of the disease and recovery of the patient. rarely, during the icteric phase, extensive necrosis of the liver occurs. these patients show a sudden increase in body temperature, marked abdominal pain, vomiting, jaundice, and the development of hepatic encephalopathy associated with coma and seizures, all signs of fulminant hepatitis. death occurs in 70-90% of patients with fulminant hepatitis. 16 experiments showed that hepatitis a causes disease only in humans, chimpanzees, several species of south american marmosets, stump-tailed monkeys, and owl monkeys via the oral or intravenous (iv) routes. [17] [18] [19] [20] it is known that cynomolgus macaques are infected with hepatitis a virus in the wild. 21 amado et al. used cynomolgus macaques (macaca fascicularis) for experimental hepatitis a infections. 17 the animals did not exhibit clinical signs of disease, but viral shedding was observed in saliva and stool as early as 6 h postinoculation (pi) and 7 days pi, respectively. although mildto-moderate hepatic pathology was observed in all macaques, seroconversion and mildly increased alanine aminotransferase (alt), an enzyme associated with liver function, were observed in some of them. because this study had a very small group of animals (four macaques), the data should not be considered as conclusive, and more studies are needed to better define the cynomolgus macaque model. although hepatitis a virus is transmitted by the oralfecal route, studies in chimpanzees and tamarins showed that the iv route was much more infectious than oral route was. there was no correlation between dose and development of clinical disease for either species or experimental routes, and similar to cynomolgus macaques, none of these species showed clinical signs of disease. 20 inoculation of common marmosets (callithrix jacchus) with hepatitis a virus did not produce clinical signs of disease as seen in other nhp models. 22, 23 liver enzyme levels increased on day 14 pi, and monkeys had measurable antihepatitis a antibodies by day 32 pi. an experimental study with cell culture-adapted hepatitis avirus in guinea pigs challenged by oral or intraperitoneal routes did not result in clinical disease, increase in liver enzymes, or seroconversion. 24 viral load was detected in stool and serum between days 14 and 52 and 21 and 49 days, respectively. liver pathology showed mild hepatitis. furthermore, histopathology indicated that virus replicated in extrahepatic tissues such as spleen, regional lymph nodes, and intestinal tract. in summary, none of the animal models for hepatitis a infection is suitable for studying pathogenesis of the virus because all clinical and most of the laboratory parameters remain within normal range or only slightly increased after the infection. one possibility is to test the safety of vaccines against hepatitis a virus in those models with demonstrable viral shedding. noroviruses, of which norwalk is the prototypic member, are responsible for up to 85% of reported food-borne gastroenteritis cases. in developing countries, this virus is responsible for approximately 200,000 deaths annually. 25 a typical disease course is self-limiting, but there have been incidences of necrotizing enterocolitis and seizures in infants. 26, 27 symptoms of infection include diarrhea, vomiting, nausea, abdominal cramping, dehydration, and fever. incubation normally is for 1-3 days, with symptoms enduring for 2-3 days. 28 viral shedding is indicative of immunocompromised status within an individual with the elderly and young having a prolonged state of shedding. 29 transmission occurs predominately through the oral-fecal route with contaminated food and water being the major vector. 30 a major hindrance to basic research into this pathogen is the lack of a cell culture system. therefore, animal models are used not only to determine the efficacy of novel drugs and vaccines but also for understanding the pathogenesis of the virus. therapeutic intervention consists of rehydration therapy and antiemetic medication. 31 no vaccine is available, and development of one is expected to be challenging given that immunity is short lived after infection. 32 nhps including marmosets, cotton-top tamarins, and rhesus macaques infected with norwalk virus can be monitored for the extent of viral shedding; however, no clinical disease is observed in these models. disease progression and severity are measured exclusively by assay of viral shedding. 33 it was determined that more virus was needed to create an infection when challenging by the oral route than when challenging by the iv route. chimpanzees were exposed to a clinical isolate of norwalk virus by the iv route. although none of the animals developed disease symptoms, viral shedding within the feces was observed within 2-5 days postinfection and lasted anywhere from 17 days to 6 weeks. viremia never occurred, and no histopathological changes were detected. the amount and duration of viral shedding were in line with what is observed upon human infection. 34 a recently identified calicivirus of rhesus origin, named tulane virus, was used as a surrogate model of infection. rhesus macaques exposed to tulane virus intragastrically developed diarrhea and fever 2 days postinfection. viral shedding was achieved for 8 days. the immune system produced antibodies that dropped in concentration within 38 days postinfection, mirroring the short-lived immunity documented in humans. the intestine developed moderate blunting of the villi as seen in human disease. 35 a murine norovirus has been identified and is closely related to human norwalk virus. however, clinically, the viruses present a different disease. the murine norovirus does not induce diarrhea nor vomiting and can develop a persistent infection in contrast to human disease. [36] [37] [38] porcine enteric caliciviruses can induce diarrheal disease in young pigs and an asymptomatic infection in adults. 39 gnotobiotic pigs can successfully be infected with a passaged clinical noroviruses isolate orally. diarrheal disease developed in 74% of the animals, and 44% were able to shed virus in their stool. no major histopathological changes or viral persistence was noted. 40 calves are naturally infected with bovine noroviruses. experimentally challenging calves with an oral inoculation of a bovine isolate resulted in diarrheal disease [14] [15] [16] the link between equine cases and the human disease was confirmed in 1938 by observing 30 cases of fatal encephalitis in children living in the same area as the equine cases. during this outbreak, eeev was isolated from the cnss of these children as well as from pigeons and pheasants. 44 eeev primarily affects areas near salty marshes and can cause localized outbreaks of disease in the summer. the enzootic cycles are maintained in moist environments such as coastal areas, shaded marshy salt swamps in north america (na), and moist forests in central america and south america (sa). 45 birds are the primary reservoir, and the virus is transmitted via mosquitoes. furthermore, forest-dwelling rodents, bats, and marsupials frequently become infected and may provide an additional reservoir in central america and sa. despite known natural hosts, the transmission cycles in these animals are not well characterized. 44 reptiles and amphibians have also been reported to become infected by eeev. eeev pathogenesis and disease have been studied in several laboratory animals. as a natural host, birds do not generally develop encephalitis except pheasants or emus, in which eeev causes encephalitis with 50-70% mortality. 46 young chickens show signs of extensive myocarditis in early experimental infection and heart failure rather than encephalitis is the cause of death. 47 besides the heart, other organs such as pancreas and kidney show multifocal necrosis. additionally, lymphocytopenia has been observed in the thymus and spleen in birds. 45 eeev causes neuronal damage in newborn mice, and the disease progresses rapidly, resulting in death. 48 similarly, eeev produces fatal encephalitis in older mice when administered via the intracerebral route, whereas inoculation via the subcutaneous route causes a pantropic infection eventually resulting in encephalitis. 49, 50 guinea pigs and hamsters have also been used as animal models for eeev studies. 51, 52 guinea pigs developed neurological involvement with decreased activity, tremors, circling behavior, and coma. neuronal necrosis was observed and resulted in brain lesions in these animals. 52 subcutaneous inoculation of eeev produced lethal biphasic disease in hamsters with severe lesions of nerve cells. the early visceral phase with viremia was followed by neuroinvasion, encephalitis and death. in addition, parenchyma necroses were observed in the liver and lymphoid organs. 51 intradermal, intramuscular, or iv inoculations of eeev in nhps cause disease but does not always result in symptoms of the nervous system. intracerebral infection of eeev results in nervous system disease and fatality in monkeys. 53 the differences in these models indicate that the initial viremia and the secondary nervous system infection do not overlap in monkeys when they are infected by the peripheral route. 54 intranasal and intralingual inoculations of eeev also cause nervous system symptoms in monkeys, but less drastic than those caused by intracerebral injections. 54 the aerosol route of infection also progresses to uniformly lethal disease in cynomolgus macaques. 55 in this model, fever was followed by elevated white blood cells and liver enzymes. neurological signs subsequently developed, and nhps became moribund and were euthanized between days 5 and 9 days postexposure. meningoencephalomyelitis was the main pathology observed in the brains of these animals. 56 similar clinical signs and pathology were observed when common marmosets were infected with eeev by the intranasal route. 57 both aerosol and intranasal nhp models had similar disease progression and pathology as those seen in human disease. a common marmoset model was used for comparison studies of sa and na strains of eeev. 57 previous studies indicated that the sa strain is less virulent than na strain for humans. common marmosets were infected intranasally with either the na or sa strain of eeev. na strain-infected animals showed signs of anorexia and neurological involvement and were euthanized 4-5 days after the challenge. although sa strain-infected animals developed viremia, they remained healthy and survived the challenge. epizootics of viral encephalitis in horses were previously described in argentina. more than 25,000 horses died from western equine encephalitis virus (weev) in the central plains of the united states in 1912. 58 weev was first isolated from the brains of horses during the outbreak in the san joaquin valley of california in 1930. although it was suspected, the first diagnosis of weev as a cause of human encephalitis occurred in 1938, when the virus was recovered from the brain of a child with fatal encephalitis. 44 in horses, the signs of disease are fever, loss of coordination, drowsiness, and anorexia, leading to prostration, coma, and death in about 40% of affected animals. 59 weev also infects other species of birds and often causes fatal disease in sparrows. weev infection occurs throughout western na and sporadically in sa as it circulates between its mosquito vector and wild birds. 44 chickens and other domestic birds, pheasants, rodents, rabbits, ungulates, tortoises, and snakes are natural reservoirs of weev. 60,61 weev has caused epidemics of encephalitis in humans, horses, and emus, but the fatality rate is lower than that for eeev. 62 predominately young children and those older than 50 years demonstrate the clinical symptoms of the disease. 63 severe disease, seizures, fatal encephalitis, and significant sequelae are more likely to occur in infants and young children. 64, 65 typically, the disease progresses asymptomatically with seroprevalence in humans being fairly common in endemic areas. species used to develop animal models for weev are mice, hamsters, guinea pigs, and ponies. studies with ponies resulted in viremia in 100% of the animals 1-5 days pi. fever was observed in 7 of 11 animals, and six exhibited signs of encephalitis. 44 after subcutaneous inoculation with weev, suckling mice started to show signs of disease by 24 h and died within 48 h. 66 in suckling mice, the heart was the only organ in which pathologic changes were observed. conversely, adult mice exhibited signs of lethargy and ruffled fur on days 4-5 postinfection. mice were severely ill by day 8 and appeared hunched and dehydrated. death occurred between days 7 and 14, and both brain and mesodermal tissues such as heart, lungs, liver, and kidney were involved. 66, 67 intracerebral and intranasal routes of infection resulted in a fatal disease that was highly dependent on dose, while intradermal and subcutaneous inoculations caused only 50% fatality in mice regardless of the amount of virus. 49 studies demonstrated that although the length of the incubation period and the disease duration varied, weev infection resulted in mortality in hamsters by all routes of inoculation. progressive lack of coordination, shivering, rapid and noisy breathing, corneal opacity, and conjunctival discharge resulting in closing of the eyelids were indicative of disease in all cases. 68 cns involvement was evident with intracerebral, intraperitoneal, and intradermal inoculations. 68 weev is highly infectious to guinea pigs. 69 intraperitoneal inoculation of weev is fatal in guinea pigs regardless of virus inoculum, with the animals exhibiting signs of illness on days 3-4, followed by death on days 5-9 (nalca, unpublished results). very limited studies have been performed with nhps. the intranasal route of infection causes severe, lethal encephalitis in rhesus macaques. 54 reed et al. exposed cynomolgus macaques to low and high doses of aerosolized weev. the animals subsequently developed fever, increased white blood counts, and cns involvement, demonstrating that the cynomolgus macaque model would be useful for testing of vaccines and therapeutics against weev. 70 venezuelan equine encephalitis virus (veev) is maintained in nature in a cycle between small rodents and mosquitoes. 45 the spread of epizootic strains of the virus to equines leads to high viremia followed by a lethal encephalitis, and tangential spread to humans. veev can easily be spread by the aerosol route making it a considerable danger for laboratory exposure. in humans, veev infection causes a sudden onset of malaise, fever, chills, headache, and sore throat. 45, 71, 72 symptoms persist for 4-6 days, followed by a 2-to 3-week period of generalized weakness. encephalitis occurs in a small percentage of adults ( 0.5%); however, the rate in children may be as high as 4%. neurologic symptoms range from nuchal rigidity, ataxia, and convulsions to the more severe cases exhibiting coma and paralysis. the overall mortality rate in humans is <1%. 45 laboratory animals such as mice, guinea pigs, and nhps exhibit different pathologic responses when infected with veev. the lymphatic system is a general target in all animals infected with cns involvement variable between different animal species. the disease caused by veev progresses very rapidly without showing signs of cns disease in guinea pigs and hamsters. mortality is typically observed within 2-4 days after infection and fatality is not dose dependent. 44 veev infection lasts longer in mice, which develop signs of nervous system disease in 5-6 days and death 1-2 days later. lethal dose in mice changes depending on the age of mice and the route of exposure. 56 in contrast to guinea pigs and hamsters, the time of the death in mice is dose dependent. mortality is observed generally within 2-4 days after infection and fatality is not dose dependent. subcutaneous/dermal infection in the mouse model results in encephalitic disease very similar to that seen in horses and humans. 73 virus begins to replicate in the draining lymph nodes at 4 h pi. eventually, virus enters the brain primarily via the olfactory system. furthermore, aerosol exposure of mice to veev can result in massive infection of the olfactory neuroepithelium, olfactory nerves, and olfactory bulbs and viral spread to brain, resulting in necrotizing panencephalitis. 74, 75 aerosol and dermal inoculation routes cause neurological pathology in mice much faster than other routes of exposure do. the clinical signs of disease in mice infected by aerosol are ruffled fur, lethargy, and hunching progressing to death. 56, 74, 75 intranasal challenge of c3h/hen mice with high dose veev caused high morbidity and mortality. 76 viral titers in brain peaked on day 4 postchallenge and stayed high until animals died on day 9-10 postchallenge. protein cytokine array done on brains of infected mice showed elevated interleukin (il)-1a, il-1b, il-6, il-12, monocyte chemoattractant protein-1 (mcp-1), ifng, mip-1a, and regulated and normal t-cell expressed and secreted levels. this model was used successfully to test antivirals against veev. 77 xi. viral disease veev infection causes a typical biphasic febrile response in nhps. initial fever was observed at 12-72 h after infection and lasted <12 h. secondary fever generally began on day 5 and lasted 3-4 days. 78 veev-infected nhps exhibited mild symptoms such as anorexia, irritability, diarrhea, and tremors. leucopenia was common in animals exhibiting fever. 79 supporting the leucopenia, observed microscopic changes in lymphatic tissues such as early destruction of lymphocytes in lymph nodes and spleen, a mild lymphocytic infiltrate in the hepatic triads, focal myocardial necrosis with lymphocytic infiltration have been observed in monkeys infected with veev. surprisingly, characteristic lesions of the cns were observed histopathologically in monkeys in spite of the lack of any clinical signs of infection. 78 the primary lesions were lymphocytic perivascular cuffing and glial proliferation and generally observed at day 6 postinfection during the secondary febrile episode. cynomolgus macaques develop similar clinical signs including fever, viremia, lymphopenia, and encephalitis upon aerosol exposure to veev. 80 chikungunya virus is a member of the genus alphaviruses, specifically the semliki forest complex, and has been responsible for a multitude of epidemics mainly within africa and southeast asia. 45 the virus is transmitted by aedes mosquitoes. given the widespread endemicity of aedes mosquitoes, chikungunya virus has the potential to spread to previously unaffected areas. this is typified by the emergence of disease for the first time in 2005 in the islands of the southwest indian ocean, including the french la reunion island, and the appearance in central italy in 2007. 81, 82 the incubation period after a mosquito bite is 2-5 days followed by a self-limiting acute phase that lasts 3-4 days. symptoms during this period include fever, arthralgia, myalgia, and rash. headache, weakness, nausea, vomiting, and polyarthralgia have all been reported. 83 individuals typically develop a stooped posture due to the pain. for approximately 12% of infected individuals, joint pain can last months after resolution of primary disease, and has the possibility to relapse. underlying health conditions, including diabetes, alcoholism, or renal disease, increase the risk of developing a severe form of disease that includes hepatitis or encephalopathy. children between the ages of 3 and 18 years have an increased risk of developing neurological manifestations. 84 there is no effective vaccine or antiviral. wild-type c57bl/6 adult mice are not permissive to chikungunya virus infection by intradermal inoculation. however, it was demonstrated that neonatal mice were susceptible, and severity was dependent upon age at infection. six-day-old mice developed paralysis by day 6, and all died by day 12, whereas 50% of nine-day-old mice were able to recover from infection. by 12 days, mice were no longer permissive to disease. infected mice developed loss of balance, hind limb dragging, and skin lesions. neonatal mice were also used as a model for neurological complications. 85, 86 an adult mouse model has been developed by injection of the ventral side of the footpad of c57bl/6j mice. viremia lasted 4-5 days accompanied by foot swelling and noted inflammation of the musculoskeletal tissue. 87, 88 adult ifna/br knockout mice also developed mild disease with symptoms including muscle weakness and lethargy, symptoms that mirrored human infection. all adult mice died within 3 days. this model was useful in identifying the viral cellular tropism for fibroblasts. 85 imprinting control region (icr) cd1 mice can also be used as a disease model. neonatal mice subcutaneously inoculated with a passaged clinical isolate of chikungunya virus developed lethargy, loss of balance, and difficulty in walking. mortality was low, 17% and 8% for newborn cd1 and icr mice, respectively. the remaining mice fully recovered within 6 weeks after infection. 86 a drawback of both the ifna/ br and cd1 mice is that the disease is not a result of immunopathogenesis as occurs in human cases, given that the mice are immunocompromised. 89 long-tailed macaques challenged with a clinical isolate of the virus developed a similar clinical disease to humans. initially, the monkeys developed high viremia with fever and rash. after this period, viremia resolved and virus could be detected in lymphoid, liver, meninges, joint, and muscle tissue. the last stage mimicked the chronic phase in which virus could be detected up to two months after infection, although no arthralgia was noted. 90 dengue virus is transmitted via the mosquito vectors aedes aegypti and aedes albopictus. 91 given the endemicity of the vectors, it is estimated that half of the world's population is at risk for exposure to dengue virus. this results in approximately 50 million cases of dengue each year, with the burden of disease in the tropical and subtropical regions of latin america, south asia, and southeast asia. 92 it is estimated that there are 20,000 deaths each year caused by dengue hemorrhagic fever (dhf). 93 there are four serotypes of dengue virus, numbered 1-4, which are capable of causing a wide spectrum of disease that ranges from asymptomatic to severe with the development of dhf. 94 incubation can range from 3 to 14 days, with the average being 4-7 days. the virus targets dendritic cells and macrophages after a mosquito bite. 95 typical infection results in classic dengue fever (df), which is self-limiting and has flu-like symptoms in conjunction with retroorbital pain, headache, skin rash, and bone and muscle pain. dhf can follow, with vascular leak syndrome and low platelet count, resulting in hemorrhage. in the most extreme cases, dengue shock syndrome (dss) develops, characterized by hypotension, shock, and circulatory failure. 94 thrombocytopenia is a hallmark clinical sign of infection, and aids in differential diagnosis. 96 severe disease has a higher propensity to occur upon secondary infection with a different dengue virus serotype. 97 this is hypothesized to occur due to antibodydependent enhancement (ade). there is no approved vaccine or drug, and hospitalized patients receive supportive care including fluid replacement. in developing an animal model, it is important to note that mosquitoes typically deposit 10 4 -10 6 pfu, and is therefore the optimal range to be used during challenge. a comprehensive review of the literature regarding animal models of dengue infection was recently published by zompi et al. 98 several laboratory mouse strains including a/j, balb/c, and c57bl/6 are permissive to dengue infection. however, the resulting disease has little resemblance to human clinical signs, and death results from paralysis. [99] [100] [101] a higher dose of an adapted dengue virus strain induced dhf symptoms in both balb/c and c57bl/6. 102, 103 this model can also yield asymptomatic infections. a mouse-adapted (ma) strain of dengue virus 2 introduced into ag129 mice developed vascular leak syndrome similar to the severe disease seen in humans. 104 passive transfer of monoclonal dengue antibodies within mice leads to ade. during the course of infection, viremia was increased, and animals died due to vascular leak syndrome. 105 another ma strain injected into balb/c caused liver damage, hemorrhagic manifestations, and vascular permeability. 103 intracranial injection of suckling mice with dengue virus leads to death and has been used to test the efficacy of therapeutics. 106 scid mice engrafted with human tumor cells develop paralysis upon infection, and are thus not useful for pathogenesis studies. 107,108 df symptoms developed after infection in nod/scid/il2rgko mice engrafted with cd34 ã¾ human progenitor cells. 109 rag-hu mice developed fever, but no other symptoms upon infection with a passaged clinical isolate and laboratory-adapted strain of dengue virus 2. 110 a passaged clinical isolate of dengue virus type 3 was recently used to create a model in immunocompetent adult mice. interperitoneal injection in c57bl/6j and balb/c caused lethality by day 6-7 postinfection in a dose-dependent manner. the first indication of infection was weight loss beginning on day 4 followed by thrombocytopenia. a drop in systolic blood pressure along with noted increases in the liver enzymes, aspartate aminotransferase (ast) and alt, were also observed. viremia was established by day 5. this model mimicked the characteristic symptoms observed in human dhf/dss cases. 111 a novel model was developed that used infected mosquitoes as the route of transmission to hu-nsg mice. female mosquitoes were intrathoracically inoculated with a clinical isolate of dengue virus type 2. infected mosquitoes then fed upon the mouse footpad to allow for the transmission of the virus via the natural route. the amount of virus detected within the mouse was directly proportional to the amount of mosquitoes it was exposed to, with four to five being optimal. detectable viral rna was in line with what is observed during human infection. severe thrombocytopenia developed on day 14. this model is intriguing given that disease was enhanced with mosquito delivery of the virus in comparison to injection of the virus. 112 nhp models have used a subcutaneous inoculation in an attempt to induce disease. although the animals are permissive to viral replication, it is to a lower degree than that observed in human infection. 113 the immunosuppressive drug, cyclophosphamide enhances infection in rhesus macaques by allowing the virus to invade monocytes. 114 throughout these preliminary studies, no clinical disease was detected. to circumvent this, a higher dose of dengue virus was used in an iv challenge of rhesus macaques. hemorrhagic manifestations appeared by day 3 and resulted in petechiae, hematomas, and coagulopathy; however, no other symptoms developed. 115 further development would allow this model to be used for testing of novel therapeutics and vaccines. although primates do not develop disease upon infection with dengue, their immune system does produce antibodies similar to those observed during the course of human infection. this has been advantageous in studying ade. sequential infection led to a crossreactive antibody response, which has been demonstrated in both humans and mice. 116 this phenotype can also be seen upon passive transfer of a monoclonal antibody to dengue and subsequent infection with the virus. rhesus macaques exposed in this manner developed viremia that was 3-to 100-fold higher than was previously reported; however, no clinical signs were apparent. 117 the lack of inducible dhf or dss symptoms hinders further examination of pathogenesis within this model. japanese encephalitis virus ( jev) is a leading cause of childhood viral encephalitis in southern and eastern asia and is a problem among military personnel and travelers to these regions. it was first isolated from the brain of a patient who died from encephalitis in japan in 1935. 118 culex mosquitoes, which breed in rice fields, transmit the virus from birds or mammals (mostly domestic pigs) to humans. the disease symptoms range from a mild febrile illness to acute meningomyeloencephalitis. after an asymptomatic incubation period of 1-2 weeks, patients show signs of fever, headache, stupor, and generalized motor seizures, especially in children. the virus causes encephalitis by invading and destroying the cortical neurons. the fatality rate ranges from 10% to 50%, and most survivors have neurological and psychiatric sequelae. 119, 120 jev virus causes fatality in infant mice by all routes of inoculation. differences in pathogenesis and outcome are seen when the virus is given by intraperitoneal inoculation. 121 these differences depend on the amount of virus and the specific viral strains used. the biphasic viral multiplication after peripheral inoculation is observed in mice tissues. primary virus replication occurs in the peripheral tissues and the secondary replication phase in the brain. 122 hamsters are another small animal species that are used as an animal model for jev. fatality was observed in hamsters inoculated intracerebrally or intranasally, while peripheral inoculation caused asymptomatic viremia. studies with rabbits and guinea pigs showed that all routes of inoculation of jev produce asymptomatic infection. 123 serial sampling studies with 12-day-old wistar rats inoculated intracerebrally with jev indicated that jev causes the overproduction of free radicals by neurons and apoptosis of neuronal cells. 124 following a study in 2010 by the same group, showed that although cytokines tumor necrosis factor (tnf)-a, ifn-g, il-4, il-6, il-10, and chemokine mcp-1 increased gradually and peaked on days 10 pi with jevin rats, the levels eventually declined, and there was no correlation with the levels of cytokines and chemokines and neuronal damage. 125 intracerebral inoculation of jev causes severe histopathological changes in brain hemispheres of rhesus monkeys. symptoms such as weakness, tremors, and convulsions began to appear on days 6-10, with indicative signs of encephalomyelitis occurring on days 8-12 postinfection for most of the animals followed by death occurring on days 8-12 postinfection for most of the animals followed by death. 126 although intranasal inoculation of jev results in fatality in both rhesus and cynomolgus monkeys, peripheral inoculation causes asymptomatic viremia in these species. 123, 127 west nile virus west nile virus (wnv) was first isolated from the blood of a woman in the west nile district of uganda in 1937. 128 after the initial isolation of wnv, the virus was subsequently isolated from patients, birds, and mosquitoes in egypt in the early 1950s 129, 130 and was shown to cause encephalitis in humans and horses. wnv is recognized as the most widespread of the flaviviruses, with a geographical distribution that includes africa, the middle east, western asia, europe, and australia. 131 the virus first reached the western hemisphere in the summer of 1999, during an outbreak involving humans, horses, and birds in the new york city metropolitan area. 132, 133 since 1999, the range of areas affected by wnv quickly extended. older people and children are most susceptible to wnv disease. wnv generally causes asymptomatic disease or a mild undifferentiated fever (west nile fever), which can last from 3 to 6 days. 134 the mortality rate after neuroinvasive disease ranges from 4% to 11%. 131, [135] [136] [137] the most severe complications are commonly seen in the elderly, with reported case fatality rates from 4% to 11%. hepatitis, myocarditis, and pancreatitis are unusual, severe, nonneurologic manifestations of wnv infection. although many early laboratory studies of wn encephalitis were performed in nhps, mice, rat, hamster, horse, pig, dog, and cat models were used to study the disease. [138] [139] [140] [141] [142] [143] [144] inoculation of wnv into nhps intracerebrally resulted in the development of either encephalitis, febrile disease, or an asymptomatic infection, depending on the virus strain and dose. viral persistence is observed in these animals regardless of the outcome of infection (i.e. asymptomatic, fever, encephalitis). 141 thus, viral persistence is regarded as a typical result of nhp infection with various wnv strains. after both intracerebral and subcutaneous inoculation, the virus localizes predominantly in the brain and may also be found in the kidneys, spleen, and lymph nodes. wnv does not result in clinical disease in nhps although the animals show a low level of viremia. 145, 146 wnv has also been extensively studied in small animals. all classical laboratory mouse strains are susceptible to lethal infections by the intracerebral and intraperitoneal routes, resulting in encephalitis and 100% mortality. intradermal route pathogenesis studies indicated that langerhans dentritic cells are the initial viral replication sites in the skin. 147, 148 the infected langerhans cells then migrate to lymph nodes, and the virus enters the blood through lymphatic and thoracic ducts and disseminates to peripheral tissues for secondary viral replication. virus eventually travels to the cns and causes pathology that is similar to human cases. [149] [150] [151] [152] tesh et al. developed a model for wn encephalitis using the golden hamster, mesocricetus auratus. hamsters appeared normal during the first 5 days, became lethargic at approximately day 6, and developed neurologic symptoms by days 7-10. 143 many of the severely affected animals died 7-14 days after infection. viremia was detected in the hamsters within 24 h after infection and persisted for 5-6 days. although there were no substantial changes in internal organs, progressive pathologic differences were seen in the brain and spinal cord of infected animals. furthermore, similar to the above-mentioned monkey experiments by pogodina et al., persistent wnv infection was found in the brains of hamsters. the etiologic agent of severe acute respiratory syndrome (sars), sars-coronavirus (cov), emerged in 2002 as it spread throughout 32 countries in a period of 6 months, infecting >8000 people and causing nearly 800 deaths. 153, 154 the main mechanism of transmission of sars-cov is through droplet spread, but it is also viable in dry form on surfaces for up to 6 days and can be detected in stool, suggesting other modes of transmission are also possible. 155 although other members of the family usually cause mild illness, sars-cov infection has a 10% case fatality with the majority of cases in people over the age of 15 years. 156, 157 after an incubation period of 2-10 days, clinical signs of sars include general malaise, fever, chills, diarrhea, dyspnea, and cough. 158 in some sars, cases, pneumonia may develop and progress to acute respiratory distress syndrome (ards). fever usually dissipates within 2 weeks and coincides with the induction of high levels of neutralizing antibodies. 159 in humans, sars-cov replication destroys respiratory epithelium, and a great deal of the pathogenesis is due to the subsequent immune responses. 160 infiltrates persisting within the lung and diffuse alveolar damage (dad) are common sequelae of sars-cov infection. virus can be isolated from secretions of the upper airways during early, but not later stages of infection as well as from other tissues. 161 sars-cov can replicate in many species, including dogs, cats, pigs, mice, rats, ferrets, foxes, and nhps. 162 chinese palm civets, raccoon dogs, and bats are possible natural hosts. no model captures all aspects of human clinical disease (pyrexia and respiratory signs), mortality (w10%), viral replication, and pathology. 163 in general, the sars-cov disease course in the model species is much milder and of shorter duration than in humans. viral replication in the various animal models may occur without clinical illness and/or histopathologic changes. the best-characterized models use mice, hamsters, ferrets, and nhps (table 38 .1). mouse models of sars-cov typically are inoculated by the intranasal route under light anesthesia. young, 6-to 8-week old balb/c mice exposed to sars-cov have viral replication detected in the lungs and nasal turbinates, with a peak on day 2 and clearance by day 5 postexposure. there is also viral replication within the small intestines of young balb/c mice. however, young mice have no clinical signs, aside from reduced weight gain, and have little to no inflammation within the lungs (pneumonitis). intranasal sars-cov infection of c57bl/6 (b6) also yield reduced weight gain and viral replication in the lungs, with a peak on day 3 and clearance by day 9. 171 in contrast, balb/c mice 13-14 interstitial pneumonitis, alveolar damage, and death also occur in old mice, resembling the age-dependent virulence observed in humans. 129s mice and b6 mice show outcomes to sars-covinfection similar to those observed for balb/c mice but have lower titers and less prolonged disease. one problem is that it is more difficult to obtain large numbers of mice older than 1 year. a number of immunocompromised knockout mouse models of intranasal sars-cov infection have also been developed. 129svev mice infected with sars-cov by the intranasal route develop bronchiolitis, with peribronchiolar inflammatory infiltrates, and interstitial inflammation in adjacent alveolar septae. 172 viral replication and disease in these mice resolve by day 14 postexposure beige, cd1ã�/ã�, and rag1ã�/ã� mice infected with sars-cov have similar outcomes to infected balb/c mice with regard to viral replication, timing of viral clearance, and a lack of clinical signs. signal transducer and activator of transcription-1 (stat1) ko mice infected intranasally with sars-cov have severe disease, with weight loss, pneumonitis, interstitial pneumonia, and some deaths. the stat1 ko mouse model is therefore useful for studies of pathogenicity, pathology, and evaluation of vaccines. syrian golden hamsters (strain lvg) are also susceptible to intranasal exposure of sars-cov. after the administration of 10 3 tcid 50 (tissue culture infective dose), along with a period of transient viremia, sars-cov replicates in nasal turbinates and lungs, resulting in pneumonitis. there are no obvious signs of disease, but exercise wheels can be used to monitor decrease in nighttime activity. some mortality has been observed, but it was not dose dependent and could have more to do with genetic differences between animals because the strain is not inbred. 163 damage is not observed in the liver or spleen despite detection of virus within these tissues. several studies have shown that intratracheal inoculation of sars-cov in anesthetized ferrets (mustela furo) results in lethargy, fever, sneezing, and nasal discharge. 167 clinical disease has been observed in several studies. sars-cov is detected in pharyngeal swabs, trachea, tracheobronchial lymph nodes, and high titers within the lungs. mortality has been observed around day 4 postexposure as well as mild alveolar damage in 5-10% of the lungs, occasionally accompanied by severe pathology within the lungs. 173 with fever, overt respiratory signs, lung damage, and some mortality, the ferret intratracheal model of sars-cov infection is perhaps most similar to human sars, albeit with a shorter time course. sars-cov infection of nhps by intransal or intratracheal routes generally results in a very mild infection, which resolves quickly. sars-cov infection of old world monkeys, such as rhesus macaques, cynomolgus macaques (cynos), and african green monkeys (agms) have been studied with variable results, possibly due to the outbred nature of the groups studied or previous exposure to related pathogens. clinical illness and viral loads have not been consistent; however, replication within the lungs and dad are features of the infections for each of the primate species. some cynos have no illness, but others have rash, lethargy, and respiratory signs and pathology. 170 rhesus have little to no disease and only have mild findings upon histopathological analysis. agms infected with sars-cov have no overt clinical signs, but dad and pneumonitis have been documented. viral replication has been detected for up to 10 days in the lungs of agms; however, the infection resolves and does not progress to fatal ards. farmed chinese masked palm civets, sold in open markets in china, were thought to be involved in the sars-covoutbreak. intratracheal and intranasal inoculation of civets with sars-covresults in lethargy, decreased aggressiveness fever, diarrhea, and conjunctivitis. 174 leucopenia, pneumonitis, and alveolar septal enlargement, with lesions similar to those observed in ferrets and nhps, have also been observed in laboratory-infected civets. common marmosets have also been shown to be susceptible to sars-cov infection. 175 vaccines have been developed for related animal covs in chickens, cattle, dogs, cats, and swine have used live-attenuated, killed, dna and viral-vectored vaccine strategies. 176 an important issue to highlight from work on these vaccines is that cov vaccines, such as those developed for cats, may induce a more severe disease. 177 as such, immune mice had th2-type immunopathology upon sars-cov challenge. 178 severe hepatitis in vaccinated ferrets with antibody enhancement in liver has been reported. 179 additionally, rechallenge of agms showed limited viral replication but significant lung inflammation, including alveolitis and interstitial pneumonia, which persisted for long periods of time after viral clearance. 180 mouse and nhp models with increased virulence may be developed by adapting the virus by repeated passage within the species of interest. ma sars and human ace2 transgenic mice are available. 181 all mammals experimentally or naturally exposed to rabies virus have been found to be susceptible. this highly neurotropic virus is a member of the lyssavirus genus and is transmitted from the bite of an infected animal to humans. 182 the virus is able to replicate within the muscle cells at the site of the bite, and then travel to the cns. once reaching the cns by retrograde axonal transport, the virus replicates within neurons creating inflammation and necrosis. the virus subsequently spreads throughout the body via peripheral nerves. 183 a typical incubation period is 30-90 days, and is highly dependent upon the location of the bite. proximity to the brain is a major factor for the onset of symptoms. the prodromal stage lasts from 2 to 10 days and is when the virus initially invades the cns. flu-like symptoms are the norm in conjunction with pain and inflammation at the site of the bite. subsequently, there are two forms of disease that can develop. in 80% of cases, an individual develops the encephalitic or furious form. this form is marked by hyperexcitability, autonomic dysfunction, and hydrophobia. the paralytic, or dumb form, is characterized by ascending paralysis. ultimately, both forms result in death days after the onset of symptoms. once the symptoms develop, there is no proven effective therapy. in the developing world, death is caused by the lack of access to medical care including postexposure prophylaxis. in na, fatal cases result because of late diagnosis. 184 syrian hamsters have been challenged with rabies virus intracerebrally, intraperitoneally, intradermally, and intranasally. all animals died as a result of the exposure, although intracerebral and intranasal inoculation led to only the furious form depicted by extreme irritability, spasms, excessive salivation, and cries. the virus used had been isolated from an infected dog brain and passaged in swiss albino mice. animals inoculated by intracerebral injection develop disease within 4-6 days, whereas all other routes of entry develop disease within 6-12 days. 185 this model has been used to study and test novel vaccine candidates. 186 mice have been extensively studied as an animal model for rabies. it was shown that swiss albino mice intracerebrally injected with a virus isolated from a dog developed only the paralytic form of disease 6 days after the initial challenge. balb/c mice are universally susceptible to intracerebral injection of rabies virus within 9 days. disease symptoms include paralysis, cachexia, and bristling appearing 1-3 days before death. 187 a more natural route of infection via peripheral injection into masseter muscles was tested on icr mice. these mice developed neurological signs including limb paralysis, and all died within 6-12 days. 188 icr mice have been instrumental in analyzing novel vaccines and correlates of protection. 189 this line of mice was also used to assess the value of ketamine treatment to induce coma during rabies infection. 190 another mouse line used is the p75 neurotrophin receptor-deficient mouse. this mouse developed a fatal encephalitis when inoculated intracerebrally with the challenge virus standard. 191 bax-deficient mice have also been used to determine the role of apoptotic cell death in the brain during the course of infection. 192 a viral isolate from silver-haired bats can also be used in the mouse model. this strain is advantageous given that it is responsible for the majority of deaths in north na. 193 early death phenomenon is typified by a decrease to time of death in a subset of individuals and animals that have been vaccinated and subsequently exposed to rabies. 194 this trend has been demonstrated experimentally in swiss outbred mice and primates. 195, 196 cynomolgus and rhesus were both infected with passaged rabies virus to create an nhp model. a high titer of virus was needed to induce disease, but exposure was found to not be universally fatal. the animals that survived beyond 4 weeks within the experiment did not develop clinical disease nor succumb to infection. primates that did develop disease refused food and had progressively less activity until death. this lasted from 24 h up to 4 days, with all animals with symptoms dying within 2 weeks. 196 bats have been experimentally challenged with rabies. vampire bats, desmodus rotundus, intramuscularly injected with a bat viral isolate displayed clinical signs including paralysis in half of the population of the study animals. of those who did develop disease, the duration was 2 days, and incubation period ranged from 7 to 30 days. regardless of disease manifestation, 89% of challenged animals died. 197 skunks can be challenged intramuscularly or intranasally with either challenge virus strain or a skunk viral isolate. interestingly, the challenge virus strain more readily produced the paralytic form, whereas the street form of rabies developed into the furious form. however, the challenge strain virus resulted in a shorter incubation period of 7-8 days in comparison to 12-14 days seen with the street virus. 198 filoviridae consists of two well-established genera, ebola virus and marburg virus (marv) and a newly discovered group, cuevavirus 199 (table 38 200 two other ebola viruses are known; ta㯠forest (tafv; previously named cote (circumflex over the 'o') d'ivoire) (ciebov) and reston (restv), which have not caused major outbreaks or lethal disease in humans. the disease in humans is characterized by aberrant innate immunity and a number of clinical symptoms such as fever, nausea, vomiting, arthralgia/myalgia, headaches, sore throat, diarrhea, abdominal pain, anorexia, and numerous others. 201 approximately 10% of patients develop petechia and a greater percentage, depending on the specific strain, may develop bleeding from various sites (gums, puncture sites, stools, etc.). 199 natural transmission in an epidemic is thought to be through direct contact or needle sticks in hospital settings. however, much of the research interest in filoviruses primarily stems from biodefense needs, particularly from aerosol biothreats. as such, intramuscular, intraperitoneal, and aerosol models have been developed in mice, hamsters, guinea pigs, and nhps for the study of pathogenesis, correlates of immunity, and for testing countermeasures. 202 because filoviruses have such high lethality rates in humans, scientists have looked for models that are uniformly lethal to stringently test efficacy of candidate vaccines and therapeutics. immunocompetent mice have not been successfully infected with wild-type filoviruses due to the control of the infection by the murine type 1 ifn response. 203 however, wild-type inbred mice are susceptible to filovirus that has been ma by serial passage. 204 balb/c mice, which are the strain of choice for intraperitoneal inoculation of ma-ebov, are not susceptible by the aerosol route. 205 for aerosol infection of immunocompetent mice, a panel of bxd (balb/ c ã� dba) recombinant inbred strains were screened, and one strain, bxd34, was shown to be particularly susceptible to airborne ma-ebov, with 100% lethality to low or high doses (w100 or 1000 pfu). these mice developed weight loss of >15% and succumbed to infection between days 7 and 8 postexposure. the aerosol infection model uses a whole-body exposure chamber to expose mice aged 6-8 weeks to ma-ebov aerosols with a mass median aerodynamic diameter (mmad) of approximately 1.6 mm and a geometric standard deviation of approximately 2.0 for 10 min. another approach uses immunodeficient mouse strains such as scid, stat1 ko, ifn receptor ko, or perforin ko with a wild-type ebov inoculum by intraperitoneal or aerosol routes. 206 mice are typically monitored for clinical disease "scores" based on activity and appearance, weight loss, and moribund condition (survival). coagulopathy, a hallmark of filovirus infection in humans, has been observed, with bleeding in a subset of animals and failure of blood samples to coagulate late in infection. liver, kidney, spleen, and lung tissue taken from moribund mice have pathology characteristic of filovirus disease in nhps. although most mouse studies have used ma-ebov or ebov, an intraperitoneal ma marv model is also available. 207 ma-marv and ma-ebov models are particularly useful for screening novel antiviral compounds. 208 hamsters are frequently used to study cardiovascular disease, coagulation disorders, and thus serve as the basis for numerous viral hemorrhagic fever models. 209 an intraperitoneal ma-ebov infection model has been developed in syrian hamsters. 210 this model, which has been used to test a vesicular stomatitis virus vectored vaccine approach, uses male 5-to 6-week-old syrian hamsters that are infected with 100 ld50 of ma-ebov. virus is present in tissues and blood collected on day 4, and all animals succumbed to the disease by day 6. detailed accounts of this model have been presented at international scientific meetings by ebihara and feldmann et al. but have not been reported in a scientific journal at the time of writing this chapter. 211 guinea pig models of filovirus infection have been developed for intraperitoneal and aerosol routes using guinea pig-adapted ebov (gp-ebov) and marv (gp-marv). 212, 213 guinea pigs models of filovirus infection are quite useful in that they develop fever, which can be monitored at frequent (hourly) intervals by telemetry. additionally, the animals are large enough for regular blood sampling in which measurable coagulation defects are observed as the infection progresses. hartley guinea pigs exposed to aerosolized gp-marv or gp-ebov become moribund at times comparable to that of nhps, generally succumbing to the infection between 7 and 12 days postexposure. by aerosol exposure, gp-ebov is uniformly lethal at both high and low doses (100 or 1000 pfu target doses) but lethality drops with low (<1000 pfu) presented doses of airborne gp-marv, and more protracted disease is seen in some animals. 213 weight loss of between 15% and 25% is a common finding in guinea pigs exposed to gp-ebov or gp-marv. fever, which becomes apparent by day 5, occurs more rapidly in gp-ebov exposed guinea pigs than with gp-marv exposure. lymphocytes and neutrophils increase during the earlier part of the disease, and platelet levels steadily drop as the disease progresses. increases in coagulation time can be seen as early as day 6 postexposure. blood chemistries (i.e. alt, ast, alkaline phosphatase (alkp), and blood urea nitrogen) indicating problems with liver and kidney function are also altered late in the disease course. nhp models of filovirus infection are the preferred models for more advanced disease characterization and testing of countermeasures because they most closely mimic the disease and immune correlates seen in humans. 214 old world primates have been primarily used for the development of intraperitoneal, intramuscular, and aerosol models of filovirus infection. uniformly lethal filovirus models have been developed for most of the virus strains in cynomolgus macaques, rhesus macaques, and to a lesser degree, in agms and marmosets. [215] [216] [217] [218] [219] low-passage human isolates that have not been passaged in animals have been sought for development of nhp models to satisfy the food and drug administration (fda) animal rule. prominent features of the infections are onset of fever by day 5 postexposure, alteration in liver function enzymes (alt, ast, and alkp), decrease in platelets, and increased coagulation times. clinical disease parameters may have a slightly delayed onset in aerosol models. petichial rash is a common sign of filovirus disease and may be more frequently observed in cynomolgus macaques than in other nhp species. dyspnea late in infection is a prominent feature of disease after aerosol exposure. a number of pronounced pathology findings include multifocal necrosis and fibrin lesions, particularly within the liver and the spleen. lymphocytolysis and lymphoid depletion are also observed. multilead, surgically implanted telemetry devices are useful in the continuous collection of temperature, blood pressure, heart rate, and activity levels. as such, blood pressure drops as animals become moribund and heart rate variability (standard deviation of the heart rate) is altered late in infection. the most recently developed telemetry devices can aid in plethysomography to measure respiratory minute volume for accurate delivery of presented doses for aerosol exposure. hendra and nipah virus are unusual within the paramyxoviridae family given that they can infect a large range of mammalian hosts. both viruses are grouped under the genus henipavirus. the natural reservoirs of the viruses are the fruit bats from the genus pteropus. hendra and nipah have the ability to cause severe disease in humans with the potential for a high case fatality rate. 220 outbreaks caused by nipah virus have been recognized in malaysia, singapore, bangladesh, and india, while hendra outbreaks have yet to be reported outside of australia. 221, 222 hendra was the first member of the genus to be identified and was initially associated with an acute respiratory disease in horses. all human cases have been linked to transmission through close contact with an infected horse. there have been no confirmed cases of direct transmission from human to human or bat to human. nipah has the distinction of being able to be transmitted by humans, although the exact route is unknown. 223 the virus is susceptible to ph, temperature, and desiccation, and thus close contact is hypothesized to be needed for successful transmission. 224 both viruses have a tropism for the neurological and respiratory tract. hendra virus incubation period is 7-17 days and is marked by a flu-like illness. symptoms at this initial stage include myalgia, headache, lethargy, sore throat, and vomiting. 225 disease progression can continue to pneumonitis or encephalitic manifestations, with the person succumbing to multiorgan failure. 226 nipah virus has an incubation period of 4 days to 2 weeks. 227 much like hendra, the first signs of disease are nondescript. severe neurological symptoms subsequently develop including encephalitis and seizures that can progress to coma within 24-48 h. 228 survivors of infection typically make a full recovery; however, 22% suffer permanent sequelae, including persistent convulsions. 229 at this time, there is no approved vaccine or antiviral, and treatment is purely supportive. animal models are being used to not only test novel vaccines and therapeutics, but also deduce the early events of disease because observed human cases are all at terminal stages. the best small animal representative is the syrian golden hamster due to their high susceptibility to both henipaviruses. clinical signs upon infection recapitulate the disease course in humans including acute encephalitis and respiratory distress. challenged animals died xi. viral disease 38 . in vivo models of viral diseases affecting humans within 4-17 days postinfection. the progression of disease and timeline are highly dependent on dose and route of infection. intranasal inoculation leads to imbalance, limb paralysis, lethargy, and breathing difficulties whereas intraperitoneal resulted in tremors and paralysis within 24 h before death. virus was detected in lung, brain, spleen, kidney, heart, spinal cords, and urine, while the brain was the most affected organ. this model has been used for vaccination and passive protection studies. [230] [231] [232] the guinea pig model has not been widely used due to the lack of a respiratory disease upon challenge. 233, 234 inoculation with hendra virus via the subcutaneous route leads to a generalized vascular disease with 20% mortality. clinical signs were apparent 7-16 days postinfection with death occurring within 2 days of cns involvement. higher inocula have been associated with the development of encephalitis lesions. intradermal and intranasal injections do not lead to disease, although the animals are able to seroconvert upon challenge. inoculum source does not affect clinical progression. nipah virus challenge only develops disease upon intraperitoneal injection and results in weight loss and transient fever for 5-7 days. virus was shed through urine and found to be present in the brain, spleen, lymph nodes, ovary, uterus, and urinary bladder. 235 ferrets display the same clinical disease as seen in the hamster model and human cases. 236, 237 upon inoculation by the oronasal route, ferrets develop severe pulmonary and neurological disease within 6-9 days including fever, coughing, and dyspnea. lesions do develop in the ferrets' brains, but to a lesser degree than seen in humans. cats have also been used as an animal model for henipaviruses. disease symptoms are not dependent upon the route of infection. the incubation period is 4-8 days and leads to respiratory and neurological symptoms. 238, 239 this model has proven to be useful in a vaccine challenge model. squirrel and agms are representative of the nhp models. within the squirrel monkeys, nipah virus is introduced by either the intranasal or iv route and subsequently leads to clinical signs similar to that in humans, although intranasal challenge results in milder disease. upon challenge, only 50% animals develop disease manifestations including anorexia, dyspnea, and acute respiratory syndrome. neurological involvement is characterized by uncoordinated motor skills, loss of consciousness, and coma. viral rna can be detected in the lung, brain, liver, kidney, spleen, and lymph nodes but is only found upon iv challenge. 240 agms have been found to be a very consistent model of both viruses. intratracheal inoculation of the viruses results in 100% mortality, and death within 8.5 and 9-12 days postinfection for hendra and nipah, respectively. the animals develop severe respiratory and neurological disease with generalized vasculitis. 241, 242 the reservoir of the viruses, gray-headed fruit bats, has been experimentally challenged. due to their status as the host organism for henipaviruses, the bats do not develop clinical disease. however, hendra virus can be detected in kidneys, heart, spleen, and fetal tissue and nipah virus can be located in urine. 243 pigs have been investigated as a model as they develop a respiratory disease upon infection with both nipah and hendra. [244] [245] [246] oral inoculation does not produce a clinical disease, but subcutaneous injection represents a successful route of infection. live virus can be isolated from the oropharynx as early as 4 days postinfection. nipah can also be transmitted between pigs. nipah was able to induce neurological symptoms in 20% of the pigs, even though virus was present in all neurological tissues regardless of symptoms. 247 within the pig model, it seemed that nipah had a greater tropism for the respiratory tract, while hendra for the neurological system. horses also are able to develop a severe respiratory tract infection accompanied with fever and general weakness upon exposure to nipah and hendra. oronasal inoculation led to systemic disease with viral rna detected in nasal swabs within 2 days. 248, 249 animals died within 4 days postexposure and were found to have interstitial pneumonia with necrosis of alveoli. 250, 251 virus could be detected in all major systems. mice, rats, rabbits, chickens, and dogs have been tested but found to be nonpermissive to infection. 232, 252 suckling balb/c mice succumb to infection if the virus is inoculated intracranially. 253 embryonated chicken eggs have been inoculated with nipah virus leading to a universally fatal disease within 4-5 days postinfection. 254 respiratory syncytial virus is responsible for lower respiratory tract infections of 33 million children under the age of 5 years, which in turn results in three million hospitalizations and approximately 200,000 deaths. 255 within the united states, hospital costs alone amount to >600 million dollars. 256 outbreaks are common in the winter. 257 the virus is transmitted by large respiratory droplets that replicate initially within the nasopharynx and further spreads to the lower respiratory tract. incubation for the virus is 2-8 days. respiratory syncytial virus is highly virulent leading to very few asymptomatic infections. 258 disease manifestations are highly dependent upon the age of the individual. primary infections in neonates produce nonspecific symptoms including the overall failure to thrive, apnea, and feeding difficulties. infants present with a mild upper respiratory tract disease that could develop into bronchiolitis and bronchopneumonia. contracting the virus at this age results in an increased chance of developing childhood asthma. 259 young children develop recurrent wheezing, whereas adults exacerbate previous respiratory conditions. 260 common clinical symptoms are runny nose, sneezing, and coughing accompanied with fever. mortality rates in hospitalized children are 1-3% with the greatest burden of disease seen in 3-4-month-olds. 261 there is no vaccine available, and ribavirin usage is not recommended for routine treatment. 262 animal models were developed in the hopes of formulating an effective and safe vaccine unlike the formalin-inactivated respiratory syncytial virus vaccine (fi-rsv). this vaccineinduced severe respiratory illness in infants who received the vaccine and were subsequently infected with live virus. 263 mice can be used to model disease, although a very high intranasal inoculation is needed to achieve clinical symptoms. 264, 265 strain choice is crucial to reproducing a physiological relevant response. 266 age does not affect primary disease manifestations. 267 however, it does play a role in later sequelae showing increased airway hyperreactivity. 268 primary infection produces increased breathing with airway obstruction. 264, 269 virus was detected as early as day 3 and reached maximum titer at day 6 postinfection. clinical illness is defined in the mouse by weight loss and ruffled fur as opposed to runny nose, sneezing, and coughing as seen in humans. cotton rats are useful given that it is a small animal disease model. the virus is able to replicate to high titers within the lungs and can be detected in both the upper and lower airways after intranasal inoculation. 270, 271 it has been reported that viral replication is 50-to 1000-fold greater in the rat model than in the mouse model. 272 the rats develop mild-to-moderate bronchiolitis or pneumonia. 273 although age does not seem to factor in clinical outcome, it has been reported that older rats tend to take longer to achieve viral clearance. viral loads peak by the fifth day, dropping to below the levels of detection by 8. the histopathology of the lungs seems to be similar to that in humans after infection. 274 this model has limited usage in modeling the human immune response to infection as challenge with the virus creates a th2 response, whereas humans tend to skew toward th1. [275] [276] [277] fi-rsv disease was recapitulated upon challenge with live virus after being vaccinated twice with fi-rsv. chinchillas have been challenged experimentally via intranasal inoculation. the virus was permissive within the nasopharynx and eustachian tube. the animals displayed an acute respiratory tract infection. this model is thought to be useful in studying mucosal immunity during infection. 278 chimpanzees are permissive to replication and clinical symptoms of respiratory syncytial virus including rhinorrhea, sneezing, and coughing. adult squirrel monkeys, newborn rhesus macaques, and infant cebus monkeys were also challenged but did not exhibit any disease symptoms nor high levels of viral replication. 279 bonnet monkeys were also tested and found to develop an inflammatory response by day 7 with viral rna detected in both bronchial and alveolar cells. 280 the chimpanzee model has proven to be useful for vaccine studies. 281, 282 sheep have also been challenged experimentally since they develop respiratory disease when exposed to ovine respiratory syncytial virus. 283 lambs were also found to be susceptible to human respiratory syncytial infection. 284, 285 when inoculated intratracheally, the lambs developed an upper respiratory tract infection with cough after 6 days. some lambs went on to develop lower respiratory disease including bronchiolitis. the pneumonia resolved itself within 14 days. during the course of disease, viral replication peaked at 6 days, and rapidly declined. studying respiratory disease in sheep is beneficial given the shared structural features between them and humans. 286, 287 the influenza viruses consist of three types: influenza a, b, and c, based on antigenic differences. influenza a is further classified by subtypes; 16 ha and 9 na subtypes are known. seasonal influenza is the most common infection and usually causes a self-limited febrile illness with upper respiratory symptoms and malaise that resolves within 10 days. 288 the rate of infection is estimated at 10% in the general population and can result in billions of dollars of loss annually from medical costs and reduced work-force productivity. approximately 40,000 people in the united states die each year from seasonal influenza. 289 thus, vaccines and therapeutics play a critical role in controlling infection, and development using animal models is ongoing. 290 influenza virus replicates in the upper and lower airways, peaking at approximately 48 h postexposure. infection can be more severe in infants and in children under the age of 22 years, people over the age of 65 years, or immunocompromised individuals in whom viral pneumonitis or pneumonia can develop or bacterial superinfection resulting in pneumonia or sepsis. 291 pneumonia from secondary bacterial infection, such as streptococcus pneumonia, streptococcus pyrogenes, and neisseria meningitides, and more rarely, staphylococcus aureus, is more common than viral pneumonia from the influenza itself, accounting for approximately 27% of all influenza-associated fatalities. 292 death, often due to ards can occur as early as 2 days after the onset of symptoms. lung histopathology in severe cases may include dad, alveolar edema and damage, hemorrhage, fibrosis, and inflammation. 288 the h5n1 avian strain of influenza has lethality rates of approximately 60% (of known cases), likely because the virus preferentially binds to the cells of the lower respiratory tract, and thus, the potential for global spread is a major concern. 293 the most frequently used animal models of influenza infection include mice, ferrets, and nhps. a very thorough guide to working with mouse, guinea pig, ferret, and cynomolgus models was published by kroeze et al. 294 lethality rate can vary with the virus strain used (with or without adaptation), dose, route of inoculation, age, and genetic background of the animal. the various animal models can capture differing diseases caused by influenza: benign, severe, superinfection and sepsis, severe with ards, and neurologic manifestations. 290 also, models can use seasonal or avian strains and models have been developed to study transmission, important for understanding the potential for more lethal strains such as h5n1 for spreading among humans. mouse models of influenza infection are very predictive for antiviral activity and tissue tropism in humans, and are useful in testing and evaluating vaccines. 295 inoculation is by the intranasal route, using approximately 60 ml of inoculum in each nare of anesthetized mice. exposure may also be to small particle aerosols containing influenza with an mmad of <5 mm. most inbred strains are susceptible, with particularly frequent use of balb/c followed by c57bl/6j mice. males and females have equivalent disease, but influenza is generally more infectious in younger 2-to 4-week-old (8-10 g) mice. mice are of somewhat limited use in characterizing the immune response to influenza. mice lack the mxa gene, which is an important part of the human innate immune response to influenza infection. the mouse homolog to mxa, mx1, is defective in most inbred mouse strains. 296 weight loss or reduced weight gain, decreased activity, huddling, ruffled fur, and increased respiration are the most common clinical signs. for more virulent strains, mice may require euthanasia as early as 48 h postexposure, but most mortality occurs from 5 to 12 days postexposure accompanied by decreases in rectal temperature. 297 pulse oximeter readings and measurement of blood gases of oxygen saturation are also used to determine the impact of influenza infection on respiratory function. 298 virus can be isolated from bronchial lavage fluids throughout the infection and from tissues after euthanasia. for influenza strains with mild-tomoderate pathogenicity, disease is nonlethal and virus replication is detected within the lungs, but usually not other organs. increases in serum alpha-1-acidglycoprotein and lung weight are also frequently present. however, mice infected with influenza do not develop fever, dyspnea, nasal exudates, sneezing, or coughing. mice can be experimentally infected with influenza a or b, but the virus generally requires adaptation to produce clinical signs. mice express the receptors for influenza attachment in the respiratory tract; however, the distribution varies and sa 2,3 predominates over sa 2,6 which is why h1, h2, and h3 subtypes usually need to be adapted to mice and h5n1, h2, h6, and h7 viruses do not require adaptation. 299 to adapt, mice are infected intratracheally or intranasally by virus isolated from the lungs, and reinfected into mice and then the process is repeated a number of times. once adapted, influenza strains can produce severe disease, systemic spread, and neurotropism. however, h5n1 and the 1918 pandemic influenza virus can cause lethal infection without adaptation. 300 h5n1 infection of mice results in viremia and viral replication in multiple organ systems, severe lung pathology, fulminant diffuse interstitial pneumonia, pulmonary edema, high levels of proinflammatory cytokines, and marked lymphopenia. 301 as in humans, the virulence of h5n1 is attributable to damage caused by an overactive host immune response. additionally, mice infected with the 1918 h1n1 influenza produces severe lung pathology and oxygen saturation levels that decrease with increasing pneumonia. 302 in superinfection models, a sublethal dose of influenza is given to mice followed 7 days later by intranasal inoculation of a sublethal dose of a bacterial strain such as s. pneumoniae or s. pyrogenes. 303 morbidity, characterized by inflammation in the lungs, but not bacteremia, begins a couple of days after superinfection and may continue for up to 2 weeks. at least one transmission model has also been developed in mice. with h2n2 influenza, transmission rates of up to 60% among cage mates can be achieved after infection by the aerosol route and cocaging after 24 h. 304 domestic ferrets (mustela putorius furo) are frequently the animal species of choice for influenza animal studies because the susceptibility, clinical signs, peak virus shedding, kinetics of transmission, local expression of cytokine mrnas, and pathology resemble that of humans. [305] [306] [307] ferrets also have airway morphology, respiratory cell types, and a distribution of influenza receptors (sa 2,6 and sa 2,3) within the airways similar to that of humans. 308 influenza was first isolated from ferrets infected intranasally with throat washes from humans harboring the infection and ferret models have since been used to test efficacy of vaccines and therapeutic treatments. 309 when performing influenza studies in ferrets, animals should be serologically negative for circulating influenza viruses. infected animals should be placed in a separate room from uninfected animals. if animals must be placed in the same room, uninfected ferrets should be handled before infected ferrets. anesthetized ferrets are experimentally exposed to influenza by intranasal. inoculation of 0.25-0.5 ml containing approximately 10 4 -10 6 egg id50 dropwise to each nostril. influenza types a and b naturally infect ferrets, resulting in an acute illness, which usually lasts 3-5 days for mildly to moderately virulent strains. 310 ferrets are more susceptible to influenza a than to influenza b strains and are also susceptible to avian influenza h5n1 strains without adaptation. 311 virulence and degree of pneumonitis caused by different influenza subtypes and strains vary from mild to severe and generally mirror that seen in humans. nonadapted h1n1, h2n2, and h3n2 have mild-to-moderate virulence in ferrets. strains of low virulence have predominant replication in the nasal turbinates. clinical signs and other disease indicators are similar to that of humans with a mild respiratory disease, sneezing, nasal secretions containing virus, fever, weight loss, high viral titers, and inflammatory infiltrate in the airways, bronchitis, and pneumonia. 312 replication in both the upper and lower airways is associated with more severe disease and greater mortality. additionally, increased expression of proinflammatory mediators and reduced expression of antiinflammatory mediators in the lower respiratory tract ferrets correlates with severe disease and lethal outcome. h5n1-infected ferrets develop severe lethargy, greater ifn response, transient lymphopenia, and replication in respiratory tract, brain, and other organs. 313 old and new world primates are susceptible to influenza infection and have an advantage over ferret and mouse models, which are deficient for h5n1 vaccine studies because there is a lack of correlation with hemagglutination inhibition. 314 of old world primates, cynomolgus macaque (m. fascicularis) are most frequently used for studies of vaccines and antiviral drug therapies. 315, 316 h5n1 and h1n1 1918 infections of cynos are very similar to those in humans. 317 cynos develop fever and ards upon intranasal inoculation of h5n1 with necrotizing bronchial interstitial pneumonia 318 nhps are challenged by multiple routes (ocular, nasal, and tracheal) simultaneously 1 ã� 10 6 pfu per site. virus antigen is primarily localized to the tonsils and pulmonary tissues. infection of cynos with h5n1 results in fever, lethargy, nasal discharge, anorexia, weight loss, nasal and tracheal washes, pathologic and histopathologic changes, and alveolar and bronchial inflammation. the 1918 h1n1 caused a very high mortality rate due to an aberrant immune response and ards and had >50% lethality (humans only had a 1-3% lethality). ards and mortality also occur with the more pathogenic strains, but nhps show reduced susceptibility to less virulent strains such as h3n2. 299 influenzainfected rhesus macaques represent a mild disease model pathogenesis for vaccine and therapeutic efficacy studies. 319 other nhp models include influenza infection of pigtailed macaques as a mild disease model and infection of new world primates such as squirrel and cebus monkeys. 320 rats (f344 and sd) inoculated with rat-adapted h3n2 developed inflammatory infiltrates and cytokines in bronchoalveolar lavage fluids, but had no lethality and few histopathological changes. 321 additionally, an influenza transmission model has been developed in guinea pigs as an alternative to ferrets. 322 cotton rats (sigmodon hispidus) have been used to test vaccines and therapeutics in a limited number of studies. 323, 324 cotton rats have an advantage over mice in that the immune system is similar to humans (including the presence of the mx gene) and influenza viruses do not have to be adapted. 325, 326 nasal and pulmonary tissues of cotton rats were infected with unregulated cytokines and lung viral load peaking at 24 h postexposure. virus was cleared from the lung by day 3 and from the nares by day 66, but animals had bronchial and alveolar damage, and pneumonia for up to 3 weeks. there is also a s. aureus superinfection model in cotton rats. 327 coinfection resulted in bacteremia, high bacterial load in lungs, peribronchiolitis, pneumonitis, alveolitis, hypothermia, and higher mortality. domestic pig influenza models have been developed for vaccine studies for swine flu. pigs are susceptible in nature as natural or intermediate hosts but are not readily susceptible to h5n1. 328, 329 although pigs infected with influenza may have fever, anorexia, and respiratory signs such as dyspnea and cough, mortality is rare. 330 size and space requirements make this animal difficult to work with, although the development of minipig (ellegaard gottingen) models may provide an easier-to-use alternative. incubation period, animals exhibit signs of fever, hepatitis, and abortion, which is a hallmark diagnostic sign known among farmers. 331 mosquito vectors, unpasteurized milk, aerosols of infected animal's body fluids, or direct contact with infected animals are the important routes of transmission to humans. 332,333 after 2-6 days of incubation period, rvfv causes a wide range of signs and symptoms in humans ranging from asymptomatic to severe disease with hepatitis, vision loss, encephalitis, and hemorrhagic fever. [334] [335] [336] depending on the severity of the disease when the symptoms start, 10-20% of the hospitalized patients might die in 3-6 days or 12-17 days after the disease onset. 334 hepatic failure, renal failure or disseminated intravascular coagulation (dic), and encephalitis are demonstrated within patients during postmortem examination. mice are one of the most susceptible animal species to rvfv infection. subcutaneous or intraperitoneal routes of infection cause acute hepatitis and lethal encephalitis at a late stage of the disease in mice. 337, 338 mice start to exhibit signs of decreased activity and ruffled fur by day 2-3 postexposure. immediately after these signs are observed, they become lethargic and generally die 3-6 days postexposure. ocular diseases or hemorrhagic form of the disease has not been observed in mice models so far. 334 increased viremia and tissue tropism were reported in mice with 338 increased liver enzymes and lymphopenia observed in sick mice. rats and gerbils are also susceptible to rvfv infection. rats' susceptibility is dependent on the rat strain used for the challenge model. there was also noted an age dependence in susceptibility of rats. although wistar-furth and brown norway strains and young rats are highly susceptible to rvfv infection, fisher 344, buffalo and lewis strains, and old rats demonstrated resistance to infection. 339, 340 similar pathologic changes such as liver damage and encephalopathy were observed in both rats and mice. there was no liver involvement in the gerbil model and animals died from severe encephalitis. the mortality rate was dependent on the strain used and the dose given to gerbils. 341 similar to the rat model, the susceptibility of gerbils was also dependent on age. so far, studies showed that rvfv does not cause uniform lethality in an nhp model. intraperitoneal, intranasal, iv, and aerosol routes have been used to develop the nhp model. rhesus macaques, cynomolgus macaques, african monkeys, and south american monkeys were some of the nhp species used for this effort. 342 monkeys showed a variety of signs ranging from febrile disease to hemorrhagic disease and mortality. temporal viremia, increased coagulation parameters (pt, aptt), and decreased platelets were some other signs observed in nhps. animals that succumbed to disease showed very similar pathogenesis to those seen in humans such as pathological changes in liver and hemorrhagic disease. there was no ocular involvement in this model. recently, smith et al. compared iv, intranasal, and subcutaneous routes of infection in common marmosets and rhesus macaques. 343 marmosets were more susceptible to rvfv infection than were rhesus macaques with marked viremia, acute hepatitis, and late onset of encephalitis. increased liver enzymes were observed in both species. necropsy results showed enlarged livers in the marmosets exposed by iv or subcutaneous routes. although there were no gross lesions in the brains of marmosets, histopathology showed encephalitis in the brains of intranasally challenged marmosets. crimean-congo hemorrhagic fever virus (cchfv) generally circulates in nature unnoticed in an enzootic tick-vertebrate-tick cycle and similar to other zoonotic agents, seems to produce little or no disease in its natural hosts, but causes severe disease in humans. cchfv transmits to humans by ixodid ticks, direct contact with sick animals/humans, or body fluids of animals/humans. 344 incubation, prehemorrhagic, hemorrhagic, and convalescence are the four phases of the disease seen in humans. the incubation period lasts 1-9 days. during the prehemorrhagic phase, patients show signs of nonspecific flu-like disease for approximately a week. the hemorrhagic period results in circulatory shock and dic in some patients. 345, 346 over the years, several attempts have been made to establish an animal model for cchf in adult mice, guinea pigs, hamsters, rats, rabbits, sheep, nhps, etc. [347] [348] [349] [350] until recently, the only animal that manifests disease is the newborn mouse. infant mice infected with cchfv intraperitoneally caused fatality around day 8 postinfection. 351 pathogenesis studies showed that virus replication was first detected in the liver, with subsequent spread to the blood (serum). virus was detected very late during the disease course in other tissues including the heart (day 6) and the brain (day 7). the recent studies using knockout adult mice were successful to develop a lethal small animal model for cchfv infection. 352, 353 bente et al. infected stat1 knockout mice by the intraperitoneal route. in this model, after the signs of fever, leucopenia, thrombocytopenia, viremia, elevated liver enzymes, and proinflammatory cytokines, mice were moribund and succumbed to disease in 3-5 days of postexposure. the second model was developed by using ifn-alpha/beta (ifna/b) receptor knockout mice. 353 similar observations were made in this model as in the stat1 knockout mouse model. the animals were moribund and died 2-4 days after exposure with high viremia levels in the liver and spleen. other laboratory animals, including nhps, show little or no signs of infection or disease when infected with cchfv. 348 butenko et al. used agms (cercopithecus aethiops) for experimental cchfv infections. except one monkey with a fever on day 4 postinfection, the animals did not exhibit signs of disease. antibodies to the virus were detected in three out of five monkeys, including the one with fever. in 1975, fagbami et al. infected two patas monkeys (cercopithecus erythrocebuserythrocebus) and one guinea baboon (papio papio) with cchfv. 347 although all three animals had low level viremia between days 1 and 5 after inoculation, only the baboon serum had neutralizing antibody activity on day 137 postinfection. similar results were obtained when horses and donkeys have been used for experimental cchfv infections. donkeys develop a low-level viremia, 354 and horses developed little or no viremia, but high levels of virus-neutralizing antibodies, which remained stable for at least 3 months. these studies suggest that horses may be useful in the laboratory to obtain serum for diagnostic and possible therapeutic purposes. 355 shepherd et al. infected 11 species of small african wild mammals and laboratory rabbits, guinea pigs, and syrian hamsters with cchfv. 349 although scrub hares (lepus saxatilis), cape ground squirrels (xerus inauris), red veld rats (aethomys chrysophilus), white-tailed rats (mystromys pumilio), and guinea pigs had viremia; south african hedgehogs (atelerix frontalis), highveld gerbils (tatera brantsii), namaqua gerbils (desmodillus auricularis), two species of multimammate mouse (mastomys natalensis and m. coucha), and syrian hamsters were negative. all species regardless of viremia levels developed antibody responses against cchfv. iv and intracranially infected animals showed the onset of viremia earlier than those infected by the subcutaneous or intraperitoneal routes. the genus hantavirus is unique among the family bunyaviridae in that it is not transmitted by an arthropod vector, but rather by rodents. 356 rodents of the family muridae are the primary reservoir for hantaviruses. infected host animals develop a persistent infection that is typically asymptomatic. transmission is achieved by the inhalation of infected rodent saliva, feces, and urine. 357 human infections can normally be traced to a rural setting with activities such as farming, land development, hunting, and camping as possible sites of transmission. rodent control is the primary route of prevention. 358 the viruses have a tropism for endothelial cells within the microvasculature of the lungs. 359 there are two distinct clinical diseases that infection can yield; hemorrhagic fever with renal syndrome (hfrs) due to infection with old world hantaviruses or hantavirus pulmonary syndrome (hps) caused by new world hantaviruses. 360 hfrs is mainly seen outside of the americas and is associated with the hantaviruses dobrava-belgrade (also known as dobrava), hantaan, puumala, and seoul. 358 incubation lasts two to three weeks and presents as flulike in the initial stages that can further develop into hemorrhagic manifestations and ultimately renal failure. thrombocytopenia subsequently develops, which can further progress to shock in approximately 15% patients. the overall mortality rate is 7%. infection with dobrava and hantaan viruses are typically linked to the development of severe disease. hps was first diagnosed in 1993 within the southwestern united states when healthy young adults became suddenly ill, progressing to severe respiratory distress and shock. the etiological agent responsible for this outbreak was identified as sin nombre virus. 361 this virus is still the leading cause within na of hps. hps due to other hantaviruses has been reported in argentina, bolivia, brazil, canada, chile, french guiana, panama, paraguay, and uruguay. 362, 363 the first report of hps in maine was recently documented. 361 andes virus was first identified in outbreaks in chile and argentina. this hantavirus is distinct in that it can be transmitted between humans. 364 the fulminant disease is more lethal than that observed for hfrs with a mortality rate of 40%. there are four phases of disease including prodromal, pulmonary, cardiac depression, and hematologic manifestation. 365 incubation typically occurs 14-17 days after exposure. 366 unlike hfrs, renal failure is not a major contributing factor to the disease. there is a short prodromal phase that gives way to cardiopulmonary involvement accompanied by cough and gastrointestinal symptoms. it is at this point that individuals are typically admitted to the hospital. pulmonary function is hindered and continues to suffer within 48 h after cardiopulmonary involvement. interstitial edema and air-space disease normally follow. in fatal cases, cardiogenic shock has been noted. 367 vaccine development has been hampered by the vast diversity of hantaviruses and the limited number of outbreaks. 368 syrian golden hamsters are the most widely used small animal models for hantavirus infection. hamsters inoculated intramuscularly with a passaged andes viral strain died within 11 days postinfection. clinical signs did not appear until 24 h before death at which point the hamsters were moribund and in respiratory distress. mortality was dose dependent, with high inoculums leading to a shorter incubation before death. during the same study, hamsters were inoculated with a passaged sin nombre isolate. no hamsters developed any symptoms during the course of observation. although an antibody response to the virus that was not dose dependent was determined via an enzymelinked immunosorbent assay. hamsters infected with andes virus were found to have significant histopathological changes to their lung, liver, and spleen. all had an interstitial pneumonia with intraalveolar edema. infectious virus could be recovered from these organs. viremia began on day 8 and lasted up to 12 days postinfection. infection of hamsters with andes virus yielded a similar clinical disease progression as is seen in human hps including rapid progression to death, fluid in the pleural cavity, and significant histopathological changes to the lungs and spleen. a major deviation in the hamster model is the detection of infectious virus within the liver. 369 lethal disease can be induced in newborn mice but does not recapitulate the clinical symptoms observed in human disease. 370 adult mice exposed to hantaan virus leads to a fatal disease dependent upon viral strain and route of infection. the disease progression is marked by neurological or pulmonary manifestations that do not mirror human disease. 371, 372 knockout mice lacing ifn-a/b were found to be highly susceptible to hantaan virus infection. 373 in a study looking at a panel of laboratory strains of mice, c57bl/6 mice were found to be most susceptible to a passaged hantaan viral strain injected intraperitoneally. animals progressed to neurological manifestation including paralyses and convulsions and succumbed to infection within 24-36 h postinfection. clinical disease was markedly different than that observed in human cases. 372 nhps have been challenged with new world hantaviruses; however, no clinical signs were reported. 374, 375 cynomolgus monkeys challenged with a clinical isolate of puumala virus developed a mild disease. 376, 377 challenge with andes virus to cynomolgus macaques by both iv and aerosol exposure led to no signs of disease. all animals did display a drop in total lymphocytes within 5 days postinfection. aerosol exposure led to 4 of 6 monkeys and 8 of 11 iv injected monkeys developed viremia. infectious virus could not be isolated from any of the animals. the family arenaviridae is composed of two serogroups: old world arenaviruses including lassa fever virus and lymphocytic choriomeningitis virus and the new world viruses of pichinde virus and junin virus. all these viruses share common clinical manifestations. 378 lassa fever virus is endemic in parts of west africa and outbreaks are typically seen in the dry season between january and april. 379 this virus is responsible for 100,000-500,000 infections per year, leading to approximately 5,000 deaths. 380 outbreaks have been reported in guinea, sierra leone, liberia, nigeria, and central african republic. however, cases sprung up in germany, the netherlands, the united kingdom, and the united states due to transmission to travelers on commercial airlines. 381 transmission of this virus typically occurs via rodents, in particular the multimammate rat, mastomys species complex. 379 humans become infected by inhaling the aerosolized virus or eating contaminated food. there has also been noted human-to-human transmission by direct contact with infected secretions or needle-stick injuries. the majority of infections are asymptomatic; however, severe disease can occur in 20% of individuals. the incubation period is from 5 to 21 days, and the initial onset is characterized by flulike illness. this is followed by diarrheal disease that can progress to hemorrhagic symptoms including encephalopathy, encephalitis, and meningitis. a third of patients develop deafness in the early phase of disease, which is permanent for a third of those affected. the overall fatality is about 1%; however, of those admitted to the hospital, it is between 15% and 25%. there is no approved vaccine, and besides supportive measures, ribavirin is effective only if started within 7 days. 382, 383 the primary animal model used to study lassa fever is the rhesus macaque. 384 aerosolized infection of lymphocytic choriomeningitis virus has been a useful model for lassa fever. both rhesus and cynomolgus monkeys exposed to the virus developed disease, but rhesus more closely mirrored the disease course and histopathology observed in human infection. 385 iv or intragastric inoculation of the virus led to severe dehydration, erythematous skin, submucosal edema, necrotic foci in the buccal cavity, and respiratory distress. the liver was severely affected by the virus as depicted by measuring the liver enzymes ast and alt. 386 disease was dose-dependent with iv, intramuscular, and subcutaneous inoculation requiring the least amount of virus to induce disease. aerosol infections and eating contaminated food could also be used, and mimic a more natural route of infection. 387 within this model, the nhp becomes viremic after 4-6 days. clinical manifestations were present by day 7, and death typically occurred within 10-14 days. 388, 389 intramuscular injection of lassa virus into cynomolgus monkeys also produced a neurological disease due to lesions within the cns. 390 this pathogenicity is seen in select cases of human lassa fever. 391, 392 a marmoset model has recently been defined using a subcutaneous injection of lassa fever virus. virus was initially detected by day 8 and viremia achieved by day 14. liver enzymes were elevated, and an enlarged liver was noted upon autopsy. there was a gradual reduction in platelets and interstitial pneumonitis diagnosed in a minority of animals. the physiological signs were the same as seen in fatal human cases. 393 mice develop a fatal neurological disorder upon intracerebral inoculation with lassa, although the outcome of infection is completely dependent upon the major histocompatibility complex (mhc) background and age of animal along with the route of inoculation. 394 guinea pig inbred strain 13 was found to be highly susceptible to lassa virus infection. the outbreed hartley strain was less susceptible, and thus, strain 13 has been the preferred model given its assured lethality. the clinical manifestations mirror those seen in humans and rhesus. 395 infection with pichinde virus that has been passaged in guinea pigs has also been used. disease signs include fever, weight loss, vascular collapse, and eventual death. 396, 397 the guinea pig is an excellent model given that it not only results in similar disease pattern as humans but also the viral distribution is similar along with the histopathology and immune response. 398, 399 infection of hamsters with a cotton rat isolate of pirital virus is similar to what is characterized in humans, and the nhp and guinea pig model. the virus was injected intraperitoneally resulting in the animals becoming lethargic and anorexic within 6-7 days. virus was first detected at 3 days and reached maximum titers within 5 days. neurological symptoms began to appear at the same time, and all the animals died by day 9. pneumonitis, pulmonary hemorrhage, and edema were also present. 400 these results were recapitulated with a nonadapted pichinde virus. [401] [402] [403] globally, diarrheal disease is the leading cause of death with rotavirus being one of the main etiological agents responsible. according to the world health organization, rotavirus alone is responsible for a third of all hospitalization related to diarrhea and 500,000-600,000 deaths per year. 404 the virus is very stable due to its three-layer capsid, which allows it to be transmitted via the oral-fecal route, depositing itself in the small intestine. rotavirus is highly contagious, and only 10 viruses are needed to cause symptomatic disease. 405 the host determinant with the greatest influence on clinical outcome is age. neonates typically are asymptomatic, which is suggested to be due to the existence of maternal antibodies. hence, the most susceptible age group is 3 months to 2 years, coinciding with a drop in these protective antibodies. 406 within this age range, children will develop noninflammatory diarrhea. virus replicates in the intestinal villus enterocytes resulting in their destruction and malabsorption of needed electrolytes and nutrients. symptoms of disease include watery, nonbloody diarrhea with vomiting, fever, and potentially dehydration that lasts up to a week. 407 there is a short episode of viremia during the course of infection. 408 mice can be used as both an infection and disease model depending upon age at challenge. mice <14 days old develop disease, whereas older mice are able to clear the infection before the onset of symptoms. 409 this halts the study of active vaccination against disease in the infection model. in the adult mouse model, the course of the infection is monitored via viral shedding within the stool. 410 infant mice, specifically balb/c, receiving an oral inoculation of a clinical strain of virus developed diarrhea within 24 h postinfection, and 95% of those exposed developed symptoms within 72 h postinfection. symptoms lasted from 2 to 4 days with no mortality. viral shedding was at its peak at 24 h and lasted up to 5 days. there were noted histopathological changes within the small intestine localized to the villi that was reversible. 407 within the adult mouse model, oral inoculation of a mouse rotavirus strain showed viral shedding by 3 days lasting up until 6 days postinfection. 410 these mouse models have been used to study correlates of protection and therapeutic efficacy including gastro-gard ã� . 409, 411, 412 rats can also be used as disease models depending upon the strain of rat. 413, 414 suckling fischer 344 rats were exposed to a simian strain of rotavirus orally. the rats were susceptible to diarrheal disease till they were 8 days old with age determining the length of viral shedding. 415 rats have mainly been used to study the correct formulation for oral rehydration. these rodents are large enough to perform in situ intestinal perfusions. within these studies, 8-day-old rats were infected with a rat strain of rotavirus by orogastric intubation. within 24 h postinfection, the rats developed diarrhea, at which point the small intestine was perfused to compare differing solutions of oral rehydration. 416 gnotobiotic pigs are also used given that they can be infected with both porcine and human strains. 417 they are susceptible to developing clinical disease from human strains up to 6 weeks of age. they allow for the analysis of the primary immune response to the virus given that they do not receive transplacental maternal antibodies and are immune competent at birth. 418 another advantage of this model is that the gastrointestinal physiology and mucosal immune system closely resemble that of humans. 419 this model has been useful in studying correlates of protection. gnotobiotic and colostrum-deprived calves have also been used as an experimental model of rotavirus infection. they are able to develop diarrhea and shed live virus. 420 gnotobiotic lambs can also develop clinical disease upon oral inoculation with clinical strains. 421 infant baboons, agms, and rhesus macaques have all proven to be infection models with severity measure by viral shedding. 422, 423 retroviridae human immunodeficiency virus type 1 the lentiviruses are a subfamily of retroviridae, which includes human immunodeficiency virus (hiv), a virus that infects 0.6% of the world's population. a greater proportion of infections and deaths occur in sub-saharan africa. worldwide, there are approximately 1.8 million deaths per year with >260,000 being children. transmission of hiv occurs by exposure to infectious body fluids. there are two species, hiv-1 and hiv-2, with hiv-2 having lower infectivity and virulence (confined mostly to west africa). the vast majority of cases worldwide are hiv-1. 424 hiv targets t-helper cells (cd4ã¾), macrophages, and dendritic cells. 425 acute infection occurs 2-4 weeks after exposure, with flu-like symptoms and viremia followed by chronic infection. symptoms in the acute phase may include fever, body aches, nausea, vomiting, headache, lymphadenopathy, pharyngitis, rash, and sores in the mouth or esophagus. cd8ã¾ t-cells are activated which kill hiv-infected cells, and are responsible for antibody production and seroconversion. acquired immune deficiency syndrome (aids) develops when cd4ã¾ t-cells decline to <200 cells per microliter; thus, cell-mediated immunity becomes impaired, and the person is more susceptible to opportunistic infections and certain cancers. humanized mice, created by engrafting human cells and tissues into scid mice, have been critical for the development of mouse models for the study of hiv infection. a number of different humanized mouse models allow for the study of hiv infection in the context of intact and functional human innate and adaptive immune responses. 426 the scidhu hiv infection model has proven to be useful, particularly in screening antivirals and therapeutics. 427 a number of different humanized mouse models have been developed for the study of hiv, including rag1ã�/ã�gcã�/ã�, rag2ã�/ ã�gcã�/ã�, nod/scidgcã�/ã� (hnog), nod/ scidgcã�/ã� (hnsg), nod/scid blt, and nod/ scidgcã�/ã� (hnsg) blt. cd34ã¾ human stem cells derived from the umbilical cord blood or fetal liver are used for humanization. 428 hiv-1 infection by intraperitoneal injection can be successful with as little as 5% peripheral blood engraftment. 429 vaginal and rectal transmission models have been developed in blt scidhu mice in which mice harbor human bone marrow, liver, and thymus tissue. hiv-1 viremia occurs within approximately 7 days pi. 430 in many of these models, spleen, lymph nodes, and thymus tissues are highly positive for virus, similar to humans. 431 importantly, depletion of human t-cells can be observed in blood and lymphoid tissues of hiv-infected humanized mice, and at least some mechanisms of pathogenesis that occur in hiv-infected humans also occur in the hiv-infected humanized mouse models. 432 the advantage of these models is that these mice are susceptible to hiv infection, and thus, the impact of drugs on the intended viral targets can be tested. one caveat is that although mice have a "common mucosal immune system," humans do not, due to the differences in the distribution of addressins. 433 thus, murine mucosal immune responses to hiv do not reflect those of humans. there are a number of important nhp models for human hiv infection. simian immunodeficiency virus (siv) infection of macaques is widely considered to be the best platform for modeling hiv infection of humans. importantly, nhps have similar, pharmacokinetics, metabolism, mucosal t-cell homing receptors, and vascular addressins to those of humans. thus, although the correlates of protection against hiv are still not completely known, immune responses to hiv infection and vaccination are likely comparable. these models mimic infection through the use of contaminated needles (iv), sexual transmission (vaginal or rectal), and maternal transmission in utero, or through breast milk. [434] [435] [436] there are also macaque models to study the emergence and clinical implications of hiv drug resistance. 437 these models most routinely use rhesus macaques (macaca mulatta), cynomolgus macaques (macaca fasicularis), and pigtailed macaque (macaca nemestrina). animals of all ages are used, depending on the needs of the study. for instance, the use of newborn macaques may be more practical for evaluating the effect of prolonged drug therapy on disease progression; however, adult nhps are more frequently used. studies are performed in bsl-2 animal laboratories, and nhps must be of simian type-d retrovirus free and siv seronegative. siv infection of pigtailed macaques is a useful model for hiv peripheral nervous system pathology, wherein an axotomy is performed and regeneration of axons is studied. 438 challenges may be through a single high dose. iv infection of rhesus macaques with 100 tcid 50 of the highly pathogenic siv/deltab670 induces aids in most macaques within 5-17 months (mean of 11 months). 439 peak viremia occurs around week 4. aids in such models is often defined as cd4ã¾ t-cells that have dropped to <50% of the baseline values. alternatively, repeated low-dose challenges are often used, depending on the requirements of the model. 440, 441 because nhps infected with hiv do not develop an infection with a clinical disease course similar to that in humans, siv or siv/hiv-1 laboratory-engineered chimeric viruses (simian-human immunodeficiency virus or shiv) are used as surrogates. nhps infected with pathogenic siv may develop clinical disease, which progresses to aids and are thus useful pathogenesis models. a disadvantage is that siv is not identical to hiv-1 and is more closely related to hiv-2. however, the polymerase region of siv is 60% homologous to that of hiv-1, and it is susceptible to many reverse transcriptase (rt) and protease inhibitors. siv is generally not susceptible to nonnucleoside inhibitors; thus, hiv-1 rt is usually put into siv for such studies. 442 sivmac239 is similar to hiv in the polymerase region and is therefore susceptible to nucleoside, rt or integrase inhibition. 443 nhps infected with sivmac239 have an asymptomatic period and disease progression resembling aids in humans, characterized by weight loss/wasting, cd4ã¾ t-cell depletion. additionally, sivmac239 uses the cxcr5 chemokine receptor as a coreceptor, similar to hiv, which is important for drugs that target entry. 444 nhps infected with shiv strains may not develop aids, but these models are useful in testing vaccine efficacy. for example, rt-shivs and env-shivs are useful for the testing and evaluation of drugs that may target the envelope or rt, respectively. 442 one disadvantage of the highly virulent env-shiv (shiv-89.6 p) is that it uses the cxcr4 coreceptor. of note, env-shivs that do use the cxcr5 coreceptor are less virulent; viremia develops and then resolves without further disease progression. 445 simian-tropic (st) hiv-1 contains the vif gene from siv. infection of pigtailed macaques with this virus results in viremia, which can be detected for three months, followed by clearance. 446 a number of routes are used for siv or shiv infection of nhps, with iv inoculation being the most common route. mucosal routes include vaginal, rectal, and intracolonic. mucosal routes require a higher one-time dose than does the iv route for infection. for the vaginal route, female macaques are treated with depo-provera (estrogen) one month before infection to synchronize the menstrual cycle, thin the epithelial lining of the vagina, and increase the susceptibility to infection by atraumatic vaginal instillation. 447 upon vaginal instillation of 500 tcid50 of shiv-162p3, peak viremia was seen around 12 days postexposure with >10 7 copies per milliliter and dropping thereafter to a constant level of 10 4 rna copies per milliliter at 60 days and beyond. in another example, in an investigation of the effect of vaccine plus vaginal microbicide on preventing infection, rhesus macaques were vaginally infected with a high dose of sivmac251. 448 an example of an intrarectal model used juvenile (2year-old) pigtailed macaques, challenged intrarectally with 10 4 tcid 50s of siv mne027 to study the pathogenesis related to the virulence factor, vpx. 449 here, viremia peaked at approximately 10 days with >10 8 copies per milliliter. viral rna was expressed in the cells of the mesenteric lymph nodes. the male genital tract is seen as a viral sanctuary with persistently high levels of hiv shedding even with antiretroviral therapy. to better understand the effect of highly active antiretroviral therapy on virus and t-cells in the male genital tract, adult (3-to 4-year old) male cynomolgus macaques were intravenously inoculated with 50 aid50s of sivmac251, and the male genital tract tissues were tested after euthanasia by pcr, ihc, and in situ hybridization. 450 pediatric models have been developed in infant rhesus macaques through the infection of siv, allowing for the study of the impact of developmental and immunological differences on the disease course. 451 importantly, mother-to-infant transmission models have also been developed. 452 pregnant female pigtailed macaques were infected during the second trimester with 100 mid 50 shiv-sf162p3 by the iv route. four of nine infants were infected; one in utero and three either intrapartum or immediately postpartum through nursing. this model is useful for the study of factors involved in transmission and the underlying immunology. nhps infected with siv or shiv are routinely evaluated for weight loss, activity level, stool consistency, appetite, virus levels in blood, and t-cell populations. cytokine and chemokine levels, antibody responses, and cytotoxic t-lymphocyte responses may also be evaluated. the ultimate goal of an hiv vaccine is sterilizing immunity (preventing infection). however, a more realistic result may be to reduce severity of infection and permanently prevent progression. strategies have included live attenuated, nonreplicating and subunit vaccines. these have variable efficacy in nhps due to the genetics of the host (mhc and terminal-repeat retrotransposon in miniature (trim) alleles), differences between challenge strains, and challenge routes. 453 nhp models have led to the development of antiviral treatments that are effective at reducing viral load and indeed transmission of hiv among humans. one preferred variation on the models for testing the longterm clinical consequences of antiviral treatment is to use newborn macaques and treat from birth onward, in some cases more than a decade. 454 unfortunately, however, successes in nhp studies do not always translate to success in humans, as seen with the recent step study that used an adenovirus-based vaccine approach. 455 vaccinated humans were not protected and may have even been more susceptible to hiv, viremia was not reduced, and the infections were not attenuated as hoped. with regard to challenge route, iv is more difficult to protect than mucosal and is used as a "worst-case scenario." however, efficacy at one mucosal route is usually comparable to that at other mucosal routes. human and animal papillomaviruses cause benign epithelial proliferations (warts) and malignant tumors of the various tissues that they infect. 456 there are >100 human papillomaviruses (hpvs), with different strains causing warts on the skin, oropharynx, nasopharynx, larynx, and anogenital tissues. approximately a third of these are transmitted sexually. of these, virulent subtypes such as hpv-16, hpv-18, hpv-31, hpv-33, and hpv-45 place individuals at high risk for cervical and other cancers. major challenges in the study of these viruses are that papillomaviruses generally do not infect any other species outside of the natural hosts and can cause a very large spectrum of severity. thus, no animal models have been identified that are susceptible to hpv. however, a number of useful surrogate models exist that use animal papillomaviruses in their natural host, or a very closely related species. 457, 458 these models have facilitated the recent development of useful and highly effective prophylactic hpv vaccines. 459 wild cottontail rabbits (sylvilagus floridanus) are the natural host for cottontail rabbit papillomavirus (crpv), but this virus also infects domestic rabbits (oryctolagus cuniculus), which are a very closely related species. 460 in this model, papillomas can range from cutaneous squamous cell carcinomas on the one end of the spectrum, and spontaneous regression on the other. lesions resulting from crpv in domestic rabbits do not typically contain infectious virus. canine oral papillomavirus (copv) cause florid warty lesions in the mucosa of the oral cavity within 4-8 weeks postexposure in experimental settings. 461 the mucosatrophic nature of these viruses and the resulting oropharyngeal papillomas that are morphologically similar to human vaginal papillomas caused by hpv-6 and hpv-11 make this a useful model. 462 these lesions typically spontaneously regress 4-8 weeks after appearing; this model is therefore useful in understanding the interplay between the host immune defense and viral pathogenesis. male and female beagles, aged 10 weeks to 2 years, with no history of copv, are typically used for these studies. infection is achieved by the application of a 10 ml droplet of virus extract to multiple 0.5 cm 2 scarified areas within the mucosa of the upper lip of anesthetized beagles. 463 bovine papillomavirus (bpv) has a wider host range than do most papillomaviruses, infecting the fibroblasts cells of numerous ungulates. 458 bpv-4 infection of cattle feeding on bracken fern, which is carcinogenic, can result in lesions of the oral and esophageal mucosa that lack detectable viral dna. bpv infections in cattle can result in a range of diseases such as skin warts, cancer of the upper gastrointestinal tract and urinary bladder, and papillomatosis of the penis, teats, and udder. finally, sexually transmitted papillomaviruses in rhesus macaques and cynomolgus macaques, rhesus papillomavirus, is very similar to hpv-16 and is associated with the development of cervical cancer. 464 mice cannot be used to study disease caused by papillomaviruses unless they are engrafted with relevant tissue, but they are often used to look at immunogenicity of vaccines. 465, 466 herpesviridae please see chapter 25. monkeypox virus (mpxv) causes disease in both animals and humans. human monkeypox, which is clinically almost identical to ordinary smallpox, occurs mostly in the rainforest of central and western africa. the virus is maintained in nature in rodent reservoirs including squirrels. 467, 468 mpxv was discovered during the pox-like disease outbreak among laboratory java macaques in denmark in 1958. no human cases were observed during this outbreak. the first human case was not recognized as a distinct disease until 1970 in zaire (the present drc) with the continued occurrence of a smallpox-like illness despite eradication efforts of smallpox in this area. during the global eradication campaign, extensive vaccination in central africa decreased the incidence of human monkeypox, but the absence of immunity in the generation born since that time and increased dependence on bush meat have resulted in renewed emergence of the disease. in the summer of 2003, a well-known outbreak in the midwest was the first occurrence of monkeypox disease in the united states and the western hemisphere. among 72 reported cases, 37 human cases were laboratory confirmed during an outbreak. 469, 470 it was determined that native prairie dogs (cynomys sp.) housed with rodents imported from ghana in west africa were the primary source of outbreak. the virus is mainly transmitted to humans while handling infected animals or by direct contact with the infected animal's body fluids, or lesions. person-toperson spread occurs by large respiratory droplets or direct contact. 471 most of the clinical features of human monkeypox are very similar to those of ordinary smallpox. 472 after a 7-to 21-day incubation period, the disease begins with fever, malaise, headache, sore throat, and cough. the main sign of the disease that distinguishes monkeypox from smallpox is swollen lymph nodes (lymphadenitis), which is observed in most of the patients before the development of rash. 471, 473 typical maculopapular rash follows the prodromal period generally lasting 1-3 days. the average size of the skin lesions is 0.5-1 cm, and the progress of lesions follows the order macules through papules, vesicles, pustules, umblication then scab and desquamation and lasts typically 2-4 weeks. fatality rate is 10% among the unvaccinated population and death generally occurs during the second week of the disease. 469, 471 mpxv is highly pathogenic for a variety of laboratory animals, and so far, many animal models have been developed by using different species and different routes of exposure (table 38. 3). because of the unavailability of variola virus to develop animal models and resulting disease manifestations in humans that are similar, mpxv is one of the pox viruses that are used very heavily to develop a number of small animal models via different routes of exposure. wild-derived inbred mouse, stat1-deficient c57bl/6 mouse, prairie dogs, african dormice, ground squirrels are highly susceptible to mpxv by different exposure routes. [474] [475] [476] [477] [478] [479] [480] [481] cast/eij mice, one of the 38 inbred mouse strains tested for susceptibility to mpxv, showed weight loss and dose-dependent mortality after intranasal exposure to mpxv. studies with the intraperitoneal route of challenge indicated a higher susceptibility to mpxv with an almost 50-fold less ld50 when compared to the intranasal route. 474 scid-balb/c mice were also susceptible to the intraperitoneal challenge route, and the disease resulted in mortality day 9 postinfection. 476 similarly c57bl/6 stat1 ã�/ã� mice were infected intranasally with mpxv, and the infection resulted in weight loss and mortality 10 days postexposure. mice models mentioned here are very promising for screening of therapeutics against pox viruses, but testing in additional models will be required for advanced development. high doses of mpxv by intraperitoneal or intranasal route caused 100% mortality in 6 days postexposure and 8 days postexposure, respectively, in ground squirrels. 480 the disease progressed very quickly, and most of the animals were lethargic and moribund by day 5 postexposure without any pox lesions or respiratory changes. a comparison study of usa mpxv and central african strain of mpxv in ground squirrels by subcutaneous route resulted in systemic disease and the mortality in 6-11 days postexposure. the disease resembles hemorrhagic smallpox with nose bleeds, impaired coagulation parameters, and hemorrhage in the lungs of the animals. because in the us outbreak the virus was transmitted by infected prairie dogs, this animal model has recently been studied much further and used to test therapeutics and vaccines compared to other small animal models. 475, 481, 491, 492 studies using intranasal, intraperitoneal, and intradermal routes of exposure showed that mpxv was highly infectious to prairie dogs. by using the west african mpxv strain, the intraperitoneal route caused a more severe disease and 100% mortality than challenge by the intranasal route. anorexia and lethargy were common signs of the disease for both exposure routes. in contrast to the intraperitoneal route, the intranasal route of exposure caused severe pulmonary edema and necrosis of lungs in prairie dogs, while splenic necrosis and hepatic lesions were observed in intraperitoneally infected animals. 481 recent studies by hutson et al. used intranasal and intradermal infections with west african and congo basin strains and showed that both strains and routes caused smallpox-like disease with longer incubation periods and generalized pox lesions. 475 therefore, this model can be used for testing therapeutics and vaccines against pox viruses. the african dormouse is susceptible to mpxv by the foodpad injection route or intranasal route. 478 mice exhibited decreased activity, hunched posture, dehydration, conjunctivitis, and weight loss. viral doses of 200 and 2000 pfu provided 100% mortality with a mean time to death of 8 days. upper gastrointestinal hemorrhage, hepatomegaly, lymphadenopathy, and hemorrhage in lungs were observed during necropsy. with the hemorrhage in several organs, this model resembles hemorrhagic smallpox. considering the limited availability of ground squirrels and african dormice, lack of reagents to these species, and resemblance to hemorrhagic smallpox disease, these models are not very attractive for further characterization and vaccine and countermeasure testing studies. nhps were exposed to mpxv by several different routes to develop animal models for mpxv. 482, 483, 485, 489, 490 during our studies by using an aerosol route of exposure, we observed that macaques had mild anorexia, depression, fever, and lymphadenopathy on day 6 postexposure. 482 complete blood count and clinical chemistries showed abnormalities similar to those of human monkeypox cases with leukocytosis and thrombocytopenia. 493 whole-blood and throat swabs had viral loads peak around day 10, and in survivors, gradually decrease until day 28 postexposure. because doses of 4 ã� 10 4 , 1 ã� 10 5 , or 1 ã� 10 6 pfu resulted in lethality for 70% of the animals, whereas a dose of 4 ã� 10 5 pfu resulted in 85% lethality, survival was not dose dependent. the main pitfall of this model was the lack of pox lesions. with the high dose, before animals can develop pox lesions, they succumbed to disease. with the low challenge dose, pox lesions were observed, but they were few in comparison to the iv model. mpxv causes dose-dependent disease in nhps when given by the iv route. 490 studies showed that with 1ã� 10 7 pfu iv, challenge results in systemic disease with fever, lymphadenopathy, macula-papular rash, and mortality. an intratracheal infection model deposits virus into the trachea, delivering directly to the airways without regard to particle size and the physiological deposition that occurs during the process of inhalation by skipping the upper respiratory system. fibrinonecrotic bronchopneumonia was described in animals that received 10 7 pfu of mpxv intratracheally. 489 although a similar challenge dose of intratracheal mpxv infection resulted in a similar viremia in nhps than with the aerosol route of infection, the timing of the first peak was delayed by 5 days in intratracheally exposed macaques compared to aerosol infection, and the amount of virus detected by qpcr was approximately 100-fold lower. this suggests that local replication is more prominent after aerosol delivery compared to that after intratracheal delivery. an intrabronchial route of exposure resulted in pneumonia in nhps. 490 delayed onset of clinical signs and viremia were observed during the disease progression. in this model, similar to aerosol and the intratracheal route of infection models, the number of pox lesions was much less than in the iv route of the infection model. a major downside of the iv, intratracheal and intrabronchial models is that the initial infection of respiratory tissue, incubation, and prodromal phases are circumvented with the direct inoculation of virus into the blood stream or into the lung. this is an important limitation when the utility of these models is to test possible vaccines and treatments in which the efficacy may depend on protecting the respiratory mucosa and targeting subsequent early stages of the infection, which are not represented in these challenge models. although the aerosol model is the natural route of transmission for human variola virus (varv) infections and a secondary route for human mpxv infections, the lack of pox lesions is the main drawback of this model. therefore, when this model is decided to be used to test medical countermeasures, the endpoints and the biomarkers to initiate treatment should be chosen carefully. hepatitis b is one of the most common infections worldwide with >400 million people chronically infected and 316,000 cases per year of liver cancer due to infection. 494 the virus can naturally infect both humans and chimpanzees. 495 hepatitis b is transmitted parenterally or postnatally from infected mothers. it can also be transmitted by sexual contact, iv drug use, blood transfusion, and acupuncture. 496 the age at which one is infected dictates the risk of developing chronic disease. 497 acute infection during adulthood is self-limiting and results in flu-like symptoms that can progress to hepatocellular involvement as observed with the development of jaundice. the clinical symptoms last for a few weeks before resolving. 498 after this acute phase, life time immunity is achieved. 499 of those infected, <5% will develop the chronic form of disease. chronicity is the most serious outcome of disease as it can result in cirrhosis or liver cancer. hepatocellular carcinoma is 100 times more likely to develop in a chronically infected individual than in a noncarrier. 500 the viral determinant for cellular transformation has yet to be determined, although studies involving the woodchuck hepadna virus suggest that x protein may be responsible. 501 many individuals are asymptomatic until complications emerge related to chronic carriage. chimpanzees have a unique strain that circulates within the population. 502, 503 it was found that 3-6% of all wild-caught animals from africa are positive for hepatitis b antigen. 504 natural and experimental challenge with the virus follows the same course as human disease; however, this is only an acute model of disease. 505 to date, the use of chimpanzees provides the only reliable method to ensure that plasma vaccines are free from infectious particles. 506 this animal model has been used to study new therapeutics and vaccines. chimpanzees are especially attuned to these studies given that their immune response to infection directly mirrors humans. 507 other nhps that have been evaluated are gibbons, orangutans, and rhesus monkeys. although these animals can be infected with hepatitis b, none develop hepatic lesions or liver damage as noted by monitoring of liver enzymes. 508 mice are not permissible to infection, and thus, numerous transgenic and humanized lines that express hepatitis b proteins have been created to facilitate their usage as an animal model. these include both immunocompetent and immunosuppressed hosts. the caveat to all of these mouse lines is that they reproduce only the acute form of disease. 495 recently, the entire genome of hepatitis b was transferred to an immunocompetent mouse line via adenovirus. this provides a model for persistent infection. 509 hepatitis b can also be studied using surrogate viruses, naturally occurring mammalian hepadna viruses. 510 the woodchuck hepatitis virus was found to induce hepatocellular carcinoma. 511 within a population, 65-75% of all neonatal woodchucks are susceptible to chronic infection. 512 a major difference between the two hepatitis isolates is the rate at which they induce cancer; almost all chronic carriers developed hepatocellular carcinoma within 3 years of the initial infection in woodchucks, whereas human infection takes much longer. 513 the acute infection strongly resembles what occurs during the course of disease in humans. there is a self-limiting acute phase resulting in a transient viremia that has the potential of chronic carriage. 514 challenge with virus in neonates leads to a chronic infection, while adults only develop the acute phase of disease. 515 a closely related species to the woodchuck is the marmota himalayan. this animal is also susceptible to the woodchuck hepadna virus upon iv injection. it was found to develop an acute hepatitis with a productive infection. 516 hepatitis d is dependent upon hepatitis b to undergo replication and successful infection in its human host. 517 there are two modes of infection possible between the viruses: coinfection in which a person is simultaneously infected or superinfection in which a chronic carrier of hepatitis b is subsequently infected with hepatitis d. 518 coinfection leads to a similar disease as seen with hepatitis b alone; however, superinfection can result in chronic hepatitis d infection and severe liver damage. 519 both coinfection and superinfection can be demonstrated within the chimpanzee and woodchuck by inoculation of human hepatitis d. 520 a recently published report demonstrated the use of a humanized chimeric mouse to study the interactions between the two viruses and drug testing. 521 the ideal animal model for human viral disease should closely recapitulate the spectrum of clinical symptoms and pathogenesis observed during the course of human infection. whenever feasible, the model should use the same virus and strain that infects humans. it is also preferable that the virus be a low passage clinical isolate; thus, animal passage or adaptation should be avoided if model species can be identified that are susceptible. ideally, the experimental route of infection would mirror that which occurs in natural disease. to understand the interplay and contribution of the immune system during infection, an immunocompetent animal should be used. the above characteristics cannot always be satisfied, however, and often virus must be adapted, knockout mice must be used, and/or the disease is not perfectly mimicked in the animal model. well-characterized animal models are critical for licensure to satisfy the food and fda's "animal rule." this rule applies to situations in which vaccines and therapeutics cannot safely or ethically be tested in humans; thus, licensure will come only after preclinical tests are performed in animal models. many fields in virology are moving toward standardized models that can be used across institutions to test vaccines and therapeutics. a current example of such an effort is within the filovirus community, where animal models, euthanasia criteria, assays, and virus strains are in the process of being standardized. the hope is that these efforts will enable results of efficacy tests on medical countermeasures to be compared across institutions. this chapter has summarized the best models available for each of the viruses described. fields' virology pathogenesis of poliomyelitis: reappraisal in the light of new data one hundred years of poliovirus pathogenesis expression of the poliovirus receptor in intestinal epithelial cells is not sufficient to permit poliovirus replication in the mouse gut present status of attenuated live-virus poliomyelitis vaccine world health organization. polio global eradication initiative annual report pathogenesis of human poliovirus infection in mice. ii. age-dependency of paralysis the poliovirus receptor protein is produced both as membranebound and secreted forms poliovirus pathogenesis in a new poliovirus receptor transgenic mouse model: agedependent paralysis and a mucosal route of infection establishment of a poliovirus oral infection system in human poliovirus receptor-expressing transgenic mice that are deficient in alpha/beta interferon receptor macaque models of human infectious disease ulnar nerve inoculation of poliovirus in bonnet monkey: a new primate model to investigate neurovirulence experimental poliomyelitis in bonnet monkey. clinical features, virology and pathology hepatitis a in day-care centers. a community-wide assessment persistence of hepatitis a virus in fulminant hepatitis and after liver transplantation acute liver failure: redefining the syndromes experimental hepatitis a virus (hav) infection in cynomolgus monkeys (macaca fascicularis): evidence of active extrahepatic site of hav replication experimental hepatitis a virus (hav) infection in callithrix jacchus: early detection of hav antigen and viral fate animal models of hepatitis a and e relative infectivity of hepatitis a virus by the oral and intravenous routes in 2 species of nonhuman primates wild malaysian cynomolgus monkeys are exposed to hepatitis a virus histopathological and immunohistochemical studies of hepatitis a virus infection in 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fever viruses protective efficacy of a bivalent recombinant vesicular stomatitis virus vaccine in the syrian hamster model of lethal ebola virus infection pathogenesis of filoviruses in small animal models program abstr 5th int symp filoviruses pathogenesis of experimental ebola virus infection in guinea pigs characterization of disease and pathogenesis following airborne exposure of guinea pigs to filoviruses manuscripts in preparation postexposure antibody prophylaxis protects nonhuman primates from filovirus disease aerosol exposure to the angola strain of marburg virus causes lethal viral hemorrhagic fever in cynomolgus macaques a small nonhuman primate model for filovirus-induced disease pathology of experimental ebola virus infection in african green monkeys. involvement of fibroblastic reticular cells pathogenesis of marburg hemorrhagic fever in cynomolgus macaques a characterization of aerosolized sudan ebolavirus infection in african green monkeys, cynomolgus macaques, and 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analysis of national databases the relationship of meteorological conditions to the epidemic activity of respiratory syncytial virus viral and host factors in human respiratory syncytial virus pathogenesis evidence of a causal role of winter virus infection during infancy in early childhood asthma respiratory syncytial virus infection in elderly and high-risk adults respiratory syncytial virus american academy of pediatrics subcommittee on diagnosis & management of bronchiolitis. diagnosis and management of bronchiolitis respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine respiratory syncytial virus induces pneumonia, cytokine response, airway obstruction, and chronic inflammatory infiltrates associated with long-term airway hyperresponsiveness in mice genetic susceptibility to respiratory syncytial virus infection in inbred mice differential pathogenesis of respiratory syncytial virus clinical isolates in balb/c mice primary 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infection and il-13 production [comparative study research support immunomodulation with il-4r alpha antisense oligonucleotide prevents respiratory syncytial virusmediated pulmonary disease chinchilla and murine models of upper respiratory tract infections with respiratory syncytial virus experimental respiratory syncytial virus infection of four species of primates respiratory syncytial virus infects the bonnet monkey serum neutralizing antibody titers of seropositive chimpanzees immunized with vaccines coformulated with natural fusion and attachment proteins of respiratory syncytial virus reduced clearance of respiratory syncytial virus infection in a preterm lamb model human respiratory syncytial virus a2 strain replicates and induces innate immune responses by respiratory epithelia of neonatal lambs respiratory syncytial virus is associated with an inflammatory response in lungs and architectural remodeling of lung-draining lymph nodes of newborn lambs structure as revealed by airway dissection. a comparison of mammalian lungs biomedical applications of sheep models: from asthma to vaccines the pathology of influenza virus infections mortality due to influenza in the united statesdan annualized regression approach using multiple-cause mortality data animal models for the study of influenza pathogenesis and therapy serious morbidity and mortality associated with influenza epidemics centers for disease control & prevention. bacterial coinfections in lung tissue specimens from fatal cases of 2009 pandemic influenza a (h1n1)dunited states human and avian influenza viruses target different cell types in cultures of human airway epithelium animal models head-to-head comparison of four nonadjuvanted inactivated cell culture-derived influenza vaccines: effect of composition, spatial organization and immunization route on the immunogenicity in a murine challenge model [comparative study interferon-induced mx protein: a mediator of cellular resistance to influenza virus in vitro and in vivo assay systems for study of influenza virus inhibitors utilization of pulse oximetry for the study of the inhibitory effects of antiviral agents on influenza virus in mice the contribution of animal models to the understanding of the host range and virulence of influenza a viruses biological heterogeneity, including systemic replication in mice, of h5n1 influenza a virus isolates from humans in hong kong distinct pathogenesis of hong kong-origin h5n1 viruses in mice compared to that of other highly pathogenic h5 avian influenza viruses effect of oral gavage treatment with znal42 and other metallo-ion formulations on influenza a h5n1 and h1n1 virus infections in mice inactivated and live, attenuated influenza vaccines protect mice against influenza: streptococcus pyogenes super-infections [comparative study research support the use of an animal model to study transmission of influenza virus infection strong local and systemic protective immunity induced in the ferret model by an intranasal virosome-formulated influenza subunit vaccine local innate immune responses and influenza virus transmission and virulence in ferrets regional t-and b-cell responses in influenza-infected ferrets human and avian influenza viruses target different cells in the lower respiratory tract of humans and other mammals live, attenuated influenza virus (laiv) vehicles are strong inducers of immunity toward influenza b virus [comparative study the ferret: an animal model to study influenza virus pathogenesis of avian influenza a (h5n1) viruses in ferrets severe seasonal influenza in ferrets correlates with reduced interferon and increased il-6 induction neuropathology of h5n1 virus infection in ferrets evaluation of three strains of influenza a virus in humans and in owl, cebus, and squirrel monkeys a computationally optimized hemagglutinin virus-like particle vaccine elicits broadly reactive antibodies that protect nonhuman primates from h5n1 infection evaluation of intravenous zanamivir against experimental influenza a (h5n1) virus infection in cynomolgus macaques aberrant innate immune response in lethal infection of macaques with the 1918 influenza virus pathology of human influenza a (h5n1) virus infection in cynomolgus macaques (macaca fascicularis integrated molecular signature of disease: analysis of influenza virus-infected macaques through functional genomics and proteomics integration of clinical data, pathology, and cdna microarrays in influenza virus-infected pigtailed macaques (macaca nemestrina kinetic profile of influenza virus infection in three rat strains the guinea pig as a transmission model for human influenza viruses influenza-induced tachypnea is prevented in immune cotton rats, but cannot be treated with an anti-inflammatory steroid or a neuraminidase inhibitor the antiviral potential of interferon-induced cotton rat mx proteins against orthomyxovirus (influenza), rhabdovirus, and bunyavirus the cotton rat as a model to study influenza pathogenesis and immunity the cotton rat provides a useful small-animal model for the study of influenza virus pathogenesis co-infection of the cotton rat (sigmodon hispidus) with staphylococcus aureus and influenza a virus results in synergistic disease pathogenicity of a highly pathogenic avian influenza virus, a/chicken/yamaguchi/7/04 (h5n1) in different species of birds and mammals domestic pigs have low susceptibility to h5n1 highly pathogenic avian influenza viruses [comparative study research support animal models in influenza vaccine testing rift valley fever: an uninvited zoonosis in the arabian peninsula prevalence of anti-rift-valley-fever igm antibody in abattoir workers in the nile delta during the 1993 outbreak in egypt rift valley fever the occurrence of human cases in johannesburg the pathogenesis of rift valley fever epidemic rift valley fever in egypt: observations of the spectrum of human illness crc handbook series in zoonoses rift valley fever virus in mice. i. general features of the infection the pathogenesis of rift valley fever virus in the mouse model the susceptibility of rats to rift valley fever in relation to age inbred rat strains mimic the disparate human response to rift valley fever virus infection the gerbil, meriones unguiculatus, a model for rift valley fever viral encephalitis experimental rift valley fever in rhesus macaques development of a novel nonhuman primate model for rift valley fever crimean-congo hemorrhagic fever: a global perspective crimean-congo hemorrhagic fever the clinical pathology of crimean-congo hemorrhagic fever experimental congo virus (ib-an7620) infection in primates crimean congo hemorrhagic fever: a global perspective viremia and antibody response of small african and laboratory animals to crimean-congo hemorrhagic fever virus infection a comparative study of the crimean hemorrhagic faverdcongo group of viruses [comparative study ribavirin efficacy in an in vivo model of crimean-congo 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c-reactive protein, creatinine, and nitric oxide in cynomolgus macaques pathology of puumala hantavirus infection in macaques viral haemorrhagic fevers caused by lassa, ebola, and marburg viruses new opportunities for field research on the pathogenesis and treatment of lassa fever imported lassa fever lassa fever. effective therapy with ribavirin lassa virus hepatitis: a study of fatal lassa fever in humans lassa virus infection of rhesus monkeys: pathogenesis and treatment with ribavirin experimental inhalation infection of monkeys of the macacus cynomolgus and macacus rhesus species with the virus of lymphocytic choriomeningitis (we) arenavirus-mediated liver pathology: acute lymphocytic choriomeningitis virus infection of rhesus macaques is characterized by high-level interleukin-6 expression and hepatocyte proliferation experimental studies of arenaviral hemorrhagic fevers lcmv-mediated hepatitis in rhesus macaques: we but not arm strain activates hepatocytes and induces liver regeneration mucosal arenavirus infection of primates can protect them from lethal hemorrhagic fever pathogenesis of lassa fever in cynomolgus macaques lassa fever encephalopathy: clinical and laboratory findings lassa fever encephalopathy: lassa virus in cerebrospinal fluid but not in serum lassa virus infection in experimentally infected marmosets: liver pathology and immunophenotypic alterations in target tissues virus taxonomy, viiith report of the international committee on taxonomy of viruses pathogenesis of lassa virus infection in guinea pigs the effect of an arenavirus infection on liver morphology and function cardiovascular and pulmonary responses to pichinde virus infection in strain 13 guinea pigs pathogenesis of pichinde virus infection in strain 13 guinea pigs: an immunocytochemical, virologic, and clinical chemistry study pichinde virus infection in strain 13 guinea pigs reduces intestinal protein reflection coefficient with compensation clinical laboratory, virologic, and pathologic changes in hamsters experimentally infected with pirital virus (arenaviridae): a rodent model of lassa fever variation between strains of hamsters in the lethality of pichinde virus infections interferon alfacon-1 protects hamsters from lethal pichinde virus infection treatment of lethal pichinde virus infections in weanling lvg/lak hamsters with ribavirin, ribamidine, selenazofurin, and ampligen [comparative study research support global mortality associated with rotavirus disease among children in minimal infective dose of rotavirus rotavirus immunoglobulin levels among indian mothers of two socio-economic groups and occurrence of rotavirus infections among their infants up to six months analyses of clinical, pathological and virological features of human rotavirus strain, yo induced gastroenteritis in infant balb/c mice epizootic diarrhea of infant mice: identification of the etiologic agent immunity to rotavirus infection in mice development of an adult mouse model for studies on protection against rotavirus a gastrointestinal rotavirus infection mouse model for immune modulation studies protection of the villus epithelial cells of the small intestine from rotavirus infection does not require immunoglobulin a rotavirus viremia and extraintestinal viral infection in the neonatal rat model [comparative study research support characterization of clinical and immune response in a rotavirus diarrhea model in suckling lewis rats development of a heterologous model in germfree suckling rats for studies of rotavirus diarrhea studies of oral rehydration solutions in animal models induction of mucosal immune responses and protection against enteric viruses: rotavirus infection of gnotobiotic pigs as a model developmental immunity in the piglet swine in biomedical research neonatal calf diarrhea induced by rotavirus characterisation of the primary local and systemic immune response in gnotobiotic lambs against rotavirus infection experimental infection of non-human primates with a human rotavirus isolate development of a rotavirus-shedding model in rhesus macaques, using a homologous wild-type rotavirus of a new p genotype reflections on 30 years of aids hivs and their replication the utility of the new generation of humanized mice to study hiv-1 infection: transmission, prevention, pathogenesis, and treatment antiretroviral pre-exposure prophylaxis prevents vaginal transmission of hiv-1 in humanized blt mice hematopoietic stem cell-engrafted nod/ scid/il2rgamma null mice develop human lymphoid systems and induce long-lasting hiv-1 infection with specific humoral immune responses hiv-1 infection and cd4 t cell depletion in the humanized rag2ã�/ã� gamma cã�/ã� (rag-hu) mouse model hiv-1 infection and pathogenesis in a novel humanized mouse model induction of robust cellular and humoral virusspecific adaptive immune responses in human immunodeficiency virus-infected humanized blt mice an aptamer-sirna chimera suppresses hiv-1 viral loads and protects from helper cd4(ã¾) t cell decline in humanized mice mucosal immunity and vaccines low-dose rectal inoculation of rhesus macaques by sivsme660 or sivmac251 recapitulates human mucosal infection by hiv-1 propagation and dissemination of infection after vaginal transmission of simian immunodeficiency virus limited dissemination of pathogenic siv after vaginal challenge of rhesus monkeys immunized with a live virulence and reduced fitness of simian immunodeficiency virus with the m184v mutation in reverse transcriptase siv-induced impairment of neurovascular repair: a potential role for vegf therapeutic dna vaccine induces broad t cell responses in the gut and sustained protection from viral rebound and aids in siv-infected rhesus macaques a nonfucosylated variant of the anti-hiv-1 monoclonal antibody b12 has enhanced fcgammariiiamediated antiviral activity in vitro but does not improve protection against mucosal shiv challenge in macaques a trivalent recombinant ad5 gag/pol/nef vaccine fails to protect rhesus macaques from infection or control virus replication after a limiting-dose heterologous siv challenge animal model for the therapy of acquired immunodeficiency syndrome with reverse transcriptase inhibitors susceptibility of hiv-2, siv and shiv to various anti-hiv-1 compounds: implications for treatment and postexposure prophylaxis use of a small molecule ccr5 inhibitor in macaques to treat simian immunodeficiency virus infection or prevent simian-human immunodeficiency virus infection shiv-1157i and passaged progeny viruses encoding r5 hiv-1 clade c env cause aids in rhesus monkeys update on animal models for hiv research limited or no protection by weakly or nonneutralizing antibodies against vaginal shiv challenge of macaques compared with a strongly neutralizing antibody macaque studies of vaccine and microbicide combinations for preventing hiv-1 sexual transmission vpx is critical for sivmne infection of pigtail macaques impact of short-term haart initiated during the chronic stage or shortly post-exposure on siv infection of male genital organs the rhesus macaque pediatric siv infection modeld a valuable tool in understanding infant hiv-1 pathogenesis and for designing pediatric hiv-1 prevention strategies perinatal transmission of shiv-sf162p3 in macaca nemestrina immune and genetic correlates of vaccine protection against mucosal infection by siv in monkeys chronic administration of tenofovir to rhesus macaques from infancy through adulthood and pregnancy: summary of pharmacokinetics and biological and virological effects efficacy assessment of a cell-mediated immunity hiv-1 vaccine (the step study): a double-blind, randomised, placebo-controlled, test-of-concept trial human papillomavirus in cervical cancer human papillomavirus research: do we still need animal models? animal models of papillomavirus pathogenesis evidence of human papillomavirus vaccine effectiveness in reducing genital warts: an analysis of california public family planning administrative claims data the rabbit viral skin papillomas and carcinomas: a model for the immunogenetics of hpv-associated carcinogenesis protection of beagle dogs from mucosal challenge with canine oral papillomavirus by immunization with recombinant adenoviruses expressing codon-optimized early genes naturally occurring, nonregressing canine oral papillomavirus infection: host immunity, virus characterization, and experimental infection regression of canine oral papillomas is associated with infiltration of cd4ã¾ and cd8ã¾ lymphocytes characterization and experimental transmission of an oncogenic papillomavirus in female macaques a multimeric l2 vaccine for prevention of animal papillomavirus infections preclinical development of highly effective and safe dna vaccines directed against hpv 16 e6 and e7 us doctors investigate more than 50 possible cases of monkeypox isolation of monkeypox virus from wild squirrel infected in nature reemergence of 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and hepatocellular carcinoma hepatitis b virus replication in primary macaque hepatocytes: crossing the species barrier toward a new small primate model animal models of hepatitis delta virus infection and disease experimental hepatitis delta virus infection in the chimpanzee expression of the hepatitis delta virus large and small antigens in transgenic mice experimental hepatitis delta virus infection in the animal model humanized chimeric upa mouse model for the study of hepatitis b and d virus interactions and preclinical drug evaluation key: cord-031279-8rckjc41 authors: enriquez, josue; mims, brianyell mc daniel; trasti, scott; furr, kathryn l.; grisham, matthew b. title: genomic, microbial and environmental standardization in animal experimentation limiting immunological discovery date: 2020-09-02 journal: bmc immunol doi: 10.1186/s12865-020-00380-x sha: doc_id: 31279 cord_uid: 8rckjc41 background: the use of inbred mice housed under standardized environmental conditions has been critical in identifying immuno-pathological mechanisms in different infectious and inflammatory diseases as well as revealing new therapeutic targets for clinical trials. unfortunately, only a small percentage of preclinical intervention studies using well-defined mouse models of disease have progressed to clinically-effective treatments in patients. the reasons for this lack of bench-to-bedside transition are not completely understood; however, emerging data suggest that genetic diversity and housing environment may greatly influence muring immunity and inflammation. results: accumulating evidence suggests that certain immune responses and/or disease phenotypes observed in inbred mice may be quite different than those observed in their outbred counterparts. these differences have been thought to contribute to differing immune responses to foreign and/or auto-antigens in mice vs. humans. there is also a growing literature demonstrating that mice housed under specific pathogen free conditions possess an immature immune system that remarkably affects their ability to respond to pathogens and/or inflammation when compared with mice exposed to a more diverse spectrum of microorganisms. furthermore, recent studies demonstrate that mice develop chronic cold stress when housed at standard animal care facility temperatures (i.e. 22–24 °c). these temperatures have been shown alter immune responses to foreign and auto-antigens when compared with mice housed at their thermo-neutral body temperature of 30–32 °c. conclusions: exposure of genetically diverse mice to a spectrum of environmentally-relevant microorganisms at housing temperatures that approximate their thermo-neutral zone may improve the chances of identifying new and more potent therapeutics to treat infectious and inflammatory diseases. use of genetically standardized mice exposed to a welldefined, pathogen free microbiota has markedly reduced the variability of disease phenotype reported by different laboratories. despite these important advancements, only 10% of promising therapeutic strategies reported in mouse models of disease have gone on to be used as clinically-effective treatments in patients [2] [3] [4] . it has been suggested that differences in murine vs. human immune systems may account for the reduced success of translation of preclinical data [3, [5] [6] [7] [8] [9] [10] [11] . it is well-known that the structure and cellular composition of the murine immune system is, in some cases, substantially different than that of humans [6, [12] [13] [14] [15] [16] . these differences are thought to contribute to differing immune responses to foreign and/or self-antigens in mice vs. humans [17] . indeed, the debate continues as to whether genomic responses of mice during inflammation mimic those in humans [18] [19] [20] . in addition to differences in immunological responses between the two species, there is a growing literature suggesting that housing inbred mice in ultra-clean conditions at temperatures well-below their thermoneutral zone, may significantly alter their response to infectious micro-organisms and tumor cells as wells as to allo-and auto-antigens. for example, inbred mice housed under specific pathogen free (spf)/ barrier conditions may respond to microbial infections or inflammatory mediators differently than do outbred mice or inbred mice colonized with more diverse communities of microorganisms [12, 15] . in addition, virtually all animal care facilities house mice at 22-24°c that is well below their thermoneutral zone. these housing temperatures create chronic cold stress that are known to alter murine immune responses to different foreign or auto antigens when compared with mice housed at their thermo-neutral body temperature of 30-32°c [15, [21] [22] [23] [24] . when taken together, it is reasonable to assume that these intrinsic and extrinsic factors may markedly affect the translational nature of preclinical studies using mouse models of infectious and inflammatory diseases. this review discusses how genetic diversity and the environment affects immunity and inflammation in mice and describes how potential modifications of current animal husbandry and experimental protocols may help to more closely model infectious and inflammatory diseases. the use of mice to study immune responses to infection and inflammation have revealed a number of fundamental principles that have been confirmed in humans. one paradigm-changing example of how mouse models have advanced our understanding of immune regulation is recognition that failure to maintain tolerance to commensal bacteria and/or autoantigens results in the development of chronic inflammatory diseases [25] [26] [27] . although these and other important studies have helped to uncover a number of novel immuno-pathogenic mechanisms underlying infectious and inflammatory diseases, relatively few of the promising therapeutic treatments or interventions reported in follow up studies have been translated into effective therapies in humans [2-4, 7, 17] . as discussed above, several reasons have been proposed to account for the low bench-to-bedside transition that have largely focused on differences in immune system composition and function in mice vs. humans. more recent studies have begun to address the likelihood that the restricted genetic diversity of inbred mice may limit their ability to express the full range of immune responses that are observed in diverse human populations. accumulating evidence suggests that certain immune responses and/or disease phenotypes observed in inbred mice may be quite different than those observed in their outbred counterparts [28] [29] [30] [31] [32] . this is not surprising given the fact that inbred strains of mice are produced by single lineage brother/sister mating for at least 20 generations. because this type of breeding scheme produces mice that are genetically identical, there is far less inter-animal variability. however, it should be noted that this type of breeding scheme produces inbreeding depression and reduced genetic diversity [33] . the use of inbred mice to model human disease is tantamount to using "multiple copies of one individual" [17] . while this tool has been crucial to our understanding of innate and adaptive immunity, it most likely does not capture the hybrid vigor and genetic diversity present in humans. historically, outbred mice have been used for toxicological, pharmacological, cancer and aging research. the most common outbred stocks used in preclinical studies include cd-1, institute for cancer research (icr), swiss webster (sw) and nih swiss mice [33] . the majority of outbred mice used in the u.s. today are derived from 2 male and 7 female, non-inbred albino mice that were imported from switzerland by dr. carla lynch to the rockefeller institute for medical research in 1926 [34] . these original stocks were collectively referred to as "swiss" mice. it should be noted that different lineages of outbred mice are referred to as "stocks" whereas inbred lineages of mice are referred to as "strains" [33] . by definition, outbred stocks are "a closed population … of genetically variable animals that are bred to maintain maximum heterozygosity" [34] . in theory, no two individuals are genetically identical within an outbred stock. siblings of inbred strains of mice are genetically identical that are maintained through brother and sister mating for 20 or more generations [33] . because outbred stocks are genetically more diverse than inbred strains, data obtained using these mice tend to be more variable requiring substantially larger numbers of animals for statistical power [34] . thus, the majority of published studies exploring the immuno-pathogenesis of infectious and inflammatory diseases have used the more genetically homogeneous inbred strains. despite the advantages of using inbred strains, there is a clear need to understand how genetic diversity influences immunity and inflammation. below, we present an overview of several studies that highlight the differences in immune responses between different strains and stocks of mice subjected to infection or inflammation. it is now well-known that the susceptibility of inbred and outbred mice to infectious micro-organisms may be quite different. thus, translation of preclinical data based upon only one strain or stock may be problematic. for example, citrobacter rodentium (c. rodentium) infection induces severe but self-limiting diarrhea, colonic epithelial hyperplasia and mild intestinal inflammation in a variety of mice on different genetic backgrounds. borenstein et al. demonstrated that infection of outbred sw mice with c. rodentium recapitulated this phenotype i.e. subclinical and self-limiting intestinal inflammation [35] . however, when inbred mice derived from the sw stock [called friend virus b (fvb)] mice were infected with c. rodentium, they developed a lethal disease characterized by severe ulcerative colitis [35] . in a more recent study, sunagar et al. compared the immune responses of sw vs. inbred c57bl/6 mice to infection with the highly virulent pathogen franscisella tularensis (ft) [36] . this tier 1 bio-threat has been extensively studied over the past several years yet no fda-approved vaccine has been developed. sunagar et al. found that outbred sw mice were more resistant to ft challenge and were better protected from the lethal effects of this pathogen following vaccination than were inbred c57bl/6 mice [36] . the authors concluded that outbred mice may more accurately reflect the genetic diversity of human immunity to this deadly pathogen. another example of differential susceptibility between inbred and outbred mice to an infectious agent was recently reported by carreras et al [37] .. these investigators found that c57bl/6 mice were more susceptible than cd1 mice to the lethal effects of sepsis induced by intravenous administration of the fungus candida albicans when compared with c57bl/6 [37] . in another study using a mouse model of streptococcus suis (s. suis)-induced sepsis and cerebral inflammation, dominguez-punaro et al. reported that while infection of different inbred strains of mice (e.g. balb/c, c57bl/6, a/j) with s. suis caused septic shock and death, it did not induce meningitis and encephalitis [38] . in contrast, these investigators showed that infection of outbred cd1 mice with s. suis induced rapid and systemic production of high levels of several different inflammatory cytokines that likely contributed to the death of 20% of the mice within the first 48 h following infection [39] . in those mice that survived, approximately 40% developed clinical signs of meningitis between days 4 and 9 post-infection. these exciting studies provided the first demonstration that hematogenous infection with s. suis produces both septic shock and cerebral inflammation. one immune cell that plays a critical role in recognizing and eliminating intracellular pathogens (e.g. viruses, bacteria, protozoan) and tumor cells is the cd8 + t cell [40] . the ability to accurately define the magnitude and kinetics as well as the phenotypic and functional characteristics of cd8 t cells following pathogen infection is not only important for our understanding of immune regulation but it is also crucial for developing new and more robust vaccination protocols. much of what we have learned about t cell-mediated immunity has come from studies using inbred mice [31, 41] . recent studies from the badovinac laboratory have shown that infection of outbred sw mice with either listeria monocytogenes (lm) or armstrong strain of lymphocytic choriomeningitis virus (lcmv) results in large animal-to-animal variations in pathogen-specific cd8 t cell responses when compared with their inbred c57bl/6 counterparts. these data suggest that genetic background may control the magnitude of cd8 t cell expansion in response to pathogen infection [31, 41] . in addition, martin et.al. found that infection of sw mice with lm or lcmv generated a number of different populations of pathogen-specific cd8 t cells and much greater variability in the kinetics of phenotypic progression of agspecific cd8 t cells when compared with c57bl/6 mice [31] . importantly, these investigators found, using an infection/reinfection protocol, that c57bl/6 mice were protected from the lethal effects of lm whereas survival of sw mice was significantly more variable. these studies illustrate the larger degree of variability of t cell responses to pathogen infection in outbred mice. allograft rejection the use of genetically restricted, inbred mice have been the mainstay for the large majority of studies exploring the immunopathogenesis of transplant rejection a variety of different tissue allografts. in one of the few studies that has assessed the effects of genetic diversity of donor and recipient on allograft survival in mice, reichenbach et al. performed vascularized, heterotopic heart transplantation in outbred and inbred mice [29] . they found that all heart allografts that were obtained from an inbred strain and transplanted into a different recipient strain (e.g. inbred→inbred) or into an outbred stock (inbred→outbred) were rejected within 6-16 days. in contrast, when heart allografts obtained from outbred donors and transplanted into an inbred (outbre-d→inbred) or outbred recipient (outbred→outbred), they observed a spectrum of outcomes that included very early rejection (< 4 days) in 30% of the recipients, acute rejection (6-24 days) in 54% of the transplanted recipients and chronic rejection (> 75 days) in 17% of the recipients [29] . the striking, very early rejection observed in 30% of the outbred→inbred and outbre-d→outbred mice did not appear to be classic hyperacute rejection since the presence of preformed, anti-donor antibodies in recipients was very low or absent. in fact, very early allograft rejection was characterized by neutrophilic vasculitis and hemorrhagic necrosis that could be abrogated by depletion of neutrophils or complement [29] . these investigators concluded that very early allograft failure was dependent on the genetic diversity of outbred donor mice and not the recipient. intestinal inflammation a number of mouse models of the inflammatory bowel diseases (e.g. ibd; crohn's disease, ulcerative colitis) have been developed to investigate the immuno-pathogenesis of these chronic inflammatory disorders. although a great deal of important mechanistic information has been obtained using these preclinical mouse models, only a small percentage (< 10%) of promising targets and therapeutic interventions have been translated to patient treatment [3, 7] . a recent study by barone et al. sought to determine how genetic diversity (and gender) affects the onset and severity of chronic colonic inflammation induced by intra-rectal administration of the haptenating agent dinitrobenzene sulfonic acid (dnbs) in c57bl/6 inbred vs cd1 outbred male and female mice [32] . colonic inflammation was induced via two intra-rectal injections of dnbs spaced 21 days apart and assessed for evidence of colonic inflammation. barone et al. found that colonic administration of dnbs significantly reduced survival of cd1 male and female mice compared with c57bl/6 mice as well as reduced body weight to a greater extent in cd1 vs. c57bl/6 mice. in addition, cd1 mice displayed greater macroscopic and histopathological evidence of colitis when compared with their inbred counterparts, suggesting that cd1 outbred mice may be more susceptible to chemically-induced colitis [32] . these findings suggest that the use of outbred mice may provide investigators with new information to better understand the immunomodulatory mechanisms associated with this model of chronic colitis. inflammatory angiogenesis inflammation and angiogenesis are important physiological responses that are required for fibrovascular tissue growth and wound healing. however, dysregulation of this proliferative response may result in the development of chronic inflammatory diseases such as atherosclerosis, rheumatoid arthritis, asthma and inflammatory bowel disease, to name just a few [42] [43] [44] [45] [46] [47] . recent studies by andrade and coworkers examined how host genetic diversity may influence the development and perpetuation of inflammatory angiogenesis and fibrogenesis. using a wellcharacterized mouse model of peritonitis, they compared the immuno-pathological responses (e.g. inflammatory cell infiltration, angiogenesis and fibrosis) in three inbred strains (dba-1, balb/c, c57bl/6) and one outbred strain (swiss) [47, 48] . these investigators observed highly variable responses among the four different groups of mice at 7 days following implantation. for example, angiogenesis and macrophage infiltration were greatest in c57bl/6 mice when compared with the other three inbred strains of mice [47] . interestingly, fibrogenesis markers, as assessed by implant-derived transforming growth factor beta-1 and collagen were consistently lower in dba/1 mice when compared with the other three inbred strains. in addition to defining the angiogenic and fibrogenic profiles among the different inbred and outbred mice, marques et al. investigated how host genetics affected the anti-platelet activity of dipyridamole (dp) in their peritonitis model [47] . overall, they found that dp treatment exerted a more generalized anti-inflammatory response in inbred strains vs outbred swiss mice with significant anti-fibrogenic effects observed only in c57bl/6 mice [47] . taken together, these data demonstrate that inflammatory angiogenesis is highly strain dependent and highlight the importance of host genetics in these models of peritonitis. neuroinflammation murine experimental autoimmune encephalomyelitis (eae) is a mouse model of t cellmediated demyelinating disease of the central nervous system (cns) that is used to study the immunepathogenesis of human multiple sclerosis (ms) [49, 50] . this autoimmune model of ms is induced in mice via immunization with different myelin-derived antigens such as myelin basic protein (mbp), myelin oligodentrocyte glycoprotein (mog) or proteolipid protein (plp) [49, 50] . it is well-recognized that different inbred strains of mice exhibit differential susceptibility to antigeninduced eae [49, 50] . one of the few published studies that have compared the immunological responses of susceptible c57bl/6 mice to those of resistant outbred cd1 mice following immunization with the mog peptide has been reported by marin et al. [51] . using identical immunization protocols, these investigators found that t cells obtained from immunized c57bl/6 mice were capable of enhanced proliferation when challenged with mog in vitro whereas t cells from cd1 mice showed little or no proliferation. in addition, marin and coworkers found that immunization of cd1 mice with mog 35-55 peptide increased the percentages of regulatory t and b cells when compared with c57bl/6 mice. the authors concluded that the resistance of cd1 mice to mog induced eae may be mediated by expansion of regulatory t and b cells via mhc-independent mechanisms. nikodemova et al. compared neuro-inflammatory responses of microglial cells obtained from inbred c57bl/ 6 vs. outbred icr/cd1 mice subjected to lipopolysaccharide (lps) administration in vitro and in vivo. for their in vitro studies, they used bv2 and n9 microglial cell lines that were originally derived from either c57bl/6 inbred mice or icr/cd1 outbred mice, respectively as well as primary microglial cells derived from the brains of the two different mice [28] . they found that addition of lps to n9 cells (derived from icr/cd1 mice) induced large and significant production of tnfα and the nitric oxide (no)-derived metabolite, nitrite that were significantly greater than those produced by the lps-activated bv2 (c57bl/6) microglial cells [28] . in addition, they found that addition of lps to icr/cd1 primary microglial cells induced greater amounts of tnfα and nitrite when compared with lpsactivated microglial cells obtained from c57bl/6 mice. furthermore, intra-parenchymal injection of lps induced greater expression of mrna for tnfα, il-6 and inducible no synthase in microglial cells from icr/cd1 vs c57bl/6 mice [28] . taken together, these data demonstrate the influence of host genetics on microglial responses to inflammatory mediators in vitro and in vivo. the use of the commercially available stocks of outbred mice has provided investigators with a greater understanding of range and variability of immune responses that may not be observed using inbred strains. nevertheless, outbred stocks may in many cases, exhibit genetic lability as well as a poorly defined genotype and genetic variation within and between different stocks [34] . in an attempt to approximate the complex interactions of genes involved in human immune responses, a genetically-diverse reference population of mice has been generated that is referred to as collaborative cross (cc) mice [52, 53] . this unique panel of genetically diverse mice was generated by combinational "funnel" breeding schemes using 8 unique and genetically diverse founder strains of mice that included 3 inbred strains (c57bl/6, a/j, 129s1/svimj), 2 inbred mouse models of disease (nod/shiltj-type 1 diabetes and new zealandobese/type 2 diabetes mice) and 3 wild-derived strains (cast/eij, pwk/phj, and watkins star line b (wsb)/ eij mice) ( fig. 1 ) [30, [53] [54] [55] . partial recombinant inbred strains of cc mice (termed pre-cc mice) were produced by three generations of funnel breeding followed by brother-sister/inbred mating for 4-5 generations. over the past few years, different strains of the pre-cc mice were interbred for at least 20 generations thereby creating numerous recombinant inbred cc strains termed cc-ri strains. both pre-cc and cc-ri strains contain genetic contributions from all eight of the original founders that display~90% of the known genetic variation present in wild mice (mus musculus) [30, 53] . each mouse within a given cc-ri strain is genetically identical containing maximum allelic variation whereas each cc-ri strain is genetically distinct from all other cc-ri strains. to date, more than 60 individual cc-ri strains have been created at the university of north carolina (http://www.csbio.unc.edu/ccstatus/index.py?run= ccv). additional cc strains of mice have been created by interbreeding two different cc-ri strains to generate f1 recombinant intercross offspring (cc-rix) that are heterozygous for the h-2b major histocompatibility complex (mhc) [30, 56] . this type of breeding scheme allows investigators to use specific reagents (e.g. mhc tetramers) to examine different aspects of t cellmediated immunity. pre-cc, cc-ri and cc-rix mice have been used to characterize immune responses to human viral pathogens (e.g. influenza, west nile and ebola), chronic inflammation and cancer [30] (and references therein). by observing the spectrum of disease phenotypes in models of infectious and inflammatory diseases using well-defined strains of cc-ri or cc-rix strains in which the genetics have been wellcharacterized, investigators may be able to identify disease susceptibility loci [54] . multiple cc strains have been used to identify promising candidate genes that influence susceptibility or resistance to viral infections [54] . the studies outlined above provide examples of how murine genetic diversity may affect immune responses to pathogens and/or inflammatory mediators. these studies suggest that inbred strains may be better suited for initial investigations to define specific immunopathological mechanisms of infectious and inflammatory diseases with follow up studies using outbred mice to address the role of genetic diversity in these models. the mammalian body is home to a plethora of microorganisms that include prokaryotes (bacteria and archaea), viruses and eukaryotes (fungi, helminths and protozoa). a recent reevaluation of the total numbers of bacteria in the human body reports that the colon contains 10 14 bacteria followed by the small bowel (10 11 ), skin (10 11 ) and saliva (10 11 ) [57, 58] . under normal, steady state conditions, these commensals play a critical role in the development, instruction and function of the host immune system [59] . although the host immune system limits translocation of commensal bacteria into tissue and the systemic circulation, emerging evidence demonstrates that bacterial components and metabolites gain access to the systemic circulation and peripheral tissues where they may modulate tissue function [60] . josefsdottir et al. recently reported that intestinal bacterial products such as microbial-associated molecular patterns are required for robust hematopoiesis [61] other groups of commensal microorganisms that are particularly prevalent in the gut lumen are eukaryote and bacterial viruses [62] . in addition to their well-known pathogenic properties, both groups of viruses are known to possess potent immunomodulatory properties. for example, studies have shown that infection of mice with certain eurkaryote viruses are capable of attenuating development of chronic inflammation in mouse models of type i diabetes and sle-like disease (see [62] and references therein). in addition, liu et al. recently reported that eukaryotic viruses are required for maintaining appropriate numbers of intraepithelial lymphocytes in the small and large bowel [63] . bacterial viruses (i.e. bacteriophage) are also thought to protect the gut and host by infecting and killing pathogenic bacteria. a study by barr et.al . demonstrated that bacteriophage are highly enriched in the gut mucus layer where they are thought to interfere with adhesion of pathogenic bacteria to the gut epithelium thereby preventing or limiting their invasion into the tissue [64] . the mammalian gut and skin are also colonized with a diverse population of fungi. although not as well studied as other commensals, data suggest that certain fungi play important roles in immune system development. zhang et al. reported that intra-gastric administration of candida tropicalis (but not trichosporon asahii nor saccharomyces cerevisiae) to immune cell-deficient germ free mice or wild type mice treated with an antifungal cocktail promoted the migration of retinaldehyde dehydrogenase positive dendritic fig. 1 generation of collaborative cross mice. a. this unique panel of genetically diverse mice was generated by interbreeding 8 unique and genetically diverse founder strains of mice that included 3 inbred laboratory strains (c57bl/6, a/j, 129s1/svimj), 2 inbred mouse models of disease (nod/shiltj-type 1 diabetes and new zealand-obese/type 2 diabetes mice) and 3 wild-derived strains (cast/eij, pwk/phj, and watkins star line b (wsb)/eij mice). each strain was assigned both a letter (a-h) and a specific color. b a combinational funnel breeding scheme was used to produce offspring with equal distribution of founder alleles. this figure illustrates the specific breeding funnel for the generation of the cc strain cc001. c color illustration of founder genome contributions to cc001 strain. reproduced from [54] with permission cells into lymph nodes (lns) draining the gut and skin thereby stimulating the development of functional lns and gut-associated lymphoid tissue [65] . in another study, markey et al. reported that colonization of mice with candida albicans protects mice from a subsequent lethal challenge with the bacterial pathogen clostridium difficile [66] . although the vast majority of all microbiome studies have focused on prokaryotes, emerging studies are demonstrating multi-cellular helminths and unicellular protozoa represent additional groups of commensal residents in the healthy gut that possess immunomodulatory activity. historically, past investigations have focused almost exclusively on the parasitic and pathogenic properties of these eukaryotes. although these multicellular organisms can produce severe disease, colonization does not necessarily result in disease [67] recent studies suggest that these organisms may in fact, represent true commensal microbiota that may possibly protect their host [68, 69] . for example, helminths are known to release a variety of excretory-secretory (es) components that have been shown to modulate the functions of several immune cells as well as induce the generation of immunosuppressive regulatory t cells (tregs) and modify the intestinal microbiota (reviewed in [68] . indeed, there is a growing literature demonstrating that colonization of animals with helminths or treatment with certain es components attenuate allergic and chronic inflammatory diseases [68, 70] . in addition to helmiths, the parasitic and disease producing properties of protozoa have been studied extensively over the past several decades. recent evidence suggests that while many protozoa are indeed pathogens, several of these unicellular microorganisms have been identified in the healthy microbiota [69] . one such commensal is blastocystis spp. this protozoa has been shown to enhance the diversity and alter the microbial composition of individuals in industrialized countries, suggesting that microorganisms may exert a beneficial effect to the host [69] . these observations are particularly important given the fact that industrialized countries have attempted to eliminate both helminths and protozoa thereby limiting parasitic infections. loss of these and possibly other commensal eukaryotes appears to be associated with dramatic increases in allergic and inflammatory diseases over the past five decades [68, 69] . a major breakthrough in our fundamental understanding of innate and adaptive immunity occurred following the generation of mice containing spontaneous or genetically-engineered alterations in specific genes related to immune system development and/or function. because many of these mutant mice were immunocompromised, it was necessary to develop animal housing conditions that would prevent the introduction of pathogens into the mouse colonies. in addition to maintaining a healthy environment for these mice, eliminating the introduction of known mouse pathogens into the vivarium allowed for more consistent breeding of mice as well as more reproducible induction disease phenotypes [71] . although the term "specific pathogen-free" (spf) was first used in the 1950s, it wasn't until the 1980s when animal care facilities began to use filter top micro-isolator cages in conjunction with meticulous biocontainment protocols to limit the introduction of murine pathogens into these facilities [71] . currently, all animal care facilities in the u.s. continuously monitor for (and exclude) known murine pathogenic viruses, bacteria and parasites to create spf conditions. in addition, mice are housed under barrier conditions that require animals to be kept in micro-isolator cages with filter top lids or in individually ventilated cages (ivc) that use hepa filtered air rather than ambient air. furthermore, barrier facilities use sterilized cages, food, and water. these ultra-hygienic housing conditions have been incredibly important for the biomedical research community because they essentially "standardized" the mouse microbiome that appeared to reduce the variability of results observed by different laboratories. however, recent evidence suggests the immune system of mice raised under spf/barrier conditions is immature when compared with their wild (i.e. feral) counterparts and to adult humans suggesting natural exposure to pathogens may be important in promoting maturation of the immune system [12, 16, 71] . the following sections will focus on how the composition of intestinal bacteria influences immunity and inflammation. infection a recent study by beura et al. demonstrated that feral (i.e. wild) or pet store-housed mice exhibited an immune cell composition similar to adult humans whereas spf/barrier-housed mice possessed an immune system that was more akin to neonatal humans (fig. 2a & b) [12] . these investigators found that spf/barrier mice and neonatal humans have many fewer antigenexperienced, cd8 + memory/effector t cells when compared with feral or pet store-housed mice or adult humans (fig. 2c & d) . these and other analyses suggested that exposure of mice to the diverse spectrum of microorganisms that are normally encountered during the wild greatly influences the maturation and cellular composition of their immune system. furthermore, these investigators showed that infection of spf/barrierhoused c57bl/6 mice with the intracellular pathogenic bacteria listeria monocytogenes (lm) had > 10,000-fold greater bacterial load than did pet store mice infected with same pathogen suggesting that exposure to diverse microbiota greatly increases resistance to pathogenic bacteria [12] . although pet store mice do in fact possess a more diverse microbiota than spf/barrier mice, it is likely to be less diverse than free-living feral mice and humans who are exposed to an even greater spectrum of viral, bacterial and parasitic micro-organisms. rosshart et al. recently tested the hypothesis that colonization of spf/barrier-housed mice with intestinal microbiota obtained from feral mice would greatly influence immune responses to viral pathogens [15] . these investigators captured over 800 feral mice from horse barns located in several different regions of maryland and washington d.c. of these, 98 mice were found to be mus musculus domesticus, the feral species of mouse that is most closely related to common laboratory mice such as c57bl/6 mice. surprising, despite being exposed to pathogens in the wild, a number of captured mice were found to be pathogen-free with no observable evidence of disease. not surprisingly, rosshart et al. determined that the intestinal microbiota of feral mice was significantly different from that of commercially available c57bl/6 mice [15] . colonization of pregnant, germ free c57bl/6 mice was performed using daily oral gavage of microbiota (for 3 days) from pathogen-free feral mice or from spf/barrier c57bl/6 microbiota. these mice were referred to as wildr and labr mice, respectively. spf/barrier-housed c57bl/6 mice (called lab mice) were used to compare to wildr and labr mice. all three groups of mice were then infected via intranasal administration of the mouse-adapted, influenza a virus (iav; puertorico/8/1934 h1n1 strain). surprisingly, 92% of the wildr mice infected with iav survived for 18 days post-infection whereas only 17% of the labr and lab mice were alive at 18 days post-infection (fig. 3a) [15] . survival of wildr mice was associated with ∼10-fold lower titers of lung-residing iav as well as significantly lower lung histopathology scores and fig. 2 mice housed under spf/barrier conditions lack differentiated memory cd8 + t cell subsets compared with feral mice, pet store mice and adult humans. a percentages of cd8 + t-cell phenotypes in laboratory/spf mouse blood, adult human blood and human neonatal cord blood. the top panels are gated on cd3 + /cd8 + cells with naïve t cells highlighted in blue, central memory t cells highlighted in green and effector memory t cells highlighted in red as defined by established lineage markers in each species. bottom panels are gated on antigen-experienced subsets as defined in the green and red quadrants above). b percentages of granzyme b + /cd8 + t-cells are much less in the antigen-experienced subsets of laboratory mice and neonatal humans when compared with adult humans. c and d percentages of naïve cd8 + t cells (cd44 lo / cd62l hi ) in pbmcs from laboratory mice are much greater than those in pbmcs from feral and pet store mice whereas percentages of antigenexperienced c8 + t cells (cd44 hi /cd62l lo ) were significantly greater in feral and pet store mice when compared those in laboratory mice. reproduced from [12] with permission inflammatory cytokine levels when compared with labr and lab mice (fig. 3b) . overall, these exciting studies strongly suggested that colonization of laboratory mice with feral microbiota protects the host from a potentially lethal viral infection observed in spf/barrier mice. fig. 3 colonization of germ free mice with microbiota obtained from feral mice protects against viral infection and inflammation-induced cancer. pregnant, germ free c57bl/6 mice were colonized via oral gavage of gut microbiota obtained from feral mice (wildr mice) or from spf c57bl/6 mice (labr mice). barrier-housed c57bl/6 mice (lab mice) were used as controls to compare to wildr and labr mice. a male and female mice in all three groups of mice were infected (intranasally) with influenza a virus (iav; puerto rico/8/1934 h1n1 strain). approximately 92% of the wildr mice infected with iav survived for 18 days post-infection whereas only 17% of the labr and lab mice were alive at 18 days post-infection. b survival of wildr mice was associated with ∼10-fold lower titers of lung-residing iav as well as significantly lower lung histopathology scores and inflammatory cytokine levels when compared with labr and lab mice. c inflammation-induced colorectal cancer was induced in all three groups of mice via a single injection (i.p.) of the mutagen azoxymethane (10 mg/kg of body weight) followed by induction of colonic inflammation via oral (ab libitum) administration of dextran sodium sulfate (2-2.5%) in the drinking water. representative images of dissected colons demonstrated greater numbers of colonic tumors (tumors indicated by red dots) in lab and labr mice when compared with wildr mice. d top panel: representative histopathology (10x magnification) of h&e-stained sections of longitudinal colon tumors with arrows indicating well-differentiated adenocarcinoma in the mucosa. histopathological analyses revealed that lab and labr developed tubular adenoma, well-differentiated tubular adenocarcinoma and mucinous carcinoma. furthermore, mucinous carcinoma cells were found invading the submucosa and muscular layer where moderate-severe inflammation was also noted. asterisks identify mucinous nodules. in contrast, wildr mice developed smaller numbers of colonic tumors, reduced tumor area/colon area, diminished tumor invasion and less inflammation when compared with the labr and lab mice that received the same treatment protocol. bottom panel: movat's staining of serial sections from the same tumors presented in the top panels clearly shows the presence of mucinous nodules (mucin stains green) containing mucinous carcinoma cells in submucosa and muscle layers of lab and labr but not wildr tumors. reproduced from [15] with permission the studies described above are consistent with two other reports that characterized the differences in immune cell composition and responses in feral vs. laboratory mice infected with different pathogens. abolins et al. foud that innate and adaptive immune cells in feral mice exist in a much higher state of activation when compared with barrier-housed laboratory mice [72] . because feral but not spf/barrier mice were found to be seropositive for several microbial, parasitic and myocoptes (mites) organisms, the authors suggested that alterations in the different immune cell populations and their responses were most likely driven by pathogen infection of these free-living rodents. these data agreed well with those reported by reese et al. who found that sequential infection of spf/barrier mice with three viral, bacterial and parasitic pathogens known to infect children, induced gene expression patterns in blood cells that were quite similar to those observed in pet storehoused mice and human adults [73] . autoimmune and chronic inflammatory diseases it is well known that the phenotype of different mouse models of autoimmune and chronic inflammatory diseases may be dramatically altered by changes in institution, housing conditions, and/or diet [74] [75] [76] [77] . this is not surprising given the evidence demonstrating that the intestinal microbiota of mice may vary greatly among different animal care facilities and animal vendors [74, 75, [78] [79] [80] . several laboratories including our own, have shown that the incidence and severity of chronic gut inflammation in different mouse models of inflammatory bowel disease is markedly altered by changing animal care facilities or by purposely altering the gut bacteria [81] [82] [83] [84] [85] [86] [87] . in addition to its effects on intestinal inflammation, alterations in gut bacteria have been found to greatly influence the onset and severity of joint inflammation. using the k/bxn t cell receptor transgenic mouse (kbn) model of autoimmune arthritis [88] , wu et al. demonstrated that when kbn mice were housed under spf/barrier conditions, they developed chronic and severe arthritis whereas housing these mice under germ free (gf) conditions exhibited greatly reduced disease [89] . in addition, when newborn kbn mice were continuously treated with vancomycin or ampicillin for several weeks, disease severity was markedly reduced suggesting that commensal microbiota were required for induction of disease [89] . ivanov et al. had previously reported that a single bacterial species within the commensal flora called segmented filamentous bacteria (sfb) was capable of inducing the generation of intestinal lamina propria (i-lp) th17 cells that are thought to play a role in the development of arthritis [79] . thus, wu et al. colonized gf mice with sfb to determine whether this one bacterial species could recapitulate the disease-producing effects of the spf microbiota. indeed, they found that colonization with sfb induced the generation of i-lp th17 cells that produced robust autoimmune arthritis [89] . similar findings were reported by lee et al. who showed that while gf mice failed to develop eae, gf mice colonized with sfb developed severe eae [90] . these important studies demonstrated that a single group of commensal bacteria is capable of inducing autoimmune disease in different tissues via the generation of a specific disease-producing t cells. although sfb are important in promoting inflammatory tissue damage in mouse models of arthritis and ms, other studies demonstrate that the presence of sfb may limit the development of other chronic inflammatory diseases such as diabetes. it has been known for more than 25 years that the incidence and severity of experimental autoimmune diabetes is markedly increased when nonobese diabetic (nod) mice are housed under gf conditions suggesting that commensal bacteria may protect against autoimmune destruction of islet cells [76, 80] . kriegel et al. took advantage of the fact that their nod mouse colony was variably colonized with sfb to examine whether the presence or absence of sfb affected the incidence and/or severity of diabetes in males and females. they found that 91% of the sfb-negative (sfb-) females developed robust disease by week 30 whereas only 16% of the sfb-colonized (sfb+) females developed hyperglycemia [80] . in contrast, both sfb+ and sfbmales developed little or no disease with an overall incidence of < 20%. furthermore, development of insulitis occurred in both sfb+ and sfb-females suggesting that the presence of sfb somehow interferes with the progression of islet inflammation to hyperglycemia. inflammation and tumorigenesis colorectal cancer (crc) is one of the most common forms of cancer in modernized (i.e. westernized) societies that is predicted to increase over the next 20 years [91, 92] . it is becoming increasingly appreciated that changes in diet in modernized societies may contribute to the development of crc by altering intestinal microbiota communities and immune responses that may promote gut inflammation [91, 93] . because rosshart et al. demonstrated that colonization of barrier mice with feral mouse microbiota protected these wildr mice from the lethal effects of the viral pathogen iav (discussed above), this same group wished to determine whether wildr mice would also be protected in a mouse model of crc [15] . to do this they used the well-characterized mutagen and inflammation model of crc in which mice were injected (i.p.) with the mutagen azoxymethane followed by induction of colonic inflammation via oral administration of dextran sodium sulfate. this model exhibits chronic colitis that progresses to high grade dysplasia and development of crc [15, 94] . they found that wildr mice developed smaller numbers of colonic tumors, reduced tumor area/colon area and diminished tumor and inflammatory cell invasion when compared with the labr and lab mice that received the same treatment protocol [15] (fig. 3c) . there is no question that spf/barrier housing has greatly enhanced reproducibility of immune responses and disease phenotypes. nevertheless, the lack of exposure of lab mice to the spectrum of naturally occurring microorganisms, produces mice with an immune system that differs quite dramatically from feral mice and humans. it is becoming increasingly apparent immune system development in both mice and humans is shaped by exposure to a multitude of diverse immunological experiences that begin at birth (fig. 4) . although there is a growing interest in the use of "dirty" mice to enhance translation of preclinical studies, the generation, use and housing of these mice may be quite difficult to implement. because feral or pet store colonized mice would likely contain a plethora of viral, bacterial and parasitic pathogens that could quickly contaminate barrier facilities, dirty mice would have to be housed in a location that is physically separated from the barrier facility [71] . for example, dirty mice could be housed in a quarantine facility or in a location that does not house rodents. in order to prevent the spread of pathogens in these facilities, several protocols would have to be implemented such as the use of dedicated equipment for caging and for sanitation of cages and water bottles. in addition, it would be necessary to carefully control the movement and hygiene of animal care and laboratory workers as well as employ directional airflow to limit the spread of airborne pathogens outside of the animal room. finally, the cost of housing these mice would undoubtedly be much greater than barrier housing. one intriguing protocol that creates mice that are colonized with natural/wild microbiota is called "rewilding". graham and coworkers recently reported that when lab mice are transferred to outdoor enclosures where they are exposed to the weather and microbiome that inhabits the soil and vegetation, they display maturation and differentiation of different t cell subsets, increased numbers of circulating granulocytes and changes in intestinal microbiota that are similar to those described above in feral and pet store mice [95] [96] [97] . when taken together, it is becoming clear that colonization of mice with diverse populations of naturally occurring microorganisms protects them against an environment that contains potentially lethal infectious microbes, inflammogens and carcinogens. fig. 4 genetic variability, immunologic experiences and antigenic challenges in humans and mice. humans are genetically more diverse and are subjected to highly variable, immunological experiences that shapes immune system development and function. immunologic experiences in mice can be manipulated using different animal housing and husbandry protocols as well as exposure to a more diverse or natural microbiota. reproduced from [71] with permission another environmental parameter that has received a great deal of attention over the past several years is the influence of housing temperature on murine physiology and immunology. virtually all animal care facilities in the u.s. house mice at 20-24°c, a temperature range that is comfortable for human caregivers. however, this temperature range induces mild but chronic cold stress in these rodents as their thermoneutral temperature (tnt) is 30-32°c [22] [23] [24] [98] [99] [100] . murine tnt is defined as "the temperature range within which the heat produced as a byproduct of normal metabolism alone, combined with blood flow movements from the core to the surface of the body, enables an animal to maintain a normal core body temperature of~37°c" [22, 23] . housing mice at standard animal care temperatures (st; 20-24°c) is known to increase their resting heart rate and basal metabolic rate when compared with mice housed at their tnt [24] . in addition, housing mice at st activates the sympathetic nervous system (sns) resulting in the release of the catecholamines epinephrine (epi) and norepinephrine (ne) as well as activates the hypothalamic-pituitary-adrenal axis (hpa) to induce production of glucocorticoids [22] . because catecholamine and glucocorticoid receptors are found on nearly all cells within the body, cold stress-induced activation of the sns and hpa produces marked alterations in the cardiovascular, skeletal-muscular and immune systems [23, 101] . (fig. 5) . furthermore, it is becoming increasing appreciated that cold stress may exert profound effects on intestinal homeostasis and microbial composition [22, 102, 103] . this inconvenient reality has prompted investigators to examine how housing temperature may influence immune responses in mice. it is well known that primary lymphoid tissue (thymus, bone marrow) as well as the spleen, lymph nodes and mucosa-associated lymphoid tissue are innervated by sympathetic nerves suggesting that ne, the major neurotransmitter released by sympathetic nerves, is involved in modulating immune responses [104] [105] [106] . indeed, it has been shown that ne-induced β2 adrenergic receptor (β2-ar) signaling in t and b cells, myeloid cells and dendritic cells (dcs) modulates antigen-processing and presentation as well as t cell activation, differentiation and recirculation [104, [106] [107] [108] [109] [110] [111] [112] [113] . a recent study by araujo et al. fig. 5 housing temperature affects the physiology, immunology and immunopathogenesis pathogenesis of mouse models. this illustration depicts a number of different experimental settings and models in which outcomes differ in mice housed under standard animal care temperatures (∼22°c) vs. housing at their thermoneutral temperature (∼30°c). reproduced from [23] with permission. references for the different studies in this figure appear in reference [23] demonstrated that the sns limits the development of mog 35-53 /cfa-induced experimental autoimmune encephalomyelitis (eae) in mice via β2-ar signaling in cd4 + t cells that constrains the generation of encephalogenic effector cells [107] . in contrast, when β2-ardeficient (β2-ar −/− ) mice were immunized with mog 35-53 /cfa, they developed more severe disease when compared with wild type mice. taken together, these data clearly demonstrate that the sns plays an important role in regulating cns autoimmunity. it will be interesting to assess the role of the sns in mouse models eae or other autoimmune disease housed at their tnt. the following section presents a brief overview of the effects of housing temperature on infection, immunity and inflammation. the reader is referred to the following references that summarize the role of housing temperature in mouse models of cancer and antitumor immunity [22, 23, 112, 114, 115] . in addition, investigators have shown that cold stress may exert profound effects on intestinal homeostasis and microbial composition [22, 102, 103] . as pointed out above, β2-ar are found on all myeloid cells as well as t and b cells. stress induced release of ne and its engagement with the β2-ar modulates immune responses to infectious microorganisms [21] . although relatively few studies have directly assessed the effects of housing temperature on infection and immunity, there are numerous studies demonstrating that β2-ar signaling is generally immunosuppressive and results in increased susceptibility of the mice to pathogen infection (the reader is referred to these reviews: [21, 104, 106, 114] . using selective β2-ar agonists and antagonists or genetic ablation of the receptor, several laboratories have described the role that β2-ar signaling pathways play in infection with different bacterial (l. monocytogenes, p. aeruginosa, s. typhimurium, k. pneumonia, e. coli) or viral pathogens (cytomegalovirus, herpes simplex, influenza, vesicular stomatitis) [21, 114] . cold stress-induced immune suppression has been suggested to account for the high mortality rate and lack of clinical translation of mouse models of infectious disease [24, [116] [117] [118] . in a recent study, rubin directly compared different immune responses to the francisella tularensis (ft) live vaccine strain in mice housed at st (22°c) to mice housed at temperatures approximating their tnt (28°c). rubin demonstrated that when mice were housed at their tnt they exhibited increased antigen-specific t-cell responses compared with mice housed at 22°c [116] . in addition, rubin found that intranasal challenge of ft to immunized mice housed at 22°c was consistently fatal whereas immunized mice housed near their tnt (28°c) survived the same intranasal challenge. when taken together, this study demonstrates that mice housed below their tnt exhibit diminished t cell responses to this intracellular pathogen resulting in animal death. furthermore, this study as well as others referenced above suggest that housing mice at their tnt may help to improve the translation of data obtained from mouse models of infectious disease and vaccine development. obesity, metabolic inflammation and atherosclerosis excessive ingestion of lipid-laden foods is known to induce low grade but chronic inflammation that is thought to play a role in the pathogenesis of different metabolic diseases such as obesity, atherosclerosis, chronic liver disease, type 2 diabetes and cancer [119, 120] . indeed, obesity has become an international epidemic that is the second most preventable cause of death in modernized societies [121] . one life-threatening disease associated with obesity is atherosclerosis. this cardiovascular disease is the leading cause of death of men and women in the u.s. [69] . histopathological examination of atherosclerotic vessels reveals the presence of cholesterol and immune cells in the arterial wall that ultimately progresses to plaque formation and occlusion of blood flow. defining the immuno-pathogenic mechanisms responsible for obesity-induced vascular plaque formation and progression has been difficult to model in mice since wild type mice do not develop atherosclerosis when placed on a high fat/high cholesterol western diet (wd). much of what we have learned has come from the use of genetically-engineered mice that lack the lowdensity lipoprotein receptor (ldr −/− ) or apolipoprotein e (apoe −/− ). data obtained from studies using these mutant mice have been important in revealing the role of lipid accumulation and immune cell infiltration in hyperlipidemia-induced vascular inflammation and plaque formation; however, neither mouse model exhibits lipid profiles and vascular pathology that are identical to patients with atherosclerosis [122, 123] . differences between these models of atherosclerosis and human disease have prompted investigators to develop additional mouse models that more closely recapitulate the pathophysiology of this vascular occlusive disease. recent studies demonstrating that housing mice at their tnt affects cardiovascular physiology, inflammation and metabolism have motivated investigators to examine how housing temperature may influence the development and progression of obesity, metabolic inflammation and atherosclerosis in mice fed a high fat diet (hfd). giles et al. reported that feeding a wd to c57bl/6 mice housed at their tnt (30°c) enhanced weight gain and fat mass when compared with mice fed a wd diet while housed at st (22°c) [124] . in addition, they demonstrated that tnt housing in combination with wd induced mild atherosclerosis that was associated with increases in serum concentrations of total cholesterol and ldl, aortic plaque formation, and greater immune cell infiltration into the vascular lesions when compared to mice fed a wd and housed at st [124] . furthermore, both giles et al. [124] and tian et al. [119] demonstrated that feeding a wd to apoe −/− mice housed at their tnt greatly enhanced the development of obesity as well as accelerated the onset and severity of atherosclerosis when compared with their st controls. thus, both studies demonstrate that housing temperature plays an important role in the development of metabolic inflammation, obesity and atherosclerosis. the development of increased weight gain and adiposity at tnt has also been reported by stemmer et al. in t cell-deficient, c57bl/6 nude (nu/nu) mice [125] . these results have important implications for investigators who wish to model the effects of obesity on human tumor development in vivo. obesity has been linked to the development of a variety of different types of cancers; however, the vast majority of human tumor xenograft studies have been performed using obesity-resistant balb/c nude mice. thus, the data reported by stemmer et al. may provide a novel mouse model for assessing the impact of obesity on human tumor growth in vivo. nonalcoholic fatty liver disease another, potentially life-threatening condition that is strongly associated with obesity is nonalcoholic fatty liver disease (nafld). this chronic condition is characterized by the accumulation of excess fat in the liver of individuals who drink little or no alcohol. the most common form of nafld is a relatively innocuous condition referred to as nonalcoholic fatty liver (nafl). although fat accumulation within hepatocytes is alarming, liver function does not appear to be impaired by this condition. some individuals with nafl may go on to develop a more serious condition called nonalcoholic steatohepatitis (nash). the livers of these individuals exhibit fat accumulation, inflammatory cell infiltration and varied levels of fibrosis. if left untreated, nash may ultimately progress to cirrhosis and hepatocellular carcinoma [126] [127] [128] [129] . although current mouse models of nafld have been important in delineating certain aspects of disease pathogenesis, they do not fully recapitulate human disease. for example, mouse models are restricted to the use of male mice since females do not develop high fat diet (hfd)-induced obesity and nafld. in addition, mouse models of nafld do not develop the fibrosis (called "bridging fibrosis") that is observed in humans. therefore, investigators are attempting to develop mouse models that more closely recapitulate nash in both male and female mice. giles et al. recently reported that feeding male c57bl/6 mice with a hfd housed at their tnt (30°c) induced an acceleration in weight gain, increased liver weight and steatosis and an increase in liver immune cell infiltration when compared with mice that were fed a hfd and housed at st (22°c) [126] . in addition, they found that hfd and tnt housing enhanced expression of chemokine and fibrosis genes as well as increased infiltration of macrophages when compared with hfd/st mice. these tnt-induced alterations were associated with increased hepatocellular injury as determined by an increase in serum levels of alanine transaminase. interestingly, the authors did not observe overt fibrosis in these mice which is not surprising given the well-known fact that c57bl/6 mice are resistant to developing hepatic fibrosis [126] . in a second series of studies, giles et al. used male akr mice, an inbred strain that has been shown to develop marked obesity and nafld when fed a hfd at st (22°c). when these mice were housed at their tnt (30°c) and fed a hfd, they developed many of the same features and histopathological characteristics of nafld as did c57bl/6 mice; however, hfd/tnt akr mice developed hepatic fibrosis suggesting that these mice may be better suited to model human nash [126] . as noted above, the vast majority of preclinical studies have been performed using male mice since a hfd does not induce obesity and nafld in females. however, the prevalence of nafld in male and female humans is virtually the same [126] . therefore, giles et al. assessed the development of nafld in female c57bl/6 mice when housed at their tnt. they found that when female mice were housed at their tnt and fed a hfd, they displayed remarkable increases in body weight, liver weight, tissue adiposity and hepatic steatosis when compared with female hfd/st mice [126] . similar to male hfd/tnt mice, females housed under the same nafld-producing conditions exhibited increased expression of genes associated with fibrosis and augmented hepatocellular injury but no overt fibrosis. nevertheless, the development of these novel mouse models may allow investigators to identify novel the immuno-pathogenic mechanisms responsible for the development and progression of nafl d in both sexes. another interesting aspect of nafld development in both mice and humans is the relationship among intestinal dysbiosis, increased intestinal permeability and hepatocellular damage. in their recent study, giles et al. also observed greater increased numbers of live bacteria in the livers of hfd/tnt mice that appeared to correlate with dysbiosis and increased intestinal permeability when compared with their hfd/st counterparts. in fact, these investigators demonstrated that the bacterial composition of the dysbiotic microbiome in hfd/tnt mice was similar to that observed in humans with nash [126] . administration of neomycin and polymixin b sulfate reduced intestinal permeability, hepatic inflammation scores and liver damage when compared with obese, antibiotic-treated hfd/st mice. taken together, these data suggest that exacerbation of nafld in hfd/tnt mice may be mediated by alterations in intestinal bacterial composition. acute graft vs. host disease allogeneic hematopoietic stem cell transplantation (hsct) is a potentially lifesaving treatment for certain blood cancers, autoimmune diseases or hematologic disorders (sickle cell disease, fanconi anemia) [130] . although allogeneic hsct is a potential cure for these diseases,~50% of patients receiving this treatment will develop a potentially lifethreatening, multi-organ inflammatory condition called acute graft versus host disease (agvhd) [131, 132] . clinically, inflammation may occur in the gastrointestinal (gi) tract, skin, liver, lungs, bone marrow and lymphoid tissue [131] [132] [133] [134] [135] [136] [137] . the immuno-pathogenesis of agvhd has not been completely defined; however, experimental and clinical studies demonstrate that donor cd4 + and/or cd8 + t cells are the major effector cells responsible for mediating inflammatory tissue injury in the different target tissues [132] . the large majority of mouse models of agvhd use lethal, whole body irradiation to ablate the immune system prior to engraftment of allogeneic bone marrow (bm) that is supplemented with allogeneic t cells [138] . adoptive transfer of bm alone serves as the control group for these studies since recipients do not develop agvhd when housed at standard animal housing temperatures of 20-22°c. it should be noted that this contrasts with clinical hsct in which great care is taken to eliminate all t cells from the donor graft as the presence of even small numbers of residual, graft-associated t cells may induce fulminant disease [132] . in a landmark study using the conventional mouse model of agvhd described above, leigh et al. demonstrated that agvhd does develop in lethally-irradiated recipients engrafted with allogeneic bm alone, provided that the mice are housed at their tnt of 30°c [98] (fig. 6 ). in contrast, these investigators confirmed numerous other studies demonstrating that agvhd fails to develop in bm-engrafted mice housed at st of 22°c. they found that st-mediated suppression of disease was due to cold stress-induced β2-ar signaling by ne [98] . treatment of bm-engrafted mice housed at 22°c with a selective β2-ar antagonist reversed the cold stress-induced, ne-mediated suppression of agvhd such that these mice developed disease that was similar to bm-engrafted mice housed at 30°c [98] . conversely, when bm-engrafted mice were housed at 30°c and then treated with a selective β2-ar agonist, little or no disease was observed demonstrating that β2-ar signaling is crucial for suppression of agvhd. a recent study by mohammadpour et al. extended these findings using different models of allogeneic and xenogeneic models of hsct. they found that adoptive transfer of allogeneic, β2-ar deficient (β2-ar −/− ) cd4 + t cells (with bm) into lethally irradiated recipients induced more severe agvhd than did engraftment with wild type (wt) allogeneic t cells [139] . the exacerbation of disease in mice engrafted with β2-ar −/− cd4 + t cells was associated with increased numbers th1 effector cells whereas transfer of wt t cells resulted in increases in tregs, th2 cells and myeloid derived suppressor cells [139] . taken together, these data suggest that selective β2-ar agonists may prove useful in attenuating agvhd. asthma approximately 70% of patients suffering from asthma have reported difficulty breathing in cold air during the winter months [140] . although investigators and clinicians have suggested that inhalation of cold air may exacerbate asthma by inducing epithelial cell injury as well as immune cell infiltration and mast cell activation in the airways [141] [142] [143] , few mechanistic studies have fig. 6 housing mice at their thermoneutral body temperature (30°c) exacerbates the development of acute graft vs. host disease (agvhd). disease was induced by adoptive transfer of allogeneic c57bl/6 bone marrow (bm) alone into lethally-irradiated balb/c recipients. mice were housed at either 22°c or 30°c and monitored for signs of gvhd such as weight loss (a) and survival (b). reproduced from [98] with permission been reported to address whether ambient air temperature may alter the onset and/or severity of this chronic respiratory disease. a recent study by liao et al. compared disease symptoms and immune responses in asthmatic mice housed at st (20°c) or at tnt (30°c). airway inflammation was induced using a well characterized mouse model of acute asthma induced by intraperitoneal sensitization of the mice with ovalbumin (ova) followed by intratracheal challenge with aerosolized ova in saline. this protocol induces airway hyper-responsiveness, eosinophil infiltration into the bronchi and overproduction of mucus [144] . these investigators found that when asthmatic mice were housed at their tnt, inflammatory cell numbers, il-4 and il-13 levels were all significantly reduced in the bronchoalveolar lavage fluid as were serum levels of ige and airway hyper-responsiveness in tnt vs st mice. these data provide some of the first evidence that housing temperature may influence the onset and/or severity of acute bronchial inflammation and dysfunction. the use of genetically standardized mice and housing conditions have greatly advanced our fundamental understanding of innate and adaptive immunity as well as increased the reproducibility of animal studies. nevertheless, translation of promising therapeutic strategies observed in mouse models of infectious and inflammatory diseases to patient treatment has been disappointing. the reasons for the poor bench-to-beside transition have not been definitely defined; however, emerging evidence suggests that exposure of genetically constrained mice to pathogen-free microbiota at suboptimal housing temperatures may impair maturation of the immune system thereby altering the animal's immune responses to infectious microorganisms and inflammatory mediators. we propose that by using genetically diverse mice housed under more immunologically-and environmentally-relevant conditions may improve the chance of identifying new and more potent therapeutics to treat human disease. the mouse ascending: perspectives for humandisease models translation of research evidence from animals to humans pharmacological intervention studies 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reconstituted with cd45rbhigh cd4+ t cells intestinal microbiota composition of interleukin-10 deficient c57bl/6j mice and susceptibility to helicobacter hepaticus-induced colitis attenuation of immune-mediated bone marrow damage in conventionally housed mice organ-specific disease provoked by systemic autoimmunity gut-residing segmented filamentous bacteria drive autoimmune arthritis via t helper 17 cells proinflammatory t-cell responses to gut microbiota promote experimental autoimmune encephalomyelitis the intestinal microbiota in colorectal cancer global patterns and trends in colorectal cancer incidence and mortality nutrients, foods, and colorectal cancer prevention a novel inflammation-related mouse colon carcinogenesis model induced by azoxymethane and dextran sodium sulfate rewilding nod2 and atg16l1 mutant mice uncovers genetic and environmental contributions to microbial responses and immune cell composition altered immunity of laboratory mice in the natural environment is 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signaling in immune cells norepinephrine controls effector t cell differentiation through beta2-adrenergic receptor-mediated inhibition of nf-kappab and ap-1 in dendritic cells beta2-adrenoreceptor agonist inhibits antigen cross-presentation by dendritic cells control of lymphocyte egress from lymph nodes through beta2-adrenergic receptors adrenergic control of the adaptive immune response by diurnal lymphocyte recirculation through lymph nodes adrenergic signaling: a targetable checkpoint limiting development of the antitumor immune response beta2-adrenergic receptor signaling in cd4+ foxp3+ regulatory t cells enhances their suppressive function in a pka-dependent manner beta-adrenergic signaling in mice housed at standard temperatures suppresses an effector phenotype in cd8(+) t cells and undermines checkpoint inhibitor therapy baseline tumor growth and immune control in laboratory mice are significantly influenced by subthermoneutral housing temperature mice housed at elevated 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nonalcoholic fatty liver disease: pathology and pathogenesis will the increased prevalence of nonalcoholic steatohepatitis (nash) in the age of better hepatitis c virus therapy make nash the deadlier disease? longterm survival and late events after allogeneic stem cell transplantation from hla-matched siblings for acute myeloid leukemia with myeloablative compared to reduced-intensity conditioning: a report on behalf of the acute leukemia working party of european group for blood and marrow transplantation murine models of steroid refractory graft-versus-host disease acute graft-versus-host disease -biologic process, prevention, and therapy bone marrow graft-versus-host disease: early destruction of hematopoietic niche after mhc-mismatched hematopoietic stem cell transplantation bone marrow graft-versus-host disease: evaluation of its clinical impact on disrupted hematopoiesis after allogeneic hematopoietic stem cell transplantation hematopoietic niches: targets of gvhd association of an impaired bone marrow microenvironment with secondary poor graft function after allogeneic hematopoietic stem cell transplantation immunological basis of bone marrow failure after allogeneic hematopoietic stem cell transplantation mouse models of graft-versus-host disease: advances and limitations beta2-adrenergic receptor activation on donor cells ameliorates acute gvhd effects of cold weather on mortality: results from 15 european cities within the phewe project inhalation of cold air increases the number of inflammatory cells in the lungs in healthy subjects exercise and airway injury in athletes mechanisms and management of exercise-induced asthma in elite athletes thermoneutral housing temperature regulates t-regulatory cell function and inhibits ovabumin-induced asthma development in mice springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations none. authors' contributions mbg was responsible for the overall conception and focus of the manuscript. je, bmm, st and kf were responsible for composing the different sections of the review. the authors wrote, read and approved the final manuscript. the datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. all experimental procedures involving the use of animals were reviewed and approved by the institutional animal care and use committee of ttuhsc and performed according to the criteria outlined by the national institutes of health. not applicable. the authors declare that they do not have competing interests. key: cord-265847-oq34lc26 authors: yagami, k.; hirai, k.; hirano, n. title: pathogenesis of haemagglutinating encephalomyelitis virus (hev) in mice experimentally infected by different routes date: 1986-11-30 journal: journal of comparative pathology doi: 10.1016/0021-9975(86)90061-7 sha: doc_id: 265847 cord_uid: oq34lc26 abstract three-day-old suckling mice inoculated intracerebrally (i.c.) with the 67n strain of haemagglutinating encephalomyelitis virus (hev) showed nervous signs and died. the virus was passaged 10 times in suckling mice and was designated the mb-67n strain. the pathogenesis of mb-67n was studied with various ages of mice and inoculation routes. all mice inoculated i.c. with a large dose of virus died regardless of age, although a smaller dose caused fatal infection only in suckling mice. by intranasal, intraperitoneal and subcutaneous inoculation, the virus also killed suckling mice under 16 days old, but not older mice, even with a large dose. the susceptibility of mice for the mb-67n strain was influenced by age and inoculation routes. high titres of virus were re-isolated from the brain of diseased mice after inoculation by any route, but not from other organs. histologically, numerous areas of severe tocal necrosis were produced in the cerebral cortex. specific immuno-fluorescence and numerous viral particles were found in the cytoplasm of nerve cells by immuno-fluorescence staining and electron microscopy. these findings indicate that the mb-67n propagates mainly in the central nervous system and nerve cells serve as a main target of virus replication. haemagglutinating encephalomyelitis virus (hev), classified as a coronavirus, causes encephalomyelitis or so-called vomiting and wasting disease in suckling piglets. some strains of hev cause fatal encephalomyelitis characterized by squealing, vomiting, constipation, progressive paralysis and nervous signs in suckling piglets up to one week of age (mitchell, 1963) . other strains have been shown to cause vomiting and wasting disease of piglets under 2 weeks of age, i.e. signs of gastrointestinal infection rather than encephalomyelitis (alexander and saunders, 1969; cartwright, lucas, cavill, gush and blandford, 1969; kershaw, 1969) . on the other hand, mengeling, boothe and ritchie (1972) have isolated hev from tonsillar swabs of asymptomatic adult pigs. several coronaviruses are known to propagate in suckling mouse brain (simpson and group& 1959; mcintosh, becker and chanock, 1967; bhatt, percy and jonas, 1972; weiner, 1973; horzinek, osterhause, wirahadiredja and der kreek, 1978) . hev, however, replicates only in porcine kidney (pk) cell culture (greig and girard, 1963; mengeling et al., 1972) . appel, greig and corner (1965) have reported that when some laboratory animals are inoculated orally with hev-1 strain, they develop no clinical signs and virus cannot be re-isolated. so far as the authors are aware, the attempt to demonstrate the growth of hev in laboratory animals has not been successful. in this report, we present some basic experiments elucidating the pathogenesis of hev in mice, and the target cell for vjral replication by immuno-fluorescent and electron-microscopic observations. random-bred ddy mice were obtained from the shizuoka laboratory animal centre, hamamatsu, japan, and bred at our laboratory. litters were weaned at 3 weeks of age and given a commercial diet and water ad libitum. et al. the 67n strain of hev used was supplied by dr w. l. mengeling, national animal disease laboratory, ames, iowa, u.s.a., and was passaged 12 times in primary pk cell cultures and 10 times in suckling mouse brain. the suckling mouse-propagated strain, designated mb-67n strain, was used for further experiments. antiserum against the 67n strain of hev was prepared in rabbits by intravenous injections of the virus propagated in pk cell cultures. inoculation of mice with the virus was made by the intracerebral (i.c.), intranasal (i.n.), intraperitoneal (i-p.) and subcutaneous (s.c.) routes. the inoculum was o-02 ml for the i.c. and i.n. routes and 0.1 ml for the i.p. and s.c. routes. the mice were observed for clinical signs for 2 weeks after inoculation. for determination of infectivity titres, lo-fold dilutions of the virus or 10 per cent tissue suspensions were made in phosphate buffered saline (pbs, ph 7.2) containing antibiotics and clarified by centrifugation at 2000 rpm for 10 min. at each dilution 0.02 ml was inoculated by the i.c. route into 3-day-old suckling mice, using 4 mice per dilution. the inoculated mice were observed daily for 10 days. the ld,, of virus suspensions was calculated by the method of reed and muench (1938) . haemagglutinating activity (ha) and haemagglutination inhibition (hi) tests were performed by the method described by hirai, chang and shimakura (1974) . a virus suspension of 0.5 ml ( 10' ld,, per 0.02 ml) was mixed with an equal volume of serial 2-fold dilutions of serum heated at 56 "c for 30 min and incubated at room temperature for 60 min. each virus-serum mixture was assayed in four 3-day-old mice by i.c. inoculation. the neutralizing antibody titre was expressed as the reciprocal of the highest serum dilution at which at least 2 of 4 mice survived. anti-hev serum was collected from a rabbit inoculated intravenously with the 67n strain. anti-rabbit igg was prepared in guinea-pigs by intravenous injections of rabbit igg, and conjugated with fluorescein isothiocyanate (fitc). the tissues were rapidly frozen in n-hexane chilled in dry ice and acetone, cut by cryostat to approximately 4pm thickness and immersed in acetone at -20 "c for 20 min. staining was by the indirect method at room temperature. the brains of suckling mice were fixed for 2 h in chilled 50 mm phosphate buffer (pb, ph 7.2) containing 2 per cent glutaraldehyde. post-fixation was carried out in 1 per cent osmium tetroxide in 50 mm pb. fixed specimens were rinsed with 200 itim pb containing 0.4 per cent sucrose, dehydrated in a graduated ethanol-water series and propylene oxide and embedded in epon 812. sections were cut on a porter blum mt 2-b ultramicrotome, stained with many1 acetate and lead citrate and examined with a hitachi model hu-12 electron microscope at an accelerating voltage of 70 kv. the 67n strain of hev propagated in pk cells was serially passaged in suckling mice by i.c. inoculation. on the first passage, 67n strain caused illness with an onset at 4 to 8 days post-inoculation (p.i.) and characterized by emaciation and weakness, followed by paralysis and death at 6 to 10 days. all 8 mice inoculated with 67n strain were affected. at the same time, 8 control mice inoculated with culture fluid of uninfected pk cells remained well. the subsequent passages were performed by i.c. inoculation with 10 per cent suspensions of pooled brain obtained from moribund or dead mice. in the course of serial passages, the incubation period decreased progressively, and death occurred consistently at 2 to 3 days p.i. control material passaged in parallel with the infected passage series produced no clinical signs. in a 10 per cent suspension of the tenth passaged brain, infectivity titres assayed in 3-day-old mice and pk cell cultures were 107'4 ld,, per 0.02 ml and 104" tcid,, per o-2 ml, respectively. the adaptation of 67n to suckling mouse brain was repeated a second time. the 67n strain of hev passaged 10 times in suckling mice was designated the mb-67n strain and was employed for further experiments. haemagglutination activity (ha) the mb-67n strain was tested for ha with erythrocytes of various species at room temperature. the virus agglutinated the erythrocytes of chicken, mouse and rat and their ha titres were 16 400, 8200 and 8200, respectively. erythrocytes of guinea-pig, rabbit, dog, goat, sheep, calf, goose, turkey and human (o-type) failed to agglutinate under test conditions,, the serological specificity of the mb-67n strain against anti-67n rabbit serum was examined by neutralization and hi tests. as shown in table 1 , the mb-67n and original 67n strains were neutralized by a 1 in 256 and a 1 in 5 12 dilution of anti-67n serum, respectively. the hi titre of anti-67n serum against the mb-67n was 160, compared with 1280 for the original 67n strain. forty 3-day-old mice were inoculated i.c. with io3 ld,, of mb-67n. three mice each were killed at 6-or 12-h intervals and a 10 per cent suspension of pooled brains was made in pbs for assays of infectivity and ha. dead mice further experiments on the pathogenesis of mb-67n were performed using various ages of mice and routes. groups of 5 to io mice aged 3, 6, 9, 12, 16, 20 and 35 days were inoculated with a large dose (io6 ld,,) of virus by the i.c., i.n., i.p. and s.c. routes. all mice inoculated by the i.c. route developed nervous signs and died regardless of age. following i.n., i.p. and s.c. inoculation, suckling mice under 16 days old died with similar signs, whereas 20-day-old or older mice failed to show signs during the 14-day observation period (table 2) . since all mice inoculated by the i.c. route with a large dose of virus died regardless of age, 3-to 35-day-old mice were inoculated with lo-fold dilutions of virus suspension by the i.c. route and infectivity titres of the virus were calculated for each age group. titres were 107'4, 106'5, 105'3, 104s, 104.3, 104'3 and 104" ld,, per 0.02 ml in 3-, 6-, 9-, 12-, 16-, 20-and 35-day-old mice by i.c. inoculation, respectively. similarly, comparative titration was performed in 3-day-old mice inoculated by the i.n., i.p. and s.c. routes. titres in 3-day-old mice by i.n., i.p. and s.c. inoculation were 10"' ld,, per 0.02 ml, 103.' and 1 03.' ld,, per 0.1 ml, respectively (table 3) . groups of five 3-and 35-day-old mice were inoculated with lo6 ld,, of mb-67n by different routes and killed when moribund or after 14 days' observation. ten per cent suspensions of pooled organs were assayed in 3-day-old mice by i.c. inoculation. as shown in table 4 , the infectivity titres of brain suspensions from 3-day-old mice inoculated by the i.c., i.n., i.p. and s.c. routes were 107", 10s5, 1 05.6 and 103'6 ld,, per 0.02 ml, respectively. low titres or no virus was re-isolated from organs other than lung following in. inoculation. in 35-day-old mice, the virus was re-isolated only from the brain after i.c. inoculation. neither signs nor virus re-isolation were detected in other routes. in the final experiment, 3 each of g-day-old mice inoculated with 107'5 ld,, of the virus by the in. route were killed at 24 h, 4% h and 72 h and viral distribution was examined. virus was detectable in the olfactory bulb, cerebrum and lung at 24 h p.i. and titres became higher in the olfactory bulb, cerebrum and cerebellum in the course of infection. viral recovery from the olfactory bulb seemed to precede the appearance of virus in cerebrum and cerebellum. similar titres of virus were detected in the lung for 72 h until the late stage of infection. in other organs, very low titres were detectable only at 72 h p.i. (table 5 ). histological examination of the brain was carried out in the late stages of infection. areas of focal necrosis were numerous in all parts of the cerebral cortex after all inoculation routes (fig. 2) . the lesions following i.c. and i.n. inoculation appeared to be more severe than those following i.p. and s.c. routes. the distribution of virus-specific fluorescence was closely correlated with that of the histological lesions (fig. 3) . a bright fluorescence was found in the cytoplasm of neurons and ependymal cells in sections at 48 h p.i. various features of the developmental process of the virus were found in brain sections from 3-day-old mice after i.c. inoculation. in the early stage, swelling of mitochondria and increase in endoplasmic reticulum were associated with accumulation of free ribosomes. particles, presumably virions, approximately 90 nm in diameter, appeared individually or in short rows in the space between membranes of endoplasmic reticulum in some areas (fig. 4) . similar particles were visible within electron-dense areas enclosed by a membrane or a double membrane (fig. 5) . in the next stage, the particles became more numerous. amorphous tubular structures and a large number of vesicles containing virions were observed in the cytoplasmic space of the nerve cells (fig. 6) . the developmental process of individual particles appeared to be that of budding from intracytoplasmic membranes (figs 7 to 9 ). in the late stage, vesicular or cellular walls were broken and the particles were frequently noted in the extracellular space. the mature particles had club-shaped projections and were 100 to 130 nm in diameter. discussion several coronaviruses have been shown to grow in suckling mouse brain (simpson and group& 1959; mcintosh et al., 1967; bhatt et al., 1972; weiner, 1973; horzinek et al., 1978) , but hev has not. hev replicates only in porcine kidney cell cultures, because of its fastidious growth requirements. appel et al. (1965) have reported that suckling mice inoculated orally with hev-i strain developed no clinical signs and that virus was not re-isolated from oral swabs. we have demonstrated the growth of the 67n strain of hev in suckling mouse brain following i.c. inoculation. to be the segment of a crescent developing on vesicular wall (short arrow i n fig. 7) . the crescent bulged further into the vesicle (fig. 8) . it bulged away from the cytoplasm into the vesicle, and devel oped a dense underlying layer on the cytoplasmic side. cl ub-shaped projections were already visible on the surface of the vesicular side of particles (fig. 9) . a mature &i on has been rel eased into the vesicles (long arrow i n fig. 7) . the underlying layer remained on the vesicular wall. et&. brain is identical in serological and morphological properties with the cell-propagated 67n strain of hev. mouse hepatitis virus (mhv), a coronavirus, is common and causes persistent infection in laboratory mouse colonies (fujiwara, takenaka and shumiya, 1976 ), but does not agglutinate erythrocytes of any species. the mb-67n strain could agglutinate the erythrocytes of chicken, mouse and rat, and react with antiserum against the original 67n strain of hev. thus, the mb-67n was confirmed to be identical to hev. hev infection of pigs is known to occur as three different types: fatal encephalomyelitis in suckling piglets (mitchell, 1963) , vomiting and wasting disease in piglets under 2 weeks of age (alexander and saunders, 1969; cartwright et al., 1969; kershaw, 1969) and asymptomatic infection of adults (mengeling et al., 1972) . in the present study, mice inoculated with the mb-67n strain by the i.c. route were shown to succumb without age difference, although a smaller dose of virus was fatal for suckling mice only. adults did not die after i.n., i.p. or s.c. inoculation, even with a large dose of the mb-67n strain. death occurred in suckling mice under 16 days old by i.n., i.p. and s.c. inoculation. these findings indicate that the susceptibility of mice for the mb-67n strain was influenced by age and inoculation routes, and appears to resemble that of hev infection in pigs. in transmission studies on piglets, virus was re-isolated from tonsil, respiratory tract, brain and spinal cord in late stages but not in the early stages of infection (appel et al., 1965; cutlip and mengeling, 1972; mengeling and cutlip, 1972) . the present results of viral distribution revealed that a high titre of virus was re-isolated from the brain of diseased mice after i.c., i.n., i.p. and s.c. inoculation with the mb-67n strain in the late stages of infection. these findings indicate that the mb-67n strain replicates mainly in certain cells of the central nervous system (cns). also, the finding that the virus found in the olfactory bulb preceded the appearance of the virus in the cerebrum and the cerebellum in in. inoculated mice, suggests neural transmission through the olfactory nerve. recently, andries, pensaert and callebaut (1978) and andries and pensaert (1980a, 1980b) h ave determined the preliminary sites of viral replication and the route of viral spread in oro-nasally infected piglets by the direct fluorescent antibody technique and viral isolation. they stated that nasal mucosa, tonsil, lung and small intestine served as primary sites of replication and the virus then progressed via peripheral nerves to the cns. the trigeminal nerve, vagal nerve and intestinal plexuses were also proposed as pathways to the cns (andries and pensaert, 1981) . our findings in mice support their concepts in pigs. furthermore, the olfactory nerve may also serve as one of the pathways to the cns in i.n. inoculated mice. the relation between age-dependent susceptibility and maturation of the nervous system, regarded as the pathway of viral spread, will be an interesting subject for future research. because of the high fatality rate after a rapid course of infection, it was possible to examine the histological changes of the disease over a short period only. these findings revealed the characteristic lesions consisting of necrosis in almost all areas of the brain inoculated by the i.c. route. similar necrosis of the brain was observed after i.n. inoculation, but the reaction was slightly milder in i.p. and s.c. routes. mcintosh et al. (1967) reported that the predominant histological finding in mouse brain affected with human coronavirus (hcv) was round cell infiltration of the meninges and perivascular spaces of the cerebral cortex. in mice inoculated with sialodacryoadenitis virus (sdav) of rats, diffuse and focal neuronal degeneration with minimal inflammatory cell response were found by bhatt et al. (1972) . the findings with the mb-67n strain are considered to be more severe than the findings cited above. the morphogenesis of the virions in the infected brain was essentially in accord with that in pk cell culture (mengeling et al., 1972) . in addition, the amorphous tubular structures found in infected cells with hcv (mcintosh et al., 1967; oshiro, scieble and lennette, 1971 ) and mhv (david-ferreira and manaker, 1965) were also observed in this study. the relationship of these structures to the developmental process of virus particles has not been elucidated. the findings by immuno-fluorescence and electron microscopy revealed the localization of large numbers of virus particles in the nerve cells. thus, the nerve cell is considered to serve as a main target of virus replication. in this study, fatal infection of suckling mice caused by hev was shown and a few similarities between infections of mice and pigs were also represented. mice infected with the mb-67n strain will be utilized as an experimental model for hev infection in pigs. summary three-day-old suckling mice inoculated intracerebrally ( 1i.c.) with the 67n strain of haemagglutinating encephalomyelitis virus (he\ ') showed nervous signs and died. the virus was passaged 10 times in suck1 ing mice and was designated the mb-67n strain. the pathogenesis of mb-67n was studied with various ages of mice and inoculation routes. all mice inoculated i.c. with a large dose of virus died regardless of age, although a smaller dose caused fatal infection only in suckling mice. by intranasal, intraperitoneal and subcutaneous inoculation, the virus also killed suckling mice under 16 days old, but not older mice, even with a large dose. the susceptibilily of mice for the mb-67n strain was influenced by age and inoculation routes. high titres of virus were re-isolated from the brain of diseased mice after inoculation by any route, but not from other organs. histologically, numerous areas of severe focal necrosis were produced in the cerebral cortex. specific immuno-fluorescence and numerous viral particles were found in the cytoplasm of nerve cells by immuno-fluorescence staining and electron microscopy. these findings indicate that the mb-67n propagates mainly in the central nervous system and nerve cells serve as a main target of virus replication. encephalomyelitis of swine caused by a hemagglutinating virus. i. case histories electron microscopic studies of coronavirus a simple method of estimating fifty per cent endpoints temperature of incubation as a critical factor in the behaviour of avian bronchitis virus in chicken embryos we wish to thank professor dr s. shimakura, department of veterinary microbiology, gifu university, for his suggestion and encouragement of this study. key: cord-256903-8lyw27gh authors: guzman, efrain; montoya, maria title: contributions of farm animals to immunology date: 2018-12-06 journal: front vet sci doi: 10.3389/fvets.2018.00307 sha: doc_id: 256903 cord_uid: 8lyw27gh by their very nature, great advances in immunology are usually underpinned by experiments carried out in animal models and inbred lines of mice. also, their corresponding knock-out or knock-in derivatives have been the most commonly used animal systems in immunological studies. with much credit to their usefulness, laboratory mice will never provide all the answers to fully understand immunological processes. large animal models offer unique biological and experimental advantages that have been and continue to be of great value to the understanding of biological and immunological processes. from the identification of b cells to the realization that γδ t cells can function as professional antigen presenting cells, farm animals have contributed significantly to a better understanding of immunity. great advances in immunology are usually supported by experiments carried out in animal models and by far, inbred lines of mice and their corresponding knock-out or knock-in derivatives, are the most commonly used animal systems in immunological studies. though with much credit to their usefulness, laboratory mice will never provide all the answers to fully understand immunological processes. also, some answers provided in mouse models are not applicable to other species of animals or humans. large animal models offer unique biological and experimental advantages that have been and continue to be of great value to the understanding of biological and immunological processes. the humble cow, the underestimated pig and the unassuming chicken have greatly influenced our current understanding of human immunology. for most immunologists dedicated to fundamental and applied research, it is easy to forget that b cells were first identified in chickens and vaccination first occurred because of a cow. although there are far too many important events to discuss in this paper, we have chosen to highlight a few of the most important contributions of farm animals to the current understanding of immunology ( table 1) . edward jenner published in 1798 a booklet entitled "an inquiry into the causes and effects of the variolae vaccinae, a disease discovered in some of the western counties of england, particularly gloucestershire and known by the name of cow pox" (1, 2) and although strictly speaking jenner did not discover vaccination, he was the first person to use scientific rigor to prove protection from disease through targeted intervention. the english dairy farmer benjamin jesty (1737-1816) was the first person known to vaccinate against smallpox (3) protecting his family against the virus even after numerous exposures (3) . however, the idea and indeed the term vaccination, only came into the light spot 100 years later thanks to louis pasteur. this time the chicken takes a privileged position and the story was beautifully explained by pasteur himself (4, 5) and has been romanticized in paul de kruif 's book "microbe hunters" (6) . in 1878 pasteur inoculated chickens with "stale" cultures of pasteurella multocida. the chickens became sick but recovered so he decided to re-inoculate them with a fresh culture. the chickens that had received the "stale" culture recovered whereas chickens that had not been pre-exposed to the stale cultures died. pasteur recognized the similarities between his studies in chickens and what jenner had published with smallpox. he coined the term "vaccine" (4, 5, 7) in honor of jenner. by the early 1880s, william smith greenfield in the uk (8, 9) and pasteur working with henri thullier, charles chamberland and pierre paul émile roux in france (10, 11) had begun developing and testing vaccines against anthrax in sheep and cattle. a decade later, the german scientists friedrich loffler and paul frosch identified the first ever filterable infectious agent in mammals: foot and mouth disease virus (fmdv) and developed a fully protective heat-inactivated vaccine against it (12, 13) ; however an effective long-lasting and broadly protective vaccine against fmdv remains elusive. pigs also played an important role in early vaccinology studies. by the late 1800s swine plague or hog cholera (later discovered to be caused by a virus now called classical swine fever virus, csfv (14) was killing hundreds of thousands of pigs across the word and was particularly of concern to the us pig producing industry, causing an impressive us$15 million a year in losses in 1875 (15) and us$20 million by 1878 1 . once again, pasteur and thullier developed a vaccine to what is now thought to be the first ever vaccine against a viral infectious disease (16) and the first mass-vaccination campaign in history. in addition, it is rarely recognized that csfv was the first animal virus ever to be cultured in vitro (17) and the techniques developed by carl tenbroek continue to be used today. horses have also contributed to the understanding of fundamental immunological mechanisms. in a series of experiments, emile roux working with alexandre yersin and followed by emil von behring and shibasaburo kitasato immunized horses to produce an "antidote" or immune sera against the diphtheria toxin that was eventually used to treat humans, an important step in understanding antibodies and humoral immunity (18) . behring won the nobel prize for medicine in 1901 for this work. another milestone in vaccine development was the generation in the 1970's of vaccines to control marek's disease (md), a naturally occurring neoplastic disease in chickens caused by an oncogenic herpesvirus (19) . md vaccines are the first examples of the use of vaccination to protect against cancer (20, 21) . with the discovery of molecular biology techniques in the 1960's and 70's, the race was on to develop recombinant vaccines against numerous infectious diseases. the first report of a biosynthesized polypeptide vaccine was published in 1981 (22) . the structural protein vp3 of fmdv was cloned 1 (1881). swine plague. science 2, 121 and expressed in e. coli and the purified protein used to vaccinate six cattle and two swine, which developed neutralizing antibodies and were protected against challenge with fmdv (22) . and new technologies have only helped to highlight the importance of farm animals in vaccine development: using a computational approach to assess protein-protein stability, kotecha and colleagues (23) used molecular dynamic ranking to predict fmdv capsid stabilities and produced stabilized fmdv capsids based on these predictions, assessed their stability using x-ray crystallography and demonstrated their improved immunogenicity in vivo by vaccinating cattle. this demonstrates the potential value of structure-based design of vaccines to develop stabilized vaccine antigens for animals and humans alike. although the innate immune system of animals is largely conserved, there are significant variations in the pattern-recognition-receptor (prr) structures of various species (24) . it has been suggested that laboratory mice have not been subjected to the selective pressures that other animals have and so innate immune studies carried out in laboratory animals do not accurately inform human biology (25) . it has been demonstrated that human and farm animal prr responses to their ligands (24, 26) are more similar to each other than human-mouse prr responses (26) (27) (28) . because prr recognition is associated with adaptive immunity, a better understanding of these molecules in farm animals is likely to better inform on their effect in these animals as well as humans. a major contribution of the chicken to fundamental innate immunity was the description in 1957 of the first interferon. chicken embryos were exposed to influenza virus by alick issacs and jean lindenmann (29) and they identified an immune soluble element responsible for regulating virus infection. this discovery was certainly one of the scientific landmarks in cell biology in the twentieth century and one which opened the doors of what we now know as innate immunity. perhaps the most recognizable contribution of the chicken to science, and immunology in particular, was in the definition of the two elements of adaptive immunity: the b-dependent and the t-dependent immunity. the avian bursa of fabricius, named after hieronymus fabricius of aquapendente (30) is a sac-like structure located in the cloacal passage of the bird and its function remained elusive until well into the twentieth century. bruce glick and timothy chang working at the poultry science department at ohio state university (30, 31) described how following the surgical removal of the bursa, chickens injected with salmonella typhimurium "o" antigen failed to develop bacteria-specific antibodies. glick and chang wrote a paper entitled: "the role of the bursa of fabricius in antibody production" and was rejected by leading scientific journals (30) and eventually published in poultry science (32) . several years later, the bone marrow in mammals was shown to be the equivalent of the bursa in birds (33) , and so the term "b-lymphocyte" originated from "bursa-derived lymphocyte". several years later, cooper et al. published a fundamental paper on the demarcation of the thymic and bursal and systems in birds and proposed the existence of equivalent systems in mammals (34) . the cannulation of lymphatic vessels was developed in the early twentieth century in rats to study the lymphatic system but due to the complexity of the surgical procedure, sheep and then cattle were used extensively in the 1960s and 70s in lymphatic cannulation studies (35) . in a series of adoptive transfers of lymph-migrating cells and fluid, hall and colleagues first identified in sheep that antibody-secreting cells (acs) encounter antigens and are activated in the lymph nodes (36), then migrate via the efferent lymphatics to the circulatory system, and that the immune response depends on an intact lymphatic system. cattle have also contributed to fundamental b cell immunology and the generation of a highly diverse antibody repertoire. most vertebrates encode a large number of variable (v), diversity (d), and joining (j) gene segments and antibody diversity is achieved by recombination of these 3 segments. in contrast, cattle only express a limited number of v genes and so it is thought that antibody diversity is achieved recombination events and endogenous mutation mechanisms in the cdr3 region (37) . another unusual feature of bovine antibodies is their exceptionally long cdr3 regions (38) . these long cdr3 and unusual mutation mechanisms result in "microfolds" within the cdr3 region that allow bovine antibodies to bind antigens that would normally be inaccessible (38) . a recent report demonstrated that cows can be immunized with a single hiv env trimer and this results in potent hivspecific nabs which are dependent on the length of the cdr3 loops of bovine ig (39) . it has been suggested that this could be an efficient way of producing super-antibodies against other human pathogens. transchromosomal cows have been engineered to produce large amounts of full human igg molecules with pathogen specificity: mers-cov (40) , hanta virus (41), veev (42) , and ebola virus (43) . this technology has the potential to generate prophylactic antibodies against emerging viral diseases. on the other hand, chickens have serum igm and iga both of which are homologs of their mammalian counterparts; in addition, they express igy, not found in mammals but thought to be an evolutionary ancestor for mammalian igg and ige. chickens however do not have either ige nor igd but instead use a distinct process for generating antibody diversity that is distinctly different to mammals (44) . engineers frequently look to nature for inspiration. antibody engineers are no exception, modeling new therapeutics on molecules found in animals such as camels and cows. indeed, 10% of bovine antibodies have unusually long heavy-chain cdr3s as part of their antigen-recognition sites. stanfield et al. (45) have solved crystal structures of three new bovine fab fragments and analyzed the five known structures to show that their ultra-long cdr h3s all adopt similar architectures composed of a knob domain containing a small conserved β-sheet connected by diverse disulfide-bonded loops that is separated from the antibody surface by a long conserved stalk. they propose that varying the length of the stalk and the positions and number of disulphide links in the knob may help drive antibody diversity. these structural insights could be leveraged to tailor antibody-based therapeutics. in contrast to all other mammals, camelids (dromedaries, camels, llamas, etc) also have an unique antibody type similar igg but with identical heavy chains lacking the ch1 domain and which do not pair with their corresponding light chains. these "heavy-chain antibodies" (hcabs) display antigenspecific variable domains or "vhh" which are structurally and functionally similar to an igg fv but have only three cdr loops defining the antigen biding sites. camel vhh domains, also called "nanobodies, " have been of great interest because of their stability and small size and strong affinity to their corresponding antigens. in fact, several camel vhh domain antibodies are in early preclinical development in oncology, infectious, inflammatory, and neurodegenerative diseases (44), the most recent example being the generation of broadly neutralizing antibodies to influenza in llamas (46) . cytotoxic and helper t cells are generally considered to be phenotypically different due to the mutually exclusive expression of the co-receptors cd8αβ and cd4 and differences in mhcrestriction (class i vs. class ii). however, between 3 (in normal individuals) and 60% (in certain pathologies) of human peripheral blood lymphocytes have been shown to be cd4/cd8 double positive (dp) t cells (47) . thymic and extra-thymic development of t cells has been studied mainly in mice and because the expression of cd8 and cd4 in mouse t cells for the most part mutually exclusive, cd4/cd8 dp lymphocytes have generally been ignored. nevertheless, the presence of cd4/cd8 dp t cells in many animals makes it impossible to ignore these cells. studies in pigs have shown that cd4/cd8 dp are a distinct subset of activated and/or memory t helper cells (48) and in humans the increase in circulating cd4/cd8 dp t cell frequency has been identified in autoimmune and chronic inflammatory diseases (49) (50) (51) (52) (53) suggesting the importance of this particular t cell population in human health. most circulating t cells in humans and mice are conventional t cells expressing the αβ t cell receptor (tcr) and either cd4 or cd8. unlike mice, other species like cattle, pigs and chickens possess a substantial proportion of t cells expressing the γδ tcr cells in the circulation suggesting that circulating γδ tcr t cells have a more important role immunity than previously thought (54) . the fact that the phenotype and frequency of circulating and tissue resident t cells is so vastly inconsistent in different species suggests that immune responses to (vaccine) antigens are also distinct. it is assumed that that all animal species have a similar immune response to a particular antigen, but this is a statement to be reviewed in light of each host particularities. in addition, the th1/th2 polarization of t cells observed in response to particular antigens is a phenomenon of certain strains of laboratory mice and not of outbred mammals including farm animals and humans (55, 56) . in fact, it has been shown that cytokine profiles defining th1/th2 responses to antigens in cattle are more similar to human responses than those observed in mice (57). dendritic cells (dc) as such, and their role in immunity were first described in the 1970s and in 1995 ralph steinman published a series of papers describing that a cellular receptor called "dec-205" (now cd205) was expressed on mouse dc, was involved in antigen processing (58, 59) and was detected by the monoclonal antibody nldc-145. in fact, it was 2 years earlier in 1993, that chris howard, a bovine immunologist working at the then called "institute for animal heath" in the uk published a series of papers identifying an important and until then uncharacterized marker expressed on pseudoafferent lymph veiled cells (also called aldc) detected by the monoclonal antibody wc9 (now cc98) (60-63). although steinman's identification of mouse cd205 helped characterize the binding of cc98 to bovine cd205 (64), the importance of cd205 in identifying dc was first evident in cattle. as mentioned above, steinman's seminal work in characterizing dc using the mouse system has been one of the most important developments in cellular immunology of the twentieth century, and one which lead to his nobel laureate. however, the idea that a component of the immune system was involved in antigen processing and presentation had been proposed many times before. as mentioned above, cannulation of the lymphatic vessels is more practical in large than small animals, and this technique has been used to investigate dc biology. afferent or peripheral lymph dc were first described in sheep in 1972 (65) as "very phagocytic dendritic macrophages that are involved in long term immunological reactions" that are very potent antigen presenting non-lymphoid cells (66) and that their phagocytic and antigen presentation capacities differed from "classical" peritoneal macrophages (67) , therefore indicating that dc and macrophages were different cell types (67) several years before steinman's observations (68) . in addition, lymphatic cannulation of sheep has revealed important ontologic, phenotypic and functional characteristics of dc subsets that are relevant in other mammals, particularly humans (69, 70) . similitudes and differences between swine and human dc/macrophage populations have recently been described (71) . in one striking example and in contrast to studies performed in mice, swine and human cdc2, which are associated with th2 responses, both express fcεriα and are localized in or next to the tracheal and bronchial epithelia. these observations have been proposed to imply that swine and humans have similar allergen responses as opposed to mice. this theory is supported by the fact that localization of cdc2 helps them access antigens such as airborne allergens, and fcεriα expression on these cells might help proliferation observed in allergic responses. as mentioned before and unlike mice, horses and humans, most other animals have a large γδ t cell compartment. for example, up to 70% of all blood lymphocytes in young calves are t cell expressing the γδ t cell receptor (tcr). although the reasons for the enlarged t cell compartment in cattle, pigs and chickens is still unknown, their large numbers and ease of collection has resulted in great advances in γδ t cell biology knowledge not only for farm animals, but also for humans. for example, apcs were shown to influence γδ t cell proliferation (72, 73) . cynthia baldwin's lab has defined antigen-specific bovine γδ t cell responses in various systems (74) (75) (76) and adrian smith's lab has done similar observations in chickens (77, 78) . it has also been shown that bovine γδ t are potent regulatory t cells (79) , an observation that is also true for a subset of human γδ t cells (80, 81) . these results in farm animals have and continue to enhance our understanding of human γδ t biology (82) . perhaps the most important one was the realization that a subset of bovine γδ t cells expressed mhc class ii and co-stimulatory molecules on their surface, a characteristic normally attributed to macrophages, b cells and dc but not t cells (83) . bovine γδ t cells were also shown to phagocyte antigens and of mhc iirestricted presentation to cd4+ t cells (83) . this function of bovine γδ t cells was subsequently reported in pigs (84) and much later in mouse (85) and human (86) (87) (88) γδ t cells. perhaps the best known contribution of any farm animal to scientific progress was the somatic cell nuclear transfer that gave origin to dolly, the sheep (89) . although nuclear transfer itself is not a direct contribution to immunology, nuclear transfer technology has directly influenced many immunological concepts underpinned by technologies such as induced pluripotent stem (ips) cells and crispr-cas systems. clustered regularly interspaced short palindromic repeats (crispr) is a rna-guided endonuclease used both in vivo and in vitro. genetically modified animals becoming more common and their availability can be exploited in many applications such as comparative immunology, physiology and disease, to generate in vivo bioreactors to produce complex proteins, or to produce genetically modified organs for transplantation in humans (90) . although the majority of pharmaceutical research is performed in laboratory mice models, it is clear that humans are not "large mice." by a large extent, studies in laboratory mice have been the victim of over interpretation; for example, by extrapolating successful pre-treatment in mice to therapeutic treatment in men. the weakness of the mouse model in pharmaceutical research was recently highlighted in a study showing that inflammatory responses in mouse models do not correlate with human inflammatory disease (91) . an additional study showed a close similarity in expression profiles of immune-related genes between humans and pigs (92) . cattle, pigs, and chickens, are useful, valid, and valuable models to study human infectious diseases and important clinical targets in their own right. both humans and cattle are the natural hosts for tuberculosis (being infected with the geneticallyrelated mycobacterium bovis and mycobacterium tuberculosis, respectively) and the bovine and human diseases share many similarities in terms of immunity and pathology (93) , whereas the mouse model of tuberculosis does not provide a faithful representation of the disease in humans (94) . similarly, bovine respiratory syncytial virus (brsv) is closely related to human (h) rsv and the pulmonary pathology, immune responses and epidemiology seen in young calves and children are very similar (95) . swine have been shown to be a more faithful model for human influenza infection and immunity studies and the same strains of influenza infect both humans and pigs because the distribution of influenza virus receptors and physiopathology are similar in both species. the transfer of maternal-derived antibodies (mda) to new born pigs enables fancy vaccine study design to elucidate the role of mda in immunity (96, 97) , vaccine efficacy and in enhancement of respiratory disease (98) . gnotobiotic piglets have been used to study various human gastrointestinal pathogens. for example, human noroviruses are antigenically and genetically related to swine noroviruses and unlike mice, humans and pigs show genetic susceptibilities to noroviruses depending on their histoblood group antigen phenotypes and the virus strain. similarly, gnotobiotic pigs have been used in rotavirus research to study disease pathogenesis and identify virus-specific iga and asc as correlates for protection and vaccine efficacy in children (99) . pigs have also been proposed to be better models than mice for many other infectious diseases including female genital infection with chlamydia trachomatis, helycobacter pylori, neisseria meningitides, and nipah virus among others because of the natural susceptibility of pigs to these pathogens (100) . endogenous retroviruses were first discovered in pig kidney cell lines in 1971 (101) and are now known to be present in most, if not all, mammalians. the presence and potential reactivation of endogenous retroviruses has very important consequences in both allo-and xeno-transplantation. immunotherapy is becoming more popular in clinical trials and vaccine efficacy studies. the success of immune cell therapies partially depends on the effective delivery of cells to target organs, a process that invariably involves the lymphatic system. dc migration in mice has not proven to be very informative, however, dc migration in pigs may be able to answer several question on dc migration that cannot be addressed otherwise. these studies demonstrate that using large animals to investigate immune cell trafficking will help improve immunotherapies in humans (102) . in 1906 the french surgeon mathieu jaboulay (1860-1913) implanted a pig's kidney into one woman and a goat's liver into another thus starting the idea of xenotransplantation; unsurprisingly, both women died (103) . the acceptance or rejection of a donor's organ or cells is fundamentally an immunological event. cellular rejection involves nk and t cells that recognize foreign antigens on the grafted tissue. using xenotransplantation models (pig-to-rat, pig-to-primate, and pigto-human), the main mechanism for organ and tissue rejection has been proposed to involve arteriosclerosis, or thickening of the arterial walls. this process if thought to be caused by activated and allo-reactive lymphocytes that migrate over time to the transplanted organ (104) . arteriosclerosis is a major cause of chronic organ rejection (103) . studies in laboratory mice have underpinned many concepts of immune tolerance and the generation of immune responses in the neonate. however, the peripheral immune system in mice remains unpopulated during pregnancy and it is only after birth that b and t cells begin to emmigrate to the periphery. in contrast, lymphoid cells circulate through the fetus in humans and large animals well-before birth and specialized lymphoid tissues are also well-developed and populated by the time of birth and are able to respond to a number of antigens (105, 106) . certainly the immune system in neonate humans and large animals is not matured, but calves, lambs and piglets can be more useful than mice in understanding immune responses during pregnancy and in new borns and these studies can be used to better inform human developmental immunology. this advantage over mice has recently been used to develop extracorporeal support technologies using neonatal lambs with the ultimate objective to use these technologies in premature children (107) . perhaps one of the most common uses of large animal models is in the development of vaccines with several advantages over mice. the serial collection of peripheral blood from animals such as pigs, cattle, chickens, and horses allows for immunokinetic studies to be possible in response to vaccination or infection at the level of the individual. these immunokinetic studies can be used to correlate immune responses generated with protection after challenge with the relevant pathogen. in vaccinology studies using mice, the typical approach would be to sacrifice groups of mice sequentially and harvest spleen and blood, so the immune response to vaccination at the individual level is not normally achieved. large animals also provide several advantages over mice when investigating mucosal immunity. when mice are vaccinated or inoculated intranasally, it is common for the inoculum to be digested because anesthetized mice can both swallow and inhale the material placed on their nose. in addition, the structure of the mucosal associated lymphoid tissue (malt) differs significantly in mice from that of large animals and humans; for example mice do not have tonsils but instead have undefined networks of malt, whereas cattle, pigs and sheep have well-defined tonsils (108) (109) (110) . in the case of vaccine delivery through the skin, cattle, and pigs appear to be better suited than mice for these studies. skin thickness, structure of the epidermis and the presence and distribution of langerhan's cells are among many characteristics that humans and cattle and pigs have in common and which are practically relevant in transcutaneous immunizations (111) . the process of selecting a relevant and appropriate animal model is a balanced and complicated exercise due to the diversity in vertebrate physiology, adaptive and innate immunity. studies in mice, for example, have shown the efficacy of vaccines against fmdv, however these efficacy studies have failed to be translated to the target species (cattle and pigs), presumably due to fundamental differences in the immune systems of model organisms and target species and the ability of the virus to mutate in these animals (112) . it has recently been shown that because immunoglobulin subclass diversification occurred after speciation (113, 114) a particular immunoglobulin subclass in one species bears no functional homology to one of the same name of another species (115) . thus, our knowledge of the functions of igg1 in mice cannot be extrapolated to other mammals. characterizing generating reagents for each animal model hinders the development and usefulness of any of these models and therefore limiting the usefulness of cows, cattle or chickens as models for human immunology. mice and rats are and will probably continue to be the chosen model organisms over farm animals. mice can be readily mutated (knock in or knock out) to study immunological pathways; so far this has been proven to be very difficult-and expensive-in large animals. as mentioned above, the availability of reagents to study immune cells and processes in mice far out competes the availability of these reagents for large animals. pharmacokinetic and toxicology studies would be prohibitively expensive in pigs, horses or cattle, so small rodents and rabbits are the best organisms to use in these studies. in addition, studies in mice have been fundamental in the discovery of antibiotics, chemotherapy agents and more recently car-t cell therapies that can be directly applied to humans. genetic homogeneity, low cost, the availability of biologically-relevant mutants and reagents make the mouse the optimal animal model for many academic and industrial researchers. farm animals have historically contributed and continue to contribute to fundamental and applied immunology. the use of these animals in research is not difficult as long as the appropriate facilities and reagents are available. dedicated housing, cost, biosecurity, and genetic variability are some of the many disadvantages confronted when using farm animals in research. however, selecting an appropriate animal model should be more than just a matter of accessibility and common practice (116) but should be based on the scientific question to be addressed and its relevance. not just a country doctor: edward jenner, scientist edward jenner and the eradication of smallpox new light in the dawn of vaccination on the germ theory on chicken cholera: study of the conditions of non-recidivation and of some other characteristics of this disease microbe hunters the life and works of louis pasteur lectures on some recent investigations into the pathology of infective and contagious diseases professor superintendent, the brown animal sanatory institution concerning the priority due to him for the production of the first vaccine against anthrax remarks on anthracic vaccination as a prophylactic of splenic fever summary report of the experiments conducted at pouilly-le-fort, near melun, on the anthrax vaccination, 1881 summarischer bericht uber die ergebnisse der untersuchungen der kommission zur erforschung der maul-und klauenseuche bei dem institut fur infektionskrankheiten in berlin. centralblatt fur bakteriologie berichte der kommission zur erforschung der maulund klauenseuche bei dem institut fur infektionskrankheiten in berlin. centralblatt fuxr bakteriologie the survival of the hog-cholera virus in laboratory animals, particularly the rat swine plague, or hog cholera correspondence of pasteur and thuillier concerning anthrax and swine fever vaccinations cultivation of the hog cholera virus remembering emil von behring: from tetanus treatment to antibody cooperation with phagocytes studies on the epidemiology of marek's disease herpesvirus in broiler flocks effect of vaccination with herpesvirus of turkeys (hvt) on horizontal spread of marek's disease herpesvirus protective efficacy of marek's disease vaccines cloned viral protein vaccine for foot-and-mouth disease: responses in cattle and swine structure-based energetics of protein interfaces guides foot-and-mouth disease virus vaccine design toll-like receptors in domestic animals variation matters: tlr structure and species-specific pathogen recognition human but not murine toll-like receptor 2 discriminates between tripalmitoylated and tri-lauroylated peptides human toll-like receptor 4 recognizes host-specific lps modifications identification of full length bovine tlr1 and functional characterization of lipopeptide recognition by bovine tlr2/1 heterodimer virus interference. i the interferon a landmark contribution to poultry science-immunological function of the bursa of fabricius growth of the bursa of fabricius and its relationship to the adrenal gland in the white pekin duck, white leghorn, outbred new-hampshire, and inbred new-hampshire the bursa of fabricius and antibody production cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells restoration of gamma globulin production in agammaglobulinemic chickens surgical techniques for the collection of lymph from unanaesthetized sheep the ultrastructure and function of the cells in lymph following antigenic stimulation maintenance of antibody to pathogen epitopes generated by segmental gene conversion is highly dynamic during long-term persistent infection reshaping antibody diversity rapid elicitation of broadly neutralizing antibodies to hiv by immunization in cows human polyclonal immunoglobulin g from transchromosomic bovines inhibits mers-cov in vivo dna vaccine-derived human igg produced in transchromosomal bovines protect in lethal models of hantavirus pulmonary syndrome antibody preparations from human transchromosomic cows exhibit prophylactic and therapeutic efficacy against venezuelan equine encephalitis virus production of potent fully human polyclonal antibodies against ebola zaire virus in transchromosomal cattle structural and genetic diversity in antibody repertoires from diverse species conservation and diversity in the ultralong third heavy-chain complementarity-determining region of bovine antibodies universal protection against influenza infection by a multidomain antibody to influenza hemagglutinin coexpression of t4 and t8 on peripheral blood t cells demonstrated by two-color fluorescence flow cytometry phenotypic maturation of porcine nk-and t-cell subsets evaluation of t cell subsets in myasthenia gravis using anti-t cell monoclonal antibodies circulating cd3+ cd4+ cd8+ t lymphocytes in multiple sclerosis circulating cd4+cd8+ t lymphocytes in patients with kawasaki disease cd4+ cd8+ double positive (dp) t cells in health and disease peripheral cd4cd8 double positive t cells with a distinct helper cytokine profile are increased in rheumatoid arthritis porcine gammadelta t cells: possible roles on the innate and adaptive immune responses following virus infection the relative magnitude of transgene-specific adaptive immune responses induced by human and chimpanzee adenovirus vectors differs between laboratory animals and a target species differences in immune responses against leishmania induced by infection and by immunization with killed parasite antigen: implications for vaccine discovery type 1 and type 2 responses in regulation of ig isotype expression in cattle the receptor dec-205 expressed by dendritic cells and thymic epithelial cells is involved in antigen processing dec-205, a 205-kda protein abundant on mouse dendritic cells and thymic epithelium that is detected by the monoclonal antibody nldc-145: purification, characterization, and n-terminal amino acid sequence summary of workshop findings for cattle (tables 1 and 2) leukocyte antigens of cattle and sheep. monoclonal antibodies submitted to the second workshop studies of monoclonal antibodies identifying two novel bovine lymphocyte antigen differentiation clusters: workshop clusters (wc) 6 and 7 phenotypic variation and functional differences within dendritic cells isolated from afferent lymph dec-205 expression on migrating dendritic cells in afferent lymph the cells of lymph and their role in immunological reactions. handbuch der allgemeinen pathologie the properties and functional activity of non-lymphoid cells from bovine afferent (peripheral) lymph some properties of dendritic macrophages from peripheral lymph identification of a novel cell type in peripheral lymphoid organs of mice. morphology, i., quantitation, tissue distribution plasmacytoid dendritic cells migrate in afferent skin lymph existence of cd8alpha-like dendritic cells with a conserved functional specialization and a common molecular signature in distant mammalian species the respiratory dc/macrophage network at steady-state and upon influenza infection in the swine biomedical model monocytes control gamma/delta t-cell responses by a secreted product bovine gamma/delta t-cell proliferation is associated with self-derived molecules constitutively expressed in vivo on mononuclear phagocytes protective killed leptospira borgpetersenii vaccine induces potent th1 immunity comprising responses by cd4 and gammadelta t lymphocytes the role of bovine γδ t cells and their wc1 co-receptor in response to bacterial pathogens and promoting vaccine efficacy: a model for cattle and humans the bovine model for elucidating the role of γδ t cells in controlling infectious diseases of importance to cattle and humans an alphabeta t-cell-independent immunoprotective response towards gut coccidia is supported by gammadelta cells γδ t cells play a protective role during infection with nippostrongylus brasiliensis by promoting goblet cell function in the small intestine bovine γδ t cells are a major regulatory t cell subset antigen-specific regulation of ige antibodies by non-antigen-specific γδ t cells a new effect of il-4 on human γδ t cells: promoting regulatory vδ1 t cells via il-10 production and inhibiting function of vδ2 t cells bovine γδ t cells: cells with multiple functions and important roles in immunity gammadelta t cells present antigen to cd4+ alphabeta t cells a sub-population of circulating porcine gammadelta t cells can act as professional antigen presenting cells mouse gammadelta t cells are capable of expressing mhc class ii molecules, and of functioning as antigen-presenting cells professional antigen-presentation function by human gammadelta t cells cross-presenting human gammadelta t cells induce robust cd8+ alphabeta t cell responses prolonged antigen survival and cytosolic export in cross-presenting 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cell lines in vivo tracking and immunological properties of pulsed porcine monocyte-derived dendritic cells xeno's paradox: why pig cells are better for tissue transplants than human cells xenoislet rejection following pig-to-rat, pig-to-primate, and pig-to-man transplantation expanding the role of peyer's patches in b-cell ontogeny induction of immune responses in newborn lambs following enteric immunization with a human adenovirus vaccine vector an extra-uterine system to physiologically support the extreme premature lamb of mice and not men: differences between mouse and human immunology revisiting the b-cell compartment in mouse and humans: more than one b-cell subset exists in the marginal zone and beyond the lymphoid system: a review of species differences transcutaneous immunization of domestic animals: opportunities and challenges laboratory animal models to study foot-and-mouth disease: a review with emphasis on natural and vaccine-induced immunity antibody repertoire development in fetal and neonatal piglets. xiii hybrid vh genes and the preimmune repertoire revisited genomic organization of the immunoglobulin light chain gene loci in xenopus tropicalis: evolutionary implications therapeutic administration of broadly neutralizing fi6 antibody reveals lack of interaction between human igg1 and pig fc receptors model organisms: there's more to life than rats and flies all authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. the authors were funded by the uk's bbsrc grants bbs/e/i/00002067 and bbs/e/i/00002014. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2018 guzman and montoya. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-254190-bxfne94u authors: tu, wenwei; zheng, jian title: application of humanized mice in immunological research date: 2015-07-07 journal: suppression and regulation of immune responses doi: 10.1007/978-1-4939-3139-2_10 sha: doc_id: 254190 cord_uid: bxfne94u during the past decade, the development of humanized mouse models and their general applications in biomedical research greatly accelerated the translation of outcomes obtained from basic research into potential diagnostic and therapeutic strategies in clinic. in this chapter, we firstly present an overview on the history and current progress of diverse humanized mouse models and then focus on those equipped with reconstituted human immune system. the update advancement in the establishment of humanized immune system mice and their applications in the studies of the development of human immune system and the pathogenesis of multiple human immune-related diseases are intensively reviewed here, while the shortcoming and perspective of these potent tools are discussed as well. as a valuable bridge across the gap between bench work and clinical trial, progressive humanized mouse models will undoubtedly continue to play an indispensable role in the wide area of biomedical research. during the past century, the application of rodent animal models , especially diverse gene-engineered mouse models , provided indispensable platforms and numerous valuable information for the advances in experimental medicine and biological research. however, the gap between species is still the most challenging obstacle for translation of results from rodents to humans . with the great advancement of technology in molecular biology and gene modifi cation, the attempt to establish "humanized" mouse models has made a leap since 1990s [ 1 -3 ] . nowadays, a wide variety of humanized mouse models have been generated and applied in nearly all fi elds of biomedical research [ 4 ] . in this chapter, we briefl y review the history and classifi cation of humanized mouse models and then summarize the current situation and recent advancement of their application in biomedical research, especially in the research of immune-related diseases. in general, the "humanized mice" are composed of three main classes: human gene-expressed transgenic mice ( human genetransgenic mice ), which are modifi ed by gene knock-in or replacement technology to express one or more human specifi c genes; humanized mice carrying human tissue, such as the liver ( humanized liver mice ) in which murine hepatocytes are completely or partly replaced by infused human-original hepatocytes; humanized mice equipped with functional human immune system ( humanized immune system mice ), which are established on immunodefi cient mice by transplanting human immune organs or cells to reconstitute human immune system in mice and thus referred to special "humanized mice." in the following section, we briefl y review the history and current advance of human gene-transgenic mice and humanized liver mice, and then focus on humanized immune system mice. human gene-transgenic mice are closer to gene engineered mice rather than "humanized mice." although the expressions of human gene or protein in transgenic mice provides the platform for studying in vivo role of specifi c human gene or molecule, the value of these data is limited in translational medicine due to the lack of human microenvironment and signal networks in these mice. the most widely used human gene-transgenic mice are human leukocyte antigen (hla)-transgenic mice [ 5 ] . these hlaexpressed transgenic mice represent for a useful tool in studying in vivo tcr-restricted immune responses and thus were adopted in the studies of immune-related diseases during the fi rst 10 years of this century. for example, hla-a0201-transgenic mice were used in inducing cd8 + t cell-restricted type i diabetes (t1d) [ 6 ] and experimental autoimmune encephalitis (eae) [ 7 ] , while hla-drb1-transgenic mice were applied in establishing cd4 + t cellmediated eae [ 8 ], system lupus erythematosus (sle) [ 9 ] and rheumatoid arthritis (ra) models [ 10 ] . more recently, the respective role of hla-dr2 and hla-dq8 in eae [ 11 ] and autoimmune diabetes [ 12 ] was also studied through transgenic mice. meanwhile, transgenic mice with distinct hla subtype expression favor the study of hla-related susceptibility on specifi c diseases, such as eae [ 13 ] , experimental autoimmune uveitis [ 14 ] , arthritis [ 15 -17 ] , allergic bronchopulmonary aspergillosis-like pulmonary responses [ 18 ] , and celiac disease [ 19 ] . although the application of hla-transgenic mice has been reduced due to the simplifi cation of diseases into specialized immune responses, the combination of hla-transgenic technology and reconstitution of human immune system in immunodefi cient mice has re-assigned them vitality in biomedical research, which will be discussed in the next section. other , which had all been blocked by the lack of optimal animal models in "pre-humanized mice time." on the other side, chen et al. tried to stabilize the function of cryopreserved human hepatocytes in immune competent mice through a novel system called "human ectopic artifi cial livers (heals)," which involved juxtacrine and paracrine signal in polymeric scaffolds. they claimed that mice transplanted with heals exhibited persistent normal liver function for weeks and thus provided a window for drugrelated investigation [ 34 ] . however, the effi cacy and value of humanized liver mice in the development of drug are still on debate due to the proposed side effects such as ongoing liver injury caused by transgenic and the infl uences on "normal metabolism" mediated by exogenous treatment [ 35 , 36 ] . apart from these, the potential application of humanized liver mice in immune-related research also deserves further exploration because liver also represents for a critical component of human immune system. the development of humanized immune system mice could be divided into three phases corresponding to the establishment of prkdc scid (protein kinase, dna activated, catalytic polypeptide; severe combined immunodefi ciency) mutation in cb17 mice, the development of nod (non-obese diabetic)-scid mice, and the generation of immunodefi cient mice homozygous for mutation at il (interleukin)-2 receptor γ chain locus [ 2 , 37 ] . each breakthrough mentioned previously signifi cantly improved the engraftment of human immune cells or pluripotent stem cells and stood as milestone on the way to "real humanized mice." the engraftment of multiple human immune components in these mice surpassed conventional human-gene knock-in in breaking the limited viewpoint of studying specifi c molecules under isolated environment. this unique advantage of humanized immune system mice favors their general application in immune-related studies, and opens a window for researchers to observe the interaction among human immune cells in vivo. in the following content, we focus on the characteristics and application of these mouse models and simply refer them as "humanized mice" if not otherwise specifi ed. currently, il2γc −/− mice established on nod/scid and recombination activating gene 2 (rag2) −/− balb/c background were most widely used strains for the reconstitution of human immune system in vivo [ 38 -40 ] . recently, by using bone marrow, liver, thymus (blt) co-transplantation, lavender et al. engrafted high levels of multi-lineage hematopoiesis and organized lymphoid tissues in c57bl/6-rag2 −/− γc −/− cd47 −/− triple-knockout mice. these humanized mice sustained human cell and tissue engraftment as long as 29 weeks post-transplantation without the development of chronic graft-versus-host diseases (gvhd), and thus represented for a new advancement in establishment of humanized mice [ 41 ] . the reconstitution of functional immune system is the key to evaluate the successful establishment of humanized mice. the graft used for reconstituting human immune system includes stem cells [ 42 ] , blt [ 41 ] , and peripheral blood cells [ 43 ] according to specifi c objectives. generally, stem cell and blt transplantation exhibit advantage in establishing stable multi-lineage hematopoietic cells but might need additional treatment for improving development of specifi c cell subpopulations. on the contrary, humanized mice established by peripheral blood cells provide a ready platform for studying the functions of mature immune cells but the length of window appropriate for research is still limited by chronic gvhd and ongoing reduced engraftment. to maximize the potential of humanized mouse model , some progresses have been made recently. firstly, pretreatment or gene-engineering of pluripotent stem cell exhibited satisfactory effects on improving engraftment of immune cells [ 38 , 42 , 44 ] . secondly, human growth factors, cytokines [ 44 , 45 ] or signal regulatory protein alpha (sirpa)expressed [ 46 ] immunodefi cient mice demonstrated superior engraftment for specifi c immune cell subpopulations as well. in the following paragraphs, we briefl y review current status of the reconstitution of specifi c immune cell subpopulations in humanized mice. lymphocytes are most important components of immune system and thus draw a major attention. although human peripheral blood mononuclear cells (pbmc) transplantation led to rapid reconstitution of human lymphocytes in humanized mice, it was found that after initial activation and induction of antibody production, human t cell lymphocytes enter an unresponsiveness status due to loss of human professional antigen-presenting cells (apc), which could be reversed by adoptive transfer of human apc [ 47 ] or activating organ-resident myeloid dendritic cells (dc) through poly(i:c) treating [ 48 ] . meanwhile, stem celltransplanted humanized mice displayed diversifi ed t cell repertoire, but the gap between hla and murine major histocompatibility complex (mhc) molecules prohibited the induction of effi cient t cell-mediated primary immune responses in vivo [ 49 , 50 ]. to overcome these problems, hla-expressed immunodefi cient mice were generated and their effi cacy has been confi rmed [ 51 ] . another concern origins from th1 and th17 immunocompetence in humanized mice [ 52 ] , which supports the utility of their application as surrogate model in transplantation rejection and autoimmunity but might cause some unwanted immune responses against murine tissue antigen as well. distinct from their t cell companion, reconstitution of functional b lymphocytes is generally poor in humanized mice and needed to improve in the future although their primary repertoire were principally unaltered by the differences between mouse and human stromal environments [ 53 ] and their ability to produce antigen-specifi c antibody was partly developed [ 54 ] . as described previously, the reconstitution of myeloid cells not only guarantees immune system intact, but also determines the development and function of both adaptive and innate lymphocytes [ 47 , 48 , 55 ] . unfortunately, monocytes and other myeloid cells usually exhibit immature phenotype and impaired function in humanized mice [ 56 ] , which could be partly rescued by human colony stimulating factor (csf)-1 [ 57 ]. however, the improvements in their survival, differentiation and even migration and residence [ 58 ] are still urgently required. besides leukocytes, other blood components also play important roles during immune response and regulation. recently, hu et al. established the full reconstitution of human platelets in humanized mice after depletion of murine macrophage [ 59 ] , which represents for an interesting attempt in constructing a more "humanized" circulation in mice. in summary, the optimization of humanized mouse model is still on the way and the advances in molecular biology, cellular biology, and system biology will defi nitely bring new era to the development of this useful tool. the applications of humanized mice cover nearly all fi elds of biomedical research and here we concentrate on immune-related studies, especially those aiming at the mechanisms and translational potentials of immune regulation and suppression . we also briefl y summarize the benefi ts brought by these potent models in tumor, infectious diseases, and vaccine studies. cd34 + cd38 lo cd1a − (early t lineage progenitors, etp), and cd34 + cd38 + cd1a + pre-t cells in liver of humanized mice by intrahepatic injection of cd34 + stem cells, establishing a wonderful platform for investigating human t cell development [ 60 ] . however, joo et al. found that human t cells educated by murine mhc in mice without a human thymus differ from normal human t cells marked as higher expression of cd45ro and promyelocytic leukemia zinc fi gure protein (plzf) regardless of similar development stages [ 61 ] . correspondingly, danner et al. generated hla-dr4-expressed nod-rag1 −/− γc −/− mice and demonstrated the critical role of hla class ii molecule for development of functional t cells by infusion with hla-dr-matched human hematopoietic stem cells [ 62 ] . meanwhile, the roles of il-12 [ 63 ] and notch [ 64 ] signals during the development of human cd4 + and cd8 + t cells were evaluated by human hematopoietic stem cell-transplanted mice. moreover, using a human stem cell factor, granulocyte-macrophage colony-stimulating factor (gm-csf) and il-3-expressed nod/scid-γc −/− mice, billerbeck et al. found the increased accumulation of human cd4 + foxp3 + t cells in blood, spleen, bone marrow and liver. most importantly, these cd4 + foxp3 + t cells exhibited potent suppressive capability on t cell proliferation, which made a signifi cant contribution to study of human regulatory t cells (treg) development in vivo [ 65 ] . as described previously, the development of human b cells in humanized mice is relatively weak compared to t cells. in 2011, choi et al. evaluated the effi cacy of busulfan, a chemotherapeutic agent, and claimed that it could effi ciently improve the reconstitution of human specifi c antibody -producing b cells, t cells, macrophage, and even dc from cd34 + cord blood cells with less toxic effects [ 66 ] . on the other hand, kim et al. found that cotransplantation of fetal bone tissue with fetal thymus could facilitate the development and reconstitution of human b cells from fetal liver-derived cd34 + cells together with t cells [ 67 ] . besides adaptive lymphocytes like t and b cells , innate lymphocytes development-related factors were also illustrated in humanized mouse model . as early as in 2008, huntington and di santo made a periodic review on the application of humanized mice in the research of nk cell development [ 68 ] . in 2011, pek et al. further confi rmed the crucial role of il-15 in nk cell development in bone marrow and liver with humanized mouse model [ 69 ] . we believe that more studies in the development of other innate lymphocytes such as nkt, γδ-t cells and innate-like t cells (ilt) will be reported in the near future. myeloid cells are generally regarded as more fragile and diffi cult to survive in "strange environment", which made it attractive and subtle to improve reconstitution of these sensitive cells. addition of human original cytokines such as gm-csf and il-4 was generally accepted as an effi cient way to improve dc maturation [ 70 ] . similarly, the effects of macrophage colony-stimulating factor (m-csf) and fms-related tyrosine kinase (flt)-3 ligand on promoting the development of macrophage [ 71 ] , and cd141 + and cd1c + dc [ 72 ] have also been confi rmed in humanized mouse model respectively. moreover, the development of megakaryocytes was replicated and used as index of dengue virusinfection in humanized mouse model recently [ 73 ] , which also supported the multi-lineage hematopoietic cell development in humanized mice. finally, transplantation of human stem cells from bone marrow of patients with bone marrow failure syndrome into humanized mice provided invaluable tools for evaluating novel gene-targeted therapy before clinical trial [ 74 ] . in summary, the reconstitution of diverse human immune cell populations from their pluripotent progenitors in immunodeficient mice has become a potent platform for investigating the development of human immune system while the next question is how to create a more "humanized" environment in mice for human cells [ 75 ] . the advances in the study of autoimmune diseases in humanized mice, especially those t cell-mediated diseases, are always correlated with development of hla-transgenic technology. in 1999, bachmaier et al. generated a cd4−cd8− double-knockout mice transgenic for human cd4 and hla-dq6 to specifi cally reconstitute the human hla-dq6/cd4 arm in mice and established a dilated cardiomyopathy model [ 76 ] , which was one of the earliest attempt for applying humanized mouse model in the study of autoimmune diseases. using similar strategy, eming et al. established a ra model in a hla-dr4/human cd4/tcr combined transgenic mice with the stimulation of a ra-related human autogenic protein hcgp-39 in 2002 [ 77 ] . however, the lack of human immune system reconstitution in these models constrained their representative for the whole map occurring during autoimmune diseases. on the other side, shultz et al. established t1d model in nod/scid-γc −/− mice by co-transplanting with human stem cell and islet cells [ 78 , 79 ] . importantly, this group pointed out the potential of hla-transgenic immunodefi cient mice in optimization of these models and provided some interesting preliminary data [ 78 ] . soon after, infl ammatory arthritis and type 2 diabetes models were established in hla-transgenic humanized mice by david [ 80 ] and schultz groups [ 81 ] respectively. as we mentioned previously, t cell-mediated immune responses were generally incomplete in humanized mice established on conventional immunodefi cient mice, which usually led to insignifi cant clinical symptoms [ 82 ] and thus limited the application of these models. the involvement of hla not only improves the effi cacy of immune responses, but also provides a platform for study of the relationship between hla subtypes and specifi c diseases susceptibility. nevertheless, the complexity and individuality of hla phenotypes in healthy donors or patients still remain as the biggest challenge in rebuild of physiopathology process in relatively limited hla-expressed humanized mice. due to relatively weak reconstitution of human b cells in humanized mice, the establishment of b cell or antibody -mediated autoimmune diseases seems to be more diffi cult than those t cellmediated autoimmune diseases. kerekov et al. rebuilt the clinical pathogenesis in humanized mice with cells transferred from sle patients and evaluated the potential of b cell-targeted therapy with a chimeric molecule containing a monoclonal antibody against human inhibitory complement receptor type i coupled to a decapeptide that mimic dna antigenicity [ 83 ] . in 2012, another group led by duffi eld recapitulated systemic vasculitis in humanized mice by treating them with anti-proteinase-3 igg isolated from patients [ 84 ] . with the improvement in reconstitution of multiple components of human immune system in humanized mice, it is predictable that the induction of diverse human b cellmediated autoimmune diseases in vivo will be accessible soon. application of humanized mice models in transplantation-related diseases arises as early as the birth of humanized mice but the process is so tortuous till now due to chronic exogenous rejection and ongoing decrease of immune cells . in above three studies, investigators planted solid grafts into immunodefi cient mice before reconstitution of human immune system and induced rejection by infusion of mature human cells. however, the longterm outcome of these models is still not clear. in order to further mimic clinical situation, human cd34 + stem cells were applied in establishing humanized mice. using this strategy, three independent groups reported allogeneic islet transplantation [ 89 ], xenogeneic islet rejection [ 90 ], and xenogeneic skin rejection [ 91 ] models during 2010-2012. unfortunately, insuffi cient development of immune cell populations in these humanized mice still stayed as an obstacle and even led to the failure of rejection [ 89 ] . to solve this problem, some other groups tried to develop a more "mature" human immune system in humanized mice by transplanting human peripheral blood cells. in 2013, our group reported a novel human allogenic gvhd model established on humanized mice reconstituted with human pbmc [ 92 ] . this model reproduced typical clinical process of acute gvhd occurring during allogeneic bone marrow transplantation without apparent interruption of exogenous reactivity. using this model, we evaluated the protective effects of human cd8 + treg induced ex vivo by allogeneic cd40-activated b cells and found that human cd8 + treg could inhibit gvhd and induce long-term tolerance without compromising general immunity and graft-versus-tumor (gvt) activity [ 92 ] . the potent regulatory activity of the cd8 + treg was mainly mediated by the expression of cytotoxic lymphocyte antigen (ctla)-4 on cell surface, while their alloantigen-specifi city and the ability to induce the long-term tolerance favor their clinical application. more importantly, this strategy might reduce clinical dependence on limited hla-match donors and largely improve the survival chance of millions of patients who are waiting for bone marrow transplantation. humanized mouse model undoubtedly brings new hope for transplantation research, but we also need to keep in mind that a lot of questions are still waiting to be answered on this way. as emphasized by brehm and shultz, keys to successful humanized mouse model included available immunodefi cient mouse strains, the choice of tissue to transplant and the specifi c human immune cell population that can be grafted [ 85 ] . besides autoimmune diseases and transplantation-related diseases, humanized mice models are also useful to study some other infl ammatory diseases. in 2002, hammad et al. compared the th2 allergic infl ammation in the lung of humanized mice reconstituted with pbmc. to induce infl ammatory reaction, dcs from home dust mite (hdm)allergic patients or healthy donors were injected intratracheally and mice were then repeated exposed to aerosol of hdm. in contrast to ifn-γ secretion induced in mice receiving normal dcs, those injected with dcs from patients induced il-4 and il-5 production accompanied with the increase of ige production, which represents characteristics of th2 response lymphadenopathy, and other infl ammatory sequelae in humanized mouse model [ 97 ] . in addition to immunopathology study, humanized mouse model was also applied in studying the underlying mechanisms of injury repair. by plating retroviral vector-modifi ed human skin on nude mice and adding human keratinocyte growth factor (kgf) to artifi cial wound in the skin, the re-epithelialization was signifi cantly accelerated [ 98 ] . although this model could not be described as "real" humanized mice because no human immune system was involved in it, this attempt initiated an innovative application of humanized mice. compared to satisfactory reconstitution of circulating blood cells, the successful reconstitution of mucosa immunity in humanized mice is still absent till now. mucosa, especially respiratory and digestive tract surface, plays indispensable role in protection and immune regulation. however, the residence and exchange of immune components in the locus are still diffi cult to rebuild in animal models because the physiological dynamics remains largely unknown the occurrence of humanized mouse model provided a perfect platform for evaluating immunotherapy against tumor. the earliest attempts of inducing antitumor immune responses in humanized mice focused on the generation of specifi c antibody but the outcome varied due to unstable humanization of models [ 104 , 105 ] . in the new century, researchers started to pay more attention on developing complete tumorigenicity, especially metastasis process and its relationship with stromal cells, in immunocompetent humanized mice, and made some signifi cant advances in multiple fi elds like human prostate cancer [ 106 ], mixed-lineage leukemia [ 109 ] . based on these progresses, some novel immunotherapy strategies were evaluated on humanized mouse models, such as inhibitory receptor ig-like transcript (ilt)-3 depletion or blockade in melanoma [ 110 ] and il-15-enhanced nk cell-mediated cytotoxicity against human breast cancer [ 111 ] . recently, our group reported a novel application of pamidronate, a phosphoantigen generally used to treat osteoporosis, in treating epstein-barr virus (ebv)-induced b cell lymphoproliferative disease in humanized mouse model reconstituted with human pbmc [ 112 ] . this "new application of an old drug" was mediated by expanding and activating human vγ9vδ2-t cells, a small cell population of human lymphocytes, which might inspire further exploration of currently available resources. more importantly, the established of donorand tissue-specifi c humanized mouse tumor models will undoubtedly play an indispensable role during the development of individual therapies in the future [ 113 ] . the development of humanized mice represents a milestone in the history of human immunodefi ciency virus (hiv) study. the new generation of humanized mice not only improved our understanding on transmission, latency, and pathogenesis of hiv [ 114 -119 ] , but also provided unprecedented platform for antiviral study. besides further exploration of effi cient virus-specifi c neutralization antibodies [ 120 -125 ] and conventional antiretroviral or antimicrobial therapies [ 126 -128 ] in these models, the effi cacy of vectored immunoprophylaxis [ 129 ] and ccr5-targetd treatment [ 130 -132 ] in preventing hiv transmission were evaluated as well. meanwhile, the crucial roles of hiv-specifi c cd8 + t cells [ 133 , 134 ] and plasmacytoid dc (pdc) [ 135 , 136 ] in the replication of virus and activation of immune responses, and their potentials in targeted therapy were also investigated. other novel immunotherapy assays performed in humanized mouse model included blockade of programmed cell death (pd)-1 receptor [ 137 , 138 ] , engineering hiv-resistant t cells from short-hairpin rna (shrna)-expressing hematopoietic stem/progenitor cells [ 139 ] , and inhibition of hiv replication by a chimera containing an rna aptamer with high binding affi nity to the hiv envelop protein gp120 and virus neutralization properties and a small interfering rna (sirna) triggering sequence-specifi c degradation of hiv rnas [ 140 ] . moreover, a preliminary study on mechanisms underlying viral controlling in hla-b*57 elite controller or suppressor (es) was completed in humanized blt mice and demonstrated that elite suppressors are capable of controlling hiv-1 due to the possession of unique host factors rather than infection with defective virus in vivo [ 141 ] . nowadays, we could even make in-depth study on the cell dynamics in hiv-infected humanized mice model with the help of intravital microscopy [ 142 ] . therefore, it is countable that the future molecular biology will bring more surprise to the efforts of gene therapy against hiv [ 143 ] . except for application in hiv-related studies, humanized mouse model also brought span-new opportunities for other human infectious diseases [ 144 ] , especially those blood-borne pathogencaused diseases such as dengue virus infection [ 145 -149 ] , ebv infection [ 150 -155 ] , hcmv infection [ 156 ] , htlv infection [ 157 ] , and malaria parasite infection [ 158 ] . on the other hand, humanized mouse models for leishmaniasis [ 159 ] , salmonella typhi infection [ 160 , 161 ] , herpesvirus infection [ 162 , 163 ] , mycobacteria infection [ 164 , 165 ] , and group b streptococcus (gbs) infection [ 166 ] have been established. these efforts fi ll in the lacks of suitable animal models for those human-specifi c pathogen-caused diseases and push forward the correlating investigations on development of prevention and treatment , although some technological obstacles like the replication of natural infection and transmission routes are still needed to resolved. in 2011, our group used pbmc-transplanted humanized mouse model to evaluate a novel therapeutic strategy by targeting the host rather than the virus for treating infl uenza virus infection. we demonstrated that aminobisphosphonate can control infl uenza disease through boosting human vγ9vδ2-t cell immunity and this benefi cial effect is active against viruses of varying subtypes and virulence [ 43 ] . nevertheless, differences in the characteristics of molecules, tissues, and organs between human and mice might impair effi ciency of pathogen infection and initiation of specifi c immune responses [ 167 ] . in 2005, lassning et al. increased the susceptibility of mice on human coronavirus by crossing aminopeptidase n (apn), the receptor for human coronavirus (hcov)-229e, and transgenic mice into signal transducer and activator of transcription (stat)-1 null mice [ 168 ] . this work, together with hla-and human cytokines/growth factor-transgenic technology [ 169 ] , provided successful examples for future studying human infectious agents in humanized mice. in the next stage, improvement of versatility and variability of human immune system in humanized mouse model and application of gene-modifi ed pathogens [ 170 ] will defi nitely enhance translational effi ciency of these models. the usage of humanized mice in the development of vaccines targeting human diseases including ebv, hiv-1, dengue virus, infl uenza virus, severe acute respiratory syndrome (sars) corona virus, and carcino-embryonic antigen (cea) has obtained outstanding achievements during the past decade, while the introduction of hla transgenic immunodefi cient mice further accelerated the advancement in this fi eld [ 171 -173 ] . with the improvement of immune cell population reconstitution, more and more novel vaccination protocol will be carried out in humanized mice. compared to conventional mice and non-human primate model, humanized mice exhibit great advantage in translational potential, reproductive capacity and data repeatability, economical and ethical concerns. the increasing applications of diverse humanized mice models in biomedical research during the past two decades significantly improved our understanding on human physiological and pathological, especially immunological process at systemic, cellular, and molecular levels. this further accelerated the development of current translational medicine signifi cantly. nevertheless, there are several major caveats on their development remain to be dealt with, including complete replacement of murine mhc with diversifi ed hla molecules and effi cient methodology to express corresponding growth factors and cytokines at specifi c time and organs [ 174 ] ; how to prolong the maintenance of human engraftment, promote the development of myeloid cells and increase relatively weak quantity and quality of immune cells [ 175 ] ; and the limited development of lymph nodes, inter-organ traffi c of immune cells, and the reconstitution of red blood cells and granulocytes [ 176 ] . in another word, the most important issue is to fi nd the convenient and cost-effective ways to construct appropriate human-like microenvironment including physical structure, intercellular contact and molecular signals transfer in humanized mice. it is foreseeable that knowledge exchange in the age of big data will bring an even more bright future to this advancing tool than ever. analysis of candidate human blood stem cells in "humanized" immune-defi ciency scid mice humanized mice in translational biomedical research mighty mice. scientists are still improving the humanized mouse model but are optimistic about its future role in evaluating aids vaccine candidates humanized mice as a preclinical tool for infectious disease and biomedical research hla transgenic mice as humanized mouse models of disease and immunity prevention of "humanized" diabetogenic cd8 t-cell responses in hla-transgenic nod mice by a multipeptide coupled-cell approach cd8 t cell responses to myelin oligodendrocyte glycoprotein-derived peptides in humanized hla-a*0201 humanized mice for studying human leukocyte integrins in vivo full reconstitution of human platelets in humanized mice after macrophage depletion human t cell development in the liver of humanized nod/scid/ il-2rgamma(null)(nsg) mice generated by intrahepatic injection of cd34(+) human (h) cord blood (cb) cells systemic human t cell developmental processes in humanized mice cotransplanted with human fetal thymus/ liver tissue and hematopoietic stem cells expression of hla class ii molecules in humanized nod. rag1ko.il2rgcko mice is critical for development and function of human t and b cells insuffi cient interleukin-12 signalling favours differentiation of human cd4(+) and cd8(+) t cells into gata-3(+) and gata-3(+) t-bet(+) subsets in humanized mice activation of notch1 promotes development of human cd8(+) single positive t cells in humanized mice development of human cd4+foxp3+ regulatory t cells in human stem cell factor-, granulocytemacrophage colony-stimulating factor-, and interleukin-3-expressing nod-scid il2rgamma(null) humanized mice human b cell development and antibody production in humanized nod/scid/il-2rgamma(null) (nsg) mice conditioned by busulfan co-transplantation of fetal bone tissue facilitates the development and reconstitution in human b cells in humanized nod/scid/il-2rgammanull (nsg) mice humanized immune system (his) mice as a tool to study human nk cell development characterization and il-15 dependence of nk cells in humanized mice gm-csf and il-4 stimulate antibody responses in humanized mice by promoting t, b, and dendritic cell maturation induction of functional human macrophages from bone marrow promonocytes by m-csf in humanized mice flt3-ligand treatment of humanized mice results in the generation of large numbers of cd141+ and cd1c+ dendritic cells in vivo inhibition of megakaryocyte development in the bone marrow underlies dengue virus-induced thrombocytopenia in humanized mice a novel lentiviral vector targets gene transfer into human hematopoietic stem cells in marrow from patients with bone marrow failure syndrome and in vivo in humanized mice humanized mice as a model to study human hematopoietic stem cell transplantation generation of humanized mice susceptible to peptideinduced infl ammatory heart disease humanized mice as a model for rheumatoid arthritis humanized nod/ ltsz-scid il2 receptor common gamma chain knockout mice in diabetes research humanized mice for the study of type 1 diabetes and beta cell function role of hla class ii genes in susceptibility/resistance to infl ammatory arthritis: studies with humanized mice humanized mice for the study of type 1 and type 2 diabetes subclinical cns infl ammation as response to a myelin antigen in humanized mice humanized scid mice models of sle anti-proteinase 3 antineutrophil cytoplasm autoantibodies recapitulate systemic vasculitis in mice with a humanized immune system targeted activation of human vgamma9vdelta2-t cells controls epstein-barr virus-induced b cell lymphoproliferative disease humanized nod-scid il2rg-/-mice as a preclinical model for cancer research and its potential use for individualized cancer therapies the utility of the new generation of humanized mice to study hiv-1 infection: transmission, prevention, pathogenesis, and treatment generation of hiv latency in humanized blt mice can humanized mice refl ect the complex pathobiology of hiv-associated neurocognitive disorders? blt humanized mice as model to study hiv vaginal transmission hiv-1 infection of hematopoietic progenitor cells in vivo in humanized mice mucosal hiv-1 transmission and prevention strategies in blt humanized mice hiv therapy by a combination of broadly neutralizing antibodies in humanized mice inhibition of in vivo hiv infection in humanized mice by gene therapy of human hematopoietic stem cells with a lentiviral vector encoding a broadly neutralizing anti-hiv antibody inhibitory effect of hiv-specifi c neutralizing iga on mucosal transmission of hiv in humanized mice hiv-1 suppression and durable control by combining single broadly neutralizing antibodies and antiretroviral drugs in humanized mice broadly neutralizing antibodies and viral inducers decrease rebound from hiv-1 latent reservoirs in humanized mice humoral immune responses in humanized blt mice immunized with west nile virus and hiv-1 envelope proteins are largely mediated via human cd5+ b cells long-acting nanoformulated antiretroviral therapy elicits potent antiretroviral and neuroprotective responses in hiv-1-infected humanized mice humanized mice recapitulate key features of hiv-1 infection: a novel concept using long-acting antiretroviral drugs for treating hiv-1 minocycline attenuates hiv-1 infection and suppresses chronic immune activation in humanized nod/ltsz-scidil-2rgamma(null) mice vectored immunoprophylaxis protects humanized mice from mucosal hiv transmission pre-clinical modeling of ccr5 knockout in human hematopoietic stem cells by zinc fi nger nucleases using humanized mice a topical microbicide gel formulation of ccr5 antagonist maraviroc prevents hiv-1 vaginal transmission in humanized rag-hu mice expansion of activated memory cd4+ t cells affects infectivity of ccr5-tropic hiv-1 in humanized nod/ scid/jak3null mice hiv-specifi c cd8(+) t-cell immunity in humanized bone marrow-liver-thymus mice rapid evolution of hiv-1 to functional cd8(+) t cell responses in humanized blt mice effi cient infection, activation, and impairment of pdcs in the bm and peripheral lymphoid organs during early hiv-1 infection in humanized rag2(-)/(-) gamma c(-)/(-) mice in vivo plasmacytoid dendritic cells suppress hiv-1 replication but contribute to hiv-1 induced immunopathogenesis in humanized mice in vivo blockade of the pd-1 receptor suppresses hiv-1 viral loads and improves cd4+ t cell levels in humanized mice pd-1 blockade in chronically hiv-1-infected humanized mice suppresses viral loads engineering hiv-1-resistant t-cells from short-hairpin rnaexpressing hematopoietic stem/progenitor cells in humanized blt mice an aptamer-sirna chimera suppresses hiv-1 viral loads and protects from helper cd4(+) t cell decline in humanized mice hla-b*57 elite suppressor and chronic progressor hiv-1 isolates replicate vigorously and cause cd4+ t cell depletion in humanized blt mice intravital microscopy in blt-humanized mice to study cellular dynamics in hiv infection gene therapy strategies for hiv/aids: preclinical modeling in humanized mice humanized mice for modeling human infectious disease: challenges, progress, and outlook mosquito bite delivery of dengue virus enhances immunogenicity and pathogenesis in humanized mice utility of humanized blt mice for analysis of dengue virus infection and antiviral drug testing enhanced humoral and hla-a2-restricted dengue virus-specifi c t-cell responses in humanized blt nsg mice dengue virus infection induces broadly cross-reactive human igm antibodies that recognize intact virions in humanized blt-nsg mice dengue virus tropism in humanized mice recapitulates human dengue fever ebna3b-defi cient ebv promotes b cell lymphomagenesis in humanized mice and is found in human tumors a novel animal model of epstein-barr virus-associated hemophagocytic lymphohistiocytosis in humanized mice epstein-barr virus induces erosive arthritis in humanized mice t cells modulate epstein-barr virus latency phenotypes during infection of humanized mice reproduction of epstein-barr virus infection and pathogenesis in humanized mice adoptive transfer of ebv specifi c cd8+ t cell clones can transiently control ebv infection in humanized mice hcmv infection of humanized mice after transplantation of g-csf-mobilized peripheral blood stem cells from hcmv-seropositive donors adult t-cell leukemia/lymphoma development in htlv-1-infected humanized scid mice of men in mice: the success and promise of humanized mouse models for human malaria parasite infections leishmania major infection in humanized mice induces systemic infection and provokes a nonprotective human immune response humanized nonobese diabetic-scid il2rgammanull mice are susceptible to lethal salmonella typhi infection humanized mice for salmonella typhi infection: new tools for an old problem modeling of human herpesvirus infections in humanized mice human herpesvirus 6a infection and immunopathogenesis in humanized rag2(-)/(-) gammac(-)/(-) mice engrafted human cells generate adaptive immune responses to mycobacterium bovis bcg infection in humanized mice cd4+ cell-dependent granuloma formation in humanized mice infected with mycobacteria humanized mice, a new model to study the infl uence of drug treatment on neonatal sepsis humanized mice for the study of infectious diseases development of a transgenic mouse model susceptible to human coronavirus 229e new generation humanized mice for virus research: comparative aspects and future prospects infectious diseases in humanized mice use of humanized severe combined immunodeficient mice for human vaccine development human immune responses and potential for vaccine assessment in humanized mice broad infl uenza-specifi c cd8+ t-cell responses in humanized mice vaccinated with infl uenza virus vaccines humanized mice: are we there yet? humanized mice: current states and perspectives humanized mice for immune system investigation: progress, promise and challenges this work was supported in part by the area of excellence program supported by the university grants committee of the hong kong sar, china (aoe/m-12/06 and aoe/m-06/08). key: cord-278136-ol2buwld authors: gonzales, natalia m.; howell, viive m.; smith, clare m. title: 29th international mammalian genome conference meeting report date: 2016-05-02 journal: mamm genome doi: 10.1007/s00335-016-9640-0 sha: doc_id: 278136 cord_uid: ol2buwld nan during november 8-15, 2015, the 29th annual international mammalian genome conference (imgc) attracted researchers from all over the world to yokohama, japan to discuss the latest advances, tools, and techniques in mammalian genetics. organized by piero carninci (riken) and the international mammalian genome society (imgs; www.imgs.org), the meeting brought together 336 scientists from 28 countries, not to mention hundreds of online participants that followed the live tweeting of talks (#imgc15). social media has become an integral part of the imgc, complementing the scientific content and facilitating ongoing discussions between scientists around the globe. the conference opened with a bioinformatics workshop, guided tours of riken laboratories, and a trainee symposium, giving ph.d. students and early-career postdoctoral researchers an opportunity to share their works in a collegiate and mentoring setting and vie for the chance to present at the main meeting. participants were officially welcomed to yokohama at the evening reception, where old friends and new were met over a smorgasbord of local cuisine. the main meeting was divided into sessions showcasing the wide-ranging research interests of imgs members. these included human disease models and immunology, neuroscience, development and stem cells, genomics and computational analysis, epigenomics and noncoding rnas, advances in genome editing, and largescale resources. the verne chapman lecture was given by professor john mattick, director of the garvan institute of medical research in sydney, and the inaugural darla miller distinguished service lectureship was awarded to janan eppig, professor at the jackson laboratory and a pioneer of the mouse genome informatics (mgi) program project. several poster sessions, a mentor lunch for trainees and workshops on bioinformatics, systems genetics, scientific literature curation, gene enrichment analysis, and fantom were also featured in the meeting program, which culminated in a feast of epic proportions. abstracts from the meeting are available at www.imgc2015.jp. the imgc has a strong reputation for advocating new scientific talent, epitomized by the 33 trainee scholarships awarded for conference travel, the mentor-trainee lunch, presentation awards, and numerous opportunities to present work and receive feedback. the trainee symposium is an integral part of the meeting, featuring oral presentations from 16 graduate students and postdoctoral researchers. the wide variety of topics presented exemplified the diversity and utility of mammalian model systems for both clinical and basic research. xenograft mouse models were utilized by hiroyuki yoda (ts-01; chiba cancer centre research institute) and takahiro inoue (ts-13; chiba cancer centre research institute) to demonstrate the in vivo anti-tumor effects of novel alkylating agents targeting the oncogenes mycn in neuroblastoma, and kras g12d/v in colorectal cancer, respectively. the power of forward genetics was demonstrated by lisa gralinski (ts-02; university of north carolina) using the precollaborative cross to identify sars-coronavirus susceptibility loci and irina treise (ts-16; german mouse clinic), exploiting n-ethyl-n-nitrosourea (enu) mutagenesis to uncover molecular mechanisms of immunodeficiency. the sanger knockout mouse resources were highlighted by kifaythullah liakath-ali (ts-12; kings college london), who conducted a phenogenomics screen to investigate the genetic basis of skin phenotypes. gennadiy tenin (ts-10; university of manchester) followed up on human genomewide association studies (gwas) of the congenital heart defect tetralogy of fallot by developing an in vitro mouse heart culture for testing candidate genes by sirna knockdown. ximena ibarra-soria (ts-07; wellcome trust sanger institute) took us on a journey through the mouse olfactory system, demonstrating that novel olfactory receptor genes can be identified, characterized, and distinguished from environmental regulators despite the system's overwhelming complexity. several speakers focused on behavioral traits, including yuki matsumoto (ts-04; national institute of genetics), who identified a locus on chromosome 11 associated with mouse tameness in wild-derived heterogeneous mouse stocks, and akira tanave (ts-09; national institute of genetics) who is exploring the etiology of anxiety and stress by studying strain differences between wild-derived msm mice and c57bl/6 mice. guzel gazizova (ts-03; kazan federal university) introduced the dormouse as a genetic model, discussing how transcriptome-level differences between two genera of dormice can be analyzed to yield insights into the evolution of hibernation and to clarify the status of the dormouse in the mammalian phylogeny. belinda goldie (ts-06; kyoto university) took us further into the world of gene expression, demonstrating the importance of extending micro-rna (mirna) analysis beyond evolutionarily conserved targets when exploring gene regulation of human neuronal synapses. hazuki takahashi (ts-14; riken) demonstrated a high-throughput system to optimize antisense long-noncoding rnas that increase translation of target mrnas, and riti roy (ts-15; university of western australia) examined expression profiles of receptors and ligands in cell lines profiled in the fantom5 project and tumors profiled by the cancer genome atlas (tcga) to understand how cancer cells communicate. mice were not the only model system represented by trainee symposium talks. pavel mazin (ts-05; skolkovo institute of science and technology) examined brain rna-seq data from humans, chimpanzees, and macaques to identify species and age-related differences in alternative splicing patterns. pavel prosselkov (ts-11; riken) branched from the mammalian tree, using the sea squirt to investigate the role of gene paralogs in cognition. finally, brandon velie (ts-08; swedish university of agricultural sciences) showcased the horse, an underutilized organism in mammalian genetics, as a model for identifying genetic factors related to locomotion, allergic diseases, and eczema. ximena ibarra-soria, akira tanave, hazuki takahashi, and irina treise were named as the lorraine flaherty awardees for their talks during the trainee symposium ( fig. 1 ; table 1 ) and received the opportunity to present their work at the main conference. several plenary sessions focused on a range of mammalian tools and resources for modeling human disease. the session showcased tools such as recombinant inbred lines (rils), outbred populations, classic crosses, and enu mutagenesis to yield new understanding and identify candidate genes for disease susceptibility, while knockout and patient-derived xenograft mice enabled further mechanistic insight. the session featured many talks utilizing the phenotypic diversity and genetic mapping power of diversity outbred (do) mice and the collaborative cross (cc), community resources *10 years in the making. fernando pardoumass medical school) showed that resistance loci underlying tuberculosis pathogenesis could be mapped in *60 cc lines and *20 lines of the incipient c57bl/ 6 9 dba/2 (bxd) cross. interestingly, bacterial modules could be mapped onto the host genome to understand how both host and pathogen genomes determine disease outcome. the mapping resolution of the do population was also highlighted in cancer, with nigel crawford (o-19; national institutes of health, bethesda, md) using quantitative trait locus (qtl) mapping in tramp 9 j:do f1 males to identify metastasis susceptibility loci for prostate cancer. the power of the enu approach was demonstrated by several talks. gaetan burgio (o-02; australian national university) identified host factors altering malaria infection outcomes that could be targeted as a novel host-directed antimalarial therapy, and kart tomberg (o-16; university of michigan) mapped thrombosis modifier genes by bulk exome sequencing mice from a sensitized enu suppressor screen. both talks featured the use of gene editing candidate mutations with crispr/cas9 to validate causative alleles. the impact of mouse models on precision oncology was showcased by carol bult (o-01; the jackson laboratory), who discussed how patient-derived xenograft models can provide a platform for testing therapeutic options to guide treatments for breast and other cancers (fig. 2) . kate ackerman (o-22; university of rochester) used inducible wt1 creert2 to determine the impact of loss of ctnnb1 at different time-points, concluding that b-catenin is critical for diaphragm development during a defined window of time. han kyu lee (o-18; duke university) analyzed polymorphisms among the ancestral haplotypes of 32 inbred mouse strains to map loci for ischemic stroke outcomes, validating the interleukin 21 receptor as a candidate by analysis of gene expression patterns and knockout models. other features of this session included a gwas of aerobic capacity in rats segregated on running ability by yu wang german center for neurodegenerative diseases tuebingen) conducted a massive forward genetic screen using human exome data, followed by systematic rnai screens in worms, flies, and human cell lines to identify genes and pathways involved in parkinson's disease. this year's imgc included a mini-session that sought to address the question of whether or not the mouse is still relevant as a model for human disease. while this particular audience needed no convincing of the fundamental scientific understanding gained through use of the mouse as a model, tsuyoshi miyakawa (o-23; fujita health university) gave a thought-provoking narration of his lab's response to a controversial publication claiming otherwise. his careful consideration of arguments made from both sides of the controversy emphasized the importance of understanding the experimental design and methods for analyzing a study before interpreting its results. miyakawa compared his re-analysis of mouse genomic data from seok et al. (2013) with the original study, which had concluded that genomic responses in mouse models poorly mimic human inflammatory diseases. miyakawa's group drew the opposite conclusion from the data, demonstrating that responses in mouse models greatly mimic human inflammatory diseases (takao and miyakawa 2015) . alterations to the seok et al. (2013) analysis included comparing genes that exist in both mouse and human, not just human disease genes that lack rodent homologs. he emphasized the need to define appropriate phenotypes and choose appropriate statistical methods. ultimately, he concluded, the 15 % overlap in gene expression between mouse and human does not mean that the mouse is a bad model; instead, the overlapping 15 % probably contain the genes that are the most important for disease. the panel responses also emphasized other advantages of mouse models, including but not limited to access to tissue, ability to measure responses at different time-points on the same background and the capacity to do epigenetic studies. the symposium also raised a conundrum of the scientific review process by highlighting ways in which the design and methods chosen to address a question can either obscure or reveal scientific truths, inciting a thoughtful series of questions about training and the process of peer review. how can we ensure that future generations of biologists are adequately trained to evaluate statistical methods? when the same data can be analyzed to show very different results, potentially affecting funding decisions on models, is the ultimate onus for publication on the journal or expert scientific reviewers? these questions were discussed in the open forum and reflect a larger conversation taking place within the wider scientific community, where they will undoubtedly continue to be discussed. this plenary session encompassed the use of mouse embryonic stem cells (mescs), gene expression analysis, and recent advances in genome engineering to address fundamental questions about development and degenerative disease. anne czechanski (o-06; the jackson laboratory) described how her experience deriving novel pluripotent mescs with the inhibitor cocktail 2i led to the unexpected observation that female cell lines experience a higher rate of attrition than male cell lines. future transcriptional profiling may uncover why the combination of x chromosome dosage and 2i culture conditions leads to attrition of female lines and will have important implications for those using mescs. sandra richardson (o-07; university of queensland) used retrotransposon capture sequencing (rc-seq) to deduce the timing and frequency of retrotransposon insertions in multigeneration c57bl/6 pedigrees. she presented data showing retrotransposition in the early embryo resulting in somatic and germline genetic mosaicism, adding to the evidence for retrotransposition as an important source of genetic diversity. patrizia rizzu (o-08; german center for neurodegenerative diseases) shared how she used fantom5 data to understand how a hexanucleotide repeat expansion influences the transcriptional profile of c9orf72, a gene involved in neurodegenerative disease. yasuhide furuta (o-09; riken) derived compound mutant mice from mescs containing multiple targeted mutations in the fibroblast growth factor (fgf) signaling system to study the role of fgf family genes in eye development. due to tight linkage between the genes of interest and reduced fertility in fgf mutant lines, it was previously impossible to generate compound mutants from an experimental cross. however, the development of crispr/cas9 allowed furuta's group to measure the extent of functional redundancy within the fgf pathway and uncover novel roles for its constituents. gabriela sanchez-andrade (o-10; wellcome trust sanger institute), recipient of this year's verne chapman young investigator award (table 1) , closed the session with a discussion of her efforts to identify new biomarkers of neurodegenerative disease in a mouse model of frontotemporal dementia and parkinsonism (ftdp) with severe olfactory deficits. olfactory dysfunction is one of the earliest and most common symptoms of neurodegenerative disease in humans, yet the underlying molecular mechanisms are unknown. sanchez-andrade's impressive analysis of the olfactory epithelium measured differential expression and protein dynamics in wild-type and transgenic mice to identify a set of candidate genes correlated with onset of ftdp. furthermore, she demonstrated that similar changes in the expression of these genes occur in other brain regions relevant to ftdp pathology, highlighting the potential of her approach to identify additional biomarkers of human disease. the imgc featured three sessions on genomics and computational analysis. although the 13 selected talks were diverse in both content and approach, each of them touched upon at least one of the following themes: advances in omic technology, molecular mechanisms underlying complex traits, and the relationship between genomic architecture and mammalian evolution. speakers in the first session discussed their experiences with single-cell rna-seq to address a series of questions that remain integral to the imgc each year; namely, what tools and technologies are driving our field forward? what exciting possibilities do they create, and what obstacles do we face in using these methods and interpreting their results? anna mantoski (o-11; roslin institute) shared her solutions to some of the analytical puzzles that technical variability can create for single-cell sequencing experiments and described how studying differences in gene in describing cap analysis of gene expression (cage), a method for capturing single-cell transcriptomes, charles plessy (o-12; riken) highlighted many of the challenges familiar to researchers using rna-seq and shared how his strategy of combining cage with techniques including ''pseudo-random'' primers to remove rrna, molecular tagging, and fragmentation can improve the quality of single-cell data. anton kratz (o-13; riken) further emphasized cage's potential as he described how his group applied translating ribosome affinity purification (trap), which can isolate the ribosome-associated transcriptome (the translatome) to purkinje dendrites to measure transcription at the subcellular level and identify biomarkers for specific cell types. much of the remaining work sought to address broad evolutionary questions dealing with the relationship between biological mechanisms, complex traits, and genomic architecture. martin taylor (o-33; university of edinburgh) discussed how the distribution of replicationassociated polymorphisms in mouse and human genomes may be explained in part by patterns of transcription factor binding and chromatin accessibility in the paternal germline. satoshi oota (o-52; riken) used enu mutagenesis in the mouse to observe evolution in real time, which allowed gaining insight into how the distribution of gc content has evolved in mammalian genomes. andrew morgan (o-34; university of north carolina chapel hill) explored the functional and evolutionary impacts of large copy number variations at a specific locus in the mouse genome. another prominent theme among the research featured in the sessions on genomics and computational analysis was the relationship between genotypes and quantitative phenotypes at both the level of the cell and the organism. jason lin (o-36; chiba cancer center research institute) discussed how a new class of molecules that combine the histone deactylase inhibitor saha and dna-binding pyrrole-imidazole polyamide (saha-pip) can induce epigenetic reprogramming and regulate pluripotency. steven munger (o-53; the jackson laboratory) used a system's genetics approach in the do to resolve the conflict between the expectation of high mrna-protein expression correlation from the central dogma of biology and recent observations suggesting a weak correlation. his work showed that while the levels of many proteins are regulated by nearby variants that influence mrna expression levels (cis-eqtl), the relative stoichiometry of proteins in stable partnerships and complexes is a key posttranslational regulator of protein abundance that can act to buffer individual member proteins against cis-acting transcriptional variation. robert young (o-35; university of edinburgh) presented evidence of divergence in the patterns of promoter gain and loss in humans and mice, offering insight into the evolutionary processes that contribute to gene expression and phenotypic diversity in both species. peter williamson (o-54; university of sydney) used a panel of inbred mouse strains to identify qtl related to metabolism, body composition, lactation, and other complex traits. a common approach featured at the imgc each year is the use of the mouse as a model for understanding how biological processes influence and respond to changes in the mammalian genomic landscape. notably, this year's sessions on genomics and computational analysis also included speakers that used a variety of other organisms in their research. in addition to the several talks mentioned above, we heard from david beier (o-32; seattle children's research institute) who analyzed the genomewide distribution of nonsense mutations in the exomes of individuals without severe mendelian disorders. he found that the strength of heterozygote selection correlated with the likelihood of recessive lethality and that many genes with established roles in developmental diseases had high heterozygote selection. martin frith (o-51; national institute of advanced industrial science and technology, tokyo) used sequence data from humans, chimps, orangutans, and dogs to classify different types of human chromosomal rearrangements, which occur in slowly evolving regions. finally, isaac adeyemi babarinde (o-37; national institute of genetics) introduced the audience to the capybara (the world's largest living rodent) and shared how he used its genome to understand the relationship between mutation rate and body size in rodents. for the first time at an imgc, two plenary sessions were devoted to epigenomics and noncoding rnas. this highlights the increasing awareness of looking beyond the traditional gene-protein relationship for understanding transcriptional control in both normal and diseased states in mammals. the influence of parental diet was featured in two presentations. joseph nadeau (o-25; pacific northwest research institute) illustrated that both folate supplementation and mutations in rna modulating genes such as dnd1, a1cf, and apobec1 bias fertilization toward wildtype genotypes. johannes beckers (o-26; helmholtz zentrum, munich) reported the epigenetic inheritance of an acquired metabolic disorder. he showed that a parental high-fat diet increased offspring susceptibility to obesity and type 2 diabetes. both speakers stressed the importance of understanding the underlying mechanisms related to diet for guiding future public health policies. different aspects of genomic location as a determinant of transcriptional regulation were also discussed in the first plenary session. through determination of the three-dimensional configuration of x chromosomes in different tissues and cells, christine disteche (o-24; university of washington) demonstrated that genes that escape x inactivation preferentially localize at the periphery of the nucleus. she also showed that the expressed alleles of imprinted genes have greater chromatin contacts than the silent alleles, suggesting that these expressed regions are under greater organizational constraints. reanalysis of the publicly available encode data led siddharth sethi mrc harwell) to the discovery that transcription factor-binding sites are highly enriched in dnase1 hypersensitive sites compared to promoter and enhancer regions. as such, dnase1 hypersensitivity data could be used as an alternative method for discovering enriched regulatory motifs with the aim of improving understanding phenotypic variability. the second plenary session continued the theme of using genomewide data to understand transcriptional control by different noncoding elements-promoters, enhancers, microrna, and l1-transposable elements. to dissect the relationship between dynamic changes in mrna and enhancer rna, erik arner (o-44; riken) measured the activities of promoters and enhancers over time in a number of cell types following different biological stimuli. he presented data supporting enhancer transcription as the earliest event in successive waves of transcriptional change. this phenomenon was observed in multiple biological systems, suggesting this may be a general feature of mammalian transcriptional regulation, contrary to models showing coexpressions of enhancers and promoters. albin sandilan (o-43; university of copenhagen) provided a clinical application, presenting rna-seq data profiling colon specimens from patients with inflammatory bowel disease. a promoter set was identified, which could accurately distinguish between the primary disease subtypes: crohn's disease and ulcerative colitis. moreover, 20,000 active enhancer regions were identified with subsets induced in general inflammation or specifically in one subtype. these enhancers had links to both known and novel genes involved in the pathogenesis of inflammatory bowel disease. michiel de hoon (o-45; riken) turned attention toward the role of mirna in gene regulation. he presented the results from a collaboration spanning 31 centers internationally, which analyzed deep, sequencing data of paired small rna and cage libraries across a wide range of cell types. this analysis revealed two classes of mirna: celltype-specific mirnas and ubiquitous mirnas. cell-typespecific mirnas are highly expressed in only a few cell types and may act as buffers of gene expression. ubiquitous mirnas are expressed in most cell types, but depleted in particular cell types and may be important in preventing inappropriate activation of transcriptional programs. valerio orlando (o-46; ircss fondazione santa lucia) presented the last talk in the plenary session focusing on the epigenetic role of retrotransposable elements, specifically long interspersed nuclear elements 1 (l1). analysis of l1 transcription followed that of enhancer elements and myogenic program in normal muscle cells but was absent in duchenne muscular dystrophy (dmd)-affected muscle cells. pharmacological rescue of the dmd phenotype by histone deacetylase inhibitors or gene therapy was accompanied by normal l1 expression. he proposed that deregulation of l1 mobilization is a key trait in loss of cell identity and disease. genome editing using crispr/cas9 technology has taken the scientific world by storm, allowing rapid and efficient editing in eukaryotic cells. the in vitro and in vivo applications of crispr/cas9 genome editing was a strong theme at this year's meeting, with every plenary session including talks that made use of this technology, as well as the plenary session being completely devoted specifically to advances in genome editing. marie-christine birling (o-29; institut clinique de la souris) kicked off the session demonstrating crispr/cas9 genome editing in rats. she described her group's effort to generate alleles with precise gene deletions and duplications of a 24-mb region by means of two different guide rnas on both sides of the target region. kazuto yoshimi (o-30; national institute of genetics) then combined crispr/cas9 ''scissors'' with single-stranded oligodeoxyribonucleotides as the ''paste'' mechanism to ligate the cut sites for efficient replacement of rat genes with human genes. finally, dave bergstrom (o-31; jackson laboratory) presented his lab's modified crispr approach to enable rapid ''humanizing'' of large segments of the mouse genome, giving the example of replacement of a mouse tumor suppressor gene with 25 kb of the orthologous human gene. inbred strains with whole-genome illumina sequencing and discussed the challenges of making alignments in complex regions. attendees were treated to the test site launch of the latest data available through the uscd genome browser (http://www.hgwdev-mus-strain.sdsc.edu/cgi-bin/hggateway). the 2015 verne chapman lecture titled ''the hidden layer of regulatory rna in mammalian genome biology'' was delivered by eminent molecular biologist john mattick (o-42; garvan institute of medical research). mattick is known for his research in revealing the central role of nonprotein-coding dna in the production of regulatory nonprotein-coding rnas (ncrnas) and his efforts to understand how ncrna regulation contributes to the staggering level of phenotypic complexity observed throughout the animal kingdom. he began his lecture with the quote ''are we letting a philosophy of the protein-coding gene control (our) reasoning? what then is the philosophy of the gene?'' he pointed out that this quote was not from this year or even this century-it was in fact a concern attributed to nobel laureate barbara mcclintock 75 years ago. he reminded us that the mammalian genome contains only *20,000 protein-coding genes, the same number as in simple nematodes. on the other hand, the extent of nonprotein-coding dna increases with increasing developmental and cognitive complexity, reaching 98.5 % in humans. he guided us through the events leading up to his discovery and the explosion of studies that followed, peppering his tale with vivid descriptions of the findings and figures that have influenced him over the years. he shifted effortlessly between concrete descriptions of data and philosophical speculations on the evolution of paradigm shifts, the mysteries of biological complexity, and the silent assumptions that inform (and occasionally hinder) scientific practice. he entertained the audience with his encyclopedic knowledge of molecular genetics, framing his research within a larger historical context that served to complement data-driven descriptions of his and others' work establishing ncrnas as prominent regulators of development, cognition, and disease in the mammalian genome. mattick also elaborated on the relevance of ncrnas to incipient areas of research and technology development in epigenomics and neurobiology, creating a memorable and thought-provoking experience for both experienced investigators and trainees (fig. 3) . this year there were two additional keynote lectures, both showcasing scientific advancements using induced pluripotent stem cells (ipscs). masayo takahashi (o-28; riken center for developmental biology) provided a historical perspective of ipscs, from basic research to clinical application. it is remarkable that the field has gone from the invention of ipsc technology to the clinic in only 7 years. dr. takahashi presented the first human application of ipsc-derived targeting age-related macular degeneration, a retinal disease. she described an impressive panel of experimental validations for retinal pigment epithelial cell sheets derived from ipscs, including whole genome, genotyping arrays, methylome, and single-cell analysis. next, her group hopes not only to treat the retinal epithelium but also to use ipscs to derive photoreceptors to completely restore vision in patients. the talk discussed the use of allogenic as well as autologous ipscs and treated the audience to an early view of potentially revolutionary treatments utilizing ipscs, highlighting the steadily growing wave of interest in this technology. hideyuki okano (o-47; keio university graduate school of medicine) then went on to discuss the use of ipscs in the study and treatment of neurological disorders. he described work to establish ipscs from patients with psychiatric disorders and characterize their pathophysiology. in a quest to investigate human psychiatric and neurological disorders more effectively, hideyuki then presented his group's impressive work generating transgenic marmoset models of parkinson's disease. the marmosets express human synuclein and recapitulate typical human diseases including sleep disturbances, lewy bodies, tremors, and gait abnormalities. in summary, the 2015 meeting showcased a variety of cutting edge mammalian genetic approaches, concepts, and results-from large-scale mapping to single gene approaches and everything in between. the meeting ended in a spectacular fashion, with attendees treated to the spa and fig. 4 a spectacular finale for an amazing conference massage facilities at the yokohama minatomirai manyo club, donning kimonos and yakutas for a traditional banquet (fig. 4) . attendees were entertained by local performers before awards were made to acknowledge exceptional trainee oral and poster presentations, and to thank the outgoing imgs secretariat and nominations and elections committee. it is certainly the first and only imgs meeting to date where ongoing scientific collaborations were made and discussed in a traditional japanese onsen: a most fitting end to a wonderful meeting. we eagerly await the next imgc, which will be part of mouse genetics 2016 at the allied genetics conference (tagc) in orlando, florida in july 2016. that conference will bring the mouse genetics community together with yeast, ciliate, c. elegans, drosophila, and zebrafish communities to highlight the importance of model systems in understanding and translating fundamental advances in genetics. updates about tagc 2016 and the following imgc in heidelberg, germany in 2017 can be found at www.imgs. org. follow us on twitter (https://www.twitter.com/imgs_ news) or facebook (https://www.facebook.com/mamma lian.genome/). genomic responses in mouse models poorly mimic human inflammatory diseases genomic responses in mouse models greatly mimic human inflammatory diseases key: cord-022393-s26d54ew authors: e. newcomer, christian; g. fox, james title: zoonoses and other human health hazards date: 2007-09-02 journal: the mouse in biomedical research doi: 10.1016/b978-012369454-6/50054-6 sha: doc_id: 22393 cord_uid: s26d54ew zoonoses refers to the infectious diseases and infestations that are transmissible directly from an animal host to humans. the biomedical literature contains numerous reports of zoonotic diseases and parasitic infestations from laboratory mice and their wild counterparts. the extended maintenance of the laboratory mouse over a number of generations under controlled and increasingly sophisticated laboratory animal housing conditions with veterinary oversight and effective infection control measures has markedly reduced the likelihood that zoonotic agents would be encountered in a modem animal care and use environment. wild caught mice that are maintained in naturalistic housing environments in the laboratory, laboratory mice that have contact with wild or feral mice, and mice kept as pets in the home environment are examples of animal management conditions that would be conducive to the expression and transmission of zoonotic diseases and other mouse-associated hazards. in addition to the zoonoses, mice are capable of inflicting bites on inadequately trained personnel and are a rich source of allergens for a substantial number of persons predisposed to develop mouse-associated allergic sensitivities. this chapter discusses the mouse-associated zoonotic diseases and other health hazards and explains the strategies that are helpful for reducing or eliminating the risk of personnel exposure to these conditions. zoonoses is derived from the greek words zoon (meaning animals), and noses (meaning disease), and refers to the infectious diseases and infestations that are transmissible directly from an animal host to humans. the biomedical literature contains numerous reports of zoonotic diseases and parasitic infestations from laboratory mice and their wild counterparts. the extended maintenance of the laboratory mouse over a number of generations under controlled and increasingly sophisticated laboratory animal housing conditions with veterinary oversight and effective infection control measures has markedly reduced the likelihood that zoonotic agents would be encountered in a modem animal care and use environment. however, when these essential animal program quality measures fail or are not incorporated into the animal facility operations, zoonotic pathogens may be unwittingly introduced and perpetuated, placing personnel at increased risk of exposure. wild caught mice that are maintained in naturalistic housing environments in the laboratory, laboratory mice that have contact with wild or feral mice, and mice kept as pets in the home environment are examples of animal management conditions that would be conducive to the expression and transmission of zoonotic diseases and other mouse-associated implications in the new world serocomplex group are present among the wild rodents endemic to the united states such as neotoma spp. and peromyscus spp. buchmeier et al. 2001; fulhorst et al. 2002) . research animal programs that import wild rodents for laboratory studies should stay abreast of the developments on the identification and host-range characteristics of the new world arenaviruses of emerging importance. this section will focus only on lymphocytic choriomeningitis (lcm), a naturally occurring viral zoonosis of the laboratory mouse. many published reports of human lcm infection are associated with laboratory animal and pet contact, particularly mice and hamsters, and these studies now span many decades (armstrong and lillie 1934; bowen et al. 1975; dykewicz et al. 1992; jahrling and peters 1992; lehmann-grube et al. 1979; rousseau et al. 1997) . there seems to be a resurgent awareness among physicians that lcm should be sought as an etiology in human neurological disease and in pediatric congenital brain disorders (barton and hyndman 2000; barton et al. 1995 barton et al. , 2002 romero and newland 2003) . lcmv is widely distributed among the wild mouse population throughout most of the world presenting a zoonotic hazard (childs et al. 1992; childs and peters 1993; morita et al. 1996; smith et al. 1993 ). surveys conducted within the urban environment of baltimore, maryland, reported that 9% of house mice and 4.7% of persons tested had measurable lcmv antibody titers (childs et al. 1991 (childs et al. , 1992 . a similar serological survey conducted in spain across urban and rural ecological settings also found a 9% prevalence in mice and a 1.7% prevalence among persons by immunofluorescence assay (lledo et al. 2003) . the recent serological detection of lcmv in five mice on the treeless, sub-antarctic, macquarie island of australia, indicates the extent of distribution of this agent to the remotest areas of our planet (moro et al. 2003) . the apparent ease with which lcmv is transmitted to humans also occurs in a variety of other laboratory animal species; hamsters, guinea pigs, swine, dogs, and nonhuman primates, especially callitrichids, which readily sustain natural infections. in the case of the callitrichids, there have been numerous reports of epizootic infectious hepatitis (callitrichid hepatitis) due to lcmv, with a high mortality rate in zoological parks in both the united states and england over the past two decades (lucke and bennett 1982; montali et al. 1989; stephensen et al. 1990 stephensen et al. , 1991 stephensen et al. , 1995 . rodent (mouse) infestations of these zoos and/or the supplementation of the diets of tamarins and marmosets with suckling mice, a common practice (richter et al. 1984) , are potentially rich sources for lcmv. in the research animal facility environment, the laboratory mouse continues to merit attention as the species of primary concern as a reservoir for cases of human lcm (dykewicz et al. 1992 ). in the laboratory mouse, and to a lesser degree the hamster, breeding colonies can become endemically infected when the virus is transmitted to pups in utero or early in the neonatal period, producing a tolerant subclinical infection characterized by chronic viremia and viruria. when infected, athymic and other immunodeficient mouse strains may be predisposed to harboring silent, persistent infections and present a higher risk to personnel (dykewicz et al. 1992) . under some circumstances lcmv also produces a pantropic infection and may be copiously present in blood, cerebrospinal fluid, urine, nasopharyngeal secretions, feces, and tissues of infected natural hosts and possibly humans. bedding material and other fomites contaminated by lcmv-infected animals can also be important sources of infection for humans, as demonstrated in a recent outbreak among laboratory animal technicians and on many previous occasions (biggar et al. 1975; dykewicz et al. 1992) . the experimental passage of tumors and cell lines contaminated with lcmv has long been recognized (haas and stewart 1956) and represents one of the biggest threats for the introduction of lcmv into animal facilities at the present time (bhatt et al. 1986; dykewicz et al. 1992; nicklas et al. 1993) . reported that 17 of 63 rodent transplantable tumors screened were positive for lcmv and identified contamination in 4 of 14 hamster tumors and 2 of 81 mouse tumors that had been propagated in animals (nicklas et al. 1993) . the growth of lcmv in insect cell lines has also been demonstrated (rahacek 1965) , and the article by hotchin summarized the work of others indicating that numerous experimentally infected, bloodsucking ectoparasites are capable of transmitting the disease to laboratory rodents (hotchin 1971) . lcm virus also has been recovered from cockroaches (armstrong and lillie 1934) . the diagnosis and control of lcmv infection in mouse colonies has been reviewed in chapter 7 of this volume. tumor and cell-line screening before animal passage, the control of wild rodent infestations in areas where animals are housed or used, and the early detection of colony infections through sound colony health surveillance practices are of critical importance to the prevention of infection in mouse colonies. once established in mouse breeding colonies, the high viral load characteristically shed by mice infected congenitally or neonatally represents a very serious hazard to personnel. most of the cases of human infection, whether involving exposure in the home, agricultural, or laboratory setting, have involved contact with live mice and their excreta or mouse carcasses (dykewicz et al. 1992; havens 1948; morbidity and mortality weekly report 1984) . several authors have emphasized the association between the actual handling of infected mice and the contraction of the disease by humans (dykewicz et al. 1992; havens 1948; smithard and macrae 1951) . several cases of human infection have suggested the possibility that infected rodent tissues can serve as a source of infection for laboratory personnel (baum et al. 1966; dykewicz et al. 1992; tobin 1968 ). humans may be infected by inhalation or by the contamination of mucous membranes or broken skin with infectious tissues or fluids from infected animals. the transmission of lcmv by the bite of an infected mouse can also occur (scheid et al. 1964) . also, hotchin reported the findings of other researchers that lcmv was transmissible experimentally through the intact skin of the guinea pig, but this finding has not been reported in humans (hotchin 1971) . airborne transmission is well documented and plays a very important role in human infections, especially through the ready dispersion and inhalation of viral-contaminated dust from the animal cage or room (biggar et al. 1975; hinman et al. 1975) . following an incubation period of 1 to 3 weeks, humans may experience asymptomatic or a mild febrile disease ranging to a serious flu-like illness characterized by anorexia, malaise, diffuse myalgia and arthralgia, fever, chills, vomiting, headache, stiff neck, and photophobia. some patients enter a second stage of the disease several days after the resolution of early mild symptoms, developing meningoencephalitis and exhibiting additional signs such as drowsiness, confusion, sensory disturbances, and motor abnormalities. patients can also develop more serious nonneurological manifestations of the disease such as maculopapular rash, lymphadenopathy, parotitis, orchitis, arthritis, and epicarditis (peters 1997 ). central nervous system involvement has resulted in death in several cases. infections during pregnancy pose a risk of infection for the human fetus (wright et al. 1997). wright et al. reported 26 cases in human infants, with lcmv confirmed serologically over a two-year period in a major u.s. medical center (wright et al. 1997) . these infants presented with ocular abnormalities, macrocephaly, and hydrocephalus with microcephaly. fifty percent of the mothers reported having had illnesses compatible with lcmv infection, and over half reported exposures to rodents during their pregnancies. the diagnosis of lcm infection in humans is currently made by serological testing using either the immunofluorescent antibody (ifa) test or the enzyme-linked immunosorbent assay (elisa) (barton et al. 2002) . both of these tests are available through the centers for disease control and prevention and are superior to the complement fixation test that is widely available commercially. although there are no proven effective antiviral therapies for lcm infection, intravenous ribavirin therapy reduces mortality in patients infected with lassa fever virus (a member of the old world arenavirus serocomplex) and may be of some benefit in patients with severe lcmv infections (andrei and de clercq 1993; mccormick et al. 1986 ). this disease can be prevented in the laboratory through periodic serological surveillance using elisa and ifa tests of newly introduced animals with inadequate disease profiles and of resident animal colonies at risk. thorough screening of all tumors and cell lines intended for animal passage using the highly sensitive mouse antibody production assay or newer pcr-based laboratory tests for the presence of lcmv is another crucial element in the program to prevent the introduction of lcmv into established animal colonies (besselsen et al. 2003 and chapter 7 of this volume). sound animal facility sanitation practices and the use of microbarrier caging systems with proper infection-control techniques should prevent or suppress the spread of lcmv if present in the environment. the elimination of wild rodent infestations in animal facilities is very important to prevent the introduction of lcmv into the animal facility environment. also, facilities with wild rodent infestations may encounter the relatively common, free-living, bloodsucking mite of the rodent, ornithonyssus bacoti, in abundance (personal communication). although the natural lcmv transmission to humans from bloodsucking ectoparasites is unproven, the control of potential ectoparasitic vectors of this type would be a prudent measure. rabies is an acute, almost invariably fatal disease that occurs worldwide with the exception of a few countries, generally island nations, and other regions that have excluded the disease through animal importation and control programs and the aid of geographic barriers. neither the laboratory mouse nor other small wild rodent hosts appear to be important as reservoirs of natural rabies infection. hence, the principal reason for our discussion of rabies as a zoonotic disease of the laboratory mouse is to provide information that should quickly allay the fears of uninformed research and animal facility staff who suffer the bite of a mouse. on the other hand, the experimental use of mice in the study and characterization of rabies virus and in rabies vaccine development is an important component of some animal care and use programs that deserves special attention by the institution during all phases of research planning and implementation. there are no known cases of human rabies from rodents in the united states (2002). the incidence in larger wild rodent species within the united states has increased in recent years, however. during the interval 1971-1984, a total of 97 cases of rabies in rodents were recorded, but from 1995 to 2000 approximately 52 cases were reported in large rodents annually (2002) . the rodents involved were woodchucks and beavers, both species presumably large enough to survive the chance encounter and attack by a wild rabid carnivore such as the raccoon, skunk, fox, or feral cat. earlier literature on the federal republic of germany summarized findings from 1961 to 1967, which indicated that three mice, one rat (species not given), nine norway rats, and three muskrats were infected with rabies and had bitten humans (scholz and weinhold 1969) . despite rare reports of this nature, rodents are not a proven source of rabies transmission to humans (johnson 1989 ). all mammals are generally regarded as susceptible to rabies. in humans, the course of the disease proceeds through several phases: incubation, prodromal, acute neurological, coma, and rarely, recovery (johnson 1989) . the incubation period varies from 9 days to over 8 months. during the prodromal stage lasting 2 to 4 days, patients experience a period of apprehension and develop headache, malaise, and fever. an abnormal, indefinite sensation at the site of a prior animal bite wound is the first specific symptom. patients also may develop intermittent periods of excitation, nervousness, or anxiety interspersed with quiet periods when the mental state appears normal. further progression of the disease involves paresis or paralysis, inability to swallow, and the related hydrophobia, delirium, convulsions, and coma. rabies produces an almost invariably fatal acute viral encephalomyelitis, with death due to respiratory paralysis. adult mice used experimentally in rabies studies usually exhibit clinical signs between 5 and 15 days following inoculation and die within 5 days of the onset of clinical signs coinciding with the period of viral shedding. clinical signs in the mouse consist of muscular tremors, incoordination, excitation, or paralysis. certain rabies virus isolates from skunks produce a spastic paralysis in adult mice followed by recovery in a high percentage of infected mice. also, infant mice inoculated with certain strains of street rabies virus are capable of full recovery (johnson 1989 ). personnel working with mice experimentally infected with rabies virus should adhere to the well-established and detailed procedures that have been described in other sources for animal inoculation, husbandry, and tissue harvest procedures (johnson 1989) . vaccination of personnel involved in rabies studies with laboratory animals also is clearly indicated, regardless of the animal species involved. four other viruses that produce natural infections in the mouse have been implicated previously or are known to be infectious for humans. these include hantavirus, sendai virus, reovirus 3, and mouse hepatitis virus. for none of these viral agents is there documented evidence of zoonotic transmission of the agent from naturally infected laboratory mice to personnel in the laboratory. hantaviruses are zoonotic viruses comprising at least 22 species that are maintained among natural rodent reservoirs, despite the presence of neutralizing antibody in the rodent host (elliott et al. 2000; meyer and schmaljohn 2000) . approximately half of the hantaviruses are known human pathogens producing virus-specific patterns of disease that include hantavirus pulmonary syndrome, hemorrhagic fever with renal syndrome, and its benign form, nephropathia endemica (mills and childs 1998) . although mus musculus can exhibit the pattern of persistent hantavirus infection in the presence of neutralizing antibody when induced experimentally (araki et al. 2003) , this does not occur in natural infections of the mouse (meyer and schmaljohn 2000) . this is the likely reason that the wild mouse apparently is not important as a natural reservoir for the hantaviruses. this interpretation is supported by serological studies of wild mus musculus that detected either no or a very low incidence of serological evidence to hantavirus exposure even when other wild rodent species in the vicinity had a high level of endemic infection (kantakamalakul et al. 2003; meyer and schmaljohn 2000; pacsa et al. 2002; zuo et al. 2004) . mus musculus is used in the laboratory as an animal model to study various aspects of hantavirus infection ranging from vaccine development (choi et al. 2003) , viral pathogenicity (ebihara et al. 2000; kim and mckee 1985) , and immunological aspects of persistence (araki et al. 2003) , and it is primarily in this context that hantavirus deserves mention as a zoonotic infection in the mouse. the control of wild rodent infestations in animal facilities, quarantine and testing of wild caught rodent species during importation, and proper observance of animal biosafety guidelines for hantavirus-infected animals would be expected to virtually eliminate the risk of this zoonosis in laboratory maintained mice. mouse hepatitis virus (mhv) remains a prevalent infection in many mouse colonies where it potentially impacts colony health and disrupts experimental studies (see chapter 6). earlier studies have demonstrated that human sera contained complement-fixing and neutralizing antibodies to mhv (hartley et al. 1964) . later studies suggested that this was most likely due to cross-reactive antibodies from human cold virus infections (bradburne 1970; mcintosh et al. 1967 ). mouse hepatitis virus and the two prototype human cold viruses (oc43 and 229e) are members of the antigenic group 2 coronaviruses, and it is now known that members of this group share four, and in some cases five, structural genes that could account for this crossreactivity (navas-martin and weiss 2003). the coronaviruses have a very narrow host range and generally replicate only in the cells of the host species (navas-martin and weiss 2003). however, under unique laboratory conditions involving persistent cell culture infection, the use of mixed cell cultures of murine and of a nonpermissive species, or the use of cells possessing modified receptors, mhv has been adapted to grow in human, nonhuman primate, and hamster cells. also, the many strains of mhv in combination with use of targeted rna recombinant system have also been very useful for experimental study of the molecular basis of coronavirus pathogenicity (masters 1999) . application of the targeted rna recombinant system to mhv for exploring of emerging coronavirus infections such as sars may delineate the molecular basis for expanding of host range. these studies may warrant the reader's future attention, but at the present, mhv can be reasonably dismissed as a zoonotic infection. sendai virus was once a prevalent agent in mouse colonies but has become a rarity in most institutions. this is due to its ease of eradication through the use of temporary cessation of breeding to eliminate a naive population that is susceptible to infection and through the use of caging systems that prevent transmission (see chapter 11). sendai virus was originally isolated and described in the 1950s during the investigation of cases of human respiratory illness (gerngoss 1957; kuroya et al. 1953a kuroya et al. , 1953b sano et al. 1953; zhandoff et al. 1957 ). in the original report involving the isolation of sendai virus from japanese newborn human infants suffering from fatal pneumonitis, lung suspensions from the newborns were inoculated intranasally into mice, producing lung consolidation and death in several cases (kuroya et al. 1953b) . in later studies, sendai virus isolates were reported to produce disease in human volunteers (kuroya et al. 1953a; yamada 1956) , and reports from many countries indicated that serological evidence of sendai virus infection was associated with outbreaks of human respiratory illness (demeio and walker 1958; gardner 1957; jensen et al. 1955). tennant et al. demonstrated that personnel working with laboratory animals had antibody titers to sendai virus, but personnel with no known exposure to laboratory animals also had significant titers to the agent (tennant et al. 1967) . recombinant sendai virus is widely used for gene transfer experiments, and these vectors can readily infect human airway epithelium and a variety of other human tissues under experimental conditions (nagai 1999; pinkenburg et al. 2004) . although these recent studies have clearly demonstrated that sendai virus is capable of infecting human tissues, the initial evidence for its role as a human pathogen remains doubtful. the mice used for the early isolations of the agent may have already been endemically infected with sendai and served as the source, or it may be that other serologically cross-reactive parainfluenza viruses, which were not characterized during this era, were responsible for producing false positive reactions to sendai virus (ishida and homma 1978) . reoviruses are generally regarded as the cause of childhood infections producing asymptomatic or very mild disease, and there are few reports linking these infections with a particular disease (tsai 2000) . reovirus 3 was originally isolated from the feces of a clinically ill child (stanley et al. 1953) , and it continues to receive attention as a possible etiology for neonatal hepatitis and extrahepatic biliary atresia in infants (richardson et al. 1994; steele et al. 1995) . reovirus 3 is highly infectious for infant laboratory mice and still receives some attention in the health-screening programs for the mouse and laboratory rodent species. jacoby and lindsey (1997) reported that mouse colonies in the united states continue to have a 5 to 20% prevalence of reovirus 3 infection. although there are no confirmed reports of reovirus 3 transmission from mice (or other laboratory animal species) to humans, the laboratory mouse should be considered a possible source for this infectious agent for humans and other susceptible species. the rickettsiae are fastidious, small pleomorphic coccobacilliary organisms maintained in nature in a cycle of infection involving mammalian hosts and arthropod vectors as reservoirs (saah 2000) . in most rickettsial infections, humans serve only as an incidental host and do not contribute to the propagation of the organism in nature. such is the case for rickettsialpox, a nonfatal, self-limiting zoonotic disease caused by rickettsia akari, which is perpetuated in a cycle of infection involving mus musculus as the primary host and a mite vector (liponyssoides sanguineus). isolation of the organisms from rats (rattus) and voles (microtus) has also been reported. the first cases of human rickettsialpox were described in patients in new york city (huebner et al. 1946a (huebner et al. , 1946b , and outbreaks of the disease have generally remained clustered within large urban areas of the united states and in rural north carolina, utah, south africa, korea, and the former soviet union . according to koss et al. approximately 800 cases of rickettsialpox have been reported in the literature, with nearly 500 of these within the three years following the original description of the disease . prior to the report by koss et al. the largest case study included 13 patients accumulated over a 10-year period. the recent report by koss et al., however, included 18 patients in new york city reporting over only a 20-month period in the wake of the september 11, 2001 attacks, suggesting that perhaps the heightened sensitivities to possible bioterrorism events stimulated an upsurge in the reporting of cases as a byproduct of increased patient concerns . authors have commented on a variety of other social and demographic factors that may also be contributing to the noticeable increase in the incidence of rickettsialpox and murine typhus in the urban environment (comer et al. 1999; paddock et al. 2003) . there are no reported cases of rickettsialpox in personnel related to exposure to naturally infected laboratory mice. the mite vector l. sanguineus has not been reported in laboratory mouse colonies either historically or contemporarily. however, the tropical rat mite (ornithonyssus bacoti) can be experimentally infected with r. akari but has not been shown to play a role in the natural cycle of infection. in the authors' experience of ornithonyssus bacoti infestations of laboratory mouse or rat colonies are still seen with some frequency in facilities that have resident wild or feral mouse populations and should be addressed in the institution's pest control and infection control programs. rickettsialpox has an incubation period of 7 to 21 days following the bite of the infected mite (saah 2000 mild to severe with an abrupt onset, and it typically presents with a classic triad of an eschar, fever, and a papulovesicular rash. the papule develops at the site of the bite and later ulcerates and progresses to an eschar, 0.5 to 3 cm in diameter, as r. akari proliferates locally in the skin and vasculitis develops saah 2000) . the rash begins with firm, generally nonpruritic, erythematous papules, 2 to 10 mm in diameter, that develop into vesicles and heal by crusting. in addition to fever, patients may experience chills and headache, and less commonly, backache, myalgia, and photophobia. the disease is mild and self-limiting within 6 to 10 days, and serious complications or death have rarely been reported (saah 2000) . patients typically respond quickly after the initiation of antirickettsial therapy with tetracycline, doxycycline, or other appropriate agent saah 2000) . however, following resolution of the infection, headache and lassitude can persist for 1 to 2 weeks. the reader should refer to koss et al. for information on other rash-producing or eschar-related diseases that should be considered in the differential diagnosis to rickettsialpox . rickettsia akari are diagnosed by complement fixation tests or the more sensitive indirect immunofluorescent antibody test. serum antibody to r. akari generally takes 2 weeks to develop, and paired sera are needed to confirm a four-fold rise in antibody titer (saah 2000) . during the acute phase of the infection, immunohistochemistry or pcr analysis can be used for a rapid diagnosis on biopsy material obtained from the papulovesicular rash or eschar paddock et al. 2003) . laboratory mice infected with r. akari develop fatal pneumonia with intranasal inoculation and severe illness and death with intraperitoneal inoculation. mice develop anorexia, depression, and dyspnea. peritonitis, splenomegaly, and lymphadenitis are found upon necropsy examination. subcutaneous inoculation produces an active infection for 1 month, with organisms being recovered from the spleen but not the feces or urine (bell 1970) . the control and eradicaton of r. akari infections depend on the prevention of wild mice and the mite vector from entering laboratory animal facilities and human dwellings. murine typhus is a rickettsial disease caused by rickettsia typhi (previously r. mooseri) that occurs worldwide with epidemics or with high prevalence in particular geographic areas (dumler and walker 2000) . in the united states, most human cases of the disease are concentrated in texas and southern california. the disease is now predominantly associated with the rat as the primary host species for the oriental rat flea (xenopsylla cheopis), which serves as the principal ectoparasitic vector transmitting the disease to humans. however, the mouse can also serve as a host for this flea, as well as for the northern rat flea (nasopsyllus fasciatus) and the mouse flea (leptosylla segnis), which also bite man and can be involved in the transmission of r. typhi (flynn 1973; pratt and wiseman 1962; yunker 1964 ). an early report in the literature indicated that x. cheopis was easily established in an animal facility inhabiting rooms used for housing laboratory mice (yunker 1964) . it is now also known that the cat and opossum and the cat flea (ctenocephalides felis) can be involved in sustaining the cycle in some geographic localities (azad et al. 1997) . clark and will (1994) reported on use of the laboratory mouse as an experimental host for rearing x. cheopis, but there have been no reports of natural infestations of mouse colonies with any of the flea vectors of r. typhi for several decades. also, r. typhi has not been isolated from natural infections in laboratory mice. murine typhus is a more serious disease than rickettsialpox and presents with fever, headache, chills, nausea, and vomiting. splenomegaly, hepatomegaly, central nervous system involvement, and multiorgan failure can occur as severe and potentially fatal complications. a skin rash, which is typically maculopapular in the case of murine typhus, occurs much less commonly than in rickettsialpox, and its absence should not dissuade the clinician from making a diagnosis of murine typhus and from promptly instituting therapy due to the potential severity of the disease (dumler and walker 2000) . the methods used for the laboratory diagnosis and treatment of the disease in humans and the principles of preventing the introduction of r. typhi into laboratory animal colonies are similar to those for r. akari. leptospirosis microorganisms were discovered in 1914 when they were isolated from jaundiced patients (inada et al. 1916) ; after further study they were named in 1917 (noguchi 1918) . leptospirosis is solely a zoonotic disease of livestock, pet and stray dogs, and wildlife, including wild rodents. rodent reservoir hosts of leptospirosis include, in addition to rats, mice, field moles, hedgehogs, gerbils, squirrels, rabbits, and hamsters (fox and lipman 1991; torten 1979) . human to human transmission is extremely rare. leptospira interrogans (comprising more than 200 serovars) have been isolated worldwide (tappero et al. 2000) . l. interrogans contains 23 serogroups with strains pathogenic for amphibians, reptiles, and mammals including humans. serovars australis, ballum, bataviae, hardjo, grippotyphosa, icterohemorrgagiae, javanica, and pomona are associated with rodent infections. leptospira serovars, including l. australis, bataviae, grippotyphosa, hebdomidis, icterohaemorrhagiae, pomona, and pyrogenes, are found in the house mouse (torten 1979) . leptospira ballum has also been reported from mice and is e. newcomer and james g. fox most commonly associated with zoonotic outbreaks (borst et al. 1948; friedmann et al. 1973; stoenner and maclean 1958) . although particular serovars usually have distinct host species, most serovars can be carried by several hosts. leptospira are well adapted to a variety of mammals, particularly wild animals and rodents. in the chronic form, the organism chronically infects the host and is shed in the urine inconspicuously for long periods of time. rodents are the only major animal species that can shed leptospires throughout their lifespan without clinical manifestations (fox and lipman 1991; torten 1979) . active shedding of leptospires by rodents can go unrecognized until personnel handling the animals become clinically infected or are infected by exposure to water or food contaminated by urine. rats and mice are common animal hosts for serotype, l. ballum, although it has been found in other wildlife as well. water can often be contaminated with infected rodent urine. the infection can persist unnoticed in laboratory rodents, though their carrier rates for laboratory-maintained rodents in the united states are unknown, but it is probably low. the organism is not routinely screened on health surveillance protocols for mouse colonies; however, there was a report of leptospirosis in 1984 in a research colony of mice in the united states being housed in a large research institution (alexander 1984) . l. icterohaemorrhagiaes antibody when compared to children living in the detroit suburbs. therefore, children living in rodent-infested tenements may be at increased risk of infection (demers et al. 1983) . in europe and more recently in north america, rodents including house mice have provided a source of leptospira infection for swine and by extension could also infect personnel working in swine production units (galton 1966; smith et al. 1992) . leptospira interrogans serovar bratislava is commonly reported in these mice. the disease may vary from unapparent infection to severe infection and death. a self-limited systemic illness is seen in approximately 90% of infected humans. the incubation period is usually 5 to 14 days. individuals infected with leptospira experience a biphasic disease (faine 1991; sanger and thiermann 1988; stoenner and maclean 1958) . they become suddenly ill with weakness, headache, myalgia, malaise, chills, and fever and usually exhibit leukocytosis. during the second phase of the disease, conjunctival suffusion and a rash may occur. upon examination, renal, hepatic, pulmonary, and gastrointestinal findings may be abnormal. intravenous penicillin is the drug of choice in treating early-onset and late-stage leptospirosis infection (faine 1991; taber and feigin 1979; watt et al. 1988 ). ampicillin and doxycycline also have been effective in treating people with mild to moderate forms of leptospirosis. because leptospirosis in humans is often difficult to diagnose, the low incidence of reported infection in humans may be misleading. from 1974 to 1979, only 498 cases were reported, for an incidence of 0.05 per 100,000 people per year (sanger and thiermann 1988) . leptospirosis was removed from the reportable disease category in the united states in 1995 because of the small number of cases reported. outbreaks have been documented in the united states from personnel working with laboratory mice (barkin et al. 1974; stoenner and maclean 1958) . in one study, 8 of 58 employees handling the infected laboratory mice (80% of breeding females were excreting l. ballum in their urine) contracted leptospirosis (stoenner and maclean 1958) . infection with leptospira most frequently results from handling infected animals (contaminating the hands with urine) or from aerosol exposure during cage cleaning (barkin et al. 1974; friedmann et al. 1973; stoenner and maclean 1958) . skin abrasions or exposure to mucous membranes may serve as the portal of entry. all secretions and excretions from infected animals should be considered infective. in one instance, a father apparently was infected after his daughter used his toothbrush to clean a contaminated pet mouse cage (boak et al. 1960) . rodent bites can also transmit the disease (looke 1986 ). in detroit, children from the inner city had a significantly higher because of the variability in clinical symptoms and the lack of pathognomonic pathologic findings in humans and animals, serologic diagnosis or actual isolation of leptospires is imperative (faine 1991) . as an aid to diagnosis, leptospires can sometimes be observed by examination or direct staining of body fluids or fresh tissue suspensions (sulzer et al. 1968 ). the definitive diagnosis in humans or animals is made by culturing the organisms from tissue or fluid samples, or by animal inoculation (particularly in 3-to 4-week-old hamsters) and subsequent culture and isolation. culture media with long-chain fatty acids with 1% bovine serum albumin are routinely used as a detoxicant (faine 1991) . serologic assessment is accomplished by indirect hemagglutination, agglutination analysis, complement fixation, microscopic agglutination, and fluorescent antibody techniques (faine 1991) . the serologic test most frequently used is the modified microtiter agglutination test. titers of 1:100 or greater are considered significant. molecular techniques including pcr and randomly amplified polymorphic dna fingerprinting are used for identification of serovars (tappero et al. 2000) . in mouse colonies infected with l. ballum, antibodies against l. ballum were detected in sera of mice of all ages, but 26. zoonoses and other human health hazards 727 leptospires could be recovered only from mature mice. progeny of seropositive females had detectable serum antibodies at 51 days of age but not at 65 days. it was also reported that progeny of seropositive female mice, which possessed antibody at birth and acquired additional antibody from colostrums, remained free of leptospires if isolated from their mothers at 21 days of age, despite exposure during the nursing period (stoenner 1957) . studies in mice experimentally infected with l. grippotyphosa demonstrated that maternal antibodies, whether passed through milk or placental transfer, conferred protection of long duration against the carrier state and shedding of leptospires. thus, serologically positive immune mothers do not transmit the disease to their offspring. however, mice born to nonimmune mothers, if infected at 1 day postpartum, become carriers with no trace of antibodies. thus a population of carrier pregnant mice without antibody could serve as a precipitator in outbreaks among susceptible mouse populations (birnbaum et al. 1972) . field surveys have supported this data in that a significant percentage of carrier mice do not have antibodies. this led to the diagnostic approach, which specifies that both serologic and isolation methods must be utilized to determine the rate of leptospiral infection in rodents (galton et al. 1962) . leptospira ballum is frequently found in the common house mouse (m. musculus) (brown and gorman 1960; yager et al. 1953) . therefore, eradication of infected colonies, use of surgically derived and barrier-maintained mice or of conventional laboratory mice free of leptospira infection, coupled with the prevention of ingress of wild rodents, should effectively preclude introducing of the organism into research and commercial laboratories (loosli 1967) . leptospira ballum has been eliminated from a mouse colony by administration of feed containing 1000 gm chlorotetracycline hydrochloride per ton for 10 days. after 7 days of antibiotic therapy, mice were transferred to clean containers and administered clean water, both having been sterilized by steam. mouse traps and rodenticides were used to destroy escaped mice and to prevent reintroduction of l. ballum by the common house mouse . commercial animal colonies maintained in research vivarium today are not routinely screened for leptospirosis, assuming that the organism has been effectively eliminated from commercial and research-maintained mice. rat bite fever can be caused by either of two microorganisms: streptobacillus moniliformis or spirillum minus. streptobacillus moniliformis causes the diseases designated as streptobacillary fever, streptobacillary rat bite fever, or streptobacillosis. haverhill fever and epidemic arthritic erythemia are diseases associated with ingestion of water, food, or raw milk contaminated with str. moniliformis. sodoku, derived from the japanese words for rat (so) and poison (doku), spirilosis, and spirillary rat bite fever are caused by another bacterium, spirillum minus. the bite of an infected rat is the usual source of infection. in some cases, other animal bites, including mice, gerbils, squirrels, weasels, ferrets, dogs, and cats, or rare traumatic injuries unassociated with animal contact, cause the infection. in a retrospective analysis coveting three decades of 45 s. moniliformis isolates (91% from humans) from the department of public health in berkeley, california, 50% of the isolates were from children < 9 years of age (graves and janda 2001) . in 75% of the human infections where a diagnosis was made, rat bite fever (rbf) was suspected; 83% of those suspected cases involved either known rat bite or exposure to rodents. two cases of rbf were attributed to exposurem in one case a squirrel, and in the second a mouse (graves and janda 2001). interestingly, > 9% of the cases could not be attributed to a rat bite or scratch, indicating that close contact with infected rodents can be sufficient to become infected (graves and janda 2001) . other reports have indicated that the disease can occur in individuals who have no history of rat bites, but reside or work in rat-infested areas or have pet rats with whom they have close contact (fordham et al. 1992; holroyd et al. 1988; rumley et al. 1987) . rat scratches can also be the source of the organism (edwards and finch 1986; shanson et al. 1985) . exposure to cats and dogs that prey on wild rodents may also be the source of the organisms. these organisms are present in the oral cavity and upper respiratory passages of asymptomatic rodents, usually rats (wilkins et al. 1988 ). mice can be infected with resulting morbidity and mortality due to arthritis and pneumonia. in one study, streptobacillus moniliformis was isolated as the predominant microorganism from the upper trachea of laboratory rats (paegle et al. 1976) . presumably the incidence of str. moniliformis is now lower in high-quality, commercially reared specific pathogen-free rats. surveys in wild mice indicate 0 to 25% infection with spirillum minus (hull 1955) . (2004). streptobacillus moniliformis incubation varies from a few hours to 2 to 10 days, whereas spirillum minus incubation ranges from 1 to 6 weeks (table 26-1) . fever is present in either form. inflammation associated with the bite and lymphadenopathy are frequently accompanied by headache, general malaise, myalgia, and chills (arkless 1970; cole et al. 1969; gilbert et al. 1971; mcgill et al. 1966) . the discrete macular rash that often appears on the extremities may generalize into pustular or petechial sequelae. arthritis occurs in 50% of all cases of s. moniliformis but is less common in spirillum minus. streptobacillus moniliformis may be cultured from serous to purulent effusion, which is recovered from affected larger joints. a total of 18 cases of endocarditis due to s. moniliformis were reported from 1915 to 2000 (shvartsblat et al. 2004 ). death has occurred in cases of s. moniliformis involving preexistent valvular disease or as a result of endocarditits in a previously healthy individual. infants can also die of the infection (sens et al. 1989) . if antibiotic treatmentmusually penicillin at doses of 400,000 to 600,000 daily for 7 daysmis not instituted early, complications such as pneumonia, hepatitis, pyelonephritis, enteritis, and endocarditis may develop (richter 1954 ). if endocarditis is present, the penicillin should be given parenterally at doses of 15 to 20 million units daily for 4 for 6 weeks. streptomycin and tetracyclines are also effective antibiotics for those individuals with penicillin-associated allergies. addition of streptomycin to standard therapy is also advised in cases where isolates of sir.. moniliformis are cell wall deficient (rupp 1992) . spirillum minus does not grow in vitro and requires inoculation of culture specimens into laboratory animals, with subsequent identification of the bacteria by dark-field microscopy. streptobacillus moniliformis grows slowly on artificial media but only in the presence of 15% blood and sera, usually 10% to 20% rabbit or horse serum incubated at reduced partial pressures of oxygen. sodium polyanethol sulfonate sometimes found in blood-based media because of its properties as a bacterial growth promoter should not be used due to its inhibitory effects on sir. moniliformis (lambe et al. 1973; shanson et al. 1985) . growth on agar consists of 1 to 2 mm gray, glistening colonies. the api zym diagnostic system can be used for rapid biochemical analysis and diagnosis. a pcr-based assay has also been described to diagnose sir. moniliformis (berger et al. 2001) . the genus salmonella are gram-negative bacteria with approximately 2000 serotypes. nontyphoidal salmonellosis is caused by any of these serotypes. other than salmonella typhi, the causative agent of typhoid fever, salmonellosis occurs worldwide and is important in humans and animals. s. typhi and salmonella choleraesuis have only one serotype, whereas the remaining 2000 serotypes are within the species salmonella enteritidis. references to the salmonella enteritidis serotypes are abbreviated such that "enteritidis" is dropped; for example, s. enteritidis serotype typhimurium is called salmonella typhimurium. salmonella typhimurium is the serotype most commonly associated with disease in both animals and humans. other serotypes most commonly reported from humans and animals are salmonella heidelberg, salmonella agona, salmonella montevideo, and salmonella newport. salmonellae are pathogenic to a variety of animals. salmonella are ubiquitous in nature and are routinely found in water or food contaminated with animal or human excreta. fecal-oral transmission is the primary mode for spreading infection from animal to animal or to humans. transmission is enhanced by crowding and poor sanitation. during the early 1900s, rodenticides containing live cultures of s. enteritidis were distributed on a large-scale basis by commercial and public health organizations in an attempt to eliminate feral rats. these cultures were known as "rat viruses" and were widely used in europe, england, and the united states as "rat poisons" (weisbroth 1979). however, enthusiasm for their use waned when it was discovered that the spread of the organisms couldn't be limited; predictably, the baiting program was implicated in several epidemics among exposed human populations (weisbroth 1979) . surprisingly, as late as the 1950s in england, s. enteritidis (serovar danzy) was isolated from adults living four miles apart. the source of infection was traced to contaminated cakes from a local bakery. mice that had acquired the (brown and parker 1957) . as with other fecal-oral transmitted diseases, control depends on eliminating contact with feces, food, or water contaminated with salmonella or animal reservoirs excreting the organism. salmonella survive for months in feces and are readily cultured from sediments in ponds and streams previously contaminated with sewage or animal feces. fat and moisture in food promote survival of salmonella. pasteurization of milk and proper cooking of food (56~ for 10 to 20 minutes) effectively destroys salmonella. municipal water supplies should be routinely monitored for coliform contamination (pavia and tauxe 1991) . clinical signs of salmonellosis in humans include acute sudden gastroenteritis, abdominal pain, diarrhea, nausea, and fever. diarrhea and anorexia may persist for several days. organisms invading the intestine may create septicemia without severe intestinal involvement; most clinical signs are attributed to hematogenous spread of the organisms. as with other microbial infections, the severity of the disease relates to the organism's serotype, the number of bacteria ingested, and the host's susceptibility. in experimental studies with volunteers, several serovars induced a spectrum of clinical disease ranging from brief enteritis to serious debilitation. incubation varied from 7 to 72 hours. cases of asymptomatic carriers, persisting for several weeks, were common (hull 1955) . salmonella are flagellated, nonsporulating, aerobic gramnegative bacilli that can be readily isolated from feces on selective media designed to suppress bacterial growth of other enteric bacteria. salmonella serotyping requires antigenic analysis (fox 1991) . salmonella gastroenteritis is usually mild and self-limiting. with careful management of fluid and electrolyte balance, antimicrobial therapy is not necessary. in humans, antimicrobial therapy may prolong rather than shorten the period that salmonella are shed in the feces (nelson et al. 1980; pavia and tauxe 1991) . in one double-blind placebo study of infants, oral antibiotics did not significantly affect the duration of salmonella carriage. bacteriologic relapse after antibiotic treatment occurred in 53% of the patients, and 33% of these suffered a recurrence of diarrhea, whereas none of the placebo group relapsed (nelson et al. 1980 ). tick-borne relapsing fever occurs primarily in foci in the western part of the united states, as well as other parts of the world. the disease is caused by at least 15 borrelia species and is transmitted to humans from a variety of rodents (chipmunks, squirrels, rats, mice, prairie dogs, hedgehogs) via soft ticks of the genus ornithodorus. of recent interest are the increasingly recognized enterohepatic helicobacter spp., which cause both hepatic and intestinal disease in mice (whary and fox 2004) . one of these, h. bilis, isolated routinely from mice, has been found using pcr-based assays in bile and gallbladder of chilean patients with chronic cholecystitis and in biliary cancers in japanese patients (fox et al. 1998; matsukura et al. 2002) . whether these new helicobacters will be linked to zoonotic transmission from wild or laboratory rodents will require further studies. pathogenic staphylococcus aureus of human phage type can cause clinical disease in mice and rats. this organism has been (davies and shewell 1964) 1 lab worker % not determined, alopecia, increased scaling on head (booth 1952) and back, 10 mice 1 bacteriologist 60 of 400, crusted or crustless plaques, circular with (cetin et al. 1965 ) prominent periphery; general alopecia; mortality in some mice 1 technician 20% colony with alopecia and scaly skin (dolan et al. 1958 ) 1 technician alopecia with crusting an erythema (povar 1965) introduced into spf barrier-maintained mouse colonies and spf rats and guinea pigs; the same phage type was isolated from their animal caretakers (davey 1962; shults et al. 1973) . colonization by normal s. aureus strains in the nasopharyngeal area of humans presumably minimizes the zoonotic potential of animal-originated s. aureus. as reviewed comprehensively in blank, reports of ringworm (favus) in the mouse began to appear in the european literature in the mid-nineteenth century and in the north american literature during the early twentieth century (blank 1957) . several of the early authors noted the similarities between the lesions of favus in the mouse and in humans. quincke, who is generally credited with isolating the causative agent which he named 3~-pilz (now trichophyton mentagrophytes), suggested that the infection in the mouse was also a source of infection of the cat, and thereby, of humans. earlier reports of murine ringworm referred to the causative agent as t. quinckeanum, but the successful crossing of t. quinckeanum with the perfect state of t. mentagrophytes, arthroderma behamiae, indicates that t. quinckeanum is not a distinct species (ajello et al. 1968) . a later study of the two varieties, t. mentagrophytes var. mentagrophytes and t. m. var. quinckeanum, noted that the conidia from both produced two morphological variants on cultivation (granular and fluffy), and these variants were a. behamiae type + and pathogenic (hejtmanek and hejtmankova 1989) . in addition to t. mentagrophytes, epidermophyton floccosum, mircrosporum gallinae, m. gypseum, m. canis, t. erinacai, t. schoenleini, and t. (keratinomyces) ajello have been reported as zoophilic dermatophytes that can infect mice and cause ringworm in humans (dvorak 1964; krempl-lamprecht and bosse 1964; marples 1967; papini et al. 1997; refai and ali 1970) . the dermatophytes are distributed worldwide and can involve a variety of small animal host species in addition to the mouse. chmel et al. (1975) conducted field studies in a wooded farm setting in czechoslovakia and detected an overall prevalence rate of 4.4% (57 positive of 1288) for t. mentagrophytes infection in 6 of 13 species sampled; the prevalence in mus musculus was 3.4%, with mice comprising 15.8% of the infections detected. of the species that harbored the infection, all frequented the barn or granary area; the seasonal incidence was highest during the winter months when the rodent carriers were more likely to seek harborage indoors. chmel et al. (1975) also analyzed patient data and demonstrated that t. mentagrophytes was the predominant isolate from those who did agricultural work, while t. verrucosum was the main isolate from individuals who worked with farm animals. also, human t. mentagrophytes infections were most common on the hands, wrist, forearm, face, and neck, unprotected skin sites readily contaminated by fodder, litter, or other materials while working in the barns. ringworm infections associated with the handling of bags of grain in which mice had been living have also been reported (alteras 1965; blank 1957) . ringworm infection in laboratory mice is often asymptomatic, remaining unrecognized until laboratory personnel become infected. early reports in the literature indicated that the prevalence of t. mentagrophytes was 80 to 90% among some laboratory mouse stocks (davies and shewell 1964; dolan et al. 1958 ). however, these reports predated the era of modem laboratory animal colony management marked by the commercial availability of cesarean-derived, barrier-maintained rodents. moreover, the modem production practices that have been universally adopted by the industry for decades and the use of microbarrier cages with appropriate technique have further reduced the opportunity for ringworm to become a significant problem in contemporary colonies. in recognition of this fact, none of the major commercial vendors in the united states survey their colonies for dermatophyte infections as part of routine health monitoring. sporadic cases of ringworm infections in rodents have been reported in the past three decades (hironaga et al. 1981; mizoguchi et al. 1986; papini et al. 1997) . programs involved in importing mice from sources that fail to meet contemporary rodent production and husbandry practices should consider screening mice for dermatophytes during the quarantine period. the ease of transmission of dermatophytes from animals to humans is well known and is a significant health hazard. laboratory mice, as well as other laboratory animal species, can harbor dermatophyte infection, with few or no visible skin lesions transmitting the infection to unsuspecting personnel (dolan et al. 1958) . transmission can occur through direct contact with the infected animal or through indirect contact with animal bedding or other materials in the environment of the contaminated animal room. rigorous facility and equipment sanitation has long been recognized as an essential element of an effective control program and should be undertaken in conjunction with efforts to treat valuable animals or to repopulate previously contaminated areas of a facility (davies and shewell 1964; dolan et al. 1958; mizoguchi et al. 1986 ). the importance of barrier protections by donning appropriate clothing, using gloves and other personal protective equipment, and modifying work practices to minimize skin exposure to dermatophytes has also been acknowledged for the prevention of transmission (dolan et al. 1958) . when prevention methods fail, allowing the introduction of dermatophyte infection into a mouse colony, and when transmission to humans occurs, clinical cases of dermatophytosis routinely respond well either to topical or systemic antifungal therapy. *found in laboratory animals that cause allergic dermatitis or from which zoonotic agents have been recovered in nature (see yunker 1964) . **wee, western equine encephalitis. +sle, st. louis equine encephalitis. ++rmse rocky mountain spotted fever. dermatophytosis or ringworm in humans can be asymptomatic and minor, often self-limiting and drawing little attention from the affected individual. the infection usually causes an expanding, scaly and erythematous inflammatory plaque on the skin that occasionally contains fissures or vesicles when severely eczematous. on the trunk and extremities, the lesion may consist of one or more circular lesions with a central clearing and sharply defined margins, forming a ring, and hence the name "ringworm" (fig. 26-1) (merlin et al. 1994) . other dermatophytes are named according to the sites of involvement on the body (e.g., tinea pedis for foot infections, tinea capitis for scalp infections). the dermatophyte infections of humans associated with direct or indirect contact from mice usually involve the body or extremities, especially the arms and hands. zoophilic t. mentagrophytes infection usually produces a highly inflammatory lesion and often undergoes rapid resolution. however, it can also produce furunculosis--deep infection of the hair follicles or widespread tinea corporism which is also seen in infections of e. f l o c c o s u m . in a laboratory-acquired infection with t. (keratinomyces) ajelloi, mice were the source of infection for a laboratory technican who developed small, grayish-white, scaly lesions on both hands. hand lesions yielded the organism, as did 2 of 250 apparently health mice (refai and ali 1970) . a. tapeworms a. reservoir and incidence although this parasite occurs in the mouse intestine, it is more commonly associated with rats and is especially common in wild norway (rattus norvegicus) and black (rattus rattus) rats throughout the world (faust and russell 1970; stone and manwell 1966; wardle and mcleod 1952 (voge and heyneman 1957) . larval development in tribolium sp. at 30~ requires 8 days. therefore, humans become infected only through ingestion of infected insects, such as flour beetles, which may contaminate rodent food or cereal marketed for human consumption. c. clinical signs the infection in humans is usually asymptomatic, but in moderate to heavy infections it may cause headaches, dizziness, abdominal discomfort, and diarrhea. the greatest length of an adult parasite removed from a patient was 1 meter. usually, adult parasites are 20 to 50 cm long and as much as 4 mm wide (markell et al. 1999 ). a. reservoir and incidence the dwarf tapeworm is a common parasite of both the wild house mouse and the laboratory mouse. as indicated earlier in the text, in most well-managed mouse colonies, r. nana incidence is low compared to earlier reports of its high incidence in rodent colonies (wescott 1982) . the estimate that 20 million humans in the world are infected was made many years ago but probably is an underestimate (markell et al. 1999) . surveys conducted in central europe report that this tapeworm in humans is more prevalent in warm than in temperate regions. an incidence of 10% has been noted in some south american countries (jelliffe and stanfield 1978) . it is most commonly diagnosed in children. diagnosis is made by observing characteristic eggs in the feces. b. mode of transmission r. nana is unique among tapeworms in that the adult worm develops after the egg is ingested. the hooked oncosphere then invades the intestinal mucosa and develops into a cysticeroid larva. rodentolepsis nana eggs can contaminate hands, be trapped on particulate matter, or be aerosolized, and then accidentally ingested. since no intermediate host is required, the eggs are readily infective for the reciprocal hosts (faust and russell 1970) . precautions against infection include strict personal hygiene, appropriate laboratory uniforms, and use of disposable gloves and face masks when handling contaminated bedding and feces. c. clinical signs the clinical picture of r. nana infection is quite cosmopolitan. in well-nourished persons, essentially no symptoms occur; the infection is noted when the proglottids or ova are seen in the stool. in other persons, the symptoms include headaches, dizziness, anorexia, inanition, pruritis of the nose and anus, periodic diarrhea, and abdominal distress. a tapeworm identified as r. nana was found in a tumor removed from the chest wall (jelliffe and stanfield 1978) . the diagnosis is based on identification of the characteristic eggs or proglottids in the stool. d. treatment praziquantfel, given orally in a single dose of 25 mg/kg body weight is the drug of choice. alternatively, niclosamide is given daily for 5 days because of the tissue phase of the parasite. the dose is 2 gm for adults and 1.5 gm for children > 34 kg, and 1.0 gm for children between 11 and 34 kg (markell et al. 1999) . recently, a parasite known to naturally colonize mice, r. microstoma, has been identified in the feces of humans living in the northwest of western australia (macnish et al. 2003) . although r. nana was the most common enteric parasite based on microscopic examination of feces, r. microstoma was identified as a mixed infection in 4 of 11 individuals by using a molecular-based assay consisting of restriction fragment length polymorphism of tapeworm dna as well as a sequencing of the pcr product of the internal transcribed spacer 1 region of ribosomal dna (macnish et al. 2002) . given that r. microstoma requires an intermediate host, tribolium confusum for its life cycle, it is understandable why it was not as common as r. nana in this study. however, given the morphological similarities of the eggs of r. nana and r. microstoma, the true prevalence of r. microstoma in humans won't be known until molecular techniques to differentiate the two species are utilized in future studies. syphacia obvelata is an ubiquitous parasite in both wild and laboratory mice. although parasitology texts report that syphacia is infectious to humans, this citation originates from a publication in 1919, in which two s. obvelata adult worms and eggs reportedly were found in the formalin preserved feces of a filipino child whose entire family of five was infected with h. nana (riley 1919) . no mention is made of the method of collecting the feces, nor is it known whether the feces could have been contaminated with murine feces or with the parasite and/or eggs. the only other report is an unpublished finding of s. muris eggs in the feces of two children and two rhesus monkeys, cited in a personal letter from dr. e. e. faust of tulane university, dated january 6, 1965 (stone and manwell 1966) . both of these cases may therefore be examples of spurious parasitism, but definitive information for that conclusion is lacking. regardless, no published information indicates that laboratory personnel have been infected by working with syphacia-infected mice. contamination of food or utensils or accidental ingestion of syphacia ova (e.g., via contaminated hands) could result in infection of humans. people working with infected mice probably ingest ova occasionally, but there is no evidence that this exposure results in active infection. because syphacia infection in humans has not been described, clinical signs have not been noted. there are striking differences in size between specimens of female s. obvelata and those of enterobius vermicularis, the pinworm, in humans (markell and voge 1965) . syphacia is 3.5 to 5.8 mm long, whereas the enterobius sp. female reaches a length of 8 to 13 mm. the male syphacia sp. measures 1.1 to 1.5 mm compared to 2.5 mm for enterobius. the size difference between the eggs of the two species is also marked: syphacia eggs are more than twice as long (125 gm versus 52/am as those of enterobius). it is unlikely therefore that syphacia sp. would be misdiagnosed as enterobius sp., assuming, of course, that the observer was aware of the size difference and measured the eggs. although many species of mites are found on laboratory mice, only ornithonyssus bacoti, the tropical rat mite, and liponysoides sanguineus, the house mouse mite, are vectors of human disease. ornithonyssus bacoti is seen in laboratory mice (fox 1982) ; l. sanguineus has been identified only on wild mice. bites from these mites, as well as from another mouse mite, haemalaelaps casalis, are responsible for allergic dermatitis, or local inflammation, in humans. ornithonyssus bacoti can be found on many rodents; the brown norway rat and the black roof rat are probably the primary host species (beaver and jung 1985) . since the time of the first report of human ornithonyssus bacoti-associated dermatitis in australia in 1913, and a 1923 report in humans in the united states, many other cases have continued to be described throughout the world (see table 26 -4) (charlesworth and clegern 1977; chung et al. 1998; dove and shelmire 1931; dowlati and maguire 1970; engel et al. 1998; fox 1982; haggard 1955; hetherington et al. 1971; riley 1940; theis et al. 1981; wainschel 1971; weber 1940) . ornithonyssus bacoti is an obligate bloodsucking parasite, usually tan but red when engorged with blood. both the male and female feed on a rodent as their preferred host. the female is 700 ~tm to 1 mm in length; the male is smaller (fig. 26-2) . eggs are laid in bedding or wall crevices by the female, which survives for about 70 days and feeds about every two days during this period. the mite has five developmental stages: adult, egg, nonfeeding larva, bloodsucking protonymph, and nonfeeding deutonymph. after feeding, the adults and protonymphs leave their host and seek refuge in cracks and crevices. the life cycle from adult to egg requires 7 to 16 days at room temperature. unfed protonymphs have survived for 43 days (brettman et al. 1981) . the mite often gains access to the premises on wild rodents and lives in crevices. if wild rodents are not readily available or are captured, the mite will seek blood elsewhere, either from the wild or laboratory rodent (if in an animal research facility) and/or humans. in some infestations, the rodent shows no clinical signs. however, in more chronic cases, dermatitis and anemia may develop. historically, this mite has been a troublesome parasite in certain laboratory animals, especially rats, mice, and hamsters (fox 1982; keefe et al. 1964 ). a. clinical signs tropical rat mites produce painful, pruritic lesions in humans. examination of patients often discloses papular lesions on the wrists, arms, abdomen, and chest. raised erythematous papules and nodules several millimeters to greater than 1 cm in size occur singly or in linear configuration ( fig. 26-3) . epidemiologically, cases usually occur in clusters that involve a common source of exposure to the mite. experimentally, cases have been shown to be a vector of pathogens. in the laboratory, mite transmission of various rickettsial species, pasteurella tularensis, and coxsackie virus between different laboratory animals has been shown (hopla 1951; petrov 1971; philip and hughes 1948; schwab et al. 1952) . affected individuals may be treated with topical lindane or treated symptomatically, given that the mite does not reside on humans for any extended periods. papular dermatitis will regress after a period of 7 to 10 days post-therapy. recurrence of ornithonyssus bacoti infestations is common unless the premises have been treated with an appropriate insecticide, and any feral rodents eradicated (engel et al. 1998 ). fleas are seldom found in laboratory mice but are common parasites of feral rodents. the oriental rat flea, xenopsylla cheopis, and another flea, nasopsyllus fasciatus, both naturally infest mice and rats; they are vectors for murine typhus. apparently, x. cheopis is easily established in animal facilities. at a midwestern u.s. university, it inhabited a room housing laboratory mice where, on two separate occasions, the flea caused distress by biting students (yunker 1964) . leptopsylla segnis, the mouse flea, bites humans and is a vector for plague and typhus, serious diseases in humans. also, l. segnis can serve as an intermediate host for the rodent tapeworms r. nana and r. diminuta, which can infect people. the flea's bite can also be irritating and cause allergic dermatitis. epidemiological perspective on the transmission of infectious diseases, principally rabies. the bite of the rat is far more powerful and more likely to be disfiguring than that of the mouse, and rat bites are known to be associated with the transmission of bacterial zoonoses such as rat bite fever and leptospirosis (see elsewhere in this chapter). one should assume that the mouse is also capable of transmitting these agents via bite. rabies transmission from small rodents in the wild occurs but is exceedingly rare (gdalevich et al. 2000) ; therefore, rabies is of concern only if experimental studies with the virus are being conducted in mice. mice can also transmit hantavirus infection and lymphocytic choriomeningitis virus infection via bites. anecdotally, most animal care and use programs report that rodent bites among personnel are a reasonably common occurrence that often are unreported to an institution's occupational medical service, despite the fact that bites inflict pain, produce anxiety, and may have significant health consequences. in addition to the hazard of zoonotic disease or local wound infection with pyogenic or toxin producing bacteria such as clostridium tetani, staphylococcus spp., streptococcus spp., escherichia coli, and bacillus subtilis, rodent bites, including those of the mouse, can induce a severe local allergic reaction or anaphylaxis in individuals previously sensitized to allergen (hesford et al. 1995; teasdale et al. 1983; thewes et al. 1999) . thus, bite wounds from mice should be immediately cleaned thoroughly and reported to the institutional occupational medical service to permit evaluation of the person's tetanus immunization status and need for additional local wound or other medical care. the need for additional training of bitten persons in animal handling may also be indicated. authoritative information on the incidence and impact of animal bites in the general population over the past several decades is scant, and reliable data on the incidence of mouse bites among personnel who work in laboratory animal facilities or among the general populace is lacking. there have been occasional studies on the occurrence and clinical characteristics of rat bites within urban populations, including a recent investigation of 622 bites over the period 1974-1996 that associated this phenomenon with urban blight, poverty, and unemployed populations (hirschhorn and hodge 1999) . traditionally, animal bites have received attention from the clinical perspective of wound management and complications and from the allergic skin and respiratory reactions to laboratory mice are very common in laboratory animal caretakers and technicians who work with these animals. a survey by lutsky (1987) demonstrated that three-fourths of all institutions with laboratory animals had animal care personnel with allergic symptoms. the prevalence of symptoms of laboratory animal-associated allergy (laa) among personnel working with laboratory animals has been estimated as between 10 and 46% in numerous recent studies, and among these individuals, approximately 10% are estimated to eventually proceed to the development of asthma (chan-yeung and malo 1994; eggleston and wood 1992; hollander et al. 1996; hunskaar and fosse 1990; knysak 1989; renstrom et al. 1994) . furthermore, other sources have suggested that among atopic individuals with preexisting allergic disease, up to 73% of persons exposed to lab animal allergens may eventually develop laa (committee on occupational health and safety in research animal facilities/national research council allergens 1997). the population at risk for work-related exposure to rodents was estimated at 90,000 (newill et al. 1986 ); this population has likely grown in the intervening years to the expanding populations of genetically modified mice that are used in contemporary biomedical research programs and require care. moreover, a recent study would seem to suggest that the risk of exposure to mouse allergens is not confined to those working in the laboratory animal facility environment. data analyzed from the first national survey of lead and allergens in housing in the united states demonstrated that 82% of homes of diverse types and income levels across geographic locations had evidence of mouse allergen; 57% had detectable levels on the kitchen floors specifically; and 22% had allergen concentrations greater that 1.6 ~tg/g of dust collected, a level previously correlated with the significantly increased rate of sensitization to mouse allergen (cohn et al. 2004) . the large number of staff at risk of exposure in the workplace or already presensitized, in combination with the substantial added costs to employers for the medical management, operational disruptions, and retraining efforts related to employees who develop laa and later proceed to asthma, should provide the impetus for many institutions to pay grater attention to this element of the occupational health and safety program (schweitzer et al. 2003) . the major allergen of the laboratory mouse is the mus m 1 protein, a member of the mouse major urinary proteins encoded by a multigene family consisting of approximately 35 genes (clark et al. 1984a (clark et al. , 1984b . the earlier literature on the subject of mouse allergy referred to the mouse urinary proteins (mups), whereas recent literature cites the specific protein (mus m 1) that is now known to be the primary offending allergen in the mup multigene family. the mus m 1 protein is in the lipocalin family of proteins that are produced in the saliva and liver and are excreted in the urine at levels 100 times higher than are present in mouse serum. lipocalins serve to bind small hydrophobic molecules and function biologically to transport vitamins, small volatile odorants, and pheromones conferring the characteristic odor to mouse urine (cavaggioni et al. 1999; flower et al. 1993; konieczny et al. 1997; santa et al. 1998; virtanen et al. 1999) . several studies have indicated that production of mus m 1 is under hormonal control and that the urine of male mice contains four-fold higher levels than the urine of female mice (hastie et al. 1979; lorusso et al. 1986; price and longbottom 1987) . in addition to being present in the saliva and urine, mus m 1 in the serum becomes incorporated into the pelt, conferring the allergenic property to mouse dander. the main allergens of many furred animals are structurally similar proteins within the lipocalin family, including those of the cow (bos d 2), horse (equ c 1), dog (can f 1), and rat (rat n 1) (virtanen et al. 1999) . the mus m 1 and rat n 1 lipocalin allergens, to which 90% of mouse and rat allergic individuals react, respectively, are closely related, sharing a 66% homology (clark et al. 1984a) . some have proposed that personnel exposed to laboratory animal allergens can be categorized into four basic risk groups based on their history of allergic disease and sensitization to animal proteins (committee on occupational health and safety in research animal facilities/national research council allergens 1997). these risk groups are (1) normal individuals, (2) atopic individuals with preexisting allergic disease, (3) asymptomatic individuals with ige antibodies to allergic animal proteins, and (4) symptomatic individuals with clinical symptoms upon exposure to animal allergens. individuals in the normal risk group do not have a history of allergic disease, and 90% will never develop symptoms of laa. if laa develops in individuals in the normal risk group, it usually appears during the first three years of exposure. however, infrequently individuals in this risk group who have remained free of laa for 10 or more years of exposure have developed a delayed onset of the condition (department of health and human services, national institute of occupational safety and health 1997). atopic individuals have a genetic predisposition for an exaggerated tendency to mount ige responses to common environmental allergens. atopic individuals have higher total levels of ige in the circulation and higher blood eosinophil counts compared to normal individuals, possibly as a result of the activation of cytokines involved in ige isotype switching, eosinophil survival, and mast cell proliferation (janeway et al. 2001 ). among atopic individuals, up to 73% of those exposed to allergenic animal proteins eventually develop symptoms (agrup et al. 1986) . in asymptomatic individuals with elevated circulating ige antibodies to animal allergens, up to 100% are at risk of developing allergic symptoms. of the individuals in risk groups that are already symptomatic for laa, approximately 33% will develop chest symptoms and 10% are likely to develop occupational asthma and face the prospect that continued exposure will result in permanent impairment. allergic rhinitis, allergic conjunctivitis, and contact urticaria are the most common disorders seen in laa (committee on occupational health and safety in research animal facilities/ national research council allergens 1997). clinically, allergic rhinitis and conjunctivitis present with the symptoms of sneezing, clear nasal discharge, nasal congestion, itchiness, and watery eyes. contact urticaria presenting as raised, circumscribed, erythematous lesions may also be present in laa patients who report an intense itchiness to the skin in the area of contact with the allergen. figs. 26-4 and 26-5 (fox and brayton 1982) illustrate the typical wheal and flare reaction on the skin provoked in an individual who had developed hypersensitivity to mouse urine over a period of several years and who was exposed by having a mouse with urine-contaminated feet walk over his arm (ohman 1978) . one large survey of laboratory animal workers summarized in the niosh alert (department of health and human services, national institute of occupational safety and health 1997) reported that of 5641 animal workers from 137 animal facilities, 23% developed allergic symptoms related to laboratory animals. of the workers reporting symptoms, 82% had nasal or eye symptoms, 46% had skin complaints, and 33% had asthma. patients who develop asthma as a more serious complication of laa manifest symptoms of wheezing, intermittent dyspnea or shortness of breath, cough, often nocturnal or in the early morning, and tightness of the chest. the key clinical sign in these patients is wheezing on auscultation, and physiological abnormalities include airflow obstruction, which may vary over time, bronchodilator responsiveness, and increased airway responsiveness (airway hyperreactivity) (tang et al. 2003) . though quite rare, generalized anaphylactic reactions that are potentially life threatening can occur in individuals highly sensitized to animal allergens. anaphylaxis may manifest as diffuse itching, hives, and swelling of the face, lips, and tongue. in some individuals, breathing becomes difficult owing to laryngeal edema, and others develop asthma and wheezing. laboratory animal-associated allergy is an example of the type i, ige antibody-mediated, immune reaction, and the reader should refer to other sources for a detailed discussion of the molecular mechanisms involved in developing this reaction (janeway et al. 2001 ). in the case of animal allergens, the usual route of initial exposure is airborne, although bite exposures (saliva) and direct contact with the skin can also become important in later clinical symptoms. in the type i reaction, upon exposure to antigen, which is often a protein or glycoprotein, the allergen is taken up and processed by cells of the innate and adaptive immune systems and by dendritic cells located in the mucosal-associated lymphoid cells, gut-associated lymphoid cells, and/or the dermis. the cytokine profiles of these cells favor the development of na'fve cd4 t cells into th2 cells that induce b cells to produce ige specific for the allergen. once the ige response is initiated, it can be further enhanced by basophils, mast cells, and eosinophils that also drive allergen-specific ige production (janeway et al. 2001) . ige is normally found only in low levels in the circulation because it binds to tissue mast cells and circulating basophils. in the sensitized individual, restimulation with the sensitizing allergen results in allergen binding to ige and the release of histamine and other chemical mediators from the mast cells and basophils. these mediators produce the array of clinical signs and symptoms that are characteristic of the allergy: itchiness, nasal congestion, sneezing, nasal and ocular drainage, coughing, wheezing, and shortness of breath. to establish the diagnosis of laa related to mouse exposure, the physician should begin by considering the strength of the history, physical examination findings, the temporal relationship between the patient symptoms and the environmental exposure to mice, and possibilities of alternative explanations for the patient's problems such as exposure to other potential allergens in the workplace or allergens of a nonoccupational nature. the development of clinical symptoms concomitant with or following exposure to an environment containing mice or mus m 1 laden mouse products should help in narrowing the number of allergens tested. the patient's family history of allergy is also very important to consider because atopy is a proven risk factor in developing laa (botham et al. 1995; meijer et al. 2002; venables et al. 1988 ). physical examination of the patient and clinical monitoring for the progression of allergic disease incorporate a number of approaches. pulmonary function tests such as bronchial hyperresponsiveness (in response to pharmacologic challenge with methacholine and not the specific allergen) and the forced expiratory volume in one second (fev1) are commonly used to evaluate the degree of airway impairment and the response to bronchodilators, glucocorticoids, and other therapeutic agents. radiographs may also be useful in patients with pulmonary involvement. routine laboratory tests may also aid in the characterization and management of the patient's condition, such as complete blood count and nasal smears for eosinophilia which is common in allergic individuals but also can be seen in those with perennial nonallergic rhinitis (dykewicz et al. 1998) . measurement of total serum ige has little value to the physician as an aid in distinguishing whether a particular patient has allergic disease, but it may offer some potential for the identifying of populations at risk for developing of laa as indicated in both prospective and cross-sectional studies of laboratory animal workers (hollander et al. 1996; renstrom et al. 1995) . use of the radioallergosorbent test (rast) for the detection of human ige antibodies of defined allergen specificity is also available for patient evaluation. however, the quality of the laboratory performing the in vitro rast assays, the specificity of the allergens used, and the potential for cross-reactivity are important considerations in adopting the rast as a diagnostic tool (hamilton 2003) . even when properly conducted, in vitro tests usually fail to detect a modest number of skin testpositive individuals, and on a per-test basis, skin tests have lower time and reagent costs (hamilton 2003) . clinicians agree that when properly performed, prick-puncture skin tests are generally considered the most convenient and least expensive screening method for detecting allergic reactions in most patients (demoly et al. 2003) . the valid interpretation of these tests relies on standardized allergens and methods, and negative prick-puncture tests may be confirmed by more sensitive intradermal techniques. even after falsepositive and false-negative tests have been eliminated, the proper interpretation of results requires thorough knowledge of the patient's history and physical findings. a positive skin test alone does not confirm a definite clinical sensitivity to an allergen in the asymptomatic patient but possibly predicts the onset of allergic symptoms. a positive skin test in conjunction with a history suggestive of clinical sensitivity strongly indicates the allergen as the cause of the disease (horak 1985) . strong positive skin tests along with a suggestive clinical history also correlate well with results of bronchial or nasal challenges with the antigen. the animal facility conditions and practices that contribute to mouse-associated laa as a serious and prevalent workplace hazard have received considerable study over the past several decades, enabling effective strategies for achieving control of exposures in most research animal care and use settings. in summary, these strategies involve exposure reduction through source reduction, containment of hazard through the use of modern equipment and engineering controls, and barrier protection with personal protective equipment. the mus m 1 allergen load in the environment is markedly increased when male mice are used in studies due to the fact that they excrete 4-fold higher levels of allergen in the urine than do female mice (lorusso et al. 1986 ). therefore, sources have recommended, that whenever scientifically possible, use of only female mice would be a means of reducing allergen load in the environment and minimizing the exposure of personnel (department of health and human services, national institute of occupational safety and health 1997; renstrom et al. 2001 ). furthermore renstrom et al. (2001) reported a three-fold higher rate of allergic sensitization in technicians who worked with male rodents. although this approach may be useful in a few studies, this method of source reduction would appear to have only very limited applicability across the broad scope of contemporary studies using mouse models. source reduction of mouse allergen is also achieved through reduction of animal density within an animal room (the number of animals per room volume) and through use of frequent, effective facility sanitation practices (department of health and human services, national institute of occupational safety and health 1997). the risk of exposure to mouse allergen varies by the type of animal-related activities conducted by personnel and by the type of animal housing systems and equipment containment devices employed in the use and maintenance of laboratory mice (gordon et al. 1997 (gordon et al. , 2001 schweitzer et al. 2003; thulin et al. 2002) . many studies have examined the different caging systems used for mouse housing, and the ability of each cage system design to reduce environmental allergen is well known (gordon et al. 2001; schweitzer et al. 2003) . the application of just a simple filter sheet top or fitted filter bonnet to an open cage is effective in reducing ambient allergen levels (reeb-whitaker et al. 1999) . however, studies indicate the clear superiority of individually ventilated caging (ivc) systems run under negative pressure for the purpose of controlling room allergen levels (gordon et al. 2001; reeb-whitaker et al. 1999; schweitzer et al. 2003) . gordon et al. (2001) suggested that the use of efficient negative ivc in combination with other engineering controls for allergen containment during procedures and waste processing would potentially produce a virtually allergen-free work environment. when negative pressure ivc housing is not available, the placement of cages in a hepafiltered, ventilated cabinet is effective at reducing room allergen loads (thulin et al. 2002) . gordon et al. (1997) reported that individuals who have direct contact with mice (animal technicians) have the highest exposure, followed by those who have intermittent contact with anesthetized animals or mouse tissues (scientists and necropsy technicians), followed by those with indirect contact (office workers or histology technicians). the specific animal husbandry activities that are known to result in high exposure of personnel to mouse allergen are cage-changing activities, including animal transfer, stacking dirty cages, and manual emptying of cages; handling animals directly (particularly males); and room sweeping (gordon et al. 1997) . for each of these activities, use of improved containment equipment and changes in equipment handling procedures are effective in the controlling the allergen hazard and should be encouraged. for example, use of ventilated cabinets or biological safety cabinets during cage changing and animal handling is effective in conjunction with the use of microisolation cages (gordon et al. 2001; schweitzer et al. 2003; thulin et al. 2002) . containment equipment has also been designed for the capture of airborne allergens generated when the bottom of one dirty cage is placed into the opening of another to stack the cages for transport to the cage wash area or when dirty bedding is removed prior to cage washing (gordon et al. 1997; kacergis et al. 1996) . room cleaning with a vacuum equipped with hepa filtration followed by mopping with a damp mop also aids in reducing the environmental allergen load and personnel exposure (kacergis et al. 1996) . use of personal protective equipment and dedicated work clothing for personnel involved in high-exposure activities is an important asset in reducing allergen exposure. it is important for the work clothing to remain at work, as evidenced by the finding that children of laboratory animal workers had a higher incidence of clinical signs during provocative testing, positive skin tests, and ige specific to laboratory rodents than did the children of parents who worked in other occupations (krakowiak et al. 1999) . full sleeve protection and gloves should be worn to prevent the urticarial reactions in persons who are highly sensitive to the mus m 1 allergen. personnel should also be provided with respiratory protection and eye or face protection when warranted. either filtering facepiece particulate respirators (n95 equivalent) or powered air purified respirators are effective in reducing exposure and alleviating clinical symptoms (schweitzer et al. 2003; thulin et al. 2002) . special attention must be paid to the selection and fitting of n95 filtering facepiece particulate respirators to ensure proper function (morbidity and mortality weekly report 1998). when the elimination of mouse allergen exposure in the workplace is not achieved through the use of engineering controls, work practices, and personal protective equipment, allergic reactions in persons sensitive to mus m 1 can be managed with pharmacological agents that have a long history of use for this condition. these include antihistamines, topical ~-adrenergic agents (bronchodilators), cromolyn sodium as a nasal spray, and intranasal potent glucocorticoids (austen 2004) . prophylaxis in patients with mild symptoms is often provided by topical cromolyn sodium on a continuous basis, supplemented with the intermittent use of antihistamines often at bedtime. the selection of the antihistamine has been an area of considerable discussion, and the reader should refer to casale et al. (2003) for further insights into this matter. in more serious cases, potent topical glucocorticoids may be necessary for alleviating clinical signs. immunotherapy, or hyposensitization, is typically reserved for patients who are unable or unwilling to escape the allergen provoking the response. although allergy to the dog or the cat can be ameliorated by immunotherapy (norman 1998 (norman , 2004 , the infrequent reports in the literature of immunotherapy for allergy to mice and other small laboratory animals have failed to establish the usefulness of this approach for the control of allergy to these species (sorrell and gottesman 1957; wahn and siraganian 1980) . the progress made in the past two decades in the evolution of health care systems responsible for the monitoring, control, and elimination of infectious diseases in laboratory mice as well as the advancements in the facilities, equipment, and techniques used to maintain mice in contemporary animal care and use programs, has vastly reduced the likelihood that personnel will encounter zoonotic diseases or other health hazards in the laboratory under most circumstances. continued program success in the control of mouse-associated hazards relies on the use of well-designed and maintained animal facilities, exclusion of wild rodents and other vermin, and quality control in animal care and veterinary care practices. in unique experimental situations that place personnel at a high risk of exposure to mouse-associated hazards, institutional review should ensure that procedures are carefully planned and conducted using personal protective equipment for worker safety. allergy to laboratory animals in laboratory technicians and animal keepers the relationship of trichophyton quinckeanum to trichophyton mentagrophytes alteras, i. 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exposure to laboratory animal allergens in a research laboratory viruses transmissible from laboratory animals to man leptospirosis. in crc handbook series in zoonoses orthoreoviruses. in principles and practices of infectious diseases laboratory animal allergy in a pharmaceutical company important animal allergens are lipocalin proteins: why are they allergenic? development of hymenolepis nana and hymenolepis diminuta (cestoda: hymenolepididae) in the intermediate host tribolium confusum efficacy and specificity of immunotherapy with laboratory animal allergen extracts rat mite bite the zoology of tapeworms placebo-controlled trial of intravenous penicillin for severe and late leptospirosis bacterial and mycotic diseases helminths natural and experimental helicobacter infections rat-bite fever in a gerbil breeder congenital lymphocytic choriomeningitis virus syndrome: a disease that mimics congenital toxoplasmosis or cytomegalovirus infection natural occurrence of leptospira ballum in rural house mice and in an opossum on the biological character of hvj akitsugu strain and its pathogenicity in human beings as revealed after experimental inoculation of it in volunteers infections of laboratory animals potentially dangerous to man: ectoparasites and other arthropods, with emphasis on mites influenza d in early infancy key: cord-287527-ep6ug9c3 authors: algaissi, abdullah; agrawal, anurodh s; han, song; peng, bi-hung; luo, chuming; li, fang; chan, teh-sheng; couch, robert b; tseng, chien-te k title: elevated human dipeptidyl peptidase 4 expression reduces the susceptibility of hdpp4 transgenic mice to middle east respiratory syndrome coronavirus infection and disease date: 2018-09-26 journal: the journal of infectious diseases doi: 10.1093/infdis/jiy574 sha: doc_id: 287527 cord_uid: ep6ug9c3 background: the ongoing middle east respiratory syndrome coronavirus (mers-cov) infections pose threats to public health worldwide, making an understanding of mers pathogenesis and development of effective medical countermeasures (mcms) urgent. methods: we used homozygous (+/+) and heterozygous (+/−) human dipeptidyl peptidase 4 (hdpp4) transgenic mice to study the effect of hdpp4 on mers-cov infection. specifically, we determined values of 50% lethal dose (ld(50)) of mers-cov for the 2 strains of mice, compared and correlated their levels of soluble (s)hdpp4 expression to susceptibility, and explored recombinant (r)shdpp4 as an effective mcm for mers infection. results: hdpp4(+/+) mice were unexpectedly more resistant than hdpp4(+/−) mice to mers-cov infection, as judged by increased ld(50), reduced lung viral infection, attenuated morbidity and mortality, and reduced histopathology. additionally, the resistance to mers-cov infection directly correlated with increased serum shdpp4 and serum virus neutralizing activity. finally, administration of rshdpp4 led to reduced lung virus titer and histopathology. conclusions: our studies suggest that the serum shdpp4 levels play a role in mers pathogenesis and demonstrate a potential of rshdpp4 as a treatment option for mers. additionally, it offers a validated pair of tg mice strains for characterizing the effect of shdpp4 on mers pathogenesis. middle east respiratory syndrome (mers) is an emerging infectious disease caused by a coronavirus (mers-cov), first identified in saudi arabia in 2012, that has since spread to 27 mostly surrounding countries, resulting in more than 2229 laboratory-confirmed cases of infection and 791 deaths (approximately 36%), as of june 2018 [1] . the pandemic potential of this infection calls for a better understanding of mers pathogenesis and the development of effective medical countermeasures (mcms) for humans. like other human covs, mers-cov uses an exoaminopeptidase, human dipeptidyl peptidase 4 (dpp4), as the entry receptor for infection of permissive cells [2] . dpp4, also known as cd26, is involved in many physiological functions via its ubiquitous expression in a variety of tissues, its propensity to interact with adenosine deaminase and other important regulatory molecules of the immune system, and its intrinsic proteolytic activity that cleaves many biologically active peptides or proteins that contain proline or alanine at the penultimate position [3, 4] . not only is dpp4 expressed as a type ii transmembrane glycoprotein, primarily on endothelial and epithelial cells and subsets of immune cells, but it is also present in a functionally intact soluble form (sdpp4) in the circulation and other body fluids [3, 4] . because wild type mice are not susceptible to mers-cov, we established a heterozygous (+/−) transgenic (tg) mouse model globally expressing hdpp4 for studies of mers pathogenesis and development of mcms against mers-cov infection [5, 6] . to ensure a steady and cost-effective supply of the animals, a tg mouse model with homozygous expression of hdpp4, designated hdpp4 +/+ tg mice, was developed through mating of hdpp4 +/− mice and used as breeders for generating offspring of both genotypes of hdpp4 tg mice. because hdpp4 is the functional mers-cov receptor, the doubling of encoded hdpp4 gene in hdpp4 +/+ tg mice could render them more susceptible than their hdpp4 +/− counterparts to mers-cov infection and disease. to our surprise, we found that hdpp4 +/+ mice are more resistant than their hdpp4 +/− counterparts to mers-cov infection. we subsequently found that an increased expression of functionally intact soluble hdpp4 (shdpp4) in the circulation of hdpp4 +/+ tg mice, relative to that of hdpp4 +/− mice, was associated with this increased resistance and might be, at least in part, accountable for the seemingly counterintuitive findings on susceptibility to mers-cov infection. this notion was supported by studies showing that elevated shdpp4 levels, brought about by administration of recombinant shdpp4 (rshdpp4), resulted in increased resistance of recipient hdpp4 +/− mice to mers-cov. together, our results indicate that manipulation of shdpp4 might serve as a strategy for counteracting mers-cov infection and disease in humans. hdpp4 +/− transgenic mice were established, as previously reported [5, 6] . hdpp4 +/+ breeder mice were derived by mating 2 parental hdpp4 +/− mice. the homozygosity was determined by quantitative polymerase chain reaction analysis of tail dna (data not shown) and verified by their subsequent mating with wildtype (wt) mice. only those mice uniformly yielding heterozygous offspring were selected as hdpp4 +/+ breeders. interbreeding between hdpp4 +/+ mice produced additional hdpp4 +/+ mice, whereas backcrossing them to wt mice generated hdpp4 +/− mice. all of the in vitro and animal studies involving infectious mers-cov were conducted at the biosafety level 3 (bsl3) laboratory and animal bsl3 facilities at the galveston national laboratory in accordance with approved protocols and the guidelines and regulations of the national institutes of health (nih) and association for assessment and accreditation of laboratory animal care (aaalac). detailed methodologies for viral infection, isolation from infected lungs and brain, and determination of infectious viral loads have been established and routinely used in our laboratory [5, 6] . the original stock of mers-cov emc-2012 strain, a gift of heinz feldmann (nih, hamilton, mt) and ron a. fouchier (erasmus medical center, rotterdam, netherlands), was expanded in vero e6 cells 3 times consecutively. passage 3 containing a titer of approximately 5 × 10 6 50% cell culture infectious dose (tcid 50 )/ml of virus was used throughout the study. the 50% lethal dose (ld 50 ) values for hdpp4 +/+ and hdpp4 +/− mice was determined by using traditional virus dilution assays and the reed-muench method, as we previously described [6] . briefly, groups of 4 young (6-8 weeks) or old (7-10 months) hdpp4 +/+ and hdpp4 +/− mice were inoculated, via intranasal route, with dosages of emc-2012 mers-cov in 10-fold decrements from 10 2 to 10 -1 tcid 50 in a volume of 60 µl. mice were monitored daily for clinical manifestations (weight loss) and mortality for at least 21 days postinfection (dpi). ld 50 values for each strain of mice were estimated based on the ratio of the surviving mice to the total inoculated mice, as previously described [6] . those surviving for more than 21 days were also evaluated for specific antibody responses to mers-cov receptor binding domain (rbd) protein by enzymelinked immunosorbent assay (elisa) [6] . only those showing specific antibody to rbd were considered as "mers-cov infected. " to quantify the circulating shdpp4 in the sera of naive dpp4 +/+ , dpp4 +/− , and dpp4 −/− mice, a commercial elisa-based assay was used, following the manufacturer's instructions (ebioscience catalog no. bms235). absorbance at 450 nm in 96-well plates was read in an elisa plate reader (molecular device). elisa-based and vero e6 cell-based microneutralization assays, previously described [7] , were used to determine the titers of mers-cov rbd-specific serum igg and neutralizing antibodies in hdpp4 tg mice in response to mers-cov infection. purified insect cell-derived human dpp4 ectodomain (residues 39-766; genbank accession no. np_001926.2) containing an n-terminal human cd5 signal peptide and a c-terminal his6 tag, as we previously described and characterized [7, 8] , was prepared and used for treatment studies. testing binding specificity of the rshdpp4 to the rbd proteins of mers-cov and severe acute respiratory syndrome coronavirus (sars-cov; both rbds were generous gifts of drs du and jiang at new york blood center, ny) was determined in elisa-based assays [7, 9] . for determining the capacity of rshdpp4 to inhibit mers-cov infection in vitro we initially used our standard microneutralization procedure with cytopathic effect (cpe) inhibition as the endpoint. these assays revealed a dose-dependent reduction of cpe at 72 hours, ranging from less than 5% for 100, 50, and 25 μg/ml and gradually increased to approximately 30% for 12.5 μg/ml of rshdpp4. in addition to standard microneutralization assay with a cpe endpoint, we measured the antiviral effect of rshdpp4 using virus yield of each rshdpp4 dilution from 100 to 0.8 μg/ml, expressed as log 10 tcid 50 /ml. the effect of rshdpp4 for inhibiting mers-cov infection in tg mice was determined using hdpp4 +/− mice in 2 pilot studies with 2 different batches of rshdpp4 showing similar, but not identical, binding capacity to mers-cov rbd and in vitro neutralizing activity. briefly, groups of hdpp4 +/− mice (n = 3 per group) were treated twice with either 100 µg or 400 µg of rshdpp4 or phosphate-buffered saline (pbs) as control, via the intraperitoneal route 2 hours before (−2 hours) and 24 hours after (+24 hours) infection (intranasal) with 10 3 tcid 50 of mers-cov. mice were sacrificed at 3 dpi to assess infectious viral loads and histopathology in the lungs. inflated lung specimens and brain tissues were fixed in 10% neutral buffered formalin for 48 hours before paraffin embedding and processing for routine hematoxylin and eosin stain (h&e) to assess the histopathology, as we previously described [5, 6] . statistical analyses were performed using graphpad prism software. neutralizing antibody titers and virus titers were averaged for each group of mice and compared using students t test, 1-way anova, or others as indicated. for the initial comparison of the susceptibility of hdpp4 +/+ and hdpp4 +/− mice to mers-cov, we determined the ld 50 values for mice 7-10 months of age, as we previously described [6] . because hdpp4 is the functional receptor of mers-cov, we anticipated that dpp4 +/+ mice might be more, or at least equally, permissive as dpp4 +/− mice to mers-cov infection. to our surprise, we found that hdpp4 +/+ mice were more resistant than hdpp4 +/− mice as indicated by ld 50 values of 4.3 and 32.4 tcid 50 of mers-cov for hdpp4 +/− and hdpp4 +/+ mice, respectively. to confirm this seemingly counterintuitive finding and rule out any potential effect of age and gender, we repeated the study using age-(6-8 weeks old) and sex-matched tg mice of both genotypes. shown in figure 1a is a representative of 2 independently performed experiments that confirmed the ld50 difference; values for the hdpp4 +/− and hdpp4 +/+ mice were 7.7 and 70.0 tcid 50 of mers-cov, respectively, indicating that hdpp4 +/+ mice are more resistant than their age-and sex-matched hdpp4 +/− counterparts to mers-cov infection. using sera collected 21 dpi from each strain of tg mice that survived the lower challenge dosages, we quantified mers-cov rbd-specific igg antibodies by elisa. we found that infection had occurred in tg mice of both strains. infection rates for those given 100 tcid 50 were similar (1/1 for dpp4 +/− mice and 2/2 for dpp4 +/+ mice) and 10 tcid 50 (2/2 for each strain) but were greater for dpp4 +/− mice (3/3) than dpp4 +/+ mice (1/4) for 1 tcid 50 . the difference in infection rates is consistent with the increased resistance of dpp4 +/+ mice described earlier. to further verify the difference in susceptibility to mers-cov infection, we infected (intranasal) hdpp4 +/+ (n = 9) and hdpp4 +/− (n = 11) tg mice with an equal dose of mers-cov (10 3 tcid 50 /per mouse) and monitored them daily for morbidity (weight loss) and mortality. three mice of each strain, unless indicated otherwise, were euthanized at 3, 5, and 7 dpi to assess infectious viral titers and the histopathology of lungs and brains. in contrast to dpp4 +/− mice, which exhibited marked weight loss, starting at 3-4 dpi, and 2 deaths at 6 dpi (data not shown), infected hdpp4 +/+ mice exhibited minimal weight changes and uniformly survived through 7 dpi when the experiment was terminated ( figure 1b) . when the viral loads were measured at 3 dpi, we readily recovered infectious virus from the lungs, but not the brains, of all 3 hdpp4 +/− mice examined, but from the lung of only 1 of 3 hdpp4 +/+ mice. although we usually recover virus from lungs of some mice, efforts to recover infectious virus from both lung and brain specimens of both strains of tg mice at 5 dpi were unsuccessful (data not shown); however, we were able to retrieve infectious virus from the brain (but not lungs) of the sole hdpp4 +/− survivor and from all 3 hdpp4 +/+ mice that survived to 7 dpi. the virus titer in the brain was 10 6.2 /g for the single hdpp4 +/− mouse, a titer significantly higher than the average of 10 3.7 /g for 3 hdpp4 +/+ mice ( figure 1c ). this ability to recover infectious virus from the lungs approximately 2-3 days earlier than from the brains is consistent with the pattern, kinetics, and tissue distribution of mers-cov infection in dpp4 tg mice we have previously reported [6] . we also compared the histopathology of lungs and brains, 2 of the prime targets of mers-cov infection of hdpp4 tg mice [5] . although infected dpp4 +/− mice elicited mild-to-moderate histopathological changes within the lungs at 3 dpi after a dose of 10 3 tcid 50 of mers-cov infection, as in our earlier study [6] , infected dpp4 +/+ mice exhibited reduced or no lung histopathology (data not shown). brain histopathology at 7 dpi for the sole hdpp4 +/− survivor had infiltrations of mononuclear cells in the meninges ( figure 1d , below), perivascular cuffing ( figure 1d , above and below), microglial nodules ( figure 1d , below), microhemorrhage ( figure 1d , above) and cell death at the junctions of gray and white matter ( figure 1d , above) but not in dpp4 +/+ mice. collectively, the significant differences between these 2 strains of mice in their ld 50 values, seroconversion rates, viral loads, weight loss, and histopathology support the notion that dpp4 +/+ mice are more resistant than dpp4 +/− mice to mers-cov infection. . sera of naive human dipeptidyl peptidase 4 (hdpp4 +/+ ) mice contain significantly higher levels of soluble hdpp4 (shdpp4) that exhibit higher levels of neutralizing antibody-like activity than those of naive hdpp4 +/− mice. a, groups of at least 10 age-and sex-matched hdpp4 +/+ , hdpp4 +/− , and their transgene-negative (hdpp4 −/− ) littermates were subjected to retroorbital bleeding to assess the contents of shdpp4, using commercially available elisa kits that quantify specific hdpp4 (hdpp4 +/+ vs hdpp4 +/− , p < .001 t test). b, sera obtained from naive hdpp4 +/+ and hdpp4 +/− mice (n = 10 of each) and hdpp4 −/− mice (n = 6) were subjected to the standard vero e6-based microneutralization tests to determine their potential to neutralize mers-cov. data are presented as the geometric mean neutralization titers (gmt log 2 ) of 50% neutralization titer (nt 50 ). a 1-10 dilution of sera is approximately 3.4 log 2 . (* p = .026, t test and mann-whitney rank sum test, when compared to those of hdpp4 +/− mice). the gmt titers of hdpp4 −/− mice were uniformly below the limit of detection. abbreviations: elisa, enzyme-linked immunosorbent assay; mers-cov, middle east respiratory syndrome-associated coronavirus. dotted line, limit of detection. tg mice could be different, thereby contributing to their difference in susceptibility to mers-cov infection. using a commercial elisa-based analysis, we were unable to detect shdpp4 in age-and sex-matched hdpp4-negative (hdpp4 −/− ) littermates. however, as shown in figure 2a , a representative of 2 independently performed studies, an average of 8.1 ± 0.2 μg/ml (mean ± sd) and 6.2 ± 0.4 μg/ml of shdpp4 was detected in hdpp4 +/+ (n = 16) and dpp4 +/− mice (n = 10), respectively. as shdpp4 retains its binding specificity to mers-cov rbd protein [7] , the significantly different expression of shdpp4 in the sera of these 2 strains of tg mice (p < .001) prompted us to examine if elevated shdpp4 expression might relate to the higher resistance of dpp4 +/+ to mers-cov infection by possibly acting like a decoy that binds mers-cov rbd and prevents virus infection. using microneutralization tests, we noted that the 50% neutralization titers (nt 50 ), expressed as the geometric mean titers (gmt), were 3.9 ± 0.3 and 3.1 ± 0.1 for hdpp4 +/+ and hdpp4 +/− mice, respectively (p = .026), compared to those of hdpp4 −/− mice which were uniformly below the limit of detection (i.e., ~3.0) ( figure 2b ). because the significantly higher expression of shdpp4 with better neutralizing activity might contribute to the increased resistance of hdpp4 +/+ mice to mers-cov infection, we explored whether an increased shdpp4 expression might increase resistance of naive hdpp4 +/− mice to mers-cov infection. using our limited amount of insect cell-derived rshdpp4, known to specifically bind to rbd of mers-cov but not sars-cov, and to neutralize mers-cov in vitro in a dose-dependent manner ( figure 3a and 3b), we administered 100 μg of rshdpp4, via the intraperitoneal route, into each of 3 dpp4 +/− mice 2 hours before (−2 hours) and 24 hours after (+ 24 hours) infection with 10 3 tcid 50 of mers-cov. the effect of the rshdpp4 against mers-cov infection was assessed at 3 dpi by using the titers of infectious virus within the lungs as the end point for this pilot study. while all of 3 pbs-treated mice exhibited moderate titers of live virus, we were unable to recover infectious virus from any of 3 rshdpp4-treated mice ( table 1 , experiment 1). encouraged by the preliminary data, we generated another batch of rshdpp4 shown to inhibit mers-cov infection in a dose-dependent manner ( figure 3b ) to repeat the experiment. we gave groups of 3 dpp4 +/− mice either 100 or 400 μg of rsh-dpp4, or pbs (as control) 2 hours before and 24 hours after virus infection as before. to determine if administration of rshdpp4 would increase the circulating levels of shdpp4, serum levels of shdpp4 in mice prior to and 2 hours after the first administration and before challenge with mers-cov were measured. we found that titers at 0 and 2 hours of tg mice given 100 μg (6.6 ± 0.4 versus 6.6 ± 0.9 μg/ml), were similar to those of pbs-treated mice (7.5 ± 0.8 versus 7.3 ± 1.0 μg/ml). however, mice treated with 400 μg of rshdspp4 showed significantly increased titers 2 hours after treatment (6.6 ± 0.6 increased to 13.7 ± 0.8 μg/ml; p = .002, t test). these results suggest that an increased serum level of shdpp4 can be achieved by administration of rshdpp4 in a dose-dependent manner. in addition to the viral loads within the lungs at 3 dpi, the pulmonary histopathology was examined to investigate the effect of rshdpp4 on mers-cov infection. unlike the first study in which treatment with 2 doses of 100 μg (at −2 hours and +24 hours) of rshdpp4 fully protected against mers-cov infection, we were able to recover reduced titers of infectious virus from each of 3 mice given 100 μg of the second batch of rshdpp4 when compared to those of pbs-treated controls (p = .077, elisa 96-well plates, precoated with rbd protein of mers-cov or sars-cov, were used to determine the binding specificity of rshdpp4 by using the standard elisa-based assays. absorbance was measured at a wavelength of 450 nm. b, the dose-dependent neutralizing activities of 2 different batches of rshdpp4 (rshdpp4-1 and -2) against mers-cov. the modified vero e6-based microneutralization test (virus yield) was used to quantify the neutralizing antibody-like capacity of rshdpp4, as described earlier [12] and in the methods. *p < .05; **p < .01. abbreviations: elisa, enzyme-linked immunosorbent assay; od, optical density; tcid 50 , 50% cell culture infectious dose. t test). however, as shown in table 1 , experiment 2, the titers of infectious virus in mice treated with 400 μg of rshdpp4 were significantly reduced from an average of 4.3 ± 0.3 (mean ± se) in control mice to 2.6 ± 0.1 tcid 50 /g (p = .011, t test). this finding is consistent with the reduced potency of batch 2 of rshdpp4, as shown in figure 3b . however, the histopathology in mice treated with the high dose of rshdpp4 (ie, 400 μg) was reduced as well when compared to that of the pbs controls (data not shown). in this study we found that hdpp4 +/+ mice were more resistant than hdpp4 +/− mice to mers-cov infection, as evidenced by approximately 10-fold increases of ld 50 , reduced infectious viral yields, and seroconversion rates, as well as less weight loss and lower mortality than their age-and sex-matched hdpp4 +/− counterparts ( figure 1 ). we also found that hdpp4 +/+ mice had significantly higher levels of shdpp4 in their circulation than did hdpp4 +/− mice ( figure 2 ). moreover, these higher serum levels of shdpp4 exhibited higher titers of neutralizing activity against mers-cov in vero e6 cell-based assays. finally, we showed that administration with functionally active rhsdpp4 proteins (figure 3 ) enabled hdpp4 +/− mice to better resist mers-cov infection in a dose-dependent manner (table 1) , a finding in accordance with increased levels of shdpp4 in their circulation. taken together, these results support the notion that increasing the levels of shdpp4 is a potential option for counteracting mers-cov infection and disease in humans. dpp4, ubiquitously expressed on many types of cells and tissues, has been well characterized as critically involved in regulating many important physiological functions, in part through its intrinsic enzymatic activity and propensity to interact with other key regulatory molecules of the immune system [3, 13] . as the functional receptor that mediates entry of mers-cov to permissive host cells, the membrane-associated hdpp4 plays a pivotal role in mers-cov infection and disease. however, specific role(s) that shdpp4 might have in mers pathogenesis remain much less understood. while the levels of sdpp4 vary significantly, even among healthy individuals, it has been shown that the intensities of sdpp4 expression in the circulation, along with its intrinsic enzymatic activity, could be a factor in dictating the severity of many human diseases, including malignancies, autoimmune and inflammatory diseases, diabetes mellitus and other metabolic syndromes, and chronic infectious disease such as aids and hepatitis c [4, 10, 11, 14, 15] . it has been recently reported that serum levels of sdpp4 expression in confirmed mers patients were significantly reduced when compared to those of healthy individuals [16] ; however, the suggestion that these reduced levels could serve as biomarkers for susceptibility requires knowledge regarding the levels in mers cases before onset of infection and disease. in addition, further studies of the therapeutic value of shdpp4 as either a significant resistance factor or a potential countermeasure for mers-cov in humans is warranted. of note, the soluble forms of the viral receptors for several other viruses, including those caused by sars-cov, rhinovirus, and hiv, have been proposed as potentially effective antiviral therapeutics [17] [18] [19] . we showed in this study that sera derived from naive hdpp4 tg mice of either strain, especially hdpp4 +/+ , possess detectable neutralizing antibody-like activity against mers-cov ( figure 2b ). whether the significantly higher shdpp4 expression of hdpp4 +/+ mice could be solely accountable for its greater resistance to mers-cov infection through functioning as receptor decoys seems unlikely, because it took at least 12.5 μg of rsh-dpp4, a level higher that the approximately 8 μg/ml in sera of hdpp4 +/+ mice, to significantly inhibit mers-cov infection in vero e6 cells ( figure 3b ). additional studies are needed to better understand the shdpp4-related protective mechanisms against mers-cov, especially those of the immune system. however, the validated direct correlation between the level of shdpp4 and the susceptibility to mers-cov infection, as shown in this study, may provide a possible genetic basis for the observed wide spectrum of diseases, ranging from asymptomatic, mild-to-moderate, to severe infection and death, in mers patients [20, 21] . with the limited supplies of rshdpp4, we have shown in 2 independently performed proof-of-principle studies that administration of exogenous rshdpp4 might be a treatment a mice (n = 3 each group) were given either 100 μl of pbs or pbs containing 100 or 400 μg of rshdpp4/per mouse by the intraperitoneal route 2 hours before and 24 hours after intranasal challenge with 100 ld 50 (approximately 10 3 tcid 50 ) of mers-cov. lung infectious viral titers were quantified at 3 dpi using a standard vero e6 cell-based infectivity assay. b mean ± se. c none detected (limit of detection was 2.5 log 10 tcid 50 /g). d p = .038 (t test) using 2.4 as the value for nondetectable samples. e p = .011, 1-way anova. option for mers-cov infection (table 1) . additional studies are required to determine if increasing shdpp4 levels by rsh-dpp4 treatment could be a useful treatment option for human mers. the study presented in this report demonstrates the usefulness of this homozygous and heterozygous pair of hdpp4 tg mice to fully explore the interactions between hdpp4 and mers-cov infection and disease, studies that could lead to identification of novel molecular and cellular targets for mcms against mers-cov infection and disease in humans. financial support. this work was supported by the national institute of allergy and infectious diseases, national institutes of health (grant numbers r21ai113206 to c.-t. k. t. and r01ai110700 to f.l). potential conflicts of interest. all authors: no reported conflicts of interest. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. world health organization regional office for the eastern mediterranean dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc cut to the chase: a review of cd26/dipeptidyl peptidase-4's (dpp4) entanglement in the immune system unravelling the immunological roles of dipeptidyl peptidase 4 (dpp4) activity and/or structure homologue (dash) proteins generation of a transgenic mouse model of middle east respiratory syndrome coronavirus infection and disease characterization and demonstration of the value of a lethal mouse model of middle east respiratory syndrome coronavirus infection and disease a truncated receptor-binding domain of mers-cov spike protein potently inhibits mers-cov infection and induces strong neutralizing antibody responses: implication for developing therapeutics and vaccines receptor usage and cell entry of bat coronavirus hku4 provide insight into bat-to-human transmission of mers coronavirus molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 imunomodulatory activity of dpp4 dipeptidyl-peptidase iv from bench to bedside: an update on structural properties, functions, and clinical aspects of the enzyme dpp iv immunization with inactivated middle east respiratory syndrome coronavirus vaccine leads to lung immunopathology on challenge with live virus the structure and function of cd26 in the t-cell immune response a dipeptidyl peptidase-4 inhibitor directly suppresses inflammation and foam cell formation in monocytes/macrophages beyond incretins dipeptidyl peptidase-4: a key player in chronic liver disease reduction of soluble dipeptidyl peptidase 4 levels in plasma of patients infected with middle east respiratory syndrome coronavirus susceptibility to sars coronavirus s protein-driven infection correlates with expression of angiotensin converting enzyme 2 and infection can be blocked by soluble receptor prevention of rhinovirus infection in chimpanzees by soluble intercellular adhesion molecule-1 aavexpressed ecd4-ig provides durable protection from multiple shiv challenges clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia middle east respiratory syndrome key: cord-281161-u896icp9 authors: wang, jing; tricoche, nancy; du, lanying; hunter, meredith; zhan, bin; goud, gaddam; didier, elizabeth s.; liu, jing; lu, lu; marx, preston a.; jiang, shibo; lustigman, sara title: the adjuvanticity of an o. volvulus-derived rov-asp-1 protein in mice using sequential vaccinations and in non-human primates date: 2012-05-17 journal: plos one doi: 10.1371/journal.pone.0037019 sha: doc_id: 281161 cord_uid: u896icp9 adjuvants potentiate antigen-specific protective immune responses and can be key elements promoting vaccine effectiveness. we previously reported that the onchocerca volvulus recombinant protein rov-asp-1 can induce activation and maturation of naïve human dcs and therefore could be used as an innate adjuvant to promote balanced th1 and th2 responses to bystander vaccine antigens in mice. with a few vaccine antigens, it also promoted a th1-biased response based on pronounced induction of th1-associated igg2a and igg2b antibody responses and the upregulated production of th1 cytokines, including il-2, ifn-γ, tnf-α and il-6. however, because it is a protein, the rov-asp-1 adjuvant may also induce anti-self-antibodies. therefore, it was important to verify that the host responses to self will not affect the adjuvanticity of rov-asp-1 when it is used in subsequent vaccinations with the same or different vaccine antigens. in this study, we have established rov-asp-1's adjuvanticity in mice during the course of two sequential vaccinations using two vaccine model systems: the receptor-binding domain (rbd) of sars-cov spike protein and a commercial influenza virus hemagglutinin (ha) vaccine comprised of three virus strains. moreover, the adjuvanticity of rov-asp-1 was retained with an efficacy similar to that obtained when it was used for a first vaccination, even though a high level of anti-rov-asp-1 antibodies was present in the sera of mice before the administration of the second vaccine. to further demonstrate its utility as an adjuvant for human use, we also immunized non-human primates (nhps) with rbd plus rov-asp-1 and showed that rov-asp-1 could induce high titres of functional and protective anti-rbd antibody responses in nhps. notably, the rov-asp-1 adjuvant did not induce high titer antibodies against self in nhps. thus, the present study provided a sound scientific foundation for future strategies in the development of this novel protein adjuvant. the use of an adjuvant is a key element in promoting vaccine effectiveness because it can stimulate the immune system and accelerate, prolong, or enhance antigen-specific immune responses, even when used in combination with weak vaccine antigens [1] . adjuvants have been used in vaccines since the early 20th century following more than 100 years of research. in the u.s., alum remains the sole fda-approved adjuvant for general use of vaccines [2] . however, few adjuvants have been licensed for use around the world [3] . no new adjuvants have been approved in the united states since the 1930s, and only recently has the european heads of medicines agencies licensed the mf59, as03 and as04 adjuvants for use with the fluadh, fendrix tm and cervarix tm defined vaccines, respectively [4] . cervarix tm which uses as04, a combination of aluminum hydroxide and monopho-sphoryl lipid a (mpl), in its formulation was also licensed by fda on october 16, 2009 , to prevent cervical cancer caused by human papillomavirus types 16 and 18. adjuvants are important in guiding the type of adaptive response that is induced after vaccination and that is most effective against incoming infections. the development of novel adjuvants that stimulate discrete subsets of immune cells, in particular, cytotoxic t-lymphocytes (ctl), is required to unleash the full potential of new vaccines and immunotherapy strategies [5] . although tremendous progress has been made in the development of many vaccine platforms, including dna-based vaccines, recombinant subunit vaccines, viruses and conjugates, the absence of safe and effective adjuvants impedes the clinical development of such new generations of vaccines. interest in developing new adjuvants has increased significantly over the past decade, as highlighted by the following issues [6, 7] : 1) the inability of traditional approaches to develop successful vaccines against ''difficult'' organisms such as hiv and hcv; 2) the emergence of epidemics or outbreaks of new infectious diseases with high mortality, especially those causing serious threats to public health and socioeconomic stability worldwide (e.g., sars, ebola, west nile, dengue, pandemic flu and nvcjd); 3) the re-emergence of ''old'' infections like tuberculosis; 4) the continuing spread of antibiotic-resistant bacteria; and 5) the increased threat of bioterrorism. therefore, the molecular design of potently adjuvanted vaccines that would enhance antigen uptake in vivo and potentially also simplify their adjuvant requirements would be highly desirable [8] . there are many reports showing that helminth-derived molecules have potent regulatory or stimulatory effects on the immune system of their mammalian hosts (reviewed in [9] , [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] ). some of these molecules were shown to contain pathogen associated molecular pattern that bind to endocytic-pattern recognition receptors on antigen presenting cells (apcs). three helminth products have also been reported to act as adjuvants in experimental vaccine models. proteins secreted by adult nippostrongylus brasiliensis (nes) induced strong th2 responses in mice immunized with hen egg lysozyme [22] . nes actively matured dendritic cells (dc) and selectively up-regulated cd86 and ox40l, together with il-6 production, while blocking il-12p70 responsiveness in a manner consistent with th2 generation in vivo [23] . similarly, lacto-n-fucopentaose iii (lnfpiii), a carbohydrate found on the surface of the eggs of a human parasite, schistosoma mansoni, acted as a th2 adjuvant for human serum albumin when injected intranasally, subcutaneously or intraperitonealy into mice [24] . it functions as an innate th2 promoter via its action on murine dcs. its ability to drive dc2 maturation was shown to be dependent on signaling via toll-like receptor 4 (tlr4) [25] . finally, when co-administered with an inactivated antiinfluenza vaccine in both young and aged mice, a 19 aa synthetic peptide (gk-1) from taenia crassiceps cysticerci has induced increased levels of anti-influenza antibodies in aged mice, both before and after infection, reduced the local inflammation that accompanied influenza vaccination itself, and favored virus clearance after infection in both young and aged mice [26] . a recombinant onchocerca volvulus activation-associated protein-1, rov-asp-1, has been shown to be a novel protein adjuvant that can increase specific immune responses in mice, both humoral and cellular responses, against recombinant protein or peptide-based figure 1 . anti-rbd antibody responses in vaccinated mice. rbd-specific responses in mice immunized with recombinant sars-cov rbd in the presence of the rov-asp-1, alum or cpg. titer of rbd-specific igg and igg subtypes was detected by elisa using sera from mice before (preimmune) and 10 days after each vaccination. * indicates significant difference (p,0.05) among multiple comparisons; in particular between mice that were immunized in the presence of rov-asp-1 or control adjuvants vs. no adjuvant. doi:10.1371/journal.pone.0037019.g001 antigens when formulated in aqueous mixtures [14, 27, 28, 29] . we have previously suggested that these effects are probably attained through the cellular activation of apcs such as dendritic cells via tlr-2 and tlr-4 [27] . the rov-asp-1 adjuvant is able to induce balanced th2 and th1-associated igg1 and igg2a antibodies to proteins, polypeptides and small peptides. with a few antigens such as rgp41, rbd and hbsag, it also promoted a putative th1biased response based on pronounced induction of th1-associated igg2a and igg2b antibody responses and/or a significantly upregulated production of th1 cytokines, including il-2, ifn-c, tnf-a, and il-6 [14, 27, 29, 30] . its ability to augment th1associated antibody responses was further demonstrated in studies using three commercial inactivated vaccines against hemorrhagic fever with renal syndrome, flu and rabies [29] . moreover, in a novel recombinant configuration, rov-asp-1 fused to 3 copies of the highly conserved extracellular domain of the h5n1 influenza m2 protein sequence was able to induce high levels of m2especific igg, igg1, igg2a providing strong cross-protection from a lethal challenge with 3ld 50 or 10 ld 50 of h5n1 viruses of different clades (clade 1: vn/1194, or clade 2.3.4: sz/406h [28] . in this study, we further demonstrated the adjuvanticity of rov-asp-1 in sequential vaccines in mice and also confirmed its ability to be a potent innate adjuvant in nhps. the rov-asp-1 adjuvant enhances humoral and cellular responses after immunization with rrbd of sars-cov in mice to evaluate the adjuvant activity of rov-asp-1, mice were immunized with sars-cov rrbd in the presence or absence of rov-asp-1 or with alum or cpg for comparison. we previously reported that rov-asp-1 could effectively induce a mixed, but th1-skewed immune response against rs and rrbd in immunized mice [27] . as shown in figure 1 , the titers of rbd-specific igg, igg1, igg2a and igg2b antibodies increase after the first or second boost immunization in the sera of mice immunized with rrbd plus rov-asp-1, which were all significantly higher than in mice immunized with rrbd alone. the igg and igg2a responses in mice vaccinated with rrbd in the presence of alum were significantly higher only after the second boost; only the igg1 response was significantly higher after the first boost. the anti-rrbd igg response was significantly higher in the presence of cpg only after the second boost when compared to mice vaccinated only with rrbd (1:102,400 vs. 1:11,314) . notably, the rov-asp-1 augmented igg antibody response to rrbd was almost 4 times higher than the alum vaccination group (64,000 vs. 16,000) and 5.7 times higher than the cpg vaccination group (64,000 vs. 11,200) already after the first boost. further comparison of the rov-asp-1 and the alum induced rrbd antibody responses after the second boost revealed similar levels of igg1 (1,884,544 vs. 1,722,156); two-fold higher level of igg2a (935,763 vs. 512,000) as well as ten times higher level of igg2b (430,538 vs. 45,255) endpoint titers in the rov-asp-1 vaccine group. comparison of the rov-asp-1 and the cpg induced rrbd antibody responses after the second boost revealed a 27 fold increase in igg1 (1,884,544 vs. 68,000), 14.6 fold increase in igg2a (935,763 vs. 64,000), and 215 fold increase in igg2b (430,538 vs. 2,000) endpoint titers. each adjuvant/rrbd model performed differently depending on the adjuvant, e.g., a skewed th2 response with alum (igg1/igg2a = 3.3), but a mixed th1/ th2 response with cpg (igg2a/igg1 = 0.94) and rov-asp-1(igg2a/igg1 = 2), with a predominance of th1-associated antibodies (when both igg2a and igg2b are taken into account) with rov-asp-1. these results further supported our previous studies showing that rov-asp-1 can induce a more balanced antibody response with some bias towards a skewed th1associated antibody response than other adjuvants used with the same bystander antigen such as alum or cpg (this study) or the mlp plus tdm adjuvant [14, 27, 29] . to further evaluate whether the induced igg antibodies could neutralize infection of sars-cov in vitro (fig. 2) , we tested the antisera from the 10-day post-second boost immunization and found that mice immunized with rrbd in the presence of rov-asp-1 contained a very high titer of neutralizing antibodies against infection by sars-cov pseudovirus (nt 50 = 1:76,592), which was not significantly different than in neutralizing antibodies found in sera from mice immunized with rrbd plus the alum adjuvant (nt 50 = 1:64,666). the subclass of immunoglobulin induced after immunization is an indirect measure of the relative contribution of th1-type cytokines vs.th2-type cytokines. in this study, the data from the cba analyses showed that the levels of th1-and th2-type cytokine secretion were significantly higher in mice immunized with rrbd+rov-asp-1 or alum than in those who were immunized with rrbd alone ( table 1 ). the rov-asp-1 induced the production of type i proinflammatory cytokines (il-2, ifn-c, tnf-a, il-17a and il-6) to the same extent as alum, as well as the th2/regulatory cytokines il-6 and il-10. there was no significant recall induction of the th2 il-4 or il-5 cytokines by rrbd (data not shown). notably, the responses to rrbd when formulated with cpg are in comparison more ifn-c and il-17a dominant with diminished il-2, tnf-a, il-10 and il-6 responses. we found that the variation between individual mice was very low [31, 32] , and therefore we are confident that the results obtained using the pooled spleens are a good representation of what would have been the outcome if we had used individual mice. distinct from the previous studies are the responses to two rbd-specific peptides; n50 (cd8 + t cell epitope) and n60 (cd4 + t cell epitope) (table 1) . interestingly, the cytokine levels in the culture supernatants of the murine splenocytes when stimulated with either n50 or n60 were similar to those produced by stimulation with the full length rrbd in mice vaccinated with rrbd+rov-asp-1, thus further confirming the ability of rov-asp-1 to elicit rbd-specific cd8 + and cd4 + cellular responses in the tested vaccine formulation. naïve balb/c mice or mice ten weeks after immunization with rrbd in the presence of rov-asp-1 were immunized with has of influenza viruses in the absence or presence of yeast expressed rov-asp-1 (100 mg/mice). the mice that were previously immunized with rrbd in the presence of rov-asp-1 had endpoint total anti-rov-asp-1 antibody titers of 1:256,000-1:512,000 at the time of the priming with the second vaccine. as shown in table 2 , similar igg1 and igg2a humoral immune responses against the influenza viruses were induced in the mice vaccinated previously with rrbd plus rov-asp-1 adjuvant and those administered with pbs only. moreover, the igg2b was higher in the group that got the sequential vaccine (162,000 vs. 54,000). the anti-ha igg1 and igg2b antibody responses were much higher in mice that were immunized with the has vaccine in the presence of rov-asp-1 adjuvant than those immunized in the absence of rov-asp-1 adjuvant (igg1: 1,458,000 vs. 607,000 or 729,000; igg2b: 243,000 vs. 162,000 or 54,000). all levels of igg isotype responses in mice when the flu vaccine was formulated with rov-asp-1 were statistically higher than when mice were immunized with no adjuvant (p.0.05; fig. 3 ). there was no significant difference in the igg1 and igg2a responses if the flu vaccine was given to naïve mice or to mice that were previously immunized with another vaccine; rrbd of sars-cov+rov-asp-1. the adjuvanticity of rov-asp-1 was evaluated in a rhesus macaque immunization model using rrbd as the target antigen of table 2 . titers of anti-ha antibody response in rrbd+ rov-asp-1 vaccinated mice and naïve mice after vaccination with an influenza vaccine or an influenza vaccine in combination with rov-asp-1. the sars-cov vaccine. as shown in table 3 , all of the nhps vaccinated with rrbd protein plus 50 mg (n = 2), 100 mg rov-asp-1 (n = 2) or 500 mg cpg (n = 1) as the adjuvant developed rbdspecific igg antibody response with increasing antibody level after each boost. rbd-specific antibodies were not detected in the preimmune sera of the vaccinated rhesus macaques or the rhesus macaques injected with rbd+pbs control (n = 1). immunization with rrbd plus an optimized quantity of the cpg (500 mg) as the adjuvant as used in other vaccine studies [33, 34] was the most effective for the induction of rbd-specific antibodies, with endpoint igg titer of 102,400 after third boost. although this was a limited pilot experiment using only two concentrations of the e. coli expressed rov-asp-1 adjuvant for the formulation (50 mg, 100 mg), which was two or four times the amount used in our mouse model experiments (25 mg), we clearly show that immunization with 50 or 100 mg of the rov-asp-1 adjuvant exhibited a dose-dependent efficacy in the induction of rbd-specific antibodies in the two macaques per group; endpoint igg titers of 3,200 (50 mg ) or 6,400 (100 mg). notably, the anti-rov-asp-1 antibody response titers to the adjuvant itself were lower in the nhps than we might have expected based on the data in mice; the range in all the 4 immunized macaque monkeys was: 1:100-1:800 after first boost, 1:800-1:1,600 after second boost and 1:200-1;1600 after the third boost. although the anti-rbd endpoint tiers were much higher in the nhp that was immunized with cpg, the differences in the nt 50 titers were less prominent. sera from all rhesus macaques vaccinated with the rrbd+adjuvant formulation effectively neutralized the infection of sars pseudovirus in 293t cells expressing the receptor ace2 (ace2/293t) with nt 50 values of 1:3,500-1:6,392. as shown in figure 4 , sera from macaques immunized with rrbd protein plus 100 mg rov-asp-1 induced a slightly higher titer of neutralizing antibody than the 50 mg rov-asp-1 group; 4,167/5,376 vs. 3,724/3,509. the macaques that were immunized with cpg had a higher nt 50 titer at 1:6,392. in an in vitro study, as described above, we showed that mice immunized with rrbd+rov-asp-1 had a strong cytokine production ex vivo when stimulated with rrbd protein or rbd-specific peptides n50 and n60. notably, only a significant tnf-a response was obtained in nhps that were immunized with rrbd in the presence of rov-asp-1 or cpg ex vivo when pbmcs were stimulated with 5 mg/ml of rrbd ( table 4 ). the cytokine response was similar regardless of the amount of rov-asp-1 used as the adjuvant. all other secreted cytokines, such as ifn-c, il-2, il-4, il-5 and il-6 were negligible. vaccines remain the most effective means of preventing or eradicating infectious diseases, and there are ongoing efforts to apply active immunization approaches to prevent and treat table 3 . rbd-specific igg responses in nhps immunized with rrbd in the presence of rov-asp-1 or cpg adjuvant. table 4 . tnf-a response in immunized nhps. autoimmune diseases and cancer [35, 36] . adjuvants potentiate antigen-specific immune responses and can be a key element of vaccine effectiveness [1, 37, 38] . adjuvants can be broadly separated into two classes based on their principal mechanism of action: vaccine delivery systems and immunostimulation. vaccine delivery systems are generally particulate and function mainly to target associated antigens into apcs, e.g., emulsions, microparticles, iscoms, and liposomes [6, 39] . immunostimulatory adjuvants contain residues that are recognized by receptors on apcs, such as tlrs, which play an important role in the innate recognition of pathogens by dcs, and thus directly activate innate immune responses. these adjuvants are now regarded as the most effective means by which an adjuvant-antigen complex can target apcs [40] . adjuvants targeting multiple innate immune receptors may prove to be the most effective adjuvants, as they may induce different arms of the immune responses in the host. a number of microbial products, including bacterial lps, peptidoglycan, dsrna, muramyl peptides, cpg, flagellin and microbial proteins, were shown to act as vaccine adjuvants [41] [42] [43] [44] [45] . some of these immunomodulators could skew acquired immune responses towards a th1-type immune response. adjuvants can also be classified according to their capacity to stimulate either innate or adaptive immunity based on significant differences in their cellular receptors and mechanisms of action [46] . as previously noted, alum is the only adjuvant licensed in the u.s. for general use in humans. yet, alum is not effective in stimulating th1 and/or cytotoxic t cell responses to a number of pathogens and is therefore limited in its applications, in particular for new-generation vaccines [47] . some of the adjuvants being developed in clinical testing include mpl, the saponin derivative qs-21, cpg, flagellin, and combinations of some of these adjuvants [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] . one of the major concerns regarding the use of immunostimulatory adjuvants in humans is the possible increased risk of autoimmune diseases due to targeting pattern recognition receptors by such adjuvants. however, the recombinant ov-asp-1 adjuvant we studied corresponds to a secreted filarial protein, which is presented as an antigen in the o. volvulus exposed or infected individuals in africa. there is no evidence to show that this secreted filarial protein could induce autoimmune disease in the infected patients, thus excluding such a concern. polarized th1-type immunity can be achieved by the addition of complete freund's adjuvant and cpg dna to an antigen [52] [53] [54] . on the other hand, th2 antibody responses can be induced by the alum or incomplete freund's adjuvant, as indicated by increased igg1 relative to igg2a [53] [54] [55] . however, in situations where both th1 and th2 responses are required for protection, the choice of one regimen over another might be counter effective. this has led to additional research for alternative adjuvants or adjuvant combinations that promote balanced mixed th1/th2 responses. the present study clearly demonstrated that rov-asp-1 could effectively induce mixed rbd-or ha-specific th1/th2 antibody associated responses, when used as an adjuvant with recombinant subunit vaccine or as an addition to a commercial flu vaccine (fluvirin; using 20% of the dose recommended for human use). since the rov-asp-1 adjuvant is a protein, it is potentially processed and presented to the immune system and subsequently induces antibodies against self. therefore, concerns might be raised whether preexisting anti-rov-asp-1 antibodies may suppress its adjuvanticity when it is used in subsequent vaccine formulations. previously we demonstrated that antibody response to the adjuvant itself did not hindered the development of ova, rs or rrbd antigen-specific antibody responses after each boost; in all cases the responses in the presence of rov-asp-1 were more elevated than those in the presence of mpl+tmp or alum adjuvants. our present results further confirmed that antibodies induced against the adjuvant had no impact on its ability to induce immune responses against bystander antigens when used as an adjuvant in a sequential vaccine when two vaccine model systems were used: the rbd of sars-cov spike protein and a commercial influenza virus ha vaccine comprised of three virus strains. even though a high level of anti-rov-asp-1 antibodies (1:256,000-1:512,000) was present in the sera of mice before the administration of the second vaccine, the adjuvanticity of rov-asp-1 was retained with efficacy similar to that obtained when it was used as an adjuvant in a first vaccine immunization of naïve mice (table 2 and figure 3) ; no difference in the igg titers was observed between the two vaccine groups, mice pre-immunized with rrbd+rov-asp-1 or pbs. thus, we confirmed that the preexisting anti-rov-asp-1 antibodies induced by a previously administrated rov-asp-1-based vaccine do not have a significant effect on the adjuvanticity of rov-asp-1 when formulated in a subsequent vaccine. notably, immunization of nhps with rov-asp-1 did not induce high titers of anti-self-antibodies, for reasons that are not yet clear. future studies will be needed to further validate that even these reasonably low antibody responses to the adjuvant have no impact on subsequent use of this adjuvant with other vaccines also in nhps. the pilot immunogenicity studies in the nhps have provided us with extremely valuable ''proof of principal'' information in an outbred primate model. firstly, no adverse reactions were observed at the site of the immunizations, indicating the safety of rov-asp-1 as an adjuvant. secondly, using two concentration of the rov-asp-1 adjuvant, 50 or 100 mg, and rrbd as the vaccine antigen, we were able to induce after three immunizations high titers of neutralizing antibodies (1:3,500-1:6,392) that much exceed what is needed for protection against sars-cov infection in vivo (.1:500) [56] . moreover, our studies have shown that rov-asp-1 was able to support the induction of functional antibodies against a pathogen in nhps, thus clearing the way for its future development for human vaccines as well. thus, this pilot nhp study is a definitive step for demonstrating relevance of rov-asp-1 for human vaccine formulations, even though more studies will be needed to find the optimized amount of the adjuvant in vaccine formulations, which will specify the putative starting adjuvant dose for future clinical trials of rov-asp-1-based vaccines. interestingly, we did not see augmented recall rbd specific cellular responses except tnf-á. therefore, future studies will be needed to validate the adjuvanticity of rov-asp-1 in primates from the point of its ability to augment the desired types of cellular responses are associated with protective immunity against possibly other pathogens, duration of immunity and the potential to establish t cell memory. in summary, the present studies have advanced our confidence that the rov-asp-1 protein adjuvant can be further developed for human use, particularly when strong functional balanced antibody responses are needed against the pathogens. importantly, the rov-asp-1 that was used to immunize mice previously immunized with rbd+rov-asp-1 or the naïve mice with the ha vaccine was expressed in the yeast. having a functional yeast expressed adjuvant will support future development of a scalable process for the downstream manufacture of rov-asp-1, including the development of a series of critical biochemical and biophysical assays for in-process, release and stability testing, which will result in a high-yield reproducible production process and a stable product that is highly potent and stimulates the desired antibody and cellular responses to co-administered vaccine antigens in nhps for further analyses and for future clinical trials in humans. animal protocols were approved by the institutional animal care and use committee at the new york blood center (mice, protocols #255 and #194) and the tulane national primate research center (nhps, protocol #p0052). the tulane national primate research center tnprc is a usda-inspected and association for assessment and accreditation of laboratory animal care international (aaalac)-approved facility, and has an animal welfare assurance on file with the office of laboratory animal welfare. all animal studies were carried out in strict accordance with the recommendations of the american veterinary medical association (avma) guidelines and the approved protocols. the animals were handled delicately. any treatment was done with extreme care and professionalism to avoid any unnecessary discomfort for the animals. forty-two female balb/c mice aged 4-6 weeks and six purpose-bred adult male indian-origin rhesus macaques aged 6-10 years were used in this study. animal housing and environmental conditions met all applicable standards. blood was collected retro-orbitally for mice and from the femoral vein using the sarstedt s-monovette system for nhps. the animals were anesthetized using ketamine (0.1 ml/kg im), which ameliorate any suffering of the animals during the blood draw or immunization. the nhp protocol also included a full cbc and chem20 profile, which were taken at each blood drawing, with results falling within normal levels. their body weight was frequently monitored and physical examinations were performed regularly by the attending veterinarian. the rov-asp-1 protein was expressed as a histidine-tagged protein in escherichia coli and purified as previously described [27] . the purified rov-asp-1 was tested negative in a limulus amoebocyte lysate assay. a quantitative lps testing by cambrex bioscience showed that purified rov-asp-1 contained ,0.25 endotoxin units per milligram of the protein (25 pg endotoxin/ mg), and we considered it as an lps-free stock. in addition, we expressed rov-asp-1 in yeast. yeast codon optimized ov-asp-1 cdna sequence (af020586) without the region encoding the n-terminal signal peptide was cloned into the pichia expression vector ppiczaa (invitrogen) using the ecori and xbai restriction sites. the recombinant plasmid was linearized with saci digestion and transformed into pichia pastoris x33 strain as described previously [57] . the positive transformants were screened on zeocin-resistant ypd plates, and the highest expression clone was selected by scale up culturing. the expression of rov-asp-1 with 66his and c-myc tag at c-terminus was induced with 0.5% methanol and scaled up in 10 liters fermentation as described previously [58] . the rov-asp-1 was purified from the fermentation culture with sp sepharose fast flow exchange chromatography as described previously [58] . briefly, the fermentation supernatant was filtered through a 0.22 mm membrane and the ph was adjusted to 4.8 by adding glacial acetic acid. the positively charged rov-asp-1 was captured onto cation sp sepharose ff column and eluted with 50 mm sodium acetate, ph 4.8 containing 200 mm nacl. the eluted rov-asp-1 pool was then purified and buffer exchanged using gel filtration chromatography (sephadex g-25 fine) into pbs, ph 7.2 [28, 57] . the product was tested for its adjuvanticity in mice using ova as the model antigen as previously described [27] and it was established that 100 mg yeast-expressed rov-asp-1 per mouse was as effective as 25 mg per mice of the e. coli expressed rov-asp-1; both eliciting after two immunizations end point anti-ova igg titers of 1:25,600 (data not shown). thus, the e. coli or the yeast expressed rov-asp-1 at their optimal quantities can be used intermittently with assurance. mice were vaccinated subcutaneously with rrbd protein purified from culture supernatant of transfected human embryonic kidney cell-line 293t (hek293t) (atcc, va) according to the previously described protocol [31, 59, 60] using 20 mg/mouse of the protein mixed in aqueous solution with the e. coli-expressed optimized quantity of rov-asp-1 (25 mg/mouse) in 200 ml. as adjuvant controls we immunized mice with rrbd mixed with imject alum (40 mg/ml aluminum hydroxide+40 mg/ml magnesium hydroxide, thermo scientific) diluted 1:1 with the vaccine antigen in a total volume of 200 ml per mouse or with rrbd mixed with 50 mg cpg-odn1826 (invivogen, san diego ca) in a total volume of 200 ml per mouse. mice immunized with rrbd in pbs were used as the negative control. the mice were boosted twice 3 weeks apart with 10 mg/mouse of rrbd protein in pbs, rov-asp-1, imject alum or cpg. all mice were bled retro-orbitally under anaesthesia prior to immunizations and 10 days post injections. sera were stored at 280uc. to test the adjuvanticity of rov-asp-1 in a sequential vaccine model, we immunized intramuscularly naïve mice or mice that were previously immunized with the rrbd+rov-asp-1 with a commercial flu vaccine (100 ml/mouse) containing 3 mg (fluvirin, novartis) . the immunization was done in the presence or absence of the yeastexpressed optimized quantity of rov-asp-1 (100 mg/mouse). the mice were boosted once with the same dose of vaccines three weeks later. all mice were bled prior to immunization and 7 days post immunization as described above and sera were stored at 280uc. rhesus macaques were vaccinated subcutaneously with rrbd protein (50 mg) with either 50 mg (n = 2) or 100 mg (n = 2) of e. coliexpressed rov-asp-1, cpg-c-iss-odn c274 (500 mg; n = 1) (donated by dynavax technologies corporation, berkeley, ca) or pbs (n = 1). they were boosted with the same dose at 1-, 2-and 6month intervals. the macaques were bled before immunization and 7 days post-immunization. sera were collected for serological testing, and pbmcs were purified for in vitro assays. a baseline bleed was also provided. elisa was used to detect specific antibody responses induced in the vaccinated mouse or nhp. briefly, 96-well micro titer plates (costar) were coated with rrbd (1 mg/ml), rov-asp-1 (1 mg/ml) or has (2.5 mg/ml) at 4uc overnight. the plates were blocked with 2% non-fat milk in pbs-tween (0.05%) for 2 h at 37uc. after washing the plates six times with pbs-t, serial diluted sera in binding buffer (1% non-fat milk in pbs-t) were added into each well in duplicate and incubated for 1 h at 37uc. bound antibodies were detected with hrp-conjugated goat anti-mouse igg, igg1, igg2a or igg2b (molecular probes-invitrogen) (1:2000 dilution) or mouse anti-monkey igg-hrp (clone sb108a, southern biotech) (diluted 1:1000) in binding buffer. after incubation for 1 h at 37uc, plates were washed and tmb substrate (kpl) was added, and the reaction was stopped by the addition of 2 n h 2 so 4 . absorbance at 450 nm was measured using spectramax 190 (molecular devices, sunnyvale, ca). end point titers were defined as the highest dilutions giving an a 450 measurement of 0.1. this cutoff value represents the value of mean optical density (od) plus 2 standard deviations (sd) of 10 normal mouse serum samples tested at 1:100 dilutions or the 6 pre-bleeds from the normal nhp serum samples also tested at 1:100 dilutions. the neutralization assay against sars pseudovirus infection was performed as previously described [61] . in brief, plasmid dna encoding sars-cov spike protein and a plasmid dna encoding env-defective, luciferase-expressing hiv-1 genome (pnl4-3.luc.re) were co-transfected into hek293t cells (attc, ga). culture supernatant containing sars pseudovirus was collected at 72 h and used for single-cycle infection in vitro. the sars pseudovirus was incubated in the presence or absence of serially diluted antisera from vaccinated mice or nhps for 1 h at 37uc. subsequently, the antisera-virus mixtures were added to 293t cells expressing the sars receptor angiotensin-converting enzyme 2 (293t/ace2) in 96-well plates, and the infection was allowed to proceed for 48 h, followed by lysing the infected cells using cell lysis buffer included in the luciferase kit (promega). aliquots of cell lysates were transferred to 96-well costar flatbottom luminometer plates (corning costar), followed by addition of luciferase substrate (promega). relative light units were determined immediately in the ultra 384 luminometer (tecan). the neutralization of sars pseudovirus is presented as 50% neutralizing antibody titer (nt 50 ) [60] . th1/th2/th17 cytokine assay was used to estimate the corresponding cytokine production ex vivo from splenocytes of the vaccinated mice. briefly, splenocytes were harvested from the immunized mice and resuspended in rpmi 1640 medium (invitrogen, carlsbad, ca) supplemented with 10% fbs (hyclone laboratories, inc.), 2 mm l-glutamine, 10 mm hepes, and antibiotic-antimycotic (invitrogen, carlsbad, ca). the cells were plated at 4610 5 cells into 96-well u-bottom culture plates for in vitro stimulation with 5.0 mg of rrbd, or with the sars-cov rbd-specific n50 (cd8 + t cell epitope) or n60 (cd4 + t cell epitope) peptide [31] ; a concentration that was pre-determined to be optimal. cells were stimulated with or without pma (10 ng/ml) plus ionomycin (1 mg/ml) as the positive and negative controls, respectively. the plates were incubated at 37uc for 72 h, and the secreted cytokines were quantified from the culture supernatants using the mouse th1/th2/th17 bd cytometric bead array kit (bd biosciences) according to the manufacture's protocols. theoretical limit of detection data is il-2 = 0.1 pg/ml; il-4 = 0.03 pg/ml; il-6 = 1.4 pg/ml; il-10 = 16.8 pg/ml; tnfa = 0.9 pg/ml, inf-c = 0.5 pg/ml; and il-17a = 0.8 pg/ml. detection of the th1/th2 cytokine production in the vaccinated nhps was done using similar protocol as above with some modifications. pbmcs were isolated following a ficoll-hypaque density gradient (sigma). single-cell suspensions were then stimulated at 4610 5 cells with 5 mg of rrbd, n50 or n60 peptides, or pma (10 ng/ml) plus ionomycin (1 mg/ml) for positive control and culture media alone for negative control. the cells were stimulated for 5 days, and cytokines were quantified using the non-human primate th1/th2 bd cytometric bead array kit (bd biosciences) according to the manufacture's protocols. results are expressed as mean 6 sem. the data were analyzed using graphpad version 5.01 for windows (graphpad software). unpaired two-tailed student's t test or mann-whitney test was used to compare means between different groups. one-way anova with bonferroni post-test was considered appropriate for multiple comparisons. p value less than 0.05 was considered significant. alum's adjuvant action: grease is the word alum interaction with dendritic cell membrane lipids is essential for its adjuvanticity immunological mechanisms of vaccination vaccine adjuvants: putting innate immunity to work adjuvants for the future recent advances in vaccine adjuvants rationally-designed vaccine adjuvants: separating efficacy from toxicity enhancing oral vaccine potency by targeting intestinal m cells diversity and dialogue in immunity to helminths immune regulation by helminth parasites: cellular and molecular mechanisms t helper type-2 cytokine responses: potential therapeutic targets helminth antigens modulate tlr-initiated dendritic cell activation tlr11 activation of dendritic cells by a protozoan profilin-like protein rov-asp-1, a recombinant secreted protein of the helminth onchocerca volvulus, is a potent adjuvant for inducing antibodies to ovalbumin, hiv-1 polypeptide and sars-cov peptide antigens immune evasion genes from filarial nematodes helminth parasites-masters of regulation a novel therapeutic approach targeting articular inflammation using the filarial nematode-derived phosphorylcholine-containing glycoprotein es-62 es-62, a filarial nematode-derived immunomodulator with anti-inflammatory potential modulation of a heterologous immune response by the products of ascaris suum taenia crassiceps carbohydrates stimulate il-6 expression in naive murine macrophages via toll-like receptors (tlrs) a novel host-parasite lipid cross-talk. schistosomal lyso-phosphatidylserine activates toll-like receptor 2 and affects immune polarization proteins secreted by the parasitic nematode nippostrongylus brasiliensis act as adjuvants for th2 responses selective maturation of dendritic cells by nippostrongylus brasiliensis-secreted proteins drives th2 immune responses lacto-n-fucopentaose iii found on schistosoma mansoni egg antigens functions as adjuvant for proteins by inducing th2-type response maturation of dendritic cell 2 phenotype by a helminth glycan uses a toll-like receptor 4-dependent mechanism a novel synthetic adjuvant effectively enhances the immunogenicity of the influenza vaccine recombinant ov-asp-1, a th1-biased protein adjuvant derived from the helminth onchocerca volvulus, can directly bind and activate antigen-presenting cells induction of protection against divergent h5n1 influenza viruses using a recombinant fusion protein linking influenza m2e to onchocerca volvulus activation associated protein-1 (asp-1) adjuvant evaluation of recombinant onchocerca volvulus activation associated protein-1 (asp-1) as a potent th1-biased adjuvant with a panel of protein or peptide-based antigens and commercial inactivated vaccines a novel, helminth-derived immunostimulant enhances human recall responses to hepatitis c virus and tetanus toxoid and is dependent on cd56+ cells for its action priming with raav encoding rbd of sars-cov s protein and boosting with rbd-specific peptides for t cell epitopes elevated humoral and cellular immune responses against sars-cov infection intranasal vaccination of recombinant adeno-associated virus encoding receptor-binding domain of severe acute respiratory syndrome coronavirus (sars-cov) spike protein induces strong mucosal immune responses and provides long-term protection against sars-cov infection local and systemic effects of intranodally injected cpg-c immunostimulatoryoligodeoxyribonucleotides in macaques cpg-c iss-odn activation of blood-derived b cells from healthy and chronic immunodeficiency virus-infected macaques anticytokine vaccination in autoimmune diseases cancer vaccines. any future? trends in vaccine adjuvants recent developments in cancer vaccines adjuvants-a classification and review of their modes of action dendritic cell subtypes as primary targets of vaccines: the emerging role and cross-talk of pattern recognition receptors adjuvant activities of immune response modifier r-848: comparison with cpg odn targeting the innate immune response with improved vaccine adjuvants vaccination with recombinant fusion proteins incorporating toll-like receptor ligands induces rapid cellular and humoral immunity a west nile virus recombinant protein vaccine that coactivates innate and adaptive immunity the science of adjuvants vaccine adjuvants: role and mechanisms of action in vaccine immunogenicity aluminium adjuvants-in retrospect and prospect immunisation of metastatic cancer patients with mage-3 protein combined with adjuvant sbas-2: a clinical report potent immunogenicity and efficacy of a universal influenza vaccine candidate comprising a recombinant fusion protein linking influenza m2e to the tlr5 ligand flagellin effect of monophosphoryl lipid a (mpl) on t-helper cells when administered as an adjuvant with pneumocococcal-crm197 conjugate vaccine in healthy toddlers three double-blind, randomized trials evaluating the safety and tolerance of different formulations of the saponin adjuvant qs-21 cpg dna is a potent enhancer of specific immunity in mice immunized with recombinant hepatitis b surface antigen adjuvant-guided type-1 and type-2 immunity: infectious/noninfectious dichotomy defines the class of response induction of skewed th1/th2 t-cell differentiation via subcutaneous immunization with freund's adjuvant adjuvant-dependent modulation of th1 and th2 responses to immunization with beta-amyloid receptor-binding domain of sars-cov spike protein induces long-term protective immunity in an animal model biochemical characterization and vaccine potential of a heme-binding glutathione transferase from the adult hookworm ancylostoma caninum expression of the necator americanus hookworm larval antigen na-asp-2 in pichia pastoris and purification of the recombinant protein for use in human clinical trials recombinant receptorbinding domain of sars-cov spike protein expressed in mammalian, insect and e. coli cells elicits potent neutralizing antibody and protective immunity protocol for recombinant rbd-based sars vaccines: protein preparation, animal vaccination and neutralization detection a 219-mer cho-expressing receptor-binding domain of sars-cov s protein induces potent immune responses and protective immunity we would like to thank dynavax technologies corporation for donating the cpg-c iss-odn c274 used in the nhp experiment. key: cord-264408-vk4lt83x authors: ruiz, sara i.; zumbrun, elizabeth e.; nalca, aysegul title: animal models of human viral diseases date: 2017-06-23 journal: animal models for the study of human disease doi: 10.1016/b978-0-12-809468-6.00033-4 sha: doc_id: 264408 cord_uid: vk4lt83x as the threat of exposure to emerging and reemerging viruses within a naïve population increases, it is vital that the basic mechanisms of pathogenesis and immune response be thoroughly investigated. recent outbreaks of middle east respiratory syndrome corona virus, ebola virus, chikungunya virus, and zika virus illustrate the emerging threats that are encountered. by utilizing animal models in this endeavor, the host response to viruses can be studied in a more complex and integrated context to identify novel drug targets, and assess the efficacy and safety of new products rapidly. this is especially true in the advent and implementation of the fda animal rule. although no one animal model is able to recapitulate all aspects of human disease, understanding the current limitations allows for a more targeted experimental design. important facets to consider prior to an animal study are route of viral exposure, species of animal, biomarkers of disease, and a humane endpoint. this chapter covers the current animal models for medically important human viruses, and demonstrates where the gaps in knowledge exist. well-developed animal models are necessary to understand disease progression, pathogenesis, and immunologic responses to viral infections in humans. furthermore, to test vaccines and medical countermeasures, animal models are essential for preclinical studies. ideally, an animal model of human viral infection should mimic the host-pathogen interactions and the disease progression that is seen in the natural disease course. a good animal model of viral infection should allow assay of many parameters of infection, including clinical signs, growth of virus, clinicopathological parameters, cellular and humoral immune responses, and virus-host interactions. furthermore, viral replication should be accompanied by measurable clinical manifestations and pathology should resemble that of human cases such that a better understanding of the disease process in humans is attained. there is often more than one animal model that closely represents human disease for a given pathogen. small animal models are typically used for first-line screening, and for testing the efficacy of vaccines or therapeutics. in contrast, nonhuman primate (nhp) models are often used for pivotal preclinical studies. this approach is also used for basic pathogenesis studies, with most studies in small animal models when possible, and studies in nhps to fill in the remaining gaps in knowledge. the advantages of using mice to develop animal models are low cost, low genetic variability in inbred strains, and abundant molecular biological and immunological reagents. specific pathogen free (spf), transgenic and knockout mice are also available. a major pitfall of mouse models is that the pathogenesis and protection afforded by vaccines and therapeutics cannot always be extrapolated to humans. additionally, blood volumes for sampling are limited in small animals, and viruses often need to be adapted through serial passage in the species to induce a productive infection. the ferret's airways are anatomically and histologically similar to that of humans, and their size enables collection of larger or more frequent blood samples, making them an ideal model for certain respiratory pathogens. ferrets are outbred, with no standardized breeds or strains, thus greater numbers are required in studies to achieve statistical significance and overcome the resulting variable responses. additionally, spf and transgenic ferrets are not available, and molecular biological reagents are lacking. other caveats making ferret models more difficult to work with are their requirement for more space than mice (rabbit-style cages), and the development of aggressive behavior with repeated procedures. nhps are genetically, the closest species to humans, thus disease progression and host-pathogen responses to viral infections are often the most similar to that of humans. however, ethical concerns pertaining to experimentation on nhps along with the high cost and lack of spf nhps raise barriers for such studies. nhp studies should be carefully designed to ensure the fewest number of animals are used, and the studies should address the most critical questions regarding disease pathogenesis, host-pathogen responses, and protective efficacy of vaccines and therapeutics. well-designed experiments should carefully evaluate the choice of animal, including the strain, sex, and age. furthermore, depending on the pathogen, the route of exposure and the dose should mimic the route of exposure and dose of human disease. the endpoint for these studies is also an important criterion. depending on the desired outcome, the model system should emulate the host responses in humans when infected with the same pathogen. in summary, small animal models are helpful for the initial screening of vaccines and therapeutics, and are often beneficial in obtaining a basic understanding of the disease. nhp models should be used for a more detailed characterization of pathogenesis and for pivotal preclinical testing studies. ultimately, an ideal animal model may not be available. in this case, a combination of different well-characterized animal models should be considered to understand the disease progression and to test medical countermeasures against the disease. in this chapter, we will be reviewing the animal models for representative members of numerous virus families causing human diseases. we will focus on viruses for each family that are of the greatest concern for public health worldwide. norovirus, the genus of which norwalk is the prototypic member, is the most common cause of gastroenteritis in the united states (hall et al., 2013) . there are five distinct genogroups (gi-gv) and numerous strains of norwalk virus, including the particularly significant human pathogens gi.1 norwalk virus, gii.2 snow mountain virus, and gii.1 hawaii virus. in developing countries, norwalk virus, also known as "winter vomiting virus," is responsible for approximately 200,000 deaths annually (patel et al., 2008) . a typical disease course is self-limiting, but there have been incidences of necrotizing enterocolitis and seizures in infants (chen et al., 2009; lutgehetmann et al., 2012; turcios-ruiz et al., 2008) . symptoms of infection include diarrhea, vomiting, nausea, abdominal cramping, dehydration, and fever. incubation is normally 1-3 days, with symptoms persisting for 2-3 days (koopmans and duizer, 2004) . viral shedding can range from 6 to 55 days in healthy individuals (atmar et al., 2014) . however, longer illness duration can be indicative of immunocompromised status, with the elderly and young having a prolonged state of shedding (harris et al., 2008; rockx et al., 2002) . interestingly, individuals vary greatly in susceptibility to norovirus infection depending on their fucosyl transferase 2 (fut2) allele functionality and histoblood group antigen status, with type a and o individuals susceptible and types ab and b resistant (hutson et al., 2005) . transmission occurs predominately through the oralfecal route with contaminated food and water being a major vector (atmar and estes, 2001; becker et al., 2000; koopmans and duizer, 2004) . vomiting results in airborne dissemination of the virus with areas of 7.8 m 2 being contaminated and subsequent transmission from oral deposition of airborne particles or contact with contaminated fomites, which can remain contaminated for up to 42 days (makison booth, 2014; tung-thompson et al., 2015) . each vomiting event in a classroom setting elevates the risk of norovirus illness among elementary students with proximity correlating with attack rates (evans et al., 2002; marks et al., 2003) . viral titers in emesis and fecal suspensions are as high as 1.2 × 10 7 and 1.6 × 10 11 ges (genomic equivalent copies per milliliter), respectively and the 50% infectious dose is 1320 ges (atmar et al., 2014) . therefore, outbreaks can be extremely difficult to contain. therapeutic intervention consists of rehydration therapy and antiemetic medication (bucardo et al., 2008; moe et al., 2001) . no approved vaccine or therapeutic is available, and development has been challenging given that immunity is short-lived after infection, new strains rapidly evolve and the correlates of protection are not completely understood (chen et al., 2013) . however, one promising strategy utilized a virus-like particle (vlp)-based vaccine that protected or reduced infection by almost 50% in human volunteers (aliabadi et al., 2015; atmar et al., 2011) . given the relatively benign disease in adults, experimental challenge has been carried out on human volunteers (ball et al., 1999; tacket et al., 2000) . viral titers are determined by shedding in feces and sera with histopathology changes monitored by biopsies particularly of the duodenum. the ph of emesis samples collected containing virus is consistent with viral replication in the small intestine with reflux to the stomach (kirby et al., 2016) . additionally, norwalk virus has been shown to bind to duodenal tissue (chan et al., 2011) . however, this type of research is technically difficult and expensive, and thus other models have been developed. a major hindrance to basic research into this pathogen is the lack of permissive cell culture systems or animal models for norwalk virus. nhps including marmosets, cotton-top tamarins, and rhesus macaques infected with norwalk virus are monitored for the extent of viral shedding; however, no clinical disease is observed in these models. disease progression and severity is measured exclusively by assay of viral shedding (rockx et al., 2005) . incidentally, more viruses were needed to create an infection when challenging by the oral route than by the intravenous (iv) route (purcell et al., 2002) . chimpanzees were exposed to a clinical isolate of norwalk virus by the iv route (bok et al., 2011) . although none of the animals developed disease symptoms, viral shedding within the feces was observed within 2-5 days postinfection and lasted anywhere from 17 days to 6 weeks. viremia never occurred and no histopathological changes were detected. the amount and duration of viral shedding was in-line with what is observed upon human infection. as such, chimeric chimpanzee-human antinorovirus neutralizing antibodies have been explored as a possible therapeutic strategy (chen et al., 2013) . a recently identified calicivirus of rhesus origin, named tulane virus, has been used as a surrogate model of infection. unlike norwalk virus, tulane virus can be cultured in cells. rhesus macaques exposed to tulane virus intragastrically developed diarrhea and fever 2 days postinfection. viral shedding was detected for 8 days. the immune system produced antibodies that dropped in concentration within 38 days postinfection, mirroring the short-lived immunity documented in humans. the intestine developed moderate blunting of the villi as seen in human disease (sestak et al., 2012) . a murine norovirus has been identified and is closely related to human norwalk virus (karst et al., 2003) . however, clinically the virus presents a different disease. the murine norovirus model does not include observable gastrointestinal clinical signs, possibly in part because rodents lack a vomiting reflex. additionally, mice infected with norovirus develop a persistent infection in contrast to human disease (hsu et al., 2006 (hsu et al., , 2007 khan et al., 2009) . porcine enteric caliciviruses can induce diarrheal disease in young pigs, and an asymptomatic infection in adults (wang et al., 2006 . gnotobiotic pigs can successfully be infected with a passaged clinical norovirus isolate by the oral route. diarrheal disease developed in 74% of the animals and virus was detected in the stool of 44% of the animals. no major histopathological changes or viral persistence was noted (cheetham et al., 2006) . calves are naturally infected with bovine noroviruses (scipioni et al., 2008) . experimental challenge of calves by oral inoculation with a bovine isolate resulted in diarrheal disease 14-16 h postinfection. recovery of virus was achieved after 53.5 and 67 h postinfection (otto et al., 2011) . eastern equine encephalitis virus (eeev), western equine encephalitis virus (weev), and venezuelan equine encephalitis virus (veev) present with near synonymous symptoms. the majorities of human cases are asymptomatic, but can present as a flu-like illness progressing to central nervous system (cns) involvement to include seizures and paralysis. mortality rates vary among the virus, with the highest reported for eev at 36%-75% followed by weev and lastly veev at less than 1% (ayers et al., 1994; griffin, 2007; steele and twenhafel, 2010) . there are currently no licensed vaccines or therapies but a recent phase 1 clinical trial of a veev dna vaccine resulted in veev-neutralizing antibody responses in 100% of the subjects (hannaman et al., 2016) . mouse models have been developed for numerous routes of infection including cutaneous, intranasal (in), intracranial (ic), and aerosol. eeev susceptibility in mouse models is correlated with age, with younger mice being more susceptible than adults. importantly, eeev pathogenesis is dependent on route of infection with delayed progression upon subcutaneous (subq) exposure (honnold et al., 2015) . newborn mice display neuronal damage with rapid disease progression, resulting in death (murphy and whitfield, 1970) . similarly, eeev produces fatal encephalitis in older mice when administered via the intracerebral route, while inoculation via the subq route causes a pantropic infection eventually resulting in encephalitis (liu et al., 1970; morgan, 1941) . a general drawback to the usage of the mouse model is the lack of vascular involvement during the disease course (liu et al., 1970) . after subq inoculation with weev, suckling mice started to show signs of disease by 24 h and died within 48 h (aguilar, 1970) . the heart was the only organ in which pathologic changes were observed. conversely, adult mice exhibited signs of lethargy and ruffled fur on day 4-5 postinfection. mice were severely ill by day 8 and appeared hunched and dehydrated. death occurred between days 7 and 14 with brain and mesodermal tissues, such as heart, lungs, liver, and kidney involvement (aguilar, 1970; monath et al., 1978) . intracerebral and in routes of infection resulted in a fatal disease that was highly dependent on dose while intradermal (id) and subq inoculations caused only 50% fatality in mice regardless of the amount of virus (liu et al., 1970) . comparing susceptibility of inbred and outbred strains revealed that cd-1, balb/c, a/j, and c57bl6 mice were all highly susceptible to experimental infection via subq inoculation when challenged prior to 10 weeks old with cns involvement and lethality (blakely et al., 2015) . subq/dermal infection in the mouse model results in encephalitic disease very similar to that seen in horses and humans (macdonald and johnston, 2000) . virus begins to replicate in the draining lymph nodes at 4 h postinoculation. eventually, virus enters the brain primarily via the olfactory system. furthermore, aerosol exposure of mice to veev can result in massive infection of the olfactory neuroepithelium, olfactory nerves, and olfactory bulbs and viral spread to brain, resulting in necrotizing panencephalitis (charles et al., 1995; steele et al., 1998) . aerosol and dermal inoculation routes cause neurological pathology in mice much faster than other routes of exposure. the clinical signs of disease in mice infected by aerosol are ruffled fur, lethargy, and hunching progressing to death (charles et al., 1995; steele and twenhafel, 2010; steele et al., 1998) . in challenge of c3h/hen mice with high dose veev caused high morbidity and mortality (julander et al., 2008b) . viral titers in brain peaked on day 4 postchallenge and remained elevated until animals succumbed on day 9-10 postchallenge. protein cytokine array performed on brains of infected mice showed elevated il-1a, il-1b, il-6, il-12, mcp-1, ifnγ, mip-1a, and rantes levels. this model was used successfully to test antivirals against veev (julander et al., 2008a) . additionally, a veev vaccine inactivated with 1,5-iodonaphthyl azide v3526 protects against both footpad and aerosol challenge with virulent veev in a mouse model (gupta et al., 2016) . guinea pigs and hamsters have also been developed as animal models for eeev studies (paessler et al., 2004; roy et al., 2009) . guinea pigs developed neurological involvement with decreased activity, tremors, circling behavior, and coma. neuronal necrosis was observed in brain lesions in the experimentally challenged animals (roy et al., 2009) . subq inoculation of eeev produced lethal biphasic disease in hamsters with severe lesions of nerve cells. the early visceral phase with viremia was followed by neuroinvasion, encephalitis, and death. in addition, parenchyma necrosis were observed in the liver and lymphoid organs (paessler et al., 2004) . harlan sprague-dawley hamsters develop viremia and progress to respiratory, gastrointestinal, and nervous system involvement when inoculated via subq route. vasculitis and encephalitis were both evident in this model, which mirrors the human disease clinical spectrum (paessler et al., 2004) . weev is highly infectious to guinea pigs and has been utilized for prophylactic screening (sidwell and smee, 2003) . studies demonstrated that although the length of the incubation period and the disease duration varied, weev infection resulted in mortality in hamsters by all routes of inoculation. progressive lack of coordination, shivering, rapid and noisy breathing, corneal opacity, and conjunctival discharge resulting in closing of the eyelids were indicative of disease in all cases (zlotnik et al., 1972) . cns involvement was evident with intracerebral, intraperitoneal (ip), and id inoculations (zlotnik et al., 1972) . ip inoculation of weev is fatal in guinea pigs regardless of amount of virus inoculum, with the animals exhibiting signs of illness on day 3-4, followed by death on day 5-9 (nalca, unpublished results) . id, im, or iv inoculations of eeev in nhps cause disease, but does not reliably result in neurological symptoms (dupuy and reed, 2012) . intracerebral infection of eeev produces nervous system disease and fatality in monkeys (nathanson et al., 1969) . the differences in these models indicate that the initial viremia and the secondary nervous system infection do not overlap in nhps when they are inoculated by the peripheral route (wyckoff, 1939) . in and intralingual inoculations of eeev also cause nervous system symptoms in monkeys, but are less drastic than intracerebral injections (wyckoff, 1939) . the aerosol route of delivery will result in uniformly lethal disease in cynomolgus macaques (reed et al., 2007) . in this model, fever was followed by elevated white blood cells and liver enzymes. neurological signs subsequently developed and nhps became moribund and were euthanized between 5-9 days postexposure. meningoencephalomyelitis was the main pathology observed in the brains of these animals (steele and twenhafel, 2010) . similar clinical signs and pathology were observed when common marmosets were infected with eeev by the in route (adams et al., 2008) . both aerosol and in nhp models had similar disease progression and pathology as seen in human disease. very limited studies have been performed with nhps. reed et al. exposed cynomolgus macaques to low and high doses of aerosolized weev. the animals subsequently developed fever, increased white blood counts, and cns involvement, demonstrating that the cynomolgus macaque model could be useful for testing of vaccines and therapeutics against weev (reed et al., 2005) . veev infection causes a typical biphasic febrile response in nhps. initial fever was observed at 12-72 h after infection and lasted less than 12 h. secondary fever generally began on day 5 and lasted 3-4 days (gleiser et al., 1961) . veev-infected nhps exhibited mild symptoms, such as anorexia, irritability, diarrhea, and tremors. leukopenia was common in animals exhibiting fever (monath et al., 1974) . supporting the leukopenia, microscopic changes in lymphatic tissues, such as early destruction of lymphocytes in lymph nodes and spleen, a mild lymphocytic infiltrate in the hepatic triads, and focal myocardial necrosis with lymphocytic infiltration have been observed in monkeys infected with veev. surprisingly, characteristic lesions of the cns were observed histopathologically in monkeys in spite of the lack of any clinical signs of infection (gleiser et al., 1961) . the primary lesions were lymphocytic perivascular cuffing and glial proliferation and generally observed at day 6 postinfection during the secondary febrile episode. similar to these observations, when cynomolgus macaques were exposed to aerosolized veev, fever, viremia, lymphopenia, and clinical signs of encephalitis were observed but the nhps did not succumb to disease (reed et al., 2004) . a common marmoset model was utilized for comparison studies of south america (sa) and north america (na) strains of eeev (adams et al., 2008) . previous studies indicated that the sa strain is less virulent than na strain for humans. common marmosets were infected in with either the na or sa strain of eeev. na strain-infected animals showed signs of anorexia and neurological involvement and were euthanized 4-5 days after the challenge. although sa strain-infected animals developed viremia, they remained asymptomatic and survived until the end of study. chikungunya virus (chikv) is a member of the genus alphaviruses, specifically the semliki forest complex, and has been responsible for a multitude of epidemics centered within africa and southeast asia (griffin, 2007) . the virus is transmitted by aedes aegypti and aedes albopictus mosquitoes. given the widespread endemicity of aedes mosquitoes, chikv has the potential to spread to previously unaffected areas. this is typified by the emergence of disease reported for the first time in 2005 in the islands of south-west indian ocean, including the french la reunion island, and the appearance in central italy in 2007 (charrel et al., 2007; rezza et al., 2007) . the incubation period following a mosquito bite is 2-5 days, leading to a self-limiting acute phase that lasts 3-4 days. symptoms during this period include fever, arthralgia, myalgia, and rash. headache, weakness, nausea, vomiting, and polyarthralgia have all been reported (powers and logue, 2007) . individuals typically develop a stooped posture due to the pain. for approximately 12% of infected individuals, joint pain can last months after resolution of primary disease, and has the possibility to relapse. underlying health conditions including diabetes, alcoholism, or renal disease, increase the risk of developing a severe form of disease that includes hepatitis or encephalopathy. children between the ages of 3 and 18 years old have an increased risk of developing neurological manifestations (arpino et al., 2009) . there is currently no approved vaccine or antiviral. wild-type c57bl/6 adult mice are not permissive to chikv infection by id inoculation. however, it was demonstrated that neonatal mice were susceptible and severity was dependent upon age at infection. six-dayold mice developed paralysis by day 6, and all succumbed by day 12, whereas 50% of 9-day-old mice were able to recover from infection. by 12 days, mice were no longer permissive to disease. symptomatic mice developed loss of balance, hind limb dragging, and skin lesions. neonatal mice were also used as a model for neurological complications (couderc et al., 2008; ziegler et al., 2008) . an adult mouse model has been developed by injection of the ventral side of the footpad of c57bl/6j mice. viremia lasted 4-5 days accompanied with foot swelling and noted inflammation of the musculoskeletal tissue morrison et al., 2011) . adult ifnα/βr knockout mice also developed mild disease with symptoms including muscle weakness and lethargy, symptoms that mirrored human infection. all adult mice died within 3 days. this model was useful in identifying the viral cellular tropism for fibroblasts (couderc et al., 2008) . icr and cd-1 mice can also be utilized as a disease model. neonatal mice inoculated subq with a passaged clinical isolate of chikv developed lethargy, loss of balance, and difficulty walking. mortality was low, 17 and 8% for newborn cd-1 and icr mice, respectively. the remaining mice fully recovered within 6 weeks after infection (ziegler et al., 2008) . a drawback of both the ifnα/βr and cd-1 mice is that the disease is not a result of immunopathogenesis as occurs in human cases, given that the mice are immunocompromised (teo et al., 2012) . a chronic infection model was developed using recombinant activating gene 1 (rag1 −/− ) knockout mice. in this study, mice inoculated via the footpad lost weight in comparison to the control group. both footpad and subq injected mice developed viremia 5-6 days postinfection, which was detectable up to 28 days postinfection. inflammation was evident in the brain, liver, and lung of the subq inoculated animals at 28-56 days postinfection. despite minimal footpad swelling on day 2 postinfection, on day 14 there was severe muscle damage noted at necropsy, which resolved by day 28 (seymour et al., 2015) . golden hamsters serve as another option for small animal modeling. although hamsters do not appear to develop overt clinical symptoms following subq inoculation, viremia developed in the majority of animals within 1 day postinfection with clearance following from day 3 to 4. histologically, inflammation was noted at the skeletal muscle, fascia, and tendon sheaths of numerous limbs. this study was limited in the number of animals utilized, and more work is needed to further develop the hamster model (bosco-lauth et al., 2015) . nhp models of disease include adult, aged, and pregnant rhesus macaques in addition to cynomolgus macaques (broeckel et al., 2015) . differing routes of infection (subq, iv, and im) have been successfully administered, although there is not a clear understanding of the role that route of transmission plays in subsequent pathogenesis and clinical symptoms. typically, viremia is observed 4-5 days postinfection with a correlation between infectious titer and time to viremia observed in cynomolgus but not rhesus (labadie et al., 2010; messaoudi et al., 2013) . fever began at 1-2 days postinfection and persisted for 2-7 days and 3-7 days in cynomolgus and rhesus, respectively and coincided with rash (chen et al., 2010; labadie et al., 2010; messaoudi et al., 2013) . overall blood chemistries changed in conjunction with initiation of viremia, and returned to baseline 10-15 days postexposure (chen et al., 2010) . cns involvement has been difficult to reproduce in nhp models, although it was reported that high inoculum in cynomolgus did result in meningoencephalitis (labadie et al., 2010) . the nhp models have been utilized to conduct efficacy testing on novel vaccines and therapeutics (broeckel et al., 2015) . dengue virus (denv) is transmitted via the mosquito vectors a. aegypti and a. albopictus (moore and mitchell, 1997) . given the endemicity of the vectors, it is estimated that half of the world's population is at risk for exposure to denv. this results in approximately 50 million cases of dengue each year, with the burden of disease in the tropical and subtropical regions of latin america, south asia, and southeast asia (gubler, 2002) . it is estimated that there are 20,000 deaths each year due to dengue hemorrhagic fever (dhf) (guzman and kouri, 2002) . there are four distinct serotypes of denv, numbered 1-4, which are capable of causing a wide clinical spectrum that ranges from asymptomatic to severe with the development of dhf (world health organization, 1997) . incubation can range from 3 to 14 days, with the average being 4-7 days. the virus targets dendritic cells and macrophages following a mosquito bite (balsitis et al., 2009) . typical infection results in classic dengue fever (df), which is self-limiting and has flu-like symptoms in conjunction with retroorbital pain, headache, skin rash, and bone and muscle pain. dhf can follow, with vascular leak syndrome and low platelet count, resulting in hemorrhage. in the most extreme cases, dengue shock syndrome (dss) develops, characterized by hypotension, shock, and circulatory failure (world health organization, 1997) . thrombocytopenia is a hallmark clinical sign of infection, and aids in differential diagnosis (gregory et al., 2010) . severe disease has a higher propensity to occur upon secondary infection with a different denv serotype (thein et al., 1997) . this is hypothesized to occur due to antibody dependent enhancement (ade). there is no approved vaccine or drug, and hospitalized patients receive supportive care including fluid replacement. in order to further progress toward an effective drug or vaccine, small human cohort studies have taken place. however, to provide statistically relevant results, testing must progress in an animal model. in developing an animal model, it is important to note that mosquitoes typically deposit 10 4 -10 6 pfu, and is considered the optimal range during experimental challenge . denv does not naturally replicate effectively in rodent cells, creating the need for mouse-adapted strains, engineered mouse lines, and a variety of inoculation routes to overcome the initial barrier. several laboratory mouse strains including a/j, balb/c, and c57bl/6 are permissive to dengue infection. however, the resulting disease has little resemblance to human clinical signs, and death results from paralysis (huang et al., 2000; paes et al., 2005; shresta et al., 2004) . a higher dose of an adapted denv strain induced dhf symptoms in both balb/c and c57bl/6 souza et al., 2009) . this model can also yield asymptomatic infections. a mouse-adapted strain of denv 2 introduced into ag129 mice developed vascular leak syndrome similar to the severe disease seen in humans (shresta et al., 2006) . passive transfer of monoclonal dengue antibodies within mice leads to ade. during the course of infection, viremia was increased and animals died due to vascular leak syndrome (balsitis et al., 2010) . another mouse-adapted strain injected into balb/c caused liver damage, hemorrhagic manifestations, and vascular permeability (souza et al., 2009) . ic injection of suckling mice with denv leads to death by paralysis and encephalitis, which is rare in human infection (lee et al., 2015; parida et al., 2002; zhao et al., 2014a) . immunocompromised mice have also been used to gain an understanding of the pathogenesis of denv. the most well-defined model is ag129 which is deficient in ifnα/β and γ receptors and can recapitulate dhf/dss if a mouse-adapted strain is utilized yauch et al., 2009) . scid mice engrafted with human tumor cells develop paralysis upon infection, and thus are not useful for pathogenesis studies (blaney et al., 2002; lin et al., 1998) . df symptoms developed after infection in nod/scid/il2rγko mice engrafted with cd34 + human progenitor cells (mota and rico-hesse, 2011) . rag-hu mice developed fever, but no other symptoms upon infection with a passaged clinical isolate and labadapted strain of denv 2 (kuruvilla et al., 2007) . a passaged clinical isolate of denv 3 was used to create a model in immunocompetent adult mice. ip injection in c57bl/6j and balb/c caused lethality by day 6-7 postinfection in a dose dependent manner. the first indication of infection was weight loss beginning on day 4 followed by thrombocytopenia. a drop in systolic blood pressure along with noted increases in the liver enzymes, ast and alt, were also observed. viremia was established by day 5. this model mimicked the characteristic symptoms observed in human dhf/dss cases (costa et al., 2012) . vascular leakage was also observed when c57bl/6 were inoculated with denv 2 (st john et al., 2013) . a murine model was developed that utilized infected mosquitoes as the route of transmission to hu-nsg mice. female mosquitoes were intrathoracically inoculated with a clinical isolate of denv 2. infected mosquitoes then fed upon the mouse footpad to allow for transmission of the virus via the natural route. the amount of virus detected within the mouse was directly proportional to the number of mosquitoes it was exposed to, with 4-5 being optimal. detectable viral rna was in line with historical human infection data. severe thrombocytopenia developed on day 14. this model is notable in that disease was enhanced with mosquito delivery of the virus in comparison to injection of the virus (cox et al., 2012) . nhp models have used a subq inoculation in an attempt to induce disease. although the animals are permissive to viral replication, it is to a lower degree than that observed in human infection (marchette et al., 1973) . the immunosuppressive drug, cyclophosphamide enhances infection in rhesus macaques by allowing the virus to invade monocytes (marchette et al., 1980) . throughout these preliminary studies, no clinical disease was detected. in order to circumvent this, a higher dose of denv was used in an iv challenge of rhesus macaques. hemorrhagic manifestations appeared by day 3 and resulted in petechiae, hematomas, and coagulopathy; however, no other symptoms developed (onlamoon et al., 2010) . a robust antibody response was observed in multiple studies (marchette et al., 1973; onlamoon et al., 2010) . marmosets also mirror human dengue infection, developing fever, leukopenia, and thrombocytopenia following subq inoculation (omatsu et al., 2011 (omatsu et al., , 2012 . nhps are able to produce antibodies similar to those observed during the course of human infection, making them advantageous in studying ade. sequential infection led to a cross-reactive antibody response which has been demonstrated in both humans and mice (midgley et al., 2011) . this phenotype can also be seen upon passive transfer of a monoclonal antibody to dengue and subsequent infection with the virus. rhesus macaques exposed in this manner developed viremia that was 3-to 100-fold higher than previously reported, however, no clinical signs were apparent (goncalvez et al., 2007) . the lack of inducible dhf or dss symptoms hinders further examination of pathogenesis within this model. west nile virus (wnv) was first isolated from the blood of a woman in the west nile district of uganda in 1937 uganda in (smithburn et al., 1940 . after the initial isolation of wnv, the virus was subsequently isolated from patients, birds, and mosquitoes in egypt in the early 1950s (melnick et al., 1951; taylor et al., 1953) and was shown to cause encephalitis in humans and horses. wnv is recognized as the most widespread of the flaviviruses, with a geographical distribution that includes africa, the middle east, western asia, europe, and australia (hayes, 1989) . the virus first reached the western hemisphere in the summer of 1999, during an outbreak involving humans, horses, and birds in the new york city metropolitan area (centers for disease control and prevention, 1999; lanciotti et al., 1999) . since 1999, the range of areas affected by wnv quickly extended. older people and children are most susceptible to wnv disease. wnv generally causes asymptomatic disease or a mild undifferentiated fever (west nile fever), which can last from 3 to 6 days (monath and tsai, 2002) . the mortality rate following neuroinvasive disease ranges from 4% to 11% (asnis et al., 2000; hayes, 1989; hubalek and halouzka, 1999; komar, 2000) . the most severe complications are commonly seen in the elderly, with reported case fatality rates from 4% to 11%. hepatitis, myocarditis, and pancreatitis are unusual, severe, nonneurologic manifestations of wnv infection. inoculation of wnv into nhps intracerebrally resulted in the development of either encephalitis, febrile disease, or an asymptomatic infection, depending on the virus strain and dose. viral persistence is observed in these animals regardless of the outcome of infection (i.e., asymptomatic, fever, encephalitis) (pogodina et al., 1983) . thus, viral persistence is regarded as a typical result of nhp infection with various wnv strains. after both intracerebral and subq inoculation, the virus localizes predominantly in the brain and may also be found in the kidneys, spleen, and lymph nodes. wnv does not result in clinical disease in nhps although the animals show a low level of viremia (lieberman et al., 2009; pletnev et al., 2003) . this is mirrored in new zealand white rabbits in that they only develop fever and low levels of viremia following inoculation via footpad (suen et al., 2015) . id inoculation of both marmosets and rhesus macaques did not yield any clinical signs of disease including fever. viremia was detected in both nhp species, but marmosets developed a higher titer for a greater duration than rhesus (verstrepen et al., 2014) . wnv has also been extensively studied in small animals. all classical laboratory mouse strains are susceptible to lethal infections by the intracerebral and ip routes, resulting in encephalitis and 100% mortality. id route pathogenesis studies indicated that langerhans dendritic cells are the initial viral replication sites in the skin (brown et al., 2007; johnston et al., 1996) . the infected langerhans cells then migrate to lymph nodes and the virus enters the blood through lymphatic and thoracic ducts and disseminates to peripheral tissues for secondary viral replication. virus eventually travels to the cns and causes pathology that is similar to human cases (byrne et al., 2001; cunha et al., 2000; diamond et al., 2003; fratkin et al., 2004) . the swiss mouse strain was inoculated ip in order to screen a variety of viral lineages to assess differences in pathogenesis (bingham et al., 2014) . tesh et al. developed a model for wn encephalitis using the golden hamster, mesocricetus auratus. hamsters appeared asymptomatic during the first 5 days, became lethargic at approximately day 6, and developed neurologic symptoms between days 7 and 10 . many of the severely affected animals died 7-14 days after infection. viremia was detected in the hamsters within 24 h after infection and persisted for 5-6 days. although there were no substantial changes in internal organs, progressive pathologic differences were seen in the brain and spinal cord of infected animals. furthermore, similar to the previously mentioned monkey experiments by pogodina et al. (1983) , persistent wnv infection was found in the brains of hamsters. zika virus recently came to the forefront of public health concerns with the outbreak in brazil at the end of 2015. the clinical disease spectrum is highly variable with reports of a flu-like illness accompanied by rash, guillan-barre syndrome, and microcephaly in newborns (ramos da silva and gao, 2016) . to date, a correlation between gestational age at which exposure to the virus occurs and severity of microcephaly is not fully understood (brasil et al., 2016) . however, a recent study of pregnant women in columbia found that infection with zika virus during the third trimester was not associated with any obvious structural abnormalities of the fetus (pacheco et al., 2016) . transmission of the virus occurs via the bite from an infected a. aegypti or a. albopictus (ramos da silva and gao, 2016) . other reported routes of exposure include sexual transmission and blood transfusion (cunha et al., 2016; d'ortenzio et al., 2016; hills et al., 2016; mccarthy, 2016) . the emergence of this virus with no approved vaccine or therapy, and few diagnostic options demonstrates the utility of well-characterized animal model development. it was first demonstrated in 1956 that experimentally infected mosquitoes could be used to transmit the virus to mice and nhps (boorman and porterfield, 1956) . a129 mice were susceptible to nonadapted zika virus infection following subq inoculation of the limbs. mice began to lose weight 3 days postinfection and met euthanasia criteria by day 6. microscopic lesions within the brain were noted upon necropsy. in conjunction, viral rna was detected in the blood, brain, ovary, spleen, and liver of the infected mice. wild-type 129sv/ev mice were also challenged with no observable clinical disease. however, viral rna was detected at day 3 postinfection in the blood, ovary and spleen, and then remained at detectable levels in the ovaries and spleen on day 7 (dowall et al., 2016) . footpad inoculation of the virus leads to a fatal disease in ag129 mice by day 7 postinoculation with significant histopathological changes in the brain noted at necropsy (aliota et al., 2016) . ag129 mice were also observed to develop neurologic disease by day 6 postexposure (rossi et al., 2016) . immunocompetent mice are resistant to infection via the subq route (rossi et al., 2016) . recently, a mouse model was identified to verify vertical transmission of the virus. pregnant c57 mice were injected either ip or in utero into the lateral ventricle of the fetal brain. ip inoculation induced transient viremia in the pregnant mice on day 1. viral rna was detected in five out of nine placentas on day 3 postinfection. the virus was able to infect the radial glia cells in the fetal brain and leads to a reduction in the cortical neural progenitors . viral exposure via cerebroventricular space/ lateral ventricle of the fetal brain exhibited small brain size at day 5 postexposure in addition to cortical thinning (cugola et al., 2016; li et al., 2016a) . ifnar1 −/− pregnant mice exposed to the virus had nonviable fetuses. in the same study, wild-type mice were given an anti-ifnar antibody prior to and during infection resulting in detectable virus in the fetal head with mild intrauterine growth restriction (miner et al., 2016) . all of these murine studies will further study of the pathogenesis of vertical transmission and the resulting neurological disorders in conjunction with screening novel countermeasures. nhp studies are currently ongoing for animal model development. numerous viruses from the coronavirus (cov) family exist that infect a wide range of animals. six species have been identified that can infect humans. two of these are alpha coronavirues: hcov-229e and hcov-nl63. four are beta coronavirueses: hcov-oc43, hcov-hku1, hcov-sars, and mers-cov. hcov-229e and hcov-oc43 were first detected in the 1960s from the nasal passages of humans with the "common cold" (gaunt et al., 2010) . hcov-nl63, which was first isolated in 2004, causes upper and lower respiratory infections of varying intensity and has been continuously circulating among humans (van der hoek et al., 2006) . hcov-hku1, first isolated in 2002, has been identified more sporadically but also causes respiratory infections (lau et al., 2006) . a significant portion of common cold infections in humans are caused by coronaviruses. in 2002 and 2012, two human coronaviruses, sars-cov and mers-cov, emerged that caused a great deal of alarm since these infections have resulted in nearly 10 and 40% fatality, respectively (assiri et al., 2013; peiris et al., 2004) . the etiologic agent of severe acute respiratory syndrome (sars), sars-cov, emerged in 2002 as it spread throughout 32 countries in a period of 6 months, with 8437 confirmed infections and 813 deaths (roberts and subbarao, 2006; world health organization, 2003) . no additional cases of community acquired sars-cov infection have been reported since 2004. the natural reservoir of sars-cov is the horseshoe bat and the palm civet is an intermediate host (lau et al., 2005) . the main mechanism of transmission of sars-cov is through droplet spread, but it is also viable in dry form on surfaces for up to 6 days and can be detected in stool, suggesting other modes of transmission are also possible (pearson et al., 2003; rabenau et al., 2005; rota et al., 2003) . sars-cov infection has a 10% case fatality with the majority of cases in people over the age of 15 (peiris et al., 2003; wang et al., 2004) . after an incubation period of 2-10 days, clinical signs of sars include general malaise, fever, chills, diarrhea, dyspnea, and cough (drosten et al., 2003) . in some sars cases, pneumonia may develop and progress to acute respiratory distress syndrome (ards). fever usually dissipates within 2 weeks and coincides with the induction of high levels of neutralizing antibodies (tan et al., 2004) . in humans, sars-cov replication destroys respiratory epithelium, and a great deal of the pathogenesis is due to the subsequent immune responses (chen and subbarao, 2007; perlman and dandekar, 2005) . infiltrates persisting within the lung and diffuse alveolar damage (dad) are common sequelae of sars-cov infection (perlman and dandekar, 2005) . virus can be isolated from secretions of the upper airways during early, but not later stages of infection as well as from other tissues (cheng et al., 2004) . sars-cov can replicate in many species, including: dogs, cats, pigs, mice, rats, ferrets, foxes, and monkeys (roper and rehm, 2009) . no model captures all aspects of human clinical disease (pyrexia and respiratory signs), mortality (∼10%), viral replication, and pathology (roberts et al., 2008) . in general, the sars-cov disease course in the model species is much milder and of shorter duration than in humans. viral replication in the various animal models may occur without clinical illness and/or histopathologic changes. the best-characterized models utilize mice, hamsters, ferrets, and nhps. mouse models of sars-cov typically are inoculated by the in route under light anesthesia (roberts et al., 2005) . young, 6-to 8-week-old balb/c mice exposed to sars-cov have viral replication detected in the lungs and nasal turbinate, with a peak on day 2 and clearance by day 5 postexposure (mcauliffe et al., 2004) . there is also viral replication within the small intestines of young balb/c mice. however, young mice have no clinical signs, aside from reduced weight gain, and have little to no inflammation within the lungs (pneumonitis) . in sars-cov infection of c57bl/6 (b6), also yields reduced weight gain and viral k. viral disease replication in the lungs, with a peak on day 3 and clearance by day 9 (glass et al., 2004) . in contrast, balb/c mice 13-14 months of age show weight loss, hunched posture, dehydration, and ruffled fur on day 3-6 postexposure (bisht et al., 2004) . interstitial pneumonitis, alveolar damage, and death also occur in old mice, resembling the age-dependent virulence observed in humans. 129s mice and b6 mice show outcomes to sars-cov infection similar to those observed for balb/c mice but have lower titers and less prolonged disease. while the aged mouse model is more frequently used then young mice, it is more difficult to obtain large numbers of mice older than 1 year (table 33 .1). a number of immunocompromised knockout mouse models of in sars-cov infection have also been developed. 129svev mice infected with sars-cov by the in route develop bronchiolitis, with peribronchiolar inflammatory infiltrates and interstitial inflammation in adjacent alveolar septae . viral replication and disease in these mice resolves by day 14 postexposure. beige, cd1 −/− , and rag1 −/− mice infected with sars-cov have similar outcomes to infected balb/c mice with regard to viral replication, timing of viral clearance, and a lack of clinical signs (glass et al., 2004) . stat1 ko mice infected in with sars-cov have severe disease, with weight loss, pneumonitis, interstitial pneumonia, and some deaths . the stat1 ko mouse model is therefore useful for studies of pathogenicity, pathology, and evaluation of vaccines. angiotensin converting enzyme 2 (ace2) and cd209l were identified as cellular receptors for sars-cov, with affinity for the spike (s) protein of the virus (jeffers et al., 2004) . the variations in the ace2 sequence across animal species could partially explain the differences in infection severity (li et al., 2016b; sutton and subbarao, 2015) . since mice in particular have a greater number of sequence differences in ace2, transgenic mice were created that express human ace2 (mccray et al., 2007; netland et al., 2008; yang et al., 2007) . unlike other murine models of sars-cov, mice expressing hace2 had up to 100% mortality, with severity correlating to the level of hace2 expression (tseng et al., 2007) . with high levels of hace2 expression, mice developed a severe lung and brain infection. however, cns k. viral disease infection is only rarely observed in humans infected with sars-cov. syrian golden hamsters (strain lvg) are also susceptible to in exposure of sars-cov. after the administration of 10 3 tcid 50, along with a period of transient viremia, sars-cov replicates in nasal turbinates and lungs, resulting in pneumonitis (roberts et al., 2005) . there are no obvious signs of disease, but exercise wheels can be used to monitor decrease in nighttime activity. limited mortality has been observed, but it was not dose dependent and could have more to do with genetic differences between animals because the strain is not inbred (roberts et al., 2008) . damage is not observed in the liver or spleen despite detection of virus within these tissues. several studies have shown that intratracheal (it) inoculation of sars-cov in anesthetized ferrets (mustela furo) results in lethargy, fever, sneezing, and nasal discharge (skowronski et al., 2005) . clinical disease has been observed in several studies excluding one, perhaps due to characteristics of the inoculating virus (kobinger et al., 2007) . sars-cov is detected in pharyngeal swabs, trachea, tracheobronchial lymph nodes, and high titers within the lungs. mortality has been observed around day 4 postexposure as well as mild alveolar damage in 5%-10% of the lungs, occasionally accompanied by severe pathology within the lungs (martina et al., 2003; ter meulen et al., 2004) . with fever, overt respiratory signs, lung damage, and some mortality, the ferret intratracheal model of sars-cov infection is perhaps most similar to human sars, albeit with a shorter time course. sars-cov infection of nhps by intransal or it routes generally results in a very mild infection that resolves quickly. sars-cov infection of old world monkeys, such as rhesus macaques, cynomolgus macaques (cynos), and african green monkeys (agms) have been studied with variable results, possibly due to the outbred nature of the groups studied or previous exposure to related pathogens. clinical illness and viral loads have not been consistent; however, replication within the lungs and dad are features of the infections for each of the primate species. some cynos have no illness but others have rash, lethargy, and respiratory signs and pathology martina et al., 2003; mcauliffe et al., 2004; rowe et al., 2004) . rhesus have little to no disease and only have mild findings upon histopathological analysis (rowe et al., 2004) . agms infected with sars-cov have no overt clinical signs but dad and pneumonitis has been documented (mcauliffe et al., 2004) . viral replication has been detected for up to 10 days in the lungs of agms; however, the infection resolves, and does not progress to fatal ards. farmed chinese masked palm civets, sold in open markets in china, were involved in the sars-cov outbreak. it and in inoculation of civets with sars-cov results in lethargy, decreased aggressiveness, fever, diarrhea, and conjunctivitis . leucopenia, pneumonitis, and alveolar septal enlargement, with lesions similar to those observed in ferrets and nhps, have also been observed in laboratory-infected civets. squirrel monkeys, mustached tamarinds, and common marmosets have not been susceptible to sars-cov infection (greenough et al., 2005; roberts et al., 2008) . vaccines have been developed for related animal covs in chickens, cattle, dogs, cats, and swine, and have included live-attenuated, killed, dna and viral-vectored vaccine strategies (cavanagh, 2003) . an important issue to highlight from work on these vaccines is that cov vaccines, such as those developed for cats, may induce a more severe disease (perlman and dandekar, 2005; weiss and scott, 1981) . as such, immune mice had th2type immunopathology upon sars-cov challenge (tseng et al., 2012) . severe hepatitis in vaccinated ferrets with antibody enhancement in liver has been reported (weingartl et al., 2004) . additionally, rechallenge of agms showed limited viral replication but significant lung inflammation, including alveolitis and interstitial pneumonia, which persisted for long periods of time after viral clearance (clay et al., 2012) . mouse and nhp models with increased virulence may be developed by adapting the virus by repeated passage within the species of interest. mouse-adapted sars with uniform lethality was developed from 15 serial passages in the lungs of young balb/c mice (mccray et al., 2007; roberts et al., 2007; rockx et al., 2007) . middle east respiratory syndrome (mers-cov) emerged in saudi arabia and is associated with fever, severe lower respiratory tract infection, and oftentimes renal failure (al-tawfiq et al., 2016; omrani et al., 2015) . mers patients can also occasionally manifest with neurological symptoms. mers-cov infection has a high fatality rate. infections in humans can also be asymptomatic. as of october 2015, there were 1589 confirmed cases and 567 deaths (li et al., 2016b) . bats serve as the likely natural reservoir since virus with 100% nucleotide identity to the index case was isolated from egyptian tomb bats (memish et al., 2013) . spread to humans likely comes from infected dromedary camels (adney et al., 2014; azhar et al., 2014) . the host range for mers-cov is dependent on the binding of the viral s protein to the host receptor, which is human dipeptidyl peptidase four (hdpp4), also known as cd26 (raj et al., 2013) . the expression and distribution of dpp4 in the human respiratory tract has recently been well characterized (meyerholz et al., 2016) . interestingly, dpp4 expression is preferentially localized to alveolar regions, perhaps explaining why mers predominantly manifests as an infection of the lower respiratory tract. humans with preexisting pulmonary disease have increased dpp4 expression in alveolar epithelia. small animals typically used for viral disease research, such as mice, hamsters, guinea pigs, and ferrets are naturally nonpermissive to mers-cov infection due to a low binding efficiency of the viral s protein to the host dpp4 (sutton and subbarao, 2015). in contrast the rhesus macaque and common marmoset have complete homology to human dpp4, allowing productive mers-cov infection to occur falzarano et al., 2014; munster et al., 2013; yao et al., 2014) . new zealand white rabbits can be infected with mers-cov, and virus was isolated from the upper respiratory tract, but there were no clinical symptoms or significant histopathological changes (haagmans et al., 2015) . due to the lack of strong binding affinity of the mers-cov s protein to the murine dpp4 receptor, wildtype mice are not susceptible to mers-cov infection. as such, several approaches have been used to create susceptible murine animal models of mers-cov infection by inducing the expression of hdpp4. one approach utilized an adenovirus vector expressing hdpp4 to transduce mice (zhao et al., 2014b) . these mice developed pneumonia but survived mers-cov infection. in mers-cov infection of mice with global expression of hdpp4 resulted in id 50 and ld 50 values of <1 and 10 tcid 50 , respectively (tao et al., 2016) . thus, mers-cov infection of these transgenic mice can be either sublethal or uniformly lethal depending on the dose. inflammatory infiltrates were found in the lungs and brain stems of mice with some focal infiltrates in the liver as well. another strategy uses transgenic mice expressing hdpp4 under either a surfactant protein c or cytokeratin 10 promoter (li et al., 2016b) . in mers-cov infection in these mice resulted in a uniformly lethal disease characterized by alveolar edema and microvascular thrombosis and mononuclear clear cell infiltration in the lungs. the brain stem was also impacted by the infection. dpp4 expression with an ubiquitously expressing promoter from cytomegalovirus also had a uniformly lethal infection with predominant lung and brain involvement, but numerous other tissues were also impacted and contained virus (agrawal et al., 2015) . common marmosets infected with 5.2 × 10 6 tcid 50 (emc-2012) mers-cov by the combined in, oral, ocular, and it routes capitulate the severe disease in human infections (falzarano et al., 2014) . the animals manifested moderate to severe clinical disease, with interstitial infiltration of both lower lung lobes. two of nine animals became moribund between days 4 and 6. viral rna was detected in nasal and throat swabs, various organs, and in the blood of some animals, indicating a systemic infection. histologically, animals showed evidence of acute bronchial interstitial pneumonia as well as other pathological defects. infection of rhesus macaques with mers-cov results in a mild clinical disease characterized by a transient lung infection with pneumonia. rhesus macaques were inoculated with at least 10 7 tcid 50 (emc-2012) mers-cov either by the it route or a combined in, it, oral, and ocular inoculation . the result was a mild respiratory illness including nasal swelling and a short fever with all animals surviving. viral rna was recovered from nasal swab samples and replicating virus was found in lung tissue . mild pathological lesions were found only in the lungs. radiographic imaging of the lungs revealed interstitial infiltrates, which are signs of pneumonia (yao et al., 2014) . interestingly, mer-cov infection is more severe in marmosets compared to rhesus macaques (falzarano et al., 2014) . this is despite the finding that both species have complete homology with humans within the dpp4 domain that interacts with the viral s protein. other host factors influencing disease severity have not yet been identified. transgenic mouse models expressing hdpp4 are ideal for initial development and screening of mers-cov countermeasures, and marmosets can be used for final selection and characterization. filoviridae consists of three genera, ebolavirus and marburgvirus, and a newly discovered group, cuevavirus (kuhn, 2008) . it is thought that various species of bats are the natural host reservoir for these viruses that have lethality rates from 40% to 82% in humans. there is evidence that the egyptian rousette bat (rousettus aegyptiacus) is the natural reservoir for marburgviruses but may not be for ebolaviruses (jones et al., 2015) . marburg virus (marv) first emerged in 1967 in germany when laboratory workers contracted the virus from agms (chlorocebus aerthiops) that were shipped from uganda. ebolaviruses sudan and zaire (sudv and ebov) caused nearly simultaneous outbreaks in 1976 in what is now the democratic republic of congo (drc). the most recent outbreak of ebov in west africa was by far the largest with over 28,000 suspected, probable and confirmed cases and over 11,000 deaths. bundibugyo virus (bdbv) first emerged in 2007 in bundibugyo, uganda with 56 confirmed cases . two other ebolaviruses are known: taï forest (tafv) (previously named cote d'ivoire) (ciebov) and reston (restv), which have not caused major outbreaks or lethal disease in humans. filovirus disease in humans is a characterized by aberrant innate immunity and a number of clinical symptoms: fever, nausea, vomiting, k. viral disease arthralgia/myalgia, headaches, sore throat, diarrhea, abdominal pain, and anorexia as well as numerous others (mehedi et al., 2011; wauquier et al., 2010) . approximately 10% of patients develop petechia and a greater percentage, depending on the specific strain, may develop bleeding from various sites (gums, puncture sites, stools, etc.) (table 33 .2). natural transmission in an epidemic is through direct contact or needle sticks in hospital settings. however, much of the research interest in filoviruses primarily stems from biodefense needs, particularly from aerosol biothreats. as such, im, ip, and aerosol models have been developed in mice, hamsters, guinea pigs, and nhps for the study of pathogenesis, correlates of immunity, and for testing countermeasures . since filoviruses have such high lethality rates in humans, scientists have looked for models that are uniformly lethal to stringently test efficacy of candidate vaccines and therapeutics. one issue to take note of in animal model development of filovirus infection is the impact of particle to plaque-forming unit (pfu) ratios on lethality, wherein it is possible that increasing the dose could actually decrease infectivity due to an immunogenic effect produced by inactive virions in the stock. additionally, the plaque assay used to measure live virions in a stock may greatly underestimate the true quantity of infectious virions in a preparation (alfson et al., 2015; smither et al., 2013a) . immunocompetent mice have not been successfully infected with wild-type filoviruses due to the control of the infection by the murine type 1 interferon response (bray, 2001) . however, wild-type inbred mice are susceptible to filovirus that has been mouse adapted (ma) by serial passage in mice (bray et al., 1999) . marv angola was particularly resistant to adaptation, but after 24 serial passages in scid mice, infection caused severe disease in balb/c and c57bl/6 mice when administered in or ip (qiu et al., 2014) . these mice had pathology with some similarities to infection in humans including lymphopenia, thrombocytopenia, liver damage, and viremia. balb/c mice, which are the strain of choice for ip inoculation of ma-ebov, are not susceptible by the aerosol route (bray et al., 1999; zumbrun et al., 2012a) . for aerosol infection of immunocompetent mice, a panel of bxd (balb/c x dba) recombinant inbred strains were screened and one strain, bxd34, was particularly susceptible to airborne ma-ebov, with 100% lethality to low or high doses (approximately 100 or 1000 pfu) ( zumbrun et al., 2012a) . these mice developed weight loss of greater than 15% and succumbed to infection between days 7 and 8 postexposure. the aerosol infection model utilizes a whole-body exposure chamber to expose mice aged 6-8 weeks to ma-ebov aerosols with a mass median aerodynamic diameter (mmad) of approximately 1.6 µm and a geometric standard deviation (gsd) of approximately 2.0 for 10 min. another approach uses immunodeficient mouse strains, such as scid, stat1 ko, ifn receptor ko, or perforin ko with a wild-type ebov inoculum by ip or aerosol routes (bray, 2001; lever et al., 2012; zumbrun et al., 2012a) . mice are typically monitored for clinical disease "scores" based on activity and appearance, weight loss, and moribund condition (survival). coagulopathy, a hallmark of filovirus infection in humans, has been observed, with bleeding in a subset of animals and failure of blood samples to coagulate late in infection (bray et al., 1999) . liver, kidney, spleen, and lung tissue taken from moribund mice have pathology characteristic of filovirus disease in nhps (zumbrun et al., 2012a) . while most mouse studies have used ma-ebov or ebov, an ip mouse-adapted marv model is also available (warfield et al., 2007 (warfield et al., , 2009 ). ma-marv and ma-ebov models are particularly useful for screening novel antiviral compounds (panchal et al., 2012) . recently, a model was created using immunodeficient nsg [nonobese diabetic (nod)/scid/il-2 receptor chain knockout] mice with transplanted human hematopoietic stem cells from umbilical cord blood. these mice were susceptible to lethal wt (nonadapted) ebov by ip and in exposure (ludtke et al., 2015) . the transplanted mice had all of the cellular components of a fully functional adaptive human immune system and upon ebov (brannan et al., 2015; lever et al., 2012) . interestingly, inoculation of infa/br −/− mice with tafv and restv does not result in clinical signs. yet another strategy uses knockout mice lacking possible receptors for filovirus entry, such as niemann-pick c1 and c2 (npc1 and npc2). npc2 (−/−) mice were fully susceptible to infection with ebov but npc1 (−/−) mice were completely resistant (herbert et al., 2015) . hamsters are frequently used to study cardiovascular disease, coagulation disorders, and thus serve as the basis for numerous viral hemorrhagic fever models (gowen and holbrook, 2008; herbert et al., 2015) . an ip ma-ebov infection model has been developed in syrian hamsters gowen and holbrook, 2008; herbert et al., 2015; tsuda et al., 2011) . this model, which has been used to test a vesicular stomatitis virus vectored vaccine approach, utilizes male 5-to 6-week-old syrian hamsters which are infected with 100 ld 50 of ma-ebov. virus is present in tissues and blood collected on day 4 and all animals succumbed to the disease by day 6. infected hamsters had severe coagulopathy and uncontrolled host immune responses, similar to what is observed in primates. (ebihara et al., 2010) guinea pig models of filovirus infection have been developed for ip and aerosol routes using guinea pigadapted ebov (gp-ebov) and marv (gp-marv) (choi et al., 2012; connolly et al., 1999; twenhafel et al., 2015; zumbrun et al., 2012c) . guinea pig models of filovirus infection are quite useful in that they develop fever, which can be monitored at frequent intervals by telemetry. additionally, the animals are large enough for regular blood sampling in which measurable coagulation defects are observed as the infection progresses. a comparison of ip infection of outbred guinea pigs with guinea pig-adapted marv angola and marv ravn revealed similar pathogenesis (cross et al., 2015) . infection with either strain resulted in features of the disease that are similar to what is seen in human and nhp infection, such as viremia, fever, coagulopathy, lymphopenia, elevated liver enzymes (alt and ast), thrombocytopenia, and splenic, gastrointestinal and hepatic lesions. gp-marv-ravn had a delayed disease progression relative to gp-marv-ang. hartley guinea pigs exposed to aerosolized gp-ebov develop lethal interstitial pneumonia. this is in contrast to subq infection of guinea pigs, aerosol ebov challenge of nhps, and natural human infection (twenhafel et al., 2015) . both subq and aerosol exposure of guinea pigs to gp-ebov resulted in only mild lesions in the liver and spleen. by aerosol exposure, gp-ebov is uniformly lethal at both high and low doses (100 or 1000 pfu target doses) but lethality drops with low (less than 1000 pfu) presented doses of airborne gp-marv and more protracted disease is seen in some animals (our unpublished observations) (zumbrun et al., 2012c) . weight loss of between 15% and 25% is a common finding in guinea pigs exposed to gp-ebov or gp-marv. fever, which becomes apparent by day 5, occurs more rapidly in gp-ebov exposed guinea pigs than with gp-marv exposure. lymphocytes and neutrophils increase during the earlier part of the disease, and platelet levels steadily drop as the disease progresses. increases in coagulation time can be seen as early as day 6 postexposure. blood chemistries (i.e., alt, ast, alkp, and bun) indicating problems with liver and kidney function are also altered late in the disease course. transmission of ebov has been documented from swine to nhps via the respiratory tract (kobinger et al., 2011) . as such, guinea pigs have been used to establish transmission models (wong et al., 2015a,b) . nonexposed guinea pigs were placed in the cages with infected guinea pigs 1 day postexposure to gp-ebov. guinea pigs challenged intanasally were more likely to transmit virus to naive cagemates than those that were exposed by the ip route. nhp models of filovirus infection are the preferred models for more advanced disease characterization and testing of countermeasures because they most closely mimic the disease and immune correlates seen in humans (dye et al., 2012) . old world primates have been primarily used for development of ip, im, and aerosol models of filovirus infection ( twenhafel et al., 2013) . uniformly lethal filovirus models have been developed for most of the virus strains in cynomolgus macaques, rhesus macaques, and to a lesser degree, agms (alves et al., 2010; carrion et al., 2011; davis et al., 1997; hensley et al., 2011a; reed et al., 2007; zumbrun et al., 2012b) . low-passage human isolates that have not been passaged in animals have been sought for development of nhp models to satisfy the food and drug administration (fda) animal rule. ebov-makona, the strain responsible for the recent large outbreak in west africa, was compared to the "prototype" 1976 ebov strain (marzi et al., 2015) . the disease in cynos was similar for both viruses, but disease progression was delayed for ebov-makona. this delay as well as the lower fatality rate in the 2014 epidemic compared to the 1976 outbreak suggest that ebov-makona is less virulent. the large number of cases in the 2014-15 ebov outbreak brought to light previously underappreciated eye pathology and ocular viral persistence in survivors. while survivors of nhp filovirus infection are infrequent, necrotizing scleritis, conjunctivitis, and other ocular pathology has been observed in ebov-infected animals (alves et al., 2016) . prominent features of the filovirus infections in nhps are onset of fever by day 5 postexposure, viremia, lymphopenia, tachycardia, azotemia, alteration in liver function enzymes (alt, ast, and alkp), decrease in platelets, and increased coagulation times. petechial rash is a common sign of filovirus disease and may be more frequently observed in cynomolgus macaques than in other nhp species (zumbrun et al., 2012b) . immunological parameters have been evaluated and t, b, and natural killer cells are greatly diminished as the infection progresses (fernando et al., 2015) . a cytokine storm occurs with rises in ifnγ, tnf, il-6, and ccl2 (fernando et al., 2015) . however, there is also evidence from transcriptional profiling of circulating immune cells that the early immune response is skewed toward a th2 response (connor et al., 2015) . strikingly, animals surviving challenge may have a delay in the production of inflammatory cytokines and chemokines (martins et al., 2015) . clinical disease parameters may have a slightly delayed onset in aerosol models. dyspnea late in infection is a prominent feature of disease after aerosol exposure (zumbrun et al., 2012b) . aerosol filovirus infection of nhps results in early infection of respiratory lymphoid tissues, dendritic cells, alveolar macrophages, blood monocytes, and fibroblastic reticular cells followed by spread to regional lymph nodes then multiple organs (ewers et al., 2016; twenhafel et al., 2013) . a number of pronounced pathology findings include multifocal hepatic necrosis and fibrin accumulation, particularly within the liver and the spleen. for aerosolized marv infection of rhesus, the most significant pathology included destruction of the tracheobronchial and mediastinal lymph nodes (ewers et al., 2016) . lymphocytolysis and lymphoid depletion are also observed (alves et al., 2010) . multilead, surgically implanted telemetry devices are useful in continuous collection of temperature, blood pressure, heart rate, and activity levels. as such, blood pressure drops as animals become moribund and heart rate variability (standard deviation of the heart rate) is altered late in infection (zumbrun et al., 2012b) . the most recently developed telemetry devices can also aid in plethysmography to measure respiratory minute volume for accurate delivery of presented doses for aerosol exposure. standardized filovirus-infected nhp euthanasia criteria have also been developed to enhance reproducibility for studies that evaluate therapeutic and vaccine countermeasures (warren et al., 2014) . filovirus infection of common marmosets (callithrix jacchus) is also a viable model to study the disease course. respiratory infection of marmosets with marv results in a lethal infection with fever, hemorrhaging, transient rash, disseminated viral infection, increases in liver function enzymes, coagulopathy, hepatitis, and histological lesions particularly in the kidney and liver (smither et al., 2013b) . marmosets are similarly susceptible to infection with ebovkikwit (smither et al., 2015) . thus, ebov or marv infection of marmosets produces features of the disease that are very similar to that of other nhps and humans. hendra and nipah virus are unusual within the paramyxoviridae family given that they can infect a large range of mammalian hosts. both viruses are grouped under the genus henipavirus. the natural reservoirs of the viruses are the fruit bats from the genus pteropus. hendra and nipah have the ability to cause severe disease in humans with the potential for a high case fatality rate (rockx et al., 2012) . outbreaks due to nipah virus have been recognized in malaysia, singapore, bangladesh, and india, while hendra virus outbreaks have yet to be reported outside of australia (luby et al., 2009a,b) . hendra was the first member of the genus identified and was initially associated with an acute respiratory disease in horses. all human cases have been linked to transmission through close contact with an infected horse. there have been no confirmed cases of direct transmission from bat to human. nipah has the distinction of transmission among, although the exact route is unknown (homaira et al., 2010) . the virus is susceptible to ph, temperature, and desiccation, and thus close contact is hypothesized as needed for successful transmission (fogarty et al., 2008) . both viruses have a tropism for the neurological and respiratory tracts. the incubation period for hendra virus is 7-17 days and is marked by a flu-like illness. symptoms at this initial stage include myalgia, headache, lethargy, sore throat, and vomiting (hanna et al., 2006) . disease progression can continue to pneumonitis or encephalitic manifestations, with the person succumbing to multiorgan failure (playford et al., 2010) . nipah virus has an incubation period of 4 days to 2 weeks (goh et al., 2000) . much like hendra, the first signs of disease are nondescript. severe neurological symptoms subsequently develop including encephalitis and seizures that can progress to coma within 24-48 h (lo and rota, 2008). survivors of infection typically make a full recovery; however, 22% suffer permanent sequelae, including persistent convulsions (tan and chua, 2008) . at this time, there is no approved vaccine or antiviral, and treatment is purely supportive. animal models are being used to not only test novel vaccines and therapeutics, but also deduce the early events of disease because documentation of human cases is at terminal stages. the best small animal model is the syrian golden hamster due to their high susceptibility to both henipaviruses. clinical signs upon infection recapitulate the disease course in humans including acute encephalitis and respiratory distress. challenged animals died within 4-17 days postinfection. the progression of disease and timeline is highly dependent on dose and route of infection. in inoculation leads to imbalance, limb paralysis, lethargy, and breathing difficulties whereas ip resulted in tremors and paralysis within 24 h before death. virus was detected in lung, brain, spleen, kidney, heart, spinal cords, and urine, with the brain having the highest titer. this model is used for vaccination and passive protection studies (guillaume et al., 2009; rockx et al., 2011; wong et al., 2003) . the guinea pig model has not been widely used due to the lack of a respiratory disease upon challenge (torres-velez et al., 2008; williamson et al., 2001) . inoculation with hendra virus via the subq route leads to a generalized vascular disease with 20% mortality. clinical signs were apparent 7-16 days postinfection with death occurring within 2 days of cns involvement. higher inoculum has been associated with development of encephalitis and cns lesions. id and in injection does not lead to disease, although the animals are able to seroconvert upon challenge. the inoculum source does not affect clinical progression. nipah virus challenge only causes disease upon ip injection and results in weight loss and transient fever for 5-7 days. virus was shed through urine and was present in the brain, spleen, lymph nodes, ovary, uterus, and urinary bladder (hooper et al., 1997) . ferrets infected with hendra or nipah virus display the same clinical disease as seen in the hamster model and human cases (bossart et al., 2009; pallister et al., 2011) . upon inoculation by the oronasal route, ferrets develop severe pulmonary and neurological disease within 6-9 days including fever, coughing, and dyspnea. lesions do develop in the ferret's brains, but to a lesser degree than seen in humans. cats have also been utilized as an animal model for henipaviruses. disease symptoms are not dependent upon the route of infection. the incubation period is 4-8 days and leads to respiratory and neurological symptoms (mungall et al., 2007; johnston et al., 2015; westbury et al., 1996) . this model has proven useful for vaccine efficacy studies. squirrel and agms are representative of the nhp models. for squirrel monkeys, nipah virus is introduced by either the in or iv route and subsequently leads to clinical signs similar to humans, although in challenge results in milder disease. upon challenge, only 50% of animals develop disease manifestations including anorexia, dyspnea, and acute respiratory syndrome. neurological involvement is characterized by uncoordinated motor skills, loss of consciousness, and coma. viral rna can be detected in lung, brain, liver, kidney, spleen, and lymph nodes but is only found upon iv challenge (marianneau et al., 2010) . agms are very consistent model of both viruses. it inoculation of the viruses results in 100% mortality, and death within 8.5 and 9-12 days postinfection for hendra and nipah viruses, respectively. the animals develop severe respiratory and neurological disease with generalized vasculitis rockx et al., 2010) . the reservoir of the viruses, gray-headed fruit bats, has been experimentally challenged. due to their status as the host organism for henipaviruses, the bats do not develop clinical disease. however, hendra virus can be detected in kidneys, heart, spleen, and fetal tissue, and nipah virus can be located in urine . pigs develop a respiratory disease upon infection with both nipah and hendra viruses (berhane et al., 2008; li et al., 2010; middleton et al., 2002) . oral inoculation does not produce a clinical disease, but subq injection represents a successful route of infection. live virus can be isolated from the oropharynx as early as 4 days postinfection. nipah virus can also be transmitted between pigs. nipah virus was able to induce neurological symptoms in 20% of the pigs, even though virus was present in all neurological tissues regardless of symptoms (weingartl et al., 2005) . within the pig model, it appeared that nipah virus had a greater tropism for the respiratory tract, while hendra for the neurological system. horses are also able to develop a severe respiratory tract infection accompanied with fever and general weakness upon exposure to nipah and hendra viruses. oronasal inoculation led to systemic disease with viral rna detected in nasal swabs within 2 days (marsh et al., 2011; williamson et al., 1998) . animals died within 4 days postexposure and have interstitial pneumonia with necrosis of alveoli (murray et al., 1995a,b) . virus could be detected in all major systems. mice, rats, rabbits, chickens, and dogs have been tested but are nonpermissive to infection (westbury et al., 1995; wong et al., 2003) . suckling balb/c mice succumb to infection if the virus is inoculated intracranially (mungall et al., 2006) . in exposure with nipah does not induce a clinical disease; however, there is evidence of a subclinical infection in the lungs following euthanasia of the mice (dups et al., 2014) . in addition, a human lung xenograph model in nsg mice demonstrated that the human lung is highly susceptible to nipah viral replication and damage (valbuena et al., 2014) . embryonated chicken eggs have been inoculated with nipah virus leading to a universally fatal disease within 4-5 days postinfection (tanimura et al., 2006) . annually, respiratory syncytial virus (rsv) is responsible for the lower respiratory tract infections of 33 million children under the age of 5, which in turn results in 3 million hospitalizations and approximately 200,000 deaths (nair et al., 2010) . within the united states, hospital costs alone amount to over 600 million dollars per year (paramore et al., 2004) . outbreaks are common in the winter (yusuf et al., 2007) . the virus is transmitted by large respiratory droplets that replicate initially within the nasopharynx and spreads to the lower respiratory tract. incubation for the virus is 2-8 days. rsv is highly virulent leading to very few asymptomatic infections (collins and graham, 2008) . disease manifestations are highly dependent upon the age of the individual. rsv infections in neonates produce nonspecific symptoms including overall failure to thrive, apnea, and feeding difficulties. infants present with a mild upper respiratory tract disease that could develop into bronchiolitis and bronchopneumonia. contracting rsv at this age results in an increased chance of developing childhood asthma (wu et al., 2008) . young children develop recurrent wheezing while adults have exacerbation of previously existing respiratory conditions (falsey et al., 2005) . common clinical symptoms are runny nose, sneezing, and coughing accompanied by fever. mortality rates from rsv in hospitalized children are 1%-3% with the greatest burden of disease seen in 3-4 month olds (ruuskanen and ogra, 1993). hematopoietic stem cell transplant patients, solid organ transplant patients, and copd patients are particularly vulnerable to rsv infection and have mortality rates between 7.3% and 13.3% upon infection (anderson et al., 2016) . although there are almost 60 rsv vaccine candidates which are in preclinical and clinical phases, there is no licensed vaccine available and ribavirin usage is not recommended for routine treatment (american academy of pediatrics subcommittee on diagnosis and management of bronchiolitis, 2006; higgins et al., 2016; kim and chang, 2016) . animal models of rsv were developed in the hopes of formulating an effective and safe vaccine unlike the formalin-inactivated rsv (fi-rsv) vaccine. this vaccine induced severe respiratory illness in infants whom received the vaccine and were subsequently infected with live virus (kim et al., 1969) . mice can be used to model rsv infection, although a very high in inoculation is needed to achieve clinical symptoms (jafri et al., 2004; stark et al., 2002) . strain choice is crucial to reproducing a physiological relevant response (stokes et al., 2011). age does not affect primary disease manifestations (graham et al., 1988) . however, it does play a role in later sequelae showing increased airway hyperreactivity . primary rsv infection produces increased breathing with airway obstruction (jafri et al., 2004; van schaik et al., 1998) . virus was detected as early as day 3 and reached maximum titer at day 6 postinfection. clinical illness is defined in the mouse by weight loss and ruffled fur as opposed to runny nose, sneezing, and coughing as seen in humans. a humanized mouse model was recently developed by in inoculation. the challenged mice experienced weight loss and demonstrated a humoral and cellular immune response to the infection (sharma et al., 2016) . cotton rats are useful given that rsv is able to replicate to high titers within the lungs and can be detected in both the upper and lower airways after in inoculation (boukhvalova et al., 2009; niewiesk and prince, 2002) . viral replication is 50-to 1000-fold greater in the cotton rat model than mouse model (wyde et al., 1993) . the cotton rats develop mild to moderate bronchiolitis or pneumonia (grieves et al., 2015; prince et al., 1999) . although age does not appear to factor into clinical outcome, it has been reported that older cotton rats tend to take longer to achieve viral clearance. viral loads peak by the 5th day, dropping to below the levels of detection by day 8. the histopathology of the lungs appears similar to that of humans after infection (piazza et al., 1993) . this model has limited use in modeling the human immune response to infection as challenge with the virus induces a th2 response in cotton rats, whereas humans tend to have a response skewed toward th1 (culley et al., 2002; dakhama et al., 2005; ripple et al., 2010) . fi-rsv disease was recapitulated upon challenge with live virus after being vaccinated twice with fi-rsv. chinchillas have been challenged experimentally with rsv via in inoculation. the virus was permissive within the nasopharynx and eustachian tube. the animals displayed an acute respiratory tract infection. this model is therefore useful in studying mucosal immunity during infection (gitiban et al., 2005) . ferrets infected by it were found to have detectable rsv in throat swabs up to day 7 postinfection, and positive qpcr up to day 10. immunocompromised ferrets were observed to have higher viral loads accompanied with detectable viral replication in the upper respiratory tract (stittelaar et al., 2016) . chimpanzees are permissive to replication and clinical symptoms of rsv including rhinorrhea, sneezing, and coughing. adult squirrel monkeys, newborn rhesus macaques, and infant cebus monkeys were also challenged but did not exhibit any disease symptoms or high levels of viral replication (belshe et al., 1977) . bonnet monkeys were developed an inflammatory response by day 7 with viral rna detected in both bronchial and alveolar cells (simoes et al., 1999) . the chimpanzee model has been proven useful for vaccine studies (hancock et al., 2000; teng et al., 2000) . sheep have also been challenged experimentally since they develop respiratory disease when exposed to ovine rsv (meyerholz et al., 2004) . lambs are also susceptible to human respiratory syncytial infection (olivier et al., 2009; sow et al., 2011) . when inoculated intratracheally, the lambs developed an upper respiratory tract infection with cough after 6 days. some lambs went on to develop lower respiratory disease including bronchiolitis. the pneumonia resolved itself within 14 days. rsv replication peaked at 6 days, and rapidly declined. studying respiratory disease in sheep is beneficial given the shared structural features with humans (plopper et al., 1983; scheerlinck et al., 2008) . the influenza viruses consist of three types: influenza a, b, and c, based on antigenic differences. influenza a is further classified by subtypes; 16 ha and 9 na subtypes are known. seasonal influenza is the most common infection and usually causes a self-limited febrile illness with upper respiratory symptoms and malaise that resolves within 10 days (taubenberger and morens, 2008) . the rate of infection is estimated at 10% in the general population and can result in billions of dollars of loss annually from medical costs and reduced work-force productivity. approximately 40,000 people in the united states die each year from seasonal influenza (dushoff et al., 2006) . thus, vaccines and therapeutics play a critical role in controlling infection, and development using animal models is ongoing (braun et al., 2007b) . influenza virus replicates in the upper and lower airways, peaking at approximately 48-h postexposure. infection can be more severe in infants and children under the age of 22, people over the age of 65, or immunocompromised individuals where viral pneumonitis or pneumonia can develop or bacterial superinfection resulting in pneumonia or sepsis (barnard, 2009; glezen, 1982) . pneumonia from secondary bacterial infection, such as streptococcus pneumonia, streptococcus pyogenes, and neisseria meningitides, and more rarely, staphylococcus aureus, is more common than viral pneumonia from the influenza virus itself, accounting for ∼27% of all influenza associated fatalities (alonso et al., 2003; ison and lee, 2010; speshock et al., 2007) . death, often due to ards can occur as early as 2 days after onset of symptoms. lung histopathology in severe cases may include dad, alveolar edema and damage, hemorrhage, fibrosis, and inflammation (taubenberger and morens, 2008) . the h5n1 avian strain of influenza, has lethality rates of around ∼50% (of known cases), likely because the virus preferentially binds to the cells of the lower respiratory tract, and thus the potential for global spread is a major concern (matrosovich et al., 2004; wang et al., 2016) . h7n9 is another avian influenza a strain that infected more than 130 people and was implicated in 37 deaths. approximately 75% of infected people had a known exposure to birds. there is no evidence of sustained spread between humans but these viruses are of great concern for their pandemic potential (zhang et al., 2013) . the most frequently used animal models of influenza infection include mice, ferrets, and nhps. a very thorough guide to working with mouse, guinea pig, ferret, and cynomolgus models was published by kroeze et al. (2012) . swine are not frequently utilized but are also a potentially useful model for influenza research since they share many similarities to human anatomy, genetics, susceptibility, and pathogenesis (rajao and vincent, 2015). lethality rates can vary with virus strain used (with or without adaptation), dose, route of inoculation, age, and genetic background of the animal. the various animal models can capture differing diseases caused by influenza: benign, severe, super infection, and sepsis, severe with ards, and neurologic manifestations (barnard, 2009) . also, models can utilize seasonal or avian strains and have been developed to study transmission, important for understanding the potential for more lethal strains, such as h5n1 for spreading among humans. mouse models of influenza infection are very predictive for antiviral activity and tissue tropism in humans, and are useful in testing and evaluating vaccines (gilbert and mcleay, 2008; hagenaars et al., 2008; ortigoza et al., 2012) . inoculation is by the in route, utilizing approximately 60 µl of inoculum in each nare of anesthetized mice. exposure may also be to small particle aerosols containing influenza with a mmad of <5 µl. most inbred strains are susceptible, with particularly frequent use of balb/c followed by c57bl/6j mice. males and females have equivalent disease but influenza is generally more infectious in younger 2-to 4-week-old (8-10 g) mice. mice are of somewhat limited use in characterizing the immune response to influenza. most inbred laboratory mice lack the mxa gene which is an important part of human innate immune response to influenza infection. the mouse homolog to mxa, mx1 is defective in most inbred mouse strains (staeheli and haller, 1987) . mice with the knocked-in mx1 gene have a 1000-fold higher ld-50 for an influenza a strain (pr8) than wildtype background c57bl/6 mice (grimm et al., 2007) . weight loss or reduced weight gain, decreased activity, huddling, ruffled fur, and increased respiration are the most common clinical signs in influenza infected mice. for more virulent strains, mice may require euthanasia as early as 48 h postexposure, but most mortality occurs from 5 to 12 days postexposure accompanied by decreases in rectal temperature (sidwell and smee, 2000). pulse oximeter readings and measurement of blood gases for oxygen saturation are also used to determine the impact of influenza infection on respiratory function (sidwell et al., 1992) . virus can be isolated from bronchial lavage (bal) fluids throughout the infection and from tissues after euthanasia. for influenza strains with mild to moderate pathogenicity, disease is nonlethal and virus replication is detected within the lungs, but usually not other organs. increases in serum alpha-1-acidglycoprotein and lung weight also frequently occur. however, mice infected with influenza do not develop fever, dyspnea, nasal exudates, sneezing, or coughing. mice can be experimentally infected with influenza a or b, but the virus generally requires adaptation to produce clinical signs. mice express the receptors for influenza attachment in the respiratory tract; however, the distribution varies and sa 2,3 predominates over sa 2,6 which is why h1, h2, and h3 subtypes usually need to be adapted to mice and h5n1, h2, h6, and h7 viruses do not require adaptation (o'donnell and subbarao, 2011). to adapt, mice are infected intratracheally or intranasally by virus isolated from the lungs, and reinfected into mice and then the process is repeated a number of times. once adapted, influenza strains can produce severe disease, systemic spread, and neurotropism. h5n1 and the 1918 pandemic influenza virus can cause lethal infection in mice without adaptation (gao et al., 1999; taubenberger, 2006) . h5n1 infection of mice results in viremia and viral replication in multiple organ systems, severe lung pathology, fulminant diffuse interstitial pneumonia, pulmonary edema, high levels of proinflammatory cytokines, and marked lymphopenia ( dybing et al., 2000; gubareva et al., 1998; lu et al., 1999) . as in humans, the virulence of h5n1 is attributable to damage caused by an overactive host immune response. additionally, mice infected with the 1918 h1n1 influenza virus produce severe lung pathology and oxygen saturation levels that decrease with increasing pneumonia (barnard et al., 2007) . reassortment influenza viruses of the 2009 h1n1 virus and a low-pathogenicity avian h7n3 virus can also induce disease in mice without adaptation . in superinfection models, a sublethal dose of influenza is given to mice followed 7 days later by in inoculation of a sublethal dose of a bacterial strain, such as s. pneumoniae or s. pyogenes (chaussee et al., 2011) . morbidity, characterized by inflammation in the lungs, but not bacteremia, begins a couple of days after superinfection and may continue for up to 2 weeks. at least one transmission model has also been developed in mice. with h2n2 influenza, transmission rates of up to 60% among cagemates can be achieved after infection by the aerosol route and cocaging after 24 h (schulman, 1968). rats (f344 and sd) inoculated with rat-adapted h3n2 developed inflammatory infiltrates and cytokines in bronchoalveolar lavage fluids, but had no lethality and few histopathological changes (daniels et al., 2003) . additionally, an influenza transmission model has been developed in guinea pigs as an alternative to ferrets (lowen et al., 2006) . cotton rats (sigmodon hispidus) have been used to test vaccines and therapeutics in a limited number of studies (eichelberger et al., 2004) . cotton rats have an advantage over mice in that the immune system is similar to humans (including the presence of the mx gene) and influenza viruses do not have to be adapted (eichelberger et al., 2006; ottolini et al., 2005) . nasal and pulmonary tissues of cotton rats were infected with unregulated cytokines and lung viral load peaking at 24 h postexposure. virus was cleared from the lung by day 3 and from the nares by day 66, but animals had bronchial and alveolar damage, and pneumonia for up to 3 weeks. there is also a s. aureus superinfection model in cotton rats (braun et al., 2007a) . coinfection resulted in bacteremia, high bacterial load in lungs, peribronchiolitis, pneumonitis, alveolitis, hypothermia, and higher mortality. domestic ferrets (mustela putorius furo) are frequently the animal species of choice for influenza animal studies because the susceptibility, clinical signs, peak virus shedding, kinetics of transmission, local expression of cytokine mrnas, and pathology resemble that of humans (lambkin et al., 2004; maines et al., 2012; mclaren and butchko, 1978) . like humans, ferrets exclusively express neu5ac, which acts as a receptor for influenza a virus, a feature likely contributing to the susceptibility of ferrets to human-adapted influenza a virus strains (ng et al., 2014) . the glycomic characterization of ferret respiratory tract tissues demonstrated some similarities and some differences to humans in terms of the potential glycan binding sites for the influenza virus (jia et al., 2014) . ferrets also have airway morphology, respiratory cell types, and a distribution of influenza receptors (sa 2,6 and sa 2,3) within the airways similar to that of humans (van riel et al., 2007) . influenza was first isolated from ferrets infected in with throat washes from humans harboring the infection and ferret models have since been used to test efficacy of vaccines and therapeutic treatments (huber et al., 2008; lambkin et al., 2004; maines et al., 2012) . when performing influenza studies in ferrets, animals should be serologically negative for circulating influenza viruses. infected animals should be placed in a separate room from uninfected animals. if animals must be placed in the same room, uninfected ferrets should be handled before infected ferrets. anesthetized ferrets are experimentally exposed to influenza by in inoculation of 0.25-0.5 ml containing approximately 10 4 -10 6 egg id 50 dropwise to each nostril. however, a larger inoculum volume of 1.0 ml has also been explored as being more appropriate, yielding more severe and consistent respiratory tract pathology, likely because the larger inoculum is more widely distributed in the lower respiratory tract (moore et al., 2014) . video tracking to assign values to activity levels in ferrets can aid ferret studies, eliminating the need for collection of subjective and arbitrary clinical scores (oh et al., 2015) . viral replication in the upper respiratory tract is typically measured by nasal washes, but virus can also be measured in bronchoalveolar lavage fluid using a noninvasive technique (lee et al., 2014) . influenza types a and b naturally infect ferrets, resulting in an acute illness, which usually lasts 3-5 days for mild to moderately virulent strains (maher and destefano, 2004) . ferrets are more susceptible to influenza a than influenza b strains and are also susceptible to avian influenza h5n1 strains without adaptation (zitzow et al., 2002) . however, the localized immune responses within the respiratory tract of ferrets infected with influenza a and b have been characterized and are similar (carolan et al., 2015) . virulence and degree of pneumonitis caused by different influenza subtypes and strains vary from mild to severe and generally mirrors that seen in humans (stark et al., 2013) . nonadapted h1n1, h2n2, and h3n2 have mild to moderate virulence in ferrets. the sequencing of the ferret genome has allowed for the characterization of the ferret host response using rnaseq analysis . distinct signatures were obtained depending on the particular influenza strain to inoculate the ferrets. also helpful is the sequencing and characterization of the influenza ferret infectome during different stages of the infection in naïve or immune ferrets (leon et al., 2013) . since influenza infection is particularly devastating to the elderly population, an aged ferret model of h1n1 influenza infection was developed (paquette et al., 2014) . features associated with increased clinical disease are weakened hemagglutinin antibody generation and attenuated th1 responses. pregnant and breastfeeding women and infants are also susceptible to more severe illness from influenza virus. to study this dynamic, a breastfeeding mother-infant ferret influenza infection model was created (paquette et al., 2015) . notably, the mammary gland itself harbored virus and transcript analysis showed downregulation of milk production genes. in support of the development of therapies, the ferret influenza model for pharmacokinetic/pharmacodynamics studies of antiviral drugs as also been developed (reddy et al., 2015) . critical to this model is ensuring pronounced clinical signs and robust viral replication upon influenza infection. strains of low virulence have predominant replication in the nasal turbinates of ferrets. clinical signs and other disease indicators in ferrets are similar to that of humans with mild respiratory disease, sneezing, nasal secretions containing virus, fever, weight loss, high viral titers, and inflammatory infiltrate in the airways, bronchitis, and pneumonia (svitek et al., 2008) . replication in both the upper and lower airways is associated with more severe disease and greater mortality. additionally, increased expression of proinflammatory mediators and reduced expression of antiinflammatory mediators in the lower respiratory tract of ferrets correlates with severe disease and lethal outcome. h5n1-infected ferrets develop severe lethargy, greater interferon response, transient lymphopenia, and replication in respiratory tract, brain, and other organs (peng et al., 2012; zitzow et al., 2002) . immunocompromised humans have influenza illness of greater duration and complications. immunocompromised ferrets infected with influenza similarly had prolonged virus shedding (van der vries et al., 2013) . interestingly, antiviral resistance emerged in both humans and ferrets with immunocompromised status infected with influenza. alveolar macrophage depleted of ferrets infected with 2009 pandemic h1n1 influenza also had a more severe disease with greater viral replication in the lungs and greater induction of inflammatory chemokines (kim et al., 2013) . a superinfection model resembling that of mice has been developed by in instillation of influenza in 6-to 8-week-old ferrets followed by in inoculation of s. pneumonia 5 days later (peltola et al., 2006) . this typically resulted in otitis media, sinusitis, and pneumonia. transmission models in ferrets have recently met with worldwide media attention and controversy with regard to the study of h5n1 (enserink, 2013; fouchier et al., 2012; herfst et al., 2012; oh et al., 2014) . in general, some subtypes, such as the 2009 h1n1, can transmit easily through aerosol and respiratory droplets (munster et al., 2009) . of concern, h7n9 isolated from humans was more pathogenic and readily transmissible between ferrets by larger respiratory droplets and smaller particle aerosols (kreijtz et al., 2013; richard et al., 2013; zhang et al., 2013) . h5n1 became transmissible by adopting just four mutations, spreading between ferrets in separate cages (imai et al., 2012) . transmission occurs more readily at the height of pyrexia, but for the 2009 h1n1 in particular, can occur before fever is detected (roberts et al., 2012) . ferret-to-ferret transmission of a mouseadapted influenza b virus has also been demonstrated (kim et al., 2015) . since ferrets can be expensive and cumbersome, influenza infection has been characterized and a transmission model developed in the guinea pig; however, this is a newer model with infrequent utilization thus far (lowen et al., 2014) . old and new world primates are susceptible to influenza infection and have an advantage over ferret and mouse models which are deficient for h5n1 vaccine studies because there is a lack of correlation with hemagglutination inhibition (murphy et al., 1980) . of old world primates, cynomolgus macaque (macaca fascicularis) is most frequently utilized for studies of vaccines and antiviral drug therapies (stittelaar et al., 2008) . h5n1 and h1n1 1918 infection of cynos is very similar to humans (rimmelzwaan et al., 2001) . cynos develop fever and ards upon in inoculation of h5n1 with necrotizing bronchial interstitial pneumonia . nhps are challenged by multiple routes k. viral disease (ocular, nasal, and tracheal) simultaneously 1 × 10 6 pfu per site. virus antigen is primarily localized to the tonsils and pulmonary tissues. infection of cynos with h5n1 results in fever, lethargy, nasal discharge, anorexia, weight loss, nasal and tracheal washes, pathologic and histopathologic changes, and alveolar and bronchial inflammation. the 1918 h1n1 caused a very high mortality rate due to an aberrant immune response and ards and had more than 50% lethality (humans only had a 1%-3% lethality) (kobasa et al., 2007) . ards and mortality also occur with the more pathogenic strains, but nhps show reduced susceptibility to less virulent strains, such as h3n2 (o'donnell and subbarao, 2011) . influenza-infected rhesus macaques represent a mild disease model for vaccine and therapeutic efficacy studies (baas et al., 2006) . host microarray and qrt-pcr proved useful for analysis of infected lung tissues. other nhp models include influenza infection of pigtailed macaques as a mild disease model and infection of new world primates, such as squirrel and cebus monkeys (baskin et al., 2009) . domestic pig models have been developed for vaccine studies for swine flu. pigs are susceptible in nature as natural or intermediate hosts but are not readily susceptible to h5n1 (isoda et al., 2006; lipatov et al., 2008) . while pigs infected with influenza may have fever, anorexia, and respiratory signs, such as dyspnea and cough, mortality is rare (van der laan et al., 2008) . size and space requirements make this animal difficult to work with, although the development of minipig models may provide an easier to use alternative. cat and dog influenza models have primarily been utilized to study their susceptibility to h5n1 with the thought that these animals could act as sentinels or could serve to transmit the virus to humans (giese et al., 2008; rimmelzwaan et al., 2006) . these models are not generally used to better understand the disease in humans or for testing vaccines or antivirals. rift valley fever virus (rvfv) causes epizootics and human epidemics in africa. rvfv mainly infects livestock, such as sheep, cattle, goats, etc. after 2-4 days incubation period, animals show signs of fever, hepatitis, and abortion, which is a hallmark diagnostic sign known among farmers (balkhy and memish, 2003) . mosquito vectors, unpasteurized milk, aerosols of infected animal's body fluids, or direct contact with infected animals are the important routes of transmission to humans (abu-elyazeed et al., 1996; mundel and gear, 1951) . after 2-to 6-day-incubation period, rvfv causes a wide range of signs and symptoms in humans ranging from asymptomatic to severe disease with hepatitis, vision loss, encephalitis, and hemorrhagic fever (ikegami and makino, 2011; laughlin et al., 1979; peters and linthicum, 1994) . depending on the severity of the disease when the symptoms start, 10%-20% of the hospitalized patients might die in 3-6 days or 12-17 days after the disease onset (ikegami and makino, 2011) . hepatic failure, renal failure or dic, and encephalitis are demonstrated within patients during postmortem examination. live domestic animals especially sheep and goats were used to develop animal models of rvfv (weingartl et al., 2014) . this study indicated that goats were more resistant to the disease compared to sheep. the viremia in goats was lower and had a shorter duration with only some animals developing fever. the susceptibility is influenced by route of infection, breed of animals, the rvfv strain, and growth conditions as well as the passage history. therefore, it might be difficult to establish an animal model with domestic ruminants. mice are one of the most susceptible animal species to rvfv infection. several mouse models including balb/c, ifnar −/− , mbt/pas, 129 and c57bl/6 were exposed to rvfv via parental or aerosol routes of infection (ross et al., 2012) . subq or ip routes of infection cause acute hepatitis and lethal encephalitis at a late stage of the disease in mice (mims, 1956; smith et al., 2010) . mice start to show signs of decreased activity and ruffled fur by day 2-3 postexposure. immediately following these signs, they become lethargic and generally die 3-6 days postexposure. ocular disease or the hemorrhagic form of the disease has not been observed in mouse models so far (ikegami and makino, 2011) . increased viremia and tissue tropism were reported in mice with (smith et al., 2010) increased liver enzymes and lymphopenia observed in sick mice. aerosolized rvfv causes faster and more severe neuropathy in mice compared to the parental route (dodd et al., 2014; reed et al., 2014) . the liver is a target organ following aerosol exposure and liver failure results in fatality. rats and gerbils are also susceptible to rvfv infection. the rat's susceptibility is dependent on the rat strain utilized for the challenge model and route of exposure. there is also noted age dependence in the susceptibility of rats. while wistar-furth and brown norway strains, and young rats are highly susceptible to rvfv infection, fisher 344, buffalo and lewis strains, and old rats demonstrated resistance to infection via subq route of infection (findlay and howard, 1952; peters and slone, 1982) . similar pathologic changes, such as liver damage and encephalopathy were observed in both rats and mice. the recent study by bales et al. (2012) showed that aerosolized rvfv caused similar disease outcome in wistar-furth and aci rats while lewis rats developed fatal encephalitis which was much more severe than the subq route of infection. there was no liver involvement in the gerbil model and animals died from severe encephalitis. the mortality rate was dependent on the strain used and the dose given to gerbils (anderson et al., 1988) . similar to the rat model, the susceptibility of gerbils was also dependent on age. natural history studies with syrian hamsters indicated that the liver was the target organ with highly elevated alt levels and viral titers (scharton et al., 2015) . lethargy, ruffled fur, and hunched posture were observed in hamsters by day 2 post-subq inoculation and the disease was uniformly lethal by day 2-3 postexposure. this model has been successfully used to test antivirals against rvfv (scharton et al., 2015) . studies thus far showed that rvfv does not cause uniform lethality in a nhp model. ip, in, iv, and aerosol routes have been utilized to develop nhp model. rhesus macaques, cynomolgus macaques, african monkeys, and south american monkeys were some of the nhp species used for this effort . monkeys showed a variety of signs ranging from febrile disease to hemorrhagic disease and mortality. temporal viremia, increased coagulation parameters (pt, aptt), and decreased platelets were some other signs observed in nhps. animals that succumbed to disease showed very similar pathogenesis to humans, such as pathological changes in the liver and hemorrhagic disease. there was no ocular involvement in this model. smith et al. compared iv, in and subq routes of infection in common marmosets and rhesus macaques (peng et al., 2012) . marmosets were more susceptible to rvfv infection than rhesus macaques with marked viremia, acute hepatitis, and late onset of encephalitis. increased liver enzymes were observed in both species. necropsy results showed enlarged livers in the marmosets exposed by iv or subq routes. although there were no gross lesions in the brains of marmosets, histopathology showed encephalitis in the brains of in challenged marmosets. a recent study by hartman et al. (2014) demonstrated that aerosolized rvfv only caused mild fever in cynomolgus macaques and rhesus macaques, while agms and marmosets had encephalitis and succumbed to disease between days 9 and 11 postexposure. in contrast to other lethal models, the brain was the target organ in agms and marmosets. although no change was observed in ast levels, alp levels were increased in marmosets. little or no change was observed in hepatic enzyme levels in agms. lack of information regarding human disease concerning the aerosol route of exposure makes it difficult to evaluate these animal models. crimean-congo hemorrhagic fever virus (cchfv) generally circulates in nature unnoticed in an enzootic tick-vertebrate-tick cycle and similar to other zoonotic agents, appears to produce little or no disease in its natural hosts, but causes severe disease in humans. cchfv transmits to humans by ixodid ticks, direct contact with sick animals/humans, or body fluids of animals/humans (ergonul and whitehouse, 2007) . incubation, prehemorrhagic, hemorrhagic, and convalescence are the four phases of the disease seen in humans. the incubation period lasts 1-9 days. during the prehemorragic phase, patients show signs of nonspecific flu-like disease for approximately a week. the hemorrhagic period results in circulatory shock and dic in some patients (mardani and keshtkar-jahromi, 2007; swanepoel et al., 1989) . over the years, several attempts have been made to establish an animal model for cchf in adult mice, guinea pigs, hamsters, rats, rabbits, sheep, nhps, etc. (fagbami et al., 1975; nalca and whitehouse, 2007; shepherd et al., 1989; smirnova, 1979) . until recently, the only animal that manifests disease is the newborn mouse. infant mice ip infected with cchfv resulted in fatality around day 8 postinfection (tignor and hanham, 1993) . pathogenesis studies showed that virus replication was first detected in the liver, with subsequent spread to the blood (serum). virus was detected very late during the disease course in other tissues including the heart (day 6) and the brain (day 7). the recent studies utilizing knockout adult mice were successful to develop a lethal small animal model for cchfv infection (bente et al., 2010; bereczky et al., 2010) . bente et al. infected stat1 knockout mice by the ip route. in this model, after the signs of fever, leukopenia, thrombocytopenia, viremia, elevated liver enzymes and proinflammatory cytokines, mice were moribund and succumbed to disease in 3-5 days postexposure. the second model was developed by using interferon alpha/beta (ifnα/β) receptor knockout mice (ifnar −/− ) (bereczky et al., 2010) . similar observations were made in this model as in the stat1 knockout mouse model. animals were moribund and died 2-4 days after exposure with high viremia levels in liver and spleen. characterization studies with ifnar −/− mice challenged with different routes (ip, in, im, and subq) showed that cchfv causes acute disease with high viral loads, pathology in liver and lymphoid tissues, increased proinflammatory response, severe thrombocytopenia, coagulopathy, and death, all of which are characteristics of human disease . proinflammatory cytokines and chemokines, such as g-csf, ifnγ, cxc-cl10, ccl2 increased dramatically day 3 postchallenge and gm-csf, il-1a, il-1b, il-2, il-6, il-12p70, il-13, il-17, cxcl1, ccl3, ccl5, and tnf-α concentrations were extremely elevated at the time of death/euthanasia. this model is also utilized to test therapeutics, such as ribavirin, arbidol, and t-705 (favipiravir) successfully (oestereich et al., 2014) . experimental vaccines developed for cchf were evaluated in this model provided protection compare to unvaccinated mice (buttigieg et al., 2014; canakoglu et al., 2015, p. 725) . thus, the ifnar −/− mouse model would be a good choice to test medical countermeasures against cchfv, although they have an impaired ifn and immune response phenotype. other laboratory animals, including nhps, show little or no sign of infection or disease when infected with cchfv (nalca and whitehouse, 2007) . butenko et al. utilized agms (cercopithecus aethiops) for experimental cchfv infections. except one monkey with a fever on day 4 postinfection, the animals did not show signs of disease. antibodies to the virus were detected in three out of five monkeys, including the one with fever. fagbami et al. (1975) infected two patas monkeys (erythrocebus patas) and one guinea baboon (papio papio) with cchfv. whereas all three animals had low-level viremia between days 1 and 5 after inoculation, only the baboon serum had neutralizing antibody activity on day 137 postinfection. similar results were obtained when horses and donkeys have been used for experimental cchfv infections. donkeys develop a low-level viremia (rabinovich et al., 1972) and horses developed little or no viremia, but high levels of virus-neutralizing antibodies, which remained stable for at least 3 months. these studies suggest that horses may be useful in the laboratory to obtain serum for diagnostic and possible therapeutic purposes (blagoveshchenskaya et al., 1975 (blagoveshchenskaya et al., ). shepherd et al. (1989 infected 11 species of small african wild mammals and laboratory rabbits, guinea pigs, and syrian hamsters with cchfv. whereas scrub hares (lepus saxatilis), cape ground squirrels (xerus inauris), red veld rats (aethomys chrysophilus), white-tailed rats (mystromys pumilio), and guinea pigs had viremia; south african hedgehogs (atelerix frontalis), highveld gerbils (gerbilliscus brantsii), namaqua gerbils (desmodillus auricularis), two species of multimammate mouse (mastomys natalensis and mastomys coucha), and syrian hamsters were negative for virus. all species regardless of viremia levels developed antibody responses against cchfv. iv and intracranially infected animals showed onset of viremia earlier than those infected by the subq or ip routes. the genus hantavirus is unique among the family bunyaviridae in that it is not transmitted by an arthropod vector, but rather rodents (schmaljohn and nichol, 2007) . rodents of the family muridae are the primary reservoir for hantaviruses. infected host animals develop a persistent infection that is typically asymptomatic. transmission is achieved by inhalation of infected rodent saliva, feces, and urine (xu et al., 1985) . human infections can normally be traced to a rural setting with activities, such as farming, land development, hunting, and camping as possible sites of transmission. rodent control is the primary route of prevention (lednicky, 2003) . the viruses have a tropism for endothelial cells within the microvasculature of the lungs (zaki et al., 1995) . there are two distinct clinical diseases that infection can yield: hemorrhagic fever with renal syndrome (hfrs) due to infection with old world hantaviruses or hantavirus pulmonary syndrome (hps) caused by new world hantaviruses (nichol, 2001) . hfrs is mainly seen outside of the americas and is associated with the hantaviruses dobrava-belgrade (also known as dobrava), hantaan, puumala, and seoul (lednicky, 2003) . incubation lasts 2-3 weeks and presents as flu-like in the initial stages that can further develop into hemorrhagic manifestations and ultimately renal failure. thrombocytopenia subsequently develops which can further progress to shock in approximately 15% patients. overall mortality rate is 7%. infection with dobrava and hantaan viruses are typically linked to development of severe disease. hps was first diagnosed in 1993 within southwestern united states when healthy young adults became suddenly ill, progressing to severe respiratory distress and shock. the etiological agent responsible for this outbreak was identified as sin nombre virus (snv) (centers for disease control and prevention, 1993) . this virus is still the leading cause within north america of hps. hps due to other hantaviruses has been reported in argentina, bolivia, brazil, canada, chile, french guiana, panama, paraguay, and uruguay (padula et al., 2000; stephen et al., 1994) . the first report of hps in maine was recently documented (centers for disease control and prevention, 1993). andes virus (andv) was first identified in outbreaks in chile and argentina. this hantavirus is distinct in that it can be transmitted between humans (wells et al., 1997) . the fulminant disease is more lethal than that observed of hfrs with a mortality rate of 40%. there are four phases of disease including prodromal, pulmonary, cardiac depression, and hematologic manifestation (peters and khan, 2002) . incubation typically occurs 14-17 days following exposure (young et al., 2000) . unlike hfrs, renal failure is not a major contributing factor to the disease. there is a short prodromal phase that gives way to cardiopulmonary involvement accompanied by cough and gastrointestinal symptoms. it is at this point that individuals are typically admitted to the hospital. pulmonary function is hindered and continues to suffer within 48 h after cardiopulmonary involvement. interstitial edema and air-space disease normally follow. in fatal cases, cardiogenic shock has been noted (hallin et al., 1996) . syrian golden hamsters are the most widely utilized small animal model for hantavirus infection. hamsters inoculated im with a passaged andes viral strain died within 11 days postinfection. clinical signs did not appear until 24 h prior to death at which point the hamsters were moribund and in respiratory distress. mortality was dose dependent, with high inoculums leading to a shorter incubation before death. during the same study, hamsters were inoculated with a passaged snv isolate. no hamsters developed any symptoms during the course of observation. however, an antibody response to the virus that was not dose dependent was determined via elisa. hamsters infected with andv have significant histopathological changes to their lung, liver, and spleen. all had an interstitial pneumonia with intraalveolar edema. infectious virus could be recovered from these organs. viremia began on day 8 and lasted up to 12 days postinfection. infection of hamsters with andv yielded a similar clinical disease progression as is seen in human hps including rapid progression to death, fluid in the pleural cavity, and significant histopathological changes to the lungs and spleen. a major deviation in the hamster model is the detection of infectious virus within the liver . normally, snv does not cause a disease in hamsters (wahl-jensen et al., 2007) . but a recent study showed that immunosuppression with dexamethasone and cyclophosphamide in combination causes lethal disease with snv in hamsters (brocato et al., 2014) . the disease was very similar to the disease caused by andv in hamsters. lethal disease can be induced in newborn mice, but does not recapitulate the clinical symptoms observed in human disease (kim and mckee, 1985) . the disease outcome is very much dependent on the age of the mice. younger mice are much more susceptible to virus than the adult mice. adult mice exposed to hanta virus leads to a fatal disease dependent upon viral strain and route of infection. the disease progression is marked by neurological or pulmonary manifestations that do not mirror human disease (seto et al., 2012; wichmann et al., 2002) . knockout mice lacking ifnα/β are highly susceptible to hanta virus infection (muller et al., 1994) . in a study of panel of laboratory strains of mice, c57bl/6 mice were most susceptible to a passaged hanta viral strain injected ip. animals progressed to neurological manifestation including paralyses and convulsions, and succumbed to infection within 24-36 h postinfection. clinical disease was markedly different from that observed in human cases (wichmann et al., 2002) . in a recent study, 2-weekold icr mice was exposed to htnv strain aa57 via the subq route (seto et al., 2012) . mice started to show signs of disease by day 11 postinoculation. piloerection, trembling, hunching, loss of body weight, labored breathing, and severe respiratory disease were observed in mice. studies to develop nhp models were not successful until recently. nhps have been challenged with new world hantaviruses; however, no clinical signs were reported (hooper et al., 2006; mcelroy et al., 2002) . cynomolgus monkeys challenged with a clinical isolate of puumala virus developed a mild disease (klingstrom et al., 2002; sironen et al., 2008) . challenge with andv to cynomolgus macaques by both iv and aerosol exposure led to no signs of disease. all animals did display a drop in total lymphocytes within 5 days postinfection. four of six aerosol exposed monkeys and 8 of 11 iv injected monkeys developed viremia. infectious virus could not be isolated from any of the animals. in a recent study, rhesus macaques were inoculated by the intramuscular route with snv passaged only in deer mice (safronetz et al., 2014) . characteristics of hps disease including rapid onset of respiratory distress, severe pulmonary edema, thrombocytopenia, and leukocytosis were observed in this promising model. viremia was observed 4-10 days prior to respiratory signs of the disease that were observed on days 14-16 postinoculation. with all aspects, this animal model would be very useful to test medical countermeasures against hanta virus. the family arenaviridae is composed of two serogroups: old world arenaviruses including lassa fever virus and lymphocytic choriomeningitis virus and the new world viruses of pichinde virus and junin virus. all of these viruses share common clinical manifestations (mccormick and fisher-hoch, 2002) . lassa fever virus is endemic in parts of west africa and outbreaks are typically seen in the dry season between january and april (curtis, 2006) . this virus is responsible for 100,000-500,000 infections per year, leading to approximately 5000 deaths (khan et al., 2008) . outbreaks have been reported in guinea, sierra leone, liberia, nigeria, and central african republic. however, cases have sprung up in germany, netherlands, united kingdom, and the united states due to transmission to travelers on commercial airlines (amorosa et al., 2010) . transmission of this virus typically occurs via rodents, in particular the multimammate rat, mastomys species complex (curtis, 2006) . humans become infected by inhaling the aerosolized virus or eating contaminated food. there has also been noted human-to-human transmission by direct contact with infected secretions or needle-stick injuries. the majority of infections are asymptomatic; however, severe disease occurs in 20% of individuals. the incubation period is from 5 to 21 days and initial onset is characterized by flu-like illness. this is followed by diarrheal disease that can progress to hemorrhagic symptoms including encephalopathy, encephalitis, and meningitis. a third of patients develop deafness in the early phase of disease that is permanent for a third of those affected. the overall fatality is about 1%; however, of those admitted to the hospital it is between 15% and 25%. there is no approved vaccine and besides supportive measures, ribavirin is effective only if started within 7 days (mccormick et al., 1986a,b) . the primary animal model used to study lassa fever is the rhesus macaque (jahrling et al., 1980) . aerosolized infection of lymphocytic choriomeningitis virus has been a useful model for lassa fever. both rhesus and cynomolgus monkeys exposed to the virus developed disease, but rhesus mirrored more closely the disease course and histopathology observed in human infection (danes et al., 1963) . iv or intragastric inoculation of the virus led to severe dehydration, erythematous skin, submucosal edema, necrotic foci in the buccal cavity, and respiratory distress. the liver was severely affected by the virus as depicted by measuring the liver enzymes ast and alt (lukashevich et al., 2003) . disease was dose dependent with iv, intramuscular, and subq inoculation requiring the least amount of virus to induce disease. aerosol infections and eating contaminated food could also be utilized, and mimic a more natural route of infection (peters et al., 1987) . within this model, the nhp becomes viremic after 4-6 days. clinical manifestations were present by day 7 and death typically occurred within 10-14 days (lukashevich et al., 2004; rodas et al., 2004) . intramuscular injection of lassa virus into cynomolgus monkeys also produced a neurological disease due to lesions within the cns (hensley et al., 2011b) . this pathogenicity is seen in select cases of human lassa fever (cummins et al., 1992; gunther et al., 2001) . a marmoset model has recently been defined utilizing a subq injection of lassa fever virus. virus was initially detected by day 8 and viremia achieved by day 14. liver enzymes were elevated and an enlarged liver was noted upon autopsy. there was a gradual reduction in platelets and interstitial pneumonitis diagnosed in a minority of animals. the physiological signs were the same as seen in fatal human cases (carrion et al., 2007) . mice develop a fatal neurological disorder upon intracerebral inoculation with lassa, although the outcome of infection is dependent on the mhc background, age of the animal, and inoculation route (salvato et al., 2005) . stat1 knockout mice inoculated ip with both lethal and nonlethal lassa virus strains develop hearing loss accompanied by damage to the inner ear hair cells and auditory nerve (yun et al., 2015) . guinea pig inbred strain 13 was highly susceptible to lassa virus infection. the outbred hartley strain was less susceptible, and thus strain 13 has been the preferred model given its assured lethality. the clinical manifestations mirror those seen in humans and rhesus (jahrling et al., 1982) . infection with pichinde virus passaged in guinea pigs has also been used. disease signs include fever, weight loss, vascular collapse, and eventual death (lucia et al., 1990; qian et al., 1994) . the guinea pig is an excellent model given that it not only results in similar disease pattern, viral distribution, histopathology, and immune response to humans (connolly et al., 1993; katz and starr, 1990) . infection of hamsters with a cotton rat isolate of pirital virus is similar to what is characterized in humans, and the nhp and guinea pig models. the virus was injected ip resulting in lethargy and anorexia within 6-7 days. virus was first detected at 3 days, and reached maximum titers within 5 days. neurological symptoms began to appear at the same time, and all animals died by day 9. pneumonitis, pulmonary hemorrhage, and edema were also present (sbrana et al., 2006) . these results were recapitulated with a nonadapted pichinde virus (buchmeier and rawls, 1977; gowen et al., 2005; smee et al., 1993) . the lentiviruses are a subfamily of retroviridae, which includes human immunodeficiency virus (hiv), a virus that infects 0.6% of the world's population. a greater proportion of infections and deaths occur in subsaharan africa. worldwide, there are approximately 1.8 million deaths per year with over 260,000 being children. transmission of hiv occurs by exposure to infectious body fluids. there are two species, hiv-1 and hiv-2, with hiv-2 having lower infectivity and virulence (confined mostly to west africa). the vast majority of cases worldwide are hiv-1 (de cock et al., 2011) . hiv targets t-helper cells (cd4+), macrophages, dendritic cells (fields et al., 2007) . acute infection occurs 2-4 weeks after exposure, with flu-like symptoms and viremia followed by chronic infection. symptoms in the acute phase may include fever, body aches, nausea, vomiting, headache, lymphadenopathy, pharyngitis, rash, and sores in the mouth or esophagus. cd8+ t-cells are activated which kill hiv-infected cells, and are responsible for antibody production and seroconversion. acquired immune deficiency syndrome (aids) develops when cd4+ t-cells decline to less than 200 cells/µl; thus cell-mediated immunity becomes impaired and the person is more susceptible to opportunistic infections as well as certain cancers. hiv has a narrow host range likely because the virus is unable to antagonize and evade effector molecules of the interferon response (thippeshappa et al., 2012) . humanized mice, created by engrafting human cells and tissues into scid mice, have been critical for the development of mouse models for the study of hiv infection. a number of different humanized mouse models allow for the study of hiv infection in the context of an intact and functional human innate and adaptive immune responses (berges and rowan, 2011) . the scidhu hiv infection model has proven useful, particularly in screening antivirals and therapeutics (denton et al., 2008; melkus et al., 2006) . a number of different humanized mouse models have been developed for the study of hiv, including rag1 −/− γc −/− , rag2 −/− γc −/− , nod/scidγc −/− (hnog), nod/scidγc −/− (hnsg), nod/scid blt, and nod/scidγc −/− (hnsg) blt (karpel et al., 2015; li et al., 2015; shimizu et al., 2015) . cd34+ human stem cells derived from umbilical cord blood or fetal liver are used for humanization (baenziger et al., 2006; watanabe et al., 2007) . hiv-1 infection by ip injection can be successful with as little as 5% peripheral blood engraftment (berges et al., 2006) . vaginal and rectal transmission models have been developed in blt scid hu mice in which mice harbor human bone marrow, liver, and thymus tissue. hiv-1 viremia occurs within approximately 7 days postinoculation . in many of these models, spleen, lymph nodes, and thymus tissues are highly positive for virus, similar to humans (brainard et al., 2009) . importantly, depletion of human t-cells can be observed in blood and lymphoid tissues of hivinfected humanized mice and at least some mechanisms of pathogenesis that occur in hiv-infected humans, also occur in the hiv-infected humanized mouse models (baenziger et al., 2006; neff et al., 2011) . the advantage of these models is that these mice are susceptible to hiv infection and thus the impact of drugs on the intended viral targets can be tested. one caveat is that while mice have a "common mucosal immune system," humans do not, due to differences in the distribution of addressins (holmgren and czerkinsky, 2005) . thus, murine mucosal immune responses to hiv do not reflect those of humans. another strategy uses a human cd4-and human ccr5-expressing transgenic luciferase reporter mouse to study hiv-1 pseudovirus entry (gruell et al., 2013) . hiv-1 transgenic (tg) rats are also used to study hiv related pathology, immunopathogenesis, and neuropathology (lentz et al., 2014; reid et al., 2001) . the clinical signs include skin lesions, wasting, respiratory difficulty, and neurological signs. brain volume decreases have been documented and the hiv-1 tg rat is thus used as a model of neuropathology in particular. there are a number of important nhp models for human hiv infection (hessell and haigwood, 2015) . an adaptation of hiv-1 was obtained by four passages in pigtailed macaques transiently depleted of cd8(+) cells during acute infection (hatziioannou et al., 2014) . the resulting disease has several similarities to aids in humans, such as depletion of cd4(+) t-cells (kimata, 2014) . simian immunodeficiency virus (siv) infection of macaques has been widely used as a platform for modeling hiv infection of humans (demberg and robert-guroff, 2015; walker et al., 2015) . importantly, nhps have similar, pharmacokinetics, metabolism, mucosal tcell homing receptors, and vascular addressins to those of humans. thus, while the correlates of protection against hiv are still not completely known, immune responses to hiv infection and vaccination are likely comparable. these models mimic infection through use of contaminated needles (iv), sexual transmission (vaginal or rectal), and maternal transmission in utero or through breast milk (keele et al., 2009; miller et al., 2005; stone et al., 2009) . there are also macaque models to study the emergence and clinical implications of hiv drug resistance (van rompay et al., 2002) . these models most routinely utilize rhesus macaques (macaca mulatta), cynomolgus macaques (m. fasicularis), and pigtailed macaques (macaca nemestrina). all ages are used, depending on the needs of the study. for instance, use of newborn macaques may be more practical for evaluating the effect of prolonged drug therapy on disease progression; however, adult nhps are more frequently employed. female pigtailed macaques have been used to investigate the effect of the menstrual cycle on hiv susceptibility (vishwanathan et al., 2015) . studies are performed in bsl-2 animal laboratories and nhps must be simian type-d retrovirus free and siv seronegative. siv infection of pigtailed macaques is a useful model for hiv peripheral nervous system pathology, wherein an axotomy is performed and regeneration of axons is studied (ebenezer et al., 2012) . exposure in model systems is typically through a single high-dose challenge. iv infection of rhesus macaques with 100 tcid 50 of the highly pathogenic siv/ deltab670 induces aids in most macaques within 5-17 months (mean of 11 months) (fuller et al., 2012) . peak viremia occurs around week 4. aids in such models is often defined as cd4+ t-cells that have dropped to less than 50% of the baseline values. alternatively, repeated low dose challenges are often utilized, depending on the requirements of the model (henning et al., 2014; moldt et al., 2012; reynolds et al., 2012) . since nhps infected with hiv do not develop an infection with a clinical disease course similar to humans, siv or siv/hiv-1 laboratory-engineered chimeric viruses (shivs) are used as surrogates. nhps infected with pathogenic siv may develop clinical disease which progresses to aids, and are thus useful pathogenesis models. a disadvantage is that siv is not identical to hiv-1 and is more closely related to hiv-2. however, the polymerase region of siv is 60% homologous to that of hiv-1 and it is susceptible to many reverse transcriptase (rt) and protease inhibitors. siv is generally not susceptible to nonnucleoside inhibitors, thus hiv-1 rt is usually put into siv for such studies (uberla et al., 1995) . sivmac239 is similar to hiv in the polymerase region and is therefore susceptible to nucleoside, rt, or integrase inhibition (witvrouw et al., 2004) . nhps infected with sivmac239 have an asymptomatic period and disease progression resembling aids in humans, characterized by weight loss/wasting, cd4+ t-cell depletion. additionally, sivmac239 utilizes the cxcr5 chemokine receptor as a coreceptor, similar to hiv, which is important for drugs that target entry (veazey et al., 2003) . nhps infected with shiv strains, may not develop aids, but these models are useful in testing vaccine efficacy . for example, rt-shivs and env-shivs are useful for testing and evaluation of drugs that may target the envelope or rt, respectively (uberla et al., 1995) . one disadvantage of the highly virulent env-shiv (shiv-89.6 p), is that it uses the cxcr4 coreceptor. of note, env-shivs that do use the cxcr5 coreceptor are less virulent; viremia develops then resolves without further disease progression (humbert et al., 2008) . simian-tropic (st) hiv-1 contains the vif gene from siv. infection of pigtailed macaques with this virus results in viremia, which can be detected for 3 months, followed by clearance (haigwood, 2009) . a number of routes are utilized for siv or shiv infection of nhps, with iv inoculation the most common route. mucosal routes include vaginal, rectal, and intracolonic. mucosal routes require a higher one-time dose than the iv route for infection. for the vaginal route, female macaques are treated with depo-provera (estrogen) 1 month before infection to synchronize the menstrual cycle, thin the epithelial lining of the vagina, and increase susceptibility to infection by atraumatic vaginal instillation (burton et al., 2011) . upon vaginal instillation of 500 tcid 50 of shiv-162p3, peak viremia was seen around 12 days postexposure with greater than 10 7 copies/ml and dropping thereafter to a constant level of 10 4 rna copies/ml at 60 days and beyond. in another example, in an investigation of the effect of vaccine plus vaginal microbicide on preventing infection, rhesus macaques were vaginally infected with a high dose of sivmac251 (barouch et al., 2012) . an example of an intrarectal model utilized juvenile (2-year-old) pigtailed macaques, challenged intrarectally with 10 4 tcid 50s of siv mne027 to study the pathogenesis related to the virulence factor, vpx (belshan et al., 2012) . here, viremia peaked at approximately 10 days with more than 10 8 copies/ml. viral rna was expressed in the cells of the mesenteric lymph nodes. the male genital tract is seen as a viral sanctuary with persistent high levels of hiv shedding even with antiretroviral therapy. to better understand the effect of haart therapy on virus and t-cells in the male genital tract, adult (3-to 4-year-old) male cynomolgus macaques were intravenously inoculated with 50 aid50s of sivmac251 and the male genital tract tissues were tested after euthanasia by pcr, ihc, and in situ hybridization (moreau et al., 2012) . pediatric models have been developed in infant rhesus macaques through the infection of siv, allowing for the study of the impact of developmental and immunological differences on the disease course (abel, 2009) . importantly, mother-to-infant transmission models have also been developed (jayaraman et al., 2004) . pregnant female pigtailed macaques were infected during the second trimester with 100 mid 50 shiv-sf162p3 by the iv route. four of nine infants were infected, one in utero and three either intrapartum or immediately postpartum through nursing. this model is useful for the study of factors involved in transmission as well as the underlying immunology. nhps infected with siv or shiv are routinely evaluated for weight loss, activity level, stool consistency, appetite, virus levels in blood, and t-cell populations. cytokine and chemokine levels, antibody responses, and cytotoxic t-lymphocyte responses may also be evaluated. the ultimate goal of an hiv vaccine is sterilizing immunity (preventing infection). however, a more realistic result may be to reduce severity of infection and permanently prevent progression. strategies have included live attenuated, nonreplicating, and subunit vaccines. these have variable efficacy in nhps due to the genetics of the host (mhc and trim alleles), differences between challenge strains, and challenge routes (letvin et al., 2011) . nhp models have led to the development of antiviral treatments that are effective at reducing viral load and indeed transmission of hiv among humans. one preferred variation on the models for testing the long-term clinical consequences of antiviral treatment is to use newborn macaques and treat from birth onward, in some cases more than a decade (van rompay et al., 2008) . unfortunately, however, successes in nhp studies do not always translate to success in humans, as seen with the recent step study which used an adenovirus-based vaccine approach (buchbinder et al., 2008) . vaccinated humans were not protected and may have even been more susceptible to hiv, viremia was not reduced, and the infections were not attenuated as hoped. with regard to challenge route, iv exposure is more difficult to protect than mucosal exposure and is used as a "worst case scenario." however, efficacy at one mucosal route is usually comparable to other mucosal routes. human and animal papillomaviruses cause benign epithelial proliferations (warts) and malignant tumors of the various tissues that they infect (bosch and de sanjose, 2002) . there are over 100 human papillomaviruses, with different strains causing warts on the skin, oropharynx, nasopharynx, larynx, and anogenital tissues. approximately one third of papillomaviruses are transmitted sexually. of these, virulent subtypes, such as hpv-16, hpv-18, hpv-31, hpv-33, and hpv-45 place individuals at high risk for cervical and other cancers. up to 35% of head and neck cancers are caused by hpv-16, particularly oropharyngeal cancers. major challenges in the study of these viruses are that papillomaviruses generally do not infect any other species outside of the natural hosts and can cause a very large spectrum of severity. thus, no wild-type animal models have been identified that are susceptible to hpv. however, a number of useful surrogate models exist which use animal papillomaviruses in their natural host or a very closely related species (borzacchiello et al., 2009; brandsma, 1994; campo, 2002) . these models have facilitated the recent development of useful and highly effective prophylactic hpv vaccines (rabenau et al., 2005) . wild-type inbred mice cannot be used to study disease caused by papillomaviruses unless they are engrafted with relevant tissue, orthotopically transplanted or transgenic, but they are often used to look at immunogenicity of vaccines (jagu et al., 2011; oosterhuis et al., 2011) . transgenic mice used for hpv animal modeling typically express the viral oncogenes e5, e6, e7, or the entire early region of hpv-16 from the keratin 14 promoter which is only active in the basal cells of the mouse epithelium (chow, 2015) . cancers in these models develop upon extended estrogen exposure (maufort et al., 2010; ocadiz-delgado et al., 2009; stelzer et al., 2010; thomas et al., 2011) . transgenic mice with constitutively active wnt/b-catenin signaling in cervical epithelial cells expressing the hpb oncoprotein e7 develop invasive cervical squamous carcinomas (bulut and uren, 2015) . the tumors occur within 6 months approximately 94% of the time. another model uses c57bl/6 mice expressing the hpv16-e7 transgene which are then treated topically with 7,12-dimethylbenz(a)anthracene (dmba) (de azambuja et al., 2014) . these mice developed benign and malignant cutaneous lesions. cervical cancers can also be induced in human cervical cancer xenografts transplanted onto the flanks of athymic mice and serially transplanted thereafter ( hiroshima et al., 2015; siolas and hannon, 2013) . a wild-type immunocompetent rodent model uses m. coucha, which is naturally infected with mastomys natalensis papillomavirus (mnpv) (vinzon et al., 2014) . mnpv induces papillomas, keratoacanthomas, and squamous cell carcinomas and provides a means to study vaccination in an immunocompetent small animal model. wild cottontail rabbits (sylvilagus floridanus) are the natural host for cottontail rabbit papillomavirus (crpv), but this virus also infects domestic rabbits (oryctolagus cuniculus), which is a very closely related species ( breitburd et al., 1997) . in this model, papillomas can range from cutaneous squamous cell carcinomas on one end of spectrum, and spontaneous regression on the other. lesions resulting from crpv in domestic rabbits do not typically contain infectious virus. canine oral papillomavirus (copv) causes florid warty lesions in mucosa of the oral cavity within 4-8 weeks postexposure in experimental settings (johnston et al., 2005) . the mucosatrophic nature of these viruses and the resulting oropharyngeal papillomas that are morphologically similar to human vaginal papillomas caused by hpv-6 and hpv-11 make this a useful model (nicholls et al., 1999) . these lesions typically spontaneously regress 4-8 weeks after appearing; this model is therefore useful in understanding the interplay between the host immune defense and viral pathogenesis. male and female beagles, aged 10 weeks to 2 years, with no history of copv, are typically used for these studies. infection is achieved by application of a 10 µl droplet of virus extract to multiple 0.5 cm 2 scarified areas within the mucosa of the upper lip of anesthetized beagles (nicholls et al., 2001) . some investigators have raised concerns that dogs are not a suitable model for high-risk hpv-induced oral cancer (staff, 2015) . bovine papillomavirus (bpv) has a wider host range than most papillomaviruses, infecting the fibroblasts cells of numerous ungulates (campo, 2002) . bpv-4 infection of cattle feeding on bracken fern, which is carcinogenic, can result in lesions of the oral and esophageal mucosa that lack detectable viral dna. bpv infections in cattle can result in a range of diseases, such as skin warts, cancer of the upper gastrointestinal tract and urinary bladder, and papillomatosis of the penis, teats, and udder. finally, rhesus papillomavirus (rhpv), a sexually transmitted papillomaviruses in rhesus macaques and cynomolgus macaques is very similar to hpv-16 and is associated with the development of cervical cancer ( ostrow et al., 1990; wood et al., 2007) . monkeypox virus (mpxv) causes disease in both animals and humans. human monkeypox, which is clinically almost identical to ordinary smallpox, occurs mostly in the rainforest of central and western africa. the virus is maintained in nature in rodent reservoirs including k. viral disease squirrels (charatan, 2003; khodakevich et al., 1986) . mpxv was discovered during the pox-like disease outbreak among laboratory monkeys (mostly cynomolgus and rhesus macaques) in denmark in 1958. no human cases were observed during this outbreak. the first human case was not recognized as a distinct disease until 1970 in zaire (the present drc) with continued occurrence of a smallpox-like illness despite eradication efforts of smallpox in this area. during the global eradication campaign, extensive vaccination in central africa decreased the incidence of human monkeypox, but the absence of immunity in the generation born since that time and increased dependence on bush meat have resulted in renewed emergence of the disease. in the summer of 2003, a well-known outbreak in the midwest was the first occurrence of monkeypox disease in the united states and western hemisphere. among 72 reported cases, 37 human cases were laboratory confirmed during an outbreak (nalca et al., 2005; sejvar et al., 2004) . it was determined that native prairie dogs (cynomys sp.) housed with rodents imported from ghana in west africa were the primary source of outbreak. the virus is mainly transmitted to humans while handling infected animals or by direct contact with the infected animal's body fluids, or lesions. person-to-person spread occurs by large respiratory droplets or direct contact (jeézek and fenner, 1988) . most of the clinical features of human monkeypox are very similar to those of ordinary smallpox (breman and arita, 1980) . after a 7-to 21-dayincubation period, the disease begins with fever, malaise, headache, sore throat, and cough. the main sign of the disease that distinguishes monkeypox from smallpox is swollen lymph nodes (lymphadenitis), which is observed in most of the patients before the development of rash (di giulio and eckburg, 2004; jeézek and fenner, 1988) . a typical maculopapular rash follows the prodromal period, generally lasting 1-3 days. the average size of the skin lesions are 0.5-1 cm and the progress of lesions follows the order: macules, papules, vesicles, pustules, umblication then scab, and desquamation and lasts typically 2-4 weeks. the fatality rate is 10% among the unvaccinated population and death generally occurs during the 2nd week of the disease (jeézek and fenner, 1988; nalca et al., 2005) . mpxv is highly pathogenic for a variety of laboratory animals and many animal models have been developed by using different species and different routes of exposure (table 33 .3). due to unavailability of variola virus (smallpox) to develop animal models and similar disease manifestations in humans that are similar, mpxv is one of the pox viruses that are utilized very heavily to develop a number of small animal models via different routes of exposure. wild-derived inbred mouse, stat1-deficient c57bl/6 mouse, icr mouse, prairie dogs, african dormice, ground squirrels, and gambian pouched rats are highly susceptible to mpxv by different exposure routes (americo et al., 2010; falendysz et al., 2015; hutson et al., 2009; osorio et al., 2009; sbrana et al., 2007; schultz et al., 2009; sergeev et al., 2016; stabenow et al., 2010; tesh et al., 2004; xiao et al., 2005) . cast/eij mice, one of the 38 inbred mouse strains tested for susceptibility to mpxv, showed weight loss and dose dependent mortality after in exposure to mpxv. studies with ip route of challenge indicated a 50fold higher susceptibility to mpxv when compared to in route (americo et al., 2010) . scid-balb/c mice were also susceptible to the ip challenge route and the disease resulted in mortality on day 9 postinfection (osorio et al., 2009) . similarly, c57bl/6 stat1 −/− mice were infected in with mpxv and the infection resulted in weight loss and mortality 10 days postexposure. recently sergeev et al. (2016) showed that in challenge of icr mice with mpxv resulted in purulent conjunctivitis, blepharitis, and ruffled fur in these mice although there was no death. the mouse models mentioned here are very promising for screening therapeutics against poxviruses but testing in additional models will be required for advanced development. high doses of the mpxv by ip or in routes caused 100% mortality in 6 days postexposure and 8 days postexposure, respectively, in ground squirrels (tesh et al., 2004) . the disease progressed very quickly and most of the animals were lethargic and moribund by day 5 postexposure without any pox lesions or respiratory changes. a comparison study of usa mpxv and central african strain of mpxv strains in ground squirrels by the subq route resulted in systemic disease and mortality in 6-11 days postexposure. the disease resembles hemorrhagic smallpox with nosebleeds, impaired coagulation parameters, and hemorrhage in the lungs of the animals. another study by sergeev et al. (2017) showed that in challenge with mpxv caused fever, lymphadenitis, and skin rash in ground squirrels 7-9 days postexposure. mortality was observed in 40% of the animals 13-22 days postexposure (sergeev et al., 2017) . since mpxv was transmitted by infected prairie dogs in the us outbreak, this animal model has been more thoroughly studied and utilized to test therapeutics and vaccines compared to other small animal models ( hutson et al., 2009; keckler et al., 2011; smith et al., 2011; xiao et al., 2005) . studies using in, ip, and id routes of exposure showed that mpxv was highly infectious to prairie dogs, ip infection with the west african mpxv strain caused a more severe disease and 100% mortality than challenge by the in route. anorexia and lethargy were common signs of the disease for both exposure routes. in contrast to ip route, the in route of exposure caused severe pulmonary edema and necrosis of lungs in prairie dogs, while splenic necrosis and hepatic lesions were observed in ip-infected animals (xiao et al., 2005) . hutson et al. (2009) african and congo basin strains and showed that both strains and routes caused smallpox-like disease with longer incubation periods and most importantly generalized pox lesions. therefore, this model has the utility for testing therapeutics and vaccines against pox viruses. furthermore, mpxv challenged prairie dogs were used to perform in vivo bioluminescent imaging (bli) studies (falendysz et al., 2015) . bli studies showed real time spread of virus in prairie dogs as well as potential routes for shedding and transmission. the african dormouse is susceptible to mpxv by a footpad injection or in routes (schultz et al., 2009) . mice had decreased activity, hunched posture, dehydration, conjunctivitis, and weight loss. viral doses of 200 and 2000 pfu provided 100% mortality with a mean time to death of 8 days. upper gastrointestinal hemorrhage, hepatomegaly, lymphadenopathy, and lung hemorrhage were observed during necropsy. with the hemorrhage in several organs, this model resembles hemorrhagic smallpox. in a recent study, comparison of the disease pathogenesis was performed by using live bioluminescence imaging in the cast/eij mouse and african dormouse challenged with low dose of mpxv (earl et al., 2015) . following in challenge, mpxv dissemination occurred through the blood or lymphatic system in dormice compared to dissemination that was through the nasal cavity and lungs in cast/eij mice. the disease course was much faster in cast/eij mice (earl et al., 2015) . considering the limited availability of prairie dogs, ground squirrels and african dormice, lack of reagents specific for these species, and not having commercial sources of these species, these small animal models are as attractive for further characterization and vaccine, and countermeasure testing studies. nhps were exposed to mpxv by several different routes to develop animal model for mpxv (edghill-smith et al., 2005; johnson et al., 2011; nalca et al., 2010; stittelaar et al., 2006; zaucha et al., 2001) . during our studies using an aerosol route of exposure, we observed that macaques had mild anorexia, depression, fever, and lymphadenopathy on day 6 postexposure (nalca et al., 2010) . complete blood count and clinical chemistries showed abnormalities similar to human monkeypox cases with leukocytosis and thrombocytopenia (huhn et al., 2005) . whole blood and throat swabs had viral loads peak around day 10, and in survivors, gradually decrease until day 28 postexposure. since doses of 4 × 10 4 pfu, 1 × 10 5 pfu, or 1 × 10 6 pfu resulted in lethality for 70% of the animals, whereas a dose of 4 × 10 5 pfu resulted in 85% lethality, survival was not dose dependent. the main pitfall of this model was the lack of pox lesions. with the high dose, animals succumbed to disease before developing pox lesions. with the low challenge dose, pox lesions were observed but they were few in comparison to the iv model. a recent study also evaluated the cytokine levels in aerosol challenged animals. (tree et al., 2015) . tree et al. (2015) showed that ifnγ, il-1rα, and il-6 increased dramatically on day 8 postexposure the day that death was most likely to occur, and viral dna was detected in most of the tissues. these results support the idea of a cytokine storm causing mortality in monkeypox disease. mpxv causes dose dependent disease in nhps when given by the iv route (johnson et al., 2011) . studies showed that a 1 × 10 7 pfu iv challenge results in systemic disease with fever, lymphadenopathy, macula-papular rash, and mortality. an it infection model skips the upper respiratory system and deposits virus into the trachea, delivering the virus directly to the airways without regard to particle size and the physiological deposition that occurs during the process of inhalation. fibrinonecrotic bronchopneumonia was described in animals that received 10 7 pfu of mpxv intratracheally (stittelaar et al., 2006) . although a similar challenge dose of it mpxv infection resulted in a similar viremia in nhps to the aerosol route of infection, the timing of the first peak was delayed by 5 days in intratracheally exposed macaques compared to aerosol infection, and the amount of virus detected by qpcr was approximately 100-fold lower. this suggests that local replication is more prominent after aerosol delivery compared to the it route. an intrabronchial route of exposure resulted in pneumonia in nhps (johnson et al., 2011) . delayed onset of clinical signs and viremia were observed during the disease progression. in this model, similar to aerosol and it infection models, the number of pox lesions was much less than in the iv infection model. a major downside of the iv, it, and intrabronchial models is that the initial infection of respiratory tissue, incubation, and prodromal phases are circumvented with the direct inoculation of virus to the blood stream or to the lung. this is an important limitation when the utility of these models is to test possible vaccines and treatments in which the efficacy may depend on protecting the respiratory mucosa and targeting the subsequent early stages of the infection, which are not represented in these challenge models. although the aerosol model is the natural route of transmission for human varv infections and a secondary route for human mpxv infections, the lack of pox lesions is the main drawback of this model. therefore, when this model is used to test medical countermeasures, the endpoints and the biomarkers to initiate treatment should be chosen carefully. hepatitis b virus (hbv) is one of the most common infections worldwide with over 400 million people chronically infected and 316,000 cases per year of liver cancer due to infection (lee, 1997) . the virus can naturally infect both humans and chimpanzees (guha et al., 2004) . hbv is transmitted parenterally or postnatally from infected mothers. it can also be transmitted by sexual contact, iv drug use, blood transfusion, and acupuncture (lai et al., 2003) . the age at which one is infected dictates the risk of developing chronic disease (hyams, 1995) . acute infection during adulthood is self-limiting and results in flu-like symptoms that can progress to hepatocellular involvement as observed with the development of jaundice. the clinical symptoms of hbv infection last for a few weeks before resolving (ganem and prince, 2004) . after this acute phase, lifetime immunity is achieved (wright and lau, 1993) . of those infected, less than 5% will develop the chronic form of the disease. chronicity is the most serious outcome of the disease as it can result in cirrhosis or liver cancer. hepatocellular carcinoma is 100 times more likely to develop in a chronically infected individual than a noncarrier (beasley, 1988) . the viral determinant for cellular transformation has yet to be determined, although studies involving the woodchuck hepatitis virus suggest that x protein may be responsible (spandau and lee, 1988). many individuals are asymptomatic until complications emerge related to chronic hbv carriage. chimpanzees have a unique strain that circulates within the population (hu et al., 2000; . it was found that 3%-6% of all wild-caught animals from africa are positive for hbv antigen ( lander et al., 1972) . natural and experimental challenge with the virus follows the same course as human disease; however, this is only an acute model of disease (prince, 1972) . to date, chimpanzees are the only reliable method to ensure that plasma vaccines are free from infectious particles (prince and brotman, 2001 ). this animal model has been used to study new therapeutics and vaccines. chimpanzees are especially ideal for these studies given that their immune response to infection directly mirrors humans (nayersina et al., 1993) . recent regulations by the national institute of health (nih) and restrictions to use great apes as animal models forced researches to find alternate models for hbv infection. other nhps that have been evaluated are gibbons, orangutans, and rhesus monkeys. although these animals can be infected with hbv, none develops hepatic lesions or liver damage as noted by monitoring of liver enzymes (pillot, 1990 ). mice are not permissible to infection, and thus numerous transgenic and humanized lines that express hbv proteins have been created to facilitate their usage as an animal model. these include both immunocompetent and immunosuppressed hosts. the caveat to all of these mouse lines is that they reproduce only the acute form of disease (guha et al., 2004) . recently, the entire genome of hbv was transferred to an immunocompetent mouse line via adenovirus. this provides a model for persistent infection (huang et al., 2012) . another model that has been developed is hydrodynamic injection of hbv genomes in the liver of mice (liu et al., 1999; yang et al., 2002) . although this model is very stressful to mice and has liver toxicity, it is successfully used to evaluate antivirals against hbv (mccaffrey et al., 2003) . liver chimeric mouse models are an additional set of surrogate models for hbv infection (dandri and lutgehetmann, 2014) . in these models human hepatocytes are integrated into the murine liver parenchyma (allweiss and dandri, 2016) . this model might be used to test antivirals as well as to study the molecular biology of hbv infection. hbv can also be studied using surrogate viruses, naturally occurring mammalian hepadna viruses (mason et al., 1982) . the woodchuck hepatitis virus induces hepatocellular carcinoma (summers et al., 1978) . within a population, 65%-75% of all neonatal woodchucks are susceptible to chronic infection (cote et al., 2000) . a major difference between the two hepatitis isolates is the rate at which they induce cancer; almost all chronic carriers developed hepatocellular carcinoma within 3 years of the initial infection in woodchucks, whereas human carcinogenesis takes much longer (gerin et al., 1989) . the acute infection strongly resembles what occurs during the course of human disease. there is a self-limiting acute phase resulting in a transient viremia that has the potential of chronic carriage (tennant, 2001) . challenge with virus in neonates leads to a chronic infection while adults only develop the acute phase of disease (buendia, 1992) . a closely related species to the woodchuck is the marmota himalayan. this animal is also susceptible to the woodchuck hepadna virus upon iv injection. the marmot himalayan develops an acute hepatitis with a productive infection (lucifora et al., 2010) . hepatitis d virus (hdv) is dependent upon hbv to undergo replication and successful infection in its human host (gerin, 2001) . there are two modes of infection possible between the viruses: coinfection where a person is simultaneously infected or superinfection in which a chronic carrier of hbv is subsequently infected with hdv (purcell et al., 1987) . coinfection leads to a similar disease as seen with hbv alone; however, superinfection can result in chronic hdv infection and severe liver damage (guilhot et al., 1994) . both coinfection and superinfection can be demonstrated within the chimpanzee and woodchuck by inoculation of human hepatitis d (ponzetto et al., 1991) . a recently published report demonstrated the use of a humanized chimeric upa mouse to study interactions between the two viruses and drug testing (lutgehetmann et al., 2012) new models ranging from nhps to small animals and representing the disease characteristics in humans are necessary to study viral and host factors that drive disease pathogenesis and evaluate medical countermeasures. the ideal animal model for human viral disease should closely recapitulate the spectrum of clinical symptoms and pathogenesis observed during the course of human infection. whenever feasible, the model should use the same virus and strain that infects humans. it is also preferable that the virus is a low passage clinical isolate thus animal passage or adaptation should be avoided if model species can be identified that are susceptible. ideally, the experimental route of infection would mirror that occurs in natural disease. in order to understand the interplay and contribution of the immune system during infection, an immunocompetent animal should be used. the aforementioned characteristics cannot always be satisfied; however, and often virus must be adapted, knockout mice must be used, and/or the disease is not perfectly mimicked in the animal model. well-characterized animal models are critical for licensure to satisfy fda "animal rule." this rule applies to situations in which vaccine and therapeutic efficacy cannot safely or ethically be tested in humans; thus licensure will come only after preclinical tests are performed in animal models. many fields in virology are moving toward standardized models that can be used across institutions to test vaccines and therapeutics. a current example of such an effort is within the filovirus community, where animal models, euthanasia criteria, assays, and virus strains are in the process of being standardized. the hope is that these efforts will enable results of efficacy tests on medical countermeasures compared across institutions. this chapter has summarized the best models available for each of the viruses described. the rhesus macaque pediatric siv infection model-a valuable tool in understanding infant hiv-1 pathogenesis and for designing pediatric hiv-1 prevention strategies prevalence of anti-rift-valley-fever igm antibody in abattoir workers in the nile delta during the 1993 outbreak in egypt common marmosets (callithrix jacchus) as a nonhuman primate model to assess the virulence of eastern equine encephalitis virus strains replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels. emerg generation of a transgenic mouse model of middle east respiratory syndrome coronavirus infection and disease pathological changes in brain and other target organs of infant and weanling mice after infection with nonneuroadapted western equine encephalitis virus particle-to-pfu ratio of ebola virus influences disease course and survival in cynomolgus macaques progress toward norovirus vaccines: considerations for further development and implementation in potential target populations characterization of lethal zika virus infection in ag129 mice experimental in vitro and in vivo models for the study of human hepatitis b virus infection a model of meningococcal bacteremia after respiratory superinfection in influenza a virus-infected mice middle east respiratory syndrome coronavirus: current situation and travel-associated concerns aerosol exposure to the angola strain of marburg virus causes lethal viral hemorrhagic fever in cynomolgus macaques necrotizing scleritis, conjunctivitis, and other pathologic findings in the left eye and brain of an ebola virus-infected rhesus macaque (macaca mulatta) with apparent recovery and a delayed time of death american academy of pediatrics subcommittee on diagnosis and management of bronchiolitis identification of wild-derived inbred mouse strains highly susceptible to monkeypox virus infection for use as small animal models the gerbil, meriones unguiculatus, a model for rift valley fever viral encephalitis morbidity and mortality among patients with respiratory syncytial virus infection: a 2-year retrospective review chikungunya and the nervous system: what we do and do not know the west nile virus outbreak of 1999 in new york: the flushing hospital experience hospital outbreak of middle east respiratory syndrome coronavirus diagnosis of noncultivatable gastroenteritis viruses, the human caliciviruses norovirus vaccine against experimental human norwalk virus illness determination of the 50% human infectious dose for norwalk virus an epizootic attributable to western equine encephalitis virus infection in emus in texas evidence for camel-to-human transmission of mers coronavirus integrated molecular signature of disease: analysis of influenza virus-infected macaques through functional genomics and proteomics disseminated and sustained hiv infection in cd34+ cord blood cell-transplanted rag2 −/− gamma c −/− mice choice of inbred rat strain impacts lethality and disease course after respiratory infection with rift valley fever virus rift valley fever: an uninvited zoonosis in the arabian peninsula recombinant norwalk virus-like particles given orally to volunteers: phase i study tropism of dengue virus in mice and humans defined by viral nonstructural protein 3-specific immunostaining lethal antibody enhancement of dengue disease in mice is prevented by fc modification animal models for the study of influenza pathogenesis and therapy effect of oral gavage treatment with znal42 and other metallo-ion formulations on influenza a h5n1 and h1n1 virus infections in mice macaque studies of vaccine and microbicide combinations for preventing hiv-1 sexual transmission early and sustained innate immune response defines pathology and death in nonhuman primates infected by highly pathogenic influenza virus hepatitis b virus. the major etiology of hepatocellular carcinoma transmission of norwalk virus during football game vpx is critical for sivmne infection of pigtail macaques experimental respiratory syncytial virus infection of four species of primates pathogenesis and immune response of crimean-congo hemorrhagic fever virus in a stat-1 knockout mouse model crimean-congo hemorrhagic fever virus infection is lethal for adult type i interferon receptor-knockout mice the utility of the new generation of humanized mice to study hiv-1 infection: transmission, prevention, pathogenesis, and treatment hiv-1 infection and cd4 t cell depletion in the humanized rag2 −/− gamma c −/− (rag-hu) mouse model bacterial infections in pigs experimentally infected with nipah virus evaluation of a mouse model for the west nile virus group for the purpose of determining viral pathotypes severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice study of susceptibility to crimean hemorrhagic fever (chf) virus in european and long-eared hedgehogs. tezisy konf manipulation of host factors optimizes the pathogenesis of western equine encephalitis virus infections in mice for antiviral drug development genetic basis of attenuation of dengue virus type 4 small plaque mutants with restricted replication in suckling mice and in scid mice transplanted with human liver cells chimpanzees as an animal model for human norovirus infection and vaccine development a simple technique for infection of mosquitoes with viruses; transmission of zika virus human papillomavirus research: do we still need animal models? human papillomavirus in cervical cancer development of a hamster model for chikungunya virus infection and pathogenesis a neutralizing human monoclonal antibody protects against lethal disease in a new ferret model of acute nipah virus infection the cotton rat model of respiratory viral infections correlates of immunity to filovirus infection filovirus vaccines induction of robust cellular and humoral virusspecific adaptive immune responses in human immunodeficiency virus-infected humanized blt mice animal models of human-papillomavirus-associated oncogenesis interferon alpha/beta receptor-deficient mice as a model for ebola virus disease zika virus outbreak in rio de janeiro, brazil: clinical characterization, epidemiological and virological aspects co-infection of the cotton rat (sigmodon hispidus) with staphylococcus aureus and influenza a virus results in synergistic disease effectiveness of influenza vaccination the role of the type i interferon response in the resistance of mice to filovirus infection a mouse model for evaluation of prophylaxis and therapy of ebola hemorrhagic fever the rabbit viral skin papillomas and carcinomas: a model for the immunogenetics of hpv-associated carcinogenesis the confirmation and maintenance of smallpox eradication a lethal disease model for hantavirus pulmonary syndrome in immunosuppressed syrian hamsters infected with sin nombre virus nonhuman primate models of chikungunya virus infection and disease tissue tropism and neuroinvasion of west nile virus do not differ for two mouse strains with different survival rates pediatric norovirus diarrhea in nicaragua efficacy assessment of a cell-mediated immunity hiv-1 vaccine (the step study): a double-blind, randomised, placebo-controlled, test-of-concept trial variation between strains of hamsters in the lethality of pichinde virus infections hepatitis b viruses and hepatocellular carcinoma generation of k14-e7/n87betacat double transgenic mice as a model of cervical cancer limited or no protection by weakly or nonneutralizing antibodies against vaginal shiv challenge of macaques compared with a strongly neutralizing antibody a novel vaccine against crimean-congo haemorrhagic fever protects 100% of animals against lethal challenge in a mouse model interleukin-1beta but not tumor necrosis factor is involved in west nile virusinduced langerhans cell migration from the skin in c57bl/6 mice animal models of papillomavirus pathogenesis immunization of knock-out alpha/beta interferon receptor mice against high lethal dose of crimean-congo hemorrhagic fever virus with a cell culture based vaccine characterization of the localized immune response in the respiratory tract of ferrets following infection with influenza a and b viruses lassa virus infection in experimentally infected marmosets: liver pathology and immunophenotypic alterations in target tissues a small nonhuman primate model for filovirus-induced disease severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus outbreak of acute illness-southwestern united states outbreak of west nile-like viral encephalitis-new york, 1999. mmwr morb. mortal in vitro whole-virus binding of a norovirus genogroup ii genotype 4 strain to cells of the lamina propria and brunner's glands in the human duodenum animal models for studying dengue pathogenesis and therapy us doctors investigate more than 50 possible cases of monkeypox mechanism of neuroinvasion of venezuelan equine encephalitis virus in the mouse chikungunya outbreaks-the globalization of vectorborne diseases inactivated and live, attenuated influenza vaccines protect mice against influenza: streptococcus pyogenes super-infections pathogenesis of a genogroup ii human norovirus in gnotobiotic pigs the immunobiology of sars* induction of tetravalent protective immunity against four dengue serotypes by the tandem domain iii of the envelope protein norovirus infection as a cause of diarrhea-associated benign infantile seizures comparative pathogenesis of epidemic and enzootic chikungunya viruses in a pregnant rhesus macaque model development of norwalk 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wild-type mice epidemiology and clinical presentations of the four human coronaviruses 229e, hku1, nl63, and oc43 detected over 3 years using a novel multiplex real-time pcr method development of an acute and highly pathogenic nonhuman primate model of nipah virus infection animal models of hepatitis delta virus infection and disease hepadnavirusinduced liver cancer in woodchucks experimental infection and natural contact exposure of dogs with avian influenza virus (h5n1). emerg megaribavirin aerosol for the treatment of influenza a virus infections in mice discovery of novel human and animal cells infected by the severe acute respiratory syndrome coronavirus by replication-specific multiplex reverse transcription-pcr chinchilla and murine models of upper respiratory tract infections with respiratory syncytial virus mechanisms of host defense following severe acute respiratory syndromecoronavirus (sars-cov) pulmonary infection of mice studies on the virus of venezuelan equine 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cotton rat (sigmodon hispidus) experimental hepatitis delta virus infection in the chimpanzee relative infectivity of hepatitis a virus by the oral and intravenous routes in 2 species of nonhuman primates cardiovascular and pulmonary responses to pichinde virus infection in strain 13 guinea pigs establishment and characterization of a lethal mouse model for the angola strain of marburg virus stability and inactivation of sars coronavirus possibility of extracting hyperimmune gammaglobulin against chf from doneky blood sera dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc swine as a model for influenza a virus infection and immunity zika virus: an update on epidemiology, pathology, molecular biology, and animal model oseltamivir population pharmacokinetics in the ferret: model application for pharmacokinetic/pharmacodynamic study design aerosol exposure to western equine encephalitis virus causes fever and encephalitis in cynomolgus macaques severe 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transmission of influenza virus infection experimental infection of an african dormouse (graphiurus kelleni) with monkeypox virus animal noroviruses human monkeypox infection: a family cluster in the midwestern united states the possibility of using the icr mouse as an animal model to assess antimonkeypox drug efficacy using the ground squirrel (marmota bobak) as an animal model to assess monkeypox drug efficacy experimental inoculation of juvenile rhesus macaques with primate enteric caliciviruses a rodent model of chikungunya virus infection in rag1 −/− mice, with features of persistence, for vaccine safety evaluation respiratory syncytial virus (rsv) pulmonary infection in humanized mice induces human anti-rsv immune responses and pathology viremia and antibody response of small african and laboratory animals to crimean-congo hemorrhagic fever virus infection early activation of natural killer and b cells in response to primary dengue virus infection in a/j mice murine model for 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viruses in ferrets lethal crimean-congo hemorrhagic fever virus infection in interferon alpha/beta receptor knockout mice is associated with high viral loads, proinflammatory responses, and coagulopathy the pathogenesis of western equine encephalitis virus (w.e.e.) in adult hamsters with special reference to the long and short term effects on the c.n.s. of the attenuated clone 15 variant development of a murine model for aerosolized filovirus infection using a panel of bxd recombinant inbred mice a characterization of aerosolized sudan ebolavirus infection in african green monkeys, cynomolgus macaques, and rhesus macaques characterization of disease and pathogenesis following airborne exposure of guinea pigs to filoviruses manuscripts in preparation opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the us army. key: cord-021499-up5vftj4 authors: brayton, cory; mähler, michael; nicklas, werner title: viral infections date: 2007-09-02 journal: the laboratory mouse doi: 10.1016/b978-012336425-8/50076-5 sha: doc_id: 21499 cord_uid: up5vftj4 nan in interpreting the microbiological status of laboratory animals, it must be understood that infection and disease are not synonymous. infection refers to the invasion and multiplication of microorganisms in body tissues and may occur with or without apparent disease. disease refers to interruption or deviation from normal structure and function of any tissue, organ, or system. many of the infections with which we are concerned may not cause discernable disease in many strains of mice. however, they may cause inapparent or subclinical changes that can interfere with research. such interference often remains undetected, and therefore modified results may be obtained and published. the types of interference of an agent with experimental results may be diverse. there is no doubt that research complications due to overt infectious disease are significant and that animals with clinical signs of disease should not be used for scientific experiments. but also clinically inapparent infections may have severe effects on animal experiments. there are numerous examples of influences of microorganisms on host physiology and hence of the interference of inapparent infections with the results of animal experiments. many microorganisms have the potential to induce activation or suppression of the immune system or both at the same time but on different parts of the immune system, regardless of the level of pathogenicity. all infections, apparent or inapparent, are likely to increase inter-individual variability and hence result in increased numbers of animals necessary to obtain reliable results. microorganisms, in particular viruses, present in an animal may contaminate biological materials such as sera, cells, or tumours (collins and parker, 1972; nicklas et al., 1993) . this may interfere with in vitro experiments conducted with such materials and may also lead to contamination of animals (lipman et al., polymerase chain reaction (pcr) testing of biologics to be inoculated into mice is an important component of a disease prevention programme. finally, latent infections may be activated by environmental factors, by experimental procedures, or by the combination and interaction between various microorganisms. for all these reasons, prevention of infection, not merely prevention of clinical disease, is essential. unfortunately, research complications due to infectious agents are usually considered artefacts and published only exceptionally. information on influences of microorganisms on experiments is scattered in diverse scientific journals, and many articles are difficult to detect. to address this problem, several congresses were held on viral complications on research. the knowledge available was summarized in conference proceedings (melby and balk, 1983; bhatt et al., 1986b; hamm, 1986) and has later repeatedly been reviewed (lussier, 1988; national research council, 1991; baker, 1998; nicklas et al., 1999) . this chapter covers only viral infections of laboratory mice. viral infections of mice have been studied in detail, and comprehensive information on their pathogenic potential, their impact on research, and the influence of host factors such as age, genotype, and immune status on the response to infection is available. bacterial agents may be similarly important, but with few exceptions (e.g. helicobacter species) little is known about their potential to influence host physiology and experiments. even less is known about most parasites in this regard. among fungal agents, only pneumocystis carinii can be expected to play a significant role in contemporary mouse colonies. the nomenclature and taxonomy of viruses described are based on recent nomenclature rules by the international union of microbiological societies (2000) and the universal virus database of the international committee on the taxonomy of viruses (http://www.ictvdb.iacr.ac.uk). retroviruses are not covered in this chapter because they are not included in routine health surveillance programmes and cannot be eradicated with presently available methods. this is because most of them are incorporated in the mouse genome as proviruses and thus are transmitted via germline. the ability to accurately determine whether or not laboratory animals or animal populations have been infected with virus depends on the specificity and sensitivity of the detection methods used. most viral infections in immunocompetent mice are acute or short-term, and lesions are often subtle or subclinical. the absence of clinical disease and pathological changes has therefore only limited diagnostic value. however, clinical signs, altered behaviour, or lesions may be the first indicator of an infection and often provide clues for further investigations. serology is the primary means of testing mouse colonies for exposure to viruses, largely because serological tests are sensitive and specific, are relatively inexpensive, and allow screening for a multitude of agents with one serum sample. they are also employed to monitor biological materials for viral contamination using the map test. serological tests detect specific antibodies, usually immunoglobulin g (igg), produced by the host against the virus and do not actually test for the presence of virus. an animal may have been infected, mounted an effective antibody response, and cleared the virus, but remains seropositive for weeks or months or forever, even though it is no longer infected or shedding the agent. active infection can only be detected by using direct diagnostic methods such as virus isolation, electron microscopy, or pcr. meanwhile, pcr assays have been established for the detection of almost every agent of interest. they are highly sensitive and depending on the demands, they can be designed to broadly detect all members of a genus or only one species. however, good timing and selection of the appropriate specimen is critical for establishing the diagnosis. in practice, combinations of diagnostic tests are often necessary including the use of sentinel animals or immunosuppression to get clear aetiological results or to avoid consequences from false-positive results. reports on the prevalence of viral infections in laboratory mice throughout the world have been published frequently. in general, the microbiological quality of laboratory mice has constantly improved during the last decades, and several agents (e.g. herpes-and polyomaviruses) have been essentially eliminated from contemporary colonies due to advances in diagnostic methodologies and modern husbandry and rederivation practices (jacoby and lindsey, 1998; zenner and regnault, 2000; livingston and riley, 2003) . they may, however, reappear, since most have been retained or are still being used experimentally. furthermore, the general trend towards better microbiological quality is challenged by the increasing reliance of biomedical research on genetically modified and immunodeficient mice, whose responses to infection and disease can be unpredictable. increasing numbers of scientists are creating genetically modified mice, with minimal or no awareness of infectious disease issues. as a consequence, they are more frequently infected than 'standard' strains of mice coming from commercial breeders, and available information on their health status is often insufficient. frequently, they are exchanged between laboratories, which amplifies the risk of introducing infections from a range of animal facilities. breeding cessation strategies that have been reported to eliminate viruses from immunocompetent mouse colonies may prove to be costly and ineffective in genetically modified colonies of uncertain or incompetent immune status. it must also be expected that new agents will be detected, although only occasionally. infections therefore remain a threat to biomedical research, and users of laboratory mice must be cognizant of infectious agents and the complications they can cause. two members of the family herpesviridae can infect mice (mus musculus). mouse cytomegalovirus 1 (mcmv-1) or murid herpesvirus 1 (muhv-1) belongs to the subfamily betaherpesvirinae, genus muromegalovirus. murid herpesvirus 3 (muhv-3) or mouse thymic virus (mtv) has not yet been assigned to a genus within the family herpesviridae. both viruses are enveloped, doublestranded dna viruses that are highly host-specific and relatively unstable to environmental conditions such as heat and acidic ph. both agents are antigenically distinct and do not cross-react in serological tests, but their epidemiology is similar (cross et al., 1979) . seropositivity to mcmv-1 was reported in less than 5% of specified pathogen-free (spf) mouse colonies in the usa in 1996 (jacoby and lindsey, 1998) , and some institutions reported to have mice 'on campus' that were positive for mtv. in a more recent study, a low rate (0.1%) of samples was found to be positive for mcmv-1 whereas no sample tested positive for mtv (livingston and riley, 2003) . the data available suggest that the prevalence of both viruses in contemporary colonies and thus their importance for laboratory mice is negligible. however, both mcmv-1 and mtv are frequently found in wild mice, which may be coinfected with both viruses (national research council, 1991; singleton et al., 2000) . natural infection with mcmv-1 causes subclinical salivary gland infection in mice. the virus persists in the salivary glands (particularly in the submaxillary glands) and also in other organs (osborn, 1982; kercher and mitchell, 2002; lenzo et al., 2002) . most information concerning the pathogenesis of mcmv-1 infection is based on experimental infection studies. these results are very difficult to summarize because the outcome of experimental infection in laboratory mice depends on various factors such as mouse strain and age, virus strain and passage history, virus dose, and route of inoculation (osborn, 1982) . in general, newborn mice are most susceptible to clinical disease and to lethal infection. virus replication is observed in newborn mice in many tissues (for details, see osborn, 1982) and appears in the salivary glands towards the end of the first week of infection when virus concentrations in liver and spleen have already declined. resistance develops rapidly after weaning between days 21 and 28 of age. experimental infection of adult mice results in mortality only in susceptible strains and only if high doses are administered. not even intravenous or intraperitoneal injections of adult mice usually produce signs of illness in resistant strains (shanley et al., 1993) . mice of the h-2 b (e.g. c57bl/6) and h-2 d (e.g. balb/c) haplotype are more sensitive to experimental infection than are mice of the h-2 k haplotype (e.g. c3h), which are approximately 10-fold more resistant to mortality than are those of the b or d haplotype (osborn, 1986) . subclinical or latent infections can be activated by immunosuppression (e.g. with cyclophosphamide or cortisone). reactivation of mcmv-1 occurs also after implantation of latently infected salivary glands into prkdc scid mice (schmader et al., 1995) . immunodeficient mice lacking functional t cells or natural killer (nk) cells, such as foxn1 nu and lyst bg mice, are more susceptible than are immunocompetent animals. experimental infection in prkdc scid mice causes severe disease or is lethal, with necrosis in spleen, liver, and other organs, and multinucleate syncytia with inclusion bodies in the liver (reynolds et al., 1993) . similar to aids patients infected with human cytomegalovirus, athymic foxn1 nu mice experimentally infected with mcmv also develop adrenal necrosis (shanley and pesanti, 1986) . the virus also replicates in the lungs leading to pneumonitis whereas in heterozygous (foxn1 nu / ϩ ) littermates replication and disease are not seen (shanley et al., 1997) . the most prominent histological finding of cytomegaloviruses is enlarged cells (cytomegaly) of salivary gland epithelium with eosinophilic nuclear and cytoplasmic inclusion bodies. the inclusion bodies contain viral material and occur in other organs such as liver, spleen, ovary, and pancreas (osborn, 1982) . depending on inoculation route, dose, strain, and age of mice, experimental infections may result in inflammation or cytomegaly with inclusion bodies in a variety of tissues, pneumonitis, myocarditis, meningoencephalitis, or splenic necrosis in susceptible strains (national research council, 1991; osborn, 1982; percy and barthold, 2001) . virus is transmitted oronasally by direct contact and is excreted in saliva, tears, and urine for several months. wild mice serve as a natural reservoir for infection. the virus is most frequently transmitted horizontally through mouse-to-mouse contact but does not easily spread between cages. it is generally assumed that mcmv-1 has a very low prevalence in contemporary colonies of laboratory mice. the risk of introduction into facilities housing laboratory mice is very low if wild mice are strictly excluded. monitoring is necessary if populations of laboratory mice may have been contaminated by contact with wild mice. as for other viruses, enzyme-linked immunosorbent assay (elisa) and indirect immunofluorescence assay (ifa) are the most appropriate tests for detecting antibodies. as the virus persists, direct demonstration of mcmv-1 in infected mice is possible by pcr (palmon et al., 2000) or by virus isolation using mouse embryo fibroblasts (3t3 cells). although mcmv-1 does not play a significant role as a natural pathogen of laboratory mice, it is frequently used as a model for human cytomegalovirus infection (bolger et al., 1999) . however, the virus is known to influence immune reactions in infected mice and may therefore have impact on immunological research (osborn, 1986; national research council, 1991; baker, 1998) . mouse thymic virus was detected during studies in which samples from mice were passaged in newborn mice. unlike other herpesviruses, the virus can not yet be cultured in vitro and is propagated by intraperitoneal infection of newborn mice. the thymus is removed 7-10 days later, and thymus suspensions serve as virus material for further studies. the prevalence of mtv is believed to be low in laboratory mice, and for this reason and also due to the difficulties in virus production for serological assays, it is not included in many standard diagnostic or surveillance testing protocols. limited data are available indicating that it is common in wild mice, and it is also found in laboratory mice (osborn, 1982; morse, 1987; national research council, 1991) . further, mtv obviously represents a significant source of contamination of mcmv-1 (and vice versa) if virus is prepared from salivary glands since both viruses cause chronic or persistent salivary gland infections and can coinfect the same host. all mouse strains are susceptible to infection, but natural or experimental infection of adult mice is subclinical. gross lesions appear only in the thymus and only if experimental infection occurs at an age of less than about 5 days. virus is present in the thymus but may also be found in the blood and in salivary glands of surviving animals. salivary glands are the only site yielding positive virus isolations if animals are infected as adults. mouse thymic virus also establishes a persistent infection in athymic foxn1 nu mice, but virus shedding is reduced compared to euthymic mice and virus recovery is possible only in a lower percentage of mice (morse, 1988) . pathological changes caused by mtv occur in the thymus, and reduced thymus mass due to necrosis in suckling mice is the most characteristic gross lesion (percy and barthold, 2001) . lymphoid necrosis also may occur in lymph nodes and spleen (wood et al., 1981) , with necrosis and recovery similar to that in the thymus. in mice infected during the first 3 days after birth, necrosis of thymus becomes evident within 3-5 days, and its size and weight are markedly reduced at day 12-14. intranuclear inclusions may be present in thymocytes between days 10-14 post infection. the thymus and the affected peripheral tissues regenerate within 8 weeks after infection. regardless of the age of mice at infection, a persistent infection is established in the salivary glands, and infected animals shed virus for life. several alterations of immune responses are associated with neonatal mtv infection. there is transient immunosuppression, attributable to lytic infection of t lymphocytes, but activity (e.g. response of spleen cells to t cell mitogens) returns to normal as the histological repair progresses (wood et al., 1981) . selective depletion of cd4 ϩ t cells by mtv results in autoimmune disease (morse and valinsky, 1989; morse et al., 1999) . information about additional influences on the immune system is given by osborn (1982 ), national research council (1991 ), and baker (1998 . in experimentally infected newborn mice, oral and intraperitoneal infections similarly result in thymus necrosis, seroconversion, and virus shedding suggesting that the oral-nasal route is likely to be involved in natural transmission (morse, 1989) . the virus spreads to cage mates after long periods of contact. it is transmitted between mice kept in close contact, and transmissibility from cage to cage seems to be low. mouse thymic virus is not transmitted to foetuses by the transplacental route, and intravenous infection of pregnant mice does not lead to congenital damage, impairment in size or development, or abortion (st-pierre et al., 1987) . mouse thymic virus and mcmv-1 do not crossreact serologically (cross et al., 1979) . serological monitoring of mouse populations for antibodies to mtv is possible by ifa testing, which is commercially available; elisa tests have also been established (morse, 1990b) . elisa and complement fixation yield similar results . it must be noted that the immune response depends on the age at infection. antibody responses are not detectable in mice infected as newborns whereas adult mice develop high titres that are detectable by serological testing. if neonatal infection is suspected, homogenates of salivary glands or other materials can be inoculated into pathogen-free newborn mice followed by gross and histological examination of thymus, lymph nodes, and spleens for lymphoid necrosis (morse, 1987) . alternatives to the in vivo infectivity assay for detecting mtv in infected tissues include a competition elisa (prattis and morse, 1990) and map testing, although this is slightly less sensitive than infectivity assays (morse, 1990a) . very little experience exists on eradication methods for mtv due to its low prevalence in contemporary mouse colonies. methods that eliminate other herpesviruses likely will eliminate mtv. procurement of animals of known negative mtv status is an appropriate strategy to prevent infection. strict separation of laboratory mice from wild rodents is essential to avoid introduction into laboratory animal facilities. mousepox (ectromelia) virus (ectv) is a member of the genus orthopoxvirus belonging to the family poxviridae. it is antigenically and morphologically very similar to vaccinia virus and other orthopoxviruses. poxviruses are the largest and most complex of all viruses with a diameter of 200 nm and a length of 250-300 nm. mousepox (ectromelia) virus contains one molecule of double-stranded dna with a total genome length of 185,000 nucleotides. it is the causative agent of mousepox, a generalized disease in mice. experimental transmission to young rats (up to 30 days of age) is possible (jandasek, 1968; buller et al., 1986) . the virus is resistant to desiccation, dry heat, and many disinfectants. it is not consistently inactivated in serum heated 30 min at 56њc (lipman et al., 2000b) and persists for 26 weeks when maintained at 4њc in foetal bovine serum (bhatt and jacoby, 1987a) . effective disinfectants include vapour-phase formaldehyde, sodium hypochlorite, and iodophores (small and new, 1981; national research council, 1991) . historically, ectv has been an extremely important natural pathogen of laboratory mice. the virus was widespread in mouse colonies worldwide and can still be found in several countries. between 1950 and 1980 almost 40 individual ectromelia outbreaks were reported in the usa. the last major epizootic in the usa occurred in 1979-80 and has been described in great detail (e.g. wagner and daynes, 1981) . severe outbreaks were also described in various european countries (deerberg et al., 1973; owen et al., 1975; osterhaus et al., 1981) . a more recent outbreak in the usa, which resulted in the eradication of almost 5000 mice in one institution, was described by dick et al. (1996) . the most recent well-documented case of mousepox was published by lipman et al. (2000b) . few additional but unpublished cases of ectromelia have been observed thereafter. in a recent survey conducted in the usa, one population was reported to be seropositive for mousepox (jacoby and lindsey, 1998) . natural infections manifest differently depending on many factors. mousepox may occur as a rapidly spreading outbreak with acute disease and deaths, or may be inconspicuous with slow spreading and mild clinical signs. the mortality rate can be very low in populations in which the virus has been present for long periods. the infection usually takes one of three clinical courses: acute asymptomatic infection, acute lethal infection (systemic form), or subacute to chronic infection (cutaneous form ; fenner, 1981 fenner, , 1982 manning and frisk, 1981; national research council, 1991; dick et al., 1996) . the systemic or visceral form is characterized clinically by facial oedema, conjunctivitis, multisystemic necrosis, and usually high mortality. this form is less contagious than the cutaneous form because the animals die before there is virus shedding. the cutaneous form is characterized by typical dermal lesions and variable mortality. the outcome of infection depends on many factors including strain and dose of virus; route of viral entry; strain, age, and sex of mouse; husbandry methods; and duration of infection in the colony. while all mouse strains seem to be susceptible to infection with ectv, clinical signs and mortality are strain-dependent (fenner, 1982; wallace and buller, 1985, 1986; brownstein et al., 1989b) . acute lethal (systemic) infection occurs in highly susceptible inbred strains such as dba/1, dba/2, balb/c, a, and c3h/hej. immunodeficient mice may also be very susceptible (allen et al., 1981) . outbreaks among susceptible mice can be explosive, with variable morbidity and high mortality (ͼ80%). clinical disease may not be evident in resistant strains such as c57bl/6 and akr, and the virus can be endemic in a population for long periods before being recognized. furthermore, females seem to be more resistant to disease than males, at least in certain strains of mice brownstein et al., 1989b) . the mechanisms determining resistance versus susceptibility are not fully understood but appear to reflect the action of multiple genes. the genetic loci considered to be important include h-2d b (termed rmp-3, resistance to mousepox, on chromosome 17; o'neill et al., 1983) , the c5 genes (rmp-2, on chromosome 2), rmp-1, localized to a region on chromosome 6 encoding the nk cell receptor nkr-p1 alloantigens (brownstein and gras, 1997) , the nitric oxide synthase 2 locus on chromosome 11 (karupiah et al., 1998) , and the signal transducer and activator of transcription 6 locus on chromosome 10 (mahalingam et al., 2001) . clearance of the virus by the immune system is absolutely dependent upon the effector functions of cd8 ϩ t cells while nk cells, cd4 ϩ t cells, and macrophages are necessary for the generation of an optimal response (niemialtowski et al., 1994; delano and brownstein, 1995; karupiah et al., 1996) . mousepox (ectromelia) virus usually enters the host through the skin with local replication and extension to regional lymph nodes (fenner, 1981 (fenner, , 1982 wallace and buller, 1986; national research council, 1991) . it escapes into the blood (primary viraemia) and infects splenic and hepatic macrophages resulting in necrosis of these organs and a massive secondary viraemia. this sequence takes approximately 1 week. many animals die at the end of this stage without premonitory signs of illness; others develop varying clinical signs including ruffled fur, hunched posture, swelling of the face or extremities, conjunctivitis, and skin lesions (papules, erosions, or encrustations mainly on ears, feet, and tail; figure 23 .1). necrotic amputation of limbs and tails can sometimes be seen in mice that survive the acute phase, hence the original name of the disease 'ectromelia' (meaning absent or short limbs; figure 23 .2). common gross lesions of acute mousepox include enlarged lymph nodes, peyer's patches, spleen, and liver; multifocal to semiconfluent white foci of necrosis in the spleen and liver; and haemorrhage into the small intestinal lumen (allen et al., 1981; fenner, 1982; dick et al., 1996; percy and barthold, 2001) . in animals that survive, necrosis and scarring of the spleen can produce a mosaic pattern of white and red-brown areas that is a striking gross finding. the most consistent histological lesions of acute mousepox are necroses of the spleen (figure 23 lymph nodes, peyer's patches, thymus, and liver (allen et al., 1981; fenner, 1982; dick et al., 1996; lipman et al., 2000b; percy and barthold, 2001) . occasionally, necrosis may also be observed in other organs such as ovaries, uterus, vagina, intestine, and lungs. the primary skin lesion, which occurs about a week after exposure at the site of inoculation (frequently on the head), is a localized swelling that enlarges from inflammatory oedema. necrosis of dermal epithelium provokes a surface scab and heals as a deep, hairless scar. secondary skin lesions (rash) develop 2-3 days later as the result of viraemia (figure 23 .1). they are often multiple and widespread and can be associated with conjunctivitis. the skin lesions also can ulcerate and scab before scarring. mucosal and dermal epithelial cells may have characteristic intracytoplasmic eosinophilic (cowdry type a) inclusion bodies ( figure 23 .4). basophilic (cowdry type b) inclusions may be found in the cytoplasm of all infected cells, especially in hepatocytes. natural transmission of ectv mainly occurs by direct contact and fomites (fenner, 1981; wallace and buller, 1986; national research council, 1991) . the primary route of infection is through skin abrasions. faecal-oral and aerosol routes may also be involved (werner, 1982) . in addition, the common practice of cannibalism by mice may contribute to the oral route of infection (bhatt and jacoby, 1987b) . intrauterine transmission is possible at least under experimental conditions (schwanzer et al., 1975) . virus particles are shed from infected mice (mainly via scabs and/or faeces) for about 3-4 weeks, even though the virus can persist for months in the spleen of an occasional mouse (bhatt and jacoby, 1987b; national research council, 1991) . cage-to-cage transmission of ectv and transmission between rooms or units is usually low and largely depends on husbandry practices (e.g. mixing mice from different cages). importantly, the virus may not be transmitted effectively to sentinel mice exposed to dirty bedding (lipman et al., 2000b) . various tests have been applied for the diagnosis of ectromelia. previous epidemics were difficult to deal with because of limited published data and information on the biology of the virus and the lack of specific and sensitive assays (wallace, 1981) . in the 1950s, diagnosis relied on clinical signs, histopathology, and animal passages of tissues from moribund and dead animals. culture of the virus on the chorioallantoic membrane of embryonated eggs was also applied. serology is currently the primary means of testing mouse colonies for exposure to ectv. the methods of choice are elisa and ifa; they are more sensitive and specific than the previously used haemagglutination inhibition (hi) assay (collins et al., 1981; buller et al., 1983; aclad, 1991) . both tests detect antibodies to orthopoxviruses and do not distinguish between ectv and vaccinia virus. vaccinia virus is commonly used as antigen for serological testing to avoid the risk of infection for mice. thus, false-positive serological reactions may be found after experimental administration of replication-competent vaccinia virus. it has been shown that even cage contact sentinels may develop antibodies, and vaccinia virus leading to seroconversion may even be transmitted by dirty bedding (gaertner et al., 2003) . confirmation of positive serological results is important before action is taken because vaccinia virus is increasingly prevalent in animal facilities as a research tool (e.g. for vaccination or gene therapy). as observed in different outbreaks, serological testing is of little value in the initial stages of the disease. for example, in the outbreak described by dick et al. (1996) depopulation was nearly completed before serological confirmation was possible. for this reason, negative serological results should be confirmed by direct detection methods (pcr, immunohistochemistry, virus isolation) or by histopathology, especially when clinical cases suggestive of mousepox are observed. polymerase chain reaction assays to detect different genes of poxviruses in infected tissues have been described by dick et al. (1996) , neubauer et al. (1997) , and lipman et al. (2000b) . the key to prevention and control of mousepox is early detection of infected mice and contaminated biological materials. all institutions that must introduce mice from other than commercial barrier facilities should have a health surveillance programme and test incoming mice. perhaps even more important than living animals are samples from mice (tumours, sera, tissues). the virus replicates in lymphoma and hybridoma cell lines (buller et al., 1987) , and such cells or material derived from them may therefore be a vehicle for inadvertent transfer between laboratories. the last two published outbreaks of ectromelia were both introduced into the facilities by mouse serum (dick et al., 1996; lipman et al., 2000b) . lipman et al. (2000b) found that the contaminated serum originated from a pooled lot of 43 l that had been imported from china. because mouse serum commonly is sold to the end user in small aliquots (few millilitres), it has to be expected that aliquots of the contaminated lot are still stored in numerous freezers. both cases provide excellent examples of why map or pcr testing should be performed on all biological materials to be inoculated into mice. eradication of mousepox usually has been accomplished by elimination of the affected colonies, disinfection of rooms and equipment, and disposal of all infected tissues and sera. while culling of entire mouse colonies is the safest method for eradication of mousepox, it is not a satisfactory method due to the uniqueness of numerous lines of genetically modified animals housed in many facilities. several studies indicate that mousepox is not highly contagious bhatt and jacoby 1987a,b ) and that it may be self-limiting when adequate husbandry methods are applied. therefore, strict quarantine procedures along with cessation of breeding (to permit resolution of infection) and frequent monitoring with removal of clinically sick and seropositive animals are a potential alternative. the period from the last births before the break until the first matings after the break should be at least 6 weeks (bhatt and jacoby, 1987b) . sequential testing of immunocompetent contact sentinels for seroconversion should be employed with this option. in the past, immunization with live vaccinia virus was used to suppress clinical expression of mousepox. vaccination may substantially reduce the mortality rate, but it does not prevent virus transmission or eradicate the agent from a population bhatt and jacoby, 1987c) . after vaccination, typical pocks develop at the vaccination site, and infectious vaccinia virus is detectable in spleen, liver, lungs, and thymus (jacoby et al., 1983) . vaccination also causes seroconversion so that serological tests are not applicable for health surveillance in vaccinated populations. it is therefore more prudent to control mousepox by quarantine and serological surveillance than by relying on vaccination. mortality and clinical disease are the major factors by which ectv interferes with research. severe disruption of research can also occur when drastic measures are taken to control the infection. the loss of time, animals, and financial resources can be substantial. murine adenoviruses (madv) are non-enveloped, double-stranded dna viruses of the family adenoviridae, genus mastadenovirus. two distinct strains have been isolated from mice. the fl strain (madv-1) was first isolated in the usa as a contaminant of a friend leukaemia (hartley and rowe, 1960) ; the k87 strain (madv-2) was first isolated in japan from the faeces of a healthy mouse (hashimoto et al., 1966) . both strains are now considered to represent different species (hamelin and lussier, 1988; jacques et al., 1994a,b) . in laboratory mice, seropositivity to adenoviruses was reported in 2% of spf colonies and in 8% of non-spf colonies in the usa (jacoby and lindsey, 1998) . antibodies were also detected at a low prevalence rate in french colonies (zenner and regnault, 2000) , but the virus strain used as antigen is not mentioned. a similar range of positive samples was reported by livingston and riley (2003) . antibodies to madv were also found in wild mice and in rats (otten and tennant, 1982; smith et al., 1986) . both viruses are not known to cause clinical disease in naturally infected, immunocompetent mice. however, madv-1 can cause a fatal systemic disease in suckling mice after experimental inoculation (hartley and rowe, 1960; heck et al., 1972; wigand, 1980) . disease is characterized by scruffiness, lethargy, stunted growth, and often death within 10 days. experimental infection of adult mice with madv-1 is most often subclinical and persistent (richter, 1986 ) but can cause fatal haemorrhagic encephalomyelitis with neurological symptoms, including tremors, seizures, ataxia, and paralysis, in susceptible c57bl/6 and dba/2j mice (guida et al., 1995) . balb/c mice are relatively resistant to this condition. athymic foxn1 nu mice experimentally infected with madv-1 develop a lethal wasting disease (winters and brown, 1980) . similarly, prkdc scid mice succumb to experimental infection with madv-1 (pirofski et al., 1991) . gross lesions in response to natural madv infections are not detectable. occasional lesions observed after experimental infection with madv-1 include small surface haemorrhages in the brain and spinal cord of c57bl/6 and dba/2j mice (guida et al., 1995) , duodenal haemorrhage in foxn1 nu mice (winters and brown, 1980) , and pale yellow livers in prkdc scid mice (pirofski et al., 1991) . histologically, experimental madv-1 infection of suckling mice is characterized by multifocal necrosis and large basophilic intranuclear inclusion bodies in liver, adrenal gland, heart, kidney, salivary glands, spleen, brain, pancreas, and brown fat (heck et al., 1972; margolis et al., 1974; national research council, 1991; percy and barthold, 2001) . in experimentally induced haemorrhagic encephalomyelitis, multifocal petechial haemorrhages occur throughout the brain and spinal cord, predominantly in the white matter, and are attributed to infection and damage to the vascular epithelium of the central nervous system (cns; guida et al., 1995) . histopathological manifestations in madv-1-infected prkdc scid mice are marked by microvesicular fatty degeneration of hepatocytes (pirofski et al., 1991) . in contrast to madv-1, the tissue tropism of madv-2 is limited to the intestinal epithelium. naturally or experimentally infected mice develop intranuclear inclusions in enterocytes, especially in the ileum and caecum (takeuchi and hashimoto, 1976; otten and tennant, 1982; national research council, 1991; percy and barthold, 2001) . transmission of madv primarily occurs by ingestion. madv-1 is excreted in the urine and may be shed for up to 2 years (van der veen and mes, 1973). murine adenovirus-2 infects the intestinal tract and is shed in faeces for only a few weeks in immunocompetent mice (hashimoto et al., 1970) ; immunodeficient mice may shed the virus for longer periods (umehara et al., 1984) . murine adenovirus infections are routinely diagnosed by serological tests. however, there is a one-sided cross reactivity of madv-1 with madv-2 (wigand et al., 1977) . serum from mice experimentally infected with madv-1 yielded positive reactions in serological tests with both viruses while serum from mice infected with madv-2 reacted only with the homologous antigen . smith et al. (1986) reported that sera may react with madv-1 or madv-2 or both antigens. occasional reports of mice with lesions suggestive of adenovirus infections and negative serology (with madv-1) indicate that the infection may not be detected if only one virus is used as antigen (luethans and wagner, 1983) . it has therefore become standard practice to test sera for antibodies to both madv-1 and madv-2. the common methods are ifa and elisa, and both are more sensitive than the previously used complement fixation test. the low prevalence in colonies of laboratory mice indicate that madv can easily be eliminated (e.g. by hysterectomy derivation or embryo transfer) and that barrier maintenance has been very effective in preventing infection. the low pathogenicity and the low prevalence in contemporary mouse populations are the main reasons why adenoviruses are considered to be of little importance. however, immunodeficient mice are increasingly used and candidates for natural infections and wasting disease (richter, 1986) , and the viruses might easily be spread by the exchange of genetically modified mice and therefore re-emerge. only few influences on research attributable to madv have been published. for example, it has been shown that madv-1 significantly aggravates the clinical course of scrapie disease in mice (ehresmann and hogan, 1986) . natural infections with madv could also interfere with studies using adenovirus as a gene vector. polyomaviridae are enveloped, double-stranded dna viruses. two different agents of this family exclusively infect mice (mus musculus), and both belong to the genus polyomavirus. murine pneumotropic virus (mptv) has formerly been known as 'newborn mouse pneumonitis virus' or 'k virus' (named after l. kilham who first described the virus). the second is murine polyomavirus (mpyv). both are related but antigenically distinct from each other (bond et al., 1978) . they are enzootic in many populations of wild mice but are very uncommon in laboratory mice. even older reports indicate that both have been eradicated from the vast majority of contemporary mouse colonies, and their importance is negligible (national research council, 1991) . seropositivity to these viruses was not reported in a survey conducted in the usa (jacoby and lindsey, 1998) . in a retrospective study in french facilities, antibodies to mpyv were found in 1 of 69 colonies, and all samples tested for mptv were negative (zenner and regnault, 2000) . comparable data were reported by livingston and riley (2003) . due to their low prevalence, both viruses are not included in the list of agents for which testing is recommended on a regular basis by felasa (nicklas et al., 2002) . although polyomavirus genes, especially those of sv40 are used widely in gene constructs for insertional mutagenesis, very few reports have been published on spontaneous or experimental disease due to mpyv or mptv in the last 10-15 years. the reader is therefore referred to previous review articles for details (eddy 1982; parker and richter, 1982; richter, 1986; shah and christian, 1986; national research council, 1991; orcutt, 1994; porterfield and richter, 1994) . natural infections with mptv are subclinical. the prevalence of infection is usually low in an infected population. the virus may persist in infected animals for months and perhaps for life depending on the age at infection and is reactivated under conditions of immunosuppression. virus replicates primarily in endothelial cells, but renal tubular epithelial cells are the major site of viral persistence (greenlee et al., 1991 (greenlee et al., , 1994 . clinical signs are observed only after infection of infant mice less than 6-8 days of age. infected pups suddenly develop respiratory symptoms after an incubation period of approximately 1 week, and many die within a few hours of onset of symptoms with an interstitial pneumonia caused by productive infection of and damage to pulmonary endothelium. endothelial cells in other organs are involved in virus replication also (ikeda et al., 1988; greenlee et al., 1994) . in older suckling mice, mptv produces a more protracted infection, and the virus or viral antigen can be detected for as long as 4 months. in adult animals, the virus produces a transient asymptomatic infection. even in immunodeficient foxn1 nu mice, experimental infection of adults is clinically asymptomatic although virus is detectable for a period of several months (greenlee, 1986) . in vitro cultivation of mptv is difficult. no susceptible permanent cell line is known to support growth. it can be cultured in primary mouse embryonic cells, but viral titres are not sufficient for use in serological assays (greenlee and dodd, 1987) . for this reason, the hi test using homogenates of livers and lungs of infected newborn mice is still frequently used, but ifa and elisa tests are also available (groen et al., 1989) . furthermore, a pcr test for demonstration of mptv in biological samples has been published (carty et al., 2001) . murine polyomavirus was first detected as a contaminant of murine leukaemia virus (mulv) when sarcomas developed in mice after experimental inoculation of contaminated samples. it has later been shown to be a frequent contaminant of transplantable tumours (collins and parker, 1972) . natural infection of mice is subclinical, and gross lesions including tumours are usually not found. tumour formation occurs if mice are experimentally infected at a young age or if they are inoculated with high virus doses. development of tumours may be preceded by multifocal necrosis and mortality during the viraemic stage (percy and barthold, 2001) . parotid, salivary gland, and mammary tumours are common, and sarcomas or carcinomas of kidney, subcutis, adrenal glands, bone, cartilage, teeth, blood vessels, and thyroid occur also. virus strains vary with regard to the tumour types or lesions that they induce, and mouse strains vary in their susceptibility to different tumour types. those of c57bl and c57br/cd lineage are considered to be the most resistant strains; athymic foxn1 nu mice are considered to be most susceptible; c3h mice are particularly susceptible to adrenal tumours and a mice tend to develop bone tumours. immunosuppression or inoculation into immunodeficient strains (e.g. foxn1 nu ) also support the growth of tumours. on the other hand, experimental infection of adult immunocompetent mice does not result in tumour formation because the immune response suppresses tumour growth, and newborn immunocompetent mice develop runting only if inoculated with high virus doses (atencio et al., 1995) . after experimental intranasal infection, mpyv initially infects the respiratory tract followed by a systemic phase in which liver, spleen, kidney, and the colon become infected (dubensky et al., 1984) . the virus is shed in faeces and in all body fluids, and transmission occurs rapidly by direct contact between animals, but also between cages in a room. further, intrauterine transmission has been documented after experimental infection (mccance and mims, 1977) . murine polyomavirus persists in all organs in prkdc scid mice while viral dna is detectable in immunocompetent mice after experimental infection for only a limited period of about 4 weeks (berke et al., 1994) . however, virus may persist and can be reactivated by prolonged immunosuppression (rubino and walker, 1988) or during pregnancy, at least in young mice (mccance and mims, 1979) . biological materials of mouse origin are likely to be the most common source of contamination of laboratory mice emphasizing the importance of map or pcr screening of biological materials to be inoculated into mice. the most frequently used tests for health surveillance of mouse colonies are elisa and ifa (aclad, 1991) ; in addition, the hi test is still used. latent infections can be detected by intracerebral inoculation of neonate mice or by map testing, but direct demonstration of virus in biological samples is also possible by pcr testing (porterfield and richter, 1994; carty et al., 2001) . parvoviruses are non-enveloped small viruses (approximately 20 nm in diameter) with a single-stranded dna genome of approximately 5000 nucleotides. murine parvoviruses are members of the family parvoviridae, genus parvovirus. they are remarkably resistant to environmental conditions like heat, desiccation, acidic and basic ph-values. two distinct serotypes infect laboratory mice: the mice minute virus (mmv) and the mouse parvovirus 1 (mpv). nonstructural proteins (ns-1 and ns-2) are highly conserved among both viruses whereas the capsid proteins (vp-1, vp-2, vp-3) are more divergent and determine the serogroup (ball-goodrich and johnson, 1994) . both viruses require mitotically active cells for replication. severe infections are therefore not found in mature animals due to the lack of a sufficient number of susceptible cells in tissues. general aspects of rodent parvovirus infections and their potential effects on research results have been reviewed (tattersall and cotmore, 1986; national research council, 1991; jacoby et al., 1996) . already in the mid-1980s mouse colonies were identified that gave positive reactions for mmv by ifa but not by hi tests. it was subsequently shown that these colonies were infected with a novel parvovirus, initially referred to as 'mouse orphan parvovirus'. the first isolate of mpv was detected as a contaminant of cultivated t-cell clones interfering with in vitro immune responses (mckisic et al., 1993) and was named 'mouse parvovirus'. it does not replicate well in currently available cell cultures, and sufficient quantities of virus for serological tests are difficult to generate. hitherto, only very few isolates of mpv have been cultured and characterized on a molecular basis (ball-goodrich and johnson, 1994; besselsen et al., 1996) . at present, mpv is among the most common viruses in colonies of laboratory mice. the prevalence of sera positive for parvoviruses was nearly 10% in a study from livingston et al. (2002) , with the majority of sera being positive for mpv. this is consistent with a recent survey conducted in the usa showing that almost 40% of non-spf colonies were seropositive (jacoby and lindsey, 1998) . similar results were obtained for genetically modified mice in japan (yamamoto et al., 2001) , in contrast to earlier studies indicating that the infection was rare in japan (ueno et al., 1998) . clinical disease and gross or histological lesions have not been reported for mice naturally or experimentally infected with mpv. infections are subclinical even in newborn and immunocompromised animals . in contrast to many other viruses infecting mice, viral replication and excretion is not terminated by the onset of host immunity. tissue necrosis has not been observed at any stage of infection in infected infant or adult mice . humoral immunity to mpv does not protect against mmv infections and vice versa (hansen et al., 1999) . serological surveys have indicated that mpv naturally infects only mice. differences in mouse strain susceptibility to clinical mpv infection do not exist. however, seroconversion seems to be strain-dependent. after experimental infection, seroconversion occurred in all c3h/hen mice, fewer balb/c, dba/2, and icr mice, and seroconversion could not be detected in c57bl/6 mice (besselsen et al., 2000) . diagnosis of mpv infection by pcr testing of small intestine and mesenteric lymph nodes also depended on the mouse strain. mpv dna was detected in all mouse strains evaluated except dba/2 even though seroconversion was detected in these mice. after oral infection, the intestine is the primary site of viral entry and replication. the virus spreads to the mesenteric lymph nodes and other lymphoid tissues, where it persists for more than 2 months , and seems to be excreted via the intestinal and the urinary tract. after experimental inoculation of weanling mice, mpv is transmitted to cagemates by direct contact for 2-4 weeks , and transmission by dirty bedding is also possible. these results implicate a role for urinary, faecal, and perhaps respiratory excretion of virus. another study showed that naturally infected mice may not transmit the virus under similar experimental conditions (shek et al., 1998) . serology is a useful tool to identify mpv infections in immunocompetent hosts, but reaching a diagnosis based on serological assays may be difficult and requires a good knowledge of the available techniques. neither the virion elisa nor hi are practical screening tests for mpv because they require large quantities of purified mpv which is difficult to obtain. diagnosis of mpv infections has long been made on the basis of an mmv hi-negative result coupled with an mmv ifapositive result. a generic rodent parvovirus elisa using a recombinant ns-1 protein as antigen has been developed , but mpv ifa and mpv hi assays are more sensitive techniques than the ns-1 elisa and the mmv ifa (besselsen et al., 2000) . recently, elisa tests have been described that use recombinant vp-2 and provide sensitive and serogroupspecific assays for the diagnosis of mpv infections in mice (ball-goodrich et al., 2002; livingston et al., 2002) . in immunodeficient mice that do not generate a humoral immune response, pcr assays can be used to detect mpv (besselsen et al., 1995; redig and besselsen, 2001 ) and other parvoviruses. mpv has been shown to persist for at least 9 weeks in the mesenteric lymph nodes . this tissue is considered the best suited for pcr analysis, but spleen and small intestine can also be used with good success (besselsen et al., 2000) . the virus persists sufficiently long in mesenteric lymph nodes so that pcr assays may also be used as a primary screening tool for laboratories that do not have access to specific mpv antigenbased serological assays. polymerase chain reaction is further a good confirmatory method for serological assays and has also been described for the detection of parvoviruses in cell lines and tumours (yagami et al., 1995) . in addition, the map test has been reported as a sensitive tool to detect mpv (shek et al., 1998) . given the high environmental stability of the virus and the potential fomite transmission together with the long virus persistence in infected animals, spontaneous disappearance from a mouse population (e.g. by cessation of breeding) is very unlikely. eradication of infection is possible by elimination of infected animals and subsequent replacement with uninfected mice, and the agent can be eliminated from breeding populations only by embryo transfer or by hysterectomy. although there are few published reports of confounding effects of mpv on research, it is lymphocytotropic and may perturb immune responses in vitro and in vivo. infections with mpv have been shown to influence rejection of skin and tumour grafts (mckisic et al., , 1998 . mice minute virus is the type species of the genus parvovirus. the virus was formerly called 'minute virus of mice' (mvm) and was renamed recently (international union of microbiological societies, 2000) . it was originally isolated by crawford (1966) from a stock of mouse adenovirus, and this prototype isolate was later designated mvmp. its allotropic variant was detected as a contaminant of a transplantable mouse lymphoma (bonnard et al., 1976) and designated mvmi because it exhibits immunosuppressive properties in vitro. both variants have distinct cell tropisms in vivo and in vitro. the mmvp infects fibroblast cell lines and does not cause clinical disease (kimsey et al., 1986 , brownstein et al., 1991 . the mmvi grows lytically in t cells and inhibits various functions mediated by these cells in vitro. both strains are apathogenic for adult mice, but the immunosuppressive variant is more pathogenic for neonatal mice than is mmvp. serological surveys show that the mouse is the primary natural host (parker et al., 1970; smith et al., 1993b; singleton et al., 2000) , but the virus is also infective for rats, hamsters (garant et al., 1980; ward and tattersall, 1982) , and mastomys (haag et al., 2000) during foetal development or after parenteral inoculation. natural infections are usually asymptomatic in adults and infants, and the most common sign of infection is seroconversion. kilham and margolis (1970) observed mild growth retardation a few days after experimental infection of neonatal mice with mmvp. studies of transplacental infection yielded no pathological findings in mice (kilham and margolis, 1971 ). the immunosuppressive variant but not the prototype strain is able to produce a runting syndrome after experimental infection of newborn mice (kimsey et al., 1986) . depending on the host genotype, experimental infections of foetal and neonatal mice with mmvi produce various clinical presentations and lesions. infection in c57bl/6 mice is asymptomatic, but the virus causes lethal infections with intestinal haemorrhage in dba/2 mice. infection of strains such as balb/c, cba, c3h/he, and sjl is also lethal and mice have renal papillary haemorrhage (brownstein et al., 1991) . the mmvi also infects haematopoietic stem cells and mediates an acute myelosuppression (segovia et al., 1991 (segovia et al., , 1995 . due to their dependency on mitotically active tissues, the foetus is at particular risk for damage by parvoviruses. mice minute virus and other parvoviruses may have severe teratogenic effects and cause foetal and neonatal abnormalities by destroying rapidly dividing cell populations, often resulting in foetal death. adult prkdc scid mice develop an acute leukopenia 1 month after experimental infection with mmvi and die within 3 months. the virus persists lifelong in the bone marrow of these mice (segovia et al., 1999) . mice minute virus is shed in faeces and urine. contaminated food and bedding are important factors in viral transmission because the virus is very resistant to environmental conditions. direct contact is also important and the virus does not easily spread between cages. routine health surveillance is usually conducted by serological methods. unlike mpv, mmv can easily be cultured in cell lines so that antigen production for hi and elisa (using whole purified virions) is easy. haemagglutination inhibition is a highly specific diagnostic test whereas ifa always exhibits some degree of cross reactivity with mpv and other closely related parvoviruses. enzyme-linked immunosorbent assay is probably the most frequently used test, but depending on the purity of the antigen preparation, cross reactions with mpv may occur due to contamination with nonstructural proteins that are common to both viruses. this problem can be avoided by the use of recombinant vp-2 antigen (livingston et al., 2002) . viral detection is also possible by pcr in biological materials and in organs (intestines, kidney, spleen) from infected animals (yagami et al., 1995; chang et al., 1997; redig and besselsen, 2001) . in contrast to mpv, pcr is not appropriate as a confirmatory method for serology because mmv has not been shown to persist in immunocompetent animals for sufficiently long periods. the virus can be eliminated from infected breeding populations by caesarean derivation or by embryo transfer. in experimental colonies, elimination of infected animals and subsequent replacement with uninfected mice is practical if careful environmental sanitation is conducted by appropriate disinfection procedures. it is important that reintroduction is avoided by exclusion of wild mice and by strict separation from other infected populations and potentially contaminated materials in the same facility. admission of biological materials must be restricted to samples that have been tested and found free from viral contamination. both allotropic variants of mmv have been used as models for molecular virology, and their small size and simple structure have facilitated examination of their molecular biology and expedited understanding of cell tropism, viral genetics, and structure. the significance for laboratory mouse populations was considered low or uncertain because natural infections are inapparent. however, various effects on mouse-based research have been published (tattersall and cotmore, 1986; jacoby et al., 1996; baker, 1998; nicklas et al., 1999) . due to their predilection for replicating in mitotically active cells, they are frequently associated with tumour cells and have a marked oncosuppressive effect (rommelaere and cornelis, 1991) . special attention is also necessary for immunological research and other studies involving rapidly dividing cells (embryology, teratology). in addition, mmv is a common contaminant of transplantable tumours, murine leukaemias, and other cell lines (collins and parker, 1972; nicklas et al., 1993; garnick, 1996) . lactate dehydrogenase-elevating virus (ldv) is a single-stranded rna virus of the genus arterivirus belonging to the family arteriviridae. lactate dehydrogenase-elevating virus has repeatedly been detected in feral mice (mus musculus), which are considered to be a virus reservoir (rowson and mahy, 1975; li et al., 2000) . only mice and primary mouse cells are susceptible to infection with ldv. after infection, virus titres of 10 10 -10 11 particles per ml serum are found within 12-14 h after infection. the virus titre drops to 10 5 particles per ml within 2-3 weeks and remains constant at this level for life. lactate dehydrogenase-elevating virus replicates in a subpopulation of macrophages in almost all tissues and persists in lymph nodes, spleen, liver, and testes tissues (anderson et al., 1995a) . the virus can be stored in undiluted mouse plasma at ϫ70њc without loss of infectivity, but it is not stable at room temperature and is very sensitive to environmental conditions. lactate dehydrogenase-elevating virus was first detected during a study of methods that could be used in the early diagnosis of tumours (riley et al., 1960) . it produces a persistent infection with continuous virus production and a lifelong viraemia despite ldv-specific immune reactions of the host ( van den broek et al., 1997) . lactate dehydrogenase-elevating virus has been found in numerous biological materials that are serially passaged in mice such as transplantable tumours including human tumours (nicklas et al., 1993; ohnishi et al., 1995) , monoclonal antibodies or ascitic fluids (nicklas et al., 1988) , or infectious agents (e.g. haemoprotozoans, k virus, clostridium piliforme). these materials are contaminated after passage in an infected and viraemic animal. contamination with ldv leads to the infection of each sequential host and to transmission of the virus by the next passage and remains associated with the specimen. it is therefore the most frequently detected contaminant in biological materials (collins and parker, 1972; nicklas et al., 1993) . infection with ldv is usually asymptomatic, and there are no gross lesions in immunocompetent as well as in immunodeficient mice. the only exception is polyomyelitis with flaccid paralysis of hind limbs developing in c58 and akr mice when they are immunosuppressed either naturally with aging or experimentally (anderson et al., 1995b; monteyne et al., 1997) . it has been shown that only mice harbouring cells in the cns that express a specific endogenous mulv are susceptible to poliomyelitis (anderson et al., 1995c) . the characteristic feature of ldv infection is the increased activity of lactate dehydrogenase (ldh) and other plasma enzymes (brinton, 1986; national research council, 1991) , which is due to the continuous destruction of permissive macrophages that are responsible for the clearance of ldh from the circulation. as a consequence, the activity of plasma ldh begins to rise by only 24 h after infection and peaks 3-4 days after infection at 5-10-fold normal levels, or even be up to 20-fold in sjl/j mice. the enzyme activity declines during the next 2 weeks but remains elevated throughout life. antigen-antibody complexes produced during infection circulate in the blood and are deposited in the glomeruli (brinton, 1986; national research council, 1991) . in contrast to other persistent virus infections (e.g. lymphocytic choriomeningitis virus lcmv), these complexes do not lead to immune complex disease and produce only a very mild glomerulopathy. the only gross finding associated with ldv infection is mild splenomegaly. microscopically, necrosis of lymphoid tissues is visible during the first days of infection. in mouse strains that are susceptible to poliomyelitis, ldv induces lesions in the grey matter of the spinal cord and the brain stem (brinton, 1986) . lactate dehydrogenase-elevating virus is not easily transmitted between mice, even in animals housed in the same cage. fighting and cannibalism increase transmission between cage mates most likely via blood and saliva. infected females transmit the virus to their foetuses if they have been infected few days prior to birth and before igg anti-ldv antibodies are produced, but developmental and immunological factors (e.g. gestational age, timing of maternal infection with ldv, placental barrier) are important in the regulation of transplacental ldv infection (haven et al., 1996; zitterkopf et al., 2002) . maternal immunity protects foetuses from intrauterine infection. immunodeficient prkdc scid mice transmit virus to their offspring also during chronic infection (broen et al., 1992 ). an important means of transmission is provided by experimental procedures such as mouse-to-mouse passage of contaminated biological materials or the use of the same needle for sequential inoculation of multiple mice. in principal, serological methods such as ifa may be used for detecting ldv infection (hayashi et al., 1992) but they are not of practical importance. circulating virus-antibody complexes interfere with serological tests (aclad, 1991) , and sufficient quantities of virus for serological tests are difficult to generate because ldv replicates only in specific subpopulations of primary cultures of murine macrophages and monocytes for one cell cycle (brinton, 1986) . therefore, diagnosis of ldv infection is primarily based on increased ldh activity in serum or plasma of mice. lactate dehydrogenase-elevating virus activity in serum or plasma can be measured directly, or samples (e.g. plasma, cell or organ homogenates) are inoculated into pathogen-free mice and the increase in ldh activity within 3-4 days is measured. an 8-10-fold increase is indicative of ldv infection. detection of infectivity of a plasma sample by the induction of increased ldh activity in the recipient animal is the most reliable means of identifying an infected animal. however, it is important to use clear nonhaemolysed samples because haemolysis will (falsely) elevate activities of multiple serum or plasma enzymes, including ldh. while this assay may be included in a commercial 'map test', it does not involve antibody detection. persistent infection makes ldv an ideal candidate for pcr detection in plasma or in organ homogenates (van der logt et al., 1994; chen and plagemann, 1997) . however, reports exist that pcr may produce false-negative results and should be used cautiously (lipman et al., 2000a) . similarly important as detecting ldv in animals is its detection in biological materials. this may be done by assay for increased ldh activity after inoculation of suspect material into pathogen-free mice (collins and parker, 1972; nicklas et al., 1993) or by pcr (goto et al., 1998; bootz and sieber, 2002) . lactate dehydrogenase-elevating virus spreads slowly in a population because direct contact is necessary. therefore ldv-negative breeding populations can easily be established by selecting animals with normal plasma ldh activity. embryo transfer and hysterectomy derivation are also efficient. the presence of ldv in experimental populations is indicative of contaminated biological materials. in such cases, it is essential that the virus is also eliminated from these samples. this is easily achieved by maintenance of cells by in vitro culture instead of by animal-to-animal passages (plagemann and swim, 1966) . due to the extreme host specificity of the virus, contaminated tumour samples can also be sanitized by passages in nude rats or other animal species. lactate dehydrogenase-elevating virus is a potential confounder of any research using biological materials that are passaged in mice. once present in an animal, the virus persists lifelong. the most obvious signs are increased levels of plasma ldh and several other enzymes. lactate dehydrogenase-elevating virus may also exhibit numerous effects on the immune system (thymus involution, depression of cellular immunity, enhanced or diminished humoral responses, nk cell activation, development of autoimmunity, and suppression of development of diabetes in nod mice; cafruny and hovinen, 1988; nicklas et al., 1988; takei et al., 1992; markine-goriaynoff et al., 2002; gomez et al., 2003) and enhance or suppress tumour growth (brinton, 1982; baker, 1998; nicklas et al., 1999) . lymphocytic choriomeningitis virus is an enveloped, segmented single-stranded rna virus of the genus arenavirus, family arenaviridae. its name refers to the condition that results from experimental intracerebral inoculation of the virus into adult mice and is not considered to be a feature of natural infections. mice (mus musculus) serve as the natural virus reservoir (salazar-bravo et al., 2002) , but syrian hamsters are also important hosts (ackermann, 1977) . additional species such as rabbits, guinea pigs, squirrels, monkeys, and humans are susceptible to natural or experimental infection. infection in hamsters is considered to be asymptomatic (national research council, 1991) . natural infection of callitrichid primates (marmosets and tamarins) leads to a progressive hepatic disease that is known as 'callitrichid hepatitis' (montali et al., 1995; asper et al., 2001; lukashevich et al., 2003) . antibodies to lcmv have been found in wild mice in europe (ackermann et al., 1964) , africa (el karamany and imam, 1991), asia (morita et al., 1991 (morita et al., , 1996 , australia , and america (childs et al., 1992) . thus, it is the only arenavirus with worldwide distribution. infection with lcmv is rarely found in laboratory mice (smith et al., 1984) . seropositivity to lcmv was reported in approximately 5% of non-spf mouse colonies in the usa in 1996 (jacoby and lindsey, 1998) and in 4% of french colonies in 1996-97 (zenner and regnault, 2000) . recent studies confirm that only a small percentage of mice tested are positive for lcmv (livingston and riley, 2003) . in addition to laboratory mice and other vertebrate hosts, the virus has frequently been found in transplantable tumours and tissue culture cell lines from mice and hamsters (bhatt et al., 1986a; nicklas et al., 1993) . despite the low prevalence in laboratory mice, seropositivity to this zoonotic agent should raise serious concern for human health. lymphocytic choriomeningitis virus is frequently transmitted to humans from wild mice (childs et al., 1991) and is also endemic to a varying degree in the human population (childs et al., 1991; marrie and saron, 1998; lledo et al., 2003) due to contact with wild mice. lymphocytic choriomeningitis virus is further transmitted to humans by domestic syrian hamsters rousseau et al., 1997) . in addition, infected laboratory mice (dykewicz et al., 1992) and contaminated biological materials are important sources of infections for humans, and several outbreaks of lcm among laboratory personnel have been traced to transplantable tumours biggar et al., 1977; mahy et al., 1991) . in mice, clinical signs of lcmv infection vary with strain and age of mouse, strain and dose of virus, and route of inoculation (lehmann-grube, 1982; national research council, 1991) . two forms of natural lcmv infection are generally recognized: a persistent tolerant and an (acute) nontolerant form. the persistent form results from infection of mice that are immunotolerant. this is the case if mice are infected in utero or during the first days after birth. this form is characterized by lifelong viraemia and shedding. mice may show growth retardation, especially during the first 3-4 weeks, but appear otherwise normal. infectious virus is bound to specific antibodies and complement, and these complexes accumulate in the renal glomeruli, the choroid plexus, and sometimes also in synovial membranes and blood vessel walls. at 7-10 months of age, immune complex nephritis develops with ruffled fur, hunched posture, ascites, and occasional deaths. this immunopathological phenomenon is called 'late onset disease' or 'chronic immune complex disease'. the incidence of this type of disease varies between mouse strains. gross lesions include enlarged spleen and lymph nodes due to lymphoid hyperplasia. kidneys affected with glomerulonephritis may be enlarged with a granular surface texture or may be shrunken in later stages of the disease process. microscopically, there is generalized lymphoid hyperplasia and immune complex deposition in glomeruli and vessel walls, resulting in glomerulonephritis and plasmacytic, lymphocytic perivascular cuffs in all visceral organs (percy and barthold, 2001) . the nontolerant acute form occurs when infection is acquired after the development of immunocompetence (in mice older than 1 week). these animals become viraemic but do not shed virus and may die within a few days or weeks. natural infections of adults are usually asymptomatic. surviving mice are seropositive and in most cases clear the virus to below detection levels of conventional methods. however, virus may persist at low levels in tissues (particularly spleen, lung, and kidney) of mice for at least 12 weeks after infection as determined by sensitive assays such as nested reverse transcriptasepolymerase chain reaction (rt-pcr) or immunohistochemisty (ciurea et al., 1999) . such nonlethal infection leads to protection against otherwise lethal intracerebral challenge. protection from lethal challenge is also achieved by maternally derived anti-lcmv antibodies through nursing or by the administration of anti-ldv monoclonal igg2a antibodies (baldridge and buchmeier, 1992) . in experimentally infected animals, the route of inoculation (subcutaneous, intraperitoneal, intravenous, intracerebral) also influences the type and degree of disease (lehmann-grube, 1982; national research council, 1991) . intracerebral inoculation of adult immunocompetent mice typically results in tremors, convulsions, and death due to meningoencephalitis and hepatitis. neurological signs usually appear on day 6 postinoculation, and animals die within 1-3 days after the onset of symptoms or recover within several days. the classic histological picture is of dense perivascular accumulations of lymphocytes and plasma cells in meninges and choroid plexus. while infection following subcutaneous inoculation usually remains inapparent, reaction of mice to intraperitoneal or intravenous inoculation depends on the virus strain and on the mouse strain. infection by these routes primarily causes multifocal hepatic necrosis and necrosis of lymphoid cells. athymic foxn1 nu mice and other immunodeficient mice do not develop disease but become persistently viraemic and shed virus. as a general rule, all pathological alterations following lcmv infection are immune-mediated; and mice can be protected from lcmv-induced disease by immunosuppression (gossmann et al., 1995) . lymphocytic choriomeningitis virus disease is a prototype for virus-induced t-lymphocyte-mediated immune injury and for immune complex disease. for detailed information on the pathogenesis of lcmv infection, the reader is referred to a recent review article by oldstone (2002) . extensive information on the clinical and pathological features of lcmv infection in mice has been assembled by lehmann-grube (1982) . in nature, carrier mice with persistent infection serve as the principal source of virus. intrauterine transmission is very efficient, and with few exceptions all pups born from carrier mice are infected. furthermore, persistently infected mice and hamsters can shed large numbers of infectious virions primarily in urine, but also in saliva and milk. the virus can replicate in the gastric mucosa after intragastric infection (rai et al., 1996 (rai et al., , 1997 . gastric inoculation elicits antibody responses of comparable magnitudes as intravenous inoculation and leads to active infection with lcmv indicating that oral infection is possible, e.g., by ingestion of contaminated food or cannibalism. a self-limiting infection frequently results from infection of adult mice. the virus does not spread rapidly after introduction in populations of adult mice, and the infectious chain usually ends. however, if the virus infects a pregnant dam or a newborn mouse, a lifelong infection results, and soon a whole breeding colony of mice may become infected if the mice live in close proximity (which is the case under laboratory conditions). lymphocytic choriomeningitis virus is most commonly diagnosed by serological methods. methods of choice are ifa and elisa, which have replaced the relatively insensitive complement fixation test. it is important that bleeding of mice is done carefully because of a potential risk due to viraemic animals. historically, direct viral detection was performed by inoculating body fluids or tissue homogenates into the brain of lcmv-free mice or by subcutaneous injection into mice and subsequent serological testing (map test). more recently, pcr assays have been developed for the direct detection of viral rna in clinical samples or animals (park et al., 1997 . both map test and pcr can also be used to detect contamination of biological materials (bootz and sieber, 2002) . vertical transmission of lcmv by transuterine infection is efficient so this virus cannot reliably be eliminated by caesarean rederivation. caesarean derivation may be effective if dams acquired infection after the development of immunocompetence (nontolerant acute infection) and subsequently eliminated the virus, but such a strategy is difficult to justify in light of lcmv's zoonotic potential. in breeding colonies of great value, virus elimination might be possible soon after introduction into the colony by selecting nonviraemic breeders. this procedure is expensive and time consuming and requires special safety precautions. fortunately, infections of laboratory mice with lcmv are very uncommon. however, once lcmv has been detected in animals or in biological materials, immediate destruction of all contaminated animals and materials is advisable to avoid risk of human infection. foxn1 nu and prkdc scid mice may pose a special risk because infections are silent and chronic (mahy et al., 1991) . cages and equipment should be autoclaved, and animal rooms should be fumigated with disinfectants such as formaldehyde, vaporized paraformaldehyde, and hydrogen peroxide. appropriate precautions are necessary for experiments involving lcmv, or lcmv-infected animals or materials. biological safety level (bsl) 2 will be considered to be sufficient in most cases. biological safety level 3 practices may be considered when working with infected animals owing to the increased risk of virus transmission by bite wounds, scratching, or aerosol formation from the bedding. animal biosafety level (absl) 3 practices and facilities are generally recommended for work with infected hamsters. appropriate precautions have been defined for different bsls or animal biology safety levels by cdc (1999) . lymphocytic choriomeningitis virus is an important zoonotic agent. it has been transmitted to humans working with infected animals or with contaminated biological materials and can cause mild to serious or fatal disease in humans (dykewicz et al., 1992; barton et al., 1995; barton and hyndman, 2000) . congenital infection in humans may result in hydrocephalus, or foetal or neonatal death (barton et al., 2002) . lymphocytic choriomeningitis virus is also frequently utilized as a model organism to study virus-host interactions, immunological tolerance, virus-induced immune complex disease, and a number of immunological mechanisms in vivo and in vitro (slifka, 2002; zinkernagel, 2002) . accidental transmission may have a severe impact on various kinds of experiments (for details, see lehmann-grube, 1982; bhatt et al., 1986b; national research council, 1991; baker, 1998; nicklas et al., 1999) . mammalian orthoreoviruses (mrv) are nonenveloped, segmented double-stranded rna viruses of the family reoviridae, genus orthoreovirus. they have a wide host range and are ubiquitous throughout the world. the designation reo stands for respiratory enteric orphan and reflects the original isolation of these viruses from human respiratory and intestinal tract without apparent disease. the term 'orphan' virus refers to a virus in search of a disease. mammalian orthoreovirus can be grouped into three serotypes (1, 2, 3). mammalian orthoreovirus-3 (synonyms: hepatoencephalomyelitis virus; echo 10 virus) infection remains prevalent in contemporary mouse colonies and has been reported in wild mice barthold, 1997a) . seropositivity to mrv-3 was found in less than 5% of spf colonies and in approximately 20% of non-spf mouse colonies in the usa in 1996 (jacoby and lindsey, 1998) . a study in france reported antibodies to mrv-3 in 9% of mouse colonies examined (zenner and regnault, 2000) . more recently, a study in north america found a low rate (0.2%) of mouse sera to be positive for antibodies against this virus (livingston and riley, 2003) . in addition, contamination of mouse origin tumours and cell lines by mrv-3 has been reported many times (national research council, 1991; nicklas et al., 1993; barthold, 1997a) . experimentally, mrv-3 infection of infant mice has been used to model human hepatobiliary disease, pancreatitis, diabetes mellitus, and lymphoma (kraft, 1982; national research council, 1991; fenner et al., 1993) . the literature on mrv-3 infections in mice is dominated by studies on experimentally infected animals. the virus can cause severe pantropic infection in infant mice (kraft, 1982; tyler and fields, 1986; barthold, 1997a) . after parenteral inoculation, virus can be recovered from the liver, brain, heart, pancreas, spleen, lymph nodes, and blood vessels. following oral inoculation, reoviruses gain entry by infecting specialized epithelial cells (m cells) that overlie peyer's patches. the virus then becomes accessible to leukocytes and spreads to other organs by way of the lymphatic system and the bloodstream. neural spread to the cns has also been well documented (morrison et al., 1991) . the mechanisms of viral pathogenesis and their interactions with the host cell are reviewed in detail by and . natural infection by mrv-3 in a mouse colony usually is subclinical although diarrhoea or steatorrhoea and oily hair effect in suckling mice may be noted (kraft, 1982; tyler and fields, 1986; national research council, 1991; barthold, 1997a; percy and barthold, 2001) . the latter term has been used to describe the matted, unkempt appearance of the hair coat that results from steatorrhoea due to pancreatitis, maldigestion, and biliary atresia. in addition, runting (attributed to immune-mediated destruction of cells in the pituitary gland that produce growth hormone), transient alopecia, jaundice (due to excessive bilirubin in the blood, which is attributed to the liver pathology, especially biliary atresia), and neurological signs such as incoordination, tremors, or paralysis may develop. when present in natural infections, clinical signs and lesions are similar to but milder than in experimental neonatal infections. early descriptions of naturally occurring disease may have been complicated by concurrent infections such as mhv or murine rotavirus a (murv-a)/epizootic diarrhoea of infant mice (edim) virus that contributed to the severity of the lesions especially in liver, pancreas, cns, and intestine. the outcome of mrv-3 infection depends on age and immunological status of mouse, dose of virus, and route of inoculation. adult immunocompetent mice typically show no clinical signs and have no discernible lesions even in experimental infections. mucosal and maternally conferred immunity are considered to be important in protection from or resolution of disease (cuff et al., 1990; barthold et al., 1993b) . experimental infection of adult prkdc scid mice is lethal (george et al., 1990) . depending on the route of inoculation, experimental infection of adult foxn1 nu mice is subclinical or results in liver disease (carthew, 1984; george et al., 1990) . histological findings reported to occur after experimental mrv-3 infection of neonatal mice include inflammation and necrosis in liver, pancreas, heart, adrenal, brain, and spinal cord; lymphoid depletion in thymus, spleen, and lymph nodes; and hepatic fibrosis with biliary atresia (papadimitriou and robertson, 1976; tyler and fields, 1986; barthold et al., 1993b; barthold, 1997a; percy and barthold, 2001) . transmission of reoviruses probably involves the aerosol as well as the faecal-oral route (national research council, 1991) . fomites may play an important role as passive vectors because reoviruses resist environmental conditions moderately well. serological screening with elisa or ifa is in widespread use for detection of antibodies to mrv-3 in diagnostic and health surveillance programmes. both elisa and ifa detect cross-reacting antibodies to heterologous mrv serotypes that can infect mice (aclad, 1991) . the hi test does not detect such cross-reacting antibodies but is prone to give false positive results due to nonspecific inhibitors of haemagglutination (kraft and meyer, 1986; van der logt, 1986; aclad, 1991) . reverse transcriptase-polymerase chain reaction methods for the detection of mrv-3 rna (steele et al., 1995) or mrv rna (leary et al., 2002) are also available. reports on contamination of mouse origin tumours and cell lines by mrv-3 and its interference with transplantable tumour studies (bennette, 1960; nelson and tarnowski, 1960) emphasize the importance of screening of biological materials to be inoculated into mice by map test or pcr. natural seroconversion to mrv-3 without clinical disease is also observed in laboratory rats, hamsters, and guinea pigs (national research council, 1991; barthold, 1997a) . caesarean derivation and barrier maintenance have proven effective in the control and prevention of mrv-3 infection (kraft, 1982; national research council, 1991) . the virus may interfere with research involving transplantable tumours and cell lines of mouse origin. it has the potential to alter intestinal studies and multiple immune response functions in mice. in enzootically infected colonies, protection of neonates by maternal antibody could complicate or prevent experimental infections with reoviruses. it could further complicate experiments that require evaluation of liver, pancreas, cns, heart, lymphoid organs, and other tissues affected by the virus. the term murine hepatitis virus (mhv; commonly referred to as 'mouse hepatitis virus') designates a large group of antigenically and genetically related, singlestranded rna viruses belonging to the family coronaviridae, genus coronavirus. they are surrounded by an envelope with a corona of surface projections (spikes). murine hepatitis virus is antigenically related to rat coronaviruses and other coronaviruses of pigs, cattle, and humans. numerous different strains or isolates of mhv have been described. they can be distinguished by neutralization tests that detect strain-specific spike (s) antigens. the best studied strains are the prototype strains mhv-1, mhv-2, mhv-3, jhm (mhv-4), a59, and s, of which mhv-3 is regarded as the most virulent. murine hepatitis virus, like other coronaviruses, mutates rapidly, and strains readily form recombinants, so that new (sub)strains are constantly evolving. strains vary in their virulence, organotropism, and cell tropism (homberger, 1997) . based on their primary organotropism, mhv strains can be grouped into two biotypes: respiratory (or polytropic) and enterotropic. however, intermediate forms (enterotropic strains with tropism to other organs) exist. murine hepatitis virus is relatively resistant to repeated freezing and thawing, heating (56њc for 30 min), and acid ph but is sensitive to drying and disinfectants, especially those with detergent activity (national research council, 1991) . mus musculus is the natural host of mhv. it can be found in wild and laboratory mice throughout the world and is one of the most common viral pathogens in contemporary mouse colonies. while polytropic strains have historically been considered more common, this situation is thought to have reversed. a survey conducted in the usa in 1996 reported antibodies to mhv in more than 10% of spf mouse colonies and more than 70% of non-spf colonies (jacoby and lindsey, 1998) , though very recent monitoring results for research institutions across north america indicate that the prevalence of mhv has decreased during the past few years (livingston and riley, 2003) . a retrospective study in france covering the period from 1988 to 1997 reported antibodies to mhv in 67% of mouse colonies examined (zenner and regnault, 2000) . suckling rats inoculated experimentally with mhv had transient virus replication in the nasal mucosa and seroconversion but no clinical disease . similarly, deer mice seroconverted but showed no clinical disease after experimental infection (silverman et al., 1982) . murine hepatitis virus is also a common contaminant of transplantable tumours (collins and parker, 1972; nicklas et al., 1993) and cell lines (sabesin, 1972; yoshikura and taguchi, 1979) . the pathogenesis and outcome of mhv infections depend on interactions among numerous factors related to the virus (e.g. virulence and organotropism) and the host (e.g. age, genotype, immune status, and microbiological status; kraft, 1982; barthold, 1986; national research council, 1991; compton et al., 1993; homberger, 1997; percy and barthold, 2001) . murine hepatitis virus strains appear to possess a primary tropism for the upper respiratory or enteric mucosa. those strains with respiratory tropism initiate infection in the nasal mucosa and then may disseminate via blood and lymphatics to a variety of other organs because of their polytropic nature. respiratory (polytropic) strains include mhv-1, mhv-2, mhv-3, a59, s, and jhm. infection of mice with virulent polytropic mhv strains, infection of mice less than 2 weeks of age, infection of genetically susceptible strains of mice, or infection of immunocompromised mice favour virus dissemination. virus then secondarily replicates in vascular endothelium and parenchymal tissues, causing disease of brain, liver, lymphoid organs, bone marrow, and other sites. infection of the brain by viraemic dissemination occurs primarily in immunocompromised or neonatal mice. additionally, infection of adult mouse brain can occur by extension of virus along olfactory neural pathways, even in the absence of dissemination to other organs. in contrast, enterotropic mhv strains (e.g. livim, mhv-d, and mhv-y) tend to selectively infect intestinal mucosal epithelium, with no or minimal dissemination to other organs such as mesenteric lymph nodes or liver. all ages and strains are susceptible to active infection, but disease is largely age-related. infection of neonatal mice results in severe necrotizing enterocolitis with high mortality within 48 h. mortality and lesion severity diminish rapidly with advancing age at infection. adult mice develop minimal lesions although replication of equal or higher titres of virus occurs compared with neonates. the age-dependent decrease in severity of enterotropic mhv disease is probably related to the higher mucosal epithelium turnover in older mice, allowing more rapid replacement of damaged mucosa. another factor that is of considerable importance to the outcome of mhv infections is host genotype. for example, balb/c mice are highly susceptible to enterotropic mhv disease while sjl mice, at the other end of the spectrum, are highly resistant (barthold et al., 1993a) . unlike in polytropic mhv infection where resistance is correlated with reduced virus replication in target cells (barthold and smith, 1987) , enterotropic mhv grows to comparable titres in sjl and balb/c mice at all ages (barthold et al., 1993a) . therefore, the resistance of the sjl mouse to disease caused by enterotropic mhv seems to be mediated through an entirely different mechanism than resistance to polytropic mhv. furthermore, mouse genotypes that are susceptible to disease caused by one mhv strain may be resistant to disease caused by another strain (barthold, 1986) . it is therefore not possible to strictly categorize mouse strains as susceptible or resistant. the genetic factors determining susceptibility versus resistance in mhv infections are as yet poorly understood. both polytropic and enterotropic mhv infections are selflimiting in immunocompetent mice. immune-mediated clearance of virus usually begins about a week after infection, and most mice eliminate the virus within 3-4 weeks (barthold, 1986; barthold and smith, 1990; barthold et al., 1993a) . humoral and cellular immunity appear to participate in host defences to infection, and functional t cells are an absolute requirement (williamson and stohlman, 1990; kyuwa et al., 1996; lin et al., 1999; haring and perlman, 2001) . therefore, immunodeficient mice such as foxn1 nu and prkdc scid mice cannot clear the virus (barthold et al., 1985; compton et al., 1993) . similarly, some genetically modified strains of mice may have deficits in antiviral responses or other alterations that allow the development of persistent mhv infection (rehg et al., 2001) . recovered immune mice are resistant to reinfection with the same mhv strain but remain susceptible to repeated infections with different strains of mhv (barthold and smith, 1989a,b; . similarly, maternal immunity protects suckling mice against homologous mhv strains but not necessarily against other strains . however, maternal immunity, even to homologous strains, depends on the presence of maternally acquired antibody in the lumen of the intestine . therefore, the susceptibility of young mice to infection significantly increases at weaning. most mhv infections are subclinical and follow one of two epidemiological patterns in immunocompetent mice (national research council, 1991; homberger, 1997) . enzootic (subclinical) infection, commonly seen in breeding colonies, occurs when a population has been in contact with the virus for a longer period (e.g. several weeks). adults are immune (due to prior infection), sucklings are passively protected, and infection is perpetuated in weanlings. epizootic (clinical) infection occurs when the virus is introduced into a naive population (housed in open cages). the infection rapidly spreads through the entire colony. clinical signs depend upon the virus and mouse strains and are most evident in infant mice. typically, they include diarrhoea, poor growth, lassitude, and death. in infections due to virulent enterotropic strains, mortality can reach 100% in infant mice. some strains may also cause neurological signs such as flaccid paralysis of hind limbs, convulsions, and circling. adult infections are again usually asymptomatic. as the infection becomes established in the colony, the epizootic pattern is replaced by the enzootic pattern. in immunodeficient (e.g. foxn1 nu and prkdc scid ) mice, infection with virulent polytropic mhv strains often is rapidly fatal while less virulent strains cause chronic wasting disease (compton et al., 1993) . in contrast, adult immunodeficient mice can tolerate chronic infection by enterotropic mhv, with slow emaciation and diarrhoea, or minimal clinical disease (barthold et al., 1985; barthold, 1986) . subclinical mhv infections can be activated by a variety of experimental procedures (e.g. thymectomy, whole body irradiation, treatment with chemotherapeutic agents, halothane anaesthesia) or by co-infections with other pathogens (e.g. eperythrozoon coccoides, k virus; reviewed by kraft, 1982; national research council, 1991) . in most natural infections, gross lesions are not present or are transient and not observed. gross findings in neonates with clinical signs include dehydration, emaciation, and in contrast to edim, an empty stomach (ishida et al., 1978; barthold et al., 1982; kraft, 1982) . the intestine is distended and filled with watery to mucoid yellowish, sometimes gaseous contents. haemorrhage or rupture of the intestine can occur. depending on the virus strain, necrotic foci on the liver (ishida et al., 1978; kraft, 1982; percy and barthold, 2001) and thymus involution godfraind et al., 1995) may also be seen in susceptible mice. liver involvement may be accompanied by jaundice and haemorrhagic peritoneal exudate. splenomegaly may occur as a result of compensatory haematopoiesis (fox et al., 1977) . histopathological changes in susceptible mice infected with polytropic mhv strains include acute necrosis with syncytia in liver, spleen, lymph nodes, gut-associated lymphoid tissue, and bone marrow (kraft, 1982; barthold, 1986; national research council, 1991; percy and barthold, 2001) . neonatally infected mice can have vascular-oriented necrotizing (meningo)encephalitis with demyelination in the brain stem and peri-ependymal areas. lesions in peritoneum, bone marrow, thymus, and other tissues can be variably present. mice can develop nasoencephalitis due to extension of infection from the nasal mucosa along olfactory pathways to the brain, with meningoencephalitis and demyelination, the latter of which is thought to be largely t cell-mediated (haring and perlman, 2001) . this pattern of infection regularly occurs after intranasal inoculation of many mhv strains but is a relatively rare event after natural exposure. syncytia arising from endothelium, parenchyma, or leukocytes is a hallmark of infection in many tissues including intestine, lung, liver, lymph nodes, spleen, thymus, brain, and bone marrow. lesions are transient and seldom fully developed in adult immunocompetent mice, but they are manifest in immunocompromised mice. highly unusual presentations can occur in mice with specific gene defects. for example, granulomatous peritonitis and pleuritis were found in interferon-␥-deficient mice infected with mhv (france et al., 1999) . histopathological changes caused by enterotropic strains of mhv are mainly confined to the intestinal tract and associated lymphoid tissues (kraft, 1982; barthold, 1986; national research council, 1991; percy and barthold, 2001) . the most common sites are terminal ileum, caecum, and proximal colon. the severity of disease is primarily age-dependent, with neonatal mice being most severely affected. these mice show segmentally distributed areas of villus attenuation, enterocytic syncytia (balloon cells), and mucosal necrosis accompanied by leukocytic infiltration. intracytoplasmic inclusions are present in enterocytes. erosions, ulceration, and haemorrhage may be seen in more severe cases. lesions can be fully developed within 24-48 h, but are usually more severe at 3-5 days after infection. surviving mice may develop compensatory mucosal hyperplasia. mesenteric lymph nodes usually contain lymphocytic syncytia, and mesenteric vessels may contain endothelial syncytia. pathological changes in older mice are generally much more subtle and may only consist of transient syncytia. an occasional exception seems to occur in immunodeficient animals such as foxn1 nu mice, which can develop chronic hyperplastic typhlocolitis of varying severity (barthold et al., 1985) , but other agents such as helicobacter species may have been involved. in general, enterotropic mhv strains do not disseminate, but hepatitis and encephalitis can occur with some virus strains in certain mouse genotypes. murine hepatitis virus is highly contagious. it is shed in faeces and nasopharyngeal secretions and appears to be transmitted via direct contact, aerosol, and fomites (kraft, 1982; national research council, 1991) . vertical (in utero) transmission has been demonstrated in experimental infections (katami et al., 1978) but does not seem to be of practical importance under natural conditions. diagnosis during the acute stage of infection can be made by histological demonstration of characteristic lesions with syncytia in target tissues, but clinical signs and lesions can be highly variable and may not be prominent. suckling, genetically susceptible or immunocompromised mice are the best candidates for evaluation. active infection can be confirmed by immunohistochemistry (brownstein and barthold, 1982) or by virus isolation. virus recovery from infected tissues is difficult but can be accomplished using primary macrophage cultures or a number of established cell lines such as nctc 1469 or dbt (aclad, 1991) . these cells, however, may not be successful substrates for some enterotropic mhv strains. virus in suspect tissue can also be confirmed by bioassays such as map testing or infant or foxn1 nu mouse inoculation (de souza and smith, 1989; aclad, 1991) . amplification by passage in these mice increases the likelihood of detection of lesions and antigen, or virus recovery. other direct diagnostic methods that have been successfully utilized to detect mhv in faeces or tissue of infected mice include monoclonal antibody solution hybridization assay (casebolt and stephensen, 1992 ) and a number of rt-pcr assays (homberger et al., 1991; kunita et al., 1992; yamada et al., 1993; besselsen et al., 2002) . because of the transient nature of mhv infection in immunocompetent mice, serology is the most appropriate diagnostic tool for routine monitoring. enzyme-linked immunosorbent assay and ifa are well established and sensitive, and all known mhv strains cross-react in both tests (smith, 1983; aclad, 1991) . the magnitude of antibody response depends on mhv strain and mouse genotype (nakanaga et al., 1983; barthold and smith, 1987) . dba/2 mice are poor antibody responders whereas c57bl/6 mice produce a high antibody titre and are therefore good sentinels. antibody titres remain high over a period of at least 6 months (barthold and smith, 1989b; . infected mice may not develop detectable antibodies for up to 14 days after initial exposure (smith, 1983 ). in such cases, a direct diagnostic method as discussed above may be useful. another drawback of serology is that mice weaned from immune dams can have maternal antibodies until they are 10 weeks of age (homberger, 1992) . this may impact serological monitoring because the possibility must be considered that low positive results are due to maternally-derived passive immunity. because the virus can be transmitted by transplantable tumours and other biological materials from mice, including hybridomas (holmes et al., 1986) and embryonic stem cells (okumura et al., 1996; kyuwa, 1997) , these materials should also be routinely screened for mhv contamination. mouse inoculation bioassay, map test, and rt-pcr can be used for this purpose. the best means of mhv control is to prevent its entry into a facility. this can be accomplished by purchase of mice from virus-free sources and maintenance under effective barrier conditions monitored by a welldesigned quality assurance programme. control of wild mouse populations, proper husbandry and sanitation, and strict monitoring of biological materials that may harbour virus are also important measures to prevent infection. if infection occurs, the most effective elimination strategy is to cull the affected colony and obtain clean replacement stock. however, this is not always a feasible option when working with valuable mice (e.g. genetically modified lines, breeding stocks). caesarean derivation or embryo transfer can be used to produce virus-free offspring, and foster-nursing also has been reported to be effective (lipman et al., 1987) . quarantine of an affected colony with no breeding and no introduction of new animals for approximately 2 months has been effective in immunocompetent mice (weir et al., 1987) . the infection is likely to be terminated because mhv requires a constant supply of susceptible animals. this method works best when working with small numbers of mice. large populations favour the development of new mhv strains that may result in repeated infections with slightly different strains (adami et al., 1995) . it may be practical to select a few future breeders from the infected population and quarantine them for approximately 3 weeks (compton et al., 1993) . this can be achieved in isolators, or in individually ventilated cages if proper handling is guaranteed. after this interval, breeding can resume. the 3-week interval should permit recovery from active infection, and the additional 3-week gestation period effectively extends the total quarantine to 6 weeks. it is advisible to select seropositive breeders because the possibility of active infection is lower in such animals. the breeding cessation strategy may not be successful if immunodeficient mice are used because they are susceptible to chronic infection and viral excretion (barthold et al., 1985) . genetically engineered mice of unclear, unknown or deficient immune status pose a special challenge because they may develop unusual manifestations of infection or may be unable to clear virus. rederivation likely is the most cost effective strategy in such situations. along with the measures described, proper sanitation and disinfection of caging and animal quarters as well as stringent personal sanitation are essential to eliminate infection. careful testing with sentinel mice should be applied to evaluate the effectiveness of rederivation. if transplantable tumours are contaminated with mhv, virus elimination can be achieved by passage of tumours in athymic whn rnu rats (rülicke et al., 1991) . murine hepatitis virus is one of the most important viral pathogens of laboratory mice and has been intensively studied from a number of research perspectives (e.g. as a model organism for studying coronavirus molecular biology or the pathogenesis of viral-induced demyelinating disease). numerous reports document the effects of natural and experimental infections with mhv on host physiology and research, especially in the fields of immunology and tumour biology (reviewed by barthold, 1986; national research council, 1991; compton et al., 1993; homberger, 1997; baker, 1998; nicklas et al., 1999) . murine pneumonia virus, commonly referred to as 'pneumonia virus of mice' (pvm), is an enveloped, singlestranded rna virus of the family paramyxoviridae, genus pneumovirus. it is closely related to human respiratory syncytial virus (hrsv). the virus name is officially abbreviated as 'mpv' according to the international union of microbiological societies (2000); however, the former designation 'pvm' will be used in this chapter to avoid confusion with the official abbreviation of mouse parvovirus 1 (mpv). 'pneumonia virus of mice' infection is relatively common in colonies of mice and rats throughout the world. seropositivity to pvm was reported in less than 5% of spf mouse colonies and in approximately 20% of non-spf mouse colonies in the usa (jacoby and lindsey, 1998) . a serological survey in france demonstrated antibodies to pvm in 16% of mouse colonies examined (zenner and regnault, 2000) . in a more recent study in north america, such antibodies were found in only 0.1% of mice monitored (livingston and riley, 2003) . antibodies to pvm have also been detected in hamsters, gerbils, cotton rats, guinea pigs, and rabbits (parker and richter, 1982; richter, 1986; national research council, 1991) . experimentally, pvm infection of mice is used as a model for hrsv infection (domachowske et al., 2000) . in immunocompetent mice, natural infection with pvm is transient and usually not associated with clinical disease or pathological findings (parker and richter, 1982; national research council, 1991; brownstein, 1996b) . however, natural disease and persistent infection may occur in immunodeficient mice (carthew and sparrow, 1980; richter et al., 1988; weir et al., 1988) . in particular, athymic foxn1 nu mice seem to be susceptible to pvm infection, which can result in dyspnoea, cyanosis, emaciation, and death due to pneumonia (richter et al., 1988; weir et al., 1988) . similar clinical signs have been reported for experimentally infected, immunocompetent mice (cook et al., 1998) . necropsy findings in naturally infected foxn1 nu mice include cachexia and diffuse pulmonary oedema or lobar consolidation (weir et al., 1988) . pulmonary consolidation (dark red or grey in colour) also has been found after experimental infection of immunocompetent mice (brownstein, 1996b) . histologically, natural infection of foxn1 nu mice with pvm presents as interstitial pneumonia (richter et al., 1988; weir et al., 1988) . experimental intranasal inoculation of immunocompetent mice can result in rhinitis, erosive bronchiolitis, and interstitial pneumonia with prominent early pulmonary eosinophilia and neutrophilia (brownstein, 1996b; domachowske et al., 2000) . hydrocephalus may result from intracerebral inoculation of neonatal mice (lagace-simard et al., 1980) . susceptibility to infection is influenced by age of mouse, dose of virus, and a variety of local and systemic stressors (parker and richter, 1982; national research council, 1991) . pneumonia virus of mice is labile in the environment and rapidly inactivated at room temperature (parker and richter, 1982; national research council, 1991) . the virus is tropic for the respiratory epithelium (carthew and sparrow, 1980; cook et al., 1998) , and transmission is exclusively horizontal via the respiratory tract, mainly by direct contact and aerosol (parker and richter, 1982; national research council, 1991) . therefore, transmissibility in mouse colonies is low, and infections tend to be focal enzootics. serology (elisa, ifa, or hi) is the primary means of testing mouse colonies for exposure to pvm. immunohistochemistry has been applied to detect viral antigen in lung sections (carthew and sparrow, 1980; weir et al., 1988) , however, proper sampling (see chapter on health monitoring) is critical for establishing the diagnosis due to the focal nature of the infection. an rt-pcr assay to detect viral rna in respiratory tract tissues has also been reported . however, the use of direct methods requires good timing because the virus is present for only up to about 10 days in immunocompetent mice (brownstein, 1996b) . embryo transfer or caesarean derivation followed by barrier maintenance can be used to rear mice that are free of pvm. because active infection is present in the individual immunocompetent mouse for only a short period, strict isolation of a few (preferably seropositive) mice with the temporary cessation of breeding might also be successful in eliminating the virus (richter, 1986; national research council, 1991) . pneumonia virus of mice could interfere with studies involving the respiratory tract or immunological measurements in mice. in addition, pvm can have devastating effects on research using immunodeficient mice because they are particularly prone to develop fatal disease (richter et al., 1988; weir et al., 1988) or become more susceptible to the deleterious effects of other agents such as pneumocystis carinii (roths et al., 1993) . murine rotavirus-a/edim (commonly referred to as 'mouse rotavirus' or 'epizootic diarrhoea of infant mice virus') is a nonenveloped, segmented double-stranded rna virus of the family reoviridae, genus rotavirus. it is antigenically classified as a group a rotavirus, similar to rotaviruses of many other species that cause neonatal and infantile gastroenteritis (fenner et al., 1993) . murine rotavirus-a/edim infection remains prevalent in contemporary mouse colonies and appears to occur worldwide. seropositivity to murv-a/edim was reported in approximately 5% of spf colonies and in almost 30% of non-spf mouse colonies in the usa in 1996 (jacoby and lindsey, 1998) . more recently, livingston and riley (2003) found a low rate (1%) of mouse sera to be positive for antibodies against murv-a/edim. experimentally, murv-a/edim infection in mice is used as a model for human rotavirus infection, especially in investigations on the mechanisms of rotavirus immunity and in the development of vaccination strategies (ward and mcneal, 1999) . clinical symptoms following murv-a/edim infection range from inapparent or mild to severe, sometimes fatal, diarrhoea. 'epizootic diarrhoea of infant mice' describes the clinical syndrome associated with natural or experimental infection by murv-a/edim during the first 2 weeks of life (kraft, 1982; sheridan and vonderfecht, 1986; national research council, 1991; barthold, 1997b; percy and barthold, 2001) . diarrhoea usually begins around 48 h after infection and persists for about 1 week. affected suckling mice have soft, yellow faeces that wet and stain the perianal region. in severe instances, the mice may be stunted, have dry scaly skin, or are virtually covered with faecal material. morbidity is very high but mortality is usually low. gross lesions in affected mice are confined to the intestinal tract. the caecum and colon may be distended with gas and watery to paste-like contents that are frequently bright yellow. the stomach of diarrheic mice is almost always filled with milk, and this feature has been reported to be a reliable means to differentiate diarrhoea caused by rotavirus from the diarrhoea caused by mhv infection. histopathological changes may be subtle even in animals with significant diarrhoea. they are confined to the small intestine and are most prominent at the apices of villi, where rotaviruses infect and replicate within epithelial cells. hydropic change of villous epithelial cells is the hallmark finding of acute disease. the villi become shortened, and the cells that initially replace the damaged cells are less differentiated, typically cuboidal instead of columnar, and lack a full complement of enzymes for digestion and absorption, resulting in diarrhoea due to maldigestion and malabsorption. undigested milk in the small intestine promotes bacterial growth and exerts an osmotic effect, exacerbating damage to the villi. intestinal fluid and electrolyte secretion is further enhanced by activation of the enteric nervous system (lundgren et al., 2000) and through the effects of a viral enterotoxin called nsp4 (for nonstructural protein 4; ball et al., 1996) . it is hypothesized that nsp4 is released from virusinfected cells and then triggers a signal transduction pathway that alters epithelial cell permeability and chloride secretion. susceptibility to edim depends on the age of the host and peaks between 4 and 14 days of age (kraft, 1982; sheridan and vonderfecht, 1986; national research council, 1991; barthold, 1997b; percy and barthold, 2001) . mice older than about 2 weeks can still be infected with murv-a/edim, but small numbers of enterocytes become infected, there is little replication of virus, and diarrhoea does not occur. the exact reason for this age-related resistance to disease is unknown. pups suckling immune dams are protected against edim during their period of disease susceptibility (rosé et al., 1998) . in general, the infection is selflimiting and resolves within days. successful viral clearance is promoted by an intact immune response (feng et al., 1997; mcneal et al., 1997; rosé et al., 1998) , and some immunodeficient mice (e.g. prkdc scid and rag2 tm1fwa mice) may shed virus for extended periods or become persistently infected (riepenhoff-talty et al., 1987; franco and greenberg, 1995) . protection against murv-a/edim reinfection is primarily mediated by antibodies (feng et al., 1997; rosé et al., 1998) . murine rotavirus-a/edim is highly contagious and transmitted by the faecal-oral route (kraft, 1982; sheridan and vonderfecht, 1986; national research council, 1991) . dissemination of the virus occurs through direct contact or contaminated fomites and aerosols. murv-a/edim is stable at ϫ70њc but otherwise tends to be susceptible to extreme environmental conditions, detergents, and disinfectants. enzyme-linked immunosorbent assay and ifa are in widespread use for detection of serum antibodies to murv-a/edim in diagnostic and health surveillance programmes; other assay systems such as those using latex agglutination are also utilized (ferner et al., 1987) . rotazyme ii is a commercially available elisa for detection of rotavirus antigen in faeces; however, great care must be used in interpreting the results because some feeds have been reported to cause false positive reactions (jure et al., 1988) . electron microscopy of faeces of diarrheic pups should reveal typical wheelshaped rotavirus particles, 60-80 nm in diameter. reverse transcriptase-polymerase chain reaction also can be used to detect rotavirus rna in faecal samples (wilde et al., 1990) . good timing is critical for establishing the diagnosis from faeces because virus is shed for only a few days in immunocompetent mice. embryo transfer or caesarean derivation followed by barrier maintenance is recommended for rederivation of breeding stocks (kraft, 1982; national research council, 1991) . in immunocompetent mice in which infection is effectively cleared, a breeding suspension strategy combined with excellent sanitation, filter tops, and conscientious serological testing of offspring may also be effective. murine rotavirus-a/edim has the potential to interfere with any research utilizing suckling mice. it may have a significant impact on studies where the intestinal tract of neonatal or infant mice is the target organ. the infection also poses a problem for infectious disease and immune response studies, particularly those involving enteropathogens in infant mice (newsome and coney, 1985) . in addition, runting could be interpreted erroneously as the effect of genetic manipulation or other experimental manipulation. sendai virus (sev) is an enveloped, single-stranded rna virus of the family paramyxoviridae, genus respirovirus. it is antigenically related to human parainfluenza virus 1. the virus was named for sendai, japan, where it was first isolated from mice. infections of mice and rats are relatively common and occur worldwide. in addition, there is evidence that hamsters, guinea pigs, and rabbits are susceptible to infection with sev (machii et al., 1989; aclad, 1991; national research council, 1991; percy and palmer, 1997) ; however, some apparently seropositive guinea pigs may in fact be seropositive to other parainfluenza viruses instead of sev. seropositivity to sev was reported to be absent from spf mouse colonies and to be approximately 20% in non-spf mouse colonies in the usa (jacoby and lindsey, 1998) . a study in france reported antibodies to sev in 17% of mouse colonies examined (zenner and regnault, 2000) . a low rate of seropositive mice (0.2%) was found in a recent survey in north america (livingston and riley, 2003) . furthermore, sev can contaminate biological materials (collins and parker, 1972) . sendai virus is pneumotropic and the leading cause of viral respiratory disease in mice. the pneumotropism is partially a consequence of the action of respiratory serine proteases such as tryptase clara, which activate viral infectivity by specific cleavage of the viral fusion glycoprotein (tashiro et al., 1999) . in addition, the apical budding behaviour of sev may hinder the spread of virus into subepithelial tissues and subsequently to distant organs via the blood. two epidemiologic patterns of sev infection have been recognized, an enzootic (subclinical) and epizootic (clinically apparent) type (parker and richter, 1982; national research council, 1991; brownstein, 1996a) . enzootic infections commonly occur in breeding or open colonies, where the constant supply of susceptible animals perpetuates the infection. in breeding colonies, mice are infected shortly after weaning as maternal antibody levels wane. normally, the infection is subclinical, with virus persisting for approximately 2 weeks, accompanied by seroconversion that persists for a year or longer. epizootic infections occur upon first introduction of the virus to a colony and either die out (self-cure) after 2-7 months or become enzootic depending on colony conditions. the epizootic form is generally acute, and morbidity is very high resulting in nearly all susceptible animals becoming infected within a short time. clinical signs vary and include rough hair coat, hunched posture, chattering, respiratory distress, prolonged gestation, death of neonates and sucklings, and runting in young mice. breeding colonies may return to normal productivity in 2 months and thereafter maintain the enzootic pattern of infection. factors such as strain susceptibility, age, husbandry, transport, and copathogens are important in precipitating overt disease. dba and 129/j strains of mice are very susceptible to sev pneumonia whereas sjl/j and c57bl/6/j strains and several outbred stocks are relatively resistant. a/j, balb/c, and swr/j are among the strains that show intermediate susceptibility. there is no evidence for persistent infection in immunocompetent mice, but persistent or prolonged infection may occur in immunodeficient mice and can result in wasting and death due to progressive pneumonia (ward et al., 1976; iwai et al., 1979; percy et al., 1994) . clearance of a primary sev infection is mediated by cd8 ϩ and cd4 ϩ t cell mechanisms (kast et al., 1986; hou et al., 1992) . heavier than normal, consolidated, plum-coloured or grey lungs are a characteristic gross finding in severe sev pneumonia (parker and richter, 1982; national research council, 1991; brownstein, 1996a; percy and barthold, 2001) . lymphadenopathy and splenomegaly reflect the vigorous immune response to infection. histologically, three phases of disease can be recognized in susceptible immunocompetent mice: acute, reparative, and resolution phases (brownstein, 1996a; percy and barthold, 2001) . lesions of the acute phase, which lasts 8-12 days, are primarily attributed to the cell-mediated immune response that destroys infected respiratory epithelial cells and include necrotizing rhinitis, tracheitis, bronch(iol)itis, and alveolitis. epithelial syncytiae and cytoplasmic inclusion bodies in infected cells may be seen early in this phase. alveoli contain sloughed necrotic epithelium, fibrin, neutrophils, and mononuclear cells. atelectasis, bronchiectasis, and emphysema may occur as a result of damage and obstruction of airways. the reparative phase, which may overlap the acute phase but continues through about the third week post infection, is indicated by regeneration of airway lining epithelium. adenomatous hyperplasia and squamous metaplasia (with multilayered flat epithelial cells instead of normal columnar cells) in the terminal bronchioles and alveoli are considered to be a hallmark of sev pneumonia. mixed inflammatory cell infiltrates in this phase tend to be primarily interstitial rather than alveolar as they are in the acute phase. the resolution phase may be complete by the fourth week post infection and lesions may be difficult to identify subsequently. residual, persistent lesions that may occur include organizing alveolitis and bronchiolitis fibrosa obliterans. alveoli and bronchioles are replaced by collagen and fibroblasts, foamy macrophages, and lymphoid infiltrates, often with foci of emphysema, cholesterol crystals, and other debris, which represent attempts to organize and wall off residual necrotic debris and fibrin. lesions are more severe and variable when additional pathogens such as mycoplasma pulmonis are present (national research council, 1991) . otitis media has also been reported in natural infections with sev although some of these studies have been complicated by the presence of other pathogens (ward, 1974) . sendai virus has been detected in the inner ear after experimental intracerebral inoculation of neonatal mice (shimokata et al., 1977) . sendai virus is extremely contagious. infectious virus is shed during the first 2 weeks of infection and appears to be transmitted by direct contact, contaminated fomites, and respiratory aerosol (parker and reynolds, 1968; parker and richter, 1982; national research council, 1991) . serology (elisa, ifa, or hi) is the approach of choice for routine monitoring because serum antibodies to sev are detectable soon after infection and persist at high levels for many months, although active infection lasts only 1-2 weeks in immunocompetent mice. the short period of active infection limits the utility of direct methods such as immunohistochemistry (carthew and sparrow, 1980) and rt-pcr (hayase et al., 1997; wagner et al., 2003) . although sev is considered to be highly contagious, studies have shown that dirty bedding sentinel systems do not reliably detect the infection and that outbred stocks may not seroconvert consistently (dillehay et al., 1990; artwohl et al., 1994) . mouse antibody production test and rt-pcr can be used to detect sev in contaminated biological materials. sendai virus infection in mouse colonies has proven to be one of the most difficult virus infections to control because the virus is highly infectious and easily disseminated. depopulation of infected colonies probably is the most appropriate means to eliminate the virus in most situations. embryo transfer followed by barrier maintenance has also been used successfully in eliminating the virus (national research council, 1991) . a less effective alternative is to place the infected animals under strict quarantine, remove all young and pregnant mice, suspend all breeding, and prevent addition of other susceptible animals for approximately 2 months until the infection is extinguished and then breeding and other normal acitivities are resumed (parker and richter, 1982; national research council, 1991) . vaccines against the virus have been developed (brownstein, 1986; national research council, 1991) , but these probably do not represent a practical means to achieve or maintain the seronegative status of colonies that is in demand today. sendai virus has the potential to interfere with a wide variety of research involving mice. reported effects include interference with early embryonic development and foetal growth; alterations of macrophage, nk cell, and t and b cell function; altered responses to transplantable tumours and respiratory carcinogens; altered isograft rejection; and delayed wound healing (reviewed by national research council, 1991; baker, 1998; nicklas et al., 1999) . pulmonary changes during sev infection can compromise interpretation of experimentally induced lesions and may lead to opportunistic infections by other agents. they could also affect the response to anaesthetics. in addition, natural sev infection would interfere with studies using sev as a gene vector. theiler's murine encephalomyelitis virus (tmev) or murine poliovirus is a member of the genus cardiovirus in the family picornaviridae. members of this genus are nonenveloped viruses with singlestranded rna. the virus is rapidly destroyed at temperatures above 50њc. it is considered to be a primary pathogen of the cns of mice and can cause clinical disease resembling that due to poliomyelitis virus infections in humans. antibodies to tmev have been identified in mouse colonies and feral populations worldwide, and mus musculus is considered to be the natural host of tmev (lipton et al., 2001) . the most well-known and most frequently mentioned tmev strain is gdvii, which is virulent for mice. infant or young hamsters and laboratory rats are also susceptible to intracerebral infection. the original isolate is designated to (theiler's original) and represents a group of tmev strains with low virulence for mice. many additional virus strains have been isolated and studied, and they all fall in the broad grouping of to and gdvii. a similar virus strain has also been isolated from rats, but in contrast to mouse isolates this virus is not pathogenic for rats and mice after intracerebral inoculation (hemelt et al., 1974) . recently, another rat isolate has been characterized and shown to be most closely related to but quite distinct from other tmev viruses (ohsawa et al., 2003) . antibodies to tmev (strain gdvii) have been detected in guinea pigs and are considered to indicate infection with another closely related cardiovirus (hansen et al., 1997) . seropositivity to tmev was reported in approximately 5% of spf mouse colonies and approximately 35% of non-spf mouse colonies in the usa (jacoby and lindsey, 1998) . zenner and regnault (2000) reported a prevalence rate of 9% in french mouse colonies in a retrospective study, and it has been one of the most common virus infections in rodent colonies. in a recent study, antibodies were found in 0.2% of mice monitored (livingston and riley, 2003) indicating that tmev, like most viruses, has meanwhile been eliminated from the majority of mouse colonies. theiler's murine encephalomyelitis virus is primarily an enteric pathogen, and virus strains are enterotropic. in natural infections, virus can be detected in intestinal mucosa and faecal matter, and in some cases it is also found in the mesenteric lymph nodes. however, histological lesions in the intestine are not discerned. virus may be shed via intestinal contents for up to 22 weeks, sometimes intermittently (brownstein et al., 1989a) , and transmission under natural conditions is via the faecal-oral route by direct contact between mice as well as by indirect contact (e.g. dirty bedding). the host immune response limits virus spread, but it does not immediately terminate virus replication in the intestines. virus is cleared from extraneural tissues, but it persists in the cns for at least a year. clinical disease due to natural tmev infection is rare, with a rate of only 1 in 1000-10,000 infected immunocompetent animals (percy and barthold, 2001) . in immunodeficient mice, especially in weanlings, clinical signs may be more common and mortality may be higher (rozengurt and sanchez, 1993) . this group of viruses usually causes asymptomatic infections of the intestinal tract. they may spread to the cns as a rare event where they cause different neurological disease manifestations. the most typical clinical sign of tmev infection is flaccid paralysis of hind legs. the animals appear otherwise healthy, and there is no mortality. experimental infection in mice provides models of poliomyelitis-like infection and virus-induced demyelinating disease including multiple sclerosis (mcgavern et al., 2000) . after experimental infection, tmev causes a biphasic disease in susceptible strains of mice. the acute phase is characterized by early infection of neurons in the grey matter. encephalomyelitis may develop during this phase and may be fatal, but most animals survive and enter the second phase of the disease at 1-3 months after the acute phase. this phase is characterized by viral persistence in the spinal cord white matter, mainly in macrophages, and leads to white matter demyelination. persistence and demyelination occur only in genetically susceptible mouse strains while resistant strains clear the infection after early grey matter encephalomyelitis through a cytotoxic t lymphocyte response. for this reason, the nude mutation (foxn1 nu ) confers susceptibility on mice with an otherwise resistant background. the severity and nature of disease depend on virus strain, route of inoculation, host genotype and age (downs, 1982; lipton and rozhon, 1986; national research council, 1991; percy and barthold, 2001) . in general, virus isolates with low virulence produce persistent cns infection in mice whereas virulent strains are unable to cause persistent infection. intracerebral inoculation results in the most severe infections, but the intranasal route is effective also. experimental intracerebral infections with virulent fa and gdvii strains of tmev are more likely to cause acute encephalomyelitis and death in weanling mice 4-5 days after inoculation ('early disease'). death may be preceded by neurological manifestations of encephalitis such as hyperexcitability, convulsions, tremors, circling and rolling, and weakness. animals may develop typical flaccid paralysis of hind limbs, and locomotion is possible only by use of the forelimbs. interestingly, the tail is not paralysed. experimental infections with low virulence virus strains (e.g. to, da, ww) are more likely to cause persistent infection with development of mild encephalomyelitis followed by a chronic demyelinating disease after a few months ('late disease'). these virus strains infect neurons in the grey matter of the brain and spinal cord during the acute phase of viral growth, followed by virus persistence in macrophages and glial cells in the spinal cord white matter. sjl, swr, and dba/2 strains are most susceptible to this chronic demyelinating disease. cba and c3h/he are less susceptible strains, and strains a, c57bl/6, c57bl/10, and dba/1 are relatively resistant (lipton and dal canto, 1979) . differences in humoral immune responses play a role in resistance to tmev infection (pena rossi et al., 1991a) , but genetic factors are also important. several genetic loci implicated in susceptibility to virus persistence, demyelination, or clinical disease have been identified, including the h-2d region of the major histocompatibility complex (brahic and bureau, 1998) . furthermore, the age at infection influences the severity of clinical disease. in infant mice, intracerebral infection with low virulence virus strains (e.g. to) is often lethal. young mice develop paralysis after an incubation period of 1-4 weeks while adult mice often show no clinical signs of infection (downs, 1982) . the only gross lesions are secondary to the posterior paralysis and may include urine scald or dermatitis due to incontinence of urine and trauma to paralysed limbs, or wasting or atrophy of the hind limbs in long term survivors. theiler's murine encephalomyelitis virus infects neurons and glial cells, and histological changes in the cns include nonsuppurative meningitis, perivasculitis, and poliomyelitis with neuronolysis, neuronophagia, and microgliosis in the brainstem and ventral horns of the spinal cord (percy and barthold, 2001) . demyelination in immunocompetent mice is considered to be immune-mediated. susceptible strains develop a specific delayed-type hypersensitivity response which is the basis for inflammation and demyelination. this reaction is mediated by cytotoxic t lymphocytes (lindsley et al., 1991; pena rossi et al., 1991b) and by the activation of cytokines as a consequence of infection of macrophages and other cells of the cns (rubio and capa, 1993; sierra and rubio, 1993; palma et al., 2003) . protection from chronic demyelinating disease is possible by vaccination with live virus given previously by subcutaneous or intraperitoneal inoculation (crane et al., 1993; kurtz et al., 1995) . early immunosuppression at the time of infection, e.g. by treatment with cyclophosphamide or antithymocyte serum, inhibits or diminishes demyelination. immunosuppression in mice chronically infected with tmev leads to remyelination of oligodendrocytes (rodriguez and lindsley, 1992) . further details related to the pathogenesis of tmev infections and the role of immune mechanisms have been reviewed by yamada et al. (1991) . experimental infection of foxn1 nu mice results in acute encephalitis and demyelination. demyelination associated with minimal inflammation and neurological signs including the typical hind limb paresis develop 2 weeks after inoculation, and most animals die within 4 weeks. in foxn1 nu mice, demyelination is caused by a direct lytic effect of the virus on oligodendrocytes (rosenthal et al., 1986) . demyelination and lethality are reduced after administration of neutralizing antibodies (fujinami et al., 1989) . histopathological changes in prkdc scid mice are very similar to those in foxn1 nu mice (rozengurt and sanchez, 1992) . young mice born in infected populations usually acquire infection shortly after weaning and are almost all infected by 30 days of age. intrauterine transmission to foetuses is possible during the early gestation period, but a placental barrier develops during gestation and later prevents intrauterine infection (miyamae, 1990; abzug et al., 1991) . all tmev isolates are closely related antigenically and form a single serogroup as determined by complement fixation and hi (lipton and rozhon, 1986) . hemelt et al. (1974) demonstrated cross reactions among four strains used in experimental infections, but differences were evident in homologous and heterologous titres. the viral strain most commonly used as antigen for serological testing is gdvii. this strain agglutinates human type 0 erythrocytes at 4њc, and hi has been the standard test for routine screening of mouse populations. meanwhile, hi has been replaced by elisa or ifa, both of which are more sensitive and specific. virus isolation is possible from brains or spinal cords of mice with clinical disease or from the intestinal contents of asymptomatic mice. pcr techniques also are available to test for virus-specific nucleotide sequences in biological samples (trottier et al., 2002) . mice that have been shown to be free from tmev by serological testing can be selected for breeding populations. if the virus is introduced into a mouse population, depopulation of infected colonies may be the most appropriate means to eliminate tmev. embryo transfer or caesarean derivation are the methods of choice for eliminating virus from valuable breeding populations. foster-nursing has been reported to be effective in generating virus-free offspring (lipman et al., 1987) although transplacental transmission has been demonstrated with experimental infection early in gestation. lesions of demyelination in cns of mice with clinically inapparent chronic infection may interfere with investigations that require evaluation of the cns (krinke and zurbriggen, 1997) . conceivably, such lesions also could affect neuromuscular responses or coordination, and affect neurological and behavioural evaluations. viral and mycoplasmal infections of laboratory rodents: effects on biomedical research monographs on pathology of laboratory animals: digestive system monographs on pathology of laboratory animals: digestive system viral and mycoplasmal infections of laboratory rodents: effects on biomedical research the mouse in biomedical research viral and mycoplasmal infections of laboratory rodents: effects on biomedical research viral and mycoplasmal infections of laboratory rodents: effects on biomedical research monographs on pathology of laboratory animals: respiratory system monographs on pathology of laboratory animals: respiratory system biosafety in microbiological and biomedical laboratories (bmbl), 4th edn. u.s. department of health and human services proc. natl. acad. sci. usa 96 the mouse in biomedical research the mouse in biomedical research the mouse in biomedical research veterinary virology complications of viral and mycoplasmal infections in rodents to toxicology research and testing viral and mycoplasmal infections of laboratory rodents: effects on biomedical research virus taxonomy. seventh report of the international committee on taxonomy of viruses the mouse in biomedical research the mouse in biomedical research viral and mycoplasmal infections of laboratory rodents: effects on biomedical research proc. natl. acad. sci. usa the importance of laboratory animal genetics, health, and the environment in biomedical research proc. natl. acad. sci. usa national research council, committee on infectious diseases of mice and rats manual of microbiologic monitoring of laboratory animals the mouse in biomedical research viral and mycoplasmal infections of laboratory rodents: effects on biomedical research the mouse in biomedical research the mouse in biomedical research pathology of laboratory rodents and rabbits proc. natl. acad. sci. usa manual of microbiologic monitoring of laboratory animals viral and mycoplasmal infections of laboratory rodents: effects on biomedical research lactic dehydrogenase virus viral and mycoplasmal infections of laboratory rodents: effects on biomedical research viral and mycoplasmal infections of laboratory rodents: effects on biomedical research viral and mycoplasma infections of laboratory rodents: effects on biomedical research viral and mycoplasmal infections of laboratory rodents: effects on biomedical research viral and mycoplasmal infections of laboratory rodents: effects on biomedical research handbook of animal models of infection the mouse in biomedical research key: cord-320172-qw47pf9r authors: greaves, peter title: vii digestive system 1 date: 2000-12-31 journal: histopathology of preclinical toxicity studies doi: 10.1016/b978-044450514-9/50007-3 sha: doc_id: 320172 cord_uid: qw47pf9r publisher summary this chapter deals with the digestive system. the major and minor salivary glands and their secretions also represent and integral part of the protective mechanism of the oral cavity, and derangement of saliva production may lead to loss of integrity of the oral mucosa. drug-induced abnormalities of taste sensation are also well-described phenomena occurring in man although human studies are necessary for the detection of these effects. inflammation of the oral cavity may involve the buccal mucosa, the gingiva (gingivitis), the tongue (glossitis), and the peridontal tissues (peridontitis). therapeutic agents can induce inflammatory lesions in the tongue. moreover, a protective layer of mucus, a visco-elastic material containing high molecular weight glycoproteins produced by the major and minor salivary glands, covers the stratified squamous mucosa of the oral cavity. salivary secretions also possess digestive enzyme activity although in herbivores and carnivores, it is usually low in contrast to high digestive enzyme activity in omnivorous species. the oral mucosa can be damaged by excessive local trauma from foreign materials, hard fragments in food and damaged teeth may produce ulceration of the mucosa with subsequent infection. however, the oral mucosa may show manifestations of local or systemic disease or derangement produced by therapeutic agents. excessive contact by therapeutic agents such as aspirin, potassium supplements and corticosteroids have been reported to produce local ulceration in the mouth (zentler-monro and northfield, 1979) . the increased use of mouthwashes over the last 20 years has resulted in a number of reported adverse effects to the buccal mucosa in people (gargari and kabani, 1995) . systemic disorders produced by anticoagulants or chemotherapeutic drugs may also be evident by bleeding or ulceration in the oral cavity (goepp, 1982) . buccal ulceration is also described as part of a generalised hypersensitivity reaction to drugs (zentler-monro and northfield, 1979) . the major and minor salivary glands and their secretions also represent and integral part of the protective mechanism of the oral cavity and derangement of saliva production may lead to loss of integrity of the oral mucosa. drugs that effect motor co-ordination can give rise to drooling and disruption of cricopharyngeal co-ordination (wyllie et al., 1986) . drug-induced abnormalities of taste sensation are also well-described phenomena occurring in man although human studies are necessary for the detection of these effects. indeed, many alterations in the oral mucosa are those that are more readily detected by careful clinical and macroscopic observation rather than exhaustive histopathological examination of the buccal mucosa in laboratory animals -provided the basic toxicity profile of a novel agent is adequately assessed in the usual preclinical studies. oral irritation studies are used in the testing of products for use in the oral cavity, mainly for surgical, dental and hygiene purposes but also therapeutic agents administered by the sublingual route. this route may be selected for substances that are broken down in the stomach or show a rapid first pass effect. as it is technically not feasible to perform full preclinical toxicity studies by the sublingual route, conventional oral or parenteral routes are preferred for systemic toxicity studies on such compounds. the choice of the best route will to a large extent be dictated by pharmacokinetic considerations. however, it is necessary to assess local irritancy potential to oral mucosa using a laboratory animal model. test species for oral irritation studies are usually rats, hamsters (cheek pouch), guinea pigs, dogs or primates using gross and histopathological assessment. a similar scheme to that employed in the histological assessment of skin irritancy is appropriate. inflammation of the oral cavity (stomatitis) may involve the buccal mucosa, the gingiva (gingivitis), the tongue (glossitis) and the peridontal tissues (peridontitis). although inflammatory lesions are found sporadically in untreated laboratory rodents, dogs and primates, stomatitis can be induced by systemic administration of high doses of therapeutic agents. anticancer and antimitotic agents are particularly liable to induce stomatitis. a notable example is bleomycin that is capable of producing stomatitis as part of its general effect on squamous cells (thompson et al., 1972) . in humans, the adverse effects on therapeutic ionising radiation on the salivary glands may also give rise to inflammatory changes in the oral cavity (fox, 1998) diuretics and other agents, which are capable of producing severe electrolyte disturbances and uraemia at excessive doses, can also produce stomatitis when then are administered in high doses to laboratory animals (garthoff et al., 1982) . these lesions may be analogous to the well-described association of ulcerative stomatitis and uraemia in man and laboratory animals (boyd, 1978; barker and van dreumel, 1985) . the dog appears very sensitive to the ulcerogenic effects of uraemia in the oral cavity, although as there is a poor correlation between actual levels of blood urea and stomatitis, other biochemical factors are undoubtedly involved. compounds, which effect pigmentation of the skin, can produce similar changes in pigmented oral mucosa. a number of drugs including chlorpromazine, quinacrine, chloroquine, amodiaquine and pyrimethamine cause pigmentation of the oral mucosa in man notably over the hard palate. chloroquine and pyrimethamine have also been shown to significantly increase numbers of active melano-cytes within the palatal mucosa of pigmented da rats when treated orally for 12 weeks (savage et al., 1986) . melanocytes in treated rats were shown to be enlarged and packed with melanin pigment and to possess extensive arborisation of cell processes between squamous cells. an experimental inhibitor of platelet aggregation, which produced pigment loss in the dark hair of long-evans rats and the skin of beagle dogs, also induced pallor of the normally pigmented oral mucous membranes in dogs (gracon et al., 1982; walsh and gough, 1989) . apart from loss of pigment, the histology of the mucous membranes and skin was normal. the tongue is conveniently sectioned for histological study, although reliance is often placed on careful visual inspection, because the usefulness of systematic histological examination of the tongue in routine preclinical safety studies has not been clearly established. a few lesions occur which are fairly specific to the tongue. amyloid may become deposited in the muscular and connective tissue of the tongue in amyloid-prone species, particularly mice (dunn, 1967) . mice, especially dba and dba/2ncrj strains, are liable to develop calcification in the lingual muscle spontaneously, even at a young age (imaoka et al., 1986) . calcified lesions are seen in the longitudinal muscle under the dorsolateral epithelium and the central part of the tongue, which, when severe, are associated with inflammation, granulation tissue, polypoid change, hyperplasia of the overlying squamous epithelium and ulceration. the histogenesis of this lesion is uncertain. in the dba/2ncrj mice, mineralisation of the tongue is associated with myocardial and aortic mineralisation (doi et al., 1985) . therapeutic agents can induce inflammatory lesions in the tongue. an example is provided by the investigational anticancer immunotoxin, zd0490, a mouse monoclonal antibody (c242) against colorectal carcinoma antigen conjugated to recombinant ricin a-chain. when administered to wistar-derived rats, this agent produced myocyte necrosis and inflammation specifically located below the ventral subepithelial surface of the tongue (westwood et al., 1996) . as the changes were different to the low grade myositis seen elsewhere in treated animals, these authors speculated that the changes in the tongue may have been related to the particular receptor profile of this area targeted by the monoclonal antibody. in common with other changes induced in the digestive tract of rats and cynomolgus monkeys by the administration of recombinant human epidermal growth factor, the tongue showed squamous epithelial hyperplasia characterised by a uniform increase in the thickness of the squamous epithelium in both species (breider et al., 1996; reindel et al., 1996) . at high doses, the squamous epithelium of the tongue of the primates was twice the thickness of control mucosa associated with elongation of rete pegs. teeth are usually only inspected by naked eye in conventional toxicity studies and this is appropriate for the assessment of a mature dentition. however, there has been an increasing awareness of dental lesions in toxicity studies, particularly as the teeth are visualised when the maxilla is examined histologically in inhalation studies. study of the rodent dentition in inhalation studies has shown that spontaneous lesions of the dentition are quite common. in one laboratory, malformations (dental dysplasia) of the maxillary incisors were observed in 3% of female and 9% of male cd-1 mice and 14.5% female and 10.5% sprague-dawley rats in 24 and 18 month inhalation studies respectively (losco, 1995) . the rat incisor and its pathology has been the subject of an excellent review (kuijpers et al., 1996) . unlike in humans, the rodent incisor continues to grow and differentiate throughout life and is renewed every 40-50 days. located at the centre of the tooth is the vascular pulp. this is surrounded by proliferating ondotoblasts which form predentin which when calcified becomes dentin. surrounding ameloblasts when induced by the presence of dentin produce the overlying enamel layer. it is these active cellular layers, which can be modified or damaged by xenobiotics, vitamin deficiencies, calcium, phosphate or magnesium deficiency, parathyroidectomy, hypophysectomy, hyperparathyroidism, adrenal insufficiency and fluorosis (kuijpers et al., 1996) . although in humans the mature dentition is no longer growing, in children the dentition is in a growth phase that starts in utero and lasts into the second decade. as increasing numbers of children survive malignant disease, damage to the mature dentition can occur as a result of cytotoxic therapy during childhood. clinical study of the teeth of children treated for malignancy have shown increased incidence of enamel hypoplasia and missing teeth (welbury et al., 1984) . histological examination of teeth from children treated with vincristine or combination chemotherapy for malignant disease has demonstrated prominent incremental lines in dentine correlating with the number of times the intravenous cytotoxic agents were administered (macleod et al., 1987) . it has also been shown that vincristine, a drug which interferes with the assembly of microtubules and reduces secretory activity in a number of cells including osteoblasts and chondroblasts, also effects dentine formation in the rat incisor (stene and koppang, 1976) . two weeks following a single intravenous dose of vincristine to young adult rats, a faint incremental line in the dentine was observed, probably a reflection of a direct effect of the drug on the dentinogenic tissue at the time of injection. at higher doses, focal niche-like or punched out defects in dentine were observed, expression of more severe injury to highly sensitive dentinogenic populations at the time of injection (stene and koppang, 1976 ). the precise mechanism of damage is not fully understood although decreased secretion of dentine matrix by odontoblasts has been demonstrated. calcification appears unaltered (stene and koppang, 1980) . administration of the alkylating anticancer agent cyclophosphamide or a sin-gle exposure to ionising radiation, produces localised niche-like or punched out defects in the rat incisor, rather than the more diffuse changes induced by vincristine. this presumably reflects more localised injury to a sensitive subpopulation of dentinogenic cells (koppang, 1973; vahlsing et al., 1997) . anticonvulsant drugs also produce changes in the dentition of man and experimental animals. in humans, reported alterations include tooth root resorption, small teeth, delayed shedding of deciduous teeth and retarded eruption of permanent teeth, features similar to those found in hypoparathyroid or pseudohypoparathyroid conditions (robinson et al., 1983) . tooth root alterations were also reported in a study in which young male wistar rats were treated with diphenylhyantoin for 1 month. treated rats showed evidence of molar root resorption lacunae that penetrated the cementum and involved the dentine. the lacunae contained a dense infiltrate of cells contiguous with similar cells in the surrounding periodontal ligament. robinson and harvey (1989) showed that the changes were similar to those occurring in parathyroidectomized rats but not those in rats made hypocalcaemic with a calcium deficient diet. they suggested that the changes induced by diphenylhydantoin in rats were similar to those in pseudohypoparathyroidism in which resistance of tooth roots to resorption is reduced. discoloration of teeth and bone is a well-described side effect of tetracycline administration and it has also been reported in patients treated with the semisynthetic derivative, minocycline (cale et al., 1988) . interestingly the ameloblastic epithelium of the enamel forming tissues of growing incisors in wistar rats treated with high doses of human recombinant epidermal growth factor showed hyperplasia characterised by pseudostratification, increased nuclear-cytoplasmic ratio and increased cytoplasmic eosinophilia (breider et al., 1996) . this finding is consistent with the presence of epidermal growth factor receptors in the cells of the enamel organ (martineau et al., 1991) . periodontitis is a common and important disease in man and animals although overt cases are not usually seen in toxicity studies. however, periodontitis of a degree sufficient to disrupt chronic rat toxicity and carcinogenicity studies has been reported. robinson (1985) described periodontitis in alpk/ap rats in which there were erosive granulomatous cavities adjacent to molar teeth with fistulas opening into the nasal cavity. these changes were associated with penetrating food fibres in the gingival sulcus and it was suggested that the presence of long pointed food fibres in the powdered diet was the main reason for occurrence of periodontitis. peridontitis in rodents also results from the effects of dental pathology such as fractures, malformation or malposition of incisors (losco, 1995) . drug-induced overgrowth of the gingival tissues is a well-described phenomenon in both humans and laboratory animals including dogs, cats, and rats. in man, these changes have been associated with diphenylhydantoin (phenytoin) (beghi et al., 1986) nifedipine, calcium channel blockers (ledermann et al., 1984) , cyclosporin a (barthold, 1987) and valproic acid (syrjamen and syrjamen, 1979) . cyclosporin a, diphenylhydantoin and calcium channel blockers have been associated with similar changes in laboratory animals (do'nascimento et al., 1985; latimer et al., 1986; waner et al., 1988) . in most instances there is swelling of the gingiva by firm nodular overgrowths around the teeth. histologically, these overgrowths are characterised by marked acanthosis of the squamous epithelium overlying connective tissue that is infiltrated by large numbers of chronic inflammatory cells. fibrovascular proliferation may be marked. in patients treated with cyclosporin, myxomatous degeneration is described in association with dense infiltration of plasma cells and lymphocytes (barthold, 1987) . secondary acute inflammation in association with food debris and hair shafts is described in dogs treated with oxodipine (waner et al., 1988) . the forces behind these changes are unclear. studies of changed induced by nifedipine and hydantoin have shown increases in extracellular ground substance and increased numbers of fibroblasts containing sulphated acid mucopolysaccharides (kantor and hassel, 1983; lucas et al., 1985) . these drugs may alter fibroblastic proliferative and synthetic activity, possibly by selection of a subpopulation of fibroblasts (hassel et al., 1976) . it has also been suggested that an underlying mechanism in phenytoin-induced gingival hyperplasia involves the decrease in salivary iga that develops in some patients (beghi et al., 1986) . study of cyclosporin a-induced changes have suggested that impairment of t lymphocyte function may permit overgrowth of oral bacteria and bacterial products which may influence fibroblast function (barthold, 1987) . a spontaneous form of gingival hyperplasia has been described in non-human primates (macaca mulata). this is characterised by an enlargement of the marginal and alveolar gingiva by connective tissue consisting of relatively poorly cellular bundles of collagen fibres. the lesions show little inflammatory alterations and the overlying squamous epithelium shows mild hyperkeratosis only (schiã¸dt et al., 1994) . this pathology is similar to hereditary gingival fibromatosis in humans. sessile or pedunculated squamous papillomas and infiltrating squamous carcinomas are occasionally found in the oral cavity of most laboratory animals including rodents (odashima, 1979; emminger and mohr, 1982; leiniger and jokinen, 1994; takahashi and okamiya, 1996; mohr, 1997) , rabbits (sundberg et al., 1985; sundberg and everitt, 1986) , and beagle dogs (watrach et al., 1970) . the microscopic structure of these neoplasms in rodents resembles those occurring in squamous epithelium in other sites. although a number of agents induce squamous neoplasms in the oral cavity, spontaneous squamous carcinomas are generally uncommon spontaneous lesions in laboratory animals. however, some strains of rodent may develop squamous neoplasms more commonly. for instance, in life time studies ad libitum fed brown-norway rats, 21% of males and 32% females developed oral squamous cell carcinomas although only 9% and 10% in food-restricted animals respectively (thurman et al., 1997) . it was suggested that certain pedigrees possessed a genetic predisposition to these neoplasms. papillomas occurring in rabbits and dogs are of note because they can occur in quite young animals, apparently as a result of infection with viruses of the papilloma group. viral inclusions may be seen in histological sections. the implications of papilloma viruses in laboratory species are that the progression of virally induced papillomas to malignant squamous carcinomas can be potentiated by nonviral factors including application of xenobiotics (howley et al., 1986) . in rabbits, the prevalence of oral papillomas varies considerably but they are quite common in some laboratory strains. they are overlooked because of their small size and a distribution limited to the ventral surface of the tongue (sundberg et al., 1985) . microscopically, they are typical squamous papillomas composed of irregular acanthotic squamous epithelium and a fibrovascular stalk of variable size. squamous cells at the margins of papillomas at the junction with normal mucosa, often show large, oval nuclei, marginated chromatin and central, basophilic, intranuclear inclusions, which electron microscopic examination shows to contain viral particles. oral papillomas in dogs develop as multiple growths, regressing spontaneously after a few months. they are also caused by a virus of the papilloma group, which possesses a high degree of specificity for the mucosa of the oral cavity and adjacent skin (watrach et al., 1970) . histologically, they are composed of proliferative masses of epithelial cells, keratinised on the surface and resting on an irregular connective tissue stroma or pedicule. large vesicular cells with basophilic intranuclear inclusions are also found in the granular cell layer, identifiable as virus arrays by electron microscopy (cheville and olson, 1964) . malignant change has been described in these canine lesions and this can occur in young beagle dogs (watrach et al., 1970) . although many types of papilloma viruses have been identified in both man and animals (pfister, 1984) , common antigenic determinants exist between viruses in different species. this immunological cross-reactivity can be exploited in the immunocytochemical localisation of papilloma viruses in epithelial lesions of many animal species. papilloma virus antigen has been demonstrated in oral papilloma of dogs and rabbits using antisera to bovine papilloma virus type i (sundberg et al., 1984) . cells positive for virus and viral inclusions are located in the upper layers of the epithelium, especially within cells of the granular layer. spontaneously developing odontogenic tumours are rare in rodents but they have been induced in laboratory animals given carcinogens such as nitrosoureas or exposed to ionising radiation (gã¶ssner and luz, 1994) . a range of tumours originating from dental tissues with epithelial, mesenchymal or mixed appearances has been reported in rodents (kuijpers et al., 1996) . the classification of odontogenic tumours is complex and confusing. they range from benign anomalies and cystic structures through to malignant neoplasms. the ameloblastoma comprises cords, nests, anastomosing strands or islands of ondontogenic epithelial cells within a fibrous stroma. the tumour cells resemble ameloblasts with the cords of spindle shaped cells similar to the stellate epithelium bounded by a peripheral layer of cuboidal or columnar cells resembling the inner enamel epithelium. other tumours of the odontogenic epithelium show induction of the mesenchymal elements or develop a complete sequence of odontogenic epithelium, odontogenic mesenchyme and dental hard tissues including dentine, enamel and cementum. in the rat these have been classified as odontoma characterised by the presence of all dental hard tissues and odontogenic fibroma composed of undifferentiated or primitive mesenchymal cells of developing dental tissue (mohr, 1997) . odontogenic tumours developing in fischer rats treated with aflatoxin were located in the upper jaw associated with the incisor teeth and were composed of proliferating fibroblast-like cells within which ovoid calcified bodies resembling cementum were seen (cullen et al., 1987) . occasional inclusions of solid epithelial nests were also seen. no metastatic deposits were found although the neoplasms were locally aggressive. in addition, squamous tumours and neoplasms of mesenchymal origin typical of other organs, bones and soft tissues are found in this region. although salivary glands may not represent vital organs in the same sense as the kidneys or heart, severe derangement of their secretions can alter both the quality and quantity of saliva. depending on the particular glands and cells affected, dry mouth, mucositis, and dental caries may develop . the severe oral complications of irreversible salivary damage and dysfunction, which can occur patients with head and neck cancer as a consequence of local irradiation, may have a significant impact on the efficacy of therapy, quality of life and survival (fox, 1998) . a protective layer of mucus, a visco-elastic material containing high molecular weight glycoproteins produced by the major and minor salivary glands, covers the stratified squamous mucosa of the oral cavity. these mucins usually contain more than 50% carbohydrate in the form of neutral and acidic oligosaccharide chains, o-glycosidically linked to threonine or serine. mucins possess several roles including mechanical flushing of the oral cavity, protection and lubrication of soft and hard tissues, modulation of oral microbial flora, buffering activity, regulation of calcium/phosphate equilibrium, digestion and extracellular post translation processing of molecules present in saliva . the heterogeneity of salivary glycoproteins suggests that they act as a defence against pathogenic microorganisms by competing with microbial binding sites of similar structure on the surface of cells lining the digestive tract (schulte, 1987) . minor salivary glands may also play an important part in the local immunosurveillance of the oral cavity for their ducts are anatomically closely associated with lymphoid tissue (nair and schroeder, 1986; nair et al., 1987) . salivary secretions also possess digestive enzyme activity although in herbivores and carnivores, it is usually low in contrast to high digestive enzyme activity in omnivorous species (junqueira et al., 1973) . the phylogenetic association of the salivary glands with the thyroid gland is evident functionally because salivary glands are capable of concentrating iodide in their secretions, although this is not under control of thyroid stimulating hormone (ingbar, 1985) . it has been shown that thyroxine accelerates the differentiation of the granular convoluted tubule cells and the appearance of epidermal growth factor in the submandibular gland of the neonatal mouse (chabot et al., 1987) . the structure of the salivary glands differs among laboratory species, between different glands in the same species and between sexes. it is usually considered that there are three major salivary glands, the parotid, the sublingual and the submandibular (submaxillary) glands. minor salivary glands are scattered in other locations throughout the mouth and oropharynx. in dogs and other carnivores, the zygomatic (infra-orbital) gland, located just below the zygomatic arch and the buccal (molar) gland are also often referred to as major salivary glands. microscopically, salivary glands are composed of secretory glands or 'endpieces' attached to a connecting system of intralobular and extralobular (secretory) ducts. secretory endpieces may be acinar or tubulo-acinar in nature. the secretory cells have been subdivided into serous, mucous, seromucous and special serous types. controversy remains about the precise nature of the secretory cells found in the various salivary glands of different species and this makes critical interspecies comparisons difficult (see detailed discussion of this problem by pinkstaff, 1980 ). the duct system is less complex. this comprises an intercalated duct which leads from the secretory endpiece into a striated (secretory or intralobular) duct, so termed because their lining cells are striated by delicate eosinophilic cytoplasmic rods. the striated ducts converge into interlobular ducts and a main excretory duct system. in rats, mice and hamsters, an overall similarity in gross and microscopic anatomy of the various salivary glands exists although there are histochemical differences (munhoz, 1971; glucksmann and cherry, 1973; dawe, 1979; pinkstaff, 1980; emmiger and mohr, 1982) . moreover, it has been demonstrated that salivary glands in rodents as well as a number of other species show morphological and histochemical sexual dimorphism (pinkstaff, 1998) . the sublingual gland in rats, mice and hamsters is composed principally of mucous acini, with indistinct serous demilunes. acini open into fairly long intercalated ducts lined by flat or cuboidal cells devoid of granules. the parotid gland is composed of serous-type secretory cells containing zymogen granules and prominent hyperchromatic basal cytoplasmic poles. the submandibular gland is anatomically the most complex salivary gland in rodents. secretory endpieces are composed of small or moderately sized cells with foamy cytoplasm and basophilic basal poles. the most striking feature is the presence of an additional duct segment interposed between the intercalated and striated ducts. this segment is lined by cylindrical epithelium with basal nuclei and eosinophilic cytoplasm containing secretory granules. this duct segment is termed the granular duct or granular convoluted tubule. these granular cells are of special interest because they contain a large number of heterologous biologically active peptides including nerve growth factor, epidermal growth factor, renin, and kallikrinins (barka, 1980; mori et al., 1983 ). the precise physiological role of many of these peptides in salivary gland remains uncertain. epidermal growth factor was originally isolated from the mouse salivary gland. it initiates premature eyelid opening and incisor eruption when injected into the neonatal mouse (cohen, 1962) . study of the mouse submandibular gland has shown that both epidermal growth factor and nerve growth factor are released into saliva following the administration of phenylephrine, sympathomimetic amine acting mainly on î±receptors and isoprenaline (isoproterenol), a î²-adrenergic agent (murphy et al., 1980) . immunohistochemical study also demonstrates that epidermal growth factor becomes depleted in mouse salivary tissue following administration of phenylephrine and similar agents (tsukitani and mori, 1987) . phenylephrine has been shown to cause marked secretory activity accompanied by loss of granules from granular cells, as well as loss of immune reactive carbonic anhydrase, an enzyme which participates both in membrane transport of bicarbonate ions into saliva and glandular secretion (noda, 1986) . morphological studies have shown that both acinar and granular tubular cells participate in this response to adrenergic agents (murphy et al., 1980) . this is in contrast to the effects of pilocarpine, a cholinergic agent, which elicits the secretion of saliva deficient in serous proteins with little or none of the growth factors, as its effects are more limited to acinar cells. glycoprotein secretion of rodent salivary glands has stimulated histochemical study using both conventional mucin histochemical techniques and labelled lectins which possess affinity for specific sugars or sugar sequences (tables 1 and 2 , pages 340 and 342). studies of rat, mouse and hamster salivary glands using batteries of labelled lectins have shown a greater heterogeneity of oligosaccharides in salivary glands than seen by classical histochemical techniques. there are considerable species differences and variations between murine strains and sexes of the same strain as well as heterogeneity among morphologically similar cells within one gland (schulte and spicer, 1983, 1984; schulte, 1987) . the results of histochemical studies are in excellent agreement with studies using biochemical methods but suggest a significant influence of genetic and hormonal factors on the synthesis of salivary glycoproteins. less attention has been paid to the structure and cytochemistry of the dog salivary glands. there appears to be little variation between the structure of salivary tissues between beagles and other strains although variation with age has been reported (reifel and travill, 1972; nagoyo and tandler, 1986) . munhoz (1971) has described the histochemical features of the dog parotid gland. the dog parotid is of seromucinous type secreting both acidic and neutral mucosubstances, in contrast to the more neutral mucosubstances secreted by rodent glands. the salivary glands of non-human primates are similar to those in man. they possess parotid glands of serous or seromucous type, submandibular glands with both serous and mucous acini and sublingual glands of mainly mucous type. the salivary glands of the non-human primate react to adverse stimuli such as ionising radiation in a similar manner to human salivary tissue . focal chronic inflammation of the salivary glands occurs sporadically in untreated rats, mice, hamsters, dogs or primates employed in toxicology although severity and prevalence is variable. sialoadenitis as a result of a corona virus, the sialodacryoadenitis virus, is a well-known and fairly ubiquitous condition in rats, first described by innes and stanton (1961) . the condition is characterised histologically by oedema and congestion of submandibular and parotid salivary glands as well as extra-orbital lachrymal and harderian glands. it is accompanied by inflammation of variable severity and chronicity in both glandular and connective tissue as well as degeneration and necrosis of duct epithelium (fig. 44) . the regenerative hyperplasia of the duct epithelium may be quite intense about a week after infection but all changes regress after about 2 weeks and glands are essentially normal after 3 or 4 weeks (carthew and slinger, 1981; percy and wojcinski, 1986) . there may be a delay in the appearance of inflammatory cells and the onset of repair in rats immunosuppressed with cyclophosphamide (hanna et al., 1984) . depletion of salivary gland epidermal growth factor also occurs during the infection (percy et al., 1988) . suppurative infections in the neck region of the rat such as those produced by klebsiella aerogenes also cause acute and chronic inflammation of salivary glands with fibrosis and glandular proliferation of salivary tissue (arseculeratne et al., 1981) . sialadenitis occurs spontaneously in autoimmune-prone strains of mice such as the nzb/nzw and sl/ni strains and it has been reported in ageing female, but not male bdf1 mice (hayashi et al., 1988) . the non-obese diabetic mouse known for its spontaneous insulin-dependent diabetes mellitus also develops immune mediated damage to submandibular glands (fujino-kurihara et al., 1985; tã¶rnwall et al., 1999) . in ageing bdf1 females the submandibular gland was shown to be involved by a destructive inflammatory process characterised by an intense infiltration by small and medium sized lymphocytes, associated with mild inflammation in other organs such as the parotid and sublingual glands, pancreas and kidney. immunocytochemistry showed that most of the lymphocytes were t cells (thy-1.2 and lyt-1 positive) of the helper/inducer subset (l3t4 or cd4 positive) and less than 10% were of suppresser/cytotoxic (lyt-2 or cd8 fig. 44 . section from salivary tissue from a sprague-dawley rat during an infection with the sialodacryoadenitis virus showing intense ductular inflammation. (he, ã�25.) positive) type (see haemopoietic and lymphatic systems, chapter iii). circulating anti-salivary duct antibody of igg was also detected in afflicted mice. it was suggested that helper/inducer t cells played a key role in the production of this change, unlike induced autoimmune sialoadenitis in which cytoxic t-cell subsets may directly destroy glandular tissue. it has been suggested that this process in ageing females is related to the decline in the number of splenic lyt-2 cells in mice with advancing age (hayashi et al., 1988) . these cells are believe to be the most susceptible to ageing (see haemopoietic and lymphatic systems, chapter iii) in the non-obese diabetic strain of mouse derived from jcl-icr mice, a periductal chronic inflammatory infiltrate is found in the submandibular gland at about the same time that immune-mediated insulitis is most marked. this suggests that there is an extension of the autoimmune process to salivary tissue (fujino-kurihara et al., 1985) . it is probable that helper/inducer cd4 t cells are essential components of this infiltrate and a number of cytokines and their receptors such as ip-10 (interferon-î³ inducible protein 10) and rantes (regulated upon activation normal t cell expressed and secreted) may have an important role (tã¶rnwall et al., 1999) . an autoimmune type of sialoadenitis can also be experimentally induced certain strains of mice. crj:cd1 mice, thymectomized at 3 days, a time point at which lyt-2 positive cells (cd8 suppresser t lymphocytes) can be maximally reduced, followed by immunisation at 28 and 42 days after birth with homogenates of salivary gland and complete freund's adjuvent, develop a distinctive sialoadenitis in the submandibular and to some extent the parotid glands (hayashi et al., 1985) . this sialoadenitis is characterised by degenerative changes in salivary glandular tissue associated with an extensive and intense infiltrate of small and medium sized lymphocytes. these cells appear shortly after immunisation but increase in number with time. immunocytochemical study has shown that many of these cells are reactive to antisera to thy-1.2 and lyt-2 (cd8) features of suppresser/cytotoxic t lymphocytes. later appearing cells demonstrate features of plasmacytoid lymphocytes and contain immunoglobulin of mainly igg class (hayashi et al., 1985) . these authors therefore suggested the sialoadenitis appeared as both a result of cytotoxic/suppresser t-cell activity and an antibody-dependent cell-mediated cytotoxicity. in the hamster salivary glands, interstitial infiltrates of lymphocytes and plasma cell are quite common and may become more marked with advancing age (mcmartin, 1979) . whereas necrosis of the parotid gland of uncertain aetiology sometimes occurs in the dog, mild focal chronic inflammation is quite a common incidental finding in canine salivary glands and has been reported in about 5% of normal beagle dogs (kelly et al., 1982) . although the inflammation in salivary tissue which results from ionising radiation is only indirectly relevant to drug safety evaluation, it is of interest in view of the notable species differences in sensitivity to this form of insult. serous acinar cells in man and rhesus monkey appear least resistant to the effects of ionising radiation, where damage is characterised by widespread degranulation and degeneration of acini, infiltration by polymorphonuclear cells followed by lymphocytes, plasma cells and subsequent atrophy and fibrosis . these changes contrast with the lesser effects of ionisation radiation on the rodent salivary glands in which there is little or no acute inflammatory response. lymphoid bodies are sharply circumscribed collections of lymphoid cells generally located between the parotid and sublingual glands close to a cervical lymph node in mice. they are apparently normal aggregates of lymphoid tissue. like many other glandular organs, the size of the secretory tissue of the salivary gland is responsive to functional demand and is subject to age-related changes. in man, the gland parenchyma frequently becomes atrophic and replaced by connective tissue or fat with advancing age, possibly partly related to vascular changes (waterhouse et al., 1973; scott, 1977) . in ageing rats, the extent and height of granular ducts and their content of mature secretory granules has also been shown to decrease with age (sashima, 1986) . dietary factors influence salivary gland size. decreased food consumption or protein starvation can reduce the weight of salivary glands in rats. there is shrinking of mucous and serous glands and loss of zymogen granules associated with decreased rna but unchanged dna content, attributable to the reduced requirements for protein synthesis , mcbride et al., 1987 . as salivary gland function is responsive to adrenergic stimulation, it is not surprising that atrophy occurs following adrenergic blockade. the weights of the submandibular gland in mice were shown to decrease following administration of the î²-adrenergic blocking agent, propranolol (smith and butler, 1977) . this was associated with a reduction in stainable neutral mucins and a decrease in the thickness of the acinar cells making the gland lumens appear larger than normal. the cytotoxic agent, alloxan, known primarily for its specific effect on pancreatic b cells, has also been shown to produce weight loss of the rat submandibular gland, associated with lipid inclusions in the acinar cells, capillary basement membrane thickening and reduced salivary flow (reuterving et al., 1987) . it is probable that alloxan exerts a cytotoxic effect on the acinar cells of the rat submandibular gland (sagstrã¶m et al., 1987) . methotrexate, a folic acid antagonist, has also been reported to cause vacuolization of acinar and ductular cells with reduction of secretory granules in rat salivary glands (mcbride et al., 1987) . ligation of the main excretory ducts has frequently been used as an ex-perimental model for study of salivary gland atrophy as well as the regeneration that follows removal of the ligature. there is marked atrophy of all cell types but most markedly the acinar cells through apoptosis. although overt necrosis has been reported following ligation of the excretory duct, it appears that this may have been the result of constriction of the vasculature for acinar cells are relatively intolerant to a decrease in oxygen and nutrient (denny et al., 1997) . a number of therapeutic agents increase salivary gland size in man, although the scarcity of biopsy data precludes a critical assessment of the precise mechanism in many cases. drugs reported to produce salivary gland enlargement in man include iodide-containing radiological contrast media, isoprenaline and anti-inflammatory agents phenylbutazone and oxyphenbutazone. enlargement may also occur after endotracheal anaesthesia and upper gastrointestinal tract endoscopy in man (riddell, 1982) . some of these agents and procedures may produce spasm of large salivary ducts and retention of secretions. several pharmacological agents, particularly sympathomimetic amines, have been shown to produce increases in salivary gland size in rodents following repeated dosing (brenner and stanton, 1970) . there is an intimate relationship of sympathomimetic amines with the control of the secretory process in salivary tissue. whereas a single injection of isoprenaline (isoprotorenol) in the range of 20-200 mg/kg induces discharge of preformed secretory granules followed by gradual re-synthesis and reconstitution, repeated injections produces an increase in the size of salivary glands (simson et al., 1974) . histologically, the enlarged glands are composed of secretory cells of increased size that contain increased amounts of secretory substances in the cytoplasm (simson et al., 1974) . although these histological features are principally those of diffuse cellular hypertrophy, the increase in dna content and radioactive thymidine uptake described in the salivary tissue following repeated administration of isoprenaline suggests that a degree of hyperplasia also occurs (barka et al., 1972) . these effects do not depend on the integrity of the autonomic nerves because they occur after ablation of the autonomic ganglia (barka et al., 1972) . they appear to be mediated by an effect on adrenergic î²-receptors. the effects can be blocked by propranalol, a î²-receptor antagonist but not by phenoxybenzamine, an î±-receptor antagonist (brenner and stanton, 1970) . as theophylline and caffeine also elicit salivary gland enlargement in rats, a role for cyclic 3',5-adenosine monophosphate (camp) in salivary gland enlargement has been postulated (brenner and stanton, 1970) . detailed study of hypertrophy, protein synthesis, and intracellular camp activity in the salivary glands of rats treated for 10 days with isoprenaline (isoproterenol), a series of î²-adrenergic receptor agonists and the phosphodiesterase inhibitors, theophylline and caffeine, showed that similar effects occurred with all agents although differences in the degree of hypertrophy, the nature of pro-tein and glycoprotein synthesis and golgi membrane enzyme activity were recorded (wells and humphreys-beher, 1985) . the parotoid gland showed the most pronounced hypertrophy followed by the submandibular gland but the sublingual gland appeared to be unaffected by treatment. the degree and nature of the changes induced by the various î²1/î²2 receptor agonists suggested that most of these effects were mediated through î²1 receptors which are present in greatest numbers on the parotid and salivary cells. it was suggested that the effects of î²-adrenergic agonists on salivary gland are produced by a receptor-mediated stimulation of adenylate cyclase activity causing an increase in levels of intracellular camp. however, other factors may be important for wells and humphreys-beher (1985) also showed that although isoproterenol and caffeine increased salivary cell camp to comparable levels, the hypertrophy was greater with isoproterenol. cardioactive phosphodiesterase inhibitors were shown to produce submaxillary hypertrophy in rat subacute toxicity studies (rogers et al., 1985; jayasekara et al., 1986; smith et al., 1988) . parotid and submaxillary glands were those most affected by the inotropic phosphodiesterase inhibitor ici 153,110 (westwood et al., 1991) . as the agents produced their positive inotropic action via selective inhibition of the cardiac phosphodiesterase subfraction iii specifically requiring camp as its substrate, it was suggested that the salivary gland hypertrophy was a result of phosphodiesterase inhibition (smith et al., 1988) . other classes of drugs can also produce salivary gland enlargement in rats in repeated dose studies. doxylamine, a representative of the widely used ethanolamine group of antihistamines, has been reported to produce marked cytomegaly in the fischer 344 rat parotid gland. enlarged cells were characterised by a basophilic and coarsely granular or vacuolated cytoplasm (jackson and blackwell, 1988) . the b6c3f1 mouse did not develop these changes after a similar treatment schedule. in view of the presence of considerable amount of epidermal growth factor in salivary glands, it is of interest to note the effects of its administration to laboratory animals. salivary gland weights were increased in rats and cynomolgus monkeys infused with high doses of recombinant human epidermal growth factor (breider et al., 1996; reindel et al., 1996) . however histological features seen are primarily those of ductular epithelial hyperplasia (see below under hyperplasia). epithelial cells characterised by abundant granular eosinophilic cytoplasm as a result of the accumulation of mitochondria are often referred to as oncocytes, a term used by hamperl (1950) to describe similar cells in hã¼rthle tumours of the thyroid gland. they may be found in various focal nodular and neoplastic states of the salivary glands in both man and laboratory animals. the precise significance of these cells is uncertain. the mitochondria usually appear unremarkable except for lack of dense granules and it has been suggested that the mito-chondrial changes represent an adaptive phenomenon or compensatory hyperplasia (ghadially, 1982) . in human salivary tissue their prevalence seems to increase with advancing age and they can be associated with hyperplastic lesions or neoplasms such as oxyphil adenomas and adenolymphomas. eosinophilic cells also occur in the salivary glands of certain strains of aged rats (bogart, 1970) and in mice with experimentally induced autoallergic sialoadenitis (takeda et al., 1985) . in the study of takeda et al. (1985) the eosinophilic cells appeared to arise predominantly in the secretory (glandular) ducts of the submandibular glands, although eosinophilic cells can apparently develop from either duct or acinar cells. well-defined, unencapsulated foci of enlarged acinar cells occur spontaneously in the salivary glands, particularly the parotid of rats, mice (chiu and chen, 1986) , and hamsters although their reported incidence varies between laboratory. the enlarged cells possess greatly expanded cytoplasmic volume that retains a vesicular, vacuolated or foamy appearance or possesses a pale eosinophilic granular texture. the basal parts of the cells usually stain intensely blue in haematoxylin and eosin stained sections and contain large, dense, irregular hyperchromatic or pyknotic nuclei showing little evidence of mitotic activity. although there has been little ultrastructural study of these foci, the cytoplasmic alterations appear to be distinct from those of so-called oncocytes that characterised by granular eosinophilic cytoplasm packed with mitochondria. the biological nature of these foci is uncertain. the lack of any prominent mitotic activity, cell proliferation or expansive growth suggests that they are most aptly regarded as hypertrophic lesions (chiu and chen, 1986 ). although they increase in prevalence with increasing age in certain strains of rat, there is no evidence to suggest that they represent pre-neoplastic lesions or possess any relationship with development of neoplasia in salivary tissue (dawe, 1979) . hyperplasia and squamous metaplasia of the salivary ducts are common features of many inflammatory and reactive conditions in the salivary glands of rodents, dogs, non-human primates and man and can be associated with the presence of stones and calculi with the duct system. squamous metaplasia and regenerative change in the ducts occurs in rats afflicted with sialodacryoadenitis (carthew and slinger, 1981) . it is also described specifically located in the ducts of the sublingual glands in the wistar rat in the absence of obvious sialodacryoadenitis or evidence of any specific disease. similar regenerative hyperplastic duct changes are also seen in necrotic and inflammatory conditions in the dog salivary gland . detailed morphological examination with immunocytochemical study of epi-dermal cytokeratins of the rat salivary gland after arterial ligation has shown that the acinar units can also undergo squamous metaplasia (dardick et al., 1985) . it appears that the acinar-intercalated duct complexes can rapidly reprogram to produce epidermal cytokeratin filaments in ischaemic or inflammatory states. hyperplasia of the ductular epithelium appears to be the principle result of the administration of epidermal growth factor to rats and cynomolgus monkeys. in rats histological features were primarily of ductular epithelial hyperplasia without evidence of significant acinar hyperplasia (breider et al., 1996; reindel et al., 1996) . in primates the changes were most striking in the interlobular and large intralobular ducts where the epithelium showed multilayered and papilliform projections. however, mitotic activity was evident throughout the duct epithelia and acinar cells showed hypertrophy with depletion secretory granules and the presence of large vesicular nuclei . focal duct and acinar hyperplasia, showing minimal compression of the surrounding parenchyma and distinct from focal hypertrophy is also described in the classification of rat salivary lesions (mohr, 1997) . primary neoplasms of salivary glands are uncommon in the usual strain of rats and mice employed in carcinogenicity bioassays (haseman et al., 1998) . acinar and tubular adenomas and adenocarcinomas as well as squamous carcinomas are reported in rats (mohr, 1997) , mice (frith and heath, 1994) and hamsters (takahashi and okamiya, 1996) . some carcinomas showing squamous or glandular differentiation may be observed infiltrating the salivary gland that originate in other local structures of the head and neck region. occasionally, salivary gland neoplasms show adenomyomatous differentiation. mixed glandular and lymphoid tissue patterns resembling wartin's tumour in man are also sometimes seen. neoplasms of soft tissues also develop in and around the major salivary gland in rodents (see integumentary system chapter i). in humans the oesophagus is not considered a common site for drug-induced injury although some studies have suggested that medication-induced changes are more prevalent than previously supposed (bonavina et al., 1987) . severe damage can occur following prolonged contact between mucosa and ingested tablets or capsules which results in local high concentrations of potentially irritant substances (bott and mccallum, 1986; brors, 1987; kikendall, 1999; levine, 1999) . damage as a result of local contact may be more common in elderly subjects as the amplitude of oesophageal contractions decrease with age and capsules more liable to lodge in the lumen of the oesophagus (bonavina et al., 1987) . however patients of all ages may be affected. women have been injured more frequently than men probably because of the greater likelihood of their being treated with potentially injurious drugs (kikendall, 1999) . the shape and surface coating of tablets may influence their tendency to adhere to the mucosa and lodge in the oesophagus (marvola et al., 1983) . a wide variety of drugs have been implicated. in the united states the majority of cases appear to be caused by ingestion of tetracycline or doxacycline (levine, 1999) . some of the causative agents such as potassium chloride, aspirin and other non-steroidal anti-inflammatory drugs are also implicated in ulceration lower in the gastrointestinal tract. over recent years the bisphosphonate, alendronate has been one of the most commonly reported causes of adverse effects in the oesophagus with severe injury being reported. although injury is linked to ingestion without water or failing to remain upright after swallowing the medication, alendronate is particularly caustic (kikendall, 1999) . oesophagitis due to candida albicans is a well-described complication of antibiotic therapy. administration of immunosuppressive drugs may predispose to viral infections in the oesophagus. a number of agents affecting neuromuscular co-ordination may also predispose to gastro-oesophageal regurgitation and reflux oesophagitis (bott and mccallum, 1986) . in laboratory rodents spontaneous lesions of the oesophagus are occasionally seen. oesophageal impaction has been described in untreated srl:bhe rats. this is characterised by massive dilatation of the oesophagus with food or bedding (ruben et al., 1983) . histologically, the muscle fibres in the wall of the oesophagus show varying degrees of degeneration including swelling or shrinking of fibres, myofibrillar fragmentation, cytoplasmic vacuolation and mineralisation. so-called megaoesophagus, characterised by enlargement of the oesophagus, degeneration of muscle fibres and ganglion cells in the myenteric plexus has also been described in certain strains of rats and mice (harkness and ferguson, 1979; randelia and lalitha, 1988) . its cause is unknown. a commonly occurring lesion reported in fischer 344 rats is oesophageal hyperkeratosis, which occurs at all ages . in the study by maeda et al. (1985) , it occurred more commonly in rats fed a protein-restricted, calorie unrestricted diet than in rats fed ad libitum with normal diet. it was suggested that the particular high prevalence of oesophageal hyperkeratosis observed in all groups in this particular study was related to acidification of drinking water . another pathological findings in rodents is perforation of the oesophagus as a result of a gavage accident. under these circumstances there is a variable inflammatory and purulent exudate localised around the perforation or spread within the pleural or occasionally the pericardial cavities. the oesophagus and surrounding tissues need careful examination by the pathologist for it is not always clear from clinical findings that oesophageal damage has occurred. spontaneous oesophageal lesions are uncommon in laboratory beagles, even though emesis and vomiting are frequent responses of this species following dosing in toxicity studies. local oesophageal irritancy potential of drugs has been assessed in a number of animal models, notably the cat and pig (carlborg and densert, 1980; olovson et al., 1983) . in these models, the test drugs are placed in the upper oesophagus using endoscopic techniques for periods of several hours to allow dissolution of the preparation. subsequently, the animals are followed for 3-6 days and histopathological assessment performed on the oesophagus. the degree of inflammation, erosion of mucosa or deep ulceration is recorded in a semiquantitative manner. the degree of ulcerogenic activity of drugs in these models seems to correlate with reported ulcerogenic activity in the human oesophagus (carlborg et al., 1983) . systemic administration of drugs with radiomimetic or antimitotic activity can cause hypoplastic changes in the oesophageal mucosa as well as the remaining gastrointestinal tract mucosa (tucker et al., 1983) . conversely, hyperplasia with increased keratinization has been reported in the oesophagus of the rat following chronic high dose administration of alcohol (mascrã¨s et al., 1984) . acanthosis with hyperkeratosis and parakeratosis has been reported in the oesophagus but not stomach of rats treated for up to 18 months with mesuprine hydrochloride, a î²-adrenergic receptor stimulator (nelson et al., 1972) . as part of its effects on the gastrointestinal tract, the oesophagus in rats and primates has been reported to develop uniform hyperplasia of the squamous epithelium following infusion of recombinant epidermal growth factor (breider et al., 1996; reindel et al., 1996; vinter-jensen, 1999) . in the rat, mouse and hamster the forestomach occupies about two-thirds of the proximal stomach area and is lined by cornified stratified squamous epithelium. the limiting ridge is a distinct elevated mucosal fold at the junction between the forestomach and the mucosa of the glandular part of the stomach. as humans lack a forestomach, the relevance of changes produced by drugs and chemicals in the rodent forestomach is disputed. studies in rats in which the forestomach has been removed have suggested that the forestomach acts as a storage organ releasing relatively undigested food into glandular stomach in response to energy demand (gã¤rtner and pfaff, 1979) . hence, the forestomach mucosa may be exposed to xenobiotics mixed in undigested food for far longer periods than elsewhere in the gastrointestinal tract. the interpretation of forestomach changes should take into account physiological factors, residence time and exposure differences to drugs between the rodent forestomach and human oesophagus. however the squamous mucosa lining the oesophagus in species without a forestomach may react to xenobiotics in a similar way to the forestomach mucosa of rodents if equivalent exposure levels are attained. inflammation and ulceration of the forestomach mucosa are some of the commonest spontaneous gastrointestinal lesions in laboratory rats, mice and hamsters. the prevalence of these gastric lesions varies between species, strains of laboratory rodents as well as between different laboratories. the precise causes of forestomach ulceration remain unclear although a variety of factors have been associated with its development including advanced age, infection, parasitism, diet, feeding regimens and stress. in rats, conflict-induced ulceration occurs in the forestomach and there is an age-related susceptibility, older rats developing more ulcers than younger rats (sawrey and sawrey, 1966) . in rats and mice dying of spontaneous disease, ulceration of the forestomach is also quite frequently observed. protein restriction or starvation has also been shown to produce forestomach ulceration in rats . ulceration of the forestomach in rodent toxicology studies may be incidental, particularly if the lesions are few and show no clear relationship to dose. if limited to high dose groups, ulceration may be a result of non-specific toxicity and stress-related. however, administered chemicals may have direct local effects of sufficient severity to cause focal damage to the forestomach mucosa. histological features of ulcers and inflammatory lesions of the forestomach are similar in rats, mice and hamsters. in mild cases, a scattering of acute inflammatory cells is seen in the intact squamous mucosa. ulcers can be single or multiple and are characterised by loss of squamous epithelium with a variable accumulation of neutrophils, mononuclear cells, cellular debris, fibrin and hair fragments in the ulcer crater. the inflammatory process may extend deeply into the stomach wall and be associated with intramural inflammation, oedema, endarteritis and fibrosis. haemosiderin pigment is also found in the ulcer margins. profuse haemorrhage may follow erosion of large blood vessels and complete perforation of the stomach wall with peritoneal involvement also occurs (greaves and faccini, 1992) . in long-standing cases of ulceration, hyperplasia of the adjoining squamous epithelium occurs, characterised by irregular acanthosis and down-growths of squamous epithelium into the submucosa . xenobiotics may produce inflammatory changes in the forestomach mucosa following initial dosing but subsequently, repair occurs even though treatment continues. an example of this phenomenon is illustrated by butylated hydroxyanisole. after 1 week of administration of this agent in a 2% mixture in diet to rats, a vesicular inflammatory reaction characterised histologically by the presence of subepithelial vesicles containing inflammatory cells and exudate was seen (altmann et al., 1985) . after further treatment, only hyperplasia of the squamous epithelium was evident, presumably as an adaptive response to the effects of the continued insult. hyperkeratosis associated with hyperplasia of the squamous epithelium is seen sporadically in untreated aged rodents. these changes may be localised to the margins of chronic forestomach ulcers or they can be associated with diffuse inflammation of the mucosa. occasionally, the forestomach mucosa of untreated, aged rodents exhibits hyperkeratosis with hyperplasia without inflammation (fig. 45 ). such changes may be diffuse or focal, but they are often localised to the zone adjoining the glandular stomach mucosa. there may be evidence of basal cell proliferation and downgrowth of the epithelium into the underlying stroma. dietary factors also influence the thickness of the forestomach mucosa. vitamin a deficiency, known to produce squamous metaplasia in glandular tissues may produce forestomach hyperplasia and hyperkeratosis in rats. when spf fischer 344 rats were maintained in a vitamin a deficient state for over 3 months, hyperplasia with hyperkeratosis, not unlike that produced by known carcinogens was reported (klein-szanto et al., 1982) . administration of a wide range of both industrial chemicals, therapeutic agents including both genotoxic and non-genotoxic carcinogens produces hyperkeratosis and hyperplasia of the forestomach epithelium which may be followed by preneoplastic lesions and squamous carcinoma (see below). histologically, the changes are characterised by hyperkeratosis, parakeratosis with varying degrees of acanthosis and papillomatosis (greaves and faccini, 1992) . the changes can be florid and it may be difficult to make a clear distinction between severe hyperplasia and neoplasia. nevertheless, it has been shown that the florid hyperplasia of the forestomach epithelium without evidence of cellular atypia can be completely reversible following the withdrawal of an inciting stimulus, ethyl acrylate (ghanayem et al., 1991) . hence, a critical feature may be the presence of cellular atypia in view of its association with agents with potent (genotoxic) carcinogenic activity. neoplasms arising in the forestomach of rodents are usually squamous carcinomas although basaloid features are also seen (fukushima and leiniger and jokinen, 1994; mohr, 1997; tatematsu, 1997) . squamous carcinomas, as at other sites, show variable differentiation being composed of proliferating squamous epithelium with moderate to marked cellular atypia, pleomorphism and mitotic activity with clear evidence of invasion into the muscularis. although they are relatively uncommon spontaneous lesions in aged rodents, they can be induced in rodents by administration of nitroso compounds (tatematsu 1997) as well as a range of non-genotoxic agents (see below). some authors report basal cell carcinoma when basaloid features are pronounced (tatematsu, 1997) . a wide range of agents is capable of producing squamous hyperplasia of the rodent forestomach and a number of these also induce squamous carcinomas. in 1986 kroes and wester reviewed over 60 genotoxic and non-genotoxic compounds that were reported to produce hyperplasia and carcinoma in the forestomach of rats, mice or hamsters. a well-studied example is butylated hydroxyanisole (bha) an important food antioxidant (reviewed by whysner and williams, 1996) . structurally related phenols and acids produce similar changes (rodrigues et al., 1986) . ethyl acrylate, used in the production of materials for dental and medical devices is also capable of inducing marked squamous hyperplasia, papillomas and carcinomas after long-term treatment of f344 rats and b6c3f1 mice (ntp, 1986; ghanayem et al., 1991) . sk&f 93479, an experimental histamine h 2 receptor antagonist produced atypical forestomach hyperplasia in rats following administration by gavage for 1 year by a mechanism which appeared unrelated to the inhibition of the h 2 receptor (betton and salmon, 1984) . other therapeutic agents associated with squamous hyperplasia and neoplasia include the 3-hydroxy-3-methylglutaryl coenzyme a (hmg-coa) reductase inhibitors (kloss et al., 1991; bueld et al., 1996; akiba et al., 1998; physicians' desk reference, 1999) and aristolochic acid (gã¶ggelmann et al., 1982; schmeiser et al., 1988) . some cytoprotective prostaglandins appear capable of inducing hyperkeratosis and hyperplasia without neoplasia presumably through a mechanism related to their pharmacological activity (levin, 1988) . this occurs in rats treated with misprostol, a synthetic prostaglandin e1 methyl ester analogue with gastric anti-secretory and anti-ulcer activity (kotsonis et al., 1985) , cl115,574, a synthetic analogue of prostaglandin e1 type (kramer et al., 1985) and 16,16-dimethyl prostaglandin e2 (reinhart et al., 1983) . even the extensively used antibiotic, ampicillin has been associated inflammation, ulceration with acanthosis and hyperkeratosis in mice but not rats treated for 2 years (national toxicology program technical report, 1987) . sodium saccharin is also reported to produce hyperplasia without neoplasia of the forestomach in f344 rats (hibino et al., 1985) . among its wide range of pharmacological effects on the gastrointestinal tract, the forestomach has also responds to recombinant epidermal growth factor when infused into rats (breider et al., 1996; vinter-jensen, 1999) . histological examination showed hyperkeratosis and hyperplasia of the squamous epithelium. the large body of studies performed with butylated hydroxyanisole illustrates the various factors that can influence the development of treatment-induced hyperplasia and neoplasia of the rodent forestomach and subsequent assessment of human risk. butylated hydroxyanisole possesses little or no mutagenic activity in vitro but when administered to rats for 2 years as a 2% mixture in the diet, it produced squamous hyperplasia, squamous papillomas and squamous carcinomas of the forestomach. at 0.5% in the diet butylated hydroxyanisole induced only hyperplasia . it also produces proliferative lesions in the forestomach of both mouse and hamster (ito et al., 1986) . studies in which butylated hydroxyanisole was fed in the diet to rats for shorter periods have shown that squamous epithelial hyperplasia occurs after only 1 week of treatment preferentially over the lesser curvature, the site at which carcinomas developed in the 2-year studies (altmann et al., 1985) . after 13 weeks' treatment, mucosal hyperplasia characterised by pronounced hyperkeratosis, parakeratosis and acanthosis most pronounced over the lesser curvature, was present in rats given 2% butylated hydroxyanisole in diet but not in rats given 0.5, 0.25 and 0.1% mixtures . abundant mitoses were found in the basal cells layers and tritiated-thymidine labelling confirmed that the changes were accompanied by a high rate of cell proliferation. following cessation of administration of butylated hydroxyanisole after 13 weeks, the tritiatedthymidine labelling index rapidly reverted to control levels within about 1 week although hyperplasia took longer to regress. nearly complete regression of the hyperplasia occurred after about 9 weeks of normal diet . the distribution of squamous hyperplasia induced in the rodent stomach by butylated hydroxyanisole is influenced by the mode of administration. whereas following feeding of rats with butylated hydroxyanisole mixed in the diet lesions tended to be located near the limiting ridge, altmann et al. (1985) showed that gavage of butylated hydroxyanisole in corn oil produced similar changes at the apex of the forestomach. it was suggested that this difference was due to incomplete mixing of butylated hydroxyanisole in the stomach lumen when given by gavage and prolonged contact of the gavage mixture with the upper segment of the forestomach (altmann et al., 1985) . more recently, it was shown that fischer 344, shr, lewis and sprague-dawley rats differ in their response to the hyperplastic and carcinogenic effects of 2% butylated hydroxyanisole in pelleted diet. the most sensitive was the shr strain followed by the f334 rats and the differences correlated with the cytotoxic effects of butylated hydroxyanisole in the different strains (tamano et al., 1998) . it was suggested that the presence of vascular damage in the stomachs of the shr rats might have contributed to the response to cytotoxicity and subsequent carcinogenicity. residence time of administered compounds in the forestomach may influence the development of lesions. although it has been demonstrated that butylated hydroxyanisole does not produce hyperplasia in the oesophagus of animals without a forestomach, have shown that high-doses given to primates are capable of producing an increase in mitotic activity in the lower end of the oesophagus similar to that occurring at equivalent exposure levels in the rat. the implication is that these interspecies differences may simply be a question of differences in exposure of the squamous mucosa to compound. this underlines the fact that mechanisms of action and exposure levels of xenobiotics attained in the gastrointestinal tract of rodent and non-rodent species as well as of man need to be carefully assessed when hyperplastic changes are induced in the forestomach mucosa of rodents. such information can be helpful in facilitating regulatory decisions in this area (moch, 1988) . on balance, the evidence suggests that the tumour development by butylated hydroxyanisole in rodents represents an epigenetic phenomenon related to largely reversible cytotoxicity and increased cell proliferation (whysner and williams, 1996) . in view of the low levels of exposure to butylated hydroxyanisole that occurs with the usual use of this agent, carcinogenic hazard for the human stomach is therefore probably very small. similar phenomena have also been reported in studies of phenols and acids that are structurally related to butylated hydroxyanisole (rodrigues et al., 1986) . these agents include n-butyl and n-propyl-4-hydroxybenzoic acid esters, propionic acid and 4-methoxyphenol. however, these studies suggested that certain areas of the forestomach epithelium react differently to structurally related chemicals, possibly due to the variable levels of activating enzymes within different zones of the forestomach epithelium. co-administration of acetylsalicylic acid was shown to abrogate some of these effects, suggesting that prostaglandin synthetase may be involved in the hyperplastic response (rodrigues et al., 1986) . a number of hmg-coa reductase inhibitors with different chemical structures including marketed products such as lovastatin, simvastatin and fluvastatin are also associated with the development of squamous hyperplasia of the rodent forestomach. the hyperplasia is time and dose dependent and may be associated with oedema and some inflammation of the submucosa. some, but not all of these agents are also capable of producing squamous neoplasia of the forestomach mucosa of rats, or mice or both after long-term treatment (kloss et al., 1991; bueld et al., 1996; akiba et al., 1998; physicians' desk reference, 1999) . the mechanism of action remains unclear although the degree of hyperplasia seems related to pharmacological potency. their carcinogenic potential in rodent bioassays does not seem to relate to the degree of hyperplasia in shortterm studies. moreover, the development of hyperplasia depends on local high concentrations of drug because when administered by non-oral routes, hyperplasia does not occur (kloss et al., 1991) . as most of these drugs are non-mutagenic, these findings are presumably also epigenetic in origin and possess relatively little risk for humans when given in the usual therapeutic doses. a contrasting example is provided by aristolochic acid, a nitrophenanthrene derivative of the ancient medicinal plant aristolochia clematis which was used as an anti-inflammatory component in a number of medicinal preparations in germany until 1982 (gã¶ggelmann et al., 1982; schmeiser et al., 1988) . aristolochic acid is a direct acting mutagen in salmonella typhimurium. when fed to rats at doses of 1.0 and 10 mg/kg/day, aristolochic acid produced severe papillomatosis of the entire forestomach within a period of 3 months. this was characterised histologically by the presence of branched squamous papillomas up to 6 mm high with focal dysplastic features. invasive squamous carcinomas with metastases were found subsequently, 3 or 6 months later without further treatment (mengs et al., 1982) . even at a low dose of 0.1 mg/kg/day papillomas and squamous carcinomas developed 9 months after a 3-month period of treatment. quite clearly the complexity of the hyperplastic response of the rodent stomach to xenobiotics, the association of hyperplasia induced by non-mutagenic compounds with the development of forestomach carcinomas, and the similarity of response in the forestomach to that of the oesophagus, dictates the need for a careful analysis of hyperplasia induced by novel drugs in the forestomach. the prelude to this assessment is careful histopathological characterisation of the changes. unlike the mouth and oesophagus through which tablets, capsules, gavage fluids and drug/diet mixtures pass relatively rapidly, the human stomach mucosa remains in contact with high local concentrations of administered compounds for much longer periods of time. administration of compounds in liquid or solid form, particle size, fasting and feeding all affect the gastric motility pattern. in the fasted state there is a cyclical pattern of motility consisting of three main phases. the first is a quiescent phase, followed by a phase of irregular contractions that increase in amplitude and frequency to reach a maximum in a third phase. feeding results in the replacement of this cyclic pattern by regular tonic contractions that move food towards the antrum and mix it with gastric secretions. these patterns have been well studied in both dog and man and appear to be qualitatively similar in the two species (sarna, 1985) . these motility patterns may have an impact on the length of time drugs remain in contact with stomach mucosa. for instance, the residence time of large non-disintegrating capsules or tablets administered in the fasting state is more dependent on the frequency of powerful phase iii contraction than if drugs are given as fluids or mixed with diet. for dosage forms released in the stomach, gastric residence time will influence drug supply to the main absorptive surfaces in the small intestine, which in turn may affect drug absorption (dressman, 1986) . gastric acid is also important in making ingested salts soluble. although the presence of food in the stomach is a stimulus of acid production, the ph in the forestomach of rats is highest in full stomachs and lowest when empty, presumably as a consequence of the buffering action of food (ward and coates, 1987) . the glandular stomach is conveniently divided into the fundus characterised by mucosal folds or rugae and the smoother antrum, which opens into the pylorus and duodenum. in species devoid of a forestomach, the proximal stomach mucosa or cardia is also lined by glandular mucosa. the glandular mucosa is covered by surface epithelium of regular columnar cells that extends downwards to form small gastric pits or foveolae. the gastric glands are simple tubular structures usually considered to comprise three segments. the base is the deepest part, the neck the mid-region, and the most superficial is the isthmus, continuous with the gastric pit. the upper part of the gastric gland contains mucous neck cells. small cuboidal chief or zymogenic cells, which secrete pepsinogen and stain blue or purple in haematoxylin and eosin sections, are located in deeper parts of the gland. the eosinophilic-staining parietal (oxyntic) cells, which produce hydrochloric acid, are distributed more randomly throughout the gastric glands. parietal cells can also be visualised by immunocytochemical staining with antibodies directed at h + k + -atpase (canfield et al., 1996) . the gastric glands situated near the limiting ridge in rodents, show a modified structure. in species not endowed with a forestomach, the mucosa near the cardia is composed of simplified branched glands lined by columnar epithelium. the antral mucosa is covered by a surface epithelium with gastric pits similar to that of the fundus but mucous secreting columnar glands line the glands. the stomach mucosa is richly endowed with endocrine cells, not all of which have been well characterised. enterochromaffin cells are quite numerous in the basal parts of the gastric glands of the fundus, particularly in the rat . they are generally argyrophilic, staining with silver staining techniques such as that of grimelius (grimelius, 1968; grimelius and willander, 1980 ) that utilise exogenous reducing agents. these cells contain histamine and histamine-related enzymes such as histidine decarboxylase in the rat and other species . endocrine cells which are argentaffin in type stain with silver stains such as that of masson (1914) because of the presence of endogenous reducing substances including 5-hydroxytryptamine and catechol-amines are also reported in the mucosa of the fundus of some species including man but apparently not in the rat . enterochromaffin cells are characterised ultrastructurally by the presence of numerous rounded or oval, vesicular, electron-lucent granules frequently containing a small eccentric electron dense core. gastric enterochromaffin cells can also be stained by immunocytochemical techniques using antisera to histamine and histidine decarboxylase as well as to non-specific enolase and chromogranin a betton et al., 1988) . immunocytochemical study of the rat fundus using a battery of antisera to a variety of gastrointestinal peptides has shown somatostatin containing (d) cells and glucagon staining cells but no cells with gastrin (g cells) or serotonin reactivity (bishop et al., 1986) . gastrin or g cells possess apical processes reaching the stomach lumen believed to be important in stimulation of gastrin release as a result in increases in antral lumen ph or the presence of amino acids or peptides . glucagon and serotonin containing endocrine cells have also been located in the rat antral mucosa (bishop et al., 1986) . increased gastric acid secretion is initiated by activation of central vagal efferent pathways but acid secretion is maintained by both neural and endocrine reflexes activated by the presence of food in the stomach. gastrin secreted from the g cells of antrum is the main stimulant of acid secretion. somatostatin is secreted from antral d cells when the luminal ph falls to below 3.5 to act by a paracrine mechanism to suppress g cell function thus forming a negative feedback loop (dockray, 1999) . the two main endocrine cell types from the body mucosa integrate neurohumoral stimuli rather than respond to luminal chemicals. although gastrin is capable of stimulating parietal cells directly, it has an even greater effect through stimulation of enterochromaffin cells to release histamine, a potent paracrine stimulator of parietal cells (hinkle and samuelson, 1999) . gastrin stimulates release of histamine from enterochromaffin cells of the body mucosa, which increases acid secretion through activation of parietal cell histamine-h 2 receptors. both parietal and enterochromaffin cells are inhibited by somatostatin released from the d cells of the body mucosa in response to a variety of neurohumoral stimuli such as noradrenaline, vasoactive intestinal peptide, calcitonin gene-related peptide, and cholecystokinin. it should also be noted that gastrin has a role in kinetics and differentiation of both parietal and enterochromaffin cells (dockray, 1999) . gastrin acts at the gastrin/cholecystokin-b receptor that is expressed by gastric epithelial cells and by neurones in the central nervous system (kopin et al., 1992; wank, 1995) . the gastrin receptor is simply the cholecystokinin-b receptor located in the stomach. the other cholecystokinin receptor, cholecystokin-a has high affinity for cholecystokinin. stimulation of this receptor in the stomach mediates secretion of pepsin from gastric chief cells and release of somatostatin from d cells resulting in inhibition of acid secretion. in the central nervous system cholecystokinin and its receptors contribute to the regulation of satiety, anxiety, analgesia and dopamine-related behaviour (wank, 1995) . finally, it is worth recording that both progesterone and oestrogen receptors have been identified in both normal and pathological gastric tissues of humans (wu et al., 1992) . generative cells in the gastric mucosa as shown by uptake of tritiated thymidine for dna synthesis are distributed principally in the isthmus (inokuchi et al., 1983) . tracing of cells using thymidine labelling have shown that most of the cells in the generative zone migrate in a successive manner to the mucosal surface to form columnar epithelium. the life span of surface epithelium in the stomach of rats, mice and hamsters has been calculated to be about 3-4 days. studies of cell cycle and dna synthesis time in the proliferative zones in the stomach of rat, hamster and man have suggested that the generative cells in the isthmus undergo mitoses at about 30-hour intervals in rodents and 40-hour intervals in man (inokuchi et al., 1983) . although this process of migration from the proliferating cell zone of the isthmus renews surface epithelial cells rapidly, cell migration to the lower parts of the gastric glands is much slower and more complex. detailed studies have show that undifferentiated cells in the region of the isthmus represent a common source for surface mucous cells and mucous neck cells (karam and leblond, 1995) . electron microscopic and ultrastructural cytochemistry has suggested note: saccharide binding specifications are much more complex than the inhibition by simpler sugars outlined above suggests. see review by nicholson (1974) . *source: nicholson, 1974; goldstein and hayes, 1978; schulte and spicer, 1983; giannasca et al., 1994. that chief cells in the adult rat stomach develop from undifferentiated stem cells in the isthmus (suzuki et al., 1983) . graft experiments in mice have also suggested that immature cells of the isthmus differentiate into chief cells as well as parietal cells (matsuyama and suzuki, 1970) . studies in transgenic mice have shown that mature parietal cells influence the fate of other gastric epithelial cells because targeted degeneration of parietal cells is associated with loss of chief cells suggesting interactions between these cell populations in determining their differentiation (li et al., 1996; canfield et al., 1996) . gastrin is also an important regulator of parietal cell and enterochromaffin differentiation and number (dockray, 1999; montgomery et al., 1999) . labelling experiments in the hamster stomach have shown that both chief and parietal cells possess a similar but quite long life span of about 200 days (hattori, 1974; hattori and fujita, 1976) . it has been suggested that the relative distribution of chief and parietal cells in the gastric gland represents an expression of their different migration patterns downwards from the proliferative zones in the isthmus. this type of migration pattern in which cells are able to overtake each other has been termed a 'stochastic flow system' (inokuchi et al., 1983) . the origin and kinetics of endocrine cells of the stomach has also been the subject of debate but the available morphological, cytochemical and kinetic evidence suggests that the majority of these cells develop from the same stem cells as the other non-endocrine cells of the gastric mucosa, although self replication also occurs (matsuyama and suzuki, 1970; inokuchi et al., 1983; solcia et al., 1986) . much of our knowledge about mucins produced by the epithelial cells lining the gastrointestinal tract has been obtained using histochemical techniques and these approaches continue to be helpful in the understanding of spontaneous and drug-induced gastrointestinal disease (sheahan and jarvis, 1978; filipe, 1979; tsiftsis et al., 1980; jass and roberton, 1994) . for these reasons, mucin histochemical techniques represent useful tools for the characterisation and elucidation of experimentally or drug-induced changes in the glandular mucosa of gastrointestinal tract. techniques commonly employed are presented in table 2 . the physiochemical properties of gastrointestinal mucins are dependent on their glycoprotein constituents. these glycoproteins are high molecular weight compounds with large numbers of sugar chains attached to a polypeptide backbone by o-glycosidic linkages between n-acetylgalactosamine and serine or threonine (berger et al., 1982) . the principle monosaccharides present are fucose, galactose, n-acetylgalactosamine, n-acetylglucosamine and sialic acid. traces of mannose may be present and ester sulphate residues are common (filipe, 1979) . due to this extensive glycosylation, mucins have a filamentous conformation, which is often negatively charged. this is believed to be important in forming a protective barrier to the cell. however, this property is a two-edged sword because when opposing cells have specific receptors for mucins, adhesion may become the predominant factor (van klinken et al., 1995) . although mucins are important in the gastrointestinal tract, it should be remembered that other products secreted by goblet cells might be important in mucosal defence. it has been recently recognised that trefoil proteins, a family of small proteins secreted by goblet cells and present on the mucosal cell surface, can also protect against a variety of deleterious agents, including bacteria, toxins and drugs (podolsky, 1999) . there are considerable regional variations in glycoprotein constituents in the gastrointestinal tract and these differences are probably related to physiological and functional factors. furthermore, synthesis and secretion of glycoproteins alter with changes in cell differentiation. alterations also occur in mucins in various inflammatory and neoplastic disease states as well as following administration of certain drugs and chemicals (ishihara et al., 1984) . terminal sugars or sugar sequences can be demonstrated histochemically by the use of labelled lectins, mostly plant proteins which combine non-enzymatically with particular sugar molecules, see table 1 (goldstein and hayes, 1978; debray et al., 1981; rudiger, 1982) . magenta: all mucosubstances containing hexoses schiff d-pas (pearse, 1968) and deoxyhexoses with vicinal glycol groups. some non-sulphated acid mucosubstances. neutral mucosubstances. periodate-borohydride/ magenta: pas activity following periodate borosaponification/pas, hydride/potassium hydroxide indicates pb/koh/pas presence of o-acylated sialic acids. (reid et al., 1973; periodate borohydride reduces periodate culling et al., 1974; generated aldehydes. potassium hydroxide removes o-acylesters from potential vicinoldiols and sialic residues linked glycosidically to a potential vicinoldiol. alcian blue ph 2.5 basophilia: weakly sulphated mucins. carboxyl (pearse, 1968) groups of sialomucins alcian blue ph 1.0 basophilia: sulphated mucins (lev & spicer, 1964) alcian blue ph 2.5 -magenta: neutral mucins periodic acid schiff, ab/pas basophilia: acid mucins (mowry & morard, 1957) purple-blue: neutral and periodate reactive acid mucins high iron-diamine, brown-black: sulphated mucins hid (spicer, 1965) unstained: sialomucins high iron-diamine-alcian brown-black: sulphated mucins blue ph 2.5, hid/ab basophilia: non-sulphated acid mucins (sialomucins) (spicer, 1965) source: adapted from filipe, 1979. when gastrointestinal mucins were studied in several species using histochemical techniques under uniform conditions, species differences were most obvious in the stomach and duodenum (sheahan and jarvis, 1976) . neutral mucins generally predominate in the stomach, contrasting with acid mucins in the small intestine, and sulphated mucins in the colon. in the stomach neutral mucins staining purple with the pas/alcian blue stain, predominate in the surface and foveolar mucosa, whereas mucous neck cells and antral glands contain acidic mucins that stain blue with pas/alcian blue procedure. sulphated mucins, as shown by the high iron diamine technique (hid) are also found in the deep glandular mucosa of the antrum in rat, mouse and man (filipe, 1979; jass, 1980; greaves and boiziau, 1984) . extremely heterologous staining patterns are seen in the gastric mucosa with labelled lectins, each lectin staining quite different cell populations. there are considerable interspecies differences in staining patterns with the same lectins (kuhlmann et al., 1983; suganuma et al., 1984) . the so-called paradoxical concanavalin a stain, in which conjugated concanavalin a is used to label mucins before and after periodate oxidation, has also been used to classify the alterations in mucins in proliferative and neoplastic conditions of the rat stomach mucosa (kobayasi et al., 1991; tatematsu 1997 ). although gastric erosions and ulcers in the glandular mucosa occur quite commonly in laboratory animals in toxicity studies, it is often difficult to determine whether such lesions in treated animals indicate a real ulcerogenic risk for the test compound. there is little that is histologically specific to drug-induced ulceration of the gastric glandular mucosa. mucosal haemorrhage, depletion of mucin, erosions and ulcers with or without inflammation may all be found. erosions represent mucosal breaks superficial to the muscularis mucosa. ulcers are lesions that extend through the muscularis mucosa. whilst the histopathological features of gastric erosions and ulcers are themselves relatively non-specific, it is important to look for any associated pathology in the stomach such as mucus depletion, epithelial hyperplasia or dysplasia, intestinal metaplasia and vascular lesions (see below). in humans, biopsy data suggests that drug-induced ulceration is characteristically devoid of an inflammatory component, but the most usual histological appearances are those of underlying gastric pathology (riddell, 1982) . formation of gastric and duodenal ulcers is dependent on the presence of both acid and peptic activity in gastric juice because acid without pepsin appears to have little digestive power. important predisposing factors in human patients with peptic ulceration include helicobacter pylori (campylobacter pylori) infection of the antrum, cigarette smoking and ingestion of non-steroidal anti-inflammatory drugs (soll, 1990) . helicobacter pylori is believed to infect over half the human population and its presence in the gastric mucosa is associated with chronic atrophic gastritis and peptic ulceration (cover and blaser, 1996) . it is a microaerophilic, gram-negative organism that possesses potent urease activity crucial for its survival at acidic ph. genome sequence analysis has shown that helicobacter pylori has well developed sequences for motility, scavenging iron and dna restriction and modification systems used by bacteria to degrade foreign dna. the link between helicobacter pylori infection and peptic ulceration is related to increases in gastrin release, perhaps through bacterial products or cytokines released from activated lymphocytes (richter-dahlfors et al., 1998) . although helicobacter pylori can infect other species, apart from non-human primates, the usual laboratory animal models do not appear to develop the inflammatory disease seen in humans (nedrud, 1999) . erosions and ulcers also develop following stress, reflux of intestinal contents and bile, changes in acid secretion and hypoxia, all of which may develop under the conditions occurring in high-dose toxicity studies. the requirement to give the test compound in high doses may also dictate the need to administer exceedingly high concentrations of test agent. this may produce damaging high local concentrations on the mucosa not relevant to therapeutic doses used in clinical practice. it has been demonstrated that hyperosmolar solutions of quite innocuous substances such as glucose can cause haemorrhage, erosions and ulcers of the rat gastric mucosa (puurunen et al., 1980) . the well-known association of gastric erosions and haemorrhage with uraemia may also be manifest following administration of high doses of drugs such as diuretics which severely derange fluid and electrolyte balance (garthoff et al., 1982) . stress ulceration may be linked to temporary ischaemia of the mucosa (dubois, 1987) . synergism between the ulcerogenic action of drugs and stress is a well-described phenomenon (rainsford, 1975; beattie, 1977) . protein depletion and starvation is also capable of inducing gastric ulceration in rats . although gastric pathology represents the largest cause of morbidity and mortality in man following therapy with non-steroidal inflammatory agents (fowler, 1987) , the reasons for this are probably multifactorial. the acidic properties of some of these drugs may cause direct local damage of gastric epithelial cells, demonstrable by the fact that appropriate formulation can reduce gastric toxicity of these agents in man (brors, 1987) . anti-inflammatory agents are capable of decreasing synthesis of glycoproteins and this may adversely influence protective mucus production of gastric mucosa (azuumi et al., 1980; ishihara et al., 1984) . it has also been suggested that non-steroidal anti-inflammatory agents cause cellular damage to the gastric mucosa by back-diffusion of gastric acid into mucosal tissues (davenport, 1964) or by causing damage to the gastric capillary bed with subsequent mucosal infarction (robins, 1980) . the theory that has gained widespread acceptance is that the ulcerogenic potential of non-steroidal anti-inflammatory drugs is related to their pharma-cological activity. vane (1971) proposed that the ulcerogenic potential of these agents was largely a result of their ability to inhibit prostaglandin synthetase, thereby reducing the protective effects of prostaglandins. pharmacokinetic factors may also be important. lipid solubility in the low ph environment of the stomach may influence local penetration into the mucosa (mccormack and brune, 1987) . moreover, it has been proposed that certain anti-inflammatory agents may possess lesser ulcerogenic potential in man because their inhibition of prostaglandin production is more limited to sites of inflammation, sparing gastric mucosa (whittle et al., 1980; whittle and vane, 1984) . it has also been demonstrated that factors altering the enterohepatic circulation of drugs can influence the expression of gastric damage (overvold et al., 1987) . comparative studies of the ulcerogenic activity of indomethacin in beagle dogs and domestic pigs has suggested that the dog may be an excessively sensitive species as a result of extensive enterohepatic circulation of indomethacin in this species (hanhijarvi et al., 1986) . prediction of ulcerogenic potential for man based on data from animal models is clouded by the lack of good comparative data on the relative ulcerogenic potential of non-steroidal anti-inflammatory agents in man because of extensive differences in side effect reporting (fowler, 1987) . moreover proper comparisons in man require not only equivalent therapeutic doses but also comparable dosage forms (brors, 1987) . in laboratory animals, a variety of different patterns of drug-induced gastric damage have been described. the study by shriver et al. (1975) in which a wide variety of different anti-inflammatory drugs were administered to fasted sprague-dawley rats under identical conditions, suggested the drugs could be divided into three groups based on their profiles of gastrointestinal toxicity. immunological agents such as azathiaprine, cyclophosphamide, methotrexate and d-penicillamine produced gastric mucosal haemorrhage whereas aspirin and related agents produced gastric mucosal haemorrhage and ulcers. the powerful nonsteroidal anti-inflammatory drugs indomethacin and phenybutazone produced gastric mucosal erosion and ulcers as well as small intestinal damage. comparative single oral dose studies of several different non-steroidal antiinflammatory agents at three different dose levels by suwa et al. (1987) in the rat using histology and measurement of faecal blood loss with 51 cr-labelled blood cells have also shown that different patterns of ulceration can be produced by different agents when administered under identical conditions. single oral doses of some non-steroidal anti-inflammatory drugs including aspirin produced widespread superficial damage and desquamation of gastric epithelium with little or no inflammation at 6 hours following dosing which completely healed 2 weeks later. this damage was associated with transient faecal blood loss. by contrast, indomethacin and ibuprofen produced both gastric damage and circumscribed, penetrating ulcers along the mesenteric border of the jejunum and ileum. furthermore, ulcers were still present after 2 weeks and were associated with prolonged or biphasic blood loss (see small intestine). in addition feeding conditions can influence the distribution of erosions and ulcers in laboratory animals. in fasted rats, erosions due to indomethacin treatment are found in the body of the stomach whilst in conventionally fed rats they are most prominent in the small intestine. detailed studies by satoh et al. (1981) showed that rats fed for 1 hour after a 24 hour-fast and given a single dose of indomethacin within 2 hours of re-feeding developed erosions and ulcers in the antrum primarily along the lesser curvature. indomethacin given to fasted rats produced erosions in the body mucosa. a further factor that needs to be kept in mind is that chronic administration of ulcerogenic compounds may produce quite different pathological appearances to those found following single dose administration. administration of aspirin to rats for 4 weeks has been shown to stimulate epithelial proliferation of the gastric body but not antral mucosa, possibly by an effect on cyclic adenosine 3',5' monophosphate (cyclic amp) or though increasing the rate of epithelial exfoliation (eastwood and quimby, 1982) . such a response may be the basis for increased resistance of the gastric mucosa to the chronic affects of these agents. it also may explain the tendency for ulcers to occur in the antrum following chronic administration of aspirin-like drugs as the proliferative response and presumably the adaptive potential appears less in this part of the gastric mucosa. both interspecies variations and strain differences have been reported in the response to ulcerogenic compounds. rainsford et al. (1982) showed that extravasation of red blood cells and greater vascular damage was observed in rats treated with aspirin or benoxprofen than in pigs given similar doses. sprague-dawley rats appear less susceptible to the ulcerogenic effects of cold-restraint stress than wistar rats (goldenberg, 1973) . diuretics and some angiotensin converting enzyme (ace) inhibitors and angiotensin ii antagonists have been associated with the development of gastric erosions and ulceration when administered in high doses to laboratory animals ( fig. 46 ) (imai et al., 1981; garthoff et al., 1982) . however, these effects appear related to the severe electrolyte disturbances produced by excessive doses of these drugs. this is perhaps analogous to the well-known association of gastrointestinal tract erosion and haemorrhage with uraemia. dogs appear to have a particular predisposition to this effect where it may be associated with deposition of basophilic ground substance and mineral in connective tissues and blood vessels in the mucosa (barker and van dreumel, 1985) . although inflammatory conditions due to microorganisms are generally uncommon in the stomach, gastritis is reported in laboratory rhesus monkeys in association with the presence of helicobacter organisms (reed and berridge, 1988) . as in the analogous condition in man, the stomach of affected animals shows an infiltration of the central mucosa by small lymphocytes and plasma cells, associated with reactive or atrophic changes in the mucosa and the presence of small curved bacteria in glands, visualised best with the warthin-starry stain. infiltration of the stomach by lymphocytes in rats treated with human recombinant interleukin-2 without ulceration was reported as part of a multisystem involvement induced by this agent (anderson and hayes, 1989 ). decrease in gastric mucus secretion may accompany both spontaneous inflammatory conditions and drug-induced lesions in the stomach of man and experimental animals. mucus depletion is characterised histologically by the presence of an intact epithelial layer in which cells show loss of the normal clear cytoplasm replete with mucous substances by more basophilic cells that contain little or no mucin. qualitative changes in mucus composition can also accompany mucus depletion. gastric epithelium in man may show decreases in sulphated mucosubstances following stress, high alcohol consumption or after aspirin administration (filipe, 1979) . similar changes occur in laboratory animals subjected to ulcerogenic regimens. stress ulceration in the rat is accompanied by decreased sulphation of gastric glycoproteins, presumably an expression of the changes in gastric cellular activity accompanying stress (lambert et al., 1969) . administration of aspirin and other anti-inflammatory agents including adrenocortical steroids to laboratory animals also reduces the content of sulphomucins in the gastric mucosa, probably by reducing their synthesis (denko, 1958; gerard, 1965; ishihara et al., 1984) . rather surprisingly, administration of histamine h 2 -receptor antagonists and fig. 46 . section from the glandular stomach from a rat treated with ah high dose of an angiotensin ii antagonist that shows superficial degeneration and ulceration (erosion) of the mucosa. (he, ã�30.) proton pump inhibitors associated with reduction of gastric acid output and increases in gastrin secretion have also been associated with alterations in gastric mucus. administration of omeprazole or famotidine to rats for 4 weeks was shown to inhibit prostaglandins pge 2 as well as the synthesis of both total and sulphated glycoprotein synthesis along with histochemical evidence of reduction in pas staining of the surface mucus (yoshimura et al., 1996) . although the mechanism for this change is unclear, the reduction in mucus, particularly sulphated mucus that is believed to be particularly resistant to peptic digestion, may have implications for mucosal defence. intestinal metaplasia of the stomach is characterised by the presence of differentiated epithelium, which resembles small intestine on the basis of light microscopic and ultrastructural morphology, mucin patterns and enzyme histochemistry (morson, 1955; planteydt and willighagen, 1960; lev, 1966; goldman and ming, 1968; . it develops in man in gastric mucosa altered by chronic atrophic gastritis and its significance is due to the fact that a link exists between intestinal metaplasia and gastric cancer. although intestinal metaplasia is found much less commonly in laboratory animals, it has also been reported to occur in association with gastric cancer induced by polychlorinated biphenyls (ward, 1985) . in view of this association with gastric cancer, it has been suggested that intestinal metaplasia represents a pre-neoplastic lesion. however, over recent years prospective clinical studies and experimental data have suggested that it is an epiphenomenon, coexisting with, but unrelated to the development of cancer. in man, several forms of intestinal metaplasia have been described. these variants fall into two main groups, an incomplete type and a complete form (teglbjaerg and nielson, 1978; jass and filipe, 1979; jass, 1980) . complete intestinal metaplasia is characterised by the presence of goblet cells, paneth cells and absorptive cells with brush borders and variably developed intestinal villi. incomplete forms are more heterogeneous characterised by goblet and mucous columnar cells but no absorptive cells and variable patterns of mucin. the routine alcian blue: ph 2.5, periodic acid-schiff stain (ab/pas) ( table 2) distinguishes between the intestinal acid mucins (blue) from the neutral mucins of gastric type. however, variable sialomucin and sulphomucin staining patterns are seen in intestinal metaplasia in man with the high iron-diamine/alcian blue stain (hid/ab) (jass, 1980) . the incomplete form of intestinal metaplasia, showing marked sulphomucin secretion, has been found more commonly in association with gastric cancer in man (jass and filipe, 1979; jass, 1980; wells et al., 1982) . however, prospective studies have tended to indicate that intestinal metaplasia with sulphomucin secretion may be an age-related form of chronic atrophic gastritis and not a premalignant lesion (ectors and dixon, 1986) . it has been suggested that intestinal metaplasia represents an adaptive response to long-standing chronic inflammation and reduced acid secretion. it may also represent an adaptive defensive response to long-standing helicobacter pylori infection because intestinal mucosa is more resistant to these organisms (steer, 1984; ectors and dixon, 1986) . intestinal metaplasia has been found in association with gastric cancer in laboratory animals. fischer 344 rats treated with the polychlorinated biphenyl, aroclor 1254, mixed in the diet for 2 years developed foci of intestinal metaplasia in the stomach epithelium in association with gastric adenocarcinomas (ward, 1985) . these lesions were characterised by abundant mucin-containing cells and alkaline phosphatase activity typical of the small intestine (morgan et al., 1981; ward, 1985) . similar, but more diffuse intestinal metaplasia was reported in the stomach of primates treated with polychlorinated biphenyls, although unassociated with gastric neoplasia (allen, 1975; mcconnell et al., 1979) . intestinal metaplasia is also found in the stomach of laboratory animals treated with powerful genotoxic gastric carcinogens. although tsiftsis et al. (1980) showed hyperplasia and foci of atypical changes (dysplasia) but little or no intestinal metaplasia in rats following administration of n-methyl-nâ´-nitro-n-nitroguanidine, tatematsu et al. (1983) were able to show intestinal metaplasia in rats treated with the same agent. however, intestinal metaplasia can be induced in rodents by a variety of different procedures that are not associated with the development of gastric cancer. intestinal metaplasia can be induced in the glandular stomach of rodents by fractionated, localised, ionising radiation (watanabe, 1978; watanabe et al., 1980) , injection of xenogenic stomach antigens as well as propantheline bromide and the non-carcinogen, iodoacetamine shirai et al., 1985) . the characteristics of intestinal metaplasia in laboratory rodents are similar to those seen in man with early increases in intestinal enzyme activity (alkaline phosphatase, lactase, trehalase, sucrose and maltase), development of goblet cells containing neutral, sialo-, or sulphomucins, and intestinal crypts with or without paneth cells. both the fundus and antrum can show changes although as in man, males appear more prone to develop intestinal metaplasia than females . based on these experimental findings, have also proposed that intestinal metaplasia is not a precancerous condition but an adaptive response to a chronic elevation in ph in gastric secretion due to the early loss of parietal cell mass brought about by these various procedures. on balance therefore, the evidence to date suggests that although intestinal metaplasia is associated with cancer and may consequently be considered a helpful morphological feature in the evaluation of human gastric biopsies, the finding of isolated intestinal metaplasia in safety studies does not indicate a preneoplastic state. finally, a form of metaplasia in which hepatocytes have been found has been reported as rare incidental findings in the glandular stomach of mice sacrificed at the end of 2-year carcinogenicity bioassays (leiniger et al., 1990) . histologically, focal accumulation of well-differentiated hepatocytes were found in the submucosa and lamina propria adjacent to the limiting ridge with dilated adjacent gastric glands showing epithelial hyperplasia and mineralisation with herniation into the submusosa. whilst these foci were not believed to be related to treatment with xenobiotics, it is not clear whether represented metaplasia or congenital ectopia. the gastric glandular epithelium is predisposed to the deposition of calcium possibly as it is a site at which marked ion exchange normally takes place. focal aggregates or concretions of densely blue-staining mineral are fairly commonly observed in haematoxylin-stained sections from the stomachs of aged rats where they are associated with cystic dilatation of the gastric glands (greaves and faccini, 1992) . mice and hamsters occasionally show similar changes. small concretions are also observed in gastric glands in the beagle dog. these appear to represent aggregates of calcium around mucoid material. gastric mineralisation may become marked in rodents and dogs when there is disturbance of mineral metabolism, particularly in association with renal pathology. this has been well described in rats with severe renal disease (glomerulosclerosis) and parathyroid hyperplasia (snell, 1967) . a similar phenomenon has been described in the stomach of dogs in uraemic states (cheville, 1979) . identical changes result from the administration of drugs that induce prolonged azotemia or electrolyte disturbances. these changes are characterised by diffuse deposition of mineral in the intestinal tissue of the mucosa of the gastric body but not cardia, antrum or pylorus. mineral deposits develop around basement membranes surrounding epithelium and blood vessels. the lamina propria becomes expanded by oedema and fibroplasia of the interstitium also develops. the gastric glands themselves become distorted with swelling and degeneration of parietal cells and atrophy of chief cells. erosion of the glandular epithelium with haemorrhage occurs presumably as a result of the ischaemia caused by diffuse vascular injury and altered parietal cell function. focal atrophy of the gastric glandular mucosa is a sporadic occurrence in laboratory rodents, usually as a result of previous focal gastric inflammation, ulceration, mineralisation or vascular occlusion. these changes, characterised histologically by focal fibrosis of the mucosa, gastric glandular dilatation and atrophy variably accompanied by polymorphonuclear cells and mast cells are common in certain strains of rats when 2 years or more in age (anver et al., 1982) . whereas diffuse mucosal atrophy occurs following severe inflammatory insult, diffuse atrophy of the stomach glandular mucosa without inflammation can be a result of surgically or drug-induced reduction in trophic factors necessary for the maintenance of normal gastric morphology and function. this is observed in man and experimental animals following antrectomy because this removes the peptide-producing cells of the antrum (gjurldsen et al., 1968; neilsen et al., 1972) . in the rat, antrectomy is accompanied by hypogastrinaemia, reduced weight and height of the oxyntic mucosa and a reduced number of argyrophil cells (hã¥kanson et al., 1976 . this is in contrast to procedures such as antral exclusion that lead to hypergastrinemia and increased thickness of the oxyntic mucosa. mice with genetic deletion of the gastrin gene also show reduction in the thickness of the gastric mucosa. whilst all cell types are present, there is a most pronounced decrease in the numbers of parietal cells as well as enterochromaffin cells associated with an increase in surface mucous cells. these changes are linked to a profound decrease in acid secretion, which becomes unresponsive to histaminergic, cholinergic and gastrinergic stimulation (hinkle and samuelson, 1999) . analogous atrophic changes have been reported following pharmacological removal of trophic stimuli. for instance, administration of the cholecystokinin-b/gastrin receptor antagonist, ci-988 to cynomolgus monkeys for periods of up to 13 weeks was associated with an initial phase of multifocal degeneration of gastric glands primarily in the fundus followed by diffuse reduction in the thickness of the glandular mucosa with little or no qualitative changes to the cell populations (dethloff et al., 1997) . although bilateral vagotomy produces profound functional changes in the stomach, notably reduction of gastric acid secretion, morphological changes in the fundal mucosa are not marked either in experimental animals or in man (crean et al., 1969; aase and roland, 1977) . studies in the rat have shown that diffuse atrophy of the gastric glands characterised by a decrease in the number and size of parietal, chief and mucous cells occurs transiently following truncal vagotomy but histological features return to normal by about 1 month after surgery (nakamura, 1985) . by contrast, unilateral vagotomy in the rat leads to marked and persistent atrophy of the oxyntic zone on the denervated side. this is characterised histologically by reduced height of the mucosa and reduced numbers and staining intensity of argyrophil cells (hakanson et al., 1984) . hã¥kanson and his colleagues argued that this unilateral atrophy was due to the removal of the trophic action of the vagus. the lack of lasting atrophy after bilateral but not unilateral vagotomy was explained by the subsequent rise in gastrin that occurs after bilateral vagotomy as a result of lack of acid feedback inhibition of gastrin release (hã¥kanson et al., 1984) . removal or reduction in extra-gastric trophic factors or hormones may also reduce the thickness of the gastric mucosa. this is has been demonstrated in the rat by hypophysectomy which causes a reduction in thickness of oxyntic and antral mucosa, compared with pair-fed controls. although there was little or no change in peptic:parietal cell ratios, a significant decrease in cell volume and secretory activity of gastric glandular cells were demonstrated which suggested a widespread disturbance of synthesis and secretory mechanisms (bastie et al., 1985) . atrophic changes in the chief cells were observed in rats treated for 6 months with high doses of omeprazole, an inhibitor of acid secretion. the findings were considered to represent disuse atrophy secondary to the inhibition of acid secretion (hansson et al., 1986) . another inhibitor of gastric acid secretion, the tricyclic agent pirenzepin, also produced atrophy of the fundic mucosa of rats following 3 months but not 1 month of treatment (lehy et al., 1978) . the atrophy was characterised by reduction in parietal cell numbers associated with lower numbers of gastrin-containing cells in the antrum, features unlike those following prolonged treatment with histamine h 2 -receptor antagonists. an increase in the thickness of the gastric mucosa can be the result of hypertrophy or hyperplasia of the mucosal cells and this occurs both spontaneously or following administration of drugs and chemicals. in view of the different cell populations in the gastric mucosa and the variety of morphological alterations that occur, it is difficult to make a clear distinction between hypertrophy and hyperplasia without morphometric techniques. morphometric techniques have shown that hypertrophy of some mucosal cells can coexist with hyperplasia of other gastric cell populations. a distinction also needs to be made between diffuse or uniform hyperplasia involving one or more of the cell populations from the hyperplasia associated with proliferative or adenomatous overgrowth. adenomatous hyperplasia also needs to be evaluated for atypical cytological features (dysplasia), which are linked to development of gastric carcinoma (see below). cells of gastric glandular mucosa undergo increases in size or number in response to the effects of gastrointestinal trophic hormones or their synthetic analogues. similar changes also follow administration of compounds that inhibit gastric acid secretion or modify other trophic hormones or growth factors. when gastrin or its synthetic analogue, pentagastrin is administered subcutaneously to rats and mice for several weeks, there is both an increase in the number and size of parietal cells without concomitant increase in zymogenic chief cells (willems and lehy, 1975; crean et al., 1978; balas et al., 1985) . in addition, diffuse hyperplasia of enterochromaffin cells also occurs. by contrast, cholecystokinin, a trophic peptide found in the duodenum and sharing the same c-terminal tetrapeptide sequence as gastrin, increases in the number of chief cells but not parietal cells when administered to mice under similar conditions . drugs which inhibit or neutralise gastric acid secretion such as histamine h 2antagonists, proton pump inhibitors and antacids also induce hypertrophy or hyperplasia of the parietal cell population (witzel et al., 1977; crean et al., 1978; mazzacca et al., 1978; kaduk and hauser, 1980; betton et al., 1988; white et al., 1998) . these agents are associated with a rise in serum gastrin levels, probably as a result of loss of feedback inhibition of low antral ph on gastrin-producing g cells (witzel et al., 1977) . not all histamine h 2 antagonists produce identical effects. other cytological changes have been reported with famotidine, another h 2 -receptor antagonist. this agent produced a dose-related increase in the prevalence and degree of eosinophilic granularity in chief cells of the stomach in toxicity studies in rats but not dogs (burek et al., 1985) . electron microscopy showed an increase in electron density of zymogen granules and it was argued that these effects were the result of secondary inhibition of pepsin secretion or turnover due to inhibition of acid secretion. cytoprotective agents of prostaglandin type produce different forms of diffuse gastric hyperplasia. rats treated with 16,16-dimethyl prostaglandin e2 hourly for 3 weeks, not only developed forestomach alterations (see above) but also thickening of both the body and antral mucosa. in the body mucosa, these changes were the result of a proportional increase in the total mass of surface and foveolar mucous cells, mucous neck cells, chief cells, parietal and endocrine cells as well as connective tissue. this was largely as a result of increase in cell number, although parietal cells also increased in size (reinhart et al., 1983) . unlike treatment with gastrin and gastrin analogues there was an increase in number of surface and foveolar mucous cells associated with increase in mucus content. misprostal, a synthetic prostaglandin e1 methyl ester analogue also produced diffuse glandular hyperplasia, characterised by lengthening of gastric pits and increased mucous secretion in the preclinical safety studies in dogs and rats (kotsonis et al., 1985) . this glandular hyperplasia not only affected the body but also the antral mucosal. studies with tritiated thymidine showed that the labelling index was reduced in rats treated with misprostal, suggesting hyperplasia following administration of prostanoids is a result of an increase in cell survival and decrease in cell shedding rather than an increase in cell proliferation (fich et al., 1988) . levin (1988) has reviewed the effects of prostaglandins of the e series on the gastrointestinal tract of dogs and rodents. a dose-related diffuse hyperplasia of the gastric glandular mucosa has been reported in both rats and cynomolgus monkeys given human recombinant epidermal growth factor. the gastric mucosa was thickened and there was a increase in the number of undifferentiated cells particularly in the neck region and upper part of the gastric glands (breider et al., 1996; reindel et al., 1996) . mitotic figures were also numerous in the upper reaches of the mucosa. the lower parts of the gastric glands were generally less affected. the large increase in the number of undifferentiated cells may have a functional effect on gastric acidity and function (vinter-jensen, 1999) . administration of recombinant growth hormone has also been reported to induce thickening of the gastric glandular mucosa in dog toxicity studies along with typical growth hormone-induced changes in other organs, body weight increases and insulin-like growth factor (prahalada et al., 1998) . the pyloric and fundic mucosa showed histological evidence of hyperplasia of the mucous neck cells. thickening of the gastric glandular mucosa as a result of an irregular proliferation and cystic dilatation of gastric glands associated with inflammation characterises a number of non-neoplastic conditions in the stomach of man and laboratory animals. cystic change with chronic inflammation and foveolar hyperplasia is observed in biopsies taken from the edge of chronic gastric ulcers in man (franzin and novelli, 1981) . mã©nã©trier's disease ('polydadenomes en nappes'), a rare disease found primarily in middle-aged men is also characterised by enlarged gastric folds, foveolar hyperplasia and gastric glandular cystic dilation (berenson et al., 1976; wilkerson et al., 1998) . although its pathogenesis remains elusive, increased expression of transforming growth factor î± (tgfî±) and the epidermal growth factor receptor has been described (demsey et al., 1992) . as tgfî± is an epithelial cell mitogen that inhibits gastric acid secretion and increases gastric mucin, and transgenic mice that overexpress tgfî± in gastric mucosa develop a similar condition (see below), it was suggested by demsey et al. (1992) that tgfî± might have an important role in this condition. similar changes have been observed in animals in association with infestation of the gastrointestinal tract by parasites (jubb and kennedy, 1970; cook et al., 1981) . laboratory rodents may develop a similar pattern of changes spontaneously with advancing age, although the cause of this change remains uncertain. the distinction between adenomatous hyperplasia and adenoma is not clear cut. nevertheless, adenomas are usually defined as localised or focal proliferative lesions with well-ordered glandular patterns with a clear boundary with the surrounding normal mucosa. they are usually exophytic or polypoid in nature but adenomas with localised downward growth are described (mohr, 1997) . proliferation of the gastric glandular mucosa has been well characterised in the laboratory mouse because certain strains have a particular tendency to develop this condition spontaneously with advancing age (stewart and andervont, 1936; rowlatt et al., 1969) . hyperplasia also occurs spontaneously in conventional laboratory strains employed in carcinogenicity bioassays. its prevalence can be influenced by environmental factors such as housing (chvã©doff et al., 1980) , food restriction (rehm et al., 1987) and the administration of xenobiotics (poynter et al., 1985; betton et al., 1987) . similar gastric changes have also been reported to occur in mice thymectomised shortly after birth (suzuki et al., 1981) . histologically, these changes in mice are characterised by hyperplasia of the foveolar and neck regions of the body mucosa (fig. 47) . in advanced cases this is accompanied by elongated, tortuous, or dilated glands lined by simple columnar or cuboidal epithelium, devoid of parietal or chief cells. the abnormal cells show only mild cellular pleomorphism and mitotic activity. the abnormal glands dis-place normal glandular tissue and may penetrate through the muscularis mucosa to reach the muscularis externa and serosa. step sections demonstrate continuity between these glandular elements and a total absence of metastatic spread in the adjacent tissues and lymph nodes. the lamina propria also shows increased amounts of smooth muscle and collagen accompanied by variable numbers of lymphocytes and other chronic inflammatory cells. oedema may be observed and blood vessels are often dilated. the antral mucosa remains relatively unaffected. histochemistry has shown variable mucin secretion of the altered glands. some glands are devoid of mucin, others show an increase in sulphomucin as revealed by the high-iron diamine technique (greaves and boiziau, 1984) . it has been suggested that these features are similar to mã©nã©trier's disease in man and might have a similar pathogenesis (dempsey et al., 1992; takagi et al., 1992) . the aetiology of the spontaneous condition in the mouse is uncertain. the occurrence of similar lesions in thymectomized mice has given rise to the suggestion that autoimmune damage to the gastric mucosa may be responsible (kojima et al., 1980) . the presence of circulating anti-parietal antibodies and the decrease in the number of parietal cells in thymectomized mice suggested that autoimmune damage can occur to parietal cells with compensatory chronic stimulation and proliferation of the generative zones (suzuki et al., 1981) . however, based on findings in female han nmri mice, rehm et al. (1987) showed that this proliferative condition can develop in mice in the absence of antiparietal antibodies. they demonstrated that this change is associated with an increase in the number of antral gastrin cells, raising the possibility of a hormone or paracrine mechanism. a similar proliferative form of gastropathy has been reported in mice which overexpression transforming growth factor î± (tgfî±), a potent mitogen and member of the epidermal growth factor family of peptides. tgfî± acts by binding to and activating the tyrosine kinase of the epidermal growth factor receptor. transgenic mice overexpressing tgfî± develop severe adenomatous and cystic hyperplasia of the gastric glandular mucosa starting from about 2 months of age along with loss of mature parietal cell numbers and a diminution in gastric acid production (takagi et al., 1992) . the degree of change was to some extent dependent on the genetic background on which the transgene operated. an increased prevalence of similar changes have been reported in cd-1 mice treated with the novel histamine h 2 -receptor antagonist sk&f 93479 for 21 months (betton et al., 1987; . although treated mice developed hyperplasia of gastric neuroendocrine cells similar to that observed in rodents treated with other antisecretory agents, they also showed an increase in the severity of glandular hyperplasia. like the spontaneous condition, these changes were characterised by thickening of the mucosa by hyperplasia of the foveolar and neck regions, and downward proliferation of glandular elements into gastric glands (betton et al., 1988) . poynter et al. (1985) have also reported similar glandular hyperplasia in the mouse stomach associated with histamine h 2 -blockade with the agent ioxtidine. these findings were similarly associated with hyperplasia of neuroendocrine cells. adenomatous polyps have also been reported in the pyloric antrum of c57bl/ 10j mice treated for 52 weeks with the synthetic progestin, cyproterone acetate (tucker et al., 1996) . these were single, pedunculated and well-differentiated lesions showing little evidence of dysplasia. the mechanism for the induction of these polyps is unclear although they may have been hormonally mediated as progesterone receptors have been identified in gastric tissue (wu et al., 1992) . although usually less prevalent and less exuberant than in mice, the aged rat also develops proliferative gastric glandular changes spontaneously. these changes are characterised by hyperplasia of the foveolar and mucin-secreting cells of the body mucosa, development of cystic glands lined by simple mucous or flattened cells, accompanied by chronic inflammatory cells, prominent blood vessels and smooth muscle in the lamina propria (greaves and faccini, 1992) . the antrum remains relatively unaffected. proliferative alterations can be induced by administration variety of xenobiotics as well as following surgical procedures that induce chronic reflux of normal intestinal contents. for instance, hyperplasia of the gastric mucosa, notably over the lesser curvature has been described in rats following the so-called bilroth ii gastrectomy that allows reflux of intestinal and biliary secretions into the stomach (kobayashi et al., 1991) . a proliferative condition of the gastric mucosa has been shown to develop following long-term treatment of rats with an ulcerogenic regimen of aspirin. female sprague-dawley rats given 250 mg/kg of aspirin in 1% methylcellulose once daily orally by gavage for 6 months followed for periods of up to 18 months without treatment, developed focal proliferative changes at the sites of healed ulcers, mainly in the mucosa of antrum or antral-body junction (st john et al., 1977) . these lesions were characterised by the presence of proliferating gastric glands lined by columnar, cuboidal or flattened epithelial cells in the mucosa, which also extended through the muscularis mucosa. mucus content of these glands was variable but when present was principally acidic in type, as shown by staining with alcian blue at ph 2.5. the lesions were accompanied by increased collagen in the lamina propria, endarteritis and an infiltration of lymphocytes, plasma cells and mast cells. the lesions were not associated with the development of carcinoma following 18 months' observation and it is probable that they were the result of the chronic damage and repair induced by aspirin treatment. hyperplasia of the gastric glandular mucosa also occurs in rats following the administration of powerful genotoxic carcinogens, although characteristically in association with of atypical histological changes and ultimately carcinoma. these changes have been best characterised in sequential studies with the rat using the carcinogen n-methyl-nâ´-nitro-n-nitrosoguanidine at doses low enough to avoid overt gastric ulceration and regenerative hyperplasia. it was shown that hyperplasia developing under these conditions occurs diffusely both in the body and antral mucosa. furthermore, the changes occurred earlier in the antrum than in the body and were focal or polypoid in character (tsiftsis et al., 1980) . involvement of the antrum in this way is quite unlike the spontaneous hyperplasia of the rat gastric mucosa. histologically, this form of hyperplasia is characterised by lengthening of the foveolae and neck regions both in the antrum and body. hyperplastic pits or foveolae show increased secretion of sialomucins and sulphomucins with a concomitant loss of neutral mucins. polychlorinated biphenyls such as arochlor 1254, which produce intestinal metaplasia and adenocarcinoma in the stomach of rats, also induce proliferative alterations characterised by proliferative cystic lesions in the mucosa associated with inflammation and fibrosis (morgan et al., 1981) . in common with lesions induced by genotoxic agents, these changes are found primarily in the antrum and pyloric regions, zones of predilection for the development of gastric carcinoma in man and experimental animals. it is important to distinguish between the various hyperplastic and adenomatous conditions found in the gastric glandular mucosa in laboratory animals that are not associated with neoplasia from those which precede the development of carcinoma. this distinction is complicated by the fact that proliferative changes associated with the development of cancer both in man and laboratory animals possess features in common with lesions not associated with neoplasia. however, a key distinctive feature is the degree of epithelial dysplasia. dysplasia is considered to be the lesion common to gastric conditions in man such as atrophic gastritis and gastric polyps that have been linked with a significantly increased risk of gastric cancer. although the term dysplasia may be less widely employed in experimental pathology, similar dysplastic changes to those occurring in man have been characterised in laboratory animals in which precancerous gastric lesions have been studied (tsiftsis et al., 1980) . it therefore represents a unifying concept in the assessment of proliferative changes in the gastric glandular mucosa of laboratory animals. as defined by an international group concerned with the diagnosis of preneoplastic conditions in the stomach of man, the principle features of dysplasia are (i) cellular atypia; (ii) abnormal differentiation; and (iii) disorganised mucosal architecture (morson et al., 1980; nagayo, 1981) . cellular atypia is characterised by nuclear pleomorphism, hyperchromasia and stratification of nuclei, increased nuclear-cytoplasmic ratio and loss of cellular and nuclear polarity. abnormal differentiation is shown by reduction or alteration in the normal secretory products of the mucosa. disorganised mucosal architecture is shown by irregularity of crypt structure, back-to-back glands, budding and branching of crypts and intraluminal and surface papillary growths. it is important to assess gastric mucosa very carefully for the features of dysplasia when hyperplastic gastric changes are found in treated animals. in the rat gastric cancer model employing the carcinogen n-methyl-nâ´-nitro-n-nitrosoguanidine, dysplastic changes were shown to start in the proliferating neck region of hyperplastic zones (tsiftsis et al., 1980) . these changes were characterised histologically by irregular growth patterns of glandular cells showing reduced mucin secretion, numerous mitoses and enlarged pleomorphic nuclei. these atypical glands were observed to extend downwards, eventually replacing normal gastric glands and ultimately penetrating the muscularis mucosa forming infiltrating adenocarcinomas of variable differentiation. the antrum developed these changes earlier than the body mucosa (tsiftsis et al., 1980) . these considerations were important in the safety evaluation of the histamine h 2 receptor antagonist, tiotidine (ici 125,211) a guanidino-thiazole derivative that also produced proliferative gastric lesions in the stomach of rats in a 24month carcinogenicity study (streett et al., 1984; . these changes were found mainly in the pyloric region and were characterised histologically by superficial erosions and irregular pyloric glands lined by cells with basophilic cytoplasm and enlarged hyperchromatic nuclei. some atypical glands penetrated the muscularis mucosae. dysplastic lesions situated primarily in the pyloric region were also associated with the development of invasive carcinoma in some rats. extensive histological sectioning of the stomach in rats treated with tiotidine for only 6 months also revealed evidence of early proliferative changes (streett et al., 1988) . therefore, these lesions produced by tiotidine possessed more in common with those induced by powerful carcinogens such as n-methyl-nâ´-nitro-n-nitrosoguanidine than the benign, species-specific proliferative change of little or no relevance for human safety. interestingly, mice treated for 18 months with tiotidine were devoid of dysplastic changes in the gastric mucosa (streett et al., 1988) . one of the most remarkable examples of drug-induced gastric alterations in rodent bioassays is the hyperplasia of enterochromaffin cells and development of carcinoid-like neoplasms in the stomach of rats treated with omeprazole (havu, 1986) . omeprazole is a substituted benzimidazole which inhibits gastric acid secretion by blocking the enzyme h + , k + -atpase, the proton pump of the parietal cells, in a specific and dose-dependent manner (fellenius et al., 1981) . although in rats there is a increase in number of gastric argyophilic cells with increasing age, rats treated with omeprazole for 104 weeks showed a marked, dose-related and diffuse increase of argyophilic, non-argentaffin cells in the basal half of the oxyntic fundal mucosa (havu, 1986) . these changes were more marked in female than in male rats but were not observed in the bioassay in which cd-1 mice were treated with similar doses of omeprazole for 78 weeks. these diffuse changes in the rat stomach were associated with focal hyperplasia of argyrophilic cells. these focal lesions were also associated with a doserelated increase in larger focal nodular lesions of argyrophilic cells, some of which were undoubtedly locally infiltrating carcinoid tumours. these nodular argyrophil lesions posed the usual problems of differential diagnosis of endocrine hyperplasia and neoplasia (see endocrine system, chapter xiii) and distinction of hyperplasia from neoplasia was uncertain. histologically, nodular lesions were composed of multifocal anastomosing solid or pseudoacinar cords of proliferating, regular cells with uniform nuclei and moderately abundant fine granular pale cytoplasm. these nodules showed little or no cellular pleomorphism or mitotic activity but clear evidence of submucosal infiltration without involvement of the muscularis externa was observed in some cases. the overall light microscopic features were similar to those of gastrointestinal carcinoid tumours reported in man. the incidence of gastric carcinoids was reported to be as high as 40% in females in the high dose group but only a few cases observed in similarly treated males (ekman et al., 1985; havu, 1986) . electron microscopy of the altered argyophil cells confirmed the presence of electron-lucent, vesicular granules, frequently with small irregular dense cores characteristic of enterochromaffin cells of the stomach. immunocytochemical study showed that these cells contained histidine decarboxylase which is found normally in gastric enterochromaffin cells which produce and store histamine . other findings reported in rats treated with omeprazole have been a proportional increase in the number and size of non-endocrine cells of the fundus (blom, 1986) , an increase in the number and immunostaining properties of the antral gastrin-containing g cells and hypergastrinaemia (bishop et al., 1986; creutzfeldt et al., 1986) . all functional and morphological changes following treatment for 60 days were fully reversible after 42 days' drug withdrawal (creutzfeldt et al., 1986) . as a result of these treatment-related increases of these normally rare gastric carcinoids in the rat bioassay with omeprazole, clinical trials with this agent were suspended until it was agreed that the endocrine alterations were a result of prolonged drug-induced achlorhydria. it was postulated that omeprazole causes a prolonged inhibition of acid secretion in the rat, which causes activation, and subsequently hyperplasia of antral gastrin cells and marked hypergastrinaemia. hypergastrinaemia in turn stimulates enterochromaffin cells of the fundus, which in time results in enterochromaffin hyperplasia . this argument is supported by the fact that similar morphological findings are reported in chronic atrophic gastritis and other achlorhydric sites in man (solchia et al., 1986; mã¼ller et al., 1987) and that antrectomy in the rat prevents the appearance of enterochromaffin hyperplasia following treatment with omeprazole . although mild dose-related gastric argyrophil cell hyperplasia was noted in dogs treated with omeprazole for 1 year, neoplasms of the stomach were not observed during this time period. why mice neither developed neither argyrophil hyperplasia nor gastric carcinoids with a similar treatment regimen is not clear, as the mechanism of action of omeprazole is similar in rat, dog and mouse. however, as the duration of action of omeprazole is shorter in the mouse, it was postulated that sustained inhibition of gastric acid secretion over 24 hours is necessary to activate increased gastrin secretion from antral cells (havu, 1986) . it has also been suggested that the mouse possesses fewer gastric enterochromaffin cells than the rat and shows a much lower serum gastrin response to omeprazole treatment (ekman et al., 1985) . duration of action or potency may also be the explanation for the lack of reports of carcinoid neoplasms in rats following inhibition of gastric acid secretion by the histamine h 2 -receptor blockers cimetidine and ranitidine. neither of these drugs completely inhibits gastric acid secretion in the rat for 24 hours (leslie and walker, 1977; larsson et al., 1986) , although mild gastric neuroendocrine hyperplasia has been recently described in cimetidine-treated rats (hirth et al., 1988) . however, the long-acting h 2 -receptor antagonist sk&f 93479 produced gastric carcinoid neoplasms when administered at a high dose (1000 mg/kg) to rats for 2 years (figs. 48 and 49) (betton et al., 1987; . although this dose level of sk&f 93479 did not entirely suppress gastric acid secretion and control gastric ph over 24 hours, plasma gastrin levels remained elevated at 3-4 times control values over this period. in a 21-month oral carcinogenicity study in cd-1 mice at the same dose level (1000 mg/kg), a diffuse neuroendocrine cell hyperplasia and multifocal glandular hyperplasia or neoplasia was also observed (betton et al., 1987) . similarly, loxitidine, a potent, non-competitive, insurmountable histamine h 2 -antagonist produced hyperplasia of neuroendocrine cells and carcinoid tumours in the gastric fundus of both rats and mice after 2 years' treatment in diet and drinking water, respectively (poynter et al., 1985 (poynter et al., , 1986 . other histamine antagonists bl-6341 and ici 162846 have been reported to produce neuroendocrine neoplasms in the stomach of rats and rats and mice, respectively (hirth et al., 1988; streett et al., 1988) . wormsley (1984) reviewed the considerations in the risk-benefit analysis of agents intended for long-term administration for peptic disease. drugs of other classes also cause hyperplasia of gastrin-containing cells. immunocytochemical study using antigastrin antibody revealed increased gastrin cell numbers in the antral mucosa of dogs given high doses of adrenocorticosteroids for 4 weeks and these changes were accompanied by enhanced serum and tissue gastrin levels (delaney et al., 1979) . these results suggest that adrenocorticosteroids have a trophic effect on gastrin-containing cells. in human patients hypergastrinaemia is also produced by pharmacologically induced hypochlorhydria although this is usually only slight and hyperplasia of enterochromaffin cells has not been observed (soll, 1990) . a complicating factor in drug safety evaluation is the association of gastric cancer in both man and laboratory animals with n-nitroso compounds. some of the most effective stomach carcinogens in laboratory animals have proved to be nnitroso compounds particularly since sugimura and fujimura (1967) induced gastric adenocarcinomas in rats with n-methyl-nâ´-nitro-n-nitrosoguanidine dissolved in drinking water. furthermore, epidemiological evidence associating nnitroso compounds with human cancer is also fairly strong for the stomach (corea et al., 1975; pocock, 1985) . the formation of n-nitroso compounds is theoretically possible with a variety of compounds that contain amino groups. it has been suggested that the formation of nitrosamines occurs in vivo under the acidic conditions in the stomach following dietary ingestion of nitrite, nitrates and secondary amines (mirvish, 1975 (mirvish, , 1983 . low levels of preformed nitrosamines are also present in some commercial pelleted diets for laboratory animals, principally derived from fishmeal (edwards et al., 1979) . calculations based on dietary intake and nitrosatability of precursors and carcinogenicity of derivatives have suggested that the risk that arises from endogeneous nitrosation is highly variable but highest from ureas and aromatic amines (shephard et al., 1987) . a number of drugs in widespread clinical use have been shown to produce nnitroso products in acidic aqueous media, although the extent to which this occurs in actual therapeutic use is unclear (gillatt et al., 1985) . some clinical evidence suggests that nitrosation of therapeutic agents can occur in clinical practice. for instance piperazine, a cyclic secondary amine, widely used as an antihelmintic drug, has been shown to form small quantities of n-mononitrosopiperazine in the human stomach as measured by gas chromatography-thermal energy analysis (bellander et al., 1985) . however, n-mononitrosopiperazine has not been shown to be carcinogenic in rodents (love et al., 1977) . n,nâ´-dinitrosopiperazine, carcinogenic to the upper gastrointestinal tract in rodents (lijinsky and taylor, 1975) , was not detected in man after administration of piperazine under the same circumstances (bellander et al., 1985) . the possibility of nitrosation is not usually taken into account in the testing of carcinogenic potential of novel drugs as bioassays are usually only performed with parent compound. however, concerns about nitrosation have arisen in subsequent clinical practice. an example of this was the proposal that a few gastric cancers found in patients whilst being treated with the histamine h 2 receptor antagonist cimetidine, were the result of treatment (elder et al., 1979; reed et al., 1979; hawker et al., 1980) . it now seems likely that all those observed cancers associated with cimetidine were incidental (penston and wormsley, 1986) . however, at that time concerns were increased by the theoretical possibility that cimetidine has the potential be nitrosated in vivo (elder et al., 1982) . a further factor was the concept that the treatment-induced gastric secretory inhibition with subsequent bacterial colonisation of the stomach rendered the conditions conducive to the generation of n-nitroso compounds from normal dietary constituents (reed et al., 1981; penston and wormsley, 1986) . these concerns appear to be unfounded. long-term surveillance studies with cimetidine have shown no causal link between its clinical usage and gastric malignancy (colin-jones et al., 1965; langman, 1985) . in addition, carcinogenicity bioassays performed with cimetidine, cimetidine plus nitrite and nitrosocimetidine have not shown any tumorigenic effect in the gastric mucosa (anderson et al., 1985) . a 7-year study in dogs in which multiple gastric biopsies were taken at intervals of approximately 6 months have also shown no indication of gastric hyperplasia, dysplasia, intestinal metaplasia or neoplastic change . although complacency is certainly not warranted with respect to the nitrosation of therapeutic agents in vivo, the risks of development of gastric malignancy from such drugs when administered on a short-term basis are probably very small (world health organisation, 1978) . even for gastric antisecretory agents which may be administered for longer periods of time, the balanced view would also permit development of novel agents provided they are not obviously mutagenic or carcinogenic in the usual preclinical studies and are not particularly liable to undergo rapid nitrosation. most carcinomas of the glandular mucosa are adenocarcinomas, whether induced by the potent genotoxic carcinogens or therapeutic agents. these tend to develop in the antral region in rodents (streett et al., 1984; szentirmay and sugar, 1985) . they range from those with well differentiated tubular or papillary features to poorly differentiated forms with trabecular, mucoid or signet ring features (tatematsu, 1997) . squamous metaplasia within adenocarcinoma can also be observed. stroma may be abundant with pronounced chronic inflammatory infiltration and hyalinisation. metaplastic cartilage and bone has also been described (szentirmay and sugar, 1985; mohr, 1997) . gastric adenocarcinomas induced in dogs by n-methyl-nâ´-nitro-n-nitrosoguamide show similar histological features although their reported distribution in the stomach appears more variable (fujita et al., 1974) . histological criteria for the diagnosis of invasive adenocarcinoma in experimental animals may vary between individual pathologists. some retain the old criteria of stewart and co-workers (1961) who defined invasive cancer as a neoplastic growth reaching the serosa. it is now considered more appropriate to apply criteria of use in human diagnostic pathology (see mohr, 1997) . unequivocal invasion of the submucosa is sufficient evidence of an invasive and therefore malignant process (greaves and faccini, 1992) . the small intestine is of major importance in drug safety evaluation for it represents the primary site of drug absorption. in view of its length and the presence of villi, it possesses an enormous surface area of specialised absorptive epithelium. furthermore, ingested substances have an extended residence time in this part of the gastrointestinal tract. the canine model has been one of the most popular for the study of drug absorption because the dimensions of the canine gastrointestinal tract permit administration of dosage forms intended for clinical use in humans. for this reason, factors, which influence drug absorption, have been better studied in dog and man than many other species. however, data from dog and man not only suggest that diverse factors influence drug absorption from the small intestine but that there are considerable species differences. residence time is of particular importance for drugs, which are incompletely absorbed because differences in mucosal contact time can be expected to result in differences in the fraction absorbed. dressman (1986) has shown using the heidelberg capsule technique that small intestine transit time in dogs is varies from between 15 and over 200 minutes whereas in man equivalent times are between 180 and 300 minutes. these results suggest that absorption of poorly absorbable drugs is likely to be quantitatively less although more variable in dogs than in man. however, these differences do not explain why some poorly lipophilic drugs such as chlorothiazine, acyclovir and phosphalinic acid are more extensively absorbed in dogs than in man. intestinal ph is consistently higher in dogs than in man so that drugs with half maximal absorption ph in the range ph 5-7 may also be expected to be absorbed at different rates in man and dog (dressman, 1986) . physiological and anatomical differences in the small intestine of other test species particularly rodents and humans are also likely to have an impact on drug absorption although many of these factors are still poorly understood. in addition to the small intestine acting as an absorptive surface, it is becoming increasingly obvious that it plays an important part in the metabolism of drugs (breckenridge, 1987) . although monoxygenase activity is relatively low in the gut compared with the liver, conjugation mechanisms are efficient and activity of udp-glucuronosyltransferase and glutathione-s-transferase are as high or even higher than in the liver (hã¤nninen et al., 1987) . in addition, the gastrointestinal microflora not only possesses metabolic capacity itself but also can influence the turnover rate of mucosal cells and subsequent exfoliation and release of enzymes into the lumen (hã¤nninen et al., 1987) . gastrointestinal metabolising activity is important because that the mucosa is exposed to high concentrations of xenobiotics in toxicity studies, and this can influence their overall bioavailability (chhabra and eastin, 1984) . studies in untreated rats have shown that the concentration of total cytochrome p450 in small intestinal microsomes is only about 10% of that found in liver microsomes (bonkovsky et al., 1985) . however, it exists in at least two forms and as in the liver, its activity can be induced by xenobiotics. it has been shown that in the rat, the concentration of cytochrome p450 and drug metabolising enzyme activity increases in intestinal epithelial cells as they move from crypt to villous tips and they are found in greater concentration in the proximal two-thirds of the small intestine than in the distal third (hoensch et al., 1976; bonkovsky et al., 1985) . bonkovsky et al. (1985) also showed that the phenobarbital-inducible form of cytochrome p450 represents less than 5% of total p450 in the small bowel, but as in the liver, phenobarbital treatment can increase this form to about 50% of total cytochrome p450 in small intestine cells. furthermore, it has been shown that drug metabolising activity in the tips of the villi in the duodenum is greater in rats fed a conventional diet than a semisynthetic diet and that the activity depends critically on the absorption of iron from the intestine (hoensch et al., 1976) . glutathione is also present throughout the entire mucosa, although in rats, cells at the tips of the villi contain less than cells located more basally, whereas related enzymes î³-glutamyl transpeptidase and glutathione-s-transferase show highest activity in the villous tip region (ogasawara et al., 1985) . the fact that these enzyme activities are highest in the duodenum and lowest in the terminal ileum suggests that detoxification systems for exogenous compounds are greater in the proximal small intestine. the small intestinal mucosa is constructed not only to act as an absorptive surface but also as a barrier to potentially pathogenic substances and microorganisms. although the main cell population of the epithelium is composed of absorptive cells, other major epithelial cell types, the mucous (goblet) cells, paneth cells and endocrine cells have important protective functions. in addition, specialised epithelial cells, the microfold (membranous or m) cells are located in the epithelium over peyer's patches. these cells form part of the other important protective system of the intestine, the gut associated lymphoid tissue (galt) or mucosal associated lymphoid tissue (malt). the mucosal lining is in a constant state of renewal. enteric epithelium possesses the fastest rate of turnover of any tissue exceeded only by a few rapidly growing neoplasms . in normal circumstances, the constant turnover of small bowel mucosa is maintained by equilibrium between cell production in the crypts and cell loss at the tips of the villi. there are intrinsic controls within the mucosa itself. exogeneous substances, intraluminal secretions, mechanical and neural factors as well as alterations in blood flow all possess potential to influence mucosal cell kinetics . all main epithelial cell types are believed to arise from undifferentiation columnar cells at the crypt base (cheng and leblond, 1974) , although mucous cells may also arise by proliferation of partly differentiated mucous cells in the crypts. as cell division is limited to crypts, the cell population in the crypts have high activities of enzymes such as thymidine kinase that are involved in nucleic acid synthesis (imondi et al., 1969) . the complete cell cycle lasts about 10-17 hours in rodents and at least 24 hours in man. enteric epithelium is completely replaced within 2-3 days in mice and rats and within 3-6 days in man . after two or more divisions in the crypt cells migrate to the villus, lose ability to incorporate thymidine and differentiate into mature cells equipped with enzymes associated with nutrient absorption (imondi et al., 1969) . cell migration in the rat is completed more rapidly in the ileum than in the jejunum principally as a result of the lower villous height in the ileal mucosa (altman and enesco, 1967). migration terminates by loss of cells from the tip of the villi. surrounding the crypt is a sheath of fibroblastic cells. these cells also undergo synchronous division and migration with the epithelial cells, maintaining the intimate relationship between the epithelium and supporting tissues (parker et al., 1974) . mature absorptive cells are important in the active and passive transport of nutrients as well as in the endocytosis of macromolecules. they are characterised by the presence of a striated or brush border which is seen in haematoxylin and eosin-stained sections as a refractile bi-laminar band. the inner, wider lamina corresponds to the microvillous region that is associated with the presence of neutral mucosubstances in most species. the outer, thinner band corresponds to the glycocalyx, which is composed principally of acidic mucosubstances (sheahan and jarvis, 1976) . this outer band of the brush border shows histochemical staining predominantly for sulphomucins in most species including mouse, hamster, dog and rhesus monkey, although in the duodenum of the rats and in the entire human small bowel sialomucins predominate in this layer. electron microscopy of the absorptive cells shows that the surface of absorptive cells is covered by tightly packed and well developed microvilli approximately 1 âµm long and 0.1 âµm wide. these are considered the first site of entry of food substances into the cell. the plasma membrane of microvilli is associated with fine filamentous projections, which probably represent the polysaccharide chains of the glycocalyx (bennett, 1969) . as the glycocalyx is composed of a network of polysaccharides, it has been suggested that it may behave like an ionexchange resin, be able to bind certain lectin-like molecules or trap substances in its matrix so providing a site for efficient intraluminal digestion (bennett, 1969; goldberg et al., 1969; king et al., 1986 ). the plasma membrane shows a trilamina structure at ultrastructural level. freeze fracture replicas from the microvilli which cleave this membrane through the plane of apposed non-polar groups of the lipid bilayer, demonstrate smooth complementary surfaces studded with small particles. these particles represent integral globular proteins of the plasma membrane. some of these intramembranous particles, mostly those of 10 nm diameter show irregular outlines with a central pit and are believed to represent gap junctions or transport channels (yamamoto, 1982) . a particularly important aspect of the absorptive cell membrane is its high concentration of disaccharidases such as sucrase, maltase and lactase, related to the absorption of sugars. alkaline phosphatase activity is also abundant on the surface of absorptive cells, although its precise role here uncertain (owen and bhalla, 1983) . immunocytochemical demonstration of alkaline phosphatase provides a useful tool to examine the effects of xenobiotics on intrinsic membrane glycoproteins in the small intestine (hasegawa et al., 1987) . enterokinase, the glycoprotein enzyme, which initiates the activation of pancreatic zymogens by converting trypsinogen to trypsin, is also present in the brush border and glycocalyx of the small intestinal epithelium, both in man and animals. immunocytochemical studies have demonstrated that in man this enzyme is located in the duodenum and proximal jejunum but not ileum, colon and stomach (hermon-taylor et al., 1977) . these enzymes constitute integral structural proteins of the cell membrane with active sites protruding from the cell surface. they are synthesised by the absorptive cells (blok et al., 1984) . the lateral surfaces of absorptive cells are in direct contact with neighbouring cells and firmly attached to each other by terminal bars or junctional complexes. a terminal bar comprises an apically situated tight junction or zonula accludens, a central zone, the zonula adherens, below which is situated a desmosome or macula adherens. the junctional complexes are relatively impermeable to macromolecules. studies with labelled tracers in the rat jejunum have shown that horseradish peroxidase (molecular weight 40,000, diameter 5 nm) and ferri-tin (molecular weight 100,000, diameter 10 nm) do not penetrate junctional complexes (yamamoto, 1982) . below the junctional complexes, the cell membranes interdigitate and the intercellular space widens towards the cell base, a feature that may be important in the movement of electrolytes and water across the intestinal epithelium (rhodin, 1974) . the cytoplasm of absorptive cells contains smooth and granular endoplasmic reticulum, free ribosomes and mitochondria. the golgi apparatus is located above the nucleus. the apical part of the cytoplasm is devoid of organelles except for a tight meshwork of filaments called the terminal web. filaments of actin within the microvilli are linked to the terminal web and this is believed to be important in the movement of microvilli (moosker and tilney, 1975; moosker et al., 1978) . goblet cells are much fewer in number than absorptive cells in the small intestine but they increase in number from the duodenum to the lower ileum. they are important in the production of mucus, which remains on the surface of the mucosa as a viscous layer and acts as the first line of defence against intestinal pathogens. goblet cells are characterised by the presence of abundant mucous droplets formed by the golgi complex and which accumulate in the apical part of the cell cytoplasm. histochemical study shows that neutral mucosubstances are present in the goblet cells found in crypts and on the villi in the entire small bowel mucosa of most species including man but there is an interspecies variation in the population of sialo-and sulphomucins (sheahan and jarvis, 1976) . in the mouse, sulphomucins predominate but among rats considerable individual variation in the proportion of sialo-and sulphomucins is reported. in the hamster, sulphomucins are more prominent in the proximal and sialomucins in the distal small bowel. in the dog, both sulphomucins and sialomucins are found with predominance of one or other in individual animals. staining for acidic mucins is less intense in the goblet cells of the small intestine in man compared with non-human primates but sialomucins are predominant in both species. a few goblet cells in the distal ileum in man also contain sulphated mucins (sheahan and jarvis, 1976) . paneth cells remain located near the crypt base throughout the small intestine (sandow and whitehead, 1979) . they are found in rodents and man but typically not in carnivores such as dog and cat (rhodin, 1974; . they are characterised by the presence of numerous eosinophilic cytoplasmic secretory granules between about 1.0 and 2 âµm diameter that contain various enzymes and mucosubstances. particular care is needed in fixation and staining for optimal demonstration of paneth cells for they rapidly degranulate after death and granules are destroyed by acetic acid fixation. formalin and mercuric fixatives appear appropriate methods and they permit staining with methylene blue, lendrum's phloxine-tartrazine and masson's trichrome (lewin, 1969) . the apical parts of paneth cells show glucose-6-phosphatase, carbonic anhydrase and monoamine oxidase activity and they have been shown to contain lysozyme and immunoglobulins, particularly iga (speece, 1964; riecken and pearse, 1966; ghoos and vantrappen, 1971; . staining for lysozyme appears to be the most practical immunocytochemical stain for the detection of paneth cells in formalin fixed paraffin wax embedded tissue sections. however, other immunocytochemical reagents have been employed in human and rodent tissues. these include antibodies to the cd15 antigen (ariza et al., 1996) and to a rat paneth cell zinc binding protein (sawada et al., 1994) as well as the use of pokeweed lectin for murine paneth cells (evans et al., 1994) . the paneth cell granules not only contain lysozyme but also a range of antimicrobial peptides such as secretory phospholipid a 2 , î±-defensins, also called cryptins. these are believed not only to possess antimicrobial activity but also important in the regulation of cell volume, chemotaxis, mitogenesis and inhibition of natural killer cell activity (ouellette, 1997) . it has been shown that these substances are released from paneth cells when germ free rats are dosed with the intestinal flora from specific pathogen free rats (satoh and vollrath, 1986; . however, it has been shown that paneth cells develop under germ free conditions and do not require luminal bacteria or dietary material for their development (ouellette, 1999) . endocrine cells are also scattered throughout the small intestinal mucosa. they are of both argentaffin and argyrophil types and are situated predominately in crypts. immunocytochemical study shows that they contain a variety of different peptides although gastrin, secretion and serotonin-containing cells have been those most extensively studied (inokuchi et al., 1983) . in addition to the barrier formed by mucus and epithelial cells, lymphocytes, plasma cells, macrophages, dendritic cells and mast cells also form part of the protective function of the small intestine. some lymphocytes are located within the epithelium mostly, above the basal lamina but below epithelial nuclei (pabst, 1987) . these lymphocytes are termed intraepithelial lymphocytes and are predominantly of t-suppresser/cytotoxic type in man and laboratory animals (selby, 1981; martin et al., 1986; pabst, 1987) . most lymphocytes in the lamina propria are also t cells but t-helper (cd4 positive) cells outnumber the t-suppresser/cytotoxic or cd8 positive phenotype (hirata et al., 1986; pabst, 1987; bruder et al., 1999) . intraepithelial lymphocytes are also mainly t cells (bruder et al., 1999) . many plasma cells present in the lamina propria produce iga, the major immunoglobulin of mucosal secretions (michalek et al., 1975) . iga represents another important component of the mucosal barrier between the gastrointestinal mucosa and intraluminal antigens. the main function of iga is to effect immune exclusion by intimate co-operation with non-specific defence mechanisms (brandtzaeg et al., 1985) . plasma cells in the lamina propria produce dimeric iga with two dimeric molecules joined by a joint (j) piece. a secretory component (sc), a glycoprotein expressed on the basolateral surface of epithelial cells acts as a receptor for dimeric iga and as a transport system for iga to the gut lumen where monomeric iga is secreted (brandtzaeg et al., 1985) . morphometric analysis of iga-containing immunocytes in the rat ileal mucosa using immunocytochemical staining has shown that the number of these cells varies with alterations in the microbiological status of intestinal contents (rodning et al., 1983) . a significant reduction in iga-containing lymphocytes and plasma cells was observed following microbial reduction associated with gnotobiosis, probably reflecting decreased microbial antigenic stimulation. experimental studies using labelled mesenteric lymphocytes also suggests that local microenvironments are important in the distribution of there cells in the intestinal wall (mcdermott et al., 1985) . peyer's patches are the most prominent aggregates of lymphoid tissue in the gastrointestinal tract and constitute important sites at which antigens from the gut lumen encounter immune competent cells which are responsible for the initiation of immune responses. peyer's patches are located on the ante-mesenteric wall of the small bowel and consist principally of collections of lymphoid follicles. in man, peyer's patches are more common in the ileum (cornes, 1965) but in mice they are more uniformly distributed (owen and neumanic, 1978) . in rats they are also more numerous in the distal than in the proximal small intestine and the number of follicles in patches usually varies from 2 to 6 but sometimes many more may be seen in any particular section (martin et al., 1986) . peyer's patches vary between rat strains. a comparative study showed that in fischer 344 rats they are smaller than those in wistar rats (bruder et al., 1999) . particular care in selection and orientation of tissue blocks is therefore essential for any form of quantitative or semiquantitative assessment of peyer's patches. peyer's patches are more than simple aggregates of lymphoid follicles. they consist of lymphoid follicles surrounded by a corona of small lymphocytes principally of b-cell type. the interfollicular area contains post-capillary venules with specialised cobblestone type epithelium (yamaguchi and schoefl, 1983 ) and many t lymphocytes. beneath the epithelium, over the bulging follicles (dome area), mixtures of t and b lymphocytes, plasma cells and macrophages can be seen (pabst, 1987) . immunohistochemical study in rats demonstrates the presence of w3/13-positive t (cd43) cells in the interfollicular area. in the rat, many cells with macrophage morphology also stain with the w3/25 (cd4) monoclonal antibody in the interfollicular zone (bland and warren, 1985; martin et al., 1986) . immunocytochemical study of the peyer's patches in the mouse has also shown considerable heterogeneity of staining patterns, particularly under the dome epithelium (ermak and owen, 1986) . similar patterns have been observed following immunohistochemical study of peyer's patches in man (spencer et al., 1986) . the epithelium overlying the peyer's patch follicles (dome area) contains specialised epithelial cells, called microfold, membranous or simply m cells. these cells have been identified in many species including rats, mice, hamsters, dogs, monkeys and man (owen and bhalla, 1983; wolf and bye, 1984) . these cells differ functionally from other enterocytes by their ability to transport large molecules such as ferritin and horseradish peroxidase and particulate matter from the lumen to the underlying lymphoid tissue (owen, 1977; jeurissen et al., 1985) . they have also been shown to be the site of penetration of reoviruses into the epithelium and they can transport vibrio cholerae and other organisms (wolf and bye, 1974; smith et al., 1987) . m cells therefore form weak points in the intestinal wall which transport intact antigen and macromolecules to the follicles where they can be processed and be transported to lymph nodes with consequent iga immune responses. this contrasts with the uptake of soluble antigens, which can be taken up by ordinary epithelial cells and transported in the circulation of the villi to be ultimately trapped in the spleen possibly to evoke an igm/ igg response (jeurissen et al., 1985) . understanding of these cellular and molecular characteristics is critical for the design of mucosal vaccines for pathogens that exploit this pathway (neutra, 1998) . no simple, specific histological markers for m cells have been identified for use in routine histological sections although many specialist techniques can be applied (reviewed by gebert et al., 1996) . this has hampered their study because they are still best identified on the basis of their ultrastructural characteristics or ability to capture and transport macromolecules (hamzaoui and pringault, 1998) . the m cell shares tight junctions and desmosomes with adjacent epithelial cells but it has fewer and shorter microvilli than absorptive cells. there is lack of a well organised terminal web. vesicles are abundant in the apical cytoplasm but lysosomes are reduced in number. attenuated cytoplasmic processes may be seen embracing lymphocytes (owen and nemanic, 1978; wolf and bye, 1984) . a cardinal feature is the presence of a intraepithelial pocket on the basal membrane which acts as an internal docking site for lymphocytes, such that they are filled by b and cd4 positive lymphocytes, macrophages and dendritic cells that move between underlying follicles and the epithelium (ermak et al., 1990; farstad et al., 1994) . with care, these attenuated microvilli can be seen at light microscopy in semithin plastic sections (wolf and bye, 1984) . cytochemical analysis has also demonstrated that they can be distinguished at light microscopic level by markedly reduced alkaline phosphatase activity on their terminal surface, in contrast to the dense reaction product produced by other enterocytes (owen and bhalla, 1983) . as the glycoproteins on the surface of m cells are different to those on surrounding cells, they can be selectively stained by labelled lectins, although these patterns are different in different species. for instance, mouse m cells are labelled by the fucose-specific probe ulex europaeus and to some extent anguilla anguilla (giannasca et al., 1994) . mucosal mast cells also appear to be involved in the immunological defence of the gastrointestinal tract. they respond by proliferation, migration and discharge of granules during nematode infestations (miller, 1980) . it has been shown in the rat that mucosal mast cells of the gut differ in several ways from connective tissue mast cells. these differences result in poor preservation of mast cells of the gut if the usual metachromatic staining techniques employed for the demonstration of mast cells in tissue sections (wingren and enerbã¤ck, 1983) . histochemical study suggests that mucosal mast cells differ from connective tissue mast cells by a lower degree of sulphation of glycosaminoglycans and different spatial relationships of protein and glycosaminoglycans in their granules. these cross link following formalin fixation in a way, which is sufficient to prohibit cationic dye binding. wingren and enerbã¤ck (1983) showed that these staining difficulties can be surmounted in tissues fixed in formaldehyde by staining in toluidine blue for prolonged periods of time (5-7 days), a procedure which allows adequate penetration of the toluidine blue molecule. optimal histopathological study of the small intestine is complicated by its length and mucosal fragility. it is important to avoid vigorous washing procedures or any form of excessive manipulation of the unfixed bowel, as artefact caused by washing may confound interpretation of changes induced by xenobiotics (roe, 1984) . combination of artefact due to washing, autolysis and the presence of neutrophils can produce a histological appearance that mimics in vivo damage. although careful visual inspection of the intestine and sampling of appropriate segments for histological examination is usually sufficient for routine examination, various forms of 'swiss roll' techniques are helpful for more complete study. rolling the unfixed, opened rodent intestine around a wooden stick prior to freezing or fixation is one proposed method (moolenbeck and ruitenberg, 1981) , although this method risks undue manipulation of the unfixed tissue. another more versatile technique applicable to rodent, large animal and human intestine can be performed after fixation. the unfixed opened bowel is pinned flat on a cork or board and fixed in a bath of formal saline. after fixation, the full thickness of rodent intestine can be rolled, transfixed by a pin and embedded in paraffin wax. likewise the mucosa of the intestine of large animal species or humans can be rolled after fixation by separating it from the muscularis externa (filipe and branfoot, 1974) . inflammation and ulceration of the mucosa occurs as a result of stress, infection with bacteria, viruses, and infestation by parasites or as a direct result of the effects of xenobiotics or ionising radiation. antimitotic or radiomimetic agents as well as ionising radiation are liable to adversely affect the rapidly dividing cells of the small intestine with resulting breakdown of the mucosal barrier. the ulcerogenic activity of non-steroidal anti-inflammatory drugs may also be expressed in the small bowel mucosa. different agents also act in synergy to enhance damage to the small bowel mucosa. an important example is the effect of drugs that depress the immune system and permit the development of pathological infections by microorganisms of the opportunistic type in the small intestine. the histological features of the inflammatory process in the small intestine are not usually specific for a particular agent. it is important to search for evi-dence of microbiological organisms and viral inclusions, which can indicate the cause of intestinal inflammation and ulceration. associated features in non-ulcerated mucosa such as morphology of the villi, accumulation of abnormal cells or foreign substances and changes in lymphoid cells or blood vessels are also important in the assessment of these changes. a number of organisms including those, which are normal residents of the gastrointestinal tract, can cause inflammatory changes in the intestinal mucosa of laboratory animals. with the notable exception of non-human primates, inflammatory bowel disease caused by microbiological organisms is not usually evident or of concern in most toxicity or carcinogenicity studies. however, when animals are treated with antibiotics, immunosuppressive agents or other drugs, which alter the normal intestinal flora, conditions may favour the proliferation of potentially pathogenic organisms in sufficient quantities to cause overt damage to the mucosa. certain bacterial flora may also act synergistically with intestinal protozoans to produce pathological changes (boorman et al., 1973) . in non-human primate colonies, gastrointestinal disease remains one of the most important causes of death . in contrast to other laboratory species, histological evidence of intestinal infectious disease is relatively common and may confound the interpretation of gastrointestinal alterations occurring in toxicity studies. although the majority of potentially pathogenic organisms affect the primate colon, a number of bacteria, protozoa and metazoa occur in the small intestine. a detailed review of the protozoa and metazoa occurring in the primate gastrointestinal tract has been published (toft, 1982) . owen (1993) has reviewed the parasites of laboratory animals including those of the gastrointestinal tract and the principle reference for identification of metazoa in tissue sections remains that of chitwood and lichtenfels (1973) . organisms which can cause inflammatory disease in the small bowel but which are primarily agents that produce inflammatory disease in large bowel are reviewed under 'colon' (see below). bacillus piliformis is the agent responsible for tyzzer's disease produces intestinal inflammation and ulcers in rats, mice and hamsters. susceptibility of different species and strains to experimental infection with bacillus piliformis is variable. for instance, c57bl, balb mice and f344 rats appear more resistant to infection than outbred syrian hamsters (waggie et al., 1987) . lesions of variable severity usually occur in the ileum but may also extend into the caecum and colon. severe infections are characterised histologically by ulceration of the mucosa, oedema and acute inflammation of the submucosa and muscle coats. muscle may also show focal necrosis. non-ulcerated mucosa is typically infiltrated by polymorphonuclear cells and crypt abscesses form. there is blunting and fusion of villi and reactive hyperplasia and mucin depletion of the overlying epithelium . mucosal lymphoid tissue may also show reactive changes or hyperplasia. filamentous bundles of bacillus piliformis can usually be found in the cytoplasm of both epithelial cells and smooth muscle cells at the edges of necrotic zones. methylene blue, giemsa or silver impregnation techniques such as warthin-starry or levaditi stains are the best stains for the demonstration of these organisms although with care they can be visualised in haematoxylin and eosin stained sections (weisbroth, 1979) . they are gram negative and pas positive. intestinal infections due to salmonella species are relatively common in the mouse but also occur in the hamster and rat . salmonella typhimurium and salmonella enteritidis are regarded as the organisms typical of murine salmonellosis (weisbroth, 1979) . lesions occur in the ileum and may extend into the jejunum and caecum. they are characterised by the presence of ulcers covered by fibrinous exudate and associated with diffuse infiltration of the adjacent mucosa by macrophages, neutrophils and lymphocytes. intact crypt epithelium shows mucin loss and reactive proliferative changes. a characteristic feature is the presence of poorly defined granulomatous lesions composed of macrophages mainly in associated lymphoid tissue or peyer's patches. clostridia species, especially clostridia difficile which cause a pseudomembranous colitis in man and laboratory animals (especially hamsters) may also produce inflammation and ulceration in the terminal ileum with histological features similar to those found in the colon (see following). proliferative ileitis (transmissible ileal hyperplasia) is a striking lesion of hamsters affecting the distal segment of the ileum that is associated with intracellular invasion of the intestine mucosa epithelium by bacteria. the organism has not been cultivated, but has been suggested that it is a campylobacter (jacoby, 1985) although other organisms have been identified in this condition (fox et al., 1993; peace et al., 1994) . although it is characterised by hyperplasia of the ileal mucosa in its early stages, an inflammatory phase intervenes in which there is focal necrosis and haemorrhage of the mucosa, crypt abscesses and infiltration of the lamina propria by acute inflammatory cells and macrophages. the histological features of the associated hyperplasia are characteristic. the mucosa is covered by immature, mucin-depleted pseudostratified hyperchromatic epithelium with mitoses extending to the tips of villi and densely basophilic intracytoplasmic inclusions (jacoby, 1985) . helicobacter jejuni (campylobacter jejuni) is a common cause of diarrhoea in man and may be the causative agent in small intestinal inflammation in laboratory dogs and primates. campylobacter species may be more prevalent in beagle dogs and primates than commonly appreciated. it is important to recognise that dogs colonised with these agents may be susceptible to stress-induced, acute onset gastroenteritis (fox et al., 1988; reed and berridge, 1988) . in man this form of bacterial disease is characterised histologically by mucin-depletion, flat-tening and reactive changes in the small bowel epithelium, crypt abscesses, oedema and infiltration of the mucosa by neutrophils, lymphocytes and plasma cells. similar histological findings have been reported in dogs infected with this organism (prescott and monroe, 1982) . the organisms are gram-negative curved, slender rods, which can be visualised in tissue sections with the warthin-starry stain, a recognised technique for spiral bacteria. the carbol fuchsin technique of gimenez (1964) , first used for the identification of ricketsiae in yolk sac culture and a cresyl fast violet technique are also useful methods for the identification of campylobacter species in paraffin sections (burnett et al., 1987; mcmullen et al., 1987) . another campylobacter-like organism, helicobacter pylori (campylobacter pylori) has been identified in human patients with gastritis, gastric and duodenal ulcers. it is an aetiological or predisposing factor in these forms of gastrointestinal disease (marshall and warren, 1984; rouvroy et al., 1987; richter-dahlfors et al., 1998) . spironucleus muris (hexamitis muris) is also a cause of inflammation in the small bowel of rats, mice and hamsters. during overt infestation, organisms are seen extracellularly in crypts and intervillous spaces associated with blunting of intestinal villi, epithelial degeneration and mucin-depletion, reactive epithelial hyperplasia, oedema and leucocyte infiltration (boorman et al., 1973; wagner et al., 1974) . the morphological expression of damage is accompanied by decreased levels of disaccharidases such as maltase, sucrase and lactase, which may represent a direct effect of the trophozoites on the brush border enzymes (gillon et al., 1982) . trophozoites are characteristically elongated symmetrical flagellates approximately 2-5 âµm wide, 12-20 âµm long. giardia species represent marginally pathogenic flagellates, which are found in the upper gastrointestinal tract. they are opportunistic agents that can become important in both animals and man with depressed immune function. studies in mice infected experimentally with giardia muris have shown that an early response is an increased infiltration of the epithelium by lymphocytes, predominantly t cells (gillon et al., 1982) . this has led to the suggestion that the response to infection by these parasite is primarily a cell-mediated immune reaction similar to experimental graft versus host reaction in the small intestine of mice (mowat and furguson, 1981; gillon et al., 1982) . depression of the immune response by treatment with corticosteroids has been shown not only to increase parasite numbers in murine giardiasis but also cause recrudescence of occult infections (nair et al., 1981) . it has also been suggested that decreased gastric acidity can predispose to giardiasis in man (nalin et al., 1978) . giardia muris (lamblia muris) is sometimes found in the small intestine of rat and mouse but it is common in the hamster (fig. 50) . trophozoites appear in histological sections as crescent-shaped structures on the brush border of the intestinal mucosa or in the adjacent lumen. mucosal lesions may be totally ab-sent or there may be blunting of villi, reactive epithelial hyperplasia. a typical feature is increased infiltration of the epithelium and lamina propria by mononuclear cells (boorman et al., 1973) . another important finding is that lactase, sucrase and maltase levels have been shown to decrease in the small intestine in mice infested with giardia muris (gillon et al., 1982) . giardia lamblia may colonise the small intestine of non-human primates and of man and produce similar morphological appearances to those found with infestations in rodents by giardia muris. other flagellates such as tritrichomonas muris are also be found in the small intestine of mice, rats and hamsters. the coccidian protozoan parasite cryptosporidium represents a striking example of the close relationship between some human and animal diseases. this organism was first recognised in the gastric glands of mice by tyzzer in 1907 and has since been confirmed as a cause of diarrhoea in animals and as a human pathogen. it causes mild diarrhoea in normal subjects especially children and young adults but it can produce severe intestinal disease in immunocompromised individuals (casemore et al., 1985) . histological examination of the small intestine of laboratory animals infested by cryptosporidium reveals the presence of organisms attached to the mucosal surface, often associated, as in man, with other parasites or infections. they are rounded, weakly basophilic structures 1-4 âµm diameter in haematoxylin and eosin stained sections but are strongly basophilic following romanowsky staining. transmission electron microscopy reveals the detailed internal structure of cryptosporidium attached to the microvillous surface of the epithelial cells. the various stages in the life cycle have been visualised by light and electron microscopy. infection starts with ingestion of an oocyst containing four sporozoites, which are probably released by the action of digestive enzymes. these attach themselves to the intestinal mucosa and undertake their life cycle attached to the epithelial cells (casemore et al., 1985) . these organisms have been demonstrated in laboratory species including mice, hamsters, rabbits, dogs and nonhuman primates (cockrell et al., 1974; rehg et al., 1979; toft, 1982; fukushima and helman, 1984; davis and jenkins, 1986) . hymenolepis nana (dwarf tapeworm) and hymenolepis diminuta (rat tapeworn) are described in the intestine of rats, mice, hamsters, non-human primates and man (hsu, 1979) . a variety of other metazoan patients are found in the small intestine of non-human primates, see review by toft (1982) . certain viruses produce inflammatory small bowel changes in mice. mouse hepatitis virus (lethal intestinal virus of infant mice) can cause mucosal epithelial necrosis and inflammation with characteristic compensatory epithelial hyperplasia and the formation of epithelial syncytia . murine rotavirus (epidemic diarrhoea of infant mice) produces swollen enterocytes of small and large bowel with fine cytoplasmic vesiculation with little or no inflammation but dilated lymphatics and vascular congestion. cytoplasmic acidophilic inclusions, 1-4 âµm diameter, are characteristic findings . electron microscopic examination shows cytoplasmic vesicles arising from the rough endoplasmic reticulum, which contain virus particles and electron dense granular material . a mouse adenovirus may also produce large basophilic intranuclear inclusions in the epithelial cells of the small intestine and caecum. k virus infection produces lesions in the jejunal and ileal mucosa of mice. histological features are characterised by mild polymorphonuclear infiltration, with ballooning of occasional endothelial cells within intestinal villi. intranuclear inclusions can be demonstrated in these endothelial cells by light microscopy using appropriate fixation (greenlee, 1985) . a variety of viruses have been isolated from the gastrointestinal tract of nonhuman primates including viruses of man (kalter, 1982) . however, they appear to be relatively infrequent causes of gastrointestinal disease and when disease is caused by these viruses it usually affects other organs. not only can non-steroidal anti-inflammatory agents such as indomethacin and phenylbutazone produce gastric ulceration but also penetrating ulcers of the small bowel of laboratory animals (shriver et al., 1975) . imaging studies with 111 indium-labelled leukocytes in man have also suggested that subclinical intestinal inflammation is associated with long-term therapy with non-steroidal anti-inflammatory drugs (bjarnason et al., 1987) . it has been postulated that indomethacin-induced intestinal ulcers in rats and dogs are produced by a prostaglandin-independent mechanism, different from the manner in which gastric ulceration is induced (tabata and okabe, 1980; whittle, 1981) . conversely, satoh et al. (1981) have suggested that similar mechanisms are responsible for both gastric and intestinal ulceration. they showed that indomethacin-induced gastric ulceration developed in the body mucosa in fasted rats but in the antrum and small intestine in rats given indomethacin 30 minutes after a 1-hour period of refeeding following a 24-hour fast. there was good temporal correlation between the development of intestinal ulcers and inhibition of prostaglandin synthesis (satoh et al., 1981) . the ulcers in the small intestine were morphologically similar to those occurring in the stomach and they were distributed mainly in the mucosa on the mesenteric aspect of the bowel wall. single-dose studies with indomethacin and ibuprofen in rats conducted by suwa et al. (1987) have demonstrated differences between pathology of induced gastric and intestinal damage. gastric damage was superficial, occurred within 6 hours and was fully repaired 2 weeks after dosing. ulcers in the jejunum and ileum reached a maximum area at 48-72 hours after dosing, occurred on the mesenteric border, penetrated through the muscularis mucosa and were accompanied by inflammation and oedema. ulcers were still present 2 weeks later. rainsford (1978) has shown that potent intestinal ulcerogens such as indomethacin inhibit the incorporation of radioactive 35 s sulphate into glycoproteins of the upper intestinal mucosa as well as the stomach of rats. this may decrease the capacity of the mucus in the intestine to act as a buffer for hydrogen ions. indomethacin given orally to dogs in doses of 2.5 mg/kg/day for 1-23 days was also shown to produce intestinal ulceration. these ulcers were deep, punchedout lesions, many of which were lying over peyer's patches (stewart et al., 1980) . some ulcers involved the whole circumference of the small intestine wall. histologically, the ulcers were associated with an intense inflammatory response principally of mononuclear cells, which infiltrated the bowel wall to the serosa particularly adjacent to peyer's patches. it was suggested that this distribution of ulcers was a result of an exaggerated immune response to normal intestinal antigens. these antigens may have been produced by inhibition of suppresser cells in peyer's patches, following depression of prostaglandin synthetase by indomethacin (stewart et al., 1980) . special dye techniques, scanning and transmission electron microscopy have also shown that non-steroidal anti-inflammatory drugs also produce adverse effects on the small intestine mucosa without overt pathological changes being evident by light microscopy (brodie et al., 1970; djaldetti and fishman, 1981) . following administration of aspirin to mice for 5 weeks, shortening and erosion of microvilli and increased numbers of goblet cells were only demonstrated in the duodenum and jejunum by scanning and transmission electron microscopy (djaldetti and fishman, 1981) . morphometric studies of the intestinal mucosa of indomethacin-treated mice have also shown widespread alterations to columnar cells, goblet cells and paneth cells suggesting generalised effects on mitotic activity and crypt loss (ettarh and carr, 1996) . such submicroscopic findings support the idea that non-steroidal anti-inflammatory agents may induce damage to the small intestine more commonly than supposed. although non-steroidal, anti-inflammatory drugs are the best-known drugs with adverse effects on gastrointestinal mucosa, small intestinal inflammation and ulceration can also produced by other agents through different mechanisms. anticancer drugs and other therapeutic agents, which affect cell proliferation, depress the bone marrow or the immune system can also produce intestinal mucosal necrosis, haemorrhage, inflammation and opportunistic gastrointestinal overgrowth when administered to dogs or rodents in high doses (fig. 51 ) bregman et al., 1987) . lymphoid infiltrates without tissue fig. 51 . section of small intestine from a wistar rat treated with a 5-day course of an antiproliferative anticancer drug. the mucosa shows mucus-depletion, loss of villous architecture as well as a reparative and inflammatory response. (he, ã�25.) damage were also reported in the small intestine of rats treated with human recombinant interleukin-2 (anderson and hayes, 1989) . agents of particular toxicological interest are cysteamine, propionitrile and their structural analogues as well as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (mptp) which are capable of producing ulcers of chronic type in the duodenum of rats and mice (szabo and cho, 1988) . these compounds vary in their ulcerogenic capacity but they are all able to produce ulcers of chronic type with crater formation, granulation tissue and reactive changes in adjacent mucosa in the anterior and posterior wall of the proximal segment of the duodenum of rodents. although these different agents influence gastric acid secretion in different ways, structure-activity relationships suggest that they produce duodenal dysmotility, decrease bicarbonate production and reduce its delivery from the distal to proximal duodenum. these factors decrease the neutralisation of gastric acid in the first part of the duodenum and this may contribute to the development of ulceration (szabo and cho, 1988) . furthermore, these effects can be attenuated or prevented by dopamine agonists or their precursors whereas dopamine antagonists can potentiate their effects. this suggests that the central or peripheral dopamine-mediated actions of these agents may be involved in the pathogenesis of duodenal ulceration (szabo, 1979; . using appropriate fixation and staining procedures, fine granular lipid droplets can be normally visualised in the apical parts of epithelial cells covering the upper third of small intestinal villi. administration of drugs and chemicals may produce an excessive accumulation of lipid through specific effects on lipid metabolism or as part of general cellular toxicity. in the preclinical toxicity studies with 2,6-di-tert-butylamino-3-acetyl-4-methylpyridine (sa h51-055), an inhibitor of glucose transport intended for use as an anti-obesity drug, lipid accumulation occurred in the lamina propria of the small intestinal villi of sprague-dawley rats and guinea pigs but not in dogs or primates (visscher et al., 1980) . after administration of sa h51-055 to rats, there was progressive accumulation of lipid droplets in the epithelial cells over the tips of the duodenal villi demonstrable by osmium tetroxide staining. ultrastructural examination revealed uniform electron-lucent droplets within profiles of the smooth endoplasmic reticulum and golgi apparatus. lipid droplets increased with time, accumulated and coalesced to form large droplets in the lamina propria. larger droplets were phagocytosed by macrophages in the lamina propria but there was no evidence of epithelial damage or necrosis. changes were most pronounced in the duodenum but were also noted to a lesser extent in jejunum and ileum but not in colon or stomach. sequential studies using electron microscopy showed that lipid rapidly accumulated within several hours in the profiles of smooth endoplasmic reticulum and golgi apparatus of the epithelial cells and formed droplets or chylomicra in the intercellular space. the absence of any other subcellular changes or evidence of derangement of protein synthesis suggested that sa h51-055 altered the pathways of lipid resynthesis or transport. this was consistent with the distribution of the lipid in the upper third of the jejunal villous epithelium, a zone most active in lipid absorption, resynthesis and transport (dobbins, 1969) . it was suggested fatty change might have taken place because of alterations in the sugar moiety of chylomicra brought about by interference with glucose transport (visscher et al., 1980) . lipid droplets which stained with oil-red-o in formalin-fixed frozen sections and showed uniform electron density characteristic of neutral lipid were also observed in the epithelial cells and macrophages in the lamina propria of jejunum and duodenum and mesenteric lymph nodes in rats given a synthetic 2'-dodecyl glutaramide ester of erythromycin (gray et al., 1974) . unlike the erythromycin base, rats poorly tolerated this ester. it appeared that the ester was absorbed unhydrolyzed and converted to chylomicron-like droplets, which then accumulated in the macrophages of the lamina propria and local mesenteric lymph nodes, without overt damage to epithelial cells. accumulation of lipid in epithelial cells of intestinal villi has been observed in rats following administration of puromycin (friedman and cardell, 1972) and ethionine (hyams et al., 1966) , agents which have inhibitory effects on protein synthesis. detailed morphological study of the intestinal epithelial cell in rats treated with puromycin have shown that there is concomitant accumulation of lipid with a decrease in the quantity of rough endoplasmic reticulum and golgi membranes (friedman and cardell, 1972) . these changes were in keeping with the concept that lipid accumulates as a result of inhibition of the synthesis of membrane components of the golgi by the rough endoplasmic reticulum which are important for the transport of lipid. in addition to lipid droplets forming as a result of altered lipid metabolism, they may form in the epithelial cells of the intestinal mucosal as a result of a direct toxic effect of the ingested drugs on the small intestinal mucosa. in such instances atrophy of villi and degenerative changes in the epithelial cells may also be observed (see following). the small intestinal mucosa is also one of the many sites at which drug-induced accumulation of polar lipids form laminated structures (myeloid bodies) or crystalloid structures within lysosomes. this form of lipid storage disorder is produced by diverse amphiphilic cationic drugs in both man and laboratory animals probably as a result of drug interaction with polar lipids rendering them difficult to digest (lã¼llmann-rauch, 1979) . species differences in susceptibility and tissue distribution of phospholipid are probably not only related to physiochemical characteristics of the inducing drugs which influences their ability to permeate selective biomembranes and react with different lipids, but also to tissue concentrations of drugs achieved and the ability of organs to metabolise parent drug to less amphiphilic products. in general terms, this form of disorder is characterised by membrane-bound, acid phosphatase-positive cytoplasmic inclusions, which on ultra-structural study are seen as lamellated or crystalloid structures in lysosomes. these appearances are characteristically reversible on cessation of treatment with the inciting agent. an example of this phenomenon occurring in the small intestine is provided by the iodinated amphiphilic drug, amiodarone, which has been used clinically in europe for the past 20 years in the treatment of angina and more recently in the control of supraventricular cardiac arrhythmias. its adverse effects in man are believed to be the result of accumulation of drug in lysosomes particularly in liver, skin and eye (d'amico et al., 1981; shepherd et al., 1987) . when high doses of amiodarone were administered orally to rats and beagle dogs, multilamellated lysosomal inclusion bodies accumulated first in the jejunal mucosa and mesenteric lymph nodes before becoming widely distributed in other organs particularly in the lungs (mazuã© et al., 1984) . in both rats and dogs the small intestinal lesions were characterised by the presence of foamy macrophages with pale finely vacuolated cytoplasm and condensed eccentric nuclei within the lamina propria of the jejunal villi (mazuã© et al., 1984) . mesenteric lymph nodes were also involved early after the onset of treatment. in the dog, jejunal villi were somewhat flattened and widened or showed a variable degree of villous atrophy, most marked in the proximal and middle jejunum (vic et al., 1985) . electron microscopy confirmed the presence of lamellated lysosomal bodies distending macrophages. the early accumulation of foam cells in the jejunal macrophages was probably a reflection of the disposition of drug following oral absorption. although similar lipidosis was seen in many organs following intravenous administration in dogs, more lipidosis was seen in the jejunum after oral dosing. moreover, there were species differences in sensitivity to these changes, baboons being relatively insensitive compared to dogs. fischer 344 rats were very sensitive to these changes compared to sprague-dawley rats and wistar rats were almost completely resistant to lipidosis induced by amiodarone under similar conditions (mazuã© et al., 1984) . similar cytological changes have been reported in cells of some organs in patients treated with amiodarone (d'amico et al., 1981; shepherd et al., 1987) . villous shortening or stunting results when the proliferative activity of the crypt epithelium is reduced or under circumstances in which crypt cell proliferation is insufficient to compensate for increased cell loss as a result of mucosal cell damage. decreased cell proliferation can be seen segments, which are surgically bypassed, or following decreased food intake, parenteral nutrition, hypophysectomy or thyroidectomy bastie et al., 1982) . as adrenergic factors are important in the control of small intestinal epithelial cell division, drugs that alter î± or î² adrenoreceptor activity may influence the proliferative capacity of the epithelium. in mice, increased î±1 or î² receptor stimulation by appropriate agonists (e.g. phenylephrine) diminishes proliferation of crypt cells but proliferation is increased by stimulation of î±2 receptor activity (kennedy et al., 1983) . yohimbine, a î±2-antagonist also reduces cell proliferation in the same animal model. some of the effects of these agents may be mediated by changes in splanchnic blood flow . the detailed morphological study of the small intestinal mucosa in the rat following hypophysectomy by bastie et al. (1982) has shown a reduction in the height of the small intestinal villi associated with reduction in mitoses in the crypt epithelium. the number of goblet cells was shown to fall particularly in the jejunum and the number of paneth cells increase in the ileum. ultrastructural examination showed decreased height of the microvilli of absorptive cells and a lower number of their intracytoplasmic organelles and ribosomes. there were also significant decreases in brush border enzyme activities of alkaline phosphatase, aminopeptidase, maltase and lactase reported about 1 week following hypophysectomy. substances which reduce mitotic activity and therefore lower regenerative capacity of the intestinal epithelium also produce shortening or stunting of small intestinal villi and eventually flattening of the mucosa. a wide variety of anticancer agents and antiviral drugs with radiomimetic properties interfere with cell division in the crypts thereby reducing the number of epithelial cells produced. histologically, the effects of such agents are characterised by blunting, shortening or complete atrophy of villi. mitotic activity is reduced in the crypts and the crypts become dilated and lined by flattened cells. the overlying epithelium loses its normal regular arrangement and cells show pleomorphic nuclei with irregular chromatin patterns. increased numbers of inflammatory cells may infiltrate the lamina propria and epithelium. ulceration, haemorrhage and secondary infection of the gut wall ensue if there is overwhelming cell damage. comparison of the gastrointestinal toxicity expressed by antimitotic anticancer drugs of different classes in rodents, dogs, non-human primates and man have suggested that there is a higher degree of correspondence between effects in man and dog than between man and other species (owens, 1962; schein et al., 1970) . in studies with the antiviral agent acyclovir, a radiomimetic effect was noted in the gastrointestinal tract of dogs at high doses but not rodents (tucker et al., 1983) . another example is the villous atrophy described in rats following treatment with an antibacterial agent ici 17,363. this was believed to arise as a result of both interference with cell division and a direct effect on the surface epithelial cells (murgatroyd, 1980) . the effects of ici 17,363 were characterised by atrophy of villi with dilatation of crypts and atypical features in the crypt epithelium suggestive of an effect on mitotic activity. in addition, vacuolated lipid-laden epithelial cells were observed over the tips of villi accompanied by reductions in the numbers of goblet cells and reduced activity of acid and alkaline phosphatase, esterase, adenosine triphosphatase, glucose-6-phosphatase and succinic dehydrogenase, compatible with a direct adverse effect on superficial mature epithelial cells. a variety of factors stimulate cell proliferation in the small intestinal epithelium. these include enterectomy, increased feeding and stimulation of autonomic nerves. administration of neurotransmitters, thyroxine, growth hormone, corticosteroids, testosterone, gastrin, glucagon and recombinant epidermal growth factor may also stimulate epithelial cell proliferation breider et al., 1996; reindel et al., 1996; vinter-jensen, 1999) . in rats hypothalamic damage, hyperthyroidism, tube feeding, diabetes mellitus and insulin injections have been shown to produce intestinal hyperplasia (mackay et al., 1940; levin and smyth, 1963; jarvis and levin, 1966; forrester, 1972) . most causes of greater cell production lead to increased villous height and mucosal hyperplasia, although intense crypt cell proliferation as a compensatory regenerative response can be associated with villous atrophy (see previous). the compensatory response to the surgically resected or bypassed intestine has been the focus of the most detailed studies of increased cell renewal in the small intestine. partial resection in both rats and man is accompanied by increased villous height and crypt length (hanson et al., 1977) . this is primarily the result of hyperplasia for it has been shown that the numbers of cells per unit length of villus remains unchanged (hanson et al., 1977) but there is an overall increase in the cell population of villus and crypt (hanson and osborne, 1971) . dna/rna ratios also remain largely unaltered . no gross changes in villous shape have been reported after resection and the total number of crypts remains constant. although increased intestinal uptake of substances from the bowel lumen occurs in hypertrophied segments per unit length of bowel, disaccharide and dipeptidase activities are normal or even decreased after resection suggesting a comparative immaturity of cells in the residual mucosa. functional adaptation therefore is achieved by a larger number of cells, the individual absorptive capacity of which is not increased . increased numbers of specific goblet cell populations are also seen in hyperfunctional states. following jejuno-ileal bypass operations in rats, increased numbers of pas-positive goblet cells develop in the villi and crypts of the hyperfunctional segments of the duodenum, jejunum and ileum (olubuyide et al., 1984) . mucin histochemistry using the high-iron diamine and alcian blue techniques have shown that the goblet cells in the hyperfunctional segments contain increased sialomucins in the villi and crypts of the jejunum and ileum but not in the duodenum and increased sulphomucins in the distal ileal segment. sialomucin production may reflect relative cellular immaturity of the more rapidly proliferating cells under these circumstances. however, as sialic acid conveys more viscoelastic properties to mucin, it has been suggested that the goblet cells change following intestinal bypass fulfils a protective function against the increased flow of gastrointestinal contents (olubuyide et al., 1984) . a number of nutritional factors, particularly dietary fibre, can influence the proliferative characteristics of the small bowel mucosa. carefully controlled studies in rats given different forms of dietary fibre have shown that the prolif-erative characteristics of the small intestine can be modified by both the quantity and the quality of the fibre. one study has shown a decrease in the length of villi, crypt cell hyperplasia and shorter transit times in rats fed pectin-supplemented diet but an increase in mucosal growth without alteration in relative differences in crypt and villous length with guar supplementation compared with rats fed fibre-free diet (jacobs, 1983) . another rat study has shown that pectin feeding leads to increased mucosal area and height associated with an increase muscle mass (stark et al., 1996) . these different effects may be the result of differences in solubility, gel formation, water holding capacity, effect on transit time and ion exchange activity or bile acid adsorption of the different fibres. interactions between dietary constituents are complex. for instance, in rats the effects of 2% dietary cholestyramine, a non-absorbable ion exchange resin, on small intestinal histomorphology have be shown to depend on interaction of dietary factors (burkhardt et al., 1998) . administration of an inhibitor of cholesterol biosynthesis, 5î±-cholest-8-(14)en-3î²-ol-15-one, to rats for up to 9 days was also shown to produce enlargement of the small intestine in a way which was morphologically similar to the changes found following intestinal bypass (smith et al., 1989) . the enlargement was most marked in the proximal segment of the small intestine and progressively diminished towards the ileocaecal junction, sparing the stomach, caecum and colon. histological examination and morphometric analysis revealed an increase in smooth muscle mass, lengthening of the villi as well as an increase in the depth and cellular proliferation in the crypts of lieberkuhn without evidence of cell damage or fatty change. like the changes following jejunal bypass procedures, there was also an increase in acid mucosubstances in the goblet cell population overlying the villous mucosa (smith et al., 1989) . the mechanism for this change in the rat was not clear, particularly as intestinal hyperplasia was not seen in baboons treated with this 15-ketosterol for long periods. however, it was suggested that it was an adaptive response, possibly related to inhibition of cholesterol metabolism and cholesterol absorption from the diet, particularly as the laboratory diet employed in the rat study was particularly low in cholesterol. local and systemic changes in hormones and various transmitter substances also influence the number of cells in the small intestinal epithelium. morphometric studies of the small intestinal mucosa in mice following gastrin administration have shown increases in villous area associated with decreases in microvillous area, increased number of goblet cells and paneth cells . studies in which rats were treated with the prolactin-inhibitor, ergocryptine, have shown that the total number of mucous cells and the number staining with alcian blue at ph 1.0 increase in the ileal crypts, possibly as a result of increased synthesis of sulphated mucosubstances (gona, 1981) . in this context, it is of interest that chronic treatment with the rauwolfia neuroleptic, reserpine, causes an increase in the sulphation of goblet cell mucin in the small intestine as demonstrated by alcian blue staining at ph 1.0 and the high iron diamine technique without changes in the goblet cell numbers (park et al., 1987) . agents which affect activity of the sympathetic nervous system can also alter epithelial cell proliferation in the small (and large) intestine. treatment of rats with adrenaline, isoprenaline, phenylephrine, phentolamine and yohimbine all result in decreased mitotic activity of jejunal and colonic crypt cells (tutton and helme, 1974; kennedy et al., 1983) . by contrast, administration of metaraminol, clonidine, propranolol, prazosin and labetolol as well as simultaneous injection of propranolol and adrenaline all resulted in an increased rate of crypt cell proliferation (kennedy et al., 1983) . these results suggest that agents that stimulate î±2 adrenergic receptor activity and those that are î±1-antagonists and î²-adrenergic receptor antagonists increase proliferative activity in the rodent intestinal mucosa. caffeine is also reported to produce an increase in thickness of the intestinal mucosa when administered in high doses to rats (lachance, 1982) . this raises the possibility that intestinal mucosal hyperplasia can be produced by phosphodiesterase inhibition and resultant increases in intracellular camp in a similar way to the hypertrophy induced in salivary tissue. this is supported by recent findings in rats treated for periods of up to 6 months with the inotropic vasodilator, ici 153,110, a phosphodiesterase inhibitor intended for treatment of congestive cardiac failure. administration of high doses not only produced salivary gland hypertrophy but also marked thickening of the small and large intestinal mucosa. this was characterised histologically by an increase in villous length and deepening of intestinal glands, with a relatively unchanged number of epithelial cells per unit length of gland or villus (westwood et al., 1990) . although prostaglandin e analogues produce most of their effects in the stomach, increased thickness of the small intestine characterised by longer villi, deeper crypts and increase in cell size have been reported in rats treated with these agents (levin, 1988) . focal hyperplasia, focal avillous hyperplasia, focal atypical hyperplasia, duodenal plaque, polypoid hyperplasia, polyp -mouse irregular, atypical single or multiple foci of glandular hyperplasia may be found in the small intestinal mucosa of several strains of aged, untreated mice. the lesions are usually located in the first part of the duodenum where they form discrete, raised plaques composed of elongated, irregular or branched glands which replace the normal villous structure of the mucosa (rowlatt and chesterman, 1979) . the glands are lined by hyperchromatic columnar cells, which show marked pseudostratification and proliferative activity. paneth cells and mucinsecreting goblet cells may also be prominent. some glands are cystic and the stroma is fibrous and infiltrated by chronic inflammatory cells. the lesions become pedunculated or polypoid in appearance and show a fibrovascular core that is infiltrated by inflammatory cells. they resemble adenomatous polyps described in man. the cause of these changes in the mouse small intestine is unknown but their prevalence can be altered by dietary fibre and panthothenic acid deficiency as well as by administration of drugs and chemicals (hare and stewart, 1956; seronde, 1965; ito et al., 1981) . in their study of dba mice, hare and stewart (1956) considered that the lesions were not genuine neoplasms since they were composed of a mixture of cell types, which normally populate the mucosa. furthermore, they suggested that the presence of an inflammatory component in the stroma and the fact that the prevalence of these lesions was increased in mice fed a high roughage diet were consistent with the concept that they represent an inflammatory adenomatoid hyperplasia. seronde (1965 seronde ( , 1970 reported these lesions in mice fed purified diets, particularly when deficient in panthothenic acid. panthothenic acid deficiency was also associated with inflammation and deep penetrating chronic ulcers of the duodenum in affected mice, compatible with an inflammatory aetiology of the lesions. an increase in the prevalence of these duodenal changes was described in cd-1 mice treated with the synthetic prostaglandin e1 analogue, misoprostol for 21 months . these authors suggested that the findings posed no real concerns for the safety of patients treated with misoprostol on the grounds that the mouse was unique in this aspect of the response to misoprostol because the mouse had a particular liability to develop such changes in the small intestine. the proliferative lesions were found in a few control cd-1 mice in the same study. in addition, it was also argued that the lesions were neither neoplastic nor preneoplastic in nature . they were not seen in rats treated with misoprostol for 2 years . lesions characterised by such intense proliferative activity may be difficult to distinguish from neoplastic lesion. indeed chronic administration of hydrogen peroxide to c57bl/6j mice in drinking water was not only shown to potentiate the development of a similar type of duodenal hyperplasia but also to produce frankly invasive adenocarcinomas (ito et al., 1981) . anatomically the large intestine is broadly similar in man and laboratory animals but there are significant functional differences. the rat colon is probably one of the best studied of the laboratory animal species because the rat is widely used for experimental work on colon carcinogenesis . as the canine model is popular for oral dosage-form testing, differences in colonic physiology between dog and man may be better understood than between man and many other species (dressman, 1986) . in man, as well as in the non-human primates, the large intestine can be divided anatomically into caecum, appendix, ascending colon, transverse colon, sigmoid colon rectum and anal canal. like the small bowel, the colon comprises mucosa, submucosa, muscularis mucosa and serosa. mucosal plicae are only found in the rectum although plicae semilumaris, formed by folds of the entire thickness of the bowel wall, are found in the colon. the large intestine of the dog resembles that of man more than that of most other domestic species. it is a simplified tubular structure only slightly larger in diameter than the small intestine. the colon of the dog is divided anatomically into ascending, transverse and descending parts, but there is no well-defined sigmoid segment. the caecum in dog is a small diverticulum, similar to that found in other carnivorous species and it communicates directly with the colon. the colon of the rat and mouse is shaped like an inverted v that can be divided into ascending and descending segments. there is no clearly defined transverse colon. a characteristic feature in both rat and mouse is the presence of a curved kidney-shaped caecum. its size is intermediate between the large and anatomically complex caecum of herbivores such as the rabbit and the small caecum of carnivorous species. this probably reflects the omnivorous nature and flexibility of the rat and mouse in their dietary habits, particularly their ability to breakdown cellulose (rerat, 1978) . the caecum of the rat and mouse is a blind pouch from which the colon and ileum exit in close proximity and in which antiperistaltic movements occur. this structure and the presence of bacteria undoubtedly contribute to its ability to function as a fermentation organ in which breakdown of substances can occur in a reasonably controlled milieu (snipes, 1981) . in rat and mouse, the caecum is the site of absorption of many substances including calcium, magnesium, water and electrolytes vitamin k and fatty acids (snipes, 1981) . caecectomy has been shown to decrease digestion of carbohydrates and protein and increase loss of faecal water in these species (ambuhl et al., 1979) . the activity of intestinal microflora in the metabolism of both endogenous and exogenous substances has been demonstrated in the rodent caecum rowland, 1988) . the usual stock diets for rodents contain abundant plant fibre which provides bulk and fermentable carbohydrate for the microbial population in the caecum. rats fed stock diets have been shown to possess high levels of reductive and hydrolytic enzyme activity (e.g. azoreductase, nitroreductase, nitrate reductase, î²-glucosidase and î²-glucuronidase) in their caecal contents compared with rats fed purified fibre-free diets . intestines of germ free animals have thinner lamina propria, lower cell turnover, enlarged caecum, altered metabolism of cholesterol, bilirubin and bile salts and larger amounts of mucin in faeces compared with animals possessing normal gastrointestinal microflora (midtvedt, 1987) . species differences in microflora are also reported. comparative studies with human and rat intestinal microflora have suggested that each population of organisms possesses a degree of autonomous self-regulation and capable of responding quite differently to dietary changes (mallett et al., 1987) . comparative studies have shown large differences in the numbers of facultative anaerobic gram-negative bacteria in the gastro-intestinal tract of mice from three, major specific pathogen free units in australian laboratories. it was shown that these differences could influence the immune system, susceptibility to infection and experimental results (o'rourke et al., 1988) . the colon and caecum in man and laboratory animals are lined by a fairly uniform mucosa devoid of villi. columnar cells of two main types cover the surface epithelium. these are absorptive and mucous cells similar to those found in the small intestine. intestinal glands or crypts of lieberkuhn extend downwards from the surface generally as simple, unbranched tubules lined principally by mucous cells with smaller populations of absorptive, endocrine and undifferentiated cells. the mucosa in man and laboratory animals is not entirely flat but shows a slightly corrugated or uneven pattern, which varies with the particular site within the colon. in histological sections of the colon in man, this corrugated pattern is seen as an anthemion-like structure of crypts reminiscent of a greek architectural feature (filipe and branfoot, 1974) . this is also seen in larger laboratory animal species. in rats and mice the crypts of the caecal mucosa are wider near the lumen than in the crypt base and crypts may be branched, features which may be related to the absorptive function of this zone (snipes, 1981) . the proliferative zone in the large bowel is found in the lower part of the gland and mitotic figures are normally limited to this zone. as in the small bowel, multipotent, undifferentiated stem cells situated in the gland base give rise to the principle cell types which migrate to the cell surface with subsequent differentiation and alteration of their enzyme activities and morphological features (chang and leblond, 1971 ). in studies with mouse aggregation chimaeras in which mosaic cell populations of the intestinal epithelium were localised immunocytochemically, it was demonstrated that the entire epithelium of each adult gland descended from a single progenitor cell (ponder et al., 1985) . the single progenitor may itself give rise to several stem cells responsible for the cell renewal in the complete crypt. absorptive cells are found most commonly in the surface epithelium but also to a lesser extent in the glandular epithelium. they are morphologically similar to those in the small intestine each possessing apical plasma membranes with uniform microvilli and a well-formed glycocalyx. microvilli of absorptive cells have been shown by electron microscopic study to become longer and denser with increasing distance distally in the gastro-intestinal tract. thus, they are longer and more dense in the ileum than in the caecum and least dense and shortest in the colon, perhaps reflecting their relative absorptive capacity (snipes, 1981) . this is reflected at light microscopic level by the less conspicuous brush border in the large intestine compared with that in the small intestine. there are species and regional differences in the glycoconjugates found in the brush border of the large intestine, although they generally contain predominantly acidic mucosubstances. in the mouse and rat, sialomucins with some neutral mucins are found. in hamsters, dogs, non-human primates and man both sulphomucins and sialomucins may be seen in the brush border (sheahan and jervis, 1976) . the glycocalyx is important in the protective function of the colonic mucosa for its disruption by agents such as salicylates has been shown to increase absorption of xenobiotics from the rat rectal mucosa (sithigorngul et al., 1983) . although there has been some debate about the precise nature of the mucous cell populations based on structural studies in mice and rats (chang, 1981; , for practical purposes two principle types of mucous cell can be defined. one of these is the typical goblet cell with cytoplasmic mucous droplets forming a goblet shape, which is found both in glandular and surface epithelium. the other type, the so-called vacuolated cell or mucous cell, is found only in the crypts (chang and leblond, 1971; thomopoulos et al., 1983) . these vacuolated or mucous cells show empty-appearing vacuoles in the cytoplasm which rather than being empty contain abundant sialomucins of a type different from those in goblet cells (spicer, 1965; wetzel et al., 1966) . detailed structural studies and cytochemistry using lectin probes have suggested that these vacuolated cells are able to differentiate into absorptive cells with the cellular apparatus to produce the cell surface glycoconjugates of the glycocalyx (thomopoulos et al., 1983) . sulphomucins, as demonstrated by the high-iron diamine technique (table 2) , generally predominate in the distal colonic segment. in both rat and mouse, goblet cells of the proximal colonic mucosa contain largely sialomucins in the lower parts of the crypts with sulphomucins predominating in the upper parts of the crypt. the distal colon contains largely sulphomucins. the only difference between rat and mouse appears to be the fact that the mouse caecum contains almost exclusively sulphomucins but sulphomucins and sialomucins are found in the rat caecal mucosa. in hamsters, the entire colon contains predominantly sulphomucins. neutral mucins and sulphomucins predominate throughout the dog colon with occasional goblet cells containing sialomucin. in non-human primates, neutral mucins, sialomucins and sulphomucins are seen throughout the colon with sialomucins generally more prominent in the proximal colon and sulphomucins in the distal segment. in man, neutral mucins are found mostly in the caecum. in the caecum and ascending colon, sulphomucins are found in the upper crypts and sialomucins in the crypt base. the converse occurs in the distal colon where sulphomucins predominate in the lower two-thirds of the glands and sialomucins in the upper third of the glands and in the surface epithelium. lectin-labelling (table 1) shows even greater heterogeneity of mucins in the colonic mucosal cell population, probably reflecting differentiation patterns and changes in glycosyltransferase activity as cells migrate upwards (freeman et al., 1980; thomopoulos et al., 1983) . it has been suggested by jass and roberton (1994) that the two principle changes in pathological processes in the human colonic mucins are loss of oacylation substituents at sialic acid c 4 and c 7,8,9 and increased sialylation, neither being specific for neoplasia. the colon, like many other tissues also possess drug metabolising activity, although less than in the liver. it has been shown that the activity of cytochromes p450 involved in hydroxylation of benzo[a]pyrene in microsomes prepared from the colons of sprague-dawley rats retain their activity and responsiveness to inducers better than those in the liver with advancing age (sun and strobel, 1986) . the lamina propria of the large bowel is arranged in a similar way to that of the small bowel. by virtue of the presence of lymphocytes, plasma cells, macrophages and dendritic cells as well as scattered small lymphoid aggregates or patches, it forms an integral and important part in the mucosal immune defence system. most of the lymphocytes in the lamina propria of the human colonic mucosa, like that of the ileum, have been shown to be t cells with helper t cells out numbering the t-suppresser phenotype (hirata et al., 1986; pabst, 1987) . this contrasts with intra-epithelial lymphocytes of the human colonic mucosa which are also t cells but more than 80% of which possess characteristics of the suppresser/cytotoxic phenotype and only 10-20% being helper t cells. this distribution of lymphocyte subsets is seen immunocytochemically in the rat colon using the monoclonal antibodies w3/13, w3/25 and mrc ox8 (see haemopoietic and lymphatic systems, chapter iii). the pan t-cell marker w3/ 13 shows the presence of t lymphocytes in the lamina propria and most of these are labelled by w3/25 demonstrating their cd4 helper phenotype (bland and warren, 1985) . mrc ox8 demonstrates that few lymphocytes in the lamina propria are of suppresser/cytotoxic (cd8) type, which contrasts, with the high proportion of mrc ox8 positive lymphocytes in the colonic epithelium (bland and warren, 1985) . the monoclonal antibodies mrc ox6 and mrc ox17, specific for the rat ia antigen, also label numerous cells with macrophage and dendritic cell morphology in the large bowel of the rat (bland and warren, 1985; martin et al., 1986) . mature, small b lymphocytes are relatively uncommon in the colonic lamina propria of man and laboratory animals. however, the lamina propria contains large numbers of plasma cells, which are mainly of iga type, followed by smaller numbers of igm an igg subtypes (pabst, 1987) . a feature of the colonic mucosa is the presence of lymphoid aggregates also called lymphoid nodules, patches, lymphoid-glandular complexes or microbursa. these are similar to peyer's patches of the small intestine as they are composed principally of lymphoid cells of the b-cell series arranged in follicles with germinal centres with interfollicular and perifollicular zones composed of t cells (pabst, 1987) . they are distributed along the entire length of the colonic mucosa although they are generally smaller than peyer's patches. in sprague-dawley rats, lymphoid aggregates are usually about 5 mm diameter except in the distal colon where they attain sizes of up to 10 mm in maximum diameter (martin et al., 1986) . unlike peyer's patches which are characteristically not associated with crypts or villi, the colonic lymphoid aggregates frequently contain irregular atypical mucosal glands which may enter deeply in the lymphoid tissue and penetrate below the muscularis mucosa both in man and laboratory animals scott, 1982; martin et al., 1986) . in some strains of rat, cells in these glands express the ia antigen, unlike the other parts of the colonic epithelium (martin et al., 1986) . these glandular structures, which are intimately associated with lymphoid tissue, may be important in the immune protection of the colonic mucosa, perhaps by acting as a special local receptor for antigens . it has been proposed that these glandular structures represent sites of predilection for the spread of inflammatory disease to the submucosa by allowing microorganisms to pass through the muscularis mucosa (scott, 1982) . it has also been suggested that they constitute physical weak points in the bowel wall and may play a part in the pathogenesis of diverticular disease of the colon in man . colonic carcinomas induced by dimethylhydrazine in the rat also appear to develop more commonly in the lymphoid aggregates than in other zones (martin et al., 1986) . m cells have been described over the lymphoid aggregates in the caecum of the mouse (owen and nemanic, 1978) , see small intestine. although microorganisms are important causes of inflammatory disease in the large intestine of man and animals, among laboratory animals they are usually only significant problems in non-human primates and hamsters. in the strains of rats and mice and in beagle dogs commonly employed in drug safety evaluation, spontaneous disease of the colon as a result of infectious agents is uncommon. nevertheless, treatment with some therapeutic agents may alter the normal bacterial flora to permit overgrowth of pathogenic organisms or disturb the normal balance between antigens in the lumen or control mechanism to evoke inflammation. inflammation induced by organisms may also confound the histological assessment of drug-induced changes in the colon. ulceration and inflammation of the colon as a direct result of administration of potential therapeutic agents is reported in humans although less commonly that in the small intestine. it has been suggested from studies of the effects of anticancer compounds on neoplastic colonic cells that intestinal cells may possess inherent protective properties in the form of an accelerated efflux pump which can serve to protect them from potentially damaging agents (klohs and steinkampf, 1986) . ulceration and inflammation can be induced by the local application of drugs and vehicles to the rectal mucosa. assessment of these effects in an appropriate animal model is important in the safety evaluation of preparations designed for use in man as rectal suppositories. although inflammatory conditions of the large bowel may possess morphological features typical for some inducing agents, inflammation of the large intestinal mucosa is usually characterised by non-specific histological features. in early or mild inflammation, the surface and glandular mucosa remains intact but shows mucin depletion. this is characterised by reduction in the mucus in goblet cells and increased cytoplasmic basophilia. scattered neutrophils may be seen in the epithelium and adjacent lamina propria. in more severe cases, crypts become filled or distended with acute inflammatory cells (crypt abscesses). the lamina propria is variably hyperaemic and congested and contains increased numbers of mononuclear cells. severe changes are characterised by attenuation or frank erosion of the epithelium and the formation of penetrating ulcers filled with fibrinous exudate and surrounded by intense inflammation, granulation tissue and eventually fibrosis. residual glands may be dilated and lined by flattened epithelium or show reactive changes and mitotic activity. regenerative hyperplasia, which can become florid in chronic ulcerative conditions, is characterised by lengthening, irregularity and cystic dilatation of glands which are often lined by hyperplastic epithelial cells and goblet cells distended with mucin. where ulcerative damage has destroyed glands and supporting stroma, regeneration of glands may not occur in the normal regular fashion and branching of crypts may be evident. clostridium difficile may cause inflammatory changes in the colon of laboratory animals, particularly hamsters, and this may extend into the distal ileum. as in man this form of colitis, often referred to as pseudomembranous colitis, is usually associated with antibiotic therapy. in man it was originally associated with lincomycin and clindamycin therapy but other antibiotics have been implicated. it has been shown that both in man and the hamster experimental model that the enteritis is the result of the toxin produced by clostridium difficile (bartlett et al., 1977; milligan and kelly, 1979) . in man this condition is histologically characterised by the presence of plaques or pseudomembranes on the colonic mucosal surface. the pseudomembrane is composed of mucus, fibrin, blood cells, inflammatory cells and cell debris, which has an appearance of streaming from the underlying mucosa. the mucosa may be partly necrotic or mucosal glands are dilated and lined by flattened or hyperplastic cells. the ileal mucosa may show similar changes (milligan and kelly, 1979) . similar features are observed in the antibiotic-treated hamster although the pseudomembrane is less prominent and it may be distributed more proximally with involvement of the terminal ileum (rehg, 1985) . in the hamster, the condition is characterised histologically by erosion of the colonic epithelium and the variable presence of a pseudomembranous plaque of mucin and cell debris. intact but affected mucosa is thickened with reactive changes accompanied by mucin loss in the epithelium and infiltration of a hyperaemic and oedematous lamina propria and submucosa by polymorphonuclear cells (rehg, 1985) . although most instances of this form of clostridia colitis in the hamster have been associated with antibacterial therapy, it has also been reported in untreated hamsters (rehg and lu, 1982) and those treated with antineoplastic drugs (cudmore et al., 1980) . similar changes have been reported in antibiotic-treated guinea pigs and rabbits (rehg and lu, 1981; rehg and pakes, 1981) . guinea pigs are particularly sensitive to antibiotics especially those active against gram-positive organisms (young et al., 1987) . as in man, these drugs are believed to alter the intestinal flora, permitting overgrowth of clostridium difficile as well as gram-negative organisms, resulting in a severe and frequently fatal enterocolitis. a study of the disposition of ampicillin administered parenterally to guinea pigs showed that this drug was rapidly eliminated from the systemic circulation and excreted in urine and bile, possibly favouring this effect on flora in the colon (young et al., 1987) . citrobacter freundii, a gram-negative, short, plump rod and member of the family of enterobacteriaceae, is the causative agent of naturally occurring transmissible colonic hyperplasia of mice. this agent usually produces a mild or even asymptomatic enteritis in susceptible mouse populations, although it is a cause of rectal prolapse in mice (ediger et al., 1974) . marked strain differences have been noted in mice infected with this organism. nih swiss mice show the most severe histological changes and c57bl/6j mice appear the least affected (barthold et al., 1977) . rats and hamsters seem to be unaffected by citrobacter freundii (barthold et al., 1977) . microscopic changes are found primarily in the descending colon, although proximal segments of the colon and the caecum may also be affected. an important morphological feature is epithelial hyperplasia, which occurs maximally 2-3 weeks after experimental inoculation with citrobacter freundii (barthold et al., 1977) . the colonic glands are elongated and lined by cells that show mucin depletion or loss of goblet cells, considerable immaturity and mitotic activity. the surface epithelium may be covered with numerous coccobacilli, which can be visualised in routine haematoxylin, and eosin stained sections. crypt abscesses, inflammatory cells in the lamina propria, mucosal erosions and ulceration are also features (barthold et al., 1976; . in regressing lesions there is a rebound increase in goblet cells, which are often distended with mucin. the colonic glands may be branched or irregular (barthold et al., 1978) . most laboratory animals are naturally resistant to shigella infections but this is not the case for most non-human primates (takeuchi, 1982) . in infections with shigella, the colon shows a superficial acute inflammatory reaction comprising oedema, congestion, haemorrhage and infiltration by acute inflammatory cells. the surface epithelium shows mucin loss and formation of microulcers where total destruction of the epithelium has occurred. ulcers can extend into the lamina propria but in general terms the inflammatory process remains relatively superficial (takeuchi, 1982) . organisms are also located predominantly in the superficial epithelium. another bacterial infection of the gastrointestinal tract, which affects the colon in primates, is that produced by non-tuberculous mycobacteria . large intestinal lesions are characterised by massive accumulation of epitheloid macrophages in the lamina propria, which may extend into the submucosa and muscular layers and along lymphatics to involve mesenteric lymph nodes. small intestinal lesions may also occur, characterised by the presence of similar large macrophages in the lamina propria of villus tips. superficial ulcers may occur in severely affected segments of intestine . acid-fast bacteria are typically found within macrophages. other organs, including spleen, liver, bone marrow and lungs, may also be involved by focal accumulations of bacteria-laden macrophages or occasionally discrete granulomas with multinucleated giant cells. numerous protozoa and metazoa have been described as inhabitants of the caecum and colon of the non-human primate (toft, 1982) . far fewer are observed in the usual laboratory rodents and beagle dogs. amoebiasis caused by entamoeba histolytica is a widespread disease among non-human primates. it is characterised histologically by the presence of necrotizing ulcers, which reach the muscularis mucosa to form typical flank-shaped ulcers containing or surrounded by trophozoites. extensive haemorrhage may be seen as well as an inflammatory infiltrate composed of neutrophils and mononuclear cells (toft, 1982) . the ciliate, balantidium coli, can also cause an ulcerative process in the colon of primates, characterised by ulcers which extend down to the muscularis mucosa accompanied by lymphocytic infiltrate and balantidium coli trophozoites of up to 150 âµm in greatest diameter (toft, 1982) . a variety of metazoan parasites can be observed in the primate colon and usually can be reasonably well identified in tissue sections (see review by chitwood and lichtenfels, 1973) . the nematode of species strongyloides is an important parasite, which may be observed in the intestinal mucosa of primates. oxyurids commonly known as pinworms are essentially innocuous parasites seen in man, non-human primates and rodents. enterobius vermicularis is found in the large intestine and appendix of man and non-human primates, syphacia muris and syphacia obvelata in rodents. oesophagostomum species (nodular worms) are especially common nematode parasites of non-human primates forming characteristic nodules up to 5 mm diameter most frequently on the serosal surface of the large intestine and caecum and adjoining mesentery as well as in other sites in the peritoneal cavity. histologically, the nodules are composed of parasite cell debris surrounded by fibrous tissue and a variable mantle of chronic inflammatory cells and occasional foreign-body giant cells. they are frequently found in close proximity to small arteries and arterioles in the submucosa and subserosa of the colon and may be associated with a local granulomatous arteritis (lumb et al., 1985) . the inflammatory process may spread to surrounding or draining tissues, particularly if nodules rupture. mild periportal hepatic chronic inflammation is sometimes associated with the presence of this parasite in the mesentery, which may confound interpretation of drug-induced hepatic changes in the non-human primate. although the stomach and to a certain extent the small intestine remain the primary sites of predilection for the ulcerogenic action of non-steroidal anti-inflammatory, the colonic mucosa may become involved under certain conditions. less common complications of non-steroidal anti-inflammatory drugs and potassium chloride therapy in humans are colonic strictures. it appears that nonsteroidal anti-inflammatory drugs produce local inflammation followed by focal scarring of the submucosa with constriction and formation of a mucosal diaphragm whereas potassium causes segmental full thickness scarring and constriction (fellows et al., 1992; haque et al., 1992; van velzen et al., 1996; wolfe et al., 1999) . another form of induced colon damage has been reported in children with cystic fibrosis, the majority of who take high strength pancreatic-enzyme supplements to control malabsorption (smyth et al., 1994; fitzsimmons et al., 1997) . this condition has distinctive pathological features. there is involvement a long segment of ascending colon by a fusiform stenosis primarily as a result of submucosal thickening by deposition of mature collagen. the mucosa appears relatively spared but shows some ulceration and reparative changes (van velzen et al., 1996) . although it has been suggested that the changes may have been linked to the methylacrylate copolymer used for enteric coating of the high-strength preparations, a case-control study showed a strong relation between high daily doses of the enzyme supplements, accentuated by more recent availability of high dose forms (fitzsimmons et al., 1997) . in view of their usage for over 50 years, preclinical data on this material is scarce. colonic damage can be induced experimentally by administration of therapeutic agents. dogs administered 2.5 mg/kg indomethecin orally each day for periods of up to 23 days developed not only gastric and small intestinal ulceration but also scattered haemorrhagic erosions in the colon and rectum. histologically, these lesions were characterised by loss of superficial epithelial cells, mucus-depletion of glandular epithelium, crypt abscesses, frequently with acute inflammation in adjacent lymphoid aggregates in the submucosa (stewart et al., 1980) . an example of chemically induced colitis of relevance to safety assessment of therapeutic agents is that induced by degraded carrageenans or synthetic sulphated dextrans. carrageenans are a heterogeneous group of sulphated polysaccharides composed mainly of long chains of d-galactose subunits (d-galactan) derived from red seaweed species which are widely used as food emulsifiers, stabilisers, thickeners and gelling agents (ishioka et al., 1987) . when carrageenans are degraded by acid hydrolysis into smaller molecular weight fragments of about 20,000-40,000 and administered orally in high doses (e.g. 10% of diet) to rats, mice, guinea pigs, rabbits and rhesus monkeys, colitis results (sharratt et al., 1970; marcus and watt, 1971; benitz et al., 1973; fath et al., 1984; kitano et al., 1986) . similarly, colitis has been induced in rats following administration of a 5% dietary admixture of dextran sulphate sodium, a sulphated polymer of glucose (a d-glucose) of molecular weight of 54,000 (hirono et al., 1981) and a very high molecular weight d-glucan, amylopectin sulphate (ishioka et al., 1987) . although histological features of this form of induced colitis vary between study, species and strain, the colitis is generally characterised mucosal ulceration mainly in the caecum but also in the distal ileum, distal colon and rectum. there is mucus-depletion with variable acute inflammatory infiltrate of the in-tact epithelium, crypt abscesses, inflammatory infiltrate of the lamina propria with oedema, hyperaemia and even vascular thrombosis in the submucosa (hirono et al., 1981; fath et al., 1984) . increased proliferative activity of the mucosa is confirmed by an increase in the tritiated thymidine index compared with controls (fath et al., 1984) . in the caecum of rats, ulcers are linear but often circulating the entire circumference of the intestinal wall with subsequent scarring and stricture formation (oohashi et al., 1981) . ulcerating lesions in the rectum and at the anal margin are associated with squamous metaplasia. both the squamous metaplasia and the regenerative hyperplasia of the columnar epithelium have been shown to progress even after cessation of treatment (oohashi et al., 1981) . foamy macrophages containing metachromatic material, presumably polysaccharide, are also seen in the lamina propria, submucosa, regional lymph nodes, liver and spleen (hirono et al., 1981; oohashi et al., 1981) . the cause of this colitis is unclear. low dose levels, which may be expected to mimic human exposure, do not produce colitis. dextrans, carrageenans and other polysaccharides of molecular weights outside the range 20,000-60,000 tend not to incite colitis. an exception to this is the agent amylopectin sulphate, which has a far higher molecular weight. however, amylopectin is composed of polysaccharide chains, which can be degraded by amylase, and therefore smaller molecular weight fragments may be formed in vivo (ishioka et al., 1987) . it has been suggested that colonic disease produced by these agents is in some way linked to induced changes in intestinal microflora (marcus and watt, 1971) although the evidence for this is conflicting (ishioka et al., 1987) . a recent study in guinea pigs and rats using permeability markers of different molecular weights has suggested that degraded carrageenans enhance intestinal permeability in the absence of overt ulceration (delahunty et al., 1987) . it was therefore proposed that carrageenan-induced colitis could be the result of increased intestinal permeability to antigenic or inflammatory substances normally resident in the large intestine. moreover, long-term administration of high doses of these agents to rats leads to the development of colorectal cancer despite their being devoid of any mutagenic potential (see below). the only obvious features, which are common to a number of these non-genotoxic agents, is chronic inflammation and increased proliferative activity. the rectal administration of therapeutic agents and surfactants may also induce similar ulcerative and inflammatory changes. chemical colitis resembling pseudomembranous colitis has been reported in man as a result of chemical cleaning agents accidentally induced by endoscopic examination (jonas et al., 1988) . cellular degeneration, with loss of mitotic activity and mucin depletion can also occur in the colon following treatment with antimitotic drugs. lymphoid infiltrates without tissue damage were reported in the large bowel of rats treated with human recombinant interleukin-2 (anderson & hayes 1989) . melanosis coli is a well-described phenomenon in man associated with chronic ingestion of anthraquinone purgatives. it is considered to be due to the excessive accumulation of lipofuscin-like pigment in the macrophages of the colonic lamina propria (schrodt, 1963; ghadially and parry, 1966; steer and colin-jones, 1975) . this pigment probably originates from organelles of epithelial cells or macrophages, which are damaged by treatment. similar morphological changes have been induced in laboratory animals (guinea pigs) by treatment with anthraquinones (walker et al., 1988) . as a result of these animal studies, walker et al. (1988) suggested that the primary process is a treatment-induced increase in apoptotic bodies in the surface colonic epithelium that are phagocytosed by intraepithelial macrophages and transported to the lamina propria. lipofuscin and iron pigment is occasionally observed in the lamina propria of untreated rodents, notably hamsters, presumably a result of ageing, previous inflammatory processes and haemorrhage. as in other glandular epithelial tissues, hyperplasia may be focal or diffuse with or without atypical cellular features. the term used for hyperplasia with atypical features is atypical hyperplasia in the iarc classification (mohr, 1997) although others use the term dysplasia. like small intestine, cell proliferation in the large intestinal mucosa can be stimulated by a variety of different factors although these functional adaptive responses have been less well studied. physical stimulation by distension or increased dietary bulk is sufficient to initiate hyperplasia including thickening of the muscle coats (dowling et al., 1967; stragand and hagemann, 1977) . one of the most clearly documented forms of compensatory hyperplasia is that which occurs as a response to surgical resection or bypass of a segment of the colon. following resection of a segment of colon in rats, barkla and tutton (1985) showed that the remaining proximal segment of the right side of the colon showed an increase in the thickness of the mucosa and the muscularis externa as well as enlargement of lymphoid aggregates. histologically, the mucosa of the right side of the colon was uniformly thickened showing accentuated folds, elongated mucosal glands with increased height of the surface columnar cells. the changes were most marked up to 30 days following surgery but were less pronounced after 72 days. there was also a significant increase in the mitotic index in the proximal segment at 7 days although at 14 days and later the mitotic index had returned to normal. the distal, down-stream segment showed little or no morphological change but rather a long-lived increase in mitotic activity. it was suggested that these differences were related to the different embryological origin of the segments (barkla and tutton, 1985) . a similar form of uniform colonic hyperplasia affecting principally the caecal and right-sided colonic mucosa also occurs in rats following oral administration of sulphated dextrans of molecular weight of approximately 400,000 (figs 52 and 53). oral administration of a wide range of compounds such as raw and chemically modified starches, various dietary fibres, caramels, sugar alcohols (lactitol, sorbitol, mannitol, xylilol), lactose, a synthetic polydextrose, polyethylene glycol and magnesium sulphate to rats or hamsters has also been linked to an increase caecal size and colonic mucosal hyperplasia (leegwater et al., 1974; roe and bã¤r, 1985; newberne et al., 1988; stark et al., 1996) . the characteristic histological appearance of the caecum following administration of these agents is lengthening of the mucosal glands which are lined by epithelium composed of increased numbers of enlarged epithelial cells (i.e. hypertrophy and hyperplasia) showing increased proliferative activity and more rapid incorporation of tritiated thymidine (newberne et al., 1988) . in addition, mucosal and submucosal oedema has been reported in association with the administration of lactose and increased mucosal lymphoid aggregates following lactose or xylitol feeding (newberne et al., 1988) . changes in the colon due to fibre are complex. morphometric analysis has shown that changes to the mucosa depend on the fibre type (stark et al., 1996) . there may also be an interaction between dietary fibre content and colonic microflora that influences mucosal growth, although the mechanism is unclear (whiteley et al., 1998) . hypertrophy of the muscularis external is also reported in rats fed high fibre diets (stark et al., 1996) . as the increase in caecal size and mucosal hypertrophy appears generally related to the osmotic activity of the caecal contents in rodents treated with these agents, it has been postulated that the changes represent a physiological adaptation to increased osmotic forces, irrespective of the contributing compounds (leegwater et al., 1974) . treatment of rodents with antibiotics also causes caecal enlargement or dilatation without significant histopathological changes, probably as a result of changes in caecal microflora. it has been suggested that the enlargement relates to accumulation of urea as a result of inhibition of bacterial ureases (juhr and ladeburg, 1988) . however, histochemical studies of the intestinal mucosa of rats treated with neomycin have also shown treatment-related reductions in activities of nad tetrazolium reductase, succinate dehydrogenase, esterase, alkaline and acid phosphatase in the distal ileum, suggesting that some antibiotics also posses the potential to directly influence absorption and metabolic functions of mucosal cells (van leeuwen et al., 1986) . long-term administration of 16,16-dimethyl prostaglandin e2 to rats also produced thickening of the proximal colonic mucosa, although this was less fig. 53 . similar area of colonic mucosa to that seen in fig. 52 at the same magnification but from a rat treated with 10% dextran (molecular weight 500,000) in the diet for 2 weeks. this shows diffuse hyperplasia of the mucosa with elongation of colonic glands that are lined by relatively normal epithelial cells with abundant mucin and prominent vesicular nuclei. (he, ã�40.) marked than in the stomach and duodenum (reinhart et al., 1983 ) and similar changes have been reported in rats treated with other prostaglandin e analogues (levin, 1988) . as in the small intestine, administration of epidermal growth factor to rats and cynomolgus monkeys induces hyperplasia of the colonic mucosa characterised histologically by hyperplasia and increased mitotic activity of crypt cells and reduction in goblet cell numbers with an increase in crypt depth and a slight increase in the numbers of infiltrating neutrophils (breider et al., 1996; reindel et al., 1996) . in common with other epithelial surfaces, atypical hyperplasia is associated with the development of colonic cancer in both man and laboratory animals. the early alterations observed in rats treated with colonic carcinogens are similar to those found in the immediate vicinity of human colorectal carcinomas. the changes are characterised by lengthening, dilatation and branching of glands. the epithelium lining these glands shows mucous cell hyperplasia (goblet cell hyperplasia, see fig. 54 ), goblet cells containing predominantly sialomucin instead of the normal sulphomucin (filipe and branfoot, 1974; filipe, 1975; olubuyide et al., 1985) . despite mucin alterations, activities of glucose-6-phosphatase, glucose-6-phosphate dehydrogenase and glyfig. 54 . section from the colon of an aged hamster from a colony that developed intestinal inflammation and neoplasia spontaneously. this shows focal mucous cell hyperplasia characterised by enlargement and lengthening of the colonic glands with lining cells replete with mucins. (he, ã�25.) ceraldehyde-3-phosphate dehydrogenase were shown to be normal in this epithelium in rats treated with 1,2-dimethylhydrazine (mayer et al., 1987) . in man, this form of hyperplastic mucosa associated with cancer, has been termed 'transitional mucosa' (filipe and branfoot, 1974) . as lesions become more atypical, these dilated, branched glands become more complex and lined by epithelium that shows increasing pseudostratification and vesicular cell nuclei. for example in rats treated with the carcinogens azoxymethane or 1,2-dimethylhydrazine, crypts show diminution of mucus secretion, increased cytoplasmic basophilia, prominent, rounded or enlarged nuclei which show variable degrees of pseudostratification and which eventually develop into frankly invasive glands . in contrast to goblet cell hyperplasia, these atypical zones show increased activity of glucose-6-phosphate, glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase (mayer et al., 1987) . described similar alterations in rats treated with azoxymethane and the non-genotoxic agent dextran sulphate. these authors also demonstrated that these atypical foci could be identified by low power microscopy as aberrant crypt foci by translumination of the whole mounts of the fixed mucosa stained with methylene blue. note: some compounds may induce qualitative and quantitative changes in mucin content in the colonic mucosa without marked morphological alterations. an example of this phenomenon was described in rats treated with reserpine for 7 days. the colonic mucosa showed an increase in sulphomucin (high-iron diamine positive) containing goblet cells in the surface epithelium (park et al., 1987) . adenomas and adenocarcinomas of the small and large intestine are infrequent spontaneous neoplasms in laboratory animals compared with man where colorectal carcinoma is one of the most prevalent neoplasms in the western world. adenomas and adenocarcinomas probably occur spontaneously in older dogs more than in any other animal species and as in man these are located most frequently in the distal colon and rectum (lingeman and garner, 1972) . in nonhuman primates glandular neoplasms of the intestine occur with increasing age in the ileum and in the colon with a predilection for the zones near the ileocaecal valve (depaoli and mcclure, 1982) . in rats and mice, spontaneous intestinal neoplasms are uncommon although adenocarcinomas are occasionally observed in the ileum or colon in mice and rats used in chronic toxicity studies and carcinogenicity bioassays (burn et al., 1966; wells, 1971; maeda et al., 1985; greaves and faccini, 1992; zwicker et al., 1992) . most of these arise in the small intestine and appear to originate in the distal part of the small intestine, caecum and right side of the colon. they may produce metastases, mostly to liver and lungs. in a review of spontaneous adeno-carcinomas developing over a 17-year period in wistar rats, vandenberghe et al. (1985) identified 17 adenocarcinomas, all in ascending colon. in 15 of these cases there appeared to be an intimate relationship with campylobacter-like organisms together with diverticulae and chronic inflammation. these authors suggested that the organisms and the associated inflammation were involved in the pathogenesis of these cancers. in view of the importance of colon cancer in humans, a number of new genetic mouse models predisposed to colon cancer have been developed over recent years (heyer et al., 1999) . some hamster colonies, liable to develop inflammatory bowel disease (see above), also have a high incidence of small and large intestinal polyps, adenomas and adenocarcinomas (fortner, 1957; van hoosier et al., 1971; personal observations) . poorly differentiated carcinomas may infiltrate local lymph nodes and it may be difficult to locate the primary neoplasm. polyps are predominantly adenomatous in nature although inflammatory or regenerative polyps are observed (van hoosier et al., 1971) . adenocarcinomas are induced experimentally in the rodent intestine by the carcinogens 1,2-dimethylhydrazine and azoxymethane. the histogenesis of these induced carcinomas has been extensively studied and it is generally accepted that they resemble human colorectal cancer (ward, 1974; freeman, 1983) . however, there are differences between reported studies. some have shown that these experimental carcinomas arise from pre-existing adenomas consistent with the 'adenoma-carcinoma sequence' theory (ward, 1974; ward et al., 1977) . others suggest that they arise 'de novo' from altered mucosa as microinvasive carcinomas (sunter et al., 1978; maskens and dujardin-loits, 1981; rubio et al., 1986) . these differences may be partly the result of different dosage schedules. rubio et al. (1988) have shown that a single dose of 1,2-dimethyhydrazine produces non-polypoid, micro-invasive carcinomas, particularly in the mucosa overlying lymphoid aggregates, whereas in their earlier studies using multiple doses in the same strain of rat, an adenoma-carcinoma sequence was more evident. in addition, there are undoubtedly species and strain differences in the response to these agents. teague et al. (1981) demonstrated clear differences in the distribution and both macroscopic and histological types of adenomas and adenocarcinomas between three different inbred strains of rat given a similar dosage regimen of 1,2-dimethylhydrazine. in general, many of these carcinomas develop in the distal colonic segments similar to the distribution of human colorectal cancer, although tumours also develop in the proximal colon and in the ileum in rats treated with this agent (ward, 1974; teague et al., 1981) . neoplasms occurring in the rat colon following administration of high doses of degraded carrageenans and sulphated dextran also commonly occur in the distal colon and rectum and are commonly polypoid adenomas and adenocarcinomas (hirono et al., 1981; oohashi et al., 1981; ishioka et al., 1987) . however, in these models adenomas and adenocarcinomas also occur in the caecum and proximal colon and squamous carcinomas are sometimes seen in association with squamous metaplasia at the colorectal junction (oohashi et al., 1981) . the pathogenesis of neoplasms induced by carrageenans and dextrans remains unexplained. although they are biologically active compounds, they are non-mutagenic in the usual short-term tests (ishioka et al., 1987) . it has been suggested that carrageenans act as tumour promoters as they potentiate the appearance of carcinomas in rats treated with standard intestinal carcinogens hirono et al., 1981) . conversely it has been proposed that these agents are tumour initiators based on the development of carcinomas in rats treated with degraded carrageenans for only 2 months (oohashi et al., 1981) . however, despite only a short period of treatment, inflammation, regenerative changes and squamous metaplasia persisted throughout a period of 18 months after treatment was withdrawn before development of cancer in these rats. the only consistent association of carrageenans with development of carcinoma in rats is that of chronic inflammation and increased cell proliferation. although dose levels needed to produce inflammation are far higher than any exposure likely to be achieved in man, interpretation of this inflammation-cancer sequence in rats remains a challenge in safety assessment for similar xenobiotics. this situation is interest in view of the unquestionable risk of carcinogenesis in ulcerative colitis in man (riddell et al., 1983) . a similar range of neoplasms can be defined histologically in both human and experimental pathology. it is appropriate, to use the same classification for all species including man. lingeman and garner (1972) who reviewed a range of tumours from domestic and laboratory animals were able to employ the classification for human gastrointestinal neoplasms. a similar approach has been used in the iarc classification of rat intestinal tumours (mohr, 1997) . this classification can be summarised as follows: these represent localised, sessile or polypoid neoplasms composed of proliferating tubular glands, which show varying degrees of nuclear hyperchromatism, pseudostratification and cellular pleomorphism. a useful scheme for grading the carcinogenic potential of hyperplastic mucosa and adenomatous polyps in man based on the degree of epithelial pseudostratification has been proposed by kozuka (1975) . although experimental neoplasms may not always show the full spectrum of these changes reported in man, this grading provides a useful baseline concept for the assessment of these non-invasive proliferative lesions. with increased nuclear pseudostratification and atypical branching of the glandular structures of these polyps becomes more prominent. if neoplastic cells or glands are seen in the stroma of the stalk or base the diagnosis of carcinoma is made. villous adenoma is a form of adenoma in which the epithelial proliferation takes the form of elongated villi with a sparse fibrovascular stroma. they can be graded in a similar way to other adenomas. these are glandular neoplasms of variable differentiation, sometimes originating in adenomatous polyps or villous adenomas but which show infiltration of the intestinal wall, i.e. beyond the boundary of the muscularis mucosa. squamous carcinomas also occur in the anorectal zone but are similar to those which occur in squamous epithelium elsewhere. similarly, mesenchymal neoplasms also are found in the small and large intestinal wall (see integumentary system, chapter i). light and electron microscopical studies of parietal cells before and one year after proximal vagotomy in duodenal ulcer patients six-month repeated oral toxicity study of nk-104 in rats response of the non-human primate to polychlorinated biphenyl exposure cell number as a measure of distribution and renewal of epithelial cells in the small intestine of growing and adult rats induction of early lesions in the forestomach of rats by 3-tert-butyl-4-hydroxyamisole (bha) effects of caecetomy in the young adult female rat on digestibility of food offered and libitum and in restricted amounts toxicity of human recombinant interleukin-2 in rats. pathologic changes are characterized by marked lymphocytic and eosinophilic proliferation and multisystem involvement effects of cimetidine, cimetidine plus nitrite, and nitrosocimetidine on tumors in mice following transplancental chronic lifetime exposure age-associated lesions in barrier-reared male sprague-dawley rats: a comparison between hap: (sd) and crl:cobs[r] cd[r] (sd) stocks expression of cd15 in normal and metaplastic paneth cells of the digestive tract an epizootic of klebsiella aerogenes infection in laboratory rats correlation of quantitative changes of gastric mucosal glycoproteins with aspirin-induced gastric damage in rats long-term comparative effect cholecystokinin and gastrin on mouse stomach, antrum, intestine, and exocrine pancreas the effect of 6-hydro-xydopamine on rat salivary glands and on their response to isoproterenol biologically active peptides in submandibular glands the alimentary system proliferative and morphologic changes in rat colon following bypass surgery cyclosporin and gingival overgrowth the etiology of transmissible murine colonic hyperplasia dietary, bacterial, and host genetic interactions in the pathogenesis of transmissible murine colonic hyperplasia transmissible murine colonic hyperplasia mouse hepatitis virus infection, intestine, mouse murine rotavirus infection, intestine, mouse adenovirus infection, intestine, mouse clindamycinassociated colitis due to a toxin-producing species of clostridium in hamsters antibiotic-associated pseudomembranous colitis due to toxin-producing clostridia histological variations jejunal and ileal mucosa on days 8 and 15 after hypophysectomy in rat: morphometric analysis in light and electron microscopy comparative study of histological and kinetic variations of the digestive mucosa and pancreatic parenchyma after hypophysectomy in the rat effect of drugs on rats exposed to cold-restraint stress adverse effects of anticonvulsant drugs: a critical review formation of n-mono-nitrosopiperazine in the stomach and its secretion in the urine after oral intake of piperazine intestinal effects of carrageenans in the rhesus monkey (macaca mulatta) the cell surface: components and configurations mã©nã©trier's disease. serial morphological, secretory, and serological observations structure, biosynthesis and functions of glycoprotein glycans. experientia pathology of the forestomach in rats treated for 1 year with a new histamine h2-receptor antagonist, sk&f 93479 trihydrochloride fundic mucosal ecl cell hyperplasia and carcinoids in rodents following chronic administration of the histamine h2-receptor antagonist sk&f 93479 and other antisecretory agents gastric ecl-cell hyperplasia and carinoids in rodents following chronic administration of the h2 antagonist sk&f 93479 and oxmetidine and omeprazole gastric regulatory peptides in rats with reduced acid secretion non-steroidal anti-inflammation in humans immunohistologic analysis of the t-cell and macrophage infiltrate in 1,2-dimethylhydrazine-induced colon tumors in the rat turnover of brush-border glycoproteins in human intestinal absorptive cells: do lysosomes have regulatory function? alterations in gastric mucosal morphology induced by long-term treatment with omeprazole in rats the effect of aging on the rat submandibular gland. an ultrastructural, cytochemical and biochemical study drug-induced esophageal strictures cytochrome p450 of small intestinal epithelial cells. immunocytochemical characterization of the increase in cytochrome p450 caused by phenobarbital synergistic role of intestinal flagellates and normal intestinal bacteria in a post-weaning mortality of mice medication-induced oesophageal injury. survey of the literature resistance to starvation in albino rats fed from weaning on diets containing from 0 to 81% of protein as casein diseases of the kidney the human gastrointestinal secretory immune system in health and disease clinical aspects: an overview single-dose and multiple-dose intravenous toxicity studies of bmy-25282 in rats cellular hyperplasia in rats following continuous intravenous infusion of recombinant human epidermal growth factor adrenergic mechanisms responsible for submandibular salivary glandular hypertrophy in the rat aspirin: intestinal damage in rats gastrointestinal mucosal lesions: a drug formulation problem intestinal t lymphocytes of different rats strains in immunotoxicity effects of propionic acid and pravastatin on hmg-coa reductase activity in relation to forestomach lesions in the rat famotidine: summary of preclinical safety assessment effects of cholestyramine and diet on small intestinal histomorphology in rats spontaneous carcinoma of the colon of the rat cresyl fast violet staining method for campylobacter-like organisms pigmentation of the jawbone and teeth secondary to minocycline hydrochloride therapy genetic ablation of parietal cells in transgenic mice: a new model for analyzing cell lineage relationships in the gastric mucosa esophageal lesions caused by orally administered drugs. an experimental study in the cat tetracycline induced esophageal ulcers. a clinical and experimental study diagnosis of silodacryoadenitis virus infection of rats in a virulent enzootic outbreak cryptosporidium species a 'new' human pathogen thyroxine accelerates the differentiation of granular convoluted tubule cells and the appearance of epidermal growth factor in the submandibular gland of the neonatal mouse. a fine structural immunocytochemical study renewal of the epithelium in the descending colon of the mouse. i. presence of three cell populations: vaculated-columnar, mucous and argentaffin two types of mucous cells in the colon crypt origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine. iii entero-endocrine cells cytology of the canine oral papilloma uremic gastropathy in the dog intestinal absorption and metabolism of xenobiotics in laboratory animals parasitological review. identification of parasitic metazoa in tissue section spontaneous basophilic hypertrophic foci of the parotid glands in rats and mice effects of housing conditions on food intake, body weight and spontaneous lesions in mice. a review of the literature and results of an 18-month study cryptosporidiosis in the intestines of rhesus monkeys (macaca mulatta) isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening in the newborn animal post marketing surveillance of the safety of cimetidine: mortality during second, third, and fourth years of follow-up hyperplastic gastropathy in the rat due to taenia taeniaeformis infection: parabiotic transfer and hypergastrinaemia. gastroenterology number, size and distribution of peyer's patches in the human small intestine a model for gastric cancer epidemiology helicobacter pylori infection, a paradigm for chronic mucosal inflammation: pathogenesis and implications for eradication and prevention the effects of vagotomy on the gastric mucosa of the rat the effect of prolonged administration of large doses of cimetidine on the gastric mucosa of rats effect of short-and long-term feeding of omeprazole on rat gastric endocrine cells clostridial enterocolitis produced by antineoplastic agents in hamsters and humans odontogenic tumours in fischer rats the histo-chemical demonstration of o-acylated sialic acid in gastrointestinal mucins: their association with the potassium hydroxide-periodic acid-schiff effect a new histochemical method for the identification and visualization of both side chain acylated and non-acylated sialic acids amiodarone keratopathy, drug-induced lipid storage disease salivary gland components involved in the formation of squamous metaplasia gastric mucosal injury by fatty and acetylsalicylic acids cryptosporidosis and proliferative ileitis in a hamster specificity of twelve lectins towards oligosaccharides and glycopeptides related to n-glycosylproteins intestinal permeability changes in rodents. a possible mechanism for degraded carageenan-induced colitis adrenal corticosteroids cause gastrin cell hyperplasia possible role of transforming growth factor alpha in the pathogenesis of menetrier's disease: supporting evidence from humans and transgenic mice the effect of hydrocortisone and cortisone on fixation of 35s in the stomach the effect of phenylbutazone and its derivatives, oxyphenbutazone and sulfinpyrazole, on 35s sulfate incorporation in cartilage and stomach salivary glands: a paradigm for diversity of gland development gastrointestinal neoplasms in non-human primates: a review and report of new cases gastric gland degeneration induced in monkeys by the cck-b/gastrin receptor antagonist ci-988 the effect of aspirin on small intestinal mucosa morphologic aspects of lipid absorption gastric and gastric epithelial physiology two-year evaluation of misprostol for carcinogenicity in cd sprague-dawley rats distribution and incidences of calcified lesions in dba/2ncrj and balb/canncrj mice interaction of phenytoin and inflammation induces gingival overgrowth in rats the intestinal response to high bulk feeding in the rat comparison of canine and human gastrointestinal physiology stress ulceration-clinical relevance of animal and human studies pathology of laboratory rats and mice effect of chronic aspirin ingestion on epithelial proliferation in rat fundus, antrum and duodenum the prognostic value of sulphomucin positive intestinal metaplasia in the the development of gastric cancer colitis in mice with high incidence of rectal prolapse volatile nitrosamine contamination of laboratory animal diets toxicological studies on omeprazole possible role of cimetidine and its nitrostated products in human stomach cancer cimetidine and gastric cancer tumours of the oral cavity, check pouch, salivary glands, oesophagus, stomach and intestines differential distribution of lymphocytes and accessory cells in mouse peyer's patches phenotypically distinct subpopulations of t cells in domes and m-cell pockets of rabbit gut-associated lymphoid tissues morphometric analysis of the small intestinal epithelium in the indomethacin-treated mouse expression of pokeweed lectin binding in murine intestinal paneth cells heterogeneity of m-cell-associated b and t cells in human payer's patches degraded carrageenan-induced colitis in cf1 mice. a clinical, histo-pathological and kinetic analysis substituted benzimidazoles inhibit acid secretion by blocking (h ++ k + ) atpase nonsteroidal anti-inflammatory drug induced jejunal and colonic diaphragm disease: a report of two cases effect of chronic misoprostol ingestion on rat gastric morphology and turnover abnormal patterns of mucous secretion in apparently normal mucosa of large intestine with carcinoma mucous secretion in rat colonic mucosa during carcinogenesis induced by dimethylhydrazine. a morphological and histo-chemical study mucins in the human gastrointestinal epithelium: a review. invest high-dose pancreatic-enzyme supplements and fibrosing colonopathy in children with cystic fibrosis the number of villi in rat's jejunum and ileum: effect of normal growth, partial enterectomy and tube feeding spontaneous tumors including gastrointestinal neoplasms and malignant melanoma, in syrian hamster aspirin, paracetamol and non-steroidal anti-inflammatory drugs. a comparative review of side effects campylobacter jejuni/coli in commercially reared beagles. prevalance and serotypes antigen specificity and morphological characteristics of chlamydia trachomatis, strain sfpd, isolated from hamsters with proliferative ileitis acquired salivary dysfunction. drugs and radiation gastritis cystica profunda application of lectins for detection of goblet cell glycoconjugate differences in proximal and distal colon of the rat lectin histochemistry of 1,2-dimethylhydrazine-induced rat colon neoplasia effects of puromycin on the structure of rat intestinal epithelial cells during fat absorption morphological aspects on pancreatic islets of non-obese diabetic (nod) mice carcinoma and related lesions in dog stomach induced by oral administration of n-methyl-nâ´-nitro-n-nitrosoguanidine cryptosporidiosis in a pup with distemper. vet.pathol squamous cell carcinoma, forestomach, rat adverse effects of mouthwash use. a review. oral surg.oral med.oral pathol tyzzer's disease, intestine, mouse, rat, hamster salmonellosis, intestine, mouse, rat, hamster adequate substitution with electrolytes in toxicological testing of 'loop' diuretics in the dog the forestomach in rats and mice, a food store without bacterial protein digestion m cells in peyer's patches of the intestine histochemie de la muqueuse gastrique fundique du chien traitã© par des drogues ulcã©rigã¨ne an electron-microscope and histo-chemical study of melanoisis coli mitochondria. in: ultrastructural pathology of the cell and matrix sustainability of forestomach hyperplasia treated with ethyl acrylate for 13 weeks and regression after cessation of dosing the cytochemical localization of lysozyme in paneth cell granules regional differences in glycoconjugates of intestinal m cells in mice: potential targets for mucosa vaccines susceptibilities of drug to nitrosation under simulated gastric conditions features of small intestinal pathology (epithelial cell kinetics, intra-epithelial lymphocytes, disaccharidases) in a primary giardia muris infection staining rickettsiae in yolk sac cultures alterations of gastric mucosa following a graded partial gastrectomy tumours of the salivary glands the oral cavity aristolochic acid is a direct mutagen in s. typhimurim studies of the binding of trypsin and chymotrypsin by human intestinal mucosa study of cold plus restraint stress gastric lesions in spontaneously hypertensive, wistar and sprague-dawley rats mucins in normal and neoplastic gastrointestinal epithelium the lectins: carbohydrate-binding proteins of plants and animals prolactin and ergocryptine effects mucus glycoproteins of the rat ileum tumours of the jaws hypopigmentary changes with a platelet aggregation inhibitor (abstract no. 2526). fed.proc drug induced enteropathy characterized by lipid in macrophages altered patterns of mucin secretion in gastric hyperplasia in mice digestive system. in: rat histopathology. a glossary for use in toxicity and carcinogenicity studies k virus infection, intestinem mouse a silver nitrate stain for alpha-2 cells in human pancratic islets silver stains in the study of endocrine cells of the gut and pancreas effects of antrectomy or porta-aval shunting on the histamine-storing endocrine-like cells in oxyntic mucosa of rat stomach. a fluorescence histochemical, electron microscopic and chemical study the vagus exerts trophic control of the stomach in the rat activation and hyperplasia of gastrin and enterochromaffin-like cells in the stomach gastrin and the trophic control of gastric mucosa onkocytes and so-called hã¼rthle cell tumor interaction of microorganisms, epithelium, and lymphoid cells of the mucosa-associated lymphoid tissue dog and swine as models for testing indomethacin-induced gastrointestinal irritation sialodacryoadenitis in the rat: effects of immunosuppression on the course of the disease role of gut in zenobiotic metabolism epithelial cell kinetics in the small intestine of the rat 60 days after resection of 70 percent of the ileum and jejunum compensation by the residual intestine after intestinal resection in the rat proceedings of the lst international symposium on omeprazole a cecal diaphragm associated with the use of nonsteroidal anti-inflammatory drugs chronic gastritis of the glandular stomach, adenomatous polyps of the duodenum, and calcareous pericarditis in strain dba mice idiopathic megaoesophagus in rat immunocytochemical localization of alkaline phosphatase in absorptive cells of rat small intestine after colchicine treatment spontaneous neoplasm incidences in fischer 344 rats and b6c3f1 mice in two-year carcinogenicity studies: a national toxicology program update diphenyldydantoin (dilantin) gingival hyperplasia: drug-induced abnormality of connective tissue on cell proliferation and differentiation of the fundic mucosa of the golden hamster. fractographic study combines microscopy and 3 h-thymidine autoradiography tritiated thymidine autoradiographic study on cellular migration in the gastric gland of the golden hamster enterochromaffin-like cell carcinoids of gastric mucosa in rats after life long inhibition of gastric secretion gastric cancer after cimetidine in a patient with two negative pre-treatment biopsies induction of experimental allergi sialadenitis in mice spontaneous development of auto-immune sialodenitis in aging bdf1 mice i mmunofluorescent localization of enterokinase in human small intestine mouse models for colorectal cancer morphologic changes in the urinary bladder and stomach after long-term administration of sodium saccharin in f344 rats lessons from genetically engineered animal models iii. lessons learned from gastrin gene deletion in mice immunohistological characterization of intra-epithelial and lamina propria lymphocytes in control ileum and colon and inflammatory bowel disease induction of intestinal tumors in rats by dextran sulphate sodium gastric enterochromaffin-like hyperplasia and neoplasia in the rat: an indirect effect of the histamine h2-receptor antagonist bl-6341 oxidative metabolism of foreign compounds in rats small intestine: cellular localization and dependence on dietary iron clinicopathological studies of gastrointestinal disease in macaques non-tuberculous myobacterial disease in rhesus monkeys on human papillomaviruses the laboratory rat. biology, diseases inhibition of intestinal protein synthesis and lipid transport by ethionine experimental toxicity studies with captopril, an inhibitor of angiotesin 1-converting enzymes 2. one month studies of chronic toxicity of captopril in rats development of spontaneous tongue calcification and polypoid lesions in dba/2ncrj mice changes in enzyme levels accompanying differentiation of intestinal epithelial cells textbook of endocrinology acute disease of the submaxillary and harderian glands (sialodacryoadenitis) of rats with cytomegaly and no inclusion bodies cellular kinetics of gastrointestinal mucosa, with special reference of gut endocrinecells changes of gastric mucus glycoproteins with aspirin administration in rats induction of colorectal tumours in rats by sulpated polysaccharides induction of duodenal tumors in mice by oral administration of hydrogen peroxide carcinogencity of butylated hydroxyanisole in f344 rats modifying effects anti-oxidants on chemical carcinogenesis a 13 week feeding study of butylated hydroxyanisole: the subsequent regression of the induced lesions in male fischer 344 rat forestomach epithelium an 85-day study of butylated ydroxyanisole in the cynomolgus monkey subchronic studies of doxylamine in fischer 344 rats effects of dietary fiber on mucosal growth and cell proliferation in the small intestine of the rat: a comparison of oat, bran, pectin, and guar with total fiber deprevation transmissible ileal hyperplasia, hamster anatomic adaption of the alimentary tract of the rat to the hyperphagia of chronic alloxan-diabetes a variant of intestinal metaplasia associated with gastric carcinoma: a histochemical study role of intestinal metaplasia in the histogenesis of gastric carcinoma colorectal mucin histochemistry in health and disease: a critical review uptake of particulate and soluble antigens in the small intestines of the rat chemical colitis due to endoscopic cleaning solutions: a mimic of pseudomembranous colitis pathology of domestic animals intestinal accumulation of urea in germ-free animals: a factor in caecal enlargement digestive enzymes in the parotid and submandibular glands of mammals morphologishe verã¤nderungen der magenmukosa von ratten nach chronischer antazidagabe enteric viruses of non human primates increased accumulation of sulfated glycoaminoglycans in cultures of human fibroblasts from phenytoin-induced gingival overgrowth new insights into the stem cells and the precursors of gastric epithelium colonic lymphoid-glandular complex (microbursa): nature and morphology lymphoid tissue and lymphoid-glandular complexes of the colon: relation to diverticulosis histology of salivary gland infarction in the dog adrenergic factors involved in the control of crypt cell proliferation in jejunum and descending colon of mouse pill esophagitis immunogold localization of ingested kidney bean (phaseolus vugaris) lectins in epithelial cells of the rat small intestine epithelial dysplasia of the rabbit colon induced by degraded carrageenan hyperkeratinization and hyperplasia of the forestomach epithelium in vitamin a deficient rats intrinsic resistance of colon tumors to anthrapyrazoles and antracyclines may be linked with a detoxification mechanism of intestinal cells (abstract no. 1040) studies on the effects of 3-hydroxy-3-methylglutaryl coenzyme a reductase inhibitors on the rodent forestomach reversibility of adenomatous hyperplasia in the gastric stump after diversion of bile reflux in rats experimental production of possible autoimmune gastritis followed by macrocytic anemia in athymic mice expression cloning and characterization of the canine parietal cell gastrin receptor histomorphologic investigations on the effect of cyclophosphamide on dentinogenesis of the rat incisor. scand preclinical toxicology profile of misoprostol premalignancy of the mucosal polyp in the large intestine: i. histologic gradation of the polyp on the basis of epithelial pseudostratification and glandular branching morphologic changes in the gastric mucosa of rats and dogs treated with an analog of 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ulceration as a model for human ulcerative colitis. lancet, ii comparative histochemistry of gastro-intestinal mucosubstances assessment of risk of formulation of carcinogenic n-nitroso compounds from dietary precursors in the stomach granular cells as a marker of early amiodarone hepatotoxicity: a pathological and analytical study marked epithelial hyperplasia of the rat glandular stomach induced by long-term administration of iodoacetamide a profile of the gastrointestinal toxicity of drugs used to treat inflammatory diseases morphology and cyto-chemistry of rat salivary gland acinar secretory granules and their alteration by isoproterenol. i. parotid gland effects of sodium salicylate on epithelial cells of the rectal mucosa of the rat: a light and electron microscopic study the effects of long-term propranolol on the salivary glands and intestinal mucosa of the mouse effects of cardiotonic phosphodiesterase inhibitors on rat salivary gland. presented at the 39th annual acvp meeting inhibitors of sterol synthesis. morphological studies in rats after dietary administration, administration of 5 î±-cholest-8(14)-en-3î²-ol-15-one, a potent hypocholesterolemic compound m cell numbers increase after transfer of spf mice to a normal animal house environment strictures of ascending colon in cystic fibrosis and high-strength pancreatic enzymes pathology of laboratory rats and mice anatomy of the cecum of the laboratory mouse and rat gastric carcinoids and related endocrine growths pathogenesis of peptic ulcer and implications for therapy histochemical distribution of lysozyme activity in organs of normal mice and radiation chimeras human peyer's patches: an immunohistochemical study diamine methods for differentiating mucosubstances histochemically metabolic and morphometric changes in small and large intestine in rats fed high-fiber diets melanosis coli: studies of the toxic effects of irritant purgatives surface morphology of the gastroduodenal mucosa in duodenal ulceration the effect of vincristine on dentino-genesis in the rat incisor. scand autoradiographic investigation of dentine production in rats incisosrs after vincristine administration. scand acute and late radiation injury in rhesus monkey parotid glands. evidence of interphase death unique radiosensitivity of serous cells in rhesus monkey submandibular glands pathologic observations on the adenomatous lesions of the stomach in mice of strain i carcinoma of the glandular stomach of rats ingesting n,nâ´2 ulcerative enterocolitis in dogs induced by drugs aspirininduced glandular dysplasia of the stomach. histologic and histochemical studies in rats effect of lumenal contents on colonic cell replacement pathologic findings in the stomach of rats treated with the h2-receptor antagonist tiotidine morphologic stomach findings in rats and mice treated with the h2-receptor antagonists, ici 125211 and ici 162846 lectin-peroxidase reactivity in rat gastric mucosa tumour production in glandular stomach of rat by nmethyl-nâ´nitro-n-nitrosoguanidine ageing affects the drug metabolism systems of rat liver, kidney, colon and lung in a differential fashion oral papillomatosis in new zealand white rabbits diagnostic exercise: lingual growths in rabbits immunoperoxidase localization of papillomaviruses in hyperplastic and neoplastic epithelial lesions of animals hypergastrinemia after blockade of acid secretion in the rat. trophic effects pathological features of the colonic tumors induced in rats by the administration of 1,2-dimethylhydrazine comparative studies on the gastrointestinal lesions caused by serveral non-steroidal anti-inflammatory agents in the rats cells intermediate between mucous neck cells and chief cells in rat stomach fine structure of giant hypertrophic gastritis developed in thymectomized mice hyperplastic gingivitis in a child receiving sodium valproate treatment dopamine disorderin dodenal ulceration biology of disease. pathogenesis of duodenal ulcer disease from cysteamine to mptp: structure-activity studies with duodenal ulcerogens digestive system. monographs on pathology of laboratory animals role of salivary mucins in the protection of the oral cavity effects of 16,16-dimethyl prostaglandin e2-methyl ester on aspirin-and indomethacin-induced gastrointestinal lesions in dogs hypertrophic gastropathy resembling menetrier's disease in transgenic mice overexpressing transforming growth factor î± in the stomach tumours of the oral cavity, buccal pouch, oesophagus, forestomach and salivary glands nodular hyperplasia of oncocytes in mouse submandibular glands early colonic lesions in experimental shigella infections in rhesus monkeys: revisited variation in susceptibility to the induction of forestomach tumours by butylated hydroxyanisole among rats of different strains pathology of neoplasia and preneoplasia in rodents independent induction of intestinal metaplasia and gastric cancer in rats treated with n-methyl-nâ´-nitro-n-nitrosoguanidine the response of three inbred strains of rat to the carcinogen 1,2-dimethylhydrazine small intestinal type' and 'colonic type' intestinal metaplasia of the human stomach and their relationship to the histogenetic types of gastric adenocarcinoma light and electron microscopic cytochemistry of glycoconjugates in the recto-sigmoid colonic epithelim of the mouse and rat preclinical toxicologic evaluation of bleomycin (nsc 125 006), a new anti-tumor antibiotic oral squamous cell carcinoma in ad libitum-fed and food restricted brown-norway rats the pathoparasitology of the alimentary tract and pancreas of non-human primates: a review t cell attractant chemokine expression initiates lacrimal gland destruction in nonobese diabetic mice altered patterns of mucin secretion in the precancerous lesions induced in the glandular part of the rat stomach by the carcinogen n-methyl-nâ´nitro-n-nitrosogaunidine immunohistochemistry and radioimmunoassay of egf in submanidbular glands of mice treated with secretogogues carcinogenicity of cyproterone acetate preclinical toxicology studies with acylovir: acute and sub-chronic tests the influence of adrenoreceptor activity on crypt cell proliferation in rat jejunum a sporozoan found in the peptic glands of the common mouse cyclophosphamide-induced abnormalities in the incisors of the rat spontaneous adencarcinoma of the ascending colon in wistar rats: the intracytoplasmic presence of a campylobacter-like bacterium inhibition of prostaglandin synthesis as a mechanism of action of aspirin-like drugs spontaneous hyperplastic and metaplastic duct epithelium in the sublingual salivary glands of wistar rats spontaneous tuors of the syrian hamster: observations in a closed breeding colony and a review of the literature mucin gene structure and expression: protection vs. adhesion morphological effects of high dose neomycin sulphate on the small and large intestine comparative and experimental pathology of fibrosing colonopathy intestinal pathology in the dog induced by sublethal doses of amiodarone pharmacological effects of epidermal growth factor (egf) with focus on the urinary and gastrointestinal tracts chemically induced lipidosis of the small intestinal villi in the rat lesions of experimentally induced tyzzer's disease in syrian hamsters, guinea pigs, mice and rats hexamitis in laboratory mice, hamsters, and rats melanosis coli: a consequence of anthraquinone-induced apoptosis of colonic epithelial cells safety evaluation of cimetidine: report at the termination of a seven-year study in dogs hypopigmentation in dogs treated with an inhibitor of platelet aggregation gingival hyperplasia in dogs induced by oxodipine, a calcium channel blocking agent cholecystokinin receptors gastrointestinal ph measurement in rats: influence of microbial flora, diet and fasting morphogenesis of chemically induced neoplasms of the colon and small intestine in rats natural history of intestinal neoplasms induced in rats by a single injection of methyl (acetoxymethyl) nitrosamine proliferative lesions of the glandular stomach and liver in f344 rats fed diets containing aroclor 1254 experimentally induced intestinal metaplasia in wistar rats by x-ray irradition. gastroenterolgy effect of dietary undegraded carrageenan on colon carcinogenesis in f344 treated with azoxymethane or methyl-nitrosourea induction of intestinal metaplasia in the rat gastric mucosa by local x-irradiation the effect of sex difference on induction of intestinal metaplasia in rats distribution of the histaminergic neuron system in the central nervous system of rats; a fluorescent immunohistochemical analysis with histidine decarboylase as a marker replacement of functional parenchymal cells by fat and connective tissue in human submandibular salivary glands. an age related change canine papilloma: progression of oral papilloma to carcinoma bacterial and mycotic disease dental health of survivors of malignant disease analysis of protein synthesis in rat salivary glands after chronic treatment with î²-receptor agonists and phosphodiesterase inhibitors mucinous carcinoma of the ileum in the rat mucin histochemistry of gastric intestinal metaplasia long-term effects of an inotropic phosphodiesterase inhibitor (ici 153,110) on rat salivary gland, hardarian gland and intestinal mucosa the synovial membrane, liver, and tongue: target organs for a ricin a-chain immunotoxicin (zd0490) ultrastructural localization of acid mucosubstances in the mouse colon with iron-containing stains quantitation of glandular gastric changes in rats given a proton pump inhibitor for 3 months with emphasis on sampling scheme selection aberrant crypt foci in the colonic mucosa of rats treated with genotoxic and nongenotoxic colon carcinogen the interactions of diet and colonic microflora regulating colonic mucosal growth selective inhibition of prostaglandin production in inflammatory exudates and gastric mucosa temporal relationship between cyclooxygenase inhibition, as measured by prostacyclin biosynthesis, and the gastro-intestinal damage iinduced by indomethacin in the rat a biochemical basis for the gastro-intestinal toxicity of non-steroid antirheumatoid drugs butylated hydroxianisole mechanistic data and risk assessment: conditional species-specific cytotoxicity, enhanced cell proliferation, and tumor promotion menetrier's disease presenting as iron deficiency anaemia radioautographic and quantitative studies on parietal and peptic cell kinetics in the mouse: a selective effect of gastrin on parietal cell proliferation intestinal adaptation. structural, functionaland cytokinetic changes instestinal adaptation. mechanisms of control mucosal mast cells of the rat intestine: a re-evaluation of fixation and staining properties with special reference to protein blocking and solubility of the granular glycosaminoglycan effect of mixtures of dietary fibres on the enzyme activity of the rat caecal microflora effect of prolonged metiamide medication on the fundic mucosa the membraneous epithelial (m) cell and the mucosal immune system medical progress: gastrointestinal toxicity of nonsteroidal antiinflammatory drugs nitrosatable drugs: an assessment of the risks assessing the safety of drugs for the long-term treatment of peptic ulcers the mechanism of mitrazepam-induced drooling and aspiration blood vessels of the peyer's patch in the mouse. iii high endothelial venules ultrastructural basis of intestinal absorption the effects of omeprazole and famotidine on mucin and pge 2 release in the rat stomach forestomach ulcers in crj:b6c3 (c57bl/6ncrj ã� c3h/hencrj) f1 mice forestomach ulcers in crj:b6c3 (c57bl/6ncrj ã� c3h/hencrj) f1 mice an evaluation of ampicillin pharmacokinetics and toxicity in guinea pigs drug-induced gastro-intestinal disease naturally occurring intestinal neoplasms in aged crl:cd â® br rats key: cord-265299-oovkoiyj authors: hickman, d.l.; johnson, j.; vemulapalli, t.h.; crisler, j.r.; shepherd, r. title: commonly used animal models date: 2016-11-25 journal: principles of animal research for graduate and undergraduate students doi: 10.1016/b978-0-12-802151-4.00007-4 sha: doc_id: 265299 cord_uid: oovkoiyj this chapter provides an introduction to animals that are commonly used for research. it presents information on basic care topics such as biology, behavior, housing, feeding, sexing, and breeding of these animals. the chapter provides some insight into the reasons why these animals are used in research. it also gives an overview of techniques that can be utilized to collect blood or to administer drugs or medicine. each section concludes with a brief description of how to recognize abnormal signs, in addition to lists of various diseases. the mouse is a small mammal that belongs to the order rodentia ( fig. 7 .1). the house mouse of north america and europe, mus musculus, is the species most commonly used for biomedical research. it is likely that the mouse originated in eurasia and utilized its commensal relationship with humans to spread through to other continents as humans explored and colonized. mouse fanciers around the turn of the 20th century are the source of the majority of the laboratory mice that are in use today. a summary of the overarching categories of mouse models that are available is presented in table 7 .1. physiology; and the possibility for breeding genetically manipulated mice and mice that have spontaneous mutations. mice have been used as research subjects for studies ranging from biology to psychology to engineering. they are used to model human diseases for the purpose of finding treatments or cures. some of the diseases they model include: hypertension, diabetes, cataracts, obesity, seizures, respiratory problems, deafness, parkinson's disease, alzheimer's disease, various cancers, cystic fibrosis, and acquired immunodeficiency syndrome (aids), heart disease, muscular dystrophy, and spinal cord injuries. mice are also used in behavioral, sensory, aging, nutrition, and genetic studies. this list is in no way complete as geneticists, biologists, and other scientists are rapidly finding new uses for the domestic mouse in research. mice are mammals and their organ systems are very similar to organ systems in humans in terms of shape, structure, and physiology. basic physiologic data are presented in table 7 .2. mice have very long loops of henle in the kidneys, thus allowing for maximal concentration of their urine. as a result, urine output in mice usually consists of only a drop or two of highly concentrated urine at a time. they also excrete large amounts of protein in their urine with sexually mature male mice excreting the largest levels of protein possibly as pheromones. mice have only two types of teeth, incisors and molars. the incisors are openrooted and erupt (i.e., grow) continuously throughout their lives. this predisposes mice to malocclusion if not given feeds or objects such as nylon bones to help wear down the teeth during mastication. the molars are rooted and, thus, do not continuously erupt. the stomach has two compartments with the proximal portion completely keratinized and the distal portion entirely glandular. their intestines are simple, but the rectum is very short (1e2 mm) and hence is prone to prolapse, especially if the animal has colitis. the gastrointestinal flora consists of more than 100 species of bacteria that form a complex ecosystem that aids digestion and health of the mouse. mice have no sweat glands but have a relatively large surface area per gram of body weight. this results in dramatic changes in physiology and behavior in response to fluctuations in ambient temperature. when too cold, mice will respond by nonshivering thermogenesis (i.e., metabolism of brown adipose tissue). in addition to the lack of sweat glands, they cannot pant or produce large amounts of saliva to aid in cooling their body temperature. therefore, when exposed to very hot situations, mice increase the blood flow to their ears to maximize heat loss; and in the wild, they move into their burrows which are at cooler temperatures. the thermoneutral zone, the range of ambient temperatures at which the mouse does not have to perform regulatory changes in metabolic heat production or evaporative heat loss to maintain its core temperature, is about 85.3 fe86.9 f (29.6 ce30.5 c). the female reproductive system is comprised of paired ovaries and oviducts, uterus, cervix, vagina, clitoris, and paired clitoral glands. pregnant female mice have hemochorial placentation, similar to humans (i.e., maternal blood is in direct contact with the chorion, the outermost layer of the fetal placental membranes). the female mouse also has five pairs of mammary glands. the male reproductive system consists of paired testes, penis, and associated sexual ducts and glands. the inguinal canals are open in the male mouse, and the testes can retract easily into the abdominal cavity. both sexes have well-developed preputial glands, which can become infected. males have a number of accessory sex glands, including large seminal vesicles, coagulating glands, and a prostate. secretions from these glands make up a large part of the mouse's ejaculate. when mice ejaculate, the semen forms a coagulum or copulatory plug. mice breed continuously throughout the year unless conditions are very unfavorable to them (e.g., lack of food). their reproductive potential can be affected by a number of external influences such as noise, diet, light cycles, population density, or cage environment. genotype also can affect reproductive performance as it is common knowledge that some inbred strains of mice are poor breeders, and if pups are born they may receive poor maternal care. additional reproductive physiologic data are presented in table 7 .3. mice can be bred using a one-on-one system (one male to one female; monogamous) or in a harem mating system (polygamous mating). in a monogamous system, the male and female are always left together, but at weaning the pups are removed from the cage. this system allows for maximal use of the postpartum estrus and the maximum number of litters for the females involved, and facilitates recordkeeping and monitoring of specific breeders in the colony. in a harem mating system, multiple males are placed with multiple females, usually at a ratio of one male to two to six females. usually, females are removed to separate cages just before parturition and the postpartum estrus is underutilized. mouse pups are born hairless, blind, and deaf and require extensive parental care that is provided mainly by the mother. due to the ruddy coloration of the skin of the hairless pups, they are also known as "pinkies." while mouse pups can increase their body temperature through the metabolism of brown fat stores, they are unable to adequately conserve body heat until they develop an adequate fur coat. thus, inclusion of nesting materials in the cage is highly recommended as huddling inside the nest can provide much needed warmth and safeguard against temperature-associated neonatal losses. the most reliable method for determining the sex of a mouse is by measuring the length of the anogenital distance, i.e., the distance from the anus to the genitalia. this distance can be measured with a ruler or animals assessed side by side with the rear ends held up by their tails. the anogenital distance is longer in males than females. in sexually mature animals one can also determine the sex of mice by the presence or absence of testicles in a testicular sac. mice are social creatures and can be group housed easily. their main method of communication is via pheromones. they use these olfactory cues to establish a pecking order (i.e., a hierarchical system of social organization). these chemicals are so important that when cage environments are changed, such as by simple cleaning or with bedding changes, a bout of fighting may occur until scent marking of the cage is completed as a way to reestablish the pecking order and social organization in that cage. pheromones also play a vital role in reproduction of these animals. this is demonstrated by the whitten effect and the bruce effect. the whitten effect occurs when a group of female mice that are not cycling are exposed to male urine, which contains a large quantity of pheromones. the females will all resume cycling as a group soon after the introduction of the male. in contrast, the bruce effect is characterized by abortion of litters when pregnant females are exposed to the urine of a strange male. as with most rodents, mice are nocturnal animals exhibiting peak levels of activity at night. because mice are a prey species, they display thigmotactic behavior or wall hugging. they avoid open spaces where they might be easily caught by predators. despite this, mice are very curious about any new objects in their territory and will often examine them at length. mice not only have poorly developed eyesight but they are also color blind. a number of inbred strains (e.g., fvb/n and c3h/he) are functionally blind by weaning. they rely on their very sensitive hearing to escape detection and on their sense of smell and taste to detect food (and possibly avoid poisons). mice can hear over a range of frequencies between 0.5 and 120 khz; however, normal mice are most sensitive to frequencies of 12e24 khz. it is important to note here that some inbred strains of mice, e.g., c57bl/6, suffer significant hearing loss before 1 year of age. mice can climb, swim, and jump (up to a foot), though they normally prefer to avoid swimming, if possible. under certain conditions they display stereotypies, which are obsessiveecompulsive behaviors. the behaviors may be strain-related, environment-related, or study-related and include wire gnawing, circling, jumping, and aggression. the use of environmental enrichment items such as cardboard tubes or other structures offer the animal an area for retreat from cage mates and add complexity to the environment. aggression is another important behavior that commonly occurs in group-housed male mice. it can also occur in group-housed females and mixed-sex cages. indications that there is an aggressive animal in the cage include bite wounds on the tail, rump, ears, and shoulders of mice ( fig. 7 .2). the wounds can be so severe as to cause significant blood loss and abscess formation at bite sites. aggression has been shown to be influenced by strain, age, and prior encounters. in terms of strains, the more aggressive strains are balb/c, c57bl/10, c57bl/6, dba/2, and outbred swiss. methods to prevent or reduce aggression include use of properly designed enrichment devices; provision of adequate space and shelter for each animal; grouping of mice before they reach puberty; use of docile strains; and removal of dominant animals as soon as possible. a common manifestation of social organization in group-housed mice is barbering, a behavior in which a dominant mouse will trim, by chewing, the hair or whiskers of other mice in the cage. barbering is also sometimes referred to as the dalila effect. it is usually instigated by a dominant male or female mouse. the dominant animals retain their whiskers and full hair coats, while their cage mates have "shaved faces and bodies" (fig. 7. 3) . although barbering does not generally result in any physical harm to the animal, removing the dominant mouse (the nonbarbered one) from the cage is a good approach to control. general types of housing mice in a laboratory setting include: conventional, specific pathogen free (spf), and germ free. in conventional housing, no attempt is made to keep out adventitious microbial and parasitic organisms. mice housed in this manner can be found in open-topped cages. room air, along with any airborne contaminants, is allowed to freely circulate into the mouse's cage. in addition, the food and water are not sterilized, though it should be noted that microbial contaminants may enter into the mouse population in this way. spf mice are raised in barrier conditions to ensure that they remain free of a specific list of pathogens. care is taken to ensure that adventitious microbes and parasites are excluded from the animals. spf mice are typically raised in specialized caging such as microisolator cages. these cages contain a 0.22 mm filter top that aids in the exclusion of microbes and parasites. individually ventilated caging systems include a rack of microisolator cages, each of which receives a filtered air supply ( fig. 7.4) . under spf conditions, everything that comes into contact with the animal should be sterilized or disinfected. this includes, but is not limited to, the water, food, bedding, and caging. special care must be taken by anyone handling the mice, including researchers, to ensure that handling and experimental procedures do not introduce potential pathogens into the colony. thus, all handling and procedures done on spf mice are often performed under hepa-filtered air conditions, such as within a biosafety cabinet. placing the mouse in an unfiltered environment ("room" air), even for a moment, is enough to potentially colonize the mouse with a whole host of adventitious microorganisms, thus destroying its spf status. once a contaminated mouse is placed back into the colony, the entire colony is at risk for infection. germ-free, or axenic, mice are raised to contain no microbes or parasites whatsoever. raising germ-free mice requires strict barrier maintenance. usually, this requires the use of flexible film isolators which provide hepa-filtered air to the mice within the isolator. additionally, any materials (e.g., food, water, and bedding) must be sterilized or thoroughly disinfected prior to being moved into the isolator unit so as not to contaminate the living space of the germ-free rodents with adventitious microorganisms. all procedures performed within the flexible film isolator must utilize strict aseptic technique for the same reason. in addition to the microbiological environment of the animal's housing systems, mice need to be housed at specific environmental parameters otherwise they may experience stress. the guide for the care and use of laboratory animals, 8th edition (national research council, 2011) is an internationally accepted document that outlines and discusses globally accepted environmental parameters for housing different species of animals including the mouse. table 7 .4 outlines the specific environmental requirements listed in this document for housing mice. mice are omnivorous and coprophagic with at least one-third of their diet being the consumption of their feces. in the laboratory setting mice are fed a clean, wholesome, and nutritious pelleted rodent diet ad lib. there are many commercially formulated diets for the various stages of life and for animals with specific induced diseases such as diabetes mellitus or hypertension. these diets may be available as autoclavable or irradiated forms to prevent transmission of disease via contaminated feed. there are also a variety of "pet" treats available for mice. however, the treats should not make up more than 5e10% of the daily diet. mice should be provided with a continuous supply of water daily. if the animals do not get enough water daily, their food consumption will decrease. the animals will also look scruffy and unhealthy. mice can be provided with water from water bottles or pouches, automatic watering systems with nipples, or water-based gel packs. some general signs of ill health include: weight loss, depression or lethargy, anorexia, obesity, diarrhea, scruffiness or ruffled coats, abnormal breathing, sneezing, weakness, dehydration, enlarged abdomen, discolorations (e.g., yellow for jaundiced animals or very pale for anemic animals), masses or swellings, and abnormal posture or gait. body condition scoring is an objective measure to truly assess how fat or thin the animal is and can be used for accurate determination of endpoints in studies where animals are expected to lose or gain weight ( fig. 7 .5) (ullman-cullere and foltz, 1999) . some of the more commonly found diseases of mice are presented in table 7 .5. noninfectious disorders are presented in table 7 .6. based on their genetic and physiologic makeup, mice can be either immunocompetent or immunodeficient. immunocompetent means that the mouse has a normal functioning immune system and can stage an immune response to any insult or injury. in contrast, immunodeficient means that some component or components of the mouse's immune system is not working or functioning normally, and so they cannot stage an adequate immune response and are more susceptible to infectious disease. immunosuppressed mice are mice that have a complete immune system but because of a drug or chemical or disease state, the immunological response is attenuated. rats and humans have a long history of coexistence. the origins of the laboratory rat, also known as the norway rat, stretch back centuries to the areas of modern day china and mongolia (burt, 2006; song et al., 2014) . the dispersal of the norway rat has occurred across the centuries and its natural habitat stretches from the mediterranean across southeast asia and down into australia and new guinea . unfortunately, most people associate rats with disease and destruction. throughout history, outbreaks of bubonic plague, typhus, and hantaviruses have had an unwitting accomplice in the rat (zinsser, 1935; benedictow, 2004; firth et al., 2014) . over the centuries, rats have also been used for food (e.g., in imperial china), companionship, and sport (gorn and goldstein, 2004; hopkins et al., 2004; burt, 2006) . ratting, a vicious blood sport where people laid bets on the dog that could kill the most rats in a given period of time was especially popular in both the victorian england and american underworld (thomas and mayhew, 1998; gorn and goldstein, 2004) . at the turn of the 20th century, breeding rats as a hobby or for companionship (i.e., "fancy rats") was recognized by the addition of "rat" to both the name and mission of national mouse club in the united kingdom (american fancy rat and mouse association, 2014). however, as interest in pet rats waned over the following years, the club reorganized and dropped "rat" from its name. a similar club, the american fancy rat and mouse association, was founded in the united states in 1983 (american fancy rat and mouse association, 2014). the first recorded use of rats as research subjects occurred in 1828 (hedrich, 2000) , and the first known rat breeding experiments occurred in the late 1800s (lindsey and baker, 2006) . the first major effort to perform research in the united states using laboratory rats occurred at the wistar institute of philadelphia, the oldest independent research institute in the united states, in 1894 (lindsey and baker, 2006) . rattus norvegicus constitutes one of the most commonly used laboratory species ( fig. 7 .6), second only to the laboratory mouse. because rats and mice are not included under the animal welfare act regulations, the precise number of these species used per year within the united states is unavailable. however, examining the data collected within the european union can give some indication of their use relative to other common laboratory animal species. in 2011, rats accounted for just fewer than 14% (1.6 million) of the total animals (11.5 million) used in research within the european union (european commission, 2013) . this contrasts to mice, which constituted 61% (6.9 million) of the total animals used within the european union (european commission, 2013). rats possess a number of qualities which make them a highly suitable and much preferred animal model. like mice, these traits include relatively small size; known genetic background; short generation time; similarities to human disease conditions; and known microbial status. their tractable nature makes them easier to handle in a laboratory setting than many other rodents. rats rarely bite their handlers unless extremely stressed or in pain. rats have been used as animal models in numerous areas of research from space exploration to answering more basic scientific questions regarding nutrition, genetics, immunology, neurology, infectious disease, metabolic disease, and behavior. perhaps their largest use is in drug discovery, efficacy, and toxicity studies. in the united states, the approval of any new drug for use in humans or animals usually necessitates that toxicity testing be done in at least one small animal species (e.g., rodents) and one large animal or target species (e.g., dog, nonhuman primate). there are known physiologic differences between the numerous outbred stocks and inbred strains of rats. the rat genome database (rgd) is an extensive, free resource filled with information regarding the different phenotypes, models, and genomic tools used in rat research (laulederkind et al., 2013) . vendors of commercially available rat stocks and strains are often good resources for normal physiologic data of these strains. many provide stock-and strain-specific data directly on their websites such as growth curves, complete blood count and serum biochemistry panels, and spontaneous lesions seen on histopathology. a summary table of normal physiologic references is in table 7 .7. sexual dimorphism exists between male and female rats. sexing of adult rats is most easily done by examining the perineal area of the rat and identifying the external reproductive structures such as the penis, testes, or vagina. in addition, male rats are typically larger and weigh significantly more than their age-and strainmatched female counterparts. sexing of rat pups is most easily performed by examining the distance between the anus and genital opening in the pup. males have a greater anogenital distance than females. male rats possess paired testicles that descend from the abdomen into the scrotal sac at approximately 15 days of age (russell, 1992) . due to the lack of closure of the inguinal rings, the testes may be retracted into the abdominal cavity even as an adult. the male rat also possesses a number of accessory sex glands. a four-lobed prostate is present along with four other paired glands, including: the seminal vesicles, coagulating glands, ampullary glands, and bulbourethral glands (noted in some texts by the older name, cowper's gland) (popesko et al., 1992) . due to the unusual bihorned shape of the closely associated coagulating gland and seminal vesicles, individuals unfamiliar with rodent male anatomy may initially mistake these structures for the female uterus. however, the apices of these glands are freely mobile and easily exteriorized from the abdominal cavity unlike the uterus, which is attached to the dorsal body wall bilaterally via paired ovaries and their respective ovarian ligaments. the reproductive anatomy of the female rat contains some distinct features. the uterus of the female rat is classified as a duplex uterus, because the vagina is separated from the uterus by two individual cervices with each cervix leading to a separate uterine horn (popesko et al., 1992) . the placentation of the pregnant rat is hemotrichorial (three layers) rather than the hemomonochorial (single layer) placentation present in humans (wooding and burton, 2008) . the rest of the reproductive anatomy (e.g., ovary, oviduct) is structurally and functionally similar to other mammals. a summary of basic reproductive physiology is presented in table 7 .8. rat pups are born hairless, blind, and deaf and require extensive parental care that is provided mainly by the mother. as with mice, the skin of the hairless rat pups has a pink coloration, thus they are also referred to as "pinkies." the inclusion of nesting materials in the cage is recommended to assist the rat pups with thermal regulation until they have a full hair coat (whishaw and kolb, 2005). like other rodents, rattus norvegicus is a nocturnal species with the highest level of activity occurring during the dark phase. behaviors exhibited by rats include grooming, nesting, eating, and other social behaviors. nesting behavior serves several purposes among rats and mice (gaskill et al., 2012 (gaskill et al., , 2013a . nests allow for better thermoregulatory control within a given environment as well as protection against predation (gaskill et al., 2013c) . recent work in mice suggests that energy not diverted to thermoregulation can be shunted to other activities as seen via improved feed conversion and breeding performance (gaskill et al., 2013c) . however, anecdotal evidence suggests that nest building in rats is largely a learned behavior, and it appears that there is a developmental period in young rats whereupon if exposed to nesting materials during this time they will begin using the materials to build at least rudimentary nests (gaskill, 2014) . at a minimum, rats benefit from having a structural shelter or nest box into which they may rest away from prying eyes ( fig. 7 .7). rats emit alarm vocalizations during times of distress. however, these negatively associated vocalizations typically register in the ultrasonic wavelengths (approximately 22 khz), well outside of the human hearing range (burman et al., 2007; parsana et al., 2012) . rodents may also emit high-pitched audible vocalizations when extremely alarmed, distressed, or in pain (jourdan et al., 1995; han et al., 2005) . as discussed in chapter 5, rats exhibiting abnormal behaviors and stereotypies can create variables in the research findings and should not be used in a study unless abnormal behavior is the object of the study subjects (baenninger, 1967; callard et al., 2000; garner and mason, 2002; cabib, 2005; ibi et al., 2008) . examples of stereotypies seen in rats include: bar-gnawing, pawing behavior, repetitive circling, and backflipping. it is critical that rats should be provided some form of environmental enrichment to stimulate positive species-typical behaviors. the housing of rats in a laboratory setting is similar to that described previously for mice: conventional, spf, and germ free. as rats are social animals, at minimum, they should be housed in pairs whenever possible. there is a preponderance of evidence that shows the differences in affiliative versus aggressive behavior, biochemical changes, and changes in learning between rats raised and housed in social isolation versus those housed in social groups (baenninger, 1967; einon and morgan, 1977; robbins et al., 1996) . enrichment items, such as a hut, nesting box, or similar type of shelter may be included in the cage to provide a visual barrier between the rat and the rest of the animal room. evidence suggests that rats prefer shelters made from opaque plastic (patterson-kane, 2003) . rats also spend a lot of time in the wild chewing either for eating or for manipulating objects for nest building. providing objects made of safe materials in the cage allows the rats to exhibit this natural behavior and encourage the normal wear of the rat's incisors, minimizing the incidence of malocclusion of the teeth. rodents can benefit from frequent gentle handling by the researcher and animal care staff. this concept is also known as "gentling" and has been demonstrated to reduce the stress experienced by rats during experimental handling and procedures (hirsjarvi et al., 1990; van bergeijk et al., 1990) . another positive interaction between humans and rats is found in the "tickling" of rodents. based on ultrasonic vocalization data, rodents find tickling a pleasurable experience (burgdorf and panksepp, 2001; panksepp, 2007; hori et al., 2014) . tickling may also decrease the stress response seen in rodents after experimental manipulations like intraperitoneal injections (cloutier et al., 2014 ). a multilevel cage with an intracage shelter. this style of caging provides opportunities for exercise for the rats. photo provided by melissa swan. as for mice, some general signs of ill health would include: weight loss, depression or lethargy, anorexia, obesity, diarrhea, scruffiness or ruffled coats, abnormal breathing, sneezing, weakness, dehydration, enlarged abdomen, discolorations (e.g., yellow for jaundiced animals or very pale for anemic animals), masses or swellings, and abnormal posture or gait. when assessing animals, a body condition score can be used as an objective measure or scale to truly assess how fat or thin the animal is; and it allows for the accurate determination of endpoints in studies where animals are expected to lose or gain weight (hickman and swan, 2010) (fig. 7.8) . some of the more commonly found diseases of rats are presented in table 7 .9. the ancestral home of the european rabbit (oryctolagus cuniculus) is the iberian peninsula (hardy et al., 1995) . the earliest archeological evidence of the coexistence of humans and rabbits can be found in excavation sites dated at approximately 120,000 years bce in nice, france (dickenson, 2013) . in antiquity, romans used rabbits as a food source and are thought to be responsible for their dispersal throughout europe, although there is no evidence that they attempted to actually domesticate them (dickenson, 2013) . domestication and selective breeding is thought to have begun in france in the middle ages where monks began to breed rabbits in their monasteries (dickenson, 2013) . the rabbits were kept confined in enclosures called "clapiers"(dickenson, 2013). they were kept largely as a source of food for the monks especially since 600 ce when pope gregory i officially classified them as "fish" and thus eligible to being eaten during lent. however, rabbit wool production soon became a welcome by-product of these domestication efforts. european rabbits have been used in research since the middle of the 19th century. early work with the species was concentrated on the comparative anatomy of the rabbit with other species, such as the frog, and the unique features of the rabbit's heart and circulatory system (champneys, 1874; roy, 1879; smith, 1891) . louis pasteur used rabbits in a series of experiments that led to the development of the world's first rabies vaccine (rappuoli, 2014) . while there are numerous so-called "fancy" breeds of rabbits available in the pet trade, the list of breeds routinely used in research is much shorter. the new zealand white (nzw) rabbit is the most frequent breed of used in research (fig. 7.9 ). the california and dutch-belted rabbit breeds are also occasionally used. researchers have developed genetically inbred rabbit strains for particular research applications. for example, the watanabe heritable hyperlipidemic (whhl) and the myocardial infarction-prone whhl rabbit (whhlmi), both developed by researchers in japan, are used to explore diseases associated with dyslipidemia such as atherosclerosis (shiomi et al., 2003; shiomi and ito, 2009) . rabbits have been used as a model of human pregnancy and for the production of polyclonal antibodies for use in immunology research (hanly et al., 1995; ema et al., 2010; ito et al., 2011; fischer et al., 2012) . rabbits are routinely used in (southard et al., 2000; arslan et al., 2003; mcmahon et al., 2005; castaneda et al., 2008; habjanec et al., 2008; manabe et al., 2008; xiangdong et al., 2011; panda et al., 2014; sriram et al., 2014; wei et al., 2014; zhou et al., 2014) . the production of polyclonal antibodies is preferentially performed in the rabbit due to its relatively large blood volume compared to rodents (hanly et al., 1995) . their tractable nature and larger body size also make them suitable for surgical implantation of biomedical devices (gotfredsen et al., 1995; swindle et al., 2005; ronisz et al., 2013) . additionally, rabbits are a favored model in pharmacologic studies for teratogenicity testing of novel pharmaceutic compounds (gibson et al., 1966; lloyd et al., 1999; foote and carney, 2000; jiangbo et al., 2009; oi et al., 2011) . while much of the anatomy of the rabbit is similar to other mammalian species, it should be noted that there are a number of key differences. for example, the skin of the rabbit is quite thin and fragile. care should be taken when restraining a rabbit or shaving a rabbit's fur (e.g., in preparation for surgery) to avoid tearing the skin. unlike rodents and other laboratory animals, rabbits do not have pads on their feet; rather, the plantar surface is covered with fur (quesenberry and carpenter, 2012). new zealand white rabbits are commonly used in research. photo provided by kay stewart. the long ears of the rabbit serve several purposes. the most obvious is for hearing. in addition, the central ear artery and marginal ear veins are easily accessible for both intravenous administration and blood sampling (diehl et al., 2001; parasuraman et al., 2010) (fig. 7.10) . the ears also serve as a means of thermoregulation, as excess heat may be exchanged across the large surface area of the ears (sohn and couto, 2012) . the skin of rabbits lacks sweat glands and is therefore unable to sweat; panting is insufficient to dissipate the excess heat (sohn and couto, 2012) . thus, the ears play a vital role in maintaining proper body temperature. other unique features of the skin and adnexa are the presence of chin and inguinal glands used in scent marking. additionally, the female rabbit (doe) is noted by the presence of a large skin fold filled with subcutaneous fat just under the chin (the dewlap) (sohn and couto, 2012) . the skeleton of rabbits makes up only 8% of the body weight by mass (brewer, 2006) . this is in contrast to other similarly sized mammals. for example, the cat skeleton makes up 12e13% of body weight (brewer, 2006) . the small skeletal mass of the rabbit coupled with strong back muscles mean that the back is prone to traumatic fracture (meredith and richardson, 2015) . proper holding and restraint techniques are necessary to avoid this undesirable outcome. there are several unique features of both the respiratory and cardiovascular systems of rabbits. for example, rabbits are obligate nose breathers (varga, 2014) . this is especially important during procedures involving anesthesia and placement of an endotracheal tube. with respect to the cardiovascular system, the rabbit heart is unique in that the right atrioventricular (av) valve has only two leaflets instead of three (brewer, 2006) . additionally, due to the similarity to humans with respect to the neural anatomy of the ventricles, the rabbit is the species of choice for purkinje fiber research (brewer, 2006) . rabbit teeth are "open-rooted" meaning that they continue to erupt and grow throughout life. this applies to all of the teeth in the rabbit dental arcade (i.e., incisors, premolars, and molars; rabbits do not have canines). this contrasts with rodents, where the incisors are the only open-rooted (or hypsodontic) teeth (sohn and couto, 2012) . thus, rabbit teeth are subject to overgrowth. another unique feature of rabbit dentition that sets them apart from rodents is the presence of a second set of incisors just behind the first set of upper incisors known as "peg" teeth (sohn and couto, 2012) . they are thought to aid in tearing off the succulent leaves of plants while grazing. as an obligate herbivore, the gastrointestinal tract of rabbits differs greatly from that of carnivores and omnivores. rabbits require a high fiber diet of between 14 and 20% (sohn and couto, 2012) . the small intestine is divided up into three main sections: the duodenum, jejunum, and ileum. the ileum connects to the cecum via a structure called the sacculus rotundus. the presence of lymph follicles suggests that the sacculus rotundus has immunological functions. it is sometimes referred to as the ileocecal "tonsil" (jenkins, 2000) . in the rabbit, gastric associated lymphoid tissue is also present in the small intestine and the vermiform appendix (lanning et al., 2000) . the cecum, a large distensible outpouching of the large intestine, holds up to an estimated 40% of the total ingesta (sohn and couto, 2012) . rabbits are considered to be "hindgut fermenters." bacteria present in the cecum ferment the digestible fiber found within the diet. the product of this fermentative process becomes cecotrophs (also known as "night feces"). cecotrophs are excreted roughly 8 h after the initial foodstuffs are ingested (sohn and couto, 2012) . they are softer and more mucoid in appearance than the hard, dry "day feces" produced just 4 h after consuming food (sohn and couto, 2012) . the bulk of day feces consists of the indigestible fiber found in the diet. the sorting of foodstuffs destined to become either day feces or cecotrophs and the timing of their relative excretion is largely dependent upon the neural input of the fusus coli, also termed the "pacemaker" of the colon (sohn and couto, 2012) . the fusus coli is anatomically located between the ascending and transverse colons of the rabbit (popesko, 1992) . consumption of cecotrophs by rabbits is an important part of the digestive process in rabbits as they are rich in b vitamins, such as niacin and b12, and vitamin k (hã¶rnicke, 1981) . while cecotrophs are known colloquially as "night feces," rabbits produce and eat them at all hours of the day (sohn and couto, 2012) . rabbits are agile enough to eat these night feces directly from their anus (sohn and couto, 2012) . those researchers performing digestive research (e.g., fecal collection via metabolism cages) should keep this in mind. sexing of adult rabbits is aided by the sexual dimorphism present in the species. mature females are readily identified by the presence of the dewlap (sohn and couto, 2012) . females have 8e10 nipples, while in males these nipples are present, but rudimentary (sohn and couto, 2012) . nzw rabbits reach sexual maturity between 5 and 7 months (suckow et al., 2002) . reproductive data are summarized in table 7 .10. mature bucks have paired testicles enclosed in paired hairless scrotal sacs (sohn and couto, 2012) . like rodents, the inguinal rings do not close. accessory sex glands include several bilobed organs: the seminal vesicle, vesicular gland, prostate, and paraprostatic gland. the bulbourethral glands of bucks are paired (sohn and couto, 2012) . female rabbits are induced ovulators (dal bosco et al., 2011; sohn and couto, 2012) . that is, the egg does not ovulate spontaneously from the ovary, rather manual stimulation via copulation is required. ovulation occurs approximately 10 h postcopulation (sohn and couto, 2012) . because they are induced ovulators, does do not have a defined estrous cycle. rather, they have periods of sexual receptivity lasting approximately 14e16 days followed by 1e2 days of nonreceptivity. nonfertile matings may result in a period of pseudopregnancy of up to 15e16 days (sohn and couto, 2012) . fertile matings result in pregnancy lasting 31e32 days (sohn and couto, 2012) . the placenta of rabbits is classified as hemodichorial; this is in contrast to humans which have a hemomonochorial placenta (furukawa et al., 2014) . birthing (i.e., parturition; also known as "kindling") occurs most often during the early morning hours (sohn and couto, 2012) . kits are born deaf and blind. by 7 and 10 days of age they can hear and see, respectively (quesenberry and carpenter, 2012) . amazingly, does suckle their young ones daily, usually during the dark phase, and for approximately only 5e6 min (sohn and couto, 2012) . the kits are able to drink about 30% of their entire body weight in that time. wild and domesticated does both follow this nursing behavior. rabbit kits may be weaned between 5 and 8 weeks of age (suckow et al., 2002) . earlier weaning should not be attempted as there may be profound detrimental effects on the functioning of the gastrointestinal system (bivolarski and vachkova, 2014). rabbits are very social, nocturnal creatures. scent marking is a normal and important part of their behavior repertoire. rabbits will rub the secretions from their chin scent glands against inanimate objects, other rabbits, and human handlers in a process called "chinning" (sohn and couto, 2012) . dominance hierarchies are established behaviorally. dominant animals may mount, "barber," or scent-mark subordinates (sohn and couto, 2012) . barbering is the act of chewing the hair of a subordinate animal, usually on the neck and back in the case of rabbits, very close to the skin giving the appearance of having been cut or "barbered" (bays, 2006). rabbits will "thump" one or both back feet on the ground when frightened or as an alarm call to other rabbits (bays, 2006) . highly stressed rabbits may actually emit a loud, piercing scream, especially when roughly caught by an untrained individual (bays, 2006) . it is important to approach rabbits calmly and quietly. relaxed, content rabbits may be heard making a purring sound (bays, 2006) . rabbits benefit by repeated, positive interactions with people similar to the concept of "gentling" in rats (see section 7.2). changes in behavior are often first indication that an animal is in pain. given that rabbits are a prey species, it is evolutionarily speaking not in a rabbit's best interest to display signs of pain. thus, these behaviors are most often quite subtle in nature and may be easily missed if particular attention is not paid. the first sign often seen in a rabbit experiencing pain is a decreased appetite resulting in little to no food intake (sohn and couto, 2012) . rabbits will often grind their teeth (i.e., bruxism) when experiencing pain (sohn and couto, 2012) . other rabbits may simply appear very dull and inactive. as with rodents, rabbits can develop stereotypies. due to the sensitivity of the rabbit's nose and lips many stereotypies involve chewing behaviors. bar chewing, chewing on the water bottle, and self-barbering are all stereotypical behaviors seen in rabbits (gunn and morton, 1995; chu et al., 2004) . in addition, rabbits may engage in "nose sliding" against solid surfaces like the cage walls and head swaying (sohn and couto, 2012) . animals that exhibit stereotypies do not make good research animals. efforts should be made, where possible, to prevent these behaviors through the use of environmental enrichment. enrichment may be in the form of chew-resistant objects (such as plastic dumbbells and stainless steel rattles) and food treats (poggiagliolmi et al., 2011). as a prey species, rabbits benefit from the inclusion of a hut in the cage or at least a visual barrier into which they may retreat when psychologically stressed (baumans, 2005) . breeding females should always have access to a nest box to allow for the necessary expression of normal nesting behavior (baumans, 2005) . being social creatures, ideally rabbits should be housed in compatible pairs or trios unless contraindicated by the research objectives or by incompatibility of the animals (sohn and couto, 2012) . stable social groups formed shortly after weaning, where animals are not added or removed, is most beneficial (boers et al., 2002) . structurally, rabbits benefit from housing that has both adequate vertical and horizontal space (boers et al., 2002) . one recommendation on the space requirements of laboratory rabbits stipulates 16 in. as the minimum vertical cage height (national research council, 2011) . at a minimum, rabbits must be able to comfortably sit upright in the cage without their ears bending over (national research council, 2011) . laboratory rabbits are typically housed in easily sanitized stainless steel cage racks ( fig. 7.11 ). slatted flooring allows for urine and feces to fall through the slats onto special pans fixed below the cage, thus providing for easier sanitation of the cages. however, care must be taken that the slats are of sufficient width so as to prevent a condition known as bumblefoot (see section 7.3.7). dog runs with elevated, slatted flooring or a solid floor with bedding have also been used by some investigators in group-housed rabbits with success (personal observations). again, attention should be paid to the flooring and its effect on foot health. commercially available research diets specifically formulated for rabbits are available. these diets are preferred to so-called "natural diets" and feeding individual vegetables. this is because rabbits tend to be very selective eaters which can lead to nutritional imbalances (fraser and girling, 2009 ). additionally, the use of fresh vegetables may lead to the introduction of unwanted pathogens like salmonella (varga, 2014) . commercial diets are available in maintenance and reproductiveperformance dietary formulations as well as presterilized diets for rabbits housed under spf conditions. rabbits are very easily heat stressed and thus must be kept at significantly lower temperatures than other laboratory animals like rats and mice. noise is another significant stressor to rabbits (verga et al., 2007) . sudden, high-pitched, sharp noises are most disruptive. however, in general, noise within the animal rooms should be avoided as much as possible. for this reason, rabbits should not be housed, even temporarily for short procedures, near areas of high noise. problems in rabbits related to the gastrointestinal system are relatively common. these problems can become serious very quickly. therefore, it is critical that abnormalities seen (e.g., rabbit not eating or abnormal feces) be reported to the veterinary care staff immediately. even if a researcher is unsure if there is a problem, it is best to report suspicions because without prompt intervention, seemingly minor problems can escalate to potentially life-threatening conditions. some of the most commonly seen clinical conditions in rabbits are summarized in table 7 .11. the zebrafish, danio rerio of the cyprinidae family, is a small, dark blue and yellow striped, shoaling, teleost fish, popular among aquarium enthusiasts, and increasingly among the research community (fig. 7.12) . the adult fish are 4e5 cm in length, with an incomplete lateral line and two pairs of barbels (laale, 1977) . males have larger anal fins and more yellow coloration; females have a small genital papilla just rostral to the anal fin (laale, 1977; creaser, 1934) . zebrafish are hardy, fresh water fish originating from a tropical region with an annual monsoon season. the fish are generally found among slow moving waters of rivers, streams, and wetlands, across the south asia region of india, bangladesh, example of rabbit caging for a laboratory setting. photo provided by deb hickman. and nepal (engeszer et al., 2007; spence et al., 2008) . the waters tend to be shallow, relatively clear with substrates of clay, silt, or stone of varying size (mcclure et al., 2006; engeszer et al., 2007) . the fish feed mostly on insects and plankton, with evidence of feeding along the water column as well as water surface (mcclure et al., 2006; engeszer et al., 2007; spence et al., 2008) . gastrointestinal upset generally secondary to the use of broad spectrum antibiotics, such as penicillin. misalignment and subsequent overgrowth of the continuously growing teeth pododermatitis ("bumblefoot") infection of the underside of the feet pasteurella multocida common cause of respiratory infections and abscesses male zebrafish have a more stream-lined body with darker blue strips while the females have a white protruding belly. photo provided by kay stewart. the small size of zebrafish, the ease of keeping large numbers, frequent spawning, large egg clutches, translucent nonadherent eggs, rapid development and complex sequencing of the zebrafish genome are all key components that make the zebrafish an attractive research model. interestingly, approximately 70% of zebrafish genes have at least one orthologous human gene (howe et al., 2013) . publications on the use of zebrafish in research are cited as early as the 1930s (creaser, 1934) . until the early 1970s, the use of zebrafish stayed fairly low, with the number of articles published staying below 20 per year. in the mid-1970s, publications increased to about 40 per year, doubling again in the 1980s, increasing to almost 200 articles per year in the early 1990s, and rapidly expanding to 1929 publications by 2012. developmental biology was the initial focus of zebrafish research use. however, in recent years, use of the zebrafish in research related to biochemistry and molecular biology, cell biology, neurological sciences, and genetics has been rapidly increasing. zebrafish are known to live for only a year in the wild (spence et al., 2008) . for most of the year, the fish reside in shallow streams. with the onset of monsoon rains, they move to flooded, highly vegetated shallow wetlands and floodplains, including rice paddies, with little to no current and often silt bottoms for spawning (engeszer et al., 2007) . the offspring then develop in these waters until the seasonal waters diminish (engeszer et al., 2007) . zebrafish rapidly mature, reaching sexual maturity as early as 2 months postfertilization . the zebrafish continues to grow throughout life, which is much longer in captivity, with a mean lifespan of 3.5 years in captivity (gerhard et al., 2002) . in nature, spawning behavior occurs within small groups of three to seven fish. males within the group pursue females, with spawning occurring along the substrate (spence et al., 2008) . similar behaviors are noted in laboratory zebrafish, with spawning often occurring with the first light of day. courtship behavior involves a rapid chase of the female, the male swimming around the female, nudging her, or swimming back and forth working the female to the spawning site. interestingly, zebrafish prefer spawning near artificial plants. once there, the male remains close to the female, extending his fins to bring his genital pore in line with the female. the male may also rapidly undulate his tail against the side of the female to initiate egg release by the female, coinciding with sperm discharge by the male. the female produces eggs in batches of 5e20 over several encounters with the male for up to an hour. most eggs are released within the first 30 min, with a peak in production during the first 10 min (darrow and harris, 2004; spence et al., 2008) . zebrafish produce large clutches of eggs, from 150 to 400 eggs per clutch (laale, 1977) . the eggs, approximately 0.7 mn in diameter, are transparent and protected within a chorionic membrane (kimmel et al., 1995) . first body movements and beginning stages of organ development occur 10e24 h postfertilization (kimmel et al., 1995) . as development continues, the larva hatches from the egg two to three days postfertilization (kimmel et al., 1995) . the early larva has special secretory cells within multicellular regions of the head epidermis that allow the larvae to attach to various hard surfaces and plants until the swim bladder inflates 4 or 5 days postfertilization (laale, 1977; kimmel et al., 1995) . once the air bladder inflates, the fish can maneuver through the water column. in captivity, zebrafish can breed year round. the presence of males, or even just the male pheromones, is needed to induce ovulation (gerlach, 2006) . if females are housed away from males for an extended period, they can retain the eggs resulting in egg-associated inflammation, which can be lethal (kent et al., 2012a) . to accommodate the fish life cycle, zebrafish are typically housed in static spawning cages to allow for fertilized egg production. spawning cages include a housing tank containing a clear slotted bottom insert, and a plastic plant. the insert is often placed in the holding tank at an angle to create a shallow region for spawning and the slotted bottom of the insert allows for ease of egg collection (lawrence and mason, 2012; nasiadka and clark, 2012) (fig. 7.13 ). the embryos are then incubated at around 28.5 c in a petri dish for at least 3e4 days postfertilization (wilson, 2012) . the fish are then kept in static or slow water flow containment and can be fed paramecium, rotifer, a powdered food, or a combination of these feed types. unfortunately, other than the need for essential fatty acids in their diet, little is yet known on the nutritional requirements of zebrafish. zebrafish in research settings are typically fed live feed like artemia (brine shrimp), rotifers, bloodworms example of a zebrafish spawning system. the system is designed to allow eggs to fall beneath a slotted insert to the bottom of the tank as a way to prevent the adult fish from consuming the eggs. photo provided by robin crisler. (chironomid larvae), commercial feed, or a combination of all (lawrence, 2007) . the size of the feed is necessary to suit the gape size of the larvae, approximately 100 mm (lawrence, 2007; wilson, 2012) . water flow and feed size increase with development, with transition of the feed to artemia (brine shrimp), and/or use of a larger particle commercial feed during the 8e15 days postfertilization (wilson, 2012) . once the juvenile stage is reached, around 29 days postfertilization, the fish are housed more like adult fish, with more frequent feeding and slower water flow to accommodate their remaining development and smaller size, respectively (wilson, 2012). as early as 2 months of age the fish are sexually mature . adult zebrafish can be housed in traditional glass aquaria or elaborate computerized and automated systems that monitor and control water quality parameters such as temperature (typically 28.5 c), ph, water hardness, salinity, dissolved oxygen, and nitrogenous wastes (lawrence, 2007; lawrence and mason, 2012) . whether maintained manually or computerized, these parameters are important to monitor and maintain at appropriate levels to maximize the health of the fish. poor water quality can lead to disease in the fish (kent et al., 2012b) . many of the organisms that cause disease in zebrafish are opportunists in the environment and remain subclinical until the fish is stressed, often due to problems with husbandry. appropriately maintained housing, combined with a healthy water quality, avoidance of overcrowding, and a functional quarantine and health surveillance program are key components to avoiding stress and disease. to date, there are currently no known viruses documented in zebrafish as naturally occurring disease concerns (kent et al., 2012b) . mycobacterium infections are the most frequently documented bacterial infections (kent et al., 2012a,b) . class reptilia is made up of four orders classified as chelonia, rhynchocephalia, squamata, and crocodilia (frye, 1991) . in class amphibia, animals more commonly encountered in research setting are in the order anura, containing frogs and toads (such as xenopus, bufo, rana, hyla, and dendrobates spp.); and in the order caudata, containing salamanders such as the tiger salamander, ambystoma tigrinum and the axolotl, ambystoma mexicanum (national research council, 1974) (figs. 7.14 and 7.15). snakes and lizards are in class reptilia, order squamata; chelonians (turtles, tortoises, terrapins) are in order chelonia; and alligators, caimans, and crocodiles are in order crocodilia. in contrast to research in mammals, there is a tendency for reptile and amphibian research to be more oriented to studying evolution and ecology as opposed to basic science evaluating models of human disease (pough, 1991) . salamanders and frogs are important for studying embryonic development, metamorphosis, regeneration, figure 7.14 a commonly used amphibians in research is the axolotl, ambystoma mexicanum. provided by chris konz. african clawed frogs, xenopus laevis, a commonly used amphibian. provided by randalyn shepherd. physiology, and climate change (burggren and warburton, 2007; hopkins, 2007; pough, 2007) . reptiles are often studied because of their more simple cardiovascular systems as well as for evaluating mechanisms of immune responses, hormonal controls, and unique reproduction methods such as parthenogenesis (frye, 1991) . of the amphibians, xenopus laevis (south african clawed frog) and xenopus tropicalis (western clawed frog) are commonly studied in the research setting. x. laevis is a prominent research model in comparative medicine and developmental studies, and is the most commonly studied species in the genus xenopus (denardo, 1995; schultz and dawson, 2003; o'rourke, 2007) . advantages include large-sized eggs for ease of observing embryo development, as well as the wealth of published literature in areas of research such as evolution, neurobiology, regeneration, endocrinology, and toxicology (koustubhan et al., 2008; gibbs et al., 2011) . rana catesbeiana (bullfrogs) have been used for developmental and toxicological studies, and for infectious disease study of the chytrid fungus batrachochytrium dendrobatidis (alworth and vazquez, 2009) . a. mexicanum, in particular, is studied to understand the regenerative ability of the blastema of amputated limbs at the molecular level (gresens, 2004; rao et al., 2014) . ambystoma tigrarium has been studied in regard to general amphibian decline in north america, environmental contaminants such as pesticides and effects of infection with a. tigrarium virus (sheafor et al., 2008; kerby and storfer, 2009; chen and robert, 2011; kerby et al., 2011) . a variety of snakes, crocodiles, lizards, and turtles have been studied in research. for example, anolis carolinensis (the green anole) has been used for the study of reproduction biology (lovern et al., 2004) . caiman crocodilus and alligator mississippiensis (crocodiles), trachemys scripta elegans (red-eared sliders) represent a few other examples of reptiles used in research (o'rourke and schumacher, 2002). amphibians and reptiles are considered to be ectotherms (greene, 1995) . unlike mammals and birds, ectotherms are unable to internally regulate body temperatures above that of the ambient environment through metabolism and require complex behavioral and thermoregulatory adaptations to regulate temperature (pough, 1991; seebacher and franklin, 2005) . in captivity, ectotherms typically require supplemental sources of heat to mimic the thermoregulatory effects of basking in the sun. some amphibians and reptiles are aquatic (xenopus frog spp.) whereas others are semiaquatic or terrestrial. x. laevis and x. tropicalis are from geographically distinct areas and have different temperature requirement depending on life stage, with x. tropicalis adults generally around 25 c in their natural habitat versus about 20 c for x. laevis (tinsley et al., 2010) . the skin of amphibians is permeable to water and some adults (semiterrestrial tree frogs in family hylidae, arboreal and terrestrial toads in bufonidae) may receive a significant portion of their daily water requirement via absorption through a vascular-rich area on the pelvic area termed the pelvic patch (pough, 2007; ogushi et al., 2010) . the skin of some amphibians contains toxins which can cause arrhythmias in human handlers, for example, alkaloids from dendrobatid frogs, and bufotoxins from toads of the genus bufo (denardo, 1995) . the toxins serve to keep predators away but, as with xenopus, may harm the animals themselves by continued direct contact or diffusion through the water (tinsley et al., 2010; chum et al., 2013) . the skin of amphibians is easily damaged thus to protect the animal during handling powder-free gloves should be worn (gentz, 2007) . researchers and animal care providers should investigate the natural environment of each species within their care and critically evaluate what features are required for normal behavior and physiology to provide the essential elements in the research setting (pough, 1991) . in the wild, amphibians and reptiles live in ecological environments that span a range of diversity from topical forest areas to dry desert. they may be arboreal, aquatic, or terrestrial. they are often secretive and hide when in natural habitats, preferring to hide under vegetation or in crevices. parameters from the natural habitat to evaluate include temperature, humidity, nutritional requirements, natural diet, nocturnal versus diurnal behavior, and housing density. temperature and lighting gradients should be established so animals can choose to move toward or away from the heat source as a way to avoid overheating. most amphibian species in the wild are nocturnal (pough, 2007; tinsley et al., 2010) . amphibians and reptiles are sensitive to chemicals in the environment. water quality parameters (such as ph, hardness, ammonia, nitrate/nitrite, salinity, conductivity) should be regularly monitored. chloramine and chloramines are often present in municipal water supplies and are toxic to aquatic species. water should be treated prior to use for aquatic species with an agent like sodium thiosulfate, since chloramine does not readily dissipate (browne et al., 2007) . ammonia is a breakdown product between the chloramine and sodium thiosfulfate reaction and is a concern for aquatic animals (browne et al., 2007; koustubhan et al., 2008; o'rourke and schultz, 2002) . a wide variety of caging materials may be used for housing such as glass, plastic, stainless steel, or fiberglass but should be free of contaminants or harmful chemicals like bisphenol a that could leach from the caging into the water (levy et al., 2004; browne et al., 2007; bhandari et al., 2015) . agents used to sanitize caging should be chosen to minimize likelihood of harmful residues. environmental enrichment should be provided to encourage natural behaviors and can include providing cage mates for social interaction, cage accessories that serve as hiding spots or shelters (fig. 7 .16) as well as providing a variety of food treats in changing locations for foraging opportunities (hurme et al., 2003) . scents, sounds, and color choices may also be incorporated into enrichment strategies provided that they are carefully evaluated to ensure that they are beneficial and do not cause stress. for example, the tortoise, chelonoidis denticulata, may show a color preference for red-colored enrichment items (passos et al., 2014) . pvc tubes are another example of enrichment that has been provided to x. laevis for use as hiding cover (koustubhan et al., 2008) . some species may require haul-out ramps, areas for sun basking, floating rest areas, or enrichment devices along the water's surface to help prevent drowning. one should consider the possibility of ingestion, as reptiles and amphibians may attempt to consume the substrates provided to them. the degree to which amphibians are social varies significantly depending on the species and is not always well understood. they use visual and olfactory discrimination to help them find food, forage, and avoid predators (vitt and caldwell, 2014) . both in the wild and in captivity, reptiles and amphibians may exhibit excitatory behavior when fed (sometimes described as a "feeding frenzy") which may result in animal injury where animals are in close proximity (divers and mader, 2006; tinsley et al., 2010) . overcrowded tanks can result in competition for food and subsequent trauma. thus, when placed together for the first time, animals should always be observed for compatibility; and only members of the same species should be housed together. many reptiles and amphibians are escape artists and prevention of escape and injury is a critical factor when considering housing design. species that are prone to jumping must have secured lids on their enclosures. the diets of amphibians and reptiles are highly variable in the wild and are species dependent. commercially prepared pelleted diets may be available and accepted by reptiles and aquatic amphibians, however, terrestrial amphibians and many use of a rabbit feeder for xenopus enrichment. photo by randalyn shepherd. reptiles may prefer live diets (pough, 2007) . it is not unusual for some species to go several days of fasting between meals in nature (pough, 1991) . consultation with those experienced at successful housing and feeding the species in question (zoos, nutritionists, herpetologists) is recommended. there are many different types of infectious agents such as bacteria, viruses, fungi, and parasites that can cause health problems in amphibians and reptiles in addition to noninfectious conditions such as those resulting from nutritional imbalances, metabolic disease, neoplasia, trauma, and other spontaneous maladies. although significant advances in knowledge have been made over the past 100 years regarding disease in these species, much still remains unknown. it is not possible to go into detail here, but there are excellent reference texts for diseases in amphibians and reptiles that can be consulted (jacobson, 2007; frye, 1991; wright and whitaker, 2001) . from a taxonomic standpoint, birds are placed into class aves which includes multiple orders based on anatomical, physiological, and genetic characteristics. passeriformes is the largest order and contains songbirds and perching birds such as the finch, canary, and cardinal ( fig. 7.17 ). order columbiformes contains pigeons and doves; order psittaciformes contains budgies and parrots such as the african gray; and order galliformes contains domestic fowl such as the chicken and quail (proctor and lynch, 1993; ritchie et al., 1994) (fig. 7.18 ). birds have been used as research models of human disease and are important in evaluation of aging, memory, parasitology, atherosclerosis, reproduction, and infectious disease among other topics (austad, 1997 (austad, , 2011 maekawa et al., 2014) . the genomes of several avian species have now been sequenced (jarvis et al., 2014) . historically, chickens (gallus domesticus) are the most common bird species studied in biomedical and agricultural research and are a classic model in areas such as immunology, virology, infectious disease, embryology, and toxicology (scanes and mcnabb, 2003; kaiser, 2012) . chickens are also studied to evaluate reproductive development and retinal disease. embryonated chicken eggs have been used to commercially produce vaccines (such as for human influenza), studied for developmental analysis, and are now being treated with viral vectors like lentivirus to produce transgenic embryos. inbred lines with improved disease resistance are being developed and transgenic technology in the future may allow embryos to be used as bioreactors to produce therapeutic proteins of interest and potentially to generate transgenic chickens which have improved resistance to pathogens (bacon et al., 2000; scott et al., 2010) . because chickens develop spontaneous ovarian cancers at an incidence of up to 35%, they are also a prominent model of ovarian cancer in humans (bahr and wolf, 2012; hawkridge, 2014) . quail the zebra finch is a common avian species used in research. from http://www.redorbit.com/news/science/1112751282/male-zebra-finches-fake-song-121912/. the domesticated chicken commonly used in research. provided by kay stewart. (coturnix coturnix and coturnix japonica) have been studied in many of the same research disciplines as chickens, but offer advantages because of their smaller size and because they are among the shortest-lived bird species (austad, 1997) . japanese quail (c. japonica) have been selected as a model to evaluate reproductive biology and social behaviors such as mate selection because they readily show sexual behavior in captivity (ball and balthazart, 2010) . as with the chicken, methods to study transgenic quail are now becoming available and offer a useful tool to study gene function (seidl et al., 2013) . of the psittaciformes, amazon parrots and budgies (melopsittacus undulates) are among the most commonly studied, with research topics including veterinary medicine, diagnostics, behavioral, cognition, aging, and sensory studies (austad, 2011; kalmar et al., 2010) . the african gray parrot has been studied for its cognition and communication abilities (hesse and potter, 2004; harrington, 2014) . of the passerines studied in laboratory research, the most commonly evaluated include the zebra finch (taeniopygia guttata), european starling (sturnus vulgaris and sturnus roseus), and house sparrow (passer domesticus) (bateson and feenders, 2010). zebra finches and other songbirds are commonly studied in regard to aging and neurogenesis in addition to speech, learning, and memory because of their ability to learn and communicate intricate bird songs (harding, 2004; scott et al., 2010; austad, 2011; mello, 2014) . the most popular songbird species for neurobiological research include the zebra finch, canary, and other types of small finches such as lonchura striata domestica (schmidt, 2010) . zebra finches are favored in research settings since they are easy to house due to their small size, for their compatibility in groups, and proclivity for breeding. they are also studied for their biologic features such as sexual dimorphism, year-round singing in captivity, age-dependent period of song-learning propensity, and for ease of measurement with respect to their bird song (fee and scharff, 2010; mello, 2014) . pigeons (columba livia) have been evaluated in areas such as comparative psychology, neuroanatomy, neuroendocrinology, and atherosclerosis (santerre et al., 1972; austad, 1997; shanahan et al., 2013) . they are studied to understand their navigational skills and memory which allow homing, vision and discrimination ability. barn owls (tyto alba) are an example of a nocturnal avian species and are studied for neuroanatomy, vision, hearing, and for understanding learning mechanisms during auditory space mapping (pena and debello, 2010; rosania, 2014) . birds are warm-blooded vertebrates that have feathers for the purpose of flight and plumage. their respiratory system includes avascular air sacs, some of which attach to the lung and bronchi, but do not serve as sites for gas exchange as does the lung (maina, 2006; ritchie et al., 1994) . air sacs serve as internal compartments which hold air and facilitate internal air passage to allow birds to have a continuous flow of large volumes of air through the lungs as a way to increase oxygen exchange capacity and efficiency. birds lack a functional diaphragm and use muscles of the thorax to assist with respiration (ritchie et al., 1994) . care must be taken to ensure that use of physical restraint does not interfere with respiratory movement, cause the bird to struggle, or become stressed. the skeletal system includes pneumatic bones which are lined with air sac epithelium and are considered pneumatized by connection to the respiratory system (frandson et al., 2009 ). the specific bones which are pneumatized depend on the species but typically include the humerus, cervical vertebrae, sternum, sternal ribs, and sometimes the femur (ritchie et al., 1994) . the esophagus in birds leads to the crop, which is an outpocketing where food is held temporarily, and then continues to the proventriculus (also called the true stomach) which produces enzymes to break down food. food travels from the proventriculus to the ventriculus (gizzard) and then on into the small and large intestines. the presence or absence of a gallbladder is species dependent (tully et al., 2009; kalmar et al., 2010) . the rectum and urinary tract terminate in the cloaca, resulting in excreta where the fecal portion of waste is mixed with urate (white and/or creamy component). there are many additional unique and complex anatomic and physiologic adaptations of birds. other excellent references are available in the literature (scanes, 2015). housing requirements of birds held in captivity vary significantly depending on the particular species. basic parameters that apply to all birds include the necessity to provide an enclosure which is safe and permits species-specific behaviors to the greatest extent possible. consideration should be given to ensure that the type of structure is nontoxic, as some birds such as parrots have a powerful beak with the ability to chew through substrates. enclosures may be made of metals or durable plastic, but it is important to note that zinc wire, as well as leaded paint, can be toxic to birds and is best avoided. bar spacing on caging should be appropriate to prevent escape and injury based on the size of the bird. caging size varies and can include large aviaries where full short-distance flight is possible, to individual housing in smaller sized cages where flight may not be feasible. use of environmental enrichment and provision of opportunity for interaction is important to include as part of the cage structure, complexity, and social dynamic. some types of birds are considered social, polygamous, and benefit from group housing, whereas others such as those that pair-bond (such as new world quail) may prefer housing with a single mate (ritchie et al., 1994) . some species, genders, or individuals show aggression and may not be compatible. for example, sexually active male quail may injure each other and are generally considered incompatible (huss et al., 2008) . to help reduce aggression, housing densities should be kept low and multiple points of access to resources, such as feed and perches, should be provided. enrichment in the form of manipulanda can take the form of toys and food items. some types of birds may demonstrate foraging behavior in nature and may like to manipulate their feed. parrots, for example, typically grasp their food with their feet and may peel or strip the outer portion of the foodstuff prior to ingesting. toys should be size appropriate for the species, easily sanitized, free from sharp edges, and replaced once wear shows. birds can become easily caught in items that hang from the cage and as toys deteriorate they can become a hazard. for example, rope toys may begin to fray and become a hazard, causing entrapment; and some types of toys contain weights which pose a choking hazard or may be made of toxic materials such as lead. some types of birds spend considerable time perching and require perches, which vary in diameter, for comfort and to prevent pressure sores from developing on their feet. the respiratory system of the bird is very sensitive and caution must be taken by animal care staff to avoid exposure of birds to aerosols from chemicals that may arise from disinfectants used in the laboratory animal facility. scented cleaners, perfumes, hairspray, and emissions from teflon-coated materials are all examples of products which can be especially harmful to birds and may cause death. feeding requirements vary by species and life stage, but commercial pelleted diets designed to meet the nutritional needs can generally be provided. although many birds are seed eaters, a diet of seeds alone is unlikely to provide adequate or balanced nutrition. many birds have a requirement for dietary calcium, especially those that are reproductively active, and should be provided with calcium supplementation in the form of soluble grit such as cuttlebone or crushed oyster shells (sandmeier and coutteel, 2006; tully et al., 2009 ). birds often display neophobic behavior and may require long acclimation periods before fully accepting novel foodstuffs. for this reason, dietary changes should not be made abruptly and daily intake should be closely monitored. for birds in the laboratory setting, clean, fresh water should be provided daily either by use of nonbreakable bowls or sipper tubes. water intake will vary by species and environmental housing conditions. birds can mask disease and are easily stressed. it is best to first observe the bird in its normal home environment whenever possible and only perform restraint for physical exam or collection procedures when indicated. general indications of sickness may include decreased appetite, depressed behavior, loose stools, distended abdomen, ruffled feathers or unkempt appearance, skin lesions, openmouth breathing, abnormal respiratory sounds such as wheezing or sneezing, or signs of dehydration such as reduced skin turgor and sunken eyes. a healthy bird should have well-groomed feathers, appear alert, active and inquisitive, and should show species-typical behaviors. its eyes should be clear and bright. no evidence of discharge should be present from the eyes, nares, mouth, or urogenital area. numerous types of infectious (example, fig. 7.19 ) and noninfectious disease presentations are described in birds. additional reference resources should be consulted for in-depth information (ritchie et al., 1994; tully et al., 2009; doneley, 2010) . to provide the reader a broader view of animal use in research, descriptions of some less commonly used small mammal models follow. guinea pigs (cavia porcellus) are rodents, related to porcupines and chinchillas in the suborder hystricomorpha (fig. 7.20) . they originate from the mountain and grassland regions along the mid-range of the andes mountains in south america. they are small, stocky, nonburrowing, crepuscular herbivores with short legs and little to no tail, ranging from 700 to 1200 g, females being smaller than males (harkness et al., 2010) . guinea pigs have a long-standing historical role in research stretching as far back as the 1600s, when they were first used in anatomical studies (pritt, 2012) . further, they were used by louis pasteur and robert koch in their example of skin pox on the feet of a dark-eyed junco (junco hyemalis). photo from randalyn shepherd. investigations of infectious disease, and have contributed to the work of several nobel prize worthy studies (pritt, 2012) . specifically, the guinea pig has been used as a model for infectious diseases such as tuberculosis, legionnaires disease, sexually transmitted diseases such as chlamydia and syphilis, and one of the more common causes of nosocomial infections in people, staphylococcus aureus (padilla-carlin et al., 2008) . guinea pigs have also been useful tools in researching cholesterol metabolism, asthma, fetus and placental development and aspects of childbirth, as well as alzheimer's disease (bahr and wolf, 2012) . guinea pigs have many similarities to humans hormonally, immunologically, and physiologically. unlike other rodents, and more like primates (including people), guinea pigs are prone to scurvy if they do not receive adequate vitamin c, typically in their diet (gresham et al., 2012) . guinea pigs are housed similarly to other rodents, although they require more room than the smaller rodents. hamsters are of the rodentia order, suborder myomorpha along with the mouse and the rat. there are over 24 species of hamsters described in the literature, with the most common hamster used in research being the golden or syrian hamster, mesocricetus auratus (harkness et al., 2010) (fig. 7.21) . originating from the northwest region of syria, golden hamsters are thought to be descendants of only three or four littermates collected from syria in 1930 (adler, 1948; smith, 2012) . as their name implies, the typical wild-type coat is reddish gold along their dorsum, with a gray underside. they are granivores and insectivores, weighing 85e150 g, females weighing more than males, with short legs and short tail, and large cheek pouches (harkness et al., 2010) . specific anatomical and physiological features including their susceptibility to disease and infection make them a useful model for study. initially hamsters were utilized in studies of infectious disease, parasitology and dental disease, transitioning into cancer research in the 1960s (smith, 2012) . hamsters are still used in many areas of research, including investigations into metabolic diseases like diabetes mellitus (hein et al., 2013) , cardiovascular disease (russell and proctor, 2006) , reproductive endocrinology (ancel et al., 2012) , and oncology (tysome et al., 2012) . guinea pigs have also been used as models for infectious disease associated with bacteria, parasites, and viruses, such as leptospirosis (harris et al., 2011 ), leishmaniasis (gomes-silva et al., 2013 , and severe acute respiratory syndrome (sars) and ebola viruses (roberts et al., 2010; wahl-jensen et al., 2012) . other species of hamsters used have been used in research. for example, chinese and african hamsters have been used for investigations into diabetes mellitus (kumar et al., 2012) ; european and turkish hamsters have been useful to evaluate aspects of hibernation (batavia et al., 2013) ; and siberian and turkish hamsters have been used to study circadian rhythm and pineal gland activity (butler et al., 2008) (fig. 7.22) . chinchillas (fig. 7.23 ) are in the order rodentia, suborder hystricomorpha, as are the guinea pig and the degu. there are the long-tailed chinchilla, chinchilla lanigera, and the short-tailed chinchilla, chinchilla chinchilla. chinchillas originate from the andes mountains of south america (martin et al., 2012) . they are 400e800 g in size, females weighing more than males, with compact bodies and long, strong hind limbs and dense fur coats (alworth et al., 2012) . the lushness of the coat is what led them close to extinction in the wild due to excessive hunting siberian hamsters. photo from greg demas. chinchilla. photo from bill shofner jr. in the early to mid-1900s (jimenez, 1996) . the chinchilla has a large head, large eyes and ears. the large inner ear anatomy is of specific note as chinchillas are the traditional model for auditory studies (shofner and chaney, 2013) and otitis media (morton et al., 2012) . the gerbil is a rodent, suborder myomorpha, used in research. there are over 100 species of gerbil-like rodents documented, but the mongolian gerbil (meriones unguiculatus) is the species most commonly used in the united states (fig. 7.24 ). mongolian gerbils originate from a desert terrain in mongolia and northeast china. they are long-tailed, burrowing, herbivorous rodents, 55e130 g in size, males being larger than females (harkness et al., 2010) . due to anatomical variations in the blood supply to the brain in an anatomical region known as the "circle of willis," gerbils have been used most notably as a model for cerebral ischemia or stroke (small and buchan, 2000) . an interesting animal model to note among the small mammals is the nine-banded armadillo (dasypus novemcinctus), a new world mammal ranging from the southeastern half of north america, extending south through the americas to the northern region of argentina (balamayooran et al., 2015) . armadillos have a banded carapace, and, importantly, a low core body temperature of 33e35 c. the breeding season is in the summer, but embryo implantation is delayed until late fall, at which gerbil. photo used with permission of american association for laboratory animal science. point identical quadruplicates are always formed (balamayooran et al., 2015) . the armadillo's low body temperature, and susceptibility and physiologic response to the infectious organism, mycobacterium leprae, have made it an ideal model for studying leprosy (balamayooran et al., 2015) . the consistent polyembryony of the species has also made the animal a model of interest in understanding various aspects of twinning (blickstein and keith, 2007) . choosing the correct animal model is an essential component to the success of biomedical research. each species used in biomedical research must be provided with adequate housing and care to ensure the well-being of the animals. because good science and good animal care go hand in hand, it is important to understand and address the biological and behavioral needs of the animals being studied. origin of the golden hamster cricetus auratus as a laboratory animal chinchillas: anatomy, physiology and behavior a novel system for individually housing bullfrogs american fancy rat and mouse association the effects of osteoporosis on distraction osteogenesis: an experimental study in an 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amphibians and reptiles use of the syrian hamster as a new model of ebola virus disease and other viral hemorrhagic fevers pseudomonas aeruginosa infectious keratitis in a high oxygen transmissible rigid contact lens rabbit model. invest. ophthalmol amphibian medicine and captive husbandry animal models for the atherosclerosis research: a review construction of corneal epithelium with human amniotic epithelial cells and repair of limbal deficiency in rabbit models rats, lice, and history key: cord-262445-54ng7m92 authors: gabellini, davide; musarò, antonio title: 16th meeting of the interuniversity institute of myology (iim) assisi (italy), october 17-20, 2019: foreword, program and abstracts date: 2020-09-15 journal: eur j transl myol doi: 10.4081/ejtm.2020.9345 sha: doc_id: 262445 cord_uid: 54ng7m92 the 16th meeting of the interuniversity institute of myology (iim), october 17-20, 2019, assisi, italy brought together scientists, pharma and patient organization representatives discussing new results on muscle research. internationally renowned keynote speakers presented advances on muscle development, homeostasis, metabolism, and disease. speakers selected among submitted abstracts presented their new, unpublished data in seven scientific sessions. the remaining abstracts were showcased in two poster sessions. young trainees where directly involved in the selection of keynote speakers, the organizing scientific sessions and roundtables discussions tailored to the interests of their peers. a broad italian, european and north-american audience participated to the different initiatives. the meeting allowed muscle biology researchers to discuss ideas and scientific collaborations aimed at better understanding the mechanisms underlying muscle diseases in order to develop better therapeutic strategies. the active participation of young trainees was facilitated by the friendly and inclusive atmosphere, which fostered lively discussions identifying emerging areas of myology research and stimulated scientific cross-fertilization. the meeting was a success and the iim community will continue to bring forward significant contributions to the understanding of muscle development and function, the pathogenesis of muscular diseases and the development of novel therapeutic approaches. here, we report abstracts of the meeting illustrating novel results of basic, translational, and clinical research, which confirms that the myology field is strong and healthy. -2 -associated with muscle diseases. 1 leading experts of the muscle field, along with speakers selected from submitted abstracts, provided important insights into the extracellular agonists, receptors, protein kinases, intermediate molecules, transcription factors, and epigenetic mechanisms involved in the modulation of specific cellular responses in embryonic and adult myocytes/myofibers. [2] [3] [4] [5] olivier pourquié demonstrated that, mimicking key signaling events leading to muscle formation in the embryo, it is possible to efficiently produce striated, millimeter-long muscle fibers together with satellite-like cells from human pluripotent stem cells (es/ips), avoiding the requirement for genetic modifications or cell sorting. bente klarlund pedersen discussed the important role of skeletal muscle as an endocrine organ, which has the capacity to produce hundreds of myokines, providing a conceptual basis for understanding how muscles communicate with other organs such as adipose tissue, liver, pancreas, bones, and brain. alessandra sacco discussed the dynamics of muscle stem cells during tissue growth and regeneration, whereas bénédicte chazaud discussed the roles of macrophages in normal skeletal muscle regeneration versus muscle disease. muscle regeneration recapitulates many aspects of embryonic myogenesis and it is an important homeostatic process of adult skeletal muscle, which, after development, retains the capacity to regenerate in response to appropriate stimuli, activating the muscle compartment of stem cells, namely satellite cells, and other precursor cells. 3, [6] [7] [8] skeletal muscle stem cells (musc), also known as satellite cells, are the main source of skeletal muscle growth and regeneration. within their native tissue microenvironment, or stem cell niche, they are exposed to signals emanating from neighboring cells, as well as the extracellular space that instruct their cell fate decisions, whether to undergo self-renewal or commitment and differentiation upon cell division. by utilizing genetic and pharmacological tools, sacco and co-workers have identified stat3 signaling as a critical regulator of musc fate decisions. the inflammatory response of injured skeletal muscle plays an important and critical role in muscle homeostasis and regeneration and consists in the recruitment of specific myeloid cell populations within the injured area. 3, 7, 8 macrophages rapidly increases within 24 hours after injury, are the predominant inflammatory cell type within the injured area, detectable at the level of perimysium and epimysium. because of their high versatility and their impact on their environment, they sustain both the mounting and the dampening of the inflammatory response. in a context of adult tissue damage, that is post-injury skeletal muscle regeneration, accumulating lines of evidence of the beneficial and pleiotropic roles of macrophages in tissue repair indicate that inflammation should not be considered as a bad or detrimental process. conversely, as discussed by chazaud, it should be viewed as a dynamic process of which sequential steps must be tightly coordinated in space and time to be fully efficient to support skeletal muscle regeneration. based on the submitted abstracts, thirty five contributions were selected for oral communications that were grouped in seven sessions chaired by young trainees: 1. genetic and epigenetic alterations in muscle dystrophies and myopathies; 2. satellite cells and muscle regeneration in healthy muscle and in diseases; 3. biophysics and e-c coupling in the pathophysiology of neuromuscular diseases; 4. signaling in muscle growth, homeostasis and disease; 5. metabolic alterations and muscle diseases; 6. muscle fibrosis, sarcopenia and cachexia; 7. therapeutic approaches for muscle diseases. twenty seven poster presentations, always on display during the entire meeting, completed the program. presentations provided breakthroughs in the muscle field, at a fundamental and therapeutic level, and stimulated lively and exciting scientific discussions, which were continued through spontaneous gatherings of several groups at coffee breaks and after lunch and dinner leading to the development of new collaborations. on the first evening of the meeting, there were three roundtable discussions organized by young trainees and moderated by the invited speakers and selected members of the iim. the participants discussed about new ideas concerning muscle development & regeneration; inflammation, metabolism; oxidative muscle damage. the roundtables allowed to discuss muscle research in an informal way, over food and drinks provided by local producers. on the second evening of the meeting, guided tours were organized to the historic section of the city of montefalco and the "cantina lungarotti" winery, terminating with a wine tasting and banquet dinner. finally, an award ceremony was held on the last evening of the meeting to congratulate marco rosina, martina sandonà and valentina taglietti, winners of prices for best poster, oral and active participation awards, respectively, selected by an international panel composed by the invited speakers and key members of the iim. for the first time, the iim meeting was joined with a high training course on "advanced myology", organized together with the university of perugia and reserved to young trainees. the highlight of the course was on october 20th with lectures on muscle development and response to exercise by international speakers taking place in palazzo bernabei, a beautiful 1400 building located in front of the st. francis basilica. the meeting and the course were very successful and instrumental to foster new ideas and facilitate collaborations among the different stakeholders. taken together, the attendees of the iim and of the paduamuscledays (pmds, a meeting more oriented to advanced translational myology) and the authors of papers e-printed in the european journal of translational myology (ejtm) are a substantial part of the international community of myologists. 9,10,11 here, the muscle as an endocrine organ skeletal muscle works as an endocrine organ with the capacity to produce hundreds of myokines. this finding provides a conceptual basis for understanding how muscles communicate with other organs such as adipose tissue, liver, pancreas, bones, and brain. some myokines are thought to induce anti-inflammatory responses with each bout of exercise and other mediate long-term exercise-induced improvements in cardiovascular risk factors, having an indirect anti-inflammatory effect. myokines also mediate anticancer effects and contribute to regulate metabolism and cognitive function. the myokine il-6 is released into the blood during exercise and it has been shown that il-6 has multiple immunologic and metabolic effects. recent advances regarding the physiology of il-6 will be discussed. human studies show that il-6 infusion delays gastric emptying, reduces postprandial glucose concentrations and reduces insulin secretion, whereas experimental studies suggest a role for il-6 in appetite regulation. evidence is also accumulating for a central role of il-6 in training-induced loss of visceral adipose tissue mass in humans. moreover, recent experimental studies in mice show that voluntary exercise suppresses tumor growth through epinephrine-and il-6-dependent mobilization and redistribution of cytotoxic nk cells. it has been known for a while that il-6 is a pleiotropic molecule, however, recent advances suggest that the physiological roles of il-6 involve multiple aspects of metabolism as well as a role in tumor defense. bente klarlund pedersen, md mdsc, is professor of integrative medicine and a specialist in infectious diseases and internal medicine. she is the director of the centre of inflammation and metabolism (cim) and the centre for physical activity research (cfas) funded by trygfonden. cim and cfas count 17 senior researchers/postdocs, 17 phd students, 21 other academic and technical personnel, 5 pre-graduate students and an administration of 5 persons (http://aktivsundhed.dk). she has supervised 47 phd projects and been a mentor of 5 doctoral theses. the research group has identified skeletal muscle as an endocrine organ that produces and releases so-called "myokines". the identification of myokines provides a conceptual basis for understanding how muscles communicate with other organs. through translational research, the aim is to develop targeted exercise training regimes for specific disease groups by applying a translational strategy: "from bedside to bench and back". bkp has had many positions of trust and is a member of the royal danish academy of sciences and letters. bkp has more than 650 scientific publications, > 41.000 citation and her "h"-index is 107 (web of science). inflammation after a tissue damage encompasses several sequential phases including: (1) the mounting of the inflammatory response, characterized by the infiltration of immune cells to the site of injury and the release of proinflammatory effectors; (2) the resolution of inflammation, characterized by a shift from a proinflammatory environment to the establishment of the anti-inflammatory phase of inflammation; and (3) tissue repair/regeneration including angiogenesis, matrix remodeling and return to homeostasis. almost all immune cell types participate in this inflammatory process. however, macrophages are present in high number and during all the sequences of the inflammatory response. because of their high versatility and their impact on their environment, they sustain both the mounting and the dampening of the inflammatory response. in a context of adult tissue damage, that is post-injury skeletal muscle regeneration, accumulating lines of evidence of the beneficial and pleiotropic roles of macrophages in tissue repair indicate that inflammation should not be considered as a bad or detrimental process. conversely, it should be viewed as a dynamic process of which sequential steps must be tightly coordinated in space and time to be fully efficient to support skeletal muscle regeneration. particularly, macrophages exert specific actions on muscle stem cells during the sequential phases of myogenesis, as they first support the expansion of muscle stem cells, then their differentiation and fusion into new functional myofibers. conversely, in chronic degenerative muscle diseases, such as in muscle dystrophies, phenotypes and functions of macrophages are altered, participating to the pathogeny of the disease. our recent investigations indicate that the inflammatory status of macrophages may be a therapeutic target to alleviate muscle dystrophies. dr. bénédicte chazaud is director of research class 1 (eq. of professor) and leader of the team "stem cell environment and skeletal muscle homeostasis" at institut neuromyogène (inmg) inserm u1217-cnrs 5310, université claude bernard in lyon, france. the research of her team is dedicated on the role of environmental cells on adult muscle stem cell behavior in skeletal muscle regeneration and during myopathies. the chazaud lab was pioneer in bringing the concept of a stromal support for these cells to promote an efficient muscle regeneration. particularly, they have been extensively studying macrophage biology in this process. they described that beyond their role in innate immunity through scavenging debris, macrophages directly act on muscle stem cells to regulate adult myogenesis. similarly, while vessels are usually considered only as oxygen and nutriment providers, the work from dr. chazaud identified new interactions between endothelial or peri-endothelial cells with muscle stem cells that regulate their fate. the goal of these studies is to decipher the cell interactions that allow a proper myogenesis after muscle injury and to identify the molecular mechanisms underlying these interactions. these investigations are pursued in both normal muscle regeneration as well as in the context of degenerating myopathies. in vitro modeling of human muscle development and disease progress toward finding a cure for muscle diseases has been slow because of the absence of relevant cellular models and the lack of a reliable source of muscle progenitors for biomedical investigation. we have developed an optimized serum-free differentiation protocol to efficiently produce striated, millimeter-long muscle fibers together with satellite-like cells from human pluripotent stem cells (es/ips) in vitro. by mimicking key signaling events leading to muscle formation in the embryo, this directed differentiation protocol recapitulates the developmental sequence of myogenesis and avoids the requirement for genetic modifications or cell sorting. we engineered a series of human ips reporter lines including pax7-yfp, myog-yfp and pax7-yfp/myod1-cherry that we used to characterize the differentiation of the myogenic lineage in human myogenic cultures. we are particularly interested in the ontogeny of the human pax7-expressing lineage that leads to the adult satellite cells. we show that human pax7-yfp cells produced in vitro exhibit characteristics of fetal satellite cells. we performed single cell rna sequencing of the pax7-yfp cells generated in vitro and identified the major steps of the differentiation of this lineage. this work provides a framework to study early stages of human myogenesis which are poorly accessible in the embryo. olivier pourquie is professor in the department of genetics at harvard medical school and professor of pathology at the brigham and women's hospital. he is an associate member of the broad institute and a principal faculty member at the harvard stem cell institute. he was the director of the institute for genetics and molecular and cellular biology (igbmc) in france and before that a howard hughes medical institute investigator at the stowers institute for medical institute in kansas city. the pourquie laboratory is a world leader in vertebrate musculo-skeletal axis development. using chicken and mouse embryos as model systems, they combine developmental biology and genomic approaches to study patterning and differentiation of the precursors of muscles and vertebrae. they also develop quantitative approaches at the interface with physics to study morphogenesis of the vertebral column. while most of this work is being carried out in vivo, they also develop protocols to recapitulate these early developmental processes in vitro using mouse and human embryonic or reprogrammed stem cells. they are also turning to translational approaches, using their understanding of the early development to produce cells of the muscle and vertebral lineages in vitro from pluripotent cells to study human diseases of the musculo-skeletal axis and for cell therapy approaches. dr. pourquie graduated as an engineer in france and trained with nicole le douarin in paris. he authored more than 100 peer-reviewed publications. he is an elected member of the european molecular biology organization and of the academia europea. his work on the segmentation clock that controls the periodicity of vertebrae was recognized as one of the milestones in developmental biology of the 20th century by nature magazine. amyotrophic lateral sclerosis (als) is a fatal neurodegenerative disease, characterised by muscle atrophy, weakness, and progressive degeneration of motor neurons, leading to death by respiratory failure within 2/5 years from the onset of clinical symptoms. diagnosis of als relies only on clinical, electrophysiological and neuropathological examination, and is based on the exclusion of alternative related pathologies. nowadays specific molecular markers for als diagnosis or prognosis are not available. micrornas are a class of highly stable molecules, detectable in serum samples, recently proposed as biomarkers for several human diseases, such as cancer and muscular disorders. the aim of the present study was to identify circulating micrornas as serum molecular markers of als disease. indeed, we performed an ngs analysis on a sub-cohort of patients, and we identified several differentially expressed micrornas. moreover, we analysed a subset of micrornas preferentially expressed in skeletal muscle (myo-mirnas), involved in myogenesis, and skeletal muscle differentiation and regeneration. here we provide a longitudinal study on serum samples of 27 spinalonset als patients, analysed every three months by absolute rt quantification. we associated serum levels of the identified micrornas with the disease stage, evaluated by als -health state scale (als-hss), and we observed that some micrornas significantly changed during disease progression, while the others were good biomarkers for early and late stages of the disease. furthermore, since als disease progression is variable in terms of velocity and aggressiveness, we divided patients cohort into two subgroups with a different disease progression rate (slow and fast), evaluated by als -functional rating scale (als-frs). we identified a group of micrornas differentially modulated in slow and fast progressing patients, that resulted as good prognostic biomarkers for als patients at the moment of the diagnosis. overall the results of our study can contribute to define a molecular signature of als phenotypes, and can allow to better characterise patients to enrol in clinical trials. exon skipping is one of the promising strategies to treat duchenne muscular dystrophy. recently we have studied the case of a duchenne patient (gs44) characterized by a slower progression of the pathology and healthier conditions, if compared to other age-matched duchenne patients. studies in our lab revealed that his situation is due to a natural exon skipping occurring in his cells. briefly, muscles of this subject lack the expression of celf2a splicing enhancer. this protein is involved in splicing of exon 45, and when is absent, like in gs44, this exon isn't properly recognized and, in a sub-population of dystrophin transcripts, is skipped. this phenomenon, in a dmd 44 context, like the gs44 one, allows the restoration of the frame of the transcript and the production of a small amount of the dystrophin protein. starting from this evidence, it seemed interesting to deepen the study of the regulation of this protein since its inhibition could be a treatment for those patients in which the skipping of exon 45 could recover the dystrophin mrna frame. at first, we investigated the reason of the absence of the protein in this individual. genetic mutations were identified nor in the promoter neither in the coding sequence. moreover, ipsc derived from gs44 fibroblasts and differentiated to myotubes, regain the expression of celf2a. these data suggest that an aberrant epigenetic control doesn't allow celf2a expression in the patient. in order to elucidate this regulation, we decided to perform chip-seq and atac-seq in gs44 versus control myoblasts. the first was performed to study common histones modifications, the latter to take a snapshot of the genome wide conformation of the chromatin. in this way we discriminate loci characterized by opened and actively transcribed chromatin and closed and inactive regions. celf2a regulatory region is marked by closed chromatin and repressive histone labels in gs44 (h3k27me3), while in control is open and shows histone modifications that are usually enriched at active chromatin regions (h3k4me3 and h3k27ac), in line with our observations. to further understand the reason of the missing expression of celf2a in the patient, rna-seq analysis was performed on gs44 and control myoblasts, in order to find a regulator of the factor. no differences in transcription factor expression, between the two condition, were found. instead we identified some long noncoding differentially expressed. among these, one is particular interesting: some experiments correlated its presence in the patient to the absence of celf2a. we hypothesize that this long noncoding could repress celf2a expression through the fine-tuning of chromatin accessibility. at the same time, fibroblasts derived from a 44 patient were reprogramed to ipsc and clones knock out for celf2a were obtained through crispr/cas9 technique. interestingly these clones are able to skip exon 45 thus confirming the therapeutical potential of the inhibition of celf2a. histone deacetylase 4 (hdac4) is a member of class ii hdacs which, by cooperating with class i hdacs, deacetylates proteins, thereby mediating the response to different stimuli in skeletal muscle. recently its protective, essential role in maintaining skeletal muscle homeostasis after long-term denervation (1) or in amyotrophic lateral sclerosis (2) and in satellite cell proliferation and differentiation (3) has been clarified. further, hdac4 is crucial for skeletal muscle regeneration by mediating soluble factors that influence musclederived cell proliferation and differentiation after injury (4). duchenne muscular dystrophy (dmd) is a fatal inherited muscle-wasting disease, caused by mutations in the dystrophin gene (5) and characterized by progressive muscle weakness and degeneration. the membrane repair response, which involves multiple proteins such as dysferlin, mg53 and annexin 1, is enhanced to actively maintain membrane integrity in dmd (6) . the pan-hdac inhibitor givinostat is presently in phase iii clinical trial for the treatment of dmd, despite no efficacy in skeletal muscle function has been registered in dmd patients (7) , highlighting the needs to study the hdac functions in skeletal muscle in dmd further. while the function of class i hdacs in dmd has been partially elucidated (8) , little is known about the role of class ii hdacs. to shed light on additional functions of hdac4 in skeletal muscle, we are currently studying the role of hdac4 in dmd by using a genetic approach. we generated dmd mice with hdac4 deleted in skeletal muscle (mdx;hdac4mko), by crossing mice with a skeletal muscle-specific deletion of hdac4 (hdac4 mko) with mdx mice, a mouse model of dmd. to determine hdac4 functions, muscular dystrophy progression has been analyzed over time, by histological and functional analyses. the deletion of hdac4 in skeletal muscles exacerbates muscle degeneration, increases circulating creatine kinase levels and decreases muscle functionality over time. further investigations have highlighted an impaired membrane repair mechanism in mdx;hdac4mko mice that may underpin the more pronounced progression of the pathology. from our results, we conclude that hdac4 is important for maintaining skeletal muscle integrity in dmd mice. ongoing studies are necessary to define the molecular signaling modulated by hdac4 in dmd in order to provide the experimental basis for a more efficacious pharmacological therapy. cerebral palsy (cp) is the single largest condition leading to childhood physical disability. this neurodevelopmental disorder is characterized primarily by neural deficits caused by a non-progressive lesion in the immature brain, and secondly by musculoskeletal problems that progress with age. alterations at the level of the muscle have been observed in comparison to normal developing children on both macroscopic (e.g. reduced muscle volume, shorter muscle belly, longer tendons) and microscopic (e.g. hypertrophic extracellular matrix and fibrotic tissue accumulation) levels. unfortunately, the onset and the development of these muscle alterations are not well understood, since data on young cp patients are still lacking. the skeletal muscle is a highly dynamic tissue and resident stem cells are the postnatal actors of repair and remodelling processes. therefore, to better understand the impaired development of the muscle and the underlying mechanisms responsible for the alterations reported in cp children, we aim to comprehensively describe the features of the stem cells from cp children compared to stem cells from typically developing children (td). as a starting point for an extended research project on the macroscopic and microscopic muscle properties of these children, the current study reports the pilot results on the microscopic level. a minimally invasive needle microbiopsy (16g) was applied to obtain muscle biopsies from the medial gastrocnemius, during interventions that required general anaesthesia. a total of 10 cp patients with gross motor function classification system level i-iii were included within a range from 2 to 9 years old. biopsies on td children are currently ongoing. the microbiopsy technique was well-tolerated. the explant technique resulted in a reasonable number of cells for all the planned in vitro examinations. satellite cells (scs), mesoangioblasts and fibro-adipogenic progenitor-like cells were isolated using fluorescence-activated cell sorting based on respectively cd56, alkaline phosphatase and pdgfra protein expression. all cell types were characterised by their myogenic and/or adipogenic differentiation potential. six-day differentiation of scs led to heterogeneous levels of differentiation and maturation based on the expression of myosin heavy chain protein and on fusion indexes. in addition, on these myotubes, disturbed nuclear migration was observed. in conclusion, microbiopsy proved to be harmless and efficient for our purposes. in ongoing research, a higher number of patients and multiple age-matched td children are being included to confirm our preliminary observations. future analysis will focus on nuclear migration and other impaired features that will be further assessed in the upcoming months. mitochondrial calcium uptake plays a key role in modulating cell metabolism, cell survival and other crucial cell functions. calcium accumulates into the mitochondrial matrix through the mitochondrial calcium uniporter (mcu). few years ago a mcu homolog has been discovered, that was called mcub. mcu and mcub share a 50% sequence and structure similarity although some conserved differences in the primary sequence prevent mcub from forming a ca 2+ -permeable channel, thus acting as a dominant-negative subunit. interestingly, mcub/mcu expression ratio varies greatly among tissues, suggesting that it might contribute to the spatiotemporal control of mitochondrial calcium uptake. here we show that mcub is highly expressed during skeletal muscle regeneration, a process that is mainly regulated by phenotypic skewing of tissue resident and freshly recruited macrophages. we have demonstrated that mcub expression is specifically induced in macrophages, the most abundant cell population in a regenerating muscle that drive both the proliferation and differentiation of the muscle stem cells, satellite cells, thus affecting the progression of healing after muscle damage. in vitro and in vivo experiments demonstrated that the absence of mcu affects macrophages skewing from a pro (m1) to an anti-inflammatory profile (m2). indeed, macrophages of mice lacking mcub are more prone to acquire a pro-inflammatory m1 profile. since m2 macrophages have a proregenerative role and promote myogenic precursor cell differentiation, we hypothesized that mcub absence, by affecting m2 polarization, might influence the correct remodeling and reorganization of skeletal muscle structure after damage. our results clearly show that regenerating muscles lacking mcub show a decrease in the expression level of myogenic regulatory transcription factors involved in satellite cells activation and differentiation. we also observed a decrease in the percentage of regenerating myofibers and a decrease in collagen deposition. in addition, co-culture experiments demonstrated that macrophage-conditioned medium from m2 mcub ko macrophages drastically decreases satellite cell differentiation. altogether these results demonstrate the role of mitochondrial ca 2+ uptake in promoting the polarization of macrophages towards an m2 pro-regenerative profile during skeletal muscle regeneration. further experiments will help to clarify the role of mitochondrial calcium during skeletal muscle regeneration. dystrophic muscle is characterised by chronic injury, and a steady recruitment of inflammatory ly6c hi monocytes. recent studies have identified the spleen as the dominant reservoir of these cells during chronic inflammation. here we investigated the hitherto unexplored contribution of splenic ly6c hi monocytes to dystrophic muscle pathology. using the mdx mouse model of muscular dystrophy, we show that ly6c hi monocytes accumulate in great numbers in the spleen over the course of the disease. the chemokine receptor ccr2 was upregulated before disease onset, enabling their egress from the spleen. splenectomy performed before disease onset significantly reduced the number of ly6c hi monocytes infiltrating dystrophic limb muscle. moreover, in the absence of splenic ly6c hi monocytes there was a significant reduction in dystrophic muscle inflammation and necrosis, along with improved regeneration during early disease. however, during late disease, lack of splenic ly6c hi monocytes adversely affected muscle fiber repair, caused a delay in the phenotypic shift of pro-inflammatory f4/80 + /ly6c hi /cd206 lo to anti-inflammatory f4/80 + /ly6c lo /cd206 + macrophages, and was associated with increased fibrosis. overall, we show that the spleen is an indispensable source of ly6c hi monocytes in muscular dystrophy, and that splenic monocytes are critical players in both muscle fiber injury and repair. muscular dystrophies (mds) are severe genetic disorders mainly due to mutations in structural proteins, causing contraction-induced damages. previous attempts to treat these diseases raised from the idea that accelerating muscle growth and regeneration would exert beneficial effects. we recently demonstrated that slowing down the degeneration-regeneration cycles and switching muscle fibers towards a slow-twitching phenotype by silencing the transcription factor nfix leads to a morphological and functional amelioration of the dystrophic phenotype. on the basis of the identification of the molecular pathways regulating nfix expression, we are now developing a pharmacological approach to inhibit nfix in mds which is based on the inhibition of the erk pathway by using two fda-and ema approved drugs for cancer. at the same time, another strategy considered is the use of antioxidants to protect myofibers from oxidative stress generated by muscular contraction. different evidences are indeed establishing the importance of dietary anthocyanin's for preventive and ameliorative strategies against chronic diseases. we therefore tested the therapeutic benefit of a cyanidinenriched diet in the progression of the md. interestingly, dystrophic mice fed with a cyanidin-enriched diet show a morphological and functional amelioration of the dystrophic phenotype through mechanisms that involve both cell survival and anti-inflammatory pathways. all this evidence, strongly demonstrate that promising therapeutic and supporting strategies to slow down the disease progression in dystrophic patients is to reduce, the oxidative stress, muscle regeneration and muscle contraction achievable through different and synergistic strategies. profiled both the cytokines and the evs at different time points during muscle regeneration of the cardiotoxin injured mouse model. we performed a multiplex elisa assay of the mononuclear cell compartment lysate to analyze the expression kinetics of 90 cytokines. in addition, by profiling the rna expression profile of four cell populations, we determined the cell types that have the potential to secrete the different cytokines and those that synthetize the specific receptors. based on the kinetic of their expression during damage recovery, we selected 15 cytokines to be tested in vitro on fibro-adipogenic progenitors (faps). using this approach we identified cytokines that influence fap proliferation and differentiation decisions. in addition, by culturing injured muscles ex-vivo, we obtained the evs secreted by the tissue during the regeneration process. to analyze their cargo, we characterized the vesicle heterogeneity by using a flow cytometry approach. specifically, we identified and quantified the rnas-and enzymes-containing evs. finally, we investigated their ability to interact with different muscle-derived cell populations, influencing their proliferation and differentiation potential. our data, combined with in silico modeling, will increase our understanding of cell-cell communication during muscle regeneration. the neuromuscular junction (nmj) is a chemical synapse localized between the terminal branches of the spinal motor neurons and the skeletal muscle fibers. in the past two decades co-culture systems to recreate the nmj in vitro were developed to address concerns about animal models, nevertheless the complexity of its highly specialized structure makes the in vitro modeling a challenging task. however, a further improvement in this issue was recently provided by microfluidics that, unlike in mass co-cultures, allow spatial and temporal control over different microenvironment by manipulating either neural cells or muscle cells populations independently. this enables the investigation of mechanisms involved in the formation and the maintenance of a stable and functional nmj. therefore, exploiting an organ-on-a-chip approach, our aim is to obtain a reliable and predictive in vitro human model of nmj in physiological and pathological conditions, to investigate the occurrence of synapse detriment in neuromuscular diseases. for this purpose, motor neurons derived from human ipscs and human skeletal muscle cells derived from either perivascular muscle progenitors, namely pericytes, or immortalized myoblasts are seeded in two separated chambers of a microfluidic device. the two side of the device are linked together through microchannels that enable the axonal outgrowth to the muscle side, but not cell bodies migration, allowing the compartmentalization of the cell populations without interrupting cell-cell communication. while being designed as a reliable platform to investigate the molecular actors of nmj processes, the setup is versatile enough to host patient-specific cells and perform functional and molecular analysis. spinal and bulbar muscular atrophy (sbma) is caused by polyglutamine (polyq) expansions in the ar gene. although clinical and experimental evidence highlight a primary role for skeletal muscle in the onset, progression, and outcome of sbma, the pathophysiological and molecular processes underlying sbma muscle atrophy are poorly understood. we show that polyq-expanded ar alters intrinsic muscle force generation before denervation. reduced muscle force was associated with a switch in fiber-type composition, disrupted muscle striation, altered calcium (ca++) dynamics in response to muscle contraction, and aberrant expression of excitation-contraction coupling (ecc) machinery genes in transgenic, knock-in and inducible sbma mice and patients. acute suppression of ar activation by surgical castration elicited similar ecc gene expression changes in normal mice, suggesting that ar regulates the expression of these genes in physiological conditions. importantly, treatment to suppress polyq-expanded ar expression restored ecc gene expression back to normal. bioinformatic analysis revealed the presence of androgen-responsive elements on several genes involved in muscle function and homeostasis. experimental evidence showed ardependent regulation of expression and promoter occupancy of the most up-regulated gene from transcriptomic analysis in sbma muscle. these observations reveal an unpredicted role for ar in the regulation of expression of genes involved in muscle contraction and ca++ dynamics, a level of muscle function regulation that is disrupted in sbma muscle, yet restored by pharmacologic treatment. the aim of this project is to dissect the identity and localization of the nascent muscle proteome in vivo. the tool of choice is the transgenic mouse metrs, expressing a mutant methionyl-trna synthetase together with a green fluorescent protein. we crossed this mouse with a skeletal muscle specific cre-expressing mouse to obtain a tissue specific expression. the mutated methionyl-trna synthetase can integrate a synthetic amino acid (anl) instead of a methionine in a nascent peptide chain. the synthetic amino acid side chain ends with an azide, allowing the formation of a covalent bond with an alkyne by a click chemistry reaction. to visualize the gfp-2a-metrs expression, a western-blot with anti gfp antibody has been performed as well as immunohistochemistry on cryosections. the flexibility of the click reaction allows the visualization of nascent peptide chains either by western blot or immunofluorescence. intraperitoneal injections of anl for different time periods resulted in a significant labelling of the muscle proteome after 1, 2 and 7 days. after the protein extraction, each whole cell lysate has been clicked with biotin labelled alkyne in the presence of copper (ii) sulphate and ascorbic acid and labelled proteins were revealed by western blotting. labelled proteins can also be revealed by ihc performing funcat (fluorescent non-canonical amino-acid tagging) on cryosections. this way we were able to identify that a specific proteome was synthesized during a determined amount of time. these preliminary studies will give us the necessary tools to better understand the skeletal muscle proteome in physiological and pathological conditions. mitochondria change distribution across cells following a variety of pathophysiological stimuli. the mechanisms presiding over this redistribution are yet undefined. in a murine model overexpressing drp1 specifically in skeletal muscle, we find marked mitochondria repositioning in muscle fibres and we demonstrate that drp1 is involved in this process. drp1 binds klc1 and enhances microtubule-dependent transport of mitochondria. drp1-klc1 coupling triggers the displacement of kif5b from kinesin-1 complex increasing its binding to microtubule tracks and mitochondrial transport. high levels of drp1 exacerbate this mechanism leading to the repositioning of mitochondria closer to nuclei. the reduction of drp1 levels decreases kinesin-1 activation and induces the partial recovery of mitochondrial distribution, thus linking mitochondrial movements to drp1 levels. this aberrant positioning of mitochondria is accompanied by the collapse and aggregation of desmin confirming that the loss of mitochondria anchoring system may contribute to mitochondria distribution. we conclude that desmin alterations accompanied by increased drp1 levels enhance microtubule-dependent trafficking promoting changes in mitochondrial positioning unravelling a hitherto unknown function of drp1 in the regulation of kinesin-1 complex. valentina taglietti the "ubiquitous" ccaat-binding transcription factor nf-y is a trimer formed by the histone-like nf-yb/nf-yc and the sequence-specific nf-ya, present in two isoforms. all three subunits are required for dna-binding and function. the role of nf-y in muscle physiology is not clear: if overexpressed, the "long" nf-ya isoform favors differentiation, yet it is negatively regulated during the process. we targeted nf-ya exon 3 by crispr-cas9 in mouse c2c12, thus ablating the "long" nf-ya: despite expression of nf-ya "short", differentiation is blocked. to understand the nf-y regulome in a more physiological system, we turned to primary mouse fetal myoblasts, performing chip-seq, rna-seq after nf-yb rnai, and monitoring gene expression from myoblasts to myotubes. nf-y locations are similar before and after differentiation, despite far lower levels of nf-ya. notably, the nf-y regulome is devoid of binding sites of mrfs -muscle regulatory factors-and myotubes-specific genes are not targeted. instead, we observe a positive functional partnership with srebps, master regulators of lipids biosynthesis, and a negative one on cell-cycle genes. srebp-2 is active in differentiation and bound to promoters of genes co-regulated with nf-y. these data establish that nf-y, and specifically the "long" nf-ya isoform, is essential for myotubes formation, but marginally, if at all, through direct activation of muscle-specific genes. instead, it teams up with srebps to activate crucial steps of lipids metabolic pathways required for the process. the nf-y/srebps partnership will be discussed. ck2 is a tetrameric protein-kinase, composed of two catalytic ( and/or ') and two regulatory β-subunits. our study provides the first molecular and cellular characterization of the different ck2-subunits highlighting their individual roles in skeletal muscle specification and differentiation. analysis of c2c12 cells knockout for each ck2-subunit reveals that: i) ck2β is mandatory for the expression of the muscle master-regulator myod in proliferating myoblasts, thus controlling both myogenic commitment and subsequent muscle-specific gene expression and myotube formation; ii) ck2 is involved in the activation of the muscle-specific gene-program; iii) ck2'-activity regulates myoblast fusion by mediating plasma membrane translocation of fusogenic proteins essential for membrane coalescence, like myomixer. accordingly, ck2' overexpression in c2c12 cells and in mouse regenerating muscle is sufficient to increase myofiber size and myonuclei content via enhanced satellite cell fusion. consistent with these results, pharmacological inhibition of ck2-activity substantially blocks the expression of myogenic markers and muscle cell fusion both in vitro, in c2c12 and primary myoblasts, and in vivo, in mouse regenerating muscle and zebrafish development. concluding, our work describes the specific and coordinated functions of ck2-subunits in orchestrating muscle differentiation and fusogenic activity, highlighting ck2 relevance in the physiopathology of skeletal muscle tissue. fibro/adipogenic progenitors (faps) are muscle interstitial progenitors mediating pro-myogenic signals that are critical in muscle homeostasis and regeneration. in pathological conditions, including ageing and myopathies, the autocrine/paracrine constraints controlling fap adipogenic potential are released causing the formation of fat infiltrates. to reveal pathways that transduce the niche messages controlling fap adipogenesis, we have screened a library of inhibitors targeting a large fraction of the human/murine kinome, looking for molecules with a potential to modulate fap differentiation. hit selection criteria enrich compounds targeting glycogen synthase kinase 3 (gsk3), thus unveiling its crucial role in fap adipogenesis. consistently, we show that the pharmacological blockade of gsk3, by using the high-selective inhibitor ly2090314, severely impedes fap adipogenesis ex vivo while limiting intramuscular fat infiltrations upon glycerol-induced muscle injury, in vivo. by exploiting high-dimensional single-cell mass cytometry, we demonstrate that -catenin down-regulation is critical for ppar expression. ly2090314, by targeting gsk3, counteracts this process and prevents fap adipogenesis. by combining computational strategies based on the interrogation of single-cell transcriptomes, in silico network modeling and rnaseq data integration, we conclude that: i) faps are the main source of wnt ligands in the muscle and that the release of these cytokines may trigger autocrine/paracrine responses; ii) the wnt pathway has a dominant role in regulating the adipogenic network of faps; iii) wnt5a expression is impaired in faps from dystrophic mice. finally, we show that exogenous treatment of dystrophic faps with wnt5a is sufficient to repress ppar expression and the adipogenic conversion of faps into adipocytes. these results suggest that by modulating the wnt pathway either by targeting gsk3 or by restoring autocrine wnt5a signaling in faps might be a strategy to counteract intramuscular fat infiltrations in myopathies. in skeletal muscle, mitochondrial ca 2+ uptake plays important roles in organ homeostasis, ranging from control of metabolism to regulation of fiber trophism. still debated is whether in muscle mitochondria can buffer cytosolic ca 2+ increases. to answer to this question, we explored the effect of removing parvalbumin (pv), the most important muscle cytosolic ca 2+ buffer. by using a pv knockout (ko) mouse model, we are investigating whether the absence of pv induces compensatory mechanisms on the expression and function of the mitochondrial ca 2+ uptake machinery (mcu). the data obtained so far confirm that the absence of pv induces an increase of mitochondrial ca 2+ uptake and this is accompanied by the induction of the expression of the mcu coin vitromplex components. moreover, electron microscopy analysis performed on extensor digitorum longus (edl) muscle of wt and pv ko mice, demonstrated that pv ko mitochondria are significantly larger than wt controls and with a different redistribution in the muscular tissue, suggesting a strict connection and regulation between pv expression and mitochondrial morphology and function in muscle cells. furthermore, cytosolic ca 2+ transients in pv ko muscle fibers show that the time to reach the peak upon stimulation and the time to half relaxation are prolonged in pv ko muscles. to verify whether the increase of mitochondrial ca 2+ uptake in ko fibers is due to mitochondrial adaptation and whether mcu is responsible of buffering cytosolic ca 2+ increase, we decided to silencing mcu and measure [ca 2+ ]cyt on fdb muscles of wt and pv ko animals. [ca 2+ ]cyt was almost unaffected by the absence of mcu in wt animals while, in pv ko fibers, [ca 2+ ]cyt was significantly higher upon stimulation. furthermore, since pv is one of the most downregulated atrogenes, the genes commonly up-and down-regulated during several types of atrophy, and that mitochondrial ca 2+ controls skeletal muscle trophism, we decided to study the role of pv on muscle mass through denervation experiments. when pv is absent, loss of muscle mass is reduced compared to wt fibers, demonstrating that pv can partially protect muscles from denervation-induced atrophy. overall, our results indicate that pv plays an important role in spatiotemporal control of cytosolic ca 2+ responses on mitochondrial ca 2+ uptake and have a profound impact on skeletal muscle trophism. the second messenger ca 2+ regulates a broad repertoire of cellular processes. upon physiological stimuli, skeletal muscle mitochondria rapidly and efficiently accumulate ca 2+ into their matrix via an electrogenic pathway, that relies on the driving force of a steep electrochemical gradient. a large [ca 2+ ]mt peak occurs dynamically in parallel to agonist-induced [ca 2+ ]cyt increases, thanks to the activity of the mitochondrial calcium uniporter (mcu), the highly selective channel responsible for mitochondrial ca 2+ accumulation. mcu positively regulates myofiber size in physiological conditions, and counteracts pathological loss of muscle mass. we have previously demonstrated that skeletal muscle-specific mcu deletion (mcu -/-) inhibits myofiber mitochondrial ca 2+ uptake, impairs muscle force and exercise performance. mitochondrial ca 2+ uptake is required for effective glucose oxidation and efficient mitochondrial activity. nonetheless in mcu -/myofibres, impaired oxidative capacity is partially sustained by increased fatty acid (fa) oxidation. the main trigger of this metabolic rewiring is the decreased pyruvate dehydrogenase activity which is tightly controlled by mitochondrial ca 2+ accumulation. here, we have investigated the role of mitochondrial ca 2+ uptake during skeletal muscle aging. in detail, we show that mitochondrial ca 2+ accumulation is decreased in 24 months old mice and this condition is accompanied by a decreased pyruvate dehydrogenase activity. in this scenario we demonstrate a rewiring of skeletal muscle metabolism where mitochondrial activity is sustained by fa oxidation rather than glucose. further studies are needed to evaluate whether the restore of glucose as the main fuel for oxidative metabolism will be sufficient to counteracting aging process, including force and performance, in skeletal muscles. dystrophin deficiency causes chronic wasting of the skeletal muscle tissue leading to patient respiratory or heart failure and, finally, death. in addition to fiber fragility, the absence of the dystrophin protein, as in duchenne muscular dystrophy (dmd), causes a variety of poorly understood secondary effects. notably muscle fibers of dystrophic individuals are characterized by mitochondrial dysfunctions, as revealed by a reduced atp production rate and by defective oxidative phosphorylation (oxphos). we recently discovered that in a mouse model of dmd (mdx), the interstitial fibro/adipogenic progenitor (fap) cells are also characterized by a dysfunctional mitochondrial metabolism which correlates with an increased adipogenic differentiation potential. using high-sensitivity mass spectrometry-based proteomics, we highlighted that a short-term high-fat diet regimen reprograms dystrophic fap metabolism in vivo. by combining our proteomic dataset with a literature-derived signaling network, we discovered a high-fat dependent modulation of the crucial hub protein, -catenin, which controls follistatin expression. our results reveal that a short-term highfat diet restores the key role of faps in enhancing the myogenic activity of the skeletal muscle stem cells in dystrophic mice. consistently, we observe that muscle regeneration in the mdx mouse is significantly improved by the short-term high-fat diet. our results support metabolic reprogramming of muscle interstitial progenitor cells as a novel approach to alleviate some of the adverse outcomes of duchenne muscular dystrophy. if we concentrate homo sapiens existence in just 24 hours, the time he started to be totally sedentary is just 30 seconds ago, while his life expectation exceeded the 60-year during the last 25 seconds. the combination of a novel life-style with an increased life-span, two conditions that our species never experienced before, resulted in the onset of many chronic and metabolic diseases. nevertheless, emerging evidence shows that readopting a "more active" life-style is associated with healthier and longer life-span. whole-body beneficial effects of physical activity are the consequence of adaptive metabolic changes established in the entire organism to sustain the high energy expenditure of contracting skeletal muscles. during last years, many efforts have been made to dissect the molecular mechanisms explaining why physical activity sustains and improves life quality. recently, our group identified the trascription-factor eb as a muscular master metabolic regulator that translocates into myonuclei in response to exercise promoting the expression of genes for different fuel disposal and utilization. these findings strongly support tfeb activity as crucial for the beneficial effects of exercise. starting from this evidence we decided to discover new uncharacterized genes that may mechanistically explain adaptive effects of tfeb on skeletal muscle. by crossing gene expression data of tfeb transgenic and trained muscles, we found a gene of unknown function belonging to the riken cdna collection that we called exe-riken.exe-riken encodes a putative 125 residues protein highly conserved among mammals; interestingly, exe-riken coding sequence translates in a real protein when expressed in vitro and in vivo. based on gene expression databases, brown adipose and gut are the tissues with higher basal exe-riken expression in mice. on the other hand, skeletal muscle transcript levels are very low, type of muscle specific and finely tuned with different exercise protocol. in addition, exe-riken protein sequence presents a nuclear export signal (nes) and nuclear localization signal (nls), suggesting a potential shuttling behavior upon appropriate stimuli. supporting this idea, in vivo experiments demonstrate exe-riken nuclear translocation upon exercise. finally, overexpression studies in adult tibialis anterior muscle showed an increased sdh activity and a decreased pas staining, suggesting its impact in muscle metabolism. overall these findings highlight a possible role of this new gene in controlling metabolic adaptations during physical activity. cholecalciferol, or vitamin d3 (vd hereafter for brevity), besides its well-known role in regulation of calcium and phosphate homeostasis, impacts on skeletal muscle homeostasis as well, and vd deficiency has been correlated with decrease in muscle mass, function, and performance in elderly subjects, as well as in cachectic cancer patients. while vd supplementation was able to restore muscle strength and prevent muscle mass loss in frail elderly subjects, it was ineffective in the case of cancer cachexia-associated muscle wasting. investigating the direct effects of the main cholecalciferol-derived metabolites -namely 1,25-(oh)2-vd and its precursor 25-oh-vd-on skeletal muscle cells, we found divergent effects of these molecules in promoting/preserving from atrophy induced by pro-cachectic cytokines. given the anti-atrophic action of 25-oh-vd, we wondered if also the pro-hormone vd could have a direct activity on skeletal muscle cells. for this purpose, we used an in vitro model of cytokine-induced atrophy and found that also vd has a protective action. intriguingly, the activity of vd is notmediated by an intracellular conversion in the protective form 25-oh-vd. our recent study revealed the existence of a novel embryonic isoform of cavβ1 (cavβ1e), the beta subunit of cav1.1, which expression increases in adult muscle after denervation. the increase of cavβ1e expression boosts downstream gdf5 signaling to counteract muscle atrophy due to nerve damage. we further reported that aged muscle expresses significantly reduced levels of cavβ1e and that its overexpression improves mass wasting in aging muscle by increasing gdf5 expression. crucially, we also identified the human cavβ1e analogous and showed a correlation between the decrease of cavβ1e expression and the age-related muscle mass decline in people. altogether, our data open a new field in the development of strategies against muscle mass loss during senescence. here, we have preliminary data indicating that the systemic administration of the recombinant gdf5 protein (rgdf5) represents a promising therapeutic approach to improve age-related muscle wasting. in a pilot study, old mice (90 weeks old) were treated with rgdf5, by systemic delivery, for ten weeks. strikingly, rgdf5 implementation induced a significant increase of lean mass and a decrease of the fat mass. indeed, muscle/body-weight ratios of tas and quadriceps (quad) were significantly increased in the rgdf5-100 weeks old mice compared to vehicle-treated old mice. in order to evaluate the possible mechanisms behind rgdf5 effects in muscle, we analyzed: a) myostatin (gdf8) pathway, involved in protein degradation; b) neuromuscular junction (nmj) status by measuring the expression of acetylcholin receptor subunit epsilon (chrnε). we found a significant decrease in myostatin pathway activation in muscle treated with rgdf5, suggesting his protective role against myostatin-dependent protein degradation. furthermore, while chrnε transcription was significantly decreased during aging revealing an alteration of nmj, rgdf5 administration led to chrnε re-expression suggesting an improvement of nmj state. all together these data show that rgdf5 implementation efficiently counteracts age-related muscle wasting and, more generally, could ameliorate neuromuscular decline and protein degradation underlying several (neuro) muscular diseases. cachexia is a highly debilitating multifactorial syndrome associated with various diseases and is characterized by severe muscle wasting leading to pronounced weight loss, impaired quality of life, reduced response to therapy, and premature death. vitamin d binding protein (vdbp), also known as group-specific component (gc-globulin), is a multifunctional serum glycoprotein synthesized by hepatocytes that belongs to the albumin gene family. besides the binding and transport of vitamin d metabolites in blood, vdbp has other activities, including binding and clearance of monomeric g-actin released from dead cells and acting as a chemotactic cofactor for c5a.proteomic analysis of samples from patients affected by pathologies susceptible to progressive muscle loss or cachexia, including several types of cancers, shows upregulation of vitamin d binding protein (vdbp), raising the hypothesis that vdbp might play a role in cancer cachexia-associated muscle loss. we found that vdbp expression increases in tumor-bearing mice undergoing cachexia, further supporting the notion that vdbp could play a role in the onset/progression of cancer-associated muscle wasting. we explored the direct action of vdbp in skeletal muscle and found that vdbp indeed induces atrophy in c2c12 myotubes and that mice devoid of vdbp are protected from cancer-associated cachexia. our preliminary results demonstrate that vdbp is not a simple carrier that modulates the bioavailability of vitamin d but an active hormone in itself with pro-cachectic effects on skeletal muscle cancer cachexia is a devasting multifactorial syndrome characterized by the progressive unintentional body weight loss mainly due to skeletal muscle wasting and atrophy that occurs in the majority of terminally-ill cancer patients. notably, cancer-induced muscle wasting markedly results in a drastic worsening of patient prognosis and quality of life. furthermore, cancer-driven cachexia also limits the therapeutic options, as cachectic patients usually manifest reduced tolerance and response to antineoplastic treatments. aside from this, cachexia directly accounts for nearly 20% of all cancer-related deaths, as the cachectic skeletal muscle wasting also affects survival needed muscles such as diaphragm and cardiac muscle, leading to respiratory and cardiac failure. in the last decade, the pivotal role of cancer-driven inflammation both in tumour progression and cachexia onset has clearly emerged, as well as cancer cachexia is associated to a broad range of metabolic and endocrine impairments leading to tissue function disruptions. nevertheless, the potential role of muscle metabolic alterations in the activation and development of cancer cachexia has been little studied so far. here, we report that conditioned media (cm) from murine and human carcinoma cell lines able to trigger cachexia in vivo, induce cachexia in c2c12 myotubes that is associated to a metabolic shift towards fermentation. our findings suggest that this metabolic shift is crucially involved in the activation of cachexia in c2c12 myotubes, since the abolishment through glycolysis block or lactate dehydrogenase inhibition, prevents the onset of the cachectic phenotype. furthermore, our evidences suggest that cm from cancer cell lines able to induce cachexia express high levels of ifn-γ and il-6 and that these two proinflammatory cytokines could be involved in the metabolic shift towards fermentation that occurs in cm-treated myotubes, thus leading to cachectic phenotype. overall, our results show the possible key role of the metabolic reprogramming towards fermentation in the induction of cachexia in myotubes, thus opening the study to new drugs that could counteract the metabolic shift and inhibit the cachectic phenotype. aging is associated with a progressive loss in skeletal muscle mass and strength, known as sarcopenia. sarcopenia results in a decrease in mobility and an increased risk of developing chronic metabolic disease, thus it represents a major socio-economical problem. age-related muscle loss cannot be consistently prevented by physical therapy and a pharmacologic therapy does not exist, probably because the molecular basis of this condition is still largely unknown. many factors such as mitochondrial dysfunction, oxidative stress, inflammation, changes in the innervation of muscle fibers probably play an important role in age-related muscle decline. pin1 is a widely expressed peptydyl prolyl cis/trans isomerase, involved in post-phosphorylation control of the function of multiple target proteins. many evidences indicate that pin1 controls signaling pathways involved in skeletal muscle wasting. our results indicate that skeletal muscle of pin1 ko mice is protected against muscle loss and weakness during aging. at the molecular level, we found 1) an increased expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (pgc1α), a transcription factor that promotes mitochondrial biogenesis, in skeletal muscle of pin1 ko mice. coherently with their resistance to muscle loss, we also found 2) an increase in the expression of the protein synthetic signaling proteins p70 ribosomal s6 kinase (s6k) and of its phosphorylated form in aged skeletal muscle of pin1 ko mice compared to the wild type controls, suggesting that in these mice protein synthesis is maintained efficient. we also found 3) a significant decrease of myostatin levels in skeletal muscle of aged pin1 ko mice. it is well known that myostatin is up regulated sarcopenia; it activates smad2/3 signaling and contributes to protein degradation and muscle atrophy. the transcriptional effects of pin1 depletion on pgc1α and s6k genes must be mediated by a transcription factor. a putative candidate to mediate these effects is represented by the transcription factor myocyte enhancer factor 2c (mef2c), a known pin1 target. in skeletal muscle cells a specific splice variant of mef2c, mef2c α1, activates the increase of skeletal muscle mass by activating the expression of igf1 and s6k. these activities are repressed by its phosphorylation, that renders it a target for the inhibitory effect of pin1 on its protein stability and activity. coherently with these premises, we found 4) a decrease of mef2c protein phosphorylation levels in aged ko mice compared to the control animals. this might at least partially contribute to the increased expression of pgc1α and of s6k. our results indicate that pin1 could represent a valuable pharmacological target to counteract age-related muscle loss, simultaneously modulating multiple targets in a concerted way. skeletal muscle (skm) atrophy is determined by several physiological and pathological conditions, such as cancer and neuromuscular disorders. biomolecular characteristics of most types of skm atrophy are the suppression of protein synthesis and the increase in protein degradation. although the signalling pathways involved in the control of skm waste are multiple, the regulatory role of lipids is only in part known. sphingolipids (sls) represent a class of bioactive molecules capable of modulating the destiny of many cell types, including skm cells. here, we investigated the role of ceramide kinase (cerk), the enzyme involved in the phosphorylation of ceramide (cer) to ceramide 1-phosphate (c1p). we found that the expression of cerk was comparable in c2c12 myoblasts and in terminally differentiated myotubes excluding that cerk expression modulators may be required for the achievement of myogenic differentiation. however, cerk inhibition promotes myoblast cell growth arrest and myotubes reduction in size suggesting a role of cer/cerk axis in the maintenance of normal mature phenotype. notably a drastic decrease of cerk protein expression was observed in skm tissues obtained from animals bearing the c26 tumor, a well-characterized experimental model of cancer cachexia, and in c2c12 myotubes treated with the glucocorticoid dexamethasone. these findings provide the evidence that cer/cerk axis acts as a molecular regulator of skm atrophy, thereby representing a new possible target for therapy in pathophysiological muscle conditions. cachexia is a debilitating syndrome affecting the majority of patients with advanced cancer, and directly responsible for about 20% of all cancer-associated deaths. the major clinical feature of cachexia is skeletal muscle atrophy that leads to pronounced weight loss, drastically dampens patients' quality of life, reduces the response and tolerance to chemotherapy, and is associated with poor prognosis and outcome (1). the identification of reliable biomarkers of cachexia is of great importance to identify patients in the different phases of the disease and to monitor the treatment outcome (2) . rage (receptor for advanced glycation end-products) is a multiligand receptor of the immunoglobulin superfamily physiologically involved in skeletal muscle development and homeostasis (3). in cancer conditions, the increase of rage ligands levels leads to hyperstimulation of the receptor translating into systemic inflammation and muscle wasting, and reducing mouse survival (4). we analysed rage expression in muscle tissue of different preclinical mouse models of cancer cachexia [lewis lung carcinoma (llc)-bearing c57bl/6 and colon adenocarcinoma (c26-adk)-bearing balb/c mice] in the absence or presence of voluntary endurance exercise (wheel), which has been reported to partially antagonize muscle wasting in mice (5), and correlated rage expression with myofiber crosssectional area (csa) and hallmarks of muscle atrophy (body and muscle weights, protein degradation extent, and activation of proteolytic systems). we also analysed rage expression in muscle biopsies from cancer patients. we found that: i) llc-and c26-adk-bearing mice express rage in myofibers in coincidence with reduced body and muscle weights and induction of proteolysis; ii) an inverse relationship exists between rage expression in muscles and tumor masses and the beneficial effects of endurance exercise in llc-bearing mice; iii) rage expression increases in muscles during cachexia progression; iv) rage is not expressed in myofibers of athymic-nude mice injected subcutaneously with llc or melanoma a375 cells, which do not develop cachexia; and v) muscles of cachectic patients express higher amounts of rage than non-cachectic subjects. altogether, our results suggest that rage might represent a biomarker to monitor the cachectic stage at muscle level. tissue engineering (te) and regenerative medicine approaches are designed to increase skeletal muscle damage response. scientific advances in biomaterials and stem cells allowed the opportunities to generate tissues from combinations of engineered extracellular matrices, cells and biologically active molecules in order to promote muscle regenerative response. therefore, functional contractile properties as close as possible to that of healthy muscles are requested to allow for a good compatibility and a proper functional contribution. the x-met is an engineering vascularized skeletal muscle tissue able to recapitulate the complex morphological properties, the architecture and the function of skeletal muscle. the x-met shows biomechanical properties like muscles, can contract spontaneously as well as to respond to electrical stimulation. the aim of our study is to define the functional plasticity of x-met subjected to mechanical stimuli. interestingly, preliminary evidences suggest that different mechanical tensions induce a functional remodeling of x-met toward a non-skeletal muscle phenotype. in this work, we demonstrated that x-met show preserved cellular communication junctions, endogenous extracellular matrix (ecm), and integrative adhesive agents that could create a favorable environment for survival and integration of the x-met in the host tissue. for this features, x-met can be considered a useful experimental tool for in vitro and in vivo studies to improve drug discovery technologies, to supersede current preclinical animal models of disease and to restore the lost function of muscles. sertoli cells (sec) are the major component of the seminiferous tubules in the testes, where they secrete a plethora of trophic and immunomodulatory factors necessary for development of germ cells and to protect developing germ cells against the immune system attack, respectively. naked or encapsulated sec have been widely used as a therapeutic approach to many pre-clinical studies [1, 2] . a single intraperitoneal (i.p.) injection of microencapsulated porcine sec (sec-mc) translated into recovery of muscle morphology and performance in dystrophic (mdx) mice in the absence of pharmacological immunosuppression, thanks to a double sec effect, i.e., an antiinflammatory action and heregulin beta 1-dependent induction of utrophin at the sarcolemma [3] . this opened a new route of treatment for duchenne muscular dystrophy (dmd) patients. however, it is still debating if sec exert immunomodulatory or immunosuppressive effects, which is of relevance in view of their application in humans. here we show that mice previously injected i.p. with sec-mc were more able to solve pulmonary infection by aspergillus fumigatus, one of the most common species causing disease in immunodeficient individuals, compared to control mice injected with empty microcapsules (e-mc), as evaluated by analysis of bronchoalveolar lavage, fungal growth, histology (pas and grocott staining), and inflammatory chemokines and cytokines expression (rt-pcr) in lungs. moreover, mice previously injected i.p. with sec-mc and later injected subcutaneously with llc (lewis lung carcinoma) cells showed similar tumor growth over time to mice injected with e-mc, further suggesting that sec do not exert an immunosuppressive role. this was also confirmed by the observation that one year from i.p. injection sec-mc-injected mdx mice did not show neither adverse effects nor increased incidence of tumor formation. altogether, our data suggest that sec exert immunomodulatory rather than immunosuppressive effect, further supporting the use of i.p. injection of sec-mc as a potential treatment of dmd patients and diseases characterized by an inflammatory or autoimmunity environment. duchenne muscular dystrophy (dmd), caused by genetic mutations leading to the absence of dystrophin, is the most common and severe muscular dystrophy, characterised by progressive muscle degeneration leading to premature death by the age of 20-30 years old. in dmd skeletal muscle, the repeated episodes of muscle fiber injury and necrosis, and the subsequent fibrosis are driven to a large extent by complex interactions between dystrophin deficiency and the host immune response. this has been demonstrated by removing macrophages and other inflammatory cell types in the mdx mouse model of dmd as well as through the inhibition of key mediators of inflammation. the approved therapies, based on corticosteroids, support the evidence of the major role of inflammation in this disease. nevertheless, due to their limitations in terms of efficacy and adverse events, they encourage us to find alternative approaches derived from the identification of novel suitable therapeutic targets. to this end we have been investigating, in dmd progression, the pathway of the sphingolipid metabolising enzyme, acid sphingomyelinase (a-smase), which has recently been demonstrated to be involved in inflammatory related disorders. our results in mdx mice muscles revealed an upregulation of a-smase expression and activity strongly connected to inflammation. muscle regeneration in dmd can be impaired due to an imbalance in m1 (pro-inflammatory) and m2 (anti-inflammatory) macrophage phenotypes. our studies in a-smase -/mice, to investigate deeper the role of a-smase show that this enzyme is fundamental for m1 macrophage polarization and that following injury in a-smase -/mice there is a short m1 macrophage phenotype retention phase and a corresponding enhanced m2 macrophage phenotype function. as a proof-of-concept of pharmacological inhibition of a-smase as a potential strategy for dmd we analysed a-smase expression or activity in mice treated with drugs already proven to be beneficial for mdx mice: the anti-inflammatory drugs naproxen/naproxcinod and amitriptyline, a functional inhibitor of a-smase (fiasma). we found that the anti-inflammatory drugs inhibited expression and activity of a-smase while amitriptyline reduced the enzyme activity. our next step is to test the in vivo efficacy of other fiasmas (i.e. the ssri fluoxetine and sertraline) with a better safety profile than amitriptyline. the advantage of this strategy is manifold: i. these drugs are already approved by the ema and the fda, confirmed minimally toxic and potentially rapidly available for clinical use as repositioned drugs; ii. they can also alleviate the depressive symptoms often displayed by patients; iii. they can synergise with the gene-based therapies currently under investigation. the skeletal muscle tissue exhibits regenerative capabilities related to damage extension. in fact, it is able to restore limited injuries while, following volumetric muscle loss (vml), the recovery is poor accompanied by scar formation and functional detriment. this condition negatively affects the quality of life of patients affected by vml, making necessary reconstructive therapeutic approaches. even if surgical autologous transplantation is a standardized procedure, the outcomes are often unsatisfactory. hence, the pressing need to develop engineered artificial tissues to replace wasted muscle. tissue engineering (te), exploiting stem cells embedded in biomimetic scaffolds, aims to mimic organogenesis by building artificial tissues to replace the damaged ones. skeletal muscle te is an up-and-coming biotechnology with great potential for muscle repair, but no conclusive strategy has been conceived yet. reconstructing the skeletal muscle architecture and function is still a challenge requiring the parallel alignment of myofibrils arranged into organized sarcomeres. recently we demonstrated the great potential of a hybrid biomimetic matrix, namely peg-fibrinogen, for enhancing the engraftment of myogenic cell progenitors by providing a suitable 3d environment for mouse muscle reconstruction. starting from these observations, we chose to use a novel approach for the regeneration and/or reconstruction of skeletal muscle tissue combining muscle progenitors with 3d bio-printing technology to guarantee a functional architecture. in particular we have developed a microfluidic wet-spinning system that allows to fabricate rapidly macroscopic yarns of cell-laden hydrogel microfibers that closely mimic the structure of skeletal muscle tissue. in vitro and in vivo characterization of cell-laden constructs showed enhanced myogenesis and positive myo-structure alignment. oxidative stress and mitochondrial dysfunction play a crucial role in the pathophysiology of muscular dystrophies. we recently reported that the reactive oxygen species (ros) produced by the mitochondrial enzyme monoamine oxidase b (mao-b) are causally involved in duchenne pathophysiology, as demonstrated by the results we obtained by treating mdx mice with the mao-b specific inhibitor safinamide. we now show that a novel drug, pxs, designed to inhibit both mao-b and the amino oxidase vap-1 (a glycoprotein found on the plasma membrane of several cell types), provides comparable or superior effect to safinamide, but at a much lower dosage. specifically, we found that a 30-day treatment with pxs in one-and three-month-old mdx mice greatly reduced fibrosis, inflammation and oxidative stress in both diaphragm and tibialis anterior, while also improving muscle contractile properties in diaphragm and gastrocnemius. importantly, we have also obtained preliminary indications that a 90-day treatment with pxs led to a decrease in heart fibrosis in ninemonth-old animals. considering that mao-b inhibitors have been in clinical use for many years and that pxs has already passed all toxicity tests in several animal models, our findings indicate that pxs could be a promising candidate for the treatment of dmd. lurbinectedin (pm01183, pm) is a synthetic alkaloid derivate of trabectedin (et743, et), a marine-derived anticancer agent. pm is a dna minor groove covalent binder that has been tested in different phase i-iii trials. it affects tumor microenvironment by limiting the production of inflammatory cytokines. some of these cytokines are elevated in various cancers characterized by rapid body wasting with muscle, fat and cardiac tissue depletion (i.e. cachexia). differing from et, pm displays less liver toxicity, and less endothelial inflammation at the site of injection. mice injected with murine colon adenocarcinoma c26 cells display cachexia, increased circulating levels of inflammatory cytokines, acute phase response activation and subsequent splenomegaly. thus, we tested whether pm, at doses with no antitumoral activity on c26 colon adenocarcinoma, has any beneficial effects in mice against c26-induced cachexia. 10-weeks old balb/c mice were injected subcutaneously with c26 cells and three days later randomized to receive into their tail vein, three times a week for three weeks 0.07 mg/kg pm (n = 8) or vehicle (n = 8). c26-carrying mice showed decreased body weights and premature death. strikingly, pm was able to extend the lifespan of c26-bearing mice by about 85% from a median survival time of 20 days (range day 10-31) to a median survival time of 37 (range day 14-41). this occurred without affecting tumor growth or food intake or the number of lung metastases. another set of mice were sacrificed at 10-13 days from c26 implant. pm did not grossly protect multiple tissues (fat, muscle, heart) from cachexia. preliminary data showed that c26-induced splenomegaly was inhibited by pm administration as long as pm treatment lasted. in c26-bearing mice, this effect exerted by pm seems to be associated also to restrained circulating levels of m-csf, but not of other inflammatory cytokines (i.e. il-6, g-csf or gm-csf). further studied are necessary to correlate the improved survival with the pharmacodynamic effect observed. oxytocin, classically known for its effects on uterus, lactation, cns, is also a powerful regulator of myogenic differentiation and muscular homeostasis. previous works have shown that the addition of ot stimulates differentiation of myogenic precursors and induces myotube hypertrophy. interestingly, in sarcopenia (senile muscular atrophy) exogenous ot antagonizes skeletal muscle atrophy and restores skeletal muscle trophism in mice. neoplastic cachexia has a strong negative prognostic significance in cancer patients, being associated with a reduction in quality of life and response to therapies. several pharmacological and hormonal treatment have been proposed to counteract cachexia, but this syndrome is still incurable. oxytocin (ot) plays a physiological role in the maintenance of muscle homeostasis in aging. therefore, we propose to study whether the administration of ot counteracts skeletal musculature atrophy in cancer-cachexia. to mimic cancer cellmediated effects on muscle cells, we incubated l6 myoblasts with c26 tumor-conditioned medium (c26 cm). preliminary observations suggest that in vitro the inhibition of differentiation by c26 cm is reversed by the addition of ot in the culture medium of myogenic cells. we also showed that in vivo, ot counteracts the tnf effects on muscle regeneration of the tibialis muscle following freeze-injury. since hampered muscle regeneration and satellite cell function is a key feature of cachexia, contributing to muscle wasting, our preliminary data suggest that indeed ot treatment may have a beneficial effect on muscle homeostasis in tumor bearing mice. thanks to the fact that ot is already approved for clinical use, this work seems promising to prevent cancer-cachexia skeletal muscle atrophy in patients. -41 -ameliorate muscle wasting. in addition, we recently described the essential role of autophagy in driving msc function toward efficient muscle regeneration. the established role of autophagy in maintaining muscle mass and tissue homeostasis together with the emerging role of stat3 in regulating the autophagic process inspired the rationale behind this work which resides in the study of the autophagic contribution in mediating stat3 signalling toward skeletal muscle repair, a function that is compromised during aging. our hypothesis is that stat3 might have a role in regulating myogenic lineage and regeneration process by affecting the autophagic process thereby restoring the pro-myogenic niche that support muscle regeneration. we show that the autophagic process influences msc activation and proliferation suggesting that autophagy might exert different functions depending on msc proliferative vs. myogenic state and in muscle fibers. likely, a combination of autophagy impairment in both compartments cooperates in muscle wasting during sarcopenia. our further analysis indicates that stat3 displays nuclear localization in conditions of active autophagy -i.e. young mice-while stat3 localize in the cytoplasm in aged muscles, characterized by reduced autophagic process. altogether, these evidences suggest that the nuclear/cytoplasmic compartmentalization of stat3 regulates the autophagic process and the regenerative drive, highlighting potential biological targets that preclude an efficient regenerative response in aged mice. ageing, in humans, is characterized by a progressive loss of muscle mass and strength, known as sarcopenia. in the sarcopenic muscle, the regenerative capacity of satellite cells (scs), adult muscle stem cells, is compromised. the scs are responsible for the postnatal muscle growth and the maintenance of muscular mass in the adulthood. these cells are the main source of muscle stem cells able to regenerate the skeletal muscle tissue. during aging, the myogenic capability of scs decreased and it results in the failure to complete the differentiation program. however, the mechanisms behind the impaired myogenic differentiation process of elderly are not fully understood. moreover, during ageing in the skeletal muscle, there is an imbalance between reactive oxygen species (ros) production and antioxidant enzyme activity and alterations in the gene expression 1 . the oxidative stress is harmful for the maintenance of skeletal muscle structure and function, and is considered one of the main contributors causing cellular aging. an effective exercise protocol might be particularly useful to counteract the detrimental decline that occurs in sarcopenic muscle. indeed, the exercise could promote cellular and molecular adaptation mediated by micrornas modulation 2 . furthermore, we showed that during aging activated scs display a complex picture of impaired regenerative potential, linked to altered ros production and mir-1 and mir-133 dysregulation 3 . the aim of this study was to determine whether endurance and resistance training and neuromuscular electrical stimulation (nmes) affect muscle regeneration modulating micrornas and oxidative management in healthy elderly. to achieve this goal, healthy male elderly subjects (76.6±3.7 years; n=50) volunteers were recruited and randomly assigned to different training and control groups; training plans consisted in 12 weeks, 3 sessions/w. functional evaluation tests revealed specific improvements of elderly performance. furthermore, at the volunteer were done the skeletal muscle biopsy before (pre-) and after (post-) training. in detailed 1) pre-and post-scs were isolated and characterized; 2) myo-micrornas (mir133a, mir133b, mir-1, mir206) were analyzed; 3) oxidative status were assessed. in conclusion, our data suggested that endurance and nmes protocols promoted a higher antioxidant status. strikingly, we found that physical intervention is able to modify myo-micrornas expression. indeed, they were specifically down-or up-regulated by the training typologies. -42 -glycosylation inhibition on myoblast differentiation. c2c12 cells were treated with the n-glycosylation inhibitor tunicamycin (tm) (0.01µg/ml) and the mrna expression of ccnd1, pax7, myogenin, myod, mrf4 and desmin was quantified in proliferative myoblasts and differentiated myotubes. pcna, myosin (mf20), igf-1r, igf-1r pathway activation and myogenic index were also quantified by western blot and fluorescent imaging, respectively. finally, we tested whether the sirna knockdown of the pmm2 gene produces effects on myoblast differentiation resembling the tm administration. as expected, ccnd1 and pax7 were expressed in control myoblasts and were downregulated in myotubes, while myogenin and desmin showed the opposite trend. tmtreated myoblasts failed to down-regulate ccnd1 mrna after induction of differentiation and the expression of ccnd1 and pax7 remained higher in tm-treated compared to control myotubes. conversely, tm-treated myotubes expressed lower level of myogenin and desmin compared to control. moreover, mf20 protein and myogenic index were significantly reduced in tm-treated myotubes. glycosylation inhibition also decreased the igf-1r level and markedly attenuated the igf-1-induced erk-1/2 and akt phosphorylation. interestingly, 70% mrna knockdown of pmm2 increased ccnd1 in myoblasts and reduced myogenin, myod and mrf4 mrnas and mf20 expression in myotubes. in conclusion, we found a reduction of myoblast differentiation after n-glycosylation inhibition by tm and pmm2 silencing. tm administration also affected the igf-1r pathway reducing the igf-1r expression and inhibiting erk-1/2 and akt activation. the decrement in the expression of differentiation markers observed here after pmm2 silencing, suggests that a deficit in muscle cell differentiation could be present in cdg patients. duchenne muscular dystrophy (dmd) is an x-linked genetic disorder resulting from mutations and deletions of the dystrophin gene that leads to severe degeneration of muscle tissue that is progressively substituted by fat and fibrous tissue. among the cytokines involved in this process, tgf- is a key player polypeptide that orchestrates the crosstalk between parenchymal, inflammatory and collagen-expressing cells with a recognized pro-fibrotic role. in dmd patients, tgf- is elevated both in plasma and in muscles and its expression correlates with increased fibrosis. in vitro, it has been found highly expressed in dmd myotubes and in muscle derived fibroblasts. tgf- activity is strictly controlled at various levels and the cytokine is secreted as a latent complex where it is tightly bound to the so-called latent tgf- binding protein (ltbp) and released as active polypeptide upon specific proteolytic cleavage. among the ltbps described, the ltbp4 gene was discovered as a modifier of murine muscular dystrophy. studies in dmd patients revealed that four ltbp4 snps were predictive of the age at onset of loss of ambulation and of dilated cardiomyopathy. further studies in mice and cell culture demonstrated that more tgf-β is released from the ltbp4 vttt variant compared to the iaam-containing ltbp4 variant, the latter being associated with older age at loss of ambulation and less tgf-β signaling. histone deacetylases (hdacs) are involved in fibrogenesis and reduction of hdac activity by inhibitors targeting both class i and ii, hinders tgf-β induced fibrosis. moreover, it was observed that our proprietary hdac inhibitor givinostat significantly reduced fibrosis in mdx mice and in dmd boys. the aim of this study was to investigate the molecular effects of givinostat on tgf-β induced fibrotic gene program in an in vitro model of human healthy and dmd-derived skeletal muscle cells. in agreement with its role, tgf-β induced the expression of pro-fibrotic genes such as alpha smooth muscle actin (sma), fibronectin (fn1) and collagen iii, both in healthy and dmd cells. givinostat clearly reduced the upregulation of sma and fn1 in healthy cells while the effect on dmd cells was negligible for sma, and fn1 expression was further increased. downregulation of collagen iii was observed in both cell types. a different epigenetic regulation of key genes in the tgf-β pathway was also highlighted by the effects on the expression of tgf-β receptor subunits tgf r1 an r2. thus, tgf r1 was upregulated by the cytokine and downregulated by givinostat in both cell types while tgf r2 was downmodulated by tgf-β in dmd cells only and givinostat increased its expression in both cell types. a differential regulation of matrix metalloproteinases and their inhibitors was also observed. these data emphasize how a different epigenetic regulation of key fibrotic genes takes place in healthy and dmd cells. furthermore, they also highlight the importance of having an in vitro model whereby both healthy and dmd cells are used to study the specific pathways that can be targeted in diseased cells to more accurately advance drug candidates into in vivo studies. introduction: cancer-associated cachexia is a multifactorial syndrome characterized by anorexia and body weight loss, mainly due to muscle and fat wasting (1) . micrornas (mirnas) are a class of non-coding posttranscriptional regulators that play, among other functions, a central role in appropriated muscle cell commitment, proliferation and differentiation by modulating the translation of specific genes (2) . the present study was aimed to evaluate the possibility to use dysregulated mirnas as biomarkers of cancer cachexia. methods: total rna was extracted from both skeletal muscles and plasma-derived microvesicles from controls and c26 tumor-bearing balb/c mice. next-generation sequencing (ngs) was used to sequence whole mirna transcriptome. those mirnas differentially regulated were validated by real time pcr. results: clear differences in mirna expression between the two experimental groups were observed. in particular, mir-21a-5p, mir-29a-3p, mir-29c-3p, mir-185-5p, mir-185-3p, mir-223-3p were statistically upregulated in the tumor-bearing mice. by contrast, mirnas from microvesicles were poorly modulated in the c26 host, only three mirnas (mir-181a-5p, mir-375-3p, mir-455-5p) were found dysregulated at ngs analysis, suggesting that their use as a circulating biomarker might be limited. conclusion: the results obtained in the present study suggest that modulations of the skeletal muscle mirnaome can be used as effective biomarkers in the pathogenesis of cancer-associated cachexia in murine models. -44 -decreases in c26 tumor bearing mice, and that exercise can rescue. indeed, we showed a decrease of srf expression at the protein level in cancer cachexia. consistently, we showed a decrease in the expression of srf target genes such as myod and sk-actin, which suggest a decrease of the srf transcriptional activity. these effects were counteracted by wheel running. since we observed opposite effects of tumor and exercise on myod and pax7 we hypothesized the involvement of a myogenic program in stem cell recruitment to muscle fibers upon exercise: indeed, we observed the recruitment of nuclei within the muscle fibers in response to exercise, which could contribute to muscle homeostasis. our preliminary results suggest that physical activity rescues srf expression as well as its transcriptional activity, highlighting the importance of genetic activation induced by skeletal muscle activity for muscle rescue and homeostasis. these effects could be extended to the fiber microenvironment, including myogenic stem cell activity. vitamin (vit) d is a pleiotropic hormone synthesized mainly in the skin via a uv-dependent reaction. vitd circulates in the blood bound to vitamin d-binding proteins, reaching its target tissues, where exerts its endocrine actions through the vitd3 receptor (vdr), a member of the nuclear receptor family expressed in different tissues, including skeletal muscle. some vitd actions have been shown to be at long term and others at more immediate term, suggesting the engagement of nuclear vdr for gene expression regulation and of membrane-associated vdr for the control of specific signalling pathways. vitd deficiency has been shown to cause abnormalities in skeletal muscle, such as reduced actomyosin content and decrease in mitochondrial ca 2+ levels. moreover, in vitro experiments suggest that vdr is likely to play an important role during myoblast differentiation, regulating the expression of myogenin. despite several evidence demonstrating a protective role of vitd in skeletal muscle cells, the characterization of biomolecules that positively affect vdr expression /function are limited. in the present study we investigate the role of sphingolipids in the regulation of vdr expression in c2c12 myoblasts/myotubes and the molecular mechanisms by which sls metabolites, in particular sphingosine-1-phosphate, determine an increase in vdr expression in skeletal muscle cells. oxidative stress and chronic inflammation have been proposed as important interconnected mechanisms affecting skeletal muscle homeostasis under pathologic conditions, being mutually induced processes. although reactive species are physiologically formed inside of skeletal muscle by inflammatory cells as well as myofibers, the endogenous antioxidant defense is able to maintain reactive radicals at functional levels. instead, under pathological conditions the excess of reactive species can overwhelm antioxidant defense leading to oxidative damages and to the activation of pro-inflammatory pathways. on the other hand, inflammatory response is considered one of the principal determinants of degenerative processes in degenerative disorders. however, the clear connection between the extent of oxidative stress and inflammation has still to be understood. hence the needs of identifying and studying key factors involved in the intimate relation between these two important pathogenic mechanisms that might allow relevant advances in the field of chronic diseases. among factors playing a critical role in skeletal muscle physiopathology interleukin-6 (il-6) is an elective candidate. here we reported that increased levels of circulating il-6 are sufficient to alter the physiologic redox balance of skeletal muscle tissue and to induce the establishment of an inflammatory milieu. we revealed that high levels of il-6 in the bloodstream enhance the production and accumulation of reactive species in diaphragm muscles of adult nse/il-6 mice, by impinging the muscle redox balance and impairing the nrf2-mediated antioxidant response. in addition, the occurrence of pro-oxidant conditions in muscles exposed to abnormal amounts of il-6 stimulates the establishment of a delicate inflammatory balance within muscle environment, characterized by the induction of pro-inflammatory mediators. thus, uncontrolled levels of this cytokine, which characterize chronic degenerative conditions, can alter muscle milieu, linking the generation of oxidative stimuli to the promotion of inflammatory changes. background: facioscapulohumeral muscular dystrophy (fshd) is a frequent neuromuscular disease caused by epigenetic alterations on the 4q35 chromosome leading to gain of expression of the double homeobox 4 (dux4) gene. our laboratory previously showed that the long non-coding rna dbe-t is required for aberrant dux4 expression in fshd. using affinity purification followed by proteomics, we identified the chromatin remodeling protein wdr5, a core component of the mll/set1 epigenetic activation complex, as a novel dbe-t interactor. the goal of this work is to analyze the role of wdr5 on the regulation of dux4 in fshd. methods: primary myoblast isolated from both fshd and healthy control subjects were differentiated in myotubes in presence of sirnas. 24 hours after induction of differentiation, myotubes have been transfected with sirna on-target wdr5 and sirna non-targeting as control. four days after differentiation in mature myotubes the expression level of dux4 and its known targets have been assessed with qrt-pcr. moreover, the myogenic differentiating level and the fusion index have been assessed through immunofluorescence analysis of the myosin heavy chain (mhc) differentiation marker. results: we found that the expression level of dux4 and its target mbd3l2, trim43 and trim48 are significantly reduced by silencing wdr5. similar results were obtained using a wdr5 small molecule inhibitor. remarkably, wdr5 knockdown leads to rescue of the myogenic defects of fshd muscle cells at both the differentiation level and the fusion index. conclusion: these results show the pivotal role played by wdr5 in dux4 expression and candidate wdr5 as potential therapeutic target for fshd treatment. . the purpose of this work has been to study the skeletal muscle strength development, the main feature of this tissue, when sedentary adult mice were exposed to elf-emfs. male c57bl/6 sedentary adult mice were exposed to elf-emfs (0.1 or 1 mt) generated by a solenoid for 1h/die up to 5 weeks and fed with a standard diet or a n-acetyl-cysteine (nac) enriched diet. grip strength and body weight were measured weekly, and after 5 week exposure the mice were sacrificed and skeletal muscle from hind limbs were isolated and collected for biochemical analyses. the elf-emfs did not affect the body weight and, conversely, increased the grip strength in exposed mice in comparison to non-exposed ones. in addition, both the expression levels of pax7 and of myosin heavy chain were found increased in muscle isolated from exposed mice. the elf-emfs failed to trigger their effects when the mice were fed with an nac-enriched diet. these preliminary data suggest that elf-emfs may provide some benefits improving the muscle fitness in sedentary adult mice. the current leading cause of death in duchenne's muscular dystrophy (dmd) patients is cardio-respiratory failure, mainly due to heart and diaphragm (dia) damage and fibrosis during the late stage of the disease. given that fibrosis is considered a consequence of muscle inflammation, the role of the immune response in dmd heart and diaphragm should be considered, to better understand the pathological events during and prior to the onset of fibrosis. indeed, very little is known about the kinetics of immune cells infiltration in dystrophic heart and diaphragm, that likely contributes to the fibrosis and chronic inflammation. the treatment of choice in dystrophic patient is the use of corticosteroids, supporting an important role of the inflammatory compartment in the disease progress. the several and severe adverse effects of long-term corticosteroid treatment also highlight the need for other anti-inflammatory approaches. our group recently found that pharmaceutical -46 -inhibition of protein kinase c theta prevented damage in limb skeletal muscle and ameliorated disease when administered in young (2week-old) mdx mice, acting predominantly through inhibition of early t cell infiltration of dystrophic muscle. we now plan to study in detail the inflammatory compartment of dystrophic heart and dia using the mouse model of dmd, mdx. we will characterize in detail the kinetics and quality of inflammatory cell populations in mdx heart and dia, using our established 9 color cytofluorimetric protocol, analysing mice from 4 weeks of age up to 11 months. morphological analysis on muscle fibrosis, necrosis and organization will be performed, as well as molecular analyses to assess cytokines levels and fibrosis markers. given that mdx mice often show a poor heart pathology, we are currently defining an exercise protocol in order to worsen the dystrophic phenotype, in order to mirror the human pathology. the data we will collect in this part of the study will be instrumental to design new pharmacological treatments using specific inhibitors (as we did for skeletal muscle) instead of broad-spectrum anti-inflammatory compounds. we hope that our study will help developing more effective and fine targeted treatments, with less adverse effects for patients thyroid hormone (th) has a major role in the control of systemic metabolism, influencing carbohydrate, protein and lipid metabolism. moreover, t3 regulates mitochondrial function and turnover controlling mitochondrial biogenesis, proton leak, oxphos and ros generation. the deiodinases enzymes enable intracellular th activation or inactivation, regardless of circulating hormone levels. to address the physiological function of deiodinases in skeletal muscle metabolism, we used muscle-specific gain-of-function approaches. here, we show that muscle-specific hypothyroidism in mice by d3 overexpression (tg-d3 mice) induces a metabolic reprograming of muscle fibers reducing glycolytic and lipid metabolism. d3 overexpression in muscle also reduces mitochondrial dynamics. immunofluorescence analysis revealed a structural alteration of mitochondria. facs analysis highlighted a reduced mitochondrial density with increased size. however, the increased mitochondrial size did not correlate with increased activity. consistently with these findings, mitochondria turnover genes drp-1, mfn-1 and opa-1, as well as pgc-1α, were reduced in tg-d3 muscles. these data suggest that metabolic differences between tg-d3 and control muscles are associated with an alteration in number and function of the mitochondria. interestingly, the overexpression of pgc-1α in tg-d3 muscles rescued the mitochondrial defects, reactivating the expression of genes involved in mitochondrial fusion and fission. altogether these results indicate that the mitochondrial dysfunction of tg-d3 muscles is mediated by the downregulation of pgc-1α, the master regulator of mitochondrial biogenesis. our findings indicate that muscle-specific hypothyroidism via d3 overexpression potently impacts on mitochondrial metabolism, identifying the deiodinases as critical metabolic regulators. understanding the mechanisms of action of deiodinases in metabolism might be relevant for therapeutic treatment of metabolic disorders. background: physical activity ameliorates the prognosis of cancer patients, also by contrasting the associated muscle wasting (i.e. cachexia). since aerobic exercise seems to be the most effective to preserve muscles during cancer, we asked whether it promotes secretion of proteins by muscles (i.e. myokines) that may contrast cachexia. methods: to mimic aerobic exercise, we infected c2c12 myotubes with pgc1α expressing adenoviruses. in vitro we evaluated the effects of supernatants from gfp or pgc1α-overexpressing cells on protein synthesis and degradation of atrophying myotubes and in lucassay. by microarray, we identified putatively secreted proteins inducible by pgc1α and confirmed by q-pcr. we measured by q-pcr their expression in tibialis anterior (ta) muscle of c26-bearing mice during cachexia and plasma levels by elisa. to induce aerobic exercise adaptations, mice were run on treadmill. anaerobic exercise-like effects were obtained in vivo in overloaded plantaris muscle and in vitro in myotubes expressing myristoylated akt. results: our microarray and q-pcr analyses showed musclin as a pgc1α-induced myokine. conversely, its expression was unchanged in myotubes hypertrophying because of activated akt. dexamethazone-treated myotubes or constitutively active (ca)foxo3-expressing ones undergo atrophy as measured by increased proteolysis and murf1 induction. unlike to gfp, musclin restrained the dexamethazone-induced murf1 expression in luciferase assays. consistently, musclin-containing supernatants reduced the cafoxo3-induced rates of long-lived protein degradation. among the newly identified pgc1α-induced myokines, we found that -47 -only musclin (and its receptor npr3) was strongly downregulated in cachectic muscles and plasma of c26bearing mice. thus, we electroporated ta of c26-bearing mice with musclin or npr3-encoding plasmids and found either musclin or npr3 to preserve fiber area. interestingly, treadmill exercise protected c26-bearing mice from muscle loss, with no effect on tumor growth, and rescued the c26-induced downregulation of musclin in muscles and plasma. by contrast, musclin expression did not change in overloaded plantaris of adult mice. conclusions: musclin is a myokine induced specifically by pgc1α, typically increased upon aerobic exercise and preserves muscles from wasting during c26 growth or myotubes from atrophy. overall, musclin could be beneficial to cancer patients that cannot exercise and are at risk of developing cachexia. reconstruction of the skeletal muscle tissues after damage relies on the activation of satellite cells (scs), a population of resident muscle stem cells. several transcription factors are involved in scs activation and proliferation and ensure proper temporal and spatial expression of muscle-specific genes during muscle regeneration. the transcription factor nf-y, composed by nf-ya, nf-yb and nf-yc subunits, has an important role in the regulation of cellular proliferation and differentiation in different cell types, among which muscle cells. while nf-ya, the dna binding subunit of nf-y, is down-regulated in the adult muscle of wt mice, its expression is observed in the mdx mouse and correlates with euchromatic markers and expression of nf-y target genes controlling cell growth. with the aim to investigate the role of nf-ya in the scs proliferation and differentiation, we generated and characterized a conditional knock out mouse model in which nf-ya is deleted in pax7+ scs by tamoxifen induction in adult nf-ya flox/flox :pax7 creer mice (nf-ya cko). cellular and molecular analysis carried out on isolated myofibers and scs from wt and nf-ya cko mice highlighted that nf-y activity is important for the maintenance of scs homeostasis. nf-ya loss depletes pax7+ scs pool and impairs their proliferation. moreover, scs-mediated regeneration following muscle damage induced by cardiotoxin is delayed in nf-ya cko. the effect of nf-ya abrogation was also explored in post-natal muscle growth. immunohistological analysis showed defects in muscle morphology and a decrease in scs number in 3 weeks aged nf-ya cko mice, period of major increment of muscle mass by scs-mediated myonuclear accretion. the molecular mechanism underlying the impairment of scs activity following nf-ya loss was investigated by gene expression profiling. overall, our results highlight a role of nf-y in muscle regeneration and in scs fate, whose modulation could be useful to improve stem cell based therapies to treat muscular dystrophies. thyroid hormone (th) is a key metabolic regulator that acts by coordinating short-term and long-term energy needs. accordingly, significant metabolic changes are depending on thyroid status. although it is established that hyperthyroidism augments basal energy consumption, thus resulting in enhanced metabolic state, the net effects on cellular respiration and generation of reactive oxygen species (ros) remain unclear. to elucidate the effects of augmented th signal in muscle cells, we generated a doxycycline-inducible cell line, in which the expression of the th-activating enzyme, type ii deiodinase (d2) is reversibly turned on by the "tet-on" system. interestingly, increased intracellular th caused a net shift from oxidative phosphorylation (oxphos) to glycolysis and a consequent increase in extracellular acidification rate. as a result, the mitochondrial ros production, and both the basal and doxorubicin-induced production of cellular ros were reduced. importantly, the expression of a set of antioxidant genes was up-regulated, and, among them, the mitochondrial scavenger sod2 was specifically induced at transcriptional level, by d2-mediated th activation. finally, we observed that the attenuation of the oxidative stress and increased levels of sod2 are key elements of the differentiating cascade triggered by the th and d2, thereby establishing that d2 is essential in coordinating metabolic reprogramming of myocytes during myogenic differentiation. in conclusion, our findings indicate that th plays a key role in oxidative stress dynamics by regulating ros generation. our novel finding that th and its intracellular metabolism act as mitochondrial detoxifying agents sheds new light on metabolic processes relevant to muscle physiology the functional exhaustion of muscle stem cells (muscs) contributes to duchenne muscular dystrophy (dmd) progression by compromising the compensatory regeneration of diseased muscles. muscs activity is influenced by functional interactions with cell types that compose their niche, including fibro-adipogenic progenitors (faps) that regulate the regenerative ability of skeletal muscles in physiological and pathological conditions. we have recently discovered that fap-derived extracellular vesicles (evs) support functional interactions with muscs, and contribute to the beneficial effect of hdac inhibitors (hdaci) on dmd muscles. fap-derived evs mediate microrna transfer to muscs, and that exposure of dystrophic faps to hdac inhibitors (hdaci) increases the intra-ev levels of a subset of micrornas (mirs), which cooperatively target biological processes of therapeutic interest, including regeneration, fibrosis and inflammation. in particular, increased levels of mir206 in evs released from faps of muscles from dmd patients or mdx mice exposed to hdaci correlated with improvement of key histological parameters, such as compensatory regeneration and inhibition of fibrosis. we found that evs from hdaci-treated dystrophic faps stimulated musc activation and expansion ex vivo, and promoted regeneration, while inhibiting fibrosis and inflammation of dystrophic muscles, upon intramuscular transplantation, in vivo. these data reveal a potential for pharmacological modulation of fap-derived ev's content as novel strategy for local therapeutic interventions in muscular diseases. sarcopenia is the age-related loss of muscle mass, strength and function. muscle mass decrease is directly responsible for functional impairment with loss of strength, increased likelihood of falls and loss of autonomy. satellite cells are myogenic progenitors and sarcopenia is predicted to be the result of reduced satellite cell number and/or function. despite several investigations, the precise molecular mechanisms underlying stem cell dysfunctions in the age-associated muscle decline remains unclear. recently cutting-edge studies highlighted the role of epigenetic mechanisms in muscle senescence and sarcopenia. among the biological mechanisms involved in the regulation of heterochromatin conformation, we recently described a functional and evolutionary conserved crosstalk between the nuclear lamin a/c and the pcg proteins. this functional interplay is required for the maintenance of the pcg repressive functions at the onset of muscle differentiation -49 -and does not work properly in the pathological emery dreifuss muscular dystrophy. based on these findings we hypothesize that in aged satellite cells an altered lamin/pcg axis could determine a pathological remodelling of the heterochromatin organization leading to the loss of muscle stem cell functions. taking advantage of a new technique developed in our laboratory, named sequential analysis of macromolecules accessibility (sammy-seq) we were able to map lamina associated heterochromatic regions in small amount of primary cells. the protocol is based on the sequential extraction of multiple chromatin fractions, corresponding to increasingly compacted and less accessible chromatin regions, which are mapped along the genome using high-throughput sequencing. we will show results obtained in muscle tissue or stem cells derived from mice and human at different stage of muscular aging and sarcopenia with the purpose to highlight age-related chromatin structural changes, opening up the possibility to identify coding and/or noncoding genomic regions, dependent or not by polycomb proteins, involved in premature senescence of sarcopenic muscles. telocytes (tcs) represent unique stromal cells characterized by a small cell body and distinctive extremely long, thin and moniliform cytoplasmic extensions called telopodes alternating slender segments (podomers) with dilatations (podoms). tcs have been identified in the skeletal muscle interstitium with their telopodes being strategically positioned in the close vicinity of striated myofiber s with regenerative features, nerve endings, small blood vessels, and often of resident muscle stem cells, namely satellite cells (scs). a "nursing" role for tcs in sc-mediated skeletal muscle regeneration has been supposed; however, to date there is no experimental evidence demonstrating a morpho-functional interaction between these two cell types in an injured skeletal muscle. hence, the aim of present morphological study was to explore the presence and the behavior of tcs in an ex vivo murine model of skeletal muscle (extensor digitorum longus) damage induced by forced eccentric contraction (ec) in isometric condition, focusing on their interaction with scs. ec-damaged skeletal muscles showed evidence of structural and ultrastructural injury as judged by light microscopic and transmission electron microscopy (tem) examination and along with significant electrophysiological changes of sarcolemnic properties. specifically, we observed a resting membrane potential depolarization, an increase of membrane capac itance, a reduction of membrane resistance and a reduction of the outward k + current amplitude, leading to an overall alteration of myofiber excitability. tcs were identified in both control and ec-injured muscles within the interstitium, alongside the myofibers and in close vicinity of vascular structures either by cd34/cd31 double confocal immunofluorescence staining (i.e. tcs: cd34 + /cd31 -; endothelial cells: cd34 + /cd31 + ) or by tem. of note, in ec-damaged muscles, the telopode network was more extended and arranged around activated scs displaying nuclear positivity for pax7, the most reliable marker of scs, and for myod, the sc activation marker. tem analysis clearly demonstrated the presence of tcs invading the sc niche passing with their telopodes a broken basal lamina to contact the underlying activated sc that exhibited a swollen appearance and an euchromatic nucleus. the interaction between tcs and scs was confirmed by in vitro experiments performed by culturing single living endomysial sheath-covered myofibers isolated from ecdamaged muscle and the derived stromal cells/tcs and scs. interestingly, tcs from ec -damaged myofibers showed an increased expression of vascular endothelial growth factor (vegf) -a, whose role in promoting myoblast proliferation and differentiation is documented. scs isolated from the same samples exhibited an increased myod expression as well as a major tendency to fuse into myotubes. these findings establish for the first time a morphological interaction between tcs and scs in a damaged muscle and suggest a juxtacrine-paracrine cell-cell interaction involving vegf-a, which worth investigating further. facioscapulohumeral muscular dystrophy (fshd), the third most common hereditary myopathy, is not due to a classical mutation within a protein-coding gene. instead, almost all fshd patients carry deletions of an integral number of tandem 3.3-kilobase repeat units, termed d4z4, located on chromosome 4q35. d4z4 deletion leads to modifications of chromatin structure and inappropriate overexpression of 4q35 genes. studies have proposed several candidate genes within this genomic region and several mouse models have been generated. among these models, mice overexpressing facioscapulohumeral muscular dystrophy region gene 1 (frg1) present a progressive myopathy that recapitulate features of human disease. frg1 encodes for an rna binding protein whose biological function is not well understood. to investigate the molecular mechanism triggered by frg1 overexpression leading to overt myopathy we analyzed molecular changes occurring during disease development. at first, gene expression profiles of skeletal muscles of mice overexpressing increasing levels of frg1 were examined at 28 days (dystrophy onset) and at 96 days (full dystrophy). we found a profound transcriptional deregulation correlating the severity of the muscle phenotype and frg1 expression. gene set enrichment analysis and gene ontology revealed alterations in pathways related to muscle function, energy metabolism and inflammation. indeed, genes associated with adult and normal myogenesis were down-regulated with a significant enrichment of genes specifically expressed during embryogenesis. by contrast, we observed the incremental activation of inflammatory pathways through time. we found that frg1 overexpression causes the global perturbation in the mechanisms that guide postnatal muscle maturation. this process includes the anomalous expression of embryonic/neonatal proteins fundamental for muscle structures and metabolic pathways, the lack of mature proteins and the lag of muscle growth throughout postnatal life. in particular at 7 days and 14 days the expression of the embryonic isoforms of myosin remain high in frg1 mice instead of following the physiological down-regulation occurring in wt mice, meanwhile the expression of the mature isoforms is reduced. starting from 14 days we observed the deceleration of body weight growth curve and a reduction of muscle cross-sectional area. moreover, frg1 muscles displayed the significant reduction of atp and the phosphocreatine in association with the transcriptional downregulation of glut4, hk2 and aldoa. our results indicate that frg1 overexpression induce the impairment of muscle maturation and energy metabolism that precedes dystrophy. our study opens new perspectives on the molecular mechanisms at the basis of muscular dystrophies. p. 25. effects of aerobic, resistance and combined exercises on 24-hr glucose variability, metabolism and muscle signalling pathways regulation in type 1 diabetics giosuè annibalini a# , dean minnock b# , giacomo valli a , serena contarelli a , roberta saltarelli a , carel leroux c , vilberto stocchi a , elena barbieri a,d , giuseppe de vito b ; # equally contributing authors duchenne muscular dystrophy (dmd) is a neuromuscular disease caused by a deficiency of the dystrophin protein. currently no cure is available for the disease and numerous pharmacological approaches are being studied to extend the regenerative phase of dystrophic muscles. in this project we aim to investigate if the modulation of the epi-transcriptome could represent a new pharmacological tool for dmd. in recent years, genetic and biochemical studies have revealed how post-transcriptional modifications of rna influence various cellular mechanisms, in particular splicing, mirna processing and transcripts stability. one of the most important changes in rna is n6-methyladenosine (m6a). this modification is added from a complex containing the mettl3 / mettl14 heterodimer and removed from rna demethylases such as fto and alkbh5. we have recently observed an increase in m6a levels in skeletal muscles derived from dystrophic mice compared to wilde type animals, suggesting that under pathological conditions the n6-methyladenosine modification could influence muscle regeneration through the modulation of various cellular pathways. to validate this hypothesis, we conducted several experiments both in vivo and ex vivo using pharmacological and genetic approaches, aimed at modulating the expression and activity of components of the methylation machinery. in the pharmacological approach we have treated dystrophic mice with a drug approved by the fda that has recently shown to increase rna methylation through the inhibition of fto demethylase; the genetic approach instead involves the use of sirna against metll3 in primary myoblasts. our preliminary results showed that the rna methylome (m6a) is dynamically modulated during skeletal muscle differentiation and regeneration, pointing at the epi-transcriptome as a new potential target for therapeutic interventions. in addition, we identified new mettl3 direct targets in muscle cells through rna-seq experiments. finally, increasing rna methylation by fto inhibition modulates the regenerative response in dystrophic mice. altogether, the results derived from these studies will set up the basis for a new therapy based on the modulation of the epi-transcriptome for dmd. italy muscular dystrophies are a group of heterogenous genetic neuromuscular pathologies, usually manifesting a progressive muscle degeneration accompanied by strength detriment. despite muscular dystrophies are present in more than 40 forms, many of these affect the dystrophin associated glycoprotein complex (dagc) being responsible for phenotype affection. concretely, the involvement of sarcoglycan (sg) complex, dystrophin, or α-dystroglycan, present a relative common clinical manifestation affecting proximal muscles differing on pathological severity. nowadays, no cure is available for these disease, however different strategies are emerging against muscle degeneration likewise gene and cell therapy, or anti-inflammatory treatments. these strategies aim to reestablish the genetic disorders and muscle integrity, or at least to reduce fibrosis and stop fat replacement. nevertheless, spontaneously several muscle groups resist degeneration and wasting process as it occurs with tongue and calf in human patients. we analyzed mdx and α-sarcoglycan mouse model revealing differences on myofibers degeneration among muscle groups of the same subject, presenting common affection frequency. our objective is to go deeper on characterization of these mice models epigenetic tuning of mir in fap-derived extracellular vesicles promotes regeneration and inhibits fibrosis in dystrophic muscles clinical r&d italfarmaco spa, cinisello balsamo. -50 -60, and mainly extracts proteins from multimeric structures. in particular, by interacting with ufd1 or p47, it facilitates the rapid degradation of myofibrillar proteins during muscle atrophy caused by denervation or fasting. the aim of this study was to investigate if p97 (and through which of its adaptors) plays a role also during cancer cachexia and if this is modulated by physical exercise. to induce cachexia, we injected subcutaneously one million of colon adenocarcinoma (c26) cells in balb/c mice. this tumour causes massive muscle depletion with premature death in mice. interestingly, by microarrays, we found that 8 out of 58 p97-binding proteins were induced in cachectic tibialis anterior (ta) from c26 mice 24 the rna binding protein frg1 controls transcription landscape regulating muscle maturation and metabolism mattia forcato a , ernesto picardi b , elena germinario c , bert blaauw c , graziano pesole b , giuseppe d'antona d , rossella tupler a . a school of public health physiotherapy and sports science t1dm patients tend to avoid exercise to reduce the risk of hypoglycaemia [2] and losing the beneficial effects of exercise. furthermore, t1dm patients show an impairment of the skeletal muscle growth, energy metabolism and repair mechanisms interstitial glucose (ig) was measured with a flash glucose monitoring (fgm, abbott freestyle libre) for 24-hr pre and post exercise. the mean amplitude of glycaemic excursions (mage), standard deviation (sd) and coefficient variation (cv) of ig were calculated from fgm data. muscle biopsies were collected immediately before and after each session. myogenesis-(myogenin, myod, mrf4, myf5), inflammation-(il-6, tnfα, mcp1), oxidative capacity/metabolism-(pgc-1α1, pgc-1α4, glut-4) and muscle growth-related genes (igf-1 isoforms) were quantified by real-time pcr. the exercise-induced skeletal muscle signalling pathways (phosphorylation of akt, p38, erk1/2, eef2, ampk) were quantified by western blot. mage, sd and cv decreased 24-hr post comb. all exercise sessions reduced the cv over a 6 to12-hr period after exercise. no severe hypoglycaemic events occurred. both res and comb increased the activation of the igf-1/akt/mtor signalling pathway (akt and p38mapk phosphorylation), while eef2 activation (dephosphorylation) and gene expression of myogenin and mrf4 increased only after comb and res. both aer and comb promoted the splicing of igf-1ea variant and increased pgc-1α isoforms expression. the expression of inflammation-related genes (tnf-α, il-6 and mcp-1) increased after all exercise sessions diabetes care diabetes care n6-methyladenosine as a novel pharmacological target involved in muscle regeneration costantin heil a , francesco millozzi a , luca madaro b , marco de bardi c , daniela palacios a a. laboratory of epigenetics and signal transduction, irccs fondazione santa lucia, via del fosso di fiorano identification of a phytotherapy formulation to counteract muscle atrophy skeletal muscle atrophy is a loss of muscle mass and strength associated with several pathologies including diseases characterized by systemic chronic inflammation the increase of a catabolic state resulting in the breakdown of myofibrillary proteins, especially myosin heavy chain (myhc), is recognized as the main process promoting muscle atrophy [3]. recently, the interest in plant extracts/metabolites active in the pathophysiology of muscle is obtaining growing attention [4]. starting from one hundred hydroalcoholic extracts from medical plants, our work was aimed to individuate a herbal formulation efficient in counteracting muscle atrophy. by using well-characterized in vitro experimental models mimicking muscle atrophy, i.e., treatment of myotubes from c2c12 myoblasts with proinflammatory cytokines (tnfα/ifnγ) or excess gcs (dexamethasone) [3], we firstly identified fifteen extracts able to rescue myotube atrophy, by morphological analysis after may-grunwald/giemsa staining. ten extracts displayed an extremely potent protective effect against myotube atrophy and myhc degradation, as indicated by measurement of myotube diameters after immunofluorescence staining for myhc-iia, even if at different extent in the presence of cytokines or dexamethasone. based on their anti-atrophic potential and nmr-based metabolic profiles, we selected six extracts (withania somnifera dunal, panax ginseng meyer, silybum marianum gaertner, peumus boldus molina, trigonella foenum-graecum l. and urtica dioica l.) to obtain twenty combinations of three extracts to test on myotube cultures. three herbal formulations showed a surprising ability to counter the reduction of myotube diameters and degradation of myhc in the presence of different atrophic stimuli the emerging awareness that cell populations are heterogeneous has changed our criteria to define a cell type. among the non-genetic sources of cell to cell variability, a spread in a bell-shaped distribution of a feature defines a condition called micro-heterogeneity. the effect at cellular and physiological level of this type of heterogeneity remains to be investigated. we chose skeletal muscle regeneration and fibro/adipogenic progenitors (faps) to address this issue. faps are a population of mesenchymal progenitors that resides in the interstitium of skeletal muscle and are identified by the expression of sca1. in the mdx mouse, an animal model of duchenne muscular dystrophy (dmd), faps are one of the populations responsible for the development of ectopic tissues, such as intramuscular adipose tissue (imat) and fibrotic tissue. taking advantage of a multiplex flow cytometry analysis we defined two fap cell states characterised by high and low sca1 expression (sca1 high and sca1 low). these two sub-populations are differentially represented in wild type and mdx mouse. next, we isolated the two fap sub-populations by fluorescence activated cell sorting (facs) from mdx mouse and we studied their differentiation potential ex vivo. we found that sca1 high faps have a higher capability to express ppar-gamma and to differentiate into mature adipocytes. whereas, sca1 low faps differentiate readily into myofibroblasts in a pro-fibrogenic environment. moreover, sca1 high faps exhibit a higher proliferation rate than sca1 low cells. overall, we demonstrate that micro-heterogeneity contributes to determine the fate of a mesenchymal population in vitro. these findings raise several questions about its interaction with in vivo microenvironment and its impact in pathology progression. cancer-cachexia results in severe muscle tissue wasting affecting patients' quality of life and survival. recent studies showed that physical activity increased survival in cancer patient and animal models. the underlying mechanisms, however, are still largely unknown. to identify signaling pathways involved in exercisedependent maintenance of muscle mass and function in cachexia, we investigated the role of serum response factor (srf), a transcription factor of the mads-box family, having a major role in muscular growth, differentiation and regeneration. we hypothesize that the expression and/or transcriptional activity of srf key: cord-348091-pnvn0x4q authors: nolte, thomas; brander-weber, patricia; dangler, charles; deschl, ulrich; elwell, michael r.; greaves, peter; hailey, richard; leach, michael w.; pandiri, arun r.; rogers, arlin; shackelford, cynthia c.; spencer, andrew; tanaka, takuji; ward, jerrold m. title: nonproliferative and proliferative lesions of the gastrointestinal tract, pancreas and salivary glands of the rat and mouse date: 2016-02-13 journal: j toxicol pathol doi: 10.1293/tox.29.1s sha: doc_id: 348091 cord_uid: pnvn0x4q the inhand (international harmonization of nomenclature and diagnostic criteria for lesions in rats and mice) project is a joint initiative of the societies of toxicologic pathology from europe (estp), great britain (bstp), japan (jstp), and north america (stp) to develop an internationally accepted nomenclature and diagnostic criteria for nonproliferative and proliferative lesions in laboratory animals. the purpose of this publication is to provide a standardized nomenclature and diagnostic criteria for classifying lesions in the digestive system including the salivary glands and the exocrine pancreas of laboratory rats and mice. most lesions are illustrated by color photomicrographs. the standardized nomenclature, the diagnostic criteria, and the photomicrographs are also available electronically on the internet (http://www.goreni.org/). sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. content includes spontaneous and age related lesions as well as lesions induced by exposure to test items. relevant infectious and parasitic lesions are included as well. a widely accepted and utilized international harmonization of nomenclature and diagnostic criteria for the digestive system will decrease misunderstandings among regulatory and scientific research organizations in different countries and provide a common language to increase and enrich international exchanges of information among toxicologists and pathologists. the digestive tract is the entry site into the body for orally administered test articles. an irritant test article may lead to local acute lesions at this first site of contact to the body and the digestive tract has been identified as the organ most commonly affected in british patients admitted to hospital with adverse drug reactions (pirmohamed et al. 2004 ). on the other hand, orally administered test articles may be highly toxic to other organs yet having little or no noticeable effect on the digestive tract. a distinctive feature of the digestive tract is the high proliferative rate of the epithelium, making it particularly sensitive to agents interfering with cell division, but resulting also in a high regenerative capacity. the villi enhance the surface of the intestine to 400-500 m² in humans. because of the large surface area of these tissues, accurate assessment of potential treatment effects is almost entirely dependent on a thorough gross examination and sampling of focal lesions. standardized nomenclature and diagnostic criteria are essential to harmonize the classification and reporting of nonproliferative as well as proliferative histopathological lesions. they should facilitate communication between different research groups and regulatory authorities. in this way they should reduce time and efforts in the review of histopathology data by regulators and minimize clarifying questions to the sponsor. furthermore, standardized diagnostic criteria are a prerequisite for the generation of any historical control database of histopathological lesions. discussions about the future need of concurrent control groups highlight the importance of these standards. the inhand project (international harmonization of nomenclature and diagnostic criteria for lesions in rats and mice) is a joint initiative of the societies of toxicologic pathology from europe (european society of toxicologic pathology -estp), uk (british society of toxicological pathologists -bstp), japan (japanese society of toxicologic pathology -jstp), and north america (society of toxicologic pathology -stp) to unify, update and complete the existing who/ iarc and stp/ssndc nomenclature systems. the inhand nomenclature and the related diagnostic criteria should represent the future international standard in toxicologic pathology. they represent a consensus of senior toxicologic pathologists and were reviewed by the inhand-gesc (inhand-global editorial and steering committee) for compliance with in-hand principles. all members of the societies of toxicologic pathology involved in the inhand process had the opportunity to comment to the draft version during a 6-week period. however, these recommendations for diagnostic criteria and preferred terminology may not be applicable in all situations. purposes of specific experiments or the specific context of a given study may require deviation from this standardized nomenclature and diagnostic criteria. the appropriate diagnoses are ultimately based upon on the discretion of the senior toxicologic study pathologist. the present publication provides a set of standardized terms, diagnostic criteria and example images for the upper and lower digestive tract as well as for the salivary glands and the pancreas. the nomenclature of liver lesions has been published separately (thoolen et al. 2010 ) and that of tooth lesions is in preparation. like all other inhand publications, the nomenclature and diagnostic criteria for the digestive tract are also available online (website.goreni.org/). the online version contains additional images and useful links to differential diagnoses characterizing it as a practical tool for diagnostic work. the recommended nomenclature is generally descriptive rather than diagnostic. the diagnostic criteria used require standard hematoxylin and eosin stained paraffin sections only. histochemical or immunohistochemical staining characteristics may be addressed in the comments section of the respective lesion. such special techniques may be required in some situations, but a comprehensive discussion of these methods is outside the scope of this publication. lesions of disseminated organs like blood vessels, soft tissues or peripheral nerves are generally described in separate publications , greaves et al. 2013 . they are described here only in cases where the digestive tract is a major site of manifestation, e.g. leiomyoma of the intestine. lesions included in this nomenclature system may be further specified by modifiers. criteria are given for modifiers that are considered to be of particular relevance. these modifiers should be consistently applied. it is upon the discretion of the pathologist to use additional modifiers not suggested in this nomenclature system. such modifiers may describe the location, tissue type or duration among others. further principles of the inhand nomenclature have been published separately (mann et al. 2012) . findings that can reliably be diagnosed grossly are also not covered by this monograph. this applies in particular to some congenital malformations like intestinal duplication (elangbam et al. 1998; tamai et al. 1999 ). this nomenclature system describes lesions that occur in conventional (wildtype) strains of rats and mice. previous rodent nomenclatures for the digestive tract have been published by our societies and international who and other collaborative committees (betton et al. 2001; deschl et al. 1997; deschl et al. 2001; frantz et al. 1991; leininger and jokinen 1994; takahashi and hasegawa 1990; whiteley et al. 1996) . lesions occurring in specific genetically engineered mouse models only are not covered in this document. for more information on these specific lesions the reader should refer to relevant publications of workshops on genetically engineered mouse models (e.g. boivin et al. 2003; hruban et al. 2006 ). the oral cavity/pharynx, tongue, and esophagus are components of the upper alimentary tract, and each has a mucosa lined by a stratified squamous epithelium with a keratinized surface. incidental nonproliferative or proliferative microscopic findings may occur in these tissues as well as microscopic findings that are associated with effects of the test article (systemic or direct contact exposure) or, rarely, changes related to injury from the dosing (gavage) procedure. the borders of the oral cavity are defined by several general landmarks, including the hard and soft palate/pharynx dorsally; the gingiva, teeth, and mucosal surfaces of the lips/ cheeks laterally; and the floor of the mouth/tongue ventrally. oral cavity/pharynx is typically not a protocol-required tissue for microscopic evaluation, but portions may be sampled for examination due to a clinical sign or gross finding at necropsy. the oral cavity/pharynx tissue samples may include minor salivary glands (palatine, buccal), adjacent muscle, bone, teeth, or haired skin, in addition to the vascular and connective tissue of the keratinized, squamous epithelium of the mucosal surface. the tongue is comprised of the interlacing bundles of striated muscle interspersed with adipocytes, scant to numerous mast cells, nerves, and vascular tissue lined with a squamous mucosal surface. in the tongue of aged rats, the amount of adipose tissue has been reported to be decreased with a relative increase in its fibrous connective tissue as compared to that in younger animals (brown and leininger 1994) . the squamous mucosa of the dorsal tongue surface contains numerous papillae and a large circumvallated papilla, while the lateral/ventral surface is a simple stratified squamous mucosa (hebel and stromberg 1976; brown and hardisty 1990) . the thin, compact lamina propria of the mucosa of the tongue is less dense on the ventral surface nearer the root attachment (frenulum) to the oral cavity surface. in the more caudal, ventral area, there are large lymphatic vessels, veins, and ducts from the minor salivary glands in the adjacent oral mucosa. the esophagus consists of a muscle (tunica muscularis) layer with longitudinal and circular-oriented striated muscle fibers and a squamous mucosa with a keratinized surface that typically has small aggregates of bacterial coccoid organisms within the superficial layers of keratin. the physiology of the oral cavity, tongue, and esophagus is related primarily to their function as a conduit for food. effects on function with the development of megaesophagus have been reported in mice with abnormal myogenic plexus (randelia and lalitha 1988; randelia et al. 1990) , and esophageal obstruction, secondary to effects of scopolamine on salivary secretion and the swallowing reflex, had been described for rats (ntp tr 445 1997) . the routine necropsy includes gross examination of the oral cavity (oropharynx), tongue, and esophagus. although sampling and histologic examination of the oral cavity/pharynx are generally limited to necropsy observations, portions of these tissues may be included with routine nasal or tooth sections required for possible histopathology examination. in those circumstances when oral cavity structures may be considered as potential targets sites for nonneoplastic or neoplastic changes, whole-head fixation (calvarium and brain removed), may be the better approach for consistent examination of required tissues. with whole-head fixation, sectioning of the nasal/oral cavities, mandible, and associated tissues can be performed at various levels and result in consistent orientation for sectioning of desired tissue sites in these special circumstances. unlike the oral cavity/pharynx, routine histologic sections of the tongue and esophagus are usually protocol-required for microscopic evaluation in all animals from toxicity and carcinogenicity studies. for microscopic evaluation, the tongue may be prepared as a longitudinal histologic section (see rita trimming guide ruehl-fehlert et al. 2003 , website//reni.item. fraunhofer.de/reni/trimming) that provides for examination of lingual glands; it often may prepared as a transverse section congenital/developmental lesions in the oral cavity/pharynx, tongue, and esophagus have been rarely reported in mice and rats from toxicity and carcinogenicity studies. this is most likely due to their uncommon occurrence, exclusion of animals with these findings prior to study initiation, and minimal routine sampling of this location in the absence of a gross observation. in developmental and reproductive studies, congenital/ developmental lesions are more commonly observed. (figures 1 and 2 ) synonym: fordyce's granules pathogenesis: this developmental change consists of aggregates of ectopic dermal sebaceous glands (fordyce's granules) in the oral cavity of rats (yoshitomi et al. 1990 ) and is associated with gingival mucosa. this is most common in the f344 strain and seen primarily in males. • may appear grossly as white nodule or cyst in the gingival mucosa. • most commonly observed in gingival mucosa of upper incisors. • consists of normal sebaceous gland acini with ducts opening to mucosal surface. • presence of cysts (dilated ducts) with or without inflammation may be components of these ectopic glands. • sebaceous gland adenoma: absence of common duct/lumen and presence of mitotic figures; adenoma of ectopic sebaceous gland in the gingiva of rats has not been reported. comment: this finding is more likely observed in routine nasal cavity sections that may include teeth and gingival tissue. with the exception of its correlation to a gross observation when a cyst/inflammation has developed, this finding is of little or no apparent pathologic significance. synonyms: palatoschisis; congenital malformation pathogenesis: defect in the fusion of the bone and overlying mucosa of the hard palate. • midline space defect in oral mucosa and hard palate. • visible grossly or microscopically. • no evidence of trauma. • trauma: evidence of necrosis, fracture, or inflammatory response. comment: cleft palate is a longitudinal defect in bone and mucosa of the midline of the hard palate resulting from failure of fusion of the lateral palatine shelves from the maxillary processes (jones et al. 1997) . cleft palate in mice and rats has been attributed to maternal treatment with high doses of vitamin a (kalter and warkany 1961) and recently been reported as a genetic mutation in the mouse (stottmann et al. 2010) . this condition has also been produced in mice (era et al. 2009 ) and rats with in utero chemical exposure or as an effect from puncture of the amniotic sac (ferguson 1981; schuepbach and schroeder 1984) . diagnosis is based primarily on the gross observation. synonyms: distention; megaesophagus; dilatation; impaction pathogenesis: may be spontaneous/idiopathic, due to food impaction, or secondary to an alteration of the esophageal neuromuscular function, decreased secretion of the salivary glands or an altered swallowing reflex. • thinning of the muscle and mucosal layers as result of distention of the esophagus with food/bedding material. differential diagnosis • none. comment: this condition is typically related to a grossly distended esophagus containing food/bedding material and has also been diagnosed as impaction because of the gross appearance. this has been described as a spontaneous or idiopathic change (harkness and ferguson 1979; ruben et al. 1983) in rats and also attributed to feeding a powdered food diet (brown and hardisty 1990) . esophageal dilation associated with obstruction occurred in rats administered scopolamine (national toxicology program 1997a) and as a background finding in several mouse strains mahler et al. 2000) , in particular in mice with an abnormal myogenic plexus (randelia and lalitha 1988; randelia et al. 1990) . esophageal dilation, described as megaesophagus, has also been reported as an induced finding in mice with intrauterine exposure to diethylnitrosamine (ghaisas et al. 1989 ) and in rats secondary to effects of scopolamine on salivary secretion and the swallowing reflex (national toxicology program 1997b). synonym: pharyngoesophageal diverticulum pathogenesis: usually caused by mechanical or biochemical disruption of esophageal wall structures resulting in loss of extracellular matrix integrity and internal projection of surface lining into deeper layers; may alternatively represent a congenital abnormality. • irregular cyst-like extension of the mucosal lumen into or through the muscularis layer in the esophagus. • lumen of diverticulum that opens to mucosal surface. • focal. • cyst: isolated from mucosal surface. comment: diverticulum of the esophageal mucosa has been reported in rats (brown and hardisty 1990) . pathogenesis: rare developmental defects have been reported for the esophagus. duplication of the esophagus has been reported in an adult male rat as a spontaneous occurrence (canpolat et al. 1998 ) and tracheal-esophageal malformation represents one of many teratogenic effects reported in mice with intrauterine exposure to adriamycin (dawrant, et al. 2007 ). • gross observation of congenital/developmental structural alterations. differential diagnosis • none. pathogenesis: epithelial cysts may occur as downgrowth of entrapped surface mucosa or a dilated salivary duct in the oral cavity/pharynx, tongue, or esophagus and have been observed in the oral cavity/pharynx of rats with dilated glands/ ducts associated with ectopic sebaceous glands (yoshitomi et al. 1990) . • dilated gland/ducts lined by simple squamous epithelium. • lumen of cyst may contain sebaceous secretion or keratin debris. • inflammation associated with larger or ruptured cysts. • esophageal diverticulum: lumen opens into the esophagus lumen, epithelial connection to the surface mucosa of the esophagus. pathogenesis: decrease in normal thickness/cellularity of squamous mucosa of tongue, pharynx or esophagus. • decreased thickness of mucosal epithelial layer. • focally extensive or diffuse lesion. • erosion/ulcer: focal or focally extensive absence of superficial or, in severe cases, of all epithelial layers with extension of the alteration into the muscularis. unlike atrophy, this finding is typically associated with surface cellular debris and an inflammatory cell infiltrate in mucosa or as a surface exudate. synonyms: erosion; ulcer; ulceration pathogenesis: localized disruption in the uniform thickness/continuity of the squamous epithelium in the oral cavity/pharynx, tongue, or esophagus with either partial (erosion) or full penetration of the squamous epithelium (ulcer). • focal or focally extensive loss of surface epithelium in the oral cavity/pharynx, tongue, or esophagus. • basal lamina may remain intact (erosion) or in more severe lesions the defect in the mucosa may extend into the muscularis (ulcer). • intraepithelial inflammatory cell infiltrate frequently associated with more severe lesions. • squamous cell carcinoma: may present with erosion/ulcer appearance on the mucosal surface. the presence of this neoplasm would preclude diagnosis of an associated erosion/ulcer. • atrophy, epithelial: reduced thickness of all layers of the squamous epithelium or, in severe cases, absence of basal germinative layers; usually no inflammatory cell infiltrate. comment: although there are specific morphologic criteria to differentiate the more superficial lesion (erosion; confined to the epithelial surface) from the deeper ulcer (extension of the epithelial defect through basal cell layer/basal lamina), the plane of section through smaller, focal lesions often impacts on the appearance (depth of the epithelial loss) of this finding. in most instances it is not practical to separate these changes and they are typically diagnosed under the single, combined term of erosion/ulcer with an appropriate severity grade. erosion/ulcer usually is the result of epithelial necrosis. because of its unique topography adjacent to the lumen and often involving multiple organ layers, which uniquely affects the progression and resolution of the lesion (e.g. margin hyperplasia, granulomatous tissue, inflammation), it is useful to record erosion/ulcer separately from epithelial necrosis. a deep ulcer of the esophagus may be perforating if it destroys all layers of the esophagus wall, and may be designated as such by a free text entry. however, a perforation resulting from a physical insult, e.g. gavage-related, typically is a macroscopic lesion and generally not recorded on the light microscopic level. pathogenesis: gene regulated, energy dependent process leading to formation of apoptotic bodies which are phagocytosed by adjacent cells; typically associated with cytotoxic chemotherapeutics that affect the mucosal epithelium of the tongue, esophagus and/or pharynx. • single cells or small clusters of cells. • cell shrinkage. • hypereosinophilic cytoplasm. • nuclear shrinkage, pyknosis, karyorrhexis.intact cell membrane. • apoptotic bodies. • cytoplasm retained in apoptotic bodies. • phagocytosis of apoptotic bodies tissue macrophages or other adjacent cells. • necrosis, epithelium: the morphologic features of cell death clearly fit with necrotic diagnostic criteria (cells and nuclei swollen, pale cytoplasm etc.). • apoptosis/necrosis, epithelium: both types of cell death are present and there is no requirement to record them separately or a combined term is preferred for statistical reasons. the term is also used when the type of cell death cannot be determined unequivocally. • erosion/ulcer: focal or focally extensive absence of superficial or, in severe cases, of all epithelial layers with extension of the alteration into the muscularis. the approach for the nomenclature and diagnostic criteria of cell death applied here is based on a draft recommendation of the inhand cell death nomenclature working group. apoptosis is not synonymous with necrosis. the main morphological differences between these two types of cell death are cell shrinkage with nuclear fragmentation and tingible body macrophages in apoptosis versus cell swelling, rupture and inflammation in necrosis; however, other morphologies (e.g. nucelar pyknosis and karyorrhexis) overlap. in routine h&e sections where the morphology clearly represents apoptosis or single cell necrosis, or whereby special procedures (e.g. tem or ihc for caspases) prove one or the other, individual diagnoses may be used. however, because of the overlapping morphologies, necrosis and apoptosis are not always readily distinguishable via routine examination, and the two processes may occur sequentially and/or simultaneously depending on intensity and duration of the noxious agent (zeiss 2003) . this often makes it difficult and impractical to differentiate between both during routine light microscopic examination. therefore, the complex term apoptosis/necrosis may be used in routine toxicity studies. a differentiation between apoptosis and single cell necrosis may be required in the context of a given study, in particular if it aims for mechanistic investigations. transmission electron microscopy is considered to represent the gold standard to confirm apoptosis. other confirmatory techniques include dna-laddering (easy to perform but insensitive), tunel (false positives from necrotic cells) or immunohistochemistry for caspases, in particular caspase 3. these techniques are reviewed in detail by elmore (elmore 2007) . some of these confirmatory techniques detect early phases of apoptosis, in contrast to the evaluation of h&e stained sections, which detects late phases only; overall interpretation of findings should take into account these potential differences. thus, low grades of apoptosis may remain unrecognized by evaluation of h&e stained slides only. certain studies may require the consideration of forms of programmed cell death other than apoptosis which requires specific confirmatory techniques (galluzzi et al. 2012) . synonyms: oncotic cell death; oncotic necrosis; necrosis pathogenesis: unregulated, energy independent, passive cell death with leakage of cytoplasm into surrounding tissue and subsequent inflammatory reaction; may be induced by direct contact with a test article after oral uptake/administration. • cells swollen with pale eosinophilic cytoplasm. • loss of nuclear basophilia, pyknosis and/or karyorrhexis that affects aggregates of cells. • in more severe cases, there may be clefting and detachment of the epithelium from the submucosa. • typically, the presence of degenerative cells is a component of necrosis. • minimal or slight inflammatory cell infiltrates may be present as a feature of necrosis. single cell • only single cells affected. • apoptosis, epithelium: the morphologic features of cell death clearly fit with apoptosis (cell and nucleus shrunken, hypereosinophilic cytoplasm, nuclear pyknosis etc.) and/or special techniques demonstrate apoptosis. • apoptosis/necrosis: both types of cell death are present and there is no requirement to record them separately or a combined term is preferred for statistical reasons. the term is also used when the type of cell death cannot be determined unequivocally. atrophy, epithelium: reduced thickness of all layers of the squamous epithelium or, in severe cases, absence of basal germinative layers; usually no cellular degeneration or necrosis. • erosion/ulcer: focal or focally extensive absence of su-perficial or, in severe cases, of all epithelial layers with distention of alteration into the muscularis. comments: epithelial necrosis usually develops into erosion/ ulcer and in the past has frequently been recorded as such. a separate recording of necrosis as the initial event in this process is recommended when the structure of the mucosa is still intact. synonym: cell death pathogenesis: unregulated, energy independent, passive cell death with leakage of cytoplasm into surrounding tissue and subsequent inflammatory reaction (single cell necrosis) and/ or gene regulated, energy dependent process leading to formation of apoptotic bodies which are phagocytosed by adjacent cells (apoptosis); typically associated with cytotoxic chemotherapeutics that affect the mucosal epithelium of the tongue, esophagus and/or pharynx. • both types of cell death are present and there is no requirement to record them separately or a combined term is preferred for statistical reasons. • the type of cell death cannot be determined unequivocally. • apoptosis, epithelium: the morphologic features of cell death clearly fit with apoptosis (cell and nucleus shrunken, hypereosinophilic cytoplasm, nuclear pyknosis etc.) and/or special techniques demonstrate apoptosis and there is a requirement for recording apoptosis and necrosis separately. • necrosis, epithelium: the morphologic features of cell death clearly fit with necrotic diagnostic criteria (cells and nuclei swollen, pale cytoplasm etc.) and there is a requirement for recording apoptosis and necrosis separately. • erosion/ulcer: focal or focally extensive absence of superficial or, in severe cases, of all epithelial layers with distention of alteration into the muscularis. comments: necrosis and apoptosis are not always readily distinguishable via routine examination, and the two processes may occur sequentially and/or simultaneously depending on intensity and duration of the noxious agent (zeiss 2003) . this often makes it difficult and impractical to differentiate between both during routine light microscopic examination. in these cases, the combined term apoptosis/necrosis may be used. it is recommended to explain in detail the use of this combined term in the narrative part of the pathology report. degeneration/necrosis, muscle (figure 12) pathogenesis: degeneration and necrosis of muscle fibers in the tongue or esophagus. • myocyte degeneration/necrosis of muscle fibers characterized typically by the presence of both swollen or contracted hyperchromatic or pale eosinophilic degenerative fibers and necrotic fibers with coagulative necrosis and nuclear pyknosis or lysis. • usually a focal change of minimal severity. • amphophilic muscle fibers consistent with a regenerative response may be present and a primary feature of this finding. differential diagnosis • degeneration/necrosis or regeneration of muscular layer in conjunction with inflammation, edema and/or hemorrhage associated with gavage-related injury (discussed below under inflammation, mixed cell, gavage-related). comment: this change can occur as a focal lesion in the esophagus that also variably includes the presence of several degenerative and/or regenerative muscle fibers. while potentially a test article-related or incidental finding, it may be related to very minor physical effects from the gavage procedure. morphologic features more conclusive of gavagerelated injury such as perforation and inflammation are not present. in the absence of such clear, gavage-related inflammatory changes, degeneration/necrosis is the preferred diagnosis for this esophagus finding. degeneration/necrosis of muscle fibers in the tongue may be related to test article administration (greaves 2012) . synonyms: amyloidosis; amyloid deposition pathogenesis: extracellular deposits of polypeptide fragments of a chemically diverse group of glycoproteins. • affects medium or larger vessels (arterioles) of the tongue. • accumulates as a thin layer of homogenous and pale eosinophilic amyloid material in basal lamina of mucosa in the tongue and esophagus. • green birefringence using polarized light with congo red stain. • fibrinoid change (necrosis) in vessel walls: deeply eosinophilic homogenous or coagulative appearance of the vascular media. comment: amyloid deposition occurs in the tongue and esophagus of mice, although much less frequently at these locations as compared to other locations (e.g. kidney, intestine). the characteristic morphologic appearance and location of amyloid in h&e sections that is usually adequate to make this diagnosis. confirmation of the deposits as amyloid can be accomplished with light microscopy using special stains such as congo red. amyloid appears apple green under polarized light with this stain. the term "hyaline" should be used in case of uncertainties about the nature of extracellular deposits of hyaline material in h&e sections and the unavailability of a congo red stain. hyaline is also the appropriate term for homogenously eosinophilic extracellular deposits that react negative with congo red. synonyms: calcification; mineral deposition pathogenesis: mineralization of blood vessel walls and muscle fibers of the oral cavity/pharynx, tongue, or esophagus. • basophilic granular deposits in the wall of arteries as well as within individual muscle fibers. • mineral deposits usually easily identified in routine sections but can be further characterized by special stains (alizarin red, von kossa). differential diagnosis • none. pathogenesis: increased pigment has been described in the tongue muscle of aging rats (bodner et al. 1991 ) and in the oral mucosa of rats (ag strain) given antimalarial drugs (savage et al. 1986 ). • yellow/brown pigment (lipofuscin) in muscle fibers of the tongue. • brown/black pigment (melanin) within oral mucosa. • none. comment: pigment (increased pigmentation) is not a common finding in the oral cavity/pharynx, tongue, and esophagus. when this is present, histochemistry, immunohistochemistry, ultrastructural, or other laboratory methods may be required to characterize this material. synonyms: hpyerkeratosis, orthokeratotitc may have been identified as hyperkeratosis hyperkeratosis, parakeratotic may have been identified as parakeratosis pathogenesis: increased production and/or retention of keratin by squamous epithelium of the mucosa with normal (orthokeratotic) or abnormal (parakeratotic) maturation of the keratin layer. • increase in keratin on the surface of the mucosa. • focal or diffuse. • often accompanied by hyperplasia of squamous epithelium. orthokeratotic • thickened keratin layer with non-nucleated keratinized cells. parakeratotic • thickened keratin layer with nucleated keratinized cells. • normal: keratin layer that is normally less than the thickness of the mucosal squamous epithelium. hyperplasia, squamous cell: proliferation and thickening of the stratum spinosum. comment: parakeratosis of the esophagus has been described in zinc-deficient rats (barney et al. 1968 ). it may also be induced by vitamin imbalances or hydroxymethylglutaryl-coa reductase inhibitors (sigler et al. 1992 ). it is recommended that when possible, descriptive terminology should be used to best characterize the type (e.g., neutrophil, mixed cell, mononuclear cell, lymphocyte, etc.) of cellular component(s)/infiltrates rather than a specific type/duration of inflammation (e.g., acute, ulcerative, suppurative, subacute, chronic) for changes in the oral cavity, pharynx, tongue and esophagus. the presence of various inflammatory cell infiltrates in the absence of other features of inflammation should be diagnosed as an 'infiltrate' with predominate/appropriate cell type(s) identified. however, when other prominent morphologic features (congestion, hyperemia, hemorrhage, edema, necrosis, fibrosis, foreign material, bacterial organisms, etc.) are present, it is preferable to characterize the change as an inflammatory process (inflammation) with specific descriptors. the reader is referred to the nomenclature and review of basic principles document for discussion on the preferred use of descriptive, rather than diagnostic terminology (mann et al. 2012) . listed below are descriptive diagnostic terms and examples of inflammatory cell infiltrates or types of inflammation. many of the examples provided in this manuscript are more consistent with inflammation, as indicated by figure legends. subsequently we describe common types of inflammation in an exemplary manner, while we give only a general description of inflammation for other parts of the digestive tract. (figures 17 and 18 ) synonyms: infiltrate inflammatory; infiltrate inflammatory cell; infiltration (plus modifier); infiltration, inflammatory; infiltration, inflammatory cell modifiers: type of inflammatory cell that represents the predominant cell type in the infiltrate pathogenesis: infiltrate of lymphocytes, plasma cells, macrophages, neutrophils, eosinophils or mixtures thereof within the mucosa or muscle layers of the oral cavity/pharynx, tongue, or esophagus without other histological features of inflammation as noted above. • focal, multifocal or diffuse cellular infiltrate in mucosa/ muscle layers. • presence of mononuclear or polymorphonuclear leucocytes but no other histological criteria of inflammation. • lesion may be associated with overlying ulceration, distended/ruptured cyst, self-induced traumatic lesions or dysplasia/neoplasia of the teeth in the oral cavity/pharynx. • inflammation: in addition to the infiltrate, other morphologic features of inflammation such as edema, hemorrhage, necrosis and/or fibroplasia will be present. • granulocytic leukemia: infiltration of neutrophil precursors and/or abnormal neutrophils will probably be present along with mature neutrophils; infiltration of similar neoplastic cells is likely present in other organs. • lymphoma: infiltration of monomorphic population of lymphocytes (often with atypical, increased and/or abnormal mitoses); infiltration of similar neoplastic cells is likely present in other organs. • fibrosis may be a prominent component in a more chronic inflammatory change. • may be associated with overlying ulcer, distended/ruptured cyst, or self-induced traumatic lesions. in the oral cavity/pharynx, the process may be associated with dysplasia/neoplasia of the teeth. • infiltrate, lymphocytes or infiltrate mononuclear cell: other morphological features of inflammation such as edema, hemorrhage, necrosis and/or fibroplasia will be absent. • lymphoma: infiltration of monomorphic population of lymphocytes (often with atypical, increased and/or abnormal mitoses); infiltration of similar neoplastic cells is likely present in other organs. comment: when a mononuclear cell infiltrate is considered to represent an inflammatory process (inflammation, mononuclear cell) and other morphologic features (fibrosis, mineralization) associated with the infiltrate suggest an ongoing/ continuing inflammatory process, the modifier 'chronic' may be included in the diagnosis (i.e. inflammation, mononuclear cell, chronic) to better characterize the finding. pathogenesis: inflammatory cell response within the mucosa or muscle layers of the oral cavity/pharynx, tongue, or esophagus. • mixed inflammatory cell infiltrate consisting of variable numbers of neutrophils, lymphocytes or macrophages without predominance of one cell type. • edema, congestion/hemorrhage, and/or cell debris are components of the mixed cell infiltrates more consistent with an inflammatory process. • may be associated with overlying ulcer, distended/ruptured cyst, self-induced traumatic lesions or dysplasia/ neoplasia of the teeth in the oral cavity/pharynx. • in the oral cavity/pharynx, the inflammatory process may be associated with dysplasia/neoplasia of the teeth. reactive/regenerative hyperplasia of the overlying mucosal epithelium may also be a feature of this inflammatory response. • infiltrate, neutrophils: other morphological features of inflammation such as edema, hemorrhage, necrosis and/ or fibroplasia will be absent. comment: when a mixed cell infiltrate is considered to represent an inflammatory process (inflammation, mixed cell) with the presence of other morphologic features (fibrosis; granulomatous inflammation, granulation tissue and a prominence of neutrophils), the modifier 'chronic' may be included in the diagnosis (inflammation, mixed cell, chronic) to better characterize the finding. inflammation, mixed cell is commonly observed with gavage-related injury (figures 21 and 22 ). it may have variable histopathological appearance, dependent on extent of damage and duration/onset of the lesion to include: perforation (typically seen macroscopically), bacterial organisms, or foreign material in the pharyngeal / esophageal or peripharyngeal / periesophageal tissue with the variable presence of degeneration/necrosis and/or regeneration of muscular layer in conjunction with inflammation, edema or hemorrhage. if the lesion is interpreted to be secondary to gavage-related injury, this may be addressed in the pathology narrative, or alternatively may be diagnosed as inflammation, mixed cell, gavage-related to distinguish from spontaneous and/or test article-related findings. pathogenesis: inflammatory cell response to presence of foreign material; observed primarily in the tongue. • focal inflammatory lesion, most often seen in mucosa, ducts of minor salivary glands or muscle layers of the tongue. • mixed inflammatory cell infiltrate (macrophage/multinucleated giant cell, lymphocyte, neutrophil) sometimes associated with an imbedded hairshaft fragment or other ingested foreign material (food/bedding material). • abscess: circumscribed and grossly visible globular process with characteristic zonation of inner cell debris, neutrophils, and outer capsule of fibrosis/fibroplasias with numerous capillaries (granulation tissue). • inflammation, mixed cell: the inflammatory process lacks foreign material and/or multinucleated giant cells. synonyms: arteritis; vasculitis; polyarteritis pathogenesis: inflammation of vascular wall of the oral cavity/pharynx, tongue, or esophagus. • typically affects muscular wall of arteries in tongue. • variable mixed inflammatory cell infiltrate within and around vessel. • possible presence of fibrinoid necrosis of vessel wall. • proliferation of endothelium and thickening (fibrosis) of vessel wall. • infiltrate, inflammatory cell: may occur perivascular, but the vessel wall itself is unaffected. comment: more information and differential diagnoses are given in the inhand publication of nomenclature and diagnostic criteria of the cardiovascular system (berridge b et al. in preparation) . pathogenesis: accumulation of tissue fluid in the interstitium resulting from increased vascular permeability. • increased eosinophilic fluid (interstitial fluid) within interstitium. • inflammation, neutrophils: usually associated with tissue and vascular damage resulting in exudative edema and inflammatory cell infiltrate. edema accompanying an inflammatory process should be recorded separately if it is a predominant lesion. pathogenesis: increased vascular permeability (diapedesis) or rupture of blood vessels. histogenesis: basal layer of the stratified squamous epithelium. • proliferation of the basal cell layer, basophilic staining increased. • focal or diffuse. • endophytic growth pattern. • no alteration of basement membrane integrity. • hyperplasia, squamous cell: thickening of the epithelium with all normally existing layers. • carcinoma, squamous cell: evidence for lost basement membrane integrity, spinous cells and keratinized cells proliferate, cellular atypia. comment: basal cell hyperplasia and squamous cell hyperplasia may occur concurrently. isolated nests of basal cells may occur in the lamina propria, dependent on the plane of section through the rete peg structures. however, their discrete border indicates an intact basement membrane. neoplastic lesions in the oral cavity/pharynx, tongue, and esophagus are usually correlated to a gross observation. in addition to the proliferative lesions of squamous cell origin, neoplasms of the bone, tooth, or adjacent soft tissues (malignant schwannoma, zymbal's gland tumor) may extend into the oral cavity and be associated with a gross observation at this location. these tumors are described in separate inhand publications on "musculoskeletal system", "soft tissue", and "mammary, zymbal's, and clitorial glands". histogenesis: epithelial squamous cells of the mucosa in the oral cavity/pharynx, tongue, and esophagus. • central fibrovascular stalk with multiple finger/frondlike projections covered with variably thick squamous epithelium. • squamous epithelium often heavily keratinized. • cells show orderly maturation. • when occurring on the tongue, are generally dorsally located. • aggregates, granular cell (although there are no reports this has been observed in the tongue, this likely could occur early in the process, as described in the inhand cns/pns and female reproductive systems): few scattered cells to small aggregates with minimal disturbance of normal tissue architecture and no compression of adjacent tissue. • tumor, granular cell malignant (although there are no reports this has been observed in the tongue, there is the potential for this lesion to progress to malignancy, as described in the inhand cns/pns and female reproductive systems): prominent pleomorphism and/or invasion of surrounding tissue. comment: benign granular cell tumor (gct) of the tongue is rare and has not been previously reported in rodents. we are currently aware of several cases of granular cell tumors in the tongue of wistar rats (4 females and one male) and one case in a sprague dawley rat (male) but are unaware of this neoplasm occurring in the tongue of mice. gcts have been reported in the tongue as well as other sites in the dog, cat, horse, and a bird (patnaik 1993) . although uncommon, gcts most frequently occur in the head and neck region (often in the tongue) of humans with a slight predominance in females (becelli et al. 2001; van de loo et al., 2015) . gcts in the meninges of rats and the reproductive tract (uterus/cervix/vagina) of rats and mice have also been described (dixon et al, 2014) but their relationship to the gct observed in the tongue of rats is unknown. the rodent stomach is morphologically different from the stomach of other laboratory species and from that of humans. in the rat and mouse the proximal portion, the nonglandular stomach (synonym forestomach), is lined by stratified squamous epithelium and comprises approximately half of the total area of the stomach. it is separated from the distal glandular stomach by the limiting ridge (squamocolumnar junction), where the squamous epithelium is thicker than elsewhere in the stomach. as humans lack a nonglandular stomach, the relevance of test article-related changes in this tissue for humans can be challenged. however, it is likely that the squamous mucosa of the esophagus (in species without the nonglandular stomach), will react to test articles in a similar way as the nonglandular stomach epithelium, if equivalent exposure levels are attained. therefore, interpretations of changes in the nonglandular region need to take account of retention time of a test article in the stomach and physiological functioning of the stomach (greaves 2012) . exposure to the rodent nonglandular stomach by gavage can mimic aspects of skin painting studies, i.e. direct contact of test chemical to the keratinized squamous epithelium. the glandular stomach of rodents has histological and functional similarities to that of other mammals with both corpus and antral regions. it anatomically differs from other non-rodent laboratory animal species and humans by the lack of a notable cardiac region in the glandular mucosa and the absence of the dorsal/anterior pouch in the glandular region known as the fundus. in rodents, the analogous dorsal/anterior pouch is nonglandular although the term glandular fundus is frequently used for the corpus. the term "fundic gland" has been generically applied to the human stomach to denote glands containing parietal (oxyntic) and/or chief (zymogenic) cells, regardless of anatomic site. rodents do not have glands in their fundus; however, the ubiquitous use of the term in medical pathology has led to its frequent use in rodent studies. similarly, pylorus is sometimes used to denote the antrum; however, its use should be limited to designating the portion of the antrum adjoining the gastroduodenal junction. the surface epithelium of the glandular mucosa is a simple columnar mucous epithelium that extends into the gastric pits; the depth of the gastric pit varies based on location in the corpus or antrum, being most shallow close to the limiting ridge and becoming deeper approaching the antrum. accordingly, the proportion of fundic glands (synonym: oxyntic glands) contributing to the thickness of the corpus mucosa is greatest near the limiting ridge on the greater curvature and progressively diminishes toward the corpus/antral junction. the fundic glands are continuous with the gastric pit and have a simple tubular structure with isthmus, neck and base regions. there are 4 cell types in the corpus: mucous neck cells which produce mucus; parietal or oxyntic cells which produce hydrochloric acid and intrinsic factor; chief or zymogenic cells which produce pepsinogen; and argentaffin or neuroendocrine cells that produce a variety of endocrine and paracrine hormones. the isthmus region serves as the site of proliferation with mucous neck cells acting as progenitor cells that differentiate luminally to become mucous epithelium and basally to become the specialized cells of the fundic glands (karam, 1999) . this is reflected in differences of the turnover rate, which is at around 3 days in mucous cells compared to 194 day in oxyntic and 54 day in parietal cells (karam, 1999) . in the antrum, the glands are lined by mucous cells and neuroendocrine cells including those that produce gastrin. the turnover rate of mucus cells is much faster at around 3 days than oxyntic or parietal cells (about 194 and 54, respectively) (karam, 1999) . the mucus phenotype of gastric epithelium and glandular cells can be identified by histochemical methods, e.g. the combination alcian blue ph 2.5 followed by pas stain ( table 1) . the mucosa is supported by the connective tissue of the lamina propria and the continuous muscle layer of the muscularis mucosa. beneath the muscularis mucosa is the relatively loose connective tissue of the submucosa, which contains larger blood vessels, lymphatics and nerves, and the tunica muscularis. the external surface is covered by the serosal membrane. the esophagus enters into the nonglandular region of the rodent stomach. functionally, the nonglandular stomach serves as a temporary storage organ and, due to prolonged exposure, is potentially a major site for interaction with test articles entering the body via oral ingestion. as a result of this storage function, the presence of irritants in the diet may quickly lead to damage and inflammation in this area. the lower ph in the nonglandular stomach when compared to the esophagus may be of major importance, as it may influence the diffusion of test articles into the lipophilic squamous epithelium. in the stomach, food undergoes mechanical and chemical breakdown to form chyme which then passes into the duodenum for further digestion. mechanical breakdown is achieved through the churning action of the tunica muscularis and chemical breakdown results from the action of digestive juices secreted from the glandular mucosa. the glandular mucosa produces an acidic watery secretion containing pepsinogen. pepsinogen is converted into active pepsin in the acid environment which then hydrolyses proteins into polypeptide fragments. self digestion of the stomach mucosa is prevented by a layer of surface mucous which has a higher ph than the gastric juice due to secretion of bicarbonate ions by the surface mucous cells. changes in, or loss of, the mucous layer by test articles may quickly lead to erosion and ulceration of the glandular epithelium. on the other hand complete inhibition of acid secretion by test articles will lead to hypergastrinemia which, if maintained, leads to proliferation of the enterochromaffinlike cells. attention should be paid to the amount and nature of the stomach contents at necropsy. a stomach abnormally distended with food, particularly in a rodent fasted overnight, may indicate that the test article and/or vehicle is inhibiting gastric function, and this may have lead to reflux of stomach contents and entry of gastric contents into the respiratory tract (damsch et al. 2011 ). conversely, a stomach grossly distended with gas is often an indication in a rodent that there has been a blockage in the upper respiratory tract resulting in dyspnoea, mouth breathing and aerophagia. in its natural configuration, the non-distended stomach has an irregularly folded mucosa and submucosa. consistent opening and positioning during fixation and trimming are necessary to obtain standardized sections of both glandular corpus and antrum and nonglandular stomach with correct orientation of the muscularis, submucosa and mucosa. at necropsy, the stomach should be opened along its greater curvature to the proximal part of the duodenum, any ingesta washed off with isotonic saline and then pinned to a rigid surface (e.g. cork). the stomach should be gently but sufficiently stretched to produce a flattened surface of both nonglandular and glandular regions but excessive stretching should be avoided. alternatively, the pylorus may be ligated and the stomach inflated with fixative for a short period, before opening and flattening between filter paper prior to further fixation (mahler et al. 2000) however, an excessive volume must not be used to avoid excessive stretching. trimming of stomach should be undertaken as specified in the goreni trimming guide (website.reni.item.fraunhofer.de/ reni/trimming) so that standardized sections of nonglandular epithelium, corpus, antrum and pyloric-duodenal junction are obtained. the ratios of parietal and chief cells in the fundic glands and the height of the mucosa vary between lesser and greater curvature so inter-animal variation in trimming needs to be avoided and a sensitivity to topography maintained. furthermore it is important to recognize that the glandular corpus mucosa may be minimal or absent along the lesser curvature with the antral mucosa extending to the limiting ridge. in the following sections, lesions that may affect the nonglandular and glandular regions in a similar manner are described only once. congenital/developmental lesions occur occasionally in rodents. most occur as isolated cases and the pathologist must distinguish such background changes from test article-induced lesions. synonym: cyst, epithelial pathogenesis: invagination of stratified squamous epithelium into the wall of the nonglandular stomach. • most commonly seen in nonglandular stomach or near the limiting ridge in glandular mucosa. • keratinized stratified squamous epithelium. • mucous cells occasionally present. • may extend into submucosa/tunica muscularis. • lumen frequently contains keratinized material which may be mineralized. • may be surrounded by chronic inflammation. • carcinoma, squamous cell: epithelial atypia and invasive growth patterns with penetration of basement membrane by single cells or nests of neoplastic cells. comment: while squamous cysts are most likely to be of congenital origin they may potentially form during life sec-ondary to derangement of the epithelium e.g. in an inflammatory process. if post-inflammatory extension is the suspected pathogenesis, the pathologist may choose to interpret the focal change as a diverticulum, rather than a congenital cyst. ectopic tissue (figures 37, 38, 39 and 40) modifiers: hepatocytes, pancreas pathogenesis: presence of pancreatic or hepatic tissue in the mucosa or submucosa. • normally formed pancreatic acini in mucosa or submucosa. islets of langerhans may also be present. • normally formed hepatocytes present in cords or nests in mucosa or submucosa; binucleate cells may be present. • adenocarcinoma: formation of hepatocyte-like cells has been reported in human gastric adenocarcinoma. comment: ectopic pancreatic acini or hepatocytes are rare incidental findings in rats and mice (maekawa et al. 1996; brown and hardisty 1990) , but ectopic differentiation may be a feature of some engineered mice (fukuda et al. 2006) . in one publication, hepatocytes accompanied fundic gland abnormalities in the mouse and it was not clear whether they represented metaplasia or congenital heterotopia ; the same conundrum may apply to ectopic pancreatic foci. metaplasia to a pancreatic cell type was reported as part of a hypertrophic gastropathy in a strain of rat (wtcdfk) which has a deletion of the potassium channel kcnq1 gene (kuwamura et al. 2008) . ectopic pancreatic tissue also may occur rarely in the pyloric submucosa of mice (maekawa et al. 1996) . pathogenesis: decrease in normal thickness/cellularity of squamous mucosa. • focally extensive or diffuse lesion. • decreased thickness of mucosal epithelial layer. • erosion/ulcer: focal or focally extensive absence of superficial or, in severe cases (ulcer) of all epithelial layers. • artifact: excessive stretching due to food contents or stretching stomach at necropsy leads to decreased thickness of all layers of the gastric wall. vacuolation, squamous epithelium ( figure 43 ) pathogenesis: degenerative change in the squamous epithelial cells which may precede necrosis, erosion or ulceration of the nonglandular epithelium. • cells of squamous epithelium have a pale staining, vacuolated appearance. • may be accompanied by edema in the submucosa and vacuolation of the muscularis. differential diagnosis • artifacts. comment: epithelial vacuolation and vesiculation along with submucosal edema was described in rats after administration of ethyl acrylate as a forerunner to necrosis and erosion/ ulcer (ghanayem et al. 1985) . certain compounds may specifically cause vacuolation of the squamous epithelium adjacent to the limiting ridge. pathogenesis: gene regulated, energy dependent process leading to formation of apoptotic bodies which are phagocytosed by adjacent cells; typically associated with cytotoxic chemotherapeutics that affect the mucosal epithelium of the nonglandular stomach. • necrosis, epithelium: the morphologic features of cell death clearly fit with necrotic diagnostic criteria (cells and nuclei swollen, pale cytoplasm etc.). • apoptosis/necrosis, epithelium: both types of cell death are present and there is no requirement to record them separately or a combined term is preferred for statistical reasons. the term is also used when the type of cell death cannot be determined unequivocally. • erosion/ulcer: focal or focally extensive absence of superficial or, in severe cases, of all epithelial layers with extension of the alteration into the muscularis. the approach for the nomenclature and diagnostic criteria of cell death applied here is based on a draft recommendation of the inhand cell death nomenclature working group. apoptosis is not synonymous with necrosis. the main morphological differences between these two types of cell death are cell shrinkage with nuclear fragmentation and tingible body macrophages in apoptosis versus cell swelling, rupture and inflammation in necrosis; however, other morphologies (e.g. nucelar pyknosis and karyorrhexis) overlap. in routine h&e sections where the morphology clearly represents apoptosis or single cell necrosis, or whereby special procedures (e.g. tem or ihc for caspases) prove one or the other, individual diagnoses may be used. however, because of the overlapping morphologies, necrosis and apoptosis are not always readily distinguishable via routine examination, and the two processes may occur sequentially and/or simultaneously depending on intensity and duration of the noxious agent (zeiss 2003) . this often makes it difficult and impractical to differentiate between both during routine light microscopic examination. therefore, the complex term apoptosis/necrosis may be used in routine toxicity studies. a differentiation between apoptosis and single cell necrosis may be required in the context of a given study, in particular if it aims for mechanistic investigations. transmission electron microscopy is considered to represent the gold standard to confirm apoptosis. other confirmatory techniques include dna-laddering (easy to perform but insensitive), tunel (false positives from necrotic cells) or immunohistochemistry for caspases, in particular caspase 3. these techniques are reviewed in detail by elmore (elmore 2007) . some of these confirmatory techniques detect early phases of apoptosis, in contrast to the evaluation of h&e stained sections, which detects late phases only; overall interpretation of findings should take into account these potential differences. thus, low grades of apoptosis may remain unrecognized by evaluation of h&e stained slides only. certain studies may require the consideration of forms of programmed cell death other than apoptosis which requires specific confirmatory techniques (galluzzi et al. 2012) . synonyms: oncotic cell death; oncotic necrosis; necrosis pathogenesis: unregulated, energy independent, passive cell death with leakage of cytoplasm into surrounding tissue and subsequent inflammatory reaction; may be induced by direct contact with a test article after oral uptake/administration. • cells swollen with pale eosinophilic cytoplasm. • loss of nuclear basophilia, pyknosis and/or karyorrhexis that affects aggregates of cells. • in more severe cases, there may be detachment of the epithelium from the submucosa. • typically, the presence of degenerative cells is a compo-nent of necrosis. • minimal or slight inflammatory cell infiltrates may be present as a feature of necrosis. single cell • only single cells affected. • apoptosis, squamous epithelium: the morphologic features of cell death clearly fit with apoptosis (cell and nucleus shrunken, hypereosinophilic cytoplasm, nuclear pyknosis etc.) and/or special techniques demonstrate apoptosis. • apoptosis/necrosis, squamous epithelium: both types of cell death are present and there is no requirement to record them separately or a combined term is preferred for statistical reasons. the term is also used when the type of cell death cannot be determined unequivocally. • atrophy, squamous epithelium: reduced thickness of all layers of the squamous epithelium or, in severe cases, absence of basal germinative layers; usually no cellular degeneration or necrosis. • erosion/ulcer: focal or focally extensive absence of superficial or, in severe cases, of all epithelial layers with distention of alteration into the muscularis. comments: epithelial necrosis usually develops into erosion/ ulcer and in the past has frequently been recorded as such. a separate recording of necrosis as the initial event in this process is recommended when the structure of the mucosa is still intact. synonym: cell death pathogenesis: unregulated, energy independent, passive cell death with leakage of cytoplasm into surrounding tissue and subsequent inflammatory reaction (single cell necrosis) and/or gene regulated, energy dependent process leading to formation of apoptotic bodies which are phagocytosed by adjacent cells (apoptosis); typically associated with cytotoxic chemotherapeutics that affect the mucosal epithelium of the nonglandular stomach. • both types of cell death are present and there is no requirement to record them separately or a combined term is preferred for statistical reasons. • the type of cell death cannot be determined unequivocally. • apoptosis, squamous epithelium: the morphologic features of cell death clearly fit with apoptosis (cell and nucleus shrunken, hypereosinophilic cytoplasm, nuclear pyknosis etc.) and/or special techniques demonstrate apoptosis and there is a requirement for recording apoptosis and necrosis separately. • necrosis, squamous epithelium: the morphologic features of cell death clearly fit with necrotic diagnostic criteria (cells and nuclei swollen, pale cytoplasm etc.) and there is a requirement for recording apoptosis and necrosis separately. • erosion/ulcer: focal or focally extensive absence of superficial or, in severe cases, of all epithelial layers with distention of alteration into the muscularis. comments: necrosis and apoptosis are not always readily distinguishable via routine examination, and the two processes may occur sequentially and/or simultaneously depending on intensity and duration of the noxious agent (zeiss 2003) . this often makes it difficult and impractical to differentiate between both during routine light microscopic examination. in these cases, the combined term apoptosis/necrosis may be used. it is recommended to explain in detail the use of this combined term in the narrative part of the pathology report. pathogenesis: localized loss of mucosa with either partial mucosal penetration (erosion) or with full penetration of the mucosa to the muscularis mucosa (ulcer). • focal or multifocal. • ulcers will be associated with acute/chronic inflammatory cell infiltration in submucosa. • basal lamina may remain intact (erosion) or in the more severe cases defect in the mucosa and inflammation will extend to the tunica muscularis and serosa (ulcer). • hyperplasia is often present in the surrounding epithelium. • hemorrhage may be seen with larger ulcers. • carcinoma, squamous cell: may show secondary ulceration, but always penetration of basement membrane by single cells or nests of neoplastic cells. • necrosis, squamous epithelial: no superficial or deep loss of epithelial cells. • artifacts: mucosal loss due to manual or processing artifact -inflammation will be absent. comment: ulcers in the nonglandular epithelium are often seen macroscopically as multiple small dark depressed areas surrounded by raised areas of whitened epithelium (maekawa 1994) . whilst ulcers have been induced by a wide range of irritant agents, the cause of ulceration in control animals is often unclear, although advanced age, parasitism, infection, diet, feeding regimen, generalized debility and stress can play a role (maekawa 1994; maekawa et al. 1996; greaves 2012) . protein restriction and starvation have also been shown to produce ulcers (boyd et al. 1970) . although there are specific morphologic criteria to differentiate the more superficial lesion (erosion; confined to the epithelial surface) from the deeper ulcer (full penetration of the mucosa to the muscularis mucosa), the plane of section through smaller, focal lesions often impacts on the appearance (depth of the epithelial loss) of this finding. in most instances it is not practical to separate these changes and they are typically diagnosed under the single, combined term of erosion/ulcer with an appropriate severity grade. erosion/ulcer usually is the result of epithelial necrosis. because of its unique topography adjacent to the lumen and often involving multiple organ layers, which uniquely affects the progression and resolution of the lesion (e.g. margin hyperplasia, granulomatous tissue, inflammation), it is useful to record erosion/ulcer separately from epithelial necrosis. a deep ulcer may be perforating if it destroys all layers of the gastric wall, and may be designated as such by a free text entry. however, a perforation resulting from a physical insult, e.g. gavage-related, typically is a macroscopic lesion and generally not recorded on the light microscopic level. hyperkeratosis (figure 45, 46 and 47) synonyms: hpyerkeratosis, orthokeratotic may have been identified as hyperkeratosis hyperkeratosis, parakeratotic may have been identified as parakeratosis pathogenesis: keratinization of squamous epithelium faster than desquamation of keratinized layers with normal (hyperkeratotic) or abnormal (parakeratotic) maturation of the keratin layers • increase in the thickness of the keratin layer on the luminal epithelial surface. orthokeratotic • thickened keratin layer with non-nucleated keratinized cells. parakeratotic • thickened keratin layer with nucleated keratinized cells. • hyperplasia, squamous cell: proliferation and thickening of the stratum spinosum. comments: hyperkeratosis occurs often in association with hyperplasia of underlying epithelium. alternatively, it may be observed in the absence of hyperplasia under circumstances suggesting anorexia and a presumptive lack of mechanical abrasion. hyperkeratosis can be a frequent observation attributable to local irritation of orally administered test articles in toxicity studies (til et al. 1988 ). the local irritant effect is presumably potentiated by increased duration of exposure associated with intragastric storage and increased contact, if test article precipitates in the gastric compartment. comment: glandular cysts may be congenital but most are acquired as their incidence increases with age in both rats and mice (brown and hardisty 1990; maekawa et al. 1996) . in the fischer rat they are more common in the antrum than in the fundus (brown and hardisty 1990) . when cysts extend through the muscularis mucosae, the diagnostic term "diverticulum, cystic" or "diverticulum, atypical, cystic" should be used. modifiers: mucosa or specific cell type (e.g., chief cell, parietal or mucous cell) pathogenesis: decreased number and/or size of epithelial cells reducing the mucosal thickness and function of the glandular mucosa. in the fundic glands, it may affect one or all cell types. in most instances chief cells are lost before other cell types. • decreased numbers of a one or more cell types. remaining cells may be small and/or poorly differentiated. • reduced mucosal thickness in advanced cases, although compensatory hyperplasia of less differentiated epithelial cells can mask this. • dilatation of atrophic glands. • focal areas of atrophy may be seen with aging. • artifact: over stretched stomach during 'pinning' at necropsy leading to a reduction in thickness of all layers of the gastric wall. • secretory depletion, mucus: reduction of mucous cell size due to reduced amounts of mucus but no loss of cells. comment: at least two different types of atrophy can be distinguished. in one type, atrophy broadly affects all mucosal cell types resulting in an overall reduction in mucosal height with maintenance of relatively normal cellular composition. under other circumstances a specific cell type may be atrophic with the loss of specific functionality. atrophy affecting all cell types of the gastric mucosa can be produced by starvation, antrectomy or deletion of the gastrin gene which removes the trophic stimulation of gastrin (greaves 2012) . analogous changes may be produced by test articles that block the secretion or activity of gastrin (dethloff et al. 1997) . in contrast, chronic inflammation and some xenobiotics can cause loss of specific cell types (greaves 2012 ). in addition, in the lesions associated with helicobacter infections in mice it has been noted that chief cells are lost before parietal cells (rogers and houghton 2009; rogers 2012) . focal or diffuse atrophy of the mucosa can occur with age in rodents where there is partial replacement of the glands by fibrous connective tissue (brown and hardisty 1990) . modifiers: mucosa or specific cell type (e.g., chief cell, parietal or mucous cell) pathogenesis: vacuolation with/without degeneration of epithelial cells. may affect all cell types or be primarily chief, parietal or mucous cell. • cytoplasmic vacuolation, swelling. • occasional apoptosis/single cell necrosis may also be present. • autolysis: destruction and loss of cell types but often occurs preferentially at the luminal surface. • depletion, mucus: only mucous cells affected, no other cytological changes. comment: vacuolation with no or minimal evidence of cell loss can be induced by agents that inhibit gastric acid secretion (dethloff et al. 1997; karam and alexander 2001) . in addition, degeneration is observed with anticancer cytotoxic agents, ulcerogenic agents and agents that decrease mucosal blood supply, or test articles that sequester in parietal cells (ito et al. 2000; bertram et al. 2013; greaves 2012) . depending on the nature and severity of the insult, degeneration will be followed by necrosis, erosion/ulcer of the epithelium, inflammation and/or hemorrhage. pathogenesis: decreased mucus content of mucous cells. • intact epithelial layer. • replacement of normal clear cytoplasm with more basophilic cytoplasm containing little or no mucus. • degeneration, epithelial: additional cytological features apart from reduction in mucus. • atrophy, mucous cell: reduced number of mucus cells in addition to mucus depletion. comment: mucus depletion may develop secondary to spontaneous inflammatory lesions and drug-induced lesions (greaves 2012) . qualitative changes in mucus composition may accompany depletion as described following administration of aspirin, anti-inflammatory agents and adrenocortical steroids (ishihara et al. 1984; bertram et al. 2013; greaves 2012) . these substances may also alter the phospholipids present in the mucus layer thereby reducing the protective hydrophobic barrier properties and a loss of functional integrity. administration of histamine h2 receptor antagonists and proton pump inhibitors that reduce gastric acid secretion have also been shown to reduce total and sulphated glycoproteins along with reduction in mucus (yoshimura et al. 1996) . pathogenesis: transformation of any glandular epithelial cell to produce hyaline cytoplasm. • intensely pink-red cytoplasmic droplets and/or crystals in surface/mucous cells, most often adjacent to the limiting ridge. • may extend into and/or replace fundic glands in advanced cases. • cells are usually hypertrophic. • can be single or multiple (focal or multifocal). • crystals if present may be intracellular or extracellular. • none. comment: mucous epithelial cells distended with bright, hyaline eosinophilic cytoplasm occur infrequently in rodents either as a spontaneous change or associated with another lesion e.g. inflammation (especially eosinophilic) or lymphoma infiltration (bertram et al. 1996) . they are usually observed in mucus neck cells of the glandular mucosa near the limiting ridge and occasionally may be accompanied by hyaline eosinophilic crystals. eosinophilic globules may be considered a precursor of these crystals. formation of these cells is also associated with the administration of gastric antisecretory agents although the incidence is not proportional to the neuroendocrine cell hyperplasia which also occurs with these agents (betton et al. 1988 ). the eosinophilic granules are similar to those seen in other epithelia e.g. upper and lower respiratory tract or pancreas (leininger et al. 1999; renne et al. 2009 ). once thought to be accumulation of mucosubstances or pepsinogen, they have been shown to be composed of a ym1/ym2 a chitinase-like protein that may be produced in response to mucosal irritation rogers and houghton 2009) . the use of the term hyalinosis for this lesion will potentially cause confusion in safety assessment as the same term may also be used for a distinctly different clinical disorder in humans and can be used to describe vascular and renal glomerular changes. pathogenesis: gene regulated, energy dependent process leading to formation of apoptotic bodies which are phagocytosed by adjacent cells. • necrosis, mucosa: the morphologic features of cell death clearly fit with necrotic diagnostic criteria (cells and nuclei swollen, pale cytoplasm etc.). • apoptosis/necrosis, mucosa: both types of cell death are present and there is no requirement to record them separately or a combined term is preferred for statistical reasons. the term is also used when the type of cell death cannot be determined unequivocally. • erosion/ulcer: focal or focally extensive penetration of the mucosa, either partial (erosion) or full to the muscularis mucosa (ulcer). the approach for the nomenclature and diagnostic criteria of cell death applied here is based on a draft recommendation of the inhand cell death nomenclature working group. apoptosis is not synonymous with necrosis. the main morphological differences between these two types of cell death are cell shrinkage with nuclear fragmentation and tingible body macrophages in apoptosis versus cell swelling, rupture and inflammation in necrosis; however, other morphologies (e.g. nucelar pyknosis and karyorrhexis) overlap. in routine h&e sections where the morphology clearly represents apoptosis or single cell necrosis, or whereby special procedures (e.g. tem or ihc for caspases) prove one or the other, individual diagnoses may be used. however, because of the overlapping morphologies, necrosis and apoptosis are not always readily distinguishable via routine examination, and the two processes may occur sequentially and/or simultaneously depending on intensity and duration of the noxious agent (zeiss 2003) . this often makes it difficult and impractical to differentiate between both during routine light microscopic examination. therefore, the complex term apoptosis/necrosis may be used in routine toxicity studies. a differentiation between apoptosis and single cell necrosis may be required in the context of a given study, in particular if it aims for mechanistic investigations. transmission electron microscopy is considered to represent the gold standard to confirm apoptosis. other confirmatory techniques include dna-laddering (easy to perform but insensitive), tunel (false positives from necrotic cells) or immunohistochemistry for caspases, in particular caspase 3. these techniques are reviewed in detail by elmore (elmore 2007) . some of these confirmatory techniques detect early phases of apoptosis, in contrast to the evaluation of h&e stained sections, which detects late phases only; overall interpretation of findings should take into account these potential differences. thus, low grades of apoptosis may remain unrecognized by evaluation of h&e stained slides only. certain studies may require the consideration of forms of programmed cell death other than apoptosis which requires specific confirmatory techniques (galluzzi et al. 2012) . synonyms: oncotic cell death; oncotic necrosis; necrosis pathogenesis: unregulated, energy independent, passive cell death with leakage of cytoplasm into surrounding tissue and subsequent inflammatory reaction; may be induced by direct contact with a test article after oral uptake/administration. • cells swollen with pale eosinophilic cytoplasm. • loss of nuclear basophilia, pyknosis and/or karyorrhexis that affects aggregates of cells. • in more severe cases, there may be detachment of the epithelium from the submucosa. • typically, the presence of degenerative cells is a component of necrosis. • minimal or slight inflammatory cell infiltrates may be present as a feature of necrosis. single cell • only single cells affected. • apoptosis, mucosa: the morphologic features of cell death clearly fit with apoptosis (cell and nucleus shrunken, hypereosinophilic cytoplasm, nuclear pyknosis etc.) and/or special techniques demonstrate apoptosis. • apoptosis/necrosis, mucosa: both types of cell death are present and there is no requirement to record them separately or a combined term is preferred for statistical reasons. the term is also used when the type of cell death cannot be determined unequivocally. • atrophy: reduced thickness of all layers of the squamous epithelium or, in severe cases, absence of basal germinative layers; usually no cellular degeneration or necrosis. • erosion/ulcer: focal or focally extensive penetration of the mucosa, either partial (erosion) or full to the muscularis mucosa (ulcer). comments: epithelial necrosis usually develops into erosion/ ulcer and in the past has frequently been recorded as such. a separate recording of necrosis as the initial event in this process is recommended when the structure of the mucosa is still intact. synonym: cell death pathogenesis: unregulated, energy independent, passive cell death with leakage of cytoplasm into surrounding tissue and subsequent inflammatory reaction (single cell necrosis) and/ or gene regulated, energy dependent process leading to formation of apoptotic bodies which are phagocytosed by adjacent cells (apoptosis). • both types of cell death are present and there is no requirement to record them separately or a combined term is preferred for statistical reasons. • the type of cell death cannot be determined unequivocally. • apoptosis: the morphologic features of cell death clearly fit with apoptosis (cell and nucleus shrunken, hypereosinophilic cytoplasm, nuclear pyknosis etc.) and/or special techniques demonstrate apoptosis and there is a requirement for recording apoptosis and necrosis separately. • necrosis, mucosa: the morphologic features of cell death clearly fit with necrotic diagnostic criteria (cells and nuclei swollen, pale cytoplasm etc.) and there is a requirement for recording apoptosis and necrosis separately. • erosion/ulcer: focal or focally extensive penetration of the mucosa, either partial (erosion) or full to the muscularis mucosa (ulcer). comments: necrosis and apoptosis are not always readily distinguishable via routine examination, and the two processes may occur sequentially and/or simultaneously depending on intensity and duration of the noxious agent (zeiss 2003) . this often makes it difficult and impractical to differentiate between both during routine light microscopic examination. in these cases, the combined term apoptosis/necrosis may be used. it is recommended to explain in detail the use of this combined term in the narrative part of the pathology report. pathogenesis: loss of superficial mucosa with preservation of the muscularis mucosa (erosion) or with penetration of muscularis mucosa and exposure of submucosa (ulcer). • focal or multifocal. • epithelial cells are absent but muscolaris mucosa is intact (erosion). • epithelial cells are absent and muscularis mucosa has been destroyed (ulcer). • ulceration will be associated with acute/chronic inflammatory cell infiltration in submucosa. • in the more severe cases ulceration and/or inflammation will extend into the tunica muscularis and serosa. • hyperplasia is often present in the surrounding epithelium. • may be associated with foreign bodies (plant fibers) in submucosa. • hemorrhage may be seen with larger ulcers. • adenocarcinoma: may show secondary ulceration, but penetration of basement membrane by single cells or nests of neoplastic cells always present. • artifacts: mechanical loss of mucosa during necropsy; plane of section due to orientation of tissue in block. • autolysis: all tissues in stomach affected but there is often preferential destruction and loss of cells at the luminal surface. comment: erosions and ulcers develop in rodents following stress, bile reflux, changes in acid secretion and hypoxia (bertram et al. 2013; greaves 2012; haschek et al. 2010) . certain types of xenobiotics e.g. non-steroidal anti-inflammatory drugs and ethyl alcohol are well known ulcerogenic agents but ulcers have also been induced by innocuous substances e.g. saline glucose when given as hyperosmolar solutions (puurunen et al. 1980) . ulcers can also reflect systemic disease e.g. associated with uremia. small focal erosions can occur in the glandular corpus of both rats and mice following overnight fasting. although there are specific morphologic criteria to differentiate the more superficial lesion (erosion; confined to the epithelial surface) from the deeper ulcer (full penetration of the mucosa to the muscularis mucosa), the plane of section through smaller, focal lesions often impacts on the appearance (depth of the epithelial loss) of this finding. in most instances it is not practical to separate these changes and they are typically diagnosed under the single, combined term of erosion/ulcer with an appropriate severity grade. erosion/ulcer usually is the result of epithelial necrosis. because of its unique topography adjacent to the lumen and often involving multiple organ layers, which uniquely affects the progression and resolution of the lesion (e.g. margin hyperplasia, granulomatous tissue, inflammation), it is useful to record erosion/ulcer separately from epithelial necrosis. a deep ulcer may be perforating if it destroys all layers of the gastric wall, and may be designated as such by a free text entry. however, a perforation resulting from a physical insult, e.g. gavage-related, typically is a macroscopic lesion and generally not recorded on the light microscopic level. pathogenesis: enlargement of mucous cells that line gastric glands, accompanied by loss of parietal and/or chief cells. the mucous cells may manifest foamy cytoplasm. • parietal and/or chief cells in corpus mucosa replaced by round, foamy, lightly basophilic cells resembling duodenal brunner's gland cells. concurrent loss of oxyntic glandular cells implicates an effect on the differentiation of the glandular epithelium. • increased numbers of such cells may be present (hyperplasia). • histochemical staining with alcian blue/pas ph 2.5 shows that cells express increased gastric-type neutral mucins (red) as well as antral gland and intestinal-type acid mucins (blue). differential diagnosis • none. comment: evaluation in h&e sections limits the characterization of this mucosal change to hypertrophy, hyperplasia and/or vacuolation. this may occur spontaneously or in response to chronic injury. histochemical stains and immunochemical markers facilitate further characterization of the change as a metaplastic shift in the mucous secretory phenotype of the cells. several classes of metaplasia have been identified based on these additional techniques, which may have relevance for modeling human gastric pathogenesis. true intestinal metaplasia (metaplasia with development of a genuine intestinal phenotype) is very rare in rodents but has been induced by pcbs in mice (morgan et al. 1981) . "pseudopyloric" metaplasia where the cells resemble antral cells morphologically and histochemically is much more common. authentic intestinal metaplasia is best confirmed by immunohistochemistry for the intestinal-specific nuclear protein cdx2 (fox et al. 2007 ). pseudopyloric/intestinal metaplasia occurs in the rodent stomach as a change associated with some experimentally induced cancers (e.g. following administration of polychlorinated biphenyls), localized ionizing radiation, injection of xenogenic stomach antigens and the non carcinogen, iodoacetamide (greaves 2012) , as well as infectious models in mice such as h. felis and h. pylori (leininger et al. 1999; rogers and fox 2004) . pathogenesis: extracellular deposits of polypeptide fragments of a chemically diverse group of glycoproteins, in various tissues including stomach mucosa. • deposition of pale amorphous, homogenous eosinophilic material. • location is extracellular in connective tissue and/or blood vessel walls. • green birefringence with congo red stain when viewed under polarized light. • hyaline connective tissue: congo red negative. • fibrinoid necrosis of blood vessels: other evidence of tissue damage in section. comment: amyloid deposition may occur in the stomach mucosa as part of systemic amyloidosis but it is not one of the predilection sites. amyloidosis is extremely rare in rats but common in some strains of mice. in susceptible strains a number of factors including gender, diet, housing conditions, stress, endocrine status and microbiological status can influence the occurrence of amyloidosis (lipman et al. 1993) . for as yet unidentified reasons the previously susceptible cd-1 strain in europe now rarely develops amyloidosis. the characteristic morphologic appearance and location of amyloid in h&e sections that is usually adequate to make this diagnosis. confirmation of the deposits as amyloid can be accomplished with light microscopy using special stains such as congo red. amyloid appears apple green under polarized light with this stain. the term "hyaline" should be used in case of uncertainties about the nature of extracellular deposits of hyaline material in h&e sections and the unavailability of a congo red stain. hyaline is also the appropriate term for homogenously eosinophilic extracellular deposits that react negative with congo red. pathogenesis: deposition of mineral in muscle, fibrous or elastic tissues secondary to necrosis (dystrophic mineralization) or hypercalcemia (metastatic mineralization). • mineral may be found in the muscle layers, blood vessels, basement membranes and/or in the interstitium of mucosa and submucosa. • interstitial mineral occurs predominantly in a band paralleling the parietal cell-rich region of the corpus mucosa. • acellular basophilic material. • mucosal mineralization can be limited to small focal deposits. • diffuse mineralization of the corpus mucosa may be accompanied by degenerative changes or mucous cell hyperplasia in neighboring glands. • artifact: hematoxylin stain precipitate. • bacteria: colonies of bacteria may be confused with mineral due to their basophilic staining. ante mortem bacterial invasion will be accompanied by necrosis and usually inflammation; postmortem bacterial invasion will be accompanied by autolysis. • osseous metaplasia: osteoid present. comment: presence of mineral can be confirmed with von kossa or alizarin red stains but these are rarely needed to make a diagnosis. multifocal or diffuse mineralization occurs secondary to chronic renal disease in rodents (leininger et al. 1999; brown and hardisty 1990) and in situations where serum calcium and/or phosphate balances are perturbed e.g. following intravenous administration of salts of rare earth metals such as gadolinium chloride (rees et al. 1997) . mineralization can sometimes be observed in combination with inflammation and neoplasia. infiltrate ( figure 64 ) synonyms: infiltrate inflammatory; infiltrate inflammatory cell; infiltration (plus modifier); infiltration, inflammatory; infiltration, inflammatory cell modifiers: type of inflammatory cell that represents the predominant cell type in the infiltrate pathogenesis: infiltration of lamina propria and/or submucosa with neutrophils (infiltrate, neutrophil), eosinophils (infiltrate, eosinophil), mononuclear cells (infiltrate, mononuclear cell) or a combination of more than one type (infiltrate, mixed) without other histological features of inflammation e.g. hemorrhage, edema, fibroplasia. • focal, multifocal or diffuse infiltration of cells in the lamina propria and/or submucosa. • presence of mononuclear or polymorphonuclear leucocytes but no other histological criteria of inflammation. • inflammation: other histological features of inflammation such as edema, hemorrhage, necrosis and/or fibroplasia will be present. • granulocytic leukemia: infiltration of neutrophil precursors and/or abnormal neutrophils will probably be present along with mature neutrophils; infiltration of similar neoplastic cells is likely present in other organs. • lymphoma: infiltration of monomorphic population of lymphocytes (often with atypical, increased and/or abnormal mitoses); infiltration of similar neoplastic cells is likely present in other organs. comment: a minimal/mild infiltration of eosinophilic leucocytes is common in the submucosa of the corpus of rats and the infiltrate may also extend into the non-glandular region near the limiting ridge (mcinnes 2012). the stimulus for this infiltration is unknown. modifiers: type of inflammatory cell that represents the predominant cell type in the infiltrate pathogenesis: infiltration of lamina propria and/or submucosa with neutrophils (inflammation, neutrophil) or mononuclear cell (inflammation, mononuclear cell) or a combination (inflammation, mixed) with additional histological features of inflammation e.g. hemorrhage, edema, fibroplasia. propria and/or submucosa. • presence of other histological criteria of inflammation e.g. hemorrhage, edema, fibroplasia. • infiltrate, inflammatory cell: other morphologic features of inflammation such as edema, hemorrhage, necrosis and/or fibroplasia will be absent. • granulocytic leukemia: infiltration of neutrophil precursors and/or abnormal neutrophils will probably be present along with mature neutrophils; infiltration of similar neoplastic cells is likely present in other organs. • lymphoma: infiltration of monomorphic population of lymphocytes (often with atypical, increased and/or abnormal mitoses); infiltration of similar neoplastic cells is likely present in other organs. comment: inflammation in the both glandular and nonglandular stomach is most often seen in combination with erosion or ulceration. pathogenesis: presence of helicobacter spp. in mice. • spiral shaped bacteria in the glands primarily usually near border of antrum and corpus. • infection by h. felis and h. pylori in some strains of mice results in inflammatory, dysplastic and eventually neoplastic changes. • proliferation of gut associated lymphoid tissue (galt), resembling human malt lymphoma, may occur in certain strains of mice (e.g. balb/c). • inflammation: non-helicobacter opportunists may colonize the achlorhydric rodent stomach (e.g. gastrin-deficient mice) and invoke a similar chronic inflammatory disease. comment: the progression from infection by h. pylori and h. felis to the formation of gastric adenocarcinoma in various mouse strains has been well described (rogers and fox, 2004; rogers and houghton 2009) . host immunity plays a crucial role in determining the outcome of gastric helicobacter infection. mouse strains such as c57bl/6 that mount a strong th1 responses to helicobacter infection demonstrate lower colonization but increased inflammation and hyperplasia/dysplasia compared to strains such as balb/c with th2 predominant responses (rogers and houghton 2009 ). silver stains e.g. wartin-starry can be used to confirm the presence of helicobacter in tissues section. pathogenesis: infection of the glandular stomach in compromised host, e.g. immunodeficiency. • presence of a layer of round yeast-like structures along the glandular stomach epithelial surface lining and in the gastric contents. • maybe be large numbers of yeasts or few. • budding maybe seen. • associated inflammation is usually not observed but may be seen. • stains with giemsa stain. • bacteria: do not show budding. • cryptosporidia: are more within or intergrated with the surface epithelium. comments: this infection appears to be rare histologically but may be more commonly found by culture (mackinnon 1959, savage and dubos 1967) . a variety of similar organisms have been reported in many species (kurtzman et al. 2005) . these organisms usually are not pathogenic but may be under specific conditions such as immunodeficiency. edema and hemorrhage of the stomach are similar to those described for the upper digestive tract. other vascular lesions in the stomach and mesentery are dealt with in the inhand monograph on the cardiovascular system. histogenesis: squamous epithelium of the nonglandular stomach. diagnostic features • proliferation and thickening of the stratum spinosum, often in association with a thickened keratin layer (parakeratotic or hyperkeratotic). • focal, multifocal or diffuse. • exophytic (papillary hyperplasia) or endophytic growth pattern. • papillary projections not or only minimally branched and without fibrovascular stroma. • broad-based hyperplastic foci might have multiple straight or minimally branched projections. • polarity of differentiation to keratinocytes is orderly and complete. • rete peg formation may be exaggerated, i.e. papillary body may show undulation due to enlarged germinal layers. • endophytic growth with cyst-like structures may occur; cysts lined by well-differentiated squamous epithelium and filled with keratin; may extend into the submucosa, but basement membrane is intact. • mitotic activity may be increased. • dysplasia of spinous and basal cells may occur. • reactive as a descriptive term: evidence for a primary irritating process, e.g. ulcer, irritating chemical, foreign material. • papilloma, squamous cell: papillary projection with a single and complex branching fibrovascular connective tissue core. • hyperplasia, basal cell: endophytic proliferation of basal cells; cytoplasm of proliferative cells is basophilic. • carcinoma, squamous cell: true invasion, i.e. lost basement membrane integrity; in the poorly differentiated type: lost polarity of differentiation to keratinocytes. comments: there may be a morphologic continuum from hyperplasia to papilloma and the distinction between severe hyperplasia and papilloma may in these cases be subjective. the squamous epithelium at the border of nonglandular and glandular stomach (limiting ridge) is generally thickened relative to the adjacent squamous mucosa. this area may be folded and, thus, cut tangentially or obliquely, if the stomach was not properly stretched prior to fixation. hyperkeratosis or parakeratosis alone should be distinguished from squamous hyperplasia. acanthosis is a synonym for hyperplasia of the squamous cell epithelium of the skin and should not be used in this organ. diffuse squamous cell hyperplasia is relatively common in oral gavage studies when the test article has an irritating potential. the modifier "reactive" should be used if proliferation of stratified squamous epithelium is associated with or secondary to local erosion, ulcer or inflammation. the absence of an ulcer or erosion in the plane of section does not preclude the presence of such in adjacent unexamined tissue. florid hyperplasia of the nonglandular stomach epithelium without evidence of cellular atypia can be completely reversible following the withdrawal of an inciting stimulus. nonglandular stomach carcinogens may induce dysplasia of spinous and basal cells, especially if the hyperplasia is a precursor to a papilloma or squamous cell carcinoma. squamous cell hyperplasia and basal cell hyperplasia may occur concurrently. this change has been observed in association with test article exposure (e.g. butylated hydroxyanisole) or chronic vitamin a deficiency (klein-szanto et al. 1982) . histogenesis: basal layer of the stratified squamous epithelium. • proliferation of the basal cell layer, basophilic staining increased. • focal or diffuse. • endophytic growth pattern. • no alteration of basement membrane integrity. • papillary body shows marked undulation but rete peg structure still present. • hyperplasia, squamous cell: thickening of the epithelium with all normally existing layers. • carcinoma, squamous cell: evidence for lost basement membrane integrity, spinous cells and keratinized cells proliferate, cellular atypia. • tumor, basal cell, benign: circumscribed proliferation of basal cells with loss of rete peg structure and leading to compression of surrounding tissue or prominent elevation of overlying epithelial layers. • tumor, basal cell, malignant: basal cells predominate, keratinization is absent, evidence for lost basement membrane integrity. comment: basal cell hyperplasia and squamous cell hyperplasia may occur concurrently. isolated nests of basal cells may occur in the lamina propria, dependent on the plane of section through the rete peg structures. however, their discrete border indicates an intact basement membrane. basal cell hyperplasia and dysplasia are more frequently associated with chemicals (e.g., butylated hydroxyanisole) that result in neoplasms of the nonglandular mucosa. dysplasia of basal cells, characterized by keratinization within the basal layer, may be observed. foci of basal cell hyperplasia may be found in the mucosa of the glandular stomach in the vicinity to the limiting ridge ( figure 49b and c). due to their similarity to basal cell hyperplasia of the nonglandular stomach and their specific location they are considered to originate from the nonglandular stomach. such lesions should be recorded under "nonglandular stomach, hyperplasia, basal cell". papilloma, squamous cell ( figure 72 ) histogenesis: stratified squamous epithelium. • solitary or multiple. • exophytic or endophytic (usually exophytic). • formation of a single complex branching fibrovascular core by exophytic tumors; the stroma typically forms secondary branches or finger-like projections which blend with the underlying lamina propria. • well-differentiated squamous cell layer (orderly maturation of the epithelium), often with prominent hyperkeratosis or parakeratosis. • penetration of muscularis mucosae is possible (papillomas extend into the submucosa), but no evidence for lost basement membrane integrity. • lack of cellular atypia. • mitotic figures are rare. • local inflammatory reactions are common (lymphocytic infiltrations in the stromal stalk). • hyperplasia, squamous cell: papillary projections are minimally branched at the most and lack a fibrovascular stromal stalk. • carcinoma, squamous cell: evidence for lost basement membrane integrity is obligatory; invading tumor cells generally with varying degrees of structural and cellular atypia. comment: there is a morphologic continuum from hyperplasia to papilloma and the distinction between severe hyperplasia and papilloma is sometimes subjective. also comparative diagnosis and distinction of well differentiated squamous cell carcinomas from papillomas may be difficult: the tumor tissue of papillomas may extend into the submucosa; a growth pattern suggestive of intact basement membranes and the lack of cellular and structural atypia distinguishes it from squamous cell carcinomas. invasive squamous cell carcinomas may have papillary lesions on their luminal surface. in carcinomas arising within papillomas, invasion of the fibrovascular connective tissue stalk is present. the spontaneous papilloma in the nonglandular stomach is a rare event in rats, but more common in mice and in some lines of genetically-engineered mice (sundberg et al. 1992 ). papillomas occur more often in gavage studies than with other routes of exposure. histogenesis: stratified squamous epithelium. • exophytic and/or endophytic growth pattern. • penetration of basal membrane by single cells or nests of neoplastic cells. • loss of cellular differentiation, anaplasia. • submucosa, muscle layer and serosa can be infiltrated. • well-differentiated type: nearly normal squamous epithelium with irregular structure of the papillary body, frequently showing foci with central hyperkeratinization (keratin pearls); in areas of invasion, predominance of polygonal and pleomorphic cells with varying degrees of keratinization. poorly differentiated type (anaplastic): solid sheets or strands of spindle cells leading to varying degrees of desmoplastic appearance; keratinization may be difficult to detect. • cells show varying size (frequently larger than normal) and shape; nuclei are hyperchromatic, enlarged and have prominent nucleoli. • mitotic figures may be numerous. • superficial ulceration and inflammation or fibroplasia of the lamina propria or submucosa are common features. • may metastasize to the abdominal cavity, regional lymph nodes, or the lung. • papilloma, squamous cell: no evidence of lost basement membrane integrity or invasion and no cellular atypia. • tumor, basal cell, benign or tumor, basal cell, malignant: exclusively endophytic growth pattern and a typical arrangement of the small basophilic basal cells in garlands or rows; poorly differentiated squamous cell carcinomas can have a prominent basal cell component but this is often associated with extensive invasion and a scirrhous response. • hyperplasia, squamous cell: no evidence of lost basement membrane integrity or of invasive growth although muscularis mucosae may be displaced; no evidence of cellular atypia. • hyperplasia, basal cell: can form round or oval clusters which extend into the underlying lamina propria; these clusters usually have discrete borders (suggestive of maintained basement membrane integrity) and no cellular atypia. comment: depending on the plane of section, it can be difficult to distinguish a well-differentiated carcinomas from a papilloma. papillomas may also extend into the submucosa and thereby displace the muscularis mucosa. the presence of carcinomatous foci in an otherwise benign appearing papilloma is indicative of malignancy (fukushima and ito, 1985; tsukamoto et al. 2007 ). in general, the lack of cellular atypia allows a distinction to be made. many squamous cell carcinomas in the nonglandular stomach are exophytic which can result in partial or total occlusion of the lumen. the exophytic portion can be papillary resembling papilloma. chemically-induced (e.g. butylated hydroxyanisole, 1,2-dibromo-ethane) squamous cell carcinomas may contain mostly basal or spindle cells with little squamous differentiation. these tumors are considered a morphological variant of the more typical squamous cell carcinoma. ultrastructurally, squamous cell carcinomas contain typical desmosomes and bundles of electron-dense tonofilaments. some chemicals especially when given by gavage cause highly malignant squamous cells carcinomas in rats which metastasize to the peritoneal cavity and lungs (olson et al. 1973) . histogenesis: basal layer of the stratified squamous epithelium. • endophytic growth pattern. • closely packed small or oval cells from the stratum germinativum forming garlands or nests with peripherally palisade-like cells. • loss of rete peg structure. • growth pattern suggests that basement membrane is intact. • compression of surrounding tissue or elevation of overlying epithelium which itself does not show neoplastic alteration. • no keratinization. • hyperplasia, basal cell: proliferation of basal cells with preservation of rete peg structure; no or only slight compression of surrounding tissue or elevation of overlying epithelial layers. • carcinoma, basal cell: growth pattern suggests that basement membrane integrity is lost. • carcinoma, squamous cell: growth pattern suggests that basement membrane integrity is lost, spinous cells and keratinized cells proliferate. comment: the absence of differentiation and therefore the predominant proliferation of the basal cell is a criterion for the basal cell tumor diagnosis. this tumor type is very rare, and no images were available at point of publication. synonym: carcinoma, basal cell histogenesis: basal layer of the stratified squamous epithelium. • endophytic growth pattern. • superficial layers almost normal, but sometimes ulcerated, while neoplastic basal cells infiltrate into the lamina propria of the mucosa and submucosa. • round to oval closely packed nonspinous cells with dark nuclei and little cytoplasm, often forming palisade-like garlands or chains. • keratinization generally absent. • growth pattern suggests that basement membrane integrity is lost. • frequent mitotic figures. • carcinoma, squamous cell: well differentiated squamous cell carcinomas with prominent keratinization; poorly differentiated variants can have a prominent basal cell component but this is generally associated with extensive invasion and a scirrhous response. • hyperplasia, basal cell: proliferation of basal cells with preservation of rete peg structure; no or only slight compression of surrounding tissue or elevation of overlying epithelial layers; growth pattern indicates that basement membrane integrity is maintained. • tumor, basal cell, benign: growth pattern indicates that basement membrane integrity is maintained. comment: the absence of differentiation and therefore the predominant proliferation of the basal cell is a criterion for the basal cell tumor diagnosis. this tumor type is very rare, and no images were available at point of publication. diverticulum (figures 75, 76, 77, 78, 79 and 88) synonyms: cystic adenomatous hyperplasia; diverticulosis; diverticulum, atypical, may have been identified as: atypical cystic hyperplasia; cystic hyperplasia with growth into the gastric wall; cystic adenomatous hyperplasia; herniation atypical; "pseudoinvasion", atypical; hamartoma, atypical pathogenesis: usually caused by mechanical or biochemical disruption of gastric wall structures resulting in loss of extracellular matrix integrity and internal projection of surface lining into deeper layers; may alternatively represent a congenital abnormality. • extension of glands through muscularis mucosae, into submucosa and further in some cases. • morphology of epithelial lining is variable, ranging from single layer cuboidal or columnar cells to complete mucosa. • epithelium may show features of regeneration like increased basophilia, increased nucleus-to-cytoplasm ratio and gradual loss of polarity, but atypia is minimal at the most. • basement membrane integrity is always maintained. • more often seen in the antrum of mice. • often accompanied by inflammation and regenerative / reparative processes. • may contain ingesta, hair, or other foreign material. cystic: • rounded cystic structures in lamina propria or deeper layers. • compression of surrounding tissue, some degree of compression atrophy of the epithelium lining the cyst. • may contain mucus. atypical: • extension of atypical/dysplastic mucosal glands into or through muscularis mucosa, submucosa, and deeper in some cases. • hyperplastic atypical (dysplastic) epithelial cells forming cystic glands that may appear to be "invading" into deeper layers of the stomach. • atypical change often focal in an otherwise normal appearing epithelium. differential diagnoses • adenoma: polypoid or papillary growth into gastric lumen obligatory, but pseudo-invasion into stalk may be seen. • adenocarcinoma: multiple layers of lining epithelium with varying degrees of dysplasia. true infiltrative growth with loss of basement membrane integrity and often with a scirrhous stromal reaction. comment: glandular diverticula of the stomach, frequently observed in mice (especially in genetically-engineered mice and in mice with chronic experimental gastric infections), may be associated with local mucosal defects (e.g. postulcerative repair), suggesting opportunistic expansion, but these have also been observed within submucosal veins in the absence of mucosal defects, suggesting there may be a vascular route of penetration from the mucosa. these structures are typically lined by a simple cuboidal mucus epithelium, which may have an antral or intestinal mucus phenotype that can be characterized by histochemical stains (e.g. alcian blue). these focal gastric lesions do not resemble diverticulosis in human colon which may be focal, multifocal or diffuse. they do not appear to be neoplastic, although they do create an increased risk of rupture and septic peritonitis. it is not recommended to record "diverticulum" (plus modifier) as a separate finding, if it occurs in association with another proliferative process (e.g. hyperplasia, adenoma). small glandular diverticula lined by a single layer of cuboidal or columnar epithelium resemble "crypt herniation" of human gastrointestinal pathology (e.g. rex et al. 2012) . such herniation of crypts through the muscularis mucosae is interpreted as "pseudoinvasive" or "inverted" growth pattern. although the process-related term "crypt herniation" is common in literature (e.g. betton et al. 2001) , the term "diverticulum" is considered to better fit to the inhand principle to use descriptive terms and to avoid process-related ones (mann et al. 2012) . also the misnomer "adenomatous diverticulum" has been used to describe these structures and the prefix "adenomatous" should be avoided. atypical diverticula must be distinguished from genuine neoplastic invasion (e.g. loss of basement membrane and other signs of malignancy in the latter). they are associated with preexisting mucosal atypical hyperplasia/dysplasia as seen under specific experimental conditions, tumor-associated gastric toxins, genetically-engineered mice and chronic helicobacter pylori infection (andersson et al. 2005; boivin et al. 1996; boivin et al. 2003; fernandez-salguero et al. 1997; miyoshi et al. 2002; rogers and houghton 2009 ). whereas they often do not progress to unequivocal adenocarcinoma, atypical glandular and cellular features are consistent with neoplastic potential. atypical diverticula may be associated with atypical hyperplasia and adenoma, but usually not with adenocarcinoma. as noted above for the simple diverticula, they should not be recorded as a separate diagnostic entity when associated with another proliferative process. synonyms: regenerative hyperplasia, focal hyperplasia, focal (mucosa), atypical may have been identified as: adenomatous hyperplasia, dysplasia, or gastric intraepithelial neoplasia (gin) histogenesis: glandular and/or surface epithelial cells. • focal or multifocal due to underlying injury (e.g. ulcer); mucosal defects/injury and inflammation may be observed in tandem with proliferative changes. • confined to the gastric mucosa. • no significant compression of adjacent tissue. • proliferation may be manifested as an increase in mucosal thickness or penetration of glands through the muscularis mucosae to the submucosa, tunica muscularis, or serosa forming cystic or glandular diverticula. these structures are typically lined by a simple cuboidal mucus epithelium. • with the exception of diverticula, no distortion of regular mucosal structure by the proliferative process itself. • may be associated with a reduction of parietal cells. • eosinophilic globules may be present in the epithelial cell cytoplasm. • mitoses may be increased. atypical: • abnormal structure of gastric glands. • focal penetration by glandular diverticula into lamina propria or deeper layers but basement membrane is always intact. • hyperchromatic foci with cellular atypia and pleomorphism. • loss of epithelial polarity. • adenoma: typically arise as singular foci in the distal antral/pyloric region, often with polypoid architecture. nodular (polypoid, papillary) lesions growing into the lumen of the stomach; distortion of glandular architec-ture and in case of non-papillary adenomas also compression of surrounding tissue. • adenocarcinoma: invasion of nests and cords of neoplastic cells into lamina propria or deeper layers, indicating loss of basement membrane integrity; cellular atypia. comment: focal hyperplasia may be accompanied by metaplasia of the mucus epithelium, characterized by a shift from a neutral mucin phenotype of the surface epithelium to an acidic antral or intestinal mucin phenotype. this may be associated with a reduction in parietal cells (atrophy) and a presumptive shift in local ph. distinct patterns of hyperplastic changes may arise in the glandular stomach because of the cellular complexity, regional differences in architecture, and selectivity of stimuli. focal atypical hyperplasia represents an altered proliferation of mucosal epithelium. it may be associated with chronic inflammation. an example is the helicobacter pylori mouse model. the lesion may progress into an adenoma or adenocarcinoma. although frequently considered as preneoplastic by nature, this needs to be proven in a given case in order to be classified as such. focal intra-epithelial atypical proliferations resembling morphologically an invasive adenocarcinoma have been found in specific genetically modified mouse lines and after chemical induction. they have been termed gastric intraepithelial neoplasia (gin). other terms occasionally used but not preferred as synonyms are microadenoma, carcinoma in situ, and intra-epithelial carcinoma. synonyms: regenerative hyperplasia diffuse; hypertrophic gastritis; mucosal hypertrophy histogenesis: glandular and/or surface epithelial cells. • may be diffuse within a specific regional zone (corpus, antrum, pylorus). • mitotic activity may be increased. • within a region of mucosa, thickening of the mucosa may affect the full thickness or may be compartmentalized within discrete glandular zones or cell types. • with increasing severity, basophilia, decreased epithelial differentiation, and structural disorganization may be observed. • metaplasia of the mucus epithelium may arise, characterized by a shift from a neutral mucin phenotype to an intestinal mucin phenotype. this may be associated with a reduction in parietal cells and a presumptive shift in local ph. • a cause may be evident (irritating substance, gavage material). • adenoma: nodular lesions growing exophytic (polypoid, papillary). comment: diffuse hyperplasia may be induced by chemicals (e.g. misprostal) or by larval (taenia taeniaeformis) infestation in the liver. the latter results in hypergastrinemia and diffuse glandular mucosal hyperplasia. the incidence of mitotic figures at the base of the gastric pit and the ratio of the lengths of the foveolus (gastric pit) to the deeper oxyntic region can be indices of proliferative activity; however, the latter will normally increase moving distally from the squamocolumnar junction. diffuse fundic hyperplasia is a common finding in aged mice, where it is often accompanied by degenerative changes like glandular cysts or diverticula. synonym: enterochromaffin-like (ecl) cell hyperplasia histogenesis: neuroendocrine cells of the gastric mucosa. the enterochromaffin-like (ecl) cell is the predominant neuroendocrine cell of the oxyntic mucosa. • focal: aggregates of neuroendocrine cells no greater than 3 glands in diameter. • growth pattern often polypoid, sessile, or papillary with or without a fibrovascular stalk; in polypoid adenomas of the antral mucosa, gland architecture may be retained. • focal penetration by glandular diverticula into lamina propria or deeper layers may be present but basement membrane is always intact. • cells are a basophilic, less differentiated glandular epithelium, but with little atypia; polarity is maintained. • epithelial nuclei are unilayered or organized with varying degree of stratification. • hyperplasia, focal (mucosal): linear mucosal orientation without polypoid or papillary architecture. • adenocarcinoma: infiltration as evidenced by penetration into lamina propria or deeper layers with a growth pattern indicative of lost basement membrane integrity; scirrhous stromal reaction. • tumor, neuroendocrine cell, benign: discrete intramucosal foci, which typically arise in hyperplastic areas in the oxyntic mucosa (i.e. corpus region). closely organized clusters of neuroendocrine cells with eosinophilic cytoplasm, separated by thin fibrovascular stroma. argyrophilic and characteristic immunohistochemical phenotype. comment: well-differentiated, hyperplastic glands may be entrapped in the adjoining lamina propria or submucosa, such structures originate from herniation of glands, are well demarcated, lack a scirrhotic stromal response and should not be interpreted as neoplastic infiltration. adenomas, as well as atypical hyperplasia, may exhibit some degree of dysplasia and distortion of pre-existing architecture. macroscopic visibility has been suggested as a differential diagnostic criterion by some authors. this criterion is not used in the context of this description of histologic criteria. any lesion exhibiting a papillary or polypoid growth pattern is an adenoma, irrespective whether it was grossly visible or not. an intra-epithelial small focus with cytological characteristics seen in adenomas should not be diagnosed as an adenoma but as atypical hyperplasia. an adenoma must possess a polypoid, sessile or papillary growth pattern, whether grossly visible or not. gastric adenomas are an uncommon observation in untreated rodents, but may be frequent in some genetically modified mouse strains miyoshi et al. 2002) . they are associated with specific mutations, which may be similar to those of humans with gastric polyps. • hyperplasia focal (mucosal): proliferation with maintained mucosal structure, except of glandular diverticula; no atypia in simple hyperplasia; in case of glandular diverticula, localized and discrete penetration into lamina propria or deeper layers with maintained basement membrane integrity and surrounded by pre-existing mature tissues lacking a scirrhotic stromal response. • hyperplasia focal (mucosal) atypical: limited to the mucosal lining, no infiltrative growth; focal penetration by glandular diverticula into lamina propria or deeper layers may be present but basement membrane always intact. • diverticulum: localized and discrete penetration into lamina propria or deeper layers with maintained basement membrane integrity and surrounded by pre-existing mature tissues lacking a scirrhotic stromal response. • adenoma: no infiltrative growth; focal penetration by glandular diverticula into lamina propria or deeper layers may be present but basement membrane always intact. ▪ tumor, neuroendocrine cell, malignant: typically arise in hyperplastic areas in the oxyntic mucosa (i.e., corpus region); closely organized clusters of neuroendocrine cells with eosinophilic cytoplasm, separated by thin fibrovascular stroma; argyrophilic and characteristic immunohistochemical phenotype. comment: gastric adenocarcinomas are uncommon spontaneous tumors in rodents, except of some genetically modified mouse strains. chemically-induced tumors in routine mouse/rat strains are not common except for induction by specific chemicals, e.g. n-methyl-n'-nitrosoguanidine, nmu, mnng in rats, while mice are more resistant (tsukamoto et al. 2007 ). the morphologic type may depend on the etiology / carcinogen. synonyms: enterochromaffin-like cell tumor; apudoma [apud = amine precursor uptake and decarboxylase]; intramucosal neuroendocrine cell tumor histogenesis: neuroendocrine cells of the gastric mucosa; the enterochromaffin-like (ecl) cell is the predominant neuroendocrine cell of the oxyntic mucosa. • discrete, well-demarcated aggregates of neuroendocrine cells, which may arise from hyperplastic areas in the oxyntic mucosa (i.e. corpus region). • size of the aggregates exceeds 3 gastric glands in diameter. • restricted to intramucosal involvement. • compression or necrosis of adjoining mucosa glands may be observed. • hyperplasia, neuroendocrine cell: scattered distribution of individual or small clusters of well-differentiated cells; discrete foci no more than 3 gastric glands in diameter. • tumor, neuroendocrine cell, malignant: infiltration through muscularis mucosae by neoplastic, often less differentiated cells arranged in cords or nodules or serosal deposits or distant metastases. • ectopic hepatocytes: absence of neoplastic behavior or cytologic alteration. absence of characteristic markers for neuroendocrine cells (e.g. argyrophilia, immunohistochemical profile). comment: neuroendocrine cell hyperplasia and neoplasia may be seen in response to hypergastrinemia following antisecretory drug treatment (spencer et al. 1989 ). fundic mucosal hyperplasia is usually also evident. small numbers of normal neuroendocrine cells can be observed in the submucosa and should not be categorized as neoplasia. synonyms: neuroendocrine carcinoma; carcinoid; enterochromaffin-like cell tumor; apudoma [apud = amine precursor uptake, decarboxylase]; intramucosal neuroendocrine cell tumor (benign) histogenesis: neoplastic transformation of neuroendocrine cells. • aggregates of neuroendocrine cells, which may arise from hyperplastic areas in the oxyntic mucosa (i.e., corpus region). • growth is intramucosal as nodules of pale staining or eosinophilic cells, sometimes with larger submucosal nodules. • infiltration through muscularis mucosae by neoplastic, often less-differentiated, cells arranged in cords or nodules. • metastasis to regional lymph nodes or liver may be present. • hyperplasia, neuroendocrine cell: scattered distribution of individual or small clusters of well-differentiated cells. discrete foci are less than 3 gastric glands in diameter. • ectopic hepatocytes: absence of neoplastic behavior or cytologic alteration. absence of characteristic markers for neuroendocrine cells (e.g. argyrophilia, immunohistochemical profile). comment: neuroendocrine cell hyperplasia and neoplasia may be seen in response to hypergastrinemia following antisecretory drug treatment (spencer et al. 1989 ). fundic mucosal hyperplasia is usually also evident. small numbers of normal neuroendocrine cells can be observed in the submucosa and should not be categorized as neoplasia. interlacing bundles and whorls of uniform spindle cells arranged in criss-cross pattern; blunt-ended, fusiform nuclei with hyperchromasia, pleomorphism, and high mitotic index; phosphotungstic acid-hematoxylin positive myofibrils. • schwannoma: cd117 negative, s-100 positive; indistinct cell borders, elongate cells with eosinophilic cytoplasm; nuclei sometime arranged in palisades (i.e., antoni a pattern). comment: gist will generally not be easily differentiated from leiomyomas / leiomyosarcomas or other soft tissue tumors. however, immunohistochemical differentiation of the different tumor types, including gist, needs to be performed in case of imbalances in the incidence of smooth muscle tumors in a given study. experimentally-induced gist in the glandular stomach of rats and a spontaneous gist in rat nonglandular stomach have been reported mukaisho et al. 2006 ). cecal gist have been identified in gem mice overexpressing a mutant kit (nakai et al. 2008 ). the question arises whether some conventionally diagnosed rodent stromal tumors (e.g., leiomyoma) were actually gist. synonyms: interstitial cajal cell tumor, malignant; gastrointestinal pacemaker cell tumor, malignant histogenesis: specialized smooth muscle cells (i.e. interstitial cells of cajal) in the tunica muscularis or myenteric plexus. • locally infiltrative growth pattern. • cells may be arranged in bundles with storiform architecture, myxoid areas may be present. • spindle, epithelioid, or pleomorphic cell morphology. indistinct cell borders and fibrillary cytoplasm. • spindle-or irregular-shaped nuclei. • typically cd117 positive (cytokine receptor encoded by c-kit). • distant metastases may be present. • gastrointestinal stromal tumor, benign: expansive growth pattern, no distant metastases. • leiomyoma: cd117 negative, desmin positive; interlacing bundles and whorls of uniform spindle cells arranged in criss-cross pattern; blunt-ended, fusiform nuclei, minimal nuclear pleomorphism; phosphotungstic acid-hematoxylin positive myofibrils. • leiomyoscarcoma: cd117 negative, desmin positive; interlacing bundles and whorls of uniform spindle cells arranged in criss-cross pattern; blunt-ended, fusiform nuclei with hyperchromasia, pleomorphism, and high mitotic index; phosphotungstic acid-hematoxylin positive myofibrils. • schwannoma: cd117 negative, s-100 positive; indistinct cell borders, elongate cells with eosinophilic cytoplasm; nuclei sometime arranged in palisades (i.e. antoni a pattern). comment: gist will generally not be easily differentiated from leiomyomas / leiomyosarcomas or other soft tissue tumors. however, immunohistochemical differentiation of the different tumor types, including gist, needs to be performed in case of imbalances in the incidence of smooth muscle tumors in a given study. experimentally-induced gist in the glandular stomach of rats and a spontaneous gist in rat nonglandular stomach have been reported mukaisho et al. 2006 ). cecal gist have been identified in gem mice overexpressing a mutant kit (nakai et al. 2008 ). the question arises whether some conventionally diagnosed rodent stromal tumors (e.g. leiomyoma) were actually gist. the small intestine in rodents, as in other mammals, is subdivided into duodenum, jejunum and ileum and is designed for the digestion and absorption of ingested products (treuting and dintzis, 2012) . the large intestine is composed of cecum, colon and rectum. the cecum is a large blind end sac with considerable fermentation potential. the colon has an ascending section, a short transverse section and a descending section leading to the rectum which terminates at the anus. all parts, except the terminal rectum, are connected to the body wall by the mesentery which functions as a conduit for vascular, lymphatic and nervous supply. the terminal rectum lies outside the pelvic cavity and consequently lacks a surrounding serosal membrane. very rarely, congenital developmental abnormalities can occur, e.g. intestinal duplication (elangbam et al. 1998) . such pathologic conditions that can be reliably diagnosed grossly are not covered by this monograph. histologically, the mucosa of the small intestine is composed of basal crypts (also known as crypts of lieberkuhn) and villi which extend into the lumen. the base of the crypt is lined by stem cells; the remainder of the crypt is composed of proliferating epithelial cells which either migrate up the villi as they differentiate into absorptive cells with microvilli or goblet cells, with a turnover time of about 3 days, or remain in the crypt and differentiate into paneth cells. the villous epithelium is composed of tall columnar cells with surface microvilli (the main absorptive cells), goblet cells containing mucus and scattered neuroendocrine cells covering a lamina propria composed of connective tissue with a rich vascular and lymphatic network. the lamina propria contains variable numbers of lymphocytes, macrophages, plasma cells, eosinophils and/ or mast cells whilst occasional intraepithelial lymphocytes can also be observed. paneth cells, which have an exocrine serous morphology and produce antimicrobial peptides, are present in higher numbers in the crypts of jejunum and ileum compared to the duodenum. brunner's glands, which produce a bicarbonate rich secretion, are present in the submucosa immediately following the pyloric-duodenal junction. villi in the duodenum are taller than those in jejunum and ileum. jejunum and ileum can be hard to differentiate in cross section except that the terminal ileum has two serosal attachments (mesentery and ligamentum ileocecale) compared to the single mesenteric attachment of the jejunum. with age, the duodenal villi tend to become shorter and thicker due to increased amounts of fibrous connective tissue in the core, whilst those in the ileum become longer probably related to reduced absorption in the proximal intestine resulting in greater amounts of nutrients passing into the distal intestine (shackelford and elwell 1999) . in the jejunum and ileum there are well defined aggrega-tions of lymphoid tissue (peyer's patches) located opposite the mesenteric attachment; these may show formation of germinal centers. similar aggregations are present in other parts of the gastrointestinal tract and are collectively referred to as gastrointestinal-associated lymphoid tissue (galt). villi are absent from the large intestine and instead the mucosa is thrown into transverse or longitudinal folds. in mice and rats the folds of the proximal colon have a distinctive spiral orientation. the mucosa is lined by a single layer of three cell types: absorptive cells, goblet (mucous) cells and neuroendocrine cells. there is a zone of proliferation in each gland and cells either migrate outwards and differentiate into absorptive or goblet cells or remain in the basal part of the gland as secretory cells. the turnover time of the absorptive and outer goblet cells is 4.6 days, that of the deep crypt secretory cells 14 to 21 days (karam 1999) . aggregates of lymphoid cells may be found in the mucosa and submucosa of all parts of the large intestine, but these are not limited to the antimesenteric aspect as in the small intestine. the mucosa in both small and large intestines is supported by the connective tissue of the lamina propria and the continuous muscle layer of the muscularis mucosa. beneath the muscularis mucosa is the relatively loose connective tissue of the submucosa which contains larger blood vessels, lymphatics and nerves and the tunica muscularis (also known as muscularis propria or muscularis externa). the external surface, except for the terminal rectum, is covered by the serosal membrane. the rectum also is enclosed by a notably thicker tunica muscularis. the enteric nervous system in the intestine is in the form of the submucosal and myenteric plexi, the latter being located between the longitudinal and circular layers of the tunica muscularis. the primary function of the small intestine is to complete the chemical digestion of food by pancreatic enzymes and bile salts and allow for efficient absorption of the products of digestion. the primary functions of the large intestine are to allow for further digestion by fermentation in the cecum, absorption of water, and temporary storage of waste material in the rectum before defecation. disease processes or test articles that interfere with these processes will often produce diarrhea. less commonly muscle function is compromised leading to impaction of rectum and colon and clinical constipation. the mucosa of the small intestine is a site of high enzyme activity and conjugation reactions and consequently orally administered test articles may be metabolized, activated or deactivated in the intestine. the galt plays a primary role in the defense of the body against invasion by micro-organisms in the food and those normally resident in the lumen. the small intestine is the primary site for absorption of test articles given orally so it must be carefully evaluated in safety evaluation studies. due to variations in morphology along the length of both small and large intestine it is important there is consistent sampling at necropsy and consistent orientation on the slide. the goreni trimming guide (website.reni.item. fraunhofer.de/reni/trimming) specifies the following sites for routine examination: 1) duodenum: 1 cm distal to the pyloric sphincter; 2) jejunum: central section; 3) ileum: 1 cm proximal to cecum; 4) cecum; 5) colon: central section; and 6) rectum: 2 cm proximal to the anus. for investigative studies involving induction of intestinal tumors, the entire intestine can be opened, lesion location and size recorded and individual tumors may be fixed for sectioning in additional to the 6 standard sections. alternatively the whole length of the intestine can be fixed using the swiss roll method. however, opening the intestine and washing away contents with normal saline brings the risk of damaging the delicate mucosa which degenerates quickly due to post mortem autolysis; tissue orientation may be less reliable during subsequent tissue trimming and blocking using this method. in addition, it is important to note the amount and quality of ingesta/digesta present in the segments of intestine sampled for histological examination as this may affect the height of the mucosa. attempts should be made at necropsy to sample peyer's patches (which are visible macroscopically) from the jejunum or ileum so that microscopic evaluation of galt can be undertaken on the routine section of small intestines. toxicological evaluation of the intestine should also include examination of the tunica muscularis and the submucosal and myenteric nerve plexi which occasionally show changes related to test article administration. ectopic tissue ( figure 99 ) pathogenesis: presence of pancreatic cells in the mucosa or submucosa. • normally formed pancreatic acini in mucosa or submucosa. islets of langerhans may also be present. • present near the site of mesenteric attachment. • none. comment: presence of ductular (biliary or pancreatic) tissue in the duodenal wall is a normal finding in the anterior duodenum (shackelford and elwell 1999) . cyst, squamous ( figure 100 ) pathogenesis: presence of a cyst(s) lined by squamous cells in the mucosa, muscularis or serosa. • cyst lined by keratinized stratified squamous epithelium. • lumen frequently contains keratinized material. • diverticulum, simple, cystic or diverticulum, atypical, cystic: lined by glandular epithelium; no keratin formation. comment: while squamous cysts are most likely to be of congenital origin they may potentially form secondary to derangement of the epithelium e.g. in an inflammatory process. squamous cysts been observed very occasionally in the muscularis of the colon and rectum of b6c3f1 mice (shackelford and elwell 1999) . they are thought to be associated with focal weakness or discontinuity of the muscularis resulting in herniation through the submucosa and mucosa to the serosa. if post-inflammatory extension is the suspected pathogenesis, the pathologist may choose to interpret the focal change as a diverticulum, rather than a congential cyst. atrophy, brunner's gland ( figure 101 ) pathogenesis: reduction in size and number of brunner's glands. • reduction in size and number of the acini that comprise the brunner's glands. • may be accompanied by an inflammatory reaction. • may be accompanied by fibrosis. • none. atrophy ( figure 102 ) pathogenesis: atrophy of the mucosa related to reduced mitotic capabilities. in the small intestine, there is a reduction in villus height and crypt length; in the large intestine there is a decrease in size and number of mucous cells leading to a reduction in mucosal thickness. • villi show reduced height but increased width. • reduced depth of crypts in small intestine and mucous glands in large intestine. • reduced mitotic rate and/or increased cell loss through apoptosis or necrosis. • epithelium loses normal regular arrangement and may be flattened, cuboidal or vacuolated in appearance. • dilatation: dilatation of the intestine by fluid or gas will reduce villous height and thickness of the large intestinal mucosa by mechanical means. • autolysis: preferential destruction and loss of cells at the tips of villi; no inflammatory reaction. • normal topography: villi overlying brunner's glands may be smaller/fewer than in neighboring areas of the duodenum. comment: villous atrophy can be induced by withdrawal of food, administration of antimitotic cytotoxic agents, secondary to chronic inflammation or following rotavirus infection (maekawa 1994; shackelford and elwell 1999; percy and barthold 2007; kramer et al. 2010; haschek et al. 2010 ). in addition, villous atrophy can be induced experimentally by hypophysectomy, thyroidectomy and in segments of surgically bypassed intestine (greaves 2012) . villous atrophy may be accompanied by other degenerative changes e.g. lipidosis (murgatroyd 1980) . villous and mucosal atrophy without damage to the underlying lamina propria or to the proliferative cell population is reversible. however, if there is a sustained effect on proliferation e.g. with cytotoxic agents, the atrophy will be followed by erosion/ulceration, hemorrhage and secondary infection. mucosal atrophy induced by agents that interfere with the spindle apparatus may also show nuclear abnormalities including karyomegaly. hypertrophy (figures 103 and 104) synonyms: hyperplasia mucosa diffuse pathogenesis: increased in height of villi and depth of crypts. • increased length of villi in the small intestine. • increased mucosal thickness in the large intestine. • increased mitotic activity may be observed. • artifacts: villi are longer in the duodenum compared to jejunum and ileum, so inconsistency of sampling at necropsy could lead to an erroneous diagnosis. comment: increased mucosal thickness or villous length reflects hyperplasia of all cellular elements. villous hypertrophy has been induced by test articles (e.g. alloxan, polychlorinated aromatics, thyroid mimetic test articles), and by hormonal changes e.g. lactation and hyperthyroid states (bertram et al. 1996; greaves 2012) . villous length can also be influenced by nutritional factors e.g. amount of dietary fiber (burkhardt et al. 1998) . pathogenesis: reduction in paneth cell granules and/or loss of paneth cells in small intestine. differential diagnosis • none; however, it is necessary to verify the location of the intestinal specimen because paneth cell density varies based on topography with considerably fewer cells present in the duodenum. comment: recent literature identifies paneth cells as particularly sensitive targets of endoplasmic reticulum stress responses and disruption of their function is a risk factor in inflammatory bowel disease (ouellette 2010) . reduction in paneth cells is observed in norovirus infection in certain transgenic mice strains (cadwell et al. 2010) . pathogenesis: enlargement of paneth cells in small intestine. • hypertrophic paneth cells. • may accompany villous atrophy. • paneth cell hyperplasia may also be present. differential diagnosis • none. comment: hypertrophy and hyperplasia of the paneth cells, accompanying villous atrophy, were induced experimentally by surgical isolation of ileal loops (keren et al. 1975) . pathogenesis: presence of paneth cells in the large intestinal mucosa. differential diagnosis • none. comment: in colitis induced by the administration of dextran sodium sulphate and 1,2-dimethylhyadrazine, paneth cell metaplasia occurred 4 weeks after induction of inflammatory lesions (imai et al. 2007 ) paneth cells showed increased expression of ß-catenin (imai et al. 2007 ). paneth cell differentiation is also seen in tumors that develop in this model. accumulation of intracytoplasmic lipid in villous epithelial cells has been reported with several xenobiotics, including glucose transport inhibitors, puromycin, ethionine and vanadium compounds which alter lipid synthesis or transport (murgatroyd 1980; greaves 2012; imura et al. 2013) . in addition, lipid droplets were described where an ester of erythromycin was administered and the compound appeared to be absorbed unhydrolysed and converted to chylomicron-like droplets which accumulated in macrophages (visscher et al. 1980) . a fine cytoplasmic vacuolation, resulting in foamy appearance, may give the suspicion of phospholipidosis. in such cases, the modifier "foamy" should be used. if the change reflects phospholipidosis, increases in size and number of lysosomes can be identified by histochemistry (for alkaline phosphatase), immunostains (for lysosomal membrane proteins, e.g. lamp-2) or by transmission electron microscopy. with the later technique, concentric multilaminated structures (myeloid bodies/lamellar bodies) are observed in the lysosomes. the term phospholipidosis should only be used when confirmation by one of these techniques has been achieved. in the intestines, changes due to phospholipidosis are most commonly observed in macrophages, but epithelial cells, myocytes and neuronal ganglia can also be affected depending on the particular cellular distribution of the test article (reasor et al. 2006) . affected cells will probably be present in other organs and tissues of the body, but the intestines are likely to be one of the more severely affected sites following oral administration of a phospholipidosis-inducing test article (mazue et al. 1984) . the intestines can also be affected following intravenous administration of such test articles depending on the tissue distribution of the test article in the body. the classical definitive diagnostic feature is the identification of lysosomal lamellar bodies by electron microscopy, but immunohistochemical staining for lysosomal membrane proteins can clearly distinguish lysosomes from neutral fat vacuole membranes stained immunohistochemically for adipophilin (obert et al. 2007 ). pathogenesis: degeneration/necrosis of brunner's glands. • degeneration and necrosis of epithelial cells. • disorganisation of gland acini with disruption of basement membranes. • glands may be dilated and lined with flattened, atrophic epithelial cells. • accompanied by an inflammatory reaction. • may be accompanied by reactive hyperplasia of the duodenal epithelium. • atrophy, brunner's glands: reduction in size and number of cells is present but necrosis and inflammation are absent. comment: degeneration/necrosis of brunner's glands in rats has been described following administration of receptor tyrosine kinase (rtk) inhibitors directed against vascular endothelial growth factor (vegf) (inomata et al. 2014) . degeneration and necrosis of the glands is followed by disruption of the basement membrane leading to dilation of the glands and a neutrophilic inflammatory reaction. in the most severe lesions, inflammation extends into the surrounding mucosa and muscle layers. with more prolonged administration, dilated brunner's glands lined with flattened atrophic epithelium are observed and these may form cystic spaces which can extend into the muscle layers. a mixed neutrophilic and lymphocytic inflammatory reaction with fibrosis and reactive hyperplasia of neighbouring duodenal epithelium accompanies these brunner's gland changes after prolonged administration. lymphangiectasis ( figure 110 ) pathogenesis: lacteals are dilated with lipid. • dilated lacteals are present in the villi. • subcapsular sinuses of the mesenteric lymph node may also be dilated. • may be accompanied by minimal vacuolation of epithelial cells and/or macrophages in the lamina propria. differential diagnosis • none. comment: lymphangiectasis may be primary, due to a metabolic alteration affecting the formation of chylomicrons or transit of lipid in the lacteals, or secondary to blockage or inflammatory conditions e.g. neoplasia, fibrosis, granulomatous infections. lymphangiectasis in the small intestine due to the accumulation of large lipid droplets was described in rats administered indole-3-carbinol, possibly due to an impairment of lipid transport in the lacteals (boyle et al. 2012) . although there were vacuolated macrophages and giant cells in the lamina propria, the epithelial cells were unaffected (boyle et al. 2012) . pathogenesis: distrubance of cell division (cytokinesis), e.g. after infection by enterotropic strains of mouse hepatitis virus (mhv), a coronavirus. • formation of enterocyte syncytia (balloon cells occasionally containing eosinophilic intracytoplasmic inclusions). • villous atrophy and mucosal necrosis also present, particularly in young mice. differential diagnosis • none. comment: epithelial syncytia are common in mhv infection and mhv infection should be considered as an etiological agent when epithelial syncytia are observed. with enterotropic mhv strains lesions are limited to the intestines and are generally located in ileum, cecum and proximal colon (percy and barthold 2007; shackelford and elwell 1999) . lesions are most severe in young mice and are limited to syncytia in adult mice. compensatory hyperplasia of the intestinal epithelium develops in mice that survive the infection. with respiratory strains, enteric lesions are rare with infection largely restricted to galt. pathogenesis: gene regulated, energy dependent process leading to formation of apoptotic bodies which are phagocytosed by adjacent cells. • single cells or small clusters of cells. • necrosis, mucosa: the morphologic features of cell death clearly fit with necrotic diagnostic criteria (cells and nuclei swollen, pale cytoplasm etc.). • apoptosis/necrosis, mucosa: both types of cell death are present and there is no requirement to record them separately or a combined term is preferred for statistical reasons. the term is also used when the type of cell death cannot be determined unequivocally. • erosion/ulcer: focal or focally extensive penetration of the mucosa, either partial (erosion) or full to the muscularis mucosa (ulcer). the approach for the nomenclature and diagnostic criteria of cell death applied here is based on a draft recommendation of the inhand cell death nomenclature working group. apoptosis is not synonymous with necrosis. the main morphological differences between these two types of cell death are cell shrinkage with nuclear fragmentation and tingible body macrophages in apoptosis versus cell swelling, rupture and inflammation in necrosis; however, other morphologies (e.g. nucelar pyknosis and karyorrhexis) overlap. in routine h&e sections where the morphology clearly represents apoptosis or single cell necrosis, or whereby special procedures (e.g. tem or ihc for caspases) prove one or the other, individual diagnoses may be used. however, because of the overlapping morphologies, necrosis and apoptosis are not always readily distinguishable via routine examination, and the two processes may occur sequentially and/or simultaneously depending on intensity and duration of the noxious agent (zeiss 2003) . this often makes it difficult and impractical to differentiate between both during routine light microscopic examination. therefore, the complex term apoptosis/necrosis may be used in routine toxicity studies. a differentiation between apoptosis and single cell necrosis may be required in the context of a given study, in particular if it aims for mechanistic investigations. transmission electron microscopy is considered to represent the gold standard to confirm apoptosis. other confirmatory techniques include dna-laddering (easy to perform but insensitive), tunel (false positives from necrotic cells) or immunohistochemistry for caspases, in particular caspase 3. these techniques are reviewed in detail by elmore (2007) . some of these confirmatory techniques detect early phases of apoptosis, in contrast to the evaluation of h&e stained sections, which detects late phases only; overall interpretation of findings should take into account these potential differences. thus, low grades of apoptosis may remain unrecognized by evalu-ation of h&e stained slides only. certain studies may require the consideration of forms of programmed cell death other than apoptosis which requires specific confirmatory techniques (galluzzi et al. 2012) . synonyms: oncotic cell death; oncotic necrosis; necrosis pathogenesis: unregulated, energy independent, passive cell death with leakage of cytoplasm into surrounding tissue and subsequent inflammatory reaction; may be induced by direct contact with a test article after oral uptake/administration. • cells swollen with pale eosinophilic cytoplasm. • loss of nuclear basophilia, pyknosis and/or karyorrhexis that affects aggregates of cells. • in more severe cases, there may be detachment of the epithelium from the submucosa. • typically, the presence of degenerative cells is a component of necrosis. • minimal or slight inflammatory cell infiltrates may be present as a feature of necrosis. single cell • only single cells affected. • apoptosis: the morphologic features of cell death clearly fit with apoptosis (cell and nucleus shrunken, hypereosinophilic cytoplasm, nuclear pyknosis etc.) and/or special techniques demonstrate apoptosis. • apoptosis/necrosis, mucosa: both types of cell death are present and there is no requirement to record them separately or a combined term is preferred for statistical reasons. the term is also used when the type of cell death cannot be determined unequivocally. • atrophy, mucosa: reduced thickness of all layers of the squamous epithelium or, in severe cases, absence of basal germinative layers; usually no cellular degeneration or necrosis. • erosion/ulcer: focal or focally extensive penetration of the mucosa, either partial (erosion) or full to the muscularis mucosa (ulcer). comments: epithelial necrosis usually develops into erosion/ ulcer and in the past has frequently been recorded as such. a separate recording of necrosis as the initial event in this process is recommended when the structure of the mucosa is still intact. synonym: cell death pathogenesis: unregulated, energy independent, passive cell death with leakage of cytoplasm into surrounding tissue and subsequent inflammatory reaction (single cell necrosis) and/or gene regulated, energy dependent process leading to formation of apoptotic bodies which are phagocytosed by adjacent cells (apoptosis). • both types of cell death are present and there is no requirement to record them separately or a combined term is preferred for statistical reasons. • the type of cell death cannot be determined unequivocally. • apoptosis: the morphologic features of cell death clearly fit with apoptosis (cell and nucleus shrunken, hypereosinophilic cytoplasm, nuclear pyknosis etc.) and/or special techniques demonstrate apoptosis and there is a requirement for recording apoptosis and necrosis separately. • necrosis, mucosa: the morphologic features of cell death clearly fit with necrotic diagnostic criteria (cells and nuclei swollen, pale cytoplasm etc.) and there is a requirement for recording apoptosis and necrosis separately. • erosion/ulcer: focal or focally extensive penetration of the mucosa, either partial (erosion) or full to the muscularis mucosa (ulcer). comments: necrosis and apoptosis are not always readily distinguishable via routine examination, and the two processes may occur sequentially and/or simultaneously depending on intensity and duration of the noxious agent (zeiss 2003) . this often makes it difficult and impractical to differentiate between both during routine light microscopic examination. in these cases, the combined term apoptosis/necrosis may be used. it is recommended to explain in detail the use of this combined term in the narrative part of the pathology report. erosion/ulcer (figure 114 and 124) pathogenesis: localized loss of mucosa with either partial mucosal penetration (erosion) or with full penetration of the mucosa to the muscularis mucosa (ulcer). • epithelial cells are necrotic or absent but muscularis mucosa is intact (erosion). • epithelial cell are necrotic or absent and muscularis mucosa has been destroyed (ulcer). • submucosal edema may be present, particularly with ulceration. • hemorrhage and hemosiderin may be present, particularly with ulcers. • epithelial cells adjacent to an erosion or ulcer may be necrotic, cuboidal to flat or basophilic. • inflammatory cell infiltration is present, except in recently formed lesions. • chronic lesions will show chronic inflammatory cell infiltration, granulation tissue, fibrosis and regenerative epithelial hyperplasia which results in irregular, cystic glands in the mucosa and submucosa. • artifact: mechanical loss of mucosa during necropsy; plane of section related to presentation of tissue in block. • autolysis: preferential destruction and loss of cells at the luminal surface; no inflammatory reaction. the most likely cause of erosions and ulcers in immunocompetent barrier maintained rodents is the administration of xenobiotics (greaves 2012) . acute erosion and ulceration of the distal small intestine has been described following administration of non-steroidal anti-inflammatory agents, the newer inhibitors of cyclooxygenase 2 (cox-2) and cytotoxic anticancer drugs, whereas ionizing radiation produces acute lesions followed by fibrosis and arteriolar sclerosis (langberg and hauer-jensen 1996; haschek et al. 2010) . a variety of agents e.g. cysteamine, proprionitrile and 1-methy-4-pheny-1,2,3,6-tetrahydropyridine (mptp) are capable of producing chronic ulcers in the duodenum possibly by altering gastric acid secretion, bicarbonate production and its delivery to the duodenum, changes in duodenal motility and/or dopamine inhibition (szabo and cho 1988) . some viral and bacterial infections may induce erosion and ulceration, particularly in immunodeficient mice or rats (haines et al. 1998 ). erosion/ulcer usually is the result of epithelial necrosis. because of its unique topography adjacent to the lumen and often involving multiple organ layers, which uniquely affects the progression and resolution of the lesion (e.g. margin hyperplasia, granulomatous tissue, inflammation), it is useful to record erosion/ulcer separately from epithelial necrosis. pathogenesis: extracellular deposits of polypeptide fragments of a chemically diverse group of glycoproteins, in various tissues including intestinal mucosa. • deposition of pale amorphous, homogenous eosinophilic material. • extracellular location in connective tissue and/or blood vessel walls. • green birefringence with congo red stain when viewed under polarized light. • hyaline connective tissue: congo red negative. • fibrinoid necrosis of blood vessels: other evidence of tissue damage in section. comment: amyloidosis is extremely rare in rats but common in some strains of mice in which the lamina propria of the intestinal villi is a common site for deposition of amyloid. in susceptible strains of mice a number of factors including sex, diet, housing conditions, stress, endocrine status and microbiological status can influence the occurrence of amyloidosis (korenaga et al. 2004; lipman et al. 1993) . for as yet unidentified reasons the previously susceptible cd-1 strain in europe now rarely develops amyloidosis. the characteristic morphologic appearance and location of amyloid in h&e sections that is usually adequate to make this diagnosis. confirmation of the deposits as amyloid can be accomplished with light microscopy using special stains such as congo red. amyloid appears apple green under polarized light with this stain. the term "hyaline" should be used in case of uncertainties about the nature of extracellular deposits hyaline material in h&e sections and the unavailability of a congo red stain. hyaline is also the appropriate term for homogenously eosinophilic extracellular deposits that react negative with congo red. mineralization ( figure 116 ) pathogenesis: deposition of mineral in muscle, fibrous or elastic tissues secondary to necrosis (dystrophic mineralization) or hypercalcemia (metastatic mineralization). • mineral may be found in muscle layers, blood vessels, basement membranes and/or in the interstitium of mucosa and submucosa. • amorphous basophilic material. • mucosal mineralization can be limited to small focal deposits. • artifact: hematoxylin stain precipitate. • bacteria: colonies of bacteria may be confused with mineral due to their basophilic staining. ante mortem bacterial invasion will be accompanied by necrosis and usually inflammation; postmortem bacterial invasion will be accompanied by autolysis. • osseous metaplasia: osteoid present. comment: presence of mineral can be confirmed with von kossa or alizarin red stains but these are rarely needed to make a diagnosis. diffuse superficial mineralization of the colonic and rectal mucosa has been observed following administration of xenobiotics that decrease peristalsis and cause fecal retention (gopinathet al. 1987) . multifocal or diffuse mineralization occurs secondary to chronic renal disease in rodents and in situations where serum calcium and/or phosphate balance are perturbed, but the intestines are not as susceptible as the glandular mucosa of the stomach. (figure 117 ) pathogenesis: presence of osteoid. • presence of mineralized or non-mineralized bone matrix. • mineralization: osteoid absent. • sarcomas: osseous metaplasia may be present in stroma of sarcomas. comment: osseous metaplasia has been described in the submucosa and lamina propria of old rats in association with chronic inflammation, ulceration or regenerative hyperplasia (elwell and mcconnell 1990; maekawa 1994; bertram et al. 1996) . dilatation ( figure 118 ) synonyms: dilation, megaileus; cecomegaly; megacolon pathogenesis: dilatation of the lumen due to a defect in neuromuscular function. • dilatation of the lumen is observable macroscopically. • mucosa, submucosa and muscularis are thin due to stretching. • degeneration of muscle fibers and ganglia cells in the nerve plexus may be present. • meteorism: dilatation of the lumen due to the accumulation of air occurs as the result of mouth breathing and consequent aerophagia follows obstruction of nasal turbinates and/or nasopharynx in rodents as they are obligate nose breathers; the stomach will be more severely affected than the intestines. • obstruction: dilatation of the lumen due to simple mechanical obstruction e.g. proximal to a tumor. comment: megaileus with inflammation (megaileitis) is often present in rats with tyzzer's disease (infection with clostridium piliforme); the lesions are characterized by a necrotizing transmural ileitis with necrosis of the mucosa and muscularis, edema and infiltration primarily of mononuclear cells (percy and barthold 2007) . cecomegaly is observed following administration of some antibiotics, starches, polyols, lactose, fibers and vehicles which have an osmotic effect (newberne et al. 1988 ). megacolon as a result of constipation was induced in rats by a calcium channel antagonist; secondary mucosal necrosis, hemorrhage and inflammation were present in some rats (nyska et al. 1994) . tribromoethanol (avertin) administered intraperitoneally as an anesthetic has been associated with ileus in rodents (king and russell 2006) . figure 119 ) pathogenesis: invagination of a one portion of intestine into its immediate distal section ('telescoping'). • observable macroscopically. • invagination of a portion of intestine into its immediate distal section. • intestinal lumen proximal to the site of intussusception may be dilated whilst distally the lumen may be contracted and devoid of contents. • histologically ischemic necrosis, inflammation, congestion and hemorrhage will often be present in the involved segments; the degree of such changes will depend on the length of time that the intussusception has been formed. • adhesions may develop between the invaginating intussusceptum and the receiving intussuscipiens. • associated with intestinal tumors, especially polyps. • artifacts, post mortem invagination of the intestine: no reactive changes present. comment: intussusceptions are very rare in barrier maintained rodents but may occur in animals with intestinal tumors and rarely with high nematode infestations (percy and barthold 2007) . very occasionally an intussusception may be induced by a test article (gopinath et al. 1987 ). although intussusception is primarily a gross pathological term, it is recommended to use it also microscopically, as an unequivocal macroscopic diagnosis is not always possible. pathogenesis: eversion of rectum through the anus. • observable macroscopically. • eversion of rectum through the anus. • microscopic lesions of mucosal necrosis, hemorrhage, edema and inflammation. differential diagnosis • none. comment: rectal prolapses are rare in barrier maintained rodents but may occur secondarily to inflammatory conditions in the colon and rectum such as occur with citrobacter and helicobacter infection in mice, weakening of anal sphincter and with a high nematode load percy and barthold 2007; mcinnes 2012; miller et al. 2014 ). the diagnosis of prolape is usually possible by gross pathological examination. nevertheless it is recommended to confirm the gross observation histopathologically. infiltrate ( figure 123 ) • inflammation: other morphologic features of inflammation such as edema, hemorrhage, necrosis and/or fibroplasia will be present. • granulocytic leukemia: infiltration of neutrophil precursors and/or abnormal neutrophils will probably be present along with mature neutrophils; infiltration of similar neoplastic cells is likely present in other organs. • lymphoma: infiltration of monomorphic population of lymphocytes (often with atypical, increased and/or abnormal mitoses); infiltration of similar neoplastic cells is likely present in other organs. comment: the number of mononuclear cells is the lamina propria of the large intestine (particularly cecum) is variable in healthy animals and care must be taken not to 'over diagnose' infiltration of inflammatory cells. modifiers: type of inflammatory cell that represents the predominant cell type in the inflammation pathogenesis: infiltration of the lamina propria and/or submucosa with neutrophils (inflammation, neutrophil) or mononuclear cell (inflammation, mononuclear cell) or a combination (inflammation, mixed) with additional histological features of inflammation e.g. hemorrhage, edema, fibroplasia. diagnostic features • focal, multifocal or diffuse infiltration of cells in the lamina propria and/or submucosa. • presence of other histological criteria of inflammation e.g. hemorrhage, edema, fibroplasia. • infiltrate, inflammatory cell: other morphologic features of inflammation such as edema, hemorrhage, necrosis and/or fibroplasia will be absent. • granulocytic leukemia: infiltration of neutrophil precursors and/or abnormal neutrophils will probably be present along with mature neutrophils; infiltration of similar neoplastic cells is likely present in other organs. • lymphoma: infiltration of monomorphic population of lymphocytes (often with atypical, increased and/or abnormal mitoses); infiltration of similar neoplastic cells is likely present in other organs. features of specific bacterial and viral infections have been discussed in the previous sections. nematode and protozoa are dealt with separately below. pathogenesis: presence of protozoan or nematode parasitess in the intestinal lumen. • presence of cross sections of protozoa in large intestinal lumen; morphology will depend on whether ameboid, ciliate or flagellates are present. • inflammatory cell infiltration and/or edema may be present in lamina propria. nematodes • presence of cross sections of parasites in large intestinal lumen. • rarely submucosal granulomata may be present. • none. comment: it is now uncommon for intestinal parasites to be present in barrier-reared and maintained rodents. protozoa that have been reported in laboratory rodents include spironucleus muris, giadia muris and cryptosporidia muris, but infections are often subclinical especially in adults of immunocompetent strains (percy and barthold 2007) . the reader is referred to standard texts for a full list of intestinal protozoan that may be present in non-barrier maintained or wild rodents (percy and barthold 2007; baker 2007) . the most likely nematodes to be seen in the event of a barrier break down in a rodent unit are the pinworms syphacia muris, syphacia obvelata and/or aspicularis tetrapertera which inhabit the large intestine (percy and barthold 2007) . the reader is referred to standard texts for a full list of intestinal parasites that may be present in non-barrier maintained or wild rodents (percy and barthold 2007; baker 2007) . histogenesis: degenerative changes in neurons of the myenteric/submucosal plexi. differential diagnosis • none. the degenerative changes and inclusions have only been seen with flaviviral infections (ward, personal observation). viral antigen is abundant in these ganglion cells. edema and hemorrhage of the intestine are similar to those described for the upper digestive tract. other vascular lesions in the intestine and mesentery are dealt with in the inhand monograph on the cardiovascular system. diverticulum (figures 122, 129, 130 and 131) synonyms: cystic adenomatous hyperplasia; diverticulosis; herniated crypt; crypt herniation diverticulum, atypical, may have been identified as: atypical cystic hyperplasia; cystic hyperplasia with growth into the gastric wall; cystic adenomatous hyperplasia; herniation, atypical; "pseudoinvasion", atypical pathogenesis: usually caused by mechanical or biochemical disruption of intestinal wall structures resulting in loss of extracellular matrix integrity and internal projection of surface lining into deeper layers; may alternatively represent a congenital abnormality. • extension of crypts through muscularis mucosa, into submucosa and further in some cases. • morphology of epithelial lining is variable, ranging from single layer cuboidal or columnar cells to complete mucosa. • epithelium may show features of regeneration like increased basophilia, increased nucleus to cytoplasm ratio and gradual loss of polarity. • basement membrane integrity always maintained. • often accompanied by inflammation and regenerative / reparative processes. • may contain ingesta, hair, or other foreign material. cystic: • rounded cystic structures in lamina propria or deeper layers. • compression of surrounding structures, some degree of compression atrophy of the epithelium lining the cyst. • may contain mucus. • extension of atypical/dysplastic mucosal glands into or through muscularis mucosa, submucosa, and deeper in some cases. • hyperplastic atypical (dysplastic) epithelial cells forming cystic glands that may appear to be "invading" into deeper layers of the intestine. • basement membrane always intact. • adenoma: proliferation beyond the confinements of the intestinal mucosa. • adenocarcinoma: true infiltrative growth with loss of basement membrane integrity and often with a scirrhous stromal reaction. comment: these focal intestinal lesions are primarily formed by extensions of mucosal epithelium and do not involve evaginations of the other mural layers; as such they do not resemble diverticulosis in human colon, which may be focal, multifocal or diffuse. diverticula do not appear to be neoplastic, although they do create an increased risk of rupture and septic peritonitis. these structures are typically lined by a simple cuboidal mucus epithelium. it is not recommended to record "diverticulum" and its modifier as a separate finding, if it occurs in association with another proliferative process (e.g. hyperplasia, adenoma). small glandular diverticula lined by a single layer of cuboidal or columnar epithelium resemble "crypt herniation" of human gastrointestinal pathology (e.g. rex et al. 2012) . such herniation of crypts through the muscularis mucosa is interpreted as "pseudoinvasive" or "inverted" growth pattern. although the process-related term "crypt herniation" is common in literature (e.g. betton et al. 2001) , the term "diverticulum" is considered to better fit to the inhand prin-ciple to use descriptive terms and to avoid process-related terms (mann et al. 2012 ). spontaneous and induced intestinal diverticula occur more frequently in mice than in rats. they have been reported in the colon of cdx-2 knockout mice due to a developmental defect (tamai et al. 1999) . in this model they appeared as polyps with herniation and due to the congenital etiology the authors termed it hamatoma. atypical diverticula must be distinguished from genuine neoplastic invasion (e.g. loss of basement membrane and other signs of malignancy in the latter). they are associated with preexisting mucosal atypical hyperplasia/dysplasia as seen in specific experimental conditions, tumor-associated intestinal toxins and genetically-engineered mice. whereas they often do not progress to unequivocal adenocarcinoma, atypical glandular and cellular features are consistent with neoplastic potential. atypical diverticula may be associated with atypical hyperplasia and adenoma, but usually not with adenocarcinoma. as noted above for the simple diverticula, they should not be recorded as a separate diagnostic entity when associated with another proliferative process. synonyms: hyperplasia, regenerative synonyms for atypical hyperplasia may include dysplasia, gastrointestinal intraepithelial neoplasia (gin, mice), early neoplastic lesion, atypical crypts, dysplastic crypts, dysplastic foci, or aberrant crypt foci. histogenesis: enterocytes of the intestinal mucosa. • focal or diffuse process accompanying epithelial damage; no evidence of compression. • villous and glandular architecture is not altered by the proliferative process itself but in regenerative hyperplasia may have been altered by the initiating event (degeneration and necrosis). • focal penetration by glandular diverticula into lamina propria or deeper layers may be present but basement membrane is always intact. • no cellular or nuclear atypia. • focal lesion of the duodenum in aging mice. • smooth luminal surface lacking intestinal villi. • hyperplastic crypts may often be interspersed between hyperplastic brunner's glands. • goblet cells may be reduced or increased. • crypt dilatation and diverticula may be present in larger lesions. • frequently accompanied by submucosal edema and inflammatory cell infiltration. propria or deeper layers may be present but basement membrane is always intact. • cellular atypia and pleomorphism as evidenced by hyperchromatism with increased cytoplasmic basophilia and lost polarity, nuclear pleomorphism, increased n/c ratio, hyperchromasia, increased mitotic activity. goblet cell: • increased proportion of goblet cells, predominantly in crypts; crypts exhibit normal length or are enlaged. differential diagnosis • adenoma: exophytic or endophytic growth pattern extending beyond the confinement of the mucosa; compression of adjacent tissue if growth pattern is not exclusively polypoid. comment: reactive or reparative (regenerative) hyperplasias often follow injury due to chemicals, infectious agents and irradiation. focal or diffuse reactive hyperplasia is seen in the cecum, colon and rectum of immuno-compromised mice following helicobacter infection. diffuse reactive hyperplasia is observed in the distal colon of mice infected with the gram negative bacterium citrobacter freundii (transmissible murine colonic hyperplasia). occasionally the transverse and ascending colon and the cecum are affected. in such cases of hyperplasia the surface epithelium is covered by myriads of cocobacillary bacteria. the lamina propria shows diffuse leukocytic infiltration. diffuse non-reactive hyperplasia of the mucosa with increased height of the villi and depth of the crypts is synonymous with hypertrophy, mucosa. hypertrophy is the recommended term for such lesions. an increased proportion of goblet cells has been reported in the literature as both goblet cell hyperplasia or metaplasia. we recommend the term hyperplasia, as goblet cells are normal constituents of the intestinal mucosa. hyperplasia of goblet cells has been induced in the small and large intestine of rats by treatment with a gamma secretase inhibitor (aguirre et al. 2014). focal hyperplasia of the proximal duodenum, adjacent to the pylorus, occurs in aged mice has been referred to in the literature as avillous hyperplasia (facinni, et al. 1990; mc-innes 2012) . it may be visible macroscopically as a thickened plaque and is characterised by hyperplasia of all cells in the epithelium including brunner's glands, but with loss of villi. the epithelium can show erosion/ulcer formation with associated inflammation. in the more severe cases dilated mucosal glands can extend towards the submucosa and be interspersed with dilated brunner's glands. focal atypical hyperplasia is a frequent finding in mice treat-ed with test articles which affect the intestinal mucosa or in genetically-engineered mice. under such conditions they often occur in multiple foci. it has not been described in untreated wild type animals so far. the term gin or dysplasia has been used in genetically-engineered mice. other terms occasionally used but not preferred as synonyms are microadenoma, carcinoma in situ, intra-epithelial carcinoma. dysplasia is often used for similar lesions in human colon (bosman et al. 2010 ). colon crypts exhibiting focal atypia ("aberrant crypt foci") can be demonstrated by the examination of methylene blue stained colon whole mounts with a dissecting microscope (raju 2008) . they are usually not seen grossly until 1 mm in size. atypical hyperplasia may be low-grade to high-grade, but this grading is usually not included in the diagnosis. under conditions of experimental intestinal carcinogenesis, focal atypical hyperplasia is considered as a preneoplastic lesion that may progress to an adenoma or adenocarcinoma. rectal prolapse often due to helicobacter sp or citrobacter sp infection, often shows severe degrees of simple hyperplasia, atypical hyperplasia and the appearance of a invasivelike growth pattern. these cases should be recorded as rectal prolapse. histogenesis: cells of brunner's glands. • brunner's glands cells are often hypertrophic with more extensive cytoplasm, larger nuclei, or both. • glands are more branched and coiled, compared with normal glandular structure. • brunner's glands cells and underlying stroma may form pedunculated structures within the spaces of cystic glands. • cellular polarity is maintained. • cells are tall columnar, with extensive, poorly-stained, or somewhat eosinophilic-stained cytoplasm with dense, basally-located nuclei. • glands may expand into the underlying circular muscle layer, but basement membrane integrity always maintained. • diverticulum: localized and discrete penetration of intestinal crypts into lamina propria or deeper layers with maintained basement membrane integrity and surrounded by pre-existing mature tissues lacking a desmoplastic stromal response. • carcinoma, brunner's glands: effacement of brunner's glands structures, prominent desmoplasia; invasive growth. comment: brunner's glands hyperplasia has been observed in transgenic mice (hemizygous rash2) after dosing for one month with a receptor tyrosine kinase inhibitor that targets multiple cellular kinase receptors, including those of vascular endothelial growth factor (vegf), platelet-derived growth factor (pdgf), stem cell factor receptor (kit), fmslike tyrosine kinase 3 (flt-3) and glial cell line-derived neurotrophic factor rearranged during transfection (ret). progression from preneoplastic hyperplasia to carcinoma/ adenocarcinoma can occur. to appropriately capture the area of interest in the rodent, it is important to retain the gastroduodenal junction in a manner that allows a comprehensive evaluation of the area of interest and prevents folding of the stomach and gastroduodenal region that can obscure brunner's glands. this can be accomplished by opening the stomach along the greater curvature, leaving the duodenum attached, and pinning the tissue flat for fixation. synonyms: squamous metaplasia. histogenesis: colon or rectum epithelium. • focal, multifocal or diffuse • replacement of normal epithelium with squamous epithelium. • frequently associated with chronic inflammation. • carcinoma, squamous cell: invasion, areas with moderate to poor differentiation or anaplasia, ususally prominent mitoses. • extension of anal mucosa into rectum: usually in proximity to the anus and without concomitant inflammatory reaction. comment: squamous metaplasia is often seen in chronic colitis models in mice, usually in the dextran sulfate model. squamous cell carcinomas have not been reported to arise in the lesions (seamons et al. 2013 ). adenoma (figures 138, 139 • branching villi or tubular crypt proliferation can be present. • squamous metaplasia and focal mineralization may be present. • may be associated with glandular or cystic diverticula, but basement membrane always intact. • one cell type is predominant and goblet cells are absent or reduced in number. • the epithelium may contain foci of varying degrees of dysplasia. • signs of cellular atypia / dysplasia include cellular basophilia, loss of cellular polarity, hyperchromatic and pleomorphic nuclei, and nuclear stratification. • hyperplasia, atypical: circumscribed proliferation with varying degree of atypia, but limited to the confinements of the intestinal mucosa. • diverticulum: extension of crypts through muscularis mucosa, into submucosa and further in some cases. morphology of epithelial lining is variable, ranging from single layer cuboidal to columnar cells to complete mucosa. epithelium may show features of regeneration like increased basophilia, increased nucleus to cytoplasm ratio and gradual loss of polarity. often accompanied by inflammation and regenerative / reparative processes. • adenocarcinoma: true invasion by individual, groups or areas of tumor cells, with or without a stromal response. the term "polyp" was avoided, because of implied uncertainty about the neoplastic nature of this proliferative lesion. benign neoplasms may develop dysplastic foci and evolve into malignant neoplasms. chemicals causing benign intestinal tumors often also cause intestinal adenocarcinomas (chandra et al. 2010; pandiri et al. 2011; ward 1974) . adenomas, as well as atypical hyperplasia, exhibit some degree of dysplasia and distortion of pre-existing architecture. macroscopic visibility has been suggested as a differential diagnostic criterion by some authors. this criterion is not used in the context of this description of histologic criteria. any lesion exhibiting a papillary or polypoid growth pattern is an adenoma, irrespective whether it was grossly visible or not. an intra-epithelial small focus (compatible with atypical hyperplasia) with cytological characteristics seen in adenomas should not be diagnosed as an adenoma. the frequency of spontaneous intestinal adenomas varies with the intestinal segment. incidences up to 51% are reported for the duodenum and anterior part of the jejunum (hare and stewart 1956) . in the other intestinal segments the incidence of adenomas is below 5%. however, the detection frequency is highly dependent on the mode of macroscopic examination of the intestines, e.g. random blocks, naked eye inspection of mucosal surface, transillumination or swiss roll technique. the determination of the malignancy status of a papillary or polypoid intestinal tumor requires a good fixation and trimming protocol, to obtain an optimal section through the middle of the tumor with its connecting area to the normal intestine. if there is no connection to the intestine, it is impossible to determine if invasion is present. the cytology of a non-invasive adenoma may include high grade dysplasia. in most rat and mouse chemically-induced or genetically-engineered tumor models, true invasion of the stalk is unusual but may occur commonly in other models. adenocarcinoma (figures 143, 144, 145 • diverticulum: localized and discrete penetration into lamina propria or deeper layers with maintained basement membrane integrity and surrounded by pre-existing mature tissues lacking a scirrhotic stromal response. • hyperplasia atypical and adenoma: may be associated with glandular or cystic diverticula, but basement membrane always intact. ▪ rectal prolapse (mice): associated with helicobacter sp or citrobacter sp infection in mice; hyperplastic epithelium extending as diverticula through muscularis mucosa and into submucosa, with associated inflammation. comment: an intestinal adenocarcinoma may arise de novo from normal epithelium, but more commonly from a preneoplastic lesion such as atypical hyperplasia or from an ad-enoma. the histogenesis, morphology and biology of tumor progression often depend on the etiology, i.e. the chemical carcinogen or the kind of genetic change in a gem model. in contrast to humans, spontaneous rodent adenocarcinomas show a predilection for the small intestine, especially the jejunum and ileum, rather than the large intestine. the descriptions available indicate that the morphology of spontaneous lesions does obviously differ from the induced ones and that the etiology may determine the morphologic phenotype. in addition, induced adenocarcinomas have their preferred localization in the distal colon and show high invasiveness combined with a low tendency to metastasize. helicobacter sp has been shown to be a colon co-carcinogen in some experimental mouse models involving gem or immunodeficient mice (erdman and poutahidis 2010) . inflammatory mouse models of colon adenocarcinoma are often scirrhous and mucinous (erdman and poutahidis 2010; kosa et al. 2012) . metastases are often associated with a specific method of tumor induction. in rats, the mucinous adenocarcinoma induced by some carcinogens commonly metastasizes, predominantly to the lung. mucinous intestinal adenocarcinomas are often the most malignant type of intestinal tumor in rats and mice . histogenesis: cells of brunner's glands. • there is effacement of brunner's glandular structures and the gastroduodenal junction. • desmoplasia is prominent and can be the most dominant feature. • glandular structure is diminished or completely lost. • the mitotic index is usually low. • there is moderate pleomorphism of brunner's glandular cells. • cells have well-stained cytoplasm that may be eosinophilic, amphophilic or basophilic. • attenuated glandular cells may line cystic areas that contain mucus. • invasion through the muscularis mucosa and/or through the circular and longitudinal muscles, as well as the serosa, can be extensive. the basement membrane of the glands is breached or not evident. • hyperplasia, brunner's glands: glandular architecture may be slightly altered, but still recognizable; cell polarity maintained; glands may penetrate into the underlying circular muscle layer, but basement membrane maintained. • diverticulum: localized and discrete penetration of intestinal crypts into lamina propria or deeper layers with maintained basement membrane integrity and surround-ed by pre-existing mature tissues lacking a desmoplastic stromal response. comment: brunner's glands carcinoma is a rare tumor, although a high occurrence can be induced with certain test articles. brunner's glands carcinoma has been observed in transgenic mice (cb6/f1/jic tg rash2 hemizygous) and sprague-dawley rats after dosing for 6 and 24 months, respectively, with a receptor tyrosine kinase inhibitor that targets multiple cellular kinase receptors (see brunner's glands hyperplasia; comment). the investigations in the transgenic model indicated that the rapid occurrence and high incidence of brunner's glands carcinoma is more consistent with altered transcriptional regulation rather than a genotoxic mechanism. knowledge of the site of the incipient lesion or the use of immunohistochemical methods are often required to diagnose the origin of the change because invasion of a gastric glandular cell or duodenal enterocyte carcinoma can have similar morphologic features to those of a malignant neoplasm of brunner's glands that has progressed extensively. histogenesis: smooth muscle cells from tunica muscularis. • intramural involvement. • interlacing bundles and whorls of uniform spindle cells arranged in criss-cross patterns of bundles. • nuclei typically blunt-ended or cigar shaped. • eosinophilic cytoplasm contains longitudinal myofilaments and perinuclear clear space and show immunoreactivity for desmin. • leiomyosarcoma: shows mitotic activity and cellular pleomorphism. histogenesis: smooth muscle cells from tunica muscularis. • intramural involvement. • similar to leiomyoma, but more pleomorphic and with mitotic activity. • necrosis, cystic change and mineralization are common features. • distant metastases. • leiomyoma: shows little or no cellular pleomorphism or mitotic activity. synonyms: interstitial cajal cell tumor, benign; gastrointestinal pacemaker cell tumor, benign histogenesis: specialized smooth muscle cells (i.e., interstitial cells of cajal) in the tunica muscularis or myenteric plexus. • well demarcated tumor, expansive growth. • cells may be arranged in bundles with storiform archi-tecture, myxoid areas may be present. • spindle, epithelioid, or pleomorphic cell morphology. indistinct cell borders and fibrillary cytoplasm. • spindle-or irregular-shaped nuclei. • typically cd117 positive (cytokine receptor encoded by c-kit). • gastrointestinal stromal tumor, malignant: infiltrative growth, distant metastases may be present. • leiomyoma: cd117 negative, desmin positive; interlacing bundles and whorls of uniform spindle cells arranged in criss-cross pattern; blunt-ended, fusiform nuclei. minimal nuclear pleomorphism; phosphotungstic acid-hematoxylin positive myofibrils. • leiomyoscarcoma: cd117 negative, desmin positive; interlacing bundles and whorls of uniform spindle cells arranged in criss-cross pattern; blunt-ended, fusiform nuclei with hyperchromasia, pleomorphism, and high mitotic index; phosphotungstic acid-hematoxylin positive myofibrils. • schwannoma: cd117 negative, s-100 positive; indistinct cell borders, elongate cells with eosinophilic cytoplasm; nuclei sometime arranged in palisades (i.e., antoni a pattern). comment: gist will generally not be easily differentiated from leiomyomas / leiomyosarcomas or other soft tissue tumors. however, immunohistochemical differentiation of the different tumor types, including gist, needs to be performed in case of imbalances in the incidence of smooth muscle tumors in a given study. cecal gist have been induced in gem mice over-expressing a mutant kit (nakai et al. 2008 ). the question arises whether conventionally classified stromal tumors (e.g., leiomyoma) previously were actually gist. synonyms: interstitial cajal cell tumor, malignant; gastrointestinal pacemaker cell tumor, malignant histogenesis: specialized smooth muscle cells (i.e., interstitial cells of cajal) in the tunica muscularis or myenteric plexus. • locally infiltrative growth pattern. • cells may be arranged in bundles with storiform architecture, myxoid areas may be present. • spindle, epithelioid, or pleomorphic cell morphology. indistinct cell borders and fibrillary cytoplasm. • spindle-or irregular-shaped nuclei. • typically cd117 positive (cytokine receptor encoded by c-kit). • distant metastases may be present. • gastrointestinal stromal tumor, benign: expansive growth pattern, no distant metastases. • leiomyoma: cd117 negative, desmin positive; interlacing bundles and whorls of uniform spindle cells arranged in criss-cross pattern; blunt-ended, fusiform nuclei. minimal nuclear pleomorphism; phosphotungstic acid-hematoxylin positive myofibrils. • leiomyoscarcoma: cd117 negative, desmin positive; interlacing bundles and whorls of uniform spindle cells arranged in criss-cross pattern; blunt-ended, fusiform nuclei with hyperchromasia, pleomorphism, and high mitotic index; phosphotungstic acid-hematoxylin positive myofibrils. • schwannoma: cd117 negative, s-100 positive; indistinct cell borders, elongate cells with eosinophilic cytoplasm; nuclei sometime arranged in palisades (i.e., antoni a pattern). comment: gist will generally not be easily differentiated from leiomyomas / leiomyosarcomas or other soft tissue tumors. however, immunohistochemical differentiation of the different tumor types, including gist, needs to be performed in case of imbalances in the incidence of smooth muscle tumors in a given study. cecal gist have been induced in gem mice over-expressing a mutant kit (nakai et al. 2008) . the question arises whether conventionally classified stromal tumors (e.g., leiomyoma) previously were actually gist. the salivary glands consist of major and minor glands and develop from the endodermal lining of the embryonal gut. the major salivary glands are grossly apparent and include the parotid, sublingual and submandibular glands. the minor salivary glands are microscopic and are distributed throughout the oral cavity encompassing the lingual (von ebner's glands around the vallate and foliate papillae, weber's glands at base of the tongue), sublingual, buccal, palatine, laryngeal and pharyngeal tissues. the submandibular gland matures with age and progress from rudimentary lobules of mesenchyme with ductular structures at birth to acinar development and complete maturation by about 30 days postnatally in rats (proctor and carpenter, 2007) . the sublingual salivary glands are mature at birth unlike the parotid and submandibular salivary glands. the major salivary glands are located on the ventral and lateral areas of the cervical region and extend to the base of the ear and are closely associated with the extraorbital lachrymal gland and mandibular lymph nodes. the largest of the three major salivary glands, the submandibular (mandibular or submaxillary) salivary gland is a tan, multilobular gland that is located in the ventral cervical region. it extends rostrally to the mandibular lymph nodes, caudally to the thoracic inlet, and is bordered bilaterally by the parotid glands. the sublingual glands consist of tan-brown bilateral single lobes located on the rostro-lateral surface of each of the submandibular glands and directly below the mandibular lymph nodes. the parotid glands are bilateral and consist of pink to off white, multiple flattened lobes extending from the submandibular gland ventrally, to the base of the ear dorsally, to the extraorbital lacrimal gland rostrally, and to the clavicle caudally. the parotid glands drain into the parotid duct while the submandibular and sublingual glands drain into the oral cavity via wharton's duct that terminates in small papillae near the incisors. the submandibular gland is a seromucinous (mixed) compound tubuloacinar gland. in the mouse it is predominantly seromucinous, where as, in the rat it is mostly mucinous with a small serous component (gresik, 1994; ozono et al. 1991; tucker, 2007) ). the submandibular gland is comprised of several adenomeres with large pyramidal mucous cells that are flanked basally by variable numbers of cresentric serous demilunes. the large pyramidal mucous cells have abundant vesicular basophilic cytoplasm and basally located nuclei and the cresentric serous demilunes have scant eosinophilic cytoplasm and hyperchromatic nuclei. the acinar secretions from the adenomeres empty into intercalated ducts lined by low cuboidal to columnar epithelial cells. few oncocytes are interspersed between the cuboidal epithelial cells of the intercalated ducts of aged rats. these cells are characterized by abundant granular eosinophilic cytoplasm (abundant mitochondria) and a central hyperchromatic nucleus. thin fusiform myoepithelial cells are located between the basal lamina and epithelial cells of acini and intercalated ducts (bogart, 10970; sashima, 1986) . only in the submandibular gland, the intercalated ducts continue into granular (convoluted) ducts. the granular ducts are lined by tall columnar epithelial cells with abundant eosinophilic granular cytoplasm. the granulation of these ducts is much more prominent in sexually mature males when compared to females (figures 156 and 157) . these ducts continue into striated (intralobular) ducts that are lined by a single layer of tall columnar epithelial cells with marked basal infoldings (hence, striated cells) and central to apically located nuclei. several of the striated (intralobular) ducts merge into interlobular excretory ducts that are lined by tall columnar epithelial cells with more apically located nuclei and prominent cytoplasmic striations. several interlobular excretory ducts continue into one main excretory duct that expands into a diverticulum lined by simple columnar epithelium and end as squamous epithelium at the oral surface. the sublingual gland is predominantly mucous secreting and has the largest acini. they are comprised of large pyramidal mucus cells with basally located nuclei and are infrequently capped by serous demilunes. the acinar secretions empty into intercalated ducts that in turn empty into striated (intralobular) ducts. the multiple striated ducts empty into the main excretory duct. the microscopic anatomy of the ducts is similar to their counterparts within the submandibular gland. the parotid gland contains serous acini, which are morphologically very similar to the exocrine pancreatic acini (wolff et al. 2002) . the acinar cells are pyramidal with broad basophilic base and tapered apices. the eosinophilic granular cytoplasm contains zymogen granules, the nuclei are basally located. the duct system of the parotid gland is similar to the submandibular gland. the minor salivary glands lack a true connective tissue capsules and are usually located within the submucosa or are interspaced between connective tissue stroma or muscle fibers. the glands are morphologically diverse and are organized into distinct serous (von ebner's glands), mucous (anterior and posterior buccal glands and minor sublingual glands) or mucous glands with serous demilunes (glossopalatal, palatal, and weber's glands, figure 155 ) glands (redman 2011) . the acini are organized into lobule-like structures and collections of secretory end pieces and ducts open onto the mucosal surface. the salivary glands secrete a hypotonic sero-mucinous saliva containing electrolytes and enzymes that aid in lubrication and partial digestion of food. the serous secretions contain water, electrolytes (rich in k + and hco3 -and poor in na + and clcompared to plasma), iga and enzymes (ptyalin (amylase), lysozyme, lipase) and the mucinous secretions contain water, and glycoproteins. the minor salivary glands contribute about 14% protein and 1% amylase of the total salivary secretions, as determined by pilocarpine stimulation (blazsek and varga 1999) . the salivary secretions are tightly regulated by the sympathetic (adrenergic) and parasympathetic (cholinergic) nervous system (garrett 1987) . most cell types within the submaxillary and parotid glands have both sympathetic and parasympathetic innervations while the sublingual glands have scant sympathetic innervation limited to striated ducts and blood vessels (garrett et al. 1991 ). the parasympathetic system and the α-adrenergic sympathetic system regulate the water and inorganic ion phase of the primary secretory fluid, whereas the β-adrenergic sympathetic system effects primarily the macromolecular (enzymes) phase. parasympathetic stimulation is mainly responsible for stimulation of the salivary acini resulting in copious secretion of saliva, however, the sympathetic stimulation does not inhibit salivary secretion but it modulates the saliva composition and causes salivary secretion to a lesser extent (proctor and carpenter 2007) . the sympathetic and parasympathetic stimulation of the myoepithelial cells surrounding the acini results in contraction of acini and directing the saliva into the ducts (tucker 2007) . surprisingly, it is the sympathetic stimulation but not parasympathetic stimulation that results in morphologically obvious depletion of storage granules within the acini (garrett et al. 1991) . β-adrenergic sympathomimetic agonists such as isoprenaline (selye et al. 1961 ) and terbutaline (sodicoff et al. 1980 ) first cause rapid depletion of secretory granules and later promotes re-synthesis of secretory material leading to acinar cell hypertrophy and hyperplasia. on the other hand, β-adrenergic antagonists like propranolol suppress salivary secretions and eventually cause acinar atrophy (fukuda 1968) . the sexual dimorphism of the submandibular gland salivary glands is dependent on testosterone levels (harvey 1952) . the increased granularity in males ( figure 156) is accompanied by an about 10 times higher aminotransferase activity compared to females that have very little if any ductal secretory granules ( figure 156 ) (hosoi et al. 1978 ). a typical approach for toxicological examination includes all the three grossly visible major salivary glands, i.e., submandibular, sublingual, and parotid gland. at necropsy, all three major salivary glands are removed in one piece, usually together with the mandibular lymph nodes. trimming of the salivary glands is then undertaken as specified in the rita trimming guide (website//reni.item.fraunhofer.de/reni/trimming): the salivary glands are trimmed longitudinally and through the largest surface. all three salivary glands should be present in the section. the typical approach may be varied according to study protocol requirements, e.g. determination of organ weights and tissues required for pathology examination. the microscopic minor lingual glands are routinely examined if the entire tongue, including its base, is removed at necropsy and trimmed longitudinally. the other minor salivary glands located within the oral cavity and pharynx are examined only occasionally during microscopic evaluation of these tissues. ectopic tissue (figures 158 and 159) pathogenesis: remnant of embryonic cell rests. diagnostic features • focal. • circumscribed normal parotid, submandibular or sublingual gland acini within a heterotopic salivary gland. • none. comment: care should be taken to differentiate sectioning artifacts ("floaters") from the ectopic parotid gland in the sublingual glands. vacuolation ( figure 160 ) synonyms: hydropic change, cloudy swelling, hydropic degeneration, fatty degeneration, lipidosis, lipid accumulation pathogenesis: accumulation of substances of different character, including fluids, , lipids, phospholipids, and glycoproteins within cells. • distribution may be focal or multifocal and involve several lobules or a diffuse change involving all the lobes. • cells may be swollen with pale eosinophilic cytoplasm and have intracytoplasmic vacuoles with variably margins. • the size of vacuoles may vary from small (microvesicular) to large when multiple small vacuoles coalesce together (macrovesicular) often resulting in nuclear displacement. • variable loss of zymogen granules or mucinous secretions within the affected cells • lobular architecture of gland is retained. • hypertrophic cells with foamy cytoplasm characterized by clear vesicles with eosinophilic fine granules differential diagnoses • artifact: medium-sized clear vesicles within the cytoplasm of acinar cells, especially at the periphery. no positive staining by fat stains. comments: based solely on light microscopic evaluation of hematoxylin and eosin stained tissue sections, it is not possible to conclusively characterize the nature of the intracytoplasmic vacuoles. hence, vacuolation, cytoplasmic is the preferred terminology of the lesion that was previously diagnosed as fatty change, hydropic degeneration, cloudy swelling etc. any chemical/factor affecting the integrity of the cell membranes can potentially cause poorly defined intracytoplasmic vacuoles that was previously diagnosed as hydropic degeneration (sela et al. 1977; simson, 1972) . care should be taken to discriminate artifactual vacuolation induced by processing from the lesion induced by treatment. some metabolic insults cause disruption of normal lipid transport and metabolism and subsequently cause accumulation of lipid within the cytoplasm. in the streptozotocin-diabetes rat model, the parotid is the major salivary gland most susceptible to cytoplasmic vacuolation (anderson 1998) . cytoplasmic vacuolation within the parotid was observed in doxylamine treated f344 rats (jackson and blackwell 1988) . cytoplasmic vacuolation within the parotid but not submandibular glands was noted in aged wistar rats (andrew 1949 a fine cytoplasmic vacuolation, resulting from foamy appearance, may give the suspicion of phospholipidosis. in such cases, the modifier "foamy" should be used. if the change reflects phospholipidosis, increases in size and number of lysosomes can be identified by histochemistry (for alkaline phosphatase), immunostains (for lysosomal membrane proteins, e.g. lamp-2) or by transmission electron microscopy. with the later technique, multilamellated structures (myeloid bodies/lamellar bodies) are observed in the lysosomes. the term phospholipidosis should only be used when confirmation by one of these techniques has been achieved. pathogenesis: unknown. • multifocal to coalescing accumulations of adipocytes within the interstitium. • compression of the adjacent acini, ducts and lobes. • moderate replacement (loss) of acini in rare cases. • disruption of lobular structure. • vacuolation due to storage of lipids: moderate to abundant intracytoplasmic lipid vacuoles; acinar and ductular architecture is retained. • atrophy: reduction in the number/size of acini is the predominant finding. comment: the accumulation of adipocytes within salivary glands is an uncommon lesion and it may be an age related change. it occurs predominantly in parotid and less commonly in submandibular and sublingual salivary glands (andrew 1949) . apoptosis ( figure 162 ) pathogenesis: gene regulated, energy dependent process leading to formation of apoptotic bodies which are phagocytosed by adjacent cells. • necrosis: the morphologic features of cell death clearly fit with necrotic diagnostic criteria (cells and nuclei swollen, pale cytoplasm etc.). • apoptosis/necrosis: both types of cell death are present and there is no requirement to record them separately or a combined term is preferred for statistical reasons. the term is also used when the type of cell death cannot be determined unequivocally. the approach for the nomenclature and diagnostic criteria of cell death applied here is based on a draft recommendation of the inhand cell death nomenclature working group. apoptosis is not synonymous with necrosis. the main morphological differences between these two types of cell death are cell shrinkage with nuclear fragmentation and tingible body macrophages in apoptosis versus cell swelling, rupture and inflammation in necrosis; however, other morphologies (e.g. nucelar pyknosis and karyorrhexis) overlap. in routine h&e sections where the morphology clearly represents apoptosis or single cell necrosis, or whereby special procedures (e.g. tem or ihc for caspases) prove one or the other, individual diagnoses may be used. however, because of the overlapping morphologies, necrosis and apoptosis are not always readily distinguishable via routine examination, and the two processes may occur sequentially and/or simultaneously depending on intensity and duration of the noxious agent (zeiss 2003) . this often makes it difficult and impractical to differentiate between both during routine light microscopic examination. therefore, the complex term apoptosis/necrosis may be used in routine toxicity studies. a differentiation between apoptosis and single cell necrosis may be required in the context of a given study, in particular if it aims for mechanistic investigations. transmission electron microscopy is considered to represent the gold standard to confirm apoptosis. other confirmatory techniques include dna-laddering (easy to perform but insensitive), tunel (false positives from necrotic cells) or immunohistochemistry for caspases, in particular caspase 3. these techniques are reviewed in detail by elmore (elmore 2007) . some of these confirmatory techniques detect early phases of apoptosis, in contrast to the evaluation of h&e stained sections, which detects late phases only; overall interpretation of findings should take into account these potential differences. thus, low grades of apoptosis may remain unrecognized by evaluation of h&e stained slides only. certain studies may require the consideration of forms of programmed cell death other than apoptosis which requires specific confirmatory techniques (galluzzi et al. 2012) . synonyms: oncotic cell death; oncotic necrosis; necrosis pathogenesis: unregulated, energy independent, passive cell death with leakage of cytoplasm into surrounding tissue and subsequent inflammatory reaction; may be induced by direct contact with a test article after oral uptake/administration. • focal affecting cell groups, lobular, diffuse. • cells swollen with pale eosinophilic cytoplasm. • loss of nuclear basophilia, pyknosis and/or karyorrhexis that affects aggregates of cells. • in more severe cases, there may be detachment of the epithelium from the submucosa. • typically, the presence of degenerative cells is a component of necrosis. • minimal or slight inflammatory cell infiltrates, edema and fibrin may be present as a feature of necrosis. single cell • only single cells affected. • apoptosis: the morphologic features of cell death clearly fit with apoptosis (cell and nucleus shrunken, hypereosinophilic cytoplasm, nuclear pyknosis etc.) and/or special techniques demonstrate apoptosis. • apoptosis/necrosis: both types of cell death are present and there is no requirement to record them separately or a combined term is preferred for statistical reasons. the term is also used when the type of cell death cannot be determined unequivocally. • inflammation, acute: infiltrates of inflammatory cells, edema, and fibrin predominate; necrosis may be present, but is a minor component. comments: necrosis is usually accompanied by inflammatory cell infiltrate and is usually a significant component of acute inflammation (levin et al. 1999) . necrosis of salivary glands may be due to chemical toxicity or emboli. synonym: cell death pathogenesis: unregulated, energy independent, passive cell death with leakage of cytoplasm into surrounding tissue and subsequent inflammatory reaction (single cell necrosis) and/ or gene regulated, energy dependent process leading to formation of apoptotic bodies which are phagocytosed by adjacent cells (apoptosis). • both types of cell death are present and there is no requirement to record them separately or a combined term is preferred for statistical reasons. • the type of cell death cannot be determined unequivocally. • apoptosis: the morphologic features of cell death clearly fit with apoptosis (cell and nucleus shrunken, hypereosinophilic cytoplasm, nuclear pyknosis etc.) and/or special techniques demonstrate apoptosis and there is a requirement for recording apoptosis and necrosis separately. • necrosis: the morphologic features of cell death clearly fit with necrotic diagnostic criteria (cells and nuclei swollen, pale cytoplasm etc.) and there is a requirement for recording apoptosis and necrosis separately. comments: necrosis and apoptosis are not always readily distinguishable via routine examination, and the two processes may occur sequentially and/or simultaneously depending on intensity and duration of the noxious agent (zeiss 2003) . this often makes it difficult and impractical to differentiate between both during routine light microscopic examination. in these cases, the combined term apoptosis/necrosis may be used. it is recommended to explain in detail the use of this combined term in the narrative part of the pathology report. (figures 164 and 165 ) synonym: degranulation, acinar cell pathogenesis: decrease of acinar cell zymogen granules or mucin leading to shrunken acinar cells. • focal, lobular or diffuse. • reduced acinar diameter. • partial or complete loss of acinar cell zymogen granules or mucin leading to a reduced cell size and increased basophilia. • lack of fibrosis or adipocyte infiltration. • atrophy, acinar cell: complete degranulation of acini may be accompanied by fibrosis, mononuclear cell infiltrates, prominent intra-and inter-lobular ducts. the morphology of the acini may be altered due to several physiological conditions, such as anorexia/inanition. degranulation of the acini may also be noted in rodents treated with sympathomimetic agents like isoprenaline (selye et al. 1961 ) and terbutaline (sodicoff et al. 1980) . synonym: feminization of the granular duct; degranulation granular duct pathogenesis: decrease of granulated duct cell serous granules leading to shrunken granulated duct cells. • may be focal or multifocal, but usually diffuse. • affects males, not females. • partial or complete loss of serous granules leading to a reduced cell size. • reduced cell diameter of the granulated duct cell. • lack of fibrosis or adipocyte infiltration. • may be accompanied by degranulation of the acinar cells. differential diagnosis • none. the morphology of the granular ducts is androgen dependent. secretory depletion and subsequent atrophy of granular ducts may be observed in male rats and mice with decreased androgen levels. it is also observed in the streptozotocin-induced chronic diabetes rat model. the acinar cells are less susceptible compared to the granular ducts. the atrophy of the granular ducts in chronic diabetic rats may be due to the hypogonadal effect of diabetes. in cases of sympathetic but not parasympathetic nerve stimulation, extensive degranulation may be observed in both acinar (betaadrenergic) and granular duct (alpha-adrenergic) cells. pathogenesis: increased androgenic stimulus in females. • diffuse, limited to the submandibular gland of females. • prominent large granular ducts with increased eosinophilic granules within female animals. • increased granularity of the granular duct. • increased diameter of the granular cells. • except for the change in the granular duct, rest of the histomorphology is normal. differential diagnosis • none. the submandibular salivary gland is sexually dimorphic and shows increased granularity of the convoluted (granular) ducts in males compared to females. when female rats or mice are treated with androgenic test articles (e.g. androstenedione or oxymetholone) or have increased endogenous androgen levels, their granular ducts of the submandibular glands acquires male morphology. pathogenesis: spontaneous or experimentally induced. • focal, lobular or diffuse. • variable reduction in the number and/or size of acini to total loss of acini. • degranulation (parotid), reduced mucin (submandibular and sublingual). • relatively more numerous, prominent, occasionally dilated, intra-and inter-lobular ducts lined by cuboidal or flattened epithelial cells. • pyknotic and karyorrhectic nuclei, apoptotic bodies may be present,. • variable degree of interstitial fibrosis with/without lymphocytes and plasma cells. • scattered to coalescing adipocytes may be present within the interstitium. • may be accompanied by mineralization of luminal contents, salivary calculus, ductular foreign bodies. • secretory depletion, acinar cell: variable loss of serous or mucinous granules resulting in reduced cellular size, lack of fibrosis or adipocyte infiltration, or lack of chronic inflammatory cell infiltrate. • accumulation, adipocytes: accumulation of adipocytes is the predominant finding. comment: atrophy more commonly affects the submandibular and parotid glands than the sublingual gland. it may be an age related lesion or may be induced experimentally, usually as a secondary response after obstruction of excretory ducts or after alteration of trophic factors. examples for obstruction of excretory ducts include ligation (bhaskar et al. 1956; standish and shafer 1957) , sialoliths and foreign bodies. testosterone, adrenocorticoids, and sympathetic stimulation are important trophic factors for the salivary glands. any experimental condition that acts on their homeostasis may potentially induce diffuse salivary gland atrophy. this has been shown for hypophysectomy (koerker 1967) , adrenalectomy, denervation and α-and β-adrenergic antagonists. also decreased food consumption, and protein starvation lead to salivary gland atrophy. atrophy of salivary glands due to liquid diet affects parotid and submandibular glands but the sublingual glands are apparently spared (hall and schneyer 1964; scott and gunn 1991) . also, the sublingual glands lack sympathetic innervation and thus are spared in situations where the chemicals act on the sympathetic nervous system. salivary gland atrophy was noted in f344 rats treated with sodium dichromate dihydrate (ntp, 2008) . often, salivary atrophy is accompanied by mild chronic inflammation and mild interstitial fibrosis. pathogenesis: deposition of mineral either secondary to cellular necrosis (dystrophic mineralization) or secondary to hypercalcemia (metastatic mineralization). • focal, lobular, diffuse. • affected structures are ducts and arteries (metastatic mineralization) or areas of degeneration / necrosis (dystrophic mineralization). • dark basophilic granular material (on h&e) replacing acini, ducts or interstitium. • artifacts: hematoxylin stain deposits (distribution unrelated to tissue structures), acid hematin (birefringent in polarized light). • pigment: lipofusin and porphyrin stain brown to golden brown on h&e and may further be differentiated from mineralization by special stains. • calculi (concretions, sialoliths): localization in the lumen of ductules or ducts. comment: dystrophic calcification occurs in necrotic/injured tissue even in animals with reference range serum calcium levels, whereas metastatic calcification occurs throughout the body, in hypercalcemic animals, especially within interstitial tissues and blood vessel walls. special stains like alizarin red s and von kossa can help identify calcium salts within tissue sections. amyloid ( figure 172 ) pathogenesis: extracellular deposits of chemically diverse complex insoluble polypeptide fragments. • light eosinophilic amorphous extracellular material within the tunica media of blood vessels, within the interstitium and basement membrane. • variable degree of atrophy and loss of intervening acini and ducts may occur, if amyloid deposition is prominent. • green birefringence using polarized light with congo red stain. • necrosis: loss of cellular detail, but without extracellular congo red positive material. • fibrinoid change (necrosis) in vessel walls: deep eosinophilic homogenization of vascular media. comment: amyloid and amyloid-like material has been observed in various tissues, including salivary glands of aged rodents, especially mice. rats are much more resistant to develop amyloidosis than mice. the characteristic morphologic appearance and location of amyloid in h&e sections that is usually adequate to make this diagnosis. confirmation of the deposits as amyloid can be accomplished with light microscopy using special stains such as congo red. amyloid appears apple green under polarized light with this stain. the term "hyaline" should be used in case of uncertainties about the nature of extracellular deposits hyaline material in h&e sections and the unavailability of a congo red stain. hyaline is also the appropriate term for homogenously eosinophilic extracellular deposits that react negative with congo red. infiltrate ( figure 173 ) diagnostic features • focal, multifocal or diffuse. • presence of mononuclear or polymorphonuclear leukocytes but without other histological features of inflammation like edema, congestion or necrosis. • mild atrophy of surrounding cells may be present. • usually no acinar cell degranulation or mucus depletion. • inflammation: an inflammatory cell infiltrate associated with additional morphologic features of inflammation such as edema, hemorrhage, necrosis, and/or fibroplasia. • granulocytic leukemia: infiltration of neutrophil precursors and/or abnormal neutrophils will probably be present along with mature neutrophils; infiltration of similar neoplastic cells is likely present in other organs. • lymphoma: infiltration of monomorphic population of lymphocytes (often with atypical, increased and/or abnormal mitoses); infiltration of similar neoplastic cells is likely present in other organs. comment: in toxicity studies, inflammatory cell infiltrates are encountered more commonly than inflammation (sialadenitis). the term inflammation (or sialadenitis) should not be confused with or used in lieu of inflammatory cell infiltrate. the term inflammatory cell infiltrate should be used rather than sialadenitis (or inflammation) when there is no tissue in-jury/reaction associated with the inflammatory cell infiltrate. also, these inflammatory cell infiltrates should be qualified with the type of inflammatory cells that are predominant, such as mononuclear, lymphocyte, neutrophil, etc. the use of terminology summarizing the nature of the infiltrates as acute or suppurative (in lieu of neutrophilic), granulomatous (in lieu of histiocytic), and chronic (in lieu of lymphocytic, plasmacytic, mononuclear, etc.) is not recommended since these terms may imply inflammation. the term inflammation (or sialadenitis) should be reserved for lesions where the inflammatory cell infiltrate is associated with obvious tissue injury/reaction associated with inflammation such as edema, congestion, acinar (parotid) cell degranulation, hemorrhage or necrosis. inflammation (figures 174, 175, 176 and 177) modifiers: type of inflammatory cell that represents the predominant cell type in the inflammation pathogenesis: infiltration of the lamina propria and/or submucosa with neutrophils (inflammation, neutrophil) or mononuclear cell (inflammation, mononuclear cell) or a combination (inflammation, mixed) with additional histological features of inflammation e.g. hemorrhage, edema, fibroplasia. • focal, multifocal, or diffuse. • infiltrate of mononuclear or polymorphonuclear leukocytes into the gland parenchyma. • presence of other histological criteria of inflammation, e.g. hemorrhage, edema, fibroplasia inflammation, neutrophil • infiltrate predominantly neutrophilic; frequently accompanied by edema and congestion, parotid acinar cell degranulation, acinar and ductular degeneration and necrosis. • infiltrate predominantly mononuclear cell; frequently accompanied by acinar cell atrophy and mild acinar cell degeneration, ductal proliferation, interstitial fibrosis; mineralization or nonkeratinizing squamous metaplasia of acini and ducts may be present. • infiltrate, inflammatory cell: other morphologic features associated with inflammation such as edema, hemorrhage, necrosis, and/or fibroplasias will be absent. • granulocytic leukemia: infiltration of neutrophil precursors and/or abnormal neutrophils will probably be present along with mature neutrophils; infiltration of similar neoplastic cells is likely present in other organs. • lymphoma: infiltration of monomorphic population of lymphocytes (often with atypical, increased and/or abnormal mitoses); infiltration of similar neoplastic cells is likely present in other organs. comment: when a mononuclear or mixed cell infiltrate is considered to represent an inflammatory process (inflammation, mononuclear cell or inflammation, mixed) and other morphologic features (fibrosis, mineralization) associated with the infiltrate suggest an ongoing/continuing inflammatory process, the modifier 'chronic' may be included in the diagnosis (i.e. inflammation, mononuclear cell, chronic) to better characterize the finding. inflammation, neutrophilic of the salivary glands is uncommon in toxicological evaluation of tissues. infection with murine cytomegalovirus (mcmv) and sialodacryoadenitis virus (sdav) were once common causes of acute sialodacryoadenitis in mice and rats (jacoby et al. 1975 ), respectively, but are now rare in barrier bred and maintained colonies. mcmv predominantly affects the submandibular glands and in rare cases the parotid glands. sdav has a tropism for tubuloalveolar glandular tissue of serous (parotid) or mucous/serous (submandibular) glands. the sublingual gland is spared in sdav infections in rats. sialadenitis may be differentiated by their respective pathognomonic clinical signs and lesions. bacterial infections due to klebsiella aerogenes and staphylococcus aureus may also cause severe acute necrotizing sialadenitis as well as abscesses. spontaneous autoimmune sialadenitis is seen in several strains of mice (nod, nzb/nzw, sl/ni, bdf females) and rats (percy and barthold 2007) . the infiltrating lymphocytic population is predominantly cd4+ t cells and less than 10% cells are c8+ t cells. variable degree of chronic inflammation of the salivary glands may be seen occasionally in aged animals of other strains. chronic infections of mcmv and sdav or regenerative lesions from acute mcmv and sdav infections may appear morphologically similar to chronic inflammatory lesions (ohyama et al. 2006) . rats treated with sympathomimetics like isoproterenol present with mild chronic inflammation in the submandibular salivary gland (cohen et al. 1992 ). refer also to the inhand monograph on the cardiovascular system. pathogenesis: accumulation of fluid in salivary interstitium resulting from increased vascular permeability due to damage to capillaries and blood vessels, increased hydrostatic pressure in vasculature, decreased oncotic pressure, or impaired lymphatic drainage. • diffuse. • increased clear spaces between acini and interstitium. • increase in total lobe volume. • rare inflammatory cell infiltrate secondary to diapedesis may be present. • inflammation, neutrophil: tissue and capillary damage accompanied by exudative edema, granulocytic infiltration, fibrin, congestion and hemorrhage. comment: edema within the salivary glands is very uncommon. it may be exudative (inflammatory edema due to capillary injury secondary to leakage of enzymes) or transudative (hydrodynamic derangement secondary to increased hydrostatic osmotic pressure and decreased oncotic pressure). the diagnosis of edema should be reserved for a transudative type of edema since exudative edema is usually accompanied by abundant inflammatory cell infiltrate and possibly tissue injury. hence, the primary diagnosis in cases of exudative edema should be inflammation, neutrophil. chemical induced salivary gland edema has been noted in mice treated with p-nitrophenol and rats administered d & c yellow no. 11 in us-ntp studies. pathogenesis: increased vascular permeability (diapedesis) or rupture of blood vessels. • presence of erythrocytes in parenchymal or interstitial tissue or lumina of ductules and ducts. • angiectasis: blood present within dilated vascular lumina. • artifact: erythrocytes on the surface of tissues as a result of necropsy procedures. synonyms: sialoliths; sialolithiasis; salivary concretion; duct concretion; salivary calculus; inspissated material pathogenesis: precipitation of secretory components after reabsorption of the water component by the ductular cells. • basophilic or eosinophilic inspissated material within the ductular lumen. • ductular epithelium may be low to flattened cuboidal epithelium. • may be accompanied by mild periductular mononuclear inflammatory cell infiltrate. • ectasia, duct: salivary calculi are absent. • foreign body: foreign body within the duct is usually associated with some tissue damage and elicits an inflammatory response. a diagnosis of ductular ectasia should not be used in the presence of the ductular calculi, even though they share other morphologic features. synonyms: dilatation; luminal distension pathogenesis/cell of origin: luminal dilatation due to obliteration of the intralobular or interlobular ducts. • solitary or multiple tortuous irregularly enlarged lumina that may or may not contain gland-specific secretory material. • lined by flat cuboidal epithelial cells. • may be accompanied by mild interstitial infiltration of lymphocytes and plasma cells. • calculus, ductular: salivary calculi are present. comment: usually ectasia results from blockage of inter-and intra-lobular ducts either proximally or distally due to rare inspissated secretory material (sialolithiasis), sialodacryoadenitis tumors, or foreign bodies. however, a diagnosis of ectasia is used only in the absence of the calculi or the foreign body within the histologic section. fibrosis ( figure 181 ) pathogenesis: collagen deposition by fibroblasts following inflammation, necrosis or hemorrhage. • focal, lobular, or diffuse. • increase in the interstitial tissue due to collagen deposition and fibroblast cell proliferation. • amyloid: there is abundant extracellular eosinophilic amorphous ground substance that exhibits apple green birefringence in congo red stained sections under polarized light and there is an absence of fibroblast cell proliferation. • inflammation, mixed cell, chronic: fibrosis associated with inflammatory cell infiltrates and degenerative parenchymal changes. comment: fibrosis may be minimal to marked depending on the severity and duration of insult. it is usually a significant component of chronic inflammation and may be a component of healing after acute toxic insults. mature collagen in h&e sections is birefringent under polarized light. pathogenesis: usually secondary to increased functional demand or due to sympathomimetic stimulation. • focal, multifocal or diffuse. • acinar structure not altered, no encapsulation, no compression. • enlarged cells with abundant cytoplasm of normal staining pattern. • large nuclei with euchromatin and prominent nucleoli. • relative reduction in the number of secretory ducts due to separation by enlarged acini. • basophilic hypertrophic focus (parotid gland): discrete non-compressing foci of hypertrophic cells with increased basophilia within discrete non-compressing foci. • hyperplasia, non-regenerative: may be associated with hypertrophy, but always increase in cell number; minimal compression or distortion of architecture may be present. • hyperplasia, regenerative: may be associated with hypertrophy, but always increase in cell number and cytoplasmic basophilia; minimal compression or distortion of architecture may be present. comment: acinar hyperplasia is often associated with hypertrophy. in these cases, it is recommended to record hyperplasia, but not hypertrophy. hypertrophy (and hyperplasia) of the parotid and submandibular glands has been observed after administration of certain drugs such as isoproterenol (selye et al. 1961, brenner and stanton, 1970) , methoxamine and pilocarpine (inanaga et al. 1988 ), terbutaline (sodicoff et al. 1980) , methacholine, epinephrine, phenylephrine, reserpine, furosemide (scarlett et al. 1988 ), alloxan (sagstrom et al. 1987 , thyroxine, and dexamethasone (sagulin and roomans 1989) . single or repeated mandibular incisor tooth amputation also resulted in enlargement of submandibular glands, presumably due to neural regulation (wells 1963) . this enlargement was due to abundant intracytoplasmic accumulation of mucus along with calcium, amylase and other protein. (figures 182 and 183) synonyms: focus, basophilic; basophilic focus; basophilic hypertrophic focus; focus, basophilic, hypertrophic; focus of cellular alteration; pathogenesis/cell of origin: unknown. diagnostic features • focal, multifocal or diffuse. • discrete unencapsulated, noncompressing foci involv-ing one or multiple acini. • enlarged cells with increased cytoplasm and occasionally enlarged nuclei. • apical regions of the acinar cells have an eosinophilic granular or fine vesicular cytoplasm on h&e stain. • basal regions of the acinar cells have intensely basophilic cytoplasm with large hyperchromatic nuclei. • the enlarged cells can be diffusely basophilic in extreme cases where most of the parotid is affected. • pyknotic nuclei or mitoses may be observed. • in diffusely affected cases, the acinar enlargement is not as prominent as seen in smaller focal basophilic foci. • relative reduction in the number of secretory ducts due to separation by enlarged acini. • hyperplasia, non-regenerative or hyperplasia, regenerative: increase in cell number that may or may not be associated with hypertrophy; minimal compression or distortion of architecture may be present. • hypertrophy, acinar cell: cells in single or multiple acini (foci) enlarged without increased cytoplasmic basophilia. comment: in untreated animals, basophilic hypertrophic foci are more common in rats than in mice (chiu and chen 1986) . the incidence of basophilic hypertrophic foci slightly increases with age. in addition, several chemicals such as doxylamine (jackson and blackwell 1993) , triprolidine (greenman et al. 1995) , glyphosate (ntp, 1992a), methyleugenol (ntp, 2000) , and diethanolamine (ntp, 1992b) induce basophilic hypertrophic foci in rodents. these foci are considered adaptive hypertrophic lesions rather than precursors of neoplasia. pathogenesis: transdifferentiation of one fully differentiated salivary gland epithelium into a different type of fully differentiated salivary gland epithelium. • focal to multifocal scattered distribution. • usually accompanied by prior tissue injury. • presence of fully differentiated epithelial cells that are not native to the tissue, e.g. presence of parotid gland epithelial cells within the submandibular gland. ectopic tissue: usually focal, circumscribed and not associated with prior tissue injury. comment: documenting prior injury and the corresponding chronic inflammation as well as the multifocal nature of the lesion supports the diagnosis of metaplasia instead of ectopic tissue. (figures 185 and 186 ) synonyms: squamous metaplasia; ductal squamous metaplasia histogenesis: ductular epithelial structures of the salivary glands. • cuboidal ductular epithelium replaced by squamous epithelium. • squamous epithelial cells may be uni-or multilayered. • keratinization may be present. • may be accompanied by duct hyperplasia. • carcinoma, squamous cell: local invasion of adjacent tissue, distant metastasis, or marked cellular atypia. • hyperplasia: hyperplastic cells cuboidal to columnar in shape and unilayered. • hyperplasia, atypical: multiple layers of cuboidal to columnar epithelial cells with lost polarity and increased basophilia; no horizontal flattening as in squamous metaplasia. comment: ductal squamous cell metaplasia is usually seen as a part of the regenerative response following necrosis of the ductal epithelium. in rats, a common cause of this lesion in parotid and submandibular glands is due to sialodacryoadenitis viral (sdav) infection. the sublingual glands are reportedly spared from pathologic changes in sdav infections (jacoby 1985) . however, the sublingual excretory ducts may have ductal squamous metaplasia in the absence of sdav infections. vitamin a deficient diet induced squamous metaplasia in the ducts of parotid, submandibular and sublingual glands in sprague-dawley rats (horn et al. 1996) . squamous cell metaplasia in the submandibular gland was described in mice after treatment with dmba and was proven to be of ductal segment origin by the presence of keratin proteins (takai et al. 1986 ). light and electron microscopy studies have demonstrated that the principal portion of salivary gland tissue undergoing squamous metaplasia is the acinar-intercalated duct cell complex (dardick et al. 1985) . though, squamous cell metaplasia of the ductular epithelium has been considered as a preneoplastic condition progressing to neoplasia (neuenschwander and elwell 1990) , the potential for progression of hyperplasia and squamous metaplasia of the ductular epithelium is unclear. these changes in the sublingual gland of wistar rats, often associated with inflammation/fibrosis, were not associated with neoplasia at that site (van esch et al. 1986 ). in addition, treatment-related squamous cell metaplasia of the salivary gland in a chronic f344 rat study with iodinated glycerol was not associated with neoplastic findings in the salivary gland after two years (ntp, 1990) . squamous cell metaplasia was induced with chlorodibromomethane in f344 rats but not in b6c3f1 mice after 13-week treatment, but no tumor progression was noted after 2 years treatment (dunnick et al. 1985) . on the other hand, transition of squamous metaplasia into squamous cell carcinoma in the submandibular gland via a non-genotoxic, proliferation-dependent mechanism was postulated after 2-year treatment with potassium iodide in f344 rats (takegawa et al. 1998 ). in addition, in experiments with implanted sponge pellets containing dmba inducing squamous cell carcinomas in sprague-dawley rats, the authors concluded that ductal segments undergoing squamous cell metaplasia may have participated in the genesis of neoplasia during experimental carcinogenesis (cao et al. 2000 , sumitomo et al. 1996 . hyperplasia ( figure 187 ) modifiers: acinar, ductal, reactive, atypical histogenesis: ductal and/or acinar epithelium of the salivary glands. diagnostic features • focal, multifocal or diffuse. • glandular architecture is preserved or only minimally altered. • minimal compression of the surrounding tissue may be present. • often rounded circumference, but maintenance of normal acinar or ductal architecture. • no capsule. acinar: • acinar cells with atypia may occur. ductal: • ducts may show dilated lumina lined by flattened epithelium. reactive: • acinar or ductular proliferation with cytoplasmic basophilia; evidence of an underlying process, e.g. inflammation, fibroplasias, ductal obstruction or foreign material. atypical: • focal or multifocal, not diffuse. • multiple layers of acinar / ductular epithelial cells. • gradual loss of cellular polarity and differentiation, cytoplasm may be amphophilic, increased nucleus to cytoplasm ratio. differential diagnoses • adenoma: loss or distortion of the normal acinar/ductular structure; well-demarcated and distinct compression of the surrounding tissue. • ectopic parotid gland: foci of serous acini between mucous acini of the sublingual gland. • hypertrophy: enlarged acinar cells without increase in cell number; no compression, no alteration of glandular architecture. • focus, hypertrophic, basophilic: enlarged acinar structures with increased basophilia; no compression. comment: hyperplastic or preneoplastic changes associated with spontaneously occurring neoplasms have not been identified in f344 rats (neuenschwander and elwell. 1990) . spontaneous hyperplastic and metaplastic duct epithelium was described in the sublingual glands of wistar rats (van esch et al. 1986 ). hyperplasia of the salivary ducts is a common feature of many inflammatory and reactive conditions in the salivary glands of rodents (greaves 2012) . multifocal ductal cell hyperplasia was induced in the intercalated duct cells of the submandibular salivary glands of wistar rats after chronic treatment with a steroidal test article comprising prostagenic and estrogenic properties (de rijk et al. 2003) . spontaneous hyperplastic changes of the salivary gland have not been observed in b6c3f1 mice (botts et al. 1999) . transgenic mice models were reported to show hyperplasia of the submandibular glands in serous acinar cells expressing a retinoid acid receptor (bérard et al. 1994) and ductular cells expressing simian virus 40 t antigen (ewald et al. 1996) , respectively. spontaneous and primary tumors of the rodent major salivary glands are rare (elwell and leininger 1990; frith and heath 1985; greaves 2012) . salivary gland adenomas in rats and mice may arise from either acinar or ductular components (neuenschwander and elwell 1990; botts et al. 1999) . few carcinomas and adenocarcinomas have been reported in mice and rats (botts et al. 1999; hosokawa et al. 2000; nishikawa et al. 2010; tsunenari et al. 1997) . malignant tumors in the salivary glands may be induced with chemical substances in rats (neuenschwander and elwell 1990; sumitomo et al.1996; cao et al. 1999; zaman et al. 1996) and mice (botts et al. 1999; takegawa et al. 1998; takai et al. 1986; yura et al. 1995) or in various transgenic animal models (dardick et al. 2000; declercq et al. 2005; nielsen et al. 1991; nielsen et al. 1995) . adenoma (figures 188, 189, 190, 191 and 192) modifiers: acinar, tubular, papillary, solid, mixed acinar/ tubular histogenesis: ductal or acinar epithelium of salivary glands. • well demarcated. • compression of the adjacent parenchyma or connective tissue. • alteration of normal acinar or ductular architecture. • may be surrounded partially or completely by a thin capsule. • nuclei are hyperchromatic and basally oriented when cytoplasmic mucus present. • mitotic figures are rare. • growth pattern may be acinar, tubular, papillary, solid, or mixed. acinar: • show some signs of secretory activity. minor areas with solid growth pattern may be present; excretory ducts are usually absent. tubular: • consists of lobules or aggregates of tubules lined by squamous to cuboidal or columnar epithelium, separated by a variable amount of fibrous stroma. some ducts may have dilated lumina. papillary: • papillary projections dominate and cystic dilatation may be present. solid: • diffuse sheet of epithelial cells without acinar or ductular structure. mixed acinar/tubular: • a mixture of acinar and tubular forms is not infrequent. • hyperplasia: acinar structure preserved or only minimally altered; no or only minimal compression, no capsule; in case of reactive hyperplasia evidence of an underlying process, e.g. inflammation, fibroplasia, ductal obstruction or foreign material. • hyperplasia, atypical: focal proliferation of acinar or tubular epithelium with no or only minimal compression, with multiple epithelial layers and cellular atypia; no capsule. • adenocarcinoma: cellular pleomorphism and cytoplasmic amphophilia; numerous mitotic figures, invasive growth. • tumor, mixed, benign: consists of two proliferative cell types, one myoepithelium and the other glandular acinar epithelium. • adenoma of the mammary gland (anterior part): glandular structure is not as compact; staining characteristics of epithelium different. comment: in mice, only acinar adenomas of the salivary glands have been described, while rats may develop the different growth patterns listed as modifiers. multinodular acinar adenomas may be difficult to distinguish from well-differentiated adenocarcinomas. salivary gland adenomas in rats may arise from either acinar or ductular components (neuenschwander and elwell 1990) . few adenomas have been observed in mice and it is unclear if they arise from ducts or acini (botts et al. 1999) . therefore it is recommended to use the modifiers acinar and tubular not in the sense of histogenesis but as a description of the predominant growth pattern. mice transgenic for the pleomorphic adenoma gene (plag1 or plag2) developed a high incidence of submandibular gland adenomas. these tumors developed pleomorphic characteristics, similar to human pleomorphic adenomas of the salivary glands. they were composed of epithelial and myoepithelial structures. the epithelial component showed various growth patterns, including keratinization. the spindle-shaped myoepithelial cells were embedded in a myxoid stroma (declercq et al. 2005) . adenocarcinoma (figures 193, 194, 195 and 196) modifiers: acinar, tubular, papillary, solid, mixed acinar/tubular/solid histogenesis: ductal or acinar epithelium of salivary glands. diagnostic features • grows in whorls or spindle pattern. • necrosis frequently present. • areas of squamous differentiation may be present. • cells are large, pleomorphic and polyhedral with amphophilic cytoplasm. • nucleus to cytoplasm ratio high. • nuclei are large vesicular and have multiple nucleoli. • mitotic figures numerous. • invasion of the surrounding tissue present. • metastasis to the lung may be present. • growth pattern may be acinar, tubular, papillary, solid, or mixed. acinar: • show some signs of secretory activity. • minor areas with solid growth pattern may be present, excretory ducts are usually absent. tubular: • differentiation pattern variable, ranging from wellformed tubular structures to poorly formed tubules or nodular masses of anaplastic epithelial cells. • some ducts may have dilated lumina. • diffuse sheet of epithelial cells without acinar or ductular structure. mixed acinar/tubular/solid: • may have different patterns in one tumor: acinar, tubular and solid. papillary: • papillary projections dominate and cystic dilatation may be present. • hyperplasia: no evidence of invasion, glandular structure preserved or only slightly altered (acinar hyperplasia; in case of reactive hyperplasia evidence of an underlying process like inflammation, fibroplasia, ductal obstruction or foreign material. • hyperplasia, atypical: focal proliferation of acinar or tubular epithelium with multiple layers and cellular atypia; no invasion of surrounding tissue. • adenoma: minimal cellular pleomorphism; acinar cells well differentiated with secretory granules; no or only minimal pleomorphism; non-invasive; mitotic figures rare. • adenocarcinoma of the mammary gland: cytoplasmic staining differences and lipid vacuolation may be present. comment: multinodular acinar adenomas may be difficult to distinguish from well-differentiated adenocarcinomas. spontaneous tumors of the major salivary glands are rare in the rat (elwell and leininger 1990; glucksmann and cherry 1973) . poorly differentiated carcinomas of the parotid gland of young sprague-dawley rats (tsunenari, et al. 1997; nishikawa et al. 2010) and papillary cystadenocarcinoma in the parotid gland of a f344 rat (hosokawa et al. 2000) were reported. induction of adenocarcinomas in the submandibular gland was reported after intraglandular injection of dmba in female wistar rats (zaman et al. 1996) and female mice (yura et al. 1995) . adenocarcinomas have rarely been reported in mice (botts et al. 1999) . a naturally occurring mucoepidermoid carcinoma in the salivary gland associated with both myoepithelium and duct epithelium was described in two mice (ishikawa et al. 1998 ). submandibular gland adenocarcinomas of intercalated duct origin were induced in smgb-tag mice (dardick et al. 2000) . in these mice the oncogene sv40 t antigen is expressed from the neonatal submandibular gland secretory protein b (smgb) gene promoter. development of pleomorphic adenoma with malignant characteristics and lung metastasis was described in the submandibular gland of transgenic mice with plag1 proto-oncogene over expression (declercq et al. 2005) . transgenic mice expressing a human ha-ras oncogene developed adenosquamous carcinomas arising from the serous areas from the submandibular gland (nielsen et al. 1991) . in wap-ras transgenic mice adenocarcinomas arising from the submandibular gland were reported to develop spontaneously by one year of age (nielsen et al.1995) . (figures 197, 198 and 199) synonym: carcinoma, epidermoid histogenesis: probably ductular epithelial structures of the salivary glands. • well-differentiated islands and cords with keratin formation. • variable mitotic activity. • invasion of adjacent tissues or metastasis. • high grades of anaplasia may occur. • tumors may be extensively keratinized. comment: spontaneous squamous cell carcinomas in rodents are rare, but have been induced with chemicals in rats (neuenschwander and elwell 1990; sumitomo et al. 1996; cao et al. 1999) and mice (botts et al. 1999; takegawa et al.1998; takai et al. 1986) , and occur spontaneously in mice with the v-ha-ras oncogene (cardiff et al. 1993) . transgenic mice expressing a human ha-ras oncogene developed adenosquamous carcinomas arising from the serous areas of the submandibular gland (nielsen et al.1991) . also adenosquamous carcinomas of the mammary gland, which are not uncommon in mice, need to be considered as a differential diagnosis. squamous cell carcinomas of the salivary glands should be distinguished from invasive zymbal's gland carcinomas with squamous differentiation by their location in situ (neuenschwander and elwell 1990) . (botts et al. 1999; frith and heath 1994; dawe 1979 (botts et al. 1999; frith and heath 1994) . the differentiation between the mixed malignant tumor and the malignant myoepithelioma with epithelioid and spindeloid growth pattern may be difficult based on hematoxylin and eosin stained slides. confirmatory immunohistochemistry may be required which in case of the mixed tumor would demonstrate two different tissue compartments that show either epithelial or mesenchymal immunophenotype, but usually not a mixed one. in contrast, mixed epithelial and mesenchymal antigenic expression would be demonstrated in case of the myoepithelioma. histogenesis: myoepithelial cells, extraglandular ductal origin, salivary glands (possibly other glandular tissues). • unencapsulated tumor; may show invasive cords of solid tissue extending into adjacent structures. • in larger tumors, central degeneration and necrosis may form pseudo-cysts, which may be filled by brown, mucoid material or cell debris. comment: myoepitheliomas of the salivary glands have not been described in rats. they are rare in most strains of mice but occur more often in the balb/c, a/j, and c58 strains (mainly females). the most frequent locations are the parotid and submandibular glands (frith and ward 1988 ), but they are not limited to salivary glands or the cervical region. neoplastic myoepithelial cells have been reported to stain positive for cytokeratin and for keratins k5 and k14 and vimentin. also ptah staining will aid in confirmation of the intracytoplasmic fibrils within the myoepithelial cells (botts et al. 1999; sundberg et al. 1991) . they are negative for smooth muscle cell alpha-actin immunohistochemistry, in contrast to normal myoepithelial cells; furthermore, sundberg et al. has suggested that the neoplasms arise from a subset of extraglandular ductal myoepithelial cells that are negative for smooth muscle cell alpha-actin (sundberg et al. 1991) . such confirmatory stains may be required for differentiation of myoepitheliomas from scirrhous/anaplastic squamous cell carcinomas or mixed malignant tumors. myoepitheliomas in mice preferably occur in the ventral neck region (sundberg et al. 1991) . although most of these tumors have been ascribed to the salivary glands, it needs to be taken into account that they may occur from any other glandular tissue, e.g. the mammary gland or extraorbital lacrimal gland. the frequent poor encapsulation, pleomorphic morphology, and central necrosis are compatible with a malignant interpretation; however, sundberg et al. suggested few are malignant based on stringent criteria of metastasis to the lung or infiltration of underlying bone (sundberg et al. 1991) . the morphology of tumors with confirmed metastases is not unique, as compared to those without, and the clinical appearance of the neoplasms may lead to culling prior to development of readily defined metastases, so it is recommended that they should be regarded as potentially malignant. during embryonic development the pancreas is formed by fusion of its dorsal and ventral buds derived from the septum transversum at the embryonic foregut/midgut junction. the pancreas consists of both exocrine and endocrine components. the exocrine pancreas forms the bulk of the pancreas and produces numerous enzymes (in proenzyme form) that aid in digestion. the pancreas of rat and mouse is classified as mesenteric type (versus compact type) since discrete pancreatic tissues are diffusely distributed in the mesentery of the duodenal loop, transverse colon, and greater omentum adjacent to stomach and spleen. the large interlobular ducts open into a common hepatic (biliary)/pancreatic duct that enters the duodenum. in addition, there are some ducts that open directly into the duodenum boorman and sills 1999) . the exocrine pancreas is classified as a compound tubuloalveolar or compound acinar gland. the pancreatic acinus consists of a single layer of pyramidal acinar cells arranged concentrically around a lumen. the apex of the pyramidal cells facing the lumen contains eosinophilic zymogen granules and the base of the pyramidal cells is basophilic due to abundant rough endoplasmic reticulum and also contains the nucleus. each pancreatic acinus is surrounded by a thin basal lamina, scant reticular stroma, and pancreatic stellate cells (akin to the hepatic stellate cells). located centrally within the acinus, the centroacinar cells form an interface between the acinus and the intercalated duct. the intercalated duct continues into intralobular ducts formed by the ductular cells. the intralobular ducts fuse to form the interlobular ducts that finally open into the pancreato-hepatic (biliary) duct boorman and sills 1999) . the exocrine pancreas is physiologically and morphologically compartmentalized into peri-and tele-insular regions. the peri-insular acinar cells are in the immediate proximity of the islets of langerhans and are larger due to more abundant cytoplasm, larger zymogen granules and larger nuclei than the tele-insular acinar cells that are farther from the islets. due to differences in sizes of the peri-and tele-insular acinar cells, at lower magnification, the islets of langerhans appear to be surrounded by "halos" (peri-insular halos) (greaves 2012) . the histologic constituents of the pancreatic tissue such as the exocrine acini, centro-acinar cells, ductal cells and the endocrine cells within the islets have a unique ability to de-differentiate and transdifferentiate into other pancreatic histologic cell types based on the severity and type of injury, distinct transcriptional and protein alterations and epigenetic factors (stanger and hebrok 2013) . as a result, tissue injury in exocrine pancreas may resolve by forming a histologic constituent that may be similar or different from the damaged tissue type. after certain types of pancreatic tissue injury, the exocrine acini undergo de-differentiation and may attain a ductular morphology and in some cases even attain an endocrine phenotype. notch, wnt and hedgehog signaling pathways play important roles in these de-differentiation and transdifferentiation pathways (stanger and hebrok 2013) . the pancreas is a dual function gland with both exocrine (acini) and endocrine (islets of langerhans) functions. the acinar cells store the digestive enzymes in heterogeneously composed vacuoles and the release of these enzymes occurs in a cyclic and secretagogue-specific fashion. the exocrine acinar secretions collect within the acinar lumen and then accumulate within the interlobular ducts that subsequently drain into the duodenum via the pancreato-biliary duct. hormones like gastrin (from g-cells in the stomach) and cholecystokinin (from i-cells in the duodenum) stimulate the exocrine pancreas to secrete inactive proenzymes (zymogens like trypsinogen, chymotrypsinogen, elastase and carboxypeptidase) as well as active enzymes such as lipase, amylase and nuclease. in addition, the duodenal hormone secretin stimulates the centroacinar cells (in addition to the brunner's glands) to secrete bicarbonate ions that aid in neutralizing acidic chyme from the stomach and stabilizing the enzymes. the intestinal enteropeptidases activate the trypsinogen to trypsin that in turn cleaves the rest of trypsinogen and chymotrypsinogen to their active forms resulting in protein digestion. lipase and amylase aid in the digestion of lipids and carbohydrates, respectively (greaves 2012) . the exocrine pancreas has great reserve capacity and no clinical evidence of exocrine pancreatic insufficiency is noted with up to 90% destruction of exocrine pancreas. exocrine pancreatic function deficits > 90% results in improper digestion of food and malabsorption of nutrients (hotz et al. 1973 ). at necropsy, the whole left lobe of the pancreas (located in the omentum close to the spleen) is removed for trimming. for microscopic evaluation, a large part of the left lobe is cut in longitudinally in the horizontal plane, making the cut surface as large as possible. the right lobe is removed together with the adjacent small intestine (ruehl-fehlert et al. 2003 ). ectopic tissue (figure 202 ) pathogenesis: congenital abnormalities resulting in splenic or liver tissues in pancreas. • usually focal distribution. • fully differentiated non-pancreatic tissue in the pancreas. • usually circumscribed and not wholly integrated into the 'host' tissue. • no evidence of dysplasia or history of tissue injury. • metaplasia: the metaplastic tissue is usually integrated into the histological pattern of the host tissue and is usually a result of tissue reaction and adaptation. comment: ectopic tissues are usually a rare finding in toxicity studies and care should be taken to properly interpret them and not be confused with metaplasia or neoplasia. vacuolation, acinar cell ( figure 203 ) comments: based solely on light microscopic evaluation of hematoxylin and eosin stained tissue sections, it is not possible to conclusively characterize the nature of the intracytoplasmic vacuoles. hence, vacuolation is the preferred terminology of the lesion that was previously diagnosed as fatty change, hydropic degeneration, cloudy swelling etc. acinar cell vacuolation is a reversible change and is usually a reflection of hypoxia and metabolic insults resulting in damage to mitochondria, endoplasmic reticulum, protein machinery, and plasma membrane. oil red o and sudan black stains on frozen sections help to identify the neutral lipids within the micro/macro vesicles. it is useful to differentiate from cytoplasmic vacuoles due to water accumulation (hydropic change) resulting from cell injury due to hypoxia. marked vacuolation of the exocrine pancreatic acini has been documented in female sprague-dawley rats exposed to dioxins and dioxin-like compounds (yoshizawa et al. 2005) . a fine cytoplasmic vacuolation, resulting from foamy appearance, may give the suspicion of phospholipidosis. in such cases, the modifier "foamy" should be used. if the change reflects phospholipidosis, increases in size and number of lysosomes can be identified by histochemistry (for alkaline phosphatase), immunostains (for lysosomal membrane proteins, e.g. lamp-2) or by transmission electron microscopy. with the later technique, multilamellated structures (myeloid bodies/lamellar bodies) are observed in the lysosomes. the term phospholipidosis should only be used when confirmation by one of these techniques has been achieved. pathogenesis: replacement of exocrine parenchyma after age-related or chemically-induced atrophy or in case of obesity. • multifocal to coalescing adipocytes within the interstitium. • compression of the adjacent exocrine tissue only occasionally. • multifocal to diffuse atrophy of exocrine acini and replacement by adipose tissue. • islets of langerhans unaffected. • vacuolation, acinar cell: moderate to abundant intracytoplasmic vesicles with a multifocal to diffuse distribution. the exocrine acinar architecture is retained. comments: fatty infiltration of exocrine pancreas is an uncommon lesion which may be age related. the pathogenesis is not usually apparent but may be secondary to atrophy or is the cause of atrophy. synonyms: hyalinosis; eosinophilic change pathogenesis: storage of hyaline material within the cytoplasm of ductal cells. • focal or multifocal. • intensely pink-red cytoplasmic droplets and/or crystals in ductal cells. • cells are usually hypertrophic. • crystals, if present, may be intracellular or extracellular. • none. comments: aged mice of the c57bl/6, 129 and b6,129 strains develop a high incidence of eosinophilic globules in various epithelia, including glandular stomach, respiratory tract, bile duct, gall bladder and pancreatic duct . the protein isolated from the characteristic eosinophilic lesions has been identified as ym1/ym2 a member of the chitinase family that may be produced in response to mucosal irritation rogers and houghton 2009) . the use of the term hyalinosis for this lesion will potentially cause confusion in safety assessment as the same term may also be used for a distinctly different clinical disorder in humans and can be used to describe vascular and renal glomerular changes. synonyms: autophagy, acinar cell pathogenesis: increased autophagocytosis by segregation of cytoplasmic organelles or cytoplasmic contents in sublethally injured acinar cells. diagnostic features • multifocal. • retained lobular architecture. • intracytoplasmic hypereosinophilic to basophilic globules surrounded by a thin clear halo. • apoptosis: apoptotic bodies also have a hypereosinophilic cytoplasm and are surrounded by a clear halo but they always contain basophilic condensed nuclear fragments. apoptosis may be differentiated from autophagy by using tunel staining that marks the nuclear fragments (zhang et al. 2014 ). comments: autophagy is a homeostatic mechanism that plays a major role in the catabolism by lysosomal digestion of organelles and/or aged cytoplasmic contents. autophagy occurs at basal levels in all tissues to maintain cellular homeostasis. however, it is rapidly increased during energy depletion, hypoxia, endoplasmic reticulum stress, high temperature, hormonal stimulation, or cellular remodeling to combat oxidative stress. alterations in autophagy may lead to disruption of cellular homeostasis and can result in pancreatitis and cell death (helin et al. 1980; gukovskaya and gukovsky 2012) . increased autophagic vacuoles in a treated group should always be considered in the context of the control group. autophagy and apoptosis are not necessarily mutually exclusive entities. there is evidence that apoptosis can occur simultaneously with autophagy (maiuri et al. 2007 ). the exocrine acini are very sensitive to agents affecting autophagic flux such as vinblastine (enhances autophagic segregation) or cycloheximide (regression of the autophagic compartment). autophagy may be detected by electron microscopy, by the fluorescent localization pattern of lc3 puncta (intracytoplasmic granular fluorescent material) or by immunohistochemistry using antibodies against lc3-ii (processed lc3). the autophagosomes within the cytoplasm, under light microscopy, appear as discrete vacuoles with some dark eosinophilic or amphophilic contents and under electron microscopy, remnants of mitochondria or endoplasmic reticulum may be observed. it is important to note that accumulation of autophagosomes is not always indicative of autophagy induction since it could be the result of blockage of autophagosomal maturation or increased generation of autophagosomes. autophagic flux assays may measure lc3 turnover, levels of autophagic substrates, mrfp-gfp-lc3 color change, free gfp generated from gfp-lc3, and lysosome-dependent long-lived protein degradation (iovanna and vaccaro 2010) . pathogenesis: gene regulated, energy dependent process leading to formation of apoptotic bodies which are phagocytosed by adjacent cells. and there is no requirement to record them separately or a combined term is preferred for statistical reasons. the term is also used when the type of cell death cannot be determined unequivocally. the approach for the nomenclature and diagnostic criteria of cell death applied here is based on a draft recommendation of the inhand cell death nomenclature working group. apoptosis is not synonymous with necrosis. the main morphological differences between these two types of cell death are cell shrinkage with nuclear fragmentation and tingible body macrophages in apoptosis versus cell swelling, rupture and inflammation in necrosis; however, other morphologies (e.g. nucelar pyknosis and karyorrhexis) overlap. in routine h&e sections where the morphology clearly represents apoptosis or single cell necrosis, or whereby special procedures (e.g. tem or ihc for caspases) prove one or the other, individual diagnoses may be used. however, because of the overlapping morphologies, necrosis and apoptosis are not always readily distinguishable via routine examination, and the two processes may occur sequentially and/or simultaneously depending on intensity and duration of the noxious agent (zeiss 2003) . this often makes it difficult and impractical to differentiate between both during routine light microscopic examination. therefore, the complex term apoptosis/necrosis may be used in routine toxicity studies. a differentiation between apoptosis and single cell necrosis may be required in the context of a given study, in particular if it aims for mechanistic investigations. transmission electron microscopy is considered to represent the gold standard to confirm apoptosis. other confirmatory techniques include dna-laddering (easy to perform but insensitive), tunel (false positives from necrotic cells) or immunohistochemistry for caspases, in particular caspase 3. these techniques are reviewed in detail by elmore (elmore 2007) . some of these confirmatory techniques detect early phases of apoptosis, in contrast to the evaluation of h&e stained sections, which detects late phases only; overall interpretation of findings should take into account these potential differences. thus, low grades of apoptosis may remain unrecognized by evaluation of h&e stained slides only. certain studies may require the consideration of forms of programmed cell death other than apoptosis which requires specific confirmatory techniques (galluzzi et al. 2012) . acinar cell apoptosis is classically manifested in many cases of xenobiotic-induced injury. it is considered a "preferred" response to injury, as it does not lead to subsequent inflammation. an inverse relationship between apoptosis and necrosis in acinar cell injury has been described for various experimental models. stimulation of apoptosis seems to protect against acute necrotizing reactions, while inhibition of apoptosis leads to necrosis and acute inflammation (wallig and sullivan 2013) . exocrine pancreatic acinar apoptosis is observed in rodents treated with synthetic cholecystokinin analog caerulein (reid and walker 1999) , ethionine (fitzgerald and alvizouri 1952; walker et al. 1993) , physical blockage of the pancreas by ductal ligation doi et al. 1997) , duct obstruction involution following soy flour-induced hyperplasia (oates et al. 1986 ) and zinc toxicants (kazacos and van vleet 1989) , copper-deficient diet supplemented with a copper-chelating agent (rao et al. 1993) , azaserine (woutersen 1996) , lipopolysaccharide (laine et al. 1996) and pan cdk inhibitor (ramiro-ibáñez et al. 2005 ). in addition, exocrine pancreatic apoptosis is also observed in some genetically modified mice such as serpini2-deficient mouse model for pancreatic insufficiency (loftus et al. 2005) . non-treatment associated sporadic apoptosis may be noted in pancreas from rodents and this is part of the normal tissue homeostasis and is considered as a background change. acinar cell apoptosis may be increased in fasted or chronically anorectic animals. the degree of apoptosis is in these cases lower than that of acute pancreatic injury. synonyms: oncotic cell death; oncotic necrosis; necrosis pathogenesis: unregulated, energy independent, passive cell death with leakage of cytoplasm into surrounding tissue and subsequent inflammatory reaction; may be induced by direct contact with a test article after oral uptake/administration. • focal affecting cell groups, lobular, diffuse. • cells swollen with pale eosinophilic cytoplasm. • loss of nuclear basophilia, pyknosis and/or karyorrhexis that affects aggregates of cells. • in more severe cases, there may be detachment of the epithelium from the submucosa. • typically, the presence of degenerative cells is a component of necrosis. • minimal or slight inflammatory cell infiltrates, edema and fibrin may be present as a feature of necrosis. single cell • only single cells affected. • apoptosis: the morphologic features of cell death clearly fit with apoptosis (cell and nucleus shrunken, hypereosinophilic cytoplasm, nuclear pyknosis etc.) and/or special techniques demonstrate apoptosis. • apoptosis/necrosis: both types of cell death are present and there is no requirement to record them separately or a combined term is preferred for statistical reasons. the term is also used when the type of cell death cannot be determined unequivocally. • inflammation, acute: infiltrates of inflammatory cells, edema, and fibrin predominate; necrosis may be present, but is a minor component. comments: necrosis is usually accompanied by an inflammatory cell infiltrate and may be a significant component of acute inflammation. usually, either necrosis or acute inflammation will be recorded. however, in the context of a specific study it may be more appropriate to record both processes separately. in the pancreas, dead cells quickly pass through the stage of coagulation necrosis to liquefaction because of the high content of hydrolytic enzymes activated and released into the interstitial tissue. necrosis of acinar tissue develops very rapidly and is best recognized within 12-48 hours after the insult (wallig and sullivan 2013) . pathogenesis: unregulated, energy independent, passive cell death with leakage of cytoplasm into surrounding tissue and subsequent inflammatory reaction (single cell necrosis) and/or gene regulated, energy dependent process leading to formation of apoptotic bodies which are phagocytosed by adjacent cells (apoptosis). • both types of cell death are present and there is no requirement to record them separately or a combined term is preferred for statistical reasons or • the type of cell death cannot be determined unequivocally. • apoptosis: the morphologic features of cell death clearly fit with apoptosis (cell and nucleus shrunken, hypereosinophilic cytoplasm, nuclear pyknosis etc.) and/or special techniques demonstrate apoptosis and there is a requirement for recording apoptosis and necrosis separately. • necrosis: the morphologic features of cell death clearly fit with necrotic diagnostic criteria (cells and nuclei swollen, pale cytoplasm etc.) and there is a requirement for recording apoptosis and necrosis separately. comments: necrosis and apoptosis are not always readily distinguishable via routine examination, and the two processes may occur sequentially and/or simultaneously depending on intensity and duration of the noxious agent (zeiss 2003) . this often makes it difficult and impractical to differentiate between both during routine light microscopic examination. in these cases, the combined term apoptosis/necrosis may be used. it is recommended to explain in detail the use of this combined term in the narrative part of the pathology report. pathogenesis: decrease of acinar cell zymogen granules leading to shrunken acinar cells with increased basophilia. • focal, lobular, or diffuse lesion. • reduced acinar diameter. • partial or complete loss of acinar cell zymogen granules leading to a reduced cell size and increased basophilia. • nuclei of acinar cells inactive • lack of fibrosis or adipocyte infiltration. • islets of langerhans are unaffected. • atrophy, acinar cell: loss of acinar cell basophilia and decreased zymogen granules resulting in small acini lined by small columnar cells almost completely devoid of cytoplasm and with a small and inactive nucleus; may be accompanied by fibrosis and minimal mononuclear cell infiltrates. • focus, basophilic: basophilic cells of normal size and commonly slightly enlarged; nulcei slightly enlarged and with prominent nucleoli. • peri-insular halos: tele-insular acinar cells have relatively less zymogen granules when compared to periinsular acinar cells. the morphology of the exocrine pancreatic acini may be altered due to several physiological conditions, such as anorexia/inanition. the volume of the zymogen granules and rer is reciprocally related. during protein synthesis, rer is increased with a proportionate decrease in zymogen granules. in moribund and/or anorexic animals with protein deficiency, the zymogen granules are decreased and the cells are shrunken with apparently increased basophilia due to relative predominance of the basally located basophilic cytoplasm (longnecker and wilson 2002) . pathogenesis: decrease in number and/or size of acinar cells may be due to spontaneous or experimentally induced degenerative changes, apoptosis, or a sequel of chronic inflammation. • focal, lobular, or diffuse. • reduction in the number and/or size of acini. • loss of acinar cell basophilia and decreased zymogen granules resulting in small acini lined by small columnar cells almost completely devoid of cytoplasm and with a small and inactive nucleus. • relatively prominent intra and inter-lobular ducts. • pyknotic and karyorrhectic nuclei, apoptotic bodies, and mitotic figures may be present. • scattered to coalescing adipocytes within the interstitium. • variable degree of interstitial fibrosis. • variably dilated cyst-like or duct-like acini and/or ducts lined by cuboidal or flattened epithelial cells. • transitional structures containing mixtures of normal acinar cells, atrophic acinar cells, and cuboidal ductal cells. • may be accompanied by minimal to mild infiltration of lymphocytes or macrophages. • islets of langerhans are unaffected. • degranulation, acinar cell: diffuse reduction of zymogen granules but maintenance of the basophilic basal cytoplasm; no fibrosis or adipocyte infiltration. • peri-insular halos: tele-insular acinar cells have relatively less zymogen granules and more rer when compared to peri-insular acinar cells. in certain planes of section, areas with more tele-insular regions may give a false impression of atrophy. • inflammation, chronic: infiltration of mononuclear inflammatory cells and interstitial fibrosis are frequently accompanied by acinar cell atrophy of variable degree. comment: exocrine acinar atrophy is the most common spontaneous degenerative change in pancreas of both rats and mice. acinar atrophy may range from focal atrophic exocrine acini with no inflammation or fibrosis to diffuse atrophy of exocrine acini with replacement by adipose tissue and residual ducts, vasculature and islets of langerhans. acinar atrophy frequently represents the sequel of chronic inflammation and, as such, is often accompanied by infiltrates of mononuclear cells and fibrosis. it is recommended to record in standard toxicity studies for such lesions either atrophy or chronic inflammation, but not both. inflammation, chronic, should be recorded only, if the inflammatory changes are the predominant component. in rats, the acinar atrophy increases with age and the incidence varies with sex (males > females) and strain (bn/bi/wag/rij(f1) > crl:cd(sd) br = bn/bi > slc:wistar > hap:(sd) > f344 > osborne-mendel). in b6c3f1 mice, the incidence of spontaneous exocrine pancreatic atrophy is 1-2% in 2-year studies (boorman and sills 1999) . in rats, chronic protein or essential amino acid deficiency, copper deficiency, zinc deficiency, ethionine, hypophysectomy or high doses of glucagon induce loss of zymogen granules and pancreatic atrophy (svoboda et al. 1966; kitagawa and ono 1986; rao et al. 1987; koo and turk 1977) . malonaldehyde caused atrophy of exocrine pancreas in both male and female mice (ntp, 1988) . acinar atrophy has also been induced by pancreatic duct ligation eustis et al. 1990) . pathogenesis: deposition of mineral either secondary to cellular necrosis (dystrophic mineralization) or secondary to hypercalemia (metastatic mineralization). • focal, lobular, or diffuse. • deposits of dark basophilic granular material (on h&e) within exocrine acini, ducts or interstitium. • artifacts: hematoxylin stain deposits (distribution unrelated to tissue structures), acid hematin (use of unbuffered formalin; dark brown to black, tropism for erythrocytes). • pigment: lipofuscin and porphyrin appear brown to golden brown in h&e slides and may further be differentiated from mineralization by special stains. comment: dystrophic calcification occurs in necrotic/injured tissue even in animals with reference range serum calcium levels, whereas metastatic calcification occurs in hypercalcemic animals throughout the body, especially within interstitial tissues and blood vessel walls. the two commonly used stains for detecting calcium are alizarin red and von kossa. pathogenesis: extracellular deposits of chemically diverse complex insoluble polypeptides. • light eosinophilic homogenous amorphous extracellular material within the tunica media of blood vessels, within the interstitium and along basement membranes. • variable degree of atrophy and loss of intervening acini and ducts may occur, if amyloid deposition is prominent. • green birefringence using polarized light with congo red stain. • inflammation: the inflammatory cell infiltrate is associated with additional morphological features of inflammation such as edema, hemorrhage, necrosis, and/or fibroplasia. • granulocytic leukemia: infiltration of neutrophil precursors and/or abnormal neutrophils will probably be present along with mature neutrophils; infiltration of similar neoplastic cells is likely present in other organs. • lymphoma: infiltration of monomorphic population of lymphocytes (often with atypical, increased and/or abnormal mitoses); infiltration of similar neoplastic cells is likely present in other organs. in toxicity studies, inflammatory cell infiltrates are encountered more commonly than inflammation (pancreatitis). the term pancreatitis (or inflammation) should not be confused with or used in lieu of infiltrate (plus cell type). the term infiltrate (plus cell type) should be used instead of inflammation (or pancreatitis) when there is no tissue injury/reaction associated with the inflammatory cell infiltrate. also, the inflammatory cell infiltrate should be qualified with the type (name) of the cells that are predominant, such as lymphocytic, neutrophilic, etc. the use of terminology summarizing the nature of the infiltrates as suppurative (in lieu of neutrophilic), granulomatous (in lieu of macrophage), and chronic (in lieu of lymphocytic, plasmacytic, mononuclear) is not recommended since these terms imply inflammation. the term inflammation (pancreatitis) should be reserved for lesions where the inflammatory cell infiltrate is associated with tissue injury/reaction such as edema, congestion, acinar cell degranulation, necrosis, vascular fibrinoid necrosis, hemorrhage, fat necrosis or saponification (mann et al. 2012) . modifier: type of inflammatory cell that represents the predominant cell type in the inflammation pathogenesis: infiltration with neutrophils (infiltrate, neutrophil), eosinophils (infiltrate, eosinophil), mononuclear cells (infiltrate, mononuclear cell) or a combination of more than one type (infiltrate, mixed) with additional histological features of inflammation, e.g. hemorrhage, edema, fibroplasia. in control rats, the most common cause of inflammation is spontaneous arteritis involving the pancreatoduodenal artery (coleman et al. 1977) . for pancreatitis induced by xenobiotics, the pathogenesis usually involves release of inactive proenzymes from exocrine pancreas and activation of these digestive enzymes locally and systemically. • focally extensive or diffuse, involving predominantly the interstitium, but also acinar cells and intra-or interlobular ducts. • infiltrate of mononuclear or polymorphonuclear leukocytes into the gland parenchyma. • presence of other histological criteria of inflammation, e.g. hemorrhage, edema, fibroplasias. • islets of langerhans may be entrapped within the inflammation. inflammation, neutrophil • infiltrate predominantly neutrophilic; frequently accompanied by edema and congestion; acinar cell degranulation and necrosis, fibrinoid necrosis of small vessels and hemorrhage as well as fat necrosis and saponification may be present. • infiltrate predominantly mononuclear cell; frequently accompanied by acinar cell atrophy and mild acinar cell degeneration, ductal proliferation, interstitial fibrosis; mineralization of acini may be present. • infiltrate, inflammatory cell: other histologic features of inflammation such as edema, hemorrhage, necrosis, and/ or fibroplasias will be absent. • granulocytic leukemia: infiltration of neutrophil precursors and/or abnormal neutrophils will probably be present along with mature neutrophils; infiltration of similar neoplastic cells is likely present in other organs. • lymphoma: infiltration of monomorphic population of lymphocytes (often with atypical, increased and/or abnormal mitoses); infiltration of similar neoplastic cells is likely present in other organs. • postmortem autolysis: uniform and often diffuse change in acinar cells starting with loss of basophilia, gradual loss of definition and final lysis; no inflammatory cell infiltration, congestion or edema. • atrophic, acinar cell: minimal to mild infiltrates of mononuclear cells and fibrosis, but atrophy is the predominant component. comment: when a mononuclear cell or mixed infiltrate is considered to represent an inflammatory process (inflammation, mononuclear cell or inflammation, mixed) and other morphologic features (acinar cell atrophy and mild acinar cell degeneration, ductal proliferation, interstitial fibrosis; mineralization of acini) associated with the infiltrate suggest an ongoing/continuing inflammatory process, the modifier 'chronic' may be included in the diagnosis (i.e. inflammation, mononuclear cell, chronic) to better characterize the finding. spontaneous inflammation, neutrophilic or mixed of the pancreas is very uncommon in control mice and rats. the severity of inflammation within pancreas can range from mild edematous inflammation to severe necrotizing inflammation. acute inflammation is observed in humans and some rodents treated with several classes of drugs belonging to antimicrobials, estrogens, corticosteroids, diuretics, antibiotics, analgesics/anti-inflammatory agents, statins, ace inhibitors, and highly active antiretroviral therapy (haart) drugs (badalov et al. 2007) . several animal models are used for studying pancreatitis such as choline-deficient ethionine supplemented diet; intraperitoneal injection of arginine; intraperitoneal or intravenous administration of secretogogues such as cholecystokinin analogue (caerulein), muscarinic receptor agonist (carbachol), anticholinesterases (organophosphate); sepsis (lps); vascular compromise (hypovolemic shock, occlusion of pancreatoduodenal artery, occlusion of splenic and/or gastroduodenal vein occlusion); closed duodenal loop procedure; and obstruction of pancreatic duct or common biliopancreatic duct (chan and leung 2007) . acute pancreatitis and necrosis are also seen in cases of zinc toxicity, copper deficiency and extension of inflammation from other lesions such as gastritis secondary to oral gavage of irritating chemicals (boorman and sills 1999; greaves 2012) . spontaneous chronic inflammation is sporadically found in aged rats and mice. chronic inflammation occurs more commonly in diabetic (bb) wistar rats than their non-diabetic counterparts (wright et al. 1983) . chronic exposure to manganese causes chronic pancreatitis, fibrosis and acinar atrophy in rats (scheuhammer 1983) . in many cases, chronic inflammation progresses into pancreatic atrophy and subsequent multifocal to diffuse fatty infiltration. acinar atrophy frequently represents the sequel of chronic inflammation and, as such, is often accompanied by infiltrates of mononuclear cells and fibrosis. it is recommended to record in standard toxicity studies for such lesions the predominant morphology, either atrophy or chronic inflammation, not both. inflammation, chronic, should be recorded only, if the inflammatory changes are the predominant component. refer also to the inhand monograph on the cardiovascular system. synonyms: arteritis; vasculitis; polyarteritis pathogenesis: spontaneous or induced inflammation of vascular wall in the pancreas. • typically affects muscular wall of small to medium sized arteries or veins. • variable mixed inflammatory cell infiltrate within and around vessel. • possible presence of fibrinoid necrosis of vessel wall. • proliferation of endothelium and thickening (fibrosis) of vessel wall. • inflammation, acute: inflammatory processes predominantly within the parenchyma; vascular involvement only secondary and representing a minor proportion of the inflammatory process. • infiltrate, inflammatory cell: lack of vascular orientation, no other features of inflammation such as hemorrhage or fibrinoid necrosis. comment: inflammation of the vasculature, if present, is frequently noted in histological sections of pancreas due to the prominent pancreato-duodenal artery and other small caliber interlobular pancreatic arteries, and mesenteric vessels in the vicinity. arteritis lesions may be due to immune-mediated etiology as seen in (nzbxnzw)f1 hybrid, and mrl/mp mice or due to hypertension as seen in spontaneously hypertensive rat (shr) strains. arteritis due to drug/chemical treatment is occasionally observed and may depend on the species, strain and sex. examples include vasoactive agents like angiotensin, norepinephrine, dopamine agonists (like fenoldopam mesylate) and xanthine compounds which cause focal to diffuse medial necrosis and hemorrhage especially within the small and medium-caliber mesenteric, pancreatic and renal arteries; and non-vasoactive compounds like 2-amino-5-nitrophenol, nitrofurantoin, or phenacetin. pathogenesis: accumulation of tissue fluid in the interstitium resulting from increased vascular permeability due to release of pancreatic enzymes, increased hydrostatic pressure in vasculature, decreased oncotic pressure, impaired coagulation system, or impaired lymphatic drainage. • focally extensive or diffuse. • distension of the interstitial tissue forming clear spaces or spaces filled with lightly eosinophilic material. • minimal inflammatory cell infiltrate may be present in some cases. • increase in total pancreatic volume and weight. • inflammation, acute: usually associated with tissue and vascular damage resulting in exudative edema and inflammatory cell infiltrate. comment: edema within the exocrine pancreas may be exudative (inflammatory edema due to capillary injury secondary to leakage of pancreatic enzymes) or transudative (hydrodynamic derangement secondary to increased hydrostatic osmotic pressure and decreased oncotic pressure). the exudative edema is usually accompanied by inflammatory cell infiltrate and tissue injury. examples of chemical induced pancreatic edema have been documented in mice treated with p-nitrophenol and rats treated with d & c yellow no. 11 and pcb mixtures in us-ntp studies. pathogenesis: hemorrhage can occur from acinar necrosis, inflammation, vascular injury, or tumors. • presence of erythrocytes in interstitial tissue, acini, ductules, or ducts. • angiectasis: blood present within dilated vascular lumina. • artifact: erythrocytes on the surface of the pancreas sample or infiltrating from the surface into interstitial tissue; a result of necropsy procedure. comment: islet hemorrhage has been described as a spontaneous finding in sprague-dawley rats (imaoka et. al. 2007 ). if prominent, this may involve peri-insular exocrine tissue. pathogenesis: increased size of pancreatic exocrine acinar cells due to increased trophic factors. • usually diffuse distribution. • enlarged acinar cells with more abundant cytoplasmic volume and more numerous zymogen granules. • no increase in the number of cells per acinus. • larger nuclei with prominent nucleoli. • increase in pancreas to body weight ratios. • halos, peri-insular, increased: characteristic distribution pattern; exocrine acini (peri-insular) surrounding the islets of langerhans have more zymogen granules and slightly larger nuclei than their distal (tele-insular) counterparts. • hyperplasia, acinar cell: increased number of cells per acinus. comment: exocrine pancreatic acinar hypertrophy results from dietary and trophic factors. feeding rats with raw but not heat-treated soy flour (lack trypsin inhibitors) caused acinar hypertrophy initially. however, with continued feeding, the hypertrophic change progressed to hyperplasia with marked increase in dna content. this hypertrophic change is attributed to the presence of trypsin inhibitors within the raw soy flour and its ability to stimulate the secretion of cholecystokinin by feed back mechanism (crass and morgan 1982; folsch et al. 1978) . similarly, administration of gastrin (but not secretin analogues) like pentagastrin, pancreozymin, and peptavlon also caused pancreatic acinar cell hypertrophy (rothman, and wells 1967) . administration of isoprenaline (isoproterenol) also cased an increase in pancreatic weight due to acinar cell hypertrophy and increase in zymogen granules (sturgess and reid 1973) . however, pancreas weight and pancreas to body weight ratios are not commonly recorded or calculated in toxicity studies. synonyms: eosinophilic change; focal eosinophilic hypertrophic cells; hypertrophy, peri-insular pathogenesis: hypertrophy of pancreatic exocrine acinar cells surrounding the islets of langerhans. • hypertrophy of exocrine pancreatic acini surrounding the islets of langerhans. • acinar cells with more abundant cytoplasmic volume with larger zymogen granules than tele-insular acinar cells (located distantly from the islets). • larger nuclei with more nucleoli than tele-insular acinar cells. • hypertrophy, acinar cell: no local association with islets, usually multifocal to diffuse distribution. the difference in the size of peri-and tele-insular acinar cells is greater in mice than in rats, resulting in more prominent peri-insular halos in mice. the peri-insular acinar cells are more resistant (3 hours) to pilocarpine-induced degranulation than the tele-insular acinar cells (1 hour). the size of peri-insular halos is markedly reduced in alloxaninduced diabetic pancreata compared to control non-diabetic rats. these features of the peri-insular halos are due to the hormones (mainly ghrelin and insulin) secreted by the betacells in islets of langerhans that are locally enriched due to the insulo-acinar capillary anastomoses or due to diffusion. the peri-insular halos are not recorded in routine toxicity studies but recording any alterations within these halos may provide important information about chemicals affecting islet cells such as alloxan or about chemicals directly affecting the exocrine pancreas like pilocarpine. pathogenesis: degranulation of the peri-insular acinar cells following loss or decrease in trophic factors released from the surrounding the islets of langerhans. • decreased zymogen granules within exocrine pancreatic acini surrounding the islets of langerhans. • loss of size distinction between the peri-insular acini located adjacent to the islets of langerhans (usually larger) and the tele-insular acini located distantly from the islets (usually smaller than peri-insular acini) comment: the difference in the size of peri-and tele-insular acinar cells is greater in mice than in rats, resulting in more prominent peri-insular halos in mice. the peri-insular acinar cells are more resistant (3 hours) to pilocarpine-induced degranulation than the tele-insular acinar cells (1 hour). the size of peri-insular halos is markedly reduced in alloxaninduced diabetic pancreata compared to control non-diabetic rats. these features of the peri-insular halos are due to the hormones (mainly insulin) secreted by the β-cells in islets of langerhans that are locally enriched due to the insulo-acinar capillary anastomoses or due to diffusion. the peri-insular halos are not recorded in routine toxicity studies but recording any alterations within these halos may provide important information about chemicals affecting islet cells such as alloxan or about chemicals directly affecting the exocrine pancreas like pilocarpine. synonyms: basophilic-atypical acinar cell focus; focal cellular change; focal basophilic cellular change; cytological alteration pathogenesis: focal formation of morphologically atypical pancreatic exocrine acinar cells. • tinctorially distinct focus with decreased zymogen granules and increased basophilia due to abundance of rer. • affects single or multiple contiguous acini with oval to irregular shape without encapsulation and with retained angular shape of the pancreatic lobe. • no compression or displacement of adjacent pancreatic acini • cellular hypertrophy is commonly seen. • slightly enlarged basal to parabasal nuclei with prominent nucleoli. • hyperplasia, acinar cell, focal: increased number of cells per acinus, usually no increase of cytoplasmic basophilia. • zymogen granules decreased, acinar cell: reduced cell size due to loss of zymogen granules, nuclei inactive, the incidence of basophilic foci is 6.6% in control and 5.4% in corn oil gavage historical control male f344 rats in the ntp database . basophilic foci occur also in mice, but at a much lower incidence than in rats (roebuck et al. 1980) . there is an increase in the incidence of basophilic foci in rats treated with 4-hydroxyaminoquinoline-1-oxide (4haqo), 7,12-dimethyl-benzanthracene (dmba), n5-(n-methyl-n-nitrosocarbamoyl)-l-ornithine (mnco), raw soy bean flour, plant trypsin inhibitors, and azaserine. the proliferative ability of exocrine acinar cytological alterations as measured by tritiated thymidine autoradiography and mitotic index indicated that basophilic foci have similar proliferative ability as the surrounding normal exocrine acini (rao et al. 1989 ). thus, unlike focal hyperplasia, the basophilic foci should not be considered as preneoplastic . the cytologic appearance of acinar cells within the basophilic foci may indicate an altered capacity for, or a defect in, the synthesis of secretory proteins. basophilic foci stained positively with gammaglutamyltranspetidase (ggt) while eosinophilic foci stained negatively. a microscopic morphological entity described as spontaneous pancreatic acinar hypertrophic foci in rats by chiu (chiu 1983 ) may indeed be basophilic foci because they share several features such as increased incidence with age, and they are non-hyperplastic and non-neoplastic. pathogenesis: periductal oval cells or acinar/endocrine intermediate transitional cells transdifferentiate into foci morphologically identical to hepatocytes within the liver. • polygonal cells with central nuclei and abundant finely granular eosinophilic cytoplasm (morphologically identical to hepatocytes). • located adjacent to ducts and islets of langerhans. • usually well integrated into surrounding tissue. • hepatocytes have well developed bile canaliculi • hypertrophy, acinar cell: pancreatic exocrine acini; focal to lobar distribution. • halos, peri-insular: pancreatic exocrine acini; almost always surrounding islets. • ectopic liver tissue: usually circumscribed and not wholly integrated into the 'host' tissue; no evidence of dysplasia or history of tissue injury. comment: the incidence of metaplastic hepatic foci is less than 1% in control rats and mice. these lesions are occasionally observed in the rat pancreas and less frequently in the mouse pancreas. the pathogenesis of these sporadic lesions is not known. experimentally, these metaplastic hepatic foci within the rat exocrine pancreas may be induced by several protocols such as copper depletion followed by copper repletion (s), ciprofibrate feed model , and multiple subcutaneous injections of cadmium chloride (konishi et al. 1990) . metaplastic hepatic foci in the pancreas were also induced in a transgenic mouse model where kgf (fgf7) was over-expressed in the pancreas under the control of the rat insulin promoter (krakowski et al. 1999 ). an increase in metaplastic hepatic foci in toxicological studies may also be due to the result of test article-induced pancreatic atrophy and subsequent hepatic transdifferentiation. synonym: tubular complexes pathogenesis: acinar cells transdifferentiate or take the appearance of ducts especially in areas of chronic pancreatitis. • focal, circumscribed lesions surrounded by normal exocrine acini. • variable caliber ductules lined by flat cuboidal epithelial cells. • located in the acinar compartment rather than in the native ducts. • intermixed with pale atrophic exocrine acini. • islets not usually involved. • hyperplasia, ductular: do not have intermixed pale exocrine acini. • atrophy, acinar cell: ductular metaplasia may a component in some cases of atrophy and is considered a reparative process. comment: exocrine acinar ductular metaplasia is usually secondary to chronic pancreatitis and atrophy and is considered a reparative process. the diagnosis of ductular metaplasia is not commonly used in toxicologic studies since it is one of the features of atrophy. in dmba implantation studies in rats that resulted in pancreatic ductular adenocarcinoma, these lesions were considered preneoplastic; however, in the majority of cases these lesions do not progress to neoplasia and are of reparative or adaptive nature. the diagnosis of ductular metaplasia may be considered in those studies where a progression to ductular hyperplasia and neoplasia is noted. synonyms: dilatation; luminal distension; cystic duct; ductal cyst pathogenesis: luminal dilatation due blockage of the intralobular or interlobular ducts secondary to fibrosis or due to calculi. • solitary or multiple tortuous irregular enlarged lumina. • lined by flattened cuboidal epithelial cells. • may be accompanied by mild interstitial infiltration of lymphocytes and plasma cells. • inspissated secretions may be present. comment: duct ectasia is the most common age-associated ductal change in albino and gray norway, august hooded, fisher 344, hap:(sd), and crl:cobs cd (sd) rats (denda et al. 1994) . the lesions of duct ectasia or interlobular cystic ducts are uncommon and the incidence is usually less than 1% in rats and mice. these lesions usually result from blockage of inter-and intra-lobular ducts either proximally or distally due to chronic pancreatitis or rare inspissated secretory material. pathogenesis: pancreatic stellate cells reaction to acute or chronic inflammation or toxicity. • focal, lobular, or diffuse. • increase in the interstitial connective tissue due to pancreatic stellate cell proliferation and collagen deposition. • may be accompanied by inflammatory cell infiltrates. • amyloid: abundant extracellular eosinophilic amorphous ground substance that exhibits apple green birefringence in congo red stained sections under polarized light and absence of fibroblast cell proliferation. comment: collagen may be identified by its birefringence under polarized light and can be stained histochemically using masson's trichrome, and van gieson protocols. in some adult control rats, there may be some peri-insular interstitial fibrosis that extends for short distance into exocrine pancreas. this should be distinguished from fibrosis resulting from tissue insults based on the severity, distribution and the accompanying chronic inflammatory cell infiltrates. the lesions described here cover the currently known spectrum of proliferative findings in conventional strains of (wildtype) rats and mice. the majority of proliferative lesions in transgenic mouse models of human pancreatic cancer resemble the lesions of wildtype animals. lesions that occur only in specific transgenic mouse models, like mucinous cystadenomas with ovarian-like stroma, are not described here (hruban et al. 2006) . hyperplasias and tumors of the exocrine pancreas are rare and age-related conditions in control rats and mice (longnecker and millar 1990; boorman and sills 1999) . the separation of acinar hyperplasia and adenoma can be difficult and in the end may be arbitrary as these lesions seem to represent a continuum. hyperplasia of ducts in circumscribed fibrous areas is often found in aged rats. duct-like structures do not indicate an origin from pancreatic ducts as is the case in humans. although hyperplasia of ducts may be seen in aged rats, it is still questionable if true ductular neoplasia occurs in rats such as humans (cystadenoma, cystadenocarcinoma) (greaves and faccini 1992) . in mice, no spontaneous ductal cell hyperplasia or neoplasia has been described. animal models with carcinogens revealed that rats develop acinar cell carcinomas whereas hamsters develop ductal carcinomas with the same carcinogen. this suggests that the species is a major determinant of the phenotype in carcinomas of the pancreas (longnecker 1986; longnecker 1994) . mice are relatively insensitive to the induction of pancreatic tumors with chemicals that are known to be tumorigens in rats (boorman and sills 1999) . hyperplasia, acinar cell (figures 228 and 229) synonym: focus, eosinophilic histogenesis: acinar cells. • focal lesions usually larger than an average, unchanged islet. • increased number of cells per acinus. • may exhibit tinctorial variation from surrounding tissue. • merge usually imperceptibly with adjacent tissue; no or only minimal compression or displacement of adjacent acinar tissue. • usually non-encapsulated. • acinar architecture usually maintained or slightly altered at the most. • low to high mitotic index, nuclear polymorphism and nuclear crowding possible. • adenoma, acinar cell: distinct demarcation from the surrounding tissue with compression and sometimes encapsulation; altered architecture. • focus, basophilic: enlarged acinar structures with increased basophilia due to abundance of rer; number of cells per acinus not increased; no compression, acinar structure not altered. • hypertrophy, acinar cell: enlarged acinar cells with increased zymogen granules; number of cells per acinus not increased; usually diffuse distribution. comment: the separation of acinar hyperplasia and adenoma can be difficult and in the end may be arbitrary as these seem to be a continuum. some foci of hyperplasia may be just at the edge of an adenoma and thus categorized as such due to plane of section. the grade of alteration of acinar structure and compression of adjacent tissue are the most important differential diagnostic criteria. a sub-category of focal acinar hyperplasia with cell en-largement due to an increase of zymogen granules has been termed "focus, eosinophilic". eosinophilic foci are thought to be part of a continuum of lesions that subsequently progress into adenomas and adenocarcinoma (rao et al. 1989; scarpelli et al. 1984) . in contrast to basophilic foci, these foci are positive for atpase and glutathione-s-transferase µ histochemical stains. the incidence of eosinophilic foci is 2.6% in male f344 rats (eustis and boorman 1985) . eosinophilic foci occur also in mice, but at a much lower incidence than in rats (roebuck et al. 1980 ). due to the difficulties to differentiate eosinophilic foci from focal hyperplasia in h&e stained sections, it is recommended to diagnose eosinophilic foci not separately but to collect them under focal hyperplasia in this nomenclature system. acinar hyperplasias have a low spontaneous incidence in rats and mice that increase with age (longnecker and millar 1990; boorman and sills 1999) . histogenesis: intra-and inter-lobular pancreatic ducts. • multiple cross section profiles of tortuous hyperchromatic ductular cells within the acinar parenchyma. • ducts with increased number of lining epithelial cells often forming papillae. • protruding papillae may cause partial obliteration of the lumen. • varying levels of cellular pleomorphism and atypia may be present. • dilated tubules lined by flattened epithelium may occur. • mucus metaplasia in hyperplastic duct epithelium may occur in transgenic mouse models of pancreatic neoplasia. • adenoma, ductal cell: complex of ductular structures; focal acinar atrophy leading to increased numbers of ducts/unit area. comment: hyperplasia ductal cell is more frequently noted within the intrapancreatic ducts than in the extrapancreatic ducts. hyperplasia of ducts in circumscribed fibrous areas is often found in aged rats. in mice, no spontaneous ductal cell hyperplasia or neoplasia has been described. ductal phenotype may occur under experimental conditions after administration of dmba (wendt et al. 2007 ), infection with reovirus 3 (greaves 2012), feeding a western-style diet (xue et al. 1996) , and in transgenic mice (kras oncogene) (grippo et al. 2003) . transgenic mouse models for human pancreatic cancer may develop atypical ductal hyperplasia with mucin accumulation within tall columnar cells lining the ducts (figure 231 ) (hingorani et al. 2003; aguirre et al. 2003) . following human pathology, these preneoplastic lesions are termed murine pancreatic intraepithelial neoplasia (mpanins) (hruban et al. 2006 ). morphologically they range from ducts lined with an increased number of monomorphic tall columnar mucinous cells to multilayered lesions with cribriform growth and loss of cell polarity (figure 232) . a grading scheme of panin has been introduced (hruban et al. 2006) . adenoma, acinar cell (figures 233 and 234) histogenesis: acinar cells • compression of adjacent tissue often present. • encapsulation may occur but is often not present. • sharp demarcation from surrounding normal pancreatic tissue. • architecture is increasingly altered with increasing size. • cells usually well-differentiated. • exhibit variable degree of mitotic index, and nuclear polymorphism. • hyperplasia, acinar cell: no encapsulation or sharp demarcation, no or only slight alteration of acinar structure. • adenocarcinoma, acinar cell: local invasion or metastases. comment the separation of acinar hyperplasia and adenoma can be difficult and in the end may be arbitrary as these seem to be a continuum. some foci of hyperplasia may be just at the edge of an adenoma and thus categorized as such due to plane of section. histogenesis: intrapancreatic ducts. • complex of ductular structures lined by a high cuboidal epithelium resembling that of normal ductules. • hyperplasia, ductal cell: ducts with hyperplastic cells, only partly obliterating the lumen, found within focal fibrous areas. comment: duct-like structures do not indicate an origin from pancreatic ducts such as occurs in humans. although hyperplasia of ducts may be seen in aged rats, it is still questionable if true ductular neoplasia occurs in rats as it does in humans (cystadenoma, cystadenocarcinoma) (greaves and faccini 1992) . in mice, no spontaneous ductal cell hyperplasia or neoplasia have been described. pancreatic neoplasm with ductal phenotype may occur under experimental conditions after administration with dmba (wendt et al. 2007 ), infection with reo-virus 3 (greaves 2012), feeding a western-style diet (xue et al. 1996) , and in transgenic mice (kras oncogene) (grippo et al. 2003) . histogenesis: acinar cells. diagnostic features • glandular, trabecular or solid growth pattern. • loss of acinar architecture. • cellular pleomorphism and anaplasia. • local invasion or distant metastases. • may be associated with scirrhous reactions. • adenoma, acinar cell: no local invasion or metastases. comment: animal models of chemical carcinogenesis revealed that rats develop acinar cell carcinomas whereas hamsters develop ductal carcinomas with the same carcinogen. this suggests that the species is a major determinant of the phenotype in carcinomas of the pancreas (longnecker 1986; longnecker 1994) . azaserine is an effective pancreatic carcinogen in rats which initiates a sequence of focal proliferative changes in acinar cells that develop into carcinomas. the acinar phenotype is mostly retained but focal development of duct-like structures has been noted in a few carcinomas (longnecker et al. 1992) . mice are relatively insensitive to the induction of pancreatic tumors with chemicals that are known to be tumorigenic in rats (boorman and sills 1999) . squamous cell carcinoma of the exocrine pancreas has been experimentally induced in mice and has morphologic features of squamous cell carcinoma that occurs at other sites (dixon and maronpot 1994) . the transgenic mouse model bearing the elastase promoter-sv40-early antigen construct (ela-1-sv40 t) develop focal acinar cell proliferation that progresses into acinar cell carcinomas while mostly retaining acinar differentiation, although in some cases they can develop into undifferentiated tumors (glasner et al. 1992) . a consensus report and recommendations for a standardized nomenclature with definitions and associated images was developed for the pathology of genetically-engineered mouse models of pancreatic exocrine neoplasia (hruban et al. 2006) . histogenesis: intrapancreatic ducts. • malignant features, invasive growth. • cell atypia. • duct-like structures often accompanied by a dense fibrous stroma. • adenocarcinoma, acinar cell: normally no ductular structure. comment: duct-like structures do not indicate an origin from pancreatic ducts such as occurs in humans. although hyperplasia of ducts may be seen in aged rats, it is still questionable if true ductular neoplasia occurs in rats as it does in humans (cystadenoma, cystadenocarcinoma) (greaves and faccini 1992) . pancreatic adenocarcinomas induced by dmba implantation in rats showed strong keratin, cytokeratin 19 and cytokeratin 20 expression by immunohistochemistry, consistent with a ductal phenotype (jimenez et al. 1999) . implantation of dmba in rats induced transdifferentiation of acinar cells to ductal cells and provided precursor lesions (tubular complexes) for the development of ductal adenocarcinoma (bockman et al. 2003) . in mice, no spontaneous ductal cell hyperplasia or neoplasia has been described. pancreatic intraepithelial precursor lesions in small caliber pancreatic ducts and ductal adenocarcinoma were induced by dmba implantation in mice (wendt et al. 2007 ). aggressive pancreatic ductal adenocarcinomas may occur in transgenic mice (kras oncogene) after blockage of tgf-β signalling (ijichi et al. 2006) . a consensus report and recommendations for a standardized nomenclature with definitions and associated images was developed for the pathology of genetically-engineered mouse models of pancreatic exocrine neoplasia (hruban et al. 2006) . in genetically-engineered mice, panin has been described as a preneoplastic lesion for adenocarcinomas, but conclusive histopathogenesis has not been shown. we thank dr. rupert kellner for manuscript review and ms. beth mahler, epl inc., for image editing. in addition, we acknowledge the tremendous help provided by ms. emily singletary, epl inc., in providing several images from the national toxicology program image database. we also thank dr. kyathanahalli janardhan, ils inc., for providing immunohistochemistry and hematoxylin & esoin images of gastrointestinal stromal tumors and gastric neuroendocrine tumors. figure 67 indicating maintained polarity of epithelium and complete differentiation to keratinocytes. figure 73 ---rat nonglandular stomach. carcinoma squamous cell, arising from diffuse severe hyperplasia, squamous cell. foci of hyperplastic basal cells in the glandular mucosa close to the limiting ridge. as this lesion is considered to originate from nonglandular stomach it should be collected under the term "nonglandular stomach, hyperplasia, basal cell". figure 71 ---higher magnification of figure 70, showing the basal cell character of the lesion. figure 72 ---mouse nonglandular stomach. papilloma, squamous cell. figure 127 --mouse colon. protozoa, probably trichomonas muris. figure 128 --mouse colon. neuronal degeneration, myenteric plexus with formation of hyaline inclusions in ganglion cells (flavivirus infection). figure 129 --mouse colon. diverticulum, characterized by extension of intact mucosa into subserosal tissue. figure 130 --mouse colon. multiple diverticula, some of them cystic (arrows) inflammation, mononuclear cell. the vacuolation of acinar cells and other degenerative processes are covered by this term; viral etiology. figure 176 --mouse parotid gland. inflammation, mononuclear cell. intranuclear inclusion bodies, some of them indicated by arrows figure 178 --mouse parotid gland. edema. figure 179 --rat parotid gland. calculi, ductular, accompanied by atrophy, accumulation adipocytes and fibrosis. figure 180 --rat submandibular gland. ectasia, duct. figure 181 --mouse sublingual gland. fibrosis, accompanied by atrophy and angiectasis. figure 182 --rat parotid gland. focus, hypertrophic, basophilic. figure 183 --rat parotid gland. focus, hypertrophic, basophilic, exaggerated lesion. figure 184 --rat submandibular gland. metaplasia, acinar cell (arrows) aquiring the morphology of parotid acinar cells. figure 185 --rat submandibular gland. metaplasia squamous cell. figure 186 --rat sublingual gland. metaplasia, squamous cell. figure 199 --mouse parotid gland. carcinoma, squamous cell. higher magnification of figure 197. figure 200 -mouse submandibular gland. tumor mixed, malignant. figure 201 --mouse submandibular gland. myoepithelioma, malignant. pancreas figure 202--mouse pancreas. ectopic tissue, spleen. figure 203 --rat pancreas. vacuolation, acinar cell. figure 204 -mouse pancreas. accumulation adipocytes after severe atrophy, acinar cell. figure 223 --mouse pancreas. halo, peri-insular. figure 224--rat pancreas. focus, basophilic. figure 225 --rat pancreas. metaplasia,hepatocytic at the interface between endocrine and exocrine tissue apoptosis 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related proteins and their potential as markers of pancreatitis key: cord-022353-q2k2krnm authors: w. quimby, fred; h. luong, richard title: clinical chemistry of the laboratory mouse date: 2007-09-02 journal: the mouse in biomedical research doi: 10.1016/b978-012369454-6/50060-1 sha: doc_id: 22353 cord_uid: q2k2krnm the frontier of clinical chemistry in the mouse has advanced and expanded because of two major events such as, the increasing reliance on mice in biomedical research, and increasing availability of practical yet sophisticated techniques and instrumentations that have allowed for the detection of a wider variety of biomarkers of disease. the progression of these two events is partially driven by the increasing regulatory demands related to safety/toxicity assessment of novel drug development. the availability of inbred strains has led to major breakthroughs in cancer, biology, and immunology. in addition, outbred stocks continue to be utilized in a wide variety of studies but particularly in the fields of toxicology and pharmacology. the power of these models to elucidate the genetic basis of disease cannot be overemphasized. this provided complete nucleotide sequences for each genome allowing investigators to quickly develop the equivalent murine model for many of the inherited human diseases. transgenic and knockout mice have helped clarify disease pathogenesis in virtually every area of medicine and often elucidated biochemical pathways, previously unknown, which are now subject to testing and quantification. it has been more than 20 years since publication of the first edition of the mouse in biomedical research, and since that time new emphasis has been placed on the mouse as a model for the pathophysiology and treatment of human diseases. during this time, the frontier of clinical chemistry in the mouse has advanced and expanded because of two major events: the increasing reliance on mice in biomedical research, and increasing availability of practical yet sophisticated techniques and instrumentations that have allowed for the detection of a wider variety of biomarkers of disease. the progression of these two events has been partially driven by the increasing regulatory demands related to safety/toxicity assessment of novel drug development. abbreviations used in this chapter are summarized in appendix 6-1. for the last quarter century, mice have been the most frequently used mammal in biomedical research, rising from 61% of all mammals used in 1983 to 71% in 1993 (national center for research resources 1997 . the nature of their use has changed dramatically during this time. during the first half of the 20th century, a great deal of emphasis was placed on the development of inbred strains. the availability of inbred strains has led to major breakthroughs in cancer, biology, and immunology (quimby 2002) . by 1970 there were approximately 250 inbred strains available. in addition, outbred stocks continue to be utilized in a wide variety of studies but particularly in the fields of toxicology and pharmacology. during the second half of the 20th century, investigators developed congenic lines and recombinant inbred strains, each having an impact on elucidating the role of individual genes and assigning new traits to linkage groups respectively (paigen 2003a ). however, beginning in the early 1980s scientists began to genetically engineer mice by either adding a new gene (transgenic) or deleting a normal mouse gene (knockout, ko) (paigen 2003b) . the power of these models to elucidate the genetic basis of disease cannot be overemphasized. over the past 15 years, publications citing transgenic and ko mice have increased exponentially and at the time of this writing the mutant mouse resource of the jackson laboratory listed more than 10,000 such unique lines. during this same period the results of both the human and mouse genome projects were published (international human genome sequencing consortium 2001; mouse genome sequencing consortium 2002) . this provided complete nucleotide sequences for each genome allowing investigators to quickly develop the equivalent murine model for many of the inherited human diseases. transgenic and ko mice have helped clarify disease pathogenesis in virtually every area of medicine and often elucidated biochemical pathways, previously unknown, which are now subject to testing and quantification. as a result, clinical chemistry in the mouse has grown from evaluation of 15-20 analytes found in plasma (or urine), to hundreds of biomarkers that can monitor disease status at the cellular level. due to the massive amount of new information characterizing each of the standard strains and mutant lines of mice, a mouse phenome database has been developed to manage data and provide researchers with the ability to explore raw phenotypic data (including clinical chemistry) and summary analyses. the resource allows investigators to select the appropriate murine model for physiology testing, drug discovery, toxicology studies, mutagenesis, modeling human disease, quantitative trait loci (qtl) analyses, identification of new genes, and unraveling the influence of environment on genotype (bogue and grubb 2004) . by far, the most important new technology in the rapidly growing and changing field of clinical chemistry is the discipline of proteomics. proteomics combines the disciplines of molecular biology, biochemistry, and genetics to the analysis of the structure, function, and interactions of proteins produced by the genes of a particular cell, tissue, or organism. the field of proteomics has grown mainly due to the development of new instruments that are based on sophisticated techniques, yet amenable to practical implementation. for example, the development of surface-enhanced laser desorption/ionization (seldi) platform time-of-flight (tof) mass spectroscopy (ms) allowed petricoin et al. (2002a) to identify five peptides in the sera of women with ovarian cancer that were not found in women without ovarian cancer, even though the structure and function of some of these peptides were not actually known. building on these findings, zhang et al. (2004) combined the seldi-tof ms with protein separation procedures to develop a multianalyte immunoassay for rapidly screening potential cancer patients. this process of identifying proteins (biomarkers) that are predictive for a disease is known as plasma protein profiling. similar technology has been used to develop plasma protein profiles for neoplastic conditions in humans, such as prostate cancer petricoin et al. 2002b; qu et al. 2002) and bladder cancer (adam et al. 2001; vlahou et al. 2001) , as well as a variety of non-neoplastic conditions, such as ischemic versus hemorrhagic stroke , severe acute respiratory syndrome (kang et al. 2005 ), alzheimer's disease (carrette et al. 2003) , and creutzfeldt-jakob disease (sanchez et al. 2004) . plasma protein profiling has also begun in mouse models. xiang et al. (2004) used a combination of two-dimensional gel electrophoresis (2d-ge) and matrix-assisted laser desorption/ionization (maldi) tof ms to quantify serum protein profiles of c57bl/6 mice harboring the lewis carcinoma with and without treatment using acetazolamide. they found upregulation of many peptides associated with tumor growth and metastasis, and many of these same peptides were modified during treatment. two specific targets of acetazolamide antitumor activity were subsequently identified as histone h2b and croci (a ubc-like peptide). park et al. (2004) used similar technology to determine plasma protein profiles for high (c57bl/6) and low (c3h) atherosclerosis prone strains of mice on normal or atherogenic diets. they identified 30 proteins in which the levels had changed after eating an atherogenic diet. of these, 14 were differentially changed in c57bl/6 mice and an additional 16 were changed in both strains. in addition, 28 proteins were differentially expressed between the two strains regardless of diets. four of these proteins were upregulated in c57bl/6 and 11 were upregulated in c3h. nine of those protein markers were definitively identified and their roles in the pathogenesis of atherosclerosis discussed in the "atherosclerosis" section. plasma protein profiling in mice has the potential to become an important discovery tool for translational research, particularly in identifying cancer-associated plasma biomarkers in humans. for example, juan et al. (2004) also used 2d-ge and maldi tof ms to identify plasma biomarkers in balb/c-nude mice harboring human xenotransplanted tumors. in mice bearing a human stomach cancer cell line, serum amyloid a (saa) was elevated. the authors then went back to screen the sera of human stomach cancer patients and were able to demonstrate that, when compared to controls, humans with stomach cancer also had significantly elevated levels of saa. all of these examples illustrate the power of proteomics. proteomic pattern diagnostics offers a means to look at molecular diagnostic information in human or mouse serum, without preconceived assumptions about the existence or identity of the biomarkers. this allows for the development of sensitive and specific peptide assays for identified biomarkers of disease. practical applications of proteomics have been facilitated by the creation of instrumentation that can analyze multiple analytes from small sample volumes with fast through-put (such as multiplex technology, which are discussed in the "multiplex technology" section). in an attempt to manage all the experimental data arising from the fields of proteomics, metabolomics and transcriptomics (gene array), the chemical effects in biological systems (cebs) knowledge base was developed (xirasagar et al. 2004 ). this is a useful, searchable database that integrates experimental findings from all three disciplines, by biosource identification (animal, test article, genotype, and investigator). by making the cross correlation from data arising from three different streams it is hoped that combination groups of predictive markers for disease and toxicity will emerge. the cost of bringing a new drug to market is estimated at $0.8-1.7 billion (food and drug administration 2004), of which a large proportion is spent on safety/toxicity assessments during the preclinical phase of the development of a drug. although expensive, safety/toxicity regulations are necessary, as illustrated by the fact that a new drug entering phase i clinical trials has only an 8% chance of reaching the marketplace due to toxicity issues. the ability to decrease the costs of drug development due to safety/toxicity issues is becoming increasingly reliant on the use of the laboratory mouse as a model for human disease and the identification of sensitive biomarkers for toxicity and disease. the underlying basis of using laboratory mice as models for human disease was demonstrated by everett and harrison (1983) , who showed that the predictive reliability of mice for quantitative toxicity of five chemotherapeutic drugs in humans was at least as good, if not better, that the predictive reliability demonstrative by dogs and monkeys. more recently, newell et al. (2004) presented data on predictive performance of rats and mice to demonstrate qualitative human toxicity when given 39 novel cancer chemotherapeutic agents. they claim that nonrodent species are unnecessary for identification of a safe phase 1 starting dose for human trials. furthermore, the two rodent species (rats and mice) used gave similar quantitative and qualitative results. the arrival of genetically engineered mice has further enhanced the relevance of using mice in research, especially in terms of basic mechanistic research and applied screening for genotoxicity and carcinogenicity. transgenic mice carrying a human gene and expressing the protein are said to be "humanized." these animals are invaluable in drug assessment especially when the drug interacts only with the human protein (bolon 2004) . zambrowicz and sands (2003a) showed that the ko phenotype of mice correlated well with the molecular targets of the 100 best selling drugs available to the u.s. market. zambrowicz et al. (2003b) compared the physiology of ko mice, where the deleted gene was known to produce a novel target for each of the 24 new drugs in the developmental pipeline of the 10 largest pharmaceutical companies, and found that 85% of these targets demonstrated a sound biologic rationale for the selected disease. transgenic and ko mice have been used successfully to screen new drug candidates for safety and to elucidate basic mechanisms of toxicity. a large number of drug metabolizing enzyme (dme) ko lines have been employed in safety screening. dmes may be involved in the safe metabolism of a drug or they may generate toxic intermediates. removal of the dme gene may result in the animal being more sensitive to the test article, and it may provide protection against toxic effects of the drug. for instance, ko mice lacking the nqo-1 gene have increased menadione toxicity, and mice lacking the cyp2e1 gene are resistant to acetaminophen hepatotoxicity (henderson and wolf 2003) . murine models are also available for evaluation of chemical mutagenicity (big blue mouse; stratagene, la jolla, ca and muta mouse; covance research products, denver, pa). these marker genes are present in mice of different genetic backgrounds (bolon 2004) . ko lines have been created with the increased sensitivity to chemically induced carcinogenesis. mice carrying a single p53 allele, or over expressing ha-ras, or having a complete deletion of the xpa (xeroderma pigmentosum) gene, have all been used for screening xenobiotics in vivo (bolon 2004) . these same transgenic and ko models are useful in screening environmental chemicals for toxicities (jacobson-kram et al. 2004) . it is clear that the mouse will continue to be essential for discovery and in evaluating the safety of new drugs. equally relevant is the development of clinical chemistry assays needed to study the associated metabolic events in mice (especially in transgenic and ko lines) and assess serum/plasma biomarkers. these biomarkers should either be quantitative measures of the biologic effects (which provide informative links between mechanism of action and clinical effectiveness) or surrogate markers (which are quantitative measures that predict effectiveness). in this arena, the field of proteomics is likely to make a dramatic impact on clinical chemistry. although we hope all readers of this chapter will benefit from this section on assays and instruments, the primary purpose of this chapter is to briefly introduce the reader to areas where methods in clinical chemistry are changing and provide sources of information for services, reagents (including test kits), and instrumentation, specifically for testing biomarkers (including traditional analytes) in mouse serum, plasma, or urine. those seeking a detailed description of clinical chemistry methods and instruments should refer to the fundamentals of clinical chemistry (burtis and ashwood 2001) . some dramatic changes have occurred over the past two decades that have revolutionized the discipline of clinical chemistry. historically, most analytes were measured by a colorimetric end point assay (based on the binding of an analyte to another molecule creating a new substance that absorbs light of a specific wavelength). some of these analytes and the newer biomarkers are now measured by enzymatic tests that have higher specificities and sensitivities. another change has been the substitution of electrochemical assays, such as ion-selective electrodes, for flame photometry used in the quantitation of sodium and potassium. perhaps the change that has had the largest impact on clinical chemistry is the development of monoclonal antibodies and their use as reagents in immunoassays (to be discussed later). many commercial companies now offer services that measure analytes and biomarkers in the blood of mice using a combination of colorimetric-, enzymatic-, electrochemical-, and immunologic-based methods, and many of the instruments used are capable of running multiple assay types simultaneously. the use of laboratories that offer validated assays specifically for mouse blood is important to ensure accurate and precise results. a summary of a few laboratories that provide validated murine assays is presented in table 6 -1. the increasing availability of mouse-specific reagents has resulted in many new assay techniques that provide a high degree of sensitivity and specificity. growth in the number of reagents capable of quantitating analytes in mouse serum is illustrated in table 6 -2. techniques employing nonisotopic labels for detection such as enzyme cascade, fluorescence, chemiluminescence, and electrochemiluminescence, are rapidly replacing older radioimmunoassay (ria) techniques. enzyme immunoassays, such as enzyme-linked immunosorbent assay (elisa), enzyme-multiplier immunoassay technique (emit), and cloned enzyme donor immunoassay (cedia), provide quantitative results based on photometric methods. enzyme immunoassay is popular because it generates compounds that can be quantitated photometrically. typical enzymatic labels include ~-galactosidase, horseradish peroxidase, alkaline phosphatase, and glucose-6-dehydrogenase. in addition these elisa test kits are compact, easy to use, and quantitated with inexpensive instruments. these kits (usually in a well format) are available for quantitation of a wide range of mouse serum and plasma biomarkers (table 6-3) . fluoroimmunoassay (fia), which utilizes a fluorescent molecule as an indicator label, was previously subject to problems associated with background fluorescence. today this problem has been largely resolved by using chelates of lanthanide as a label. modifications of fia have eliminated the need to separate free from bound label (homogenous assay). chemiluminescent and electrochemilumiscent immunoassays are similar to enzyme immunoassays, except that quantitation of results is based on the emission of light after a chemical label is exposed to an oxidation reaction (chemiluminescence) or to an electrochemical reaction (electrochemiluminescence). the number of biomarkers that can be quantified by commercially available test kits based on immunoassays is likely to grow at unparalleled rates as a result of proteomics research where mice are the favored model. mulitplex immunoassay is a unique technology that combines four distinct components in a manner that allows for simultaneous analysis of up to 90 different analytes from 50 ktl of serum/plasma. the core component is an inexpensive, consumable, 5.6 ~tm diameter polystyrene microsphere that are encoded into 100 different fluorescent color sets using two fluorophores (red and infrared) at multiple concentrations. the second component is a biologic assay (combined with an mmp-3 (matrix metalloproteinases) pro-mmp-9 mmp-9 tissue inhibitor of metalloprotease 1 (timp-1) orange fluorescent reporter molecule) that is built onto the surface of the microspheres. a diverse range of biologic assays can be built onto the microsphere surface, including immunoassays, nucleic acid assays, enzymatic reaction assays, or receptor-ligand analysis assays. the third component is a flow cytometer that focuses the microspheres into a single file in front of a two interrogating lasers, which allow for high throughout. one laser is a red diode emitting at 635 nm, which illuminates each microsphere. the resulting red and infrared fluorescence provides classification information for that particular microsphere set. the other laser is a green yag diode emitting at 532 nm that excites the orange fluorescent reporter molecules of the surface of the microsphere, providing a quantitative signal for that particular biologic assay. the last component is digital signal processing data acquisition hardware that provides the speed necessary to read the microspheres at up to 5000 per second. because multiplex technology can analyze a wide range of biomarkers simultaneously, dynamic reference results (plasma profiles) can be developed based on the changing concentrations of the biomarkers during the course of a disease. the known and potential applications of developing plasma profiles of diseases are powerful. plasma profiles can be used to screen and identify diseases (especially during the early development of a disease) and characterize the efficacy of drugs targeted against these diseases. multiplex technology can help elucidate new biomarkers of disease. furthermore, plasma profiles could also potentially be developed to monitor the blood concentrations of drugs as well. investigators seeking additional information on availability of reagents and test kits should refer to "clinical laboratory reference" (nelson 2006 ) and the linscott's directory of immunologic and biologic reagents (linscott 2005) . sections of these two references provide the names and addresses of sources. the catalogs of individual companies may be fruitful, but many reagents for use in human blood and have not been validated for use in other species (including the mouse). each of the various instruments mentioned in this section may be found in the 8th annual analyzers buyer's guide (advance for administrator of the laboratory 2003). this guide lists all manufacturers and gives detailed information on each instrument including technology platforms, methods used, through-put capability, purchase price, maintenance costs, reagent package cost, as well as a variety of options available. contact information, including web site, is available for each manufacturer. additional information regarding clinical chemistry on animal blood is available on the web site of the division of animal clinical chemistry at the american association of clinical chemistry (www.aacc.org/divisions/animal). with a mean body size of 35 g, adult mice have approximately 2.4 ml of blood volume, allowing 75 ~tl to be removed weekly without consequence to health and welfare (loeb 1997; loeb and quimby 1999) . four blood collection sites for acquiring 75 ~tl of blood repetitively from adult mice include the orbital plexus, the tail vein, the jugular vein, and cardiac puncture (quimby 1999b) . general anesthesia is recommended for orbital plexus and cardiac puncture, although terrilrobb et al. (1996) claim that topical application of proparacaine hydrochloride is an acceptable procedure for collecting orbital plexus blood. the jugular vein is punctured using a lancet directed at the rear of the jaw, exposing the jugular vein and its tributaries and the submandibular and facial veins (golde et al. 2005) . collection of blood from any of these veins at this site works well, and the animal does not require anesthesia or a restraint device. the blood should be collected with a small centrifuge tube or capillary tube. nerenberg and zedler (1975) describe a vacuum apparatus used to collect larger volumes of blood from the tail vein. lewis et al. (1976) found that heparinization of mice before tail bleeding increases the yield. prewarming the mouse under a lamp or through immersion in warm water facilitates tail bleeding. the use of heparinized microhematocrit tubes, during tail or orbital plexus bleeding can maximize the plasma volume with minimal hemolysis (quimby 1999b) . hem and smith (1998) recommend a saphenous vein prick method over the orbital plexus method for collecting repeated small volumes from conscious mice. macleod and shapiro (1988) used indwelling fight atrial catheters for repetitive bleeding of conscious, unstressed mice. disadvantages of specific procedures have been described. sakaki et al. (1961) reported sympathetic nervous system stimulation associated with tail bleeding and the mixing of venous and arterial blood with the orbital plexus method. patrick et al. (1983) found that, compared to jugular vein collection, cardiac puncture was associated with higher plasma glucose concentrations and lower creatine kinase (ck) activity. plasma glucose concentrations are higher in blood collected from the orbital sinus compared to the tail vein. differences associated with the method of anesthesia are also important. halothane, methoxyflurane, isoflurane, and pentobarbital sodium have been widely used and are considered safe. however, caution should be exercised in the interpretation of certain chemistry values. higher plasma glucose levels are reported with tail vein sampling in mice anesthetized with pentobarbital or methoxyflurane compared with conscious mice (chuang and luo 1997) . plasma glucose levels are higher after collection from the orbital plexus if pentobarbital or proparacaine hydrochloride is administered. methoxyflurane has no glucose elevating effect on samples collected from the orbital plexus. cunliffe-beamer (1983) claims that carbon dioxide narcosis provides sedation and analgesic for 1-2 minutes and is an appropriate anesthetic for orbital plexus bleeding. when greater volumes of blood are required, four collection sites can be used that require anesthesia and subsequent euthanasia of the mouse. these sites include the jugular vein (ambrus et al. 1951) , the abdominal aorta (lushbough and moline 1961) , the brachial artery (young and chambers 1973) , and the heart (cubitt and barrett 1978; mitruka and rawnsley 1977) . blood collection from a newborn mouse can be accomplished by decapitation. injection of two units of heparin subcutaneously several minutes before decapitation may allow collection of up to 40 ~tl of blood (20 ~tl of plasma). although collection of urine is possible in mice, it is impractical due to anatomic and physiologic restrictions. the total urinary bladder volume is less than 0.5 ml, and total urinary output per day is less than 2 ml (jung et al. 2003) , meaning that mice frequently micturate. this restricts the amount urine that can be collected at any one time point and, therefore, will also restrict the number of analytes that can be assessed from any one urine sample. to be able to collect enough urine for assessment from a single live mouse, mice can be placed in a plastic cage without absorbent bedding material and urine then collected from the bottom of the cage after 4-6 h. however, a disadvantage of this technique is that the urine is exposed to the contamination by feces and other environmental contaminants and organisms. pooled urine samples collected from a group of live mice using the same technique can increase the total amount of urine collected but will not allow specific results to be related back to specific individuals from that group. mice removed quickly from their cage and held over a piece of parafilm will frequently void and the parafilm helps prevent the urine from spreading. urine can also be collected directly from the urinary bladder from mice after euthanasia. this technique allows for a sterile sampling, but the volume that can be collected is restricted by total urinary bladder volume (0.5 ml). often, the volume is much less because mice typically void urine from the urinary bladder at the time of death. reference ranges refer to the range of an analyte or biomarker in a population that has not been selected for the presence of disease or abnormality. reference ranges are usually generated from a large number of individuals from a population, so reference ranges for the same analyte or biomarker can vary between two different populations. table 6-4 lists the reference ranges for selected analytes in two inbred strains and an outbred stock. in some situations, reference ranges that have been published or generated by laboratories cannot be relied on to accurately some variables known to adversely affect the host include environmental factors, pathogens, and shipment. in addition, nutrition, time of sample collection, and storage techniques may all contribute to variability. age has an effect on analytes in the mouse. in an early study, barrett et al. (1975) found that serum calcium levels in 4-monthold c3h/fg and a/fg inbred mice were significant higher than 7-month-old mice of the same strains. more recent and more comprehensive analyses completed by loeb et al. (1996) reinforces the effect that age has on clinical chemistry parameters in five inbred strains and two f1 hybrids, including serum protein. the effect sex has on chemistry parameters is most demonstrable with sex hormones, including follicle-stimulating hormone (fsh), luteinizing hormone (lh), and progesterone. table 6 -3 illustrates these differences between male and female mice, and between female mice in estrus and out of estrus. strain-associated changes appearing in healthy animals have been documented for complement components (goldman and goldman 1976) , cholesterol (dunnington et al. 1981; meade and gore 1982) , testosterone (ivanyi et al. 1972) , cortisol-binding protein (goldman et al. 1977) , and serum protein (borovkov and svirdov 1975; loeb 1997) . cyclic biorhythms, whether circadian or ultradian, influence blood levels of various analytes in mice. blood levels of adrenocorticotropic hormone (acth), corticosterone, growth hormone (gh), and lh may peak one or more times daily, and thus special attention must be given to both the time of collection and the order of sampling individuals between groups if between-group comparisons are to be made (loeb 1997) . the degree of hydration, exposure to noise, degree of confinement, and environmental temperatures has all been shown to affect serum chemistry analytes (quimby 1999b) . diet is known to influence the blood levels of many analytes. perhaps the best studied is the effect of atherogenic diets on serum cholesterol. similarly, significant differences in both serum cholesterol and urea nitrogen are seen in mice maindetermine the presence or absence of disease or abnormality, tained on a semipurified (ain-76) diet. the mouse is unique these situations include evaluating analytes and biomarkers in j among mammals because murine muscles do not contain transgenic and ko mouse populations (especially in experiments in which population sizes are small) and evaluating certain analytes and biomarkers (such as those assessing immune function). instead, baseline data compiled from controls (the population of wild-type mice from which the transgenic and ko mice were derived from) should be used. the usefulness of compiled baseline data depends on controlling a large number of variables known to influence chemistry determinations. several studies have demonstrated significant differences in selected serum analyte concentrations with age difference in the same strain, between sexes in the same strain, and among strains (everett and harrison 1983) . carnosine or anserine. carnosine serves as a source of histidine when histidine is restricted. unlike other mammals, mice on histidine-free diets show signs of histidine deficiency. fasting may also affect the levels of certain analytes (quimby 1999b) . the presence or absence (axenic mice) of intestinal microbial flora is associated with dramatic changes in immunoglobulin (ig) levels and may be associated with changes in other analytes as well. for example, axenic mice have significantly lower levels of iga compared to conventional mice (moreau et al. 1982) . the presence of pathogens may be associated with dramatic changes in various analytes, even with subclinical infection. for instance, infection with lactic dehydrogenase-elevating virus (ldv) is associated with major elevations in serum lactic dehydrogenase, isocitric dehydrogenase, malic dehydrogenase, aspartate aminotransferase, and glutathione reductase activities (quimby 1999b ). asummarized from loeb and quimby (1999) . alterations in clinicopathologic parameters can be attributed to stress in mice. landi et al. (1982) found that plasma corticosterone concentrations in mice tested within 48 h after arrival (by plane or truck transport) were significantly higher than tested after 48 h post arrival. mice sensitized on arrival with sheep red cells as an antigen had significantly lower antibody titers, fewer plaque-forming cells, and a decreased delayed type of hypersensitivity reaction when compared to normal mice allowed to acclimate to the facility for 48 h before being sensitized. elevated serum corticosterone levels can arise from excessive handling of mice prior to blood collection. the effect of sample storage at various temperatures and for varying periods has been described. falk et al. (1981) evaluated the effect of storage time (after freezing) on 20 serum analytes in six laboratory species. they found that in the mouse, only creatine kinase activity changed significantly with storage up to 28 days. hemolysis is a common problem during blood collection in mice. hemolyzed samples are associated with changes in various enzymes, such as increased ck activity and decreased lipase activity. to minimize this, everett and harrison (1983) recommend heparinized plasma and careful selection of collection sites for routine chemical determinations. the effect of lipemia, various anticoagulants, and pharmacologic agents on clinical chemistry values has been reviewed for domestic animals (meyer and harvey 1998) . using several methods. regardless of the distribution of data, it is generally useful to describe the limits that include 95% of the test results in a disease-free population. for values exhibiting a gaussian distribution, parametric methods (such as mean and standard deviation) are appropriate. for gaussian distributed data, this is the range that includes two standard deviations above and below the mean. certain murine analytes have non-gaussian distributions and must be evaluated using nonparametric methods. a variety of methods are available, including the percentile method and logarithm-transformed data analyzed with parametric methods. the method of percentile estimates is more vulnerable to bias due to extreme values (outliers) than is the log-transformed parametric method. boyd (1985) asserts that a sample size of at least 120 is required to give 90% confidence intervals using the percentile method, whereas a sample size of 50 may give reliable ranges if parametric analyses are used. neither statistical method just described will replace raw data in certain situations, such as when assessing analytes for immune function, nor when using data derived from wild-type mice as comparison baseline data for transgenic and ko mice. the issue of quality control and test validity has been thoroughly discussed for common domestic animals and is applicable to chemistry determinations in mice (meyer and harvey 1998) . quality assurance in clinical chemistry determinations is important to ensure that consistently accurate and precise results are achieved. everett and harrison (1983) stressed the importance of a clinical pathology quality assurance program that includes regular assays of pooled and commercially prepared pre-assayed sera. in addition, they encourage participation in a subscription quality assurance program, such as with a veterinary laboratory association. the within-day and day-to-day coefficient of variation should be known for each analyte measured. due to the tremendous number of variables known to influence clinical chemistry values in mice, it is often prudent to test adequate numbers of control specimens along with the experimental samples. this technique is often impractical when tests are being conducted strictly for diagnostic purposes, and in those situations, compiled values that are controlled for as many variables as possible may be sufficient. the values that define a reference range for a particular analyte or biomarker in normal or healthy mice may be described this section is intended to serve as a review of specific tests for routine analytes. additionally, a more comprehensive discussion is presented for analytes and biomarkers involved in two areas of translational research of intense interest (obesity/diabetes and atherosclerosis) and novel biomarkers of disease (such as immune function tests). only analytes and biomarkers for which there are currently available commercial tests for mice serum, plasma, and/or urine are discussed; certain analytes and biomarkers for which commercially available tests are not available for the mouse (such as calcitonin) are not covered. for more complete information for the routine analytes, please refer to loeb and quimby (1999) . glucose is the main source of energy in mice. blood glucose concentrations depend on the rates of entry and the removal rate from the blood. the rate of entry is dependent on intestinal absorption of dietary sources of glucose, the breakdown of body glycogen stores (glycogenolysis), and synthesis from gluconeogenic metabolites (gluconeogenesis). the removal rate is mainly dependent on insulin, which is released from [3-cells of the pancreatic islets. insulin promotes cellular uptake of blood glucose (mainly in muscle, liver, and fat) by stimulating the translocation of glucose transporters, glut-1 to glut-7 to the cell membrane. when removed from circulation, blood glucose may either be utilized (to maintain cell function) or converted to fat and glycogen in liver and muscle as an energy store. however, the effect of insulin is modulated by other hormones (such as glucagon, corticosterone, gh, epinephrine, somatostatin, and amylin) that ultimately result in the tight control the levels of blood glucose, depending on tissue demands for energy. glucagon is released from t~-cells of the pancreatic islets in response to low circulating glucose, stimulating the liver to increase circulating glucose through glycogenolysis and gluconeogenesis. corticosterone and gh antagonize the action of insulin. epinephrine suppresses insulin release and stimulates glucagon release and glycogenolysis. somatostatin suppresses both insulin and glucagon secretion, whereas amylin increases blood glucose, blood insulin, and insulin resistance (burtis and ashwood 2001; kaneko 1999) . glucose determinations are generally conducted on fresh serum. however, plasma glucose determination is acceptable if delay of greater than 30 minutes before separation of erythrocytes is anticipated. in this case, fluoride should be used as the anticoagulant because it inhibits glycolysis by erythrocytes. blood glucose in mice is measured using the hexokinase or glucose oxidase. these tests may be performed as an analytical method or by using glucose oxidase coated test strips (for urine glucose), or small portable analyzers (seidelmann et al. 2005) . assessment of long-term average blood glucose levels in mice is also available by rias measuring glycosylated hemoglobin and glycosylated serum proteins (collectively known as fructosamines) (gould et al. 1986 ). the mouse phenome database lists serum glucose levels of 41 strains with blood collected after a 4-h fast on 7-9 week old males and females on a standard laboratory diet. for all mice, the overall mean was 179 + 30.9 mg/dl (naggert et al. 2003) , but blood glucose levels varied with age, sex, and strain. females of a strain tended to have lower levels than males, with lp/j mice showing the lowest values (f = 125 + 22.0 and m = 146 _+ 18.8 mg/dl) and c57b1/10j mice showing the highest values (f = 230 _+ 25.5; m = 263 _+ 57.3 mg/dl). in mice serum glucose levels decrease between the 3rd and 12th month of age and in c57bl/6 and balb/c strains the glucose level rises again after 24 months (loeb 1997) . serum or plasma glucose levels may also vary depending on the site of collection and anesthetic used (quimby 1999b) . patrick et al. (1983) found that compared to jugular vein collection, cardiac puncture was associated with higher blood glucose. differences associated with the method of anesthesia are also important. higher plasma glucose levels are reported after tail vein sampling under either pentobarbital or methoxyflurane compared to sampling conscious mice (chuang and luo 1997) . plasma glucose levels were higher after orbital plexus sampling of mice if pentobarbital or proparacaine hydrochloride was provided. methoxyflurane had no glucose elevating effect on samples collected from the orbital plexus; however, in conscious animals, plasma glucose levels were higher in blood collected by retro-orbital sinus compared to tail bleeding. hyperglycemia (increased blood glucose levels) in the mouse can be due to increased peripheral resistance of tissues to insulin (such as with exogenous corticosteroid administration, increased endogenous corticosterone release, glucagons administration, and gh administration), and diabetes (see discussion later). hypoglycemia (decreased blood glucose levels) can be due to excessive circulating insulin (such as with excessive insulin administration and transgenic mice with [3-cell tumors of the pancreatic islets), reduced glycogen stores (such as with advanced liver disease), excessive glucose use (such as with pregnancy, septicemia, and neoplasia), and reduced glucose intake (such as with starvation and diseases of malabsorption). diabetes in mice is defined as persistent blood glucose levels of greater than 300 mg/dl in fasting mice. this level also corresponds to the renal threshold for glucose urine excretion in mice, so urine glucose assessment is useful in this regard for mice. nonobese diabetic mice (a model of type 1 diabetes) have nonfasting plasma glucose levels in young prediabetic mice between 130-180 mg/dl, which rises to >300 mg/dl between 10-30 weeks of age (leiter 1997) . genetically modified mice have been identified for virtually every ligand and receptor regulating glucose metabolism and, depending on the gene mutation, may exhibit altered levels of plasma glucose. for example, adenosine monophosphate-activated protein kinase (ampk) is a critical enzyme in energy metabolism (including cellular glucose uptake and fatty acid oxidation in muscle, and fatty acid synthesis and gluconeogenesis in hepatocytes). using ko mice, shaw et al. (2005) demonstrated that kinase lkb 1 is an important activator of ampk in the liver under energy-stress conditions, and that ko mice deficient in kinase lkb1 were persistently hyperglycemic. the authors also demonstrated that kinase lkb 1 is the target of the type 2 diabetic therapeutic drug, metformin. glucose tolerance tests (gtt) have been performed in mice. one-, 3-, and 4-h tests have been conducted. in the 1-h test, glucose concentrations are evaluated before and 1 h following the administration of 2 mg/g glucose administered intraperitoneally (ip) (oldstone et al. 1984) . the 3-h test compares pre-injection serum to serum collected at 15, 30, 60, and 120 minutes following administration of 3 mg/g glucose given ip (hotamisligil et al. 1996) . a sensitive 4-h gtt has also been described in which mice are given 10 ml/kg of 10% glucose orally (gates et al. 1972) . the hypoglycemic response to insulin in mice has also been described (hotamisligil et al. 1996) . kaku et al. (1988) measured glucose, glycosylated hemoglobin and insulin levels in six inbred strains undergoing gtt (either in fed or fasted mice). to estimate the number of genes involved in phenotypic differences in glucose tolerance, the least glucose tolerant strain (c57bl/6) was bred to the most tolerant strain (c3h/hej) and f1 hybrids and backcross animals tested. the authors concluded that glucose tolerance in six commonly used inbred strains is a polygenic trait. thrifty genes have been hypothesized to give early human hunter-gatherers a survival advantage by providing an economic mechanism to store energy during periods of famine (zimmet and thomas 2003) . because the most efficient mechanism for energy storage is through promotion of adipose tissue, it seems reasonable that at least some of these "thrifty" genes may function in this manner. another hypothesis holds that during periods of famine it is essential to conserve glucose for use by the brain and that the mechanism responsible involves insulin resistance in peripheral tissues (neel 1962) . should both hypotheses be true it is easy to envision an association between obesity and type 2 diabetes, especially where sedentary lifestyle and unrestricted access to food occur together (lazar 2005) . one candidate thrifty gene encodes the hormone leptin. leptin is produced by adipose tissue, and its absence leads to obesity and insulin resistance. during times of high adipose storage blood levels of leptin are high and it promotes energy metabolism and inhibits food intake. the opposite occurs during starvation. but leptin is only one of a number of adipokines, secreted by adipose tissue, which aid in the regulating appetite and metabolism. in fact the location of the adipose tissue, the size of average adipocytes, and adipocyte metabolism of glucose and corticosteroids each modify the endocrine function of adipose tissue (lazar 2005) . among the proteins secreted by adipose tissue are adiponectin, adipsin, resistin, visfatin, tumor necrosis factor-a (tnf-a), interleukin-6 (il-6), macrophage chemoattractant protein-1 (mcp-1), plasminogen activator inhibitor-1 (pai-1), angiotensinogen, saa, and a-acid glycoprotein. like adipocyte-derived free fatty acids, which have been shown to contribute to insulin resistance in liver and muscle, most of these proteins are capable of modulating glucose metabolism and insulin action. adiponectin and visfatin work synergistically with insulin to enhance glucose uptake by muscle and block glucose synthesis by the liver (hug and lodish 2005) . blood levels of visfatin increase in obesity and the cytokine can bind to and stimulate the insulin receptor (fukuhara et al. 2005) . blood levels of adiponectin negatively correlate with body mass and are lower in obese humans and mice, suggesting that reduced mrna expression of the adiponectin gene may be involved with obesity (masaki et al. 2004) . increases in adiponectin downregulate the hepatic expression of tnf-a. it mediates its antidiabetogenic effects via receptors on peripheral tissues, especially liver. tnf-a, resistin, and il-6 each induce resistance to insulin. however tnf-a also suppresses expression of adipocyte specific fred w. quimby and richard h. luong t genes; resistin maintains glucose during fasting; and il-6 production increases in the obese. the cytokines tnf-~ and il-6 are proinflammatory, are also produced by monocytes, and act on the liver to produce acute phase reactants. they also induce suppressor of cytokine signaling-3 (socs-3), an intracellular signaling molecule that impairs neuronal signaling by leptin and insulin, and thus causes resistance to the central actions of both hormones (schwartz and porte 2005) . resistin mediates its effects principally by decreasing the expression of gluconeogenic enzymes in the liver (banerjee et al. 2004 ). certain cytokines, increased endoplasmic reticulum stress, chronic hyperglycemia, chronic hyperlipidemia, and oxidative stress may all induce apoptosis of insulin producing ~l-cells in the islets of the pancreas (rhodes 2005) . insulin receptor substrate-2 (irs-2) is a key molecule promoting ~-cell growth and survival. these molecules act immediately downstream from surface receptors for insulin and insulin-like growth factor-1 and inhibition of irs-2 has been shown to lead to insulin resistance. inhibition may result from accumulation of reactive oxygen species in ~-cells chronically exposed to increased glucose metabolism or from chronic exposure to elevated fatty acid (which through production of long chain acyl-coa active protein kinase c-isoforms degrade irs-2). leptin has been shown to modulate interleukin-l~ (il-i~), a potent inducer of apoptosis. tnf-t~ and il-6 induce ~-cell apoptosis by activating the transcription factor nuclear factor k:i] (nf-~:[3). centrally the actions of both insulin and leptin take place in the mediobasal hypothalamus, where the neurons exert potent effects on food intake and energy expenditure. here neurons co-express neuropeptide y (npy) and agouti-related peptide (agrp), which stimulate food intake and reduce energy expenditure. leptin and insulin inhibit these neurons. under conditions of reduced leptin and insulin signaling, npy increases, inducing hyperphagia, weight gain, insulin resistance, and glucose intolerance. the anabolic effects of agrp arise from its antagonism of the melanocortin receptors mc3r and mc4r, which serve to limit food intake. blockage of mc3r and mc4r leads to weight gain and insulin resistance. precursor proopiomelanocortin (pomc) and pomc neurons in the arcuate nucleus are stimulated by leptin and insulin and the resultant production of melanocortin and its binding to mc3r and mc4r inhibits food intake and promotes weight loss (schwartz and porte 2005) . however, in the absence of leptin, as seen in lep ~176 mice, neurons of the arcuate nucleus of the hypothalamus are permanently disrupted and treatment in adulthood cannot reverse this defect (bouret et al. 2004) . orexin a (hypocretin-1) and orexin b (hypocretin-2) are neuropeptides produced in the lateral hypothalamus by neurons with axonal projections to many sites including those that control feeding behavior and sleep/wakefulness (taylor and samson 2003) . orexin ko mice develop hypophagia with obesity and insulin-resistant diabetes (hara et al. 2001 ). ghrelin, a peptide made predominantly by the stomach, is also known to act centrally and affect food intake and increase secretion of gh (ghigo et al. 2004; korbonits et al. 2004 ). in the periphery leptin has been shown to specifically repress rna levels and enzymatic activity of hepatic stearoyl-coa desaturase-1 (sld-1), which catalyzes the biosynthesis of monounsaturated fatty acids. this effect was found to be an important metabolic action of leptin (cohen et al. 2002) . leptin resistance, a common feature of obesity in mice and humans, has also been shown to result, in part, from the shedding of membrane-bound hepatic leptin receptors into the plasma, where soluble receptors modulate circulating leptin levels and possibly its biologic activity (cohen et al. 2005) . thus the connections between factors regulating obesity and insulin resistance (diabetes) are complex and occur both centrally and peripherally. further investigations in mice will involve quantifying glucose, adipokines, insulin, leptin, soluble leptin receptor, and sld-1. for further discussion of mouse models of obesity and diabetes please refer to chapter 19 in this text. metabolism and food intake insulin, leptin, amylin (also called islet amyloid polypeptide, iapp), glucagon, ghrelin, obestatin, orexin a, orexin b, gh, and corticosterone have been measured in mice, and ria and elisa kits are commercially available (see table 6 -3). in addition, these hormones may be measured as part of a multiplex panel (see "multiplex technology" section). leptin values in c57bl/6, 129, and fvb/n strains range from 1-3 ng/ml, insulin values range from 2.2-5.2 ~t iu/ml, corticosterone levels range from 5-40 ~tg/dl, and gh ranges from 1-90 ~tg/ml. epinephrine is measured by ria, and the range for mice is 0-200 pg/dl (depaolo and masoro 1989). adipokines secreted entirely by adipose tissue include adiponectin, adipsin, and resistin. adiponectin and resistin have been measured in mouse serum and commercial elisa test kits are available for this species. a polyclonal antibody that cross-reacts with a conserved sequence of mouse adipsin has been created (searfoss et al. 2003) . tnf-ct, il-6, and visfatin are adipokines that are synthesized also by macrophages and lymphocytes; as such they provide a common link between regulation of obesity, resistance to insulin and inflammation (lazar 2005) . both tnf-t~ and il-6 may be measured by commercially available elisa kits (see the "cytokines and chemokines" section). a method has also been published for measurement of visfatin (fukuhara et al. 2005 ). reagents for quantifying the soluble leptin receptor and sdl-1 are not commercially available at this time, although methods for measurement of these analytes in mouse serum have been published (cohen et al. 2002; cohen et al. 2005 ). the four main types of lipids in plasma are free cholesterol, esterified cholesterol, triglycerides, and phospholipids. lipids are derived from the diet (mainly from long-chain fatty acids), although endogenous recycling (mainly in the liver) occurs. plasma lipids have poor water solubility; thus, they require water-soluble protein molecules for their transport in plasma. the complex of plasma lipids and proteins are known as apoproteins (also known as apolipoproteins), which contain a core of nonpolar lipids surrounded by a surface layer of phospholipids, free cholesterol, and apoproteins. apoproteins are classified according to physical and chemical parameters, and include (in order of increasing density) chylomicrons, very low-density lipoproteins (vldl), intermediate density lipoproteins (idl), low-density lipoproteins (ldl), and high-density lipoprotein (hdl). particle density is proportionally related to the amounts of phospholipid, protein, and triglycerides they contain; increasing particle density corresponds with increasing proportions of phospholipid and protein, and decreasing density corresponds with decreasing proportions of triglyceride. chylomicrons and vldl are often referred to as triglyceride-rich lipoproteins. apoproteins on the surface of the particles serve as ligands for receptors, cofactors of enzyme interaction and structural components (wagner et al. 1999 ). table 6 -5 lists the mouse apoproteins and describes their known function. the largest lipoprotein particle is the chylomicron, which transports dietary lipids in the form of triglycerides from the intestines. chylomicrons contain apoproteins a and b48 and are absorbed into the lymphatics from the intestine, eventually entering the blood. they deliver fatty acids to the tissues with assistance from lipoprotein lipase (lpl) and release glycerol into the blood. as chylomicrons lose triglycerides they become smaller and are called remnants. these remnants acquire apoprotein e from plasma hdl and are rapidly cleared by the liver by the apoprotein e receptor or chylomicron remnant receptor. vldl is the second most prevalent lipoprotein particle in normal mouse blood (after hdl) and it transports triglyceride from liver to extrahepatic tissues. vldl is synthesized in the liver with apoprotein b 100, c, and e attached. lpl aids in the release of fatty acids (from triglycerides) to peripheral tissues. ldl, which is present in very low concentrations in normal mice, carries cholesterol to extrahepatic tissues. ldl contains apoprotein b 100 and is the primary source of cholesterol deposited in the intima of arteries in mice. apoprotein b 100 binds to the ldl receptor (ldlr). hdl is synthesized in the liver and contains large amounts of free cholesterol and apoproteins a, c, and e. during metabolism hdl free cholesterol is esterified by the action of lecithin:cholesterol acyltransferase (lcat). the exchange of cholesterol ester for triglycerides (from vldl) results in a less dense hdl subfraction that is completely removed from the circulation by the liver. exchange of esterified cholesterol for triglycerides is mediated by cholesteryl ester transfer protein (cetp) in humans; however, this activity is absent in mice. phospholipid transfer from vldl to hdl is mediated by phospholipids transfer protein (pltp). high levels of soluble hepatic lipase are found in mouse plasma (lusis, 2000; wagner et al. 1999) . hdl is thought to transport cholesterol from peripheral tissue to liver by a process known as reverse cholesterol transport (rct). this process is believed to be facilitated by the adenosine triphosphate (atp)binding cassette transporter a1 (abca1), which transports phospholipids and cholesterol to the acceptors apoprotein a-1 and apoprotein e (aiello et al. 2002) . hdl also serves as a reserve of apoprotein c and apoprotein e necessary for vldl metabolism. table 6 -6 lists the apolipoprotein receptors found in mice and describes their ligands and functions. atherosclerosis is a major factor in heart disease, stroke, and peripheral vascular disease in humans, and as such, is the principal cause of death in western countries. the etiology is complex, involving both genetic and environmental components. although there are some examples of single gene defects in humans that lead to atherosclerosis, these conditions are rare and do not explain disease prevalence. atherosclerosis is characterized by the formation of plaques in the intima of large and medium size arteries, usually in locations where blood flow is disturbed. the plaques contain a variety of cells including endothelial cells, monocytes or macrophages, smooth muscle cells, and lymphocytes, which secrete products that modulate progression of plaque formation. in addition plaques contain a complex mix of collagen, proteoglycans, occasional cartilaginous tissue with calcification and lipoprotein (primarily ldl). disease risk correlates directly with elevated circulating levels of ldl cholesterol and risk is indirectly correlated to levels of hdl. thus, although ldl promotes atherosclerosis, hdl protects against it (national cholesterol education program 1993). the combination of high ldl and low hdl is termed dyslipidemia and is found in human patients with insulin resistance syndrome. mice do not develop spontaneous atherosclerosis, because they have low circulating levels of ldl and lack cetp activity. however, genetically engineered mice that over-or under-express genes involved in lipid metabolism have contributed greatly to our understanding of lipoprotein metabolism (marschang and herz 2003) . in particular, transgenic mice have been developed with plasma lipid profiles that are similar to those of humans with atherosclerosis (breslow 1994 (breslow , 1996 . in humans and transgenic mice with elevated ldl (or low hdl), ldl passively diffuses across the endothelium, especially in areas where endothelial cell morphology has been altered by the sheer forces generated by blood turbulence. once ldl is within the intima there is an interaction between ldlapoprotein b and matrix proteoglycans resulting in ldl trapping. ldl undergoes modifications associated with oxidation, lipolysis, proteolysis, and aggregation that contribute to inflammation and uptake of ldl by tissue macrophages. lipoxygenases (los) insert molecular oxygen into polyenoic fatty acids producing hydroperoxyeicosatetraenoic acid (hete). endothelial cells release hete into the vessel wall, where it initiates oxidation of ldl. further oxidation of ldl is aided by myeloperoxidase and sphingomyelinase. mice lacking los have diminished atherosclerosis. macrophages and monocytes recognize oxidized ldl via their surface scavenger receptors and become loaded with cholesterol ester, forming a fatty streak appearing along the vessel wall. hdl, on the other hand, removes excess cholesterol from peripheral tissue and inhibits lipoprotein oxidation. hdl carries paraoxonase, which degrades oxidized phospholipids (lusis 2000) and protects against subsequent oxidative damage. oxidized ldl stimulates endothelial cells to express adhesion molecules (such as icam, vla-4, and vcam) and monocyte colony-stimulating factor (m-csf) on their luminal surface, which attracts additional monocytes and lymphocytes. oxidized ldl also inhibits nitric oxide production. infiltrating thymic-derived lymphocytes (t cells) and macrophages initiate migration of smooth muscle cells from the media to the intima, where they synthesize matrix components. mcp-1 is also released by macrophages, inducing monocyte differentiation, migration, and scavenger receptor expression. the accumulation of excess free cholesterol can be inhibited by activation of acyl coa:cholesterol acyltransferase (acat)mediated cholesterol esterification and cellular cholesterol effiux. one mechanism responsible for cholesterol effiux is secretion of apoprotein e by macrophages that promotes effiux via hdl. if this fails, as in the case of cholesterol-laden macrophages in plaques, apoptosis of the macrophages ensues. loading of the endoplasmic reticulum with free cholesterol activates er resident protein kinase and the unfolded protein response (upr) that initiates apoptosis through activation of caspase-12 (feng et al. 2003a) . as macrophages undergo apoptosis they create a necrotic core in the plaque, promoting extracellular cholesterol cleft formation. smooth muscle cells create a fibrous cap over the plaque near the luminal surface. the lesion advances as t cells, activated by cd40-cd40l ligation, release cytokines such as interferon-3, (ifn-y) and tnf-ct. these substances activate matrix degrading proteases and adhesion molecules, promoting additional inflammation. as the inflammation progresses the fibrous cap becomes compromised, leading to physical rupture and the generation of a thrombogenic surface. oxidized ldl increases the production of tissue factor that, on the thrombogenic surface, initiates the coagulation cascade and thrombus formation. each of these details have been elucidated using genetically modified mice (auerbach et al. 1992; choudhury et al. 2004; feng et al. 2003b; lusis 2000; reardon and getz 2001; tailleux et al. 2003; trigatti et al. 2004) . for a further discussion of these models readers are urged to read chapter 16 in this book. for measurements of plasma lipids, serum is the preferred sample. serum can be stored at 4~ for 5-7 days without adverse effect on measurements. a. serum cholesterol and triglycerides serum cholesterol in mice can be measured using the enzymatic oxidation method of roschlay (meade and gore 1982) , the lipid research clinic program protocol (morrisett et al. 1982) , or the abell technique (mitruka and rawnsley 1977) . the most common enzymatic method employs cholesterol ester hydrolase (to convert cholesterol ester to cholesterol), cholesterol oxidase (which oxidizes cholesterol to hydrogen peroxide and cholest-4-en-3-one) and peroxidase (which catalyzes a reaction involving hydrogen peroxide, phenol and 4-aminoantipyrine to form the dye quinonelmine). all of these components of this test are combined in a single reagent mix. the dye absorbance is measured at 500 nm (choudhury et al. 2004) . triglyceride (tg) levels, which are mainly reflective of the tg content of chylomicrons and vldl in the mouse, are quantified using a variety of enzymatic methods. the most popular method combines lipoprotein lipase, glycerol kinase, and glycerol-phosphate oxidase with peroxidase and 4-aminoantipyrine (usually in two reagents). in this method, serum tgs are converted to glycerol by lpl via hydrolysis. next the glycerol is phosphorylated in an atp-dependent reaction catalyzed by glycerol kinase. glycerol-phosphate is catalyzed to dihydroxyacetone and hydrogen peroxide by glycerophosphate oxidase and peroxidase catalyzes the final reaction in which peroxide and 4-aminoantipyrine are converted to a stable dye and absorbance can be read at 500 nm (tsimikas et al. 2000) . reagents for both total cholesterol and total tgs may be used in an automated analyzer or purchased directly from a chemical company with instructions for manual laboratory analysis. total serum cholesterol and tg levels vary by strain, gender, age, length of fast, and diet. jiao et al. (1990) studied total plasma cholesterol (tpc) and tg levels of various inbred strains fed a standard diet. after 18-20 h of fasting, tpc levels ranged from 55 mg/dl (akr/j) to 128 mg/dl (nzb/b 1nj), and tg levels ranged from 13 mg/dl (c57bl/6) to 67 mg/dl c3h/hej; each much lower than seen in normal humans. albers and piagen (1999) quantified total cholesterol levels in 15 strains give a standard laboratory diet and fasted for 4 h before blood collection. levels (in 6-to 8-week-old mice) varied greatly between strains with c57blks/j females having the lowest level (39 mg/dl) and nzb/b 1nj males having the highest levels (127 mg/dl). for a given strain, males always had higher levels than females. trends in total hdl levels in the same strains paralleled total cholesterol levels. triglyceride levels also varied greatly among strains but did not parallel cholesterol levels. among the strains tested, c57bl/6j mice had the lowest tg levels (71-80 mg/dl) and c3h/hesnj the highest (f, 162 mg/dl; m, 231 mg/dl). differences between genders of the same strain were less pronounced, compared to cholesterol and males generally had higher levels than females. a transient cause of plasma lipid elevation (hyperlipidemia) in the mouse is related to recent feeding (postprandial hyperlipidemia), especially on a high-fat diet. causes of persistent hyperlipidemia include diabetes, sustained feeding of a high-fat diet, and nephrotic syndrome. b. hdl, ldl, idl, and vldl levels of individual apolipoproteins can be measured by density gradient ultracentrifugation combined with either electrophoretic, immunologic, chemical, or morphologic analyses. although published reference ranges are not available, the distribution and characterization of murine apoproteins has been reported (camus et al. 1983 ). similar methods were used to evaluate plasma apoproteins in mice consuming atherogenic diets (moltisett et al. 1982) . more recently the profile of murine plasma lipoprotein cholesterol has been determined by fast protein liquid chromatography with on-line post-column analysis of superose 6 gel-filtration eluates (sehayek et al. 2003; strauss et al. 2001) . in contrast to humans, mice normally have low levels of ldl in their plasma (15 mg/dl), with the major plasma lipoprotein being hdl. the low ldl concentration is due to editing of 70% of apoprotein b mrna transcripts in mouse liver leading to apoprotein b48 containing particles that are cleared much faster than ldl in humans (lusis 2000) . in contrast, human apoprotein b mrna transcript editing only occurs in the intestine. a much simpler methodology has been developed to measure hdl and non-hdl lipoproteins in mice, based on precipitation and enzymatic analysis. when phosphotungstate and magnesium salt is added to serum (or plasma), all lipoprotein, except hdl, is precipitated and can be removed. the remaining solution is then tested for cholesterol as described previously. the non-hdl-cholesterol component is calculated by subtracting hdl-cholesterol from total cholesterol in the nonprecipitated sample (sehayek et al. 2003) . some strains develop elevated cholesterol levels associated with increases in non-hdl levels after consuming high fat diets (breslow 1994) . c. mouse apoproteins commercial antibodies against mouse apoprotein a1 and apoprotein a2 are available and elisas have been described for the measurement of these apoproteins in plasma and in hdl fractions of plasma (dansky et al. 1999) . plasma apoprotein a1 levels can also be measured by multiplex analysis. an assay for mouse apoprotein j (apoj or clusterin) has also been described (navab et al. 1997) . apoj, which is a ubiquitous glycoprotein postulated to have multiple functions, is associated with hdl and is the amyloid-associated protein associated with amyloid plaque formation in alzheimer's disease in humans. d. other analytes associated with lipid metabolism and atherosclerosis in mice elisa kits are commercially available for the quantitation of many mouse coagulation proteins including: fibrinogen, factor vii, d-dimer, tissue factor, and von willebrand's factor antigen. elisa kits are also available to quantify many murine products of arachidonic acid metabolism including hete. reagents, elisa kits, and multiplex assays are available for murine inflammatory cytokines and chemokines (see the "cytokines and chemokines" section), as well as monocyte colony stimulating factor. dansky et al. (1999) have described assays for the quantitation of murine aryl esterase and paraoxonase. table 6 -7 lists the mouse enzymes involved in lipid metabolism with references that describe their measurement. the innate and adaptive immune responses have been extensively studied (see volume 4 of this series) and therefore we will not attempt to describe all the features of mouse immunity in detail here. readers are directed to volume 4, molecular and cellular immunology of the mouse, for a general overview, and the 15 chapters that follow for a more detailed description. this section describes the growing number of quantifiable soluble serum proteins and lipids associated with immunity and inflammation in the mouse (see table 6 -3). furthermore, in addition to immunodeficiency, autoimmunity, and allergy, investigations of atherosclerosis, obesity, diabetes, cancer, as well as infectious diseases, each have immunologic and inflammatory components. 1. immunoglobulins (ig) as in humans, mouse immunoglobulins (ig) are molecules composed of four polypeptide chains; two of lower molecular weight called light (l) chains, and two with higher molecular weight called heavy (h) chains. disulfide bonds link one l chain to one h chain, and the two h chains to each other. immunoglobulin h chains are composed of four to five domains, including an n-terminal variable region domain and four constant-region domains. in addition, structural differences in the constant-region domains of the heavy chain are used to classify the five different classes of immunoglobulin, igm, igg, iga, ige, and igd. l chains are composed of only two domains and structural differences in these domains are used to classify l chains as either kappa or lambda type. in the mouse, 95% of serum ig has kappa l chains. any individual antibody secreting b cell (or plasma cell) will make a single ig molecule composed of two identical h chains and two identical l chains. mice make four subtypes of igg: igg1, igg2a, igg2b, and igg3. certain strains, c57bl/6, c57b1/10, sjl, and nod, do not make igg2a but rather make a novel igg2c. the igg subtypes in mice are not exact homologues of human subtypes (mestas and hughes 2004) . prenatal (transplacental) transfer of maternal ig as well as postnatal transfer across the intestinal epithelium is limited to the igg2a, 2b, and 3 subclasses of immunoglobulin and is mediated by neonatal fc receptors (fcrn) located in the placenta or on the intestinal brush border of the proximal small intestine (bankert and mazzaferro 1999) . mouse igg2b fixes complement by the classical pathway and igg 1 and igg2a fix complement by the alternative pathway. ige and igg1 are homocytotropic antibodies capable of binding to receptors on mast cells and basophils and mediate immediate hypersensitivity reactions. although igg and ige circulate in the mouse as monomers, igm circulates as a pentamer and iga circulates as a polymeric molecule. normally very little igd can be detected in serum. serum levels of igm, iga, igg, and ige are influenced by the rate of synthesis and rate of catabolism. like humans, the rate of igg catabolism in mice is directly proportional to the serum concentration of the subclass. the average half-life of murine igg is 4.5 days. the catabolic rate of iga is independent of serum concentration. mice synthesize from 50-130 mg/kg/day of total ig, although this is dependent on strain and level of antigenic stimulation. certain strains have a propensity to develop specific t-helper (th, cd4+) cell subclasses in response to antigenic stimulation, and these strains are referred to as having principally a th1 or th2-1ike phenotype. typically cd4+ lymphocytes modulate immune responses by the cytokines they secrete. those secreting il-1, ifn-y, and lymphotoxin are generally referred to as th1 (and are favored responses for immunity against viruses and intracellular pathogens) and those secreting il-4, il-5, il-10, and il-13 are referred to as th2 (and enhance humoral immunity while suppressing cell mediated immunity). when confronted with the same antigen, balb/c mice exhibit a th2-dominant response, and c57bl/6 mice exhibit a thl-dominant response. il-4 participates in immunoglobulin (antibody) class switching (see volume 4, chapter 5). consequently, th2 strains are the models of choice for investigations of allergic inflammation because they produce higher concentrations of il-4 induced immunoglobulin classes (ige and igg1). the cba/n strain is deficient in its ability to produce igm and igg3. immunoglobulin levels are also greatly reduced in germ-free mice, offspring of mice on zinc-deficient diets, and mice on protein-deficient diets (quimby 1999b) . quantifying the various classes and subclasses of murine ig can be done using various immunoassays including: radial immunodiffusion, ria, or enzyme immunoassay. both ria and enzyme immunoassay have the higher degrees of sensitivity that are needed to accurately quantitate levels of ige and igd in murine serum. enzyme immunoassay has become the favored assay to avoid isotope handling and disposal. the reported concentrations of normal balb/c mice are: igg1 (6.5 mg/ml), igg2a (4.2 mg/ml), igg2b (1.2 mg/ml), igg3 (0.1-0.2 mg/ml), iga (0.7 mg/ml), igm (1.0 mg/ml), and ige and igd are both less than 0.01 mg/ml (bankert and mazzaferro 1999) . in addition, there are many commercial sources of enzyme-linked antibodies that supply reagents (and instructions) for developing assays to measure antigen-specific antibody concentrations by subclass of antibody. the complement system is composed of 40 or more chemically and immunologically distinct proteins capable of interacting with antibodies, certain bacterial products, and cell membranes. a brief summary of this system is described later. please refer to two recent publications (quimby 1999a; turnberg and botto 2003) for more details about the structural and functional aspects of each protein of the complement system. the role of the complement system in mouse immunity is described in volume 4 (overview) of this series. the sequential activation of individual complement proteins from inactive to active substances is a dynamic event called the complement cascade. the ability of the first component of complement, c1, to bind specific sites on the heavy chain of mouse igg2b and activate a sequence of reactions leading to production of a molecular unit capable of lysing a target cell membrane has established the complement system as the primary mediator of antibody-antigen reactions. recent findings suggest that the pentraxins, c-reactive protein (crp), serum amyloid protein (sap), and pentraxin 3 (ptx3) can bind to complement component clq and activate the classical pathway. this may be an important mechanism for removal of apoptotic cells that might otherwise predispose to autoimmune disease (nauta et al. 2003) . each protein of the complement system is normally present in the circulation as an inactive molecule. although the complement cascade may be activated by any of four separate pathways, the central event for each is activation of c3 to c3b yielding a small c3 cz chain fragment. the two c3 convertases are c4b2a (for the classical, lectin, or pentraxin pathways) and c3bbb for the alternative pathway. each c3 convertase cleaves c3 and adds the c3b fragment to the convertase complex forming c5 convertase. cleavage of c5 leads to the membrane attack complex (mac) common to all activation pathways (goldsby et al. 2003) . assemblage of the mac on the surface of a target cell leads to the formation of a large channel enabling ions and other small molecules to diffuse out leading to cell death. the activated components of complement also participate in chemotaxis, phagocytosis, cell adhesion, and b-cell differentiation. there are many notable differences between mice and humans regarding expression of complement components and regulation of cascade activation. mice have both an active and inactive form of circulating c4 and the genes (both on chromosome 17) are designated ss and slp, respectively. many inbred strains do not synthesize active c5 (e.g., dba/2 and a/j strain) due to a post-translational defect and therefore they cannot generate a mac. some strains are deficient in the production of c8 (e.g., dba/2j strain). complement component 6 exists as two allelic forms in mice with 90 and 100 kda molecular weights. similarly there are several notable differences in the regulators of complement activation. most of these regulators are found in the regulator of complement activation (rca) locus on murine chromosome 1 and, as in humans, restrict assembly and stability of convertase enzymes. in mice, decay acceleration factor (daf) is encoded by two genes. however, only the product of daf-1 is widely dispersed on all tissues. this membrane-anchored protein inhibits c3 cleavage by accelerating decay of c3 convertases. in mice it is the major regulator of c3 in the skin (not kidney) and is a ligand for cd97 which, on cross-linking, leads to lymphocyte activation. cd59 is a membrane-anchored inhibitor of c5b-9 formation (mac) and prevents c9 from binding the c5b-8 complex. deficiencies of cd59 in humans lead to hemolytic anemia but not in mice. membrane cofactor protein (mcp) is a major cofactor for factor i in humans causing cleavage of c3b and c4b deposited on self-tissue. in mouse mcp is only expressed in the testis. complement receptors 1 and 2 are encoded by separategenes in humans but are produced by alternative splicing of a single gene in mouse. mice have a complement receptor 1 (cr-1) related gene/protein y (crry) not found in humans, which is a membrane-anchored c3 inhibitor. it is the major regulator of c3 in mouse kidney and has some of the functions as mcp and cr1 in humans. crry has daf activity and serves as a cofactor for factor i (which enzymatically cleaves c3b). the mouse has been widely used in studies on the biosynthesis and molecular biology of individual components of complement. the genes encoding 39 components, subcomponents, receptors, and inhibitors have been identified in mice. the complement components may be quantified by assays designed to measure the functional properties of these proteins or their antigenic properties. tests designed to measure antigenic properties of complement are generally simpler, less subject to error, and less expensive; however, they have the disadvantage of measuring both active and inactive forms of their proteins and therefore may not correlate well with functionally active protein. functional assays measure the ability of the entire classical or alternative pathway, or individual components of pathways to lyse (hemolyze) antibody-coated (sensitized) or noncoated (for the alternative pathway) red cells in suspension or in agarose gel. these assays are precise and sensitive (quimby 1999a) . antigenic assays include radial immunodiffusion, electroimmunodiffusion (rocket electrophoresis), automated immunoprecipitation, crossed immunoelectrophoresis, and more recently elisa (quimby 1999a) . elisa kits are commercially available for quantifying murine c lq, c3, c4, c3a (desarg), and c3a. in addition antibodies are commercially available for these as well as murine c4a, c4d, c5, and c6. complement components c2, c5, c6, c7, and factor b may be quantified using functional assays (quimby 1999a) . membrane-bound complement regulators, crry, cd59, and daf, can all be detected using previously published antibodies (lin et al. 2001 (lin et al. , 2002 . the principle mediator of the lectin activation pathway is mannose-binding lectin (mbl). after binding to mannose residues on the surface of microorganisms, two mbl-associated serine proteases (masps), masp-1 and masp-2, bind to mbl. this complex causes cleavage and activation of c4 and c2 (masp1 and 2 mimic the activities of c14 and cls). commercially available elisa kits are available for murine mbl-a and mbl-c quantitation. other activators of complement include members of the pentraxin family. in mice this may include crp or sap, although the concentration of crp in normal mice is very low. immunologic reagents are available to quantify both pentraxins in mice (gentry 1999; quimby 1999b ). circulating immune complexes (cic) are multimolecular substances composed of antigen, antibody, and activated complement components. in mice, igm, igg1, igg2a, and igg2b have complement activation regions. although cic may form following exposure to circulating foreign antigens, such as those associated with microorganisms, they are also common manifestations of spontaneous autoimmune disease such as that seen in (nzb x nzw)f1, mrl/lpr, bxsb, and krn strains. cic are cleared from the circulation by both cr-1, cr-2, and fcr. tissue deposition of immune complexes may lead to vasculitis. cic have been quantified in mice by capitalizing on their binding to various complement receptors, by precipitation of clq with polyethylene glycol, or by immunoassay. commercially available elisa kits are commonly employed today (quimby 1999b ). more than 100 inbred strains or mutant lines spontaneously develop autoimmune disease or are susceptible to autoimmune disease induction. the details of many of these lines may be found in volume 4, chapters l l and 12 in this series. autoimmune diseases in mice include: thyroiditis, rheumatoid arthritis, sj6gren's syndrome (ss), hemolytic anemia, lupus erythematosus, type 1 diabetes mellitus, experimental allergic encephalitis, oophoritis, orchitis, gastritis, ulcerative colitis, and polyendocrine disease (boyton and altmann 2002; ravirajan and isenberg 2002; sakaguchi 2000) . antibodies directed to self-antigens in the mouse have been quantified using a wide range of methods from immunofluorescence to elisa. table 6 -3 lists the commercially available elisa kits used to quantify murine autoantibodies. those antigenic targets associated with systemic lupus erythematosus include: cardiolipin, double-stranded deoxyribonucleic acid (dsdna), single-stranded deoxyribonucleic acid (ssdna), histone, [~-2 glycoprotein, proliferating cell nuclear antigen (pcna), neutrophil cytoplasmic antibody (canca), ribosomal p, and smith. antigenic targets for arthritis include: rheumatoid factor (rf), collagen type 1, collagen type 2, and ssdna. antibodies against insulin and glutamic acid decarboxylase are seen in type 1 (juvenile) diabetes. mixed connective tissue diseases are characterized by antibodies to ribonuclearprotein (rnp). antibodies to myeloperoxidase (mpo) may be observed in vasculitis. mice with ss develop autoantibodies to ss antigens a and b (ssa and ssb, respectively). panels containing 14 autoantigens are available as multiplex assays for quantifying murine autoantibodies. a. interleukins (il) (ils are cytokines that are secreted by leukocytes and act on other leukocytes. interleukins have been classified based on the secretory cell type (i.e., monokines vs. lymphokines), and they have been classified based on whether they are primarily involved in innate (il-1, il-6, il-12, tnf-t~, ifn-t~), or adaptive (il-2, il-4, il-5, il-10) immunity. commercially available elisa kits are available to quantify most murine interleukins, as demonstrated in table 6 -3. i. interleukin-1 (il-1) il-1 is a name for two proteins, il-lt~ and il-1 [3, that are encoded by separate genes. along with il-1 receptor antagonist, il-18, il-6, and tnf-t~, these proteins modulate acute inflammation. the effects of il-1 are pleotrophic and involve bone remodeling, insulin secretion, appetite regulation, fever induction, neuronal development, and many others. both il-1 o~ and il-1 [3 are secreted as 269-271 amino acid (aa) pro-cytokines that are enzymatically cleaved into bioactive 17-kda segments. unlike il-1 [3, the intact procytokine of il-1 ct is also bioactive, both within the cytoplasm and on the cell surface, where it is anchored to the cell membrane via a mannose glycosylation residue that attaches to the membrane-associated lectin. there is 78% sequence identify between mouse and human il-i~ genes and 58% identity between mouse and human il-1 t~ genes. a third gene encodes il-1 receptor antagonist, a soluble 25-kda molecule with 19% sequence homology to il-lt~ and 26% homology with il-113. mouse il-lra is 75% homologous with human il-lra. there are two il-1 receptors, types i and ii, but only il-1ri is capable of signal transduction. il-lra inhibits the action of il-lt~ and il-1b by binding il-1ri. a 60-kda form of il-1ri has also been described that is soluble and preferentially binds il-lra. il-1rii has no signal transducing associated protein and serves to modulate levels of il-1 t~, il-i[3, and il-lra by binding them on the cell surface. it can also occur as a soluble receptor. with the recent finding of six new members in the il-1 ligand family (in humans), a revised nomenclature for both il-1 ligands fred w. quimby and richard h. luong and il-1/il-18 receptor families has been developed (sims 2002) . further details may be found in volume 4 (overview and chapter 8) of this series. ii. interleukin-2 (il-2) il-2 is a lymphokine secreted by activated t-helper cells. it acts in an autocrine fashion to induce the expression of il-2 receptor on t cells, resulting in t-cell proliferation (cogoli-greuter et al. 2004) . il-2 also acts in a paracrine fashion modulating the activities of b cells, natural killer (nk) cells, and lymphocyte activated killer (lak) cells. il-2 is a glycoprotein of 133 amino acids (in humans) with 63% homology between mouse and human. the il-2 receptor (il-2r) is a multisubunit cellular receptor belonging to the class 1 cytokine receptor family (hematopoietin receptor family). the il-2r has a-, [~-, and y-chains. [3-and y-chains interact to transduce the il-2r signal and the y-chain is shared with receptors for il-4, il-7, il-9, and il-15. iii igg1 in mice and is responsible for the downstream events leading to differentiation and activation of th2 cells. il-4 also induces expression of adhesion molecules like vcam, th2 cytokines such as il-5, il-6, and il-9 and chemokines like eotaxin-1 and-2. il-4 primes mast cells and basophils leading to enhanced activation during allergic challenge (mueller et al. 2002) . homology between mouse and human molecules is low (25%) and each is species-specific in its biologic activity. it induces the growth of b-1 progenitors and igm production by b-1 cells. il-5 induces class switch, favoring production of iga, igg1, and ige. on eosinophils, il-5 induces iga and igg receptors and stimulates leukotriene (lt), c4, and paf secretion, in addition to inducing eosinophil growth and maturation. the receptors for il-5 consist of a ligand binding tx-subunit and a non-ligand binding (common) signal transducing ~-subunit that is shared by receptors for il-3 and granulocyte-monocyte colony-stimulating factor (gm-csf) (sato and miyajima 1994) . vi. interleukin-6 (il-6) il-6 is secreted by a wide variety of cells including t cells, b cells, monocytes, fibroblasts, hepatocytes, keratinocytes, astrocytes, and endothelial cells. it has broad pleiotropic effects on host defense, acute phase responses, immune responses, and hematopoiesis. il-6 is classified as an inflammatory cytokine and based on a helical cytokine structure and subunit makeup, il-6 is the prototypic member of a family of molecules that includes leukemia inhibitory factor (lif), oncostatin m (osm), ciliary neurotrophic factor (cntf), cardiotrophin (ct-1), and il-11. mouse il-6 is 25 kda and contains four cysteines and contains o-glycosylation sites and shares 40% homology with the human molecule (van snick 1990). the il-6 receptor has two subunits, a nonsignal transducing subunit binding with low affinity (~-subunit), and a signal transducing subunit (~-subunit) that does not bind il-6 by itself but participates in high-affinity binding. the soluble il-6r~ chain binds il-6 and the complex induces expression of mcp-1, which attracts monocytes into areas of inflammation (kaplanski et al. 2003) . vii. interleukin-7 (il-7) il-7, previously called lymphopoietin-1, is expressed by stromal cells, especially in the bone marrow and thymus, where it promotes thymopoiesis of t cells and the differentiation of pro-b cells into pre-b cells (aspinall et al. 2004; goldsby et al. 2003) . mouse il-7 has 65% amino acid sequence homology with human il-7 and both proteins exhibit cross-species activity. viii. interleukin-8 (il-8) il-8 is not expressed in the mouse; however, another protein, kc, is secreted and has many properties of the human chemokine, gro, which is known to bind the il-8 receptor (see the "cytokines and chemokines" section). ix. inrelclevlcln-io (il-io) il-10 is the prototypic member of the il-10 cytokine family comprising ill0, il-19, il-20, il-22, il-24 (fisp), and il-26. il-10 is a 178 amino acid protein with an 18 amino acid signaling sequence. both mouse and human il-10s have two intrachain disulfide bonds and form non-disulfide linked homodimers. mouse and human il-10 are 72% homologous. il-10 is a th2 cytokine, which inhibits ifn-y and gm-csf production by th1 cells. additionally it induces cd8 + t-cell chemotaxis, inhibits t-cell apoptosis, participates in iga class switch in b cells, induces histamine release from mast cells, and promotes tnf-tx and gm-csf production by nk cells. il-10 inhibits secretion of the neutrophil chemokines mip-lt~ and mip-i~ and blocks production of il-1~ and tnf-ct by neutrophils. it is immunosuppressive to dendritic cells and induces the differentiation of a subset of regulatory cd4 + t cells (trl) (grouz and cottrez 2003; morel et al. 1997 ). x. il-11, also known as adipogenesis inhibitory factor (agif), is a pleiotropic cytokine with effects that overlap that of il-6. il-11 is a member of the il-6 cytokine family and as such has a four-helix bundle fold motif. it binds to the multimeric il-11 receptor that shares the promiscuous gpl30 signaling ~-subunit with other receptors in this family. the il-11r ~ chain is unique and binds il-11 but does not have a cytoplasmic domain; instead binding leads to homodimerization of the ~-chain that activates the janus kinases. il-11 stimulates proliferation and differentiation of monocytes and megakaryocytes causing thrombopoiesis. it also activates osteoclasts and enhances bone resorption, decreases new bone formation, and stimulates chondrocyte and synoviocyte production. il-11 protects small intestinal epithelial cells from chemotherapy and radiation injury and ameliorates inflammatory bowel disease (schwertschlag et al. 1999) . il-11 inhibits adipogenesis, regulates neuronal differentiation, and regulates t-cell function (enhances th2 and inhibits th1 cytokine production). overexpression of il-11 in the lung causes airway remodeling, fibrosis, and mononuclear nodules analogous to the clinical picture in chronic asthma. il-11 is also required for the uterine decidualization response (robb et al. 2002; zheng et al. 2001) . xi. il-12, also known as natural killer cell stimulatory factor (nksf), is a heterodimeric cytokine composed of a 40-kda (p40) subunit and a 35 kda (p35) subunit. the p40 subunit is shared by il-23, a cytokine with similar activities. macrophage, monocytes, and dendritic cells produce il-12 after activation of toll-like receptors (tlr) on these cells by bacterial ligands. il-12 induces production of ifn-7by th1 and nk cells and intact il-12 skews the balance between th subsets in favor of th1 cells. il-12 binds to the il-12 receptor that is composed of two subunits, [31 and ~2, on the surface of nk and th1 cells. il-12p40 interacts with il-12r[31, and il-12p35 binds il-12r[32. negative feedback regulation of il-12 production involves down regulation of tlr signaling by phosphoinositide 3-kinases (piks). thus il-12 is centrally involved at the interface of innate and adaptive immunity (fukao and koyasu 2003" ottenhoff et al. 2002) . xit, il-13, along with il-4 and il-5, is a member of the type-2 cytokine family and as such is involved in inflammation, mucus production, tissue remodeling, and fibrosis. this single-chain protein shares 58% amino acid sequence homology with human il-13. il-13 is produced by activated t cells, mast cells, and nk cells and promotes th2 responses including synthesis of ige. signaling is mediated by the type-2 il-4 receptor which consists of il-4r~ and il-13rc~i chains. another il-13 binding protein, il-13r~2, strongly inhibits the activity of il-13 (goldsby et al. 2003; mentink-kane and wynn, 2004) . xiii. il-17, also known as cytotoxic t lymphocyte-associated antigen-8 (ctla-8), is produced by t cells and is pleiotropic in activity. il-17 is a 158 amino acid residue polypeptide with a 21 amino acid signal sequence and a mature polypeptide of 137 amino acids. it is a disulfide-linked homodimer. based on the presence of spatially conserved cysteine residues in the il-17 family of proteins, there are six family members in humans and mice, il-17, il-17b, il-17c, il-17d, il-17e, and il-17f (aggarwal and gurney 2002) . like il-17 itself, several of the family members appear to modulate immune function. produced by mouse cd4 ⧠t cells, il-17 induces il-6, mcp-1, prostaglandin-e2 (pge2) and granulocyte colony-stimulating factor (g-csf) by fibroblasts, keratinocytes, epithelial cells, and endothelial cells. it induces icam-1 surface expression, proliferation of t cells, and the differentiation of cd34 + marrow progenitors into neutrophils (fossiez et al. 1998 ). the ubiquitously distributed receptor is a type 1 transmembrane glycoprotein of 830 amino acids in length. xiv. il-18 is a 24-kda, nonglycosylated polypeptide that lacks a classical signaling sequence. its structure resembles il-1 and the propeptide undergoes proteolytic cleavage by interleukin-1 [3-converting enzyme (ice) or another caspase to produce an 18-kda bioactive molecule. there is 64% sequence homology between mouse and human il-18. il-18 induces the production of ifn-7 by t cells and nk cells and the expression of fas ligand (fasl) on a variety of all types. il-18 activates nf-~:[3 and the induction of various chemokines. il-18 plays an important role in the early antibacterial host response (weijer et al. 2003) . xv. and il-20 induces keratinocyte differentiation and proliferation. il-21 is a four-helix-bundle cytokine similar in structure to il-15 and sharing sequence homology with il-2 and il-4. murine il-21 is 57% homologous to human il-21. the il-21 receptor utilizes the common 7-chain. il-21 is produced by the th2 cells. the actions of il-21 are pleiotropic and seen on b cells, t cells, nk cells, and dendritic cells. il-21 induces apoptosis in resting and activated b cells, an effect counteracted by activation of cd40. it also upregulates production of igg1 and inhibits ige, in fact it inhibits many il-4 activities. il-21 also inhibits dendritic cell differentiation. il-21 enhances the activity of activated nk cells and mediates the proliferation and expansion of t-cell subsets. il-22 induces acute phase reactants by hepatocytes and reduces il-4 production by th2 (mehta et al. 2004) . recombinant antigens and antibodies are commercially available for murine il-20, il-21, and il-22. b. the transforming growth factor-~ superfamily (tgf-~sf) of cytoi~nes members of tgf-~ family share 25-40% sequence homology with tfg-~i and a monomeric structure that consists of two antiparallel pairs of [3-strands forming a flat curved surface, a separate long cz-helix, and a disulfide rich core with a characteristic cysteine knot. most tgf-~sf members are disulfide-linked homodimers; however, three members lack the seventh conserved cysteine residue and are not covalent homodimers. members of the tgf-[3sf include tgf-[31, tgf-132, tgf-133, activins, inhibins, bone morphogenic proteins (bmp), growth differentiation factors (gdf), glial-derived neutrophic factors (gdnf), and mtillerian inhibiting substance (mis). tgf-[31 is stimulatory for cells of mesenchymal origin and inhibitory for cells of epithelial or neuroectodermal origin. in the murine immune system tgf-[31 is the mediator of immune suppression via cd4+cd25+tr cells and, at least in the case of suppressing cd8 + effector t cells, involves tgf-[3 receptor ii on these cells (powrie 2004; von boehmer 2005) . in addition tgf-[3 is known to inhibit b-cell proliferation and it promotes isotype switch to iga. oral tolerance to th2 responses (against food allergins) is mediated by tgf-~i1 (mucida et al. 2005 ). tgf-[3 has a wide range of effects on cell growth differentiation and malignant transformation (letterio 2005) . murine tgf-~i 1 may be quantified in serum or plasma using commercially available elisa kits. antibodies are also available which specifically bind murine bmp, activin a, activin c, and gdf-1,-3,-5,-8, and-9, although they are not recommended for elisa development. c. the tumor necrosis factor superfamily (tnfsf) tnf-related ligands share many features but high amino acid sequence homology is not one of them. with the exception of nerve growth factor and tnf-~, all ligands are type ii tramsmembrane proteins (extracellular c-terminus) that contain a short cytoplasmic segment and a long extracellular region. tnf-~ is fully secreted and has a nonfunctional transmembrane segment. tnfsf members form trimeric structures and their monomers are composed of ~-strands oriented into a twosheet structure. receptors for the tnfsf ligands also belong to a superfamily, tnfrsf (gruss and dower 1995) , and are characterized as type i transmembrane proteins (with their amino termini outside of the cell), with extracellular cysteine-rich structural motifs. tnfrsf members exist both as membrane and soluble forms. commercially available elisa kits or matched antibody for development of assays are available to quantify the murine receptors and ligands of the tnfsf discussed in this section. i. tumor necrosis factor-ix (tnf-ix) tnf-tx is expressed as a 26-kda membrane glycoprotein and the soluble glycoprotein is generated by proteolytic cleavage via tnf-ct converting enzyme (tace). the 17-kda homotrimer cleavage product circulates. mouse tnf-tx has 79% sequence homology with human. tnf-tx is expressed widely on tissues throughout the body (goetz et al. 2004) . tnf-tx is a strong mediator of inflammation and immune function, or regulates on growth and differentiation, and is cytotoxic for many transformed cells. ii iii. cd40l cd40l (tnfsf5, cd154) is a 39-kda, type ii, transmembrane glycoprotein that can be proteolytically cleaved to 15-to 18-kda soluble forms with full biologic activity. it forms natural trimeric structures and the mouse cd40l shares 73% sequence identity with human cd40l. cells expressing cd40l include b cells, cd4 + and cd8 ⧠t cells, monocytes, nk cells, and y t cells (toubi and shoenfeld 2004) . on binding cd40, the complex initiates signals important for cell proliferation or apoptosis. cross-linkage between t and b cells allows cd40 to transduce the tyrosine kinases lyn and syk, and activate phospholipase c, ip3, and dag. when combined with other cytokines, ligation of b-cell cd40 provides the second signal allowing differentiation of b cells to plasma cells (goldsby et al. 2003) . iv. cd30l cd30l (tnf5f8, cd153) is a 40-kda glycoprotein with 72% sequence homology between murine and human molecules. cd30l is expressed on monocytes, macrophages, b cells, activated t cells, neutrophils, megakaryocytes, resting cd2 ⧠t cells, erythroid precursors, and eosinophils. ligation to cd30 can induce either proliferation or apoptosis. v. fas ligand (fasl) fasl (also known as tnfsf6), is a 40-kda glycoprotein that, after cleavage, forms a 70-kda homotrimer that is active only in membrane form in the mouse. polymorphisms in fasl also exist and a single amino acid substitution in position 273 (phe to leu) results in the generalized lymphoproliferative disease (gld) mutation. fasl is expressed on cells of the adaptive and innate immune systems, as well as cells of the lung and intestine. there is 77% sequence homology between murine fasl and human fasl (lynch et al. 1994) . ligation of fas by fasl on mature t cells leads to activation of the caspase cascade and apoptosis. this is a major homeostatic mechanism regulating the size of the t-cell pool and for eliminating t cells that repeatedly encounter self-antigens (goldsby et al. 2003) . vi. tnf-relateo activation-induced cytokines (trance) trance, also called rank ligand and osteoprotegerin ligand (opgl), is an osteoclast differentiation factor. mouse and human share 85% sequence homology, and trance is expressed on t cells and t-cell rich organs such as thymus and lymph nodes. vii. tnf-receeror superfamily (tnfrsf) tnfrsf members mediate the cellular effects of tnfsf members. tnfri and tnfrii bind tnf-~ and it appears tnf-~ complexes with lt-[3 and the complex binds to tnfri or lt-[~ receptor. cd40 is associated with b-cell proliferation but is expressed on many cells throughout the body. mouse cd40 shares 62% sequence homology with human cd40; however, the mouse molecule has a 28 amino acid extension of its cytoplasmic tail. cd30 has 480 amino acid residues and a 90 amino acid deletion in the extracellular region compared to human. it is expressed on cd4 + and cd5 + t cells and ligation results in production of il-5. murine fas lacks 8 amino acid residues found in human and shares only 50% sequence homology. soluble forms result from alternative gene splicing and circulate as dimers or trimers. fas is expressed by cd34 stem cells, fibroblasts, nk cells, keratinocytes, hepatocytes, b and t cells (and their precursors), and eosinophils. osteoprotegerin (opg) inhibits the action of osteoclasts and is a secreted member of the tnfrse although it has no transmembrane segment and circulates as a disulfide-linked homodimer. murine troy, also named toxicity and jnk inducer (taj) and tnfrsf19, shares 92% homology with human in its extracellular domains. d. interferons interferons are a group of related but distinct proteins that share more than 95% amino acid sequence homology. members of the type i interferon family share a common cell surface receptor composed of two subunits. commercially available elisa kits may be used to quantify murine io interferon-~ (ifn-~) ifn-~ is induced in a wide variety of cells, including monocytes and macrophages, in response to viral infection. one known inducer is double stranded ribonucleic acid (dsrna). induce resistance to viral replication by binding the ifn-~/~ receptor, which activates the jak-stat pathway, inducing several genes. one of those genes is ribonuclease, which degrades viral rna. binding of ifn-~ to nk cells enhances their lytic activity for virally infected cells. ifn-~ is secreted by leukocytes. iii inreturelcon-'t(ifn-y) ifn-y is secreted by th1 cells, nk cells, and cytotoxic t cells (tc), which activates macrophages to secrete tnf-ct, express class ii major histocompatibility complex (mhc) molecules, and produce antimicrobial activities. ifn-y secretion by th1 also induces antibody-class switch to igg2a, which supports phagocytosis and complement fixation. ifn-y promotes differentiation of tc from cd8 + precursors that will be involved in the effector response to viral infections and intracellular pathogens. ifn-y also inhibits the expansion of th2 cells. ifn-y secretion is induced by successful stimulation of t cells by antigen presenting cells. e. chemo~s chemokines, along with adhesion molecules, are the principle controllers of leukocyte migration and as such directly affect leukocyte retention and relocation during hematopoiesis and at sites of immune defense and inflammatory disease (moser et al. 2004) . chemokine-induced signaling is via g-protein coupled cell surface receptors. although most chemokines are secreted proteins, two chemokines, cxcl16 and cx3cl 1, are membrane bound. two primary subfamilies are recognized based on the arrangement of two nh2-terminal cysteine residues that are either located adjacent to each other (cc) or are separated by a single amino acid (cxc). two minor subfamilies include chemokines with a single cysteine resides (xcl1, xcl2) and a chemokine with three amino acids separating the cysteine residues (cx3cl1). functionally conserved regions of the n-terminus of each member mediate receptor binding and extracellular matrix fixation (or binding cell surface glycosaminoglycans). many chemokines are designated with a name given at the time of their identification; all have also been assigned a name based on their structural motif (cc, cxc, xc, cx3c) followed by l for ligand. receptors are heterotrimers and their activated g-protein subunits stimulate phospholipase c[3, piks, and c-src tyrosine kinases (moser et al. 2004) . receptors are designated by the type of chemokine they bind (i.e., cxc, cc, xc, or cx3c), followed by r. mice lack the cxcr1 family of chemokines and receptors found in humans. table 6 -8 lists the murine chemokine receptors and the known ligands. table 6 -3 lists the murine chemokines for which there are either commercial test kits or matched antibodies for kit development. two main functional groups define chemokines. inflammatory chemokines recruit effector leukocytes to sites of infection, inflammation, and repair. homeostatic chemokines control the navigation of leukocytes during hematopoiesis in the bone marrow and thymus, control homing of cells to spleen, lymph nodes, and peyer's patches during the adaptive immune response and control immune surveillance in peripheral tissues. some chemokines participate in both inflammation and homeostasis and are called dual-function chemokines. many dual function chemokines are highly selective for the recruitment of t cells. other chemokines have ill-defined functions regarding homeostasis and inflammation but participate in other vital activities such as the role of pf4 (cxcl4) in thrombosis and the role of cxcl 10 in gut epithelial cell turnover (cliffe et al. 2005) . most inflammatory chemokines are thought to be induced and the variety of stimuli that induce their expression is broad. by contrast most homeostatic chemokines are thought to be constitutively expressed. an exception is the inducing effect of lymphotoxin and tnf-~ on b-cell attracting chemokine-1 (bca-i, cxcl13), ccl19, and secondary lymphoid tissue chemokine (slc, ccl21), which also participate in inflammation. leukocytes also release several kinds of proteases that degrade chemokines at their n-terminus, resulting in the loss of receptor binding, antagonist generation, or enhancing their biologic function. leukocyte cd26, dipeptidyl peptidase iv is known to act on cxcl9, cxcl10, cxcl11, and cxcl12. murine sulphostin inhibits the action of cd26 and stimulates g-csf and granulopoiesis (abe et al. 2005) . matrix metalloproteases (mmps) are enzymes that degrade extracellular matrix proteins, including stromal cell derived factor-1 (sdf-1, cxcl12) and mcp-1. table 6 -3 lists the murine mmps that can be quantified by commercially available elisa kits. in addition to mmps, cathepsins have been shown to modify chemokines. table 6 -9 lists the murine chemokines and the proteases that degrade them and stimuli that induce them. certain chemokines may antagonize the activity of other chemokines; for instance, three agonists for the receptor cxcr3 are also antagonists for receptor ccr3 (which is agonized by eotaxin [ccl11]). because cxcr3 and ccr3 are differentially expressed on th1 and th2 cells, these chemokines [monokine induced by ifn-y (mig/cxcl9), ifninducible protein-10 (ip-10/cxcl10), and ifn-inducible t-cell chemoattractant (i-tac/cxcl11)] modulate the th subpopulation allowed to enter tissue sites favoring th1 immune polarization (moser et al. 2004) . table 6 -10 summarizes some known effects of murine chemokines. because chemokines and their receptors are known to modulate many inflammatory diseases including the autoimmune diseases, they have become target for new therapeutics. the chemokine antagonist, met-rantes, has been shown to be an effective inhibitor of allergic airway disease in mice. likewise, deficiencies of chemokines and their receptors in mice have modified disease progression in atherosclerosis, autoimmunity, and also prolong allograft survival (mackay 2001 ). in addition, many viral genomes are known to encode structural genes for chemokine antagonists, which appears to be a principal mechanism used by many viruses to evade the host immune system. these present another target for drug intervention for viral infections. finally, pepducins, derived from the intracellular loops of cxcr1, cxcr2, and cxcr4, specifically inhibit receptor g-protein signaling in mice and prevent fatal sepsis and disseminated intravascular coagulation (kaneider et al. 2005 ). certain growth factors (see table 6 -3) and cytokines activate phospholipases after binding their cell surface receptors. these phospholipases act on membrane phospholipids to release arachidonic acid, a precursor for several eicosanoids. arachidonic acid is metabolized by any one of three biochemical pathways: the cyclooxygenase (cox) pathway, which forms pgs and thromboxane, the lo pathway, which forms hetes and leukotrienes (lts), and the cytochrome p-450 monooxygenase pathway, which forms epoxides and hetes. the cox enzymes, cox-1 and cox-2, catalyze the first step in the synthesis of pgs by converting arachidonic acid to prostaglandin h 2 (pgh2). pgh 2 is chemically unstable and is the precursor for enzymatic and nonenzymatic production of pgd 2, pge 2, and pgfza. thromboxane synthetase converts pgh 2 to thromboxane a 2 (txa2) that is quickly converted to thromboxane b 2 (txb2). in vascular tissue, pgh 2 is converted to pgi 2 or prostacyclin by prostacyclin synthetase (natarajan and nadler 2004; reimers 1999) . although cox-1 is constitutively expressed in most tissues, cox-2 is induced by bacterial lipopolysaccharides, il-i~, il-ll~, and tnf-~ (see the "cytokines and chemokines" section). in the circulation, pge 2 and pgi 2 cause vasodilation, whereas pgfza and txb 2 are potent vasoconstrictors. in the kidney, pge1, pge2, pgd2, pgg2, pgi2, and pgh2 produce vasodilation, increased renal blood flow, and urinary excretion of sodium. renal production is increased by pgd2, pge2, and pgi2. pgg2, pgh2, and txa 2 modulate platelet aggregation and, following platelet adhesion, they release catecholamines, serotonin, and adenosine diphosphate, which enhance platelet aggregation. pgi 2 is a potent inhibitor of platelet aggregation. pge1 increases, whereas pge2 decreases, the ability of red cells to pass through capillaries. pge2 and pgfza inhibit the activities of t and b cells and the production of ils and chemokines, which attract monocytes. pgd 2 is a potent inducer of chemotaxis for th 2 cells and plays a major role in allergic airway disease. through the varied activities of vasodilation, vascular permeability, and leukocyte migration, the pgs are potent modulators of inflammation. in addition pge1 inhibits mackay 2001 rossi et al. 1999 niess et al. 2005 mackay 2001 huang and xiang 2004 yamaguchi et al. 2005 insulin secretion and release after glucose challenge (see the "glucose and carbohydrate metabolism" section), and it inhibits the lipolytic effects of glucagons, adrenocorticotropic hormone, and epinephrine (see the lipid metabolism section) and inhibits the secretion of corticosterone, prolactin (prl), gh, thyroid stimulating hormone (tsh), and lh. in fact, pgs have a multitude of effects associated with female reproduction (reimers 1999 table 6 under the action of 5-lo, arachidonic acid is converted to 5-hydroxyperoxyeicosatetraenoic acid (5-hpete) and then leukotriene a4 (lta4), which is unstable. lta4 is metabolized to ltb4 by lta4 hydrolase or to cysteinyl leukotrienes (ltc4, ltd4h, and ltc45) by ltc4 synthase. 5-lo expression is limited to neutrophils, eosinophils, monocytes, and mast cells; however, lta4 hydrolase is expressed in erythrocytes, t cells, platelets, airway and intestinal epithelial cells, fibroblasts, heart, kidney, and adrenal cortex. because the latter cells and tissues do not express 5-lo, lta4 must be delivered to them via myeloid cells by a process known as transcellular biosynthesis (maclouf 1993) . receptors for ltb4 (blt1 and blt2) have been identified and are either widely expressed (blt2) or are confined to myeloid cells, t cells, and lung cells (blt1). in addition to attracting myeloid cells to sites of inflammation, ltb4 is a potent inducer of th1 and th2 t effector cell chemotaxis (as potent as cxcl12 in the mouse) and cd8+t-effector cell chemotaxis (as potent as rantes). like pgd2, ltb4 plays a major role in allergic airway disease (luster and tager 2004) . additional information on murine prostanoids and their receptors is available in recent reviews (jala and haribabu 2004; kobayashi and narumiya 2002) . all the arachidonic acid metabolites discussed in this section may be quantified using commercially available elisa kits (see table 6 -3). e. enzymes ap is an inducible enzyme in which serum activity is increased due to increased synthesis. the exact physiologic function of ap is not known but is thought to transport metabolites across cell membranes. quantification of serum ap is based on a reaction between ap and a suitable phosphorylated substrate that is susceptible to ap activity. as in other species, there are two major forms of ap in mice: intestinal ap (lap) and tissue nonspecific ap (tnap). unlike other domestic animal species, iap activity contributes to serum ap activity, in addition to tnap. lap is located along the brush-border of the enterocytes. intestinal ap activity was shown to vary four-fold between two strains of swiss mice (nayudu and moog 1967) . this difference in activity was under polygenic control and influenced by a strain-specific factor in milk. tnap is found in various tissues, and in each location, post-translation modifications may result in a different isoenzyme. hoshi et al. (1997) used immunohistochemistry to localize these isoenzymes in mice to the following locations: bone tissue (specifically the entire cell surface of preosteoblasts and the basolateral cell membrane of osteoblasts), cartilage (mostly in chondrocytes of the proliferative and hypertrophic zones), the incisors (particularly the cells of the stratum intermedium, the subodontoblastic layer, the proximal portion of secretory ameloblasts, and the basolateral portion of odontoblasts), kidneys (on the brush borders of proximal renal tubules in kidney), liver (on cell membrane of the biliary canaliculi), and the placenta (on trophoblasts). serum ap activity can vary due to the type of assay used, age, sex, and strain. different ap assays vary in the type of substrate used, the ph of the reaction, and the incubation temperature of the reaction, all of which can affect quantitation of ap activity. for example, the hausamen technique can detect renal and intestinal ap activity, but not hepatic ap activity (hausamen et al. 1967 ). loeb et al. (1996 ) and frith et al. (1980 demonstrated that serum ap activity decrease significantly after 3 months of age in balb/c and c57bl/6 mice of both sexes but increase again in very old (36 month old) mice. the high levels of serum ap seen in juveniles is related to increases in the bone ap isoenzymes, which is associated with osteoblastic activity due to rapid growth. picketing and picketing (1984) showed that serum ap activity is lower in males than in females. quantification of serum ap measures the total ap activity from all sources, and therefore elevations in ap activity can be a nonspecific determinant of tissue dysfunction, depending on the tissue (and therefore ap isoenzyme) involved. for example, changes in the serum activity of ap due to liver disease only occur if cholestasis is concurrently involved. mice lacking the gene that modifies tnap into the bone isoenzyme suffer from skeletal hypomineralization (anderson et al. 2004 ). alt is a cytoplasmic enzyme in which serum activity is increased due to leakage across damaged cytoplasmic membranes. alt (also known as glutamic pyruvic transaminase [gpt]) reversibly catalyzes the conversion of alanine to pyruvate. quantification of serum alt is based on a reaction between alt and a suitable substrate (such as alanine). in mice, alt is found in the highest concentrations in the liver, although activity has also been demonstrated in intestine, kidney, heart, muscle, and brain (clampitt and hart 1978) . despite its widespread tissue distribution, alt is mostly used as an analyte to assess hepatocellular damage (masaki et al. 2005; taieb et al. 2005 ). an 11,000% increase in serum alt activity has been reported following infection with mouse hepatitis virus, and significant increases follow infection by helicobacter hepaticus (mccathey et al. 1997 ). ast is a cytoplasmic and mitochondrial enzyme in which serum activity is increased due to leakage across damaged cytoplasmic and mitochondrial membranes. ast (also known as glutamic oxaloacetate transaminase [got]) reversibly catalyzes the conversion of aspartate to oxaloacetate. quantification of serum ast is based on a reaction between ast and a suitable substrate (such as aspartate). in mice, ast is found in a variety of tissues, including liver, skeletal muscle, cardiac muscle, erythrocytes, blood vessels, brain, intestine, kidney, lung, testes (papadimitriou and van duijn 1970) . the highest specific activity of ast is found in mouse cardiac muscle and lowest in skeletal muscle (herzfeld and knox 1971) . in the liver, ast is found mainly in periportal hepatocytes based on histochemical studies (papadimitriou and van duijn 1970) . activity in lung, kidney, intestine, and skeletal muscle is very low when measured by the technique of bergmeyer and bernt (1974) . loeb et al. (1996) demonstrate significant age-associated increases in serum ast activity in two inbred and two f1 hybrid strains. although widely distributed, alt is mainly used to assess hepatocellular damage, cardiac muscle damage (naraoka et al. 2005; ray et al. 2005) , and testicular injury (santos et al. 2005) . alt activity has been used as an indicator of hepatic injury of mice infected with mouse hepatitis virus (fassati et al. 1969 ). ldh is a cytoplasmic enzyme in which serum activity is increased due to leakage across damaged cytoplasmic membranes. ldh reversibly catalyzes the conversion of pyruvate to lactate. quantification of serum ldh is based on a reaction between ldh and a suitable substrate (such as pyruvate or lactate). as in other species, ldh in the mouse is a tetrameric enzyme consisting of either a or b subunits. there are five isoenzymes of ldh (based on differences subunit a and b composition), and these are ldh-1 (b4), ldh-2 (a1b3), ldh-3 (a2b2), ldh-4 (a3b1), and ldh-5 (a4). specific isoenzyme distribution depends on differential expression of either the a and b subunits (quimby 1999b) . for instance, all embryonic murine tissues contain ldh-5 because the a subunit is only expressed during early fetal development. as the embryo matures, subunit expression can involve both a or b subunits in different tissues, meaning that by birth and sexual maturity, each tissue contain a characteristic ldh subunit profile. in adult mice, the heart and erythrocytes contain ldh-1 and ldh-2, whereas most other tissues have ldh-3. skeletal muscle and hepatocytes fail to express subunit b and therefore are composed predominantly of ldh-5. serum ldh activity can vary due to age and sex. ldh levels have been shown to be higher in males versus females of the balb/c strain (frith et al. 1980) . serum ldh levels increase with age in balb/c and c57bl/6 mice of both sexes (frith et al. 1980) . serum ldh can also be elevated falsely by hemolysis. quantification of serum ldh measures the total ldh activity from all sources. however, damage to a particular tissue will result in increased activity of that isoenzyme in serum. in general, the highest activity of ldh in the mouse is in skeletal muscle, with decreasing activity in the heart, liver, kidney, and intestine. serum ldh-5 activity rises within 72 h after inoculating mice with the mouse hepatitis virus (fassati et al. 1969) . mice infected with the ldh virus (ldv) exhibit increased serum concentrations of ldh, isocitric dehydrogenase, malic dehydrogenase, phosphohexase isomerase, and ast (notkins 1965) . along with ast and ck, ldh is considered an excellent marker for cardiac injury (naraoka et al. 2005; ray et al. 2005) . otc is a mitochondrial enzyme in which serum activity is increased due to leakage across damaged mitochondrial membranes. otc is found primarily in the liver of mice, and there increases in serum activity reflect severe injury to hepatocytes. abnormal otc activity has been described in mice having the sparse-fur (spf/y) mutation. they serve as a model for the most common inborn error of urea synthesis in humans. assays for mouse otc have been developed to monitor activity levels following gene transfer or liver transplantation (batshaw et al. 1999; ye et al. 2001 ). ck is a cytoplasmic and mitochondrial enzyme in which serum activity is increased due to leakage across damaged cytoplasmic and mitochondrial membranes. ck reversibly catalyzes the phosphorylation of adenosine diphosphate (adp) to ate using creatine phosphate as the donor for the phosphate group. quantification of serum ck is based on a reaction between ck and a suitable substrate (such as creatine phosphate). as in other species, cytoplasmic ck is a dimeric enzyme consisting of either m or b subunits. there are three isoenzymes of ck (based on differences subunit m and b composition), and these are ck-1 (bb), ck-2 (mb), and ck-3 (mm). brain contains ck-1, skeletal muscle contains ck-3 (mm), and cardiac muscle contains ck-1, ck-3, and ck-2 (mb) (quimby 1999b ). in the mouse, the greatest ck activity is found in skeletal muscle, with much less activity found in the heart and brain. mitochondrial ck (ck-mt) is found in mitochondria of many tissues. serum ck activity is affected by age, sex, and method of collection and anesthesia. patrick et al. (1983) found that compared to jugular vein collection, cardiac puncture was associated with lower ck activity. levels of serum ck activity have been reported for balc/cann mice and c57bl/10 mice of varying ages and sex (sub et al. 1994) . serum ck activity is a useful and specific marker enzyme of muscle injury, because ck in central nervous tissue does not cross the blood-brain barrier. sub et al (1994) compared normal c57bl/10 mice with heterozygous male and homozygous female mice carrying the mdx (mutant dystrophin) allele, and found that homozygous females have 12-to 15-fold increases and heterozygous males (mdx/y) have 30 fold increases in plasma ck compared to wild-type mice. plasma ck levels correlated with skeletal muscle necrosis in these dystrophic mice. mice have been engineered that lack cytoplasmic ck and ck-mt . mitochondria from heart or skeletal muscle from double kos had higher adp concentrations compared to wild-type animals, suggesting the higher concentrations contribute to the control of the reduced cytosolic atp free energy potentials seen in double kos. aldolase is a cytosolic enzyme that can alter its distribution between soluble and particulate forms, according to the metabolic status of the tissue. in adult mice, nine aldolase isoenzymes are known to occur in tissues with significant activities in the muscle, brain, liver, kidney, and spleen. in the mouse liver aldolase, together with fructokinase and triokinase, metabolize fructose (hagopian et al. 2005) . everett and harrison (1983) report no apparent advantages in mice in the measurement of aldolase over other enzymes known to have specific liver or muscle activity. sdh is a cytoplasmic and mitochondrial enzyme in which serum activity is increased due to leakage across damaged cytoplasmic and mitochondrial membranes. sdh (also known as iditol dehydrogenase [idh]) reversibly catalyzes the conversion of fructose to sorbitol. quantification of serum sdh is based on a reaction between sdh and a suitable substrate (such as fructose). sdh is located primarily liver, kidney, and seminal vesicles. the activity of sdh is usually low in the serum and rises during hepatic injury. however, the labile nature of sdh during handling makes is less suitable overall as a indicator of hepatic dysfunction compared to over liver-specific enzymes (such as ast). mice with the gene for sdh knocked out have been used to study the role of sorbitol accumulation in diabetic albuminuria (ii et al. 2004 ). amylase is a cytoplasmic enzyme in which serum activity is increased due to leakage across damaged cytoplasmic membranes. amylase hydrolyzes complex carbohydrates to form maltose and glucose in the presence of free calcium ions. quantification of serum amylase is based on a reaction between amylase and a suitable substrate (such as starch). similar to humans and pigs (but not dogs, cats, cattle, and horses), expression of amylase in mice is related to two distinct but closely linked loci (meisler et al. 1983) . salivary amylase is the gene product of amy-1 (salivary), and appears to be a single enzyme. pancreatic amylase is the gene product of amy-2, and based on electrophoretic studies in inbred mice, there appear to four isoenzyme classes: a1, a2, b 1, and b2. similar to other domestic animal species, pancreatic amylase is filtered through the glomerulus, but unlike other domestic species, pancreatic amylase is not resorbed by renal tubular epithelial cells and is excreted rapidly in the urine. therefore normal serum amylase activity in mice consists mainly of salivary amylase (mackenzie and messer 1976) . despite this, elevations in serum amylase activity is usually considered a reliable marker for pancreatitis in mice (nathan et al. 2005) . ross et al. (1974) reported two-to three-fold increases in serum amylase activity in mice infected with coxsackievirus of salivary and pancreatic trophism. alterations in the activity of specific pancreatic isoenzymes have been shown in streptozotocin-induced diabetes in mice (quimby 1999b) . there are two pancreatic lipase isoenzymes in mice. serum lipase activity has been used to monitor cerulean-induced acute pancreatitis in mice (cuzzocrea et al. 2004) . recently a new colipase-dependent lipase has been described in suckling mice (d'agostino and lowe 2004) . the enzyme 5'-nucleotidase was measured in the serum of normal mice using a simple one-step kinetic method (dooley and racich 1980) . a reference range of 10.9 + 4.5 (sd) u/1 has been recorded in 100 mice, and it is thought but not proven to be a good indicator of hepatic injury (clampitt and hart 1978) . glutamate dehydrogenase (gdh) has been measured in the tissues and serum of mice. gdh is known to play a key role in insulin secretion (carobbio et al 2004) . the activity of gdh is fivefold greater in the liver than in the kidney and brain, and the authors speculated that its measurement in serum would be a sensitive indicator of hepatic cell injury. serum gdh activity is also elevated in mice on caloric restriction (hagopian et al. 2003 ). corticosterone is the major glucocorticoid secreted by the adrenal cortex of mice. it functions as a regulator of carbohydrate, protein, and fat metabolism and modifies the host response to stress. the male mouse has a well-defined diurnal concentration pattern, with a maximum concentration of 9 ktg/dl at the start of the dark cycle and a minimum concentration of 5 btg/dl shortly before the end of the dark cycle (ottenweller et al. 1979) . in contrast, female mice have a minimum concentration of 13.5 ktg/dl at the beginning of the dark cycle and a maximum of 40 ~tg/dl well into the dark period (scheving et al. 1983) . the length of the dark cycle was different in each study. it may be measured using radioimmunoassay, elisa, or fluorometric assay in mouse serum or plasma. corticosterone circulates in both free and protein bound forms. in the mouse, greater than 99% circulates bound to cortisol-binding globulin (cbg) and albumin. the diurnal variation in corticosterone levels is paralleled by the diurnal pattern of cbg. urinary and salivary corticosterone is derived only from the free plasma fraction. corticosterone synthesis and secretion may be influenced by many drugs (woodman 1997) . measurements of corticosterone in unrestrained mice using indwelling catheters have elucidated the necessity of eliminating stress for accurate interpretation of data (macleod and shapiro 1988) . both handling and crowding laboratory mice can cause elevations in corticosterone (balcombe et al. 2004; fullwood et al. 1998) . c57bl/6 ob/ob mice have elevated corticosterone levels that increase markedly after 40 weeks of age, preceding elevations in serum glucose (garthwaite et al. 1980) . lean c57bl/6 controls had lower serum concentrations of corticosterone that declined between 5 and 20 weeks of age. food restriction (to 60% of ad libitum) profoundly affects the diurnal increase in plasma corticosterone in mice. at 20:00 hours (8 p.m.), the daily maximum in this study, food restricted balb/c mice had 300% more corticosterone compared to controls. these mice also had significantly reduced thymic weight and inflammation in response to the injection of carrageenan subcutaneously. the authors report that the magnitude of carrageenan-induced inflammation fluctuates with a diurnal trough, which coincides with peak corticosterone levels (klebanov et al. 1995) . corticosterone is synthesized in response to acth, which is made in the anterior pituitary. acth release is episodic (not at fixed intervals) and does not involve steroid feedback. acth concentration peaks in the early evening in mice and can be reversed by reversing the light cycle. acth is highly conserved with the mouse acth differing from human by only two amino acid residues. ria may be used to measure acth in the mouse (daily levels vary from 2.6-5.4 ng/ml) and to demonstrate rhythmic cycling. samples must be collected at 5-to 30-minute intervals. commercial elisa kits for acth are also available (quimby 1999b) ko mice incapable of synthesizing corticotrophin releasing hormone or acth may require corticosterone supplementation in drinking water and are particularly susceptible to hypoglycemia during fasting. fetuses of homozygous ko crh null mothers must be supplemented from gestational day 12 to weaning with 30 mg/ml corticosterone in drinking water (mugila et al. 1995) . oxytocin gene ko mice respond to a psychogenic stressor with more anxiety-related behavior and more corticosterone production (amico et al. 2004 ). lh promotes follicular development, increases estradiol secretion in the preovulatory follicle, causes follicular rupture, converts the preovulatory follicle into a corpus luteum, increases progesterone production by the corpus luteum, and, in the male, increases production of testosterone from leydig cells (woodman 1997) . lh is a glycoprotein composed of t~-and ~-chains. the amino acid sequence of the t~-chain is identical to that of follicle-stimulating hormone (fsh); however, the ~l-chain is specific and confers the receptor binding properties on the hormone. a homologous ria for rat lh using antisera to rat lh and purified rat lh has been employed to measure lh in mice (quimby 1999b) . lh and folliclestimulating hormone (fsh) are secreted by the same anterior pituitary cell in response to stimulation by gonadotrophinreleasing hormone (gnrh), which is synthesized in the hypothalamus. gnrh is identical in mouse and humans, and its differential stimulatory effect on gonadotropes, producing fsh or lh is controlled through gnrh pulse frequency. pulsatile secretion of lh leads to great variations in blood concentration. in female mice, there is a 10-to 25-fold increase in basal lh levels in the afternoon of proestrus. in a study comparing young cycling c57bl/6 female mice to old females (40% of which not cycling), flurkey et al. (1982) found lower lh levels in the older group and a more rapid rise in lh among younger cycling mice. these authors found that reproductive failure in male mice correlated with the loss of episodic release of lh. female homozygous ko mice lacking the gene for the immediate early transcription factor ngfi-a were infertile secondary to lh-[3 deficiency. although ovaries from these mice had a similar number of follicles, they lacked corpora lutea (lee et al. 1996) . basal levels of serum progesterone were lower in ngfi-a females (6.3 + 2.4 ng/ml) compared to wild type (11.3 +_ 3.6 ng/ml), whereas serum estradiol levels were similar in deficient (50.7 + 36.9 pg/ml) and wild-type (52.0 _+ 34.3 pg/ml) mice. decreased amounts of mrna encoding lh-[3, but not fsh-[3, were observed in ngfi-a-deficient mice; no changes were observed in mrna levels for prl or gnrh receptor between deficient and wild-type mice. an ovariectomy normally removes gonadal feedback inhibition, allowing for increased amounts of lh-~ and fsh-[3 in the pituitary; however, ovariectomy of ngfi-a deficient female mice lead to an increase in fsh-~ only. homozygous ngfi-a deficient males did make lower amounts of lh-~ compared to wild-type males, but they made significantly more lh-[3 than did ngfi-a-deficient females. the levels of lh in males appeared to be sufficient for fertility, and they had normal serum testosterone levels. these results suggest that ngfi-a acts synergistically with transcription factor sf-1 to regulate the promoter for lh-[3 gene expression. the simultaneous activation of both sf-1 and ngfi-a by gnrh appears to confer the specificity of lh-~ synthesis. female transgenic mice overexpressing lh are anovulatory, develop granulosa cell tumors, and undergo precocious puberty (mann et al. 2003) . excellent reviews detailing the use of transgenic and ko mice in elucidating the secretion and function of lh have been published (bums and matzuk 2002; huhtaniemi et al. 2002; wells and murphy 2003) . fsh stimulates the growth and maturation of ovarian follicles in the female and promotes the latter stages of spermatogenesis in males. it acts principally on the sertoli cells of the seminiferous tubules of the testis and induces the secretion of androgen-binding protein and inhibin. in females, fsh and lh have distinct secretory patterns that are synchronized to the estrous cycle; mouse estrous cycles have a 4-to 5-day periodicity. in addition to estrous cycle dependent rhythms, both lh and fsh have ultradian pulses and show circadian periodicity, with highest levels occurring during the dark period of the light cycle. testosterone, estrogen, and progesterone all provide feedback regulation of lh and fsh secretion (woodman 1997) . because rat and mice share considerable sequence homology for the fsh-~ chain, the rat ria kit from national institute of diabetes and digestive and kidney diseases (niddk) can be used to measure fsh in the mouse (dalterio et al. 1981) . a mouse specific ria is commercially available. in cycling female mice, the plasma levels of fsh increase from 80 to 120 ng/ml during proestrus and 250-300 ng/ml during estrus. although bronson and desjardins (1977) found age associated decreases in serum lh and testosterone in male cbf 1 mice experiencing decreased sperm production, no similar decreases were observed in fsh concentration. no significant differences in the twofold increased fsh levels were seen among young versus old males following castration. this appears to differ from results obtained from young versus old rats (finch et al. 1977) . the regulation and function of fsh has been studied in transgenic and ko mice (cooke and saunders 2002; wells and murphy 2003) . testosterone promotes spermatogenesis and provides feedback regulation of gonadotrophin synthesis. through its action on the epididymis, proteins necessary for spermatozoal maturation are synthesized. dihydrotestosterone (dht) promotes the growth and differentiation of accessory sex organs (depaolo and masoro 1989) . in addition to their effects on reproductive organs, testosterone and dht have physiologic functions on the central nervous system, cardiac tissue, and the liver. in the mouse, testosterone stimulates the growth of kidneys and synthesis of erythropoietin (woodman 1997) . lh stimulates synthesis of testosterone from cholesterol by the interstitial leydig cells of the testis. in target tissues, the enzyme 5r converts testosterone to dht. in the fetal testes, leydig cells first become identifiable during the rise of testosterone. in most mammalian species, leydig cells disappear when testosterone levels fall during gestation. however, the mouse is an exception and leydig cells do not undergo regression postnatally (aoki 1970) . testosterone and dht circulate in both free and protein bound forms. ninety-eight percent of total testosterone is bound to protein, mainly albumin. mice, unlike humans, dogs, monkeys, and cats, do not have circulating sex hormone-binding globulin. in mice, a pulse release pattern may be seen within their diurnal rhythm of testosterone. testosterone can be measured in mice using ria or elisa. plasma testosterone was not found to change during the average lifespan of c57bl/6 and dba/2j mice (finch et al. 1977) . this is in contrast to cbf1 mice, wistar and long-evans rats, and humans, which experience greater than a 30% decrease in testosterone at midlife (bronson and desjardins 1977) . in contrast to testosterone levels in older mice, castration-induced elevation of lh was impaired in 28-month-old c57bl/6j mice compared to 12-month-old mice. androgen receptor ko mice have been described (cooke and saunders 2002) . 5. estradiol (e2) the granulosa cells of the mature graafian follicle are the main source of 17[3-estradiol (e2) in mammals. fsh stimulates the activation of aromatase in follicles, which is responsible for the conversion of androgens to e2. e 2 is also synthesized by the testis, adrenal, liver, and skin, although in much lower amounts than by the ovary. e2 provides negative feedback control over lh secretion, and it stimulates prl secretion in mice. e 2 promotes the growth and development of the female reproductive tract, external genitalia, and the ductal system of the mammary glands. in addition, e 2 sensitizes the follicle to fsh. e 2 is measured in mice using ria and elisa. high circulating levels of e 2 precede the preovulatory surge in lh. ryan and schwartz (1980) reported basal levels of e 2 in mice of 1 to 5 pg/ml. holinka et al. (1978) studied circulating levels of progesterone and estradiol in young and old c57bl/6 female mice during gestation. they found that the old multiparous females have delayed and reduced preparturitional rise is plasma e 2 compared to young females. because preparturitional e 2 is thought to regulate uterine progesterone levels, the decline in e 2 seen in older females coupled with elevated progesterone may delay the onset of labor and lead to prolonged gestation. when sex steroid hormones such as e 2 bind their receptor, they induce a conformational change that allows the complex to bind dna response elements on nuclear target genes and associate with coactivators and transcription factors to form an active transcriptional complex. this complex is responsible for initiating the transcription of the target gene. one coactivator that associates with the active transcriptional complex involving sex hormones is steroid receptor coactivator-1 (src-1). to better understand its physiologic role during sex hormone-receptor binding, ko mice that were deficient in src-1 were created (xu et al. 1998 ). deficient male mice had a 34% reduction in testosterone-stimulated prostate growth and small testes compared to wild-type mice. deficient females had a half-normal uterine growth response to exogenous e 2 and only a partial ductal growth response (in the mammary gland) following exogenous e 2 and progesterone. serum concentrations of e 2 and testosterone were elevated 1.2 to 1.5 times wild-type levels, indicating an abnormality in the endocrine feedback control system. although src-1 was shown to clearly be necessary for optimal sex hormone-induced cellular activation, the lesion created in this ko mouse was not nearly as profound as that seen in mice with disrupted e 2 receptors (korach et al. 1996) . mice with the genes for the e 2 receptors and aromatase knocked out have been described (simpson et al. 2005 synthesized by the corpus luteum before implantation and by both corpus luteum and placenta following implantation, progesterone is necessary for preparing the uterus for implantation and maintaining pregnancy. in addition, a small amount of progesterone is synthesized by the adrenal gland. activation of the corpus luteum to secrete progesterone requires both lh and prl. progesterone provides negative feedback control over lh. in mice, progesterone is measured using ria or elisa. in cycling mice, the levels of progesterone increased from 5 to 35 ng/ml on proestrus and returned back to baseline on estrus. flurkey et al. (1982) compared the plasma levels of lh, progesterone, and prl in cycling young (10-week) and old (8month) female c57bl/6j mice. they showed age-related deficits in the preovulatory levels of all three hormones. the lh elevations during the ascending and descending portions of the preovulatory surge were smaller; the slower rises in lh during the ascending phase of the surge correlated with decreased progesterone in older females. there was no correlation between lh or progesterone level and length of estrus cycle. holinka et al. (1978) observed changes in plasma progesterone levels in young and old female mice during gestation. older mothers had a much slower decline in circulating progesterone than younger mothers between gestational days 17-23. because a major decrease in plasma progesterone is thought to be essential for the onset of uterine contractions and parturition in mice, the authors hypothesized that the attenuated decline in older mothers may account for their prolonged gestation. surgimoto et al. (1997) engineered mice lacking the pgfzareceptor (fp) and found that deficient female mice had normal estrous cycles, ovulation, fertilization, and implantation but did not undergo parturition. these pregnant females were characterized by a decline in preparturition progesterone levels due to delayed luteolysis and failure to develop labor. close examination revealed that near term there was no expression of the oxytocin receptor in the uterus of fp-deficient mice. when the ovaries of fp-deficient pregnant females were removed on day 19 of pregnancy, progesterone levels fell, uterine oxytocin receptors were expressed, and pups were born alive within 24 h. this study demonstrated that the role of pgf2~ is to initiate luteolysis, resulting in an immediate decline in progesterone levels. the subsequent induction of oxytocin receptors and their activation by bound oxytocin initiate labor. it is feasible that the age-associated prolongation of gestation in old female mice may involve a defect in pgfz,~-induced luteolysis. this mechanism for the induction of labor also explains the well-known observation that aspirin-like drugs (that inhibit cox metabolism) may delay parturition in women. prl, a 23-kda single-chain polypeptide, is principally made by the pituitary gland but may be also be found in brain, thymus, spleen, lymph nodes, mammary gland, myometrium, sweat gland, lacrimal gland, and bone marrow. more than 300 functions have been attributed to prl, involving reproduction and lactation, growth and development, endocrinology and metabolism, brain and behavior, immunomodulation, and electrolyte balance. it mediates effects by endocrine, paracrine, and autocrine mechanisms. in mice, prl is of major importance for maintenance of the structure, life span, and function of the corpora lutea and the development and maintenance of the mammary gland and lactation. however, prl-receptor ko males have no major defect in fertility. prl plays a role in anxiety-related behaviors, bone development, and abdominal fat deposition in both sexes (kelly et al. 2001) . its role in immune function is more controversial, although it appears to modulate immunity during times of stress (dorshkind and horseman 2000) . prl mediates its effects by binding the prl receptor (prlr). mice have two major forms of prlr that are created via alternative splicing of a single gene. the prlrs are members of the class i cytokine receptor superfamily. although most functions of prl are mediated by the unmodified 23-kda peptide, post-translational modification allows variants of prl to bind its receptor eliciting transcription of genes necessary for tissue specific changes (harris et al. 2004) . prl secretion is under inhibitory control by dopamine and among its secretagogues are thyrotropin-releasing hormone (trh), vasoactive intestinal peptide, gastrin, serotonin, ~-endorphin, oxytocin, angiotensin ii, gnrh, and arginine vasopressin. prl in mice is quantified using ria using the nih rp-1 reference standard. male mice measured during the light phase have <1 ng/ml whereas the range during the dark phase was 10-20 ng/ml (depaolo and masoro 989). oxytocin, a 21-kda nonapeptide, is synthesized as a prohormone in neurons whose bodies are located in the supraoptic and paraventricular nuclei at the base of the hypothalamus. once synthesized, the prohormone is packaged into neurosecretory granules and transported down the axons of these neurons transport synthesized oxytocin to the pars nervosa of the pituitary gland. during transport, the molecule is cleaved to yield the 10-kda carrier proteins, neurophysin i, and the l l-kda oxytocin. the primary stimulus for oxytocin release is mechanical stimulation of the mammary gland and distention of the reproductive tract (reimers 1999) . there is a single oxytocin receptor (otr). investigations using ko mice have shown that oxytocin is required for milk let down by the mammary gland as well as for postpartum alveolar proliferation. in males, oxytocin is required for normal spermiation and sperm transfer. in both genders, oxytocin helps control normal blood pressure and salt intake. kos display reduced aggression and a striking deficit in the ability to recognize a previously encountered mouse (young and gainer 2003) . oxytocin may be quantified in mice using commercially available elisa kits or by species-specific ria. values in one recent publication showed plasma oxytocin at 1.5 _+ 0.6 pg/ml in male c57bl/6 mice ). avp is synthesized and released by the same neurons previously described for oxytocin, although the two secretory granules rarely colocalize in the same neuron. the carrier protein for avp is neurophysin ii. avp is released from neuron secretory granules by electrical signals from osmoreceptors measuring the osmolarity of extracellular fluid. action potentials, generated in the receptors, cause calcium influx into axon terminals and exocytosis of ave avp is transported in the blood to the kidneys, where it binds receptors in the distal segment of the nephron and collecting ducts leading to increased resorption of water (reimers 1999) . there are three avp receptors, v l ar, vlbr, and v2r; and these are all g-protein coupled receptors. nonsense mutations of the avp gene (as seen in the brattleboro rat) or kd mice develop neurohypophysial (central) diabetes insipidus. mice with the v2r knocked out develop nephrogenic diabetes insipidus characterized by polyuria, polydipsia, and failure to concentrate urine. when the v1 ar gene is knocked out, mice develop an enhanced proliferation of splenic b cells and enhanced igg1 and igg2b production to thymicdependent antigens. when v lbr is knocked out the mice display a marked reduction in social aggression and a deficit in social recognition (young and gainer 2003) . avp can be measured by ria or elisa and the reported values in normal male c57bl/6 mice are 5.1 + 1.5 pg/ml in plasma ). tsh is a 28-kda glycoprotein made by the anterior pituitary gland on stimulation by thyrotropin-releasing hormone (trh). trh is made in the hypothalamus. tsh is secreted in a pulsatile manner, similar to acth. tsh secretion is regulated by triiodothyronine (t3), which acts on the hypothalamus to inhibit secretion of trh and acts on the pituitary gland to inhibit tsh synthesis (reimers 1999) . tsh plays a role in stimulating the thyroid gland to produce thyroxine and it influences the outcome of t-cell development in thymus and intestine. in addition to the thyroid gland, tsh receptors are found on many different populations of hematopoietic cells in bone marrow, subsets of dendritic cells, monocytes, and lymphocytes in the spleen and lymph nodes (klein 2003; scofield et al. 2005) . ko mice have been created in which genes for trh (wells and murphy 2003; yamada et al. 2003) and tsh receptors (biesiada et al. 1996) have been deleted. in addition, spontaneous mutations in the pitl gene lead to deficiencies of gh, prl, and tsh (yeap and leedman 1999) . tsh is measured by ria and reference ranges established around a 3.1 u/mg reference standard were 1-90 ng/ml (depaolo and masoro 1989). 11. thyroxine (t4) and triiodothyronine (t3) t 4 is synthesized entirely in the thyroid gland, whereas t 3 is produced primarily by conversion of t 4 to t 3 in extrathyroidal tissues. in the thyroid, thyroglobulin is synthesized and stored, and later hydrolyzed to form t 4. the tyrosine residues, held in peptide linkage within thyroglobulin, are first iodinated and later iodotyrosines are chemically coupled to form hormonally active t 4 and t 3. production of t 4 depends on adequate supplies of iodine. both t 3 and t 4 circulate in the blood primarily bound to albumin and m-globulin; however, mouse transthyretin does not bind t 4. approximately 85% of t3, the active thyroid hormone, in blood is made through the monodeiodination of the outer ring of t 4 in a variety of tissues. certain drugs, food deprivation, and illness can all effect monodeiodinase activity (reimers 1999) . t3 actions are mediated via two t3 receptors, tr~x and tr[3, which act as hormone-inducible transcription factors and belong to a large superfamily of nuclear hormone receptors including the steroid hormone, vitamin d, retinoic acid, and peroxisomal proliferator receptors (yen 2003) . in mouse and human tr~ and tr[3 encode nine mrna isoforms through alternative splicing; four isoforms tr~i, trc~2, tr[31, and tri]2, are expressed as proteins. trc~ ~ ko mice, which lack all products from the tr~ locus, are fertile and have normal basal thyroid status but have increased sensitivity to thyroid hormones in the pituitary and liver following provocative testing with increasing doses of t3. tr~x -/-have a disruption of the first coding exon in the tr~x locus, which prevents transcription of tr~i and try2 mrnas but not tra~i or tra~2. these pups die shortly after weaning unless supplemented with t3 for the first 2.5 months of life, after which they develop normal thyroxine levels. however, both genders are infertile. try2 -/-overexpresses tr~i and are hypothyroid but have inappropriately normal tsh levels. they exhibit some signs of hyperthyroidism, such as increased heart rate, weight loss, and elevated body temperature. tr~ -/-lack all tr[3 isoforms and display resistance to thyroid hormones demonstrating the key role of tr~ in set-point control of the pituitary-thyroid feedback axis. tr[32 -/-ko mice have resistance to thyroid hormones and elevated t3, t4, and tsh. they have defective tsh suppression by t3. several double kos (both tr~ and tri]) have been developed with profound resistance to t3. these targeted mutants have helped to elucidate the full function of thyroid hormones involving: bone formation and mineralization, abnormal development of skeletal muscle, disrupted development of the cones in the retina, abnormalities in the auditory system, cochlear and vestibular structures, delayed small intestine development, impaired thermogenesis, and altered development of the central nervous system and immune system (o'shea and williams 2002). both total t 4 and t3 can be measured by ria in mice, and reference ranges vary greatly by strain and age. although t3 is the active hormone, its serum levels are so low that it is a less reliable indicator of thyroid status. total t 4 ranges in swiss webster and icr mice are 5.5 _+ 0.7 and 4.7 + 0.3 jag/ml, respectively. total t3 levels range from 65-85 ng/100 ml in both sw and icr stocks (depaolo and masoro 1989). many factors, including autoantibodies, are known to interfere with thyroid hormone assays (despres and grant 1998; reimers 1999) . 12. parathyroid hormone (pth) pth is synthesized by the chief cells of the parathyroid gland and secreted as an 84 amino acid peptide. circulating levels of ionized calcium induce synthesis of pth. low calcium stimulates pth synthesis, and high calcium inhibits secretion. the primary function of pth is to control calcium concentrations in extracellular fluid and prevent hypocalcemia by increasing calcium resorption from bone, glomerular filtrate, and intestines (reimers 1999) . pth mediates its effect via its g-protein coupled receptor, pth1r. interestingly a second protein, pth related protein (pthrp), which is secreted by a variety of tissues and acts by autocrine and paracrine signaling, uses the same pth1r. pthrp modulates a wide range of physiologic and developmental responses (goltzman and white 2000) . mice with targeted mutations of the pth, pthrp, and pth1r genes have demonstrated the critical role of these proteins in regulating both the switch between proliferation and differentiation of chondrocytes and their replacement by bone cells (schipani and provost 2003) . serum levels of pth may be quantified in mice using a commercially available elisa kit, which can also be used to quantify the protein in rats. 13. other hormones elisa kits are commercially available to quantify many additional hormones in the serum of mice, including insulin, leptin, gh, epinephrine, orexin a, orexin b, adiponectin, adipsin, and resistin. each of these hormones have been briefly discussed in the "glucose and carbohydrate metabolism" section. the liver has many complex functions including protein metabolism, carbohydrate metabolism, and lipid metabolism. the liver is involved with the synthesis of many plasma proteins (including albumin), the conversion of ammonia to urea, and the production of glucose from glucogenic amino acids). the liver is implicated with the regulation of blood glucose levels (via glycogenolysis and gluconeogenesis, as discussed in the "glucose and carbohydrate metabolism" section), the removal of glucose from the blood via glut-1 and glut-2 membrane transporters, and storage in the form of glycogen. the liver is also involved in cholesterol and triglyceride synthesis, the formation of lipoproteins (discussed in the "lipid metabolism" section), and the synthesis of carbohydrates from fats. there are also other more specific functions that the liver facilitates, including the synthesis of heme, the synthesis of many coenzymes, detoxification/biotransformation of exogenous and endogenous substances via the cytochrome p-450 microsomal enzymes, and the synthesis of bile. the role of the liver in these functions for various animals has been reviewed (tennant 1999; woodman 1988 ). the elevation of plasma or serum enzymes usually confined to the cytosol or mitochondria of hepatocytes is helpful in elucidating liver injury. many factors influence the duration of elevated serum enzyme levels including molecular size, intracellular location, rate of plasma clearance, rate of enzyme inactivation, and hepatic production. hepatic necrosis is associated with elevations of alt, ast, sd, and otc in mice, and extrahepatic cholestasis is associated with elevated ap (tennant 1999) . please refer to the "enzymes" section for a description of each enzyme. bilirubin is a pigment that is produced by the degradation of the hemoglobin by cells of the mononuclear phagocyte system. there are two main types of bilirubin. unconjugated bilirubin is a non-water soluble molecule that is transported in blood bound to albumin. hepatocytes uptake unconjugated bilirubin, where it undergoes glucuronidation to produce the water-soluble form, conjugated bilirubin. conjugated bilirubin is then excreted via the hepatobiliary system and excreted in bile. quantification of serum bilirubin levels is based on the van den bergh or diazo reaction. diazo reacts directly with conjugated bilirubin, and with unconjugated bilirubin only after addition of alcohol to the reaction. therefore, measuring serum bilirubin involves the following steps. first, the level of conjugated bilirubin is measured. second, alcohol is added to the reactants, which allows quantification of total bilirubin levels. finally, the level of unconjugated bilirubin is determined by subtracting the conjugated measurement from the total measurement. the excretory capacity of the liver may be assessed by measuring serum bilirubin. elevated levels of unconjugated bilirubin are usually observed in situations of increased erythrocyte breakdown, such as in hemolytic diseases. unconjugated hyperbilirubinemia is also seen in disease in which hepatic uptake, conjugation, and secretion of bilirubin are diminished. increased levels of conjugated bilirubin are usually associated with intrahepatic cholestasis or extrahepatic bile duct obstruction. conjugated bilirubin in blood is normally filtered by the glomerulus in small amounts. conjugated hyperbilirubinemia may result in bilirubinuria (tennant 1999) . bilirubin clearance from blood and the role of the constitutive androstane receptor in this process has been studied in normal and transgenic mice (huang et al. 2003; . studies documenting the bilirubin-metabolism/detoxifying enzymes, their regulatory nuclear receptors, and lipid transporters in mice have been reported (wagner et al. 2005) . bile acids are cholesterol breakdown products that are secreted by hepatocytes into the hepatobiliary system, and ultimately into the intestinal tract. bile acids aid digestion by emulsifying dietary lipid aggregates, and solubilizing and transporting lipids in an aqueous environment (in particular fat-soluble vitamins). in the liver, bile acids also play a role regulating the secretion of apolipoprotein b (elzinga et al. 2003) . extensive enterohepatic recirculation results in almost 99% return of bile acids secreted by the liver from the intestinal tract. absorption occurs mainly in the ileum under the influence of the apical na ⧠bile acid transporter on epithelial cells (kida et al. 2003) . some absorption also takes place in the large intestine. absorbed conjugated bile acids (from ileum) pass unaltered to the portal circulation where they are removed by na+-taurocholate cotransporting polypeptide located on the basolateral hepatocyte membrane (jung et al 2004) . there are two types of bile acids, cholic acid and chenodeoxycholic acid. in mice, they are conjugated with taurine before secretion by the liver. the liver-specific enzymes, bile acid coa ligase and bile acid-coa:amino acid n-acyltransferase, are responsible for conjugation (inoue et al. 2004 ). stedman et al. (2004) described normal serum bile acid levels in normal and transgenic mice. elevated serum bile acid levels in mice is usually due to decreased bile acid recycling by the liver, and mainly includes diseases associated with decreased hepatic functional mass and cholestasis (tennant 1999) . hoda and green (2003) measured increased levels of serum bile acids after bile duct ligation. albumin is a nonglycosylated protein and is the most abundant protein in plasma. it serves as the most important determinant of plasma oncotic pressure, and it is a major transport protein for both endogenous metabolites and xenobiotics. it is made exclusively by the liver. serum albumin may be measured by radial immunodiffusion, dye binding reactions (using bromcresol green or bromcresol purple), electrophoresis, and elisa. serum albumin levels decrease with age in many inbred strains (quimby 1999b) . hypoalbuminemia is usually reflective of decreased hepatic synthesis (due to hepatic disease, inflammation, and malabsorptive/maldigestive diseases), increased loss due to hemorrhage or via the intestinal tract (protein-losing enteropathies) and kidneys (protein-losing nephropathies) (tennant 1999) . most of the proteins associated with coagulation are synthesized by the liver (except factor viii), and measurement of fibrinogen or prothrombin time has served as a marker for decreased synthesis due to hepatic injury (tennant 1999) . prothrombin time will also be increased due to consumption of clotting components and in vitamin k deficiency. fibrinogen is also an acute phase reactant, and plasma elevations are seen during inflammation (tennant 1999) . fibrinogen may be quantified in mouse plasma by elisa. ceruloplasmin is copper-containing acute phase reactant and iron transport protein made exclusively in hepatocytes of mice. its serum levels may be decreased during hepatic injury and increased in response to inflammatory stimuli (min et al. 1991; shim and harris 2003) . many of the complement components are also made in the mouse liver, especially c2, c3, c4, and factor b. decreased circulating levels may be seen due to hepatic injury or due to consumption during activation. please refer to the "complement" section for further details on complement function and measurement. two dyes, sulfobromophthalein and indocyanine green, have been used to assess hepatocellular or bile tract function. following an intravenous bolus, these dyes are rapidly cleared from the plasma by hepatocytes and excreted into the bile. both dyes have been used to assess liver function in mice (hurwitz et al. 1994; huang and vore 2001) . the kidney plays a complex role in maintaining homeostasis in the body and is involved in such functions as water and electrolyte balance, nutrient conservation, maintenance of blood ph, and removal of the end products of nitrogen metabolism (such as urea, creatinine, and allantoin). in addition, the kidney produces and responds to a variety of hormones. however, assessing renal function is complicated by the large functional reserve of the kidneys. for instance, increases in urea nitrogen do not occur until 70-75% of renal mass has become functionally compromised. additionally, assessment of analytes in urine is difficult due to the small size of mice (as discussed in the "sampling" section). urea is produced in the liver as a waste product of protein catabolism (specifically a breakdown product of ammonia). urea is a small molecule that freely diffuses across cell membranes, and therefore the urea concentration is the same in blood, serum, and plasma. traditionally, urea concentrations is measured in terms of urea nitrogen (the amount of nitrogen contained within urea). determination of urea nitrogen is usually made from serum, but whole blood can be used (hence the term blood urea nitrogen [bun] ). quantification of urea nitrogen is based on spectrophotometry assays, measuring the amount of urea that is hydrolyzed by urease. frith et al. (1980) investigated urea nitrogen levels in balb/c and c57bl/6 mice. urea nitrogen levels decreased after 3 months of age but increased again after 12 months in both sexes. in balb/c mice, levels were higher in males than females, whereas in c57bl/6 mice, females had significantly higher levels than males. elevated urea nitrogen (azotemia) can be caused by prerenal, renal, and postrenal conditions. prerenal causes include increased protein catabolism (such as with inflammation, starvation, and high-protein diets). renal causes usually are associated with conditions that compromise more than 70-75% of functional renal mass and include conditions such as renal amyloidosis, glomerular immune complex disease, and polycystic disease. postrenal include any cause that results in obstruction of the lower urinary system. decreased urea nitrogen is found with disease associated with hepatic insufficiency and low-protein diets. creatinine is a degradation product of creatine and creatine phosphate and represents an end-product of muscle metabolism. quantification of creatinine is based on spectrophotometry assays. baseline serum levels are directly related to muscular conditioning and total muscular mass, which varies between individuals. pathologically elevated serum creatinine levels are caused by the same prerenal, renal, and postrenal causes that elevate urea nitrogen in serum. therefore, quantification of serum creatinine offers no interpretative advantage over urea nitrogen (everett and harrison 1983) . proteinuria is a common finding in normal mice, and includes rodent-specific proteins such as uromucoid, small quantities of c~-and [3-globulins, and a family of prealbumins known as major urinary protein (mup). the mup has three electrophoretic variants, designated as mup-1, mup-2, and mup-3, that are under both genetic and hormonal control. a regulatory locus designated mup-1 with codominantly expressed alleles a and b (located on chromosome 4) controls the urinary levels of the three variants. the mup is synthesized in the liver, secreted into blood, and excreted into urine. males are have higher levels of proteinuria than are females, with levels of 5 mg/ml, and age-related increases are seen in mice of both sexes. increases in other urinary proteins have been associated with a variety of renal diseases in mice. the concentration of urine (amount of solutes dissolved in urine) can be measured by urine specific gravity (usg) or osmolality. urine concentration in healthy mice is very high. watts (1975) determined the usg of healthy cba mice, which ranged from 1.060 to 1.080. the author also found the usg increased from 20 to 80 days of age. the urine concentrating ability of transgenic mice expressing human sickle cell hemoglobin (kos for mouse hb) was assessed by ryan et al. (1997) . they found that sickled cells caused vascular, tubular, and glomerular changes, as well as corresponding hyposthenuria (4-h water deprived osmolality of affected mice was 807 + 285 mosm compared to 1541 + 360 mosm in controls). i. electrolytes 1. sodium, potassium, chloride, and phosphorus sodium and potassium levels are easily measured in murine serum using flame photometry (lithium reference) or ion-specific electrodes. serum sodium levels are slightly higher in mice than in most other mammalian species, with reported values of 174 + 23 (sd) meq/1 (finch and foster 1973) , 147 + 15 (sd) meq/l (everett and harrison 1983) , and 155-161 meq/1 (loeb et al. 1996) . no differences in serum sodium were seen during aging, between sexes, or among strains. serum chloride has been measured using mercuric thiocyanate and the chloridimetric or ion-specific electrode techniques, and inorganic phosphorus in mice has been measured using the phosphomolybdate technique. serum inorganic phosphorus levels decreased as mice aged between 1 and 12 months. this change was documented in balb/c and c57bl/6 strains, as well as in outbred mice (loeb et al. 1996) . total serum calcium reflects both ionized (active calcium) and protein-bound calcium (mainly bound to albumin). ionized calcium is biologically active in bone formation, neuromuscular activity, cellular biochemical processes, and blood coagulation. decreased serum albumin levels are also expected to decrease total calcium levels, by decreasing the amount of protein-bound calcium. however, hypoalbuminemia does not result in clinical signs of hypocalcemia. in mice, total serum calcium has been measured using the sodium alizarin sulfonate technique or atomic absorption spectrometry. two reports, using different techniques, list similar reference ranges of 9 + 1 (sd) mg/dl, whereas a third report lists reference ranges of 5.6 + 0.4 (sd) mg/dl for male and 7.4 + 0.50 (sd) mg/dl for female albino mice. the latter values reported for male mice were significantly lower than those for six inbred strains of mice in two different age-groups using the same techniques (quimby 1999b) . likewise, no differences associated with sex were reported by everett and harrison (1983) , who also examined random bred albino mice. however, bonella et al. (1968) demonstrated significantly higher levels in female swiss albino mice. serum calcium levels appear to decline between 3 and 12 months of age in the balb/c and c57bl/6 strains as well as in outbred mice . frith et al. (1980) found that female c57bl/6 mice had lower calcium levels than males. bonella et al. (1968) demonstrated significantly elevated calcium levels when blood was collected by orbital puncture versus cardiac puncture. hypercalcemia in mice can results as a response to increased levels of pth (such as with hyperparathyroidism) or pthrp (such as with certain neoplasms like multiple myeloma). in mice, the response of parathyroid chief cells to hypercalcemia appears different from other species. grone et al. (1992) studied this response in nude mice with pthrp secreting tumor cells or infusions of pthrp, and found prominent membranous whorls and increased cytoplasmic area of chief cells in mice with hypercalcemia (17.0 + 3.1 mg/dl), which marked these cells as distinctly different from parathyroid chief cells of other species. hypocalcemia can be due to hypoalbuminemia (as previously discussed), renal disease, pancreatitis, and hypomagnesemia. alcock and shils (1974) found that mice fed magnesium-deficient diets developed hypocalcemia, as did mice receiving intramuscular injections of heparin. total serum protein has been evaluated in mice using either the lowry or biuret methods, although the lowry method is preferred for analysis of small volume samples. hypoproteinemia is seen in hepatic injury (also see the "liver function tests" section) or during protein-losing enteropathies or nephropathies. hyperproteinemia is seen during dehydration. age-related changes have been described in a number of strains, such as increased total serum protein in older balb/c and b6 mice and lower levels in dba/2 mice (loeb et al. 1996) . each of the major classes of serum protein ((xl-, (x2-, ~-, and y-globulins) in mice can be distinguished by electrophoresis. changes in serum levels have been associated with a wide variety of diseases in mice. for instance, hypergammaglobulinemia was found in scid mice injected with human cag multiple myeloma cells . the acute phase reactants ceruloplasmin and fibrinogen have been discussed in the "serum proteins" section and crp and sap in the "complement" section. saa is an acute phase reactant synthesized by the liver and may be induced by the inflammatory cytokines il-1 or il-6 (see the "cytokines and chemokines" section). normally saa is quickly cleaved to a small molecular weight product, but during chronic infection or failure in the degradation pathway, tissue deposition of amyloid may occur. differences in serum concentrations have been described in various mouse strains and during disease. commercial ria and elisa kits are available for its quantitation in mice (quimby 1999b) . alpha-1-fetoprotein (afp) is encoded by the afp gene which is nearly identical in its organization to the mouse albumin gene, albl. it is likely afp arose due to gene duplication. afp is present in fetal serum but the synthesis declines shortly after birth. serum levels are regulated by two loci, afr-1 and afr-2, which are not linked to afp. the action of these genes may be modified by tgf-~ and p53 (wilkinson et al. 2005) . levels in mouse serum have been quantified by immunoelectrophoresis (zizkovsky 1975 ) and both polyclonal antibodies and recombinant dna-derived mouse afp are available for assay development (boismenu et al. 1997) and are elevated in mice with certain neoplasms and during hepatic regeneration (jin et al. 2005; quimby 1999b ). sulphostin, a novel inhibitor of dipeptidyl peptidases iv (dppiv) that stimulates hematopoiesis in mice proteomic approaches to biomarker discovery in prostate and bladder cancers 8th annual analyzers buyer's guide il-17: prototype member of an emerging cytokine family increased atherosclerosis in hypeflipidemic mice with inactivation of abca-1 in microphages diet-induced changes in plasma phospholipids transfer protein activity, lipids, and lipoproteins. mpd: 801, 802, 828. mouse phenome database web site comparison of magnesium deficiency in the rat and mouse apoc-1 and apoc-iii as potential plasmatic markers to distinguish between ischemic and hemorrhagic stroke comparison of methods for obtaining blood from mice anxiety and stress responses in female oxytocin deficient mice impaired calcification around matrix vesicles of growth plate and bone in alkaline phosphatase-deficient mice hormonal control of leydig cell differentiation interleukin-7 an interleukin for rejuvenating the immune system a spectrophotometric microtiter-based assay for the detection of hydroperoxy derivatives of linoleic acid mast cell lineage development and phenotypic regulation laboratory routines cause animal stress regulation of fasting blood glucose by resistin biochemistry of immunoglobulins variations in serum calcium between strains of inbred mice correction of ureagenesis after gene transfer in an animal model and after liver transplantation in humans with ornithine transcarbamylase deficiency biology of the congenitally hypothyroid hyt/hyt mouse the chemokine stromal cell-derived factor-1 regulates the migration of sensory neutron progenitors methods of enzymatic analysis the mouse phenome project purification and characterization of human and mouse recombinant alpha-fetoproteins expressed in escherichia coli genetically engineered animals in drug discovery and development: a maturing resource for toxicologic research effects of heparin on serum calcium in mice albumin content in blood of inbred mice strains lentiviral shrna silencing of murine bone marrow cell ccr2 leads to persistent knockdown of ccr2 function in vivo trophic action of leptin on hypothalamic neurons that regulate feeding the interpretation of serum biochemistry test results in domestic animals transgenic models of autoimmune disease lipoprotein metabolism and atherosclerosis susceptibility in transgenic mic mouse models of atherosclerosis reproductive failure in aged cbf male mice: interrelationships between pituitary gonadotropic hormones, testicular function, and mating success stromal cell derived factor 1/cxcl12 selectively counteracts inhibitory effects of myelosuppressive chemokines on hematopoieteic progenitor cell proliferation in vitro minireview: genetic models for the study of gonadotropin actions recruitment of cxcr3 and ccr5 t-cells and production of interferon-gamma inducible chemokines in rejecting human arteries tietz fundamentals of clinical chemistry distribution and characterization of the serum lipoproteins and apolipoproteins in the mouse, mus musculus disparate lymphoid chemokine expression in mice and men: no evidence for ccl21 synthesis by human high endothelial venules insulin secretion profiles are modified by over expression of glutamate dehydrogenase in pancreatic islets a panel of cerebrospinal fluid biomarkers for the diagnosis of alzheimer's disease normal, benign, preneoplastic, and malignant prostate cells have distinct protein expression profiles resolved by surface enhanced laser desorption/ionization mass spectrometry chemokine-cytokine cross talk. the elrt cxc chemokine lix (cxcls) amplifies a proinflammatory cytokine response via a phosphatedy linositol 3-kinase-nf-kappa b pathway rapid neurosecretory and cardiovascular response to osmotic stimulation in conscious mice high density lipoproteins retard the progression of atherosclerosis and favorably remodel lesions without suppressing indices of inflammation or oxidation influence of blood collection sites and use of anesthesia on plasma glucose concentration in mice the tissue activities of some diagnostic enzymes in ten mammalian species accelerated intestinal epithelial cell turnover: a new mechanism of parasite expulsion signal transduction in t cells: an overview role of stearoyl-coa desaturase-1 in leptinmediated weight loss induction of leptin receptor expression in the liver by leptin and food deprivation mouse models of male infertility the primary germinal center response in mice a comparison of serum calcium levels obtained by two methods of cardiac puncture in mice biomethodology and surgical techniques reduction in the development of cerulean-induced acute pancreatitis by treatment with m40401, a new selective superoxide dismutase mimetic pancreatic lipase-related protein 2 is the major colipase-dependent pancreatic lipase in suckling mice effects of cannabinoids and female exposure on the pituitary testicular axis in mice: possible involvement of prostaglandins genetic background determines the extent of atherosclerosis in apoe-deficient mice endocrine hormones in laboratory animals antibody interference in thyroid assays: a potential for clinical misinformation a new kinetic determination of serum 5'-nucleotidase activity, with modifications for a centrifugal analyzer the roles of prolactin, growth hormone, insulin-like growth factor-l, and thyroid hormones in lymphocyte development and function; insights from genetic models of hormone and hormone receptor deficiency selection for serum cholesterol, voluntary physical activity, 56-day body weight, and feed intake in random bred mice. ii. correlated response ccr2 expression by brain microvascular endothelial cells critical for macrophage transendothelial migration in response to ccl2 inhibition of apolipoprotein b secretion by taurocholate is controlled by n-terminal end of the protein in rat hepatoma mcardle-rh7777 cells clinical biochemistry the effect of freezing on various serum chemistry parameters of common laboratory animals alkaline phosphatase, lactate dehydrogenase, and aspartate aminotransferase and their isoenzymes as indicators of the development of experimental hepatitis in mic the endoplasmic reticulum is the site of cholesterol-induced cytotoxicity in macrophages niemann-pick c heterozygosity confers resistance to lesional necrosis and macrophage apoptosis in murine atherosclerosis hematologic and serum electrolyte values of the c57bl/6j male mouse in maturity and senescence hormone production by the pituitary and testes of male c57bl/6j mice during aging age effects of luteinizing hormone, progesterone, and prolactin in proestrus and acyclic c57bl/6j mice innovation~stagnation: challenge and opportunity on the critical path to new medical products interleukin-17 hematologic and clinical chemistry findings in control balb/c and c57bl/6 mice pi3k and negative regulation of tlr signaling visfatin: a protein secreted by visceral fat that mimics the effects of insulin floor space needs for laboratory mice: c57bl/6males in solid-bottom cages with bedding a longitudinal hormonal profile of the genetically obese mouse return to normal of blood-glucose, plasma insulin, and weight gain in new zealand obese mice after implantation of islets of langerhans acute phase proteins ghrelin: more than a new frontier in neuroendocrinology tumor necrosis factors a rapid, simple, and humane method for submandibular bleeding of mice using a lancet palatal cytosol cortisol-binding protein associated with cleft palate susceptibility and h-2 genotype relationship in the functional levels of early components of complement to the h-2 complex of mice developmental and tissue-specific regulation of parathyroid hormone (pth)/pth-related peptide receptor gene expression ccl1-ccr8 interactions: an axis mediating the recruitment of t-cells and langerhans-type dendritic cells to sites of atopic skin inflammation measurement of glycosylated haemoglobins and glycosylated plasma proteins in animal models with diabetes or inappropriate hypoglycaemia effects of humoral hypercalcemia of malignancy on the parathyroid gland in nude mice the complex role of interlukin-10 in autoimmunity tumor necrosis factor ligand superfamily: involvement in the pathology of malignant lymphomas caloric restriction increases gluconeogenic and transaminase enzyme activities in mouse liver fructose metabolizing enzymes from mouse livers influence of age and caloric restriction genetic ablation of orexin neurons in mice result in narcolepsy, hypophagia, and obesity prolactin and the prolactin receptor: new targets of an old hormone optimal conditions for the determination of serum alkaline phosphatase by a new kinetic method blood collection in mice using the saphenous vein: an alternative to retroorbital collection transgenic analysis of drug-metabolizing enzymes: pre-clinical drug development and toxicology the distribution of aspartate aminotransferases in normal and neoplastic rat and mouse tissues hepatic canalicular membrane transport of bile salt in c57l/j and akr/j mice: implications for cholesterol gallstone formation prolonged gestation, elevated preparturitional plasma progesterone, and reproductive aging in c57bl/6j mice mild dyslipidemia in mice following targeted inactivation of the hepatic lipase gene immunolocalization of tissue non-specific alkaline phosphatase in mice uncoupling of obesity from insulin resistance through a targeted mutation in a p2, the adipocyte fatty acid binding protein synergistic effect of lymphotactin and interferongamma-inducible protein-10 transgene expression in t-cell localization and adoptive t-cell therapy of tumors multidrug resistance p-glycoprotein 2 is essential for the biliary excretion of indocyanine green induction of bilirubin clearance by the constitutive androstane receptor a traditional herbal medicine enhances bilirubin clearance by activating the nuclear receptor car visfatin: a new adipokine transgenic and ko mouse models for the study of luteinizing hormone and luteinizing hormone reception function loperamide effects on hepatobiliary function, intestinal transit, and analgesia in mice redox state-dependent and sorbitol accumulationindependent diabetic albuminaria in mice with transgene-derived human aldose reductase and sorbitol dehydrogenase deficiency hepatocyte nuclear factor 4 alpha is a central regulator of bile acid conjugation genetic association between h-2 gene and testosterone metabolism in mice use of transgenic mice in carcinogenicity hazard assessment leukotrienes and atherosclerosis: new roles for old mediators endothelial lipase and hdl metabolism targeted mutation of plasma phospholipids transfer protein gene markedly reduces high density lipoprotein levels genetic heterogeneity of lipoproteins in inbred strains of mice: analysis by gel permeation chromatography afp gene expression after acute diethylnitrosamine intoxication is not afr2 regulated identification of tumor-associated plasma biomarkers using proteomic techniques: from mouse to human role of liver enriched transcription factors and nuclear receptors in regulating the human, mouse, rand rat ntcp gene expression of urea transporters in potassium-depleted mouse kidney genetic analysis of glucose tolerance in inbred mouse strains. evidence for polygenic control reversing systemic inflammatory response syndrome with chemokine receptor pepducins carbohydrate metabolism proteomic fingerprints for potential application to early diagnosis of severe acute respiratory syndrome il-6: a regulator of the transition from neutrophil to monocyte recruitment during inflammation implications of multiple phenotypes observed in prolactin receptor ko mice. front neuroendocrino122 vectorial transport of bile acids in immortalized mouse bile duct cells hyperadrenocorticism, attenuated inflammation, and the life prolonging action of food restriction in mice physiological relevance of thyroid stimulating hormone and thyroid stimulating hormone receptor in tissues other than the thyroid genetic modifiers of atherosclerosis in mice function of prostanoid receptors: studies in ko mice estrogen receptor gene disruption: molecular characterization and experimental and clinical phenotypes ghrelin--a hormone with multiple functions effects of shipping on immune functions of mice how obesity causes diabetes: not a tall tale luteinizing hormone deficiency and female infertility current protocols in immunology disruption of the tgf-i] pathway and modeling human cancer in mice a new technique for obtaining blood from mice decay-accelerating factor confers protection against complement-mediated podocyte injury in acute nephrotoxic nephritis tissue distribution of products of the mouse decay-accelerating factor (daf) genes: exploitation of a daf-1 knockout mouse and site-specific monoclonal antibodies linscott's directory of immunological and biological reagents clinical biochemistry of laboratory rodents and rabbits. in clinical biochemistry of domestic animals clinical biochemistry the clinical chemistry of laboratory animals improved terminal bleeding method t-cell trafficking in asthma: lipid mediators grease the way the mouse fas-ligand gene is mutated in gld mice and is part of a tnf family gene cluster chemokines: immunology's high impact factors studies on the origin and excretion of serum alpha-amylase in the mouse repetitive blood sampling in unrestrained mice using a chronic indwelling right atrial catheterization apparatus transcellular biosynthesis of arachidonic acid metabolites: from in vitro investigations to in vivo reality consequences of elevated luteinizing hormone on diverse physiological systems: use of the lh beta ctp transgenic mouse as a model of ovarian hyperstimulation-induced pathophysiology mouse models as tools for dissecting disorders of lipoprotein metabolism ccl19 and ccl21 induce a potent proinflammatory differentiation program in licensed dendritic cells the role of histamine hi receptor and h2 receptor in lps-induced liver injury adiponectin protects lps-induced liver injury through modification of tnf-t~ in kk-ay obese mice in vivo administration of helicobacter hepaticus cytotoxin is associated with hepatic inflammation and necrosis an h-2-associated difference in murine serum cholesterol levels tissue-specific expression of the human gene for lecithin: cholesterol acyltransferase in transgenic mice alters blood lipids, lipoproteins, and lipases towards a less atherogenic profile biology of il-21 and the il-21 receptor multiple genes encode mouse pancreatic amylases opposing roles for il-13 and il-13 receptor ct2 in health and disease of mice and not men: differences between mouse and human immunology veterinary laboratory medicine: interpretation and diagnosis induction of hepatic metallothionein by non-metallic compounds associated with the acute phase response in inflammation clinical biochemistry and hematological reference values in normal experimental animals relationship between the development of the intestinal iga immune system and the establishment of microbial flora in the digestive tract of young holoxenic mice split activity of interleukin-10 on antigen capture and antigen presentation by human dendritic cells: definition of a maturative step genetic susceptibility and resistance to diet-induced atherosclerosis and hyperlipoproteinemia chemokines; multiple levels of leukocyte migration control initial sequencing and comparative analysis of the mouse genome oral tolerance in the absence of naturally occurring tregs structure, binding, and antagonists in the il-4/il-13 receptor system corticotropinreleasing hormone deficiency reveals major fetal, but not adult, glucocorticoid need diet effects on bone mineral density and content, body composition, and plasma glucose, leptin and insulin levels. mpd: 143. mouse phenome database web site evaluation of h-fabp as a marker of ongoing myocardial damage using hgh transgenic mice lipid inflammatory mediators in diabetic vascular disease transgenic expression of pancreatic secretory trypsin inhibitor-1 ameliorates secretagogue-induced pancreatitis in mice summary of the second report of the national cholesterol education program (ncep) expert panel on detection, evaluation, and treatment of high blood cholesterol in adults recognition and clearance of apoptotic cells: a role for complement and pentraxins mildly oxidized ldl induces an increase apolipoprotein j/paraoxonase ration the genetic control of alkaline phosphatase activity in the duodenum of the mouse diabetes mellitus: a "thrifty" genotype rendered detrimental by "progress"? cx3crl-mediated dendritic cell access to the intestinal lumen and bacterial clearance clinical laboratory reference sequential blood samples from the tail vein of rats and mice obtained with modified liebig condenser jackets and vacuum the cancer research uk experience of pre-clinical toxicology studies to support early clinical trials with novel cancer therapies lactic dehydrogenase virus insight into the physiological actions of thyroid hormone receptors from genetically modified mice ccr7 governs skin dendritic cell migration under inflammatory and steady state conditions viral pertubation of endocrine function: disordered cell function leads to disturbed homeostasis and disease targeted disruption of hormone-sensitive lipase results in male sterility and adipocyte hypertrophy, but not in obesity genetics, cytokines, and human infectious disease: lessons from weekly pathogenic mycobacteria and salmonellae circadian rhythms of plasma corticosterone binding activity in the rat and mouse one hundred years of mouse genetics: an intellectual history. i. the classical period one hundred years of mouse genetics: an intellectual history. ii. the molecular revolution the ultrastructural localization of the isoenzymes of aspartate aminotransferase in murine tissues proteomic analysis of diet-induced hypercholesterolemic mic clinical chemistry values of the n:nih(s) mice and parameter variations due to sampling techniques use of proteomic patterns in serum to identify ovarian cancer lessons from kitty hawk: from feasibility to routine clinical use for the field of proteomic pattern diagnostics alkaline phosphatase activity of the mouse immune regulation in the intestine boosted decision tree analysis of surface enhanced laser desorption/ionization mass spectral serum profiles discriminates prostate cancer from non-cancer patients animal models in biomedical research complement the mouse transgenic models of tolerance and autoimmunity: with special reference to systemic lupus erythematosus short-term and long-term in vivo exposure to an ephedra-and caffeine-containing metabolic nutrition system does not induce cardiotoxicity in b6c3f1 mice model models of atherosclerosis hormones type 2 diabetes: a matter of beta-cell life and death leukemia inhibitory factor and interleukin-ll: cytokines with key roles in implantation virus induced pancreatic disease; alterations in concentrations of glucose and amylase in blood lungkine, a novel cxc chemokine, specifically expressed by lung bronchoepithelial cells changes in serum hormone levels associated with male-induced ovulation in group housed adult female mice ko transgenic mouse model of sickle cell disease animal models of autoimmunity and their relevance to human diseases hematological comparison of the mouse blood taken from the eye and tail cystatin c as a potential cerebrospinal fluid marker for the diagnosis of creutzfeldt-jakob disease efficacy of 2, 3-dimercapto-l-propanesulfonic acid (dmps) and diphenyl diselenide on cadmium induced testicular damage in mice multimeric cytokine receptors: common versus specific functions bilateral lesions in the suprachiasmatic nuclei affect circadian rhythms and h-thymidine incorporation into deoxyribonucleic acid in mouse intestinal tract, mitotic index of corneal epithelium, and serum corticosterone pthrp, pth, and the pth/pthrp receptor in endochondral bone development diabetes, obesity, and the brain hematopoietic, immunomodulatory and epithelial effects of interleukin-11 intestinal tsh production is localized in crypt enterocytes and in villus 'hotblocks' and is coupled to il-7 production: evidence for involvement of tsh during acute enteric virus infection adipsin, a biomarker of gastrointestinal toxicity mediated by a functional gamma-secretase inhibitor loci controlling plasma non-hdl and hdl cholesterol levels in a c57bl/6j x casa/rk intercross quantitative trait loci mapping of genetic modifiers of metabolic syndrome and atherosclerosis in low-density lipoprotein receptor-deficient mice the kinase lkb 1 mediates glucose homeostasis in liver and therapeutic effects of metformin genetic defects in copper metabolism estrogen: the good, the bad, and the unexpected il-1 and il-18 receptors, and their extended family b-cell-and monocyte-activating chemokine (bmac), a novel non-elr ct-chemokine feed-forward regulation of bile acid detoxification oby cyp3a4: studies in humanized transgenic mice adenovirus-mediated rescue of lipoprotein lipase-deficient mice breeding of the gad-mdx mouse: influence of genetically induced denervation on dystrophic muscle fibers failure of parturition in mice lacking the prostaglandin f receptor inactivation of stress protein p8 increases murine carbon tetrachloride hepatotoxicity via preserved cyp2e1 activity murine models to investigate pharmacological compounds acting as ligands of ppars in dyslipidemia and atherosclerosis the other side of the orexins: endocrine and metabolic actions assessment of hepatic function mitochondrial affinity for adp is twofold lower in creatine kinase ko muscles. possible role in rescuing cellular energy homeostasis evaluation of proparacaine hydrochloride as a topical anesthetic for the collection of blood from the orbital sinus of mice mitochondrial affinity for adp is twofold lower in creatine kinase knockout muscles. possible role in rescuing cellular energy homeostasis the role of cd40-cd154 interactions in autoimmunity and the benefit of disrupting this pathway scavenger receptor class b type 1 in high-density lipoprotein metabolism, atherosclerosis, and heart disease: lessons from gene targeted mice cxc chemokine receptor cxcr2 is essential for protective innate host response in murine pseudomonas aeruginosa pneumonia in vivo uptake of radiolabeled mda2, an oxidation-specific monoclonal antibody, provides an accurate measure of atherosclerotic lesions rich in oxidized ldl and is highly sensitive to their regression the regulation of the complement system: insights from genetically-engineered mice interleukin-6: an overview development of a novel proteomic approach for the detection of transitional cell carcinoma of the bladder in urine mechanisms of suppression by suppressor t-cells lipids and lipoproteins car and pxr agonists stimulate hepatic bile acid and bilirubin detoxification and elimination in mice pneumocystis carinii activates the nf-kappa[~ signaling pathway in alveolar epithelial cells a simple capillary tube method for the determination of the specific gravity of 25 and 50 micro 1 quantities of urine interleukin-18 facilitates the early antimicrobial host response to escherichia coli peritonitis transgenic studies on the regulation of the anterior pituitary gland function by the hypothalamus cxcr3 and its ligand participate in the host response to bordetella bronchiseptica infection of the mouse respiratory tract but are not required for clearance of bacteria from the lung a direct intersection between p53 and transforming growth factor beta pathways targets chromatin modification and transcription repression of the alpha-fetoprotein gene assessment of hepatic function and damage in animal species laboratory animal endocrinology investigation of antitumor effects of synthetic epothilone analogs in human myeloma models in vitro and in vivo the protein profile of acetazolamidetreated sera in mice bearing lewis neoplasm cebs object modes for systems biology data, sysbio-om partal hormone resistance in mice with disruption of the steroid receptor coactivator-1 (src-1) gene platelet factor 4 gene transfection into tumor cells inhibits angrogenesis tumor growth and metastasis mice lacking the thyrotropin-releasing hormone gene: what do they tell us? differences in the human and mouse amino-terminal leader peptides of ornithine transcarbamylase affect mitochondrial import and efficacy of adenoviral vectors combined pituitary hormone deficiency: lessons from the murine models molecular basis of resistance to thyroid hormone a mouse bleeding technique yielding consistent volume with minimal hemolysis transgenesis and the study of expression, cellular targeting, and function of oxytocin, vasopressin and their receptors kos model the 100 best-selling drugs: will they model the next 100? predicting drug efficacy: kos model pipeline drugs of the pharmaceutical industry three biomarkers identified from serum proteomic analysis for the detection of early stage ovarian cancer cxc chemokine ligand 129 stromal cell-derived factor 1 alpha and cxcr4-dependent migration of ctls toward melanoma cells in organotypic culture il-ii: insights in asthma from overexpression transgenic modeling genotype, obesity, and cardiovascular disease: has technical and social advance outstripped evolution? specific fetal serum proteins of 13 mammalian species t3 t4 tace taj tc tg tgf-i3 tgf-~isf th tlr tnap tnf tnfrsf tnfsf tof tpc trance trh tsh tx upr usg vldl serum amyloid a serum amyloid protein standard deviation stromal cell-derived factor-1 sorbitol dehydrogenase surface-enhanced laser desorption/ionization secondary lymphoid tissue chemokine stearoyl-coa desaturase-1 suppressor of cytokine signaling-3 steroid receptor coactivator-1 sj6gren's syndrome single-strand deoxyribonucleic acid triiodothyronine thyroxine tumor necrosis factor-a converting enzyme toxicity and jnk inducer cytotoxic t-cells triglyceride transforming growth factor-j3 transforming growth factor-13 superfamily t-helper toll-like receptors tissue nonspecific alkaline phosphatase tumor necrosis factor tumor necrosis factor receptor superfamily tumor necrosis factor superfamily time-of-flight total plasma cholesterol tumor necrosis factor-related activation-induced cytokines thyrotropin-releasing hormone thyroid-stimulating hormone thromboxane unfolded protein response urine specific gravity very low-density lipoprotein key: cord-256998-or73in8m authors: nguyen, khue g.; vrabel, maura r.; mantooth, siena m.; hopkins, jared j.; wagner, ethan s.; gabaldon, taylor a.; zaharoff, david a. title: localized interleukin-12 for cancer immunotherapy date: 2020-10-15 journal: front immunol doi: 10.3389/fimmu.2020.575597 sha: doc_id: 256998 cord_uid: or73in8m interleukin-12 (il-12) is a potent, pro-inflammatory type 1 cytokine that has long been studied as a potential immunotherapy for cancer. unfortunately, il-12's remarkable antitumor efficacy in preclinical models has yet to be replicated in humans. early clinical trials in the mid-1990's showed that systemic delivery of il-12 incurred dose-limiting toxicities. nevertheless, il-12's pleiotropic activity, i.e., its ability to engage multiple effector mechanisms and reverse tumor-induced immunosuppression, continues to entice cancer researchers. the development of strategies which maximize il-12 delivery to the tumor microenvironment while minimizing systemic exposure are of increasing interest. diverse il-12 delivery systems, from immunocytokine fusions to polymeric nanoparticles, have demonstrated robust antitumor immunity with reduced adverse events in preclinical studies. several localized il-12 delivery approaches have recently reached the clinical stage with several more at the precipice of translation. taken together, localized delivery systems are supporting an il-12 renaissance which may finally allow this potent cytokine to fulfill its considerable clinical potential. this review begins with a brief historical account of cytokine monotherapies and describes how il-12 went from promising new cure to ostracized black sheep following multiple on-study deaths. the bulk of this comprehensive review focuses on developments in diverse localized delivery strategies for il-12-based cancer immunotherapies. advantages and limitations of different delivery technologies are highlighted. finally, perspectives on how il-12-based immunotherapies may be utilized for widespread clinical application in the very near future are offered. interleukin-12 (il-12) is a potent, pro-inflammatory type 1 cytokine that has long been studied as a potential immunotherapy for cancer. unfortunately, il-12's remarkable antitumor efficacy in preclinical models has yet to be replicated in humans. early clinical trials in the mid-1990's showed that systemic delivery of il-12 incurred dose-limiting toxicities. nevertheless, il-12's pleiotropic activity, i.e., its ability to engage multiple effector mechanisms and reverse tumor-induced immunosuppression, continues to entice cancer researchers. the development of strategies which maximize il-12 delivery to the tumor microenvironment while minimizing systemic exposure are of increasing interest. diverse il-12 delivery systems, from immunocytokine fusions to polymeric nanoparticles, have demonstrated robust antitumor immunity with reduced adverse events in preclinical studies. several localized il-12 delivery approaches have recently reached the clinical stage with several more at the precipice of translation. taken together, localized delivery systems are supporting an il-12 renaissance which may finally allow this potent cytokine to fulfill its considerable clinical potential. this review begins with a brief historical account of cytokine monotherapies and describes how il-12 went from promising new cure to ostracized black sheep following multiple on-study deaths. the bulk of this comprehensive review focuses on developments in diverse localized delivery strategies for il-12-based cancer immunotherapies. advantages and limitations of different delivery technologies are highlighted. finally, perspectives on how il-12-based immunotherapies may be utilized for widespread clinical application in the very near future are offered. keywords: interleukin-12 (il-12), cancer immunotherapy, cancer vaccine, cytokine delivery system, localized delivery, intratumoral administration overview of il-12-based immunotherapies a brief history of cytokine and il-12 immunotherapies since the discovery of an "endogenous pyrogen, " now known as il-1, in 1953, scientists have anticipated the use of exogenous cytokines to manipulate a patient's immune system in an effort to control malignant neoplasms (1) . early obstacles to cytokine-based immunotherapy centered on difficulties achieving reproducible manufacture of a sufficient and pure supply of cytokines for clinical trials. in the early 1980s, recombinant dna technology and advances in the biochemical characterization of proteins combined to overcome this hurdle. finally, in 1986, ifn-α broke through as the first cytokine to win fda approval as a single agent cytokine therapy for cancer (2) . since then, hundreds, if not thousands, of studies have evaluated more than 40 cytokines against a range of preclinical tumor models. a number of promising cytokines, including gm-csf, il-1, tnfα, ifn-γ, and il-12, subsequently entered clinical trials as single agents but failed to provide clinical benefit. currently, only 2 of 40+ identified cytokines are approved as single agent immunotherapies for a limited number of indications ( table 1) . another fda approved cancer immunotherapy, talimogene laherparepvec (t-vec; imlygic tm ) is an oncolytic herpes simplex virus that uses gm-csf expression as an immune enhancer (7) . perhaps the greatest disappointment in cytokine immunotherapy development thus far is il-12. il-12 is a potent, pro-inflammatory cytokine produced by antigen presenting cells typically in response to microbial pathogens. it is comprised of two subunits, p35 and p40, that are linked by three disulfide bridges to form a p70 heterodimer (7) (8) (9) (10) . il-12 is chiefly responsible for the induction and enhancement of cell-mediated immunity. among its diverse functions, il-12 has been shown to: (i) induce t h 1 cell differentiation; (ii) increase activation and cytotoxic capacities of t and nk cells; and (iii) inhibit or reprogram immunosuppressive cells, such as tumor associated macrophages (tams) and myeloid-derived suppressor cells (mdscs) (11) (12) (13) (14) (15) (16) . il-12 also induces the production of large amounts of ifnγ which itself is cytostatic/cytotoxic (17, 18) , anti-angiogenic (19, 20) and can upregulate mhc i and ii expression on tumor cells for enhanced recognition and lysis (21) . not surprisingly then, il-12 has demonstrated remarkable antitumor effects against a range of malignancies in preclinical studies (22) (23) (24) (25) . these effects are largely dependent on cd8 + t cells, nk cells, and nk t cells (23, 26, 27) . in clinical studies, il-12 has been evaluated as an experimental treatment for numerous malignancies (28) (29) (30) (31) (32) (33) (34) (35) (36) (37) (38) . unfortunately, the efficacy of il-12 at tolerated doses has been minimal (29, 30, 33) . atkins and colleagues were the first to employ il-12 immunotherapy in a clinical trial (28) . this phase i study enrolled 40 patients, including 20 with renal cancer and 12 with melanoma, to investigate intravenous administration of recombinant hil-12 (rhil-12). one melanoma patient experienced a transient complete response and one renal cancer patient had a partial response (28) . subcutaneous rhil-12 was employed in a separate pilot study that enrolled 10 advanced melanoma patients (29) . in this study, a fixed dose of rhil-12 (0.5 µg/kg) was given to patients on days 1, 8, and 15 for two sequential cycles of 28 days. no partial or complete responses were reported. minor tumor shrinkages involving some subcutaneous metastases and hepatic metastases were observed (29) . in yet another early melanoma study, the administration of il-12 was found to induce a striking peripheral burst of hla-restricted ctl precursors directed to autologous tumors and to multiple immunogenic tumor-associated antigens (39) . significantly, the infiltration of cd8 + t cells with an effector-memory phenotype was identified in posttreatment metastatic lesions, but not in pretreatment metastatic lesions of three patients (39) . il-12 has also been shown to induce productive antitumor responses against cutaneous t cell lymphoma variants (38) , aids-related kaposi sarcoma (37) , and non-hodgkin's lymphoma (38) . although il-12 has demonstrated robust antitumor activity in preclinical studies and potent immune-stimulating potential in humans, systemic administrations of il-12 have been shown to be exceedingly toxic. in one phase ii trial, a maximal dose of 0.5 µg/kg per day resulted in severe side effects in 12 out of 17 enrolled patients and the deaths of two patients (40) . interestingly, the same dose of 0.5 µg/kg il-12 per day was found to be well-tolerated in patients that were enrolled in a previous phase i study. a change in dosing schedule accounted for the differences in toxicity between the phase i and phase ii trials. in the phase i trial, a single tester dose of il-12 was administered 1 week before a multiple-dose regimen. the tester dose was found to blunt the toxicity induced by subsequent doses (41) . overall, severe toxicities in early clinical trials, including 2 on-study deaths (42) due to frequent systemic injections of il-12, together with disappointing clinical responses in large phase 2 studies (43, 44) , dampened enthusiasm for il-12-based immunotherapy. the disappointing antitumor responses in clinical trials raised the possibility that il-12 is simply less active in humans. however, the severe toxicities outlined above indicate that il-12 has potent biological activity in humans. another possibility for the limited clinical efficacy is insufficient delivery of il-12 to the tumor microenvironment in humans. il-12, like most cytokines, functions locally through paracrine and autocrine mechanisms. the ideal targets of il-12 immunotherapy are not lymphocytes in circulation, but rather immune cells within the tumor and nearby lymph nodes, including activated but exhausted t cells, nk cells, tams, and mdscs. therefore, maximizing the amount of il-12 that reaches the tumor seems critical for a robust antitumor response. we and others have noted that il-12 immunotherapeutics would be more effective and less toxic if delivered and maintained in the tumor through the use of novel delivery technologies. there are five benefits of local, persistent il-12 delivery. the first is enhanced spatiotemporal distribution of il-12 compared to systemic delivery. the failure of il-12-based immunotherapies to achieve widespread clinical success may be at least partially attributed to the inability of a tolerated dose of systemically administered il-12 to reach therapeutic concentrations within human tumors. in mice, implanted or induced tumors are disproportionately large. for example, a 1.5-g tumor (∼1.25 cm 3 ) comprises 6% of the body weight of a 25-g mouse. a comparably sized tumor in a 70 kg (154 lbs) human would weigh 4.2 kg (9.2 lbs). furthermore, rapidly growing murine tumors are highly vascularized relative to their human counterparts (45, 46) . taken together, a significantly larger fraction of a systemically administered il-12 dose can be expected to reach the tumor in a mouse compared to a human. localized delivery strategies, on the other hand, are capable of enhancing il-12 concentrations in the tumor microenvironment by one or more orders of magnitude (47) (48) (49) (50) . a second benefit of localized il-12 delivery is the ability to generate systemic antitumor immunity from a locally initiated intravenous injection (4, 5) talimogene laherparepvec (t-vec) unresectable advanced melanoma intratumoral injection (6) * subcutaneous and intramuscular injections are local deliveries. however, they result in systemic cytokine distribution and are utilized in the clinic as systemic therapies. immune response. as cancer metastasizes and becomes a "systemic" disease, conventional wisdom has suggested that metastases must be treated with systemic therapies such as i.v. administered chemotherapies or immune checkpoint inhibitors. however, systemic delivery increases the frequency of adverse events through off-target interactions. for instance, systemic il-12 therapy has the potential to cause activation and/or differentiation of all circulating t cells whereas activation/differentiation of only tumor-specific or tumor antigen-experienced t cells is preferred. fortunately, a growing mountain of evidence demonstrates that localized il-12 can generate systemic, adaptive immunological memory capable of controlling anestic tumors, inhibiting metastases, and preventing tumor recurrences (51) (52) (53) . in particular, local administration of il-12 has been shown to activate or reactivate tumor infiltrating cd8 + t cells, improve antigen presenting machinery and subsequently cause the expansion of tumor-specific cd8 + effector t cells. this often leads to enhanced infiltration of contralateral untreated tumors (54) . research from our own lab demonstrated that local/intravesical administration of il-12 eliminated untreated flank tumors only when a primary orthotopic bladder tumor was treated. these data indicated that t cells must be educated with antigens from a primary tumor in order to find and eliminate abscopal tumors. similarly, intratumoral injections of il-12 neoadjuvant to resection have been shown to inhibit metastases by multiple groups in a t cell, nk cell, or ifn-γ dependent manner (52, 55) . taken together, the convincing evidence demonstrating that localized il-12 can induce abscopal immunity renders systemic il-12 delivery unnecessary even for the treatment of metastatic disease. third, as mentioned above, il-12 is a pleiotropic cytokine with context dependent consequences. when il-12 is administered systemically, it induces rapid increases in pro-inflammatory cytokines, such as ifn-γ, tnf-α, and il-6 (56). this "cytokine storm" combined with rapid decreases in peripheral blood lymphocytes, monocytes, and neutrophils can be lethal (42) . however, when controlled locally, pleiotropic cytokines have the potential to engage multiple antitumor effector mechanisms. for instance, il-12 increases the activation and cytolytic capacity of cd8 + t cells and nk cells and induces the production of ifn-γ. ifn-γ, in turn, may kill tumor cells directly, inhibit angiogenesis (57) (58) (59) (60) , and stimulate nk cells, ctls (61, 62) , and macrophages (63) while upregulating mhc i and ii molecules (64) on the surfaces of tumor cells. fourth, high levels of locally administered il-12 can reverse tumor-supporting immunosuppression. the immunosuppressive tumor microenvironment is a major hindrance to the clinical efficacy of all cancer immunotherapies. in fact, the cancer vaccine literature teaches that the majority of patients in clinical studies are able to mount significant antigen-specific t cell responses, yet few patients experience clinical benefit (65) (66) (67) . similarly, the extraordinary activity of car t cells against hematologic malignancies becomes less than ordinary against solid tumors. many solid tumors lack the chemokines and inflammation necessary to recruit cytotoxic t cells (68) . moreover, dense tumor stroma prevents t cell penetration while immunosuppressive factors released by tumor cells, suppressor t cells and tams can cause t cell anergy. regarding the latter, many tumors bathe in a cocktail of immunosuppressive factors such as tgfβ, il-10, ido, and larginase. fortunately, high intratumoral concentrations of il-12 can cause apoptosis and elimination of cd4 + cd25 + foxp3 + suppressor t cells in tumors (69) . in addition, the tumor suppressive phenotype of tams can be converted to a cytotoxic, antitumor phenotype in the presence of localized il-12 (11) . finally, il-12 has been shown to modulate and alter the suppressive activities of tumor-associated mdscs (12) . lastly, and perhaps most importantly, activation of t cells in the presence of il-12 can not only enhance ctl function, but also reduce negative regulatory mechanisms such as pd-1/pd-l1 signaling and autocrine ifnγ-induced apoptosis. this "protective" effect has been observed mostly in the cellular immunotherapy literature. standard protocols for ex vivo expansion of tumor infiltrating lymphocytes for adoptive cell therapy (act) traditionally used high dose il-2 to facilitate t cell proliferation (70) . the inclusion of il-12 in conditioning/expansion media has been explored recently because it had been shown previously to result in optimal t cell priming (71) . indeed, adoptive transfer of tumor-specific cd8 + t cells primed ex vivo in the presence of il-12 resulted in enhanced antitumor responses (72, 73) , increased persistence of infused t cells (73, 74) , as well as increased expression of il-2rα (cd25], icos, ox40, granzyme b, and ifnγ (73) . importantly, cytotoxic t lymphocytes (ctls) stimulated with il-12 were more effective in controlling tumors following adoptive transfer than ctls stimulated with ifnα (75) . il-12-stimulated t cells expressed lower levels of pd-1 and higher levels of ifnγ and il-2 compared to ifnα-stimulated t cells (75) . il-12 conditioning caused downregulation of ifnγr2 with a concomitant decrease in susceptibility to ifnγ-induced apoptosis of tumor-infiltrating cd8 + t cells (74, 76) . il-12 delivery strategies can be divided into three general approaches. the first involves fusion of a targeting moiety to il-12 in order to facilitate accumulation in a tumor following a systemic injection. the most common of class of fusion molecules are immunocytokines, which involve linking a tumor binding antibody fragment to a cytokine. the second approach involves delivery of genetic material encoding il-12 directly to the tumor or a tissue of interest. this category can be further divided based on the type or method of gene delivery. plasmids, mrna, viruses, and transduced cells are all capable of expressing and delivering il-12 after a local injection. the third major approach involves controlled release of recombinant il-12 protein from a sustained delivery system. here, the cytokine delivery system is injected or implanted directly in a tumor or tissue of interest. the remainder of this section will present and discuss the most relevant preclinical and clinical data pertaining to each il-12 delivery strategy. a summary of current clinical trials utilizing localized il-12 delivery is presented in table 2 . as mentioned above, immunocytokines are part or whole cytokines that have been engineered to contain antibody fragments or other targeting moieties. these "targeted" cytokines are administered systemically but are expected to accumulate within tumors at higher levels compared to non-targeted cytokines. various tumor-related features have been targeted by immunocytokines including: (1) tumor antigens which are overexpressed or uniquely expressed by tumor cells; (2) cryptic extracellular matrix epitopes found only in tumors; and (3) neovasculature markers as tumors require angiogenesis for growth. developments in immunocytokines are discussed below. the pan-carcinoma antigen, epithelial cell adhesion molecule (epcam), is highly expressed by cancer cells of epithelial origin such as colon, prostate, breast, and lung carcinomas. huks-il-12 is an immunocytokine of il-12 fused to the fc fragment of a humanized antibody that recognizes epcam. in a murine prostate cancer model, huks-il-12 was found to suppress experimental metastases in scid mice reconstituted with activated human t and nk cells lymphocytes (78) . another immunocytokine, hu14.18-il-12, which is irrelevant in this system due to its targeting of ganglioside gd2, was found to be somewhat less effective than huks-il-12, although differences in antitumor activity were not statistically significant (78) . dual immunocytokines in which both il-2 and il-12 were fused to the huks1/4 antibody fragment were found to eliminate epcam-expressing llc flank tumors following intratumoral (i.t.) injection (79) . interestingly, a mixture of huks-il-2 and huks-il-12 was less effective than the dual immunocytokine if delivered i.t., but exhibited similar in antitumor activity if administered i.v. (79) . the epidermal growth factor receptor her2/neu is overexpressed in roughly a third of breast and ovarian cancers, with high expression correlating with poor prognosis. trastuzumab, a monoclonal antibody targeting her2, has been approved for the treatment of certain breast cancers for more than 20 years. a mouse single chain il-12 fused to an anti-her2/neu igg3 (mscil-12.her2.igg3) retarded the growth of ct26-her2/neu tumors in immunocompetent mice (80) . a direct comparison demonstrated that mscil-12.her2.igg3 and free il-12 induced similar activities against ct26-her2/neu tumors (81) . follow up studies revealed that mscil-12.her2.igg3 also displayed robust antitumor activity against mc38/her2/neu and d2f2/e2 tumors (82, 83) . a more recent study revealed that disruption of the heparin binding domain in the mscil-12.her2.igg3 immunocytokine, reduced il-12 bioactivity (84) . this result was consistent with recent studies showing that heparin and heparan sulfate bind to and enhance the activity of il-12 (85) (86) (87) (88) (89) . while eliminating heparin binding reduces il-12 activity, we speculate that this reduction could be counterbalanced by an enhancement in tumor targeting as the il-12 immunocytokine may no longer bind to ubiquitous sulfated glycosaminoglycans in non-targeted tissues. mesothelin is a differentiation antigen that is highly expressed in a number of human cancers including mesotheliomas, pancreatic and lung adenocarcinomas, and ovarian and breast carcinomas. to direct il-12 to mesothelin expressing cancer cells, a scfv, called ss1, that specifically binds to mesothelin was fused to the p35 subunit of a single-chain il-12 (90) . human peritoneal mesotheliomas established in nude mice were significantly inhibited by i.p. injections of il12-ss1 (90) . that these studies were successful in nude mice seems to imply a prominent role for nk cells in this model. ca166-9 is a cancer antigen that is expressed in about half of human ovarian cancers (91) . a scfv of the 6b11 monoclonal antibody that binds to ca166-9 was fused to mil-12 (92) . systemically (i.v.) administered 6b11scfv-mil-12 was found to inhibit the growth of subcutaneously implanted id8 ovarian tumors more effectively than non-targeted mil-12 (92) . cd30 is expressed by activated lymphocytes and thus serves as a useful target for several types of lymphoma. a cd30-targeted il-12 fusion protein was developed for cd30 + hodgkin's lymphoma therapy (93) . the immunocytokine was found to induce activation of t and nk cells and secretion of proinflammatory cytokines resulting in enhanced cytotoxicity of cd30 + mc38 cells. interestingly, a cd30-targeted il12-il2 fusion protein outperformed targeted il-2 or il-12 alone in all in vitro measures. the dual cytokine construct induced regression of cd30 + but not cd30-mc38 tumors in vivo (93) . whether the dual cytokine fusion protein was better than the single cytokine constructs is not known as the latter were not evaluated in vivo. many solid tumors overexpress extracellular matrix (ecm) which serves as transport barrier to the penetration of therapeutics and immune cells. in ecm-rich solid tumors, it may frontiers in immunology | www.frontiersin.org targeting ecm proteins instead of cancer cells, therefore, is a promising strategy to encourage immunocytokine accumulation in tumors. there are two immunocytokines, hubc1-il12 and il-12-l19, that have been developed to target the splice variant extra domain b (ed-b) of fibronectin, which is highly expressed in tumor tissues but undetectable in normal adult tissues with the exception of endometrium (94) . bc-1 is a monoclonal antibody that recognizes the ed-b isoform, thus a hubc1-il12 immunocytokine has been constructed from two molecules of il-12 fused to each of the igg heavy chains of humanized bc-1. systemic administration of hubc1-il12 was found to eliminate experimental pc3 metastases and suppress the growth of multiple human tumor lines in immunocompromised mice more effectively than il-12 alone (95) . a phase i trial evaluated the safety of weekly infusions of as1409 (hubc1-il12) in 13 renal carcinoma and malignant melanoma patients (96) . the maximum tolerated dose (mtd) was found to be 15 µg/kg. in contrast, the mtd of twice weekly i.v. il-12 was previously found to be 0.5 µg/kg (97) . dose limiting toxicities, including fever, fatigue, and elevated transaminase levels, were consistent with known toxicities of il-12 (96) . the second ed-b targeted immunocytokine, il-12-l19, is comprised of the ed-b-binding l19 scfv and il-12 (98) . l19-targeted cytokines have been shown to selectively accumulate in tumors following i.v. administration (99, 100) . intravenous administration of il-12-l19 every 48 h was found to control the growth of primary c51 colon adenocarcinomas, f9 teratocarcinomas as well as experimental pulmonary c51 metastasis (101) . biodistribution studies confirmed that a greater percentage of the injected dose of il-12-l19 was found in tumors as compared to an il-12-fusion negative control. il-12-l19 also demonstrated synergistic antitumor activity when combined with l19-tnfα (102) . most recently, il-12 was fused to the collagen-binding proteoglycan lumican and mouse serum albumin (msa), to create il12-msa-lumican (103) . lumican binds to collagen types i and iv, components of the thick fibrotic capsule surrounding tumors and perivascular basement membrane, respectively. in mice bearing established subcutaneous flank b16f10 tumors, treatment with il12-msa-lumican resulted in prolonged tumor control and longer survival. significant weight loss was observed following il12-msa compared to il12-msa-lumican treatment, indicating that collagen targeting may reduce systemic toxicities of il-12. finally, the combined treatment of lumican-msa-il2 and il12-msa-lumican potentiated anti-pd-1 increasing survival in multiple models and completely protecting cured mice from live tumor rechallenge (103) . another collagen-binding immunocytokine comprised of the a3 cbd of von willebrand factor fused to both subunits of il-12 was also recently developed (104) . systemic (i.v.) administration of this cbd-il12 was found to accumulate in emt6 mammary carcinomas at significantly higher levels and induce higher rates of complete tumor regression against 1-week old b16f10 and emt6 tumors compared to il-12 (104) . inclusion of the cbd resulted in a 5-6-fold decrease in plasma half-life despite the larger size of cbd-il12. the distributions of cbd-il12 and il-12 in normal tissues following i.v. injection were surprisingly similar although typical sites of collagen targeted drugs, e.g., bone and skin, were not examined. most importantly, although elevated liver enzymes were observed, levels following cbd-il12 at an impressive dose of 50µg/mouse were similar to 10µg/mouse of il-12 (104) . in general, the key advantage of systemically administered immunocytokines is their ability to preferentially accumulate within a site of disease, e.g., a tumor. however, immunocytokines retain complete cytokine activity in circulation which allows them to interact with circulating lymphocytes and induce similar cytokine-induced toxicities as parental cytokines (105) . one clever strategy has been developed to reduce adverse effects associated with systemic il-12 by separating the targeted delivery of the p35 and p40 subunits. this split-immunocytokine approach involves first delivering a bivalent p35-based antibody fusion protein (f8-p35s-f8). f8 binds to the alternatively spliced extra domain a (ed-a) domain which is present on the subendothelial extracellular matrix of tumor neovasculature (106) . after allowing time for binding and clearance of unbound f8-p35s-f8, a subsequent administration of p40, which has no activity by itself, interacts with p35 to recover il-12 activity. quantitative biodistribution investigation in f9 teratocarcinomas bearing mice showed that both targeted subunits accumulated in the tumor (106) . furthermore, the recombined subunits displayed robust il-12 activity in terms of ifnγ production and stat4 phosphorylation (106) . tumor necrosis is a common feature of most advanced solid tumors. approaches to target dna strands that become uniquely exposed in necrotic foci are under investigation. the monoclonal antibody, chtnt-3, recognizes single-stranded dna (107) . a fusion between the variable heavy chain of chtnt-3 and hil-12 forms the necrosis-targeting immunocytokine, chtnt-3/hil-12 (94) . chtnt-3/hil-12 was retained in a subcutaneous tumor after i.v. injection and resulted in a significant inhibition of du145 prostate tumors in human pbl-engrafted scid mice (94) . another necrosis-targeting il-12, capitalizes on the specificity of the nhs76 antibody for ssdna and dsdna (108, 109) . nhs-il12 is comprised of the full length nhs76 antibody fused to 2 single-chain il-12 molecules. systemic administration of a murine analog, nhs-muil12, has been shown to delay the growth of mc38-cea+ colorectal carcinomas in cea.tg mice (108) . furthermore, tumorbearing mice treated with nhs-muil12 developed cd8 + t cell responses against an endogenous tumor antigen, p15e. in vivo imaging studies have shown that nhs-muil12 accumulated in flank tumors following a s.c. injection (108) . subcutaneous administrations of nhs-muil12 were also recently shown to provide significant reductions in orthotopic mb49luc bladder tumors (110) . tumor control was associated with a noticeable reduction in markers of immunosuppression, e.g., mdscs, macrophages and tumor-associated tgf-β (110) . the combination of nhs-muil12 with avelumab, an anti-pd-l1 antibody, resulted in improved control of both mc38 and mb49 flank tumors with higher frequencies of cd8 + t cells and enhanced t cell activation compared to either agent alone (111) . against orthotopic emt-6 mammary tumors (∼100 mm 3 ) the combination of nhs-muil12 and avelumab induced complete regression in 7 of 8 mice (112) . the same treatment was shown to delay, but not completely regress, the growth of 350-400 mm 3 established emt-6 tumors. importantly, nhs-muil12 plus avelumab was shown to induce protective immunity as all cured mice resist an emt-6 tumor challenge but not a 4t1 challenge. furthermore, treatments enhanced cytotoxic nk and cd8 + tcell proliferation, t-bet expression, plasma cytokine levels, and innate and adaptive immune genes (112) . combining nhs-il12 with fcil-7 or il-2mab602 resulted in improved antitumor immunity, increased survival, and longterm remission in sarcoma-bearing mice (113) . fcil-7 is a fusion of interleukin-7 and an fc fragment while il-2mab602 is a fusion of il-2 and a monoclonal antibody against il-2, mab602. separately, the combination of nhs-il12 with local tumor irradiation was shown to increase treatment efficacy (114, 115) . in preparation for first-in-human clinical trials, a comparative oncology study in client-owned dogs with melanoma revealed that s.c. injections of nhs-il-12 induced transient increases in serum ifnγ and il-10. two of 7 dogs in a dose escalation cohort experienced a partial response while 5 of 7 dogs had increased levels of tumor-infiltrating cd8 + t cells (116) . nhs-il12 is currently in phase i clinical studies either as a monotherapy (nct01417546) or in combination with avelumab (nct02994953). in the former study, nhs-il12 induced transient lymphopenia and elevated liver transaminases, but was otherwise well-tolerated with a mtd of 16.8 µg/kg (117) . no objective tumor responses were observed, however, 5 of 59 patients experienced stable disease. immune assays revealed that nhs-il12 treatment increased nk cell frequencies and broadened the tcr diversity of tumor-infiltrating t cells (117) . systemically administered immunocytokines can significantly reduce but are unlikely to completely avoid il-12-related toxicities. as mentioned above, in circulation, immunocytokines will interact with immune cells and induce signaling outside of the tumor. in addition, all targeting moieties are susceptible to non-specific binding and distribution in normal, untargeted tissues. for example, radiolabeled nhs76 has been found in all major tissues in mice for 2-3 days after i.v. administration (109) . furthermore, substantial amounts of il-12-l19 were found in the livers of treated animals, likely leading to hepatotoxicity (101) . on-target/off-tissue specific binding may create additional concerns. in the case of nhs-targeting moieties, cancer patients have high levels of circulating cell-free dna that is shed from tumors (108). it is not clear how circulating dna impacts nhs targeting. in the case of neovasculature targeting moieties, angiogenesis is a normal process of wound healing and promotes collateral circulation for atherosclerotic blood vessels. disrupting non-cancerous angiogenesis could induce hypertension and cardiac ischemia which are among the adverse events associated with anti-angiogenic agents, such as bevacizumab. moreover, the potential immunogenicity of a nonendogenous immunocytokine is another factor that may limit therapeutic potential. as non-native proteins, immunocytokines could contain immunogenic epitopes against which an immune response, likely an antibody response, could be raised. antiimmunocytokine antibodies could induce pharmacological abrogation, therapeutic alteration, or hypersensitivity reactions (118) . because of the potential for anti-immunocytokine antibodies, novel immunocytokines should be engineered to minimize the presence of immunogenic epitopes. overall, although immunocytokines remain capable of inducing il-12-related adverse events, the use of targeting moieties may improve biodistribution enough to expand the therapeutic window of il-12-based immunotherapies. intratumoral (i.t.) injections of dna and rna encoding il-12 have the potential to localize and sustain the production of il-12 in the tumor microenvironment. nucleic acids are much easier to produce, purify and manipulate than recombinant cytokines. however, mammalian host cells are not easy to transfect and typically require chemical, physical, or electrical assistance to achieve reasonable transfection rates. this section will highlight progress in nucleic acid-based approaches both preclinically and clinically. around the same time that recombinant il-12 was failing in clinical trials, a limited number of preclinical and clinical studies explored i.t. injection of plasmid dna encoding il-12 (pil-12) as a potentially less toxic approach. preclinically, i.t. pil-12 inhibited but did not eliminate b16 melanomas (119) . in this study, il-12 was not detected in the serum following i.t. injection. in another study involving gray horses with metastatic melanoma, i.t. pil-12 resulted in detectable levels of pil-12 in the serum for up to 36 h (120). however, it is not clear if systemic dissemination of pil-12 resulted in significant systemic increases in serum il-12 or ifnγ as these were not measured (120) . in a phase i/ii trial of intralesional injections with pil-12, 3 of 9 and 8 of 9 patients with stage iv malignant melanoma experienced clinical and local responses, respectively (121) . in a phase i/ib study, i.t. pil-12 was found to reduce the size of treated lesions by at least 30% in 5 of 12 malignant melanomas and renal cell carcinomas (122) . pil-12 injections were welltolerated as no patient in either study experienced a significant treatment-related adverse event. despite successful safety studies, the use of naked pil-12 for cancer immunotherapy has not progressed, mostly likely due to poor transfection efficiency. the application of pulsed, high electric fields to facilitate cellular uptake and expression of genes has been a part of the molecular biologist's toolbox for decades. intratumoral injection of pil-12 immediately followed by electroporation, referred to here as pil-12+ep, has been explored in several murine tumor models (123) (124) (125) (126) (127) (128) (129) (130) . as expected, the benefit of adding electroporation was immediately apparent as one early study showed pil-12 alone had no effect on b16f10 tumor growth while pil-12+ep significantly inhibited tumors and extended survival (123) . importantly, the increase in antitumor efficacy was not associated with an increase in systemic il-12 levels (123) . among the more notable responses in other early preclinical studies, nearly half of mice bearing established b16f10 melanomas experienced complete tumor regression following 2 weekly treatments with pil-12+ep (124) . in a follow up study, pil-12+ep induced tumor regression in up to 80% of mice, whereas i.t. injections of pil-12 alone delayed but could not eliminate b16f10 primary tumors (125) . cured mice displayed protective immunity as 20 of 21 rejected a b16f10 challenge (125) . in the sccvii squamous cell carcinoma (scc) model, complete regressions were observed in 40% of mice following pil-12+ep (127). furthermore, 3 of 6 cured mice resisted a tumor challenge containing five times the original dose of tumor cells (127, 128) . against bjmc3879 murine mammary adenocarcinomas, ct26 murine colon adenocarcinomas and renca renal cell carcinomas, pil-12+ep significantly suppressed, but did not eliminate implanted tumors (129, 131) . against murine sa-1 fibrosarcomas, pil-12+ep suppressed tumor growth and induced complete regression in 90% of treated mice with 11 of 18 becoming resistant to tumor rechallenge (132) . in this study, il-12 and ifnγ were detected in the serum of treated mice, however, no side effects were observed (132) . abscopal responses have been documented in several studies. against bilateral sa-1 tumors, pil-12+ep treatment consistently eliminated primary, treated tumors, while slowing the growth of secondary, untreated tumors (132) . similarly, pil-12+ep treatment of mh134 hepatocellular carcinomas, inhibited both treated and untreated tumors while preventing spontaneous pulmonary metastases (133) . a recent study using bilateral b16 tumors demonstrated that an optimized pil-12+ep protocol (134) was capable of regressing treated lesions while inhibiting the growth of contralateral untreated tumors (54) . injecting pil-12 directly into tumors is important as multiple studies have confirmed that i.t. pil-12+ep treatments were significantly more effective than either peritumoral or intramuscular (i.m.) routes (124, 125, 128, 132) . the i.m. route also resulted in significantly more il-12 and ifn-γ in the serum (124, 128) . in terms of mechanism, multiple studies agreed that pil-12+ep treatment was associated with increased t cell infiltration, increased ifnγ expression and decreased angiogenesis (124, 127, 128, 131, 133, 135) . these findings are consistent with known antitumor mechanisms of il-12. more recent mechanistic studies have focused on changes in immune cell phenotype and function. for example, b16f10 tumor regression following pil-12+ep was mediated via the perforin/granzyme lytic pathway while antigen-specific cd8 + t cell responses were directed against tyrosinase-related protein epitope trp2 [180] [181] [182] [183] [184] [185] [186] [187] [188] (136) . in another study, pil-12+ep-induced elimination of b16f10 tumors was associated with increased tumor infiltration and polarization of macrophages toward an m1 phenotype (137) . another group found that pil-12+epinduced antitumor responses against b16f10 tumors were correlated with a reduction in pd-1 expression on cd4 + and cd8 + t cells (138) . yet, another group found that the treatment of bilateral b16f10 tumors induced a unique population of cd8 + effector t cells with low pd-1 expression in both untreated tumors and systemically (54) . this finding suggested that a subset of cd8 + effectors generated by pil-12+ep may be protected or "armored" against checkpoint-mediated exhaustion (54) . a unique feature of pil-12+ep immunotherapy is the potential to manipulate electric field parameters to enhance transfection, and therefore, efficacy. an exploration of electric field parameters demonstrated that pil-12+ep-induced cures ranged from 65 to 80% in b16f10 tumor-bearing mice depending on pulsing conditions (138) . about half of these cured mice resisted a b16f10 rechallenge (138) . by further enhancing electric field intensity and/or pulse length, it is possible to directly kill tumor cells and release tumor antigens via irreversible electroporation. in one recent study, partial-irreversible electropermeabilization (pire) administered after peritumoral electrotransfection with pil-12 caused complete regression of about 40-50% of treated b16f10 tumors (139) . two of 4 cured mice completely resisted tumor rechallenge while the remaining two experienced delayed tumor growth from the rechallenge. this pil-12 plus pire approach was found to delay, but not eliminate distant, untreated tumors in about half of the mice (139) . there have been several attempts to enhance antitumor activity through the incorporation of additional cytokineencoding plasmids. of note, two studies have demonstrated that ep using a combination of il-12 and il-18 plasmids outperformed il-12 alone in terms of antitumor activity (140, 141) . the rationale to combine these two cytokines is wellsupported given that il-12 and il-18 synergize to enhance th1 responses and ifn-γ production. however, adverse events are also enhanced as systemic co-administration of recombinant il-12 and il-18 proteins leads to lethal toxicity in mice (142) . in one study, addition of pil-18 to pil-12 increased serum il-12 and ifn-γ levels for at least 6 days after ep although no inflammation was observed in liver, lung, and intestine samples (140) . in a second study, pil-18 did not increase il-12-induced serum ifn-γ, but intratumoral ifn-γ was significantly higher (141) . several notable canine clinical studies have explored pil-12+ep in dogs with naturally occurring tumors. in one such study, pil-12+ep resulted in a 13-83% reduction in mast cell tumor volume (143) . treated nodules displayed increases in leukocytic inflammation and decreases in the number of malignant mast cells (143) . in beagles with canine transmissible venereal tumors (ctvts), pil-12+ep induced complete regression of all treated lesions (144) . contralateral untreated tumors were also significantly inhibited. serum il-12 levels peaked 7 days after treatment; however, relevant blood chemistries, i.e., liver and kidney enzymes, as well as cell counts were not different from those of control dogs (144) . another study investigated pil-12+ep for the treatment of canine oral malignant melanoma (omm). there were no differences in the percentages of helper cd4 + and cd8 + cells before and after treatment, while t reg frequencies declined from 1.2 to 0.3%. one month post treatment, the objective response rate was 67% (6/9) but by the end of the observation period, all but one of the dogs developed progressive disease (145) . a more recent study in 9 dogs with a range of spontaneous cancers, demonstrated that pil-12+ep induced immunostimulatory and anti-angiogenic effects (146) . administration of three pil-12+ep treatments every other day caused significant systemic toxicities, including anemia and thrombocytopenia. after switching to a weekly schedule, treatments were well-tolerated, however, all treated tumors continued to progress (146) . several human clinical trials, mainly against advanced melanoma, have investigated the safety and efficacy of pil-12+ep. in a phase i study, 24 patients with stage iii or iv melanoma received i.t. pil-12+ep (six 100 µs, 1,300v/cm pulses) on days 1, 5, and 8 during a single 39-day cycle. fifty three percent of patients experienced a systemic response, defined as either stable disease or regression of untreated lesions, following pil-12+ep (147) . most notably, 2 of 19 patients showed complete regression of all metastases. no grade 3 or higher adverse events were observed and neither il-12 nor ifnγ was detectable in serum samples. in a follow up phase ii study, 29 patients with in-transit or m1a melanoma were treated with up to four 12-week cycles of pil-12+ep as described above (148) . intermediate results revealed an objective response rate of 33% with 11% complete responses. the treatment was found to increase nk cell levels both intratumorally and systemically (148) . no grade 3/4 drug-related adverse events were noted. a subsequent analysis of clinical samples revealed that responses were associated with increased intratumoral infiltration of cd3 + t cells (148) . in addition, t cell receptor beta chain (tcrβ) sequencing revealed a focusing of the tcr repertoire following treatment. however, there were no differences in t cell clonality between responders and non-responders to pil-12+ep (148) . a second study from the same trial, nct01502293, found a complete response rate of up to 17.9% and a best overall response rate of 35.7% in patients with stage iii/iv melanoma (149) . nearly half of these patients experienced regression of at least one anenestic lesion. an analysis of transcripts in melanoma biopsies found increases in t cell trafficking, immune activation, and antigen presentation (149) . genes associated with adaptive resistance, e.g., pd-l1, tgfβ, and trail, were also increased (149) . recent results from a safety study in 3 patients with locoregional merkel cell carcinoma (mcc) and 12 patients with metastatic mcc received 1 or up to 4 cycles of pil-12+ep, respectively (150) . the overall response rate in the metastatic mcc cohort was 25% (3/12). of 10 patients with measurable untreated lesions, 3 experienced abscopal regressions. in addition, 2 patients experienced clinical responses lasting 16 and 55+ months, respectively. two of the locoregional mcc patients, all of whom were treated with definitive surgery after pil-12+ep, were recurrence-free at 44+ and 75+ months, respectively (150) . serum il-12 levels were not measured, but treatments were well-tolerated, and no serious adverse events were observed. lipoplexes, polyplexes, and lipopolyplexes are complexes of lipids, polymers, and lipids plus polymers, respectively, with dna. these complexes are under investigation to enhance the delivery and transfection efficiency of plasmids encoding genes of interest, including pil-12. numerous studies have explored a range of different materials to create novel pil-12 complexes (151) (152) (153) (154) (155) . studies demonstrating antitumor efficacy following local or targeted delivery of pil-12 complexes are highlighted below. polyethyleneimine (pei), a highly cationic polymer that readily complexes with negatively charged dna, has been widely used to enhance gene delivery. pei protects dna from degradation in vivo, encourages interaction with negatively charged cell membranes, and enhances release from lysosomes by acting as proton sponge (156) . pei:il-12 complexes were shown to transfect lung tissue following delivery via nebulization (157) . this approach led to production of il-12 in the lungs which was not detectable in the plasma of treated mice (157, 158) . weekly or twice weekly administration of aerosolized pei:il-12 was found to suppress or eliminate experimental pulmonary metastases of saos-2 human osteosarcomas in athymic nude mice (157) . recent attempts to enhance uptake and il-12 production have focused on modification of pei with tetraiodothyroacetic acid (tetrac) which binds the α v β 3 integrin receptor that is overexpressed in some tumors (159) or diethylene triamine penta-acetic acid (dpta) which can reduce the surface charge of pei:il-12 complexes (160, 161) . as of this writing, no in vivo data using either modification have been published. polyvinylpyrridilone (pvp) is another cationic polymer that readily complexes with dna. twice weekly i.t. injections of pil-12/pvp complexes were found to eliminate 30 and 50% of 8-10 mm 3 renca and ct26 tumors, respectively (162) . most mice that were cured of a primary tumor rejected a subsequent rechallenge (162) . in a follow-up study, pil-12/pvp was found to be more effective than pifnα/pvp in controlling preclinical tumors, while the combination of pil-12/pvp and pifnα/pvp synergized to eliminate 100% of renca and 50% of ct26 tumors (163) . in both studies, cd8 + t cells but not cd4 + t cells were identified as primary effectors. complexation with poly-a-(4-aminobutyl)-l-glycolic acid (paga), a biodegradable polyester, enhanced transfection efficiency of pil-12 and expression of il-12 in vitro and in vivo (164, 165) . however, t cell infiltration of injected ct26 colon adenocarcinomas and antitumor activities following repeated injections of paga/pil-12 and naked pil-12 were similar (164) . encapsulation of pil-12 in nanoparticles comprised of poly-(d,l-lactic-co-glycolic acid) (plga) and 1,2-dioleoyl-3-(trimethylammonium) propane (dotap) demonstrated complete regression of up to 75% of established heterotopic bnl hepatocarcinomas following a single i.t. injection (166) . importantly, treatment with encapsulated pil-12 was more effective than treatment with nanoparticles with pil-12 adsorbed to the surface (166) . long term expression of inflammatory cytokines could be a concern as both il-12 and ifnγ were detected in the serum for up to 30 days after treatment (166) . a similar, so-called dmp nanoparticle, comprised of dotap and methoxy-poly(ethylene glycol)-poly(lactide) (mpeg-pla), has been developed to facilitate gene delivery (167) . complexation with pil-12 resulted in inhibition of ct26 tumors with no signs of systemic toxicity, as determined by appearance, body weight, fecal output, and urinary excretion (167) . a slightly different dmp nanoparticle that uses polycaprolactone (pcl) instead of pla, inhibited the growth of intraperitoneal c26 colon carcinomas and ll/2 lewis lung carcinomas (168) . in addition to delaying tumor growth, dmp/il-12 particles resulted in high il-12 gene expression and t cell infiltration although the treatment regimen consisted of 7 daily or every other day treatments (168) . plasmids complexed with mannosylated chitosan (mc) are under development as a method to target mannose receptors on i.t. dcs. chitosan is a linear co-polymer of β-linked dglucosamine and n-acetyl-d-glucosamine. it is primarily derived from the structural polysaccharide, chitin, found in shells of crustaceans. i.t. injection of mc/pil-12 complexes elicited modest growth delay of ct26 tumors, which was associated with increased tumor cell apoptosis and decreased angiogenesis (169) . lipopolymers, which incorporate a lipid tail on a polymer backbone, are also under exploration for non-viral gene delivery. water soluble lipopolymers (wslp) comprised of cholesterol conjugated to pei have been developed to increase cell membrane permeability and reduce pei-mediated toxicity (170) . wslp/p2cmvmil-12 dna complexes inhibited the growth of ct26 tumors and improved survival following a single i.t. injection (170) . however, the antitumor efficacies of wslp/pil-12 complexes and naked pil-12 appeared similar. in a later study, wslp/p2cmvmil-12 complexes injected every 4 days outperformed single injections and multiple injections with naked pil-12 or pei/pil-12 complexes (171) . the vast majority of injected wslp/p2cmvmil-12 was found in the tumor for up to 24 h with small but increasing accumulation in the liver and blood (171) . subsequent studies demonstrated that i.t. wslp/p2cmvmil-12 significantly suppressed the growth of primary and metastatic 4t1, tsa, and emt-6 mammary carcinomas (172, 173) . polytraxane (prx) is a composite molecule made of polyethylene glycol (peg) and cationic cyclodextrin (cd) that self assembles with dna into a spherical particle (174) . a 4-arm configuration, rather than a linear configuration, had a higher accumulation in mc38-luc tumors and lower accumulation in the lungs following systemic delivery. the 4-arm prx/pil-12 also produced higher levels of il-12 and significantly slowed mc38-luc tumor growth after five i.v. injections of the complex starting 10 days after tumor implantation. (174) systemic injections of prx/pil-12 complexes induced moderate lymphopenia, but no elevation of liver enzymes (174) . pil-12 complexed with a polyethyleneglycolpolyethylenimine-cholesterol (ppc) lipopolymer was shown to inhibit 4t1 and sccvii tumors (175) and increase survival in mice with intracranial gl261 gliomas (176) following localized injections. of note, although the i.p. route is frequently used to deliver drugs systemically, i.p. injections of pmil-12/ppc for treatment of id8 ovarian carcinomas resulted in high levels of il-12 and ifn-γ in ascites but low levels in serum (177) . in this study, i.p. pmil-12/ppc was well-tolerated with no significant changes in serum chemistries (177) . in a phase i study, phil-12/ppc was administered i.p. to 13 women with chemo-resistant recurrent ovarian cancer (178) . escalating doses of phil-12/ppc were well-tolerated with no dose-limiting toxicities. five of the 13 treated patients reported a serious adverse event, however, only one was possibly related to the phil-12/ppc (178) . similar to preclinical studies, no detectable increase in serum il-12 was found following phil-12/ppc treatment (178) . a subsequent phase ii study in 20 patients with platinum-resistant recurrent ovarian cancer demonstrated similar safety following weekly i.p. phil-12/ppc, however, with no objective clinical responses observed (179) . a different phase i trial evaluated the safety of phil-12/ppc in ovarian cancer patients when combined with carboplatin and docetaxel chemotherapy (180) . while there were no dose limiting toxicities, grade 3 adverse events included manageable abdominal pain and cytokine release syndrome. two of 12 patients experienced complete response while 4 of 12 experienced a partial response (180) . a more recent phase i trial combined weekly i.p. pil-12/ppc with i.v. pegylated liposomal doxorubicin in 14 patients with persistent or recurrent platinum-resistant ovarian or peritoneal cancers (181) . although increased levels of il-12, ifn-γ, and tnf-α were found in peritoneal fluid, no dose limiting toxicities were observed and a maximum tolerated dose was not reached. the best partial response (28.6%) and stable disease (57.1%) rates were found at the highest dose (36 mg/m 2 ) of pil-12/ppc (181) . pil-12 complexed with a cationic lipid (+/-)-n-(2-hydroxyethyl)-n,ndimethyl-2,3-bis(tetradecyloxy)-1propanaminium bromide/dioleoylphosphatidylethanolamine (dmrie/dope), and injected i.t. was found to inhibit and eliminate ct26 and renca tumors while protecting up to 96 and 100%, respectively, of mice from rechallenge (182) . interestingly, a direct comparison between naked pil-12 and dmrie/dope/pil-12 revealed no difference in antitumor activity (182) . polyphosphazene particles were modified with hydrophobic n,n-diisopropylethylenediamine (dpa) and hydrophilic monomethoxy poly-(ethylene glycol) (mpeg) to create weakly cationic particles to complex with pil-12 (183) . mpeg/pil-12 polymersomes delayed ct26 tumor growth when administered i.v. body weights were unaffected by mpeg/pmil-12 treatments. tumor il-12 levels steadily increased, reaching about 80 pg/g tumor on day 16, while serum il-12 concentration remained on average about 40 pg/ml after day 9 and continued to day 16. the concentration of ifn-γ in the tumor reached a maximum of 350 pg/g tumor on day 9, while serum levels of ifn-γ slightly increased from day 9 to day 16, reaching a concentration of only 4 pg/ml. while mpeg/pmil-12 polymersomes did not affect cd3 + cd4 + cells in the tumor, there was a 2-fold increase of cd3 + cd8 + cells and significant increases of cd3 − nk1.1 + and cd3 + nk1.1 + cells in the tumor. in another study, all-trans-retinoic acid (atra) was incorporated in cationic liposomes and complexed with pil-12 (184) . atra was previously found to increase the expression of tnf receptor 1 and mediate apoptosis of lung cancer cells via tnfα (185, 186) . i.v. injections of atra-cationic liposome/pil-12 reduced lung nodules and extended survival compared to cationic liposome/pil-12 treatment in an experimental pulmonary metastasis model using c26 cells expressing luciferase (184) . concentration of il-12 in the lungs reached a maximum of 12 pg/mg protein at 6 h post injection, while levels of il-12 in the spleen and liver were significantly lower and nearly eliminated by 24 h. interestingly, the incorporation of atra reduced liver enzymes levels and thus hepatic toxicity suggesting a possible anti-inflammatory role (184) . recently, mrna delivery platforms have received tremendous attention, most notably as front running vaccines against sars-cov-2. mrna, like dna, can encode an unlimited number of proteins and polypeptides. although mrna-based platforms are less stable than dna-based platforms, mrna can be protected from digestion through encapsulation in polymeric or lipidbased micro-or nanoparticles. a key advantage of mrna is their ability to directly translate encoded proteins in the cytoplasm. in contrast, dna must first translocate to the nucleus to be transcribed to mrna before translation in the cytoplasm. regarding the use of mrna to deliver il-12 locally, a recent study demonstrated that weekly i.v. delivery of lipid nanoparticles (lnp) loaded with mrna encoding il-12 reduced tumor burden in a myc-driven transgenic mouse model of hepatocellular carcinoma (hcc) (187) . the tumor inhibition and extended survival were attributed to increased infiltration of cd3 + cd4 + cd44 + immune cells and not suppression of myc (188) . similarly, moderna/astrazeneca has developed, medi1191, an lnp formulation with il-12 mrna. medi1191 is currently in phase i clinical trials for intratumoral injection of advanced solid tumors in combination with durvalumab ( table 2 ) (189) . in preclinical studies, a single intratumoral injection of mrna encoding murine il-12 (mil-12) increased ifnγ expression and genes associated with a th1 response in mc38 tumor-bearing mice (190) . when combined with anti-pd-l1, enhanced t cell infiltration and expanded tumor-specific t cell subsets were observed (190) . in another phase i clinical trial, sanofi and biontech are testing sar44100 (bnt131), an mrna platform encoding a cocktail of il-12sc, il-15sushi, ifnα, and gm-csf for intratumoral injection as a monotherapy and in combination with cemiplimab (191) . to our knowledge, no preclinical data with sar44100 have been disclosed. in general, nucleic acid-based il-12 delivery approaches are limited by variable transfection rates as well as unregulated production of gene products. in other words, it is easy to control the amount of pil-12 or il-12 mrna delivered but it is not easy to control the dose of recombinant il-12 that each subject receives. variable transfection rates may be responsible for conflicting reports on whether pil-12+ep does (131-133, 140, 143, 144) or does not (124, 126, 130) produce significant increases in serum il-12 and ifn-γ. fortunately, lethal il-12-related toxicities have not been reported in the any of the preclinical or clinical studies detailed above. nevertheless, the potential for severe il-12-related adverse events caused by continued and/or unregulated production of il-12 remains. strategies to enhance safety and efficacy, either by localizing gene-based il-12 though incorporation of an anchoring or binding domain or by incorporation of an inducible safety switch to turn off il-12 production, will help improve the therapeutic window of promising nucleic-acid-based il-12 delivery technologies. another potential limitation that must be considered, is any type of adverse reaction against components of the delivery vehicle or against the nucleic acid vector itself. regarding the former, foreign delivery components have the potential to induce immune responses which could influence il-12 delivery. most notably, it has been reported that about 7 in 10 humans have circulating anti-peg antibodies (192) . the high prevalence of anti-peg antibodies could limit the efficacy of any peg-based delivery vehicle. regarding the immune responses against il-12 vectors, nucleic acids, particularly dna that is found in the cytoplasm have the potential to stimulate the cgas-sting pathway, which may induce its own inflammatory response. thus, studies utilizing nucleic acid-based delivery must take care to decouple the effects of il-12 from sting activation. lastly, although an exceedingly rare event, dna vectors could become integrated within a cell's genome. depending on the site, such integration could have deleterious or even transforming effects. given that only transient il-12 expression is desirable, strategies capable for preventing integration, like the use of circular instead of linearized plasmids, should be preferred. adenoviruses, herpes simplex viruses, semliki forest viruses, poxviruses, and other viral vectors have been engineered to express biologically active il-12. these engineered viruses injected directly into a tumor are able to infect cancer cells and induce expression of il-12 within the tumor microenvironment. furthermore, many viruses have the unique ability to selectively lyse cancers cells after infection. such oncolytic viruses take advantage of defective cell cycle and interferon signaling pathways that are hallmarks of cancer cells but not normal cells (193) . oncolytic viruses can kill compromised cancer cells in a variety of ways from direct virus-mediated cytotoxicity to indirect destruction of tumor-feeding blood vessels (194) . the following sections discuss the progress and limitations of il-12encoding viral vectors. adenoviruses are the most well-studied among the il-12 expressing vectors (47, (195) (196) (197) (198) (199) (200) (201) . in preclinical studies, i.t. injections of adenoviruses encoding il-12 (ad-il-12) have mediated regressions of murine colorectal carcinomas (202) (203) (204) , breast carcinomas (47, 201, 202) , prostate carcinomas (205, 206) gliomas (207, 208) , bladder carcinomas (209) , fibrosarcomas (202, 210) , laryngeal squamous cell carcinoma (211) , hepatomas (212) and hepatocellular carcinomas (213, 214) medullary thyroid carcinomas (215) , thyroid follicular cancer (216) , and ewing's sarcoma (217) . among the more robust responses, ad-il-12 induced complete regression of subcutaneous neuro-2a neuroblastomas in nearly half of mice receiving a single i.t. injection (218) . mice becoming tumor-free also rejected a subsequent tumor rechallenge (218) . similar results were found against ct26 colon adenocarcinomas with more than three-fourths of mice completely eliminating their tumors and all cured mice rejecting a tumor rechallenge (203) . the antitumor immune response was mediated primarily by cd8 + t cells. impressively, 3 of 7 mice with bilateral tumors experienced complete regression of an untreated tumor (203) . against 6-23 rat medullary thyroid carcinomas, i.t. injection of adtcpmil-12 caused complete regression of more than 60% of treated tumors (215) . all cured rats rejected a tumor rechallenge while separate experiments showed that treatment of a single tumor resulted in inhibition of a distant untreated tumor (215) . while liver infection following i.t. adtcpmil-12 injection was documented, no toxicity was observed (215) . a follow up study found similar antitumor and abscopal responses against rat thyroid follicular cancer (216) . in the pymt-derived transplanted mammary carcinoma model, adenoviral vectors encoding il-12 induced complete regression in 31% and partial regression in 47% of mice (47) . ten of 11 tumor-free mice completely rejected a tumor rechallenge. in contrast with similar studies using ad vectors expressing il-2, no obvious toxic side effects due to admil-12 were noted (47) . in another difficult model, a single i.t. injection of ad.5/3.crgd-mil12p70 resulted in >60% long term survival of mice with intracranial gl261 gliomas (207) . in a murine model of ewing's sarcoma (tc71), twice weekly i.t. injections of ad.mil-12 significantly delayed treated tumors as well as untreated tumors (217) . ad.mil-12 also induced complete regression in all treated mice bearing heterotopic mb49 bladder carcinomas (209) . although body weights were not affected, serum ifn-γ levels due to i.t. ad.mil-12 were maximal from 2 (∼3,500 pg/ml) to 5 days (∼1,500 pg/ml) post injection (209) . in a useful comparison against other cytokines, one study demonstrated that ad-ifn-γ had no greater antitumor activity than an empty ad vector, whereas admil-12 induced complete regressions of p815 mastocytomas in >80% of treated mice (219) . similarly, ad-gm-csf inhibited the growth of frtl-tc rat thyroid tumors, however, adil-12 was found to be much more effective (220) . adil-12 also generated systemic immunity capable of inhibiting the growth of distant tumors (220) . an adenoviral vector expressing a single chain il-12 (scil-12) was developed to enhance bioactivity over the native heterodimeric form (221) . long-term tumor-free survival was observed in up to 90% of rats with established mh-7777a hepatocellular carcinomas following i.t. infections with ad.scil-12 (221) . all tumor-free mice were protected from tumor rechallenge. however, despite i.t. injections, il-12 and ifn-γ were detected in the serum of treated mice (221) . an oncolytic adenovirus expressing single-chain il-12 (ad-dhscil12) was more effective than non-replicating (nononcolytic) adenoviruses expressing il-12 at controlling liver metastases of pancreatic cancer in hamsters (222) . however, il-12 levels in serum and non-tumor tissues were similar (222) . in clinical studies, i.t. ad.il-12 was well-tolerated with no dose-limiting toxicities in a phase i trial in 21 patients with advanced pancreatic, colorectal, or primary liver malignancies (223) . serum ifnγ levels peaked 1 day after ad.il-12 administration and was likely responsible for the 16 grade 3 adverse events observed. one patient had a partial response and 29% of patients experienced stable disease. four of 10 assessable patients experienced increases in tumor infiltrating cd4 + and cd8 + cells. delayed-type hypersensitivity tests with inactivated adenovirus indicated that all patients developed an immune response against the adenovirus (223) . despite robust antitumor immune responses, interest in ad-il-12 waned in the mid-2000s due to the aforementioned lethal toxicities associated with systemic administration of ril-12 and the inability of ad-il-12, even if administered intratumorally, to prevent systemic dissemination of the cytokine. in recent efforts to mitigate systemic il-12 dissemination and associated toxicities, two strategies have been developed. the first involves engineering il-12 to prevent its dissemination. this has been accomplished either by anchoring il-12 to the surface of tumor cells via fusing a transmembrane domain or glycosylphosphatidylinositol (gpi)-anchored signal sequence to the cytokine (224, 225) or by deleting the n-terminal signal peptide and thus preventing il-12 secretion (226) . using the former technology, i.t. injection of an adenoviral vector encoding membrane-anchored il-12 (ad/scil-12-b7tm) eliminated the majority of primary ct26 tumors and suppressed the growth of untreated contralateral tumors (5 out of 5 mice), in which complete regression occurred in 1 out of 5 mice (227). importantly, negligible il-12 was found in the circulation of mice treated with ad/scil-12-b7tm. the second technology deletes the signal peptide from the p35 subunit of il-12 to prohibit il-12 secretion (226) . newly designed oncolytic adenoviral vectors with three genes deleted, i.e., triple deletion (td), and encoding either wild-type il-12 (ad-td-il-12) or a non-secreting il-12 (ad-td-nsil-12) were evaluated in syrian hamster models of pancreatic cancer. six i.t. injections of either ad-td-il-12 or ad-td-nsil-12 were found to eliminate subcutaneous hpd1nr tumors in all mice (226) . against peritoneally disseminated shpc6 tumors and orthotopic hap-t1 pancreatic tumors, ad-td-nsil-12 outperformed ad-td-il-12 in terms of overall survival (226) . most importantly, ad-td-nsil-12 resulted in significantly lower serum il-12 levels and reduced systemic inflammatory cytokine expression (226) . the second strategy to mitigate systemic il-12 dissemination involves conditional expression of il-12. the rheoswitch therapeutic system r (rts) is an ecdysone receptor-based gene regulation platform in which a transcription factor becomes activated only in the presence of a synthetic small molecule ligand (228) . an adenoviral vector encoding the rts switch and mil-12 (ad-rts-mil-12) and controlled by the oral activator, veledimex (vdx) was recently shown to extend survival in mice bearing intracranial gl261 gliomas (229) . intratumoral ad-rts-mil-12 plus oral vdx was found to induce il-12 expression in a dose-dependent manner. local il-12 expression correlated with increases in tumor-infiltrating lymphocytes. il-12 and ifnγ were detected in the sera of treated animals, albeit at an order of magnitude lower than levels found in tumors (229) . several clinical studies utilizing the regulatable ad-rts-il-12 platform are underway ( table 2) . recent results from a phase i study in 31 patients undergoing resection of recurrent highgrade glioma demonstrated that vdx induced il-12 expression in a dose-dependent manner (230) . likewise, the frequency and severity of adverse events, including grade 3 cytokine release syndrome, also increased with vdx dose. demonstrating the advantage of the inducible system, all serious adverse events were reversible with vdx discontinuation. interestingly, the use of corticosteroids negatively impacted survival (230) . at the optimal dose, and in the absence of corticosteroids, the median overall survival of 17.8 months was encouraging (230) . while the localized injection of ad-rts-hil-12 in the resected tumor bed induced il-12 expression in a recurrent tumor microenvironment, systemic dissemination of il-12 and its resultant toxicities could not be avoided. herpes simplex viruses (hsvs) are another family of viruses that have been widely explored for localized il-12 delivery. wild-type hsv are cytolytic and thus must be significantly attenuated or rendered replication-incompetent to avoid systemic infection. injection of replication-incompetent hsv-il-12 into established hepatomas prior to partial hepatectomy inhibited the engraftment of an intraportal tumor cell challenge in preclinical studies (231) . importantly, no changes in serum il-12 were detected in treated buffalo rats (231) . in order to capitalize on their lytic potential, hsvs have been engineered, through deletion or mutation of genes responsible for viral replication such that only cancer cells are lysed. not surprisingly, these oncolytic hsvs (ohsvs) have been shown to exhibit greater antitumor potential than their replicationincompetent, parental counterparts. for instance, treatment of established hepatomas with a non-cytokine encoding ohsv exhibited significant antitumor activity which was further increased with an il-12 insert (232). the ohsv-il-12 treatment was also more effective than ohsv at protecting animals from a tumor rechallenge (232) . similar antitumor responses to ohsv and increased efficacy with ohsv-il-12 were observed against flank ct26 and sccvii tumor models (233) (234) (235) . a single i.t. injection was able to delay the growth of established sccvii tumors whereas multiple injections induced complete regressions in 5 of 6 treated mice (235) . in contrast, multiple injections of ohsv-gm-csf cured only half of treated mice (235) . in the ct26 model, ohsv-il-12 inhibited or eliminated injected 5 mm tumors while inhibiting non-injected, contralateral tumors (236, 237) . when low dose mil-12 was added to i.t. ohsv-il-12, both treated and untreated ct26 tumors were eliminated in more than two-thirds of mice (237) . the importance of t cells in this model was established as ohsv-il-12 had no antitumor effect in ct26-bearing nude mice (236, 237) . intraperitoneal administration of ohsv-il-12 also increased survival in misiir-tag mice bearing ovarian carcinomas (238) . treatment was associated with tumor antigen-specific cd8 + t cell infiltration (238) . against flank sarc-043 and sarc-045 sarcomas, ohsv-il-12 extended survival compared to saline injections (239) . although there was no difference is survival between control and il-12 encoding ohsv, the ohsv-il-12 was found to increase tumor-infiltrating effector t cells while decreasing immunosuppressive mdsc and t reg populations (239) . in an interesting comparison of cytokines, ohsv-il-12 was more effective at inhibiting flank prostate tumors, tramp-c2 and pr14-2, than ohsv encoding gm-csf (ohsv-gm-csf) which was no more effective than non-cytokine encoding ohsv (240) . only 1 of 18 mice treated with ohsv-il-12 exhibited an increase in serum il-12 4 days after treatment (240) . ohsv-il-12 also significantly outperformed ohsv-gm-csf in a model of ct26 metastasis (241) . in addition to localizing il-12, i.t. injections may be key in assuring safety as intrasplenic injections of ohsv-il-12 induced concerning increases in il-12 and ifnγ in both serum and liver specimens (241) . frequent and worthwhile targets of ohsv-il-12 immunotherapy are the various forms of brain cancer. in one early study, ohsv-il-12 was found to extend survival and cure approximately one-fourth of mice bearing 5-day-old intracranial neuro-2a neuroblastomas (242) . treatment was associated with an influx of cd4 + and cd8 + t cells as well as macrophages. the inclusion of il-12 was critical, as median survivals of mice treated with the parental, non-cytokine encoding ohsv or saline were similar (242) . likewise, ohsv-il-12 but not non-cytokine ohsv was effective at extending survival against intracerebral mouse 005 glioblastomas (243) . in an intracranial 4c8 glioma model, ohsv-il-12 was significantly more effective than ohsv at extending survival and curing mice (244) . in a model of breast cancer metastasizing to the brain, i.t. ohsv-il-12 modestly extended the survival of mice bearing intracranial sck tumors (245) . intracerebral injections of ohsv-il-12 in owl monkeys resulted in neither histopathological changes in brain tissue nor clinical evidence of toxicity, as assessed by changes in temperature, neurologic performance, feeding or social behavior, or weight (246) . in addition to encoding cytokines, hsvs can be engineered to target cancer-associated antigens. four i.t. injections of a her2-targeted ohsv-il-12 was significantly more effective at inhibiting both day 3 and day 10 tumors than the non-cytokine encoding parental hsv (day 3: 15/22 ohsv-il-12 vs. 7/20 hsv becoming tumor free; day 10: 3/18 ohsv-il-12 vs. 1/12 hsv becoming tumor free) (247) . immune responses to ohsv-il-12 included elevated levels of ifnγ, il-2, granzyme b, tbet, and tnfα as well as th1 polarization and nk activation (247) . this her-2 targeted ohsv-il-12 was also found to induce complete remission in more than one-fourth of treated mice bearing orthotopic high grade gliomas (hgg) expressing her2 (248) . cured mice were protected from hgg rechallenge regardless of her2 expression (248) . this is an important finding given the heterogeneity of her2 expression among and within tumors. the clinical precedence for hsv has been established with talimogene laherparepvec, which encodes for gm-csf and is approved for intratumoral injection in patients with advanced, non-resectable melanoma. a clinical grade preparation of hsv-1 encoding hil-12 (m032) induced no adverse clinical signs after intracerebral injection in non-human primates (249) . a phase 1 clinical trial exploring the safety of m032 in patients with recurrent or progressive glioblastoma multiforme, anaplastic astrocytoma, or gliosarcoma is currently recruiting [nct02062827]. semliki forest virus (sfv) is an alphavirus that was first isolated from mosquitos in uganda and has a broad range of hosts, making it ideal for translation (250) . in addition, modified sfv can produce higher levels of recombinant protein than retroviral vectors, and expresses protein more stably than adenoviral vectors (251) . sfv is also less pathogenic in humans, and induces apoptosis of tumor cells at the end stage of virulence (250) . sfv encoding il-12 (sfv-il-12) was found to extend the survival of mice with established orthotopic 203 gliomas (252) . because sfv infection induces apoptosis, uptake of infected tumor cells by dendritic cells in the presence of il-12 is posited as a potential mechanism of enhanced antitumor activity. in a b16 brain tumor model, the same group demonstrated a prolonged median survival by 5 days when immunizing with sfv-il-12 pulsed dendritic cells, compared to a retroviral vector encoding il-12 (253) . in a woodchuck hcc model, induced by chronic infection with woodchuck hepatitis virus (whv), using a single intratumoral treatment with sfv-enhil-12, which included 10 separate injections at different sites of one tumor, partial tumor regressions of up to 80% occurred in 5 out of 6 animals (254) . although all tumors regrew after treatment, injections of sfv-enhil-12 resulted in a favorable safety profile with only transient reductions in body weight (254) . in a transgenic mouse model of spontaneous hcc, i.t. sfv-il-12 treatments achieved 100% survival for at least 135 days (255) . interestingly, sfv-il-12, which induces transient infection and expression of il-12, was found to be more effective and less toxic than long-term il-12 expression induced by a plasmid encoding il-12 which was delivered hydrodynamically (255) . i.t. sfv-il-12 inhibited tumor growth and extended survival in mice bearing orthotopic 4t1 mammary carcinomas (256) . when administered neoadjuvant to resection and combined with attenuated salmonella (lvr01) as a post-surgery adjuvant, an impressive 90% long-term tumor free survival was achieved (256) . while there is consensus on its benefits, the mechanism of sfv-il-12-induced tumor regression is debated. initial studies performed in a subcutaneous b16 melanoma model did not find a significant decrease in tumor regression when using t cell-deficient nude or nk cell-deficient beige mice. rather, this group suggested that inhibition of angiogenesis mediated by ifnγ production causes massive tumor necrosis (257) . a decade later, the same group further supported this conclusion using inos deficient mice that express high levels of vegf (258) . the effect of il-12 was even more pronounced, with fewer tumor vessels in inos −/− mice than in wild-type mice given the same treatment with sfv-il-12 (258) . however, they did find immunohistochemical evidence that nk cell activation and recruitment is correlated with murine endothelial cell death (258) . more recently, the efficacy of sfv-il-12 was found to be dependent on type i interferons produced by macrophages and dendritic cells (259) . furthermore, the type i interferon receptor, ifnar, is necessary for il-12-dependent cd8 + t cell expansion (259) . different routes of administration were compared using a subcutaneous p815 model and demonstrated that i.t. injection of sfv-il-12 was superior in producing ifnγ compared to either s.c. or i.v. routes (260) . importantly, none of the sfv-il-12 injections resulted in increased serum levels of ifnγ (260) . the combination of sfv-il-12 with monoclonal antibodies or adjuvants is another strategy that has been studied. by administering sfv-il-12 and anti-cd137 to provide costimulation to t cells, survival was improved in both s.c. b16-ova and s.c. tc-1 models with a maximum longterm survival of 75% in both models (261) . in the bilateral b16-ova model, 90% of treated and 22% of untreated tumors experienced complete regression with 10 8 sfv-il-12 viral particles and anti-cd137 (261) . cd8 + t cells were crucial for this tumor regression and sfv-il-12 increased the ratio of cd8 t/t regs compared to anti-cd137 alone. a subsequent paper further demonstrated that sfv-il-12 induces pd-l1 expression on b16-ova cells and therefore combined pd-1/pd-l1 blockade with the viral construct (262) . this combination significantly enhanced survival compared to individual components using b16-ova and mc38 models, with long-term survival >75% (262) . modifications to improve the performance of sfv-il-12 vectors have also been explored. an enhanced (sfv-enhil-12) vector with separate promoters for each subunit of il-12 increased il-12 expression 8-fold over the single promoter construct (251) . one i.t. injection of 10 8 viral particles using either the original or enhanced constructs resulted in >80% longterm tumor free survival of mc38 tumor-bearing mice (251) . lower doses of sfv-enhil-12 induced tumor regression more efficiently than sfv-il-12, although the enhanced vector induced higher levels of serum il-12 (251) . a separate enhanced sfv vector with 10x higher gene expression, called psfv10-e-il12, has also been developed (263) . this vector also included separate promoters for p40 and p35 with an additional capsid enhancer to drive enhanced expression of the recombinant protein. two helper vectors with instructions for structural proteins were cotransfected with the enhanced sfv to produce virus-like particles (vlps). intratumoral injections in subcutaneous k-balb and ct26 tumors caused complete regression and furthermore inhibited primary tumor growth and reduced metastases in a heterotopic 4t1 model (263) . in an effort to limit undesirable virus-mediated cytotoxicity for intracranial applications, sfv vlps expressing il-12 have been developed (264) . low dose (5 × 10 7 vlps) treatment decreased orthotopic rg2 rat glioma volumes by 70% and extended survival by a little more than 20% over vehicle controls. high dose (5 × 10 8 vlps) treatment resulted in enhanced tumor reduction but also caused cns inflammation, necrosis, and treatment-related death (264) . the broad infectivity of sfv-based vectors is a major limitation to their use in cns applications. vaccinia virus (vv), which was used extensively during the eradication of smallpox, has a large capacity for gene insertion, a wide host range and high gene expression efficiency. vv expressing il-12 (vv-il-12) has been found to inhibit, but not eliminate, c6 gliomas in nude mice following i.t. injection (265) . low vv-il-12 doses, 10 2 -10 4 pfu, and high vv-il-12 doses, 10 5 -10 7 pfu, resulted in similar levels of tumor inhibition (265) . all doses above 10 pfu resulted in cytokine-associated toxicities punctuated with a 40% mortality rate in the high dose group (265) . similar tumor inhibition and mortality were found in a subsequent publication which also correlated high plasma levels of ifnγ and tnfα with toxicity (266) . vv-il-12 and il-2 expressing vv (vv-il-2) exhibited similar inhibition of c6 gliomas (265, 266) however, against ae17 mesotheliomas, vv-il-12 cured 80% of mice compared to only 20% of mice treated with vv-il2 (267) . recently, an oncolytic vv-il-12 was shown to increase lymphocytic infiltration and inhibit llc and b16f10 tumors with 14-25% complete responses (268) . antitumor efficacy was enhanced by the addition of an il-7 expressing vv (vv-il7) as well as immune checkpoint inhibitors (268) . the combination of vv-il-12 and vv-il7 did not affect mouse body weight (268) and appears to be better tolerated than previous vv-il-12 treatments which utilized different vv strains (265, 266) . a recombinant canarypox virus (alvac) encoding il-12 has been shown to induce complete regression of ts/a mammary carcinomas after six i.t. injections, whereas a single i.t. injection of alvac-il-12 had no effect on tumor growth (269) . of the mice that were rendered tumor-free, 70% were protected from a contralateral rechallenge. antitumor activity was driven by il-12 as the alvac vector itself displayed very limited tumor inhibition. in a phase i study in 9 patients with unresectable metastatic melanoma, i.t. injections of alvac-il-12 resulted in increased intratumoral il-12 mrna expression in only 4 of 9 patients (270) . three patients experienced modest increases in serum il-12 and ifnγ levels and no dose-limiting toxicities were observed. one patient had a complete response of an injected lesion and an uninjected in-transit metastases (270) . in a similar phase i study performed by the same group, only 1 of 4 evaluable patients exhibited an increase in intratumoral il-12 mrna after alvac-il-12 administration (271) . attenuated measles viruses (mevac) have been used safely as a vaccine to prevent measles in billions of people. mevac is another type of oncolytic virus which makes it an attractive potential cancer therapy. mevac encoding il-12 (mevac fmil-12) achieved complete regression of carcinoembryonic antigen (cea) expressing mc38 tumors in 90% of mice treated i.t. (272) . mev encoding anti-pd-l1 or igg-fc induced complete regressions in 60 and 40% of treated mice, respectively. antitumor activity was cd8 + t cell dependent and correlated with high levels of intratumoral ifnγ. all mice experiencing complete regressions following mevac fmil-12 also complete rejected tumor rechallenge (272) . a follow up study demonstrated that mevac fmil-12 was more effective at controlling tumors than mev encoding il-15 in two murine tumor models (273) . against mc38-cea tumors, mevac fmil-12 eliminated tumors in all treated mice whereas mev-il15 induced complete tumor regressions in 60% of mice (273) . effects appear to be model dependent as treatment of b16 tumors expressing human cd46 with mev-il-12 and mev-il15 only modestly extended survival (273) . vesicular stomatitis virus (vsv) is another potent oncolytic virus under exploration for cancer therapy. vsvs engineered to express il-12 (vsv-il-12) were recently evaluated in an orthotopic floor of mouth model of sccvii murine squamous cell carcinoma (274) . multiple i.t. injections of vsv-il-12 were more effective at eliminating tumors and induced higher longterm survival rates (40 vs. 0%) compared to non-cytokine encoding vsv (274) . moloney murine leukemia virus (momlv) is a positive sense, single-stranded rna (ssrna) retrovirus. a momlv expressing il-12 was engineered to display an anti-erbb2 scfv against the her2 receptor (275) . eight daily i.t. injections of 6-day-old flank mbt2 tumors was found to result in inhibition and complete regression in about one-fifth of mice treated with the her2targeted momlv-il-12 (275) . a hybrid viral vector which pairs the high infectivity of adenoviruses with the high gene expression of sfv has been engineered to specifically infect and express il-12 in hepatocellular carcinoma cells (276) . in buffalo rats bearing orthotopic mca-rh7777, the adv-sfv hybrid vector expressing il-12 induced complete regression in 4 of 12 treated animals. while the non-hybrid sfv-il-12 induced complete regression in 50% of treated rats, treatment was accompanied by elevated liver enzymes indicating hepatotoxicity (276) . in contrast, liver enzyme levels following administration with the tissue-specific, hybrid vector were no different than levels in saline-treated control rats (276) . newcastle disease virus (ndv) is an oncolytic, enveloped, negative-sense, single-stranded rna virus. ndvs have been engineered to express checkpoint inhibitor molecules and checkpoint inhibitor-cytokine conjugates (277) . although the antitumor activity of i.t. ndv expressing il-12 alone was not tested, 5 i.t. injections of ndv-anti-cd28-mil-12 plus i.p. anti-ctla-4 or ndv-antipdl1-mil-12 plus i.p. anti-ctla-4 resulting in complete responses in 77 or 70%, respectively of mice bearing 9 day-old b16f10 flank tumors (277) . the same treatments of one of two bilateral tumors resulted in 10 and 62% complete responses, respectively, of untreated tumors (277) . a major benefit of virus-based il-12 delivery is high transfection rates, particularly when compared to non-viral vectors. however, virus-based delivery is limited in several aspects. first, neutralizing immune responses including anti-viral antibody production (271, 278, 279) and dth responses (223) may preclude repeated injections with the same vector. however, the extent to which anti-viral immunity limits repeated injections is unclear. mice previously exposed to a viral vector demonstrated a 2.4-fold reduction in intratumoral transgene expression. this level of reduction did not affect antitumor activity; however, the potential exists for reduced antitumor immune responses when lower doses of virus are used (280) . on the positive side, viral dissemination and transgene expression in non-targeted liver tissue was reduced by more than 1,000-fold in mice pre-exposed to the viral vector (280) . secondly, viral il-12 delivery requires transfection of host cells, which has been shown to be highly heterogeneous due to inherent susceptibility or previous exposure to related vectors (223) . in an extreme example of susceptibility, a dose of adcmvmil-12 that was found to be lethal in 100% of treated c57bl/6 mice did not result in a single death in the balb/c strain (281) . livers of c57bl/6 mice exhibited much higher levels of adenovirus transduction (281) . the current coronavirus outbreak further illustrates the point that some individuals appear to be more susceptible than others to viral infections. third, although clinical trials have shown that recombinant viruses are tolerated, viral dissemination, and infection in liver cells following i.t. injection were reported at an alarming degree (282) (283) (284) . even when small volumes (10-20 µl) of recombinant adenoviruses were injected i.t., transgene expression was found in the liver, intestine, spleen, kidney, and brain. as much as 34% of the injected adv infected non-targeted normal tissues (284) . similar levels of transgene expression was found in the liver and tumor 3 days after an i.t. injection (280) . fourth, some viral vectors, such as momlv and mev, can insert their genetic information into an infected cell's genome causing undesirable mutations and uncontrolled longterm expression of il-12. the benefits of using such integrating vectors must be weighed against these risks as other viruses, such as adv, hsv, vsv, and ndv, are non-integrating and induce transient expression (285) . lastly, and most relevant to the topic of localizing il-12, even local injections of viruses encoding il-12 are not able to prevent il-12 dissemination (47, 202, 282, 286, 287) . consequently, antitumor responses and il-12-mediated adverse events are strongly correlated with virus dose. similar to plasmidbased il-12 delivery, novel strategies that anchor il-12 to an injection site or prevent its secretion appear best suited to break this correlation and widen the therapeutic window of virusencoding il-12. dendritic cells (288, 289) , fibroblasts (290, 291) , macrophages (292, 293) , tumor cells (294, 295) , and mesenchymal stem cells (mscs) (296) (297) (298) have all been engineered to express il-12. transduced dcs, fibroblasts, tumor cells, and macrophages have been directly injected into tumors, while mscs have been injected systemically for migration to tumor beds (296, 297) . a much larger number of preclinical and clinical studies have used il-12-transduced tumor cells for s.c. immunization. these studies are not covered in this review as il-12 in this context is functioning as a vaccine adjuvant following injection of transduced cells in healthy, non-cancerous s.c. tissue rather than a strategy to localize il-12 to a tumor. dendritic cells and macrophages have been engineered to express a variety of cytokines including il-12. in addition to supplying the tumor microenvironment with il-12, transduction of these antigen presenting cells increases their ability to induce robust antitumor immune responses. following i.t. injection, dcs and macrophages are capable of capturing tumor antigen, migrating to draining lymph nodes, presenting antigen and priming tumorspecific t cell responses. transfection of antigen presenting cells with il-12 helps polarize t cells to a th1 phenotype and facilitate robust tumor-specific ctl responses (288) . in an early preclinical study, bone marrow-derived dendritic cells (bmdcs) expressing il-12 were more effective than dcs expressing il-2 in controlling the growth of b16f10 melanomas and generating tumor-specific ctl activity (299) . il-12-transduced bmdcs were also found to significantly suppress established mca205 fibrosarcomas, b16 melanomas, d122 adenocarcinomas, and 178-2 bma prostate carcinomas following a single i.t. injection (288, 300, 301) . in one study, 36% of treated mca205 tumors were completely rejected. tumor-free mice also rejected a subsequent mca205 rechallenge (288) . the growth of contralateral, non-treated tumors was also suppressed, implying that systemic tumor-specific immunity can be induced following i.t. injection. il-12-transduced bmdcs were substantially more effective than il-12-transduced fibroblasts or non-transduced bmdcs at inducing tumor-specific ctl activity and ifnγ production by draining lymph node cells and splenocytes (288) . enhanced trafficking of dcs to regional lymph nodes provides a major advantage over non-migratory cells such as il-12transduced tumor cells or fibroblasts. dcs transfected with adenovirus to express il-12 (dc-adcmvil-12) were found to eliminate ct26 and mc38 colon adenocarcinomas in 67-84% and 50% of mice, respectively (289, 302) . mice experiencing complete regression exhibited tumor-specific ctl activity and rejected tumor rechallenge. i.t. injections of adcmvil-12-infected fibroblasts or allogeneic dc-adcmvil-12 were not effective (289) . in a similar study, dc-adcmvil-12 reversed immune suppression mediated by tbj neuroblastomas and induced complete tumor regression (303) . dcs engineered to co-express both il-12 and il-18 displayed synergistic antitumor activity that was greater than either dcencoding cytokine alone (304) . all mice experienced complete regression of cms4 tumors while treated metha tumors were substantially reduced (304) . while serum ifn-γ levels reached 400 pg/ml 2 days after dc administration, no toxicities were noted (304) . bmdcs transfected by simian virus 40 (sv40) to express il-12 or il-15 demonstrated complete regression of ct26 colon adenocarcinomas in 40 or 73% of mice, respectively (305) . there was no synergy between il-12 and il-15 when delivered together. interestingly, maturation of dcs with lps prior to i.t. injection impaired antitumor activity, presumably due to reduced antigen loading in the tumor. however, this hypothesis was not evaluated (305) . transfection of bmdcs isolated from cms4 sarcoma-bearing mice with an adenoviral vector expressing il-12 was shown to restore antigen-presenting and allostimulatory functions (306) . direct injections of the rescued bmdcs were shown to eliminate intrahepatic cms4 tumors and induce protective immunity (306) . dcs can also be isolated from mouse spleens prior to transduction with an il-12 encoding vector. a recent study demonstrated that splenic dcs overexpressing il-12 can inhibit the growth of melanomas in mice (307) . il-12-transduced macrophages are less well-studied, however, like dcs, macrophages have antigen presenting capabilities that can be enhanced by il-12. a single i.t. injection of peritoneal exudate-derived macrophages transduced by admil-12 induced suppression of primary and metastatic 178-2 bma orthotopic prostate tumors (293) . serum il-12 levels peaked 3 days after injection and remained elevated for at least 2 weeks. antitumor activity was correlated with increased leukocytic infiltrate and cytolytic activity (293) . the admil-12-transduced macrophages were found to migrate to draining lymph nodes following i.t. injection. similar to some viral constructs, to reduce the potential for toxicity, il-12 expression can be placed under the control of an inducible promoter. one inducible system consists of 2 cassettes including gal4-ecr;vp16-rxr and il-12. first, gal4-ecr and vp16-rxr are expressed under the constitutive ubiquitin c promoter. the heterodimerization of gal4-ecr and vp16-rxr are triggered by addition of small-molecule ligand, e.g., pharmacologically inert diacylhydrazine. gal4-ecr-vp16-rxr heterodimers bind to a synthetic promoter containing gal4 binding site and induce the expression of il-12 (308) . dcs with inducible il-12 demonstrated inhibition of renca and metha tumors following i.t. administration (309) . combining il-12expressing dcs with either il-21-or ifnα expressing dcs did not improve antitumor efficacy (309) . the ability to turn cytokine expression "on" and "off " is an attractive feature which may potentially allow for better control over cytokine delivery. to date, only one clinical study has been published. in a phase i study, autologous dendritic cells transfected to express il-12 were administered i.t. to patients with metastatic gastrointestinal carcinomas (310) . of the 17 treated patients, one experienced a partial response and two experienced stable disease. serum ifnγ, but not il-12, levels were elevated following treatment. four instances of transient grade 3 lymphopenia were observed, but no grade 4 toxicities were observed. in vitro il-12 production was highly variable, most likely due to differences in susceptibility of dendritic cell infection with adenoviruses encoding il-12 (310) . fibroblasts expressing il-12 caused the elimination of 7-day mca207 tumors in 70-100% of mice after a single peritumoral injection (291) . in addition, local il-12-mediated regression was also able to control tumors on the contralateral flank. as has been shown in other systems, i.t. injections were more effective than distant s.c. injections at controlling tumor growth. repeated injections of il-12-engineered fibroblasts were welltolerated, with no abnormalities detected in liver and renal function tests (291) . a similar peritumoral implantation of alginate encapsulated nih3t3 fibroblasts expressing il-12 was found to inhibit ct26 tumor growth (311) . in a phase i study, peritumoral injections of il-12-transduced autologous fibroblasts induced transient tumor reductions in four of nine patients with disseminated cancers (312) . treatment with fibroblasts capable of secreting up to 5,000 ng of il-12 per day was well-tolerated (312) . mesenchymal stem cells (mscs) are multipotent adult stem cells that divide and differentiate in order to repair skeletal tissues, such as bone, cartilage, and muscle. as such, mscs are predisposed to traffic from bone marrow to a wound site based on expression of soluble factors and unique receptors in damaged tissues. tumors can express many of the same factors and receptors which results in mscs trafficking to, and infiltrating tumors following systemic injections (313) . thus, using il-12 transduced mscs as a "trojan horse" to target tumors and deliver il-12 is a promising approach. in one preclinical study, ad.il-12-transduced mscs inhibited the growth of tc71 human ewing sarcomas in mice (314) . one potential concern about il-12-expressing mscs (msc/il-12) is their tropism for some normal tissues. in the above study, msc/il-12 were found in tumor, liver, lung and spleen tissues but not kidney, heart, or brain tissues 10 days after i.v. injection (314) . in a different study, high levels of msc/il-12 were found in the tumor but not in the liver, lung, and spleen 20 days after administration (297) . earlier time points were not presented. il-12 levels were high in the tumor; however, significant serum il-12 levels were found to persist for at least 2 weeks after msc/il-12 administration (297) . these high levels of intratumoral il-12 resulted in the inhibition of 786-0 renal cell carcinomas in nude mice (297) . in a study that illustrates the importance of tropism, mscs that were non-virally transfected with pil-12 significantly inhibited b16f10 lung metastases but not subcutaneous b16f10 tumors following i.v. administration (315) . only when administered i.t., were msc/il-12 cells able to inhibit s.c. b16f10 tumors (315) . despite the tumor-homing potential of mscs, published data suggest that msc/il-12 may be most effective at treating tumors in tissues where mscs have a natural tropism, e.g., lung, liver and bone (315) . several other studies have favored local over systemic administration of msc/il-12 to treat various tumors. intracranial gl26 glioma-bearing mice treated with i.t. ucb-msc-il12m generated from human umbilical cord blood experienced significantly prolonged survival (316) . specifically, 70% of ucb-msc-il12m treated mice survived more than 90 days post tumor implantation whereas all mice treated with mscs expressing gfp succumbed within 65 days (316) . cured mice were completely protected from ipsilateral and contralateral tumor rechallenge (316) . in another study, bone marrow derived mscs were transfected with a lentivirus to express mil-12 (317) . these lenti-mil-12-mscs reduced the volume of h22 and metha ascites and increased survival when administered i.p. 2 and 7 days after tumor inoculation (317) . no pathological abnormalities were noted on biopsies taken from internal organs of lenti-mil-12-msc treated mice (317) . human mscs transduced with a retroviral vector expressing a modified il-12 (mscs/il-12m) inhibited b16f10 melanomas following five i.t. injections over 3 weeks (298) . safety was implied as no il-12 was detected in the serum following treatment. interestingly, the antitumor activities elicited by i.t. injections of mscs/il-12m and il-12 plasmid dna were similar (298) . in a rare comparison of cytokine delivery technologies, i.t. injections of mscs transfected with rad/il-12m inhibited b16f10 primary and metastatic tumors more effectively than i.t. injections of rad/il-12m alone (318) . despite the homing feature tsa mammary carcinoma cells transduced to secrete il-12 were found to cure 10 of 40 mice bearing 1-day established tsa tumors following local injection (294) . when tumors were allowed to establish for seven days prior to treatment, antitumor efficacy dropped, with only 2 of 40 mice experiencing tumor regression. local delivery of 9l gliosarcoma cells engineered to express il-12 (9l-il12) significantly prolonged the survival of rats challenged intracranially with wild-type 9l tumors (295) . similar results were observed whether the 9l-il12 treatment was given at the same time or 5 days later than the tumor inoculation. the authors noted that only mice receiving the delayed treatment were protected from tumor rechallenge (295) . natural killer (nk) cells and chimeric antigen receptor (car) t cells have also been transduced to produce il-12. in one recent study, primary nk cells isolated from c57bl/6 mice and transduced to express il-12 (nk il−12 ) increased mhc class i expression in ova-transfected melanoma cells (b16m05) compared to mock plasmid transduced nk cells (319) . combination treatment studies involving tumor-specific cytotoxic t lymphocytes (ctls), pegylated il-2, and activated nk il−12 revealed that only groups containing activated nk il−12 demonstrated statistically significant prolonged survival over mock constructs, untreated, and ctl alone controls in b16-ova tumor bearing mouse models. in particular, nk il−12 plus il-2 treatment increased survival by 11.5 days, while nk il−12 plus ctl and il-2 prolonged survival for 16.5 days. nevertheless, all mice eventually succumbed to the disease and no stable tumor regressions were reported (319) . car t cells have shown remarkable efficacy against hematologic malignancies but have yet to achieve clinical benefit against solid tumors. engineering car-t cells to express il-12 is under investigation to improve antitumor efficacy against solid tumors. in one study, i.v. infusion of il-12 expressing anti-vegfr2 car t cells induced curative regressions in 40-80% of mice in five different tumor models including 10-12-day-old b16f10 melanomas, mca-205 sarcomas, mc38 colorectal adenocarcinomas, mc17-51 sarcomas or 12-14 days old ct26 colon adenocarcinomas (320) . t cells singularly transduced with either anti-vegfr-2 or il-12 were markedly less effective (320) . because constitutive systemic expression of il-12 can be toxic, inducible il-12 expression strategies have been developed. by using an nfat-responsive promoter, il-12 could be expressed only after tcr engagement (320) (321) (322) . this modification reduced toxicity but required lymphodepletion to achieve durable tumor regressions (320) . the efficacy of car t cell therapy in general is dependent upon lymphodepletion via chemotherapy or radiotherapy. in an attempt to eliminate the need for lymphodepleting preconditioning, cd19 car t cells expressing il-12 (cd19/il-12) have been investigated (323, 324) . indeed, treatment with cd19/il-12 car t cells significantly enhanced the survival of cd19-el4 tumor-bearing mice vs. treatment with cd19 car t cells (75 vs . 0% survival at day 60 after tumor inoculation) (323) . similarly, second generation cd19/il-12 car t cells produced a 25% long-term survival rate in mice with established a20 b cell lymphomas. without the il-12 insert, all mice treated with cd19 car t cells succumbed to disease (324) . 4h11-28z/il-12 car t cells, which are specific for the muc-16 ecto antigen and secrete il-12, controlled skov3 human ovarian carcinomas in scid-beige mice following i.p. infusion 2 or 4 weeks after tumor implantation (325) . specifically, ∼80 or 100% of mice treated 4 or 2 weeks, respectively, after tumor implantation with 4h11-28z/il-12 car t cells survived for 90 days. in contrast, ∼0% and 20% of mice treated 4 or 2 weeks, respectively, after tumor implantation with 4h11-28z car t cells, not containing the il-12 gene, survived for 90 days (325). glypican-3 (gpc3)-specific car t cells with inducible expression of il-12 (gpc3-28z-nfat-il-12 car t) significantly inhibited or eliminated established plc/prf/5 and huh-7 human hccs (326) . the il-12 secreting car t cells resulted in decreased t reg infiltration. serum ifnγ and il-12 levels were elevated although neither pathological changes to critical organs nor significant loss in body weight were observed (326) . it should be noted, however, that these studies were performed in immunocompromised mice which is not a reliable model for immune-related adverse events. from a clinical standpoint, administrations of il-12-transduced cells have been well-tolerated, with patients experiencing only transient symptoms of lymphopenia, fever, and malaise; however, significant, durable clinical responses have been elusive. from a manufacturing perspective, the transduction of allogeneic and xenogeneic cells is easier and more reproducible. however, these cells are rapidly recognized and rejected by the host immune system. thankfully, recent advances in autologous cell transduction due to the emergence of car t cell immunotherapy has eliminated many concerns related to technical feasibility and patient heterogeneity (310) . local administration of recombinant il-12 protein is the most direct and most quantifiable strategy in terms of ensuring the accuracy and reproducibility of a delivered dose. however, recombinant cytokines disseminate rapidly from local injection sites into the systemic circulation (327) . thus, in order to maintain high levels of recombinant il-12 in the tumor microenvironment while minimizing systemic exposure, sustained delivery technologies, capable of locally releasing proteins and cytokines over extended periods of time following direct injection, are under development (328) . the encapsulation of pharmaceuticals in polymeric microspheres for sustained delivery has been explored for several decades (329, 330) . the use of microspheres to deliver cytokines is a more recent trend. synthetic polymers, such as poly-lactic coglycolic acid (plga), poly-caprolactone (pcl), and poly-lactic acid (pla), that have been widely explored for drug delivery, are being adapted to encapsulate and deliver various cytokines including il-12 (55, (331) (332) (333) (334) (335) . human il-12-loaded pcl:pla microspheres, when co-delivered with human peripheral blood lymphocytes (pbls), were shown to prevent engraftment of human tumors in scid mice (333) . peg-il-2 also suppressed tumor engraftment and growth, but not as effectively as il-12-loaded microspheres. although high levels of il-12 and ifn-γ were found in sera after administration of il-12-loaded microspheres, this localized delivery strategy was more effective at preventing tumor engraftment than repeated i.p. injections of il-12 (336) . similarly, a single i.t. injection of il-12-loaded pcl:pla microspheres outperformed bolus injections of il-12 in the inhibition of human head and neck tumor tissue xenografts containing human pbls (337) . il-12-loaded microspheres completely suppressed engraftment in 50% of mice, whereas il-12 alone slowed but could not eliminate tumor growth (337). in the above xenograft studies, antitumor activity was mediated by cd8 + t cells and cd56 + natural killer cells (333, 337) . in yet another study, lung tumor xenografts using non-disrupted human lung tumor tissue were completely suppressed when injected with il-12-loaded pla microspheres 1 week after implantation (338) . mechanistically, quiescent cd4 + effector memory t cells present within the tumor microenvironment were reactivated upon exposure to high local levels of il-12 (339) . the proliferation of reactivated cd4 + cells and their production of ifn-γ facilitated tumor destruction (339) . in murine tumor models, i.t. injections of il-12-loaded pla microspheres eliminated up to 70% of line-1 lung alveolar carcinomas (332) . in contrast, i.t. injections of free il-12 induced a complete response in only one of five treated mice. interestingly, mice experiencing complete tumor regression following microsphere administration were more resistant to tumor rechallenge than mice vaccinated with either live or irradiated line-1 cells co-injected with il-12-loaded microspheres (332) . in other studies, i.t. injections of il-12loaded pla microspheres significantly reduced the incidence and number of spontaneous pulmonary tumor nodules (331, 340) . when administered neoadjuvant to tumor resection, il-12-loaded microspheres inhibited local recurrence, reduced pulmonary metastases, and improved disease-free survival as compared to resection alone (331) . a similar study using more advanced line-1 tumors, which were 1,000-1,300 mm 3 at study onset, demonstrated that neoadjuvant i.t. injections of il-12-loaded microspheres prevented both local recurrence and pulmonary metastases better than surgery alone (341) . the addition of gm-csf-loaded pla microspheres enhanced antitumor activity and survival. however, more cytokines were not always better, as the addition of low dose il-2 to neoadjuvant immunotherapy with il-12-and gm-csf-loaded microspheres abrogated antitumor immunity (341) . a theme common to nearly all il-12-based immunotherapies was that il-12-loaded microspheres induced cd8 + t cell and nk cell activation, including increases in cytolytic function and ifn-γ expression. in-depth effector mechanisms are elegantly described in a series of papers by egilmez and kilinc et al. (69, 340, (342) (343) (344) (345) . in addition to inducing a strong effector response, a potentially more important feature of i.t. injected il-12-loaded microspheres is their potential to reverse immunosuppression in the tumor microenvironment. for example, a single i.t. injection of il-12-loaded microspheres with and without gm-csf-loaded microspheres induced apoptosis and elimination of tumor-infiltrating cd4 + cd25 + foxp3 + t suppressor cells (69, 344) . il-12-loaded microspheres were also shown to reprogram immunosuppressive tumor-infiltrating macrophages (tims) and tumor-associated macrophages (tams) to a proinflammatory, antitumor phenotype (11, 346) . microsphere platforms are well-suited to explore codelivery of multiple cytokines. the combination of il-12-and tnfα-loaded pla microspheres was shown to be more effective than il-12 and gm-csf-loaded microspheres at eliminating mt-901 mammary carcinomas (347) and b16 melanomas (55) . nearly 70 and 40% of mt-901 tumor-bearing mice were rendered tumor-free following i.t. injections with dual cytokine loaded microspheres of il-12/tnfα and il-12/gm-csf, respectively (347) . dual cytokine loaded microspheres of il-12/tnfα also induced increases in tumor-specific cytokine release from effector cells as compared to microspheres containing either cytokine alone (334) or the combination of il-12 and gm-csf (55, 347) . a single i.t. injection of il-12-and il-18-loaded pla microspheres outperformed injections of either cytokine-loaded microsphere alone in terms of delaying the growth of b16 melanomas (348) . unlike previous studies demonstrating the elimination of 70% of line-1 tumors (332) and impressive reductions in metastatic lesions (331, 341) , il-12-loaded microspheres had no impact on either primary b16 tumor growth or pulmonary metastases (348) . interestingly, the triple combination of il-18/il-12/tnfα loaded in microspheres was found to reduce antitumor activity in the 4t1 model (335) . in her-2/neu transgenic mice, which develop spontaneous, multifocal mammary carcinomas, il-12-and gm-csf-loaded microspheres injected intratumorally caused complete regression of primary tumors in up to 40% of mice (349) . unfortunately, responses were temporary, and all tumors recurred. interestingly, chronic immunotherapy with cytokine-loaded microspheres could not control tumor burden due to a progressive decline in the intensity of antitumor t cells and an increase in tumor-infiltrating cd4 + cd25 + foxp3 + t suppressor cells with repeated injections (349, 350) . subsequent studies have shown that administration of cyclophosphamide can block the postimmunotherapy increase in t suppressor cells (351, 352) . recently, intratumoral delivery of il-12 microspheres (il-12 ms) in combination with stereotactic body radiation (sbrt) resulted in remarkable tumor regression in several types of preclinical pancreatic ductal adenocarcinoma mouse models (353). this study showed that intratumoral levels of ifnγ were enhanced following the treatment of sbrt/il-12 ms, which in turn increased cd8 + t cell activation. sbrt/il-12 ms treatments also established systemic tumor immunity that was confirmed by antitumor effects on liver metastases (353). chitosan (cs) is a bioadhesive polysaccharide derived primarily from the exoskeletons of crustaceans. it is nontoxic, biodegradable, and non-immunoreactive (354) with an established safety profile in humans. co-formulation of il-12 in cs (cs/il-12) has been shown to increase the i.t. residence of il-12 from 1-2 days to 1-2 weeks (23) . cs is believed to hinder il-12 diffusion through viscous and electrostatic interactions. the sustained presence of cs/il-12 induced complete regression of 80-100% of mice bearing mc38, panc02, ct26, e0771, and mb49 tumors (23, 355) . administration of cs/il-12 neoadjuvant to 4t1 tumor resection resulted in complete clearance of lung metastases and long-term survival in about two-thirds of mice. in contrast, all mice treated with surgery alone died within 38 days and mice receiving il-12 without cs neoadjuvant to resection had a median survival of 46 days (52). cured mice rejected a subsequent challenge with live 4t1 cells, but not live renca cells illustrating the specificity of the antitumor immune response. in another demonstration of systemic tumorspecific immunity from localized il-12, 70% of mice bearing both flank and orthotopic mb49 bladder tumors were able to clear the flank tumor following intravesical administration of cs/il-12 (53) . in contrast, mice with only a flank tumor, did not benefit from intravesical cs/il-12 in a naïve bladder. these data indicate that intravesical cs/il-12 induced systemic tumor-specific immunity only when il-12 was localized in or near a tumor. in a subsequent study, orthotopic bladder tumor regression following intravesical cs/il-12 immunotherapy was associated with a rapid infiltration of macrophages and granulocytes followed by increases in cd4 + and cd8 + effector t cells along with a reduction of immunosuppressive t regs and mdscs (14) . importantly, local administrations of cs/il-12 did not result in significant toxicity, such as elevated liver enzymes, commonly observed following regular, free il-12 injections (52) . a polysaccharide gel matrix, designated f2 gel, is comprised of 70% deacetylated poly-n-acetyl glucosamine (p-glcnac) coformulated with lactate salt. p-glcnac gels were evaluated as slow-release cytokine depots for gm-csf, il-2, and il-12 (356) (357) (358) (359) (360) . in particular, i.t. injections of il-2 in p-glcnac gel delayed the growth of ova-transfected ae17 malignant mesotheliomas (359) . interestingly, gm-csf and il-12 depots had no impact on mesothelioma growth, although only a single i.t. injection was given. the p-glcnac gel by itself induced a strong, transient inflammatory response which was thought to be due to a foreign body reaction or toxic species that may have been contained within the gel (361, 362) . human il-12 has been encapsulated in multilamellar liposomes for sustained release after i.t. injection (363) . antitumor activity on xenografts of human lung tissues indicated that liposomal encapsulation is a promising approach capable of eliminating tumor cells and inducing lymphocyte infiltration 2 weeks after i.t. injection. liposomal leakage and/or il-12 liposome dissemination could be problematic following delivery of large doses (10 µg/mouse) as significant sustained levels of il-12 and ifn-γ were found in sera of treated mice (363) . capitalizing on the tumor disruption caused by cationic liposomes, il-12-and tlr4 ligand monophosphoryl lipid a (mpl)-loaded cationic liposomes were injected i.t. to treat 4t1 tumors. this combination produced an abscopal effect which arrested growth in both treated and distal tumors but did not result in tumor elimination (364) . to date, sustained release technologies for recombinant il-12 have not been evaluated in clinical studies. while sustained release technologies are designed to maximize il-12 concentrations in the tumor microenvironment, some amount of delivered il-12 can be expected to reach the systemic circulation. however, due to the infrequency of administration for these long-acting platforms, they are unlikely to induce the same lethal toxicities that were observed following the frequent systemic il-12 administrations in early clinical trials (42) . nevertheless, it will be essential to perform rigorous pharmacokinetic and toxicology studies prior to clinical translation of any sustained recombinant il-12 platform. a possible limitation of the microsphere encapsulation technology is the use of organic solvents during the encapsulation process. organic solvents can denature cytokines immediately or adversely affect long term storage. one study indicated that cytokines lose about half of their bioactivity during microencapsulation (365) . loss of bioactivity appears to be cytokine dependent (366) . specific activity of il-12 after encapsulation in pla microspheres was 90%. however, more than 80% of the bioactivity of il-12 was lost when pla/il-12 microspheres were stored for 8 days (366) . in addition, because many commonly used biodegradable polymers are comprise of acidic monomers, polymer degradation often forms highly acidic microenvironments that may inactivate cytokines (188) . a number of techniques are under investigation to either raise ph (367) or stabilize encapsulated cytokines (368) . a common limitation of liposomal carriers is their inherent leakiness, and therefore the choice of lipid and modifications to stabilize the bilayer are critical for sustained release. high temperature phase-transition lipids, such as dspc, increase the retention of cargo and enhance drug circulation time. incorporation of cholesterol into the lipid bilayer increases the rigidity and further decreases leakage of interior cargo (369) . another major limitation of liposomes is opsonization by serum proteins and clearance by phagocytes of the reticuloendothelial system (res). pegylation can reduced res clearance, however, peg can sterically inhibit ligand-cell interaction (370) . if administered i.t., loss of tumor targeting is likely not detrimental. while some localized il-12 immunotherapies have shown limited efficacy in clinical studies, a long-awaited breakthrough leading to widespread application of localized il-12 as a monotherapy does not appear imminent. however, there are a number of near-term opportunities to combine localized il-12 with traditional or experimental cancer management approaches. and in fact, most contemporary experimental therapies for aggressive or advanced cancers involve combination approaches. thus, near future opportunities involving translatable combination approaches are explored in the following sections. the majority of cancer patients with solid tumors undergo tumor resection as part of their treatment. surgery, by itself, is unable to prevent recurrence and/or metastasis which is responsible for 9 out of 10 cancer-related deaths. patients at high risk of recurrence receive adjuvant chemotherapy and/or radiotherapy following resection. while chemotherapy and radiotherapy may help reduce the risk of recurrence, they induce life-altering side effects and fail in a significant fraction of patients. as a result, most metastatic cancers arise from previously treated non-metastatic disease (371) (372) (373) . administration of localized il-12 to the tumor microenvironment prior to surgery has the potential to reduce recurrence rates and/or eliminate occult metastases through the induction of systemic, tumor-specific immunity. as mentioned above, our own investigation demonstrated that neoadjuvant i.t. injections of cs/il-12 prior to resection can improve overall survival from 0 to 65% in a highly metastatic 4t1 breast carcinoma model (52) . cured mice demonstrated tumor-specific immunity via tumor rechallenge, ctl activity, and elispot assays. if proven to be safe, localized il-12 can be administered neoadjuvant to any solid tumor resection, not just in high risk patients. in fact, at the time of surgery, it is likely that a significant fraction of patients already has occult metastases that cannot be detected via radiographic imaging. the median primary breast tumor size for which 100% of patients had micrometastases was found to be only 1.8 cm (374) . while most immune-oncology trials focus on developing therapies for metastatic disease, an approach, such as neoadjuvant localized il-12, capable of preventing metastasis in the first place, may be a more effective strategy to reducing cancer mortality. the combination of il-12-based immunotherapy and chemotherapy ("biochemotherapy") is also under exploration. several clinical studies have shown significant clinical benefit when certain cytotoxic drugs are combined with il-12 (181, 375, 376) . there are three distinct advantages for combining chemotherapy with il-12 immunotherapy. first, chemotherapy provides a debulking benefit with the elimination of some or most of the tumor. second, some chemotherapeutics can deplete immunosuppressive cells. cyclophosphamide has been shown to deplete t regs while 5-fluorouricil and gemcitabine have been shown to deplete mdscs. third, some chemotherapies cause upregulation of t cell-attracting chemokines in the tumor (377) . for example, melanoma-bearing mice treated with temozolomide, cisplatin, or dacarbazine increase expression of ccl5, cxcl9, and cxcl10 (378) . the timing of chemotherapy relative to local il-12 immunotherapy is expected to be critical. unlike the neoadjuvant resection approach mentioned above, chemotherapy should be administered prior to il-12 therapy to avoid elimination of beneficial proliferating immune cells. chemotherapy may synergize with il-12 by reducing tumor burden and creating a source of tumor antigen for in situ vaccination. in fact, certain chemotherapies can induce an immunogenic cell death that is capable of priming an antitumor immune response (379) . to date, several examples demonstrate the promise of chemotherapy plus local il-12 delivery. antitumor efficacy of pil-12/ppc is improved with paclitaxel, cyclophosphamide, or taxol/paraplatin (172, 175, 177) . the antitumor effect of il-12 lipoplexes against intracranial glioma was substantially improved when combined with fda-approved carmustine (bcnu) wafers (176) . finally, a phase-1 clinical trial found that intraperitoneal il-12 gene therapy in combination with intravenous pegylated liposomal doxorubicin resulted in greater clinical benefit relative to the chemotherapeutic alone (57.1% vs. 40-45.3%) in patients (n = 11) with platinum-resistant epithelial ovarian cancer (181) . standard-of-care radiotherapy of inoperable tumors can be administered prior to local il-12 delivery. radiation-induced tumor cell death can supply a source of tumor antigens, while radiation has been shown to upregulate co-stimulatory molecules on the surface of tumor cells which makes them more susceptible to immune-mediated killing (380) . the addition of tumor irradiation after pil-12+ep was found to improve antitumor activity (130) . similarly, radiotherapy improved the antitumor activity of i.t. oncolytic adenoviruses expressing il-12 and gm-csf (381) . it has also been found that radiotherapy can induce infiltration of mscs in the tumor site. through a combination of radiation and il-12-expressing mscs, both primary tumor growth and metastases were reduced in hca-i and hepa 1-6 hepatoma models (382) . in another study, radiation along with an i.t. injection of an il-12-encoding adenoviral vector greatly reduced tumor growth and metastasis compared to either treatment alone in a bnl-p2 hepatocellular carcinoma model, while a near-complete abscopal response occurred in a ct26 colorectal cancer model (383) . an abscopal response was also induced in a fully humanized rhabdomyosarcoma xenograft model. mice with bilateral tumors were treated with a combination of single-tumor radiation and systemic nhs-il12, which resulted in significantly lower tumor burdens than either individual treatment (114) . cryoablation, radiofrequency ablation (rfa), microwave ablation (mwa), embolic microspheres and high intensity focused ultrasound (hifu) are routinely used in the clinic to destroy some operable and many inoperable solid tumors. irreversible electroporation is another ablative technology in clinical and preclinical development (384) (385) (386) (387) . all of these strategies have the potential to induce in situ vaccinations through the release of tumor antigen, although abscopal responses or robust anti-tumor immune responses are rare. among the ablation strategies, cryoablation appears distinct from most other ablation approaches that kill via high temperatures and result in coagulation, enzyme inactivation, and antigen denaturation. cryoablation is thought to mostly preserve tumor antigen structure (388, 389) and has been shown to induce greater levels of tumor antigen uptake by dendritic cells compared to rfa (390) . in addition, cryoablation also induces higher levels of inflammatory cytokines, such as il 1, il 6, and tnfα, compared to rfa and mwa (391, 392) . perhaps most importantly, cryoablation was recently shown to induce expansion of oligoclonal t cells both in tumor tissues and in peripheral blood of kidney cancer patients (393) . the addition of localized il-12 to the above ablation strategies may enhance tumor control as well as antitumor immunity. our recent preclinical data, demonstrated that the combination of cryoablation and cs/il-12 could eliminate multiple tumor types and induce abscopal immunity in a bilateral flank model (394) . ongoing studies are aimed at determining mechanisms of systemic tumor control. immune checkpoint inhibitors are ineffective against noninflamed, "cold" tumors. localized il-12 induces profound intratumoral infiltration that can be expected to synergize with checkpoint inhibitors. indeed, the addition of anti-pd-1 or anti-ctla-4 improved the antitumor activity of l19-il-12 in multiple murine tumor models (395) . similarly, systemic anti-ctla-4 in combination with intratumoral il-12 induced synergistic antitumor responses in gl-26 glioblastoma and sma-560 astrocytoma models (396) . also, as mentioned in a previous section, i.t. injections of vv-il-12 and vv-il7 enhanced the responsiveness of poorly immunogenic tumors to checkpoint inhibition (268) . finally, a recent phase ii study of i.t. pil-12+ep and systemic pembrolizumab in patients with non-infiltrated melanomas resulted in a 41% objective response rate with 36% complete responses (397) . correlative studies demonstrated increased antigen cross-presentation along with enhance t cell infiltration and activity leading to higher than expected clinical responses (397) . il-12 enhances cytotoxic t and nk cell activity while reversing tumor-induced immunosuppression, inhibiting angiogenesis, increasing lymphocyte trafficking and antigen presentation either directly or through induction of ifnγ (398) . based on these diverse mechanisms as well as its profound antitumor activity in numerous preclinical studies, il-12 is arguably one of, if not, the most potent antitumor cytokine evaluated to date. unfortunately, the much-anticipated clinical translation of il-12-based immunotherapies suffered a tremendous setback in the late 1990s/early 2000s due to severe clinical toxicities associated with systemic il-12 injections. through the development of several clever approaches to localize il-12 to the tumor microenvironment while limiting systemic exposure, il-12-based immunotherapies are making a comeback. several clinical studies evaluating localized il-12 have been initiated ( table 2 ) with more on the way. while each delivery strategy has limitations, the approaches reviewed above may retain enough il-12 in the tumor while eliminating the need for frequent systemic injections. by reducing the potential for il-12-mediated toxicities associated with systemic injections, localized il-12 can expand the therapeutic window and finally allow il-12 to fulfill its considerable potential. as exemplified by the current immune-oncology clinical trial landscape, combination approaches appear to be most effective for accelerating clinical impact. several promising preclinical combination approaches with localized il-12 are likely to be translated in the near future. there is unprecedented interest in finding immunomodulators that can enhance lymphocytic infiltration in order to improve the efficacy of checkpoint inhibitors. localized il-12, based on its ability to drive th1 responses, enhance cytolytic activity, and protect t cells from pd-1/pd-l1 exhaustion and ifnγ-induced apoptosis may be an ideal partner for checkpoint inhibitors. if safety concerns are assuaged, localized il-12 could be used in earlier stage cancer patients as a neoadjuvant to resection. for tumors that are inoperable, combining localized il-12 with ablation may help increase local as well as distant tumor control. with the interesting combination approaches, as well as the uptick in il-12-based immunotherapies in clinical trials, there is reason to believe that localized il-12 may play a major role in cancer immunotherapy in the near future. studies on the pathogenesis of fever. ii. characterization of fever-producing substances from polymorphonuclear leukocytes and from the fluid of sterile exudates anticancer cytokines: biology and clinical effects of interferon-alpha2, interleukin (il)-2, il-15, il-21, and il-12 melanoma: therapeutic options with recombinant interferons results of treatment of 255 patients with metastatic renal cell carcinoma who received high-dose recombinant interleukin-2 therapy high-dose recombinant interleukin 2 therapy for patients with metastatic melanoma: analysis of 270 patients treated between 1985 and (1993) talimogene laherparepvec improves durable response rate in patients with advanced melanoma coexpression of two distinct genes is required to generate secreted bioactive cytotoxic lymphocyte maturation factor identification and purification of natural killer cell stimulatory factor (nksf), a cytokine with multiple biologic effects on human lymphocytes cloning of cdna for natural killer cell stimulatory factor, a heterodimeric cytokine with multiple biologic effects on t and natural killer cells cloning and expression of murine il-12 il-12 rapidly alters the functional profile of tumor-associated and tumor-infiltrating macrophages in vitro and in vivo the role of interleukin-12 on modulating myeloid-derived suppressor cells, increasing overall survival and reducing metastasis interleukin-12: a master regulator immunological mechanisms of intravesical chitosan/interleukin-12 immunotherapy against murine bladder cancer. oncoimmunology differential effects of il-12 on tregs and non-treg t cells: roles of ifn-gamma, il-2 and il-2r il-12 triggers a programmatic change in dysfunctional myeloidderived cells within mouse tumors antiproliferative effects of four different cytokines on renal carcinoma cell lines ifn-gamma induces apoptosis in ovarian cancer cells in vivo and in vitro inhibition of angiogenesis in vivo by interleukin 12 inhibition of tumor-induced angiogenesis by the administration of recombinant interferon-gamma followed by a synthetic lipid-a subunit analogue (gla-60) cellular responses to interferon-gamma interleukin-12 in anti-tumor immunity and immunotherapy intratumoral immunotherapy of established solid tumors with chitosan/il-12 effect of interleukin 12 on tumor induction by 3-methylcholanthrene therapeutic effect of interleukin 12 on mouse haemangiosarcomas is not associated with an increased antitumour cytotoxic t-lymphocyte activity the anti-tumor activity of il-12: mechanisms of innate immunity that are model and dose dependent antitumor and antimetastatic activity of interleukin 12 against murine tumors phase i evaluation of intravenous recombinant human interleukin 12 in patients with advanced malignancies pilot study of subcutaneous recombinant human interleukin 12 in metastatic melanoma phase i trial of subcutaneous recombinant human interleukin-12 in patients with advanced renal cell carcinoma interleukin-12 therapy of cutaneous t-cell lymphoma induces lesion regression and cytotoxic t-cell responses phase i study of intraperitoneal recombinant human interleukin 12 in patients with mullerian carcinoma, gastrointestinal primary malignancies, and mesothelioma phase 1 study of the intravesical administration of recombinant human interleukin-12 in patients with recurrent superficial transitional cell carcinoma of the bladder a phase ii trial of interleukin-12 in patients with advanced cervical cancer: clinical and immunologic correlates. eastern cooperative oncology group study e1e96 nk and cd8+ t cell-mediated eradication of poorly immunogenic b16-f10 melanoma by the combined action of il-12 gene therapy and 4-1bb costimulation intratumoral recombinant human interleukin-12 administration in head and neck squamous cell carcinoma patients modifies locoregional lymph node architecture and induces natural killer cell infiltration in the primary tumor activity of subcutaneous interleukin-12 in aids-related kaposi sarcoma phase ii clinical trial of interleukin-12 in patients with relapsed and refractory non-hodgkin's lymphoma and hodgkin's disease peripheral burst of tumor-specific cytotoxic t lymphocytes and infiltration of metastatic lesions by memory cd8+ t cells in melanoma patients receiving interleukin 12 after initial setback, il-12 regaining popularity interleukin 12: still a promising candidate for tumor immunotherapy? effects of single-dose interleukin-12 exposure on interleukin-12-associated toxicity and interferon-gamma production evaluation of recombinant human interleukin-12 in patients with recurrent or refractory ovarian cancer: a gynecologic oncology group study randomized multicenter phase ii trial of subcutaneous recombinant human interleukin-12 versus interferon-alpha 2a for patients with advanced renal cell carcinoma selective ablation of immature blood vessels in established human tumors follows vascular endothelial growth factor withdrawal patterns of vasculature in mouse models of lung cancer are dependent on location direct intratumoral injection of an adenovirus expressing interleukin-12 induces regression and long-lasting immunity that is associated with highly localized expression of interleukin-12 co-delivery of antigen and il-12 by venezuelan equine encephalitis virus replicon particles enhances antigen-specific immune responses and antitumor effects right time and place for il12: targeted delivery stimulates immune therapy new insights into il-12-mediated tumor suppression gene therapy of cancer based on interleukin 12 neoadjuvant immunotherapy with chitosan and interleukin-12 to control breast cancer metastasis intravesical chitosan/interleukin-12 immunotherapy induces tumorspecific systemic immunity against murine bladder cancer characterization of abscopal effects of intratumoral electroporation-mediated il-12 gene therapy neoadjuvant intratumoral cytokine-loaded microspheres are superior to postoperative autologous cellular vaccines in generating systemic anti-tumor immunity repeated administrations of interleukin (il)-12 are associated with persistently elevated plasma levels of il-10 and declining ifn-gamma, tumor necrosis factor-alpha, il-6, and il-8 responses transcriptionally active stat1 is required for the antiproliferative effects of both interferon alpha and interferon gamma ifn-gamma regulates donor cd8 t cell expansion, migration, and leads to apoptosis of cells of a solid tumor ifn-gamma-mediated upmodulation of mhc class i expression activates tumor-specific immune response in a mouse model of prostate cancer ifn-gamma-mediated inhibition of tumor angiogenesis by natural killer t-cell ligand, alpha-galactosylceramide interferon: a cytotoxic t lymphocyte differentiation signal il-2 and ifn-gamma are two necessary lymphokines in the development of cytolytic t cells gamma interferon enhances macrophage transcription of the tumor necrosis factor/cachectin, interleukin 1, and urokinase genes, which are controlled by short-lived repressors interferon and major histocompatibility complex genes: a model to analyse eukaryotic gene regulation? placebo-controlled phase iii trial of patient-specific immunotherapy with mitumprotimut-t and granulocyte-macrophage colony-stimulating factor after rituximab in patients with follicular lymphoma evaluation of ipilimumab in combination with allogeneic pancreatic tumor cells transfected with a gm-csf gene in previously treated pancreatic cancer cancer immunotherapy comes of age cancer immunotherapy strategies based on overcoming barriers within the tumor microenvironment reversing tumor immune suppression with intratumoral il-12: activation of tumor-associated t effector/memory cells, induction of t suppressor apoptosis, and infiltration of cd8+ t effectors use of tumor-infiltrating lymphocytes and interleukin-2 in the immunotherapy of patients with metastatic melanoma. a preliminary report il-12 priming during in vitro antigenic stimulation changes properties of cd8 t cells and increases generation of effector and memory cells synergy of brief activation of cd8 t-cells in the presence of il-12 and adoptive transfer into lymphopenic hosts promotes tumor clearance and anti-tumor memory ex vivo interleukin-12-priming during cd8 + t cell activation dramatically improves adoptive t cell transfer antitumor efficacy in a lymphodepleted host ex vivo conditioning with il-12 protects tumor-infiltrating cd8 + t cells from negative regulation by local ifn-gamma cutting edge: il-12 and type i ifn differentially program cd8 t cells for programmed death 1 re-expression levels and tumor control switching on of the proliferation or apoptosis of activated human t lymphocytes by ifn-gamma is correlated with the differential expression of the alpha-and beta-chains of its receptor clinical trial listed in this table meet the following four criteria: 1) involve a delivery technology; 2) be administered locally, i.e. intratumoral or peritumoral; 3) use il-12 as an effector and 4) demonstrate antitumor activity of il-12. clinical trials listed in this table include: 1) nhs-il12 for solid tumors 14) a first-in-human dose escalation and expansion study to evaluate intratumoral administration of sar441000 as monotherapy and in combination with cemiplimab in patients with advanced solid tumors antibody-il-12 fusion proteins are effective in scid mouse models of prostate and colon carcinoma metastases bifunctional cytokine fusion proteins for gene therapy and antibody-targeted treatment of cancer a single-chain il-12 igg3 antibody fusion protein retains antibody specificity and il-12 bioactivity and demonstrates antitumor activity mechanism of antitumor activity of a single-chain interleukin-12 igg3 antibody fusion protein (mscil-12.her2.igg3) cytokines fused to antibodies and their combinations as therapeutic agents against different peritoneal her2/neu expressing tumors long-term immunity elicited by antibody-cytokine fusion proteins protects against sequential challenge with murine mammary and colon malignancies amino acid residues involved in the heparin-binding activity of murine il-12 in the context of an antibody-cytokine fusion protein modulation of interleukin-12 activity in the presence of heparin molecular mechanisms of heparin-induced modulation of human interleukin 12 bioactivity species-specific differences in heparin-induced modulation of il-12 family cytokines efficient production and purification of recombinant human interleukin-12 (il-12) overexpressed in mammalian cells without affinity tag novel immunocytokine il12-ss1 (fv) inhibits mesothelioma tumor growth in nude mice prognostic value and characterization of the ovarian cancer-specific antigen ca166-9 construction, expression, and function of 6b11scfv-mil-12, a fusion protein that attacks human ovarian carcinoma an il12-il2-antibody fusion protein targeting hodgkin's lymphoma cells potentiates activation of nk and t cells for an anti-tumor attack expression of the extra domain b of fibronectin, a marker of angiogenesis, in head and neck tumors hubc1-il12, an immunocytokine which targets edb-containing oncofetal fibronectin in tumors and tumor vasculature, shows potent anti-tumor activity in human tumor models a phase 1 study of as1409, a novel antibody-cytokine fusion protein, in patients with malignant melanoma or renal cell carcinoma phase i trial of twice-weekly intravenous interleukin 12 in patients with metastatic renal cell cancer or malignant melanoma: ability to maintain ifn-gamma induction is associated with clinical response an engineered antibody-interleukin-12 fusion protein with enhanced tumor vascular targeting properties enhancement of the antitumor properties of interleukin-2 by its targeted delivery to the tumor blood vessel extracellular matrix engineered vascular-targeting antibody-interferon-gamma fusion protein for cancer therapy enhancement of the antitumor activity of interleukin-12 by targeted delivery to neovasculature synergistic therapeutic effects of a tumor targeting antibody fragment, fused to interleukin 12 and to tumor necrosis factor alpha anchoring of intratumorally administered cytokines to collagen safely potentiates systemic cancer immunotherapy collagen-binding il-12 enhances tumour inflammation and drives the complete remission of established immunologically cold mouse tumours a dose-escalation and signal-generating study of the immunocytokine l19-il2 in combination with dacarbazine for the therapy of patients with metastatic melanoma targeted reconstitution of cytokine activity upon antigen binding using split cytokine antibody fusion proteins a new chemically modified chimeric tnt-3 monoclonal antibody directed against dna for the radioimmunotherapy of solid tumors the immunocytokine nhs-il12 as a potential cancer therapeutic characterization of a phage display-derived human monoclonal antibody (nhs76) counterpart to chimeric tnt-1 directed against necrotic regions of solid tumors temporal changes within the (bladder) tumor microenvironment that accompany the therapeutic effects of the immunocytokine nhs-il12 enhanced antitumor effects by combining an il-12/anti-dna fusion protein with avelumab, an anti-pd-l1 antibody combination therapy with nhs-muil12 and avelumab (anti-pd-l1) enhances antitumor efficacy in preclinical cancer models cancer-targeted il-12 controls human rhabdomyosarcoma by senescence induction and myogenic differentiation tumortargeted il-12 combined with local irradiation leads to systemic tumor control via abscopal effects in vivo. oncoimmunology enhanced binding of necrosis-targeting immunocytokine nhs-il12 after local tumour irradiation in murine xenograft models defining the pharmacodynamic profile and therapeutic index of nhs-il12 immunocytokine in dogs with malignant melanoma first-in-human phase i trial of a tumor-targeted cytokine (nhs-il12) in subjects with metastatic solid tumors immunogenicity to biotherapeutics -the role of anti-drug immune complexes tumor regression of human and murine melanoma after intratumoral injection of il-12-encoding plasmid dna in mice in vivo induction of interferon gamma expression in grey horses with metastatic melanoma resulting from direct injection of plasmid dna coding for equine interleukin 12 intratumoral injection of dna encoding human interleukin 12 into patients with metastatic melanoma: clinical efficacy intratumoral injection of il-12 plasmid dna-results of a phase i/ib clinical trial effective tumor therapy with plasmid-encoded cytokines combined with in vivo electroporation il-12 plasmid delivery by in vivo electroporation for the successful treatment of established subcutaneous b16.f10 melanoma il-12 gene therapy using an electrically mediated nonviral approach reduces metastatic growth of melanoma evaluation of toxicity following electrically mediated interleukin-12 gene delivery in a b16 mouse melanoma model regression of tumor growth and induction of long-term antitumor memory by interleukin 12 electro-gene therapy administration route-and immune cell activation-dependent tumor eradication by il12 electrotransfer intratumoral delivery of interleukin 12 expression plasmids with in vivo electroporation is effective for colon and renal cancer radiosensitizing effect of intratumoral interleukin-12 gene electrotransfer in murine sarcoma in vivo electrogene transfer of interleukin-12 inhibits tumor growth and lymph node and lung metastases in mouse mammary carcinomas local and systemic antitumor effect of intratumoral and peritumoral il-12 electrogene therapy on murine sarcoma electroporation-mediated interleukin-12 gene therapy for hepatocellular carcinoma in the mice model improving therapeutic efficacy of il-12 intratumoral gene electrotransfer through novel plasmid design and modified parameters systemic administration of interleukin-12 can restore the antitumor potential of b16 melanoma-draining lymph node cells impaired at a late tumor-bearing state intratumoral electroporation of il-12 cdna eradicates established melanomas by trp2(180-188)-specific cd8+ ctls in a perforin/granzyme-mediated and ifn-gamma-dependent manner: application of trp2(180-188) peptides gene electrotransfer of plasmid-encoding il-12 recruits the m1 macrophages and antigen-presenting cells inducing the eradication of aggressive b16f10 murine melanoma il-12 gene electrotransfer triggers a change in immune response within mouse tumors pre-clinical investigation of the synergy effect of interleukin-12 gene-electro-transfer during partially irreversible electropermeabilization against melanoma in vivo electroporation-mediated transfer of interleukin-12 and interleukin-18 genes induces significant antitumor effects against melanoma in mice combination of il-12 and il-18 of electro-gene therapy synergistically inhibits tumor growth coadministration of interleukin-18 and interleukin-12 induces a fatal inflammatory response in mice: critical role of natural killer cell interferongamma production and stat-mediated signal transduction electrogene therapy with interleukin-12 in canine mast cell tumors electroporation-mediated il-12 gene therapy in a transplantable canine cancer model a combination of electrochemotherapy, gene electrotransfer of plasmid encoding canine il-12 and cytoreductive surgery in the treatment of canine oral malignant melanoma intratumoural interleukin 12 gene therapy stimulates the immune system and decreases angiogenesis in dogs with spontaneous cancer phase i trial of interleukin-12 plasmid electroporation in patients with metastatic melanoma intratumoral plasmid il12 electroporation therapy in patients with advanced melanoma induces systemic and intratumoral t-cell responses intratumoral delivery of tavokinogene telseplasmid yields systemic immune responses in metastatic melanoma patients intratumoral delivery of plasmid il12 via electroporation leads to regression of injected and noninjected tumors in merkel cell carcinoma integrin-targeted nanocomplexes for tumour specific delivery and therapy by systemic administration conjugation of poly(amidoamine) dendrimers with various acrylates for improved delivery of plasmid encoding interleukin-12 gene preparation and characterization of gelatin nanoparticles containing pdna encoding il-12 and their expression in ct-26 carcinoma cells preparation and characterization of chitosan/beta-cyclodextrin nanoparticles containing plasmid dna encoding interleukin-12 efficient gene delivery by egf-lipoplexes in vitro and in vivo polyethylenimine: a versatile, multifunctional nonviral vector for nucleic acid delivery aerosol gene therapy with pei: il-12 eradicates osteosarcoma lung metastases eradication of osteosarcoma lung metastases following intranasal interleukin-12 gene therapy using a nonviral polyethylenimine vector tetraiodothyroacetic acid-conjugated polyethylenimine for integrin receptor mediated delivery of the plasmid encoding il-12 gene enhanced delivery of plasmid encoding interleukin-12 gene by diethylene triamine penta-acetic acid (dtpa)-conjugated pei nanoparticles delivery of plasmid encoding interleukin-12 gene into hepatocytes by conjugated polyethylenimine-based nanoparticles intratumoral delivery of il-12 gene by polyvinyl polymeric vector system to murine renal and colon carcinoma results in potent antitumor immunity combination of interleukin 12 and interferon alpha gene therapy induces a synergistic antitumor response against colon and renal cell carcinoma biodegradable polymer-based interleukin-12 gene delivery: role of induced cytokines, tumor infiltrating cells and nitric oxide in anti-tumor activity soluble biodegradable polymer-based cytokine gene delivery for cancer treatment in vivo targeted gene delivery by cationic nanoparticles for treatment of hepatocellular carcinoma modified nanoparticle mediated il-12 immunogene therapy for colon cancer local and systemic delivery of interleukin-12 gene by cationic micelles for cancer immunogene therapy mannosylated chitosan nanoparticle-based cytokine gene therapy suppressed cancer growth in balb/c mice bearing ct-26 carcinoma cells intratumoral delivery of p2cmvmil-12 using water-soluble lipopolymers tumor regression by repeated intratumoral delivery of water soluble lipopolymers/p2cmvmil-12 complexes combination of local, nonviral il12 gene therapy and systemic paclitaxel treatment in a metastatic breast cancer model local, non-viral il-12 gene therapy using a water soluble lipopolymer as carrier system combined with systemic paclitaxel for cancer treatment development of self-assembled multi-arm polyrotaxanes nanocarriers for systemic plasmid delivery in vivo synthesis and application of a non-viral gene delivery system for immunogene therapy of cancer a safety and efficacy study of local delivery of interleukin-12 transgene by ppc polymer in a model of experimental glioma treatment of disseminated ovarian cancer using nonviral interleukin-12 gene therapy delivered intraperitoneally phase-i clinical trial of il-12 plasmid/lipopolymer complexes for the treatment of recurrent ovarian cancer a phase ii trial of intraperitoneal egen-001, an il-12 plasmid formulated with peg-pei-cholesterol lipopolymer in the treatment of persistent or recurrent epithelial ovarian, fallopian tube or primary peritoneal cancer: a gynecologic oncology group study phase i trial of a formulated il-12 plasmid in combination with carboplatin and docetaxel chemotherapy in the treatment of platinum-sensitive recurrent ovarian cancer a phase i trial of intraperitoneal gen-1, an il-12 plasmid formulated with peg-pei-cholesterol lipopolymer, administered with pegylated liposomal doxorubicin in patients with recurrent or persistent epithelial ovarian, fallopian tube or primary peritoneal cancers: an nrg oncology/gynecologic oncology group study intratumoral injection of interleukin-12 plasmid dna, either naked or in complex with cationic lipid, results in similar tumor regression in a murine model cationic polyphosphazene vesicles for cancer immunotherapy by efficient in vivo cytokine il-12 plasmid delivery enhanced growth inhibition of metastatic lung tumors by intravenous injection of atra-cationic liposome/il-12 pdna complexes in mice retinoic acid receptors and cancers all-trans-retinoic acid upregulates tnf receptors and potentiates tnf-induced activation of nuclear factors-kappab, activated protein-1 and apoptosis in human lung cancer cells lipid nanoparticles that deliver il-12 messenger rna suppress tumorigenesis in myc oncogene-driven hepatocellular carcinoma visual evidence of acidic environment within degrading poly(lactic-co-glycolic acid) (plga) microspheres a study of medi1191 in sequential and concurrent combination with durvalumab in subjects with advanced solid tumors abstract 5017: medi1191, a novel il-12 mrna therapy for intratumoral injection to promote t<sub>h</sub>1 transformation of the patient tumor microenvironment a first-in-human dose escalation and expansion study to evaluate intratumoral administration of sar441000 as monotherapy and in combination with cemiplimab in patients with advanced solid tumors analysis of pre-existing igg and igm antibodies against polyethylene glycol (peg) in the general population viruses as anticancer drugs oncolytic virotherapy adenovirus-mediated interleukin-12 gene therapy for metastatic colon carcinoma construction of a double recombinant adenovirus vector expressing a heterodimeric cytokine: in vitro and in vivo production of biologically active interleukin-12 adenovirus-mediated expression of interleukin-12 induces natural killer cell activity and complements adenovirus-directed gp75 treatment of melanoma lung metastases construction and characterization of a recombinant adenoviral vector expressing human interleukin-12 intranasal therapy with an adenoviral vector containing the murine interleukin-12 gene eradicates osteosarcoma lung metastases antitumor mechanism of intratumoral injection with il-12-expressing adenoviral vector against il-12-unresponsive tumor the antitumor effects of adenoviralmediated, intratumoral delivery of interleukin 23 require endogenous il-12 induction of antitumor immunity by direct intratumoral injection of a recombinant adenovirus vector expressing interleukin-12 regression of colon cancer and induction of antitumor immunity by intratumoral injection of adenovirus expressing interleukin-12 role of nk and t cells in il-12-induced anti-tumor response against hepatic colon carcinoma adenovirusmediated interleukin-12 gene therapy for prostate cancer: suppression of orthotopic tumor growth and pre-established lung metastases in an orthotopic model attenuation of the glucocorticoid response during ad5il-12 adenovirus vector treatment enhances natural killer cell-mediated killing of mhc class i-negative lncap prostate tumors depletion of myeloid-derived suppressor cells during interleukin-12 immunogene therapy does not confer a survival advantage in experimental malignant glioma in situ adenoviral interleukin 12 gene transfer confers potent and longlasting cytotoxic immunity in glioma eradication of murine bladder carcinoma by intratumor injection of a bicistronic adenoviral vector carrying cdnas for the il-12 heterodimer and its inhibition by the il-12 p40 subunit homodimer a single intratumoral injection of a fiber-mutant adenoviral vector encoding interleukin 12 induces remarkable anti-tumor and anti-metastatic activity in mice with meth-a fibrosarcoma growth suppression of human laryngeal squamous cell carcinoma by adenoviral-mediated interleukin-12 enhanced antitumor effect of combined replicative adenovirus and nonreplicative adenovirus expressing interleukin-12 in an immunocompetent mouse model adenovirus-interleukin-12-mediated tumor regression in a murine hepatocellular carcinoma model is not dependent on cd1-restricted natural killer t cells gene therapy of orthotopic hepatocellular carcinoma in rats using adenovirus coding for interleukin 12 effective gene therapy for medullary thyroid carcinoma using recombinant adenovirus inducing tumor-specific expression of interleukin-12 gene therapy of a rat follicular thyroid carcinoma model with adenoviral vectors transducing murine interleukin-12 intratumor murine interleukin-12 gene therapy suppressed the growth of local and distant ewing's sarcoma neuroblastoma regression and immunity induced by transgenic expression of interleukin-12 high frequency of specific cd8+ t cells in the tumor and blood is associated with efficient local il-12 gene therapy of cancer thyroid cancer immuno-therapy with retroviral and adenoviral vectors expressing granulocyte macrophage colony stimulating factor and interleukin-12 in a rat model low-dose adenoviral immunotherapy of rat hepatocellular carcinoma using single-chain interleukin-12 treatment of pancreatic cancer with an oncolytic adenovirus expressing interleukin-12 in syrian hamsters phase i trial of intratumoral injection of an adenovirus encoding interleukin-12 for advanced digestive tumors membrane-tethered proteins for basic research, imaging, and therapy tumor therapy applying membrane-bound form of cytokines re-designing interleukin-12 to enhance its safety and potential as an anti-tumor immunotherapeutic agent cancer immunotherapy using a membrane-bound interleukin-12 with b7-1 transmembrane and cytoplasmic domains the rheoswitch system for inducible up-and down-regulation of gene expression regulated intratumoral expression of il-12 using a rheoswitch therapeutic system((r)) (rts((r))) gene switch as gene therapy for the treatment of glioma regulatable interleukin-12 gene therapy in patients with recurrent highgrade glioma: results of a phase 1 trial neoadjuvant interleukin-12 immunogene therapy protects against cancer recurrence after liver resection in an animal model neoadjuvant treatment of hepatic malignancy: an oncolytic herpes simplex virus expressing il-12 effectively treats the parent tumor and protects against recurrence-after resection interleukin 12 secretion enhances antitumor efficacy of oncolytic herpes simplex viral therapy for colorectal cancer angiogenesis inhibition by an oncolytic herpes virus expressing interleukin 12 cytokine gene transfer enhances herpes oncolytic therapy in murine squamous cell carcinoma in situ cancer vaccination: an il-12 defective vector/replication-competent herpes simplex virus combination induces local and systemic antitumor activity augmentation of antitumor immune responses by multiple intratumoral inoculations of replicationconditional hsv and interleukin-12 il-12 expressing oncolytic herpes simplex virus promotes anti-tumor activity and immunologic control of metastatic ovarian cancer in mice newly characterized murine undifferentiated sarcoma models sensitive to virotherapy with oncolytic hsv-1 m002 enhanced therapeutic efficacy of il-12, but not gm-csf, expressing oncolytic herpes simplex virus for transgenic mouse derived prostate cancers cytokine-secreting herpes viral mutants effectively treat tumor in a murine metastatic colorectal liver model by oncolytic and t-cell-dependent mechanisms engineered herpes simplex virus expressing il-12 in the treatment of experimental murine brain tumors multifaceted oncolytic virus therapy for glioblastoma in an immunocompetent cancer stem cell model increased efficacy of an interleukin-12-secreting herpes simplex virus in a syngeneic intracranial murine glioma model preclinical evaluation of oncolytic deltagamma(1)34.5 herpes simplex virus expressing interleukin-12 for therapy of breast cancer brain metastases preclinical evaluation of a genetically engineered herpes simplex virus expressing interleukin-12 a fully-virulent retargeted oncolytic hsv armed with il-12 elicits local immunity and vaccine therapy towards distant tumors eradication of glioblastoma by immuno-virotherapy with a retargeted oncolytic hsv in a preclinical model evaluation of the safety and biodistribution of m032, an attenuated herpes simplex virus type 1 expressing hil-12, after intracerebral administration to aotus nonhuman primates the molecular pathogenesis of semliki forest virus: a model virus made useful? semliki forest virus vectors engineered to express higher il-12 levels induce efficient elimination of murine colon adenocarcinomas induction of a therapeutic antitumor immunological response by intratumoral injection of genetically engineered semliki forest virus to produce interleukin-12 marked enhancement of antitumor immune responses in mouse brain tumor models by genetically modified dendritic cells producing semliki forest virus-mediated interleukin-12 semliki forest virus expressing interleukin-12 induces antiviral and antitumoral responses in woodchucks with chronic viral hepatitis and hepatocellular carcinoma short-term intratumoral interleukin-12 expressed from an alphaviral vector is sufficient to induce an efficient antitumoral response against spontaneous hepatocellular carcinomas neoadjuvant administration of semliki forest virus expressing interleukin-12 combined with attenuated salmonella eradicates breast cancer metastasis and achieves long-term survival in immunocompetent mice transfer of the murine interleukin-12 gene in vivo by a semliki forest virus vector induces b16 tumor regression through inhibition of tumor blood vessel formation monitored by doppler ultrasonography the anti-angiogenic activity of il-12 is increased in inos-/-mice and involves nk cells strict requirement for vector-induced type i interferon in efficacious antitumor responses to virally encoded il12 immunotherapy with recombinant sfv-replicons expressing the p815a tumor antigen or il-12 induces tumor regression immunotherapeutic synergy between anti-cd137 mab and intratumoral administration of a cytopathic semliki forest virus encoding il-12 virotherapy with a semliki forest virus-based vector encoding il12 synergizes with pd-1/pd-l1 blockade regression of mouse tumours and inhibition of metastases following administration of a semliki forest virus vector with enhanced expression of il-12 semliki forest virus-mediated gene therapy of the rg2 rat glioma low-dose vaccinia virus-mediated cytokine gene therapy of glioma evaluation of cytokine toxicity induced by vaccinia virus-mediated il-2 and il-12 antitumour immunotherapy cytokine-armed vaccinia virus infects the mesothelioma tumor microenvironment to overcome immune tolerance and mediate tumor resolution intratumoral expression of il-7 and il-12 using an oncolytic virus increases systemic sensitivity to immune checkpoint blockade canarypox virus-mediated interleukin 12 gene transfer into murine mammary adenocarcinoma induces tumor suppression and long-term antitumoral immunity intratumoral administration of a recombinant canarypox virus expressing interleukin 12 in patients with metastatic melanoma phase i study of the intratumoral administration of recombinant canarypox viruses expressing b7.1 and interleukin 12 in patients with metastatic melanoma oncolytic measles virus encoding interleukin-12 mediates potent antitumor effects through t cell activation. oncoimmunology immunological effects and viral gene expression determine the efficacy of oncolytic measles vaccines encoding il-12 or il-15 agonists interleukin-12 expression enhances vesicular stomatitis virus oncolytic therapy in murine squamous cell carcinoma enhancement of antitumor activity of gammaretrovirus carrying il-12 gene through genetic modification of envelope targeting her2 receptor: a promising strategy for bladder cancer therapy increased efficacy and safety in the treatment of experimental liver cancer with a novel adenovirus-alphavirus hybrid vector engineering newcastle disease virus as an oncolytic vector for intratumoral delivery of immune checkpoint inhibitors and immunocytokines immunotherapy of established tumors in mice by intratumoral injection of an adenovirus vector harboring the human il-2 cdna: induction of cd8 + t-cell immunity and nk activity effect of viral dose on neutralizing antibody response and transgene expression after aav1 vector re-administration in mice pre-existing immunity to adenovirus does not prevent tumor regression following intratumoral administration of a vector expressing il-12 but inhibits virus dissemination genetic heterogeneity in the toxicity to systemic adenoviral gene transfer of interleukin-12 systemic vector leakage and transgene expression by intratumorally injected recombinant adenovirus vectors systemic dissemination of viral vectors during intratumoral injection in vivo cancer gene therapy with a recombinant interleukin-2 adenovirus vector gene therapy leaves a vicious cycle intratumoral delivery of adenovirus-mediated interleukin-12 gene in mice with metastatic cancer in the liver preclinical toxicology of oncolytic adenovirus-mediated cytotoxic and interleukin-12 gene therapy for prostate cancer induction of systemic and therapeutic antitumor immunity using intratumoral injection of dendritic cells genetically modified to express interleukin 12 intratumoral injection of bone-marrow derived dendritic cells engineered to produce interleukin-12 induces complete regression of established murine transplantable colon adenocarcinomas fibroblasts genetically engineered to secrete interleukin 12 can suppress tumor growth and induce antitumor immunity to a murine melanoma in vivo cancer immunotherapy of established tumors with il-12. effective delivery by genetically engineered fibroblasts therapeutic effects of gelatin matrix-embedded il-12 gene-modified macrophages in a mouse model of residual prostate cancer macrophages transduced with an adenoviral vector expressing interleukin 12 suppress tumor growth and metastasis in a preclinical metastatic prostate cancer model antitumor efficacy of adenocarcinoma cells engineered to produce interleukin 12 (il-12) or other cytokines compared with exogenous il-12 paracrine delivery of il-12 against intracranial 9l gliosarcoma in rats expression of interleukin-12 by adipose-derived mesenchymal stem cells for treatment of lung adenocarcinoma. thorac cancer therapeutic potential of human mesenchymal stem cells producing il-12 in a mouse xenograft model of renal cell carcinoma anti-tumor activity of mesenchymal stem cells producing il-12 in a mouse melanoma model enhancement of antitumor immunity against b16 melanoma tumor using genetically modified dendritic cells to produce cytokines route of administration influences the antitumor effects of bone marrowderived dendritic cells engineered to produce interleukin-12 in a metastatic mouse prostate cancer model augmented anti-tumor effect of dendritic cells genetically engineered by interleukin-12 plasmid dna upregulation of natural killer cells functions underlies the efficacy of intratumorally injected dendritic cells engineered to produce interleukin-12 interleukin-12 transduced dendritic cells induce regression of established murine neuroblastoma intratumoral delivery of dendritic cells engineered to secrete both interleukin (il)-12 and il-18 effectively treats local and distant disease in association with broadly reactive tc1-type immunity intratumoral injection of dendritic cells transduced by an sv40-based vector expressing interleukin-15 induces curative immunity mediated by cd8+ t lymphocytes and nk cells injection of il-12 gene-transduced dendritic cells into mouse liver tumor lesions activates both innate and acquired immunity intratumoral injection of dendritic cells overexpressing interleukin12 inhibits melanoma growth conditional interleukin-12 gene therapy promotes safe and effective antitumor immunity therapeutic effect of intratumoral administration of dcs with conditional expression of combination of different cytokines intratumoral injection of dendritic cells engineered to secrete interleukin-12 by recombinant adenovirus in patients with metastatic gastrointestinal carcinomas continuous release of interleukin 12 from microencapsulated engineered cells for colon cancer therapy interleukin 12 gene therapy of cancer by peritumoral injection of transduced autologous fibroblasts: outcome of a phase i study concise review: mesenchymal stem cell tumorhoming: detection methods in disease model systems murine bone marrow-derived mesenchymal stem cells as vehicles for interleukin-12 gene delivery into ewing sarcoma tumors reversal of tumor growth by gene modification of mesenchymal stem cells using spermine-pullulan/dna nanoparticles gene therapy of intracranial glioma using interleukin 12-secreting human umbilical cord blood-derived mesenchymal stem cells mesenchymal stem cells genetically modified by lentivirus-mediated interleukin-12 inhibit malignant ascites in mice the effects of mesenchymal stem cells injected via different routes on modified il-12-mediated antitumor activity adoptive transfer of natural killer cells promotes the anti-tumor efficacy of t cells local delivery of interleukin-12 using t cells targeting vegf receptor-2 eradicates multiple vascularized tumors in mice il-12 release by engineered t cells expressing chimeric antigen receptors can effectively muster an antigen-independent macrophage response on tumor cells that have shut down tumor antigen expression improving adoptive t cell therapy by targeting and controlling il-12 expression to the tumor environment tumor-targeted t cells modified to secrete il-12 eradicate systemic tumors without need for prior conditioning cd19 car t cells expressing il-12 eradicate lymphoma in fully lymphoreplete mice through induction of host immunity il-12 secreting tumor-targeted chimeric antigen receptor t cells eradicate ovarian tumors in vivo armored inducible expression of il-12 enhances antitumor activity of glypican-3-targeted chimeric antigen receptor-engineered t cells in hepatocellular carcinoma chitosan solution enhances the immunoadjuvant properties of gm-csf sustained release technology and its application in environmental remediation: a review designing hydrogels for controlled drug delivery recent advances in hydrogel based drug delivery systems for the human body neoadjuvant therapy with interleukin-12-loaded polylactic acid microspheres reduces local recurrence and distant metastases in situ tumor vaccination with interleukin-12-encapsulated biodegradable microspheres: induction of tumor regression and potent antitumor immunity cytokines delivered by biodegradable microspheres promote effective suppression of human tumors by human peripheral blood lymphocytes in the scid-winn model synergistic effect of intratumoral il-12 and tnf-alpha microspheres: systemic antitumor immunity is mediated by both cd8+ ctl and nk cells intratumoral delivery of encapsulated il-12, il-18 and tnf-alpha in a model of metastatic breast cancer antitumor immune response of human peripheral blood lymphocytes coengrafted with tumor into severe combined immunodeficient mice interleukin-12 delivered by biodegradable microspheres promotes the antitumor activity of human peripheral blood lymphocytes in a human head and neck tumor xenograft/scid mouse model human cd4+ t cells present within the microenvironment of human lung tumors are mobilized by the local and sustained release of il-12 to kill tumors in situ by indirect effects of ifn-gamma human cd4+ effector memory t cells persisting in the microenvironment of lung cancer xenografts are activated by local delivery of il-12 to proliferate, produce ifn-gamma, and eradicate tumor cells transient activation of tumor-associated t-effector/memory cells promotes tumor eradication via nk-cell recruitment: minimal role for long-term t-cell immunity in cure of metastatic disease cancer immunotherapy with interleukin 12 and granulocyte-macrophage colony-stimulating factor-encapsulated microspheres: coinduction of innate and adaptive antitumor immunity and cure of disseminated disease dichotomous effects of ifn-gamma on dendritic cell function determine the extent of il-12-driven antitumor t cell immunity central role of tumorassociated cd8+ t effector/memory cells in restoring systemic antitumor immunity activated cd8+ t-effector/memory cells eliminate cd4+ cd25+ foxp3+ tsuppressor cells from tumors via fasl mediated apoptosis central role of ifngamma-indoleamine 2,3-dioxygenase axis in regulation of interleukin-12-mediated antitumor immunity rapid release of cytoplasmic il-15 from tumor-associated macrophages is an initial and critical event in il-12-initiated tumor regression intratumoral il-12 and tnf-alpha-loaded microspheres lead to regression of breast cancer and systemic antitumor immunity generation of a tumor-specific systemic response after intratumoral injection of il-12 and il-18-loaded polylactic acid microspheres il-12 + gm-csf microsphere therapy induces eradication of advanced spontaneous tumors in her-2/neu transgenic mice but fails to achieve long-term cure due to the inability to maintain effector t-cell activity chronic immune therapy induces a progressive increase in intratumoral t suppressor activity and a concurrent loss of tumor-specific cd8+ t effectors in her-2/neu transgenic mice bearing advanced spontaneous tumors chronic chemoimmunotherapy achieves cure of spontaneous murine mammary tumors via persistent blockade of posttherapy counterregulation effect of chitosan properties on immunoreactivity intravesical immunotherapy of superficial bladder cancer with chitosan/interleukin-12 characterization of a sustained-release delivery system for combined cytokine/peptide vaccination using a poly-n-acetyl glucosamine-based polymer matrix sustained release of granulocyte-macrophage colony-stimulating factor from a modular peptide-based cancer vaccine alters vaccine microenvironment and enhances the antigen-specific t-cell response paracrine release of il-12 stimulates ifn-gamma production and dramatically enhances the antigen-specific t cell response after vaccination with a novel peptide-based cancer vaccine intratumoral poly-n-acetyl glucosamine-based polymer matrix provokes a prolonged local inflammatory response that, when combined with il-2, induces regression of malignant mesothelioma in a murine model using poly-n-acetyl glucosamine gel matrix to deliver il-12 with anti-schistosomasis vaccination evaluation of the biocompatibility of a chitosan scaffold in mice biocompatibility evaluation of crosslinked chitosan hydrogels after subcutaneous and intraperitoneal implantation in the rat il-12 delivered intratumorally by multilamellar liposomes reactivates memory t cells in human tumor microenvironments adjuvant cationic liposomes presenting mpl and il-12 induce cell death, suppress tumor growth, and alter the cellular phenotype of tumors in a murine model of breast cancer cytokine immunotherapy of cancer with controlled release biodegradable microspheres in a human tumor xenograft/scid mouse model characterization of cytokine-encapsulated controlled-release microsphere adjuvants stabilization of proteins encapsulated in injectable poly (lactide-co-glycolide) biodegradable microand nanoparticles as long-term delivery vehicles for interferon-alpha challenges in development of targeted liposomal therapeutics advances and challenges of liposome assisted drug delivery surgery for cancer: a trigger for metastases chemotherapy-induced metastasis in breast cancer radiotherapy targeting cancer stem cells "awakens" them to induce tumour relapse and metastasis in oral cancer biological behavior of human breast cancer micrometastases a novel synergistic combination of cyclophosphamide and gene transfer of interleukin-12 eradicates colorectal carcinoma in mice a phase i clinical trial of adoptive t cell therapy using il-12 secreting muc-16(ecto) directed chimeric antigen receptors for recurrent ovarian cancer the next challenge in cancer immunotherapy: controlling t-cell traffic to the tumor chemotherapy induces intratumoral expression of chemokines in cutaneous melanoma, favoring t-cell infiltration and tumor control immunogenic effects of chemotherapy-induced tumor cell death radiation-induced effects and the immune system in cancer a novel combination treatment of armed oncolytic adenovirus expressing il-12 and gm-csf with radiotherapy in murine hepatocarcinoma irradiation-induced localization of il-12-expressing mesenchymal stem cells to enhance the curative effect in murine metastatic hepatoma combination of radiation and interleukin 12 eradicates large orthotopic hepatocellular carcinoma through immunomodulation of tumor microenvironment the role of irreversible electroporation (ire) for locally advanced pancreatic cancer: a systematic review of safety and efficacy irreversible electroporation reverses resistance to immune checkpoint blockade in pancreatic cancer systematic review of surgical and percutaneous irreversible electroporation in the treatment of locally advanced pancreatic cancer high-frequency irreversible electroporation is an effective tumor ablation strategy that induces immunologic cell death and promotes systemic anti-tumor immunity immunologic response to cryoablation of breast cancer thermal ablation of tumours: biological mechanisms and advances in therapy synergy between in situ cryoablation and tlr9 stimulation results in a highly effective in vivo dendritic cell vaccine changes in interleukin-1beta and 6 after hepatic microwave tissue ablation compared with radiofrequency, cryotherapy and surgical resections image-guided thermal ablation of tumors increases the plasma level of interleukin-6 and interleukin-10 characterization of the cryoablation-induced immune response in kidney cancer patients abscopal immunity achieved via in situ vaccination using a novel combination of cryoablation and interleukin-12 the antibody-based delivery of interleukin-12 to solid tumors boosts nk and cd8 + t cell activity and synergizes with immune checkpoint inhibitors intratumoral il-12 combined with ctla-4 blockade elicits t cell-mediated glioma rejection phase ii trial of il-12 plasmid transfection and pd-1 blockade in immunologically quiescent melanoma revisiting interleukin-12 as a cancer immunotherapy agent abbreviations a-nk il−12 or activated nk il−12 , activated primary natural killer (nk) cells transduced to express il-12 adcmvil-12 or adcmvmil-12, recombinant defective adenovirus expressing il-12 or mil-12 under control of a cmv promoter adenoviral vector expressing murinedil-12 protein under the control of the hcmv promoter and sv40 polyadenylation sequences; ad-dhscil12, adenovirus with replication dependent on hypoxia-inducible factor (hif) activity expressing single-chain il-12; ad-il-12 or adil-12 or ad.il-12 or admil-12 or ad.mil-12 inducible adenoviral vector engineered to express il-12 under the control of the rheoswitch therapeutic system r (rts) gene switch adtcpmil-12, recombinant replication-defective adenoviral vector expressing murine interleukin-12 (mil-12), driven by a modified calc-i promoter oncolytic triple-deletion (td) adenoviral vector encoding wild-type il-12 oncolytic triple-deletion (td) adenoviral vector encoding non-secreting il-12 antibody-dependent cellular cytotoxicity; ad.scil-12, adenoviral vector expressing single-chain il-12 ad5-ycd/muttksr39rep-hil12, a replication-competent oncolytic adenovirus encoding the murine pro-inflammatory cytokine interleukin-12 (il-12) gene and two suicide fusion genes, a yeast cytosine deaminase (ycd) and a mutant form of herpes simplex virus type 1 thymidine kinase /3.crgd-mil12p70, a replication-deficient double targeted ad5 backbone-based vector carrying a chimeric ad5/3 fiber with integrin-binding rgd motif incorporated in its ad3 knob domain expressing murine il-12p70 adenoviral vector encoding membrane-anchored murine single-chain il-12 with b7-1 transmembrane and cytoplasmic domains atra-cationic liposome/il-12 pdna, pil-12 complexed with cationic liposomes incorporating all-trans-retinoic acid chimeric antigen receptor; cbd, collagen binding domain; cbd-il-12, collagen-binding immunocytokine comprised of a3 cbd of von willebrand factor fused to both subunits of il-12 chtnt-3/huil-12, necrosis-targeting immunocytokine comprised of huil-12 fused to the variable heavy chain of chtnt-3 antibody dendritic cells transfected with adenovirus expressing il-12 under control of the cmv promoter enzyme-linked immune absorbent spot fda, food and drug administration; gm-csf, granulocyte-macrophage colony-stimulating factor glypican-3-specific car t cells with nfat-inducible expression of il-12 herpes simplex viruses (hsv) encoding il-12; hubc1-il12, immunocytokine comprised of two molecules of il-12 fused to each of the igg heavy chains of humanized bc-1 antibody immunocytokine comprised of il-12 fused to the fc fragment of humanized ks antibody; hu-14.18-il-12, immunocytokine comprised of il-12 fused to gd2 targeting hu14 dmp/il-12, pil-12 complexed with dmp cationic micelles 3-dimyristyloxypropyl)-n,n-dimethyl-(2-hydroxyethyl)ammonium bromide/dioleoyl phosphatidylethanolamine (dmrie/dope) cationic lipids; il-12, interleukin-12; il-12m, modified il-12; an n-glycosylation mutant of il-12 at asn220; il12-il2, immunocytokine comprised of il-12 and il-2 fused to anti-cd30 scfv antibody immunocytokine comprised of il-12 fused to l19 scfv antibody; il12-msa, il-12 fused to mouse serum albumin (msa) il-12 fused to mouse serum albumin (msa) and lumican, a collagen-binding proteoglycan immunocytokine comprised of p35 subunit of single-chain il-12 fused to ss1, a mesothelin-binding single-chain variable fragment lenti-mil-12-mscs, bone-marrow derived mscs transfected with lentivirus to express il-12 mc/pmil-12, pmil-12 complexed with mannosylated chitosan (mc) myeloid-derived suppressor cells mevac fmil-12, attenuated measles viruses (mevac) encoding il-12 histocompatibility complex; momlv-mil-12, moloney murine leukemia virus (momlv) expressing il-12; mpeg/pmil-12, polymersomes comprised of pmil-12 complexed with polyphosphazene particles modified with dpa and mpeg; mscs, mesenchymal stem cells; mscil-12.her2.igg3, mouse single-chain il-12 fused mesenchymal stem cells (mscs) expressing il-12; l19-mil12, immunocytokine comprised of murine il-12 fused to l19 scfv antibody; nhs-il12, necrosis-targeting immunocytokine comprised of two single-chain il-12 molecules fused to fulllength nhs76 antibody; nhs-muil12, murine analog of nhs-il12; nk cells a biodegradable polyester; pbl, peripheral blood lymphocytes; pd-1, programmed cell death protein 1; pd-l1, programmed death-ligand 1; pei:il-12, il-12 complexed with polyethyleneimine (pei); pil-12 enhanced sfv vector with 10x higher gene expression of recombinant il-12 protein; rad/il-12m, recombinant adenovirus expressing il-12m; rndv-anti-cd28-mil-12, newcastle disease virus (ndv) expressing immunocytokine comprised of anti-cd28 antibody fused to mil-12; rndvanti-pdl1-mil-12, newcastle disease virus (ndv) expressing immunocytokine comprised of anti-pdl1 antibody fused to mil-12; rsfv/il12, recombinant semliki forest viruses (sfv) encoding il-12; rvsv-il12, recombinant vesicular stomatitis virus (vsv) encoding il-12; rvv-mil-12 severe combined immunodeficient; scil-12, single-chain il-12; sfv-il12, semliki forest viruses encoding il-12 sfv-il-12, semliki forest viruses encoding il-12 tumor associated macrophages; tlr, toll-like receptor; tnf, tumor necrosis factor; ucb-msc-il12m, human umbilical cord blood-derived mesenchymal stem cells (ucb-mscs) expressing il-12m vv-il-12, vaccinia virus (vv) expressing il-12 il-12 expression plasmid complexed with water-soluble lipopolymers car t cells which are specific for the muc-16ecto antigen and secrete il-12; 6b11scfv-mil-12, immunocytokine comprised of mil-12 fused to scfv of 6b11 antibody key: cord-267671-ys43n672 authors: whary, mark t.; baumgarth, nicole; fox, james g.; barthold, stephen w. title: biology and diseases of mice date: 2015-07-10 journal: laboratory animal medicine doi: 10.1016/b978-0-12-409527-4.00003-1 sha: doc_id: 267671 cord_uid: ys43n672 today’s laboratory mouse, mus musculus, has its origins as the ‘house mouse’ of north america and europe. beginning with mice bred by mouse fanciers, laboratory stocks (outbred) derived from m. musculus musculus from eastern europe and m. m. domesticus from western europe were developed into inbred strains. since the mid-1980s, additional strains have been developed from asian mice (m. m. castaneus from thailand and m. m. molossinus from japan) and from m. spretus which originated from the western mediterranean region. laboratory animal medicine development of the 'modern' laboratory mouse. research use of mice has grown exponentially during the past and current century with the recognition of the power of the mouse for gene and comparative mapping and have made the laboratory mouse, in genetic terms, the most thoroughly characterized mammal on earth (silver, 1995; lyon et al., 1996; morse, 2007a) . the current ability to create highly sophisticated, genetically engineered mice by inserting transgenes or targeted mutations into endogenous genes has also made the laboratory mouse the most widely and heavily used experimental animal. historical reviews have documented the origins of the laboratory mouse, which extend thousands of years into antiquity (keeler, 1931; morse, 1978; silver, 1995) . the laboratory mouse belongs within the genus mus, subfamily murinae, family muridae, superfamily muroidea, order rodentia, and within the m. musculus clade collectively called the 'house mouse' (lundrigan et al., 2002) . anatomic features of molar teeth and cranial bones were traditionally used by zoologists to identify over 100 different species within the genus, and to differentiate them from other murids. because of considerable phenotypic variation within a single mus species, this approach has proven to be inaccurate, and given way to contemporary genetic analysis. the native range of the genus mus is eurasia and north africa. members of this genus are generally classified as aboriginal, consisting of species that live independent of humans, or commensal, which includes taxa that have coevolved and geographically radiated with human civilization since the dawn of agriculture 12,000 years before present (bp). this close association with human agrarian society gave rise to the genus name, derived from sanskrit, mush: to steal. the commensal group is known as the 'house mouse' clade, consisting of several subspecies of mus musculus, including m. m. domesticus, m. m. musculus, m. m. castaneus, m. m. bactrianus , and a lesser known lineage, m. m. gentilulus (prager et al., 1998) . the japanese house mouse, m. m. molossinus, is a natural hybrid of m. m. musculus and m. m. castaneus. the progenitor of the m. musculus clade arose in the northern indian subcontinent and diverged into genetically isolated and distinct species or subspecies due to geographic barriers (mountain ranges). there is debate whether these taxa are species or subspecies, and some have referred to them as 'incipient species,' but their genetic divergence is now blurring as they colonize the world and hybridize. the native ranges of these taxa are important for understanding the origins of various laboratory mice, whose genomes are mosaics derived from m.m. domesticus (~60%), m.m. musculus (~30%), and m.m. castaneus (~10%) (wade and daly, 2005; wade et al., 2002) . it is now apparent that the m.m. musculus and m.m. castaneus contributions to the laboratory mouse genome were primarily derived from m.m. molossinus japanese fancy mice (takada et al., 2013) . mus m. domesticus is indigenous to western europe and southwest asia, m.m. musculus to eastern europe and northern asia, m.m. castaneus to southeast asia, and m.m. molossinus to japan and the korean peninsula. the cohabitation of humans with commensal mice gave rise to captive breeding for coat color and behavioral variants in china over 3000 years bp. by the 1700s, mouse 'fanciers' in asia had created many varieties of fancy mice, as did european fanciers, who subsequently acquired asian stocks, particularly japanese fancy mice (m.m. molossinus) , to mix with european (m.m. domesticus) fancy mouse varieties. this genetic mixing for fancy variants was also occurring in the united states, and these mouse lines contributed to many of the major laboratory mice used today. meanwhile, the european colonial expansion era contributed to the worldwide dissemination of m.m. domesticus, which now occupies every continent of the world. it is well documented that wild-caught m.m. domesticus also contributed to the genetic composition of fancy and laboratory mice on multiple occasions. despite their diverse genetic origins and phenotypic differences, most laboratory mouse strains are closely related, since many were derived from a genetically mixed but small number of fancy mice from a single mouse breeder (abbie lathrop's granby mouse farm, massachusetts) at the beginning of the 20th century. most inbred laboratory mice share a common maternal mitochondrial genome derived from m.m. domesticus (ferris et al., 1982; yu et al., 2009) , and a common y chromosome contributed by m.m. musculus (bishop et al., 1985) through its contribution to the genome of m.m. molossinus (nagamine et al., 1992) . thus, the most inclusive name that can be assigned to the genetically mosaic laboratory mouse is m. musculus, the over-arching name for the entire commensal clade. there are exceptions, however. c57bl/6 mice contain minor genetic elements derived from m. spretus (hardies et al., 2000) , and a number of wild aboriginal species that are not members of the m. musculus clade, including m. spretus, m. caroli, and others, have been established as inbred lines of mice. genetic mapping in mice began in the early 1900s with a focus on inheritance of coat color. the first autosomal genes, albino and pink-eyed dilution, were linked in 1915 (haldane et al., 1915) . extensive linkage maps and an impressive array of inbred strains are now available to expedite genetic research (table 3 .1) (lyon et al., 1996) . mice have 20 pairs of telocentric chromosomes that are differentiated by their size and patterns of transverse bands. the chromosomes are designated by arabic numbers in order of decreasing size. during the 1970s, chromosome rearrangements were used to assign known genetic linkage groups -identified by roman numerals -to specific chromosomes and for determining locus order with respect to the centromere. genes can be located physically on chromosomes by fluorescent in situ hybridization (fish). development of quantitative trait loci (qtl) methodology for mapping genes and the similarity between mouse and human genomes have made the mouse invaluable for identifying genes and underlying complex traits that are inherent to the most common human genetic diseases (moore and nagle, 2000) . for more information on comparative genomics, see chapter 35 animal models in biomedical research, subsection c. one of the most thoroughly studied genetic systems of the mouse is the histocompatibility complex. histocompatibility (h) loci control expression of cell surface molecules that modulate critical immune responses, such as the recognition of foreign tissue. for example, the time, onset, and speed of skin graft rejection are controlled by two groups of h loci. the major group is located in the major histocompatibility complex (mhc, h2) on chromosome 17. the h2 complex contains several loci, including k, d, l, i-a, and i-e. inbred strains of mice, being homozygous, each have unique sets of h2 alleles, termed h2 haplotypes. for example, the balb h2 haplotype is h2 d and the c57bl h2 haplotype is h2 b . the international immunogenetics (imgt) information system provides details on h2 haplotypes for various inbred mice (www.imgt.org/imgtrepertoiremhc/polymorphism/ haplotypes/mouse/mhc/mu_haplotypes.html). minor h loci groups are scattered throughout the genome and are responsible for delayed graft rejection. genes associated with the h2 complex also control other immunological functions, such as cell-cell interactions in primary immune responses and the level of response to a given antigen. immune-mediated responses to infectious agents such as viruses and complement activity are influenced directly or indirectly by the h2 complex (stuart, 2010) . non-mhc or minor histocompatibility systems also are under active study (roopenian et al., 2000) . mouse genomics have accelerated tremendously in the last two decades, heralded by the development of a robust physical map and high-quality genome sequence of the c57bl/6j mouse in 2002 by the international mouse genome sequencing consortium (waterston et al., 2001) . the mouse genomes project/wellcome trust sanger institute is extending this effort to include the genomic sequences of 17 key mouse strains. completed and evolving sequence data are available through the european nucleotide archive (www.ebi.ac.uk/ena/home). the burgeoning numbers of inbred mouse strains, natural mutants, induced mutants, transgenic lines, and targeted mutant lines of mice are cataloged in the mouse genome informatics (mgi) database: http://www.informatics.jax.org/mgihome). the growing number of mutant mice has fostered the development of a number of mouse repositories, from which specific mice can be located and acquired. in the united states, there are four regional national institutes of health (nih)-supported mutant mouse regional resource centers (http://www.mmrrc.org), which link to international repositories in europe, japan, china, australia, and canada, as well as additional resource programs in the united states through the international mouse strain resource (imsr; http://www.informatics.jax.org/ imsr/index.jsp) for depositing, archiving, and distributing mutant mouse and embryonic stem cell lines to the scientific community. in addition to numerous mutant mice produced independently by scientists in various academic institutions, three major targeted gene knockout programs, all utilizing c57bl/6n embryonic stem cells, are under way internationally, and funded by the nih, the european community, and genome canada (collins et al., 2007; skarnes et al., 2011) . these include the knock out mouse project (komp; http://www.knockoutmouse.org), the european conditional mouse mutagenesis program (eucomm; http://www.eucomm.org), and the north american conditional mouse mutagenesis project (norcomm; http://norcomm.phenogenomics.ca/index. htm). these mouse lines will be available through laboratory animal medicine three distribution centers: the german resource center for genome research (rzpd; http://www.rzpd.de), the komp repository (https://komp.org), and the canadian mouse consortium (cmc; http://www.mousecanada. ca/index.htm). the repositories are all linked to the imsr, and provide access to mice, germplasm, genomic detail, and phenotypic data. genetic, genomic, and biological data are also available through the international mouse phenotyping consortium (impc; www.mousephenotype. org) and the mouse genome database (mgd; http://www. informatics.jax.org) (eppig et al., 2012) . inbreeding is a fundamental genetic tool applied to the laboratory mouse and detailed information is available on the web (table 3 .2). the first inbred strain (dba) was developed by c.c. little in 1909, with the subsequent creation of over 1000 inbred strains and stocks of mice (festing, 1996) . genetic origins, basic characteristics, references, and breeding performance of inbred strains of mice are available through michael festing's online version of inbred strain characteristics (http:// www.informatics.jax.org/external/festing/mouse/ strains.shtml). overviews of genetic manipulation for the creation of different types of mice are available (lyon et al., 1996; silver, 1995) . inbred mouse lines are termed strains, and are achieved by 20 or more brother × sister (filial; f) generations (table 3 .3). mice within an inbred strain, for practical purposes, are genetically identical (syngeneic or isogenic) to other mice of the same strain and sex. because of residual heterozygosity, a strain is not fully inbred until after 60 f generations. most commonly used inbred mouse strains represent 200 or more f generations, providing a high degree of experimental reproducibility. the mouse genome is not static, so when branches of an inbred strain are separated, spontaneous mutations, residual heterozygosity, and retroelement integrations result in genetic differences. therefore, if branches of an inbred strain are separated before f 40 , if branches have separated for 100 generations, or if genetic differences arise, the different branches become substrains. the same holds true if branches of a substrain diverge, resulting in substrains of the inbred substrain. when two inbred mouse strains are crossed, the f 1 hybrids are genetically identical to one another (isogenic), but maximally heterozygous (with chromosomes of each chromosomal pair separately contributed by each parental strain), whereas f 2 hybrids are maximally genetically diverse from one another (with chromosomes of both chromosomal pairs containing a mixture of contributions from each parental strain). with each subsequent f generation, mice once again approach inbred status. this technique is used for creating recombinant inbred (ri) strains. ri strains are sets of inbred strains of mice derived from crossing two inbred strains, and developed by singlepair random matings of sibling mice from the f 2 generation, thereby creating separate breeding lines. each line created is maintained separately, and then propagated by brother-sister matings for 20 generations, with each line becoming a separate inbred strain, but belonging to a set of ri strains. ri mice are useful for mapping phenotypic or quantitative traits that differ between the progenitor strains (bailey, 1971) . ri sets are generally limited to two parental strains. an ongoing international effort has been undertaken to increase allelic diversity among ri strains by creating the collaborative cross (cc) in which a panel of ri strains are being generated mixing the genomes from eight disparately related inbred (octo-parental) mouse strains, including a/j, c57bl/6j, 129s1/svimj, nonobese diabetic (nod)/shiltj, nzo/ hlltj, cast/eij, pwk/phj, and wsb/eij. these eight strains capture nearly 90% of the known genetic variation present among laboratory mice. future applications of the cc will utilize ri intercrosses of pairs of ri cc lines (threadgill and churchill, 2012; welsh et al., 2012) . recombinant congenic strains are sets of inbred strains derived in a manner similar to that for ri sets, except that one or more backcrosses to one parental strain (designated the background strain) are made after the f 1 generation, before inbreeding is begun. the other parental strain is designated as the donor strain. the proportion of background and donor genomes is determined by the number of backcrosses preceding inbreeding (demant and hart, 1986) . advanced intercross lines (ails) are another type of ri lines. they are made by producing an f 2 generation between two inbred strains and then, in each subsequent generation, intercrossing mice but avoiding sibling matings. the purpose is to increase the possibility of recombination between tightly linked genes. when a mutation arises spontaneously or is induced within an inbred strain, that mutant mouse becomes co-isogenic with the parental inbred strain, being virtually identical except for the single mutant allele. frequently, a mutation that arose in one inbred strain may be desired within the genetic background of another inbred strain. this can be accomplished by backcrossing, in which an f 1 hybrid is created by mating the donor mutant strain to the desired background strain, with subsequent matings to the background strain while retaining the mutant locus. after 10 backcross generations (n generations), the mutant mouse line is now congenic to the background inbred strain. backcrossing to create congenic strains of mice has been used extensively when targeted mutations have been induced in 129 embryonic stem cells, with backcrossing onto c57bl/6 inbred mice. congenic mice are never co-isogenic, as the preserved locus in a congenic mouse is invariably surrounded by flanking dna, which may significantly influence phenotype (linder, 2006) . in contrast to inbred mice, outbred mice are genetically heterogeneous and are maintained by breeding systems that intentionally minimize inbreeding. outbred mice are called stocks, which are defined as a closed population (for at least four generations) of genetically variable mice that are bred to maintain maximal heterozygosity. outbred mice may be used when high genetic heterogeneity is desired or for experiments requiring large numbers of mice. outbreeding can be achieved only in a large breeding population using a systematic breeding scheme, or randomized selection of breeders from the population. a small breeding population or passage through the genetic 'bottleneck' of rederivation to improve health status will reduce genetic heterogeneity and lead eventually to some degree of inbreeding. in a population of 25 breeding pairs, e.g., heterozygosity will decrease at 1% per generation with standard randomization techniques. random breeding involves the statistically random selection of breeders by using a random numbers table or computer program. an outbreeding program that is easy to manage is the circular pair mating system, in which each pair is mated only once. conceptually, cages are visualized in a circle, and each cage contains one breeding pair in the nth generation. another 'circular' set of cages serves as the breeding nucleus for the n + 1 generation. each mated pair in the nth generation contributes one female and one male to the n + 1 generation. outbreeding is accomplished by assigning the female and male derived from each nth generation cage to different cages in the n + 1 generation. most outbred mouse stocks are of 'swiss' origin, derived from nine mice imported to the united states in 1926, and are therefore quite homogeneous genetically (chia et al., 2005) . various lines of these mice have been maintained at different institutions, giving rise to numerous closely related stocks. although considered outbred, they have a high degree of homozygosity, exemplified by the fact that many swiss mouse stocks are blind due to the homozygous recessive rd1 allele (serfilippi et al., 2004b) . it is preferable to ensure genetic heterogeneity by intercrossing multiple inbred strains to achieve heterogeneity with known genetic input. in that regard, the diversity outbred mouse has been developed, which is a heterogeneous stock derived from the same eight founder inbred strains of the cc . additional types of inbred mice are utilized in research, including consomic and conplastic strains. consomic strains, also known as chromosome substitution strains, are inbred mice that are congenic for entire chromosomes, and are useful for studying polygenic traits (singer et al., 2004) . conplastic mice are inbred mice that are congenic for different mitochondrial genomes (mtdna) contributed by other inbred strains, other subspecies, or other species of mus (yu et al., 2009 ). in addition to spontaneously occurring mutations that are maintained as co-isogenic strains (such as the c57bl/6 beige mouse), mutant lines of mice have been created by radiation mutagenesis, chemical mutagenesis, or transgenesis. radiation was one of the earlier methods for in vivo mutagenesis (silver, 1995) , but in vitro radiation of embryonic stem (es) cells is also performed (thomas et al., 1998) . chemical mutagenesis involves in vivo treatment of male mice or in vitro treatment of es cells with mutagenic chemicals such as ethylmethanesulphonate (ems) or n-ethyl-n-nitrosourea (enu) , which induce point mutations in dna (o'brien and frankel, 2003; justice et al., 1999 justice et al., , 2000 . technically, a transgenic mouse is any mouse in which foreign dna has been integrated into its genome, regardless of method. however, the term transgenic commonly refers to mice that are genetically altered by additive transgenesis through microinjection of foreign dna into the pronucleus of a fertilized egg. each ensuing embryo results in a genetically different founder mouse, since the transgene is integrated in random sites of the genome of each founder mouse. since the injected dna is not homologous to the mouse genome and is not an allele, transgenic founders are hemizygous (rather than heterozygous) for the transgene until the mice carrying the transgene are bred into homozygosity for the transgene. transgenes typically integrate as tandem repeats, copy numbers affect phenotype of each founder, and may be lost in subsequent generations, thereby changing the phenotype of the mouse line (tinkle and jay, 2002) . transgenes are often constructed with an upstream promoter, which confers widespread (ubiquitous) or tissuespecific expression of the cdna, so that the transgene expression pattern reflects the expression pattern of the promoter. transcriptional regulation of the transgene can be inducible by drug-dependent regulatory control, such as the widely used tetracycline (tet) regulatory system, in which treatment of mice with tetracycline or doxycycline induces up-or down-regulation of the transgene (jaisser, 2000) . es cells are used for the less efficient integration of genetic material by homologous dna recombination, but allow large-scale screening of es cell clones for transformation. integration can be achieved in a random fashion by gene trapping, or by targeted mutation. both methods involve homologous dna recombination. gene trapping is a high-throughput approach that randomly introduces insertional mutations within the genome. vectors contain a gene trapping cassette with a promoter-less reporter gene and/or selectable genetic marker flanked by an upstream 3′ splice site and a downstream termination sequence. when inserted into an laboratory animal medicine intron of an expressed gene, the gene trap is transcribed from the endogenous promoter of that gene. gene traps simultaneously inactivate and report the expression of the trapped gene at the insertion site, and provide a dna tag for the rapid identification of the disrupted gene (skarnes et al., 2011) . targeted gene mutations are achieved by homologous recombination of specific sites within the genome of es cells. homologous sequences flank the upstream and downstream regions of the targeted gene, and the construct between the flanking sequences may inactivate (knock out) or replace (knock in) a gene, and typically contains a reporter gene to track the integration. a variation on this approach is site-specific recombinase (ssr) technology. two of the most common recombinases are cre from the coliphage p1 and flp from saccharomyces cerevisiae. cre and flp mediate recombination between target sites, termed loxp and frt, respectively. for example, cre loxp target sites are engineered to flank the gene target, which can be used in different ways to achieve different outcomes (conditional mutations), depending upon the orientation and location of the flanking loxp sites. if the loxp sites are oriented in opposite directions, cre recombinase mediates inversion of the floxed segment. if the loxp sites are on different chromosomes (trans), cre recombinase mediates a chromosomal translocation. if the loxp sites are oriented in the same direction on the same chromosome (cis), cre recombinase mediates deletion of the floxed segment. once the floxed mutation is created in es cells, the transformed es cells are developed into a mouse with the conditional mutation. the conditional mutant mouse is then genetically crossed with a cre transgenic mouse, in which cre recombinase is under the control of a ubiquitous or tissue-specific promoter. wherever and whenever cre is expressed, cre recombinase will recognize and recombine the loxp sites. this approach can include insertion of reporter genes and selectable markers, and can be under the control of inducible gene expression systems (http:// www.eucomm.org/docs/protocols/mouse_protocol_1_ sanger) (nagy, 2000) . es cells are pluripotent with the full genetic capacity to develop into mice when implanted into the blastocyst of a developing embryo. interest in 'embryonal carcinomas' (teratomas) that arose in relatively high frequency in the testes of 129 mice and early gene transfer experiments in the late 1970s and early 1980s led to the development of es cell lines derived from several different 129 strains. this early emphasis on teratomas prompted creation of 'better' 129 mouse lines that were more prone to development of testicular teratomas, resulting in genetic corruption of the 129 mouse (simpson et al., 1997; threadgill et al., 1997) . this realization gave rise to the need to revise 129 mouse nomenclature (festing et al., 1999) . this was necessary because genetic variation significantly impacts homologous recombination in order to match genome sequence of the es cell line with the mouse from which it was derived. es cells can be created from any mouse strain or hybrid, but 129 es cell lines have been commonly used. recent international knockout mouse program efforts use c57bl/6n es cells. transformed es cells are microinjected into the inner cell mass of recipient blastocysts, which are then implanted into the uteri of pseudopregnant surrogate mothers. the pups that are born are composed of a mixture of cells derived from recipient blastocysts and the transformed es cells (chimeras). the goal is for male chimeric progeny to produce spermatozoa of es cell origin (containing the mutation), in order to create f 1 progeny by mating the chimera with the desired background strain (http://www.eucomm.org/docs/protocols/mouse_protocol_1_sanger). for this reason, most es cell lines are xy, which favors 129 male chimerism. if the es cells are of 129 (or other) strain origin, the chimeras are often bred to a desired background mouse strain (commonly c57bl/6) and backcrossed for n10 generations, thereby creating congenic inbred mouse lines. recent international knockout mouse efforts utilize c57bl/6n es cells, so that chimeric males are bred directly with c57bl/6 mice, thereby creating co-isogenic lines. the latter approach saves time and money, and creates a more genetically refined mutant mouse. an alternate approach is to allow es cells to aggregate with a developing embryo to form blastocysts in culture (aggregation chimera), then implant the chimeric blastocysts (tanaka et al., 2001) . rna interference (rnai), which functions through short double-stranded rna (dsrna), has also been utilized to produce transgenic mice, known as gene knockdown mice (gao and zhang, 2007; peng et al., 2006) . the dsrna is enzymatically processed into small molecules, termed small interfering rna (sirna), which find homologous target mrnas, resulting in interference. this phenomenon is believed to be a self-defense mechanism against viral infection. in order to adapt this approach to generation of transgenic mice, small hairpin rna (shrna) can be expressed in the same way as other transgenes in mice, resulting in processing of the shrna into sirna with gene-silencing effects. constructs are introduced into mouse es cells by electroporation or lentiviral infection. this method can be embellished conditionally, as with other transgenes. although rnai knockdown mice are genetically stable, rnai-mediated transgenesis is never complete, has variable tissue expression, and cannot induce point mutations (peng et al., 2006) . recent advances in engineered endonuclease (ee) technology, including zinc finger nucleases (zfns), laboratory animal medicine transcription activator-like effector nucleases (talens), and rna-guided endonucleases (rgens), have revolutionized the field of transgenics (sung et al., 2014; wijshake et al., 2014; gaj et al., 2013) . zfns and talens consist of engineered proteins that target dna fused to the nonspecific endonuclease, fok1 (cathomen and joung, 2008; joung and sander, 2013) . zfns are comprised of three to six tandem zinc finger proteins, each of which targets a specific 3 bp nucleotide sequence. paired zfns are generated, with each half of the pair targeting opposite dna strands, allowing dimerization of fok1 which is required for introduction of double-stranded breaks (dsbs) in the dna of interest (cathomen and joung, 2008) . talens function similarly, but are composed of tandem repeats of 33-35 amino acids, each with nucleotide specificity occurring in two hypervariable amino acids, the 'repeat variable di-residue (rvd)', at positions 12 and 13 (joung and sander, 2013) . in contrast to zfns and talens, clustered regularly interspaced short palindromic repeats (crisprs) paired with crispr-associated (crispr/cas) systems are rgen systems that target specific dna sequences. cas proteins, rather than fok1, produce dsb (hsu et al., 2014) . dsb generated by ee are repaired by host cells by either nonhomologous end joining (nhej) or, less commonly, by homologous recombination (hr). nhej is an error-prone repair system and results in insertions or deletions (indels) with a relatively high frequency, which can result in gene disruption. hr is a less common repair pathway, but certain manipulations of the engineered nucleases can increase hr efficiency. for example, nucleases can be engineered to generate a break in a single strand of dna rather than inducing dsb, and the resulting nickases increase the incidence of hr with high fidelity (gaj et al., 2013; wijshake et al., 2014) . hr allows for the introduction of donor dna to generate knock-ins, specific point mutations, or for the generation of larger modifications such as insertions of loxp sites (brown et al., 2013; wijshake et al., 2014) . vectors encoding the ee can be injected into mouse embryos by pronuclear injection of dna, intracytoplasmic injection of rna, or transfection of mouse es cells (sung et al., 2014; wijshake et al., 2014) . one advantage of ee technologies over more traditional transgenic methods is the ability to target dna and induce mutations in any background strain of mouse negating the need to backcross onto the desired strain. multiple genes can be targeted with crisprs simultaneously, thus avoiding the need to cross single knockout animals (zhou et al., 2014) . in addition, it is possible to obtain bi-allelic mutations in some cases, allowing for the generation of functional gene knockout animals in a single generation (zhou et al., 2014; wijshake et al., 2014) . vectors for generating ee are available through plasmid repositories; websites are available to assist in identifying appropriate dna sequences to target; and multiple websites post protocols for generating the various types of engineered endonucleases (xie et al., 2014; sander et al., 2010; bae et al., 2014; reyon et al., 2011; herscovitch et al., 2012; wolfson, 2013) . crisprs tend to be particularly cost effective and easy to design, with minimal restrictions for targeting specific dna sequences. there are currently more than 1000 separate outbred stocks and traditional inbred strains, often with multiple substrains (table 3 .4). in addition, there are thousands of induced mutant strains. therefore, it is critical that strain or stock designations be complete and accurate to avoid semantic and genetic confusion, and to ensure reproducibility of research results. as an example of substrain variation that makes precise nomenclature important, cba/j mice are homozygous for the retinal degeneration allele (rd1), whereas cba/caj mice do not carry this allele. the international committee on standardized genetic nomenclature for mice and rats, established in the early 1950s, is responsible for genetic nomenclature rules. the rules are available online at the mgi website (http:// www.informatics.jax.org/mgihome/nomen). inbred mouse strains are designated by a series of capital letters and/or numbers, which often provide a shorthand description of the origin and history of the strain. the c57bl/6j mouse serves as an example. the inbred strain c57bl originated from abbie lathrop's female 57 (and male 52) at the cold spring harbor laboratory (c), and was the black (bl) line from this female. early in their history, inbred c57bl mice split into major substrains, e.g., c57bl/6 and c57bl/10. substrains are identified by appending a forward slash (/) after the inbred strain name. since 1950, uniform international nomenclature has been built upon these historical names, so that substrains of an inbred strain are now designated using lab codes that are registered in the international laboratory code registry maintained at the institute for laboratory animal research (ilar) of the national academies (dels. nas.edu/global/ilar/lab-codes). laboratory codes are composed of one to five letters that identify an institute, laboratory, or investigator. each lab code starts with an uppercase letter, followed by lowercase letters if more than one letter is used (such as n, j, jci, crl, and tac). the j in c57bl/6j means it is a substrain maintained at the jackson laboratory (j). another common substrain of c57bl/6 mice is c57bl/6n, which is maintained at nih (n). substrains can be cumulative, reflecting the genetic history of the mouse strain. for example, there are a number of c57bl/6j substrains (such as c57bl/6jjci and c57bl/6jjmsslc), and a number of c57bl/6n substrains (such as c57bl/6njci, c57bl/6ncrlcrlj, dba/2j inbred strain named for its characteristic coat color genes (using their original gene symbols), dilute (d), brown (b), and nonagouti (a); it is the second of two sublines separated before 20 generations of brother × sister breeding and is the subline maintained at the jackson laboratory (j) c3h/hesn-ash/+ co-isogenic segregating inbred mutant strain carrying the ashen (ash) mutation, which arose on c3h/hesn c57bl/6j-tyrc-2j /+ co-isogenic segregating inbred mutant strain carrying the albino 2j mutant allele of the cloned tyrosinase gene (tyr) aej/gnj-a e /a w-j inbred strain segregating for two alleles at the agouti gene congenic inbred strain in which the b haplotype at the h2 complex was transferred from c57bl/6j (b6) to the akr background b6.cba-d4mit25-d4mit80 congenic strain in which the chromosomal segment between d4mit25 and d4mit80 was transferred from cba to b6 b6.cg m lepr db /++ congenic inbred strain in which the linked mutant genes misty (m) and diabetes (lepr db ) were transferred from multiple, mixed, or unknown genetic backgrounds to b6 and are carried in coupling, i.e., on the same chromosome b6.cg-m +/+ lepr db congenic inbred strain in which the m and lepr db mutations are carried in repulsion bxd-1/ty recombinant inbred (ri) strain number 1 in a set of ri strains derived from a c57bl/6j (b) female mated to a dba/2j (d) male and made by taylor (ty) recombinant congenic (rc) strain number 1 in a set made by crossing the balb/c (c) and sts (s) strains, backcrossing one or two times to balb/c and then inbreeding as with ri strains and c57bl/6ntac). significant differences may exist among these substrains (mekada et al., 2009 ). thus, a string of substrain designations indicate the genetic progression of the substrain, which can be identified when reading the entire strain name. this nomenclature is highly nuanced, as c57bl/6ncrlcrlj mice, whose last letter is a lowercase j, are not a substrain maintained at the jackson laboratory (j), but rather at charles river japan (crlj), underscoring the importance of upper-and lowercase lettering in rodent nomenclature. balb/c mice are another popular inbred strain with numerous substrains. like the '6' in c57bl/6, the 'c' that follows laboratory animal medicine of the mutational event as a superscript. for example, cftr tm1unc is a targeted mutation (tm), first line (1) congenic mice are often derived from 129 es cells, backcrossed onto a background strain, such as c57bl/6. under such circumstances, when the backcross generation is at n10, the '.' symbol is used between the background inbred strain and the donor strain (e.g., c57bl/6n.129p2/olahsd-abc tm1zzz , abbreviated as b6.129-abc tm1zzz . when backcrossing is incomplete but at the n5 generation, the mouse is an incipient congenic, designated with a ';' in lieu of a '.': b6;129-abc tm1zzz . if the background strain is mixed genetic origin, it is designated stock.129-abc tm1zzz . if the donor strain is mixed origin, it is designated 'cg'. for example, b6.cg-abc tm1zzz outbred stock that meets specific criteria is designated by placing the lab code before the stock symbol, separated by a full colon (':'). for example, hsd:icr designates an icr (swiss) outbred stock maintained by harlan sprague dawley (hsd). the above overview covers the nomenclature of commonly encountered types of mice. there are numerous additional specifications for nomenclature of mice. details are available at the mgi website (http://www. informatics.jax.org/mgihome/nomen). optimum housing conditions and husbandry practices for research mice should be guided by program requirements to ensure biosecurity, occupational health, efficient use of equipment, labor and financial resources, behavioral needs of mice, and investigator needs for consistent colony maintenance, including standardized husbandry practices and nutrition. the emerging interest in the mouse microbiome in combination with the immune competency of diverse genetically engineered mouse strains demands high standards of mouse care. mouse colonies are optimally maintained as specificpathogen-free (spf) which obligates veterinary and facility management to exclude specific organisms. housing options for spf immunocompetent mice typically include static or individually ventilated microisolator cages, which differ significantly in cost and labor required to maintain. severely immunodeficient strains such as nod.cg-prkdcscid il2rgtm1wjl/szj (nsg) mice require staff training, caging systems and husbandry practices that minimize risk for opportunistic infections the '/' in balb/c is a lowercase letter because of historical precedent. subsequent substrains follow accepted nomenclature, e.g., balb/cbyj and balb/cann. hybrids of two inbred strains are often used in research, and are particularly common with engineered mutations that are created in 129-derived es cells, followed by intercrossing the 129 chimeric mice with c57bl/6 or other background strains of mouse. when an f 1 hybrid is created, the female partner is listed first, e.g., a c57bl/6j × 129s2/svpas hybrid would be designated: c57bl/6j129s2/svpasf1. ri strain sets that are derived from two parental inbred strains are identified by an x between the two parental strains followed by a hyphen designating the specific ri line, e.g., c57bl/6jxdba/2j-1, c57bl/6jxdba/2j-2, etc. cc ri strains do not use the x between the parental strains because they are derived from eight parental strains, so they are designated cc-1, cc-2, etc. in order to simplify the complexity of this nomenclature, abbreviations are used for common inbred strains and substrains of mice (table 3 .4), but it is important to include the full genetic nomenclature in publications. using the abbreviated nomenclature, c57bl/6j129s2/svpasf1 mice would be b6129f1 and c57bl/6jxdba/2j-1 ri mice would be bxd-1. parental order is an important consideration in nomenclature, as a b6129 mouse is genetically different from a 129b6 mouse due to mitochondrial dna (from the female) and y chromosome (from the male) differences. mutant genes are designated by a brief abbreviation for the mutation (e.g., bg for beige which arose at the jackson laboratory, j). the symbol for the parent gene is noted in italics, starting with an uppercase letter (e.g., lyst) and the mutant allele is designated in superscript (e.g., lyst bgj ). thus, the beige mutation arose in c57bl/6j mice, so that c57bl/6j beige mice, which are co-isogenic with c57bl/6j mice, are designated c57bl/6j-lyst bgj . a transgenic strain is designated by the strain and substrain name, followed by a symbol for the transgene. transgene symbols take the form tg(yyy)#zzz, where 'tg' indicates transgenic, yyy defines the transgene as a brief description of the inserted dna (such as a gene symbol), '#' is the assigned number in the series of events generated using a given construct, and 'zzz' is the lab code. for example, fvb/n-tg(mmtv-erb2)1led mice are inbred fvb/n mice in which the rat erb2 gene was introduced under control of the mouse mammary tumor virus (mmtv) ltr promoter (mmtv-erb2), the first line (1) created in the laboratory of phil leder (lab code led). when a transgene causes an insertional mutation in an identified endogenous gene, the mutant allele of the gene is designated by using the gene symbol and an abbreviation for the transgene as a superscript (-abc tg1zzz ). a targeted mutation, or knockout, is designated by the mutated gene with the identification laboratory animal medicine (foreman et al., 2011) . barrier practices and microisolator techniques may include autoclaved or irradiated feed and bedding, autoclaved or acidified water, cage-tocage transfer of mice using disinfected forceps, positive displacement change hoods, and verified sanitation of caging and equipment through tunnel or rack washers to prevent fomite transmission of infectious agents (compton et al., 2012) . in addition to husbandry staff, it is critical to maintenance of colony health status that investigators who handle cages are also trained in these techniques. the microenvironment for mice is the cage which will vary in design, size, and composition. vendors often successfully house production colonies in open-top cages to expedite detection of pathogen transmission should a break occur. end-users usually prefer filter-top microisolator cages which prevent (at least) gross contamination between cages by fecal contamination and aerosolized debris. the objective is to keep mice in an uncrowded, socially compatible, low-odor, dry and clean environment. ambient temperature should minimize any confounding impact on the animal model and energy expenditure for the mice, while also being suitable for staff and investigators. shoebox static cages made of polycarbonate, polypropylene, or polystyrene plastic (in order of decreasing cost and durability) with filtered microisolator tops continue to be used for housing and breeding mice. older cage designs are being rapidly supplanted by individually ventilated caging systems that promote the advantages of increasing housing capacity, decreasing labor costs, and mitigating exposure of mice to noxious gases such as ammonia and exposure of humans to allergens. as more advanced caging systems are developed, the level of biosecurity may be increased but at the cost of increased health surveillance efforts to detect the source of an infectious outbreak (shek, 2008) . disposable, recyclable polyethylene caging is a recent innovation, particularly for facilities not equipped with a cage wash facility. animal care programs should carefully consider the necessity for housing mice on wire-mesh flooring because of injury risk to limbs and thermoregulation issues in neonates and hairless mice which are more difficult to maintain without nesting material. solidbottom cages should contain sanitary bedding, such as hardwood chips, paper products, or ground corn cob. criteria for selecting bedding vary with experimental and husbandry needs. it may be preferable to irradiate or autoclave bedding, but if this is not done, the bedding should be used only after its origin and microbial content have been evaluated (table 3 .5). germfree and gnotobiotic mice require positive pressure isolators, most usually flexible film, with additional protection provided by sterile air through high-efficiency particulate air (hepa) filters. this equipment can be negatively pressurized when the objective is to contain known or unknown pathogens. animal care programs should establish enrichment policies which for mice should include social housing when mice are compatible and experiments do not require single housing. species-specific behaviors are encouraged by nesting material and hiding places such as tubes or shacks. nutrient requirements for the mouse are influenced by genetic background, disease status, growth rate, pregnancy, lactation, and environmental factors such as ambient temperature. the best current estimate of nutritional requirements is shown in table 3 .6. nutritional requirements for laboratory mice are also published periodically by the national research council and have been reviewed by knapka and coworkers (knapka et al., 1974; knapka, 1983) . feed intake and weight gain data are used to estimate the nutritional needs of a particular stock or strain. mice consume about 3-5 g of feed per day after weaning, and maintain this intake throughout life. outbred mice tend to gain weight faster than inbred mice and are heavier at maturity (figs. 3.1 and 3.2). diet is often neglected as a variable in animal-related research. diet can influence responses to drugs, chemicals, or other factors and lead to biased research results. therefore, diet must provide a balance of essential kraft (1980) . knapka (1983) . b linoleic acid: 0.6% is adequate. c john and bell (1976) . d theuer (1971) . e knapka et al. (1974 ). f nutrition (1977 . g hurley and bell (1974) . h pleasants et al. (1973) . nutrients, and contaminants must be kept to a minimum (see also chapter 29). natural-product commercial diets for mice are usually satisfactory for breeding and maintenance. animal care programs should avoid using fresh produce, grains, fish meal, or other supplements to minimize exposure of colonies to pathogens or harmful chemicals such as pesticide residues or phytoestrogens (guerrero-bosagna et al., 2008) . mouse diets can be purchased as open-formula, fixedformula, constant nutrition, and closed-formula which laboratory animal medicine are designed to reduce variation in experimental data attributable to diet (reviewed in barnard et al. (2009) ). diets are supplied in standard, irradiated, or autoclavable formulations. irradiated diets will be virtually free of live microorganisms but have the risk of residual, radio-resistant bacteria. autoclavable diets are higher in heat-labile nutrient content. many programs use sterilized mouse chow exclusively to minimize risk of opportunistic infections. because commercial diets vary in nutrient content, diets should be selected for optimal maintenance of adult mice or for growth and reproduction in breeding colonies. mice should have continuous access to potable water even if a high-moisture diet is fed. water is needed for lubrication of dry food and for hydration. adult mice drink 6-7 ml of water per day. decreased water intake will decrease food consumption. water imbalance may occur immediately post weaning and weanlings on automatic watering systems need extra attention. water intake will decrease in sick mice. therefore, dosing mice with medicated water requires careful assessment of hydration and clinical or experimental efficacy of the compound administered. the main reference used to update this section of the 3rd edition is volume iii; normative biology, husbandry and models in the mouse in biomedical research, 2nd edition, aclam series published by academic press. normative data on the mouse are presented in table 3 .7, and clinical chemistry reference ranges are summarized in table 3 .8. mice have a relatively large surface area per gram of body weight. this results in dramatic physiologic changes in response to fluctuations in the ambient temperature (t a ). the mouse responds to cold exposure, e.g., by nonshivering thermogenesis. a resting mouse acclimated to cold can generate heat equivalent to triple the basal metabolic rate, a change that is greater than for any other animal. a mouse must generate about 46 kcal/m 2 per 24 h to maintain body temperature for each 1°c drop in t a below the thermoneutral zone. mice cannot tolerate nocturnal cooling as well as larger animals that have a greater heat sink. therefore, it is not advisable to conserve energy in animal quarters at night by lowering t a . because of the ratio of evaporative surface to body mass, the mouse has a greater sensitivity than most mammals to water loss. its biological half-time for turnover of water (1.1 days) is more rapid than for larger mammals. water conservation is enhanced by cooling of expired air in the nasal passages and by highly efficient concentration of urine. the conservation of water can preempt thermal stability. if the mouse had to depend on the evaporation of body water to prevent elevations of body temperature, it would go into shock from dehydration. the mouse has no sweat glands, it cannot pant, and its ability to salivate is severely limited. mice can partially compensate for changes in t a increases from 20°c to 35°c. it adapts to moderate but persistent increases in environmental temperature by a persistent increase in body temperature, a persistent decrease in metabolic rate, and increased blood flow to the ears to increase heat loss. its primary means of cooling in the wild is behavioral -retreat into a burrow. in the confinement of a cage, truck, or plane, mice do not survive well in heat and begin to die at an ambient temperature of 37°c or higher. thus, the mouse is not a true endotherm. in fact, the neonatal mouse is ectothermic and does not have well-developed temperature control before 20 days of age. the thermoneutral zone for mice varies with strain and with conditioning but is about 29.6-30.5°c, narrower than that of any other mammal measured thus far. thermoneutrality should not be equated with comfort or physiological economy. recent data have suggested that mice housed under routine vivarium conditions are chronically cold-stressed. mice maintained at 21°c were shown to expend more energy compared with mice housed at intermediate (26°c) and a higher temperature (31°c) with an increase in glucose utilization and activation of brown adipose tissue (david et al., 2013) . in contrast, other studies report that mice in a t a range of 21-25°c grow faster, have larger litters, and have more viable pups than those maintained in the thermoneutral zone. the respiratory tract has three main portions: the anterior respiratory tract consists of nostrils, nasal cavities, and nasopharnyx; the intermediate section consists of larynx, trachea, and bronchi, all of which have cartilaginous support; and the posterior portion of the respiratory tract consists of the lungs. the left lung is a single lobe. the right lung is divided into four lobes: superior, middle, inferior, and postcaval (cook, 1983) (fig. 3.3 loeb and quimby (1999) . a mouse at rest uses about 3.5 ml o 2 /g/h, which is about 22 times more o 2 /g/h than is used by an elephant. to accommodate for this high metabolic rate, the mouse has a high alveolar p o 2 ; a rapid respiratory rate; a short air passage; a moderately high erythrocyte (rbc) concentration; high rbc hemoglobin and carbonic anhydrase concentrations; a high blood o 2 capacity; a slight shift in the o 2 -dissociation curve, enabling o 2 to be unloaded in the tissue capillaries at a high p o 2 ; a more pronounced bohr effect, i.e., the hemoglobin affinity for o 2 with changes in ph is more pronounced; a high capillary density; and a high blood sugar concentration. the kidneys, ureters, urinary bladder, and urethra form the urinary system. the paired kidneys lie against the dorsal body wall of the abdomen on either side of the midline. the right kidney is normally located anterior to the left kidney. kidneys from males of many inbred strains are consistently heavier than kidneys from females. the glomeruli of mice are small, about 74 μm in diameter, or about half the size of glomeruli in rats. there are, however, 4.8 times as many glomeruli in the mouse, and the filtering surface per gram of tissue is twice that of the rat. mice excrete only a drop or two of urine at a time, and it is highly concentrated (table 3 .8). the high concentration is made possible by long loops of henle and by the organization of giant vascular bundles (vasa recta) associated with the loops of henle in the medulla. the mouse can concentrate urine to 4300 mosm/l, whereas humans can concentrate to a maximum of 1160 mosm/l. mice normally excrete large amounts of protein in the urine. taurine is always present in mouse urine, whereas tryptophan is always absent. creatinine is also excreted in mouse urine, a trait in which mice differ from other mammals. the creatinine/creatine ratio for fasting mice is about 1:1.4. mice excrete much more allantoin than uric acid. the submaxillary salivary gland, a mixed gland in most animals, secretes only one type of saliva (seromucoid) in the mouse. the tubular portion of the gastrointestinal (gi) tract consists of esophagus, stomach, small intestine, cecum, and colon. the esophagus of the mouse is lined by a thick cornified squamous epithelium, making gavage a relatively simple procedure. the proximal portion of the stomach is also keratinized, whereas the distal part of the stomach is glandular. gastric secretion continues whether or not food is present. the gastrointestinal flora consists of (at least) 1000 species of bacteria that begin to colonize the alimentary canal selectively shortly after birth. the ceca of normal mice contain up to 10 11 bacteria/g of feces. the bacteria throughout the gastrointestinal tract form a complex ecosystem that provides beneficial effects, such as an increase in resistance to certain intestinal pathogens, production of essential vitamins, and homeostasis of important physiological functions. gnotobiotic animals colonized with known microbiota have been used to great advantage as models for biomedical research (see chapter 39). for certain studies, it is desirable to colonize germfree mice with a defined microbiota. in the mid-1960s, schaedler was the first to colonize germfree mice with selected bacteria isolated from normal mice (schaedler and orcutt, 1983) . he subsequently supplied animal breeders with this group of microorganisms. these defined bacteria included aerobic bacteria and some less oxygen-sensitive anaerobic organisms. the so-called extremely oxygen-sensitive (eos) fusiform bacteria, which make up the majority of the normal microbiota of rodents, were not included, because of technical difficulties in isolation and cultivation. of the defined microbiotas later used for gnotobiotic studies, the one known as the 'schaedler flora' was the most popular. in 1978, the national cancer institute (nci) decided to revise the schaedler flora, or 'cocktail' consisting of eight bacteria, in order to standardize the microbiota used to colonize germfree rodents. the new defined microbiota, now known as the 'altered schaedler flora' (asf), consisted of four members of the original schaedler flora (two lactobacilli, bacteroides distasonis, and the eos fusiform bacterium), a spiral-shaped bacterium, and three new fusiform eos bacteria. studies have quantified the regional colonization of the asf strains along the gastrointestinal tract (sarma-rupavtarm et al., 2004) (fig. 3.4) . individual strain abundance was dependent on oxygen sensitivity, with microaerotolerant lactobacillus murinus asf361 present at 10 5 -10 7 cells/g of tissue in the upper gastrointestinal tract and obligate anaerobic asf strains being predominant in the cecal and colonic flora at 10 8 -10 10 cells/g of tissue. it is difficult to monitor a gnotobiotic mouse colony with a defined microbiota. it is necessary to demonstrate that microorganisms of the specified microbiota are present and that adventitious microorganisms are absent. in the past, monitoring relied on bacterial morphology, limited evaluation of biochemical traits, and growth characteristics. with the advent of polymerase chain reaction (pcr) technology, the eight asf strains were identified taxonomically by 16s rrna sequence analysis . three strains were previously identified as lactobacillus acidophilus (strain asf 360), l. salivarius (strain asf 361), and bacteroides distasonis (strain asf 519), based on phenotypic criteria. 16s rrna analysis and genome sequencing indicated that each of the strains differed from its presumptive identity (wannemuehler et al., 2014) . the 16s rrna sequence of strain asf 361 is essentially identical to the 16s rrna sequences of the type strains of l. murinus and l. animalis (both isolated laboratory animal medicine from mice), and all of these strains probably belong to a single species. strain asf 360 is a novel lactobacillus that clusters with l. acidophilus and l. lactis. strain asf 519 is a parabacteroides sp. the spiral-shaped strain, strain asf 457, is in the flexistipes phylum, exhibits sequence identity with rodent isolates of robertson, and has been formally named, mucispirillum schaedleri (robertson et al., 2005) . the remaining four asf strains, which are eos fusiform bacteria, group phylogenetically with the low-g + c content gram-positive bacteria (firmicutes, bacillus-clostridium group) (asf 492 -eubacterium plexicaudatium; asf 500 -firmicutes bacterium; asf 502 and asf 356 -clostridium sp.) (fig. 3.5) . the 16s rrna sequence information was determined by dewhirst et al. (1999) and draft genome sequences for each member of asf were recently published (wannemuehler et al., 2014) . this genetic data will permit detailed analysis of the interactions of asf organisms during development of intestinal disease in mice that are coinfected with a variety of pathogenic microorganisms. the lymphatic system consists of lymph vessels, thymus, lymph nodes, spleen, solitary peripheral nodes ( fig. 3.6) , and intestinal peyer's patches. mouse lymph nodes are numerous but typically are small, reaching only a few millimeters. the typical lymph node is beanshaped and consists of a cortex and a medulla. the cortex is divided into b lymphocyte domains, called primary follicles, and t lymphocyte domains, known s2 i1 i2 i3 i4 i5 i6 c1 c2 l1 l2 l3 e1 s1 s2 i1 i2 i3 i4 i5 i6 c1 c2 l1 l2 l3 e1 s1 s2 i1 i2 i3 i4 i5 i6 c1 c2 l1 l2 l3 e1 s1 s2 i1 i2 i3 i4 i5 i6 c1 c2 l1 l2 section of the gi tract bacteroides sp. asf519 the sections are taken from the esophagus (esop.) (section e1), stomach (sections s1 and s2), small intestine (sections i1 to i6), ileocecal junction and apical cecum (sections c1 and c2, respectively), and colon (sections l1 to l3). from sarma-rupavtarm et al. (2004). as the diffuse cortex. the mouse does not have palatine or pharyngeal tonsils. the spleen lies adjacent to the greater curvature of the stomach. different strains of mice have varying degrees of accessory splenic tissue. age, strain, sex, and health status can affect the size, shape, and appearance of the spleen. male spleens, e.g., may be 50% larger than those of females. most lymphocytes enter and leave the spleen in the bloodstream. the so-called white pulp of the spleen is organized along the central arteriole and is subdivided into t-and b-cell zones. the periarteriolar sheath is composed mainly of cd4 + and cd8 + t cells, and lymph follicles, which often contain germinal centers, are located at the periphery. the red pulp consists of sinusoids and hemoreticular tissue. cellular and humoral components of immunity are distributed to the bloodstream and tissues by efferent lymphatic vessels and lymphatic ducts, which empty into the venous system. the thymus is a bilobed lymphoid organ lying in the anterior mediastinum. it reaches maximum size around the time of sexual maturity and involutes between 35 and 80 days of age. the thymus plays a major role in maturation and differentiation of t lymphocytes. this function is not complete in newborn mice. thymectomy is routinely performed in immunological research for experimental manipulation of the immune system. thymectomy of newborn mice causes a decrease in circulating lymphocytes and marked impairment of certain immune responses, particularly cellular immune responses. thymectomy in adult mice produces no immediate effect, but several months later mice may develop a progressive decline of circulating lymphocytes and impaired cellular immune responses. the mutant athymic nude mouse is a powerful experimental tool in the study of the thymus in immune regulation (fogh, 1982) . the mucosa-associated lymph tissue (malt) contains more lymphoid cells and produces greater amounts of immunoglobulin than both the spleen and the lymph nodes. the term malt designates all peripheral lymphoid tissues connecting to cavities communicating with the external milieu. they include the peyer's patches, the cecal lymphoid tissue, and the lymphoid tissue in upper and lower respiratory tract, as well as the respiratory and genitourinary system. lymphatics drain these lymphoid-rich areas, thus providing a direct link with lymph nodes and the bloodstream. bone marrow and splenic red pulp produce erythrocytic, granulocytic, and megakaryocytic precursors over the life of the mouse. bone marrow is located in the protected matrix of cancellous bone and is sustained by reticular tissue rich in blood vessels and adipose cells (pastoret et al., 1998) . normal hematologic values are listed in table 3 .7. bone marrow-derived mononuclear phagocytes remove particulate antigens and act as antigen-presenting cells for lymphocytes. tissue macrophages, which often function in a similar way, are found in many tissues, including peripheral lymphoid tissues, lung, liver, intestine, and skin. the cardiovascular system of mice is reviewed extensively by hoyt et al. in the 2nd edition of volume iii; normative biology, husbandry and models in the aclam series the mouse in biomedical research (hoyt, 2007) . the heart consists of four chambers, the thin-walled atria and the thick-walled ventricles ( fig. 3.7) . mice conditioned to a recording apparatus have mean systolic blood pressures ranging from 84 to 105 mmhg. an increase in body temperature does not lead to an increase in blood pressure. heart rate, cardiac output, and the width of cardiac myofibers are related to the size of the animal. heart rates from 310 to 840/min have been recorded for mice, and there are wide variations in rates and blood pressure among strains. the skeleton is composed of two parts: the axial skeleton, which consists of the skull, vertebrae, ribs, and sternum, and the appendicular skeleton, which consists of the pectoral and pelvic girdles and the paired limbs. the normal vertebral formula for the mouse is c7t13l6s4c28, with some variations among strains, especially in the thoracic and lumbar regions. normal mouse dentition consists of an incisor and three molars in each quadrant. these develop and erupt in sequence from front to rear. the third molar is the smallest tooth in both jaws; the upper and lower third molar may be missing in wild mice and in some inbred strains. the incisors grow continuously and are worn down during mastication. the mouse brain has a typical mammalian structure as documented by a detailed study of the neuroanatomy of the c57bl/6j mouse (sidman et al., 1971) . more recently, gene expression patterns have been used to study the functional anatomy of the mouse brain (bohland et al., 2010) . use of wild-type and genetically modified mice in behavior, learning, and memory paradigms has exponentially increased over the last decade. the male reproductive organs consist of paired testes, urethra, penis, prostate and associated ducts and glands ( fig. 3.8) . the female reproductive organs consist of paired ovaries and oviducts, uterus, cervix, vagina, clitoris, and paired clitoral glands ( fig. 3.9 ). the clitoral glands are homologous to the male preputial glands and secrete a sebaceous substance through ducts entering the lateral wall of the clitoral fossa. the female mouse normally has five pairs of mammary glands, three in the cervicothoracic region and two in the inguinoabdominal region ( fig. 3 .10). the mammary glands are often not appreciated for how far they extend over the cervical, axillary, and inguinoabdominal flank regions which become the following section summarizes normal reproduction in the mouse. the reader is referred to a more comprehensive text in the aclam series (pritchett and taft, 2007) and online resources such as the jackson laboratories publication of the biology of the laboratory mouse (http://jaxmice.jax.org/jaxnotes/509/509j.html). external influences, such as noise, vibration, diet, light cycle, and cage density, and intrinsic factors, such as health status, genetics, and parity impact reproductive success by directly or indirectly influencing the hypothalamic-pituitary axis for hormonal control of ovarian and testicular function. genotype also dramatically affects the reproductive performance of the mouse. coincident with the explosion in the number of mouse strains, each with unique induced or spontaneous mutations, a sound breeding program must include training of care staff to recognize anticipated and unanticipated breeding performance and strain or stock characteristics. in the new age of genomics, older methods of confirming genetic purity of mouse lines are being replaced with formal genetic monitoring by comparing strain-specific panels of single-nucleotide polymorphisms (snps). follicle-stimulating hormone promotes gametogenesis in both sexes. luteinizing hormone promotes the secretion of estrogen and progesterone in the female and androgen in the male. prolactin promotes lactation and development of the ovary during pregnancy. these gonadal hormones also ensure proper maintenance of the reproductive tract and modulate behavior to promote successful mating. the hypophysis is usually responsive to hormonal influence by day 6 in the male and day 12 in the female. ovarian follicle development begins at 3 weeks of age and matures by 30 days. rising levels of gonadotropins evoke signs of sexual maturity at about the same age. in the female, estrogen-dependent changes such as cornification of vaginal epithelium at the vaginal opening can occur as early as 24-28 days. puberty is slightly later in the male (up to 2 weeks). sexual maturation varies among strains and stocks of mice and is subject to seasonal and environmental influences. mating behavior and the ability to conceive and carry fetuses to parturition are under complex hormonal control mediated by the anterior pituitary. the mouse is polyestrous and cycles every 4-5 days. in the first two phases (proestrus and estrus), active epithelial growth in the genital tract culminates in ovulation. degenerative epithelial changes occur during the third phase, followed by diestrus, a period of quiescence or slow cell growth. the cycle can be followed by changes in the vaginal epithelium that are often used to determine optimum receptivity of the female for mating and fertilization (table 3 .9). patency of the vaginal orifice and swelling of the vulva are useful signs of proestrus and estrus ( fig. 3.11) . irregularities of the estrous cycle occur during aging. seasonal and dietary factors, such as estrogenic substances found in a variety of feeds, and genetic backgrounds also influence estrous cycles. estrus is routinely observed in mice at about 14-24 h after parturition (postpartum estrus). however, cornification of the vagina is not complete, and fertile matings are not as frequent compared with normal estrus. mice are spontaneous ovulators. ovulation does not accompany every estrus, and estrus may not coincide with every ovulation, because estrus is dependent on gonadal hormones, whereas ovulation is responsive to gonadotropin. the cyclicity of estrus and ovulation is controlled by the diurnal rhythm of the photoperiod. mating, estrus, and ovulation most often occur during the dark phase of the photoperiod. reversing the timing cook (1983) . laboratory animal medicine of the light-dark cycle reverses the time of estrus, ovulation, and mating. pheromones (table 3 .10) and social environment also affect the estrous cycle. for example, estrus may be suppressed in group-housed female mice and reentry into estrus can be synchronized by exposure to pheromones in male mouse urine ('whitten effect'). once exposed to male urine, most female mice will be in estrus within 3 days with a second estrus in about 11 days. hence, estrus can be synchronized by group-housing females prior to pairing with males. in contrast, pheromones from a strange male mouse, particularly of a different strain, may prevent implantation or pseudopregnancy in recently bred females and is known as the 'bruce effect'. see section ii.c on behavior for more detail on the effect of pheromones on mouse reproductive behavior. mating is normally detected by formation of a vaginal plug (a mixture of the secretions of the vesicular and coagulating glands of the male) whose prevalence is highly strain dependent. the plug usually fills the vagina from cervix to vulva (fig. 3.11 ). plug detection is often coupled with vaginal cytology to evaluate fertility and conception. when the cervix and vagina are stimulated physically during estrus, prolactin is released from the anterior pituitary to enable the corpus luteum to secrete progesterone. secretion continues for about 13 days. if fertilization has occurred, the placenta takes over progesterone production. if fertilization does not occur, a pseudopregnant period ensues, during which estrus and ovulation do not occur. fertilization usually takes place (1) testis, (2) head of epididymitis, (3) caudal epididymitis, (4) vas deferens, (5) testicular vein, (6) ampullary gland, (7) seminal vesicle, (8) anterior prostate, (9) ureter, (10) bladder, (11) ventral prostate, (11') dorsal prostate, (12) urethra, (13) bulbourethral muscle, (14) ischiocavernosus, (15) bulbourethral gland, (16) diverticulum of bulbourethral gland, (17) penis, (18) preputial gland, (19) glans penis, (20) prepuce, (21) testicular artery, and (22) vas deferens artery. adapted from (komarek, 2007) . fallopian tube, (3) uterine horn, (4) endometrium, (5) cervix, (6) vagina, (7) vaginal vestibulum, (8) clitoris, (9) clitoral gland, (10) urethra, (11) bladder, (12) medial ligament of bladder, (13) lateral ligament of bladder, (14) left ureter, (14') right ureter, (15) mesovarium, (16) mesometrium, (17) ovarian artery, (18) uterine horn artery, and (19) ovarian artery and vein. adapted from (komarek, 2007) . adapted from komarek (2007) . in the ampulla or the upper portion of the oviduct. ova can be fertilized to produce normal embryos for 10-12 h after ovulation. gestation is usually 19-21 days. because of postpartum estrus, lactation and gestation can occur simultaneously. lactation can delay gestation because of delayed implantation. this may cause prolongation of gestation for up to 12-13 days in certain inbred strains. the effective reproductive life of some inbred strains approaches 2 years where optimum environmental conditions are maintained, but litter size usually decreases as the female ages. therefore, females are usually retired by 6 months of age. average litter size is strain dependent and commonly ranges from 1 to 12 pups. maternal care can account for about 70% of the variation in body weight of neonatal mice. nursing females usually lactate for 3 weeks. milk production increases up to 12 days postpartum and then declines until weaning at 21 days. interestingly, oxytocin is required for nursing but is not essential for parturition or reproductive behavior (nishimori et al., 1996) . some transmission of humoral immunity from dam to progeny occurs in utero, but the majority of antibody is transferred through colostrum. transmission of passive immunity by colostral antibodies has been demonstrated to a wide variety of antigens, including viruses, bacteria, and parasites. antibodies continue to be secreted in the milk throughout lactation. decay of maternally acquired immunity occurs within several months after weaning. loss of maternal immunity increases susceptibility to infection and warrants continued care of weaned mice under barrier conditions. mice are socially gregarious animals with strong family bonds who communicate through complex olfactory, auditory, tactile, and visual signals. wild mice aggregate into groups called demes with low exchange of individuals between different groups. each deme consists of kinrelated members with a high degree of natural inbreeding, higher mutation rates compared to other mammals, and a wide range of developmental flexibility based on early life experience, which all contribute to their remarkably successful environmental adaptability. the deme is composed of a dominant breeding male, a hierarchy of females, subordinate males, and juveniles. wild mice occupy territories measuring just a few square meters when food is abundant to several square kilometers. mice are crepuscular (active during the twilight hours of dawn and dusk), strongly territorial, and omnivorous. coprophagy contributes to approximately one-third of their ingesta as an essential nutritional activity. aside from territoriality, social interactions, breeding, burrowing (when conducive substrates are available), and nest building are major activities. in managing laboratory mice, it is important to understand the complex behavioral biology of their free-living counterparts (latham and mason, 2004) . chemo-olfactory communication is mediated through extremely diverse chemical factors that trigger innate (non-learned) social responses among conspecifics, known as pheromones (table 3 .10). pheromones have been traditionally divided into two broad categories: releaser pheromones, which elicit an immediate behavioral response, and primer pheromones, which mediate a slowly developing and longer-lasting endocrine response. this original definition of pheromone categories has been expanded to another category, termed signaler pheromones, which convey individual or group identity, as well as mediating parent-offspring recognition and mate choice. the biology and genetics of pheromone signaling is being extensively studied in the mouse as a model of mammalian pheromone communication (brennan and zufall, 2006; rodriguez and boehm, 2009) . mouse pheromones are excreted in the urine, as well as plantar, salivary, lacrimal, preputial, and mammary glands. in the urine, major urinary proteins (mups), small peptides, mhc class i peptides, volatile chemicals, and sex hormones all contribute to chemosignals that communicate dominance, kinship, diversity, and gender. wild mice possess a great deal of individual variations of roberts et al. (2012) . c ferrero et al. (2013) . laboratory animal medicine these elements, providing a 'bar code' that distinguishes individuals. inbreeding of laboratory mice has reduced individual variation, but each inbred strain possesses a characteristic array of signals, and to a certain extent, unique signals exist among individuals within a strain (sharrow et al., 2001; sturm et al., 2013) . pheromones are detected by sensory neurons in the vomeronasal organ, the olfactory epithelium, and the lesser known septal organ of masera within the olfactory epithelium, and the gruenberg ganglion, which is located at the anterior end of the nasal cavity (breer et al., 2006; chamero et al., 2012; liberles and buck, 2006; restrepo et al., 2004) . neuronal signals are transmitted to the ganglion layer of the olfactory bulb, and thence to the brain. mups are important components of chemosensory communication in mice, and also an important occupational hazard to human handlers. chromosome 4 contains a cluster of 21 mup genes, plus a number of pseudogenes. mups are small soluble proteins known as lipocalins, which bind small organic chemicals (pheromones) with high affinity, and function as pheromone transporters and stabilizers (thereby contributing to slow release), but also act as protein pheromones themselves. they are synthesized in the liver and excreted in the urine, as well as nasal mucosa, lacrimal glands, and salivary glands. their endogenous role on metabolic activity is not yet understood. male mice excrete significantly more mups in the urine than females. one wellcharacterized mup is 'darcin', named after fitzwilliam darcy, the romantic hero in pride and prejudice. as its name implies, it is a female attractant. mups also act as kairomones, which function as chemical signals between species. for example, cat and rat mups invoke fear in mice. mups are important in the laboratory animal management context, as they are excreted in copious amounts (1-5 mg/ml in urine) and are potent allergens for humans, particularly mus m 1 (ag1 or ma1), which is encoded by the mup 17 gene (sharrow et al., 2001) . chemosensory communication has numerous behavioral effects that influence mouse social interactions. one of the most studied behavioral effects is the bruce effect, or pregnancy block, which is a complex physiologic response in which recently conceived females resorb fetuses during early pregnancy in the presence of an unrelated male, particularly a dominant male. the continued presence of the original mate protects the female from this effect (bruce, 1959) . the vandenbergh effect results in acceleration of puberty of juvenile females in response to male urine (vandenbergh, 1973) . the lee-boot effect occurs among group-housed females that are isolated from males, in which there is suppression of estrus cyclicity (van der lee and boot, 1955) . the whitten effect results in synchronization of estrus among a group of females in response to a male (whitten et al., 1968) . the lee-boot and whitten effects are utilized in the laboratory to assist in induction of synchronized timed pregnancy, but the bruce effect can have deleterious consequences on breeding colonies when foreign males are introduced to a breeding colony, as pheromone communication can occur in the absence of direct contact. the above effects are well-defined pheromone-driven behavioral responses, but chemosensory communication has a myriad of other effects. estrus, pregnant, or lactating females also accelerate puberty among juvenile females. females use odor cues to avoid parasite-laden males, males prefer odors of estrus females, and estrus females prefer odors of dominant males. mice have strong mating and social preferences based upon mhc proteins, which indicate genetic relatedness. maternal recognition of young is also mhc-related, and pups prefer nest odors of maternal and sibling pups based upon mhc relatedness. male aggression against unrelated males is also a strong mhc-related phenomenon. mhc haplotypes determine not only mhc proteins in the urine, and mhc-specific olfactory receptors, but also the composition of volatile chemicals in the urine (kelliher and wersinger, 2009) . the complexity of social communication extends to auditory stimuli as well. male mice utilize ultrasonic 'birdsong' to vocally communicate and attract females. mouse vocalization patterns are largely genetically innate and unique to each strain of mouse, but they can also be modified, or learned, to a limited extent (arriaga et al., 2012) . the behavioral biology of the mouse is highly complex, and depends upon genetic, physiologic, social, and environmental variables, which all impact on how laboratory mice can best be managed in captivity. it is clear that this rich complexity cannot be fully addressed under laboratory conditions, but that does not mean that basic needs, such as nest building, burrowing, foraging, and olfactory environments, cannot be provided. for example, intermale aggression, which is particularly apparent in some strains of mice such as balb/c and swiss-origin stocks and strains, can be minimized by maintaining males from infancy as sibling groups, since adult siblings tend not be aggressive to one another. this sibling bond, however, can be easily broken by short-term separation. environmental enrichment often features provision of plastic houses, which may make vivarium managers feel good, but maximal enrichment can be provided by provision of nesting material, which includes structural scaffolding, such as crinkled cardboard, which facilitates construction of three-dimensional nests. mouse nests are replete with 'appeasement' pheromones, thereby contributing to harmony within the cage, whereas introduction of dirty bedding has the opposite effect. frequent cage changing, including removal of established nests, is highly stressful and disruptive to social harmony within a cage. provision of appropriate and adequate amounts of bedding material that is conducive to burrowing is desirable. it is important to remember that mice are socially gregarious, and that mouse welfare is optimally enriched by other mice within a socially harmonious deme (latham and mason, 2004; van loo et al., 2003) . a laboratory mouse ethogram, defined as an operationalized list of mouse behaviors, arranged by their adaptive meaning to the animal, is available on the web: www. mousebehavior.org. behavioral phenotyping, particularly of transgenic mice, is used extensively in genomic research. a wide variety of standardized test batteries and approaches are used, depending upon the focus of research (reviewed in crawley 2008) . initial behavioral evaluations include general health, body weight, body temperature, appearance of the fur and whiskers, and neurological reflexes assessment. specific tests include observations of home cage behaviors, righting reflex, acoustic startle, eye blink, pupil constriction, vibrissae reflex, pinna reflex, digiscan open field locomotion, rotarod motor coordination, hanging wire, footprint pathway, visual cliff, auditory threshold, pain threshold, and olfactory acuity. novel and complex environmental enrichment in animal housing conditions facilitates enhanced sensory and cognitive stimulation as well as physical activity. environmental enrichment and exercise have beneficial effects such as cognitive enhancement, delayed disease onset, enhanced cellular plasticity, and associated molecular processes in animal models of brain disorders (pang and hannan, 2013) . the immune system of the mouse is very similar to that of humans. the availability of inbred mouse strains, in which each individual animal expresses identical mhc alleles so that tissues and cells can be transplanted without tissue rejection, greatly simplifies and indeed enables functional analyses of immune system components not possible with any other outbred mammalian species. in addition, the ability to genetically manipulate the mouse genome, adding to, altering, and deleting existing genes, enables unprecedented in vivo analysis of immune cell functions. it is for these reasons that the mouse is the primary animal model for immunology research. the immune system is an unusual organ system in that it consists of both solid tissues and various migrating cell populations. the bone marrow and thymus are considered primary lymphoid organs, as sites of hematopoiesis and b-and t-lymphocyte development, respectively. lymph nodes, spleen, and intestinal peyer's patches are considered secondary lymphoid tissues, as sites of immune response initiation. lymph nodes and spleen are analyzed frequently for studies of immune responses and as organs for immune cell isolation. tertiary lymphoid tissue sites are those that form in other solid organs in response to an insult or microbial exposure. among them are the lymphoid cell aggregates of the gastrointestinal and respiratory tract, also called 'gut-associated lymphoid tissue' (galt) and bronchusassociated lymphoid tissues (balt). leukocytes are classified as belonging to the innate or adaptive immune system. the innate immune system responds rapidly to an antigen insult via recognition of pathogen-associated molecular patterns (pamps), such as lipopolysaccharide, bacterial flagellin, single (s)-and double-stranded (ds) rna, and non-methylated dna, via extracellular or intracellular pattern recognition receptors (prrs). receptors include the toll-like receptors (tlrs), such as tlr4 (recognizing lps), tlr3/7 (ss and dsrna) and tlr9 (dna), nod-like receptors (nod1/2), and rig-like receptors (rig-i, mda-5) among others (takeuchi and akira, 2010) . cells of the innate immune system are monocytes/macrophages, granulocytes and dendritic cells as well as innate-like lymphocyte populations (ilc) 1, 2 and 3, which include natural killer (nk) cells (spits et al., 2013) . cells of the adaptive immune system (t and b lymphocytes) express a highly antigen-specific receptor that has arisen through gene rearrangement (t-cell and b-cell receptors, respectively). b cells of the b-1 lineage and γδ t cells are regarded as innate-like cells, as they express a rearranged antigen receptor but seem to respond in an innate-like manner. leukocytes are identified and classified by sets of monoclonal antibodies (mab) against uniquely expressed surface receptors, typically measured by flow cytometry. identification of a unique receptor by one or more mab of the same specificity leads to the assignment of a receptor name, as a 'cluster of differentiation (cd)'. for example, t cells are differentiated into two subsets based on their expression of either cd4 or cd8. cd4 + t cells (t helper cells) recognize peptides presented in mhc class ii and promote b-lymphocyte activation and activate and regulate cellular immune responses via secretion of differing cytokines (see below). cd8 + t cells recognize antigenic peptides presented in mhc class i and serve as cytotoxic cells during the cell-mediated immune response where they can destroy infected cells (e.g., against cells containing infectious agents). the major function of b cells is to respond to an encounter with an antigen/pathogen with the production of highly antigen-specific immunoglobulins (ig; antibodies), which can bind to and inactivate pathogens and toxins. activation of b cells can lead to their differentiation to plasma cells, which produce large amounts of ig. five laboratory animal medicine classes or ig 'isotypes' can be distinguished, which differ in effector function: igm, igg, iga, ige, and igd. the latter is expressed only on the surface of b cells in mice. the igg class, the most abundant antibody class in the serum, is further divided into subtypes: igg 1 , igg 2a/c , igg 2b , and igg 3 . polymorphisms exist on the ig locus such that some strains of mice produce the igg2a subtype (e.g., balb/c), whereas others produce igg2c (e.g., c57bl/6) (zhang et al., 2012) . additional allelic polymorphisms of the locus also exist. for example, balb/c and 129sv mice express the igh-a allotype, whereas c57bl/6 mice express the igh-b allotype. recombinant inbred strains of mice exist for both balb/c and c57bl/6, which harbor the reciprocal igh locus (i.e., igh-b for balb/c and igh-a for c57bl/6 mice). these mice are useful tools for tracking b cells following adaptive cell transfer via allotype-specific mab (see below). immunoglobulin isotype production varies according to the type of immunogen used to evoke the response. igm is secreted short term after initial exposure to an antigen, followed by the other ig isotypes. in viral and intracellular bacterial infections, igg 2a/c is dominant, whereas in extracellular bacterial infections igg 1 dominates the response. igg 2b and igg 3 are usually induced to carbohydrate or lipid antigens. ige is linked to parasitic infections and to allergy. serum antibodies specific for an immunogen can often be measured for the life of the animal. while serum iga levels are low, iga is the highest produced ig in mice. iga production, however, occurs in plasma cells lodged in the lamina propria of mucosal tissues, from where the iga is actively transported in dimeric form onto the luminal surface of mucosal tissues as 'secretory' iga (brandtzaeg, 2009 ). cytokines are secreted signaling molecules involved in cell-cell communication in a complex biological system (table 3 .11). these include the large family of interleukins (ils, currently il-1 to il-37), tumor necrosis factors (tnfs), interferons (type i, ii, and iii) and growth factors such as granulocyte-macrophage colonystimulating factor (gm-csf) and stem cell factor (scf). cytokine secretion often occurs in response to recognition of antigen via prr or tcr. because of their importance in modulating immunity to antigenic stimuli, mice with specific deletions or overexpression of individual cytokines have been made and have contributed to a detailed understanding of many of their often pleotropic functions (akdis et al., 2011) . chemokines are a similarly large group of small, secreted molecules that regulate cell trafficking to sites of antigen encounter but also facilitate cell-cell contact by acting as chemoattractants. chemokines are grouped according to the number of cysteines and disulfide bonds in the molecule into c-x-c-, c-c, c, and cx 3 cl chemokine ligands (l) and receptors (r) and designated accordingly as cxcr1-7/cxcl1-16 and ccr1-10/ccl 1-28 (allen et al., 2007) . immune responses must be coordinated to provide the most appropriate effector functions for the type of pathogen/antigen encountered. immune effector responses differ depending on the life cycle (facultative or obligate intracellular, extracellular, localized, systemic, etc.) and antigen types displayed by the encountered antigen/ pathogen, because this affects the type of prr engaged and activated. prr engagement leads to cytokine and chemokine responses by the first responders, i.e., epithelial cells, local macrophage populations and other innate cells. the type of cytokines and chemokines produced then dictates the types of cells recruited to the site of infection and their subsequent differentiation and functions. the prr engagement also leads to antigen uptake, activation and migration of dendritic cells (dcs) from the site of insult to the regional lymph nodes, where dcs present antigen peptides on mhc molecules to t cells. in addition, the dcs secrete cytokines induced by the initial prr activation, which cause the differentiation of cd4 t cells towards a particular effector response. for example, secretion of il-12 in response to activation of tlr2 or 4 will result in the induction of interferongamma (ifn-γ) production by cd4 t cells, whereas il-6 and tgf-β production by dc will induce cd4 t cells to secrete il-17 (kara et al., 2014) . because the dc translates signals from prr at the site of infection into differentiation signals for t cells in the lymph tissues, these cells are regarded as a 'bridge' between the innate and adaptive immune systems. the specific ig isotype secreted in response to a pathogen depends to a large degree on the type of cytokine produced by cd4 t cells that provide 't-cell help' for b cells. t cells that interact with b cells are identified as a discrete subset termed 't follicular helper cells (t fh )' and it is their cytokine profile that directs b cells to secrete a particular ig isotype (kara et al., 2014) . the classic t h 1/t h 2 dichotomy outlined above was in part shaped by the observation that ifn-γ production will lead to switching of b cells to secrete igg2a/c, whereas production of il-4 leads to the secretion of igg1. interestingly, it appears that the cytokine profile induced by the effector t-cell population is mirrored by the innate immune response. innate-like lymphocytes also have effector phenotypes that correspond to those of cd4 t cells and are induced by the same signals and transcriptional regulators (spits et al., 2013) and the same appears to be true also for macrophages and other innate immune cells (sica and mantovani, 2012) . while initial studies identified two particular antagonistic effector response types (termed t h 1 and t h 2 and classified by t-cell production of ifn-γ and il-4, respectively), more recent studies now demonstrate a much wider array of effector responses in which innate and adaptive immunity acts together to reinforce an immune response phenotype as well as modulate its size by induction of t regulatory cells (t regs ) that generate inhibitory cytokines (kara et al., 2014; sica and mantovani, 2012; spits et al., 2013) . the use of cytokine-deficient and reporter mice that enabled the identification of cytokine-producing cells via expression of a fluorescent reporter was particularly valuable for the development of this more nuanced view of the quality of immune responses. spontaneous mouse models of immune deficiencies have been used extensively in research. their use, plus the expanding number of knockout, transgenic, and dominant negative mouse mutants, has advanced understanding of human immune deficiency diseases as well as basic understanding of the immune system (table 3 .12). interbreeding of multiple immune-deficient mice has allowed the development of 'humanized' mice in which immune cells of the mouse are replaced with those of humans. while many challenges remain to fully replenish mice with components of the human immune system, the use of immune-deficient nod/severe combined immunodeficient (scid)/il-2rγ -/recipients for transfer of human peripheral blood lymphocytes, cordblood or bone marrow-derived cd34+ stem cells with human liver and thymus (blt-mice) is yielding promising results (akkina, 2013) . investigators using genetically engineered mice are constantly reminded that phenotypic analysis of these animals must be done cautiously because the immune system may be profoundly affected and in ways that are not always anticipated. this may make it difficult to determine whether a given gene product is directly involved or may be secondary to a more global dysregulation of the immune system. as with other biological systems, compensation mechanisms also may mask the phenotype. experimental approaches are being increasingly used to refine the knockout technology by restricting a specific genetic deficiency to a particular tissue of interest using the cre-lox system, in which tissue-specific or temporal restricted expression of the cre recombinase induces the deletion of a 'floxed' gene (mak et al., 2001) . transgenic mice are available that restrict cre expression to various hematopoietic cells or tissue or drive cre recombination following injection of tamoxifen. other approaches are the generation of 'bone marrow irradiation' chimeras. here, inbred wild-type mice or mice deficient in certain immune cells (table 3 .12) are lethally irradiated by exposure to a gamma-irradiation source to deplete the hematopoietic stem cells. these are then replaced by transfer of bone marrow cells to the irradiated mice. reconstitution of the hematopoietic system is usually achieved within about 6 weeks, during which time mice are provided with antibiotic-containing drinking water to avoid infections of these temporarily immune-compromised animals. transfer of bone marrow from a congenic knockout restricts the genetic defect to the hematopoietic system. a mix of bone marrow from two sources is also often used to generate tissue-specific knockouts. for example, mixing a bone marrow from t-cell-deficient mice (75%) with that of a gene knockout (25%) generates 'mixed bone marrow chimeras' in which all t cells only develop from the knockout, thus lack the gene of interest, whereas most of the other cells t from the wild-type source, effectively constraining the genetic defect to the t-cell population. sets of congenic mice with defined allotypic differences are often used to confirm the source of individual cells. such markers include the gene locus cd45.1/cdc45.2 or cd90.1/cd90.2 (thy1.1/thy1.2). alternatively, cells may express a fluorescent transgene, such as green-fluorescent protein (gfp). identification is usually performed by flow cytometry, or less commonly by immunofluorescence or immunohistochemistry. generation of bone marrow chimeras circumvents the time-consuming breeding of cre recombinase-expressing flx/flx mice. however, numerous controls are needed to exclude off-target effects due to irradiation damage. repeat injection of antibodies targeting specific cell populations is another rapid approach that avoids the potential for irradiation damage and allows short-term depletion of individual cell subsets. its main disadvantage is the need to identify mab that bind to surface receptors uniquely expressed by a cell subset of interest and the verification of the efficacy of the depletion. frequently used is antibody treatment for the short-term depletion of t-cell subsets using mab against cd4 or cd8 as well as individual cytokines. contemporary knowledge about diseases of laboratory mice has developed primarily from examining the effects of disease on traditional strains and stocks. the widespread use of genetically engineered mice is likely to modify current concepts because of novel or unpredictable interactions among genetic alterations, the genetic backgrounds on which they are expressed, and exogenous factors, such as infectious agents. because the number of combinations is extraordinarily high, clinical and laboratory diagnosticians should be alert to the potential for altered disease expression in genetically engineered mice and not be misled by unexpected signs, lesions, and epizootiology. many microbial agents have the potential to cause disease in mice or interfere with mouse-based research. housing and husbandry in microbiologically sheltered environments are designed to reduce the risks of disruptive infection, especially among immunologically dysfunctional mice, but must be accompanied by effective microbiological surveillance. surveillance should encompass resident mice and mouse products (serum, cell lines, transplantable tumors) procured from external sources. because surveillance strategies will vary with research needs and operating conditions, it is prudent to consult a number of sources, such as the federation of european laboratory animal science associations (felasa) (nicklas et al., 2002) and commercial laboratories, for guidance. detailed discussion of microbial quality control is provided in chapter 10. there are also recommendations regarding specific agents in following sections. diagnostic methods involve gross and microscopic pathology, parasitology, microbial isolation and culture, serology, and pcr. serology is particularly important for viral surveillance, and now relies principally on enzyme-linked immunosorbent assay (elisa), multiplex fluorescent immunoassay (mfi) for simultaneous detection of antibodies to multiple agents (hsu et al., 2007) , indirect fluorescent antibody (ifa) assay, or hemagglutination inhibition (hai), with the latter two methods generally used for confirmation (livingston and riley, 2003; pritchett-corning et al., 2009) . mouse antibody production (map) testing has been historically used for testing biological materials for contamination by infectious agents. pcr panels for murine infectious agents are now commercially available and have cost and time-saving advantages as well as improved assay sensitivity and specificity. beyond the classic bacterial and viral murine infections, pcr assays are now available for endo-and ectoparasites (see chapter 11). etiology mousepox is caused by ectromelia virus (ectv), an orthopoxvirus that is antigenically and genetically closely related to a number of other poxviruses, including vaccinia, variola, and cowpox viruses. the original isolate of ectv, known as the hampstead strain, was discovered by j. marchal in 1930 (marchal, 1930 as the cause of epizootic disease among laboratory mice in england. the disease featured amputation of extremities, which marchal termed ectromelia (from the greek, ectro, amputation and melia, limb). other strains of the virus include moscow, nih-79, washington university, st. louis 69, beijing 70, ishibahsi i-iii, and naval (nav) strains, which vary in virulence, but are essentially indistinguishable genetically and serologically, suggesting a common origin. virus can be isolated from infected tissues by inoculation of cell cultures (bs-c-1, hela, l cells) or embryonated eggs. the natural host (and original source of infection of laboratory mice) of ectv remains unknown. clinical signs the expression of clinical signs reflects an interplay among virus-related factors, including virus strain, dose and portal of entry, and host-related factors, including age, genotype, immunological competence, and gender (brownstein et al., 1991a) . during natural epizootics, it was observed that a, bc, dba/1, dba/2, and cba strains developed acute fatal infections, whereas c57bl/6 mice were resistant to severe disease (briody, 1966) . experimental studies have shown that all strains of mice are susceptible to infection, but balb/c, a, dba/2, and c3h/he mice were highly susceptible, akr and sjl mice were moderately susceptible, and c57bl/6 mice were highly resistant to lethal infection (bhatt and jacoby, 1987c; wallace and buller, 1985) . the mechanisms of genetic resistance are not fully understood but appear to reflect multiple genes, some of which appear to be expressed through lymphoreticular cells, including nk cells (brownstein et al., 1991a; jacoby et al., 1989) . the nuances of cytokine and cellular immune responses to ectv infection have received recent attention (reviewed in buller and fenner (2007) and esteban and buller (2005) ). outbreaks among susceptible mice are often volatile, with variable morbidity and high mortality in susceptible strains of mice. clinical signs such as ruffled fur or prostration may occur for only a few hours before death. mice that survive acute infection may develop chronic disease characterized by a focal or generalized rash anywhere on the body ( fig. 3 .12). conjunctivitis also may occur. skin lesions usually recede within several weeks, but hairless scars may remain. additionally, severe viral infection of the feet and tail during the rash syndrome can lead to necrosis and amputation. epizootiology mousepox is not a common disease. outbreaks occur sporadically and recent outbreaks have been traced to the importation of contaminated mice or mouse products. for example, contaminated mouse serum was responsible for recent outbreaks in the united states (dick et al., 1996; . natural exposure is thought to occur through direct contact and skin abrasions. cage-to-cage transmission is low and can be virtually nil if filter-topped cages are used (bhatt and jacoby, 1987b) . ectromelia virus is highly stable at room temperature, especially under dry conditions, leading to the potential for prolonged environmental contamination in infected colonies (bhatt and jacoby, 1987d) . aerogenic exposure is not a major factor in natural outbreaks, and arthropod-borne transmission does not appear to occur. virus-free progeny can be obtained laboratory animal medicine from immune dams (bhatt and jacoby, 1987b) . however, intrauterine infection and fetal deaths, albeit rare, have been reported. natural transmission is facilitated by intermediately resistant mice, which survive long enough to develop skin lesions that can shed virus for relatively long periods of time. the risks for transmission are further increased by persistence of infectious virus in excreta and exfoliated scabs. although virus excretion typically lasts for about 3 weeks, virus has been found in scabs and/or feces for up to 16 weeks. resistant mouse strains also are dangerous because they can shed virus during subclinical infections. however, infections in resistant mice tend to be short-lived. highly susceptible mice are a relatively small hazard for dissemination of infection, if properly discarded, because they die before virus shedding becomes prominent. thus, juxtaposition of resistant or intermediately resistant infected mice with highly susceptible mice can provoke explosive outbreaks. infant and aged mice are usually more susceptible to lethal infection than young adult mice. maternal immunity among enzootically infected breeding mice may perpetuate infection by protecting young mice from death, but not from infection. such mice may subsequently transmit infection by contact exposure. pathology the classic descriptions of ectv pathogenesis by fenner remain timely, including the frequently cited and reproduced figure summarizing the pathogenesis of infection ( fig. 3 .13) (fenner, 1948b) . interest in smallpox has renewed the interest in ectv as a model of host response to infection (esteban and buller, 2005) . ectv multiplies in the cell cytoplasm and produces two types of inclusion bodies. the a type (marchal body) is well demarcated and acidophilic in histological sections. it is found primarily in epithelial cells of skin ( fig. 3 .14) or mucous membranes and can also be found in intestinal mucosa. the b type of inclusion is basophilic and can be found in all ectromelia-infected cells. however, it is difficult to visualize unless cells are stained intensely with hematoxylin. ectv antigen can be readily visualized by immunohistochemistry on formalin-fixed, paraffin-embedded tissue sections (esteban and buller, 2005; jacoby and bhatt, 1987) . following skin invasion, viral multiplication occurs in the draining lymph node and a primary viremia ensues. splenic and hepatic involvement begin within 3-4 days, whereupon larger quantities of virus are disseminated in blood to the skin. this sequence takes approximately 1 week and, unless mice die of acute hepatosplenic infection, ends with the development of a primary skin lesion at the original site of viral entry. the primary lesion is due to the development of antiviral cellular immunity. severe hepatocellular necrosis occurs in susceptible mice during acute stages of mousepox. white spots indicative of necrosis develop throughout the liver ( fig. 3.15 ). in nonfatal cases, regeneration begins at the margins of necrotic areas, but inflammation is variable. splenic necrosis in acute disease commonly precedes hepatic necrosis but is equally or more severe. necrosis and scarring of red and white pulp can produce a macroscopic 'mosaic' pattern of white and red-brown the primary skin lesion, which occurs 6-10 days after exposure, is a localized swelling that enlarges from inflammatory edema. necrosis of dermal epithelium provokes a surface scab and heals as a deep, hairless scar. secondary skin lesions (rash) develop 2-3 days later as the result of viremia. they are often multiple and widespread and can be associated with conjunctivitis, with blepharitis, and, in severe cases, with buccal and lingual ulcers. the skin lesions also can ulcerate and scab before scarring. diagnosis mousepox can be diagnosed from clinical signs, lesions, serological tests, and demonstration of virus or viral antigen in tissues. observation of characteristic intracytoplasmic eosinophilic inclusions aids detection of infection. several serological tests are available to detect mousepox. historically, the standard test was hai, using vaccinia antigen as a source of hemagglutinin. elisa is more sensitive and specific and has replaced hai for serological monitoring among nonvaccinated mice (buller et al., 1983) . ectv infection also can be detected by ifa (buller et al., 1983) and pcr. serological differentiation of mousepox from vaccinia infection in vaccinated mice is based on the lack of hemagglutinin in the vaccine strain of virus. thus, serum from vaccinated mice may react by elisa but should not react by hai. differential diagnosis mousepox must be differentiated from other infectious diseases associated with high morbidity and high mortality. these include sendai pneumonia, mouse hepatitis, and tyzzer's disease. the latter two can be expressed by acute necrosis in parenchymal organs, but they can be differentiated by morphological, serological, and virological criteria. the skin lesions of chronic mousepox must be differentiated from other skin diseases caused by opportunistic or pathogenic bacteria, ascariasis, and bite wounds. prevention and control mousepox is a dangerous disease because of its virulence for susceptible mice. therefore, infected colonies should be quarantined immediately. depopulation has been used as a primary means for control, but confirmation of infection should be obtained before exposed mice are destroyed. tissues, supplies, instruments, or other items that have had potential contact with infected mice should be disinfected by heat or chemicals such as formalin, sodium hypochlorite, or chlorine dioxide. materials should be autoclaved or, preferably, incinerated. disinfected rooms should be challenged with susceptible sentinel animals that are observed for clinical signs and tested for seroconversion after several weeks. depopulation and disinfection must be carried out vigorously. because modern housing and husbandry methods based on the use of microbarrier caging are effective for containing infection, testing and culling properly isolated mice is a potential alternative, especially for irreplaceable breeding mice. such mice can be quarantined along with cessation of breeding to permit resolution of infection (bhatt and jacoby, 1987b) . sequential testing with contact-exposed sentinels should be employed with this option. additionally, maternal immunity from fully recovered dams can protect mice from infection, thereby enhancing opportunities to derive virus-free mice from previously infected dams, with the caveat that progeny will be transiently seropositive with maternally derived antibody. vaccination can control or prevent clinically apparent mousepox. the hemagglutinin-deficient strain of vaccinia virus (ihd-t) is used to scarify skin on the dorsum of the tail. 'takes' should occur in previously uninfected mice by 6-10 days, but not in infected mice (bhatt and jacoby, 1987a) (fig. 3.17 ). infected mice should be quarantined separately or eliminated. vaccination may not prevent infection, although infection in vaccinated mice is often transient. furthermore, vaccinia virus can be shed from scarification sites for at least several days. therefore, other preventive measures, such as strict controls on the entry of mice or mouse products, combined with periodic serological monitoring, should not be relaxed until diagnostic testing has confirmed the elimination of vaccinia and ectromelia virus. additionally, seroconversion evoked by vaccination must be taken into account in serological monitoring of vaccinated colonies. finally, vaccinia virus is a human pathogen, so vaccination procedures should include personnel protective measures to prevent exposure. research complications the primary threat from mousepox is mortality in susceptible mice. the loss of time, animals, and financial resources can be substantial. (shellam, 2007, 96) mice are naturally susceptible to two herpesviruses from the subfamily betaherpesvirinae and in the genus muromegalovirus, the two species murid herpesvirus 1 (of which one of the members is mouse cytomegalovirus (mcmv)) and murid herpesvirus 3 (of which one of the members is mouse thymic virus (mtv)). they are species-specific viruses and distinct from each other and from other rodent herpesviruses. mctv has received considerable attention as a model of human cmv infection. etiology mouse cytomegalovirus (mcmv) is a mouse-specific betaherpesvirus. it can, however, replicate in cell cultures from several species, including mouse (fibroblasts and 3t3 cells), hamster, rabbit, sheep, and nonhuman primate. cocultivation may be required to rescue latent virus. clinical signs mcmv causes subclinical infection in adult immunocompetent mice, but experimental inoculation of neonates can cause lethal disease due to multisystemic necrosis and inflammation. epizootiology the prevalence of mcmv in laboratory mice is probably uncommon but undefined, since infection is clinically silent and serological surveillance is not widely practiced. wild mice are commonly infected and serve as a natural reservoir for infection, which implies that the entry of virus into a modern vivarium is most likely to occur from contaminated animal products. persistence is a central feature of nonlethal infection. persistently infected mice excrete virus in saliva, urine, and tears for many months, resulting in horizontal transmission through mouse-to-mouse contact. virus also can infect prostate, testicle, and pancreas, implicating other modes of excretion. vertical transmission does not appear to be a common factor in natural infection. further, maternal immunity protects sucklings from infection. pathology mouse cytomegalovirus can replicate in many tissues, and viremia commonly occurs. lesions are not remarkable during natural infection and may be limited to occasional enlarged cells (megalocytosis) containing eosinophilic intranuclear and/or cytoplasmic inclusions associated with lymphoplasmacytic interstitial inflammation, especially in the cervical salivary glands. susceptibility to experimental infection varies with age, dose, route, virus strain, and host genotype. infection can occur in young and adult mice. however, the pathogenicity of mcmv for mice decreases with age. neonates are highly susceptible to lethal infection, but resistance to disease develops by the time mice are weaned. immunodeficient mice, however, remain susceptible to pathogenic infection as adults. persistent infection often affects the salivary glands and pancreas. the persistence of salivary gland infection appears to be dose dependent. there is experimental evidence that mcmv can produce latent infection of b cells, probably t cells as well as aforementioned tissues. persistent infection may lead to immune complex glomerulonephritis. latent persistent infection can be reactivated by lymphoproliferative stimuli and by immunosuppression. diagnosis mcmv antigens appear to be weak stimuli for humoral antibody production, which is consistent with the fact that cellular immunity is critical for protection against infection. neutralizing antibody titers are low during acute infection and difficult to find during chronic infection. serology and pcr-based diagnosis are available, but neither is widely used because of assumptions that infection has a very low prevalence. detection of enlarged cells with intranuclear inclusions, especially in salivary glands, is diagnostic, if they are present. in situ hybridization can be used as an adjunct to routine histopathology. differential diagnosis mcmv infection must be differentiated from infection with mtv. the latter virus can produce necrosis of thymic and peripheral lymphoid tissue when infant mice are experimentally inoculated. lytic lesions of lymphoid tissues are not a hallmark of mcmv. the viruses can also be distinguished from each other serologically. sialoadenitis with inclusions can occur during infection with mouse polyoma virus. like mcmv, mtv infects the salivary gland as its primary target organ. prevention and control control measures for mcmv have not been established, because it has not been considered an important infection of laboratory mice. cage-to-cage transmission has not been demonstrated, but horizontal infection from contaminated saliva must be considered. the exclusion of wild mice is essential. research complications mcmv can suppress immune responses. apart from the potential for interfering with immunology research, it can exacerbate the pathogenicity of opportunistic organisms such as pseudomonas aeruginosa. etiology mouse thymic virus (mtv) is a herpesvirus (murid herpesvirus 3) that is antigenically distinct from mcmv. no suitable in vitro method for cultivation has been developed; therefore, viral propagation depends on mouse inoculation. clinical signs natural infections are subclinical. epizootiology the prevalence of mtv is thought to be low. mice can be infected at any age, although lesions develop only in mice infected perinatally. mice infected as infants or adults can develop persistent infection of the salivary glands lasting several months or more. excretion of virus in saliva is considered the primary factor in transmission. seroconversion occurs in adults but does not eliminate infection. infection in neonates may not elicit seroconversion, rendering such mice serologically negative carriers. the mode of infection is obscure, but virus is excreted in saliva, suggesting that transmission from infected dams to neonatal mice occurs by ingestion. mtv also has been isolated from the mammary tissue of a lactating mouse, suggesting the potential for transmission during nursing. prenatal transmission has not been found. pathology mtv causes severe, diffuse necrosis of the thymus and lymphoid tissue with tropism for cd4 + t cells in mice inoculated within approximately 1 week after birth. the severity of thymic and lymph node necrosis can be mouse strain-dependent. grossly, the thymus is smaller than normal. infected thymocytes display mtv-positive intranuclear inclusions. necrosis is followed by granulomatous inflammation and syncytium formation. reconstitution of lymphoid organs takes 3-8 weeks. diagnosis thymic necrosis associated with intranuclear viral inclusions is the hallmark lesion. viral antigen can be detected by immunohistochemistry. serologic detection is effective, but generally not utilized, and is potentially negative in neonatally exposed mice. suspicion of infection in seronegative mice can be tested by inoculation of virus-free neonatal mice with homogenates of salivary gland or with saliva. inoculated mice should be examined for thymic necrosis 10-14 days later. pcr or the mouse antibody production (map) test can also be used to detect infection. differential diagnosis reduction of thymus mass can occur in severe mouse coronavirus infection, during epizootic diarrhea of infant mice, or following stress. prevention and control because mtv induces persistent salivary infection, rederivation or restocking should be considered if infection cannot be tolerated as a research variable. research complications mtv transiently suppresses cellular and humoral immune responses because of its destructive effects on neonatal t lymphocytes. parvoviruses are among the most common viral infections in contemporary laboratory mouse populations (livingston et al., 2002) (pritchett-corning et al., 2009) , and pose major challenges to both detection and control. the mouse parvoviruses are composed of two antigenically and genetically distinct but related groups, including minute virus of mice (mvm) and mouse parvovirus (mpv), with each group containing a number of strains. the international committee on taxonomy of viruses classifies mpv, mvm, and several other rodent parvoviruses into one genus, protoparvovirus, and species, rodent protoparvovirus 1, but these viruses will be treated separately herein. etiology minute virus of mice (mvm) is a small (5-kb) single-stranded dna virus. the prototypic strain is designated mvmp. an allotropic variant with immunosuppressive properties in vitro is named mvmi, and additional laboratory animal medicine named strains include mvmc and mvmm. the genome encodes two nonstructural proteins, ns-1 and ns-2, which are highly conserved among the rodent parvoviruses and account for prominent cross-reactivity in serological assays that utilize whole virus antigen. the viral capsid proteins, vp-1 and vp-2, are virus-specific and form the basis for serological differentiation of mvm from mpv. mvm has a broad in vitro host range. it replicates in monolayer cultures of mouse fibroblasts (a9 cells), c6 rat glial cells, sv40 (simian virus 40)-transformed human newborn kidney (324k cells), t-cell lymphomas (el4), and rat or mouse embryo cells, producing cytopathic effects that can include the development of intranuclear inclusions. clinical signs natural mvm infections are subclinical. neonatal mice of some inbred strains are experimentally susceptible to lethal renal and/or intestinal hemorrhage during mvmi infection, but this syndrome has not been reported in natural outbreaks. experimental inoculation of adult c.b-17-prkdc scid (scid) mice with mvmi results in lethal infection (lamana et al., 2001; segovia et al., 1999) , and similar severe illness has been noted in naturally infected b-cell-deficient nod. cg-h2h4-igh6 null mice (naugler et al., 2001) . epizootiology mvm is a common virus that naturally infects laboratory mice, but appears to be less common than mpv (besselsen et al., 2006; livingston et al., 2002) . mvm is moderately contagious for mice, its only known natural host. virus can infect the gastrointestinal tract and is excreted in feces and urine. the resistance of rodent parvoviruses to environmental inactivation increases the risks of transmission after virus is excreted. therefore, contamination of caging, bedding, food, and clothing must be considered a risk for the spread of infection. transmission occurs by oronasal exposure, but viral contamination of biologicals used for experimental inoculation, such as transplantable tumors, also can be a source of infection. continuous contact exposure to infected animals or soiled bedding usually induces a humoral immune response within 3 weeks, but limited exposure may delay seroconversion. young mice in enzootically infected colonies are protected by maternal antibody, but actively acquired immunity develops from infection sustained after the decay of maternal immunity. mvm, in contrast to mpv, is not thought to cause persistent infection; infection in immunocompetent adult mice usually lasts less than 3 weeks (smith, 1983; smith and paturzo, 1988 ). infection appears to last less than 1 month, even in oronasally inoculated neonatal mice, but immunodeficient mice may be persistently infected. there is no evidence that mvm is transmitted in utero. pathology natural infections or experimental inoculation of adult mice appears to be nonpathogenic. contact-exposed neonates have been reported to develop cerebellar lesions, but these are very rare. experimental infection of neonatal balb/c, swr, sjl, cba, and c3h mice with mvmi can cause renal hemorrhage and infarction (brownstein et al., 1991b) . dba/2 mice also developed intestinal hemorrhages and accelerated involution of hepatic hematopoiesis. c57bl/6 neonates are resistant to vascular disease. this lesion has been attributed to viral infection of endothelium. infection of immunodeficient mice, including scid and b-cell-deficient mice, results in lethal damage to granulomacrophagic, megakaryocytic, and erythrocytic hematopoietic tissue with severe leukopenia (lamana et al., 2001; naugler et al., 2001; segovia et al., 1999) . intranuclear viral inclusions and viral antigen have been observed in splenic mononuclear cells of b-cell deficient mice (naugler et al., 2001) . diagnosis serology is the primary method of detecting infection, which utilizes recombinant mvm and mpv major capsid viral proteins (vp2) as antigens, which discriminate between the two groups of mouse parvoviruses. in contrast, the conserved nonstructural protein, ns1 can be used to detect antibody to both groups, but is less sensitive than vp2 assays (livingston et al., 2002) . mvm infection also can be detected by pcr, in situ hybridization, and immunohistochemistry. pcr assays can be used to detect mvm-or mpv-specific vp2 or all rodent parvovirus group specific ns1 exons (besselsen, 1998; besselsen et al., 2006) . mvm can be isolated from the spleen, kidney, intestine, and other tissues by inoculation of the c6 rat glial cell line. it also can be detected by the mouse antibody production test. prevention and control because mvm does not persist in immunocompetent mice, control and elimination should exploit quarantine combined with thorough disinfection of the environment, because parvoviruses are resistant to environmental inactivation. mpv has been shown to be successfully eliminated by a cage-bycage test (serology and fecal pcr) and cull approach, although there are no published reports confirming the success of this strategy for eliminating mvm (macy et al., 2011) . cesarean rederivation or embryo transfer may also be used to rederive virus-free progeny. prevention of mvm infection depends on strict barrier husbandry and regular surveillance of mice and mouse products destined for use in vivo. research complications mvm contamination of transplantable neoplasms can occur; therefore, infection can be introduced to a colony through inoculation of contaminated cell lines. failure to establish long-term cell cultures from infected mice or a low incidence of tumor 'takes' should alert researchers to the possibility of mvm contamination. mvmi has the potential to inhibit the generation of cytotoxic t cells in mixed lymphocyte cultures. etiology mouse parvovirus (mpv) is among the more common viruses detected within contemporary laboratory animal medicine mouse colonies, and is more common than mvm (livingston et al., 2002; livingston and riley, 2003; pritchett-corning et al., 2009) . mpv was initially isolated following its detection as a lymphocytotropic contaminant in in vitro assays for cellular immunity. the virus grew lytically in a cd8 + t-cell clone designated l3 and inhibited the proliferation of cloned t cells stimulated with antigen or interleukin 2 (il-2) (mckisic et al., 1993) . molecular analysis of mpv indicates that regions encoding the ns proteins are similar to those of mvm (and other rodent parvoviruses). however, they differ significantly in regions encoding the capsid proteins, accounting for their antigenic specificity. the prototype isolate was first called an 'orphan' parvovirus of mice because its biology and significance were obscure, but it has subsequently been named mouse parvovirus (mpv). immortalized t cells (l3) are the only cells found thus far to support replication of mpv. there are three genetically distinct variants of mpv, including mpv-1, mpv-2, and mpv-3. mpv-1 includes a number of closely related variants, including mpv-1a, mpv-1b, and mpv-1c. in addition, a hamster parvovirus isolate is closely related to mpv-3, which is infectious to mice and likely to be of mouse origin (besselsen et al., 2006; christie et al., 2010) . clinical signs mpv infection is clinically silent in infant mice and adult immunocompetent or immunodeficient mice (besselsen et al., 2007) . immunologic perturbations are the most likely signs of infection (mckisic et al., 1993) . epizootiology mpv causes persistent infection in infant and adult mice, a property that differentiates it from mvm. in situ hybridization has identified the small intestine as a site of viral entry and early replication, but respiratory infection cannot be excluded. experimental studies following inoculation of neonatal balb/c and c.b-17-prkdc scid (scid) mice revealed that balb/c mice shed high levels of virus for 3 weeks, with transmission to sentinels exposed during the first 2 weeks of infection. thereafter, balb/c mice shed extremely low virus intermittently. in contrast, scid mice shed high levels of virus until weaning, but lower levels at 6 weeks of age, yet they effectively transmitted infection to sentinels at all stages of infection (besselsen et al., 2007) . others have shown that transmission of mpv by sencar mice inoculated as infants was intermittent up to 6 weeks, whereas transmission by mice inoculated as weanlings occurred during the first 2 weeks of infection . transmission to balb/c progeny from infected dams was shown to occur, but embryo transfer rederivation was found to be successful in experimentally infected scid mice (besselsen et al., 2008) . humoral (e.g., passively or maternally acquired) immunity can protect against mpv infection. however, immunity to mvm may not confer cross-immunity to mpv (hansen et al., 1999) . pathology mpv appears to enter through the intestinal mucosa, which is a site of early virus replication ( fig. 3.18 ). acute infection is widespread but mild, involving the lung, kidney, liver, and lymphoid organs. histological lesions are not discernible. lymphocytotropism is a characteristic of acute and persistent mpv infection in infant and adult mice. during acute infection, virus is dispersed within lymph nodes, but during persistent infection virus localizes in germinal centers (fig. 3.19) . diagnosis because infected mice do not manifest signs or lesions and the virus is very difficult to propagate in cell culture, detection and diagnosis rely on serology and molecular methods. serology that utilizes mpv vp2 as antigen is a sensitive and specific assay that differentiates mpv from mvm (livingston et al., 2002) . the map test also can be used to detect parvovirus infections but is relatively time-consuming and expensive. as noted for mvm, pcr for murine parvoviruses, using nucleoprotein gene sequences that are conserved among murine parvoviruses, can be used as a screening test. pcr also can be used to detect mpv-specific sequences in the vp2 gene. although diagnostic pcr is sensitive and specific, it is effective only in actively infected animals. it can be used on feces to detect virus shedding, or applied to tissues, such as mesenteric lymph nodes, obtained at necropsy. differential diagnosis mpv infection must be differentiated from mvm infection. because both viruses are enterotropic and lymphocytotropic, serology and pcr must be used to distinguish between them. prevention and control the persistence of mpv in individual mice, its potential for provoking immune dysfunction, and the resistance of murine parvoviruses to environmental inactivation favor active control and prevention of mpv infection. quarantine of infected rooms is appropriate. elimination (depopulation) of infected mice should be considered if they are an immediate threat to experimental or breeding colonies and can be replaced, but a cage-by-cage test and cull approach has been shown to be successful under natural conditions (macy et al., 2011) . for mice that are not easily replaced, virus persistence in the absence of transplacental transmission favors cesarean rederivation or embryo transfer as relatively rapid options to eliminate infection. control of infection also should include environmental decontamination. chemical disinfection of suspect animal rooms and heat sterilization of caging and other housing equipment are prudent steps. prevention is based on sound serological monitoring of mice and surveillance of biologicals destined for inoculation of mice. with the increasing use of mouse germplasm, it is important to note that mouse sperm, oocytes, ovarian tissue, and preimplantation embryos from enzootically mpvinfected mouse colonies may have a high prevalence of mpv contamination, based upon pcr (agca et al., 2007) . research complications murine parvoviruses can distort biological responses that depend on cell proliferation. for mpv, such effects are seen on immune function and include augmentation or suppression of humoral and cellular immune responses. etiology adenoviruses are nonenveloped dna viruses that produce intranuclear inclusions in vitro and in vivo. two adenovirus species in the genus mastadenovirus have been associated with mice: murine mastadenovirus a (with the representative strain being mav-1 or fl) and murine mastadenovirus b (with the representative strain being mav-2 or k87). both strains replicate in mouse kidney tissue culture but are antigenically distinct. clinical signs mav-1 can cause severe clinical disease after experimental inoculation of infant mice. signs include scruffiness, lethargy, stunted growth, and often death within 10 days. mav-2 virus is enterotropic and is responsible for virtually all naturally occurring infections in contemporary mouse populations. infection is usually subclinical in immunocompetent mice, with the possible exception of transient runting among infant mice. wasting disease can occur in athymic mice infected with mav-1. epizootiology the prevalence of adenovirus infection in mouse colonies is low, particularly mav-1 riley, 2003, pritchett-corning et al., 2009) . transmission occurs by ingestion. adult mice experimentally infected with mav-1 may remain persistently infected and excrete virus in the urine for prolonged periods. adult mice experimentally infected with mav-2 excrete virus in feces for at least 3 weeks but eventually recover. athymic mice can shed mav-2 for at least 6 weeks and episodically for at least 6 months. pathology infection with mav-1 causes multisystemic disease characterized by necrosis. infant mice are especially susceptible to rapidly fatal infection characterized by necrosis of brown fat, myocardium, adrenal cortex, salivary gland, and kidney, with the development of intranuclear inclusions. more mature mice usually develop subclinical infection leading to seroconversion; however, athymic and scid mice can develop intestinal hemorrhage and wasting, with fatal disseminated infection (lenaerts et al., 2005) . infection with mav-2 produces amphophilic, intranuclear inclusions in intestinal epithelium, especially in the distal small intestine (fig. 3.20) . inclusions are easier to detect in infant mice than in adults. infection of c.b-17-prkdc scid mice with mav-2 results in enteric infection, but also hepatic lesions resembling reye's syndrome (pirofski et al., 1991) . diagnosis although mav strains can be isolated in tissue culture, routine diagnosis depends on detection of infection by serological assay and/or demonstration of adenoviral inclusions, most commonly in the intestinal mucosa. cross-neutralization tests have revealed that antiserum to mav-2 neutralizes both strains, but antiserum to mav-1 neutralizes mav-2 weakly at best. therefore, mav-2 antigen should be used for the serological detection of adenovirus infection irrespective of the assay employed. mav also can be detected by pcr. differential diagnosis intranuclear adenoviral inclusions in intestinal epithelium are pathognomonic laboratory animal medicine and differentiate mav-2 infection from other known viral infections of mice. infection may resemble rotavirus infection, with runting and abdominal bloating in infant mice. prevention and control prevention requires serological monitoring of mice and examination for contamination of animal products such as transplantable tumors. because mav-2 infection appears to be transient in individual mice, segregation of infected colonies may be effective for control. however, rederivation coupled with subsequent barrier housing is a more conservative approach. research complications mav infection is unlikely to affect research using immunocompetent mice. however, it has the potential for pathogenicity in immunodeficient mice. mice can incur natural infection with two polyomaviruses: polyoma virus (pyv) and k virus. these viruses belong to the family polyomaviridae. k virus belongs to the genus polyomavirus and the species murine pneumotropic virus, while the classical polyoma virus belongs to the species murine polyomavirus. etiology polyoma virus (pyv) is a small dna virus that derives its name 'polyoma' (many tumors) from its ability to experimentally induce multiple types of tumors in mice experimentally infected as neonates. its primary importance stems from use in murine models of experimental oncogenesis, with natural infection being rare. the transformative activity is mediated by 't' (tumor) antigens, encoded by large t, middle t, and small t genes, with middle t (mt) being considered the major viral oncogene, and as a result has been used extensively in transgenic constructs. clinical signs natural infections in immunocompetent mice are usually subclinical. however, tumor induction, neurological disease, and wasting can occur in naturally exposed immunodeficient mice (mccance et al., 1983; sebesteny et al., 1980) . epizootiology modern husbandry and health care have essentially eliminated natural exposure in laboratory mice. pyv is used for experimental studies and thus can inadvertently be introduced to mouse colonies. inoculation of mice with contaminated biologicals or cell cultures is a potential source of entry and spread. natural transmission occurs via the respiratory route. exposure of neonatal mice results in persistent infection and shedding of the virus in urine, feces, and saliva, thereby contaminating the environment for spread to other mice. infection of adult mice is transient, with minimal virus shedding, although pcr has revealed infection lasting up to 5 months in cba mice inoculated with virus as adults (berke and dalianis, 1993) . maternal antibody is highly effective at preventing infection of newborn mice, but as maternal antibody wanes, mice are partially susceptible, with transient virus shedding. thus, the natural cycle of transmission in enzootically infected populations requires contamination of bedding and nesting material in order to infect and be inefficiently transmitted, which is readily precluded by modern husbandry. intrauterine infection also can occur, and persistent renal infection, contracted neonatally, can be reactivated during pregnancy. as in immunologically immature neonatal mice, pyv infection can persist in adult immunodeficient mice. pathology pyv-induced tumors are essentially a laboratory phenomenon, optimized by virus strain and mouse strain, with akr, c3h, c58, cba, swr, and others being most susceptible, and c57bl/6 being among the most resistant to pyv oncogenesis. intranasal inoculation of neonatal mice results in initial replication in pulmonary respiratory epithelium (gottlieb and villarreal, 2000) followed by viremic dissemination and acute, lethal disease. tumors appear 2-12 months after inoculation of surviving mice. tumors of both epithelial and mesenchymal origin arise in multiple organs, particularly mammary carcinomas, basal cell tumors of the skin, carcinomas of salivary glands, thymomas, and various types of sarcomas. athymic mice can develop cytolytic and inflammatory lesions, followed by multisystemic tumor formation. intranuclear inclusions may be present in cytolytic lesions. demyelinating disease and skeletal tumors have been reported in experimentally . laboratory animal medicine inoculated and naturally exposed athymic mice, and myeloproliferative disease has been reported in experimentally inoculated c57bl/6-scid mice (szomolanyi-tsuda et al., 1994) . diagnosis pyv can be isolated in mouse fibroblast cell lines, but infection is ordinarily detected serologically. additionally, pcr and immunohistochemistry can be used. differential diagnosis wasting in athymic mice can be caused by other infectious agents, including coronaviruses, sendai virus (sv), and pneumocystis. intranuclear inclusions can occur in infections caused by mouse adenovirus, mouse cytomegalovirus, and k virus. prevention and control control depends on elimination of infected mice and material, together with prevention of airborne spread. biological material destined for mouse inoculation should be tested for pyv by the map test or molecular diagnostics. research complications pyv infection can affect experiments by inadvertent contamination of cell lines or transplantable tumors, leading to infection of inoculated mice and the potential for epizootic spread. k virus infection k virus has historical importance, and is apparently absent from contemporary mouse populations (livingston and riley, 2003; pritchett-corning et al., 2009) , but it continues to be tested for, adding to the expense of infectious disease surveillance. oral inoculation of neonatal mice results in initial infection of capillary endothelium in the intestine, followed by viremic spread. vascular endothelium is the primary target in affected tissues, which often include the lung, liver, spleen, and adrenal glands. dyspnea occurs from pulmonary infection because of edema and hemorrhage. infection of immunocompetent adult mice is subclinical and results in a vigorous immune response. however, both adults and infant mice develop persistent infection. the primary organ for persistence is the kidney, with shedding of virus from tubular epithelium, and shedding can be reactivated by immunosuppression (greenlee et al., 1991) . additionally, infection of athymic mice can lead to clinical signs and lesions akin to those described for neonatally inoculated mice. gross lesions are limited to pulmonary hemorrhage and edema. histologically, intranuclear inclusions, which are visualized more easily using immunohistochemistry, are present in vascular endothelium of infected tissues. mild hepatitis with hepatocyte degeneration also may develop. infection can be detected by serology or pcr. prevention and control measures, if ever to be found within a mouse population, are similar to those described for pyv. etiology lactate dehydrogenase-elevating virus (ldv) is a mouse-specific small enveloped rna virus belonging to the family arteriviridae. infected mice are persistently viremic, resulting in increased concentration of several serum enzymes, most notably lactate dehydrogenase (ldh). infection is common among wild mice, but is now rare in contemporary laboratory mouse populations. however, surveys of biologic material indicate that ldv may be a common contaminant of biologic materials (nicklas et al., 1993) . clinical signs infection is subclinical. however, poliomyelitis has occurred in immunosuppressed c58 and akr mice inoculated with ldv, and has recently been observed in icr-scid mice following inoculation with contaminated biologic material (carlson-scholz et al., 2011) . epizootiology the primary mode of mouse-tomouse transmission is mechanical transfer from aggressive behavior (e.g., bite wounds). inoculation of mice with contaminated animal products such as cell lines, transplantable tumors, or serum is probably the most common source of induced infection. it is important to note, with respect to mechanical transmission, that infection induces lifelong viremia. natural transmission between cagemates or between mother and young is rare even though infected mice may excrete virus in feces, urine, milk, and probably saliva. pathology viremia peaks within 1 day after inoculation, then persists at a diminished level. the elevation of enzyme levels in blood is thought to result primarily from viral interference with clearance functions of the reticuloendothelial system. ldv selectively targets mature f4/f8-positive macrophages, which are continually produced by uninfected progenitor cell populations, thereby maintaining persistent infection. virus also escapes immune clearance by evolution of neutralizing antibody-resistant quasi-species. no lesions are seen in naturally infected mice. the only significant lesion that can arise from experimental infection is poliomyelitis. this syndrome requires a combination of immunosuppression (due to age, genetics or induced means), mouse strain (c58, akr, c3h/fg, and pl), neurotropic ldv strains, and endogenous ecotropic murine leukemia virus. the mouse strain-dependent element is homozygosity for the fv-1 n allele, which permits replication of endogenous n-tropic ectotropic murine leukemia virus. mice develop spongiosis, neuronal necrosis, and astrocytosis of the ventral spinal cord and brain stem, with axonal degeneration of ventral roots. lesions contain both ldv and retrovirus. although this syndrome is largely experimentally induced, a natural outbreak of poliomyelitis has been reported in fv-1 homozygous icr-scid mice following inoculation with contaminated biologic material (carlson-scholz et al., 2011) . diagnosis plasma ldh levels are elevated, a response that is used to detect and titrate ldv infectivity. of the five isoenzymes of ldh in mouse plasma, only laboratory animal medicine ldh-v is elevated. sjl/j mice in particular show spectacular increases in ldh levels (15-20 times normal), a response controlled by a recessive somatic gene. ldv is detected by measuring ldh levels in mouse plasma before and 4 days after inoculation of specific pathogenfree (spf) mice with suspect material. it is important to use nonhemolyzed samples because hemolysis will produce falsely elevated readings. plasma enzyme levels are measured in conventional units/ml, 1 conventional unit being equivalent to 0.5 international units (iu). normal plasma levels are 400-800 iu, whereas in ldv infection, levels as high as 7000 iu can occur. ldv also interferes with the clearance of other serum enzymes and results in their elevation in serum. in a recent survey, 6000 serum samples were tested by serum ldh enzyme assays, among which 10% were deemed potentially positive. however, pcr revealed that all were false-positives (pritchett-corning et al., 2009) , emphasizing the inaccuracy of traditional enzyme assays. infection provokes a modest humoral antibody response, but it is difficult to detect because of formation of virus-antibody immune complexes. molecular diagnostics also can be used to diagnose infection in mouse tissues and serum and biologic materials. however, inhibitory factors in cells and serum may cause falsenegative results in pcr testing, so appropriate quality control measures are essential if this method is used (lipman and henderson, 2000) . prevention and control transplantable tumors have been a common source of ldv historically. therefore, tumors or cell lines destined for mouse inoculation should be monitored for ldv contamination. although ldv can contaminate tumor cell lines, it does not replicate in the tumor cells. therefore, one can attempt to free tumors of virus by passaging them through athymic nude rats, which are not nonpermissive to ldv but are permissive to xenografts. research complications ldv has numerous potential effects on immunological function. it may reduce autoantibody production, cause transient thymic necrosis and lymphopenia, suppress cell-mediated immune responses, and enhance or suppress tumor growth. etiology the house mouse is the natural host for lymphocytic choriomeningitis virus (lcmv), an old world member of the arenaviridae family that has spread worldwide along with m. musculus. lcmv virions are pleomorphic, containing single-stranded rna, and bud from the cell membrane. disease associated with infection is due to host immune response to the otherwise non-cytolytic virus. its name is derived from the immune-mediated inflammation resulting from the intracerebral inoculation of virus into immunologically competent mice. lcmv is a zoonotic virus that may cause a variety of clinical manifestations in humans, including meningitis. it has been extensively studied as an experimental model of virus-induced immune injury, using a number of closely related strains, including ones that have been selected for their relative neurotropism or viscerotropism. lcmv can be propagated in a variety of mammalian, avian, and even tick cell lines, with minimal cytopathic effect. these characteristics favor its propensity to persistently and silently contaminate biologic products, such as tumor cell lines. clinical signs natural infection in immunocompetent adult mice is usually self-limiting and subclinical. during enzootic infection of a mouse population, lcmv is transmitted in utero from persistently infected dams to their fetuses or to neonates, which are persistently infected and immunologically tolerant to lcmv. since lcmv is non-cytolytic in and of itself is minimally pathogenic, congenitally infected mice grow into adulthood, reproduce, and therefore transmit infection to the next generation. however, with age, immune tolerance breaks down, and mice develop a syndrome known as 'late disease' in which mice will progressively lose weight and die. in utero infection results in a low level of fetal mortality and maternal cannibalism of infected pups. the immune tolerance to lcmv is virus-specific, with the mice capable of eliciting effective immune responses against other agents. clinical signs following experimental inoculation of lcmv vary with age and strain of mouse, route of inoculation, and strain of virus. when virus is inoculated intracerebrally into immunocompetent adult mice, mice develop immune-mediated lymphocytic choriomeningitis, characterized by illness beginning 5-6 days after inoculation. sudden death may result or subacute illness associated with one or more of the following signs may develop: ruffled fur, hunched posture, motionlessness, and neurological deficits. mice suspended by the tail display coarse tremors of the head and extremities, culminating in clonic convulsions and tonic extension of the rear legs. spontaneous convulsions also can occur. animals usually die or recover in several days. a visceral form of infection can occur in adult mice inoculated by peripheral routes with 'viscerotropic' strains. it can be subclinical or lead to clinical signs, including ruffled fur, conjunctivitis, ascites, somnolescence, and death. if mice survive, recovery may take several weeks. surviving mice may have immune exhaustion due to consumption of lymphoid tissue, in contrast to immune tolerance that occurs when mice are infected in utero or as neonates. runting and death from lcmv infection may occur in neonatally infected mice and can lead to transient illness or to death. clinical signs are nonspecific, recovery is slow, and survivors may remain runted. this early form of disease is attributed to endocrine dysfunction caused laboratory animal medicine by lcmv infection. late-onset disease can occur in previously subclinical carrier mice that develop immune complex glomerulonephritis. it is usually the result of prenatal or neonatal infection and occurs in persistently infected mice when they are 9-12 months old. clinical signs are nonspecific and include ruffled fur, hunched posture, weight loss, proteinuria, and ascites. epizootiology lcmv is distributed widely in wild m. musculus throughout the world. among common laboratory species, mice, hamsters, guinea pigs, and nonhuman primates are susceptible to infection, but only the mouse and the hamster are known to transmit virus. lcmv infection is rare in laboratory mice produced and maintained in modern quarters (livingston and riley, 2003; pritchett-corning et al., 2009) . infection is usually introduced through inoculation of virusinfected biologicals, such as transplantable tumors, or by feral mice. wild mice are a natural reservoir of infection and a potential threat to research colonies if they gain entry inadvertently. naturally infected carrier mice can have persistently high concentrations of virus in many organs, thereby facilitating virus excretion in saliva, nasal secretions, and urine. persistently infected neonates usually reach breeding age and can perpetuate infection in a breeding colony. thus introduction of a single lcmv carrier mouse to a breeding colony can eventually result in a high prevalence of persistently infected mice. infection in adult mice, in contrast, is often acute because of the onset of effective immunity, and the spread of virus is halted. horizontal spread of infection is enhanced by close contact, but rapid horizontal spread is not characteristic. mice can transmit lcmv to hamsters, which can remain viremic and viruric for many months, even if they contract infection as adults. infected hamsters can transmit virus to other hamsters and mice and are the primary source of human lcmv infection. persistent infection in immunodeficient mice may carry greater risks for viral excretion and zoonotic transmission. pathology lcmv disease is a prototype for virusinduced, t-lymphocyte-mediated immune injury, noncytolytic endocrine dysfunction, and immune complex disease. however, lesions comparable to experimentally induced disease are rare during natural infection. intracerebral inoculation of virus into immunocompetent adult mice induces nonsuppurative leptomeningitis, choroiditis, and focal perivascular lymphocytic infiltrates. host tissues are damaged during the course of the cellular immune response to the virus. the character of visceral lesions depends on virus strain and mouse strain; the ratio of cytolytic to proliferative responses in lymphoid organs is mouse strain-dependent. in severe infection, nonsuppurative inflammation can occur in many tissues. the severity of accompanying cytolytic lesions seems to parallel the intensity of cellular immunity. liver lesions can include hepatocyte necrosis accompanied by nodular infiltrates of lymphoid cells and kupffer cells, activated sinusoidal endothelium, an occasional granulocyte or megakaryocyte, and fatty metamorphosis. cytolysis, cell proliferation, and fibrinoid necrosis can develop in lymphoid organs. necrosis of cortical thymocytes can lead to thymic involution. lesions of late-onset disease are characterized by formation of immune complexes and associated inflammation. renal glomeruli and the choroid plexus are most severely affected, but complexes may also be trapped in synovial membranes, blood vessel walls, and skin. lymphoid nodules can form in various organs. lesions associated with early deaths in neonatally infected mice have not been thoroughly described but include hepatic necrosis. the lesions of acute and persistent lcmv infection reflect separate immunopathologic processes. in adult mice with acute lcmv infection, virus multiplies in dcs, b cells, and macrophages, whereas t cells are resistant. internal viral epitopes induce humoral immune responses, but surface epitopes elicit cell-mediated immunity and neutralizing antibodies. thus, elimination of virus and virus-associated immunological injury are both t-cell-mediated. this apparent paradox has been explained by the view that prompt cellular immunity limits viral replication and leads to host survival, whereas slower cellular immune responses permit viral spread and increase the number of virus-infected target cells subject to attack once immunity is fully developed. antibody can be detected by 1 week after infection but does not play a significant role in eliciting acute disease. lesions of lcmv infection appear to develop from direct t-cell-mediated damage to virus-infected cells and may involve humoral factors released from immune effector t cells. lcmv also can suppress humoral and cellular immunity in acutely infected mice. persistent infection commonly evolves from exposure early in pregnancy, and virus has been demonstrated in the ovaries of carrier mice. prenatal or neonatal infection induces immunological tolerance to lcmv, which can then replicate to high titer in many tissues. nevertheless, persistently infected mice develop humoral antibody to lcmv. antibody can complex with persistent virus to elicit complement-dependent inflammation in small vessels. immune complex glomerulonephritis exemplifies this process, as noted above. diagnosis lcmv infection can be diagnosed serologically. whereas immunocompetent adult mice will normally seroconvert after exposure, carrier mice may develop poor humoral immune responses. therefore, testing must avoid false-negative results. employment of adult contact sentinel mice is a useful strategy for detecting lcmv infection by seroconversion. tissues, including biologic products and cell lines, can be tested laboratory animal medicine by pcr. a traditional method for detection involved collection of small blood samples from persistently infected live suspects, which are often viremic, and using them to inoculate cultured cells or adult and neonatal mice. intracerebral inoculation of lcmv-positive tissues should elicit neurological signs in adult mice within 10 days, whereas infant mice should remain subclinical. histological examination of brains from affected adults may reveal nonsuppurative inflammation, but lesions may be minimal in mice infected with viscerotropic isolates. immunohistochemistry can be used to detect viral antigen in brains of suckling and adult mice. intraperitoneal inoculation of adult mice may yield short-lived infection with seroconversion, i.e., the map test. virus can be grown and quantified in several continuous cell lines, including mouse neuroblastoma (n-18) cells, bhk-21 cells, and l cells. application of immunofluorescence staining to detect lcmv antigen in inoculated cultured cells yields results more quickly than animal inoculation. of course, all diagnostic procedures involving potential contact with live virus should be carried out under strict containment conditions to avoid infection of laboratory personnel (see chapter 29). the use of in vitro detection has the added advantage, in this regard, of reducing biohazardous exposure and the use of live animals for testing. differential diagnosis neurological signs must be differentiated from those due to mouse hepatitis virus, mouse encephalomyelitis virus, and meningoencephalitis from bacterial infection. trauma, neoplasia, and toxicities also must be ruled out in neurological disease with low prevalence. late-onset disease is associated with characteristic renal lesions, including deposition of viral antigen in tissues. early-onset disease must be differentiated from other causes of early mortality, such as mouse hepatitis virus, ectromelia virus, reovirus 3 infection, tyzzer's disease, or husbandry-related insults. prevention and control adequate safeguards for procurement and testing of animals and animal products are essential to prevent entry. because mouse-to-mouse spread is slow, selective testing and culling for seropositive or carrier mice is possible. if mice are easily replaced, however, depopulation is a safer and more reliable option. valuable stock can be rederived, but progeny must be tested to preclude in utero transmission. because infected hamsters can excrete large quantities of virus, exposed hamsters should be destroyed and hamsters should not be housed with mice. infection of immunodeficient mice poses similar risk. lcmv can be transmitted to human beings, who can contract flu-like illness or severe cns disease. more frequently, human infection is subclinical. the zoonotic potential of lcmv infection makes it especially important to detect and eliminate carrier animals and other potentially contaminated sources, such as cell cultures, transplantable neoplasms, and vaccines to prevent human exposure. serum banking and periodic serological testing of highrisk human populations, such as those working with lcmv experimentally, are recommended. research complications lcmv may stimulate or suppress immunological responses in vivo and in vitro, and it can replicate in cells used as targets or effectors for immunological studies. introduction of immune cells to a carrier animal may elicit an immunopathological response. immune complex disease can complicate longterm experiments and morphological interpretations. illness and death in mice and zoonotic risk to humans are obvious research-related hazards. etiology sv is a paramyxovirus that is antigenically related to human parainfluenza virus 1. viral particles are pleomorphic, contain single-stranded rna, and have a lipid solvent-sensitive envelope that contains glycoproteins with hemagglutinating, neuraminidase, and cell fusion properties. sv grows well on embryonated hens' eggs and in several mammalian cell lines (e.g., monkey kidney, baby hamster kidney , and mouse fibroblast [l]). virus replicates in the cytoplasm and by budding through cell outer membranes. once common in laboratory rodent populations, sv is now rare or absent (livingston and riley, 2003; pritchett-corning et al., 2009) . clinical signs clinically affected adult mice often assume a hunched position and have an erect hair coat. rapid weight loss and dyspnea occur, and there may be chattering sounds and crusting of the eyes. although highly susceptible adults may die, lethal infection is more common in suckling mice. sex differences in susceptibility have not been found. genetically resistant mice usually have subclinical infection. athymic mice and immunodeficient mice are at high risk for development of a wasting syndrome. they develop illness later than their immunocompetent counterparts, since clinical signs in immunocompetent mice are related to immune-mediated destruction of respiratory epithelium. opportunistic infections can complicate the clinical presentation. for example, secondary bacterial infections of the ear can cause vestibular signs. epizootiology sv is transmitted by aerosol and is highly contagious. morbidity in infected colonies is commonly 100%, and mortality can vary from 0% to 100%, partly because strains of mice vary greatly in their susceptibility to lethal sv infection. for example, c57bl/6 mice are highly resistant to clinically apparent infection, whereas dba/2 mice are highly susceptible. aerogenic infection is promoted by high relative humidity and by low air turnover. prenatal infection does not occur. enzootic infection is commonly detected in postweaned mice (5-7 weeks old) and is associated with seroconversion within 7-14 days and the termination of infection. therefore, entrenched infection is perpetuated by the introduction of susceptible animals. there is no evidence for persistent infection in immunocompetent mice, but prolonged infection is common in immunodeficient mice. maternally acquired immunity protects young mice from infection, and actively acquired immunity is thought to be long-lived. rats, hamsters, and guinea pigs also are susceptible to sv infection. therefore, bidirectional cross-infection is a risk during outbreaks. pathology viral replication is nominally restricted to the respiratory tract and peaks by the first week after infection. gross lesions feature partial to complete consolidation of the lungs (fig. 3.21) . individual lobes are meaty and plum-colored, and the cut surface may exude a frothy serosanguinous fluid. pleural adhesions or lung abscesses caused by secondary bacterial infection are seen occasionally, and fluid may accumulate in the pleural and pericardial cavities. sv targets airway epithelium and type ii pneumocytes. type i pneumocytes are less severely affected. histologically, the pattern of pneumonia is influenced by mouse genotype. susceptible mice usually have significant bronchopneumonia and interstitial pneumonia, whereas the interstitial component may be less prominent in resistant mice. typical changes begin with inflammatory edema of bronchiolar lamina propria, which may extend to alveolar ducts, alveoli, and perivascular spaces. necrosis and exfoliation of bronchiolar epithelium ensue, frequently in a segmental pattern (fig. 3.22 ). alveolar epithelium also may desquamate, especially in severe disease, and necrotic cell debris and inflammatory cells can accumulate in airways and alveolar spaces. alveolar septae are usually infiltrated by leukocytes to produce interstitial pneumonia (fig. 3.23) . lymphoid cells also invade peribronchiolar and perivascular spaces. the lymphocytic response to sv infection reflects the fact that cellular immunity contributes both to lesions and to recovery. local immunoglobulin synthesis by infiltrating cells also occurs. the extent of inflammatory cell infiltration corresponds to the level of genetic resistance expressed by the infected host, with clinically susceptible hosts mounting a more florid immune response than resistant hosts. additionally, strain-related differences in the severity of infection may reflect differences in airway mucociliary transport. multinucleated syncytia are occasionally seen in affected sucklings and scid mice, and inclusion bodies have been reported in infected athymic mice. regeneration and repair begin shortly after the lytic phase and are characterized by hyperplasia and squamous metaplasia of bronchial epithelium, which may extend into alveolar septae. proliferation of cuboidal laboratory animal medicine epithelium may give terminal bronchioles an adenomatoid appearance. repair of damaged lungs is relatively complete in surviving mice, but lymphocytic infiltrates, foci of atypical epithelium, and mild scarring can persist. acute phase lesions are prolonged in immunodeficient mice, which can lead to wasting and death. aged mice also have a prolonged recovery phase accompanied by focal pulmonary fibrosis (jacoby et al., 1994) . diagnosis sv is notable for its ability to cause epizootics of acute respiratory distress in adult genetically susceptible strains. serology is an effective means to detect infection in all strains of immunocompetent mice. antibody can be detected by 7 days postinfection and coincides with development of clinical signs related to the immune-mediated necrotizing bronchiolitis and alveolitis. repeated serologic sampling over several weeks can help stage infection within a population. alternatively, sentinel animals can be added to seropositive colonies to detect active infection. irrespective of serologic results, histopathology, immunohistochemistry (which can be performed on formalin-fixed, paraffin-embedded sections), and, where possible, virus isolation should be used to confirm infection. virus can be isolated from the respiratory tract for up to 2 weeks, with peak titers occurring at about 9 days postinfection. nasopharyngeal washings or lung tissue homogenates are most reliable and should be inoculated into embryonated hens' eggs or bhk-21 cell monolayer cultures. sv infection of cultured cells is non-cytolytic, so erythrocyte agglutination or antigen detection methods must be used. rt-pcr also can be used to detect virus in infected lungs. differential diagnosis respiratory infection caused by pneumonia virus of mice (pvm) is generally milder or subclinical. histologically, necrosis of airway epithelium is less severe. bacterial pneumonias of mice, including murine respiratory mycoplasmosis, are sporadic and can be differentiated morphologically and by isolation of causative organisms. because sv pneumonia may predispose the lung to opportunistic bacterial infections, the presence of bacteria should not deter evaluation for a primary viral insult. control and prevention sv infection is self-limiting in surviving immunocompetent mice. suckling mice from immune dams are protected from infection by maternal antibody until after weaning. control and eradication measures must eliminate exposure of susceptible animals, so that infection can 'burn out.' this is most easily accomplished by a quarantine period of 4-6 weeks wherein no new animals are introduced either as adults or through breeding. control also is aided by the fact that sv is highly labile. barrier housing is preferred for prevention and for control of transmission. vaccination with formalin-killed virus can provide short-term protection of valuable mice but is not commonly used for prevention. research complications sv can cause immunosuppression and can inhibit growth of transplantable tumors. this effect has been attributed to virus-induced modification of tumor cell surface membranes. pulmonary changes during sv pneumonia can compromise interpretation of experimentally induced lesions and may lead to opportunistic infections by other bacteria. they also have been associated with breeding difficulties in mice. this sign is thought to be an indirect effect due to stress, fever, or related changes during acute infection. clinical signs natural pvm infection in mice is subclinical. therefore, its name is clinically misleading, being derived from pneumonic illness that occurred after serial passage of the agent in mice. however, dyspnea, listlessness, and wasting may develop in immunodeficient mice infected with pvm (weir et al., 1988) . pvm is used experimentally as a model to study acute respiratory infection, using highly pathogenic strains of the virus (dyer et al., 2012) . epizootiology pvm causes natural infections of mice, rats, hamsters, and probably other rodents and may be infectious for rabbits. serological data indicate that pvm was once common, but is now relatively uncommon (livingston and riley, 2003; pritchett-corning et al., 2009 ). pvm appears to spread less rapidly than sv. intimate contact between mice is probably required for effective transmission. this characteristic may reflect the fact that environmental inactivation of virus occurs rapidly. infection is acute and self-limiting in immunocompetent mice but may persist in immunodeficient mice. pathology pvm replicates exclusively in the respiratory tract and reaches peak titers in the lung 6-8 days after infection. although pulmonary consolidation can occur in experimentally infected mice, gross lesions are rare during natural infection. histological lesions can occur in the upper and lower respiratory tract. they consist of mild necrotizing rhinitis, necrotizing bronchiolitis, and interstitial pneumonia, which usually occur within 2 weeks after exposure to virus and are largely resolved by 3 weeks. the predominant inflammatory infiltrate is comprised of mononuclear cells, but some neutrophils laboratory animal medicine are usually present. immunohistochemistry on paraffinembedded tissues can be used to detect viral antigen in bronchiolar epithelium, alveolar macrophages, and alveolar epithelium during acute infection. residual lesions include nonsuppurative perivasculitis, which can persist for several weeks after acute infection has ceased. severe progressive pneumonia, with wasting, can occur in immunodeficient mice. it is characterized by generalized pulmonary consolidation that reflects severe interstitial pneumonia with desquamated alveolar pneumocytes and leukocytes filling alveolar spaces (fig. 3.24) . diagnosis diagnosis is based primarily on serological detection that can be supplemented by histopathology, immunohistochemistry, in situ hybridization, and virus isolation. virus replication in bhk-21 cells is detected by immunofluorescence or other antigen detection methods. virus also can be detected in tissues by rt-pcr. differential diagnosis because pvm is antigenically distinct from other murine viruses, serology is the most useful method to separate pvm infection from other respiratory infections of mice. however, in immunodeficient mice, where clinical signs and lesions are typical, it must be differentiated from other pneumonias, especially those due to sv and pneumocystis. additionally, pvm can coexist with and exacerbate pneumocystis infection in immunodeficient mice (bray et al., 1993) . prevention and control pvm infection is acute and self-limiting in immunocompetent mice, but persistent in immunodeficient mice. seropositive mice should be viewed as either immune or in the final stages of acute infection. therefore, control and prevention follows guidelines applicable to sv infection. research complications pvm can exacerbate pneumocystosis, as noted above. (ward et al., 2007) two members of the family reoviridae infect laboratory mice: reovirus per se (species: mammalian orthoreovirus) and murine rotavirus (species: rotavirus a), also known as epizootic diarrhea of infant mice (edim) virus. etiology reoviruses of mammals, although taxonomically considered one type species, have been divided into three cross-reacting prototypic serotypes: reovirus 1, 2 and 3, which can be differentiated by cross-serum neutralization. mice can be infected with any serotype, but reovirus 3 is emphasized because it has been associated with naturally occurring disease. natural infections in mice are usually not caused by pure serotypes, because reoviruses actively recombine. a number of wild-type and laboratory strains have been characterized, and related viruses have been recovered from virtually every mammal tested, as well as birds, reptiles, and insects. the virion contains segmented, double-stranded rna and is relatively heat stable. reoviruses replicate well in bhk-21 cells and other continuous cell lines, as well as in primary monolayer cultures from several mammals. clinical signs clinical disease is rare and age dependent. acute disease affects sucklings at about 2 weeks of age, whereas adults have subclinical infection. signs in sucklings include emaciation, abdominal distension, and oily, matted hair due to steatorrhea. icterus may develop and is most easily discerned as discoloration in the feet, tail, and nose. incoordination, tremors, and paralysis occur just before death. convalescent mice are often partially alopecic and are typically runted. alopecia, runting, and icterus may persist for several weeks, even though infectious virus can no longer be recovered. infants born to immune dams are protected from disease by maternal immunity. epizootiology the prevalence of reovirus 3 infection in contemporary mouse colonies is rare (livingston and riley, 2003; pritchett-corning et al., 2009 ). reoviruses are highly contagious among infant mice and can be transmitted by the oral-fecal or aerosol routes, but mechanical transmission by arthropods has also been documented. additionally, virus may be carried by transplantable neoplasms and transmitted inadvertently by injection. transmission is inefficient among adult mice. there is no evidence that vertical transmission is important or that genetic resistance or gender influence expression of disease. infection in immunocompetent mice appears to be self-limiting, lasting up to several weeks but terminating with the development of host immunity. the course of infection in immunodeficient mice should be considered prolonged, but the duration has not been determined. pathology reovirus 3 can cause severe pantropic infection in infant mice. after parenteral inoculation, virus can be recovered from the liver, brain, heart, pancreas, spleen, lymph nodes, and blood vessels. following ingestion, reoviruses gain entry by infecting intestinal epithelial cells (m cells) that cover peyer's patches. virus can be carried to the liver in leukocytes, where it is taken up by kupffer cells prior to infecting hepatocytes. in acute disease, livers may be large and dark, with yellow foci of necrosis. the intestine may be red and distended, and, in infants, intestinal contents may be bright yellow. myocardial necrosis and pulmonary hemorrhages have been reported. myocardial edema and necrosis are especially prominent in papillary muscles of the left ventricle. the brain may be swollen and congested. central nervous system lesions have a vascular distribution, and are most prevalent in the brain stem and cerebral hemispheres. neuronal degeneration and necrosis are followed quickly by meningoencephalitis and satellitosis. severe encephalitis may evoke focal hemorrhage. in the chronic phase, wasting, alopecia, icterus, and hepatosplenomegaly may persist. orally infected suckling mice can develop multifocal hepatocyte necrosis, which may include the accumulation of dense eosinophilic structures resembling councilman bodies. hepatocytomegaly, kupffer cell hyperplasia, and intrasinusoidal infiltrates of mononuclear cells and neutrophilic leukocytes also can develop. in experimentally inoculated mice, necrotic foci can persist in the liver for at least 4 weeks. chronic active hepatitis may develop after acute infection and result in biliary obstruction. acinar cells of the pancreas and salivary glands can undergo degeneration and necrosis. because pancreatic duct epithelium is susceptible to infection, parenchymal lesions in the pancreas may be caused by obstruction rather than by viral invasion of parenchyma. pulmonary hemorrhage and degeneration of skeletal muscles also have been observed. both humoral and cellular immunity seem to participate in host defenses, but it is unclear how host immunity may influence the course of chronic infection. oronasal inoculation of infant mice with reovirus 1 results in a similar distribution but significantly milder lesions compared to reovirus 3. in contrast, reovirus 2 is highly enterotropic, inducing mild enteritis without lesions in other tissues, similar to epizootic diarrhea of infant mice (edim) . diagnosis serology uses reovirus 3 as antigen, which detects seroconversion to all serotypes, and viral rna can be detected by rt-pcr. a presumptive diagnosis of reovirus infection is aided clinically by detection of the oily hair effect, accompanied by jaundice and wasting. the presence histologically of multisystemic necrosis is consistent with severe reovirus 3 infection but should be confirmed by immunohistochemistry or virus isolation. differential diagnosis reovirus infection must be differentiated from other diarrheal diseases of infant mice, including those caused by mouse coronaviruses, edim virus, salmonella spp., or clostridium piliforme. prevention and control although surviving mice appear to recover completely from infection, the potential for a carrier state is unresolved. therefore, it may be necessary, after adequate testing for the continued presence of virus by the use of sentinels, map testing, or other appropriate means, to rederive or replace infected stock. prevention depends on adequate barrier husbandry coupled with adequate serological monitoring. research complications reovirus 3 infection can interfere with research in several ways. infections in breeding colonies can result in high mortality among sucklings from nonimmune dams. virus has been commonly recovered from transplantable neoplasms and is suspected of being oncolytic. the potential exists for interference with hepatic, pancreatic, cardiovascular, or neurological research. etiology rotaviruses are double-stranded, segmented rna viruses that have a wheel-like ultrastructural appearance. edim virus is a group a rotavirus that replicates in differentiated epithelial cells of the small intestine by budding into cisternae of endoplasmic reticulum. currently, only a single antigenic strain is recognized, but antigenically distinct variants may exist. edim virus shares an inner capsid antigen with rotaviruses of rabbits, fowl, nonhuman primates, human beings, and domestic and companion animals. these agents tend to be species-specific under natural conditions and can be differentiated by serum neutralization tests. cultivation of edim virus requires the presence of proteolytic enzymes to cleave an outer capsid polypeptide. clinical signs clinical signs occur in infant mice less than 2 weeks old. this age-related susceptibility also applies to infection in immunodeficient mice. furthermore, clinical signs occur only in offspring of nonimmune dams, because maternal immunity protects infants until they have outgrown susceptibility to clinical disease. the cardinal signs are bloated abdomens with fecal soiling of the perineum, which may extend to the entire pelage in severe cases. despite high morbidity, mortality is low because affected mice continue to nurse. transient weight loss does occur, and there may be a delay in reaching adult weight. recovery from infection usually occurs in about 2 weeks and, once weight is regained, is clinically complete. epizootiology edim virus appears to be infectious only for mice and occurs episodically in mouse colonies, and infection is probably widespread geographically (livingston and riley, 2003; pritchett-corning laboratory animal medicine et al., 2009) . all ages and both sexes can be infected, but genetic resistance and susceptibility have not been determined. the virus is highly contagious and is transmitted by the oral-fecal route. subclinically infected adult mice can shed virus in feces for at least 17 days, an interval that may be extended in immunodeficient mice. after oral inoculation, virus is essentially restricted to the gastrointestinal tract, although small amounts of virus may be present in the liver, spleen, kidney, and blood. nursing dams can contract infection from their litters. transplacental transmission has not been demonstrated. pathology gross lesions occur primarily in the gastrointestinal tract, but thymic involution can result from infection-related stress. the intestine is often distended, flaccid, and filled with gray-green gaseous liquid or mucoid fecal material that soils the pelage. the stomach contains curdled milk, except in terminal cases with anal impaction due to caking of dried feces. virus preferentially infects terminally differentiated enterocytes in the small and large intestines, which accounts for the agerelated susceptibility to disease; the number of such cells decreases as the intestinal tract matures. characteristic histological lesions are often very subtle, but are most easily discerned in the small intestine in mice less than 2 weeks old. they consist of vacuolation of villar epithelial cells with cytoplasmic swelling, which give villi a clubbed appearance (fig. 3.25) . the vacuoles must be differentiated from normal absorption vacuoles in nursing mice. the lamina propria may be edematous, but necrosis and inflammation are not prevalent. diagnosis edim virus infection is readily detected serologically. clinical disease is diagnosed from signs and typical histological lesions in the intestine, which can be confirmed by immunohistochemical or ultrastructural demonstration of virus in the intestine or in intestinal filtrates or smears. rotavirus antigen can be detected in feces by elisa, but certain dietary ingredients can cause false-positive reactions. infection can also be diagnosed by rt-pcr. differential diagnosis edim virus infection must be differentiated from other diarrheal diseases of suckling mice such as intestinal coronavirus (mouse hepatitis virus) infection, reovirus 3 infection, tyzzer's disease, and salmonellosis. the presence of milk in the stomach can be helpful in differentiating edim virus infection from more severe enteric infections, such as those caused by pathogenic coronaviruses, during which cessation of nursing often occurs. the possibility of dual infections must also be considered. thymic necrosis in edim virus-infected mice, although nonspecific, must be differentiated from that due to mouse thymic virus (mtv) infection or other stressors. prevention and control the spread of edim can be controlled effectively by the use of microbarrier cages and good sanitation. because infection appears to be acute and self-limiting, cessation of breeding for 4-6 weeks to allow immunity to build in adults while preventing access to susceptible neonates also is recommended. alternatively, litters with diarrhea can be culled, in combination with the use of microbarrier cages. the duration of infection in immunodeficient mice has not been determined, but it is reasonable to assume that chronic infection occurs. therefore, such animals should be eliminated. litters from immune dams are more resistant to infection. if edim virus is allowed to become enzootic within a colony, clinical signs will disappear within the population, which may be an appropriate management approach in conventional colonies. prevention of edim virus infection depends on maintenance of sanitary barrier housing with adequate serological surveillance. research complications the research complications of edim infection pertain to clinical illness with diarrhea and retarded growth. transient thymic necrosis may perturb immunological responses. infection (barthold, 1997a,b) etiology coronaviruses are large, pleomorphic, enveloped rna viruses with radially arranged peplomers (spikes). in mice, early clinical and laboratory investigations emphasized their potential to induce hepatitis, so their original designation, which is still used actively, is mouse hepatitis virus (mhv). during that time, enteritis in infant mice was recognized as a separate entity caused by an uncharacterized virus, known as lethal intestinal virus of infant mice (livim). subsequent studies revealed that hepatitis-causing mhv and enteritis-causing livim were closely related coronaviruses, now collectively termed mhv. mhv isolates differ in biologic behavior according to their organ tropism into two biotypes: enterotropic strains, which infect primarily the intestinal tract, and polytropic strains, which initially infect the respiratory tract but may progress to multisystemic dissemination, including the liver and brain. these differences are often reflected in their cell tropism in vitro. however, natural isolates may contain features of both biotypes. several prototype polytropic strains have been extensively studied as experimental models of hepatitis and encephalitis. they include jhm (mhv4), mhv-1, mhv-3, mhv-s, and mhv-a59. numerous additional strains have been identified that differ in virulence, tissue tropism, and antigenicity. differentiation by strain, particularly under natural conditions, is irrelevant, since mutation is common among coronaviruses, and even named prototype strains differ significantly depending upon passage history. although mhv isolates and strains share internal antigens (m and n), they can be distinguished by neutralization tests that detect strainspecific spike (s) antigens. mhv shares antigens with the coronaviruses of rats, a finding that has been exploited to develop heterologous antigens for serological tests. mhv also is related to human coronavirus oc43. a number of established cell lines may be used for propagating polytropic mhv strains in vitro. however, field isolates are difficult to maintain in vitro. nctc 1469 mouse liver cells are useful for growing many polytropic strains. mhv can also be grown in mouse macrophages, cells that have been used for genetic studies of resistance and susceptibility to infection. enterotropic strains, because of their tendency to be strictly enterotropic, have been grown in cmt-93 cells derived from a rectal carcinoma in a c57bl mouse, but are generally difficult to propagate in cell culture. irrespective of cellular substrate used for isolation or propagation, syncytium formation is emblematic of mhv infection (fig. 3.26) . clinical signs clinical signs depend primarily on the age, strain, and immunological status of infected mouse and strain and tropism of virus. as with many murine viruses, infection is often clinically silent among immunologically competent mature mice. clinical morbidity is most often associated with suckling mice less than 2 weeks old or with immunodeficient mice. suckling mice infected with enterotropic mhv develop inappetence, diarrhea, and dehydration, often terminating in death (fig. 3.27) . epizootics of enterotropic mhv have been known to result in 100% mortality among neonatal mice in a breeding colony. older mice (2-3 weeks of age) may have ruffled pelage and runting. neurotropic strains such as mhv-jhm may induce flaccid paralysis of the hindlimb, but this sign is rarely encountered alone during natural infection. conjunctivitis, convulsions, and circling may be seen occasionally. enterotropic strains may not cause acute disease in athymic mice when exposed as adults, whereas mildly pathogenic polytropic strains can cause a progressive wasting syndrome that may be accompanied by progressive paralysis. epizootiology mhv infection, despite constant surveillance and preventive programs, continues to be a common threat to laboratory mouse populations (livingston and riley, 2003; pritchett-corning et al., 2009 ). there are no reports of natural transmission from mice to other species, but suckling rats have been found to develop necrotizing rhinitis after intranasal inoculation with mhv-s. mhv is highly contagious, with natural transmission occurring by respiratory or oral routes. mouse appears normal and has a milk-filled stomach. lower mouse is runted and dehydrated and has an empty stomach. from barthold et al. (1982) . enterotropic biotypes predominate in natural infections in contemporary laboratory animal facilities, since they tend to be the most contagious due to copious excretion of virus in feces, whereas polytropic strains generally spread by direct respiratory contact. natural vertical transmission has not been demonstrated. introduction of mhv through injection of contaminated biologicals can be an important factor in epizootics, especially because some isolates infect b lymphocytes and, by implication, hybridomas nonlytically. infection in immunocompetent mice is self-limiting. immune-mediated clearance of virus associated with seroconversion usually begins about a week after infection, and mice recover fully within 3-4 weeks. humoral and cellular immunity participate in host defenses to infection, and t-cell-dependent immunity is an absolute requirement. thus, age-related resistance to mhv correlates with maturation of lymphoreticular tissues, but intestinal proliferative kinetics are critical determinants of disease susceptibility with enterotropic mhv. enzootic infection had been construed to include persistent infection in individual mice. current evidence suggests, however, that enzootic infection results either from the fresh and continuous introduction of immunologically naive or deficient mice or from the recurrent infection of immune mice with mhv variants that arise by natural mutation. mutation is favored by immune pressure in enzootically infected colonies as well as missteps during natural replication, which include copying errors and recombination. thus, mice that have developed immunity to one strain of mhv can remain susceptible to one or more genetically and antigenically divergent strains, resulting in reinfection. this caveat has practical importance for breeding colonies. maternal immunity protects suckling mice against homologous mhv strains but not against antigenically variant strains. however, maternal immunity, even to homologous strains, depends on the presence of maternally acquired antibody in the lumen of the intestine. therefore, the susceptibility of young mice to infection increases significantly at weaning. strain differences in resistance and susceptibility to polytropic mhv can be inherited as an autosomal dominant trait. for example, dba/2 mice are highly susceptible to mhv-3 and die acutely even as adults, whereas a/j mice develop resistance to lethal infection shortly after weaning. however, genetic resistance is also virus strain-dependent. therefore, mice resistant to one strain of mhv may be susceptible to another strain. it also is worth noting that the expanded use of genetically altered mice with novel or unanticipated deficits in antiviral responses may alter the outcome of virus-host interactions unpredictably. this pertains to mhv as well as other agents. for example, mhv infection has presented as granulomatous peritonitis and pleuritis in interferongamma (ifn-γ) knockout mice (france et al., 1999) . pathology polytropic strains replicate initially in the nasal mucosa, where necrotizing rhinitis may occur. viremic dissemination can follow if virus gains access to regional blood vessels and lymphatics. thus, viremia leads to secondary infection of vascular endothelium and parenchymal tissues in multiple organs including liver, brain, lymphoid organs, and other sites. mice also may develop central nervous system disease by direct extension of infection from the olfactory mucosa along olfactory tracts. at necropsy, yellow-white foci indicative of necrosis can occur in multiple tissues, with the involvement of the liver as the classical lesion. liver involvement may be accompanied by icterus and peritonitis. histologically, necrosis can be focal or confluent and may be infiltrated by inflammatory cells (fig. 3.28) . syncytia commonly form at the margin of necrotic areas and, in mild infections, may develop in the absence of frank necrosis. syncytia formation is a hallmark of infection in many tissues, including the intestine (fig. 3.26) , lung, liver, lymph nodes, spleen, thymus, brain, and bone marrow and in vascular endothelium in general. although syncytia are transient in immunocompetent mice, they are a persistent feature in chronically infected, immunodeficient mice ( fig. 3.29) . neurotropic variants cause acute necrotizing encephalitis or meningoencephalitis in suckling mice, with demyelination in the brain stem and in peri-ependymal areas secondary to viral invasion of oligodendroglia. convalescent mice may have residual mononuclear cell infiltrates around vessels or as focal lesions in the liver. immunodeficient mice can develop progressive necrotic lesions in the liver and elsewhere. compensatory splenomegaly may occur because of expansion of hematopoietic tissue. enterotropic strains infect primarily the intestine and associated lymphoid tissues, although some may also figure 3.28 necrosis, inflammation, and syncytium in the liver of a mouse infected with mhv. courtesy of s.w. barthold. cause systemic lesions, especially in the liver and brain. the most common sites are terminal ileum, cecum, and proximal colon. the severity of disease is age-related, and dependent upon intestinal proliferative kinetics, similar to edim, with young infants being at highest risk for lethal infection. pathogenic strains can cause lesions ranging from villus attenuation to fulminant necrotizing enterotyphlocolitis, which can kill suckling mice within a few days (fig. 3.27) . the stomach is often empty, and the intestine is filled with watery to mucoid yellowish, sometimes gaseous contents. syncytia are a consistent feature in viable mucosa (fig. 3.30) and not only are formed in intestine but also may be present in mesenteric lymph nodes and endothelium of mesenteric vessels. enterocytes may contain intracytoplasmic inclusions, but they are not diagnostic. surviving mice develop compensatory mucosal hyperplasia, which eventually recedes, but may contribute to clinical signs due to osmotic, secretory, and malabsorptive diarrhea. older mice are equally susceptible to infection, but are resistant to severe disease due to their mature (more rapid) intestinal proliferative kinetics. pathology may be subtle, consisting of transient syncytia without necrotic lesions. in adult mice, syncytia can be found most often in the surface mucosal epithelium of the ascending colon. the exception occurs in immunodeficient mice, such as athymic and scid mice, which can develop chronic proliferative bowel disease of varying severity with mhv antigen in mucosal epithelium (figs. 3.31 and 3.32). this may not always be present, as athymic nude mice exposed as adults may only manifest a few enterocytic syncytia without hyperplasia. diagnosis because mhv infection is often subclinical, serological testing is the most reliable diagnostic tool. many animal resources rely on sentinel mouse protocols for continuous serological surveillance. serology is well established, sensitive, and reliable. neutralization tests are used to differentiate individual virus strains in the research laboratory but are inappropriate for routine use, because of cost, technical complexity, and serologic identification per se does not predict biological behavior, including virulence or tissue tropism. serology also can be used in the context of map testing in which adult mice are inoculated with suspect tissues to elicit seroconversion. rt-pcr protocols to detect virus in tissues or excreta are available. the detection of syncytia augmented, when possible, by immunohistochemistry to laboratory animal medicine detect mhv antigen is a useful and practical means to confirm infection. this strategy should attempt to select mice that are in early stages of infection, because necrosis in infant mice or seroconversion in older mice may reduce the chances of detecting syncytia or viral antigens. the option of using immunodeficient mice as sentinels can be considered, because they sustain prolonged infection. however, they should be securely confined because they also amplify virus loads. if properly controlled, amplification in immunodeficient mice can, however, facilitate subsequent virus isolation in tissue culture. differential diagnosis mhv infection must be differentiated from other infectious diseases that cause diarrheal illness, runting, or death in suckling mice and wasting disease in immunodeficient mice. these include edim, mousepox, reovirus 3 infection, tyzzer's disease, and salmonellosis. neurological signs or demyelinating lesions must be differentiated from mouse encephalomyelitis virus infection or noninfectious cns lesions, such as neoplasms, including polyoma virus-induced tumors in athymic mice. prevention and control control and prevention of mhv infection can be difficult because of the numerous variables that influence its expression. perhaps the most important factor is the duration of infection in individual mice and in mouse colonies. there is evidence that infection in an individual immunocompetent mouse is acute and self-limiting. such mice can be expected to develop immunity and eliminate virus within 30 days. therefore, selective quarantine at the cage (not room) level with the temporary cessation of breeding can be used effectively to eliminate infection. quarantine at the room basis is likely to fail, since mutations arise and continually reinfect the mouse population. additionally, maternally derived immunity can protect infant mice from infection until they are weaned and moved to uncontaminated quarters. careful testing with sentinel mice should be used to assess the effectiveness of quarantine or 'natural rederivation,' as just described. immunodeficient mice, in contrast, are susceptible to chronic infection and viral excretion. mice with unrecognized or unanticipated immune dysfunction or with selective immune dysfunction may impact on mhv infection and its control. such colonies, which may contain highly valuable or irreplaceable mice, may be rescued by cesarean rederivation or embryo transfer if vertical transmission of mhv infection is subsequently ruled out. although rodent coronaviruses are not viable for extended periods in the environment, excreted virus may remain infectious for up to several days, so proper sanitation and disinfection of caging and animal quarters as well as stringent personal sanitation are essential to eliminate infection. the prevention of mhv requires procurement of animals from virus-free sources and maintenance under effective barrier conditions monitored by a well-designed quality assurance program. control of feral mouse populations, proper husbandry and sanitation, and strict monitoring of biological materials that may harbor virus (e.g., transplantable neoplasms, cell lines) are also important strategies to prevent adventitious infection. research complications numerous research complications have been attributed to mhv, and the unpredictable outcome of infection in genetically altered mice is likely to lengthen the list. for example, apart from its clinical impact, mhv may stimulate or suppress immune responses, contaminate transplantable neoplasms, and be reactivated by treatment of subclinically infected animals with several classes of drugs, including immunosuppressive agents, and by intercurrent infections. it also can alter tissue enzyme levels. additionally, the ubiquitous threat of mhv infection and uncertainty about its potential effects on a given research project provoke concerns that may exceed its true impact. for example, transient infection with a mild enterotropic strain is unlikely to disrupt systemic immune responses, whereas infection with a polytropic strain may be highly disruptive. this is not to say that subclinical or strictly enterotropic infection should be taken lightly but simply to caution against overreaction in assessing the impact of an outbreak. etiology mice are susceptible to infection by two members of the cardiovirus genus within the theilovirus. emcv has a less selective host range and can infect wild mice, but is not known to infect laboratory mice. tmev is a small, nonenveloped, rna virus that was discovered by max theiler during experimental studies of yellow fever virus in mice. established prototype strains include to (theiler's original), fa, da, and gd vii, the last of which is named after george martine (george's disease), an assistant in theiler's laboratory. tmev is rapidly destroyed by temperatures over 50°c and by alcohol but not by ether. it can be cultivated in vitro in several continuous cell lines, but bhk-21 cells are routinely used for isolation and propagation. tmev is antigenically related to emcv. as with other nonenveloped viruses, tmev is resistant to environmental inactivation, a factor that must be considered in control and prevention of infection. clinical signs the development of clinical disease depends on virus strain, mouse strain, and route of exposure, but natural disease is exceedingly rare (estimated at 0.1-0.01% of infected mice). when clinical signs occur, they are expressed as neurological disease. the characteristic sign is flaccid posterior paralysis, which may be preceded by weakness in the forelimbs or hindlimbs, but in mice that are otherwise alert (fig. 3.33) . some mice may recover, but death frequently ensues, often because of failure to obtain food or water. furthermore, mice that recover from the paralytic syndrome are disposed to a chronic demyelinating phase, which is expressed as a gait disturbance. epizootiology infection occurs primarily in laboratory mice with the exception of the mgh strain, which has been isolated from laboratory rats and is pathogenic in mice and rats after experimental inoculation. the prevalence of tmev in mouse colonies is low, a reflection of the slow rate at which virus is transmitted from mouse to mouse, but it continues to be among the more common viral contaminants of mouse colonies (livingston and riley, 2003; pritchett-corning et al., 2009) . tmev infection is acquired by ingestion and replicates primarily in the intestinal mucosa. enteric infection can persist after the development of host immunity and can result in chronic or intermittent excretion of virus in feces over several months . mice often become infected shortly after weaning, but virus is seldom recovered in mice over 6 months of age. however, neurologic infection can persist in the brain and spinal cord for at least 1 year. immunity to one strain of tmev provides cross-protection to other strains. there are no reports of differences in mice with respect to susceptibility to infection under natural conditions. prenatal transmission has not been found. pathology intestinal tmev infection does not cause lesions, but virus can be detected in enterocytes by immunohistochemistry or in situ hybridization. poliomyelitis-like disease, the syndrome that may be encountered during natural infections, is characterized by acute necrosis of ganglion cells and neurons, neuronophagia, and perivascular inflammation, which occur particularly in the ventral horn of the spinal cord gray matter but also can involve higher centers such as the hippocampus, thalamus, and brain stem. during the subsequent demyelinating phase, mononuclear cell inflammation develops in the leptomeninges and white matter of the spinal cord, accompanied by patchy demyelination. the white-matter lesions are due to immune injury. spontaneous demyelinating myelopathy, affecting the thoracic spinal cord and associated with mev infection, has also been reported in aged mice. virulent strains may cause acute encephalitis after experimental inoculation, whereas less virulent isolates produce acute poliomyelitis followed by chronic demyelinating disease. diagnosis infection is usually detected serologically or by pcr of feces, but virus shedding from infected mice may be intermittent. clinical signs are striking, if they occur, but are too rare to rely on for routine diagnosis. histological lesions in the cns and especially the spinal cord are characteristic when present. differential diagnosis neurotropic variants of mhv may, on occasion, cause similar neurological signs. injury or neoplasia affecting the spinal cord can also produce posterior paralysis. polyoma virus infection in athymic mice can induce tumors or demyelination in the cns, which may result in clinical signs resembling those of tmev infection. prevention and control disease-free stocks were originally developed by foster-nursing infant mice. this technique, cesarean rederivation, or embryo transfer can be used successfully to eliminate infection. in either case, foster mothers should be surveyed in advance to ensure their mev-free status. selective culling can be considered as an option to eliminate infection, because laboratory animal medicine infection spreads slowly. however, the virus is hardy in the environment and resists chemical inactivation, so it may be prudent to depopulate and disinfect rooms if the presence of infection is unacceptable. research complications the principal hazard from tmev for research relates to its potential effects on the cns. noroviruses are nonenveloped rna viruses that belong to the family caliciviridae. they are notoriously resistant to environmental inactivation, and cause significant gastrointestinal morbidity in humans. noroviruses are species-specific, including mnv, which exclusively infects mice. until the discovery of mnv, replication of noroviruses in vitro has not been possible. for this reason, mnv has emerged as an important small animal model of norovirus pathogenesis. mnv was relatively recently discovered in 2003, and subsequent surveillance has revealed that it is the most common adventitious virus infection in laboratory mice (hsu et al., 2005; pritchett-corning et al., 2009) . over 35 mnv isolates have been found in mouse research colonies around the world, which display nearly 90% genetic identity, comprising a single genetic cluster. although genetically homogeneous, significant biological differences exist among mnv strains (thackray et al., 2007) . mnv effectively replicates in macrophages and dendritic cells, including the mouse macrophage-like raw264.7 cell line, as well as a microglial cell line (wobus et al., 2004) . clinical signs clinical signs of infection in immunocompetent mice are usually absent, but infection leads to systemic disease with high mortality in interferon αβγ receptor and stat1 null mice. affected mice have loss of body weight, ruffled fur, and hunched posture (ward et al., 2006) . experimental infection of 129 and c3h mice with mnv-1 caused mild diarrhea (kahan et al., 2011) . epizootiology mnv is transmitted by the fecaloral route, and contaminates the environment as an environmentally resistant virus. for this reason, it can efficiently infect sentinel mice with soiled bedding (manuel et al., 2008) . duration of infection varies with mnv strain, mouse immunocompetence and mouse genotype. experimental studies have revealed that several mnv strains persist in various tissues of c57bl/6j, hsd:icr, and jcl:icr and c.b-17-prkdc scid mice, with fecal shedding for at least 35-60 days (goto et al., 2009; hsu et al., 2006; thackray et al., 2007) . although not clinically ill, rag1 null mice are unable to clear infection . comparative studies with mnv-1 and mnv-3 have shown differences in virus replication and shedding (kahan et al., 2011) . mnv has a tropism for macrophages and dendritic cells, and virus can be detected in the intestine, intestinal lymphoid tissue, liver, and spleen (hsu et al., 2006; kahan et al., 2011; wobus et al., 2006) . pathology naturally and experimentally infected stat1 or ifnγr null mice may develop splenomegaly and multifocal pale spots on the liver. microscopic findings include varying degrees of hepatitis, focal interstitial pneumonia, vasculitis, peritonitis, and pleuritis (karst et al., 2003; ward et al., 2006) . encephalitis, cerebral vasculitis, pneumonia, and hepatitis have also been described in intracerebrally infected stat1 null mice (karst et al., 2003) . infection of immunocompetent mice may be associated with mild inflammation of the intestine, splenic hypertrophy, and lymphoid hyperplasia of spleen and lymph nodes (mumphrey et al., 2007) . diagnosis mnv infection can be detected by serology or rt-pcr. sentinel mouse surveillance, using soiled bedding, is an effective strategy for detecting mnv (manuel et al., 2008) differential diagnosis the mild change in fecal consistency associated with mnv in adult mice may mimic rotavirus, coronavirus, helicobacter spp., citrobacter rodentium, or other enteric diseases. disseminated lesions in stat1 or ifnγr null mice must be differentiated from other polytropic viral diseases in immunodeficient mice, including mhv. prevention and control depopulation and decontamination has been shown to be effective at eliminating mnv from an enzootically infected colony, whereas testand-removal of positive mice was found to be ineffective (kastenmayer et al., 2008) . embryo transfer and cesarean rederivation are also effective (goto et al., 2009; perdue et al., 2007) . neonatal mice are resistant to infection, so that cross-fostering neonates onto uninfected dams is another effective means of rederivation mnv-free mice (artwohl et al., 2008; compton, 2008) . research complications the tropism of mnv for macrophages and dendritic cells is likely to modify immune responses, and mnv infection may interfere with studies involving enteric disease. hantaviruses are rna viruses belonging to the very large bunyaviridae family. they differ from other members of this family by not being arthropod-borne. each hantavirus is antigenically distinct and maintained within single or at most a few rodent or insectivore hosts, but are infectious for other hosts. infection is lifelong, and virus is transmitted by shedding of virus in urine, feces, and saliva. several hantaviruses are zoonotic and may cause severe disease in humans. although there is overlap, hantaviruses in asia and europe cause hemorrhagic fever with renal syndrome (hfrs) in humans, a multisystem disease with significant renal involvement, and hantaviruses that are endemic in the americas cause laboratory animal medicine hantavirus pulmonary syndrome (hps) in humans, which is a multisystem disease with pulmonary involvement. among the better-known old world hfrs hantaviruses are hantaan, seoul, puumala, and dobrava-belgrade viruses. sin nombre virus is the best known new world phs hantavirus, among many others. most notably from the perspective of laboratory animal medicine, the norway rat, rattus norvegicus, serves as a reservoir host for hantavirus in the wild, but infection has also been associated with laboratory rats. in addition to being endemic in wild rats in asia, it has been found to be endemic in wild rats in the eastern united states and associated with human cases of hfrs (childs et al., 1988; leduc et al., 1984; tsai et al., 1985) . over 120 cases of hantavirus infection have been transmitted to humans from laboratory rats in japan, belgium, and the united kingdom (desmyter et al., 1983; kawamata et al., 1987; lloyd et al., 1984; umenai et al., 1979) . m. musculus is not considered to be a primary reservoir host, but hantavirus infection has been documented serologically in conventional and barrier-maintained laboratory mice and rats in korea (won et al., 2006) , infection of wild m. musculus has been documented in the united states (baek et al., 1988) , and infection of wild mice in europe has been associated with human exposure (diglisic et al., 1994) . hantaviruses are difficult to culture in vitro. infection in rodents is subclinical and is detected by serology or rt-pcr. the main research complication from natural infection is the zoonotic risk and potentially subclinical effects on the immune response associated with viral defenses such as cd8 + t cell (taruishi et al., 2007) and nk function as demonstrated in human studies (braun et al., 2014) . the mouse is host to a number of enveloped rna viruses of the family retroviridae, subfamily orthovirinae, including the two type species (and their variants) mouse mammary tumor virus (mmtv) and murine leukemia virus (mlv). these viruses belong to a diverse assemblage of related mobile dna elements that are integrated into the host genome, and collectively termed 'retroelements', which include retrovirus-related elements and nonviral elements. during cell division, retroelements are transcribed into rna, and subsequently reverse-transcribed into dna copies that become integrated into a new location within the genome. this process utilizes reverse transcriptase, which is encoded by the retroelement. over millennia, retroelements have been repeatedly integrated within the genome in large numbers, comprising approximately 40% of the mammalian genome. various families of mouse retroelements share sequence similarity, despite their random distribution throughout the mouse genome, and the majority of them are truncated, mutated, and methylated to become incapable of infectivity. nevertheless, many of them continue to be mobile within the genome. noninfectious retrovirus-related retroelements include iap, vl30, musd, and etn elements. replication-competent retroviruses represent the pinnacle of the retroelement constellation and are best considered as the most evolutionarily recent members. these include mmtv and mlv. mtv and mlv share similar genetic structure, except that the long terminal repeat (ltr) region of the mmtv genome encodes an additional superantigen (sag). both mmtv and mlv include exogenous viruses, which are horizontally transmitted, replication-competent viruses, and endogenous viruses, which are closely related to exogenous viruses, encoded within the mouse genome, and transmitted by mendelian inheritance. exogenous mmtv and mlv exist in wild mouse populations, but have been eliminated from contemporary laboratory mice. however, they may continue to be used experimentally, including bittner mmtv, and gross, friend, moloney, and rauscher mlvs. in particular, mouse colonies may be purposely infected with mmtv for mammary cancer research and are termed 'mmtv-positive', reflecting their exogenous virus status, even though the mice may also carry endogenous mmtv. the genomes of all inbred strains of mice encode one or more (over 50 in some mouse strains) endogenous mmtv loci, the distribution of which is unique to each inbred strain of mouse. most mmtv genomic loci do not encode infectious virus or are transcriptionally inactive, except for mouse strains (dba, c3h, grs) that carry mtv1 or mtv2 loci. these loci encode infectious virus, which can be visualized as b-type particles by electron microscopy. likewise, all mouse strains carry endogenous mlv loci within their genome, but not all mice carry replication-competent mlv sequences. some endogenous mlvs encode infectious virus, which can be visualized as c-type particles by electron microscopy. mice have often evolved mechanisms to counter the deleterious effects of retroviruses by preventing reentry or replication of virus into other cells. if an endogenous retrovirus is still infectious to other mouse cell targets, it is termed ecotropic, whereas if it is no longer infectious for mouse cells, but can infect cells of other species, it is termed xenotropic. viruses capable of infecting cells of mice as well as other species are termed polytropic. the combinations of endogenous replication-competent mlvs and cell tropism factors are a reflection of selective breeding of mouse strains for susceptibility to various types of cancer. clinical signs mice were originally inbred for specific phenotypes, including mammary tumors and lymphomas. thus, some strains of mice were genetically selected for unique combinations of endogenous mmtv and mlv in concert with susceptibility factors laboratory animal medicine that favored their expression and disease manifestations. in addition, noninfectious retroelements continue to reintegrate randomly within the genome during cell division as retro transposons. these ongoing integrations contribute to genetic drift, spontaneous mutations, and well-recognized mouse strain phenotypes, including the athymic nude allele, the hairless allele, and the rodless retina allele, among others. epizootiology exogenous mmtv and mlv are horizontally transmissible, primarily through the milk of lactating females. endogenous retroviruses and retroelements are inherited through the genome. replicationcompetent endogenous mmtvs and mlvs are also transmissible like their exogenous counterparts, but differ by being integrated within the genome of the mouse. pathology replication-competent mmtv and mlv, regardless of their exogenous or endogenous origin, are usually clinically silent. their ability to cause neoplasia is a reflection of genetic selection for susceptibility factors that are genetically encoded within individual mouse strain genomes. mmtv derives its name from its association with induction of mammary carcinomas in mammary cancer-susceptible strains of mice. mlv is associated with lymphomas, the pattern of which is mouse strain specific. for example, akr mice develop 100% prevalence of thymic lymphoma between 6 and 12 months of age, whereas aging balb/c mice commonly develop multicentric lymphoma. in these strains of mice, multiple endogenous mlvs are coexpressed in tissues and undergo recombination events that allow them to target and transform cells into neoplasia. despite its name, mmtv can induce lymphomas in some strains of mice, such as sjl mice which develop lymphomas arising from enteric lymphoid tissue and mesenteric lymph nodes. diagnosis exogenous retroviruses have been eliminated from contemporary mouse populations, unless purposely introduced for experimental purposes. because endogenous retroviruses and retroelements are encoded within the genome, and reflect the unique genetic composition of each strain of mouse, they are not targets of diagnostic pursuit. differential diagnosis patterns of some types of neoplasia within individual inbred strains of mice are a reflection of their endogenous retroviral integration. prevention and control exogenous retroviruses have been eliminated from laboratory mice by cesarean rederivation and foster nursing. mmtv-s, the 'bittner agent', continues to be purposely maintained in some mouse breeding populations, but can be eliminated by foster-nursing or other means. caution is advised when re-deriving such mouse colonies for other purposes, as elimination of exogenous mmtv will be an unintended consequence. research complications endogenous retroviruses and retroelements influence the life span of individual strains of mice, and random integrations during cell division can give rise to spontaneous mutations and genetic drift. it is estimated that significant mutations may arise due to mobile retroelement integrations every 50 generations. astroviruses are small, nonenveloped, singlestranded rna viruses that have been associated with human gastroenteritis and detected in association with other enteric pathogens. the viral family astroviridae is split into two genuses: avastrovirus for those astroviruses infecting avians and mamastrovirus for those infecting mammals. astrovirus infection has been detected in research mice (muastv) using metagenomic analyses and appears to have a wide geographical, institutional, and host strain distribution. clinical signs none reported. epizootiology pcr screening has found muastv infection in up to 22% of a variety of mouse strains housed in vendor and academic facilities in the united states and japan. the virus has been detected most commonly in immunocompromised mice (nsg, nod-scid, nsg-3gs, c57bl6-timp-3 −/− , and upa-nog), but also in immuncompetent strains (b6j, icr, bash2, and balb/c). both immunodeficient and immunocompetent mice are susceptible to muastv, but adaptive immunity is required to clear the virus. based on human epidemiology indicating children are at highest risk for infection, the virus may preferentially infect young mice. pathology immunodeficient mice showed no sign of pathology based on histopathology. diagnosis pcr data has indicated that muastv causes a systemic, chronic infection in immunocompromised mice, indicating samples from most tissues will be pcr positive. yokoyama et al. (2012) detected high viral load (up to 10 9 genome copies) per fecal pellet from immunocompetent mice. differential diagnosis none, in the absence of lesions and clinical disease. prevention and control because immunocompetent mice clear the infection, quarantine may be successful but lack of routine screening for muastv in laboratory mice will allow for uncontrolled spread of the infection. research complications based on limited surveys, muastv may have a high prevalence in laboratory mice. the impact of infection on both innate and adaptive immune responses warrants further investigation to assess the potential for confounding research data. this section briefly describes the etiology, clinical signs, epizootiology, pathology, diagnosis, differential diagnoses, prevention and control, and research complications of the most common bacterial diseases encountered in research colonies of mice. as sequencing technology becomes more available, the number and genus/species classification of bacteria potentially responsible for infections, in particular, opportunistic infections, will grow (benga et al., 2014) . potential candidates include members of pasteurellacaeae, bordetella hinzii, streptococcus danielae, acinetobacter spp., and others, for which little is currently known about their pathogenic potential. etiology lawsonia intracellularis, an obligate intracellular bacterium and the causative agent of proliferative enteropathy, is not a pathogen encountered in research colonies of mice but has been reported to infect wild mice and rats in close contact with infected livestock (collins et al., 2011) . clinical signs none reported but should consider lawsonia as a differential in necropsy cases with gross or histologic evidence of proliferative lower bowel lesions. epizootiology although mice are experimentally susceptible to infection and develop classic lesions of hyperplastic ileitis and typhlocolitis (murakata et al., 2008) , susceptibility varied with mouse strain and source of inoculum from rabbits or swine, suggesting important differences in l. intracellularis strains. pathology lawsonia infection may result in hyperplastic ileitis, typhlitis and/or colitis, and hemorrhagic intestines may be noted (percy and barthold, 2007) . diagnosis lawsonia spp. has been diagnosed using a variety of techniques, including pcr, immunohistochemistry, in situ hybridization, and warthin-starry silver stains. differential diagnosis bacterial infections associated with hyperplastic intestinal epithelium, including c. rodentium and enterohepatic helicobacter species in susceptible (typically immunodeficient) mouse strains. prevention and control species separation from hosts more commonly associated with natural infection (hamsters, ferrets, pigs). research complications none reported. the following section describes infection due to mycoplasma pulmonis and summarizes infections associated with other murine mycoplasmas including m. arthriditis, m. neurolyticum, m. collis, and m. muris. antigenic cross-reactivity among these species, and especially between m. pulmonis and m. arthriditis, mandates that reliable diagnostic strategies incremental to serology (elisa, ifa, mfia) such as culture (often false negative) and pcr be employed to distinguish potentially pathogenic infections. when screening cell lines for opportunistic pathogens, pcr is the most efficient method to discriminate between m. pulmonis and mycplasma contaminants associated with cell culture. etiology m. pulmonis is a pleomorphic, gram-negative bacterium that lacks a cell wall and has a single outer limiting membrane. it causes murine respiratory mycoplasmosis (mrm). clinical signs mice are relatively resistant to florid mrm; thus, subclinical infection is more common. when clinical signs occur, they reflect suppurative rhinitis, otitis media, and chronic pneumonia. affected mice may display inactivity, weight loss, and ruffled hair coat, but the most prominent signs are 'chattering' and dyspnea, due to rhinitis and purulent exudate in nasal passages. otitis media may cause a head tilt, whereas suppurative inflammation in the brain and spinal cord, although rare, can cause flaccid paralysis. experimental infection of the genital tract can cause oophoritis, salpingitis, and metritis, which may lead to infertility or fetal deaths. experimental inoculation of scid mice has caused systemic infection accompanied by severe arthritis (evengard et al., 1994) . epizootiology mrm historically was a common infectious disease of mice, but improved housing, husbandry, and health surveillance have reduced its prevalence dramatically. serologic data from a large diagnostic laboratory indicated m. pulmonis infection affects about 0.01% of conventionally housed mouse colonies in the united states and 0.16% in europe (pritchett-corning et al., 2009) . m. pulmonis infection is contracted by inhalation and can occur in suckling and adult mice. therefore, infection should be considered highly contagious. mice injected with cells harvested from m. pulmonis contaminated cell cultures may develop disease. m. pulmonis can also be transmitted venerally; in utero infection has been demonstrated in rats but not in mice. because transplacental infection occurs in rats, the same route may be possible in mice, particularly immunocompromised strains. concomitant viral pneumonia (sv, mouse coronavirus) or elevated environmental ammonia concentrations may increase susceptibility to mrm. m. pulmonis also infects rats, hamsters, guinea pigs, and rabbits. among these species, only rats are significant reservoirs of infection for mice. pathology m. pulmonis is an extracellular organism that colonizes the apical cell membranes of respiratory epithelium. attachment occurs anywhere from the anterior nasal passages to the alveoli and may be mediated by surface glycoproteins. the organism may injure host cells through competition for metabolites such as carbohydrates and nucleic acids or by release of toxic substances such as peroxides. ciliostasis, reduction in the number of cilia, and ultrastructural changes leading to laboratory animal medicine cell death have also been described. detrimental effects on ciliated epithelium can lead to disrupted mucociliary transport, which exacerbates pulmonary disease. experimental infection of mrm is dose dependent. doses of 10 4 colony-forming units (cfus) or less cause mild, transient disease involving the upper respiratory tract and middle ears, whereas higher doses often lead to acute, lethal pneumonia. additionally, m. pulmonis strains can differ in virulence. survivors of severe infection may develop chronic bronchopneumonia with bronchiectasis and spread infection to other mice. intravenous inoculation of m. pulmonis can cause arthritis in mice, but arthritis is not a significant feature of natural infection. host genotype also is a major factor in the outcome of infection, with resistance being expressed phenotypically through the bactericidal efficiency of alveolar macrophages. strains derived from a c57bl background appear to be resistant to pathogenic infection, whereas balb/c, c3h, dba/2, swr, akr, cba, sjl, and other strains have varying degrees of increased susceptibility (cartner et al., 1996; lai et al., 1993) . the initial lesion of mrm is suppurative rhinitis, which may involve the trachea and major airways. early inflammatory lesions, if not quickly resolved, progress to prominent squamous metaplasia. transient hyperplasia of submucosal glands may occur, and lymphoid infiltration of the submucosa can persist for weeks. syncytia can sometimes be found in nasal passages, in association with purulent exudate (fig. 3.34) . affected mice also develop suppurative otitis media and chronic laryngotracheitis with mucosal hyperplasia and lymphoid cell infiltrates. pulmonary lesions are typified by bronchopneumonia, which spreads from the hilus. lymphoid cells and plasma cells accumulate around bronchi which often contain neutrophils in their lumen. chronic lung disease features suppurative bronchitis, bronchiolitis, and alveolitis (fig. 3.35) . chronicity also increases the prevalence of bronchiectasis and abcessation. diagnosis accurate diagnosis should exploit the complementary use of clinical, serological, microbiological, molecular, and morphological methods. clinical signs are variable but can be characteristic when they occur. serology is sensitive but although antibodies do not clear the infection, seroconversion may be weak or take months and may not accurately differentiate between m. pulmonis infection and m. arthriditis infection (cassell et al., 1981) . therefore samples for culture and pcr of the upper respiratory tract should be obtained to confirm diagnosis. buffered saline or mycoplasma broth should be used to lavage the trachea, larynx, pharynx, and nasal passages. specimens for culture from the genital tract are warranted if this site is suspected. mycoplasma spp. may be difficult to grow, so it is prudent to confirm that the relevant expertise and quality control exist in the diagnostic laboratory. speciation can be accomplished by immunofluorescence or immunoperoxidase staining or by growth inhibition. immunohistochemistry should be considered to supplement basic histopathologic examination. immunofluorescence and immunoperoxidase techniques are available to identify mycoplasma antigens in tissue sections or in cytological preparations of tracheobronchial or genital tract lavages (brunnert et al., 1994) . pcr assays for m. pulmonis at veterinary diagnostic laboratories and pcr kits to screen cell culures for mycoplasma are readily available. differential diagnosis mrm must be differentiated from bronchopneumonia associated with ciliaassociated respiratory (car) bacillus. silver stains may reveal car bacilli adherent to the respiratory epithelium. sv also can cause bronchopneumonia in mice but can be detected by serology and immunohistochemistry. other causes of respiratory infection include pvm, corynebacteriosis and, in immunodeficient mice, pneumocystis murina infection. combined infections with known pathogens or secondary opportunists also must be considered. prevention and control mice mount an effective immune response to m. pulmonis, as measured by their recovery from mild infection and their resistance to infection after active or passive immunization (cartner et al., 1998) . antibodies of various classes are produced locally and systemically, but clearance of the infection has been attributed to innate immune responses (love et al., 2010; sun et al., 2013) . there is some evidence that antibody may facilitate phagocytosis of m. pulmonis. t-cell responses, however, appear to exacerbate m. pulmonis in mice, because immunity cannot be transferred with immune cells. in addition, athymic and neonatally thymectomized mice are not more susceptible than immunocompetent mice to m. pulmonis pneumonia. nude and scid mice develop less severe respiratory disease than immunocompetent mice but infection becomes systemic and they may develop suppurative disease in multiple organs and joints (arthritis). host immunity aside, effective control and prevention of mrm depend primarily on maintenance of mycoplasma-free colonies under barrier conditions supported by careful surveillance for infection by serology, microbiology, pcr, and histopathology. cesarean or embryo rederivation may eliminate infection, although vertical transmission may occur in immunocompromised mice. treatment with tetracycline suppresses clinical disease but does not eliminate infection. earlier interest in developing dna-based vaccines against m. pulmonis has not achieved clinical application (lai et al., 1997) . research complications m. pulmonis can interfere with research by causing clinical disease or death. experiments involving the respiratory tract, such as inhalation toxicology, can be compromised by chronic progressive infection. additionally, affected mice are at greater risk during general anesthesia. m. pulmonis may alter immunological responsiveness. for example, it is mitogenic for t and b lymphocytes and can increase nk cell activity. perhaps one of the most important complications of mycoplasma infection is contamination of cell lines and transplantable tumors. other murine mycoplasmas cell lines are often contaminated with mycoplasma species such as m. arginini, m. hyorhinis, m. orale, or m. fermentans that can distort the results of in vitro assays (garner et al., 2000) . initial evidence of a contamination is often by pcr evidence of mycoplasma at the genus level when cell lines are pcr screened for opportunistic murine pathogens prior to use in mice. other than m. pulmonis, these mycoplasmas are not normally considered mouse pathogens in immunocompetent mice. in contrast, injection of mycoplasma contaminated cells into immunodeficient mice (e.g., xenografts) may result in clinical disease or confounding effects on immune responses (peterson, 2008) . mycoplasma contamination of murine embryonic stem cells has adversely affected germline transmission and postnatal health of chimeric progeny (markoullis et al., 2009) . mycoplasma arthritidis is antigenically related to m. pulmonis. therefore, serological evidence of mycoplasma infection must be supplemented by other diagnostic tests, as outlined above, to differentiate between these agents. differentiation is important because m. arthritidis, though arthritogenic in mice after intravenous inoculation, is nonpathogenic during natural infection. mycoplasma collis has been isolated from the genital tract of mice but does not appear to cause natural disease. mycoplasma neurolyticum is the etiological agent of rolling disease, a rare syndrome which occurs within hours after intravenous inoculation of m. neurolyticum exotoxin. characteristic clinical signs include spasmodic hyperextension of the head and the raising of one foreleg followed by intermittent rolling on the long axis of the body. the rolling becomes more constant, but mice occasionally leap or move rapidly. after 1-2 h of rolling, animals become comatose and usually die within 4 h. all published reports of rolling disease are associated with experimental inoculation of m. neurolyticum or exotoxin. large numbers of organisms are needed to produce disease, and there is no indication that, under natural conditions, organisms replicate in the brain to concentrations required for the induction of these signs. because animals are frequently inoculated with biological materials by parenteral routes, contamination with m. neurolyticum may induce rolling disease inadvertently. diagnosis can be made from the appearance of typical clinical signs, astrocytic swelling, and isolation of the causative organism. clinical signs must be differentiated from rolling associated with pseudomonas-and p. pneumotropica-caused otitis. m. pulmonis has been recovered from the brain of mice but does not seem to cause overt neurological disease. hemotropic mycoplasmas ribosomal rna sequencing has reclassified hemobartonella muris and eperythrozoon coccoides as mycoplasma hemomuris and mycoplasma coccoides, respectively (neimark et al., 2005; percy and barthold, 2007) . distinct from the mycoplasmas just discussed, these agents are trophic for red blood cells and cause anemia and hemolytic disease. these laboratory animal medicine infections could be encountered in wild mice but are rarely found in research mice. diagnosis is by morphologic assessment of blood smears and pcr. clinical signs mice infected with m. coccoides may remain clinically normal or develop febrile, hemolytic anemia and splenomegaly, which can be fatal. hepatocellular degeneration and multifocal necrosis have been recorded in acute infections. hemotropic mycoplasma infections are long-lived and are expressed clinically in one of two ways: acute febrile anemia and latent or subclinical infection that can be reactivated by splenectomy. the carrier state may be lifelong. epizootiology the primary natural vector of m. coccoides, historically, is the mouse louse, polyplax serrata. infection was associated with primitive housing and husbandry conditions that no longer occur in modern vivaria. although the risks for infection have been reduced substantially by modern animal care procedures, m. coccoides can be transmitted to mice from contaminated biological products such as transplantable tumors or blood plasma. diagnosis splenectomy or inoculation of test material into splenectomized mice is the most sensitive means of detecting m. coccoides infection. these procedures provoke mycoplasmemia, usually within 2-4 days. because mycoplasmemia may be transient, blood smears stained by the romanowsky or indirect immunofluorescence procedures of the blood should be prepared every 6 h, beginning at 48 h after splenectomy of index animals or inoculation of test specimens into splenectomized animals to ensure that mycoplasmemia is not missed. prevention and control treatment of m. coccoides infection is not practical. control is based on culling or rederivation of infected stock. if replacement animals are readily available, euthanasia is a more prudent course. suspect biological materials destined for animal inoculation should be screened for mycoplasma contamination by inoculation of splenectomized mice. research complications subclinical infection can be reactivated by irradiation, immunosuppressive therapy, or intercurrent disease. conversely, m. coccoides may potentiate coincident viral infections in mice. this effect has been clearly demonstrated for mouse coronavirus and has been suspected for lymphocytic choriomeningitis virus and ldv. active infection also may suppress interferon production. etiology car bacillus is a slender, gram-negative, non-spore-forming bacillus, which, in rats, produces clinical disease and lesions that closely resemble those of mrm (see chapter 4). clinical signs chronic respiratory disease has been produced in mice by experimental inoculation, but natural clinical disease is rare (griffith et al., 1988; pritchett-corning et al., 2009) . furthermore, putative natural cases were reported in mice that were seropositive for sv and pneumonia virus of mice. therefore, car bacillus may exacerbate respiratory disease as an opportunist rather than as a primary pathogen. on balance, it is assumed that mice contract natural infection, but attributing severe chronic respiratory disease in mice solely to car bacillus should be supported by screening for other respiratory pathogens. epizootiology car bacillus is transmitted by direct contact; dirty bedding transfer to sentinel mice may not reflect colony infection status. pathology lung lesions are typically mild in mice and are similar to respiratory mycoplasmosis. uncomplicated car bacillus infection results in peribronchiole cuffing with lymphocytes and plasma cells. severe bronchiolitis and pneumonia are possible (fig. 3.36) . fatal bronchopneumonia was reported in ob/ob mice (griffith et al., 1988) . diagnosis an elisa for serological screening is routinely used; pcr and histology are used for definitive diagnosis. in active infection, histologic assessment using warthin-starry or similar stains will reveal argyrophilic bacilli adherent to the apical membranes of bronchial respiratory epithelium along with the presence of peribronchial lymphocytes (fig. 3.37) . alternatively, immunohistochemistry assays have also been used successfully to detect infection. recovery of car bacillus requires cell culture or culture in embryonated eggs. differential diagnosis respiratory mycoplasmosis, bordetella (avium, hinzii). prevention and control given car bacillus does not form spores, disinfection of the environment should be effective. treatment using sulfamerazine (500 mg/l) in drinking water may eradicate infection (matsushita and suzuki, 1995) but culling or embryo rederivation is recommended. research complications infection is most often subclinical, but like other infectious agents for mice, may confound studies particularly when mice are immunocompromised (griffith et al., 1988) . etiology the causative agent of transmissible murine colonic hyperplasia, c. rodentium (formerly citrobacter freundii strain 4280), is a nonmotile, gramnegative rod that ferments lactose but does not utilize citrate or does so marginally (barthold, 1980; schauer et al., 1995) . clinical signs c. rodentium infection can be a selflimiting colitis with sterilizing immunity or lead to severe colitis with life-threatening dehydration. clinically apparent infection is characterized by retarded growth, ruffled fur, soft feces or diarrhea, rectal prolapse, and moderate mortality in older suckling or recently weaned mice (barthold et al., 1978) . epizootiology c. rodentium is not detected in the gastrointestinal flora of normal mice, and therefore, there is not a carrier state. it is thought to be introduced by contaminated mice, food, or bedding, from which it spreads by contact or additional fecal contamination. c. rodentium shares several pathogenic mechanisms, such as attaching and effacing lesions mediated by the intimin receptor, with select escherichia coli (reviewed in collins et al. (2014) ). c. rodentium is used experimentally to model colitis caused by enteropathogenic (epec) and enterohemorrhagic e. coli (ehec) in humans (mallick et al., 2012; collins et al., 2014) . host genotype can influence the course and severity of disease (barthold et al., 1977) . for example, dba, nih swiss, and c57bl mice are relatively resistant to mortality, whereas c3h/hej mice are relatively susceptible both as sucklings and as adults. interestingly, c57bl mice obtained from different commercial sources have varying susceptibility to c. rodentium (ostensibly due to the presence or absence of segmented filamentous bacteria). diet also can modulate infection, but specific dietary factors responsible for this effect have not been identified. pathology c. rodentium attaches to the mucosa of the descending colon and displaces the normal flora. attachment is accompanied by effacement of the microvillus border and formation of pedestal-like structures (attaching and effacing lesions) (schauer and falkow, 1993; newman et al., 1999) . colonization results in prominent mucosal hyperplasia, by unknown mechanisms. the characteristic gross finding is severe thickening of the descending colon, which may extend to the transverse colon and lasts for 2-3 weeks in surviving animals ( fig. 3.38) . affected colon segments are rigid and either are empty or contain semiformed feces. histologically, accelerated mitotic activity results in a markedly hyperplastic mucosa, which may be associated with secondary inflammation and ulceration (fig. 3.39 ). lesions subside after several weeks. intestinal repair is rapid and complete in adults but slower in sucklings. diagnosis diagnosis depends on clinical signs, characteristic gross and histological lesions, and isolation of c. rodentium from the gastrointestinal tract or feces. the organism can be cultured on macconkey's agar during early phases of infection, whereas the intestine may be free of c. rodentium during later stages of the disease. c. rodentium also can be detected by molecular hybridization (schauer et al., 1995) . barthold et al. (1978) . diagnosis transmissible murine colonic hyperplasia must be differentiated from other diarrheal diseases of mice, including infections caused by coronavirus, rotavirus, adenovirus, reovirus, salmonella, c. piliforme, and helicobacter spp. prevention and control some success in curtailing epizootics has been achieved by adding antimicrobials to the drinking water (barthold, 1980; silverman et al., 1979) . because c. rodentium may contaminate food, bedding, or water, proper disinfection of such materials is prudent before they are used for susceptible animals. additionally, the employment of microbarrier caging can reduce transmission. surveillance for c. rodentium should be incorporated into quality-assurance programs, and the organism screened for during quarantine of incoming mice from atypical sources. research complications the potential effects on research of colonic hyperplasia as a clinically severe disease are obvious. colonic hyperplasia has been shown to increase the sensitivity of colonic mucosa to chemical carcinogens and to decrease the latent period between administration of carcinogen and the appearance of focal atypical cell growth (barthold and beck, 1980) . c. rodentium infection has been incriminated in immune dysfunction, poor reproductive performance, and failure to thrive in t-cell receptor transgenic mice (maggio-price et al., 1998) . immunocompromised mice infected with c. rodentium will die from sepsis. etiology pseudomonas aeruginosa is a motile, gramnegative rod. clinical signs p. aeruginosa infections are almost always silent, but immunologically compromised animals are prone to septicemia (brownstein, 1978) . p. aeruginosa can, e.g., cause severe or lethal infections in athymic and scid mice. sick mice may have equilibrium disturbances, conjunctivitis, serosanguinous nasal discharge, edema of the head, weight loss, and skin infections. immunosuppressed mice may also develop gastrointestinal ulcers. generalized infection is associated with severe leukopenia, especially neutropenia. neurologic signs are rare, but there are reports of central nervous system infection. chronic proliferative inflammation in the cochlea and vestibular apparatus with dissolution of surrounding bone may cause torticollis. epizootiology p. aeruginosa is not considered a component of the normal flora. however, it is an opportunist that inhabits moist, warm environments such as water and skin. once established in a host, it may be found chronically in the nasopharynx, oropharynx, and gastrointestinal tract, all sites from which additional environmental contamination or direct transmission to susceptible mice can occur. pathology pathogenic infection is most common in immunodeficient mice. organisms enter at the squamocolumnar junction of the upper respiratory tract and, in some cases, the periodontal gingiva. bacteremia is followed by necrosis or abscess formation in the liver, spleen, or other tissues. if otitis media occurs, the tympanic bullae may contain green suppurative exudate. the bowel may be distended with fluid, and gastrointestinal ulceration has been reported. diagnosis infection is diagnosed on the basis of history (e.g., immune dysfunction or recent immunosuppression), clinical signs, lesions, and isolation of p. aeruginosa from affected mice. carrier mice can be detected either by nasal culture or by placing bottles of sterile, nonacidified, nonchlorinated water on cages for 24-48 h and then culturing the sipper tubes. p. aeruginosa can also be cultured from feces. differential diagnosis pseudomoniasis must be differentiated from other bacterial septicemias that may occur in immunodeficient mice. these include, but are not limited to, corynebacteriosis, salmonellosis, colibacillosis, staphylococcosis, and tyzzer's disease. prevention and control infection can be prevented by acidification or hyperchlorination of the drinking water (homberger et al., 1993) . these procedures will not, however, eliminate established infections. entry of infected animals can be prevented by surveillance of commercially procured colonies. maintenance of pseudomonas-free animals usually requires barrierquality housing and husbandry. p. aeruginosa has a long history in the literature of antibiotic resistance and resistance to quaternary amine disinfectants. research complications p. aeruginosa infection is not a substantial threat to immunocompetent mice but can complicate experimental studies by causing fatal septicemia in immunodeficient mice. viral infections that alter host defense mechanisms, such as mcmv may enhance susceptibility to pseudomoniasis. (lindsey et al., 1991a; percy and barthold, 2007) etiology pasteurella pneumotropica is a short, gramnegative rod. clinical signs many early observations concerning the pathogenicity of p. pneumotropica are questionable because they were made on colonies of mice with varying levels of bacterial and viral contamination. infection is usually subclinical. therefore, p. pneumotropica is most properly viewed as an opportunistic pathogen. studies of experimental p. pneumotropica suggest that it may complicate pneumonias due to mycoplasma pulmonis or sv. it has also been associated with suppurative or exudative lesions of the eye, conjunctiva, skin, mammary glands, and other tissues, especially in immunodeficient mice or in mice with a predisposing primary infection. epizootiology p. pneumotropica is a ubiquitous inhabitant of the skin, upper respiratory tract, and gastrointestinal tract of mice. litters from infected dams can become infected during the first week after birth. pathology infections can cause suppurative inflammation, which may include abcessation. dermatitis, conjunctivitis, dacryoadenitis, panophthalmitis, mastitis, and infections of the bulbourethral glands have been attributed to p. pneumotropica. preputial and orbital abscesses also occur, especially in athymic mice (fig. 3.40 ). its role in metritis is unclear, but it has been cultured from the uterus, and there is some evidence that it may cause abortion or infertility. cutaneous lesions can occur without systemic disease. they include suppurative lesions of the skin and subcutaneous tissues of the shoulders and trunk. diagnosis diagnosis requires isolation of the organism on standard bacteriological media. although infection can be detected serologically by elisa (wullenweber-schmidt et al., 1988; boot et al., 1995a, b) , subclinical carriers often do not seroconvert. pcr assays also are available (dole et al., 2010) and have shown that p. pneumotropica did not transmit from infected mice to contact or dirty bedding sentinels (ouellet et al., 2011; dole et al., 2013) . differential diagnosis suppurative lesions in mice may be caused by other bacteria, including staphylococcus, streptococcus, corynebacterium, klebsiella, and mycoplasma. treatment antibiotic sensitivity testing in vitro indicated p. pneumotropica was significantly more sensitive than p. aeruginosa to enrofloxacin (sasaki et al., 2007) . enrofloxacin in the drinking water at 85 mg/kg daily for 7 days eliminated clinical signs and infection in a closed breeding colonic of transgenic mice and after 14 days of treatment there were no detectable carriers when the colony was screened 4 weeks later (matsumiya and lavoie, 2003) . prevention and control because p. pneumotropica is an opportunistic organism, it should be excluded from colonies containing immunodeficient mice and from breeding colonies. achieving this goal will normally require barrier housing supported by sound microbiological monitoring. rederivation should be considered to eliminate infection in circumstances where infection presents a potential threat to animal health or experimentation. research complications clinically severe infection in immunodeficient mice is the major complication. although clinically silent, experimental evidence has shown that p. pneumotropica infection in immunocompetent mice (c57bl/6) stimulated transcription of multiple proinflammatory cytokines for at least 7 days with residual elevation detectable 28 days later (patten et al., 2010) . pioneering studies conducted in the 1990s first linked a novel microaerobic bacterium, helicobacter hepaticus, with chronic active hepatitis and hepatic tumors in a/jcr mice (fox et al., , 2011 ward et al., 1994) . the organism could be visualized by electron microscopy in the bile canaliculi of the liver in susceptible mouse strains (fig. 3.41 ). subsequently, it was associated with inflammatory bowel disease in several murine models (table 3 .13) which were further developed to examine the role of immune cell subsets, such as t regulatory cells, in the pathogenesis of inflammatory bowel disease (ibd) and colon cancer (fig. 3.42 ). helicobacteriosis is laboratory animal medicine now appreciated to be a common infection of laboratory mice. it is caused by a growing list of helicobacter spp. that vary in clinical, pathologic, and epidemiologic significance (whary and fox, 2004; fox et al., 2011) . because recognition and investigation of helicobacteriosis continues to evolve, many important questions about the impact of this infection on mice remain unresolved. h. hepaticus infection is emphasized here, because it is among the most prevalent causes of helicobacteriosis and has been studied more extensively than other murine enterohepatic helicobacter spp. (ehs) (fox et al., , 2011 ward et al., 1994; suerbaum et al., 2003) . however, current information about other murine helicobacters is summarized in the concluding section. etiology helicobacter spp. are gram-negative, microaerophilic, curved to spiral-shaped organisms that have been isolated from the gastrointestinal mucosa of many mammals, including humans and mice whary and fox, 2004) . to date, the genus includes 20 formally named helicobacter spp. assigned on the basis of 16s rrna analysis, complemented by biochemical, molecular, and morphological characteristics. the organisms can be grown on freshly prepared antibiotic impregnated blood agar or in broth supplemented with fetal bovine serum in a microaerobic atmosphere (5% co 2 , 80% n 2 , 10% h 2 ). there are currently 11 formally named helicobacter species have been isolated from laboratory mice, as well as several other novel helicobacter spp. awaiting formal naming. species isolated from mice include h. hepaticus, h. bilis (which also infects rats), h. muridarum, h. rappini, and h. rodentium, h. ganmani, h. mastomyrinus, h. magdeburgensis, and h. typhlonius , each of which cahill et al. (1997) tcrα, β mutants defective t-receptors typhlocolitis chin et al. (2000) scid icr-defined flora b t-and b-cell deficient typhlocolitis shomer et al. (1998 ), shomer et al. (1997 c57bl/il-10 −/−c lacks il-10 typhlocolitis burich et al. (2001) , kullberg et al. (2001) , kullberg et al. (2006) , kullberg et al. (2002) c57blrag2 mice infected with h. bilis also developed ibd (shomer et al., 1997) . c ibd also developed in c57bl/il-10 −/− mice experimentally infected with a novel urease-negative helicobacter spp. now named h. typhlonius (franklin et al., 2001) ; also ibd produced with h. trogontum (whary et al., 2006) and h. cinaedi (shen et al., 2009) . d h. bilis produces ibd (maggio-price et al., 2002 and colon cancer (maggio-price et al., 2006) . have been formally named (except for h. rappini) (fox and lee, 1997; franklin et al., 1999; whary and fox, 2004) . most recently, helicobacter pullorum, a human pathogen, has been isolated from commercial, barriermaintained mice (boutin et al., 2010) . these ehs are most commonly urease-, catalase-, and oxidase-positive. however, h. rodentium, h. typhlonicus, and another novel helicobacter sp. are urease-negative. clinical signs helicobacteriosis in adult immunocompetent mice is usually asymptomatic. liver enzymes are elevated in h. hepaticus-infected a/jcr mice (fox et al., 1996a) . infection of immune-dysregulated mice with h. hepaticus can cause inflammatory bowel disease, which may present as rectal prolapse and/or diarrhea (miller et al., 2014) . epizootiology recent surveys and anecdotal evidence suggest that helicobacteriosis is widespread among conventional and barrier-maintained mouse colonies (shames et al., 1995; fox et al., 1998b; taylor et al., 2007; lofgren et al., 2011) . furthermore, h. hepaticus (and probably other helicobacters) can persist in the gastrointestinal tract, particularly the cecum and colon, and is readily detected in feces. these results indicate that transmission occurs primarily by the fecal-oral route and imply that carrier mice can spread infection chronically in enzootically infected colonies. pathology helicobacter spp. colonize the crypts of the lower bowel, where, depending on host genotype, the organisms can be pathogenic or nonpathogenic. h. hepaticus and h. bilis, e.g., can cause inflammation in the gastrointestinal tract, which is expressed as ibd and colon cancer in immunodeficient mice or typhlitis in a/jcr mice infected with h. hepaticus (ward et al., 1996; knutson et al., 2013; shomer et al., 1997; erdman et al., 2003b; nguyen et al., 2013) . thickening of the cecum and large bowel develops because of proliferative typhlitis, colitis, proctitis, and lower bowel carcinoma. these lesions can occur without coincident hepatitis. indeed, helicobacter spp. induced ibd and colon carcinoma are increasingly popular models to study pathogenesis of the disease in humans (table 3 .13). helicobacter spp. also can cause liver disease. bacterial translocation is thought to occur and results in colonization of the liver and progressive hepatitis. it is characterized by angiocentric nonsuppurative hepatitis and hepatic necrosis (fig. 3.43) . inflammation originates in portal triads and spreads to adjacent hepatic parenchyma. hepatic necrosis also may occur adjacent to intralobular venules, which can contain microthrombi. additionally, phlebitis may affect central veins. this lesion has been linked to the presence of organisms in bile canaliculi by silver stains and electron microscopy. age-related hepatocytic proliferation can develop in infected livers, a response that is more pronounced in male mice than in female mice (fox et al., 1996a) . this lesion may laboratory animal medicine increase susceptibility to hepatomas and hepatocellular carcinomas among aged male a/jcr and b6c3f1 mice from infected colonies. an increased incidence of hepatic hemangiosarcoma also has been noted in h. hepaticusinfected male b6c3f1 mice. in this context, a/jcr, c3h/ hencr, and sjl/ncr mice are susceptible to hepatitis, whereas c57bl/6 mice are resistant (ward et al., 1994) . the finding of severe liver disease and tumor induction in b6c3f1 mice infected with h. hepaticus infers that genetic susceptibility to h. hepaticus-induced neoplasia has a dominant pattern of inheritance. studies with h. hepaticus in recombinant inbred mice also indicate that disease susceptibility has multigenetic properties (hailey et al., 1998; fox and lee, 1997; ihrig et al., 1999; franklin, 2006; hillhouse et al., 2011) . diagnosis rapid generic diagnosis can be accomplished by pcr detection of the highly conserved 16s rrna region of the helicobacter genome in feces or tissues, using suitable oligonucleotide primers (fox et al., 1998a; shames et al., 1995; beckwith et al., 1997) . however, genus-specific pcr does not differentiate among different helicobacter spp. molecular speciation can be accomplished by 16s rrna sequencing, restriction fragment length polymorphism analysis of the pcr product or use of species-specific pcr assays. this procedure requires suitable skill and experience to avoid technological pitfalls and should be performed by qualified laboratories. an igg elisa using the outer membrane protein as the antigen has been proposed for serological diagnosis, but shared antigens among ehs create lack of specificity for the assay. as noted above, helicobacters can be isolated on antibiotic-impregnated blood agar under microaerobic conditions and can then be speciated biochemically, and by helicobacter species-specific pcr. isolation of h. hepaticus and from other helicobacter spp. with spiral to curved morphology from feces should be preceded by passing slurried samples through a 0.45-μm filter before plating. if infection with larger fusiform helicobacters (h. bilis, h. rappini) is suspected, filtration at 0.65 μm is preferred. helicobacters grow slowly and require prolonged incubation of cultures (up to 3 weeks) before they can be deemed negative. signs (rectal prolapse) and lesions (hepatitis, typhlocolitis), depending on host genotype, can be suggestive of infection. histopathological examination should include silver stains, especially of liver, to attempt to visualize spiral or curved organisms (whary and fox, 2004) . differential diagnosis clinically apparent helicobacteriosis must be differentiated from other gastrointestinal or hepatic infections of mice. coronavirus infection, clostridium piliforme, and salmonella spp. can cause enterocolitis and/or hepatitis. c. rodentium also causes colonic hyperplasia, which can present as rectal prolapse. infections caused by other helicobacters of mice h. bilis has been isolated from the livers and intestines of aged mice and experimentally induces ibd in scid mice as does h. hepaticus. h. bilis also experimentally produces lower bowel cancer in immunocompromised mice (nguyen et al., 2013) . helicobacter muridarum colonizes the ileum, cecum, and colon. it appears to be nonpathogenic, although it can colonize the stomach of mice and induce gastritis under certain circumstances. h. 'rappini' has been isolated from the feces of mice without clinical signs. h. rodentium also colonizes the intestine and may be a component of normal flora. a dual infection of h. bilis and h. rodentium was noted in a natural outbreak of ibd in immunocompromised mice (shomer et al., 1998) . a novel urease negative helicobacter, which has been named h. typhlonius, causes ibd in il-10 −/− and scid mice (franklin et al., 1999 (franklin et al., , 2001 fox et al., 1999) . decreased reproductive efficiency has been reported in il10 knockout mice infected with h. rodentium and/or h. typhlonius (sharp et al., 2008) . prevention and control eradication of infection from small numbers of mice, such as quarantine groups, can be achieved by standard rederivation or intensive antibiotic therapy. the best results have been obtained by triple therapy with amoxicillin, metronidazole, and bismuth given for 2 weeks (del carmen martino-cardona et al., 2010) . this strategy requires repeated daily gavage rather than administration in drinking water, but it has successfully eliminated h. hepaticus from naturally infected mice. antibiotic impregnated wafers have been used to eradicate helicobacter spp. in mouse colonies (kerton and warden, 2006) . wide-scale, eradication of enzootic helicobacteriosis can be expensive and time-consuming, without guarantee of success. careful husbandry procedures can limit infection within a colony (whary et al., 2000) . therefore, strategies have to be weighed carefully against risks of enzootic infection for the health and use of mice. in contrast, infection should be avoided in immunodeficient mice, including genetically engineered mice with targeted or serendipitous immune dysfunction. lastly, the outcome of opportunistic helicobacteriosis has not been thoroughly examined. this condition could occur during simultaneous infection with two or more helicobacter species or during combined infection with an intestinal virus (e.g., coronavirus) and helicobacter spp. if highly valuable animals are exposed, antibiotic therapy or rederivation may be warranted. research complications chronic inflammation of the liver and or gastrointestinal tract may be injurious to health. additionally, it may impede the development and assessment of noninfectious disease models, such as ibd models in mice with targeted deletions in t-lymphocyte receptors (fox et al., 2011) . h. hepaticus infections provoke a strong th1 proinflammatory response, which may perturb other immunological responses. h. hepaticus infection also has been incriminated as a cofactor or promoter in the development of hepatic neoplasia in a/ jcr, b6c3f1, ab6f1, b6af1, and carko mice (hailey et al., 1998; fox et al., 1998a; garcia et al., 2008 garcia et al., , 2011 h. salmonellosis (ganaway, 1982; lindsey et al., etiology the genus salmonella contains two species, s. bongori which infects mainly poikilotherms and rarely, humans, and s. enterica which includes approximately 2500 serovars and are a major cause of food-borne illness in humans (fookes et al., 2011) . the salmonella of historical importance in mice that are now rare include s. enterica subsp. enterica serovar typhimurium (aka s. typhimurium) and serovar enteritidis (s. enteritidis). s. enteritidis is a motile, gram-negative rod that rarely ferments lactose. the genomes of many strains have been sequenced. virulence factors carried on pathogenicity islands and plasmids include antimicrobial resistance genes, type iii secretion systems, vi antigen, lipopolysaccharide and other surface polysaccharides, flagella, and factors essential for a intracellular life cycle in macrophages (de jong et al., 2012) . pathogenassociated molecular patterns (pamps) unique to salmonella interact with tlrs and nod-like receptors (nlrs) which recruit neutrophils and macrophages leading to inflammasome formation and release of pro-inflammatory il-6, il-1β, tnf-α, and ifn-γ. clinical signs acute infection is especially severe in young mice (casebolt and schoeb, 1988) . it is characterized by anorexia, weight loss, lethargy, dull coat, humped posture, and occasionally conjunctivitis. gastroenteritis is a common sign, but feces may remain formed. subacute infection can produce distended abdomens from hepatomegaly and splenomegaly. chronic disease is expressed as anorexia and weight loss. enzootic salmonellosis in a breeding colony can produce episodic disease with alternating periods of quiescence and high mortality. the latter can be associated with diarrhea, anorexia, weight loss, roughened hair coat, and reduced production. epizootiology s. typhimurium is commonly used experimentally and cross-contamination in a mouse facility is a risk. modern production and husbandry methods have reduced the importance of salmonellosis as a natural infection of mice. however, the organisms are widespread in nature. therefore, cross-infection from other species or from feral mice remains a potential hazard. salmonellas are primarily intestinal microorganisms that can contaminate food and water supplies. infection occurs primarily by ingestion. salmonella have a broad host range and vermin, birds, feral rodents, and human carriers are potential sources of infection. other common laboratory species such as nonhuman primates, dogs, and cats also can serve as carriers. conversely, murine salmonellosis presents a zoonotic hazard to humans. the induction and course of infection are influenced by the virulence and dose of the organism, route of infection, host sex and genetic factors, nutrition, and intercurrent disease. suckling and weanling mice are more susceptible to disease than mature mice. immune deficiency, exposure to heavy metals, and environmental factors such as abnormal ambient temperatures can increase the severity of disease. nutritional iron deficiency has an attenuating effect on salmonella infection in mice, whereas iron overload appears to promote bacterial growth and enhance virulence. resistance to natural infection is increased by the presence of normal gastrointestinal microflora. resistance to infection also can be an inherited trait among inbred strains. among the most important considerations is that mice that recover from acute infection can become subclinical carriers and a chronic source of contamination from fecal shedding. pathology the virulence of s. enteritidis depends on its ability to penetrate intestinal walls, enter lymphatic tissue, multiply, and disseminate. organisms reach peyer's patches within 12 h after inoculation and spread quickly to the mesenteric lymph nodes. bacteremia results in spread to other lymph nodes, spleen, and liver within several days. in chronic infections, organisms persist in the spleen and lymph nodes as well as in the liver and gallbladder and from the latter are discharged into the intestinal contents. bacteria reaching the intestine can reinvade the mucosa and can be shed intermittently in the feces for months. s. enteritidis infection also has been associated with chronic arthritis. acute deaths may occur without gross lesions, but visceral hyperemia, pale livers, and catarrhal enteritis are more common. if mice survive for up to several laboratory animal medicine weeks, the intestine may be distended and reddened, whereas the liver and spleen are enlarged and contain yellow-gray foci of necrosis. affected lymph nodes are also enlarged, red, and focally necrotic. focal inflammation can develop in many organs, including the myocardium (percy and barthold, 2007) . histologic lesions reflect the course of disease and the number of bacteria in affected tissues. during acute infection, necrotic foci are found in the intestine, mesenteric lymph nodes, liver, and spleen. neutrophilic leukocytes and histiocytes accumulate in lymphoid tissues. thrombosis from septic venous embolism may occur, especially in the liver. granulomatous lesions are particularly characteristic of chronic salmonellosis, especially in the liver. diagnosis diagnosis is based on isolation of salmonellas together with documentation of compatible clinical signs and lesions. in mice with systemic disease, bacteria may persist in the liver and spleen for weeks. during acute stages, bacteria can also be isolated from the blood. subclinically infected animals can be detected by fecal culture using selective enrichment media (selenite f broth plus cystine followed by streaking on brilliant green agar). culture of the mesenteric lymph nodes may be more reliable, because fecal shedding can be intermittent. isolates can be speciated with commercial serotyping reagents. alternatively, isolates can be sent to a reference laboratory for confirmation. antibodies to salmonellas can be detected in the serum of infected mice by an agglutination test. however, this method is not entirely reliable, because serological crossreactivity is common even among bacteria of different genera. pcr-based assays are also available. differential diagnosis salmonellosis must be differentiated from other bacterial diseases, including tyzzer's disease, helicobacter spp., pseudomoniasis, corynebacteriosis, c. rodentium, and pasteurellosis. viral infections that cause enteritis or hepatitis must also be considered, especially infections caused by coronavirus, ectromelia virus, and reoviruses. among noninfectious conditions, mesenteric lymphadenopathy is an agingassociated lesion in mice and is not indicative of chronic salmonellosis. prevention and control salmonellosis can be prevented by proper husbandry and sanitation. contact between mice and potential carriers, such as nonhuman primates, dogs, and cats, should be prevented. diets should be cultured periodically to check for inadvertent contamination. contaminated colonies should be replaced to eliminate infection and its zoonotic potential. research complications apart from the clinical manifestations, the zoonotic potential for salmonellosis is a major concern. this includes transmission among laboratory species, but especially between mice and the personnel working with them. i. streptobacillosis (lindsey et al., 1991e; percy and barthold, 2007) etiology streptobacillus moniliformis is a nonmotile, gram-negative, pleomorphic rod that can exist as a nonpathogenic l-phase variant in vivo. however, it can revert to the virulent bacillus form. clinical signs streptobacillosis generally has an acute phase with high mortality, followed by a subacute phase and finally a chronic phase that may persist for months. signs of acute disease include a dull, damp hair coat and keratoconjunctivitis. variable signs include anemia, diarrhea, hemoglobinuria, cyanosis, and emaciation. cutaneous ulceration, arthritis, and gangrenous amputation may occur during chronic infection. the arthritis can leave joints deformed and ankylosed. hindlimb paralysis with urinary bladder distention, incontinence, kyphosis, and priapism may occur if vertebral lesions impinge on motor nerves. breeding mice may have stillbirths or abortions. epizootiology streptobacillosis has historical importance as a disease of rats and mice, but modern husbandry, production, and health surveillance strategies have reduced its impact dramatically (wullenweber, 1995) . subclinical, persistently infected rats are the most likely source of dissemination to mice, but mouse-tomouse transmission then ensues. transmission may occur from aerogenic exposure, bite wounds, or contaminated equipment, feed, or bedding. s. moniliformis is also pathogenic for humans, causing rat bite fever (haverhill fever). pathology during acute disease, necrotic lesions develop in thoracic and abdominal viscera, especially in the liver, spleen, and lymph nodes. histological lesions include necrosis, septic thrombosis of small vessels, acute inflammation, fibrin deposition, and abscesses. chronically infected mice may develop purulent polyarthritis because of the organism's affinity for joints. diagnosis diagnosis depends on clinical and pathological evidence of septicemia and isolation of the organism on blood agar. the organism has been recovered from joint fluid as long as 26 months after infection. isolation from chronic lesions requires serumenriched medium. s. moniliformis as a cause of septic joints in humans has been diagnosed using pcr and electrospray-ionization followed by mass spectrometry (mackey et al., 2014) . differential diagnosis clinical signs must be differentiated from septicemic conditions, including mousepox, tyzzer's disease, corynebacteriosis, salmonellosis, mycoplasmosis, pseudomoniasis, and traumatic lesions. prevention and control control is based on exclusion of wild rodents or carrier animals such as latently infected laboratory rats. bacterins and antibiotic therapy are not adequately effective. the potential laboratory animal medicine for cross-infection is a reason not to house rats and mice in the same room. research complications infection can be disabling or lethal in mice and has zoonotic potential for humans. j. corynebacteriosis (lindsey et al., 1982; weisbroth, 1994; percy and barthold, 2007) etiology corynebacteria are short gram-positive rods. corynebacterium kutscheri is the cause of pseudotuberculosis in mice and rats. corynebacterium bovis has been associated with hyperkeratosis, especially in immunodeficient mice (clifford et al., 1995; scanziani et al., 1998; dole et al., 2013) . clinical signs c. kutscheri infection is often subclinical in otherwise healthy mice. active disease is precipitated by immunosuppression or environmental stresses and is expressed as an acute illness with high mortality or a chronic syndrome with low mortality. clinical signs include inappetence, emaciation, rough hair coat, hunched posture, hyperpnea, nasal and ocular discharge, cutaneous ulceration, and arthritis. c. bovis infection causes hyperkeratotic dermatitis characterized by scaly skin, which is accompanied by alopecia in haired mice. severe infection may cause death. corynebacterial keratoconjunctivitis has been reported in aged c57bl/6 mice (mcwilliams et al., 1993) . epizootiology subclinically infected animals harbor c. kutscheri in the upper alimentary tract, colon, respiratory tract, regional lymph nodes, middle ear, and preputial gland. c. bovis colonizes skin and is shed in feces. therefore, transmission is by direct contact, fecaloral contact, and aerosol. resistance to infection appears to be under genetic control in some mouse strains. rats are susceptible to c. kutscheri, so cross-infection to mice may occur. pathology lesions caused by c. kutscheri develop from hematogenous spread to various internal organs and appear as gray-white nodules in the kidney, liver, lung, and other sites. cervical lymphadenopathy and arthritis of the carpometacarpal and tarsometatarsal joints also may occur. septic, necrotic lesions often contain caseous material or liquefied exudate. histologic lesions are characterized by coagulative or caseous necrosis bordered by intense neutrophilic infiltration. colonies of gram-positive organisms with 'chinese letter' configurations can usually be demonstrated using tissue gram stains of caseous lesions. mucopurulent arthritis of carpal, metacarpal, tarsal, and metatarsal joints are related to bacterial colonization of synovium accompanied by necrosis, cartilage erosion, ulceration, and eventually ankylosing pan arthritis. c. kutscheri is not a primary skin pathogen, but skin ulcers or fistulas follow bacterial embolization and infarction of dermal vessels. subcutaneous abscesses have also been reported. hyperkeratotic dermatitis caused by c. bovis is characterized grossly by skin scaliness and alopecia. microscopically, skin lesions consist of prominent acanthosis and moderate hyperkeratosis accompanied by mild nonsuppurative inflammation (fig. 3.44) . hyperkeratosis is typically more severe in glabrous athymic mice than in haired mice. organisms can be demonstrated in hyperkeratotic layers by gram stain. diagnosis c. kutscheri is usually diagnosed by culture and tissue gram stains on lesions from clinically apparent cases. agglutination serology is available, and immunofluorescence, immunodiffusion, and elisa tests have been reported (boot et al., 1995a) . pcr of skin swabs or feces is a sensitive and specific method for the detection of c. bovis infection in mice (dole et al., 2013) . differential diagnosis the caseous nature of c. kutscheri-induced lesions helps separate them from necrotic changes or abscesses caused by other infectious agents of mice. thus, they can be differentiated from streptococcosis, mycoplasmosis, and other septicemic bacterial infections in which caseous necrosis does not occur. because mice can sustain natural infections with mycobacterium avium, histochemical techniques for acidfast bacilli and appropriate culture methods for mycobacteria should be considered if nodular inflammatory lesions of the lung are detected. diffuse scaling dermatitis in athymic nude mice is classic for c. bovis infection; however, in one case report staphylococcus xylosus was instead isolated in high numbers from the skin lesions (russo et al., 2013) . hyperkeratotic dermatitis caused by laboratory animal medicine c. bovis must be differentiated from scaly skin caused by low humidity in glabrous mice. prevention and control c. kutscheri infection occurs sporadically and infected colonies should be culled or rederived into an spf facility as treatment is not curative and control is difficult. c. bovis can be endemic in athymic nude mouse colonies. prevention and control are difficult because both immunocompetent and athymic mice as well as humans can carry c. bovis on the skin and in the upper respiratory system, respectively. c. bovis readily contaminates the environment as aerosolization within a class ii biosafety cabinet was shown to spread the bacterium during cage-change procedures (burr et al., 2012) . antibiotic treatment has been unrewarding (burr et al., 2011) research complications corynebacteriosis can cause morbidity and mortality, especially among immunodeficient mice. dermatologic disease in suckling mice can be fatal but is less severe and transient in weanling mice. etiology staphylococci are gram-positive organisms that commonly infect skin and mucous membranes of mice and other animals. the two most frequently encountered species are staphylococcus aureus, which can be highly pathogenic, and s. epidermidis, which is generally nonpathogenic. species subtypes are identified by phage typing and biochemistry profiles. pathogenic staphylococci are typically coagulase-positive, although s. xylosus has caused serious infections and is coagulasenegative (gozalo et al., 2010) . clinical signs staphylococcosis causes suppurative conjunctivitis, periorbital and retroorbital abscesses, preputial adenitis, and pyoderma in mice, particularly in immunocompromised strains such as nude mice. some evidence suggests that staphylococci can produce primary cutaneous infections, but they are more likely opportunistic organisms that induce lesions after contamination of skin wounds. eczematous dermatitis develops primarily on the face, ears, neck, shoulders, and forelegs and can progress to ulcerative dermatitis, abscessation (including botryomycotic granulomas), and cellulitis. because lesions are often pruritic, scratching causes additional trauma and autoinoculation. staphylococcal infection in the genital mucosa of males may produce preputial gland abscesses. these occur as firm, raised nodules in the inguinal region or at the base of the penis and may rupture to spread infection to surrounding tissues. male mice also may develop septic balanoposthitis secondary to penile self-mutilation. retrobulbar abscesses caused by s. aureus are frequently noted in athymic mice. sjl mice, which are nk cell deficient, are prone to necrotic dermatitis on the tail secondary to s. xylosus infection. epizootiology staphylococci are ubiquitous and can be carried on the skin and in the nasopharnyx and gastrointestinal tract. they also can be cultured from cages, room surfaces, and personnel. the prevalence of staphylococcal dermatitis appears to be influenced by host genotype, the overall health of the animal, and the degree of environmental contamination with staphylococcus spp. c57bl/6, c3h, dba, and balb/c mice are among the most susceptible strains. age may also influence susceptibility, with young mice being more susceptible than adults. immunodeficient mice (e.g., athymic mice) contaminated with staphylococci often develop abscesses or furunculosis (fig. 3.45 ). as noted above, behavioral dysfunction resulting in selfmutilation, including scratching and trichotillomania, is a likely predisposing factor. once virulent staphylococci contaminate the environment, colonization of the gastrointestinal tract can occur and produce a carrier state. phage typing can help to determine the source of infection. human phage types of staphylococci can infect mice, but the zoonotic importance of this connection is not clear. pathology gross lesions are typified by suppurative, ulcerative and necrotic dermatitis involving the head and neck but may extend to the shoulders and forelegs (percy and barthold, 2007) . superficial or deep abscesses may occur in conjunction with dermatitis or separately, as, e.g., in the external male genitalia. histologically, acute skin infections result in ulceration with neutrophils in the dermis and subcutis. chronic lesions contain lymphocytes, macrophages, and fibroblasts. deep infections appear as coalescing botryomycotic pyogranulomas with necrotic centers containing bacterial colonies. infected athymic mice may develop laboratory animal medicine furunculosis of the muzzle and face accompanied by regional lymphadenitis. diagnosis diagnosis is made by documenting gross and histological lesions, including gram staining of suspect tissues, complemented by isolation of grampositive, coagulase-positive (s. aureus), or coagulasenegative staphylococcus species. differential diagnosis staphylococcosis must be differentiated from other suppurative infections of mice, including pasteurellosis, streptococcosis, corynebacteriosis, and pseudomoniasis. ectoparasitism, fight wounds, and self-mutilation per se should also be considered. prevention, control, and treatment removal of affected animals, sterilization of food and bedding, and frequent changing of bedding may limit or reduce transmission. in affected animals, nail trimming can reduce self-inflicted trauma. conditions that facilitate aggressive or self-mutilating behavior should be avoided. research complications staphylococcosis can cause illness and disfigurement in mice. immunodeficient mice are at increased risk. etiology streptococci are ubiquitous commensal gram-positive organisms and in some cases, primary pathogens. pathogenic streptococcal infections in laboratory mice are caused by β-hemolytic organisms in lancefield's group c, but epizootics caused by group a streptococci have occurred, and group g organisms have been isolated occasionally. group d has been reclassified as an enterococcus. alpha-hemolytic streptococci can cause systemic disease in scid mice, and group b streptococcus sp. infection has been reported to cause meningoencephalitis in athymic mice (schenkman et al., 1994) . additionally, streptococcus dysgalactiae subsp. equisimilis has lancefield group g or c antigens and was isolated from visceral abscesses of immunocompetent mice (greenstein et al., 1994) . clinical signs cutaneous infections can cause ulcerative dermatitis over the trunk, which may appear gangrenous, whereas systemic infections may be expressed as conjunctivitis, rough hair coat, hyperpnea, somnolescence, and emaciation. epizootiology mice can carry streptococci subclinically in their upper respiratory tracts. lethal epizootics can occur, but factors leading to clinical disease are unknown, although some infections may be secondary to wound contamination. pathology systemic lesions reflect hematogenous dissemination and include abscessation, endocarditis, splenomegaly, and lymphadenopathy (percy and barthold, 2007) . streptococcal cervical lymphadenitis can lead to fistulous drainage to the neck complicated by ulcerative dermatitis. infection with α-hemolytic streptococci can cause inflammatory lesions affecting kidney and heart. diagnosis diagnosis and differential diagnosis depend on isolation of organisms from infected tissues, combined with histopathologic confirmation. differential diagnosis streptococcosis must be differentiated from other suppurative infections of mice, including staphylococcosis, pasteurellosis, corynebacteriosis, and pseudomoniasis. prevention and control removal of affected animals, sterilization of food and bedding, and frequent changing of bedding may limit or reduce transmission. research complications immunodeficient mice are at increased risk for streptococcosis. etiology e. coli is a small gram-negative rod that is a normal inhabitant of the mouse intestine. epizootiology infection is considered nonpathogenic in immunocompetent mice. however, hyperplastic typhlocolitis resembling transmissible murine colonic hyperplasia has been reported in scid mice infected with a non-lactose-fermenting e. coli (waggie et al., 1988; arthur et al., 2012) . clinical signs affected mice develop lethargy and fecal staining. pathology gross lesions consist of segmental thickening of the colon or cecum, which may contain blood-tinged feces. microscopically, affected mucosa is hyperplastic and may be inflamed and eroded. diagnosis diagnosis depends on demonstrating lesions and isolating non-lactose-fermenting e. coli. differential diagnosis this condition must be differentiated from proliferative and inflammatory intestinal disease caused by lawsonia intracellularis, c. rodentium, or enterotropic mouse hepatitis virus, especially in immunodeficient mice. colibacillosis provides an example of the morbidity associated with a nominally innocuous organism when it affects an immunocompromised host. prevention and control removal of affected animals and disinfection of caging and equipment will limit or reduce transmission. research complications clinical illness may develop in immunodeficient mice. historically, klebsiella pneumoniae is a ubiquitous gram-negative organism that is a natural inhabitant of the mouse alimentary tract. most commercial vendors have excluded it from their barriers. it can be pathogenic for the respiratory and urinary tract of mice after experimental inoculation but is not a significant cause of naturally occurring disease. etiology klebsiella oxytoca is an opportunistic pathogen implicated in various clinical diseases in animals and humans. epizootiology k. oxytoca also is purported to be an etiological agent of antibiotic-associated hemorrhagic colitis (aahc) in adult humans and adolescents. in animals, k. oxytoca has been isolated from apparently healthy sentinel rodents being monitored for pathogens in health surveillance programs and from utero-ovarian infections including suppurative endometritis, salpingitis, perioophoritis, and peritonitis in aged b6c3f1 mice (davis et al., 1987; rao et al., 1987) . a model of aahc has been developed in rats by administering amoxicillinclavulanate followed by orally infecting rats with a strain of k. oxytoca cultured from a patient with aahc. studies in humans suggest that k. oxytoca exerts its pathogenicity in part through a cytotoxin. recently, authors have showed that several animal isolates of k. oxytoca, including clinical isolates, produced secreted products in bacterial culture supernatant that display cytotoxicity on hep-2 and hela cells, indicating the ability to produce cytotoxin. using mass spectroscopy techniques, they also confirmed tilivalline as the cytotoxin present in animal k. oxytoca strains. tilivalline may serve as a biomarker for k. oxytoca-induced cytotoxicity (darby et al., 2014) . clinical signs k. oxytoca has been cultured from cases of suppurative otitis media, urogenital tract infections, and pneumonia in c3h/hej and nmri-foxn1 (nu) mice (bleich et al., 2008) . additionally, k. oxytoca was recently cultured from three breeding colonies of nod. cg-prkdc scid il2rg tm1wjl /szj (nsg) mice with chronic renal inflammation and ascending urinary tract infections (foreman et al., 2011) . differential diagnosis other bacterial infections capable of causing suppurative lesions, including staphylococci, streptococci, pasteurella sp., and e. coli, among others are considered a differential diagnosis. research complications morbidity and mortality from spontaneous infections can affect ongoing research. etiology clostridium difficile was identified as the etiology of antimicrobial-associated pseudomembranous colitis in humans and currently a considerable cause of morbidity in hospitalized patients who acquire nosocomial infections. in the early 2000s, an increased interest in c. difficile infection (cdi) resulted from the emergence of a hyper-virulent strain (nap1/bi/027) associated with frequent recurrences and more severe clinical disease (abou chakra et al., 2014; mcfarland, 2009; kuijper et al., 2006) . c. difficile has also been implicated in antibioticassociated colitis in syrian hamsters (bartlett et al., 1977) , guinea pigs (lowe et al., 1980) , rabbits (thilsted et al., 1981; ryden et al., 1991) , prairie dogs (muller et al., 1987) , ostriches (frazier et al., 1993) , and horses (diab et al., 2013) . c. difficile is a rod-shaped strict anaerobe. cycloserinecefoxitin-fructose agar (ccfa) is a commonly used selective medium for c. difficile. cultures are incubated under anaerobic conditions at 35-37°c. when grown on blood agar, c. difficile colonies are nonhemolytic and gray, and have a slightly raised umbonate profile with filamentous edges and a ground-glass appearance. colonies grown on blood agar have fluorescence under ultraviolet light. c. difficile forms acid from glucose and fructose, but is negative on lactose, maltose, and sucrose. two closely related exotoxins, toxin a and toxin b, are produced by c. difficile. recent taxonomic classification support placement of c. difficile and its close relatives within the family peptostreptococcaceae. the authors suggested renaming it peptoclostridium difficile (yutin and galperin, 2013) . epizootiology it is estimated that c. difficile spores germinate and establish infection less than 10 h after ingestion. spores rapidly transit through the upper gastrointestinal tract and colonize the colon and cecum. spore shedding begins less than 2 h postingestion. when c57bl mice were challenged with 10 8 cfu of c. difficile spores, severe cdi signs developed and all mice were clinically affected by 48 h postchallenge (chen et al., 2008) . specific methods to control and prevent c. difficile infections in mice have not been described. given the method of transmission of c. difficile and c. perfringens are via ingestion or spores, these clostridia can probably be excluded from mouse colonies by maintaining strict husbandry practices, robust sanitation, and use of autoclaved feed, bedding, cages, and cage accessories. sudden dietary changes should be avoided and antibiotics should be used judiciously to minimize disruption of the normal gut microbiota of mice. diagnosis of c. difficile-associated disease is generally based on detection of cytotoxin using a tissue culture cytotoxicity assay. pcr assays for detection of both c. difficile and its cytotoxins have been developed (eastwood et al., 2009) . there are no published regimens specifically for the treatment of natural c. difficile infections in mice. oral doses given twice daily of 2 mg vancomycin for 7 days to experimentally infected gnotobiotic mice caused a 2-to 3-log decrease in vegetative bacterial cell count and no detectable cytotoxin. bacterial counts and cytotoxin levels returned to previous levels after treatment was discontinued. clinical signs untreated mice are relatively resistant to infection with c. difficile and do not develop fatal infections, although these mice can become asymptomatic carriers that persistently shed low numbers of spores (lawley et al., 2009) . susceptibility of mice to infection must be induced by disrupting the microbiota through antibiotic treatment. brief exposure to environmental laboratory animal medicine spore contamination is sufficient for transmission of c. difficile to naïve but susceptible mice. the cdi transmission model has been used to demonstrate that clindamycin treatment of asymptomatic carriers of c. difficile can inadvertently trigger the excretion of high levels of spores (lawley et al., 2009) . a c57bl mouse model of recurrence/relapse cdi has been reported (sun et al., 2011) . the primary bout of cdi induced little or no protective antibody response against c. difficile toxins and mice continued shedding c. difficile spores. antibiotic treatment of surviving mice induced a second episode of diarrhea. a simultaneous reexposure of mice to c. difficile bacteria or spores elicited a full clinical spectrum of cdi similar to that of the primary infection. immunosuppressive agents resulted in more severe and fulminant recurrent disease. vancomycin treatment only delayed disease recurrence; however, neutralizing polysera against both tcda and tcdb completely protected mice against cdi relapse (sun et al., 2011) . a recent study in c57bl mice demonstrated that antibiotic-mediated alteration of the gut microbiome favors a global metabolic profile, and therefore increases susceptibility to c. difficile clinical diseases (theriot et al., 2014) . c. difficile is not tissue invasive and only toxigenic strains are associated with disease. experimental c. difficile infections include diarrhea, cecitis, polymorphonuclear cell infiltration of the lamina propria, inflammation, pseudomembrane formation, and death. differential diagnosis c. difficile-induced diarrhea is most often associated with antibiotic treatment. other clostridial diseases in mice must be ruled out as well as other enteric pathogens in mice causing diarrhea and mortality. salmonella spp. and c. rodentium should be considered in the differential diagnosis. etiology clostridium perfringens is associated with a number of diseases in domestic animals and humans. c. perfringens is a nonmotile, rod-shaped, encapsulated, anaerobic bacterium measuring 4-8 µm in length and 0.8-1.5 µm in diameter (murray et al., 2002) . c. perfringens grow rapidly on blood agar, and colonies are smooth, round, and grayish in color, and are surrounded by a double zone of hemolysis. c. perfringens is grouped into five types based on the production and secretion of four major toxins. c. perfringens produces a number of other virulence-enhancing toxins and hydrolytic enzymes. the most significant of these is probably enterotoxin, released with the bacterial spore after cell lysis. epizootiology c. perfringens is most likely acquired by the ingestion of spores that originated in the soil or in the intestinal tract of a carrier animal. the organism can be a member of the normal microbiota in human and domestic animals. factors that have been associated with the proliferation of the organism of these species include poor husbandry and sudden dietary changes (quinn et al., 2002) . methods to control and prevent c. perfringens infections have not been evaluated in mice. because the bacterium is most likely acquired by the ingestion of spores, it can probably be excluded from mouse colonies by maintaining good sanitation and sterilizing feed, bedding, cages, and cage accessories. sudden dietary changes have also been associated with proliferation of the organism and should be avoided if possible (quinn et al., 2002) . clinical signs only a few reports in the literature exist describing clinical disease associated with c. perfringens infection in mice (matsushita and matsumoto, 1986; rozengurt and sanchez) . disease has been observed in mice of both sexes, from 2 to 32 days old, and in female mice of breeding age. clinical signs have included hunched posture, ruffled hair coat, enlarged painful abdomen, soft or impacted feces, hindquarter paralysis, and dyspnea. sudden death without premonitory signs has also been reported. the toxin types of c. perfringens isolated from these cases were reported to be non-type a (matsushita and matsumoto, 1986) , type b (rozengurt and sanchez, 1999) , and type d (clapp and graham, 1970) . mucosal necrosis in both the large and small intestine is a consistent finding on microscopic examination of tissues from mice with clinically apparent c. perfringens infections. differential diagnosis c. perfringens produces a number of major and minor toxins. different types of the bacterium produce different toxins which account for different disease outcomes. c. perfringens type a is a constituent of the normal microbiota of the intestine of humans and other animal species. bacterial culture should be obtained from live or recently dead animals, and placed in anaerobic transfer medium for transport to a microbiology laboratory and should be cultured soon after their arrival. a presumptive diagnosis for c. perfringens can be based on the presence of large grampositive rods in fecal smears or in histologic sections of intestines (quinn et al., 2002) . definitive diagnosis is based on toxin identification. mice treated with chlortetracycline hydrochloride in drinking water at a level of 11 mg/l for 2 weeks have eliminated c. perfringens-associated disease (matsushita and matsumoto, 1986) . penicillin g in the diet or changing the diet has also been reported to be effective in disease remission. c. perfringens treatments in domestic species include ampicillin, amoxicillin-clavulanate, tylosin, clindamycin, metronidazole, and bacitracin (marks, 2013; mcgorum et al., 1998) . commercially available bacterins for use in mice were not effective in controlling the disease (clapp and graham, 1970) . research complications clostridia are large, rodshaped, gram-positive anaerobic bacteria. naturally occurring clostridial infection in mice is rare. epizootics of c. perfringens type d infection with high mortality laboratory animal medicine have been reported in a barrier colony where heavy mortality occurred in 2-to 3-week-old suckling mice. clinical signs included scruffy hair coats, paralysis of the hindquarters, and diarrhea or fecal impaction. however, attempts to reproduce the disease experimentally with clostridia isolated from naturally infected animals were unsuccessful. c. perfringens also has been isolated from sporadic cases of necrotizing enteritis in recently weaned mice. clostridium piliforme -tyzzer's disease (fujiwara and ganaway, 1994; ganaway, 1982; ganaway et al., 1971; percy and barthold, 2007) etiology tyzzer's disease is named for ernest tyzzer, who first described it in a colony of japanese waltzing mice. the causative organism, c. piliforme (formerly bacillus piliformis), is a long, thin, gram-negative spore-forming bacterium that appears to require living cells for in vitro growth. it has not been grown successfully on cell-free media, but it can be propagated by inoculation of susceptible vertebrates, in select cell lines, the yolk sac of embryonated eggs, or hepatocyte cell cultures obtained from mice (ganaway et al., 1985; kawamura et al., 1983) . clinical signs clinical disease occurs as unexpected deaths that may be preceded by diarrhea and inactivity. although outbreaks can be explosive and mortality is usually high, morbidity varies. additionally, subclinical infections can occur, accompanied by the development of antibodies to c. piliforme. stresses, such as overcrowding, high temperature and humidity, moist food, and immunosuppression, and young age, may predispose mice to tyzzer's disease. susceptibility and resistance also are influenced by host genotype. it has been shown, e.g., that c57bl/6 mice are more resistant than dba/2 mice to tyzzer's disease (waggie et al., 1981) . resistance to severe infection appears to be due, in part, to b-lymphocyte function. the role of t cells in resistance is not clear, because susceptibility among athymic mice appears to vary (livingston et al., 1996) . however, the involvement of t cells can be inferred by the fact that several interleukins modulate resistance and susceptibility. depletion of neutrophils or nk cells also increases susceptibility to infection. epizootiology current prevalence rates, reservoirs of infection, carrier states, and the mechanism of spread remain speculative. tyzzer's disease occurs in many species of laboratory animals and in domestic and free-living species. some strains appear capable of cross-infecting mice, rats, and hamsters, whereas others have a more restricted host range (franklin et al., 1994) . therefore, the risks for cross-infection depend on the strain causing a given outbreak. although the vegetative form of c. piliforme is unstable, spores can retain infectivity at room temperature for at least 1 year and should be viewed as the primary means of spread. natural infection is probably due to ingestion of organisms, which are subsequently shed in feces. feces-contaminated food and soiled bedding are the most likely sources of environmental contamination. prenatal infection can be induced by intravenous inoculation of pregnant mice, but its importance in the natural transmission of infection has not been determined. pathology infection begins in the gastrointestinal tract, followed by bacteremic spread to the liver and, to a smaller extent, the heart. the lesions are characterized by necrosis in these tissues and in the mesenteric lymph nodes. grossly, segments of the ileum, cecum, and colon may be red and dilated, with watery, fetid contents, whereas the liver, mesenteric lymph nodes, and heart often contain gray-white foci. histologically, intestinal lesions include necrosis of mucosal epithelium, which may be accompanied by acute inflammation and hemorrhage. in the liver, foci of coagulation necrosis are generally distributed along branches of the portal vein, a finding compatible with embolic infection from the intestine. peracute lesions are largely free of inflammation, but neutrophils and lymphocytes may infiltrate less fulminant lesions. myocardial necrosis is sporadic in natural infection. diagnosis tyzzer's disease is diagnosed most directly by the demonstration of characteristic intracellular organisms in tissue sections of liver and intestine. bundles of long, slender rods occur in the cytoplasm of viable cells bordering necrotic foci, especially in the liver (fig. 3 .46) and intestine. they are found more easily during early stages of infection. organisms in tissue sections do not stain well with hematoxylin-eosin stain. silver stains, giemsa stains, or periodic acid-schiff stains are usually required for visualization of the organism. pcr and serologic assays are readily available at diagnostic laboratories. older supplemental procedures included inoculation of cortisonized mice or embryonated eggs laboratory animal medicine with suspect material, followed by histological or immunocytochemical demonstration of organisms in tissues. differential diagnosis the histological detection of organisms is essential for differentiating tyzzer's disease from other infections that can produce similar signs and lesions, especially mousepox, coronaviral hepatitis, reoviral hepatitis, helicobacteriosis, and salmonellosis. it also is important not to misconstrue extracellular rods as c. piliforme. prevention and control barrier housing and husbandry that incorporate sanitation measures to avoid the introduction or buildup of spores in the environment are the bases for control or prevention of tyzzer's disease. if infection occurs, spore formation will make control or elimination by antibiotic therapy problematic. therefore, strict quarantine, followed by replacement of affected or exposed stock, must be considered. rederivation by embryo transfer or cesarean section should take the potential for prenatal transmission of infection into account in housing and testing offspring. thorough decontamination of the environment with an oxidizing disinfectant must be included in any control program. additionally, procurement of food and bedding from suppliers with thorough quality assurance and vermin control programs is essential for both prevention and control. husbandry supplies should be stored in vermin-proof quarters, and the option of heat sterilization of food and bedding should be considered. research complications research complications stem from clinical morbidity and mortality. mice with immune dysfunction are at increased risk. there is recent evidence that infection causes elevations in selected cytokines (van andel et al., 2000) . etiology two mycobacteria are known to be pathogenic for laboratory mice: mycobacterium avium-intracellulare and m. lepraemurium. both are acid-fast, obligate intracellular bacteria. epizootiology mycobacteria are widespread in water and soil. their presence in laboratory mice would indicate a significant break in husbandry practices. infection with m. avium-intracellulare should be considered extremely rare, with the only published report describing an episode in a breeding colony of c57bl/6 mice . the source of the outbreak was presumed to be drinking water. mycobacterium lepraemurium has been isolated from healthy laboratory mice and can persist as a latent infection, but its significance is primarily historical, as a model for human leprosy. it is highly unlikely to encounter this infection in a modern, well-managed mouse colony. clinical signs m. avium-intracellulare infection is typically subclinical but mice have developed granulomatous pneumonia . pathology lesions are classically a chronic granulomatous disease with granulomas, langhans giant cells, and concurrent presence of acid-fast bacteria in various organs including the lungs, liver, spleen and lymph nodes. m. lepraemurium may cause alopecia, thickening of skin, subcutaneous swellings, and ulceration of the skin. disease can lead to death or clinical recovery. gross lesions are characterized by nodules in subcutaneous tissues and in reticuloendothelial tissues and organs (lung, spleen, bone marrow, thymus, and lymph nodes). lesions can also occur in the lung, skeletal muscle, myocardium, kidneys, nerves, and adrenal glands. the histologic hallmark is perivascular granulomatosis with accumulation of large, foamy epitheloid macrophages (lepra cells) packed with acid-fast bacilli. diagnosis acid-fast bacilli in lesions are the hallmark of presumptive diagnosis of mycobacteriosis. definitive diagnosis results from positive culture which takes days to weeks to rule out or positive pcr assays which are more time-efficient but require associated expertise. differential diagnosis other bacterial species that cause granulomatous lesions in mice. research complications natural infection is very rare. etiology proteus mirabilis is a ubiquitous gram-negative organism that can remain latent in the respiratory and intestinal tracts of normal mice (percy and barthold, 2007) . epizootiology proteus mirabilis colonizes the intestinal tract of most humans and is commonly found in research mice unless specifically excluded. clinical signs clinical disease can occur following stress or induced immunosuppression. immunodeficient mice have a heightened susceptibility to pathogenic infection. pathology proteus has been associated with ulcerative lesions in the gastrointestinal tract of immunodeficient mice. infected animals lose weight, develop diarrhea, and die within several weeks. if septicemia develops, suppurative or necrotic lesions, including septic thrombi, may be found in many organs, but the kidney is commonly affected. proteus pyelonephritis is characterized by abscessation and scarring. ascending lesions may occur following urinary stasis, but hematogenous spread cannot be ruled out. proteus mirabilis and pseudomonas aeruginosa have been isolated concomitantly from cases of suppurative nephritis or pyelonephritis. infection in immunodeficient mice is typified by splenomegaly and focal necrotizing hepatitis. pulmonary lesions include edema and macrophage activation. septic thrombi can occur, however, in many tissues. diagnosis culture recovery of proteus mirabilis as a predominant or single isolate confirms an opportunistic local or systemic infection. differential diagnosis gram-negative bacterial infections. research complications natural infections are typically isolated cases. etiology leptospirosis remains one of the most common zoonoses transmissible from rodents (desvars et al., 2011) but is exceedingly rare in laboratory mice. infection with leptospira interrogans serovar ballum has been reported on several occasions (see chapter 29). epizootiology leptospira are gram-negative organisms that, after a septicemic phase, establish persistent infection in the renal tubules and are periodically excreted in the urine. clinical signs natural infection is subclinical and causes no significant lesions. experimental infections can result in severe vascular, hepatic and renal lesions dependent on serovar, mouse strain and immunocompetency. diagnosis diagnosis requires isolation of organisms in kidney culture. serological testing should be used with caution because neonatal exposure can lead to persistent infection without seroconversion. histologic examination of kidney using silver stains can also be attempted. pcr assays are reliable for preliminary diagnosis. differential diagnosis not applicable in research colonies. research complications persistent murine infections associated with active shedding present a zoonotic hazard for humans; therefore, infected mice should be culled. elimination of infection from highly valuable mice requires rederivation. (percy and barthold, 2007) etiology chlamydia trachomatis is an intracellular organism that produces glycogen-positive intracytoplasmic inclusions (elementary bodies). c. trachomatis causes ocular and urogenital disease in humans. however, at least one strain historically referred to as the 'nigg agent' after clara nigg, is most recently classified as chlamydia muridarum and is used experimentally to model human chlamydia infection. epizootiology mice are susceptible to natural infection and experimental infection with c. trachomatis and chlamydophila psittaci, especially immunodeficient mouse strains. clinical signs natural infections are typically subclinical but persistent. pathology c. muridarum is also known as the 'mouse pneumonitis agent' due to severe acute infection which is characterized by ruffled fur, hunched posture, and labored respiration due to interstitial pneumonitis and death in 24 h. mice with more chronic infections may develop progressive emaciation and cyanosis of the ears and tail. experimental infections to model human venereal chlamydia infections will develop hydrosalpinx, cervical, and vaginal infections in female mice and urethritis in male mice. diagnosis chlamydia can be diagnosed by impression smears stained with giemsa or macchiavello stains, cell culture, or inoculation of embryonated eggs. pcr and sequencing can be used to speciate the type of chlamydia. differential diagnosis c. muridarum, c. trachomatis, and c. psittaci are included in the differential diagnosis. research complications chlamydia is a rare spontaneous infection in research mice; its potential significance is low. etiology pneumocystis murina (pm) is a common opportunistic organism of laboratory mice and other mammals. when first described by chagas in 1909, p. carinii was misidentified as trypanosoma cruzi and was considered a protozoan (chagas, 1909) . it was renamed as a new species, p. carinii, when observed in a rat in 1912 (delanoë, p. and delanoë, m. 1912) . p. carinii, however, has now been grouped taxonomically with the fungi based on dna analysis and the homology of p. murina housekeeping genes with those found in fungi (edman et al., 1989; stringer et al., 2002; wakefield et al., 1992) . these dna studies and apparent differences of host susceptibility prompted a new name, p. jiroveci, for pneumocystis isolated from humans (stringer et al., 2002; frenkel, 1999) . p. carinii is now used to name the organism in rats and p. murina, the organism in mice. clinical signs pm infection is subclinical in immunocompetent mice. however, it can be clinically severe in immunodeficient mice, because an adequate complement of functional t lymphocytes is required to suppress infection (roths et al., 1990; shultz and sidman, 1987; walzer et al., 1989; weir et al., 1986) . b cells have also been shown to be critical to clearance of infection and the mechanism appears only partially related to igg and has a more important role in promoting activation and expansion of t cells (lund et al., 2003) . b cells may also protect early hematopoietic progenitor activity during systemic responses to pneumocystis infection (hoyt et al., 2014) . infection proceeds slowly, but relentlessly in immunodeficient mice leading to clinical signs of pneumonia, usually within several months. primary signs include dyspnea and hunched posture, which may laboratory animal medicine be accompanied by wasting and scaly skin. severe cases, such as those that occur in advanced disease in scid mice, may be fatal. epizootiology pm is known to infect a number of mammalian hosts, including ferrets, rats, mice, and humans. pm is a ubiquitous organism that is often present as a latent infection. although firm prevalence data are not available, because detection methods are not simple to apply, infection is assumed to be present in mouse colonies unless ruled out by extensive surveillance. although these organisms appear morphologically similar, there are antigenic and genetic differences among p. murina isolated from different hosts (weinberg and durant, 1994; cushion, 1998) . furthermore, studies indicate that p. carinii isolated from one host species is unable to survive and replicate after inoculation into a different immunodeficient host species (gigliotti et al., 1993b) . pm infection also occurs in human beings, but transmission between rodents and human beings has not been documented. pm is transmitted by aerosol and establishes persistent, quiescent infection in the lungs of immunocompetent mice. prenatal infection has not been demonstrated. pathology pm is normally not pathogenic but can be activated by intercurrent immunosuppression. activation fills the lung with trophic and cystic forms. gross lesions occur in the lungs, which are often rubbery and fail to deflate (fig. 3.47 ). histopathological changes are characterized by interstitial alveolitis with thickening of alveolar septa from proteinaceous exudate and infiltration with mononuclear cells (fig. 3 .48) (roths et al., 1990) . alveolar spaces may contain vacuolated eosinophilic material and macrophages. special stains are required to visualize pm. silver-based stains reveal round or partially flattened 3-to 5-mm cysts in affected parenchyma (fig. 3.49) . in florid cases, alveolar spaces may be filled with cysts, but cysts may be sparse in mild cases. disease can be especially severe when subclinically infected immunodeficient mice are reconstituted with competent immune cells that subsequently promote pneumonitis. diagnosis respiratory distress in immunodeficient mice should elicit consideration of pneumocystosis. pathologic examination of the lung, including silver laboratory animal medicine methenamine staining, is essential to confirm a presumptive clinical diagnosis. past infections of immunocompetent mice also can be detected by elisa (furuta et al., 1985) . pcr can be used to detect active infection (gigliotti et al., 1993a; reddy et al., 1992) and is particularly useful for screening immunodeficient mice. differential diagnosis pneumocystosis must be differentiated from viral pneumonias of mice. it is worth noting, in this regard, that pneumonia virus of mice has been shown to accelerate the development of pneumocystosis in scid mice (bray et al., 1993; roths et al., 1993) . prevention and control pm infection is a significant disease threat to immunodeficient mice. its widespread distribution strongly suggests that susceptible mice should be protected by microbarrier combined, where possible, with macrobarrier housing. husbandry procedures should include proper sterilization of food, water, and housing equipment and the use of hepa-filtered change stations. infected colonies can be rederived by embryo transfer or cesarean methods, because infection does not appear to be transmitted in utero. research complications pneumonia in immunodeficient mice is the major complication of pm infection. trichophyton mentagrophytes is the most common fungal agent of mice. however, infection rarely causes clinical disease. clinical signs include sparse hair coats or well-demarcated crusty lesions, with a chalky surface on the head, tail, and legs (favus or ringworm). skin lesions are composed of exfoliated debris, exudate, mycelia, and arthrospores with underlying dermatitis. invasion of hair shafts is not characteristic. diagnosis depends on effective specimen collection. hairs should be selected from the periphery of the lesion, and hairless skin should be scraped deeply to obtain diagnostic specimens. t. mentagrophytes rarely fluoresces under ultraviolet light, and hyphae must be differentiated from bedding fibers, food particles, and epidermal debris. histological sections should be stained with a silver stain or schiff's reagent to reveal organisms. trichophyton also can be cultured on sabouraud agar. plates are incubated at room temperature (22-30°c), and growth is observed at 5-10 days. ringworm is not easily eradicated from laboratory mice. the use of antifungal agents to treat individual mice is time-consuming, expensive, and variably effective. rederivation is a more prudent course. cages and equipment should be sterilized before reuse. concurrent infection with ectoparasites also must be considered during eradication steps. candida albicans and other systemic mycoses are not important causes of disease in mice, but they can be opportunistic pathogens in immunodeficient mice. etiology giardia muris is a pear-shaped, flagellated organism with an anterior sucking disk. it inhabits the duodenum of young and adult mice, rats, and hamsters. clinical signs infection is often subclinical, unless organisms proliferate extensively, and can cause weight loss, a rough hair coat, sluggish movement, and abdominal distension, usually without diarrhea. additionally, immunodeficient mice may die during heavy infestation. epizootiology the contemporary prevalence of affected mouse colonies is not well documented, but surveys during the 1980s found the rates exceeding 50%. transmission occurs by the fecal-oral route. crossinfection between mice and hamsters after experimental inoculation of organisms has been demonstrated, whereas rats were resistant to isolates from mice and hamsters (kunstyr et al., 1992) . c3h/he mice are particularly susceptible to giardiasis, whereas balb/c and c57bl/10 mice are more resistant. additionally, female mice appear to be more resistant to infection than male mice (daniels and belosevic, 1995) . c57bl/6 females, e.g., have lower trophozoite burdens and for a shorter interval than male mice. females also shed cysts later than male mice. these differences may be related to a more potent humoral immune response to giardia in female mice. pathology gross lesions are limited to the small intestine, which may contain yellow or white watery fluid. histopathology reveals organisms in the lumen that often adhere to microvilli of enterocytes or reside in mucosal crevices or mucus. the crypt/villus ratio may be reduced, and the lamina propria may have elevated numbers of inflammatory cells. diagnosis diagnosis is based on detection of trophozoites in the small intestine or in wet mounts of fecal material. organisms can be recognized in wet preparations by their characteristic rolling and tumbling movements. ellipsoidal cysts with four nuclei also may be detected in feces. infection also can be detected by serology (daniels and belosevic, 1994) and by pcr (mahbubani et al., 1991) . treatment, prevention, and control murine giardiasis can be treated by the addition of 0.1% dimetridazole to drinking water for 14 days. prevention and control depend on proper sanitation and management, including adequate disinfection of contaminated rooms. research complications accelerated cryptal cell turnover and suppression of the immune response to sheep erythrocytes have been observed in infected mice. the potential for severe or lethal infection in immunodeficient mice was noted previously. etiology spironucleus muris is an elongated, pearshaped, bilaterally symmetrical flagellated protozoan that commonly inhabits the duodenum, usually in the crypts of lieberkühn. it is smaller than giardia muris and lacks an anterior sucking disk. clinical signs s. muris infection is usually subclinical in normal adult mice. it is more pathogenic, however, for young, stressed, or immunocompromised mice (kunstyr et al., 1977) . additionally, clinical morbidity may indicate an underlying primary infection with an unrelated organism. clinically affected mice can have a poor hair coat, sluggish behavior, and weight loss. mice at 3-6 weeks of age are at notably higher risk for clinically evident infection. they can develop dehydration, hunched posture, abdominal distension, and diarrhea. severe infections can be lethal. epizootiology transmission occurs by the fecaloral route and can occur between hamsters and mice as well as between mice. it does not appear to be transmitted between mice and rats (schagemann et al., 1990) . the most recent surveys, which are somewhat dated, indicated that prevalence rates exceeded 60% among domestic mouse colonies in the mid-1980s. there is some evidence that inbred strains vary in their susceptibility to infection and their rate of recovery (baker et al., 1998; brett and cox, 1982) . pathology gross findings associated with infection include watery, red-brown, gaseous intestinal contents. however, it is essential to rule out primary or coinfection by other organisms before attributing these lesions to spironucleosis. microscopically, acute disease is associated with distension of crypts and intervillous spaces by pear-shaped trophozoites and inflammatory edema of the lamina propria. organisms can be visualized more easily with periodic acid-schiff staining, which may reveal invasion of organisms between enterocytes and in the lamina propria. chronic infection is associated with lymphoplasmacytic infiltration of the lamina propria and occasional intracryptal inflammatory exudate. diagnosis diagnosis is based on identification of trophozoites in the intestinal tract. they can be distinguished from giardia muris and tritrichomonas muris by their small size, horizontal or zigzag movements, and the absence of a sucking disk or undulating membrane. pcr-based detection also is available (rozario et al., 1996) . it is not clear whether duodenitis is a primary pathogenic effect of s. muris or represents opportunism secondary to a primary bacterial or viral enteritis. therefore, it is prudent to search for underlying or predisposing infections. treatment, prevention, and control treatment consists of adding 0.1% dimetridazole to drinking water for 14 days, as described for giardiasis. prevention and control require good husbandry and sanitation. research complications as with giardiasis, infection can accelerate enterocytic turnover in the small intestine. there is some evidence that infected mice may have activated macrophages that kill tumor cells nonspecifically and that infection can diminish responses to soluble and particulate antigens. additionally, infected mice also have increased sensitivity to irradiation. such effects should, however, be interpreted cautiously in order to rule out intercurrent viral infections. tritrichomoniasis t. muris is a nonpathogenic protozoan that occurs in the cecum, colon, and small intestine of mice, rats, and hamsters. no cysts are formed, and transmission is by ingestion of trophozoites passed in the feces. it can be detected by microscopy or by pcr (viscogliosi et al., 1993) . coccidiosis eimeria falciformis is a pathogenic coccidian that occurs in epithelial cells of the large intestines of mice. it was common in european mice historically but is seldom observed in the united states. heavy infection may cause diarrhea and catarrhal enteritis. klosiella muris causes renal coccidioisis in wild mice but is rare in laboratory mice. mice are infected by ingestion of sporulated sporocysts. sporozoites released from the sporocysts enter the bloodstream and infect endothelial cells lining renal arterioles and glomerular capillaries, where schizogony occurs. mature schizonts rupture into bowman's capsule to release merozoites into the lumen of renal tubules. merozoites can enter epithelial cells lining convoluted tubules, where the sexual phase of the life cycle is completed. sporocysts form in renal tubular epithelium and eventually rupture host cells and are excreted in the urine, but oocysts are not formed. infection is usually nonpathogenic and subclinical. gray spots may occur in heavily affected kidneys and are the result of necrosis, granulomatous inflammation, and focal hyperplasia. destruction of tubular epithelium may impair renal physiology. diagnosis is based on detection of organisms in tissues. prevention and control require proper sanitation and management techniques. there is no effective treatment. cryptosporidiosis cryptosporidium muris is a sporozoan that adheres to the gastric mucosa. it is uncommon in laboratory mice and is only slightly pathogenic. cryptosporidium parvum inhabits the small intestine and is usually nonpathogenic in immunocompetent and athymic mice (ozkul and aydin, 1994; taylor et al., 1999) . athymic mice may develop cholangitis and hepatitis, however, if organisms gain access to the biliary tract. entamoebiasis entamoeba muris is found in the cecum and colon of mice, rats, and hamsters throughout the world. organisms live in the lumen, where they feed on particles of food and bacteria. they are considered nonpathogenic. encephalitozoonosis encephalitozoon cuniculi is a gram-positive microsporidian that infects rabbits, mice, rats, guinea pigs, dogs, nonhuman primates, humans, and other mammals. infection is extremely rare among laboratory mice. the life cycle of the organism is direct, and animals are infected by ingesting spores or by cannibalism. spore cells are disseminated in the blood to the brain and other sites. infection can last more than 1 year, and spores shed in the urine serve as a source of infection. vertical transmission has not been confirmed in mice. e. cuniculi is an obligate intracellular parasite, but infection usually elicits no clinical signs of disease. organisms proliferate in peritoneal macrophages by asexual binary fission. they have a capsule that accepts giemsa and goodpasture stains but is poorly stained by hematoxylin. fulminating infection can cause lymphocytic meningoencephalitis and focal granulomatous hepatitis. in contrast to encephalitozoonosis in rabbits, affected mice do not develop interstitial nephritis. infection is diagnosed by cytological examination of ascitic fluid smears, histopathologic examination of brain tissues stained with goodpasture stain, and elisa serology. no effective treatment has been reported. prevention and control require rigid testing and elimination of infected colonies and cell lines. pcr-based assays may also be useful. toxoplasmosis toxoplasma gondii is a ubiquitous gram-negative coccidian parasite for which the mouse serves as a principal intermediate host. however, the prevalence of natural infection is negligible because laboratory mice no longer have access to sporulated cysts shed by infected cats, which were historically the major source for cross-infection. toxoplasmosis can cause necrosis and granulomatous inflammation in the intestine, mesenteric lymph nodes, eyes, heart, adrenals, spleen, brain, lung, liver, placenta, and muscles. diagnosis is based on elisa serology, histopathology, and pcr. control and prevention depend largely on precluding access of mice to cat feces or to materials contaminated with cat feces. oocytes are very resistant to adverse temperatures, drying, and chemical disinfectants; therefore, thorough cleaning of infected environments is required. b. cestodiasis baker, 2007) etiology hymenolepis (rodentolepis) nana, the dwarf tapeworm, infects mice, rats, and humans although the zoonotic risk has been questioned (macnish et al., 2002) . adults are extremely small (25-40 mm) and have eggs with prominent polar filaments and rostellar hooks (fig. 3.50) . clinical signs young adult mice are most frequently infected. signs and lesions include weight loss and focal enteritis, but clinical disease is rare unless infestation is severe. epizootiology the life cycle may be direct or indirect (r. nana is the only cestode known that does not require an intermediate host). the indirect cycle utilizes arthropods as intermediate hosts. liberated oncospheres penetrate intestinal villi and develop into a cercocystis stage before reemerging into the intestinal lumen 10-12 days later. the scolex attaches to the intestinal mucosa, where the worm grows to adult size in 2 weeks. the cycle from ingestion to patency takes 20-30 days. pathology cysticerci are found in the lamina propria of the small intestine and sporadically in the mesenteric lymph nodes, whereas adults, which have a serrated profile, are found in the lumen. inflammation is not a feature of infection. diagnosis infection can be diagnosed by demonstrating eggs in fecal flotation preparations or by opening the intestine in petri dishes containing warm tap water to facilitate detection of adults. r. nana can be differentiated from another species of rodent tapeworm, h. diminuta, by the fact that r. nana has rostellar hooks and eggs with polar filaments. however, h. diminuta requires an intermediate arthropod host, so it is rarely found in contemporary mouse colonies. treatment, prevention, and control drugs recommended for treatment and elimination include praziquantel (0.05% in the diet for 5 days), albendazole, mebendazole, and thiabendazole. although the benzimidazoles have an excellent activity against cestodes and nematodes in rats, they have not been tested extensively in mice. the potential for successful treatment is high, however, because eggs do not survive well outside the host and because the prevalence of infestation is low in caged mice kept in properly sanitized facilities. because r. nana can directly infect humans, proper precautions should be taken to avoid oral contamination during handling of rodents (see chapter 28). hymenolepis microstoma is found in the bile ducts of rodents and could be confused with r. nana in the mouse. however, the location of the adult as well as the large size of h. microstoma eggs compared with those of r. nana make differential diagnosis relatively simple. the mouse and the rat are intermediate hosts of the cestode taenia taeniaformis. the definitive host is the cat. this parasite should not be found in laboratory mice housed separately from cats. c. nematodiasis (wescott, 1982) syphacia obvelata (mouse pinworm) infestation etiology syphacia obvelata, the common mouse pinworm, is a ubiquitous parasite of wild and laboratory mice. the rat, gerbil, and hamster are also occasionally infected. female worms range from 3.4 to 5.8 mm in length, and male worms are smaller (1.1-1.5 mm). eggs are flattened on one side and have pointed ends (fig. 3.51 ). the nucleus fills the shell and is frequently at a larval stage when eggs are laid. clinical signs infestation is usually subclinical, although heavily infested mice can occasionally sustain intestinal lesions, including rectal prolapse, intussusception, enteritis, and fecal impaction. epizootiology pinworm infestation is one of the most commonly encountered problems in laboratory mice. a national survey revealed that more than 30% of barrier colonies and about 70% of conventional colonies were affected (jacoby and lindsey, 1997; carty, 2008) . syphacia obvelata infestation can occur unexpectedly in commercial barrier murine colonies, resulting in widespread dissemination of the parasite into academic mouse colonies. the epizootiological impact of pinworm infestation is increased by the airborne dissemination of eggs, which can remain infectious even after drying. the life-cycle is direct and completed in 11-15 days. females deposit their eggs on the skin and hairs of the perianal region. ingested eggs liberate larvae in the small intestine and they migrate to the cecum within 24 h. worms remain in the cecum for 10-11 days, where they mature and mate. the females then migrate to the large intestine to deposit their eggs as they leave the host. there is unconfirmed speculation that larvae may reenter the rectum. infestation usually begins in young mice and can recur, but adult mice tend to be more resistant. syphacia infestation often occurs in combination with aspiculuris tetraptera. because the life cycle of syphacia is much shorter than that of aspiculuris, the number of mice that are apt to be infected with s. obvelata is correspondingly greater. there is evidence that resistance to infestation may be mouse strain-specific (derothe et al., 1997) . pathology gross lesions are not prevalent, aside from the presence of adults in the lumen of the intestine. diagnosis infestation is diagnosed by demonstrating reniform-shaped eggs in the perianal area or adult worms in the cecum or large intestine. four-to 5-weekold mice should be examined because the prevalence is higher in this age group than in older mice. because most eggs are deposited outside the gastrointestinal tract, fecal examination is not reliable. eggs are usually detected by pressing cellophane tape to the perineal area and then to a glass slide that is examined by microscopy. aspiculuris tetraptera eggs are not ordinarily found in tape preparations and are easily differentiated from eggs of s. obvelata (see below). adult worms can be found in cecal or colonic contents diluted in a petri dish of warm tap water. they are readily observed with the naked eye or with a dissecting microscope. an elisa also is available to detect serum antibodies to s. obvelata somatic antigens (sato et al., 1995) . pcr assays are increasingly being used to augment traditional diagnostic methods and to discriminate between pinworm species (dole et al., 2011) . pcr panels for pinworm detection using fecal pellets are available from commercial diagnostic laboratories. treatment, prevention, and control pinworm infestation can be treated effectively by a number of regimens, which include the use of anthelmintics such as piperazine, ivermectin, and benzimidazole compounds alone or in combination (klement et al., 1996; le blanc et al., 1993; lipman et al., 1994; flynn et al., 1989; wescott, 1982; zenner, 1998) . because some of the recommended therapies have the potential for toxicity, it is prudent to keep mice under close clinical observation during treatment (davis et al., 1999; skopets et al., 1996; toth et al., 2000) . fenbendazole diets can be fed with 1 week on/1 week off rotation with normal chow although the potential impact on experimental data must be considered (duan et al., 2012; gadad et al., 2010; landin et al., 2009) . prevention of reinfestation requires strict isolation because syphacia eggs become infective as soon as 6 h after they are laid, and they survive for weeks, even in dry conditions. strict sanitation, sterilization of feed and bedding, and periodic anthelmintic treatment are required to control infestation. the use of microbarrier cages can reduce the spread of infective eggs. syphacia muris is the common rat pinworm. it can potentially infest mice but is not found in well-managed colonies. it can be differentiated from s. obvelata because s. muris eggs are smaller. treatment is the same as for pinworms of mice. etiology aspiculuris tetraptera is the other major oxyurid of the mouse and may coinfect mice carrying s. obvelata. females are 2.6-4.7 mm long, and males are slightly smaller. the eggs are ellipsoidal (fig. 3.52) . clinical signs ingested eggs hatch, and larvae reach the middle colon, where they enter crypts and remain for 4-5 days. they move to the proximal colon about 3 weeks after infection of the host. because the life cycle is 10-12 days longer than in s. obvelata, infestations appear in somewhat older mice; heaviest infestation is expected in 5-6 weeks after initial exposure. infection is usually subclinical, but heavy loads can produce signs similar to those discussed for s. obvelata. light to moderate loads do not produce clinical disease. epizootiology as noted under s. obvelata, pinworm infestation is highly prevalent and contagious in laboratory mice. the life cycle is direct and takes approximately 23-25 days. mature females inhabit the large intestine, where they survive from 45 to 50 days and lay their eggs. the eggs are deposited at night and are excreted in a mucous layer, covering fecal pellets. they require 6-7 days at 24°c to become infective and can survive for weeks outside the host. pathology see s. obvelata (section iii, a,5,c). diagnosis aspiculuris tetraptera eggs can be detected in the feces, and adult worms are found in the large intestine. eggs are not deposited in the perianal area; therefore, cellophane tape techniques are not useful. measures for treatment, prevention, and control are similar to those described for s. obvelata. because a. tetraptera takes longer to mature and because eggs are deposited in feces rather than on the host, adult parasites are more amenable to treatment by frequent cage rotations. immune expulsion of parasites and resistance to reinfection are hallmarks of a. tetraptera infection. research complications see s. obvelata (section iii, a,5,c). several species of mites infest laboratory mice. they include myobia musculi, radfordia affinis, myocoptes musculinus, and, less commonly, psorergates simplex. the common murine mites are described below, while less frequently encountered species are listed in table 3 .14. these include the mouse mite trichoecius romboutsi, which resembles myocoptes and ornithonyssus bacoti, the tropical rat mite, which can infect laboratory mice. characteristics of specific infestations are described after a general introductory section. clinical signs mites generally favor the dorsal anterior regions of the body, particularly the top of (jacoby and lindsey, 1997; carty, 2008) reported mite infestations in 40% of colonies. acarids spend their entire lives on the host. populations are limited by factors such as self-grooming, mutual grooming, the presence of hair, and immunological responses, which tend to produce hypersensitivity dermatitis. inherited resistance and susceptibility also affect clinical expression of acariasis. mite populations, e.g., vary widely among different stocks and strains of mice housed under similar conditions. pathology gross lesions include scaly skin, regional hair loss, abrasions, and ulcerations. histologically, hyperkeratosis, acanthosis, and chronic dermatitis may occur. long-standing infestation provokes chronic inflammation, fibrosis, and proliferation of granulation tissue. ulcerative dermatitis associated with acariasis may have an allergic pathogenesis but often results in secondary bacterial infections. lesions resemble allergic acariasis in other species and are associated with mast cell accumulations in the dermis. diagnosis classic methods of detection include direct observation of the hair and skin of dead or anesthetized mice. hairs are parted with pins or sticks and examined with a dissecting microscope. examination of young mice, prior to the onset of immune-mediated equilibrium, is likely to be more productive. alternatively, recently euthanized mice can be placed on a black paper, and double-sided cellophane tape can be used to line the perimeter to contain the parasites. as the carcass cools, parasites will vacate the pelage and crawl onto the paper. sealed petri dishes can also be used. cellophane tape also can be pressed against areas of the pelt of freshly euthanatized mice and examined microscopically. skin scrapings made with a scalpel blade can be macerated in 10% koh/glycerin or immersion oil and examined microscopically. this method has the disadvantage of missing highly motile species and low-level populations of slower moving immature forms. it is important to remember that mite infestations may be mixed, so the identification of one species does not rule out the presence of others. detecting mites in sentinels exposed to dirty bedding from colony animals has been reported to be unreliable (lindstrom et al., 2011) . thus, pcr assays offered by commercial diagnostic laboratories are increasingly being used to augment traditional diagnostic methods and to test individual animals or equipment using a swabbing technique; samples can be pooled to decrease cost (jensen et al., 2013) . gross anatomical features facilitate differentiation of intact mites. myocoptes has an oval profile with heavily chitinized body, pigmented third and fourth legs, and tarsal suckers (fig. 3.54) . myobia and radfordia have a similar elongated profile, with bulges between the legs. myobia has a single tarsal claw on the second pair of legs (fig. 3.55) , whereas radfordia has two claws of unequal size on the terminal tarsal structure of its second pair of legs (fig. 3.56) . histopathological examination of skin is helpful for diagnosing unique forms of acariasis, such as the keratotic cysts associated with psorergates simplex infestation. treatment, prevention, and control ivermectin can be used topically, in drinking water or as a medicated feed and often is the first-choice approach for attempting eradication although cost and potential toxicity are concerns. because of potential differences in laboratory animal medicine blood-brain barrier permeability to ivermectin, pilot treatments should be evaluated. for large facilities, ivermectin medicated feed may be an attractive option (ricart arbona et al., 2010) . for valuable lines of mice, rederivation may be cost-and time-effective. control and prevention programs should be carried out on a colony-wide basis, which includes thorough sanitation of housing space and equipment to remove residual eggs. research complications hypersensitivity dermatitis has the potential to confound immunological studies (jungmann et al., 1996) , especially those involving skin, and has been shown to elevate serum ige (morita et al., 1999) . heavy mite infestations can cause severe skin lesions and have been associated with weight loss, infertility, and premature deaths. chronic acariasis also may provoke secondary amyloidosis due to long-standing dermatitis. myocoptes musculinus this is the most common ectoparasite of the laboratory mouse but frequently occurs in conjunction with myobia musculi. the life cycle includes egg, larva, protonymph, tridonymph, and adult stages. eggs hatch in 5 days and are usually attached to the middle third of the hair shaft. the life cycle may range from 8 to 14 days. transmission requires direct contact, for mice separated by wire screens do not contract infestations from infested hosts. bedding does not seem to serve as a vector. neonates may become infested within 4-5 days of birth, and parasites may live for 8-9 days on dead hosts. myocoptes appears to inhabit larger areas of the body than myobia and tends to displace myobia during heavy infestations. it has some predilection for the skin of the inguinal region, abdominal skin, and back, but it will also infest the head and neck. it is a surface dweller that feeds on superficial epidermis. infestation can cause patchy thinning of the hair, alopecia, or erythema. lesions can be pruritic, but ulceration has not been reported. chronic infestations induce epidermal hyperplasia and nonsuppurative dermatitis. myobia musculi this is a common mite of laboratory mice. the life cycle of myobia can be completed in 23 days and includes an egg stage, first and second larval stages, protonymph, deutonymph, and adult. eggs attach at the base of hair shafts and hatch in 7-8 days. larval forms last about 10 days, followed by nymphal forms on day 11. adults appear by day 15 and lay eggs within 24 h. myobia are thought to feed on skin secretions and interstitial fluid but not on blood. they are transmitted primarily by contact. mite populations increase during new infestations, followed by a decrease to equilibrium in 8-10 weeks. the equilibrated population can be carried in colonies for long periods (up to years). population fluctuations may represent waves of egg hatchings. because mites are thermotactic, they crawl to the end of hair shafts on dead hosts, where they may live for up to 4 days. infestation may result in hypersensitivity dermatitis, to which c57bl mice are highly susceptible. clinical signs vary from ruffled fur and alopecia to pruritic ulcerative dermatitis. therefore, lesions can be exacerbated by self-inflicted trauma. radfordia affinis radfordia is thought to be common in laboratory mice, but it closely resembles myobia and may occur as a mixed infestation. therefore, its true prevalence is conjectural. additionally, its life cycle has not been described. it does not appear to cause clinical morbidity. psorergates simplex this species has not been reported as a naturally occurring infection in well-managed colonies for several decades, but it is unique in that it inhabits hair follicles. its life cycle is unknown, but developmental stages from egg to adult may be found in a single dermal nodule. transmission is by direct contact. invasion of hair follicles leads to development of cyst-like nodules, which appear as small white nodules in the subcutis. histologically, they are invaginated sacs of squamous epithelium, excretory products, and keratinaceous debris. there is usually no inflammatory reaction, but healing may be accompanied by granulomatous inflammation. diagnosis is made by examining the subcuticular surface of the pelt grossly or by histological examination. sac contents also can be expressed by pressure with a scalpel blade or scraped and mounted for microscopic exam. mesostigmoid mites rarely, blood-sucking ornithonyssus bacoti and laelaps echidnina, normally limited to wild rodents, can also infect laboratory rodent colonies (watson, 2008; fox, 1982) . these mites may also transiently bite humans and can transmit zoonotic infections (see chapter 29). unlike the more common rodent fur mites, mesostigmoid mites live off the host and can travel a long distance in search of a blood meal. they access research colonies via contaminated supplies or wild rats and mice gaining access to the facility. polyplax serrata, the mouse louse, is encountered in wild mice but no longer is a significant issue in research colonies. eggs are deposited at the base of hair shafts and nymph stages and adults can be found principally on the dorsum. p. serrata causes pruritus with associated dermatitis, anemia and debilitation and historically is the vector for mycoplasma coccoides. amyloidosis is caused by the deposition of insoluble (polymerized), mis-folded amyloid protein fibrils in organs and/or tissues. primary amyloidosis is a naturally occurring disease in mice, associated with the deposition of amyloid proteins consisting primarily of immunoglobulin light chains. secondary amyloidosis is associated with antecedent and often chronic inflammation. it results from a complex cascade of reactions involving release of multiple cytokines that stimulate amyloid synthesis in the liver (falk and skinner, 2000) . primary amyloidosis is common among aging mice (lipman et al., 1993) but also may occur in young mice of highly susceptible strains such as a and sjl or somewhat older c57bl mice. other strains, such as balb/c and c3h are highly resistant to amyloidosis (percy and barthold, 2007) . secondary amyloidosis is usually associated with chronic inflammatory lesions, including dermatitis resulting from prolonged acariasis. it can be induced experimentally, however, by injection of casein and may occur locally in association with neoplasia or in ovarian corpora lutea in the absence of other disease. in reactive amyloid a (aa) amyloidosis, serum aa (saa) protein forms deposits in mice, domestic and wild animals, and humans that experience chronic inflammation. aa amyloid fibrils are abnormal β-sheet-rich forms of the serum precursor saa, with conformational changes that promote fibril formation. similar to prion diseases, recent findings suggest that aa amyloidosis could be transmissible in mice and other species (murakami et al., 2014) . amyloid fibrils induce a seeding-nucleation process that may lead to development of aa amyloidosis. amyloidosis can shorten the life span of mice and can be accelerated by stress from intercurrent disease. amyloid appears histologically as interstitial deposition of a lightly eosinophilic, acellular material in tissues stained with hematoxylin and eosin. however, it is birefringent after staining with congo red when viewed with polarized light. deposition patterns vary with mouse strain and amyloid type. although virtually any tissue may be affected, the following sites are common: hepatic portal triads, periarteriolar lymphoid sheaths in spleen, renal glomeruli and interstitium (which can lead to papillary necrosis), intestinal lamina propria, myocardium (and in association with atrial thrombosis), nasal submucosa, pulmonary alveolar septa, gonads, endocrine tissues, and great vessels (fig. 3.57) . naturally occurring mineralization of the myocardium and epicardium and other soft tissues is a common finding at necropsy in some inbred strains of mice. although this condition is usually an incidental finding at necropsy, interference with organ function such as the heart cannot be ruled out if lesions are severe. it occurs in balb/c, c3h, and especially dba mice (eaton et al., 1978; brownstein, 1983; brunnert et al., 1999) . it is found in the myocardium of the left ventricle ( fig. 3.58) , in the intraventricular systems, and in skeletal muscle, kidneys, arteries, and lung and may be accompanied by fibrosis and mononuclear inflammatory infiltrates. dba mice also can develop mineralization in the tongue and cornea. dietary, environmental, disease-related, and endocrine-related factors are thought to influence the prevalence of this lesion. ectopic mineralization is associated clinically with skin and vascular connective tissue conditions in humans and mouse models have been developed to study metastatic and dystrophic tissue mineralization (li and uitto, 2013) . pseudoxanthoma elasticum (pxe), a heritable ectopic mineralization disorder in humans, is caused by mutations in the abcc6 gene. knockout abcc6 −/− mice model the histopathologic and ultrastructural features of pxe, notably with mineralization of the vibrissae dermal sheath, serving as a biomarker of tissue mineralization (benga et al., 2014) . other inbred mouse strains, including kk/hlj and 129s1/svimj, also develop vibrissae dermal mineralization and have an snp (rs32756904) in the abcc6 gene associated with low levels of abcc6 protein expression in the liver. dba/2j and c3h/hej mice have the same polymorphism and low abcc6 protein levels; however, these mice only develop tissue mineralization when fed an experimental diet enriched in phosphate and low in magnesium. a reye's-like syndrome has been reported in balb/ cbyj mice (brownstein et al., 1984) . the etiology is unknown; however, antecedent viral infection may be involved. affected mice rapidly become lethargic and then comatose. they also tend to hyperventilate. high mortality ensues within 6-18 h, but some mice may recover. lesions are characterized grossly by swollen, pale liver and kidneys. the major histopathological findings include swollen hepatocytes with fatty change and nuclear swelling among astrocytes in the brain. hepatic lesions resembling changes in reye's syndrome have been reported in scid mice infected with madv-1 (pirofski et al., 1991) . deficiencies (tobin et al., 2007) vitamin deficiencies in mice have not been thoroughly described. unfortunately, much of the information that does exist reflects work carried out 30-50 years ago; thus, the reliability and specificity of some of these syndromes is questionable. vitamin a deficiency may produce tremors, diarrhea, rough hair coat, keratitis, poor growth, abscesses, hemorrhages, and sterility or abortion. vitamin a is recognized for its importance in development of the immune system (ross, 2012) and knockout mouse models have been used to demonstrate genetic polymorphisms in humans that negatively regulate intestinal β-carotene absorption and conversion to retinoids in response to vitamin a requirements for growth and reproduction (von lintig, 2012) . vitamin e deficiency can cause convulsions and heart failure, as well as muscular dystrophy and hyaline degeneration of muscles. two knockout mouse models of severe vitamin e deficiency were independently developed and lack α-tocopherol transfer protein (α-ttp), a gene that controls plasma and tissue α-tocopherol concentrations by exporting α-tocopherol from the liver. ttpa −/− mice have very low to undetectable levels of α-tocopherol and are infertile. the phenotype includes neuronal degeneration associated with progressive ataxia and age-related behavioral defects (yu and schellhorn, 2013) . deficiency of b complex vitamins produces nonspecific signs such as alopecia, decreased feed consumption, poor growth, poor reproduction and lactation, as well as a variety of neurological abnormalities. choline deficiency produces fatty livers and nodular hepatic hyperplasia, as well as myocardial lesions, decreased conception, and decreased viability of litters. folic acid-deficient diets cause marked decreases in red and white cell blood counts and the disappearance of megakaryocytes and nucleated cells from the spleen. pantothenic acid deficiency is characterized by nonspecific signs, such as weight loss, alopecia, achromotrichia, and posterior paralysis, as well as other neurological abnormalities. thiamin deficiency is associated with neurological signs, such as violent convulsions, cartwheel movements, and decreased food consumption. dietary requirements for ascorbic acid have not been shown in mice, and mouse diets are generally not fortified with ascorbic acid. the gulonolactone oxidase knockout mouse (gulo −/− ) on the c57bl/6 background requires vitamin c supplementation although the plasma ascorbate concentration of gulo −/− mice fed a vitamin c-deficient diet is maintained at 15% of wild-type concentrations, suggesting an uncharacterized pathway to generate a small amount of ascorbate (yu and schellhorn, 2013) . the gulo −/− mouse has become the model of choice in studying the role of vitamin c in complex diseases. vitamin c production has been successfully restored in gulo −/− mice using adenovirus vectors, making it possible to robustly manipulate physiological ascorbate concentrations in an inbred mouse. mineral deficiencies have been described only for several elements, and the consequences of the deficiencies are similar to those observed for other species. for example, iodine-deficient diets produce thyroid goiters; magnesium-deficient diets may cause fatal convulsions; manganese deficiency may cause congenital ataxia from abnormal development of the inner ear; and zinc deficiency may cause hair loss on the shoulders and neck, emaciation, decreased liver and kidney catalase activity, and immunosuppression. chronic essential fatty acid deficiency may cause hair loss, dermatitis with scaling and crusting of the skin, and occasional diarrhea. infertility has also been associated with this syndrome. mice have an absolute requirement for a dietary source of linoleic and/or arachidonic acid. (sundberg, 1994; ward, 2000) the significant syndrome of ulcerative dermatitis (ud) is a common idiopathic skin lesion that causes morbidity and early euthanasia losses in c57bl/6 and related lines of mice. significant pruritus leads to skin trauma associated with opportunistic bacterial infection and deep dermal ulcerations. initial signs include alopecia and papular dermatitis, which usually occur over the dorsal trunk (fig. 3.59) . progressive inflammation can be halted, sometimes reversed, by nail trimming and therapy with a wide spectrum of topical or systemic antibiotics, steroids, and other drugs such as vitamin e and aloe, all of which speak to the frustrating search for a primary etiology. treatment should be based on microbiological culture and sensitivity and screening for ectoparasites as hypersensitivity to acariasis has been proposed. seasonal fluctuation in the incidence of disease suggests that environmental factors may play a role. the incidence appears to increase during periods of significant seasonal changes in temperature and humidity, i.e., the onset of winter and early spring. there is some evidence that incidence is related to dietary fat with mice on high fat or ad libitum diets being more susceptible than those on restricted diets (neuhaus et al., 2012) . ileus associated with high mortality has been reported to occur in primiparous female mice during the second week of lactation (kunstyr, 1986) . this disorder has been described as acute intestinal pseudo-obstruction (ipo) in c57bl/6 mice free of known pathogens (feinstein et al., 2008) . lactating mice are either found dead or becoming moribund. segments of the small intestine become distended with fluid contents and histologically there is apoptosis of the villus epithelium of the small intestine and superficial epithelial cells of the large intestine. the enteric nervous system appears morphologically normal but necrotic enterocytes, mucosal erosions, and acute mucosal inflammation are commonly observed. there is no strong evidence for metabolic issues such as hypocalcemia or low blood glucose. the direct cause is unknown but death probably results from sepsis secondary to loss of barrier function reflected in apoptosis of the gut epithelium during peak lactation. environmental variables can affect responses of mice in experimental situations. changes in respiratory epithelial physiology and function from elevated levels of ammonia, effects of temperature and humidity on metabolism, effects of light on eye lesions and retinal function, and effects of noise on neurophysiology are examples of complications that can vary with the form of insult and the strain of mouse employed. mice do not easily acclimatize to sudden and dramatic changes in temperature. therefore, they are susceptible to both hypothermia and hyperthermia. mice also are susceptible to dehydration. poorly functioning automatic watering system valves or water bottles, resulting in spills (hypothermia) or obstructed sipper tubes (dehydration), are a significant cause of husbandry-related morbidity. shipping mice between facilities, irrespective of distance, warrants institutional guidelines to minimize exposure to temperature extremes. reheat coils should be designed to fail in the closed position to avoid overheating holding rooms. ringtail is a condition associated with low relative humidity. clinical signs include annular constriction of the tail and occasionally of the feet or digits, resulting in localized edema that can progress to dry gangrene ( fig. 3 .60). it should be differentiated from dryness and gangrene that may occur in hairless mice exposed to low temperatures and perhaps other environmental or nutritional imbalances. necrosis of legs, feet, or digits also can occur in suckling mice because of disruption of circulation by wraps of stringy nesting material such as cotton wool. corneal opacities can result from acute or chronic keratitis, injury (unilateral) and developmental defects; the latter may occur in combination with inherited microphthalmia in c57 black mice (koch and gowen, 1939) . there is some evidence that the buildup of ammonia in mouse cages may contribute to inflammatory keratitis, because it can be controlled by increasing the frequency of cage cleaning. corneal opacities and anterior polar cataracts are a developmental defect in inbred c57 black mice (pierro and spiggle, 1967) . corneal opacity may be associated with keratolenticular adhesions involving a persistent epithelial stalk of the lens vesicle, which normally disappears around day 11 of gestation (koch and gowen, 1939) . typically noted in runted or cachectic mice soon after weaning, malocclusion of the open-rooted, continually growing incisor teeth is an inherited trait expressed as poorly aligned incisors, especially of the lower incisors causing osteomyelitis, soft tissue abscesses, or necrosis in the lips or oral cavity. the incidence of inherited malocclusion varies with mouse strain (petznek et al., 2002) . malocclusion in older mice may be the result of trauma or oral neoplasia. overgrown molar teeth have been associated with trauma to developing tooth buds. skin lesions can be caused by fighting, tail biting, and overgrooming such as whisker chewing. barbering of facial hair and whiskers in subordinate mice by a dominant cagemate is common and may be solved by removing the dominant, normally haired mouse. hair or whisker chewing (barbering) has long been interpreted to be a manifestation of social dominance. apparent dominant animals retain whiskers, whereas cagemates have 'shaved faces' (fig. 3.61 ). chronic hair chewing can produce histological abnormalities such as poorly formed or pigmented club hairs. once chewing has ceased, many mice regrow previously lost hair in several weeks. both sexes may engage in this activity, and sometimes females may be dominant. barbering of whiskers and fur-plucking behavior in mice has been suggested to model human trichotillomania (compulsive hair plucking) because of similarities including elevated serotonin levels (dufour et al., 2010) , 'barbers' predominately pluck hair from the scalp and around the eyes and the genitals; the behavior is female biased, and begins during puberty and is impacted by genetic background (garner et al., 2004) . fighting is more common in male mice and more aggressive in some strains (sjl, fvb, balb/c) with bite wounds typically located on the head, neck, shoulders, perineal area, and tail. often one mouse in the cage is free of lesions and is the likely aggressor. removal of the unaffected male may end the fighting or simply reorder the dominance order. removing males for breeding and then regrouping them often results in fighting. for programs that produce sentinel mice in-house, castration is an option to reduce aggression in group-housed male sentinels (lofgren et al., 2012) . regional alopecia, especially around the muzzle, may result from abrasion against cage surfaces. improperly diluted disinfectants may also cause regional hair loss. ear tags used for identification may cause pruritis and self-induced trauma. hair removal products or clipping prior to imaging or application of experimental compounds to the skin may cause pruritus and can augment lesions that interfere with test results. dermatophytosis, ectoparasitism, or idiopathic hair loss must be considered in the differential diagnoses for muzzle or body alopecia. (burek et al., 1982; percy and barthold, 2007) common idiopathic lesions in aging mice include cardiomyopathy (with or without mineralization or arteritis), chronic nephropathy (frequently with mineralization), myelofibrosis (fibrotic change in the bone marrow) especially in female mice, melanosis in the meninges, ovarian atrophy (with or without hyaline material), pigment (ceroid-lipofuscin), tubular or stromal hyperplasia, cystic endometrial hyperplasia, testicular tubular degeneration or mineralization, prostate atypical epithelial hyperplasia, gastric glandular epithelial hyperplasia, pancreatic islet cell hyperplasia, dental dysplasia of incisor teeth, pituitary hyperplasia of pars intermedia and pars distalis, cataracts, increased extramedullary hematopoiesis in spleen, and lymphocytic infiltrates or other inflammatory changes in various tissues, including harderian gland, salivary gland, kidney, liver, gall bladder, nasal, trachea, thyroid, periovarian fat, epididymis, and urinary bladder. lymphoma is also very common . spontaneous atrial thrombosis is rare in mice (<1% in 2-year-old mice) and appears to be strain-related, with a high prevalence in rfm mice. it also is more common in aged mice affected by kidney disease and amyloidosis. organizing thrombi will be found usually in an enlarged, hyperemic left atrium and auricle and may be accompanied by amyloidosis. affected mice may display signs of heart failure, particularly severe dyspnea. induction of atrial thrombosis in b6c3f1 mice has been used to assess cardiovascular risk of chemical exposures (yoshizawa et al., 2005) . myocardial and epicardial mineralization is described above (section iii,b,2). periarteritis, also known as arteritis, polyarteritis, or systemic arteritis, impacts older mice and lesions may be observed in multiple tissues, including the spleen, heart, tongue, uterus, testes, kidney, and urinary bladder. the media of the affected vessels is homogenous and intensely eosinophilic with hematoxylin and eosin stain. fibrosis and mononuclear cells infiltrate the vessel wall. experimental coronary arteritis with cardiac hypertrophy has been model in dba/2 and other strains by intraperitoneal administration of mannoprotein-beta-glucan complex isolated from c. albicans (nagi-miura et al., 2006) . hyperplasia of alveolar or bronchial epithelium occurs in old mice and must be differentiated from pulmonary tumors. pulmonary histiocytosis, acidophilic macrophage pneumonia, and acidophilic crystalline pneumonia are synonymous morphologic descriptions of an idiopathic lung lesion that can be incidental or the cause of significant morbidity. incidence varies with mouse strain or stock, with c57bl, 129s4/svjae and swiss mice and older mice in general particularly susceptible. histologically, alveoli and bronchioles are filled with varying quantities of macrophages containing eosinophilic crystalline material . the crystalline material consists of ym1 and/or ym2 chitanases and can be found in other tissues including the upper respiratory tract, stomach, gall bladder, and bone marrow where it is described as hyalinosis (nio et al., 2004) . gastric lesions include crypt dilatation, submucosal fibrosis, adenomatous gastric hyperplasia, mineralization, and erosion or ulceration. gastric ulcers have been induced by cold stress, food restriction (rehm et al., 1987) , chemical injury (yadav et al., 2013) , and gastritis and gastric tumors by helicobacter infection (fox et al., 2003) . germfree mice have reduced muscle tone in the intestinal tract. cecal volvulus is a common cause of death in germfree mice and is caused by rotation of the large, thin-walled cecum. age-associated lesions are common in the livers of mice. cellular and nuclear pleomorphism, including binucleated and multinucleated cells, are detectable by 6 months. mild focal necrosis occurs with or without inflammation, but an association of mild focal hepatitis with a specific infectious disease is often hard to confirm. other geriatric hepatic lesions include biliary hyperplasia with varying degrees of portal hepatitis, hepatocellular vacuolization, amyloid deposition (especially in periportal areas), strangulated or herniated lobes, hemosiderosis, lipofuscinosis, and fibrosis. extramedullary hematopoiesis occurs in young mice and in response to anemia. exocrine pancreatic insufficiency has been reported in cba/j mice. acinar cell atrophy is common but is strain-and sex-dependent. blood-filled mesenteric lymph nodes may occur in aged mice, especially c3h mice. this condition is an incidental finding and should not be confused with infectious lymphadenopathy such as that associated with salmonellosis. aggregates, or nodules of mononuclear cells, are found in many tissues of aged mice, including the salivary gland, thymus, ovary, uterus, mesentery and mediastinum, urinary bladder, and gastrointestinal tract. these nodules should not be mistaken for lymphosarcomas. grossly observable black pigmentation in the spleen of c57bl/6 is normal and is melanosis caused by melanin deposition (weissman, 1967) . the spleen is subject to amyloidosis and hemosiderin deposition. lipofuscin deposition is common, especially in older mice. the thymus undergoes age-associated atrophy. a variety of genetic immunodeficiencies have been described in mice, many of which increase susceptibility to infectious diseases. perhaps the most widely known of these is the athymic nude mouse that lacks a significant hair coat and, more importantly, fails to develop a thymus and thus has a severe deficit of t-cellmediated immune function. additionally, scid mice, which lack both t and b lymphocytes, are used widely and are highly susceptible to opportunistic agents such as pneumocystis murina. specific immune deficits have become excellent models for studying the ontogeny and mechanisms of immune responsiveness (table 3 .12). age-associated osteoporosis or senile osteodystrophy can occur in some mice. it is not associated with severe renal disease or parathyroid hyperplasia. nearly all strains of mice develop some form of osteoarthrosis. it is generally noninflammatory, affects articulating surfaces, and results in secondary bone degeneration. glomerulonephritis is a common kidney lesion of mice. it is more often associated with persistent viral infections or immune disorders rather than with bacterial infections. its prevalence in some strains approaches 100%. nzb and nzb × nzw f 1 hybrid mice, e.g., develop immune complex glomerulonephritis as an autoimmune disease resembling human lupus erythematosus, whereas glomerular disease is relatively mild in nzb mice (nzb mice have a high incidence of autoimmune hemolytic anemia). renal changes occur as early as 4 months of age, but clinical signs and severe disease are not present until 6-9 months. the disease is associated with wasting and proteinuria, and lesions progress until death intervenes. histologically, glomeruli have proteinaceous deposits in the capillaries and mesangium. later, tubular atrophy and proteinaceous casts occur throughout the kidney. immunofluorescence studies show deposits of immunoglobulin and the third component of complement, which lodge as immune complexes with nuclear antigens and antigens of murine leukemia virus in glomerular capillary loops. mice infected with lcmv or with retroviruses can also develop immune complex glomerulonephritis. mice also can develop chronic glomerulopathy characterized by progressive thickening of glomerular basement membrane by pas-positive material that does not stain for amyloid. this lesion can be accompanied by proliferation of mesangial cells; local, regional, or diffuse mononuclear cell infiltration; and fibrosis. advanced cases may lead to renal insufficiency or failure. interstitial nephritis can be caused by bacterial or viral infections but may also be idiopathic. typical lesions include focal, regional, or diffuse interstitial infiltration of tubular parenchyma by mononuclear cells, but glomerular regions also may be involved. severe lesions can be accompanied by fibrosis, distortion of renal parenchyma, and intratubular casts, but not by mineralization. if renal insufficiency or failure ensues, it can lead to ascites. some strains of mice, such as balb/c, can develop polycystic kidney disease, which, if severe, can compromise normal renal function. urinary tract obstruction occurs as an acute or chronic condition in male mice. clinical signs usually include wetting of the perineum from incontinence. in severe or chronic cases, wetting predisposes to cellulitis and ulceration. at necropsy, the bladder is distended, and proteinaceous plugs are often found in the neck of the bladder and proximal urethra. in chronic cases the urine may be cloudy, and calculi may develop in the bladder. additionally, cystitis, urethritis, prostatitis, laboratory animal medicine balanoposthitis, and hydronephrosis may develop. this condition must be differentiated from infectious cystitis or pyelonephritis and from the agonal release of secretions from accessory sex glands, which is not associated with an inflammatory response. hydronephrosis also may occur without urinary tract obstruction. ascending pyelitis occurs in mice secondary to urinary tract infection. parvovarian cysts are observed frequently and may be related to the fact that mouse ovaries are enclosed in membranous pouches. amyloidosis is also common in the ovaries of old mice. cystic endometrial hyperplasia may develop unilaterally or bilaterally and may be segmental. in some strains, the prevalence in mice older than 18 months is 100%. endometrial hyperplasia is often associated with ovarian atrophy. mucometra is relatively common in adult female mice. the primary clinical sign is abdominal distension resembling pregnancy among mice that do not whelp. testicular atrophy, sperm granulomas, and tubular mineralization occur with varying incidence. preputial glands, especially of immunodeficient mice, can become infected with opportunistic or pathogenic bacteria. spontaneous lesions in prostate, coagulating gland (anterior prostatic lobe), seminal vesicles, and ampullary glands were described in control b6c3f1 mice from 12 national toxicology program 2-year carcinogenicity and toxicity studies conducted in one of four different laboratories (suwa et al., 2002) . lymphocytic infiltration, inflammation, edema, epithelial hyperplasia, mucinous cyst, mucinous metaplasia, adenoma, adenocarcinoma, granular cell tumor, and glandular atrophy were variously observed in accessory sex glands. accessory adrenal cortical nodules are found in periadrenal and perirenal fat, especially in females. these nodules have little functional significance other than their potential effect on failures of surgical adrenalectomy. lipofuscinosis, subcapsular spindle cell hyperplasia, and cystic dilatation of cortical sinusoids are found in the adrenal cortices of aged mice. some inbred strains have deficiencies of thyrotropic hormone, resulting in thyroid atrophy. thyroid cysts lined by stratified squamous epithelium and generally of ultimo-branchial origin may be seen in old mice. amyloid can be deposited in the thyroid and parathyroid glands as well as in the adrenal glands. spontaneous diabetes mellitus occurs in outbred swiss mice and genetic variants of several strains such as nod mice (lemke et al., 2008) . high levels of estrogen in pregnancy may influence postpartum hair shedding. various endocrine effects on hair growth have also been described. abdominal and thoracic alopecia have been reported in b6c3f 1 mice. symmetrical mineral deposits commonly occur in the thalamus of aged mice. they may also be found in the midbrain, cerebellum, and cerebrum and are particularly common in a/j mice. lipofuscin accumulates in the neurons of old mice. age-associated peripheral neuropathy with demyelination can be found in the nerves of the hindlimbs in c57bl/6 mice. deposits of melanin pigment occur in heavily pigmented strains, especially in the frontal lobe. a number of neurologically mutant mice have been described. they commonly have correlative anatomical malformations or inborn errors of metabolism. a seizure syndrome in fvb mice has been described (goelz et al., 1998) and can be spontaneous or associated with tail tattooing, fur clipping, and fire alarms. mice are most often female with a mean age of 5.8 months (range, 2-16 months) and can exhibit facial grimace, chewing automatism, ptyalism with matting of the fur of the ventral aspect of the neck and/or forelimbs, and clonic convulsions that may progress to tonic convulsions and death. ischemic neuronal necrosis was consistently observed in these mice and is consistent with status epilepticus in humans. unilateral and bilateral microphthalmia and anophthalmia are frequent (as high as 12%) developmental defects in inbred and congenic strains of c57bl mice, especially impacting the right eye and female mice. these conditions may first be recognized due to ocular infections, secondary to inadequate tear drainage. other common findings include central corneal opacities, iridocorneal and corneal-lenticular adhesions, abnormal formation of the iris and ciliary body, cataracts, extrusion of lens cortical material with dispersion throughout the eye, failure of vitreous development, and retinal folding. these syndromes can be reproduced by exposure to alcohol at critical stages of embryogenesis when the optic cup and lens vesicle are developing and impacting normal development of other ocular structures, including the iris, ciliary body, vitreous, and retina . retinal degeneration can occur as either an environmental or a genetic disorder (chang et al., 2007) in mice. nonpigmented mice, both inbred and outbred, can develop retinal degeneration from exposure to light, with the progression of blindness being related to light intensity and duration of exposure. mouse genetics have laboratory animal medicine been shown to be more important than potential light associated tissue injury (serfilippi et al., 2004a) . other strains such as c3h, cba, and fvb are genetically predisposed to retinal degeneration because they carry the rd gene, which leads to retinal degeneration within the first few weeks of life and has been used extensively as a model for retinitis pigmentosa (farber and danciger, 1994) . presence of the rd gene in some mouse strains highlights that impaired vision must be a consideration when selecting strains for behavioral assays that rely on visual clues (garcia et al., 2004) . blindness does not interfere with health or reproduction and blind mice cannot be distinguished from non-blind mice housed in standard caging. cataracts can occur in old mice and have a higher prevalence in certain mutant strains. vestibular syndrome associated with head tilt, circling, or imbalance can result from infectious otitis or from necrotizing vasculitis of unknown etiology affecting small and medium-sized arteries in the vicinity of the middle and inner ears. (jones et al., 1983 (jones et al., -1993 maronpot et al., 1999; percy and barthold, 2007) neoplasms of lymphoid and hematopoietic tissues are estimated to have a spontaneous prevalence of 1-2%. there are, however, some strains of mice that have been specifically inbred and selected for susceptibility to spontaneous tumors. leukemogenesis in mice may involve viruses and chemical or physical agents. viruses associated with lymphopoietic and hematopoietic neoplasia belong to the family retroviridae (type c oncornaviruses) and contain rna-dependent dna polymerase (reverse transcriptase). these viruses are generally noncytopathogenic for infected cells, and mice appear to harbor them as normal components of their genome. although they may be involved in spontaneous leukemia, they are not consistently expressed in this disease. recombinant viruses have recently been discovered that can infect mouse cells and heterologous cells and are associated with spontaneous leukemia development in high leukemia strains such as akr mice. their phenotypic expression is controlled by mouse genotype. endogenous retroviruses are transmitted vertically through the germ line. horizontal transmission is inefficient but can occur by intrauterine infection or through saliva, sputum, urine, feces, or milk. the leukemia induced by a given endogenous virus is usually of a single histopathological type. loss of function in nucleic acid-recognizing, tlr3, tlr7, and tlr9 can result in spontaneous retroviral viremia and acute t-cell lymphoblastic leukemia (yu et al., 2012) . chemical carcinogens, such as polycyclic hydrocarbons, nitrosoureas, and nitrosamines, and physical agents such as x-irradiation can also induce hematological malignancies in mice. the most common hematopoietic malignancy in the mouse is lymphocytic leukemia that originates in the thymus. disease begins with unilateral atrophy and then enlargement of one lobe of thymus as tumor cells proliferate. cells can spread to the other lobe and then to other hematopoietic organs, such as the spleen, bone marrow, liver, and peripheral lymph nodes. clinical signs include dyspnea and ocular protrusion. the latter sign is due to compression of venous blood returning from the head. tumor cells spill into the circulation late in disease. most of these tumors originate from t lymphocytes or lymphoblasts, but there are leukemias of b-lymphocyte or null cell lineage. in the last two syndromes, the lymph nodes and spleen are often involved, but the thymus is generally normal. reticulum cell sarcomas are common in older mice, especially in inbred strains such as c57bl/6 and sjl. primary tumor cell types have been divided into several categories based on morphological and immunohistochemical features. histiocytic sarcomas correspond to the older dunn classification as type a sarcomas and are composed primarily of reticulum cells. the tumor typically causes splenomegaly and nodular lesions in other organs, including the liver, lung, kidney, and the female reproductive tract. follicular center cell lymphomas correspond to dunn type b sarcomas. they originate from b-cell regions (germinal centers) of peripheral lymphoid tissues, including the spleen, lymph nodes, and peyer's patches. typical tumor cells have large vesiculated, folded, or cleaved nuclei and ill-defined cytoplasmic borders. tumors also often contain small lymphocytes. type c reticulum cell tumors often involve one or several lymph nodes rather than assuming a wide distribution. they consist of reticulum cells with a prominent component of well-differentiated lymphocytes. myelogenous leukemia is uncommon in mice and is associated with retrovirus infection. disease begins in the spleen, resulting in marked splenomegaly, but leukemic spread results in involvement of many tissues including the liver, lung, and bone marrow. leukemic cells in various stages of differentiation can be found in peripheral blood. in older animals, affected organs may appear green because of myeloperoxidase activity, giving rise to the term chloroleukemia. the green hue fades on contact with air. affected mice are often clinically anemic and dyspneic. erythroleukemia is rare in mice. the major lesion is massive splenomegaly, which is accompanied by anemia and polycythemia. hepatomegaly can follow, but there is little change in the thymus or lymph nodes. erythroleukemia can be experimentally induced in mice by friend spleen focus-forming virus (sffv) which initially activates the erythropoietin (epo) receptor and the receptor tyrosine kinase sf-stk in erythroid cells, resulting in proliferation, differentiation, and survival. in a second stage, sffv activates the myeloid transcription factor pu.1, blocking erythroid cell differentiation, and in conjunction with the loss of p53 tumor suppressor activity, results in the outgrowth of malignant cells (cmarik and ruscetti, 2010 ). mast cell tumors are also very rare in mice. they are found almost exclusively in old mice and grow slowly. they should not be confused with mast cell hyperplasia observed in the skin following painting with carcinogens or x-irradiation. natural plasma cell tumors are infrequent in the mouse. they can, however, be induced by intraperitoneal inoculation of granulomatogenic agents such as plastic filters, plastic shavings, or a variety of oils, particularly in balb/c mice. mineral oil-induced plasmacytomas in balb/c mice produce large amounts of endogenous retroelements such as ecotropic and polytropic murine leukemia virus and intracisternal a particles. associated inflammation may promote retroelement insertion into cancer genes, thereby promoting tumors (knittel et al., 2014) . similar to other spontaneous cancers, plasmacytoma development in mice is inhibited by innate immune responses of nk cells which when activated by viruses will release γinf (thirion et al., 2014) . mammary tumors can be induced or modulated by a variety of factors, including viruses, chemical carcinogens, radiation, hormones, genetic background, diet, and immune status. certain inbred strains of mice, such as c3h, a, and dba/2, have a high natural prevalence of mammary tumors. other strains, such as balb/c, c57bl, and akr, have a low prevalence. among the most important factors contributing to the development of mammary tumors are mammary tumor viruses. several major variants are known. the primary tumor virus mmtv-s (bittner virus) is highly oncogenic and is transmitted through the milk of nursing females. infected mice typically develop a precursor lesion, the hyperplastic alveolar nodule, which can be serially transplanted. spontaneous mammary tumors metastasize with high frequency, but this property is somewhat mouse strain dependent. metastases go primarily to the lung. some mammary tumors are hormone dependent, some are ovary dependent, and others are pregnancy dependent. ovary-dependent tumors contain estrogen and progesterone receptors, whereas pregnancy-dependent tumors have prolactin receptors. ovariectomy will dramatically reduce the incidence of mammary tumors in c3h mice. if surgery is done in adult mice 2-5 months of age, mammary tumors will develop, but at a later age than normal. grossly, mammary tumors may occur anywhere in the mammary chain. they present as one or more firm, welldelineated masses, which are often lobular and maybe cystic (fig. 3.62) . histologically, mammary tumors have been categorized into three major groups: carcinomas, carcinomas with squamous cell differentiation, and carcinosarcomas. the carcinomas are divided into adenocarcinoma types a, b, c, y, l, and p. most tumors are type a or b. type a consists of adenomas, tubular carcinomas, and alveolar carcinomas. type b tumors have a variable pattern with both well-differentiated and poorly differentiated regions. they may consist of regular cords or sheets of cells or papillomatous areas. these two types are locally invasive and may metastasize to the lungs. type c tumors are rare and are characterized by multiple cysts lined by low cuboidal to squamous epithelial cells, and they have abundant stroma. type y tumors, which are also rare, are characterized by tubular branching of cuboidal epithelium and abundant stroma. adenocarcinomas with a lacelike morphology (types l and p) are hormone dependent and have a branching tubular structure. the control or prevention of mammary neoplasms depends on the fact that some strains of mammary tumor virus are transmitted horizontally, whereas others are transmitted vertically. although horizontally transmitted virus such as mmtv-s can be determined by cesarean rederivation or by foster nursing, endogenous strains of tumor virus may remain. fortunately, these latter tumor viruses have generally low oncogenicity relative to the bittner virus. mammary tumors are increased in frequency in c57bl apc +/− female mice infected with h. hepaticus (rao et al., 2007) . mice develop an assortment of liver changes as they age, including proliferative lesions which can progress from hyperplastic foci to hepatomas to hepatocellular carcinomas. almost all strains of mice have a significant prevalence of hepatic tumors, some of which appear to result from dietary contamination or deficiency and h. hepaticus infections in susceptible strains of mice such as the a/jcr male mouse ward et al., 1994) . the prevalence of spontaneous liver tumors in b6c3f 1 hybrids is increased by feeding choline-deficient diets or when infected with h. hepaticus (hailey et al., 1998) . tumors also can develop in mice exposed to environmental chemicals, many of which are carcinogenic or potentially carcinogenic (hoenerhoff et al., 2009) . spontaneous liver tumors in mice occur grossly as gray to tan nodules or large, poorly demarcated darkred masses. they are usually derived from hepatocytes, whereas cholangiocellular tumors are rare. hepatomas are well circumscribed and well differentiated, but they compress adjacent liver tissue as they develop. hepatocellular carcinomas are usually invasive and display histopathological patterns ranging from medullary to trabecular. large carcinomas also may contain hemorrhage and necrosis. carcinomas also may metastasize to the lungs. primary respiratory tumors of mice occur in relatively high frequency. it has been estimated that more than 95% of these tumors are pulmonary adenomas that arise either from type 2 pneumocytes or from clara cells lining terminal bronchioles. pulmonary adenomas usually appear as distinct whitish nodules that are easily detected by examination of the lung surface. malignant alveologenic tumors are infrequent and consist of adenocarcinomas and squamous cell carcinomas. they invade pulmonary parenchyma and are prone to metastasize. the prevalence of spontaneous respiratory tumors is mouse straindependent. for example, the prevalence is high in aging a strain mice but low in aging c57bl mice. the number of tumors per lung is also higher in susceptible mice. pulmonary tumors often occur as well-defined gray nodules. microscopically, adenomas of alveolar origin consist of dense ribbons of cuboidal to columnar cells with sparse stroma. adenomas of clara cell origin are usually associated with bronchioles. they have a tubular to papillary architecture consisting of columnar cells with basal nuclei. pulmonary adenocarcinomas, though comparatively rare, are locally invasive. they often form papillary structures and have considerable cellular pleomorphism. given the rapid development of mouse strains genetically predisposed to neoplasia, the mouse tumor biology database maintained by jackson laboratory is a valuable centralized resource for the most current tumor descriptions. the database contains information on more than 4400 strains and substrains, 348 tissues and organs, over 42,000 tumor frequency records, and nearly 4800 histopathological images and descriptions. risk factors for recurrence, complications 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host genotype an exteroceptive block to pregnancy in the mouse comparison of polymerase chain reaction and immunohistochemistry for the detection of mycoplasma pulmonis in paraffin-embedded tissue chromosomal localization of the loci responsible for dystrophic cardiac calcinosis in dba/2 mice evaluation of an enzymelinked immunosorbent assay for the detection of ectromelia (mousepox) antibody mousepox selected non-neoplastic diseases helicobacter-induced inflammatory bowel disease in il-10-and t cell-deficient mice strategies to prevent, treat, and provoke corynebacteriumassociated hyperkeratosis in athymic nude mice corynebacterium bovis: epizootiologic features and environmental contamination in an enzootically infected rodent room inflammatory bowel disease: an immunity-mediated condition triggered by bacterial infection with helicobacter hepaticus poliomyelitis in mulv-infected icr-scid mice after injection of basement membrane matrix contaminated with lactate 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alphabeta mutant mice experimental infection of mice with hamster parvovirus: evidence for interspecies transmission of mouse parvovirus 3 the diversity outbred mouse population an experience with clostridium perfringens in cesarean derived barrier sustained mice hyperkeratosis in athymic nude mice caused by a coryneform bacterium: microbiology, transmission, clinical signs, and pathology friend spleen focus-forming virus activates the tyrosine kinase sf-stk and the transcription factor pu.1 to cause a multi-stage erythroleukemia in mice colonisation and shedding of lawsonia intracellularis in experimentally inoculated rodents and in wild rodents on pig farms a mouse for all reasons citrobacter rodentium: infection, inflammation and the microbiota prevention of murine norovirus infection in neonatal mice by fostering transmission of mouse parvovirus by fomites anatomy lactate dehydrogenase-elevating virus behavioral phenotyping strategies for mutant mice genetic heterogeneity of rat-derived pneumocystis serum antibody responses by male and female c57bl/6 mice infected with giardia muris comparison of the course of infection with giardia muris in male and female mice cytotoxic and pathogenic properties of klebsiella oxytoca isolated from laboratory animals the hidden cost of housing practices: using noninvasive imaging to quantify the metabolic demands of chronic cold stress of laboratory mice the role of klebsiella oxytoca in utero-ovarian infection of b6c3f1 mice behavioral effects of ivermectin in mice host-pathogen interaction in invasive salmonellosis sur les rapports des kystes de carini du poumon des rats avec le trypanosoma lewisi eradication of helicobacter spp. by using medicated diet in mice deficient in functional natural killer cells and complement factor d recombinat congenic strains -a new tool for analyzing genetic traits by more than one gene comparison between patterns of pinworm infection (aspiculuris tetraptera) in wild and laboratory strains of mice, mus musculus laboratory rat associated outbreak of haemorrhagic fever with renal syndrome due to hantaan-like virus in belgium animal leptospirosis in small tropical areas phylogeny of the defined murine microbiota: altered schaedler flora clostridium difficile infection in horses: a review mousepox outbreak in a laboratory mouse colony isolation of a puumala-like virus from mus musculus captured in yugoslavia and its association with severe hemorrhagic fever with renal syndrome assessment of rpob and 16s rrna genes as targets for pcrbased identification of pasteurella pneumotropica comparison of traditional and pcr methods during screening for and confirmation of aspiculuris tetraptera in a mouse facility pathogenicity and genetic variation of 3 strains of corynebacterium bovis in immunodeficient mice use of fenbendazole-containing therapeutic diets for mice in experimental cancer therapy studies nutritional up-regulation of serotonin paradoxically induces compulsive behavior the pneumonia virus of mice (mpnv) model of acute respiratory infection comparison of nine commercially available clostridium difficile toxin detection assays, a real-time pcr assay for c. difficile tcdb, and a glutamate dehydrogenase detection assay to cytotoxin testing and cytotoxigenic culture methods dystrophic cardiac calcinosis in mice: genetic, hormonal, and dietary influences isolation and expression of the pneumocystis carinii dihydrofolate reductase gene the mouse genome database (mgd): comprehensive resource for genetics and genomics of the laboratory mouse typhlocolitis in nf-kappa b-deficient mice cd4+ cd25+ regulatory t lymphocytes inhibit microbially induced colon cancer in rag2-deficient mice cd4(+)cd25(+) regulatory lymphocytes require interleukin 10 to interrupt colon carcinogenesis in mice nitric oxide and tnf-alpha trigger colonic inflammation and carcinogenesis in helicobacter hepaticusinfected, rag2-deficient mice ectromelia virus: the causative agent of mousepox intranasal inoculation of mycoplasma pulmonis in mice with severe combined immunodeficiency (scid) causes a wasting disease with grave arthritis the systemic amyloidoses: an overview inherited retinal degenerations in the mouse molecular detection of novel picornaviruses in chickens and turkeys fatal acute intestinal pseudoobstruction in mice the epizootic behaviour of mouse-pox (infectious ectromelia) the pathogenesis of acute exanthema. an interpretation based on experimental investigations with mouse-pox (infectious ectromelia of mice) studies in mousepox, infectious ectromelia of mice; a comparison of the virulence and infectivity of three strains of ectomelia virus studies in mousepox, infectious ectromelia of mice; the effect of age of the host upon the response to infection a juvenile mouse pheromone inhibits sexual behaviour through the vomeronasal system evidence from mtdna sequences that common laboratory strains of inbred mice are descended from a single female origins and characteristics of inbred strains of mice revised nomenclature for strain 129 mice congenic strains treatment of syphacia obvelata in mice using ivermectin the nude mouse in experimental and clinical research opportunistic bacterial infections in breeding colonies of the nsg mouse strain outbreak of tropical rat mite dermatitis in laboratory personnel the role of helicobacter species in newly recognized gastrointestinal tract diseases of animals helicobacter hepaticus sp. nov., a microaerophilic bacterium isolated from livers and intestinal mucosal scrapings from mice chronic proliferative hepatitis in a/jcr mice associated with persistent helicobacter hepaticus infection: a model of helicobacterinduced carcinogenesis persistent hepatitis and enterocolitis in germfree mice infected with helicobacter hepaticus hepatic helicobacter species identified in bile and gallbladder tissue from chileans with chronic cholecystitis comparison of methods of identifying helicobacter 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polyomavirus in lungs of intranasally infected neonatal mice spontaneous staphylococcus xylosus infection in mice deficient in nadph oxidase and comparison with other laboratory mouse strains genetics and probability in animal breeding experiments the major site of murine k papovavirus persistence and reactivation is the renal tubular epithelium isolation of streptococcus equisimilis from abscesses detected in specific pathogen-free mice ciliaassociated respiratory (car) bacillus infection of obese mice epigenetic and phenotypic changes result from a continuous pre and post natal dietary exposure to phytoestrogens in an experimental population of mice impact of helicobacter hepaticus infection in b6c3f1 mice from twelve national toxicology program two-year carcinogenesis studies pathology of aging b6;129 mice reduplication in mice humoral immunity and protection of mice challenged with homotypic or heterotypic parvovirus line-1 (l1) lineages in the mouse addgene provides an open forum for plasmid sharing quantitative trait loci in a bacterially induced model of inflammatory bowel disease a review of the molecular mechanisms of chemically induced neoplasia in rat and mouse models in national toxicology program bioassays and their relevance to human cancer control of pseudomonas aeruginosa infection in mice by chlorine treatment of drinking water mouse phyisology b cells modulate systemic responses to pneumocystis lung infection and protect on-demand hematopoiesis via t cell-independent, innate mechanism when type-i-ifn-signaling is absent development of a microsphere-based serologic multiplexed fluorescent immunoassay and a reverse transcriptase pcr assay to detect murine norovirus 1 infection in mice persistent infection with and serologic cross-reactivity of three novel murine noroviruses multiplex fluorescent immunoassay for the simultaneous detection of serum antibodies to multiple rodent pathogens development and applications of crispr-cas9 for genome 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activation of th-2 lymphocytes mouse enu mutagenesis effects of enu dosage on mouse strains comparative murine norovirus studies reveal a lack of correlation between intestinal virus titers and enteric pathology tailored immune responses: novel effector helper t cell subsets in protective immunity stat1-dependent innate immunity to a norwalk-like virus eradication of murine norovirus from a mouse barrier facility control of laboratory acquired hemorrhagic fever with renal syndrome (hfrs) in japan growth of tyzzer's organism in primary monolayer cultures of adult mouse hepatocytes the laboratory mouse. its origin, heredity, and culture olfactory regulation of the sexual behavior and reproductive physiology of the laboratory mouse: effects and neural mechanisms review of successful treatment for helicobacter species in laboratory mice on the maximum avoidance of inbreeding an oral ivermectin regimen that eradicates pinworms (syphacia spp.) in laboratory rats and mice nutrition effect of open and closed formula rations on the performance of three strains of laboratory mice insertional hypermutation in mineral oil-induced plasmacytomas chemical and cytokine features of innate immunity characterize serum and tissue profiles in inflammatory bowel disease gross anatomy the manufacture, shipping and receiving and quality control of rodent bedding materials emergence of clostridium difficile-associated disease in north america and europe helicobacter hepaticus-induced colitis in interleukin-10-deficient mice: cytokine requirements for the induction and maintenance of intestinal inflammation bacteria-triggered cd4(+) t regulatory cells suppress helicobacter hepaticus-induced colitis il-23 plays a key role in helicobacter hepaticus-induced t cell-dependent colitis paresis of peristalsis and ileus lead to death in lactating mice experimental spironucleosis (hexamitiasis) in the nude mouse as a model for immunologic and pharmacologic studies host specificity of giardia muris isolates from mouse and golden hamster genetic control of resistance to mycoplasma pulmonis infection in mice therapeutic effect of dna immunization of genetically susceptible mice infected with virulent mycoplasma pulmonis in vitro and in vivo susceptibility of mouse megakaryocytic progenitors to strain i of parvovirus minute virus of mice effects of fenbendazole on the murine humoral immune system from house mouse to mouse house: the behavioral biology of free-living mus musculus and its implications in the laboratory antibiotic treatment of clostridium difficile carrier mice triggers a supershedder state, spore-mediated transmission, and severe disease in immunocompromised hosts use of topical ivermectin treatment for syphacia obvelata in mice hantaan-like viruses from domestic rats captured in the united states obesity and non-insulin-dependent diabetes mellitus in swiss-webster mice associated with late-onset hepatocellular carcinoma mouse adenovirus type 1 infection in scid mice: an experimental model for antiviral therapy of systemic adenovirus infections mineralization/anti-mineralization networks in the skin and vascular connective tissues a second class of olfactory chemosensory receptors in the olfactory epithelium genetic variables that influence phenotype mycoplasma and other bacterial diseases of the respiratory system pasteurella pneumotropica pseudomonas aeruginosa salmonella enteritidis staphylococcus aureus streptobacillus moniliformis streptococcus pneumoniae soiled bedding sentinels for the detection of fur mites in mice false negative results using rt-pcr for detection of lactate dehydrogenase-elevating virus in a tumor cell line eradication of pinworms (syphacia obvelata) from a large mouse breeding colony by combination oral anthelmintic therapy mousepox resulting from use of ectromelia virus-contaminated, imported mouse serum husbandry factors and the prevalence of age-related amyloidosis in mice cardioviruses: encephalomyocarditis virus and theiler's murine encephalitis virus diagnostic testing of mouse and rat colonies for infectious agents a novel presentation of clostridium piliforme infection (tyzzer's disease) in nude mice serodiagnosis of mice minute virus and mouse parvovirus infections in mice by enzyme-linked immunosorbent assay with baculovirus-expressed recombinant vp2 proteins hfrs outbreak associated with laboratory rats in uk the clinical chemistry of laboratory animals lack of commensal flora in helicobacter pylori-infected ins-gas mice reduces gastritis and delays intraepithelial neoplasia castration eliminates conspecific aggression in group-housed cd1 male surveillance mice toll-like receptor 2 (tlr2) plays a major role in innate resistance in the lung against murine mycoplasma clostridium difficile-associated cecitis in guinea pigs exposed to penicillin clearance of pneumocystis carinii in mice is dependent on b cells but not on p-carinii-specific antibody phylogenetic relationships in the genus mus, based on paternally, maternally, and biparentally inherited characteristics genetic variants and strains of the laboratory mouse direct detection of indirect transmission of streptobacillus moniliformis rat bite fever infection fenner's veterinary virology failure to infect laboratory rodent hosts with human isolates of rodentolepis (=hymenolepis) nana detection and control of mouse parvovirus diminished reproduction, failure to thrive, and altered immunologic function in a colony of t-cell receptor transgenic mice: possible role of citrobacter rodentium helicobacter bilis infection accelerates and h. hepaticus infection delays the development of colitis in multiple drug resistance-deficient (mdr1a −/−) mice helicobacter infection is required for inflammation and colon cancer in smad3-deficient mice detection of giardia cysts by using the polymerase chain reaction and distinguishing live from dead cysts knockout mice: a paradigm shift in modern immunology a novel murine infection model for shiga toxin-producing escherichia coli infection-induced colitis in mice causes dynamic and tissue-specific changes in stress response and dna damage leading to colon cancer soiledbedding sentinel detection of murine norovirus 4 infectious ectromelia. a hitherto undescribed virus disease of mice mycoplasma contamination of murine embryonic stem cells affects cell parameters, germline transmission and chimeric progeny clostridium perfringens and clostridium difficile-associated diarrhea pathology of the mouse from sexual attraction to maternal aggression: when pheromones change their behavioural significance an outbreak of pasteurella pneumotropica in genetically modified mice: treatment and elimination spontaneous necrotic enteritis in young rfm/ms mice prevention and treatment of ciliaassociated respiratory bacillus in mice by use of antibiotics a paralytic disease in nude mice associated with polyomavirus infection renewed interest in a difficult disease: clostridium difficile infections -epidemiology and current treatment strategies use of metronidazole in equine acute idiopathic toxaemic colitis identification and propogation of a putative immunosuppressive orphan parvovirus in cloned t cells corynebacterium species-associated keratoconjunctivitis in aged male c57bl/6j mice genetic differences among c57bl/6 substrains isolation of helicobacter spp. from mice with rectal prolapses complex trait analysis in the mouse: the strengths, the limitations, and the promise yet to come fur mites induce dermatitis associated with ige hyperproduction in an inbred strain of mice, nc/kuj origins of inbred mice: proceedings of a workshop building a better mouse: one hundred years of genetics and biology retroelements in the mouse a mammalian herpesvirus cytolytic for cd4+ (l3t4+) t lymphocytes prairie dog model for antimicrobial agent-induced clostridium difficile diarrhea murine norovirus 1 infection is associated with histopathological changes in immunocompetent hosts, but clinical disease is prevented by stat1-dependent interferon responses transmission of systemic aa amyloidosis in animals infection of different strains of mice with lawsonia intracellularis derived from rabbit or porcine proliferative enteropathy the musculus-type y chromosome of the laboratory mouse is of asian origin lethal and severe coronary arteritis in dba/2 mice induced by fungal pathogen, caws, candida albicans water-soluble fraction cre recombinase: the universal reagent for genome tailoring manual of microbiological monitoring of laboratory animals reduced fecundity and death associated with parvovirus infection in b-lymphocyte deficient mice phylogenetic analysis and description of eperythrozoon coccoides, proposal to transfer to the genus mycoplasma as mycoplasma coccoides comb. nov. and request for an opinion experimental analysis of risk factors for ulcerative dermatitis in mice citrobacter rodentium espb is necessary for signal transduction and for infection of laboratory mice colitis and colon cancer in waspdeficient mice require helicobacter species contamination of transplantable tumors, cell lines, and monoclonal antibodies with rodent viruses recommendations for the health monitoring of rodent and rabbit colonies in breeding and experimental units cellular expression of murine ym1 and ym2, chitinase family proteins, as revealed by in situ hybridization and immunohistochemistry oxytocin is required for nursing but is not essential for parturition or reproductive behavior report of the american institute of nutrition ad hoc committee on standards for nutritional studies moving forward with chemical mutagenesis in the mouse implementation of a pcr assay of pasteurella pneumotropica to accurately screen for contaminated laboratory mice natural cryptosporidium muris infection of the stomach in laboratory mice enhancement of cognitive function in models of brain disease through environmental enrichment and physical activity handbook of vertebrate immunology perturbations in cytokine gene expression after inoculation of c57bl/6 mice with pasteurella pneumotropica a transgenic approach for rna interference-based genetic screening in mice mouse pathology of laboratory rodents and rabbits naturally occurring murine norovirus infection in a large research institution from bench to cageside: risk assessment for rodent pathogen contamination of cells and biologics reduced body growth and excessive incisor length in insertional mutants mapping to mouse chromosome 13 congenital eye defects in the mouse. i. corneal opacity in c57black mice murine adenovirus infection of scid mice induces hepatic lesions that resemble human reye syndrome improved lactation in germfree mice following changes in the amino acid and fat components of a chemically defined diet a systematic method of breeder rotation for noninbred laboratory animal colonies genetic variation and phylogeography of central asian and other house mouse mice, including a major new mitochondrial lineage in yemen reproductive biology of the laboratory mouse helminth parasites of laboratory mice contemporary prevalence of infectious agents in laboratory mice and rats clostridium species. veterinary microbiology and microbial disease uteroovarian infection in aged b6c3f1 mice breast cancer: should gastrointestinal bacteria be on our radar screen? detection of pneumocystis carinii in a rat model of infection by polymerase chain reaction spontaneous nonneoplastic gastric lesions in female han:nmri mice, and influence of food restriction throughout life emerging views on the distinct but related roles of the main and accessory olfactory systems in responsiveness to chemosensory signals in mice zfngenome: a comprehensive resource for locating zinc finger nuclease target sites in model organisms treatment and eradication of murine fur mites: iii. treatment of a large mouse colony with ivermectin-compounded feed pheromonal induction of spatial learning in mice mucispirillum schaedleri gen. nov., sp. nov., a spiral-shaped bacterium colonizing the mucus layer of the gastrointestinal tract of laboratory rodents pheromone sensing in mice minor histocompatibility antigens: from the laboratory to the clinic vitamin a and retinoic acid in t cell-related immunity spontaneous pneumocystis carinii pneumonia in immunodeficient mutant scid mice. natural history and pathobiology lethal exacerbation of pneumocystis carinii pneumonia in severe combined immunodeficiency mice after infection by pneumonia virus of mice primary structure and phylogenetic relationships of glyceraldehyde-3-phosphate dehydrogenase genes of free-living and parasitic diplomonad flagellates duodenal adenomas in balb/-c mice monoinfected with clostridium perfringens diffuse scaling dermatitis in an athymic nude mouse clostridium difficile typhlitis associated with cecal mucosal hyperplasia in syrian hamsters zifit (zinc finger targeter): an updated zinc finger engineering tool spatial distribution and stability of the eight microbial species of the altered schaedler flora in the mouse gastrointestinal tract comparison of the in vitro susceptibility of rodent isolates of pseudomonas aeruginosa and pasteurella pneumotropica to enrofloxacin antibody production in syphacia obvelata infected mice hyperkeratosis-associated coryneform infection in severe combined immunodeficient mice gastrointestinal microflora host specificity of cloned spironucleus muris in laboratory rodents the eae gene of citrobacter freundii biotype 4280 is necessary for colonization in transmissible murine colonic hyperplasia genetic and biochemical characterization of citrobacter rodentium sp. nov outbreak of group b streptococcal meningoencephalitis in athymic mice demylination and wasting associated wigh polyomavirus infection in nude (nu/nu) mice severe leukopenia and dysregulated erythropoiesis in scid mice persistently infected with the parvovirus minute virus of mice assessment of retinal degeneration in outbred albino mice assessment of retinal degeneration in outbred albino mice identification of widespread helicobacter hepaticus infection in feces in commercial mouse colonies by culture and pcr assay helicobacter infection decreases reproductive performance of il10-deficient mice pheromone binding by polymorphic mouse major urinary proteins role of housing modalities on management and surveillance strategies for adventitious agents of rodents murine cytomegalovirus and other herpesviruses cytolethal distending toxin promotes helicobacter cinaediassociated typhlocolitis in interleukin-10-deficient mice manual of microbiological monitoring of laboratory animals helicobacter bilis-induced inflammatory bowel disease in scid mice with defined flora helicobacter bilis/helicobacter rodentium co-infection associated with diarrhea in a colony of scid mice genetically determined murine models of immunodeficiency macrophage plasticity and polarization: in vivo veritas atlas of the mouse brain and spinal cord mouse genetics: concepts and applications a natural outbreak of transmissible murine colonic hyperplasia in a/j mice genetic variation among 129 mouse substrains and its importance for targeted mutagenesis in mice genetic dissection of complex traits with chromosome substitution strains of mice a conditional knock out resource for the genome-wide study of mouse gene function ivermectin toxicity in young mice response of weanling random-bred mice to inoculation with minute virus of mice explant cultures for detection of minute virus of mice in infected mouse tissue in vivo studies with an 'orphan' parvovirus microphthalmia and associated abnormalities in inbred black mice mouse adenoviruses innate lymphoid cells -a proposal for uniform nomenclature a new name (pneumocystis jiroveci) for pneumocystis from humans major histocompatibility complex (mhc): mouse. els mouse urinary peptides provide a molecular basis for genotype discrimination by nasal sensory neurons the complete genome sequence of the carcinogenic bacterium helicobacter hepaticus mouse relapse model of clostridium difficile infection dendritic cells are the major antigen presenting cells in inflammatory lesions of murine mycoplasma respiratory disease chronic ulcerative dermatitis in black mice generation of knockout mice using engineered nucleases spontaneous lesions in control b6c3f1 mice and recommended sectioning of male accessory sex organs acute, lethal, natural killer cell-resistant myeloproliferative disease induced by polyomavirus in severe combined immunodeficient mice the ancestor of extant japanese fancy mice contributed to the mosaic genomes of classical inbred strains pattern recognition receptors and inflammation aggregation chimeras: combining es cells, diploid and tetraploid embryos analysis of the immune response of hantaan virus nucleocapsid protein-specific cd8+ t cells in mice the pathogenesis of experimental infections of cryptosporidium muris 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analysis of transgene integration nutrition toxicity evaluation of prophylactic treatments for mites and pinworms in mice serological and virological evidence of hantaan virus-related enzootic in the united states korean hemorrhagic fever in staff in an animal laboratory prolonged perturbations of tumour necrosis factoralpha and interferon-gamma in mice inoculated with clostridium piliforme acceleration and inhibition of puberty in female mice by pheromones spontaneous pseudopregnancy in mice male management: coping with aggression problems in male laboratory mice phylogeny of trichomonads based on partial sequences of large subunit rrna and on cladistic analysis of morphological data il-7 deficiency prevents development of a non-t cell non-b cell-mediated colitis provitamin a metabolism and functions in mammalian biology genetic variation in laboratory mice the mosaic structure of variation in the laboratory mouse genome a study of mouse strains susceptibility to bacillus piliformis 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tract of 129s4/svjae and wild-type and cyp1a2-null b6, 129 mice pathology of immunodeficient mice with naturally occurring murine norovirus infection reoviridae protozoa initial sequencing and comparative analysis of the mouse genome new building, old parasite: mesostigmatid mites -an ever-present threat to barrier facilities genetic diversity of pneumocystis carinii derived from infected rats, mice, ferrets, and cell cultures spontaneous wasting disease in nude mice associated with pneumocystis carinii infection respiratory disease and wasting in athymic mice infected with pneumonia virus of mice arthropods manual of microbiological monitoring of laboratory animals genetic and histochemical studies on mouse spleen black spots status and acces to the collaborative cross population helminths natural and experimental helicobacter infections monitoring sentinel mice for helicobacter hepaticus, h rodentium, and h bilis infection by use of polymerase chain reaction analysis and serologic testing rapid onset of ulcerative typhlocolitis in b6.129p2-il10tm1cgn (il-10−/−) mice infected with helicobacter trogontum is associated with decreased colonization by altered schaedler's flora estrus-inducing pheromone of male mice. transport by movement of air endonucleases: new tools to edit the mouse genome replication of a norovirus in cell culture reveals a tropism for dendritic cells and macrophages murine norovirus: a model system to study norovirus biology and pathogenesis addgene: the bank that gives points for (plasmid) deposits microbiological contamination of laboratory mice and rats in korea from streptobacillus moniliformis -a zoonotic pathogen. taxonomic considerations, host species, diagnosis, therapy, geographical distribution an enzyme-linked immunosorbent assay (elisa) for the detection of antibodies to pasteurella pneumotropica in murine colonies sgrnacas9: a software package for designing crispr sgrna and evaluating potential off-target cleavage sites inhibition of tnf-alpha, and nf-kappab and jnk pathways accounts for the prophylactic action of the natural phenolic, allylpyrocatechol against indomethacin gastropathy adaptive immunity restricts replication of novel murine astroviruses chemical-induced atrial thrombosis in ntp rodent studies nucleic acid-sensing toll-like receptors are essential for the control of endogenous retrovirus viremia and erv-induced tumors recent applications of engineered animal antioxidant deficiency models in human nutrition and chronic disease dissecting the effects of mtdna variations on complex traits using mouse conplastic strains a genomic update on clostridial phylogeny: gram-negative spore formers and other misplaced clostridia effective eradication of pinworms (syphacia muris, syphacia obvelata and aspiculuris tetraptera) from a rodent breeding colony by oral anthelmintic therapy possible allelic structure of igg2a and igg2c in mice one-step generation of different immunodeficient mice with multiple gene modifications by crispr/cas9 mediated genome engineering key: cord-023143-fcno330z authors: nan title: molecular aspects of viral immunity date: 2004-02-19 journal: j cell biochem doi: 10.1002/jcb.240591009 sha: doc_id: 23143 cord_uid: fcno330z nan mechanisms of t-cell mediated clearance of viruses from the central nervous system are poorly understood, but likely to differ from those employed in the periphery because the cns lacks lymphatic drainage and constitutive expression of mhc class i antigen, and the unique structure of the cns vasculature imposes constraints on access by leukocytes and soluble immune mediators. to study the mechanism by which viruses are cleared from neurons in the central nervous system, we have developed a mouse model involving infection with a neurotropic variant of mouse hepatitis virus (oblv60). grew preferentially in the olfactory bulbs of balbk mice. using in situ hybridization, we found viral rna localized primarily in the outer layers of the olfactory bulb, including neurons of the mitral cell layer. virus was cleared rapidly from the olfactory bulb between 5 and 11 days. athymic nude mice failed to eliminate the virus demonstrating a requirement for t lymphocytes. immunosuppression of normal mice with cyclophosphamide also prevented clearance. both cd4+ and cd8+ t-cell subsets were important as depletion of either of these subsets delayed viral clearance. gliosis and infiltrates of cd4+ and cd8+ cells were detected by immunohistochemistry at 6 days. the role of cytokines in clearance was investigated using an rnase protection assay for il-la, il-lp, il-2, il-3, il-4, il-5, il-6, tnfa, tnfp and ifny. in immunocompetent mice there was upregulation of rna for il-la, il-lp, il-6, tnfa and ifny at the time of clearance. nude mice had comparable increases in these cytokine messages with the exception of ifny. induction of mhc-i molecules on cells in infected brains was demonstrated by immunohistochemistry in normal and nude mice, suggesting that ifny may not be necessary for induction of mhc-i on neural cells in vivo. luca g. guidotti, kazuki ando, tetsuya ishikawa, lisa tsui and francis v. chisari. the scripps research institute, la jolla, ca 92037 although cytotoxic t lymphocytes (ctl) are known to clear viral infections by killing infected cells, recent studies suggest that they can also suppress the replication of certain viruses by noncytolytic mechanisms. we have examined this area by monitoring the immunopathological and antiviral consequences of antigen recognition by hepatitis b virus (hbv) specific ctl in hbv transgenic mice that express the viral gene products in their hepatocytes. we have shown that intravenously injected ctl rapidly trigger their target hepatocytes to undergo apoptosis, but that the direct cytopathic effect of the ctl is minimal in comparison with the cytopathic effects of the antigen-nonspecific intrahepatic inflammatoly response that they activate. in addition to killing the hepatocyte, the same ctl also downregulate hbv gene expression and completely abolish hbv replication in the hepatocytes that they don't destroy. this noncytolytic antiviral ctl effect is mediated by at least two distinct processes in these animals. first, the ctl cause a quantitative reduction in the steady state content of all hbv mrna species in the hepatocyte, and this is followed by disappearance of all of the corresponding viral proteins in the liver and serum. the ctl initiate this process by secreting ifny and tnfa when they are activated by antigen recognition. since the regulatory effect of the ctl can he prevented completely by prior administration of the corresponding antibodies. nuclear run-on experiments reveal that viral mrna transcription is unaffected despite the profound reduction in hbv mrna content in the liver, suggesting that the ctl-derived cytokines accelerate viral mrna degradation in the hepatocyte. a second noncytolytic antiviral pathway is also activated by the ctl. we have recently shown that hbv nucleocapsid particles, and the replicative hbv dna intermediates that they contain, disappear from the transgenic mouse liver following either ctl administration or partial hepatectomy. the latter of which triggers hepatocellular regeneration without any change in hepatocellular hbv mrna content. these results suggest that preformed hbv nucleocapsid particles may be actively degraded during hepatocyte turnover, and they raise the possibility that similar events might also occur in nondividing hepatocytes that are activated by noncytolytic signals delivered by the ctl. we propose that, in addition to their pathogenetic effect, the comhined effects of the ctl response at die hbv mrna. nucleocapsid and rcplicative dna levels may represent a curative antiviral stimulus during hbv infection. since the virus must contain molecular elements that iespond to these ctl-induced antiviral signals. inactivating mutations at these loci could be very efficiently selected by immune pressure, because a single mutation could abrogate the antiviral effect of a wide spectrum of t cell responses, irrespectrve of epitope specificity. identification of these viral response elements and the intracellular pathways that interact with them may lead to the development of new strategies for antiviral drug design. human fibroblasts infected with hsv are resistant to lysis by cd8+ cytotoxic t lymphocytes (ctl), yet human b cell lines can be efficiently lysed by these ctl. the effect on human fibroblasts is rapid (within 2 hr of infection of cells), occurring before synthesis of mhc class i is altered by virus infection. a recombinant hsv, f-usbmhc, which expresses mouse mhc class i proteins does not render human fibroblasts sensitive to lysis by mouse ctl. mhc class i molecules are retained in the er of hsv-infected fibroblasts i n a misfolded, unstable form and stability of the mhc complex can be restored by addition of exogenous peptides. using a panel of hsv mutants and ad expression vectors we demonstrated that the hsv ie protein icp47 was both necessary and s f i c i e n t to cause retention of class i and icp47 expression in fibroblasts caused the cells to resist lysis by cd8+ t lymphocytes. icp47 is a soluble, cytosolic protein and we have found no evidence of membrane association. therefore, it appears that icp47 inhibits cytosolic stages of the antigen presentation pathway so that antigenic peptides do not reach the er. to date, polyclonal and monoclonal antibodies directed to icp47 have not specifically precipitated any of the previously described components of the antigen presentation pathway and we have not found icp47 associated with tap transporter proteins or proteosomes i n these experiments. the effects of icp47 are being assessed in proteosome and tap transporter assays. gst-icp47 fusion proteins tightly bind a 8.5 kda cytosolic cellular protein which is found in a number of adherent human cell lines but not lymphocytes. the protein has been purified and sequencing is in progress. in addition, radiolabelled icp47 binds to a single cellular protein of =55 kda on ligand blots. these proteins are good candidates as cellular targets of icp47 and as novel components of the antigen presentation pathway. preliminary experiments support the hypothesis that icp47 is very effective i n blocking cd8+ t lymphocyte responses in vivo, perhaps explaining the predominance of cd4+ vs. cd8+ anti-hsv ctl i n vivo. we expect that icp47 may be very useful, not only to elucidate antigen presentation pathways, but also to prevent immune recognition of gene transfer vectors and a s a immunosuppressive agent. susceftibility to polyoma virus-induced tumors is conferred by an endogenous mmtv superantigen. aron e. lukacherl, yupo ma2, john p. carroll2, sara r. abromson-leeman2, joseph c. laning2, martin e. dorf2, and thomas l. benjamin2. idepartment of pathology, emory university school of medicine, atlanta, ga 30322, and 2department of pathology, harvard medical school, boston, ma 02115. susceptibility to tumors induced by mouse polyoma virus varies among inbred mouse strains. we have previously shown that polyoma tumor susceptibility is controlled by products of mhc as well as non-mhc genes. in crosses between mhc-nonidentical strains differing in tumor susceptibility, resistance correlates with dominantkodominant inheritance of the resistant h-2 haplotype. we have observed the opposite pattern of inheritance of susceptibility in crosses between mhc-identical strains. in crosses between the highly susceptible c3wbida mouse and the highly resistant but mhc-identical (h-2k) c57bwcd.i mouse, polyoma tumor susceptibility is conferred by a single autosomal dominant gene, which we have designated pyvs. pyj does not encode cell receptors for the virus, affect viral dissemination or anti-viral antibody responses, or affect intracellular events essential for productive infection or cell transformation by the virus. whole-body irradiation renders cs7bwcd.i mice fully susceptible to polyoma-induced tumors, indicating an immunological basis for this strain's resistance. we hypothesized that p y j encodes an mtv superantigen (sag) that confers susceptibility to c3wbida mice by deleting precursors of polyoma-specific t cells. we found that tumor susceptibility in (c3wbida x c57bwcd.i) x c57bwcdj backcross mice cosegregated with mtv-7. inheritance of mtv-7 showed perfect concordance with absence of peripheral vp6+ t cells. genotyping of backcrossmice using markers of simple sequence repeat polymorphisms flanking mtv-7 showed no evidence of recombination between pyvs and mtv-7. strongly biased usage of vp6 by (a) polyoma-specific cd8+ ctl from virus-infected c57bwcdj mice and by @) cd8+ t cells infiltrating a polyoma tumor in a virus-immune c57bwcd.i host provide further evidence that t cells bearing this mtv-7 sag-reactive vp domain are critical anti-polyoma tumor effector cells. these results indicate identity between p y j and mtv-7 sag, and demonstrate a novel mechanism of inherited susceptibility to virus-induced tumors based on effects of an endogenous superantigen on the host's t cell repertoire. infection of mice with lymphocytic choriomeningitis virus (lcmv) causes a transient to longlasting immunosuppression dependent upon virus-isolate dose of virus and age, h-2, non h-2, level of cd4+ t cells, of cd8+ t cells and kinetics of neutralizing antibodies of the host. the immunohistological analysis suggests that cd8+ t cell dependent disappearence of marginal zone macrophages of follicular dendritic cells and of virus infected cells in general correlates with immunosuppression. the details of mechanisms responsible for these findings are now being analysed. a role of this cd8+ t cell dependent immunosuppression in the establishment of a lcmv carrier state in immunocompetent mice is suggested by the following experiments: the otherwise slow and low neutralizing antibody response agamst lcmv is accelerated and enhanced by cd8+ t cell depletion at the time of infection, suggesting virus-specific immunopathology being responsible at least partially. the elisa antibody response is not significantly altered under the same conditions but is abrogated if lcmv-specific t cell receptor transgenic mice are infected with high doses of lcmv, indicating, that suppression of the specific antibody response depends upon the relative kinetics of ctl versus antibody responses. whether exhaustion of specific ctl responses is enhanced by similar mechanisms remains to be tested. the role of interleukins of the relative distribution of virus in the mouse and in the various aspects of immunosuppression are now being studied. immunosuppression, caused by cd8+ t cell-dependent immunopathology, may also be operational in hiv infection in humans. such a pathogenesis of hiv-triggered aids could explain several aspects of the disease process not readily fitting the (unproven) conventional idea that hiv is causing immunodeficiency via direct viral pathogenicity. the cellular immunity against two dna tumor viruses (i.e. human adenovirus type 5 (ad 5) and human papillomavirus type 16 (hpv16)) was studied with respect to possible immune escape mechanisms and to the development of ctl epitope based peptide vaccines. after identifying an immunorelevant ctl epitope in the ad 5e1a protein to which ctl clones were directed that could eradicate ad 5e1 induced tumors in nude mice, an amino acid replacement study of this epitope revealed a point mutation that totally eliminated the possibility to recognize the mutant peptide by the ctl clones directed against the wild-type peptide sequence. new viral constructs were made that contained this point mutation and used to transform mouse embryo cells. however, these mutant tumor cells were still immunogenic and ctl clones specific for these mutant tumor cells were shown to react with a peptide derived from the ad 5e1b protein. these ad 5eib specific ctl clones, however, were as effective as the ad 5e1a specific ctl clones in the eradication of ad 5e1 induced tumors in nude animals, indicating that a choice can be made of immunorelevant epitopes to which an immunization strategy could be developed. in addition, we discovered that by supertransfection of ad 5e1 induced tumor cells with the activated ras oncogen the possibility of ad 5e1b specific ctl to recognize the ad 5e1 induced tumors was eliminated whereas the ad 5e1a specific ctl could still kill these tumor cells. this might indicate a new mechanism of tumors to escape ctl. in an hpv16 induced mouse tumor model an immunosubdominant ctl epitope was identified in the e7 protein that could, upon immunization with that peptide,protect mice against a subsequent challenge with hpv16 induced tumor cells. by changing the anchor residues in that peptide an even more immunoprotective peptide could be generated. combined, these data indicated a successful use of a ctl epitope based peptide vaccine in the prevention of hpv16 induced tumors in mice. subsequently this led us to identify relevant ctl epitopes of hpv16, that is highly associated with cervical carcinoma in humans, for the major hla-a alleles (i.e. hla-a *0101, a *0201, a"0301, a*1101 and a *2401). together these alleles cover a majority of all humans. ctl epitopes were identified through peptide-mhc binding assays followed by in v i m peptide immunizations with high affinity binding peptides to induce primary ctl responses and immunogenicity studies in hla-a transgenic mice. thereafter, memory ctl responses were measured in cervical cancer patients against selected peptides. combined, these data led us to develop a ctl epitope based peptide vaccine that could be of use in hpv16 induced cervical cancer patients. a clinical trial for this disease is scheduled to start in the fall of 1994. class ii presentation of an endogenously synthesized glycoprotein. carol s. re is^'.'.^, shirley m. bartido', miriam stein', and stephanie diment3.4, biology department', and center in contrast to class i presentation which is well characterized to use peptide fragements of proteins sythesized in the cytoplasm, exogenously administered experimental antigens enter the class ii mhc pathway through endocytosis. we have been studying the recognition of the glycoprotein of vesicular stomatitis virus (vsv) which can enter either the exogenous or endogenous pathways for presentation to cd4 + t cells. investigations of the intracellular sites involved, the proteolytic processes involved in the epitope generation, will be discussed. the glycoprotein studied in detail is a truncated form of the wt type 1 glycoprotein, termed poison tail (gpt) . expressed with a vaccinia virus vector, the gpt remains endo h sensitive and never becomes endo d sensitive, indicating that it is restricted to the endoplasmic reticulum. gpt is degraded in the er, and w e believe the degradation products include the immunogenic epitopes recognized by a panel of lad and i-ed t cell clones and hybridomas. lmmunofluorescence studies have confirmed the er localization. flow cytometric evaluations s h o w that the gpt never appears on the cell surface, in contrast to the wt g. the peptides generated are not secreted; using an innocent bystander assay, gpt-infected cells are incapable of sensitizing 5'cr-labeled uninfected apc. this contrasts with the rapid ability of supernatants from wt g-vaccinia virus-infected cells to sensitize apc for t cell recognition. investigations of the characteristics of the enzymes contributing to the degradation of the gpt have shown that a reducing environment is essential, as diamide treatment of cells prevents degradation. lysosomotropic drugs (eg. nh,ci and leupeptin) d o not alter the half-life of the protein, but do prevent presentation of the peptides; this is inconsistent with an autophagic component to the proteolysis. ph optima are physiological, as ph8 environment inhibits the enzyme activity. inhibitors of enzyme classes are consistent with a trypsin-like, and not cystein-, cathepsin b-, or chymotrypsin-like class. supported by nih grant al 18083 to csr. (emcv) and mengovirus are related members of the cardiovirus genus of picomaviruses. their rna genomes encode a large polyprotein which is cleaved proteolytically in co-and post-translational reactions to yield all mature viral proteins necessary to establish an infection. although originally thought to be exclusively murine in host range, both viruses actually infect a wide range of mammals. emcv has caused devastating epizootics in captive primates (eg: macaques, chimps and baboons), domestic pigs and exotic zoo collections (elephants, lions and tigers). death, following ingestion of virus-contaminated material, is rapid, and caused by extensive meningoencephalitis and virus-induced damage to the cns. myocarditic lesions are common in older animals. when administered intracerebrally, the ld,, for emcv strain r is about 1 pfu. we are studying the pathogenesis of emcv and mengo with engineered cdna plasmids containing infectious viral sequences. many plasmids contain h'uncated versions of the unusual 5' noncoding homopolymeric poly(c) tract that is a hallmark of these cardioviruses. short poly(c) mengoviruses grow very well in tissue culture but are 106-10'z fold less pathogenic to mice than the wild-type strains. animals receiving sublethal doses of short-tract mengo strains develop high titers of neutralizing antibodies, exhibit potent ctl responses and acquire lifelong protective immunity against challenge with wild-type virus. the genetic stability of the short-tract strains, even upon serial brain passage, mark them as safe, efficacious live vaccines. currently, we believe the poly(c) phenomenon is due to interference by the wild-type virus sequences (long poly(c) tract) with normal cellular cytokine induction mechanisms (ie: ifa and ifp) during the initial stages of animal infection. the targeted cells are probably macrophages, and their singular ability to correctly respond or not respond to poly(c) tract length during the first few hours of infection determines whether an inoculated animal will live (protectively vaccinated) or die. the short-tract viruses probably induce if in the macrophages, and are consequently killed then rapidly cleared from the host in related experiments we've found that attenuated mengo strains can easily carry large heterologous insertions within their genomes, and express these sequences into protein them during replication in animals. the resulting immune response (b cell and ctl) to the chimeras is directed towards the foreign sequences (epitopes) as well as towards the mengo proteins. a chimeric hiv vaccine, a rabies vaccine and an lcmv vaccine have been developed and tested. the lcmv chimera seems especially effective, as a single pfu of this engineered mengo strain, administered orally to a mouse, is sufficient for complete immunogenic protection against intracerebral challenge with wild-type lcmv virus. rsv is the most common cause of serious viral lower respiratory tract disease in infants and children. we have recently renewed our efforts to generate a safe and effective live attenuated rsv vaccine for topical administration that will overcome the deficiencies of previously studied live and non-living rsv vaccines. this vaccine will be a bivalent vaccine consisting of subgroup a and b live attenuated virus components. since the peak incidence of severe disease caused by rsv is in the 2-month old infant, an rsv vaccine will need to be effective when given to 1-month old infants. based on the success of live poliovirus vaccines given early in infancy, it is anticipated that the intranasally administered live virus vaccine will infect and induce a protective local and systemic immune response even in infants with passively acquired maternal antibodies. the main approach that we have taken in this effort to develop the live rsv vaccine is to introduce one or more ts mutations by chemical mutagenesis into a cold-passaged virus (cprsv) that had been partially attenuated by the acquisition of host-range mutations selected by passage in cells of a heterologous host species. we have developed a large set of cprsv subgroup a rs mutants (termed cprs mutants) that contain the host-range mutations selected during cold passage and two or more ts mutations introduced by chemical mutagenesis. these mutants have been evaluated in virro for their level of temperature sensitivity and in vivo in rodents, chimpanzees, and humans. a large set of rsv subgroup b cpts mutants has been similarly produced and evaluated. the immunogenicity and protective efficacy of three candidate live attenuated rsv vaccine strains that represent a specaum of attenuation were evaluated for protective efficacy in chimpanzees. prior to infection some of these animals were given rsv immune globulin by the iv route to simulate the condition of the very young infant who possesses passively-acquired maternal rsv antibodies. the three candidate vaccine strains were immunogenic and induced significant resistance to rsv challenge in both groups of chimpanzees. interestingly, the chimpanzees infused with rsv antibodies prior to immunization were primed more effectively for an unusually high serum neutralizing antibody response to infection with challenge virus than chimpanzees which did not receive such antibodies. this high booster response occurred despite marked reshiction of replication of the challenge virus. the evaluation of two candidate vaccines in seronegative human infants will also be described. rs virus is immunologically interesting for at least t w o reasons: 1) upper respiratory reinfection occurs despite previous exposure and demonstrable immunological memory: 2) humans or rodents previously immunised against virus infection can show enhanced disease during reinfection. others have shown that passive transfer of antiviral antibody either protects against virus infection or has no effect, and there is no evidence of antibody enhancement of disease in vivo. by contrast, t cell immunity appears closely associated with disease augmentation. we have focused on examining the immunological mechanisms of disease enhancement in mice. initial studies showed that transfer of cd8+ cytotoxic t lymphocytes (ctl) causes rapid virus clearance from the lungs of rs virusinfected mice, but also increased disease severity with alveolar haemorrhage and polymorphonuclear (pmn) cell recruitment to the lung. this disease (reminiscent of shock lung) could sometimes be fatal, whereas normal mice recover well from similar doses of rs virus. next, we compared the effects of cd4' and cd8+ t cells, using polyclonal t cells separated immunomagnetically from mixed lines grown in vitro with viral antigen. cd4' t cells were more pathogenic than cd8+ t cells in a dose-for-dose comparison, but that the type of pathology varied depending on the type of cell injected. while testing recombinant vaccinia viruses expressing single rs viral proteins for their ability to protect mice against infection, we observed that animals sensitised t o the major surface glycoprotein g (attachment protein) developed lung eosinophilia after challenge with rs virus intranasally. t cell lines from the spleens of mice sensitised with various recombinant vaccinia viruses were established. those form mice primed with the m 2 (22k) protein were predominantly cd8' ctl, and that produced few cytokines. those from mice primed with fusion protein (f) generated mixed t cell lines with both t h l cd4+ t cells, and ctl. mice primed to g protein gave rise to predominantly cd4' t cells producing th2 cytokines. ln vivo transfer of these cell lines into na'ive rsv infected mice reproduces the patterns of disease seen in mice sensitised in vivo with the respective antigens. the mouse model of rs virus disease therefore has excellent potential for illustrating mechanisms of lung immunopathology. the eye is a complex organ whose function is to transmit light images through different cell and tissue layers and liquid media to a neurosensory retina. elements as could occur when invading pathogens arrive and an inflammatory response with its swelling, plasma protein extravasation, leukocyte infiltration and tissue damage results. inflammatory responses when possible and rely on immune defenses which do not involve tissue distortion and damage. restricting tissue damaging responses is not always effective and the process is best developed in response to agents delivered to locations such as the anterior chamber. where an inflammatory response is initiated which may result in ocular impairment. such herpetic stromal keratitis (hsk) is a common cause of blindness in man. animal model studies indicate that hsk is a multi-step process initiated by virus in an avascular structure. hsk fails to occur in the absence of t cells or replicating virus. disappears several days before a visible inflammatory response becomes evident. evidence will be presented that the secondary agonists which drive the inflammatory response may not be viral antigen(s) per s e . multiple cell types are involved in hsk, with the respective role of functional sets of lymphocytes changing according to the clinical phase of the disease. in addition, nonspecific inflammatory cells such as neutrophils and nk cells also influence the severity of lesions. basically the reaction begins with t cells that produce type one cytokines, particularly ifn-y, dominating the scene, but during remission type 2 cytokines, notably il-10, appear as mechanistically involved. from the use of knockout mice for various immunological parameters, evidence will be presented that numerous mechanisms of pathogenesis may be at play during hsk. damage to corneal tissues in all systems appear to involve tnfo. a second ocular damaging event in which immunopathology is at least partially involved is herpetic retinal necrosis. evidence that this disease may involve the immunopathological role of cd4 t cells and protective effects by cd8' t cells will be presented, as will be suggestions by which the pathological events are mediated at the molecular level. thus it is in the eye's functional interest to limit acute viral infections and live vaccines often confer long-term immunity the nature of t and b cell memory is different. b cell memory is manifested not only by the presence of memory b cells but also by continuous antibody production in contrast, the effector phase of the t cell response i s shortlived and long-term t cell memory is due to the presence of 'quiescent' antigen specific memory t cells that are present at higher frequencies and are able to respond faster upon re-exposure to virus due to increased levels of adhesion molecules in this talk i w i l l present our results on. (i) the bone marrow as a major site of long-term antibody production after acute viral infection, (ii) the role of c d 4 ' t cells and b cells (immune complexes) in maintaining cd8+ t cell memory, (iii) the role offas antigen in regulating t cell responses, and (iv) the efficacy ofvarious antigen delivery systems in inducing long-term t cell memory sendai virus is a natural respiratory viral pathogen of mice. intranasal infection of mice with the virus provokes a virus specific antibody-forming-cell reaction that exhibits a distinct kinetic pattern in the lymph nodes that drain the respiratory tract, in the spleen, and in the bone marrow. the bone marrow afc population is extremely long-sustained, and supports an active humoral response that essentially persists for the lifetime of the infected animal. thus the conventional categories of "primary" and "secondary" response may not apply to the humoral response of mice naturally exposed to respiratory viruses. paradoxically, the population of b cells that reacts most rapidly to sendai virus infection does not itself secrete antibody, but can he demonstrated by the recovery of hyhridomas that secrete "polyspecific" antibodies. the activation of this polyspecific b cell population is, like the humoral response, extremely persistant. viral infection thus sets in train multiple b cell "memory" processes. variation in the rules of development and turnover of different b cell populations constrains the mechanisms that may operate to generate these different forms of memory. establishment and maintenance of t cell memory to respiratory viruses, peter c. doherty, sam hou, christine ewing, david topham, anthony mcmickle, james houston, and ralph tripp, department of immunology, st. jude children's research hospital, memphis, tn 38105. the analysis of the development and memory phases of the cd4+ "helper" n h ) and cd8+ cytotoxic t lymphocyte (ctl) responses to the respiratory pathogens, influenza virus and sendai virus (parainfluenza type 1) have been characterized by a combination of limiting dilution analysis (lda) for determining th and ctl precursor @) frequency and facs separation of lymphocytes with different activation phenotypes. the interpretation at this stage, largely based on the analysis of the ctl response, is that the development phase of t cell memory and the primary response are synonymous. virus-specific ctlp are produced in considerable excess of the numbers required to provide the effector ctl that terminate the primary infection, with only a fairly small proportion localizing to the target organ (the lung) that supports virus growth. even when many of the proliferating ctlp are killed by administration of a small dose (20 mgkg) of the dna-targeted drug cyclophosphamide (cy), there is no indication of immune exhaustion. the cd4+ th response has, at this stage, not been analyzed through the course of the primary infection. use of the lda approach to determine thp frequencies is inherently more difficult, as the "read-out'' is lymphokine production and there is considerable "bystander" activation in these primary responses to respiratory viruses. memory thp and ctlp are characterized initially by the expression of an "activated" phenotype: cd44-high, l-selectin-low, cd49d (vla-4) high. after some months, an increasing proportion of the memory t cells revert to the l-selectin-high cd49d-low form typical of naive ctlp. the change, which is never absolute, seems to occur first with cd49d and the rate varies for different viruses. current experiments are addressing the possibility that intercurrent infection, particularly with the mouse y-herpesvirus 68 which causes persistent infection of lymphoid tissue, may be inducing a switch back to the activated pattern, as a consequence of "bystander" effects, or "low affmity" stimulation via the clonotypic tcr in responding lymphoid tissue. the question of such cross-reactivity and/or exposure to "high lymphokine" environments for the long-term maintenance of memory is also being addressed. to study the factors which regulate the generation and persistence of specific t cell memory we have used model systems utilizing t cell receptor transgenic mice as a source of enriched naive cells which can be either cultured in vitro to generate effector populations or restimulated in adoptive hosts. in either case one can visualize the development of an expanded effector population. we have documented that the proliferation and il-2 production of the naive t cells depends on their activation by apc expressing high levels of co-stimulatory molecules. we find that b7.1 and icam-i as costimulators strongly synergize and that increased t cell receptor triggering can both increase the magnitude of the response and decrease its dependence on costimulation. when cytokines il-4 vs il-i2/ifny are present at the initiation of the response of either cd8 or cd4 cells they dictate that the effectors generated will be polarized either towards il-4 and il-5 secretion or il-2 and ifny secretion, respectively. the fate of the effector population generated and followed in vitro, also is tightly regulated by ag, cytokines and probably by costimulation. cd4 effector cells not re-exposed to ag, produce no cytokines and they die within 3-4 days. effectors restimulated with ag make massive amounts of cytokines, regardless of the presence of cytokines, at low densities of ag and with little dependence on costimulation. when there is little il-2 produced and no cytokines added, effectors die rapidly by apoptosis. however the combination of il-2 and tgfp block apoptosis and support expansion of the effector population which is greatly enhanced by periodic ag stimulation. some conditions favor the reversion of effector-like cells to a more resting memory phenotype and these are being further explored. we have also examined the development and maintenance of memory after transfer of effector cells to adoptive hosts. long-lived polarized memory populations are generated from the polarized effectors and these persist for prolonged period in the absence of apparent ag stimulation. this supports the idea that factors other than antigenic stimulation, present in situ can support the expansion and maintenance of memory cells. the rabies glycoprotein (g) is the only external protein of the virion and is therefore responsible for any interaction that rabies makes with the host cell during the first steps of the virus cycle. the g protein is also the target of neutralizing antibodies. there are around 450 trimers of g at the virion surface which constitute the spikes visible by electron microscopy. upon exposure to slightly acidic ph, the glycoprotein undergoes a conformational change which results in ion er and less regular spikes. strikingly and quite differently from influenza hemagglutinin, this conformational change is reversible: if the p d is risen back to 7.0, the s ikes re ain their neutral configuration (1). probably as a consequence, the viral infectivity is totally preserved after an exposure of 2 hours at p 8 6.4 an cf 37t, which induces the conformational change, followed by an incubation at neutral ph. since the conformational change is reversible, there is a ph-dependant equilibrium between the native and the low-ph conformation: the higher the ph, the more spikes are in their native configuration. two main antigenic sites and several minor sites have been identified on the native rabies glycoprotein (2). specific amino acids belonging to each of the two major antigenic sites are important or essential for viral virulence. for instance a lysine in position 198, which is part of antigenic site 11, is important, although not essential, for the viral virulence. similarly, the arginine 333, which belongs to antigenic site 111, is essential for pathogenicity while dispensable for multiplication in cell culture (reviewed in 3). viral strains mutated at arginine 333 have lost the capability to penetrate certain categories of neurons, suggesting that this mutation affected the recognition of specific receptors or subsequent interactions necessaly for the penetration of the virus at nerve terminals. therefore the two main antigenic sites are regions of the glycoprotein which also interact specifically with neurons in the animals. we have found that neutralization requires the fixation of at least one or two igg for every three spikes, irrelevant of the anti enic site recognized by the antibody (4). most neutralizing antibodies recognize conformational epitopes which are accessible on the native configuration of the protein. some epitopes remain accessible also on the acidic configuration while others are not. in addition, a minority of antibodies recognize epitopes which are only accessible on the acidic conformation. this is not unlikely in view that each spike has a certain probability to undergo a conformational change, even at neutral ph. in consequence the surface of the virus probably fluctuates and g epitopes which are not accessible on the native glycoprotein could be transiently exposed. conformational flexibility at neutral ph and physiological temperatures has also been observed for poliovirus (5). structural flexibility of external proteins could have important implications in virus-host interactions. katpus, norlhwestern university medical school, chicago, il 6061 1 theiler's murine encephalomyelitis viruses (tmev) are endemic enteric pathogens of wild and colony-reared mice. lntracerebral inoculation of susceptible mouse strains leads to a chronic, progressive inflammatory demyelinating disease of the central nervous system (cns) characterized clinically by an abnormal gait, progressive spastic hind limb paralysis and urinary incontinence, and histologically by parenchymal and perivascular mononuclear cell infiltration and demyelination of cns white matter tracts. demyelination is related to persistent cns viral infection. due to the similarity in clinical and histological presentation, tmev-induced demyelination is considered to be a highly relevant model of multiple sclerosis (ms). our current interests are in determining the phenotype, fine specificity, lymphokine profile and tcr usage of cns-infiltrating cells involved in the effector stages of tmev-induced demyelination. based on a variety of experimental evidence, it is clear that demyelination induced in sjuj mice by infection with the bean strain of tmev is a thl-mediated event: (a) disease induction is suppressed in t cell-deprived mice and by in vivo treatment with anti-i-a and anti-cd4 antibodies; (b) disease susceptibility correlates temporally with the development of tmev-specific, mhc-class il-restricted dth responses and with a predominance of anti-viral lgg2a antibody; (c) activated (le., ll-2rc) t cells infiltrating the cns are exclusively of the cd4+ phenotype, and (d) proinflammatory cytokines (ifnq and tnf-p) are predominantly produced in the cns. we have mapped the predominant thl epitope on the virion to amino acids 74-86 of the vp2 capsid protein. a thl line specific for vp274-86 exacerbates the onset of demyelination in recipient mice infected with a suboptimal dose of tmev. tmev-infected sjuj mice fail to exhibit peripheral dth and t cell proliferative responses to the major myelin proteins, mbp and plp, and pre-tolerization with neuroantigens has no affect on the incidence or severity of tmev-induced demyelinating disease, whereas neuroantigen-specific tolerance prevents the induction of relapsing experimental autoimmune encephalomyelitis (eae). in contrast, tolerance induced with intact tmev virions specifically anergizes virus-specific thl responses and results in a dramatic reduction of the incidence and severity of clinical disease and cns dernyelination in sjuj mice subsequently infected with tmev. these results have important implications for a possible viral trigger in ms as they indicate that chronic demyelination in tmev-infected mice is initiated in the absence of demonstrable neuroantigen-specific autoimmune responses and are consistent with a model wherein early myelin damage is mediated via primarily by mononuclear phagocytes recruited to the cns and activated by pro-inflammatory cytokines produced by tmev-specific thl cells. the concept that prions m novel pathogens which are different fium both viroids and viruses has received increasing support from many avenues of investigation over the past decade. enriching fractions from syrian hamster (sha) brain for scrap= prion infectivity led to the discovery of the prion protein 0. prion diseases of animals include scrapie and mad cow disease; those of humans present as inherited, sporadic and w o r n neurodegenemive disorders. the inhecited human pion diseases m genetically linked to mutatim in the prp gene that result in non-conswative amino acid substitutions. transgenic v g ) mice expressing both sha and m o w @lo) prp genes were used to demonstrate that the "specie9 bank?' for -pie prions resides in the primary structure of pip. this concept was strengthened by the results of studies with mice expressing chimeric mdsha transgenes &om which "artificial" prions have been synthesized. similar chimeric mdhuman (hu) rp transgenes were constructed which differ from m o w by 9 amino acids between residues 96 and 167. au of the tg(mhu2m) mice developed neurologic drsease -200 days after inmulation with brain homogenates from three patients who died of creutzfeldt-jakob disease (cjd). inoculation of tg(mhu2m) mice with cjd prims produced mhu2mprpsc, inoculation with mo prions produced moprw. ihe patterns of meluzmprpc and mom% accumulation in the brains of tg(mhu2m) mice wen differenl about 10% of tg(huprp) mice expressing huf" and non-tg mice developed neurologic diseane >500 days after inoculation with cn, prions. the different susce@uies of tg(hw) and tg(mhu2m) mice to human prions indiate that additional species specific factors such as chaperone proteins are involved in prion replicaton. diagnosis, prevention and treament of human @on diseases should be faciliated by tg(mhu2m) mice. in other sindies, tg mice were compared expressing wt and mutant moprp. overexpression of the wtmoprp-a aansgene -8-fold was not deleterious to themiw but it did shorten scrapie incubation times from -145 d to -45 d after inoculation with murine m p i e pnons. in contrast, overexpression at the same level of l morp-a transgene mutated at codon 101 (corresponding to codon 102 in hurp) pmdnced spontaneous, fatal neurcdegeneration between 150 and 300 d of age in two lines of tg(mohp-pio1l) mice designated 2866 and 2247. genetic crosses of tg(moprp-p101l)2866 mice with gene targeted mice lacking both rp alleles ( p m -p ) produced anhats with a highly synchronous onset of illness between 150 and 160 days of age. the t g~o p r p -p l o l l ) 2 8 6~~ mice had numerous prp plaques and widespread spongiform degeneration in contrast 10 the tg2866 and 2247 mice that exhibited spongifonn degeneration but only a few prp amyloid plaques. another line of mice designated tg2862 overexpress the mutant transgene -32-fold and develop fatal neurodegeneration behveen 200 and 400 d of age. tg2862 mice exhibited the most severe spongiform degeneration and had numerous, large pip amyloid plaques. while mutant moprpccploll) clearly produces neurodegeneration, wtmoprpc profoundly modifies both the age of onset of illness md the mumpathology for a given level of transgene expression. our tidigs and those from other smdies suggest that mutant and wtprp interact, phaps through achaperone-like protein as noted above in sndies of tg(mhu2m) mice, to modify the pathogenesis of the dominantly inhe&ed prion diseases. anton, heidi t. link, and jonathan w. yewdell, laboratory of viral diseases, niaid, bethesda, md 20892-0440. cd8' lymphocytes (tcd8+) play an important role in host immunity to viruses and other intracellular parasites. virus-specific tcdi+ recognize mhc class i molecules in association with peptides of 8 to 10 residues derived from viral proteins. this presentation will focus on how and where antigenic peptides are generated by cells. to begin to characterize the nature of proteases involved in the generation of antigenic peptides from cytosolic proteins, we used a panel of recombinant vaccinia viruses expressing different forms of influenza virus nucleoprotein (np). we found that the efficiency of generation of two np peptides is related to the metabolic stability of the source gene product. there has been considerable speculation that such short lived proteins are degraded by proteasomes in a ubiquitin-targeted process. our observations, however, call into question the importance of ubiquitin targeted-proteolysis in generating antigenic peptides from exogenously provided or endogenously synthesized viral proteins. we also examined the extent to which antigenic peptides can be generated in the endoplasmic reticulum (er). we found that antigenic peptides could be produced from short precursors (17 residues) hut not from a number of full length proteins (influenza virus hemagglutinin, np, ovalbumin) that are targeted to the er by a nh2-terminal signal sequence. peptides were generated much more efficiently from the cooh-terminus of the 17 residue precursor than from the nh2-terminus. these findings indicate that the er has a much more limited capacity than the cytosol to generate antigenic peptides, but that er proteases (particularly aminopeptidases) could perform the final proteolytic steps in the generation of class i binding peptides from precursors imported from the cytosol by tap, the mhc encoded peptide transporter. potential advantages of synthetic peptide or engineered recombinant vaccines are that they can be limited to contain only the specific antigenic determinants for desired responses without other determinants that elicit unwanted responses, and that the sequences of the determinants themselves can be modified to enhance potency or breadth of crossreactivity. however, they can have the disadvantage that any single determinant may be presented by only a limited selection of major histocompatibility complex (mhc) molecules of the species. to overcome the problem of mhc polymorphism, we have identified determinants presented by multiple mhc molecules, and have also located multideterminant regions of the hiv-1 envelope protein that contain overlapping determinants each presented by different class i1 mhc molecules, so that the whole multideterminant region is presented by multiple mhc molecules of both mouse and human. we have made use of "cluster peptides" spanning these multideterminant regions of the hiv-1 envelope to provide help for neutralizing antibody (ab) and cd8+ cytotoxic t lymphocyte (ctl) responses to peptides attached to these helper regions. these synthetic peptide vaccine constructs containing the p18 peptide from the v3 loop of hiv-1 iiib or mn, elicited both neutralizing ab and ctl in multiple strains of mice. the cluster peptides inducing helper t cells were essential for elicitation of ab and ctl to the p18 segment of both iiib and mn strains of hiv-1 in mice of several mhc haplotypes. several adjuvants were compared for their ability to elicit both ctl and ab simultaneously, without one response inhibiting the other. a single formulation in incomplete freund's adjuvant (ifa) could elicit all 3 responses, neutralizing ab, ctl, and th1 helper cells. the ctl specific for the mn strain p18 peptide crossreacted with strains sc, sf2,2321, and cdc4. the peptides in f a also elicit high titers of antibodies in rabbits. boosting was found to enhance ctl responses as well as ab responses. these constructs are being prepared for a human immunotherapy trial. these vaccine constructs are potent and also avoid sites on gp160 that are known to elicit enhancing antibodies or autoimmune responses that might conmbute to disease pathogenesis. however, we can potentially improve on these by tinkering with the internal structure of the individual epitopes. we have found that replacing a negatively charged glutamic acid residue with an uncharged amino acid in one of the helper determinants makes it 10 to 100-fold more potent in binding to the class i1 mhc molecule and in eliciting murine helper t cells that still recognize the natural hiv-1 sequence. thus, such a modified peptide should be more potent as a vaccine, while retaining the ability to elicit t cells that will respond to hiv proteins that of course do not have the altered sequence. we are currently mapping the critical residues for presentation of one of these peptides by human hla-a2, with the intent of developing modified peptides that will be more potent as components of a human vaccine. thus, by leaming how these peptides bind to mhc molecules and tcell receptors, we can design internally modified determinants to construct more potent or more crossreactive second generation vaccines. we are testing these vaccine approaches in a mouse model in which mice can be protected against tumor cells expressing hiv proteins as would an hivinfected cell. dna vaccines, comprised of non-replicating plasmids encoding viral proteins, are capable of generating protective immunity in animal models of several viral diseases. in preclinical models of influenza infection, reduced viral shedding was observed in dna-vaccinated ferrets after challenge with the human clinical virus strain, a/georgia/93. cross-strain protection was conferred by dna encoding the major internal proteins (nucleoprotein, np, and matrix, m1) and the surface protein haemagglutinin (ha) from the antigenically-distinct previous virus strains, a/beijing/89 and a/hawaii/91. this protective efficacy was greater than that seen by immunization with the widely-used clinical vaccine composed of killed a/beijing/89 virus. thus, compared to a killed virus vaccine, protection seen with the dna vacane against a drifted virus strain was greater. we previously demonstrated that immunization of mice with np dna generated mhc class i-restricted cytotoxic t lymphocytes. mice likewise were protected from death and morbidity following cross-strain challenge'. ha dna vaccines generated neutralizing antibodies in mice, ferrets and primates, and provided protection in m i d and ferret models of influenza. in animal models of other viral diseases, immune responses and protection against viral challenge have been seen after immunization with dna encoding viral proteins. dna encoding hiv gp120 generated ctl and neutralizing antibodies in monkeys. antigen-specific proliferative responses and, in mice, secretion of high levels of yifn relative to levels of il-4, months after immunization were also observed . immunization of rabbits with dna encoding l1, the major viral capsid protein of cotton tail rabbit papilloma virus (crpv), resulted in neutralizing antibodies and protected against the development of warts after inoculation with crpv. mice immunized with dna encoding the glycoprotein gd from herpes simplex virus type 2 (hsv-2), developed neutralizing antibodies and were protected from death when subsequently challenged with hsv-2. dna vaccines were protective in animal models of various viral diseases. neutralizing antibodies, helper t cells (thl) and cytotoxic t cells were generated. cross-strain protection due to cellular immunity was demonstrated. ' science, 1993 2593745-1749 , 2dna cell biol, 1993 the profile of a neurovirulent virus is determined by its mechanism of entry into the cns (neuroinvasion), the type of cns cell in which it replicates (neurotropism) and its ability to cause pathologic effects in the brain (neurovimlence). whereas neuroectodermal cells, especially neurons, are the target cells of most neurovirulent viruses, the main target cell in the brain for siv and other lentiviruses is the macrophage. infection in, expression of viral antigens by and products of siv replication exported from these cells result in inflammation and degenerative changes in the brain and concomitant loss of neurons. siv strains that are mainly t-cell tropic cause transient activation of t-cells and during this period, infected t-cells cross the blood brain barrier and localize in the brain causing persistent hut minimally productive infection and minimal neuro pathologic effects. viral proteins but not virions are produced continuously. by virtue of the tropism of the virus for cd4 t cells, many infected animals eventually become immunosuppressed and develop aids, but not classical ueurological disease. viruses which are macrophage tropic invade the brain presumably also in t lymphocytes and the viruses infect macrophages in the brain. however, productive virus replication is minimized by antiviral cd8 t cells which suppress (kill?) all virus producing cells throughout the body, including the cns. productive virus replication in brain macrophages and accompanying inflammatory changes develop only when cd8 cells fail i.e. after profound immunosuppression sets in. the neurological disease that results from productive virus replication in macrophages in the brain therefore depends on presence of an appropriate macrophage-tropic viral phenotype invading the neuropil and development of immunosuppression in the host. the neurological disease could therefore be defined as one of the aids syndromes. the adenovirus (ad) early transcription region (e3) codes for more than 7 polypeptides, four of which have already been shown to alter the immune response to ad infection. the amount of the class i major histocompatibility complex (mhc) on the plasma membrane can be reduced by the binding of the ad e3 gpl9k protein to the mhc heavy chain, which prevents transport of the complex out of the endoplasmic reticulum. this process interferes with presentation of viral peptides to cytotoxic t lymphocytes. cytolysis by tumor necrosis factor-o (tnf) is inhibited by 4 distinct viral polypeptides, 3 of which (the ad e3 14.7k or the complex of the 10.4k and 14.5k proteins) are coded in the e3 region. the e3 polypeptides are translated from a family of viral mrnas, that are synthesized from a single viral promoter and processed by alternative splicing. we have studied the functions of the e3 polypeptides in several murine models. the goals of these experiments were to determine the effects of the ad e3 polypeptides in acute and persistent viral infections as well as in a transplantation model designed to measure whether these viral immunoregulatory proteins would abrogate allogeneic graft rejection. in a vaccinia virus (v.v.) pneumonia model, in which the isolated ad e3 14.7k or ad e3 gpl9k genes were inserted into the v.v. pathogen, the ad anti tnf polypeptide increased viral virulence but the ad anti mhc had no effects. in addition to manipulating the ad e3 genes in viral constructs, several transgenic mouse lines containing the ad e3 genes have been constructed for these experiments. the e3 genomic dna behind the rat insulin promoter (rip) has been used to generate transgenic animals. islets from rip-e3 transgenic animals (h-2b'd) have been transplanted allogeneically to h-2d recipients and remained viable, secreting insulin until the end of the experiment at 94 days; in contrast, control nontransgenic islets of the same genotype were rejected by 21-28 days. the e3 genes behind the native e3 promoter have been inserted into mouse embryos to generate transgenic animals, and the expression of the transgene monitored in multiple organs. the e3 promoter of the transgene is responsive to stimulation by the ad e1a following infection with an e3 minus ad 7001 and can also be upregulated by administration of bacterial lipopolysaccharide. the effects of this transgene on ad pathogenesis are currently being studied. thus, these viral immunoregulatory genes have been shown to alter viral pathogenicity during acute infection and to downregulate the host immune response sufficiently to permit islet cell transplantation. these results on manipulating the ad e3 genes for the control of the host immune response also have implications for designing adenovirus vectors for gene therapy. "emerging" infections can be defined as infectious diseases that either have newly appeared in the population, or that are rapidly increasing their incidence or expanding their geographic range. recent viral examples include aids, ebola, and hantavirus pulmonary syndrome (fnst identified in a 1993 outbreak in the southwestern u.s.). emerging viral infections show a number of common features. most "new" viruses derive from existing viruses that move into new areas or acquire new hosts ("viral traffic"). many are zoonotic (originating from animal sources) (even pandemic influenza appears usually to be a reassortant originating in wildfowl). ecological or environmental changes (either natural, or, often, man-made) may precipitate emergence of new diseases by placing people in contact with a previously unfamiliar zoonotic reservoir or by increasing the density of a mtud host or vector of a pathogen, increasing the chances of human exposure. upon introduction into a human population from a zoonotic reservoir, the newly introduced virus may cause localized outbreaks of disease. some may show rapid variation and evolution upon introduction, and some evidence suggests a role for immune selection in this process. a few viruses (such as hiv) may succeed in establishing themselves and disseminating in the human population, becoming truly "human" infections. human activities can also play an important role in establishment and dissemination. migrations from rural areas to cities, now an accelerating worldwide phenomenon, or other displacements, can introduce remote viruses to a larger population; the virus may then spread along highways and (globally) by air travel. the development of an effective system of surveillance and rapid response is essential, but resources for this are presently inadequate. vaccine development, production, and deployment problems also need to be addressed. immunopathology may be a key feature of many of these infections, a number of which manifest as hemorrhagic fevers. many of the life threatening complications are due to increased vascular permeability. the resemblances to septic shock suggest that cytokines (such as tnf) are likely to be important in the pathogenesis of these infections. the response of cells, such as the macrophage, that induce or synthesize key cytokines, may be an important element, and the ability to infect these cells may be one common denominator. why some viruses elicit this response, while other closely related viruses do not, cannot yet be predicted from molecular data. better understanding of these aspects of the immune response should lead to additional therapeutic strategies. (supported by nih grant roi rr03121.) genetic approaches have been used to detect and characterize numerous previously unidentified hantaviruses. puumalarrospect hilvsin nombre-like viruses or virus variants are present throughout north and south america, europe and russia. several of the american viruses identified are associated with the newly recognized hantavirus pulmonary syndrome (hps), a severe respiratory illness with high mortality. the genetic relationships of these and previously characterized hantaviruses have been studied by phylogenetic analysis of the nucleotide sequence differences located in pcr bgments amplified from the g2 encoding region of the virus m segments. the relationships observed are consistent with a long-term association of viruses with their primary rodent reservoirs and suggestive of coevolution of host and virus. a sin nombre virus isolate is now available and its genetic characterization has been completed. various virus antigens have been expressed and are being used to probe the interaction of the virus with the host immune system. hantaviruses cause significant morbidity and mortality throughout the world. more than 200,000 cases of hemorrhagic fever with renal syndrome (hfrs) are reported annually in asia, europe and scandinavia. the etiologic agents of hfrs are hantaan, seoul and puumala viruses, with hantaan virus causing the most severe form of the disease. in 1993, a new hantavirus was discovered in the united states (initially termed four comers virus), and was identified as the etiologic agent of hantavirus pulmonary syndrome (hi's). vaccines for hantaviruses are not readily available, although a number of inactivated viral preparations have been made and tested in asia. recurrent problems with inactivated hantaviral vaccines have been lot to lot variability, the need for repeated immunizations, and their inability to elicit long-lasting neutralizing antibody responses in immunized volunteers. because of such limitations on traditional vaccine development for these viruses, as well as the viruses' hazardous nature and slow, low-titer replication in cell culture, we used a recombinant dna approach to develop a vaccine for i-ifrs. our vaccine is a recombinant vaccinia virus expressing the m segment of hantaan virus under control of the vaccinia virus 7.5 k promoter and the s segment under control of the 11 k promoter. the m segment, which encodes the g1 and g2 envelope proteins, was included because of our findings that: (1) immunization with vaccinia or baculovirus-expressed g1 and g2 induced a neutralizing and protective immune response in hamsters; and, (2) neutralizing antibodies to g1 or g2 could passively protect hamsters from challenge with virulent virus. the s segment, which encodes the nucleocapsid protein (n), was included because of our finding that hamsters immunized with baculovirus-expressed n also were protected from subsequent infection. although the protective immune response to n is probably cell-mediated, the importance of such a response is presently not well defined. assessment of our vaccine in preclinical studies, indicated that immunized hamsters developed neutralizing antibodies and were protected from displaying viral antigen in their lungs after challenge. in a phase i, dose escalation, clinical study, the vaccine induced neutralizing antibodies in individuals immunized subcutaneously with approximately lo7 pfu of the recombinant virus. in addition to humoral responses, immunized volunteers developed a cell-mediated immune response as indicated in lymphocyte proliferation assays. larger clinical studies, including alternate routes or booster immunizations, are planned. based on these studies, we anticipate that the vaccine will be efficacious for preventing hfrs caused by hantaan and the antigenically closely related seoul virus. we are studying the cross-protective properties of this vaccine with more distantly related hantaviruses such as puumala virus. although we expect this vaccine to be safe as well as effective, we also are investigating the use of more attenuated pox-viruses as vaccine vectors. infection of mice with lymphocytic choriomenigitis virus (lcmv) results in a profound expansion in the number of spleen cd8 t cells and in the induction of virus-specific ctl activity. thereafter, the cd8 t cell number declines, and the ctl activity diminishes, though the frequency of lcmvspecific precursor ctl per cd8 cell, as assessed by limiting dilution assays (lda), is remarkably stable throughout long-term immunity. the decline in t cell and total spleen leukocyte number at the late stages of acute infection is associated with high levels of apoptosis, as detected by the in situ nucleotidyl transferase assay. apoptosis occurred in both the t cell and b cell populations, with the b cells dying in clusters. this apoptosis was also seen in tfansgenic mice ectopically expressing bcl-2 in the t and b cells and in c57bl/6 ipr/@r mice, which have a mutation in the fas gene. t cells from the infected animal underwent apoptosis in vitro when stimulated through the tcr with anti-cd3, thereby explaining some of the immunosuppression seen during acute viral infections. memory cells persisted for over a year and could be found in blast-size cell populations. challenge of lcmv-immune mice with either pichinde virus, vaccinia virus, or murine cytomegalovirus led to the reactivation of the lcmv-specific ctl response. lda analyses showed unexpectedly that these heterologous viruses crossreacted with subpopulations of lcmv-specific memory t cells. this memory t cell response to virus from an earlier infection was associated with enhanced immunopathology and enhanced clearance of virus during a heterologous virus challenge. over the course of the acute infection, ctl specific for the second virus were preferentially expanded over the crossreactive ctl, and after the acute infection, when the t cell response had subsided, ctl memory to the first infection had decreased. there is therefore a network of memory t cells which contribute to and are modulated by infections with putatively unrelated viruses, and apoptosis plays a homeostatic role in the course of these t cell responses. immune responses to live attenuated retroviral vaccines, r. paul johnson*?, cara wilsont, kelledy mansons, michael wyands, bruce walker?, ronald c. desrosiers* *new england regional primate research center, southborough, ma 01772 thfectious disease unit, massachusetts general hospital, boston, ma 021 14 â§tsi/mason, worcester, ma immunization of rhesus macaques with live attenuated retroviruses deleted in nef can induce protective immunity against challenge with pathogenic siv. development of protective immunity in these vaccinated animals occurs only after several months of infection, with maximal protection observed after one year. the specific immune responses responsible for mediating protection have not been defined, and little is known about the cellular immune responses in animals vaccinated with these live attenuated retroviruses. we have analyzed cellular and humoral immune responses in rhesus macaques and chimpanzees infected with live attenuated retroviruses. siv-specific neutralizing antibodies were present in vaccinated animals, but did not clearly correlate with protection against challenge. ctl specific for envelope and gag were identified in vaccinated macaques studied 12 or more months after vaccination. quantitation of siv-specific ctl activity in one of these animals using limiting dilution analysis revealed a relatively high precursor frequency of cytotoxic t lymphocytes, up to moo0 for gag and 1/8500 for envelope. cd8+ lymphocytes obtained from vaccinated macaques were also able to suppress siv replication in autologous cd4+ cells. suppression mediated by unstimulated cd8 autologous cells was maximal when cells were in direct contact with siv-infected lymphocytes, but cd8+ cells activated by an anti-cd3-specific monoclonal antibody were able to release. a potent soluble inhibitor of siv replication. in contrast to the relatively vigorous ctl response present in vaccinated macaques, we were not able to detect consistent ctl activity in chimpanzees infected with a hiv-1 molecular clone (nl43) or attenuated viruses at periods up to one year after infection, despite the use of a variety of stimulation techniques. proliferative responses to hiv p24 and gp160 were observed in chimpanzees infected with n u 3 and attenuated variants. although the relative contribution of these immune responses to protective immunity is not known, the relative vigor of the cellular immune responses observed in vaccinated macaques suggest they may play a role in mediating resistance to challenge. obiectives: to analyze the magnitude and specificity of the ctl response to hiv-1, and to determine the tcr usage by clonal ctl responses in infected persons, including persons with documented infection of up to 15 years with cd4 cells > 500/mm3. methods: hiv-l-specific ctl activity was evaluated in pbmc as well as in pbmc stimulated in vitro with hn-1 infected autologous cd4 cells, using target cells infected with recombinant vaccinia viruses expressing hn-1 proteins. ctl epitopes recognized by these individuals were determined using cloned effector cells. quantitative cultures were performed by endpoint dilution, and viral quantitation was determined by qc-pcr tcr analysis was performed by pcr, using both family-specific primers and anchored pcr, followed by sequencing. sequence analysis of ctl epitopes in autologous viruses was determined by pcr amplification and sequencing. clonal frequency was analyzed in pbmc by oligonucleotide probe to the cdr3 region of the tcr. studies performed in long-term non-progressing persons indicate the presence of a vigorous and broadly directed ctl response. detailed epitope mapping in a person infected for 15 years, who by qc-pcr had <i67 viral rna molecules per ml and who tested negative by bdna assay, revealed ctl clones directed at six different epitopes, with detectable recognition of three of these epitopes using freshly isolated pbmc as effector cells. the ctl clones were broadly reactive against many hown hiv-1 variants, and both the p17 and p24 epitopes were cross reactive with siv. sequence analysis of autologous virus within the three dominant ctl epitopes revealed the lack of immune escape variants in pbmc, plasma, and virus obtained from coculhue. supernatant. analysis of tcr usage by clones to defined epitopes in persons at different disease stages revealed heterogeneity in tcr usage and heterogeneity in recognition of virus variants withii these epitopes. analysis of clonal frequency using an oligonucleotide probe to the cdr3 region of the tcr in an individual with a vigorous response to gp41 suggested that approximately 6% of circulating t cells were a single hiv-l-specific clone. conclusions: our results indicate that cytotoxic t lymphocytes are part of the immune response in persistent non-progressive hiv-1 infection, and that prolonged infection can occur without the development of viral immune escape variation within defined ctl epitopes. in addition, vigorous circulating ctl responses can occur in the setting of an e x m e l y low viral load. heterogeneity in the ability of clones from different individuals to recognize sequence variation within ctl epitopes may be important in the pathogenesis of infection. ctl to conserved epitopes or ctl which are broadly reactive with multiple viral mains may be important in conmbuting to maintenance of the asymptomatic state. factor-i (irf-1) gene is essential for the transcriptional activation of inducible nitric oxide synthase (nos) in murine macrophages. it also plays an important role in the activation of interferon and il-6 and il-7 receptor genes. to investigate the role of irf-1 related systems of defense against viral myocarditis, the transgenic mice were innoculated with l o 5 pfu of coxsackie virus 83 (cvb3). homozygous irf-1 (+) and heterozygous irf-1 (+/-) mice were randomized into groups to determine heart and spleen viral titres, cardiac pathology and mortality rates at early and late stages of the disease. the mortality was dramatic in the irf-1 -imice. the hearts of the homozygous animals also had increased mononuclear cell infiltration, necrosis and viral replication. we condude that irf-1 regulated gene products such as inos and ifn are essential for the early defense against cvb3-induced myocarditis by attenuating viral replication and inflammatory responses. the flu season is a yeariy problem, especially in the elderly population. annual vaccination lacks broad protection and antibody titers induced in the elderly are frequently lower than those acheived by younger vaccinees. using the mouse model, we are investigating the differences in immune response between old and young mice to influenza vaccine and at the same time testing the ability of a potent adjuvant to boost the response in the the elderly. our studies show defects in the immune response of the elderly. antibody titers after immunization are lower in the elderly for both seronegative and seropositive mice. cmi studies show that helper t-cell response (proliferation) to in-vitro antigen stimulation is also decreased in the elderly. facs analysis of wbcs show the elderly mice have lower leukocyte and lymphocyte populations, lower cd4 and b-cell populations and a higher proportion of macrophages and cd8 cells than young mice. it was also noted that the young mice had very consistent wbc profiles while the elderly profiles had greater variability. serum cytokine studies show lower levels of il2, il4 and il5, but higher levels of il6 and ifng in the elderly as compared to the young mice. the addition of mf59 to the immunization increased the antibody titers of both naive and seropositive young and old mice. the helper t-cell response of the young mice was unaffected, while the response of the elderly group was increased. cytokine profiles show an increase in il2, il4 and il5 levels for both young and old, while il6 and ifng levels went up for young animals but remained constant for the elderly mice. coxsackievirus 8 3 (cvb3), a member of the picornavirus family, is a common cause of acute or chronic human myocarditis. however, whether cell damage results primarily from direct virusmediated effects or from immune-mediated destruction of the heart tissue during the infection remains unclear. we used a cardiovirulent variant of cvb3 which is able to infect several strains of mice. the infection results in a disease which strongly implicates immunopathogenetic mechanisms in myofiber necrosis. to characterize the role of the immune system in this disease we used various transgenic knockout (ko) mice with different immune defects. cvb3 is able to infect normal c57bl/6 mice and immunocompromised (cd4 ko and 62m ko) mice and causes a lethal disease after i.p. injection in a viral dose dependent manner. we found elevated ld5os in immunocompromised mice, which also die slightly later than normal mice. this virus replicates in the hearts of all mice used with maximal titers 3-5 days pi.. large pathological changes were detectable only in heart tissue of cd4 ko mice, and were maximal at 1-2 weeks after infection. these results suggest the possibility that cd4' t cells may limit tissue damage by an as yet undefined mechanism; the roles of cd4+ and cd8' t cells in this disease are under investigation. research supported by a grant of the daad, germany. mice lacking p56lck are resistant to coxsackievirus b3 induced heart disease. tamara martino, karen aitken, joseph penninger, jan0 nikhilanandhan, tak mak, fayez dawood, wen-hu wen, lily wee, martin petric, and peter liu, the toronto and princess margaret hospitals, university of toronto, canada. coxsackievirus 83 (cvb3) causes severe heart disease in adult mice, and is a useful model for the human cardiac conditions of myocarditis and dilated cardiomyopathy. in this study, we have demonstrated that transgenic mice lacking the t-cell signalling molecule p56kk are resistant to cvb3 induced heart disease. the virus replicates in the heart, and is cleared from the myocardium at a rate comparable to "normal" littermates, but there is only minimal mortality, or damage to the cardiac tissue. however, in ick-deficient mice depleted of natural killer (nk) cells prior to viral infection, there is increased cvb3 replication, and some infiltration by mononuclear cells in the myocardium. to further elucidate the role of the immune system in cvb3induced heart disease, cytokine expression in the hearts of cvb3 infected normal, ick-deficient, and nk-cell depleted mice is under investigation. we conclude that t-cell activation is critical to produce substantive myofiber necrosis after cvb3 infection, that nk-cells play an fundamental role in protecting the mice from severe virus infection, and that ick transgenic mice serve as an important model for understandina the dathogenic mechanisms involved. we now show that cd4+ t cells from 62m-/-mice exhibit antigen specific release of serine esterase and interferon-gamma. further, as quantitated by flow cytometry, the early decrease in cd4+ t cells seen in normal mice 3 days after lcmv infection, previously presumed to be due to the activity of cd8+ ctl, still occurs in 62m-/-mice. however, 621114mice show an earlier rise in percentage and absolute numbers of cd4+ t cells by 7 days after infection. cd4+ t cells from am-/mice rise to above baseline levels with kinetics paralleling the early rise in cd8+ t cells in normal mice after lcmv infection. cd4+ ciz activity is lower in 62m-/mice and have a later peak than cd8+ ctl from normal mice. these differences may explain the lower mortality and longer time course of immunopathologic meningitis in 621114-mice. mice can assume some of the cytotoxic functions of the cd8+ cell population from normal mice, including immune-mediated pathology. further, cd4+ t cells from 62m-/-mice have the capacity to release serine esterase, and show kinetics similar to cd8+ t cells from normal mice. mice genetically engineered to be deficient in 62-microglobulin in conclusion, these data suggest that cd4+ t cells from am-/australia. molecule is an essential component of the t cell help required for an antibody response. the interaction between cd40 and its ligand, in the presence of b cell acting cytokines, such as interleukin 4 (il-4), is sufficient to trigger b cells to proliferate and differentiate and to induce immunoglobulin class switching. a recombinant vaccinia virus encoding both factors, cd40l and il-4, did not, however, stimulate an enhanced antibody response in mice infected with the construct. instead, the antibody levels were lower than those of mice infected with a control virus. the reduced antibody response reflected the diminished growth of cd4ol-expressing viruses, to the extent that immunocompromised mice were able to resolve these infections. the kinetics of virus clearance indicate that the anti-viral mechanism of cwol is rapidly activated and has generalized tissue distribution. the cwolmediated clearance of virus is independent of the anti-viral cytokines, tnf and ifn-y. the anti-viral activity of c w l may represent a surprising and potent effector mechanism of t cells activated during a virus infection. mary's medical school, norfolk place, london w2 lpg, u. k. and *nationallnstirute formedica1 research, london, nw7 1aa. respiratory syncytial (rs) virus is the most important respiratory pathogen of infants, causing the majority of cases of bronchiolitis, it can be life threatening in very young infants and those with underlying cardiopulmonary disease or immunodeficiencies. t cell immunodeficiency can lead to prolonged infection in children and t cell transfers clear virus in the murine disease model. mice can be readily infected with rs virus and have proved a valuable model of its pulmonary pathology and the t cell response to it. il-3 has a broad spectrum of activities on proliferation and terminal differentiation of early blast cells and committed progenitors of many lineages. less is known about the effects of il-3 on t cell development and proliferation and its effect on the host response to infection by common pathogens. the il-3 gene was disrupted in 129/sv embryonic stem (es) cells by homologous recombination after transfection with omega-type constructs flanked with a herpes simplex thymidine kinase gene. these cells were used to produce a mouse strain deficient of il-3. mice were infected intranasally with a 2 strain rs virus; pathological changes in the lung were monitored by cytology of bronchial lavage, and virus persistence by nested rt-pcr of lavage fluid and lung homogenate. no differences were found in pathology or virus persistence between knockouts and normal mice. il-3 deficiency is therefore compatible with apparently normal responses to viral infection. recombinant adenoviruses are an atuactive vehicle for gene therapy to lung in the treatment of cystic fibrosis (cf). first generation viruses deleted of ela and elb transduce genes into airway epithelial cells in vivo, however, expression of the transgene is transient and associated with substantial inflammatory responses, and gene transfer is significantly reduced following a second administration of the virus. in this study, we have used mice deficient in immunological effector functions in combination with adoptive and passive nansfer techniques to define antigen-specific cellular and humoral immune responses that underlie these important limitations. our studies indicate that mhc class i resmcted cd8+ cytotoxic t lymphocytes (ctls) are activated in response to viral antigens leading to destruction of virus infected cells and loss of transgene expression. mhc class ii associated presentation of viral antigens activates cd4+t helper (th) cells of the -1 subset and, to a lesser extent, of the tm subset. ldv establishes a life long viremic infection in mice which is not controlled by anti-ldv immune responses. anti-ldv antibodies are rapidly generated but they neutralize ldv infectivity only poorly. here we show that ldv-specific cytotoxic t cells (ctls)are generated within 7 days pi. c t l s were detected by h-2 specific lysis of ldv-specific target cells. these target cells were generated by transfection of 3t3-nm cells with a retrovirus vector carrying the gene (ow 7) for the ldv nucleocapsid (n) protein. spleen cells from ldv-infected mice were incubated in vitro with ldv-infected macrophages or transfected 3t3-nih cells and cultured for 5 days in the presence of il-2. the ctls had largely disappeared in mice by 30 days pi. the following experiments were conducted to further explore the mechanism of immune escape. in situ hybridization was used to localize ldvinfected cells in tissues of infected mice. these were found in persistently infected mice only in the liver. testis, and lymphoidal tissues. in addition large amounts of virion or virion debris accumulated in the germinal centers of the spleen and lymph nodes beginning about 3 days p.i. the accumulation of large amounts of ldv antigen in the germinal centers may be causally related to the suppression of the ctl response as well as to the polyclonal activation of b cells associated with ldv infection. to determine whether immune selection plays a role in ldv immune escape, ldv was reisolated from nude and immunocompetent balb/c mice at various times pi. the ows for the nand the two envelope glycoproteins were r t k r amplified and sequenced. the role of cytokines in initiating the immune response to venezuelan equine encephalitis (vee) was investigated. mice were infected in the left rear foot pad with either cloned virulent vee (v3000) or an isogenic single-site attenuated vee mutant (v3032) (single amino acid change at e2 glycoprotein position 209 from glu-lys) and at 1,6,24,48,72, and 96 hours post-infection, the popliteal lymph nodes and spleen were harvested and examined for tnf-a, il-12, ifn-y, il-2, il-4, il-6, and il-10 gene expression using a quantitative rt-pcr. in both strains elevations of tnf-a, il-12, ifn-y, il-10, and il-6 were detected in the lymph node, with no changes in either il-2 or il-4; however, whereas cytokine elevations were detected by 6 hours after injection of v3000, elevations were delayed until 24 hours following infection with v3032. cytokine mrna elevations in the spleen were less substantial, but elevated levels of ifn-y, il-10, and il-12 were also observed. these findings suggest that vee infection induces an early highly specific cytokine pattern associated with simultaneous elevations in both ifn-y and il-10 and that a single amino acid mutation can induce temporal changes in this pattern without alterring either the actual cytokines induced or the degree of their elevation. we are currently performing intervention experiments to investigate the effect of intravenous anti-il-12 andor anti-ifn-alp antibody administration on this early in vivo cytokine response to examine in particular whether these cytokines may be required for the observed elevations in either ifn-y or il-10. to confirm the importance of the immune system in clearing rotavirus infection we infected rag 2 knockout mice with murine rotaviruses. both knockout p u p s and adults developed chronic infections. to determine if a t cell independent antibody could be mediating the clearance of rotavirus infection in nude mice we measured igg, igm and iga in faeces and serum of rotavirus infected mice. a low and transient igm response was found in serum but not faeces of infected nude mice but not in age matched naive nude mice. the protein specificity of these antibodies is being determined. to determine if a primary infection in n u d e mice would induce protection (memory) against a secondary infection, we challenged six week old mice that had been infected as pups with the murine ew strain of rotavirus with the murine cambridge strain of rotavirus. the previously infected mice, as well as naive nude mice, both shed very low but equal levels of rotavirus in the stools. interestingly adult naive heterozygous littermates shed more virus than the nudes. the factors responsible for this relative resistance of adult nude mice to rotavirus infection as well as the mechanism for viral clearence in nude mice will be discussed. symp. 1990, 121:149-160) . the great majority of the poxvirus encoded proteins actively mediating the evasion of host defense are secretory proteins termed "virokines". viruses produce proteins which can either compete with or antagonizz molecules critically involved in generating immune responses. the vaccinia virus complement control protein (vcp) was the first virokine to be reported that has structural similarity to humadmouse complement control proteins, with the greatest similarity to the human complement 4b binding protein (bc4-bp) and the first protein to have a postulated role in the viral evasion of host defense (kotwal and moss, nature 1988, 355:176-179) . bioactive vcp, purified from serumfree medium has been shown to be more potent than the hc4bbp ( kotwal, am. biotech. lab., 1994) and has also been shown to bind to two important complement components, c3 and c4, and block the complement cascade at multiple sites in vitro (kotwal et al., science 1990, 2502327-830; mckenzie, kotwal et al., j. inf. dis. 1992 , 1661245-1250 . the importance of this protein was demonstrated in vivo using recombinant virus lacking an intact dna coding for vcp (isaacs, kotwal and moss, pnas 1992, 89:628-672) and further underscored by the smallpox virus genomic sequence analysis, indicating the presence of a highly conserved homolog of vcp ( massung et al., nature 1993,366:748-751) . in order to determine the precise effects of vcp at the site of infection, a mouse model has been developed. measurement of the specific swelling response and microscopic examination of the histological changes in balb/c and dbn2 mice has revealed that vcp is capable of regulating inflammatory response in vivo (jayaraman and kotwal, 1993 mol. immunol. 30:20) . in order to ensure that the possiblity of the unrelated mouse strains sharing identical h-2 haplotypes but differing at numerous other loci was not overlooked, we performed similar experiments in congenic strains. well characterized c5deficient mice injected with poxviruses in comparison to normal mice, produce a significant inflammatory response, accompanied with severe ulceration that persists for several days. the data suggests that the host complement response is a critical factor in egulating poxvirus pathogenesis. previous studies have suggested that immune response was one of the major factors that contributed to the host resistantlsusceptible mechanism to sendai virus infection (brownstein, 1981) . various immune regulatory molecules were investigated in inbred susceptible 129/j (129) mice and resistant c57bl/6j (b6) mice which share the same mhc haplotype. when they were given 200 eid, of sendai virus intranasally, 129 mice showed histologically diffuse interstitial pneumonia compared to the local moderate inflammation in 86 mice. virus was eliminated from the infected lung 3 days earlier in b6 mice than in 129 mice. the cytokine levels and cytokine-producing cells were investigated in the acute pneumonic lung. also the recall cytokine production in the draining mediastinal lymph node (mln) were studied. the maximal numbers of il-2, ifn-y, il-10, and il-4 producing cells were comparable for each strains of mice, while more il-6 producing cells were present in the lung of 129 mice. however, the numbers of these cytokine producing cells peaked in 129 mice 3 days later than in b6 mice. analysis of soluble cytokines in bronchoalveolar lavage fluid showed that at least two-fold higher levels of ifn-y, il-10, and il-6 were present in 129 mice than in 86 mice. recall cytokine production in the draining mln showed only the presence of il-2, ifn-y, il-10, and il-6, while no il-4 was detected. generally, higher levels of these cytokines were detected throughout the whole infection process in 129 mice. therefore, the susceptibility of 129 mice to sendai virus is not due to insufficient cytokine production in these two anatomical sites, yet heavier and prolonged virus burden in susceptible mice may drive the more rigorous and protracted induction of immune regulatory molecules. cytokines play a decisive role in determining the type of immune response elicited by an antigen, both in driving th cell differentiation along thl or th2 pathway and, ultimately, the effector and regulatory activity of these subsets. we have studied the role of cytokines in virus infections by constructing recombinant virus vectors encoding cytokine genes. during replication in mice, these vectors produce the encoded factor which is secreted from the infected cells with profound immunological consequences. cytokines studied todate fall into two categories; either altering viral pathogenesis or selectively stimulating specific immune responses. the expression of il-2, ifn-y, tnf-a or il-7 in recombinant vaccinia virus (rvv) dramatically alters pathogenicity, such that immunodeficient mice resolve the normally lethal infection. on the other hand, expression of il-4 by rvv suppresses the induction of cytotoxic t cells (ctl) inhibiting the clearance of virus which leads to an increase in viral pathogenicity. the expression of il-4 by rvv was shown to inhibit the host il-2 response required for the development of ctl's. no production was also significantly suppressed by il-4. rvvs expressing il-5 or il-6, although not affecting pathogenicity, preferentially stimulates iga reactivity to coexpressed antigens. encoding cytokines in recombinant viruses has clearly demonstrated the important role of cytokines as anti-viral effector molecules and in regulating appropriate immune responses. in mice infected with lymphocytic choriomeningitis virus (lcmv), interleukin-i2 (il-12) doses of >i00 ngld induce profound immunotoxicities characterized as almost complete inhibition of virus-induced cd8+ t cell expansion and ctl activation, and up to 2 log increases in viral replication [orange, wolf, and biron, j. immunol. 152:1253, 19941 . serum tumor necrosis factor (tnf) is also observed under these conditions. the studies reported here further characterize the expression and function of tnf in this context. northern blot and in sifu hybridization analyses demonstrated that il-12 induced tnf-cx expression and that lcmv infection synergized with il-12 for this induction. administration of antibodies neutralizing tnf reversed the il-12-induced immunotoxicities in lcmv-infected mice and restored anti-viral defenses. the tnf-mediated immunotoxicities appeared to result from an induced cellular sensitivity to the factor, as splenic leukocytes and cd8+ t cells isolated from lcmv-infected mice were more sensitive to tnfmediated cytotoxicity in culture than were equivalent populations prepared from uninfected mice. additional physiological changes were observed in il-12-treated uninfected mice and were dramatically elevated in il-12-treated virus-infected mice, including: 1) decreases in body weights; 2) elevation of circulating glucocorticoid levels: and 3) decreases in thymic mass. these changes were also reversed by anti-tnf. the results delineate a unique tnf-mediated immunotoxicity and have significant implications concerning detrimental consequences of in vivo tnf andlor il-12 for protective anti-viral responses. lactate dehydrogenase-elevating virus (ldv), a naturally occurring virus, causes a persistent infection in mice and presents an ideal model for the study of immune modulation during acute and persistent virus infections. within a few days following infection with ldv there is a pronounced polyclonal activation of b cells followed by the suppression of primary b cell responses to t-dependent ag. we investigated the effect of acute and persistent ldv infection on the development of a memory b cell response to the model protein antigen, horse cytochrome c (cyt), by employing a modification of the splenic fragment assay. about a 50% decrease in the frequency of responding agspecific memory b cells was observed in balb/c mice infected with ldv, whether the mice were immunized with cyt at the time of ldv infection or three weeks later. this may be due in part to a defect in t cell help, since in cultures of normal memory b cells and t cells derived from ldv acutelyinfected mice the frequency of responding b cells was also decreased two-fold. in situ hybridization using a cdna probe specific for ldv revealed two patterns of ldv rna within the spleen. twenty-four hr p.i. ldv rna was located within the marginal zone, surrounding each follicle. this pattern is consistent with permissive macrophages. during persistence viral rna could no longer be detected in the marginal zone, but was located within the follicles. the absence of ldv-permissive cells within the follicular region suggests that the source of ldv rna is not due to ongoing viral replication. one possibility is that circulating virus is trapped by a specific cell population within the follicle. the effect of virus trapping within the spleen provides a mechanism by which ldv and other viruses can modulate immune cell function during persistent infections. ifn-y can be produced by activated nk cells. this cytokine enhances immune responses by augmenting macrophage antigen presentation. viral infection induces ifn-dp and nk cell activation. changes in splenic architecture, cell trafficking, and cytokine expression were examined during viral infections of c57bu6 mice. at times coinciding with ifn-dp production and nk cell activation, there was a redistribution of nucleated cells from red pulp to white pulp regions in spleens isolated from mice infected with either lymphocytic choriomeningitis virus (lcmv) or murine cytomegalovirus (mcmv). cell transfer experiments with dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate-or pkh26-gl-labeled bone marrow cells isolated from normal mice demonstrated an infection-induced accumulation of non-t/non-b cell populations along recipient splenic marginal zones. flow cytometric analyses demonstrated that approximately 10% of the transferred bone marrow cells accumulated in spleens after 20 hrs and 30% of these expressed the nk cell marker, nkl.l+. in vivo antibody treatment procedures, to eliminate cell subsets in donor mice, demonstrated that the cells localizing at the marginal zone were derived from agmi+ and n k l . l + populations. a small subpopulation of marginal zone cells in infected mice were shown to be expressing high levels of ifn-7 mrna by in sifu hybridization. treatment with anti-agmi or anti-nk1 .i antibodies eliminated both endogenous nk cells and the ifn-y mrna positive cells. these data demonstrate that newly derived nk cells accumulate along marginal zones. the results also suggest that this trafficking pattern may act to enhance immune responses by facilitating delivery of cytokines to specialized antigen presenting cells. david segal, janet ruby, alistair ramsay and ian ramshaw. depamnent of cell biology, john curtin school of medical research, po box 334, canberra, act, 2601 australia cytokine expression has been shown to correlate with protective or ineffective immune responses in a number of disease models. recently there has been the suggestion that immunity to some retroviruses is associated with the production of ceaain patterns of cytokines. to explore this further we have have used rauscher murine leukemia virus (r-mulv) infection of c57bu6 (resistant) and balb/c (susceptible) mice to elucidate the role of cytokines immunity to retroviruses. initially the in viho proliferation of spleen and lymph node cells from infected mice was examined. in response to stimulation with immohilised anti-cd3 antibodies the proliferation of spleen hut not lymph node cells from infected mice. was found to he rapidly suppressed. suceptible balb/c mice exhibited a much greater suppression than resistant c57bu6 mice. the cause of this suppression is under investigation however, the immunosuppressive molecules nitric oxide and prostaglandins are not involved. in vitro cytokine production by spleen and lymph node cells from r-mulv infected mice was determined. in response to stimulation with immobilised anti-cd3 antibodies, spleen cells from infected balb/c mice produced diminishing amounts of ifn-7 and il-2. in contrast spleen cells from infected c57bu6 mice produced ifn-y and l-2 to levels that were only slightly less than uninfected controls. a-6 production by spleen cells from infected mice of both strains was at levels higher uninfected controls. anti-cd3 stimulated lymph node cells from infected mice produced elevated ifn-1 suggesting that suppressed cytokine production is spleen specific. expression of cytokine genes in vivo is currently being investigated using rt-pcr to detect cytokine mrna in the spleens of infected mice. we have previously shown that primary resting murine b lymphocytes are non-permissive for vesicular stomatitis virus (vsv), however, a productive infection can be induced when infected b cells are activated with anti-immunoglobulin (a-lg) plus il-4 or lipopolysaccharide (lps). we posit vsv in unactivated primary b cells provides a paradigm of persistently infected lymphocytes and activation dependent recall of an active infection. analysis of the behavior of virus in unstimulated b cells during long term culture and the requirements for subsequent induction of productive infection has been limited by the poor survival of primary cells in culture. we circumvented this limitation by using highly purified small b cells from mice transgenic for the bcl-2 proto-oncogene, expression markedly extends in vitro survival of unstimulated primary b cells. overexpression of bcl-2 does not alter b cell infection or induction of a productive infection by activators during acute infection. infection does not effect b cell survival in culture. unstimulated virus infected b cells produce primary viral mrnas but not viral proteins or infectious particles (pfu) during culture. persistently infected b cells stimulated with a-lg plus il-4 produced a fully productive vsv infection at all times analyzed, up to 3 weeks post infection. in contrast, vsv production in persistently infected b cells activated with lps markedly declined relative to acutely infected activated cells (50-1 00 fold by week 1 and 1,000 fold by week 2). cells were not completely refractory to lps activation as vsv protein was produced. the selective lps deficiency is unique to persistently infected cells as uninfected cultured b cells proliferate and differentiate to produce antibody upon lps activation. these data show that a persistent infection may selectively alter the host cell response to previously productive activators which may as a consequence interfere with immune regulation. rsv-g glycoprotein specific t cells preferentially secrete il-5 and predispose to pulmonary eosinophillia., anon srikiatkhachorn. and thomas j. braciale, the beirne b. carter center for immunology research and the departments of microbiology, pathology, and pediatrics, university of virginia health sciences center, charlottesville, va 22908 we studied the immune responses to two different glycoproteins of respiratory syncytial virus (rsv) in a murine model. balb/c mice were immunized with recombinant vaccinia virus expressing either rsv-fusion glycoprotein ( vac-f), attachment glycoprotein (vac-g) or 8-galactosidase (as a control). these mice were given rsv intranasally three weeks after priming and then sacrificed 5 or 14 days later. spleens and bronchial lymph nodes were harvested for in vitro culture and lungs were harvested for histologic studies . we found that bulk cultures obtained from both vac-f and vac-g immunized animals secreted both thl and th2 type cytokines when stimulated with rsv infected spleen cells . however, the levels of 11-5 and 1fn-y were higher in bulk cultures derived from vac-g primed animals while the levels of il-2 were higher in the bulk culture from vac-f primed animals. the il-4 and il-5 production was relatively short lived since spleen cells and bronchial lymph node cells obtaind form mice sacrificed 14 days after intranasal inoculation produced much lower levels of il-4 and 11-5 while the levels of il-2 and ifn-y production were comparable to bulk cultures obtained from mice at the peak of infection. there was little inflammatory response in the lungs obtained from mice immunized with the control vaccinia. in contrast , lungs from mice immunized with vac-f or vac-g showed significant infiltration of inflammatory cells. there was a striking infiltration of eosinophils in the lungs from mice primed with vac-g. these eosinophils could be detected aroud major bronchi and blood vessels, as well as, in some cases, in lung parenchyma. this study suggests that the immune responses to different viral glycoproteins may be distinct and may play important roles in viral pathogenesis. during infection of normal mice with lymphocytic choriomeningitis virus (lcmv), nk cell responses peak on day 3 and subside as cd8+ t cell responses are activated at day 7 post-infection. in contrast, 02m-/-mice, lacking cd8+ t cells, have dramatically elevated nk cell responses on day 7 postinfection. the 02m-/-response is evidenced by increased nk cell activity, as well as up to 5-fold increases in blast and total nki.i+cd3-cell numbers. nk cell responses in normal mice are cyclosporin a (csa)-resistant and interleukin (il)-2independent, whereas day 7 nk cell responses in 02m-/-mice are csa-sensitive and il-2-dependent. to investigate the role of additional cytokines in regulating cellular responses during acute viral infections, production and function of il-4 and transforming growth factor4 (tgf-0) were examined. induction of il-4 mrna, at late times post-infection of normal mice, was shown by in situ hybridization of t cell-enriched splenic leukocytes and polymerase chain reaction (pcr) amplification of cdna from rna. ellsas of media cor.aitioned with cells isolated on days 0, 3, 5, 7, 9, and 14 post-infection demonstrated delayed induction of il-4 protein as compared to ctl activation. tgf-0, evaluated in biological and elisa assays, was induced maximally at days 7 to 9 post-infection. the kinetics of tgf-0 production by cells from infected 82m-/mice was similar to that of normal mice. however, cells from 02m-/-mice produced il-4 at early but not at late times postinfection. together, these results suggest that either il-4 is a critical cytokine for shutting off nk cells during normal responses to viral infection, or that the 02m-l context modulates responsiveness of nk cell subsets to other late cytokines. studies are in progress to distinguish between these two possible mechanisms. the induction of fever in response to infection is an important host defense mechanism that enhances aspects of the immune response and restricts the replication of some microorganism. vaccinia virus, a member of the poxvirus family, is a complex cytoplasmic dna virus that encodes a variety of proteins that interfere with host immune functions, such as complement regulatory factors and soluble receptors for il-lp, tnf and ifny. here we show that expression of the vaccinia virus il-1p receptor (vil-lpr) in the w r strain prevents the febrile response and reduces the severity of infection in intranasally inoculated mice. fever was recorded on days 1-6 after infection of mice with a vil-lpr deletion mutant, but not in animals infected with wild type wr or a virus revertant. these studies were extended to other virus strains that were used as smallpox vaccines, and expression of the vil-lpr was consistently found to prevent the onset of fever. vaccinia virus induced a severe hypothermia after 6 days in infected mice that was independent on vil-lpr expression and correlated with virus replication in the brain, the organ that controls body temperature. these results represent the first example of a virus mechanism to inhibit the host febrile response and suggest a central role for soluble il-lp in the induction of fever in poxvirus infections. measles virus (mv) infection can depress cell-mediated immune responses for months following clinical disease. mv is known to infect the thymus during human illness and this may contribute to immune suppression. we have used the scid-hu m o w with co-implants of human fetal thymus and liver to determine the effect of virulent and avirulent strains of mv on the thymus. scid-hu mice were. infected by direct inoculation of the graft with 103 pfu of either a wild type strain of mv(chicago-1,chi-1) or an attenu-ated strain (moraten, mor) and sacrificed at intervals over 28 days. peak viral titers, as judged by plaque assay on vero cells, were reached by chi-1 on d4 (105.7 pfu/ third of implant), and moron d21 (103.2 pfu/ third of implant). hematoxylideosin stained sections of chi-1-infected thymuses showed marked distortion of the cortex and medulla by d4 with thymocyte poilolosis and decreased cellularity. by d14, these. implants were mostly devoid of normal thymocytes. mor-infected thymuses showed relatively preserved architecture and cellularity. suspensions of the cells from implants stained with mabs to cd3,cd4 and cd8 were analyzed by flow cytomehy. there were significant decreases in the cd4+cd8+ cell pop-ulation by d10 with complete loss of all such cells by d28 with chi-1, and only modest reductions with mor. immune fluorescence staining of sections with a mv mab to hemagluttinin(ha) and abs for either human cytokera-tins(ael/ae3) or cd15 co-localized mv predominantly to epithelial and monocytic cells. additionally, mv antigen was present diffusely by d4 in both cortex and medulla in chi-i infection whereas mor-infected implants had only patchy distribution by d21. only rare cells stained both with mv ha and cd2 or cd4. mv ha was not expressed over background on any cd4+ cells judged by facs. we conclude that mv replicates in the scid-hu thymic implant primarily in epithelial and monocytic cells, and that the attenuated virus reproduces more slowly and with less cellular disruption. little mv ha could be demonstrated in thymocytes, therefore the data suggest that significant infection of the thymic epithelial stroma disrupts the thymic microenvironment which normally supports and aids in selection of immature t cells. part of the long-term immune suppression seen in mv infection may be due to infection of the thymic epithelial stroma with subsequent loss of thymocytes. it is becoming increasingly evident that many poxviruses contain genes that enable the virus to evade the host's immune system. myxoma virus is a leporipoxvirus and is the causative agent of myxomatosis, a rapidly lethal disease in the european rabbit (oryctolagus cuniculus). one possible mechanism of immune evasion is virus-induced downregulation of cell-surface receptors important for an immune response. cell-surface levels of several receptors on a rabbit t cell lymphoma cell line (rl-5) were monitored by flow cytometry. following infection with myxoma virus, cellsurface levels of cd4 were found to drop dramatically. other cell surface antigens such as cd18, cd43, and cd45 were unaffected during infection with myxoma virus. further more, the downregulation of cd4 by myxoma virus could be inhibited by treating cells for an extended period of time with pma, suggesting that the downregulation was not simply a masking of the epitope via viral antigens. analysis of cd4 levels in the presence of cytosine arabinoside indicates that late gene expression is not necessary for the modulation. since the tyrosine specific protein kinase p56lck associates with the cytoplasmic domain of cd4 we have also examined the association of p56lck with cd4 as well as steady state levels of p56lck during viral infection. the modulation of surface cd4 has also been described in hiv infected t cells suggesting that the loss of cell-surface cd4 may be a common viral immune evasion tactic by lymphotrophic viruses. i n addition, stably-transfected cell l i n e s expressing e i t h e r u s 1 1 o r us2-6 gene products s i g n i f i c a n t l y reduced l e v e l s of mhc class i heavy chain. studies are i n progress t o f u r t h e r d e f i n e t h e mechanism by which t h e s e v i r a l gene products a l t e r immune recognition. cytotoxic t lymphocytes (ctl) may play a significant role in containing the spread of hiv in infected individuals. although hiv-infection is associated with immune suppression, a vigorous ctl response has been detected in infected adults. hiv can be transmitted from mother to child. one third of vertically infected children has a rapid evolution toward disease, with onset of aids before 18 months. the other two thirds remain asymptomatic for years. the bimodal course of disease evolution in hiv-infected children could be related to differences in the host immune control of viral replication. hiv-specific ctl response from fresh and in vitro activated pbmc of hiv-infected children was measured. the vast majority of infected chidren had detectable hiv-specific ctl, which where cds+cd8+. we previously showed that among children with a slow disease progression, fresh ctl were more frequent in the p2a(paucisymptomatic) group than in the pl(asymptomatic) and the p2b-f groups (symptomatic group). the cohort of children has now been followed during 4 years, and 46 children have been tested at least once. we found that ctl responses were less frequent in the children with a rapid disease progression than in the children with a slow disease progression at the same age. our data suggest that ctl response is an important factor in delaying disease evolution. we, as well as others. have proposed that sag function is critical to the ability of milk-borne m m n to infect mice. to determine whether this is the case, we created transgenic mice (hyb pro/cla) with a frameshift mutation int the sag gene. young hyb pro/cla mice (c 10 weeks of age) showed no deletion of their cognate vp14* t cells, unlike transgenic mice carrying a functional sag gene however, a slow, progressive loss was seen in the hyb prolcla mice as they aged, indicating that it was due to expression of wild type sag protein. thus, as the hyb pro/cla mice aged, there was production of virus that appeared to lose the cla mutation. the hyb pro/cla mice produced transgene rna in their lactating mammary gland and shed virus in their milk. their nontransgenic offspring of showed infection with transgene-encoded mmtv because they had the typical slow deletion of vp14+ t cells characteristic of c3h mmtv infection and because we detected transgene-derived m m n rna in their mammary glands. cloning and sequencing of the viral rna produced by the nontransgenic offspring of the hyb pro/cla mice showed that recombination between the mtv-1 endogenous viral rna and the transgene-encoded rna occurred, such that the frameshift introduced by the cia mutation was repaired. these results show that there is selection of infectious virus that contains a functional sag gene. thus, it appears that the only virus that is capable of being transmitted by the milk borne infection pathway is that which encodes a functional sag protein. hepatitis b virus (hbv) causes acute and chronic liver diseases and is closely associated with hepatocellular carcinoma. in order to understand the cellular immune response against hbv in chronic hbv infection, t cell proliferation, cytotoxicity and cytokine production were studied. we found that although the majority of asymptomatic hbsag carriers and patients of chronic hepatitis b (chb) had no proliferative response to hbsag, some individuals in both groups showed significant t cell proliferation against hbsag. in contrast, the proliferative t cell response to hbcag in asyrnpatomatic hbsag carriers was significantly stronger than that in patients of chb with acute exacerbation. in addition, the frequency of hbcag-reactive t cell precursors measured by limiting dilution assay was much higher in asymptomatic hbsag carriers than in patients of chb. therefore, t cell responses against hbsag and hbcag are regulated differently in chronic hbv infection. furthermore, we demonstrated hbsag-and hbcag-specific cytotoxic t lymphocyte (ctl) activity in asymptomatic hbsag carriers, using autologous hbsag-and hbcag-expressing lymphoblastoid cell lines (lcl) as target cells, respectively. the cloned ctl were able to produce ifn-y, tnf-a or gm-csf after stimulation. these findings demonstrate that t cell response to hbv is not completely suppressed in asymptomatic hbsag carriers. most of them have strong hbcag-specific response and some of them have hbsag-specific response. transcription and tax the human t-lymphotropic virus type i (htlv-i) promoter contains the structural features of a typical rna polymerase i1 (pol 11) template. the promoter contains a tata box 30 bp upstream of the transcription initiation site, binding sites for several pol i1 transcription factors, and long poly a+ rna is synthesized from the integrated htlv-i proviral dna in vivo. consistent with these characteristics, htlv-i transcription activity was reconstituted in v i m using tbp, tfiia, rtfiib, rtfiie, rtfiif, tfiih and pol 11. in hela whole cell extracts, however, the htlv-i ltr also contains an overlapping transcription unit (otu). htlv-i otu transcription is initiated at the same nucleotide site as the rna isolated from the htlv-i-infected cell line, mt-2, but was not inhibited by the presence of a-amanitin at concentrations which inhibited the adenovirus major late pol i1 promoter (6 pglml). htlv-i transcription was inhibited when higher concentrations of a-amanitin were used (60 pglml), in the range of a typical polymerase in (pol 111) promoter (va-i). purified tax, transactivates this promoter 5-to 10-fold in v i m . interestingly, basal and tax,-transactivated transcriptional activity of the htlv-i ltr could be reconstituted with the 0.5 m phosphocellulose fraction. these observations suggest that the htlv-i ltr contains overlapping tax,responsive promoters, a typical pol i1 promoter and a unique pol i11 promoter which requires a distinct set of transcription factors. tax, further in vifro transactivates a polymerase i1 template containing the 21 base pair repeats cloned upstream of the ovalbumin promoter and g-free cassette. tax,-transactivated transcription was concentration dependent and inhibited by low concentrations of a-amanitin. flaviviruses are arthropod-borne viruses whose route of infection is via the skin. they are mostly neurotropic and responsible for significant human morbidity and mortality. the classic cell-mediated immune response to a viral infection may be influenced by the ability of these viruses to modify expression of cell-surface molecules involved in the presentation of antigen to, and activation of, t cells. the skin langerhans cell is the prototypic nonlymphoid dendritic cell and as such is uniquely placed to participate in a response against epidermally-acquired viral infections. the migratory properties of these cells contribute to their role as initiators of t cell-mediated immune responses within the draining lymph node. we have previously shown infection of epidermal cells in vifro by the flavivirus west nile (wnv) results in an increase in mhc class i and i1 expression on the majority of epidermal cells and langerhans cells respectively. in this study a technique for infecting the epidermis with wnv in vivo was developed. tme-dependent increases in the surface expression of a number of antigens which are involved either directly or in a co-stimulatory capacity in initiating a cell-mediated immune response, were detected on both the majority of epidermal cells and the langerhans cell population using flow cytometry. these increases were detectable as early as 16 hours after infection. a significant decrease in the percentage of langerhans cells remaining in the epidermis was observed within 48 hours of infection. the phenotypic changes observed in vivo are analogous to those described following in vifro culture of langerhans cells. these results, together with the reduction in langerhans cell numbers, may represent the in situ maturation and concomitant migration of these. cells as a consequence of virus-induced cytokines within the skin microenvironment. which cause a wide variety of illnesses with high morbidity and mortality in humans throughout the world. their high genomic stability argues for a survival strategy related more to interaction with the vertebrate host immune response, than a dependence on viral genetic mutation. our previous work has shown that west nile virus (wnv) infection of many cell types directly induces functional increases in class i and 11 mhc expression. we report here that wnv infection of human embryonic fibroblasts (hef) results in the increased expression of cd54 by two distinct mechanisms. an early, direct cytokine-independent mechanism operates within 2 h of virus infection, while an indirect mechanism, regulated by type 1 interferon (ifn), operates within 24 h of virus infection. cd54 expression increased by 4-5 fold within 2h of wnv infection on hef, and by 6-7-fold within 24h. wnv-inactivated, conditioned supematants removed from infected hef cultures after 4 h incubation did not alter cd54 expression on unqimulated hef. whereas conditioned supernatants from 24 h-infected cultures increased cd54 expression by about 1.5-2-fold after incubation for 24 h, but not after 4 h, similar to cd54 induction by 200ulml of ifn-p. increased cd54 expression on hef by wnv was also cell-cycle dependent. cd54 increased only in quiescent, contact-inhibited infected hef in go phase. in contrast, induction of cd54 by types 1 and 2 ifn was not cell-cycle dependent. other viruses, including double-stranded dna viruses, vaccinia, and adenovirus 2 and 5, and the single, positive-stranded rna alphavirus, semiliki forest virus, did not induce cd54 expression on hef after 24 h. another alphavirus, ross river, was able to induce cd54 but only by the indirect mechanism of type 1 ifn-dependent release. poly i.c, also, increased cd54 expression to the same extent as ifn-p after 24 h, making it unlikely that the early increase was due to a nonspecific viral effect. the closely related flavivirus, kunjin, induced increased cd54 expression in a manner similar to wnv. the ability of flavivhses to induce increased cd54 expression directly within a few hours of infection may be an important virus-host survival strategy promoting cell-cell adhesion and hence possible further viral infectiodreplication. recognition of viral peptides presented on the cell surface in association with class i mhc molecules leads to lysis by cytotoxic t cells (ctl) and forms an important part of the immune response to hiv infection. hiv virus has a high mutation rate and variation in the region of the viral epitope may allow evasion of this immune response. variation could theoretically affect processing of the antigen, binding of the epitope to the hla molecule or recognition of the presented epitope on the cell surface. we have studied proviral sequence variation in gag and ctl responses in a number of hla b8 patients infected with hiv. amino acid substitutions, such as a lysine to arginine change at position 3 of the pi7 gag nonamer cckkkyklk, lead to loss of recognition of the peptide by ctl from the patient whose provirus contained this sequence. these variant peptides bind to hla 68 with comparable affinity to the index peptide suggesting that this loss of recognition is likely to be caused by changes in the interaction between the hla-peptide complex and the t cell receptor. other changes, such as lysine to arginine or glutamine at position 7, not only cause loss of recognition, but also lead to inhibition of lysis of targets bearing the index peptide. thus it appears that in addition to loss of recognition by cytotoxic t cells, naturally occurring epitope variants may act as "antagonists", as has been demonstrated in mhc class ii systems. antagonism may be an important mechanism allowing immune escape by the hiv virus. genes. subsequent complex formation between peptide, class i and p2microglobulin in the er results in stable cell surface expression of the trimeric mhc-1 molecule. in previous studies we showed that in hpv-16 positive cervical carcinomas there was a loss of mhc-1 protein expression, which correlated at the single cell level with loss of tap protein. in this study we investigated whether loss of tap and mhc-1 is mediated by an hpv-16 encoded protein. human keratinocytes were transfected withvarious hpv-16 constructs including pat16, the full length genome, pat16esx the full length genome with a premature stop codon in e5, puc.et16, the e6 and e7 oncogenes only, and pkve5, expressing e5 from mouse moloney ltr the different constructs were transfected into primary keratinocytes, cloned cells grown in medium supplemented with and without y-interferon ( y -a r ) for 48 hours. cells were harvested and total rna and protein harvested for northern and western blots respectively. western blots showed very low steady state levels of tap-1 and mhc-1 heavy chains in the cells with pat16 as well as those containing es alone, which was marginally increased by y-lfn. in contrast, primary keratinocytes, pat16esx and puc.et16 lines showed comparable tap-1 and mhc-1 protein levels, which increased a & y-ifn treatment. northem blots showed no differences in the amounts of tap-1 and mhc-1 mrna between the different cell lines. the data indicate that expression ofhf'v-16 e5 leads to post-transcriptional loss of mhc-1, presumably by interfering with tap. to map and characterize functional differences between e1a of ad5 and adl2, we previously constructed a series of hybrid ad5/12 e1a genes and used them with ad12 e1b to transform primary hooded lister rat kidney cells. at least two regions within the first exon of ad12 e1a were identified which influenced tumorigenicity. this study further examines the role of these regions in tumorigenicity by analyzing their affect on cell surface mhc class i expression and sensitivity to class i-restricted cd8+ as well as to non-class irestricted nks. the bcrfl open reading frame of epstein-barr virus exhibits remarkable sequence homology with the coding sequences of interleukin-10 from a variety of organisms. many of the numerous immunological properties ascribed to interleukin-10 are shared by the product of bcrfl and this has led to it being termed viral interleukin-10. in order to investigate the activity of viral interleukin-i0 (vil-10) and its interactions with the human interleukin-10 receptor we have expressed the protein in a bacterial and the eukaryotic cos-7 expression systems. the bacterially expressed vil-i0 was partially purified and used to set up two assays to measure i l l o activity: i)the increase in igm secretion from an ebv transformed b cell line -mt4.l and ii)the downregulation of class ii hla expression on the human monocytic cell line thp-1. a series of deletion mutants (both n-and c-terminal as well as an internal deletion to remove a putative heparin binding domain) were constructed to identify possible domains within the vil-10 protein that interact with the hil-10 receptor and confer its biological activity. a number of these mutants have been expressed in the cos-7 expression system and their structure and biological activity are currently being assessed. the identification of the domains within vil-10 that interact with the receptor or accessory proteins may aid in the understanding of the possible role of vil-i0 within the ebv life cycle and in the pathogenesis of the numerous diseases associated with the virus. generation. to further test the role of ctl in ad pathogenesis, viruses lacking the cll epitopes were tested when mutants that lack the immunodominate ctl epitope in eia where used, a second immun-ssive epitope in elb becorns the predominate target of clu. these findings arc important since human ad is currently being tested as a vector for gene therapy of cystic fibrosis. our data suggest that when consuucting ad vectors to be. used for gene therapy, one must retain either the 10.4k or 14.7k genes to decrease pathology and that meting the genes that encode the antigens that a n recognized by clu does not prevent the generation of ad specific clu. the interferons (ifns) a n ? a family of cytokines whose functions include the protection of cells against viral infection. type i ifns include the 15 ifna subtypes and ifnp that compete for binding to the same cell surface receptor, while type ii ifn (ifny) binds to a different receptor. the orthopoxviruses, of which vaccinia virus (vv) is the prototypic member, have developed a number of anti-ifn strategies. the vv e3l protein competitively binds dsrna and prevents the activation of ifninduced and dsrna-activated protein kinase (pkr), while the vv k3l protein shows sequence similarity to the eukaryotic initiation factor 2a (eif2a) that is phosphorylated and inactivated by pkr. the k3l protein competitively binds the kinase and blocks host eif2a phosphorylation and hence ifn-induced inhibition of host protein synthesis. onhopoxviruses also suppress cytokine action by expressing soluble cytokine receptors that bind and sequester the ligand; to date soluble receptors for interleukin-18, tumour necrosis factor and ifny have been described. supernatants from vv-infected cells were found to contain a soluble inhibitor of type i ifn that was conserved in most of the orthopoxviruses tested. the inhibitor was produced early in infection and did not inhibit ifny. the ifna/p inhibitor was mapped and the gene expressed from recombinant baculovirus. the inhibitor blocked the binding of 125i-ifna to u937 cells and binding of 125i-ifna to supernatants from baculovirus and vv-infected cells demonstrated that the inhibitor functioned as a soluble receptor for 1fnc1fp. direct binding of 1251-ifna to vv wr supernatants revealed that the soluble ifna/p receptor had a high affinity for type i ifn. deletion of the gene from the vv genome and ligand blotting of the soluble receptor demonstrated that ifn binding was encoded by a single protein. competitive binding curves using ifna from other species revealed that the poxvirus soluble ifndp receptor bound human and bovine ifn with high affinity but murine ifn with relatively low affmity. interestingly, the soluble ifncrip receptor is highly conserved in variola virus. given the importance of ifn in antiviral defense it is likely that the soluble ifndp receptor plays an important role in the virulence of the orthopoxviruses. endogenous processing of a viral glycoprotein for presentation t o cd4+ t cells has defined a previously under-investigated pathway in antigen processing and presentation. it may be important not only for pathogens, but also for self-proteins, and thus may be involved in self-tolerance. we have been characterizing the processing o f the er-restricted gpt glycoprotein of vesicular stomatitis virus (vsv) biochemically and enzymatically, by cellular localization using confocal immunofluorescence, cellular fractionation, and by t cell recognition assays. by flow cytometry, gpt is undetected on the plasma membrane; in contrast, the wild type protein (g) is readily found following infection of a20 cells with a vaccinia virus vector, leading t o endogenous synthesis. the gpt can be found exclusively in the er compartment using co-localization with markers for er (signal peptide binding protein, calnexin), and not in the golgi compartment (a-mannosidase 11, wheat germ agglutinin), endosome, lysosome, or surface plasma membrane. this is consistent with the characteristics o f the localization of the proteases which appear to be responsible for its degradation. work is in progress to localize the site of peptide binding to mhc heterodimers. supported by nih grant a118083 t o csr. presentation of an out-of-frame class i restricted epitope. t.n.j.bullock and l.c.eisenlohr, department of immunology, thomas jefferson university, philadelphia, pa 19107. antigen presentation by class i mhc molecules is thought to require the degradation of fully formed proteins in the cytosol. this degradative process supplies oligopeptide epitopes for transport into the endoplasmic reticulum (er) where they can interact with and stabilize class i molecules. stable class i molecules, associated with p2-microglobulin, can then proceed to the cell surface where they present the epitopes to t cell receptors. the generally accepted model for protein translation, the scanning hypothesis proposed by ko&, is thought to describe the traditional method of translation for the majority of proteins. we wished to test the hypothesis that any internal methionine that is in good translation initiation context can be a source of short peptides, which may then be processed into class i epitopes. nucleoprotein gene (np), the target of the ctl response of several inbred mouse strains. np contains three class i restricted epitopes at amino acids 50-57 (h2-kk), 147-155 (h2-kd) and 366-374 (h2-db). the frameshift was introduced 26 amino acids upstream of the h2-kd epitope. the mutated genes were then recombined with vaccinia virus and tested for presentation using ctl restricted to each of the epito s described above. we found that, whilst presentation of the h2-i@ epitope was unaffected by the frame shift, the epitope proximal to the frameshift (h2-kd) was no longer presented to appro riately restricted ctl. however, presentation of the distal h2-dg epitope was retained. therefore we have shown, using a viral protein and a viral expression system, that out-of-frame epitopes can be processed and presented to ctl. work is ongoing to c o n f m that internal methionines are capable of providing a platform for the initiation of translation for in-frame and out-of-frame epitopes. we have created a frameshift mutation in the influenza pr8 the fine specificity of t cell recognition of peptide analogues of the influenza nucleoprotein epitope np 383-391 srywairtr was studied using hla b27-restricted influenza-specific cytotoxic t cell (ctl) clones, of defined t cell receptor (tcr) usage, derived from unrelated individuals following natural infection. synthetic analogue peptides were synthesized containing single amino acid substitutions, and tested both for binding to hla b'2705 in vitro, and for presentation to ctl clones by hla 827positive targets. even conservative amino acid substitutions of the peptide residues p 4 , 7, and 8 profoundly influenced ctl recognition, without affecting binding to hla 8'2705. these amino acid side chains are thus probably directly contacted by the tcr. ctl clones which used the tcr v a l 4 gene segment (but not those using tcr va12) were also sensitive to p1 substitutions, suggesting that the tcr alpha chain of these clones lies over the n terminus of bound peptide, and that the "footprint" of certain tcrs can span all exposed residues of a peptide bound to mhc class 1. these results, taken together with previous structural and functional data, suggest that, for nonarner peptides bound to hla 827, p i , p4 and p8 are "flag" residues with tcr accessible side chains. the e3/19k protein of human adenovirus type 2 (ad2) is a resident transmembrane glycoprotein of the endoplasmic reticulum. its capacity to associate with class i histocompatibility (mhc) antigens abrogates cell surface expression and the antigen presentation function of mhc antigens. at present, it is unclear exactly which structure of the e3/19k protein mediates binding to mhc molecules. apart from a stretch of approximately 20 conserved amino acids in front of the transmembrane segment, e3/19k molecules from different adenovirus subgroups (b and c) share little homology. remarkably, the majority of cysteines is conserved. in this report, we examined the importance of cysteine residues (cys) for structure and function of the ad2 e3/19k protein. we show that e3/19k contains intramolecular disulfide bonds. by using sitedirected rnutagenesis, individual cysteines were substituted by serines and alanines, and mutant proteins were stably expressed in 293 cells. based on the differential binding of monoclonal antibody tw1.3 and cyanogen bromide cleavage experiments, a structural model of e3/19k is proposed, in which cys 11 and cys 28 as well as cys 22 and cys 83 are linked by disulfide bonds. both disulfide bonds (all four cysteines) are absolutely critical for the interaction with human mhc antigens. this was demonstrated by three criteria: loss of e3/19k coprecipitation, lack of transport inhibition and normal cell surface expression of mhc molecules in cells expressing mutant e3/19k molecules. mutation of the three other cysteines at position 101, 109 and 122 had no effect. this indicates that a conformational determinant based on two disulfide bonds is crucial for the function of the e3/19k molecule, namely, to bind and to inhibit transport of mhc antigens. previous studies have suggested that several abundant cmv proteins are major immunogenic targets in seropositive adults. we are interested in defining the major viral protein targets of a cd8' ctl response, in order to derive a vaccine strategy for individuals who are unable to mount immune responses which are lymphokinedependent because of immunosuppression. hla-typed and cmv-pgsitive normal volunteers who have hla-a alleles that represent -75% of the u.s. population are being tested to determine which of 5 abundant cmv proteins they recognize by a cd8' ctl response: p28, p65, p150, ie, and gb. t cell lines will be derived in order to unambiguously determine the hla restriction of the cd8' ctl response to each of these proteins. proteins which are recognized by the most hla diverse population will be further characterized in terms of mapping of class 1 epitopes through the use of t cell clones derived from the polyclonal cell lines by limiting dilution. the defined epitopes will form the basis of a vaccine strategy to augment the memory responses of seropositive volunteers against cmv. these epitopes will be used to boost the ctl precursor frequency of bone marrow transplant donors as a means to transfer cellular immunity to immunosuppressed hematologic transplant recipients. an alternative strategy is to immunize seropositive individuals with recombinant viral proteins as a means to boost immunologic memory. we are pursuing that strategy in a transgenic murine model of hla-a2.1 developed by dr. l. sherman (scripps institute, la jolla). we are vaccinating the transgenic mice with two well defined cmv proteins, p65 and gb together with either of two lipid-based adjuvants, commercially available d0tapm (bcehringer-mannheim) or mf5gth (chiron, emeryville, ca). our preliminary studies with hsv-2 gb demonstrate that both adjuvants are effective at eliciting murine class i restricted responses against the protein. current studies are evaluating the recognition properties of the adjuvant-cmv protein complexes by hwa2 as a restriction element in the transgenic model. the ctl response to sendai virus in c57by6 mice is directed almost exclusively to a single h-2kb-restricted epitope derived from the virus nucleoprotein, npj24-332 (sev-9). analysis of 18 independent t cell hybridomas generated from c57by6 mice following primary sendai virus infection has shown that a very diverse repertoire of tcr is selected in response to this epitope. crystallographic analysis of sev-9 bound to kb has shown that the side chaiis of peptide residues phpi, gl484, a d s , and alaps protrude towards the solvent and are potentially available for recognition by the tcr notably, residues gi484 and a d 5 protrude prominently from the peptide binding site due to their l o c a l i o n on a bulge in the center of sev-9. to determine the importance of each of these residues for t cell recognition, we analyzed hybridoma responses to sev-9 analogs substituted at each of these four positions. preliminary data showed there generally appeared to be dominant recognition of glyp4 and asnm. however, individual hybridomas exhibited distinct patterns of fine specificity for residues phep1 and alaps. thus, individual hybridomas were dependent on one, both, or neither of these residues for recognition of sev-9. these data are consistent with a critical role for the gi94 and a d 5 in governing tcr-sev-9eb recognition and suggest a structural basis for the diversity of the tcr repertoire selected by this @tope. previous results from this laboratoty demonstrated that the dominant influenza a epitope recognized by hla42.1 restricted ctl from hla-a2.1 uansgenic mice was the m1 peptide epitope that is immunodominant in human ctl responses. however, analysis of a large number of ctl lines revealed a subset of influenza a/pr/8/34-specific murine ctl that recognized an hla-a2.1 restricted epitope distinct from m1. using recombinant vaccinia viruses encoding werent influenza gene segments, the epitope recognized by these ctl was shown to be derived from the a/pr/8 nsl protein. because these ctl did not recognize targets infected with the a/alaska/6/77 saain of influenza, candidate peptide epitopes were synthesized based on sequences that included an hla-a2.1 specific binding motif and that differed between a/pw and nalaska all of these ctl recognized a nonamer and a decamer peptide which contained a common 8 amino acid sequence and two distinct sets of bmding mtif residues. however, the n0name.r peptide was able to sensitize ctl for half maximal lysis at 80-2500 fold lower doses than either the octamer or decamer. the homologous peptide derived from nalaska nsl contained conservative amino acid changes at positions 4 and 8 and was not recognized at any tested concentration, although it bound with higher &ity to hla-a2.1 than the peptide from a/pw8. the a/pr/8 nsl nonamer epitope was also recognized by human influenza a specific ctl derived from two individuals. these results substantiate the general utility of hla class i aansgenic mice for the identification of human cn epitopes for other pathogens. furthemore, the recombinant dhfr was functional in the induction of gb epitope-specific ctl response upon immunization of c57bv6 mice. these results indicate that an viral epitope expressed in a cellular protein can be. efficiently processed, presented and recognized by epitope-specific ctl, and suggest that the cellular proteins can be used to express ctl epitopes for induction of cd8+ immune responses. virus-specific cytotoxic t lymphocytes (ctl.) were generated a day later at this site. to determine which apc was capable of stimulating virusspecific ctl precursors in the mln, b, t and dendritic cells from the mln of influenza-infkcted mice were separated and examined for the presence of virus. the predominant cell type which contained infectious virus was the dendritic cell. b and t cells from the mln contained little, ifany, virus. the apc capacity ofthese populations was tested by their ability to stimulate vir~~-~pecific t cell hybridomas. only dendritic cells from the mln of influenza-infected mice were able to stimulate virusspecific t cell hybridomas, althwgh all apc populations from both naive and influenza-infected mice were effective stimulators after in y h pulsing with the appropriate intluenza peptide. potential apc populations were also separated from the lung. v i s was detected in bronchioalveolar macrophages and dendritic cells but not b or t cells. both macrophages and dendritic cells isolated from intlum-infected lungs could stimulate virus-specific t cell hybridomas. the ability of the mln and lung apc populations to stimulate naive cd8' t cells and generate virus-specific ctl is currently being examined. virus infected cells present only a very limited number of peptides intracellularly processed from a viral protein to ctl even when many peptides hearing the mhc class i-restricted binding motif are present in the protein. infection of h-2b mice w i t h lymphqtic choriomeningitis virus (lcmv) induces a cd8+ ctl response directed against three wellcharacterized epitopes presented by h-2db molecules: "396-404 (fqpq-ngqfi), gp33-43 (kavynfatcgi) and gp276-286 (sgven-pggycl). the h-2db motif is characterized by a sequence of 9 to 11 a.a. with two anchor residues: asn at position 5 and hydrophobic (met, ile, leu) at the c-terminus. the lcmv np and gp proteins contain thirly-one other peptides exhibiting the db motif. however, no ctl response against one (or more) of these peptides has been characterized. peptide binding to mhc is a critical step in antigen presentation. the aim of this study was therefore to analyze the binding properties of the potential db lcmv peptides. the 34 lcmv peptides and 11 known db-selective peptides were synthesized and their mhc binding affinities measured in two db-specific binding assays. most of the lcmv peptides (28/34) did not bind to db. the other 6 (including the 3 epitopes) and all the known db peptides showed good affinity. comparison of the sequences (good vs. non binders) allowed the identification of auxilliary anchors required for high binding affinity or of negative elements hampering mhc binding. in addition to the main anchors, the positive and negative factors at secondary residues play a crucial role in governing peptidemhc interactions. knowledge of such factors might he of importance for the prediction of mhcrestricted ctl epitopes. etienne joly, andrea gonzalez, carol clarkson, jonathan c. howard and geoffrey w. butcher. laboratory of immunogenetics, department of immunology, the babraham institute, cambs cb2 4at, uk. tap transporters from rats can be divided into two allelic groups, depending on their capacity to provide the rt1.aa molecule with an appropriate level of suitable peptidesl. recent results suggest that this might correlate with the rt1.aa molecule requiring arginine-ended peptides (powis et al., manuscript submitted), which the tapb allele of the transporter is unable to translocate across the er membrane efficiently2~3. rt1.a alleles are naturally linked with the tapa or the tapb allelic group4. we have set out to characterise various alleles for the rt1.a molecule, and find that, for the majority of tapaassociated rt1.a molecules, 3 acidic residues line the c/e pocket, dictating arginine as c-terminal anchor residue for the bound peptides. on the other hand, in tapb-associated rt1.a molecules, one acidic residue at the most is found in the c/e pocket, which certainly results in a different anchor residue for the bound peptides. the selective pressure of viral infections must have driven this coevolution which affects dramatically the array of peptides presented to cytotoxic t lymphocytes. cytotoxic t lymphocyte responses in hiv infection can be impaired due to variation in the epitope regions of viral proteins such as gag. we show here an analysis of variant epitope peptides in three gag epitopes presented by hla b8. seventeen variant peptides were examined for their binding to hla b8; all but one bind at concentrations comparable to known epitopes. all except two could be seen by ctl clones grown from hla b8 positive hiv-1 infected patients and were therefore immunogenic. however, in one haemophiliac patient studied in detail, there was a failure to respond to some of the peptides that represented virus present as provirus in his peripheral blood. in one case his ctl had previously responded to the peptide. thus there was a selective failure of the ctlresponse to variant epitopes. this impaired reaction to new variants and failure to maintain responses to some epitopes late in hiv infection could contribute to the loss of immune control of the infection. pira, anna ferraris, daniele saverino, peifang sun and annalisa kunkl; dept. immunology, san martino hosp. univ. of genoa, 16132 genoa, italy. th epitopes present on viral proteins can be recognized by specific th cells if appropriately expressed by antigen presenting cells (apc) as a result of uptake and processing. since viral epitopes are not simply present in the context of viral proteins, but also in the context of whole viral particles, it is important to determine the role of the molecular and/or structural context on antigen uptake-processing-presentation. therefore we have generated panels of cd4+ human t cell lines and clones specific for different hiv antigens (gp120, p66, p24), in order to test their ability to respond to the same epitopes present within synthetic peptides, recombinant proteins or inactivated virions (provided by g. lewis, dept. microbiology, univ. maryland, baltimore). we could identify t cell lines and clones that were able to discriminate the molecular and structural context of the epitops. certain t cells, in fact, responded to peptides and proteins, but not to viral particles, whereas other t cells were also able to proliferate when challanged in vitro with autologous apc and viral particles. the data suggest that in the human th cell repertoire specific for viral antigens t cells exist that can discriminate the molecularstructural context of th epitopes. it will be interesting to ascertain whether t cells specific for epitopes that can only be recognized when provided in the context of a soluble molecule, but not of a viral particle, have any relevance in viva protection, or are a simple by-product of the cellular immune response. eric g. pamer, merceditas s. villanueva, section of infectious diseases, yale university school of medicine, new haven, ct 06520 listeria monocytogenes is a gram positive bacterium that infects macrophages and secretes proteins into host cell cytosol. the murein hydrolase p60 is secreted by l. monocytogenes and is required for complete bacterial septation. in the infected macrophage secreted p60 is processed by the host cell into the nonamer peptide p60 217-225 and is presented to cytotoxic t lymphocytes by the h-2kd mhc class i molecule. we have used strains of l. monocytogenes that secrete different amounts of p60 to show that the rate of p60 217-225 production is proportional to the amount of antigen secreted into the host cell cytosol. p60 is degraded in the host cell cytosol with a half life of 90 minutes. the appearance of p60 217-225 is coupled to the degradation of newly synthesized p60. we have determined the rate of intracellular p60 secretion and by accounting for the rate of p60 degradation we estimate that approximately 35 p60 molecules are degraded to produce one p60 217-225 epitope. this ratio is maintained over a range of intracellular antigen concentrations. our findings provide an estimate of the efficiency of antigen processing and demonstrate the remarkable capacity of the mhc class i antigen processing pathway to accommodate new epitopes. we have isolated and characterized three cytotoxic t lymphocyte (ctl) clones from the peripheral blood of two acute seroconversion patients and one patient in the first trimester of pregnancy. these clones were cd8+ and class i hlarestricted by the b7 molecule. all three clones recognized lllb and rf but not mn strains of hiv-1. using vaccinia vectors expressing truncated versions of the hiv-1 envelope, the clones were found to recognize an epitope within amino acids 287-364, but not including 312-328 of gp120. further mapping of the epitope with synthetic 20-mer peptides overlapping by 10, or 25-mers overlapping by 8, was unsuccessful. the sequence of the region of gp120 recognized by these clones was compared to the predicted hla-87 peptide binding motif and a possible matching region was found. using shorter peptides corresponding to this potential epitope recognition site, the minimum epitope recognized by the clones was determined to be the 10 aa sequence rpnnntrksi spanning amino acids we have further pursued a strategy to define a minimal cytotoxic epitope for a vaccine against cmv infection using t cell clones derived from individuals who have the mhc 835 gene (kind gifts of drs. riddell and greenberg, fred hutchinson cancer research center and dr. robert siliciano, johns hopkins university medical center). we tested by chromium release assay (cra) the recognition of a series of 835 allelic variants of ebv-lcl. by 835 restricted and cmv or hiv-specific t cell clones. several conclusions quickly became apparent. the previously described 8'3501 peptide epitope from pp65 was not able to prime the autologous 835 ebv-lcl for killing by the pp65-specific ctl, whereas a recombinant vaccinia virus expressing whole pp65 could cause the same cell line to be recognized and killed in the same experiment. in addition, an hiv gp41-specific cd8' ctl which has a defined minimal cytotoxic epitope will only recognize and kill a subset of 835 ebv-lcl. the two t cell clones will not recognize each other's autologous ebv-lcl. the resolution of this interesting phenomena comes from sequence analysis of the hla class i b genes from both ebv-lcl. ebv-lcl which contain the b'3502 allele are recognized and killed by the pp65-specific t cell clone, and cell lines carrying 8'3501 alleles are recognized by the hiv gp41-t cell clone. we conclude that the reported cmv pp65 b"3501 restricted epitope is not correct, since the ctl in question will only recognize 6'3502 alleles in combination with the correct pp65 epitope. fragments with or without a signal sequence sensitize rma-s/kd to a similar limited extent. this data i s consistent with an inefficient movement of peptides from the cytoplasm into the er by a tap independent mechanism and does not reveal a processing competent compartment within the secretory pathway. peptide transport by the transporter associated with antigen processing (tap) was studied using a microsome system as previously reported by heemels et. al.. in this system, a radiolabeled synthetic peptide which can be n-link glycosylated is used as the indicator peptide for the transport studies. the transport efficiency of synthetic peptides corresponding to antigenic peptides restricted to the murine kd molecule was measured by inhibition of labeled peptide transported into the microsomes. the transport efficiency of three kd epitopes in the type a influenza virus "147-155, ha204-212 and ha210-219 was found to be similar. an 11 amino acid peptide corresponding to ha204-214 which contains the 204-212 epitope was transported at a similar efficiency as the 9 amino acid minimum epitope. however, when the peptide sequence is further extended by one amino acid to residue 215, this peptide is poorly transported. these results suggest that the flanking region of an epitope can dramatically influence the transport of the epitope. when the transport kinetics of tap was studied using the microsome system, the vmax for transporting the indicator peptide (a variant of np epitope that has the sequence tynrtrali) was found at 260.8 fmolelminute (+/-30.5). the km for this peptide was found to be 231.9nm(+/-31.8). bypassing a block in antigen processing for class i-restricted cytotoxic t cell recognition. amy j. yellen-shaw and laurence c. eisenlohr. thomas jeferson universitv. hiladelphia, pa., 19107. previous work from our laboratory showed that processing of an influenza nucleoprotein (np) epitope (amino acids 147-155) expressed endogenously from a recombinant vaccinia virus "minigene" is severely impaired when a flanking sequence (the dipeptide threonine-glycine) is appended to the cterminus of the construct (147-158/r-). the inhibition of processing is overcome by placing the unprocessed peptide in the context of the fulllength np molecule, demonstrating that regions of a protein outside the epitope itself critically affect the ability of the proteolytic machinery to fragment the protein appropriately. to determine the requirements for bypassing the block in antigen processing, we have constructed an array of "minigene"-expressing vaccinia recombinants in which the unprocessed epitope is extended by varying lengths toward either the c-terminus or the n-terminus of the np molecule. our results show that while an extension of the c-terminus by only one amino acid restores processability, a much longer extension of the n-terminus (75 < n < 100 amino acids) will also allow the substrate to be processed. it is therefore clear that a full-length, properly folded molecule is not required for liberation of the blocked epitope, and that probably more than one mechanism can contribute to enhancement of substrate proteolysis. we hypothesize that the c-terminal extension allows recruitment of an endopeptidase versus exopeptidase ("trimming") activity which is capable of cleaving the difficult bond. we considered the possibility that the n-terminal extension rescues processing by recruitment of the ubiquitin-dependent degradation system. to address this possibility we replaced all available ubiquitination sites (lysine residues) in one of the rescued constructs (50-158/r-) to see if the construct would still be processed and presented. the six available lysine residues were changed to arginine using pcr-based mutagenesis. the resulting construct (termed 6r) was recombined into vaccinia virus and tested for presentation to np-specific ctl. the 6r construct was presented at a level equivalent to that seen with the wild-type 50-158/rconstruct. this result provides clear evidence that entry into the ubiquitindependent degradation pathway is not responsible for rescue of presentation in this system and more importantly, that ubiquitination is not required for processing of all large substrates. chia-chi ku, li-jung chien ,and chwan-chuen king, institute of epidemiology, national taiwan university, taipei, taiwan, r.o.c. dengue virus (den) can cause dengue fever (df) and dengue hemorrhagic fever (dhf) i dengue shock syndrome (dss) and den-2 was the most common serotype found in dhf outbreaks globally. current hypotheses suggested that dhf may be associated either with antibodydependent enhancement (ade) or with viral virulence. den can replicate predominantly in monocytedmacrophages (mim), but whether peripheral blood lymhocytes (pbls) are the target cells of den still remain controversial. in order to compare whether various clinically derived den-2 will interact with mim and lymphocytes in different manners, we used two isolates --plo46 strain (obtained from a df patient during taiwan 1981 outbreaks) and 16681 strain (isolated from a dhf patient in thailand by cdc, usa) to infect primary mim and lymphocytes as well as several types of cell lines. primary lymphocyte culture was nonadherent cells obtained after 24 hr adherence of pbmcs, whereas the primary mim culture was collected by depletion of lymphocytes using anti-cd3icdi9 mab and complement prior to adherence procedure and the purity of mim culture was checked by cd14 surface marker staining. supernatants (sn) of virus were harvested at various time points post infection after with several or without treatments. our prelimanary data showed that dhf-associated den-2 strain had higher viral yield in certain age of mim and a promonocytic cell line (hl-cz) than taiwan df-associated den2 strain. in addition, this dhf-den2 strain was more likely to infect the promonocytic (hl-cz) than well differentiated monocytic (ctv-1) and lymphocytic (h9) cell lines and also had higher peak yields than den-i virus in hl-cz cells. interestingly, dhf-den2 strain replicated much more efficiently in primary lymphocytes no matter these cells were activated with pha or not, whereas taiwan df-den2 strain virus was hardly detectable in sn of both activated and non-activated lymphocyte cultures. therefore we conclude that (1) different strains of dengue virus could orchestrate quite differently with immune cells, (2) different stage of mim differentiation might be an important permissive determinants for dengue virus infection and replication, and (3) den virus strain virulence -a more important factor than lymphocyte activation status -seemed to determine whether this strain would infect human pbls. further studies should be focused on searching for detaied mechanisms of virus and immune cell interactions. (2) when viral yields were enhanced early than day5 post infection, it provided tremendous opportunity to attack the immune system and finally may lead to severe disease. hiv-1 using recombinant immunoglobulin molecules, marie-claire gauduin, graham p. allaway, paul j. maddon, carlos f. barbas, dennis r. burton, and richard a. university school of medicine, new york. ny 10016. primary isolates of hiv-1 have been shown to be less sensitive to neutralization by immune sera, monoclonal antibodies and cd4-based molecules than t cell line-adapted strains of hiv-i. we studied two immunoglobulin molecules for ability to neutralize primary isolates of hiv-i. lgg12 is an immunoglobulin molecule created from a combinatorial phage expression library and reacts with the cd4 binding site (cd4-bs) on gp120. cd4-lgg2 is a recombinant molecule in which the variable domains of both heavy and light chains of lgg2 were replaced with the first and second immunoglobulin-like domains of human cd4. both molecules have been previously shown to effectively neutralize hiv-i in vitro. ex vivo neutralizations were performed as follows: lgg12 and cd4-lgg2 were added at 25 pg/ml to wells containing serial dilutions of plasma from hiv-i-infected patients and phastimulated peripheral blood mononuclear cells from seronegative donors. p24 production was measured over 14 days of culture and an end-point titer of hiv-1 in the presence and absence of added antibody was determined. both igg12 and cd4-lgg2 were found to reduce the original hiv titer from seven plasma samples with high virus titer (>250 tcid50/ml) by up to 625-fold. this is in comparison to soluble cd4 which only reduced viral infectivity by 55-fold at the same concentration. in vitro binding and neutralization assays on isolates recovered from plasma confirm the potency and breadth of neutralization by these two molecules. these studies suggest that recombinant antibodies directed at the cd4-bs of hiv-1 gp120 are able to effectively neutralize primary isolates of hiv-1 and may be useful in dissecting the mechanisms of resistance to neutralization by other antibodies. dillner and p. heino, microbiology & tumor biology center, karolinska institute, stockholm, sweden hpv 16 the major cause of anogenital precancers in man. the search for neutralizing epitopes that could form the basis for a preventive vaccine has shown that the surface-exposed imunodominant epitopes of the capsid are strongly conformationdependent, which has precluded detailed epitope analysis. similarly, immunization with whole, denatured capsid proteins has only identified linear immunodominant epitopes positioned on the inside of the capsid. reasoning that linear surface-exposed epitopes should exist, but might be cryptic, a set of 66 overlapping synthetic peptides corresponding to the entire hpv16 capsid proteins was used to generate hyperimmune sera. several antisera against 3 different peptides were reactive with intact hpv16 capsids at titers up to 1:150.000. hiv-1 serum antibodies and mucosal iga. basil golding, john inman, paul beining, jody manischewitz, robert blackburn and hana golding. div. of hematology and viral products, cber, fda, and lab. of immunology, niaid, bethesda md 20892. previously, we showed that hiv-1 proteins conjugated to 8. abortus (ba) could generate anti-hiv-1 neutralizing antibodies in mice even after depletion of cd4* t cells. in this study a 14-mer peptide from the v3 loop of hiv-1 (mn) was synthesized 013) and coupled to ba and klh. balb/c mice were immunized twice i.p. with these conjugates at two week intervals. v3-klh induced mainly igg1, whereas v3-ba induced all igg isotypes but lgg2a predominated. fecal extracts from mice immunized with v3-ba were shown by elsa to contain iga antibodies. sera from these mice bound gp120, expressed on the surface of infected cells. sera from mice immunized with v3-ba inhibited syncytia formed between cd4' t cells and chronically infected [hiv-i (mn)] h9 cells. inhibition of syncytia, formed by other hiv-1 lab. strains correlated with the degree of their homology with the v3 region of hiv-i (mn). to mimic the efffect of hiv-1, mice were depleted of cd4' cells using anti-l3t4 at the time of primary or secondary immunization. following primary immunization, cd4+ t cell depletion abrogated v3-klh antibody responses, whereas responses to v3-ba were retained and sera from these mice were able to inhibit gp-120mediated syncytia. in secondary responses, cd4' t cell-depletion prevented boosting to v3-klh, but v3-ba increased anti43 and syncytia-inhibiting antibodies. these results suggest that: 1. 8. abortus, can provide carrier function for a peptide and induce both serum and mucosal antibody responses, and 2. that infection with hiv-1 with subsequent impairment of cd4' t cell function would not abrogate anti-hiv-1 antibody responses if 8. abortus is used as a carrier to stimulate memory responses. nucleotide sequence analysis of the vh genes revealed the usage of one particular vh germline element (vh61-1p) in all clones. this finding allowed the determination of somatically mutated positions in the vh regions. two vsv-ind neutralizing antibodies expressed vh and vl genes in complete germline configuration whereas the rest of the clones showed somatic mutations which obviously were antigen dependently selected for. however, binding affinities of mutated and unmutated antibodies were comparably high. in order to determine the influence of somatic point mutations on one single antibody we generated a monovalent single chain antibody (fv-ck) of a mutated clone and reversed it stepwise to germline configuration by means of site directed mutagenesis. surprisingly, already the germline configuration of fv-ck could neutralize vsv-ind, even though the binding affinty was lower than that of the mutated fv-ck. every single somatic point mutation tested improved the binding avidity although some mutations reduced affinity. thus, during the course of vsv-ind infection some antibodies are subjected to avidity maturation although this is not required for the generation of high affme, efficiently virus neutralizing antibodies, lisa hyland'", sam hou'.~, and peter c. doherty'. 'department of immunology, st. jude children's research hospital, memphis, tn 38 10 i, 2departments of immunology and microbiology, and 'pathology, university of otago,dunedin, new zealand. the b and t cell responses in c57bl/6j(b6) mice treated with the mab mel-14 to l-selectin have been analysed following i.n. infection with sendai virus. mel-14 treatment caused a 70-90% decrease in the lymphocyte recruitment to the mediastinal (h4ln) and cervical (cln) lymph nodes following infection with sendai virus. the cellularity of the spleen was unchanged. the clonal expansion of cd8+ ctl precursors in the mln was slightly delayed, but potent ctl effectors were present in the virusinfected lung by day 10 after infection and the overall magnitude of the response was not compromised. the prevalence of iga antibody forming cells (afcs) was greatly increased in both the mln and the cln of the mice given the mel-14 antibody. the igm response was prolonged and the igg response, particularly iggl, was delayed compared to controls. the altered pattern of the antibody response may reflect the limited availability in mel-14-treated mice of th cells secreting lymphokines which are involved in ig class switching, by blocking the entry of cd4+ th precursor cells into lymph nodes. facs sorting for l-selectin+, 8220+, and l-selectin-, b220+ cell populations from the mln and the cln of normal b6 mice 9 days post sendai virus infection, showed that the afcs were from the l-selectin-, b220+ cell population, a population which comprised 6-10% ofthe total cell population. we have distinguished targets of broadly neutralizing antibodies present in hiv-1 infected individuals by imunoselection in vitro and by the use of chimeric virus. one target of neutralizing antibodies, defined by an escape mutant with an ala to thr substitution at position 582 in gp41, is resistant to human monoclonal antibodies that map to a site closely congruent with that for cd4 binding. substitution of gly, ser, and val fail to confer resistance. a second, defined by an ala to val substitution at position 281, upstream from the v3 loop, does not involve the same site and does not involve v3. substitution of thr or ile also confers resistance. replacement of the v3 loop of hiv-l(mn) into a clone of hiv-l(iiib) allows the detection of two other broadly neutralizing targets. one recognizes the v3 peptide of mn but is affected by regions outside v3. the other appears to be conformational and outside v3, but its functional recognition is influenced by the v3 loop. all of these sites seem to depend on the overall conformation of the envelope protein rather than a single discrete linear epitope. antibodies against amino acids 579-613 of the hiv transmembrane (tm) glycoprotein have been shown to enhance hiv infection in vitro in the presence of complement. there has been no study demonstrating that enhancing antibodies to this region of hiv, despite increasing levels of infectious virus 10 to 100 fold in vitro, adversely affect disease pathogenesis. in two separate studies reported herein, it is shown that animals which have high levels of antibody against this region of siv, amino acids 603-622 of the envelope, fair poorly compared to animals with lower antibody levels against this region when subsequently challenged with siv. when actively immunized with a synthetic peptide from this region of siv, animals died earlier and failed to clear antigen at two weeks after infection compared to animals that received a control peptide (p<0.05). when animals were passively immunized with antibodies from a longterm survivor of siv infection, those animals that received higher levels of antibody against the tm peptide died within six months compared to longer intervals for those animals that had lower levels of antibody to this region. when taken together, these data suggest that antibody to the tm region of siv and hiv in general, and to this highly conserved peptide in particular, are detrimental to the host. therefore, immunization strategies that minimize the immune response against tm or treatment protocols that decrease antibody levels against tm may lead to prolonged survival following exposure to lentiviruses. we have developed a mouse model to examine the immune response to hpv 16 proteins when these proteins are presented to the immune system via the epithelial route. in this model animals are grafted with keratinocytes expressing hpv e6 a n d e7 genes using a transplantation procedure which permits epithelial reformation. animals so grafted when challenged intradermally with e7 either as protein or via a recombinant vaccinia virus exhibit a delayed type hypersensitivity response which is e7-specific and cd4+ t cell mediated. animals grafted with a sub optimal priming inoculum of cells develop immune non-responsiveness and have an abrogated dth response when challenged subsequently with a priming cell graft. in the present study w e have examined the antibody status in these animals. the e7 protein of hpv 16 was expressed in e. coli as a maltose binding fusion protein using the plasmid vector pmalc. after cleavage and affinity purification this protein was used in a n elisa assay to measure antibody levels in 4 groups of mice (1) those not challenged with e7 (2) mice not grafted but challenged with e7 protein in the ear (3) mice primed by grafting with 107 hpv e7 expressing cells and challenged with e7 protein (4) mice primed by grafting with 5 x 105 hpv 16 e7 cells on day 7, grafted again with lo7 hpv 16 e7 cells on day 14 and challenged with e7 protein in the ear. mice optimally grafted and challenged (group 3) exhibited high titres of igg antibodies, particularly elevated levels of iggza. mice sub-optimally grafted (group 4) exhibited igg antibody levels comparable to the control group (1). the possible mechanisms of this immune attenuation are discussed. the hepatitis c virus is a frequent cause of chronic liver disease. a proposed mechanism responsible for virus persistence is evasion of the host immune response through a high mutation rate of crucial regions of the viral genome. the portion of hcv genome coding for the amino-terminal part of the putative envelope protein (gp70) undergoes frequent mutation during the course of infection. we have cloned and sequenced the hypervariable region (hvri) of the virus isolated from an hcv asymptomatic patient at three time points during 18 months follow up. sequence analysis has allowed the identification of variants of this region and multiple antigenic peptides (map), corresponding to three hvrl variants, sequentially foundin the blood stream of the patient, have been synthesized. maps have been used as antigens for detection of specific antibodies in elisa. our results show that anti-hvri antibodies and their cognate viral sequence coexist in the blood stream but a viral sequence becomes undetectable when the specific antibodies reach maximum levels of reactivity. thus humoral immunity against the hvrl may play a role for virus clearence. the presence of anti-hvr1 antibodies was also investigated in 100 hepatitis c viremic individuals and 25 non-viremic patients. a high frequency of positive reaction (90%) against at least one of the three hvrl variants analysed in this study was detected in the viremic patients. finally, competition experiments show that antibodies crossreacting with more than one hvrl variant are produced by hcv infected individuals. this results suggest that complex cross-reactivity exist between hcv isolates for antibodies against the hvrl region as described for antibodies against the gp120 v3 loop of hiv. we propose as mechanism for viral escape in hcv chronic infections the one described as the "original antigenic sin", observed firstly in influenza, in togavirus, paramixovirus, enterovirus, and recently in hiv infection. using an adult mouse model to study active immunity against rotavirus infection, it was previously shown that oral immunization with some, but not all, animal rotavirus strains induced protection against subsequent infection following oral challenge witb the murine rotavirus strain edim (ward et al., 1992) . to determine i f a specific rotavirus protein could be associated with protection in this model, mice were immunized with a series of 18 reassortants between the fully protective edim strain and a partially protective heterologous rotavirus strain (rrv-g). reassortants that contained genes for edim proteins responsible for protection were anticipated to provide complete protection; however, no edim proteins were found to be both necessary and sd3cient for full protection. instead, protection was found to be highly correlated with viral shedding (p = ,005) and with serum rotavirus iga titers stimulated by the different reassortants (p < ,001). this indicated that protection was related to the intestinal replication properties of the different reassortants rather than to specific immunogenic properties of edim proteins. this conclusion was supported by the finding that the titers of serum rotavirus i& but not igg, stimulated in mice following oral immunization with a series of animal rotaviruses was directly related to protection against edim. if these findings can be extended to humans, they suggest that the efficiency of intestinal replication following oral inoculation with a live rotavirus vaccine candidate may be the primary determinant of successful immunization. h a l l medical center, 55 individuals with adequate serum samples were identified as either rapidly progressing (rp) or slowly progressing (sp) by clinical and surrogate marker criteria. anti-v3 profiles were determined using synthetic proteins derived from the amino acid sequences of the v3 region of 5 laboratory strains of hiv-1 in standard capture elisa format. serum obtained from each patient at multiple different time points was screened against these peptides. the majority of individuals in both groups demonstrated broad recognition, with reactivity to peptides corresponding to the v3 regions of mn, sf2, ny5 and han/sc. less than 50% of individual in each group recognized the v3 peptide derived from iiib, @=ns, between groups). as the rp progressed to aids there was significant nonspecific narrowing of response, while the sp remained broadly reactivity (p< .001). in v i m neutralizing activity of the homologous laboratory isolates was determined with cytotoxicity, cytopathic effect and p24 ag inhibition assays. although most patient serum was capable of inhibiting p24 ag production in homologous lab strains while aids-free, there was no relationship with the ability to inhibit homologous virus effects on target cells and anti-v3 profiles. model, we show that after resolution of the acute infection, when antiviral plasma cells in the spleen decline, a population of virus-specific plasma cells appear in the bone m w and constitute the major sou~ce of longterm antibody production. following infection of adult mice, wspecific antibody secreting cells (asc) peaked in the spleen at 8 days postinfection, but were at this time undetectable in the bone marmw. the infection was essentially cleared by 15 days and the asc numbers in the spleen rapidly declined while an increasing population of lcmv-specific asc appeared in the bone marrow. when compared to the peak response at 8 days post-infection, timepoints from 30 days to more than one year later demonstrated greater than a l@fold reduction in splenic asc. in contrast, jltvlv-specific plasma cells in the bone marrow remained at high numbers and correlated with the high levels of antiviral serum antibody. the prewnce of antiviral plasma cells in the bone marrow was not due to a persistent infection at this site, since virus was cleared from both the spleen and bone marrow with similar kinetics as determined by infectivity and pcr assays. the igg subclass profile of antibody m e t i n g cells derived from bone manuw and spleen correlated with the igg subclass distribution of lcmv-specific antibody in the serum. upon rechallenge with w , the spleen exhibited a substantial increase in virus-specific plasma cell numbers during the early phase of the secondary response, followed by an equally sharp decline. bone marrow asc populations and lcmv-specific antibody levels in the serum did not change during the early phase of the reinfection but both increased about 2-fold by 15 days post-challenge. after both primary and secondary viral infection, lcmv-specific plasma cells were maintained in the bone marrow showing that the bone marrow is a major site of long-term antibody production after acute viral infection. "memory" t cells, and associated with responsiveness to soluble and recall antigens. cd4+ lymphocytes staining bright, dim, or negative (equivalent to an isotype control) for cd29 were evaluated in 49 uninfected controls (group l), 84 hiv-1 positive patients with 220% cd4+ t cells (group 2), and 47 hiv-i-infected patients with ~2 0 % cd4+ t cells (group 3). most of these subjects also had 3-color staining for cd4\cd45ro\cd45ra. the appearance of positive cd29 and cd45ro on hivinfected and uninfected cells correlated well (r=.82 p<.ool). the percentage of cells staining cd4+\cd29+.(bright plus dim) was 43.3 (95%cl 37.3-49.4) in group 1, 28.9 (27.5-30.4 ) in group 2, and 10.2(8.6-11.9) in group 3. the respective values for these groups that were cd4+\cd2gbwm was 30.6 (26.9-34.3), 20.7 (1 9.3-22.2), and 7.4(6.3-8.6). values for cd4+\cd45ro+ were 33.7 (31.8-35.5), 21.8 (20.5-23.1), and 9.9 (8.5-11.3), respectively. in single factor discriminate function tests, the %cd4+\cd29+ cells best predicted subject group (87% correct), proving to be a better discriminator than %cd4+\cd29b'h' (77% ~orrect),cd4+\cd29~"" (5 l%), cd4+\cd45ro+ (75%) and cd4+\cd45ro+\cd45ra-(63%). overall, no advantage was seen to splitting the cd4+\cd29+ cells into bright and dim positive subsets in the subjects studied for the purpose of stratifying early vs. late hiv infection. likewise, splitting the cd4+\cd45ro+ compartment into cd45ra+ subsets did not improve the ability to distinguish between uninfected and early or late hiv-1 infected patients. the relationship between the virus-specific cytotoxic response in hiv infected patients and disease progression support the concept that a vaccine candidate should also induce a virus-specific ctl activity. immunization of uninfected adult volunteers by a hiv-gpl60 recombinant canarypox virus was carried out in a phase i trial.two injections of a recombinant canarypox expressing the hiv-l/mn gp160 were performed at month 0 and 1 and two boosts of recombinant gpl60mn/lai at month 3 and 6 in alum or incomplete freund adjuvant(1fa). hiv-envelope specific cytotoxic activities were detected from ctl lines derived from pbmc stimulated by specific stimulation with autologous hiv infected blasts. ctl lines were obtained from 18 out of 20 donors : seven out of eighteen (39%) were found to present envelope specific cytotoxic activity at months 2, 4, 7 or 12 post immunization ; this activity was characterized as a cd3+,cd8+, mhc class-i restricted cytotoxic activity, and for at least two volunteers, this activity was still present two years after the first canari-pox/env injection. because avian poxviruses are incapable of complete replication and undergo abortive replication in mammalian cells , this is a n example of the persistence of long term memory cd8+ cytotoxic t lymphocytes in the absence of the priming antigen, indicating that t-cell memory might be independent of continued antigenic exposure. the university of alabama at birmingham, al 35294. mhc class i restricted cd8' ctl activity plays an important role in the control of influenza virus infection as indicated in studies in mice and humans. cytokines such as il-2 and ifn-y regulate the generation of virus-specific ctl responses. we recently demonstrated a good correlation between the induction of influenza virus-specific ctl activity and the production of ifn-y by the cd8' t cells at the single cell level using an if?-specific elispot assay, secreted ifn-y by an elisa, and ifn-y specific mrna expression by rt-pcr. several recent studies have characterized cd4+ and cd8' t cells by their expression on the surface of distinct d45r isoforms. cd45ra is expressed on naive or virgin t cells, while cd45ro is expressed on memory t cells. in the present study, pbmc of healthy young adult subjects were stimulated with influenza a virus and then enriched for cd8+ t cells. the cd8' cells were stained for cd45ro' (pe) and cd45ra' (fitc) cells and sorted. ctl activity against virus-infected autologous target cells was determined in a 4 hour 'lcr release assay while ifn-y production and expression was assessed by elispot and quantitative rt-pcr, respectively. cds+/cd45ro+ (memory) cells exhibited significant mhc class i ctl while cds+/cd45ra+ cells exhibited no lytic activity. no activity was exhibited by freshly isolated or unstimulated cd8+/cd45ro+ t cells. similarly, cd8+/cd45rot t cells contained significantly higher numbers of ifn-y spot forming cells and higher quantity of ifn-y-specific mrna than cd8+/cd45rac cells. these data support our previous findings that ifn-y may serve as a useful surrogate marker for influenza virus-specific ctl activity in humans. in studying the kinetics of the cd8+ t cell response in lcmv infection we have observed a profound activation and proliferation of cd8+ t cells with a 10-40 fold increase in total number peaking at day 8-9 post infection. in c57bw6 mice, most of the viral antigen is cleared by day seven, and after day 9 the total cd8+ number per spleen drops about 10-fold. however, the relative specificity of the viral peptidespecific precursor ctl frequencies @ctwf) per cd8+ cell remains remarkably stable between day 7-8 of the acute infection and for many months thereafter. thus, the decline in the cd8' t cell number is not a function of the tcr specificities but is rather an across-the-board event. in contrast, we found that subsequent to the decline of the ctl response to a second heterologous virus infection such that the mouse was in a "resting, immune'' state, there often was a reduction in pctl/f to the first virus. for example, infections with w or mcmv substantially reduced the pctuf to lcmv or pv in all memory compartments, including spleen, lymph nodes, peritoneal exudate cells. reinfection with the original virus substantially elevated its pctuf and restored the pctuf that had been reduced by a heterologous viral infection. analyses of the progression of ctl responses during a heterologous virus challenge of a virus-immune mouse indicated a high frequency of crossreactive ctl appearing early during infection, but as the infection progressed there was a higher proportion of ctl specific only for the second virus. thus, we believe that when the across-the-board apoptosis of t cells occurs late in the infection, ctl specific for the first virus are diluted by those responding to the second virus. this may cause the reduction in memory to the first virus and may be one of the mechanisms contributing to the waning of secondary immune responses to certain viruses over time if there is no re-exposure to the original infectious agent. t-cells which arise after virus infection will aid our understanding of tcell memory and be useful in the design of vaccines which augment the memory response. to estimate the sendai virus specific precursor frequency in memory mice, cd4+ cells from c57bl6 female mice which had been infected with sendai virus intranasally (i.n.) more than two months earlier were subjected to limiting dilution analysis. responder cell populations were enriched for cd4+ cells either by magnetic bead depletion of non-cd4+ cells, or by facs after staining with anti-cd4 monoclonal antibody these enriched (>90% cd4+) responders were cultured with sendai virus-infected, irradiated, t-cell depleted splenic antigen presenting cells (apc). supernatants from these cultures were tested for activity on the cytokine-dependent ctll cell line. duplicate cultures of responders on uninfected apc were used to set the level of rejection (mean cpm + 3x std. dev.). using this type of analysis we were able to demonstrate a frequency of memory thp at 111600 cd4+ cells, compared to a frequency greater than 1/1ooooo in naive controls. the memory cd4+ cells were further characterized as cd45rb-low (1/472) , cd44-high (1/294), lselectin-low (1/364), and cd49d-high (vla-4-high) (v102). this is close agreement with other phenotyping studies on cd4+ memory cell specific for soluble antigens. t cells, ralph a. tripp, sam hou, anthony mcmickle, james houston and peter c. doherty, department of immunology, st. jude children's research hospital, memphis, tn 38105. the immune response of influenza a and sendai-virusspecific, memory cd8' cytotoxic t lymphocyte precursors (ctlp) have been analyzed in c57bu6 mice infected intranasally with unrelated or cross-reactive respiratory viruses. the numbers of influenza a-specific memory t cells increased in the regional lymph nodes (ln), spleen and bronchoalveolar lavage through the course of an irrelevant infection (influenza b). memory t cells showed evidence of enhanced steady-state activation. profiles of ctlp recruitment were analyzed in association with t cell proliferation and activation to determine whether signaling via the t cell receptor is necessary to induce "bystander" stimulation of the memory t cell pool. the extent of t cell proliferation was addressed by treating mice with low doses of cyclophosphamide (cy). "resting" sendai virus-specific memory t cells were unaffected by cy treatment, however upon challenge with influenza and treated 5 or 6 days later, the emergence of influenzaspecific ctlp was severely diminished. cell cycle analysis showed that cy eliminated the majority of cd8' t cells from the ln and spleen resulting in dna fragmentation of 12-18% ofthis lymphocyte subset. a decrease (though smaller) in the numbers of sendai virus-specific ctlp indicated that some of the cycling cells killed by cy were memory t cells, presumably activated in a "bystander" manner. the decrease in ctlp numbers for both influenza and sendai virus-specific ctlp was still apparent 9 days after cy treatment, long after the viral elimination. thus, immune responses to unrelated antigens may be a mechanism involved in maintaining the pool of memory t cells. experimentally vsv can result in an acute cns infection of mice. data from our in vitro experiments indicate that no has inhibitory effect on productive vsv infection. vsv infection at neuroblastoma nb41 a3 cells was significantly inhibited by loopm of a no donor s-nitro-n-acetylpencillamine (snap), while 1oopm of the control compound n-acetylpencillamine (nap) had no effect. when vsv infected nb41a3 cells were treated with 500pm of a constitutive no synthase (cnos) activator n-methyl-d-aspartate (nmda), a significant inhibition of vsv production was observed. inhibition by 500pm of nmda was reversed by 300pm of nos inhibitor n-methyl-l-arginine (l-nma). work is in progress to determine the effects of inducible nos (inos) in a glioma cell line c6 on vsv infection. levels of no and expressions of both cnos in neurons and inos in glial cells in the cns following vsv will be further investlgated. supported by nih grant a118083 to carol s. reiss. pediatrics, university of iowa, iowa city, ia. 52242 mouse hepatitis virus, strain jhm (mhv-jhm), is a neurotmpic coronavirus which causes acute encephalitis and acute and chronic demyelinating encephalomyelitis in susceptible rodents. 40.90% of suckling c57bu6 (kbdb) mice inoculated intranasally with mhv-jhm at 10 days and nursed by dams immunized against the virus develop a chronic demyelinating encephalomyelitis characterized clinically b hindlimb paralysis, at 3-8 weeks postinoculation. the chronic demyelinating encephalomyelitis nor the clinical symptoms. recently, it was shown that lymphocytes isolated from the central nervous system (cns) of c57bu6 mice both acutely and persistently infected with mhv-jhm display a cytotoxic t lymphocyte (ctl) response to the s protein of mhv-jhm. this response was further characterized by identifying the ctl epitopes that are recognized by a bulk population of ctls from the cns of mhv-jhm infected c57bv6 mice. three epitopes were identified using synthetic peptides and truncated forms of the s protein in primary ciz assays. the epitopes recognized were amino acids 510-518 (cslwngphl, db), 598-605 (rcqifani, kb), and 1143-1151 (nfcgngnhi, db). thus, the results indicate that cytotoxic t lymphocytes responsive to the s protein of mhv-jhm in c57bu6 mice recognize both kb and db-restricted cil epitopes. ctl lines and clones specific to these peptides and the entire s protein are being developed to test their biological significance in vivo with respect to the acute encephalitis and chronic demyelinating disease caused by mhv-jhm. a marked change in susceptibility to some neurotropic viruses during the first few postnatal weeks has long been recognised in rodents. infection of neonatal or suckling mice with the neurotropic alphavirus, semliii forest virus results in lethal encephalitis. infection of weaned animals is not lethal. earlier investigations focusing on changes in specific immunity have shown this not to be the explanation. infection of 3-4 week old mice with severe combined immunodeficiency does not result in acute rapidly fatal encephalitis. we have studied mortality, neuroanatomical distribution and spread of infection in mice of different ages and the effect of gold compounds on rendering infection of 3-4 week old mice lethal. neuroanatomical distribution of infection correlates with synaptogenesis. as this is completed in different systems within the first two weeks postnatal, systems no longer transmit virus and infection switches from disseminated to focal and restricted. complete productive replication and transmission of infection require smooth membrane synthesis which is present in neurones undergoing synaptogenesis, absent in mature neurones but inducible by administration of gold compounds. infection of neurones undergoing synaptogenesis is productive and virus is transmitted along neuralpathways, infection spreads rapidly around the brain, destroys cells and animlas die of a fulminant encephalitis. in mice infected after 14 days of age replication in mature neurones is restricted, nonproductive, cannot be transmitted, does not spread, is non-destructive and non-lethal. as a consequence, in the absence of immune responses virus can persist in isolated cns cells for life and can even be detected by reverse transcriptase pcr in immunocompetent mice months after infection. in the presence of an immune response, cd8+ t-cells recognise and destroy infected glial cells leading to dem yelination. a~k e r m a n n ,~ virology swine,' virology cattle,' and avian diseases3 research units, national animal disease center, usoa, agricultural research service, ames, ia 5001 0 a recombinant pseudorabies virus (prv) (lltbap) was constructed which contains a 3.0 kb deletion spanning the standard recombination junction of the unique long and internal repeat sequences replaced by e lacz expression cassette. this deletion interrupted the large latency transcript gene (llt) and truncated one copy of the diploid immediate early iel80 gene. replication and viral gene expression of lltbaz in madin-darby bovine kidney cells was similar to that of the parental virus and a virus rescued for the deleted sequences (lltbres). when inoculated intranasally in 4-week-old or 4-day-old pigs, lltba2 replicated efficiently at the site of inoculation yet caused markedly reduced fatality when compared to the parent or lltbres viruses. in particular, the lltba2-infected pigs did not exhibit neurological symptoms characteristic of prv infection. to further examine the pathogenesis of lltba2, 4-day-old pigs were infected intranasally with lltpa2 or lltbres and necropsied at various times postinfection. virus isolation from the nasal turbinate, tonsils, and trigeminal ganglia was comparable between the two viruses. although both viruses spread to the brain and induced an inflammatory response in cns tissues, virus isolation from brain tissues was reduced about 20-fold for lltpa2. abundant prv antigen was detected in the cerebrum and cerebellum of lltpresinfected pigs, but only a few antigen positive neurons were observed in the cerebrum of lltba2-infected pigs. while replication of lltbres in the brain progressed until death at 7 days post-infection, replication of lltpa2 in the brain ceased by 9 days post-infection and the pigs exhibited only mild clinical signs. since lltba2 is capable of spread to the cns, reduced neurovirulence of lltbaz is likely the result of its decreased ability to replicate in cns tissues. the cns is a target for hiv infection, and in individuals with aids this can lead to a devastatin dementia. only certain viral variants appear capable 07 invading the cns and infecting microglia and brain macrophages. in order to determine whether the virus entering the brain may be particularly pathogenic to the cns, we isolated microglia from the brains of siv-infected rhesus monkeys. transfer of these cells into naive animals indicated that productive siv infection could indeed be transferred. furthermore, cns infection occurred within a relatively short time span, and was associated with viral gene expression in the brain and pathology characteristic of hiv encephalitis. serial transfer of microglia into additional animals also resulted in successful transfer of infection, neuroinvasion, and neuropathology. behavioral analysis in a trained group of animals is ongoing. this result demonstrates that neuropathogenic virions partition into the cns during natural siv infection, likely driven by mutational events that occur during the course of infection. molecular characterization of the microglia-associated virus has revealed that a distinct pattern of sequence changes in the envelope gene occurs concomitantly with this in vivo selection. our approach will allow the dissection of functional neuropathogenic elements present in these viruses. in non-specific host defense mechanisms. ifn-y-induced nitric oxide (no) in murine macrophages was previously shown to inhibit the replication of poxviruses and herpes simplex virus type 1 (hsv-1) . we now demonstrate that murine macrophages activated as a consequence of vaccinia virus (vv) infection in viva express inducible nitric oxide synthase (ios). the vvelicited macrophages were resistant to infection with w and efficiently blocked the replication of w and hsv-1 in infected bystander cells of epithelial and fibroblast origin. this inhibition was arginine dependent, correlated with no production in cultures and was reversible by the nos inhibitor nqjmonomethyl-l-arginine. the mechanism of no mediated inhibition of virus replication was studied by treating vv-infected 293 cells with the noproducing compound, s-niuoso-n-afetyl-penicillamine. antibodies specific for temporally expressed viral proteins, a vv-specific dna probe and transmission electron microscopy were employed to show that no inhibited late gene protein synthesis, viral dna replication and virus particle formation, but not expression of the early proteins analyzed. further, we have also identified putative enzymatic targets of inactivation by no that results in inhibition vv replication. although antiviral ctl are important for virus elimination. they can only halt further virus spread, and cannot reduce the number of infectious particles already present. the beneficial effect of ctl-mediated lysis is apparent only if infected cells are lysed before assembly of progeny virus. if infectious virus was released from infected cells in solid tissues before the generation of neutralizing antibody or in sites where antibody did not readily penetrate, then recruitment of mononuclear phagocytes, which phagocytose and destroy infectious material and/or become non-productively infected, would definitely help control virus dissemination. in this context, inos induction in macrophages may be an important antiviral strategy. in addition, the inhibition of virus replication in infected contiguous cells by inos-expressing macrophages at infectious foci would prevent release of mature viral particles after lysis by nk cells and ctl. since viral early proteins are expressed in such infected cells, their recognition and subsequent lysis by ctls will not be hindered. cns persistence, tropism and genetic j. pedro s i s , anthony a. nash and john k. fazakerley, department of pathology, university of cambridge, cb2 iqp, uk theiler's murine enchephalomyelitis virus, a natural occuring enteric pathogen of mice, is a picomavirus belonging to the curdovirus genus. following intracerebral inoculation of 3-4 week old cba or balb/c mice, the bean strain causes a chronic persistent cns demyelinating infection in a proportion of the cba that survive acute infection. balb/c mice are resistant to chronic demyeliating disease. we have studied the tropism, persistence and genetic variability of bean, in cba and balb/c mice in the chronic phase of this disease. by in situ hybridisation and reverse transcription (rt) pcr and southern blot analysis, no viral rna could be detected in the cns of any balb/c mice later than day 60 post-infection. in contrast, in a large group of cba mice studied up until 393 days post-infwtion, viral rna could be detected by both techniques in 50% of mice until as late as 268 days post-infection. by employing a combination of, in situ hybridisation for viral genome followed by immunocytochemistry for cell phenotypic markers, bean rna was observed predominantly in oligodendrocytes and occasionally in astrocytes during persistent infection, in both brain and spinal cord. in the persistently infected mice, the striking total destruction of the pyramidal layer of the hippocampus, substantia nigra and anterior thalamic nuclei indicated that these were the mice that had had greatest dissemination of virus and highest virus titers during the preceeding acute phase of infection. direct pcr t h d cycle sequencing of uncloned rt-pcr products, revealed that during persistent infection, loops i and ii of the vpi capsid protein gene did not undergo any genetic variability. furthermore, no changes were detected in this region in sequenced pcr products amplified from the cns of mice with severe combined immunodeficiency in which no selective immunological pressure would have been operative. infection, thomas e. lane, michael j. buchmeier, dorota jakubowski, debbie d. watry, and howard s. fox, department of neuropharmacology, the scripps research institute, la jolla, ca 92037 our laboratory is interested in the effects of siv infection in the central nervous system of rhesus macaques. to enrich for neuroinvasive and neurovirulent viruses, microglia were isolated from infected monkeys and used t o infect new, uninfected monkeys. such microglia-mediated infection resulted in the production of neuropathological changes, including giant cells, macrophage infiltrates and microglial nodules in recipient animals within 4 months. microglial cells isolated from siv-infected monkeys produced virus in vitro as measured by reverse transcription (rt) and p27 production. treatment of microglia with recombinant human interferon alpha (rhulfn-a) resulted in a sharp decrease in viral activity (both rt and p27 production) suggesting that rhulfn-a is able t o modulate viral activity in infected microglia. we have analyzed slvenv sequences by pcr amplification directly from microglia dna preparations from monkeys. nucleotide sequence analysis results in an enrichment of unique sequences in the v1 region of the siv env gene. the majority (>95%) of nucleotide changes encoded amino acid changes, indicating that these envelope sequences evolved as a result of selection. moreover, sequential passage of sivassociated microglia resulted in an increase in potential n-linked glycosylation sites within the v1 region of the env gene when compared with the parental virus. these data suggest that sequential passage of microgliaassociated siv may select for neuroinvasive, neurovirulent variants. the adoptive transfer of ctl specific for an ld-restricted epitope within the nucleocapsid protein of the jhmv strain of mouse hepatitis virus both protect from acute infection and reduce virus replication in the mhc class 1 positive cells within the cns. the source of these ctl and the route of their delivery is critical in the outcome of this protection. for example, 10 fold less spleen cells activated in vitro with the pn peptide are required for protection via the direct i.c. route than the i.v. route. in addition, ctl clones are unable to protect via the i.v. route and are very efficient via the i.c. route. these data suggested the possibility that the cd4+ t cells within the polyclonal activated spleen cell population derived from in vitro culture on the pn peptide were facilitating access to the cns. to examine this question, polyclonal pn-specific t cells were either depleted of cd4+ t cells prior to transfer to infected recipients or untreated cells were transferred to recipients depleted of cd4+ t cells with monoclonal antibody gk1.5. both of these treatments eliminated the ability of the ctl to reduce virus replication within the cns, suggesting that cd4+ t cells in the peripheral compartment are required for the entry of ctl into the parenchyma of the cns during acute cns encephalomyelitis. division of retrovirology, walter reed army institute of research and henry m. jackson foundation, rockville, md 20850; department of retrovirology, armed forces research institute of medical sciences, bangkok, thailand background the hn-1 epidemic in thailand is largely due to two highly divergent subtypes of virus, b and e. dual infection with distinct hn-1 subtypes, which has not been reported previously, would suggest that antiviral immunity evoked by one subtype can be incompletely protective against a second. merhoak: pcr typing and serologic typing were used to screen a panel of non-random convenience specimens from hiv-1 infected subjects in thailand. specimens that showed dual subtype reactivity in these assays were subjected to differential pmbe hybridization and nucleotide sequence analysis of multiple molecular clones to c o n f m the presence of dual infection. results. two individuals were shown to simultaneously harbor hiv-1 of env subtypes b and e (table) . additionally, both subtypes were identified in co-cultured pbmc from one individual. conclusions. these data provide the fmt evidence of dual hiv-i infection in humans and reinforce the need for polyvalent vaccines. infection by herpes simplex virus wsv) induces in man and in mice cytolytic t lymphocytes (ctl) which recognize the immediateearly protein icp27. because of its early expression during the hsv replication cycle, lcp27 represents a prime target for specific t cell responses susceptible of controlling virus replication. we have expressed in e. coli a remmbinant construct coding for a fusion protein consisting of a fragment of influenza virus non-structural protein4 (nsi) and the lcp27 sequence of hsv-2. the nsi-icp27 protein was purified by preparative eleclmplmresis and formulated in oil-in-water emulsions with monophosphoryl lipid a (mpl) and qszl adjuvants. balwc mice were immunized by two intrafootpad injections of formulations containing 5 pg of nsi-icp27. responder cells obtained from draining lymphnodes were re-stimulated in vitro with p815 cells lransfected with icp27 and then lesled for cytolytic activity on icp27-p815 and control p815. the induction of icp27 specific ctl by different formulations was observed and will be discussed. the induction of heterologous cytotoxic t lymphocytes (ctl) using cassettes of multiple conserved t cell epitopes derived from different proteins and/or virus strains is envisioned as a promising vaccine approach. to study the effects of antigen processing on peptide presentation from chimeric epitope precursors we are using a model system comprising two distinct viral epitopes which are immunodominant in the h-2d haplotype: a dd restricted epitope from the gp160 protein of hiv-1 and an ld restricted epitope from the murine hepatitis virus nucleocapsid protein (mhv n). the influence of proximity and flanking sequences of epitopes on antigen presentation was analyzed using vaccinia virus (vv) recombinants in which the epitopes were expressed as chimeras containing the individual epitopes in reverse order or separated by different spacer residues. whereas individually expressed epitopes were efficiently recognized by protein-specific ctl, recognition of peptides derived from tandem constructs varied significantly with closer epitope proximity and sequential order. following immunization with the recombinant viruses, the chimeras were all able to induce antiviral ctl specific for the native proteins. however, cn, frequency analysis indicated that the number of responder cells to the same epitope dramatically depends on its context within the chimera and correlates with antigen recognition in vitro. the profound effect of flanking regions on ctl induction suggests that the context of an epitope will require careful evaluation in the design of recombinant multivalent minigene vaccines to induce an optimal t cell mediated immune response. and robert e. johnston', departments of 'microbiology & immunology and %iochemistxy, univ. of north carolina, chapel hill, nc 27599 a hll-length cdna clone of venezuelan equine encephalitis virus w e ) has been altered to contain two strongly attenuating mutations and a second subgenomic rna promoter immediately downstream of the structural gene region. expression ofthe influenza ha protein from this second promoter in baby hamster kidney (bhk) cells was approximately 50?? of the level in influenza virus-infected cells, as measured by immunoprecipitation. fourweek-old cd-1 mice were inoculated subcutaneously with 2 x 10' p h of the ha vector, vector alone or diluent. expression of ha mrna was detected in the draining lymph node of ha vector-inoculated mice by in situ hybridization, consistent with the organ tropism of vee. mice were challenged three weeks after imnmization by intranasal administration of lo5 ed, of influenza h s . au 24 corn1 mice suffered severe disease and 50% died. only one of 12 ha vector-inoculated mice died, and another exhibited signs of disease for one day and recovered. the geometric mean elisa titer of anti-ha serum igg in the ha-vector inoculated mice was 246, while only three control mice had measurable serum reactivity, and that was at the lowest dilution tested, 150. in a parallel experiment, no influenza infectivity was detected in the lungs of 12 ha vector immunized mice at 4 days postchallenge. in contrast, 8/12 pbs-inoculated mice and 5/12 inoculated with vector alone were positive for influenza infectivity and had geometric mean titers of 3.04 and 1.93 x lo6 pwgm, respectively. this vector also has been used to express the h n w c a protein in a form recognized by patient sera and a specific antibody on western blots. these experiments demonstrate the feasibility of using vectors based on attenuated vee cdna clones for protective immunization against heterologous human and animal pathogens. dose/response curves have been used to compare different routes of immunization with plasmid dna encoding the h1 hemagglutinin glycoprotein of influenza virus. routes of inoculation included intramuscular, intradermal and gene gun delivery of dna. from 100 to 0.1 ug of dna was inoculated by intramuscular and intradermal routes. from 0.4 ug to 0.0004 ug of dna was inoculated by gene gun. each route was evaluated for single and boosted immunizations. antibody titers were followed over a 20 week period, following which animals were evaluated for protection against a lethal challenge. each of the routes raised both antibody and protective responses. gene gun-delivery of dna required 250 to 2,500 times less dna to raise responses than the intramuscular and intradermal inoculations. boosts did not have much of an effect on antibody titer or protection except at low dose inoculations (4 ng and lower for the gene gun). for each of the routes, antibody responses showed good persistence over the 20 weeks of the experiment. inoculation of mice with plasmid vectors carrying a microbial gene under the control of an appropriate promoter results in a full spectrum of immune responses to the vectorencoded antigen. using a murine rabies model a plasmid termed psgsrab.gp expressing the full-length rabies virus glycoprotein regulated by an sv40 promoter was shown to induce upon inmuscular inoculation a rabies virus specific t helper cell response. of the thl type, cytolytic t cells and virus neutralizing antibodies resulting in protection against a subsequent challenge with live rabies virus given either peripherally or directly into the cenaal nervous system. a response comparable in magnitude was also induce upon inoculation of a vector expressing a secreted form of the rabies virus glycoprotein. the immune response to the dna vaccine could be modulated by co-injection of the rabies virus glycoprotein-expressing vector with plasmids expressing mouse cytokines. inoculation of mice with the psg5rab.gp vector and a vector expressing granulocyte/macrophage colony stimulating factor (gm-csf) enhanced both the t helper and the b cell response to rabies virus thus improving vaccine efficacy. co-inoculation with vectors expressing interferon-g failed to improve the response. co-inoculation of the antigen-expressing vector with a plasmid encoding mouse i l 4 caused a reduction of both the t helper cell response and the b cell response to rabies vlns. hpvl6 e7 hpv associated cervical cancer cells express hpv16e7 protein and antibody to hpv16 e7 can be detected in the blood of cancer patients, yet the twnours are. not rejected. a mouse transgenic for the e7 protein of hpv16, and expressing e7 protein in the skin, has recently been described (1) and these mice develop spontaneous humoral immunity to e7 protein similar to patients with cervical cancer(2). to determine whether immunisation could induce immunity to e7 sufficient to allow tumour rejection, we firstly demonstrated that immunisation of h-zb mice with hpv16e7 protein with quil a as adjuvant could induce cytotoxic t cells able to kill hpv16 e7 expressing tumour cells in nrro. we then used similar immunisation with e7iquil a to induce e7 specific immunity in fvb (h-29) mice. h-2qskin @s expressing e7 were not rejected by e7 immunised h-zq mice, though immunisation induced antibody to e7, and similar grafts were rejected, as expected, across an doantigen mismatch in h-zb mice. we conclude either that hpvl6 e7 lack a tc epitope in the context of h-zq, or that expression ofe7 in the skin from the e7 transgenic mice is insufficient for recognition by primed effector cells, and further experiments will address this distinction. cervical carcinoma is strongly associated with infection by human papillomavirus (hpv) types 16 or 18, and continued expression of the e6 and e7 gene products. this provides an opportunity for an immunotherapeutic approach to the treatment of cervical carcinoma by activation of immune reponses directed against these virally encoded tumour specific antigens. we have constructed a recornbinant vaccinia virus expressing e6 and e7 from hpv16 and 18 with the aim of inducing e6 and e7 specific hla class i restricted cytotoxic t lymphocytes (ctl). the sequences have been inserted into the wyeth vaccine strain of vaccinia virus at a single locus in the form of two separate fused e6/e7 reading frames, each under the control of an early v a d n i a promoter, and each modified to inactivate the rb binding site. the virus has been characterised with respect to its ability to synthesise the expected hpv proteins, its genetic stability, and growth and virulence in a mouse model prior to use in human clinical trials. analysis of hpv16 â�¬7 specific ctl from c57bu6 mice immunised with this recombinant virus show the response to be equivalent to that generated by a control vaccinia recombinant expressing non-modified hpvi 6 e7 alone, with similar recognition of the defined immunodominant h-2db restricted epitope, e7 residues 49-57. ability of mice to resist influenza challenge, arthur friedman, douglas martinez, john j. donnelly and margaret a. liu, department of virus and cell biology research, merck research laboratories, west point, pa 19486 mice infected with the laboratory strains of a/pr/8/34 (hln1) or the mouse adapted a/hk/68 (h3n2) show complete protection against challenge with a different strain of influenza a. humans, however, undergo multiple influenza infections as previous infections appear to provide weak or short-lived protection against the continual antigenic change of strains. we have previously shown that immunization of naive mice with dna encoding the conserved internal antigen nucleoprotein (np) provides protection against both h1 and h3 strains of a/influenza. although such mice became infected they were resistant to weight loss and death this differed substantially from a/pr8 and a/hk recovered mice which were resistant to subsequent infection. to produce a more representative model of human infection, we infected the lungs of mice with currently circulating strains of human influenza. mice that had been given lung infections with a/beijing/92 were susceptible to subsequent infection with the a/hk/68 strain although they were resistant to weight loss and death. other strains such as a/beijing/89 or a/georgia/93 provided only marginal protection against weight loss and death against a/hk challenge. mice that were immunized with np dna had greater resistance to weight loss and death after a/hk/68 challenge than mice previously infected with a/bei/89 and a/ga/93, and were similar to mice that had been previously infected with a/bei/92. thus, infection with different virus strains provide various levels of cross strain protection and the level of protection provided by immunization with dna can exceed that induced by live influenza infection. the development of sendai virus-specific cytotoxic t lymphocyte (ctl) effectors and precursors (p) has been compared for mice that are homozygous (-/-) for a disruption of the h-21-ab class ii major histocompatibility complex (mhc) glycoprotein, and for normal (+i+) controls. the generation of cd8+ ctlp was not diminished in the (-/-) mice, although they failed to make virusspecific igg class antibodies. while the cellularity of the regional lymph nodes was decreased, the inflammatory process assayed by bronchoalveolar lavage (bal) of the infected lung was not modified and potent ctl effectors were present in bal populations recovered from both groups at day 10 after infection. there was little effect on virus clearance. as found previously with cd4-depleted h-2b mice, the absence of a concurrent class il-mhc-restricted response does not compromise the development of sendai virus -specific cd8+ t cell-mediated immunity. the importance of cytoxic t lymphocytes in defense against acute and chronic viral infections is gaining increasing recognition. our approach to investigating the structure-function relationship between immunogens and their in vivo ability to elicit cytotoxic t lymphocyte responses has been to formulate simple, well-defined structures that vary in their ability to introduce associated antigens directly into the cytoplasm of antigen presenting cells. we have introduced methods for the preparation of unique, lipid-matrix based immunogens, which are highly effective in mice and monkeys for stimulating strong cd8+ cytotoxic t cell responses, (ctl). antigens used have been proteins or peptides derived from influenza, parainfluenza, and hiv viruses, and whole formalin-fixed siv. ctl can be induced by parenteral as well as oral administration. comparing the physical and chemical nature of our formulations with those from other laboratories which have reported the use of subunit preparations to induce cd8+ ctl, leads us to propose that a minimal immunogenic formulation capable of eliciting cd8+, mhc class i restricted cytotoxic t lymphocytes includes: i) a peptide that represents a mhc class i epitope; ii) a component that enhances the aftinity of the immunogen for mhc class i positive antigen presenting cells ; iii) properties that can compromise the integrity of a lipid bilayer, facilitating delivery of the antigen directly into the cytoplasm for class i presentation. cd8+ responses to peptides, glycoproteins, and even whole fixed viruses, makes them attractive candidates for diseases where clearance of infected cells is important in protection and recovery. cani ne rabies is uncontrolled. rabies also is epizootifally active in several species in most areas of the wor!d. thm, vaccination of animals, both wild and domestic, as well as postexposure treatment of humans remains a global concern. unfortunately, in those countries in which,people most need postexposure prophylaxis, the best vaccines are expenswe and in limited supply, whereas available vaccines are of questionable immunogenic efficiency, are otten contaminated and may produce neurological complications. the goal of this study was to determine whether a rabies vaccine for global use is complete one round of replication have the potential to be used as vaccines. we have previously reported the abiliity of a ghdeleted herpes simplex virus type 1 (hsv-1) to protect mice and guinea-pigs from subsequent challenge with wild-type hsv. this virus, which we have called disc (disabled infectious single cycle) virus, can infect normal cells but the absence of gh in the progeny virus prevents further rounds of infection. as disc hsv clearly has potential as a vaccine, it is important to determine the durability of the immune response elicited by this virus. we have investigated the ability of disc hsv-1 to protect mice from a wild-type virus challenge six months post vaccination using the ear model of hsv infection. two immunisations on day 0 and day 21 resulted in a considerable reduction in virus titres in the challenged ears, and an almost complete absence of virus in the dorsal root ganglia. hsv-specific antibody titres as determined by neutralisation and ellsa were maintained for the six months period. it was possible to demonstrate an hsvspecific cytotoxic t-cell response in the disc hsv-1 vaccinated mice following challenge; this ctl activity was similar to that observed in mice vaccinated with wild-type virus and challenged after the same time period. animals vaccinated with inactivated virus or control mock-vaccinated mice showed a low level of ctl activity typical of a primary ctl response following challenge. these results indicate that an effective cell-mediated and humoral anti-hsv immune response can be maintained for at least six months following vaccination with disc hsv-1. viruses which lack an essential gene and thus can only the lmmunogenicity of two ctl @topes. influenza npl47-158 and plasmodium berghei cs protein 252-260 were studled in balblc mice. paptides were formulated as a) a iipopepudepeplkle conlugated to irlpalmltoyl-sgly~~l cysteine (pam3cyj) and dissolved in a 1% dmsolglycerol solution. b) micmparticles prepared with poly @.l ladide-coglywlide) using a solvent evaporation technique. the micropaltides were administered as a suspension in phosphate buffered saline or c) an emulsion prepared wilh egg lecithin and 10% soya oil in water. 1 wpg of peptide or controls (the welght equivalent of blanks) were administered to groups of 3 mice intra-peritoneally or subcutaneously at 1.10 and 20 days. 7 days following the last immunization splenocytes were cukured in vlro in the presence of appropriate pepwe or wntml m h rat con a supematant as a source of omwth fadors. ctl adivity was measured in a standard 4 hour chromium release assay and results expressed as % specific lysis. ctl could be elicited in vivo with all three formulations. at an ewedoctarget ratio of 1w:l the plasmodium berghei peptide encapsulated in micmpartides gave 47% iysls on peptide pulsed target calls. levels of lysis were similar for the peptide in emulslons. the iipopeplide p3ccs252-260 gave a level of lysis of 82% at an e:t ralioof1w1. these results demonstrate that peplides edminldered in a variety of formulations can induce a systemic ctl response in vivo. peplide vaccines using such formulations wuld be used to stimulate ctl responses as part of a prophyladic vaccines or as immunotherapeulics. attenuated the attenuated sabin strains of poliovirus have been used for many years to elicit protective immunity to poliovirus. oral vaccination with replicating polioviruses generates both mucosal and systemic immunity. therefore, use of recombinant polioviruses expressing heterologous antigens as vaccine delivery vectors should provide a system for generating protective immunity to those antigens. cdna copies of the poliovirus genome has been used to construct vectors containing a multiple cloning site for insertion of heterologous genes. a pilot enhanced-potency inactivated poliovirus vaccine (ejpv) with assumably improved immunogenicity containing win-treated type 3 poliovirus (shah sauketf) together with the regular type 1 and type 2 canponena was subjected to s*mdard safety and potency tesls in the labmatory and laken through wase i and i1 clinical aials. in balb/c mice, the lrypin-mcdit%d e-if' v cfryipv) was found to induce antibodies targeted ouaide the uypsin-sensitive bc-loop of capsid protein vp1. as previously shown for hypsin-mated type 3 poliovirus @vm alone. trypsin used to modify the type 3 component at the bulk phase was removed by the vaccine manufacturer (rivm) in the regular purification process. absence of uypsin in the final product was further confumed by immunizing mice and rabbits with 10-fold concentrated type 3 component of tryipv. assays for lrypin antibodies using eia and westem blot techniques were newve. in the clinical phase 1 aial six adult volunteers with existing immunity to poliovirus were given increasing doses of tryipv. already one tenth of the regular dose induced a booster effect in neuarlizing antibodies to both intact and mypsin-treated type 3 poliovirus. no unexpected sideeffects were recorded phase i1 trials comprised 50 adult volunteers with at least 5 years since the last dose of poliovirus vaccine and 50 children who were due to receive the third dose of the regular immunization schedule at about 2 years. in both groups. 25 individuals received tryipv and 25 were injected with the regular enhanced potency ipv (e-ipv). serum specimens drawn before injection and one month after were tested for neunalizing antibodies using standard microneuwlization assays (all mutypes) and the racina test (intact and uypsin-mated type 3 poliovirus). in all volunteers tryipv was at least as immunogenic t w the regular e-ipv according to all assays. no statistically significant differences in side effects were reported. a murine/influenza virus model has been used to evaluate the longevity of antibody and protective responses raised by gene gun delivery of a hemagglutinin-expressing dna. mice were immunized and boosted at one month with 0.4 ug of an h1 expressing plasmid dna (pcmvri1). antibody responses and protection against a lethal challenge were followed over the next year. antibody responses had good longevity exhibiting comparable titers at one year post boost as at 10 days post boost protection against the lethal challenge was complete at 10 days, 1 month and four months post boost, but only partial at one year. a transgenic mouse model for identifing htlv-1 t-cell epitopes: generation of hla-b*3501-restricted ctl directed against synthetic peptides and naturally processed viral antigens, christian schiinbach*, ai kariyone*, kiyoshi nokiharaa+, karl-heinz wiesmulle6 and masafumi takiguchil, departments of tumor biology* and immunology#, institute of medical science, university of tokyo, tokyo "tokyo university of agriculture and technology, tokyo +biotechnology instruments department, shimadzu corp., kyoto, japan $natural and medical science institute at the university of tubingen, reutlingen, germany the majority of human t-cell leukemia virus type-i (htlv-l), hla class i-resmcted t-cell epitopes have been identified by cloning htlv-1 patient-derived t cells. here we describe for the frst time a rapid method (reverse immunogenetics) for identifing t-cell epitopes, together with a transgenic mouse model as a guide for testing the cellular immune response to a mixture of the lipohexapeptide immunoadjuvant pamgcys-ser-(lys)4 and synthetic htlv-1 peptides which seem suitable for vaccine design. htlv-1 amino acid sequences were searched for eight to 14mer patterns carrying the anchor residues of the hla-b*3501 peptide motif at positions two and eight to fourteen. 65 candidate peptides were synthesized according to the matched sequence patterns. their hla-b*3501 affinity was quantitatively analyzed in an indirect immunofluorescence peptide binding assay using rma-s-b*3501 cells. the fourth group (controls) were inoculated with h3n2 (in) thereby providing heterotypic ctl immunity in the context of a natural infection without the confounding effects of humoral immunity against surface antigens. all four types of inoculations have been shown to protect normal (class i expressing) mice from a lethal challenge with influenza, presumably mediated by class i restricted cytotoxic t cells. the two groups inoculated via the intranasal route may gain additional protection by activating the mucosal immune system (iga). none of these types of inoculations has been evaluated in the context of class i1 restricted cytotoxic t cells, the only ctls found in class i deficient mice. for all four types of inoculations, mhc class i deficient mice lost significantly more weight than the class i expressing control groups (seven mice per group) indicating the importance of class i restricted t-cells in protection. within the class i expressing groups, there was no significant difference between the four types of inoculations; within the class i deficient groups the vac-np im immunized mice lost significantly more weight than the h3n2 group;the other two groups, vac-np in and genetically immunized groups had intermediary results. these data lend support for a protective role for mucosal immunity. results on both class i and class i1 ctl activity for the four types of inoculations will also be presented. we tested the pbmcs of patients participating in two vaccine therapy trials for their ability to recognize overlapping peptides of the gp120 la1 sequence. seventeen patients participating in a phase i gp160 protocol and 13 patients participating in a phase i gp120 protocol had their pbmcs isolated by ficoll separation of heparinized venous blood. the fresh pbmcs were plated, in triplicate, into 96 well plates containing peptides overlapping the la1 sequence of gp120, pulsed on day 7 with tritiated thymidine and harvested and counted on day 8. results: the percentage of patient's pbmcs from each trial with an lsi 2 5 to each peptide are depicted below. conclusions: the pbmcs of hiv-infected volunteers who have been multiply immunized with either gp160 or gp120 proliferate to multiple peptides within the gp120 molecule. reactivity from the end of c1 through early c2 (lai #i 12-21 1) is particularly prominent and contains previously undescribed th epitopes (asterisks). conspicuously missing is reactivity to the v3 loop peptide (la1 #300). although the percent reactivity to the entire gp120 molecule is similar between the immunization groups, there is differential recognition of some of the individual peptides, particularly peptides in early c3 (lai #319-348). the intracytoplasmic lifecycle of listeriu mmcytogenes (lm) enables it to be a convenient vaccine vehicle for the introduction of foreign proteins into the mhc class i pathway of antigen presentation. taking advantage of these properties, we have inserted the nucleoprotein (np) gene from lymphocytic choriorneningitis virus (lcmv) into the lm chromosome by site specific homologous recombination. infection of mice with recombinant lm expressing lcmv-np elicited a virus-specific ctl response. we were able to recover lcmv-np specific ctl precursers from recombinant lm vaccinated mice as shown by vigorous secondary ctl responses after in vitro stimulation. in contrast to mice immunized with wild type lm, mice vaccinated with np-recombinant lm were protected against challenge with immunosuppressive lcmv variants. protection was demonstrated by reduced viral titers or complete clearance of lcmv from serum and various organs including, spleen, liver, lung, kidney, and brain. the kinetics of the lcmv challenge indicate that mice vaccinated with recombinant lm were able to arrest viral growth early in the infection due to a strong ctl response and did not exhibit the immunopathology associated with infection of naive mice. since lm not only delivers antigens into the mhc class i pathway but also induces il-12 production, it has the potential to function simultaneously as a vehicle for expressing foreign antigens and as an adjuvant promoting cell mediated immunity. , latent membrane protein [lmp] 1 and 2a) in chromium release assays. we were fortunate in identifying one child from whom cryopresetved pbmc samples were available before. and during ebv seroconversion. ebv-specific ctl activity was demonstrated concurrent with initial detection of virus in the peripheral blood by ebv-dna pcr. in the absence of detectable serum antibody. ctl lines from all nine children recognized one or more ebv latent gene prcduct(s). all children demonstrated ctl responses against one or more ebna 3 proteins (3a, 3b, 3c). and ebna 3c was recognized most frequently. no ctl responses were detected against the ebv latent proteins ebna 1, 2, lp or lmp 1. the ebv-specific ctl lines expressed cd3/cd8 and mab blocking experiments demonstrated that the majority of target cell lysis was inhibited by antibody against mhc class-i but not antibody against mhc class-11. these results represent one of the first reports characterizing ebv-specific ctl responses in young children. the striking similarity between ebv-specific ctl responses described here in young children and those reported for adults suggests that the ebna 3 family of proteins and lmp 2a should be considered for inclusion in candidate ebv vaccines. evaluation of cellular immune responses to adenovirus vectors in the cotton rat. soonpin yei,' gary kikuchi,' ke tang' and bruce c. trapnell.' departments of virology' and immunology,2 genetic therapy, inc., gaithersburg, maryland 20878 replication deficient recombinant adenovirus (av) vectors are efficient gene delivery vehicles currently being developed for a variety of in vivo gene therapy strategies such a s for the fatal pulmonary component of cystic fibrosis. the cotton rat (sigmodon hispidus) is one of the most widely accepted animal models for studying these av vectors because wild type human adenovirus replicates in cotton rats and because of the histopathologic similarities of infected respiratory epithelial tissues from humans and cotton rats. despite this, methods for studying immunologic responses in the cotton rat have not been developed. importantly, recent studies in the cotton rat (gene tber. 1 :192-200; 1994) in our laboratory suggest that a dose-dependent specific immune response to av vectors can limit expression of the transgene. in this context, w e have established methods to evaluate cytotoxic lymphocyte (ctl) responses to av vectors in the cotton rat. to accomplish this, a ctl target cell line was established consisting of primary cotton rat lung fibroblasts (crlf). splenocytes from cotton rats exposed previously to an av vector were harvested, cultured in virro with irradiated, addb274nfected crlf. cultured (effector) splenocytes were then incubated with s'cr-labelled crlf (target) cells a t effectoctarget (e:t) ratios of 100, 50 and 10. in parallel, splenocytes from naive cotton rats served as negative controls. results demonstrated vector-specific ctl lysis of target cells significantly greater than controls: 80.3 f 1.3% vs 6.2* 0.5%. 49.6*1.6% v s 5.7*0.4%. and 22.8*3.5% vs4.8*0.5% (meanrts.e.m., n=3; p<o.ol, all comparisons) a t e:t ratios of 100, 50 and 10, respectively. thus, a specific ctl response does develop in cotton rats following in vivo av vector administration. this observation will be useful in guiding vector modifications in the rational development of improved vectors for clinical use. the identification of ctl epitopes is essential for subunit vaccine design against aids. a major portion of the anti-viral ctl responses after infection with htv in humans or siv in rhesus macaques is made up by anti-gag specificities. here, effector cell populations were established from hiv seropositive individuals by specific stimulation of pbl with pfa fixed autologous b-lcl infected with tw gag. expanded cultures were then tested for ctl activity against autologous b-lcl pulsed with overlapping 20-mer peptides derived from p24. two individuals (008 and 617) had ctl against p24.14 (a(mino) a(cids) 263-282, krwiilglnknrmysftsi), two others (038 and 157) recognized p24.17 (aa293-312, frdwdrfyktlraeqasqd). similarly we found two siv infected rhesus macaques reacting against the analogous aa sequences of siv; it. p26.14 (8653) and p26.17 (ivy). this led us to further fine map the human and rhesus ctl reactivities using sets of 9 aa overlapping 10-mers and to test for hiv-1isiv crossreactivity. both two p24.14 epitopes (possibly restricted by hla-a2) and the p26.14 epitope were non-overlapping, and all three epitopes were non-crossreactive. finemapping of the p24.17 epitopes is in progress. the sn p26.17 epitope was also non-crossreactive, despite only one aa difference (position 6, s+t) between the siv and hiv sequence. others have also mapped cl'l epitopes to the same regions of p24 in humans (johnson ji 147,1512 ,1991 , buseyne jv 67,694,1993 and to p26.17 in cynomolgus macaques (gotch jmprim 22.1 19,1993) . in conclusion, multiple non-cross reactive ctl epitopes are clustered within corresponding regions of hiv and siv gag. ctl hotspots may be important for inclusion into vaccines. forming virus) with cd-4 and cd-8 1-cells, b-cells and adherent cells, while an average of 5% of virus was associated with lps-stimulated 8-cells and adherent cells. in the second experiment the combined function of antigen presenting cells, 1-helper cells and 8-cells (humoral immune response) to a third party antigen (srbc) during the acute phase of enterovirus induced disease was increased at day 0, and decreased at days 2, 3 and 4 post-cvb3, infection relative to unlnlected animals. conclusions: these data indicate that cvb3, associates with and replicates in rnurine splenic immune cells, and that infection of susceptible mice with cvb3, modifies the immune response to a third party antigen during the acute stages of disease. an understanding of such cvb3,-i mmune cell interactions is criitical for gaining insight into the pathogenesis and persistence of enteroviral infection both within and outside of the immune system. understanding the pathogenic mechanisms of virus infections depends upon identification of viral gene products involved in host-virus interactions in vivo. many viral gene products which affect pathogenesis in vivo are nonessential for virus replication in vitro. therefore, we constructed insertion or deletion mutants in the hind i11 j and i regions of murine cytomegalovirus (mcmv) to assess the importance of these genes in in vivo replication of virus as well as interaction with immune regulatory and effector cells. three mutants replicated in nih 3t3 fibroblasts as efficiently as wild-type virus, thus confirming the nonessential nature of the genes. deletion of 2.8 kb in hind i11 j and of 7.7 kb in hind i11 j and i resulted in mutants with reduced replication in target organs in vivo. an insertion in hind i11 j had no significant effect on replication in mouse salivary gland, but curtailed growth of the virus in footpad fibroblasts and subsequent priming of mcmvspecific cpl precursors in the draining lymph node. in addition, the 7.7 kb deletion mutant was unique in its failure to replicate in a permissive, differentiated macrophage cell line. current studies aim to further define the role of this gene region in hcmv pathogenesis. that in vv5-4 progression o f v v life cycle is blocked at the the uncoating ii or replication stage. furthermore, host protein synthesis in v v 5 -4 cells is not shut-off after v v infection suggesting uncoatingll/replication is important for determining cell fate. since this mutant clone vv5-4 was isolated b y retroviral insertional mutagenesis. a cellular gene that was integrated by single retrovirus was isolate and codes for a 5kb transcript in northern blot analysis. a 2 kb cdna was isolated and codes f o r a novel polypeptide o f 800 amino acids that show no homology with known proteins. experiments are in progress to understand t h e role of this cellular gene in vv infection. in addition, a two-hybrid system will be employed to identify a n y viral gene product interacting with this cellular target u s i n g o u r laboratory's s t a n d a r d s t r a i n o f a/japan/305/57 in in uitro assays of viral infectivity of the mouse b-lymphoma a20-1.11. we have found that although infection sensitizes the a20 cells for recognition by both class i and class i1 restricted ctl.'s and leads t o high surface expression levels o f hemaglutinin (ha), the infected a20 cells grow through t h e infection, ha expression declines, and few of the infected cells die. in contrast, using a second s t r a b of a/japan/305/57 from a different passage history, we find that n o t only are the infected a20 cells sensitized for lysis by both class i and class ii restricted ctl's, exhibiting even higher levels o f ha surface expression, but in addition, most cells in the infected population die with characteristics of apoptosis. the two virus strabs appear to be highly related because in various assays we have found t h e m antigenically indistinguishable in both their t-and b-cell epitopes. using these two virus strains in a n in uiuo dose response assay, we have found that o u r laboratory's standard strain will kill all mice infected with a 10-2 the therapeutic and prophylactic antiviral efficacy of interleukin-12 (il-12) was studied using murine models of herpes simplex virus (hsv) and murine cytomegalovirus (mcmv) infection. therapeutic intraperitoneal administration of il-12 commenced 6 hours after mice were infected w i t h hsv, and was continued daily for a total of 5 days. il-12 therapy improved the survival rates of mice w i t h systemic hsv infection, compared to that of placebo treated infected mice. since neutrophilia and thrombocytopenia were common in dengue patients,we are investigating whether bm may be accompanied with abnormal hematopoiesis. using ficoll-hypaque separation, mononuclear cells prepared from the total bm cells. 1 .0x106we~ells/well of both adherent and nonadherent cell types were infected with two different den-2 virus strains : (1) thiland strain isolated from the dengue hemorrhagic fever(dhf1 patient and (2) taiwan strain isolated from the dengue fever(df) patient at moi=l and collected the suprnatants at various time points for assay. the preliminary data showed that : (a) adherent cells were infected with den2 and dhf virus strain replicated much more efficiently (2.3x104pfu/ml on day 3 p.i.1 than df strain; (b) nonadherent cells were 10 times less efficient to produce viral yields than adherent cells (dhf and2 df strains peaked at 6.3~10' pfu/ml and 1.5~10 pfu/ml on day 3 p.i., respectively); (c) addition of gm-csf to nonadherent cells increased virus infectivity and productivity. in conclusion, den viruses were able to infect bm cells and the more virulent virus strain isolated from dhf patients had better replication capability in human bone marrow cells. future studies on cytokines and immunologic evaluation are in progress. number of gene products whose function is to modify host responses. recent evidence suggests that a subclass of poxvirus-encoded host response modifiers interferes with host defense responses, and thus can be termed immune or inflammatory response modifiers (irms). several poxvirusencoded irms possess homology to mammalian proteins which are associated with host defenses, while other poxvirus irms have little sequence homology to immune-effector molecules or their receptors. the ultrecht strain of rabbitpox (rpv) virus induces a large reddish pock on the chorioallantoic membrane (cam) of the chicken embryo. rpv mutants lacking the spi-2/crmn38k gene trigger an inflammatory response by 36-48 hr after inoculation, similarly to that found for cowpox virus (brighton red strain) mutants. rpv mutant viruses with a deletion of the ps/hr gene, a gene which has recently been associated with virus host range and virion maturation, elicited an inflammatory response on the chicken embryo cam at 48-72 hr after virus inoculation. thus, another poxvirus-encoded gene product has been identified that has irm functional activity which would not have been predicted by sequence analyses. poxviruses have recently been found to possess a herbert w. virgin iv, center for immunology and division of infectious diseases, washington university school of medicine, st. louis, mo 63110 murine cytomegalovirus (mcmv) follows three stages of infection in the immunowmpetent host. following the initial acute infection, a persistent infection remains in which lytic virus resides in the acinar cells of the salivary glands. finally a third stage of infection exists which is characterized by a lack of detectable infectious virus. it is not known whether mcmv remains persistent at some low level during this thiid stage or establishes molecular latency. to test this, spleens, kidneys and salivary glands from mice 2-8 months post infection were evaluated by two sensitive assays. sensitivity was tested using virus co-cultured on murine embryo fibroblasts (mefs) for 14 days. by this method, 1 pfu mcmv was reproducibly detected in the presence of sonicated spleen, kidney and salivary glands diluted 1:lo. scid mice were used as a second detection assay for mcmv. using 46 scid mice and different doses of virus, the lds, of mcmv in scid mice was 2-3 pfu. mcmv infections were detected when 10-25 pfu were added to sonicated spleens, kidneys, and salivary glands prior to injection into scid mice. using both co-culture with mefs and injection of tissue into scid mice, we have tested a total of 34 spleens, 34 kidneys, and 37 salivary glands and have detected no persistent virus. while no preformed virus existed in these tissues, reactivation from explanted spleen, kidney and peritoneal exudate cells occurs in culture after 10-40 days. in addition, lytic mcmv was detected in scid mice 28 days after injection with 2(10)' kidney cells from latently infected mice. following identical transfers of latent spleen cells, mcmv was not detected in scid mice although explants from the same organs reactivated in vitro. using facs sorting, panning, magnetic bead separation, and reactivation assays, individual cell types have been analyzed for the presence of mcmv genome and the ability to reactivate virus. heat shock proteins (hsp) are expressed in all organisms in response to a variety of stresses, including virus infection. however, since viruses are not known to encode hsp genes, this represents expression of cellular hsps. we have found a dramatic induction of the 72kda hsp in the ovaries of w-infected mice, and using primary ovarian fibroblasts, demonstrated hsp72 mrna within virus-infected cells. the physiological role of hsps expressed during virus infection is unknown, although hsps act as molecular chaperones to facilitate protein folding and assembly in unstressed cells, and a number of bacterial hsps are essential for bacteriophage replication and morphogenesis. in order to determine whether hsp72 expression was beneficial to vv replication, we constructed a recombinant vv which encoded murine hsp72, but found that this virus grew to similar titres as control viruses, both in vitro and in vivo. in particular, kinetic studies of virus growth in an hsp72-negative cell line showed no difference from control viruses, and the presence of hsp72 did not influence the virulence of infection in immunocompromised nude mice. these results are interesting given that hsp70 proteins act as molecular chaperones and that hsp72 can be found in association with vv proteins. we suggest that this association may simply reflect the inherent affinity of hsp70 for non-native proteins, and the close physiological proximity of hsp and nascent viral proteins within the virus infected cell. our results indicate that the functional significance of these interactions are minimal with respect to the outcome of virus infection. furthermore, although the expression of hsw2 in vv-infected mice coincides with the peak anti-viral cellular immune response, hsp72 does not appear to be immunologically significant, as we cannot demonstrate any immunological responses to hsp72 during the course of vv infection. the effect of in vivo neutralisation of ifn-y on cytokine production during influenza infection was compared in cs7/bl6 and balb/c mice. similar cytokine profiles were obtained on in v i m restimulation of mln or spleen cells from virus-infected mice of each strain. at days 7 and 10 postinfection, mln or spleen cells from both strains of mice produced il-2, il-6, il-10 and ifn-y, but little or no il-4 or il-5. however, following in vivo treatment with anti-ifn-y antibody, production of 1l-4 and il-s was induced in mln and spleen cells of balb/c mice whereas those from cs7/bl6 mice showed little change in cytokine profile. an increase in il-6 and 1l-10 production was also observed on in virro restimulation of splenocytes from balb/c mice. however, significant amounts of 1l-2 and ifn-y were still secreted in these cultures. in v i m neutralisation of ifn-y in the restimulated cultures had little effect on the production of other cytokines, regardless of whether this cytokine had also been neutralised in vivo .despite the change in cytokine profile, anti-ifn-y treatment had no effect on the kinetics of viral clearance in balb/c mice. a similar situation was seen for c57/bl6 mice. congenic strains of mice are currently being used to determine whether the effect on cytokine profiles is associated with the mhc haplotype. wunner, and steven l. spitalnik, department of pathology and laboratory medicine, university of pennsylvania and the wistar institute, philadelphia, pa 19104 rabies virus glycoprotein (rgp) is the only glycoprotein on the viral surface, is the target of viral neutralizing antibodies, and is the viral protein that interacts with the host cell receptor. rgp of the era strain is a 505 amino acid type i membrane glycoprotein with 3 potential n-glycosylation sites at asn37, asn247, and asn319. due to rgps central role in the immunology and biology of rabies, it is important to determine its three-dimensional structure. towards this end we produced a recombinant form of rgp that may be more amenable to analysis by x-ray crystallography. to avoid the use of detergents, we constructed a soluble 434 amino acid form of rgp lacking a transmembrane domain (rgpt434). rgpt434 was secreted by transfected cells and was appropriately glycosylated, assembled, antigenic, and immunogenic. since n-glycosylation was required for rgpt434 secretion, we minimized the number of n-glycans by site-directed mutagenesis. interestingly, rgpt434 was secreted only when asn3l 9 was n-glycosylated. in contrast, full-length rgp was expressed at the cell surface a any of the three potential sites was n-glycosylated. soluble inhibitors of n-glycosylation were used to simplify the structures of the n-glycans on rgpt434. although inhibiting alpha-glucosidases with castanospermine blocked secretion, inhibiting any further processing with deoxymannojirimycin had no effect on secretion. finally, to simplify the purification process, a tail of 6 his residues was added to the cooh-terminus of rgpt434. this modification did not affect secretion, antigenicity, of assembly of the recombinant glycoprotein, but did allow purification using ni+2-agarose chromatography. in summary, we constructed a soluble form of rgp with a minimal number of homogeneous n-glycans and an "epitope" tag. this form of rgp is structurally similar to the extracellular domain of the full-length protein and may be amenable to analysis by x-ray crystallography. endogenous retroviral genes (ev) are well characterized in chickens. our laboratory has established chicken lines with single and multiple ev loci and, compared to ev negative birds, these lines exhibit a delayed and suppressed titer of neutralizing antibody in responses to avian leukosis virus (alv) challenge. to address the effects of ev genes on the cell mediated immune response we have developed an in-vitro assay to monitor the in-vivo induction of mhc restricted cytotoxic t cells (ctl) induced by alv. using this assay, we find the ctl response in birds expressing the ev-21 locus is reduced 85 to 95% compared to ev negative birds having nearly identical (eighth backcross) genetic backgrounds. other ev loci (1,6,10 and 11 ) also reduce the ctl response to alv in mhc matched chickens with different genetic backgrounds. ' national public health institute. helsinki. finland universities of tampere, qulu and 'helsinki, finland coxsackie b virus infections have been associated with clinical iddm in seveml previous studies but their initiating role in the slowly progressing beta-cell damage was for the fmt time directly implied in our recent prospective. nationwide dime study. siblings of the index iddm cases who progressed to clinical iddm experienced enterovim infections more frequently than nonprogressing siblings during prospective follow-up period. enterovim infections were initially demonstrated by detecting significant increases in serum class-specific cross-reactive enteroviral antibodies by using radioimmunoassay (ria) with the heavy-chain capture principle. causal association benveen these infections and the pathogenesis of iddm was suggested by temporal association of sercconvenions of viral antibodies with increases in islet cell antibody levels which are considered to be markers ok ficell damage. the possibility of the existmce of specific diabetogenic s & s of enternviruses was then considered. to identify the serotypzs responsible for the infections, successive sera from subjects exhibiting seroconvenion of autoantibodies coinciding with an increase in enteroviral antibodies were anlyzed by plaque n e u n a t i o n assay using a set of different enterovhes. the results indicate that several enternvirus serotypes may be associated with the progressive prediabetic p~ocess. not only different coxsackie b but also coxsackievirus a9 -infections were found to coincide with seroconvenions of islet-cell and insulin auwantibodies. it is known that an immunogenic region of gad 65. one of the major isletcell antigens, shows a high degree of homology with the 2c protein of cbv-4. studies on potential immunological cross-reactions between enteroviruses and two significant autoantigens, hsp60 and gad65, are in progress. this is carried out by using immunohistochemistry in infected cell lines and in frown sections of human pancreas with antibodies induced by synthetic peptides and natural and expressed viral proteins, the role of p53 alterations in human malignant pleural mesotheliomas (mpm) is unclear. immunostaining (ipox) of snap frozen mpm specimens revealed p53 expression in 31 of 52 (60%). p53 detection by ipox is usually associated with mutated p53. sscp for exons 5-9 revealed p53 alterations in 2 of 25 specimens, and loh at exon 4 was seen in only 2 of 12 evaluable specimens. thus, most of these specimens appeared to contain wild-type (wt) p53. we recently reported that sv40 induces mesotheliomas in rodents, and that sv40-like sequences are present and expressed in mpm. of note, sv40 large t antigen (tag) binds and inactivates wt p53 i n ! , abolishing its tumor suppressor function. we theorized that the immunohistochemical persistence of p53 may result from its physicdl binding with tag. tag was detected by ipox in 32 (62%) of these mpm specimens. expression of tag was significantly associated with coexpression of wt p53 (p2 = 0.008, fisher's exact text), and preliminary experiments suggest co-precipitation of p53 and tag. finally, we demonstrate that waf-1 is not detectable in mpm expressing wt p53 suggesting that p53 is inactive. we speculate that tag expression in mpm binds to and inactivates p53 contributing to the malignant phenotype. human papilloma virus (hpv) infection is involved in 90% of cervical carcinomas and possibly 95% of cervical intraepithelial neoplasia (cin). at present it is unclear whether patients infected with the transforming hpv types can mount efficient t cell responses. to address this issue we examined the ctl response of peripheral blood lymphocytes from patients attending a cervical neoplasia outpatient clinic. all were diagnosed hpv positive with various stages of c m and nine patients were selected for hla-a2 expression by facs analysis. pbmc were stimulated with caski, a cervical carcinoma cell line demonstrated to be hf' v type 16' and hla-a2'. culture media consisted of rpmi-10% human serum supplemented in three cases with 15 u/ml il-7, in another three cases with 3 u/ml il-2 and 15 u/ml il-7, and in the final three cases with 10 u/ml il-2 and 15 u/ml il-7. the ctl were screened for reactivity to caski and in addition the cervical carcinoma cell line c33a (hpv, hla-a2') transfected with either the hpv type 16 e6 or e7 genes. one patient's cells maintained with 10 uiml il-2 and 15 ulml il-7 showed hpv e7 protein specific cytotoxicity in a hla-a2 restricted fashion. these ctl lysed the caski stimulator cell line but not the nk sensitive target k-562. the ctl efficiently lysed a c33a-e7 clone but in contrast a vector only transfectant was not lysed. the lysis of c33a-e7 was blocked with a-cd3 and a-cd8 antibodies at 66% and 41% respectively. facs analysis determined that this ctl population was 92.8 % cd3'cds' and 7.3% cd3'cd4'. limited e6 recognition was also observed but has not been hlly characterized to our knowledge this report represents the first demonstration of hpv e7 responsive ctl isolated from the peripheral blood of hf' v infected patients. we have previously shown that proviral variants found in the peripheral blood mononuclear cells (pbmc) of hiv-1 infected individuals can interfere with recognition by autologous cytotoxic t-lymphocytes (ctl). abolition of recognition could be caused by interference with t h e processing of viral peptides, by a failure of the variant peptides to bind to mhc class-i molecules or by a failure of the mhc class-vpeptide complex to efficiently activate the tcr (t-cell receptor). these mechanisms could allow the virus to escape the immune surveillance by ctl. most studies so far have addressed the question of viral variation in t-cell epitopes by analysing proviral dna, extracted from the pbmc of hiv-1 infected individuals. however, the analysis of virion-associated rna offers several advantages. sequences found in viral rna are likely to be functional, a t least u p to the point of packaging into virus particles. furthermore, rna sequences represent virus which is currently actively produced. here, we present the sequence variation i n two hla-b8 restricted epitopes: p17-3 gag and amino acids 18-26 of the pol gene. both proviral dna from pbmc and viral rna sequences from plasma are discussed. the ability of viral variants to hind to mhc class-i molecules was assessed and the recognition of variant peptides by ctl was tested. variants were also tested for their ability to antagonize recognition of the index sequences. we show that viral variants with the ability to interfere with recognition by ctl are not only found in proviral dna but also in virion associated rna. objective: clinical course of hiv-1 infection may be influenced by an effective host immune response against hiv-1 and/or by viral properties. to investigate the cellular immune response to hn-1 we analyzed ctl activity against different hiv-1 proteins in long-term asymptomatic hiv-1 infection as well as during rapid progression to aids. methods: 10 longterm asymptomatic individuals with cd4 counts >500 celllpl after more than 8 years of infection were selected from the amsterdam cohort study on aids versus 10 subjects who progressed to aids < 5 years. ctl activity was measured on "cr labelled hla matched or autologous b-lcl, infected with rvv expressing hiv-1 ag. both bulk and limiting dilution ctl assays were performed longitudinally with pbmc after ag-specific stimulation. sequences of ctl epitopes were determined in homologous virus isolates resulrs: different kinetics of anti-gag ctl responses were observed in rapid progressors. in any case ctl responses disappeared during progression to aids. in long-term asymptomatic subjects persistent ctl responses were observed together with low viral load. conclusions: sustained, broad anti-hiv cellular immunity may correlate with maintenance of the asymptomatic state in long-term survival by controlling viral replication. enteroviruses are a large group of positive stranded rna viruses known to be responsible for a number of distinct disease entities. recombination is thought to be capable of generating new enterovirus strains that cause significant morbidity. for example, enterovirus 70 which was responsible for a pandemic of haemorrhagic conjunctivitis and poliomyelitis is thought to have originated by recombination between a coxsackie b like virus and another unidentified enterovirus. we are studying a group of echovirus 11 isolates from an outbreak of disease in southem india. sequence analysis within the 5' untranslated region reveals that these isolates fall into two groups that differ by -20% (equivalent diversity to that seen between between published sequences of poliovirus 1 and coxsackie a 9 virus). these two groups of viruses also differ in their cell tropism. isolates defined as group 1 by their 5'utr sequence grow equally well on ht29 cells (a human colon carcinoma cell line) and vero cells. isolates of group 2, with one exception, grow only on ht29 cells. analysis of the structural proteins of these isolates revealed differences in migration that correlated with their cellular tropism. thus, significant genotypic and biological diversity exists amongst these virus isolates. one virus isolate had the 5' untranslated region sequence of a group 1 virus but the protein profile and cellular tropism of a group 2 virus. the best explanation of these findings is that this anornolous isolate is a natural recombinant between the parented strains. both the ease with which viable recombinants are generated and the diversity present within this one enterovirus serotype increase the potential for the production of novel pathogenic enterovirus strains. dominant susceptibility to polyoma tumors in inbred wild mice, sharon r. nahill, yupo ma, john carroll and thomas l. benjamin, department of pathology, harvard medical school, boston, ma 021 15 polyoma virus (f' y) is a mouse dna tumor virus which, under appropriate conditions, causes tumors in a wide variety of cell types. generation of tumors is a function of both the viral and host genomes. lukacher et al. have recently described a dominant gene, pyv', carried by the c3-i mouse strain, which confers susceptibility to py-induced tumors mapping and immunological analyses indicate that py4 is the mouse mammary tumor virus 7 superantigen (mtv 7 sad gene, which deletes t cells required for py tumor immunosurveillance in h-2' mice. to determine the generality of endogenous superantigens as determinants of susceptibility and to reveal potentially novel mechanisms of susceptibility, we have looked for dominant susceptibility @ s ) gene(s) id newly established and genetically diverse inbred wild mouse strains, czech i1 and pedatteck (peru). both strains are susceptible to py as 100% of infected animals develop a full profile of tumors. crosses between cs7br, whose resistance is contributed by the major histocompatibility (mhc) locus, and susceptible peru or czech 11, yield f1 progeny which are fully susceptible, indicating a dominant inheritance pattern of susceptibility the incidence of tumor-bearing backcross animals [((peru x cs7br) x c57br) and ((czech i1 x cs7br) x c57br)i suggests that ds is due to at least one, but not more than two genes. amplification of genomic dna from the czech i1 and peru mice by pcr using primers specific for mtv 7 sag indicates that both strains are negative for proviral mtv 7 sag. furthermore, the mechanism ofds in these mice may be independent of all mtv sag as pcr using primers specific for the highly conserved region of mtv sag is unable to amplify mtv dna from peru or czech i1 genomic dna. these results indicate that, like the c3hibi, the pedatteck and czech i1 contain gene(s) which overide the resistance to py-induced tumors contributed by the mhc of the c57br parent and which may cause tumors via a novel, mtv sag-independent mechanism. we have initiated efforts to map the ds in peru and czech i1 mice using pcr and primer pairs flanking simple sequence length polymorphisms. fis-2 is a low leukemogenic, but relatively strong immunosuppressive variant of friend murine leukemia virus (f-mulv). this variant was originally isolated from t-helper cells of flc-infected adult nmrl mice. compared to f-mulv, fis-2 suppresses primary antibody response more efficiently in infected mice. some of the fts-2 infected adult nmri mice developed a disease resembling the acquired immunodeficiency syndrome induced by hiv. restriction mapping and nucleotide sequence analysis of fis-2 show a high degee of homology between this variant and the prototype f-mulv clone 57. in this study we have attempted to localize the genomic determinant of fis-2 which is responsible for induction of a strong suppression of primary antibody response. six chimeric viruses of fis-2 and f-mulv were constructed. the primary antibody response of the mice infected with these chimeric viruses were investigated. the results of these experiments will be presented. anti-fmdv antibodies, as measured in an elisa capture assay, were cross reactive. b) cellular: proliferative (cd4) t cell responses of peripheral blood mononuclear cells (pbmc) were low or undetectable during primary responses to vaccine or virus, and frequently low during secondary responses. for good t cell proliferation in vitro, multiple immunisation is required. this may reflect preferential stimulation of the th2 cd4 t cell subset. interestingly, when cd4 responses were observed, cd8 tcell responses were also detectable. 2 . recognition of individual viral proteins a) expression cloning: structural and non-structural protein pseudogenes were cloned from cdna by pcr. expressed in pgex-3xuc. and purified by sds-page. b) humoral: structural and non-structural proteins were recognised by infected animals. a good anamneetic antinon-structural response was only observed when the boosting serotype differed from the serotype stimulating the primary response. c) cellular: both structural and non-structural proteins were recognised and some were cross reactive. interestingly, vp1 was strain specific, and the polymerase (3d) was the most immunogenic and cross reactive. d) a construct comprising 3d and the immunodominant vp1 epitopes was prepared and tested. in common with other herpesviruses, the envelope glycoproteins of equine herpesvirus 1 (ehv-1; equine abortion virus) are major determinants of the infectious process and pathogenicity, and are inducers of humoral and cell-mediated immune responses. as such, they are candidates for components of subunit vaccines against ehv-1. to generate useful amounts of individual ehv-1 glycoproteins, we have constructed recombinant badoviruses capable of expressing glycoproteins c, d, h (gc, gd, gh ) in insect cells, and have evaluated the recombinant products as innnunogens in a murine model of ehv-1 infection. au three glycoproteins induced serum (elisa) antihodies to ehv-1, and ehv-1 gc and gd also induced neutralizing antibody responses. following intranad challenge with infectious ehv-1, protective immunity, as demonstrated by acelerated clearance of virus fiom respiratory tissues to below detectable levels, was evident in mice immunized with either recombinant gc or gd. in contrast, gh-immmkd mice did not develop detectable neutralizing antibody, and did not clear challenge virus more rapidly than controls. delayed type hypersensitivity and lymphoproliferation responses to ehv-1 antigen were observed for each of the ehv-1 glycoproteins, and in experiments with gdimmunized mice, a role for cell mediated immunity in protection was confirmed by adoptive transfer and t-cell depletion experiments. the data provide support for the potential of glycoproteins c and d as a subunit vaccine against ehv-1. molecular pathogenesis of ural infeetiom 52-331 enterovirus-immune cell interactions: implications in enterovirus-induced diseases we have also evaluated the effect of virus infection on the humoral immune response to cvb3, infection in adolescent c3h/hesnj mice. antigen presenting cell, 1-helper cell and 8-cell function were evaluated utllizlng a sheep red blood cell (srbc) plaque assay. mice were injected intrapentoneally (ip) with lo5 plaque forming units of cvb3, at day 0 and with lo7 srbc's at days 0, 2, 3 and 4 post-cvbb, infection. splenocytes were harvested 4 days post-srbc injection, mixed with target srbc's and guinea pig complement and incubated. plaques were then quantitated. results: cvb3, was associated with 12.9% to 17.4% of cd-8 positive t-cells and w a 11 % to 26% of adherent splenocytes. after mitogen (lps and con a) stimulation, b-cells and adherent cells were demonstrated to be permissive for viral replication. a 248% and 738% under non-stimulated conditions. an average of 1 % of virus is cell-associated (plaque north america. bruce anderson, teny yates, norah torrez-martinez, wanmin song, brian hjelle. university of new mexico, albuquerque, n.m.we recently identified a new species of hantavirus (hmv) associated with the harvest mouse reithrodontomys megalotis (hjelle b et al, j. viroj. 1994, in press ). an arizona woodrat (neotoma mexicana) was found to he infected with hmv, presumably through "spillover". hmv is most closely related to the four comers hantavirus (fcv) of deer mice (genus peromyscus). the nucleocapsid gene and protein of hmv differ from those of fcv by 24% and 15% of residues, and the 1896 nt s genome is shorter by 163 nt. we surveyed 174 reithrodontomys animals captured in the u.s. and mexico for hantavirus antibodies; 27 (15.6%) were positive. s segment cdnas were amplified and sequenced from seropositive animals captured in california (4), arizona (3), new mexico (l), and mexico (2). a monophyletic clade of hmv-like agents was identified at all sites, although an r. megalotis infected with an fcv-like virus was also identified in the state of zacatecas, mexico. nucleotide sequence distances among members of the hmv clade were up to 15.5%. but amino acid distances were less than 2%. hmv is enzootic in harvest mice throughout much of north america, and can also infect wood rats. htlv i-associated myelopathy/tropical spastic paraparesis (hamnsp) is a slowly pro ressive neurological disease characterized by perivascuaar mononuclear infiltrates in the cns. htlv i-specific cd8+ ctl are found in pbl and csf of infected patients with htlv i-associated neurological disease but not in htlv i seropositive individuals without neurological involvement. previous studies have shown that in hla-a2+ patients, htlv i-specific cd8+ ctl restricted by hla-a2 recognize a peptide derived from the htlv i tax protein (tax 11-19 llfgypvw). in the present study, we have analyzed the potential of these tax-specific ctl to recognize addtional peptides. our results demonstrate that a subpopulation of high affinity cd8' tax 11-19 specific ctl clones cross-react on a self peptide derived from the se uence of myelin-associated glycoprotein (mag 556-564 vl&sdfri) presented by hla-a2. these obsenatlons suggest that the demyelination process in hamltsp may be,due, in part, to virus-specific ctl recognition of a self myelin component that is independent of htlv i infection. development of pathology varies widely between different strains of mice after intracerebral inoculation with the so-called 'docile' isolate of lymphocytic choriomeningitis (lcm) virus. the c3hebfej and blo.br/sgsnj mouse strains have been of special interest because they display autoimmune hemolytic anaemia with varying degrees of apparent immunological involvement. in this study, we examined the role of cd4+ t helper cells in this autoimmune response by treating mice with the cw-specific gk1.5 monoclonal antibody. we also determined if polyclonal activation of b lymphocytes, induced either by lcm virus or by lactate dehydrogenase-elevating virus, another well known b cell activator, correlated with the development of anaemia in these mice. our results strengthened the central role of the immune system in the anaemia in c3h mice by showing that depletion of cd4+ cells largely, if not completely, abrogated this anti-erythrocyte autoimmune reaction. as reported by others, we found that the anaemia was more mild in b 1o.br mice than in c3h mice. however, we could not confirm the difference in the degree of b lymphocyte polyclonal activation between these mice. furthermore, lactate dehydrogenase-elevating virus had no apparent effect on erythrocytes, even though this virus also induced a sharp increase in plasma igg levels. one of the two class i mhc (h-pkd)-restricted immunogenic sites identified on the influenza strain aijapanl57 (h2n2) hemagglutinin (ha) encompasses two distinct partially overlapping epitopes, mapping to residues 204-212 and 210.219. when we investigated the magnitude of the ctl responses of balwc mice to the two overlapping epitopes, we found that while the nhrterminal nonamer epitope is immunodominant, eliciting vigorous ctl responses in njapanl57-immunized balb/c mice, the ctl responses to the cooh-terminal decamer epitope are weak and variable. the c-terminal epitope subdominance seems to be due to factors other than inefficient processing of the epitope in vivo because ctls generated by priming mice with recombinant sindbis viruses expressing only one of the ha 204-219 subsites displayed patterns of responsiveness similar to that of influenza virus primed ctls. limiting dilution ctl assays showed that the ctl precursor frequency (pctl) of the nterminal epitope is at least ten fold higher than the pctl of the cterminal epitope, implying that the low and variable pattern of cterminal specific responsiveness was due to the limited t cell precursors in the c-terminal specific ctl repertoire. this was further confirmed by the limited heterogeneity in the cross reactivity patterns displayed by the c-terminal specific ctl for an ig vh fragment and the ha 210-219 epitope of influenza strain a i m 5 7 in short term bulk cultures, and the facs analysis of tcr vg chain usage. taking these together with our previous observation that some jha 210-219 specific ctls can also crossrecognize an ig vh fragment. these studies had provided a strong evidence that ig gene products may influence t lymphocyte function and repertoire development. we have previously described the identification of homologous regions in the c-terminus of hiv-1 gp41 and in the n-terminus of hla class i1 beta chains. forty percent of patients infected with hiv-i virus were shown to have antibodies which bind to the homologous sequences, as well as to native hla class i1 molecules. affinity purified crossreactive antibodies (crab) were shown to have direct blocking effects on normal t cell responses to recall antigens, and could mediate adcc of hla class ii+ cell lines.in order to determine the contribution of such antibodies to disease progression, we obtained longitudinal plasma samples from patients in the macs study. in a first study, it was found that the presence of high titers crabs correlated with a more rapid disease progression (p = 0.027 by fisher two tail analysis)in a second, 7 year-longitudinal study of 12 progre.ssors and 12 stable patients we found: (1) the production of crab was seen in 70 -80% of rapid progresson, while the true stables produce only infrequent low-titers crab. (2) in rapid pmgressors, production of crab preceded by 2-3 years the marked drop in cd4 counts. (3) crab production did not correlate with the degree of hyperglobulinemia in these patients. (4) the presence of crab during the asymptomatic stage correlated with early loss of t-helper responses to recall antigens.we are currently establishing whether periodic measurements of crab in patients sera could be valuable in predicting a drop in cd4 counts and disease progression. the lymphokine ifn-y is i pleiotropic insnunomodulator and possesses intrinsic antiviral activity. we studied its significance in the development of antiviral immune responses using ifn-7 receptor deficient (ifn-yr-'.) mice. after inoculation with live attenuated pseudorabies virus (prv) the mutant mice showed no infectivity titers in various tissues and transient viral ag expression only in the spleen similar as in wild-type mice. however, the absence of the ifn-yr resulted in increased proliferative splenocyte responses. the prv-immune animals showed a normal ifn-1 and 11-2 production, without detectable 11-4, and with decreased 11-10 secretion in response to viral ag or con a. immunohistochemically, an increased ratio of ifny/i1-4 producing spleen cells was found. after immunization with either live attenuated or inactivated prv, ifn-yr"' mice produced significantly less antiviral antibody (ab), and more succumbed to challenge infection than the intact control animals. the reduction in ah titers in the mutant mice correlated with lower protection by their sera in transfer experiments. thes? findings are in line with the strong enhancing effect of exogenous ifn-y on rabies virusand prv-specific igg responses. our data demonstrate that a physiological ifn-y system is surprisingly not critical for the generation of antiviral th-i-type and the suppression of th-2-type cytokine responses. the lymphokine, however, is an important mediator in the generation of protective antiviral ab. key: cord-023055-ntbvmssh authors: nan title: immunogenicity date: 2004-02-19 journal: j cell biochem doi: 10.1002/jcb.240410506 sha: doc_id: 23055 cord_uid: ntbvmssh nan ia moyecules with respect to their roles as peptide receptors and target structures for tcr interaction. particular attention has been paid to distinguishing between local and distant effects of amino acid substitutions on ia function and to determining which residues interact with peptide antigen and which (if any) with the tcr. this ex erimental approach has led to the identification of several regions of the pofvorphic amino-terminal domains of the a and p chains as playing critical roles in chain-chain association and quaternary ia conformation. the a1 and p l putative helical regions have been found to have distinct degrees of structural lability, with the a1 helix showing much greater susceptibility to conformational change due to allelic variation in other re ions of the molecule. allelically polymor hic residues in the a1 and p 1 domainstave been shown to play important roles in &e activity of the assembly/folding control regions, and hence, analysis of local binding roles of specific residues in ia molecules must take this additional effect of substitutions at these positions into account. by controlling for large scale conformational effects, individual residues in the p chain have been assigned to desetopic ( eptide interaction) and histotopic (tcr interaction) roles. in the cytochrome c molel, a putative peptide bindin "pocket" involvin residues from both the postulated p l a helix and also the p-stran% floor has been defined, residues controlling both the extent of binding and the orientation of the bound peptide have been identified, and at least one residue with tcr interaction potential without obvious peptide binding properties has been localized. combining these data with those of other investigators leads us to propose a general model of class i1 mhc structure-function relationships. we have shown previously that memory b cells transferred into k-allotype distinct congenic rats in the absence of any priming antigen are deleted from the adoptive host within a matter of weeks (half-life of 1-2 weeks). in contrast co-injection of antigen with the cells facilitates their survival and the maintenance of a donor response for periods in excess of one year. in the experiments reported here we ask if the persistence of t cell memory is also dependent on antigen. 10. carrier (klh) primed t cells were transferred in the presence or absence of antigen into irradiated, k-allotype distinct adoptive host. a t various times after transfer these rats were injected with 2x10' hapten-carrier (dnp-klh) primed b cells together with 50 ig of soluble dnp-klh. this limiting number of b cells makes a secondary type response only if carrier-specific memory t cells survive in the adoptive host. we found that already at 6 weeks following transfer without antigen, no memory t cell help was available for these b cells. in contrast t cells transferred together with 10 ~g klh provided help for secondary type donor responses at 6 and 12 weeks after transfer. we conclude that longterm memory at both the t and b cell levels does not reside with small, very long-lived, resting cells but. with active clones that are maintained by small amounts of antigen that may persist for long periods. once antigen is lost from lymphoid tissues both t and b cell memory wanes within a relatively short time. t cells recognize antigen in the form of short peptides associated to class i or class i1 mhc molecules. each mhc molecule has the ability to bind a large number of peptides and peptides with unrelated sequences can compete for binding to the same mhc molecule, as well as in vitro. in vivo competition strictly correlates with the capacity of the competitor peptide to bind to the mhc molecule presenting the antigenic peptide and its extent dependes on the molar ratio between antigen and competitor. competition among different peptides derived by processing of hen egg-white lysozyme (hel) appears to exert a major influence on the immunodominance of antigenic determinants recognized by t cells. thus, the h l peptides 1-18 and 25-43 are both generated by hel processing and are both able to bind to the i-e molecule but only 1-18 becomes immunodominant because it has th ability to compete in vivq with other hel peptides, such as 25-43, for the available sites on the i-e molecule. however, two immunodominant t cell epitopes, such as those in hel peptides 51-66 and 112-129, both interacting with i-ak molecules, do not compete with each other when injected together at equimolar concentrations. such a coexistance is anticipated between peptides that bind with relatively high affinity to the presenting molecule and thus have both the chance to occupy a number of binding sites sufficient for t cell activation. r v s e iii xen ic tr lantation. in v i m lnvesti$ation uslng mocloml a n t m i e s r e 4 e -t e x e y skin gracs-val on m i c e w a s significantl pr:lrd l g anti-antihdy trea-t but n o t a b anti-antibody: w i d e r the saue animals but a n t i c d 4 antibody did prolong minor a n t w -d i allqrdts. in v i m studies r m that primary proliferation a n f z . 2 prcdwtion & -f i cells in response to mmkey stimulators was weak conpared to allogeneic reqonses. secondary responses t o xenogeneic stinulation were strong after in v i m priming but required the presence of responder nc's. assays for c y g t s t cell effectors in m i c e which had rejected monkey skin revealed few such cells. zhese results est that widely d i a t e xencgeneic processing and presentation, since xenogeneic antigens require that such presentation be in association with the.= antigens of regponder apc's, the xenogeneic r a f t s have a functional similarity t o aff2 leted allografts. shoved t h a t f e t a l r e n a l and f e t a l and p o s t n a t a l testis a l l o g r a f t s survived longer than corresponding a d u l t t i s s u e i n non-immunosuppressed outbred r a t hosts. the c u r r e n t study a s k s v h e t h e r t h e d i f f e r e n c e i n s u r v i v a l betveen r e n a l and t e s t i c u l a r g r a f t s and between g r a f t s of d i f f e r e n t ages is r e l a t e d t o d i f f e r e n t i a l t i s s u e expression of class i and class i1 mrna t r a n s c r i p t s or s u r f a c e antigens. and i f t h e s e p a t t e r n s change w i t h t r a n s p l a n t a t i o n . congeneic mice w e found t h a t prolonged s u r v i v a l of c57bl/6 f e t a l r e n a l (n=42; p<0.008) and f e t a l (n=14; ~( 0 . 0 5 ) and p o s t n a t a l (n= 8; ~( 0 . 0 5 ) t e s t i s mouse a l l o g r a f t s t r a n s p l a n t e d beneath t h e r e n a l c a p s u l e of a d u l t r e c i p i e n t bio.a mice and t h i s s u r v i v a l c o r r e l a t e s i n v e r s e l y w i t h t h e expression of class i and class i1 mrna (northern a n a l y s i s ) and p r o t e i n s (immunohistochemistryy) and t h a t both p r o t e i n and mrna increased throughout ontogeny f o r both t h e testis and kidney. after t r a n s p l a n t a t i o n t h e r e vas a marked i n d u c t i o n of mhc mrna t r a n s c r i p t s f o r both testis (n=207) and kidney (n=320). implanted f e t a l kidney t i s s u e t h a t survives. however. f a i l e d t o express d e t e c t a b l e mhc p r o t e i n , i n d i c a t i n g t h a t some p o s t -t r a n s c r i p t i o n a l modification i n t h i s t i s s u e occurs. t o a f f o r d it p r o t e c t i o n from r e j e c t i o n . implanted testis shoved i n d u c t i o n of both mrna and p r o t e i n v e l l above i t s much lower baseline. i n d i c a t i n g t h a t i t s r e g u l a t i o n , i n c o n t r a s t t o t h e kidney may be t r a n s c r i p t i o n a l . thus t h e f e t u s may lower t h e mhc burden as a s t r a t e g y t o escape r e j e c t i o n e i t h e r by p o s t t r a n s c r i p t i o n a l modification of p r o t e i n expression a s i n t h e kidney or by t r a n s c r i p t i o n a l modification of mrna as i n t h e testis. culture of thymus tissue in 2-deoxyguanosine (2dgua) is thought to reduce tissue immunogenicity by selectively depleting highly immunogenic, thymic immigrants of bone marrow origin. in the mouse 2dgua treated thymus tissue survival is markedly enhanced compared to untreated tissue when transplanted under the kidney capsule of allogeneic recipients. these experiments were repeated in the rat. as expected, strain da neonatal thymus tissue was rejected when transplanted under the kidney capsule of normal allogeneic strain pvg rats. surprisingly. acute rejection occurred even when the tissue was cultured for 14 days in 4 mm pdgua (3x the effective dose in mice). by in vitro criteria this dose was very effective in destroying thymocytes. to test whether residual marrow derived cells that escaped pdgua treatment were responsible for inducing rejection we "parked" the 2dgua treated da tissue in t cell depleted pvg rats. our working hypothesis was that the few remaining donor derived cells of marrow origin would be overgrown by host type cells. when pdgua-treated da thymus tissue was transplanted into t cell depleted pvg recipients graft rejection did not occur. however da pdgua treated thymus tissue, parked for as long as 200 days in t cell depleted pvg rats, was acutely rejected when retransplanted into normal pvg recipients. we interpret these results to suggest that rat thymic epithelium devoid of marrow derived cells is innately immunogenic. c 104 corinne amiel, violaine gugrin, thierry may, philippe canton, gilbert c faure, laboratoire d'immunologie and maladies infectieuses, chu de nancy, facult6 de mgdecine, 54500 vandoeuvre les nancy, france. lfal is a dimeric membrane molecule composed of a specific alpha chain (cdlla) and a beta chain (cd18) common to three members of the lfa family. lfal is physiologically expressed on all white blood cells, while other molecules of the lfa family (with cdllb and cdllc alpha chains) are restricted to cells of myeloid lineage. a defective expression of lfal has been described in some congenital immune deficiency and in aids. we investigated the lfal defect on peripheral blood lymphocytes from 100 hiv+ patients. three different monoclonal antibodies were used, respectively directed to chain-specific epitopes of cdlla (spvl7, sanbio) and cd18 (iot18, immunotech) and to a conformational epitope involving both chains (iot16, immunotech). cell suspensions were stained in indirect immunofluorescence and a flow cytometer (epics profile, coultronics) was used to assess the percentages of stained cell, the fluorescence intensity and the shape of fluorescent peaks. our data suggest that lfal expression is impaired in hiv+ patients both through the quantitative expression of each chain and through conformational alterations. the adhesion molecule lfa-1 is known to be important in antigen presentation. we have previously shown that both monocyte and t cell lfa-1 play a role in the interaction between these two cells (eji d; 943, 1987) . antibody to icam-1 (known to act as a ligand for lfa-1) also inhibits antigen presentation, although icam-1 is not thought to be expressed on resting t cells (eji 18: 35, 1988) . we have looked at the expression of icam-1 on t cells after incubation with 12 cytokines and found that only il-2 consistently effects an increase in both the percentage of icam-1 positive cells and in the level of expression. in addition we have found that a proportion of resting t cells express very low levels of icam-1. double labelling experiments have shown that these cells are part of the memory t cell population as defined by antibodies to uchli, lfa-3 and lfa-1, and furthermore that icam-1 negative cells are unable to respond to to antigens such as ppd and flu but are able to respond to pha. this suggests that icam-1 represents an additional marker on the memory t cell population which more precisely defines the subset able to respond to recall antigens icam-1 expression on t cells, anne-marie buckle and nancy hogg, macrophage lab. icrf, lincolns inn fields, london, wc2a 3px, u.k. immunization, francis r. carbone and michael j. bevan, department of immunology, research institute of scripps clinic, la jolla, ca 92037. ctl recognize peptide forms of processed, foreign antigens in association with class i molecules of the mhc and are usually directed against endogenously synthesized "cellular antigens" such as those expressed by virusinfected cells. in vifro studies have shown that small exogenous peptides can directly associate with class i molecules on the cell surface and mimic the target complex derived by intracellular processing and presentation. we have recently generated ova-specific, h-2kb-restricted ctl by immunizing c57bl/6 mice with a syngeneic tumor line transfected with the ova cdna. the ctl recognize the ova transfectant eg7-ova and the synthetic peptide ova but fail to recognize the native protein. we reasoned that given the potential for direct peptide/class ?'&$%ation observed in vifro, ova2s,-2ra may induce ctl after in vivo priming. however, we found that this is not the case. ova,,,-,,, and peptides of increasing lengths up to which are all able to form the target complex in vitro, are inefficient at priming ~% -%~~ specific ?h%sponses following intravenous injection. this is also true for both native and denatured ova. in contrast to these results, the synthetic peptide ova22g:z76 corresponding to a peptide in a partial tryptic digestion of ova can efficiently prime c57bl/6 mice in vrvo following intravenous injection. this peptide elicits ctl which appear identical to those derived from animals immunized with syngeneic cells producing ova endogenously. it is now well established that human t lymphocytes can be activated via the t cell specific cd2 antigen. in order to determine if a factor@) other than the single cd2 polypeptide is involved in cd2 mediated signal transduction, we have stamy transfected murine l cells with the human cd2 cdna. we report that such transfectants expressed hah levels of cd2 at the cell surface. formed sheep erythrocyte rosettes and expressed the three cd2 epitqm previously defined on human t lymphocytes, including the "activation associated' t i 13 epitope. the latter observation unequivocally demonstrating that expression of the ti13 epitope, in contrast to a previous report, is entirely independent of t cell specific factors. combinations of cd2 mabs that are potent stimulators of human t cells. however, failed to elicit either an increase in the concentration of intracellular free calcium or augment [3h]-thymidine inmrporatbn in the transfectants. these results provide both formal identification of the cd2 cdna and dearly demonstrate that the single cd2 polypeptide expressed in an heterokgous cell system devoid of t cell specific factors, cannot alone transduce intracellular signals in response to stimulatory combinations of cd2 mabs. the results are therefore consistent with the notion. that the functional cd2 antigen expressed in human t lymphocytes, requlres the association of another, as yet, undefined factor@). this conclusion was based on several lines of evid nce incl ding the observation that mabs specific for the class i a3 domain of either h-zl8 or l 2 d b interfered with t e generation of cd8-dependent (low substitution at position 227 in the a3 domain are not lysed by cd8-dependent primary ctl but are lysed by secondary cd8-independent (high affinity) ctl generated in the presence of antibody to the a3 domain. populations of ctl. we have isolated and characterized a c d w , cd4-da-specific cpl line. this line is cd8-independent and is capable of lysing the addition, we are currently generating clones from primary $-specific ctl cultures to obtain cd8-dependent (low affinity) ctl. directed rnutagenesis are being tested with the cd8-dependent and cd8-independent clones to define additional residues important for cd8 recognition. the comparison of ctl clones with different cd8 dependencies will allow us to more precisely define the role of cd8 in t cell recognition. percolle from the buffy coat of one unit ot blood. these cells (=400x10 ) are introduced into a curame 5000 elutriation centrifuge (rotor speed of 3000 rpm; loading flow of 10 ml/min). nine fractions can be obtained. the first three containing >90% lymphocytes; fraction 4 (3000 rpm-18 ml/min) and fraction 5 (2900 rpm-18 ml/min! contain both lymphocytes and monocytes and the next three fractions contain >90% monocytes; the finat fraction (rotor off) contains monocytes + granulocytes. cells from each fraction (5x10 /well) are incubatee for five days with tetanus toxoid (1.5lf/well) and an enriched population of t cells (5x10 /well). quadriplicate samples are then pulsed for 16 hours with 'h methyl thymidine. maximum apc activity is found in fractions 4 and 5 representing 4 to 7% of the mononuclear cells. apc activity for these two fractions can be further purified by selective absorption of the cells onto gelatin coated surfaces that have been preincubated with plasma. the non adherent lymphocytes are rgmoved after two hours. after overnight incubation spontaneously released cells (1-5x10 ) can be harvested which have a higher apc activity than cells rotated by elutriation alone. these methods are now highly reproducible in our laboratory, so we can now begin to characterize and study these cells. the male s p e c i f i c h-y a n t i g e n h a s been shown t o behave as a minor histocompatibility a n t i g e n in man and mouse. i n t r a n s p l a n t a t i o n , male t i s s u e may t r i g g e r t h e c l o n a l expansion of h-y reactive hhc r e s t r i c t e d effector cells of female o r i g i n . although male epidermal cells (ec) can induce an anti-h-y t cell response in female mice, so far in v i t r o techniques have f a i l e d t o i d e n t i f y t h e cell-defined h-y a n t i g e n on murine ec (1). here w e developed a 51cr release assay t o use human c u l t u r e d k e r a t i n o c y t e s (k) as t a r g e t cells for hla-a2 specific and ma-a2 r e s t r i c t e d h-y s p e c i f i c t cell clones. hla-a2+ but n o t h l a -a t k were l y s e d by anti-ma-a2 ctls i n a dose dependent manner. low but d e t e c t a b l e l e v e l s of anti-h-y k i l l i n g were found a g a i n s t ma-a2+ male k b u t n o t a g a i n s t h l a -a t male or hla-a2+ female k. both l e v e l s of a l l o r e a c t i v e and h-y s p e c i f i c l y s i s were d r a m a t i c a l l y enhanced after exposure of k t o ifn gamma. these r e s u l t s s t r o n g l y suggest t h a t h m a n male s k i n cells are d i r e c t l y s u s c e p t i b l e for h-y d i r e c t e d t c e l l k i l l i n g through t h e expression of f u n c t i o n a l h-y/hla complexes on t h e i r cell s u r f a c e . i n view of t h e s e f i n d i n g s , t o g e t h e r w i t h our r e c e n t s t u d i e s on t h e expression of h-y ctl determinants on h m a n hematopoietic p r o g e n i t o r c e l l s (21, t h e r o l e of h-y a s a t a r g e t s t r u c t u r e f o r c e l l mediated immunity i n o l i n i c a l t r a n s p l a n t a t i o n should be s e r i o u s l y taken i n t o account. 1. steinmuller d. and burlingham w.j. t r a n s p l a n t a t i o n 19@4,37,1,22. 2. voogt p.j., goulmy e., fibbe w.e., e t a l . j . clin. invest. sept.1988. c 116 diphteria toxoid (dt) presentation by hla dr7 transfected murine fibroblasts bismuth, laboratory of c e l l u l a r and t i s s u l a r immunology, chu p i t i e s a l p b t r i b r e , p a r i s , france and veterans medical c e n t e r , iowa c i t y , usa. l t r a n s f e c t a n t s e x p r e s s i n g s i n g l e type of human mhc c l a s s i1 molecules produced by dna conjugate formation has been studied with cloned t cell lines and a b cell hybridoma and with t cells and b cells from normal mice. resting t cells and b cells do not form appreciable numbers of conjugates but conjugates are formed between t cells stimulated with alloantigen for four days and b cells activated by 24 hour culture with lps. irrelevant lymphocytes do not affect the rate of specific conjugate formation in suspensions of cells agitated by gentle rocking but impair conjugate formation when cells are allowed to settle in round bottom tubes. in further experiments, it was shown that the conditions for the induction of lymphokine secretion by the t cell were not indentical to the conditions for conjugate formation.the significance of these and other observations for the interaction of t cells and b cells in vivo will be discussed. of the primary mixed leukocyte reaction (mlr) and that this reaction occurs in multicellular dendritic cell-cd4+ t cell clusters [cellular immunology 111, 183-195(1988) dendritic cells are able to contact, cluster, and retain allogeneic t cells and induce these alloreactive cells to proliferate and divide. tions labeled with a vital flvorescent dye, we show that only dendritic cells efficiently form stable clusters. labeled monocytes and b cells do not form clusters with t cells. when labeled monocytes and unlabeled dendritic cells are used to stimulate t cells, unlabeled clusters form. labeled monocytes do not move into the clusters until the third day of the mlr. significant levels of il-2 and a-ifn appear in the culture supernatant by the first or second day. blast transformation by the second day of the mlr as demonstrated by giemsa staining of cluster cytopreps. also been studied by immunoperoxidase staining. it is known that human peripheral blood dendritic cells are potent stimulators using purified dendritic cell populathe distribution of certain adhesion molecules within clusters has c1m microbiology and immunology, emory university, atlanta, ga 30322. immunization of sjl/j mice with myelin basic protein (mbp) induces the t cell-mediated autoimmune central nervous system disease, experimental allergic encephalomyelitis. response against a dominant epitope (residues 89-101) leads to disease. lymph node t cells from mbp-immune mice react against several epitopes in addition to 89-101 indicating that the i-as molecule is able to form immunogenic complexes with several mbp peptides. the question asked in these studies was whether subdominant epitopes from the same molecule would compete with the dominant epitope for binding sites on the i-as molecule. to address this question two t cell clones, one specific for 89-101 (sp4.2) and a second specific for a second epitope present in peptide 89-170 (sp4.7) were tested for responsiveness when cultured with the dominant epitope alone or with mixtures of peptides containing dominant and subdominant epitopes. reactivity of sp4.2 against peptide 89-101 was inhibited by peptides 1-37 and 43-88. reactivity of sp4.7 against peptide 89-170 was not inhibited by peptide 89-101 although peptides 1-37 and 43-88 were inhibitory. controls indicated that inhibitory reactivity was not due to toxicity at high concentrations of peptides. these findings imply that subdominant epitopes are able to compete with dominant epitopes of mbp for binding sites on i-as molecules. linda r. gooding, frances c . rawle, david i . kusher, w i l l i a m s . m. wold+ and barbara knowles*. department of microbiology and immunology, emory university school of medicine, atlanta, ga 30322, 'institute f o r molecular virology, s t . louis university school of medicine, s t . louis, mo 63110 and *the wistar i n s t i t u t e , philadelphia, pa 19104. i n several v i r u s systems e a r l y non-structural proteins localized predominantly i n the nucleus of infected cells are major t a r g e t antigens f o r cytotoxic t lymphocytes (ctl). whether early synthesis o r nuclear l o c a l i z a t i o n are important factors i n immunodominance is not known. w e have recently developed a system f o r studying the ctl response t o human group c adenoviruses i n mice. by us ng both transfected t a r g e t s and virus deletion mutants w e have shown t h a t , response t o wild type ad5. there are two e1a t r a n s c r i p t s , 12s and 1 3 s . which both encode major e a r l y nuclear antigens d i f f e r i n g by a 46 amino a c i d insertion: both antigens are recognized equally w e l l by ctl. the e3 encoded 19k glycoprotein (gpl9k) of ad5 binds t o mhc c l a s s i antigens i n the endoplasmic reticulum preventing t h e i r translocation t o the c e l l surface and strongly inhibiting l y s i s by ad5 specific ctl. however, the presence of gpl9k i n the priming v i r u s does not a f f e c t the s p e c i f i c i t y of the ctl generated f o r e l a , so the immunodominance of t h i s protein cannot be due t o the fact t h a t i t is the only major protein synthesized before gpl9k i n the course of infection. using virus deletion mutants we are investigating whether ctl s p e c i f i c f o r other ad5 antigens can be induced i n the absence of ela, and whether e1a is also the dominant antigen recognized i n mice of other mhc haplotypes. respond to antigens present on non-replicative virions. in contrast, we have obtained balc/c i-erestricted t hybridomas specific for the neuraminidase (na) glycoprotein of a/pr8 influenza which recognize infectious, but not non-replicative virus, closely resembling recognition requirements observed for most class i mhc-restricted responses to influenza. recognition correlated with the rte nova synthesis of viral na within antigen-presenting cells, but did not depend strictly upon the amount of na present in cultures, since high na concentrations could be achieved by addition of non-replicative virus without being stimulatory for na-specific t cells. recognition of a neo-antigen was ruled out, since, in high concentration, na isolated from purified virions, even if reduced and alkylated, was recognized by the t hybridoma clone. isolated na was recognized when added to pre-fixed apc, suggesting that this form of antigen was able to bypass the usual processing pathway of exogenous proteins. this suggests that endogenously-synthesized antigen may use different pathways to achieve class 11-associated presentation. t lymphocyte activation is a complex event which is influenced by a variety of distinct cell surface molecules. in order to determine the role of individual molecules in the activation process, we have developed an efficient methodology for generating cell variants in which expression of molecules is selectively inhibited by expression of anti-sense rna from an epstein-barr virus episomal replicon. in a previous study, we reported that marked inhibition of cd8 cell surface expression could be achieved in a human t cell clone using this approach. we have now extended this strategy to another t cell surface molecule, cd2, as a first ste towards ascertaining its role in t cell activation. to this end, we s nthesized a &-mer oli onucleotide corresponding to a sequence in. the 5: end of the c d i n g re ion of human cd'i and inserted it in an anti-sense orientation into this replicon. this a-c%2/rep3 construct was electroporated into jurkat cells. analysis of stable a-cd2irep3 transfectants by immunofluorescence staining and flow cytometry demonstrated complete and selective inhibition of cd2 expression. in contrast to the nontransfected arent, this cd2-variant demonstrated a partial loss in its ability to form conjugates a n 8 to secrete interleukin 2 when stimulated with anti-cd2 monoclonal antibodies. however, stimulation of the cd2-variant with a23187 and pma did result in interleukin 2 secretion. several observations suggest that cd8 functions not only as an adhesion molecule recognising mhc class i on the adjacent cells but also potentiate the transducting capacity of the tcr/cdg complex. comparison of the mouse ly 2 protein sequence with the homolog rat ox8 and human t8 sequences revealed most highly conserved regions in the membrane and cytoplasmic part of the molecule. the conservation of the transmembrane and cytoplasmic sequences in different species may be significant for the function of the cd8 molecule. in order to initiate the functional dissection of the cd8-molecule we constructed mutations in different parts of the molecule. by transfecting the a and b chain genes donated by a cd8 dependent cytotoxic t cell clone(kb5 c20) into the mhc class i1 restricted agd cd4 t cell hybridoma do-11.10 we were able to reconstitute the ability to respond to k only if the transfer was done with the ly 2 molecules (gabert et al., 1987. cell, 50. 545-554) . in this system surface expression of mutated and non mutated ly-2 molecules were checked by facs-analysis and the molecuar size of the proteins were analysed by immunoprecipitation with the anti-ly-2 monoclonal antibody 19/lj8. finally functional effects of the mutations were investigated in response towards the k alloantigen. we have simulated graft versus host and host versus graft reactivity in vitro by studying primary anti-minor h responses in a limiting dilution culture system. the ability of bmm and peripheral blood mononuclear cells (pbm) to stimulate and respond in this system were compared by estimating the number of proliferating cells. in gvh-direction the combination of donor-bmm (d-beim) and host-pbm (h-pbm) was 2 to 15 times more effective in stimulating proliferation than any other combination; the same applied to the combination h-pbm/d-bmm in hvg-direction.-using these combinations the median frequency of proliferating cells in gvh-direction was 1/5300 (range 1/59c-1/18100) in 10 pairs, in hvg-direction (7 pairs) 1/2330 (range 1/115-1/6100). 85% of the proliferating cells had the phenotype of mature t-cells.-using the same combination of responder/stimulator cells we have also estimated the number of cytotoxic cells specific for the hla-identical target cell. in gvh-direction the median estimate (n=8) was 1/10300 (range 1/66o-1/45700), in hvg-direction (n=5) 1/2250 (range 1/400-1/6500). by split well analysis similar or higher frequencies of cytotoxic cells with specificity for nk-targets were detected (gvhr: 1/5750, hvgr: 1/3620). it was however possible to identify a significant number of minor h-specific clones by segregation analysis; their specificity could be confirmed after clonal expansion. the clones had the phenotype of typical cytotoxic t-cells.-the relevance of the two cytotoxic subpopulations described above to clinical events such as gvhd, graft rejection and relapse needs to be clarified.molecular cloning of murine icah-1, k.j. horley, b. baker, and f. takei, terry pathology, university of british columbia, vancouver, b.c., canada. we have previously reported a novel cell surface antigen expressed on activated and proliferating murine lymphocytes. the antigen, termed hala-2, is absent or present at low densities on thymocytes, lymph node cells, and fibroblast cell lines, indicating it is not a universal proliferation antigen. some cells of the spleen and bone marrow express mala-2 at a high density possibly representing in vivo proliferation in these tissues. apparent molecular weight of 95-100 kd under both reducing and nonreducing conditions, and is susceptible to endo f digestion. the monoclonal antibody yn1/1.7 that reacts with this antigen, profoundly inhibits mlr. a xgtlo cdna library was constructed from ns-1 cells that express a high level of mala-2, and screened with synthetic oligonucleotides resulting in the isolation of a full length cdna clone (-3.2 kb). the cdna sequence has high homology with the human icau-1 sequence, indicating that hala-2 may be the murine homologue of this characterized protein. hines, trudeau i n s t i t u t e , inc., p.o. box 59, saranac lake, ny 12983 a tumor c e l l l i n e , et-5, has been derived from an apparent fibrosarcoma t h a t arose i n a c57bl/6 male mouse. antigens. mice t h a t have r e j e c t e d et-5 become imnune t o these minor h antigens, judged by accelerated s k i n g r a f t r e j e c t i o n , and t h i s imnunity can be t r a n s f e r r e d t o imnunod e f i c i e n t mice w i t h lymphoid c e l l s . however, spleen c e l l s from mice t h a t have r e j e c t e d according to the widely accepted view, cd2 (t11, sheep erythrocyte receptor) is the first t cell-specific antigen to appear on differentiating thymocytes during ontogeny. it follows that cd2 should be expressed on all immature and mature t cells. using two-color cytofluorometry i have here identified subsets of cd2-cd3+ t cells both in fetal human thymus or spleen and in adult peripheral blood. cd2-cd3+ t cells constitute 1-25% of fetal thymocytes and 0.1-0.8% of peripheral blood t cells. il-2-dependent longterm clones of cdi-cdj+ cells do not react with a panel of monoclonal antibodies (mab) directed against the t1ll, tlll or t113 epitopes of cd2 and do not transcribe cd2 mrna. fetal tissue-derived clones react with the tigammaa mab and thus express a functional tcr gamma chain, while cd2-cd3+ clones from peripheral blood are bha031+ and express a full-length 1.3 kb tcr c s .ria. the clones established here are currently being characterized with respect to functional capacities. i conclude that expression of cd2 is not an absolute prerequisite for the expression of the cd3/tcr molecular complex on human t cells. if they are added after 24 hours. these interactions are bidirectional. since both cdlla and cd18. and t h e i r ligand i-cam 1. are expressed on the presenting c e l l s as well as the t cells. however, a l l such e a r l y adhesion related events are not bidirectional since anti-cdz and anti-lfa-3. which are expressed d i f f e r e n t i a l l y on t c e l l s and presenting c e l l s respectively are also effective as inhibitors. antibodies, a n t i cd4 and a n t i cd25 antibodies do not i n h i b i t clustering but do i n h i b i t p r o l i f e r a t i o n , and t h i s i s seen irrespective o f when the antibodies are added i n t o the assay. our findings suggest t h a t there are two mechanisms involved i n dendritic c e l l -t c e l l interaction, f i r s t l y an inrnediate cell-cell adhesion step and l a t e r a secondary signal transduction process possibly mediated v i a cytokines. the q u a l i t a t i v e d i fferences between dendritic c e l l and b c e l l induced i m n o g e n l c i t y may thus l i e i n e i t h e r o f these two steps. king, department o f eathology, the bland-sutton i n s t i t u t e . university college and middlesex school i n contrast, a n t i class i1 m c lmmunogenicity c130 cultvred tissue is capable of stimulating an rggwwse when l " s p l m l e d ~e n e i c w y , robert j. ketchum and orion d. hegre, dept. of cell biology and neumanatoay, university of uinnesota, minneapolis hn 55455. neonatal rat islets derived by culture-isolation have teen shown to k free of mlc class 11+ cells, and are immunologically silent when transplanted to either syngeneic or allogeneic hosts. allogeneic transplantation of cultured neonatal non-islet pancreatic tissue, which is known to contain class 11+ cells, results in rapid allograft rejection. unexpectedly, m i c transplantation of cultured non-islet ductal tissue also resulted in lononuclear lm,me cell (hnc) infiltration of the graft in 86% of grafts examined. highly purified syngeneic islets and ductal elements grafted syngeneically at remote sites display an i u n e response in the ductal element graft, while the islet graft is free of any imnme cell infiltrate. this syngeneic imune response does not result from the use of xenogeneic serum in the medium, since cultures carried out using syngeneic rat serum supplemented medium yielded identical results. uncultured neonatal pancreatic tissue grafted syngeneically does not result in iqk: infiltrate, thi6 i.rmne response to a syngeneic stimulus correlates with the presence of class 11+ (antigen presenting) cells. in grafts free of class 11+ cells (culture-isolated islet grafts) no i.rmne responrrc to syngeneic stimulus was observed, while a response was present when syngeneic ductal elements, known to include class 11+ cells, were grafted. this indicates a need for cells capable of antigen presentation to stimulate this syngeneic rerrponne, and suggests that either a modified self antigen or a nomally sequestered antigen is being presented. this syngeneic imune response demonstrates many of the same characteristics of, and may be analagous to, the in vitro syngeneic, or autologous, mixed lymphocyte reactions. indicating this response is not to developnental antigen. c 132 the presence of "self" mhc class i1 (ma-dr) antigens determines whether blood transfusions ihmunise or suppress. el lagaaij, a termijtelen. e goulmy, & jj van rood, leiden university hospital, the netherlands. blood transfusions can immunise the recipient, as well as induce prolonged allograft survival. it is not known what makes that some transfusions inrrmnise the recipient whereas others induce immune suppression. we investigated if certain mhc compatibilities or differences between recipient and transfusion donor and organ donor are required to induce the beneficial "transfusion effect" in man. we studied graft survival and blood transfusion induced changes in cellular and humoral immunity in 4 different patient groups. the patients received a single blood transfusion of a randomly choosen donor. we found in all 4 groups that to induce a beneficial "transfusion effect" compatibility for at least 1 hla-dr antigen between recipient and transfusion donor is required. if the transfusion and recipient are mismatched for both ma-dr antigens, the recipient is immunised, resulting in an increased antibody production (p=o.ool), an increased cytoxicity (cml) (p=o.oos), an increased mixed lymfocyte reaction (mlr) (p=o.oos) and a decreased graft survival (p-0.003). after a beneficial (ma-dr sharing) transfusion. the in vitro test remain unchanged or decrease. graft survival increases with the number of shared antigens between transfusion donor and organ donor (p=o.o2), suggesting that a donor specific suppression is induced. recent experiments have revealed a direct interaction between the cd4 molecule and hla-dr antigen. to address the nature of this interaction we have used a xenogeneic system in which a human cd4 cdna was expressed in the murine cd4-and cd8-negative hybridoma 3dt52.5.8. the tcr of 3d3752.5.8 recognizes the murine class i molecule od. a class i 1 expressing dd-positive cell line was obtained by cotransfection of the human class i1 cdnas together with the murine od gene int.0 the murine fibroblast line dap3. coculture of 3dt52.5.8 and dap3 expressing dp-dd resulted in a 20 fold increase in il-2 production and in rosette formation only when both cd4 and dp were present on the responding t hybridoma and the presenting cell, respectively. we are using this system to map regions of the cd4 molecule that interact with the class i1 mhc ag. the cd4 molecule has also been shown to be the receptor for the human immunodeficiency virus (hiv) via the gp120 molecule. since gp120 and class i1 both interact kith cd4, we have used our functional assay to verify if gp120 exerts an inhibitory function on cd4 class i 1 interaction. recombinant gp120 inhibits the functional interaction and rosette formation in a concentration dependent fashion with maximal inhibition at about 10 pg/ml.. this inhibition is specific since it can be reversed by recombinant soluble cd4. the fact that recombinant. gp120 can inhibit the functional interaction between cd4 and its physiological ligand (class i1 ags) suggests that the use of gp120 on a vaccine against hiv infection could alter the immune response of such individuals. this work was supported by src, mrc and nci. t lymphocytes discern self from non-self molecules through the interaction of their antigen-specific receptors and proteins encoded by the mhc. although the nature of this association is not well-defined, a model has been proposed whereby the v-segments of the t cell receptor interact with residues along the 2 alpha helices of the class i antigen (davis et al.; 334:395, 1988) . we have recently shown that ctl generated against the class i molecule qiod crossreact on several unrelated murine class i antigens containing the shared qiod residues at amino acid positions 152, 155, and 156 (mann et al.; j x 168:307, 1988) . these residues contributed by the a-2 domain occur in the alpha helical portion of the class i molecule and amino acids 152 and 155 could interact directly with the t cell receptor. to further characterize the role of these amino acids, we are in the process of determining whether insertion of these 3 residues by site-directed mutagenesis into a human class i molecule will allow for the antigen's recognition by anti-910 ctl. here the t cell repertoire becomes restricted, so that foreign antigen can be recognized only when associated with the mhc products of the host, and mature t cells are tolerized to self antigens, a process which also seems to be mhc-restricted. thus, t cells should be non-reactive to self antigens when they are associated with mhc products present on the tolerance-inducing thymic cells, whereas they may still react to the same self antigens when associated with different mhc products. to examine mhcrestricted tolerance in vivo, a model system must have: a) self antigen in the context of one mhc haplotype. and b) tolerance to both that and a second mhc haplotype. chimeras were prepared by aggregation of preimplantation embryos of two strains of mice, c57bl/6 (b6) and balb/c. the thymus of such chimeras should be composed of two distinct and completely intermixed populations of cells, one from each parental strain (isozyme analysis indicates no detectable fusion of cells). thus, t cells maturing in the chimeric thymus should be exposed to and tolerized to minor histocompatibility antigens (mhas) of one parental strain only in association with the mhc of that strain. for example, mice might be expected to express b6 mhas only with h-2b (the 8 6 mhc). however, our chimeras were fully tolerant to f1 skin grafts, which have "hybrid" combinations of mhas and mhc (e.g., b6 mhas with h-2d). these results are most consistent with either, a) "wholesale" antigen processing and presentation of all mhas by the tolerizing thymic cells, and/or, b) functional sharing of mhc products between the parental thymic cell populations. many of the events critical to the maturation of t lymphocytes occur in the thymus. in the case of t-cell responses against viruses such response defects are associated with a marked increase in disease susceptibility as illustrated by class i mhc controlled susceptibility to lethal pneumonia induction by sendai virus. certain class i or class i1 mhc determined tc response defects (four out of six tested by us) can be restored by imunization in vivo and/or restimulation in vitro with dc. dc are the most effective apc. their superior apc capacity is due to 1) a very high absolute number of class i and class i1 mhc molecules, and 2 ) a low degree of sialylation of mhc and other surface molecules, reducing negative charge and facilitating access of the t-cell receptor to the mhc groove presenting the antigenic peptide and/or improved clustering with t cells. the more effective antigen presentation by dc allows a more prominent role for a cd4+ q cell independent pathway of cd8+ tc activation. it is postulated that the more effective direct triggering of cd8+ tc precursors lowers the threshold for il-2 production by cd8+ cells, reducing the requirement for il-2 production by the cd4+ cells. failure of dc to overcome certain mhc-linked specific tc response defects probably reflects complete failure of any foreign peptide derived from the processed antigen to interact efficiently with the mhc or a true tc repertoire defect. donald pious, department of pediatrics, university of washington, seattle, w 98195 mhc class i1 molecules bind inmunogenic peptides derivsd fro13 soluble antigen and the complex is recognized by specific t cells. ue have isolated eight independent mutant b -l u clones which are altered in their ability to present antigen. in standard proliferation assays using four different soluble protein antigens, the mutants are unable to stimulate the majority of t cell clones restricted to hlfl dr or dp. cllthough unable to present *hole hepatitis b surface antigen (hbdg), they effcctively present a hbdg peptide to a dprestricted t cell clone. the fact that both dr and dp restricted antigen presentation is abnormal in these mutants made it likely that the class i 1 structurual genes are unaltered. this hypothesis is supported by the finding that dno sequences from the dr genes of one mutant are norml. however, two observations indicate that the uture class i1 di-expmsccd by the mutants are structurally altered. binding to the mutants with tuo polymorphic anti-dr antibodies and one anti-dp antibody is reduced, although the level of cell surface class i1 expression is normal. second, the class i1 diners from the mutants dissociate into no-rs under in vitro conditions (sds-pwe) which preserve dimers in the progenitor line. together, these functional and structural data suggest that the mutants are defective in a molecule that either associates with or post-translationally modifies class i1 molecules and is required for the physiologic formation of an twc/antigen complex. they do not function as restriction elements presenting foreign antigens to t-cells. to investigate the nature of this functional defect we have constructed 3 different recombinant class i genes using dna segments from the b10 q9 (qa-2) or h-2db genes. structural protein encoded by the recombinant genes is derived entirely from the q9 gene whereas the cis-acting transcriptional regulatory elements or the dna segment encoding the membrane anchoring domain is derived from the h-2db gene. into fertilised cba/ca embryos by microinjection and transgenic lines were established. to date we have established 13 transgenic lines. we have shown that the q9 (qa-2) antigen encoded by the recombinant genes behaves as a major transplantation antigen in skin grafts and provokes strong secondary cytotoxic t-cell responses in grafted animals irrespective of the tissue distribution or mode of membrane anchorage of the q9 antigen. at present, we are investigating whether the q9 antigens encoded by the recombinant genes are able to present influenza virus or mouse minor histocompatibility antigens to t-cells during immune responses and hence whether they can function as restriction elements. is a distinctive system because it permits an analysis of the activation requirements for antigen specific, resting t cells. been isolated following culture with anti-ig-sepharose and compared to dendritic cells as stimulators of cd4' t cells in the mix. i1 mhc products and independently stimulated the 1 ' mlr and the production of several t derived lymphokines, including il-2 and 11-4. however, the relative potencies of dendritic cells and anti-ig blasts as 1'mir stimulators varied in a strain dependent fashion. times more active in stimulating hls-mismatched. mhc-matched t cells, relative to syngeneic t cells. anti-ig blasts when stimulating acroas an mhc barrier and were likewise more effective in binding iqic-disparate t cells to form the clusters in which the mix was generated. dendritic cell-t cell clustering was resistant to anti-lfa-1 mab, while b blast-t cell clustering was totally blocked. thus, anti-ig b lymphoblasts and dendritic cells, two cell types which differ markedly in phenotype, also differ in efficiency and mechanism for initiating responses in allogeneic t cells. only anti-ig blasts could stimulate across an mls barrier, being at least 100 in contrast, dendritic cells were 10-30 times more potent than the as the lymphocyte f u n c t i o n antigen 3 (lfa-3) and the i n t r a c e l l u l a r adhesion molecule (icam). we i n t e r p r e t t h i s as an increase i n the membrane expression o f these s t r u c t u r e s f o l l o w i n g incubation. the increase i s blocked by the t r a n s l a t i o n i n h i b i t o r , cycloheximide, implying t h a t p r o t e i n synthesis i s involved. helper t cell responses to soluble globular proteins require processing of the protein by ia-expressing antigen presenting cells (apc). antigen is internalized into acidic vesicles, proteolyzed, and peptides containing t ceu antigenic determinants are transported to the apc surface where they are recognized by the antigen-specific t cell in conjunction with ia. most ia-"pressing cells are competent apc, however, only b cells have antigen-specilic receptors on their surface auowing bound antigen to be processed and presented at 1/lw the antigen concentration required by nonspecific apc little is known about b cell antigen processing function during differentiation, or if ig-mediated apc function is altered at different maturational stages, thus allowing regulation of b cell-helper t cell interactions. neonatal acquisition of apc function was examined in mice ages day 1 to day 15. splenic cells from d l to d10 mice process and present pigeon qochrome $, pg at 0-20% of adult levels. by d15 neonatal spleen cells acquire the ability to process and present soluble pg at 3 5 4 of adult levels. the ability to internalize antigen through ig rwptors was determined using an antigen-antibody conjugate, p$-&(ab')z. neonatal spleen cells acquire the ability to process antigen through ig simultaneously with the ability to process soluble antigen. lack of prowsing by neonatal spleen cells prior to d10 is not attributable to insufficient levels of surface ia. since d3 neonate spleen cells are able to activate t cell hybrids to 50% of adult levels when provided with p$ 81-104, containing the t cell determinant. dlod15 neonate presentation of p@1-104 is indistinguishable from adult levels. b cell maturation into memory b cells was identified by the loss of the jlld differentiation marker. splenic jlld'o b cells increase from 540% following immunization and return to nonimmune levels after 4 weeks. during antigen-induced b cell maturation, jllb b cells are indistinguishable from splenic b cells in the ability to present antigen introduced into the processing pathway either pinocytotically or via surface ig. p p antibody conjugates specific for mouse f(ab')z i n , igd, or igg are presented equally well by both splenic and jll@ b cells. thus, acquisition of b cell processing function appears to be developmentally regulated and may play an important role in b cell tolerance meshanisms. once b cells have acquired the ability to process antigen this function is maintained and is not regulated during maturation into memory b cells. we are currently investigating the role of ig isotype during neonatal acquisition of antigen processing. (supponed by nih grants ai-18939, ai-12001, and ai-2317) as the preliminary studies suggested that carrier sc (apc) for ts vs. tcs activation might be distinct, studies were done to directly address this possibility by assessing tha ability of s3 coupled to various cell populations to activate ts and tcs. the results indicated that ts activation required that s3 be coupled to plastic adherent cells which bear both i-a and i-j determinants. these cells are nonadberent to anti-ig and nonfunctional in cyclophosphamide (cy) treated mice. i n contrast activation of tcs required coupling of s3 to plastic non-adherent and anti-ig adherent cells. these cells are functional in cy treated mice and bear the b cell markers jlld and i-a but not 1-3. thus s3-specific ts are activated by i-a+ i-j+ adherent cells (presumably macrophages) whereas tcs are activated when antigen is presented by b cells. (nih grant ca25054.) interleukin-2 activated killer (iak) lymphocytes (also known as lak cells) which destroy a broader spectrum of tumors invitro than nk cells have been used sucessfully in an adoptive immuno-therapy protocol for the treatment of patients with a variety of advanced cancers. the cell suface molecule(s) on tumor cells that a r e involved in specific binding to iak cells and in programming iak cells for cytolysis (iak acceptor molecules) have not been characterized. inorder to identify such acceptor molecules a crude membrane digest of the lung carcinoma cell line a549 was biotinylated and adsorbed to iak cells or to unstimulated human peripheral blood lymphocytes (upbl) (each from the same person). proteins from the washed solubilized cells were separated by page, western blotted and probed with streptavidin-alkaline phosphatase. several experiments demonstrated that different tumor membrane proteins bound to iak cells compared to upbl. the unstimulated cells bound one tumor membrance protein (about 40kd) not found on the iak-adsorded blot. the iak cells bound three tumor proteins (approximately 30,46 & 50kd) not found on the upbl-adsorded blot. three other proteins (about 35,44 & 55kd) were found to adhere equally well t o iak cells and upbl. utilizing a streptavidin affinity column, solubilized tumor membrane proteins that bound to iak cells could be separated from solubilized iak membrane proteins. the isolated tumor membrane proteins that adsorded to iak cells inhibited iak mediated lysis of a549 tumor cells by >85%. these studies suggest that specific cellular adsorption techniques may be useful in isolating and characterizing tumor membrane proteins involved in interactions unique to cytolytic lymphocyte-tumor cell target binding and lysis. activation of human t lymphocytes occurs via the t cell receptor-cd3 complex but can also be induced through the non-antigen-specific cd2 molecule. selected combinations of mabs or the soluble cd2 ligand, namely lfa-3 and a unique anti-cdz mab (cd2.1) induce human t cell activatlon. cd4 is an accessory molecule implicated in t h e activation of human t lymphocytes. this molecule may exert this function by increasing intercellular avidity through binding to mhc class i1 molecules and/or by transmitting intracellular signals. we have investigated the action of mabs directed against different epitopes on the cd4 molecules in the activation of human t cells via the cdz pathway. we show that anti-cd4 mabs inhibit cd2 induced t cell proliferation in an epitope-depe dent fashion. this inhibition does not appear to be linked to the lower cd2 mediated [ c a ' + ] response induced by anti-cd4 mabs, since [ca"] response is equally affected by anti-cd4 mabs whether or not they inhibi ed t cell proliferation. in conclusion, the partial inhibition of the cd2 induced [cb'] response of t cells by various anti-cd4 mabs suggest that : 1) this inhibition does not totally account for the inhibitory effect of anti-cd4 mabs, 2) and the proliferation induced by anti-cdz mabs may not be completely ascribed to the [ca2+] response of t cells. rosenberg. and alfred singer, experimental immunology branch, nci, nih, bethesda, md 20892 . we have devised a model to study the in vivo generation of suppressor cells by using mice congenic at qa-1, a class i-like molecule encoded to the right of h-2d. disparate tail skin grafts (tsg) unless a second graft with additional helper determinants was also present. without any source of additional help, failed to reject their qa-1 graft, and were unable to reject them even upon the subsequent addition of exogenous help. thus, exposure to qa-1 disparate grafts, in the absence of additional help, either led to qa-1 specific tolerance or suppression. mice failing to reject qa-1 allografts revealed the presence of qa-1 specific suppressor cells that inhibited the in vivo activation of antigen specific effector cells capable of rejecting qa-1 bearing allografts. experiments using t cell subpopulations should allow for further characterization of these qa-1 specific suppressor cells. we found that b6 mice did not reject qa-1 however, animals engrafted with a qa-1 graft alone, in the thymus, the t-cell receptor genes are rearranged, the t cells learn to recognize their own major histoconpatibilitp complex "hc). and they learn to respond to foreing hec. these events seem to be linked to the interaction between t-cell precursors and the stromal cells of the thyms. thus increasing evidence points to an essential role for the thymus epithelial cells (te cells) in development of at least hec class i1 recognition by the t cells. to be able to study the importance of te cells in t cell maturation. we have developed a method for growing murine t cells in serum-free pediun with well defined constituents. the m d i u a allows far growth of te cells without concomitant growth of bone marrow derived cells as macrophages and fibroblasts. data obtained by en and lmmunocytochenistry showing the epithelial nature of the cultured cells, as well as autoradiographic data on the growth pattern, and characterization of tb cell supernatants will be provided in addition to results obtained from co-culture of te cells and t-cell precursors (cm-cds-thymytes). lmmunogenicity c 152 branch, national cancer institute, bethesda, md 20892 the effector limb mediating skin allograft rejection is highly antigen specific, rejecting cells that express allogeneic mhc antigens while sparing those which fail to express allogeneic mhc determinants. disparate skin grafts are completely rejected in spite of the fact that only a small percentage of the cells within the graft express ia antigens. thus, it is possible that mhc class i1 disparate grafts are rejected by a mechanism that does not assess the expression of mhc determinants on each cell. we assessed the specificity of the rejection of ia disparate grafts by using allophenic skin grafts in an adoptive transfer system and concluded that skin grift rejection across an mhc class i1 disparity required recognition of allo-ia determinants expressed by every cell in the graft. therefore, we reasoned that mhc class i1 antigens must be induced on these ia negative populations. indeed, injection of mice with gamma interferon dramatically induced ia antigens on previously negative keratinocytes. we next tested whether the induction of allogeneic ia determinants on keratinocytes was necessary for graft rejection by engrafting parental strain mice with skin from f >parent bone marrow chimeras. such grafts failed to be rejected, in spite of the speciic rejection of the allogeneic langerhans cells, indicating that the failure of keratinocytes to express allogeneic class i1 determinants leads to graft preservation. conclusion, mhc class i1 disparate skin allografts are rejected in a highly antigen specific fashion, secondary to the induction of mhc class i1 antigens on skin cells that fail to constitutively express them. to address whether the extensive polymorphism characteristic of class i molecules influences cd8 binding, we have screened a panel of transfectants expressing individual class i mhc alleles. of 18 alleles tested, only aw68.1 did not bind. all other molecules dld bind, including a2.1 and aw69, which differ by 13 and 7 amino acids respectively from aw68.1. position 245 in the alpha 3 domain was identified by sitedirected mutagenesls as the critical residue differing between a2.1 and aw68.1 which determines binding. a mutant aw68.1 molecule containin alanine at position 245 bound cd8, while a mutant of a2.1 with valine at 245 did not. alanine is found at position 245 of all human and murine class i molecules sequenced to date except aw68.1 and aw68.2, which have valine at that position. bulk cultures of a2-allospecific ctl were also sensitive to this substitution, and preferenhally recognized both molecules with alanine at 245. this study shows that aw68.1 differs from other class i molecules in its capacity to bind cd8, and raises the possibility that aw68.1 may not function as a restriction element as effectively as other class i alleles. t cell hybridomas derived from h-zd lnc recognked s and pres2 antigens in a i-ad restricted way, while t cell hybridmas from h-2k lnc manifested a specificity for either pres2 in association w i t h i-ak or for s in association with i-!& the activation of lhese hybridomas by antigen and antigen presenting cells (af'c), as measured by il-2 secretion, was found to be sensitive to prostaglandines and could be completely inhibited by anti-lfa-1 moncclonal antihxlies. different aft populations were tested for their capacity to present spresz particles to these t cell hybridomas. various macmphage like populations such as resident, con a induced, thiiglycolate induced peritoneal exudate cells as well as splenic adherent cells were found to present efficiently the spres2 antigen. in conhast b cells and la+ b cell lines (ta3, m12.4) could not function as accessory cells in the spresz specific stimulation of these t cell hybridomas. the inability of these cells to present this antigen was not due t o inhibitory effects since these cells did not inhibit the presentation capacity of other potent apc's. funhcnnore addition of apc's of a different haplotype could not complement for the defective presentatiw of spresz by b cells and b ceu lines indicating that mhc independent accessory factors are not implicated in this process. hence it is clear that maaophage-like apc's and b cells d i f f a in their capacity to process and present spdz antigens. since spresz is a very stable particle composed of lipids and proteins it is conceivable that such antigen quires a smng degradation and swh plocessing might occur in cenain -phage-like apc's but not in b cells. recombinant human insulin biosynthetically labeled with k n d " s at several amino acids was used as an antigen and was exposed for varyillg lengths of time to ta3 mouse b cell apc. subcellular fractionation and hplc chromatography permitted several of the processed peptides distributed throughout the insulin molecule to be monitored. many insulin peptides localized to both the extracellular (8 peptides) and intracellular (4 peptides) compartments of ta3 cells were detected. membrane-associated peptides is in progress. many of the peptides processed by ta3 apc in situ co-elute with those obtained upon digestion i n vitro by the insulin-specific insulin degrading enzylg5 (ide). f o r the processing of 51-labelled human insulin suggest that insulin may be processed in b cell apc into immunogenic peptides by an enzyme(s) present on the plasma-membrane, intracellularly and extracellularly. (supported by mrc and cda) . we investigated the pathway of antigen processing 5 itu in cell apc. hurine ctl clones specific for hia-a2 were generatef with the human cell line jy. four of five ctl clones were found to lyse a2k transfected murine cells more effectively than a2 transfectants. anti-cd8 specific mab inhibited t 3 lysis by these four clones, and this inhibition was more pronouncgd for a2k one clone, which lysed a2 and a2k transfectants equivalently, was shown to be insensitive to anti-cd8 antibody inhibition. these findings indicate that a2-specific t r i n e ctl clones possess greater avidity for murine target cells expressing the a2k hybrid molecule relative to those expressing the a2 molecule. this implies that a cd8 interaction with the same molecule seen by the t cell receptor is important for target cell recognition. hla class i1 antigens are highly polymorphic cell surface proteins involved in initiation and regulation of the immune response. allelic sequence variation primarily affects the structure of the first external domains of the a and j3 component chains. here we provide evidence for other types of allelic polymorphism for these genes. the sequences of two cdna clones corresponding to the hla-dqp mrnas from an hla homozygous cell line exhibit both alternative splicing and readthrough of polyadenylation. furthermore, the alternative splicing event is associated with only a subset of hla-dqb alleles, while the polyadenylation site readthrough is found in a larger subset. this suggests that polymorphic & acting elements within the hla-dqp gene control both processing steps. proteins, presumably encoded by the alternatively spliced mrnas lacking transmembrane exons, are immunoprecipitated with a monomorphic monoclonal antibody directed against hla-dq. these proteins are found in supernatants of cultured cell lines for which secretion is predicted, but not in those of cell lines which do not contain the alternatively spliced mrnas. such secretion class of i1 allelic products could profoundly affect interactions between effector and target cells in an immune response. departments of biology and chemistry and the cancer center, q-063, university of california at san diego, la jolla, ca 92093. we are studying the murine icam-1 gene and the effects of icam-1 on antigen recognition. using the human icam-i cdna, we have isolated cdna and genomic clones encoding the murine homologue. the murine icam-1 gene is a single-copy gene that consists of multiple exom spanning 25kb of dna and encodes a 2.5kb mrna that is expressed at high levels in a wide variety of different cell types. sequence analysis indicates that murine icam-1 is 50% and 6096 homologous to human icam-1 on the protein and dna levels, respectively, and is a member of the immunoglobulin gene superfamily, consisting of several v-like domains linked tanddy. we are also studying the effects of icam-1 on antigen recognition. the t-cell clone 14d m s f 1988). this response is blocked with the anti lfa-1 antibody fd441.8 when antigen is presented by blo.a(2r) spleen cells but not when antigen is presented by dcek, a fibroblast transfected with i-ek. we are currently aansfecting the murine icam-1 cdna into dcek in order to determine if we can enhance the 14d response and to determine if the enhancement is lfa-1 dependent. wth an alp a beta tcr that recognizes moth the t cell differentiation anti en, cd4, is expressed by mhc class i1 restricted t lymphocytes. mhc class i1 products. the association between cd4 expression and restriction by mhc class i1 rfioducts has led to the hypothesis that cd4 may interact with monomorphic determinants of a large body of experimental evidence suggests that cd4 interaction with mhc class i1 molecules leads to an increase in the binding avidity of t cell-stimulator cell interactions. a direct test for a functional cd4-mhc class i1 interaction in t cell activation requires a separate evaluation of cd4-ia interactions from tcr-a /ia recognition. however, a separate evaluation proves difficult since the t cell receptor and cb4 may interact with the same mhc class i1 molecule. in this report, we use a t cell activation protocol, where tcr-ag/ia recognition is replaced by tcr complex-antlcd3 antibodies interactions. using this activation protocol, we pave analyzed the effects of monoclonal anti-mhc class i1 antibodies on the activation o$a cd4 hybridoma in the absence of its tcr restr$tinj mhc class i1 molecule (ie ) but in the presence of unrelated mhc class i1 molecules (ie ,ia ). the data obtained clearly indicate a functional role for cd4-mhc class i1 interactions in t cell triggering. we have targeted hen egg lysozyme (hel) to murine b cells using heterocrosslinked antibodies which specifically bind to surface igd or different mhc molecules. occurred more quickly with targeting to igd than to mhc structures as assessed by fixation and pronase stripping experiments. hel was internalized quickly into acidic compartments when targeted to igd but was detected much later when targeted to mhc molecules, as assessed by shifts of fluorescent signal of internalized fitc-hel. however, the data indicate that not all endocytosed hel entered low ph (<5.5) compartments. degraded hel was released from b cells following endocytosis of 125-i-hel. this release was detected earlier with targeting to igd than to mhc structures. interestingly, the total amount of internal 125-i-hel decreased with time after endocytosis via igd, but the internal 125-i-hel was almost entirely whole undegraded hel at all times following endocytosis. these data and those of chloroquine and leupeptin inhibition studies indicate differences in the fate of antigen entering b cells via igd or mhc structures, and support the notion of a neutral ph storage compartment for antigen endocytosed via surface igd on normal splenic b cells. internalization and presentation of hel to hybridoma t cells laboratories, department of surgery, university o f iowa, iowa city, ia 52242 target cell lysis by cd8' ctl is a highly specific phenomenon in vitro, as we have confirmed repeatedly in reverse labelling tests by showing that admixed "third party" target cells are not lysed in the presence of specific ctl-mediated cytolysis. however, when mixtures of ctl and their specific targets are inoculated into the skin of hosts syngeneic to the ctl, host cells at the site of inoculation are destroyed, often to an extent that results in grossly observable, full -thickness necrotic lesions. we have evoked these "innocent bystander" reactions in mice with ctl directed against single and multiple non-h-2 antigens and tnphapten and influenza a virus-specific antigens. thus, the ability to trigger bystander tissue destruction appears to be a general characteristic of ctl-target cell interaction in vivo. our current evidence suggests that host inflamatory cells recruited and activated by factors stimulated by ctl-target cell recognition actually mediate the tissue destruction. these ctl-initiated bystander reactions may be the basis of the non-specific tissue destruction that contributes to allograft rejection and that is observed in many serious virus infections and in intense dth reactions and contact dermatitis. rong h a lin. baael i n s t i t u t e f o r i u m l o g y , baael, snitzerland. w e have investigated the b a d e for i n u n i t y or tolerance t o a m a e aezw proteinthe f i f t h caponent of c o l p l a c n t (a). i n c5 deficient nice t h i s protein is absent from s e r m and as a cnrwquence they are not tolerized t o cs. c5 deficient dca generate uy bearing t c e l l s which recognize c5 i n the c m t e x t of clasa 11. i n contrast, c5 s u f f i c i e n t mice i n which c5 protein is continuously probced d m t mtmt t c e l l reaponsea againmt u. we have tested i f t h i s self protein i s proceseed and presented with clase i1 i n n o m l mice and can be recognized by c5 specific t c e l l s i n the absence of exogenewsly added antigen. a l l clasa i1 bearing c e l l s fm c5 s u f f i c i e n t l i c e activated c5 specific t c e l l clonea without additional antigen. presentation was mt a cansaquence o f c5 secretion by macrophages i n culture but was a h t o be derived prom endqlenewaly generated cs/cl.ss i1 caplexea. thus t h i s self protein is e f f i c i e n t l y preaented ,inin and available f o r tolerance in&xtion. although c5 deficient dca cannot secrete c5 they s t i l l synthesize a precursor mlecule, pro-c5, i n accumulating evidence from a number of models suggests that unique subsets of antigenpresenting cells a r e responsible for the induction of specific t cell-mediated responses. w e have previously described an age-dependent maturational defect in the ability of the sjl strain of mice to activate dth-inducer t cells to a wide variety of antigenic stimuli. none of the other 14 strains tested exhibited a similar defect and all other accessory cell dependent responses were unaffected in the dth unresponsive sjl. w e have also shown that the adoptive transfer a macrophage from older dth responsive sjl or other dth responsive las strains can overcome this defect in dth responsiveness. w e have recently found that a subpopulation with the mac-i+, mac-3' and mac-2-surface phenotype a r e able to transfer responsiveness. facs analysis indicate that the mac-3 phenotype is expressed on less than 20% of macrophages. titrations of the mac-3' cells isolated by facs indicate that adoptive transfer of o ly 100 mac-3' cells can overcome the defect in dth responsiveness. by contrast, transfer of 10 mac 3-or mac 2' cells were unable to overcome the defect. our data suggest that the induction of cd4' antigen specific cells dth-inducer t cells is mediated by a phenotypically unique small subset of macrophage accessory cells. in our studies, we have examined the effect of administering fab' fragments of anti-l3t4 moab (fabl-gk1.5) on the inhibition of humoral immunity. treatment of klh-primed mice with 0.5 mg fabl-gk1.5 depleted l3t4' cells from lymph node tissue while leaving other lymphocyte subpopulations intact. after injection of klh in complete freund's adjuvant, these t,depleted mice were unable to produce anti-klh antibodies. long-lasting unresponsiveness against klh (12 weeks) was observed despite the apparent regeneration of the t, population of the lymph node. the results obtained using either fab' or intact gk1.5 antibody were comparable and suggest that a transient depletion of t, does not account entirely for the long-term humoral unresponsiveness. the aim of this study was to gain a more detailed insight into the molecular aspects of antigen processing during the imune response. as a first approach, endosomal vesicles were isolated from bovine alveolar macrophages and their proteolytic activity with respect to a model protein antigen, sperm whale myoglobin (mb), was characterized. during the first stage of digestion of mb by the endosomes, a limited number of fragments were preferentially released from the antigen. we have isolated and identified these fragments. the digestion of myoglobin is completely prevented by pepstatin, a specific inhibitor of aspartic proteinases, and only marginally by other proteinase inhibitors. when mb fragments preferentially released upon digestion with purified bovine cathepsin d, an aspartic proteinase abundant in macrophages, were identified, almost all coincided with the fragments released by the endosomes. to define in more detail the selectivity of cathepsin d under the mild conditions applied, other protein antigens were similarly treated with the enzyme and the peptides released were identified. the location of the preferential cleavage siteswhen related to known t-cell epitopessuggests a dominant role for cathepsin d in the processing of protein antigens to yield fragments for presentation to t-cells. possibly, the observed selectivity of the enzyme may account for the structural similarities among t-cell epitopes, noted by others. actively acquired tolerance in mice to the antigens of the mhc (h-2) is induced by exposure of the animals to allogeneic lymphocytes within 24 hours of birth. actively acquired tolerance to the mhc in humans (hla) cannot be studied in the same way. however, we have evidence for the existence of actively acquired tolerance in humans in a study of 26 highly sensitized patients waiting for a renal allograft. they had developed complement dependent antibodies to the hla antigens of almost all unrelated caucasoid donors. the sera of these highly sensitized patients were tested against a panel of lymphocytes that were mismatched for only one hla class i antigen. we found for these 73 patients hla class i antigens that, although different from those present in the recipient, did not lead to a positive crossmatch. we called such antigens "permissible mismatches" and show that they often included those hla antigens of the patient's mother that the patient had not inherited (noninherited maternal antigens; nima). in 15 of the 26 patients, the permissible class i mismatches included the nimaa. the noninherited paternal antigens (nipas) were analyzed as a control; only two of the 25 nipas tested were acceptable mismatches, which emphasized the preferential nonresponsiveness to nima. recent experiments indicate that what holds true for antibody formation also holds true for t cell activation. of hla class i and hia class i1 allospecific cd8-positive ctl clones. monoclonal antibodies (mcab) directed against the cd8 structure were only found to inhibit antigen-specific cytotoxicity of a series of class i allospecific cd8-positive ctl clones and not of a class i1 allospecific cd8-positive ctl clone. however cytotoxicity induced by cd3 mcab (used at suboptimal concentrations) or cd2 mcabs in both types of ctl clone was blocked by cd8 mcabs. the absence of cd8 mcab blocking of antigen-specific cytotoxicity of the class-11-specific cd8positive ctl clone may be explained by assuming that it results from a triggering signal which is to strong to be overcome by the down-regulatory signal of the cd8 antigen. these combined findings clearly suggest a functional involvement of cd8 not only in tcr/cd3 activation, but also in tcr/cd3 controlled alternative activation routes, such as the cd2 activation pathway. moreover it shows that even an hla class i1 allospecific cd8-positive ctl clone expresses a functional active cd8 antigen. the absence of hia class i expression on the target cells (daudi cells) used in the experiments described indicate that the cd8 antigens not act solely in an adhesion-like fashion, but exhibit also a more general regulatory function in t-cell activation. this regulatory role of cd8 may be explained by assuming the induction of a threshold for activation, which is triggered after binding of cd8 mcab or binding to its natural ligand, hla class i. in our view, cde-mediated regulation of t-cell activation could therefore prevent non-specific triggering of cytotoxicity by interactions of insufficient affinity. *this study was supported by a grant from the dutch kidney foundation. the cd4 t-cell surface antigen 1s felt to have the dual functlon of stabilizlng the interaction of the t-lymphocyte with the antigen presenting cell (apc) a s well as transduclng an independent signal that can potentiate the actlvatlon related alteratlons generated through the t-cell receptor. we have found that upon antibody-mediated cross-linklng of the cd4 molecules of cloned murlne t-lymphocytes there is a time and temperature dependent decrease in the abundance of the lymphocyte-speciflc tyroslne klnase p66lok. this co-modulation is speclflc for cd4 and p66lck slnce cross-linking of other t-cell surface antlgens (cd3. t200, thyl.2) does not result in detectable alteratlons in the abundance of the lck protein and slnce cd4 cross-llnklng does not induce any alteratlon in the abundance of p60*.. another srcrelated tyrosine klnase highly expressed in t-cells. such data suggest that cd4 and the internal membrane lck protein are in close proximity within the cell. further analysls has revealed that slgnlflcant amounts of lck can be immunopreclpitated by antl-cd4 antibodies. in addltlon. cd4 can be speciflcally preclpltated by anti-lck antibodies. our data imply that cd4 and p661ok are physically associated in cd4+ t-lymphocytes. the flndlngs that cd4 is msoclated to the lck proteln in either murlne or human t-cells and that cd8 is also complexed to p66lck ln cd8* t-cells suggest that the lck tyroslne klnase is involved in the functlon of the cd4 and cd8 accessory molecules. these apc do not appear to present processed klsa determinants. in light of these findings, of apparent interest is the issue of which cells types are responsible for hlsa-specific t cell tolerance induction. studies in mice treated from birth with anti-p antibodies suggest an important but perhaps not exclusive role for b cells in this process. we are currently pursuing the identity of other cell types which may be involved. in addition, lmmunogenlclty c173 university of texas southwestern medical center at dallas, dallas, texas75235 qlo is a soluble class i-like major histocompatibility antigen produced specifically by the liver. previously, it has been shown that mice possessing soluble qlo can generate anti-q10 cytotoxic t lymphocytes (ctl), suggesting that this soluble molecule does not function as a tolerogen. we have recently constructed c3h transgenic animals which express an exon shuffled q10 (al, p2)/ld (03, tm) molecule. this qio/ld molecule is expressed specifically in the liver on hepatocytes but not on nonparenchymal liver cells, spleen, thymus, kidney or brain. the expression of qio/ld in the transgenic hepatocytes is equivalent to la expression on balb/c hepatocytes, suggesting the animals are expressing physiologic levels of the transgene. the presence of membrane bound qio/ld in c3h animals has not caused anti-910 ctl precursors to be deleted, however, because primary in vitro ctl assays show these transgenic animals can specifically lyse qio/ld targets. histopathologic examination of the livers of these animals does not show extensive lymphocytic infiltration or inflammation. in addition, serum levels of alanine aminotransferase. aspartate aminotransferase, and alkaline phosphatase are also normal, confirming that these animals do not show overt signs of liver rejection. results are ampatable with a t least two different pathways of antigen hardling, a pathway for degradaticm of antigen, and a "pmcessiq" pathway for antigen presenbtim. my+ l monocytcq enes appear t o i n t e r f e r e with t h e processing pathway, either by i n h i b i t i n g production of antigenic material t h a t can associate with ia o r by i n h i b i t i n g putative intracellular event@) imr0lvb-q the binding of ia to processed antigen and tmnsprt of annplexes to the cell surface the immunogenicity and antigenicity of synthetic peptides (sp) derived from the sequences of a streptococcal antigen were investigated in macaque monkeys. immunization with the free peptides of 17 and 21 residues failed to elicit serum antibodies or t cell responses. however, both serum antibodies and lymphocyte responses were elicited by immunization with the sp linked to tetanus toxoid (t) as a carrier. indeed, spl7-lt and sp21-tt elicited serum antibodies and proliferative responses of lymphocytes, not only to the sp but also to the native strtptococcal antigen. recall of sp17-tt or sp21-1t immunized monkeys w i t h suboptimal doses of the native srreptococcal antigen resulted in a significant increase in antibodies, both to the sp and native antigen, confirming that the two sp share antigenic epitopes with the native antigen. the b and t cell epitopes were then determined and the b cell epitopes resides in residue 8-13, whereas the t cell epitope overlaps and consists of residue 7-15. the t cell epitope has an amino-terminal leucine and carboxy-terminal glycine and alanine added to residue 8-13 of the b cell epitope. in spite of the b and t cell epitopes being expressed in sp17 (residues 1-15), the monomer failed to induce serum antibodies without a carrier. however, immunization with dimers of peptide-linked or disulphide-linked residues 1-15, without a canier, elicited both serum antibodies and proliferative responses of lymphocytes. the results suggest that the monomeric sp17 is not immunogenic, whereas the dimeric peptide elicits both antibodies and t cell responses. the minimal t cell-b cell structure required for immunogenicity is now being determined. lmmunogenlclty section a departments of immunology and rheumatology, mayo clinic, rochester, pin 55905. susceptibility to collagen induced arthritis (cia) in mice maps to the i-a loci in h-29 mice. however, swr (h-29) mice are cia resistant, suggesting a role of non-mhc genes. we have recently shown gene complementation between h-29 from swr and tcr v genes from several non-susceptible strains. cia has been induced in b10, c3h.a and a fackcrosses with swr with similar high incidences; 63, 71 and 67% respectively. c57l shares a similar background with b10 and is h-2b, but has the same v tcr mutation as swr. backcrosses showed a very low incidence (17%) of cia, and tte arthritis observed was of a much milder and transient nature. the invariant chain associated with hla class i1 molecules is a 31-33 kd glycoprotein implicated in antigen processing and assembly and intracellular transport of class i1 molecules. class i1 molecules and invariant chain are expressed primarily by b lymphocytes and antigen-presenting cells such as macrophages and can be induced by interferon-y in a variety of cell types. to define sequences involved in the human invariant chain gene regulation, 790 bp 5 ' to the initiation of transcription were subcloned upstream of the cat gene. transfection into invariant chain-producing cell lines and non-producing cell lines demonstrated that this 5 ' region displayed tissue specificity and responsiveness to interferon-y. deletion mutants were constructed to ascertain the functional properties of specific regions of the invariant chain upstream regulatory regions. these deletion mutants have led to the identification of 3 putative regulatory regions: 394 to 239, 2 3 to 216, and 216 to 165 bp 5 ' to the cap site of the invariant chain gene. deletion of any one of these 3 regions results in decreased cat activity. protein-dna interactions of these sequences have been characterized by mobility gel shift assay and dnase i footprinting. two regions have been identified that exhibit cell type dependent binding of nuclear proteins. two color flow cytometry was used to characterize the surface phenotypes of human bronchoalveolar lymphocytes (n=32). the cd4/cd8 ratio was highly variable (0.3-6.6, mean-2.1). a high proportion of the t cells expressed hla-dr (9-38%, mean=2l%) indicative of t cell activation. however, detectable levels of the i+-2 receptor were expressed on <3% of the cells. cd45r was absent from cd4 cells in most preparations (0-10% mean=3%) suggesting that the cells are inducers of ig synthesis. uchl1, a marker of memory cells was present on 68-100 % of lung t cells. uchll+ cd45r-lung lymphocytes responded poorly to pha and cona but did respond to il-2 in the presence of accessory cells. together these data suggest that lung lymphocytes are recently activated memory cells. il-2 induced lung t cell lines were also characterized for antigen expression and w ( activity. high lak activity was obtained in preparations containing a high proportion of cd8 cells. these cultures appeared to be suicidal. in contrast, lines with a high proportion of cd4+ had low or absent lak activity but proliferated in the presence of il-2 for at least 3 months expressing a cd2 cd45r-phenotype. this abstract is a proposed presentation and does not necessarily reflect epa policy. the polymorphic second exons of the hla-dp, and dpd genes have been specifically amplified in vitro by the polymerase chain reaction (pcr) method, using the thermostable dna polymerase of aauaticus. sequence analysis of mi3 clones containing the amplified dp sequences from a panel of thirty-four df typed cell lines revealed only the two previously characterized alleles for dp, . fourteen allelic variants were defined for dpw eight of these are associated with the t-celldefined dpwl-6 types; two subtypes were found for both dpw2 and dpw4. six additional dp alleles which were previously typed in the t cell assay as blanks were also idenlfied. based on this sequence information, non-isotopic sequence specific oligonucleotide probes have been developed and used to type a margarita betz, dominic dordai, brian e. lacy, and barbara s. fox. department of medicine, university of maryland school of medicine, baltimore md 21201. murine type 2 helper t cells (th2) secrete interleukin 4 (il 4) in response to antigen. despite the likely importance of these cells, little is known about their priming and expansion in vivo. we have demonstrated il 4 production in response to a cytochrome p peptide following t cell expansion in vitro. this antigen has not previously been shown to induce th2 cells. bio.a mice were primed with a peptide fragment of pigeon cytochrome c in cfa. lymph node cells were restimulated in vitro with antigen for 5-7 days, ficolled and rested for 3 days without antigen. cells were then tested by limiting dilution for the presence of antigen-specific il 4 producing cells. il 4 was detected using the il 4 sensitive cell line ct4s (provided by dr. w. e. paul, nih). the specificity of the response was confirmed by blocking with the anti-ll 4 antibody 11 b1 1. following in vitro restimulation of the primed lymphocytes with antigen, il 4 production was detectable from as few as1 03 cells per well. il 4 secretion was antigen dependent and required both in vivo priming and restimulation in order to be detected. it is not clear why primed lymph node cells, placed in limiting dilution culture directly after removal from the animal, failed to secrete detectable amounts of il 4 in response to antigen. suppression is an unlikely mechanism as fresh primed lymph node cells were unable to inhibit il 4 production by restimulated cells. we are now investigating the factors that may regulate the development of il 4 producing t cells. mark r. boothby, ellen gravallese, hsiou-chi liou. and laurie h. glimcher, department o f cancer biology, harvard school o f public health, boston, ma 02115. regulated pattern, and normally expression is limited to certain cell types such as 6 cells and macrophages. cells is accompanied by the loss o f class i 1 mhc expression. these genes also respond to external stimuli such as the cytokine il-4, which increases b cell ia. a region o f the aa mhc gene activated expression of a cat reporter gene in a b lymphoma cell line but not in a myeloma cell line. a nuclear protein that bound to two sites within this region was found. this binding activity was present in spleeiis that lack t cells and in b cell lines, but it was absent from all three myeloma cell lines tested. il-4 treatment of normal and athymic mouse spleen cells greatly increased the binding of this nuclear protein to its a a target sites, concomitant with increased aa transcription; "thus, 6 cells contain a sequence-specific binding activity regulated both by il-4 and by differentiation. the differentiation o f b cells to plasma lmmunogenicity c210 peter van den elsen3. ldepartment of immunology, the netherlands cancer institute, amsterdam, zdepartment of immunology, erasmus university, rotterdam, 3department of immunohaematology, academic hospital, leiden, the netherlands. human tcr y6 occurs in disulphide-linked (type 1) or non-disulphide-linked (type 2) forms, dependent on the use of the cyl or cy2 gene segment. the cyz gene segment can contain a duplication or triplication of exon 2 , which gives rise to different protein forms (types 2bc or zabc). it is not known whether functional differences exist between these receptor types. protein chemical analysis of type 1 and type 2bc receptors on functional human t cell clones derived from peripheral blood (pb) has indicated that not only the y chains, but also the 6 chains have a molecular mass and charge which set apart type 1 and q p e 2bc receptors. two sets of fifteen clones were randomly generated from pb of two normal donors after selection with the anti-tcr y6-1 mab, which recognizes all receptor types. dna rearrangement and mrna expression analysis of y and 6 genes allowed us to map the specificity of the anti-tcr y6 mabs 6tcs-1 and tiya to the v6l and vy9 gene segments respectively. subsequently it could be concluded from the analysis of random clones that the majority of type 1 receptors use vy9, while this preference seems absent in type 2 receptors. the great majority of type 1 receptors do not use v6l. while the majority of type 2 receptors do. this was confirmed by fluorescence analysis of pbl of a large panel of normal donors. we conclude that vy and v6 gene segments in functional tcr y6 in pb are used in non random combination and that their expression is correlated with rearrangement of the y gene to cyl or cy2. we have previously shown that polymorphic residues in the nh2-tenninal half of the p1 domain (amino acids 1-48; hypervariable regions 1 and 2 [phvl and 21) determine with which allelic or isotypic a chain a particular b chain can achieve efficient cell surface beterodimer expression. this result might be understood in terms of the current model for ia smcture which predicts that w v l would lie adjacent to region. therefore, to examine the role of ahvl residues in conmlling hetcroduncr expression. a mutant a d cdna was created in which the codon for amino acid 11 was mutated to code for the auk residue at this position. in addition, recombinant a d and a& cdnas, in which the segments encoding the three a hypervariable regions were exchanged between the two alleles, were used to study the connibutions of other a chain polymorphisms to this process. interestingly, the polymorphic residues in ahv2 are predicted to lie in a region of the a a chain a-helix which is adjacent to the phv4 region of the p chain a-helix. allelic substitutions in this latter region of ad have been shown to similarly affect surface ia h e t e r o d i i expression. taken together, these results suggest that there are at least two spatially separate areas in which the a and p chains interact and that these interactions are affected by polymorphic rtsidues in both areas, conmbuting to the efficiency of heteroditner expression and, most likely, ia quartcmary conformation. the aim of this project is to identify contact residues of the t cell receptor (tcr) with antigen and/or mhc class i1 molecules. as a model system, a vp17-containing tcr has been chosen since the majority of vb17' t cell hybrids react with ie molecules of the k,s,d, and b haplotype. t cell hybrids have been made which have a dual reactivity: they are vp17+ and recognize ie molecules but also show reactivity towards a known antigen, namely chicken ovalbumin (ova). one such hybrid has been mutagenized with ethyl methane sulfonate (ems). mutants were selected on the basis of their survival after stimulation by either antigen or ie. it was expected that mutations in all different kind of genes involved in t cell recognition and t cell activation would be found. mutants obtained fall into two major groups: 1) loss variants of tcr a or fl chains,t3 or l3t4: 2) mutants with point mutations in one of these genes. we are currently analyzing the mutants biochemically and functionally in order to identify the particular gene affected. point mutations in the a and p genes of tcr mutants will be localized using the polymerase chain reaction in combination with dideoxy sequencing. activation of t lymphocytes requires the intracellular fragmentation of foreign antigens and their presentation by class i or class i1 major histocompatibility complex glycoproteins. the direct binding of peptides to class i1 molecules has been shown in a number of experimental systems and its specificity compared to that of t cell activation. in contrast, direct binding of peptides to class i molecules has been difficult to detect; although peptide sensitization experiments and the crystallographic structure of hla-a2 persuasively argue for its occurence and importance. in this study, we demonstrate specific binding to hla-a2 of an influenza matrix peptide (flu-m1 residues 56-68) that has previously been shown to act as a target for certain hla-a2 restricted influenza-specific cytotoxic t lymphocytes. we estimate that less than 0.3% of the purified hla-a2 molecules were able to bind the added peptide. we and others have shown that allorecognition by cytolytic t lymphocytes (ctl) is analogous to t cell recognition of foreign antigens in that both can occur via presentation of antigenic peptides by products of the major histocompatibility complex. we have used peptides corresponding to the alphal helix of selected hla molecules to analyze t cell recognition of this polymorphic region. the alpha alpha helix of hla-b44 and -b13 are identical, and show a high degree oh homology with those of hla-bw58, -b47, and -b27. peripheral blood lymphocytes from 5 normal donors were stimulated in vitro with targets expressing hla-b44 to derive allospecific ctl lines and clones. in some individuals, the allospecific response was almost totally directed against the alphal helix. the ability of peptides corresponding to the alphal helix of these hla molecules to inhibit and induce lysis as well as to modify other assays of t cell activation will be discussed. diseases and *national institute of child health and human development, d bethesda, md 20892 . classical transplantation antigens are constitutively expressed on cells of all tissues exce t brain. transaiption is regulated by the interaction of nuclear factors with 5' flanking regions tiat include the class i re latory element (cre). previously, the cre has been divided mto 3 re om on the basis of nuclear t%or blndin . several studies have implicated the nuclear protein (ri) wfich binds to the inverted repeat geggattcccca) of re 'on i as necessary for gene transai tion although region i is identicarin all se uencedouse $d and l enes, it is not conservefin qa r y genes. a comparison of the c& from h-2ld with that of 610, a qa region gene expressed o y in the liver and fetal olk sac, shows that there are two changes within the inverted r eat se uence (tgaggactcc$a). these differences disru t the dyad symmetry. another nugotide diierence between h-2l and qlo falls within re 'on ifof the cre. however, qlo can bind to the nuclear factor (rii) that binds to region ii of the fit2ld cre, whereas qlo region i can not bind to the nuclear factor (ri) that binds to the region i inverted re at. to test whether the differences m region i contribute to the restricted tissue e ression of q l r w e have used site-directed in vitro mutagenesis to make the inverted repeat of]cglo region i like that of the classical class i genes. a change at either base enhances transcription as measured in a transient transfection system. either change also allows binding of the nuclear factor that binds to the classical r alterations in the cre regon i contribute to the limited tissue expression of310. the presence of disrupted cre region i in other region genes likely contributes to their tissue restricted expression. on i sequence. thus, molecular analysis of t cell receptor structure/function in sperm whale myoglobin specific t cell clones. jayne s.danska, alexandra m. livingstone, toshi isihara and c. garrison fathman, stanford university medical school, stanford, ca. we have undertaken structural characterization of the t cell receptors (tcr) utilized by a well defined panel of murine dba/2 t cell clones that recognize epitopes within the 110-120 peptide of sperm whale myoglobin (sp wmb) presented by i-ad or i e . only 2 of 14 independent clones show alloreactivity for 10 whc haplotypes. using the polymerase chain reaction (pcr) and dna sequencing of the tcr a and fc chains from matched sets of clones bearing either whc restriction or epitope specificity in common, we are addressing structural relationship between tcr and mhc/antigen for this model system. among 6 i-ed restricted t cell clones reactive with spwmb 110-120, all have highly homologous tcr 6 chains associated with a minimum of three different tcr a chains, some of which are derived from novel v gene families. to further characterized the specificity of these clones we are generating substituted peptides to identify residues within the epitope important for interaction with tcr or restricting mhc molecule. functional verification of the relationship between given tcr primary sequences, and recognition capability will be addressed by transfer of the a and/or 8 chafns cdnas created by pcr amplification into t-cell hybridomas expressing endogenous tcr genes of known sequence and specificity. with mhc fine specificity. the differential impact of substitutions with the n-terminal and c-terminal portions of the apl domain is consistent with models of auap structure in which the n-terminus interacts with peptide while the c-terminus interacts with both peptide and the tcr. or aauapu-resmcted t cell clones. the antigens tested were l-tymsine-p-lmmunogenicity c 218 expression of the q4p gene, patricia m. day, katherine e. lapan and jeffrey a. frelinger, department of microbiology and lmmunolog university of north carolina at chapel hill, chapel hill, nc 27599. generally, the transcription of class i genes tom the q a a region is limited to tissues of hematopoietic origin. previous work in our lab demonstrated widespread transcription of the q4 gene in the 81 0.p mouse, with high levels of mrna found in liver, lung, lymph node, spleen, testes and thymus. less rna was present in muscle and brain tissues. however,,it is not known whch individual cell types within these tissues are responsible for the transcription of the q4 gene. we raised polyclonal antisera against a synthetic peptide, derived from the predicted amino acid sequence of the q4p transmembrane region. we selected this region since it is the most locus specific. this antisera immunoprecipitates a class lsized protein. a monoclonal antibody, directed against the same peptide, has also been produced. sv40-transformed h-2p fibroblasts show an abundance of q4 message. suprisingly, indirect immunoluorescent staining with the monoclonal antibody reveals a cytoplasmic localization of the protein with a perinuclear concentration. different patterns have been observed in examination of the h-2b em onal carcinoma cell lines 402ax and pcc4. qgspecific antibodies allow us to identify the cell ty es which express the24 gene product. the application of in situ hybridization techniques will correlate the cellular site ofmrna synthesis and protein detected by antibodies. understanding the paltern of expressim of the q4 gene is the first step in determining the so far elusive function of these mhc genes. and il2r mrna a f t e r mitogenic stimulation. antibodies against cd45r, but not against c045 common determinants, synergise with suboptimal doses o f mitogen t o induce il2 and il2r mrna expression, suggesting t h a t cd45r molecules are operative i n transmembrane s i g n a l l i n g i n immature thymocytes. there i s also an i n d i c a t i o n from northerns using cd45 probes t h a t cd45p180 mrna i s not induced i n activated cd348-thymocytes as i t i s i n mature t c e l l s . these r e s u l t s support the idea t h a t cd45r+ molecules are essential f o r generating signals required f o r c e l l survival w i t h i n the productive intrathymic lineage. we examined a panel of thl and th2 t cell clones for the ability to induce antibody synthesis in a mishell-dutton culture system under cognate b-t cell conditions: our findings indicate that both thl and th2 t cells are heterogeneous, i.e., some but not all thl and some but not all th2 clones have the capacity to induce antibody under these conditions. we examined the effect of 7-irradiation or rnitomycin-c pretreatment of our thl and th2 clones on their ability induce antibody synthesis. asano et al. (j. immunol 138:667) have reported that th2 clones are exceedingly sensitive to 7-irradiation, with doses as low as 500 rads abrogating the ability of th2 clones to induce antibody synthesis. we found that while the helper activity of th2 clones was very radiation sensitive, helper activity in thl clones was very radiation resistant. thl clones given 2 0 0 0 rads of irradiation were as effective as unirradiated clones in inducing anti-tnp plaque forming cells (pfc). moreover, when used at higher t cell/b cell ratios in culture, irradiated thl clones were more effective than unirradiated clones in inducing antibody synthesis. the effect of 7irradiation on th2 clones was not simply due to inhibition of proliferation, since mitomycin-c pretreatment of the clones had little effect on helper activity. conservation, alexander l. dent, pamela j. fink and ste hen m. hedrick, department of biology, university of california, san diego, 8a 92093. the p chain gene of the murine t cell receptor has been shown previously to have an alternative1 spliced form of message. this message contains a novel exon, termed c& which is inserted between the vdj and constant region exons. we have studi6d expression of the cpo exon at the mrna level by rnase protection. we have found that about 1% or less of of p messages in normal t cell clones contain the cgo exon, whereas p m e s a es. in the thymus contain the exon at 10-20 fold higher levels. to address t i e importance of the c 0 exon in the immune ,system, we have undertaken a phylogenetic approach. \y cloning and sequencin the rat analogue of cpo, we have found that while the rat exon is very simifar to the mouse exon, both donor and acceptor rna splice signals are defective in the rat cpo gene. this implies that rat cpo cannot be spliced into rat p m e s a 8s. furthermore, we have sequenced the analo ous region to mouse cpo in 8 e human p chain locus, and have found no stretct of sequence remotel homolo ous to cpo. because cpo is not conserved evolutionarily, we beieve that ! he cpo gene element does not sewe an important function to the immune system of most vertebrates. 1988) . we found that one arrdno acid substitution at a vdjp juntional region position found to be highly conserved in pigcon cytochrome c-specific tcr's results in a change in antigen fine specificity, while an* change abolishes all detectable responses characteristic of the d6 tcr. we will present the results of mutagenesismansfection analyses of two other pigeon cytochrome c-specific tcr's. the murine ctl response to human class i molecules is 1-2 orders of magnitude lower than the response to murine alloantigens, due to structural differences between human and murine homologs. we investigated whether this discrepancy could be overcome by exposure of developing t cells to human class i molecules in transgenic c57bl/6 mice expressing hla-a2.1. ! c222 zhe bdixujiar basis of . lbve digiustn ard ed p d l u b 2 r , divof s ch mice expressed hla-a2.i in spleen, bone marrow and thymus at levels similar to those of endogenous h-2 molecules. however, the frequency of ctl specific for other human alloantigens remained similar to that of normal mice. the frequency of hla-a2.1 restricted influenza specific ctl was 1-2 orders of magnitude less than the frequency of h-2 restricted ctl. these results indicate that the poor response of murine ctl to human class i antigens is not determined by selection in the thymus, but by species-specific constraints on the interaction of mhc antigens with t-cell recognition structures. while the mice are tolerant to hla-a2.i expressed on murine cells, they still respond to hla-a2.1 expressed on human cells. the epitopes defined by such clones are present on hla-a2.1 positive human cells derived from several different tissues. such epitopej are not dependent upon the species of p2m associated with the class i molecule, nor upon the structure of the attached carbohydrate. the results suggest that one or more highly conserved normal human proteins contribute to the formation of such epitopes, and provide an explanation for the failure of ctl raised against class i molecules on human cells to recognize the same molecules expressed on murine transfectants. this suggests that normal endogenously expressed molecules may also be important in the formation of epitopes on class i antigens recognized by allospecific ctl. we previously demonstrated t h a t several subclones derived from a c03+, cd4-/cd8-t-cel i l i n e have undergone secondary rearrangements a t t h e t-cell receptor (tcr) a locus w h i l e maint a i n i n g i t s o r i g i n a l tcrb and igh d-j rearrangements (marolleau e t . al., i n press). these secondary rearrangements r e s u l t i n t h e j o i n i n g of germline va and j a gene segments which replace the p r e -e x i s t i n g va-jacmplexes of t h e parental t-cell line. i n an e f f o r t t o examine t h e molecular mechanism responsible f o r these va-ja gene replacements, t h e s t r u c t u r e s o f tcra cdnas prepared from both t h e parental and subcloned t-cell l i n e s were determined. i n addition, northern b l o t and southern b l o t analyses were performed on both t h e parental and subcloned t-cell l i n e s using a panel o f va and j a probes. our r e s u l t s i n d i c a t e t h a t : 1) secondary rearrangements r e s u l t i n both productive and non-productive va-ja j o i n s , 2) the mechanism whereby secondary rearrangements occur i s a d e l e t i o n event t h a t involves germline va genes 5 ' t o t h e p r e -e x i s t i n g va-ja complex j o i n i n g t o j a the class ii major histocompatibility complex (mhc) antigens are a family of integral membrane proteins whose expression is tissue-specific and developmentally regulated. a pair of consensus sequences, x and y, separated by an interspace element, is found upstream to all class ii genes. deletion of each of these sequences eliminates expression of class ii genes in vitro or in transgenic mice (1-3). furthermore, the absence of a specific binding protein for the hla dr a x box in patients with severe combined immunodeficiency disease whose cells lack class ii suggests a critical role for these proteins in class ii gene transcription (4). report the cloning of a agtll cdna encoding a dna binding protein (human x-box binding protein, hxbp-1) which, like the proteins in whole nuclear extract, recognizes both the x box and interspace elements of the human dra and murine aa genes. the hxbp-1 cdna hybridizes to two rna species, 2.2 kb and 1.8 kb in human, that are expressed in both class ii positive and class ii negative cells. hxbp-1 does not cross-hybridize to two murine aa x box binding cdnas recently isolated in our laboratory which also recognize the dra and a ax boxes. these observations provide evidence for the existence of multiple x box binding proteins which recognize a common or overlapping motif. chromosome mapping studies demonstrate that hxbp-1 arises from a multi-gene family two of whose members map to human chromosomes 5 and 22. taken together, these data suggest a high degree of complexity in the transcriptional control of the class ii gene family. france . in an attempt to analyze positive or negative in vivo regulation of clonal expansion of cytolytic t lymphocytes (ctl), we immunized blo.br mice with the kb specific ctl clone kbs-cu). and we tested whether t cells obtained from such mice would influence the in vitro development of the ctl clone kbs-c?o. a clone-specific helper effect has k e n observed, which is mediated by cd4+ splenic cells from immunized mice. control immunizations of b1o.br mice with ti negative variants of suggest that this growth regulation involves the recognition of the ti of kbsc20. the precise nature of the antigen recognized on the ctl clone, the possible involvement of ti determinants with or without mhc products u e now under investigation. we have shown that thy-]+ dendritic cells present in the epidermis of mice (dec) express cd3 associated v73 and v61 gene products. we have produced a monoclonal antibody directed against v73 and found that a wave of cells appearing at the earliest stages of fetal thymic development express v73. phenotypic and functional analysis of v73+ cells in the early fetal thymus indicates that they have characteristics in common with the v73+ dec. both populations express high levels of ly-1 and are ly-6c+. neither express cd4 or cd8. interestingly, the v73+ fetal cells express elevated levels of il-2 receptor, indicating that they may have been activated. functional analysis demonstrated that, unlike other fetal thymocytes, the v73+ cells can be stimulated to produce lymphokines and lyse a panel of target cells which are also lysed by the adult thy-l+ dec. these results raise the intriguing possibility that the first receptor-bearing component of the t cell system to appear in ontogeny might give rise to the thy-]+ dec. (3ritical to an understanding of the function of cells bearing the gamma-delta t cell receptor will be an understanding of when and where such cells function. in order to investigate this, we have used a variety of techniques (in situ hybridisation, cdna cloning, and pcr) to examine m a s of tcr gamma delta gene expression. one conspicuous site of expression is the intestinal epithelium, which is by contrast almost devoid of tcr alpha beta expression. interestingly, the v gene segment usage in this location is quite specific and is different to the specifcity that we have found in the spleen and in the thymus, and that others have found in the skin. this specificity suggests in turn that expression of the resmcting elements recognised by gamma delta may also be spatially nxticted. extensive analysis of junctional diversity can pmvide infomation on the diversity of antigen nxogniscd by gamma delta. the basis for selective expnssion of v gene segments may in part lie in different requkments of the v-gannna gene pmoters. to examine this, the wnscriptional capabilities of the various gamma gene promoters in t cells murine tcr gamma genes: distinct spatial restriction of v-gene segment usage, adrian thyday*, susan kyes*, simon carding#, charles a. janevay, being compared by linkage to the chloramphenicol acetyl transferase gene. biochemistry, university of wisconsin-madison, madison, 11 53706. murine strain a sublines a/j and a/wysnj have a genetic polymorphism that regulates serum immunoglobulin responses to several protein antigens. strain a/j secondary igg1 responses to bovinv gamma globulin, oralbumin, hemocyanin and galactosidase l-ere 50-, l o -, i -and 4-fold greater, respectively, than a/wysnj responses. subline a/hej is a low responding strain like .l/wysnj. analysis of h-2 class 1 and class i 1 molecules provided no evidence for a breeding error to account for the genetic polymorphism. instead, an important immune response gene outside h-2 may hare been heterozygous when the sublines diverged, and the polymorphism resulted from sezregation and differential allele fixation. a mutation subsequent to subline divergence is also a possible source of the polymorphism, but is less likely. the high responder phenotype inheritance pattern in (a/wysnj x a/j)fl, f2, and backcross mice was consistent with segregation of a single, recessive gene. we named this locus l a for the strain a sublines that define it; strain a/j represents the k a h allele and strains a/yysnj and a/hej represent the allele. hayes, keith d. hanson, faye nashold, and david j . miller, department of the secondary iggza responses were also affected. several different proteolytic digests of denatured seb have been tested for their ability to stimulate t cell hybrids to produce il-2. a tryptic digest that retains activity has been fractionated by hplc and the stimulatory component is being analyzed. examination of the amino acid sequence of seb and the proteolytic cleavage sites has led us to synthesize several peptides for analysis. these peptides, and their analogues, will be tested for their function in vivo and in vitro. to understand the interactions involved in the famation of peptide-mhc complexes, an assay has been developed to detect dr specific binding of peptide analogues of t e l l determinants to cell surfaces. ebv msformcd b cell lines (bcls) w m incubated with biotinylated peptide followed by fltc conjugated streptavidin, and then anaiysed by flow cytomctg. a panel of bcls homoygous for diffmnt dr types bound analogues of peptide 307-319 from influenza virus haemagglutinin (previously shown to be a helper t cell determinant restricted through dr1) to varying degrees, w h m no binding was observed to the dr-bcl rj225. binding could be specifically inhibited by the natural unbiotinylated t cell determinant or other drl restricted determinants. competition by a range of peptides revealed quantitative diffmnces in their ability to bind drl. the assay is currently being used to generate a detailed model of the complex formed betweenha307-319anddrl. and trp for leul 6. and hla-a2.3 differs from hla-a2.i by the substitutions of thr for ala 4 9 glu fo??a1152 and trp for zeu156. residues 9 and 95 in the 8-sheet of the molecule, and residues 149, 152, and 156 in the a-helix are thought to interact with bound peptide or the tcr. to evaluate the role of these residues on ctl-defined epitopes, two genes were constructed that encoded novel molecules which differ from hla-a2.1 only at residues 9, 43, and 95, or at residue 156. the effect of a-helix substitutions on serologic and ctl-defined epitopes that varied between hla-a2.i and hla-a2.3 were evaluated by constructing genes that encoded the individual differences at residues 149, 152, and 156, as well as additional non-naturally occuring substitutions at these same positions. hla-a2.1 specific ctl were found that were: (1) insensitive to substitutions at either residues 9, 43, and 95, or residue 156, but were lost when all four positions were changed; (2) dependent upon the residues 9, 43, 95, but not residue 156; (3) dependent upon residue 156, but not residues 9. 43, and 95; and (4) dependent upon residues 9, 43, 95, and residue 156. further epitope mapping with the a-helix mutants demonstrated that a substitution at residue 152 often destroys an epitope not affected by substitution at residue 156. even conservative substitutions at position 152 were more disruptive than nonconservative changes at residue 156. residue 149, while important in defining an mab epitope, had no effect on any ctl epitopes. these results indicate that spatially separate residues in the a-helix and 8-sheet of the molecule can contribute to the epitope recognized by a given ctl. furthermore, considerable complexity must exist in the spectrum of t cell receptors utilized to recognize hla-az, as 28 ctl clones exhibited 21 distinct fine specificity patterns. to follow the evolution of these class i types, to discern the chief selective pressures on its members and thus indicate the probable functional properties of the antigens. a cosmid library was screened for class i genes. 91 clones were mapped and could be grouped into 17 clusters of contiguous dna spanning 1,264 kb. by hybridisation studies, 61 class i genes/ gene fragments could be distinguished. transfection analysis revealed that 10 genes could be expressed as cell surface antigens: two genes, in a block of duplicated dna encoded serologically defined rt1.c products, the other 8 genes gave rise to novel class i antigens detected by the xeno-antibody 0x18. using region specific probes, we could detect clear rat homologues of the mouse qa and h-2 genes, however there were only two rat genes with limited homology to the mouse tla genes. the analysis showed extensive remodelling of the class i region in the evolutionary gap between rat and mouse. while the immunological role of t cells bearing the ap t cell receptor (tcr) has been well characterized, much less is known about the function of t cells bearing the $3 tcr. we investigated the role of tcr $cells in the immune response to complete freund's adjuvant (cfa). after immunizing mice with cfa, we observed a greater than 26-fold increase in the number of tcr $3 cells present in lymph nodes draining the sites of immunization, compared to a 3-4-fold increase in the number of tcr ap cells. there were at least three different species of 3tcr's expressed on these cells in the draining lymph nodes, including two protein products derived from the rearrangements of cyl and cp, and one product derived from cy4. 37% of tcr ys cells from immunized lymph nodes expressed the il-2 receptor in vivo. and these cells constituted roughly 50% of the proliferative response of total lymph node t cells to 11-2. tse, et al. (j.lmmun..vol.l25, p.491.1980) have demonstrated that at least three cell types are involved in the t cell proliferative response to antigen, including an antigen specific-t cell, an antigen-presenting cell, and a t cell that is found in unprimed lymph nodes or spleen, which has been termed the recruitable cell. we have utilized their approach of analyzing the slope of log cell number-log response curves to examine whether tcr @ cells can function as "recruitable" cells. we found that tcr ys cells as well as tcr ap cells can function as recruitable cells in this system. these data suggest that tcr $3 cells can participate in the immune response without being specific for the antigen. analysis of the membrane associated phosphoprotein profiles of b cells harvested from cultures of resting cells exposed to il-4 for 18-24 hrs reveals the presence of phosphoprotein with an mr in the range 75-80,000. destroy the autoradiographic signal from this phosphoprotein suggesting that it is phosphorylated upon tyrosine residues. appearance of this molecule, and lps also apparently fails to result in the presence of a 75kd structure in the phosphoprotein profiles. anti-il-4 antibody. 11811, in the cultures prevents the appearance of the 75kd phosphoprotein. the genes for t n f -a and tnf-p are tandemly arranged on mouse chromosome 17, with only 1.1 kb separating the 3' end of the tnf-p mrna from the 5' end of the tnf-a mrna. yet, the two genes are independently regulated. in vitro transcription and nuclear run-on experiments indicate that the two genes are transcribed from independent promoters. in macrophages, which express tnf-a but not tnf-p, only the tnf-a promoter is active. in t lymphocytes, which can synthesize both proteins, both promoters are active. activation of either cell type results in a moderate (up to 10-fold) increase in the level of transcription, while mrna levels increase more than 1wfoid under the same conditions. interestingly, the tnf-p gene is aanscribed 10-fold less than the tnf-a gene in t lymphocytes, although the corresponding mrna is more abundant. these results indicate that the accumulation of both tnf-a and tnf-p mrna after cell activation and their relative steady state levels are controlled mostly at a post-transcriptional step. acanomycin d chase experiments reveal that tnf-a mrna stability in macrophages is not significantly altered after activation by lf's, and therefore that stabilization done cannot account for the observed accumulation of tnf-a mrna. in order to examine more closely which elements are required for the regulation of tnf-a and tnf-p mrna abundance, we constructed hybrid genes combining putative control regions of tnf-a and tnf-p with known constitutive control elements. results obtained from the transfection of these hybrid genes into various cell types indicate that elements located both 5' and 3' of the coding sequence are required for the proper regulation of tnf-a and tnf-p mrna abundance. celiac disease is characterized by small intestinal mucosal injury and malabsorption. disease is activated when a genetically susceptible host ingests wheat gliadin or similar proteins (i.e., prolamins) in rye and barley. d region specif icities -dr3 and -dqw2. class i1 d-region haplotype associated with celiac disease is extended and also includes genes in the hla-dp subregion. chain gene with those encoding dr3 and dqw2 may indicate that the hla haplotype associated with celiac disease exhibits an unusual degree of linkage disequilibrium or, alternatively, that disease susceptibility involves the gene products of more than one hla locus. to characterize possible hla structural variants unique to celiac disease, the polymorphic second exons of the expressed dr, dq and dp genes were amplified from genomic dna of celiac disease patients, and their nucleotide sequences determined. our studies indicate the presence of a unique constellation of d region genes associated with the celiac haplotype, and exclude the presence of a disease specific dr, eq or dp structural gene variant in this disease. disease susceptibility is strongly associated with the hla class i1 we recently determined that the hla this same population of t cells contains a high frequency 0 1 % ) of cells which will respond to a given allogeneic mhc protein, or to differences at two other genetic loci termed mls, in conjunction with mhc. we have transfered the a and b chain genes from a pigeon cytochrome c/el specific, alloreactive. and mis' specific murine t cell clone into an unrelated host t cell. we demonstrate that the genes encoding a single a b receptor chain pair can transfer the recogntion of self mhc molecules c m p l e x e d with fragments of antigen, allogeneic mhc molecules. and an m1sc (hls-2) encoded determinant. in this case the transfer of antigen specificity and alloreactivity requires a specific a8 receptor chain combination, whereas mlsc reactivity can be transfered with the 6 chain alone into a recipient expressing a randomly selected a chain. site directed mutagenesis of the ja region has also been performed in an attempt to identify sites involved in the alloreactivity of this t cell clone. in addition. we demonstrate that a single amino acid change in the v-j junction of the a b receptor can alter mhc restriction a s well a s antigen fine specificity. department of genetics, washington university school of medicine, st. louis, i(0 63110. the s49 tumor sublines are variants isolated from a sing1 parent balb/c tumor which demonstrate locus-specific shut-off of their kd, dd and l8 genes. four phenotypically different sublines were characterized at the dna and rna level. southern blot analysis indicated that no major chromosomal deletions have occurred, and treatment of the sublines with 5-azacytidine had no effect on class i expression. between loci are unlikely. none of the repressed class i antigens could be induced with interferon even though the expressed antigens were fully inducible. northern blot analysis revealed message only for the expressed antigens, showing that the repression mechanism is acting at the transcriptional level. rnase protection analysis confirmed this result and demonstrated that the transcriptional repression is exquisitely specific for the kd, dd and ld genes as other "class i-like'' messages are present in. all the cell lines. expressing class i antigens from both fusion partners, but the negative class i antigens originating from the s49 partner were not expressed. lymphokine gene expression was examined in a panel of 116 short-term murine t lymphocyte clones derived by single-cell micromanipulation from allogeneic mixed leukocyte cultures. about 30% of clonable t cells, including both cd4+cd8-and cd4-cdw cells, could be expanded for assay at an average of 22 days after cloning. following stimulation with concanavalin a or anti-cd3 antibody, all clones secreted detectable granulocyte-macrophage colony stimulating factor (gi(-csf), interleukin-2 (il-2) and il-3, but cd4+ clones on average secreted higher 'levels of each lymphokine than cd8+ clones. clones (85%-96%) expressed detectable gm-csf, interferon-y and il-3 mrna and 11% expressed il-4 mrna. when the frequencies of co-expression of any pair of lymphokine mrnas vere determined, all were found to correspond to the values predicted for random assortment of the individual frequencies. for example, among 13 il-4-positive clones, 11 also transcribed interferon-y, giving the frequency of double-positive clones expected for random association (9.6% 10.8%). expression of the four lymphokine genes therefore segregated independently among the clones and did not allow the division of t cells into subsets vith distinct patterns of lymphokine synthesis. greater than 20-fold in the adult liver cell line, 2 to 3 fold in the macrophage cell line and just slightly in l-cells. we have subcloned the region 5' to the li gene which contains sequences that may be important to regulating expression of the li gene. this region includes a 15-mer (cctagaaacaagtga) which occurs 5' to many ifn?i regulated genes. current research has been directed towards identifying and comparing proteins from nuclear extracts prepared from control and ifn-7 treated cells which bind to this region (-260 to -1 1). this data indicates the li molecule may be expressed in cells not known to be directly involved in the immune response. although there has been considerable interest in the recently identified gamma, delta t cell receptor, relatively little is known as to its function. during our studies of the human immune response to autologous b cell lymphomas, we generated cytotoxic t lymphocytes (ctl) specific for tumor idiotype. these ctl lysed only autologous tumor cells and none of a large panel of other autologous and allogeneic cells. inhibitable by anti-idiotypic and anti-immunoglobulin antibodies but not by a panel of classical anti-mhc antibodies. phenotypic analyses showed that these ctl were cd3+, cd4-, cd8-, and express the delta, and presumably gamma! t cell receptor. such ctl can be used to gain new insights into the function of the gamma, delta t cell receptor and t cell recognition of immunoglobulin, and may prove clinically useful in adoptive immunotherapy. tumor lysis was for ebv-induced antigens. furthermore, lcl variant .221, which does not express any hla -a, -b. or -c determinants. is killed by cultures primed to lcl-.180. antibody blocking experiments suggested that this killing was mediated by t cells, and was not restricted by known class i antigens. depletion of leu 19 positive cells from the effector population did not eliminate cytotoxicity on lcl-.221. cold-target blocking studies further suggested that the class 11-nonexpressing lcl-.180 and the class i-nonexpressing lcl , 2 2 1 share residual deterninant(s) other than hla class i or class i1 that can restrict cytotoxic t cell responses to ebv-induced antigens. national jewish center for immunology and respiratory medicine, denver, co 80206 it is uncertain to what extent lymphokines can be differentially produced by activated primary t cell populations. to determine if il4 and ifnr were differentially regulated in uncloned human t cells from adults (ad) and neonates (nt), these mrnas vere measured by in situ hybridization after maximal stimulation by ionoaycin and pma. il4 mrna was detected in 1.2% of total (tl), 3.5% of cd4', 10% of cd4' cd45r-, and 0.1% of cdb' ad t cells, but in none of the tl, cd4+, or cd8' nt t cell populations (virtually all nt t cells were cd45r'). in contrast, ipnr mrna was found in 39.42% of tl, 34.36% of c d 4 ' , 51% of cd4' cd45r-, and 58% of cd8+ ad t cells, but only 2.3% of tl, 2% of c d 4 ' , and 4% of cd8' nt t cells. these results agreed with other estimates of il4 and ifnr production based on ria of cell culture supernatants, rna blotting, and gene transcription assays. in contrast to il4 and ipnr, il2 was expressed in similar amounts by ad and nt t cell fractions, as well as the ad cd4' cd45r' and cd45r' subsets. thus, the capacity for increased il4 and ifnr production by ad t cells appears attributable, in large part, to the postnatal acquisition of the cd45r' subset (putative memory t cell population). aowever, additional mechanisms exist which act transcriptionally to limit il4 production by both neonatal and adult t cells. such selective expression may be important for restricting the potentially pleiotropic effects of certain lymphokines t o appropriate responder cells. we observed significant inhibition (>70% at 800 ng/ml) of the presentation of wova and of ova 323-339 by the anti-ap 57-78 peptide mab's. exhibited significant inhibition. 61 peptide mab's. after incubation with antigen +/-mab, indicate that the inhibition occurs at the level of antigen presentation. dg11, was also observed for the anti-p chain peptide mab's and to a lesser extent by the anti-a chain peptide mab's. peptide sequences are capable of interfering with antigen presentation, in vitro. supported by nih grant, ai-14764. the ovalbumin (ova) i-ad restricted t cell hybridoma, do1l.10 was used to the anti-i-ad mab, mkd6, also much less inhibition was observed with the anti-% 43-experiments with glutaraldehyde fixation of the b1d.p cells before or inhibition of i-ad allorecognition by the t cell hybridoma. these results indicate that mab's generated against class i1 rijllinghoff, institute for clinical microbiology, university of erlangen-nurnberg, 8520 erlangen, f.r.g. and the *institute for clinical immunology and rheumatology, university of erlangen-niirnberg, 8520 erlangen. f.r.g. recently we have shown that cloned l . major-specific l1/1 t-helper cells of type 2 (th2cells), when stimulated with antigen, are able to induce polyclonal b-cell proliferation (1). we here present evidence demonstrating that this process is dependent on a direct cellcell interaction between t-and b-cells. which in the effector phase, i.e. during stimulation of the b-cells by activated t-cells, can be mediated by a mechanism other than cognate interaction. this conclusion is derived from experiments, in which highly purified resting b-cells were polyclonally stimulated by l1/1 t-cells triggered by an anti-t3 monoclonal antibody, in the absence of antigen. the triggering process was independent of the presence of the fc part of the antibody and occurred in cultures devoid of macrophages. thus, the well established cognate recognition does not appear to be the only way of b-cell induction by t-helper cells of type 2. studies show that a proportion of the peripheral blood cd3' t lymphocytes do not express cd4 or cd8 and are called double negative t cells. they normally have a 1 6 tcr. however, another population of double negative t cells exists that expresses the a@ heterodimer. w e have purified and expanded such a population isolated from the peripheral blood of a healthy individual and studied i t s phenotypical and functional characteristics. the c e l l s are cd3' cd4-cd8-, positive for wt31 and negative for the nk markers. they express a and p mrna b u t lack ymrna. from surface iodinated cells were precipitated w i t h monoclonal pf1 two closely running bands (46 & 48 kd) . functional studies demonstrate that they proliferate to anticd3 and pha, t h i s response was blocked by cyclosporin a. there was no nk lysis b u t anticd3 induced l y s i s of target cells. the cells responded t o il-2 and il-4 as previously shown for other t c e l l s , b u t also t o il-3, a lymphokine thought t o affect mainly stem cells and not previously shown t o stiaulate growth of mature cells. long term growth of these c e l l s was also maintained by these cytokines . roberto biassoni , silvano ferrini , rafck p. sekaly , and eric 0. long , laboratory of immunogenetics, national institute 2f allergy and infectious diseases, nih, beth-md 20892, and istituto nazionale per la ricerca sul cancro , 16132 genova, italy. cd3-cells grown in vim in the presence of il-2 acquire the ability to l~s e a wide variety of tumor cells in an mhc-unrestricted manner. we have previously shown that cd3-16 clones expressed the cd3 epsilon gene but no functional transcript from cd3 gamma, cd3 delta, tcr alpha, tcr beta and tcr gamma genes. this result suggested that these cd3-16' cells represented an early stage in t cell differentiation. to test for expression of the tcr delta gene in these cells, rna from a panel of cd3-16' clones and from three highly enriched populations was hybridized with several dna fragments of the delta locus. abundant transcripts were detected with a c delta probe and a j delta 1 probe in 6 out of 8 clones and in all three populations. at least four different transcripts were present with sizes similar to those found in cd3' tcr gamma-delta' cells. however, the tcr delta transcripts in cd3-16' cells are most likely derived from unrearranged genes because no rearrangement could be detected in dna from an enriched population using a j delta 1 probe, and because these aanscripts hybridized to a dna fragment corresponding to the unrearranged genomic sequence 5'-upstream of j delta 1. expression of unrearranged tcr delta genes in cd3-cells provides further evidence that these cells belong to the t cell lineage. functional capabilities and by differential release of either il2 or il4 upon activation. we have produced a new monoclonal antibody to cd45 which has allowed us to separate normal murine cd4+ cells into two populations based on the density of expression of cd45 epitope. the separated populations seem to be analogous of subsets found in cloned t cell lines. cd4+ t cells with high density of cell surface cd45 after polyclonal activation produce il2 and mrna encoding ifw and il2. it does not produce il4 or il4 mrna. cd45 low density population on the other hand transcribes mrna for il4 and secretes il4 protein. data will be presented to demonstrate that the two subsets of normal cd4+ cells also differ in their proliferative response to mitogenic stimuli and to exogenously added growth factors. the substitution of v to l at 95 was the only change that could be discriminated by 2 of 9 allospecific ctl lines. suggesting that those 2 ctl lines recognize a2.1 plus a peptide whose presentation andlor binding is affected by the v to l substitution in the floor of the peptide binding site. in contrast, the l to w substitution at 156 (but not the other 2 substitutions) abolished the ability of the a2 molecule to present the viral peptide to 24 out of 25 peptide-specific a2.1-restricted ctl lines, suggesting that this substitution alters the presentation of the influenza matrix peptide but does not inhibit the ability of the peptide to bind to the a2 molecule. although y8tcr.s have a great potential for diversity, it remains to be determined whether this potential is realized in terms of expressed y6tcrs. preliminary studies in several laboratories have indicated that y6tcrs expressed in earlg t h r c y t e s and adult epithelial tissues are more restricted in diversity com ared to adult tc expressin thymocytes. we have derived a panel of cloned dendritic epigrmal t cells (jetc, lines and5ybridomas that express at least three types of y6 receptors -c 8, cy26 and c n . immunoprecipitation, northern and southern blot analyses, and sequence anazses of l gt 10 cloned cdna or olymerase chain reaction ( k r ) amplified cdna segments have been used to anal ze in detail &e extent of diversit in the expressed y and 6 chains and whether restricted airin o?y and 6 chains occurs. our resu& indicate that for this panel of cloned cell lines 7 and ! irkg is nonrandom and that variability in certain types of receptors appears to be restricted. %owever, we have observed significant 6 chain diversity in these cells that is obtained by the use of multiple v-regions, and n-region and junctional diversity. we are investigating whether the observed y and 6 chain pairing, and pattern of 6 chain diversity are present in other $tcr bearing cells or whether they are only characteristic of detc. activation of ctl precursors from murine unprimed spleen cells with ril-2 or ril-4 results in distinct lytic spectra, depending on which lymphokine is present. we have used allo-stimulation in limiting dilution analysis with subsequent testing on an allo-specific target (a20) and an mhcdeficient, non-specific target (rle). in the presence of ril-4 exclusively allo-specific ctl are generated, while ril-2 supports a proximately equal numbers of precursors that k~ll a20 and rie targets. dose response analysis of ril-2-supported killing activity indicates that the lytic spectrum is independent of the amount of ril-2 used, and therefore this il-2 effect is intrinsic in its activity on unprimed spleen cells. mixing experiments indicate that ril-4 can partially override the effect of il-2 on the generation of non-specific killer cells. split well analysis and cold target inhibition experiments are in rogress to ascertain the actual proportion of specific killer cells which can be generated with ril-2. be. are also testing the ability of cofactors, such as il-1 and il-6, to optimize the response of il-4 generated ctl. we conclude that il-4, not il-2, must be used when ctl are generated from unprimed spleen cells in mice. t r a n s c r i p t s i n y/6 tcr populations. i n t e r e s t i n g l y , these same v genes, as well as a further+crosshybridizing v gene previously designated va7.2, are expressed by peripheral a& tcr c e l l s as 1.6kb tcra transcripts. these data suggest t h a t b2a2-dn th represent a developmentally unique subset i n which both v6 and vg segments are non-randomly expressed. furthermore they i n d i c a t e t h a t there i s considerable overlap between the v a and v6 gene repertoires . indianapolis, in 46285 in order to detect the small amounts of lymphokines generated in vivo following antigen stimulation, we developed a co-culture system which allows for detection of il-2/4, il-3/csf and tnf from ln cells stimulated in vivo with picryl chloride (pcl). utilizing thb system in combination with facs analysis and receptor binding studies, we examined the production of these lymphokines in primary and secondary immune responses. during a primary immune response, the production of il-2 was not readily detectable on dl, peaked on d3 and was gone by d5. at no time were we able to demonstrate the presence of il-4. alternatively, the presence of il-3/csf and tnf was w i l y detected on dl, but olso peaked on d3. in comparison to primary responses, secondary immunization lead to at least two alteraticns. (i) peak production of all lymphoki es shifted towards dl. (2) although most lymphokines did not demonstrate increasea in the amount produced/lo cells, the amount of lymphokine generated/ln was vastly increased due to an increased number of cells. utilizing single and dual color facs analysis we also examined the ln cells for alterations in t cell subpopulations. during the course of the primary response: (i) the percentage of thy i+ and l3t4+ cells decreased until d3 and then began to recover, (2) the percentage of thy-i+, t4-,t8-cells peaked at the time of greatest lymphokine production (i.e.-d3) and (3) the il-2 receptor was expressed solely on thy-i+ cells, was detected on both t4+ and t8+ subsets and peaked on d3. most of these alterations also occurred during the secondary response, but their timecoune was shifted so that maximal effects occurred earlier (e.g., dl). finally. the maximal binding of radiolabeled il-2 by the ln cells following both primary and secondary sensitization correlated with the expression of the il-zr as detected by facs analysis. in addition, binding of radiolabeled il-4 demonstrated similar patterns except for the detection of significant binding on dl. these results demonstrate that (i) an ordered timecourse of lymphokine production occurs in vivo following exposure to antigen and (2) the secondary immune response to pcl is characterrd by an accelerated tempo of lymphokine production, rather than an increased level of lymphokine production/lo cells. activation as direct g-protein activation by a1f4 pi-hydrolysis using phorbol diester stimulation of pkc restores the inhibi$$ble phenotype and the ability to upregulate c-fos. even more interesting, sig-linked ca responses by vs2.12-c1.2 are equivalent to those observed in the wildtype wehi-231. resul$g suggest that contrary to current thought, sig-generated signals may not be coupled to ca fluxes via inositol phospholipid hydrolysis. thus, vs2.12-c1.2 is a new and powerful tool with which to analyze signalling through sig at the molecular level. unlike the wildtype, crosslinking of sigm on vs2.12-c1.2 did the signaling defect in vs2.12-cl.2-appears to be proximal to phospholipase c triggers pi-hydrolysis and bypassing these latter lmmunogenicity c 302 analysis of t cell receptor 7 chains from adult cd4-,cd8-thymocytes mark w. moore, i. nicholas crispe and michael j. bevan, department of immunology, research institute of scripps clinic, la jolla, ca 92037. the role of tcr 7 genes in t cell development has not been determined. to extend out understanding of the repertoire of tcr 7 expression, we prepared a cdna library from cd4-,cd8-adult balb/c thymocytes and cloned and sequenced 15 tcrq genes from this cdna library. we found that 2 clones were transcripts of the unrearranged c7, gene and that 3 clones terminated in the j7, region. nine of the remaining clones were v71,2-j7 c7 genes and five of these were in frame. only one clone corresponded to c71 and was v7 -jy c7jofned'in frame. sds-page analysis of the 7-chain proteins from the surface of both balb/c anzcdbl)6 adult cd4-,cd8-thymocytes did not detect the 32,000 mw vy c7 protein, but did detect the 35,000 mw v7c7 protein. these results suggest that despite the abundanc$%f pull-length, functionally joined, v7 c7 transchpts in the thymocyte subset, the protein product is not expressed on the cell surface as the prehfcted 32,000 mw 7 protein. finally, our analysis of the v-j jointing of the 7 genes reveals both flexibility at the v-j junction and extensive n-region nucleotide addition that lead to diversity of the predicted protein sequence. il5 in response to the same stimuli. e identification of these two subsets of cd4' helper cells is mostly based on studies performed with long-term cultured t cell lines and it is not clear whether these two subsets exist in vivo and represent distinct lineages of t cells. in particular, the frequency, tissue distribution and ontogeny of cells capable of secreting il4 in vivo is not known. these studies have been hampered by the fact that freshly isolated t cells from unprimed animals failed to secrete detectable amounts of ila and il5 when stimulated in vitro by lectins or alloantigens, whereas iu is readily detectable in these same cultures. data presented here indicate that freshly isolated t cells from unprimed animals can be induced to produce il4 in a receptor-de endent, antigen-independent manner upon stimulation by anti-cd3 antibodies. our results also stow that only cd4' and not cdst cells can be induced to secrete il4 and that cross-linking of the receptor is required for o timal activity. we believe that this approach will be useful in identifying in vivo cells recomittefto the th2 pathway and study their ontogeny, activation requirements and tissue distriiution. hlb, brussels, belgium. we have studied the murine tcr repertoire against the c-terminus of cytochrome c in association with certain alleles of the mhc class i1 molecule, eakepk (iek) and eakepb (ieb). for mice possessing these alleles, the majority of responsive t cells utilize one member of the variable val1 gene family in conjunction with a limited set of vp genes. as an extension of these studies, we have examined ie specific, alloreactive hybridomas derived from ie non-expressing (eab) cytochrome c non-responder mice to determine their usage of va and vp genes. tion assay showed that fourteen utilized the same val1 gene segment used by the majority of cytochrome c specific, ie restricted t cells and eight utilized a closely related val1 gene that also is associated with this antigen response. element most commonly used by cytochrome c-specific t cells was not found among the alloreactive hybridomas tested, @ genes less frequently used in the cytochrome response were expressed by seven of the 22 alloreactive hybridomas whose va segments were defined by rnase protection. determining recognition of ie molecules both in mhc-restricted, antigen specific immune responses and in alloreactive responses. the t-helper cells of seven mouse strains, representing 5 class i1 haplotypes (ias, ia4, iab, iakiek, iadied) were responsive to immunization and restimulation with parent peptide. the ied determinant was shown to be a presenting element by monoclonal antibody blocking and by use of l-cell-transfectants as af'cs to purified t cells and to t cell hybridomas. a series of overlapping synthetic peptides identified two minimal t-cell sites within the parent peptide: mice expressing ia and ie responded to a fragment at the n-terminus of the parent peptide (site 1) while mice expressing only ia responded to a distinct but overlapping fragment at the c-terminus (site 2 ) . these minimal sites identified in vitro could be used to immunize mice in vivo in an mhc-restricted manner. the human 6 tcr locus is strategically located within the atcr complex between the cluster of va/v6 region and the ja segments. which can be spliced to ca in pre t cells, separates 6 from the ja segments. pulse field gel mapping as we11 as molecular cloning link diversity (ds), j g , c,5 and tea within 35 kb. considerable 6 tcr diversity is generated despite the predominant use of one v 6 and j 6 segment. d61 and d62 are 9 and 13 bp long, are frequently recombine as d,1/d62? and reveal exonucleolytic trimning with extensive "n" segment addition. specialized 5' and 3 ' 6 deleting elements, 6 rec and p j a , separate the 6 locus from the a locus. cells with 6 rec/$b ja recombinations comprise most 6 deletion events although 6 rec recombines with 2 other major acceptor sites in fetal and post-neonatal thymic dna. the 5' 6 deleting element ( 6 rec) is evolutionarily conserved in the mouse and functional comparisons are underway. delete the 6 locus may prove to be the pivotal event establishing separate y6 and ae lineages. to study the mechanism of t-cell tolerance, transgenic mice were generated that expressed the mlsa reactive t-cell receptor (tcr) o-chain vb8 1 on -90 % of peripheral t-cells. in transgenic mice bearing mlsd, the numbers of high tcr expressing thymocytes and of thy 1.2+ peripheral t-cells were reduced. the cd4/cd8 ratio of peripheral t-cells was decreased fourfold compared to negative littermates. both mlsa and mlsb tcr &transgenic mice were able to mount a t-cell dependent antibody response against viral antigens whereas the capacity to generate alloreactive and virusspecific cytotoxic t-cells was impaired in tcr &transgenic mlsa, but not in transgenic mlsb mice. rna analysis and immunof luorescence with tcr vb-specific mab further revealed, that the expression of endogenous tcr 0-genes in these mice was suppressed. tolerogen-reactive lymphocytes, as measured in the mlr, in spite of their long-term acceptance of a skin graft bearing the tolerated antigens. lymphokine production by mlr+ tolerant lymphocytes is different from that of syngeneic normal lymphocytes. normal lymphocytes produce only il-2 in primary response to tolerogen, while tolerant lymphocytes produce il-2 and il-4. using limiting dilution analysis, we have to estimated the frequencies of pil-2 and pil-4 (precursor) cells in these cultures. after primary k vitro stimulation, normal responders have a low but measurable frequency of pil-4 cells, while tolerant responders have a much higher pil-4 frequency. however, following subsequent & restimulations, the pil-4 frequency of normal responders rises and begins to approach that of the tolerant responders, such that the two populations are indistinguishable based on pil-4 frequencies following the third round of in vitro stimulation. these data suggest that the high frequency of il-4 producers (presumably t,, cells) among the tolerant lymphocytes resembles unexpectedly a "primed" state, rather than "unprimed"as in nontolerant responders (where th1, dominate the early response). the existence of "primed" t cells in phenotypically tolerant animals raises the possibility that precocious activation of tr1 (by neonatal exposure to tolerogen?) suppresses the later emergence of t,,, which would be expected to contain the cells responsible for graft rejection. a large number of cd4+ t-cell clones, obtained from peripheral blood t lymphocytes by direct limiting dilution, allowed us to address the question whether functional heterogeneity exists within the human cd4+ t-cell subset. six out of 12 cd4+ clones were able to lyse daudi or p815 cells in the presence of anti-cd3 antibodies. the remaining 6 cd4+ tcell clones tested did not acquire this cytotoxic capacity during a culture period of 20 weeks. in the absence of anti-cd3 mab, no lytic activity against daudi. p815 and k562 target cells was observed under normal culture conditions. these two types of cd4+ t cells showed high reactivity with anti-cdw29 (4b4) mab and no reactivity with anti-cd45r (284) mab. the cd4+ clones without anti-cd3 mediated cytotoxic activities (th2) consistently showed a higher expression level of cd28 antigens. th1 cd4+ clones did produce il-2, ifngamma and tnf-alpha.beta. whereas the th2 t-cell clones produced minimal amounts of il-2. ifn-gamma and tnp-alpha. beta in response to anti-cd3 mab and pma. not all cd4+ clones did release il-4, but there was no correlation with cytotoxic activity. moreover, as compared to the th1 cd4+ clones, tr2 cd4+ clones proliferated moderately in response to anti-cd3 mab. however, anti-cd3 mab induced proliferation of only the th2 cd4+ t-cell clones was enhanced by anti-cd28 mab. both cd4+ subsets provided help for polyclonal b-cell activation with anti-cd3 mab. our data suggest that the human cd4+ subset, in analogy to the murine system, comprises two functionally distinct t-cell subpopulations. in the mouse, when la antigens are isolated immunochemically, the predominant species isolated are the isotypic matched pairs, aaap and eaep. however, when la ap dimer expression is studied using an l cell transfection model, it is found that the isotype-mismatched dimer apdea is readily expressed at the cell surface. these results suggest that differences in assembly andl or transport of different la pairs may be most readily visualized in a competitive environment where multiple distinct la chains are available. to investigate this possibility, the relative efficiency of inter-and intra-isotypic dimer formation and expression was evaluated using a sequential l cell transfection system. l cells already expressing an ap dimer on the cell surface (apdea or apdaad) were supertransfected with a third la gene (aad or ea, respectively). synthesis of this second a or p protein led to competition for the unique partner chain. individual clones were scored for cell surface expression of the distinct dimers (e.g., apdea vs epdea or apdaad vs apdea) using facs analysis with chain specific monoclonal antibodies. in addition, each species of mrna was quantitated by northern blot hybridization using bcus specific probes. our results indicate that in the h-2d haplotype. isotype-matched dimers are expressed with 3-4x the efficiency of isotype-mismatched dimers. this result suggests that, regardless of the cell type studied, if each of the four murine la genes is expressed at equivalent levels, intraisotypic dimers will be expressed to the virtual exclusion of the interisotypic dimers. however, if chain synthesis asymmetry occurs, the isotype mismatched pairs may be expressed at immunologically relevant levels. differential we have identified a series of discrete stages among the cd3-double negatives which seem to form a sequence, with tcr gene rearrangement and rna expression gradually progressing, but with potential for expansion and repopulation of irradiated thymuses diminishing along the series. on this pathway cd3 must be expressed late or after the acquisition of cd4 and cd8. cell cycle analysis shows the highest rates of cell division to be among the rsa+ il-2r-pgp-1-population which probably precedes the transition to cd4+cd8+ and tcr expression. thus it seems unlikely that tcr/antigen interactions play a role in cellular events occurring among the double negative cells which lead on to mainstream t-cell development. egr-l is a murine early growth factor inducible gene which encodes a protein with zinc fingers. its expression was investigated in murine b-lymphocytes stimulated through their antigen receptor (sig) with anti-recptor antibodies (anti-ig) . rapid (by 15 minutes) upregulatlon of egr-l mrna expression was observed at doses of anti-ig sufficient to drive the majority of go cells into cell cycle. agonists and inhibitors of protein kinase c (pkc) showed that expression was coupled to the pkc component of receptor immunoglobulin transmembrane signalling. interestingly, signalling through sig on the murine b lymphoma wehi-231 did not upregulate egr-l expression even though similar signalling pathways are associated with this receptor in these cells. southern analysis showed that egr-l is not deleted or translocated in this cell line. importantly, cell growth and proliferation of wehi-231 is inhibited by anti-ig stimulation suggesting a relationship for egr-l expression and differential processing of receptor ig signals. this notion is further supported by the finding that murine b lymphomas whose proliferation is not inhibited by anti-ig showed receptor immunoglobulin coupled egr-l expression. reeulation of exdression of a class 1 wc transeene. dinah s . the expression of the transgene product. the patterns of expression of the transgene parallels that observed in situ, indicating that regulatory elements necessary for normal patterns of expression are contained within the injected 9 kb dna segment, and that trans acting factors involved in its regulation function between species. included among these elements are those specifying preferential expression in b cells relative to t cells. in vivo treatment of transgenic mice with a/@-interferon results in increased expression of the transgene in a number of tissues. the response parallels that observed for the endogenous h-zkb, but differs markedly from qa-2. analysis of the chromatin structure of the transgene reveals a single constitutive dnase i hypersensitive site present in both spleen and thymus, which is not altered by interferon. both a novel negative and positive regulatory elements have been identified in the 5'flanking region of the transgene. the negative regulatory element reduced the activity of both the homologous class i promoter and a heterologous viral promoter. in vivo competition experiments indicated that the functions of the positive and negative elements are mediated by distinct cellular trans-acting factors. the negative regulatory element requires the presence of a positive regulatory element to function. this interaction between elements represents a novel mechanism for regulating gene expression. mcdevitt. department of microbiology and immunology, stanford university school of medicine, stanford, ca 94305. published data show that encephalitogenic h-zu murine t cell clones with specificity for the n-terminal eleven amino acid peptide of myelin basic protein display a restricted fine specificity when tested on substituted analogs of the native peptide. for example, substitution of alanine at certain positions in the peptide totally abolishes the response of each clone (acha-orbea et al.. 1988. cell 54:263) . recent experiments also have shown that the ability of some peptide analogs to bind to h-zu i-a gene products does not always correlate with their ability to stimulate the t cell clones (see accompanying abstract by david c. wraith and hugh 0. mcdevitt). this suggests that h-2u mice may lack a t cell repertoire capable of recognizing these peptides complexed to h-2u i-a gene products. to test this possibility, h-2u mice were immunized with a panel of peptide analogs, as well as the native peptide. the in vitro t cell proliferative response to each of the peptides then was measured. the results show that in vivo immunogenicity of the peptide analogs also does not strictly correlate with their capacity to stimulate the t cell clones. in this way, the polyclonal t cell repertotre of h-zu mice for the myelin basic protein peptide analogs was examined, and could be compared with the i-a binding characteristics of the peptides. terms of antigen and mhc recognition. this response involves a limited repertoire of t cells which crossreact on species variants of the antigen. in addition, t cells specific for the antigen in association with syngeneic mhc can recognize antigen on similar allogeneic mhc molecules. the groupin of clones by functional phenotypes defined by these crossreactivities allowed us to corrcfate tcr gene usage with either antigen or mhc recognition. some of the pigeon cytochrome c-specific clones within one functional phenotype use receptors that differ by as few as two amino acid residues. other clones e y s s very different tcrs but exhibit similarities in antigen/mhc reco nition. the efect of these tcr differences on recognition was assessed using a pane? of anti en analogs with single amino acid substitutions presented on different mhc molecufes. each clone exhibited a unique pattern of res onse to the antigen analog panel, even clones with very similar receptors. also, eace residue in the antigenic region of the peptide was critical for interaction with at least one t cell receptor. therefore, the antigen must either be a linear molecule with each residue available to interact with the tcr or be able to assume several conformations to interact with mhc and the tcr. lmmunogenicity c323 thy-1+ cd3+ ly-5(b220)+ cd4-cd8-tcrx-6' helper cells. anne i. we have found that these cells can be preferentially stimulated to proliferate when cocultured with the b lymphoma, ch12. one to 2% of nylon wool non-adherent, ia-, jlld-, and cd8-lymph node cells from normal unimmunized mice have the phenotype thy-l', cd3', cd4-. and cd8-. these cells proliferate when co-cultured with a syngeneic surface ig' lymphoma, ch12, even in the absence of any added antigen, mitogen, or fetal calf serum. prior to stimulation we find that approximately 30% of thy 1.2' cd3' cd4-cd8-express the marker ly-5(b220), however after culture with ch12 the majority of cells with this phenotype express the marker ly-5(b220). after ch12 dependent proliferation the ly-5(b220)' t cells are able to provide help for secretion of ig by fresh ch12 b cells. surface labelling and precipitation of t cell receptor molecules reveals that most of the thy-1' cd3' ly-5(b220)* cd4-cd8-cells express tcr(r-6). furthermore, cd3 precipitation shows that as many as four different 7-6 heterodimers are utilized within the entire responding population. this suggests that a heterogeneous population of double negative tcri-6 cells are involved in the response to ch12. college of kedicine at east tennessee state university, johnson city, tn 37614 interferon-producing (t 1) and interleukin 4 (il4) producing (t 2) clones were assayed for their ability to diregtly induce cytostatic activity in macro:hages generated from splenic myeloid precursors (m -c). in the presence, but not in the absence, of antigen, t 1 clones activated the m -c to inhibit the growth of p815 tumor cells in vitro. th2 cjlones were not able to activate such effector activity in the i4 -c. effectively present antigen to the t 2 clones as evidenced by the proliferation of t 2 cells cultured with antigen in the pfesence, but not in the absence, of m -c. thereyore, although both t 1 and t 2 were activated by cognate interaction with antigen presenting (ba) or nippostrongylus brasiliensis (nb). spleen cells from these mice were cloned at limiting dilution with alloantigen stimulation, and every two weeks, lk production in response to con a was measured. clones derived from, and stimulated with, cells from unimmunized mice initially tended to secrete low lk levels, with few clearly defined th1 or th2 clones. by 56 days after cloning, some clones had acquired th1 or th2 patterns. cfa, ba and nb-imnunized mice gave rise to clones that were mostly th1 or th2 even at early times. cfa and ba immunizations induced almost exclusively th1 clones, whereas nb induced more th2 clones. these results are consistent with a model in which resting, previously unstimulated t cells produce low amounts of lks, and progress through stage(s) where they secrete both th1 and th2 lks before finally differentiating into th1 and th2 cells. the results with cfa, ba and nb-primed mice suggest that this process occurs in vivo as well as in vitro. strains as carriers of melioidosis antigens to the immune system, deja tanphaichitra, mahidol university, p.o. box 4-217, bangkok 10400, thailand the attenuated gale mutant, salmonella typhi strain, tyzla, served as the recipient in a conjugal dna transfer experiment. conjugal dna transfer was obtained by the mating procedure on an appropriate blood agar medium. were examined serologically. one selected strain was found to have the serological characteristics of the recipient s . typhi, tyfla strain and also expressed the pseudomonas the donor strain was a pseudomonas pseudomallei mu107. the resulting antigen clones were repurified by restreaking on the medium and pseudomallei antigen. the s. typhi transconjugant strain is due to the presence of the pseudomonas pseudomallei plasmid. a group of subjects when received four doses of this bivalent vaccine strain in this study it appears that pseudomonas pseudomallei synthesis in developed antibodies against pseudomonas pseudomallei up to 70%. pseudomallei, an intracellular pathogen, produces a characteristic antigen probably to be plasmid coded, we considered that the gale salmonella typhi tyzla oral vaccine strain, highly effective against typhoid fever, might be modified so as to be protective also against melioidosis due to pseudomonas pseudomallei. terminal deoxynucleotidyl transferase (tdt) is a lymphoid-specific nuclear enzyme present in early lymphocytes. to investigate the regulation of tdt gene expression, pre-b and pre-t cells were treated with phorbol 12-myristate 13-acetate (pma) o r three analogs, and tdt steady-state mrna levels were determined by northern blot analysis. treatment of early lymphocytes with pma results in a rapid and reversible decline in steady-state tdt mrna levels within six hours. this rapid decline can be blocked by pretreatment of the cells with a protein kinase c inhibitor, implicating protein kinase c activation in the decline of tdt mrna. nuclear run-off studies demonstrate that tdt transcription is rapidly down-regulated within 45 minutes after pma treatment, indicating that this regulation occurs mainly at the level of transcription. furthermore, cycloheximide blocks the decline in tdt in rna showing that new protein synthesis is required for transcriptional inactivation. the nucleoprotein gene from the influenza virus a/nt/60/68 was stably cloned into the attenuated aroa-strain of salmonella typhimurium sl3261. nucleoprotein purified from pnp -3261 was tested for the ability to generate virus-specific immunity. immunization with recombinant derived nucleoprotein induged immunity to all type a influenza tested but not against type b viruses. cd4 helper t cells were primed but no evidence was found for priming of class i restricted ctc. mice immunized with recombinant nucleoprotein were protected against a subsequent challenge of influenza virus. the information obtained from the study of the immunity and protection generated by the purified recombinant protein was then used to design experiments to investigate the possibility of using the attenuated salmonella vector to deliver the nucleoprotein molecule to the immune system by the parenteral or enteral routes. we characterized the extrachrom-1 circular i n i s in 19-day-fetal and 4-week-old m u r i n e thpmcytes and 8-week-old m u r i n e splenocytes. f popllation of circular chias was clone3 into the kgtll phase vector. we screened ca. 10 tna cl-by plaque hybridizations with all far kirds of tcr gene probes derived from jal , val 0, db1, db2, jyl , j61 and 562 loci. cut of 10,000 cna cl-from fetal and 4-week-ld thymocytes, 30 hybridized with tcr aprobes and 5 hybridized with tcr &probes. positive cl-with tcr yand 6probes were 3 to 7 in fetal thymocyte4erived library, but few in 4-week-old thymocyte. of 7 fetal tcr 6 clcnes analyzed, 6 cl-had dd or vd reciprocal joints and 1 clcne had vd ar dd d i n g joint. relative frequencies of circular dna clones for four different tcr genes are consistent with the order of the expression of the genes the t cell developnent. of 10,000 tna clsignalling could be studied. llzmambxane signalling was maasured by 9 ability to t?z3nslocate fkc frcrm the cytcplasa to the nw2leus after surface i-a was banrl by a or p dxdn specific monoclcnal antibody. i(pmwing either 6 or 12 amino rids fmn the a chain cvtoplasnic (cy) damin did not affect the ability of tkse i-a r m l d e s to trarslocate pkc to the nucleus. normal splenic b c e l l s were rendered non-responsive t o subsequent challenge w i t h lps, as measured by a decreased a b i l i t y t o generate antibody forming c e l l s (afc), by incubation overnight (18-24 hours) w i t h 10 ug/ml a n t i -i g . both i n t a c t and f(ab)', a n t i -i g , as well as monoclonal anti-igm (bet2 and b-7-6) , were able t o induce 6 c e l l non-responsiveness t o subsequent lps challenge, suggesting t h a t sig/fcr i n t e r a c t i o n s are not necessary i n the induction o f lps non-responsiveness. i n contrast, induction o f nonresponsiveness t o subsequent challenge w i t h fitc-prucella abortus required i n t a c t a n t i -i g . the a b i l i t y o f mitogenic a n t i -i g (rab f(ab)', o r 6-7-6 northern blot analysis and bioassay data were used to analyze 9 separate lymphokines as well as the il-2 receptor (murine tac). northern blot comparison of fresh and primedt4 enriched rna revealed that primed t cells produced 10-fold more lymphokine than the fresh t cells. the only lymphokine that showed equal amounts of mrna for both fresh and primed t cells was il-2. a time course of fresh and primed t4+ cell lymphokine production was also analyzed. the primed cells produced a short burst of lymphokine mrna that peaked between 7.5 and 13 hr after con a stimulation and declined after 18 hr. the fresh t cells produced a longer burst of lymphokine mrna that peaked 18-44 hr after stimulation. the il-2 receptor @-2r) mrna time course from activated primed cells showed different kinetics than lymphokine mrna. this suggested that molecular regulation of the il-2r might be different than lymphokine regulation. to further examine molecular regulation in the primed t cells polysome profiles were evaluated for lymphokines, l 2 r , and other cellular genes. the recently developed method of gene amplification by the polymerase chain reaction (pcr) has proven to be particularly suited for the analysis of t cell receptor (tcr) genes. we adopted existing methods for the preparation of cytoplasmic rna from as little as 1000 cells and used this material as template for first strand c-dna synthesis. pcr amplification of this c-dna, using v-and c-specific oligonucleotide primers yielded enough material to produce single-stranded dna in a second pcr which could then be sequenced without cloning. in case of unknown v-usage, the pcr was employed for screening for v-beta elements by sequential reactions with different v-beta specific primers. we have used this method to reinvestigate the h-2b restricted cytotoxic t cell response to tnp in c57b116 mice. beta chain sequences of 26 ctl clones obtained by direct cloning of immune spleen cells were compared to sequences of 11 clones obtained by cloning of individual short-term in vitro ctl lines. it was found that a) in vitro bulk-stimulations reduced the heterogeneity of the beta-chain responses to tnp, b) similarities between different tcr-beta-chains concentrated on the usage of certain jb-elements ( jb2.6, 2.5, 2.1 ) rather than v-region or nid-region sequences, and c) the majority of jb2.6 containing beta-chains was associated with alpha-chains expressing v-segments of the val 0 family. these expression of genes which encode the t cell antigen receptor is cenval to the generation of the t cell repemire. our labomtory has been investigating genes for both the alpha and beta chains of this receptor in inbred strains of runus norvqicus (the laboratory rat), a species in which several autoimmune disease models have been developed. and which is used extensively in transplantation studies. using genomic southern blots and mouse probes specific for five different v a subfamilies, we have estimated the size of the v a repertoire in ten inbred strains of rat. results show a significant increase in the size of one subfamily and suggest increases in two others in all ten strains. the rat v a l subfamily has about twice as many members as the mouse, while the va2 and vu5 subfamilies, depending upon the enzyme used, show a similiar duplication. the va6 and va9 subfamilies have a comparable number of members in both species. these data are most easily explained by a single duplication event in the rat invoking at least one and perhaps three subfamilies, but not encompassing the entire v a locus. this implies that the val subfamily (perhaps together with va2 and va5 subfamilies) is regionally clustered and not interspersed with either the va6 or va9 subfamily. based on restriction fragment length polymorphisms, we find evidence for six distinct v a haplotypes in the ren strains tested. we have also cloned eight unique germline v a l gene segments. one of these has been sequenced. and has a coding region 87% identical to the most closely related mouse v a l sequence. this degree of relatedness is similiar to ra#nouse vg homologues. which share 8548% nucleotide sequence similarity. we are using these clones to generate angle copy probes from flankiig regions to further map the v a l locus. current approaches to mhc-peptide binding studies require either large quantities of highly purified mhc protein and/or the use of sophisticated detection apparatus. i n order to simplify detection of peptide-mhc interactions we have investigated the use of photosensitive-crosslinkers. two reagents have been successfully tested. a benzophenone derivative of peptide 1-16 from rat myelin basic protein (rmbp) was only effective after the introduction of a glycine spacer residue between peptide and crosslinker. an azido-nitro-benzoyl derivative of peptide 7.4, a heteroclitic analog of rmbp 1-11 (1). had a high affinity and bound specifically to the peptide binding site. the 7.4 photoaffinity probe has been used to test the binding properties of other analogues of rmbp 1-11 and is currently being used to define (a) the kinetics, (b) ph and (c) temperature dependence of the binding event. this particular photoaffinity conjugate retains both the mhc binding and biological properties of the original peptide and is helping us to define the roles of "determinant" versus "t cell repertoire" selection in the mhc linked autoimmune response to mbp the antigen-specific t cell repertoire is diverse in its ability to recognize a wide universe of foreign antigens. this t cell repertoire is composed of a set of clones each of which is specific for a given foreign antigen. therefore the precursor frequency of t cells specific for any give foreign antigen is extremely low. however, two prominent exceptions to this general rule exist, and these are the t cells present at high precursor frequency which are specific for foreign hhc products or for the products of the minor lymphocyte stimulatory (mls) genes in the mouse. the present studies were undertaken in order to examine factors involved in t cell repertoire formation by assessing the relationship between t cell repertoire for conventional foreign antigens and for mls products. studies indicate a striking degree of overlap between the set of t cells specific for pigeon cytochrome c and the set of t cells specific for mlsc gene products. demonstrate that the basis for this overlap lies in the predominant expression of one tcr vp gene, vbs, by those t cells which recognize mlsc. involvement of specific tcr afl dimers in recognition of mlsc and further suggest that t cell reactivity to these gene products may play an important role in establishing the t cell repertoire for foreign antigens. conclude that, rather than destruction of some essential apc structure, ecdi fixation prevents the apc from actively responding during the encounter with the t cell. this results in a failure to express new structures (probably located on the apc plasma membrane) that appear to be essential for stimulating t cell proliferation. these structures are distinct from ia or il1. the induction of these structures during t-apc interaction occurs in six hours, requires protein synthesis, and can be elicited by il1, il4 or lps, but not ifn-gamma. in the absence of these induced structures, the apc stimulates a partial t cell response, il4 release, but the t cells fail to proliferate. these induced structures on the apc may be either adhesion molecules that stabilize the t-apc interaction, or they may provide additional stimuli to the t cell. were not c m n t o the three s t r a i n s o f mice (balb/c, regions 1, 3, 5, 6 and 7; c3h/he, regions 2, 3, 5, 6, 6', 7 and 7'; and c57bl/6, regions 2, 4, 5, 6 and 7). immunisation with type i1 collagen (cii) leads to development of arthritis in mice with certain mhc haplotypes and is associated with an immune response against cii. we have been studying the t-cell response in the arthritis susceptible strain dbm1 (h-2q) . analysing the proliferative response in cultures of lymph node cells from immunised mice a s well as t-cell lines and clones established from such cultures it was found t h a t li the t-cell response after immunisation with heterologous cii was preferentially directed against foreign determinants on the cii molecule with little o r no crossreactivity against autologous cii. 2 ' both the primary response and the reactivity of established lines and clones were directed against the cbll fragment of the cii molecule, using c b l l fragments prepared from chick, bovine or rat cii. 3/ pepsin present in cii preparations after using pepsin digestion for solubilisation of the collagen is strongly immunogenic even in very small amounts and it was therefore necessary to use cii prepared from lathyritic cartilage without pepsin digestion for immunisation. in contrast to the pattern in lymph node cultures from immunised mice we found that when culturing spleen cells from unimmunised mice there was a t-cell response against collagen that was preferentially directed against autologus cii. since we earlier have found that autologus cii may induce an immune response and also arthritis in dbn1 mice we conclude that there exist t-cells capable of reacting with autologus collagen and inducing an immune response as well as arthritis but that these cells are under regulation so that they not readily can be activated into proliferation but may be induced to perform certain effector functions. tested. the characterization of these two cd6 rdsas will be presented. analysis of hla polymorphism using sequence specific oligonucleotide probe hybridization to amplified dna, lee ann baxter-lowe, jay b . hunter, and jack gorski, the blood center of southeastern wisconsin, milwaukee, wisconsin 5 3 2 3 3 . hla polymorphism plays a key role in antigen:mhc interaction. the polymorphism of the first domain encoding exon of the hla-dr p chain has been studied by in vitro dna amplification and use of sequence specific oligonucleotide probe hybridization (ssoph) to detect polymorphic sequences. a 230 bp segment of genomic dna was amplified and hybridized with synthetic oligonucleotide probes (12-19 bases) under conditions that detect single base pair mismatches. identification of these mismatches can be used to predict micropolymorphism in the protein products, including single amino acid changes. haplotype specific patterns of oligonucleotide probe hybridization were defined for a panel of homozygous typing cells. analysis of family data demonstrated the expected inheritance patterns. most known serological specificities are encoded by multiple allelic forms of dr p chains and ssoph can identify these differences. this was exemplified by detection of unique ssoph profiles for subtypes of dr4, drw6 and drw52 alleles. this procedure was also used for analysis of hla-dr polymorphism in large numbers of heterozygous individuals, including an hla-deficient scid patient. the ssoph data were correlated with serological specificities and will be useful for delineation of hla restriction in alloand autoimmunity. different cell membrane receptors have been shown to be involved in human t lymphocyte activation induced by either monoclonal antibodies or mitogenic lectins. these t cell surface molecules can be divided into two categories : a) the t cell antigen receptor (tcr) associated with the non-polymorphic cd3 antigen b) t cell differentiation molecules not linked to cd3/ti such as cd2 (t11) and tp44 (9.3). monoclonal antibodies directed against these t cell surface structures triggered different t lymphocytes functions : mitogenesis, il-2 receptor expression, il-2 secretion. our knowledge about early events involved in t cell membrane activation is not complete, especially involving the transduction mechanism mediated by gtp-binding proteins ; nevertheless, numerous authors have demonstrated that cd3/ti complex triggering induces the activation of phospholipase c, leading to the phosphoinositide cascade associated with an increase of free cytoplasmic calcium ions. in the present report, we show that different activating cell molecules (con a , pha and pma) can trigger oxygen free radical liberation when incubated with the human jurkat tumor t cell line. since membrane oxidative metabolism has been shown to be related to the stimulation of the phospholipase a2, and to be the final consequence of a membrane nadphoxidase : this could represent a previously undescribed pathway of t lymphocyte activation. high affinity monoclonal antibodies (mab), specific for staphylococcal nuclease (nase), were produced and characterized. competitive inhibition assays were conducted resulting in a series of complementation groups that define eight overlapping epitopes. it is estimated that these epitopes account for 70% or more of the accessible surface of nase. mutagenesis of the coding sequences for nase was carried out to produce a series of variant molecules (each differing from wild-type. nase and from each other by a single amino acid) that will enable mapping of nase epitopes, determination of residues involved in antibody binding, and the contribution of various physical and chemical factors to affinity and fine specificity. screening some of these mutants with the panel of mab enabled us to map several nonoverlapping epitopes and further subdivided some of the mab complementation groups. oligonucleotide-directed mismatch mutagenesis has been done on codons encoding the original amino acid residue and other surface residues in its immediate vicinity. determination of enzyme activity and structural analysis by cd spectropolarimetry of several of the mutant proteins suggests that any structural changes that may occur are local and not global. supported by grants ai20745, l32ca09109 and s07rr05431 from the national institutes of health. activation of t cell proliferation is believed to occur via the hydrolysis of inositol phos holipids, which, through the second messengers inositol-1,4,5-tris hosphate and diacylglycerof(dag), promotes the elevation of intracellular calcium levels anjactivation of protein kinase c (pkc), respectively. the role of pkc in t cell activation was investigated by comparing the effects of stimulation by 12-0-tetradecanoyl phorbol acetate (tpa), and the dag, oleoylacetyl glycerol (oag), on a >99% pure population of t cells cultured in rpmi 1640 medium containing 10% autologous serum. treatment with either tpa or oag caused down-regulation of the t cell rece tor, a consequence of its hosphorylation, but only tpa, in syner leuiin 2 receptor (il2-r), expression and, sgsequently, proliferation. immunohistochemical staining with antisera specific for the pkc subspecies a, pi, pii and 7 shows that restin t cells express a, pi and pii pkc subspecies which are diffusely distributed throughout the celt. after 20 minutes treatment with either oag or tpa all three subspecies are redistributed to a focal area within the cell. the redistribution is transient in oag stimulated cells, where the pkc distribution is similar to that in untreated cells after 1 hour of treatment. in tpa stimulated cells, however, the pkc redistribution is prolonged, becoming more marked until mitosis occurs after 48-72 hours of treatment. these results suggest that transient intracellular redistribution ofpkc causes phosphorylation and down-regulation of the t cell receptor, but that prolonged redistribution is required or t cell proliferation. sm is a nucleoprotein complex associated with small rna molecules in eukaryotic cells. the spontaneous generation of anti-sm antibodies is specific for patients with systemic lupus erythematosus (sle) and develops in 2 5 % of mrl mice. the response has been shown to be t-cell dependent in mrl/lpr mice. t-cells specific for sm are found only in mrl (h-2k) mice and mice bearing h-2s and h-2f haplotypes (which do not develop anti-sm antibodies). we are currently working to define the variable regions of the t-cell receptor genes used in the sm response. a series of t cell hybridomas from mrl mice has been generated and are being screened for sm positivity. a technique has been designed to amplify specific alpha and beta chain tcr genes using the polymerase chain reaction allowing for a more rapid sequence analysis. it is also our intention to locate the sm specific epitopes of the t-cell hybridomas. d. bloom, p.l. cohen, and s.h. clarke, department medical institute and experimental immunology branch, nci. nih, bethesda. md 20892. to determine whether prior activation history affects t cell receptor mediated activation of t cell clones, the murine type i helper clone ae7 was maintained in tissue culture by stimulation every ten days with either (1) antigen (cytochrome c), irradiated h-zk spleen cells. and il-2 or (2) il-2 alone. ae7 cells grown with antigen and antigen presenting cells (ae7-ag) proliferated and produced t cell growth factor activity (tcgf) in its culture supernatants following stimulation with immobilized anti-t3 antibody. the tcgf activity was shown by bioassay using indicator cell lines and specific blocking antibodies to be almost entirely due to gm-csf with little or no il-2 activity detectable. detectable il-2 mrna levels. (ae7-ilz) displayed substantially greater anti-t3 induced proliferation than did ae7-ag cells. in contrast to ae7-ag cells, ae7-il2 cells produced large quantities of il-2 in response to anti-t3 stimulation. furthermore. one cycle of stimulation of clone ae7-ag with il-2 in the absence of antigen and irradiated spleen cells was sufficient to cause this clone to produce substantial amounts of il-2 upon subsequent anti-t3 stimulation. these data suggest that t cell receptor mediated stimulation of t cell clones by specific antigen and antigen presenting cells inhibits subsequent anti-t3 induced il-2 production. t cell proliferative responses and sera antibody levels of myasthenic patients to several synthetic peptides representing different epitopes of the human achr were examined. we detected significant differences in the humoral and cellular responses of mg patients compared to healthy controls to peptides of the human achr alpha-subunit with sequences p195-212, p257-269 and ~310-327. proliferative responses of lymphocytes from myasthenic patients to p195-212 and to p257-260 correlated significantly with hla-dr5 and with hla-dr3, respectively. in order t o investigate further the immune responsiveness to selected sequences of the human achr, t cell lines and clones specific for peptides p195-212 and p259-271 were established from lymph nodes of c3h.sw mice. the recognition specificities of these lines were tested by examining crossreactivity to a series of shortened and/or extended peptides of the above sequences. deletions of amino acids in positions 211 and 212 (211=p, 212=l) resulted in a decrease of the peptides' stimulatory activity on the ~195-212 specific t cell line, whereas deletion up to position 200 on the n-terminal end had no effect on the triggering potential of the peptides. similar results were obtained when deleting residues 270 and 271 (270=v, 271=p) in stimulation assays of the p250-271 specific t cell line. help in determining important t cell epitopes on the human achr. the role of guanine nucleotide binding regulatory proteins (g proteins) in the regulation of phosphorylation of the y subunit of the cd3 antigen has been examined. cd3 y chain phosphorylation in isolated t cell microsomes or permeablised t cells was stimulated by the g protein activator, guanosine 5'-0 thiotriphosphate (gtpys), but other nucleotides such as camp or gdpbs were ineffective. dependent. these data are consistent with the involvement of a g protein in the signalling mechanisms that regulate the phosphorylation of the cd3 y chain. the regulatory effects of calcium and gtpys were compared in normal peripheral blood derived t cells and jurkat cells. there were differences regarding g protein regulation of cd3 y chain phosphorylation in normal t cell and jurkat cells and current models explaining these differences will be described. expression of the gamma-delta t cell receptor has been thought to first occur in a population of thymocytes shortly after their precursors populate the thymus between 11 and 13 days of gestation. in the course of our studies investigating the ontogeny of t cell receptor expression in the mouse embryo we have identified an extrathymic site of gammadelta expression in a population of cells present at distinct times of gestation. evidence will be presented demonstrating two periods of activity of the murine gamma locus in the developing embryo. are colonizing the thymus from the liver and the gene segment useage detected is different to that first expressed in cells of the developing thymus. around the time of birth) involves the functional rearrangement and expression of a gamma gene segment corresponding to the initial functional rearrangements detected earlier in gestation in the thymus, which can occur independently of thymic influence. demonstrate a new site of gamma-delta receptor expression in the liver of newborn mice that can occur in the absence of any thymic influence. the primary (in vivo) response of cs7bl/6 animals to the class i antigen qa-1 is a helper (th) dependent event as indicated by the requirement for copriming with a distinct antigen capable of activating helper cells. in contrast, the secondary (in vitro) response to qa-1 demonstrates no need for costimulation with the helper antigen. in attempts to more closely examine the helper requirements for activation of primed ctlp, we have observed that depletion of l3t4 cells from spleens of qa-1 primed mice abrogates the in vitro generation of anti-qa-1 effectors. the response is restored by the addition of concanavalin a induced supernatant (cas) or by the addition of syngeneic but not qa-1 allogeneic l3t4 cells. indeed, even in the presence of cas, l3t4 cells expressing the qa-1 alloantigen specifically suppress the activation of anti-qa-1 ctl in a manner reminiscent of that seen with lyt-2 veto cells. although the mechanism whereby l3t4 cells exert suppression is unclear, we have determined that ctlp are susceptible to veto only within approximately the first 48 hours of culture, after which they resist suppression. results from further studies of the nature of suppression and the l3t4 veto cell will be presented. group i1 proteins induce ige ab responses in -90% of mite allergic patients. murine mab and human igg and ige ab. unrelated. crossreactive epitopes on gpi and gpii allergens from different mite species. in contrast, igg ab in balb/c mice immunized with loug specific" epitopes and <1% was a n t i -u i (a gpi homologue, with -80% amino acid sequence homology to i). four non-over lapping epitopes were defined by mab, with one species specific immunodominant site on each gpi allergen. cross-reactive gpi epitope and this mab could inhibit human ige ab binding by -40%. specificity of the murine anti-gpi response was not h-2 restricted, but could be altered by immunizing balb/c mice with lower ag doses (lug) in alum or 8 . dertussis. using these regimes, up to 52% of the murine igg ab responses was gpi cross-reactive. responses to gpii allergens appear to be strain dependant. unresponsive to gpii. however, balb/b, a/j, cba, c3h 6 c57b16 all produce gpii cross reactive igg ab. anitgenic sites on gpi allergens are conformational, whereas those on gpii may be sequential. known to affect ige expression in mice may also affect the epitope specificity of igg ab. we have compared the b cell epitopes on these allergens using panels of however, ag binding ria on 73 sera showed that human igg and ige ab recognize the gpi and gpii allergens are antigenically i in cfa was directed against "species murine ab balb/c are completely thermal denaturation and reduction and alkylation expts suggest that the results with the gpi allergens suggest that immunization regimes which are c425 this report demonstrates for the the exclusive recovery of 72-84-specific t cell clones c 426 further molecular analysis should identify and characterize achr reactive autoimmune clones and/or suppressor cells. cohplex, mogens h. claesson, p e t e r bkams a n d s t e e n d i s s i n g , l a b . e x p . h e m a t . immunol., d e p t . med. anatomy a, a n d d e p t . g e n e r a l p h y s the ly-6 alloantigens represent a family of phosphatidylinositol anchored proteins that function as accessory molecules in the process of t lymphocyte activation. the expression of these alloantigens is often induced on t and b lymphocytes after activation by mitogens or antigens. previous studies have shown that the induction of ly-6 alloantigens in t cells is at least in part due to the action of ifn-a/b or ifn-7. in the present study, we have demonstrated that ifn-7 also induced ly-6 molecules on b lymphocytes and bone marrow cells. furthermore, we now show that tnf also participates in the induction of at least one of the ly-6 proteins, ly-6a/e. tnf was found to synergize with ifn-7 to induce ly-6a/e expression in thymocytes, t lymphocytes, and bone marrow cells, but not b cells. for t lymphocytes, the synergistic induction of ly-6a/e by tnf was restricted to cells from the ly-6.1 haplotype whereas ifn-7 was sufficient to fully induce ly-6a/e expression in cells from the ly-6.2 haplotype. this result is consistent with the notion that there is more complex regulation of the ly-6afe molecules in t cells obtained from the ly-6.1 haplotype. for t cells from balb/c (ly-6.1) mice, ly-6a/e, but not ly-cc, molecules were induced by ifn-7 and tnf. furthermore, when compared to ly-6a/e, the regulation of mhc class 1 molecules in these t cells by tnf was minimal. the induction of ly-6afe molecules on balbfc t cells resulted in an enhanced capacity to activate these cells through the ly-6 t cell activation pathway. one transformed t cell line, 5.1.2. was also identified whose ly-6a /e molecules were synergistically induced by ifn-7 and tnf. optimal expression of ly-6a/e molecules on 5.1.2 cells required continuous culture of this cell line with these two cytokines and resulted in the detection of optimal levels of cytoplasmic ly-6afe mrna by northern blot analysis. this latter result suggests that ifn-7 and tnf regulate ly-6a /e at the level of transcription and/or mrna stabilization. ut southwestern medical center, dallas, texas 75235. an igm antlcd3 mab (38.1) was found to modulate cell surface cd3 on highly purified human t cells within 5 hours in the absence of a secondary antibody or accessory cells. inhibition could be overcome with accessory cells or il2. the inhibitory effects of 38.1 could be mimicked by briefly pulsing cells with the calcium ionophore, ionomycin. 38.1 or ionomycin pulsed cells were inhibited in their subsequent capacity to resp nd to pha even when exposures were carried out in the presence of egta to prevent increases in [cap*]. from extracellular sources. inhibition was not the result of an inability to respond to pha 'by increasing [ca2+] .. moreover the newly expressed cd3 molecules were capable of generating increases in [ca2*].' after reacting the cells with anti-cd3 + a cross-linking secondary antibody. these studies dem'onstrate that a state of nonresponsiveness in resting t cells can be induced by modylating cd3 with an anti-cd3 mab in the absence of co-stimulatory signals. a brief increase in [ca ' 1. resulting from the mobilization of intracellular calcium stores appears to be sufficient to induce'this state of t cell nonresponsiveness. cd3. laurie s. davis, mary c. wacholtz, and peter e. lipsky, dept. of internal medicine, lmmunogenicity c a central lab.blood transf.service, lab. of exp. and clin.immunology of the univ. of amsterdam, amsterdam. the netherlands monoclonal antibodies (mab) directed against the human cd3 molecular complex induce a strong proliferation of t cells, when immobilized on microtiter wells. this activation system, that was shown to be independent of accessory cells, accessory-cell derived factors or lfa-1 mediated intercellular adhesion (1). allows one to study the requirements for t-cell proliferation and differentiation in a well defined manner. il-2 and ifn-gamma but no il-4 could be detected i n culture supernatants of coated anti-cd3 stimulated t cells. the addition of ril-1 or ril-2 had only a moderate effect on t-cell proliferation, vhereas helper activity for ig production was strongly enhanced in the presence of these factors. in this system differentiation of precursors to cytotoxic t lymphocytes (ctl), as measured in anti-cd3mediated cytotoxicity, could be demonstrated within 2 days after initiation of the activation. allospecificity of the induced effector ctl was demonstrated using a panel of hla class-i p815-transfectants. in this system the regulatory role of the cd28 molecule in tcell activation and differentiation was studied. addition of anti-cdz8 mab to t cells stimulated with coated anti-cd3 mab enhanced il-2 production, proliferation as well as ig production. interestingly, pctl differentiation was also enhanced by anti-cd28 mab. this system seems valuable for the analysis of requirements for differentiation of human t cells subsets. 1. van noesel et al., nature 333, 850-852, 1988 analysis of the requirements for human t-cell differentiation, rolien de jong. vivienne rebel, g i j s van seventer. miranda brouwer, frank miedema, rene' van lier, the newly described t cell receptor (tcr) 6 locus is located inside the tcr a locus between va and ja . despite this unique situation, a highly efficient regulatory mechanism results in the complete independence of these two loci. we have recently described, in humans, a site specific recombination which joins a 5' deleting element (srec) to the send of the ja's (yja) resulting in the deletion of the tcr-6 locus in t lymphocytes expressing the a/b tcr. rearrangements of the tcr as well as immunoglobulin genes are mediated by a unique recombination machinery and therefore, the specificity of these rearrangements is thought to be the result of a differential accessibility of the dna involved in the recombination process. as a consequence (and/or cause) of the opening of a segment of dna, the region involved is fxst transcribed as a sterile transcript prior to the rearrangement. in that regard, we have found that the 2 kb of dna u p s a u~n of yja are actively transcribed ('t early a" transcript, tea) early during fetal development. the presence of the tea transcript presumably reflects the opening of the tea sequence prior to the tcr-6 deletional rearrangement. in order to better understand the mechanisms involved in the dna accessibility model, we started to look for dna-binding proteins which might play a putative role in the opening or blocking of the tea sequence. by the technique of "gel shift assay" we found such a negative regulatory protein in the nuclear extract from a non-lymphoid cell. the binding activity appeared to be specific as it was competed out by an excess of unlabeled autologous dna and not by an excess of irrelevant dna. further studies are now in progress to determine first wether the presence of this binding activity can be correlated with a "closed" configuration of the tea region and second to determine the precise location of the dna binding region. cohen, laboratory of chemical biology, niddk, national institutes of health, bethesda, md 20892. mabs retard lymphoproliferation as well as autoimmunity. interesting, so does the adminisuation of a mab to l3t4 , thus suggesting that the t helper subset, which is not part of the unusual expanding population, is required for initiating the pathology in these animals. as a means of characterizing the expanding population of abnormal cells as well as the phenotypically mature (l3t4+) cells that may be associated with them, i have generated a series of t cell hybridomas from the enlarged lymph nodes and spleen of m p r mice. in parallel, i have derived a series of control (non-lprflpr) hybridomas from mrulpr x balb/c f1 animals (which show no sign of pathology), and a series from mrl, mice (which have a delayed onset of autoimmunity without lymphadenopathy). very few hybridomas (20-30) were obtained in the non-lpr derived fusions. when i con a stimulated the lymphocytes from non-lpr mice prior to fusion however, many more hybridomas were obtained(l20-220). this is in contrast to the fusion efficiency obtained from lprnpr mice which did not require in viuo lymphocyte stimulation to obtain a comparable number of hybrids. this result suggests that the m u p r lymphocytes are activated in situ. in addition, while less than 15% of the lymphoid mass is comprised of t helper (l3t4+) cells , over 50% of the hybrids are l3t4+. the fact that a dispropomonate number of t helper cells are rescued by fusion suggests that the cells activated in situ may be autoreactive t helper cells. currently i am characterizing these t helper cells for their lymphokine production, t cell receptor gene usage and auto-specificity and will compare them to the hybridomas obtained from non-diseased animals. goodnow, s. gilfillan, h-j. garchon, j. erikson and m. davis. stanford university, stanford, ca 94305. we have made transgenic mice bearing gene constructs encoding the t cell receptor a and p chains from a cytochrome c-reactive t cell hybridoma. despite a lack of tissue-specificity in mrna expression, cell surface expression of uansgene-encoded protein was limited to t cells, presumably because both chains require cd3 proteins in order to assemble on the cell surface. in mice carrying only the a chain consuuct, the transgene was expressed in the thymus as early as day 15 of fetal life, 1-2 days before endogenous a chain mrna. the first detectable cell surface expression of a transgene was on 25% of day 15 fetal thymocytes. this vast increase in up-bearing cells in fetal thymus was due to pairing of transgenic a chains with endogenous p chains, of which a substantial number are. normally rearranged by day 15 of fetal liie. the balance between ap-expressing t cell supopulations was grossly disturbed in these mice, the most marked abnormality being an increase in the number of l3tnyt-2-cells both in thymus and in peripheral lymphoid organs. it therefore appears that premature expression of surface ap t cell receptor may disturb t cell differentiation pathways by allowing t cells to leave the thymus without expressing l3t4 or lyt-2. mice carrying the p construct showed no increase in surface expression of t cell receptor in fetal life, since endogenous a chain rearrangement was limiting. in mice carrying either the a or p chain mansgenes, the number and surface phenotype of t cells expressing y& t cell receptors was unaffected in early fetal liie. suggesting that the a p and y5 t cell lineages diverge before thc rearrangement and expression of the appropriate subset of t cell receptor genes. department of microbiology and immunology, institute and the university of pennsylvania, philadelphia, pa. 19104.the thymic stroma plays a major role in initiating the colonization, organization and differentiation of precursor stem cells into functionally mature t cells. a variety of cell types including thymic nurse cells, cortical and medullary epithelial cells, nonepithelial dendritic cells, and macrophages, combine to form the thymic stroma. the differential role of such cells in thymic development is unclear. we have isolated a number of morphologically distinct stromal cell lines from the thymuses of sv40 transgenic mice. several of the cell lines are of epithelial origin, while others have features consistent with non-epithelial "dendritic" cells. we have focused on one of these cell lines, bearing the phenotype of a cortical epithelial cell, for its ability to support the growth and differentiation of stem cells from the fetal liver and fetal thymus, and cloned pre-t cells obtained from adult mice. the cortical epithelial cell line produces factors that induce the dramatic proliferation of fetal liver and thymic stem cells . in addition, fetal liver cells cocultured with this cell line are induced to rearrange and express their t cell receptor (tcr) genes. a cloned pre-t cell line is also induced to rearrange its tcr oenes in resoonse to sianals mediated bv this cell line. gugrin, marie c. b6n6, corinne amiel, nadia coniglio, jacques leclsre, laboratoire d'immunologie and clinique endocrinologique, chu de nancy and facult6 de mgdecine, 54500 vandoeuvre les nancy, france. the lfal molecule, an adhesin of the lfa family involved in cell-cell interactions, is physiologically expressed on all white blood cells. it is absent in some congenital immune deficiencies ((id), and is expressed on a decreased number of peripheral blood lymphocytes (pbl) in aids. we investigated its presence on pbl from 60 patients with auto-immune disorders of the thyroid. a monoclonal antibody (iot16, immunotech) directed to a conformational epitope involving both chains of lfal was used in indirect immmunofluorescence on pbl from blood drawn at a similar time in all patients. a calibrated flow cytometer (epics profile, coultronics) was used to measure the percentage and numbers of positive cells, as well as the mean fluorescence (mf) and shape of the fluorescent peak. data were correlated with clinical information,therapeutic, and other pbl features such as the cd4icdb ratio. the percentage of lfa1+ cells was significantly decreased in patients with graves' disease, hypothyroidism and hyperthyroidism. the mf was lower and the shape of the fluorescent peak seldom displayed the bimodal characteristic noted in controls. these data suggest the participation of the altered expression of lfal in the pathogenesis or evolution of auto-immune diseases. pt=ciilat.pd that excess hla r l a s s tt expression, ciinmonly found i n a i t i v e human nutoinimiine tlisrases maintdin:? the *rctivatinn of dutnreactivr t c e l l s which in turn prnducr mediators which maintain r.la:;s i t expression. this hypothesis has been tested i n many ways i n t h y r o i d i t i s . crj t i c a l l y autoreactive t cell:? are â�¬nilrid i n thyroid autoinnline tissues which a r e rrstimnlatrd hy thyroid f o l l j c u l a r r r l l s . more rrcently we have been exploring the s p e c i f i c i t y of the autoantigen reactive t c e l l s in hashinoto's t h y r o i d i t i s where thyroy-lobulin s p e c i f i c clones have been found, i n contrast t o graves' disease, where thyrocyte recognizing clones do not react wi.th tliyroglnhulin. tn rheumatoid a r t h r i t i s , collagen type i1 clones have been found, persistently in the activated (il-2r') t c r l l pmil over several years i n t.he same p a t i e n t . to verify t h a t antigen present,ation is involved i n rhrumatoid art.hritis ( r a ) a disease i n which, unlike thyroi.dj t i s the nature of the major antigen presenting c e l l (apc) is unknown, thc e f f e c t of ,~nticl.ass i1 dntibodies a t 1-oncentration which block *ari;j vation of t r-el is mi the synthesis n â�¬ rla-dr mrna wa:j waluat.rd. the inhibitory effect supports d i.rifira1 role nf an d s yet iiriknown apc i n maintaining the i:hronj.ci t y o f r a conversely, all the clones were unable to respond to a substitution at 91 (tyr to asp). nase mutant proteins were constructed with the same single amino acid substitution and t cell responses to peptides and mutants were compared. preliminary evidence suggests that the mutant proteins like the peptides, substituted at residue 88 and 91, will not induce t cell clone responsiveness. these data suggest that the overall structure of the protein will not compensate for the lose of a particular amino acid which is necessary for t cell recognition. medicine, baltimore, md 21201. to explore the variables important in t cell priming, an adjuvantfree immunization regimen was developed. bio.a mice were primed subcutaneously with syngeneic spleen cells that had been pulsed with high concentrations (100pm) of the peptide 81-104, a cnbr cleavage fragment of pigeon cytochrome e. the t cell response was assessed using a sensitive limiting dilution assay that measures lymphokine production with the ctl-l cell line. the precursor frequency of antigen-specific cells in the draining lymph nodes of mice primed with antigen-pulsed spleen (aps) was 1 in 4000, indistinguishable from the frequency of 1 in 3400 found in mice primed in the footpads with 10 nmol of 81-104 in complete freund's adjuvant (cfa) (data are given as geometric means, n=5, s.e.m = x/t 1.7 and 1.3, respectively). despite the apparent similarity in the t cell compartment of mice primed using these different regimens, antibody induction was strikingly different. mice primed with 81-104 in cfa developed serum igm and igg responses against the peptide, with antibody detectable in an ellsa assay at a 1 :3000 dilution. mice primed with 81 -1 owaps, however, produced no detectable anti-peptide antibodies. maximal t cell clonal expansion therefore appears to be possible in the absence of antigenspecific b cells. these data argue against the hypothesis that antigen-specific b cells play an obligate role in t cell proliferation in vivo. the reasons for the lack of antibody induction are currently under investigation. cell receptor (tcr) complex of jurkat cells. the coprecipitation of these peptides with tcr requires treatment with monoclonal antibodies (mabs) directed against tcr (c305 or r140) prior to cell lysis and immunoprecipitation. treatment of jurkat cells with mabs directed against cd2 (9-1 or 9.6) or hla (w632) does not not induce the association of these peptides with tcr. the signal-transduction mutant cell lines, j.cam1 and j.cam2, have previously been described (1,z). these cell lines, derived from jurkat, fail to activate the inositol-phopholipid second-messenger pathway in response to anti-tcr mabs. treatment with mab c305 induces the association of the 35 and 39 kd peptides with tcr in j.cam2 cells but not in j.cam1. j.cam1 modulates tcr normally in response to anti-tcr mab treatment (1). hence, these observations suggest that the two peptides are involved in the signal-transduction pathway of the t cell receptor complex rather than receptor internalization. sle is an autoimmune disorder associated with several different hla class i1 antigens. we studied a large sle patient population by sequencing of the pcr amplified first domain of the dqb and dqa chains and by sequence specific oligonucleotide probes to further define these associations. shared dqb sequences at amino acid positions 26=1eu, 30=tyr, and 57fasp may predispose some individuals with hla dr1,2,4, or 6 to develop sle. a novel dqb sequence found in two drw6 dqwl sle patients shares these amino acids in the dqb hypervariable regions. the association of the drz dqwl.azh gene was greatly increased in the sle patients with lupus renal disease. the hla association may be directly due to structural aspects of the hla genes. when parent -+ f1 chimeras are prepared with supralethal irradiation (1300 rad + 900 rad), the donor-derived cd4+ cells differentiating in the chimeras show partial tolerance to host-type h-2 determinants, despite the apparent absence of host-type apc. donor-derived cd4+ cells give only low proliferative responses to host-type apc in primary mixedlymphocyte reactions (mlr); furthermore, in i-e-+ i-e+ combinations, the donor cd4+ cells show molecules. this finding implies that tolerance is induced intrathymically, presumably through contact with a non-marrow-derived component of the thymus, e.g. epithelial cells. in support of this possibility, thymectomized & + ( a x b)f1 chimeras given strain j? marrow cells and a strain thymus graft (irradiated) show no detectable tolerance to host-type strain b determinants: the strain & cd4+ cells differentiating in these chimeras give strong mlr to strain b and do not show deletion of v 11+ cells. = 70% deletion of cd4+ cells expressing i-e-reactive v 11 t cell receptor b measured by these two parameters applies not only to lymph node (ln) cd4+ also to cd4+ cells recovered from the thymus. interphotoreceptor retinoid-binding protein) is a glycoprotein of 1264 residues (bovine) which localizes in the retina and pineal gland and induces inflammatory changes in these organs (eau and eap, respectively) in immunized animals. the experimental disease is considered a model for certain uveitic condiiions in man. we have recently shown that irbpderived synthetic peptides can also induce eau/eap in lewis rats. the present study compared two such peptides, "r4" (residues 1158-1 180) and "r14" (1 169-1 191). peptide r14 was found to be immunodominant, shown by its being recognized by lymph node cells (lnc) or line cells sensitized against whole irbp. in contrast, peptide r4 was not recognized by the whole irbp-specific lymphocytes and is considered nondominant. in addition, lnc sensitized to r14, but not to r4, responded to intact irbp. r14 was superior to r4 in producing eau/eap and cellular immunity (minimal doses: 0.06 vs 67 pg/rat). on the other hand, the two peptides were comparable in their capacity to stimulate presensitized lymphocytes. moreover, lnc sensitized against r4 were similar to those sensitized against r14 in their capacity to adoptively transfer eau/eap to naive recipients. this study thus provides a unique system in which both immunodominant and nondominant peptides produce autoimmune disease and can be compared for their immunological features. the age-related diminution in immune responsiveness has been shown to result from increased regulatory mechanisms and not from a paucity of immunobgical recruitment (aging: immunology and infectious disease 1,47, 1988). we present evidence based on "libraries" of monoclonal antibodies (mabs) omained from young and aged donors that there occurs with aging an increase in autoimmunity which is possibly the result of the accumulation of liielong "original antigenic sins". the resultant increased connectivity of the immune system is represented by mabs obtained from aged donors which are multiply anti-self cross-reactive. furthermore increased connectivii is supported by the evidence that anti-2.4,6-trinitrophenyl mabs are ad8 positive, 2d3 positive as determined by i n h b i i n studies using mabs anti-idiitypic reagents. analysis of the vh and vk region genes utilized by these mabs indicate a nonrandom gene usage. life bng stochastic immunological events lead to a pattern of cross-reactiities and non-random usage of vh genes. these immnological events lead to the emergence of the patterns which are partially elucidated by the data presented. these patterns mimick those seen early in ontogeny, but indicate a possible convergence to an ever-increasing connectance of the idiitypic repertoire expression. in other words, life-long immunological experiences contribute to a down-regulation resulting in both paucity of drimaw immune reswnses and an increase in autoimrmnity which are both the earmarks of immunity in aging. (supported by usphs grants ag-04042 to eag and al 18316 to cab) lmmunogenicity c449 stimulation of these cell lines in suspension with saturating levels of mab okt3 produces total and fractional inositol phosphate accumulation linearly related to receptor number, (r > 0.9 ). this technique also allows an approximation of the minimal number of reccptors which must be engaged for second messenger generation in this system, which we estimate as 6.5~102 receptors per cell. or terminate t cell activation. since this molecule plays an important role in human t cell development we sought to identify the murine homologue of cd28 in order to determine its expression on murine t cell and its role in activation. we have used a human cd28 cdna clone to isolate a full length cdna encoding the murine equivalent of cd28 from an el4 t cell lymphoma library. this clone shows similar domain organization and a high degree of homology to the human cd28 molecule. the murine cdna clone has been used to examine mrna expression of cd28 in normal and activated murine t cells, and in various t cell tumors. peptides generated from the translated sequence will be used to produce antisera to correlate the surface expression of cd28 with mrna expression, and to biochemically and functionally characterize this molecule. pat happ and ed palmer, basic sciences division, dept. of pediatrics, national jewish center for immunology and respiratory medicine, denver, co 80206. we have attempted to determine the frequency of rearrangement and expression of the individual a and g chain v gene segments that make up an unselected, untolerized t cell repertoire. in order to do this we generated over 500 t cell hybridomas from freshly-isolated thymocytes of newborn c57blllo mice and subjected rna from these hybrids to northern dot blot analysis using 11 va, 16 vp. cy and c6 probes. comparison of the expressed repertoire of vp gene segments in this newborn thymocyle population with similar data previously generated from an adult peripheral t cell population reveals two vg genes, vp12 and vpl5, whose expression is decreased in the periphery, possibly due to the effects of tolerance. two additional vp gene segments were expressed more frequently in the peripheral population than in the newborn thymus, vp5 (4.5 times higher in the periphery) and val0 (2 times higher). it is possible that these represent ewo instances of positive selection of t cells which is determined primarily by the receptor's vg gene segment. va gene segments were expressed in only 15% of newborn thymocyte hybridomas (compared to 58% expressing vp) and determination of va rearrangement frequencies was complicated by the unexpectedly large number (67%) of hybrids expressing cs mrna. further examination revealed that several va gene probes were actually detecting rearrangements to cs. the most notable of these was va7, which accounted for approximately 34% of the expressed va repertoire but was rearranged exclusively to cs. barbara bergman, brenda bradley, kevin lafferty and mary portas. barbara davis center for childhood diabetes, u. colo. health sci. ctr., denver, co 80262. we have produced a panel of islet-specific t cell clones by culturing lymphoid cells obtained from non-obese diabetic (nod) mice in the presence of nod islet cell antigen and antigen-presenting cells (apc). these clones were selected to the panel on the basis of (a) their antigen-specific reactivity to islet cells and apc in an in vitro proliferation assay and (b) their ability to mediate islet graft rejection in vivo in a tissue-specific manner. we have further characterized these lines for cell surface phenotype, 11-2 production, and proliferative response to non-nod islet antigen. all of the clones tested to date are of the cd4 phenotype and make il-2 in response to islet antigen and nod apc. nearly all of the clones we have tested also make good proliferative responses to islet cell antigen obtained from mouse strains other than the nod or to a mouse beta cell tumor line. preliminary results indicate that at least one of these clones can lead to islet cell damage in a disease transfer experiment in which the cloned t cells are injected into a non-diabetic nod f1 recipient. we are currently carrying out tests to further characterize lymphokine production by these cloned cell lines, to analyze differences in antigen recognition and mhc restriction requirements among the clones, and to determine their effectiveness in mediating the disease process in nondiabetic animals. in an attempt to identify the epitopes on class i mo ecules recognized by alloreactive cytotoxic t lymphocytes (ctl) we have examined k -specific ctl for tpir recognition of synthetic peptides with sequences derived from the native k molecule. co secutive overlapping peptides molecule were tested for their capacity to inh&bit k -specific ctl clones in their recognition of cells expressing the native k molecule. in these studies inhib9ion by peptide was found to be an extremely rare event, although one peptide (k 111-122) did inhibit recognition by a particular ctl clone (clone 13). in a separate set of experiment8 it was observed that clone 13 could recopize kblll-122 when presented by h-2 class i molecules. as clone 13 was of h-2 origin, this finding led to conclude that inhibition may be due to class i-restricted recognition of the k pegtide on the surface of the ctl clone, peptide and native kb for the t cell receptor. we present evidence in favor of this conclusion. the pepscan method is used for the systematic identification of sequential b cell epitopes i n protein molecules (geysen, meloen, barteling. pnas 81: 3998; 1984) . it was designed for the synthesis and subsequent testing for antibody binding of large numbers of overlapping peptides directly on their solid supports. the mhc dependent presentation required for t cell recognition seemed prohibitive for the use of pepscan to identify t cell epitopes. however we now have shown that by a novel modification the peptides can be recovered from their solid supports and used in t cell assays. holmdahl, department of medical and physiological chemistry, box 575 uppsala university. s-75123 uppsala, sweden. both autoreactive t cells and autoantibodies play an important role in the pathogenesis of type i 1 collagen (cii) induced arthritis in mice. we have earlier reported that only strains with h-2q , h-2w3, h-2w17 and h2-r were responders to autologous mouse cii and only these strains developed arthritis after immunization with autologous or heterologous cii. however, heterologous cii induced a more acute and severe disease and a more pronounced autoantibody response. this findings indicate that 1) the ability to mount an immune response against autologous cii is a prerequisite for the susceptibility to collagen arthritis and 2) that a crossreactive autoantibody response after immunization with heterologous cii may further enhance development of arthritis. we have now studied activation of autoreactive b cells after primary immunization of den1 mice with rat cii. in hybridoma collections, obtained 9-11 days after immunization, 30.80% of the hybridomas produced igg reactive with autologous cii, 10-15% produced multispecific igm and a significant number produced igg rheumatoid factors. the anti-cii antibodies recognized at least 5 different epitopes on the cii molecule and originated from many different vh and v kappa gene families. furthermore, none out of 6 investigated anti-cii hybridoma expressed cd5 rna message. we therefore suggest that the primary anti-cii autoantibody response involves activation of memory b-cells. these memory b cells have most likely been earlier activated by cii autoreactive t cells. in these aspects the origin of the anti-cii autoantibody response is principally different from the origin of "natural" autoantibodies. t cell receptors (tcr) recognize antigen in association with self mhc molecules, usually following processing to smaller peptides. the t cell repertoire to an antigen, therefore, reflects not only the ability of a given mhc molecule to interact with antigen, but also the affects of initial repertoire selection by self mc. we have t#tn analyzing the tcr repertoire specific for beef insulin (61) in balb/c mice (h-2 ), which are high responders to the antigy. these studies revealed that vp.3 is dominantly used in the tcr's specific for bi/a and our preliminary data suggests that the vgb.3 chain may be involved in mhc restriction. we have now obtained several t cell hybridmas specific for bi from (balb/cxa/j) f1, animals. a/j mice (h-2a) are low responders to bi while the f 1 m ce a e high responders. most of the balb/cxa/j hybridmas were restricted to the hin the balb/c hybridmas. interestingly, the analysis of v gene usage demonstrated that vg8.3 was not used in the balb/cxa/j hydridonas. the relevance of these results to the development of the tcr repertoire in different mouse strains will be discussed. this work was supported by the mrc of canada. ctl specific for the q k molecule were generated from normal splenocytes by & vitro culture with a2k bearing stimulators. these ctl have been shown to lyse transfected targets expressing hla-a2 regardless of their murine haplotype, and they specifically kill a2 bearing human target cells. furthermore. the effector function of these ctl can be inhibited with an hla-a2 specific monoclonal antibody. thus, the transgene product functions correctly as a tolerogen and is recognized directly as a class i antigen. although transgenic mice have been shown to be tolerant to a2k expressed by murine cells, transgenic ctl specific for hla-a2 on the surface of human cells have been generated. these the major virulence factor of m.pneumoniae was shown t o be a 168 kda protein which is located in the tip structure membranes of these cells. beside the adhesin function this protein is also involved in first massive humoral and cellular responses of the human host during the acute phase of upper respiratory tract infections and interstitiel pneumonia. intranasal inoculation of guinea pigs with the isolated 168 kda protein led to lympho-histiocyte infiltrations around bronchi and small vessels of the lungs which are characteristic infiltrations after an infection with live m.pneumoniae cells. furthermore one peptide ( 1 7 amino acids long) which was synthesized according to the amino acid sequence of the adhesin, showed a proliferative activity to in vitro cultivated t-cells of bronchial washings, whereas synthetic peptides with th e sequences of the direct neighbourhood showed no in vitro activity. most interestingly this t-cell proliferative activity is located on a surface loop of this protein which is also responsible for the adhesin f uction. christopher a. smith, gwyn t. williams, rosetta kingston and john j.t. owen. department of anatomy, university of birmingham, medical school, vincent drive, birmingham 815 2tj, uk. rearrangement of t-cell receptor a and b chain gene segments during t-cell development results in a diverse array of receptor specificities. to avoid auto-immune responses, cells that have generated self reactive receptors are thought to be eliminated or inactivated, to produce self tolerance. recent studies have provided compelling evidence that clonal deletion of immature receptor bearing cells within the thymus makes an important contribution to this process, although the mechanisms involved are not uderstood. of immature mouse thymocytes with anti-cd3 antibodies added to thymus organ cultures, induces dna degradation and cell death through the endogenous pathway of apoptosis. is in marked contrast to the activation of mature t-cells by the same anti cd3 preparation and is specific to the extent that apoptosis is not induced by either anti-cd-4 or anti-thy-1. to organ cultures suggesting a role for changes in intra-cellular c a w levels in the signalling pathway leading to the induction of apoptosis in immature cells binding. thus activation of the process of apoptosis in immature cells binding self antigens may be the mechanism responsible for the selective deletion of cells that could generate an auto-reactive response if allowed to mature. we have now obtained evidence that engaging the cd3/t-cell receptor complex this in addition, calcium ionophore (ionomycin) also causes apoptosis when added anderson cancer center. houston, tx 77030 immunization of patients with bcg and irradiated tumor cells induces specific delayed-type hypersensitivity (dth) to tumor cells and not to normal colon cells. since igg antibodies may require t-cell help, we wished to characterize the igg-defined tumor-associated autoantigens (taaa) of human crc so as to define a subset of the t-cell repertoire for crc. western blots of detergent extracts of 73 primary and metastatic human colorectal carcinomas and paired normal tissues were probed with autologous igg. nine taaa were recognized by 20% or more of the sera: 74, 72, 58, 52, 45, 41, 38, 29 . and 26 kda. these taaa may be normal colon differentiation antigens, since they were present in extracts of normal colon. autoantibodies are more frequently present to the 41 kda antigen in patients with metastases (79%) than in primary tumors (47%. p<o.o5) while there is more reactivity to the 38 kda antigen in primary tumors (38%) than in metastases (7%, pso.05). only 1 of 8 patients sensitized with bcg and irradiated autologous tumor cells, developed antibody to new taaa; whereas 2 of 12 control patients developed antibody to new taaa without sensitization. the taaa in the western blots that stimulate autologous lymphocyte proliferation are greater than 200 kda. finally, immunization with a human crc failed to induce dth in mice to a syngeneic murine colon carcinoma that expresses at least 1 of the igg-defined human taaa. thus, taaa that presumably require t-cell help in patients do not appear to be part of the t-cell repertoire for dth to human crc. in several rodent species and strains, the emergence of specificity to a discrete immunodominant epitope of guinea pig basic protein (gpbp) results from selection of cell lines with whole gpbp. these epitope specificities have been found to be strain-specific and define the specificity of the t helper cells which transfer experimental autoimmune encephalomyelitis (eae) in adoptive transfer experiments. line from aci-strain rats which was encephalitogenic and specific for a new t cell epitope of gpbp. the cell line responded in vitro to the peptide cokkesponding to the region 39-54 of gpbp but not to peptides corresponding to other regions of gpbp. this cell line is restricted by i-a but not i-e and transfers a dth response to gpbp as well as eae into naive recipients. these results provide support for the hypothesis that the in vitro immunodominant epitope specificity defines the specificity of gpbp-specific encephalitogenic t cells. the definition of a new encephalitogenic epitope in the aci strain also suggests that the mechanism of eae in rats depends on strain-specific extrisic properties of antigen recognition such as the mhc class i1 genotype or the composition of the t cell antigen-receptor repertoire. we have produced a gpbp-specific t lymphocyte medical center, stanford, ca 94305. a wide variety of t lymphocyte surface antigens have been isolated and shown to play important roles in the t cell response. we outline here a strategy to isolate and characterize surface proteins unique to activated t cells. a rabbit anti-serum was generated by immunizing with allo-activated human t cells and subsequently absorbing with the t cell tumor hpb-all, a tumor that expresses all functionally relevant t cell surface antigens previously described. this absorbed antiserum was able to recognize an uncharacterized group of surface proteins on t cells that was absent from hpb-all. furthermore, the absorbed anti-serum was able to inhibit i n virro cytotoxic and mlr responses; an effect that was abolished by carrying out an additional absorption with the t cells used for the initial immunization. monoclonal antibodies to activation antigens were generated by immunizing with complexes made by adding the absorbed anti-serum to solubilized ctl. the absorbed anti-serum was also used to screen a lcgtll cdna library. one cdna clone so isolated is expressed on ctl as evidenced by northern analysis. dna sequence data indicates no similarity to other sequences in available databanks. childrens hospital of orange county, orange, ca 92668, and university of california, irvine, ca 92717. we examined the ability of cytokines to stimulate the proliferation of pbm from 41 new-onset iddm, 11 long-term iddm, 10 non-diabetic pediatric control, 5 type i1 diabetic, and 3 non-diabetic adult control subjects. initial results demonstrated the ability of human pbm culture supernatants containing peak natural il-1 activity to stimulate a significantly higher proliferative response from pbm of new-onset iddm than from pbm of long-term iddm, type i1 diabetics, or non-diabetic controls. more recent results have shown that human recombinant il-la, -lb, -3 , -4 and -6 , interferon gamma, lymphotoxin, and tumor necrosis factor, as well as con p.-stimulated rat spleen supernatants, also stimulated a significantly higher proliferative respcnse from pbm of new-onset iddm than from pbm of controls. however, human recombinant il-2 stimulated equivalent levels of proliferation from pbm of both new-onset iddm and controls. these data indicate that, in new-onset iddm, there 2.e present pbm which are in an early stage of activation not revealed by increased responsiveness to recombinant il-2. in the case of t cells, this may represent a stage prior to an il-2-dependent stage of activation. this would be consistent with the active induction of newly responsive cells during a stage of iddm when islet cells are still present. this response is not the result of the metabolic aspects of iddm but a reflection of the immunologically activated state of t cells, b cells andlor monocytelmacrophages in iddm. houghten, c. david pendleton, james l. comette, charles delisi and jay a. benofsky, metabolism branch, national cancer institute, national institutes of health, bethesda, md 20892. analysis of known helper t cell m) cpitopes suggests that th epitopes tend to be amphipathic alpha helices, that is, helices with hydrophobic residues on one side and hydrophilic residues on the other side. based on this hypothesis, a computer program "amphi" was generated which may aid in prediction of th epitopes. within residues 1 to 55 of spermwhale myoglobin (swmb), a th epitope is known to exist but has not yet been found. as an approach to further test the hypothesis, this undetermined th epitope was sought both by an unbiased test of ten evenly overlapping 15-residue peptides that span the region, and by computer analysis. of the ten peptides, only one was able to stimulate two clones that are specific for the 1-55 region. this peptide gave remarkably high stimulation index. analysis of this region by the computer program also predicted the site covered by this peptide (residues 2640) to be the most likely site for th epitope.. therefore, unbiased in v i m testing and the computer prediction correlated well. the peptide was able to prime t cells of b1o.br mice in vivo for in v i m response to the native swmb as well as to the peptide fragment of residues 1-55. immunization of mice with swmb showed that, of the ten overlapping peptides, the major site of response is to the identified peptide. finally, a peptide of residues 24-42 was made to increase the amphipathic score. this extended peptide induced greater proliferation of the clones. thus, this study has identified a new dominant epitope of swmb and confirmed the utility of the amphipathicity hypothesis in a prospective test. amino acids a t a selected site; 2 ) t-cell antigenicity, that is analysis of prirrary structures with 9-and 11-amino acid templates obtained from database of knawn y-epitopes; gly, hhydropkbic, pplar or charged amino acid (2); 4) prediction of the secondary structure according to gamier (3) to exclude sites having high turn or coil forming ptential. ccanparison of t h i s set with published program (1,4) shows that it has high predictive f r . so we used it fo search for probable t-epitopes i n hepatitis a virus capsid proand for mediators of delayed hypersensitivity (tdh ~ assayed by ear swelling induced by co-injection of responder cells with irradiated tumor cells). lymph node cells recovered 14 days post-inoculation contained tdh cells and ptc (freq 1/1,200) , but tc effector cells. by contrast, graft-infdtrating cells not only contained tdh and ptc (l/l,oao), but direct tc effector cells (21 lytic units). all three sets of t cells were antigen-spedfic. the finding of significant frequencies of ptc within the lymph nodes and the graft site, but tc q& at the latter, suggests that differentiation of tc from unprimed ptc may be a multistep process, the first of which occuis in the draining lymph nodes. the data further suggest that the terminal step@) in this process take place after the donally expanded ptc have disseminated to the graft site. we suspect that the final step requires help provided uniquely by tdh cells within the graft. the fmal step may not occur (as might have been expected) in the lymph node because priming of ptc and tdh take place at noncontiguous anatomic sites. interference & seed, pnas a, 8573, 1987) . the n-terminal amino acid sequence of normal t cell cd28 was found to match the hpb-all sequence except in residue 10 and 11 (...ts...) . in principle this might reflect somatic mutation in hpb-all or cd28-gene polymorphism: erroneous residue assignment in the gas phase sequencing cannot be excluded entirely. to investigate the significance of this sequence variation a genomic cd28 dna clone of a normal human t cell clone was analysed around residues 10. 11 and found to be identical with the hpb-all cdna sequence. sumtic mutation in hpb-all cd28 therefore can be ruled out. the molecular characterization of t cell receptor (tcr) structure in a panel of mouse t cell clones specific for sperm whale myoglobin (spw mb) has shown that 6 1-ed-restricted clones specific for the spw mb region 110-120 use highly homologous v bchains. four of these clones are indistinguishable in their response pattern to a panel of overlapping synthetic peptides spanning the spw mb sequence 110-120, yet use 3 different v a gene segments. we are currently trying to find fine specificity differences among these 4 clones, in order to correlate them with tcr expression. t cell hybridomas have been made from these clones, and are being screened on panels of substituted peptides. we hope that conservative substitutions at positions important for interaction with the tcr will reveal fine specificity differences among these clones. sloan-kettering institute, new york, ny 10021 we previously showed that clonal deletion, non-suppressor mediated immunological tolerance neonatally induced to mls allo-determinants was specific in that the frequencies of t cells responding to a variety of other stimuli were unaffected while the frequency of t cells responding to mls were drastically reduced. here we show that mls tolerance is detectable in the thymus prior to a detectable positive response in the periphery (spleen and lymph node). tolerance induced by spleen cells from mlsa congenic mice in balb/c mice is as "potent" as that induced by whole background incompatible dba/2 spleen cells, indicating that incompatibility at the ml s locus ( the ly-6 alloantigens have been shown to participate in the process of t cell activation based on the ability of anti-ly-6 monoclonal antibodies to induce il-2 production and proliferation of t lymphocytes. in the present investigation, we have demonstrated that peripheral t lymphocytes from a strain mice exhibited abnormally low proliferative responses after stimulation through ly-6a/e and ly-6c molecules when compared to responses of t cells from numerous other mouse strains. the abnormal activation of the ly-6 pathway of a strain t cells was not due to ineffective fc receptor cross-linking of the anti-ly-6 monoclonal antibodies, to inappropriate cellular expression of the ly-6a/e alloantigen in a strain t cells, or to an active suppressive phenomenon. identification of this defect provided an opportunity to compare t cell activation by the ly-6 and tcr pathways. interestingly, t lymphocytes from a strain mice proliferated normally when the cells were activated by monoclonal antibodies to thy-i or the cd3/tcr complex suggesting that this defective activation was selective for the ly-6 t cell activation pathway. cell separation studies of t cells and accessory cells demonstrated that this defect was quantitative rather than qualitative and that it was complex, residing at both the t cell and accessory cell levels. these results suggest that activation of t lymphocytes via the ly-6 molecule may involve unique signalling pathways and that activation via cd3/tcr can proceed normally in the absence of a fully functional ly-6 pathway. the h-u)d transfected l cells were recognized by the cil when they were infected with either influenza a virus or a vaccinia recombinant virus encoding the pb2. thirty-five synthetic peptides derived from pb2, which had either an amphipathic suucture or contained a rothbard' epitope, were tested for their ability to render uninfected targets susceptible to lysis by influenza specific effectors. three epitopes were identified, each of which contained a rothbard epitope. department of microbiology and inununolag, albany medical college, albany, ny 12208 our laboratozy has developed a new, powerful technique for hvestigating the inmplne ~n s = to peptide epitopes, involving the covalentcoupling of peptide to phosphatidylethanol-, follmed by amplexing with additional lipids and phospholipids to form a peptide-phoqholipid complex. these cmplexes can be used to inununize mice in the absence of protein carriers or adjuvants, thus facilitating the study of the hmne response to a mall chemically defined antigen. use of this technology has allmed us to identify two t helper cell epitopes in conserved regions of kiv gp 160 not previously identified by computer algorithims, defined by amino acids 485-518 and 585-615. inununization with these peptides in peptide-phoqholipid mmplexes results in the prduction i g and igg antibodies, which truss react with cloned fragments of the whole protein. us& this tecfinolosy we have begun to characterize the irmnune reqonse to individual peptide antigens. ?he response of h2-k mice to amino acids 494-518 of gp 160 of hiv, has been analyzed. lhe optimal dose of a peptide containing both b and t cell epitopes was found to be 15-30 ug, depending on the mute of administtation. im mhization required less antigen for optinarm antibody response than did ip. anchorage in the phospholipid cmplex is a strict requirement for an antibody response. additional variables, such as phospholipid m i t i o n and method of cross-linking have been studied and will be discussed. we believe that the use of this peptide-phospholipid ccnnplex technology will be significant both for studying the i n m u m response to single epitops and for vaccine developrent. virology, national bacteriologica; laboratory, 105 2 1 stockholm, departments of neurosurgery, virology and immunology, karolinska institute, 104 01 stockholm, johnson & johnson biomedical research, la jolla, california, pentadecapeptides, sequentially overlapping by 10 a.a., were syntesized based on the htlv-i11 sequences of gag and env proteins and used as antigens in igg-subclass elisas andt-cell stimulation assays. sera and cells were obtained from 30 asymptomatic, hiv-infected homosexuals. reactivity in all subclasses could be found to parts of the gag-protein. extensive areas of iggl and 3 reactivity were indentified mainly in the p17 and n-terminal half of p24 with an additional region in the n-terminal end of p15. the highest iggl and 3 reactivities were detected in a.a. 8-33, rich in glycine, arginine and lysine and thereby hydrophilic with a high segmental flexibility; iggz and 4 epitopes were found in the hydrophilic cooh-terminal of p1j; the second highest iggl and 3 reactivities were found in the hydro-?hilic n-terminal of p15. for gp120, the iggl epitopes identified were in the putativeloopregion (a.a. 296-331) and in the hydrophilic cooh-terminal end of gp120 (a.a.489-503). the immunodominant region of gp41 (a.a. 582-617) showed some igg2, 3 and 4 responses inaddition to igg1. t-cell proliferative responses were detected against many peptides usually in areas not binding igg. still, simultaneously t-and b-cell reactive peptides could be detected, and the showed a distinctly preference for igg1. the variation between different patients was considerable regarding both t-cell reactivity to peptides and the subclasses of reactive igg. a mapping of subclass restricted epitopes allows an elucidation of anti-viral imunological mechanisms. kristin komschlies, laboratory of experimental immunology, brmp, dct, and bcdp, program resources, inc., national cancer institute-frederick cancer research facility, frederick, md 21701 and jackson laboratories, bar harbor, me 04609. "double positive" (dp) thymocytes are precursors for cd4+ and cd8 mature subsets have been based on indirect evidence with in vivo anti-cd8 or anti-cd4 treatments. we have approached this question directly by isolating subsets of dp thymocytes from fetal or adult thymus and have shown that only a subset of dp cells ( 4 0 % of adult thymocytes) can generate donorderived thymocytes after i.t. injection into irradiated hosts. low-density dp cells are injected i.t., up to 20-40% donor-derived cells can be seen at 6 to 18 days after transfer. cd4' cells can constitute up to 30% of these donor-derived cells at day 10. in contrast, an equivalent i.t. transfer of dull ly-l/cd5 (dly-1). early precursor thymocytes would not generate cd4' cells until days 12-13 but would produce some cd8' cells by 18 days. thus we have detected differentiation of cd4+ cells and not cd8' cells from adult dp cells. depleted host and recovered donor-derived thymocytes 8 to 10 days after i.t. transfer of dly-1 cells, when most of the donor-derived cells are dp. when retransferred, these dp cells displayed a more rapid progression of thymic localization patterns than dly-1 cells, but were unable to produce enough donor thymocytes to analyze subsets. thus cd4' cells appear to differentiate via a very minor (low density) subset of dp thymocytes. may not be generated from these intermediate dp precursors but directly from dly-1 cells. the intrathymic differentiation process by which bone marrow-derived progenitor cells develop into mature, immunocompetent t lynyhocytes remains poorly understood. the majority of all intrathymic lymphocytes are cd4 8 "double positive" cells that have no well-documented function in thymic differentiation, but have been proposed to be either a critical intermediate between cd4-8precursor cells and mature t cells, or, alternatively, a "dead-end" cell type. furthermore, although signals mediated through the tcr complex are critical for t cell tolerance induction and repertoire selection, the functions of the cd4 and co8 differentiation antigens expressed by the vast majority of thymocytes are unknown. in the present study we addressed the possibility that cd4 functions as a signalling molecule during t cell development. we engaged cell surface cd4 on murine thymocytes by in vivo injection of anti-cd4 mab, and monitored the effects of that engagement on distinct thymocyte subsets. c d 4 ' 8 + cells respond to cd4 cross-linking by dramatically increasing their cell surface expression of cd3 and tcr, and decreasing their cell surface expression of cd4. in contrast, mature cd4'8-cells respond to cd4 engagement by decreasing their cell surface expression of both cd3 and cd4. these results provide clear evidence that cd4 engagement regulates cd3/tcr expression on developing thymocytes, and, most interestingly, results in increased tcr expression by cd4+8+ double positive thymocytes. thus, cd4 engagement is a membrane event to which c d 4 ' 8 ' thymocytes respond significantly, indicating that cd4 may be a signalling molecule in t cell development and may promote t cell differentiation by inducing increased cd3/tcr cell surface expression on cd4+8+ thymocytes in vivo. f.g. terpstra. p.th.a. schellekens and r.a.w. van lier, central lab.blood transf.service, lab.exp.clin. immunol. of the univ.of amsterdam, the netherlands t cells can be activated through several membrane molecules that may use distinct intracellular signalling pathways. t-cell activatiep threygh both the cd3/tcr complex and cd2 is accompanied by riaea in free intracellular ca (ca ) and pkc activation. in contrast, evidence from our laboratory suggests that triggering h a cdz8 occurs through a pkc-independent route. in this study we used mab to various t-cell membrane antigens to localize the activation defect in t cells from asymptomatic hiv-infected men that we previously showed to have an intrinsic defect in their proliferative response to soluble anti-cd3 nab. compared to hiv-homosexual controls, the monocyte-dependent response of t cells from hiv+ men to soluble anti-cd3 and a mitogenic combination of anti-cdz mab was decreased as was the monocyte-independent resgpnse to immobilized low-dose anti-cd3. in t cells from both groups a normal rise in (ca ) . was induced by anti-cd3 and anti-cdz mab. however, t cells from hiv-infected men respondad poorly to direct pkc activation by pma in the presence of healthy donor monocytes, whereas control t cells responded vigorously. in contrast, induction of proliferation by anti-cd28 mab was comparable in t cells from hiv+ and hiv-men. our results indicate in hiv+ men a selective qualitative t-cell defect in activation routes that are dependent on pkc activation, whereas pkc-dependent alternative activation appears to be unaffected. these findings will be discussed with repect to hiv immunopathology and normal t-cell physiology. we have studied the role of the protein kinase c (pkc) in the activation of a prototype th-2 cell, d10.g4.1. blocking of this enzyme with the drug h-7 [1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazinet leads to an increase in cellular proliferation of d10 cells activated with specific antigen or mitogen. n addition, an increase in the amount of il-4 and il-5 mrna is seen under these conditions compared to the level observed in mitogen activated cells in the absence of h-7. this superinduction of il-4 and il-5 lymphokine mrna is also detected in mitogen activated d10 cells that are depleted of pkc protein by pretreatment of the cells with phorbol esters. in contrast, mitogen activation of s lenic t cells measured by proliferation is blocked in the presence of h-7. these data indicate that jkc has a regulatory role in th-2 cells by exerting a negative feedback on the events that occur after cell activation avoiding an uncontrolled response. this effect is opposite to that seen in a bulk t cell population that represents mainly th-1 type cells. the mechanism of signal transmission is, therefore, distinct in the two types of cd4 + t cells. aberrant expression of class i1 mhc proteins on non-immune somatic tissue has been implicated in several autoimmune diseases, but it was unknown whether this expression was involved in the initiation of disease or resulted from induction by the immune reaction. we ha e engineered several transgenic mouse lines expressing tissues using eith r the pancreatic elastase promoter or the class i kb gene promoter. the i-as on the pancreas is fully capable of presenting peptide antigens to i-ad restricted hybridomas. h-zb mic expressing i-ad on pancreatic acinar cells but not in thymus are not tolerant to i-as in mixed lymphocyte reactions in vitro, but do not spontaneously produce an immune respon e to the pancreas in vivo. peripheral antigen presentation, i-a restricted immune response, i-a expression by t-cells, and widespread somatic i-ad expression. expression, whereas another has only medullary expression. the effect that these different patterns have on mhc restriction is being examined. ctr., stanford, ca 94305. a transient increase in the intracellular cat+ ion concentration has been shown to occur rapidly following activation of t lymphocytes. this response i s a more immediate and direct indication of t cell activation than either production of interleukins or proliferation. we examined ca++ flux analysis using indo-l and the facs to measure ag-speclfic t cell activation and also to sort for an ag-specific t lymphocyte subpopulation. human t cell clones were exposed to elther allostlmulatory 6 lymphoblastoid cells (which cause proliferation of the t cells) or nonstimulatory 6 lymphoblastoid cells. in both cd4+ and cd8+ clones, we observed an ag-specific cat+ flux response similar t o an lonomycin positive control. we then attempted to enrich for antigen specific t cells from a mixed population. two phenotypically different human t cell clones were mixed in equal amounts. after coculture of the cell mixture w i t h 6 lymphoblastoid cells shown to stimulate only one of the t cell clones, the cell mixture was sorted on the facs based on specific cat+ flux. a ten fold enrichment was observed for the ag-specific clone. this approach allows rapld observation of t cell activation and provides a means of sorting antlgen reactive t cells from a heterogeneous cell population. previously attemps to characterize the function of beta-2-microglobulin (beta-2-m) have used anti-beta-2-m antibodies, to block the functional activity of the protein. we now demonstrate that human beta-2-m and the proteolytic derivates beta-2-m lys58 and beta-2-m deslys58 added in nanomolar concentrations to allogeneic, pha and con-a stimulated mouse lymphocyte cultures modulate the response induced. thus beta-2-m and especially beta-2m deslys58 when added day 0 and 1 augment the generation of specific cytotoxic t lymphocytes in mlc, problably due to stimulation of endogeneous lymphokine production in the culture. both the con-a and pha induced response were modulated. in the presence of beta-2-m and its proteolytic derivates the proliferation was optimal at 48 hours, as opposed to the 24 hours optimum in the abscence of beta-2-m. these results strongly suggest an active role of beta-2-m or proteolytic derivates hereof in the t cell activation and generation of specific cytotoxic t lymphocytes. a (1.2 fold) . the increase peaked at 2 min and returned almost to baseline by 10 mi?. igm b cells showed random x inactivation, while more mature igg and iga b cells had only the normal x active. we conclude that the xla gene product is required for b cell development, while the xscid gene product is required for both t and b cell maturation. gene mapping has placed xscid in xq13-21.3, linked to phosphoglycerate kinase (pgk), lod 3.1, 8=0. mouse xid, which affects only b cells, is also linked to mouse pgk, suggesting the possibility that xscid and xid belong to related gene families. c 530 institute at denver, national jewish center, denver, co 80206. mapping data for the bxd and akxd recombinant inbred strains shows that the mhc and the products of at least two other genes control the expression of vp3. one of these is linked to ly-7, and the second for the akxds is linked t mtv-13 on chromos me 4. a backcross of b1o.br x (c3h x b1o.br)fl (mls-2 x ( m l~-2~ x mls-2 ) ) has been used to confirm the linkage of one of these controlling genes to ly 7. using the vp3-specific monoclonal antibody, exceptions have been found to the general rule, that strains which have eliminated t cells expressing a particular vp specific for a defined self antigen in the thymus, also express this self antigen on their spleen cells, and so these cells are capable of stimulating t cell hybridomas bearing the particular vp in quest ion. the t cell receptor (tcr) of the alloreactive t cell clone 2c recognizes the h-2ld specificity. the cloned a (va 3.1) and p (vg 8.2) tcr cdnas have been inserted into plj retroviral vectors containing ltr and regions of the momulv and neomycin resistance genes. three different constructs have been used to transfect v2 cells. g418-resistant clones showing the highest titer of viral particle psoduction have been selected, and injected subcutan ously lacking the h-2l vt cells producing retroviruses carrying the a gene have been examined for the expression of the new specificity on the infected t cells. northern blot analysis using the va and the neo-probes showed, in the lymphoid tissues, a transcript with an expected size of 3.6 kbs hybridizing with both probes. in the spleen of treated balbfc mice, but not in dm2 mice, the expression of the exogenous va 3 resulted in proliferation of infected t cells, as assessed by an auto-mlr proliferative assays. into newborn balb/c mice (h-2 l +) o r into the congenic strain dm2 loose most of their lytic activity as well as the ability to form conjugates with specific tc. preliminary experiments show that spdp-anti-cd3-derivatization of specific tc overcomes the trypsin effect, so that both conjugation and lysis are observed between trypsin-treated pel and sdpd-anti-cd3-derivatized specific tc. many investigators, the ti/cd3 complex on pel surface is central to specific lysis. the observed inhibition of trypsin may be due to its action upon it. since trypsin-treated pel could be triggered toward conjugation with and lysis of tc by anti-cd3, the cd3 at least must have remained intact and is the important molecule for both the critical conjugation step and lysis. trypsin treatment possibly disturbs the interaction of tc with ti/cd3 of pel so as to prevent proper engagement of the cd3. concanavalin a (con a) treatment of tc appears not able to bring back the lytic activity or the ability to form conjugates of trypsin-treated cells, indicating a dichotomy in the mechanism of killing by derivatization with anti-cd3 versus con a mediation. comparing pel and their blasts ipeb). unlike pel, peb do not show ldcc or lytic activity against spdp-anti-cd3-derivatized nonspecific tc. ldcc by con a has been proposed to function via ti/cd3 interaction. unlike pel, peb also do not show fluorescence. it may be that the ti/cd3 complex on peb differs from the one on pel. at the same time, total lack of cd3 is contraindicated for peb kill specifically. the region important in ti interaction for ldcc and that recognized by anti-cd3 may be within the aberrant area of cd3, so neither the con a nor the anti-cd3 trigger could be transmitted on peb. the importance of the cd3 is indicated further by immunolabeling both cells with anti-cd3 revealed that c533 combination of cd4+ and cd8+ islet specific t cell clones are pathogenic in the nod mouse, eva-pia reich and charles a. janeway. jr., department of pathology, section of imunobiology, yale university school of medicine, 310 cedar st., new haven, ct 06510. although diabetes in the nod muse is thought to be initiated by t lymphocytes, thus far no t cell clone has been derived from nod islets. we therefore isolated islets from acutely diabetic nod mice and islet derived lymphocytes were propagated in il-2 and stimulated every 2 weeks with fresh mitomycin-c treated nod islets to maintain islet specificity (i.e. a proliferative response to nod islets). t cell clones were generated by limiting dilution followed by recloning by single-cell manipulation. facs analysis showed that all clones were positive for thy-l+ and cd3+, confirming that receptor bearing t cells had been cloned. cd4+cd8-and cd4-cd8+ clones were isolated that proliferated in the presence of nod islets. respond to nod but not balblc or b1o.br islets, while the two c d 8 ' clones respond to nod and balb/c islets but not blo.br. islet-specific, i-anod restricted, while the c d e clones are islet-specific and dd restricted. injection of individual cloned lines into young, irradiated nod mice leads to no overt or microscopic disease. a syndrome of dehydration, rapid deterioration and death about 10-14 days after transfer. mice sacrificed at this time show lymphocytic infiltration around the islets. this suggests that the cd4+ clones are however, mixture of c d 4 ' and cd8+ cloned lines causes these cloned lines may be useful in the search for islet cell autoantigens. dusseldorf 1 (frg). autoantibodies to nucleolar components are a common serological feature of patients suffering from scleroderma, a collagen vascular autoimmune disease. while animal models which spontaneously develop abundant anti-nucleolar antibodies have not yet been described, high titers of such antibodies may be induced by treating susceptible strains of mice with mercuric chloride. we have identified the nucleolar autoantigen against which the hgci2-induced igg autoantibodies from mice of strain b1o.s are directed. it is a protein of apparent molecular weight of 36 kda and pi value of about 8.6 which is associated with the nucleolar snrna u3, and by these criteria, is identical with a polypeptide called fibrillarin. it is striking that scleroderma patients spontaneously form autoantibodies against the same u3 rnp protein. the hgc12-induced murine and the scleroderma-specific human anti-u3 rnp autoantibodies were indistinguishable in their reactivities towards fibrillarin. they further resemble each other in so far as both recognize epitopes on the 36-kda protein which have been highly conserved throughout evolution. our results provide a basis to investigate at the molecular level whether similar immunoregulatory dysfunctions may lead to the preferential a n t i 4 3 rnp autoantibody formation in the animal model and in scleroderma patients. forty bp-specific cd4+ t-cell clones were isolated from the peripheral blood o f a patient with multiple sclerosis. studies with a panel of xenogeneic bps of known amino acid sequence and with laree fragments of the bp molecule demonstrated the existence of at least 10 epitopes recognized by human t-cells. at least 7 of these epitopes were potentially clustered in the middle or at the c terminus of the molecule. in studies with synthetic peptides corresponding to residues 86-105 and 152-170 of human bp (hbp), 9 clones proliferated in response to 86-105 and 19 recognized 152-170. ctl assays were carried out with targets consisting of an autologous b-cell line coated with hbp. nineteen of the 40 clones demonstrated bp-specific ctl activity; these were the same 19 that had recognized peptide 152-170 i n proliferation assays. these clones also killed targets coated with peptide 152-170 but not targets coated with 86-105. clones that proliferated in response to peptide 86-105 or to epitopes located outside of these two regions failed to kill hbpcoated tarpets. to a lack of binding of 86-105 to the target cell, as the same b-cell line successfully presented peptide 86-105, in proliferation assays, to clones that had previously recognized this peptide when presented by e-cells. activity against bp is more restricted than is the specificity demonstrated for proliferative activity. the lack of killinp of targets coated with 86-105 could not be ascribed prystowsky, department of pathology and laboratory medicine, university of pennsylvania, philadelphia, pa as an approach to studying regulatory factors important during t cell proliferation, the expression of genes induced during il2-stimulated t cell growth was examined. to identify genes induced by il2, a cdna library was made from il2-stimulated cloned t cells in late g1 of the cell cycle and was screened by differential hybridization, selecting those clones which had higher steady-state mrna levels after il2 stimulation. of 40,000 clones originally screened, 90 positive clones were identified, which represented 21 different genes. when partially sequenced and compared with sequences in the nih and embl databases, 6 clones were identified as cdnas encoding glycolytic enzymes, 4 were cdnas of cytoskeletal genes, 3 were cdnas coding for proteins important in protein synthesis, and 8 clones were not identified. the maximal steady state mrna levels generally occurred during late gl/early s phase, and the induction of most m a s was not inhibited by cycloheximide at a dose that inhibited cell proliferation. nuclear run-on transcription demonstrated an increase in the rate of transcription for gapdh. in addition, there is post-transcriptional regulation of expression, as some mrnas were stabilized upon il2 stimulation. having cloned these mrnas, we can now begin to study the regulation of expression of selected genes. supported by nih grants. cyciosporin a (csa). a potent immunosuppressive drug, caused organ-spedfic autoimmune disease, such as gastritis with anti-parietal cell autoantibodies or oophoritis with anti-oocyte autoanliies, in balwc mice when the drug was administered daily for one week to newborns. administration to adult mice did not. csa abrogated the production of l3t4' t cells and lyl-2' t cells in the thymus. consequently, these t cells were substantially depleted from the peripheral lymphoid organs, especially when the drug was administered from the day of birth. autoimmune disease was prevented when csa-treated newborn mice were inoculated with splenic t cells from normal syngeneic mice. on the other hand, removal of the thymus immediately after neonatal csa treatment produced autoimmune disease with a higher incidence and in a wider spectrum of organs, i.e.. thyroiditis, sialoadenitis of the salivary gland, gastritis. insulitis of the endocrine pancreas, adrenalitis, oophoritis, or orchitis. each autoimmune disease was accompanied by the development of autoantibodies specific for the corresponding organ antigens. it is likely that csa caused organ-specifc autoimmune disease, when administered to newborns, by depleting certain regulatory t cells (suppressor t cells) controlling self-reactive clones. (this work was supported by a grant from the lucille p. markey charitable trust.) artur scherf, philippe dubois, marika plat and luis perelra da sllva, instltut pasteur, 25-28, rue du dr. roux. 75015 paris, 'u93 inserm, hopltal st. louis. 2, place du dr. alfred fournier. 75010 paris, france. we isolated from a genomlc expression library of the malarla parasite p. falciparum a clone coding for an antigen associated with the membrane of infected erythrocytes. intensive sequence analysis of this gene, called 11-1, showed that the major part of this molecule consists of at least 200 repeats of nine amino acids. the consensus sequence of these degenerated repeats is e-e-v-v-e-e-v-v-p and secondary structure analysis predicts t h a t they have a high tendency t o form amphipathic alpha helices. the 8-and t-cell response t o the two most frequent types of repeat were analysed in five different h-2 congenlc mice strains using two synthetic peptides (pqa: y-p-(e-e-v-v-e-e-v-v-p)n-k: pqb: the 8-cell response t o both peptides is restricted to the h-2d and h-2k xaplotype. pca and pqb specific antibodies do not dlscriminate between the two peptides (measured by elisa). the mhc restriction was confirmed by anaiyslng the tcell response to both peptides. t-cell lines speclfic for each of the peptides pqa/pqb were derived from h-2d responding mice. although the peptides differed in four positions of the hydrophoblc part of the helices (valine replaced by isoleucine and leucine) no signiflcant dlfferences were observed in the antigen presentation between peptide pqa and pqb. in contrast, both peptides were unable to induce a proliferation of the heterologous t-cell ilne. suggesting t h a t the variable hydrophobic amino acids of the repeats are involved in tcell recognition. we are currently investigating if other variant repeats of the 11-1 antigen are restricted t o a different mhc haplotype and that the totality of repeats may be subjected to no restriction. glven the type of amino acid replacements within the two repeats tested, the t-cell receptor appears t o discriminate between very similar amino acids. auphan, claude boyer, annick guimezanes, claire langlet, cenae dimmunologie inserm-cnrs de marseille-luminy, case 906, 13288 marseille, cedex 9, france. the yinterferon gene is activated upon cell surface triggering of the aansmembrane t cell receptor (tcr)/cd3 complex or via phosphatidylinositol anchored thy-1 molecules cell clones. both these activation events are inhibited by antibodies directed at the lyt-2jlyt-3 molecules and are lost on tcr a-chain deletion variants (1, 2). biochemical analyses failed to reveal any association between the tcwcd3 complex and either lyt-wyt-3 or thy-1 molecules. however, such studies consistently revealed a non-covalent, cis-type association between lyt-ulyt-3 and h-2 class i products as well as between thy-1 and h-2 class i products. the structural features of the molecules required for such interactions are being determined using hybrid engineered molecules and possible implications of such interactions for t cell activation are being evaluated. (1) schmitt-verhulst, a.-m. et al. nature 325, 628-631 (1987) . (2) guimezanes, a. et al. cellular immunol. 113,435-446 (1988) . i t i s increasingly apparent that the association of both chains is critical to reconstitute t,he 4g-mhc binding site. combinational mechanisms for generating diversity play a major role in increasing the total size of the repertoire. however, evidence for preferential associations have been described between other heterodiaeric proteins of the immunoglobulin gene superfamilg. i t is therefore important to determine if association between different sets of vas and ybs occur at random or if, alternatively, preferential associations exist. we have devised a system which should enable us to determine, by dna mediated gene transfer of cloned cdnas encoding different vas o r vbs, if such preferential associations do exist. we hare also used a panel of monoclonal antibodies specific for a particular va or vb to derive t cell clones expressing these vas or vbs. in a polyclonally activated t cell population expressing a specific va or vb chain, we will present data indicating that the association between these chains is preferential or random. !nteractions between t cells and mhc molecules are thought to select a t-cell repertoire skewed towards recognition of antigens in the context of self mhc molecules. in addition, t cells which react strongly to self mhc molecules are eliminated by a process called self-tolerance. we have recently described generation o f transgenic mice expressing the a6 t-cell receptor (tcr) from the cytotoxic t lymphocyte 2c (sha et al., 1988 nature 335:271) . the clone 2c was derived from a ealf3.e (h-zb) anti-eale/c (h-2d) mlc and is specific for the ld class i mhc antigen. in transgenic h-2b mice, a large fraction of t cells in the periphery expressed the 2c tcr. these t cells were predominantly cd4-cd8+ and were able to specifically lyse target cells bearing ld. in the periphery of transgenic mice expressing ld, functional t cells bearing the 2c tcr were deleted. this elimination of autoreactive t cells appears to take place at or before the c04+cd8+ stage in thymocyte development. in addition, we report that in h-2s mice, a non-autoreactive target haplotype, large numbers of cd8' t cells bearing the 2c tcr were not found providing strong evidence for the positive selection o f the 2c tcr specificity by h-26 molecules. the requirements for cd4 expression during t cell differentiation in fetal thymus organ culture woc) were investigated by analysis of the effects of addition of anti-cd4 mab. control etoc cult& for 5 days contained predominantly cd4+cd8+ cells as well as cd4 or cd8 single positive cells expressing high levels of cd3. t cells from ftw cultured for 5 days in the presence of anti-cd4 m a b expressed markedly diminished cd4 expression. when anti-cd4 m a b was removed 20 hours prior to phenotypic analysis, it was found that cd4 was recxpressed on cd4+ cd8+ t cells. after 12 days of culture, control roc contained high frequencies (23-3046) of cd4+cd8and cd4-cd8+ thymocytes expressing high levels of cd3. in contrast, anti-cd4 mated cultures, while containing cd4-cd8+, cd3+ t cells, were well depleted of cells which reacted with anti-cd4 antimy. when anti-cd4 mab was removed 20 hrs prior to analysis, cd4 was reexpressed on cd4+cd8+ cells, and low frequencies (6%) of cd4+ cd8-t cells were detected, but these cd4 single-positive cells did not express high levels of cd3 or detectable ap heterodimrs. while normal frequencies of cd4-cd8+,cd3+ cells expressed vg8 determinants, the a n t i -w mated cultures were relatively enriched in cd4-cd8+cd3and cd4-cd8-cd3-t cells compaml to control 12 day eoc. these data demonstrate that while cd8+cd3+ t cells can differentiate in the presence of anti-cd4 mab, differentiation of cd4+cd3+ cells is inhibited and cell surface cd4 is down-modulated. cd4+cd8+ cells can develop in the presence of anti-cd4 m a b and do express t cell receptors. these results suggest that cd4 expression, while not required for development of cd3+cd4+cd8+ t cells, is necessary for differentiation of mature cd4+cd8-cd3+ t cells, and that the interaction of 0 4 with its ligand is critical for thymus selection of class-ii specific t cells. uppenkamp, and alfred singer, experimantal immunology branch, nih. bethesda, md 20892. in order to investigate the role of the t cell receptor (tcr) in thymocyte maturation, we have studied thymocytes from the immunodeficient c.b-l7/scid mouse, which due to a genetic defect, is unable to express tcr genes. despite this mutation, scid thymocytes were functional as they produced lymphokines and proliferated in response to stimuli (pma, ionomycin, il-2, il-4). phenotypic analysis revealed that, like normal immature thymocytes, sci+dd'&mocytes were large, thy 1.2+, cd4-, cd8-, tcr-and were enriched in cd5 , il-zr+, pgp-lf, and hsa+ cells. however other tcrpopulations present in normal mice, (i .e. cd4-8+tcr-and c d 4 ' 8 ' t c r -cells) were absent from scid mice. in order to determine if tcr expression was required for cd4 and cd8 expression, we analyzed thymi from the occasional scid mouse which possessed small numbers of tcr-bearing thymocytes. interestingly, expression of cd4 or cd8 was detected only in those scid thymi that possessed tcr' thymocytes, although cd4 and cdb expression was not confined to those tcr-bearing cells. further, the introduction of tcr' cells from thy 1 congenic normal mice into tcr-scid mice consistently promoted the expression of cd4 and cdb (but not tcr) by scid thymocytes. moreover, scid thymocytes from these chimeras possessed cd4+8+ cells, however no mature, single positive thymocytes of scid origin were detected. together, these results suggest that tcr-bearing cells within the thymic milieu are required for the expression of cd4 and cd8 and for the differentiation into cd4+8+ cells. however, final maturation into mature single positive thymocytes requires that the thymocyte itself express tcr. tcr in thymocyte differentiation: evidence from the immunodeficient the cd4 t-cell surface receptor has been implicated in the stabilization of lntercellular interactlons. in addition, it is felt to mediate the transduction of an independent lntracellular signal. we have recently shown (veillette et al.. in press) that the cd4 molecule expressed in t-lymphocytes is complexed to the internal membrane tyrosine kinase p66'ck. suggestlng that alterations in the properties of thls enzyme mediate the cd4 related signal. to gain further insight into the structure and function of the cd4-lck complex, we have generated nih3t3 flbroblasts that co-express the cd4 and lck proteins. nih3t3 flbroblasts expressing a murine lck cdna were transfected by calclum-phosphate preclpitatlon with a construct containing a murine cd4 cdna and the neomycin resistance gene. stably transfected clones were selected for resistance to the amlnoglycoslde c418. we have now demonstrated that the cd4 and ~6 6 '~~ expressed in these non-lymphoid cells can form a stable non-covalent complex. the structure and function of the cd4-lck complex expressed in murine fibroblasts is currently being evaluated. h-2b t c e l l s from h-2b+hh-2bxd responder animals fail to generate one (type a) of these two major clonotypes. environment i s permissive for the t cell specificities of both poly-18 clonotypes (type a and b) and also rovides the determinants for t h e i r selection. the absence of type a clones in the h-2b+h-2gxd chimeras can therefore only be attributed to the failure of h-2b t c e l l s to generate the appropriate t cell receptors. these results suggest a genetic hole in the t cell recognition repertoire of h-2b mice for the lysine-containing epitope of the poly-18 antigen. we have used oligonucleotide-primed gene amplification to study the association between hla class i1 alleles and autoimmune disease. pemphigus vulgaris (pv) is a severe autoimmune skin disease highly associated with dr4 and drw6. we have shown that the nucleotide sequence of the first (and most polymorphic) domain of a new dq allele is found in 1 3 / 1 3 israeli drw6 pv patients but only 1/13 or& healthy israeli controls. this allele differs at only position 57 from two other dq alleles not associated with pv. further, we show that a shared setuence in the third hypervariable region of the first domain of two common subtypes o f dr4 and drw6 cannot be the sole determinantdrgf susceptibility to pv. none of 7 dr4 /drw6+ pv patients had this epitope. we also report a new dr first domain sequences from a drw6 pv patient. the frequencly of t h ! $ allele in patients and controls is being assessed. similar studies to identify disease associated class i1 sequences are underway in patients with multiple sclerosis and rheumatoid arthritis. the frequency of these autoimmune responses is determined genetlcally. and is essentially 100% in the projeny derived in crosses d transgenic mice to some inbred strains of mlce [e.g. those of h-2d haplotype). and yet only 1096 in others (e. g. h-2b) . recently. we initiated an analysis of the i m m~~l~g i d nonresponsiveness in two lineages of insulin/t antigen transgenic mice with early onset of expression of the transgene (rip-1 tag #2 and rir tag #2). while the presence of the transgenic protein durlng the prenatai/neonatal period is the primary requirement for the nonresponsive phenotype, it further appears that the levels of t antigen expression durlng embrydnic development quantjtatively influence the subsequent nonresponstvmess in adult me. an additional modification is exerted by a genetic component whlch seems to map to the mouse major histocompatibility complex [h-2). interesiingly, it appears that the h-2d haplotype. whlch is associated with the most complete impairment of immune responses when t antigen is expressed prenatally. is also assodated with the highest frequency of autoimmune responses when the developmental expression of the transgene is delayed. this correlation is considerably strengthened by the observation that outcrosses to c57b1/6j m-zb) produce mlce with either a weaker tolerance in the case of the early expressers. or a less hquent and weaker autoimmune response in lines with a delayed onset of expression of the transgene. thus. the two distinct biolcgical consequences of the presentation of this antigen (tolerance or autolmmunity) are &ly to result from differences of the developmental maturity of the immune system, and both effects appear to be similarly mhc restricted. lmmunogenicity c 549 mapping the pzb geneti2 contribution t o lupus-like autoimmune disease. i-division o f basic science, national jewish center for immunology and respiratory medicine, 1400 jackson street, denver co 80206; 2-the jackson laboratory, bar harbor, me. nzb mice carry a geneb) that contributes to the lupus-like autoimmune disease seen in (nzw x nzb) fi hybrids. however, the precise locus of this gene has yet t o be determined. to map i t s location, we are breeding a panel of nzbxsm/j recombinant inbred (ri) with nzw mice. by determining which of the ri strains produce fi animals that develop autoimmunity, the location of this nzb gene can be deduced based on the strain distribution pattern of the known genetic markers among the nzbxsmlj ri strains. cells were then fractionated on a percoll gradient to obtain small dense 'resting" b celb and large 'activated' b cells. the small dense b celb demonstrated increased inhibition at every dose of ic examined, when compared to large b cells. for instance, pulsing with 10 ug of ic resulted in a 96% reduction in anti-fl, but not anti-trinitrophenyl, plaque forming cell (pfc) response for small dense b cells. in contrast, the pfc response of large "activated' b cells was reduced only 66%. in addition, when subtolemgenic doses of ic were used (10 ng) prostaglandin â�¬2 (5x10-8y) could function as a potent secondary negative signal rendering these b cells susceptible to negative signalling. we concluded that small dense b cells are more sensitive to ic induced unresponsiveness and that prostaglandin â�¬2 may further sensitize b cells along this pathway. .5 were developed in the mouse against antigens present on humans, hamster and rat nk and t lymphocytes. pre-incubation of lymphocytes with moab did not block the ability of the nk cells to bind to targets but did block their ability to lyse nk sensitive targets. the pre-incubation media contained lytic factors that lysed nk sensitive targets in a 20 h chromium release assay. interestingly pre-incubation of thymus cells or resting peripheral t lymphocytes with the moab induced the t lymphocytes to release lytic factors and acquire nk activity against nk-sensitive targets. lytic factors and acquired lytic activity were unable to lyse lak targets. signalling events did not involve cross linking or fc receptor participation since factors were immunoprecipitation and western blot analysis using the moabs defined a molecule of 27 kd on the surface of thymus or lung mononuclear cells. isolation and amino acid sequence analysis is being done to assist in the characterization of the g1. we have been studying in vitro activation of cd4-, cd8-thymocytes. in the present studies, highly purified double negative thymocytes were cultured with il 1 with or without il 2. following a 72 hour culture with il 1 + il 2 (in the absence of mitogens) significant proliferation was detected, although all cells remained cd4-, cd8-. the frequency of cells staining for cd3 cell surface protein increased from 10% to 60% and tcr vbeta8 from 4% to 40%. facs sorted cd3-cells from the cd4-, cd8population showed similar increases in cell surface tcr suggesting differentiation. northern blot analyses showed an increase in c -m rna at 24 hours, no change in cd3 delta or epsilon expression at 24, 48, and 72 hours, and a marked increase in the ratio of 1 . 3 to 1.0 tcr beta transcript between 24 and 48 hours. these data suggest that il 1 + il 2 can induce cd3 cell surface protein expression on progenitor thymocytes. furthermore, this induction is not associated with increased cd3 transcription, but rather is associated with an increased proportion of functional tcr beta transcripts. the p28 antigen of schistosoma mansoni has been shown to be able to induce protective immunity against schistosomiasis in rodents and primates. synthetic peptides were used to localize the major b and t-cell epitopes of this protein. the primary structure of the p28 molecule was analyzed using various predictive algorithms : surface localization, hydrophilicity, flexibility, secondary structure, amphipathicity of predicted a-helical regions, rothbard pattern. according to these criteria, several peptides were synthesized and tested in immunological assays in vitro and in vivo. correlations between these results and the physico-chemical predictions will be shown. simian virus 40 (sv40) t antigen is recognized by cytotoxic t lymphocytes (ctl) in association with class i major histocompatibility antigens.we have identified four distinct antigenic sites on sv40 t antigen defined by h-2db restricted ctl clones. we have therefore attempted to map these antigenic sites precisely by using overlapping synthetic peptides corresponding to sv40 t antigen amino acids. the synthetic peptides of 15-20 amino acid length were incubated for five hours with 5lcr labelled-non-sv40 transformed h-2b cells in the presence of ctl clones specific for each of the four antigenic sites; and the released radioactivity was used as a measure of specific recognition of synthetic peptides by the ctl clones. the results demonstrated that the antigenic site i resides between amino acids 205-219; sites ii and 111 between amino acids 220-233. site 111 can be separated from site ii as it was found to reside between amino acids 225-239. the antigenic site v was localized between amino acids 489-503. these results show that sv40 t antigen is the target protein for ctl and the regions of t antigen recognized by ctl are tightly clustered in the amino terminal half. attempts to identify amino acids that selectively interact with the class i antigen and the t cell receptor are in progress. peripheral blood lymphocytes of cancer patients demonstrate an early humoral and specific response to application of tumoral extracts. the response is measured as a change in the spectrum of fluorescence polarization of fluorescein molecules. this is probably a measure of sol-gel transformations induced in the cytoplasm upon cellular activation of resting lymphocytes. this measurement is carried by epifluorescent microscopy on single lymphocytes, sitted on a regular holes matrix that is scanned under computer control. it is possible to follow changes within each cell in a population of upto ten thousand cells. since the cells are sitted safely in their holes, it is possible to rinse them with various reagents and follow the kinetics of individual responses by repeated scanning. further studying of the stimulative process might be useful in identifying the responding lymphocytes and the tumoral reagents. this test of the immune system is intended for early and specific detection of cancer and for follow-up of immunotherapeutic manipulations as well. c 559 katharine s. ullman, j.p. shaw, elizabeth emmel, and gerald crabtree. stanford university, stanford, ca 94305. following antigenic stimulation, t cells undergo an orderly series of changes that result in cell division and immunologic function. in hopes of working backward in the cascade which links the membrane events to the genes essential for these differentiated functions, we have begun an analysis of the nuclear proteins responsible for gene activation in stimulated t cells. in previous studies, using a panel of internal deletion mutations of the 1l-2 enhancer, we found that when the region from -13 to -82 was mutated, the level of induction dropped 20-fold. this region is protected in a dnaase footprinting assay by a protein, nf-ilz-a. although the protection is seen with nuclear extracts from both stimulated and resting jurkat cells, in vivo this is a site of dnaase hypersensitivity found only in activated jurkats and peripheral blood t cells. a tetramer of the protected site directs transcription from an unrelated promoter in jurkat cells only when the cells are stimulated by lectin or antibodies to the antigen receptor. furthermore this activation can be blocked by 1 ng/ml of cyclosporin a. as a means of detecting more subtle changes in protein binding following stimulation. the methylation-interference patterns of stimulated and non-stimulated jurkat cell nuclear extracts were compared and found to be identical. we propose that though the dna-binding protein, nf-il2-a, is present constitutively, it becomes functional only following specific signalling through the antigen receptor. further characterization of this protein will help to clarify its particular role in the activation of il-2 expression following t-cell stimulation. the negative e f f e c t of f l a n k i n g sequences on t c e l l lmmunogenicity c561 central lab. blood transf.service, lab. exp. and clin. immunology of the univ. of amsterdam, the netherlands the hydrolysis of phosphatidylinositol-diphosphate and the formation of inositol-triphosphate (ip3) and diacylglycerol (dg) re among the first events that can be monitored after t-cell triggering. ip3 mobilizes c a 2 ' from intracellular stores, whereas dg is the physiological activator of protein kinase c (pkc). although it is suggested that activation of pkc is essential in the induction of mitogenic t-cell responses, no direct evidence for this hypothesis has been presented yet. in this study we used two novel pkc inhibitors, 1.e. 1-alkyl 2 methyl-glycerol (amg) and staurosporine to assess the role of pkc activation in human t-cell stimulation. we observed that the inhibl$ors did not interfere with the anti-cd3 or anti-cdz monoclonal antibody (mab) induced ca rises in peripheral blood t cells, whereas cytoplasmatic alkalinization initiated by the same stimuli or the phorbol ester pma was strongly reduced. amg as well as staurosporine gave a dose dependent inhibition of both interleukin 2 (il-2) production in jurkat cells and t-cell proliferation. in contrast, the induction of il-2 receptor expression was highly resistent to the action of both compounds. it was shown that anti-cd28 mab were able to partly overcome the action of both inhibitors. these studies support the important role of pkc-activation in human t-cell triggering. moreover, since anti-cd28 mab were able to counteract the action of the pkc-inhibitors, it is suggested that mitogenic stimuli generated via different t-cell membrane molecules may use distinct intracellular signal transduction pathways. a f t e r incubation w i t h ndlal-26 o r al-17, t a r g e t s o f @ phenotype were lysed by a n t i -a k i l l e r c e l l s . the a n t i t h e s i s was a l s o observed: a phenotype t a r g e t s were lysed by a n t i -8 t c e l l s a f t e r incubation w i t h ndlgl-17. ndla4 we have developed a monoclonal antibody that recognizes an apparently t cell-specific antigen, ltac, expressed only in the late stages of t cell activation. when peripheral blood lymphocytes are activated with allogeneic stimulators or pha and examined by facs, ltac is expressed only after day 6 or 7. ltac is expressed by all t cell clones tested, both cd4' and cd8'. a wide range of cell types were tested and found negative for ltac expression, including b cells, fibroblasts, endothelium, and neural cells; thus ltac expression seems to be restricted to the t cell lineage. immunoprecipitation of cell-surface labelled proteins and sds-page revealed a 160 kd band under reducing conditions and three bands of 240, 180, and 160 kd under non-reducing conditions. ltac is distinct from all other reported t cell activation antigens on the basis of its pattern of expression and molecular weight. we have purified ltac protein using wheat germ agglutinin-agarose chromatography followed by monoclonal antibody-agarose chromatography, and have used it to produce an antiserum suitable for screening bacterial cdna expression libraries. the regulation and function of molecules expressed in the late stages of t cell activation is an important frontier in understanding the immune system, and reagents such as monoclonal antibodies may have important diagnostic and therapeutic uses. balb.h.p7 mice were capable of responding to all 24 antigens tested but the magnitude of the response to some of these antigens (1 1 of 24 antigens) was reduced 2-5 fold when compared with balb/c mice. responses to the remaining antigens were either comparable (11 of 24 antigens) or occasionally even enhanced (2 of 24 antigens) compared to balwc mice. these results suggest that jp2 and dg2 elements are required to maintain the strength of the t cell repertoire and that, in the wild, mice carrying this deletion a u l d be at a significant selective disadvantage. the identification of 11-1 was carried out in radiolabeled murine macrophages by immunoprecipitation and sds-page, using antibodies to were predominately of mr 31.000 and 37.000, respectively. a s assessed by estimating radioactivity in each band, 11-larwas about 4 fold more abundant than 11-16. most of the cell associated 11-1 activity, as measured by the d10.g4.1 proliferation assay, could be inhibited by anti 11-iswhereas no inhibitory effect was observed with anti 11-1b. after removal of 11-ltc from cell lysates by immunoadsorbtion the residual activity accounted for only about 5 % of the total 11-1 activity, and was completely neutralized by anti 11-1b. however, in culture supernatants anti i1-1r reduced the 11-1 activity by approx. 50 % whereas a combination of both, anti 11-leand anti 11-10 completely neutralized the activity to background levels. interestingly, only one radiolabeld polypeptide of m, 37.000 was precipated from the extracellular medium by the anti 11-16 antibody. these data therefore suggest, that externalization is required in order to generate bioactive 11-18 molecules, but apparently a proteolytic processing step of the precursor is not involved. moreover, in view of previous reports, demonstrating that cellular interactions during antigen presentation can be mediated via a membrane form of 11-1, o u r experiments substantiate the hypothesis that membrane-associated, biologically active 11-1 is predominantely i1-1q. david l. brandon and joseph w. corse. usda agricultural research service, western regional research center, albany, ca 94710 in order to prepare antibodies which would recognize 0-glucosides of cytokinins (purine phytohormones), we investigated an alternative to carbodiimide reagents. carbodiimides induce the formation of amide bonds, and thus can be used to couple haptens containing carboxyl or amino groups to proteins. the use of water-soluble carbodiimides is limited by the sparing solubility of some haptens in water and by the formation of the intermediate 0-acylisourea, which can be less soluble than the hapten and which can itself act as an unwanted hapten. to overcome these problems, we have used 2-morpholinoethyl isocyanide and a suitable catalyst to effect amide bond formation. these reagents are the basis of recently developed chemical procedures for peptide synthesis (aigner et al.. 1980) . the reaction conditions permit coupling compounds which are soluble or insoluble in water to proteins in a simple, homogeneous system. we have demonstrated that this procedure is widely applicable for preparing enzyme-labeled and immunogenic conjugates of cytokinins and other biologically relevant compounds with an available carboxyl group. we hypothesize that conjugates made with these reagents will not induce the allergic responses and unwanted crossreactivities associated with the use of carbodiimides. supported, in part, by bard grant us-711-83. mlman r i g 2 , not uwuse rig2, uxluces a furctional1y-btut-e subset of mdlse thymqtes to. secrete -4, shichin turns iniuces cells w i t h i n this subpapllation to becane cytotcouc. nolmal mxls~ thymocvtes beaw ctl in to con a * il-2, and that the cplresponses induced by-agentsm blocked canpletely by the addition of the anti--4 nyib 1lb11. in addition, normal muse thymxytes produced il-4 in response to con a f i t 2 . the fijmtionally-ture lobster agglutinin (mgl)-positive t h y r e p subset responded to con a f i g 2 in a similar mmer. ylhen the functionally-nunature subset of mgl-negative thymzyk subsets was incubated with con a f muse r i g 2 , they did not becane ctl and &d not prcduce il-4. haever, when wl-negative thymcytes were cultured w i t h con a f human r 1~2 , cells w i t h i n this subpsxllatim developea gccd ctl activity and also secreted 'ihese data qmnstrate that cpl generatim and i g 4 pmauction occur mnccanitantly i n dl thymqte m a t i o r r s studied. momever, stirmli which f a i l to induoe cn generation also f a i l tostinulate erd0qencu.s iir4 praduction. immunity to the typhus group of rickettsiae is largely dependent on the effector function of several classes of t lymphocytes including those which produce gamma interferon. since the surface protein antigen (spa) derived from typhus group rickettsiae has been shown to be an effective immunogen in animal models, human t cell clones specific for the spa of rickettsia tvoh were isolated and tested for their antigenic specificity as well as for their ability to produce gamma interferon. eighteen cm-positive clones specific for the spa of r. t v p b exhibited considerable diversity in their response to the spas derived from two strains of p. orowa zekii and from r. c a n a b . the vast majority of clones also recognized the spa's from & or0 w w but not from r. c w . two heteroclitic clones demonstrated significantly higher proliferative responses to the spa's derived from one or both of the of r. orowazek i i strains than to the spa of r., and one clone demonstrated a significantly higher response to the spa of r. t v o k as compared to the other spas. all 18 clones produced gamma interferon in response to spa stimulation. one of the clones demonstrated significant proliferative responses to a fragment of the spa produced in e. coli infected with a recombinant lamda gt 11 phage containing a 1 kb fragment of the spa gene from r.. we conclude that the spa's from typhus group rickettsiae can elicit both a diverse t cell response in humans as well as the efficient stimulation of gamma interferon-mediated immunity. a monoclonal anti-mouse rbc antibody (g-8) has been prepared which appears to represent a pathogenic autoantibody related to those that arise spontaneously in aging nzb mice and which cause autoimmune hemolytic disease (aihd). this autoantibody recognizes native erythrocytes from mice but not from other species, and northern blot analysis revealed strong hybridization with the j558 probe but not with the 5 other vh gene family probes tested. thus, g-8 is clearly distinct from autoantibodies that recognize bromelain-treated mrbcs and which belong to a unique vh gene family. when g-8-producing hybridoma cells were grown as tumors in balb/c mice, the mice developed aihd characterized by a decrease in the number of erythrocytes (hematwrit) and the development of coombs-positivity. an anti-idiotypic antibody (e8) was prepared against g-8 and was found to recognize an idiotypic determinant present on most autoantibody forming cells derived from old (coombs-positive) nzb mice. previously, we demonstrated that treatment of spleen cells from young nzb mice with g-8 + c or with anti-ly2 antibody + c resulted in the elimination of suppressor cells and the generation of afc specific for mrbc. treatment with a control nzb igm mab (g-4) + c or with a rabbit anti-mrbc antiserum + c had no effect on the afc response to mrbc. similar deletion of suppressor cells was obtained following treatment of spleen cells from balb/c mice with g-8 + c. greater than 80% of the resulting afc expressed the g-8 idiotype. the results indicate that the response to self-erythrocytes is comprised of autoantibodies that express a recurrent idiotype. in an attempt to determine the optimal molecular parameters for the synthesis of anti-pathogen vaccines, we have studied the murine antibody responses to two types of model vaccines. the vaccines were: (1) purified polysaccharide type 3 derived from pneumococcal bacteria, and (2) synthetic conjugates made from a peptide region of the circumsporozoite coat protein of the murine malaria pathogen, p. berghei, coupled with dextran of high molecular weight. in both cases the molecular size was systematically vaned, and an optimal size was found to be important in determining the level of immune responsiveness. in the case of the malaria peptide-dextran conjugate, epitope multiplicity (epitope density) was also found to be an extremely important parameter, immunogenicity increasing with increasing epitope density. the james p. allison. cancer research laboratory, university of california, berkeley, ca. 94720. we have produced an antiserum to a synthetic peptide corresponding to a portion of the murine cd2 gene product. this antiserum reacted specifically with cells expressing cd2. flow cytometric analysis of normal lymphoid tissues revealed that thymocytes were heterogenous in the level of cd2 expression while splenic t cells expressed cd2 at uniformly high levels. cd2 expression with that of other t cell differentiation antigens (il-2r, j l l d , cd5 and cd3) in the murine young thymus and during fetal ontogeny was analyzed using three-color flow microfluorometry. our results indicate that cd2 expression is correlated with the stage of thymocytes maturation and demonstrate that il-2r expression in the thymus is not induced via stimulation of the cd2 pathway. klinische argeitsgruppen fur rheumatologie/immunologie,schwabachanlage 10, 8520 erlangen. accessory cell depleted resting (la) t cells are not able to produce autocrine growth factors when stimulated via the t cell receptor complex (tcr), although il-2 receptor expression takes place. the addition of accessory cells restores the proliferative capacity. we have tested a series of recombinant lymphokines and growth factors for their ability to substitute this accessory cell function for human cdb and cd4 t cells. triggering via the tcr was achieved by a combinatorial stimulation with anti-cd3 and either anti-cd4 o r anti-cd8 that gives rise to il-2 receptor expression only on the corresponding t cell subpopulation. ll-3, tnfa, if+, il-1, il-4, ll-6 and gm-csf were either negative or induced only a minor proliferative response. however, the combination of il-1 with il-6 was nearly as efficient as il-2 in cd4 t cells. cd8 t cells were much less stimulated by il-1+ il-6. effect, which is in contrast to some reports on accessory-cell depleted mouse t cells whose function require costimulation with il-1. this observation might be important for the understanding of t cell proliferation in chronic inflammatory tissues in which il-1 as well as il-6 is produced in large amounts. structural and serological studies of these secreted effector molecules have suggested that they are disulfide-linked dimers bearing t cell receptor u and 6 chain d terminants. a t cell hybridoma, mts 79.1, constitutively antibody staining indicated mts 79.1 expresses a t cell receptor containing a vg8 element. northern and southern analyses indicated mts 79.1 used v68 and va4 genes to encode the t cell receptor. the secreted mts 79.1 suppressor molecule was bound by monoclonal anti-v68 antibodies and by antibodies specific for constant region determinants of the u chain. to assess the role of a and 6 chain genes in encoding the secreted suppressor molecule, we have generated v68-and vu4-negative variants from the mts 79.1 parent. experiments performed to date indicate that the loss of the v68 gene results in the inability to produce the suppressor molecule. the results are consistent with the idea that the t cell receptor u and 6 chain genes are used to encode these hapten-specif ic/mhc-restricted secreted suppressor molecules. producing a dnp-specific/h-zk 5 -restricted suppressor molecule was generated and studied. sensitivity to clonal deletion and selection for mhc restriction a m to be a feature of a particular stage of thymqte &el*, specifically l a t e mrtical cw+8+ cells. in mcent studies we have slwm that early events i n signal trarrwluction i n respnse to antigen r e c p x crosslinkiq differ in the bmdture cw+8+ p p l a t i o n and the mature cw'8-or a>rl-8 p p l a t i o n . m t s indicate that antigen receptolg on hath inmature and mature -itive t cells tmnsdwe signals via calcium mobilization, h-er the maqnitu3e of fnflux of e&acellular q'+ wfiich follckfi birding of antireceptor a n t q d i f f e r s tetmen these pqulaticns. specifically, imnature cells shcw a m& reduced q influx espcnse carpared to mature cells. we dmw here that dligation of ~~n p has different amepexe w i t h regard to q'+ nnbilization in mature and inmnture cells, no sucfi difference is seen f o l l a d r q ligaticm of the receptnr's transducer, a. ihe zpsults suggest that the signall* cascade leading to the influx of extracellular is intact hen c d~ is ligated, but is inccnplete w i e i i m p , the physiological liw, is ligated. in addition, ligation of cw or cd8 on bmdture t cells i n a~~ influx of extracellular a ' + canparable to that sem i n mature t cells. a clonal population has been isolated frcm inmnture thymocytes whir31 has the characteristic signal tramdmtion pxqerties of the tulk of inmnture thymdcytes. 'ihese f i r d h p suggest that "signal" transfer frcm xpnp to 03 may be inefficient in cd4+8+ cells. struchual analysis of the xpnp/cd3 canplex in hnature and mtum t cells is in progress. flood and alan friedman, dept. of pathology, yale university school of medicine, new haven, ct, 06510. protective immunity to the ultraviolet (w) light-induced sarcoma 1591-re is directed toward a single tumor-specific transplantation antigen expressed by the 1591-re tumor cells. termed the a antigen. a progressive variant line of 1591-re, termed 1591-pro4, lacks only the expression of this a antigen. immunization of mice with 1591-re tumor cells haptenated with trinitrophenyl (tnp-1591-re) leads to the subsequent rejection of tnp-haptenated progressive tumors and to increased delayed-type hypersensitivity and ctl responses to tnp in normal, immunocompetent syngeneic mice. however, little or no humoral immunity to tnp is seen in animals injected with tnp-1591-re tumor cells. pro4 did not exhibit tnp-specific tumor protective or cell-mediated immunity, but rather exhibited tolerance to subsequent immunizations with more immunogenic forms of tnp. biochemical and molecular genetic studies have revealed the a antigen to exist on the cell surface as a complex of 3 class i mhc-like molecules. transfection studies with dna encoding each of these molecules into 1591-pro4 reveals that the expression of one, and only one, of these three molecules mediates this increased immunity. these experiments suggest that an mhc-like antigen expressed on the 1591-re sarcoma acts as a natural adjuvant to increase cellmediated but not humoral immunity to linked antigens. the mechanism of this increased immunity is discussed. efforts to immunize cattle against economically important gastrointestinal nematodes showed that inrmunity is manifested by: 1) a response that reduces the fecundity of established and subsequently acquired worms. and/or 2 ) a reduction in the number of worms developing upon challenge infection. however. the ability of individuals to mount such immunity is highly variable. extending these studies to naturally infected populations indicate that there is a great difference in the number of eggs excreted by individual young calves on pasture. to delineate whether these differences were the result of host genetics and to begin to elucidate the mechanisms of resistance to parasite infection, a genetically defined cattle herd was assessed for parasite levels by determining fecal eggs per gram. three years of sampling of the calves during their first grazing season indicates that: 1) certain individuals in the herd will consistently excrete high or l o w numbers of parasite eggs, 2 ) the high or low phenotype is significantly controlled by the genetic make-up of the calf, and 3 ) the high or low phenotype is highly heritable (heritability -40). susceptibility is currently under investigation. calves have been determined and mhc class i1 typing is currently in progress. information is being used to assess the role of the bovine mhc in controlling immunoresponsiveness to parasite antigens. we have been studying the differential effects of il 1 + il 2 versus il 4 on the growth and differentiation of cd 4-, cd 8-thymocytes. culture of highly purified cd 4-, cd 8-thymocytes with il 1 (4 u/ml) + il 2 (100 u/ml) resulted in marked proliferation and increased cell size without change from the cd4-, cd0-phenotype. culture with il 1 or il 2 alone did not cause proliferation. a substantial contribution to the proliferation was secondary il 4 release: addition of anti-il 4 (11b11) blocking antibody inhibited proliferation induced by culture with il 1 + il 2 . il 4 mrna was demonstrated by northern blot analyses after 4 8 and 7 2 hours of culture with il 1 + il 2 whereas none was detected at culture initiation and very little was present at 24 hours. effects of il 1 + il 2 on il 4 transcription rate will also be reported. despite a marked inhibition of proliferation with anti-il 4 , there was no affect on expansion of cd 3+ cells following culture with il 1 + il 2 (increase from 10% to 60% in 7 2 hours). thus, in this system, il 4 enhances proliferation of progenitor thymocytes but does not contribute to induction of t cell receptor. carplex. lhese cwplexes can be used to inmmize mice in the absence of protein carriers or adjuvants, thus facilitating the study of the inmum to a sml1 ckmically defined antigen. use of this tecfinology has allawed us to identify two t helper cell epitopes in cclllserved regions of hn gp 160 not previcusly identified by computer algorithims, defined by amino acids 485-518 and 585-615. inummization with these peptides in pptide-@nqhlipid cwplexes results in the prpauction i* and i* antibodies, which cioss react with cloned fragmnts of the w l e protein. us& this &logy we have begun to characterize the innume response to individual peptide antigens. ?he reqonse of h2-k mice to amino acids 494-518 of 9p 160 of hiv, has been analyzed. lhe optimal dose of a peptide containing both b and t cell epitopes was found to be 15-30 ug, depending on the route of administration. im d z a t i o n requir& less antigen for opthum antibody resporrsf, than did ip. ment for an ant-response. additional variables, as phosfholipid carpasition and method of cross-linking have been studied anl will be . webelievethattheuseof this peptide-phospholipid canplex tehmlogy will be significant both for studying the innwe response to single epitcpes and for vaccine develolment. based on assays in which t cell proliferation was induced via oxidative mitogenesis and exposure to mhc alloantigens, it has been reported that langerhans cells (lc) isolated from normal mouse skin aquire maximum capacity to activate t cells only after 72 hours of culture. we have studied lc from balb/c mouse skins for their capacity to present ovalbumin (ova) and ia doantigens to unprimed t cells and to antigen-specific t cell hybridomas. the data reveal that both fresh and cultured lc presented ova and alloantigens with equal efficiency to previously primed responders (and with 10 fold greater efficiency than spleen cells or the b cell lymphoma a20.1-11). by contrast g& cultured lc displayed the capacity to present antigen to ynorimed t cells. we propose that the antigen presenting potential of freshly prepared and cultured langerhans cells, respectively, reflect the in vivo functional properties of intraepidermal lc and of lc that have picked-up antigen in the epidermis and migrated via dermis to the regional lymph node. if so, these data suggest that resident epidermal lc are fully prepared to present cutaneous antigens to memory/effector t cells (efferent limb), whereas resident lc must leave the influence of the epidermis in order to develop the capacity to meet the more stringent conditions required for antigen presentation to porimed t cells (afferent limb). we have investigated the structural restrictions placed on residues contained within a minimal t cell determinant, using the balb/c class i1 restricted t cell response to the site 1 determinant of the influenza hemagglutinin molecule as a model system. to delineate which of the residues comprising the site 1 determinant are involved in interaction with the t cell receptor, we have determinaed the response of a large panel of site 1 specific t cell hybridomas to a collection of peptide analogs differing by single conservative or non-conservative substitutions at 9 positions. the fine specificity patterns of the t cell panel is extremely diverse; t cells varied in both the location and number of residues within the antigenic peptide that effected recognition. our results implicate at least 6 out of 9 residues within the antigenic pepetide as being involved in interaction with the t cell receptor. this result suggest that peptides comprising the site 1 determinant do not form alpha helical structures when in association with mhc molecules. rubella-specific isotype and igg subclass responses were evaluated using elisa techniques in 44 rubella ha1 seronegative adult females undergoing rubella immunization (ra 27/3 strain). responses were evaluated prior to immunization and at 1,2,3,4,5,6,12 and 24 wks post-immunization. pre-immunization sera showed detectable levels of rubella-specific antibody in the igg class (17/44); iga class (32144) and in one or more of igg subclasses (22/44). post-immunization, i 1 subjects failed to develop igm class responses by the ha1 (sdg) technique while 43/44 developed igm antibody by elisa techniques. iga responses were detected at low levels in all vaccinees beginning at 3-4 wks and declining by 24 wks post-immunization. antibody in iggl and igg3 subclasses by 2-3 wks post-vaccine with sustained iggl levels but significant decline in igg3 levels noted between 6 and 24 wks post-immunization. no seroconversion was noted in the igg2 subclass although 9 individuals had detectable pre-immunization iggz rubella antibody present. igg4 levels were detected in all vaccinees post-vaccine with a delayed and progressive rise over the study period. subsequent correlation was then performed between rubella-specific antibody responses and the presence or absence of adverse joint reactions occurring in association with rubella vaccine administration. all individuals produced detectable c624 t lymphocyte responses to varicella zoster virus. anthony hayward, abbas vafai, roger giller & eileen villanueba. departments of pediatrics and microbiology, university of colorado school of medicine, denver co 8 0 2 6 2 61 university of iowa school of medicine, iowa city i0 the proliferative response of blood lymphocytes from varicella zoster virus (v2v)-immune donors to live vzv, extracted vzv antigens or purified glycoproteins is predominantly by cd4+, hla-d restricted t cells but little is known of the specificies of the responder cells. we restimulated t cells cloned by limiting dilution from vzv-stimulated cultures with purified vzv glycoproteins gpi, gpii and gpiii and found that t cell clones with specificity for each of these mediated both help for antibody responses and hla-dr restricted vzv-specific cytotoxicity. 5 polypeptides of 10 to 14 amino acids length corresponding to predicted amphipathic sequences in the primary structures of g p i, gp i1 and gp iv were synthesised. proliferative responses were observed to 3 of these peptips (one from each glycoprotein) with responder cell frequencies in the 1:lo blood t cells range. the gp i peptide additionally defined an epitope recognised by serum antibody. an immunomodulatory approach to treating hsv-1 corneal disease, hendricks rl, departments of ophthalmology, and microbiology/immunology, university of illinois school of medicine, chicago, il 60612 herpes simplex virus type i (hsv-1) corneal infections are a leading cause of blindness worldwide. we and others have demonstrated that the cellular immune response to hsv-1 contributes to the elimination of virus from the cornea, but in doing so causes the tissue destruction that is responsible for the blinding complications of the disease. we have demonstrated that specifically suppressing the cytotoxic t lymphocyte (ctl) response to hsv-1 renders mice resistant to corneal disease following topical corneal hsv-1 infection. in agreement with this observation was our recent finding that in vivo depletion of wt4' (t helper/inducer, and most dth effector cells) neither reduced susceptibility to corneal disease, nor increased susceptibility to disseminated disease. the corneal lesions in wt4 depleted mice contained numerous lyt-2 (t suppressor/cytotoxic) cells, and no l3t4 cells. the wt4 depleted mice exhibited normal hsv-specific ctl precursor frequencies. experiments designed to determine the effect of in vivo lyt-2 depletion on susceptibility to corneal disease are in progress. our goal is to identify cellular immune responses to hsv-1 that maximize protection, while minimizing immunopathology in the cornea, and identify hsv-1 epitopes that preferentially activate those responses. supported we found that affinity purified antibodies to bsa, klh and diptheria toxoid all contain a substantial amount of specific anti-idiotypic activity. against bsa react with mouse anti-bsa antibodies, which suggests that we are dealing with internal image antibodies. 9 mrl-lpr/lpr mice develop spontaneous autoimmunity. we found that these mice make anti-anti-(self h-2) antibodies prior to making appreciable amounts of pathological autoantibodies such as anti-dna, anti-rnp.sm, and rheumatoid factor. the anti-anti-self antibodies are detected using an inhibition of antibody mediated cytotoxicity assay, that also detects anti-anti-(self h-2) in ordinary allogeneic anti-sera. the antibodies are not rheumatoid factors, although the animals do make rheumatoid factors later in the development of the disease. anti-self activity is fully developed at 2 months, when the other autoantibodies are typically barely detectable. important role in the etiology of the disease. the anti-we conclude that anti-anti-self antibodies could play an c 627 feedback regulation of 11-1 synthesis in monccytes by t cell products: dual effect of 11-4. mikko hurme, tessa palkama and marja sihvola, department of bacteriology and immunology, university of helsinki, sf-00290, helsinki, finland. il-i production of human monocytehnacrophages is regulated by several cytokines some of which are themselves able to activate the il-1 production (e.g. tnf and il-2) while others (e.g. ifn-y) modulate the production activated by other signals. we have now examined the effect of 11-4 on the 11-1 synthesis. 11-4 alone did not induce any 11-1 bioactivity or il-la or 11-1 0 mrna expression in freshly isolated peripheral blood adherent cells. in contrast, il-4 effectively suppressed the lps induced 11-1 production. this suppression took place without any decrease in the steady-state levels of il-la and il-i 0 mrna, suggesting that this downregulative effects is posttranscriptional. monocytehnacrophages are known to rapidly loose their ability to produce il-i when cultivated in vitro. if ifn-yis present in the culture fluid, the cells remain capable of producing 11-1. as ifn-yand have been reported to have similar "priming" effects on macrophages (e.g. increasing the tumoricidal capacity and mhc class ii antigen expression) we cultivated monocytes for 24 h in the presence of either ifn-y or 11-4, and after washing the cells they were stimulated with lps. il-1 activity could be detected both in the ifnyand il-4 preincubated cultures (but not in the cultures preincubated with medium alone). these data suggest that il-4 can also display a similar upregulatory function in il-i production as ifn-y. gahreston, tx 77550 development of immunity to members of the spotted fever group of rickettsiae is a t-cell dependent response. we have used t-cell hybridomas and cloned t-cell lines from immune animals and convalescent humans to identify the rickettsial antigens that induce antigen-responsive t-cells. in these studies we found that the 155 kda antigen of rickettsia tickettsii. the causative agent of rocky mountain spotted fever, is one of the immunodominant tcell antigens. t-cells from immune animals and humans were responded in culture to a recombinant 155 kda antigen. both sources of t-cells were of the t-helper type (l3t4* and 04' respectively) and produced 1l-2 and interferon. it was found that soluble antigenic material of b. rlckettsii obtained by extraction with hypotonic buffer maximally stimulated the t-cell lines. this material was enriched far the high molecular weight polypeptides of 1s5 kda and 120 kda. also. ethirwwi ' will induce a long-lived immunity against infection with r. rickettsii. infected guinea pigs develop a minimally cross-reactive antibody response to b. nckettsii. in contrast. a strong cross-reactive t-cell proliferative response is produced. studies are in progress to determine the nature of the common protective antigen of r. humans infected with the parasitic nematode ascaris lumbricoides vary considerably in antibody responsiveness to a 14 kda component of the parasite. this molecule is secreted by the parasite, and is also abundant internally. this heterogeneous reactivity has been modelled in laboratory rodents, and the antibody response to it is h-2-and rt1-restricted in mice and rats, respectively. using inbred and congenic animals, only mice of h-2' and rats of rtiu were, so far, found to be responders, and this restriction only operated in the context of infection. the specificity of the ige response in these animals was assayed by passive cutaneous anaphylaxis, and in an ige-specific elisa assay. the data show that the above mhc restriction also applied to the specificity of the reaginic antibody response, although animals of all mhc haplotypes responded to other ascaris allergens. amino acid analysis of the 14 kda equates it to a previously identified "allergen a" of the parasite, and we now have its sequence available. these findings have implications for the genetic control of allergic responses in general, and, in particular, to the hypersensitivity responses which are such a feature of infections with parasitic nematodes. there are also implications for the generation of hypersensitivity responses by recombinant vaccines involving certain parasite antigens. the cns. immunohistochemial analysis of both frozen sections prepared from the brains of animals immunized in this manner and of highly enriched glial cell subpopulation cultures for viral gp70 expression indicated that oligodndrocytes and possibly a subset of astrocytes were the targets of this infection. further, microscopic analysis of frozen sections failed to reveal any overt signs of gross pathologic changes associated with the viral infection. we have been able to demonstrate the presence of virus specific antibody in the serum of these mice as well as virus specific cytolytic t cells in the peripheral lymphoid organs. ments are currently underway to determine whether the lack of pathology associated with wb91 infection in light of the previously shown virus specific immune responses in these mice is due to a failure of antigen presentation within the cns or some other form of immunoregulatory phenomena. the t lymphocyte proliferative response to pigeon cytochrome 5 in bio.a mice is restricted to the egk:e,k ia molecule and specific for the c-terminal determinant comprised of residues 93-104. blo.a(3r) and blo.a(sr) mice are nonresponders to pigeon cytochrome 2 nonetheless, the t cell repertoire of blo.a(3r) or (5r) contains some t cell clones capable of recognizing and proliferating to pigeon cytochrome c w h e n presented by bio.a antigen-presenting cells (aft). therefore, one would expect to stimulate such clones in allogeneic bone marrow chimeras of the type bio.a +blo.a(3r) or (5r) b1o.a apcs and a blo.a(3r) or (5r) t cell reperto4!6~.~espectively. ,en(isea9c22rzave were primed with pigeon cytochrome cytochrome 5, they showed a good antigen specific proliferative response in vitro. surprisingly, however, if pigeon cytochrome 5 was used for priming, no response was detected, even at priming doses as high as 400 nmol per mouse. 81-104 could only be achieved by treating the allochimeras with an anti-cdb monoclonal antibody in vivo during the priming step. clones specific for purified protein derivative (ppd) in the same chimera. thus the regulation which involves cd8 positive cells is antigen specific. transfer of pigeon cytochrome 81-104 primed lymph node cells from the chimera into naive bio.a mice prevented priming of the recipient for a t cell proliferative response to pigeon 81-104, but not priming to the moth synthetic fragment. chimeras of an antigen-specific suppression mechanism involving cd8 positive cells. faculty of medicine, kyoto university, kyoto 606, and department of oncology, nagasaki university school of medicine, nagasaki 852, japan. sera from b6 mice immunized with a syngeneic ctl specific for fbl-3 tumor of b6 origin blocked the cytotoxic activity of only the immunizing ctl clone. therefore, a monoclonal antibody (mab) n9-127 was produced by fusion of the b6 spleen cells immune to a syngeneic fbl-3-specific ctl clone (no. 8). the specificity of the mab n9-127 was confirmed by immunoprecipitation, blocking of cytolytic activity, stimulation of proliferation, and induction of tcr-mediated nonspecific cytolysis of the ctl clone no. 8. in some b6 mice, 3-13% of the anti-fbl-3 mltc cells were positive for this n9-127-defined idiotype, and formed a well demarcated population upon examination by flow cytomehy. even in mice in which no such population was observed some ctl clones established by limiting dilution culture were also positive for this idiotype (10 out of 89 clones from 3 mice). the cytotoxic activities of these ctl clones were blocked by n9-127, which in turn induced the nonspecific cytolysis in redirected assay. however, no positive cells were detected in non-cultured normal or fbg3-immune spleen and lymph node cells. this indicates the presence of cross-reactive (dominant) idiotype in the b6 anti-fbl-3 cytotoxic t cell responses and may provide a potent tool for analyzing the idiotype-mediated regulation of the anti-tumor immune responses. slade andsylvie gillard, max-planck-institut fur immunbiologie, d-7800freiburg, federal republic of germany t cells play a n essential role in t h e protective immune response to malaria and a r e associated with s o m e of t h e pathological consequences of t h e disease. however, t h e n a t u r e of their responses and t h e antigens t o which they respond a r e not well defined. w e have developed a limiting dilution assay system in which specific t cell responses to malaria antigens c a n be monitored a t t h e clonal level. i t is possible to determine t h e nature of t h e responding t cell by t h e growth f a c t o r s they s e c r e t e and by their ability to a c t as helper cells for t h e antibody response to malaria antigens. our d a t a suggest t h a t t h e t cell response changes during t h e primary infection and in hyperimmupe animals. o n e to t w o weeks a f t e r initiation of a blood s t a g e infection t h e major cd4+ t cell which proliferates in response t o parasite antigens s e c r e t e s il-2 and ifn-y but is not a n efficient helper cell for antibody responses. in c o n t r a s t l a t e r in infection and in immune animals t h e r e is an e f f e c t i v e helper cell response and many of t h e s e cells a r e distinct from those secreting ifn-y and il-2. we a r e currently investigating whether these cells retain these phenotypes when grown in long-term in vitro culture and whether defined antigens of t h e erythrocytic parasite elicit different t cell responses. we have localized linear neutralization epitopes on the coronaviruses ibv, mhv, fipv and tgev. the results can be summarized as follows: 1. linear epitopes of the spike proteins (1162-1452 residues) could be mapped to a resolution of a single residue by expression of gene fragments in the prokaryotic pex plasmids and/or pepscan peptide synthesis. 2. the length the epitopes varied from 4 to at least 20 amino acid residues. we present evidence that the larger epitopes, although conformation-independent according to operational criteria, are nevertheless discontinuous. 3 . in ibv, we localized several overlapping but different epitopes within an immunodominant region of 30 residues. this region is recognized by all polyclonal antisera tested. we propose that its immunodominancy is a consequence of its structure and function and does not depend on antigen presentation or idiotypic networks. an immune response against the mouse testis-specific antigen ldh-c4 reduces fertility by 70 percent in female baboons. an immune reaction to human ldh-c4 would be expected to be more effective in primates. since the human testis enzyme is not readily available in large quantities, recombinant dna technologies were uscd to create a source of human ldh-c4. antibodies to mouse ldh-c4 were used to screen a xgtll human testis cdna expression library. a full length human ldh-c clone was identified, sequenced, and the ldh-c cdna was engineered for expression in e.coli. the 5' and 3' untranslated sequences were removed by restriction enzyme digestion, and synthetic linkers were added adjacent to the start and stop codons of translation. the modified cdna was subcloned into the prokaryotic expression vector pkk223-3 and introduced into w3110 l a c iq cells. cells were grown to mid-log phase, and induced with iptg for positive regulation of the strong hybrid tac promoter. induced cells overexpressed the 35 kd subunit which spontaneously formed the enzymatically active 140 kd tetramer. human ldh-c4 was purified 196-fold from liter cultures of cells by two step affinity chromatography to a specific activity of 90 i.u./mg. the 20 n-terminal amino acids sequenced were identical to those predicted from the nucleic acid sequence. antibodies to synthetic peptide epitopes of human ldh-c4 cross-reacted with the enzyme produced in e.coli. two mg human ldh-c4 were expressed per liter of bacterial cells. the purified protein is now available for innunogenicity and fertility studies. it is now generally accepted that the principal effector mechanism in the host's defence against leishrnaniasis is gamma-interferon (ifn-y) which activates infected-macrophage to eliminate intracellular parasites. mice by prior sublethal whole body irradiation or treatment with anti-igm or anti-cd4 antibody. protection can also be induced by repeated intravenous or intraperitoneal immunisation with killed parasites or purified antigens. ly immunised mice produce little or no il-3 or il-4 but substantially elevated levels of ifn-y when stimulated with leishmania1 antigens in vitro. lymphoid cells from balb/c mice with progressive disease can inhibit the maf (macrophage activating factor) and leishmanicidal activities of the culture supernatant of lymphoid cells from mice recovered from l. major infection. maf appears to be ifn-y, whereas the maf inhibiting factors are il-3 and il-4. system can be reproduced with recombinant ifn-y, il-3 and il-4 and the maf inhibiting activity of the suppressive supernatant can be reversed by specific anti-il-3 and anti-il-4 antibodies. the disease by influencing the ability of macrophage to kill the intracellular parasite. the development of efficacious vaccines against malaria requires an understanding of the mechanisms involved in protective immunity. f'revious studies with plasmodium berghei demonstrated that sporozoite immunity is dependent upon antibody responses specific for the repeat region of the circumsporozoite (cs) protein and cell mediated mechanisms involving cd8+ t cells. in this study we analyzed the splenic t cell repertoire directed against epitopes on the cs protein of p. berghei and determined whether sporozoite-immune cd4+ and cd8+ t cells respond to shared or distinct epitopes. sporozoite-immune spleen cells, cd4+ and cd8+ enriched t cell populations of balb/c (h-m), c3h @i-%), and c57bv6 (h-2b) mouse strains were cultured in the presence of irradiated sporozoites or synthetic peptides representing 70% of the complete cs protein. surprisingly, none of the cultures proliferated to any of the peptides tested, although proliferative responses to sporozoites were observed in unfractionated spleens and cd4+ t cell populations. cd8+ t cells did not respond to any of the antigens tested, even in the presence of exogenously added 11-2. titration of cd8+ cells into proliferating cd4+ cell cultures did not suppress the anti-sporozoite response. the lack of anti-peptide reactivity contrasts with uniform responses to sporozoites and may be the result of the context in which cs antigens are presented to t cells. functional analysis of accessory splenic b cells and macrophages revealed that while the anti-sporozoite proliferative responses were not affected by the removal of macrophages, sporozoite-primed b cells were essential for the responses. these data suggest that the cs protein on sporozoites is not processed extensively by macrophages to yield many potential t cell epitopes, but instead is presented by immuncdominant b cells that resmct responses to a limited number of t cell clones. of the primary infection and is also required for optimum protection against reinfection. current studies have demonstrated that relatively few of the viral antigens tested to date ( 7 viral envelope glycoproteins or 2 nonsmctural nuclear proteins) are recognized by hsv-1 immune ctl populations generated in several different strains of mice (h2 haplotypes h2b, h2d. or h2k). this failure of hsv specific ciz to recognize the cloned gene products in in v i m assays was demonstrable at the clonal level and could not be attributed to a peculiarity of the recombinant vaccinia conshucts used because studies with adenovims vectors or tranfected l cell constucts yielded the same results. surprisingly, despite their inability to be recognized by hsv specific ciz in vim, when used to immunize mice several of the vaccinia virus constructs would induce memory ciz populations capable of lysing hsv-1 infected autologous cells. for example, hsv-1 glycoprotein c (gc) was recognized by h2b restricted but not h2k restricted hsv specific ctl. however, immunization of either haplotype of mice with a vaccinia gc recombinant induced ctl populations which upon in v i m restimulation with hsv-1 would lyse histocompatible cells infected with hsv-1. this demonstrates that despite the presence of suitable epitopes (intrinsic factors) the context of the immunogen (extrinsic factors) will also influence it's ability to induce ctl. the results of further studies into the nature of these extrinsic factors will be presented and discussed with relevance to future sub-unit vaccine design. w d ~upponrd by public ~~l t h service g~mu, ai 14981 md ai 24471 fran the ti-^ ~n,litu= md lnrcniour d ,~~~~~~. infection of mice with hsv-1 induces a brisk ciz response which is necessary for the subsequent resolution we have investigated the structural basis for antigen mimicry by anti-idiotypic internal image antibodies. two mouse monoclonal antibodies (mabs) that bear internal images of a well-defined protein epitope, i.e., the rabbit immunoglobulin (ig) a1 allotype, were produced and the variable region sequences were determined by rna primer extension sequencing. the results showed that the mab light chains did not contain any allotype-related residues; however, both heavy chain v regions contained a unique sequence homologous to the nominal antigen but in opposite orientation. this reversed sequence was expressed within cdr2 of both mabs. synthetic peptides corresponding to the putative antigenic regions of rabbit ig and the mab internal images, respectively, were tested for the ability to mimic the al-like determinant. although the homologous residues were presented in opposite orientations, both peptides completely inhibited at similar concentrations the binding of rabbit ig to anti-a1 antibody. a paired thr and clu was necessary for expression of the a1 epitope as revealed by conservative substitutions in the peptide sequence. computer-generated, energy-minimized models of rabbit ig and the mabs revealed that the critical a1 residue side chain placements could be almost superimposable in either context. thus, it appears that an antigenic epitope can be determined solely by md 20892. proliferation of murine type i cd4+ t cell clones quires simultaneous occupancy of the t cell antigen receptor and delivery of an accessory cell-duived costitnulamy signal in contrast, isolated t cell receptor occupancy induces the cell into a state of reduced proliferative responsiveness to antigen. based on the observation that pkc-activating phorbl esters can at times substitute for the p s e n c e of accessory cells in t cell proliferative response. to mitogens or anti-cd3 mnodonal antibodies, we investigated the requkment for accessory cells in the antigen-and con a-induced hydrolysis of p m and activation of pkc. the presence of normal accessory cells was found to be unnecessary for the development of pkc-dependent phosphorylations and the addition of normal accessory cells had no effect on the activity of pkc. cell il-2 synthesis and proliferation presents a paradox. we have studied the effects of ueatment with a calcium ionophore and p h h l ester on t cells and find that increased [ca2+]i and pkc activation are in fact insufficient biochemid second messengers in the induction of proliferation. while pliferation was induced at high t cell density in response. to these stimuli, incubation of t cells at decrrased cell density drmonsuated markedly reduced proliferation, and single t cells failed to divide. this suggested that cellular interactions were. q u i r e d in the response. additions of either il2 or normal accessory cells allowed p l i f d o n at low density, consistent with a requirement for an accessory cell-derived costimulatoq signal in the induction of i l 2 synthesis, even in the plifcrative response to ionomycin and pma. this result underscons the importance of an accessory cell-duived costimulatory signal, acting independently of t cell receptor-mediated increases in [caz+] i and pkc activation, in the induction of t cell proliferation. we describe experiments designed to determine the molecular requirements for recognition by fluorescein-specific ctlps and ctls derived both from n a i v e and from immunized mice. we the production of prostaglandin e, a major immunesuppressor secreted by the macrophages was inhibited by the addition of 0.1 m indomethacin to the cultures of monocytes harvested from patients suffering from pulmonary tuberculosis and those from equal number of normal controls. the il-i activity was estimated i n the supernatants of these cultures by their ability to proliferate mice thymocytes. it was found that the supernatants from cultures with indomethacin showed a greater il-i activity than the ones without it(44% p 0.001). this indicates the possibility of pge offering a negative feedback control over il-i production. the defective cell mediated immunity i n patients with pulmonary tuberculosis may be explained through the inhibition on il-i production by pge whose enhanced production is reported i n our earlier studies. the results and our hypothesis on the autoregulation of il-i production w i l l be presented and discussed. in variant viruses which differ from the parental virus (gv) at specific epitopes recognized by monoclonal antibodies directed against the env gene product, gp70. biological clones isolated from gv express the gv phenotype suggesting that the loss of specific epitopes is the result of selective de novo processes in the immunocompetent host. additionally, inoculation of adult mice with a biological clone expressing the gv phenotype also results in similar variant viruses. however, inoculation of gv into neonatal or nonlethally irradiated mice results in a population of viruses expressing only the gv phenotype suggesting that the emergence of antigenic variants may be influenced by neutralizing antibodies and/or cellular host res sds-page analysis of immunoprecipitates of sg~s~~,elled lysates of fibroblasts infected with clones expressing gv or variant pehnotypes shows a size difference of the gp70 precursor. additionally, the recognition of a neutralizing epitope (e-55) associated with gp70 by mab55 is dependent on the appropriate native conformation of the epitope which appears to require glycosylation for expression. experiments are in progress to further examine the immunogenetic basis for the generation of these variants and to determine the molecular changes in the virus genome responsible for changes in epitope expression. investigated the capacity of murine splenict cells depleted of acceso cells ( ac ) t o proliferate in response t o stimulation by con a, @ cd3 ab and activated t cells. the zepletion procedures consisted of carbonyl iron treatment, 2x "panning " on anti-ig coated flasks, 2x anti-la cytotoxic treatments and percoll gradient purification of small resting t la-cells. the appropiate concentration of con a (10 ng/ml ) and plastic-bound (pb) @cd3 lg or its f (ab)' 2 fra ments induce proliferation, 112 r expression and 112 (but not 114 secretion in t la-cellscultured for 48% at 5x105 cells/well . responsiveness of tlacells t o con a and dcd3 in low density cultures (5x104 cells/well) is restored by the addition of irradiated th2 cloned cells but not thl ,splenic cellsor r l l l + rll2 + r114. likewise, responsiveness t o non activating doses of con a (lnglml) or soluble @cd3 is restored by the addition of irradiatedth2 costimulatory cells . these experiments demonstrate that the ability of t cells t o proliferate in the absence of ac is critically dependent on t-t interactions. t cell subsets prepared by either negative (l3t4-and ly2'cells) or positive selection proliferate in response t o pb @cd3 lg . although the proliferative responses of both l3t4-and ly2-cells are maximal at 48h, the l3t4-cells require l o x more pb @cd3 lg for maximal stimulation and their res onses decline much faster than those of ly2cells. in addition, l3t4-cells are not stimulated by pb &cd3 f(ab)'2 fra ments and their responses t o @cd3 i are inhibitable by anti fcr as well as anti-lfa abs . re onsesof%oth l3t4-and ly2-cells are !nhibita%le by @i12 and @i12 r abs but not by @l3t4, @ly2 or b l l 4 . these experimentsdocument interesting differences in the triggering requirements of l3t4-and ly2 'cells. supported by nih grants po1 ca 19266, t 326m-07183 and gm 07281. the 19k glycoprotein encoded in the e3 region of ad2 and ad5 (gpl9k) binds to class i mhc antigens in the endoplasmic reticulum and prevents their translocation to the cell surface. this has been proposed as a mechanism by which virus infected cells can avoid recognition by the host cytotoxic t lymphocyte (ctl) response. we have shown that gpl9k can inhibit target cell lysis by adenovirus specific ctl, but the effectiveness of this inhibition varies greatly between different mouse strains. this is due in part to differences in the affinity of gpl9k for different mhc class i molecules, but this cannot account for a l l the variation observed. i t has been shown that cd4+8+ immature thymocytes fail to secrete il-2 or express il-2 receptors in response to activation signals. furthermore. they cannot induce il-2 gene transcription. several tumor lines have now been characterized which have a cd4+8+ phenotype and fail to secrete i l -2 or express il-2 receptors in response to stimulation with ionomycin plus pma. these cells also do not express il-2 mrna after stimulation, as determined by northern blotting and rnase protection. to determine the molecular mechanism for this lack of transcriptional activity, nuclear extracts were analysed for the presence of the d n a binding factor nfat-i. this nuclear factor i s present only in ac we have undertaken an mhc analysis using the polymerase chain-reaction (pcr) and dot blot analysis of the amplified lyme arthritis patients dna with allele specific oligonucleotide (aso) probes. genomic dna for the first domain of the dq beta chain and of the dr/pi from 20 patients with lyme arthritis has been amplified and we are analyzing the distribution of dridrr and w a l l e l e s in this population to test the hypothesis that the mhc class i1 genes might be involved in presentation of selected spirochete epitopes whose recognition by t lymphocytes leads to lyme arthritis. most inbred strains of mice do not respond to porc insulin (pins). experiments were conducted to elucidate the mechanism of the non-responsiveness in h-zk mice: 1) purification of cd-4' t c e l l s from pins-immune b1o.mbr mice revealed pins-specific t helper (th) cells, 2) these pins specific th cells could be activated by i-ak and i-ek expressing l929-fibroblasts. therefore, both i-ak and i-ek molecules can present pins in an immunogenic manner and activate pins-specific th cells. by means of different cell-fractionation procedures, it was found that antigen-specific t suppressor (ts) cells regulated the pins immune response. these ts cells were of the fcr-, cd-4-, cd-8'. thy-1' phenotype, and they were present in normal mice. we believe that these experiments indicate that antigen-specific ts cells exist and are important regulators of immune and autoimmune responses. the possibility of functional inactivation of cd4+ clones by ts-cells was investigated. m leprae-responsive cd4+ clones were preincubated with ts cd8+ clones, apc and antigen for 1 ; hours. after which the cd8+ cells were removed from culture. the cd4+ clones were then restimulated with e. leprae and apc. cd4+ clones incubated with cd8+ cells and antigen were unresponsive to restimulation by antigen, although they were not killed and could respond well to il-2. addition of il-2 in the preor post-incubation culture neither prevented the induction of unresponsiveness nor reversed it. earlier models of tolerance have suggested that receptor occupancy in the absence of second signals induces tolerance in b-and t-cells. we would suggest that in the presence of ts-cells. a second signal may be negated leading to th-cell unresponsiveness. university of texas southwestern medical school, dallas, tx 75235 graft versus host disease (gvhd) poses a serious threat to the survival of patients with bone marrow transplants. the state of immunosuppression established in gvhd results in a variety of immunological abnormalities at the humoral and/or cellular level. we have developed a murine model of chronic gvhd across a minor histocompatibility (mh) barrier. in this model, immunosuppression develops. spleen cells from mice undergoing this type of gvhd are unable to respond to the polyclonal activators lipopolysaccharide and concanavalin a . however, the response against the b cell leukemia bcll remains intact. the protective immune response against bcll is directed towards the mh antigen h-40 and is mediated by cytotoxic t lymphocytes. thus, the specific t cell response against a mh antigen can occur in the presence of chronic gvhd despite the absence of a polyclonal b and t cell response lymphocytic choriomeningitis virus (lcmv), a member of the arenavirus family, has a biseqmented rna genome which encodes at least three polypeptides. the smaller rna segment encodes two virus structural proteins, the slycoprotein (gp) and the nucleoprotein (np). upon infection of mice with lcmv a cytotoxic t cell immune response directed against these proteins is measurable in vitro and in vivo. it can be demonstrated that depending on the haplotype of the mice, one or the other protein may play a major part in the immune response. in order to define the immunogenic epitope(s) of the nucleoprotein which are recognized specifically by the t cell receptors of cytotoxic t cells, stepwise 3 ' truncated qene fraaments encodina the nucleoprotein were cloned and expressed in vaccinia virus. with these recombinant vaccinia viruses, protection experiments in mice aqainst lcmv infection were performed in parallel with in vitro studies, namely specific recoonition of target cells expressing truncated fragments of the nucleoprotein by lcmv primed spleen cells. cdna clones encoding the mouse and human t cell il-1 receptors have been isolated and expressed in mammalian cells. the recombinant receptor binds il-1 indistinguishably from the natural il-1 receptor, and is functional in signal transduction. deletion of the cytoplasmic portion of the receptor abolishes its signal transduction abilities. sequence and secondary structure analysis suggest that the cytoplasmic segment of the il-1 receptor binds a nucleotide. experiments designed to test this hypothesis and to examine the mode of signal transduction will be presented. also to be discussed are the mechanism of triggering of the receptor by il-i. and the nature of il-1 receptors expressed in other cell types such as b cells. it became evident that these subsets reflect different stages of helper t cell maturation before and after activation. therefore, these t cell subsets have been designated as naive t cells (cd45r/2h4+, cdw29[4b4]-) and memory t cells (cd45r/2h4-, cdw29[4b4]+). we analysed the expression of these antigens in dermal lymphohistiocytic infiltrates from different benign skin diseases and cutaneous t cell lymphomas (chronic contact dermatitis (n=14), parapsoriasis en plaques (n=5), lymphomatoid papulosis (n=4), mycosis fungoides (n=15), sezary's syndrome (n-2), pleomorphic t cell lymphoma (n=6) and high grade t cell lymphomas (~4)). in almost all cutaneous t cell infiltrates memory t cells were preferentially found whereas in the peripheral blood both subsets are equally distributed. this implicates, that t cells infiltrating the skin already have had contact with their respective antigen. where the switch from naive to memory t cells takes place can not be answerded by our findings, as we have investigated rather longstanding skin diseases. however, these memory t cells, which can be activated more easily, make diseased skin more e f f e c t i v e in the nmdlification of an immune resnonse. organs and after several days of stimulation with antigen or mitogen and lymphokines. we find that fresh th synthesize and secerte -iu,ifng,il3 and gmcsf but very little il4 or il.5 within 24-48 hours. this pattern resembles the pattern of lymphokines secreted by thl cell lines. the th responsible for this secretion are cd4 positive t cells which are long-lived since they disappear very slowly following adult thymectomy. they are also sensitive to the in vivo administration of ats(antithyrn0cyte serum) and they express high levels of pgp-1. the kinetics of lymphokine secretion and the phenotype of the cells suppon the hypothesis that lymphokine secretion from fresh lymphoid cells comes from a population of memory cells. in contrast we fmd that we can also stimulate a separate, ats-resistant population to become lymphokine-secreting cells after four days of in v i m priming.these primed cultures rapidly synthesize an secrete large amounts of i u and il5 in addition to ifng , il3 and gmcsf( a phenotype which could be combination of both thl and th2 helpers), when they are restimulated with ag or mitogen. the cells whic are responsible are cd4 positive and have a shorter liespan since they decline considerably after adult thymectomy. we suggest that the lymphokine secreting cells detected after priming come from a population(s) of helper t cell precufiofi which have differentiated to become effectors. this generation 0: effectors requires lymphokines, especially e-4 andlor ilz and apcs. thus the development of helper th appears to follow a similar pathway as that of cells of the b cell lineage developing into ab-secreting cells and cd8 positive t cells which develop into cytotoxic effectors from precursors. we have studied the secretion of lymphokines by helper t cells freshly obtained from lymphoid interleukin-2 production by t cells has been shown to be required for both humoral as well as cell-mediated imune responses. thus, il-2 production was measured in syphilitic rabbits as a function of their imune response. maximal il-2 production induced by con a at 10-14 days post-infection was only l/2 that observed for uninfected rabbits and this correlated well with a decrease in t cell proliferation ( < 35% that of normal rabbits) upon stimulation with con a . this decrease in il-2 production in infected rabbits was restored upon removal of most of the adherent cells. furthermore, the il-2 production by 10-14 day infected spleens was restored above normal levels upon addition of indomethacin. this decrease in il-2 levels was not due to an increase in the ability of infected spleen cells to adsorb il-2. finally, studies assessing il-2 production at various times postinfection indicated that at 4 days post-infection il-2 levels were higher than normal, however as early as 10-14 days after infection il-2 levels decreased below normal levels and continued to be depressed as late as 30 days post-infection. these results may explain why all organisms are not eliminated during primary infection w i t h ' . pallidum and why secondary and tertiary phases of the disease may develop. has been shown that especially the antigenic presentation of the fusion protein is important for eliciting a functional immune response. to study the immunolo ic properties of the f protein, we expressed the f gene in e.coli as a $galactosidase-f-fusion protein after insertion in a pex vector. we constructed deletion mutants with fragments generated with restriction enzymes and with the polymerase chain reaction method. using a panel of monoclonal antibodies a rough epitope mapping has been performed. two arears were found on the protein, with one area two monoclonal antibodies react and with an other area four monoclonal antibodies react. both area's were found in f1, the c-terminal part of the protein. the pepscan method was used to fine map the epitopes of the monoclonal antibodies reacting with the second area on the primary sequence. in at least one viral system, cd8+ effector cells can be induced in animals lacking cd4+ t cells. since cd8+ effector cells are important in immunity to malaria sporozoites, we wished to know if they,too, could be generated without help from cd4+ cells. we depleted balb/c mice of their cd4+ t cells by injection of an anti-cd4 monoclonal antibody, and then tried to immunize them with irradiated plasmodium yoelii sporozoites. when challenged with infectious sporozoites, these mice were not protected against malaria infection. although they did not make antibodies to sporozoites, passive transfer of hyperimmune serum into these animals still did not protect them against a sporozoite infections. cd8+ t cells from these animals functioned normally in in vitro assays against tnp labelled targets. it appears that, unlike viral systems, the generation of cd8+ effectors in malaria requires cd4+ helper cells. thus both cd4 and cd8 epitopes should be included in any synthetic vaccine against malaria sporozoites. univ. pennsylvania, philadelphia, pa 19104 migrate from fetal liver or bone marrow. rearrange t cell receptor (tcr) genes, express tcr. undergo thymic selection and finally emerge as mature single positive t lymphocytes. most studies of thymic t cell development have been performed by using polyclonal populations of t lymphocytes, which have made the interpretation of the results complicated. cells (0 clone) from nude mice by culturing nylon wool non-adherent cd4-cd8-spleen and lymph node cells in the presence of wehi3 supernatant and con a supernatant. clone was thy-1-cd3-cd4-cd8-il2r(il2 receptor)-and they have been maintained more than 16 months without changing phenotype. when the c9 clone was stimulated with il4. ili/il2. illnl6. gm-csf, the cells were induced lo express thy-i, tcr and il2r proteins. however, culture of the cells with gm-csffll2 did not induce the expression of these molecules. southern blotting of the dna isolakd from gm-csffll2 culture suggested that they have undergone partial db 1 -jp 1 rearrangement. the cultured cells were then recloned twice by limiting dilution. the cloned cells were again shown to induce expression of cd3 complex by the stimulation of il4 enriched medium. therefore. we have established a system in which to induce differentiation of cloned pre t cell line into tcr+ cells in vitro. the human lymphocyte differentiation antigen cd8 is encoded by a single gene which gives rise to a 32 kda glycoprotein expressed on the cell surface as a dimer, and in higher molecular weight forms. we demonstrate that the mrna is alternatively spliced such that an exon encoding a transmembrane domain is deleted. that is secreted and exists primarily as a monomer. messenger rna corresponding to both forms is present in peripheral blood lymphocytes,(pbl), con a activated pbl and three cd8+ t cell lines with the membrane form being the major species. ratio of mrna for membrane cd8 (mcd8) and secreted cd8 (scd8) exist. in addition, the splicing pattern we observe differs from the pattern found for the mouse cd8 gene. this mrna is also alternatively spliced, but an exon encoding a cytoplasmic region is deleted giving rise to a cell surface molecule which differs in its cytoplasmic tail from the protein encoded by the longer mrna. neither protein i s secreted. this is one of the first examples of a different splicing pattern between two homologous mouse and human genes giving rise to very different proteins. this represents one mechanism of generating diversity during speciation. cd4' t cells in the rat can be divided into two non-overlapping subsets by theii reactivity with the monoclonal antibody mrc ox-22 which binds some of the high molecular weight forms of the cd45 antigen. recent work, to be described has shown that the two subsets represent different stages of t cell maturation, with distinct t cell functions. the lymphokine repertoire of the memory t cell pool will be discussed with reference to the antigenic environment fmm which the cells are obtained. pancreatic islet allografts, gill, ronald g. and lafterty, kevin j., barbara davis center for childhood diabetes i univ. colo. health sciences center, 4200 e. 9th ave, box b-140, denver, co. 80262 we studied the cellular interactions requkd for the rejeaion of cultured mhc class i-dispiuate islet allografts. this model was suitable for studying t-t collaboration in that islet allograft immunity is cd4 dependent but rejection of the cultured islet graft is mediated by the cd8 cell. recipient c57by6 (b6) mice were grafted with mhc class idisparate b6.c-h-2bml (bml) islets beneath the mal capsule. islet grafts wen preaated for 7 days in 95% oxygen culture to reduce immunogenicity. thirty days after grafting, recipient mice wen immunized with 1oe6 live spleen cells from the strains indicated below. rejection of the established graft was not trig-by challenge with donortype bml spleen cells, indicating that the mhc class i stimulus was insufficient to initiate all@ immunity. further, immunization with a mixture of 1 m bml and 1oe6 mhc class ii-disparate b6.c-h-2bm12 (bml2) spleen cells failed to trigger host immunity. however, challenge with loe6 (bml x bml2)fl spleen cells t r i g g d acute rejection of the established bml islet grafts. the requirement for l i i presmtatidrecognition of class i and class 11 all* antigens to trigger allograft immunity indicates that the antigen-presenting (apc) plays an essential role for t-t collaboration in vivo. uature hla-dr complexes are purported to spontaneously internalize from and recycle to the plasma membrane of b but not t lymphocytes. using a neuraminidase protection assay, we have radiolabeled surface class i1 antigens on intact cells and cultured these cells under conditions which permit or prevent endocytosis; subsequently, surface glycoproteins on viable cells were desialylated and class i1 molecules were analyzed by iamunoprecipitation and two-dimensional gel electrophoresis. a panel of buman b lymphoblastoid cell lines and activated tonsillar b cell blasts failed to exhibit any internalization of class i1 complexes; control transferrin receptor molecules were endocytosed as ascertained by insensitivity to neuraminidase digestion. class ii+ pba blasts and sezary cells of the t lineage were also deficient in detectable hla-dr internalization. results did not vary regardless of the time allowed for efficient endocytosis (1251 ok 3~saethionine). or the addition of anti-class i1 monoclonal antibody during the chase period for endocytosis. therefore. within the limits of sensitivity of this assay, class 11 complexes do not appear to be internalized, either spontaneously or uben crosslinked by antibody. recycling represent a dynamic pathvay for regulating surface expression of class i1 antigens or a means of associating with and presenting foreign antigenic peptides. supported in part by usphs grants #5 t32 cao9058-14 and #5 eo1 a123081-03. in previous studies of antigen-specific t cell responses in two distinct models of autoimmune tubulointerstitial nephritis (tin), viiia villosa lectin binding (vv+) t cells have been shown to be necessary for effector t cell expression and mediation of tin. in anti-tubular basement membrane disease, antigen-specifi vv+ t cells direct the phenotypic selection of cd8+ nephritogenic t cells in susceptible mouse strains (j. immunol. 141, nov. 1,1988) . this function is mediated by an antigen-binding, i-js+ soluble protein factor. current studies investigate the role of the t cell glycoprotein which binds vv lectin in mediating w+ t cell function. using the previously described effector t cell induction assay, we found that n-acetyl-d-galactosamine (galnac) (at 25 mm but not 2.5 mm) inhibits vv+ t cell function and cd8+ effector t cell selection. when cd8+ effector t cell differentiation occurs in the presence of soluble factors derived from antigen-primed vv+ t cells, galnac is not inhibitory. these studies suggested that soluble gal nac may competitively bind to a soluble protein which stimulates vv+ t cells, in part by binding to the w lectin receptor, to synthesize andlor secrete their biologically active soluble factor. as an additional test of this hypothesis, we prepared detergent solubilized membranes from vv+ t cells and purified vv lectin binding proteins by affinity chromatography. like galnac. these membrane derived lectin binding proteins also inhibit vv+ t cell function and cd8+ effector t cell selection. inhibition by soluble 'lectin receptors' is dose dependent and is demonstrable with lectin binding glycoproteins derived from 15-20 x lo6 cells, in an assay utilizing 10 x106 vv+ cells. we are now further characterizing the lectin receptor and its endogenous ligand. elementary bodies and outer membranes of chlamydia trachomatis produce a high-titered igg response in rabbits and mice as measured by elisa and microscopic immunofluorescence assays. western blot analysis of total elementary body protein identifies a 40kd major outer membrane protein (momp) as the predominant antigen. to identify the cbmical structure of the epitope, purified momp was subjected to chemical and enzymatic fragmentation and the resulting peptides were purified by hplc and assayed for immunoreactivity. an immunoreactive 6kd cyanogen bromide peptide was amino-terminal sequenced and a series of overlapping synthetic peptides were synthesized and assayed for immunoreactivity. sequential single amino acid deletions at both the nhz and cooh termini allowed us to identify the precise epitope as a 12 amino acid peptide spanning residues 291-302 of momp. two amino acid substitutions at positions 293 (phe-gly) and 300 (pro-gly) completely eliminated antibody binding. the 12-amino acid synthetic peptide is a potent immunogen producing high-titered antibody responses that are specific for the momp molecule. analysis of an independently derived mutant harboring the same defect has shorn that this trans-acting gene is not required for transport of class i1 molecules. class i heavy chain is synthesized in this cell line and associates with b2m. transport of the class i appears to be blocked in the er or cis4olgi as the majority of the class i glycoproteins are not processed to the endo b resistant form. the ability of the cell to significantly increase expression of surface class i when the incubation temperature is lowered from 37oc to 22oc suggests that this gene may function to stabilize a particular conformation of the protein. consistent with this is the increased sensitivity of class i molecules in the mutant as compared to the parent to degradation when cell lysates are incubated at elevated temperatures. the inability to immunoprecipitate class i antigens in the mutant is possibly due to the action of endogenous proteases present in these lysates. two complementing approaches are being employed to isolate this gene and further analyze its role in class i biosynthesis. the first involves inactivation of the trans-acting gene by insertion of a retroviral vector and subsequent pcr amplification of regions flanking the vector. in another approach a cdna library will be introduced into the mutant cell line and the cdna will be reisolated from cells reexpressing surface class i. houston. tx. we have induced a panel of highly immunogenic (imm+) vanants of the murine fibrosarcoma mca-f using i-methyl-3niml-niuasoguanidine (mnng). 5-aza-2'-deoxycytidine (5-azacdr). and uv radiation. these tumors grew m immunosuppressed mice. but were complelely rejecled by normal syngeneic hosts. mice thac had rejected large numbers of imm+ also developed a smng, tumor. specific immunity to the parental mor. lmmunizalion with low numbers of lmm+ engendered only variant-specific immunity. the frequency of imm+ variant g e n e d o n was similar for the three induction different protocols (64% to 92%), suggesling lhat generauon of imm+ was more closely relalcd to the cell line used than to the inducing agent however, the swngth of the imm+ phenaypc was related lo ihe agent used, since mnng induced clones had the m g e s t immunogeniciues and uv-b ihe weakest the smng neoantigens expressed by mnng induced imm+ were varunt-sppifc. while uv and s -d d r induced clones displayed significant cross-reactivilies not attributable to the parental t u n a antigen. increased or inappropriate expression of class-l mhc antigens did not correlate with the imm+ phenotype. we investigated the phenotypes of the spleen cells medrating tumor rejccuon using the local adopllve lransfer a s a y (lata). variant-specific immunity w mnng, 5-azacdr and uv induced imm+ were all m d a t c d by thy1.2+. l3t4+. lyc2.1-t cells. afrw immunization with high numbers of imm+ w engender both anti-lmm+ and anu-parental immunity. both cd4+ and cdw effectors rejecled the imm+ in lata, while only ihe c w + t cells could wnsfer resistance w the parent immunity 10 the parenlal tumor anugen engendered by the imm+ suggeslcd associative recognition of the parental and neoantigem wgelher on ihe cell surface. this hypochfsis was supported by failure of lmm+ u) pmlect againu an antigenically disunct tumor (mca-d) admixed with it, either at the lime of immunization or at challenge. fusion of ihe h m * vanant with mca-d yielded a unique, hybrid parental umor antigen that was associatively recognized with rhc original imm+ neoanugen, demomirating the importance of antigen cocxpression. grant rr-5511-23. w e have recently demonstrated and reported that substitution of anionic side chain carboxylic groups with aminoethylamide groups on protein antigens exhibits a pattern of enhanced immunogenicity both in vivo and in vitro. this enhanced immunogenicity was also observed in low responder strains of mice and we investigated the mechanism by which it is achieved. we examined antigen processing and presentation of native (nbsa) and modified bsa (mbsa) to t helper c e h isolated from c57/bl low responder mice. a greatly reduced amount of mbsa than nbsa was required to activate both nbsa and mbsa primed th. proliferation of nbsa and mbsa primed t cells increased in proportion to the amount of time of exposure of the antigen presenting cells (apc) to nbsa, peaking at 8h. conversely, apc required less than 30 min exposure to mbsa to achieve optimal activation, indicating rapid uptake of mbsa. paraformaldehyde fixed apc recognized mbsa without a lag phase processing, indicating that this event also occurred quite rapidly. apc processed nbsa w a s presented to primed t cells more effectively than the soluble antigen m shown by the increased rate of t cell proliferation. in contrast, mbsa was equally well presented to th cells by apc m in soluble unprocessed form. our data demonstrate that the reduced response in low responders is greatly enhanced by a modified antigen which is rapidly taken up and processed by apc. b cells which bear surface innunoglobulin (sig) receptors specific for a particular antigen are abile t o present fragments of that antigen very efficiently t o t cells. this i s due. in part, t o the high affinity of the receptor, which facilitates antigen binding at low concentrations. using tnp-abc and specific antigen, we have demonstrated that the tnp-abc process antigen very effectively. w e have compared specific antigen with i t s polyclonal analog, anti-ig, and demonstrated differences in the kinetics of degradation of anti-ig and tnp-antigens by tnp-aex. both antigen and anti-ig bound by tnp-abc are degraded into small fragments which are released into the supernatant. however, the following differences have been found: 1) the rate of release of small fragments of tnp-antigen parallels the rate at which these cells become able to directly conjugate with t cells (a lneasure of antigen presentation), reaching a plateau between 4 and 6 hours. in contrast, the degradation of anti-ig and release of fragnents continues for 12 hours. 2) analysis of initial kinetics demonstrated that release of fragments of tnp-antigen begins 15 minutes after binding; there i s no significant release of anti-ig fragrnents u n t i l about 30 minutes. 3) in contrast to anti-ig where there i s significant accumulation of degradation intermediates within the cells, there i s very l i t t l e intracellular accumulation of intennediate-size fragments of tnp-antigen. thus, we propose that the processing of antigen bound via specific sig may involve a specific intracellular pathway and that intracellular routing may be determined either by the degree of cross-linking of sig induced by antigen vs anti-ig or the mode of interaction of the various ligands with sig. alt*, departments of biochemistry (*) and medicine (+), college of physicians and surgeons of columbia univerity, new york, new york 10032. we have recently analysed the structure of the 7 / 6 t cell receptor (tcr) expressed by the normal human thymocyte clone cii. cii expresses a c ' 2 constant region that is a polymorphic form lacking a copy of an izternal exon; the sequence of this constant region accounts for the size of the 7 chain and noncovalent linkage of 7 and 6 chains in the cii tcr. in order to elucidate its role, this 7/6 tcr will be reconstituted in immortalized t-cell lines. in addition, the productively rearranged human 7 / 6 receptor will be transgenically introduced into mice in order to assess the effect of the complete receptor on the development of t cells. the humoral immune response to human immunodeficiency virus has been shown to contain antibodies which act to mediate the uptake of virus through fc receptor mediated mechanisms. it is therefore possible that vaccination with the entire envelope polypeptide may present immunologic determinants that enhance infection. one means by which to generate an immune response to hiv that shall possess neutralizing activity in the absence of infection enhancing activity is to generate anti-idiotypic abs that bear the internal image of neutralizing human antibodies directed against hiv. we affinity purified human antibodies from hiv+ patients on a viral lysate column. we have produced 15 monoclonal anti-idiotypic antibodies directed against these abi's. two of these monoclonals were shown to he ag inhibitable by their ability to inhibit the binding of p o l y e l o n a l human antisera to hiv viral lysate on ortho hiv ab t,est, wells. one monoclonal, 0 b 0 , when coupled t.o klh and used to immunize mice, produced an abs that. bound to viral lysate in an elisa assay. an affinity column containing rbs was used to purify an abi that was shown to bind to p24 and p17 hy western blot analysis. these data suggest that 8br may be a potential vaccine candidate. we have recently described a transgenic mouse model which co-expresses the tcr u and fl chains from the 2c cell line (recognized by the 1b2 anti-clonotype). t celh bearing the transgenic clonotype are positively selected by elements of the h-zb mhc for expression on cd8' cells. thus in the periphery of h-zb animals 40-80% of the t cells are 1bz1/cd8*. the same peripheral expression is observed when the transgenes are expressed in f1 animals bearing a "neutral" mhc haplotype (eg. h-zb'*). however, when the transgene hi expressed in f1 animals which also express the h-2ld gene product, negative selection occurs by clonal deletion. however, this deletion is functional rather than structural as the 1b2 clonotype is present on 10-40% of peripheral t cells. these cells are unusual in that they express neither of the characteristic peripheral molecules cd4 or cd8. the absence of cd8 expression on the 1b2* cells appears to allow these potentially self-reactive clones to exist without evidence of autoimmunity. the original clone as well as 1b2+/cd8+ cells from h-2b animals are strongly inhibited by anti-cd8 reagents. in an effort to understand the process of negative selection and self-tolerance we have examined the capacity of these cells to be activated directly by the anti-clonotype rather than antigen (h-2ld). the results demonstrate that the clonotype is fully functional on these double negative cells, indicating a normal maturation in the thymus. further examination of their surface phenotype also supports the conclusion that these are fully mature cells which are phenotypically distinct from double negative cells which exist in the thymus of h-2b animals. of imunohematology. azl, leiden, the netherlands;2praxis biologics, rochester, new york, usa and 'university of southampton, uk. immunity to disease caused by neisseria menineitidis is associated with the presence of bactericidal and opsonic antibodies to the capsular polysaccharide (cps), lipopolysaccharide and to outer membrane proteins (omps). the cps of group a and c meningococci are proven efficacious vaccines, although the immunogenicity in infants is poor and the immunity is of short duration. the combination with t-helper epitopes will certainly improve the immunogenic properties of these t-independent (ti,) antigens. the group b cps is poorly immunogenic in humans probably because of tolerance due to structural similarity to host glycopeptides and/or glycolipids. we have focused our research onto the class 1 omps which show limited heterogeneity amongst meningococci. murine monoclonal antibodies to these proteins are highly bactericidal in vitro and will be used to map b-cell epitopes. t-epitopes have been identified by theoretical prediction of immunodominant sites by analysis of the amino acid sequence of the omp followed by their solid phase synthesis and subsequent testing for polyclonal activation of t-lymphocytes obtained from hl4-typed volunteers immunized with the omp. in addition human t-cell clones are generated with omps and maintained with antigen, ebv-transformed b-cells, fresh feeders and ril2. the clones are tested for antigen specificity, io vitro helper function, mhc restriction element, expression of surface markers and recognition of common meningococcal t-cell epitopes. c 642 demonstration o f p-azobenzene-arsonate-l-tyrosine (aba-tyr) speclfic t cells in low responder h-26 mice by il-1 supported t cell proliferatlon previous studies have shown that h-zb mice immunized with aba-tyr fail to produce aba specific delayed-type hypersensitivity and show little or no t cell proliferation in vitro to aba-tyr. these observations suggest that h-zb mice are deficient in th1 cells that respond to aba-tyr. by contrast, immunization of h-2b mice with tnp conjugates of aba-tyr revealed good cognate help, suggesting that these mice do possess aba-tyr specific th2 cells and that such cells are not revealed in conventional lymphoproliferative assays. because such assays are widely used to evaluate ir gene control and to map t cell epitopes, the databases generated from such studies may seriously under represent the total number of responder phenotypes and t cell epitopes. because of this concern, we established culture conditions that wlii support aba-tyr specific t cell proliferation in h-2b mice. in these studies, c57bu6.l mice were immunized s.c. with aba-tyr and 7 to 14 days later the draining lymph nodes were cultured with varying doses of aba-tyr or with varying doses of aba-tyr and varying doses of recombinant il-1 alpha (rll-la), a known costimulator of th2 cells. culture with aba-tyr alone produced no proliferation. by contrast, culture with aba-tyr and rll-la revealed t cell proliferation that titrated with the dose of aba-tyr and the dose of rll-la specific for conalbumin presented on ryngeneic antigen presenting cells and dependent on il-1 for its proliferation, was used a s an indicator cell for the ability of neonatal murine spleen cells to present antigen and produce il-1 and il-2.the antigen presenting capacity of neonatal spleen cells is low. during antigen presentation there is an augmentation of il-1 and il-2 production by the antigen presenting spleen cell population. however, neonatal spleen cells do not respond as well a s adult cells. the low levels o f il-1 can not be attributed t o a low potential for producing il-1 since neonatal cells produce high levels of il-1 after induction by a crude il-1 inducer factor (il-1-if).the this impairment leads t o a decreased stimulus of the 1-helper cell to produce inducer factors which leads t o low levels of il-1 and il-2 production by the neonatal cells during antigen presentation. no suppressor mechanisms responsible for the l o w interleukin production were detected. human or murine class i genomic dna was transfected into a b-lymphohlastoid x t-lymphoblastoid hybrid cell line. this fusion hybrid has lost both t cell derived copies of chromosome six and contains deletions spanning the class i1 region on both copies of chromosome six derived from the b-cell parent. previous data have described a transacting factor within this region that is responsible for class i antigen expression. hla-bw58 and b7 glycoproteins, although synthesized, were not transported to the plasma membrane in the hybrid. were surface expressed. these data suggest a fundamental difference between human and mouse histocompatibility antigens in their requirements for intracellular transport. the role of glycans in this transport dicotomy is currently under investigation. in addition, hybrid human-murine genes are being used to identify regions of the class i molecules involved in this transport phenomenon. we probed t h e means by which t h e a n t l g e n p r e s e n t l n g c e l l (apc) handles t h e a n t i g e n produce p e p t i d e s t h a t bind t o mhc-molecules. we propose t h e e x i s t a n c e o f a new type o f i n t e r n a l image i n which immunoglobulin v-region peptides. formed by processing, imitate peptides from conventional a n t i g e n s . w e r e f e r t o such denatured i n t e r n a l images as r e s i d u e internal images, since t h e y a r e a s s o c i a t e d w i t h t h e r e s i d u e of p e p t i d e s remaining a f t e r processing. in some cases, r e s i d u e internal images may be actual sequence images, i . e . , the v-region sequence m y be i d e n t i c a l t o the conventional a n t i g e n sequence.to be class i h-2ka-restricted cytolytic t lymphocytes (ctl) are directed against two immunodominant sites on the a/jap/305/57 influenza hemagglutinin (ha) that can be mimicked by synthetic oligopeptides spanning residues 202-221 in the ha1 and 523-545 in the hydrophobic, transmembrane region. analysis of the fine specificity of hal-specific ctl clones demonstrated that these ctl clones can be subdivided into at least two group based on their patterns of recognition of closely related influenza h2n2 field strains and a monoclonal antibody derived variant of a/guiyang/4/57. using a series of nested synthetic peptides spanning the 202-221 region, the minimal amino acid residues necessary for recognition by the two groups of ctl clones were defined and found to consist of two separate but overlapping sites. sequence comparison of the ha of the a/jap/305/57, the influenza field isolates and the monoclonal antibody derived variant has identified two amino acids, asn at position 207 and gly at position 215, that are critical for t cell recognition. thus, animo acid substitutions induced either by antigenic drift or by monoclonal antibody selection can affect class i ctl recognition. pretreatment with a n t m e s reactive with class 11 mhc anti-has previously been reported to be successful in exov~~kj a n t i g e n -w i r q dendritic cells (m) fran rodent tissue grafts. we have extended these exper-to inta? whole organ grafts. ilia pnmxses also demcnstrated a prolonged survival (17 f 2 days) ccapared to controls (11 t 1 days) (p < 0.01) tihen v l a n t e d into streprozatocin treat& da recipients. antigenic variation in the haemagglutinin (ha) of influenza a viruses frequently introduces new oligosacchekide attachment sites ( aan-x-serlthreo) and carbohydrate addition prevents antibody recognition by steric hindrance. acid substitution in mutant viruses of the h3n2 subtype (ha1 63 asp+asn), that introduces an n-glycosylation site (hn6gcys6&thr65), abrogates antibody and cd4+ t recognition. infected with x31 virus recognise a synthetic peptide corresponding to antigenic site e, ha1 56-76, and are sensitive to a single substitution (ha1 63 asp-basn) in mutant viruses. virus infected target cells, thereby confirming that carbohydrate addition prevents cd4' t cell recognition. here ve show that an amino cell i-ad restricted, ha specific t cell clones f r m balblc mice-reviously recognition of mutant viruses is restored however by tunicamycin-treatment of key: cord-015147-h0o0yqv8 authors: nan title: oral communications and posters date: 2014-09-12 journal: inflamm res doi: 10.1007/bf03353884 sha: doc_id: 15147 cord_uid: h0o0yqv8 nan the drosophila host defense is a multifaceted process which involves reprogramming of gene expression, activation of proteolytic cascades in the blood and uptake of microrganismsby professionalphagocytes. most of the recent studies have focused on challenge-induced expression of antimicrobial peptides and have addressed the following questions : (1) which genes are upregulated during various types of bacterial, fungal or viral infections and what are their functions? (2),what is the nature of the intracellular signalling cascades which lead togene transcription during these infections; (3) how does drosophila recognize infections and does it discriminate between various types of aggressors (e.g. fungal versus bacterial or viral) to mount an appropriate response. over the last ten years we have gained significant insights into these various aspects and the presentation will review our current understanding of the drosophila immune response and put it into a phylogenetic perspective, namely by drawing some stringent parallels with the mammalian innate immune response. there is strong evidence that autoimmunity to myelin antigens plays a major role in the development of multiple sclerosis. several myelin-derived autoantigenic targets have been described and include myelin basic protein (mbp), proteolipid protein and myelin oligodendrocyte glycoprotein. there has been a particular focus on mbp for at least two reasons: mbp-specific cd4+ tcell receptors (tcrs) have been found in multiple sclerosis brains, and cells presenting an immunodominant mbp(85-99) peptide in complex with hla-dr2b have been shown to be present in multiple sclerosis lesions. also, humanized mice expressing the hla-dr2b gene and a human t-cell receptor (tcr) that recognises the mbp85-99 peptide in the context of hla-dr2b either spontaneously or after immunization with mbp85-99 develop experimental autoimmune encephalomyelitis, which has several features in common with multiple sclerosis. this talk will focus on, how humanized mice recently has been used to study the interplay between genetic and environmental risk factors in multiple sclerosis. to resolve the pathogenic mechanisms of rheumatoid arthritis (ra) we need to identify the causative genetic and environmental factors. this has however proven to be complex, with many factors and genes interacting. inbred animals are useful for studies of the identification of genes associated with complex traits and diseases such as ra. animal models offer a possibility to better define the traits, and to segregate the genes in a controlled way enabling linkage analysis. there are several arthritis models, which each may reflect various variants of the heterogeneity of ra in humans. examples are collagen induced arthritis (cia) and pristane induced arthritis (pia), which both fulfills the clinical diagnostic criteria for ra. both diseases are genetically complex and the susceptibility is, as ra, dependent on many polymorphic genes operating in concert. so far 2 genes in this concert have been identified; the mhc class ii ab gene in the mouse (1) and the ncf1 gene in the rat (2) and in the mouse (3) . the ncf1 protein is a part of the nadph oxidase complex mediating oxidative burst. the discovery of the ncf1 polymorphism led to a new proposed pathway in which oxygen radicals modify antigen presentation and the resulting activation of autoreactive t cells. mice with the deficient ncf1 allele are more susceptible to cia, and also developed a chronic form of arthritis. interestingly, the immune response to cii was enhanced by the ncf1 deficiency linking the ncf1 pathway to the adaptive immune response. oxidation of t cell membranes seem to be a key event in the pathogenic mechanism as reduction of t cell membranes induces arthritis in rats (4). on the basis of these findings a new type of therapy myasthenia gravis is a prototypic autoimmune disease, caused in most cases by autoantibodies to the muscle acetylcholine receptor (achr) at the neuromuscular junction. the antibodies reduce the number of achr leading to failure of neuromuscular transmission and muscle weakness. the achr antibodies as measured in conventional immunoprecipitation assays are igg, high affinity, polyclonal and specific for human achr. they reduce the numbers of achr by antigenic modulation and by complement-mediated damage to the neuromuscular junction. myasthenia gravis has a very intriguing relationship with the thymus gland. in many younger patients, the thymus is hyperplastic with immune cell infiltrates and germinal centre formation. around the germinal centres, within the medulla, there are rare muscle-like cells called myoid cells that express achrs. there are many b cells and plasma cells and thymic lymphocyte preparations synthesise achr antibodies. the possibility that the thymic achr induces the germinal centre formation and achr antibody synthesis is supported by much evidence. some patients, however, have thymic tumours and in these the role of the thymus is less clear. moreover, older patients with typical myasthenia usually have thymic tissue which is normal for their age. there are up to 20% of myasthenia patients that do not have the typical achr antibodies. some of these have instead antibodies to muscle specific kinase, a receptor tyrosine kinase that is restricted to the neuromuscular junction. the pathogenic mechanisms by which the antibodies cause disease are not yet clearly identified and the evidence will be discussed. finally, among the patients who have neither achr nor musk antibodies by conventional testing, we have evidence for lower affinity antibodies to achr which can now be detected using molecular approaches which will be described. arry-886 (azd6244) is an inhibitor of mek1/2 currently in development for cancer. phase 1 determined the msd (100 mg) and the safety of the compound given continuously. in decreasing frequency, common treatment-related side effects were rash, diarrhea, nausea, peripheral edema, and vomiting. paired pre-and postdose tumor biopsies showed a reduction in perk (-83%) and proliferative index (-46%). the trough plasma concentration (400 ng/ml) corresponded to~40% inhibition of perk. about 45% of pts had stable disease after 2 months. these results demonstrate that arry-886 (azd6244) is well-tolerated, hits its target, and produces a high incidence of long-lasting stable disease. there are several on-going phase 2 studies, in melanoma, colorectal, lung and pancreatic cancer. arry-162 is another potent, selective mek 1/2 inhibitor, currently in development for inflammatory diseases. when human whole blood was stimulated with tpa, arry-162 inhibited tnfa, il-1b and il-6 (ic50s of 23, 21 and 21 nm, respectively). 50% inhibition of perk required 280 nm. arry-162 was highly efficacious in cia and aia rat models, with ed50s of 3 and~10 mg/ kg, respectively. in normal volunteers arry-162 was well-tolerated and there was a dose-proportional increase in drug exposure. in ex vivo blood samples, there was a dramatic time-and concentration-dependent inhibition of tpa-induced tnfa and il1b. an on-going multiple ascending dose clinical study is further exploring the pharmacokinetics, pharmacodynamics and tolerability of arry-162 monotherapy. in addition, we have initiated a clinical trial designed to evaluate arry-162 in combination with methotrexate in patients with rheumatoid arthritis. rho kinases (rock) are involved in many physiological and pathological processes including smooth muscle contraction, cytoskeletal arrangement, cell adhesion, migration and proliferation.rocks prominent role in cytoskeletal architecture suggests that rock inhibitors should have therapeutic impact in oncology and fibrotic diseases which require cytoskeletal rearrangement to progress.we have synthesized small molecule inhibitors of rock which are specific for the rock-2 isoform.these rock-2 inhibitors, typified by slx-2119 and slx-3060, are potent (ic50 < 100 nm), selective for rock-2 (>100 fold selectivity for rock-2 over rock-1) and exhibit good oral bioavailability.this talk will focus on several areas in oncology and fibrotic disease where the ability to demonstrate an in vitro effect on the cytoskeleton translates into activity in the disease model in vivo.slx-2119 inhibits cell proliferation and migration in several tumor cell lines including ht-1080, panc-1 and mda-mb-231. moreover in xenograft studies using nude mice, slx-2119 significantly inhibited tumor growth with these same cell lines. in liver fibrosis, slx-2119 prevents the differentiation of rat primary hepatic stellate cells into myofibroblasts and inhibits the proliferation of myofibroblasts as well as inhibiting hepatic steatosis in an atherosclerosis model.slx-3060 is an effective antifibrotic agent in the kidney unilateral urethral obstruction model and inhibits renal fibroblast differentiation and proliferation.these data suggest that rock-2 selective inhibition of cytoskeletal modification in key cell types (e.g. tumor cells, stellate cells and fibroblasts) by compounds such as slx-2119 and slx-3060 will provide effective treatment for oncology and fibrotic disease. cyclooxygenases (cox) catalyze the first step in the synthesis of prostaglandins (pg) from arachidonic acid.cox-1 is constitutively expressed.the cox-2 gene is an immediate early-response gene that is induced by variety of mitogenic and inflammatory stimuli.levels of cox-2 are increased in both inflamed and malignant tissues.in inflamed tissues, there is both pharmacological and genetic evidence that targeting cox-2 can either improve (e.g., osteoarthritis) or exacerbate symptoms (e.g., inflammatory bowel disease).multiple lines of evidence suggest that cox-2 plays a significant role in carcinogenesis.the most specific data that support a cause-and effect relationship between cox-2 and tumorigenesis come from genetic studies.overexpression of cox-2 has been observed to drive tumor formation whereas cox-2 deficiency protects against several tumor types.selective cox-2 inhibitors protect against the formation and growth of experimental tumors.moreover, selective cox-2 inhibitors are active in preventing colorectal adenomas in humans.increased amounts of cox-2-derived pge2 are found in both inflamed and neoplastic tissues.the fact that pge2 can stimulate cell proliferation, inhibit apoptosis and induce angiogenesis fits with evidence that induction of cox-2 contributes to both wound healing and tumor growth.taken together, it seems likely that cox-2 induction contributes to wound healing in response to injury but reduces the threshold for carcinogenesis. (1), k hagihara(2), t nishikawa(1), j song(1), a matsumura (2) (1) health care center, osaka university, japan (2) osaka university graduate school of medicine, japan it is still less known about the actual pathogenic role of il-6 in the inflammatory status. to know the pathogenic role of il-6 and the efficacy of il-6 blockade in inflammation, a humanized anti-il-6 receptor antibody, tocilizumab, was used for the treatment in chronic inflammatory diseases, such as castlemans disease, rheumatoid arthritis and crohns disease. since il-6 blocking therapy improved the clinical symptoms and the laboratory findings, the il-6 function in inflammation could be analyzed for the induction of inflammatory molecules, such as serum amyloid a (saa). saamrna induction, saa promoter activity and assembling of transcriptional factors on saa promoter were analyzed by the real time rt-pcr, gel shift assay and dna affinity chromatography in hepatocyte stimulated with the proinflammatory cytokines, il-6, il-1 and tnf-alpha. in result, il-6 was an essential cytokine in induction of saamrna through the activation of stat3 which constructed the complex with nf-kappab p65 and a cofactor p300. although there was no stat3 consensus region on saa promoter, stat3 bound at the 3 site of nf-kappab re. the above research proved that il-6 signal is essential on the synergistic induction of saa via newly discovered stat3 transcriptional mechanism, suggesting the presence of this stat3 mechanism in inflammation, and confirming the normalization of serum saa level by il-6 blocking therapy in inflammatory diseases. this research method develops a subsequent therapy for serious aa amyloidosis by inhibition of saa production, and elucidates the cytokine mechanism on immunopathogenesis of chronic inflammatory diseases. takashi wada(1), k matsushima(2), s kaneko (1) (1) kanazawa university, japan (2) university of tokyo, japan accumulating evidence indicates that chemokine/chemokine receptor system plays a key role in the pathogenesis of various renal diseases via leukocyte migration. pathophysiological impacts of chemokines have shed light on molecular mechanisms of leukocyte trafficking and their activation in the inflammatory aspects of progressive renal injury.locally expressed chemokines are proven to be capable of inciting leukocyte migration to the kidney, resulting in initiating and promoting chronic kidney diseases.the possible positive amplification loop from cxc chemokines to cc chemokines may contribute to progressive renal injury, resulting in sclerosis/fibrosis.it is of note that monocyte chemoattractant protein (mcp)-1/ monocyte chemotactic and activating factor (mcaf)/ ccl2, a prototype of cc chemokines, promotes and escalates chronic kidney diseases with any etiologies via the infiltration and activation of monocytes/macrophages, proteinuria and collagen synthesis.interactions between infiltrated inflammatory cells and resident renal cells eventually lead to the progression of fibrosis. new insights into renal fibrosis have been uncovered by the regulation of fibrocytes dependent on chemokine system. in addition, recent studies demonstrate that chemokines have been expanding their universe beyond leukocyte migration to the kidney, including homeostasis, development and repair of the kidney.the selective intervention of chemokines might have the therapeutic potential to alter inflammatory responses, thereby halting the progression of renal injury. we focus on recent progresses on the role of chemokines and their cognate receptors in renal injury in this symposium. (1), p lacamera (1), b shea (1), g campanella (1), b karimi-shah (1) , n kim (1), z zhao(2), v polosukhin (3), y xu(2), t blackwell (3) aberrant wound-healing responses to injury have been implicated in the development of pulmonary fibrosis, but the mediators directing these pathologic responses remain to be fully identified.here we demonstrate that lysophosphatidic acid (lpa) is induced by lung injury in the bleomycin model of pulmonary fibrosis, and that mice deficient for one of its receptors, lpa1, are dramatically protected from pulmonary fibrosis and mortality following bleomycin challenge. the absence of lpa1 markedly reduced fibroblast responses to the chemotactic activity present in the airspaces following bleomycin, and attenuated the subsequent accumulation of fibroblasts in the lung.the increase in vascular permeability caused by lung injury was also markedly reduced in lpa1-deficient mice, whereas bleomycin-induced leukocyte recruitment was preserved.these results demonstrate that lpa1 links pulmonary fibrosis to lung injury by mediating fibroblast recruitment and vascular leak, two of the wound-healing responses that are thought to be inappropriately excessive when injury leads to fibrosis rather than repair.lpa1 therefore represents a new target for lung diseases in which aberrant responses to injury contribute to the development of fibrosis, such as idiopathic pulmonary fibrosis and the acute respiratory distress syndrome. we have reported that inflammation is detrimental for survival of new hippocampal neurons early after they have been born. our data now show that microglia activation, as an indicator of inflammation, is not pro-or antineurogenic per se but the net outcome is probably dependent on the balance between secreted molecules with pro-and antiinflammatory action. we have found that a substantial fraction of the new hippocampal neurons formed after status epilepticus survive despite chronic inflammation. we have started to explore the role of tnf-alpha for adult neurogenesis. infusion of an antibody to tnf-alpha was shown to reduce survival of new striatal and hippocampal neurons generated after stroke, probably by interfering with action of the ligand on the tnf-r2 receptor. we have shown that tnf-r1 is a negative regulator of progenitor proliferation in basal and insult-induced hippocampal neurogenesis. we have also used patch-clamp technique to explore whether a pathological environment influences the synaptic properties of new granule cells. rats were exposed to either a physiological stimulus, i.e., running, or a brain insult, i.e., status epilepticus which is associated with inflammation. we found that new granule cells in runners and status epilepticus animals had similar intrinsic membrane properties. in contrast, the new neurons which had developed in the physiological and pathological tissue environments differed with respect to tonic drive and short-term plasticity of both excitatory and inhibitory afferent synapses. the role of inflammation for these differences is currently being explored. proteinase-activated receptor-2 (par-2) is cleaved within its aminoterminal extracellular domain by serine protei-nases such as trypsin, unmasking a new aminoterminus starting with sligkv that binds intramolecularly and activates the receptor. par-2 is implicated in innate defense responses associated with lung inflammation. we showed that par-2 is expressed by human alveolar (a549) and bronchial (16hbe) epithelial cell lines, and is activated by trypsin and by the activating synthetic peptide sligkv-nh2. in cystic fibrosis patients, airspaces are invaded by polymorphonuclear neutrophils that release elastase and cathepsin g, two serine proteinases, and by pseudomonas aeruginosa that secretes an elastolytic metalloproteinase.we demonstrated that these three proteinases do not activate par-2, but rather disarm this receptor, preventing its further activation by trypsin but not by sligkv-nh2. preincubation of a par-2 transfected cell line with either proteinases leads to the disappearance of the cleavage/activation epitope. proteolysis by these three proteinases of synthetic peptides representing the aminoterminal extracellular domain encompassing the cleavage/activation sequence of par-2, generate fragments that would not by themselves act as receptor-activating ligands and that would not yield receptor-activating peptides upon further proteolysis by trypsin. our data indicate that neutrophiland pathogen-derived proteinases can potentially silence the function of par-2 in the respiratory tract, thereby altering the host innate defense mechanisms. caspase-3 -dependent killing of host cells and to disrupt intestinal barrier function, which, at least in the case of giardiasis, ultimately causes lymphocyte-dependent intestinal malfunction, and the production of diarrheal symptoms. ongoing research is investigating whether par agonists and microbial pathogens may cause epithelial apoptosis, increased permeability, and overall epithelial malfunction in the gastrointestinal tract, via common or intersecting pathways. the intestinal epithelium is exposed to a variety of proteases in both health and disease, including digestive proteases such as trypsin.given that protease-activated receptor 2 (par2) responds to trypsin and is expressed on intestinal epithelial cells, we investigated the effect of trypsin on intestinal epithelial barrier function.scbn, caco-ii and t84 epithelial cells were grown to confluence on filter supports and mounted in ussing chambers to study short circuit current (isc) and transepithelial resistance (rte).cell monolayers were incubated with inhibitors of transcellular ion transport in order to isolate the contribution of the paracellular pathway to rte.apical exposure to serine proteases including trypsin, elastase and chymotrypsin caused a rapid and sustained increase in rte and decreased the transepithelial flux of a 3000mw dextran.interestingly, the effect of trypsin could not be replicated by activators of pars 1, 2 and 4, suggesting that the effect on rte was not due to activation of pars.subsequent experiments showed that trypsin activated phosphatidylinositol-dependent phospholipase c.a trypsin-induced increase in intracellular calcium was not involved.inhibition of pkc-zeta, but not of typical pkcs, also blocked the response.our data point to a role for postprandial trypsin that extends beyond that of a digestive enzyme; it is also a participant in cellular pathways that control tight junction permeability. physiologically, the trypsin-induced increase in resistance could augment transcellular transport by reducing passive paracellular back-diffusion of ions. further studies will assess how these pathways might be disrupted in the barrier dysfunction characteristic of intestinal inflammation. clustering of inflammatory bowel disease in large families and the observation of an increased concordance between monozygotic twins suggests heritable components in these disorders. the high concordance in monozygotic twins (>55%), which is not seen in dizygotic twins (<5%) points to strong contribution of genetic susceptibility to the overall risk for disease. ibd represents a "complex disease" and may involve a large number of interacting disease genes. crohns disease has become an example for the successful molecular exploration of a polygenic etiology. crohns disease was not known before 1920. incidence has increased since now leading to a lifetime prevalence of up to 0.5% in western industrialized countries. the current hypotheses propose unknown trigger factors in the life style of western industrialized nations that interact with a polygenic susceptibility. it appears that increased expression and production of tnf and an enhanced state of activation of the nfkb system are main drivers of the mucosal inflammatory reaction. the exploration of inflammatory pathophysiology of crohns disease using full genome, cdna and oligonucleotide based arrays, respectively, has generated large sets of genes that are differentially expressed between inflamed mucosa and normal controls. while this may lead to new targets for a pathophysiology oriented therapy, it appears, however, that the dissection of the inflammatory pathophysiology does not allow to identify the multifactorial etiology of the disease. genome-wide linkage analysis has demonstrated eight confirmed susceptibility regions with the one on chromosome 16 being most consistent between different populations. in 2001 three coding variations in the card 15 gene were identified that are highly associated with development of the disease. all variants affect a part of the gene that codes for the leucin rich part of the protein, that appears to be involved in bacteria induced activation of nfkb in macrophages and epithelial cells. interestingly, the three disease associated snps are never found on the same haplotype. in compound heterozygotes or homozygotes they result in a rr of > 35 to develop crohns disease as an adult. a particular subphenotype with localization of the disease in the ileocecal region is highly associated with the variants in the card 15 gene. variations in the card 15 gene do not fully explain the linkage finding in the pericentromeric region of chromosome 16. after stratification for card 15 variants, the broad linkage peak is reduced to two more defined peaks on 16p and 16q, respectively. while the exploration of these regions has led to several association signals that are subject to further fine mapping a further disease gene progress has been greater in the other linkage regions (i.e. on chromosomes 10 and 5, respectively). dlg-5 is the example of a low-risk susceptibility gene with a modest associated odds ratio (1.2-1.5) . interestingly, the associa-tion signal appears to be confined to young males. slc22a4/5 which encode the kation-transporters octn1 and 2 have been suggested to represent the disease gene in the 200+ kb haplotype block on chromosome 5q31. mdr1 has also been implicated as a disease gene in ibd. although the human association studies have resulted in highly controversial findings a knockout mouse with a colitis phenotype makes mdr1 likely as a low risk susceptibility gene. with the advent of high-density, genome wide association studies enormous progress has been made to discover the remaining disease genes. recently a 330k illumina scan has been published identifying il-23r as a further disease gene. we used a genome wide candidate gene approach (with appr. 20.000 csnps) to identify atg16l1 as a further disease genes. both genes were confirmed and a further regulatory snp involving ptger4 was annotated by a belgian genome wide scan. by the time of presentation three further genome wide snp scans in crohn disease will most likely have entered the public domain. the further exploration of crohns disease (and other inflammatory conditions of barrier organs) will have to annotate the function and pathophysiologies based on genetic risk maps that are completed with amazing speed. the creation of a medical systems biology of disease will lead to new models and eventually new therapies. the chemokine receptor ccr9 plays a pivotal role in mediating the migration of t cells to the gastrointestinal mucosa. the ligand for ccr9, teck, s highly expressed in the gi tract. the pathogenicity of intestinal ccr9+/ cd4+ t cells has been demonstrated in animal models and this cell population is substantially increased in the peripheral circulation of crohns and celiac disease patients. ccx282-b is a highly selective and potent, orally bioavailable, small molecule antagonist of ccr9.the compound proved to be highly efficacious in the tnf-aare and mdr1a-/-murine models of inflammatory bowel disease (ibd). in phase i trials, ccx282-b was well-tolerated, and no drug-related saes were reported.a 28-day placebo-controlled phase ii study was recently completed in patients with moderate to severe crohns disease. ccx282-b was shown to be both safe and to have encouraging clinical results: 56% of patients on ccx282-b (cdai !250, crp >7.5mg/l) exhibited a 70-point drop in cdai compared to 29% on placebo. furthermore, a crp decrease of 11 mg/l was seen in the ccx282-b group compared to placebo. colonic biopsy samples were analyzed for expression of several pro-inflammatory cytokines. a mean decrease from baseline in the concentrations of tnf-alpha, il-12 p70, ifn-gamma, and the chemokine rantes was shown in the ccx282-b group while levels remained stable in the placebo group. ccx282-b is the first chemokine-based inhibitor of leukocyte trafficking to be tested in ibd. the compound shows anti-inflammatory activity and encouraging evidence for clinical benefit in the treatment of crohns disease. the activating receptor nkg2d seems to be implicated in the pathogenesis of several autoimmune diseases in humans and in animal models for type 1 diabetes and multiple sclerosis. the aim of this study was to asses the role of nkg2d in a model of inflammatory bowel disease, where cd4+cd25-t cells from balb/c mice are adoptively transferred to scid mice, and to evaluate the therapeutic effect of an anti-nkg2d antibody therapy. the expression of nkg2d was evaluated by flow cytometry, immunohistochemistry and by pcr. we found a marked up-regulation of nkg2d on the cell surface as well as increased levels of nkg2d mrna in cd4+ t cells from colitic scid mice as compared to normal balb/c mice. we next studied the effect of anti-nkg2d antibody (cx5) treatment initiated either before onset of colitis, when the colitis was mild, or when severe colitis was established. cx5 treatment decreased the cellsurface expression of nkg2d and prophylactic administration of cx5 attenuated the development of colitis significantly. a moderate reduction in the severity of disease was observed after cx5 administration to mildly colitic animals, whereas cx5 did not attenuate severe colitis. thus, nkg2d may be involved in the pathogenesis of colitis in this model, particularly in the early phases, since the expression of nkg2d in cd4+ t cells increased markedly during the development of disease and since administration of cx5 early but not late in the course attenuated the disease severity. proteins are used already for more than a century in the treatment of disease. the first generation were proteins derived from animals such as antisera used to treat infectious diseases as diphtheria and tetanus and later bovine and porcine insulin for the treatment ofdiabetes. the second generation were natural proteins from human source like the plasma derived clotting factors and human growth hormone. the development of the recombinant dna and cell fusion technology in the seventies of the 20th century opened up the possibilities to produce human proteins and monoclonal antibodies in unlimited amount in microbial and mammalian host cells. in 1982 human insulin was introduced as the first recombinant dna derived biopharmaceutical and since than more than 160 have gained approval. the pipeline contains many more potential biopharmaceuticals and at present 1 in 4 new drug applications concerns a biotechnology derived product. a major problem of therapeutic proteins is the induction of antibodies. for foreign proteins such as the murine derived monoclonal antibodies thisimmunogenicity was to be expected. however the humanization of monoclonal antibodies has reduced but not solved the problem of immunogenicity. and also the proteins which are homologues of endogenousfactors such as gm-csf, interferons etc. induce antibodies, sometimes even in the majority of patients. by definition we are immune tolerant to products which are copies of endogenous proteins. the products not necessarily need to be exact copies of the natural proteins to share this immune tolerance. when human therapeutic proteins induce antibodies, they are breaking b cell tolerance, which starts with the activation of autoreactive b-cells. presenting the self-epitopes in an array form is very potent activator of these b-cells. this explains why aggregates of human proteins are the most important factor in induction of antibodies. these aggregates may not be immediately present in the product, but may appear during storage making stability and formulation an important issue in predicting the immunogenicity. there are only a few studies in experimental model systems on the properties of the aggregates which break b-cell tolerance, indicating that only multiple order aggregates (>trimers) are involved. we study the capacity of a protein product to break b-cell tolerance in mice made transgenic for the specific protein. these mice are immune tolerant and there is a good correlation of an immune response in these mice and in patients. although these models have helped to identify the factors important for breaking b-cell tolerance and also have been useful in improving the formulation of products, there is not yet enough experience to use them as absolute predictors of immunogenicity of human proteins. they also allow to study the involvement of tcells in breaking b cell tolerance. all data obtained untilnow indicate this process to be t-cell independent. contact information: dr huub schellekens, central laboratory animal institute, utrecht university, utrecht, the netherlands e-mail: h.schellekens@gdl.uu.nl biomonitor aps, and institute for inflammation research (iir), rigshospitalet, copenhagen, denmark using recombinant technology, one can now produce protein drugs which are almost identical with naturally occurring human proteins, including antibodies (abs). many have assumed that these drugs may be administered with little or no risk of triggering specific t-and/or b-lymphocyte reactivities, because patients according to immunological dogma are tolerant towards their own proteins. unfortunately, this is not the case, and even socalled 100% human biologicals are potentially immunogenic (1) (2) . i shall discuss two examples: 1) recombinant human cytokines (ifn-beta-1a and -1b), and 2) anticytokine ab constructs (anti-tumor necrosis factor (tnf)-alpha). ifn-beta has been used for treatment of patients with multiple sclerosis since the early nineties. though initially neglected as a clinical problem, ifn-beta like many other human proteins is indeed immunogenic, especially those produced by recombinant gene technologies. the reported frequencies and titers of anti-ifn-beta ab vary considerably depending upon ifn-beta preparations and administration, and the types of assays being used (2-4). it took more than 10 years of clinical use before abmediated decrease in bioactivity of ifn-beta, a condition in which the clinical effect of continued injection of rec. ifn-beta is minimized or abrogated, was universally recognized (5, 6). 2) anti-tnf-alpha human ab constructs tnf-alpha is an inflammatory cytokine of central pathogenic importance in many immunoinflammatory conditions, and measures to diminish production and/or effects of tnf-alpha have long been a goal in the treatment of these conditions. currently, there are three approved and two other anti-tnf-alpha biopharmaceuticals in clinical use. unfortunately, response failure is frequently encountered. thus, 30-40% of patients are primary non-or lowresponders to the anti-tnf constructs, and secondary response failure is commonplace, mostly due to induction of anti-abs. several different methods have been used to assess circulating levels of anti-tnf drugs as well as anti-abs. most of these have been based on elisa technology, with their inherent problems of false positivity, susceptibility to nonspecific interference, etc. interferon beta (ifnbeta) has been an important step forward in the treatment of multiple sclerosis(ms), an inflammatory disease of the human central nervous system. however, one of the problems of ifnbeta is its immunogenicity; a substantial percentage of ms patients treated this recombinant protein develop anti-ifnã� antibodies, primarily of the igg class. the level of these antibodies tends to be low in the first month or two and peaks by six to eighteen months after initiation of therapy. most studies of these antibodies have measured their ability to neutralize ifnbetas effect in vitro, using assays in which sera from ms patients inhibit the protective effect of ifnbeta on viral killing of target cells. this antibody population is called neutralizing antibodies (nabs). tests measuring binding of antibodies to ifnã� in vitro are called binding antibody (bab) assay. anti-ifnbeta antibodies detected by bab assays are present in a high percentage of ms patients, and can occur at low levels without any apparent adverse effect on ifnbeta bioactivity. the distinction between babs and nabs is artificial, and all binding antibodies are likely neutralizing, if the neutralizing assay system is adequately sensitive; i.e., the development of babs and nabs is a continuum with the assay systems simply measuring the strength of the antibody response. in many treated patients, the anti-ifnbeta antibody response is strong, despite the resemblance of the injected protein to the human homologue, and high levels of neutralizing antibodies develop. high levels of anti-ifnbeta antibodies with high affinity results in loss of ifnbeta bioactivity, a phenomenon which has been called antibody-mediated decreased bioactivity or adb. adb can be considered the in vivo correlate of the neutralizing effect of the anti-ifnã� antibody population, while the nab assay measures the in vitro neutralization of this population of immunoglobulins in the serum. the three ifnbeta preparations have different incidences of nabs and different patterns of appearance and disappearance over time of nabs. because there is no direct correlation between nab levels and bioactivity at moderate levels of nab, in vivo bioactivity assays for ifnbeta have become increasingly utilized. in a large multicenter study in the us, called the insight study, bioactivity as measured by ifnbeta induced upregulation of the ifn-response genes mxa, viperin, and ifit1, was shown to be highly correlated with nab levels, confirming a single center study (pachner, a.r., pak,e., narayan, k., multiplex analysis of expression of three ifnbeta-inducible genes in antibody-positive ms patients, neurology, 66:444-446, 2006) . multiple studies, including a large multicenter study in denmark and a recent study from our center using high resolution mri of the brain once a month, have demonstrated that nabs abolish the salutary effects of ifnbeta on clinical aspects of ms, especially inflammation. recent guidelines for european neurologists recommend stopping ifnbeta in nab-positive patients. in order to maintain bioactivity of this important medication for ms, some neurologists have attempted to use immunosuppressives either to prevent the development of nabs, or to treat them once they have developed. however, at this point in time, there is no clearly optimal way to treat nabs. major efforts have been underway to decrease the immunogenicity of ifnbeta and a new formulation of one of the higher immunogenicity products has been recently developed and tested. proteinase-activated receptors (pars). endogenous serine proteinases such as thrombin, mast cell tryptase, trypsins, kallikreins, cathepsin g, for example, as well as exogenous proteases released by mites or bacteria are involved in cutaneous inflammation, host defense or tumor cell regulation. thus, the expression of pars on keratinocytes, endothelial cells, nerves, and immune cells suggest important role of pars as a part of the communication system in the skin during inflammation and the immune response. for example, par2 activates nf-kb in keratinocytes and endothelial cells, stimulates the release of chemokines and cytokines, and is involved in proliferation and differentiation. on sensory nerves, this receptor controls neurogenic inflammation by modulating edema and extravasation via release of neuropeptides into the inflammatory site. par1 and par2 also modulate leukocyte-endothelial interactions in the skin, thereby regulating inflammatory responses such as leukocyte trafficking through the vessel wall. they also stimulate signal transduction pathways involved in cutaneous inflammation. in sum, this novel receptor family requires a paradigm shift in thinking about the role of proteases in cutaneous biology and disease. novel compounds regulating protease and par function may be beneficial for the treatment of several skin diseases such as atopic dermatitis, psoriasis or pruritus. serine proteinases are upregulated in arthritic joints where their enzymatic activity participates in the destruction of articular soft tissues. in addition to their degradative functions, serine proteinases can also act as signalling molecules by activating members of the gprotein coupled receptor family called the proteinase activated receptors (pars). these receptors are known to regulate tissue inflammation and pain, although their function in joints is unclear. our study examined the effect of par4 activation in joint inflammation and pain. male c57bl/6 mice received an intra-articular injection of either the par4 activating peptide aypgkf-nh2 or the inactive peptide yapgkf-nh2 (100 mg) into the right knee. knee joint blood flow was then measured in these mice by laser doppler perfusion imaging while joint diameter measurements gave an indication of tissue oedema. mechanical allodynia was also assessed in these animals by application of von frey filaments onto the plantar surface of the ipsilateral hindpaw and a pain score was calculated. intra-articular injection of the par4 activating peptide caused knee joint blood flow to gradually increase by up to 25% over the succeeding 2hrs. knee joint swelling was also observed as well as the development of a mechanical allodynia. all responses could be blocked by pre-treatment with the selective par4 antagonist pepducin p4pal10 (100 mg i.p.). the control peptide yapgkf-nh2 had no discernible effect on joint inflammation or pain. these experiments show that peripheral activation of par4 receptors in mice knees causes joint inflammation and pain. vincent lagente (1), e boichot (2) (1) air liquide, centre de recherche claude-delorme, jouy en josas, france (2) inserm u620, universitã� de rennes 1 matrix metalloproteinases (mmps) are a major group of proteases known to regulate the turn-over of extracellular matrix and so they are suggested to be important in the process of lung disease associated with tissue remodelling. these led to the concept that modulation of airway remodeling including excessive proteolysis damage of the tissue may be of interest for future treatment. among metalloproteinases (mmps) family, macrophage elastase (mmp-12) is able to degrade extracellular matrix components such as elastin and is involved in tissue remodeling processes in chronic obstructive pulmonary disease (copd). pulmonary fibrosis has an aggressive course and is usually fatal for an average of three to six years after the onset of symptoms. pulmonary fibrosis is associated with deposition of extracellular matrix (ecm) components in the lung interstitium. the excessive airway remodeling as a result of an imbalance in the equilibrium of the normal processes of synthesis and degradation of extracellular matrix components could be in favor of anti-protease treatments. indeed, the correlation of the differences in hydroxyproline levels in the lungs of bleomycin-treated mice strongly suggests that a reduced molar pro-mmp-9/timp-1 ratio in broncholaveolar lavage fluid is associated with collagen deposition, beginning as early as the inflammatory events at day 1 after bleomycin administration. finally, these observations emphasize those effective therapies for these disorders must be given early in the natural history of the disease, prior to the development of extensive lung destruction and fibrosis. in addition to their degradative properties, proteases can act as signalling molecules that send specific messages to cells.recent work has demonstrated that proteases are able to signal to peripheral sensory neurons, thereby participating to neurogenic inflammation processes and to the transmission or inhibition of pain messages. serine proteases cleaving specifically at an arginin site are able to activate protease-activated receptors (pars), which then send specific messages to cells. we have demonstrated that 3 members of the par family (par1, par2 and par4) are present on peripheral sensory neurons, where they can be activated by different proteases.the activation of par1 and par2 in isolated sensory neurons provokes calcium mobilization and the release of substance p and cgrp, while the activation of par4 inhibited bradykinin-and capsaicin-induced calcium signal, and neuropeptide release.thrombin and pancreatic trypsin caused inflammation respectively through a par1 and par2-dependent mechanism involving the release of neuropeptide.the extrapancreatic form of trypsin (mesotrypsin or trypsin iv) also caused neurogenic inflammation through a par2 and par1dependent mechanism, and causes inflammatory hyperalgesia and allodynia, through a par2-dependent mechanism.in contrast, activation of par4 on peripheral sensory neurons inhibited inflammatory hyperalgesia and allodynia. taken together, these results provide evidences that proteases can interfere with inflammatory and pain mechanisms through the activation of pars on peripheral sensory neurons.determining the role of each individual proteases and their receptors in sensory neuron signalling and above all inflammatory and pain mechanisms constitutes an important challenge to raise new anti-inflammatory and analgesic drugs. introduction: a scoring system for disseminated intravascular coagulation (dic) in humans has been proposed by the international society on thrombosis and haemostasis (isth). it was the objective of this study to develop and validate a similar scoring system for dic in dogs in order to establish the dog as a spontaneous animal model. methods: for the developmental study, 100 consecutive dogs admitted to the intensive care unit (icu) were enrolled prospectively (group a). blood samples were collected daily and a broad panel of coagulation assays performed. diagnosis of dic was based on the expert opinion of one human physician and two veterinarians. a multiple logistic regression model was developed with the coagulation parameters as explaining variables for the diagnosis of dic. integrity and diagnostic accuracy was subsequently evaluated in a separate prospective study according to the stard criteria. the validation study prospectively enrolled 50 consecutive dogs (group b). results: 37 dogs were excluded from group a where 23/63 dogs (37%) had dic. final multiple logistic regression model was based on aptt, pt, d-dimer and fibrinogen and had a very high diagnostic sensitivity and specificity. diagnostic accuracy of the model was sustained by prospective evaluation in group b. conclusion: based on generally available assays, it was possible to design an objective diagnostic model for canine dic, which has both a high sensitivity and specificity. such a model will provide basis for treatment optimization and make it possible to conduct multicentered therapy studies with a minimum risk of systematic misclassification of patients. in 1997, a coagulation independent change in light transmittance (biphasic waveform [bpw] ) was reported in automated activated partial thromboplastin time assays (aptt) in patients with disseminated intravascular coagulation (dic). a calcium-dependant precipitate of creactive protein and very-low-density-lipoprotein was causing the bpw. our group recently identified this phenomenon in dogs also. initially, bpw was introduced as a complementary tool to assist diagnosing dic. however, recent studies reported that bpw may have a stronger potential as a prognostic marker for survival. the aims of the study were to prospectively investigate (a) the diagnostic significance of bpw regarding dic and (b) the significance of bpw to outcome, in dogs with diseases known to predispose for dic. the study was performed as a prospective, observational study including 50 consecutive dogs with a final diagnosis known to predispose for dic (20% were finally diagnosed with dic). outcome was 28-day survival. bpw was assessed by means of a hirudin-modified aptt assay (kjelgaard-hansen et al., jvim 2006:20; 765-766) . relative risk according to bpw (rr [95% confidence interval]) for (a) a dic diagnosis and (b) 28-day mortality, were assessed. 28-day mortality in the study population was 44%. 28% were bpw positive. bpw was not a significant diagnostic factor for dic (rr=0.64 [0.16; 2.67] ), but strongly so for outcome (rr=2.57 [1.46;4.52] ) with a 79% (11/14) mortality amongst bpw positive dogs. in conclusion, bpw was observed in dogs predisposed to dic, with a strong potential as a risk factor for outcome, a finding in line with recent findings in humans. (1), b hideo (2) (1) department of molecular pathology, kumamoto university, japan (2) department of gastroenterological surgery, kumamoto university, japan aeromonas species are facultative anaerobic gramnegative rods that are ubiquitous, waterborne bacilli, most commonly implicated as causative agents of gastroenteritis. aeromonas infections often develop sepsis and disseminated intravascular coagulation syndrome (dic) is a life-threatening complication of sepsis patients, causing multiple organ failure.however, a mechanism leading to coagulation induction in the bacterial infection has not been known. to study the dic induction by aeromonas species infection, we investigated coagulation activity of a serine protease (asp) from aeromonas sobria, predominantly isolated in patients blood. proteolytically active asp shortened both activated partial thromboplastin time and prothrombin time of human plasma in a dose-dependent manner starting at an enzyme concentration of 30 nm. asp activated human prothrombin, releasing hydrolytic activity for thrombinspecific substrate boc-val-pro-arg-mca, but no enzymatic activity was produced from coagulation factors ix and x. analysis by sds-page revealed that asp released a prothrombin fragment with a molecular weight identical with that of fâ¿-thrombin in an incubation timedependent manner. western blotting using biotinylated phe-pro-arg-chloromethylketone, a thrombin inhibitor, showed that asp produced an enzymatically active fragment whose molecular weight was same as that of fâ¿-thrombin. prothrombin incubated with asp but not the protease itself caused platelet aggregation. these results indicate that asp activates prothrombin, producing fâ¿-thrombin that converts fibrinogen to fibrin clot, and suggest that asp coagulation-inducing activity contributes to dic development in sepsis caused by aeromonas sobria infection. the present study shows a link between inflammation and coagulation mediated by a bacterial protease. hemolytic episodes are often associated to high amounts of free heme in circulation (up to 20 um) and the development of an inflammatory response that may develop to a chronic inflammation. our group has shown that free heme is a prototypical proinflammatory molecule, able to induce neutrophil migration, actin cytoskeleton reorganization and nadph oxidasederived reactive oxygen species (ros) generation, as well as pkc activation and interlukin-8 expression (graã�a-souza et al., 2002) . moreover, free heme inhibits human neutrophil spontaneous apoptosis, a feature that is closely related to the impairment of resolution of inflammation and consequent promotion of chronic inflammatory status. heme protective effect requires nadph oxidase-derived ros and involves the activation of mapk, pi3k and nf-kb signaling cascades as well as heme oxygenase (ho) activity (arruda et al., 2004) . more recently, we have shown that heme antiapoptotic effect is closely related to the maintainance of mitochondrial stability, inhibition of bax insertion into mitochondria and a dramatic increase on bcl-xl/bad protein ratio in a ros-dependent manner, requiring the same signaling pathways that regulate heme anti-apoptotic effect. these findings attest to a prominent role of free heme in the onset of inflammation associated to hemolytic episodes as well as the statement of chronic inflammation related to these disorders. the recent advance on the study of free heme as a proinflammatory molecule brings up hope for the development of new strategies to ameliorate acute and chronic inflammation found during hemolytic episodes.financial support: faperj, cnpq, capes. (1) (1) university of melbourne, victoria, australia (2) monash university, victoria, australia we have previously demonstrated that mice lacking the anti-oxidative enzyme, glutathione peroxidase (gpx1), show significantly larger infarcts after stroke. recent studies have demonstrated that adhesion molecule-mediated leukocyte recruitment is associated with increased tissue damage in stroke, while mice lacking key adhesion molecules conferred neuro-protection. nevertheless, the involvement of oxidative stress in leukocyte recruitment and subsequent regulated cell injury is yet to be elucidated. to explore this, gpx1-/-mice were subjected to transient mid-cerebral artery occlusion (mcao) followed by cerebral intravital microscopy, for assessment of leukocyte-endothelium interactions in intact cerebral microvasculature. after 1hr mcao, leukocyte-endothelium interaction was significantly reduced in gpx1-/-mice compared to wt counterparts during the second hour of reperfusion. laser doppler and direct measurement of blood flow in pial postcapillary venules revealed a reduction of reperfusion in gpx1-/-mice following transient mcao. this suggests that the reduction in nutritive blood flow following stroke in gpx1-/-mice may explain the enhanced injury in these mice as well as the reduced leukocyte-endothelium interaction. furthermore, matrix metalloproteinase-9 (mmp9) which has previously been shown to be implicated in endothelial dysfunction and the pathogenesis of stroke was found to be up-regulated in gpx1-/-mice to a greater extent than in wt mice after mcao, suggesting a role for oxidative stress in cerebral microvascular injury. the data present here suggests oxidative stress may be one of the factors that contribute to reduced post-ischemic perfusion, via the disruption of the endothelial function as indicated by the increased level of mmp9. chris bolton(1), c paul(2), s barker (3), r mongru (3) (1) william harvey research institute, london, uk (2) university west of england, bristol (3) queen mary university of london adrenomedullin (am) acts as a vasodilator in many vascular beds including the cerebral circulation where the peptide is produced in larger amounts than in the periphery.in vitro work has shown that am beneficially regulates blood-brain barrier (bbb) characteristics including transendothelial electrical resistance, permeability and p-glycoprotein pump activation.our preliminary studies in acute experimental autoimmune encephalomyelitis (eae), a model of the human disease multiple sclerosis (ms), have demonstrated significant elevations in am peptide levels corresponding with am mrna changes during late, neurological disease where am production may be linked to the restoration of bbb function. however, am is not exclusively produced as result of am gene upregulation. furthermore, am peptide levels do not always match am mrna changes during other disease phases of eae.the current study has investigated, more closely, the relationship between am gene expression and subsequent levels of associated peptides. am mrna levels were determined, by rt-pcr, in the cerebellum, medulla-pons and spinal cord of normal and eae-inoculated lewis rats at the height of disease. am and proadrenomedullin peptide (pamp) levels were measured in the tissues by radioimmunoassay.all tissues examined showed an increase in am gene mrna compared to control levels.am and pamp changes were observed in the samples and differences between the peptide profiles were recorded.an understanding of alterations in the generation of am and related peptides during neuroinflammation may provide insight into mechanisms affecting bbb permeability and be of relevance to the changes in neurovascular function seen during ms. platelet-activating factor (paf) contributes to the robust inflammatory responses in acute phase and spread of secondary injury. although, paf is believed to be a potent edematous but non-painful mediator in peripheral tissues, we recently demonstrated that paf may be a mediator of noxious signaling in spinal cord in case of neuronal injury. paf-induced tactile allodynia may be mediated by atp, glutamate and the generation of nitric oxide (no). the present study elucidated down-stream signaling pathway for paf-induced tactile allodynia. paf-and glutamate-induced tactile allodynia was blocked by the pretreatment with no scavengers and inhibitors of no synthase, soluble guanylate cyclase or cgmp-dependent protein kinase (pkg). recent evidence attributes the generation of pain to specific disfunctions of inhibitory glycinergic neurotransmission. to explore the target molecule for induction of tactile allodynia, the effect of knockdown of glycine receptors containing the a3 subunit (glyr a3) by sirna spinally transfected with hvj-e vector was examined. in mice spinally transferred with sirna for glyr a3, the reduction of glyr a3 was demonstrated in superficial layer of dorsal horn by immunohistochemical analysis. pcpt-cgmp, paf, glutamate failed to induce tactile allodynia in mice spinally transferred with sirna of glyr a3, while these compounds produced tactile allodynia in mice transferred with mutant sirna of glyr a3 as a control. glycine tranporter inhibitors ameriolated paf-and pcpt-cgmp-induced allodynia. these results suggest that glutamate-no-cgmp-pkg pathway plays a key role for paf-induced tactile allodynia in spinal cord and glyr a3 may be a target molecule for pkg to induce allodynia. (1), r leite(2), ys bakhle (3) (1) federal university of minas gerais, belo horizonte, brazil (2) medical college of georgia, augusta, usa (3) imperial college, london, uk selective cyclooxygenase 2 inhibitors (coxibs) induce a characteristic increase in mechanical nociceptive threshold, referred to as "hypoalgesia", in inflammatory pain induced by carrageenan in rat paws.we have here assessed the role of the cytoskeleton in this hypoalgesia induced by celecoxib (cx). male holtzman rats (150-200g; 3-5 animals/group) were injected in the right hind paw (ipl) with a range of cytoskeletal inhibitors (selective inhibitors of microtubules (taxol, nocodazole, colchicine), of actin microfilaments (latrunculin b, cytochalasin b) or of intermediary filaments (acrylamide) (pico to nanomoles per paw) and 30 min later given cx (12mg/kg, s.c.). after a further 30 min, rats were injected (ipl) with the inflammatory stimulus, carrageenan (250 mg/paw). mechanical pain threshold was hourly measured over the next 4 h, using the randall-sellitto method. the cxinduced hypoalgesia was reversed by low doses of latrunculin b or cytochalasin (latrunculin 100% reversal = 1.26 nanomoles), higher doses of microtubule inhibitors (taxol 100% reversal = 23.4 nanomoles) with no effect of acrylamide (7 up to 141 nanomoles).we conclude that 1) local changes in (paw) cytoskeleton occurred during cxinduced hypoalgesia and 2) actin microfilaments were the cytoskeletal components most critically involved in this hypoalgesia.financial support: cnpq, fapemig and capes there are reports regarding the up-regulation of cyclooxygenase isoenzyme particularly inducible isoform i.e. cox-2 in brain during neurodegenerative or neuropsychiatric disorders.in the present study, we examined the effect of nimesulide (a preferential cox-2 inhibitor) in subchronic immobilization stress. mice were subjected to immobilization stress for 6 hrs daily for a period of seven days. nimesulide (2.5 mg/kg, i.p.) was administered daily for 7 days before challenging them to immobilization stress. behavioral analysis revealed the hyperlocomotor activity and increased anxious response. subchronic stress decreased % retention of memory and also caused hyperalgesic response in mice. biochemical analysis revealed that chronic immobilization stress significantly increased lipid peroxidation and nitrite levels and decreased the reduced glutathione and adrenal ascorbic acid levels. chronic treatment with nimesulide significantly attenuated the immobilization stress-induced behavioral and biochemical alterations. these results suggested that the use of nimesulide could be a useful neuroprotective strategy in the treatment of stress. there is accumulated evidence for ngf role as a peripheral pain mediator. ngf is upregulated in diverse inflammatory conditions and evokes hyperalgesia when injected in humans and rats. ngf increase was also observed in temporomandibular join (tmj) after cfa injection, indicating its possible involvement in local hyperalgesic states. therefore, the objective here was to evaluate if ngf participate in the tmj nociception. to test this hypothesis, the ngf was injected into the tmj alone or after carrageenan (cg) and the spontaneous nociceptive behavior of head flinches was counted for up 30min. further evidence for the ngf nociceptive activity was obtained quantifying the local production of ngf after cg injection, by elisa, and the fos-like immunolabeling in the trigeminal sensory nucleus (including the caudalis, interpolaris and oralis) after ngf injection. injections were performed in 0.25ul. ngf (0.2, 1 and 5ug) injected in the tmj challenged 1h prior by cg (100ug) induces a dose-dependent increase in the number of head flinches. this increase was reduced by k252a (1 and 2ug), indicating a trka receptor-mediated effect. we detected a significant increase in the ngf production 1 and 3h after the tmj cg (300ug) injection. the tmj injection of ngf (5ug) alone did not induce detectable spontaneous nociceptive behavior. however, the ngf (5ug) injection induces a significant increase in the fos like immunolabeling (fli) in the sensory trigeminal nucleus compared to the saline injection. these results indicate that the ngf participates in the nociceptive activity in the tmj, specially in inflammatory conditions. mif was reported as a key cytokine in the pathogenesis of rheumatoid arthritis (ra) several years ago, but it now clear that mif is also involved in the pathogenesis of systemic lupus erythematosus (sle) and atherosclerosis. mif-deficient lupus-prone mrl/lpr mice exhibit prolonged survival and reduced renal and skin disease compared to mif-expressing mice. similarly, mif-deficient atheroma-pone ldlr-deficient or apoe-deficient mice are significantly protected from disease and antimif mab therapy is beneficial. ra and sle are each characterised both by an increased prevalence of atherosclerotic vascular disease and by overexpression of mif. given the effects of mif on atherosclerosis it can be hypothesised that mif overexpression participates in the risk of atherosclerotic vascular disease in ra and sle. recent data have provided insights into mechanisms of action for mif relevant to all these concepts. firstly, the newly described role of mif in the selective recruitment of monocyte-macrophage lineage cells is of particular relevance to ra, sle, and atherosclerosis, with evidence that mif mediates macrophage recruitment in sle and atherosclerosis. secondly, glucocorticoid (gc) therapy is possible risk factor for atherosclerosis in patients with ra and sle, and it is now clear that gc increase the expression and release of mif, potentially implicating mif in gc-related increases in atherosclerosis in ra and sle. specific therapeutic targeting of mif in ra and sle may address not only primary disease pathways but also the increased risk of atherosclerosis in these diseases. to enter inflamed tissues, leukocytes must undergo adhesion molecule-mediated interactions with the endothelial surface of vessels at the site of inflammation.cytokines such as tumour necrosis factor (tnf) are established as important mediators capable of promoting leukocyte-endothelial cell interactions.however, in inflammatory diseases such as atherosclerosis and rheumatoid arthritis, elevated expression of another cytokine, macrophage migration inhibitory factor (mif) occurs, yet the role of this cytokine in leukocyte recruitment is unknown.therefore we explored the ability of mif to regulate leukocyte recruitment.this was achieved using intravital microscopy to examine the intact microvasculature in mice following local mif treatment. these experiments showed that mif induced leukocyte adhesion and transmigration in vivo, resulting in accumulation of predominantly cd68+/f4/80-ve/cd11c-ve monocyte/ macrophage lineage cells.mif did not induce upregulation of adhesion molecules p-selectin and vcam-1, although their constitutive expression contributed to recruitment.in contrast, mif-induced recruitment was blocked by antibodies to the monocyte-specific chemokine, ccl2/mcp-1, and its receptor ccr2, and in response to anti-cxcr2.this was supported by in vitro experiments showing that mif induced ccl2/mcp-1 release from cultured murine endothelial cells.finally, mice lacking cd74, the putative mif binding molecule, did not respond to mif.these data demonstrate a previously unrecognized function of this pleiotropic cytokine: induction of monocyte migration into tissues, and indicate the involvement of a pathway involving a complicated chemokine/chemokine receptor pathway with contribution from cd74.this function may be critical to the ability of mif to promote diseases in which macrophages are key participants. gm-csf and m-csf (csf-1) can enhance macrophage lineage numbers as well as modulate their differentiation and function. of recent potential significance for the therapy of inflammatory/autoimmune diseases, their blockade in relevant animal models leads to a reduction in disease activity. what the critical actions are of these csfs on macrophages during inflammatory reactions are unknown. to address this issue, adherent macrophages (gm-bmm and bmm) were first derived from bone marrow precursors by gm-csf and m-csf, respectively, and stimulated in vitro with lps to measure secreted cytokine production, as well as nf-kb and ap-1 activities. gm-bmm preferentially produced tnfa, il-6, il-12 and il-23 while, conversely, bmm generated more il-10 and ccl2; strikingly the latter population could not produce detectable il-12 and il-23. following lps stimulation, gm-bmm displayed rapid ikba degradation, rela nuclear translocation and nf-kb dna binding relative to bmm, as well as a faster and enhanced ap-1 activation. each macrophage population was also pre-treated with the other csf prior to lps stimulation and found to adopt the phenotype of the other population to some extent as judged by cytokine production and nf-kb activity. thus gm-csf and m-csf demonstrate at the level of macrophage cytokine production different and even competing responses with implications for their respective roles in inflammation including a possible dampening role for m-csf. granulocyte macrophage-colony stimulating factor (gm-csf), initially discovered for its role in the differentiation of haematopoietic cells into granulocytes and macrophages, can also affect mature cell function and may be considered proinflammatory. gm-csf is able to prime macrophages for increased pro-inflammatory responses, including the increased release of tnfa and il-12 following stimulation with, for example, lps. in addition, gm-csf has been shown in vivo, using murine disease models, to play a key role in a number of inflammatory diseases. gm-csf-/-mice have been shown to be resistant to several diseases, including arthritis, and, most notably, blockade of gm-csf with a neutralizing monoclonal antibody was effective at ameliorating arthritis when given either prophylactically or therapeutically. t cells appear to be the major cell type responsible for gm-csf production required for arthritis, and gm-csf appears important in the effector phase of disease, subsequent to t cell activation. blockade of gm-csf results in fewer inflammatory cells, particularly macrophages, and cytokines such as tnfa, at the site of inflammation. these findings suggest that blockade of gm-csf may be an effective treatment in a range of inflammatory diseases. the autoimmune disease type 1 diabetes mellitus (t1dm) is thought to be mediated by autoreactive t cells recognizing islet autoantigens, including gad65, ia-2 and proinsulin. this disease arises on a distinctive genetic background, mapping most notably to the mhc, and is also open to strong environmental influence. to investigate the pathogenesis of the disease, and in particular the prevailing paradigm that islet autoreactive t cells are important, we have developed an approach to epitope identification that is mhc allele and autoantigen specific, and operates for both cd4 and cd8 t cells. utilizing this, we have uncovered populations of islet antigen-specific t cells that have the immunological credentials to be both pathogenic (eg th1, tc1) and protective (treg) in the disease. we have cloned some of these cell types, enabling us to analyse their function and provide an insight that will be important for an understanding of disease mechanisms, as well as guiding novel therapeutic interventions. tcr transgenic targeting b:9-23 cause diabetes 2. knockouts of the insulin 2 gene (expressed in thymus as well as islets) accelerates diabetes while knockout of insulin 1 gene (islet expression) prevents 90% of diabetes 3. dual insulin knockout with transgenic insulin with altered peptide (b16:a) prevents all diabetes 4. islets with native b:9-23 sequence, but not altered sequence when transplanted into knockouts restore anti-insulin autoimmunity and diabetes transfer by t cells 5.anti-b9-23 t cells have conserved valpha and jalpha chain usage but no conservation n region or beta chain 6. alpha chain as transgene sufficient to engender anti-insulin autoantibodies 7. kay and coworkers demonstrate insulin reactivity "upstream" of igrp and igrp reactivity nonessential.future studies in nod directed at deleting specific conserved alpha chains to test diabetes prevention and develop therapeutic.in man we can now identify at birth genetic risk as high as 80% of activating anti-islet autoimmunity with mhc analysis and restricted heterogeneity suggesting dominant target.insulin autoantibodies in prospective studies such as daisy usually appear initially and levels are related to progression to diabetes.analysis of cadaveric donors is underway to elucidate primary targets. (t1d) is an autoimmune disease in which genes and environment contribute to cell-mediated immune destruction of insulin-producing beta cells in the islets of the pancreas. the holy grail of autoimmune disease prevention is negative vaccination against autoantigens to induce disease-specific immune tolerance. this has been achieved in rodents by administering autoantigen via a tolerogenic route (mucosal), cell type (stem cell or resting dendritic cell), mode (with blockade of t-cell co-stimulation molecules) or form (as an altered peptide ligand). compelling evidence demonstrates that proinsulin is the key autoantigen that drives beta-cell destruction in the non-obese diabetic (nod) mouse model of t1d, and possibly in humans. proinsulin/ insulin dna, protein or t-cell epitope peptides administered in a tolerogenic manner to the nod mouse can delay or prevent the development of diabetes, via one or more mechanisms (deletion or anergy of effector t cells, induction of regulatory t cells). administration of autoantigen via the mucosal route, which induces anti-diabetic regulatory t cells in the rodent, is the most immediately translatable approach to humans. initial human trials of vaccination with oral autoantigens lacked evidence of bioeffect, probably due to inadequate dosage in end-stage disease. recently, however, the first evidence for a therapeutic effect of mucosal autoantigen has been seen in trials of oral and nasal insulin in islet autoantibodypositive individuals at risk for t1d. combination autoantigen-specific vaccination also shows promise in combination with non-specific immunotherapy in established t1d. leukocyte extravazation is an integral process both physiologically (immunosurveillance) and pathophysiologically (inflammation). the initial paradigm of a 4-step process comprising tethering/rolling, activation, firm adhesion, and diapedesis, each involving specific adhesion molecules, has repeatedly been modified in the light of more recent findings. additionally, organ-specific differences regarding the role of distinct molecules were established. finally, the skin became a good "model" to study due to its accessability and availability of powerful animal models. in-vitro adhesion assays, flow-chamber systems, intravital microscopy, animal models for delayed-type hypersensitivity, and transplantation approaches have successfully been employed to investigate leukocyte extravazation. numerous molecular interactions such as the cutaneous lymphocyte-associated antigen and sialyl-lewisx, or icam-1 and lfa-1, have been proven sufficiently relevant to make them candidates for potential therapies. with the anti lfa-1 antibody efalizumab, approved for the treatment of psoriasis, the first therapeutic agent specifically targeting leukocyte extravazation is already on the market; other compounds are under development. moreover, novel data suggest that well-established anti-inflamamtory therapies such as fumarates also influence this process, thus contributing to their clinical efficacy. ongoing research aks for adopting a more "dynamic" view on leukocyte extravazation as several molecules obviously perform multiple tasks throughout this process rather than being limited to just one step of this multi-step cascade; this is particularly true for the so-called junctional adhesuã­on molecules which obviously mediate more than just diapedesis. finally, similarities between leukocyte extravazation and hematogenic metastases are emerging. consequently, certain anti-inflammatory compounds may turn out to also exhibit striking anti-metastatic efficacy, and vice versa. department of dermatology, heinrich-heine-university, dã¼sseldorf, germany atopic dermatitis, psoriasis vulagaris and cutaneous lupus erythematosus represent chronic inflammatory skin diseases showing distinct clinical phenotypes but sharing one aspect. the recruitment of pathogenic leukocyte subsets into the skin represents a prerequisite for their initiation and maintenance. during recent years, our knowledge of the immunopathogenesis of chronic inflammatory skin diseases increased significantly. with regard to the recruitment pathways of leukocytes, a superfamily of small cytokine-like proteins so called chemokines has attracted significant attention. here the complex interactions within the chemokine ligand-receptor network are introduced, the involvement of chemokines in memory t and dendritic cell trafficking is outlined and current concepts of their role in the immunopathogenesis of atopic dermatitis, psorasis vulgaris and cutaneous lupus erythematosus are summarized. the skin serves as a unique organ for studying general principles of inflammation because of its easy accessibility for clinical evaluation and tissue sampling. a network of pro-inflammatory cytokines including il-1 and tnf-a is known to play a key role in the pathogenesis of cutaneous inflammatory diseases through activation of specific signalling pathways. recently, progress in understanding the underlying mechanisms regulating inflammatory signalling pathways in the immunopathogenesis in skin carcinomas, psoriasis vulgaris and atopic dermatitis has been made. kinases have been identified to play a crucial role in regulating the expression and activation of inflammatory mediators in these inflammatory skin diseases. mitogen-activated protein kinases (mapks) are a family of serine/threonine protein kinases that mediate a wide variety of cellular behaviours in response to external stress signals. increased activity of mapks, in particular p38 mapk, and their involvement in the regulation of synthesis of inflammatory mediators at the transcriptional and translational level has recently been demonstrated. progress in our understanding of inflammatory signalling pathways has identified new targets for treating inflammatory diseases, but the challenge is to place a value on one target relative to another and to evolve strategies to target them. a careful examination of different signalling pathways in various inflammatory conditions is therefore needed. this presentation gathers recent advances in signal transduction in skin inflammation focusing interleukin-1, tnf-âµ, p38 mapk, msk1/2, mk2, nf-kappab and ap-1. histamine is an important inflammatory mediator in humans, and despite their relatively modest efficacy antihistamines are frequently used to treat allergic conditions, as well as other histamine-mediated reactions such as pruritus. in contrast, antihistamines are of very limited use for controlling other conditions where histamine production is abundant, including asthma. the discovery of the histamine h4 receptor (h4r) prompted us to reinvestigate the role of histamine in pulmonary allergic responses, as well as in pruritus. h4r deficient mice and mice treated with h4r antagonists exhibited decreased allergic lung inflammation in several models, with decreases in infiltrating lung eosinophils and lymphocytes and decreases in th2 responses. ex vivo restimulation of primed t cells showed decreases in th2 cytokine production, and in vitro experiments suggest that decreased cytokine and chemokine production by dendritic cells after blockade of the h4r was responsible for the the t cell effects. the influence of h4r on allergic or histamine-induced pruritus was explored in mice using selective histamine receptor antagonists and h4r deficient mice. the h4r was found to mediate the majority of histamine-mediated and allergic itching, while the contibution by the h1r was minor. surprisingly the h4r effect was independent of mast cells or other hematopoetic cells. this work suggests that the h4r can modulate both allergic responses via its influence on t cell activation, and pruritus through mechanisms that are independent of hematopoetic cells. the studies show that the h4r mediates previously uncharacterized effects of histamine and highlight the therapeutic potential of h4r antagonists. (1), bsp reddy (2) (1) nizams institute of medical sciences, hyderabad; india (2) genix pharma, india rupatidine, carries the majority of the histamine h1receptor -blocking activity and has been introducedfor the treatment of allergic rhinitis and urticaria. objectives: the aim of this study was to compare the effect of two by measure of inhibition of histamine induced wheal and flare response. methodology: 12 male volunteers were enrolled after written informed consent before to ethic committee approved protocol. in this randomised, double-blind, single oral dose, cross overstudy, they were randomized to receive either 10 mg rupatidineformulation after overnight fast. washout was 10 days. wheal and flare were induced on the forearm of the trial subjects, by histamine intradermally injection while the subject was lying comfortably with arm resting on the bed. ten minutes later, wheal and flare were visualized under a bright lamp. histamine induced wheal and flare skin test was performed before and regularly to 24hours after drug administration. results: administration of reference and test formulations of rupatidine, significantly inhibited the histamine induced cutaneous response in all the subjects. the least square mean ratio (%) t vs r for peak activity imax-% (maximum inhibition of histamine induced wheal and flare response); area under the activity time curve (auc0-24 mmsq/hr and auc 0-24 %/hr) both for untransformed and log transformed data were found to be within 80-125% of90% ci limits both formulations well tolerated. conclusions: it can thus be concluded that be concluded that test formulation of rupatidine tablet is bioequivalent to reference rupatidine tablet (1), h yoshimura(1), k ohara (1), y mastui (1), h hara (1), h inoue (1), h kitasato (1), c yokoyama(2), s narumiya (3), m majima (1) (1) kitasato university school of medicine, kanagawa, japan (2) tokyo dental medical colledge, tokyo, japan (3) kyoto university school of medicine, kyoto, japan thromboxane (tx) a2 is a potent stimulator of platelet activation and aggregation and vascular constriction. we have reported the magnitude of cytokine-mediated release of sdf-1 from platelets and the recruitment of nonendothelial cxcr4+ vegfr1+ hematopoietic progenitorsconstitute revascularization. we hypothesized that txa2 induces angiogenic response by stimulating sdf-1 and vegf which derived from platelet aggregainflamm. res., supplement 3 (2007) tion.to evaluate this hypothesis, we dissected the role of the txa2 in angiogenesis response using mouse hind limb model. recovery from acute hind limb ischemia, as assessed by the ratio between the treated ischemic limb and the untreated control right limb was assessed in wild type mice (c57bl/6 wt) , prostaglandin i2 receptor (ip) knock out(ipko) and thromboxane (tx) a2 receptor (tp) knock out(tpko). blood recovery in tp-/-significantly delayed compared to wt and ipko. immunohistochemical studiesrevealed that tp-/-mice were less stained against pecam positive cells compared to wt and ipkoplasma sdf-1 and vegf concentration were significantly reduced in tp-/-mice. we observed during in vivo fluorescence microscopic study that compared to tpko, ipko and wt significantly increased platelet attachment to the microvessels around ligated area. tpko translpanted wt bone marrow cells increased blood recovery compared to tpko transplanted tpko bone marrow cells. in addition, mice injected with txa2 synthase c-dna expressing fibroblast increased blood flow recovery compared to control mice. these results suggested that tp signaling rescues ischemic condition by inducing angiogenesis by secreting sdf-1 and vegf from platelet aggregation. purpose: the s100 calcium-binding proteins, a8, a9 and a12 are constitutively expressed in neutrophils and induced in activated macrophages. high levels are found in sera from patients with infection and several chronic inflammatory diseases. the calgranulin complex, a8/a9 is anti-microbial; a8 has oxidant-scavenging functions. a12 is chemotactic for monocytes, and recruits leukocytes in vivo by activating mast cells (mc). effects of these mediators on mc and monocyte function were compared. methods: human pbmc or murine mc were activated in vitro with s100 and mediator release and cytokine induction (assessed by quantitative rt-pcr/elisa), determined. a cys41 to ala41 a8 mutant was used to determine whether effects on mc are mediated by redox. immunohistochemistry was used to demonstrate s100s in asthmatic lung. the s100s were expressed in asthmatic lung, particularly in eosinophils and alveolar macrophages. strong reactivity occurred with an antibody recognising predominantly the hypochlorite-oxidised from of a8. a8, a9 or the a8/a9 complex had relatively low ability to induce il1, tnf, il6, and chemokines mrna from pbmc compared to a12. only a12 induced significant levels of il13; none induced il4 or gm-csf mrna compared to lps. in contrast to a12 which is activating, a8 significantly inhibited mc degranulation provoked by ige cross-linking; suppression was dependent on cys41. conclusions: the cytokine profile generated by a12 in mc and monocytes strongly supports a role the pathogenesis of asthma. in contrast, results strongly support a role from a8 in oxidant defence, particularly to hypobromite generated by activated eosinophils. (1), d mankuta(2), g gleich (3), f levi-schaffer (1) (1) hebrew university of jerusalem, israel (2) hadassah university hospital, israel (3) university of utah, usa the onset, amplitude and termination of allergic responses is regulated at the mast cell/eosinophil interface. eosinophil major basic protein (mbp), which activates mast cells in the late-chronic phase of allergic inflammation, is a central determinant in this interface. characterized more than two decades ago, the exact nature of this activation has not been clarified as yet. here we demonstrate that mbp exerts its activating effect on human mast cells and basophils through cd29 and hematopoietic cell kinase (hck). a genome-wide analysis showed that hck displays shifts in mrna levels specifically upon mbp-induced mast cell activation. hck also shows a unique priming pattern prior to this activation. cd29 is phosphorylated specifically upon activation with mbp and deploys a signaling complex that critically depends on hck. extracellular neutralization of cd29 interferes with mbp entry into the cell, and this as well as rna silencing of hck results in defective mbp-induced activation. finally, cd29 neutralization abrogates mbp-induced anaphylaxis in-vivo. these findings picture for the first time a chronic-phase specific pathway mediating eosinophil-induced mast cell activation with critical consequences for the therapy of chronic allergic inflammation. alexander robinson (1), d kashanin (2), f odowd(2), v williams(2), g walsh (1) (1) cellix ltd, institute of molecular medicine, dublin, ireland (2) university of aberdeen, scotland, uk leukocyte adhesion to endothelial cell bound proteins, such as icam-1 and vcam-1, is an initial step of the inflammatory response. we have developed an in vitro microfluidic system which mimics conditions found in blood vessels in vivo during an immune response. using this system, we can record leukocyte adhesion levels under physiologically relevant flow conditions (e.g. 0-5 dynes/cm2). the adhesion profiles of resting and pmastimulated peripheral blood lymphocytes (pbls) were recorded, with respect to vcam-1, icam-1, and bsa. images at each shear stress level were captured using a digital camera, and analysed using our in-house ducocell software package. distinct morphological changes in pma-stimulated pbls, compared to non-stimulated cells, can be observed. these include a less rounded appearance of the pma-stimulated pbls, and evidence of "uropod" formation, which anchor the t cell to the endothelium as part of the migration process. levels of adhesion to vcam-1 are high (80-90%, compared to control), but there appears to be little difference between the adhesion profiles of non-stimulated and pma-stimulated pbls.however, there is a distinct difference between the adhesion levels of non-stimulated and pma-stimulated pbls to icam-1, with pma-stimulated cells showing a higher affinity for icam-1 than nonstimulated cells (approx. 70% and 40%, respectively).pbl adhesion to bsa is negligible. we present a novel in vitro microfluidic pump system that can simulate leukocyte adhesion to the endothelium under flow conditions. this platform is a more efficient and economical system compared to those currently available, due to reduced material costs and style of construction. introduction: pulmonary aspiration of gastric contents is a common complication observed in icu patients and a potential trigger of ards. in this study we evaluated the course of lung inflammation induced by intranasal instillation of gastric juice (gj). methods: gj was obtained from donor rats (ph 1.6). male c57bl/6 were instilled with 2 ml/kg of gj. after 2 or 24 h, the animals were sacrificed and lung and balf were collected. control group consisted of non-manipulated mice. 287.3ae18.6, sg24h: 277.8ae63.8; pg/ml). discussion: gj aspiration induced an initial adherence of pmn to lung tissue that is correlated with increased tnf-a/il-10 ratio in balf at the 2nd h. the reduction of mpo activity is correlated with the decrease in tnf-a/il-10 ratio. the late increase of pmn in balf might be a consequence of the early production of tnf-a. the results are suggestive that the treatment of patients exposed to acid aspiration should be focused in the initial period of the insult and in the blockage of tnf-a. objectives: intestinal i/r is implicated as a prime initiating event in the development of acute respiratory distress syndrome (ards) after trauma and hemorrhagic shock. we investigated the effects of lps challenge to mice previously submitted to i-i/r, a two-hit model of acute lung injury. methods: male c57bl/6 mice were subjected to 45 min of intestinal ischemia and challenged with 0.1 mg/kg of intranasal lps at the 4th hour of reperfusion (two-hit). balf and culture of lung explants were performed 20 h after lps challenge. mice subjected to i-i/r or lps alone were used as controls. results: two-hit mice showed marked increase in lung evans blue dye leakage compared to i-i/r (375.5ae53.9 vs 48.8ae3.5, mg/mg). lung mpo was increased (0.30ae0.03 vs 0.15ae0.03; od 450nm) whereas the neutrophil recruitment to balf was inhibited in the two-hit group compared to lps group (11.5ae2.6 vs 28.5ae3.4; x 10e3cells/mouse). the levels of nox-in the two-hit group were significantly increased when compared to i-i/ r controls in balf (10.6ae0.5 vs 6.5ae0.8; mm) and in lung explants (3.1ae0.3 vs 1.3ae0.1; mm/mg of tissue). conclusions: intestinal i/r predisposes the animal to an exacerbated response to a low dose lps insult. the exacerbated production of nitric oxide observed in the two-hit group may cause endothelial damage, thereby explaining the major increase in vascular permeability in the two-hit group. the results are suggestive that patients exposed to systemic inflammatory response might develop ards when in contact with secondary inflammatory stimuli. nitric oxide may play an important role in this process. (1) (1) novartis institutes for biomedical research, horsham, uk (2) university of michigan, usa obligatory for using oxygen in energy transfer pathways was the simultaneous co-evolution of enzymes that detoxify the reactive species formed as by-products. thus, we hypothesized that individuals with low aerobic function will have reduced anti-oxidant capacity and, therefore, be more susceptible to smoking-related lung diseases like copd. to test this hypothesis, we exposed high capacity runner (hcr) and low capacity runner (lcr) rats to 3 months of whole-body smoke exposures.the animals, bred over successive generations on the same background strain for high or low running capacity, differ by over 500% (p<1x10-37) for exercise capacity, measured by running on a treadmill.after 3 months of exposures, inflammatory cells in bronchoalveolar lavage fluid were increased in both the hcr-and lcr-smokeexposed(se) animals compared to air-exposed controls (p<0.001); however there was a 2-3-fold increase in the number of neutrophils and lymphocytes in the lcr-over the hcr-se group (p<0.001).histopathology revealed there was greater inflammation and lung damage present in the lcr-versus hcr-se group (p<0.05). metabonomic (metabolite profiling) analysis revealed that while peroxidation of lung lipids occurred for both se groups, oxidative damage to the lung surfactant layer was significantly more extensive for the lcr-se. systemic oxidative damage was also more apparent in the lcr-se group, with metabolic profiling suggesting a reduced capacity to regenerate muscle glutathione. the metabolic data suggest that repair processes maybe more effective in the hcrs. in summary, these data support the concept that aerobic capacity may be central to ones susceptibility to developing smoking-related lung disease. (1), ap ligeiro-oliveira(1), jm ferreira-jr(4), sr almeida(1), w tavares de lima(1), shp farsky (1) (1) university of sâ¼o paulo, brazil (2) regional integrated university of alto uruguai and missã°es, brazil methods: male wistar rats were exposed to vehicle or hq (50 mg/kg; ip.;daily, 22 days, two-day interval every five days). on day 10, animals were ip sensitized with ovalbumin (oa). assays were performed on day 23. results: hq-exposed rats presented reduced number of leukocytes in the bronchoalveolar fluid and by impaired in vitro oa-induced tracheal contraction. the latter effect suggests reduction on mast cell degranulation, and it was corroborated by in vivo decreased mesenteric mast cell degranulation after topical application of oa. the oa-specificity response was confirmed by normal ability of mast cells to degranulate in both groups of animals after topical application of compound 48/80. in fact, lower levels of circulating oa-anaphylactic ige antibodies were found in hq-exposed rats. this latter effect was not dependent on number or proliferation of lymphocytes, nevertheless reduced expressions of costimulatory molecules cd45 and cd6 on oa-activated lymphocytes indicated the interference of hq exposure on signaling of the humoral response during an allergic inflammation. contact information: ms sandra manoela dias macedo, regional integrated university of alto uruguai and missã°es / university of sâ¼o p, department of clinical and toxicological analyses, sâ¼o paulo, brazil e-mail: smdmacedo@yahoo.com.br (1) (1) radboud university nijmegen, medical centre, nijmegen, the netherlands (2) university hospital, zã¼rich, switzerland toll-like receptors (tlr) are essential in the recognition of invading microorganisms. however, increasing evidence shows involvement of tlr in autoimmunity, such as rheumatoid arthritis (ra), as well. here we investigated whether synovial expression of tlr3 and tlr7 was associated with the expression of ifna, tnfa, il-1b, il-12, il-17, and il-18 and studied in what way these receptors and cytokines were associated in vitro. using immunohistochemistry, we found that tlr3 / tlr7 expression in synovial tissue was associated with the presence of ifna, il-1b and il-18, but not tnfa, il-12 and il-17. to investigate whether ifna, il-1b and il-18 could induce tlr3 / tlr7 upregulation in vitro, we incubated separate lymphocyte populations with these cytokines and subsequently determined tlr3/7 mrna expression. ifna incubation resulted in significant tlr3 /tlr7 upregulation, whereas il-1b and il-18 did not. pre-incubation with ifna and subsequent stimulation of tlr3 /tlr7 significantly enhanced il-6, tnfa and ifna/b production, indicating that the ifn-induced tlr upregulation was functional. low amounts of biologically active il-1b were produced upon stimulation with atp, but not upon tlr3 / tlr7 stimulation, although mrna levels were high. interestingly, ifna-priming significantly increased the atp-induced il-1b production. here, we demonstrated a dual role for ifna in vitro, which could explain the association between tlr and il-1b / il-18 in synovial tissue. first, involvement in tlr3 /tlr7 regulation and second, involvement in atp-induced production of biologically active il-1b. these results suggest involvement of anti-viral immune responses in ra and ifna as a key player in chronic inflammation. the pathogenesis of chronic joint inflammation remains unclear although the involvement of pathogen recognition receptors (ppr) has been suggested recently. here, we described the role of two members of the nacht-lrr (nlr) family, nod1 (nucleotide/ binding oligomerization domain) and nod2 in model of acute joint inflammation induced by intraarticular injection of tlr2 (toll-like receptor) agonist streptococcus pyogenes cell wall fragments. we found that nod2 deficiency resulted in reduced joint inflammation and protection against early cartilage damage. in contrast, nod1 gene deficient mice developed enhanced joint inflammation with concomitant elevated levels of proinflammatory cytokines and cartilage damage. to explore whether the different function of nod1 and nod2 occurs also in humans, we exposed pbmcs carrying either nod1frameshift or nod2frameshift mutation with scw fragments in vitro. both tnfa and il-1b production was clearly impaired in pbmcs carrying the nod2fs compared to pbmcs isolated from healthy controls. in line with the nod1 gene deficient mice, human pbmcs bearing the nod1 mutation produced enhanced levels of proinflammatory cytokines after 24h stimulation with scw fragments. these data indicated that the nlr family members nod1 and nod2 have a different function in controlling tlr2-mediated pathways. we hypothesize that intracellular nod1-nod2 interactions determine the cellular response to tlr2 triggers. whether lack of controlling tlr2-driven pathways by nod1 signalling is involved in the pathogenesis of autoinflammatory or autoimmune disease, such as rheumatoid arthritis (ra), remains to be elucidated. leukocyte immunoglobulin-like receptors (lilrs) are a family of receptors with potential immune-regulatory function. activating and inhibitory receptors play a role in maintaining immunological equilibrium and an imbalance may lead to the onset of autoimmune diseases such as rheumatoid arthritis (ra). ra is a chronic inflammatory disease of joints caused by mediators (i.e. tnf-a) produced by activated leukocytes. we recently demonstrated expression of activating lilra2 in synovial tissue macrophages from ra patients. the aim of this study was to determine expression and function of lilra2 in monocytes and macrophages. peripheral blood mononuclear cells (pbmc) were prepared by standard density gradient separation and in vitro-derived macrophages were generated by differentiating thp-1 cells with vitamin-d3. lilra2 expression was measured by flow cytometry before and after modulation with cytokines. differentiation to macrophages significantly up-regulated lilra2 expression (p=0.0239). treatment of macrophages with lps, tnf-a, il-1b and ifn-g but not il-10 caused significant down-regulation of lilra2 (p<0.01). function of lilra2 was assessed by cross-linking with plate-immobilised lilra2-specific mab. soluble tnf-a was measured by elisa. activation of cells elicited tnf-a production in a dose-dependent manner while time-course analysis shows maximal production at 18h. correlation between lilra2 expression and response to cross-linking indicates that level of expression may relate directly to the degree of activation. decrease expression in response to acute-phase cytokines suggests controlled regulation during inflammation. in ra, abnormal regulation of lilra2 could potentially exacerbate inflammation by inducing uncontrolled production of proinflammatory cytokines. pharmacological blocking of lilra2 could potentially provide therapeutic benefit. (1) (1) university of valencia, spain (2) northwick park institute for medical research, uk co-releasing molecules (co-rms) mimic the biological actions of co derived from heme oxygenase activity. in the present work we studied the effects of a water-soluble co-releasing molecule (corm-3) on an animal model of human rheumatoid arthritis. dba-1/j mice were treated with corm-3 (5, 10 or 20 mg/kg/day, i.p.) from day 22 to 31 after collagen-induced arthritis (cia) and sacrificed on day 32. administration of corm-3 resulted in a significant improvement of the clinical profile of this disease since it markedly reduced joint swelling and redness. histological analysis of the joints in control arthritic mice indicated the presence of granulocytes and mononuclear cells, cartilage erosion, chondrocyte death and proteoglycan depletion. all these parameters were significantly reduced by corm-3 treatment with the most pronounced protective effect observed at 10 mg/kg. the levels of pro-inflammatory mediators (pge2, il-1beta, tnfalpha, il-2 and il-6) in the hind paw homogenates were significantly inhibited by corm-3. in addition, comp levels in serum, a marker of cartilage degradation, was reduced by the co-releasing agent. our studies show that therapeutic administration of corm-3 alleviates the clinical features of murine cia at the late phase of this response. the beneficial action of co liberated from corm-3 appears to be associated with a decrease in inflammatory cytokines and reduction of cell infiltration into the synovial tissues ultimately leading to a protective effect on the cartilage. aim: to setup a bovine model for cytokine-induced articular cartilage collagen degradation, and characterize the model using a variety of compounds targeting different disease mechanisms relevant to arthritis. methods: full thickness bovine articular cartilage punches were cultured with or without 10 ng/ml il-1a, tnf-a and oncostatin m. after three weeks the cartilage and culture medium were analyzed for weight changes, water content, dna content, glycosaminoglycans (gag), hydroxyproline (hyp), damaged collagen molecules, mmp activity, ctx-ii and comp. diclofenac, dexamethasone, pioglitazone, remicade, risedronate, galardin and a77-1726 were tested for their effect on cartilage degradation. results: exposure of articular cartilage to cytokines resulted in a decreased cartilage weight, increased proteoglycans degradation, increased collagen degradation, increased percentage of denatured collagen, increased water content and increased levels of active mmps (all p < 0.01). comp release during the first week of culture showed a trend towards up regulation during the first week of culture for all three donors, this was however not significant due to the small number of donors. most of the described processes were modulated by one or more of the drugs tested, indicating that this model for articular cartilage destruction is sensitive to treatment. discussion: stimulation of bovine articular cartilage explants with a cocktail of il-1a, tnf-a and osm results in clear and consistent changes in the cartilage, highly reminiscent of cartilage destruction during arthritis. further research needs to establish whether the model is also sensitive to anabolic factors that potentially could repair the damage. toll-like receptors (tlrs) may contribute to the progression of rheumatoid arthritis through recognition of hostderived damage-associated ligands that have repeatedly been found in arthritic joints. involvement of tlr2 and tlr4 activation in the expression of arthritis was studied using interleukin-1 receptor antagonist deficient (il-1ra-/-) mice, which spontaneously develop an autoimmune t-cell mediated arthritis. spontaneous onset of arthritis was dependent on tlr activation by microbial flora, as germ-free mice did not develop arthritis. after crossing with tlr knockouts, il-1ra-/-tlr2-/-mice developed more severe arthritis compared to il-ra-/-tlr2+/+ littermates; whereas, tlr4-/-il-1ra-/-mice were protected against arthritis. to clarify the mechanism by which tlr2 and tlr4 differentially regulated the disease expression, we studied the role of these tlrs in il-23 production and th17 development, both important in il-1ra-/-arthritis. wild type bone-marrow-derived dendritic cells (bmdcs) produced similar levels of il-23 upon stimulation with tlr2 and tlr4 ligands; however, il-1ra-/-bmdcs produced less il-23 than wild type dcs upon tlr2 stimulation and more il-23 than wild type dcs upon tlr4 stimulation. furthermore, il-1ra-/-t cells produced lower amounts of il-17 when cultured with tlr2-activated apcs and higher amounts of il-17 when cultured with tlr4-stimulated apcs, both in combination with cd3 stimulation. facs analysis of th17 (cd4+/il-17+) cells from both spleen and draining lymph nodes revealed 70% reduction in il-1ra-/-tlr4-/-mice compared to il-1ra-/-tlr4+/+ littermates. specific cd3/cd28 stimulation of non-adherent splenocytes confirmed lower il-17 production in il-1ra-/-tlr4-/-. these findings suggest important roles for tlr2 and tlr4 in regulation of th17 development and expression of arthritis. prostaglandine2 (pge2) stimulates the transactivational activity of p53 through p38map kinase-dependent ser15 phosphorylation (jbc 2006) .p53 controls cell-cycle progression, in part, by differential regulation of ap-1 proto-oncogenes (jun/fos).currently we studied pge2 control of cyclin d1 promoter activity with particular attention to the role of ap-1 oncogenes.pge2 induced a 7.6 fold increase in junb mrna expression (northern blot), a 2.1-fold increase in junb promoter activity (luciferase assay), and increased ser259junb phosphorylation in human synovial fibroblasts (hsf) (western blot).c-jun was strongly inhibited while jund, c-fos, fra1/ 2, and fosb expression were upregulated by pge2.in cell-cycle experiments, transformation with a constitutively active ha-ras construct (ras g12v) resulted in a 3.9 fold increase in cyclin-d1 promoter activity, cyclin-d1 synthesis, thr202/tyr204 phosphorylation of erk1/2 (6.2 fold) and ap-1 (c-jun)-dependent transactivity (3.1 fold); cyclin d1/cdk4-6 inhibitor p16ink4a synthesis was suppressed. addition of excess rass17n dominant negative mutant construct to the plasmid mix abrogated the aforementioned processes.ectopic expression of c-jun, c-fos and especially jund expression constructs stimulated cyclin d1 promoter activity/protein synthesis, blocked p16ink4a synthesis; the latter effects were reversed by the addition of excess junb.pge2 exerted temporal and bi-phasic dose-dependent control of the cyclin d1 promoter activity, largely through differential ap-1 activation and promoted cell cycle arrest and apoptosis in hsf at high physiological concentrations.the results provide further insight into the biology of the cpla2/ cox/pges biosynthetic axis and highlight the complexity of pge2 action in terms of cell-cycle progression. di-glucopyranosylamine (diga) is an antikeratitic (roberts et al., 1990 , acvo conference, scottsdale) immunomodulatory pyranosyl disaccharide with parenteral anti-rheumatic activity (bolton et al., 2005 , inflammation res. 54(s2) s121) and unknown mechanism of action.interestingly, anti-tnf therapy is anti-keratitic.-diga hydrolyses to monoglucosylamine (mga) and glucose, which is prevented by n-acetylation (nacdi-ga).lider (1995, pnas. 92: 5037-41) showed that sulphated disaccharides are orally active, inhibit tnf synthesis and the dth reaction.we have investigated the anti-tnf and anti-rheumatic activity of the sulphated and free digas.human whole blood (hwb) was stimulated with pha (5mg/ml) to synthesise tnf.antigen induced arthritis (aia) was induced in methylated bovine serum albumin (mbsa) sensitised c57bl/6 mice challenged i.a. into the stifle joint.collagen arthritis (cia) induced in dba mice by sensitisation to bovine collagen, were boosted i.p. with collagen at day 21. hwb tnf synthesis was inhibited by diga, mga and nacdiga(ic50 <0.1mm). diga (10ml, 1mm) i.a. prevented 24 hour aia (-0.32+/-0.06mm).diga at 100mg/kg reduced aia when administered i.v. (-0.40+/-0.13mm, p<0.05) and i.p. (-0.34+/-0.13mm, p<0.05), but is hydrolysed p.o. (0.24+/-0.08mm ns). polysulphated diga (diga8s) was unstable, but stabilised by n-acetylation (nacdi-ga8s).tnf synthesis was potently inhibited by both nacdiga and nacdiga8s (ic50 <0.1mm).nacdiga8s (100mg/kg p.o.) inhibited aia (-0.43+/-0.13mm), and nacdiga8s with lower degrees of sulphation (mw 800 and 1200 kda) inhibited the development of mouse collagen induced arthritis as assessed by clinical score. sulphated diglucosylamines represent a new class of heparinoid which are potent inhibitors of tnf synthesis and possess oral anti-rheumatic activity. excessive no appears to play a key role in the pathogenesis of chronic inflammatory diseases. in this study we aimed to evaluate no synthesis in rheumatoid arthritis (ra) before and after therapy. it was performed on 92 persons, divided into 6 groups: a negative control group of healthy volunteers, a positive control group with ra, a group with ra and physiotherapy (phys), a group with ra and low doses of cimetidine (cim) + doxycycline (dox), a group with ra and combined treatment phys + cim + dox, and a group treated with usual doses of ibuprofen (ibu). serum nitrite/nitrate (griess) was measured in order to evaluate no synthesis. results: compared to the positive control group, in all the treated groups no synthesis decreased significantly. there was no significant difference between phys and cim+dox effect alone. the combined treatment, phys + cim + dox had a much better inhibitory effect on no synthesis. between the phys, cim + dox and phys + cim + dox groups and that treated with ibuprofen, there was no significant difference in reducing no synthesis. conclusions: 1) in ra phys + cim + dox treatment was as efficient as ibuprofen in reducing no synthesis. 2) the low doses of cim and dox may allow a longer treatment due to the lower side effects risk enhanced socs1 expression following exposure of murine macrophages to lps implicated socs1 in the control of lps-mediated signaling. socs1 regulates nfkb signaling in murine macrophages, blocking at the level of mal or ikba phosphorylation. we investigated the role of socs1 in regulating the production tnf by lps and pam3csk4-activated primary human monocytes. blood monocytes were isolated by centrifugal elutriation and either infected with an adenoviral vector expressing socs1 (adv-socs1), control vector (adv-gfp) or left untreated. adv-socs1 monocytes were exposed to tlr4 and tlr2 ligands, lps (500 ng/ml) or pam3csk4 (300 ng/ml). facs analysis demonstrated infection efficiencies of 50ae4% and 67ae4% (n=16, mean ae sem) of monocytes expressing adv-gfp or adv-socs1 at moi 50. adv-socs1 blocked lps and pam3csk4 induced tnf mrna and protein production in a dose-dependent manner. in contrast, il-6 and il-10 production by adv-socs1-infected monocytes was not blocked. adv-socs1 also blocked lps and pam3csk4induced tnf production by macrophages isolated from synovial fluid. infection efficiencies of 67ae1% or 72ae1% were obtained. quantitative western blot analysis revealed that the classically defined nfkb pathway was not altered at the level of ikba or p65 activation. furthermore, the kinetics of lps and pam3csk4induced ikba phosphorylation and degradation in adv-socs1 monocytes remained unaffected (n=5 and 2 donors, respectively). further, analysis of parallel mapk pathways demonstrated no block in p38 or erk mapk pathways. these data suggest that socs1 regulation of lps and pam3csk4-induced tnf production by human monocytes occurs downstream of tlrs, possibly at the level of transcription. recently, beta-nad+ has emerged as a novel extracellular player in the human urinary bladder. beta-nad+ is the natural substrate of cd38 which catalyzes the conversion of beta-nad+ to cadpr. under normal conditions in vivo, there is no or only very small quantities (submicromolar range) of extracellular beta-nad+ compared to intracellular levels (200-1000 mm). during inflammation cell lysis may cause bursts of high local beta-nad+ levels. however, the effect of beta-nad+ on the human detrusor smooth muscle cells (hdsmc) was unknown. the effect of beta-nad+ on cultured (explant technique) hdsmc was determined by: 1) measuring cytosolic free calcium ([ca2+] i) in fura-2 loaded hdsmc using spectrofluorometry and 2) force measurements in 10-20 mg detrusor strips. hdsmc responded to beta-nad+ (40-100 mm) with an immediate and transient increase in [ca2+] i. the ca2+ transient was followed by one or two much slower and transient increases in [ca2+] i, indicative of beta-nad+ enzymatic conversion into cadpr. the ca2+ responses persisted in the absence of extracellular calcium. the ca2+ responses to beta-nad+ were not affected by exposure of hdsmc to atp supporting the notion that the effects of beta-nad+ were not mediated via p2x7 purinoceptors. furthermore, beta-nad+ caused a concentration-dependent detrusor muscle relaxation. this is the first study to report that extracellular beta-nad+ affect intracellular calcium homeostasis and force in hdsmc. these powerful actions of beta-nad+ suggest a role for beta-nad+/cadpr system as a novel extracellular player in the human detrusor during inflammation. aids remains a worldwide threat more than two decades after identification of hiv as the etiological agent. its wide dissemination can be partly attributed to its successful suppression of immunity resulting in disease progression and concomitant opportunistic infections including mycobacterial and cytomegalovirus infections. hiv trans-activator (tat) is one of the regulatory proteins that mediates hiv replication and dysregulates cellular functions such as apoptosis and cytokine expression. for example, tat induces tumor necrosis factor (tnf) and enhances gp120-induced neurotoxicity. we recently showed that tat induces the overexpression of il-10 via cellular kinase pkr and activation of transcription factor ets-1. in this study, we examined whether tat plays a role in perturbing interferon-& (ifn&) signal transduction. we showed that tat impaired ifn&-induced stat1 tyrosine phosphorylation, but had no effects on the serine residue of stat1 and jak kinases in primary human blood monocytes. furthermore, we found that the nuclear translocation of phospho-stat1 was abrogated by tat. the inhibition of phospho-stat1 led to the deformation of stat1 homodimers and subsequent stat-dna complex. to investigate the cellular consequences, we measured the expression of ifn&-stimulated genes including human leukocyte antigen (hla) and 2,5oligoadenylate synthetase (2,5oas), a key enzyme in the activation of latent ribonuclease l. the results showed that tat inhibited transcriptional activation of 2,5oas and hla. taken together, we identified a new role for tat in which it impairs ifn& signal transduction and suppresses inflammation, thus crippling the immune system and contributing to hiv persistence, opportunistic infections and disease progression. caspase-1 belongs to the group of inflammatory caspases and is the activating enzyme for the pro-inflammatory cytokine interleukin-18 (il-18), a cytokine known to play an important role in the pathogenesis of psoriasis. the purpose of this study was to determine the expression of caspase-1 in psoriatic skin and the signaling mechanisms involved in stress induced activation of caspase-1 and il-18. interestingly, increased caspase-1 activity in lesional compared with nonlesional psoriatic skin was seen as determined by western blotting. in vitro experiments in cultured human keratinocytes demonstrated anisomycin induced, p38 mapk dependent increased secretion of procaspase-1 and active caspase-1. furthermore, anisomycin increased the mrna expression of il-18 through a p38 mapk dependent but caspase-1 independent mechanism, reaching a maximum level after 12 hours of stimulation. finally, anisomycin caused a rapid (4 hours) increase in the secretion of proil-18 and active il-18. secretion of active il-18 was mediated through a p38 mapk/caspase-1 dependent mechanism, whereas secretion of proil-18 was mediated by a p38 mapk dependent but capsase-1 independent mechanism. these data demonstrate that the activity of caspase-1 is increased in psoriatic skin and that il-18 secretion is regulated by a p38 mapk/caspase-1 dependent mechanism, making caspase-1 a potential target in the treatment of psoriasis. prostaglandin e2 (pge2) regulates the stability of cyclooxygenase-2 (cox-2) mrna through adenylate/uridylate-rich elements (ares) in the 3untranslated region (3utr) by a positive autocrine/paracrine feed-forward loop. the principal objective of this study was to elucidate the molecular mechanisms involved in the pge2dependent stabilization of cox-2 in human synovial fibroblasts (hsfs). transfection of well-known are binding proteins (aubps) demonstrated that tristetraprolin (ttp) potently destabilized a [luciferase-cox-2 3utr] reporter fusion mrna (71ae5.7% decrease in luciferase activity vs. control). ttp protein levels in hsfs remained constant despite il-1b-induced changes in ttp mrna levels, thus suggesting translational regulation of its expression. pge2 did not affect the transcription or translation of this gene in hsfs. western blot analysis of hsf ttp demonstrated the existence of a specific, covalent~55kda heterocomplex containing ttp (ttphcx). although ttphcxs exact composition and stoichiometry is yet to be defined, pge2 selectively regulated the amount of this heterocomplex in a time-dependent manner. furthermore, protein shuttling studies performed using real-time confocal microscopy revealed that pge2 can induce export of a small nuclear pool of ttp-gfp. finally, transfection of ttp into hsfs also influenced cox-2 gene transcription, thus enabling ttp to regulate cox-2 gene expression at both the transcriptional and post-transcriptional level. in conclusion, we have demonstrated that ttp is an rna binding protein capable of influencing cox-2 mrna stability and transcription and whose localization and interaction with other factors is regulated by pge2. these data can provide important insight into deciphering the role of pge2 in fine-tuning physiological and pathophysiological gene regulation. (1) (1) chinese academy of sciences, shanghai, pr china (2) ohio state university, usa mitogen-activated protein (map) kinases play a critical role in innate immune responses to microbial infection through eliciting the biosynthesis of proinflammatory cytokines. map phosphatases (mkp)-1 is an archetypical member of the dual-specificity phosphatase family that deactivates map kinases. induction of mkp-1 has been implicated in attenuating the lipopolysaccharide (lps) and peptidoglycan (pgn) responses, but how the expression of the mkp-1 is regulated is still not fully understood. here, we show that inhibition of p38 map kinase by specific inhibitor sb 203580 or rna interference (rnai) markedly reduced the expression of mkp-1 in lps or pgn-treated macrophages, which is correlated with prolonged activation of p38 and jnk. depletion of mapkap kinase 2 (mk2), a downstream substrate of p38, by rnai also inhibited the expression of mkp-1. the mrna level of mkp-1 is not affected by inhibition of p38, but the expression of mkp-1 is inhibited by treatment of cycloheximide. thus, p38 mapk plays a critical role in mediating expression of mkp-1 at a posttranscriptional level. furthermore, inhibition of p38 by sb 203580 prevented the expression of mkp-1 in lpstolerized macrophages, restored the activation of map kinases after lps restimulation. these results indicate a critical role of p38-mk2-dependent induction of mkp-1 in innate immune responses. the i-kb kinase (ikk) complex regulates the activation of nf-kb a key transcription factor in inflammation and immunity. whilst ikka activity is necessary for proinflammatory and anti-apoptotic gene expression, ikka has distinct roles in lymphorganogenesis and b cell maturation. here we describe a role for ikka in cell mediated immunity (cmi). paw inflammation in methylated bsa-induced cmi was significantly reduced in transgenic mice expressing a mutant ikka protein that cannot be activated (ikka aa/aa ) compared to wild-type (wt). antigen-induced il-2 and ifng production by ikka aa/aa splenocytes and ikka aa/aa t cell:dc cocultures were also significantly reduced ex vivo. this could be normalised by using wt t cell: ikka aa/aa dendritic cell (dc) but not ikka aa/aa t cell:wt dc combinations. this suggests that reduced cmi in ikka aa/ aa mice is due to a defect in ikka aa/aa t cells not dcs. this is not due to a requirement of ikka in tcrmediated activation of t cells, since anti-cd3/cd28 mediated activation of ikka aa/aa t cells was unaltered. however, lps-induced production of the important th1 cytokine il-12 is impaired in ikka aa/aa dcs. we are currently addressing the hypothesis that ikka activity may be required for the generation and maintenance of antigen-specific t cells in vivo. recently we described a role for ikkâµ in the negative regulation of innate immunity and acute inflammation, which is in contrast to its role shown here in promoting adaptive immunity and antigen-driven inflammation. ikka may represent an alternative target for the treatment of autoimmune disease which would not compromise host defence. as a latent transcription factor, nf-kb translocates from cytoplasm into nucleus upon stimulations and mediates expression of genes important in immunity, inflammation and development. although extensive studies have been done regarding how nf-kb is triggered into nucleus, little is known about how it is regulated inside nucleus. by twohybrid approach, we identify a prefolding-like protein snip that is expressed predominantly and interacts specifically with nf-kb inside nucleus. we show that rnai knockdown of snip leads to impaired nucleus activity of nf-kb and dramatically attenuates expression of nf-kb dependent genes. this interference also sensitizes cells to apoptosis by tnf-a. furthermore, snip forms a dynamic complex with nf-kb and is recruited to the nf-kb enhancesome upon stimulation. interestingly, snip protein level correlates with constitutive nf-kb activity in human prostate cancer cell lines. the presence of nf-kb within nucleus of stimulated or constitutive active cells is significantly diminished without endogenous snip. our results reveal that snip is an integral component of nf-kb enhancesome and essential for its stability in nucleus, which uncovers a new mechanism of nf-kb regulation. bone remodeling is a tightly regulated process that couples resorption of old bone by osteoclasts and the deposition of new bone by osteoblasts. an imbalance between bone formation and bone resorption can result in various metabolic bone diseases, such as rheumatoid arthritis and osteoporosis. osteoclasts are terminally differentiated cells that arise from a haematopoietic stem cell lineage, which also gives rise to monocytes and macrophages. osteoclast differentiation and regulation of this process to maintain bone homeostasis are central to the understanding of the pathogenesis and treatment of bone diseases, such as osteoporosis. in vitro, osteoclast formation from bone marrow macrophages is induced by rankl (receptor activator of nf-kappa b ligand) in the presence of m-csf (macrophage colony stimulating factor). osteoclastogenesis is markedly enhanced in bone marrow macrophages from ifnar1-/-and ifnar2-/-mice and results in increased number of multinucleated cells positive for osteoclast marker, trap (tartrate-resistant acid phosphatase). consequently, the mutant mice develop osteoporotic phenotype, characterised by reduced bone density. these findings suggest that the ifn alpha/beta system is critical for the negative feedback regulation of osteoclastogenesis and that rankl signaling is essential for the induction of osteoclast differentiation. atp acting on p2x7 receptors in macrophage is one of the main physiological signals that lead to the processing and release of the pro-inflammatory cytokine, interleukin-1beta (il-1b), their activation also leads to rapid opening of a membrane pore permeable to dyes such as ethidium. here we identify pannexin-1, a recently described mammalian protein that functions as an hemichannel when ectopically expressed, as this dye-uptake pathway and show that signalling through pannexin-1 is required for processing of caspase-1 and release of mature il-1b induced by p2x7 receptor activation. furthermore, maitotoxin and nigericin, two agents considered to evoke il-1b release via the same mechanism were studied. maitotoxin evoked dye uptake whose kinetics were similar to a slow pannexin-1-independent phase induced by p2x7 receptor activation, and this was unaltered by pannexin-1 inhibition.nigericin did not induce dye uptake.inhibition of pannexin-1 blocked caspase-1 and il-1b processing and release in response to this two stimuli.thus, while pannexin-1 is required for il-1b release in response to maitotoxin, nigericin and atp, a mechanism distinct from pannexin-1 hemichannel activation must underlie the former two processes. introduction: saa is a classic acute-phase protein upregulated during inflammatory response. saa is active on leukocytes and modulates inflammation and immunity through the induction of cytokines, including the chemokine il-8. here we verify the effect of saa on the mrna expression and release of mip-3alpha, a chemokyne involved in the recruitment of dendritic cells. methods: peripheral blood mononuclear cells (pbmc) isolated from peripheral blood by density gradient were cultured in rpmi medium in the presence of saa. mip-3alphaconcentration was determinated in the supernatant of cell cultures by elisa. mrnawas analyzed bythe ribonuclease protection assay (rpa). results: pbmc stimulated with saa (20 ug/ml) induced the expression of mip-3alpha mrna at 2, 4 and 12 hours. mip-3alpha protein was found in the suppernatant of 24 and 42 hours cultures (p<0,05) and the addition of sb 203580 (p38 inhibitor) and pd 98059 (erk 1/2 inhibitor) completely abolished the release of mip-3alpha. conclusions: saa is an inducer of mip-3alpha expression in pbmc and p38 and erk1/2 are important pathway signaling to this effect. saa is one of the factors responsible by the recruitment of dendritic cells. the p38 pathway is activated in numerous inflammatory conditions, including ra, ibd asthma, acute coronary syndrome, and copd, and its activation helps drive the production of inflammatory mediators. inhibitors of p38 decrease mediator production and therefore can produce profound anti-inflammatory effects. arry-797 is a potent inhibitor of p38 enzyme (ic50 < 5nm) with a novel pharmacophore and physiochemical properties distinct from those of other p38 inhibitors, being very water soluble. it is extremely potent in human whole blood, blocking lps-stimulated tnf production with an ic50 < 2nm.in animal models of rheumatoid arthritis (cia and aia) the compound significantly normalized histologic endpoints, such as inflammation, bone resorption and cartilage damage (ed50~3 mg/kg). a phase i single ascending dose clinical study was run in healthy volunteers. after an oral dose of 50, 100, 200, 300 or 400 mg), blood was drawn at various times, stimulated ex vivo with lps, and analyzed for cytokines and inflammatory mediatorsfj arry-797 was well-tolerated and drug exposure was proportional to dose. in ex vivo samples, there was both a time-and concentrationdependent inhibition of il1, pge2 and tnffz with >80% inhibition observed at the 50 mg dose level. the plasma concentrations of drug peaked at~20 ng/ml at the 50 mg dose and cytokine inhibition was sustained for >4 hours, showing that low doses of arry-797 produced profound effects on clinical biomarkers. further evaluation of arry-797 in patients with inflammatory diseases is planned. introduction: we demonstrated that in vivo chronicle blockage of nos (l-name, 20mg/kg; oral route; 14 days) impairs leukocyte-endothelial interactions and neutrophils migration into inflammatory focus. these effects may be depending, at least in part, on decreased expression of l-selectin on leukocytes and pecam-1 on endothelium. aimed to clarify the mechanisms involved on these inhibitory effects, we now investigated the role of l-name treatment on secretion of tnf and il-1b; by circulating leukocytes and migrated peritoneal neutrophils. methods: male wistar rats were treated with l-name (20mg/kg; oral route; 14 days) or sterile saline (control). circulating leukocytes were isolated from blood collected from abdominal aorta and migrated neutrophils were obtained 4 hours after i.p. injection of oyster glycogen (1%;10ml). no (griess reaction) and cytokines (elisa) were quantified in supernatants of 1 x 106 cultured cells before and 18 hours after lps stimulation (5m;g/ml). results: levels of no, tnf and il-1b; were reduced in circulating leukocytes from l-name-treated rats in both basal and lps stimulated conditions. on the other hand, only secretion of il-1b; was impaired by migrated neutrophils. conclusions: results show that in vivo l-name treatment, which partially reduces no production, decreases the secretion of pro-inflammatory cytokines by circulating leukocytes. however, the same pattern of inhibition is not detected if neutrophils are in vivo primed. objectives: to investigate the ability and mechanism of ifn-g to suppress interleukin-1 (il-1)-induced mmp-13 expression in articular chondrocytes. methods: human chondrocytes were treated with ifn-g or il-1beta alone or in combination. mmp-13 mrna was analyzed by rt-pcr. mmp-13 protein, phospho-stat1 and p44/42 mapk levels were measured by western blotting. mmp-13 promoter-luciferase, cmv-cbp/p300 plasmids and stat1 sirna were transfected by calcium phosphate method. ap-1 activity was monitored by elisa. stat1-cbp/p300 interaction was studied by immunoprecipitation. results: ifn-gpotently suppressed il-1-induced expression of mmp-13 and promoter activity. blockade with neutralizing ifn-gr1 antibody revealed that mmp-13 inhibition by ifn-â¼ was mediated by the ifn-â¼ receptor. ifn-beta-stimulated activation of stat1 was directly correlated with mmp-13 suppression. knockdown of stat1 gene by specific sirna or its inhibition with fludarabine partially restored the il-1 induction of mmp-13 expression and promoter activity. ifn-g did not alter activator protein (ap-1) binding ability but promoted physical interaction of stat1 and cbp/p300 co-activator. p300 overexpression reversed ifn-g inhibition of endogenous mmp-13 mrna expression and exogenous mmp-13 promoter activity. conclusions: ifn-g through its receptor activates stat1, which binds with cbp/p300 co-activator, sequesters it from the cell system and thus inhibits transcriptional induction of mmp-13 gene in chondrocytes. ifn-g and its signaling pathways could be targeted therapeutically for (1), p asmawidjaja (1), r hendriks(2), erik lubberts (1) (1) erasmus medical center, department of rheumatology, rotterdam, the netherlands (2) erasmus medical center, department of immunology, rotterdam, the netherlands the objective of this study was to identify the role of il-23 in th17 polarization in the prone autoimmune dba-1 mice with and without collagen-induced arthritis and to evaluate th17 specific cytokine and transcription factor expression. il-23 induced th17 cells in vitro from spleen cells of naã¯ve and collagen-type ii (cii) immunized dba-1 mice. the percentage of th17 cells is markedly higher in cii-immunized versus naã¯ve dba-1 mice. adding il-23 to tgf-beta/il-6 stimulated cd4 + t cells did not significantly increase the percentage of th17 cells. tgfbeta/il-6 in contrast to il-23 induced a relatively high percentage of il-17+/ifn-gamma-cells and low il-17-ifn-gamma+ cells. tgf-beta/il-6 did not increase il-23 receptor expression, which may explain why adding il-23 directly or two days after tgf-beta/il-6 did not result in an increase in the percentage of th17 cells. elevated expression of il-17a and il-17f as well as the th17 specific transcription factor rorgammat was found under il-23 as well as tgfbeta/il-6 conditions. interestingly, il-23 but not tgf-beta/il-6 is critical in the th17 cytokine il-22 expression in t cells from ciiimmunized dba-1 mice. these data show that il-23 was more pronounced in inducing il-17+/ifn-gamma-(th17) cells under cii-immunized conditions. furthermore, il-23 did not markedly increase the percentage of th17 cells induced by tgf-beta/il-6. however, il-23 is critical for the induction of il-22 expression, suggesting a unique role for il-23 in the induction of specific th17 cytokines ebi3 was initially discovered as a transcriptionally activated gene in epstein-barr virus-infected human b lymphocytes, and similar to p40 of il-12. ebi3 protein has been shown to form heterodimers with p28. p28/ebi3 termed il-27, can influence the function of multiple t cell subsets, including naive, effector, regulatory and memory t cells. however, previous studies showed that the overlapped expression of ebi3 and p28 is very limited. these data lead to the hypothesis that ebi3 may play a role independently from its association with p28. thus, to define the function of ebi3, we generated ebi3 transgenic (tg) mice expressing in multiple tissues. ebi3 tg mice exhibited no histologic abnormalities in various organs and normal numbers of naive and memory cd4+, cd8+ t cells, b cells, nk cells and nkt cells. cd4+t cells isolated from spleens of ebi3 tg mice, however, produced less ifn-g than cells from wt (wild type) control mice after in vitro stimulation with anti-cd3 and anti-cd28 antibodies. in vivo studies, delayed-type hypersensitivity (dth) and contact hypersensitivity (chs) responses were significantly reduced in ebi tg compared with wt mice. moreover, the chs responses in ebi3 tg mice were recovered with anti-ebi3 polyclonal antibody. notably, chs reaction in wt mice was increased by anti-ebi3 antibody. in contrast, anti-p28 antibody suppressed chs responses in wt mice. these data suggest that ebi3 acts in different from il-27, and reduces th1 responses. (1), o thaunat(2), x houard (1), o meilhac (1), g caligiuri(2), a nicoletti (2) (1) inserm u698 and university denis diderot-paris 7, chu xavier bichat, paris, france (2) inserm umr s 681, universitã� pierre et marie curie-paris 6, centre de recherche des cordeliers, paris, france arteries are composed of three concentric tissue layers which exhibit different structures and properties. because arterial injury is generally initiated at the interface with circulating blood, most studies performed to unravel the mechanisms involved in injury-induced arterial responses have been focused on the innermost layer (intima). in contrast, the role of the outermost tunica, the adventitia, has attracted relatively little attention and remains elusive. in the present review, we focus on involvement of the adventitia in the response to various types of arterial injury leading to vascular remodeling. several lines of evidence show that the initial insult and the early intimal response lead to the genesis of (neo-) mediators that are centrifugally conveyed by mass transport towards the adventitia. these mediators trigger local adventitial responses including angiogenesis, immuno-inflammation, and fibrosis. we propose that these three processes sequentially interact and that their net balance participates in producing each specific pathological condition. hence, an adventitial adaptive immune response predominates in chronic rejection. inflammatory phagocytic cell recruitment and initiation of a shift from innate to adaptive immunity characterize the adventitial response to proteolysis products in abdominal aortic aneurysm. centripetal adventitial sprouting of neovessels, leading to intraplaque hemorrhages, predominates in atherothrombosis. adventitial fibrosis mediated by low inflammation characterizes the response to mechanical stress and is responsible for constrictive remodeling of arterial segments and initiating interstitial fibrosis in perivascular tissues. these adventitial events thus impact not only on the vessel wall biology but also on the surrounding tissue. atherosclerosis has many of the characteristics of an inflammatory disease, and thus would classically involve endothelial cox-derived prostaglandins such as pge2 and prostacyclin acting on ep and ip receptors, respectively.activation of vascular ip receptors is especially important in limiting the atherogenic properties of thromboxane a2 acting on tp receptors.more recently, expression of ghrelin receptors has been shown to be increased in atherosclerotic plaques, and ghrelin itself has anti-inflammatory properties in addition to its classical role as a hunger hormone.as well as the complex crosstalk between g-protein-coupled receptors (gpcrs), recent evidence indicates that many gpcrs exist constitutively as homodimeric complexes, and that the formation of heterodimers not only influences the classical cell signalling pathways used by these receptors, but also affects their subcellular distribution.we have found that ep3-i, tp and ghrelin receptors readily form homodimers, but that co-transfection of hek293 cells with these receptors results in the formation of heterodimers with unpredictable effects on receptor distribution and cell signalling properties.since inflammatory conditions are thought to change the relative expression levels of gpcrs in the vasculature, and since varying the expression levels of gpcrs will affect their ability to form heterodimers, then one might predict that gpcr heterodimerization would indeed influence the reactivity of vascular tissue during inflammation. [this work was fully supported by grants from the research grants council of the hong kong special administrative region (cuhk4267/02m and 2041151 vascular inflammation leads to formation of leukotrienes through the 5-lipoxygenase pathway of arachidonic acid metabolism. leukotriene forming enzymes are expressed within atherosclerotic lesions and locally produced leukotrienes exert pro-inflammatory actions within the vascular wall by means of cell surface receptors of the blt and cyslt receptor subtypes. recent mechanistic studies have supported the notion of a major role of leukotriene signaling in atherosclerosis. leukotriene b4 (ltb4) is for example one of the most potent chemotactic mediators formed within the atherosclerotic lesion, inducing migration of a number different cell-types of both hematopoietic and non-hematopoietic origin. initially identified on neutrophils, blt receptor activation is involved in monocyte chemotaxis and adhesion as well as in vascular smooth muscle cell migration and proliferation, providing examples of potential mechanisms in ltb4-induced atherogenesis. targeting ltb4-induced activation of vascular smooth muscle cells has beneficial effects in models of intimal hyperplasia and restenosis after vascular injury. furthermore, blt receptor expression has been demonstrated on t-cells, suggesting ltb4 as a potential link between innate and adaptive immunological reactions. taken together, the local formation of leukotrienes within the atherosclerotic lesion and the potent pro-inflammatory effects of leukotriene receptor activation in target cells of atherosclerosis provide a rationale for a role leukotrienes in this disease. further experimental and clinical studies are however needed to develop therapeutic strategies of treatments targeting leukotriene signaling in atherosclerosis. in normal physiological conditions, the prostanoid (prostaglandin (pg) and thromboxane (tx)) synthesis is dependent on the constitutive isoform of cyclooxygenase (cox-1). this synthesis and release happen few minutes after cell or tissue stimulation. in vascular preparations submitted to pro-inflammatory conditions for some hours, the inducible isoform of cyclooxygenase (cox-2) and other prostanoid synthases can be observed. as an illustration of the previous experimental results, there is an increased presence of cox-2 and the inducible enzyme responsible for pge2 synthesis (mpges-1) detected by immunocytochemistry in the carotid atherosclerotic plaque with strong inflammation. in vascular cells in culture, pgi2 is the major biological active prostanoid produced in the normal physiological conditions. however, when cox-2 is induced, pgi2 and pge2 are equally produced. the role of cox-1, cox-2 and mpges-1 activities is also dependent on the expression of the various prostanoid receptors in the considered vessel. there is increasing evidence for the presence and a role of the ep receptor subtypes (ep1, ep2, ep3 or ep4) preferentially stimulated by pge2 in the vascular wall. for these reasons, we have characterized the receptors activated by pge2 in human mammary arteries. in these vessels incubated with a pro-inflammatory cytokine (interleukin-1ã¢) and lipopolysaccharides a reduced contractility to norepinephrine has been observed. this effect is abolished by treatment of the vascular preparations with a selective cox-2 inhibitor, suggesting that prostanoid synthesis and/or prostanoid receptors could be involved. rheumatoid arthritis is a syndrome which probably consists of a number of diseases for which the risk factors differ. two major processes were identified: the generation of the anti-citrullinated antigens immune response (highly sepcific for ra).we show that the different hla class ii alleles contribute to the development of anti-ccp-positive and anti-ccp negative ra.the se alleles do not independently contribute to the progression to ra, but rather contributed to the development of anti-ccp antibodies. next we determined the effect of smoking and observed that smoking only conferred risk to contract ra in the ccp-positive group and not in the anti-ccp negative group. for the risk factor ptpn22 (a gene that regulates treshold of lumphocyte activation) the allele c1858t only contributed to ccp-positive ra. in contrast to hla two other risk factors were found to be associated with both ccp-positive and ccp-negative ra. the risk factor in the fcrl-gene as has been identified in the japanese population was also tested in 931 dutch ra cases and 570 unrelated dutch controls. carrier analysis of the snp (rs7528684) revealed association of cc genotype with higher risk of developing ra as compared to tt & tc carriers (p =0.039 and or =1.31). in a meta-analysis of all studies comparing 9467 individuals, the or for the cc genotype to develop ra was 1.2 and the p-value < 0.001. in conclusion, different steps in pathogenesis of the syndrome ra can be delineated this talk will focus on recent advances in understanding primary genetic factors predisposing to inflammatory bowel disease (crohns disease and ulcerative colitis). proven genes containing genetic variants predisposing to crohns disease include ibd5/5q31, card15/nod2 and il23r. data is suggestive but not yet as convincing for many other genes. a common theme is of genetic variants influencing early innate immune responses to intestinal bacterial components, and subsequent adaptive immune responses, leading to intestinal inflammation. only for card15/nod2 is there (partial) understanding of how genetic variation influences biological function to cause chronic disease. some mouse models (gene targeted) of card15 appear to show opposite effects to other models and human systems. in humans, card15 mutations impair responses to bacterial components (muramyl dipeptide) mainly at low dose sensing. it is likely this receptor system normally maintains intestinal crypt sterility and protection from invasive infection. pathogen-recognition receptors (prrs) are key components of immune systems and are involved in innate effector mechanisms and activation of adaptive immunity. since their discovery in vertebrates, toll-like receptors (tlrs) have become the focus of extensive research that has revealed their significance in the regulation of many facets of our immune system. recently a new family of intracellular prrs, the nod-like receptors (nlrs), which include both nods and nalps have been described. mutations within the nalp3/cryopyrin/ cias1 gene are responsible for three autoinflammatory disorders: muckle-wells syndrome, familial cold autoinflammatory syndrome, and cinca/nomid. the nalp3 protein associates with asc and caspase-1 (thereby forming a molecular machine termed inflammasome that displays high proil-1beta-processing activity. macrophages from muckle-wells patients spontaneously secrete active il-1beta. increased inflammasome activity is therefore likely to be the molecular basis of the symptoms associated with nalp3-dependent autoinflammatory disorders. here we will emphasis on the ability of this protein complex to promote the development of autoinflammatory syndromes. allergic inflammation (ai) is a complex phenomenon initiated by allergen binding to ige sensitized mast cells and consequent mast cell activation. this causes the symptoms of the early phase of ai and the onset of the late phase characterized by the penetration in the inflamed tissue of inflammatory cells, notably the eosinophils. their subsequent activation is believed to cause tissue damage and to be the main responsible for the tissue remodeling, especially when the ai becomes a chronic process. we defined a novel functional unit that we termed the allergic synapse formed by mast celleosinophil couples. in the synapse these two old cellular players of ai have a cross talk via soluble mediators and receptor-ligand interactions. this results in mast celleosinophil functional synergism that consequently amplifies and prolongs the inflammatory response. in addition, mast cells and eosinophils are influenced and influence as in a sort of allergic niche the surrounding structural cells, i.e. fibroblasts and endothelial cells. we propose to view the allergic synapse/niche as a specialized effector unit worthy to be blocked for the treatment/prevention of allergic inflammation. (1) (1) erasmus mc, rotterdam, the netherlands (2) department of immunology weizmann institute of science, rehovot, israel allergic asthma is one of the most common chronic diseases in western society, characterized by reversible airway obstruction, mucus hypersecretion and infiltration of the airway wall with th2 cells, eosinophils, and mast cells. if we are to devise new therapies for this disease, it is important to elucidate how th2 cells are activated and respond to intrinsically harmless allergens. dendritic cells (dcs) are the most important antigen presenting cells in the lung and are mainly recognized for their exceptional potential to generate a primary immune response and sensitization to aeroallergens. we have shown that intratracheal injection of ovalbumin (ova) pulsed dcs induces sensitization leading to eosinophilic airway inflammation upon ova aerosol challenge. we investigated the role of dcs in the secondary immune response in a murine asthma model. ova aerosol challenge in ova-dc sensitized mice, induced an almost 100 fold increase in the number of airway dcs as well as an increase in eosinophils and t cells. to investigate the functional importance of dcs for the induction and maintenance of airway inflammation in response to allergen challenge, we conditionally knocked-out endogenous dcs in sensitised cd11c-diphtheria toxin (dt) receptor (cd11cdtr) transgenic mice by airway administration of dt 24 h before ova aerosol (4x) challenge or during an ongoing inflammation (depletion after 3x ova aerosols continued with 3 additional ova aerosols). numbers of balf eosinophils, th2 cytokine production by mediastinal lymph nodes and peribronchial and perivascular inflammatory infiltrates were dramatically decreased, illustrating an essential role for airway dcs during secondary challenge. karolinska institute, stockholm, sweden nk cells are innate lymphocytes with potent immunoregulatory functions. they are potent producers of several cytokines and chemokines, and also respond to similar molecules in the body and at inflammatory sites. even though traditionally best characterized for their role in anti-viral and anti-tumor immunity, they influence several other types of immune responses. for example, they are involved in, and affect, acute as well as chronic inflammatory responses. in the present talk, a general overview on our current knowledge of nk cell biology will be provided, with a special emphasis on the role of these cells in allergic inflammation. basophils are major effector cells in allergic reactions due to their ability to release substantial quantities of histamine and eicosanoids following activation of high affinity ige receptors (fc<epsilon>ri) with allergens. although these attributes are shared with their tissuefixed mast cell compatriots, basophils are unique in their ability to also rapidly elaborate th2-type cytokines (e.g. il-4 and il-13), subsequently supporting ige synthesis and underlying atopy. importantly, these mediators are additionally secreted following primary exposure to certain parasites (e.g. s. mansoni) and immunoglobulin superantigens, suggesting a role for basophils in innate immunity and in assisting developing th2-type adaptive immune responses. while we are beginning to understand the potential physiological functions of these cells regarding host defence, blocking their activity with respect to treating symptoms of allergic disease has remained an enigma. recent advances, however, have shed light upon the major intracellular signal transduction processes involved in fc<epsilon>ri activation and may lead to novel therapeutic strategies for inhibiting mediator secretions. an important discovery in this regard is the phosphatase ship, which downregulates pi 3-kinase signalling in both basophils and mast cells. recent data shows that ship expressions in basophils are reduced from donors with active allergic disease but that these levels may be increased, and the activity of basophils subsequently inhibited, by targeting receptors associated with ship recruitment (cd200r, cd300r). identifying the natural ligands for these inhibitory receptors may therefore pave the way for new therapies for the treatment of allergic inflammation. mitogenesis and proliferation of vsmc play an important role in atherogenesis. pro-inflammatory secretory phospholipases a2 (spla 2 ) hydrolyse glycerophospholipids of hdl and ldl and release pro-inflammatory agents, lyso-lipids, oxidized and non-oxidized fatty acids and isoprostanes.spla 2 s lipolysis products localize in vascular wall in vicinity of vsmc.we have tested the impact of spla 2 , hdl and ldl and of their hydrolysis products on mitogenesis and pge2 and ltb4 release from vsmc.mitogenesis was significantly enhanced by native hdl, and ldl, and by group v spla 2 . spla 2 hydrolysis of hdl and ldl enhanced mitogenic activity in order v>x>iia.the release of pge2 from vsmc was enhanced by group x spla 2 s but not iia or v.the greatest effect was seen for hdl hydrolysed by group v and x spla 2 .native ldl and its spla2 hydrolysis products enhanced the release of pge2 in order x>v>iia.the release of ltb4 from vsmc was markedly increased by native ldl and hdl, and hydrolysis products of group v and x, but not iia spla2.migration of vsmc was significantly enhanced by spla 2 iia and inhibited by hdl.this study demonstrates a complex interaction of hdl and ldl with pro-inflammatory spla2s, which affects mitogenesis, eicosanoid release and migration of vsmc.study of biocompatible spla2 blockers in the therapy of atherosclerosis is indicated. contact information: professor waldemar pruzanski, university of toronto, department of medicine, toronto, ontario, canada e-mail: drwpruzanski@bellnet.ca (1) (1) ipmc-cnrs umr6097, valbonne, france (2) university of washington, seattle, usa (3) inserm umrs 525, paris, france (4) university of naples, italy the superfamily of phospholipase a2 comprises at least 9 intracellular enzymes and up to 12 secreted pla2s (spla 2 s). elucidating the biological roles of each pla2 member is currently the most challenging issue in the pla2 field. the different spla 2 s are not isoforms and are likely to function either as enzymes producing key lipid mediators (eicosanoids and lysophospholipids) or as ligands that bind to specific soluble or membrane-bound proteins (like cytokines). increasing evidence suggests that spla 2 s iia, iii, v, and x are involved in inflammatory diseases including atherosclerosis. among spla2s, the human group x (hgx) enzyme has the highest enzymatic activity towards phosphatidylcholine, the major phospholipid of cellular membranes and low density lipoproteins (ldl). on human alveolar macrophages, hgx spla 2 can trigger secretion of tnf alpha, il1 and il6 in a non-enzymatic manner. on colorectal cancer cells, hgx spla 2 stimulates cell proliferation, produces potent eicosanoids including pge2, and activates the transcription of key genes involved in inflammation and cancer. the enzyme can also hydrolyze pc and platelet-activating factor (paf) of ldl particles very efficiently. finally, hgx spla 2 is present in human atherosclerotic lesions and converts ldl into a proinflammatory particle that induces macrophage foam cell formation, as well as map kinase activation, arachidonic acid release, and expression of adhesion molecules in huvec cells. some other key molecular features of spla 2 s including hgx will be presented. we have reported preferential release of polyunsaturated fatty acids during hydrolysis of lipoprotein phosphatidylcholine (ptdcho) by spla 2 s, but the mechanism of this selectivity is not known. since both sphingomyelin (sm) and lysoptdcho inhibit the activity while increasing fatty acid specificity of other pla2s, we have examined fatty acid release by spla2siia, v and x in relation to relative increases in proportion of endogenous sm and lysoptdcho during lipoprotein digestion. the analyses were performed by normal phase liquid chromatography with on-line electrospray mass spectrometry (lc/esi-ms) and lc/collision induced dissociation (cid)/esi-ms using conventional preparations ofldl and hdl. the highest preference for arachidonate release from ldl by group x spla 2 was observed when the residual sm/ pdcho molar ratio had reached 1.2 compared to a starting ratio of 0.4.group v spla 2 showed preferential release of linoleate at residual sm/ptdcho molar ratio 1.5, while at intermediate ratios, both arachidonate and linoleate were released at more comparable ratios. the relative increases in lysoptdcho and sm during the digestion with spla 2 iia were much more limited, and a preferential hydrolysis of polyunsaturated fatty acids was not observed. these results suggest a lipid phase separation as a likely basis for a differential hydrolysis of molecular species of ptdcho. the residual sm/ptdcho ratios reached during group v and x spla 2 digestion are similar to those observed for lesional ldl, which promote release of ceramides by smase leading to ldl aggregation. the above findings support a potential role of sphingomyelins in atherogenesis. although sphingomyelin (sm) is one of the most abundant phospholipids in lipoproteins and cell membranes, its physiological significance is unclear. because of its localization in the outer surface of the cells, and its structural similarity to phosphatidylcholine (pc),we proposed that it competitively inhibits phospholipolysis of cell membranes by external phospholipases (pla). we showed that sm inhibits several lipolytic enzymes including secretory pla2 iia, v, and x, and hepatic and endothelial lipases, all of which hydrolyze pc. treatment of sm in the substrate with smase c not only relieved the inhibition but also activated the pla2 reaction further, suggesting that ceramide, the product of smase c, independently stimulates pla2, possibly by disrupting the bilayer structure. smase d treatment, which produces ceramide phosphate, did not stimulate the spla2. the fatty acid specificity of pla2 is significantly affected by sm. thus spla2x exhibited enhanced specificity for the release of arachidonic acid (20:4) in presence of sm, due to a preferential inhibition of hydrolysis of other pc species. in contrast, sm inhibited the release of 20:4 by spla2v. ceramide selectively stimulated the release of 20:4 by both enzymes. only the long chain ceramides (> 10 carbons) were effective, while ceramide phosphate did not stimulate spla2 activity. sm-deficient cells released more 20:4 in response to spla2-treatment than normal cells, and pretreatment of normal cells with smase c increased their susceptibility to spla2 attack. these studies show that sm and ceramide regulate the activity and specificity of pla, and consequently the inflammatory response. secretory phospholipase a2 (spla2) types iia, v, or x, have been associated with inflammatory diseases and tissue injury including atherosclerosis in humans and mice.given the link between spla2 and atherogenesis, a mouse model of atherosclerosis (apoe-/-) was used to study the effects of a-002, an inhibitor of spla2 enzymes, on atherosclerosis and cholesterol levels over 16 weeks of treatment. mice were fed with a high-fat, high cholesterol diet alone during the study (21% fat; 0.15% cholesterol, 19.5% casein) and were treated with vehicle or a-002 bid at 30 mg/kg or 90 mg/kg by oral gavage. total cholesterol was significantly decreased after one month of treatment and remained lower throughout the study.treatment with a-002 significantly reduced aortic atherosclerotic plaque formation in apoe-/-mice fed a high fat diet when compared to the untreated control by approximately 40 %. in a different model that used angiotensin ii in conjunction with a high fat diet in a background of apoe-/-deficient mice for 4 weeks, oral dosing of a-002 (30 mg/kg bid) significantly reduced aortic atherosclerosis and aneurysm rate when compared to vehicle. these data suggest that a-002 is a potential novel therapeutic agent for the treatment of atherosclerosis. (1), s doty(2), c antonescu (3), c staniloae (1) (1) saint vincents hospital manhattan, new york, usa (2) hospital for special surgery, new york, usa (3) sloan-kettering institute for cancer research, new york, usa tnf-stimulated gene 6 (tsg-6) is induced by tnf-a during inflammation and its secreted product tsg-6 glycoprotein is involved in immune-mediated inflammatory diseases and fertility. it regulates cox-2 and prostaglandin synthesis, and participates in extracellular matrix remodeling. considering the chronic inflammatory nature of atherosclerosis we hypothesized that tsg-6 is expressed in atherosclerotic plaques and investigated tsg-6 protein expression and cellular distribution on 12 superficial femoral artery endarterectomy specimens from 6 diabetic and 6 non-diabetic patients with peripheral vascular disease. six histologically normal radial artery specimens were analyzed as control. paraffin embedded samples were studied by immunohistochemistry using a goat polyclonal anti-human-tsg-6 antibody. tsg-6 expression was consistently present in all atherectomy specimens but not in control specimens. a distinct, strong cytoplasmic staining pattern was uniformly detected in the endothelial lining of the intima, as well as in the neo-vessel proliferation of the plaque. cytoplasmic staining was also identified in the smooth muscle cell proliferation of the neo-intima. patchy tsg-6 expression was noted in the extracellular matrix. within the inflammatory plaques from diabetic patients, tsg-6 stained the foamy macrophages. tsg-6 expression was also confirmed and quantified by qrt-pcr that showed a significant up-regulation of tsg-6 gene (more that 100 fold induction compared to housekeeping genes). our study identifies for the first time the preferential expression of tsg-6 in atherosclerotic lesions and characterizes its distribution within the cellular and matrix components of the plaque. tsg-6 is a novel inflammatory mediator of atherosclerosis and a potentially new marker of endothelial / smooth muscle cell activation. (1), r krohn (1), h lue (1), jl gregory(2), a zernecke (1), rr koenen (1), t kooistra (3), p ghezzi(4), r kleemann (3), r bucala(5), mj hickey (2), c weber (1) (1) university hospital of the rwth aachen, germany (2) centre for inflammatory diseases, monash university, melbourne, australia mediators, which cannot be classified into chemokine subfamilies but share functional patterns, e.g. signaling through chemokine receptors, constitute a group termed chemokine-like function (clf)-chemokines. the pleiotropic cytokine macrophage migration inhibitory factor (mif) plays a critical role in inflammatory diseases and atherogenesis. the underlying molecular mechanisms are poorly understood, but, interestingly, mif displays structural features resembling chemokines. we have identified the chemokine receptor cxcr2 as a functional receptor for mif. mif triggered galphai/integrin-dependent arrest and chemotaxis of monocytes specifically through cxcr2, inducing rapid integrin activation. mif directly bound to cxcr2 with high affinity (kd of 1.4 nm). monocyte arrest mediated by mif in inflamed or atherosclerotic arteries involved cxcr2 as well as cd74, a recently identified membrane receptor moiety for mif. accordingly, cxcr2 and cd74 were found to occur in a receptor complex. in vivo, mif deficiency impaired monocyte adhesion to the aortic/arterial wall in atherosclerosis-prone mice, as evidenced by intravital microscopy. thus, mif displays chemokine-like functions by acting as a non-cognate ligand of cxcr2, serving as a regulator of inflammatory and atherogenic recruitment. these data harbor an intriguing novel therapeutic prospect by targeting mif in atherosclerosis and add a new dimension to mif and chemokine receptor biology. (1), r toes (3), h van bockel(2), paul quax (1,2) (1) tno bioscienses, leiden, the netherlands (2) department of vascular surgery, leiden university medical center, the netherlands (3) department of rheumatoly, leiden university medical center, the netherlands the immune system is thought to play a crucial role in regulating collateral circulation (arteriogenesis), a vital compensatory mechanism in patients with arterial obstructive disease. here, we studied the role of lymphocytes in a murine model for artiogenenesis after acutehind limb ischemia. arteriogenesis was impaired in c57bl/6 mice depleted for natural killer (nk)-cells by anti-nk1.1 antibodies and in nk-cell-deficient transgenic mice. arteriogenesis was, however, unaffected in jâµ281knockout mice that lack nk1.1+ natural killer t (nkt)cells, indicating that nk-cells, rather than nkt-cells are involved in arteriogenesis. furthermore, arteriogenesis was impaired in c57bl/6 mice depleted for cd4+ tlymphocytes by anti-cd4 antibodies, and in major histocompatibility complex (mhc)-class-ii-deficient mice that lack mature peripheral cd4+ t-lymphocytes. this impairment was even more profound in anti-nk1.1treated mhc-class-ii-deficient mice that lack both nkand cd4+ t-lymphocytes. finally, collateral growth was severely reduced in balb/c as compared with c57bl/6 mice, two strains with different bias in immune responsiveness. correspondingly, fewer cd3-positive lymphocytes accumulated around collaterals in balb/c mice. these data show that both nk-cells and cd4+ t-cells play an important role in arteriogenesis. moreover, our data hold promise for the development of novel clinical interventions as promoting lymphocyte activation might represent a powerful method to treat ischemic disease. post-interventional vascular remodeling in venous bypass grafts, seen as intimal hyperplasia (ih) and accelerated atherosclerosis, often causing graft failure. inflammation is an important trigger for these processe. complement is an important part of the immune system and participates in regulating inflammation. although involved in several other inflammatory diseases, the role of the complement cascade in vein graft remodeling is unknown. the involvement of the complement system in vein graft disease was studied here using a model in which caval veins are grafted in carotids arteries of hypercholesterolemic apoe3leiden mice. in these veins ih and accelerated atherosclerotic lesions develop within 28 days, consisting mainly of foamcells and smc. to study the functional role of complement in vein graft remodeling, cobra venom factor (cvf:20u daily) was used to deplete complement starting one day prior to vein graft surgery. cvf-treatment reduced vein graft thickening by 64% (p=0.02), when compared to saline treated controls (n=6).to confirm that the reduction by cvf was due to hampered complement function and not a direct effect of cvf, complement activation was blocked using crry-ig (inhibiting c3 convertases). crry-ig (3mg every other day) led to 50% decrease in vein graft thickening (p=0.03) compared to controls receiving non-relevant control igg. these data prove that complement activation plays a major in intimal hyperplasia and accelerated atherosclerosis in vein grafts. (1), m-c koutsing tine(1), p borgeat(2), h ong (1), sylvie marleau (1) (1) universite de montreal, quebec, canada (2) centre de recherche en rhumatologie et immunologie, canada we have previously shown that ep 80317, a growth hormone-releasing peptide (ghrp) analogue binding selectively to the scavenger receptor cd36, elicits a striking reduction in atherosclerosis development in apolipoprotein-deficient (apoe-/-), a condition associated with increased circulating numbers of primed/activated leukocytes. we investigated the effect of ghrp analogues on i/r-elicited remote lung injury in 18week-old apoe-/-mice fed a high fat high cholesterol (hfhc) diet from 4 weeks of age. at 18 weeks old, mice were treated daily with a s.c. injection of ep 80317 (300 mg/kg) for 14 days and were then subjected to unilateral hindlimb ischemia (by rubber band application) for 30 minutes, followed by 180 minutes reperfusion. our results show that ep 80317 significantly reduced leukocyte accumulation by 56% in the lungs, from 3.6 (ae 0.7) in vehicle-treated mice to 1.6 (ae 0.6) x 107 leukocytes/g lung in ep 80317-treated mice (n = 6-7 per group), as assessed by myeloperoxidase assay. this was associated with a 56% reduction of opsonized zymosan-elicited blood chemiluminescence. in contrast, neither blood chemiluminescence, nor leukocyte accumulation in the lungs were signicantly modulated in apoe-/-/cd36-/-deficient mice, from 1.9 (ae 0.5) in vehicle-treated mice to 1.9 (ae 0.8) x 107 leukocytes/g lung in ep 80317-treated mice. we conclude that ep 80317 protects i/r-elicited circulating leukocyte priming/activation and remote lung injury, possibly through a cd36-mediated pathway. glycogen synthase kinase 3beta (gsk-3beta) is a serine/ threonine protein kinase that has recently emerged as a key regulatory switch in the modulation of the inflammatory response. dysregulation of gsk-3beta has been implicated in the pathogenesis of several diseases including sepsis. here we investigate the effects of two chemically distinct inhibitors of gsk-3beta, tdzd-8 and sb216763, on the circulatory failure and the organ injury and dysfunction associated with hemorrhagic shock. male wistar rats were subjected to hemorrhage (sufficient to lower mean arterial blood pressure to 35 mmhg for 90 min) and subsequently resuscitated with shed blood for 4 h. hemorrhage and resuscitation resulted in an increase in serum levels of (a) creatinine and, hence, renal dysfunction, and (b) alanine aminotransferase and aspartate aminotransferase and, hence, hepatic injury. treatment of rats with either tdzd-8 (1 mg/kg, i.v.) or sb216763 (0.6 mg/kg, i.v.) 5 min before resuscitation abolished the renal dysfunction and liver injury caused by hemorrhagic shock. the protection afforded by these compounds was confirmed by histological observations of lung, kidney and liver samples. in addition, tdzd-8, but not sb216763, attenuated the increase caused by hemorrhage and resuscitation in plasma levels of the proinflammatory cytokine interleukin 6. neither of the gsk-3beta inhibitors however affected the delayed fall in blood pressure caused by hemorrhagic shock. thus, we propose that inhibition of gsk-3beta may represent a novel therapeutic approach in the therapy of hemorrhagic shock. (1), y ito (1), h yoshimura (1), h inoue (1), n kurouzu (1), h hara (1), y mastui (1), h kitasato (1), s narumiya(2), c yokoyama (3), m majima (1) (1) kitasato university school of medicine, japan (2) kyoto university school of medicine, japan (3) tokyo medical and dental university, japan thromboxane (tx) a2 is a potent stimulator of platelet activation and aggregation and vascular constriction. we have reported cytokine-mediated release of sdf-1 from platelets and the recruitment of nonendothelial cxcr4+ vegfr1+ hematopoietic progenitors constitute the major determinant of revascularization. we hypothesized txa2 induces angiogenic response by stimulating sdf-1 and vegf which derived from platelet aggregation. to evaluate this hypothesis, we dissected the role of the txa2 in angiogenesis response using mouse hind limb ischemia. recovery from acute hind limb ischemia, as assessed in wild type mice (c57bl/6 wt) , prostaglandin i2 receptor (ip) knock out mice (ipko) and thromboxane (tx) a2 receptor (tp) knock out mice (tpko) by using lase doppler. blood recovery in tpko significantly delayed compared to wt and ipko. immunohistochemical studies revealed that the number of cd31positive cells in the ischemic quadriceps were less stained in tpko compared to wt and ipko.plasma sdf-1 and vegf concentration were significantly reduced in tpko mice. we observed during in vivo fluorescence microscopic study that compared to tpko, ipko and wt significantly increased platelet attachment to the microvessels around ligated area. tpko translpanted wt bone marrow cells increased blood recovery compared to tpko transplanted tpko bone marrow cells. in addition, mice injected with txa2 synthase c-dna expressing fibroblast increased blood flow recovery compared to control mice. these results suggested that tp signaling rescues ischemic condition by inducing angiogenesis by secreting sdf-1 and vegf from platelet aggregation. administration of selective tp agonist may open new therapeutic strategy in regenerative cardiovascular medicine. during renal ischemia/reperfusion (i/r) injury, apoptosis has been reported as a very important contributor to the final kidney damage. the determinant role of cytoskeleton derangement in the development of apoptosis has been previously reported, but a clear description of the different mechanisms involved in this process has not been yet provided. the aim of the study is to know the role of peroxynitrite as inductor of cytoskeleton derangement and apoptosis during the inflammatory process associated to renal ischemia-reperfusion. based in a rat kidney i/r model, by experiments in which both the actin cytoskeleton and peroxynitrite generation were pharmacologically manipulated, results indicate that the peroxynitrite produced during the i/r derived oxidative stress state, is able to provoke cytoskeleton derangement and apoptosis development. thus, the control in the peroxynitrite generation during the i/r could be an effective tool for the improvement of cytoskeleton damage and reduction apoptosis incidence in the renal i/r injury. metabolomics, the global profiling of metabolites, may inform about the multiple interacting processes involved in inflammatory disease. using nmr spectroscopy we analysed metabolite fingerprints in serum from early arthritis, and at a site of inflammation, in the posterior segment of the eye. serum from 101 patients with synovitis of "t 3 months duration whose outcome was determined at clinical follow-up was used. vitreous samples were from 31 patients undergoing vitrectomy for vitreoretinal disorders. one dimensional 1h nmr spectra were acquired. principal components analysis (pca) of the processed data was conducted along with a supervised classification. with the arthritis serum there was a clear relationship between each samples score in the pca analysis and the level of crp. supervised classification of the initial samples was able to predict outcome, whether rheumatoid arthritis, other chronic arthritis or self-limiting arthritis, with high specificity and sensitivity. a similar approach using the eye fluids was able to give a clear discrimination between two pathologically similar conditions lens-induced and chronic uveitis. in this case differences were not due to a straightforward relationship with inflammatory markers (il-6, ccl2), which did not correlate with pca in these samples. similarly, certain molecules, such as lactate, were associated with ocular disease, but not rheumatoid arthritis. these results suggest that underlying inflammatory processes may differ in these conditions or may reflect predisposing metabolic patterns in individual patients. 1h-nmr-based metabolomics may provide a useful measure of outcome in inflammatory diseases and give novel insights into the pathological processes involved. (1), am artoli(2), a sequeira(2), c saldanha (1) (1) instituto de medicina molecular,faculdade de medicina de lisboa, portugal (2) cemat, instituto superior tã�cnico, universidade de tã�cnica de lisboa, portugal the recruitment of leukocytes from the blood stream and their subsequent adhesion to endothelial walls are essential stages to the immune response system during inflammation. the precise dynamic mechanisms by which molecular mediators facilitate leukocyte arrest are still unknown. in this study combined experimental results and computer simulations are used to investigate localized hydrodynamics of individual and collective behaviour of clusters of leukocytes. leukocyte-endothelial cell interactions in post-capillary venules of wistar rats cremaster muscle were monitorized by intravital microscopy. from these experiments the haemorheologic and haemodynamical measured parameters were used in time dependent three-dimensional computer simulations, using a mesoscopic lattice boltzmann solver for shear thinning fluids. the dynamics of leukocyte clusters under non-newtonian blood flow with shear thinning viscosity was computed and discussed. in this paper we present quantified distributions of velocity and shear stress on the surface of leukocytes and near vessel wall attachment points. we have also observed one region of maximum shear stress and two regions of minimum shear stress on the surface of leukocytes close to the endothelial wall. we verified that the collective hydrodynamic behaviour of the cluster of recruited leukocytes establishes a strong motive for additional leukocyte recruitment. it was found that the lattice boltzmann solver used here is fully adaptive to the measured experimental parameters. this study suggests that the influence of the leukocytes rolling on the increase of the endothelial wall shear stress may support the activation of more signalling mediators during inflammation. macrophages are essential for host defence, but when excessively and persistently activated, these cells contribute to the initiation and progression of inflammatory diseases such as rheumatoid arthritis. investigating the function of inflammatory genes in macrophages may identify novel therapeutic targets for inflammatory diseases. one family of transcripts that are highly expressed in activated macrophages are members of the schlafen (slfn) gene family; a recently identified family whose function is still unknown. this study examined the mrna expression of slfn in activated bone marrowderived macrophages in vitro, and in collagen-induced arthritis (cia) in vivo. real-time pcr expression analyses of bone marrow-derived macrophages stimulated with lipopolysaccharide (lps) over a time course, revealed differential expression of individual slfn family genes. in particular, slfn-1, slfn-2, and slfn-4 were maximally induced after 24 hours. the maximal induction of slfn-8 and slfn-9 was observed after 4 hours of lps treatment. individual members of the slfn family were also differentially expressed in cia, a model of rheumatoid arthritis. mrna levels of slfn-1, slfn-2, slfn-4 and slfn-5 were elevated in joints affected by cia. to investigate the role of slfn-4, we have generated a transgenic mouse line, which over expresses slfn-4 specifically in cells of the mononuclear phagocyte system, by using a novel binary expression based on the c-fms promoter and gal4. further characterisation of the slfn-4 over expressing mouse line will be used to assess the function of slfn-4 in macrophage biology and inflammation, and its potential as a therapeutic target. macrophages play an important role in resolving inflammation. it is known that the resolution of inflammation requires alternative activation of macrophages. but the precise events of phenotype switching in macrophages remain poorly understood. we show that lipocalin 2, lcn-2, is able to provoke a switch in macrophage activation. in an in vitro co-culture model for renal epithelial cells and macrophages, we detected by sirna technique that the presence or absence of lcn-2 determines proliferation processes in damaged renal epithelial cells. the proliferative response was dependent on proinflammatory or anti-inflammatory environment. as lcn-2 is an acute phase protein synthesized during inflammation and unregulated in a number of pathological conditions, it may play an important role in survival and regeneration. we anticipate here that our results could be relevant for further research on the mechanisms of the phenotype switch induced by lcn-2. (1), y cao (1), s adhikari (1), m wallig (2) (1) national university of singapore, department of pharmacology, singapore (2) university of illinois at urbana champaign, usa it has earlier been shown that the extent of apoptotic acinar cell death is inversely related to the severity of acute pancreatitis. our previous works have demonstrated that induction of pancreatic acinar cell apoptosis by crambene protects mice against acute pancreatitis. the current study aims to investigate the role of phagocytic receptors and the anti-inflammatory effect of phagocytosis in protecting mice against acute pancreatitis by crambene. acute pancreatitis was induced in the mouse by administering hourly injections of caerulein (50 mg/kg) for 3, 6 and 10 hours respectively. neutralizing monoclonal anti-il-10 antibody (2.5 mg/kg) was administered either with or without crambene (70 mg/kg) 12 hours before the first caerulein injection. rt-pcr, western blotting and immunostaining were performed to detect cd36 expression. apoptosis in pancreatic sections was visualized by tunel. severity of acute pancreatitis was evaluated by estimation of serum amylase, pancreatic myeloperoxidase (mpo), water content, and morphological examination. pancreatic levels of inflammatory mediators were examined by elisa. the protective effect of crambene is mediated by reducing production of pro-inflammatory cytokines such as mcp-1, tnf-a and il-1㢠and up-regulating anti-inflammatory mediators like il-10. phagocytotic clearance in mouse acute pancreatitis may be essentially through macrophage surface receptor cd36.the anti-inflammatory mediator il-10 plays an important role in crambene-induced protective action against acute pancreatitis. the release of anti-inflammatory mediator il-10 is downstream of phagocytosis. these results show that induction of pancreatic acinar cell apoptosis by crambene treatment protects mice against acute pancreatitis via induction of anti-inflammatory pathways. (1,2) (1) northern ontario school of medicine, thunder bay, ontario, canada (2) lakehead university, canada integrin receptors and their ligands are involved in adhesion and internalization of several human pathogens, including pseudomonas aeruginosa. we have recently established that beta1 integrins in lung epithelial cells (lec) provide co-stimulatory signals regulating inflammatory responses (ulanova et al, am j physiol, 2005, 288: l497-l507). we hypothesized that lec integrins serve as receptors to recognize pathogen-associated molecules and mediate the innate immune response to p. aeruginosa. to determine molecular mechanisms of integrin involvement in innate immunity, we used an in vitro model of p. aeruginosa infection of a549 cells. to investigate interactions of bacteria with lec, p. aeruginosa strain pak was chromosomally labeled with a green fluorescent protein gene using a mini-tn7 delivery system.using several fluorescence-based detection systems, we established that the natural beta1 integrin ligand, fibronectin, mediates bacterial adhesion to lec.p. aeruginosa infection caused rapid transcriptional upregulation of alphav and beta4 integrin expression followed by the increased cell surface protein expression. the surface expression of integrin beta1 increased shortly following bacterial exposure without alterations of mrna expression, suggesting rapid protein redistribution within the cells. the data indicate that p. aeruginosa are capable to modulate integrin gene/protein expression in lec, potentially using fibronectin to alleviate bacterial binding to beta1 integrins. upon their engagement, integrin receptors can initiate intracellular signaling involved in innate immune and inflammatory responses to the pathogen. integrin receptors in lec may represent significant therapeutic targets in pulmonary infection caused by p. aeruginosa. the purine nucleoside adenosine has a major modulatory impact on the inflammatory and immune systems. neutrophils, which are generally the first cells to migrate toward lesions and initiate host defense functions, are particularly responsive to the action of adenosine. through activation of the a2a receptor (a2ar) present on neutrophils, adenosine inhibits phagocytosis, generation of cytotoxic oxygen species, and adhesion. also, recent work showed that adenosine can transform the profile of lipid mediators generated by neutrophils, inhibiting leukotriene b4 formation while potentiating that of prostaglandin e2 through the up-regulation of the cyclooxygenase (cox)-2 pathway. moreover, our laboratory determined that a2ar engagement can dramatically modulate the generation and secretion of neutrophil-derived cytokines/chemokines, including tnf-and mips. in mice lacking the a2ar, migrated neutrophils expressed less cox-2 than their wild type counterpart while displaying higher mrna levels of tnf-and mip-1. mononuclear cells from a2ar knock out mice, which eventually replace neutrophils into the air pouch, also displayed a more pro-inflammatory phenotype than those from wild-type animals. signal transduction experiments, aiming to delineate the intracellular events leading to the modulation of neutrophil functions following a2ar engagement, implicate pivotal metabolic pathways such as intracellular cyclic amp, p38 and pi-3k. together, these results indicate that adenosine may have a profound and multi-pronged influence on the phenotype of neutrophils and present this cell as being pivotal in mediating adenosines anti-inflammatory effects. the newest developments regarding adenosines effects on neutrophil functions will be presented.this work is supported by the canadian institutes of health research (cihr). human skin serves not only as a physical barrier against infection, but also as a "chemical barrier" by constitutively and inducibly producing antimicrobial proteins (amps). to identify human skin amps, we analysed extracts of healthy persons stratum corneum by reversed phase-hplc and purified a novel 9 kda amp that showed sequence similarity to mouse hornerin. suggesting that it originates from the human ortholog, we cloned it. human hornerin encodes a 2850 amino acid protein that contains a s100 domain, an ef-hand calciumbinding domain, a spacer sequence and two types of tandem repeats, suggesting that it represents a novel member of the fused s100 protein family. strongest constitutive hornerin mrna expression was seen in differentiated keratinocyte cultures. to follow the hypothesis, that hornerin fragments represent amps, we recombinantly expressed three hornerin peptides, rhrnr2 (tandem repeat unit b), rhrnr3 (tandem repeat unit a) and rhrnr4 (c-terminus) and subsequently analysed their antimicrobial activity using the microdilution assay system. the rhrnr3 peptide, containing the sequence motif found in the purified natural hornerin fragment isolated from stratum corneum, exhibited antimicrobial activity at low micromolar concentrations against escherichia coli, pseudomonas aeruginosa and candida albicans. the other peptides were found to be not or nearly not antimicrobially active. our results suggest that hornerin may have a yet unknown protective function in healthy human skin as part of the "chemical barrier" with preformed amps, which are generated from parts of the tandem repeats of a hornerin precursor molecule by a yet unknown cleavage mechanism. (1), n lu(1), r jonsson(2), d gullberg (1) (1) department of biomedicine, university of bergen, norway (2) the gade institute, university of bergen, norway a11ã�1 is the latest addition to the integrin family of heterodimeric receptors for the extracellular matrix. previously, it has been shown that this collagen receptor takes part in processes such as cell migration and matrix contraction. in this study we investigated the factors that regulate mouse integrin a11ã�1 expression. specifically, we have analyzed the influence of cell passage, growth factors and the 3-d microenvironment. using sv40 immortalized as well as primary fibroblasts, we show that a11ã�1 integrin is up-regulated when these cells are cultured within stressed collagen type i lattices. however, a11ã�1is downregulated when the collagen gels are made under relaxed conditions, allowing cells contract the lattice diameter. we also show here that a11 is upregulated by tgf-a on planar substrates. these findings suggest that mechanical tension and tgf-a are important factors in the regulation of a11ã�1 that need to be to taken into consideration when evaluating the role of a11ã�1 in wound healing and fibrotic disorders. (1), n vergnolle (1), p andrade-gordon (2) (1) inflammation research network, university of calgary, canada (2) rw johnson pharmaceutical research institute, canada the objective of this study was to investigate the effects of par2 deficiency in various models of colonic inflammation in order to elucidate the role of endogenous par2 in the process of inflammation in the gut.colonic inflammation in c57bl6 wildtype and par2 -/-mice was induced by treatment with 2.5% dss (in drinking water) or tnbs (1mg or 2mg in 100ul of 40% ethanol, single intracolonic injection) or pre-sensitizing mice with 3% oxazolone (in olive oil) applied to the skin of the abdomen, and 7 days later, a single intracolonic injection of 1% oxazolone (dissolved in 50% ethanol).intravital microscopy was performed, 7 days (tnbs/dss) or 4 days (oxazolone) after induction of colitis on the colonic venules to assess changes in leukocyte rolling, adhesion and vessel diameter.lastly, various parameters of inflammation were assessed following the intravital microscopy.par2 -/-mice showed significantly lower leukocyte adherence and vessel dilation compared to the wildtype mice in dss, and tnbs challenge. in all three challenges, mpo activity, macroscopic damage score and bowel thickness were significantly higher in wild-type mice, compared to par2 -/-.our evidences indicate that deficiency in par2 attenuates inflammatory responses in the experimental models of colitis associated with either th1 (tnbs/dss) or th2 (oxazolone) cytokine profile.therefore, par2 deficiency in the gut exerts antiinflammatory properties that are independent of th1 or th2 cytokine profile.the present study further highlights par2 as a potential target for inflammatory bowel diseases. (1), n vergnolle (1), p andrade-gordon (2) (1) inflammation research network, university of calgary, canada (2) rw johnson pharmaceutical research institute, canada in a previous study, inflammatory responses induced by three different models of colitis (tnbs/dss/oxazolone) were significantly attenuated in mice deficient for par2 (par2-/-). among the inflammatory parameters observed, infiltration of granulocytes to the colon was consistently reduced by par2 deficiency. aim of this study was to assess the effects of par2 deficiency (via par2-/-mice) on the recruitment of leukocyte in colonic venules. in anaesthetized animals, leukocyte rolling/ adherence and vasodilation were induced, by topical administration of fmlp (100 mm) or paf (100 nm) or by intraperitoneal injection of tnf-a; (0.5 mg -given 3 hours before the intravital microscopy). using intravital microscopy, we evaluated the ability of various leukocyte stimuli to induce leukocyte trafficking and vasodilation in colonic venules of par2 -/-versus par2+/+ mice. fmlp and paf as well as tnf-a; induced significant vasodilation and an increase in rolling/adhesion of leukocytes in mouse colonic venules. par2 -/-mice showed significantly lower leukocyte rolling compared to the wildtype mice in response to fmlp topical administration. leukocyte adherence induced by fmlp and tnf-a; was significantly lower in par2 -/-mice compared to wild types as well. no difference was observed between par2 -/-and wildtype for leukocyte rolling/adherence-induced by paf. the lack of functional par2 attenuated leukocyte trafficking in response to fmlp and tnf-a; but not to paf. the involvement of par2 activation in mouse colon leukocyte trafficking highlights par2 as an important mediator of inflammatory cell recruitment and thereby a potential target for the treatment of inflammatory bowel diseases. (1), kk hansen(2), k chapman(2), n vergnolle (2), ep diamandis (1), md hollenberg (2) (1) advanced center for detection of cancer, mount sinai hospital, university of toronto, toronto, on, canada (2) proteinases and inflammation network, university of calgary, calgary, ab, canada kallikreins (klks) are secreted serine proteinases identified in many cancers and multiple sclerosis lesions. we have recently shown that klks can activate proteinaseactivated receptors (pars), a family of g-protein coupled receptors associated with inflammation. we hypothesized that like trypsin, kallikreins can trigger inflammation via the pars. we studied the ability of klks 6 and 14 to activate pars 1, 2 and 4 in vitro and to cause oedema in a mouse model of paw inflammation in vivo. we found that klk14 is able to activate both of pars 2 and 4 and to prevent thrombin from activating par1. on the other hand, klk6 was a specific activator of par2. kallikrein administration in vivo resulted in a paw oedema response comparable in magnitude and time to that generated by trypsin. the oedema was accompanied by a decreased threshold of mechanical and thermal nociception. our data demonstrate that by activating pars 2 and 4 and by inactivating par1, kallikreins, like klks 6 and 14, may play a role in regulating the inflammatory response and perception of pain. (1), d park (1), b short(2), n brouard(2), p simmons(2), s graves (1), j hamilton (1) (1) melbourne university, melbourne, victoria, (2) peter maccallum cancer institute. melbourne, australia mouse mesenchymal stem cell enriched populations can be isolated from bone tissue by employing lineage immuno-depletion followed by fluorescence-activated cell sorting based on the cell surface expression of the sca-1 antigen. such isolated cells can subsequently be cultured and differentiate towards the osteogenic, adipogenic or chondrogenic linage in vitro. using this model we investigated the influence of the proinflammatory cyto-kines, tnfa or il-1b, on early osteogenesis in vitro. under osteogenic conditions, il-1b was found to inhibit cell proliferation in a dose dependent manner, whereas tnfa exhibited no effect. histochemical examination revealed the presence of either tnfa or il-1b to dramatically decreased mineralization in a dose dependent manner. q-pcr analysis indicated that in the presence of il-1b, despite increased expression of bone-specific alkaline phosphatase (akp2) mrna, levels of other osteogenesis markers (runx2, col1a and sp7) were decreased. in the presence of tnfa, levels of akp2, runx2 and sp7 were all decreased. our findings indicate that the influence of early mesenchymal progenitor cells on bone remodelling may be substantially altered in the presence of proinflammatory cytokines. using are-driven and nf-kb-targeted reporter genes, transfection of the nf-kb p65 subunit and nrf2 into hepg2 or other cells, as well as sirna technique to knockdown endogenous p65 in cells, we found that nf-kb p65 subunit repressed the anti-inflammatory and anticarcinogenetic nrf2-are pathway at transcriptional level. in p65-overexpressed cells, the are-dependent expression of heme oxygenase-1 was strongly repressed. in the cells where nf-kb and nrf2 were simultaneously activated, p65 unidirectionally antagonized nrf2 transcriptional activity. the p65-mediated are inhibition was independent of the transcriptional and dna-binding activities of p65. co-transfection and rna interference experiments revealed two mechanisms which coordinate the p65-mediated repression of are: (1) p65 selectively deprives creb binding protein (cbp) from nrf2, but not mafk, by competitive interaction with the ch1-kix domain of cbp, resulting in inactivation of nrf2 transactivation domain and concomitant abrogation of the nrf2-stimulated coactivator activity of cbp; (2) p65 promotes recruitment of histone deacetylases 3 (hdac3) to are by enhancing the interaction of hdac3 with either cbp or mafk, leading to inactivation of cbp and deacetylation of mafk. this study may establish a novel pro-inflammatory and pro-carcinogenic model for the transrepression of the are-dependent gene expression by p65 subunit. since various inflammatory and tumor tissues constitutively overexpresses p65 in their nuclei, the finding in this study implies a strong repression of are-dependent gene expression must take place in those tissues. in this regard, the findings in this study may help to explain why oxidative stresses and toxic insults usually occur in those pathological loci. dendritic cells (dc) play a pivotal role in the induction of immune response and tolerance. it is less known that dc accumulate in atherosclerotic arteries, where they might activate t-cells and contribute to the progression of disease. the serine protease thrombin is the main effector protease of the coagulation cascade. thrombin is also generated at sites of vascular injury and during inflammation. hence, thrombin generation is observed within atherosclerotic and other inflammatory lesions including rheumatoid arthitis. thrombin activates various cells via protease-activated receptors (pars). immature dc do not express pars. upon maturation with lps, tnfalpha, or cd40l, only lps-matured dc expressed par1 and par3 on their surface. stimulation of dc with thrombin, par1-or par3-activating peptides elicited actin polymerization and concentration-dependent chemotactic responses in lps-, but not in tnf-alphamatured dc. the thrombin-induced migration was a true chemotaxis as assessed by checkerboard analysis. stimulation of pars with thrombin or respective receptoractivating peptides led to activation of erk1/2 and rho kinase i (rock-i) as well as subsequent phosphorylation of the regulatory myosin light chain 2 (mlc2). the erk1/2-and rock-i-mediated phosphorylation of mlc2 was indispensable for the par-mediated chemotaxis as shown by use of pharmacological inhibitors of rock, erk and mlc kinases. in addition, thrombin significantly increased the ability of mature dc to activate proliferation of naive t-lymphocytes in mixed leukocyte reactions. in conclusion, our work demonstrates expression of functionally active thrombin receptors on lps-matured dc. we identified thrombin as a potent chemoattractant for mature dc, acting via rho/ erk-signaling pathways. data concerning the role of circulating modified low density lipoproteins (modldl) in atherogenesis and other pathologies are scarce. one reason for this is the lack of suitable radiolabeling methods for direct assessment of metabolic pathways of modldl in vivo. we report a novel approach for specific labeling of human native ldl (nldl) and modldl (iron-, hypochloriteand myeloperoxidase-oxidized, nitrated, glycated, and homocysteinylated ldl) with the positron emitter fluorine-18 (18f) by either nh2-reactive n-succinimidyl-4-[18f]fluorobenzoate or sh-reactive n-[6-(4-[18f]fluorobenzylidene)-aminooxyhexyl]maleimide (radiochemical yields, 15-40%; specific radioactivity, 150-400 gbq/ mmol). radiolabeling itself caused neither additional oxidative structural modifications of ldl lipids and proteins nor adverse alterations of their biological activity and functionality in vitro. the approach was evaluated with respect to binding and uptake of 18f-nldl and 18f-modldl in cells overexpressing various lipoproteinrecognizing receptors. the metabolic fate of 18f-nldl and 18f-modldl in vivo was delineated by dynamic small animal pet studies in rats and mice. the in vivo distribution and kinetics of nldl and modldl correlated well with the anatomical localization and functional expression of ldl receptors, scavenger receptors, and receptors for advanced glycation end products. the study shows that ldl modification, depending on type and extent of modification, in part or fully blocks binding to the ldl receptor, and reroutes the modldl to tissuespecific disease associated pathways. in this line, 18flabeling of modldl and the use of small animal pet provide a valuable tool for imaging and functional characterization of these pathways and specific sites of pathologic processes, including inflammatory processes, in animal models in vivo. the p38 mapk signaling pathway, which regulates the activity of different transcriptions factors including nuclear factor-ã�b (nf-ã�b), is activated in lesional psoriatic skin. the purpose of the present study was to investigate the effect of fumaric acid esters on the p38 mapk and the down stream kinases msk1 and 2 in cultured human keratinocytes. cell cultures were incubated with dimethylfumarate (dmf), methylhydrogenfumarate (mhf) or fumaric acid (fa) and then stimulated with il-1b before kinase activation was determined by western blotting. a significant inhibition of both msk1 and 2 activations was seen after pre-incubation with dmf and stimulation with il-1b whereas mhf and fa had no effect. also, dmf decreased phosphorylation of nf-kb / p65 (ser276), which is known to be transactivated by msk1. furthermore, incubation with dmf before stimulation with il-1b resulted in a significant decrease in nf-kb binding to the il-8 kb and the il-20 kb binding sites as well as a subsequent decrease in il-8 and il-20 mrna expression. our results suggest that dmf specifically inhibits msk1 and 2 activations and subsequently inhibits nf-kb induced gene-transcriptions which are believed to be important in the pathogenesis of psoriasis. these effects of dmf explain the anti-psoriatic effect of fumaric acid esters. a humanized model of psoriasis was successfully established by transplanting non-lesional skin biopsies from psoriasis patients onto bg-nu-xid mice lacking b, t and nk cells. in this system, a psoriatic process is triggered by intradermal injection of activated autologous peripheral blood lymphocytes. inflammation is associated with the expression of activation markers and inflammatory medi-ators such as tnf-alpha, hla-dr and cd1a and this results in increased proliferation and differentiation of keratinocytes, demonstrated by increased expression of ki-67 and ck-16. epidermal hyperplasia is a typical readout in this model. in a series of studies, this model was found to be sensitive too a wide range of compounds, including inhibitors of tnf-alpha, antibodies directed against growth factors, mmp-inhibitors, calcipotriol, metothrexate, betamethasone and cyclosporine a.in addition, we showed that inhibition of fatty acid oxidation had an anti-psoriatic effect in this model (caspary et al. brit j dermatol 2005; 153, 937-944) . employing lesional skin it was demonstrated that inhibition can also be performed in a therapeutic setting.due to its humanized nature this model represents a powerful tool for the identification or validation of compounds with potential for the treatment of psoriasis. kristian otkjaer (1), e hasselager(2), j clausen(2), l iversen (1), k kragballe (1) (1) aarhus university hospital, denmark (2) novo nordisk a/s, denmark interleukin-20 (il-20) is assumed to be a key cytokine in the pathogenesis of psoriasis. increased levels of il-20 are present in lesional psoriatic skin compared with nonlesional skin where it is barely detectable. whether il-20 is derived from antigen-presenting cells or keratinocytes remains unsolved. the aim of the present study was, therefore, to characterize il-20 expression in non-lesional psoriatic skin ex vivo. 3 mm punch biopsies from nonlesional psoriatic skin were collected. biopsies were transferred to cacl enriched keratinocyte basal media and cultured with vehicle or il-1beta (10ng/ml) for 0, 1, 2, 4, 6, 12 and 24 hours, respectively. the samples were analyzed by in situ hybridisation, qrt-pcr, immunofluorescent staining and elisa. incubation with il-1beta rapidly induced il-20 mrna expression in the biopsies. the highest level of il-20 mrna was detected after 4 hours and in situ hybridisation revealed that basal as well as suprabasal keratinocytes throughout the epidermis were the only cellular source of il-20 mrna. increased levels of il-20 protein were detected in the supernatant of the il-1beta stimulated biopsies. immunofluorescent staining of the biopsies showed no il-20 protein in the keratinocytes, whereas the il-20 protein was present in epidermal cd1a positive dendritic cells. our data emphasize the keratinocyte as the cellular source of il-20 expression in human skin. interestingly, immunofluorescent staining of our cultured biopsies showed il-20 protein in epidermal dendritic cells whereas no il-20 was detected in the keratinocytes. this indicates that epidermal dendritic cells are the target for keratinocytederived il-20. one response of epidermal keratinocytes to inflammatory stress is the induction of matrix metalloproteinases (mmps) that participate in tissue remodeling. excessive proteolytic activity is associated with chronic wounds and tissue damage during persistent inflammation. calcitriol, the hormonally active form of vitamin d, is known to have beneficial effects during cutaneous inflammation. we hypothesized that one way in which calcitriol exerts its effect on inflamed skin is by attenuation of damages caused by excessive mmp proteolytic activity. our experimental model consists of hacat keratinocytes cultured with tnf to simulate an inflammatory state. pro-mmp-9 was quantified by gelatin zymography and mrna by real-time pcr. the levels and activation of signaling proteins were determined by immunoblotting. the increase in pro-mmp-9 activity and mrna levels induced by tnf was inhibited by~50% following 48h treatment with calcitriol. using specific inhibitors we established that the induction of mmp-9 was dependent upon the erk pathway, while p38-mapk and pkc inhibited, and jun-kinase, pi-3-kinase and src did not affect it. levels of c-fos, a component of ap-1 transcription complex known to mediate mmp-9 induction, were elevated by tnf and further increased by calcitriol. the induction of mmp-9 by tnf was abolished by inhibition of the egfr tyrosine kinase attesting to the requirement for egfr trans-activation. calcitriol also inhibited the induction of mmp-9 by egf. we conclude that calcitriol inhibits the induction of mmp-9 gene expression by tnf in keratinocytes by affecting an event downstream to the convergence of the egfr and the tnf signaling pathways. (1), p verzaal (1), t lagerweij (1), c persoon-deen (1), l havekes (1), a oranje (2) (1) tno pharma, department of inflammatory and degenerative diseases, leiden, the netherlands (2) erasmus medical center, department dermatology and venereology, rotterdam, the netherlands mice with transgenic overexpression of human apolipoprotein c1 in liver and skin display a strongly disturbed lipid metabolism. moreover, these mice show a loss of skin-barrier function evident from increased trans epidermal water loss. these mice develop symptoms of atopic dermatitis, i.e. scaling, lichenification, papules, excoriation and pruritus. both hyperplasia of epidermis and dermis are observed. histological analysis shows increased numbers of cd4+ t cells, eosinophils, mast cells and ige-positive cells in the dermis. serum levels of ige are increased as well. cytokine profiling of draining lymph nodes is in favor of a th2-mediated disease. development of atopic dermatis in this model was found to be sensitive to topical treatment with triamcinoloneacetonide, fluticasone-proprionate and tacrolimus. moreover, oral treatment with dexamethasone successfully inhibits the development of disease in this model. impairment of the skin barrier is most likely the underlying cause of the development of atopic dermatitis in this model.this model is useful for identifying new therapeutic strategies and obtaining new insight into the pathogenesis of atopic dermatitis. topical immunosupppressants such as elidel and protopic are highly efficacious therapeutics for the treatment of atopic dermatitis and other dermatological conditions.-when delivered topically, these calcinuerin inhibitors offer several advantages over topical steroids; however, these marketed drugs have received a controversial "black box warning" because of a potential cancer risk. we speculated that systemic exposure of these drugs over long term use may contribute to the cancer risk.accordingly, we have designed and discovered a series of "soft" cyclosporin a (csa) derivatives as potentially safer alternatives.in general, soft drugs are engineered, via medicinal chemistry, to be effective upon local delivery but upon systemic exposure they are rapidly inactivated by metabolic pathways.in this way, exposure of active drug to distal organs is greatly minimized resulting in a significant enhancement in therapeutic index.the results or our drug discovery efforts around soft csa derivatives will be presented. (1), y sawanobori (2), u bang-olsen (3), c vestergaard(4), c grã¸nhã¸j-larsen (1) background: a strain of japanese fancy-mice, nc/nga, serves as a model for atopic dermatitis. under specific pathogen-free conditions, the mice remain healthy, but when kept under non-sterile conditions, they exhibit pruritic lesions like atopic dermatitis. scratching behaviour of the mice precedes the development of dermatitis, and a correlation between registered scratching counts and expression of il-31 mrna has been shown. also, transgenic mice over-expressing il-31 exhibit increased scratching behaviour and develop severe dermatitis. consequently we decided to explore the therapeutic effect of an anti il-31 antibody on scratching behaviour and dermatitis in nc/nga mice. methods: prior to clinical manifestation of dermatitis, we commenced treatment of nc/nga mice with il-31 ratanti-mouse 10 mg/kg intraperitoneally every fifth day for seven weeks. clinical dermatitis, scratching behaviour and weight gain, was assessed throughout the intervention period. serum analysis for ige and il-13 as well as histopathological and immunohistochemistry analysis on skin biopsies were also performed at end-point. results: taken over the entire intervention period, treatment with anti il-31 antibody in nc/nga mice from age seven weeks did not meet the primary end points, which were scratch, dermatitis and body weight. however, post hoc analysis revealed a significant reduc-tion of scratch by the anti il-31 antibody treatment in the time interval day 22-43. our results suggest an anti pruritic role for il-31 antibody in an atopic dermatitis-like animal model. anti il-31 antibody is therefore a new therapeutic opportunity for the treatment of pruritus in atopic dermatitis and perhaps other pruritic diseases. (1), p ferro (1), hm asnagli (1), v ardissone (1), t ruckle (2), f altruda (3), ch ladel (1) (1) rbm merck serono/university of torino, italy (2) merck serono pharmaceutical research institute, geneva, switzerland (3) university of torino, dipartimento di genetica, biologia, biochimica, italy class-i phosphoinositide 3-kinases (pi3ks) play a critical role in modulating innate and adaptive immune responses, as they are important transducers of external stimuli to cells, such as granulocytes and lymphocytes. since pi3k-g plays a pivotal role in mediating leukocyte chemotaxis and activation, as well as mast cell degranulation, the pharmacological blockade of pi3k-g might offer an innovative rationale-based therapeutic strategy for inflammatory skin disorders. in our study the inhibitory properties of a selective pi3k-g inhibitor as605858 on inflammation was applied to murine models modeling skin diseases like psoriasis and dermatitis. two mouse models were used: the first, irritant contact dermatitis (icd), is an innate inflammatory skin condition arising from the release of pro-inflammatory cytokines in response to haptens, usually chemicals. the second, contact hypersensitivity (chs) is a t-cell dependent model, modeling in part t-cell-mediated skin diseases such as psoriasis. we demonstrated the therapeutic effect of pi3kg inhibition and subsequent inhibition of chemotaxis in models of skin diseases and showed that a selective pi3k-g inhibitor can excert an important therapeutic efficacy (dose-dependent) in models of innate immunity (icd) -effective dose 0,3mg/kg p.o. once -as well as in t-cell mediated skin pathology (chs)effective dose 1mg/kg p.o. bid. we conclude that the mechanism of action related to inhibition of pi3k-g are demonstrable after oral administration of selective inhibitors like as605858 in models of acute and chronic skin inflammation and are mediated by modulation of innate and acquired immunity. introduction: high mobility group box 1(hmgb1) has recently been identified as a late mediator of endotoxin lethality. we newly developed an extra corporeal hmgb1 absorber. the purpose of this study was to test the hypothesis that hmgb1 removal could prevent or reduce endotoxin induced lethality or tissue injury of rats. methods: all experiments were conducted in accordance with the institutional care and use committee.male wistar rats were randomly allocated into three groups; hmgb1 absorber group (group i),hmgb1 nonabsorber group (group ii), and vacant column group (group iii).we applied these columns for each groups at 6 hours after lps injection. the rats were sacrificed 48 hours after lps injection for pulmonary histology. we statistically analyzed survival rate with kaplan-meier and the levels of hmgb1 with anova. results: survival rate was 67% in the group i at 48 hours after lps injection, as compared with 0% in the group ii and 11% in the group iii. the pulmonary histology in both group ii and group iii showed acute inflammatory injuries, whereas group i showed less inflammatory changes.the level of hmgb1 in the group i was significantly lower than those of group ii and iii. discussions:these results demonstratethat specific absorption of endogenous hmgb1 therapeutically reverses lethality of established sepsis indicating that hmgb1 inhibitors and absorber can be treated in a clinically relevant therapeutic window that is significantly wider than for other known cytokines. contact information: dr hideo iwasaka, oita university, anesthesiology and intensive care unit, yufu city, japan e-mail: hiwasaka@med.oita-u.ac.jp (1) (1) department of pharmacology, national university of singapore, singapore (2) dso national laboratories, singapore hydrogen sulfide (h2s) is increasingly recognized as a proinflammatory mediator in various inflammatory conditions. in this study, we have investigated the role of h2s in regulating expression of some endothelial adhesion molecules and migration of leukocytes to inflamed sites in sepsis. male swiss mice were subjected to cecal ligation and puncture (clp) induced sepsis and treated with saline, dl-propargylglycine (pag, 50 mg/kg i.p.), an inhibitor of h2s formation or sodium hydrogen sulfide (nahs) (10mg/kg, i.p.), a h2s donor. pag was administered either 1 hour before or 1 hour after induction of sepsis while nahs was given at the time of clp. using intravital microcopy, we found that in sepsis, prophylactic and therapeutic administration of pag significantly reduced the leukocyte rolling and adherence in mesenteric venules coupled with decreased mrna and protein levels of adhesion molecules (icam-1, p-selectin and e-selectin) in lung and liver. in contrast, injection of nahs significantly upregulated leukocyte rolling and attachment as well as tissue levels of adhesion molecules in sepsis. on the other hand, normal mice were given nahs (10mg/kg i.p.) to induce lung inflammation with or without pretreatment of nf-xã�b inhibitor, bay 11-7082. h2s treatment enhanced the pulmonary level of adhesion molecules and neutrophil infiltration in lung. these alterations were reversed by pretreatment with bay11-7082. moreover, expression of cxcr2 in neutrophils obtained from h2s treated mice was significantly upregulated leading to an obvious elevation in mip-2 directed migration of neutrophils. therefore, h2s act as an important endogenous regulator of leukocyte trafficking during inflammatory response. transient receptor potential vanilloid 1 (trpv1) is primarily found on sensory nerves. we have demonstrated its pro-inflammatory potential in arthritis and now present evidence that it is protective in an endotoxininduced model of sepsis. selective trpv1 antagonists are not available for use in the mouse in vivo, thus established trpv1 knockout (-/-) mice were used. c57bl6 wt and trpv1-/-mice were matched for age and sex and injected intraperitoneally (i.p.) with lipopolysaccharide (lps). the response was monitored for 4h. blood pressure, measured before and at intervals after lps in conscious mice via a tail cuff was reduced in both wt and trpv1-/-mice, with trpv1-/-mice showing an enhanced drop at 4h. in a separate group temperature, a proposed pre-mortality marker was also reduced by 4h, again with a significantly increased drop in trpv1ko. furthermore higher levels of two inflammatory markers tnfa and nitrite (as an indicator of no) were measured in peritoneal lavage and higher levels measured intrpv1-/-as compared with wt samples. finally aspartate aminotransferase (ast) levels also enhanced in trpv1-/-versus wt mice, although markers for kidney and pancreatic damage were similar in both genotypes. we conclude that trpv1 plays a protective role in sepsis. trpv1 is known to be present on nonneuronal (e.g. vascular components) and their relative involvement in sepsis is unknown. (1), da souza-junior(2), l de paula (1), mc jamur (2), c oliver (2), sg ramos (2), cl silva (2), lucia helena faccioli (1) ( h37rv) . infected balb/c mice developed an acute pulmonary inflammation and higher levels of tnf-a, il-1, kc, mcp-1 and mip-2 were detected in the lungs by day 15. in vivo degranulation of mast cells by c48/80 led to a reduction of the inflammatory reaction associated to a marked decline proinflammatory cytokine and chemokine levels in the lungs. the magnitude of cellular immune response was also partially impaired in infected mice and treated with c48/80. histologically, the exacerbated granulomatous inflammation shown in the lung parenchyma of infected mice was attenuated in infected mice and treated with c48/80. of interest, the number of mycobacterial bacilli recovered from the lungs was 1 log higher after treatment of infected mice with c48/80. these findings suggest that mcs participate in host defense against m. tuberculosis infection through of the modulation of cytokines and chemokines, which are important for the recruitment and activation of inflammatory cells. (1), ms chadfield (2), db sã¸rensen (1), h offenberg (1), m bisgaard (1), he jensen (1) (1) department of veterinary pathobiology, faculty of life sciences, university of copenhagen, denmark (2) novo nordisk a/s, cell biology, gentofte, denmark introduction: pasteurella multocida is an important cause of pneumonia in several animal species and may spread systemically. the aim of this study was to evaluate initial inflammatory reactions and the inocula effect due to strains of p. multocida of different origin in an aerogenous murine model. materials and methods: 30 female balb/ c-j mice (app. 20 g, taconic, denmark) were infected intranasally with two clinical isolates of p. multocida of avian (vp161) and porcine (p934) origin, at three different levels of inocula concentration. after euthanasia, specimens of lung and liver tissue were collected for bacteriological and histopathological evaluation. furthermore, lung tissue samples were taken for measurement of expression of metalloproteinase mmp-9 and metalloproteinase inhibitor timp-1. results: all mice infected with the avian strain were euthanized after 24 hours. viable counts recovered from lung and liver tissue were high, and histopathology revealed pronounced acute bronchopneumonia. in the liver, disseminated necrosis with formation of microabscess was also seen. on the contrary, a dose response was observed with the porcine strain with regard to recovery of viable counts and development of lesions was apparent after 24, 48 and 72 h. furthermore, differences were seen in the nature of the lesions caused by the two strains. there was a difference in expression of mmp-9 and timp-1 between infected and noninfected mice. the model proved suitable for the evaluation of pulmonary inflammatory reactions between the two different host-derived strains as demonstrated through viable counts, histopathology and expression of mmp-9 and timp-1. (1), r molinaro (1), a franã�a (1), m bozza (1), f cunha(2), s kunkel (3) (1) universidade do rio de janeiro, brazil (2) universidade de sâ¼o paulo, brazil (3) university of michigan, usa introduction and objectives: studies reveal that regulatory t (treg) cells control immune responses; therefore these responses must be controlled to enable the effective protection against infections and cancer. ccr4 knockout mice (ccr4 -/-) are more resistant to lps shock. so, our aim is to study the mechanisms involved in the resistance of ccr4-/-subjected to severe sepsis by cecal ligation and puncture (clp) and how tregs modulate this effect. results: c57/bl6 mice were subjected to clp model, whereby the cecum was partially ligated and puncture nine times with a 21g needle. sham-operated mice were used as control. mice subjected to clp and sham surgery were treated with antibiotic since 6h after and until 3 days. ccr4 -/-mice subjected to clp presented an increase in survival rate (78%) compared with wildtype mice (17%), and a marked improvement in the innate response concern to neutrophil migration to peritoneum and lung, bacteria load and cytokine levels compared to wild-type mice. besides, tregs from ccr4-/-clp mice did not inhibit proliferation of t effector cells as observed for treg from wild clp mice, at a proportional ratio of teffector:treg. interesting, treg from ccr4-/-clp mice did not inhibit neutrophil migration to bal when co-injected with fungal challenge as secondary infection, while the treg from wild clp mice did, as expected. conclusions: these results suggest that treg cells from ccr4-/-mice did not present suppressive response and it could be an important factor in their survival. inflammation and oxidative stress are known to be one of the important causes that are responsible for many diseases. inflammation has been associated with diseases like cancer, diabetes and many other. proinflammatory cytokine (tnf -âµ and il-1ã¢) and no are considered as pivotal mediators in inflammatory conditions like rheumatoid arthritis, sepsis and cancer etc. thus inhibition of pro-inflammatory cytokines and no production are important targets for treatment of inflammatory disorders. nowadays due to the emerging side effects of cox inhibitors, these targets have been paid more attention for the treatment of these conditions. some medicinal plants such as curcuma longa (1), commiphora mukul (2) in humoral memory, antibodies secreted into serum and other body fluids protect an individual against repeated challenges of previously encountered pathogens. antibody-secreting plasmacells are mostly considered to be shortlived, terminally differentiated b lymphocytes, eliminated after a few days or weeks by apoptosis. however, in secondary lymphoid organs and in the bone marrow, plasma cells can survive for months and years, without dna-synthesis and refractory to signals from antigen or antigen-antibody complexes. the lifetime of these longlived plasmacells depends on an intrinsic competence to survive in the distinct environment of those organs, which defines a specific survival niche. the niche provides survival signals like il-6, cxcl12 and tnf. within a functional niche, the lifetime of a plasma cell is apparently not limited intrinsically. the number of niches in the body has to be limited, in order to maintain physiological concentrations of serum immunoglobulins. thus recruitment of new plasmacells to the pool of old memory plasma cells has to be competetive. this competition is probably controlled by a simple molecular mechanism, namely the dual functionality of chemokines like cxcl12, which attract newly generated plasmablasts to a survival niche and at the same time are a survival signal for the plasma cell. plasmablasts and plasma cells express cxcr4, the receptor for cxcl12. while plasmablasts migrate in response to cxcl12, plasma cells depend on it for survival in the niche, but are no longer migratory. thus once disloged from their niche, they will die. plasmablasts newly generated upon systemic secondary immunization, upon concommittant stimulation with interferon-gamma, can also express cxcr3, the receptor for the interferon-gamma-induced chemokines cxcl9, 10 and 11, which may lure the plasmablasts into inflamed tissue. the switch in the potential to migrate provides also an efficient means to eliminate plasma cells of the peak of an immune response, which as plasmablasts had migrated to the tissue inflamed in that pathogenic challenge. inflamed tissue contains survival niches for plasma cells. in the inflamed tissue, plasma cells provide high local antibody concentrations while the tissue is inflamed. upon resolution of the inflammation the plasma cells will be dislodged and die. longlived plasma cells provide longlasting antibody titers (protective memory) and leave memory b cells a role in reactive memory, generating memoryplasmacells in secondary challenges, and if serum titers are not sufficient to protect. in chronic inflammation, this mechanism can contribute to pathogenesis. thus in the nzb/w model of lupus, longlived autoreactive plasma cells are generated early in pathogenesis, which survive in bone marrow and spleen. later, in established disease, autoreactive plasma cells are shortlived and continuously generated. they do not compete with the longlived plasma cells, and both populations coexist as prominent populations. interestingly, longlived plasmacells are resistant to therapeutic immunosuppression, while the generation of shortlived plasma cells is blocked. this may be the reason for the failure to cure antibodymediated immunopathology, e.g. in autoimmunity and allergy, by conventional immunosuppression, (1), h lee (2) c5a is a potent inflammatory mediator produced during complement activation. unregulated c5a signalling through its receptor (c5ar) on neutrophils and other leukocytes is implicated in the pathogenesis of autoimmune diseases including rheumatoid arthritis and systemic lupus erythematosus. considerable effort has gone into development of c5ar antagonists for human therapy. we took neutrophils from genetically modified human c5ar knock-in mice in which the mouse c5ar coding region was replaced with human c5ar sequences and immunized wild-type mice to generate high affinity antagonist monoclonal antibodies (mabs) to human c5ar. these mabs inhibit c5a-induced neutrophil migration and calcium-flux, and bind to a region of the 2nd extracellular loop of c5ar loop that seems to be critical for receptor activity. this study investigated the effectiveness of these mabs in the k/bxn serum-transfer model of inflammatory arthritis. human c5ar knock-in mice were given 1-10 mg/kg mab intraperitoneally, before or after inflammatory arthritis developed. mice treated with anti-c5ar mab one day before serum transfer did not develop swelling or clinical signs of arthritis in contrast to controls. histopathology of the joints in anti-c5ar mab-treated mice revealed a complete block of the massive influx of leukocytes and cartilage erosion seen in controls. furthermore, and most significantly, a single 1 mg/kg dose of anti-c5ar mab given 5 days after initiation of disease completely reversed inflammation. in the collagen-induced arthritis (cia) model, injection of anti-c5ar mab after development of inflammation also reversed inflammation to baseline. these potent new antibodies to human c5ar are in preclinical development. the cytokine macrophage migration inhibitory factor (mif) participates in fundamental events in innate and adaptive immunity. the profile of activities of mif in vivo and in vitro is strongly suggestive of a role for mif in the pathogenesis of many inflammatory diseases, including rheumatoid arthritis (ra), asthma, and sepsis. mif also has a unique relationship with glucocorticoids, in that despite antagonizing their effects, the expression of mif is in fact induced by glucocorticoids. thus, mif functions as a physiological counter-regulator of the anti-inflammatory effects of glucocorticoids. therapeutic mif antagonist may therefore provide a specific means of steroid sparing. since mif are highly conserved among different species, it is hard to develop high affinity antibodies due to immune tolerance.we developed a proprietary technique to break the immune tolerance and selected high affinity mouse monoclonal antibodies against mif.the antibody can neutralize mif activity in cell based assays, and is very effective in a lps induced mouse sepsis model. using this antibody as a tool, we are studying the function of mif in comparison with the function of lps.we found that lps induced inos expression and no secretion are dependent on the secretion of mif.we also found that although both lps and mif induce g1 arrest in macrophage cell line raw264.7, their functions are independent to each other. structure-based small molecule drug design. an effective agent would be the first orally active cytokine antagonist. methods: collagen-induced arthritis (cia) was induced in dba-1 mice by immunisation with bovine type ii collagen/adjuvant on day 0 and 21. cor100140, synthesized on the basis of computer modeling of mif protein x-ray crystallographic data, was administered by daily oral gavage from day 21. etanercept (8mg/kg ip q2d) was used as a positive control. the mek-erk pathway is activated in numerous inflammatory conditions, including ra, ibd and copd. arry-162 is a potent (ic50 = 12 nm), selective, atp-uncompetitive mek1/2 inhibitor.arry-162 is highly efficacious in cia and aia rat models, with ed50s of 3 and 10 mg/kg, respectively, equal to or better than standard agents. addition of arry-162 to methotrexate, etanercept, ibuprofen or dexamethasone regimens in these models results in improved efficacy that is at least additive, if not synergistic. in tpa-stimulated human whole blood, this compound inhibited tnf, il-1 and il-6 production (ic50s of 23, 21 and 21 nm, respectively). in contrast, inhibition of perk required 280 nm to achieve a 50% reduction, demonstrating that inhibition of pro-inflammatory processes is very sensitive to perk inhibition.in clinical studies, healthy volunteers were administered a single oral dose of 10, 20, 30, 40 or 80 mg); blood was drawn at various times after dosing and stimulated ex vivo with tpa. arry-162 was well-tolerated and drug exposure was dose-proportional. in ex vivo blood samples, there was a dramatic time-and concentration-dependent inhibition of tpainduced il1 and tnffz with >80% inhibition observed at plasma concentrations of 50 and 150 ng/ml, respectively. similar inhibition of perk required 300 ng/ml. a multiple ascending dose clinical study has confirmed the pharmacokinetics and pharmacodynamics of arry-162 and helped define tolerability. clinical evaluation of arry-162 in combination with methotrexate in patients with rheumatoid arthritis is on-going. p38 (a mitogen-activated protein kinase) has been shown to play a key role in the release of cytokines such as tnfand il-1a from monocytes in signaling cascades that are initiated due to extra cellular stress stimuli. inhibition of p38 activity is expected to regulate the levels of tnf-a and il-1b thereby alleviating the effects of inflammation in ra. a new class of p38 inhibitors based on the naphthyridininone scaffold have been discovered. x-ray crystallography and site directed mutagenesis studies were critical tools that aided the evolution of the naphthyridinone lead class starting from a pyrido-pyrimidinone template. this presentation will discuss the derivation of key benchmark pre-clinical candidates in these novel scaffold classes (shown below) as influenced by structural biology studies, mutagenesis data and molecular modelling. efficacy studies in animal models for benchmark compounds will also be presented. (1), h aaes (1), w-h boehncke(2), j pfeffer(2), t skak-nielsen(1), i teige (1), k abell (1), ph kvist (1), e ottosen (1), tk petersen (1), lars svensson (1) (1) discovery, leo pharma, 55 industriparken, ballerup, denmark (2) department of dermatology, johann wolfgang goethe-university, frankfurt am main, germany p38 map kinase plays an important role in mediating an inflammatory response in mammalian cells. as a consequence of activation, several inflammatory mediators are released including il-1b] and tnfa. both cytokines have a central role in the pathogenesis of inflammatory conditions such as psoriasis. approximately 30% of psoriasis patients develop psoriatic arthritis. leo15520 is a member of newly developed class of selective p38 map kinase inhibitors. the compound was tested orally in in vivo models relevant for psoriasis and psoriatic arthritis. the in vivo models selected include the cia arthritis model, the human psoriasis xenograft scid mouse model, the uvb-induced dermatitis model, the lps induced tnfa model and a local gvh model. treatment with leo15520 led to an amelioration of the ongoing inflammation in all investigated in vivo models. in the cia model, a clear dose response effect was observed on the developing arthritis in both rats and mice (65 % reduction in mice and 77 % in rats at 10 mg/kg p.o.). in the humanised psoriasis model, leo15520 at a dose of 20 mg/kg, had an effect on both the hyperplastic epidermis (epidermal thickness reduced by 41%) and on the infiltrating inflammatory cells. the anti-inflammatory effect of leo15520 was even close to the effect of systemically delivered steroids in both models. we believe that the new highly selective class of p38map kinase inhibitors has a strong potential as an orally delivered therapy for systemic inflammation diseases such as psoriasis and arthritis. slx-2119 is a potent, selective, orally bioavailable inhibitor of the rho-kinase rock-2. its ic50 for rock-2 and rock-1 inhibition is 100 nm and >10 mm respectively. the ability of slx-2119 to inhibit septic liver injury was investigated in c57bl/6j mice challenged with lipopolysaccharide (lps) and d-galactosamine (d-gal). mice were given lps (10 mg/mouse) and d-gal (18 mg/ mouse) i.p and 5.5 hours later were sacrificed for analysis of liver injury. mice challenged with lps/d-gal had a >10 fold increase in serum alt and ast levels. this increase was reduced by >90% in mice pretreated with slx-2119 either orally (100 mg/kg, 2 and 20 hrs prechallenge) or i.p. (10-100 mg/kg, 15 min pre-challenge). slx-2119 inhibited the increase in hepatic levels of tnffalpha produced by lps/d-gal by >50%. to assess the kinetics of slx-2119s benefit, slx-2119 (100 mg/kg i.p.) was given 15 min prior to, or 15 or 60 min after the lps/ d-gal. slx-2119 was effective at inhibiting the rise in alt and ast levels at all 3 time points suggesting that inhibiting rock-2 even after the initiation of the lps/d-gal driven cascade protects against septic liver injury. in a survival study, 9 out of 10 mice given lps/d-gal were dead by 8 hrs whereas in mice given slx-2119 (100 mg/kg orally) 1 animal died at 10 hours and the remaining 9 mice were alive 48 hrs later. these results show that specific inhibitors of rock-2 may have therapeutic utility in the treatment of sepsis and subsequent liver injury. theta through three key hydrogen bond mediated interactions.they potently inhibit protein kinase c activity in vitro as demonstrated by inhibition of il-2 secretion in human purified t-cells stimulated with anti-cd3 and anti-cd28 or whole blood seb challenge.the pkc-theta inhibitors are orally bioavailable and demonstrate immunosuppressive activity in a mouse model of human delayed-type hypersensitivity responses. (1), j zhang (1), k henley (1), m white (1), d hilton (1), b kile (1) ( to investigate pathogenesis of rheumatoid arthritis, we used mice transgenic for the uniquely human fcgam-mariia in inducible and passively transferred models of arthritis. transgenic mice developed severe ra-like disease in both model systems, indicating that the transgene played a major role in arthritis pathogenesis. disease could be reduced by the administration of either specific monoclonal antibodies to fcgammariia or small chemical entities (sce) designed to bind to the fcgam-mariia dimer. to investigate the cause of this enhanced sensitivity to auto-immune stimuli, the phagocytic capacity of transgenic mice compared to c57bl/6 control mice was examined using phagocytosis of fluorescent beads coated with ova or ova/anti-ova immune complex or opsonised sheep red blood cells. in both assays macrophages from fcgammariia transgenic mice showed significantly increased phagocytosis comapred with cells from control mice at 4 hours.this difference diminished over time andwas only seen where particles were opsonised. treatment of macrophages with specific fcgammariia blocking monoclonal antibody fragments 8.7 f(ab)2 or iv.3 f(ab) reduced phagocytosis to background levels . macrophages from transgenic mice also showed significantly greater production of inflammatory cytokines tnf-alpha and il-1beta when stimulated in vitro with heat aggregated immunoglobulin (hagg). moreover, this response was also blocked by specific fcgammariia monoclonal antibody fragments or sce. thus, expression of the fcgammariia transgene in these mice leads to increased uptake of and reactivity to immune complexes, resulting in enhancement of inflammatory sequelae in the form of increased th1 cytokine secretion andamplification of the pro-inflammatory response leading to arthritic disease. harald burkhardt (1), u hã¼ffmeier (2), i kã§nig (3), j lacorz (2), a reis (2), k reich (4) (1) johann wolfgang goethe university, frankfurt, germany (2) friedrich-alexander-unisversity of erlangen-nuremberg, germany results: whereas the earlier described strong association of allele tnf*-238a with psoriasis could be confirmed, our study revealed that this association was completely dependent on carrying the psors1 risk allele. for psa, but not psoriasis vulgaris without joint involvement strong association with the allele tnf*-857t was detected (or=1.956, 95% ci 1.33-2.88; pcorr=0.0025) also in patients negative for the psors1 risk allele. our results indicate genetic differences between psoriasis vulgaris patients with and without joint manifestation. while the previously reported association between tnf*-238a and psoriasis seems to primarily reflect ld with psors1, tnf*-857t may represent a risk factor for psa independent of psors1. (1), n modi (1), m stanford (1), e kondeatis (1), r vaughan (1), f fortune (2), w madanat (3), c kanawati(4), p murray (5) methods: dna was obtained from 167 patients with bd, 61 from the uk and 106 from the middle east (me), and 159 controls individuals, 129 from the uk and 30 from me. dna was prepared by proteinase k digestion, and salt extraction and -318 and 49 snp were detected by a pcr-ssp. results: there was no significant difference in expression of -318c/t or 49a/g when all bd patients were compared to all control individuals, (p=0.3 and 0.9, respectively). as we have previously shown differences in snp expression in different patient groups we tested the uk and me patients separately. however, there was no significant difference in either snp in uk bd patients, (p=0.15, p=0.39) or me bd patients (p=0.7, p=0.6) when compared to the appropriate controls. (1), da brown (1), h johnen (1), mr qiu (1), t kuffner (1), pgm curmi (1), l brown (1), m mazzanti (2), sn breit (1) ( introduction: cerebral palsy (cp) is a nonprogressive motor disorder caused by white matter damage in the developing brain. it is often accompanied with neurocognitive and sensory disabilities. the cause and pathogenesis of cp is multifactorial and continues to be poorly understood. chorioamnionitis, clinically silent or manifest, has been reported to be a risk factor for cp both in term and preterm infants. il-16 gene is single copy gene located on chromosome 15q26.1-3 in humans. interleukin-16 is synthesized by a variety of immune (t cells, eosinophils and dendritic cells) and non-immune (fibroblasts, epithelial and neuronal) cells. it is also detected in organ-specific secretions in a number of inflammatory processes. amniotic fluid interleukin-16 concentrations decreased with advancing gestational age. but, women with preterm labor and women with chorioamnionitis have higher interleukin 16 amniotic fluid concentrations than those who delivered at term or those with sterile amniotic fluid. the aim of our study was to estimate allelic frequency for regulatory il16 -295 snp in the children with the cp. methods: dnas obtained from peripheral blood of 46 cp patients and 182 unrelated healthy volunteers were genotyped for the il16 -295 snp pcr-rflp method. results and conclusions: il16 -259 genotype fncc was more common in the population with cerebral palsy in the comparison to healthy volunteers. the significance of the association between il16 fngene polymorphisms and cerebral palsy has to be investigated in the future studies. (1), d delbro (1), e hansson (2) (1) kalmar university, sweden (2) gã§teborg university, sweden background: acetylcholine (ach) is a major signalling molecule, binding partly at nicotinic receptors (nachrs; a family of ions channels with nicotine as a selective ligand). one subtype, the alpha7nachrs, has antiinflammatory effects by way of down-regulation of tnfalpha release from macrophages. the alpha7nachrs have been demonstrated in neuronal as well nonneuronal tissues, e.g. astrocytes and microglia. aim. in rat astrocytes in primary culture, and in astrocytes cocultivated with primary microvessel cultures study: 1. the expression of alpha3nachrs and alpha7nachrs by immunofluorescence and western blot. 2. intracellular ca2+-transients spread within the astroglial networks after stimulation with nicotine. 3. pro-inflammatory cytokines, il-1b, il-6, and tnfalpha, released from microglial cells after stimulation with nicotine. results: alpha7nachrs expression was more evident in astrocytes co-cultivated with endothelial cells, suggesting that endothelial cells release factors, which increase the maturity of astrocytes. the ca2+ transient evoked by nicotine was also more pronounced in the co-cultured astrocytes. these ca2+ responses were blocked by the alpha7nachr antagonist alpha-bungarotoxin. the release of pro-inflammatory cytokines was down-regulated after stimulation by nicotine. conclusion: alpha7nachrs appear to be involved in some of the effects of nicotine administration to rat astrocytes. an anti-inflammatory action of cholinergic nerves on the astrocytes via nachrs seems probable, which may have therapeutic implications. university, department of natural sciences, kalmar, sweden e-mail: ann.pettersson@hik.se gedeon richter plc., budapest, hungary tramadol, an atypical opioid analgesic, is increasingly used for the treatment of osteoarthritis because it does not produce typical side effects of nsaids. review of clinical data show that it produces symptom relief and improves function, but these benefits are small and adverse events often cause participants to stop taking the medication (cohran database 2006). efficacy of tramadol in animal models of inflammatory pain is well established; however complex characterization of the drug regarding analgesic and side effects is missing in rats. our aim was to assess oral efficacy of tramadol at side effect free doses in rats. anti-hyperalgesic effect was determined in complete freunds adjuvant (cfa) induced thermal hyperalgesia test (th) and in model of cfa-induced knee joint arthritis. effect on inflammation was characterized in carrageenan induced paw edema test (et). for the characterization of side effect accelerating rotarod assay (arr) was used. significant impairment in arr was noted at 55 mg/kg dose, and above. in the th test weak 35% effect was seen at side effect free dose (40 mg/kg). in the arthritis model b.i.d. 40 mg/kg dose of tramadol given on days 3-7 after cfa caused a maximum 86% effect on weight bearing incapacitance (day 4). however, its effect seemed to diminish (34% on day 7) upon repeated treatment. in et tramadol had significant anti-inflammatory effect at 100 but not at 30 mg/kg dose. these results show that efficacy of tramadol in rat inflammatory pain models is limited by its side effects, in accordance with clinical data. chronic relapsing experimental allergic encephalomyelitis (cr eae) is a disease that bears striking similarities to the human condition multiple sclerosis (ms). in particular, cr eae and ms have major inflammatory events in the central nervous system (cns) that culminate in demyelination and disruption of axonal function.one group of mediators involved in the progressive cns inflammation are the prostaglandins (pgs).pg generation is regulated in experimental non-immune conditions of the cns by n-methyl-d-aspartate (nmda) receptor activation.nmda receptor-mediated events are also evident in eae and may therefore have the potential to influence pg production.the study was designed to profile pge2 and pgd2 in cns tissues during the development of cr eae and to examine the role of the nmda receptor in pg production through the use of the specific antagonist mk-801.biozzi mice were inoculated for cr eae and cns tissues were sampled during the course of disease.enzyme immunoassay of processed samples revealed pge2 and pgd2 levels within normal limits during the acute and subsequent remission phases of cr eae.in contrast, dramatic changes in pg concentrations were observed with a relapse of symptoms and a remission of disease.mk-801 was therapeutically administered to cr eae-diseased mice at the onset of relapse and changes in cns pg levels were recorded.the relapse phase of cr eae, but not the acute stage of disease, is characterised by an increase in cns pg production that may be influenced by nmda receptor activation. livia l camargo(1), lm yshii (1) (1), sk costa (1) (1) university of sâ¼o paulo, brazil (2) king's college, london, uk (3) butantan institute, sâ¼o paulo, brazil objectives: the neuropeptide substance p (sp) released by capsaicin-sensitive nerves (csn) plays a pivotal role in neurogenic inflammation. despite the prevalence of arthritis, the contribution of sp to the progression of arthritis has not been established. this study investigated the effect of csn ablation and sr140333, a sp antagonist, on knee joint inflammation and pain induced by intraarticular (i.a.) injection of kaolin (10 %, 5 h time course) in female wistar rats. the kaolin-injected knee (ipsilateral -ipsi) of vehicle-treated rats exhibited a significant, timedependent oedema as compared to the contralateral knee. in addition, increased pain score and high levels of myeloperoxidase (mpo, marker of neutrophil accumulation) and both pro-inflammatory (il-1b and il-6, but not tnfa) and anti-inflammatory (il-10) cytokines was detected in the ipsi synovial fluid of these animals. both destruction of knee joint csn fibres by neonatal capsaicin treatment and i.a. injection of rats with sr140333 (1 nmol/cavity) significantly attenuated the kaolin-induced pain score and knee oedema, suggesting that kaolin is acting, at least partially, via a neuronal mechanism. in contrast, the same treatment caused increased mpo activity and cytokine concentrations measured 5 h post kaolin injection. conclusions: peripheral release of sp after kaolin injection acts to increase pain generation, oedema formation and inflammatory cell influx. however, chronic tachykininergic depletion by capsaicin treatment up-regulates the production of pro-inflammatory cytokines that are important in triggering cell influx in the synovial cavity. (1) (1) university of sâ¼o paulo, sâ¼o paulo, brazil (2) butantan institute, sâ¼o paulo, brazil objectives: previously, intra-tracheal (i.tr.) injection of dep or 1,2-naphthoquinone (1,2-nq) evoked plasma extravasation and cell influx in rat airways. we now investigated whether simultaneous injection of these pollutants had a synergistic inflammatory action. we also determined the ability of dep and 1,2-nq-induced airway inflammation to evoke changes in the rat isolated thoracic aorta (rta) and corpus cavernosum (rcc) reactivity using organ bath assays. results: capsaicin-or vehicle-treated male wistar rats received i.tr. injection of dep (1 mg/kg) and 1,2-nq (35 nmol/kg). after 15 min, dep and 1,2-nq produced a potent (additive) plasma extravasation in the trachea and bronchi, but not lung, compared with each compound alone. in capsaicin-treated rats or treated with tachykinin antagonists, the response was inhibited, suggesting an important role for c-fibres, primarily tachykinins. increased mpo and cytokine levels were detected in bronchi of capsaicin-treated rats following 3 h treatment with pollutants. this treatment contributes to augment the ach (10-9-10-4 m)-induced relaxation in the rta but not in the rcc. in capsaicin-treated rats, the rta response to the pollutants was not affected but it was capable of evoking a marked relaxation in rcc in animals challenged with pollutants. conclusions: 1,2-nq exacerbates dep-induced plasma extravasation and mpo activity in the airways, indicating a neurogenic mechanism through tachykinins. the exposure to dep and 1,2-nq affects the endotheliumdependent response in rta without interfering with rcc. neuropeptides are unlikely to affect the pollutantsinduced changes in the rta. acknowledgements: capes, cnpq, fapesp. we thank ma alves for technical assistance. trans-resveratrol (rv) is a naturally occurring polyphenolic compound present in certain foods that has anticancer and anti-inflammatory properties. the purpose of this study was to determine the effect of rv on the production of proinflammatory cytokines and reactive oxygen species stimulated by lipopolysaccharides (lps) in glial cells. rt-pcr showed that rv (5, 25, 50 mm) dose-dependently inhibited 500 ng/ml lps induced tnfa, il-1b, il-6, mcp-1 and inducible nitric oxide synthase mrna expression. rv also inhibited lps-induced production of these cytokines (elisa), nitric oxide and reactive oxygen species in a dose-dependent manner. western blot analyses showed that resveratrol could inhibit lps-stimulated phosphorylation of erk1/2 and jnk but not p38. nf-kb reporter assay showed rv could inhibit nf-kb activation by lps in microglia and astrocytes. these results suggest that rv may inhibit lps-induced microglial and astrocyte activation through erk1/2, jnk and nf-kb signaling pathways. therefore, rv is a natural product with therapeutical potential against disease conditions in cns that involve an overproduction of proinflammatory cytokines and reactive oxygen species. (1), cs patil(2), sv padi (1), vp singh (1) (1) university institute of pharmaceutical sciences, panjab university, chandigarh, india (2) pharmacology r & d, panacea biotec ltd. lalru, punjab, india persistent stimulation of nociceptors and c-fibers by tissue injury causes hyperalgesia and allodynia by sensitization of nociceptors and facilitation of synaptic transmission in the spinal cord. the important participant in the inflammatory response of injured peripheral nerve may be nitric oxide (no). the aim of the present study was to test the sensitivity of pde5 inhibitor sildenafil in chronic constriction injury (cci) model, a rat model of neuropathic pain. sciatic nerve injury is associated with development of hyperalgesia 14 days after the nerve ligation. sildenafil (100 and 200fã� g/rat, i.t.) produced a significant decrease in pain threshold, which in lower dose did not alter the nociceptive threshold. the hyperalgesic effect of sildenafil was blocked by l-name and methylene blue (mb), which on per se treatment showed antinociceptive effect in nerve ligated rats. the results from the present study indicated that the major activation of no cgmp pathway in the chronic constriction injury model of neuropathic pain. the aggravation of hyperalgesic response might be due to the increased cgmp levels resulting in pkg-i activation and its upregulation. glycine transporter (glyt) 1 and glyt2 are expressed in glia and neurons, respectively. glyt1 make clearance of glycine released from glycinergic neuron in synapse and thus terminates the neurotransmission and also regulates over-stimulation by glycine spilled over to nmda receptors, while glyt2 supplies glycine into synaptic vesicles in glycinergic neurons. therefore, glyt inhibitors could modulate inhibitory glycinergic or excitatory glutamatergic neurotransmission. the present study examined the effects of glyt inhibitors on pain in animal models. inhibitors of glyt1 (sarcosine and org25935) and glyt2 (alx1393 and org25543) by intrathecal (i.t.) injection reduced formalin-induced nociceptive behaviors. glyt1 inhibitors reduced allodynia score and reversed the reduction of paw withdrawal threshold in complete freund adjuvant (cfa)-induced inflammation mice but the antiallodynia effects appeared after latent period. on the other hand, glyt2 inhibitors produced antiallodynia effects immediately after the i.t. injection in cfa-treated mice. these inhibitors produced the similar antiallodynia effects in the partial sciatic nerve ligationinjury or streptozotocin-induced diabetic neuropathic pain models, either by i.t. injection or i.v. injection.pretreatment of specific antagonists of glycine site of the nmda receptors disappeared the latent periods of glyt1 inhibitors and potentiated the antiallodynia effect. glycine receptor antagonist, strychnine by i.t. injected reversed the antiallodynia effect of i.v. injected glyt inhibitors. these results suggest that both glyt1 and glyt2 inhibitors by enforcing glycinergic inhibitory neurotransmission in spinal cord produce potent antinociceptive effect and may be novel candidate for medicament of pain control. the tumour microenvironment in particular tumour associated macrophages (tams) play a role in determining tumour outcome. despite strong causative links between inflammation and human gastric cancer progression, little is known of the role of tams in this disease. we have utilized our mouse model of gastric tumourigenesis, the gp130757ff mouse to assess the effect of the gp130757ff mutation on macrophage function and ascertain the role of macrophages in tumor formation. this mouse has a knockin mutation in the il-6 family cytokine receptor gp130 preventing shp2/ras/erk signaling and leading to constitutive stat3 transcriptional activation. tumour development is inflammation dependent, there is a requirement for il-11, and development is inhibited by nsaid treatment or microbial eradication, however an adaptive immune response is s410 inflamm. res., supplement 3 (2007) posters dispensable for tumorigenesis. in gastric antrum the gp130757ff mutation results, decreased erk1/2 activation and constitutive phosphorylation of stat3, abnormal signaling is replicated in macrophages. antral stat3 activation is unaffected by depletion of il-6. however in macrophages an absence of il-6 results in higher stat3 activation demonstrating the anti-stimulatory role of il-6 on macrophages. the gp130757ff mutation results in macrophages with decreased il-6 and increased inos mrna expression reflecting a more basally activated phenotype. manipulations of the gp130757ff mouse that reduce tumor size (eg. antibiotic or nsaid treatment or stat3 hemizygosity) coincidently result in reduced macrophage infiltration of antral mucosa. macrophages of gp130757ff mice display aberrant gp130 signaling potentially resulting in exaggerated response to stimuli. the key difference between mutant gastric mucosa and macrophages are changes in transcription of target genes. (1), y riffo-vasquez(2), s brain(2), s costa (1) (1) university of sâ¼o paulo, brazil (2) king's college london, uk objectives: we have previously shown that simultaneous intra-tracheal injection of diesel exhaust particles (dep) and 1,2-naphthoquinone (1,2-nq) caused a potent inflammation in rat airways partially dependent on neurogenic-mediated mechanism. this study investigates the mechanism of action of thee pollutants using inflammatory assays and histopathological approach in the lung and trachea of wild type (wt) and trpv1 knockout (ko) mice exposed to dep and 1,2-nq. dep (1 mg/kg) and 1,2-nq (35 nmol/kg)-induced airway inflammation was assessed via 125i-labelled albumin 1 h post pollutants injection. mpo assay and histopathology were performed 3 h after treatment. staining of lung and trachea specimens with h&e provided a profile of cell infiltration in both wt and ko animals. results: injection of dep and 1,2-nq evoked a potent plasma extravasation into the trachea and lung of wt, but not trpv1 ko, suggesting these pollutants act via a trpv1-mediated mechanism. in contrast, mpo activity in the airways of trpv1 ko mice was exacerbated compared to wt mice data. likewise, the histopathology revealed high numbers of leukocytes and macrophages infiltrated into the lungs and trachea of trpv1 ko mice compared to wt. conclusions: inhibition of increased microvascular permeability in the airways of trpv1 ko mice treated with pollutants suggests that these receptors are the predominant mechanism involved in the inflammation. however, neutrophils/macrophages accumulate more in trpv1 ko, indicating that the lack of trpv1 receptors up-regulates production of inflammatory cells in response to pollutants, thus supporting a protective role for trpv1 receptors. acknowledgements: capes, cnpq, fapesp. (1), n sato(1,2), y endo (1), s sugawara (1) (1) division of oral immunology, department of oral biology, tohoku university graduate school of dentistry, sendai, japan (2) division of fixed prosthodontics, department of restorative dentistry, tohoku university graduate school of dentistry, sendai, japan biotin is a water-soluble vitamin of the b complex and functions as a cofactor of carboxylases.biotin deficiency causes alopecia and scaly erythematous dermatitis.moreover, serum biotin levels are significantly lower in atopic dermatitis patients than in healthy subjects, indicating that biotin deficiency is involved in inflammatory diseases.however, immunological effects of biotin on allergic inflammation remain unclear.in this study, we investigated the effects of biotin-deficiency on metal allergy using nickel (ni)-allergy model mouse and a murine macrophage cell line j774.1. female balb/c mice (4 weeks old) received a basal diet or a biotin-deficient diet for 8 weeks.ten days after sensitization with intraperitoneal injection of lps and nicl2, the mice were challenged with intradermal injection of nicl2 into the pinnas.allergic inflammation was measured by ear swelling.the ethical board for nonhuman species of the tohoku university graduate school of medicine approved the experimental procedure followed in this study.j774.1 cells were cultured in biotin-sufficient or deficient medium for 4 weeks. ear swelling was significantly higher in biotin-deficient mice than biotin-sufficient mice.il-1 beta productions by splenocytes were significantly higher in biotin-deficient mice than in biotinsufficient mice.moreover, biotin-deficient j774.1 cells produced il-1 beta significantly higher than biotinsufficient j774.1 cells.to investigate the therapeutic effects of biotin-supplementation, biotin-deficient mice received biotin contained-water for 2 weeks.ear swelling was significantly lower in biotin-supplement mice than biotin-deficient mice.these results indicated that biotindeficiency deteriorates allergic inflammation.the augmentation of il-1 beta production is probably involved in those deteriorations. one of the most important targets of cytokine action is the blood vessels, which undergo some structural and functional changes that result in activation of endothelium. applying the elisa technique, levels of il-18, icam-1 and e-selectin were studied in 77 patients with acute pancreatitis. mediators levels were studied in arterial, venous and pancreatic ascites samples. according the atlanta criterion the mild pancreatitis was established in 33 patients and severe -in 44 patients. the highest levels of il-18 were noted in ascites and lowest in arterial samples. the highest concentration of adhesion molecules was in venous samples and lowest in ascites. it was a clear correlation between levels of il-18 and adhesion molecules and severity of pancreatitis. during first week the levels of il-18 gradually increased in patients with severe pancreatitis, while in patients with the edematous pancreatitis its levels decreased starting from the third day. icam-1 levels gradually increased during first three day with the following decrease after this term. the highest levels of e-selectin were noted at the time of admission. the clear correlation between il-18 and adhesion molecules was noted in both groups of patients. besides that, the clear strong correlation was observed between il-18 and quantity of circulating granulocytes and between e-selectin and hematocrite in patients with necrotizing pancreatitis. our study confirms the importance of activation of endothelium as a part of the systemic inflammatory response in patients with acute pancreatitis. subsequently eos infiltrate the tissues, are activated, and release mediators inducing the late phase response. this is characterized by tissue damage and repair, and in chronic reactions, tissue remodeling and fibrosis. mc/eos cross-talk by physical and non-physical contact is an essential feature of late-phase and chronic allergic reactions. we have previously described an mc/eos interaction facilitated by soluble mediators and shown to enhance allergic inflammation. still, pathways that mediate mc/eos cross-talk in allergy are not fully characterized. methods: human cord-blood derived mc were cocultured with peripheral blood eos, and activated with anti-ige. mc/eos couples in co-culture and in human nasal polyp tissue sections were specifically stained and counted using microscopy. expression of surface molecules was analyzed by facs. mc activation was measured by chromogenic assays for ã¢-hexosaminidase. relevant surface molecules were neutralized using antibodies, to assess interference with couple formation and activation. results: mc and eos physically interact, forming welldefined couples in-vitro and in-vivo. in the presence of eos, mc are more releasable under baseline or igeactivating conditions. this effect is partially mediated by cd48 and dnam-1 on mc, and 2b4 and nectin-2 on eos. we describe a novel physical interaction between mc and eos that we name "the allergic synapse". this synapse may upregulate allergic reactions, thus serving as a target for therapeutic intervention in allergic and inflammatory diseases. methods: mc were obtained from cord blood mononuclear cells (6-8 weeks with scf, interleukin-6 and prostaglandin e2, cbmc). cbmc were activated, after their culture for 5 days with myeloma ige (2mg/ml), by rabbit anti-human ige antibodies (5mg/ml), for 1, 3 and 6h at 378c. activation was measured by b-hexosaminidase release, determined by enzymatic-colorimetric assay. the expression of flip, mcl-1, bcl-2, bcl-xl, bak and bax was assessed by immunoblot analysis. results: two anti-apoptotic proteins were found to be upregulated: flip, which is involved in the extrinsic apoptotic pathway and mcl-1 that is mainly implicated in the intrinsic apoptotic pathway. in contrast, the expression of two other anti-apoptotic proteins that we have examined (i.e. bcl-2 and bcl-xl) was not altered. likewise, the expression of pro-apoptotic proteins from the bcl-2 family (i.e. bak and bax) was either undetectable or unchanged. conclusions: our findings reveal that ige-dependent activation of human mc mainly induces the selective increase in the expression of two pro-survival molecules. this may be one of the mechanisms that underlay mc hyperplasia in the chronic allergic inflammation. (1), c armishaw(2), z yang (1), h cai (1), p alewood(2), c geczy (1) (1) university of new south wales, faculty of medicne, sydney, australia (2) university of queensland, institute for molecular biosciences, st lucia, australia purpose: human s100a8, s100a9 and s100a12 are closely related proteins associated with inflammation. s100a12 is expressed in the human genome, but not in the mouse. s100a12 is a potent monocyte chemoattractant and mast cell (mc) activator. mouse (m) s100a8 and human (h) s100a8 share 58% structural identity, but the hinge domains are more divergent. the ms100a8 hinge (ms100a842-55) is a potent chemoattractant for leukocytes whereas this sequence in hs100a8 (hs100a843-56) is inactive. methods: s100a12 hinge domain and its alamine scan mutants were synthesized and their activities were tested by using thp1 cells or murine mc in vitro and mouse footpad injection. results: s100a12 hinge domain (s100a1238-53) was chemotactic for monocytes and mc and provoked mc degranulation in vitro and oedema, and leukocyte recruitment in vivo. in contrast to s100a12, the hinge domain only weakly induced cytokine production (il6, il8) by macrophages. residues essential for oedema were hydrophobic in nature (leu40, isoleu44, isoleu47 and isoleu53). n42 and i44 were essential for responses provoked by 10-12m s100a1238-53 whereas mutation of k38, l40, n46, i47, d49, k50 and i53 significantly reduced migration with 10-9m s100a1238-53. conclusions: s100a12 and ms100a8 may be functional chemattractant equivalents; s100a12 may have arisen by duplication of human s100a8. isoleucine residues in s100a12 hinge domain are essential for its proinflammatory properties. (1), g kiriakopoulou (1), e tsimara(2), a voultsou(2), k zarkadis (1) (1) general hospital of zakynthos, greece (2) medical centre of katastari, zakynthos, greece schistosome is a disease which pests in 74 tropical countries. the endemic areas are south america, far and middle east, and africa. our aim is to present an incident which concerning a parasitic infection, not endemic in greece. patient: man 30 years old who immigrated from pakistan (at the side of the aparkenar river) in greece, a year ago. he turned out with anlage, headache and weight loss. he is a swimmer. he mentioned also a fever before migrating to greece. clinically: largely-scaled paleness, stomach -mild and diffused sensitivity, with also lightly increased enteric sounds and a small degree of hepatomegaly, when palpated deeply. laboratory examination: leucocytes 9.030 eo 24.8%, hb 8.2g/dl, ht 27.2%, mcv 65,6fl, mch 19.1pg, thrombocytes 419.000, blood sedimentation 10mm, fe 10mg/ml, ferritin 2.98ng/ml. normal biochemical examinations. u/s: livers size lightly increased 16.5mm, hepatoportal vein with normal amplitude. parasitology of feces: in the immediate confection with lugol of the second sample, there were observed: big scattering oval ovules (130 -60mm) with a thin wall and quills on the side, as well as three imago worms (>10 mm): schistosoma mansoni. treatment: praziquantel was given. recheck in a months time: blood examinationleucosites 11.230, eo 9.8%, hb 10,2g/dl, ht 33.6%. parasitology of feces is negative. in the diagnosis of anemia combined with fever, to patients who are immigrants, schistosomiasis posters inflamm. res., supplement 3 (2007) should be taken into account, especially when sideropenic anemia is accompanied with intense eosinophile, because the disease is mostly a reaction of retardate supersensitivity. bronchial asthma is a chronic airway inflammatory disease caused by immune cells such as t lymphocytes and eosinophils. recently, high-sensitivity crp (hs-crp) has become available for detecting small changes in crp levels within the normal range, allowing for assessment of subclinical inflammation. this study was undertaken to investigate the relationship between hs-crp and bronchial asthma. we collected blood samples from 109 patients with bronchial asthma with or without attacks and measured serum eosinophil cationic protein (ecp) and pulmonary function as well as serum hs-crp. serum crp levels in patients without attacks (average 0.473 mg/ l; p < 0.001) and with attacks (average 0.908 mg/l; p < 0.001) were significantly higher than those of normal controls (average 0.262 mg/l). serum hs-crp levels were inversely correlated with fev1.0% in asthmatic patients (r = -0.4915, p < 0.01). in conclusion, these results indicate that serum hs-crp as well as ecp may be related to the state of asthma exacerbation and allergic inflammation. objectives: the pshr assay can be used to test the biologic activity of allergens since it mimics the effector phase of a type i hypersensitivity reaction. in this study we tested methods for removing ige-antibodies (stripping) from donor basophils. moreover a method was developed for improving the antigen specificity profile in pshr. proof-of-concept was provided using absorption with complete allergen extracts. methods: buffy coats were screened to exclude reactivity against relevant allergens. subsequently pbmcs were purified and basophils were stripped with either lactate or a phosphate buffer. cells were passively sensitized with patient sera and challenged with allergen extracts in various concentrations. released histamine was measured spectrofluorometrically (hr-test, reflab). cutoff was 10% hr. for absorption experiments patient sera (from a peanut allergic and a codfish allergic patient) were incubated with streptavidin-coated sepharose beads coupled with biotinylated allergens (peanut, and bsa as control). after centrifugation the supernatant was used to passively sensitize stripped basophils. hr was measured as described above. results: stripping experiments using the two buffers only partially removed surface ige but passive sensitization of stripped basophils was equally effective enabling determinations of sub-nanogram quantities of peanut allergen. absorption experiments showed that it was possible to specifically remove peanut specific ige from patient serum. removal of specific ige reactivity to peanut extract was verified by western blotting. conclusions: using peanut extract as a model it was demonstrated that in pshr antigen specificity can be modified. (1), sk bk(1), v sharma(2), rp bhandari (3) (1) nepal medical college teaching hospital, nepal (2) all india institute of medical sciences, new delhi, background: nepal has one of the highest maternalmortality rates in the world. this study was to evaluate the incidence, disease pattern, and risk-factors for thromboembolism in pregnant nepalese women. methods: women with thromboembolic diseases were identified and their case records retrieved and reviewed s414 inflamm. res., supplement 3 (2007) posters from january 1998 to december 2006. demographiccharacteristics were compared between women with and without thromboembolism. the total number of deliveries over the study period was 16,993, giving an incidence of 1.88 per 1000 deliveries. there were two cases of pulmonary-embolism and one resulted in a maternal-death. the others had deep-vein-thrombosis of which over 80% were limited to calf veins only. the ultrasound-examinations requested for suspected deep-venous-thrombosis before and after the event of maternal-death were 1.62 and 10.7 per 1000 deliveries (p <.001);the corresponding cases of deepvenous-thrombosis diagnosed were 0.29 and 2.94 per 1000 deliveries, respectively (p <.001). the majority (75%) of cases were diagnosed in the postpartum period, mainly after cesarean-delivery. women with venous-thromboembolism were older, had higher bmi, and a higherincidence of preeclampsia. there were approximately twice as many postpartum as antepartum events. bloodgroup a, multiple-pregnancy, caesarean-section, cardiacdisease, delivery at gestational-age of <36 weeks, a bmi of25, or more and maternal-age of 35 or over were all found to increase incidence of venous-thromboembolism. the long-standing belief that thromboembolism is rare among nepalese is at least partly because of undiagnosis most of these events are deep-veinthromboses occurring in the postpartum period and it is very essential for primary prevention developing country like nepal. (1), ch ladel (1), t ruckle(2), c rommel(2), r cirillo (1) (1) rbm-merck serono, colleretto giacosa, italy (2) serono pharmacological research institute, geneva, switzerland rheumatoid arthritis (ra) is a severe articular disease. massive leukocyte activation and infiltration into joints result in cartilage and bone destruction. blockade of pi3k signalling pathway has been demonstrated to be curative in a murine model of ra, collagen-induced arthritis (cia). in this study we explored the molecular mechanisms by which pi3k signalling inhibition resulted in clinical amelioration of disease symptoms. as605858, a novel isoform non-selective, yet specific class-i-pi3k inhibitor, administered at 15 mg/kg twice-a-day for 8 days, to mice showing signs of arthritis, paw swelling and inflamed digits, induced a significant amelioration of disease course. at the end of treatment, post-arthritic paws were removed and phosphrylation levels of akt (p-akt), downstream target in pi3k-mediated signal, were determined by semi-quantitative immunohistochemistry, also immunophenotyping of circulating cells by flowcytometry was conducted. akt phosporylation resulted to be significantly enhanced by disease induction and as605858 was able to decrease its levels down to values comparable with naã¯ve animals. controls and as605858treated mice were bled before treatment, after two days of treatment and at treatments end. no changes in cellular composition (morphology and hematology parameters) between experimental groups were observed. t cell number was not affected, however a significant decrease of natural-killer, memory and regulatory t cells was observed after as605858 administration. finally, a non-significant, moderate reduction of bcell number was also observed. these data demonstrate that efficacy of as605858 in arthritis models is mediated by direct modulation of the target resulting in a mixed anti-inflammatory (via pi3kg) and immunosuppressive (via pi3kd/a/b) activity. (1) (1) institute of biomedical science, university of sao paulo, brazil (2) ibilce -sao paulo state university, brazil epidemiologic data suggest that female sex hormones are involved in the pathophysiology of allergic asthma. we investigated in rats the immunomodulatory potential of estradiol and progesterone on the expression of allergic asthma. seven days after being ovariectomized (ovx), groups of rats were sensitized with ovalbumin (oa). fourteen days after sensitization, the animals were oachallenged and used 1 day thereafter. allergic,shamoperated animals were used as controls. some ovx animals were treated with estradiol (280 ã¬g/kg) or progesterone (200 ã¬g/kg) 24 h before being challenged. mast cell degranulation was quantified in samples of isolated, oa-challenged bronchi. the airway reactivity of inner bronchi to methacholine and the functional activity of cells we analysed. ovx caused reduction of the allergic lung inflammation and bronchial hyperresponsiveness with regard to intact female rats. estradiol reverted the reduced cellular recruitment into lungs, whereas progesterone reduced the pulmonary inflammatory response and reverted the bronchial hyperresponsiveness. cultured bal and bone marrow cells from allergic rats increased posters inflamm. res., supplement 3 (2007) s415 the release of il-10 and reduced that of il-1 and tnf. the release of il-4 by bone marrow cells was significantly reduced. these effects were reverted by estradiol, and progesterone reduced il-4 and increased il-10 production in bal and increased that of il-1 and tnf in bone marrow cells. bronchial mast cell degranulation upon direct contact with oa in ovx rats was less than in controls. it is suggested that female sex hormones can modulate the allergic lung inflammation in rats by acting on cellular migration/activity and airway responsiveness. objectives: to study the participation of fsh on modulation of e-and l-selectin, icam-1 and mac-1 expression in ovariectomized (ovx) rats made allergic. methods: female rats were sensitized (oa/alumen) after 1 (ovx-1) or 7 days (ovx-7) of ovx or sham-operated (sh). fourteen days thereafter animals were challenged (oa, 1%; aerosol) and sacrificed 24 h after. bronchoalveolar lavage (bal) was collected and flow citometry analyses oficam-1, mac-1 and l-selectin expression was studied. in parallel, lungs were frozen and sections were analysedfor e-selectin expression by immunohistochemistry. results: at day 1, e-selectin expression increased(ovx-1=1,8ae0,08) and at day 7, decreased (ovx-7=1,3ae0,1) as compared to respective controls (sh-1=1,45ae0,08; sh-7=1,9ae0,08). estrogen treatment reverted this profile in both groups (ovx-1+e=1,37ae0,09; ovx-7+e=1,8ae0,1). mean fluorescence intensity of bal cells showed increase of mac-1 expression (ovx-1=19ae0,5 vs sh-1=15,5ae1,0), icam-1 (ovx-1=18,1ae1,1 vs sh-1=13,3ae0,2) and l-selectin (ovx-1=11,2ae0,8 vs sh-1=9,5ae0,12) at day 1 i.e., ovx-1 group. on the other hand, we observed a decrease in icam-1 (ovx-7=14,2ae0,75 vs sh-7=19ae1,4) and mac-1 expression (ovx-7=20,7ae1,4 vs sh-7=27,5ae1,5) was seen in ovx-7 group. conclusions: oscillation of hormone levels during immunization with oa increased (ovx-1) and decreased (ovx-7) expression of adhesion molecules. estradiol treatment reverted this effect. this results suggest that fsh modulates the ali in rats by acting on cell (1), s lim (1), y lin (1), bp leung (1), c thiemermann (2), wsf wong (1) (1) national university of singapore (2) the william harvey research institute, london, uk glycogen synthase kinase 3b (gsk-3b) is known to regulate various cellular functions including inflammatory responses. we hypothesized that inhibition of gsk-3b may have anti-inflammatory effects in a mouse asthma model. balb/c mice were sensitized with ovalbumin (ova) and challenged with aerosolized ova. tdzd-8, a non-atp competitive gsk-3b inhibitor, was administrated by i.v. injection one hour before ova challenge. tdzd-8 significantly reduced the ova-induced eosinophilia in a dose-dependent manner and inhibited the levels of il-5, il-13 and eotaxin in bronchoalveolar lavage (bal) fluid. tdzd-8 also suppressed the mrna levels of icam-1, vcam-1 and chitinase proteins in the lung. histological studies revealed that tdzd-8 substantially reduced the inflammatory cell infiltration and mucus secretion in the lung tissue. tdzd-8 also decreased ova-specific ige level in the serum. in addition, ova-induced increase in airway resistance and reduction in dynamic compliance were attenuated by tdzd-8. our findings suggest that inhibition of gsk-3b may have therapeutic potential for the treatment of allergic airway inflammation. (1), a yildirim (1), f ercan (2), n gedik (3), m yuksel(4), inci alican (1) ( materials and methods: sprague-dawley rats (200-250 g) were exposed to 90 oc (burn group) or 25 oc water bath (control group) for 10 s under ether anesthesia. adm (100 ng/kg; s.c) was administered 10 min before burn and all rats were decapitated 24 h after the burn insult. trunk blood was collected for the measurement of tnf-alpha level and the lung, ileum and kidney samples were stored for microscopic scoring and for determination of lipid peroxidation (lp), myeloperoxidase (mpo) activity and formation of reactive oxygen metabolites (roms) using chemiluminescence assay. results: burn resulted in severe morphologic damage in tested tissues. lp increased in lung and kidney (p<0.01-0.001) and mpo activity showed a marked increase in all tested tissues (p<0.001) of the burn group. adm reversed these parameters effectively (p<0.05-0.001). luminol chemiluminescence levels showed increases in both ileum and lung (p<0.01-0.001) whereas lucigenin chemiluminescence levels increased in ileum and kidney (p<0.01) of the burn group. adm treatment was also beneficial in reducing chemiluminescence levels near to controls (p<0.01). adm reduced plasma tnf-alpha level (p<0.01) which showed a significant increase in burned animals compared to controls (p<0.001). conclusions: adm is beneficial in remote organ damage following burn insult via decreasing neutrophil infiltration, rom generation, lp, and the release of proinflammatory cytokine tnf-alpha. kalpana panday (1), sd joshi (2), kr reddy (3) ( we determined the crystal structure of human hematopoietic prostaglandin (pg) d synthase (h-pgds) as the quaternary complex with glutathione (gsh), mg2+, and an inhibitor, hql-79, with anti-inflammatory activities in vivo, at 1.45 resolution. hql-79 was found to reside within the catalytic cleft between trp104 and gsh in the quaternary complex. hql-79 inhibited h-pgds competitively against the substrate pgh2 as well as noncompetitively with respect to gsh. surface plasmon resonance analysis revealed that hql-79 bound to h-pgds with an affinity that was 10-fold higher in the presence of gsh and mg2+ than in their absence. hql-79 inhibited selectively pgd2 production by human h-pgds-expressing megakaryocytes but only marginally affected the production of other prostanoids, suggesting the tight functional engagement between h-pgds and cyclooxygenase. orally administered hql-79 inhibited antigen-induced production of pgd2, and airway inflammation in mice without affecting the production of pge2 and pgf2fâ¿. knowledge about this quaternary structure should accelerate the structure-based development of novel anti-inflammatory drugs that inhibit pgd2 production specifically. introduction: it has been proved that high levels of mechanical ventilation produce lung injury as well as local inflammation. this study was designed to evaluate how the generation of inflammatory mediators by an over-stretched lung affects the non-hyperventilated lung. methods: male wistar rats (250-275g) were anesthetized and paralyzed, and the two lungs were independently intubated. differential ventilation was applied for 3h (70 breath/min). one lung was subjected to hyper-ventilation (20ml/kg/lung) and the other was ventilated with a normal volume (5ml/kg/lung). in a control group, both lungs were ventilated with a normal volume. after sacrifice, samples of lung, plasma and liver were collected. the expression of the pro-inflammatory chemokine mip-2 was evaluated by rt-pcr and the edema was assessed by the ratio between the wet and dry weight of the lung. systemic inflammation was estimated in liver by measuring the expression of tnfa by rt-pcr as well as its levels in plasma. the hiper-ventilated lung showed an increase in the ratio between the wet and dry weight and in mip-2 expression compared to the normal ventilated lung. no differences were found in edema, neither expression of mip-2 between the normally ventilated lung and control lungs. no significant changes were observed in liver expression and plasma levels of tnfa as a consequence of unilateral lung hyper-ventilation. the over-straining caused to the hyperventilated lung leaded to a local inflammatory response without systemic effects. the normal ventilated lung is not affected by the inflammatory process triggered on the over-strained lung. (1), j-y gillon(2), v lagente (1), e boichot (1) (1) university of rennes 1, rennes, france (2) serono international s.a., geneva, switzerland macrophage elastase (mmp-12) is a metalloproteinase involved not only in emphysema but also in the inflammatory process associated with copd (chronic obstructive pulmonary disease). the mechanism of action of mmp-12 in the development of pulmonary inflammatory process is still unknown. in the present study, we investigated the effect of recombinant human mmp-12 (rhmmp12) on il-8/cxcl8 release from alveolar epithelial cell line a549 and we explored the underlying mechanisms. a549 cells were stimulated with rhmmp-12 (1.10-3-2.10-1 u/ml) during 6 hours and il-8/cxcl8 level in supernatant was determined by elisa. involvement of map (mitogen activated protein)-kinases was studied by western-blotting and also using chemical inhibitors. nfkb activation was examined with the transam nfkb p65 kit. we observed that mmp-12 elicited il-8/cxcl8 release in a dose-dependent manner. this production could be prevented by a pretreatment for 1hour with a selective mmp-12 inhibitor (as111793 1-30 mm) or with the nonselective inhibitor batimastat (1-30mm) . the il-8/cxcl8 production was also inhibited by actinomycin d (5mg/ml), erk1/2 inhibitors (u0126 5mm and pd98059 10mm) and nfkb inhibitors including bay11-7082 (10mm) and nfkb activation inhibitor (10nm), whereas p38 kinase inhibitor (sb203580) had no effect. stimulation with mmp-12 was rapidly followed by a phosphorylation of erk1/2 (5min) and an nfkb nuclear translocation and activation (1h). the nfkb activation was not inhibited by a treatment with u0126. these data suggest that alveolar epithelium is a target of mmp-12 since it upregulates gene expres-s418 inflamm. res., supplement 3 (2007) posters sion and release of il-8/cxcl8, via erk1/2 and nfkb activation, but these two pathways appears to be distinct. agents which are associated with lung inflammation, such as cigarette smoke and lipopolysaccharide (lps), induce the production of pro-inflammatory chemokines in lung epithelial cells in vitro, and the induction of interleukin (il)-8, in particular, is often used a measure of relative toxicity.in this study we have compared mrna expression and mediator release in nci-h292 human lung epithelial cells exposed to lung toxicants, namely: cigarette smoke total particulate matter (tpm), lps, bleomycin, diesel exhaust particles, residual oil fly ash (rofa), carbon black and vanadyl sulphate.polystyrene, poly(methyl-methacrylate) and the tpm vehicle, dimethyl-sulphoxide were used as negative controls.confluent monolayers of h292 cells were exposed to serial dilutions of test agents in serum-free medium for 24 hours.the conditioned medium was then removed and assayed for a range of pro-inflammatory cytokines and other selected mediators by luminex technology.the levels of gene expression of il-8, matrix-metalloprotease-1 (mmp-1), the gel-forming mucin muc5ac, heparinbinding epidermal growth factor-like growth factor (hb-egf) and the cytochrome p450s cyp1a1 and cyp1b1 were determined by quantitative-polymerase chain reaction.all of the toxicants induced similar responses whereas the negative controls were largely ineffective.-such a panel of biomarkers may enable an in vitro assessment of the potential to cause lung inflammation.-moreover the use of several biomarkers could give a more accurate picture of toxicity than the determination of il-8 alone, particularly in the case of agents such as tpm, where the conventional vehicle is found to have some biological activity. respiratory tract infections are a major public health issue. prevention in high risk populations relies mainly on vaccination. vaccination is highly recommended in decrease absenteeism. immunomodulating drugs are important tools in the treatment of infectious diseases. immunomodulatory agents are probably contributive in decreasing exacerbation rates. the authors present different classes of immunomodulators that are currently in use. the vaccine, created from a bacterial protein, reeducates the immune system to stop inflammation. by preventing infections, vaccines prevent the development of a strong t helper (th1) response. the challenge is now to inform about new possibilities of an optimal prevention respiratory tract infections. deoxypodophyllotoxin (dpt) is a medicinal herbal product that is isolated from anthriscus sylvestri. that inhibits cyclooxygenase-2 (cox-2) and cox-1dependent phases of prostaglandin d2 (pgd2) generation in bone marrow-derived mast cells (bmmc) in a concentration-dependent manner with ic50 values of 1.89mm and 65.3mm, respectively. this compound inhibited cox-1 and 2-dependent conversion of the exogenous arachidonic acid to pgd2 in a dose-dependent manner with an ic50 values of 0.01mm and 12.1mm, respectively. however, dpt did not inhibit cox-2 protein expression up to a concentration of 30mm in the bmmc, indicating that dpt directly inhibits cox-2 activity. furthermore, this compound consistently inhibited the production of leukotriene c4 (ltc4) in a dose dependent manner, with an ic50 value of 0.37mm. these posters inflamm. res., supplement 3 (2007) s419 results clearly demonstrate that dpt has a dual cox-2/5-lox inhibitory activity in vitro. therefore, this compound might provide the basis for novel anti-inflammatory drugs. in order to determine anti-allergic and antiasthmatic activity of dpt in vivo, we used rat pca model which was activated by anti-dinirophenyl ige and ovalbumin/alum induced mouse asthmatic animal model, respectively. as a result, dpt strongly inhibited pca reaction as well as it reduced infiltrated eosinophil numbers in bronchoalveolar lavage fluid. furthermore, dpt decreased the mrna levels of the th2 cytokines in a murine asthmatic model. in addition, northern blot analysis showed that dpt also reduced both the eotaxin and arginase i mrna levels in a dose dependent manner. these results suggest that dpt may be beneficial in regulating various inflammatory diseases. an imbalance of proteases and anti-proteases in the lung has been implicated in the pathogenesis of chronic obstructive pulmonary disease (copd), a smokingrelated disorder associated with accelerated lung function decline.in particular, the activity of matrix metalloproteases (mmps) have been implicated in driving both the inflammation and parenchymal destruction observed in copd patients.here, we tested whether a broad spectrum mmp inhibitor, pkf-242484, could block the inflammation induced by an acute exposure to cigarette smoke in 2 strains of mice, balb/c and c57/bl6.animals were administered the compound (1-10 mg/kg) either per os (p.o.) or intranasally (i.n.) 1 hour before and 5 hours after exposure to smoke on three consecutive days.bronchoalveolar lavage (bal) was performed and lungs were flash frozen for inflammatory marker analysis.pkf-242484 dose-dependently reduced lung neutrophilia in balb/c mice when dosed either p.o. (~45% at 10 mg/kg; p < 0.01)or i.n. (~60% at 10 mg/kg; p < 0.01).however, the compound had no clear effect on bal neutrophil infiltration in c57/bl6 mice by either route of administration.interestingly, in both strains bal macrophages dose-dependently trended towards an increase when the compound was dosed p.o. and decreased when dosed locally (p < 0.05). examination of lung tissue cytokine levels revealed that while smoke-exposure increases il-1beta, kc, and mip-2, pkf-242484 had little effect on these cytokines.these data suggests the ability of broad spectrum mmp inhibitors to inhibit smoke-induced acute neutrophil inflammation is strain-dependent, while its ability to limit macrophage infiltration may be route dependent. to investigate the role of seh in the regulation of the pulmonary inflammatory response, we have used seh deficient mice in a locally administered lps model. male seh deficient mice (ko) and their wildtype (wt) littermates were exposed to inhaled lps.four hours later they were sacrificed and bal was performed. differential counts and cytokine levels in bal were evaluated. results: lps induced a significant increase in total cell number and neutrophil number in bal in the seh deficient mice and in wt mice;no significant differences between the groups were seen (table 1 ) cytokine analysis showed significant increases in tnfalpha, il-6, kc, gm-csf, mcp-1, il-1beta and rantes in the lps-exposed wt mice. no significant differences were seen between lps exposed wt and ko mice except for a significant increase in tnfa in ko mice. our results show that seh has no pivotal role for the regulation of the acute inflammatory response to lung administered lps. fam.liliaceae. based on literature data which signaled the presence of steroidic saponins in the rhyzomes, isolation, identification and quantitative determination of these compounds were done. the antiinflammatory activity was tested in non-immune chronic inflammation model:the cotton-pellets granuloma test in rats, and in an immune chronic inflammation model:arthritis test induced by freunds adjuvant in rats. in the first test, the antiinflammatory effect of steroidic saponins 600 mg/kg is weak, statistically insignificant. in the arthritis test, steroidic saponins 600 mg/kg proved an antiinfalammatory activity, influencing especially the primary response, but also the secondary one, in the last part of the experiment (after 16 days). in both tests, the suprarenal glands weight was modified. objectives: dendritic cells (dcs) are professional antigen presenting cells. many types of dc with subtle difference in phenotype have been reported in several organs. existence of dc was reported in synovial tissue of rheumatoid arthritis (ra), however, the details in ra dc still remains unclear. in this study, we generated a new lineage of dc with gmcsf (+tnfa) and investigated their functions. furthermore the ability of osteoclastgenesis was examined. methods: monocyte-derived dcs or macrophage were generated in (1) tnfa + gm-csf (2) gm-csf (3) il-4 + gm-csf (4) mcsf. the phenotypes of these cells were analyzed by morphological examination and flow cytometry (facs calibur). cell proliferation was examined by wst-8 assay. dc functions were assessed in antigen presenting ability (mlr assay), cytokine production (elisa), and endocytosis (fitc-dextran uptake). concerning osteoclastgenesis, monocyte-derived dcs were incubated with rankl and mcsf. trap stain was performed and the resorption ability was assessed on osteologic cell culture system and dentine slices. results: these cells were dendritic-like and their surface markers were cd1a low cd11b + cd11c + cd14 + cd86 + cd83 low hla-dr + dc-sign low and different from conventional dc or macrophage. they had an antigen presenting ability to induce na*ve cd4+ t cell proliferation, il-12 production and endocytosis. in the presence of rankl and mcsf, they differentiated multinuclear trap-positive cells with bone resorption ability, which was strengthened by tnfa. we generated a new lineage of dc with gm-csf (+ tnfa). the dc seemed to play a pivotal role in inflammatory arthritis under tnf immunity. the clinical effectiveness of rituximab and other b cellattenuating rheumatoid arthritis (ra) therapies has increased interest in understanding the role of b cells in ra pathogenesis.the possible mechanisms underlying the effectiveness of rituximab were investigated by performing biosimulation research in the entelos ra physiolab platform, a mathematical model of the joint of an ra patient.the platform dynamically integrates the contributions of immune cells (t cells, b cells, and macrophages), resident cells (fibroblast-like synoviocytes and chondrocytes) and mediators to the joint inflammation and structural damage observed in ra.the b cell lifecycle is represented in the platform, as well as effector functions such as antigen presentation, mediator and autoantibody production, and immune complex formation.the dynamics of these b cell properties were calibrated to reproduce reported experimental behaviors and clinical outputs from ra patients (e.g., acr score and radiographic progression rates).an assessment of the contribution of individual b cell functions to clinical outcome suggests that plasma cell-derived immune complexes are key modulators of inflammation in ra patients. in contrast, proinflammatory cytokine production by b cells contributes minimally to synovial hyperplasia, but plays a role in the progression of structural damage.immune complex formation leads to monocyte activation, increased mediator production by macrophages and an increase in antigen availability to t cells.biosimulation research in the ra physiolab platform is advancing our understanding of the mechanisms underlying effective b cell-targeting ra therapies, and may guide the development of improved second-generation therapeutic approaches. methods: the hmscs were obtained at the operation.the mononuclear cells were extracted and the colony forming assay were performed after 2weeks. the hmscs were cultured and in passage1,the cell surface antigens of both groups were analyzed by flow cytometory.in passage2, in control group, the cells were cultured with beta glycerophosphate(bgp) and in osteogenic group, the cells were cultured with bgp and ascorbic acid(aa) and dexamethasone(dex).after 2weeks, alp staining and activity were measured in each group.after 3weeks, alizarin red s assays were performed.rna was extracted from the cells cultured with bgp and aa and dex for 2weeks and 3weeks and the gene expressions of bone formation markers were examined by real time pcr. the mann-whitney test was used for the statistical analyses. the colony forming assays showed no significant differences in oa and ra. in flow cytometory, the cell surface antigens in oa and ra were almost same.in alp activity,there were no significant differences in oa and ra.in alizarin red s, there were significant differences.in real time pcr,the gene expressions showed no significant differences in oa and ra. conclusions: the hmscs of ra will be able to use for regenerative therapy. silje vermedal hã¸gh (1), hm lindegaard (2), gl sorensen (1), a hã¸j (3), c bendixen (3), p junker (2), u holmskov (1) ( cytosolic phospholipase a2 (cpla2) plays a crucial role in eicosanoid production, by releasing an arachidonic acid from membrane phospholipids. in addition, cpla2 regulates the phagocyte nadph oxidase-releasing superoxides and that is the only isozyme responsible for the production of eicosanoid in phagocytic cells. collageninduced arthritis (cia) in mice is an experimental model of auto-immune diesease with several clinical and histological features resembling rheumatoid arthritis in humans. previous studies show that cpla2-deficient mice are resistant to cia. thus we aimed to study whether cpla2 is up-regulated during the development of cia and to detect its exact location in the inflamed joints. immunoblot analysis revealed an increase in the level of joint cpla2 protein during the development of the disease which correlates with the severity of the inflammation, as examined by paw thickness. immunohistochemistry with specific anti-cpla2 antibodies revealed low positive cpla2 protein levels in skeletal muscles, sebaceous glands and skin (epidermis, dermis) tissues of healthy paws. in the joints of the cia mice, large amounts of inflammatory infiltrate containing cpla2 were detected. in addition, robust cpla2 protein expression in the skeletal muscles surrounding the joints and strong cpla2 positive staining in sebaceous glands were detected. the high correlation between the severity of inflammation and the elevated cpla2 protein, due to an inflammatory infiltrate and increased cpla2 expression, suggests an important role of cpla2 in the development of arthritis. rheumatoid arthritis (ra) is the complex disease depending on environmental as well as genetic factors. in spite of the large research efforts we know in fact only small number of genes involved in this disease. animal models are useful tools for better understanding of the pathogenic mechanisms and genes leading to the disease process. the aim of the current project is to identify the genes and their functional role importance for arthritis. two loci associated with arthritis were identified using a cross between the b10.q (intermediate susceptible) and nod.q strains (resistant to arthritis). one on chromosome 2 a disease protective locus (cia2) and another on chromosome 1 a disease-promoting locus (cia9). nod.q allele at cia9 promotes arthritis whereas cia2, has a protective effect in contrast to the b10.q allele on chromosome 2. a promising candidate gene in cia2 locus is complement factor 5 (c5) as the nod.q allele produced defective c5 protein. cia9 locus contains several genes of potential importance for disease such as fc riib, fc riii, fc riv ncf2, fh. the results of cia (collagen induced arthritis) and caia (collagen antibody induced arthritis) experiments using the subcongenic mice generated that contains fc r region showed a significant difference in incidence and severity of arthritis. the disease is controlled in a recessive pattern. the fragment devoid of fc r region seems to be protective. the subcongenic for the cia2 locus has been generated recently which will be tested for cia and caia susceptibility. the results from these experiments will be discussed in detail. angela pizzolla, ka gelderman, r holmdahl ncf1 is a component of the nadph oxidase complex, which produces reactive oxygen species (ros) upon activation into phagosomes and extracellularly. polymorphisms in the ncf1 gene that impair the capacity to produce ros, enhance susceptibility of both mice and rats to arthritis. activation of autoreactive t cells drives arthritis development but neither ncf1 expression nor oxidative burst have been detected in t cells. we hypothesize that antigen presenting cells influence t cell activity by producing ros during antigen presentation. we aimed to clarify the role of ros produced by dendritic cells (dc) on t cell activation. dc were grown from bone marrow from ncf1 mutated and wildtype mice. we could show that ncf1 mutated dc proliferated and differentiated better, had higher expression levels of costimulatory molecules and mhc ii upon stimulation as compared to ncf1 wildtype dc. in addition, ncf1 mutated dc induced higher levels of il-2 production by hybridoma t cells. to analyze the role of ncf1 in dc on arthritis, mice were developed expressing functional ncf1 restricted to dc (b10.qdcn). these mice are characterized for burst, ncf1 expression and ability to present antigen. we published that immunization with myelin oligodendrocyte glycoprotein (mog) protein resulted in higher disease severity than immunization with mog peptide in ncf1 deficient mice. this suggests that ncf1 plays a role in the uptake and processing of posters inflamm. res., supplement 3 (2007) s423 antigens, probably by dc. this will be further investigated with the b10.qdcn mice using in vitro assays as well as in vivo models for arthritis and multiple sclerosis. purpose: macrophage migration inhibitory factor (mif) is a pro-inflammatory cytokine involved in both innate and adaptive immune responses. it is expressed in human ra synovial tissue and its suppression inhibits t or b cell dependent animal models of ra. we investigated the role of mif in k/bxn serum transfer arthritis. methods: arthritis was induced by injection of k/bxn serum on days 0 and 2 in littermate wt and mif-/-mice. arthritis was scored clinically, ankle thickness was measured using microcallipers, and joints collected for histology. sections were scored for synovitis, synovial fluid exudate, cartilage degradation and bone damage. results: wt mice exhibited arthritis as early as day 1 and 100% incidence was observed on day 7. mif -/-mice exhibited delayed arthritis, with onset on day 3 and 100% incidence on day 9. mif -/-mice exhibited significantly reduced disease severity as measured by clinical disease score ( methods: osteoarthritis was induced by bilateral transection of the medial meniscus in dunkin-hartley guinea pigs using minimal invasive surgery to avoid cartilage damage due to inflammation and/or intra-articular bleeding. results: the first signs of osteoarthritis development were macroscopically observed four weeks after meniscal transection. twelve weeks after surgery the lesions were still restricted to the medial side of the joint and did not reach into the subchondral bone. cartilage destruction due to meniscal transection was also histologically detected. however, biomarkers for cartilage destruction (ctx-ii, hp/lp ratio, comp) were not increased. treatments aiming at different processes in osteoarthritis, such as bone destruction (risedronate), inflammation (pioglitazone and anakinra), and cartilage destruction (galardin) were not effective in this model. the early degenerative changes in this transection model are probably too mild to be measured in the systemic circulation using classic biomarkers. further research into new biomarkers is needed to detect and monitor the early stages of osteoarthritis. the ineffectiveness of the compounds tested in this model underscores the urgent need for new strategies to treat the disease. the meniscal transection model might prove to be useful tool for identifying new biomarkers and treatments. livia l camargo (1), a denadai-souza (1), lm yshii (1), a schenka (2), ma barreto (1), d boletini-santos (3), c lima (3), v rioli (3), mn muscar (1), e ferro (1), sk costa (1) ( methods: aia was induced via intraarticular (i.a.) injection of methylated bovine serum albumin in immunized male rats. knee oedema and pain score were assessed daily in controls and animals treated with hemopressin (10 or 20 ã¬g/day; i.a.). histopathological changes, cell number and cytokines in the synovial fluid of aia rats were determined at day 4. results: aia rats developed a severe mono-arthritis characterized by a joint oedema and pain. at day 4, there was marked cellular infiltration, hyperplasia, pannus formation and destruction of bone and cartilage, but pro-inflammatory cytokines were undetectable by elisa. both doses of hemopressin significantly reduced the knee oedema, but only 20 mg of hemopressin attenuated the pain score. acute joint inflammation was significantly reduced by hemopressin, but failed to significantly affect chronic histopathological signs (hyperplasia, pannus etc.). conclusions: hemopressin has potential for treating acute signs of aia by reducing synovial plasma protein extravasation, alleviating pain and reducing acute joint histopathological changes, thus providing an alternative strategy for treatment of oedema and pain in arthritis. calcitriol, the hormonal metabolite of vitamin d and it synthetic analogs exert an anti-inflammatory action on psoriatic skin lesions while eliciting mild inflammation on healthy skin. the map kinase erk plays an important role in the induction of chemokines, cytokines and adhesion molecules in keratinocytes that maintain the epidermal inflammatory response.we hypothesized that the dual effect of calcitriol may be partially attributed to differential effects on erk activation in the presence or absence of inflammatory mediators. our experimental model was immortalized non-tumorigenic human hacat keratinocytes cultured in the absence of exogenous growth factors or active mediators. inflammation was mimicked by exposure to tnf. level and activation of signaling molecules were determined by immunoblotting. by using the specific egf receptor (egfr) tyrosine kinase inhibitor, ag1487, we established that tnf activates erk in an egfr-dependent and egfrindependent modes. the egfr-dependent activation resulted in the induction of the transcription factor, c-fos, while the egfr-independent activation was of a shorter duration and did not affect c-fos expression. treatment with calcitriol alone increased erk activation and c-fos induction. pretreatment with calcitriol enhanced egfr-dependent erk activation and tyrosine phosphorylation of the egfr, but completely abolished egfr-independent tnf-induced erk activation. pretreatment with calcitriol increased the rate of de-phosphorylation of activated erk, accounting for the inhibition of egfr-independent erk activation by tnf. it is possible that effects on the erk cascade underlie the dual action of calcitriol and its synthetic analogs on cutaneous inflammation. christina barja-fidalgo (1), r saldanha-gama (1), ja moraes (1), r zingali (2), c marcienkewicz (3) (1) universidade do estado do rio de janeiro, rio de janeiro, brazil (2) universidade federal do rio de janeiro, rio de janeiro, brazil (3) temple university, philadelphia, usa neutrophils adhere on vascular endothelium and directly migrate toward inflamed tissue to exert their primary defense function. integrin are receptors that drive cell adhesion and motility and interfere with cell activation, functions and survival.acting as both anchoring molecules and signaling receptor, transducing signals outsidein and inside-out, integrins are potential targets for therapeutic and diagnostic opportunities. disintegrins are a family of cystein-rich low-molecular weight peptides that usually contain an rgd sequence, a cell attachment site of ecm and cell surface proteins recognized by integrins. they are considered selective and competitive antagonists of integrins, being potent inhibitors of platelet aggregation and cell-cell/cell-ecm interactions. we reported that rgd-disintegrins, selectively interact with integrin amb2, a5b1 and/or avb3) on human neutrophils, interfering with cell functions through the activation of integrin-coupled intracellular signaling pathways. recently showed that, a selective ligand of a9/a4b1 integrins, vlo5, induces neutrophil chemotaxis, cytoskeleton mobilization and potently inhibiting neutrophil spontaneous apoptosis. these effects are mediated vlo5 interaction with a9b1 integrin, activating the focal adhesion cascade. vlo5 effects on the delay of neutrophil is modulated by pi3k, erk-2 mapk and nf-kb pathways that seems to interfere with the balance between anti-and pro-apoptotic bcl-2 family members and with mitochondrial membrane potential. data emphasize mechanistic details of the role of a9b1 integrins interactions on human neutrophils and support the use of disintegrins as prototypes to develop logical combinations of drugs to optimize or minimize the susceptibility of a selected target cell population to apoptosis during therapeutic interventions. (faperj, cnpq, capes, ifs-sweeden) (1), h serezani (2), m peters-golden(2), sonia jancar (1) '(1) university of sâ¼o paulo, brazil (2) university of michigan, usa it is been shown that leukotriene (lt) b4 and cysteinyl lt (ltc4, ltd4 and lte4) enhances fcr-mediated phagocytosis in alveolar macrophages (am), dependent on protein kinase c (pkc). in contrast, ltb4 but not cyslt effects are exclusive on syk activation. in the present study we investigated the role of specific pkc isoforms and its upstream and downstream targets involved in lt-enhanced fcr-mediated phagocytosis. to this purpose, ams were pretreated or not with inhibitors of pkc-d (rottlerin -6 um), pkc-a (ro-32-0432-9 nm), pi3k (ly290042-10 um and wortmannin-10nm), erk 1/ 2 (pd98059 -2 um), cpla2 (aacocf3-10 um), p38mapk (sb20190-10 um) and ca++ (bapta/am-10 um), before stimulation with ltb4 or ltd4 and addition of igg-opsonized red blood cells for 90 min. activation (phosphorylation) of signaling molecules by lts were analyzed by western blot. our results demonstrate that ltb4 -enhanced phagocytosis is dependent on pkc-a, while ltd4 effects are mediated by pkc-d. cell proliferation and differentiation, adhesion, cell activation and apoptosis. while galectin-1 mainly acts as an anti-inflammatory and pro-apoptotic molecule, galectin-3 is known as a strong pro-inflammatory and anti-apoptotic signal. we have recently recognized galectin-3 as a new molecular target of immunomodulatory drugs in monocyte/macrophage-like cells. in this study we investigated the effects of immunomodulatory drugs (aspirin, indomethacin, hydrocortisone and dexamethasone), applied in therapeutic ranges on the expression of galectin-1 at gene and protein level in monocytic thp-1 cells. we have also tested the effects of these drugs on both galectins in the cells activated by lipopolysaccharide from e. coli (lps). the targeted mrna level was evaluated using quantitative rt-pcr technique and the expression of both galectins in cell homogenates was determined by western-immunoblot analysis. the results showed that immunomodulatory drugs affected the expression of galectin-1 on both, gene and protein level, and that the effects were dependent on drug type and applied concentration as well as time of the exposure. the modulatory effects of the applied drugs on galectin-1 and -3 expressions were also observed in the cells activated by lps. these findings represent important step in the understanding of the effects of immunomodulatory drugs on galectin-1 and-3 expressions, as well as the role of these lectins in the physiology of monocytes. introduction: pancreatitis-associated protein (pap) has been recently described as an endogenous mechanism involved in the regulation of inflammation. in the present study, we show some of the molecular mechanisms implicated in the intracellular signaling pathways modulated by pap. the pancreatic human cell line panc1 was incubated with pap (500ng/ml) and/or tnfa (100ng/ml). total rna was obtained and the expression of tnfa was examined by rt-pcr. in addition, the effect of pap on nfkb activation was measured by inmunofluorescence in cells. western blot analysis was used to determine the expression of nfkb mediators: phosphorylated ikk, ikba and p65. results: we observed that pap administration to cells prevented nfkb translocation to the nucleus as well as the tnfa-induced tnfa gene expression. when tnfastimulated cells were treated with cycloheximide in order to block protein synthesis, the induction of tnfa gene expression was completely restored. on the other hand, pap had no effect on ikk phosphorylation or ikba degradation. conclusions:in this study we have provided evidence that pap modulates the inflammatory response by blocking nfkb translocation to the nucleus. this pap-induced nfkb inhibition requires a jak/stat-dependent de novo protein synthesis. objectives: this study investigated the inhibitory mechanism of hyaluronan (ha) on lipopolysaccharide (lps)stimulated production of proinflammatory cytokines in u937 macrophages. methods: ha was added to u937 macrophage cultures in the presence of lps, with or without pretreatment with anti-intercellular adhesion molecule-1 (icam-1) antibody. secreted levels of tumor necrosis factor a (tnfa), interleukin (il)-1b, and il-6 were determined by enzyme-linked immunosorbent assay. the phosphorylation of nuclear factor (nf)-kb, ikba, and mitogenactivated protein kinases (mapks) was analyzed by immunoblotting. results: lps stimulated production of tnfa, il-1b, and il-6. in contrast to 800 kda ha, 2700 kda ha at 1 mg/ ml inhibited lps-induced cytokine production. anti-icam-1 antibody blocked the effects of ha on the lps actions on u937 cells. lps activated nf-kb and mapk pathways, whereas ha down-regulated p65 nf-kb and ikba phosphorylation by lps without affecting mapks. inhibition studies revealed the requirement of nf-kb for lps-stimulated cytokine production. anti-icam-1 antibody reversed the inhibitory effects of ha on phosphorylation of p65 nf-kb and ikba. conclusions: ha of intrinsic molecular weight suppresses lps-stimulated production of proinflammatory cytokines via icam-1 through down-regulation of nf-kb and ikb. exogenous ha into arthritic joints could act as an anti-nf-kb agent by the mechanism demonstrated in the present study. the principal eicosanoid product of endothelial cox-2 is prostacyclin (pgi2), which is a potent vascular patency factor. induction of endothelial cox-2 under hypoxic conditions is well documented. this response, along with associated pgi2 release, is likely an important protective homeostatic response. in order to explore the role of candidate signalling agents in cox-2 expression in response to hypoxia, studies were undertaken using luciferase reporter constructs of the cox-2 promoter region in huvec. cox-2 induction under hypoxic conditions was confirmed with the wild type construct. strategic mutations of transcription factor binding sites showed that sites for hypoxia inducible factors (hifs) were more important for cox-2 expression than those for nfkb. furthermore, expression of cox-2 was increased under normoxic conditions by transfection of huvec with normoxia-stable hif mutants. emsa showed hif binding in nuclear extracts from untransfected hypoxic huvec. under these hypoxic conditions, increased release of pgi2 but not vaso-occlusive thromboxane a2 was seen. thus the putative protective induction of cox-2 in endothelial cells in response to hypoxia involves signalling by hifs. crescentic glomerulonephritis is characterized by crescent formation and rapid progress to renal failure, where the predominance of th1 immune response plays a crucial role. however, the therapeutic efficacy of the regulation of th1-predominant immune responses remains to be investigated. therefore, the effects of a th1 selective inhibitor tak-603 were investigated in a model of crescentic glomerulonephritis in wky rats. methods: tak-603 was administered orally, starting at the time of induction of glomerulonephritis. in group 1, the drug was administered daily for the initial 6 days. tak-603 was administered on day 0 only in group 2, and from day 3 to 5 in group 3. in each group, nephritic rats were killed on days 6 and 56. results: in group 1, glomerular damage, including crescent formation, was improved on day 6, with reductions in the numbers of cd4, cd8 and ed-1 positive cells, as well as in urinary protein excretion. protein and transcript levels of th1 cytokines in the diseased kidneys were markedly decreased by tak-603 treatment. renal pathology, including glomerulosclerosis and interstitial fibrosis, was ameliorated and proteinuria was markedly decreased. elevated levels of serum creatinine showed concomitant improvement. in group 3, in which treatment was initiated shortly after the appearance of glomerular abnormalities, glomerular damage was also diminished, resulting in a decrease in urinary protein excretion. treatment only on the first day in group 2, partially rescued renal dysfunction. conclusions: these results suggest that the initial inhibition of th1 immune response has an appealing therapeutic potential for crescentic glomerulonephritis. parasitic nematodes and their hosts are now known to produce a wide range of galectins. whilst host galectins have been shown to modulate the recruitment and effector function of inflammatory cells including mast cells, neutrophils and eosinophils, the role of secreted parasitic galectins is less well defined. studies at moredun have demonstrated that both the endoparasitic helminth, haemonchus contortus, and the ectoparasitic mite, psoroptes ovis, produce galectin-like factors which, in vitro, directly influence the migration and survival of eosinophils from their natural sheep host. excretory-secretory extracts from both parasites contained potent chemokinetic activity and were also able to promote eosinophil survival in the presence of dexamethasone. separation by affinity chromatography, as well as specific sugar inhibition and mass spectrometric profiling, revealed the active components to be galectins. in the case of h. contortus, there was homolgy with known est sequences, which allowed subsequent cloning and expression studies to be undertaken. a functional in vivo role for these parasitederived galectins awaits confirmation, but the possibility is raised that they could directly influence the host immune response following infection. this may have particular significance in mite infections in which exudates from the associated eosinophilc lesion appear s428 inflamm. res., supplement 3 (2007) posters to provide the primary nutrient source for their survival. moreover, the observation that two very different parasites may have evolved similar mechanisms for manipulating the host inflammatory response to their benefit, raises the further possibility that parasite galectins may provide potentially novel therapeutic targets. the aim of the study was to investigate the time course of cytokine gene expression in liver and lungs of mice with lipopolysaccharide (lps)-induced septic shock and to assess the effect of three different immunomodulatory agents on cytokine mrna levels in these tissues. male cd-1 mice were injected intraperitoneally with 50 mg/kg lps alone or concomitantly with an intravenous dose of pentoxifylline (100 mg/kg), lisofylline (100 mg/kg) or prednisolone (10 mg/kg). the tissues were harvested 0, 1, 4, 6, and 24 h following lps administration and stored at -808c. relative quantification of tumor necrosis factoralpha (tnf-alpha), interleukin-1beta (il-1beta), interleukin-6 (il-6), and interferon-gamma (ifn-gamma) mrna levels was performed by real-time rt-pcr. the highest levels of cytokine mrna were observed at 4 or 6 h after lps administration, whereas the expression of tnf-alpha and il-1beta in lungs and il-6 in liver reached the peak values at 1h and then decreased gradually. in addition, the lps effects on cytokine mrna were more pronounced in liver when compared to lungs. all administered compounds inhibited the lpsinduced tnf-alpha mrna expression (by up to approximately 50%), whereas lisofylline significantly increased ifn-gamma mrna levels in both tissues at most investigated time points. for other cytokines, the observed differences did not reached statistical significance. in conclusion, with the exception of ifn-gamma, the time course of cytokine mrnas differed considerably depending on the type of tissue. in the murine model of lps-induced septic shock, only tnf-alpha gene expression was suppressed by all compounds under investigation. maria sanz(1), m losada (1), c company (1), c lope-gines(1), l piqueras(2), j cortijo (3) the migration of leukocytes into inflamed tissues involves a cascade of molecular events finely regulated by cell adhesion molecules and chemokines. fractalkine/ cx3cl1 (fr) is a membrane-bound chemokine that functions as a mononuclear leukocyte chemoattractant and as an adhesion molecule. clinical studies and animal disease models have shown that fr is also involved in the pathogenesis atherosclerosis. we have demonstrated that angiotensin-ii (aii) has proinflammatory actions inducing the initial attachment of mononuclear cells to the arteriolar endothelium. in the present study we have investigated whether aii can cause the synthesis and expression of fr on human umbilical arterial endothelial cells (huaecs). huaecs were stimulated with ang-ii 1 microm or with tnfalpha (20 ng/ml) for 1, 4 and 24 h. fr was determined in the culture supernatants by conventional sandwich elisa. fr was only detected after 4 h and 24 h stimulation with tnfalphafnwhereas aii was unable to provoke the cleavage of the chemokine. semiquantitative rt-pcr analysis of huaecs showed increased fr mrna expression in aii-stimulated cells for 1 and 4 h. these effects were caused by the interaction of aii with its at1 receptor since they were abolished by losartan (at1 receptor antagonist). tnfalpha also increased fr mrna. immunohistochemical analysis of the cultured endothelial cells showed a clear expression of fr in huaecs stimulated with aii or tnfalpha for 4 and 24 h. these results suggest that fr could be a key chemokine in the selective adhesion of mononuclear leukocytes to the arterial endothelium elicited by aii. the lipophilic yeast malassezia is an exacerbating factor in atopic dermatitis (ad).among organisms of the malassezia species, m. globosa and m. restricta are particularly dominant on the skin of ad patients. our previous study has demonstrated that human keratinocytes responded to the two malassezia species with different th2-type cytokine profiles, i.e. m. globosa induced il-5, il-10, and il-13 secretion from the keratinocytes, whereas m. restricta induced il-4 secretion.these findings suggest that m. globosa and m. restricta play a synergistic role in triggering or exacerbating ad by stimulating the th2 immune response. pattern recognition receptors (prrs) of human keratinocytes play an important role in the induction of inflammatory and innate immune responses. in this study, we assessed the role of prrs for cytokine production by human keratinocytes in response to malassezia species. human keratinocytes were pretreated with various anti-prr monoclonal antibodies (mabs) and stimulated with m. globosa or m. restricta. cytokine secretion from keratinocytes was measured by using fast quant elisa kit. exposure of human keratinocytes to m. globosa and m. restricta resulted in enhanced secretion of il-5 and il-4, respectively. the m. globosainduced increase in il-5 secretion was inhibited by mabs against cd14 and cd91. in case of m. restricta, mabs against toll-like receptor 2 (tlr2) and cd14 suppressed significantly il-4 secretion from keratinocytes. these findings suggest that the distinct prrs interactions with fungal pathogen-associated molecular patterns (pamps) are key factors in differential cytokine secretion from keratinocytes stimulated with malassezia species. atopic dermatitis (ad) is a chronic, relapsing inflammatory skin disease associated with allergy. mdc (macrophage-derived chemokine/ccl22) and tarc (thymus and activation-regulated chemokine/ccl17) are th2type cytokines, and it has been reported that serum mdc and tarc levels are associated with ad disease. in present study, we investigated the effect of prunus yedoensis matsum barks on the inflammatory chemokines (mdc and tarc) and jak-stat pathway in hacat keratinocytes. as a result, etoac fraction and e5 sub-fraction inhibited the mrna expression and protein level of mdc and tarc in a dose-dependent manner. also, e5 sub-fraction showed inhibitory activity on the stat1 protein level. these results suggest that p. yedoensis may have an anti-atopic activity by suppressing the inflammatory chemokines (mdc & tarc). the il-1 family now consists of 11 members, most of which have assigned functions.there are 10 members of the il-1 receptor family (including the decoy receptor type ii il-1r).many of the il-1 family members possess neither a signal peptide nor an apparent prodomain, but nevertheless manage to exit the cell.the il-1 family members il-1f6, f8 and f9 signal through a complex of the il-1r family member rp2 in association with il-1r acp to activate common inflammatory pathways.the specific activity is is low, on the order of 1-10ug/ml ec50.we have found that removal of a few n-terminal amino acids from il-1f5, f6, f8 and f9 can increase their bioactivity approximately 1000-fold. the location of the n-terminus leading to increased specific activity is quite specific; removal of one more or one fewer amino acid eliminates the effect.in addition, n-terminally truncated il-1f5 is capable of antagonizing signaling via il-1rrp2, but full-length f5 is inactive. (1) university of ulsan, japan kyoto university, japan inflammation plays a pathogenic role in the development of obesity-related complications such as type ii diabetes and atherosclerosis. tumor necrosis factor alpha (tnfa) is closely associated with the enhanced inflammatory responses in obesity and the obesity-related pathologies. tr2 (hvem/tnfrsf14), which is a member of the tnf receptor superfamily and the receptor for lymphotoxins-related inducible ligand that competes for glycoprotein d binding to herpesvirus entry mediator on t cells (light/tnfsf14), is a potent mediator of inflammatory responses. the purpose of this study is to examine the hypothesis that obesity-induced inflammatory responses can be attenuated by inhibiting tr2 pathway. c57bl/6 tr2 knockout mice and their wild-type control were fed a high-fat diet for 12 weeks and the obesity phenotypes were determined in the obese tr2 knockout mice and the control. the obese tr2 mice fed a high-fat diet elicited the attenuation of body weight gain and insulin resistance relative to wild-type control mice. expression levels of inflammatory genes significantly decreased in the adipose tissue of the obese tr2 knockout mice compared with those of the control. our results demonstrate that obesity-induced inflammatory responses and insulin resistance can be attenuated in obese tr2 knockout mice fed a high-fat diet. objectives: the present study was undertaken to investigate the role of insulin on allergic airway inflammation. methods: diabetic male wistar rats (alloxan, 42 mg/kg, i.v.) and controls were sensitized with ova (100 ã¬g) and al(oh)3 (8 mg, s.c.) 10 days after alloxan or saline injection. the animals were challenged 14 days later by the intratracheal instillation of ova (1 mg/0.4 ml). the following analyses were performed 6 h thereafter: (a) total and differential cell counts in bronchoalveolar lavage (bal) fluid; (b) quantification of tnf-alpha and il-1 beta in the bal by elisa; and (c) immunohistochemistry for p-and e-selectins on lung vessels. results: compared to the control animals, diabetic rats exhibited reduced number of neutrophils (90%) and mononuclear cells (50%); reduced levels of tnf-alpha (45%) and il-1 beta (30%), and reduced p-selectin expression (30%) in response to ova challenge. these abnormalities were corrected after treatment of diabetic rats with a single dose of nph insulin (4 iu, s.c.), 2 h before ova challenge. although we did not find differences in e-selectin expression between diabetic rats and controls, expression of this molecule was amplified by insulin. conclusions: data presented show that insulin controls neutrophils migration during allergic airway inflammatory possibly by modulation of tnf-alpha and il-1 beta production and selectin expression. supported by fapesp and pronex. hormonally active vitamin d derivatives are beneficial in the treatment of cutaneous inflammatory disorders, particularly in psoriasis. their anti-inflammatory effect is usually attributed to inhibition of the activity of infiltrating immune cells.we examined whether vitamin d also interferes with the pro-inflammatory action of the keratinocytes themselves. human hacat keratinocytes cultured in the absence of exogenous growth factors or active mediators were exposed to tnf to simulate an inflammatory challenge and their response was monitored by assessing mrna levels of the cytokine tnf, the chemokine il-8 and the adhesion molecule icam-1 by real-time pcr. icam1 and il-8 were induced rapidly peaking after 2h, their mrna levels increased again from 8h to reach a plateau between 16h to 24h after exposure to tnf, whereas tnf mrna levels increased steadily between 2h and 24h. 24h pretreatment with calcitriol, the hormonal form of vitamin d, inhibited induction of il-8 but did not affect that of icam-1 or tnf 2h following exposure while calcitriol markedly inhibited the induction of all 3 pro-inflammatory genes 16h after the tnf challenge.calcitriol inhibits the activation of jun kinase (jnk) and p38 by tnf. this action was mimicked by the posters inflamm. res., supplement 3 (2007) s431 jnk inhibitor sp600125 and the p38 inhibitor sb203580.the combination of the two inhibitors fully reproduced the time and gene dependent modulatory effect of calcitriol. we conclude that vitamin d attenuates the active contribution of keratinocytes to cutaneous inflammation and that this modulatory effect is explained by inhibition of the jnk and p38 cascades. (1), cm lotufo (2), p borelli (1), zs ferreira (2), rp markus (2), shp farsky (1) (1) department of clinical and toxicological analyses, school of pharmaceutical sciences, university of sâ¼o paulo, brazil (2) department of physiology, bioscience institute,university of sâ¼o paulo, braziil introduction: we showed that endogenous glucocorticoids (eg) control neutrophil mobilizations from the bone marrow and peripheral compartment by modulating the expression of l-selectin on segmented cells. aims: we evaluated the role of eg on endothelial cells (ec) and the molecular mechanisms responsible for hormonal actions in neutrophils and ec. methods: neutrophils were collected from blood, segmented leukocytes from femoral bone marrow and ec were cultured from testis vessels. cells were obtained from adrenalectomized (adx), ru 38486-treated, shamoperated, vehicle-treated and non-manipulated (nm) wistar rats. results: circulating neutrophils and segmented cells from ru 38486-treated rats presented elevated and decreased expressions of l-selectin vs cells from control animals, respectively. the effects were not dependent on alterations of l-selectin mrna levels. ec from adx animals presented more ability to adhere neutrophils from nm rats and enhanced mrna levels and membrane expressions of icam-1, vcam-1 and pecam-1. participation of the glucocorticoid cytosolic receptor(gcr) on these effects was shown by similar results in cells from ru 38486 treated rats. nfkappab translocation in neutrophils was equivalent in all groups of animals, but it was enhanced in ec from adx or ru 38486-treated rats. conclusions: data show the participation of the gcr on events involved in neutrophil mobilizations, but nfkappab transcription is only involved on ec. (1), y naito(2), t okuda (3), k mizushima (3), t okayama (3), i hirata (3), h tsuboi (3), t suzuki (3), o handa (1) background: despite the inhalation of co at high concentrations had been considered as a toxic gas, the inhalation of co at low concentration has recently been shown the cytoprotective and anti-inflammatory effect against various animal models. however, it is unclear whether the direct exposure of co to the intestinal inflamed mucosa is effective or not. in this study, we investigated the therapeutic efficacy of the rectal co administration for rat colitis model. acute colitis was induced with trinitrobenzene sulfonic acid (tnbs) in male wistar rats. co(200ppm-10ml) was intrarectally administrated twice a day after the induction of colitis. rats were sacrificed at 3 days after the administration of tnbs. the distal colon was removed, and the ulcer lesions were measured. thiobarbituric acid (tba)-reactive substances and tissueassociated myeloperoxidase (mpo) activity were measured in the colonic mucosa as indices of lipid peroxidation and neutrophil infiltration, respectively. moreover, we evaluated the expressions of cinc-1 mrna/protein and p-p38 mapk protein. the intracolonic administration of co ameliorated tnbs-induced colonic ulceration. the increases in tba-reactive substances and mpo activity after tnbs administration were both significantly inhibited by treatment with co. moreover, the rectal administration of co significantly inhibited the increased expression of cinc-1 mrna/protein and p-p38 mapk protein after the induction of tnbs-induced colitis. the rectal administration of co protected from the intestinal inflammation in rats. based on these data, the beneficial effects of co on the intestinal mucosal injury may be attributed to its anti-inflammatory properties. alessandra gambero (1), m marã³stica (1), m saad(2), j pedrazzoli jr (1) (1) sâ¼o francisco university medical school, brazil (2) state university of campinas, brazil recent studies have shown that adipocytes produce and secrete several bioactive molecules like adipocytokines. the adipose tissue can also present short and long-term changes during inflammation and infectious pathologies. in this study, the alterations of mesenteric and perinodal mesenteric adipose tissue during experimental colitis induced by repeated intracolonic tnbs instillations were evaluated. the adipocyte size was measured after collagenase digestion. the basal lipolysis (glycerol release) and adipocytokine production was monitored after short time culture of adipose tissue. the colitis animals showed higher mesenteric fat mass (1.02+-0.06 and 0.78+-0.09 % of body weight for colitis and control, respectively; p<0.05) in despite of the lower body weight. the mesenteric adipocytes from colitis animals presented reduced diameter (31.67+-0.28 and 35.69+-0.37 um for colitis and control, respectively; p<0.01), higher basal lipolysis (41.67+-6.89 and 18.77+-3.66 ug.mg-1 for colitis and control, respectively; p<0.05) and tnf-alpha production (8.64+-0.77 and 5.51+-0.86 ng.mg-1 for colitis and control, respectively; p<0.05). perinodal mesenteric adipocytes presented normal diameters, higher basal lipolysis (72.48+-15.38 and 25.96.77+-3.05 ug.mg-1 for colitis and control, respectively; p<0.05), increased tnfalpha (46.34+-5.82 and 15.71+-2.15 ng.ml-1 for colitis and control, respectively; p<0.01), leptin (400.41+-77.10 and 201.01+-52.01 pg.ml-1 for colitis and control, respectively; p<0.05) and adiponectin production (15029+-2340 and 5918+-498 ng.ml-1 for colitis and control, respectively; p<0.01). the mesenteric adipose tissue was modified during the experimental inflammation, but some alterations were site specific. perinodal adipose tissue retained the ability to produce anti-inflammatory and pro-inflammatory cytokines, while mesenteric adipose tissue only the pro-inflammatory one. this work was financially supported by fapesp. inflammatory bowel disease (ibd) is a group of chronic inflammatory disorders of the intestine. the role of the pro-inflammatory p38 mapk signalling cascade in the pathogenesis of ibd is highly controversial. we therefore aimed to investigate the role of p38 mapk in chronic dextran sodium sulfate (dss) induced colitis, an experimental model of ibd. chronic intestinal inflammation was induced by oral cyclic administration of 3 % dss in sjl mice. clinically, the dss treatment produced episodes of colitis manifested by diarrhoea, gross intestinal bleeding, marked loss of body weight, and shortening of the colon. at the molecular level, this was accompanied by an up-regulation of tnfa, il-1ã¢, il-6, il-17, kc, cox-2, igg heavy chain, and phospho-stat3 in the dss treated mice.the clinical and molecular features described above recapitulate findings reported in human ibd. in order to assess the role of p38 mapk, the activation of the p38 mapk signalling cascade was analysed by western blot analysis. the expression and phosphorylation levels of both p38 mapk and of mk2 and hsp27, two down-stream targets, were not increased in dss treated animals compared to controls. leo 15520, a potent inhibitor of p38 activity in vivo, was dosed as pretreatment and after completion of dss treatment. pretreatment had a deteriorating effect on all measured cytokines, whereas treatment after disease induction had no effect on any measured parameters. collectively, these results strongly suggest that the p38 kinase pathway only plays a minor role, if any, in the dss model. (sp) were gmcsf differentiated, dcs purified through gr1+ cell depletion, and spleen tcells isolated by pan tcell negative selection. spdcs or bmdcs were stimulated +/-100ng/ml lps. for mlr, balb/c tcells were added for 5 days. cells were incubated with sb203580 (4-(4-fluorophenyl)-2-(4-ethylsulfinylphenyl)-5-(4-pyridyl)1h-imidazole, sb) or ml3403 ((rs)-{4-[5-(4-fluorophenyl)-2-methylsulfanyl-3h-imidazol-4-yl]pyridine-2-yl}-(1 -phenylethyl)amine, ml) and washed prior to lps stimulation (bmdcs) or cell mixing (t cells). 3hthymidine incorporation was measured, cell viability by mtt assay, tnfa and il-12 production by elisa. mlr tcell proliferation inspdcs or bmdcs was inhibited by sb (ic50 spdc 0.06mm, bmdc 1.0mm) and ml (ic50 spdc 0.03mm, bmdc 0.03mm). preincubation with dcs had no effect, despite reduced lps stimulated il-12 and tnfa synthesis by sb (ic50 il-12 1.2mm, tnf 0.16mm) and ml (ic50 il-12 0.9mm, tnf 0.11mm). preliminary data shows that preincubation of t cells with sb and ml modifies the mlr response. p38 plays a role in the interaction of dcs and t cells in antigen recognition. however, pre-incubation of drugs with dcs was ineffective. the role of t cell p38 mapk in the mlr is under investigation. p38 inhibitors may possess disease modifying properties because of reduced tcell antigen reactivity to dc antigen presentation. (1), s luik(2), s laufer(2), m seed (3), v holan(4), s fiorucci (5) (1) synovo gmbh, tã¼bingen, germany (2) university of tã¼bingen, germany in vivo anti-inflammatory activity of certain p38 kinase inhibitors is limited by bioavailability. however, it is possible that they may be useful in the therapy of ibd should it be possible to mediate there uptake in and around bowel lesions. we reasoned that activity could be especially increased if drug physical properties were altered to allow preferential partition into macrophages and neutrophils (wbc) associated with lesions. we prepared prodrugs of p38 inhibitors and screened them using whole human blood, murine spleenocytes and peritoneal macrophage. pharmacologically inert macrocycle (azilide) conjugates were assessed for enhanced efficacy in murine collagen induced arthritis either therapeutically (after onset of signs) or prophylactically (2 d post boost) or in a dss or tnbs model of ibd in the mouse. in both types of models, the prodrugs achieved improved suppression of arthritis and inflammatory score in colon sections at tolerated doses with optimal activity at 15mmolkg-1d-1. we propose that the prodrugs increase efficacy via improved pharmacokinetics partly related to biased disposition of the prodrug toward immune cells. despite the potent anti-inflammatory and immunosuppressive properties of glucocorticoids its applying in the management of severe necrotizing pancreatitis is still controversial. the plasma levels of interleukins (il-8, il-18) and adhesion molecules (e-selectin and icam-1) were measured in 36 patients with necrotizing pancreatitis. the measurement was performed immediately after admission, at the 3, 7, and 14 day. all patients were divided on two groups: first group compiled 20 patients, in which dexamethasone (16 mg/day during 7-8 days) was applied in the complex management of acute pancreatitis, and control group -16 patients that did not receive corticosteroids. all patients received the initial therapy. the increased levels of il-6, il-8, il-18, icam-1, and eselectin were noted in both groups of patients at the time of admission. the gradually increase of all proinflammatory mediators plasma levels up to seventh day was noted in patients of the control group. its levels clear correlated with the severity of mods and spreading of necrotic processes confirmed by ct. starting from the third day the gradually decrease of mediators levels were noted in the patients of the first group. the incidence of contamination of necrotic foci had no difference in both groups of patients. the ability of glucocorticoids to inhibit expression of proinflammatory mediators due to the glucocorticoids-mediated repression of nf-kappa b pathway provide the pathogenetic substantiation for the applying of glucocorticoids in the complex management of necrotizing pancreatitis. the objective of this study was to examine whether t cell specific overexpression of the th2 transcription factor gata-3 can inhibit th1/th17 cell mediated experimental mbsa arthritis. mbsa-immunized wild type mice developed joint inflammation which gradually increased in time with a maximum at day 7. at day 1, t cell specific gata-3 tg mice did not show any difference in arthritis score compared to wild type mice. however, at day 7, wild type mice had developed severe joint inflammation having the maximum arthritis score. in contrast, gata-3 tg mice showed only mild joint inflammation, suggesting that t cell specific overexpression of gata-3 protects against development of severe joint inflammation. facs analysis revealed low levels of il-17 +/ifn-gammacells in wild type as well as in gata-3 tg mice at day 1. as expected, il-4 positive cells were higher in gata-3 tg mice compared with wild type mice. interestingly, at day 7, the percentage of il-17 +/ ifn-gamma-cells were markedly increased in wild type mice but not in gata-3 tg mice, suggesting prevention of th17 expansion under gata-3 overexpression in vivo. these data revealed that t cell specific overexpression of the th2 transcription factor gata-3 protects against progression of severe joint inflammation during mbsainduced arthritis. furthermore, il-17 +/ifn-gammacells play a critical role in the progression of joint inflammation in this model and gata-3 overexpression in t cells prevents expansion of the il-17 +/ifn-gamma-t cell subset. pingping jiang (1), pt sangild(2), t thymann (2), hh-y ngai (1), w-h sit (1), k-l chan (3), jm-f wan (1) ( necrotizing enterocolitis (nec) is a severe intestinal inflammatory disease for which the disease etiology and progression remain unclear. preterm delivery and enteral milk formula feeding are factors predisposing to nec. to understand the pathophysiology of nec, two-dimensional gel electrophoresis (2d page) proteomic approach was applied in studying changes in intestinal protein pattern in preterm piglets with spontaneous nec occurring in response to 2 days of parenteral feeding followed by 1 day of enteral formula feeding. the intestinal proteomes of pigs with clinical symptoms of nec (n = 3) were compared with corresponding pigs remained healthy (n = 4). syproruby staining was used and differently expressed proteins were identified by maldi-tof ms or maldi-tof/tof ms. the proteins with significantly different expression between nec and healthy pigs involve in energy metabolism (sorbitol dehydrogenase, mitochondrial aldehyde dehydrogenase 2 and chain a, medium-chain acyl-coa dehydrogenase with 3-thiaoctanoyl-coa etc.), inflammation (peptide-binding protein 74 (pbp74) and snail homolog 3), signal transduction proteins (thyroid hormone binding protein precursor, park7 protein and chain b, structure of the rho family gtp-binding protein cdc42 in complex with the multifunctional regulator rhogdi etc.) and anti-oxidation (manganese-containing superoxide dismutase(sod)). these data underscore the significant impact of intestinal proteomics in unraveling nec pathophysiology and biomarker discovery. blood are used to monitor the progression of inflammation. the aim of our study were to investigate systemic markers of disease in a rat model of lps induced pulmonary inflammation to provide a link between preclinical in vivo research and early clinical research. animals were exposed to bacterial lipopolysacharide (e.coli 026:b6) by inhalation. the lungs were lavaged 6 hours post provocation and the level of cell influx was determined. relevant mediators of acute pulmonary inflammation were analysed with standard elisa technology in bronchoalveolar lavage fluid and in blood. in addition, measurement of changes in body temperature were performed at different time-points post provocation in order to monitor the systemic inflammatory responses to the local pulmonary inflammation manifested as alteration in body-temperature. results showing the effects of lps challenge on local and systemic parameters will be presented and the possible link to lps responses in man discussed. pulmonary inflammation models are widely used in pharmacological research. however, provocations and treatments aimed directly at the lung are often invasive which limits the possibility to perform repeated administrations of test agents and compounds. also, results derived from bronchioalvelar (bal) fluid are subject to variability if the retrieval techniques are non standardized. here we describe a non-invasive standardized method for retrieval of bal fluid to be used in mice. we present the characterisation of these techniques using the inflammatory response to lps and propose that this non invasive method can be used to refine lps and other challenge models. the objectives were to evaluate the dynamic response after a single intra-tracheal administration of of 5mg (50ml/animal) of lps (p.aeruginosa) to c57bl/6j mice. control animals received a single dose of sterile 50ml 0.9% saline/animal. the mice were terminated 4, 24, 48, 72 and 96h after instillation using a non invasive and operator independent lavage technique. results showing the effects of single lps challenge on bal parameters, excised lung gas volume and lung weight will be presented showing reliable dynamic responses. these techniques open the possibility to run repeated treatments and chronic provocations and are not subject to variability from bal fluid retrieval. the human psoriasis xenograft scid mouse model is one of the most accepted and well characterized models for screening of novel anti-psoriatic compounds. the model has primarily been applied for testing novel treatment principles via systemic or intradermal administration routes. in order to evaluate the model for topical treatment, psoriatic keratome biopsies were grafted to immune-deficient scid mice. transplanted mice were treated with daivonex /dovobet (calcipotriol) and bms (betamethasone). the results show a strong antipsoriatic efficacy after treatment with bms (epidermal thickness reduced by 68%). treatment with daivonex / dovobet also showed an anti psoriatic effect with a 31% reduction in epidermal thickness. serum did not contain test compounds, indicating that the observed effect were not due to systemic exposure. the observed effects are in concordance with clinical results of treatment of psoriasis. it is concluded that the model is useful for testing topical treatments. we have demonstrated that adult rats offspring of dams submitted to protein restriction during early lactation, presented impaired acute immune responses probably related to an imbalance in glucocorticoids and insulin secretion (barja-fidalgo; inflamm res 52 (11): 2003) . here, we evaluated the innate immunity mediated by neutrophils and host defense against infection in adult rats offspring of dams fed with either a protein free diet (un-group) or 22% protein diet (c-group) during the first 10 days of lactation. un rats showed lower number of blood pmn, though no difference in bone-marrow neutrophils number was observed. blood neutrophils from un-group presented a significantly reduced phagocytic activity against opsonized zymosan, constitutively expressed inos and spontaneously produced o2-, no and tnf-alpha. in vivo treatment with lps, at non-lethal doses, significantly increases tnf-alpha and superoxide production by neutrophils, compared with controls. lps increased no production by neutrophils from both groups, inducing inos expression in control cells, but no further increase in inos expression in un rats. nucleare nf-kb is constitutively augmented in un rats. un animals presented a higher survival rate in a model of clp-induced severe sepsis. these results indicate that a metabolic programming induced during early lactation affects the innate immune responses in adult rats, which are unable to properly mount an inflammatory response, may predispose to chronic diseases in adult life. transgenic mice over-expressing vascular endothelial growth factor (vegf) in the epidermal basal layer under the human keratin 14 (k14) promoter have previously been reported to develop a psoriasis-like inflammatory condition in the skin. important hallmarks of psoriasis are epidermal hyperplasia in association with infiltration of t-cells in the dermis and epidermis and also increased dermal angiogenesis. the aim of this study was to describe the epidermal hyperplasia and the infiltration of the skin with t-cells in transgenic k14/vegf mice. we induced a cutaneous inflammation in the ear skin by repeated topical treatments with 12-o-tetradecanoylphorbol-13-acetate (tpa), in order to investigate the inflammatory response. the in vivo pharmacological effect of topical treatment with a number of reference compounds, including betamethasone-17-valerate, was also investigated. the ear thickness was significantly increased in transgenic animals compared to wild type animals following tpa-induction. the epidermal thickness measured in histological sections of biopsies from the ear skin was also significantly increased in transgenic animals. furthermore, increased dermal vascularisation was observed in the histological sections of the ear skin. a marked infiltration with cd3-positive cells was observed in both dermis and epidermis, and this was highly correlated with the increase in epidermal thickness. finally, topical treatment with betamethasone-17-valerate significantly reduced the ear swelling and epidermal thickness. we conclude that over-expression of vegf in the epidermal basal layer plays an important role in skin inflammation and for the development of important psoriatic hallmarks. the model may furthermore be used as an in vivo screening tool for novel anti-psoriatic compounds. background and aims: the diabetes-prone bb (bbdp) rat spontaneously develops insulin-dependent diabetes resembling type 1 diabetes (t1d) in man. the bbdp rat is t-cell lymphopenic with a profound lack of regulatory t cells. the recent thymic emigrants in bbdp rapidly undergo apoptosis unless rescued from apoptosis by tcr stimulation. the increase in apoptosis is due to a frameshift mutation in gimap5 which causes a severe truncation of the protein. the mutation is the strongest genetic factor for rat t1d. we aim to detect how gimap5 affects the lifespan of t cells. results: overexpression in c58 cells of both wt gimap5 and gimap5 with the bbdp mutation causes an increase in apoptosis -the latter with a very rapid onset. reduction of human gimap5 by rna-interference in jurkat cells did not affect the number of apoptotic cells. overexpression of human gimap5 causes apoptosis in jurkat cells and primary naã¯ve t cells but not in activated t cells. finally, gimap5-mrna is upregulated in in vitro activated human primary t-cells (detected by rt-pcr). conclusions: based on the phenotype of the bbdp, rat gimap5 was expected to be anti-apoptotic. however, we report here that overexpression of both mutated and wt gimap5 causes rapid death of the cells. this suggests that gimap5 is pro-apoptotic. the results with human wt gimap5 support this conclusion: recently, much focus has been on the cellular cd38/ cadpr signaling system during inflammatory processes. the cd38/cadpr system has been shown to be regulated by interferon, estrogen and the proinflammatory cytokine il-8. to our knowledge, the expression and function of the cd38/cadpr signaling system in the human detrusor muscle have not been described. cd38 protein expression in cultured (explant technique) human detrusor smooth muscle cells (hdsmc) was demonstrated by western blot (wb) and confocal microscopy (cm). cytosolic free ca2+ concentration ([ca2+] i) in hdsmc and isometric force in human detrusor strips were measured by spectrofluorometry and myograph technique, respectively. wb and cm showed that hdsmc expressed cell surface cd38 which could be upregulated by il-8 (20 ng/ml). in hdsmc briefly activated with il-8 (20 ng/ml) cadpr induced a rapid, transient dose-dependent increase in [ca2+]i. cyclic adpr-mediated ca2+ increase was greatly reduced in ca2+ free medium suggesting ca2+ entry as well as ca2+ release. cyclic adpr -elicited ca2+ increase was mimicked by 3-deaza-cadpr, and blocked by 8-bromo-cadpr, a cadpr antagonist, but not by nifedipine or verapamil. in the presence of il-8, cadpr caused concentration-dependent relaxations of detrusor muscle. we report for the first time that 1) hdsmc express cell surface cd38, 2) the expression and function of cd38 are augmented by il-8, 3) externally added cadpr elicited a rapid, il-8 -dependent, and 8-bromo-cadpr-inhibitable ca2+ mobilization, 4) cadpr induces relaxation of human detrusor muscle. the study indicates a role of cd38/cadpr in human urinary bladder inflammation. miao lin is a formulation of sen miao san and lingzhi that consists of cortex phellodendri, atractylodisa rhizoma, radix achyranthis bidentatae, and ganoderma lucidum. these ingredients are reported to have anti-inflammatory and analgesic effects. in this study, we have investigated the anti-arthritic property of miao lin in an animal model of arthritis induced by unilateral injection of freunds complete adjuvant (fca) into rat knees. contents of the miao lin capsules were dissolved in saline and administered to the rats daily by intraperitoneal or oral route for 7 days before induction of arthritis and 7 days after. extension angle, size and blood flow of the rat knee joints were measured to give indexes of algesia, oedema, and hyperaemia, respectively. assessments of the extent of cell infiltration, tissue proliferation, and erosions of cartilage and bone provided additional indexes of the arthritis condition. single unilateral injection of fca into rat knees produced significant oedema, algesia, hyperaemia, immune cell infiltration, synovial tissue proliferation, and erosions of cartilage and bone in the ipsilateral knees compared with the contralateral saline-injected knees. intra-peritoneal injection of miao lin (50 mg/kg/day) suppressed oedema, pain and hyperaemia in the inflamed knees, and oral administration (500 mg/kg/day) suppressed oedema and hyperaemia. histological examination showed that both routes of administrations of miao lin reduced immune cell infiltration and erosions of cartilage and bone, and intraperitoneally administered miao lin also attenuated synovial tissue proliferation. these findings suggest treatment with intra-peritoneal or oral miao lin could provide significant anti-arthritic effects. an extract of the anti-arthritic thermalife cream contains 13 trace elements. diffusion studies were undertaken to assess the permeability of human epidermis to the trace elements. non-penetrating trace elements were discarded from the test formula (t2), and compared with the original formula (t1) for in vitro anti-inflammatory efficacy (tnf-a secretion in lps-challenged human monocytes). methods: human epidermis was mounted in vertical franz type diffusion cells (stratum corneum facing up). t1 cream (n=4) or no cream (n=4) was applied to the donor compartment of diffusion cells, with pbs in the receptor compartment (3.0ml ; stirred continuously at 37 c). 240 min after administration the receptor fluid was analysed for presence of metal ions by icp-ms. a replication study used a different skin donor. subsequently, human monocyte cultures (10% fcs, 5% co2) were either stimulated with 500ng/ml lps (e.coli 0111:b4,) or not in the presence of 10% t1, 10% t2, or no treatment.24 hours after incubation, culture media were collected, centrifuged, and assayed (cytokine elisa). statistical analyses used a treat by lps anova (p < 0.05). results: zinc was the only trace element to penetrate the human epidermis significantly. both formulations strongly suppressed lps-induced tnf-a secretion. t2 with zinc only was more effective than t1 (treat:f2,12 = 57.13, p < 0.0001; lps:f1,12 = 245.47, p < 0.0001; treat by lps:f2,12 = 70.01, p < 0.0001). conclusions: anti-tnf efficacy from thermalife extracts was retained with zinc chloride as the only trace element. (1) (1) osprey pharmaceuticals limited, canada (2) probetex, inc., texas, usa the ccl2/ccr2 chemokine/receptor axis, infiltrating monocytes/macrophages (m/m), th1 cells and mast cells play a pathological role in tissue damage and fibrosis in kidney diseases. the eradication of the supernumerary activated leukocytes should curb the production of inflammatory mediators and modulate chemokine communications, thus ameliorating disease. a recombinant fusion protein comprised of the human ccl2 chemokine fused to a truncated form of the enzymatically active a1 domain of shiga toxin has been developed. the ccl2 portion binds specifically to ccr2-bearing leukocytes and enters the cells, where the sa1 portion inhibits protein synthesis. the compound was tested in a model of anti-thymocyte serum (ats)-induced mesangioproliferative glomerulonephritis. male rats were injected with ats on day 0 and treated intravenously with vehicle, 50 or 100mg/kg of the recombinant protein q2d from day 2 until day 8. urine and blood collections were made prior to ats injection and on days 5 and 9. animals were sacrificed on day 9. no treatment related effects on body weight or signs of clinical toxicity were observed. urine protein levels were decreased in treated animals. histopathological analyses of kidney sections revealed maximum reductions of 40, 36, 38, and 28% for glomerular lesions, m/m count, fibronectin and âµ-smooth muscle actin, respectively. the latter two proteins are markers for extracellular matrix synthesis and mesangial cell activation, respectively. these results indicate a significant renal-protective effect in this model of nephritis. further observations suggest that different chemokine-ligand toxins may be used in the treatment of diseases modulated by other chemokine/receptor axes. inflamm. res., supplement 3 (2007) posters immuno-depletion followed by fluorescence-activated cell sorting based on the cell surface expression of the sca-1 antigen. such isolated cells can subsequently be cultured and differentiate towards the osteogenic, adipogenic or chondrogenic linage in vitro. using this model we investigated the influence of the proinflammatory cytokines, tnfa or il-1b, on early osteogenesis in vitro. under osteogenic conditions, il-1b was found to inhibit cell proliferation in a dose dependent manner, whereas tnfa exhibited no effect. histochemical examination revealed the presence of either tnfa or il-1b to dramatically decreased mineralization in a dose dependent manner. q-pcr analysis indicated that in the presence of il-1b, despite increased expression of bone-specific alkaline phosphatase (akp2) mrna, levels of other osteogenesis markers (runx2, col1a and sp7) were decreased. in the presence of tnfa, levels of akp2, runx2 and sp7 were all decreased. our findings indicate that the influence of early mesenchymal progenitor cells on bone remodelling may be substantially altered in the presence of proinflammatory cytokines. coronary artery disease (cad) is characterized by enrichment of inflammatory cells in the vessel wall. we hypothesized that an altered transmigration and activation of monocytes may contribute to plaque build up. in vivo transmigration was studied by use of a skin blister model. blisters are raised by suction and cells are analysed the following morning (0h blister) and after additional ten hours of incubation with pbs or autologous serum, corresponding to intermediate and intense blister. monocytes were analysed by flow cytometry for the expression of cd11b, before and after in vitro fmlp stimulation. chemokines in serum and blister fluid was analysed in parallel. cd11b expression on resting monocytes harvested from 0h blister was lower in patients as compared to controls (p=0.001). lower expression of cd11b in patients was also observed in the intermediate and intense blisters after stimulation with fmlp (p=0.04 and p= 0.005, respectively). the number of transmigrated cells was similar in both groups and increased with the intensity of inflammation. serum concentration of mip-1âµ was higher among patients (p=0.01) and similar levels were seen in blister fluids. concentration of mcp-1 was similar in both serum and blister fluid. we demonstrate that monocytes from patients with cad have a reduced expression and ability to up-regulate the adhesion molecule cd11b at sites of inflammation. these differences may modulate events related to the transmigration process and indicate a changed activation pattern. to which extent this feature might contribute to monocyte entrapment at the atherosclerotic site needs further studies to be delineated. in inflammation, nitric oxide (no) is produced by inducible nitric oxide synthase (inos) induced by bacterial products and cytokines, and no acts as a regulatory and proinflammatory mediator. one of the anti-inflammatory mechanisms of glucocorticoids is the inhibition of no production. the aim of the present study was to investigate the mechanisms how glucocorticoids inhibit inos expression and no production. dexamethasone and a dissociated glucocorticoid ru24858 inhibited no production, and inos protein and mrna expression in murine j774 macrophages exposed to bacterial lipopolysaccharide (lps). in the presence of a glucocorticoid receptor (gr) antagonist mifepristone, the effects of dexamethasone and ru24858 on no production were reduced. the role of histone deacetylation in the glucocorticoid effect was studied by using three inhibitors of histone deacetylases (hdacs); non-selective trichostatin a and apicidin, and hdac1 selective mc1293. hdac inhibitors reversed the effects of dexamethasone and ru24858 on inos expression or no production. stably transfected a549/8 cells containing human inos promoter were used in promoter-activity studies. cytokine-induced inos promoter activity was inhibited by dexamethasone and the inhibitory effect was reversed by trichostatin a. these results suggest that glucocorticoids inhibit inos expression and no production by a gr-mediated and gre-independent manner possibly through histone deacetylation and transcriptional silencing. we are investigating mechanisms involved in tnfainduced hyperalgesia in the mouse paw. previously, we have seen that tnfa causes a trpv1-dependent bilateral hyperalgesia. here we investigate the role of cox in this process. female cd1 mice (25-30g) were given intraplantar injections (i.pl.) of tnfa (10pmol/50microl) and tyrode (as vehicle, contralateral paw; 50microl). thermal hyperalgesic thresholds were measured using the hargreaves technique before and 1h after injection. indomethacin (20mg/kg) was co-injected with tnfa whilst contralateral paw was injected with tyrode and corresponding amounts of indomethacin vehicle (5% nahco3). another group of mice were injected with tnfa i.pl. plus 5% nahco3 with the contralateral paw injected with tyrode plus indomethacin (20mg/kg). results are expressed as mean ae s.e.m and statistical analysis performed using students t-test. tnfa (10pmol) leads to significantly reduced (p<0.05 compared to baseline values) paw withdrawal latency in both paws 1h after injection i.e bilateral hyperalgesia. however, local injection of indomethacin (20mg/kg) with tnfa prevented this reduction in paw withdrawal latency in both paws suggesting that prostaglandins are important in the development of hyperalgesia. interestingly, indomethacin co-injected with tyrode in the contralateral paw did not prevent the reduction in paw withdrawal latency in both paws. the same results were seen using the selective cox-2 inhibitor, nimesulide. in conclusion, cox-2 derived prostaglandins are important in the development of hyperalgesia. local cox-2 inhibition at the site of tnfa-induced inflammation prevents the bilateral hyperalgesia suggesting that local prostaglandin production is sufficient to cause hyperalgesia in the contralateral paw. hydrogen sulfide (h2s) is synthesized naturally in the body from cysteine by cystathionine g lyase (cse). h2s has been variously reported to exhibit both pro-and antiinflammatory activity. in an attempt to obtain further information about the role of h2s in inflammation we examined the effect of dexamethasone on lipopolysaccharide (lps)-mediated endotoxic shock. male sprague dawley rats (240-280g) were administered dexamathasone (1 mg/kg, i.p.) either 1 h before or 1 h after lps (4 mg/kg, i.p.) injection. animals were killed 3 h after lps administration and plasma and tissues harvested. as expected, lps injection significantly increased plasma tnfa and il-1b as well as liver and lung myeloperoxidase (mpo) activity. lps also increased plasma nitrate/ nitrite (nox), h2s concentration and liver and kidney h2s synthesis from exogenous cysteine indicative of upregulation of cse in these tissues. either pre-or post treatment of animals with dexamethasone reduced signs of inflammation and also reduced the increase in plasma h2s and tissue h2s synthesizing activity. in separate in vitro experiments, exposure of rat peripheral leucocytes to lps (100 ng/ml, 3 h, 37oc) resulted in upregulation of both cse and inos (measured by western blot). dexamethasone (100 nm) significantly (p<0.05) reduced expression of both cse and inos. these data provide further evidence that h2s is synthesised during endotoxic shock and suggest, for the first time, that at least part of the anti-inflammatory effect of dexamethsone may be related to inhibition of h2s production. (1), u jalonen (1), h kankaanranta (1), r tuominen(2), e moilanen (1) (1) the immunopharmacology research group, medical school, university of tampere and tampere university hospital, tampere, finland (2) the division of pharmacology and toxicology, faculty of pharmacy, university of helsinki, helsinki, finland tristetraprolin (ttp), also known as nup475, tis11, g0s24 and zfp36, is a factor that binds to 3utr of mrna of some transiently expressed inflammatory genes and regulates mrna stability. ttp has been implicated in the posttranscriptional regulation of e.g. tumor necrosis factor a and inducible nitric oxide synthase. however, the regulation of the expression of ttp itself is largely unknown. in the present study, we investigated the role of classical protein kinase c (cpkc) isoenzymes in the regulation of ttp expression. in j774 macrophages ttp expression is induced by lipopolysaccharide (lps) and this can be further enhanced by addition of 100 nm phorbol myristate acetate (pma). this additive effect of pma on ttp was abolished by a prolonged preincubation with a higher s442 inflamm. res., supplement 3 (2007) posters concentration of pma for 24 h, which also downregulated the expression of pkca, pkcbi and pkcbii isoenzymes. pkc inhibitors ro318220 (inhibits pkcb, & and e), gã�6976 (inhibits pkca, b and &) and cgp53353 (inhibits pkcbii) reduced lps + pma -induced ttp protein and ttp mrna expression. pkcbii inhibitor cgp53353 did not affect ttp mrna half-life and therefore we measured the effects of cgp53353 on the activation of transcription factors involved in ttp expression. cgp53353 had no effect on the activation of nf-kb, egr1 or sp1. in contrast, cgp53353 reduced the activation of transcription factor ap-2, which may explain its inhibitory action on ttp expression. the results suggest that pkcbii is involved in the regulation of ttp expression in activated macrophages, possibly through the activation of transcription factor ap-2. the most widespread gracilaria verrucosa in the sea of korea is the attached form of red algae growing on a rockly substrate. in this study, we isolated fourteen compounds from g. verrucosa and investigated their inhibitory effect on the production of inflammatory markers (tnf-a il-6, il-1 and no) in raw264.7 cells. among them, 10-oxooctadec-8-enoic acid and 11-oxooctadec-9-enoic acid inhibited the production of tnf-a, il-6, il-1 and no at the concentration of 20 mg. also, these two compounds showed inhibitory activity on the mrna expression and protein level of inflammatory markers (tnf-a il-6, il-1 and inos) in a dose-dependent manner. these results suggest that g. verrucosa may have anti-inflammatory activity through the inhibition of inflammatory cytokines and inos. lene jensen(1), p hjarnaa (1), j fensholdt (1), p-p elena(2), k abell (1), tk petersen (1) (1) discovery, leo pharma, ballerup, denmark (2) iris pharma, la gaude, france angiogenesis is known to play an important role in many inflammatory diseases including arthritis. additionally, inflammation is known to play a role in the angiogenesisdriven disease age-related macular degeneration (amd). we have synthesized a potent angiogenesis inhibitor, leo-a, targeting kinases related to angiogenesis, e.g. vegfr-2. additionally, leo-a has potent effects on a broad panel of other kinases, whose normal functions are related to inflammation and immunity. the compound was tested systemically in inflammatory in vivo models in mice and rats. the in vivo models selected include the cia arthritis model (mice and rats), the local gvh rat model, the lps induced tnfa model (mice and rats), the anti-cd3 induced il-2 response mouse model and the rat argon laser-induced choroidal neovascularisation (chnv) model, a model for amd. the following results were obtained after systemic treatment with doses of up to 30 mg/kg i.p. or 50 mg/kg p.o. once daily: in the local gvh model, leo-a significantly inhibited the growth of the local lymph node by 50 %. in the cia model, leo-a had a significant inhibitory effect on the progress of arthritis both in mice and in rats when dosed early (pretreatment). in the lps induced tnfa model in mice, high doses of leo-a were found to inhibit the tnfa release. in the chnv model, a significant effect was obtained following systemic treatment. in conclusion, leo-a has an interesting profile for the treatment of diseases in which inflammation and angiogenesis are involved. mice lacking pi3kg and d isoforms display severe impairment of thymocyte development, but the outcome of this developmental defect has not been investigated. we show here that mice harbor pi3kg gene depletion and pi3kd kinase-inactive mutation, pik3cgd koi, exhibited thymus atrophy, similar to previously reported pi3kg and d double knockout (p110g/d-/-) mice, and profound peripheral lymphoid depletion, markedly reduced lamda chain production and seemingly lymphopenia-provoked effector/memory t cell activity. in particular, serum igg1/ igg2a ratio and ige level were elevated in pik3cgd koi mice corresponding to a skewed th2 profile in vitro. histological analysis revealed eosionophil-and t celldominated inflammation in stomach and salivary gland as well as occasionally other organs of pik3cgd koi mice, but organ-specific auto-antibody was not detected in circulation. on the contrary, when mature wt t cells were treated with pi3k d or together with pi3k g selective inhibitors, while th1 cytokines were suppressed th2 cytokines were not augmented in vitro. thus, t cell development, but not peripheral t cell proliferation or cytokine production, requires cooperativity of pi3kg and d. genetic inactivation of these two isoforms leads to the development of severe lymphopenia, skewed type 2 ig and t cell response, and increased susceptibility to eosinophilic multiple organ inflammation; whereas pharmacological inhibition at the adult stage would probably not promote th2 reaction but attenuate th1 medicated disorders. platelet activating factor (paf) is an important mediator in several pathophysiological processes. paf receptor activation can causes a series of cellular and tissue modifications and can lead to the production and/or release of diverse molecules, including cytokines, chemokines and receptors, amongst others, which are capable of amplifying the inflammation. paf can up-regulate kinin b1 receptor expression by various mechanisms. our aim was to investigate the role for kinases in paf-induced kinin b1 receptor up-regulation. wistar rats were treated with paf, or left untreated as controls, 6h before i.d. injection of 0.1ml pbs containing des-arg9-bradykinin (dapk, 100nmol right hind paw) and 0.1ml pbs (for control, left paw). various kinase inhibitors were administered to the rats after paf treatment and oedema was measured by the use of a plethysmometer (ugo basile) 10-120 minutes after dapk-injection. oedema was expressed in ml as difference between right and left paws.additionally paw samples were taken for western blot analysis for total and phosphorylated forms of jnk and erk1/2. dabk-induced paw oedema after pafinjection is significantly inhibited by the selective jnk sp600125 and erk1/2 pd98059 inhibitors. western blot analysis shows that phosphorylation of jnk and erk1/2 is important in the up-regulation of b1 receptors. our results clearly show that the phosphorylation of both erk1/2 and jnk mapkinases is an important step for the in vivo up-regulation of b1 receptors by paf. however, the exact mechanisms (transcriptional and post-transcriptional) by which paf can trigger kinase phosphorylation and then up-regulate the b1 receptor require further investigation. continued interest in development of small molecule inhibitors of p38 mitogen-activated protein (map) kinase is based on the central role this enzyme plays in inflammatory cell signaling. activation of p38 leads to increase production of pro-inflammatory cytokines such as tnf-a and il-1b making it an prominent target for antiinflammatory drug discovery. a virtuell screening approach identified 3-(2-chlorophenyl)-6-((4-methoxyphenoxy)methyl)[1, 2, 4] triazolo [3,4-b] [1, 3, 4] thi adiazole as a potential hit. this was confirmed by synthesis and testing. to explore further sar, a first set of derivates was prepared by cyclization of the 5-substituted-4-amino-3-mercapto-4h-1,2,4-triazoles with carboxylic acids in presence of phosphorus oxychloride. the synthetic strategy used allows both variation at position 3 and 6. synthesis and sar will be presented. cytokines like il-1b and tnfa play central roles in inflammatory diseases like rheumatoid arthritis. production of cytokines in monocytes, macrophages and other cells is triggered by factors such as lps, uv-light, osmotic and cellular stress or physical and chemical attraction. in particular il-1b and tnfa are key regulators as they amplify inflammatory stimuli in cells by induction and upregulation of further cytokines. involved in this signal pathway, p38mapk as a pivotal enzyme is considered to be a validated drug target and therefore, p38mapkinhibitors are of therapeutical interest. in this study, we developed and validated an economic in vivo whole blood assay for optimization and characterization of small molecule p38mapk-inhibitors with promising in vitro activity. the assay procedure involves defined blood cell stimulation by lps and isolation of tnfa or il-1b, which are subsequently quantified by tmb-elisa technique via photometric measurement. the validation of the assay conditions involved well characterized p38mapk inhibitor sb203580 and a highly active compound developed in our lab. a data set was generated by determining 18 whole blood samples consisting of in each case three male and female individuals on three different days. statistical methods were used to analyze specificity, baseline-peak correlations, repeatability, robustness as well as gender specific intra-and interindividual differences. p38 mitogen-activated protein (map) kinase is required for the biosynthesis and release of pro-inflammatory cytokines il-1 and tnf a. inhibition of p38 map kinase could reduce the expression of these cytokines and is therefore a promising target for the treatment of many inflammatory disorders, like rheumatoid arthritis and inflammatory bowel disease. trisubstituted pyridinylimidazoles are potent inhibitors of the p38 map kinase. scope of this work was to investigate 2-thio-ether moiety as a position to link the inhibitors to macrocyclic drug carriers. we synthesised 2-alkylsulfanyl, 4-(4-fluorophenyl), 5-(2-aminopyridin-4-yl) substituted imidazoles as p38 map kinase inhibitors. as substitution at this pyridinyl moiety allows both increase the anti-inflammatory activity as well as selectivity. the synthesis and biological testing of effective the 2-aminopyridin-4-yl imidazoles with low inhibitory concentrations are described. biological data demonstrate both the imidazole derviates and the linked imidazoles lead to highly efficient inhibitors.variation at the 2-thio-ether moietywhich interacts in the phosphate binding region of the enzyme -with polar groups shows no loss of activity. studies underscored the importance of hydrogen bonding with the backbone nh group of met109, for inhibitory activity. less clear is the importance of the hydrogen bond between n3 of the imidazole ring and lys53 of p38 map kinase.to investigate the role of lys53 in interacting with the scaffold we prepared two sets of 4,5diaryl-substituted isoxazoles. these data suggest a dynamic interaction of the core heterocycle with lys53, contrary to the observation on the compound vk-19911 and p38 map kinase, that a nitrogen atom bearing a lone pair in position 3 of the imidazole ring could be necessary to avoid a repulsive interaction with the positively charged side chain of lys53 rather than to form an attractive interaction with p38 map kinase. to complete our study, we focused on the interdependency of biological effects exerted by substitution at the pyridine ring for a series of 3-substituted and unsubstituted 4,5-diarylisoxazoles investigating the interaction with the hydrophobic pocket ii of p38. these data indicate that the isoxazole has better scaffold properties compared with imidazoles, suggesting that heterocycles that are stable as regioisomers, such as isoxazole (in contrast to tautomeric imidazoles), are worthy of further investigations. despite of the intensive research effort, sepsis is still the leading cause of death in critically ill patients. it is a consequence of acute inflammatory response to lipopolysaccharide (lps), a major component of the outer membrane of gram-negative bacteria. natural products are known sources of bioactive components exerting antioxidative and anti-inflammatory effects. in this study, we investigated the effect of ferulaldehyde (fa), a natural compound of red wine, on lps-induced endotoxic shock in mice and on lps-stimulated murine macrophage-like raw 264.7 cells. treatment of c57bl/6 mice with fa significantly attenuated the lps-induced inflammatory response in the gastrointestinal tract, and decreased the level of the two major pro-inflammatory cytokines tnf-a and il-1b in the serum. the serum level of the anti-inflammatory cytokine il-10 was higher in mice treated with fa and lps compared to lps treatment alone. lps-induced phosphorylation and thereby activation of akt, and jnk was also strongly inhibited by fa treatment whereas the phosphorylation level of erk1/ 2 and p38 mapks remained unaltered. activation of nuclear factor-kappab (nf-kb) in liver of fa-treated mice were significantly suppressed. although fa had no effect on the production of inflammatory cytokines, or on inhibition of signal transduction pathways in raw 264.7 cells either, it decreased the lps-induced ros and nitrite production in a dose-dependent manner. our results suggest that fa has antioxidative and anti-inflammatory activities by enhancing antioxidative defense systems, which in turn decrease inflammatory cytokine response and suppress nf-kb activity via the down-regulation of akt and jnk. myeloperoxidase (mpo), stored in the azurophilic granules of the neutrophil granulocyte, is a heme enzyme with the unique property of oxidising chloride ion to the powerful reactant hypochlorous acid in the presence of hydrogen peroxide. therefore, it plays an important role at inflammatory loci in killing invading micro-organisms. on the other hand, hypochlorous acid reacts with a variety of biomolecules as amino acids or membrane lipids and causes therefore host tissue damage resulting in widespread diseases like atherosclerosis or rheumatoid arthritis, e.g. the formation of chloramines from taurine or ammonium ions is one possibility to reduce tissue toxicity while maintaining bactericidal properties. membrane charge alterations during apoptosis provide docking sites for the kationic enzyme myeloperoxidase and this close contact to the membrane lipids opens the possibility for lipid alteration pathways even though these reactions will normally not take place because of their slowness. we investigated alterations in phospholipids after reaction with hypochlorous acid or the myeloperoxidase-hydrogen peroxide-chloride system by matrixassisted laser desorption and ionisation time-of-flight mass spectrometry (maldi tof ms). specific reaction products play an important role in the modulation of the immune response. comparative pathobiology of the disease is also discussed within the context of current human and animal reoviral disease models. objectives: to study the safety and efficacy of infliximab plus leflunomide combination therapy in adult rheumatoid arthritis (ra). methods: twenty patients with active ra received leflunomide 100 mg for 3 days followed by 20 mg daily for 32 weeks. at week 2 all patients started infliximab 3 mg/kg, and received a further four infusions at weeks 4, 8, 16 and 24. results: the commonest adverse event was pruritis associated with an eczematous rash. there was no relationship between the serum concentration of a77 1726, the active metabolite of leflunomide, and adverse events. the mean disease activity score (das28) fell from 7.18 at week 0 to 5.18 (p<0.0001) at week 4 and remained between 3.85 and 4.85 up to week 32. in those patients remaining on treatment, more than 80% achieved an acr20 response from week 8 to week 28, and up to 46% achieved an acr70 response. conclusions: infliximab plus leflunomide combination therapy appears to be highly efficacious in the treatment of adult ra. however, widespread use may be limited by adverse events, which were common and in some cases severe. objective: the transcription factor nuclear factor-kb (nfkb) regulates the expression of proinflammatory cytokines such as tnfa and il-1 those play pivotal roles in pathogenesis in rheumatoid arthritis. parthenolide, a sesquiterpene lactone, was reported to inhibit the dna-binding of nfkb. the objective of this study is to investigate the potential of parathenolid to inhibit the pathogenesis of collagen-induced arthritis. methods: mice were injected i.p. with a cocktail of 4 anticollagentype ii mabs on day 0, followed by i.p. injection of lps on day 3 to induce anti-collagen mab-induced arthritis. the mice were orally administrated with parathenolide (50 mg/kg/day) starting on the day of first immunization (day 0) in prophylactic treatment group and after the onset of arthritis (day 4) in the therapeutic treatment group. clinical disease score, radiographic and histological scores were evaluated. mrna expression of il-1b and tnfa in the affected joints were measured by real-time pcr. results: clinical disease scores were significantly reduced both in prophylactic treatment group (7.4ae3.7) and therapeutic treatment group (7.25ae3.1) compared to untreated group (13.6ae2.7, p = 0.0163 and p = 0.0084 respectively). histological scores of joint destruction were significantly reduced in prophylactic treatment group compared to untreated mice (p<0.05). steady state mrna levels of il-1b and tnfa in isolated joints were significantly decreased in prophylactic treatment group compared to untreated mice (p<0.05). the results in this study suggest that nfkb is an important therapeutic molecular target for treatment of inflammatory arthritis. fibrinogen is a soluble plasma glycoprotein, multifunctional, that participates in haemostasis and has adhesive and inflammatory functions through specific interactions with other cells. the concentration of this glycoprotein increase in inflammatory conditions. a fundamental paradigm involved in the acute inflammatory response is neutrophil migration to the affected tissues to mount an initial innate response to the aggression. the objective of this study is to characterize how fibrinogen modulates the pattern of neutrophil activation. neutrophils from healthy donors were isolated from peripheral venous blood and loaded with the fluorescent probe dihydrorhodamine 123 (1ã¬m) to detect oxygen free radical production. the cells (1,0x 106 cell/ml) were then incubated with a range of concentrations of fibrinogen (0-400mg/dl) for 15 minutes. our results show that posters inflamm. res., supplement 3 (2007) s449 fibrinogen leads to an increase in neutrophil activation as measured by free radical production. this effect becomes evident at borderline-high concentrations (300-400mg/ dl), and in some of the individuals it was possible to differentiate two subpopulations of low-responsive and high responsive neutrophils to activation by fibrinogen. we hypothesize that, in this regard, the concentrations of fibrinogen identified as a risk factor might promote the setting of an inflammatory microenvironment in the circulation and facilitate cardiovascular disease progression. cyclooxygenases (cox-1 and cox-2) are isoenzymes involved in the first steps of the biosynthesis of prostanoids. the constitutively expressed isoform cox-1 is mainly involved in homeostatic processes, while the inducible isoform (cox-2) is associated with inflammatory reactions. various in vitro assays have been developed in order to define the selectivity against cox-1 and cox-2 of nonsteroidal anti-inflammatory drugs (nsaids). however, these in vitro assays can give discordant results related to several parameters. the aim of this study was to optimize and standardize two distinct in vitro methodologies to evaluate new nsaid candidates. first, in an enzymatic cox assay, the arachidonic acid concentration (aa; cox substrate), and the species of cox enzymes tested (ovine vs. human), two factors able to conceal the anti-cox activity of nsaids, have been evaluated and optimized. next, we developed an in vitro cell-based assay using human whole blood depleted from plasma and reconstituted in saline solution. this cell-based assay allows concomitant measurement of anti-cox-1 and anti-cox-2 effects by prostaglandin e2 (pge2) measurement after a23187 (calcimycin) and bacterial lipopolysaccharide (lps) stimulations, respectively. both assays have been calibrated and compared by testing 7 reference nsaids, selective or not for cox-1 or cox-2. fifty % inhibitory concentration (ic50) values against cox-1 and cox-2 and cox-2:cox-1 ratios obtained were in accordance with the previously described nsaid specificity and coherent between both assays (r= 0.93). in conclusion, both in vitro assays are optimized to determine the efficiency and the selectivity of new nsaid candidates against human cox-1 and cox-2. for increasing the success of preclinical drug candidate molecules, there is a need for translatable animal models. the human serum skin chamber technique and the rodent carrageenan induced air pouch model are two wellestablished methods for measuring interstitial inflammation in respective species. we aimed to study the translational aspects of these models. material and methods: in humans, epidermal skin chambers were stimulated with autologous serum for 10 hours. in rats, a dorsal subcutaneous air pouch was stimulated with autologous serum on day 6. the inflammatory response was measured after 4, 8 and 24 hours. the cellular distribution of in vivo transmigrated cells, the expression of cytokine receptors, adhesion molecules and inflammatory mediators was investigated. results: at 8/10 hours the cellular distribution was similar in air pouch and skin chambers. the major population constituted of granulocytes, followed by monocytes/macrophages and lymphocytes. both in human and rats the concentrations of mpo and mcp-1 were increased. furthermore, transmigrated cells displayed a different chemokine receptor pattern. in rats transmigrated cells expressed cd11b, were cd45lo, ssclo and rp-1+ (granulocyte marker). in humans, transmigrated granulocytes expressed cd16 and cd11b. these cells had a significantly higher cd11b expression compared to corresponding cells in peripheral circulation. our results indicate that the serum induced human skin chamber technique and rodent air pouch model translate well to each other. these models may be useful for bridgingpreclinical and clinical drug discovery. furthermore, they may work as translatable proof of mechanism (pom) models for drug candidates targeting different inflammatory components. objectives: to analyse if neopterin (a by-product of activated macrophage metabolism) is elevated in patients with systemic inflammatory insult at the time of ischemic stroke. material and methods: we investigated 86 consecutive patients with mean age 67ae7.8 years who were admitted within 24 h after ischemic stroke. a control group of 37 patients with mean age 58ae4.9 years without ischemic stroke was also tested. measurement of serum neopterin levels were performed using enzyme linked immunosorbent assay. results: patients with acute ischemic stroke had significantly higher serum levels (mean value+sd) of neopterin than those without acute ischemic stroke: 9.6ae1.2 and 7ae0.8 nmol/l. correlation analysis revealed p<0.01. discussion: immune mechanisms contribute to cerebral ischemic injury. the finding of higher serum levels of neopterin, which is regarded as a humoral component of the immune-mediated inflammatory response sustains the hypothesis that patients with ischemic stroke may show higher levels of inflammatory markers like neopterin. our results indicate increased macrophage activation after ischemic stroke. in patients with stroke it has been shown that neopterin was determinant of endothelium-dependent vascular dysfunction. however, these preliminary results need be confirmed by controlled studies. produced a marked (p<0.01) reduction in the number and duration of ventricular tachycardia (vt) during both ischemic and reperfusion phases. the total number of ischemic ventricular ectopic beats (vebs) reduced from 667ffae116 in the control to 66ffae22 at the concentration of 50 ffmg/ml (p<0.001). in the ischemic phase, cynodon dactylon (50 ffmg/ml) also decreased the incidence of vt from 100% (control) to 33%. in addition, incidences of reperfusion-induced vt, total vf and reversible vf duration were significantly lowered by the same concentration (p<0.01 for all). the results show that cynodon dactylon has a protective effect against i/r-induced cardiac arrhythmias in isolated rat hearts. regarding the presence of flavonoid glycosides confirmed during phytochemical screening of the extract and their potential role in the scavenging of oxygen free radicals, it seems that the cardioprotective effects of cynodon dactylon probably is due to its anti-inflammatory properties.key words: cynodon dactylon; arrhythmias; anti-inflammatory; isolated heart; rat objectives: intestinal ischemia-reperfusion (iri) is well known to be associated with distant organ dysfunction; but no evidences to date have focused either the brain or skeletal muscle. we thus decided to investigate the effects of iri on nos and cox isoforms, neutrophil infiltration (mpo), lipoperoxidation (tbars) and protein tyrosine nitration (nt) in different brain areas and diaphragm muscle of wistar rats. methods: iri comprised the occlusion of superior mesenteric artery during 45 min followed by 2 h of reperfusion. sham animals were submitted to the surgical procedure with no interference on the blood flow. results: iri resulted in increased expression of mrna for nnos (cortex) and cox-2 (hypothalamus) associated to a marked reduction of ca2+-dependent nos activity in cortex, hypothalamus and hippocampus (but not in cerebellum). tbars contents were also reduced in cortex and hypothalamus. neither mpo activity nor nt was altered by iri in the brain. diaphragms from animals with iri exhibited increased mpo and ca2+-dependent nos activities, as well as tbars content and nt. in contrast, enos protein expression and both gene and protein nnos expression were reduced. no effects were observed on cox isoforms or enos gene expression. conclusions: these findings suggest that, within the first 2 h of reperfusion following intestinal ischemia, an oxidative response is observed in diaphragm, involving both lipid and protein modifications. in the cns, distinctive susceptibility to the iri seems to occur in the different areas, probably as a defensive strategy aimed to counteract the iri-mediated systemic injury. anne-sofie johansson (1), h qui (2), m wang (2), i vedin (1), jz haeggstrã§m (2), j palmblad (1) ( (1), r carnuccio(2), p romagnoli (3), f rossi (1) (1) second university of naples, italy (2) university of naples, italy (3) university of florence, italy we previously found that several inflammatory markers, e.g., nuclear factor-kb (nf-kb), were increased and a neointima was formed in a model of carotid surgical injury (1) . the purpose of the present study was to determine if chronic treatment with rosiglitazone protects rat carotid artery from surgical injury induced by an incision of the vascular wall. to this aim we measured cox-2, nf-kb, platelet aggregation and neointima formation in rats administered rosiglitazone (10 mg/kg/ die, by gavage) for 7 days before carotid injury and 21 days after injury. control rats received physiological solution. 14 days after injury cox-2 expression, evaluated by western blot, was significantly lower in the treated carotid versus controls (p<0.0001). rosiglitazone also caused a significant decrease of nf-kb/dna binding activity, evaluated by electrophoretic mobility shift assay, in nuclear extracts of treated carotids at all time points considered. platelet aggregation was reduced by 30% in treated versus control carotids (p<0.0005). the influx of inflammatory cells in response to injury, monitored by electron microscopy and immunohistochemistry, was lower in treated than in control carotids starting 7 days after rosiglitazone treatment. the results indicate that rosiglitazone inhibits molecular and cellular inflammatory events induced by vascular injury. the aim of the present study was to investigate the relevance of peripheral macrophage activity for the susceptibility to the induction of experimental allergic encephalomyelitis (eae). rats of eae-susceptible dark agouti and eae-resistant albino oxford strain were immunized with guinea pig spinal cord homogenate (dagpsc and aogpsc), while non-immunized rats served as controls (danim, aonim). on day 15 after immunization rat peritoneal macrophages were tested for adherence capacity, zymosan phagocytosis and respiratory burst. macrophages from aonim rats exhibited lower adherence capacity and higher phagocytosis and h2o2 production then macrophages from danim rats. immunization decreased adherence and phagocytosis and increased h2o2 production in macrophages from ao rats, but did not influence these activities in macrophages from da rats. our results suggest that inflammatory activities of macrophages from ao rats could be considered as regulatory mechanisms connected with the resistance to eae induction ( (1), b sehnert (2), h lanig(2), s pã¤ã�ler(2), r holmdahl (3), h burkhardt (1) (1) johann wolfgang goethe university, frankfurt, germany (2) friedrich-alexander university of erlangen-nuremberg, germany (3) lund university, sweden objectives: the aim of the present study was to characterize the interaction sites between the prototypic arthritogenic murine igg mab ciic1that is highly somatically mutated and its epitope on type ii collagen (cii, aa359-369). methods: the establishment of a dynamic simulation modelling of a ciic1 single-chain fragment (scfv) in complex with the triple helical ciic1 epitope permitted structural insights into immune complex formation. the computer-based data were experimentally tested by mutations of predicted critical residues into alanine in the c1scfvs and the respective ciic1 epitope that were produced as recombinant constructs. the binding affinities of the mutated scfvs were determined by elisa and surface plasmon resonance measurements. the mutation experiments confirmed the predicted interaction sites of cii in the cdr2 and cdr3 regions of both heavy andlight chain. surprinsingly also the model prediction, that the conversion of the c1scfv sequence into the respective germline does not affect cii binding affinity (kd 3x 10-8) could be confirmed experimentally by the mutagenesis of 13(!) positions. our data indicate that potentially harmful cartilage specific humoral autoimmunity is germline encoded. the molecular modeling further demonstrate that the rigid collagen triple helix restricts the likelihood of molecular interactions with the corresponding cdrregions of the antibody considerably compared to globular antigens. these sterical constraints might provide an explanation why somatic mutations have no obvious impact on cii recognition by the arthritogenic autoantibody. moreover, the structural insights into cii-autoantibody interaction might be useful in future developments of collagenomimetic ligands for therapeutic and diagnosistic purposes. we observed a significant association between the mbp-elicited cd4+ t-cell proliferation and active brain lesions, on the one hand, and il-4, il-5 and ifn-gamma, on the other. when grown in the presence of standard serum from a healthy donor, pbmc from healthy individuals responded to mbp with a higher il-10 production than pbmc from ms patients. thus, normal pbmc respond to mbp with production of tnf-alpha, ifn-gamma and il-10, but ms is associated with enhanced tnf-alpha-, ifn-gammaand decreased il-10 responses, and disease activity is associated with mbp-induced proliferation of cd4+ t cells. (1), k goula (1), p georgakopoulos (2) (1) renal unit, st. anrdew hospital, patras, greece (2) intensive care unit, st. anrdew hospital, patras, greece background: urethritis is an infection of the urethra. most cases are sexually transmitted. haemodialysedpeople seem more prone to all kinds of urinary tract infectionsthan others. patients with underlying diabetes are also a specific population at risk. urethritis may be caused by some sexually transmitted diseases (chlamydia, gonorrhea, and ureaplasma urealyticum infections) and by the same organisms that cause urinary tract infections (e. coli or klebsiella). viral causes of urethritis include herpes simplex virus and cytomegalovirus. neisseria gonorrhoeae and c trachomatis account for most cases of urethritis in men (70%). the aim of our study was to determine all cases of urethritis of haemodialysed patients at our unit during the last five years. we also determined diabetes as a coexisting factor in the infected patients. we retrospectively reviewed all cases of urethritis of 72 maintenance haemodialysis patients at our center over the past 5 years. the diagnosis was made according to patients symptoms and signs but also using urine specimens for culture.12 patients (16.6%) from the study group were diabetic. results: 4 cases of urethritis were determined. all infected patients were diabetic. isolated microorganisms were e. coli (2 cases), enterobacter aerogenes (1 case objectives: to explore the ability to use paquinimod as a steroid sparing drug in an animal model for sle. methods: mice were initially treated with a high dose of prednisolone (2 mg/kg/day). thereafter the amount of steroid was reduced to 0.5 mg/kg/day and a low dose of paquinimod (0.2 mg/kg/day) was added. the development of glomerulonephritis was measured as hematuria during the experimental period. serum was collected for analysis of anti-dsdna antibodies. kidneys were collected and histopathological observations were performed. organ weight and lymphocyte sub-populations were assessed in the spleen. results: when treatment with high dose prednisolone was replaced by low dose prednisolone andpaquinimod a steroid sparing effect was seen in a number of variables. a significant reduction in the level of hematuria, in spleen enlargement and in the total number of cd4+, cd8+ and on cd4-cd8-t cells was observed in mice treated with paquinimod and low dose of prednisolone compared to mice treated with high dose prednisolone alone. the development of glomerulonephritis was also significantly reduced in these mice. an almost complete inhibition of anti-dsdna in serum was seen in all treated groups. conclusions: when high dose prednisolone was replaced by low dose prednisolone and paquinimod a steroid sparing effect was seen when a number of variables e.g., hematuria, t-cell sub-populations and development of glomerulonephritis were examined. this setting could be of great importance in future treatment of human sle in order to reduce the steroid dose needed in the treatment of this disease. and la(ssb). the purpose of this study was to screen for novel antibodies against cell surface antigens in primary sjs. proteins (mp) were isolated from cell membranes (hela cells), and were tested with sera from sjs patients or healthy blood donors individually in western blot (wb) at 1:100. mp were separated on 2-d gels and tested in wb (1:100) to locate the appropriate spots for mass spectrometry (ms) analysis. paraformaldehyde fixed hela cells were incubated with sera from patients or blood donors and examined by fluorescence microscopy. antigens were isolated at around 35, 50, 75, 100 kda (64 total positive/79 tested patients). the dominant antigen was at 50 kda. large quantities of endogenous proteins were obtained and the membrane fraction was enriched. one of the main obstacles to further study possible surface antigens as m3 muscarinic receptor was overcome. proteins were separated on 2d-gels and tested in wb to locate the relative spots for ms. the correct localization of the patients antibodies on the cell surface was confirmed by fluorescence microscopy. in conclusion, membrane or membrane-associated antigens were recognised by sera from sjs patients. one of them might correspond to m3 muscarinic receptor. this identification might help in developing a diagnostic assay for sjs. osamu handa, s kokura, k mizuahima, s akagiri, t takagi, y naito, n yoshida, t yoshikawa aim: various additives and preservatives are used in cosmetics, foods and medicines in order to prevent deterioration. however the precise mechanism of cytotoxicity of these additives are not known. in this study, we investigated the effects of ultraviolet-b (uvb) exposure on additives-treated human normal skin keratinocytes (hacat). most popularly used additives in cosmetics such as methylparaben (mp), octandiol (od) and phenoxyethanol (pe) were used. hacat keratinocyte was cultured in mp-containing medium for 24 h, exposed to uvb and further cultured for another 24 h. subsequent cellular viability was evaluated by fluorescent microscopy and flow cytometry using double staining method with hoechst 33342 and propidium iodide or annexin-v. same experiments were done using od and pe respectively instead of mp under same condition. in addition, gene chip analysis was performed in each group. results: uvb exposure enhances cytotoxicity of these additives even at low concentration. gene chip analysis showed that the expression of apoptosis-related genes, oxidative stress-related genes and transcription related genes were significantly upregulated in each group. these results indicate that some additives, which have been considered safe preservatives in cosmetics, may have harmful effects on human skin when exposed to sunlight. these kinases in the pathogenesis of psoriasis. recently, increased focal activation of the downstream target mitogen-and stress-activated protein kinase 1 (msk1) was demonstrated in psoriatic epidermis. the purpose of this study was to investigate msk2 and the transcription factor camp-response-element-binding protein (creb) in psoriatic skin and in cultured normal human keratinocytes. keratome and punch biopsies were taken from patients with plaque-type psoriasis. normal human keratinocytes were cultured and stimulated by interleukin-1㢠(il-1ã�) or anisomycin. some of anti-inflammatory plant flavonoids as a form of whole plant extracts have been used topically for skin inflammatory disorders. on human skin inflammation, matrix metalloproteinase-1 (mmp-1) plays a pivotal role on unbalanced turn-over or rapid breakdown of collagen molecules. in the present study, for establishing a therapeutic potential against skin inflammatory disorders, the effects of natural flavonoids on mmp-1 activity and mmp-1 expression were examined. from the results, the flavonols including quercetin and kaempferol were revealed to be strong inhibitors of human recombinant mmp-1 with the ic50s of 39.6 -43.7 ã¬m, while the flavones such as apigenin and wogonin showed weak inhibition. when the effects of flavonoids on mmp-1 induction were studied, it was found that quercetin, kaempferol, apigenin and wogonin (12.5 -25.0 ã¬m) strongly inhibited mmp-1 induction from tpa-treated human dermal fibroblasts, but naringenin (flavanone) did not. by gel shift assay, these flavonoids were also found to inhibit the activation of the transcription factor, ap-1, whereas naringenin did not. among mapks, quercetin inhibited the extracellular signal-regulated protein kinase (erk) and p38 mapk activation, and kaempferol inhibited the p38 mapk and c-jun n-terminal kinase (jnk) activation. on the contrary, the flavones and naringenin did not inhibit the activation of these three mapks. all these results indicate that the capacity of mmp-1 inhibition and mmp-1 down-regulation of flavonoids may block collagen breakdown in certain pathological conditions and certain flavonoids are useful to treat skin inflammation, especially by topical application. (1) (1) university of valencia, spain (2) istituto di chimica biomolecolare cnr, napoli, italy avarol is a marine sesquiterpenoid hydroquinone with several pharmacological properties including antioxidant, anti-inflammatory, and antipsoriatic effects. recently, its derivative avarol-3-thiosalicylate (ta) also demonstrated interesting perspectives as anti-inflammatory drug in vitro and in vivo.it is interesting to note that avarol and ta inhibited nf-ã�b activation in hacat keratinocytes. now, the effect of avarol and ta was investigated in the tpa-induced hyperplasia murine skin model, which presents some similarities with psoriatic lesions. topical treatment with ta (20 mg/ml) produced a 97 % inhibition of oedema and a strong reduction of pge2 (100 %), ltb4 (100 %) and mpo activity (65 %) in skin homogenates. the inhibitory effect of avarol at the same dose was 79 % for oedema, 90 % for pge2, and 50 % for ltb4 and mpo activity. histological study for both compounds showed a decrease in epidermal hyperplasia as well as leukocyte infiltration respect to tpa treatment. besides, the reduction of cutaneous tnf-a by avarol and ta was also detected by immunohistochemical analysis. these compounds were also capable of suppressing nf-ã�b nuclear translocation in mouse skin. in summary, our results suggest that inhibition of proinflammatory metabolites by ta and avarol might be beneficial for the treatment of the inflammatory component of psoriasis. its mechanism of action is related to the inhibition of nf-ã�b activation and can be mediated by the downregulation of intracellular signal-transduction (1), ams silva(2), cmm santos(2), dcga pinto(2), jas cavaleiro(2), jlfc lima (1) (1) requimte, departamento de quã­mica-fã­sica, faculdade de farmâµcia da universidade do porto, porto, portugal (2) departamento de quã­mica, universidade de aveiro, aveiro, portugal 2-styrylchromones are a novel class of chromones, vinylogues of flavones (2-phenylchromones), which have recently been found in nature. several natural and synthetic chromones have demonstrated to possess biological effects of potential therapeutic applications. however, the anti-inflammatory potential of 2-styrylchromones has not been explored so far. thus, the aim of this work was to evaluate the putative anti-inflammatory properties of several synthetic 2-styrylchromones by studding their influence on different systems that are related to the inflammatory process. the putative inhibitory effects of several 2-styrylchromones on the proinflammatory enzymes cyclooxygenase 1 (cox-1), cyclooxygenase 2 (cox-2) and 5-lipoxygenase (5-lox) was evaluated in vitro and compared with structurally related flavonoids. the capacity of the studied 2-styrylchromones to scavenge reactive oxygen (ros) and nitrogen species (rns) was also assessed by different in vitro assays, which allowed to identify the influence of those compounds in each reactive species, separately. from the tested 2-styrylchromones, those having a catecholic bring where shown to be the most effective scavengers of ros and rns, being, in some cases, more active than flavonoids. no considerable correlation was found between the scavenging profile of these compounds and their interactions with pro-inflammatory enzymes. the results obtained from the present study indicate that some of the tested compounds are promising molecules with potential therapeutic value. the usefulness of 2-styrylchromones in the prevention or control of inflammation can only be clarified with additional studies concerning their influence on other relevant mechanisms of this pathology. the importance of tumor-associated inflammatory cells, able to affect different aspects of neoplastic tissue, is a current matter of debate. primarily monocytes are recruited from the circulation into solid tumors and metastases where they differentiate into macrophages with several phenotypes and, e.g., may significantly contribute to uptake of certain radiotracers. we therefore sought to characterize the uptake of various radiotracers used for positron emission tomography (pet) in a well characterized in vitro model of human monocytes/macrophages in comparison with that in various human tumor cells. uptake of radiotracers 18f-fluorodeoxyglucose (fdg), 3-o-methyl-6-18f-fluoro-l-dopa (omfd), and 18f-labeled native/oxidized low density lipoproteins (nldl, oxldl) in single-or cocultivated human myeloid (monocytic) leukemia cell line thp-1 was compared with that by squamous cell carcinoma (fadu), mamma (mcf-7) and colorectal adenocarcinoma (ht29) cell lines (without or in the presence of specific inhibitiors). several thp-1 phenotypes along the monocytic pathway (monocytes, differentiated macrophages, retrodifferentiated cells) were studied before, during and after incubation with phorbol myristate acetate. differentiated thp-1 cells show, when compared with tumor cells, a comparable fdg accumulation, a considerably lower omfd uptake, and a significantly higher oxldl uptake. on the other hand, during differentiation and retrodifferentiation thp-1 cells obviously establish a distinct sequence of biological processes also reflected by considerable alterations in radiotracer uptake. the observed differences in uptake of several radiotracers in vitro in-between thp-1 phenotypes and between thp-1 phenotypes and tumor cells, respectively, stimulate studies on the contribution of macrophage radiotracer uptake to the overall uptake in neoplastic or inflammatory lesions in vivo. genomic and full-length cdna sequences provide opportunities for understanding human gene expression. determination of the mrna start sites would be the first step in identifying the promoter region, which pivotally regulates transcription of the gene. although the mrna start sites of most genes show heterogeneity, this may reflect physiological, developmental, and pathological states of the particular cells or tissues. recently, we have developed a 5-end sage (5sage) that can be used to globally identify the transcriptional start sites and frequency of individual mrnas. a strong association exists between states of chronic inflammation and cancer, and it is believed that mediators of inflammation may be responsible for this phenomenon. another important factor in tumor development seems to be the epigenetic effects on tumor suppressor genes. because of its ability to suppress tumor cell proliferation, angiogenesis, and inflammation, the epigenetic drug such as histone deacetylase (hdac) inhibitor is currently in clinical trials. however, how epigenetic drugs mediate its effects is poorly understood. to assess the effects of epigenetic drugs, the gene expression by 5sage in colon cancer cell lines treated with epigenetically affecting agents, 5-aza-2deoxycytidine, a potent inhibitor of genomic and promoter-specific dna methylation and trichostatin a, a hdac inhibitor was investigated. epigenetic modification induced not only the change of expression of several inflammation-associated genes and the cell cycle progression-associated genes in human colon cancer cells but also the gene expression with aberrant start sites. colon cancer is one of the most frequently diagnosed cancers in western societies. interleukin-6 (il-6) is a potent, pleiotropic, inflammatory cytokine that contributes to a multitude of physiological and pathophysiological processes. il-6 is produced by many different cell types. the main sources in vivo are stimulated monocytes, fibroblasts, and endothelial cells. a variety of studies have demonstrated that over expression of il-6 contributes to the pathogenesis of various inflammatory diseases as well as cancer. it has been reported that human colorectal cancer cells display a wide heterogeneity in their potential to express and produce il-6. serum levels of il-6 are elevated in patients with colorectal cancer, however serum levels of il-6 were found to be independent of il-6 mrna expression in tumor tissue. in this study we analyzed il-6 mrna expression by real-time pcr in 50 sporadic colon cancer tissue as well as corresponding normal mucous tissue. il-6 mrna expression in tumor tissue was lower than in the corresponding normal mucous tissue (p=0,0074). there was no correlation betweenil-6 mrna expression and tumor grade or stage. thus we can conclude that il-6 produced at the tumor site is not involved in sporadic colon cancer progression. (1), t aiamsa-ard (1), v chinswangwatanakul (2), ki techatraisak (3), s chotewuttakorn (1), a thaworn (1) ( material and methods: huvec were cultured as standard techniques and grown to confluence until use. serum was obtained from cholangiocarcinoma patients and normal healthy subjects. huvec were treated with 10% of serum and incubated for 24 hours. cells were analyzed by using [3h] thymidine and immunoblotting assay for cell proliferation and cox-2/nos-2 protein expression, respectively. results: serum of cca patients trend to have more effect on proliferation of endothelial cell than healthy control subjects. on the protein expression, cca serum significantly increased the expression of cox-2 but not nos-2 in hevec. however, the proliferate effect on endothelial cells by cca sera did not correlate with the expression of cox-2. conclusions: this result suggested that some factors in serum of cancer patients could induce cox-2 protein expression in huvec. the increasing of cox-2 might be one of various factors involve in the proliferation process. aim: superoxide is responsible for the neutrophil-mediated tumoricidal activity. the aim of our work was to monitor the changes of superoxide production from neutrophil attributed to tumor development from the early phase to the advanced stage, and to investigate the effects of ok-432@on neutrophil-derived superoxide production and tumor growth. methods: ah109a rat hepatocellular carcinoma cells were implanted into the hind leg of male donryu rats. pmns were harvested from rat peritoneal cavity 6 h after intraperitoneal injection of oyster glycogen. superoxide production were measured by the method of cladependent chemiluminescence, which has high sensitivity and specificity to superoxide. the counts of peripheral leukocytes were significantly increased during tumor progression, and there are significant difference between that of controls and tumor-bearing rats after 18 days of tumor inoculation. both pma and oz-induced superoxide generation derived from neutrophils became significantly reduced in the advanced stage of cancer. the suppression of neutrophil-derived superoxide generation was accompanied with tumor progression and an increased number of neutrophils in the peripheral blood. the subcutaneous administration of ok-432, a biological response modifier, prevented the suppression of neutrophil-derived superoxide generation during tumor progression, which might induce the tendency of tumor growth suppression. our results suggested that the decreased superoxide generation as well as the high leukocytes concentration in the peripheral blood could be considered as indicators of an advanced stage of cancer. furthermore, the effect of ok-432 on neutrophil-derived superoxide production in cancer-bearing rats may provide pharmacological evidence to the therapeutic effects of ok-432. (1), m jokic (2), v zjacic-rotkvic(1), s kapitanovic (2) (1) university hospital sestre milosrdnice, bucharest, romania (2) division of molecular medicine rudjer boskovic institute, bucharest, romania introduction: il-6 is a pleiotropic cytokine mapped to chromosome 7p21-24. its promoter snp -174 g/c is associated with high serum cytokine production, and according to current investigation can play a role in the development and progression of different gastrointestinal malignancies. we tested its genotypes in the gastrointestinal and pancreatic neuroendocrine tumors (gep-nets). patients and methods: dnas from 80 patients diagnosed with gep-net and 162 age and sex-matched volunteers were analyzed for -174 g/c snp of the il-6 gene. to analyze il6 -174 c/g polymorphism we used pcr -nlaiii rflp method. for statistical analysis ã�2 test and fishers exact test were used. the level of significance was 0.05. results: there were no differences observed in the frequencies of the -174 high expression (gc and gg) genotypes between the patients and healthy volunteers (p=0.5518), as well as between patients with gastrointestinal or pancreatic endocrine tumors (p=0.3762). -174 g/c genotype was statistically more frequent among patients with non-functional pancreatic endocrine tumors (pets) than in those with functional pets (p=0.0246). conclusions: high expression genotypes of il-6 -174 snp are more frequent in non-functional pets and may be a marker for the mentioned malignancies. are important in inflammation, are found around and within a variety of human tumors. their number correlates with tumor vascularity and aggressiveness and is a negative indicator for patient survival. how mast cells influence tumor growth is not well understood. the neuroendocrine peptide, neurotensin (nt) is a potent secretagogue of mc that has tumor-promoting effects in animals and promotes the growth of a variety of human cancer cells, acting via its gpcr nt-type 1 receptor (nts1). here we show that hmc-1 human mc express nt-precursor (pront) mrna and protein, and secrete immunoreactive nt when stimulated. rt-pcr on hmc-1 cell rna yielded a band with 99% sequence identity to pront and a band corresponding to the pront processing enzyme, pc5a.immunocytochemistry on hmc-1 cells showed specific staining for pront. stimulation of hmc-1 cells with a23187 + pma, pge2, c48/80 or mastoparan released immunoreactive nt.rt-pcr on hmc-1 cell rna yielded a band with 99% sequence identity to human nts1. western blotting gave bands corresponding to unglycosylated (49 kda) and glycosylated (55 kda) nts1.immunocytotochemistry on hmc-1 cells showed specific staining for nts1. these finding have significance for the role of mast cells in tumor growth. (1), j buddenkotte (1), mp schã§n(2), m steinhoff (1) (1) university hospital mã¼nster, germany (2) university hospital wã¼rzburg, germany the proteinase-activated receptor par-2 has been demonstrated to modulate tumor growth, invasion and metastasis in various tissues. however, the role of par-2 in cutaneous cancerogenesis is still unknown. here we could show a protective role of par-2 in the development of epidermal skin tumors: we established a mouse skin tumor model using chemically induced carcinogenesis. to this end, par-2-deficient and wild-type mice were painted once with dmba (7,12-dimethylbenz[a]anthracene) for sensibilization, followed by topical application of the phorbol ester pma (phorbol myristate acetate (12-o-tetradecanoylphorbol-13-acetate)) twice per week at the same sites. tumors started to appear after eight weeks. after 13 weeks, par-2-deficient mice showed a significantly increased number of skin tumors (14 per animal on the average) in contrast to the wild-type (eight tumors per mouse). analysis of possible signal transduction pathways activated upon par-2 stimulation in hacat keratinocytes showed an involvement of extracellular signal regulated kinase 1/2 (erk1/2) and profound epidermal growth factor receptor (egfr) transactivation, leading to secretion of the tumor-suppressing factor transforming growth factor-beta 1 (tgf-ã¢1). thus, our results provide the first experimental evidence for a tumor-protective role of par-2. (1), ma arbã³s (1), a fraga (1), i de torres(2), j reventã³s (1), j morote (3) ( pathogenesis of benign prostatic hyperplasia (bph) and prostate cancer (pca) is still unresolved, although chronic inflammation may play a significant role in disease progression. prostate stromal fibroblasts may be contributing to the inflammatory process through the expression and secretion of pro-inflammatory mediators, in particular proteoglycan-bound chemokines and other chemoatractants, and the interaction with inflammatory cells such as monocytes. to better understand molecular mechanisms underlying functional differences among prostate fibroblast populations, our primary objective was to characterize proteoglycan and chemokine gene expression in human fibroblasts of different histological/ pathological origin cultured in normal and monocyteconditioned media. we analysed 20 primary human fibroblast cultures from normal transition zone (tz), normal peripheral zone (pz), benign prostatic hyperplasia (bph), and pathologically confirmed prostate cancer (ca). cells of different origin displayed distinct mrna expression profiles for the core proteins of proteoglycans and both sdf1/cxcr4 and mcp1/ccr2 chemokine axis. when incubated with monocyte-conditioned medium all four cell types significantly changed sdf1/cxcr4 and mcp1/ccr2 expression in a fibroblast population dependent manner. monocyte-fibroblast cell adhesion and the chemotactic response of fibroblasts to human peripheral blood monocytes were investigated in a coculture system. monocytes adhered rapidly to fibroblasts and preferentially to bph and pz cells. in addition, chemotaxis was significantly induced in both fibroblast cultures after incubation with monocytes. our results suggest that prostatic fibroblasts have a key inflammatory role associated to a distinctive proteoglycan gene expression and chemokine induction, which is dependent on their histological and pathological source. supported by the spanish urology society (madrid, spain). we have recently shown that paf-receptor is involved in phagocytosis of apoptotic and necrotic but not viable cells, possibly through its interaction with paf-like molecules present on the surface of these cells. removal of altered cells by macrophages could modify the microenvironment at an inflammatory site, and thus influence tumor growth. in the present study we investigated the impact of apoptotic cells or treatment with paf-r antagonist on ehrlich ascitic tumor (eat, ip) and melanoma b16f10 (sc). paf-r antagonist, web2170 (5mg/kg, ip) was given daily for 6 days. we found that eat growth was significantly reduced by pretreatment with web2170, and that inoculation of apoptotic cells (thymocytes) before tumor implantation stimulated tumor growth, an effect reversed by web2170 pretreatment. eat growth was accompanied by increased production of prostaglandin e2, vegf and no which was reduced significantly by web2170 treatment. in b16f10 melanoma, web2170, alone or in association with an apoptosis inducer chemotherapeutic agent, dacarbazin (dcb, 40 ug/kg,ip) significantly reduced tumor mass volume and the number of intratumoral small vessels. in association with dcb, web-2170 reducedactive caspase-3 expression in the tumor andmarkedly increased the survival of tumor-bearing mice. the data obtained here show that during tumor growth, activation of paf-r by molecules present in the surface of apoptotic/necrotic cells, or by paf produced in the milieu, favors tumor growth and suggests that pafantagonists could be useful in tumor treatment, particularly when in association with chemotherapy. financial suport by fapesp and cnpq. (1), mt quiles (1), a figueras(2), r mangues(2), f vinals(2), jr germa(2), g capella (2) (1) institut de recerca vall de hebron, barcelona, spain (2) translational research laboratory, idibell -institut cataladoncologia, spain the malignant potential of tumor cells may be influenced by the molecular nature of k-ras mutations. we have previously shown that codon 12 mutations associate with an increased resistance to apoptosis. we hypothesized that their different malignant potential in vivo could be also related to the generation of a distinct angiogenic and inflammatory profile including vascular structure, macrophage infiltration and expression of angiogenic modulators, proteolytic mediators and the cxcl12(sdf-1)/ cxcr4 chemokine axis. to do so we have combined in vitro and in vivo studies using stable cys12 and asp13 nih3t3 transfectants. cys12 tumors showed a higher microvessel density associated with shorter latency period. prominent vessels with âµ-smooth muscle actin positives cells surrounded by f4/80 macrophages were only observed in asp13 tumors associated with a shorter growth period. asp13 tumors displayed increased vegf expression both at the rna and protein levels, mainly produced by tumor cells. tsp-1 protein levels were similarly diminished in both transfectants. higher mmp9 and mmp7 activities and expression were observed in asp13 tumors probably produced by macrophages or stromal cells. total and active mmp2 levels were higher in cys12 tumors. the expression of sdf-1 and cxcr4 remained unchanged while sdf-1g isoform was selectively induced in cys12 tumors, suggesting sdf-1a or b are induced in asp13 tumors. these results show distinct k-ras mutations induce specific angiogenic phenotypes. the differential stimulation of vegf expression, metalloprotease activities and sdf-1 expression observed is the result of the joint action of tumor cells and the local microenvironment. contact information: dr maria a arbos via, institut de recerca vall de hebron, unitat de recerca biomedica, barcelona, spain e-mail: maarbos@ir.vhebron.net incisional hernias (ihs) represent a common complication of laparotomies, involving remarkable healthcare costs. representative ih animal models are lacking and characterization of human tissue resources is scant. this limited understanding of fundamental mechanisms regulating the destruction of the abdominal wall currently limits the prevention and treatment of ihs. here, we compared tissue specimens (carefully obtained > 5cm of the defect) and primary fibroblasts cultures from fascia and skeletal muscle of subjects with/without ih hernia. the most prominent morphologic characteristics of ih tissue were: alterations of the microstructure of the connective tissue and loss of extracellular matrix (ecm), and a paucity of fibroblasts. in ih muscles, inflammatory infiltrates were observed. other significant changes were: decreased collagen type i/iii ratio; differential proteoglycans mrna expression; enhanced metalloproteinases/ endogenous inhibitors ratio (mmps/timps); and upregulation of apoptosis effectors (caspase-3 and substrates; tnf-alpha; il-6). in vitro, hernia fibroblasts (ihfs) exhibited significantly higher (2-fold) cellular proliferation and migration rates and decreased strength of adhesion as compared to control fibroblasts, even after several passages. moreover, ihfs ultrastructure analysis revealed accumulation of autophagic vacuoles, autophagolysomes-like structures and multilayered lamellar and fingerprint profiles, as well as mitochondrial swelling. based on these descriptive results in human tissues, a novel hypothesis emerges regarding ih formation. specifically we propose that inflammation-related mechanisms triggering proteolytic and apoptotic effectors regulate cell turnover and eventually contribute to atrophy and progressive tissue insuffiency. overall, this may be causally involved in the mechanisms of ecm destruction yielding ih (supported by fis pi_ 030290 and gencat_agaur_ xt_0417). (1), m spinola(2), c pignatiello(2), w cabrera (1), og ribeiro (1), n starobinas(1), t dragani (2) (1) butantan institute, sao paulo, brazil (2) istituto nazionale tumori, milan, italy airmax and airmin mice are phenotypically selected for maximal or minimal subcutaneous acute inflammatory response, respectively, and display high inter-line differences in protein exudates and neutrophil infiltration, as well as in bone marrow granulopoiesis, inflammatory cytokines, and neutrophil apoptosis. in a combined experiment of urethane-induced lung inflammatory response and lung tumorigenesis, airmin mice developed a persistent subacute lung inflammation and a 40fold higher lung tumor multiplicity than airmax mice, which showed a transient lung inflammatory response. we have analyzed gene expression profiles of these outbred lines in comparison to the lung cancer resistant c57bl/6 and lung cancer susceptible a/j mouse strains. gene expression profile analysis of urethane-treated and untreated animals was performed using the applied biosystems mouse genome survey microarray containing 32,000 mouse transcripts. mrna expression of candidate differentially expressed genes was validated by quantitative real-time pcr and the over-represented biological themes were analyzed with the ease software. urethane treatment modulated the gene expression profile in all four lines. among the confirmed genes, vanin3 (vnn3) and major histocompatibility antigen e alpha (h2-ea) resulted common to both mouse models. the most represented gene categories in air model were acute phase response, immunoresponse, electron and lipid transport, complement activation and tissue repair. mhc/antigen process and presentation and immunoresponse were the major themes in the inbred model. moreover, a gene cluster in chromosome 3 (45.0 cm) was observed. the study suggests that the expression of a subset of genes may show a strain-and line-specific modulation pattern during inflammatory response and lung tumorigenesis. inhibition of tumour induced angiogenesis constitutes very attractive anti-cancer therapeutic approach.it is well established that the vegf signal transduction pathway is one of the key drivers of deregulated angiogenesis and selective inhibition can lead to inhibition of tumour growth. however, multiple angiogenic growth factors and pathways are involved, leading to a phenomenon of redundancy and overcoming of an inhibition of vegf signalling only. we have developed a nanomolar inhibitor (compound a) of the receptor tyrosine kinase vegfr-r2 (kdr), which was subsequently shown to be a potent inhibitor of closely related kinases (vegfr-1 and -3, pdgfr, kit, csf-1r) but also unrelated soluble tyrosine kinases (src-familily of kinases and raf). compound a potently inhibits vegf stimulated endothelial cell proliferation but has no effect on non-ec proliferation, which is suggestive of a selective antiangiogenic potential. the unique kinase inhibitory profile of compound a combined with excellent oral bioavailability (87%) has translated into superior in vivo antiinflamm. res., supplement 3 (2007) posters tumour efficacy when compared to the relatively selective kdr inhibitor ptk787. thus, treatment of nude mice implanted with either commercial atcc derived tumour cells (a431 and du-145) or low passage patient derived tumors (cxf 1103; colon cancer, rxf 631; renal cancer) with compound a resulted in inhibition of tumour growth which was significantly better than for ptk787 treated mice. compound a is fairly well tolerated by rodents and extended toxicological studies have been initiated to determine the therapeutic index, which also may allow for exploration of other non-cancer indications. (1), p bobrowski(2), m shukla (3), t haqqi (3) (1) albany medical college, usa (2) rainforest nutritionals, inc, usa (3) case western reserve university school of medicine, usa background: the amazonian medicinal plant sangre de grado (croton palanostigma) has traditional applications for wound healing and inflammation. we sought to characterize an extract (progrado) in terms of safety, proanthocyanidin profile, antioxidant activity and anabolic/catabolic actions in human cartilage explants. methods: acute oral safety and toxicity was tested in rats according under oecd protocol #420. proanthocyanidin oligomers were quantified by hplc and progrados antioxidant activity assessed by the orac, norac and horac assays. human cartilage explants, obtained from surgical specimens, were treated with il-1b (10 ng/ ml) to induce matrix degradation and glycosaminoglycans (gag) release. progrado (2-10 mg/ml) was tested for its ability to maintain optimal igf-1 transcription and translation in cartilage explants and cultured chondrocytes. results: progrado displayed no evidence of toxicity (2000 mg/kg po) leading to gsh safety rating of 5/unclassifiable. oligomeric proanthocyanidin content was high (158 mg/kg) with the majority of oligomers > 10mers.progrado was a remarkably potent antioxidant and in an ex vivo model of inflammation-induced cartilage breakdown, progrado was exceptionally effective in reducing both basal and il-1b induced glycosaminoglycan release from human cartilage explants. progrado prevented il-1b induced suppression of igf-1 production from human cartilage explants as well as stimulating basal igf-1 production (p<0.05). comparable changes in igf-1 gene expression were noted in cultured human chondrocytes. conclusions: progrado has a promising safety profile, significant chondroprotective and antioxidant actions, and promotes the production of the cartilage repair factor, igf-1. this suggests that progrado may offer therapeutic benefits in joint health, wound healing and inflammation. the solvent extracts from korean fermented soybean (chungkukjang) were evaluated for their protective effects against the generation of free radicals and lipid peroxidation. the activities of chungkukjang were compared with several antioxidants and soybean isoflavones including genistein and daidzein. in addition, the protective effects against h 2 o 2 -induced cytotoxicity and oxidative dna damage in the nih/3t3 fibroblasts line were examined. the extracts from chungkukjang and soy isoflavones inhibited the generation of 1,1-diphenyl-2picryl hydrazine (dpph) radicals, and had an inhibitory effect on ldl oxidation. the extracts from chungkukjang and soy isoflavones strongly inhibited h 2 o 2 -induced dna damage in the presence or absence of endonuclease iii and fpg. furthermore, they showed cytoprotective effects against h 2 o 2 , without cytotoxicity except for the hexane extract at high concentrations (> 450 mg/ml). the ethanol and n-butanol extracts appeared to have most potent antioxidant activities. these in vitro results show that the extracts of chungkukjang may be a useful antigenotoxic antioxidant by scavenging free radicals, inhibiting lipid peroxidation and protecting against oxidative dna damage without having cytotoxic effect. moreover, the extracts of chungkukjang inhibited mda formation in the liver, dna damage assessed by comet assay and the microucleated reticulocyte formation of peripheral blood in kbro 3 -treated mice. these in vivo results were similar to those of in vitro experiments. therefore, chungkukjang containing soy isoflavones is a promising functional food that can prevent oxidative stress. (supported by bk21 project from korea research foundation). sirt1 is a histone deacetylase, involved in oxidative stress and aging. because the role of aging and exercise on sirtuins activity in rats is unknown, we investigated the effects of exercise on age-related changes in the sirt1 activity, comparing heart (h) and adipose (a) tissue of sedentary young (n10), sedentary old (n10) and trained old (n10) rats. the trained old rats performed a 8-weeks moderate training on treadmill. on h and a tissue of all rats sirt1 activity was evaluated by assay kit, peroxidative damage measuring malondialdehyde (mda) and protein-aldehyde adducts 4-hydroxynonenal (4-hne), mnsod, catalase and foxo3a by western blot, and gadd45a, cyclin d2 and foxo3a mrna by rt-pcr. aging reduced sirt1 activity in h (p<0.0001) without effects in a, producing an increase of mda (h, p<0.0005; a, p<0.0001) and 4-hne (h, p<0.005; a, p<0.0005), and a decrease of mn-sod (p<0.02) and catalase (p<0.0001) expression in both h and a. aging did not affect foxo3a protein expression in h, and foxo3a mrna in a. exercise produced an increase in h foxo3a protein expression (p<0.02) and in a foxo3a mrna, associated to higher mn-sod (h, p<0.01; a, p<0.005) and catalase (h, p<0.0001; a, p=0.01) levels in both h and a of aged rats. in heart exercise-induced higher sirt1 activity bring on decrease in cyclin d2 and increase in gadd45a mrna expression. in a we found a similar decrease in cyclin d2, without changes in gadd45a mrna expression. these findings suggest that exercise is able to increase sirt1 activity in aged rats. (1), t horiguchi(1), k abe(2), h inoue(2), t noma (1) (1) institute of health biosciences, the university of tokushima graduate school, tokushima, japan (2) minophagen pharmaceutical co. ltd, japan objectives: glycyrrhizin (gl) is a major component of glycyrrhizae radix (licorice) that is generally used for treatment of hepatitis. gl has a regulatory activity on arachidonic metabolism, immunological function, and anti-viral effects. however, the molecular mechanisms of the effects remain unclear. to analyze the molecular basis of gl signaling, we performed the microarray analysis using ccl4-induced mouse hepatitis models. methods: eight-week-old icr male mice were treated intraperitoneally with 100 fã�l/ kg bw of ccl4 w/wo 250 mg/ kg bw of gl. after 24 hours and 72 hours, livers and serum were collected and analyzed. for microarray analysis, the expression patterns of genes between 72 hour-treated-livers (ccl4 or ccl4 and gl) and no treated-livers were compared. results: gl-treatment dramatically decreased the gpt activity in plasma at 24 hours compared to that in ccl4treated plasma. however, the levels of mrna expression of inflammatory genes such as phospholipase a2, hsp47, and procollagen were still very high in gl-treated liver. after 72 hours, the mrna levels of them were significantly reduced in gl-treated mice compared to those of ccl4-treated liver. then, we screened 41,000 genes by microarray and found that 71 genes were up-regulated and 63 genes were down regulated in ccl4+gl compared to ccl4 treatment. interestingly, ros scavenger-related genes were significantly up-regulated in ccl4 + gl. detail analysis is currently ongoing. we found the unique relationship between gl activity and ros regulation. this finding suggests a novel way to treat inflammatory diseases including hepatitis. objectives: experimental autoimmune encephalomyelitis (eae) is a demyelinating autoimmune disease that results from an immunological reaction against different myelin components at the cns. it is widely employed as an animal model of human multiple sclerosis. interestingly, the number of studies relating these diseases with peripheral organs is limited. we thus investigated the consequences of eae on the degree of lipoperoxidation (tbars) and mpo activity in different rat peripheral organs (eg. lung, spleen, liver, stomach, duodenum, colon, ileum, kidney and bladder). university of waikato, hamilton, new zealand mitochondria play a fundamental role in the life and death of all eukaryotic cells. cells with dysfunctional mitochondria are known to have higher levels of a molecular stress protein (cpn60). this protein is increasingly being implicated to play a role in modulating cellular inflammation. we have developed an in vitro model cell system using thp-1 monocyte cells with compromised mitochondrial bioenergetic functions to investigate the relationships between mitochondrial dysfunction, cpn60 expression and modulation of proinflammatory cytokine responses. we have found that the ability of cpn60 to modulate tnf-a expression was strongly correlated with the loss of mitochondrial bioenergetic functions in our thp-1 cells. we also demonstrate that such modulation involves both erk1/2 and p38mapk pathways. the significance of these results in relation to the role of mitochondria as modulators of inflammation will be discussed. (1), b arnold(2), g opdenakker (3) (1) jagiellonian university, department of evolutionary immunobiology, krakow, poland (2) german cancer research center, department of molecular immunology, heidelberg, germany (3) rega institute for medical research, university of leuven, laboratory of immunobiology, leuven, belgium we showed that in mice genetically deprived of metalloproteinase 9 (mmp-9-/-) at least one compensatory mechanism operates as there are elevated levels of pge2 of cox-1 origin expressed by peritoneal macrophages during zymosan peritonitis; and this leads to increased early vascular permeability observed in those animals. also infiltration of peritoneal cavity by inflammatory neutrophils is changed in mmp-9-/-mice as at 6 hrs of inflammation, when otherwise highest numbers of neutrophils are detected in peritoneum, the cell numbers are significantly lower in the mice in comparison to their controls. in contrary, at 24 hrs of peritonitis, when normally resolution of peritonitis takes place, no decrease in neutrophil counts is observed. thus the aim of the present study was to evaluate if impairment of neutrophil apoptosis could account for this latter phenomenon in mmp-9-/-mice. for this numbers of apoptotic (annexin v) and necrotic (7-aad) peritoneal leukocytes were evaluated and levels of active caspases were tested by application of either caspase detecting antibodies or fluorochrome-labelled inhibitors; all analyses were performed by flow cytometry. the results revealed that both, numbers of apoptotic cells and levels of active caspase 3 were significantly lowered in mmp-9-/-mice while levels of caspase 7, 8 and 9 were significantly elevated in comparison to control animals. we conclude that an impairment of apoptosis is observed in mmp-9-/mice during zymosan peritonitis and it is due to the decreased levels of active caspase 3. the increased activity of other examined caspases is most probably independent of apoptosis. (1), h james (1) the selective inhibition of nitric oxide generation by inhibiting the activity of nitric oxide synthase(nos) isoforms represents a novel therapeutic target for the development of anti-inflammatory agents. the aim of this study was to evaluate the activity of nos inhibitors in experimentally induced inflammation, pain and hyperalgesia. the effect on acute inflammation was studied in carrageenan-induced paw edema in rats. the effects on carrageenan-induced hyperalgesia, tail flick response to radiant heat and acetic acid-induced writhing were also studied. nos inhibitor ng-nitro-l-arginine methylester (l-name), 10 and 20 mg/kg produced a dose-dependent inhibition of paw edema (42% and 57% at 2h; 45% and 57% at 4h). a marked reduction in paw edema was observed with ng-monomethyl-l-arginine acetate (l-nmma), 10 mg/kg(79% at 2h; 91% at 4h). selective inducible nos(inos) inhibitor aminoguanidine hemisulfate inhibited the paw edema at a dose of 20 mg/ kg(59% at 2h; 64% at 4h) but not with a dose of 10 mg/kg . the effects were comparable to nonselective cox inhibitor indomethacin 5 mg and 10 mg/kg (40% and 77% at 2h; 29% and 72% at 4h respectively) and selective cox-2 inhibitor rofecoxib, 5 mg/kg (49% and 78% respectively). nos and inos inhibitors significantly increased the pain threshold latency in the tail-flick test. these inhibited the acetic acid-induced writhes, the effect being comparable to indomethacin. however, carrageenan-induced paw hyperalgesia was not inhibited. the results suggest that nitric oxide plays a role in carrageenan-induced acute inflammation and both nosand inos inhibitors have a potential anti-inflammatoryand anti-nociceptive activity. (1), p hart(2), j edwards (1), c quirk (1) (1) molecular pharmacology limited (usa), australian division, perth, western australia (2) telethon institute for child health research, perth, western australia thermalife cream, an anti-arthritic biological product, has been successfully used off-label for sun burn recovery. a novel product, derived from thermalife, was assessed on its therapeutic potential in oxsoralen-uvb burns. as a possible mechanism for the sunburn efficacy, suppression of tnf-a and il-1㢠production by human monocytes was assessed in vitro. methods: sunburn: four sites were marked on the arm of the subject. three sites were exposed to oxsoralen (1%) plus uva/uvb light, one site was exposed to oxsoralen only. cream was applied at 5min, or at 4hrs after injury. a third injury site was not treated. photographs were taken before, 24hrs, and 7weeks after injury. cytokines: human monocyte cultures (10% fcs, 5% co2) were either stimulated with 500ng/ml lps (e.coli 0111:b4) or not in the presence of 0% or 10% active ingredient.24hrs after incubation, culture media was collected, centrifuged, and assayed (cytokine elisa). results: at 24hrs after oxsoralen-uv, the 5min treatment site showed slight erythema, the 4hr treatment site had pronounced erythema and slight blister formation, whereas the untreated site had pronounced erythema and strong blister formation. 7 weeks after injury, the 5min site was normal, the 4hr site was a dark colour, whereas the untreated site had a significant scar. oxsoralen alone had no effect on the skin. the novel product suppressed lps-induced tnf-a and il1㢠secretion by 48.7% and 71.1%, respectively. conclusions: a novel thermalife-derived product reduced total injury after oxsoralen enhanced uva/ uvb burns, which is possibly related to cytokine suppression. (1), p hart(2), j snowden (1), maud eijkenboom (1) (1) molecular pharmacology limited (usa), australian division,perth, western australia (2) telethon institute for child health research, perth, western australia a mixture of bovine plasma protein fractions and zinc chloride (bov-zn) was assessed for its ability to regulate cytokine production by lps-stimulated monocytes. dosereponse curves for tnf-a suppression were generated. further, competition with fcs in the culture medium and the metabolism of monocytes under influence of bov-zn were assessed. in all experiments the culture medium environment was similar. human monocyte cultures (10% fcs, 5% co2) were either stimulated with 500ng/ml lps (e.coli 0111:b4) or not in the presence of 0%, 2.5%, 5%, 7.5%, 10%, 20% or 40% bov-zn (two pooled experiments). 24 hours after incubation, culture media were collected, centrifuged, and assayed (cytokine elisa). a competitive inhibition design for the standard tnf-a assay was set up for 0%, 1%, 5%, 10% fcs against 0%, 2.5%, 5%, 10% bov-zn. the culture media were treated as above. metabolism of non-proliferating monocytes was measured via accumulation of bioreduced formazan (promega celltiter 96) in treated and untreated cell cultures over 0-45 hrs at intervals. the ic50 for tnf-a suppression was reached at 2.5% bov-zn in each of two experiments. fcs did not compete with bov-zn in suppressing tnf-a in lpsstimulated monocytes. at low fcs concentrations bov-zn stimulated tnf-a production in the absence of lps. this tnf-a increase was countered with increasing concentrations of fcs. metabolism of cells was not affected by 10% bov-zn. conclusions: bov-zn could reliably and effectively reduce tnf-a secretion in vitro, without competing with the fcs in the culture medium, and without disturbing the metabolism of monocytes. inflammatory diseases such as rheumatoid arthritis (ra) result from overproduction of cytokines including tnf-â£\ and il-1fã�. these cytokines are known to be regulated by the stress-activated p38fnfnmap kinase pathway. because of this, inhibition of p38 map kinase has been one of the most compelling targets for the treatment of inflammatory disease. over the last 10 years, numerous groups have reported on the development of p38 map kinase inhibitors. x-ray co-crystallization with the enzyme suggests a propensity to accommodate structurally diverse molecules. regions of the binding site are known to be unique to p38 vs other kinases, enabling the development of p38 selective molecules. inflamm. res., supplement 3 (2007) posters anti-inflammatory activities. a series of 11 labdane-type diterpenoids (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) with various patterns of substitution were tested for potential anti-inflammatory activity.of these compounds, 4 and 11 were selected to evaluate their influence in targets relevant to the regulation of the inflammatory response. these derivatives displayed good in vivo anti-inflammatory activity, and maximum inhibitions of 50 and 90 % were noted in the 12-o-tetradecanoyl-phorbol-13-acetate (tpa)-induced ear oedema in mice. in addition, inhibition of myeloperoxidase activity, an index of cellular infiltration, was also observed. the diterpenoids also reduced the production of nitric oxide, prostaglandin e2, and tumour necrosis factor-alpha in bacterial endotoxin-activated raw 264.7 macrophage cells with ic50 in the range 1-10 mm. inhibition of these inflammatory mediators was related to reductions in the expression of inducible nitric oxide synthase, and cyclooxygenase-2, as determined by western-blot analysis and rt-pcr. since nuclear factor-kappab (nf-kb) plays a central role in the transcriptional regulation of these proteins, we investigated the effects of these diterpenoids on this signalling pathway. our results indicate that both compounds interfere with the phosphorilation of ikbalpha and ikbã�, resulting in inhibition of their degradation. in summary, the anti-inflammatory effects of these labdane diterpenoids are related to the inhibition of inflammatory mediators by blocking nf-kb activation and provide potentially therapeutic perspectives in inflammatory conditions. the aim of the study was a research of mechanisms of inflammatory action of a new drug fepolen at a dosage of 100 mg/kg prepared from bee products (bee pollen and phenolic hydrophobic extract of propolis) for prostatitis treatment. to fulfill above mentioned task a model of zimozan induced oedema whose dynamics gives a possibility to estimate influence of a drug on both routs of arachidonic acid metabolism -via cyclooxiginase and lipooxigenase was used. a comparison with diclofenacum at a dosage of 8 mg/kg and substance ffw-755ã� (inhibitor of cyclooxygenase and lipooxygenase and has a high antiinflammatory effect (41%) at a dosage of 16 mg/kg) was made. the results of influence of drugs dynamics of zimozan inflammation show that anti-inflammatory action of fepolen is based on decrease of release of biogenic amines and activity of lipooxigenase and is higher then effect of diclofenacum. fepolen revealed the highest effect during first 30 min and 1 hour of inflammation that was higher then action of diclofenacum. these data proves that fepolen decreases lipooxygenase activity that is in charge for the inflammation during this period. during next hours therapeutic effects of fepolen and diclofenacum were at the same level. fepolen showed the same dynamics of anti-inflammatory action as ffw-755ã� that demonstrates a property to influence both routs of arachidonic acid metabolism. in summary with previous results in conditions of carageninic inflammation we can conclude that anti-inflammatory action of fepolen is based mostly on influence on lipooxygenase then cyclooxygenase. the aim of this study was to establish a method by which probiotic bacteria can be selected for their immuno modulatory properties, especifically the ability of certain strains to suppress an inflammatory response. the gastrointestinal inflammatory condition crohns disease involves a th1-response with increased levels of proinflammatory cytokines like tnfa and il12, and mouse models of crohns disease show that the balance of il10/12 is crucial for disease progression. we have used mouse bone marrow derived dendritic cells (bmdc) to model a proinflammatory crohns disease like condition in vitro with cocktail-induced bmdc secretion of il12, il6 and tnfaf jthe model was validated using anti-inflammatory molecules like dexamethasone and prostaglandin d2, which were able to suppress the cocktail induced il12 secretion. further validation of the model is confirmed by the fact, that probiotic strains which are able to suppress tnbs induced colitis in mice in preventive studies, also show potent anti-inflammatory activity in our model. among clinically relevant as well as novel probiotic strains, we have selected strains with potent anti-inflammatory properties, and are currently investigating the possible mechanism of action of these strains. in summary, our established model is suitable for identification of anti-inflammatory activity of probiotic strains and potentially other immune suppressing components, for rational selection of candidates for further preclinical and clinical evaluation and development. p11-12 inflammation and human incisional hernia pathophysiology maria antonia arbos via(1) eae was induced by immunization of female lewis rats with guinea-pig myelin basic protein (mbp) in complete freunds adjuvant (cfa) and the animals were studied at the stage iii of the disease (characterized by complete paralysis of the hind-limbs) compared to cfa rats, eae resulted in increased mpo activity (u/mg tissue) in kidney (280 ae 75 vs. 121 ae 14 179 ae 35; p<0.001), and higher tbars contents (nmol mda/mg tissue) in liver acknowledgements: capes, cnpq, fapesp. contact information: ms simone teixeira, university of sâ¼o paulo, department of pharmacology, campinas, brazil e-mail: mone@usp.br tolerability, investigator and subject global assessments and rescue medication consumption supported by bk21 project from korea research foundation) contact information: mr young hoon kim here we report on the development of the pge2 mimetic combination therapy (dp-837953) that inhibits basal and lps/tlr4 induced tnf-?, il-1ã�, and mmp-1, 8, 9, 13 in human and murine synovial membranes and peripheral macrophages.in a murine model of chronic synovitis (dorsal skin air pouch), dp837953 dramatically inhibited il-1ã�, tnf-?, mip2, mcp-2 and il-8 expression, delayed the profile of leukocyte/neutrophil extravasation and reduced exudate volume.in a model of inflammatory arthritis (collagen induced arthritis, cia), dp837953 markedly reversed the inflammatory pathology by reducing synovial hyperplasia, cartilage erosion and articular inflammation.in addition, tnf-?, il-1ã�, mmp-8 and to a lesser extent mmp-9 expression/synthesis levels were strongly suppressed as judged by rt-pcr and elisa measurements we have developed two rias, one for functional blood levels of the above mentioned anti-tnf-alpha constructs, and one for anti-abs (all isotypes), and we have used these methods to monitor patients treated with infliximab/remicade and etanercept/enbrel ; i shall present some of these data (7, 8 anatomy-physiology, faculty of medicine, laval university, quebec, canada neutrophils, which are often the first leukocytes to migrate at inflamed sites, can generate ltb4 from the 5-lipoxygenase pathway, pge2 through the inducible cyclooxygenase (cox-2) pathway and cytokines/chemokines as tnf-alpha, il-1beta, il-8, mip-1alpha, mip-1beta, mip-2alpha and mip-3alpha. engagement of the adenosine a2a receptor (a2ar) blocks the in vitro synthesis of ltb4 while it potentiates the cox-2 pathway in fmlp-treated neutrophils. in addition, it selectively prevents the expression and release of tnfalpha, mip-1alpha, mip-1beta, mip-2alpha and mip-3alpha in toll-like receptor-4-stimulated human neutrophils. little effect was observed on il-1beta and il-8. using the murine air pouch model of inflammation with a2ar-knockout mice, we observed that the activation of a2ar positively impacts the expression of cox-2 in vivo, with particular magnitude in inflammatory leukocytes. in mice lacking the a2a receptor, neutrophils that migrated into the air pouch 4h following lps injection expressed higher mrna levels of tnf-alpha, mip-1alpha and mip-1beta than neutrophils from wild type mice. together, these results indicate that neutrophils are important mediators of adenosines protective effects. given the uncontrolled inflammatory phenotype observed in a2ar-knockout mice and in view of the potent inhibitory actions of pge2 on inflammatory cells, an increased cox-2 expression and a prevented release of tnf-alpha, mip-1alpha and mip-1beta caused by a2ar activation, observed particularly in neutrophils, may take part in an early modulatory mechanism promoting anti-inflammatory activities of adenosine. sepsis induced by endotoxins including lipopolysaccharide (lps) is a big problem in clinical medicine. for a better insight into the molecular pathways and to assess markers of endotoxin-induced sepsis, we applied thetwo dimensional gel electrophoresis (2d-page) and maldi-tof to follow the changes of significant proteins in a murine macrophage cell line -raw 264.7 after challenged with lps (escherichia coli 026:b6) for 12, 18 and 24 hours. we identified 21 proteins from approximately 500 detected protein spots with either increased or decreased in relative abundance as a result of lps treatment. the proteins identified with increased expression are the retinoblastoma binding protein 7, capg protein, poly(rc) binding protein 1, isocitrate dehydrogenase 3 (nad+) alpha, lactate dehydrogenase 1, a chain, guanine nucleotide binding protein (g protein), beta polypeptide 2 like 1, triosephosphate isomerase 1 and proteasome alpha 5 subunit); and ones with decreased expression are the acidic ribosomal phosphoprotein p0, malate dehydrogenase, soluble, proliferating cell nuclear antigen, proteasome (prosome, macropain) subunit, alpha type 1 and rho, gdp dissociation inhibitor (gdi) beta). many of these altered proteins have interesting functions in inflammation. with the information obtained with the proteomic approach, it is possible to improve current methods of monitoring endotoxemia and to identify new therapeutic targets. the ubiquitous mitogen-activated protein (map) kinases are important enzymes in signal-tranduction cascades which regulates diverse cellular events such as cell transformation, proliferation, differentation, and apoptosis. they are therefore potential drug targets for therapeutic intervention in the treatment of inflammation, cancer, and other immune diseases. based on a virtual screening approach we identified 6-amino-1-benzyl-4-(4-bromophenyl)3-methyl-1,4-dihydropyrano[2,3c] pyrazole-5-c arbonitrile as a potential novel lead structure as p38 map kinase inhibitors. a set of compounds were prepared starting from different substituted pyrazol-5(4h)-ones via a base-catalyzed condensation with aldehydes and ch acids, such as malononitrile, to provide them for biological tests in a p38 enzyme assay. first structure-activity relationship confirm the value of this novel lead. this study was conducted to determine the physiological c-reactive protein (crp) and alpha 1-acid glycoprotein (aag) levels for two groups of beagle dogs: healthy dogs of various ages and pregnant dogs. serum crp levels were measured by elisa and aag levels were measured in healthy beagles of various ages by tia, and then separately -in pregnant beagles -by srid. serum crp levels ranged from 1.5 to 16.0 ã¬g/ml in male, and from 1.8 to 18.9 ã¬g/ml in female dogs. no significant sex-related differences were observed in the values. further, there were no significant age-related differences either. serum crp levels increased during pregnancy and peaked at 70.2-90.4 ã¬g/ml 30 or 45 days after ovulation, demonstrating two characteristic features of crp levels change in pregnant dogs. serum aag levels ranged from 40 to 960 ã¬g/ml in male, and from 47 to 833 ã¬g/ml in female dogs, without any significant sex-or age-related variation. serum aag levels increased in all pregnant beagles and peaked in the middle of gestation at 250-1,000 ã¬g/ml. despite a high value of 1,210 -1,360 ã¬g/ml being observed for serum aag levels in 3 pregnant beagles inoculated with staphylococcus aureus, its levels in umbilical cord blood were below the detection limit of srid (40 ã¬g/ml). no significant sex-/-age related differences were observed in serum both crp and aag levels and these levels increased during pregnancy. the results of aag levels in umbilical cords were below the detection levels suggest aag is not transported to the placenta. polymorphonuclear neutrophils (pmns) play a key role in the inflammatory response against infectious agents.however, they can elicit significant tissue damage and in this respect, anti-inflammatory drugs are of interest.in this study, we examined the effect of pbi-1393, a low molecular weight immunomodulatory molecule, on pmn activation by lps both in vitro and in vivo. we measured by elisa the production of tnf-a by human lps-activated pmn in the presence or absence of pbi-1393.the ability of pbi 1393 to modulate pmn activation and recruitment in vivo was assessed using a rat air pouch model of inflammation.exudates from different groups of animals (controls and pbi-1393 treated animals, n=6) were used to assess leukocyte infiltration and to measure by elisa tnf-âµ, mcp-1 and pge2 production.in vitro, pbi-1393 is able to significantly decrease by 28.8% ae 0.08% (p<0.05), tnf-a production by human lpsactivated pmn.in vivo, pbi-1393 significantly decreased the production of tnf-a (41.4% ae 7.2%; p<0.005), mcp-1 (16.3% ae 8.4%; p<0.05) and pge2 (29.2% ae 13.5%; p<0.05) induced by lps injection.however, pbi-1393 did not significantly inhibit leukocyte infiltration.these results show that pbi-1393 is able to modulate pmn activation and inflammatory response and suggest potential use as anti-inflammatory agent. (1), lj lowenstine,(2), aj norris(2), t spangler(2), lm woods (2) (1) zoological society of san diego, usa (2) department of pathology, microbiology, and immunology, university of california, davis, usa this study investigated the role of a novel reovirus in a 2004 outbreak of necrotizing typhlocolitis in american crows in california. included is a detailed characterization of the necrotizing and inflammatory characteristics of the disease, as well as a discussion of the implications of these findings upon proposed mechanisms of pathogenesis. complete histopathology including stains for lesion characterization and potential concurrent etiologic agents was performed on all outbreak crows. feces and ceca were submitted for culture, parasitology, and negative contrast electron microscopy. two control groups (n=10 each) were selected for parasitology and em (group 1), and gross and histopathology (group 2). all outbreak cases and group 2 controls tested negative for west nile virus by pcr.all outbreak crows had marked, necrotizing heterophilic typhlocolitis, fibrinonecrotizing splenitis, and variable intestinal lamina proprial necrosis and hemorrhage.two cases had multifocal hepatic necrosis. negative contrast em revealed reovirus particles in 100 % (4/4) of outbreak cases and in 0% (0/ 10) of controls. supplemental tests failed to suggest other concurrent or confounding etiologic agents.overall, the findings suggest association between the reovirus and the outbreak of typhlocolitis, and the absence of reovirus in controls suggests that it is not ubiquitous in the crow population.there was a noteable absence of similar typhlocolitis in 65 archived crows submitted to the vmth from 1994-2004, suggesting an emerging corvid disease in california, which bears further investigation. mitogen-activated protein kinase (mapks) pathways play an important role in the signalling system activated by proinflammatory cytokines. among the most important cascades the activation of erk1/2 by mek1/2 is reported to be responsible for inflammatory responses and degradation of osteoarthritic cartilage. as701820, a selective mek1 inhibitor, demonstrated anti-inflammatory properties in reducing tnf-alpha production induced by lps injection (ic50 2 mg/kg). therefore, primary aim of the present study was to assess the therapeutic strength of the as701820 in a mouse model of collagen-induced rheumatoid arthritis (cia) assessing the effect of the compound on structural changes related to the cartilage. cia is characterized by severe polyarthritis affecting peripheral joints, synovial hyperplasia with persistent inflammation and cartilage erosion. as701820 treatment was initiated when signs of arthritis were clinically visible (in terms of paw swelling and redness) and was continued for 7 days (twice daily), by oral route at the doses of 10, 30 and 100 mg/kg. as701820 at 30 and 100 mg/kg significantly reduced clinical arthritic read-outs such as clinical score and paw swelling. at histology, vehicle-treated animals showed severe inflammation and joint surface erosion. administration of as701820 significantly decreased inflammatory infiltrates and treated cartilage surfaces that presented normal levels of proteoglycan content. in conclusion, the results obtained in this study clearly demonstrate that the selective blockade of mek1 could be considered as an innovative therapeutic approach to treat rheumatoid arthritis. experimental evidences have shown that the toxicity of ni salts may involve inflammatory processes, with a subsequent overproduction of reactive oxygen species (ros) and carcinogenicity. neutrophils are the most abundant leukocytes of blood, and participate actively in the inflammatory innate host defense response. however, relatively little is known about the potential of nickel salts in activating human neutrophils.thus, the aim of the present study was to evaluate the putative stimulation of oxidative burst in isolated human neutrophils by nickel nitrate. the measurement of neutrophil burst was undertaken in vitro, by chemiluminescence, by monitoring the oxidation of luminol by neutrophil-generated ros and reactive nitrogen species (rns). enzymatic inhibitors and specific reactive species scavengers were used to evaluate which species were involved in neutrophils activation by nickel nitrate. the obtained results showed that nickel nitrate stimulates human neutrophils burst in a concentration-dependent manner, within levels that may be attained in vivo. in the present experimental conditions, the reactive species involved in neutrophils activation by nickel nitrate were superoxide radical (o2-.), hydrogen peroxide (h2o2), hydroxyl radical (ho.) and perchloric acic (hocl). the observed activation of isolated human neutrophils burst by nickel nitrate and subsequent tissue damage due to a sustained formation of reactive species may contribute for the deleterious effects attributed to this transition metal, though this assumption needs to be confirmed in vivo. (1), h spalteholz (1), u reibetanz (1), p salavei (1), m fischlechner (1), h-j glander(2), j arnhold (1) (1) university of leipzig, medical faculty, institute for medical physics and biophysics, germany (2) university of leipzig, derpartment of dermatology, andrology training centre of the european academy of andrology, germany unintentional childlessness often caused by common reasons as inflammation affects 15 -20 % of german couples. inflammations of the male genital tract lead to an infiltration of polymorphonuclear granulocytes (pmn), respectively induce a restricted spermatozoa quality associated with early triggered acrosome reaction (ar) and apoptosis as well as changes in the lipid structure and reduced mobility. stimulated pmn release the strongly cationic heme protein myeloperoxidase (mpo), which is able to bind to negatively charged membrane surfaces, e.g. apoptotic cell membranes with externalized phosphatidylserine (ps). a population of freshly prepared spermatozoa shows only a very small amount of cells with mpo binding ability as well as externalization of ps. the number of spermatozoa able to bind mpo raises considerably in samples containing predamaged cells or introducing the ar as could be observed with rhodamine b isothiocyanate (ritc)labelled mpo and antibody techniques by fluorescence microscopy as well as flowcytometry. the activation ofmpo with its substrate hydrogen peroxide (h2o2) in the presence of chloride ions generates the powerful oxidizing and chlorinating species hypochlorous acid (hocl) and enhanced markedly the number of annexin v positive and non-vital cells. components of seminal plasma as well as serum albumin can protect spermatozoa for the deleterious effects of mpo. the coincidence of ps externalization and mpo binding to spermatozoa surfaces indicates an up to now unknown role of this enzyme in recognition and removal of apoptotic cells during inflammation. recent findings suggest a crucial role of proteinaseactivated receptor-2 (par2) in inflammation and innate immunity. par2 is the second member of a novel g protein-coupled receptor subfamily with seven putative trans-membrane domains. this subfamily is characterized by a unique mechanism of receptor activation. accessible serine proteases cleave the receptor to expose a new, previously cryptic, n-terminal sequence ("tethered ligand") which further interacts with the same receptor and activates it. tryptase, trypsin, and bacterial serine proteases are capable of directly activating par2. par2 is expressed by human neutrophils, however its functions on these cells remained unclear. the data of our present study indicate that par2 agonists enhance interferon gamma (ifna)-induced up-regulation of cell surface fca;ri, one of the key receptors involved in neutrophil phagocytic activity. moreover, par2 agonists (serine proteases as well as synthetic activating peptide) and their receptor represent an additional system which controls neutrophil transendothelial migration and apoptosis in vitro. additionally, there is a significant increase of par2 expression on the neutrophil cell surface in the case of septic patients as compared to cells from healthy volunteers. together, our results indicate that par2 may be involved in the pathophysiology of acute bacteria-induced human diseases (sepsis or septic shock, for example) potentially by regulating neutrophil apoptosis, transendothelial migration and fca-receptor expression. aim: to ascertain the role of macrophages as direct inducers of regeneration after renal ischemia/reperfusion, and to establish whether inflammatory conditions contribute to the process. we determined whether adoptive transfer of macrophages at different stages of kidney inflammation after mouse renal i/r could restore reparation and assessed the influence of inflammation in the process.results: i/r provoked the increases in renal regeneration, as evaluated by inmunohistochemistry and pcr mrna of stathmin and pcna. the cytokine profile revealed the influence of the inflammatory environment on kidney repair. regeneration was macrophage-dependent, decreasing when depletion was provoked, and increasing with adoptive transfer of macrophages; however, administration of resting macrophages did not induce repair at the time points in which tissue was inflamed, and was only able to promote regeneration in the absence of inflammation (72 hours). pro-inflammatory cytokines increased at the early stages of reperfusion, coinciding with low regeneration, and anti-inflammatory cytokines increased during the longer periods of reperfusion when regeneration was more evident.conclusions: macrophages directly induce renal regeneration after ischemia/reperfusion in an inflammationdependent manner. (1), k bendtzen (1), f sellebjerg (2), ch nielsen (3) ( antibodies against myelin basic protein (mbp) are present in sera from patients with multiple sclerosis (ms), but the role of these antibodies is controversial. we collected sera from 22 ms patients and 17 healthy individuals and found that both groups contained igm anti-mbp antibodies, while ms sera contained small amounts of igg anti-mbp. however, the two groups of sera did not differ significantly with respect to the content of either antibody subclass. addition of mbp to the various sera and subsequent addition of the mixtures to normal peripheral blood mononuclear cells (pbmc) resulted in a significant deposition of igm on cd14+ monocytes, indicating that formation of mbp/igm complexes had occurred. this deposition was strongly inhibited by addition of 10 mm edta to the sera, indicating that it was complement dependent. the pbmc produced significant amounts of il-10, tnf-alpha and ifn-gamma upon stimulation with mbp, and the extent of the cytokine production did not depend upon whether sera from ms patients or from healthy controls were present. however, disruption of the tertiary structure of mbp by boiling significantly reduced the production of all three cytokines, supporting a role for antibodies in the induction of cytokine responses to mbp. we propose that natural igm autoantibodies may form complexes with mbp, facilitating the uptake of mbp by antigenpresenting cells (apc). since sera from ms patients did not enhance this uptake and the subsequent cytokine production, the mechanism may be part of an appropriate peripheral regulation of self-reactivity. we currently investigate this possibility. loredana postiglione(1), g tarantino(2), a spanã²(2), p ladogana (1), fl perrone(1), s padula(2), a riccio (2) (1) federico ii university medical school of naples, department of molecular and cellular biology and pathology l.califano, naples, italy (2) federico ii university medical school of naples, departmentof clinical and experimental medicine, naples, italybackground: hepatitis c virus (hcv) infection can induce immunological disorders with different clinical expression such asarthritis, sjogren sindrome and various form of vasculitis.aim: to study the prevalence of anti-cyclic citrullinated peptides antibodies (anti-ccp) in a group of patients affected by hcv-related arthritis and the eventual correlations with rheumatoid factor (rf) and/orantinuclear antibodies (ana), and articular involvement. study design: 30 patients with arthritis were selected in a population of 380 subjects affected by hcv infection. each patients was evaluated by clinical examination (23 denoted poliarticular and 7 mono-oligoarticualr involvement), by x-graphic aspects of joint involvement (8 patients presented join erosions), by ana, rf and anti-ccp positiveness.results: 33,3% of patients presented positivenessfor anti-ccp, without significant correlation between suchparameter and ana, rf and articular involvement. anti ccp resulted positive in 4 out of the 8 patients with joint erosions, and only in 6 out of the 22 patients without joint erosions. such frequency analyzed by chi square ended up in no significant differences. our patients presented an interesting prevalence of the positiveness for anti-ccp. these data suggest a consideration about the specificity, commonly attributed to this parameter in the diagnosis of rheumatoid arthritis. expression of nkg2d on cd4+ t cells is generally rare in both mice and humans, but has been reported in a number of inflammatory diseases, including rheumatoid arthritis, crohns disease and an animal model of type 1 diabetes. the monoclonal antibody cx5 recognizes murine nkg2d and has been shown to block ligandbinding and mediate internalization of nkg2d. furthermore, cx5 can inhibit and/or ameliorate disease in animal models of type 1 diabetes and inflammatory bowel disease. thus, it is very likely that nkg2d plays an important role in the development of inflammatory and autoimmune diseases. since little is known about the pharmacokinetics and pharmacodynamics of the cx5 antibody, we decided to study this in both regular balb/ c mice and immunodeficient cb17.scid mice. different doses of cx5 antibody was injected intraperitoneally and pk and pd was measured by elisa (anti-cx5 elisa in serum) and flow cytometry (down-regulation of nkg2d on cd49b+ nk cells) for up to two weeks after administration. we found that cx5 very efficiently down-regulate nkg2d on cd49b+ nk cells and that the effects of the antibody can be seen for more than two weeks after one single injection. finally, we propose a model which may be helpful in predicting the effects of different doses of cx5 antibody in vivo. (1), k mehta(2), n deo(2), j chaudhary(2), p bobrowski (3) (1) albany medical college, usa (2) vedic lifesciences, usa (3) rainforest nutritionals, inc, us background: the efficacy and safety of reparagen, in treating osteoarthritis was compared to glucosamine sulfate in a mumbai-based multi-center, randomized, double-blind study.methods: subjects (n=95) were screened and randomized to receive glucosamine sulfate (n= 41, 1500 mg/day) or reparagen (n=38, 1800 mg/day), a polyherbal consisting of vincaria (uncaria guianensis) and rni 249 (lepidium meyenii) administered orally, twice daily. primary efficacy variables were womac scores, visual analog score (vas) for pain, and response to treatment defined as a 20% improvement in womac pain, with assessments at 1, 2, 4, 6 and 8 weeks. secondary variables were results: subject randomization was effective and both treatments showed significant benefits in primary outcomes within one week (p<0.05), with a similar, progressive improvement over the course of the 8 week treatment protocol (42-49% reduction in total womac or vas scores). the response rate was substantial for both glucosamine (88%) and reparagen (92%), which exceeded placebo responses (55%, p <0.01) and supported by investigator and subject assessments. tolerability was excellent and safety parameters were unchanged. rescue medication use was significantly lower in the reparagen group (p <0.01), and serum igf-1 levels were unaltered.conclusions: both reparagen and glucosamine sulfate produced substantial improvements osteoarthritis symptoms. response rates were high and the safety profile was excellent, with significantly less rescue medication use with reparagen. we speculate that the high response rate to glucosamine sulfate may reflect higher baseline pain levels or synergy with dietary curcumin. inflammation accompanies and aggravates progression of all modern human chronic pathological conditions. growing evidence indicates the beneficial role of proper nutrition in controlling inflammation. we investigated the effects of selected essential nutrients in experimental inflammation and the molecular mechanisms involved. tested nutrient mixture (nm) consisted of green tea catechins, citrus flavonoids hesperidin, naringenin and quercetin, ascorbate, lysine, proline, arginine and cysteine. systemic inflammation in mice challenged with bacterial lipopolysaccharide (lps) was monitored by blood plasma levels of fourteen key inflammatory cytokines. two week supplementation with 250 mg nm/kg body weight prior to lps challenge provided significantly greater protection than did supplementation with ibuprofen. induction of interleukin-6 (il-6) and monocyte chemoattracting protein-1, two cytokines especially responsive to lps challenge, was reduced in nmsupplemented animals by 58% and 86%, respectively. corresponding reduction in ibuprofen group was 34% and 43%. protective mechanisms involved were assessed in human cultured u937 macrophages stimulated with lps.the cytokines most responsive were tumor necrosis factor alpha (71% and 17% reduction by supplementation with nm and ibuprofen, respectively) and il-12 (66% and 15% in corresponding reduction). nm supplementation dramatically reduced prostaglandin e2 secretion by stimulated macrophages along with cyclooxigenase-2 (cox2) cellular protein expression. mrna levels forcox2 and inflammatory cytokines were also dramatically reduced. quercetin was the most effective nutrient when tested individually. however, nm appeared to surplus the combined effect of individual components. we conclude that the tested combination of essential nutrients demonstrates strong beneficial effects in experimental inflammation by targeting responsible gene expression. (1), hp kim (1), kh son (2) (1) college of pharmacy, kangwon national university, south korea (2) department of food and nutrition, andong university, south korea chalcones belong to flavonoid family from plant origin and some of them possess anti-inflammatory activity. recently, several natural and synthetic chalcones were reported to inhibit inducible nitric oxide synthase (inos)-catalyzed no production in cell cultures. in the present study, to find the optimal chemical structures and to elucidate their action mechanisms, 41 synthetic chalcones having the substituent(s) on a-and b-rings were prepared and their effects on inos-catalyzed no production were evaluated using lps-treated raw 264.7 cells. among the tested compounds, 2-methoxy-3,4-dichlorochalcone (ch15), 2-hydroxy-6-methoxychalcone (ch29), 2-hydroxy-3-bromo-6-methoxychalcone (ch31) and 2-hydroxy-4,6-dimethoxychalcone (ch35) potently inhibited no production (ic 50 s, 7.0 -9.9 mm). the favorable chemical structures were found to be a methoxyl substitution in a-ring at adjacent position (2 or 6) to carbonyl moiety with/without 2-(or 6-)hydroxyl group and 3-halogen substitution in b-ring. when the cellular action mechanisms of ch15, ch31 and ch35 were further examined, it was revealed that ch15 and ch31 clearly down-regulated inos expression while ch35 did not. moreover, ch15 and ch31 were proved to suppress the nuclear transcription factor-kb activation. from the results, it is suggested that certain chalcone derivatives potently inhibit inos-catalyzed no production by the different cellular mechanisms, inos down-regulation or inos inhibition, depending on their chemical structures. these chalcone derivatives may be possibly used as lead compounds for developing new anti-inflammatory agents. an oligomeric stilbene alpha-viniferin (avf) was isolated from root of carex humilis (cyperaceae) as an inhibitor of cyclooxygenase (cox)-2 activity by bioassayguided fractionation. the avf was later found to downregulate lipopolysaccharide (lps)-induced cox-2 expression as well as to inhibit nuclear factor (nf)-kb activation, in addition to its inhibitory effect on cox-2 activity. furthermore, the compound exhibited antiarthritic effect in vivo. avf is a trimer of resveratrol and contains benzofuran moieties in its central part. starting from benzofuran and its related chemicals, 2cyclohexylimino-6-methyl-6,7-dihydro-5h-benzo [1, 3] oxathiol-4-one (lyr-64) was discovered to inhibit lpsinduced nf-kb transcriptional activity in macrophages raw 264.7. the lyr-64 reduced lps-induced dna binding activity and nuclear translocation of nf-kb as well as inhibited lps-induced degradation and phosphorylation of inhibitory kb (ikb) protein. these results suggest that lyr-64 could suppress lps signaling molecule, putatively ikb kinase (ikk) complex, upstream ikb degradation in nf-kb activating pathway. lyr-64 inhibited in vitro kinase activity, gst-ikb phosphorylation, of wild type ikkbeta or a constitutively active ikkbeta mutant (c/a, cys-179 to ala) but did not affect that of another constitutively active ikkbeta mutant (ss/ee, ser-177 and 181 to glu). therefore, lyr-64 could inhibit lps-induced nf-kb activating pathway by targeting ser-177 and/or 181 residues on the activation domain of ikkbeta. as pharmacological actions, lyr-64 prevented nf-kb-dependent expression of inducible nitric oxide synthase, cox-2, or inflammatory cytokines at the transcription level in lps-stimulated macrophages raw 264.7. furthermore, lyr-64 protected lpsinduced septic shock in vivo. faculty of medicine, institute of pharmacology, ljubljana, slovenia a part of anti-inflammatory action of antidepressants can arise from their effect on histamine elimination from the side of inflammation. in mammals histamine is mainly degraded by two enzymes: histamine-n-methyltransferase (hnmt) and diamine oxidase (dao). the aim of present investigation is to establish whether antidepressants amitriptyline and sertraline can affect histamine metabolism. their effects on enzyme activity and mrna expression were studied in guinea pig tissues. plasma and tissue homogenates were incubated with saline (control) and different antidepressant concentrations. specific enzymatic activities of dao and hnmt were determined by radiometric assay. in addition, guinea pigs were treated with saline or amitriptyline (4 mg/kg, ip), afterwards dao and hnmt mrnas were detected by pcr in different tissues. results showed thatamitriptyline, 100 nm, 50, 100 and 500 mm, increased guinea pig plasma dao activity by 5, 7, 17 and 11 %, respectively, while sertraline increased it at 30 mm (by 15 %). at higher concentrations (100 and 500 mm) sertraline decreased dao activity. in the guinea pig tissues hnmt activity changes were found only when incubated with amitriptyline; sertraline had no effect. at 10 and 50 nm amitriptyline the activity of hnmt increased by 20 and 9 %, respectively. in animals treated with amitriptyline an induction of dao and hnmt mrna expression was noticed in several tissues. our results suggest that in guinea pigs due to higher histamine metabolism antiinflammatory effects can be expected at lower concentrations of antidepressants. the effect might be the opposite with higher amitriptyline concentrations. steven hefeneider(1,2), c macarthur (1), d trune (1), s mccoy (2) (1) oregon health and science university, portland, oregon, usa (2) targeted gene delivery, inc., portland, oregon, usa engagement of toll-like receptors (tlrs) by bacterial components such as lps and dna initiates inflammation.the current study examines a novel anti-inflammatory peptide, termed p13, for treatment of inflammation induced by either lps or bacteria.peptide p13 was derived from an immunoregulatory protein of vaccinia virus, and interferes with tlr signaling.in this study we examined the efficacy of p13 to limit inflammation in a mouse model of sepsis and a model of middle ear inflammation, termed acute otitis media (aom).we demonstrate in the sepsis model, that in vivo treatment of mice with p13 inhibited lps-induced production of serum inflammatory mediators.moreover, p13 treatment, administered after initiation of inflammation, significantly increased survival of mice injected with lps.in the aom model, peptide p13 significantly reduced in vivo middle ear inflammation and fluid accumulation initiated by h. influenza.assessment of route of administration and delayed treatment studies demonstrated the efficacy of peptide p13.simultaneous injection of bacteria and peptide p13 resulted in a significant reduction in fluid accumulation, infiltrating cells, and tympanic membrane thickness.fluid accumulation within the eustachian tubes was also significantly reduced following p13 treatment.-subcutaneous and oral administrations of p13, but not intravenous administration, were also efficacious in reducing inflammation. administration of p13 after initiation of an ongoing inflammatory response was effective at reducing inflammation and fluid development.taken together, these results demonstrate the therapeutic potential of peptide p13 to limit an inflammatory response and suggest a possible new treatment strategy for bacterial-induced inflammation. (1), c zhou(2), y zhang(2), m sun(2), x wan (1), h yu(2), x yang(2), rd ye (3), j-k shen (1) formyl peptide receptor-like 1 (fprl1) is a structural homologue of fpr, which binds chemotactic peptides of as small as 3 amino acids (e.g., fmet-leu-phe, fmlf) and activates potent bactericidal functions in neutrophils. in comparison, fprl1 ligands include peptides of 6-104 amino acids, such as trp-lys-tyr-met-val-[d]met (wkymvm) and other synthetic peptides. to determine the core peptide sequence required for fprl1 activation, we prepared various analogues based on wkymvm and evaluated their bioactivities in an fprl1-transfected cell line. although substitution of d-met6 resulted in loss of activity, removal of val5 together with d-met6 produced a peptide that retained most of the bioactivities of the parent peptide. the resulting peptide, wkym, represents a core structure for an fprl1 ligand. further substitution of lys2 with nle slightly improved the potency of the tetrapeptide, which becomes a dual agonist for both fprl1 and fpr. based on these structure-activity studies, we propose a model in which the modified tetrapeptide trp-nle-tyr-met (wnleym) binds to fprl1 through aromatic interactions involving the side chains of trp1 and tyr3, hydrophobic interaction of nle2, and the thio-based hydrogen bonding of met4, with the respective residues in fprl1 which have not been identified. the identification of the core sequence of a potent peptide agonist provides a structural basis for future design of peptidomimetics as potential therapeutic agents for fprl1-related disorders.there is a growing awareness of the interaction of food constituents with the immune system. the present study aims to evaluate immunomodulatory effects of two of these nutritional components, i.e. glycine and lactoferrin. mice orally supplemented with glycine, lactoferrin or a combination were injected intradermal (in the ear) with zymosan. ear swelling, as a measure for inflammation, as well as il-1, tnf-a and il-6 levels in the ear and the number of tnf-a producing spleen cells were analyzed.-glycine and lactoferrin were able to decrease the zymosan induced inflammatory response locally (decreased ear swelling and pro-inflammatory cytokine levels) as well as systemically (reduced number of tnf-a producing spleen cells).glycine effects (20, 50 and 100 mg/mouse/day) were concentration dependent whereas for lactoferrin only the lowest doses (0.1 and 1 mg/mouse/ day) inhibited the inflammatory response significantly. surprisingly higher doses of lactoferrin (5 and 25 mg/ mouse/day) failed to influence the inflammatory reaction. a combination of both nutrients (lactoferrin 0.1mg/ mouse/day in combination with glycine 20 or 50 mg/ mouse/day) inhibited the zymosan induced ear swelling synergistically. additionally an additive effect of both components was seen on the number of tnf-a producing spleen cells. the present data show anti-inflammatory activity of glycine and lactoferrin using the zymosan induced inflammation model.moreover a combination of both components demonstrated a synergistic effect on inflammation of the skin and an additive effect on the number of tnf-a producing spleen cells. (1), p sambrook(1), k fukudome(2), m xue (1) (1) university of sydney, st leonards, nsw, australia (2) saga medical school, saga, japan objectives: to investigate the i) expression of endothelial protein c receptor (epcr) in synovial membrane and peripheral blood monocytes from patients with rheumatoid arthritis (ra) and osteoarthritis (oa) and ii) role of epcr and its ligand, activated protein c (apc), on the function of monocytes from ra patients.methods: epcr, cd68 and pc/apc in synovial tissues were detected by immunostaining and in situ pcr. monocytes were isolated from peripheral blood of patients with ra and treated with apc, lipopolysaccharide (lps), and/or epcr blocking antibody, rcr252. cells and supernatants were collected to analyze the expression/activation of epcr, nuclear factor nf-kb and tumour necrosis factor tnf-a.results: epcr was expressed by both oa and ra synovial tissues but was markedly increased in ra synovium. epcr was colocalized with pc/apc mostly on cd68 positive cells in synovium. in ra monocytes, apc upregulated epcr expression reduced monocyte chemoattractant protein-1-induced chemotaxis of monocytes by approximately 50%. apc also completely suppressed lps-stimulated nf-kb activation and attenuated tnf-a protein by more than 40% in ra monocytes. the inhibitory effects of apc were reversed by rcr252, indicating that epcr modulates the inhibitory effects of apc.conclusions: our results demonstrate for the first time that epcr is expressed by synovial tissues, particularly in ra, where it co-localizes with pc/apc on monocytes/ macrophages. in addition, apc inhibits the migration and activation of ra monocytes via epcr. these inhibitory effects on ra monocytes suggest that pc pathway may have a beneficial therapeutic effect in ra. key: cord-004879-pgyzluwp authors: nan title: programmed cell death date: 1994 journal: experientia doi: 10.1007/bf02033112 sha: doc_id: 4879 cord_uid: pgyzluwp nan it is widely held that all developmental cell death is of a single type (apoptosis) and that neuronal death is primarily for adjusting the number of neurons in a population to the size of their target field through competition between equals for target-derived factors. we shall draw on our research and on that of others to criticize these views and replace them by the following. at least three types of neuronal death occur, only one of which resembles apoptosis; a neuron can choose between several self-destruct mechanisms depending on the cause of its death. the purpose of the death is to regulate connectivity, not neuron number. competitors for trophic factors are unequal, and many losers have made axonal targeting errors. a neuron's survival and differentiation depend on multiple anterograde and retrograde signals. activity affects retrograde signals and some but not all anterograde ones. the pattern of activity is more important than the overall amount. in rodents, the period of naturally occuring cell death of motoneurons is followed by a period of supersensitivity to axonal injury. thus, in newborn rodents lesion of the facial nerve leads to a rapid degeneration of the injured motoneurons. we have tested whether overexpression, in rive, of the bcl-2 proto-oncogene was capable of preventing death of axotomized motoneurons. to address this question we used transgenic mice whose motoneurons overexpress the bcl-2 protein. one of the two facial nerves of newborn mice was transected on the 2nd-3rd post-natal day. seven days after the lesion, the morphology of the facial nuclei was analyzed. in control mice, and when compared to the intact nucleus, 70 to 80 % of axotomized motoneurons had disappeared. in contrast, in the transgenic animals, the number of motoneurons on the lesioned side remained unchanged when compared to the eontralateral nucleus. furthermore, their axons remained visible up to the distal lesion site. these experiments show that, in rive, motoneurons overexpressing the bcl-2 protein survive after axotomy, and suggest that, in rive, bcl-2 protect neurons from experimentally induced cell death and could be a target for treatment of motoneurons degenerative diseases. messmer s., mattenberger l., sager y., blatter-garin m-c., pometta d., kate a., james r.w. drpt de mrdeeine, drpt. de pharmacologie, div. de neurophysiologie clinique, facult6 de mrdecine, gen~ve. clusterin is a widely expressed glycoprotein, highly conserved across species. numerous functions have been postulated for this protein. the most important are roles in lipid transport, as elusterin is associated with apolipoprotein ai in hdl, complement regulation and tissue remodelling, in particular during cell death and differentiation. using cultures of rat spinal cord neurones (90% neurons and 5-10% non-neuronal cells), we have studied the expression of clusterin and ape e in glutamate-induced neuronal cell death to examine potential roles in lipid management. up-regulation of the two proteins was observed. clusterin and ape e appear in the conditioned medium respectively 15h and 7.5h after incubation with glutamate. control studies, in the presence of a noncompetitive nmda receptor agonist showed the secretion of clusterin and ape e to be diminished by >60%. no up-regulation of either protein was observed in complementary studies with exclusively non-neuronal cell cultures. the cellular origin of the 2 secreted proteins is presently under investigation. programmed cell death and tissue remodelling are consequences of hormonally induced restructuring of the rat ventral prostate after castration and the rat mammary gland after weaning. we used the "differential display"-method (liang and pardee, 1992, science 257:967) to detect and isolate edna fragments whose corresponding rnas are regulated either coincidentally, or in an organ specific fashion during mammary gland involution and postcastrational prostate regression. partial sequencing of 12 clones revealed high, but not absolute homology of 5 fragments with sequences, previously characterized in different biological contexts. these five encode functions which could be anticipated to be important for cell growth and/or programmed cell death, we are presently investigating the functions of several of these transcripts in cell culture and in rive. antisense oligos are being employed in vivo to determine whether these genes contribute to the phenotype of programmed cell death. b epitopes derived from the envelope gp52 glycoprotein (ep3) or from the viral superantigen of mmtv have been incorporated into inert or live vaccines. the inert vaccine consists of purified chimeric proteins which contain the b epitopes alone or fused to multimeric promiscuous t helper epitopes from tetanus toxin. mice were immunized subcutaneously with these chimeric proteins. the live vaccine consists of an avirulent strain of salmonella typhimurium which expresses the mmtv epitopes in the form of chimeric proteins fused to the nucleocapsid protein of hepatitis b virus. this vaccine is given to mice in one oral dose. the level, duration and isotype of the immune response generated by each vaccine have been measured and compared. the level of protection has been investigated by systemically challenging immunized mice with the relzovims. a reduced binding of oxytocin (ot) occurs with aging in some, but not all, areas of the rat brain (arsenijevic et al., experientia 1993, 49, a75) . the candate putamen showed the most impressive loss of ot receptors. two other regions, the hypothalamic ventromedial nucleus (vmh) and the islands of caueja (icj) had also an important deficit of ot binding sites. on the other hand, these two regions were known to be sensitive to sex steroids. in the present work, we treated from 20 month old rats during one month with testosterone propionate (2 #g/kg s.c., once every 3 days) dissolved in oil. three rats of the same age injected with oil only served as controls. we labelled ot receptors throughout the brain of old rats using a 125i-labelled ligand specific for ot receptors. analysis of autoradiograms by an image analyzer revealed that the testosterone treatment increased ot binding sites in the vmh, in the icj, and, to a lesser extent, in the bed nucleus of the stria terminalis, a region also sensitive to sex steroids, by contrast, in the caudate putamen, the disappearance of ot receptors was not compensated. in conclusion, the decrease of ot receptors occurring in vmh and icj with aging can be reversed by administration of gonadal steroids. in contrast, the loss of ot receptors in the striatum appears to depend on another mecanism. vasopressin (avp) receptors are expressed transiently in the facial nucleus during development (tribollet et el., 1991, dev. brain res., 58, 13-24) . avp may therefore play a role in the maturation of neuromuscular connexions in the neonate rat, and possibly in the restanration of these connexions after nerve lesion in the adult. in order to investigate the latter proposition, we have sectionned the facial nerve in adult rats and used quantitative autoradiography to look at avp binding sites in the facial nucleus at various postoperative times. we observed a massive and transient increase of avp binding sites on the operated side. the number of facial avp binding sites reaches a maximum about one week after nerve section, remains stable during 2-3 weeks, then begin to decrease towards control level. the induction of avp receptors is markedly delayed if the proximal stump of the nerve is ligated. to assess whether other motor nuclei would also react to axotomy by up-regulating the expression of avp receptors, we have sectionned the hypoglossal nerve and the sciatic nerve. in both cases, the binding of avp receptor ligand increases massively in the respective motor nuclei, with a time-course similar to that found in the facial nucleus. altogether, our data suggest that central avp could be involved in the process of nerve regeneration. cytotoxic t-cell mediated apoptosis schaerer,e, karapetian,o.,adrian,m. and tschopp,j. inst.de biochimie, univ.de lausanne, 1066 epalinges. an apoptotic cell death mechanism is used by cytolytic t cells (ctl) to lyse appropriate target cells. ctl harbor cytoplasmic storage compartments, containing the lytic protein perforin and serineproteases (granzymes), whose content is released upon target cell interaction. we show that these granules are multivesieular bodies and that degranulation releases these intragranular vesicles (igv) having granzymes, t-cell receptor and yet undefined proteins associated. isolated igvs and perforin induce dna breakdown in target cells within 20 minutes. microscopic analysis demonstrates that igv specifically interact with target cell via the t-cell receptor and that their contents is taken up by the target cell. already 15 min. after interaction, 3 distinct igv proteins are found in the nucleus of the target cell.one of the molecules has been identified to be granzyme a, previously reported to be involved in apoptosis. we propose that lymphocytes transfer apoptosisinducing proteins to the nucleus of the target cells using vesicles as vehicles for delivery. cytotoxic t cells kill their targets by a mechanism involving membranolysis and dna degradation (apoptosis). recently, two sets of proteins have been proposed as dna breakdown-inducing molecules in t cells: granzyme a, b and tia-i. in this study, we cloned and further characterized the tia-i mouse homologue. aa sequence comparison with the human tia-1 showed an overall identity of 93%. devoid of a signal peptide, tia is yet localized to cytotoxic granules, probably targeted via a gly-tyr-motif. as tia-i, its mouse homolcgue contains three rnabinding domains. expression of tia during development shows a very strong signal in the brain and weaker signals in thymus, heart and other organs. during embryonic development several structures that contribute to organogenesis form transiently and are later eliminated by apoptosis. this pattern of tia expression could indicate its involvement in apoptosis. prostate involution occurs after castration in rats and is associated with the death by apoptosis of a large fraction of the epithelial cells. we have isolated several genes from a prostate involution bacteriophage lambda library using differential screening methods. among these clones, one d~monstrated an especially strong signal when used as a probe against northern blots of prostate mlhna obtained before, and at different times after castration. this gene is down-regulated after castration by 40-fold within 5 days. intramuscular injection of a testosterone depot resulted in complete restoration of expression within 24 hours. upon sequencing it became apparent that this clone has a high degree of homology to a known ndah dehydrogenase encoded in mitochondrial dna. the clone failed to hybridize to any transcripts from rat organs other than prostate. we are now in the process of isolating the htm~n hc~olog to this gene for use as a biomarker in study of benign hyperplasia and developing carcinoma. this gene is a possible indicator for testosterone-independent cell populations or of cells lacking ftl~ctional testosterone receptor. during the first three postnatal weeks the rat lung undergoes the last two developmental stages, the phase of alveolarization and the phase of microvascular maturation. the latter involves a decrease of the connective tissue mass in the alveolar septa and a merging of the two capillary layers to a single one. speculating that programmed cell death may play a role during this remodeling, we searched for the presence of apoptotie cells in rat lungs between days 10 and 24. lung paraffin sections were treated with y-terminal transferase, digoxigenin-dutp, and anti-digoxigeninfluorescein-f(ab)-fragments, and the number of fluorescent nuclei was compared between sections at different days. while the number of apoptotie ceils was low until the end of the second week and at day 24, we observed an about eight fold increase of fluorescent nuclei towards the end of the third week. we conclude that programmed cell death is involved in the structural maturation of the lung. brunner, a., wallrapp, ch., pollack, i, twardzik, t. and schneuwly, s. lehrstuhl genetik, biozentrum universit~t w~rzburg, mutants in the giant lens (g/l) gene show a strong disturbance in ommatidial development. in the absence of any gene product, additional phetoreceptors, cone cells and pigment cells develop. opposite effects can be seen in flies in which the gene product of the giant lens gene can be ectopically expressed by heat shock. a second very typical phenotype is the disturbance of photoreceptor axon guidance. molecular analysis of gil shows that it encodes a secreted protein of 444aa containing three evolutionary conserved cystein-motives very similar to egf-like repeats. we propose that gil functions as a secreted signal, most likely a lateral inhibitor for the development of specific cell fates and that gil, either directly or indirectly, is involved in targeting photoreceptor axons into the brain. the decrease in cellularity during scar establishment is mediated through apoptosis desmouliere, a., redard, m., darby, i., and g. gabbiani department of pathology, cmu, 1 rue michel server, 1211 gen~ve 4 dudng the healing of an open wound, granulation tissue formation is characterized by replication and accumulation of fibroblastic cells, many of which acquire morphological and biochemical features of smooth muscle cells and have been named myofibroblasts (sch0rch et el., histology for pathologists, t992). as the wound evolves into a scar, there is an important decrease in ceuuladty, including disappearance of myofibroblasts. the question adses as to which process is responsible for myofibroblast disappearance. during a previous investigation on the expression of (z-smooth muscle actin in myofibroblasts, we have obsewed that in late phases of wound healing, many of myofibroblasts show signs of apoptosis end suggested that this type of cell death is responsible for the disappearance of myofibroblasts (darby et al., lab. invest. 63:21, 1990) . we have tested this hypothesis by means of electron microscopy and morphometry and by in situ end-labeling of fragmented dna (wijsman et al., j. histochem. cytochem. 41:7, t 993) . our results show that the number of apoptotic cells increases as the wound closes and suggest that this may be the mechanism for the disappearance of myofibroblasts as well as for the evolution of granulation tissue into a scar. (supported by the swiss national science foundation, grant n~ s01-16 r. jaggl, a. marti and b. jehn. universit~t bern, akef, tiefenaustr. 120, 3004 bern at weaning the mammary gland undergoes a reductive remodelling process (involution) which is associated with the cessation of milk protein gene expression and apoptosis of milk-produclng epithelial cells. this process can be reversed by returning the pups to the mother within 1 day. elevated nuclear protein kinase a (pka) activity was observed from one day post-lactation, paralleled by increased c-los, junb, ]und and to a lesser extent c-]un mrna levels. ap-1 dna binding activity was transiently induced and the ap-1 complex was shown to consist principally of cfos/jund. oct-1 dna binding activity and oct-1 protein were gradually lost from the gland over the first four days of involution, whereas oct-1 m_rna levels remained unchanged. comparing nuclear extracts from normal mammary glands with nuclear extracts from glands which had been cleared of all epithelial cells three weeks after birth revealed that pka activation, ap-1 induction and oct-1 inactivation are all dependent on the presence of the epithelial compartment. the increased fos/jtm expression and the inactivation of oct-1 may be consequences of the increased pka activity. when involution is reversed, both, pica activity and ap-1 dna binding activity (and fos andjun mrna levels) are reduced to basal levels. our data suggests a role for pka and ap-1 on progranlmed cell death of manlnmry epithelial ceils. bcl-2~ does not require membrane attachment for its survival activity c. borner*, i. martinout, c. mattmann*, m. irmler*, e. sch&rrer*, j.-c. martinou-j-, and j. tschopp*. * institute of biochemistry, university of lausanne, 1066 epalinges, 1 institute of molecular biology, glaxo inc.,1228 plan los ouates. 8cl-2(z is a mitochondrial or perinuclear-associated oncoprotein that prolongs the life span of a variety of cell types by interfering with programmed cell death. how it exerts this activity is unknown but it is believed that membrane attachment is required. to identify critical regions in bcl-2o~ for subcellular localization and survival activity, we created by site-directed mutagenesis, various mutations in regions which are most conserved between the different bcl-2 species. we show here that membrane attachment is not required for the survival activity of bcl-2o< a truncation mutant of bcl-2(z lacking the last 33 amino acids (t3) including the hydrophobic domain is soluble, yet fully active in blocking apoptosis of sympathetic neurons induced by ngf deprivation or l929 fibroblasts induced by tnfc~ treatment. we further provide evidence for a putative functional region in bcl-2 which lies in the conserved domains 4 and 5 upstream of the hydrophobic cooh terminal tail. the breakdown of nuclear dna is considered to be a hallmark of apoptosis. we previously identified the perinuclear membrane localized dnase i as the endonuclease involved in the formation of oligonucleosomal-sized fragments (dna ladder). it is not clear how the nuclease is activated and has access to the dna. we show that in thymocytes induced to undergo apoptosis, lamin breakdown preceded dna laddering. by transfeeting hela cells with a constitutively active cdc2 mutant, nuclear envelope breakdown and typical apoptotic features (ehromatin condensation) were observed. moreover, co-transfection with cdc2 mutant and dnase i led to dna degradation. we propose that apoptosis can be induced by wrongly timed and hence abortive mitosis leading to uncontrolled nuclear membrane disintegration. s02-01 s02-04 platelet-derived growth factor (pdgf) is thought to play an active role in fibrosing diseases. bronchiolitis obliterans-organizing pneumonia (boop) is a condition characterized by intraluminal proliferation of connective tissue inside distal air spaces. to evaluate pdgf expression in boop we performed immunohistoehemistry on lung biopsies from 20 patients and 10 controls free of fibrosis. sedal sections were stained with an antibody against either pdgf or the monoeyte/macrophage marker cd68, in both groups the pdgf ~9 cells were essentially tissue macrophages. using point counting to measure volume fraction (vv) , pdgf-pesitive cells represented 4.65+1.63% (mean+sd) of the volume occupied by lung tissue in the boop cases, and 2,12+0.65% in the controls (!0<0,001). similarily, 10.73+4.69% of the lung tissue was occupied by cd68 e~ macrophages in the boop cases, compared to 5.37:~3.73% in the controls (p<o.005). a positive linear correlation was found between the v v of pdgf ~ cells and the v v of cd68@ cells on the 30 cases (r=g.50 p<0.0o5). we conclude that boop is characterized by a recruitment of tissue macrophages and that a significant proportion of them express pdgf. as pogf can also induce the macrophage chemo-attractant mcp-1, we speculate that maerophages are involved in a positive feed back regulation, and may play a pivotal role in the pathogenesis of boop. barrier lesions in hydrostatic pulmonary edema d.wu, h.bachofen, u.gyger and e.r. weibel department of anatomy, university of bern, ch 3012, bern pulmonary edema induced in isolated perfused rabbit lungs by moderate elevation of perfusion pressure (14 tort ) results in the formation of characteristic bleb-like extensions of the thin cytoplasmic leaflet of alveolar epithelium ( bachofen et al. am rev resp dis. vo1.147. pp989-996,1993) . in this study, we show by means of electron microscopy and morphometry that the frequency of the blebs correlate with the distribution of alveolar edema fluid. we find that 5% of the epithelial surface is involved in the formation of blebs. three-dimensional reconstruction showed that blebs had near-spherical shape and that their opening toward the interstitium is irregular. these finding demonstrate a great plasticity of epithelial cell extensions when subjected to moderate pressure stress. at high pressure the cell linings demonstrate clear breaks. two major roles have been defined for no: cellcell communication mediated by the stimulation of cgmp synthesis, and cytotoxicity by direct interaction of the free radical no with cellular target. to investigate these effects in human epithelia, which constitute a major source of no in man, we introduced the murine inos sequence into sv40-t immortalized human bronchial epithelial cells (beas-2b). inos activity was higher than 100 pmol citrulline/min/ mg prot in the first passages of inos transfected cells, but it decreased by subculturing. no inos activity was detected in cells transfected with control vector. in inos transfected cells, no stimulated a cgmp synthesis and c-los expression which could be inhibited by addition of lnma. thus, in human epithelial cells no is able to significantly alter signal transduction pathway. partial pneumoneetomy, i.e. the resection of lung tissue, is known to initiate compensatory growth in the remaining lobes. under appropriate conditions this results in a complete restoration of lung parenchymal structure and function. unfortunately the molecular mechanisms controlling the onset of this compensatory growth are poorly understood. the aim of our study was to find and eharacterise genes which are up or down regulated during this time. for isolating such genes, we chose the differential display approach (p.liang and b.pardee 1992, science 257:p 967). therein a 3'end fragment of a subset of reverse transcribed mrnas is amplified by the polymerase chain reaction. the fragments can be separated on a dna sequencing gel. genes differentially expressed in pneumonectomised and sham-treated rats show different patterns on these gels. the fragments of such genes can be isolated from the gel and cloned into vectors in order to provide tools to characterise their gene. a set of such genes, some up-regulated and others downregulated following pneumonectomy, have been isolated, partially sequenced and characterised. time periods. our new plasma marker consisting of highly concentrated gold nanospheres was infused via a femoral vein catheter into the right atrium of anaesthetised and ventilated rabbits. five or 10 seconds after start of infusion, the blood flow was stopped and the lung was fixed by instillation. silver enhanced paraffin sections revealed labeled and unlabeled blood vessels. electron-microscopic sections showed various plasma gold particle concentration. morphometric analysis of electron micrographs showed an increase of the portion of marked capillaries from shorter to longer circulation times. based on our results, we conclude the existance of inhomogeneous plasma flow velocities within the pulmonary microvascular bed. (supported by snsf grant 31-30946.91) in schizosaccharomyces pombe pairing of meiotic chromosomes is not accompanied by the formation of a tripartite synaptonemal complex (sc), a structure which connects the homologs in most other eukaryotes. meiotic nuclei revealed novel structures ("linear elements") that might be related to the axial elements ($c precursors) of other organisms and function in meiotic chromosome pairing, recombination, and segregation (baler et al. (1993) jcb 121:241). to get further insight into the behavlour of meiotic chromosomes, we performed fluorescence in situ hybridizations on spread nuclei with probes from different chromosomal regions. interestingly, all centromeres and telomeres become clustered during meiotic prophase. the centl:omeres are paired already before meiosis, whereas the telomeres initiate pairing specifically during meiotic prophase, followed by pairing of interstitial regions. painting of whole chromosomes revealed that the homelogs occupy together a specific territory in the nucleus. an expression vector containing the vai gene read by rna polymerase ill, injected into xenopus zygotes, yields high levels of rna in virtually all embryonic cells. anti sense fina is produced from a segment of the xhoxla gene inserted in opposite orientation into the vai gene. immunological staining of whole mount embryos shows that target mrna level and xhoxla protein expression of xhoxla protein is diminished in about half of the antisense treated individuals. accordingly, nearly half of the embryos develop typical malformations in regions where the xitoxla gene is normally expressed. somites el the anterior trunk are heavily disorganized and mesodermal denvalives surrounding the intestinal tract are reduced or missing. antisense inhibition, through production of rna in situ from expression vectors, thus represents a useful tool to study the functional role of zygotic genes in xenopus development. cloning and characterization of the mouse homolog to s.cerevisiae sep1 encoding a dna strand exchange and homologous pairing protein vladimir bashkirov and wolf-dietrich heyer. institute of general microbiology, university of bern, baltzer-strasse 4, 3012 bern. homologous pairing and dna strand exchange are the central steps postulated in the current models of genetic recombination. to date no mammalian gene encoding a homologous pairing protein has been cloned. to get insight into the mechanism of recombination in higher eukaryotes we were interested to clone such genes. protein sequence comparison of the s.cerevisiae homologous pairing protein, sepi, and its homolog from s.pombe, p140 ex~ revealed several conserved stretches of amino acids in their nh2-terminal regions. using fully degenerate primers and mouse testes edna as a template for the polymerase-chain-reaction a product with the expected length has been amplified. this 360 bp dna was shown to encode a putative polypeptide with high degree of homology to both proteins from the two yeasts. the mouse pcr-product was used as a hybridization probe for screening a 1 gtlo mouse testis cdna library. after several rounds of successive screening a total of about 4 kb of mouse edna (msep1) was cloned and sequenced. the putative translation product revealed high homology throughout the entire sequence to its s.pombe and s.cerevisiae counterparts, 38 % and 41% of amino acid identity, respectively. the msep1 sequence was shown to be unique in the genome and the chromosomal gone is likely to contain many introns. we want to establish a functional full length edna clone of msepi and characterize the gone and protein product. we have earlier developed a cell free system to study recombinational repair of dna double strand breaks and deletions. the method allowed us to purify and characterize a high molecular weight multiprotein complex (rc-1), which catalyzes dna recombination. a dna polymerase and dna ligase as well as other enzymatic activities copurify with rc-1.we have commenced an investigation of homologous dna recombination in nuclear extracts which were prepared fzom normal and mutated mouse thymocytes. the scid mutation in mice causes an aberrant v(d)j joining reaction, an elevated sensitivity to ionizing radiation and a defect in recombinational repair. moreover, the normal mouse thymocytes were sorted into the cd4/cd4 double negative (dn), double positive (dp), and single positive (sp) subclasses and their nuclear extracts, and extracts from rag-2 -/-mice, were tested. only the scid mice and the cd4/cd8 sp thymocyte extracts were inactive in the recombinational repair reaction. this indicates a development stage specific activity and a scid specific defect of recombinational repair in thymocytes. restoration of the recombination activity was observed in thymocyte extracts derived from scid, which express a wansgene for the t-cell receptor i~ chain. a protein could be purified from normal thymus cells to near homogeneity which specifically rescues the recombination activity in scid extracts. this protein, srsp ~cid recombination stimulatory ~otein), migrates as a single band of app. 7 2 kda in sds polyacrylamide gel electrophoresis. the ade6-m26 mutation in the ade6 gene of the fission yeast schizosaecharomyces pombe gives rise to a hot spot for meiotic homologous recombination. to address whether transcription plays a role in m26 activity, the effect of the deletion of the ade6 promoter in combination with either the m26 and m375 mutations on recombination frequency was studied at the tetrad level. deletion in eis of the promoter abolishes m26 hot spot activity relative to the m375 control deletion strain. it is possible that deletion of the promoter has eliminated a sequence necessary for m26 hot spot activity, rather than lack of transcription being responsible for the loss of m26 activity. the role of transcription in m26 hot spot activity is currently being examined. we are analysing meiosis in a temperature sensitive top2 mutant. complementary temperature-shift experiments showed that topoisomerase ii is required for both meiotic divisions; early meiotic events are not impaired. in a meiotic time course, dapi staining revealed that the cells are blocked before the first meiotic division. interestingly, in meiotic spreads we found that the spindle pole body (spb) cycle is not affected by the block: the final arrest point showed 1 nucleus with 4 completely separated spbs. we want to analyse the number of spbs and spindle formation with immunofluorescence. we are planning to analyse the course of meiosis in a top2 rec7 double mutant (rec7 is recombination deficient) to see if topoisomerase ii activity is necessary for separation of recombined chromosomes. analysis of meiosis in a top2 rec8 double mutant (rec8 is recombination deficient and does not form linear elements) could give us a clue about the function of the linear elements. genetic recombination is able to induce cancer and to mediate its further progression. in cells from cancer predisposed individuals, mitotic recombination can lead to loss of heterozygous tumor suppressor genes as is e.g. observed in the hereditary form of retinoblastoma. one mechanism for gene loss is inter-or intrachromosomal recombination involving short duplicated sequences. our aim is to identify xenobiotics that are able to induce mitotic recombination and to elucidate the involved molecular mechanisms. for that purpose we have constructed transgenic d. melanogaster (1). rn.). cell lines. cells were stably transfeeted with an extrachromosomal dna element providing a putative recombination substrate+ "l%e constructed element contained two ta'uncated fragments of the neomycin resistance gene (nee r) interrupted by a hygremycin resistance marker (hygr). the hyg r insert was flanked on both sides by identical 352 bp fragments of nee r, different d. m. promoters shown to be constitutively active in various d. m. tissues were inserted 5' to both e. coil genes. restoration of a functional nee r gone may occur by deletion of the hyg r after recombination between the homologous regions. stably transfected schneider line 2 cells were obtained and preliminary results concerning chemical induction of genetic rearrangements will be shown. fleck, 0., sch~r, p. and kohli, j., institute of general microbiology, baltzerstr. 4, 3012 bern to detect mismatch-binding proteins of s. pombe we performed band shift assays with radiolabeled oligonucleotides containing a defined mismatch. we found two distinct mismatch-binding activities. one shows high affinity to most of the mismatches, but is absent for c/c. this protein might belong to the general mismatch-repair system, which is thought to be related to the bacterial muts dependent pathway. the second activity preferentially binds to those mismatches containing a cytosine and therefore represents a novel type of mismatch-binding protein. baur m., sch~r p. and kohli j., institute of general microbiology, baltzerstrasse 4, ch-3012 bern homologs of the salmonella typhimurium (mutl, s and 59 and streptococcus pneumoniae (hexa, b) genes involved in mismatch repair have been identified in several distantly related organisms. so far no mismatch repair gone is known in schizosaccharomyces pombe. in order to get more insight into the ~chanisals of mismatch repair in s. pombe we started the cloning of a mutl homolog. degenerated oligonucleotide primers based on conserved regions of mutl homologs were used in the polymerase chain reaction (fcr) to amplify a corresponding dna fragment from s. pombe. a dna fragment of about 220 bp was amplified whose deduced amino acid sequence shared a high degree of homology with paiql of saccharomyces cerevisiae and mutl, hexb. with the help of this dna fragment the gone was isolated from a genomic dna library by colony hybridization. sequence analysis of this mlhl gene (mlh = mut~ homolog) shows an orf of the expected size with three putative introns. while the deduced amino acid sequence of the 5' region shares strong homology with the procaryotic genes mutl, hexb and the eucaryotic gene pmsi, the amino acid sequenoe of the 3' region shows homology only to pmsi and hexb. the central region of the protein seems to be conserved only weakly. parisi s., and kohli j., institute of general microbiology, university of bern, ch-3012 bern rec7 and rec8 are supposed to be genes with major functions in meiotic recombination since mutations in these genes reduce the frequency of meiotic intragenic recembination ~1000 fold. these genes have been isolated and sequenced but no enzymological activity could be assigned to the protein. a first step toward enzymological and biochemical characterization of rec7 and rec8 is to make beth protein products available in sufficient quantities and in reasonable pure form. for this purpose both genes will be cloned in a s. /x~nbe over-expression system using the strong and inducible nmtl promoter.in parallel both genes will also be expressed in e. coli as gst fusion proteins in order to produce polyclonal antibodies in rats and rabbits. these antibodies can then be used for protein purification in s. pombe as well as for immunofluorescence experiments. induction of expression in e. coli produces a band of the expected size of 56kda for rec7 and of 73kda for rec8. to remove the gst carrier, the rec7 and rec8 fusion proteins can successfully be cleaved by thrombin or factor xa respectively. production of antibodies is in progress. d6bbeling, u., nobi, r,, berchtold, m.w. and kuenzle, c.c. institut fdr veterinarbiochemie, universit6t zurich, wintertharerstrasse 190, normally, only pre b-and pre t-cells can rearrange their immunoglobulin genes by v(d)j recombination, but when other cell types are transfected with the rag-i and rag-2 genes they also become able to perform v(d)j recombination. by transiently transfeeting nih 3t3 fibroblasts with both intact rag genes we found da recombination rate of 0.06%. no recombination was detected, when we transfected the intact rag-1 gene with a mutant rag-2 gene, which lacks 89 c-terminal amino acids, or the intact rag-2 gene with a rag-i gene containing a deletion in the topoisomerase iz homology region. these experiments show that these two regions of the rag genes are essential for v(d)j recombination. when the l4 fibroblast cell line, which has been rendered recombination competent by stable transfection with the rag genes was overtransfected with both wild type rag genes, we found in contrary to an expected increase of v(d)j recombination a reduction by a factor of 2-2.5. this decrease was not observed with the mutant rag genes.this result indicates, that there exists an optimal intracellular rag concentration for efficient v(d)j recombination. m. fischer, a. sailer, h. blieler, t. riilicke*, a. aguzzi +, p. autenried and c. weissmann; 1nstitut fiir molekularbiologie l universitfit ziirich, h6nggerberg, 8093 ziirich, *biologisches zentrallabor and + lnstitut fiir neuropathologie , universitdtsspital ziirich, 8091 zfirich, switzerland the infectious agent of transmissible spongiform encephalopathies such as creutzfeldt-jakob disease in man or scruple in sheep is thought to be prpsc, a posttranslationally modified form of the normal host protein prpc we have shown that mice devoid of prpc (prn-polo) are completely resistant to scrapie. here we report that also heterozygous prn-p o/+ mice with reduced levels of prp c show enhanced resistance to scrapie, as manifested by a long delay in the onset and slow progression of clinical disease. conversely, prn-po/o animals overexpressing prp transgeues exhibit shortened incubation times and accelerated progression of the disease. propagation of the scruple agent is completely abolished in prnpolo animals. high titers of infectivity, about 108 logld50 units/g of brain, are detected in prn-po/+ mice 33 weeks after inoculation (the earliest time point measured), 24 weeks or more before the animals die of scrapie. in contrast, wildtype animals survive no more than 16 weeks after reaching similar titers of infectivity. a practical implication of our findings is that moderate inhibition of prpc synthesis could mitigate disease progression in spongiform encephalopathies. the ruvb protein has been shown, biochemically and genetically, to be involved in the process of homologous recombination in e. coli. one of its functions is to promote branch migration within holliday junctions formed during the process of recombination. to investigate the mechanism by which ruvb promotes branch migration we have used conventional electronmicroscopy, cryo-electron microscopy, scanning transmission electronmicroscopy, and image analysis to study the interaction of ruvb protein with dna. we observed that ruvb forms multimeric rings on dsdna which encircle the dna. we also observed that such rings tend to localize at the holliday junctions. we propose that ruvb rings can actively move along dna using the energy of atp hydrolysis.this movement continues upon encountering a holliday junction, thus forcing branch migration along the dna. gschwind m. and huber g., pharma division, preclinical research, in some families with early onset aizheimer's disease mutations on the 8-app gene have been identified which cosegregate with the disease. so far all these pathogenic mutations have been identified on exons 16 and 17 in humans framing the sequence of the 8-amyloid itself.a genomic clone was isolated from mouse strain c57 black/6 containing the coding regions of the last three exons (16) (17) (18) according to the human sequence. sequencing of all three exons including the large 3' non coding region showed a 100% homology to the previously described balb/c mouse cdna. exon-intron boundaries were determined at the same positions as in human and rabbit genome and the flanking regions in the introns are also very similar, not only is the homology between the human and mouse f$-app very high (97.6%) but also the genomic organisation of their genes is nearly identical. therefore homologous recombination can be applied to introduce molecular 8-app changes and to study their functional consequences on the protein level. we systematically investigated the ability of reca protein to push the dna strand exchange reaction through heterologous regions. we observed that reca can push the strand exchange through substantial heteroiogy (up to 150 bp) when the heterologous insert was placed on the so called proximal end of the linear duplex. in the case of the medial beterology the strand exchange reaction could overcome heterologies of up to about 50 bp. heterology at the distal end was most difficult for reca to resolve, and already 22 bp long insert was completely blocking the progress of the strand exchange. these results, together with earlier electron microscopy studies, allow to propose a molecular mechanism by which reca can exchange strands between partially heterohigous dna molecules. institut for veterin~r-virologie, universit~t bern, ch-3012 bern bovine viral diarrhae virus is a positive-stranded rna virus of the pestivirus group. two biotypes, cytopathic and non-cytopathic in cell culture, can be distinguished. all cytopathic strains characterized so far carry insertions in their genomes. they probably result from a strand-switching activity of the viral rna polymerase during viral replication. to study the switching capacity of the viral enzyme, rna isolated from cells infected with two different strains of bovine viral diarrhea virus were analysed for homologous recombination in an rt-pcr assay. performing the assay we found that fragmented rna present during reverse transcription, the reverse transcriptase enzyme itself and viral rna present in the pcr reaction result in the production of artificial recombination during the assay. it was not possible to detect a homologous recombination activity of the viral polymerase above the background recombination frequency of the rt-pcr assay. our results strongly question the use of the rt-pcr assay for the study of recombination of rna viruses. transpositional rearrangements mediated by insertion sequence (is) elements represent an important source of genetic variation within populations 1. we analyze dna rearrangements at the level of the complete genome by rflp, using 7 is elements as probes for hybridisation to southern blots samples taken at different time points from 12 independent cuttures of e coli b grown by serial transfer for 10'000 generations 2 and stored at -70~ are used to study structure and evolution of genetic diversity within populations. the seven is elements contribute differently to genetic variation and lead to a succession of mutants affecting the genetic structure of the population. we now search for correlations between rflp patterns and the increase of relative fitness over time observed previously 2 naas t., 81o~ m, fitch w.m and arber w, (1993) the complete dna sequence of the locusta migratoria mitochondrial genome has been derived from four cloned mtdna fragments. the sequence is 15.2 kb in length, contains 13 peptide coding sequences, and genes for 22 trnas and two rrnas. the organisation of the genome shows a strong resemblance to drosophila, the gene order and orientation being the same except for the positions two trnas. this contrasts with the only other non-dipteran insect mtdna to have been sequenced, apls mellifera, which shows differences in the positions of ii trnas. this finding is discussed in relation to the at content of the different genomes. the sequences of the three mtdna genomes are also compared directly and the results related to existing knowledge concerning the evolution of insects. udem, new jersey medical school, newark, nj 07103-2714, usa nonsegmented negative strand rna viruses are so far not amenable to reverse genetics. the genomes must associate with nucleocapsid (n), phosphoprotein and polymerase proteins to form a functional rnp. as a step towards reverse genetics of measles virus (mv), plasmid vectors were constructed allowing synthesis of rnas corresponding precisely to a mv genome in which all coding and intercistronic regions are replaced by the chloram-phenicol acetyl transferase (cat) coding region, transfection of these negative polarity transcripts into mv infected hela or 293 cell lines gave rise to cat activity which could be serially transferred and massively amplified together with progeny helper virus in cell cultures. both expected mv/cat chimeric mrna and replication product were identified in the progeny. preliminary experiments indicate that only rnas in which the total number of nucleotides is a multiple of six replicate and transcribe efficiently, consistent with the data on natural and modified copyback di rna of sendal virus (calain and roux, j. virol. 67 4822 (1993) ). precise end to end encapsidation of rna, each n protein contacting six nucleotides, might be required. the embryonic tissue specificity of the human cytomegalovirus immediate-early (hcmv-ie) promoter/enhancer (-522 to +13) was examined by l~-galactosidase staining of transgenic mouse embryos carrying hcmv-ie regulated lacz genes. embryos of three transgemc lines were analyzed between 8.5 -13.5 dpc. for all lines, hcmv-ie transcription was primarily confined to subsets of neural and vascular cells. however, extents of transgene expression along the embryonic primary axis were slightly and greatly restricted in two of the lines relative to the third line at all stages analyzed. these differences most likely represent integration site-dependent chromatin constraints that affect levels of hcmv-ie-directed lxanscripfion. together, the three lines can be taken to define a graded potential for hcmv-ie transcription along the anteriorposterior axis of the embryo with greatest permissivity in mid-axial structures which include the dorsal neural tube at the level of the hindbraln and upper cervical spinal cord, the inner ear and vestibular acoustic ganglia, branchial arteries, aortic sac, and lung buds. the cell typespecificity and axially graded permissivity for hcmv-ie transcription in the mouse embryo provide a basis for understanding the pathology and relative frequencies of different types of birth defects caused by congenital hcmv infection in humans. stauffer, y., 0fford, e. and beard, p., swiss institute for experimental cancer research (isrec), ch-i066 epalinges, switzerland. hpvi8 is associated with genital carcinoma. like other human papillomaviruses, hpvi8 cannot be easily propagated in cell culture, because the viral growth cycle is dependent on the differentiation of its host cell, the keratinocyte. moreover, very little viral capsid protein is found in vivo in infected tissues. the hpvi8 l1 and l2 genes code for the major and minor capsid proteins, respectively. to obtain the viral capsid proteins we made constructs with the l1 and l2 genes for expression in e. coli (the pqe system) and in recombinant vaccinie virus (a modification of the phgs-i system) infected cells. the l1 and l2 proteins expressed in e. cell contained a histidine tag encoded by the vector. this allowed us to purify the proteins on a sickelaffinity column in order to raise antibodies. these will permit us to detect the presence of capsid proteins expressed from recombinant vaccinia viruses. we expect the l1 and l2 proteins expressed together from the vaccinia-based vector to selfassemble to form virus-like particles. to papain-like proteinases. the role of these putative catalytic residues in the activity of the proteinase was studied by site-directed mutagenesis. a minimal proteinase has also been constructed by n-and c-terminal deletion to determine the boundaries of the proteolytic domain, sequence analysis of the c-terminal cleavage product should indicate the site of cleavage. cell entry, uncoat~ng and nuclear transport of adenovirus z . u. f. greber and a. helenius. yale university school of medicine, new haven, ct, u.s.a. to enter cells, viruses must achieve three major goals: transfer their genome across the plasma membrane into the cytosol, target the genome to the replication site and decondense it for replication. adenovirus, an icosahedral nonenveloped particle with a double stranded genomic dna and ii to 15 structural proteins~ enters into human kb cells by a sequential disassembly program during receptor-mediated endocytosi~, acid-dependent penetration into the cytosol, and targeting to the nucleus. during internalization, the fibers, primarily responsible for virus binding to the cell surface, are detached. in early endosomes, the capsid-stabilizing proteins ilia and viii are released. fenton base, a vertexassociated protein implicated in penetration across the endosomal membrane, is removed in endosomes, if penetration is blocked with lysosomotroplc reagents. in the absence of inhibitors, a large fraction of penton base stays associated with the cytoplasmic capsid. the viral dna is disconnected from the shell after proteolysis of the internal protein vi~ a capsid and dna binding component. removal of protein ix, a capsid binding factor, further destabilizes the coat, and the remaining 'stripped-down' virus particles associate with nuclear pore complexes to release their dna-genomes into the nucleoplasm. this stepwise dissociation program is thought to provide the high efficiencies of the entry process needed to successfully deliver the viral dna across multiple barriers to the cell nucleus, the site of replication. ch. schmidt and w. arber; biozentrum der universit~t basel, abt. mikrobiologie, klingelbergstr. 70, ch-4056 basel bacteriophage p1, carried as a single copy plasrnid in e. col~, is widely known for its horizontal gene transfer ability (transduction) in enterobscteria. as a temperate phage, it can lysogenize its host and thereby provides it with accessory functions (restriction of foreign dna by ecop1, functions expressed by transposons carried on the p1 genome or integrative suppression). these properties reflect both symbiotic and evolutionary functions of pi. p1 regulates expression from its genes by two different mechanisms: trans(:ription of early regulatory genes is repressed by the phage encoded repressors c1 and bof, whereas transcription of its morphogenetic genes from p1 specific late promoters is induced by the early expressed gpl0. by site-directed mutagenesis the late promoter ps, composed of the -22 inverted repeat aagttactt and the -10 box, was functionally characterized. p5 and its derivatives compare well with several other late promoters of pt with regard to their sequence dependent strengths: the strong promoters ps, pdar and lpr2b all share the perfect inverted repeat around position -22, whereas the weaker promoters lpr91, lpr82a, lpr82b and lpr83 contain mismatches in their inverted repeats or the inverted repeat is positioned mere upstream relative to the transcription initiation site. homologies. however, the phages formed two immunity groups. the results may suggest a common genetic trait providing a selective advantage to lysogenic aa. by using a dna substrate with defined gap size we found that human immunodeficiency virus type 1 reverse transcriptase (hiv~rt) was able to perform strand displacement dna synthesis. this activity was not affected first by calf thymus proliferating cell nuclear antigen and replication factor c and second by escherichia coli single-stranded dna binding protein~ which together allow dna polymerase ~ to perform strand displacement dna syrlthesis (podust , v. and h~bscher, u. (1993) nucl. acids res. 21, . 3'-azido-2',3'-dideoxy-thymidine tripbosphate inhibited displacement completely indicating that dna synthesis is required for this reaction. the hiv-rt p66 polypeptide alone could perform limited strand displacement dna synthesis, whereas the hiv-rt p51 polydeptide was completely inactive, likely due to its inability to replicate extensively on a mi3 dna template, on the other hand the hiv-rt psl polypeptide e~hanced the strand displacement activity of the hiv-rt d68 subunit at a molar ratio of 4:1 mainly by chasing short products into longer ones. furthermore kinetic experiments after complementation of hiv=rt p66 with kiv-rt psl indicated that hiv-rt psl can restore rate and extent of strand displacement activity by hiv-rt p66 compared to the hiv-rt heterodimer d66/d51, suggesting a function of the 51 kda polypeptide, the mouse mammary tumor virus proviral dna contains an open reading frame in the 3' long terminal repeat which can code for a 36 kda polypeptide with a putative transmembrane sequence and five n-linked glycosylation sites. this gene is known to code for a superantigen which deletes a specific subset el co4 + t-lymphocytes in vivo, the superantigen encoded by the exogenous mouse mammary tumor virus of the gr strain acts specifically on v[314 bearing t-cells. we produced recombinant vaccinia viruses to express either the complete or a truncated orf protein after infection of primate cells in culture. the complete err gene in mammalian cells leads to the production of a 47 kda protein which is specifically detected with an anti-orf-peptide antiserum. the 47 kda protein can be labeled with d-[2-3h]mannose and its synthesis is inhibited by tunicamycin, an n-linked glycosylation inhibitor, indicating that it is a glycoprotein. the truncated orf protein beginning at the second atg of the open reading frame is also modified, but the c-terminal half of orf, starting at the fifth atg (orf5), has the expected size of the non modified polypeptide. pulse-chase experiments indicate that the 47 kda orf g}ycoprotein has a short half-life of about 1.5-2 hours in this system whereas the orf5 truncated protein is even less stable; its half-life is about 20 minutes. the cellular localization of the orf protein and the role it could play in vivo are currently under investigation. conclusion was based on a restriction fragment length polymorphism (rflp) with probes of is-elements. the mutants displayed different growth behavior. the evolutionary stability of 32 of these mutants was further tested by competing a mixture of them for ten days. polymorphism was maintained during this competition but two fitter mutants took over 60% of the population. implications of these results will be discussed in terms of generation and selection of genetic variants for the adaptation of microbial population. mouse mammary tumor virus (mmtv) is produced in the mammary gland of infected females and transmitted by the milk to suckling new-boms. the passage of mmtv from the gut to the mammary gland is still poorly understood, the nature of the tissue tropism of mmtv has not been elucidated, a single receptor present on mammary epithelial and lymphoid cells might serve for the entry of the virus. a cell-type specific entry mechanism could in part be responsible for this phenomenon. alternatively different surlace molecules might be involved in its uptake. we have studied the cell susceptibility to mmtv using a sensitive method of detection of newly acquired exogenous proviral dna sequences, prior to viral integration and transcription. thereby we confirm that mmtv infects mouse, rat and monkey cell lines of different tissue origin in vitro demonstrating the lack of a strict host range specificity in tissue culture as previously described. we have also observed the infection by mmtv of various human cell lines. furthermore various infection susceptibilities are observed for cell lines of a same species. r. cattaneo, p. spielhofer, k. kaelin, m.a. billeter mo/eku/arbio/ogie /, universit~t zorich, hsnggerberg, 8093 zorich subacute sclerosing panencephalitis (sspe), a rare, lethal disease is caused by clonally selected defective measles viruses (dmvs) characteristically mutated. this emerged from sequencing of five mv genes from sspe cases and by functional tests of the envelope glycoproteins hemagglutinin (h) and fusion protein if). these expressed h and f variants migrate poorly to the cell surface but maintain some cell fusion activity whereas the envelope associated matrix protein (m) appears dispensable. thus, the spread of dmv genomes through the brain seems to occur by membrane fusion at local cellular contacts, essentially hidden from the massive immune responses. in more than half of sspe cases, the propagated dmvs contain m genes in which up to 50% of u residues are replaced by cs. such "biased hypermutations" are likely due to the simultaneous conversion of many a residues to inosine in double stranded rna regions accidentally formed, mediated by an ubiquitously occurring adenosine deaminase. similar events at less spectacular degrees have now been observed in functional genes of several negative strand rna viruses. thus, this enzyme might be an important evolutionary driving force for mononegavirates. pull, i., wieland, s., nieder~st, b., keist, r., walter, e., and blum, h.e. department of medicine, university hospital z'tirich infection by hbv-positive organ donors remains a serious problem in transplant surgery. here we present a case of a 58-year-old woman who received a heart transplant from a hepatitis b surface antigen (hbsag) positive donor. the patient was negative for all hbv markers prior to transplantation. two months after transplantation she became hbsag positive and died from subacute liver failure about seven months later. cloning and sequencing of hbv dna revealed several hbv mutants in the serum of the recipient, each with a 108 bp in-frame deletion in the pre-s 1 region. transfection studies in huh-7 cells with the predominant pre-s 1 deletion mutant showed a higher replication competence compared to wild-type hbv. sequence analysis of pcr products from serum of the donor showed mainly wild-type hbv dna in the pre-s region without the pre-s 1 deletion. these data demonstrate that mutant viruses accumulate in immunesuppressed patients. specific mutants may play a critical role in the severe or fatal clinical course of hbv-infection in transplanted patients. in non-neuronal cells herpes simplex virus (hsv) establishes a productive lyric infection starting with transcription of its immediate early (ie) genes by corecruitment of a viral protein (vpi6) and a cellular factor (oct-l) onto characteristic oetamer-like sequence motifs ftaatgarat) in the promotors of the ie genes. in neurons, however, hsv establishes latent infections whereby transcription of the ie genes is prevented and the lyric program aborted. our efforts are focused towards determining which neuronal factors are involved in the regulation of hsv ie gene transcription and whether they may be involved in hsv latency or reactivation in neurons. in electrophoretic mobility shift assays (emsa) using mouse brain nuclear extracts we found that neuronal oct factors (noct-2, noet-3 and noct-4) bind to taatgarat sequences from the viral icp0 and icp4 promoters. an additional brain protein also binds to these sequences but not to a consensus octamer sequence (atgcaaat) as do neuronal oct factors. emsa assays with mutated taatgarat probes show that the taat sequence is crucial for this binding and we have tentatively named this brain protein taat-1. we are currently testing the noct factors in in vivo and in vitro transcription assays to study their involvement in hsv ie promoter regulation. biochemical and functional analysis of a vaccinia virus recombinant containing two copies of the p37k gene. schmutz, c., and wittek, r. institut de biologie animate, univei-sit~ de lausanne, ch-1015 lausanne. the most abundant protein of the vaccinia virus envelope is the palmitated 37k protein. this protein is required for envelopment and subsequent release of virus from infected cells. the amount of extracellular enveloped virus (eev) produced varies considerably depending on the virus strain and host cell line but remains in any case low (<30% of the virus production). if p37k is a limiting factor for envelopment, its overexpression might be a taeans to increase the production of eev. for this purpose a vaccinia virus recombinant containing a second copy of the p37k gene was enginee,'ed. western blot analysis clearly showed increased levels of this protein. titration assays revealed slightly lower yields of both intracellular naked virus (inv) and extracellular enveloped virus (eeu in ceus infected with recombinant virus. unexpectedly, with recombinant virus, the ratio of eev versus inv was significantly lower when compared to wild-type virus infection, despite the higher level of p37k expression. we are currently testing whether this is a consequence of a lower production, or a reduced infectivity of eev particles in cells infected with recombinant virus. hanspeter stalder, christian hertig and ernst peterhans institut f~r veterin~r-virologie, universit-st bern, ch-3012 bern bovine viral diarrhoea virus (bvdv) causes acute transient infection in animals exposed to virus postnataly and persistent infection in animals infected in utero in the early phase of gestation. the latter is associated with specific immunotolerance to the infecting viral strain. animals infected in utero may be born normal and remain healthy despite the persisting infection with a noncytopathic biotype of bvdv. mutation to the cytopathic biotype, or superinfection with an antigenically similar cytopathic biotype, results in lethal mucosal disease. we have pcr amplified and sequenced parts of the region coding for the surface glycoprotein e2 and p80, a nonstructural protein associated with enzymatic activities. over a time of one year, virus obtained from an immunotolerant persistently infected animal remained identical in its nucleotide sequence in the analyzed regions. contrasting with this remarkable evolutionary stasis over some 600-800 virus generations is the heterogeneity of viral isolates obtained from different persistently infected animals. we propose that the muitilineage distribution of bvdv is the result of antigenic drift in the population of acutely infected animals, combined with evolutionary stasis in immunotolerant animals. in 20 to 30% of naturally infected goats, the sevedty of disease in infected animals correlates with the antibody titers to the viral envelope protein (enw) and especially to the gp 38 transmembrane podion (tm). in order to analyze linear b epitopes of env, we produced a random library of caev env polypeptides fused to ~-galactosidase and expressed in e. coli. the complete env gene obtained from an infectious clone of caev-co was subcloned in a puc18 plasmid and dnase digested, random fragments of 200 bp were inseded in ~,gtl 1 dna and expressed in e. coil y1090. as the first step of b epitope mapping and in order to identify immunodominant group epitopes we used high titer sera from naturally infected swiss goats to screen the library. six immunoreactive clones were obtained and subsequently sequenced. all six mapped to the tm region of env. a fine mapping of these immunoreactive regions was performed using pepscan technology and elisa with synthetic peptides. four epitopes could be identified including the eysteine loop corresponding to an immunodominant epitope in other lentiviruses. these peptides were used in vitro to test the reactivity of sera from naturally and experimentally caev-infected goats and in rive to modulate the immune response of goats to tm. the minute virus of mice (mvm) has a 5kb linear single stranded dna genome. at both termini, short selfcomplementary sequences are folded into stable hairpin structures. the 5' (right) hairpin is a single stem structure with only three unpaired bases within the stem. these unpaired form a bubble structure. we examined the requirement in mvm lytic infection for the bubble structure in the 5' terminal hairpin of mvmp. mvmp rf dna containing the entire 3' extremity and extending to the hha i site downstream of the axis of symmetry of the 5' extremity was cloned into pgem-szf(+). this construct contains more than half of the 5' palindrome, however it lacks the sequence information required to form a bubble. murine fibroblast a9 cells were transfected with this dna and the 5' terminal sequence of resulting virus was analysed. a bubble was seen to be generated, however the sequence of nucleotides contributing to the bubble was of n0n-viral, plasmid origin. the nucleotides in question were removed from the original plasmid construct and the experiment repeated. virus was obtained as before. sequence analysis of the 5' end indicated that both flip and flop forms were present. the corresponding 5' hairpin deduced from these two forms contains no unpaired bases at the position where the bubble is normally located in wild type mvmp. the immediate early lie) gene promoters of herpes simplex virus type-1 share a similar sequence which in some cases overlaps with a binding site for octamer binding proteins. this sequence confers responsiveness to a viral transaetivator, vp16. on these promoters, vp16 combines with multiple cellular proteins to form an activating complex. we set up a reconstituted in vitro system consisting of hela nuclear extract and recombinant oct-1/pou domain protein (without the activation domain of oct-1 ) and vp16 where we showed that the activation of ie genes is not mediated by oct-i itself. rather, oct-1 serves to recruit vp16 that has no intrinsic dna binding capacity. vp16, however, possesses a stong acidic c terminal activation domain of -80 amino acids which is indispensable for the in vivo function of the protein. as this and other acidic activation domains possess only a relatively weak activating capability when we test them in in vitro transcription assays we are interested in investigating the role of this acidic activation domain in vitro i.e. under condition of cell free extracts and on naked template dna. we are using recombinant oct-1/pou domain protein and recombinant vp16 that has been truncated at the c terminus. additionally, we are also testing different truncations of vp16 in vitro that have been produced in cos cells. furthermore, we are also trying to see whether these different truncations already affect the assembly of the actvating multiprotein complex. we have studied the kinetics of synthesis of transcripts initiated from the vaecinia virus 7.5 kd gene early promoter. unexpectedly, after a first burst of rna synthesis early in infection, the promoter showed a second peak of activity in the late phase.reactivation of transcription was neither dependent on the presence of the sequences upstream of the early promoter, nor on the location of the promoter in the genome. reactivation seems to be dependent on virus assembly since it is prevented by rifampicin, that specifically inhibits an early step of viral morpho-g~nesis. analysis of several other early genes showed that reactivation is not unique to the 7.5 kd early promoter. analyzing, by in situ hybridization using digoxigenin-labelled riboprobes, the expression of the viral genome relative to that of an array of cytokines including tnf-(z; ifn-% il-i~, il-2, il-8 and gm-csf. initial experiments suggest that viral genome expression is restricted in the inflammatory infiltrates whereas certain cytokines, particularly tnf-oq are strongly expressed in the synovial membranes. overall, the results point to a prominent role of macrophage activation in the maintenance of chronic inflammation and possibly also in the development of arthritic lesions. the core protein of the hepatitis b virus (hbv) interacts with the viral rna pregenome and the reverse-transcriptase.jdna-polymerase to form a core particle which allows for replication of the viral genome. deletion analysis of the core protein revealed, that the arg-rich carboxyterminal domain is required for rna encapsidation and productive viral dna synthesis. we demonstrate, that the duck hepatitis b virus (dhbv) core protein can be derided into two domains comprising amino acids 1-67 (n-ter) and 67-262 (c-ter), respectively. the co-expression of these two core subdomains leads to the formation of replication competent core particles. furthermore, we demonstrate that the carboxy-terminal domain of dhbv core (c-ter) can be functionally replaced by the corresponding domain of the human hbv. the complete hbv core protein, however, does not form replication competent core particles with the dhbv pregenome and rt. these results imply that the carboxy-terminal part of the hbv core proteins define an exchangeable, functionally conserved domain. influenza ha mediates the adhesion and penetration of host cell membranes. acylation of three conserved cysteine residues in the membrane spanning region of several ha subtypes has been shown. the biological significance of this hydrophobic modification remains to be elucidated. a possible role for the membrane fusing activity has been controversially discussed in the past. removal of covalently bound fatty acid from protein by incubation with hydroxylamine is a commonly applied method. as we shall show for a number of different envelope viruses with acylated glycoproteins, this treatment leads to quite different consequences in a functional seiase depending on the virus used. as an alternative approach to study the biological significance of palmitoylation we expressed wild-type influenza ha and an hamutant lacking the fatty acid binding sites using the baculovirns system. both wild type and fa--mutant ha, after expression in insect cells bind to red blood cells, induce hemolysis, and fuse efficiently with fluoreseently labeled erythrocyte ghosts. hydroxylumine treatment of both recombinant wt-and mutant-ha leads to a loss of hemolysis and of fnsion activity. at least in the ease of fa--i-ia its inhibitory action must be related to some other effect than fatty acid cleavage. these results indicate that hydroxylamine treatment may not always be suitable for the generation of fatty acid free glycoproteius or virus particles for functional studies. gesemann and j.g. nicholls, biocenter univ. basel, 4056 basel, switzerland depolarization of leech neurons leads to growth cone collapse and neurite retraction. this response to depolarization is influenced by the substrata on which the cells are growing and is mediated by the influx of calcium (s. grumbacher-reinert and nicholls, 1992, j. exp. biol. 167:1-14; neely, 1993 , i. neurosci. 13:1292 -1301 . the morphologieal changes induced by depolarization of leech neurons growing on leech extracellular matrix extract is accompanied by a loss of microfilaments in the growth cones. leech neurons growing on a different substrate, the plant lectin coneanavalin a (coma), did not retract their neudtes or lose rnierofilaments, but continued to grow after depolarization. we attribute this to the reduced calcium influx during depolarization of neurons on cona (ross et al., 1988, pnas 85:4075-4078) . increasing the intraeellular calcium concentration by incubating neurons with the calcium-ionophore a23187, led to cessation of motility and disruption of microfilaments but not microtubutes in growth cones on coma. to investigate the mechanism by which calcium affects the organization of microfilaments we stained neurons with antibodies against gelsolin, a protein that severs microfilaments when it is activated with calcium. we have observed that these antibodies colocalize with microfilaments in leech growth cones, we are now analyzing the nature of this antigen that cross-reacts with the anti-gelsolin antibodies with the goal of establishing its potential role in the calcium-induced disruption of microfilaments in leech neuronal growth cones. this work was supported by a grant from the swiss nationalfond no. 3127814.89 to j.g. nicholls. activation of metabotropic glutamate receptors (mglurs) was studied in voltage-clamped ca3 cells in rat hippocampal slice cultures using the whole-cell pateh-clamp configuration. in saline containing 16 mm k +, 1s,3r-acpd (50 ~tm), a mglur agonist induced an inward current associated with an increase in membrane conductance. the reversal potential, assessed with a voltage ramp protocol from -80 to -60 mv and calculated by linear regression, was -15.7 â�¢ 18.7 mv, and was shifted to -53.4 â�¢ 6.4 mv when the concentration of external monovalent cations was halved. these results indicate that the conductance underlying this current is selective for cations (mainly k + and na+). the steady-state i/v curve for the 1s,3r-acpd-induced inward current showed a slight inward rectification. in most of the cells, this inward current was followed (or overlapped) by an outward current concomitant with a decrease in a k + conductance (ere v = -51.2 ~ 5.6 mv in [k+]o = 16 mm and shifted to -74.0 :t: 2.8 mv in [i4+]o = 8 ram). this k + current was voltage-independent in the membrane potential range of-120 to -20 mv. similar results were obtained with methacholine, a cholinergic muscarinic agonist. the non-specific cationic current, seen only in the presence of elevated extracellular k + concentration, could play an important role during intense neuronal activity. the c-myc protein has been implicated in the regulation of cell growth and differentiation, c-myc specifically interacts with max which in turn forms heterodimers with mad and mxil and homodimera with itself. we present evidence that this network o! proteins is regulated both at the level of relative protein concentration as well as by post-translational mechanisms. in the hematopoietic cell lines u-937, ml-1 and hl-6o e-myc mrna and protein are rapidly downregulated after induction o| differentiation whereas max expression is not significantly altered. in contrast the expression of mad is induced with properties similar to immediate early genes, taxi1 is induced to a lesser degree and with slower kinetics, in an effort to study the regulation of o-myc we have mapped all the major in vivo phosphorylation sites in both c-mye and max. two sites in the n-terminal transactivation domain are important for the regulation of the transforming potential el c-myc both in established cell lines as well as in primary rat embryo tibroblasts. however, no difference in the transactivating potential of corresponding mutants was observed. phosphorylatien of max at sites close to the basic region increases both on-and off-rates of either myc-max hetero-as well as max homodimers to dna. brain research institute, university of z~rich, ch-8029 z~rich, switzerland gaba b and adenosine receptors act via pertussis toxinsensitive g-proteins of the gi/g o class to mediate longlasting inhibition in the cns. this class of g-protein is negatively coupled to the enzyme adenylyl eyclase. our aim was to determine whether the inhibition of adenylyl nyclase mediated by these receptors has functional consequences for electrical signaling in hippocampal neurons. the calciumdependent k + current, i~, underlying adaptation of cell firing, was used as an electrophysiological bioassay for adenylyl cyclase activity, as it is very sensitive to intracellular levels of camp. accordingly, the ~-adrenergic agoniat iaoproterenol (5-500 nm), that activates g,-linked receptors, reduced the amplitude of i~ by 51.5 â�¢ 3.6% in voltage-clamped ca3 pyramidal cells in ttx (n=20). reduction of imm by isoproterenol was curtailed to 27.3 â�¢ 2.9%, (p < 0.001) in the presence of baclofen (i0 ~m), acting at gaba b receptors, or adenosine (50 nm). thus, the inhibition of adenylyl cyclase activity due to activation of gaba a and adenosine receptors can modulate responses to other neuretransmitters by an interaction occurring at the level of intracellular signal transduction. furthermore, stimulation of these receptors will tend to enhance i~, suppressing repetitive cell firing. this represents a further mechanism which will result in prolonged inhibition of hippocampal neurons. a study of the possible modulation by nitric oxide (no) of endogenous dopamine (da) release was performed in bovine retina in vitro. isolated half retinas were incubated in kkg at 37 ~ for 3 successive 30-rain incubations, and da was measured in the incubation medium by hplc with ec detection. when retinas were incubated in the presence of the no-generating compound hydroxylamine (300 ttm), a decrease in either basal or k +-(50 ram) stimulated da release was observed during the 2 nd 30-rain period. tested under similar conditions, dibutyryl cgmp (1 mm) was ineffective. the no synthase inhibitor l-ng-nitro-arginine methyl ester (l-name, 100 gm) increased basal da release during the 1 st and 2 nd incubation, whereas it did not affects the k+-stimulated da release. in addition, its effect were potentiated in calcium-free medium. finally, we have also shown in cell-free experiments that retinal no synthase activity was totally calcium-dependent and blocked by l-name. taken together, these results suggest that endogenous no regulates da release via a cgmp independent mechanism. division of endocrinology, university hospital, ch-1211 geneva 14 while the stimulation of aldosterone biosynthesis by angiotensin ii (angii) in bovine glomerulosa cells clearly depends upon a sustained influx of ca 2+, the pathway for ca 2 ⧠entry is not yet accurately defined. at least two mechanisms seem to be involved: 1) the opening of voltage-operated ca z ⧠channels, and 2) the stimulation of a "capacitative" ca 2+ entry regulated by intraocllular ca 2 ⧠stores; however the relative contribution of these pathways to the stimulation of steroidogenesis needs to be determined. two pharmacological tools were used: nieardipine, a blocker of to and l-type ca 2+ channels, and thapsigargin, which releases ca ~+ and therefore induces a capacitative ca ~+ influx. nicardipine (1 lam) completely blocked the cytosolic ca 2+ response to k +, which exclusively activates voltage-operated ca 2. channels, but only partially (22 %) the response to angli. in the presence of nicardipine, angll was unable to stimulate a ca 2+ influx aiter depletion of ca 2 ⧠stores with thapsigargin, demonstrating that angli principally stimulates a capacitative ca 2+ entry pathway. in addition, nicardipine completely blocked the steroidogenic response to k +, but only 30% of the response to angll. thapsigargin-induced aldosterone formation, as the response to angll, was markedly potentiated by low concentrations of k +. in conclusion, this study shows that in bovine glomerulosa cells, angli activates a sustained ca 2+ influx, principally through a capacitativc pathway, leading to aldostcrone formation. this finding could partially explain the poor efficiency of dihydropyridine ca 2 ⧠antagonists for preventing aldosterone secretion. harald holder and john m. lucocq*. institute of anatomy, university of berne, ch 3012 berne *depanment of anatomy and physiology, university of dundee, uk dundee dd1 4hn. subjection of hela cells to 45~ for 30 minutes leads to a complex activation pattern of map-kinases as revealed with renaturadon gels. exponentially growing hela cells show activallon of a myelin basic protein (mbp) phospborylating kinase migrating with an apparent molecular mass of around 60kda upon heat shock induction. in contrast, in cells arrested in go by serum starvation for 24h, essentially three different proteins phosphorylafing mbp are activated subsequent to heat shock treatment. these kinases migrate with apparent molecular masses of around 32kda, 44kda and 55kda. western blot analysis with antibodies recognizing the human boraologues of erkl and erk2 revealed that considerable pools of erkl and erk2 show retarded elec/rophnretic migration behavioar indicating that they are phosphorylated and probably activated in heat subjected and growth-arrested hela cells. down-regulation of protein kinase c (pkc) by a prolonged treatment with 12-o-tetradecanoyl-phothol-13-acetate (tpa) did neither change the major activation pattern observed upon separation of cell lysates on renaturation gels nor that one observed on western blots decorated with anti-erkl or anti-erk2 antibodies. these results suggest that heat shock induced activation of at least erkl, erk2 and the 55kda mbp-kinase is not mediated via activation of pkc. this work was supported by swiss nsf grant no. 31-27636.89. effects of dominant negative glucocorticoid receptor mutants in cell cultures and transgenic animals stefano brenz verca, stefan schneider, sandro rusconi, institut fiir molekularbiologie h der universitat zf~rich, winterthurerstr. 190, switzerland the glueocorticoid receptor (gr) is involved in a large number of developmental and biochemical processes. recently, we obtained a trans-dominant negative gr mutant (tdn) -called gr[aia] -by a frame shift of the cag-repeat of the rat gr (see poster by hug et al.) . the gr[ala]-mutant shows normal llgand-binding and dna-binding but cannot stimulate transcription (lanz et al., steroids; in press) . modulatable versions of the tdn-mutant have also been created by substituting the hbd of the grmutant with the hbd of sex-hormone receptors. these constructs have only been tested so far in transient cotransfection assays. therefore, we are currently trying to express them permanently in cell lines (rat hepatoma cell line fto-2b) in order to verify whether they can affect the transcription level of a known endogenous gr-dependent target-gene, like the tyrosine aminotransferase (tat) gene or a permanently transformed mtv-iacz reporter. so far no rapid test-system has been described, that allows the quantitative analysis of such effects in transient expression assays. we are about to set up such a rapid and generally applicable transient test-system. in the future we plan to express dominant negative gr mutants in a tissuespecific manner in transgenic mice. it will be particularly interesting to investigate their effects on the immune system or on liver functions. these studies investigated growth cone responses m brief local encounters with surface molecules implicated in neuronal pathfinding using chick drg neurons. we tested two interrelated hypotheses: 1) discrete points of information (guideposts) override sustained surface information and dominate growth cone behavior and 2) guidance molecules provide such information by activating second messengers instead of simply increase adhesivity. the potential guidance molecule lamialn was covalentiy coupled to polystyrene beads in order to mimic guidepost cells. encounters with laminin model guideposts significantly altered the behavior and morphology of growth cones advancing on fibronestin or polylysine. growth cones steered towards theses beads in a saltatory medea once a long lasting contact with a single filopodium had been established. subsequent to a pause at the bead, growth cones advanced with a significantly increased growth rate for the next llmin. the average increase in rate stimulated by bead contact was 5 times the basal rate on polylysine and 2.5 times that on fibronectin. positioning of multiple model guideposts along a path and within filopodial reach maintained this bead stimulated rate of outgrowth and directed growth cone advance. the laminin induced pattern of events was totally blocked in the presence of either h7 or sphingosine, two pkc inhibitors, or neomycin, a plc inhibitor. neither of these inhibitors affected neurite outgrowth on the substrata alone. our results demonstrate the necessity of filopodial contacts, the importance of spatially restricted stimuli and the activation of transducing pathways as key mechanisms in neuronal pathfinding. mechanisms of glucocorticoid receptor desactlvation by homopolymeric alanines martin hug, manuela h&fferer and sandro rusconi, institut fiir molekularbiologie 11 der universitat uz zart'ch, winterthurerstrasse 190, 8057 ziirich, switzerland the rat glucocorticoid receptor (gr) edna contains in its y-portion a (cag-repeat that encodes a siring of 22 gin residues interrupted by 1 arg. a mutant with the frame-shifted repeat which codes now for poly-ala residues (called gr[aia]) is completely trans-inactive, is more cytoplasmitally located in absence of ligund and exhibits dominant-negative properties in presence of ligand (lanz et al., steroids, in press) . we generated gr recombinants with increasing numbers of cag-triplets in three possible reading frames (oligo-gln, -ser or -ala). the gr-oligo[ala]~-~.23 mutants showed an activity comparable to wild type gr, whereas the groligo[ala]>_25 were inactive. the severity of the effect of oligo a.la su'ings seems to be dependent on the position of the repeat along the primary sequence (e.g. progressive decrease of the effects by moving toward more earboxy-position). we have also examined the effects of the oligo[ala]21, 23, 25, 27, 31 on gal4 chimeric factors and found that the inhibition by ala repeats is probably factor-specific. our results made us speculate that possible differences in post-translational modifications may be at the root of the negative effects by the ala repeats. we believe that these effects are due to transient association of the nascent gr[aia] mutant proteins with intraceuular membranes. these hypotheses are currently tested. the activation of steroidogenesls in calcium-clamped bovine glomerulosa cells and its modulation by angiotensin ii and camp, python cp, laban op, rossier mf, vallotton mb and capponi am division of endocrinology, university hospital, geneva, switzedand. the ca2+-messenger system plays a crucial role in the regulation of steroid secretion in adrenal zona glomerulosa cells, as it is known to mediate the action of angiotensin ii and potassium ion. in the present study, we used intact isolated glomerulosa cells in which the cytosolic free ca 2+ concentration ([ca2+]c) was clamped at various levels with the ca2+-ionophore, ionomycin, in order to locate the sites of action of ca 2+, to determine their sensitivity towards ca 2+ and the modulation by other physiological factors. by measuring simultaneously steroid synthesis and [ca2+]c, we show that ca2+-clamping (50-860 nm) induced a concentration-dependent increase in the production of both pregnenolone (506___70 % of the basal level) and aldosterone (255+27 % of the basal level, ec50 = 273 nm). by contrast, ca 2+ did not influence the conversion of 1 t-deoxycorticosterone into aldosterone at concentrations up to 600 rim, although at higher concentrations we observed a modest but significant inhibition. in addition, ca2+-clamping did not affect the formation of pregnenolone from a freely diffusible analogue of cholesterol, 25hydroxycholesterol, indicating that ca 2+ acts at a step upstream of cholesterol side-chain cleavage. both angiotensin ii and a membrane-permeant surrogate of camp, dibutyryl cyclic amp, potentiated the steroidogenic response to increases in [ca2+]c . in summary, these studies reveal an exquisite sensitivity of the glomerulosa cell steroidogenic pathway toward very small physiological changes in [ca2+]c. radtke, rainer heuchel, oleg georgiev, gerlinde stark#, michel aguet# & walter schaffner institut fiir molekularbiologie 11, universilat ziirlch, 8057 z'6rich # lnstitut rir molehdarbiologie i, universitiit ziirich, 8093 ztirich we have previously described and cloned a factor (mtf-1) that binds specifically to metal-responsive dna sequence elements in the enhancer/promoter region of metallothionein genes. mtf-1 is a protein of 72.5 kda that contains six zinc fingers and multiple domains for transcriptional activation. here we report the disruption of both alleles of the mtf-1 gene in mouse embryonic stem ceils by homologous recombination. the resulting null mutant cell line fails to produce detectable mounts of mtf-1. moreover, due to the loss of mtf-1 the endogenous metalinthionein-i gene is silent, showing that mtf-i is required for both basal and metal-induced transcription. accordingly, reporter genes containing either synthetic or natural metal-reslxmsive promoters were not transcribed after transfection into the null mutant ceils. cotramsfection of the mtf-i expression vector rescued both basal and heavy metal-induced transcription. these results demonstrate that mtf-1 is essential for normal metallothionein gene regulation. rat pheochromocytoma cells (pc12) were differentiated with ngf for 3-4 days, a time sufficient for the formation of growth cones and elongation of neurites. addition of the phosphatase inhibitor calyculin a (cl-a) (0.2.50.0 nm) led to a concentrationdependent collapse of growth cones and retraction of the neurites within 15 minutes. after the retraction, the cell bodies showed the appearance of a grape-like domain opposite to the cell nucleus. they recovered to normal shape within about 30-60 minutes if the inhibitor was washed out. the neurite retraction was further analysed using both low-light level fluorescence video-microscopy and fura-2 single cell microfluorimetry. the onset of retraction started in the central part of the growth cone prior to a complete collapse of filopodia. the basal intracellular free calcium concentration ([ca2+] i) remained constant during neurite retraction. as a measure for the viability of cl-a-treated cells, agonist-isduced responses of [ca2+] i were analysed. ca2+ entry across voltage-activated ca 2-~ channels was inhibited by 50% after 30 minute treatment with cl-a, while the atp-induced ca2+ entry via ca2*-permeable cation channels was not significantly reduced. however, the onset of the atp-induced rise in [ca2+]i started at the grape-like domain. the speed of the ca 2+ wave across the cell body was slower than in control cells. the addition of 0.5 gm okadaie acid, a different type of phosphatase inhibitor, caused no cell shape change or neurite retraction within 24 hours. taken the specificity of the two phesphatase inhibiters into account, the observed effects seem to be induced by inhibition of a ppl-class phosphatase. myosin in nematocytes of hydra michel nakano & robert p. stidwill dept. zoology, university of ztirich, switzerland the functions of nonmuscle myosins during cell locomotion are not clear. several models have been presented reaching from very active participation of the molecules in different parts of cells to only minor or no roles at all. there is increasing evidence that the functions of myosin ii (capable of forming bipolar filaments) are quite distinct from the ones of myosin i and generally that the different members of the growing family of myosin-like proteins may not all have identieal functions. we have attempted to demonstrate the occurrence and determine pattern of myosins in tissue of hydra and especially in migrating nematoeytes mainly for two reasons. first, nematocytes are fairly rapidly moving cells which can be studied in vitro and, second, the question is of interest from a evolutionary point of view. we have utilized several antibodies against vertebrate and invertebrate myosins for immunofluoreseenee (confocal laser scanning microscopy) and western blotting studies. in addition to these results findings on the screening of a ~.zap c-dna library are presented. the jaki protein tyrosine kinase is a member of a family of intracellular kinases characterized by having two kinase catalytic domains: a bona fide tyrosine kinase domain and an amino terminally positioned kinase-related domain. recently it has been shown that jaki and the other members of this family (jak2, tyk2) are intimately involved in cytokine and growth factor signalling. the presence of two putative nuclear localizing sequences in the amino terminus of the protein prompted us to examine the subcellular localization of this protein. immunohistochemical and biochemical analysis revealed that a part of jaki was localized to the nucleolus. metabolic labelling showed that (a) the jaki protein partitioned rapidly into the nucleus and (b) the nuclear jaki became smaller with time. the size difference was not due to either co-translational glycosylation or phospherylation. these results suggest that part of the jaki protein is rapidly translocated to the nucleoli where it becomes processed. revealed that this clone is expressed predominantly in spleen, mammary gland and thymus as two transcripts, a major species of approximately 5kb and a minor species of approximately 4kb. nucleotide sequence analysis revealed a 84% homology to the porcine syk gene. the syk gene is expressed as a major 3.0kb transcript and has been reported to be lymphoid specific. due to the discrepancy of transcript size and number, we suggest that our cdna clone represents a novel member of this family of ptks. we are currently isolating a full length sequence from a mouse spleen cdna library. in addition, we want to characterize which cell(s) in the mammary gland are responsible for the observed expression. expression and function of g-protein isoforms were studied in differenflaring (6c8) and nondifferentiaflng ((33) subclones of red-1 rauscher murine erythroleukemia cells. erythroid differentiaflon induced by the combined action of erythropoietin (epo) and dmso was associated with a selective loss (>70%) of immunoreactive gai3. simultaneously, a iruncated form of g~2 accumulated in the cytosol, while the membrane concentrations of gai2, gets and gaq/11 remained unchanged. nondifferenfiating (33 cells contained sigrtificanfly higher levels of most get subunit isoforms that remained unchanged upon epo/dmso treamaent. changes in adenylyl cyclase activity and in intracellular ca ion levels ([ca]i) resulting from activation of g-protein-coupled receptors were monitored for differentiation-dependent effects on transmembrane signaling. adp-mediated changes of [ca]i in native and differenflaflng 6c8 cells were consistent with p2t receptor coupling to gai3. thrombin receptors seemed to couple preferentially to gai in g3 cells and to gaq in 6c8 cells. our results document substantial alterations in g-protein-mediated signaling during erythroid differentiation. to investigate the involvement of protein tyrosine kinases (ptks) in the growth control of the mammary gland, we have used pcr based cloning to identify ptks expressed during normal mammary gland development. this approach led to the isolation of three members of the eph-related family of receptor ptks: myk-;, a novel member expressed predominantly in lung, heart and mammary gland; m-eck, the murine homologue of the human eck gene and mek5, the murine homologue of the chicken cek5. all three ptks were expressed in a distinct and narrow window of mammary gland development: puberty and estrus cycle. no expression was found either in late pregnancy or in the end differentiated state of lactation. 0vet-expression was found in the undifferentiated and invasive tumors of transgenic mice expressing the h-ras oncogene. in contrast, no elevated expression of all three genes was detected in the well differentiated, non-invasive mammary tumors characteristic of c-myc expressing transgenic mice. these results suggest that members of this family of ptks are involved in the control of proliferation of the mammary gland but are incompatible with its differentiation. np-tcii is a lymphocyte specific transcription factor (lattion a.-l. et el., mol. cell. biol., 1992, 12:5217-5227 a nucleotide receptor that mediates phosphoinositide and phosphatidylcholine hydrolysis by phospholipases c and d, respectively. here we report that atp and utp potently stimulate mesangial cell proliferation. both nucleotides stimulate phosphorylation and activation of map kinase and the up-stream map kinase kinase. activation of map kinase by both nucleotides is dose-dependently attenuated by the p2 receptor antagonist suramin. stimulation of protein kinase c (pkc) by phorbol ester increased map kinase phosphorylation and activation, and downregulation of pkc partially inhibited atp-and utp-induced map-kinase activation. inhibitors of pkc which display a selectivity for the ca2+-dependent isoenzymes (c~, [3, 7) , as compared to the ca2+-independent isoenzymes (5, e, ~, rl) such as staurosporine and the specific pkc inhibitor cgp 41251 did not inhibit nucleotide-stimulated map kinase phosphorylation, thus implicating the involvement of a ca2+-independent pkc isoform. in conclusion, these data suggest, that atp and utp trigger the activation of the map kinase signalling cascade in mesangial cells and this may be responsible for the potent mitogenic acitivity of both nucleotides. subcellular ion-gradients in ca signaling p. lipp & e. niggli, department of physiology, univexsity of bern recent experimental evidence has indicated an important role for submicroscopic concentration gradients during ca-signaling in various cell types. in heart muscle cells influx of ca via l-type ca channels triggers ca release from the sarcoplasmic reticulum (sr) and links electrical excitation to mechanical contraction (ec-coupling). we used a ratiometric confocal method (fluo-3 and fura-red) to follow the intraceuular ca-concentration while ha-and ca-currents (inn, ica) were elicited in the whole cell mode of the patch-clamp technique. both, icand in, were able to trigger ca release from the sr. kinetic differences between ini and ic -induced ca-transients, li-substitution experiments and the existence of a residual inn-induced ca transient in the absence of sr function suggested that these ca signals were mediated by the ha-ca exchange running in the ca influx mode. control experiments showed that ins-induced ca transients did not result from spurious activation l-type ca channels. the significant increase of the na concentration required to activate the ha-ca exchange can only occur provided cytosolic diffusion of na is restricted in the vicinity of the exchangers. this limited diffusion results in significant ha-gradients in the subsarcolemmal space. in conclusion, i~iinduced ca influx may represent a safety factor for ec-coupling while ic, ensures sustained ca-release and is the source for refilling the sr with ca. supported by snf. the regenerative ca-induced ca release (cicr) mechanism is an important amplifier in cardiac signal transducilon. the cicr contributes to the ca transient responsible for mechanical activity, but also generates ca waves propagating within the cytosol. we investigated subcellular ca signals in neonatal rat and adult guinea-pig ventricular myocytes using ratiometric confocal microscopy with a mixture of two fluorescent ca indicators. individual resting myocytes exhibited spontaneous ca release events from the sarcoplasmic reticulurn (sr) that were attributable to three distinct patterns: i) local ca release events without propagation; ll) planar ca waves propagating across the cell; iii) spiral ca waves spinning around a nucleus or shifdng along a subcellular region without entering it. transitions between these patterns were frequently observed, suggesting that a particular release pattern is not the property of an individual cell but reflects the functional state of the sr. the existence of focal release and refractory zones within a single cell indicates that the sr is not uniform on the subcellular level. the sr seems to he a network of elements with distinct functional properties. a suhcellular variability in the positive feedback of the cicr mechanism can account for the observed features of ca signaling. the degree of positive feedback exhibited by a single sr element may depend on its ca content. to characterise the intracellular signalling and regulatory properties of the cloned parathyroid hormone (pth) receptor we have stably transfected two renal epithelial cell lines with cdna's encoding the pth receptor (provided by dr g. segre). the proximal tubular, porcine (llc pk1 [clone 4]), and the distal tubular, madine darby canine (mdck) cell lines were transfected since the culture of either cell line on permeant filter supports leads to the development of polarised epithelial cell layers that mimic the epithelial organisation in vivo. transfection of the cloned pth receptor into each of these cell lines allowed us to examine, independently, the functional properties of the receptors expressed at the apical and/or the basolateral cell surface. while the basolateral application of pth produces a large increase in camp production in both cell lines, the relative efficacy of an apical application of pth differs between the cell types. the transfected llc pk1 cells exhibit a greater response to apical pth compared to the transfected mdck cells. pth-mediated regulation of intrinsic phosphate transport was also examined in polarised, transfected mdck cells. neither apical or basolateral application of pth affects intrinsic apical or basolateral ha-dependent or phosphate transport activities. similar experiments are being performed with the transfected llc pk1 cells. a. and preiss, a. biozentrum der universit&t basel, ch 4056 basel hairless, a recessive larval lethal mutation, causes dominant defects in sensory organs of adult drosophila flies.manifold genetic interactions indicate that hairless antagonizes neurogenic gene function suggesting an involvment also in neurogenesis. in order to gain insight into its function we have cloned the hairless gene which encodes an extremely basic, very serine rich protein of ii0 kd calculated molecular weight. no significant homologies to proteins of known function could be identified. therefore, we are concentrating by various means on a structure -function analysis of the hairless protein. firstly, we are testing a series of hairless deletion constructs for rescue capacity and generation of anti-hairless phenotypes after overexpression compared with the wild type gene. secondly, we are analysing hairless protein distribution throughout development, also at the subcellular level. heat induced hairless protein runs at ~ 150 kd, possibly due to phosphorylation which might be intrinsic to hairless function, a hypothesis we are currently scrutinizing. thy-1 and cd26 surface glycoproteins are both capable of delivering proliferative signals to t cells upon cross-linking with appropriate antibodies. the tyrosine phosphatase cd45 is a key regulator of t cell proliferation as it controls the activity of src family kinases p56/~k and p59fy n. associations of thy-1 with cd45, and of cd26 with cd45 have been reported after chemical cross-linking of intact cells and it is likely that such associations constitute part of the structural basis for the t-cell stimulating capacity of thy-1 and cd26 accessory molecules. we report that thy-1, cd26 and cd45 can be specifically crosslinked at the cell-surface and isolated as multimolecular complexes containing a 78 kda protein that is recognized by an anti-bip (grp78) antibody. in vitro kinase assays show the selective association of p56 ick and p59fy n with thy-1 and cd45 contained in multimolecular complexes. the multimolecular structure we describe could representa transducing unit incorporating surface receptors and regulators of tyrosine phosphorylation that are stabilized through interaction with the bip protein in the plasma membrane. structure-function-analysis of hairless, a gene involved in drosophila nervous system development maier, d., functional coupling of ppars and trs ijpenberg, a., wahli, w., desvergne, b., institut de biologie animale, 1015 lausanne we are interested in a possible functional coupling of thyroid hormone (tr) and peroxisome proliferator-activated receptors (pfar). to this end, we performed cotransfection experiments in nih 3t3 cells using cat reporter constructs containing the promoter of either the t3 responsive malic enzyme (me) gene, or the ppar regulated acyl coa oxidase (aco) gene. the me reporter construct showed very moderate response to the activated mouse ppar (mppar), whereas combination of try1 and unactivated mfpar potentialized the t3 induction. a putative ppar response element was identified within this promoter and will be further investigated. the aco reporter constructs were not responsive to trs. in constrast, a tr and t3 dependent inhibition of the fpar mediated activation was observed. the mechanism of functional coupling of these receptors, which may possibly involve the 9-cisretinoic acid receptor rxr, will be further investigated by molecular analysis. unliganded steroid receptors form a complex with hsp90 via their hormone binding domain (hbd). the receptor activation involves a hormone-induced release of hsp90. we genetically addressed the role of hsp90 in budding yeast by analyzing three kinds of hsp82 (the essential yeast homologue of hsp90) mutants. these mutations affect either the quantity of the protein (low versus normal levels), its integrity (deletion analysis) or its nature (hsp82 homologues from other species). hsp82 mutant are examined for their ability to complement a hsp82 deletion mutant. moreover, they are tested for functional interaction with a hbd. interestingly, a viable deletion of a charged region, suspected to be involved in hsp82-hbd interaction, only slightly interferes with hormonal activation of both estrogen or glucocorticoid receptors. furthermore, a short deletion in the last third of hsp82 leads to the production of a dominant negative mutant which prevents ceil growth at elevated temperature. the ability of various hsp82 homologues to palliate or not the absence of the yeast protein allows us to define, using chimeras, the regions of the protein which are necessary and sufficient to support cell life. zhang,h.,reynaud, s. and spohr,g. d~partement de biologie cellulaire, sciences iii, 30, quai ernest-ansermet, ch-1211 gen&ve 4 id cdnas expressed during early xenopus development have been isolated and sequenced. the encoded protein is homologous to the proteins described in higher vertebrates. northern blot analysis reveals that transcription starts soon after mid blastula and decreases to some extent after tailbud-stage. lower levels of transcription are detected also in adult frog tissues such as liver and heart. microinjection into oocytes of in vitro-synthesized myodor/and id-mrna, along with a cat reporter gene whose expression is under the controll of an a3-actin promoter supplemented with four e-boxes shows that myo d function is repressed by id. to investigate the importance of negative regulatory sequences we have isolated and characterised the id promoter. as a strategy for identifying cis-regulatory elements, chimeric genes consisting of different 5' elements of the promoter were attached to the cat coding sequences and microinjected into embryos. we are studying the tissue distribution of the rat peroxisome proliferators activated receptor (ppar~), a member of the nuclear hormone receptors superfamily, during development and in adults. high levels of ppar~ mrnas are detected in adult liver, kidney and heart. muscle, stomach, and intestine show moderate amounts of the ppar~, whereas brain, testis and lung present a very low expression. during postnatal development, we observe a continuous increase of the ppar~ expression in the kidney, in contrast to a steady level in the liver. anjard, c., groux, b ., gamboni, s. and reymond, c.d., institut d'histologie, unil, rue du bugnon 9, 1005 lausanne. the camp dependent protein kinase (capk) from dictyostelium is composed of a single catalytic (c) and a single regulatory (r) subunit. the c subunit we characterised, pkac is about twice the size of its mammalian counterpart. we showed that it possesses all properties of a catalytic subunit. it associates with the r subunit in absence of camp, it copurifies with capk activity and an increased activity is observed in cells over-expressing pkac. furthermore, we dissected its role during development and cell differentiation. overexpressing pkac under cell type specific promoters allowed to show the requirement for its activity during spore differentiation. pkac seems to play a more complex role in prestalk cells. we are now dissecting the functional regions of the pkac protein, making use of the known tertiary structure of the c subunits of mammalian enzymes. dna binding properties and stimulation of gene transcription of baculovirus expressed xppar~. hihi, a.k, mermod, n., medin, j., ozato, k. and wahli, w., inst. de biol. animale, uni lausanne, ch-1015 lausanne. lab. of mol. growth reg., nih, bethesda, md, 20892 usa. the peroxisome proliferators activated receptors (ppars) are members of the steroid/thyroid nuclear receptor superfamily. so far, they have been found in amphibians, rodents and humans. in xenopus laevis, 3 subtypes (xppara,~.y} have been isolated. in order to study the mode of activity of the ~ form, the xppar~ gene product was overexpressed using a baculovirus expression system. the nuclear protein obtained has the correct molecular weight of 46 kd. gel shift assays showed that nuclear extracts containing the recombinant protein are able to specifically bind the 'ppar response element' which is a direct repeat of the core aggtca motif. this dna binding activity is potentiated by another nuclear receptor, the mouse rxr~ and is inhibited by phosphatase, suggesting a role for phosphorylation in ppar binding to dna. a functional approach, using an in vitro transcription system, was chosen to define ppar and rxr action on transcription stimulation, as well as the role of their respective activators. arachidonic acid (0.5 -20 ram) induces various patterns of ca2+i signal in bronchial smooth muscle cells, as revealed by single cell dynamic video imaging of fura-2 loaded cells. most frequently, ca2+i oscillations were observed, although other patterns (a single ca2+i transient; a single peak followed by a sustained elevated level, or a sustained ca2*i elevation solely) were occasionally seen. the amplitude of ca2*i oscillations was related to the concentration of arachidonic acid. the frequency histogramm was noticeably shifted to higher frequencies by nordihydroguaiaretic acid (ndga, 0.2 rtm, a lipoxygenase inhibitor), whereas it was markedly shifted to lower frequencies by indomethacin (50 pm, a cyclooxygenase inhibitor) and by 5, 8, 11, 14-eicosatetraynoic acid (etya, 3 ~tm, a lipoxygenase and cyclooxygenase inhibitor). these observations suggest that: (1) arachidonic acid per se elicits ca2+ i oscillations in these ceils; (2) cyelooxygenase activity products modulate the frequency of these oscillations. promoter analyses of a growth factor regulated gene t. triib, m. kalousek, r. kessler, and r. klemenz dept. of pathology, university hospital, 8091 ziirich stimulation of quiescent cells to enter the cell cycle results in altered gene expression. some of the first genes to be induced (immediate early (i.e.) genes) encode transcription factors which are thought to pass on the mitotic signal to the delayed early (d.e.) genes. we have analysed the promoter elements required for growth factor mediated induction of the d.e. mouse gene t1 which encodes a secreted glycoprotein of the immunoglobulin superfamily. a 448 bp region located between 3.6 and 4.0 kb upstream of the transcription start site is sufficient for growth factor mediated inducibility. it contains an ap-1 binding site which is absolutely required for t1 gene expression. it is surrounded by 3 e-boxes. at least 2 of them are essential for efficient gene induction. thus the i.e. proteins encoded by the fos and jun gene family which form the ap-1 complex can activate the d.e. gene t1 in collaboration with helixloop-helix transcription factors binding to the e-boxes. the increase of radiolabelled inositol phosphates ([3h]ips) production in agonist-stimulated human airway smooth muscle cells (asmc) was determined by hplc. in order to observe the role of calcium, pkc and pla2 on plc activity during agonist stimulation, the effects of pharmacological agents (able to alter calcium, pkc and pla2) were tested on ip production elicited by a 5 sec stimulation with histamine. the inhibitor of pkc staurosporine increased, while pma (an activator) decreased the histamine-stimulated production of [3hi 1,4,5-ip3 and of its degradation products. an inhibition was also observed with the two pla2 inhibitors, quinacrine and p-bromophenacyl bromide. in calciumfree buffer, no difference in the ip increase was shown, but an inhibition was observed when thapsigargin, an inhibitor of the ca 2+, mg 2+-atpasc of the sarcoplasmic reticulum, was added in the same conditions. the calcium ionophore ionomycin increased the lp production in presence of external calcium, whereas it completely blocked the stimulation in calcium-free buffer. the results suggest that plc activity, in human asmc, is modulated by a positive feedback of calcium and by a negative feedback of pkc and pla2. regulation of hormone-stimulated calcium signals m. boolman, afrc, laboratory of molecular signalling, dept. zoology, cambridge, uk stimulation of cells with hormones that activate inositol 1,4,5-trisphosphate (insp~) formation, leads to an increase in the intracellular ca 2. concentration ([ca2+]~). these hormone-stimulated [ca2 ⧠signals have a complex temporal and spatial regulation, with the [ca2*]~ increase often taking the form of a series of repetitive spikes or oscillations, arising from a steady basal level. the spatial counterpart of a [ca2+]~ spike is a wave, where [ca2 ⧠is first elevated in a localised region of a cell, and then spreads throughout the cell in a regenerative manner. the [ca~*]~ spike frequency is modulated by several environmental factors including hormone concentration, extracellular calcium and thiol reagents. current evidence suggests that a cell can interpret these complex [ca2+]~ changes to regulate a diverse range of cellular activities. although many of the biochemical steps between hormonereceptor activation and the [ca2 ⧠response are known, the precise mechanism generating these complex [ca2+]~ signals is unclear. in order to understand this mechanism more clearly, we have investigated the regulation of insp~-mediated ca ~ ⧠release from intracellular stores in intact cells. our current data suggests that under certain conditions, the insp3-sensitive intracellular stores behave as functionally discrete units, and that during a ca 2 ⧠spike these stores become functionally coupled by the diffusion of ca z+ from one store to the next, triggering a regenerative ca 2+ release. phenylarsenoxide as a tool for identifying proteins involved in the secretory process wiedemann. c., schaefer, t.,*gitler, c., burger, m. m. friedrich-miescher institut, p.o.box 2543, ch-4002 basel "department of membrane research and biophysics, weizmann institute of science, rehovot 76110, israel phenylarsenoxide (pao) at 20btm blocks exocytosis in chromaffm cells of the bovine adrenal medulla. this inhibition can be reversed by small dithiois, such as dithiothrcitol or 2,3~limercaptopropanol. because trivalent arsenicals interact specifically with dithiol4containing proteins, we conclude that dithiol-proteins do play an important role in regulated secretion in chromalym cells. to identify dithiol-proteins putatively involved in the secretory process, we compare pao-binding proteins from chromaffm and pci2 cells and from fibroblasts as well as from sulx:ellular fractions by 2-dimensional gelelectrophoresis. the proteins are identified by radioactive pao bound to the dithiol in a complex that witltslands sds-page. affinity chromatography on pao-scpharose with various protein fractions is used to purify the dithiol-proteins of interest. enzymatic activity, e.g. kinase or phosphatase activity, can be measured in the eluates of such a column to give further hints at the possible function of dithiol-proteins. rac-pks (~elated to pka_ and pkcc protein kinnses) represent a serine/threonine kinase family whose catalytic domain is closely related to those of protein kinases a and c. they contain a ph (pleckstrin homology) domain n-terminal to the catalytic domain. in addition, rac-pk~ was shown to be a proto-oncogene. in order to investigate the role of rac-pk in cellular signallimg we undertook genetic and biochemical analysis of the drosophila homolog (drac-pk). the drac-pk gene encodes multiple classes of transcripts. antisera raised against the drac-pk recombinant protein specifically recognize 3 distinct molecular weight forms (68, 85 and 120 kda). all forms are expressed throughout development and are both maternally and zygotically regulated. the drac-pk gene is localized at 89b4-i0 region of the third chromosome, betwee~ the pannie~ and the stubble genes. universit6 de lausanne, rue du bugnon 9, 1005 lausanne when spinal meninges homogenates were incubated with reduced glutathione (gsh), a cofactor of prostaglandin (pg) biosynthesis, [14c] arachidonate (aa) was bioconverted into a major and a minor product which comigrated on tlc with pge2 and pgd 2 respectively. in absence of gsh: 1) pgd2 could not be detected; 2) the level of pge2 was dramatically reduced but surprisingly counterbalanced by accumulation of an unusual [i4c] aa metabolite which exhibits particular traits of migration on tlc and of retention time on hplc. this aa metabolite: 1) does not correspond to a degradation product of pge2; 2) crossreacts with pge 2 antibody; 3) fits the conditions required for a 15 hydroperoxy pge2 (chemical degradation into pge2 by hydroperoxyde reducing reagents such as snc12 or gsh-hemin). it is therefore inferred that depletion of gsh favours accumulation of hydroperoxyprostaglandins which would participate to oxidative injury. the ecdysteroid receptor (ecr) and ultraspiracle (usp) from both, chironomus tentans and drosoplu'la melanogaster, were expressed in e. coil as fusion proteins with glutathione-s-trsusferase. the identity of the expressed recombinant proteins was confirmed by western blot analysis. the drosophila ecr binds to its cognate hormone response element as a heterodimer with usp as a partner. we now want to compare the capabilities of the two receptor partners from both insect species in regard to dna binding and heterodimerization by means of gel mobility shift assays. the use of naturally occurring andartificial hormone response elements will allow to define a hormone response element consensus sequence for ecr-usp heterodimers and, if existing, for ecr and usp homodimers, respectively. for other members of this receptor family it was shown that the dna binding domain and adjacent sequences alone sufficiently defines response element specificity for both homo-and heterodimers. the dna binding domains of ecr and usp from the two species show a high similarity (92% and 85% identity, respectively). the corresponding sequences were selected for overexpression in e. coli. in combination with immtmoprecipimtion of receptor-dna complexes and pcr amplification steps, the expressed proteins will be used for the detection of naturally occurring hormone response elements within genomic dna. university of fribourg, ch-1700 fribourg. various agonist-induced cell responses in neutrophils and fibroblasts, such as chemotaxis and cytoskeletal rearrangements, have been shown to parallel the synthesis of ptdins(3,4,5)p 3. but the importance of this rise is not clear. we show here that wortmannin blocks pdgf-induced production of ptdins(3,4,5)p 3 in human foreskin fibroblasts with an ic50 of about 5 nm. a similar inhibition was observed in in vitro assays (ics0=lnm) with pi 3-kinase ii~nunoprecipitated by antibodies directed against the 85 kd subunit (p85). on the other hand, wortmannin did not affect pdgf-mediated phosphorylation of p85, indicating the correct interaction of p85 with the pdgf-~ receptor. the pl10/p85 complex of the pi 3-kinase remained intact in the presence of ~m concentrations of wortmannln. these results are consistent with a direct, specific inhibition of pi 3kinase by wortmannin at concentrations relevant for its previously reported effects on cellular responses. when stimulated with pdgf, human foreskin fibroblasts form circular membrane ruffles rich in filamentous actin. the fact that wortmannin inhibits these pdgf-mediated aotin rearrangements suggests the need of pi 3-kinase activity as a signal for this cell response. caicineurin is a calcium/calmodulin-regulated protein phosphatase which is comprised of a catalytic subunit a and regulatory subunit b. although calcineurin was discovered in brain, the calmodulin stimulated protein phosphatase which has caicineurin like structure has been described in other tissues. the protein structure show that the calcineurin b is very conserved in different tissues and species, using anti-calcineurin b monocicnal antiserum, the tissue extracts from rat have been explored. immunoreactivity was obverved in all tissues, although its intensity was different. the highest intensity was found in brain. spleen, heart and thymus had an intermediate level intensity. the results were supported by the quantitative measurement of calcineurin. the caicineurin concentration was 10 to 50 fold higher in brain than that in other tissues. furthermore, the distribution of the calcineurin activity in rat tissue was studied using dephosphorylaticn assay of calctneurin. the highest caicineurin activity was found in brain too, but was only 5 to 15 fold more than that in other tissues. the specific activity of calcineurin was different in tissues. these results indicate the caicineudn activity might be regulated in tissue specific fashion. burst and cytoskeleton arcaro a. and wymann m.p., institute of biochemistry, university of fribourg, ch-1700 fribourg chemoattractants like n-formyl-met-leu-phe (fmlp) stimulate in neutrophils a rapid and transient increase in the levels of ptdins(3,4,5)p3 (pip3) which is due to the activation of ptdlns(3)-kinase. pip 3 has been proposed to be a second messenger molecule, but its cellular targets are presently unknown.here we report that pretreatment of human neutrophils with wortmannin inhibits the fmlp-etimulated production of pip 3 in a dose-dependent manner (ic50 = 5 nm). furthermore, ptdins(3)-kinase activity immunoprecipitated from resting cells is totally abolished by i0 nm wortmannin (ic50 = 1 nm). these results show a potent and direct effect of wortmannin on ptdins(3)-kinase. wortmannin also inhibits the respiratory burst of neutrophils at nanomolar concentrations, which suggests that pip3 is involved in the signalling pathway controlling activation of the nadphoxidase.when pretreated with wortmannin and stimulated with fmlp, neutrophils display oscillatory changes in factin content that correlate with oscillations in cell ~hape. this, and the inhibition of ptdins(3)-kinase, suggest a modulatory role for pip 3 in cytoskeletal rearrangements.we are currently investigating the effects of pip 3 on the functions of gelsolinl a protein that is proposed to mediate actin polymerization and can be regulated by polyphosphoinositides and ca 2+. matthias gstaiger, walter schaffner and christopher hovens institut f'tir molekularbiologie ill der universitat z'tirich, ch 8057 ziirich the octamer motif atgcaaat plays a central role in mediating the activity of a number of both lymphoid specific and ubiquitous promoters and in the immunoglubulin heavy chain intronic enhancer. the activity of thils motif in lymphoid cells is presumably mediated by the lymphoid cellspecific factor oct-2a. when ectopically expressed in non-lymphoid ceils, oct-2a is not sufficient to mediate enhancer activity of a multimerized 50 bp enhancer core element derived from the immunoglobulin heavy chain intronie enhancer, a segment, which is however highly active in b-cells. this and other findings support the supposition that additional b-cell specific factor(s) can account for the specificity and extent of the octdependent transcription observed in lymphoid cells. to further our understanding of the molecular mechanisms involved in mediating lymphoid-specific expression, we have employed the yeast two hybrid system to identify human proteins capable of physically associating with human oet-2a. to this end, a oct-2a expressing yeast strain has been constructed which bears two integrated reporter genes, his3 and lacz under the control of the octamer motif. transformation of this strain with a cdna library has allowed us to isolate clones interacting with oct-2a. we are currently analysing these candidates to determine the veracity of these interactions in higher enkaryotic cell background. in general, untransformed nuclear hormone receptors are predominantly confined to either the nucleus or the cytoplasm of a cell. receptors of the latter class are translocated to the nucleus upon interaction with ligand. our studies of the use of the ecdysteroid receptor complex from insects, consisting of a heterodimer formed between ecr and usp, as a tool for target gene activation in vertebrate cells have revealed a novel mechanism of nuclear translocation of ecr that does not depend on hormone action. after transient transfection of respective expression plasmids into hela or cv-1 cells, the subeellular distribution of the individual receptor types was analyzed by indirect immanofluorescence staining. while ecr alone was detected in both cellular compartments, nucleus and cytoplasm, usp was predominantly found in the nucleus of either host cell. cooxpression of both ecr and usp resulted in an efficient transloeation of ecr into the nucleus. usp appears to trap ecr in the nucleus, presumably by the formation of a heterodimeric complex. thus, heterodimer formation of ecr and usp not only appears to be required for high affinity binding to cognate hormone response dements (and subsequently for transactivation of an adjacent target gene) and for binding to ecdysteroid but in addition for an enhanced translocafion of ecr into the nucleus. activation of rodent macrophages with cytokines or bacteria is accompanied by the induction of a ca2+-independent nitric oxide (no) synthase which plays a key role in antimicrobial and antitumoral activity. firm evidence for expression of a similar enzyme in other mammals or in man has been lacking. we now show that bovine bone marrow-derived macrophages produce nitrite in an l-arginine-dependent manner upon stimulation with heat-killed grampositive or gram-negative bacteria. homologous interferon-7 and tumor necrosis factor-~ induced little no 2-production, but primed macrophages for enhanced n0 2-production induced by salmonella dublin or lps. this is one of the first demonstrations of no 2-production by nonrodent macrophages and encourages a search for an involvement of reactive nitrogen species in the killing of parasites or tumor cells by macrophages from mammals other than rodents. lectins are used in many analytical methods to study the carbohydrate structure of glycoproteins. we report here a technique which combines the high resolution of two-dimensional gel electrophoresis (2-dpage) to separate plasma proteins, the specificity of lectins to bind carbohydrates and the sensitivity of chemiluminescence. this method is compared with that of a commonly used colorimetrio reaction. since the reference plasma protein map obtained by 2-d page is available (i), the technique described here allows a quick and specific identification of plasma glycoproteins. therefore any ma j or glycosylation modification which may happen in some diseases can be detected. (i) electrophoresis 1992; 13:707-714. this work was supported in part by fcar (quebec, canada). in our laboratory we use genetic and biochemical approaches to study translation initiation, in particular the initiation factor eif-4a in yeast. this factor from higher eukaryofic cells is known as an rna dependent atpase and functions as a rna helicase together with another initiation factor, elf-4b. it belongs to a family of putative rna helicases, the d-e-a-d proteins. to find new genes involved in translation initiation, suppressors of a temperature sensitive eif-4a mutant were isolated. one of the suppressors (stm1) is a single copy gene which encodes a protein resembling the human translation initiation factor eif-4b. disruption of the gene is not lethal to the cell, but affects growth. polysome analysis of strains with a deleted stm1 gene have shown that this new gene is also involved in translation initiation. it carries six repeated sequences of 25 amino acids of unknown function and has -like the human eif-4b -a rna binding domain. the second suppressor (stm2) is also a novel gene. it encodes a large protein which shows no homology to other proteins present in the data libraries. it contains high portion of charged amino acids and is rich in glutamines and serines. its function in translation initiation is currently under investigation. tobacco translationini~ationfactore~4aisencoded by a large anddiverse genefamily karl brander, therese mandel, isabelle lutziger, george owttrim and cris kuhlemeier, institute of plant physiology, university of berne, altenbergrain 21, ch-3013 berne using heterologous probes we isolated cdnas coding for translation initiation factor eif-4a from tobacco, eif-4a is encoded by a large multigene family of which at least nine members are expressed in leaves and most if not all other organs of the tobacco plant. while screening genomic libraries we found several additional genes. two of them code for genes highly homologous with eif-4a throughout the coding region, but with changes in the gkt, ptrela and dead-box motifs. a third gene is expressed exclusively in the developing male gametophyte. this eif-4a-related gene may have a role in the well-documented regulation of translation in pollen. in order to study the function of these genes we have begun altering their expression in transgenic tobacco plants. seauence reauirements for a non-aug protein initiaf~jon non-aug initiation codons are still infrequent and were initially observed in animal viruses, but a number of cellular examples have now also been reported. several years ago we reported the use of an acg in the p/c mrna of sendal virus (sen) to initiate the non-structurar c' protein. we recently described a c' protein in the human parainfluenza virus type 1 (hpiv1), which is initiated from a gug. in both cases, the c' protein is an n-terminally ebngated form of the c protein which is initiated at the second aug, in the +1 reading frame relative to the first aug. this aug is used to initiate the p protein, an essential component of the viral rna polymerase. unlike the acg in sen, the gug in hplvt is clearly not a weak start codon since c' is the most abundant protein expressed from this mrna both in rive and in vitro. it appears that not any upstream gug in an optimal context will function as a start codon. for instance, the p gene of hpiv3 cannot express a c' protein (the orf is closed by stop codons) but it does contain a gug in an optimal context upstream of the p protein aug, which could encode a p' protein. nevertheless, we have been unable to demonstrate that this gug functions as a start cedon. with the use of chimeric mrnas between hpivl and 3, we have shown that positions +5 and +6 (the first g of the gug triplet being +1) are the major determinants of the efficiency of gug as an initiaton codon for protein synthesis. we are currently investigating the possibility that these nucleotides may increase the initiation rate of weak non-augs such as the acg of sen. biological activity of the heterodimeric subunit srp9/14 of the signal recognition particle is retained in 2 fusion proteins fabrice bovia & katharina strub, d6partement de biologie cellulaire, universit6 de gen~ve, ch-1211 gen~ve 4 the targeting of secretory proteins to the membrane of the endoplasmie reticulum is mediated by a cytoplasmic ribonucleoparticle, the signal recognition particle. it is composed of 6 proteins and f rna molecule (srp rna). the 2 smallest proteins srp9 and srp14 are required for the translational control function of srp. it effects a specific arrest in the biosynthesis of er-targeted proteins. these 2 polypeptides bind to the srp rna exclusively as a heterodimer (srp9/14). we have constructed single-chain variants of this dimer using a short peptide linker of 17 aa to connect the c-terminus of the 14 kd subunit to the n-terminus of the 9 kd subunit (14-9) and vice versa (9-14). we found that both proteins bind srp rna with a similar efficiency as the wild type protein. glutaraldehyde cross-linking experiments and sedimentation analysis indicate that the 2 fusion proteins fold into a similar structure as srp9/14 and bind srp rna as a monomer. furthermore, they can functionally replace srp9/14 in the elongation arrest and translocation assay. since the 2 fusion proteins are circular permutations of each other, we can conclude from this results that n-and c-termini of both proteins have no essential role in folding, rna-binding and in mediating their biological activity. furthermore, the possibility to express an oligomeric protein as a single polypeptide facilitates the analysis of its functions as well as its structure. a24 s06-08 studer, r. and hunziker, p. e., 8iochemisches institut der universit~t z0rich, ch-8057 z0rich metallothioneins (mts) are small cys-rich proteins which are inducible by a variety of agents such as bivalent transition-metal ions, tumor promoters, cytokines and hormones. the biological role of mts is still unclear. initially postulated to serve in the sequestration of the toxic heavy metals such as cadmium and mercury, they are now believed to play a major role in the cellular metabolism of essential metals such as zinc and copper. to explore this function further we have now established a method to quantify basal mt production in several human cell lines. thus, cellular isomt contents were monitored by h.pj.c, and the rate of synthesis was followed by the incorporation of ~ss-cysteine. the results show that maximum mt synthesis and accumulation occurs when the cells enter the exponential growth phase. with increasing celldensity the mt content decreases progressively until confluence is reached. in correlation with preliminary measurements our results suggest a close relationship between the mt content, the cell growth rate and the intracellular abundance of zinc. vincent bernard, adrian etter, heinz tobler and fritz mtiller. institute of zoology, university of fribourg, 1700 fribourg (switzerland). the nematode ascaris lumbricoides undergoes chromatin diminution which causes a loss of dna in all somatic ceils. we have identified a ribosomal protein (rp) gene (coding for alep1 = ascaris lumbrieoides eliminated protein 1) which is located within the germqine specific material. alep1 shows strong homologies with the ribosomal protein s19 (rps19). the transcription of its gene takes place only in the germ-line. 2d-gel electrophoresis on germ-line and somatic purified ribosome fractions shows that the alep1 protein (rps19) is present only in the germ-line ribosome. does the alep1 germ-line specific protein have a homologue in the somatic ribosomes? we have cloned a homologue of alep1 from ascaris somatic tissue named rps19', we are currently studying the expression pattern of this rps 19'. these results indicate the existence of a developmentally regulated ribosome heterogeneity between the germ-line and somatic ceils. what is the function of rps 19? since ascaris is not suitable for genetic analyses, we isolated rps19 from the nematode caenorhabditis elegans. the c. elegans rps19 cdna shows only 60% homology at the nucleic acid level. rps19 is located on the c. elegans chromosome iv. the identification of a rps19 mutant in c. elegans will indicate its function. favre, d, l, pavlovic, j2 and michel, m.r. t 1 institute of medical microbiology, university of beme, ch-3010 berne. 2 institute for immunology and virology, ch-8006 ziirich we have successfully purified the recombinant c (recc) protein from spodoptera frngiperda (sf9) insect cells which were infected with acnpv-c (a). the recc protein retained the biological activities of native c protein obtained from wild type sfv (b). our results suggest that the c protein is responsible for the cellular shut-off of host protein synthesis by inhibiting the initiation of translation. we are currently investigating the molecular mechanisms involved in this shut-off. tif3 is a new eukaryotic initiation factor which shows 26% identity with the sequence of mammalian translation initiation factor eif-4b. the tif3 gene is not essential for growth; however, its disruption results in a slow growth and coldsensitive phenotype. in vitro translation of total yeast rna in an extract from a tif3 gene-disrupted strain is reduced as compared to a wild-type extract. the translational defect is more pronounced at lower temperatures and can be corrected by the addition of purified yeast tif3,or mammalian eif-4b. in vivo translation of ~galactosidase reporter mrnas with varying degree of rna secondary structure in the 5' leader region in a tif3 gene-disrupted strain show preferential inhibition of translation of mrna with more stable secondary structure. chloroplast protein synthesis requires the import of elongation factors ef-tu (tuf-gene) and ef-g (fusgene). it seems that the expression of both genes is light regulated. we recently cloned and sequenced a chloroplast specific nuclear fus gene in soybean (biochim.biophys acta 1174 : 191-194, 1993 . further analysis revealed that a second very similar (sequence, anatomy) nuclear fus-2 gene exists which, however, shows strong sequence diversion in the 3'trailer region. several cdna clones were partially sequenced but sofar only transcripts from the fusi gene were retrieved. we currently study the promoter regions of either gene to test possible differential expression of fus genes. comparative mapping and sequencing studies of the two genes in the 3"-region indicate that a major dna insertion occurred distal to the coding part of both fus genes what may also influence gene expression. the rna-binding domains in the 9kd and 14kd subunits of the signal recognition part~clehavea putative~-h~licalstructure. nazarena buiand katharina strub dpt de biologie cellulalre, science ill, universit4gen~ve the signal recognition particle (sr2), a cytoplasmic ribonucleoprotein is important for targeting nascent secretory proteins to the endoplasmic reticulum. srp9and srpl4are constituents of srpandare, together with the alu sequences of srp rna, required for the elongation arrest activity of the particle. srp9 and srpi4 form a heterodimer in the absence of the rna and bind the rnaexclusively as a heterodimer. the primary sequence of srp9 and srpi4 revealed that both proteins are highly basic and lack structural similarities to other characterized rna-binding proteins. we have therefore been interested in characterizing the molecular nature of rna-protein interactions. the analysis of n-and c-terminal deletion mutants of the proteins have shown that both proteins contribute to the formation of an rnabinding pocket, the rna-binding and dimerization domains in both proteins, are found in regions that have a predicted ~~helical structure. in sp~pi4, this u-helical region is located betwen aa 72and i00 at the c-terminus; it contains the rna binding and dimerization domains adjacent to each other. our results support the model that the hydrophobic face of the putative u-helix is implicatedinprotein-protein interactions and the positively charged face in rna-protein interaction. in sp~pg, the two rna-binding domains found n-and c~ terminally, also have a predicted amphipatic s-helical structure. it has been proposed that the decline in protein synthesis observed in aging organisms may result from a decrease in the protein synthesis elongation factor ef-la. john shepherd's finding that transgenic drosophila melanogaster carrying an additional copy of the ef-la gene have an extended lifespan further indicated that the ef-la gene may play an important role in determining longevity (shepherd et al., 1989) . in order to test this hypothesis, we have quantitated ef-la mrna, ef-la protein, as well as catalytic ef-la activity in john's flies. young and aging ef-hx transgenic (ef) and control lines ((2) were examined. because the the additional ef-lc~ copy is under the control of the hsp70 promoter, flies were aged either at 25~ or at 29~ the results show that transgenic ef flies do not express more ef-lct than the control flies, neither at the rna nor at the protein level. the question arises, whether the lransgene can be induced at all. this was tested by doing rnase protection assays. transgenlc message can be detected only in ef flies heat shocked at 37~ but not in ef flies grown at 29~ nor in control flies at 37~ from this experiment we conclude that the transgene is indeed inducible, but is not expressed in detectable amounts at 29~ we conclude that the difference in the lifespan does not result from the overexpression of the ef-la transgene. phenobarbital (pb) induces the expression of liver enzymes involved in the metabolism of drugs and other foreign compounds (xenobiotics). these enzymes include several cytochromes p450, nadph:p450 reductase, aldeyde dehydrogenase, epoxide hydrolase, udp-glucoronyltransferases and glutathione-s-transferases. many other proteins not yet recognized may however be induced by pb. our first approach therefore is aimed at identifying gene products which respond to pb treatment. as model systems we are using either chicken embryos (induction in ovo) or primary cultures of chicken embryo hepatocytes (induction in culture) (althaus et al., jbc 254 pp 2148 , 1979 . to differentially display mrna of induced vs non-induced cells we are using a rt-pcr technique with sets of random primers. electrophoretic separation of amplification products allows to identify mrna species which are induced (or decreased) following treatment. data obtained from these experiments and sequence information revealed from extracted pcr products indicate that pb treatment results in the transcriptional avtivation (or repression) of a variety of so far unknown genes. antithrombin -binding heparan sulfates from ovarian granulosa cells hosseini g., de agostini, a., clinique de st6rilit6, hcug, gen~ve. at the time of ovulation, several serine proteases of the plasminogen activator and coagulation cascades are activated in the ovary. these proteolytic activities are tightly controlled in time and space, and the heparin-activated serpins plaminogen-aetivator-inhibitor-1, protease nexin-1 and antithrombin (at) are present in the follicular environment. we have observed that granulosa cells produce a subset of heparan sulfate proteoglycans that bind and activate at. these at-binding heparan sulfates (ahspgs) endow the vascular endothelium with antithrombotic properties. we are now examining the role of ahspgs in the extravascular compartment formed by the ovarian follicle. using 125i-at binding assays, we have detected ahspgs on granulosa cell surfaces and culture media, in amounts comparable to those found for endothelial ceils. after 48h of stimulation by the gonadotropin fsh (1-100 ng/ml) granulosa cells increased the amounts of ahspgs they released in culture media by 2-6-fold, while keeping their cell surfaceassociated ahspgs constant. analysis of 35s-labelled hspgs revealed that heparan sulfates constitute about 40 % of the surface-associated and 10% of the soluble granulosa cell glycosamineglyeans. finally, using 125i-at autoradiography on rat ovary cryosections, we have localized ahspgs on the granulosa cell layers of large antral follicles, while ahspgs could not be detected in smaller follicles. these data suggest that granulosa cell ahspgs could be critically located to modulate proteolytic activities in the changing environment of the growing follicle. division, cibabasel, switzerland. the development of chelators which can mobilise excess storage iron (fe) and permit its excretion is necessary in the treatment of fe overloaded diseases. although oral administration of such a pharmaceutical is highly desirable, no orally active fe chelator is so far in regular clinical use. searches for orally active and safe fe chelators require appropriate animal models of fe overload in order to evaluate the potential of these drugs. an animal model is particularly useful for testing the capacity of new fe chelating ce~oounds in enhancing the mobilisation of clinically relevant storage iron. iron loading in animals was achieved by dietary supplementation with carbonyl fe, fe fumarate and 3,5,5-trimethylhexanoyl ferrocene (tmh-ferrocene). liver fe concentrations were increased 6-21 times above normal levels. tmh-ferrocene was the most efficient compound to induce stable liver fe overload. the orally given con'pounds induced a predominantly hepatocellular iron distribution and was found distributed among reticuloendothelial cells only at a later stage. this pattern of liver fe storage is similar to that observed in the early stages of primazy ha6~ochromatosis and late stage of transfusional hae~ochromatosis. eeeently, we and ethers have cloned and characterized novel transcription factors called peroxisome proliferator-actirated receptors (ppak). they belong to the superfamily of nuclear hormone receptors as the steroid, thyroid and retinoid hormone receptors. ppars are activated, beside xenobiotic peroxisome proliferators, by natural fatty acids and induce, together with the receptor for 9-cis retinoic acld (rxr), the transcription of genes involved in the ~-and ~-oxidation of fatty acids. ppar and rxr bind as heterodimers to the ppar response element (ppre), consisting of a direct repeat of aggtca-iike sequences with one intervening nucleotide. now, we show by gel retardation analysis and transient transfection assays that ppar/rxr heteredimers also bind to and transactivate gene transcription through estrogen response elements (ere). the ere is a palindrome with aggtca half-sites separated by three nucleotides. the selectivity and specificity of the transactivation through an ere was demonstrated by the testing of various related palindromic and inverted palindromic repeats of aggtca sequences, which were all inactive. the possible activation of estrogen responsive genes by fatty acids via ppars in vivo is discussed especially with regard to a debated role of fatty acids in breast cancer. the purpose of this study was to evaluate whether intermittent doxorubicin (dox) treatment in vitro selects for multidrug resistance in rpmi 8226 myeloma cells and, if so, to determine the underlying mechanism(s). cells were exposed to constant doses of dox for 4 days alternating with growth in dox-free medium for 17 days. after > 9 months of treatment, the cells developed an atypical mdr phenotype with 4-to 6-fold resistance to topo ii poisons. cross-resistance to vincristine and taxotere was < 2-fold, sensitivity to cisplatin and melphalan remained normal. efflux of mdr1 drugs was incresed with no effect of verapamit; subcellular dox distribution was normal. expression of topo ii a was found to be reduced by around 50 and 60-70% at the rna and protein level, respectively. minimum mdrt rna overexpression was detected when using rt-pcr; p-glycoprotein was not detectable. mrp rna expression was increased by 60-90% relative to the wild type cells without detectable p190. this data suggests that atypical mdr might play a role in acquired dox resistance in human myeloma. mucosal surfaces cover a vast area in the human body. they satisfy its need to communicate with its environment, e.g. in terms of nutrient uptake and sexual reproduction. however, at the same time they offer a vulnerable gate for invasion by pathogenic microorganisms. for immunoprotection of these regions, large amounts of iga antibodies are produced in the bloodstream and transported across the epithelial barrier to the lumen by means of polymeric ig receptor. there the receptor is cleaved, and its extracellular portion remains associated as secretory component (sc) to the secretory iga complex. in the context of a project aiming to mass production of secretory iga as a passive vaccine, we evaluated a series of eukaryotic expression systems for production of sc. the cdna encoding polymeric ig receptor was engineered in a way that only its extracellular domains corresponding to sc were expressed in yeast, in insect cells infected with recombinant baculovirus, and in mammalian cells infected with recombinant vaccinia virus. furthermore, a c-terminal poly-histidine tag was introduced for simple purification of the products. we will show optimization of expression levels for each of the systems as well as a quantitative and qualitative comparison of the products. recombinant vaccinia viruses (rvv) that express gone products from other organisms has become a popular and widely used laboratory expression system. our current interest is directed toward the production of secretory component (sc), a subunit of secretory iga antibody complex involved in mucosal immunity. toward this goal, we constructed rvv governing sc transcription under the control of two viral-based and the t7 bacteriophage promoters. expression of the protein was assessed in several mammalian cell lines, such as human tk-, human hela (grown in suspension or as a monolayer) and monkey cv-i. optimization of infection parameters and culture conditions were also performed. institut de biologic animale de runiversit~ de lausanne protection of mucosal surfaces is mediated by secretory iga ant/bodies (alga) constituted of dimeric iga and secretory component (sc) . toward the aim of reconstituting slga complexes in vitro and using them for passive oral immunization, sc has been produced in yeast, insect and mammalian expression systems. the gone encoding sc has been engineered so that a) the c-terminus of the protein carries a 6xhis tag and b) the newly synthesized polypeptide is released in the culture medium. culture conditions have been established to permit recovery of sc in serum-free medium. purification procedures based on nickel chelate and classical chromatographies have been optimized to yield sc under native conditions. finally, in vitro reassociation with purified dimeric igas obtained from hybddomas has been used to assess biological activity of recombinant sc. using a magnet-assisted subtraction-hybridisation technique (mast), 17 cdna clones were isolated that are expressed in nqrm~l l~nq %issue but n__d_i expressed in the corresoondina nsclc tissue, ii of the 17 edna clones are highly homologous to edna sequences for the following proteins: pulmonary surfactant proteins sp-a and sp-b; receptor for advanced glycosylated endproducts of proteins (rage); calmodulin-like protein; natural killer gone 5 (nkg5); matrix gla protein (mgp); glutamine synthetase; ubiquitin; vascular smooth muscle alpha-actin; cytoskeletal beta-actin; vimentin. the remaining 6 edna clones may represent parts of still unknown genes. the mast edna clones were derived from a patient who developed squamous adenocarcinoma. northern blot analysis of normal/tumour rna from three other nsclc patients showed that the lack of gone expression was independent of nsclc type and stage. enzymatic methylation of cytosines in mammalian dna is believed to play a fundamental role in basic gene regulation. methylation in the vertebrate genome is restricted to cpg dinucleotides at the c-5 position of cytosine. in vitro studies have shown that methylation can drastically influence the dna structure. cpg islands remain unmethylated in normal cells, whereas several immortalised, transformed cell lines contain partially methylated cpg islands. transcription of genes can be inhibited by methylation of cpg's. this modification contributes e.g. to the stable repression of genes on the inactivated x chromosome of females. the collagen vi genes show typical cpg islands in their promoter region and they were shown to be down regulated at the transcdptional level in src or myc transformed cells. given these facts we dee~ded to investigate the effects of methylation in the collagen vi gwene. e could demonstrate that the promoter activity of (x2(vi) chicken collagen gone was strongly diminished by methylation in vitro. furthermore dna methylation completely prevented binding of a novel transcription factor to the promoter, whereas binding of sp1 was not affected. to show evidence of methylation in vivo, we are currently iocalising the exact positions of methylated cpg's in normal and transformed cells by genomic sequencing. alterations of putative tumour suppressor genes in human non-small cell lung carcinoma (nsclc). r. shipman and c.u. ludwig, molecular oncology, lab 405 (zlf), basel, ch-4031 chromosomal subregion llp13 displayed non-random allelic loss in 61 nsclcs. llp13 contains the genetic loci for catalase (cat), the wilms' tumour gene (wti) and the follicle-stimulating hormone ~-chain (fsh~). 76% of the nsclcs were deleted at cat suggesting that loss of a gene(s) near cat may be involved in the progression of nsclc. yac clones containing the cat locus are being prepared for use in a direct selection procedure for cdnas encoded at this region. although 38% of the nsclcs were deleted at wti, no mutations were detected in the remaining wti allele. wti expression was subsequently demonstrated in fetal lung, fetal hematopoietic tissue, normal adult lung and nsclc. allelic deletion at 17p13, immuno-histochemical and sequence analysis of the p53 gene were also examined in our nsclcs. these analyses support the contention that aberrant expression of the p53 gene is a pivotal event in the progression of nsclc. $08-11 recently, a number of advances have been made which enable the experimentalist to study the effects of low levels of ionizing radiation. the results suggest a threshold to radiation damage exists and that above a critical level, recovery mechanisms in the cell are induced and the biological response falls. thus low levels of radiation cause greater than expected levels of biological response. this has been demonstrated in studies of mutations, cell survival and cancer induction. because the low dose-rates and the low doses per fraction result in the total doses being small, the magnitude of the biological effects induced is insufficient to warrant alarm. identification of genes associated with the revertion of the malignant phenotype of a rhabdomyosarcoma cell line. michele genini, sabina solinas toldo*, ruedi fries* and beat w. schafer, dept. of pediatrics, university of ziirich, steinwiesstr. 75, 8032 ziirich, and *institut for nutztierwissenschaften, eth ztifich, 8092 ztirich. the human rhabdomyosarcoma cell line rd is tumorigenic and differentiates very poorly despite the expression of the myogenic factors myf3 and myf4. therefore it can be regarded as natural mutant. to identify genes which are involved in suppression of tumorigenicity or enhance differentiation we applied two different approaches. since the development of human embryonal rhabdomyosarcoma has been associated with abnormalities of chromosome 11 band p15, in the first approach we transferred a normal human chromosome 11 into rd ceils via microcell fusion. the microcell hybrids did not show a higher degree of differentiation. suppression of tumorigenicity was observed in one clone but is probably independent of chromosome 11, as it was shown by chromosome 11 specific painting. ' in the second approach we applied a subtractive hybridization procedure between primary myoblasts and rd cells to find genes specific for the normal phenotype. these genes will be tested in vivo for induction of differentiation and/or tumorsuppression. deficient synthesis of the gpi anchor is the molecular basis of idiopathic paroxysmal nocturnal hemoglobinuria ("pnh") and a similar phenotype can accompany aplastie anemia cpnh-iike"). the population expressing cd48, a gpi-anehored membrane glycoprotein, was quantified by flow cytometry in samples from patient p.g. (pnh) and patient m.m. (pnh-like) and amounted to 90% (p.g.) and 78% (m.m.), respectively. t lymphocytes from both patients were isolated, expanded and cloned. from both patients, clones expressing the cd48 marker (cd48+) and cd48 deficient clones (cd48-) were obtained. takeda et al. (cell 73, 1993, 703-11) showed that in some (cd59-) bcell clones, a deletion of the p1g-a gene product is responsible for the expression of the pnh-phenotype. pcr analysis of t cell mrna from extracts of both cd48+ and cd48-clones of patient m.m. (pnh-like) using pig-a specific primers revealed a band pattern from which can he concluded that no deletion is present in the pig-a gene product. this result suggests that cd48-t lymphocytes of the pnh-like patient express a normal pig-a gene product; a disorder in regulation of pig-a expression or an unrelated biosynthetic defect may explain the cd48phenotype in these t cells. further work is aimed at defining the differences in biosynthetic defects of pnh and pnh-like cd48-t cell clones. supported by grant 31-30757-91 of the snsf to egb. w@ have recently employed an experimental approach to turn the function of "nuclear messengers" such as c-fos (and c-jun) on and off at will in polarized me/nmary epithelial cells. to this end we used constructs encoding the c-fos (and c-jun) genes fused to the hormone-binding domain of the human estrogen receptor, designated c-foser (and c-juner), we could show that short-term activation (30 mins.) of c-foser by estradiole (e2) led to the disruption of epithelial cell polarity within 24 hours, as characterized by the expression of apical and basolateral marker proteins. during the next 3-5 days, however, the cells fully regained their polarized phenotype. conversely, long-term activation (24-48 hours) caused the irreversible loss of epithelial cell polarity, leading ultimately to an epithelial-fibroblastoid cell transition. these cells underwent dramatic changes in gene expression including the complete loss or reduction of epithelial markers such as e-cadherin/uvomorulin, zo-i and cytokeratins, and the de novo expression of mesenchymal proteins like vimentin, type i collagen and fibronectin. expression of the v-ha-ras oncogene (acting upstream of c-fos) did not markedly influence epithelial cell organization when the cells were grown on plastic. however, when cultured within type i collagen gels in the presence of serum, or when injected into the mammary glands of nude mice, rapid cell growth and a clear epithelial-fibreblastoid cell transition was observed. growth properties determine the organ colonization ability of a metastatic murine melanoma cell line. the ability of metastatic tumor cells to specifically colonize distant organ sites strongly depends on the interaction of the metastatic cells with the local organ environment. we have selected a b16 routine melanoma cell fine (b16-ls9) which has an increased ability to colonize the liver, and compared its behavior with either its parental (b16-f1) or inng-specific (b16-f10) cell line. we have found that adhesion in vitro and organ lodgement in vivo were not different for ls9 and f10. in contrast, b16-ls9 grew at slower rates than the other lines both in vitro and in vivo after subcutaneous or intra-foot-pad injection. analysis of tyrosine-phosphorylated proteins indicated a higher phosphorylation activity in b16-ls9 cells, which might suggest an altered regulation of some kinase(s) in this cell line. intercellular contact with hepatocytes in vitro, or the liver in vivo could restore an efficient growth of b16-ls9, enabling thus these cells to colonize this organ much better than others. hepatocytes or liver plasma membrane (p.m.) extracts conserved the growth stimulatory activity towards b16-ls9 ceils. chromatographic fractionations of these extracts showed that a major growth sfimulatory protein in this fiver p.m. fraction turns out to be ttansferrin, which in the liver appears to exist in at least two different mature forms. protein import into mitochondria requires atp in two locations: outside the organelle, and in the matrix space. depletion of matrix atp arrests translocation of precursors across the inner membrane, but does not prevent precursors from crossing the outer membrane. as a result, proteins that reach the inner membrane or intermembrane space without passing through the matrix are often sorted normally in atp-depleted mitochondria. external atp is used to maintain precursors in a translocationcompetent state. however, some precursors can fold and still remain translocation-competent, and import of these precursors is independent of external atp. with the folded cytochrome b2 precursor, it is matrix atp rather than external atp that drives translocation across the outer membrane. thus matrix atp generates a pulling force that can drive both the translocation and the unfolding of precursor proteins. mitochondrial hsp70 probably functions as the atp-dependent import motor. arsenijcvic d, dulloo ag & girardicr l, dpt of physiology, cmu. geneva, ch. the relationship between body weight, daily energy expenditure (assessed by indirect calorimetry), and immune status (determined by lymphocyte proliferation tests) was investigated in swiss webster mice maintaining a stable body weight several weeks after infection with toxoplasma gondii(me49). following an initial period of weight loss (infection-induced cachexia), the surviving mice could be differentiated into two main groups: (i) those showing a partial regain in body weight (the infected gainers) and eventually stabilizing al a mean body weight 25% below a non-infected control group, and (if) those showing no weight regain (the infected non-gainers) and eventually stabilizing at 45% below control levels. immune function was found to be markedly suppressed in the infected non-gainers, whereas it was normal (and similar to control levels) in the group of infecled gainers. daily energy expenditure (and food intake), after normalisation for differences in body weight, was found to be the same in both infected gainers and non-gainers, but lower by 15% when compared to controls (p<0.01); thereby indicating that the infected groups were able to increase their overall efficiency of food utilization in response to the low food inlake. in conclusion, this study conducted during a weight stable phase of chronic infection shows a clear-cut association between the inability to regain body weight and impaired immune function. in contrast, no relationship was found between metabolic rate and body weight nor between metabolic tale and immune function, but the chronically infected mice exhibited the same phenomenon of enhanced metabolic efficiency characteristic of chronic underfeeding per se. identification and functional analysis of chaperonin 10, the groes homolog of yeast mitochondria. biozentrum, university of basel, klingelbergstrasse 70, ch-4056 basel, switzerland. phone ++41-61-2672171, fax ++41-61-2672148. the mitochondrial chaperonin system consists of chaperonin 60 (cpn60; also termed hsp60), which is homologous to e. cull gruel, and chaperonin 10 (cpnlo), which is homologous to e. coil groes. we have used a functional assay to identify cpnl0 of yeast mitochondria. when dimeric rubisco is denatured and allowed to bind to yeast cpn60, subsequent refolding of the enzyme is strictly dependent upon yeast cpnl0. in the presence of mgatp, yeast cpn60 and yeast epnl0 form a stable complex that can be isolated by gel filtration. the atpase activity of hsp60 is not inhibited by complex formation with cpnl0. a different result is obtained with the e, coil system, where groes is able to completely inhibit the atpase activity of gruel (1). to test the in vivo role of cpnl0, we have cloned, sequenced and disrupted the corresponding nuclear gene cpnio. this gene encodes a protein of 11,372 da that is imported into the mitochondrial matrix without detectable cleavage. haploid cells lacking a functional copy of cpnio fail to grow at temperatures between 23 ~ c and 37 ~ c (2). (1) rospert, s. et al. (1993) proc. natl. acad. sci. 90, in press. (2) rospert, s. et al. (1993) febs lett., in press. characterization of yeast mitochondrial heat shock protein 70 , l. bolliger, b. s. glick and g. schatz, biocenter, university of basel, 4056 basel. mitochondrial hsp70 (mhsp70) is thought to use the energy of atp hydrolysis to pull precursor proteins across the mitochondrial membranes. to facilitate the purification of yeast mhsp70, we used the cloned gene (a gift of dr. n. morishima) to modify mhsp70 with a six-histidine tag at its c-terminus. this construct supports growth of yeast cells lacking wild-type mhsp70. we developed a purification procedure that allows us to recover this tagged protein with a purity of about 95%. experiments are in progress to characterize the nucleotidedependent interaction of purified mhsp70 with unfolded proteins. in addition, we are looking for partner proteins that may modulate the action of mhspt0. we have copurified a protein of about 20 kd together with mhsp70. the 20 kd protein could be released from mhsp70 by atp or adp. when tryptic fragments of this protein were subjected to microsequencing, one fragment showed strong homology to the grpe protein of e.coli. the transmembrane electrical potential of mitochondria (awmit) can be assessed in single hepatocytes by using epifluorescence microscopy. for this end the rhodamine 123 fluorescence of a mitochondria-rich (fr) and a mitochondria-poor region (fp) are measured. the ratio of these signals depends on the awmit according to a modified nernst equation (reber b .f.x., somogyi r. & stucki j.w. (1990) , bba, 1018, 190-193) . to calibrate this method the cell membrane was permeabifized for monovalent cations with nystatin and gramicidin. then the cytosolie k + was replaced by na + from a k+-free bath solution. subsequent addition of valinomycin made the mitochondrial membrane permeable for k + ions. by the addition of different k + concentrations to the bath, the awmit could be clamped to defined values. the relation between the ratio fr/fp and the awmit could be well fitted to our modified nernst equation. however, the calibration curve flattens out at potentials higher than about 110 inv. therefore the detection limit of changes of a~mit within the physiological range is about 10-20mv. maximal stimulation of the na+/k + atpase by addition of nystatin to the na + containing bath medium caused a drop of awmit of about 20inv. subsequent addition of ouabain resulted in an almost complete reversal of this drop. this shows that changes in atp consumption can be assessed via changes of audmit. schneiter ph., di vetta v., j6quier e. and tappy l. institut de physiologie de l'universitg bugnon 7,1005 lausanne. the metabolic effects of an exercise performed in the fasting or fed state were studied in 8 subjects over a 8 hour period in a respiratory chamber. a mixed meal containing 13c labeled carbohydrates was ingested at 9:30 am and an exercise session (walking at 5 km/h 45 rain, slope 10 %) was performed at 8:15 am (fasting) or 11 am (postprandial). net carbohydrate (net cho ox) and lipid oxidation (indirect calorimetry) and oxidation of exogenous carbohydrates (exo cho ox, breath 13co2) were measured. glycogen breakdown was calculated as net cho ox -cho exo ox, and glycogen synthesis as cho in -cho exo ox. energy expenditure over 8 hours was similar but net cho ox was 16 % lower, lipid ox 61% higher and exo cho ox 39 % lower when exercise was performed in fasting vs fed state; moreover, glycogen synthesis was increased by 40 % (p<0.0001), while glycogen breakdown was increased by 12 % (p=0.06). in conclusion, a 45 min exercise in the fasting vs fed state a) increases utilization of endogenous lipids, b) enhances muscle glycogen turnover over a 8 hour period. hormonal regulation of e-abl tyroslne kinase by fusion to a steroid binding domain t. mattioni, o. 8chini hooft van huijsduijnen, p.k. jackson and d. picard d4partement de bielogie cellulaire, universit4 de geneve, ch-1211 geneve 4 the activities of a wide variety of proteins can be subjected to hormonal control by fusion to the hormone binding domain of steroid receptors. we show that this strategy can also be applied to a tyrosine kinase. in particular, transforming derivatives of c-abl become hormone-inducible oncogenes by fusion to a steroid binding domain. it is noteworthy that the hormone-inducible transformation of nih3t3 cells is completely reversible upon removal of the specific ugand. reversion experiments reveal another facet of abl: its ability to inhibit cell proliferation. 48 hours after hormone depletion, cells are not only morphologically reverted, but the cell cycle appears to be blocked in g1. a cytostatic effect of abl overexpression has been reported by several groups but, until today, it could only be examined by comparing different abl derivatives or the same derivatives in different cellular contexts. in contrast, our hormone-regulable system has the advantage that the two distinct functions of abl (transformation versus growth inhibition) can be studied using the same derivative expresed in the same cell line. we are using the hormone inducible system to investigate the mechanisms regulating the transtorming and the cytostatic effects of abl in a variety of cell lines. jiewu yang and herbert zuber institute for molecularbiology and biophysic, eth, 8093 z'tirich enzymes from thermophilic organisms show higher thermostability and temperature optima than those of mesophilic organisms. it has been shown that these properties are based on a specific structure of the protein. the primary structure of thermophilic (b. stearothermophilus) and mesophilic(b, megaterium) triosephosphate isomerase(tim) have been determined by cloning and sequencing of the tim genes. comparison of the primary structures of the thermophilic and rnesophilic enzyme showed specific amino acid substitutions, particularly in the c~-helices of the tim barrel, which could play a critical role with respect to thermostability. we have consmacted mutants by exchanging (x-helices between tim of b. stearothermophilus and b. megaterium. helix h1, h2 and h7 of tim from b. megaterium, corresponding to amino acid residues 13-36, 44-55. and 220-227 respectively, have been exchanged. the properties (thermostability and temperature optimum) of the hybrid-mutants were compared with the wild type enzymes. in a comparative investigation of structural and functional parameters related to the oxidative substrate metabolism in skeletal muscle cells of dogs and goats, a close relationship was found between intracellular lipid stores and mitochondria. as revealed in serial sections with subsequent em analysis, each lipid droplet was in direct contact to one or several mitochondria. quantitatively, 23% of the lipid droplet surface in goats and 40% in dogs were in direct contact to outer mitochondrial membranes or -in other terms -1% of outer mitochondrial membranes in goats and 2% in dogs were in direct contact to lipid deposits. these findings were in good agreement with the physiological data showing a 2 times higher oxidation rate of fatty acids drawn from intracellular stores for dogs than for goats. human muscle adapts to altered load with changes in the expression of enzymes involved in energy metabolism. endurance exercise in humans is known to increase the oxidative capacity of skeletal muscles (hoppeler, int. j. sports med. (1986), 7, 187) . at the ultrastructurai level, a higher mitochondriai content contributes to the higher oxidative capacity of skeletal muscles. little is known about the molecular mechanisms that lead to such adaptations in humans. the expression of mitochondriai components involves complex regulation of both mitochondriai and nuclear encoded genes. we have developed a pcr approach to quantify dna and rna from human skeletal muscle cryostat sections and addressed the question, whether the structural adaptations in endurance trained athletes are accompanied by concomitant changes in respective rna steady state levels. preliminary data indicate a higher concentration of coxiv and coxl (1.8 and 1.6 times, respectively) in trained subjects. no differences were observed for mtdna, despite a two fold higher mitochonddal content. human skeletal muscle contains three main fiber types which are based on the myosin heavy chain composition of the individual fiber, the isoforms of other contractile proteins within a given fiber type can vary leading to a continuum of different phenotypes. we are studying the fiber type specific expression of the mrnas of myosin alkali light clmins (mlc) using in situ hybridization, especially the rarer slow form mlc lsa. differences were found between shoulder muscles and leg muscles: m. deltoideus yielded stronger signals for mlc lsa and mlc lsb in the least glycolytic type i fibers (ia), weak but detectable signals for mlc lsb and mlc lf/f mrna in more glycolytic type i fibers (ib) and strong signals for mlc lf/3f in type ii fibers. in m. vast. lat. mlc lsb was strongly expressed in type ia as well as ib fibers, mlc lsa mrna was only found in some ia fibers. mlc isa has been shown to be expressed at a particular time point during muscle development and regeneration. in a biopsy of a becker dystrophy patient, we found very strong signals in some small fibers displaying the morphology of regeneralmg fibers. thus this probe could be useful to dete~t regenerative processes in pathological muscle samples. the aim of this study was to validate the complementarity of two new non-invasive methods for the assessment of autonomic regulations. reactions to a moderale exercise (75 w on a cycloergometer) were compared in cardiac transplant recipients (ctr) who modulate their heart rate (hr) by means of circulating calecholarnines, their heart being donervated, and in normal subjects where such exercise level doesn't stimulate the sympathetic system. the methods used were: 1) the spectral analysis of hr variability where the high frequency component (hf, above 0.15 t4z, related to the respiratory frequency) is a marker of the vagal activity, the low frequency component (lf; at about 0.1 hz) depends on both sympathetic and parasympathetic activities and the lf/rf ratio indicates changes in the sympatbo-vagal balance; and 2) a pure sympathetic index obtained from the amplitude of the t-wave of the ecg (twa) which is decreased by adrenergic stimulation of the myocardium. our results indicate that in both groups hr was increased by the exercise. in ctr, a net decrease in twa (-39_+ 10%, n=6) was measured. contrasting with this, in normal subjects, no change in twa (+2_+12%, n=6) but a decrease in beth hf (-67_+13%, n=7) and lf (-85_+5%, n=7) without change in lf/hf ratio (-19+_.26%, n=7) were observed. these results indicate that ctr subjects increase their hr by an adrenergic stimulation, whereas the normal subjects regulate their hr for such moderate exercise only by redudng their vagal tone. there was a significant relationship (r=0.79, p<0.0005) between ps and the ree. the slope of the regression line indicated a net cost of ps of 3.6 kcal/g. we conclude that obesity in children is associated with an absolute increase in whole-body protein turnover consistent with the increased ffm and ree. the "glucose paradox" in the anoxic and reoxygenated embryonic myocardium raddatz, e., tran, l., rochat, a.-c., kucera, p. and de ribaupierre, y. institut de physiologie de l'universitd, ch-1005 lausanne. the hearts of 4-day-old chick embryos explanted in vitro react rapidly, reversibly and reproducibly to anoxia and reoxygenation. this work deals with the effects of exogenous glucose on the mechanical activity of the hearts submitted to strictly controlled normoxia-anoxia-normoxia transitions. the anoxic periods lasted from 10 to 60 seconds. instantaneous heart rate, amplitude of contraction, velocities of contraction and relaxation of the ventricle wall were determined optically. in presence of 8 and 20 mmol/1 of glucose 1) the arrest of cardiac activity under anoxia was delayed by 60 and 110 %, respectively, 2) the duration of the postanoxic cardioplegia was reduced and 3) recovery was faster. paradoxally, these concentrations of glucose induced arrhythmias both during anoxia and reoxygenation. the incidences of such arrhythmias increased with the duration of anoxia. the absence of glucose or blockade of glycolysis suppressed arrhythmias. image analysis showed that the observed myocardial dysfunctions originated in the atrium and were sometimes associated with disturbances of propagation. the microinjeetion of okadaie acid into incompetent oocytas leads to the release of the prophase block with entry into m-phase. these g2/m-like alterations include chromatin condensation, germinal vesicle breakdown (gvbd), microtubules reorganization and formation of an abnormal (often multipolar) spindle of meiosis i, with chromosomes not aligned on the metaphase plate. the polar body is never extruded. moreover okadaic acid microiujection induces the emergence of phosphorylated cytolasmic foci as detected by the monoclonal antibody mpm-2, known to react with mitotic phosphoproteins associated with centrosomes. the kinetics of gvbd following mieroinjection is extremdy retarded (24-48hrs) when compared to spontaneous maturation (30ran-lhr) and depends on the oocyte diameter. interestingly, when inc~ompetent oocytes are cultured during 48hrs and then microinjeeted with okadaic acid meiosis resumes within a short delay (3-rhrs). mpm-2 epitopes are present on ecntrosomes only after okadaic acid injection. following gvbd one of the step depending on protein synthesis, the expression of tissue type plasminogen activator, is triggered. indeed oa-injected incompetent oocytes produce small amounts of this activator. altogether these results argue for an effect of okadaic acid favoring entry into mphase including microtubule organization, centrosomes phosphorylatiou and the recruitment of one of the masked mrnas (tpa). st~rillt~, ncb~, c~-121l g~. ~t4rs-inse~, monf~llier, france. meiotic reinitiation of the mouse oocyte is caracterized by a slow entry into metaphas~ i, b6~jinnin9 with germinal ve~ir break@own (gvbd) and ending with spindle formation. it is accompanied by a cascade of protein kinases and phosphatases increasir~g protein phosphorylation. the activation of histone hi kinase (maturation promoting factor, mpf) and of the 1342 hapk have been compared during spontaneous or okedalc acid (oa)-induced rhetoric reinitiation. in spontaneously maturing oocytes, hist6ne hi kinase activity increases before gvbd (2x) and is a~sociated with the disappearance of the upper (tyrosine phosphorylated) migrating form of p34 cdc2, following gvbd, histone hi klnase activity culminates (8x) in metaphase i. activation by phesphorylation of p42 mapk occurs after gvfld. in contrast, no increase of histone hi kinase is detec~cable in oocytes induced to reinitiate meiosis by a transient exposure %o oa, neither before gvbo nor during the following 7 hours of culture. the upper migrating form of p34 cdc2 is present for 8 houra. the activation of p42 mapk b~lins before gvbd. furthermore, when oa is applied on coclrces microinjected with p13 sucl, neither iocrease of histone hi kinase activity nor p34 cdc2 dephosphorylation are observed although gvbd is induced; p42 mapk is activated. altogether these results suggest that gvbd may or may not be associated with a detectable activation of histene hi kinase, depending on the experimental conditions. activation of ps4 cdc2 and p42 mapk are separable events. we propose that, in the mouse c~yte, oa might be able to aetivaf8 an alternative pathw~ leading to gvbd that is cdc2-independent and that involves p42 mapk activation ensuing mpf-independent phosphorylations. s 10-04 urich, k., 4002 basel, switzerland transformation of cells by polyomavirus is mediated by middle-t antigen. this viral protein is able to form complexes with src family tyrosine kinases (e.g. c-src), phosphatidylinositol-3-kinase (pi 3-k) and phosphatase 2a (pp2a). c-src associated with middle-t is constitutively dephosphorylated at tyrosione 527, a site negatively regulating the activity of this enzyme. this results in high tyrosine kinase activity in interphase and mitotic polyoma-transformed cells. middle-t is transiently hyperphosphorylated during mitosis as reflected by an increase in the apparent lvl, on sds acrylamide gels. two putative phosphorylation sites for a cyclin-dependent kinase present in middle-t, threonine 160 and threonine 291, are also phosphorylated by purified p34 ~a~ in vitro. we constructed mutant middle-t genes where individual phosphorylation sites were converted to alanine residues. mutants lacking threonine 160 were still able to associate with all cellular enzymes described above yet defective for gell transformation. interestingly, the defect of this mutation could be compensated by additional changes in the middle-t protein sequence introduced further downstream in a domain suspected to play a role in the targeting of this protein to intracellular membranes. $10-05 schmidt, s., fa~khauser, c., and viesturs, s. isrec, 1066 epalin~es, switzerland little is understood of the mechanisms which regulate cytokinesis, or how it is integrated with mitosis. we have cloned a number of genes from the fission yeast s. po~be which are required for the initiation of septation and are characterising them. defects in the cdc7 and cdcll genes result in continued nuclear division without cytokinesis, while cdcl6 mutants undergo multiple rounds of septum formation without cell cleavage. our analysis suggests that phosphorylation will play an important role in the regulation of cytokinesis and its integration with mitosis. a functional cdcl6 product is required for maintenance of p34 r activity in mitosis and the primary sequence of the cdc7 protein indicates that it encodes a protein kinase. data concerning the role of the cdc7, cdcll and cdcl6 genes will be presented. rfc was originally identified in human cell extracts as one of the cellular factors essential for the replication of sv40 origin-containing dna in vitro. it is a five subunit protein with one large subunit of 100-i40 kda and four small subunits of 36-40 kda. rfc binds specifically to primertemplate structures at the 3'-end of the primer. together with proliferating cell nuclear antigen (pcna) it serves as a primer recognition factor for dna polymerase ~ and ~. to show the invobement of rfc in cellular dna replication we are currently cloning s. cerevisiae rfc. the amino acid sequences of a number of tryptic peptides have been obtained. this information was used to amplify parts of the s. cerevisiae genes by per and to screen a genomic library with these probes. three genes were cloned so far. the large subunit, rfc1, codes for a 95 kda polypeptide that is 36% identical to the large subunit of human rfc (lirfc). the sequences for two of the small subunits were also obtained. rfc2, coding for a 40 kda polypeptide, is 50% identical to the hrfc 37 kda subunit. rfc3, coding for a 38 kda polypeptide, is 51% identical to the hrfc 36 kda subunit. there is also considerable similarity between the different subtmits. rfcj, rfc2 and rfc3, as well as the hrfc subunits, have consensus sequences for a atp/gtp binding site. all three genes are essential in s. cerevisiae. p53 is a major tumor suppressor gene which spefically interferes with the onset of e phase. we have therefore cxa~ined the possibility that p53 modulates the normal functions of enzymes and proteins directly involved in dna replication. we have shown that human wtp53 protein binds physically to calf thymus single stranded dna binding protein a, rp-a, we have also found that p53 blocks dna helicases in a typical strand displacement assay. this may reflect the fact that p53 is able to reanneal complementary dna single strands. since both wt and some mutant forms of p53 share this function, they are unlikely to be related to the p53 tumor suppressor activity. to our surprise, among the helicases which were tested we determined that the p53 inhibition could be released by rp-a in the case of cellular but not in the case of viral dna helicases. additionally, we found that p53 can partially inhibit dna polymerase ~ and s holoenzymes on singly primed mi3 dna. this inhibition, however, was only evident when e. coli ssb was substituted for rp-a. our data thus show that p53 exerts an inhibitory effect on several enzymes involved in dna replication and dna repair. the p53-rp-a interaction may function to protect replication enzymes from inhibition by p53 under certain physiological conditions. catalytic subunit showed that pol ~ is the most conserved replicative pol (cullmarm g., hindges r., berehtold m.w. and htibscher u., gene, in press). we synthesized pcr primers and cloned three fragments spanning the whole catalytic subunit. the resulting pcr products, called n-term, middle and c-term have a size of 1600, 1570 and 1280 bp, respectively. the primer for the n-term and middle fragments contained a histidin tag in order to purify the recombinant proteins by using a ni-nta column. these fragments were cloned into the expression vector pgemex174. because of the natural termination codon in the c-term fragment, the histidin tag was placed directly in the vector in front of the fusion protein, generating a new vector, prhh1s. all three fragments were expressed in e.coli. the fusion proteins could be purified in a single step and were used to raise polyclonal antibodies. finally, the entire pol 8 sequence was joined together and could be expressed. data on the characterisation of the antibodies and the proteins will be shown. hsp 70 from calf thymus can influence dna polymerase under heat shock conditions. we have generated antibodies against dnak protein, the hap70 homolog from escherichia coil, and used them for detecting of hsp70 protein during its purification from calf thymus tissue. the apparently purified protein has a molecular weight of 70kda, and has been recognized by antibodies against dnak and eucaryotic hsp72/73, it possesses a very week atpase activity, which can be stimulated by different poly-and oligopeptide substrates. results will be presented showing the influence of hsp70 on dna polymerase r from calf thymus under heat shock conditions. identification of a vertebrate cdc2 mutant which is unable to complete the g1/s transition. nicole sr and viestars simanis. chemin boveresses 155, 1066 epalinges,.isrec. s. pombe strains have been constructed which should permit the generic and molecular analysis of the events which occur in late gi when cells become committed to the mitotic cell division cycle and the initiation of dna synthesis. a number of mutants of the chicken cdc2 gene were eonsuueted during the course of analysis of phosphorylation sites some of which lead to the interesting phenotype of cold sensitivity when expressed in a cdc2 null background, when the only source of p34 cde2 is the chicken homologue of the gene, expressed from a muldcopy plasmid. in order to create a genetically tractable strain, the wild-type chicken cdc2 gone and one mutant into the s. pombe genome to create a strain in which cell cycle progression is dependent upon the chicken cdc2 gene. analysis of this strain indicates gnat the cells block predominantly before replication of dna. in order to determine whether the cells were arrested before or after the execution of the start control their ability to conjugate at the arrest point was assessed. consistent with a block before the traverse of start and contmim~ent to s-phase, cells were able to conjugate with high efficiency. further support for the view that this mutant is defective only for the g1-s transition is provided by the observation that it is able to complement a cdc2a2l mutation in trans. this mutant is defective only for (32 function and not traverse of start. tl~e double mutant is viable at the restrictive temperature of either of tile parents alld is no longer cold sensitive. further work will concentrate on cloning genes involved in the traverse of gi into s phase in fission yeast. (masson and kreis, 1993, j. cell biol. 123:357) where it is localized on a subset of microtubulea. when caco-2 cells are grown to confluency and become polarized, e-map-115 expression levels increase concomitant with a higher microtubule stability. transfection of fibroblastic cells (veto), which do not contain any detectable e-map-115, with cdna encoding this protein, results in stabilization of microtubules to nocodazole treatment. these data suggest that e-map-115 may be involved in the stabilization and reorganization of microtubules in polarizing epithelial cells. since e-map-115 is a stabilizing protein, its interaction with microtubulen should presumably be modified at the onset of mitosis to allow disassembly of the interphase microtubule network and formation of the mitotic spindle. indeed, immunolabeling for e-map-t 15 varies during mitosis. in early prophase, no e-map-115 can be detected on the microtubules of the forming mitotic spindle. the protein is observed on the spindle microtubules of metaphase cells and staining becomes bright on the new interphase microtubules of telophase cells. analysis of the protein in cells blocked in mitosis by low concentrations of nocodazole indicates that e-map-115 is hyperphosphorylated. this hyperphosphorylated form of e-map-115 is no longer able to co-sediment with microtubules in in vitro microtubule-binding assays. phosphopeptide map and phosphoamino acid analysis show the existence of novel phosphorylation sites in e-map-115 during mitosis compared to the protein during interphase, we are currently characterizing the kinases involved in the modulation of e-map-115 activity. little is known about the specific functions of calcium-binding proteins in epithelial cells ~md in particular in tumoral cells of epithelial origin. calretinin (cr) and parvalbumin (pv) are supposed to be involved in cell proliferation. we observed the endogenous expression of cr in several cell lines from human colon adenoearcinomas, and we obtained stably transfected cells for pv in a human ovarian adenocarcinoma cell line. we studied the cell cycle in correlation with the endogenous or ectopic expression of cr and pv, respeetiveiy. g1 and mitotic celts are strongly labelled with the cr-antiserum 7696, while pv immunoreactivity is dominant in interphasic, but not in mitotic cells. in cr expressing cells, the cell cycle seems to be normal and the rate of proliferation to be proportional to the percentage of positive cells. the cell cycle is inhibited at mitosis after the addition of cr antisense oligo nucleotides to the culture medium. the cell cycle is blocked in gi/s and g2 in the cells eetopically expressing pv. the results support the hypothesis that these two calcium.binding proteins, cr and pv, could intervene at different moments and probably with different mechanisms on the mechanism of the cell cycle in the tumoral cells studied. production of polyclonal antibodies against calretinin cr-22k gander, j.-ch., gotzos, v., cello, m.r. and schwaller, 8. institute of histology and gen. embryol., university; 1700-fribourg a mrna for an alternatively spliced form of calretinin, ealretinin-22k (cr-22k) was detected in widr cells (a cell line from a human adenocarcinoma). we therefore decided to produce antibodies specific for this protein. it is directed against the 14 amino-acids localized at the c-terminus of the truncated protein which are unique for this protein and not present in full-length calretinin. two different approaches have been chosen to obtain a specific antiserum. we have produced a chimeric protein containing the cterminal region of cr-22k and the (h)6(nanp)19 polypeptide as carrier. the fusion protein was expressed in e. coil and purified on a nickel chelate column. as a second method, a synthetic peptide (corresponding to the last 15 amino acids of cr-22k) was crosslinked with the carrier protein klh (keyhole limpet hemocyanin). the antigens were used to immunize two rabbits. after the first boost, the 2 sera were tested. we have observed that both antisera recognize besides the protein used for immunization also cr-22k (expressed in e. coil) in a western blot specifically. no other protein bands of either e.coli extracts or from eytosolic extracts of widr cells crossreact with the 2 antibodies. both antisera detected the protein cr-22k in immunohistochemieal stainings of widr cells confirming the presence of cr-22k in widr cells. the tctp (p23) is a growth related tumour protein which is expressed under translational control. homologous acidic proteins have been found in higher plants and in saccharomyces cerevisiae (ykl312) . the striking conservation of this polypeptide between these very divergent organisms suggests some important but still, unknown cellular function. to address this question, a null mutation was constructed in yeast, by deleting almost all the ykl312 structural gone. the deleted strain shows no detectable phenotype. therefore, we conclude that, in yeast, this gene is dispensable. the protein ykl 312has been overproduced in e. coil and injected to rabbits to raise antibodies. we are now characterizing the ykl312 expression at rna and protein level. ellenrieder,c., forrer,p., kosovsky,j., rischle, m., nueller, r. and jaussi, r., institut f~r medizinische radiobiologie, paul scherrer institut & universitgt zh, ch-5232 villigen radiation therapy of tumors could be improved by influencing cellular radiation sensitivity. yeast strains with defective cell cycle checkpoint genes (e.g. radl, rad3, radg) show increased radiation sensitivity. cross-hybridization of a probe from the tad9 gene on human mrna northern blots yielded a single prominent signal. experiments to clone the corresponding 4 kb cdna are underway. we have cloned a hamster cdna encoding the homologue of human cdk2. probably, this cdna encodes the p40 protein from hamster whose phosphorylation responds to radiation checkpoint signals and who does not bind to p13 sucl beads. treatment of cells with cdk2 antisense oligonucleotides is hoped to modulate the radiation sensitivity. proliferation of smooth muscle cells (smcs) is a major event in vascular development and atheromatous plaque formation. in order to characterize smc replicative potential, newborn rats have been injected with 3h-thymidine (3h-tdr) during their first week of life when most of smcs were proliferating; then, 3h-tdr labelling was evaluated in adult rats. depending on the number of mitosis that smcs had accomplished after the first week of life, four different smc subpopulations could be defined indicating that rat aortic smcs are heterogeneous in their replicative activity. 5-bromo-2'-deoxyuridine (brdu) incorporation after balloon induced endothelial denudation of rat aorta in adult rats showed that smcs entering into the cell cycle were mainly devoid of 3h-tdr labelling, this category could derive from two distinct smc subpopulations: smcs which were arrested in their proliferation before or just after birth or smcs which had actively replicated during development. thus, one (or two) subpopulation(s) of rat aortic smcs, characterized by a particular replicative activity during development, is (are) selectively activated after balloon induced endothelial denudation. the situation in vivo of dermal fibroblasts embedded within the connective tissue can be emulated in vitro by growing the cells in collagen gels. we have investigated how fibroblasts cultivated within a matrix react upon wounding of this dermal equivalent. human skin-derived fibroblasts (kd-cells) growing within attached collagen matrices were monitored over a period of 8 days. fluorescently labeled cytoskeletal elements (tubulin, vimentin, acfin) and fibronectin served as phenotypica/ markers. the 3d-arrangement of fibroblast microtubules, intermediate filaments and microfilaments, as well as of fibronectin was visualized by clsm and optical sectioning. cells at the wound margin, adjacent to the wound, and in non-wounded controls, up to a depth of about 100gm were analyzed. upon wounding, i.e. upon excision of a 1 mm 0 "punch biopsy" from the center of the collagen matrix, fibroblasts in the wound region oriented towards the matrix defect. layer-like structures of increased cell density evolved over time at the wound margin. the tissue defect was covered by fibroblasts, reminescent of the situation in vivo. global yeast genome expression analyzed by 2-d page after tctp homolog gene (ykl312) disruption. sanchez a translationally controlled tumor protein(tctp) was identified and microsequenced from a human liver sample. previous publications described the presence of a gene related to tctp in plants, mouse erythroleukemia and ascetic tumor, human mammary carcinoma and more recently in the yeast. tctp has no known function but its high degree of homology from plants to human underlines its probable crucial role in cell function. in order to study multifactorial chan~es in protein expression as a function of the ykl312 gene disruption m the yeast, we used high resolution two-dimensional gel electrophoresis combined with an efficient computer system (melanie). two groups of gels (ykl312 +(n=10) and ykl312 -(n=l 0)) were analyzed, compared and classified using correspondence analysis. there was no significant difference between the two populations of gels according to either qualitative and quantitative spots expression except for two spots which completely disappeared. the first one corresponded to ykl312 gsne product. the second one corresponded to a 28 kda peptide with an isoelectric point of 4.6. this ykl312 associated peptide still needs to be characterized and linked to the yeast genome. demyelinating and neurodegenerative diseases are associated with altered cellular responses including processes mediated by receptor protein tyrosine kinases. using a pcr cdna cloning approach we have identified a novel member of the eck/elk/eph genefamily in myelinating serum-free brain cell cultures. alternatively spliced cdnas encoding complete open reading frames for putative transmembrane proteins have been isolated from both rat and human brain. a recombinant protein derived from the rat cdna was demonstrated to have protein tyrosine kinase activity. an affinity purified antiserum to this protein was used to identify a glycoprotein of 120 kd molecular weight in brain cultures, and developping and adult brain tissue. the protein expression is most prominent during embryonal development and during the first three weeks of postnatal life. by in-situ mrna hybridisation the transcripts were found predominantly in restricted neuronal populations. in order to investigate systematically the effects of geometrically defhied abrupt tissue expansion on the characteristics of impulse propagation, we constructed expanding cellular structures in vitro using patterned monolayer cultures of neonatal rat heart cells (photolithographically patterned substrates.) the preparations, which consisted of narrow cell strands (40-80 tim wide) inserting into large reetangular cell areas (side lengths >1500 ~m), were stained with the voltage-sensitive dye di-g-anepps and were imaged onto a 12 x 12 photodiode matrix array (maximal spatial resolution 151.tin x 151am in the object plane). while impulse propagation was uniform in linear cell strands (average conduction velocity 0.35 m/s), a transient decrease in conduction velocity hi the transitional region was invariably observed in the suddenly expanded structures. this decrease was accompanied by marked distortions of the local action potential shapes due to bidirectional electrotonic interactions across the transition: upstream from the expansion, the repolarization phase was interrupted, a few ms after its inception, by a small secondary depolarization ("spike and dome" configuration), while, downstream, the action potential upstrokes were preceded by prominent prepotentials. in those cases where conduction failed at the tlansition, action potential durations upstream were dramatically abbreviated. these findings showed that phenomena similar to those recorded in intact purkinje-fiber-ventricular preparations can be observed in cell ensembles in vitro having comparable geometlins but consisting of cells with uniform active membrane properties. supported by swiss nsf grant 823a-028424 (sr) and usphs grant ns 16824 03ms). it is a noncollagenous glycoprotein with a molecular mass of 148 kda consisting of three identical subunits. cmp is selectively extracted with edta-containing buffer what indicates a divalent cation dependent anchorage of the protein in cartilage matrix. electron microscopy revealed the presence of three compact globular subdomains and sequence analysis indicated the presence of a coiled-coil c~-helical assembly domain formed at the c-terminal end of each subunit. the trimeric structure was retained after complete reduction under native conditions which reveals that the subunit structure of cmp is not only stabilized by disulfide bridges but also by the coiled-coil assembly domain. tissue distribution studies in mouse revealed that cmp is selectively expressed in some but not all cartilages. adult rat cardiomyoeytes (arc) in culture degenerate the myofibrillar apparatus and after attachment to the substrate they grow and reassemble new rnyofibrils. the appearance of new, well organized rnyofibrils can be observerd in several distinct regions. they appear close to the membrane proximal to the culture substrate and in the perinuclear region close to the substrate. previous studies have shown that embryonic cultured cells assemble new myofibrils which insert in adherens junctions at sites of cell-cell contacts and in sub-sarcolemmal adhesions plaques (saps) at sites of cell-substrate contacts. this saps are thought to function as the nucleation sites for myofibril formation. we have investigated the formation of this saps in cultured adult rat cardiomyocytes. using confocal light microscopy we can show that the interaction between rnyofibrils and membrans occurs close to the membrane proxirnal to the substrate and that this saps are flanking both nascent myofibrils and sfls, the latter structures being believed to serve as scaffold for myofibrillogenesis, additionally we have used electron microscopy to investigate the formation of this structures at higher resolution. $11-05 muscle satellite cells (sc) are quiescent myoblasts, ready to be activated and to regenerate new muscle fibers in case of muscle damage. in vitro, single human muscle sc, cultivated as clones, give rise in proliferating conditions to two subpopttiations of cells, cells expressing both ~-striated muscle actin and desmin (c~-sr+dsm+), and cells expressing desmin alone (dsm+). in culture conditions promoting differentiation, a clone .typically gives rise to a fusing progeny, yielding c~-sr+dsm + myotubes, and to non fusing myoblasts (nfmb). the latter are dsm +, a phenotype similar to quiescent sc found in situ. in order to determine whether nfmb are the in vitro equivalent of in situ quiescent sc, this first generation of nfmb were selectively collected and subcultured. in proliferating conditions, nfmb were able to resume proliferation and to give rise to a progeny with both c~-sr+dsm + and dsm + cells. when cultured in differentiating conditions, nfmb formed ct-sr+dsm + myntubes, and a new generation of dsm + nfmb. when nfmb of the second generation were again selectively collected and subculture& they resumed proliferation and ~re again able to yield both myetubes and a third generation of nfmb. nfmb of the first generation were also cultivated as clones, and 5 to 25% of them were able to resume extensive proliferation, yielding in their progeny both c~-sr+dsm + and dsm + cells, which differentiated into myotubes as well as nfmb. these results strongly suggest that sc have the stem cell property, of selfrenewal, yielding in their progeny both cells ready to differentiate into muscle and ceils similar to themselves. extra cellular matrix (ec~ regulates the expression of b-casein in cultured mouse mammary epithelial cells. we have developed a functional ~ epithelial cell strain, which expresses high level of milk proteins, forms alveolar-like structures when plated onto a reconstituted basement membrane and secretes casein unidirectior~lly into a it~aen. we have further shown that fflv~dependent regulation of b-casein occurs mainly at the transcriptional level and that 5' sequences play an inloortant role in this regulation. we have located a 160bp transcriptional enhancer (bcei) within the 5' flanking region of the b-casein gene. using functional assays, we show that bcei contains responsive elements for ~ and prolactin-dependent regulation. bcei placed upstream of a truncated inactive b-casein promoter reconstitutes a promoter even more potent than the intact prcmoter, which contains bcei in its natural context more than 1.5kb upstream. this small fusion promoter reconstitutes the nonaal regulation pattern. we show that bcei mediates f/24 dependent regulation even when linked to a het~rologous viral promoter. purified satellite cells (sc) were obtained from human skeletal muscle biopsies following cell sorting by flow eytometry. a chemiluminescent assay for acetylnholine (ach) revealed the presence of 1,3 nmoles/weu of 100.000 sc. in elonal cultures of proliferating sc, this intracellular level of ach was 0.l nmoles/well. when cells were cultured in the presence of an esterase inhibitor (10 ixm phospholine), the ach amount was enhanced 2 fold. conversely, cultivating the cells in presence of a potent inhibitor of choline acetyl transferase (2 gm bromoach), gave a 50% decrease of ach content finally, the release of ach was detected in the supernatant of cultured sc, following a 15 min incubation, and the measured level of ach was 3 pmoles/well/min. the presence of an ach-like compound in myogenic cells in both freshly isolated and in proliferating sc suggests that sc may contain ach in situ. in additiou, the fact that ach was present in the extracellular medium suggests that ach can be released spontaneously by the cultured sc. type xii collagen is an extracellular matrix protein associated with collagen fibrils in vivo. as observed for several other extracellular matrix proteins, the synthesis of type xii collagen by chick fibroblasts cultured in 0.1% fcs is stimulated by tgf-i~i. two splice variants of type xii collagen are known, with subunits of either 220 kda or 350 kda. by using antibodies specific for the large (350 kda) form of type xii collagen, we could show a more restricted distribution of the large variant compared to the smaller form in the embryo. while only the large variant carries chondroitin sulfate, both variants are identical in their collagenous domain. it is thought that via this domain, type xii collagen binds to collagen type i fibrils. we demonstrate here that type xii collagen can bind to collagen i fibrils also in vitro. by neutralizing acid soluble collagen type i, we could coprecipitate type xii collagen together with newly formed collagen type i fibrils. this interaction occurs in physiological saline but is highly salt dependent. in further studies we investigated wether 35s-methionine labeled type xii collagen treated with chondroitinase abc, ct-chymotrypsin, or collagenase can still be precipitated together with type i fibrils. in addition, we found that type xii collagen also affects the rate of type i collagen fibril formation. h.u. keller department of pathology, university of bern, ch-3010 bern locomoting blebbing cells colchicine (10-sm) can induce locomotion associated with blebbing in walker carcinosarcoma cells. blebs expand at a rate (about 2~m/sec) which is much faster than known rates of actin elongation. this suggest that the membrane is directly pushed forward by forces other than actin elongation, possibly by hydrostatic pressure. this interpretation is suggested by the observation that there is significant f-actin staining all along the cell membrane but not with the cytoplasmatic content the blebs. blebbing is suppressed by 0.5 m sorbitol. the finding that cytochalasin d suppresses blebbing shows that actin polymerization is nevertheless indirectly instrumental in bleb formation, possibly by generating a high intracellular pressure. a tentative model explaining locomotion of blebbing cells is presented. osteopetrosis encompasses a family of diseases with different causes and the common phenotype of impaired bone resorption. the osteopetrotic mouse mutant oo/oo is deficient in csf-1, the growth factor for the cells of the mononuclear phagocytic system. the phenotype of oo/ou mice is characterized by a low number of peritoneal macrophages and peripheral monocytes, and a virtual absence of osleoclasts, leading to the osteopetrotic phenotype. injections of csf-1 into op/oo mice reversed the osteopetrotic phenotype, proving the requirement for csf-1 during the process of osteoclastogenesis. by in situ hybridization, expression of csf-1 was demonstrated during bone development. osteoclast precursors and mature osteoclasts were identified as putative target cells for the growth factor, since these cells express csf-1 receptors, which is encoded by the proto-oncogene c-fm~. subsequently, specific binding of csf-1 to osteoclasts was shown. expression of c-fms parallels the expression of csf-1 in bone both in time and place, suggesting a local action of this growth factor in osteoclast formation, but also in the modulation of osteoclastic activity. different transcripts, raised by alternative splicing of a commmon nuclear rna precursor, have been found to encode a secreted and a membrane associated forms of csf-i. the secreted form can be modified by attachment of a glycosaminoglycan side chain, which serves as an anchor to integrate the peptide into the extracellular matrix. investigation of the role the different csf-1 forms play during recruitment of osteoclasts and regulation of their activity will provide further insights into the mechanisms governing these processes. comparative sequence alignments of known voltage-gated k + channels revealed two conserved cysteine residues in the putative transmembrane segments $2 and $6. if the cysteines were connected by a disttifide bridge, it would put a structural consllaint on possible channel models by placing $2 next to $6. we used site-directed mutagenesis to study systematically the potential roles of these invariant cysteines. fourteen of 17 substitutions in $2 (c232), and 7 of 19 substitutions in $6 (c393) maintained channel function in xenopus oocytes microinjected with mutant crna. therefore, the conserved cysteines are not essential for k + channel expression in xenopus oocytes. however, electrophysiological characterization of the cysteine replacement mutants revealed distinct roles for the two cysteines. inactivation, deactivation, and ion permeation did not considerably change in $2 mutants. in $6, in contrast, cysteine replacement by leucine, asparagine, glycine and valine accelerated inactivation and deactivation kinetics substantially, whereas serine and threonine showed opposite effects. furthermore, the voltage dependence of deactivation was differently affected. in summary, it appears that the side-chain at position 393 in $6 of kv2.1 is involved in channel gating by participating in the transitions from the open to the closed and inactivated state of the channel. (supported by grants from the nih and mda to rhj, and from the snf and the swiss foundation for medical-biological grants to rdz.) the segment between $5 and $6 of voltage-gatedk + channels, called h5 or p region, has been implicated to form part of the ion conduction pathway. little is known about the conforroation of this region although various models have been proposed including a j3 barrel-like structure formed by four adjacent antiparanel j3 sheets. to gain insight into the secondary structure of this region, we used cysteine substitution mutagenesis and sulfhydryl-specific, membrane-impermeant reagents to probe the accessibility of amino acid side chains in and around the p region of kv2.1 (drki). twemy-eighi positions from k356 to t383 were each mutated to a cysteine. after expression of mutant k + channels in xenopus oocytes, cysteine side chain accessibilities were probed by superfusion with ch3so2sch2ch2nme3 + (mtset), mtset can form a mixed disulfide with an accessible cysteine, and this may lead to current reduction if the covalently modified side chain is in or close to the ion conduction pathway. thus far, we have identified four mutations that showed k + current reduction after superfusion with 3 mm mtset. the mutants p361c, i379c, y380c, and k382c showed 87%, 99%, 39%, and 21% reduction in current amplitude, respectively. in coutrast, mutants $363c, a367c, t368c behaved like wild type kv2.1 with less than 5% current reduction. our results suggest that the side chains of p361,1379, y380, and k382 are directly accessible from the extracellular environment. taken together, these residues may, therefore, face the lumen of the ion channel pore. furthermore, the degrees of inhibition are in agreement with a model in which the ion conduction pathway narrows from residue k382 to y380 to i379. (supported by grants from nih and mda to rhj, and from the snf and the swiss foundation for medical-biological grants to rdz.) motejlek k., h,,iuselmann r., and liig:her b.; pharmakologisches institut der universitiit ziirich, winterthmtr. 190, ch-8057 zlirich comparison of 5' flanking sequences of three gabaa-receptor subunit genes (atl, ~, 8) revealed a novel conserved purine sequence dement with the consensus sequence gagaggggagaoga gagag(gg/aa)g. this element is present once each in the (zl and 72 subunit gene promoters and in seven tandem copies in the 8 gene promoter. a novel dna binding activity (bsf1) was identified that binds to various versions of this purine element. bsf1 was found ordy in brain but not in any other tissues tested. furthermore, the factor is distinct of beta, another brain-specific dna binding .protein with a purine-rich dna recognition sequence. the expression of bsf1 during differentiation of eerebeuar granule cells in vitro correlates with the expression of gabaa-receptor subunit genes. this correlation suggests a role for bsf1 as a transcriptional activator of neuronal genes. therefore, bsf1 may be important for cell ty~specific regulation of gabaa-receptor gone expression. we also found that bsf1 binds to wheat germ agglutinin, suggesting that this protein is glycosilated. taking advantage of this property, we are in the course of purifying bsf1, the question, whether bsf1 is indeed a transcription factor is being investigated in vivo and in vitro. to produce a simple method of estimating the free magnesium concentration ([mgzq) in solution, we have manufactured dip cast mg 2+ macroelectrodes using the neutral mg carrier eth 7025.we have used the elecmides to measure the dissociation constant (k~) for mgatp in a background solutions mimicking the intracellniar milieu. for the mcasmement of the big atp dissociation constant, the background solution contained 2 mmol/l n%atp. this solution was titrated with mgci 2 and the changes in [mg ~] above 0.05 retool/1 monitored with the maeroelectrode. during the titration the ph was maintained constant. fitting such titration curves with the standard hyperbolic binding equation, after correction for zero drift, gave the following mean+sd values for the ka (pmol/1 at 37~ ph 6.4, 103.3-+-25,3 (n=9); ph 7, 59.7:k-27.4 (n=6) and ph 7.4, 30.g~15.4 (n=7). elevation of the [naq to 50 m~l/1 and reducing the [kq to 1(30 mmol/l was without effect at ph 6.4 (n=6). reducing the temperature to 25~ increased the k a at 7.4 to 49.0:k21.6 (n= 5). mg =+ macroelcctrodes provide an easy way of measuring [mg z ⧠in solution. [mg ~] can also be measured in a background solution containing i mn~i/l ca, although the electrode response is reduced. interference by protein to date prevents their use in plasma. similarily to the block by zn 2+ and ca 2+, protons only partially block the channel. the ability for all mutated channels to. conduct na + current, and the partial block induced hy zn 2+, ca 2+ or h + indicate that y401 is not located deep in the pore but rather in the extracellular mouth of the channel. progesterone modulates the activity of glycine receptor expressed in colliculli neurons of neonatal rats. maury, k. & bertrand, d. dpt of physiology, cmu, 1211 geneva 11. steroids are potent narcotics and have been shown to act on ligand-gated channels activated by gaba or nicotine. however, little is known about their possible actions on other receptor types. for instance, while it was demonstrated that progesterone inhibits glycine receptors expressed by spinal cord neurons isolated from chick embryos, nothing has yet been reported for brain receptors. in this study, we have determined the properties of glycine channels expressed by brain neurons of neonatal rats using the whole-cell voltage-clamp technique. in inferior colliculi neurons, glycine elicited little-desensitizing inward currents which reversed around -40 mv. these currents were blocked by strychnine in the nanomolar range. surprisingly, however, we found that progesterone, in the micromolar range, potentiated the glycine evoked currents. these results, contrasting those obtained in chick spinal cord neurons, suggest that progesterone enhances or antagonizes glycine currents depending on the subtype of the receptor. the mammalian auditory organ (organ of corti) relies on two groups of sensory cell for its normal hearing: inner hair cell & outer hair cell. ihcs are mainly innervated by afferent fibers while the ohcs essentially receive efferent fibers. the ohcs possess unique electromofile properties which are thought to enhance the motion the basilar membrane and to refine the mapping of the sound frequency. we found, using whole ceil recordings, that 5-10% of the fleshly dissociated ohcs displays fast inward currents. the magnitude of peak currents which can be as large as 2na vary from cell to cell. further experiments have revealed that this current is sensitive to ttx and strongly reduced when extracellular sodium is replaced by choline. its kinetic is relatively slow compared to that of sodium current of typelspiral ganglion cell, and has a more negative inactivation. other voltage activated currents and ligand-gated currents are under inverstigation. these results will provide further insights in the hearing transduction mechanism. the formation and the properties of homotypic and heterotypic gap junctions were studied using two types of insect cells, c6/36 and sf9. two single cells were pushed against each other and the subsequent de novo formation of gap junction channels was assessed by means of the dual voltage-clamp method (bukauskas and weingart: pfl~g. arch. 423:152, 1993 it was symmetrical for homotypic junctions. the vj-sensitivity was less pronounced in sf9 cells. hence, the relationship for heterotypic junctions was asymmetrical. all pairs examined revealed a s-shaped relationship between gj(ss) and v, which was virtually superimposable (gj(ss) declined upon depolarization). each channel contains a vm-gate and two ~-gates. the vj-gates are operated independently. they close when their intracellular aspect is made positive. shaped curve compatible with a two-state boltzmann process. half maximal inactivation was reached at -77 mv and +66 mv, implying an asymmetrical gating behavior. in case of an asymmetrical protocol (~ and vewas stepped simultaneous, but in opposite direction), the q-dependent asymmetry disappeared (half maximal inactivation at -59 mv and +60 mv). the asymmetry in gj-gating, attributable to the pulse protocol, was also reflected in the time constants of ij inactivation (r177 cerebellar granule cell cultures were used to study the regulation of the nmda receptor expression. we have shown previously that chronic membrane depolarisation (25 him k +) or treatment with nmda {i00 ~m) promotes the functional expression of nmda receptors, as assessed by nmda-evoked 45ca2+ influx. we have now developed a rnase protection procedure for the quantitative determination of the mrna levels of the nmda receptor subunits known so far (nr-i,2a,2b,2c,2d). the growth conditions mentioned above failed to alter the mrna levels of the different subunits. at the protein level, nmda receptors were evaluated quantitatively by the labeling pattern obtained by the new photoaffinity ligand 125i-cgp55802a. the intensity of the phctolabeled bands, thought to represent nr-i and nr-2a subunits, was increased by treatment with high k + or nmda compared with control cultures. this result suggest an increase in receptor protein by high k + or nmda treatment. thus, posttranscriptional mechanisms seem to play an essential role in the modulation of the nmda receptor activity. the gene encoding the pore-forming subunit of the rat epithelial na + channel (~renac) was cloned recently and shown to belong to a novel gene family coding for cation channels (nature 361, p467-470 (1993) . transport of na + ions through these channels is the rate-limiting step of na + absorption and thereby controls osmotic balance of body fluids and secretions. in order to study the implication of ~renac in regulating sodium balance, blood volume and blood pressure and therefore its involvement in hypertension, we expressed ~renac under the control of the human cmv promoter in transgenic mice. several lines of mice showing expression in different tissues were obtained. progress in the analysis of these mice will be discussed. activation of metabotropic glutamate receptors increases the excitability of neurones in the lateral septum of the rat. to elucidate the mechanism(s) of this action, we used whole-cell patch-clamp recordings from coronal brain slices of young animals. in voltageclamped cells, the selective agonlst (is,3r)-acpd, at 1-50 /~m, had the following effects, a) it evoked a sustained inward current, which was resistant to ttx and to cadmi~am and which persisted in neurones loaded with bapta, a calcium chelator. this current displayed inward rectification and was reduced, or suppressed, when extracellular sodium was partially replaced with n-methyl-d-glucamine. b) it elicited a ttx-insensitive, voltage-dependent inward current, which activated at -50/-40 mv and which reversed at aound 0 mv. this current was suppressed in a low-calcium/high-magnesium perfusion solution and was undetectable in the presence of intracelhilar bapta. we suggest that acpd exerts a dual action on lateral septal neurones. it causes a steady depolarization by generating a sodium-dependent current and it triggers transient plateau potentials by inducing a calcium-dependent cationic current. interaction between these currents results in a powerful, self-reinforcing neuronal excitation. we have investigated the effects of protein kinase c (pkc) activators (4[~-pma, oag) and of phosphatase inhibitors (okadalc acid, calyculin a) on voltage-gated ca 2+ and k + channels in ngf-differentiated pc12 ceils. whole-cell ba 2+ and k + currents were recorded with the patch-clamp technique. by using specific ca 2+ channel blockers (co-conotoxin (cgtx), isradipine) we found 3 types of ba 2+ currents (iba): a) a ~cgtx-sensitive iba; b) an isradipine-sensitive iba; and c) a t0-cgtx plus isradipineresistant iba. 4[~-pma and oag specifically down-modulated the isradipine-sensitive iba. the inhibition of iba was prevented by staurosporine and pkc (19-31) (2 pk inhibitors). the delayed rectifier k + current was unaffected by pkc activators. applied externally, okadaic acid and calyculin a inhibited the total iba by affecting several components of the ba 2+ currents. however, the 0~-cgtx plus isradipine-resistant iba was unaffected by okadaic acid. in conclusion, our results suggest a differential modulation of voltage-gated ca 2+ and k + channels by the pkc signalling pathway in ngf-differentiated pc 12 cells. agrin, a protein isolated from basal lamina extracts of the electric organ of the marine ray, is thought to mediate the motor neuron-induced aggregation of acetylcholine receptors (achrs) in muscle fibers at their neuromuscular junction. recent cloning in the marine ray, rat and chick has revealed that agrin can be alternatively spliced at two sites in its c-terminal half. site a encodes either 0 or 4 amino adds and site b encodes either 0, 8,11 or 19 (8+11) amino acids. studies of agrin mrna expression indicate that early in synaptogenesis, chick motor neurons contain high levels of a4b19. in contrast, non-neuronal cells and muscle calls contain a4b0 and aob0 mrna. in the adult ray, electric lobe motor neurons that innervate the electric organ contain a4b8, whereas agrin mrna in the electric organ again lacks both sites (mcmahan et al. (1992) , curr. up, cell biol. 4:869-874). to investigate the functional properties of agrin isoforms in more detail, we have now compared their specific activity to aggregate achrs on cultured chick myotubes. heterologous expression of the c-terminal half in cos-7 and 293 cells revealed that chick agrin isoforms a4b8 and a4b19 were highly active, while a4bll was up to 40 fold lower in activity. no activity could be detected for the a4b0 and aob0 isoforms. in agreement with these findings the a4b8 isoform of the marine ray also showed high achr-clustering activity. therefore, we conclude that motor neurons throughout development synthesize highly active agrin isoforms while the postsynaptic target ceils do not play a primary inductive role in the formation of synapses. we have used whole-cell patch-clamp recordings and hypothalamic slices in order to characterize the effect of n-methyl-d-aspartate (nmda) on suprachiasmatic neurones. in ceils clamped at or near their resting membrane potential, nmda (50-100 #m) generated an inward current of 10-60 pa, which was insensitive to ttx and which reversed at about 0 inv. the nmda current-voltage (i-v) relation contained a region of negative slope conductance. the nmda current was reduced or suppressed by d(-)-2-amino-5-phosphonopentanoic acid (d-aps) or by mk-801. it was potentiated by reducing the extracellular magnesium concentration from 1 to 0.01 ram, or by adding glycine (10 /~m) to the perfusion solution. in a majority of neurones, lowering the extracellular calcium concentration from 2 to 0.01 mm caused a 1.5to 4-fold enhancement of the nmda current. i-v relations indicate that in the low-calcium solution, the region of negative slope conductance was attenuated. this effect was due to extraceuular calcium, since it persisted in neurones loaded with the calcium chelator bapta. we conclude that nmda channels present in suprachiasmatic neurones may be modulated by extraeellular calcium. institut, and abt. pharmakologie*, biozentrum, basel. during innervation of skeletal muscle, the myonuclei underlying the synapse begin to express acetylcholine receptor (achr} ~-subunit gene and they remain activated after the nerve is removed. our experiments show that this is due to a factor in the synaptic portion of the muscle fibre basal lamina (bl). one candidate for this factor is agrin, which regulates ache clustering in the synaptic membrane: i) unlike in untreated muscle, the level of s at synapses was strongly reduced in cultured rat muscle fibres after proteolysis of synaptic bl. 2) conversely, when rat myotubes were cultured on bl isolated from adult muscle, they preferentially expressed achr accumulations and ~-mrna at sites where they contacted the synaptic portions of the isolated bl. 3) when various substrates were impregnated locally with agrin a4big, an isoform expressed by motor neurones in fetal spinal cord, it locally induced in the myotubes the expression of s-mrna. art increase of functional nicotinic acetylcholine receptors precedes the fusion of cultured human muscle satellite cells. r. m. kranse, c.-r. bader and l. berrtheim. department of physiology and division of clinical neurophysiohigy, university medical center, 1211 geneva 4, switzerland nicotinic acetylcholine receptors (nactlr) are expressed on embryonic myoblast and acb may play a role in mechanism of fusion. our study focuses on the appearance of functional nachr in freshly isolated and cultured satellite cells (sc) from normal human muscle biopsies. sc cart be conditioned to either proliferate or fuse to form myotubes depending upon culture media. presence of ach-activated current was investigated using the whole-cell and single.channel patch-clamp technique. in freshly isolated sc (whose properties should be close to the quiescent in vivo sc/no nachr were observed (n=16). in the proliferating state, 54% (n=35) of the satellite ceils (which are also called myoblasts) displayed a small ach-activated current (10 pa/pf) and, as expected, after fusion of salellite cells into myotabes, 100% of the ceils displayed an ach-activated current (n=6; 26 pa/pf). to examine, whether the increased expression of nachr precedes sc fusion, sc were cultured in a medium which promotes fusion, but were prevented from fusing by keeping them at a low density. in this culture condition, 93% of the sc (n=15) displayed an ach-activated current and, in addition, these cells expressed a 4 times higher ach-current density. (40 pa/pf) than the proliferating sc. we also observed that, in high density cultures, a small population of sc do not fuse even atter 3 months in the medium promoting fusion. these non-fusing myoblasts (nfmb) had no nachr. our results suggest that the appearance of nachr may be related to the process of sc fusion as i) quiescent sc in vivo do not express nachll ii) nachr expression increases in conditions promoting fusion and iii) no nachr were observed in nfmb. the latter cells may be equivalent of quiescent sc in vivo. $12-20 we have investigated the sensitivity of neuronal nicotinic acetylcholine receptors (nachrs) of known subunit composition to various cholinergic antagonists. neuronal nachrs were expressed in xenopus oocytes after injection of pairwise combinations of (x2 or c~3 with either 1~2 or 1~4 crnas. the two-electrodes voltage-clamp technique was used to measure currents induced by rapid application of low concentrations of acetylcholine (ach) together with increasing concentrations of antagonists. the response of ct31~4 neuronal nachrs to ach was halfmaximally inhibited by following antagonist concentrations (ic50): hexamethonium (0.3 i.tm), mecamylamine (0.2 p.m), pentolinium (0.2 i.tm) and trimetaphan (1.2 ~tm). with (x3132 nachrs the ic50s of the same antagonists were about 10 times higher. finally, (+)-tubocurarine was a conventional competitive inhibitor of ach in ~3132 and t~2~2 nachrs. in contrast, low concentrations of (+)-tubocurarine increased the response to low ach concentrations in a3(34 and t~2~4 nachrs. these results further underline the importance of [l subunits in determining the functional properties of neuronal nachrs. supported by the hochschnlstiftung and nf grant 31.31018.91 to abc. previously, the role of the transglial tubular system was shown for the squid giant axon: in na + free (tris) solutions the series resistance increased in correlation with a decrease of the tubular opening density (tod). in the crayfish giant axon, which show similar tubules, we correlate the change in tod, measured in freeze-fracture preparations, witl] the conduction velocity. the control to'd was estimated to be 16 per #ms. after 30 and 75 rain tris-inkubation the tod decreased to 10,4 and 1,5, respectively. this reaction was reversible when these axons were reincubated in na~-c41ntaining solution. after 120 rain reincubatiou the tod was 11.8 per tam'z. to determine the influence of decreased tod on the impulse conduction velocity, the nerve was incubated for 30 min in tris solution followed by reincubation. impulse propagation then recovered in two phases. during an initial fast phase with a short time constant of 1-2 rain na + diffused back into the periaxonai space and allowed impulses to occur with a low conduction velocity. in the second phase with a long time constant of 7 min, conduction velocity recovered slowly to normal values. this time course was comparable to the recovery of tod, as estimated from recent freeze-fracture preparations after short reincubation. these results indicate that the normal tod is a necessary condition for the normal excitability of the axon and determines its conduction velocity. most spinal cord neurons respond to the inhibitory action of gaba and glycine, suggesting co-expression of gaba a-and glycine receptors 4n individual ceils. while glycine-receptors are exclusively found in post-synaptic densities, the cellular localization of gaba areceptors has not yet been characterized. in this study, the distribution of gabaa-receptors in the spinal cord was analyzed with an antiserum recognizing the (xl-subunit. double-and tripleimmunofluorescence staining were employed to identify synaptic receptors apposed to gabaergic terminals (immunoreactive for glutamic acid decarboxylase) and to assess their co-localization with glycine-receptors (visualized with an antibody to the 93 kda protein gephyrin). staining for the gabaa-receptor ctl-subunit decorated the soma and dendrites of numerous neurons in laminae iii-viii and x, revealing their morphology in clear detail. by contrast, laminae 1i and ix contained little immunoreactivity for these gabaa-receptors. most gabaa-receptor-positive cells also exhibited a prominent glycine-receptor immunoreactivity. both types of receptors had very similar distribution patterns and were frequently co-localized in sites apposed to gabaergic boutons. these results indicate that gaba aand glycine-receptors may co-exist within single post-synaptic densities, suggesting a possible synergism between gaba and glycine neurotransmission in spinal cord neurons. prenatal benzodiazepine (bdz) exposure changes both behavioral and neuiochemical parameters of developing iats. these changes are not a consequence of remaining bdz in brain tissue, but rather indicate alterations in bdz receptol binding and function. since the bdz binding site is located on the gaba~ receptor complex, it is possible that prenatal bdz treatment influences the expression of gaba~ receptor subunits. to evaluate effects of pienatal bdz exposure on mrnas expression specific for several gaba~. receptor subunits (~i, ~5, ~ & y2), we treated plegnant rats with diazepam (l.25mg/kg/d; s.c.) from gestational day 14 to 20. we then analyzed specific mrna levels in offspring at four diffeient developmental stages (gd20, pns, pni5 & adult) by in situ hybridization. pleliminary data flom optical density measurements indicate a deciease in expression of mrna in particular for ~-subunit of gab~ receptors in different brain reglons of plenatally diazepam-treated zats. processing of sensory information within the auditory system has been well characterized using histological and eleetrophysiologlcal techniques. however, relatively little is known about the neurotransmitters involved. the present study was performed to elucidate the possible role of excitatory (eaa] and inhibitory (i.e., gaba) amino acids in neurotransmission within the primary auditory cortex (all the main afferent to the ai, the ipsilatera} medial geniculate body (mgb), was electrically stimulated and evoked responses were recorded intracellularly in the ai region in halothane anesthetized cats. a seven-barrelled iontophoresis pipette glued alongside the recording electrode allowed localized application of eaaergic and gabaergic compounds onto the recorded neuron. in most ai neurons, mgb stimulation evoked a short latency, short duration epsp followed by a long-lasting ipsp. pattern, duration, and amplitude of synaptic potentia{s were highly variable and strongly dependent on stimulation intensity, reduction of gabaa receptor-mediated inhibition by iontophoretic application of either bicuculline or sr95531 markedly enhanced mgb stimulation-evoked epsps. only the late component of this enhanced epsp was reversibly blocked by the competitive nmda receptor antagonistl ap7 at currents which selectively blocked neuronal excitation induced by iontophoretic application of nmda, our results demonstrate that in the eat 1) nmda receptors in ai are activated following mgb stimulation and that 2) a dominant gaba~ receptor mediated inhibition usually masks this nmda receptor activation under our experimental conditions, a highly purified and specific cell wall degrading endo-l,5-u-l-arabinanase was isolated from an a. niger pectinase preparation by low and medium (fplc) pressure column chromatographic separation methods. the isolated enzyme was most active on linear 1,5-~-l-arabinans (-90 i.u./mg), whereas branched arabinans from sugar beets were degraded to a lesser extent (-14 i.u./mg). the enzyme was shown to be electrophoretically pure after silver staining (sds-page) and its identity was confirmed through specific binding to an antiserum directed against endo-l,5-~-l-arabinanase. the major physico-chemical characteristics of the enzyme were the following: mr 42'000, iep ~ 3.0, ph optimum 4.8, temperature optimum 55~ ph stability 3.5-8.0, temperature stability s 45oc, km = 0.205 mg/ml, vmax = 17.7.10 -3 ~unol/min. zn and hg showed potent inhibitory effects. 3.2) is a specific enzyme of the glyoxylate cycle, which plays a key role in the initiation of gluconeogenesis in germination oilseeds, this pathway is localized in glyoxysomes, single membrane organelles which are converted into peroxisomes when lipid reserves are depleted. the glyoxylate cycle enzymes have been the subjects of numerous conflicting reports typically addressing the problems of their synthesis (on free or bound ribosomes), targetting and suborganelar localization (membrane bound or matrix proteins). recent biochemical studies have shown that ms from germinating soybean cotyledons is an interconvertible enzyme (membrane bound, aggregated or soluble fomls) which is significantly affected by its ionic environment (henry et al., 1992, plant science 82:21-27) . microscopy studies have now been carried out using immunofluorescence staining or immunogold-silver staining for light microscopy, and immunogold labelling for electron microscopy. all results consistently characterize ms as a glyoxysomal mawix enzyme. chorismate mutase (ec 5.4.99.5) catalyzes the first step in that branch of the shikimate pathway which leads to the aromatic amino acids phenylalanine and tyrosine. we have isolated a cdna for this enzyme from the higher plant arabidopsis thaliana by complementing a yeast strain (aro7) with a cdna library from a. this is the first chorismate mutase cdna isolated from a plant. the a. thaliana chorismate mutase expressed in yeast revealed allosteric control by the three aromatic amino acids as previously described for plastidic chorismate mutase isozymes. an attachment of rubisco to chloroplast membranes under cu++-stress has been described for wheat (mehta et al., j. biol. chem. 267, 2810 -2816 . the displacement to the membranes has been discussed as a possible step in the degradation of this predominant stromal protein, e.g. during leaf senescence. in our recent experiments it became evident that such interactions are not restricted to rubisco. several other stroreal, and even a peroxisomal enzyme (glycolate oxidase), were also detected in the membrane fraction after 6 to 30 hours of oxidative stress induced in wheat seedlings by a treatment with 10 mm cuso 4. the velocity and the degree of membrane attachment varied for different enzymes. based on these results it was interesting to check the solubility of rubiseo and its previously described 45 kd fragment which accumulates during the incubation of bean leaf discs under oxygen deficiency (hildbrand and feller, experientia 47, a67) . neither rubisco nor the breakdown product were found to accumulate in the membrane fraction. thus, at present, the steps involved in rubiseo catabolism cannot be generalized. different mechanisms depending on the metabolic situation and on environmental conditions should be considered. overexpression of the four parsley phenylalanine ammonia-lyase (pal) isoenzymes in e. coli. characterization of the enzymes and kinetic analysis of the inhibition by 2-aminoindan-2-phosphonic acid. christoph appert, j/irg schmid, jerzy zorn* and nikolaus amrhein institute of plant sciences, eth-z/jrich, 8092 zijrich. *technical university wroclaw, 50-370 wroclaw, poland. all four phenylalanine ammonia-lyase (ec 4.3.1.5) isoenzymes of parsley (petroselinum crispum nym.) were expressed as glutathione s-transferase fusion proteins in e. coil after affinity purification, the glutathione stransferase moieties were cleaved off with factor xa and the phenylalanine ammonia-lyases were characterized. the proteins form enzymatically active tetramers, even as fusion proteins. the four isoenzymes have comparable km values for l-phenylalanine (15gm to 24.5/.tm), similar temperature (58~ and ph optima (8.5). the aminooxy-and phosphonic-analogues of l-phenylalanine are competitive inhibitors of the enzymes. 2-aminoindan-2-phosphonic acid (j. zo{a & n. amrhein, liebigs ann. chem. 1992,625) was found to be a potent slow-binding inhibitor of these and other phenylalanine ammonnia-lyases, both in vivo and in vitro. chlorophyll a fluorescence has been used to compare the drought resistance of two varieties of solanum tuherosum (avr/i)c-12st-19 and sibtema). fast fluorescence rise kinetics were measured during the first second of illumination with a time resolution of l0 ms and 1z bits in fluorescence intensity. the rise kinetics shows the typical steps called fo -j -i -p. with this values different indexes have been defind with the goal to compare the behaviour of these two varieties of potato. salicylic acid (sa) was proposed as a putative signalling molecule for the induction of systemic acquired resistance (sar) in infected plants. we studied its biosynthesis as follows. cotyledons of cucumber plants were injected with a solution containing pseudomonas lachr~ans and fed with radioactive sa precursors by injection of 14c-benzoic acid (14c-ba) or 14cphenylalanine (14c-phe). alternatively, leaf 1 of cucumber plants were inoculated with tobacco necrosis virus (tnv) and leaf disks were then vacuum-infiltrated using 14c-ba or 14c-phe. free and bound 14c-phenolies were quantified by hplc. incorporation of 14c into sa was found in the two plant-pathogen systems. i~c-ba gave mostly 14c-sa and an unknown polar compound. 14c-phe gave also some 14c-sa, in addition to 14c-labelled lignin precursors like ferulic and p-coumaric acids. the biosynthetic pathway of sa from phe through cinnamic acid and ba will be discussed. after infection with a necrotizing pathogen, systemic acquired resistance (sar) is often observed in plants. in cucumber, salicylic acid (sa) increases in the lower infected leaf as well as in the upper uninfected leaves. sa was also found in the phloem sap of infected cucumber plants and was proposed as a signalling molecule for the induction of sar. we intend to clarify whether sa is translocated from the site of infection to the upper leaf, or wether it is made in the phloem tissue upon the action of a primary systemic signal. one cotyledon of cucumber plants was first infected with pseudomonas lachrymans and then fed with 14c-sa, 14c-benzoic acid (ba) or 14c-phenylalanine. after different times, cotyledons and first leaves were collected and the radioactive ~henolics were analyzed by hplc. in some cases, 4c-sa was found in the first leaf. in addition, two unknown radioactive compounds were detected in leaf 1 after treatment with 14c-sa and 14c-ba respectively. we will discuss signalling processes for the induction of sar in cucumber. one modern approach of creating virus resistant grapevine plants is the introduction of resistance genes into existing grapevine varieties by genetic engineering. the goal of this work is to use the coat protein (cp) mediated strategy to induce resistance to nepovirus in vitis spp. as a first step, several chimeric nepovirus cp genes, were constructed by addition of various promoter regions upstream of the gflv and armv cp regions. their ability of conferring resistance to nepovirus infection in transgenic nicotiana benthamiana and n. tabacum plants will be presented. the best constructions will be used to transform several grapevine varieties in order to create nepovirus resistant grapevine plants. $13-12 the bctalains axe a class of natural pigments found only in plants of the order caryophyllales and in some fungi. the first step in betalain biosynthesis is the conversion of tyrosine to dopa. subsequently , dopa is transformed to betalamic acid, the bctalain chromophorr through the action of dopa-4,5-dioxygenase. we have purified and characterised a copper-containing enzyme of -38kda from amanita muscaria pileus that catalyses the hydroxylation of tyrosine to dopa. considering that the enzymatic activity was restricted to the coloured parts of the mushroom, we postulate that it is involved in betalain biosynthesis. the enzyme featured a broad substrate specificity, and also oxidised the diphenols to their corresponding ortho-quinones, a reaction typical for tyrosinases. the implications of our findings on betalain biosynthesis axe discussed. chlorophyll a fluorescence was measured under steady state conditions of pea and tomato leaves adapted to low light intensity (30 wm-2s -i) at different temperatures (havaux and strasser, z. naturforsch. 45c, 1133 -1141 , 1990 ). when leaves were exposed for a short period (i0 min) to heat (25 to 45~ in darkness, the level of variable fluorescence decreased. when the heat stress was imposed in presence of low light, the variable fluorescence was much less affected and virtually no effect of heat treatment was observed until 37~ this protecting mechanism by low light was absent in algae and mostly absent in submerged water plants but fully present in free floating plants on the water surface. we conclude that a mechanism has been developed for higher land plants during evolution which protects the plants against heat damage on warmer days. caryophyllales, e.g. portulaca grandiflora, and in a few fungal species, e.g. amanita muscaria. we analysed the similarity of a key enzyme of the pathway, dopa-4,5-dioxygenase, in plants and fungi both at the protein and the dna level. antibodies against purified dopa-4,5-dioxygenase from the fungus a. muscaria crossreacted with protein from p. grandiflora petals and two cdna clones were obtained from this plant. their similarity with fungal sequences and with other dioxygenases is discussed. kinetics of prollne hydroxylauonj intrucellnlur transport and c-terminal processing of the tobacco vacuolar cbltlnase. ernst frcydl, thomas boiler and jean-marc neuhaus botenisehes instimt, abt. pflanzenphysiologie, hehoistxasse 1, ch-4056 basel the vacuolar chitinase a of tobacco (nicotlana tabacum) is syndietlzed as a preproprotein with ma n-teaminal signal peptide which causes its synthesis in the er mad a c-termlnal extension, which has been idcmified as the vacuolar targeting peptide (vtp) (1) and whleh is cleaved off from the maybe chitinase. it has recently been shown that mature chltin:~e a contains several hydi'oxyprolines within the short peptide spacer that links the n-terminal cysteine-rieh chitln-bindlng domain to the catalytic domain (2). we received specific antibodies against mature chitinasr (a kind gift from dr. f.meins. f/vii) and raised antihodiea against a synthetic vtp. we performed pulse-chase experiments and cell [ractlonation with 1) stably transformed tobacco plants expressing chitinase a or mutants lacking either the chitin-binding domain and spacer (chitah) or the vacuolar targeting poptid 9 (chitavtp) and 2) transient expression of the same conswacts in nicotiana plumbaginifolia protoplasts. in both systems, proline hydroxylatino in chltinase a was detectable after 30 vain. and complete after 90-120 rain. the ehltinase intermediate forms were detected in the mlcrosomal and soluble fractions for up to 90-120 rain. the mature chitinase continuously accumulated only in the soluble fraction. in both systems chitah showed the same kinetics of intraceilular transport and processing. whereas the transport of cbitavtp seemed more rapid.these results indicate that in vivo cbitinase a is synthetized as a proprotein that is than modified by proline hydroxylation. this second intermedinte form is then transported to the oolgi apparatus and sorted to the vacuole. c-terminal processing occurs late in this pathway or in the vacuole. infection of bean roots with the soil-borne phytopathogenic fungus fusarium solani f.sp. phaseoli leads to a rapid increase of chitinase activity (~five-fold). part of this increase in enzyme activity is due to transcriptional activation of the basic class i chitinase isoenzymes. semi-native polyacrylamide gels stained for chitinase activity using the substrate glycol-chitin revealed the appearence of three additional chitinase isoenzymes that are absent from uninfected control roots. conventional protein purification techniques showed that fusarium solani infected bean roots contain two basic and two acidic chitinase isoenzyrnes. their physiochemical properties and possible biological function will be discussed. proteasomes are multicatalytic protease complexes that function as a major nonlysosomal proteolytic system. they exhibit multiple endopeptidase activities that promote the intracellular turnover of abnormal polypeptides and short-lived regulatory proteins. moss proteasome has been purified from protonemata by successive chromatography on macroprep q, bio-gel a-1.5, bio-gel a-5 and ha. the molecular mass was estimated to be 1,300 kd by gel filtration. gel electrophoresis of proteasome under nondenaturing condition gave a single band and two-dimensional gel electrophoresis identified the moss proteasome to be composed of 14 different subunits with molecular weight in the range 22-35 kd. electron microscopy in aqueous solution showed that it is a cylindrical particle composed of four stacked rings with a diameter of 9 nm and a height of 14 rim. end-on projection established a 7-fold symmetry of the outer disks. substrate specificity of proteasome indicates that it contains endopeptidase activity against substrates bearing hydrophobic, basic, acidic and glycine residues immediately preceding the cleavage site. antibodies raised against moss proteasome reacted with proteasomes of other eukaryotes, including human. $14-01 hunger r.e., hess m.w., laissue j,a,, mueller c. the nod (non obese diabetic) mouse, a widely used animal model for insulin dependent diabetes mellitus, shows in addition to mononuclear cell infiltration of the islets of langerhans, massive cellular infiltrates of the submandibular and lacrimal glands with considerable tissue damage. several lines of evidence suggest that both insulitis and sialadenitis represent autoimmune disorders, to investigate a possible role of tumor necrosis faetor-a (tnf-c0 and activated cytotoxic cells (nk cells, t cells) in the inflammatory process, tissue sections of nod mice were hybridized with radiolabeled rna probes specific for the detection of mrna for tnf-r and the serine proteases granzyme a and b (gra and gr8) and perforin which are expressed in activated cytotoxic cells. tnf-e expressing cells were mainly located in infiltrates and are absent in non affected glands, whereas gra, grb and perforin expressing cells were distributed over the whole section with highest densities in the zones adjacent to parenchyma. the finding that activated cytotoxic cells are present in early stage of disease development indicates that cell mediated cytotoxicity may play a crucial role in initial tissue destruction. the role of tnf-a in autoimmune diseases is not known yet. the appearance, however, of cells expressing this gene in the infiltrate indicates a possible role of this cytokine in the progression of the inflammatory reaction, e.g. by increasing the lymphocyte traffic to this organ. we further demonstrated that the induction of inos was at the transcriptional level. in order to understand the underlying mechanisms we characterized the promoter region of the rat nos gene. a 9enomic rat library was screened using a cdna-fragment coding for the if-end of the inos cdna (nucleotides 1-817). a 1,8 kb hinc-ii fragment containing the 5"-flanking region of the inos gene was characterized by dna-sequencing. the transcriptional start site was determined by primer extension. sequencing analysis revealed a multitude of possible cis-acting elements homologues to consensus sequences for the binding of different transcription factors including ifn~' response element (ire), nuclear factor-kb (nf-~b), tumor necrosis factor response element (tnf-re), 7f-activated sites (gas) and one x-box. nf-kb, a transcription factor involved in signaling and immediate early gene activation during inflammatory processes was induced by treatment of mesangial cells with 2 nm of il-113. this was shown by the appearence in nuclear extracts of the dna binding activity of nf-~b using electrophoretic mobility shift assays (emsas) with a 32p-labeled ~r dna probe. pyrrolidinedithiocarbamate (pdtq), an inhibitor of nf-kb, suppressed the expression of inos mrna in mesangial cells without affecting the dibutyryl-camp-triggered increases in nos mrna levels. this data suggest that the il-i ~ signalling pathway responsible for nos expression requires nf-k8 activation and is definetly different from the signalling cascade directed by camp. recent findings indicate that the multifunctional cytokine il-6, which plays a key role in irm-nune and inflammatory responses, also exerts specific effects in the central nervous system (cns). for example, il-6 promotes neuronal survival, induces differentiation and modulates neurotrophin production. using the very sensitive technique of reverse transcription combined with polymerase chain reaction the developmental profile of il-6 and its receptor mrnas was analyzed in various rat brain regions. our results indicate that both genes are expressed in a region-specific manner and are developmentally regulated. these findings support the hypothesis that il-6 is involved in differentiation and maintenance of neuronal subpopulation in the cns. interleuldn 2 (il-2) expression is strictly dependent on a signal transmitted by the t cell receptor upon antigen stimulation. this signal can be mimicked in vitro with phorbol esters and calcium ionophores. we have found that stimulation with ca-ionophore alone induces expression of il-2 mrna, but does not induce secretion of il-2. in polysome gradient fractionation of cells stimulated with phorbol esters and ionophore, the fractions containing il-2 rrtrna are found at the bottom of the gradient, bound to polysomes. however, in ionophorestimulated cells the fractions containing il-2 mrna are clearly shifted to the top of the gradient, and therefore are not lzanslated. this effect is specific for il-2, as other mrna's like 1~2-microglobulin are not regulated. this data suggests that il-2 gene is translationally regulated. in addition, in vitro experiments have shown that the lack of translation of il-2 mrna in ionophore-stimulated cells is due, most likely, to the presence of a translational regulator that specifically inhibits translation of il-2 mrna. assuming the presence of a translational regulator that specifically represses il-2 mrna translation, we can predict that, if the represser is in excess, even upon a second stimulus that is by itself able to induce secretion of il-2, there will be no translation of il-2 mrna. the resuhs of such an experiment confirmed our prediction. polysome gradients showed that after ionophore stimulation, il-2 mrna gets "stacked" with one ribosome, and no further ribosome loading takes place. if malaria is highly endemic, every febrile child is a presumptive malaria case, and there is no differential diagnosis based on clinical, immunological or parasitological grounds. we evaluated the diagnostic usefulness of interleukin-2 receptor (il-2r), tumor necrosis factor-receptors 55 (tnf-r55) and 75 (tnf-r75) . sera came from tanzanian pediatric fever patients and controls, all enrolled in the kilombero malaria project. we found that all 3 receptors marked pediatric fever episodes, whereas stability of observed levels was highest for tnf-r75 and lowest for tnf-r55. tnf-r55 levels reflected severity of the fever attack. regardless of the presence of a febrile illness, tnf-r75 levels were strongly associated with parasite density. immunological markers can be applied in rapid pre-and post-intervention morbidity assessments, validation of health interviews and monitoring of convalescence. we have recently shown that human eosinophils produce mrna for interlenkin (il)-8 when stimulated with calcium ionophom (eur. j. immunol. 23 (1993), 956). we now present evidence that human eosinophils contain preformed il-8 protein although they do not express mrna for il-8 as measured by rt-pcr. il-8 protein expression was determined using specific anti-human il-8 monoclonal antibodies by western blotting, facs analysis and immunohistochemistry. moreover, preformed il-8 was released into supernatant by 99% pure eosinophils after priming with gm-csf or il-5 and subsequent 25-min stimulation with paf, rantes, ionomycin or pma, however not with il-1 or il-2, as measured by elisa. this observation implies that activation of protein kinase c and/or inwacellular calcium mobilization am necessary events for il-8 release in human eosinophils. the determined amounts of preformed il-8 protein was higher in patients with asthma compared to normal individuals suggesting a role for eosinophil derived il-8 in asthma. since the eosinophil is the predominant cell in the asthmatic airways, we determined 1l-8 concentrations in bronchoalveolar lavage (bal) fluids from normal individuals and asthmatic patients. indeed, il-8 concentrations in bals from both groups were similar to those seen in the supematants released by activated eosinophils. the role for 1i-,-8 in asthma remains to be determined. to study the effect of canthaxanthin in vitro, the hydrophobic compound was introduced in vivo into chick high density lipoproteins (hdl) by canthaxanthin feeding. the effects of hdl and hdl associated canthaxanthin on two types of cultures have been tested: flat sedimented cell cultures of embryonic chick neuronal retina, retinal figment epithelium (rpe), brain and meninges, and in reaggregate cell cultures of the neuronal retina. at high canthaxanthin concentrations the formation of colored, birefringent entities were induced in the neuronal retina in vitro. in line with human data, parameters of cellular function and cell differentiation remained unaffected by canthaxanthin at these concentrations. by contrast, fidl was found to induce various cell type dependent effects. proliferation of meninges cells was decreased at low hdl concentrations as concluded from.light microscopic examination and indicated by protein content, and lysosomal and mitochondrial activity of the cultures (ic50:15-30 mg hdl apoprotein/l medium). these parameters, however, were increased in brain cell cultures, while they remained unaffected in cell cultures of neuronal retina and rpe. concentrations above 0.3 g. hdl apoprotein/l caused a reduction of glial cell differentiation. $14-08 tnf-c~ had close-dependent antiproliferative activity on hormone-dependent human breast cancer cells that represent an early stage of the disease, whereas hormone-independent cells representing a late stage were not affected. however, in the presence of 1000 u/ml interferon ?(inf), high concentrations of tnf-c((lnm) were able to inhibit the growth of hormone-independent cells. using specific monoclonal antibodies in ftow cytometry, no significant changes of either the p75 or the p55 tnf receptors were observed upon inf treatment of hormone-independent cells. this indicates that the increased responsiveness of these cells for tnf-c~ is not due to a change in available tnf receptors. the affinity of the receptors for tnf-r are one order of magnitude different for the two cell types. inf treatment shifted the ecs0 of hormone-independent cells towards the ecs0 of hormone-dependent cells. thus, the basis for the increased tnf-sensitivity of hormone-independent cells in the presence of inf might be the interconversion of tnf receptors from low-to high-affinity. the adhesion of tumor cells to endothelium is the first step in the processus of metastasis, and is frequently dependent of cytokinemediated activation of endothelial cells. cytokines are also involved in nitric oxide (no) production by various cells. these experiments were designed to evaluate no production during tumor cell adhesion to endothelium. rat brain-derived endothelial and rat colon carcinoma cell lines were used during these experiments. no was evaluated with the griess reagent. activation of endothelial cells with cytokines (tnf-d, and ifn-~ ), only slightly increased (10-20 %) the adhesion of tumors cells to endothelium. activation of endothelial cells with tnf-ol and ifn-~, induced a 4-8 fold increase of no production. however, addition of tumor cells to the endothelial monolayer, either directly or in a semi-permeable membrane, decreased the cytokine-induced no production. these results indicate that no production is modulated during the proeessus of adhesion of tumor cells to endothelium. is induced in rat mesangial cells by inflammatory cytokines such as interleukin 1 ]3 (il-1 ~) and requires bh 4 as a cotactor. 2,4-diamino-6-hydroxypyrimidine (dahp), a selective inhibitor of gtp cyclohydrolase i, the rate-limiting enzyme for bh 4 synthesis, potently suppressed il-i~induced nos activity, measured as nitrite production. inhibition of no synthesis by dahp was reversed by sepiapterin, which provides bh 4 via a salvage pathway. sepiapterin dose-dependently augmented il-1 i],-stimulated no synthesis, indicating that availability of bh 4 limits the production of no in cytokine-stimulated mesangial cells. n-acetylserotonin, an inhibitor of the bh 4 synthetic enzyme sepiapterin reductase, completely abolished il-lj~-induced no formation whereas methotrexate, which inhibits the pterin salvage pathway, displayed only a moderate inhibitory effect, thus suggesting that mesangial cells predominantly synthesize bh 4 tom gtp. in conclusion, these data demonstrate that bn 4 synthesis is an absolute requirement for cytokine induction of nos in mesangial cells. inhibition of bh 4 synthesis may provide new therapeutic approaches to the treatment of pathological conditions mediated by no. -1 [3) can induce a macrophage-type of nitric oxide synthase (inos). northern-blot analyses of inos mrna-levels and nuclear run-on experiments of control and il-1 ~ stimulated mesangial ceils revealed that the induction of inos was at the transcripuonal level. here we demonstrate that platelet-derived growth factor bb (pdgf-bb) can suppress the il-113 dependent inducuon of the inos gene. nortllern-blot analyses of cellular rna isolated from mesangial ceils that were coincubated with il-1 [3 (2nm) and pdgf-bb (10 ng/ml, 100 ng/ml and 300 ng/ml) revealed a dosedependent suppression of inos mrna-levels. using run-on experiments with nuclei from mesangial ceils after stimulation with il-i~ (2nm) and pdgf-bb (100 ng/ml) we demonstrate that the inhibition occurs at the transcriptional level. basic fibroblast growth factor (bfgf), on the other hand, potentiates the il-1 dependent stimulation of inos. coincubation of mesangial cells with il-113 (2nm) and bfgf (3ng/m[, 10ng/m[, 30ng/m[, 100ng/m0 dose-dependently superinduces inos mrna levels as shown by northern-blot analyses of total cellular rna form control and stimulated cells. run-on experiments with nuclei from mesangial cells stimulated with il-113 (2nm) and bfge (100ng/ml) revealed an enhanced transcriptional activity of the inos gene. thus, pdgf-bb and bfgf differentaity modulate the il-i~ dependent expression of the inos gene and are therefore an excellent model system to study the crosstalk between signal transductjon pathways. we report that ifnyr-/-mice have an increased resistance to lipopolysaccharide-induced toxicity (lps). lps-induced lymphopenia, thrombocytopenia and weight loss seen in wild type mice were attenuated in ifntr-/-mice. ifntr-/mice survived in the d-galactosamine-lps model when conditions were 100% lethal for wild type mice, correlating with serum tnf levels of up to 10 fold higher in wild type mice. bone marrow and splenic macrophages from ifnyr-/-mice had a 3 to 5 fold decreased lpsbinding capacity, with serum from these mice lowering macrophage lpsbinding by a further 50%. thus, depressed tnf synthesis, diminished expression of lps receptors and low plasma lps-binding capacity in the mutant mice likely combine to manifest in the resistant phenotype of ifny r-/mice to endotoxin. in a complementary approach we have screened the 5' portion (-7.8 kb/+4.1 kb) of the gene for tissue specific and/or inducible dnasei hypersensitive sites. in chromatin from fibroblasts or kidney epithelial cells this segment of the il2ra gene does not contain any dnasei hypersensitive sites. in contrast, in resting t-and b-lymphocytes, as well as in activated t cells, two sites (dhi, -0.05 kb; dh3, -5.8 kb) were found. a third site (dh2, -1.35 kb) is detectable in t cells that have been activated with concanavalin a and il2. this site maps to the same position as cisacting regulatory sequences that are required for the response of the il2ra gene to signals from the il2 receptor. interleukin-6 (il-6) production in cultured human dermal fibroblasts can be stimulated by interleukin-i (il-i} added to the culture medium. absence of fetal calf serum and of growth factors (insulin, egf, t3) reduced the stimulation by more than 50 %. egf and t3 and even more choleratoxin increased the il-6 production in absence of fetal calf serum. the effect was dose dependent. 2% human ab serum substituted for i0 % fetal calf serum. pretreatment of the cultures with serum or growth factors prior to stimulation with il 1 had a stimulatory effect (serum, t3, egf) or an inhibitory effect (hydrocortisone, choleratoxin). our results suggest that the il-i signal transduction to il-6 is modulated by serum components and growth factors as well as through c-amp produced by choleratoxin. interleukin-6 (il-6) is produced by a variety of cells and plays a central role in host defense mechanisms. its function include induction of il-2 and il-2 receptor expression, proliferation and differentiation in t-cells. in cocultures of human dermal fibroblasts and allogenic human t-cells a cell number dependent 10 to 100 fold increase of the il-6 production could be demonstrated after 24h and 48h. conditioned medium from tcells and from fibroblasts failed to induce il-6 secretion in fibroblast and t-cell cultures, respectively. no up-regulation of il-6 production was found in cocultures of fibroblasts with tcells killed by ethanol and in t-cell culture incubated with recombinant hll-lc~. after separation of the cells il-6 production in fibroblast and t-cells returned to precoculture levels. our results indicate that cell-cell contact more than soluble factors must be involved in the up-regulation of il-6 synthesis in cocultures of t-cells and fibroblast. using cocultures of autologes human t-cells and fibroblasts, we are currently investigating whether the cell contact essential for the il-6 production depend on immunolcical interactions of t-cells and fibroblasts. rapid growth of tumor often results in oxygen and nutritional deprivation leading to extensive necrosis, which is one of the characteristics of glinblastomas (gbl). neoplastic cells surrounding necrotic areas often present a peculiar arrangement of cells called "pseudo-palisading" -as a hallmark of this entity, together with abundant neovascularization. a special biological milieu is probably formed in these palisading areas by certain unknown cytokines and/or growth factors induced by the necrotic process. plate et al. (1992) reported the expression of vascular endothelial growth factor (vegf) in the palisading cells. we previously demonstrated that gbl cells produce il-8, which is known to be an angiogenic factor. the aim of this study is first to assess if il-8 is expressed in the palisading ceils, and second to determine if hypoxia can trigger il-8 gene expression. in rive observation using in siru hybridization and immunohistuchemistry showed that il-8 is highly expressed specifically in the palisading cells. we also demonstrated the mrna expression of il-8 receptor in the endothelial cells adjacent to necrotic area. to simulate the in vivo condition, two in-vitro models are currently developed to determine if il-8 is induced by hypoxic insult on cells. first, gbl cell lines are cultured in the absence of oxygen and il-8 expression is assessed by northern blot analysis. preliminary results have demonstrated the induction of il-8 by hypoxia. secondly gbl cells are grown in spheroids until development of central necrosis. the il-8 expression will be assessed by in situ hybridization combined with immunohistochemistry to determine if the ceils surrounding necrosis express il-8. it has been proposed by taniguchi and his colleagues that activation of the interferon (ifn)-i3 gene by virus or double-stranded rna involves induction and modification of irf-1. overexpression of irf-1 can induce expression of the ifn-b gene in certain cells. a role of irf-1 in the activation of ifn-a genes has also been proposed. furthermore, irf-1 has been claimed to play the role of a tumor suppressor gene. we have generated mice in which both irf-1 alleles were disrupted and found them to develop normally. no spontaneous tumors have been observed so far. injection of poly(i)-poly(c) resulted in the same levels oflfn in blood of wildtype and null mice, and equal ifn-ct mrna levels in spleen. induction of wild type and irf ~ embryo fibroblasts (mefs) with virus resulted in the same levels of ifn-a and ifn-i] mrna respectively. in the case of polyo)-poly(c) induction, the levels of both 1fn mrnas were higher in wild type than in mutant cells; this difference was abolished if the cells were first primed with type i ifn. we conclude that irf-1 does not play an essential role in the induction oflfn genes. mitsuhiro tada, annie-claire diserens, isabelle desbaillets, marie-france hamon, nicolas de tribolet, erwin van meir; chuv, lausanne there is increasing evidence that a variety of cytokines produced by glioblastoma (gbl) cells modulate the tumor growth by affecting the host's immune response and by stimulating neovascularization. cytokines such as il-i, il-6, il-8, mcp-i, il-10 and tgf-132, affect each other's production and receptor system and seem to form a cytokine network. among them, il-1 may play a pivotal role, since il-i can induce or increase the production of other cytokines (il-6,il-8,mcp-i,tgf-132) and modulate their receptor expression. to test this hypothesis, we investigated co-expression of cytokines and their receptors in 15 gbl cell lines and 15 gbl tissues using rt-pcr, northern blot analysis, immunnhistnchemistry and elisa quantification. a majority of the gbl cell lines and gbl tissues expressed il-i~, il-113, type i and type ii receptors, indicating the presence of an il-1 autocrine loop. il-1 stimulated growth of some of the cell lines. a positive correlation of il-6 and il-8 expressions with the presence of an il-i autocrine loop in the cell lines was found. immunohistochemical studies showed the in rive co-expression of the il-i family and the secondary cytokines (il-6, il-8) in gbls. antisense oligonucleotide to il-lc~ and/or il-113 partly suppressed this secondary cytokine expression. these results demonstrate that the il-1 autocrine loop plays a central role in the formation of the cytokine network in gbls. double-stranded rna-dependent protein kinase (pkr) is induced by type i interferon (ifn) and is implicated in the establishment of the antiviral state. upon activation, pkr phosphorylates the eukaryotic initiation factor eif-2, thereby throttling protein synthesis. it was suggested that pkr may be essential for the induction of ifn-13 gene expression by both virus and double-stranded rna and for the activation of at least some ifninducible genes. recently it was proposed that pkr is a tumor suppressor gene because 3t3 ceils overexpressing inactive human pkr gave rise to tumors in nude mice (koromilas et al., 1992; meurs et al., 1993) . we have produced homozygous pkr knockout (pkr ~176 mice and found that they develop normally and have no striking phenotype. induction of ifn-c~ and ifn-13 gene transcription by virus was unimpaired in fibroblasts derived from pkr ~ mice, as was the induction of several ifn-stimulated genes. so far, no spontaneous tumor formation was observed. pkr o/o embryonal stem ceils showed no increased malignancy in nude mice as compared to their wild type counterparts. we conclude that pkr does not play an essential role in viral induction of the ifn genes and that its tumor suppressing effect, if any, is redundant. tgf-13 plays an important role as a negative regulator of hepatocyte proliferation in liver regeneration. exposure of rodents to nongenotoxic carcinogens like cyproterone acetate (cpa), thioacetamide (ta) and phenobarbital (pb) causes abnormal hepatocyte proliferation and liver tumors after long term treatment. this suggests that these chemicals have either a direct mitotic activity or impair the negative regulatory system for growth. therefore the inhibitory activity of tgf-6 on dna-synthesis induced whith cpa was investigated in cultured rat hepatocytes. after a 48h exposure to cpa, a dose dependent increase in dna synthesis (3h-tdr incorporation) was observed. the maximum effect was attained with 12 pm cpa (up to 4.3 fold) which was stronger than with 10 ng/ml egf (up to 2.8 fold). tgf-81 and tgf-132 inhibited this response dose dependently (idso = 0.1 ng/ml) and reduced it to control levels at 1 ng/ml. these findings show that the tgf-6 mediated pathway for negative growth control in hepatocytes is still responding after cpa treatment. mouse mxl protein is an interferon (ifn) induced gtpase with intrinsic antiviral activity against influenza a viruses. it accumulates in the cell nucleus and inhibits the transcription of influenza virus genes, recently, we have shown that mxl exerts its inhibitory activity by interfering with the function of the viral polymerase subunit p82. pb2 is responsible for recruiting 5" ends of cellular mrnas as primers and for the elongation of nascent viral transcripts. to elucidate which pb2 dependent step of viral mrna synthesis is the target of mxl action, we examined the activity of recombinant mxl protein in an in vitro transcription system of influenza virus. first, we expressed wildtype and mutant mxl proteins, carrying a hlstidinetag at their n-terminus, in e. coil and purified them under nondenaturing conditions by ni-chelate affinity chromatography. mxl efficiently inhibited viral transcription in vitro, while mutant mxl proteins, lacking antiviral activity in vivo ,were inactive. moreover, the use of capped synthetic rna primers revealed, that mxl almost completely abrogated the elongation of viral transcripts, whereas the the cap-binding and endonuclease activity of pb2 was not affected. a50 $14-28 neutralization rates of tnf-a from eight animal species by a monoclonal antibody against human tnf: implication for receptor action? we have recently developed a sensitive bioassay for measuring porcine tnf-a based on homologous pk(15) cells. this bioassay is 100-to 1000-fold more sensitive than the widely used l929 bioassay, also, human tnf-a and human tnf-8 were detected with a slightly higher sensitivity in the new test compared to the l929 bioassay, we now show that also canine, ovine, bovine, equine and caprine tnf-a can be detected with the pk(15) bioassay. we tested a murine monoclonal anti-human tnf-a antibody for its capacity to neutralize tnf-a from eight different animal species. the action of this antibody revealed that neutralization of tnf bioactivity is a complex phenomenon, indicating species-specific differences in the interaction with tnf receptors. to study this differential neutralization, we are cloning the porcine tnf receptors for expression in a heterologous cell system. administration.of high dose r-tnf(~ in combination with ifn 7 in isolation and perfusion of the limbs in melanoma patients has shown to be very promising with a response rate greater that 80%. this observation prompted us to investigate the possible expression of the tnf receptors in melanoma cells using the monoclonal antibodies utr-1 specific for the tnf type a (55 kda) receptor and htr-9 specific for the tnf type b (75 kda) receptor. flow cytometric analysis of cultured melanoma cells showed the presence at low level of the tnf type a and to a slightly higher level of the type b receptor. similar results were obtained in vivo by immunohistochemistry on fresh tumor raateriai, treatmem of melanoma cells in culture with the-amp induced up to a 2 fold increase in the number of type a tnf receptors with only a minimal change in the number of type b receptors. this increased expression of tnf receptors is conflrmed by direct binding experiments using 125i-labeled tnftx. the number of cpm's bound on dbc amp treated cells was about 0.5-3 fold higher than on untreated control calls, likewise incubation of melanoma cell lines with ifny increased the specific binding of subsequently added 125i-labeled tnfct. from flow cytometric analysis using the two anti-tnf receptor antibodies it became evident that flint was able to increase the expression of both type a and b receptors depending on the cell line used. characterization of a murine tumor necrosis factor (x-lacz reporter construct tumor necrosis factor ot (tnfcq is a prominent mediator of a variety of different pathologies such as endotoxic shock syndrom and rheumatoid arthritis. it also plays a crucial role in host defence against bacterial and viral infection. up to now, only few data about signal pathways leading to tnfo~ induction are available. an easy to handle tnfc~ -lacz reporter assay will represent a useful tool to study signal transduction on the one hand and provide data about compounds with potential tnfo~ inducible activity on the other hand. in order to develop a routine macrophage cell line capable to easily report tnfc~ induction, different tnfe-lacz reporter constructs were assembled. data about functionality of the different constructs in routine macrophages will be presented in the context of tnfot expression. cloning of a novel protein binding to lymphokine, fos and myc mrna with unexpected enzyme activity j. nakagawa, h-p. waldner, s. meyer-monard, and ch. moroni inst. medizinische mikrobiolegie, univ. basel an au-rich sequence in the 3' untranslated region of lymphokines, c-los, and c-myc proto-oncogene mrna is responsible for their rapid decay. we have purified a 32 kd protein by an auuua affinity column from human brain. partial amino acid sequence was obtained and the corresponding edna has been cloned. unexpectedly the edna sequence exhibited significant homology to enoyl coa hydratase and bacterially expressed recombinant protein indeed showed the activity. while rat hydratase did not show rna binding activity, the recombinant protein bound to cfos, c-myc, auuua cluster of il-3 mrna, and adeno virus iv, but not to mutated ad-iv, nor irrelvant transcript. the binding was competed out by poly u. interestingly the protein seems to be processed from a larger precursor. we suspect that the protein plays a role in mrna turnover and perhaps in oncogenesis. institute for medical microbiology, university of basel in t cells, the immunsuppressive agent cea is known to inhibit ca 2+ dependent induction of lymphokine mrna transcription. in contrast, the immunsuppressive drug rapamycin inhibits the lymphokine induced proliferation. we have analysed the effect of these drugs on a v-h-ras induced autocrine mast cell tumor line which expresses il-3 constitutively. csa and rapa inhibited autocrine growth by different mechanisms,, because added il-3 could antagonize inhibition by csa, but not by rapa. whereas csa acted by downregulation of il-3 mrna expression, rapa blocked il-3 induced proliferation. cea also inhibited il-3 superinduction by ca-ionophore, whereas rapa did not. nuclear run on assay indicated that the mechanism is posttranscriptional. therefore, we have introduced il-3 transgenes with and without the au-rich region in the 3' utr of the mrna into a tumor line. in contrast to the wild type, the expression of the mutant construct was insensitive to csa. since the mutant lacks sequences known to regulate rapid mrna decay, we suggest that csa affects the regulation of il-3 mrna stability. some non-hematopoietic cell lines produce both scf and its receptor, the c-kit protein (c-kit). testing the hypothesis that scf may be involved in secondary growth of nb bone marrow metastasis, we found and previously communicated (beck d. et al, proc aacr, 34, 52, 1993) a low or absent expression of c-kit in nb cells. the mrna and surface expressions of scf gene in nb cell lines grown in chemically-defined medium were evaluated by northern blot analysis and flow cytometry. the scf antigenic concentrations in culture supernatants were determined by elisa and growth-inhibition experiments performed by pre-incubating nb cells with anti-scf antibodies prior to 3h-thymldine uptake assay. the scf mrna was expressed in 4 of 5 tested lines but no membrane-bound antigen could be detected on those cells. detectable levels of scf in the supernatants were found in 5/7 lines (range : 39-96 pg/ml). blocking experiments resulted in a decrease of dna synthesis in i out of 7 lines only. from these and previous results, we conclude that scf is synthesized and released by many nb cells in vitro but the functional ~ole of it remains undetermined since scf does not appear to be usually involved in autocrine or paracrine growth stimulation of nb cells (supported by swiss fnrs grant 3200-031005). expression of the v-h-ras oncogene in the il-3 dependent pb-3c mast cells leads to the generation of two classes of il-3 autocrine tumors in vivo. autocrine il-3 expression in class-1 tumors results from increased il-3 mrna stability due to the loss of negative trans-acting posttranscriptional control. this defect can be corrected by somatic cell fusion to the nontumorigenic parental pb-3c resulting in downregulation of oncogenic il-3 expression and concomitant tumor suppression. expression of the v-h-ras oncogene remains unaffected in class-1 hybrids. in contrast, class-2 tumors are characterized by transcriptional activation of the il-3 gene due to the insertion of an endogenous retroviral element (intracisternal a-particle) which cannot be overcome by ceil fusion (see hirsch et al, 1993, j.exp.medicine 178, 403-411) . although v-h-ras is required for generation of either class of tumors, expression of anti-sense ras constructs inhibited only the proliferation of class-i tumor cells. experiments are underway to characterize the target and the nature of the class-1 alteration. nitric oxide (no) promotes vasorelaxatlon of renal vessels in vitro. the purpose of the present study was to assess the role of no in regulating renal hemodynamics in vivo. renal blood flow (rbf) and glomerular filtration rate (gfr) were studied in anesthetized mechanicallyventilated newborn rabbits both before and after the administration of a no synthesis inhibitor, ng-nitro-larginine methyl ester (l-name), at a dose of 50 pg/kg followed by 10 jzg/kg x rain. such a dose of l-name did not modify mean arterial pressure or pulse rate. by contrast, the renal vascular resistance increased by 31 + 9% (p < 0.005) while rbf decreased by 20-+6% (p < 0.02). gfr and urine flow rate remained constant. the overall results suggest that in normal conditions no may play a role in decreasing the renal vascular resistance of the immature kidney, without altering systemic blood pressure. interleukin-4 (il-4) is a t-cell derived lymphokine which, in addition to its effects on the immune system, is also able to suppress the growth of certain tumor cells. il-4 reduced the growth rate of four human colon tumor cell lines up to 70% in a dose-dependent manner. three other cell lines showed no significant alteration of 3h-thymidine incorporation or colony formation in the presence of 100 u/ml il-4. responder and nonresponder tumors were immunopositive for il-4 receptor (il-4r) expression in flowcytometric analysis, using monoclonal antibodies raised against recombinant il-4r and bound biotinyated il-4. responsive tumors expressed more receptors. 11-4 binds with high affinity to a 130 kda transmembrane receptor (il-4r), through which the extracellular signal has been shown to be transduced. coimmunoprecipitation and chemical crosslinking studies have enabled us to demonstrate il-4r target proteins. tyrosine phosphorylation of various proteins between 70 and 170 kda appears to be initiated during il-4mediated signaling events in colon tumor cells. the cloning of human il-4r target proteins will contribute to the understanding of the il-4 signal transduction pathway at the initial stage. the renal effects of endothelin-1 were investigated in 16 anesthetized and meehanleally-ventilated newborn rabbits. each animal acted as its own control. in 8 newborn rabbits, a bolus injection of 5 nmol.kg "1 of endothelin-1 caused an initial fall in mean blood pressure (mbp) followed by a gradual but significant increase in mbp that lasted for 45 rain. the dramatic increase in renal vascular resistance (+ 28 -+ 4%) induced by endothelin led to a fall in glomerular filtration rate (-12 -+ 4%) and renal blood flow (-16 _+ 3%). in spite of the reduction of gfr and rbf, urine flow and sodium excretion rates increased significantly (+ 20 _+ 5% and + 49 _+ 9%, respectively). in 8 additional newborn rabbits, a bolus injection of lnmol.kgaof endothelin-1 -a dose that usually induces marked renal and systemic vasoconstriction in adult models -did not affect systemic or renal hemodynamics. in conclusion, endothelin induces renal and systemic vasoconstriction, and affects water and sodium homeostasis during the neonatal period. however, these effects occur under higher doses than those used in adult animals, possibly reflecting receptor immaturity and/or interference of high levels of counteracting hormones. ontogeny of the endocrine pancreas in xenopus laevis c, maake 1 , w. hanke 2 and m. reine~ke 1 , 1 institute of anatomy, university of z0rich, switzerland and department of zoology ii, university of kaflsruhe, f.r.g. the pancreatic islets of anurans show insulin (ins), glucagon (gluc), somatostatin (som) and pancreatic potypeptide (pp) containing cells. since only limited information exists on the ontogeny and on the presence of insulin-like growth factor 1 (igf-1), we studied the development of the gastro-entero-pancreatic (gep) system in xenopus laevis using immunohistochemical techniques. singular ins-immunoreactive (-ir) cells were observed in the pancreas as early as on stage 41. at stage 43, the first pp-ir cells were found in the gep system followed by som-ir and gluc-ir cells. the first pancreatic islets consisting of ins-ir and gluc-ir cells occurred around stage 50. between stage 54 and 60, i.e. after the onset of metamorphosis, the endocrine cells decreased in number and the islets partly disintegrated. starting with stage 61, the islets reformed and the amount of endocrine cells increased reaching the adult status at stage 63. around stage 52, igf-l-immunoreactions appeared. igf-1immunoreactivity was found in pp-ir and gluc-ir cells but not in ins-ir and som-ir cells. it is assumed that islet-derived igf-1 may p~ay an important role during metamorphosis in xenopus. t. cathomen and r. cattaneo, institut for molekularbiologie i, universit~t zorich, hfnggerberg, ch-8093 zorich the signals used for synthesis of the measles virus fusion (f) protein are poorly understood: a long (>570 bases) untranslated region is followed by three in frame augs at codons 1,4 and 15. f is a type i transmembrane protein with a postulated signal sequence of 31 amino acids. we confirmed that the 46 aminoterminal residues contain a signal peptide by transfering them to another protein. with the other envelope protein hemagglutinin, f protein induces virus-cell and cell-cell fusion. mutagenesis of the three augs indicates that proteins initiated at codon 1, 4 or 15 are all functional, but show slightly different fusion efficiencies. forms translated from aug 1 or 4 appear larger in electrophoresis than forms initiated at aug 15, suggesting that two different signal peptide cleavage sites are used. we are currently investigating whether both f protein forms are incorporated in viral particles. the production of protein isoforms with staggered amino-termini is common in cytoplasmic and type ii transmembrane proteins, but signal sequences usually preclude a similar organization of type i transmembrane proteins. beguin p., beggah a.t., rossier b.c., jaisser f. and geering k. institut de pharmacologie et de toxicologie de l'universit6, ch-1005 lausanne recent experimental evidence suggests that na,k-atpase might be a candidate for regulatory phosphorylation by protein kinases a and c (pka and pkc). to verify this hypothesis, we have mutated several serine and threonine residues in consensus phosphorylation sequences of the ~subunit of bufo marinus na, k-atpase. the mutants were expressed in x~nopus ooeytes and phosphorylation was studied in oocyte homogenates upon stimulation of oocyte, pka and pkc. our results indicate that a unique phosphorylation site for pka is located at serine 943 in the c-terminus of the ~-subunit but its phosphorylation can only be revealed in the presence of detergent. on the other hand, mutations of several serine and/or threonine residues located in consensus sequences for pkc phosphorylation did not or only partially abollsh phosphorylation by pkc. in particular, mutations close to the catalytic phosphorylation site of the ~-subunit influenced phosphorylation by pkc, suggesting that either this region contains a real phosphorylation site or is part of a conformational domain necessary for phosphorylation by pkc. the heat-stable antigen (hsa or mouse cd24) gene is differentially regulated but has a housekeeping promoter. roland h. wenger*, georges kshler and peter j. nielsen. max planck institut f(jr immunbiologie, st0beweg 51, d-79108 freiburg i. bsg. "present address: physiologisches institut der universit&t zi.)rich, winterthurerstrasse 190, ch-8057 z0rich, expression of the gpi-anchored murine glycoprotein heat-stable antigen (hsa) shows tissue-specific as well as developmental regulation. during the maturation of several hematopoietic lineages, hsa expression is generally high in immature precursor ceils and low or absent in terminally differentiated cells. we present evidence suggesting that this regulation of the hsa gene (cd24a) occurs at the transcriptional level. in addition, sequence and methylation analysis of the cd24a promoter revealed characteristics of both "housekeeping" and tissue-specific promoters, including a methylation-free, hpall tiny fragment (htf) island, multiple putative sp1 and ap-2 consensus binding sites and a tata box. functional analysis of a 0.6 kb dna fragment containing these elements fused to the cat reporter gone in transient transfection experiments showed activity in both hsa expressing and non-expressing cell lines with a strength similar to that of the hsv tk promoter. large fragments from the flanking region of the cd24a promoter did not influence the ubiquitous nature of this promoter, finally, the cd24a, cd24b and cd24c genes were mapped to mouse chromosomes 10, 8 and 14 respectively. we have cloned, sequenced and characterized the gone for the m__itochondriat nadh-cytochrome b5 reductase (mcri) of saccharomyces cerevisiae. surprisingly, this gone encodes two mitochondrial isoforms of the flavoenzyme: a 34 kda integral protein exposed on the outer surface of outer mitochondrial membrane (mcrlp34), and a 32 kda soluble protein of the intermembrane space (mcrlp32). the smaller intermembrane space isoform is generated from the larger form by the action of the inner membrane protease i (impi) on the outer surface of the inner membrane. this is the first demonstration that a single gone encodes proteins which are located in different compartments of the same organelle. with special interest in any with a mitochondrial localization. degenerate primers were designed on the basis of high homology between members of the abc family, and pcr was performed on yeast genomic dna. ten dna fragments bearing significant homology to the abc family were amplified, nine of which were previously unidentified. disruptions of five of the corresponding genes were performed. disruption of one gene led to markedly impaired growth on rich medium and cessation of growth on minimal medium. the gene was cloned and found to encode a protein of 694 amino acids, a "half-transporter" which likely forms a dimer. ibi~tlnofluorescence on yeast expressing the gene cmyc-tagged at the c-terminus reveals co-localization of the protein with porin and with mitochondrial dna, visualized by dapi staining. nycodenz-purified mitochondria are enriched for the protein by immunoblotting. additionally,the c-myc epitope is protease-protected in mitoplasts, indicating an inner membrane sub-localization and a probable orientation with the c-terminus in the matrix. the gene, termed atmi for abc transporter of mitochondria, thus encodes the first member of the large abc transporter superfamily to be localized to this organelle. present work is aimed at revealing the substrate of this transporter. cycle in cardiac myocyte. the molecular study of this protein has been difficult even after its over-expression was made possible by the cloning of the corresponding cdna. the chief problem is the lack of convenient domains which could be used in the purification of the molecule. we have previously reported the efficient expression of the cardiac na+/ca 2+ exchanger in mammalian cell lines using the t7 rna polymerase-hybrid vaccinia virus system. we have now constructed the recombinant virus for the exchanger with a 6xhis-tag at the c-terminal. this tag has enabled the purification of the protein through a ni-nta agarose column. the expressed tagged protein induced na-dependent ca uptake which was not different from that in intact cells, i.e., cells in which the exchanger did not have the tag, the most important aspects of the purification produced and those of the reconstitution of the expressed protein will be described. $15-08 the neural cell adhesion molecule l1 is involved in the formation and specification of cell contacts in the central and peripheral nervous system during development, and thus may also play a role in synaptic plasticity of the adult central nervous system, using extracellular recordings of evoked field potentials in the rat hippocampal slice, we found that locally applied polyclonal anti-l1 and fusion proteins containing the ig-domains i-vi of l1 specifically block the stabilization of ltp in the ca1 region. baseline synaptic transmission was not affected by the presence of the anti-l1 antibodies. the anti-ll-induced effect was dependent on the antibody concentration and anti-l1 antibodies had no effect on previously established ltp. ltp was also reduced by an antiserum against the recombinantly expressed ig-domains i-vi of l1, whereas controt antibodies against liver cell membranes, which bind to neuronal membranes in the hippocampus, exhibited no effect on ltp. the contribution of l1 to stabilization of ltp within the ca1 synapses suggests that members of the ig-supertamily of cell adhesion molecules are involved in the structural modifications which are associated with synaptic plasticity in the mammalian central nervous system. an increasing number of surface proteins are described as being attached to the membrane by a glycosyl-phosphatidylinositol (gp1) anchor instead of the conventional transembranous hydropbobic domain. they are initially synthesized with a coohterminal polypeptide extension which is cleaved in the e.r. and replaced by the preformed glycolipid. this processing event is directed by a signal, located in the c-terminal region of the protein, which requires a group of three small amino acids positioned 10-12 residues upstream of an hydrophobic c terminal sequence. we previously transformed the transmembfanous glycoprotein d of herpes simplex type i into a gpi-anchored molecule by deleting its cytoplasmic domain and thus creating a molecule, gdww63, which meets the requirements for anchoring via a gpi structure. we now demonstrate, using gpi-deficient cell lines and biochemical studies, that a hybrid protein consisting of the thy-i ectoplasmic domain and of the c-terminal region of gdww63 can be alternative[y expressed in a gpi-anchored or in a transmembranous form. service de p6diatrie, chuv, ch-1oll lausanne proteolipid protein (plp) is a major myelin protein in the central nervous system (cns). a number of x-linked dysmyelinating disorders have been identified as mutations in the pip gene. we have identified a novel plp mutation in paralytic tremor (it) rabbit, characterized by body tremor, spastic limb paresis and hypomyelination in the cns. reduced levels of plp protein and its mrnas were observed in the cns as well as in the pns. plp sequence analysis revealed a single nucleotide change in exon 2 which results in the substitution of a histidine by a glutamine at position 36. histidine 36 is localized on the boundary between the first transmembrane region and the intracellular part of the protein. therefore, its position can be crucial for the efficient plp interaction with other proteins and lipids, and correct incorporation of the plp molecule into the membrane. histidine 36 is also part of a short fragment of ten amino acids which is conserved in all species studied. the same amino acid is altered in another hypomyelinated mutant, shaking pup, stressing its functional importance. furthermore, the pt mutation affects the plp "premyelin" functions, including oligodendrocyte maturation. equilibrium constants for octamer dissociation as well as dissociation rates in vitro increased in correlation to the number of charged residues eliminated, e.g. mutant 4-7, in which all four charged residues in the n-terminal heptapeptide are substituted with uncharged amino acids, showed a 100-fold higher equilibrium constant for octamer dissociation and a 145-fold increased dissociation rate compared to wild type. this strongly suggests that the charged amino acids in the n-termlnal heptapeptide of mib-ck, and therefore an ionic interaction, play an important role in forming the oetameric molecule. gangliosides and neutral glycosphingolipids (gsls) cell surface molecules are involved in a variety of biological processes and are assumed to play an important role in the mechanisms of hiv entry into lymphoid and epithelial cells. the total lipid-bound sialic acid (lbsa) content and the composition of gangliosides and gsls were investigated on pbmn cells from hiv+ve and normal donors. lbsa level was quantified by a fluorimetric assay, while gangliosides and gsls were assayed with high performance thin layer chromatography (hf'tlc) after multistep lipid extraction of 100 0013 x g membrane fractions. in normal pbmn cells the lbsa content accounted for 2.6/~g/108 cells or 20 nmol/mg protein whilst in pbmn cells from hiv+ donors the lbsa level decreased to 1.6 ,ug/108 cells or 13 nmol/mg protein. the qaantitation of neutral gsls and gangliosides was carried out by scanning spots revealed on hptlc plates and the integrated areas computed with a dedicated software. our findings showed that the most relevant ganglioside present in normal pbmnc was gm3 (65-80 % of total gangliosides) followed by small amounts of od3 (5 -15 %) and other acidic glycosphingolipids. neutral gsls included gl1, gl2, gl3 and gl4-hexosylceramide. hiv+ pbmn cells were characterized by a decrease of neutral glycosphingolipids as well as gm3 content which represented only 55 % of the ganglioside fraction, indicating an alteration in the glycolipid composition in the plasma membrane of infected cells. the neuropeptide y (npy) is one of the most abundant peptides of the mammalian brain. upon stimulation of cell surface receptors, npy generates diverse physiologic responses. npy acts on the cardiovascular system. it is a vasoconstrictor by itself and in addition, it potentiates the action of vasoactive substances such as angiotensin ii or norepinephrine. npy also inhibits glucose induced insulin secretion and is a potent inducer of appetite. to facilitate the development of npy antagonists we are investigating the interaction of npy with its cell surface receptor.we first constructed a series of mutants in which negatively charged residues present in putative extracellular domains of the y1 receptor were systematically replaced by alanines. the mutant cdnas were transiently expressed in hela ceils using a vaccinia virus derived expression system and the abi}ity of the encoded proteins to bind npy was evaluated. the capacity of the mutant proteins to be recruited to the cell surface was assessed by confocal microscopy. we identified initially 4 spacially clustered extracellular acidic residues of the human y1 npy receptor essential for npy binding. subsequently, we mutated 36 additional residues. based on these data a detailed computer model of the interactions between npy and its receptor was built. functional activity to energy metabolism. l. pellerin and p.j. magistretti. institut de physiologie, universit6 de lausanne, switzerland. astrocytes play a central role in brain energy metabolism. for example glucose, the exclusive brain energy substrate, is avidly taken up by astrocytes (pnas 90:4042, 1993) . application of glutamate (glu), the main excitatory neurotransmitter, stimulates in a concentration-dependent manner 3h-2-deoxyglucose (2-19(3) uptake by astrocytes in primary culture with an ec50 of ~ 100 ~m. the effect is not receptor-mediated since it can not be reproduced by glutamatergic agonists such as (nmda, kainate, quisqualate, t-acpd) nor prevented by antagonists (apv, cnqx, ap3). instead, it involves glutamate uptake since the effect is blocked by dl-threo-j3hydroxyaspartate, a potent inhibitor of the glu transporter. replacement of na + in the medium by choline also prevents the effect, suggesting a na+ driven process. finally ouabain, a selective inhibitor of the na+/k + atpase, completely prevented the effect of glu. these observations suggest that when glutamatergic synapses are active, glutamate, taken up by astrocytes, stimulates 2-dg uptake by astrocytes whose end-feet are known to surround capillaries, i.e. the source of glucose. they also provide a simple explanation for the mechanism linking functional activity to energy metabolism. cloning and molecular characterization of the mouse astrocyte glycogen synthase. g. pellegri, c. rossier, s. van berchem, p.j. magistre~i and j.-l. martin. institut de physiologie, universit6 de lausanne, switzerland. in the brain glycogen is predominantly localized in astrocytes, although its presence has been detected in certain large neurons, in ependymal and choroid plexus cells. glycogen is synthesized by the enzyme glycogen synthase (glys) from udp-glucose. out of the different glycogen synthase isozymes, only the muscle and the liver forms of glys have been cloned. we have isolated and characterized from a mouse astrocyte ~.zapii cdna library, a cdna clone encoding the mouse cerebral cortical astrocyte glys. the 3.5 kb clone isolated contains an open reading frame of 2214 bp encoding a protein of 738 amino acids. the coding region of the mouse astrocyte glys cdna shares 87% and 86% identity with the nudeotide sequences of the human muscle and the rabbit muscle giys cdna respectively. the cellular distribution of the glys mrna studied by northern blot shows the presence of a single transcript of 4 kb in cultures of astrocytes and, to a lesser degree, of neurons. the distribution of glycogen phosphorylase, which was also examined by northern blot shows the presence of a 4.2 kb transcript both in cultured astrocytes and neurons with however higher levels expressed in astrocytes. the functional molecular size of the vacuolar h+-pyrophosphatase (h +-ppase ec 3.6.1.1) from maize (zea mays l.) roots was estimated by radiation inactivation, both for substrate hydrolysis and for proton transport. light microsomal vesicles enriched in tonoplast membranes were prepared from young (2 days) maize roots using sucrose step gradients. these vesicles were still competent for ppi-dependent proton transport after freeze-drying and rehydration of the membranes. frozen native or freeze-dried samples were irradiated with 6~ for various periods of time. after thawing the samples, the activities of glucose-6phosphate dehydrogenase (added as an internal standard) and h+-ppase (hydrolysis of pyrophosphate (ppi) and ppi-dependent proton transport) were tested. by applying target theory, the functional size for hydrolysis was found to be around mr 155 000 when the native or freeze-dried samples were irradiated. no ppi-dependent proton transport activity was measurable after irradiation of the native samples. on the contrary, the freeze-dried membranes were still pumping protons after irradiation and a target size of around mr 250 000 was measured for the transport activity. these experiments suggest that the native h+-ppase from maize roots may exist as a dimer (at least) of the mr 80 800 catalytic subunit. $15-20 the na,k-pump moves 3 na + out and 2 k + ions into the cells. the domains of the protein involved in ion translocation have not been determined. we have studied the role of the n-terminal region in the na + translocation by measuring the kinetics of charge movements under na/na exchange conditions in wild type (wt) and two n-terminally truncated forms of the bufo ~ subunit with deletions of the 31 (t31) and 40 (t40) first amino acids expressed in xenopus oocytes. we have developped a new technique to perform fast (< i ms) voltage clamp on whole oocyte. the membrane on one side of the oocyte is permeabilized by digitonin allowing to measure current flowing across the other half. we observed presteady state ouabain-sensitive currents similar to that reported by other techniques (j.gen. physiol. 101:117,1993) . the estimated forward charge translocation rate was lower in both n-truncated mutants (wt 430 â�¢ 14, t32 215 z 8, t41 124 z 3 s-l). the midpoint potential of the charge translocation (best fit to boltzman equation) was -79 , -45, and -28 mv in the wt, t31 and t40 groups, respectively. the highly charged nterminus of the q subunit has a significant role in the forward translocation of na + ions. deletion mutants of human small intestinal lactase-phlorizin hydrolase (lph) were constructed to determine the role of protein subdomains in the intracellular transport of lph. the mutant lph1646mact (236 aa deletions), which contains the membrane anchor (ma) and the cytoplasmic tail (ct), was transported to the cell surface in a fashion similar to wild type lph. by contrast, lph1646 lacking the transmembrane domain persisted as a mannose-rich polypeptidc. this suggests that the membrane anchor and/or cytoplasmic tail are implicated in the er-egress of lph. another mutant, lph1559mact (323 aa deletions), was not efficiently transported to the golgi apparatus. epitope mapping with monoclonai antibodies as well as protease-sens{tivity assays provided an evidence that the tertiary structure of lph1646mact is very similar to that lph1559mact. in view of the variations in the proportions of the complex glycosylated molecules of the two mutants we conclude that the domain between lph1646mact and lphi559mact may play an essential role along the secretory pathway of lph. containing tyrosine. hussein y. naim, and michael g. roth; dept, of biocherni~try, ut-southwestem medical center, at dallas, dallas,texas-usa we have investigated an artificial internalization signal created by site-directed mutagenesis of the short cytoplasmic sequence of the influenza virus hemagglutinin (ha). mutation of cysteine 543 to tyrosine converted ha from a protein essentially excluded from coated pits to one that internalized at rate similar to some cellular endocytic receptors. to determine whether or not the ha-y543 signal is a degenerate form of the internalization signal found in proteins such as the transferrin receptor and the mannose-6-phosphate/igfii receptor, we have mutated amino acid positions in the ha-y543 shown to be important for internalization of the two receptors. we have changed amino acids on either side of y543 to those that either fit, or break the pattern of aromatic and hydrophobic residues proposed as consensus sequence. our results indicate that the ha-y543 mutant contains a sub-optimum sequence for a tyrosine-based internalization signal similar to those found in the receptors for transferrin, ldl, and mannose-6-phosphate/igfil. however, amino acids with side chains having different chemical properties functioned well in positions that are important for the internalization signal, indicating that the consensus sequence for this signal is more variable than previously proposed. n-linked glycesylation is an essential protein modification occuring in all eukaryotic cells. core-oligosaccharides are transferred by the enzyme n-oligosaccharyl-transferase frc~ the donor glc man gicnac -dolichol to selected asparagine 9 2 . resldues of t~e nascent polypeptlde chazn. the donor is build up in a stepwise fashion at the er-membraneby the products of the alg-genes (asparagine-linked-~lycosylafion) mutants defective in this pathway are available, but often lack a phenotype suitable for isolation of the corresponding genes. we found that double mutants of alg5 or alg8 with a wbpl mutation (defective oligosaecharyl-transferase) are severely growth retarded at 30oc. the double mutant phenotype allowed the isolation of both alg5 and alg8 genes which have been refractory to cloning so far. both genes encode potential trarsmenbrane proteins and are able to complement the biochemical defects of the corresponding alg5 or alg8 mutants. the na,k-atpase produces the driving force for the regulated transport of na across epithelial ceils. it is composed of an c~-subunit which carries the catalytic and ouabain binding sites and a b-subunit. to test the role of the usubunit in transport regulation, we transfected a6 cells with the b. marinus al-subunit (txl-tbm). this subunit confers a 300-fold higher resistance to ouabain (k i = 50/~m) compared to the endogenous pump. taking advantage of this difference, stable transfectants were selected with ouabain. expression of the cd-tbm subunit was visualized by western blotting. approximately half of the positive cell lines showed a high electrical resistance similar to untransfected cells, when cultured on filters. na-transport activity, measured as isc, was lower even in the absence of ouabain, but the relative increase in response to aldosterone was maintained. functional pumps containing the exogenous or endogenous al-subunit were quantified by equilibrium ouabain binding. interestingly, cells cultured on plastic dishes had a large fraction of low affinity binding sites (txi-tbm), while most sites were of the high affinity type when the cells were grown on filters. we conclude that the ouabain-resistant na,k-atpase cd-subunit of b. marinus can be expressed in a6 cells and used as a selective marker. however, the results suggest that the expression of functional ouabain-resistant pumps containing the al-tbm subunit could be influenced by the degree of cellular differentiation. in infected cells fully functional na/pi-cotransport was found in a time dependent manner; up to a 50-fold stimulation of na/p;-cotransport compared to uninfected cells was observed aft6r 3-4 days. transport of phosphate by infected cells was highly dependent on the presence of sodium, exhibited a kmtd~ of around 0.16 mm and was dependent on extraceuular i~h'. in agreement with expressed transport of phosphate, infected cells produced an immunoreactive polypeptide of 66 kda that represents a non-glycosylated form of the 80 to 90 kda mature na/p;-cotransporter. we conclud$ that the protein napi-2/rat when expressed in sf9 cells exhibits na/p-eotransport with similar kinetic characteristics as observdd in prox ma tubular apical na/pcotransport. therefore the napi-2 protein as expressed h sf9 cells can be used for further structural and functional studies. talin interacts in vitro with actin, vinculin, integrins and bilayers of acidic phospholipids, and nucleates actin filament growth at the bilayer surface. it is therefore believed to be a key protein mediating aetin-membrane linkage. we have analyzed functional properties of proteolytic fragments of purified platelet talin. using limited proteolysis two fragments of 47 and 190 kd were generated. preliminary results, obtained using viscometry and f-aetin-nucleating assays, suggest that the 190 kd fragment contains the actin binding site. we also assessed the lipid-binding capacity of the fragments. when a mixture of the two fragments was centrifuged in the presence of large multilamellar liposomes containing phosphatidylserine, only the 47 kd fragment, but not the 190 kd domain, co-sedimented with the liposomes, suggesting the presence of a specific lipid-binding site in the 47 kd domain. interestingly, this fragment contains the n-terminus of talin which shows homologies to the membrane-binding regions of protein 4.1. we are now analyzing membrane-binding properties of talin domains in more detail using hydrophobic photolabelling. recent studies suggest a role of the neural cell adhesion molecules l1 and ncam in mechanisms of memory formation (doyle e, et al, j. neurochem. 59:1570 -1573 , 1992 scholey a.b. et al, neuroscience 55:499-509, 1993; lijthi a. et al. unpublished data and see usgeb 94) . in the present study we analyzed the effect of chronic intraventricular infusion of polyclonal antibodies against l1 (anti-u) or ncam (anti-ncam) on the performance of male wistar rats during the acquisition and retention of a spatial learning task. briefly, rats had to remember the position of a submerged escape platform in a water tank (morris water-maze). when the escape platform was removed after acquisition training (= retention test), the performance of animals chronically injected with anti-l1 showed an impaired search pattern when compared with that of salineqnjected animals (p>0.05, mann-whitney). whereas control animals spent up to 45% of their time searching for the platform in the correct (= training) quadrant, the time anti-l1 animals swam in the correct quadrant was closer to chance level (31%). although monitoring penetration of antkncam into the hippocampus was almost as efficient as that of anti-l1, the performance of the anti-ncam-group was highly variable and did not differ from the performance of controls. these results agree with recent findings on the involvement of neural cell adhesion molecules in other forms of learning. we have isolated cdnas from mouse and from xenopus encoding the ~-subunit of the gastric h,k-atpase, which is expressed in the parietal cells of the stomach mucosa. the gastric h,k-atpase transports h + into the stomach lumen with the counter transport of k +, all at the expense of atp hydrolysis. when xenopus oocytes are injected with crna encoding ~h,k from either species, as well as a gastric ~h,k subunit, the two subunit proteins are synthesized, assemble, and form functional pumps located in the plasma membrane as demonstrated by 86rb+ uptake. the sensitivity to the gastric h,k-atpase inhibitor sch28080 was determined, with the ki for beth species found to be in the low ~m range. spodoptera frugiperda ceils are often used to overexpress eucaryotle proteins using baculovirus vectors. the atomic force microscope (afm) is a device which allows the observer to obtain high resolution images of biological samples immersed in a fluid medium; this instrument has therefore the potential to visualize overexpressed proteins on the surface of living cells. however, the interpretation of the images is sometimes difficult and requires a good knowledge of the specimen topography, i.e. the "normal" plasma membrane of the cell used for the overexpressisn. on our poster we present afm images of the plasma membrane of both fixed and living spodoptera frugiperda ceils. in additon, we discuss the possibility of achieving high resolution in the imaging of dynamic changes which affect /iving systems. the deduced protein sequence consists of 295 amino acids with about 55% amino acid sequence identicy when compared to mammalian ~h,k-subunits. data from other species and from the structurally similar na,k-atpase has shown that the ~-subunit is necessary for the formation of a functional pump, which consists of an ~/~ heterodimer. following co-injection of xenopus oocytes with crna for both the ~-and ~subunits, we have observed by rb + uptake h,k-atpase activity in the plasma membrane. we are presently studying the role of the ~-subunit in the maturation and function of the h,k-atpase. the atomic force microscope (afm) is a new type of microscope which allows visualisation of inorganic and organic samples with a very high resolution. a sharp tip fixed at the end of a cantilever is used as a topographic sensor. by scanning the sample with this device, a computer records the minute deformations of the cantilever and computes the topography of the sample. the instrument allows also the measurement of the interaction between the tip and the sample as a function of the distance. the knowledge of these interactions allows us to compute several mechanical properties like indentation, elasticity and stiffness. on this poster we report the results of these measurements on cells (sfg, c131), bacteria (e. coli, b. subtilis) and proteins (actin). in addition we discuss the use of the afm as a sensor for probing the mechanical properties of biological systems at a nanometric scale. neonatal thymectomy of balb/c mice induces aig. we have cloned the ~-and ~subunits of the balb/c h,k-atpase and expressed the proton pump in crna injected oocytes. both and ~ proteins are made, form functional pumps, and are immunoprecipated using auto-immune sera. the ~-subunit can also be in~unoprecipated independent of the ~-subunit when oocytes are injected with ~ crna alone. balb/c mice will be immunized with oocyte membranes containing either the ~/~ or ~ antigens in order to study the role of each h,k-atpase subunit in triggering aig. the prion protein is expressed in both astrocytes and oligodendrocytes of the neonatal brain and is down-regulated in adulthood. m. moser, colello, r, , pott, u, and b. oesch institut for hirnforschung der universit~t zorich, august forel str. 1, 8029 zorich the infectious agent causing transmissible spongiform encephalopathies consists largely or entirely of prp sc, a modified isoform o| normal prp (prpc). prpc is most abundant in the brain. the development of the disease strictly depends on the presence of prpc. and incubation times are shortened by overexpression of prpc. the normal function of prpe is not known and its cellular iocalisation in the cns is poorly characterised, except for its evident presence in neurons. here we describe the expression of prp c mrna in both oligodendrocytes and astrocytes during neonatal development of hamster and rat. in adult animals, expression is down-regulated in gila but not in neurons. our findings provide a link to the previously described accumulation of prpsc in astrocytes and the apparently faster course of prion disease in neonatal hamster. our results argue for a major involvement of non-neuronal cell types in prion diseases. such structures we raised mab against membrane protein fractions. a ~lab against a vitronectin receptor containing fraction was found, which if applied in vitro to bi6fi mouse melanoma cells, influenced growth, adhesion and morphology. ~tnen bi6fi cells prior to tail vein injection were treated, 2 different outcomes were observed. short treatment (lh) resulted in 85% inhibition of lung colonization whereas long-ter~ treatment ( 2weeks) resulted in 10-15 times more lung colonies. a candidate molecule likely involved in these observed effects could be identified as a 150 kda cell surface protein. sequence information from peptides revealed in several cases 100 % homology to mouse centrosomin a, i.e.a 32 kda cytosolic protein involved in the organization of the spindle apparatus. these data suggest that we may have found a protein which may participate in a link between the extra-cellular space and the microtubular system. we are currently trying to clone this 150 kda protein from a bi6fi c-dna library. wahlberg, j.m., and spiess, m. dept.biochemistry,biozentrum, university of basel. signals for correct basolateral sorting of subunit hi of the human asialoglycoprotein receptor are located in the cytoplasmic tail, involving a tyrosine at position 5. removal of this residue results in nonpolarized transport of the protein to the surface in mdckii-celis. a deletion mutant of hi, lacking 30 of the normal 40 residues of the cytoplasmic tail, including the tyrosine, has been found not to be transported to the cell surface, but to be accumulated intracellularly. the mutant protein is complex glycosylated and sialylated, indicative of passage into or through the trans-golgi. immunofluorescence analyses show defined juxtanuclear, tubular structures, which do not colocalize with markers for er, early endosomes or lysosomes, but which show similar staining pattern as markers for late endosomes and tgn. to define the determinants responsible for the intracellular accumulation a series of deletions and fusions have been constructed. the results suggest that the length of the cytoplasmic domain is one important determinant for missorting of hi. salerr~ n., medilansld, j., pellegrinelli, n. and eder-colli, l. departement of pharmacology, cmu, 1211 geneva 4 in chollnergic neurons the enzyme choline acetyltransferase (chat) exists as cytosolic(80-90%) and membrane-bound activity (10-20%}. are these two activities encoded by a single mrna? is membranebound enzyme preferentially associated with a given cellular membrane? in order to investigate these questions we isolated a fulllength cdna (4.2 kb) encoding drosophila chat and used it to transfect xenopus oocytes, rat fibroblasts and human neuroblastoma cell lines. triton x-114 fractionation of transfected cells showed that hydrophilic and amphiphilic chat activity were produced. the sp act. of hydrophflic enzyme reached 2, 0.8 and 0.25 ~mol ach/h/rng protein respectively in oocytes, fibroblasts and neuroblastoma whereas the sp. act. of amphiphilic enzyme was 1.8, 0.4 and 0.125 hinol ach/h/mg protein, respectively. amphiphilic chat was less sensitive to inhibition by the product acetylcholine and to heat denaturation than was hydrophilic enzyme. similar results were observed for the native drosophila chat. amphiphillc enzyme sedimentation was affected by the type of detergent present in linear density gradients of sucrose. transfected oocytes were subjected to subfractionation. besides a large amount of soluble chat activity, a significant proportion of enzyme was found associated with a fraction enriched in endoplasmic reticulum (er). preliminary results using transfected mammalian cells indicate that apart cytosolic chat, plasma mernbranes+golgi enriched fractions as well as er-enriched fraction contain chat activity. we have characterized a drosophila melanogaster gene encoding a very hydrophobic protein. the amino acid sequence shows considerable homology (33-42% identity) to neurotransmitter transporters. the gene is, however, not expressed in neural tissue, but in the male germline. transcription and translation occurs in early spermatocytes. antibody staining data suggest the following pathway of the protein during spermatogenesis: the protein is synthesized in early spermatocytes and is transported into the nucleus during prophase of rt i. at meiosis it associates with the mitochondria and it stays associated while they fuse to the nebenkern. during individualization it is stripped off in the cystic bulge. inspection of the putative protein sequence shows that it possesses alle the necessary targeting signals to allow its transfer from the cytoplasm through the nucleus into the mitochondria: three nuclear targeting signals, a mitochondrial import signal and, moreover, two pest-sequences for rapid protein degradation are present. we shall present a, currently tested, working hypothesis. in hg-treated toad skins, water permeability is high or low depending on whether the major anion of the inner bathing medium is so~ or ci. we report here the results of experiments designed to determine if, in skins bathed with so4-ringer, net water flow (j~,) could be diminished by agents added to the outer medium. toad skins were mounted inbetween glass chambers and exposed to a standard osmotic gradient of 200 mosm. jw was continuously monitored by automatic, volumetric techniques. exposure to lmm hgclz or chzcihg (external side) led to the typical anion-induced j,, changes. re-exposure to hg caused a mild (10%), although significant (n=8, p<0.001), fall in jw. since cuso4 is a sh-reagent like hg, we looked at the effects of cuso4 (lmm). there was a 68% reduction of j, (n=7, p<0.001), an effect totally reversible by simple washing of the skins. likewise, addition of dithiothreitol (dtt, 5mm) in the presence of cu, caused a 90% reversal of the cu effect ; there was no change in jw if dtt was given before cu, or if both agents were added simultaneously. finally, in hg-treated, glutaraldehyde-fixed skins, lmm and 5ram cuso4 caused 51% and 92% jw decrements, respectively, that were totally reversible. in conclusion, cu causes a dtt-sensitive inhibition of jr, in hgtreated skins. further work is needed to establish if the cu effect is exerted on sh-groups of apical aquapories of toad skin. $15-44 the amino acid sequences of the pc-645 e-subunits and other cryptomonad biliprotein ~-subunits pose the most intriguing question about the phylogenetic origin of these unique chromopeptides. cryptomonad pc-645 ~-subunits seem not to be closely related to the known phycobiliproteins, linker polypeptides (lpps), bpe or any other light-harvesting chl polypeptides from the thylakoid. for most of them the number of identical amino acid residues was 15%-20%. sequence alignment of c-pe associated lpps with t-subunits from cyanobacteria and red algae and the cryptophytan ~-subunits however show that the identity between c-pe associated lpps and cyanobacterial x-subunits is still significant, whereas it reaches the limit of significance between cyanobacteril and eucaryotic x-subunits. nevertheless it has to be assumed that x-subunit derive from cyanobacterial c-pe associated lpps as well as cryptomonad ~-subunits may derive from t-subunits. whereas the the phycobiliprotein ~-and 8subunits remained structurally conservative during evolution (e.g. -70% identity between cyanobacterial and red algal pe-subunits) the lpps and t-subunits show drastical changes in their chromophore binding domains. $15-45 functional and evolutionary relationships of the components of the primary events in photosynthesis have been traced using amino acid sequence comparison. basically, the question was posed regarding the interrelationship of the apoproteins of different photosynthetic organisms which gather light and/or separate charges across membranes. do their protein-chemical structure and organisation decipher us certain clues and parts of the path by which they have been selected and derived from a possible primordial reaction centre polypeptide? within that context the light-harvesting-and energy transfer systems of purple bacteria offered as ideal model components. a data set of more than 70 apoprotein sequences has pointed to some keystructural elements which have been partially verified as such by limited proteolysis and genetic engineering. a careful inspection of the determined bacterial antenna-structures revealed some remarkable similarities to eukaryotic antenna and reaction centre apoproteins indicating possible basic structural motifs for complexing pigment molecules, like chlorophylls or bacteriochlorophylls in very distant representatives of phototrophs. a number of glycoproteins of the nervous and/or immune system that are involved in cellular recognition and/or adhesion share the common lz/hnk-1 carbohydrate structure. in a large proportion of patients with peripheral demyelinating neuropathy associated with paraproteinemic gammopathy (ppn), monoclonal igm antibodies (m-igm) react with the lz/hnk-1 determinant of mag and p0. we plan to test the hypothesis that anti-mag m-igm autoantibodies may alter the expression of hnk-1 bearing myelin proteins such as mag, p0, mog, ncam or pmp22. by using monoclonal antibodies to myelin proteins on tissue sections, preliminary results indicate that mbp protein content seems to be strongly downregulated, whereas galc and $100 protein do not appear to be affected by the loss of myelin in ppn nerves. gfap protein expression appear to be upregulated in demyelinating biopsy specimens from ppn patients with anti-mag m-igm gammopathy. in conclusion, the expression of some l2/hnk-1 negative proteins might be disregulated by the presence of circulating anti-mag m-igm in the serum of ppn patients. we have isolated a human liver na+-taurocholate cotransporting polypeptide (ntcp) by screening a human liver edna library with a rat edna probe derived from ntcp (pnas 88, 10629, 1991) . the cdna codes for a protein of 349 amino acids which shows 77% amino acid homology with the rat ntcp. expression of ntcp in x. laevis oocytes resulted in na+-dependent bile acid uptake which exhibited saturability with an apparent km for taurocholate of 6 ~m. ntcp mediated taurocholate uptake into oocytes was inhibited by all major bile acid derivatives, bumetanide and bromosulphophthalein. southern blot analysis of genomic dna from a panel of human/hamster somatic cell hybrids mapped the human ntcp-gene to chromosome 14. in conclusion, these studies supplement the previously suggested selective mammalian distribution of the ntcp gene family and provide the possibility for direct molecular characterization of human bile acid transporter genes. diverse cell surface molecules of the nervous system play an important role in specifying ceil interactions during development. with the aid of a polyclonal antibody against l1, a 1.skb edna clone closely related to l1 (ch-l1) was isolated from a ~. gtl 1 library derived from poly(a)* l~qa of day 8 mouse brain. using this edna as probe the remaining cdi'ia 5' sequences were cloned out of a plasmid library constructed from postnatal day 6-14 mouse brain poly (a)+ p, na. two independent full length clones of 4.2 and 4.4 kb encompassing the entire coding region of chl] were isolated. these represent putative alternatively spliced mp, nas. the deduced primary structure of chl1 reveals that, like the cell adhesion molecules (cams) mouse l1, chicken ng-cam, and nr-cam (bravo), chl1 is composed of six ig-like domains, a transmembrane domain, and a short cytoplasmatic region. in contrast to the other l1 family members, only four and a half instead of five fibronectin type hi-like repeals are found in chl1. the fibronectin type [h-like repeats were expressed in e. col[ and the protein fragment was used for immunization. as observed for li, ng-cam, and nr-cam, chl1 is susceptible to proteolytic cleavage. bands of 185kd, 165kd, 125kd, and 50kd could be detected bywestern blot analysis. by in situ hybridization, a predominant expression by neurons was found in the adult mouse brain. western blot analysis showed expression of chl1 protein in brain and heart, but not lung, kidney and intestine. in liver, a 50 kd immunoreactive band was found. the relationship of chl1 to molecules known to be involved in cell adhesion and neurite outgrowth, combined with its pattern of expression, suggests a role for this molecule in cell interactions dndng neural development. work from this laboratory revealed that in hg-treated, glutaraldehydefixed toad bladders, the water permeability coefficient (pf) is high in sod-ringer and low in ci-ringer ; in all these studies the osmolality of the serosal medium was 220mosm. as an attempt to determine the mechanism(s) underlying such pf changes, experiments were carried out with bladders whose serosal surface was exposed to [so-, hypo-or hyperosmotic media ; the outer surface was always exposed to a hyposmotic solution (22 costa). net water flows were measured continuously with automatic, volumetric techniques. after exposure to 1 mm chzcihg (10', outer medium) followed by fixation with 0.25% glutaraldehyde (5', serosal medium), the bladders were thoroughly washed. in sod-ringer, pf (/~m/sec) was as follows (n=6) we wanted to determine the molecular weight (mw) of ntcp and its topology in the plasma membrane (pm) of rat liver. a rabbit antiserum generated against the c-terminus of ntcp was used to determine its mw on western blots and its subcellular distribution in liver and in hepatocytes by immunofluorescene (if). site directed mutagenesis and deletion experiments were used to determine the n-linked glycosylation sites. ntcp encodes a 50 kd protein in rat liver pm vesicles. the basolateral localization of ntcp in liver was confirmed by if. ntop could only be immunostained in permeabilized hepatocytes suggesting an intracellular localization of the cterminus, cnda constructs showed the ntcp is glycosylated at positions 5 and ii. the data show a selective basolateral expression of ntcp in rat liver and they suggest that the two ends of ntcp are located on opposite sides of the pm. zymogen granule membranes (zgm) are known to contain proteins with nucleoside phosphatase activity, but their exact nature and function are unknown. the activities require ca ++ and mg ++ and are sensitive to atpase inhibitors but not to inhibitors of na/k-atpase activity. the aim of this study was to isolate individual proteins of pig pancreatic zgm, to attribute enzymatic activity to individual proteins and to characterize them with various substrates (gtp, atp, amp etc.), inhibitors (amp-cp, amp-cpp, gtpgs etc.) and lectins (maa, dsa, gna etc.). we have subjected highly purified zgm solubilized in chaps to ief on immobiline dry strips | (pharmacia) with s ph range 3--10.5 (ist dim.). these strips were then electrophoresed in a polyacrylamide gradient gel containing 0.2% chaps and tris/hci at ph 9.5 (2nd dim.). nucleoside phosphatase activity was detected histochemically in the 2d gel by incubation with substrate in the presence of lead nitrate. focussed strips were also subjected to sds-page for comparison. several proteins showed strong activity to gtp, atp and itp. the role of these phosphatases are discussed in the context of the role of gtp-binding proteins and the de-/phosphorylating events on the zgm in intracellular targeting and exocytosis. the disease-specific isoform of the priori protein (prp sc ) is an essential part of the infectious particle causing scrapie or bse. prp sc differs from prp of normal animals (prp c ) by its relative protease resistance. the physical nature of this difference is still unknown. here we analyzed the protease-resistance of prp sc quantitatively. prp sc was rendered completely protease-sensitive at alkaline ph or in guanidinium thiocyanate (>1.5 m). denaturation in 2m guanidinium thiocxanate (gdnscn) completely abolished protease resistance of prp bc within 15 min while denaturation in 6 m urea showed a much slower time course. denaturation cuwes were used to calculate the gibbs free energy (as d ) in dependence of gdnscn or urea at different concentrations. the linear relationship between agd and the denaturant concentration is suggestive of a two state model involving the conformational change of a single protein domain. this is also reflected in the small number of side chains (11,6) additionally exposed to the solvent upon conversion of prp sc to its proteasesensitive isoform. our results suggest that minor rearrangements of the structure of prp sc are sufficient to abolish its protease resistance. of physiology, university of zurich, winterthursrstr. 190, ch-8057 zt~rich in contrast to mammalian kidneys where inorganic phosphate (pi) is reabsorbed unidirectionally, flounder renal epithelial cells both re.absorb and secrete pi. in order to investigate on a molecular level the cellular pi handling we have cloned a re,absorptive nalp i transport system from flounder renal tubules. based on homology with the cloned na/p i cotransporter from rat we have isolated a edna clone (flounder napi-ii, 2424 bp) encoding for a protein of 637 amino acids. in the eight transmembrane spanning domains the protein is 78% identical with the corresponding system from rat kidney, in the hydrophilic regions only 30%. expression of in vitro transcribed rna in xenopus oocytes specifically stimulated na-dependent pi transport. binding properties of na and pi as well as the ph-dependency were similar to the ones found in mammalian systems (rat, human, ok cells). by northern analysis three transcripts, 1.9, 2.7, and 4.2 kb in length, could be detected. rna from flounder renal tubules contained all of the transcripts, intestinal rna only the two lower bands, and non-epithelial tissue from kidney expressed only the 1.9 kb transcript. mammalian systems share only the 2.7 kb transcript but not the related species. the close functional relationship of flounder napi-ii with the known najp i transport systems and the pronounced differences in primary structure provide the basis for structure function analysis of na/p i cotransport. the amyloid 13/a4 protein precursor (app) is a transmembrane protein expressed in different isoforms throughout the organism. our work has consisted in a detailed study of the structure and biological function of app. specifically, we have shown that the secreted form (sapp) of app-695 is involved in the growth regulation offibroblast% and also stimulates neurite extension in b 103 neurnblastoma cell line. functional mapping studies have shown that the domain responsible for both the growth-regulating and the neurotrophic activities of sapp-695 is the rerms site, arg-328 to ser-332. the presence ofsapp binding sites on the surface of b 103 ceils (through the active rerms site) indicate that the neurotrophic activity of sapp is mediated by a receptor, and the subsequent activation of a signal transduction mechanism. in addition, we have shown that the rerms site is also involved in the neuronal survival properties of sapp-695. finally, we have initiated an in vivo study of app biological function, and shown that a peptide representing the neurotrophic domain ofsapp-695 can strengthen memory retention in rat through stabilization of synaptic structures in the frontal cortex. this work was supported by grants from the swiss national science foundation (823a-028366), and nih (ag 05131). we previously showed that in smooth muscle cells, the (~i-ar is rapidly phosphorymted following exposure to agonist. to understand this biochemical event, we investigated the phosphorylation of the c(1b adrenergic receptor stably expressed in rat1 fibroblasts (3.3 pmol/mg prot.). the =(1b-ar was solubilized from 32p-labeled rat cells and immunoprecipitated with antibodies raised against the n-terminus part of the receptor. treatment of ceils with 100 ~.m epinephrine showed a rapid and significant increase in receptor phosphorylation above basal. treatment of cells with a specific pkc inhibitor (ro-318220) did not affect agonist.promoted phosphorylation while it completely abolished phorbol esterinduced receptor phosphorylation. this strongly suggests that pkc is not involved in agonist-induced c{1b-ar phosphorylation. the elb-ar contains several putative phosphorylation sites in both its third intracellular loop and c-terminus tail. to investigate the role of these domains, a trunoated receptor (lacking its last 147 amino-acids) was constructed and stably expressed in rat cells (1.3 pmol/mg prot). this receptor mutant displayed similar ligand-binding properties and abirity to activate phospholipase c as compared to the wild-type receptor. phosphorylation experiments revealed that this mutant is not at all phosphorylated, neither by agonist nor by phorbol esters. agonistinduced decrease in cell surface receptors, measured by binding of the antagonist 3h-prazosin at 4~ was however similar for both the wild-type and mutant receptor. this suggests that loss of phosphorylation sites does not affect receptor internalization. human intestinal lactase-phlorizin hydrolase (lph) is synthesized as a precursor molecule (pro-lph), which undergoes proteolytic processing during maturation. lph expressed in cos-1 cells was enzymatically active, the electrophoretic properties of lph-species synthesized by transfected cos-1 cells correspond to those in organ cultured human intestinal explants, in caco-2 ceils and in transfected mock ceils. they included the pro-lph and a proteolytically processed form, which had similar electrophoretic properties to the mature enzyme. the appearance of the putative mature form was inhibited by brefeldin a, ammonium chloride and chloroquine. in addition, only complex glycosylated pro-lph (pro-lphc) was inserted into the cell surface membrane. these data indicate that in cos-1 cells pro-lph is inserted into the cell membrane, is internalyzed and enters lysosomes where proteolytic events lead to the appearance of a mature-like enzyme. $15-59 assembly and transport of paba peptide hydrolase alpha and beta subunits in cos-1 cells eldedng, j.a., gr0nberg, j. and sterchi, e., institut for biochemie und molekularbiologie, universit&t bern. paba peptide hydrolase (pph) (ec 3.4.24.18), a metalloendopeptidase from epithelial cells of human small intestinal mucosa, consists of two subunits, ~ and 13, which form homodimers and oligomers, cdna cloning has revealed homologies with developmentally important proteins from different species and has led to the definition of a new family of metalloendopeptidases, the astacin family (j. biol. chem. 266, 21381, 1991) . here, we report on the expression of the cdnas of pphcz and pphj3 in cos-1 cells. when expressed on its own, pph(z is synthesized in a core glycosylated form only, suggesting that it is transport-incompetent and not able to exit the er. immune-fluorescence studies have confirmed that pph~ is not expressed on the surface of cos-1 cells. pphl3, on the other hand, matures and is transported to the cell surface. when the two subunits are coexpressed, ppho~ can also be detected on the cell surface, suggesting that a heterooligomeric assembly is necessary for the surface expression of pphtz. truncated forms of both pphc~ and pphi~ could be detected in the medium of transfected cos-1 cells. in our previous work, we demonstrated by immunocytochemical reaction that sensory neurons expressed nuclear triindothyronin receptors (nt3r) in embryonic as well as adult rats. in contrast, in sciatic nerve, schwann cells exhibited only transient nt3r immunoreactivity from e17 to postnatal day 10. in the present work, we attempt to demonstrate by radioautography that the detected nt3r are effectively the binding sites of [125i] labeled triiodothyronin. cryostat sections of dorsal root ganglia (drg) or sciatic nerve were incubated with 0.1 nm of l, 3,5,3'-[t25i]triiodothyronin 2200 ci/m mol (nen), while the controls were incubated with a large excess (llxm) of unlabeled trfiodothyronin (t3). in aduit rat drg, radioautographs revealed that silver grains were exclusively restricted to neuronal cell bodies, while the schwann cells ensheathing the axons were free of significant label. in newborn rat sciatic nerve, silver grains accumulated over schwann cells; in contrast in adult rat sciatic nerve, schwann ceils were free of label. in control drg or sciatic nerve sections incubated in large exceas of unlabeled "1"3, all the radioautographs exhibited only scattered silver grains. in conclusion, our results show that sensory neurons and schwann cells are able to express functional nt3r which bind thyroid hormones (snf 31-33671-92). characterization of the maturepreeursor glycophospholipid for glycosylphophatidylinositoi anchors of saccharomyces cerevisiae. many attempts to isolate the mature yeast precursor gpi glycophospholipid ready to be attached to newly translated proteins for gpi-anehoring have failed although such precursors were previously identified in mammalian cells and protozoa. here we show that metabolically labeled glycolipids of the expected structure can be observed if the incorporation, their accumulation and their extraction are optimized. two very polar, (3h)-mannose-labeled glyeolipids named cpi and cp2 were identified and purified. they were structurally characterized using phnspholipase treatments, partial deacylation with methanolie ammonia, hydrofluoric acid dephosphorylation, nitrous acid deamination, acetolysis, exoglycosidase treatments and combinations thereof to produce labeled fragments which could be analyzed by paper chromatography or thin layer chromatography. cp1 and cp2 both contain the identical core oligosaccharide mangl,2(x->po4-6)manal,2mamxl,6(y->)mana-glcn-lansitol, x and y being hydrofluoric acid-sensitive substituents (most likely ethanolamine and phospheethanolamine, respectively). the inositol is acylated although no acyllaositol is present on protein-bound yeast gpi-anchors. cp1 and cp2 can also be observed after lableling with (3h)myo-inositol, the lipid moieties of cp1 and cp2 can be completely removed by mild alcaline hydrolysis altough the protein-bound gpi-anchors made by the pmi210 cells under identical labeling conditions are completely mild base resistant. this finding reinforces the notion that the ceramide moiety typically found on the majority of yeast gpi-proteins are added only after addition of the gpi precursor glycolipid to proteins. the distributions of the mannose-6-phosphat e/igfll-recept or and a2,6slalyltransferase in hepg2 cells: no evidence for co-localization by confocal laser scanning microscopy. the organization of the trans golgi apparatus (ga) with respect to (recycling) man-6-p/igfii receptor (mpr) and (resident) c~2,6sialyltransferase (st) has been investigated by double immunofluorescence laser scanning confocal microscopy in hepg2 cells. they were chosen because biochemical evidence suggested sialylation of mpr upon recycling (duncan & kornfeld, j. cell biol. 1988, 106, 617-28) implicating colocalization of both proteins. in the steady state st was confined to the ga whereas total (endogenous) mpr was localized to coarse vesicles without evidence for colocalization by confoeal microscopy. endocytosis of surface-labeled mpr was monitored in presence and absence of brefeldin a (bfa): without bfa, early endosomes (2' of warming up) showed no, late endosomes (20') little if any colocalization with st. in presence of bfa, the st-compartment disappeared whereas late endosomes persisted with some tubular emanations. under similar conditions, in "absence of bfa, endogenous mpr concentrated in the ga region suggesting colocalization with st. in presence of bfa the mpr compartment formed a redculum whereas the st compamnem disappeared. in conclusion, within the limits of the techniques used, exogenous mpr was not detectable in the st compartment whereas endogenons mpr concentrated in the ga region but obvious co-localization was exceptional. supported by grant 31-30757.91 of the snsf to egb. cultures of dissociated striatal neurons prepared from fetal rats were grown in the presence of neurotrophin-4/5 (nt-4/5) as well as the other known neurotrophins, ngf, bdnf and nt-3. we found that acute administration of nt-4/5 to seven days old cultures stimulates the hydrolysis of phosphatidyllnositol, an event involved in neurotrophin signal transduction. growth of stdatal cultures in presence of nt-4/5 resulted in increased cell survival as indicated by elevations in total cell number, protein content and number of viable cells. nt-4/5 exposure increased gaba uptake and staining intensity in gaba immunocytochemistry in these cultures indicating atrophic action on gabaergic neurons. to further identify responsive cell populations we analyzed for calretinin, a calcium binding protein known to colocalize with gaba in a number of neuronal cells. nt-4/5 strongly increased the number of calretinin positive cells in cultures prepared from rats of embryonic day 15, as we]l as calretinin levels determined by western blot analysis. when cultures were prepared from embryonic day 18 rats, nt-4/5 very strongly increased the morphological differentiation of calretinin positive cells. while all effects produced by nt-4/5 were mimicked by bdnf with similar potency, nt-3 was found to be only marginauy effective, our findings identify nt-4/5 as a potent neurotrophie factor for striatal gabaergic neurons. fusion of cells and organelles is a general patho-biological phenomenon: muscle cells, transport vesicles, langhans cells and virus induced fusion from within (ffwi, during replication) and without (ffwo, by the particles). in vivo and after virus isolation only little fusion can be seen, only after cultivation fusing hsv can be detected; a selective pressure must be released. 6 syn loci are known on the genome; the syn 3 locus is located inside of the cell in the carboxy terminal part of glycoprotein b, all other fusion domains are outside the ceil. fusion inducing mutations are in aa 816, 853, 854, 856, 857 and 872 and were correlated to fusion from within and without as well as to selective cyclosporin a effects. a model is presented for the 3 syn locus. -by recombination the fusion capacity could be transfected to fusion negative viruses. -hsv penetrates the cellmembrane by fusion by 2 ligand-recepter interactions. ffwo+-strains penetrate at 0~ cell/cell fusion was analysed and found to need also 2 receptor-ligand interactions if induced by syn 3 mutations. timing analysis of fusion events and by certain inhibitors allow further conclusions. quenching of lhcii isolated from dark-adapted (lhcii-d) or preilluminated (lhcii-l) rye leaves was similar in both preparations, but reversibility of this process within two hours of darkness was slower in lhcii-d. no differences in fluorescence quenching and full reversibility was observed for both lhcii preparations in reverse micellar solution. xanthophyll/chl concentrations in lhcii were consi-derably decreased under this conditions. excitation difference spectra (before and after illumination) of lhcii in asolectin liposomes showed typical changes of energy transfer between carotenoids and chl. a model is proposed according the xanthophyll cycle controls reversibility of light-driven excitation quenching as well as the xa~a~q~_x~tentof ~,,~nchi~g i/~ lhc~z. myelinogenesis is a complex, developmentally regulated process involving coordinate expression of myellnafion-related proteins. the myelin forming cells are the oligodendrocytes in the central nervous system (cns) and the schwann cells in the peripheral nervous system (pns). we differentially screened a rat postnatal day 16 (p16) spinal cord edna library with probes derived from normal (plus probe) and from myelin-free (minus probe) p16 spinal cord mrnas. we succeded in the isolation of several novel edna clones whose corresponding mrnas were expressed either selectively by oligodendroeytes or by oligodendroeytes and sehwann cells. here we describe one of the edna clones (tentatively denominated ns 2) whose mrna is specifically expressed by oligodandroeytes and schwann cells. the 3.5 kd transcript is expressed at highest level during myellnation and at much lower levels in the adult as it is known for other myelin-specific mrnas. sequence analysis of the full length edna sequence showed a 50% identity to the rat liver udpglucuronosyltransferase family, involved in the detoxificafion system. the 541 amino acid open reading frame encodes a 62 kd protein with a putative signal peptide and two transmembrane domains, whether this ns 2 protein is involved in a detoxification reaction or in glycosilation of proteins and lipids with glueuronie acid is under investigation. neuroblastoma (n8) are sympathetic tumors of childhood characterized by mycn amplification in advanced stages. cd44 standard (cd44s) and splice variants (cd44v) represent a group of surface glycoproteins whose expression has been recently linked to metastasis. we analysed the expression of cd44s and cd44v on n8 tumors and cell lines by immunohistology and northern blot. results showed high levels of cd44s on 33/44 nb, 6/10 n8 cell lines and 4/4 ganglioneuromas (mature, benign sympathetic tumors). lack of cd44s staining was observed on stage iv, mycn amplified tumors only. in cell lines, expression was not detected on cells with a neuronal phenotype while high levels of cd44s were expressed by cells with a glial differentiation, independently of mycn amplification. up-regulation of cd44s was obtained with agents that induce a glial differentiation, while neuronal differentiation by retinoic acid was not accompanied by a change in cd44s expression. no gd44v were detected on tumors or cell lines. we conclude that cd44s expression is down-regulated in a subset of nb ceils and tumors and that mycn product and additional mechanisms responsible for control of differentiation are involved in this regulation. the glycoprotein gc of herpesviruses is known to be non-essential for virus replication. non-essential herpesvirus proteins are suggested to perform "luxury functions" which may influence the outcome of an infection. we are aiming to analyse the function(s) of ibc specified by bovine herpesvirus 1 (bhv1), bovine herpesvirus 5 hv5) and caprine herpesvirus 1 (caphv1). these viruses are closely related but differ in their pathogenic potential. in a first step we have cloned and sequenced the individual genes. the nucleotide and the deduced amino acid sequence homologies of two 8hv1 reference strains was found to be >98%. the sequence homologies between bhv1 and bhv5 were >75% and those between bhv1 and caphvl were >65%. comparisons on the amino acid sequence level revealed that the differences were most prominent in the n-terminal parts, whereby the potential signal sequence region might be concerned. the putative glycosylation sites, however, were not affected, and the transmembrane sequences were similar in size and location. in order to characterize the significance of these differences, we are constructing gc-deletion mutants and recombinant viruses carrying a foreign gc gene. the in vitro and in vivo behaviour of these viruses will be compared to the wildtype virus. $15-67 o. moullet and j.-l. dreyer, instimt de biochimie, unlversit6 de fribeurg, ch-1700 fribourg camp has been recently shown to promote a cell survival response by retarding apoptosis (berridge, m.v& el. (1993) exp. hematol. 21,269-276) . on the other hand quinones are widely distributed substances of often potential toxicological significance. we have tested the effects of quinones on adenylate cyclase. the enzyme is rapidly inhibited by pbenzoquinone (ic50=40-45 ktm) or dicnlorophenol-indopbennl (/6'50=200 ilm), but 2-substituted quinones are inactive. the lc50's are decreased 3-10 times after membrane soinbilization but are not affected by gtp, gdp or analogues nor by cholera and pertussis toxins. the inhibition is not mediated by a g-protein or by the activation of a defined receptor. quinone inhibition stoichiometrically competes with forskolin activation of adenylate cyclase, equimolar concentrations of beth quinone and forskolin restoring the enzyme activity to its basal value. the cytotoxicity of benzoquinone, tested in vivo on hep-g2, a human bepatocellular carcinoma cell line, could be prevented with forskolin. in plasma membranes quinones tightly bind to only one major and two minor proteins that were purified by page electrophoresis under native conditions. together these observations indicate that quinone action can be attributed to targetting to a limited number of proteins at tile plasma membrane in a highly selective way. by affecting adenylate eyclase, a modulation of the camp pool induces apoptosis as a result of the cytotoxicity via. t.congolense is an african,unicellular hemoflagellate, pathogenic for domestic animals such as cattle.within the host bloodstream,parasites adhere to the wall of the microvasculature,eausing severe organ damage.we have set up and characterized an in vitro assay in order to study the metabolic requirements,reeeptor-ligand interactions and the ultrastructure of this adhesion phenomenon.our findings suggest that adhesion is an active process requiring metabolic energy on part of the parasite, but is independent of the target cells.we also show that t.congolense possess a leetin-like domain localized at specific sites along the surface of the anterior end of the flagellum, which interacts with specific carbohydrate receptors, probably sialic acid and/or n-aeetylglueosamine residues,on the endothelial cell surface. this suggests that adhesion is likely to be mediated by a trypanosomal surface component distinct from the variable surface glycoproteins(vsgs). we also suggest that the cytoskeletal protein aetin is involved in this process. local immune response based upon intestinal parasitespecific t and b cells to giardia lamblia may be impomrant in parasite clearance. experimental infections qf mice (cr:nih:s) with g. lamblia (clone gem-h7 which e~presses a major 72kd antigen on its surface recognized b~ mab g10/4) have demonstrated that the parasite undergoes surface antigenic variation in rive. experimental i~fection of immu/%odeficient mice (nu/nu mice and sci~d mice) and oontrol nu/+ littermates provided insights into associated immunological mechanisms: dominant surfade epitope-specific slga plays a major role in modulatin6] surface antigenic variation, and thymus-dependent t-lyn~phecytes in the induction of selfcure. athymio nu/nu mioe (zu. icr-strain) were reconstituted with immune peyer:~ patch lymphocytes obtained from self-healed littermat~ nu/+ mice. intestinal b-cells from these nu/+ mice @s well as from reconstituted athymic nude mice synthesized in vitro parasite-specific iga. this iga showed a predominant immunoblet reaction with the major surface antigen (a mr 72,000 polypep-tide) characterizing the giardia clone in question. nu/nu mice reconstituted wit~ immune cells acquired the potential to decrease significantly their intestinal parasite mass. thus the hypothesis on the causative role of intestinal iga in tl~ control of intestinal g. lamblia infection has found further experimental support. stephan jentsch, heinz tobler and fritz m011er, institute of zoology, university of fribourg, p4rolles, "1700 fribourg the process of chromatin diminution in the parasitic nematode ascaris lumbricoides leads to the formation of somatic cells that contain less dna than the germ-line cells. during this process, which occurs between the third and the fifth embryonic cleavage divisions in five different blastomeres, the termini of the chromosomes are cut off and eventually degrade in the cytoplasm. to analyze this process at the molecular level we constructed a xzap somatic telomere library. the analysis of three cloned telomeres and their corresponding germ-line genomic regions revealed that chromosomal breakage takes always place within short specific chromosomal regions (cbrs) and is followed by the addition of the telomeric sequences (ttaggc)n to the breakage sites. the different cbrs do not crosshybridize to each other. a comparative nucleotide sequence analysis is now being performed to screen for the existence of conserved sequence motifs. in a parallel approach we analyze the chromatin structure of the cbrs and their flanking regions in developmental stages before and during the elimination process, putative recognition or regulation sequences important for the elimination process might be recognized as dnasel hypersensitive sites. once such sequences will be identified, it should be interesting to establish their role in the elimination process and to look for specific binding factors. chromatin diminution takes place in all presomatie ceils of the early embryo of ascaris lumbricoides and is followed by the addition of many repeats of the telomeric sequence ttaggc. chromosomal fragmentation is developmentally regulated and occurs within specific chromosomal regions (cbrs). one of these cbrs (cbr1) was analyzed in detail. agene located close to the cbr1 encodes a putative gtp-binding protein, whose promoter region is located within a distance of only 2 kb from the telomeric ttaggc repeats of the corresponding somatic chromosome. a homologous gene was isolated from c. elegans. most interestingly, the c. elegans gtp-binding protein gene is also located at a telomeric position, namely at the end of chromosome v. in ascaris, transcripts of this gene are present in all developmental stages, though they seem to be stronger expressed in oocytes and early embryos than in later developmental stages and somatic tissues. a high degree of sequence conservation of these genes between the two nematode species and man (a corresponding partial cdna clone was identified in a human heart cdna library) suggests an important biological function. currently, we are using genetic methods to determine the possible function of this gene in c. elegans and to test whether its telomeric localization in both nematode species has any functional importance. it encodes a nuclear protein which is associated with she chromatin together with other probeins of the poiyoomb group. the mechanism of gene regulation caused by pelycomb seems to be similar to that of the modifier genes of the position variegation effect which also encode structural proteins of the heterochromatin. one of these proteins is hpi which shares with polycomb a region of similarity at the amino terminus called the ~omatin ~rganisation m~difier domain (chromo domain). the particular function of the chromo domain is not yet understood, but it seems to be important for the protein-protein interactions in chromatin associated complexes. furthermore, chromobox cdntai~ing genes were found in several species, suggesting that they are widespread in the animal and plant kingdoms. here we show chat ~. clemens contains at least one chromobox containing gene (cdna: cm08h7). length and szrusture of the putative ~. elegs~ chromo domain protein are similar to those of the chromo domain containing prozeins of other species. the expression pattern of cm08h7 is s:udied with antibodies raised against the putative chromo domain protein and by injecting a 5-ga! fusion construct inns the germ line of ~. eleman$. chromatin diminution in the parasitic nematode ascaris lumbricoides is a complex molecular mechanism, involving cleavage of the chromosomes at specific breakage regions, degradation of the eliminated chromatin and new telomere formation in all presomatic cells during the early embryonic development. the aim of the present project is to reproduce this dna elimination process in vitro, step by step or all in one, by using cell-free extracts of a. lumbricoides eggs. therefore, 4-8 cell extracts were established and their quality was assessed by the ability to transcribe 5s rrna genes (pol iii) and sl rna genes (pol li). faithful extracts were then tested for cleavage activity on dna substrates derived from different chromosomal breakage regions. finally, preliminary results revealed a possible non processiv rnase sensitive telomerase activity in the in vitro extracts, capable of adding specific nucleotide sequences to an oligonucleotide primer containing four repeats of the telomeric sequence traggc. as de novo synthesis of several kilobases of somatic telomere is likely to require a strong telomerase activity, we assume that this a. lumbricoides in vitro system is a good tool to isolate the telomerase protein and its corresponding gene or other implicated factors, s16-07 university of berne, ch 3010 berne a common feature of all mycobacteria -whether pathogenic or not -is their richness and variety of lipids. among numerous lipids widely distributed, pathogenic mycobacteria exhibit a group of sulfated surface lipids thought to be determinants of virulence. therefore, we studied sulfolipid-bioslrnthesis in pathogenic and apathogenic (opportunistic) mycobacteria species by monitoring the kinetics of [35s]sulfate incorporation into cellular lipids, pathogenic mycobaeterla (two virulent m. tuberculosis strains) showed a marked sulfolipid-synthesis with highest rates during exponential growth, while apathogenic ones (an avirulent m. tuberculosis strain and an opportunistic species) manifested negligible incorporation of the label. the partial characterization of the [35s]-sulfated lipids of pathogenic mycobacteria by thin layer chromatographic separation, combined with autoradiographic detection, revealed the presence of several radiolabeled lipid components of different polarities and with altering expressionpatterns during growth. these results strengthen the hypothesis, that sulfolipids are involved in the pathogenesis of mycobacterial disease. maiada (1,2) . some features of human pf maiada are observed in mudne malaria, although the pattern of sequestration changes due to the different plasmodial species involved, in mice, we showed by immunohistocbemistry that the expression of vcam-1 and icam-1 is upregulated in brain, lung and heart of p/asmodium bergheiinfected baib/c and also in lps-pdmed 8cid mice. in $gid mice injected with labeled human erythrocytes (e), a higher rate of sequestration to brain was observed with pfe than with uninfected e. lps-pdming increased the retention of pfe in the brain and lungs significantly (t test), but decreased it in the spleen. we conclude that pfe express surface structures which allow them to adhere to the vasculature of the brain more efficiently than uninfected e. lps increases the number of adhesive molecules on the host endothelium. interestingly, sequestration in the 8cid mouse resembles that of pfe in man. f. roth, f. riniker, k. schopfer and t. burkart inst. med. microbiology, univ. of berne, ch 3010 berne it has recently been shown that bacterial lipids cancarry antigenic determinants. the study of antibody specificity to such lipid epitopes in vitro turns out to be difficult since hydrophobic antigens have to react with watersoluble molecules. we have developed a solide phase lipid antigen immunoassay (laia) that allows to quantify the antibody reactivities with an array of lipid antigens derived from mycobacterial cells. defined amounts of extracted lipids were immobilized as equidistant lines on a nitrocellulose sheet. small strips of nitrocellulose, each carrying the same sets of lipids were incubated with different human sera. bound antibodies were then reacted with [125-i]-iodine labelled antihuman antibodies and visualized by autoradiography. the reaction was quantified by densitometry. assay conditions were optimized for antigen and antibody concentrations and appropriate incubation times. in addition, the effects of different ingredients (salts, proteins, detergent) on antigen immobilization and epitope accessability were studied. the presented laia-system is rapid, sensitive and reproducible. it allows to compare the specificities of different bacterial lipid antigens and to quantify their reactivity with serumantibodies. the atomic force microscope is a very convenient tool for studying hard and flat samples with atomic resolution. biological objects, usually soft and rough, are sometimes difficult to image using this technique. in contrast bacteria, which are too small to be observed with high resolution using the optical microscope, are hard enough to be observed with an afm at a relative good level of magnification. this kind of microorganisms can be prepared for afm imaging using very rapid and simple techniques. this method could be a convenient tool to observe the effect of bioactive substances such antibiotics. we show in this poster an exemple with the action of penicillin on b. subtilis. within the family trypanosomatidae, leishmania is a protozoan pathogen producing a broad spectrum of human disease. its life cycle is divided in two stages. promastigote is the flagellated form which colonizes the gut of the sandfly vector, and amastigote is the non-flagellated form that is specialized for growth and survival in the vertebrate host macrophage. to understand gene regulation during the transition between the promasttgote and the amastigote stage, we were interested in the characterization of stageregulated genes. we isolated a gene, sw3, from leishmania major whose mrna is more abundant in amastigote compared to promastigote. structural analysis of this gene showed that an additional polypeptide could be encoded by the opposite strand of sw3. we confn'med the presence of an open reading frame on the opposite strand by in vitro translation of the sw3 antisense rna. in vivo, an amastigote sw3 antisense transcript and the corresponding polypeptide were also detected suggesting that the sw3 is transcribed in both directions and that stage specific transcripts and polypeptides could be produced. analysis of other gene segments tend to indicate that an intensive usage of the leishmania genome to produce various polypeptides could be a general phenomenon in trypanosomatidae. the four variants and / or posttranslational modifications of histone h1 of procyclic trypanosoma brucei brucei (protozoa, kinetoplastida) were purified by hplc reversed phase chromatograpy and characterized by their amino acid composition and partial amino acid sequence. differences in sequence of up to 45% as compared to histone h1 of higher eukaryotes exist. alkaline phospatase digestion and analysis of h1 by means of hpce was used to demonstrate the presence of phosphorylated modifications. the unique biochemical properties of h1 of t. b. brucei contribute to the structural differences seen in the chromatin of the parasite as compared to that of the higher eukaryote host. larvae of the parasitic wasp chelonus inanitus (braconidae, hymenoptera) develop inside embryonic and larval stages of the host spodoptera littoralis (noctuidae, lepidoptera). parasitism induces in the host a precocious onset of metamorphosis and developmental arrest in the precocious prepupa. the wasp injects, along with the egg, pdv and venom into the host egg. injection experiments with pdv and venom revealed that pdv play a crucial role in altering host development and in suppression of the host's immune system. the genome of the pdv of c. inanitus consists of at least i0 segments of double-stranded circular dna ranging in size from 7-30 kb. we analyzed the expression of viral genes in the course of parasitization with cdna made from mrna at various developmental stages of s.littoralis. we also investigated the localization of the expressed viral genes on the various segments. in addition, these bacteria were isolated and cultured from brain tissue. to identify the infectious agent we used, among other techniques, an atomic force microscope (afm). when the. isolation fluids derived from the cortex of three alzheimer's cases were examined with afm and scanning electron microscopes, we observed bacteria whose size and morphology fit to the order of spirochaetales. these images are presented and discussed on our poster. ~ottstein bruno, institute of parasitology, ch-berne. %lveolar echinococcosis ae is due to infection with the metacestode (larval stage) of echinococcus multilocularls. an immunodominant antigen em2 of e. multilocularis was characterized by mab-gii, respective studies revealed, that (a) antigen expression metacestode stage-sdeci-fic and (b} that expression was restricted to the laminated layer. the em2-antigen i s a lectin-binding carbohydrate linked to a lipid residue. the "em2positive" laminated layer proved to be a crucial factor in protecting the parasite from host immune-effector mechanisms: only e. multilocularis larval structures exp ressing em2 were able to induce secondary alveolar chinococcosis in rodents. subsequent experimental studies in resistant (c57bl/10) versus susceptible (c57bl/ 6j and akr) mouse strains showed that resistance was as-sociated with synthesis of anti-em2 igg 3 and igg i. sus-ceptibility of c57bl/6j-mice was associated anti-em2 s and no igg 3 . the parasite-specific in vitro lym-~hoproliferative i~une response remained stationary ~ith time after infection in c57bl/10 and decreased in r and aki< mice. day 30 p.i. cd4 ⧠-lymphoblast cells dominated in all meuse strains; day 90 p.i. an in-creased nu/0ber of cd4-/cd8 + and cd4+/cd8 + cells were observed. only susceptible mice exhibited a significantly ~eereased response of lymph node cells to cona-stimula-~ien with time p.i. in conclusion, subtypes of parasitespecific cellular and humoral host immune responses seem leishmania species are protozoan parasites causing a spectrum of clinical manifestations in man ranging from cutaneous lesions to morbidity and death. the insect stage (promastigote) and intracellular stage (amastigote) differ in terms of morphology, physiology, antigenicity and gone expression. it is clear that the amastigote stage is uniquely adapted for survival within the inimical environment of the macrophage phagolysosome and that mechanisms regulating these adaptations must be called into play during the early stages of infection when the parasite undergoes transformation. in an effort to elucidate these regulatory mechanisms, we have constructed a "subtracted" cdna library which is specifically enriched for parasite transcripts expressed during the first 7 hours of infection ("switch" genes). several clones have been characterized by sequence, northern, and southern analysis. three clones, sw3, sw44, and sw20, are expressed at a several-fold greater level in the amastigote stage and potentially encode proteins which interact with nucleic acids. sw3 predicted protein is homologous with histone h1 proteins and sw44 and sw20 potentially encode ribosomal proteins. analyses of transcripts from this library are yielding clues not only to mechanisms underlying the regulation of parasite transformation, but also insights into post-transcriptional and translational control of gene expression in leishmania. $16-17 microbiology, university of geneva, medical school, c.m.u., 9 avenue de champel, 1211 geneva 4. a viral rna representing the sequence of a defective interfering rna, naturally selected for efficient replication, i.e. only containing the replication promoter, was cloned under the control of the t7 rna polymerase promoter. the expressed 17 transcript was then replicated in rive by supplying in trans the replication functions, equally expressed from plasmids. this system, completely accessible to genetic manipulations, allowed us to define a basic rule directing sendal virus replication, the "rule of six" (calain and roux, j.virol. 67 : 4822-4830, ig93). a second defective rna, representing a shorter version of the viral rna, in that it contains not only the replication but also the transcription promoters, was then cloned and replicated in the same system, although with lower efficiency. the replication of this "transcribing" viral rna was found to obey the "rule of six" as well. replicationitranscdpfion promoters were then achieved to define the cissequence requirements needed for efficient replication or for replication /transcdption. results obtained with these chimerical viral rnas will be presented and their contdbufion to the comprehension of the paramyxovirus replication and transcription processes bovine t-cells that become infected with the intracellular parasite theileria parva undergo lymphoblastoid transformation and acquire the ability to proliferate continuously. they cease to require specific antigenic stimulation, become independent of exogenous growth factors and cause tumors in nude mice. the il2 and il2r genes are constitutively expressed and the transcriptional activator nfwd3 which regulates these two genes is continuously activated. these processes are all strictly dependent on the presence of the parasite in the host cell cytoplasm and cease upon the specific killing of the parasite by the theilericidal drug bw720c. we have shown that the presence of the parasite results in the activation of the protein tyrosine kinases fyn and ick, which can participate in early t cell receptor signalling, suggesting that the parasite may use this signal transducti0n pathway to induce continuous proliferation. cysteine proteinases of leishmania as targets for chemotherapy. jacques bouvier*t and judy sakanari*. *department of pathology, ucsf school of medicine, san francisco, ca, and tanimal health, ciba-geigy, ch1566 st. aubin, switzerland. the overall goal of the study is to apply the structure-based approach to develop lead compounds and design novel enzyme inhibitors as potential therapeutic agents against leishmaniases. in this regard, we have identified and characterized the cysteine proteinases of leishmania major, and showed that they consist of excellent targets for drug design. we have purified a membrane-boand cysteine pmteinase not previously described in other trypanosomatids or protozoans and obtained amino acid sequence information from the purified enzyme. in addition, we have also characterized and purified the soluble cysteine proteinases of the parasite. consequently, we have isolated two genes encoding the soluble and membrane proteinases. the soluble cysteine proteinase gene occurs in multiple copies of 2.9 kb with flanking regions of 1.2 and 2.2 kb and is localized on one chromosome of approximately 440 kb. the membranebound enzyme gene is 3.1 kb and occurs as a single copy on a chromosome of approximately 1100 kb. a three dimensional computer model of both genes wiu be generated and enable us to scan the vast chemical database for new lead componds. we akeady have identified several cysteine proteinase inhibitors that are effective compounds in killing both promastigote and amstigote stages of the parasite in the micromolar range in vitro without affecting the host cells. dollenn~ier, g., cassinotti, p. and weitz, m., institute for clinical microbiology and irananology, 9000 st .gallen/swit zerland hav is a ~r of the picornaviridae. its rna gencrne rf~y be divided into 3 coding regions (pi, p2 and p3), qhe p3 region contains a protease 3cp ro that generates mature proteins by cis-cleavage of the initial polyprotein precursor. catalytic activity of 3cp r~ has been d6~aonstrated in a vacciniavirus/t7 ~qa-polymerase hybrid expression system. (balouter aided ali~ts of hav 3cp r~ sequence with that of other picornaviruses imply that amino acid residues his-44 and/or h) the complete hav open reading frame, hav specific expression products were identified by radioj_rmmnoprecipitation and lyy sds-page. the analysis with an antibody specific for putative nonstructural protein 2b revealed a 27,5 kda peptide. this is in contrast to its predicted size (12 kda). however, analysis with anti-(putative)2a antiserum confirmed an upstream location of the 2b n-terminus. the new 2bpeptide was also identified in lyeates of hav infected mrc-5 cells. hence, recombinantly expressed hav polyprotein underwent authentic processing and the predicted p2 organization has to be modified. we are interested in the development of animal models for one of the two major pathological changes found in the brains of patients with aizheirnar~s disease, the formation of fleuroitbrillary tangles, a main component of which is abnormally phosphorylaled tau-protaln. (tau itself is a protoin associated with mlcrotubuli.) two fundamental questions may be answered with our models: first of nit we woald like to reproduce the formation of tangles, and secondly we hope to answer the question, whether the formation of these pathological structures is sufficient for the bevalopment of senile dementia ot the alzheimer type (sdat). at present there are only a tow putative candidate genes known which may be in-voned in the hyperphosphor~lation ot tau in v/vo, one of which is the map-kinase erk2. we have cloned this candidate kinase from rat txaln rna via an rt-pcr approach and expressed the cona under,the co.rat of tour different promoters in transgenis mice. these promoters were tested for maximal expression. a similar approach was chosen to express the human taut0 isofonn in the brain ot transganic mice. we have stated to analyze different tau an~ erk2 expressing lines, northern blot analysis has revealed high levels of expression for all transgenes (up to fnetoid {n comparison to endocjenous levels). the neuronal spectficry of the transgenio erk2expression was c~nfirmsd by in situ hybridization analysis. ir~ addition several tauexpi'esalog tines were analyzed in wealernblots to determine the relative amount of htau= protein in the brain. sections of the brain were stained with tau-sp~c~c ardib~iies to identify the neurons expressing tau. from intemrosses of tau-with erk2-expressing lines, we have already identified double-positive mice which we currer~y analyze by immunohistocbemistry and in western nots to examine the phosphorylaiton status of tau. we are well aware of the possibility that only animals trans~enic for both tau and erk 2 might beveiop tang{es, whereas single transgenics mlsht not. pairs of individual neurons were recorded with sharp electrodes or in whole-cell configuratio~ in hippocampal slice cultures of rat. synaptic connections were studied between pre-and postsynaptie cells in ca3 and ca1 hippneampal fields by eliciting single aetinn potential in the presynaptic cell. monosynapfic excitatory connections were highly probable between ca3 and ca1 pyramidal ceils (p=0.76, n=82) with almost no feed-forward inhibition (p=0.01). by contrast, disynaptic ipsps, blocked by cnqx or bieuculline were observed with a very high probability (9--0.6) between ca3 pyramidal cells. monosynaptic excitation was found ha 56% of cases between ca3 neurons (n=43) but only in 27% of cases (n=ll) between two ca1 cells. monosynaptie ipsps with very short lateneies (<lms) and blocked by bicuculline, but not by cnqx, were recorded within area ca3 (5/6) and within cai (2/3), but not between ca3 and cat fields (2/2). probability of transmitlzr release, studied in recordings where failures could be unambiguously distinguished from spontaneous or small psps, was found to be close to 100% at both excitatory and inhibitory synapses. specific inhibitors of presynaptie release at excitatory and hahibitory terminals (i.e. adenosine and opioid peptides, respectively) gradually reduced the anaplitude of the psps indicating that the release at excitatory and inhibitory unitary synapses is multiquantal. the cns of a neonatal opossum, monodelphis domestic& shows profuse growth of fibers across a lesion. as in other mammals, such repair is not seen in adults. experiments have been made to define the age of the animal and stage of development at which repair ceases to occur. the entire cns was removed from animals aged 3-6 days or 11-14 days after birth. the spinal cord was crushed and growth of fibers assessed 5 days later by electrical recording of impulses conducted through the crush. repair after lesions to spinal cords of younger animals occurred in 38% of the trials (45 animals, aged 3-6 days). in nervous systems from older animals (11-14 days after birth) the success rate was low (only 4 out of 38 preparations). similarly, the carbocyanine dye dil labeled numerous fibers in spinal cords of younger animals (12 out of 21), whereas only one preparation out of 15 showed scant outgrowth into lesions in the older animals. our results suggest that there is a critical period for neuronal repair; at about 12 days nerve fibers lose the ability to grow across lesions. it is an advantage that at this later stage the nervous system is still small enough to survive in vitro. features correlated with these changes are oligodendrocyte differentiation and myelin formation. with immunofluorescent staining as well as electron microscopy myelination becomes apparent at 8 days after birth. we are now using time lapse-videomicroscopy to analyze molecular mechanisms that could play a part in promoting or preventing repair. supported by grants to j.g.n from the swiss nationalfonds (31-3626292) and from the internat. res. inst. for paraplegia. i. impulse conduction in axons has integrative functions. the dorsal root ganglion (drq) of the embryonic rat put into culture forms a monolayer and is thus accessible to eonventionalmieroelectrode technique, which allows testing the above hypothesis. we have chosen a dual approach by correlating ext~rimental el~ctrophysiological data from the chlture with results obtained from a compartrhental computer model. ii. the safety factor for conduction was found to be low. thus failures of ap invasion of the drg cell soma could occur at sites of impedance mismatch when a second stimulus felt into the relative refractory period of the first or when the axon was repetitively stimulated. the electrotonic resiuues (e.rs) of the failed aps had discrete amplitude levels, suggesting that the failures were always caused at the same site along the axon. these sites of low safety factor were found to be the branch point in the unipglar drg cell and the entrance of the stem piece into the soma in both cell types, the bipolar as well as the unipolar. a systematic comparison of 15oth cell types showed that the ap conduction through the latter is more reliable.'the length of this stem piece is inversely' correlated to the delay caused at the branch point, as tile electrical load of the soma is more efficiently masked by a long stem piece. the dendrites of motoneurons (and subsequently many other neurons) have been assumed to be electrically passive since the work of coombs, ecc]es and fatt in the 1950's. however, direct evidence has been impossible to come by until recently with the advent of appropriate dyes and equipment for measuring very small and very fast optical signals. we examined the propagation of action potentials, both spontaneous and evoked, in the dendritic trees of spinal cord neurons using a 12 â�¢ 1 2 array of photodiodes and a ccd camera as weft as conventional microelectrode techniques. organotypic slice cultures of embryonic rat spinal cord were stained with a voltage-sensitive dye (di-8-anepps) or a calcium dye (fluo-3) and recordings were made from motoneurons identified by their location and morphology. the results indicate that there is in fact an increase in calcium in the dendrites that accompanies action potentials whether synaptically evoked or evoked by current injection at the soma. spikes in the dendrites up to 1 60 i~m from the soma are much larger than could be explained by models assuming passive dendritic trees. work is currently underway to test the possibility that sodium conductances might contribute to the propagation of action potentials in the dendrites. cat auditory cortex contains multiple cochlear representations in both hemispheres that are interconnected through the corpus callosum (cc). the distribution of auditory callosal axons was studied on the mediosagittal level after injections of biocytin at electrophysiologically identified locations. single injections were done in ai (2 cats), aaf, aii and paf; multiple injections in ai & aaf in one cat. an additional cat received 1 injection in sii. the mediosagittal image of cc was subdivided into 30 segments along a rostro-caudal axis. labelled axons crossing the midline were counted in coronal or horizontal sections. axons from auditory fields were found in the posterior 2/3 and those from the somatosensory cortex in the anterior third of cc. axons from a discrete injection (ca imm in diameter) spread over as much as i/5 of cc. the rostrocaudal distribution of axons at the midline reflected roughly the rostrocaudal position of the field of origin, but apparently not the tonotopic order of a given field. the major efferent projections of substantia nigra pars reticulata (snr) convey information to the thalamus, to the tegmentum, to the superior colliculus, and to the spinal cord. such a range of targets suggests a highly organized activity within snr and this study investigates the temporal organization of spike trains. in the portion of snr between +2.7 and +4.5 mm interaural levels, we recorded 40 single units along 6 electrode tracks in 4 young female wistar rats. most units (n=32) fired in a non-regular poisson process, although only a small number (7/32) was characterized by an average burst size with more than 3 spikes. crosscorrelograrns were computed for 25 pairs of units simultaneously recorded from the same electrode and for 26 pairs simultaneously recorded from distinct electrodes. most crosscorrelograms (27/51) showed a symmetrical peak centered on time zero, which indicates a tendency to synchronous firing. when units were recorded from the same electrode tip we observed that 1/8 to 1/40 of their spikes were synchronous. about half of the significant interactions were characterized by a relatively long duration (>250 ms) and are likely to correspond to a different mechanism of synchronization. these results support the hypothesis that snr cells receive common afferences of two different types and their fine temporally organized activity yields coordinated activity between the target structures. the neuronal microtubule-associated protein map2 binds to microtubules via a domain containing 3 or 4 repeats of an 18 amino acid sequence. we have previously shown that when cultured nonneuronal cells are transfected with map2 their microtubules become stiffer and are then capable of supporting process outgrowth when their cortical actin cytoskeleton is depolymerised. we hypothesise that this stiffening effect of map2 is caused by the 18 amino acid sequences binding to neighbouring tubulin subunits in the walls of the microtubules thus reducing their freedom of movement relative to one another. to investigate this further we have created map2 mutants in which 1 or 2 repeats of the tubulin-binding motif were deleted. these were tested by transfection and expression in cultured cells. the results confirm that the number of repeats affects the stability, stiffness and the cytoplasmic arrangement of cellular microtubules. we were previously able to demonstrate the presence ot kinin-like immunoreactive structures in the mouse brain. in order to characterize them, we have now performed a donble-immunofluorescence study using both polyclonai auti-srudykinin (bk) and mouse monocloual anti-neeron-specificenolase (nse) antibodies. the latter is considered to be a specific neuronal marker. after fixation with bonin-hollande solution, the brains were removed, embedded in paraffin and serially sectioned at 10 am. the sections were incubated with the antibodies, which were visualized by an indirect immunofluorescence technique using fitc for nse and by an avidin-biotin technique using texas red for bk. double-immunofluorescent cells were found in the thalamus ventralis, in the nucleus cechleuris ventralis and in the nucleus mesencephaficns nervi trigemini (v). bk-positive cells in the nucleus principalis v were nse-negative ih spite of their typical neuronal aspect. the other bk-positive structures (pia, ependyma, hnic~es) also were alwa~ nse-negatlve. most of the nsepbsifive bk-negative cells were seen in the cortex. the neuronal nature of many kinin-like immunoreacfive structures in the mouse brain is thus ascertained. in contrast to the amphibian optic nerve (on), on of adult mammals is unable to regenerate after injury. astrocytes disappear from and macrophages accumulate at the lesioned site (blaugrund et al., j. neurocytol, 118, 105, 1992) , we have followed the distribution of astrocytes and microglial cells in xenopus tadpole ons over 10 d after mechanical crush. astrocytes were stained for cytokeratin and vimentin, and microglial cells for griffonia isolectin b4. soon after crush, immunoreactivity for intermediate filament proteins was strongly increased. astroeytic processes extended into the lesion from both proximal and distal stump. at ,5 d, astrocytes had crossed the injured region. in the distal stump, some of them contained vacuoles indicating phagocytic activity. while in the normal on microglial cells were ramified and scarce, 2.5 d after crush they were roundish and vacuolized, and scarce within the lesion but numerous distal to it. at 10 d, their number was decreased and most of them had reacquired ramifications. conclusively, in the tadpole on, astrocytes are virtually never absent from the lesion site but rather extend rapidly into it. astrocytes may thus provide a guiding support for outgrowing axons, microglial cells that are frequent in the distal stump, do not accumulate at the lesion. the behaviour of the two cell types differs profoundly from that in mammals and is likely to be one of the factors that may contribute to successful neuronal regeneration of the amphibian on. ci4mence, j. f. 1, ranieri, j. p.2, aebischer, p.2, and sigrist, h. 1, 1institute of biochemistry, university of berne, ch-3012 berne; 2division autonome de recherche chirurgicale, chuv, ch-1011 lausanne. small peptidic sequences of laminin (yigsr, ikvav) are known to promote attachment and/or neurite outgrowth of various neuronal cell lines (pc12, nb2, ng108-15). new and established heterobifunctional photo crosslinkers were used to immobilize these peptides in a topically defined fashion onto biocompatible surfaces such as hydroxylated fluorinated ethylene propylene (fep-oh) and pely(vinymlcohol) (pva) . n-[m[(3-trifluoromethyl) diazirine-3-yl]phenyl]-4-maleimido-butyramide (mad) was thermochemically linked to the synthetic peptide cdpgyigsr via its n-terminal cysteine, leaving the bioactive part (yigsr) free for cell receptor interaction. mad-cdpgyigsr was radioactively labeled by reductive methylation and purified by reversed phase hplc. patterned (20 and 300 #m) peptide immobilization was attained by photocoupling onto pva and fep-oh and visualised by autoradiography. light-structured biomaterial surfaces with molecularly oriented nerve growth promoting peptides were investigated for spatially controlled neuronal cell attachment and differentiation. it is striking to observe that these inhibitory or stimulatory effects were corroborated by binduced decrease or increase in 2-deoxyglucose uptake, respectively. this gives b a putative role in the limbic regulation. the sheet-like processes of oligodendrocytes wrap themselves spirally around central axons, thus formtng the myelin sheath. a single oligodendrocyte may donate internodal segments of myelin to each of 20-30 or more adjacent axons. it is not known, if one oligodendrocyte picks out axons randomly or if it prefers axons with a certain diameter or with a certain chemical specificity. we have studied this last possibility by combining intracellular injection of a fiuorochrome, and immunohistological detection of calciumbinding proteins (cabp's), markers for the axons ef certain subpopulafions of nerve ceils. lucifer yellow was injected into oligodendrocytes of the optic nerve, of living rat brain slices. the oligodendrocytes were identified by their electrophysiologieal properties. slices containing the iniected cells were immunostained /or parvalbumin or calretinin using second antibodies labelled with texas red. using co.focal laser-scanning microscopy combined with a three*dimensional reconstruction programm, the relationship between oligodendrocyte processes and axons positive for one of the cabp's was determined. for ultrastructural confirmation, single, lucifer-yellow injected, oligodendrocytes were photoconverted, and the cabp's-positive axor~ were revealed by gold-labelled antibodies. the few oligodendrocytes injected show the classic morphology, that is of a small cell body and few processes running parallel to the course of the axons. the proximity between glial processes and positive axons can be easily appreciated in confocal laser-scanning images. a preferential assodation of oligodendrocyte processes with parvatbumin-positive axons has not as yet been found. thus, the confirmation of the hypothesis that oligodendrocytes choose their axonal targets according to chemical cues awaits further work including the injection of a lar~er number of these cells. to initiate a molecular genetic analysis of brain development in insects, we are characterizing the mechanisms of embryonic brain development in the fly drosophila. early in embryogenesis, a small number of molecularly diverse brain neuroblasts generates the neurons of the brain. subsequently, pioneering brain neurons initiate axonal outgrowth along glialbound brain compartments. these pioneering neurons establish the primary brain tracts. the pioneering brain axons express the cell-cell adhesion molecule fasciclin i dynamically during outgrowth and initial fasciculation; a subset of the pioneering axons expresses fasciclin ii. the axonal framework of the embryonic brain tracts is used for outgrowth and fasciculation by subsequently differentiating axons and, thus, prefigures the tracts of the mature brain. a comparison of early brain development in drosophila and in vertebrates reveals common mechanisms for brain development. supported by the swiss nsf. institute ot histology, university of fribourg, p~rolles, ch-1700 fribourg calretinin (cr), an ef-hand ca2+-binding protein, is expressed in cajai-retzlus cells during development of the rat cerebral cortex. these cells are located in the marginal zone and are the ear{iest cortical neurons to be generated. they have been consldered as unusual on account of their morphology and their fate during corticogenesis. the functional significance of cajai-retzius cells and their destiny in adulthood is still controversial. in order to approach these questions we have applied organotypic culturing methods. slices of neocortex from brains of 0-6 days-old rat pups were cultured for 10-21 days applying the interphase technique (stoppini etal, j. neurosci. methods 37, 1991) or the roller-tube technique (g&hwiler, j. neurosci. methods 4, i981) . the fixed cultures were immunolabe0ed with an antibody against cr. the obtained results showed that cr positive structures develop in vitro like in vivo. however, in organotyp[c cultures their distribution corresponded to a more mature stage than the situation on the day of the explantations. many cr-positive cells were seen in layer l they were horizontally oriented or had a pyriform shape. based on their morphology these ceils were identified as cajai-retzius cells. orpositive cells were numerous in layer i1-l[i and showed mostly a bipolar morphology. some muripo[ar cells could also be observed. a fine net of crpositive fibers and puncta was found in layers [ and iv. the demonstration that cajai-retzius celts are detectable in these cultures will allow us to follow the fate of these cells dynamically and [nte~ene in their function by applying exogenous factors. cervical primary afferent input to vestibule-spinal neurones projecting to the dorsal horn: a double labelling study in the rat. wheat germ agglutinin-horseradish peroxidase (wga-hrp) was injected by pressure into cervical spinal ganglia c2 or c3. in the same experiments fluorogold (fg) was iontophorefically injected into the ipsilateral dorsal horn of different cervical spinal cord segments. anterogradely labelled fibers (wga-hrp) were present mainly in the external cuneate nucleus, but also to a lesser extent in the caudal part of the medial and descending vestibular nuclei (mvn, dvn) . the fg injections, restricted to the cervical dorsal horn, revealed retrogradely labelled cells in the central part of the caudal mvn. a double exposure (fluorescent and polarisadon optics) under the microscope showed primary afferent fibers surrounding like baskets retrogradely labelled fg cells. the projection from cervical ganglia cells to the mvn represents a pathway for direct afferent information from neck receptors (in particular muscles) to vestibular nuclei and supplies the mvn with afferent information which could enable the vestibulospinal nenrones, projecting to the dorsal horn, to influence the local information processing. in the sciatic nerve, all myelinated and non-myelinated axons were snap-ir. in peripheral tissues, all nerve endings were positive. the preterminal schwann cells of motor axons were also snap-25-ir. after sciatic nerve section, many myelinating sehwann cells also expressed snap-25-ir. in conclusion, snap-25 is expressed by neurons in the pns. it seems to be also expressed by preterminal schwann cells and by myelinating schwann cells during regeneration. $17-19 repair of completely transected spinal cord of neonatal opossum in culture h.a. vischer, dept. pharmacology, 8iocenter, uni. of basel, switzerland woodward et al. (j. exp. biol. 1993 , 1993 have shown that fibers grow rapidly and profusely across a lesion made by crushing the spinal cord of the neonatal opossum monodelphis domestica. in such experiments careful controls must be made to establish that fibers were in fact broken by the lesion. tests have now been made to determine whether similar repair can occur after a more drastic lesion involving complete transection of the cord. after dissection of the entire gns from animals aged 3-8 days, the cord was cut into two with scissors at the c5 -c7 level. the two-ends were glued together with matrigel on a sylgard mold, which encompassed and held the cns. the rostral end was labeled with the fluorescent carbocyanine dye dil in order to visualize growing fibers. in 33 preparations fibers grew over the cut into the separated part of the spinal cord. such growth could extend to 500 ixm. fibers grew straight, branched and multidirectionally, or in fascicles. in another set of experiments cords were combined from different animals of the same or different ages. again, fibers could grow across the cut but they did so less frequently. these results set the stage for investigating fiber growth after rotating the two ends at a defined angle and for analyzing factors that influence the direction of growth. map la is a microtubule associated protein of 360 kd. in rat brain two monoclonal antibodies, a and bw6, recognized map la in neurons and their axonal and dendritic processes. in mouse cerebellum, monoclonal a recognized purkinje ceils and granule cells uniformly, as already described for rat brain. in contrast, monoclonal bw6 stained selectively some dendrites of purkinje cells, forming bands in the molecular layer. we compared this striation with that obtained with a monoclonal antibody, anti-zebrin ii, which recognized aldolase c. in the mouse vermis, zebrin stained in a striated pattern, but complementary to bw6. these results demonstrate that map la is present in forms which are differentially distributed in the mouse cerebellum. these observations may be explained either by differences in metabolic states of neurons, or by differences in their regional functions, or by differences in the regional stability of microtubules. this work was supported by grant no 31-33447.92 from the swiss national science foundation. serum free aggregating brain cell cultures prepared from 16 day old fetal rat telencephalon are used as a model to study brain development. in these cultures it is possible to distinguish ontogenetic events such as cell proliferation, differentiation and myelination, in the present study we examined synaptogenesis by analyzing the expression of synaptic proteins such as synaptophysin, snap-25, synapsin i, and brain spectrin. different developmental stages were analyzed by western blotting. immunoreactivity for synaptic proteins was detectable already at day 12 in culture, suggesting that the first synapses are already present at this early stage. the expression of synaptic proteins strongly increased between day 20 and day 30 in culture, reflecting a period of intense synaptogenesis. treatment of the culture with an elevated concentration of kci (30mm) increased the expression of synaptic proteins, suggesting a stimulation of synaptogenesis. these findings demonstrate the utility of this approach to study brain synaptogenesis, and possibly synaptic plasticity. drenhaus, u.i gunten, a.v., rager, g. ; institute of anatomy, university of ch-1700 freiburg the retina of tupaia comprises three types of ganglion cells (rgcs) analogous to x-, y-, and w-cells found in other mammals. these cells differ in size and in their distribution pattern: large rgcs are frequent in temporal, small rgcs in nasal retina. as the fiber diameter correlates with the size of its respective rgc, it can serve as a parameter for the topographic representation of rgcs in the nerve. to study this we analyzed the optic nerves of three tupaia. using the electron microscope we recorded the density, diameter, and the position of fibers in the nerves. we found, that fibers with the largest diameter are located dorso-temporally and close to the center of the nerve. they are surrounded by zones of smaller fibers. the diameter decreases gradually towards the periphery and is smallest in the ventro-nasal region of the nerve. since the fiber diameter distribution corresponds to that of the size of rgc-types in the retina, we assume that the topographic relationships in the nerve and the retina are similar. (supported by s 17-23 stoppini luc, s. duport, l. parisl, c. oropes~, departement of pharmacology, cmu, 1211 geneva 4 the micro environment of the central nervous system is important for neuronal function. in this context, the blood-brain (bbb) provides and maintains the extracellular medium that is compatible with normal neuronal and synaptic activity. due to the difficulties inherent using whole animal as an experimental model to study permeability and metabolic processes at the cellular level, major efforts have been engaged in recent years, in order to bring up suitable /n vitro models. we are presently developping an in vitro bbb by interfacing a coculture of endothelial cells monolayem and nervous organotypic cultures. the main idea of this study is based on the premise that the complex intercellular interactions between the different cell types pertaining to the nervous tissue and the neighbeurlng endothelial cell is of fundamental importance to promote a realistic bbb system. we expect that erganotypic culture of nervous tissue, combined to endothelial cell monolayers, will initiate and maintain a bbb phenomena/n vilro. we will test more specifically the hypothesis that neuronal activities [spontaneous or elicited) will influence gila] cell response which will in turn modify endothelial properties. preliminary results clearely show a good nervous tissue and endothelial cell survival. tight junction llke structures could be identified using freezefracture or conventional transmission electron microscopic thechniques. {work supported by chemodyne gent~ve ). the process of myelination is a prerequisite for the proper function of the brain since it enables rapid saltatory conduction of axons. in the central nervous system (cns) myelination is performed by oligodendrocytes. a differential screening approach was used to isolate new rat cdna clones that are expressed in this glial cell type. here we report the further characterization of two of these novel cdnas which appear to be expressed specifically in oligodendrocytes. the two characterized cdna clones (tentatively called cns1 and cop-25.1) share an approximately 420 bp region of sequence identity which suggests that they are dedved from the same gene by alternative splicing, within this area a putative open reading frame is located. we demonstrated by northern blot analysis and in situ hybridization that the two mrnas of the novel gene are expressed exclusively in oligodendrocytes. however, the mrnas show a different specific spatial localization within the cell. we compared mrna expression of myelin basic protein (mbp) and proteolipid protein (plp), with that of cns1 and cop-25.1 in brain and optic nerve during development. it appeared that the mrnas of the novel gene were delayed significantly as compared to mbp and plp mrnas. streit. phyaiologisches institut, btihlplatz 5, 3012 bern in organotypic slice cultures of the embryonic spinal cord, rhythmic spontaneous activity arises when the inhibitory synaptie transmission is blocked. the rhythmic activity consists of bursts of regular oscillations of activity at 4 -5 hz. this study investigates the origin of the regular oscillations. when stimulated electrically at 5hz, the synaptic transmission in the cultures showed strong depression by about 50% on average. the synaptie depression was highly variable among individual connections and tended do be less pronounced in frequently used connections. when random depression ratios with an average of 50% at 5hz were implemented in a computer model consisting of 100 randomly connected excitatory cells, regular oscillations of activity did not arise, even in the presence of synchronous pacemaker cells. however, when individual depression ratios were adapted according to the rate of activity of individual connections, regular oscillations at 4 -5 hz arose in the presence of few unsynchronized pacemakers. this finding suggests that the regular oscillations in the cultures originate in a network formed by the plasticity of synaptic depression. maier a., schlumpf m., beer h.f., sehubiger p.a. and lichtensteiger w., inst. of pharmacology, univ. of zurich, the ontogeny of expression of dopamine d 1 receptor mrna in the rat brain and binding to the receptor was studied at 12 time points from gestational day (gd) 13 to postnatal day (pn) 60 by quantitative receptor autoradiography and in situ hybridization.long evans rat pups and fetuses of time pregnant rats were frozen and sectioned coronally and sagitally at i0 um. d 1 recepto~o~inding , determined with the selective antagonist ~ji-sch 23982 was first noted on gd 18 in the developing striatum, basal ganglia and the olfactory tubercle, reaching adult levels at around pn 14. the expression of d i receptor ~na was studied by using a mixture of 3-~js-labelled oligonu-specific cleotides. on gd 13 messages were noted in the developing stiatum, olfactory tubercle and retina. this study shows a good regional correlation between the development of receptor binding and mrna, however, mrna precedes detectable binding to the receptor by several days. earlier work had shown that q~ replicase forms complexes with q~, plus strand rna via interactions at two internal binding sites, the s-and the m-site, while binding of the 3'-end is mediated by host factor. in contrast, the 3'-end of the minus strand appeared to be directly accessible by replicase. we have prepared a series of plus and minus strand variant rnas containing either internal deletions or terminal deletions extending from the 5'-end. the template activities of the variants were determined by single-round replication assays. for the plus strand we find that while deletion of the s-site remains without effect (as expected from previous results), deletion of the m-site reduces template activity to 25 % or less compared to wild-type rna; the residual activity shows a decreased dependence on the presence of host factor. the results agree with an important role of the m-site interaction both with replicase and with host factor for the formation of productive initiation complexes. the template activity of the minus strand was unexpectedly found to be strongly dependent on the presence of a segment between nt 76 and 210 from the 5'-end. this shows that recognition of the minus strand by rep(icase does not only involve interactions near the 3'-end but requires a previously unknown structural feature near the 5'-end of this template. $18-02 karsten graning and angela kr&mer d~partement de biologie cellulaire, universite de gen~ve, 30, quai emest-ansermet, 1211 gen~ve 4 splicing of nuclear pre-mrnas takes place within multicomponent complexes (spliceosomes) that are assembled by interactions between the pre-mrna, snrnps and protein splicing factors. we have purified two splicing factors that function in the formation of the pre-splicing complex. sf3a consist of three subunits of 60, 66 and 120 kda and corresponds to three proteins present in the functional 17s but not in the 12s u2 snrnp. it binds to the 12s u2 snrnp only in the presence of sf3b, suggesting a two step assembly pathway of 17s u2snrnp: binding of sf3b to the 12s u2 snrnp generates a 15s intermediate particle that is converted to the active 17s u2 snrnp by the subsequent association of sf3a. comparison of proteins enriched in the sf3b fraction with polypeptides found in the purified 15s u2 snrnp suggests that sf3b comprises at least five of the other 17s u2 snrnp specific proteins and together with sf3a promotes the u2 snrnp/pre-mrna interaction. by edna cloning, two of the subunits of sf3a have been identified as homologs of well characterized yeast proteins that function at the same stage of spliceosome assembly in yeast. splicing factor sf1, a heat-stable 75-kd protein, functions early during spliceosome assembly. tryptic peptides of sf1 were sequenced and cdna libraries were screened with degenerate oligonucleotides. the information obtained by comparing the sequences of three overlapping cdnas suggests the existence of at least three mrnas that could be derived from a common pre-mrna by alternative splicing. in agreement with this assumption mrnas of the expected sizes that are differentially expressed depending on cell line or tissue are detected by northern blotting. in theory, three polypeptides could be generated that differ by the presence or absence of 7 internal amino acids and in their ctermini. the deduced amino acid sequence is rich in prolines and contains several possible phosphorylation sites and a putative leucine zipper. proline-rich domains, which are also present in splicing factors psf and sap62, as well as leucine zippers have been found in transcription factors and could mediate protein/protein contacts, suggesting that sf1 could function during splicing by interaction with other spliceesomal proteins. pre-mrna splicing is catalyzed by a multicomponent complex (the spliceosome) that consists of small nuclear ribonucleoprotein particles (snrnps) and non-snrnp protein factors. the spliceosome is assembled in a stepwise fashion on the pre-mrna. chromatographic fractionation of hela cell nuclear extracts and subsequent reconstitution of splicing in vitro has allowed the separation and isolation of snrnps and several protein factors. sf4 triggers the transition between splicing complex b, which contains all spliceosomal snrnps but is not competent for catalysis, and complex c, the active spliceosome. this transition involves a conformational change that leads to altered base pairing interactions between snrnas and pre-mrna. the purification of sf4 has been impeded by its low abundance; however, a correlation between sf4 activity and a polypeptide of 50 kd could be established in the most purified fractions. we are currently investigating whether this protein represents sf4. interestingly an atp-dependent rna-helicase cofractionates with sf4 through several purification steps. whether or not this activity is relevant for the splicing process is under investigation. selection of iron-responsive element rnas with high affinity for iron regulatory factor henderson, b.r., menotti, e., bonnard, c. , and kith_n, l.c., isrec, ch-i066 epalinges iron regulatory factor (irf) post-transcriptionally regulates iron homeostasis via binding to mrna iron-responsive elements (ires). the ire loop sequence (5'-cagugn-3') and "bulge" cytosine are phylogenetically conserved. we prepared a pool of 16,384 ire molecules randomized at these 7 nucleotide positions, and employed in vitro selection to identify optimal sequences which bind human irf. we define two major classes of high affinity rna ligand, the optimal loop sequences of each are 5'-cagugn-3' (wild-type) and 5'-uaguan-3'. this novel finding predicts base-pairing within the loop between positions i and 5. all nucleotide substitutions in the "bulge" or at loop positions 2,3 and 4 decreased binding by 36% to 99%. in addition, binding specificity of rat irf differed with that of the related rat ire-binding protein, irf b. in vitro analysis of ire-like stem-loops identified by computer search has not yet revealed any new ire-containing genes. a73 $18-08 3' processing of histone pre-mrnas: a phylogenetic comparison reto kohler, petra duda and daniel schiimperli. zoologisehes institut der universtit/it bern, abteihing fiir entwicldungsbiologie, baltzerstr. 4, ch-3012 bern, switzerland. we are using a phylogenetic approach to study the biochemical reaction by which animal histone pm-mrnas are processed at their 3' ends. in mammals, the efficiency and specificity of this reaction is known to depend absolutely on the u7 snrna which interacts with conserved spacer sequences downstream of the proc~sing site. a highly conserved hairpin element which precedes the cleavage site serves as a binding site for an additional processing factor, but its importanea for efficient processing varies greatly between different historic genes and between extracts of different mammalian cell lines. to determine whether the relative importance of the hairpin and spacer dements have been conserved during evolution, we analysed the processing of histone genes from three different animal phyla in vitro, i.e. vertebrates (mouse), arthropods (drosophila melanogaster) and nematodes (c. elegans). in the mouse system, processing was strongly dependent on the presence of a mouse spacer element. in nuclear extracts of drosophila ke cells, processing occurred exclusively when a drosophila spacer element was present in the rna. processing efficiency was not reduced by foreign (mouse, c. elegans) hairpins. in whole cell extracts of ascaris lumbricoides embryos, an inverted situation was observed. 3' processing products were only produced by rnas that included an upstream segment derived from a c. elegans histone gene, irrespective of the spacer sequences present. these surprising results correlate with certain unique features in the c. elegans upstream element. we are currently in the process of cloning ascaris lumbricoides histone genes, which will allow us to verify our results in a homologous system. in xenopus laevls ooeytes, wild type mouse u7 rna gets assembled into functional u7 snrnps, both when transcr~ed from an injected gene or when injected as/n vitro synthesized rna. if the sm binding site of u7 rna is converted into the canonical sm sequence (u7 sm opt), the rna assembles into a particle which accumulates more efficiently in the nucleus but wbach is nonfunctional. this u7 sm off particle inhibits the function of preformed endogenous u7 snrnps, most likely by nonproductive binding to histone pre-mrnas, histone pre-mrna processing can be demonstrated in c/s if u7 rna is placed 3' of hlstone pre-mrna and injected into oocytes. only u7 sm wt sequences are active in this c/s processing. three proteins can be photoaffinity cross]inked to u7 sm wt rna, the common snrnp proteins g and b/b' and a u7-specific protein of 40 kd (50 kd in mouse). these crosstinks map to closely spaced positions within the sm binding site with the u7-specific crosslink being located most 3'. u7 sm opt rna does not become crosslinked to the u7specific protein and is also more tightly associated with sm proteins. we now investigate the in vitro assembly of u7 snrnps using these characterized u7 rnas and a preparation of highly enriched snrnp proteins. u7 sm wt and u7 sm opt rnas associate with common sm proteins in this system, but the interaction with the u7-speciflc protein could not be demonstrated. the double-stranded (ds)rna modifying enzyme, which can convert adenine to inosine, was first identified in xenopus laevis, but has since then been detected in different species and in mammalian cells. the enzyme is a specific adenine deaminase which uses dsrna as substrate and recently, it has been postulated to be responsible for a specific mrna editing reaction. we have purified this enzyme to homogeneity, approximately 400,000 fold from calf thymus whole cell extracts. the protein was purified primarily by ion exchange chromatography over six columns, with the final step being chromatography on a dsrna affinity column. the purified protein has a molecular weight of 115 kd and is the only protein present when enzymatically active fractions are visualized on an sds-polyacrylamide gel stained with silver. the dsrna modifying enzyme is a very low abundant protein. we are in the process of further characterizing the purified enzyme and of cloning cdnas coding for it. detailed mutational analysis of histone rna 3' end formation carmen spycher, andr6 furger, adrian streit, daniel albrecht, d. schiimperli, zoologlsehes iustitut der universtit/it bern, abteilung thr entwicklungsbiologie. baltzerstr. 4, ch-3012 bern, switzerland histone rna 3' ends are formed by cndonucleolytic cleavage resulting in poly(a)" mrnas with a highly conserved 3'-terminal hairpin loop structure. for the mouse h4-12 gene major and minor processing sites are located 3 and 5 nucleotides downstream of the hairpin, respectively. for the cleavage reaction, the u7 snrnp interacts with the spacer element of histone pre-mrna by base-pairing through the 5' end of its rna moiety, u7 rna. we have observed that this base-pairing potential extends further than previously recognised in either direction. we have made site-directed rnutagenesis in fltis potential hybrid region and around the processing site. rna processing and complex formation with the u7 surnp were analysed by incubation in nuclear extract from mouse k 21 cells. our results indicate that base-pairing with u7 rna both in the classical spacer element and downstream of it are very important for histone rna processing. in contrast, sequences upstream of the spacer element do not seem to be involved in base-pairing with u7 rna, but mutations in this region may affect processing by other mechanisms. systematic mutations of the highly conserved four nucleofides immediately following the hairpin show no qualitative or quantitative effects in the processing reaction. alteration of nucleotides 5 to 7 around the minor processing site, however, result in a dramatic decrease in processing efficiency and alter the specificity of the cleavage site. in further experiments we will introduce specific nuclcetidc modifications at the two processing sites, which should allow us to characterize potential cleavage factors by chemical crosslinking. evolution callaerts p., glardon s., j.,oosli f., quiring r. and gehring w. cell biology, biozentmm, basel. the pax genes encode a family of dna binding factors which share the paired-box and play an important role in the control of development. they can be subdivided into four different classes which differ in the presence or absence of other conserved sequence motifs coding for a paired type homeobox or an octapeptide. the pax-6 gene, which contains a paired-box and a homeobox, has been cloned from humans, mice, rats and zebra fish. recently, we have cloned the pax-6 homologue of drosophila melanogaster. the five genes share a very high degree of sequence identity both in the paired-domain and the homeodomain. similar to its mammalian counterparts, the drosophila gene is expressed in the eye primordia and the nervous system. in addition, mutations in pax-6 affect eye development: aniridia in humans, small eye in mouse and rat, and probably eyeless in drosophila. this would imply that the pax-6 homologues share a conserved function in eye morphogeuesis in both vertebrates and invertebrates. furthermore, this suggests that pax-6 may have been present in a common ancestor. therefore, we are now trying to isolate pax-6 homologues from other, more primitive organisms by means of the polymerase chain reaction, and to determine whether they are implicated in eye development. putative candidates have been identified from a number of species and are being characterized. their sequence similarity and some evolutionary implications will be discussed. keegan liam and gehrin~ walter j., biozentrum, university of basel, klingelbergstr. 70, ch-4056 basel, switzerland using an enhancer map screen we have identified two genes that may be direct targets of antennapedia involved in forming the adult leg. spalt major is repressed in the leg imaginal disc and tea, shirt is activated by antennapedia. we wish to determine whether the control of these genes by antennapedia is direct, spalt major is expressed in a ring in the antennal disc and is repressed by antennapedia in the leg disc. the antennal ring is repressed by ectopie antennapedia expression that transforms the antenna to a leg. we are defining an enhancer at spalt major that is required for antennal ring expression and repression by antennapedia. we are using various approaches to show that antennapedia binds to the antennal ring enhancer at spalt major. these include polytene chromosome banding studies using antibodies to antennapedia as well as in vitro dna-binding and genetic experiments. in search for novel regulators potentially involved in myogenesis, we identified a homeobox of the paired type in human muscle. subsequent cdna cloning revealed that we had cloned the full length coding region of human pax7. our human sequence extends the known mouse edna both on the 5' and the 3' end. as a first step in order to test for a functional role of pax7 in myogenesis, we analyzed its expression pattern during chicken development. transcripts are present in the neural tube and in the dermamyotome of the developing somites. therefore, the pattern of expression of pax7 is conserved between mouse and chicken. next, we assessed the expression of pax7 in different mouse and human cell lines. we found pax7 transcripts specifically in myogenic cells and not in any other cell types. interestingly, pax7 is already present at the myoblast stage. moreover, 10ti/2 fibroblasts converted to myoblasts by either transfection of myod or treatment with 5-azacytidine expressed pax7, whereas the parental cells did not. while myod was able to activate pax7 in 10t1/2 cells, expression of pax7 was not sufficient to induce the myogenic phenotype. nevertheless, our expression data are consistent with a role of pax7 during the mesodermal commitment to the myogenic lineage. the segmental organization of the drosophila head is achieved by a flow of positional information from maternal gene products to the zygotic gap genes. recent genetic analysis has identified three new gap genes involved in head development. one of these genes is the empty spiracles (ems) gene. mutations in eras cause severe defects in the head and the filzk~rper at the posterior end are missing. the eros gene encodes a putative transcription factor with a homeodomain. the eros rna is expressed in two phases of embryonic development. first expression is seen at the blastoderm stage in a single anterior band, correlating with its function as an anterior gap gene. later during embryogenesis eros is expressed in the posterior spiracles as well as lateral regions of each segment where the tracheal pits form and lateral neuroblasts originate. using g-gal fusions we could identify at least five different regulatory elements in the eros promotor region responsible for tissue specific expression of the gene. a 300 bp element responsible for the early expression which is dependent on the maternal gene bicoid, the key gene of the anterior system, and the terminal gene tailless was identified and studied in detail. this element contains two bicoid and two tailless binding sites. the effects of muations in these binding sites on eros expression will be discussed.this analysis should allow us to elucidate the molecular mechanisms by which the morphogen bicoid regulates subordinate target genes like ems in the drosophila embryo. the pax-6 gene of the mouse encodes a transcription factor with both a paired-and a homeodomain. pax-6 is affected by several allelic mutations in the small eye (sey) gene. sey mutations cause a reduction of the eyes in heterozygotes, and homozygotes lack both eyes and nasal openings and die as newborns. the corresponding mutation aniridia in humans affects eye and cranlofacial development in a similar manner. we have isolated a pax-6 homolog from drosophila (dpax-6), which shares more than 90% sequence identity in the paired-and the homeodomain with the mammalian genes. also outside of these domains we find considerable sequence conservation arguing for a true pax-6 orthologue. the drosophila gene spans ~16 kb and is expressed in the brain and the ventral nervous system of the embryo, and in the eye imagjnal discs and the brain of the larva. the gene was mapped to the eyeless (ey) locus. mutations at the ey locus cause either the partial or complete absence of the compound eyes or embryonic lethality. in one ey allele we have identified a transposable element in the first intron of dpax-6, which affects dpax-6 transcription in the eye discs, suggesting that dpax-6 is encoded by the ey gene. this implies that pax-6 (sey) in the mouse is homologous to ey in drosophila. thus, despite of the different modes of eye morphogenesis, the same gene seems to control eye development in insects and vertebrates. the drosophila homolog of the vertebrate serum response factor (srf) was isolated by low stringency-hybridization. nucleotide sequence analysis revealed that the drosophila srf homolog (dsrf) codes for a protein which displays 93% of sequence identity with human srf in the mads domain, the region required for dna binding, dimerization and interaction with accessory factors. the dsrf gene is expressed during several phases of embryonic development. both the rna and the protein are maternally provided to the egg and slowly decrease in their levels during gastrulation. after germ band retraction, high levels of zygotic expression were observed in a distinct subset of peripheral tracheal cells distributed throughout the embryo. many of these cells are at the tip of tracheal branches and are in direct contact with the target tissues these branches tracheate. the dsrf gene was mapped to position 60c on the second chromosome, and overlapping deficiencies which remove the gene were identified. analysis of tracheal development in embryos carrying these deletions revealed a degeneration of most of the major branches of the tracheal system. although the initial migration of tracheal cells was not affected in those deficiency embryos, many tracheal cells appeared not to maintain their formerly correct position and continued to migrate. thus, the dsrf gene might play a role in the proper formation and maintenance of the major branches of the trachea. s 19-08 our experiments would be expected to provide insight into such problems as the evolution and function of transcription factors with multiple dna binding domains. ideally, we would be able to mimic "gene splitting" which occurred during evolution. the poan gene plays an important role during drosophila neurogenesis. it is expressed in specific subsets of sensory mother ceils (smcs) and neuroblasts. the smcs that express poxn differentiate into polyinnervated external sense (p-es) organs. in the absence ofpoxn, smcs differentiate into mono-innervated rather than poly-innervated external sense organs. as the poxn gene contains a paired-box, it probably encodes a transcription factor. since the function ofpoxn in the central nervous system is not known and no po~n mutants are available, an ems mutagenesis screen was initiated to isolate such mutants. based on the assumption thatpoxn null alleles are lethal, we screened for lethals uncovered by the deficiency df(2r)wmg, which includes poxn, but survive over the deficiencies df(2r)xte-18 and df(2r)kl-32 flankingpoxn. in a further test, such lethals are screened for the absence of embryonic p-es organs. ifpoxn mutants are not lethal, they will escape this screen. therefore, we are inducing local hopping of p-elements that have inserted in the vicinity of poxn. the offspring of flies that display an altered eye phenotype are screened for p-element insertion into poxn. we hope that these approaches will generate mutants that will be crucial for future studies ofpoxn function in neurogenesis. chromatin structures and transcription of rdna in yeast saccharomyces eerevisiae reinhard dammaun, renzo lucchini, thee keller and jest m. sogo; institute of cell biology, eth-honggerberg, ch-8093 ziirich the chromatin structure of yeast ribosomal dna was analyzed in rive by cross-linking intact cells with psoralen. we found that in exponentially growing cultures the regions coding for the 35s rrna precursor fall into two distinct classes. one class was highly accessible to psoralen and associated with nascent rnas, characteristic for transcriptionally active rrna genes devoid of nucleosomes, whereas the other class showed a cross-linking pattern indistinguishable from that of bulk chromatin and was interpreted to represent the inactive rrna gene copies. by cross-linking the same strain growing in complex or minimal medium, we have shown that yeast ceils can modulate the proportion of active (non-nueleosomal) and inactive (nucleosomal) rrna gene copies in response to variations in environmental conditions which suggests that yeast can regulate rrna synthesis by varying the number of active gene copies, in contrast to the vertebrate ceils studied so far. whereas intergenie spacers flanking inactive rrna gene copies are packaged in a regular uucleosomal array, spacers flanking active genes show an unusual cross-linking pattern suggesting a complex interaction of regulatory factors and histories with dna. $20-04 r. felleisen & b. gottstein; institute of parasitology, university bern, bern, switzerland most patients suffering from alveolar echinococcosis (ae) respond to infection with a marked igg synthesis directed against e.multilocularis metacestode antigen ii/3, which represents a novel member of the family of cytovillin related proteins. although the respective gene basically is also present and expressed in e.granulosus, most of the cystic echinococcosis (ce) patients do not recognize the antigen. this phenomenon was tackled by generating cdna derived from full length ii/3 genes from both echinococcus species, performed by rt-pcr. sequence analysis revealed a very high degree of conservation of the primary sequence (98.6% homology), cdna fragments were expressed as recombinant proteins and were comparatively assessed in elisa respective to antibody-binding characteristics. sera from patients suffering from ce were showing no significant differences in reactivity with the antigens derived from both species. therefore, parameters others than minor differences in the primary sequence seem to be responsible for the lack of antigen recognition with respect to ce. $20-02 patrick rigoni, sandro rusconi, institut f molekularbiologie h der universitat zarich, winterthurerstr. 190, 8057 z~rich, switzerland. trinuclcotide repeats are often found in the coding sequence of transcriptional regulators in both mammals and yeast, however, their function is yet unclear and data collected in our laboratory on the gheocorticoid receptor even suggest that they may not have any, and that their presence is probably the result of a selfish replicative behavior. nonetheless, we believe that these motifs can be used as tags to identify flexible protein domains typical of modular proteins such as transcription factors. another kind or repeat (coding for a poly-glutamine/alanine) has been discovered in the yeast regulators gall1 and ssn6. from northern blots, we know that such caggcn repeats are present also in higher eukaryotes and we want to isolate them by constructing an enriched edna library using a 5' race protocol. so far, no mammalian protein bearing the motif glrdala has been characterized. among the positive clones we hope to find the homologous or paralogous of gall1 and ssn6 that have escaped so far conventional cloning techniques. meanwhile, we have been testing the effect of coexpressed yeast gall 1 on different reporter-activator combinations in mammalian cells. preliminary results show that galll but not the mutant form galllp can inhibit the activation properties of some (not all) gal4 chimeras. we are also producing antibodies directed against oligo-ala and oligo-gln and oligo-gln/ala motifs in order to better characterize natural proteins containing these repeats. we are interested in the early development of c. elegans, particularly in the contribution of genes whose homologues in vertebrates and d. melanogaster mediate positional infolzzations during development. therefore, we are working on ceh-13 which belongs to a cluster of at least 4 homeobox containing genes. this cluster is considered to be equivalent to the homeotie cluster of drosophila and to the hox clusters in vertebrates. by generating ~-galactosidase transgenie lines, we have observed that ceh-13 is expressed very early during embryogenesis, embryonic expression appears to be restricted to the posterior half of the embryo, which is rather surprising, since the ceh-13 hemologues in drosophila and vertebrates are anterior-specific. during larval stages, ceh-13 is expressed in neuronal cells. these results are now in the process of being confirmed by immunolocalization of the ceh-13 protein product with a polyclonal antiserum. in order to clarify the function of ceh-13 in the c. elegans development, we have isolated a ceh-13 mutant from a tcl insertion library, in collaboration with r. h. a. plasterk from amsterdam. from this worm, which looks wt, we are now trying to obtain a deletion derivative. previous studies have shown that bovine herpesvirus 1 (shv-1) infected cell pratein (bicp) 0 acts -depending on the promoter -as a strong transactivator or as a repressor in transient expression assays. the 81cp0 polypeptide contains a cysteine-rich zinc finger domain (c3hc4) which is conserved in a number of viral and cellular regulatory proteins including the 8icp0 homologs icr3 of herpes simplex virus type 1 and protein 61 of varicella zoster virus. this type of zinc finger (the so-called ring tinge0 was shown to bind zinc ions but functional requirement for zinc has not yet been demonstrated, a gap which we aimed to fill with the present study. transient expression assays were performed in oocytes which had been microinjected with thionein to chelate and deplete the intracellular pool of zinc. 81cp0-induced cat activity of a promoter-cat construct was 72-fold higher than the basal cat activity of the reporter plasmid, in the presence of thionein, bicp0-induced transaotivatlon was reduced 54old. with a set of control experiments we excluded that thionein might affect transcription and protein synthesis in general, to our knowledge this is the first demonstration of zinc-dependence for a member of the ring finger family. we have identified by pcr and edna cloning five pou genes expressed during early embryogenesis of zebrafislx four of these genes show extended homology to the brn-1 class of pou genes. preliminary evidence suggests that the brn-1like pou genes overlap with most of their expression domains.the gene studied in most detail so far (zp-50) is first expressed on the ventral side of the future fore-and midbrain. slightly later, expression is also found in rhombomeres 3 and 5 in the hindbraln. these rbombomeres were identified by the specific expression of the krox-20 marker using a novel in situ hybridization protocol which allows the simultaneous detection of two different transcripts using different color substrates. in the 24 hours old embryo a complex expression pattern is found involving a variety of brain structures and the spinal cord. the distribution of the transcript suggests that zp-50 is mostly expressed in glia cells. another pou gene we have identified (gp-9) defines a novel class of pou proteins. as a maternal mp, na it is initially ubiquitously present. after gastrnlation its transcript is found most notably in the developing hindbrain in rhombomeres 2 and 4 for which so far no good molecular markers were known. we are investigating the roles the identified pou genes may have in zebrafish hindbrain segmentation. we are currently attempting to manipulate gene expression in developing embryos by injection of rna or by the production of transgenic fish. we are also screening the zebrafish genome for new developmental marker genes. progress about these experiments will be presented. $20-07 willimann, t. and trueb, b. to gain insight into the regulatory mechanisms of collagen vi synthesis we have characterized the cis-acting elements of the chicken ctl(vl) collagen promoter. footprinting experiments with nuclear extracts from chicken embryos revealed three distinct elements, designated a, b and c, that were protected from dnase i digestion. the nuclear proteins that interact with the three sites were identified by gel retardation assays in combination with the use of various oligonucleotide competitiors as well as specific antibodies raised against well-characterized transcription factors. site a was found to be a target for transcriptional activator apf, whereas sites b and c were shown to be recognized each by two distinct nuclear proteins which belong to the spl multigene family. to address the question whether the three sites alone are able to direct transcription, a minipromoter construct was created in which the sequences of sites a, b and c were placed in front of a reporter gene. after transfection into chicken fibroblasts, this construct exhibited a high relative promoter activity when compared to a large genomic fragment containing the basic ctl(vi) collagen promoter. thus, the three sites are sufficient to induce transcription of this gene. octamer transcription factors (oct or otf) are a subset of the pou family of transcription factors which regulate expression of cellular and viral genes by binding to the octamer sequence atgcaaat. neurons and astroglial cells harbour, in addition to the ubiquitous oct-i factor, brain specific factors termed n-oct 2, 3, 4 and 5. in the present study we determined the chromosomal localization of the gene encoding the n-oct 3 transcription factor and characterized the structure of isolated n-oct 3 genomie clones. the chromosomal mapping was performed by analysis of somatic cell hybrid panels and radioactive and fluorescence in situ hybridization of human metaphase chromosomes. it is interesting that the 5' end of the n-oct 3 coding sequence contains repetitive cag and ggc residues. several disorders have been discovered to be related to expansion of trinucleotide repeats. we are presently investigating if the n-oct 3 cag or ggc clusters are hot spots for such mutation mechanisms and if so, which diseases could be associated with it. two dna regions upstream of the distal glucocorticoid receptor binding site interact with nuclear proteins that are tissue-specific (region a) or ubiquitous (region c). the characteristics of the dna-protein interactions have been studied in vitro. these sequences, in different combinations with the natural mmtv promoter, were tested in transfection assays of fibrcblast or mammary cell lines. we show that they are able to modulate the level of glucocorticoid-stimulated transcription. the proximal region of the mmtv promoter has binding sites for the transcription factors ctf/nf-i and oct-1. p~asmids with a deletion or mutations in the oct-1 site were tested in stable transfections, that are most representative of the state of proviral dna with respect to both number of integrated dna templates and chromatin organization. we show that the oct-1 site is important for the basal level of promoter activity. the data further indicate that the functional outcome may depend beth on the relative ratio of factors to dna templates and on the relative affinity of binding sites, as determined by oligonucleotide competition footprinting. besides the fact that telomeres represent specialised structures important for chromosome stability, the particular significance of cloned c. elegans telomeres is that they will contribute to the completion of the physical mapping of chromosomes and that they provide one set of elements for the construction of artificial c. elegans chromosomes. the cloning of c. elegans telomeres, however, is impeded by the fact that tandem repeats of the sequence ttaggc are not only located at the ends of the c. elegans chromosomes, but also at many internal chromosomal sites. therefore, we have developed a special protocol for the construction of a c. elegans endlibrary in 2~ zap. the resulting telomeric library was screened for recombinants containing ttaggc repeats and yielded about 70 positive clones. so far, 30 were analysed by partial sequencing and twelve of them satisfy all criteria required for telomeric clones. their ttaggc tandem repeats are located at the expected position, namely just adjacent to the blunt end cloning site in the polylinker. furthermore, the g-rich strand of the repeats is oriented 5' to 3' towards the end of the fragment, thus corresponding exactly to the defined orientation of eukaryotic telomeric sequences. finally, hybridisation data with one of these putative telomeric clones show that a subtelomeric fragment hybridises to bal31-sensitive fragments on a southern blot with c. elegans dna, indicating that this clone represents a c. elegans telomere. $20-09 to investigate whether chromatin structure or transcription can interfere with replication, derivatives of the yeast trp1ars1 minichromosome were constructed that contain either the ded1 or pet56 promoter firing against the origin of replication ars1. while the pet56 constructs transformed yeast at high frequencies and were maintained as high copy number minichromosomes, the ded1 constructs transformed at low frequency and the constructs were integrated in the genome suggesting that ars function was impaired. insertion of the h4-ars, a second origin of replication, rescued a ded1 construct as a minichromosome (yrpdh3).the chromatin structure mapped at low and high resolution of the ars1 region in yrpdh3 was indistinguishable to that of the pet56construct yrpft61. ananlysis of rna showed that transcription was going through ars1 in yrpdh3 and in yrpft61, but the levels of transcripts in yrpdh3 were much higher. we conclude that transcription through are1 interferes with replication and prevents extrachromosomal maintenance. dna sequence of a repeated dna segment on circular and linear plasmids of borrelia burgdorferi w.r. z%ckert and j. meyer, abt. pzmom, zahn&rztliches institut der universit~t basel a plasmid-associated repeated dna segment of b. burgdorferi strain b31 was cloned. at least two copies of this segment appear to be present on the 29 kb circular plasmid, whereas one copy is carried on the 50 kb linear plasntid of this strain. dna sequence analysis revealed a region containing a novel 906 bp putative open reading frame (orfl) on the 50 kb linear plasmid, orfl displayed extensive sequence homology (95%) to putative open reading frames present on the clones obtained from the 29 kb circular plasmid. heterogeneity is mainly caused by 3rd-base-wobbllng. flanking sequences share 85-95% homology, including the 5' end of an additional putative open reading frame irmaediately downstream of orfl. whether orfl and/or the related sequences are being transcribed and yield, in the case of orfl, an approximately 36.5 kd, lysine-rich polypeptide, is yet unknown. the repeated sequence seems to be specific for b. burgdorferi sensu lato and therefore may be useful in nucleic acidbased diagnostics of lyme disease. $20-16 similar to type ib oculocutaneous albinism in humans, overall production of pigment is greatly reduced in dark eyed albino and only obvious in eyes. we have studied the molecular basis of the c 44h mutation and show that expression of the tyrosinese gene is not affected. after sequencing tyrosinase cdna isolated from c44h/c44h homozygotes we uncovered a single base alteration from wild type leading to a serine to isoleucine exchange. the importance of this mutation was demonstrated by generating transgenic mice containing a mutated tyrosinase minigene. this showed that the single base change is sufficient to severely depress pigment production in transgenic mice. we therefore conclude that the point mutation is responsible and sufficient to generate the dark eyed albino phenotype. members of the pou-family of transcription factors are involved in developmental and differentiation processes. using a pcr-based cloning strategy with degenerated oligonucleotides we identified several pougenes from the lactating and involuting mouse mammary gland. three of these cdna clones which contained as yet unknown sequences were chosen for further investigations: clone 5.80 which has a high homology to pit-l, clone 5.54 which is related to the brn-3 gene and clone 5.48 which belongs to the class iii of pou-proteins. the expression levels were analyzed in different mouse organs and in several developmental stages of the mammary gland, including the postlactational process of involution. because of the low abundance of the specific transcripts we used a semiquantitative, reverse transcriptase-mediated pcr assay to investigate the mrna levels of the three novel pou-family members. distinct and specific expression patterns for all three members were obtained in the different investigated organs. interestingly, the expression of the pit-1 related cdna clone 5,80 was elevated in all developmental stages of the lactogenic-hormone dependent mouse mammary gland and in sceletal muscle, whereas its expression was low in other organs. repetitive sequences in dna can allow ectopic (outof-register) homologous recombination, which is often undesirable and can even lead to disease in humans. to prevent this, long homologies should often be interrupted during evolution. accelerated mutation of repetitive sequences by so-called ripping may be one of the mechanisms used to accomplish this in fungi. we are investigating if introns in vertebrate genes have an undiscovered role as interrupters of homology within and/or between genes, in addition to their established role in exon shuffling. by inserting homology-poor introns in an otherwise homology-rich region the genome could prevent ectopic homologous recombination. such a mechanism could be especially useful where there is an advantage in encoding highly repetitive protein sequences. it appears that within the collagen gene family there is indeed a correlation between repetitiveness and intron density. the influence of prenatal diazepam exposume (i,25 mg/kg/d, s.c.) from gestational day 14 to 20 on the development of the ~-opioid binding sites in striaturn, nucleus accumbens and midbrain of pn 14, pn 28 and 8 week old male and female rats was studied. 8 week old prenatally diazepam exposed male rats showed a significant decrease of k-opioid binding sites in the nucleus accumbens. a sex-dependent difference in k-opioid binding site densities could also be detected between pn 28 prenatally diazepam exposed male and female rats. we now investigate by in situ hybridisation whether changes in the level of mrna encoding fo~ prodynorphin, the precttrsor for various endogenous ~-opioid agonists, could be responsible for the decrease of ~-opioid binding sites in the nucleus accumbens of 8 week old prenatally diazepam exposed male ~ats. $21-04 distribution and morphology of nitric oxide-positive neurons in the marmoset cerebral cortex during pre-and postnatal development j.p. hornung* and j. schultz** *institute of anatomy, university of lausanne, 1005 lausanne, and **university of fdbourg, 1700 fribourg nitric oxide, synthesized by the enzyme nitdc oxide synthase (nos), is a neuroactive substance and a limited number of neurons were shown to express this enzyme either by histochemistry or by immunocytochemistry. both techniques were performed on brains of marmosets with ages ranging from 6 weeks before birth to adult. the morphology and distribution of nos-positive neurons were analyzed in all lobes of the cerebral cortex. the same pattern was revealed by histochemistry or immunocytochemistry. three populations of nos-positive neurons were revealed. first, a transient population of neurons in the molecular layer of the embryonic cortex, many having the morphology of the retzjus-cajal cells. a second population was made of large multipolar neurons in the deep layers of the cortex and the underlying white matter, which persisted from embryonic to adult life. a third, permanent, population was made of small, weakly reactive neurons in the supragranular layers appearing postnatally, this study demonstrates that no can exert its actions in the cerebral cortex through several pathways at different developmental stages. trimethyltin (tmt), a well-known neurotoxicant, was used to study the sensitivity of the different brain cell types (i.e. neurons, astrocytes, oligodendrocytes and microglial cells) to a neurotoxic insult. aggregating brain cell cultures of fetal rat telencephalon were treated during 10 days with tmt at different concentrations, ranging from 10 -10 m to 10 -6 m. microglia were found to be the most sensitive cell type, since already at 10-10m of tmt an increased number and clustering of griffonia simplicifolia-positive ceils could be observed. at 10 -8 m of tmt astrocytes showed increased staining for glial fibrillary acidic protein, characteristic of gliosis. neurons and oligodendrocytes appeared to be the least sensitive cells, since a decrease in the activity of cell type-specific enzymes was observed only at 10 -6 m of tmt. these results show that microglial cells and astrocytes respond to a toxic insult before any neuronal changes could be detected, and that the microglial reaction may be the first target of tmt. this reaction could then trigger the astrocytic response. vif is widely distributed in brain with suggested functions related to development. vip expression was studied during rat brain development using hybridization histochemistry with a 48met probe. vip mrna was found in thalamic nuclei on el7, later recognized as the ventrolateral and reticular nuclei after further maturation during prenatal period, vip mrna was found in hypothalamus, especially suprachiasmatic nucleus on el9 and its expression matured over the next days. few cortical neurons contained vip mrna on e21, they continuously increased in number and signal intensity over the perinatal period. vip gradually matured in the first three postnatal weeks and adult-like patterns were found on p 22, when cerebral cortex, ventrolateral and reticular thalamic nuclei, hypothalamus, esp. suprachiasmatic nucleus, were the regions with most prominent vip expression. these results demonstrate the relatively late appearance of vip gene expression in the rat forebrain, as compared with peptides like srif and cck, not suggesting a major role in earlv brain maturation. in primary cultures of mouse cerebral cortical astrocytes, a rapid glycogenolysis followed by a massive glycogen resynthesis ( six-to ten-fold over basal levels after 9 hr) are induced by vasoactive intestinal peptide (vip) or noradrenaline (na). both actions of these neurotransmitters are mediated by camp. since the induction of glycogen resynthesis triggered by vip or na is abolished by inhibition of transcription and translation, we applied the 2-d gel electrophoresis technique to search for astrocytic proteins induced by vip or na. the comparison of 35s-labeled proteins from primary astrocyte cultures treated or not with vip 1 ~tm revealed two newly synthesized proteins: a 36 kda protein (pi=5) and a 23/24 kda protein (pi=6). only the latter is visible on a silver stained 2-d gel, which is a prerequisite to consider its purification and microsequencing. induction of the 23/24 kda protein by vip was time-dependent with a maximum at -12 hr. preliminary results indicate a similar induction by na and isoproterenol. in humans, the central analgesic effect of tramadol (t) is only weakly reversed by the ~t opioid antagonist naloxone (nx) (br j clin pharmacol 1993; 35:73p). t analgesia may thus result from an action on monoaminergic pathways as suggested by in vitro and animal data. we therefore investigated the effect of ct2-adrenoeeptor antagonism by yohimbine (y) on t analgesia. according to a randomised double-blind crossover and placebo (p) controlled design, healthy volunteers (n=10) received t (100mg orally), followed (+ 3 h) by y (0.1mg/kg intravenously), and y + nx (0.8mg intravenously). analgesia was assessed over 8 h by subjective pain threshold (numerical scale -ns-) and objective pain threshold (rill nociceptive reflex -riii-) monitoring. peak analgesic effect was observed at ca. 3.25 h (riii +8.8 +semi.6 ma and ns +8.4 +1.8) vs p (rih -0.3 +1.2 and ns +2.3 +1.6, p<0.05) and lasted ca. 6 h. y reversed t analgesic effects during ca. 1 h (r/ii -2.9 +1.8, p<0.05 and ns +2.8 +1.5, p<0.05), whereas y + nx abolished t effects thoughout the study period (rill -1.9 4-1.1 and ns +0.6 +1.3, p<0.05), y alone tended to lower pain thresholds (rill -4.1 +1.5 and ns -1.9 +1.4, p>0.05 anova). yohimbine, an r antagonist, reverses tramadol effects, thus pointing to the major role of monoaminergic modulation in tramadol anfinociception. a monoclonal antibody (in-l)was raised against neurite growth inhibitory proteins present in rat cns myelin (caroniand schwab, neuron 1: 85, 1988) . in-1 neutralizes the inhibitory ettect of cns tissue in vitro and in vivo, thus permitting regeneration of certain lesioned cns fiber systems. we have used this antibody to immunostain cryostat sections of the nervous system of the rat. we found that in-1 stains white matter and myehnated fiber tracts in the cns. the staining pattern corresponds to that of the known myelin specific antigens mbp (myelin basic protein), mag (myelin associated glycoprotein) and mog (myelin oligodendrocyte glycoprotein). the staining of in-1 in the cns is found in regions of the adult nervous system which have been shown to be inhibitory for axonal growth. interestingly, the regions which show a high abundance of in-1 antigens are low in staining for gap-43, a marker for growth and plasticity in the developing and adult brain. in contrast, gap-43 is strongly expressed in unmyelinated fiber tracts or nuclei. prevention of oligodendrocyte development in rat spinal cord resulted in suppression of in-1 expression and elevated gap-43 levels. this suggests that inhibitory myelin proteins may negatively influence plasticity in the rat cns. to date, the existence of anp and npy in the adrenal medulla has been shown only for a few mammalian and anuran species. thus the present immunohistochemical investigation attempts to localize anp-like and npy-like peptides n the adrena gland of representative mammals, birds, reptiles, amphibia and bony fish by the use of antisera specific for mammalian anp and npy. furthermore, the catecholamine-containing cells were characterized by antisera against enzymes of catecholamine synthesis. anp-and npy-immunoreactivies were detected in adrenal chromaffin cells of all vertebrates studied while no immunoreactivities were observed in the adrenal cortex or in its homolog, the interrenal. the representatives of the different vertebrate classes exhibited marked differences in the proportions of anp-immunoreactive and npy-immunoreactive cells, in the presence of anp-and npy-immunoreactivities in noradrenalineand/or adrenaline-containing cells and in the amount of of cells containing both anp-and npy-immunoreactivities. our results give evidence for a long phylogenetie history of adrenal anp-and npy-like peptides. thus, they stress the physiological impact of these peptides as adrenal paracrine and/or endocrine hormones. human neuronal growth inhibitory factor (gie) is involved in the regulation of neuronal growth and is deficient in the brains of alzheimer's disease victims. this brain-specific metalloprotein consists of 68 amino acids out of which 20 are cys and has been found to contain copper and zinc. proteins with similar primary structure were isolated from bovine and equine brain. the amino acid sequence of these proteins shows about 70 % homology to those of mammalian metallothioneins (mt), with two conserved inserts of 1 thr and a glutamic acid rich hexapeptide in the n-and c-terminal regions, respectively. in contrast to mts, which usually contain only zn(i1), the presence of cu(i) and zn(ii) (6-7 moles total) in all gif isolations suggests the functional importance of both metals ions. to spectroscopically probe the native zn-binding sites, the zn(ii) ions were replaced by cd(ii). both bovine and equine cd/cu-gif derivatives exhibit similar absorption and cd spectra with features characteristic of cd-thiolate clusters. the release of 3h-arachidonic acid evoked by glutamate was characterized in cerebral cortical neurons in primary culture grown under conditions that prevent glial cell proliferation. in these cultures, 99% of the total ceils are immunocytochemicauy characterized as being positive for specific neuronal markers such as neuron-specific enolase and neurofilament. specific markers for astrocytes, oligodendrocytes or microglia were not detected. glutamate induces a concentration-dependent release of 3h-arachidonic acid with a ec50 of 3 pm. the pharmacological profile of this glutamate response shows the involvement of two receptor types: the nmda and the ampa ionotropic receptors. the glutamate-evoked release of 3h-arachidonic acid is phsensitive. when the incubation buffer is alkalinized from 7.25 to 7.65, both basal and glutamate evoked release of 3h-arachidonic acid are enhanced. the glutamate response is also enhanced as intracellular ph is alkalinized by pharmacological treatments: such as inhibition of c1-/hco3-antiport by dids. these results further stress the role of intracellular ph in glutamate-mediated neurotransmission. $21-09 multiceli culture system for the study of drug kinetics wechsler d., *schittny j.c. and honegger u. e., depts. of pharmacology and *anatomy, university of bern, ch-3010 bern a cell culture system was developed for the investigation of cellular drug kinetics in cultures with up to 4 cell types. it was used for the study of uptake, competitive uptake, release and redistribution of drugs. individual cell types (human skin fibroblasts, rat astrocytoma cells (c6) and rat hybfidoma ceils (oligodendrocyte x astrocytoma, roc)) were separately grown to confluency on glass circle sectors. in the same petri dish 4 sectors in various combinations were exposed to media containing radiolabelled chlorpromazine (cpz). single and repetitive uptake and release of cpz were measured in each cell type after individual exposure or exposure in any combination of cell types: in 2 hour competitive uptake studies fibreblasts reached 1.7 and 2.6 times the concentrations of c6-and roc-cells, :respectively. in redistribution experiments the exchange of cpz from preloaded to unloaded cells was tested. the exchange rate was dependent on cell type and loading period. it decreased with increasing time of exposure suggesting the formation of more stable cpz stores. we have previously demonstrated that certain neurotransmitters such as vip, noradrenaline (na) and adenosine promote glycogenolysis in mouse cerebral cortical astrocyte cultures (brain res. 563: 227-233, 1991) . the question that we then addressed was the nature of the metabolic substrate released by astrocytes following glycogenolysis. we therefore determined the presence of various metabolic substrated released from astrocytes under static conditions and in the superfusate of a laminar flow system developed in our laboratory. neither glucose nor any intermediate of the tficarboxylic acid cycle could account for the decrease in glycogen stores; astrocytes however were shown to release great quantities of pyruvate and lactate at a rate of 50 nmol/mg protein/minute. noradrenaline but not vip promotes a significant increase (42%) in lactate release. the effect of noradrenaline is mimicked by isoproterenol and inhibited by propranolol, suggesting a i~adrenergic mediation. when astrocytes are incubated in an isotonic solution containing no energy suhstrate, the glycogen stores are rapidly depleted and lactate, but not glucose, is released in the medium. in conclusion, lactate appears to play a major role in cerebral energy metabolism homeostasis, as substrate released by astrocytes to prey!de energy fuel for neurones and other neighboring cells. it is known so far that calcium-binding proteins (cabps: calbindin, pan, albumin and ca/retinin) are expressed in some cells of the pineal gland of laboratory mammals. the exact phenotype of these cells is unknown. nevertheless, according to recent studies, it seems that some of them are ganglion cells. therefore we studied the pineal gland of different species (gerbils, rats, goats, cows and humans) using calretiain (cr) antibody which is considered as marker for neurons (andressen & al, cell & tissue rss, 27t:181, 1993) , the cr-expressing cells were found in all oar species: in gerbils, rats, cows and humans they were scattered throughout the parenchyrna, whereas in goats they lined mostly the pericapillary spaces. according to our findings, it seems that most of the reactive ceils are rather neuron-like elements since morphological criteria for true neurons (axosomatic synapses, nissl bodies, bundles of neurofilaments) are missing. however one cannot exclude that among these cells some are intrapineal ganglion cells, because of ranvier's nodes found along the processes of some crpositive cells. thus, it is possible that these cells are involved in the modulatory control mechanism of the pineal neurohumoral activity and/er by accumulation of intracellular calcium in the formation of pincal calcifications. besides the excitatory amino acid transmitter glutamate, its sulfur containing analogue homocystcic acid (hca) could play a role in excitatory processes in the central nervous system. hca is present in and released from nervous tissue and is a potent neuronal excitant, predominantly activating n-methyl-d-aspartate receptors (do et al., 1992) . these properties are suggestive of a classic synaptic neurotrausmitter role. on the other hand, hca seems to be localized not in neurones but in glial cells (grandes et al., 1991; tschopp et al. 1992) . the efflux of hca is temporally delayed following activation of specific pathways (klancnik et at., 1992) ; these latter observations are not in accordance with the classic concepts of synaptically-mediated neurotransmission. a laminar flow superfnsian system of monolayer cultures was developed and the released materials from mouse cortical astrocytes, previously preincubated with [35s]-methioulne, were investigated. during application of either noradrenaline or the b-adrenergie agonlst isoproterenol, the extracellalar level of [35s]-methianine was decreased and that of labelled hca was increased. this effect was not observed with the -adrenergic agonist methoxamine, and could be blocked by the ll-adrenergic antagonist propanolol these results, combined with the delayed hca release, the glial localisatian of hca and its neuroactivity, strongly suggest a potential role of hca as a gliotransmitter, modulated by noradrenaline inputs. vasoactive intestinal peptide (vip) induces long lasting metabolic effects in the mouse cns. we have investigated a shorter neuromodulatory action of vip on the synaptic responses in coronal slices of adult mouse cingulate cortex by means of intracelhilar current clamp recordings. electrical stimulation of the underlying white matter evoked a multiphasic synaptic potential consisting of early epsp~ early ipsp, late epsp and late ipsp mediated by non-nmda, nmda, gaba a and gaba b receptors, respectively. vip (0.1 ~d~l) superfused for 5 rain. had no effect, whereas concentrations over 0.2 ixm selectively and reversibly depressed the nmda-medlated responses in 12/16 cells. in the presence of cnqx (10 [tm) and biencuuine (10 ~tm), the isolated nmda-mediated late epsp was still attenuated by 0.3 ~m vip in 8/10 cells (mean:-42% al~er 30 rain.). in 3 other cells vip induced a spreading depression (sd) and a strong, reversals inhibition of the late epsp (mean:-64 % after 10 min.).these results indicate that in the absence of gabaergic inhibition vip can induce two types of effects in the mouse cingulate cortex, a prolonged decrease of the nmdamediated syaaptic response and a striking sd. interaction between vip and glutamate on c-fos mrna expression. |.-l. martin, d. gasser and p.] . magistretti. institut de physiologie, universit4 de lausanne, lausanne, switzerland. the regulation of c-los mrna expression by vip and glutamate was examined in primary cultures of neurons originating from the mouse cerebral cortex. in primary cultures of cortical neurons, vip increases camp levels and stimulates c-los mrna expression. interestingly vip stimulation is completely blocked by mk-801, an nmda receptor antagonist but not by cnqx or ap3, which antagonize ampa/kainate and metabotropic receptors respectively, c-los expression stimulated by glutamate shares the same pharmacology. these results suggest an involvement of endogenously released glutamate in the stimulation of c-fos mrna expression evoked by vip. furthermore, when added together, vip and glutamate interact synergistically to increase cfos mrna levels. consistent with the pharmacology of vip and glutamate, the synergistic interaction between vii' and glutamate on c-fos mrna expression is also inhibited by mk-801. interestingly, this synergism is completely inhibited by h-89, a protein kinase a inhibitor, whereas staurosporin and kn-62 which block protein kinases c and ca++/calmodulin dependent respectively exhibit smaller inhibitory effects. presynaptic proteins involved in neurotransmitter release (i.e. synapsin, b-50) and some postsynaptie gaba a receptor subunits possess phosphorylation sites for camp-dependent protein kinase (pka) and/or protein kinase c (pkc). the functional consequences of phosphorylation by these kinases is not known. we have studied the action of specific activators of pkc and pka, phorbol ester (pdbu) and forskolin, respectively, on gabaergic synaptic transmission in area ca3 of hippocampal slice cultures. pdbu significantly enhanced the amplitude of evoked monosynaptic ipscs (in cnqx and ap5), and both pdbu and forskolin increased the frequency of spontaneous miniature ipscs (m[pscs) in the presence of cnqx, ap5 and ttx. this effect by pdbu was antagonized by staurosporin, a protein kinase inhibitor. pdbu and forskolin did not change the amplitude or decay of mipscs, suggesting that only presynaptic kinases are involved. furthermore, pdbu enhanced mlpsc frequency by a comparable amount in the presence of the ca 2+ channel blocker cd 2+, indicating that pdbu did not enhance gaba release from presynaptic endings by facilitating ca 2+ influx into axon terminals. valiunas, v., sc~ilinsky fluri, g. and weingart, r. arachidonic acid (aa) reversibly impairs the gap junction conductance (g~) in cardiac cells. now, we examined whether aaacts directly or via its catabolites. experiments were performed on pairs of neonatal rat myocytes using a dual voltage-clamp method. we used blockers which interfere with enzymes of various catabolic pathways. pretreatment with i0 ~mpoca (blocks carnitine acyltransferase i) did not prevent the decline in g9 by i0 ~m aa; i.e. intermediates of p-oxidation are not involved. similarly, preexposure to i0 ~m indomethacin (blocks the cyclooxygenase pathway) did not prevent aa from uncoupling; this rules out an involvement of prostaglandins and thromboxanes. exposure to 10 ~mndga (blocks the 5-1ipoxygenase pathway) per se produced a decrease in gj. likewise, exposure to i0 ~m etya (blocks the 15-1ipoxygenase pathway) also impaired gj. these effects cannot result from accumulation of endogenous aa because preexposure to 50 ~m 4bpb (blocks phospholipase a 2) did not prevent ndga-or etya-induced uncoupling. exposure to 75 ~m skf 525-a (blocks the epoxygenase pathway) also produced a decrease in gj. hence, ndga, etya and skf 525-a, compounds with different biochemical actions and of different chemical structure, act on g~ either directly or via an unknown mechanism. $21-19 potential changes associated with electrical activity in nonmyelinated nerve fibres a. robert and p. jirounek d~partement de pharmacologie, cmu, 1211 gen~ve 4 simuheneous measurements of the extracellular concentration of k + ([k+]e), and of the concomitant changes in the membrane potential (era) were measured in the rabbit vagus nerve during and after a period of activity. during a 15 hz stimulation there was a large increase in the [k+]e , accompagned by a change in era, which roughly followed the depolarization expected from the increase in [k+] e . at the end of the stimulation, although the [k+]e was still elevated, the nerve developed a posttetanic hyperpolarization (pth). the pth was generated by the electrogenicity of the na+-k + pump, but its amplitude and time-course were strongly affected by two depolarizing currents: (i) an inward ba2+-sensitive current of k + and (ii) an outward current of ci-. we hypothesize that during activity and during the early phase of recovery there was a ba2+-sensitive uptake of potassium by the schwann cells. the ci" current, by short-circuiting the na+-k ⧠pump, contributes to the control of the e m, and by this way to the driving force for the inward k ⧠current. overdose of ifosfamide has ooo/red in a canc~c patient (25g i.v. iy0/24 hours with 20 g as ~utectarfe). urir~ collece{on was obtaj/3ed in 24 hour ~ fcr t~o days. c~ was measured ~ a gas liquid d~romatogr~ method of the u5~ ~ (s~@pl. v, pag.2627-262g) ~sir~ a ~e~s ar~ ni~, sensitive ths~mi(~3ic d~. ~ u~s p~mt ~ ~ ~ ~ ~ ~ 1500 m~/24h and 207 r~/24h at day one ard day res~ec~vely. 9~ _h=~=mmtly ~ has also bsm foa~ in mti~s ~ mgh e~e ~ ~ (~/o~e). w~n ~ ~s am/nist~ to rats (i~ m~/~ ~c 200 m~f~ intraperit~neally), no ~ omild be detected in urine within 24 ht~r~ after drug administl-4(cicn. from these ~ data we conclude that c~ is extensi~_ly metabolized in rats and ~ mscbard_sm of ~ el ~mlnaticn in pati~rfc t=cj_ne m~cjlr[c reflect a flmicticrsl der~,~t of c~ m~t~0olisn in man to ifo~=~de ~tion. there are numerous reports of improved memory functions in rats following a dietary supplementation with choline. in some cases, this improvement can be related to enhanced cholinergic transmission (mack et al., behav neurosci., 103, 1234 -1241 , 1989 ). but it is not clear whether there is a general improvement in memory or whether specific aspects of learning and memory are affected. this work was aimed at analysing the effects of perinatal cholinergic supplementation on the development of spatial abilities and upon adult performance. choline supplementation (3.5 g./l. in 0.02 m saccharine solution in tap water) was maintained during two weeks before birth. additional supplementation was maintained from the 5th to the 1oth week postnatal. spatial learning capacities were studied at the ages of 26, 65 or 80 days in a cimular swimming pool (morris place navigation task) and at the age of of 7 months on a homing board. adult and aged rats were tested on a radial maze. selective aspects of the performance were markedly improved. this treatment had no specific effect upon spatial memory per se, but, instead, affected learning abilities by promoting efficient behavioural strategies hypothetically based on active representation and anticipation processes. the effects of the treatment remained evident up to 6 months following the supplementation. barcelona, e-08193 bellaterra females of both lines (n=b/grp) were injected sc daily between days 15-20 of pregnancy with vehicle (v), i(di) or 3(d3) mg/kg diazepam, or not(c). in these studies(a,b) their 5-7 mo old offspring were subjected to a 50-run session in a shuttlebox(a:12 males, 6 females of each line/condition) or a 6min session in a hexagonal tunnel maze with strategically-placed barriers and a lit central arena(b_:6-7 males of each line/condition). a: the freezing behavior of ld3 rats was reduced ~s lc, this behavior reverting more to escapes in males & avoidances in females, although v had an effect in the latter also. no within-line differences in inter-trial responses were seen, but pre-session activity was increased in female lvs and ldis, returning to lc levels for ld3s. no differences existed among the h groups. b: whereas h maze activity exceeded that of l, no wtthin-line differences appeared. hd3s entered the lit arena less than hcs or hvs, indicating an increased timidity after pnd, and a trend in the same direction existed for ldi/ld3 vs lv. two groups of abstinent female smokers, performing either a fixed rate or a subject paced version of a rapid information processing task were compared. both groups participated in two test sessions (in which the task was performed twice) to compare two different types of cigarettes which were smoked before and during the second task trial: a) the subject's habitual cigarette with a nicotine yield of at least 0.7 mg/cigarette and b) a nearly nicotine free test cigarette with a tar yield comparable to that of the habitual cigarette. reaction times to correct detections (series of three odd or even digits in a pseudorandom sequence of single digits) were longer with the subject paced version and not affected by the type of cigarette but shorter with the fixed rate version and even more decreased with the smoking of the habitual cigarette. on the other hand, the pre-to posttreatment increase in the subject paced processing rate was greater with the habitual than the test cigarette. it was concluded that the two task versions assess different cognitive functions. whereas the fixed rate version primarily assesses vigilance performance and seems to be more suitable to measure changes in reaction time, the subject paced version is more sensitive to changes in speed capabilities in rapid information processing. heart rate, physical activity, and cigarette consumption were continuously recorded under field conditions, whereas subjective craving was assessed six times per day and saliva cotinine was measured once daily. abstinence, habitual cigarettes (with a nicotine yield of at least 0.7 mg/cigarette), and nearly nicotine free test cigarettes but with a tar yield comparable to that of the habitual cigarettes were compare~) in a sample of twelve female smokers for two days each. whereas physical activity was similar for the three conditions, heart rate was highest with the habitual cigarettes, lowest on abstinence days and in between with the test cigarettes. cigarette consumption was similar for both types of cigarettes and subjective craving was higher on abstinence than on smoking days but no differentiation between the two cigarette types was obtained. saliva cotinine values were highest on the days with the habitual cigarettes but lower and similar with the test cigarettes and on abstinence days. it was concluded that heart rate and saliva cotinine depended only on the amount of nicotine absorbed, whereas subjective craving was reduced by smoking independently of the actual nicotine yield of the cigarette. (ptps) is the rate limiting enzyme in the synthesis of bh, in man, a cofactor for several hydroxylases involved in catecholamine and serotonin biosynthesis. the human and rat liver cdnas encoding the 16 kda subunit of ptps were expressed and the recombinant enzymes purified to homogeneity. the apparent k~ for the substrate, pi and heat stability of the re-combinant enzymes were similar to the native enzymes. the rat enzyme was crystallized and the x-ray structure showed a hexameric native form arranged as a sandwich of two trimers. three histidine, a glutamic acid, and -also shown by site-directed mutagenesis -a cysteine residue characterize the active center of the enzyme thought to be mg2+-dependent. according to the proposed ligands we replaced mg 2* by fe 2 ⧠or mn ~+ and observed an enhanced activity up to 100%. m. neerman-arbez, v. cirnlli and p. halban. laboratoires/eantet. centre m4dical universitaire, 1211 gen~ve 4. pci(3) and pc2, members of the mammalian family of pro-protein convertases homologous to the yeast kex 2, are both expressed in pancreatic islets of langerhans and are thought to be responsible for the conversion of proinsulin to insulin and c-peptide in beta cells. however, the insulin secreting beta ceils are not the only ceils present in these complex microorgans, prompting us to evaluate the expression of pc1 and pc2 in islet beta and non-beta cells. rat islet cells were sorted by autofluorescence activated flow cytometry to separate beta cells from non-beta ceils and conversion cndoprotease levels were analysed by western blotting. in beta cells, pc1 levels were higher than pc2 (pc1/pc2=2.6+0.2) whereas the opposite was found for non-beta cells (pc1/fc2=0.05â�¢ confirming that pc1 is important for proinsulin conversion while suggesting a role for pc2 in the conversion of proglucagon, prosomatostatin and propancreatic polypeptide. transformed murine cell lines (insulin-producing beta-tc and glucagonproducing alpha-tc) were found to faithfully reflect the primary rat cells in terms of their pc1/pc2 ratios. finally, post-translational modification of the convertases themselves was found to differ between cell-types, in particular, a precursor 75 kd form of pc2 accumulated in beta cells whereas only the fully processed 67 kd form was detected in the non-beta cells. the kringle (2+3) domains (k2+3) were successfully expressed in e. coil a n-terminal hexahistidine tag was fused to insure the isolation of k2+3 by affinity chromatography on a ni-2+-ntaagarose-column. to be able to remove the hexahistidine tag a factor xa cleavage site was introduced between the 6 histidine residues and the n-terminus of the kringle structures. the correct arrangement of the disulfid bonds was determined by amino acid-and sequence analysis. the lysine binding site was proven by affinity chromatography on iysine-bio-gel, as previously shown for k2. the dissociation constant of k2+3 was measured by intrinsic fluorescence titration with 6-aminohexanoic acid. a replication protein a copurifying dna helicsse from calf thymus anthl georgakl, narendra tuteja, blrglt sturzenegger and ulrich hobscher department of veterinary biochemistry university of z0rich-lrchel winterthurerstrasse f 90 ch-8057 z0rich, switzerland a dna helicase from calf thymus copurified with replication protein a through several steps of purification including deae-sephacel, hydroxyapatite and single stranded dna cellulose. it is finally separated from replication protein a on fplc mono q where the dna helicase elutes after replication protein a. we named this new calf thymus enzyme dna helicase f. characterization of the helicase f by affinity labeling with [a32p]atp indicated that the enzyme has a catalytic subunit of 72 kda. gel filtration experiments suggested that dna helicase f can exist both in a monomeric and an oligomedc form. the enzyme unwinds in the 5' ->3' direction in relation to the dna strand it binds. all eight deoxyribonucleoside-and ribonucleosidetriphosphates could serve as an energy source. testing a variety of dnndna substrates indicated that the dna heitcase f preferentially unwinds very short substrates and is slightly stimulated by a single stranded 3'-tail replication protein a allowed the dna helicase f to unwind longer dna substrates up to 400 bases suggesting that copurification of replication protein a with the dna helicase might be of functional relevance. 2,4,5,2',4',5'-hexachlorobiphenyl (6-cb) shows limited elimination and extensive storage in adipose tissue in vivo (lipophilie unmetabolizable compound) while chlorpromazine (cpz) is known for its lysosomal storage. uptake and release of 6-cb and cpz were studied in 3t3-preadipocytes (p) and 3t3-adipocytes (a). 3t3-cells were cultivated in dulbecco's minimal essential medium supplement with 10% fetal calf serum and grown to eonfluency. differentiation was stimulated by dexamethason and bezafibrate exposure for 48h. radiolabelled 6-cb (20~tm) or cpz (5~tm) was added to the medium. following a single dose 70-80% of cpz were taken up within an hour into p and a. in contrast, 6-cb reached an intracellular plateau of 30-50% of the dose after lh in p whereas dependent on the triglyceride content a accumulated up to 95% of the dose. 6-cb uptake into a was temperature dependent and not saturable with repetitive daily doses up to 1 week. 6-cb uptake into p was fully reversible while the release from a was limited to 10% of the accumulated drug. in contrast, the release of cpz was higher from p than from a as a consequence of the different lysosomal activities of the respective cells. the use of 3t3 cells allowed to show a remarkable difference in cellular handling of neutral and basic lipophilic drugs and may be helpful to further elucidate basic mechanisms involved. chronic granulomatous disease: an attempt to reconstitute the function of the x-linked form of the disease. chronic granulomatous disease (cgd) is a group of congenital immunodeficiencies that predispose patients to recurrent severe bacterial and fungal infections. the cause of the disease is lack or impairment of 02-production in phagocytic cells due to mutations in any of the four components of the superoxide producing system. approximately 70% of all cgd patients suffer from an x-linked form of the disease where the phagocyte oxidase gp91phox is affected. this project concentrates on the reconstitution of the deficient gene in phagocytic patient cells. expression of recombinant gp91 was assayed in vitro and in vivo. attempts to "tag" gp91 with a foreign epitope were unfortunately so far unsuccessful. the target cells for gene reconstitution are non-dividing monocytes. thus, we have chosen an adenovirus over other viral systems because of: a) its ability to infect nondividing cells, b) its genetic stability and, c) lack of human adenovirus related malignancies or side-effects. we constructed two recombinant adenoviruses: one recombinant expresses a lacz gene (control), another the gp9 lphox gene. we are trying the expression of recombinants adeno/lacz and adeno/gp91 in normal and patient-derived cells (for example ebvtransformed normal and patient b-cell lines), as well as the reconstitution of nadph oxidase function in peripheral blood monocytes of cgd patients. kinetics of molecular chaperone action schmid, d., gehring, h., *baici, a. and christen, p., biochemisches institut der universit&t z~rich, ch-8057 zorich; *rheumaklinik, universit&tsspital, ch-8091 z%rich molecular chaperones of the hsp70 type transiently sequester unfolded segments of proteins and promote their correct folding. target peptides labelled with an environmentally sensitive fluorophore allowed to follow their binding to the molecular chaperone dnak of escherichia coli in real-time. the two-step process is characterized by relaxation times z~ = 27 s and z2 = 200 s at 2 ~m dnak and 0.i ~m ligand at 25~ in the presence of atp, the formation of the complex is greatly accelerated and follows a single-exponential process with z : 0.4 s. the rate of dissociation of the complex was even more increased than that of association resulting in a decreased net affinity for ligands in the presence of atp. the binding-release cycle of dnak thus occurs in the time range of polypeptide chain elongation and folding and is too fast to be stoichiometrically coupled to the atpase activity of the chaperone (ko~ ~ = 0.13 min i at 30"c). supported by the olga mayenfisch stiftung, z%rich, and the hartmann m~ller-stiftung, z~rich. comparison of the activities of native bovine seminal ribonuclease and that secreted from e. coil schein, ch* (siat, technopark, pfingstweidstr. 30, ch8005 ziirich) and haugg, m (lab. org. chemistry, e.tm., . seminal ribonuclease (bs-rnase) differs from the pancreatic rnase a in that it forms a cysteine-linked dimer and has a relatively higher specific activity in the cleavage of double-stranded (ds-) rna substrates. we expressed bs-rn in a secretion system similar to that described previously (biochem. j. 283:377-344, 1992) using the signal sequence of routine pancreatic ribonuelease. about i mg bs-rn was isolated per liter culture supernatant. human and bovine recombinant interfcron-~ (ifn-r) inhibit the degradation of ss-and ds-rna by bovine pancreatic rnase while they activate the activity of bs-rn on the same substrates (febs letters, 270:229-332, 1990) . recombinant bovine or routine pancreatic rnase (produced in our secretion system) are inhibited by ifn-y, while the recombinant bs-rn or a cysteine-linked dimer of rnase a is also activated by ifn-y. ifn-y activates bs-rn even in the presence of inhibitory concentrations (0.1-1 ram) of mononueleotides. thus the mode of activation cannot be attributed to alleviation of product inhibition. kinetic studies using 300 bp ds-rna as substrate suggest that ifn-~ interacts directly with the bs-rn to increase the rate of highmolecular weight producffsubstrate release, as the apparent ks increases proportionately to the increase in kcat. significantly smaller movements were observed in the simulation with the liganded enzyme. the structural differences between the protein in the crystal and the ensuing calculated structures might arise from the absence of lattice forces in solution. evolutionary relationships among pyridoxal-5'-p-dependent amino acid decarboxylases sandmeier, e., hale, t.i. and christen, p. biochemisches institut der universit~t zgrich, 8057 z~rich. the pyridoxal-5'-p (plp)-dependent amino acid decarboxylases (de) can be subdivided into four apparently unrelated groups. group i is represented by glycine dc, group ii comprises glutamate, histidine, tyrosine and aromatic-l-amino acid dc, group iii procaryotic ornithine and lysine dc as well as the procaryotic biodegradative type of arginine dc, group iv eucaryotic ornithine and arginine dc as well as the procaryotic biosynthetic type of arginine dc and diaminopimelate dc. (n-l) profile analysis, a more stringent application of profile analysis [gribskov et al. (1990) methods enzymol. 183, 146-159] established the homology among the enzymes of each group. a search with the profile of group ii indicated a distant relationship with aminotransferases and thus with the ~ family of plp-dependent enzymes. no evidence was obtained that groups i, iii and iv are related with other plp-dependent enzymes or any other protein in the database. apparently, the amino acid dc are of multiple evolutionary origin, in some cases even if they have the same substrate specificity. a. lo russo, a.c. passaquin, u.t. r~eg~, ecole de pharmacie, %hiv. lausanne, c~-1015 lallsaraqe drug-induced local vasoccmstricticn appears to be respmnsible for the hypertensive side effect of the imn/nosti~pressant cyclosporin a (csa). we have therefore ex~rnir~ the [ca 2+] i increase caused by the vasoccr~trictor hormcme [ar~]vascpressin (avp) in vascular ameoth ~uscle cells (vsmc) treated with csa. [ca2+] i levels were m~nitored either with a fluoresc~qce analyser using fura 2 or by 45ca 2+ efflux. pretreatment of v~mc with csa increased the avp induced rise in [ca2+]i. a leftward and upwsxd shift of the ccncentraticm-respmnse curve of the avp-induced rise in 45ca2+ efflux was also observed after csa treatxr~%t. ilzg/cticn of csa dote~tiaticn w-as detectable after 3 rain, took 1 to 2 h to become fully established and was reversed after washout. csa potentiaticm was cc~centraticn-deperdent with a threshold at 10 -7 m. %]]is potentiating effect of csa may be the underlyin~ cause for csa-ind/ced hypert~3sicn. 92) barja, f. 1 and turian, g. i, ilab. gen. microbiology, 2dept. biochemistry, 3station exp. zoology, university of geneva, 30 quai ernest-ansermet, 1211 geneva 4 the actin has been found from the simplest forms of cell to the highly evolved ones. in higher organisms it is transcribed by multigene families but in yeast and several filamentous fungi there appears to be only one actin gene. it was therefore of interest to study the expression of actin gene in n. crassa in which three actin isoforms have been characterized (barja et al., 1991, fems left. 77:19-24) . after isolating genemic dna from wild type n. crassa a pcr was performed with primers corresponding to actin consensus sequences and an amplified fragment of the gene (verified by sequencing) was obtained. this fragment was 'used as a probe in a southern to assess the number of aetin gene(s). our results suggest that n. crassa has alsc only one actin gene. the blocking effect of the n-terminal decapeptide of msmooth muscle actin (sma) (ac.eeedstalvc) on the monoclonal antibody anti-asm-1 was compared with that of synthetic peptides modified by changing the n-terminal acetyl group or by substituting a single amino acid in position 1 to 5. using immunofluorescence or immunoblotting techniques, anti-c~sm-1 activity was abolished after incubation with the native peptide and peptides modified in position 1 (only when e~a) or 5. on immunoblots running bsa crosslinked peptides on denatured gels, only the natural peptide and peptides with substitution in position 1 or 5 were detected by the antibody. our results indicate that ac.(e)eed is the epifope for anti-c~sm-l. binding of anti-~sm-1 to sma increased actin polymedzation by decreasing the critical concentration. as control this action was not exerted on skeletal actin. this is the first example of the role of a precise n-terminal sequence in the polymerization of a single actin isoform. double immunofluorescence for msma and total actin on cultured aortic sm cells microinjected with the native peptide showed a selective disappearance of msma staining suggesting that this peptide traps a protein involved in msma polymerization. work is in progress to search for the putative actin-binding protein(s) physiologically interacting with the n-terminal sequence of o~-sma. ( within mitochondria, the mechanism of selective protein degradation is poorly understood. while the bulk of mitochondrial proteins are long-lived, some are degraded rapidly. the selective degradation of mitochondrial proteins is likely to be mediated by proteases similar to those found in bacteria; this is based on the hypothesis that mitochondria have evolved from bacterial endosymbionts, in conjunction with the finding that mammalian mitochondria contain an atp-dependent proteolytic activity similar to that in bacteria. one atp-dependent protease from bacteria, lon, is of particular interest because it is involved in regulated protein turnover. degenerate oligonucleotide primers corresponding to two regions of the bacterial lon gene were used to pcr amplify a 1 kb fragment from yeast dna that encoded an open reading frame with striking similarity to the bacterial lon protease. by screening a yeast cdna library, we isolated a 3.6 kb clone potentially encoding a protein of 1133 amino acids. haploids bearing a disruptedlon gene were unable to respire or to grow on ethanol/glycerol. fractionation of rnjtochondrial matrix from wild-type cells revealed a peak of aip-dependent proteolytic activity which was absent in the disruptant. furthermore, we have identified matrix proteins that are degraded with a half-time of ~60 min in intact wild-type ceils but are stable in the ion disruptants. electron microscopy of lon disruptants revealed the presence of many electron dense bodies in the mitochondrial matrix which are not found in lon + cells. hydrolysis of high molekular weight kin1nogen by plasma kallikrein benner, s. and duffer, h., laboratory for organic chemistry, eth h6nggerberg, ch-8093 z/inch high molecular weight kininogen (hmwk) is cleaved by the serineprotease plasma kailikrein to generate the hypotensive peptide bradykinin in human blood. we established a measuring system for the time course of the hydrolysis of hmwk in its native and deglycosylated form and in the presence or absence of c~-inhibitor. analysis of the bradykinin release and the yielded protein fragments lead to the three following results: 1. the hydrolysis did not follow plane michaelis-menten kinetics due to a hmwk-fragment operating as a competitive inhibitor. 2. addition of the ci-inhibitor stopped the hmwk-hydrolysis immediately, which is in contrast to the physiological role of plasma kallikrein in blood. 3. so far we have no evidence that the high content of o-glycosylated side-chains of the hmwk-light chain has an effect on the kinin-liberation as proposed in literature. ( tetrahydrobiopterin (bh4) is the essential cofactor for the hepatic phenylalaoine hydroxylase, but also for tyrosine and tryptophan hydroxylases that are responsible for the production of nettrofransmitter precttrsors. furthermore, bh4 is required for the deavage of giyceryl ethers aztd is i~vo[ved in the biosynthesis of nitric oxide by the nitric oxide synthase. a vazia~t type of hyperphenylalani~emia is caused by a deficiency of bh4. the most frequent form of this cofactor deficiency is due to lack of 6-pyruvoyl-tetrahydropterin synthase (ptps) activity. the human cdna for ptps was isolated, and the recombinant protein was found to be active when expressed in e. coil, thus allowing us to perform hmctional t e,3ting of mutant proteins. primary skin fibroblasts derived from ~everal ptps deficient patieilts were investigated for the molecular nature of this autosomal recessive disorder. all fibroblasts had f1"ps mrna expressed, and subsequent cdna sequence analysis revealed different mutatkons in the coding region of ptps (codon replacemenls, deletions, or nonseme codons) responsible for a lowered or no enzyme activity. in order to potentially correct ptps activity (and restore bi-i 4 biosynthesis) we are establishing a recombhtant rel~oviral-mediated gene transfer system to efficiently introduce the wild-type human ptps-cdna in these fibroblasts. the thylakoid membrane (tm) contains i0 molecular species of phosphatidylglycerol (pg). the relative amount of these species depends on the plant species and growth conditions. using phospholipase a2 (pla2) at 0~ to digest preferentially pg in the outer monolayer of spinach tm, we have shown previously that this monolayer was enriched in pg (ca 70~). the purpose of this investigation was to determine the transmembrane distribution of each pg molecular species. to this aim, we have studied the hydrolysis kinetics of each species in the presence of pla 2 in spinach and squash tm containing different relative proportion of molecular species. the hydrolysis rate and extent of the molecular species depended on the unsaturation degree of the fatty acid at the sn-i position as well as on the presence or absence of 16:1(3t) at the sn-2 position. for instance, the hydrolysis rate of pg ig:3/16:l(3t) was greater than that of pg 16:0/16:0. these results will be discussed in terms of the thylakoid transmembrane distribution of each pg molecular species. (supported by the snsf 3100-33693.92). from previous electrophysiological evidence, we concluded that metal ions (hgz+,cd2+,ag +) at low concentrations (i0-6m) increase rapidly (10-60 s) cation (na + , k + ) conductance at the apical membrane of epithelial cells. we investigated here the effects of these metals on [ca2+]i in mdck-i cultured cells, for a potential correlation between functional membrane changes and [ca2+]i . metals (10-4-10 -6 m) were added at the apical side of the epithelium, and [ca2+]i was measured with fura-2. hg 2+ induced a rapid (peak 5-10 s) and marked increase in [ca2+]i, whereas cd 2+ entailed a slower (peak 2-5 min) and marked response. the time-course of effects for both metals was similar to the electrophysiological changes. in contrast, the pattern of effects of ag + on [ca2+]i differed markedly from that on apical membrane conductance. thus, the effects of metals on [ca2+]i are conspicuous but not tightly associated with changes in membrane conductance. c. bulliard and l-l. dreyer, institut de biochimie, universit6 de fribourg, ch-1700 fribourg trans-plasma membrane oxydoreductases (pmo) play a significant role in energy transduction and post-receptor signal transmission, but the enzymes have not been characterized so far. we have achieved the purification of pmo from rat brain synaptic vesicles and from synaptic plasma membranes. the membrane enzymes were extracted by 1% lubrol xl tm, precipitated with ammonium sulfate (between 40 and 70% saturation) and purified by means of hydrophobic chromatography on butyl-agarose and gel f'fltration on superose-12, followed by page under native conditions. specific enzymatic staining of native page gels yields two homogenous diaphoraselike activities, pmo-i and pmo-ii . sds-page of the enzymes showed that pmo-i is made of two subunits of 52 kda and 40 kda whereas pmo-ii consists of a single sub-unit of 52 kda. the 52 ki)a subunits from pmo-i and pmo-[i are probably identical from their respective properties. partial n-terminal sequencing (13 aa) of the 40 kda subunit from pmo-i showed 84% homology with rat brain fructose-l,6-bisphophate aldolase. these data indicate that pmo is closely associated with the glycolytic enzyme that co-purifies in all steps under suitable conditions. the biochemical functions of pmo's remain to be established. ferredoxin:thioredoxin reductase (ftr) is the key enzyme in the ferredoxin/thioredoxin system, the light-dependent regulatory system in oxygenic photosynthesis. it is a nucleus encoded ironsulfur protein, composed of two dissimilar subunits, one of which is the catalytic subunit containing a redox-active disulfide bridge functional in the reduction of thioredoxins and a 4fe-4s cluster. using pcr we have isolated and characterized a cdna clone of 738 bp from spinach and a clone of 911 bp from corn coding for the catalytic subunits including the transit peptides. the first 31 (spinach) resp. 38 (corn) amino acids after the start exhibit typical features of chloroplast transit peptides. following the transit peptides are 113 (spinach) or 114 (corn) ami[~o acids representing the catalytic subunits. the primary structures are highly conserved between these two species with 91% similarity and 81% homology. seven of the eight cys residues found in the spinach subunit and important for the catalytic activity are at conserved positions. (snf 31-28811.90 and 31-37725.93) $23-20 r. zurbriggen and j.-l. dreyer, institut de biochimie. univarsit6 de fribourg, ch-1700 fribourg most mammalian cells display transplasma membrane oxidoreductase activity (pmo). the enzymes use intracellniar reduced pyridine nucleoticle to reduce an unspecific extracellular electron accepter as substrate. in order to set up a methodology for purifying, characterizing and quantifying pmo's, the plasma membrane from a neuroblastoma cell line, nb41a3, has been biotinylated. subsequently the biotinylated proteins were purified by immanoprecipitation with avidin, antiavidin-antibodies and insoluble protein a. the protein recovery of an immunopurified membrane preparation was <0.15% of the protein content in the cell extract. specific pmo activity was increased 15 to 20 fold compared to the activity in whole cells. this approach demonstrates the presence o1' pmo within the cell plasma membrane. this activity accounts for about one third of the total cellular diaphorase activity and cannot be attributed to an increased perraeabilization of the plasma membrane induced upon biotinylation nor to intracelluiar activity from lysed cells. pmo also promotes cell growth at low substrate concentrations (0.1-ll.tm). native gel electrophoresis of iarinobiotinylated and affinity purified plasma membrane extracts displays two diaphorase-positive bands, indicating that a homogeneous cell population may express several pmo activities at the plasma membrane. fluvastatin (fs) is a new hmg-coa reductase inhibitor. its affinity for 3 major human drug metabolizing p450 monooxygenases (cyp2c9, cyp2d6 and cyp3a4) was determined in liver microsomes. fs showed low affinity (ki > 50 ~tm) for cyp2d6 and cyp3a4 whereas cyp2c9 was selectively and competitively inhibited (ki = 0.1 ~tm). the potential for in vivo fs interactions with cyp2c9 substrates was therefore investigated in 14 volunteers using dicinfenac (df) as a probe (25 mg orally) on days 0, 1 and 8. df and 4'-ho-df kinetics were determined by hplc/uv. df cmax increased over time (0.28 [sd 0.12], 0.38 [0.20] and 0.45 [0.4] mg/l on day 0, 1 and 8, resp.). oral clearance was reduced on days 1 and 8 (14 % and 15 %, resp.). a time dependent decrease in urinary metabolic ratio (4'-ho-df/df) was noted (1.07 [0.34], 0.90 [0,23] and 0.70 [0.18] on day 0, 1 and 8, resp. (p < 0.0001)) only over the first 4 hours. fluvastatin therefore exhibits a two-phase interaction in vivo: an early transient and a late cumulative inhibition. the transient phase matches inhibition profiles predicted by quantitative models integrating in rive pharmacokinetics and in vitro inhibition data (q-dips). simple competitive inhibition by fs cannot explain the late phase. other mechanisms (i.e. fs or metabolite cumulation, mi complexation) are hypothesized. in some situations fs is expected to cause interactions with cyp2c9 substrates, which are partially predicted by quantitative modeling of in vitro data (snsf project n o 32-36600.92). 1211 gen~ve, switzerland. previously, a parvalbumin mutant, pvf~02w, was constructed with a unique reporter trp in the middle of the hydrophobie center. in the present study three new parvalbumin mutants, derived from pvft~w and containing alterations essential for ca2+-binding in either one, pv.câ�¢ and pv_m, or both, pv-co~, of the two ca2+-binding sites, were analyzed in order to study the metal binding properties of individual ca~+-binding domains and their contributions to the structure and stability of the protein. both, pv_ct, and pv.~, bind i ca ~ ⧠with affinity constants, kc,, of 1.1,107 and 3.2-106 m "~ in the absence of mg 2+. mg 2+ shifts the ca~+-binding isotherms of pv.co to higher free [ca z+] according to the competition equation with km~ = 2 9 103 m t. pv.m~ is much less effected by mg z* (kmg = 80 m "t) and can be considered as possessing a ca2+-specific site. nevertheless, in the absence of ca 2 ⧠both, pv.co and pv~, bind i mole of mg 2+ with much higher affinity than predicted from the competition studies. pv_cd;~ binds neither ca 2. nor mg 2+. trp-fluorimetry and uv-difference spectroscopy revealed that the ca~*-loaded conformations of pv_co, pv.ef and pvr~o~w are similar, with the trp-102 deeply buried in the hydrophobie core. however, striking differences between the mutants are found in the metal-free and mg~+-loaded forms. the conformation of pv~t~;~ is not affected by ca 2~ or mg z*. our results indicate that destroying the ca2+-binding of the ef loop, but not of the cd loop, strongly destabilizes the protein in the absence of ca z ⧠. biochemlsches institut der unlversitfit zfirich, switzerland, ch-8057. the sea urchin, strongylocentrotus purpuratus, metallothionein gene, mta, was constructed and expressed under the control of a heat shock promoter in a protease-deficient strain of e, coll. the nascent recombinant protein was stabilised by the addition of exogenous cd. the cd-containing protein was precipitated with ethanol and subsequently purified to homogeneity by gel permeation and ionexchange chromatography. the purified protein was identified as the desired gene product by tryptic peptide map analysis, sequence analysis and mass spectroscopy. typical yields of protein were 2mg per litre of culture. the presence of 7 cd ions per molecule was confirmed by the sh/cd ratio and by the existence of 7 h3cd nmr resonances. the apparent association constants of sea urchin mt with cd, which has a charge of -8, were found to be approximately 15 times weaker than for rabbit mt, at ph 7. (mt) are small metalloproteins displaying as their main feature clusters of d i~ metal ions bound to the thiolate ligands of the abundantly occurring cysteine residues (cys). from a set of over 85 sequences a general multialignment of 62 class i mt has been obtained on the basis of the needleman & wunsch algorithm. all cys are conserved in the man%mals and over 83% of them in other vertebrates and invertebrates. on the basis of similarity/identity scores we can isolate groups of sequences. evolutionary relationships between species and groups have been calculated with the maximum parsimony method of felsenstein and with the distance matrix method of fitch and margoliash. the divergences observed among the mammalian mts indicate a partition into four distinct subforms which all may have arisen before the separation of the species in evolution and which in part may differ in function. $23-28 bourque, a., singh, a., dykeman, a., macmahon*, a., and foster*, w. atlantic veterinary college, charlottetown, canada, and *reproductive toxicology section, environmental health centre, tunney's pasture, ottawa, canada. hexachlorobenzene (hcb) is a known environmental pollutant that is produced as an industrial by-product of certain chemicals. when administered in high dosage, hcb induces alterations in the rhesus monkey ovary. a purpose of the study was to determine a no observable adverse effect level (noael) for the compound. twenty, cynomolgus monkeys, 6-13 years old were placed in five groups. hcb was given orally in concentrations of 0.01, 0.1, 1.0, and 10 mg/kg b.w. in gelatin capsules, daily, for 13 weeks. one group of animals fed glucose only, served as the control. ovary specimens fixed in 2% buffered glutaraldehyde were prepared by conventional methods for transmission electron microscopy. ultrastructural alterations were revealed in the developing ova and follicular cells in all the ovaries from hcb-treated animals in a dose-related manner. clinical chemistry was unaffected by hcb. a noael for this reproductive toxicant is yet to be determined. a.b. was a recipient of the nserc undergraduate student award. an acidic haem-and zinc-containing protein has been isolated from bovine brain. native and sds-polyacrylamide-gel-electrophoresis reveal a monomeric protein with an apparent molecular weight of 47,2 kda. the absence of co-staining with tetramethylbenzidine implies that the haem group is non-covalently bound. the electronic absorption spectrum, with a soret-band absorption maximum at 412 nm and c~-and i~-bands at 540 and 575 nm, respectively, is similar to that of fe(ll)-myoglobin, consistent with the presence of haemiron in the ferrous oxidation state. in air the protein was easily oxidized to the fe(lll) form with a subsequent shift of the soretband maximum to 405 nm. atomic absorption measurements indicate approximately 1 mole of zinc and 1 mole of iron per mole of protein. the amino acid composition is similar to that of indoleamine-2,3-dioxygenase, although no such enzymic activity has been detected. in this study, we looked for this substance and its metabolites in young and old bovine lenses, because of their possible role in cataract formation. the substances were detected by hplc analysis. the kynurenine aminotransferase (kat) activity was determinated by the method of tobes (methods enzymol 1987~ 142:217) . the fluorescent substance formed from 3-hk was characterized by thin layer chromatography followed by reaction with rtinhydrin, uv and fluorescence spectrometry, and atom bombardment for molecular mass determination. 3-hk at concentrations of 0.08-+0.01, 0.2_+0.04, and 1_+0.4 btg/g of tissue was detected in iris/ciliary body, retina, and calf lens respectively. this substance is deaminated by kat. the activity of this enzyme was 2.6_+ 0.3, 3.7_+0.2, and 9.6+_2 ~tmol/g of tissue/hour in retina, iris/ciliary body, and lens respectively of old bovine eyes, but it was not found in calf eyes. the deamination of 3-hk resulted in the formation of a fluorescent substance which was identified as oxo-xanthurenic acid (oxa) with a molecular mass of 205 daltons. the accumulation of oxa acid possibly interacting with lens proteins could induce cataract formation. snf 32-36058.92, emdo, and hartmann-mtiller. the goal of covalent immobilization of oligonucleotide capture probes to solid supports has been accomplished by light-dependent, photolinker polymer mediated immobilization strategies. synthetic oligonucleotides were immobilized by facile layer coating procedures. the procedure does not require functionalization of the oligonucleotide probe or the matedal surface. oligonucleotides were linked to polystyrene with a biopolymer which was multiply substituted by photoactivatable diazirines. capture probe photoimmobilization (350 nm irradiation) was strictly light and diazirine dependent. using radiolabeled oligonucleotides as capture probes and polystyrene as material surface, the light dependent coupling efficiency was 40%. nonspecific oligonucleotide binding was below 2 %. photojmmobilized oligonucleotides retained the ability to form stable complexes with complementary dna strands, and target oligonueleotides of varying length were captured as well as dna fragments produced by pcr techniques. indoleamine 2,3-dioxygenase (ido), a superoxide radical scavenger, is present in transparent human lenses and produces 3-hydroxykynurenine, a uv-b filter. the synthesis of 3-hydroxykynurenine by ido and its degradation by kynurenine aminotransferase (kat) were measured in transparent lenses and cataracts. post-mortem eyes of elderly persons between 72 and 84 years of age with transparent lenses were obtained from the eye-bank of the department. fresh cataracts were obtained from cataract surgery patients. ido activity was measured by the method of yamazaki et al (biochem j 1985; 230:635) adapted for hplc. kat activity was measured by the method of tobes (methods enzymol 1987; 142:217) . ido activity found in post-mortem transparent lenses (cortex and nucleus) was 0.7_+0.3 nmol/mg protein/hour. in cataracts a low 1do activity of 0.3_+0.2 nmol/mg protein/hour was found in the cortex but not in the nucleus. kat activity, found in the eight cataracts examined, was 1.6_+0.9 nmol/mg protein/hour. inhibition of ido activity and induction of kat activity seems to be involved in human cataract formation snf 32-36058.92, emdo, and hartmarm-m011er photolinker polymer mediated covalent immobilization of antibodies and f(ab') fragments has been achieved by newly established lightdependent coupling procedures. anti alpha-l-fetoprotein monoclonal antibodies and f(ab') fragments derived from anti prostate specific antigen monoclonal antibodies were covalently linked to microplates. diazirine derivatized bovine serum albumin served as multifunctional light activatable linking agent. both immunoreagents, monoclonal antibodies and f(ab') fragments remained immunologically active after 350 nm irradiation (irradiance 0.7 mw cm -2 for 20 minutes). covalency of antibody binding was inferred from i) the photoreagent dependence, ii) the light dependence of the immobilization process, and iii) the reversibility of immunocomplexation after acid treatment. $23-33 two ~ of extracelluiar elecirical resistance in v~kwricular myocardil~ fleischhauer j.c., kl~ber a.g., dept. of physiol. bern in myocardial tissue, the electrical resistive properties of the extracellular space are important for local current flow during e~citation and therefore influence impulse conduction and the magnitude of the extracellular electrical field. to assess the r~tlti~t nature of the resistance of the extracellular space, electrical cable analysis was applied on an arterially perf~-sed rabbit papillary mascle. ~he resistivity of the intravascular space was changed (70 to 220~i~n) by variation of hematocrit frcrn 0 to 60% in the perfusate. in order to change the voltm~ and hence the electrical resistance of the interstitial space, colloidosmotic pressure in the perfusate was varied by changing dextran {m r 70000) concentration (i0 to 80g/l). altering the interstitial space volume had a marked influence on the extracellular resistance (ro) , conduction velocity and the magnitude of the extracellular field. variations of the resistive properties of the intravascular space had no significant influence on ro, conduction velocity and the magnitude of the extraeellular electrical field. our results suggest that the microvascular tree is insulated from the interstitial space and local electrical current flow during excitation in heart muscle is confined to the narrow interstitial clefts. s04-25, s 15-69 s05-18, s05-19 s 11-05, s11-06 bchini hooft van huijsduijnen s08-12 s05-30 s15-45 s08-13 s08-05, s08-06 s01-08 s15-67, $23-19 s13-03 nook, ek s01-15, s10-15 s05-02, s05-05 s 15-03, s15-29, s15-30 s19-03, s19-04, $19-05 s16-02, $16-15 s12-22 s15-48 s05-24 s03-01, s03-02, s03-03 s03-01 lrmler s05-28, s09-06 s03-07, s03-10, s03-11 s18-05 lipp, p. s05-26 so 1-02 s15-15, s15-16, $21-03, $21-11, $21-12, $21-14 $23-29,$23-30 s01-17 s 15-29, s15-30 s12-12, s12-24 molar s15-19 s08-05, s08-06 e s09-06, s09-07 s15-03, s15-29, s15-30 e. s03-05 s05-09, s05-10, s05-51 s05-52 s10-16 s08-14 s04-23, s04-29, s05-13, s05-53, $20-15 s10-16, s10-19 s09-01, s09-02 s15-68, s 17-24 stalder h. s03-19 s10-21 s09-09, s13-10, s13-13 s10-21 teheesen s01-10, s01-11, s01-18 s15-16 van dillewyn s05-32, s05-37, s05-38 s08-05, s08-06 s05-18, s05-19 huber d., ojha m. and turian g. laboratory of general microbiology, university of geneva, sciences iii, 30 quai ernest-ansermet, 1211 geneva 4, switzerland. ca2+-dependent protease in allomyces is predominantly localized in the apical region of the exponentially growing hyphae (huber and ojha, submitted) . many cytoskeletal proteins like actin and tubulin are also mainly localized in this growing region (heath, l~91). it is known that the ca2+-dependent protease proteolyzes a number of cytoskele~ ton proteins in order to regulate the plasticity of the cell (mellgren, 1987) . we have studied the proteolysis of some cytoskeletal proteins by this protease purified from the aquatic fungus allomyces erbuscula. actin, tubulin and desmin, were found to be rapidly proteolyzed in vitro at a high substrate to enzyme ratio. immunofluorescence studies of proteolyais made in situ in exponentially growing hyphae showed modification of apieally localized cytoskeletal proteins. this colocalization of the ca2+-dependent protease and cytoskeletal proteins in the same apical region is inferred in relation to hyphal elongation. jbiological databases grow exponentially. in order to provide access, as much data las needed and as little volume as possible shall be returned upon a query. the icurrentiy available dna and protein sequence databases are updated in a sophisticated network of procedures and expose a considerable amount of redundancy. research is performed on optimizing the creation of non-redundant data sets. the resulting data are disttibuted in switzerland, or made accessible to researchers who cannot utilize local resources. transparent access to the swiss resources, as well as paneuropean services, is provided with a job scheduling system which was developed in basel, the hierarchical access system for sequence libraries in europe ("hassle"), and various other computer programs which allow comprehensive browsing such as "gopher" and "www". training courses to use the resource effectively are running throughout the year. key: cord-006229-7yoilsho authors: nan title: abstracts of the 82(nd) annual meeting of the german society for experimental and clinical pharmacology and toxicology (dgpt) and the 18(th) annual meeting of the network clinical pharmacology germany (vklipha) in cooperation with the arbeitsgemeinschaft für angewandte humanpharmakologie e.v. (agah) date: 2016-02-06 journal: naunyn schmiedebergs arch pharmacol doi: 10.1007/s00210-016-1213-y sha: doc_id: 6229 cord_uid: 7yoilsho nan in vitro systems and mechanistic investigations i the demand of alternative test systems which closely mirror the in vivo situation is one of the main challenges in modern toxicity testing. the major goal is the development of in vitro systems that partly display the complexity of an organism and thus may mimick in vivo conditions. despite great efforts in the past no adequate in vitro systems are available yet. on the other hand, cell cultures from almost every organ are easily accessible and therefore may help to roughly assess the toxic potential of substances at target structures. nonetheless, the complex interactions which take place in vivo cannot be addressed in single cell cultures. in the liver, hepatocytes comprise 80% of the total liver volume while non-parenchymal cells -endothelial cells, stellate cells and kupffer cells (that is, liver resident macrophages) -contribute only 6.5% of the volume, but 40% of the total cell number (kmiec 2001) . it has been increasingly recognized that in the liver neighboring non-parenchymal cells release molecules which contribute to the inflammatory damage and even aggravate it (adams et al. 2010) . in our project a human in vitro co-culture system was established by combining a hepatic and a monocytic cell line, the latter of which can be differentiated to a macrophage-like phenotype. in this system the hepatotoxicty of substances has been analyzed, and the results were compared to single cultures and to published data from in vivo studies. using ketoconazole, an antifungal, as a known hepatotoxic substance, inflammatory markers were studied and proved to be significantly upregulated only in coculture. conversely, cultures of hepatic cells only did not display this increase in inflammatory markers. at the same time, a negative substance, caffeine, failed to show any hepatotoxic potential in the co-culture system. our results demonstrate that this novel in vitro co-culture model represents a promising tool to evaluate the hepatotoxic potential of substances. in drug research, it might help to reduce animal testing as drugs with a high dili potential can be dropped early in the development phase. question: raman spectroscopy (rs) is a highly sensitive analytical method for markerfree and non-invasive identification and characterization of cells. here, we present rs as a novel tool for gentle yet precise cell analysis in three independent experiments, focusing on monitoring cellular reactions upon treatment. we could provide evidence that rs is a suitable tool to monitor cell differentiation, analyze cell modification and study cell apoptosis after drug application. methods: in a first experiment, mesenchymal stem cells (mscs) were treated with erythropoetin (epo) for certain time points and subsequently fixed with paraformaldehyde (pfa) for raman analysis. in addition, skbr3 breast cancer cells were exposed to the anti-cancer drug herceptin (20μg/ml). cells were then fixed in pfa for rs. in a last experiment, molm-13 cells were separately cultivated in microwells and treated with thymidine for different time points prior to raman analysis. results: raman spectroscopy was able to monitor differentiation of epo treated mscs and found that around 35% of treated cells showed fibroblast like raman profiles. in case of herceptin treated skbr3 cells, rs found internal changes of the cell´s metabolism as reaction on drug application. analyzing the most prominent differences in raman spectra revealed discrimination of cells to be mainly due to changes in amid i, lipid and protein content. in the last experiment, rs was able to follow apoptosis of molm-13 cells after thymidine application and discriminate early from late apoptotic states. discussion: rs is a photonic marker for gentle yet highly specific cell analysis, which allows monitoring of single cell reaction after drug treatment. thereby, rs provides information about changes within the entire metabolome on a single cell level. raman spectra are as characteristic as a "fingerprint". rs works label-free and non-invasive and thus does not impare cell viability. this allows to gain new insights in pharmacological development and toxicological survey. acknowledgement: this project received funding from the eu 7th health program grant agreement no 279288. deutsches zentrum für herzinsuffizienz, würzburg, germany extracellular signal-regulated kinases 1 and 2 (erk1/2) are essential for the regulation of cell growth and cell survival and their kinase activity is up-regulated for example in different types of cancers and pathological cardiac hypertrophy. while inhibition of erk1/2 activity by kinase inhibitors prevents tumor growth, it can also lead to exacerbated cardiomyocyte death and impaired heart function. interestingly, we have previously identified an erk autophosphorylation at threonine 188 as a prerequisite for nuclear erk1/2 signaling and erk-mediated cardiac hypertrophy. here, we investigated an alternative strategy to interfere with erk1/2 signaling: since activation of erk1/2 triggers erk dimerization, a prerequisite for erk t188autophosphorylation, we chose the erk dimer interface as a possible target to selectively interfere with erk t188 -phosphorylation. first, we investigated the impact of monomeric erk2 on cardiac function. to address this issue, we generated mice with cardiac overexpression of monomeric erk2 ∆174-177 and performed transverse aortic constriction (tac) to induce cardiac hypertrophy. compared to wild-type mice, erk2 ∆174-177 overexpression attenuated tac-induced cardiac hypertrophy, interstitial fibrosis and mrna expression levels of collagen and brain natriuretic peptide (bnp), while cardiomyocyte survival and cardiac function were largely preserved. because of the positive effects of monomeric erk ∆174-177 in the heart, we designed a peptide to interfere with endogenous erk dimerization. cross-linking and coimmunoprecipitation experiments showed that the peptide binds to erk2 and prevents its dimerization. moreover, the peptide effectively inhibited erk t188 -phosphorylation and nuclear translocation of yfp-tagged wild-type erk2 after phenylephrine stimulation. further, adenoviral or adeno-associated virus serotype 9 (aav9)-induced overexpression of the peptide in neonatal rat cardiomyocytes (nrcm) or mouse hearts resulted in a significantly reduced hypertrophic response to phenylephrine or tac, background: g i -proteins have been proposed to be cardioprotective. it's matter of debate whether this depends on the particular g i isoform and/or on the particular conditions (e.g. cardiac "stress") only. in our study we investigated effects of a gα i2knockout on cardiac function and survival in a murine heart-failure model of cardiac β 1adrenoceptor overexpression. methods and results: β 1 -adrenoceptor overexpressing mice lacking gα i2 (β 1 -tg/gα i2 -/-) were compared to wild-type (c57bl/6) mice and littermates either overexpressing cardiac β 1 -adrenoceptors (β 1 -tg) or lacking gα i2 (gα i2 -/-). at 300 days of age mortality of mice only lacking gα i2 was higher compared to wild-type or β 1 -tg, but similar to β 1tg/gα i2 -/mice. beyond 300 days mortality of β 1 -tg/gα i2 -/mice was enhanced compared to all other genotypes (mean survival time: 363±21 days). echocardiography revealed similar cardiac function of wild-type, β 1 -tg and gα i2 -/mice, but significant impairment for β 1 -tg/gα i2 -/mice (e.g. ejection fraction 14±2% versus 40±4% in wild-type mice). significantly increased ventricle-to body-weight ratio (0.71±0.06% versus 0.48±0.02% in wild types), left-ventricular size (length 0.82±0.04 cm versus 0.66±0.03 cm in wild types) and anp and bnp expression (mrna: 2819% and 495% of wild type, respectively) clearly indicated hypertrophy. gα i3 was significantly upregulated in gα i2 -knockouts (protein compared to wild-type mice: 340±90% in gα i2 -/and 394±80% in β 1 -tg/gα i2 -/-, respectively). radioligand binding experiments confirmed cardiac overexpression of β 1adrenoceptors in β 1 -tg mice. of note, overexpression levels differed depending on the particular wild-type background. on an fvb/n background we found the overexpression level to be more than 2-fold higher (b max : 1425 ± 68 fmol/mg) than on the otherwise used c57bl/6 background. accordingly, fvb/n-based β 1 -tg mice showed a significantly impaired cardiac function at an age of 300d while c57bl/6-based β 1 -tg mice did not. conclusions: gα i2 -deficiency combined with cardiac β 1 -adrenoceptor overexpression strongly impaired survival and cardiac function. on a c57bl/6 background β 1adrenoceptor overexpression alone had not induced cardiac hypertrophy or dysfunction at day 300 while there was overt cardiomyopathy in mice additionally lacking gα i2 . we propose an enhanced effect of increased β 1 -adrenergic drive by lack of protection via gα i2 . the observed gα i3 upregulation was not sufficient to compensate for gα i2 deficiency suggesting an isoform-specific and/or a concentration-dependent mechanism. the role of gα i3 is currently addressed in a subsequent study using β 1 -tg and gα i3deficient mice. heart failure is accompanied by morphological and functional alterations (e.g. hypertrophy, decreased contractility) which are summarized by the term "cardiac remodeling". while the β-adrenergic signaling pathway is essential for short-term modulation of cardiac performance, its chronic stimulation by elevated plasma catecholamines and the subsequent activation of camp-dependent signal transduction pathways is regarded as a fundamental factor in the pathogenesis of cardiac remodeling. however, the mechanisms mediating the transition of physiological conditions of short-term to detrimental remodeling under long-term β-adrenergic stimulation are not understood in detail so far. in this context, icer, an isoform of the camp dependent transcription factor crem (camp responsive element modulator), acts as an early response gene strongly induced by beta-adrenergic stimulation via camp responsive elements (cre) in its promoter. contrary to its cre-mediated induction, icer is a strong inhibitor of cre-mediated transcription by itself. here we study the role of icer induction in the catecholamine-induced cardiac remodeling in a time dependent manner by the use of icer deficient mice (iko) and wild type (wt) controls, which were treated with isoproterenol (iso; 10 mg/kg per d) for 6 and 24 hours and 7 days. overall 7 days of iso stimulation resulted in an elevation of cardiomyocyte length in iko (in µm; 7d iso 156±1) vs. wt cardiomyocytes (7d iso 143±2). at this time point a 29 % decrease of cardiac output and a 16 % decrease of the maximal rate of rise of left ventricular pressure (dp/dt max ) in iko vs. wt animals was detectable. the maximum increase of icer mrna in wt cardiomyocytes already occurred after 6 h (75-fold), and declined after 24 h (29-fold) to 2.5 fold increase after 7 days, while icer mrna was not detectable in iko mice. this raised the hypothesis, that the early induction of icer modulates transcriptional processes after beta-adrenergicstimulation, involved in cardiac remodeling of the heart. profiling of mrna expression levels between iko vs. wt cardiomyocytes at the different time points revealed: 55 regulated genes (up-regulated: 45 %) in untreated; 103 altered genes (up 35 %) after 6h; 1437 changed genes (up 97%) after 24 h and 131 altered genes (up 19%) after 7 days of iso treatment. in summary, the absence of icer induction in myocytes resulted in an increase of cardiomyocytes length and a decrease of heart performance after 7 days of betaadrenergic stimulation. this is preceded by upregulated mrna levels of several hundred genes at 24 h, which is going along with the induction of transcriptional inhibitor icer after a few hours of beta-adrenergic stimulation. this suggested a protective role of icer by inhibiting the progression of cardiac remodeling after betaadrenergic stimulation in an early responsive manner. (supported by the dfg) the performance of the adult heart is tightly regulated by g protein-coupled receptors. adrenergic and angiotensin receptors efficiently control heart rate and contractility. muscarinic receptors, on the other hand serve as master regulators of the conduction system, which is often lost upon myocardial infarction. this function of muscarinic receptors has been well described in the adult or late embryonic heart. here we provide evidence that muscarinic receptors are crucial to constrain pacemaker cell identity. we applied subtype-specific inhibitors of muscarinic receptors to zebrafish embryos of different stages. we observed that both, early cardiac function as well as specification are specifically regulated by muscarinic m3 receptors, while m2 receptors appear to exert a heart-specific function only at later stages. continuous m3 blockage renders zebrafish with greatly altered cardiac morphology, particularly of the conduction system. furthermore, embryos with m3 inhibition display impaired ventricular function most likely due to an av-block and substantial arrhythmia in the atrium. importantly, to observe these phenotypes it was sufficient to block m3 receptors during stages of cardiac differentiation, which is long before a heart tube has formed. we corroborated our findings regarding these morphological changes using marker gene analysis. furthermore, we obtained evidence for m3 receptors preventing a transcriptional program towards the induction of pacemaker cells at the expense of av canal cells. importantly, this is not only true during heart development. a pacemaker program is also induced in adult hearts upon m3 inhibition. taken together, we postulate that muscarinic m3 receptors confine a pacemaker lineage during early steps of heart development as well as in the adult heart. our data suggests m3 receptors as potential new therapeutic targets for the regeneration of hearts with an injured sinoatrial node. systemic inhibition of mir-21 has proved effective against fibrosis of the myocardium and in other organs. mir-21 has been reported to exert detrimental effects in cardiac fibroblasts and protective roles in cardiac myocytes and other myocardial cell types. a better definition of the cell types that contribute to the beneficial effects of inhibiting mir-21 in vivo may aid the development of strategies with enhanced therapeutic efficacy. thus far, no approach to selectively manipulate micrornas in the non-myocyte population of cardiac cells in vivo has been available. in this study, we developed an icre-encoding mml virus for application in mir-21 fl/fl mice. delivery of this vector to neonates achieved targeted genetic ablation of mir-21 in non-myocyte cardiac cells. immunohistochemistry and flow cytometry confirmed that mmlv was highly selective and effective for cardiac fibroblasts and endothelial cells. in parallel, an aav9-icre vector allowed for specific and almost complete deletion of mir-21 in cardiac myocytes. when tested in a model for chronic left ventricular pressure overload, mmlv-icremediated deletion of mir-21 in cardiac fibroblasts and endothelial cells significantly reduced cardiac fibrosis and hypertrophy and improved cardiac function. the benefit of this cell-type-specific inhibition exceeded that observed upon global genetic deletion of the mir-21 gene in mice. aav9-mediated deletion of mir-21, albeit lowering cardiac hypertrophy, had no effect on fibrosis or cardiac function. taken together, neonatal delivery of engineered icre-encoding viruses enabled for the first time a differential gene targeting in non-myocyte and myocyte cells in myocardium. non-myocyte deletion of mir-21 demonstrated that mir-21 exerts its cardiac profibrotic activity directly in cardiac fibroblasts and in endothelial cells. this novel finding should encourage tailoring of antimir-21 therapy towards cellular tropism. chronic inflammatory diseases, such as psoriasis or rheumatoid arthritis, are characterized by constant leukocyte infiltration and ongoing angiogenesis in the inflamed tissue. as current anti-inflammatory pharmacotherapy is not always satisfying, there is a great demand for the discovery of new drug leads as well as novel drug targets. the synthetic carbazole alkaloid derivative c81 acts as a multikinase inhibitor. results of a thermal shift assay revealed that c81 shows by far the highest binding affinity to the bmp-2-inducible kinase (bmp2k/bike). bmp2k represents an as yet largely uncharacterized protein, which is not regulated by bmp-2 in endothelial cells. therefore, we aimed to analyze (i) the pharmacological potential of c81 and (ii) the role of bmp2k in angiogenic and inflammatory processes in the vascular endothelium. initial experiments show that only high concentrations of c81 affected the viability of human umbilical vein endothelial cells (huvecs). both c81 and the knock-down of bm2k (rnai) reduced the migratory capacity of a human microvascular endothelial cell line (hmec-1). also the proliferation of hmec-1 was reduced by c81 treatment (ic 50 : 7 µm). a tube formation assay on matrigel demonstrated that c81 significantly impaired the formation of capillary-like structures in a dose-dependent manner. interestingly, the analysis (western blot) of signaling molecules in huvecs that play a crucial role in cell proliferation (e.g. erk, akt) revealed that these pathways are not influenced, neither by c81 treatment nor by bmp2k gene silencing. in regard to inflammatory processes, c81 treatment or bmp2k silencing of huvecs decreased the adhesion of thp-1 cells, a monocytic cell line, onto the activated endothelial cells. as the interaction of leukocytes is mainly mediated by cell adhesion molecules (cams), the effect of c81 or bmp2k silencing on their expression was analyzed (flow cytometry, qpcr). while the expression of cams was strongly decreased after c81 treatment, the knock-down of bmp2k did not markedly affect their expression. furthermore, both approaches did not lead to the reduction of tnf-induced iκbα degradation (western blot) or p65 translocation into the nucleus (microscopy). our study provides first insights into the anti-inflammatory and anti-angiogenic potential of the carbazole alkaloid derivative c81 in vitro. the precise role of bmp2k in angiogenic and inflammatory endothelial processes as well as the involved pathways during bmp2k silencing and c81 treatment will be further elucidated. moreover, since the inhibition of bmp2k seems not to be responsible for all actions of c81, we will investigate the role of other kinases affected by the compound in these processes. the chemokine receptor cxcr4 is a multifunctional receptor which is activated by its natural ligand c-x-c motif chemokine 12 (cxcl12). cxcr4 seems to be part of the lipopolysaccharide sensing complex, suggesting that an intervention with cxcr4 agonists or antagonists could result in reduced tlr4 signaling. however, the role of cxcr4 and the influence of different cxcr4 ligands in acute as well as chronic inflammatory diseases are still contradictious. therefore, we aimed to characterize the systemic effects of cxcr4 activation in severe systemic inflammation and to evaluate its impact on endotoxin induced organ damages by applying a sublethal lps dose (5 mg/body weight) in mice. the plasma stable cxcl12 analog ctce-0214d was synthesized and administered subcutaneously shortly before lps treatment to ensure a delayed release and thereby a prolonged effect of the drug. 24 hours following lps administration, mice were sacrificed and blood was obtained for tnf alpha, ifn gamma and blood glucose evaluation. additionally, histopathological changes and oxidative stress in the liver and spleen were assessed and liver biotransformation capacity was determined. finally, cxcr4, cxcl12 and tlr4 expression patterns in liver, spleen and thymus tissue as well as the presence of different markers for oxidative stress and apoptosis were evaluated by means of immunohistochemistry. ctce-0214d improved the health status and distinctly reduced the lps mediated effects on tnf alpha, ifn gamma and blood glucose levels by approximately 35%, 50% or 70%, respectively. it attenuated oxidative stress in the liver and spleen tissue and enhanced liver biotransformation capacity unambiguously. ctce-0214d diminished the lps induced expression of cxcr4, cxcl12, tlr4, nf-κb, cleaved caspase-3 and gp91 phox, whereas heme oxygenase 1 expression and activity were induced above average. furthermore, tunel staining revealed anti-apoptotic effects of ctce-0214d in all organs. the cxcr4 is undoubtedly involved in inflammation. its activation was accompanied by anti-inflammatory, anti-oxidative and cytoprotective effects as ctce-0214d attenuated tlr4 signaling, induced heme oxygenase 1 activity and mitigated apoptosis. thus, the administration of cxcl12 analogs seems to be a promising treatment option to control acute systemic inflammation, especially when accompanied by a hepatic dysfunction and an excessive production of free radicals. the neurodegenerative disease friedreich ataxia (frda) is caused by a gaa triplet repeat expansion in the first intron of the frataxin gene, which results in a reduction of the corresponding mitochondrial protein. despite several cellular and animal models the exact function of frataxin is still a matter of debate, but the role of frataxin in iron sulfur cluster biosynthesis is generally accepted. however, we still don't know which primary metabolic events are caused by a frataxin deficit and until now, there is no therapeutic option available. we developed a new cellular model for frda by using the cre/loxp recombination system in mouse embryonic fibroblasts (mef). c57bl/6j mouse strains with a loxp flanked exon 4 of the frataxin gene and an tamoxifen-inducible cre-recombinase (creer t2 ) were crossed and several mef cell lines isolated. after selection by genotype and growth manner the fx-mef 2-1 (fxn -/-) and fx-mef 2-8 (fxn +/-) cell lines were finally choosed. the generation of the homozygous or heterozygous frataxin knockout was successfully tested on rna and protein level. long maintenance of the frataxin depleted fibroblasts revealed a strong growth inhibition consistent to earlier observations in other cell systems. therefore, we established a pattern of treatment over 12 days, with medium and substance changes at day 4 and 8, which allows us to get a fully functional knockout and overcome the growth inhibition problem. endpoint measurements of known metabolic phenomena from mammalian and non-mammalian models were studied at day 12 of our novel cell system. the induced total disruption of frataxin leads to a clearly reduced aconitase activity, cell division and oxygen consumption as well as an increase in ros production. in the heterozygous knockout with residual frataxin activity no such changes were observed. in addition, our pattern of treatment enables us to monitor the full and partial frataxin knockout in the course of time, to detect early and late metabolic events after frataxin disruption. therefore we analysed the mentioned parameters (with additional atp and iron content) in parallel at day 3, 5, 7 and 10 and could identify an initial event followed by secondary consequences and parameters, which seem to play only a minor role in the frda pathogenesis. on the contrary, a partial deficit of frataxin didn't result in any differences over time and suggests that there are only cellular alterations below a critical threshold. in conclusion, our new established mammalian cellular frda model mimics typical metabolic consequences of the human disease and seems to be qualified for frda research. the model shows for the first time six different metabolic events in the course of time in parallel and reveals insights into primary and secondary events of frda pathogenesis. these observations can be used to better understand the function of frataxin and can help to develop new therapeutic strategies to address the consequences of frataxin deficiency. moreover, the transfer of this cell model into 96well plates offers the possibility for a high-throughput screening of potential therapeutic substances. the disease diphtheria is caused by the diphtheria toxin (dt) which belongs to the group of single-chain ab-type bacterial protein toxins. receptor-binding of the b-domain on the target cell surface is followed by receptor-mediated endocytosis and internalization into early endosomal vesicles. endosomal acidification triggers membrane insertion and pore formation of the transmembrane (t) domain together with translocation of the (partially) unfolded catalytic (c) domain into the cytosol. herein, dta catalyzes adp-ribosylation of elongation factor 2 which leads to disruption of protein synthesis and finally causes cell death [1] . in hela cells, these events are related to cell-rounding functioning as a specific endpoint to monitor the uptake of dta into the cytosol of the host cell. as for other adp-ribosylating toxins such as c. botulinum c2 toxin, c. perfringens iota toxin and c. difficile cdt, also in case of native dt we demonstrated the involvement of several host cell factors during the translocation step of the catalytic domain across the endosomal membrane [2, 3] . in detail, we confirmed the involvement of the host cell chaperone hsp90 and the thioredoxin reductase (trxr), the latter presumably responsible for the reduction of the interchain disulfide bond between the dta and dtb moieties [4, 5, 6] . furthermore, we identified another group of protein folding helpers, the family of peptidyl-prolyl cis/trans isomerases (ppiases) including cyclophilin a (cypa), cyp40 and fk506-binding protein (fkbp)51 as required cytosolic factors for dta translocation. to characterize the role of the protein folding helpers in more detail, we investigated their interaction with purified dta in vitro by performing dot blot analysis with immobilized recombinant host cell factors, co-precipitation of cellular factors with dta from hela lysate and isothermal titration calorimetry with purified proteins therewith determining the thermodynamic parameters of the individual binding events. thereby, we detected binding of dta to hsp90, cypa, cyp40, fkbp51 and fkbp52. the data increase the knowledge of the molecular mechanisms underlying dt uptake and especially dta translocation which can be medically used to develop novel therapeutic strategies against the disease diphtheria. [1] murphy (2011) toxins 3, 294-308. [2] barth (2011) naunyn-schmied arch pharmacol 383, 237-245. [3] kaiser et al. (2012) cell. microbiol. 14, 1193-1205. [4] dmochewitz et al. (2011) cell. microbiol. 13 , 359-373. [5] ratts et al. (2003) j. cell biol. 160, 1139-1150. [6] schnell et al. (2015) novel afflictions as for example clostridium (c.) difficile associated diseases (cdad) caused by clostridium difficile are on the increase and challenging to treat. cdad most frequent occur in hospitalized patients after prolonged treatment with antibiotics. cdad includes among others diarrhea and the severe form of pseudomembranous colitis. not only the treatment of the infection, but also the treatment of the toxins has a high clinical significance. c. difficile secretes the exotoxins a (tcda) and b (tcdb), which glycosylate and thereby inactivate rho-gtpases in mammalian cells. tcda and tcdb are considered as the causative agents of cdad. in the last few years, more and more hypervirulent strains of c. difficile were described. in these hypervirulent strains, the adp-ribosyltransferase cdt was found as a third toxin in addition to tcda and tcdb. given the lack of agents effective against antibiotic-resistant bacterial strains and bacterial exotoxins, the development of novel pharmacological strategies is needed. the antimicrobial activity of naturally occurring substances is already known for a long time. one important mechanism of the innate immune system is the production of natural peptides showing antibiotic features. in recent years, it was shown that human antimicrobial peptides as important part of the native innate immune system plays a crucial role not only in inactivation of bacteria but also in inhibition of bacterial toxins (1). prompted by these result, we found that only human α-defensin-1 (hnp-1) but not human β-defensin-1 (hbd-1), both important effectors of the innate immune system, protected cultured epithelial cells from intoxication with tcda and cdt when applied prior to the toxins to the cells. moreover, α-defensin-1 prevented also the cytotoxic effects of all three c. difficile toxins tcda, tcdb and cdt combined in the medium. the combined investigation of all three toxins might be even more suitable to mimic the situation after an infection with hypervirulent c. difficile. the inhibition of the toxins was monitored by cell rounding caused by each of the toxins. currently, the molecular mechanisms underlying the inhibitory effects are still unknown and will be investigated in different cell lines. in conclusion, our results demonstrate that hnp-1 causes a loss of cytotoxicity of the c. difficile toxins and may act as novel drugs to cure c. difficile infections that contribute to cdad. neurodegenerative diseases like parkinson´s disease (pd) are accompanied by altered gene expression levels in the brain. recent studies support a role of regulatory noncoding rnas, such as micrornas (mirnas), which silence a specific set of mrnas at the post-transcriptional level. upon their aberrant expression, they are likely involved in the pathophysiology of specific neuronal loss. manipulation of neuronal gene expression is pivotal for understanding the function of proteins and the development of new therapeutic strategies. rna interference (rnai) strategies can be employed through the administration of small interfering rnas (sirna), which mediate the specific knockdown of a selected target gene. however, the main challenge is the delivery of these rnas into the neurons of interest. in this pilot study, we present a method for delivering sirnas in polymeric nanoparticles based on low molecular weight polyethylenimines (peis). their intracerebroventricular (icv) injection leads to in vivo silencing of neuronal gene expression in the brain of mice overexpressing α-synuclein (thy1-asyn mice). in a first step, pei-complexed sirna tagged with afluorescencedye were injected to track the localization and distribution after icv administration. five days later, fluorescent cells were visible throughout the brain, with the highest fluorescence intensity around the ventricles. fluorescence was also observed in large cells of the lumbar spinal cord. moreover, preliminaryresultsdemonstrate a 42.6% knockdown (p<0.05 student's t-test, n=6) of human α-synuclein (snca) in thetargetstructurestriatum upon a single icv injection of pei-complexed specific sirna compared to the control injection group (n=9). hence, our first results support the usability and efficacy of pei nanoparticle-mediated delivery of short rnas, namely sirnas, for rapidly and efficiently reducing the expression of a neuronal target gene of interest in the brain in vivo. this may allow the development of gene therapy strategies for the treatment of neurodegenerative diseases. is a propenylic alkenylbenzene found in several plants, e.g. acorus calamus. bacontaining plant materials are used to flavor foods, and are active ingredients in traditional plant medicines. thus, human exposure results primarily from the intake of bitters and teas, as well as from calamus-containing medicines and plant food supplements. although many (positive) pharmacological properties/effects of asarone isomers are described in the literature, ba was found to be carcinogenic in rodents (liver, duodenum) when given daily or in a single dosage. early experiments indicated that ba is not activated via hydroxylation and sulfonation as it is the case for known hepatocarcinogenic allylic alkenylbenzenes such as estragole or methyleugenol. because the mechanism of metabolic activation of ba in not known, we investigated the metabolism of ba in liver microsomes and human cytochrome p450 (cyp) enzymes, the mutagenicity of ba and its metabolites in the ames fluctuation assay and the dna adduct formation in primary rat hepatocytes. we found that side-chain epoxidation (leading to diols and a ketone) was by far the most dominating metabolic route of ba in liver microsomes and human cyp enzymes. ba was mutagenic in the ames test (+s9 mix), as was the synthesized ba-epoxide (-s9 mix). furthermore, we were able to synthesize and characterize a ba epoxide-derived dna adduct with deoxyguanosine. this dna adduct was formed in a concentration-dependent manner in rat hepatocytes incubated with ba. our results strongly indicate that ba is genotoxic with the side-chain epoxide being its ultimate carcinogen. morbid obesity is an independent risk factor for cardiovascular disease, type 2 diabetes mellitus and certain types of cancer. bariatric surgery -with the roux-en-y gastric bypass (rygb) being the gold standard -has become the therapeutic option of choice as a sustained weight loss and improvement of associated morbidity is achieved in the majority of patients. there is, however, a lack of evidence focusing on bariatric surgery induced sustained weight loss and its possible impact on cancer risk. we investigated the association between obesity, oxidative stress and genomic damage after roux-en-y gastric bypass surgery (rygb) or caloric restriction induced weight loss in the obese zucker rat. obese male zucker fa/fa rats were divided into three groups: sham surgery (sham), rygb and caloric restriction (cr) and were compared with lean controls (lean; zucker fa/+ rats). shams showed impaired glucose tolerance and elevated plasma insulin levels, which were less severe in rygb and cr. oxidative stress was elevated in kidney, liver and colon tissue of sham and reduced again after weight loss induced by either rygb or bwm. urine-derived oxidization products of lipids, dna and rna increased in shams and decreased after weight loss (rygb and cr). dna double strand breaks were more frequent in shams than in the weight loss groups or lean. dna damage in zucker fa/fa rats correlated with their basal plasma insulin values. obese rats showed elevated oxidative stress and genomic damage in comparison to lean rats. after body weight loss, achieved by either rygb or caloric restriction alone, oxidative stress level and genomic damage were decreased. this may indicate a reduction of the elevated cancer risk in obesity. ) mice were treated with the nocrelated compound azoxymethane (aom) followed by the administration of dextran sodium sulfate to trigger crc. tumors were quantified by non-invasive mini-endoscopy, which revealed a non-linear increase in crc formation in wt and aag -/mice. in contrast, a linear dose-dependent increase in tumor frequency was observed in mgmt -/and mgmt -/-/aag -/mice. the data was corroborated by hockey stick modeling, which yielded similar carcinogenic threshold doses for wt and aag -/mice. o 6 -meg levels and depletion of mgmt activity correlated well with the observed dose-response in crc formation. aom dose-dependently induced double strand breaks (dsbs) in colon crypts including in lgr5-positive colon stem cells, which coincided with atr-chk1-p53 signaling. intriguingly, mgmt-deficient mice displayed significantly enhanced levels of γ-h2ax, suggesting the usefulness of γ-h2ax as an early genotoxicity marker in the colorectum. this study demonstrates for the first time a non-linear dose-response for alkylation-induced carcinogenesis and reveals dna repair by mgmt, but not aag, as a key node in determining a carcinogenic threshold at low alkylation dose levels [2] . obesity is characterized as a status where the excessive accumulation of fat in adipocytes leads to local inflammation and hypoxia; both contributing to severe obesity associated co-morbidities such as cardiovascular disease and type 2 diabetes mellitus. local inflammation is mediated by macrophages, stromal vascular cells, preadipocytes, and adipocytes as well as by a number of proinflammatory cytokines and chemokines (1). in particular, the chemokines monocyte chemoattractant protein (mcp-1, ccl2), interleukin-8 (il-8/cxcl8) and stromal cell-derived factor 1 (sdf-1a/cxcl12) secreted by stromal vascular cells, preadipocytes, and adipocytes exert paracrine effects by recruiting neutrophils, monocytes/macrophages, and t-and b-cells. interestingly, deficiency in cxcl14 was shown to attenuate obesity in mice (2) . the cc chemokine ccl2 is known to stimulate ccr2 receptors, and the cxc chemokines cxcl8 and cxcl12 to activate cxcr1 and cxcr2 or cxcr4 and cxcr7, respectively. the receptor(s) activated by cxcl14 is currently unknown. a role of cxcl14 in modulating cxcr4 signaling has been proposed. we initiated our studies to determine the presence and functional significance of chemotaxin receptors in human adipocytes and their precursor cells. to this end, the mrna expression pattern of cc chemokine receptors, cxc chemokine receptors formylated peptide receptor fpr1 and the related receptor fprl1, and cc and cxc chemokines was analyzed during in vitro adipose differentiation of human simpson-golabi-behmel-syndrome (sgbs) preadipocytes, and under conditions mimicking an inflammatory response. in particular, we focused on the expression pattern of human ccr2 receptors, since previous reports indicated a role in adipogenic differentiation. however, our comprehensive analysis using different sources of adipocytes and their precursors indicated that ccr2 receptors were absent (3) . yet, the analysis revealed appreciable levels of mrna encoding ccl2, cxcl12, and cxcl14, and ccr1, cxcr2, cxcr7, fpr1, and fprl1, and cxcr4. of interest, cxcr4-and cxcr7-mrna were found to be up-regulated under the proinflammatory conditions. to analyze the responses of adipocytes and their precursors to chemokine receptor agonists, we used chemokine-mcherry fusion proteins purified from baculovirus-infected insect cells, e.g ccl2, cxcl12, cxcl14. while sgbspreadipocytes and adipocytes did not accumulate ccl2-mcherry upon stimulation, they showed a small accumulation of cxcl12-mcherry, and a strong accumulation of cxcl14-mcherry in the endosomal compartment. similar results were obtained in murine 3t3l-1 preadipocytes. using mass spectrometry analysis, we set out to identify the cxcl14-binding putative receptor protein(s) in murine 3t3l-1 preadipocytes. (1) makki, k. et al., (2013) in personalized medicine tumors are screened for several mutations in oncogenes or tumor suppressors. however, the cellular protein content not exclusively depends on the dna. we identified new rac variants generated on the mrna level in androgenindependent prostate cancer cells. all variants represent active forms of the gtpase. they are capable to suppress rhoa-induced apoptosis and additionally, mediate the synthesis of genes which are under the control of the androgen receptor. importantly, expression of the rac variants is sufficient to support tumor growth in mice. we prove the existence of the variants and verify their clinical appearance and relevance in tissue samples of a prostate cancer patient. dna analysis, however, revealed the wildtype sequence of rac. therefore, routine analysis of patient tumor tissue would miss the detection of active rac which precludes the success of therapy. the existence of active rac variants in prostate cancer tissue that promote resistance towards androgen deprivation suggest rac inhibition as an effective add on therapeutic strategy against prostate cancer. the bacterial effector protein exotoxin y (exoy) of pseudomonas aeruginosa is delivered into host cells via the bacterial type iii secretion system. once arrived in the host cell nucleotidyl cyclase activity of exoy is activated by a yet unknown cofactor and thus has a profound effect on concentrations of cyclic nucleotides: in addition to production of cyclic amp (camp) and cyclic gmp (cgmp) there is a massive synthesis of cyclic 3', and to some extent of the corresponding cytidylyl analogue ccmp 1,2 . currently, the role of cump and ccmp during the pathogenesis of p. aeruginosa infection remains unknown 3 . one of our hypotheses is that these cyclic nucleotides fulfil a role as first messengers, e.g. in the communication between individual bacteria or bacterial populations during establishment of acute or chronic infections. to test this hypothesis, the intra-and extrabacterial concentrations of cyclic nucleotides were measured via hplc-ms/ms at different time-points in liquid cultures of p. aeruginosa, either in a complete (lb medium) or a starving medium (vogel-bonner medium). additionally, we tested if supplementation of the media with extrinsic cump or ccmp had an effect on these measured concentrations. the influence of extrabacterial cyclic nucleotides on the bacterial metabolism and homeostasis was evaluated with a microarray of bacterial total cdna extracted at different time points of p. aeruginosa liquid culture with or without extrinsic cump/ ccmp. furthermore we investigated a potential function of the cyclic nucleotides in biofilm formation. cyclic ump and cyclic cmp have differential roles in bacterial metabolism and communication. for example, whereas ccmp is synthesized by p. aeruginosa when the bacteria are in a nutrient-rich environment, we could not detect bacterial cump under any tested circumstance. in our biofilm formation assays, only ccmp had a biofilmpromoting effect, but only in very high concentrations. the currently ongoing analysis of gene expression data in the presence or absence of cump may reveal a role of this cyclic nucleotide as first messenger, too. in further studies we will elucidate the signal transduction processes underlying the observed cump / ccmp effects, for example by identifying cump and ccmp binding proteins and their coupling mechanisms to intracellular signalling cascades. synapses are complex computational platforms that transmit information encoded in action potentials but also transform their functionality through synaptic plasticity. g protein-coupled receptors (gpcrs) play a major role in modulating the strength of the synapses via the second messenger camp 1 . however the spatio-temporal dynamics of the mode of action of camp underlying synaptic plasticity are still controversial. the role of this study was to investigate the dynamics of camp signaling at the drosophila neuromuscular junction, where octopamine binding to its receptors has been shown to cause camp-dependent synaptic plasticity 2 . for this purpose, we generated a transgenic drosophila expressing the camp sensor epac1-camps 3 in motor neuron. this allowed us to directly follow the octopamine-induced camp signals in real time by fluorescence resonance energy transfer (fret) in different compartments of the motor neuron (i.e. cell body, axon, boutons). we found that octopamine induces a steep camp gradient from the synaptic bouton (high camp) to the cell body (low camp), which was due by higher pde activity in the cell body. high octopamine concentrations evoked a response also in the soma. notably, these signals were independent and isolated form each other. moreover, application of octopamine by iontophoresis to single synaptic boutons induced bouton-confined camp signals. these data reveal that a motor neuron can posses multiple and largely independent camp signaling compartments, and provide new basis to explain how camp could control neurotransmission at a level of a single synapse. 1 kandel, e.r., dudai, y. & mayford, m.r. the molecular and systems biology of memory. cell 157, 163-186 (2014) . 2 koon, a.c., et al., autoregulatory and paracrine control of synaptic and behavioral plasticity by octopaminergic signaling. nat neurosci. 14 (2): p. 190-9 (2010) . 3 nikolaev vo, bünemann m, hein l, hannawacker a, lohse mj novel single chain camp sensors for receptor-induced signal propagation. j. biol. chem.279, 37215-37218 (2004) cardiovascular pharmacology 039 hyaluronic acid deposition determines engineered heart muscle characteristics and can be pharmacologically targeted to enhance function s. schlick background: engineered human myocardium (ehm) can be generated from psc derived cardiomyocytes (cms) and primary fibroblasts suspended in a collagen i hydrogel (70%:30%:0.4 mg/ml). ehm development encompasses an early consolidation phase followed by functional maturation. the presence of fibroblasts is essential for consolidation into a force-generating ehm. here we assessed the hypothesis that fibroblasts of different origin support ehm formation differentially as a function of hyaluronic acid deposition. methods and results: oscillatory rheology (1% strain, 1 hz) on cell-free and cell containing collagen i hydrogels directly after casting revealed enhanced consolidation in the presence of human foreskin fibroblasts (ffbs) compared to primary adult cardiac fibroblasts (cfbs) -change in storage modulus over time (pa/min): collagen 0.03; collagen + cms 0.03; collagen + cms + cfbs 0.09, collagen + cms + ffbs 0.2. we next generated ehm with cms and ffbs or cfbs. after 4 weeks of culture under serum-free conditions, we assessed ehm function by contraction measurements. ffb-ehms developed a significantly (p<0.01) higher force of contraction (foc) per cross sectional area (csa) than cfb-ehms (maximal foc/ csa are in mn/mm 2 : 1.8±0.1, n=29 vs. 0.3±0.1, n=20). cross sectional area (csa) of tissues was greatly increased (p<0.01) in cfb-ehms (csa in mm 2 : 1.6±0.1, n=20 vs. 0.6±0.03, n=30) and nonmyocyte content was higher in cfb-ehms (5.6±0.7, n=9 vs. 3.1±0.4, n=15; x10 5 cells/ml). histological analysis revealed that cardiomyocytes were only poorly matured in cfb-ehms compared to ffb-ehms. extending ehm functional data, principal component analysis of rnaseq data revealed distinct expression patterns for ffbs and cfbs, in which hyaluronic acid synthase 2 (has2) enzyme was significantly (p<0.01) upregulated. based on these findings, we pharmacologically intervened with has2 mediated hyaluronic acid (ha) deposition by treating cfb-ehms with hyaluronidase during all 4 weeks of culture. interestingly, ecm manipulation with low concentrations of enzyme significantly (p<0.01) reduced csa (csa in mm 2 : control 1.8±0.4, n=8; hyaluronidase of concentrations from 0.15u to 150u 0.9±0.2, n=4) with a concurrent, statistically significant (p<0.01), increase in contractile function and improved cardiomyocyte morphology on a histological level (maximal foc/csa in mn/mm 2 : control 0.2±0.05, n=8; hyaluronidase of concentrations from 0.15u to 150u 0.4±0.08, n=4). summary and conclusions: our data suggest that ehm consolidation is influenced differentially by fibroblasts of different tissue origin with hff-ehm being functionally superior to cfb-ehm. cfb-ehm could be rescued by hyaluronidase leading to reduced ha deposition. the latter demonstrates that extracellular matrix composition is centrally involved in ehm development. angiogenesis is the process of formation of new blood vessels from the pre-existing ones. vascular endothelial growth factor (vegf) is the most studied regulator of this process. by binding to its type 2 receptor (vegfr2), it has been shown to activate a variety of different signaling-pathways leading to enhanced angiogenesis. camp, on the other hand, is a versatile second messenger which regulates various endothelial functions including barrier function. it directly activates protein kinase a (pka) or the exchange protein directly activated by camp (epac) which is a guanine exchange factor (gef) for the small monomeric gtpase rap. as human umbilical vein endothelial cells (huvec) express both camp effectors (epac1 and pka), we investigated the role of camp-signaling using a spheroid based sprouting assay as an in vitro model for angiogenesis. interestingly, the activation of β-adrenergic receptors with 5 µm of isoproterenol significantly increased the cumulative sprout length. similarly, the selective activation of epac with 30 µm of the camp analog 8-pcpt-2'-o-camp (007) significantly increased the basal and the vegf-induced cumulative sprout length. in accordance, sirna-mediated depletion of epac1 in huvec decreased the basal and vegf-induced sprouting. surprisingly, 10 µm of forskolin increased basal and vegf-induced cumulative sprout length stronger than 007, indicating an additional role of pka. in accordance, 1 µm of myristoylated pki, a membrane-permeable specific pka inhibitor, significantly attenuated the forskolin-induced increase in sprouting. in all conditions tested, 50 ng/ml of vegf always showed an additive effect to the same extent on cumulative sprout length. therefore, our data indicate that the vegf-pathway is acting independently of the camp-pathway in the regulation of the sprouting angiogenesis. the β-adrenergic receptor-mediated activation of camp signaling in huvec induces angiogenic sprouting by activation of epac1 and pka. introduction: hypertension is one major risk factor for the development of chronic heart and kidney disease. mineralocorticoid receptor (mr) antagonists are a cornerstone in the therapy of heart failure and there is first evidence for a beneficial effect on the kidney as well. inflammation plays an important role in hypertensive organ injury. thus, this study was designed to evaluate and directly compare the effect of mr deletion in endothelial cells on blood pressure and cardiac vs. renal injury in a mouse model of deoxycorticosterone acetate-induced hypertension. methods and results: mice lacking the mineralocorticoid receptor in endothelial cells (mr cdh5cre ) were created using the cre/loxp system. mr cdh5cre and cre-negative littermates (mr wildtype ) underwent unilateral nephrectomy and received 1 % nacl with drinking water for 6 weeks. the mineralocorticoid deoxycorticosterone acetate (doca, 2.5 mg/d) was delivered by subcutaneous pellets. untreated mice served as controls (ctrl). ambulatory blood pressure was determined by implantable telemetry in awake mice. doca/salt treatment increased mean blood pressure in mr wildtype (141.7 ± 8.0 vs. ctrl 97.8 ± 3.2 mmhg, p<0.001) and mr cdh5cre (150.0 ± 3.0 vs. ctrl 104.1 ± 4.7 mmhg, p<0.001) without differences between genotypes. cardiac hypertrophy after doca/salt treatment was ameliorated in mr cdh5cre mice (ventricle weight 162.4 ± 4.2 mg vs. mr wildtype 189.0 ± 10.9 mg, p<0.05). doca/salt significantly increased cardiac fibrosis and the expression of fibrotic marker genes in mr wildtype but not in mr cdh5cre mice. this was accompanied by an increased expression of the vascular cellular adhesion molecule (vcam1) in mr wildtype cardiac endothelial cells. renal function was not altered by mr deletion in endothelial cells at baseline. doca/salt treatment lead to marked interstitial fibrosis in the kidneys of mr wildtype (sirius red fibrosis score: 2.4 ± 0.2 vs. ctrl 0.1 ± 0.1, p<0.001) and mr cdh5cre (2.3 ± 0.2 vs. ctrl 0.1 ± 0.1, p<0.001) mice. mrna expression of the fibrosis marker gene col3a1 (mr wildtype 4.3 ± 0.9-fold; mr cdh5cre 3.9 ± 1.1-fold vs. ctrl) was similarly increased. periodic acid-schiff staining revealed glomerular injury in both genotypes. this was associated with a marked rise in urinary albumin / creatinine ratio (mr wildtype 20.4 ± 5.7fold; mr cdh5cre 34.3 ± 12.5-fold vs. ctrl). in the kidney vcam1 mrna expression and the number of macrophages was increased by doca/salt treatment independently from endothelial mr deletion. conclusion: in conclusion, mr deletion from endothelial cells ameliorated doca/saltinduced cardiac but not renal inflammation and remodeling independently from blood pressure. these findings suggest different mechanisms for the beneficial effect of mr antagonists in hypertensive heart vs. kidney disease. platelets are relevant cells implicated in morbidity and mortality provoked by cardiovascular thrombosis. even with the actual antiplatelet therapy there is still a substantial incidence of arterial thrombosis. therefore, a better understanding of the mechanisms involved in platelet activation and aggregation is required to develop improved antiplatelet therapies. the increase in the intracellular ca 2+ concentration due to ca 2+ entry from the extracellular space is critical for platelet activation and aggregation. ca 2+ entry follows activation of plasma membrane receptors including gqcoupled receptors for adp, thromboxane a 2 (txa 2 ) or thrombin, as well as the collagen receptor glycoprotein vi (gpvi). the cellular signalling pathways downstream these receptors involve activation of phospholipase c and second messengers that are known to mediate activation of trpc channels. trpc proteins form receptor-operated cation channels, but their regulation and permeability differ depending on the cell type. it has been proposed that trpc proteins might contribute to platelet function as constituents of agonist-activated ca 2+ entry channels; however, the experimental approaches used so far and the lack of specific agonists or antagonists have not allowed to determine the individual contribution of trpc proteins for agonist-induced ca 2+ entry in platelets, aggregation and thrombosis formation. we detected the expression of trpc3 and trpc6 in human and mouse platelets. we identified that those proteins together are essential components of a system of coincidence detection in cellular ca 2+ signalling. this coincidence detection triggered by simultaneous stimulation of both thrombin and collagen receptors is required for the phosphatidylserine exposure in human and murine platelets, indicating the role of trpc3/c6 proteins for procoagulant activity. in addition, we detected the expression of s12 trpc1 transcripts in mouse platelets. therefore, we tested trpc1/c3/c6-deficient mice in an in vivo model of arterial thrombosis where they showed reduced thrombus formation. regardless of the protective effect of trpc1/c3/c6 inactivation observed in the thrombosis model, no differences were detected in tail bleeding. to evaluate the relevance of these trpc proteins in platelet aggregation we measured in vitro platelet aggregation in platelets from trpc1/c3/c6-deficient mice and we observed that the aggregation was reduced after adp (3µm) or txa 2 -analogue (1µm) stimulation, but not after collagen stimulation (10µg/ml). we are currently analyzing the in vitro aggregation and the agonist-evoked ca 2+ response in platelets from different trpc-compound and single deficient mouse lines to understand the mechanisms behind this phenotype with regard to its complementarity to actual antiplatelet therapy. background: thrombin signaling initiates inflammatory events directly and through activation of platelets. endogenous and pharmacologic inhibitors of thrombin are therefore of relevance during atheroprogression and for therapeutic intervention. the small leucine-rich proteoglycan biglycan (bgn) is such an endogenous thrombin inhibitor that acts through activation of heparin cofactor ii (hcii). here, the effect of genetic deletion of bgn on thrombin activity, inflammation and atherosclerosis was addressed. methods and results: bgn concentrations were elevated in the plasma of patients with acute coronary syndrome. in apoe -/mice, bgn was detected in the plasma as well as in the glykokalyx of capillaries. additionally, bgn expression occurred in the subendothelial matrix of arterioles as well as in atherosclerotic plaques. in line with a role of bgn in balancing thrombin activity, apoe -/-/bgn -/0 mice exhibited higher activity of circulating thrombin and increased numbers of activated platelets than did apoe -/mice. furthermore, higher concentrations of circulating cytokines in apoe -/-/bgn -/0 mice suggested a pro-inflammatory phenotype. likewise, immunohistochemistry and facs analysis of the aorta demonstrated increased macrophage content in atherosclerotic lesions of these mice. in addition, apoe -/-/bgn -/0 mice exhibited higher aortic plaque burden and larger atherosclerotic lesions at the aortic root. of note, apoe -/-/bgn -/0 mice showed progressive dilatation of the aortic arch corresponding to a decrease in collagen fibril density suggestive of an outward remodelling in the absence of bgn. no differences were evident with respect to lipid content of the aortic root plaques or circulating plasma lipids. treatment with the thrombin inhibitor argatroban reversed platelet activation and aortic macrophage accumulation in apoe -/-/bgn -/0 mice. conclusions: the present results strongly suggest a protective role of bgn during the progression of atherosclerosis by inhibiting thrombin activity and platelet activation, and ultimately macrophage-mediated plaque inflammation. the exposure to environmental or human-made xenobiotics including drugs induces the hepato-intestinal transcription of metabolizing enzymes and transporters. the time-span of induction is thought not to exceed xenobiotic exposure, in order to minimize disturbances of endobiotic metabolism. in contrast, we find cross-generational transmission of the induction of the phase i enzyme cyp2b10 (1200-fold in females, 700-fold in males) in 6-day old offspring of adult female mice exposed one week prior mating to tcpobop (3 mg/kg i.p.) , the model ligand of the xenosensing nuclear receptor car. such cross-generational effects of xenobiotics are of great clinical interest as they could have profound consequences on the health status of the offspring, including interferences with drug therapies. the multigenerational transmission of tcpobop-driven induction could be mediated by pre-uterine/pre-conceptional epigenetic changes of oocytes. alternatively, they could be brought about by direct intrauterine/post-conceptional contact with tcpobop released from long-term depots. to discriminate between these mechanisms we conducted embryo transfer experiments. both donor mothers and foster mothers were injected with tcpobop (3 mg/kg) prior to mating. the analysis of hepatic cyp2b10 expression in 6-day-old offspring is clearly consistent with a post-conceptional onset of tcpobop effects. thus, offspring of solvent-injected donor mothers transferred to tcpobopexposed foster mothers display a 700-fold induction while offspring from the reciprocal experiment show no changes. cesarean sections on day e18.5 followed by crossfostering proved transmission to be mediated predominantly via lactation (f1 hepatic cyp2b10 induction 3000-fold) and only to a minor part via intra-uterine exposure (300fold). this mechanism is consistent with the absence of induction transmission via the male germline. to analyze if tcpobop leads to functional consequences in drug metabolism of f0 and f1 generation, we conducted in vivo zoxazolamine paralysis assays taken as a functional test for cyp2b10 catalytic activity. in both tcpobop-pretreated f0 and in their f1 descendants, the induction reduced the duration of paralysis evoked by zoxazolamine by >50%. the characterization of cross-generational tcpobop-mediated effects on other processes controlled by car such as energy and bone metabolism is in progress. first tests indicate a transmission of anabolic effects on bone, as evidenced by the induction of serum osteocalcin expression by 45% in 12-weeks-old offspring. in summary, the car-mediated cyp2b10 induction by tcpobop is transmitted to the offspring mainly via lactation, resulting in lasting phenotypic consequences in drug and bone metabolism. the effects of similarly lipophilic drugs and anthropogenic environmental pollutants are currently being investigated. such compounds could affect offspring despite discontinuation of intake or exposure well ahead of pregnancy. age-related cognitive decline can eventually lead to dementia, the most common mental illness in elderly people and an immense challenge for patients, their families and caregivers. cholinesterase inhibitors constitute the most commonly used antidementia prescription medication. the standardized ginkgo biloba leaf extract egb 761 ® is approved for treating age-associated cognitive impairment and has been shown to improve the quality of life in patients suffering from mild dementia. a clinical trial with 96 alzheimer´s disease patients indicated that the combined treatment with donepezil and egb 761 ® had less side effects than donepezil alone (yancheva et al., 2009) . in an animal model of cognitive aging, we compared the effect of combined treatment with egb 761 ® or donepezil monotherapy and vehicle. we compared the effect of chronic treatment (15 days of pretreatment) with donepezil (1,5 mg/kg p. o.), egb 761 ® (100 mg/kg p. o.), or the combination of the two drugs, or vehicle in 18 -20 month old male ofa rats. learning and memory performance were assessed by morris water maze testing, motor behavior in an open field paradigm. in addition to chronic treatment, the substances were administered orally 30 minutes before testing. compared to the first day and to the control group, only the combination group showed a significant reduction in latency to reach the hidden platform on the second day of testing. moreover, from the second day of testing onwards, the donepezil, the egb 761 ® and the combination group required less time to reach the hidden platform compared to the first day. the control group did not reach the same latency reduction until day three. there were no effects on motor behavior. these results suggest a superiority of the combined treatment of donepezil with egb 761 ® compared to monotherapy. literature: yancheva, s., ihl, r., nikolova, g., panayotov, p., schlaefke, s., & hoerr, r. (2009) . ginkgo biloba extract egb 761(r), donepezil or both combined in the treatment of alzheimer's disease with neuropsychiatric features: a randomised, double-blind, exploratory trial. aging ment health, 13 (2) , [183] [184] [185] [186] [187] [188] [189] [190] institut für vegetative physiologie und pathophysiologie, göttingen, germany values are well below the plasma concentrations (3-28 µm; just et al. expert opin emerging drugs 20: 161-164, 2015) observed in patients treated with dantrolene. although not yet proven directly, oat3 may be involved in renal secretion of dantrolene and 5-oh dantrolene by mediating the first step, i.e. the uptake across the basolateral membrane of proximal tubule cells. the second step, the release of these compounds into the urine across the luminal membrane, is possibly mediated by mrp4. since oat3 was also detected in the cytoplasmic membrane of skeletal muscle cells (takeda et al. europ. j. pharmacol. 483: 133-138, 2004) , dantrolene may reach its target, the intracellular ryanodine receptor, ryr1, by influx through oat3, where it inhibits calcium efflux by ryr1 thereby preventing severe muscle contraction and malignant hyperthermia. this assumption, however, awaits a direct demonstration of dantrolene transport by oat3. in addition, we identified, besides dantrolene and 5-oh dantrolene, several further fdaapproved drugs such as tyrphostin ag 1478, ceefourin 1, glafenine, nalidixic acid, and prazosine, as inhibitors of es uptake by oat3. controls 1 with other defects and controls 2 with genetic disorders. all drugs used in the first trimester were identified from the database, and were cross-referenced against previously compiled lists of drugs with reactive intermediates and drugs with faa. drugs with reactive intermediates, with systemic absorption and with a daily dose ≥50mg were considered os-inducing drugs. when there was an association between os-inducing drugs and a group of birth defects, we further investigated two different faa exposure categories: concurrent exposure to both os-inducing drugs and faa drugs (os+/faa+) and exposure to os-inducing drugs only (os+/faa-). when the number of subjects allowed (at least five cases/controls were exposed), we examined the role of folic. odds ratios (ors) with 95% confidence intervals were adjusted for maternal smoking and alcohol use in the first trimester in controls 1 and additionally adjusted for maternal age in controls 2. results: a total of nine groups of birth defects were investigated. only nervous system defects were associated with os-inducing drugs. exposure rates were 65/464 (14.0%) for cases, 512/6033 (8.5%) for controls 1 and 130/1564 (8.3%) for controls 2 and adjusted ors (95%cis) were 1.71 (1.29-2.26) and 1.77 (1.27-2.46), respectively. this association was unchanged when we examined os+/faa+ and os+/faa-separately. the os+/faa+ category, however, had slightly higher or values than the os+/faa-(2.41 vs. 1.61 for controls 1, and 2.55 vs. 1.67 for controls 2). because of the low number of exposed subjects, we could only examine folic in relation to os+/faa-. using os-/faa-/folic+ as reference, we found the highest risk with os+/faa-/folicand a lesser magnitude with os+/faa-/folic+ (ors being 2 and 1.6 times respectively for both controls). conclusion: our study suggests an increased risk of having a child with nervous system defects in mothers who were exposed to os-inducing drugs during pregnancy, and a potential risk reduction with folic. background: inhibition of rho-gtpases with statins as well as specific inhibition of the small gtpase rac1 protects non-transformed cells from topoisomerase ii-(top2)-poisoninduced cleavable complex formation and thereof derived dna double-strand breaks. this effect rests at least partially on rac1-mediated regulation of topoisomerase ii activity. however, the link between rac1 and top2-poisoning is only poorly understood. furthermore, it is unclear whether mitochondrial or nuclear type ii topoisomerases are the most relevant target for top2-poison-induced cytotoxicity. here, we investigated the relevance of rac1-regulated actin cytoskeleton integrity as well as mitochondrial integrity in top2-poison-induced dna damage responses as well as cytotoxicity under situation of rac1 inhibition. methods: since endothelial cells are the first barrier for any kind of systemically administered chemicals and cardiomyocytes are particular sensitive to anthracyclines, endothelial cells (h5v) as well as cardiomyocytes (h9c2) were chosen as in vitro model systems for top2-poisoning. the cells were pre-treated with rac1 inhibitors, statins or actin cytoskeleton disruptors and were subsequently treated with the topoisomerase ii poisons doxorubicin or etoposide. to compare the levels of induced dna damage, γh2ax foci quantifications as well as the comet assay were employed. actin disruption was visualized by phalloidin-fitc staining. to be able to detect relevant changes in mitochondrial mass or integrity, high doses of top2-poisons had to be used in both cell lines. changes in mitochondrial homeostasis as well as integrity were detected by the jc1-assay, mitotracker assay as well as atp-assay. additionally, pcr-and gelelectrophoresis-based methods were used for detecting mitochondrial dna damages. selected components of the dna damage response machinery as well as factors of mitochondrial homeostasis were detected by western blot. results: disruption of the integrity of the actin cytoskeleton attenuated the dna damage response to a similar extent as seen by rac1 inhibition, pointing to a role of actin filaments in the dna damage response after genotoxic insults. the actin cytoskeleton seems to participate in genotoxin-induced dna damage, -repair or in the dna damage response as reflected by reduced numbers of nuclear h2ax-foci as well as the comet assay after treatment with doxorubicin. this was not related to nuclear import or export of doxorubicin. disturbance of mitochondrial homeostasis or integrity was only detectable at high doses of topoisomerase ii poisons. this was largely unaffected by pre-treatment with statins or rac1-inhibitor. top2-poison-induced raise in mitochondrial mass was slightly enhanced by the rac1-inhibitor and statins. interestingly, inhibition of rac1 counteracted doxorubicin-induced phosphorylation of the amp-kinase in endothelial cells but not in cardiomyocytes. conclusion: mitochondrial toxicity seems to play only a minor role in top2-poisoninduced cytotoxicity in h9c2 and h5v cells. the data point to a role of rac1-regulated filamentous (nuclear?) actin in the dna repair and/or dna damage response after treatment with top2-poisons. poly(adp-ribose) polymerase 1 (parp1) and the recq helicase werner syndrome protein (wrn) are important caretakers of the genome. they physically interact with each other and are both localized in the nucleus and in particular in the nucleoli. both participate in various overlapping mechanisms of dna metabolism, in particular genotoxic stress response and dna repair [1] . previously, we and others have shown in biochemical studies that enzymatic functions of wrn are regulated by parp1 as well as by non-covalent poly(adp-ribose)-wrn interaction [2] [3] [4] . furthermore, pharmacological parp inhibition as well as a genetic parp1 ablation in hela cells alters the recruitment kinetics of wrn to sites of laser-induced dna damage [5] . here we report a novel role for parp1 and poly(adp-ribosyl)ation in the regulation of wrn's subnuclear spatial distribution upon induction of oxidative stress. we could verify previous reports that wrn is transiently released from nucleoli upon induction of oxidative stress, camptothecin (cpt) treatment, and laser-induced dna damage in a time-dependent manner. while, cpt-induced translocation appears to be a parpindependent process, our results reveal that upon h 2 o 2 -induced oxidative stress, parp1 is essential for the translocation of wrn from the nucleoli to the nucleoplasm. parp1 activity only partially contributes to wrn release from nucleoli, underlining the importance of a direct wrn-parp1 interaction for subnuclear wrn redistribution. furthermore, we identified a novel par-binding motif within the wrn sequence that is located in its rqc domain, which also harbors the binding site for parp1 and is necessary for wrn's nucleolar localization under non-stress conditions. currently, we are testing corresponding wrn mutants to analyze if this region is responsible for the parp1-dependent release of wrn from nucleoli to sites of dna damage. in conclusion, we provide novel insight into the role of parp1 in wrn's spatio-temporal regulation in the nucleus during the oxidative stress response. host-cell reactivation (hcr) is an assay used to determine dna repair capacity of cells. in its canonical layout, the test utilised a virus or a plasmid with a marker gene, inactivated by uv-damage [1, 2] . among the infected or transfected host cells types, only those with functional dna repair pathway would re-activate the damaged dna, thus providing a rationale for identification of dna repair genes in the mutant screens. an obvious advantage of hcr is that repair can be measured in cells that have not been exposed to a damaging agent. however, because of multiple variables of the damage generation, transfection and interpretation of results, the assay has been hard to harmonise and develop into a widely accepted quantitative dna repair assay. over the last years, my team has developed and validated several major improvements of the mammalian hcr assay. exploiting sequence-specific nicking endonucleases and customised design of the reporter vectors, we proposed an innovative and very efficient technique for incorporation of synthetic oligonucleotides, containing single structurally defined dna base and backbone modifications, into desired gene elements [3] . this ad hoc approach allows examination of the repair in a stand-specific manner and at single nucleotide resolution. we efficiently applied hcr in its new layout for measurement of the nucleotide excision repair of various dna adducts. moreover, we demonstrated that the enhanced hcr assay can differentiate between the transcription-coupled (tc-ner) and global genome (gg-ner) subpathways of ner [4] . we further obtained new significant insights into the lesion-specific mechanisms of base excision repair of several endogenously occurring aberrant dna bases [5 -7] and plan to adapt the assay to the detection of mismatch repair and translesion dna synthesis. in addition to the applications in the dna repair field, the enhanced hcr assay provides a tool for investigation of the dynamics and transcriptional impact of the regulatory dna bases 5methylcytosine and 5-hydroxymethylcytosine as well as their derivatives (5formylcytosine and 5-carboxycytosine a coordinated and faithful dna damage response is of central importance for maintaining genomic integrity and cell survival. transcriptional activation of dna repair genes is an important regulatory mechanism contributing to the adaptation of cells to genotoxic stress and protection against genotoxin-mediated cell death. here we show that exposure to a low dose of benzo(a)pyrene 9, , the active metabolite of benzo(a)pyrene (b[a]p), which represents the most important carcinogen formed by incomplete combustion during food preparation and smoking, causes upregulation of several dna repair genes. combined induction of the nucleotide excision repair (ner) genes ddb2, xpc, xpf and xpg enhanced repair activity and protected cells against a subsequent bpde exposure. furthermore induction of the translesion polymerase polh was also involved in protection against bpde-induced apoptosis, however led to an enhanced mutation frequency in the surviving cells. activation of these dna repair pathways was also observed upon exposure to b[a]p and in vivo in buccal cells of male individuals upon smoking, indicating that this mechanism may be involved in the formation of smoking-related cancers. altogether, we could show that low-dose bpde exposure activates a complex network of transcriptional alterations, leading to protection against cell death, at the cost of increased mutation frequency, highlighting the danger of occasional smoking. poly(adp-ribosyl)ation (parylation) is an essential posttranslational modification with the biopolymer poly(adp-ribose) (par). the reaction is catalyzed by poly(adp-ribose) polymerases (parps) and plays key roles in cellular physiology and stress response by regulating physico-chemical properties of target proteins. of the 17 members of the human parp gene family, at least four have been shown to exhibit par-forming capacity. upon dna damage parp1 is catalytically activated and is thought to contribute to the bulk of the cellular par formation. parp inhibitors are currently being tested in clinical cancer treatment, in combination therapy, or as monotherapeutic agents by inducing synthetic lethality (mangerich and bürkle 2011, mangerich and bürkle 2015) . here we generated a genetic knock out of parp1 in one of the most widely used human cell systems, i.e. hela parp1 ko cells, via talen-mediated gene targeting and characterized these cells with regards to parylation metabolism and genotoxic stress response. furthermore, by reconstituting hela parp1 ko cells with a series of artificial and natural parp1 variants, we analyzed structure-function relationships of parp1 in a cellular environment without interfering with endogenously expressed wt-parp1. we confirmed that the parp1e988k mutant exhibits mono-adp-ribosylation activity and extended previous reports by demonstrating that the parp1l713f mutant is constitutively active in a cellular environment, leading to high cellular par levels even in unchallenged cells. additionally, both mutants exhibited significantly altered recruitment and dissociation kinetics at sites of laser-induced dna-damage, which can partially be attributed to non-covalent parp1-par interaction via at least one specific par binding motif located in zinc finger 2 of parp1. expression of both artificial mutants led to distinct cellular consequences, caused by the altered cellular biochemistry. while the expression of parp1l713f itself triggered apoptosis, parp1e988k expression led to a strong g2-arrest during cell cycle and sensitized cells to camptothecin treatment. interestingly, pharmacological parp inhibition with abt888 mitigated effects of the e988k mutant, suggesting distinct functions of mono-adp-ribosylation. finally, by reconstituting parp1 ko cells with a natural cancer-associated parp1 snp variant (v762a), as well as a newly identified parp1 mutant present in a patient of pediatric colorectal carcinoma (f304l-v762a), we demonstrate, that these variants exhibit altered biochemical and cellular properties, potentially supporting carcinogenesis. together, this study establishes a novel model to study parp1-dependent parylation during genotoxic stress response and reveals new insight into the structure-function relationships of artificial as well as natural parp1 variants in a cellular environment, with implications for parp research in general. mangerich, a. and a. bürkle (2011) . "how to kill tumor cells with inhibitors of poly(adpribosyl)ation." int j cancer128 (2): 251-265. mangerich, a. and a. bürkle (2015) . multitasking roles for poly (adp-ribosyl) ation in aging and longevity. parp inhibitors for cancer therapy, springer: 125-179. leukemic cells frequently overexpress the transcription factor wilms tumor 1 (wt1) and the persistence of wt1 expression after chemotherapy indicates remaining leukemic stem cells. hydroxyurea induces replicative stress by its ability to inhibit ribonucleotide reductase, an enzyme that catalyzes the synthesis of dntps from ntps. we demonstrate that the expression levels of wt1 determines the extent of dna damage and apoptosis in a panel of leukemic cells treated with hydroxyurea. accordingly, inhibiting apoptosis through chemical inhibition of caspases or by overexpression of mitochondrial anti-apoptotic bcl proteins prevents the hydroxyurea-induced depletion of wt1 and cell death. in addition, we show that an rna interference-mediated elimination of wt1 sensitizes leukemic cells to the pro-apoptotic and dna damaging effects of hydroxyurea. furthermore, such a loss of wt1 suppresses hydroxyurea-induced erythroid differentiation. pharmacological approaches that diminish wt1 also sensitize cells to hydroxyurea. these include the tyrosine kinase inhibitor (tki) imatinib or epigenetic modifiers belonging to the histone deacetylase inhibitor (hdaci) group. thus, an inhibition of wt1 is therapeutically exploitable for a targeting approach against leukemic cells undergoing replicative stress. our novel findings reveal that wt1 is a novel biological target of hydroxyurea and they suggest that wt1 has a previously unrecognized ability to prevent dna damage when replication forks halt and eventually collapse. fret (fluorescence resonance energy transfer)-based cell assays were developed to directly monitor receptor activation and receptor-stimulated camp response. mutant ß 1 ar were generated by insertion of cyan and yellow fluorescent proteins (cfp and yfp) into the third intracellular loop and the c-terminus, respectively (bornholz et al., cardiovasc res 97:472, 2013) and stably transfected to hek 293 cells (hekß 1 -fret). to monitor the camp response the epac1-based fret sensor of camp, was stably transfected alone (hekwt-e1) and together with a moderate level of native ß 1 ar to hek293 cells (hekß 1 -e1; nikolaev et al. jacc 50: 423, 2007) . fret-activity was measured with recently developed fluorescence detectors (12 channels) equipped with fast semiconductor technology, avoiding any movable optical and mechanical parts, using 438 nm for excitation and 483/540 nm for the emmission ratio. cells were cultivated in 96-format 12-well strips, incubated in physiological hepes-buffered salt solution and treated with ßar agonists of different selectivity and affinity to determine their ßar-subtype preference. catecholamines tested in hekß 1 -fret cells exhibited ec 50 -values (-log, m) which matched k d -values (-log, m) known from native heart receptor membranes (isoprenaline, iso: 6.9±0.1, adrenaline 5.7±0.1, noradrenaline 6.2±0.1). ßar-expression levels were controlled by radioligand binding with [ 3 h]-(-)-cgp12,177 resulting in different densities of ~4x10 6 and ~1.4x10 4 receptors/cell in hekß 1 -fret and hekß 1 -e1, respectively, whereas in hekwt-e1 cells only ~1000 ß 2 ar were found. surprisingly, the low level of ßar in hekwt-e1 cells allowed the measurement of the action of ß 2 -sympathomimetics (ß 2 sym), e.g. fenoterol, thereby amplifying receptor binding (pk d~6 .7) to an effective regulation of fret activity in the presence of 0.06 mm ibmx (pec 50~8 .3±0.1), nearly matching the ~100-fold amplification of iso (pk d~6 .9; pec 50~8 .9). in order to determine ß 1 ar-mediated side effects of ß 2 sym, hekß 1 -e1 cells characterized by a 14-fold higher level of ß 1 ar over ß 2 ar were assayed for fret activity. fenoterol maximally inhibited fret activity with a pec 50~8 .4 whereas the high affinity ß 2 sym salmeterol acted a partial agonist (~75% of iso maximum, pec 50~8 .6), both compounds being rather insensitive against the highly effective ß 1 ar-blockade with 1 µm cgp 20,712 a. for that reason hekß 1 -fret cells were used characterizing fenoterol as partial agonist (~25%) whereas salmeterol activated less than 10% of maximum receptor activation by iso. thus, it has to be concluded that the low level of effectively coupled wt-ß 2 ar present in hekß 1 -e1 cells precludes the exact determination of ß 1 ar-mediated side effects of ß 2 sym, and that cfp/yfp labelled receptors have to be used for the determination of the subtype specific intrinsic activity of an agonist. they account for about one third of all drug targets. their regulation from desensitization to internalization and alternative signal transduction is largely dependent on phosphorylation of intracellular serine and threonine residues of the activated receptor. even though the β 1 -adrenoceptor is of tremendous importance in a number of diseases its phosphorylation remains poorly understood. we addressed this question in a qualitative and quantitative way. by using radioactive phosphorylation assays and mass spectrometry, we were able to elucidate the phosphorylation pattern of the human β 1 -adrenoceptor in vitro. we identified ten previously unknown phosphorylation sites in the third intracellular loop and the receptor's c-terminus. labeling hek293 cells with stable heavy isotopes (silac) lead to the discovery of a stimulation-dependent regulation of several of these phosphorylation sites. furthermore, mutagenesis studies in stably transfected hek293 cells revealed the impact of phosphorylation for arrestin binding and internalization of the receptor. fluorescence resonance energy transfer experiments with β 1 -adrenoceptor variants carrying point mutations of putative phosphorylation sites identified two c-terminal phosphosites that determine arrestin recruitment. our current goal is to further investigate the functional implications of these newly identified phosphorylation sites on downstream signal transduction, with an emphasis on the map kinase pathway. a moderate increase in arrestin affinity to the β 2 -adrenergic receptor is sufficient to induce arrestin internalization. the homologous desensitization of g-protein-coupled receptors is a two-step process. initially, g-protein-coupled receptor kinases phosphorylate agonist-occupied receptors which are subsequently bound by arrestins. in many cases, the resulting receptorarrestin complex is then internalized via clathrin-coated pits. dependent on the identity of the receptor and the ligand, the complex between receptor and arrestin may exist only in the proximity of the plasma membrane or internalize into the cell interior. we constructed mutants of the β 2 -adrenergic receptor carrying three additional serine residues in various positions at the c-terminal tail. one of these mutants which carried the serine residues in close proximity to the endogenous grk phosphorylation sites (β 2 ar-sss) showed increased isoprenaline-stimulated phosphorylation and differences in arrestin-3 affinity and trafficking. the affinity of arrestin-3 to the receptor was measured by fluorescence resonance energy transfer (fret) between the receptor and arrestin-3 and by two-color fluorescence recovery after photobleaching (frap). in the fret assay, arrestin-3 dissociation from the β 2 ar-sss receptor upon agonist washout was prolonged approximately two-fold compared to the wild-type receptor. frap was performed with an n-terminally tagged receptor immobilized with an antibody against the n-terminal tag either in solution or on a micropatterned surface. in these assays, the recovery of arrestin-3 into the bleached region was prolonged between two-and fourfold for the β 2 ar-sss receptor compared to the wild-type. even though this two-to fourfold increase in affinity seemed rather modest, it resulted in the trafficking of receptorarrestin complexes to the early endosome whereas the wild-type receptor interacted only transiently with arrestin at the plasma membrane. furthermore, the increased affinity of arrestin led to more efficient internalization of the β 2 ar-sss compared to the wild-type receptor. however, recycling to the plasma membrane after agonist washout was very similar for both receptors. we conclude that even a modest change in affinity between a g-protein-coupled receptor and arrestin can lead to substantial alterations in arrestin trafficking which in turn may have effects on cellular signaling. despite recent structural research allow for better understanding of gpcr structure, the crucial aspects of the selectivity mechanism of receptor -g protein subtype coupling remain unresolved. based on the hypothesis that the affinity of the ternary complex (agonist/gpcr/g-protein) in the nucleotide-free state determines the selectivity of gpcr-g protein coupling, we set out to measure gpcr-g protein interaction in membranes of single cells. in order to quantify the affinity of gα-subunit towards gpcrs in single cell, we determined the lifetime of the receptor-g protein complex in living cells upon agonist withdrawal under conditions of gtp-depletion. therefore, we utilized förster resonance energy transfer (fret) based assays to study interactions between fluorescent muscarinic receptors and heterotrimeric g proteins in single permeabilized hek293t cells transfected with the appropriate cdnas. here we focused on muscarinic m1-, m2-, and m3-receptors and characterized the kinetics of agonist-induced binding of go/i -and gq/11-proteins to muscarinic receptors and their subsequent dissociation in the absence of nucleotides. as a measure of affinity we calculated the rate constant of g protein dissociation from the receptor after agonist withdrawal. the dissociation kinetics of go protein from m3-and m1-achrs was found to be 10-fold faster in comparison to gq. similarly, we observed a 15-fold right shift of the concentration-response curves of go proteins binding to m3-achr in comparison to gq. in order to ensure, that the affinity of the ternary complex correlates with the efficiency of g protein activation, we performed experiments on the g protein activity in intact cells expressing non-fluorescent m3-achr by using a fret-based assay. our results showed that gq activation required 10-fold lower agonist concentration compared to go activation, suggesting that indeed the stability of the ternary complex in the absence of nucleotides determines the selectivity of gpcr-g protein coupling. we further explored the subtype selectivity of m2-achr for gi family members by comparison of dissociation kinetics of gi1-, gi2, gi3-, and go-proteins from m2-achr under nucleotide depleted conditions. k off of gi1 and gi3 were found to be two-fold higher in comparison to gi2 and go proteins, indicating the higher affinity of the latter ones to m2-achr. our fret-based assay to study receptor-g-protein interactions in membranes of single cells has been proven to be a fast and reliable method to quantify the affinity of the ternary complex. the g protein subtype dependent differences in the affinity towards activated receptors correlate with the g protein coupling efficiency of this receptor. despite their tremendous pharmacological relevance and potential for the development of new drugs, our understanding of g protein-coupled receptor (gpcr) architecture and signaling mechanisms are still limited. major reasons for this are the low abundance and poor biophysical properties of gpcrs, which makes them one of the most challenging class of proteins for structural and biophysical studies. among the superfamily of gpcrs, the class b receptors comprising 15 receptors are structurally least understood because to date it has not been possible to obtain a crystal structure of this receptor class. to overcome these limitations, we have developed a method for improving functional expression and simultaneous thermo-stabilization of gpcrs by directed evolution which is based on expression of receptors in saccharomyces cerevisiae and subsequent selection of highly expressing variants by flow cytometry with fluorescent ligands. by this strategy, key residues within a receptor sequence can be rapidly identified that are responsible for improved biophysical properties without greatly affecting the pharmacological features of the receptor. we have now applied this method to the human parathyroid hormone 1 receptor, a member of the class b of gpcrs which is a major regulator of calcium homeostasis in the body and a key target for the treatment of osteoporosis. from two rounds of directed evolution in yeast we obtained several mutants of parathyroid hormone 1 receptor that exhibit strongly improved expression levels and that remain stable after solubilization in detergents. these receptor variants are ideal candidates for subsequent structural and biophysical analysis. opioid drugs exert nearly all of their clinically relevant actions through stimulation of mors (μ-opioid receptors). the molecular biology of endogenous opioid peptides and their cognate receptors has been studied extensively in vitro. for mor, signaling efficiency is tightly regulated and ultimately limited by the coordinated phosphorylation of intracellular serine and threonine residues. morphine induces a selective phosphorylation of serine 375 that is predominantly catalyzed by g protein-coupled receptor kinase 5. as a consequence, the selective morphine-induced s375 phosphorylation does not lead to a robust beta-arrestin mobilization and receptor internalization. by contrast, high-efficacy opioid agonists such as fentanyl or etonitazene not only induce phosphorylation of s375 but also drive higher order phosphorylation on the flanking residues threonine 370, threonine 376, and threonine 379 in a hierarchical phosphorylation cascade that specifically requires grk2 and grk3 isoforms. as a consequence, multisite phosphorylation induced by potent agonist promotes both betaarrestin mobilization and a robust receptor internalization. however, little is known about agonist-selective phosphorylation patterns in vivo after acute and chronic drug administration. to learn more about mor regulation in vivo we have generated a new μopioid receptor knock in mouse with an n-terminal ha-tag. using these mice, we were able to study in vivo phosphorylation of an endogenous g protein-coupled receptor using both mass spectrometry and phosphosite-specific antibodies. we were also able to address the question which of the many putative mor splice variants detected on the mrna level are indeed expressed as functional receptors in mouse brain. ion channels 061 hcn4 in thalamic relay neurons is necessary for oscillatory activity in the thalamocortical system institut für physiologie i, westfälische wilhelms-universität, münster, germany hcn channels underlie the i h current and are involved, among other functions, in the genesis of epilepsy. the significance of hcn1 and hcn2 isoforms for brain function and epilepsy has been demonstrated, however the role of hcn4, the third major neuronal hcn subunit, is not known. here we show an unexpected role of hcn4 in controlling oscillations in the thalamocortical network. hcn4 is predominantly expressed in several thalamic relay nuclei, but not in the thalamic reticular nucleus and the cerebral cortex. hcn4-deficient thalamocortical relay neurons showed a massive reduction of i h and strongly reduced intrinsic burst firing. evoked thalamic oscillations in a slice preparation were completely abolished. in vivo, brain-specific hcn4 null mutants were protected against induced spike-and-wave discharges (swd), the hallmark of absence seizures. our findings indicate that hcn4 is necessary for rhythmic intrathalamic oscillations and that the channels constitutes an important component of swd generation. ludwig-maximilians-universität, walther-straub-institut für pharmakologie und toxikologie, münchen, germany trpc4 and 5 channels are members of the classical transient receptor potential (trpc) family whose activation mechanism downstream of phospholipase c (plc) largely remained elusive until now. while trpc3/6/7 channels are directly activated by diacylglycerol (dag), trpc4 and 5 channels are commonly regarded as daginsensitive. in contrast to trpc3/6/7 channels, they contain a c-terminal pdz-binding motif allowing for binding of na + /h + exchanger regulatory factor (nherf) 1 and 2. interestingly, performing electrophysiological measurements, co-immunoprecipitations and intermolecular dynamic fret experiments, we found that dissociation of nherf proteins from the c-terminus of trpc5 confers dag-sensitivity on trpc5 channels. trpc5 channels were dag-sensitive under the following experimental conditions: inhibition of protein kinase c, amino acid exchange in the c-terminal pdz-binding motif, pip 2 depletion with and without involvement of plc, over-expression of g-protein coupled receptors, down-regulation of endogenous nherf1 and 2 proteins and overexpression of a nherf1 mutant incapable of trpc5 binding. these findings strongly argue for nherf proteins as molecular determinants for channel activation. interestingly, pip 2 depletion itself caused slight trpc5 current increases while during pip 2 depletion, the membrane permeable dag analogue oag evoked even higher trpc5 currents suggesting that pip 2 depletion induces an active and dag-sensitive channel conformation. receptor mediated pip 2 depletion also resulted in dissociation of nherf1 and 2 from the c-terminus of trpc5 thereby eliciting a dag-sensitive trpc5 channel conformation. thus, our findings suggest that dag-sensitivity of trpc5 is the result of an activation cascade starting with pip 2 depletion and subsequent dynamic dissociation of nherf1 and 2 from the c-terminus of trpc5. altogether, dagsensitivity is a unifying functional hallmark of all trpc channels. the melastatin-related transient receptor potential channel trpm3 is a heat-activated nonselective cation channel expressed in sensory neurons of dorsal root ganglia. since trpm3-deficient mice show impaired inflammatory thermal hyperalgesia, the pharmacological inhibition of trpm3 may exert antinociceptive properties. fluorometric ca 2+ assays and a compound library containing approved drugs were used to identify trpm3 inhibitors and to characterize their potency and selectivity. biophysical properties of the block were assessed using electrophysiological patch-clamp methods. microfluorometry in fura-2-loaded single cells was applied to monitor [ca 2+ ] i signals in isolated dorsal root ganglion (drg) neurons. analgesic effects were assessed applying pregnenolone sulfate (pregs)-induced chemical pain and heat stimuli at mice. in the screening approach using stably transfected hek trpm3 cells we identified the nonsteroidal anti-inflammatory drug (nsaid) diclofenac, the tetracyclic antidepressant maprotiline and the anticonvulsant primidone as highly efficient trpm3 inhibitors. the compounds exhibited half-maximal inhibitory concentrations of 0.6-6 µm. the selectivity profiles of maprotiline and primidone for trpm3 were promising with no inhibitory effects on trpm2, trpm8, trpa1, trpv1, trpc5, trpc6 and p2x7 receptor channels. primidone inhibited pregs-induced [ca 2+ ] i signals in rat drg neurones, indicating a block of native trpm3 channels. consistently, primidone attenuated nocifensive responses of mice to paw-injected pregs. furthermore, intraplantar primidone reduced nociception in healthy and hyperalgesic cfa-inflamed paws in the hot plate test. the finding that an approved drug can inhibit trpm3 at concentrations that may be therapeutically relevant and thereby can act as an analgesic, provides a method to study trpm3-related effects by acutely challenging the channel´s function. pharmacological interference with trpm3 applying an approved drug or optimised successor compounds may pave the way to better understanding of physiological functions of trpm3 in humans and may represent a novel concept for analgesic treatment. excitotoxicity, calcium deregulation, mitochondrial dysfunction and neuroinflammation contribute to progressive cell death in many neurodegenerative diseases. therefore, proteins that prevent deregulation of these pathways are considered as drug targets. potential therapeutic approaches may benefit from modulation of small-conductance calcium-activated potassium (sk) channels, since recent data supports the hypothesis that sk channel activity promotes neuronal survival against cellular stress via a dual mechanism of action: i) by controlling neuronal excitability and ii) by preventing mitochondrial dysfunction and inflammation. our previous studies showed that activation of sk channels in neurons exerted protective effects through inhibition of nmdarmediated excitotoxicity. further, we revealed recently that in a model of glutamate oxytosis, activation of sk channels attenuated mitochondrial fission, prevented the release of pro-apoptotic mitochondrial proteins, and reduced cell death. however, little is known about the function of sk channels in cell metabolism and neuroinflammatory processes in non-neuronal cells, such as microglial cells. in this study, we addressed the question whether sk channel activation affected primary mouse microglia activation upon lps and α-synuclein challenge. we found that activation of sk channels significantly reduced activation of microglia in a concentration-dependent manner, as detected by real-time xcelligence cell impedance measurements. further data on cytokine (tnf-alpha and il-6) analysis revealed that activation of sk channels attenuated α-synuclein-induced cytokine release. inhibition of glycolysis prevented microglial activation and cytokine release. although sk channel activation slightly reduced atp levels, it attenuated α-synuclein-induced no release. furthermore, glycolytic products and ampk signaling were evaluated. overall, our findings show that activation of sk channels attenuates microglial cell activation. thus, sk channels are promising therapeutic targets for neurodegenerative disorders, where neuroinflammation and cell metabolic deregulation are associated with progression of the disease. mutant of the residue pair e103/k308 can be crosslinked efficiently in both states, the closed-and open state of the p2x2r. interestingly, oxidative crosslinking of cysteine substitution mutants of each individual residue pair significantly reduced the atpinduced current amplitudes. charge reversal or swapping mutagenesis and cysteine modification by charged mts-reagents indicated the electrostatic nature of the pairwise interactions in these four residue pairs. furthermore, preliminary data from triple, tetra and penta mutant cycle analysis indicated energetic coupling between the residue pairs e84/r290, e103/k308, e167/r290 and e167/k308 and thus indicates the cooperative interaction in a larger salt bridge network. together with the markedly reduced current amplitudes following disulfide crosslinking, our data suggest that the salt bridge network serves to stabilize the closed-state conformation of the p2x2r. the comparison of the closed-state and open-state model of the rat p2x2r showed that atp promotes a marked rearrangement of the side chains of the residues r290 and k308 to enable the strong ionic coordination of the γ-phosphate oxygen of atp. in summary, our data are in line with the concept that the electrostatic interaction of r290 and k308 with atp competitively releases e84, e103 and e167 from their strong electrostatic coupling and thus initiates a destabilization of the closed-state, which favors channel opening. fig. 1 in a similarity search using sequence motifs conserved amongst various members of the trp protein family we identified three non-annotated putative membrane proteins that we initially termed tmem1, tmem2 and tmem4. expression analysis using the nanostring ncounter system, northern blotting and rt-pcr showed that murine tmem2 is expressed in various tissues including heart, brain, lung, endothelium, colon, cardiac myocytes, cardiac fibroblasts, embryonic fibroblasts, mast cells and pancreatic acinar cells. hydropathy analysis predicts that tmem2 proteins exhibit 6 to 10 plasma-membrane spanning domains, but fluorescently labeled tmem2 fusion constructs expressed in mouse embryonic fibroblasts revealed a vesicular subcellular localization pattern. in contrast to the prediction by the psort ii algorithm, tmem2-eyfp could not be identified in the plasma membrane of fibroblasts, cardiac myocytes, mast cells or pancreatic acinar cells but showed a significant colocalization with markers and fusion constructs specific for acidic compartments including lysosomes. in tmem2 -/mice, a marked elevation of amylase and lipase plasma levels was observed. we found that constitutive but not stimulated amylase secretion from tmem2-deficient acinar cells is elevated indicating a cell autonomous defect. calcium (ca ++ ) is an important signaling molecule regulating stimulated as well as constitutive secretion from pancreatic acinar cells. microfluorimetric measurements using fura-2 or indo-1 indicate higher resting ca ++ concentrations in tmem2-/-pancreatic acinar cells correlating with elevated basal enzyme secretion. in tmem2-yfp-knock-add-on mice we identified tmem2 in organelles of the apical acinar cell pole and a partial colocalisation with lamp2 proteins. furthermore, largely increased elevations in cytoplasmic ca ++ concentration were observed upon osmotic lysis of lysosomes triggered by gly-phe β-naphthylamide (gpn) or by nh 4 cl application. the role of tmem2 for ca ++ release was evaluated by stimulation with low concentration of cholecystokinin 2pm) in the absence of extracellular ca ++ using both microfluorimetric recording of cytosolic ca ++ transients as well as electrophysiological recordings of ca ++ -activated chloride currents. these measurements revealed a higher frequency of intracellular ca ++ oscillations and a larger area under the curve of ca ++ activated chloride currents upon cck-8 stimulation indicating that tmem2 inactivation leads to an enhancement of the globalization of cck-8 evoked ca2+ release from intracellular organelles. taken together, our study identifies tmem2 as a novel regulator of ca ++ release from intracellular organelles including endo-lysosomes and as a critical determinant of constitutive protein secretion in pancreatic acinar cells. while generally highlighting toxicology as a translational science that requires academic anchoring, gundert-remy and co-workers [1] have called for efforts to improve the relevance of in vitro methodologies in predicting in vivo effects. against this background, the german society of toxicology working group on alternative approaches to animal testing proposes specific quality criteria (qc) for in vitro methods and for research work using in vitro methods. these qc may serve to evaluate in vitro methods that are developed or applied in-house or that are described in work plans, peer reviewed articles, etc. for the time being, the qc focus on in vitro cell or tissue culture methods that address human health endpoints in the context of substance-related regulatory toxicity testing. nevertheless, these qc are also generally applicable to in vitro research conducted for other toxicological purposes. relevant work from, e.g., the organisation for the economic co-operation and development has been taken into account in specifying the qc that cover the following aspects:  the 3rs impact of an in vitro method in replacing, reducing (and refining) a specific animal test for a specific toxicological endpoint. this aspect also includes scientific hurdles that, in the past, had impeded the successful development of in vitro methods for the given toxicological endpoint.  scientific relevance and reliability, i.e. which fundamental requirements should an in vitro method meet to ensure that its results are relevant and reliable.  practicability and applicability, i.e. what is the expected expenditure for the in vitro method, and have relevant authorities and industrial sectors been involved in the development of the in vitro method. qc related to the scientific relevance of research work using a specific in vitro method provide a tool to justify, e.g., the suitability of the selected test system and in vitro endpoint(s) for the given purpose; the selection of test substances, positive and negative controls; the setting and control of test concentrations; and the definition of acceptance criteria to determine the relevance of test results. the proposed qc may serve as a framework to assess the relevance of in vitro methods and in vitro research work. thereby, they aim at improving in vitro predictivity of in vivo toxicological effects, which in return contributes to reducing and replacing the need for animal testing. [1] gundert-remy, u. et al. (2015) . toxicology: a discipline in need of academic anchoring -the point of view of the german society of toxicology. arch toxicol 89: 1881-93. during the past decades, considerable progress has been made in implementing 3r approaches in routine safety assessment. in spite of these achievements, animal tests still need to be conducted if legal requirements prescribe in vivo tests or if no reliable, accepted alternative method exists. efforts to foster 3r approaches and make them 'ready for use' focus on three levels: development and validation of scientific methods/strategies, regulatory acceptance of acknowledged approaches and global harmonization of standards. strong cooperation between toxicological experts from scientific bodies, national and international authorities and industry is needed to advance on all three levels for the benefit of animal welfare. 3r approaches that have already been implemented in routine safety assessment of consumer goods do not focus solely on replacement of animal testing by use of accepted alternative methods. in cases where reliable alternative approaches are not yet available, reduction in animal numbers and refinement of testing procedures can be achieved on a case-by-case basis. a tailor-made, tiered testing strategy is usually pursued that involves knowledge on specific characteristics of the test item and makes use of all available data, including details on exposure and results obtained with structural homologues. hurdles to apply alternative approaches can even occur for established methods. as legislations give different priority to alternative approaches, it remains challenging to fulfil conflicting legal requirements in different regions of the world, or even to address horizontal legislations of the same region. furthermore, successfully validated and legally implemented alternative approaches might not always provide the safety assessor with meaningful test results. with gaining experience, limitations of test systems can become evident that affect for example the applicability domain of the method, as has been the case both for some in vitro and in vivo methods. in these cases, the new information needs to be shared not only among safety assessors, but also with method developers and regulators to facilitate refinement of scientific approaches and/or amendments of regulations. leibniz-institut für arbeitsforschung (ifado), vistox, dortmund, germany two-photon microscopy facilitates imaging of biological processes in vivo. establishing this recent technique in mouse liver allowed us to record in a real-time the sequence of events during acetaminophen (apap) induced-liver damage. although apap is intensively studied and described in vivo imaging revealed so far unknown scenarios of cell death. the hepatocytes close to the central vein of a liver lobule went within hours into cell death as commonly described due to the toxic metabolite napqi. surprisingly, we observed a distinct way of cell killing at the outer border of the dead cell area which is accompanied by bile acid decompartmentalization. there, within an hour after apap administration dilatation of bile canaliculi was observed. subsequently, bile acids containing invaginations arouse from the apical side of a hepatocyte into the cytosol. these invaginations ballooned until the bile leaked into the hepatocyte volume and subsequently the plasma membrane of the affected hepatocytes lost its integrity leading to cell death. this mechanism emerged in an environment for hepatocytes where moderate napqi levels meet intracellular high bile salt concentrations of the midzonal region. in conclusion, establishing in vivo imaging in mouse liver enabled us to identify new cellular mechanisms which cannot be discovered by conventional methods. universitätsklinikum düsseldorf, institut für toxikologie, düsseldorf, germany introduction: lung inflammation and fibrosis are considered as major toxicities after thoracic cancer radiotherapy. up to now effective pharmacological interventions for normal tissue protection are largely missing. hmg-coa reductase inhibitors (statins), which are used in the clinic for lipid-lowering purpose, are reported to have multiple inhibitory effects on genotoxic stress responses. for this reason we aim to investigate the usefulness of statins to protect normal lung cells in vitro and lung tissue in vivo from damage provoked by ionizing radiation (ir). methods: according to clinically relevant anticancer radiation regimens, we used fractionated irradiation schemes (4 x 4 gy) for both in vitro as well as in vivo experiments. we analyzed the effect of lovastatin on ir-induced dna damage formation and repair, dna damage response (ddr) and cell death in non-proliferating human lung fibroblasts, epithelial as well as endothelial cells. furthermore, we established an irradiation device that is useful to selectively irradiate the right lung of mice and investigated the influence of lovastatin on lung damage following fractionated and selective irradiation of the lung in vivo (balb/c mice). results: compared to lung fibroblasts and epithelial cells, endothelial cells exhibited the highest radiosensitivity and underwent ir-induced apoptosis which was partly prevented by lovastatin. by contrast fibroblasts and epithelial cells did not undergo apoptosis upon irradiation. lovastatin did not affect initial dna damage formation in any of these cells. in all three lung cell types lovastatin enhanced the repair of dna double-strand breaks as analyzed 24 h after the last irradiation by γh2ax nuclear foci formation. depending on the cell type lovastatin affected various components of the ddr machinery in vitro. in vivo, lovastatin prevented ir-mediated increase in breathing frequency as determined two and four weeks after fractionated irradiation. moreover, statin treatment attenuated the level of residual dna damage and ir-induced apoptosis as analyzed four weeks after irradiation. these results were mimicked when eht1864, a small molecule inhibitor of the small rho-gtpase rac1, was applied in vivo, pointing to an involvement of rac1 in statin-mediated radioprotective effects. conclusion: bearing in mind that statins are well tolerated in humans, we suggest the application of statins as a promising pharmacological strategy for the prevention of irradiation-induced damage of the lung. targeted genome engineering by crispr/cas9 is an evolving tool for generating specific knockout cell lines. co-expression of crispr/cas9 allows for efficient dna cleavage and introduction of so called indel mutations (insertion/deletion point mutations) that lead to either misfolded non-functional proteins or complete knockout. we exploited this tool to generate a bid (bh3-interacting domain death agonist) knockout cell line in neuronal ht-22 cells. bid has been shown to be involved in regulated cell death pathways like oxytosis where its activation mediates mitochondrial demise, subsequent release of apoptosis inducing factor (aif) and cell death. in the cell death model of oxytosis the cystine/glutamate antiporter (x c -) is inhibited by high extracellular glutamate concentrations. following events such as increasing lipid peroxidation and ros production resemble major characteristics of another emerging cell death pathway, called ferroptosis. in this study we generated a bid crispr/cas9-knockout cell line to elucidate the role of bid as a potential link of oxytosis and ferroptosis in the ht-22 cell line. in order to investigate the potential mechanistic overlap at the level of mitochondrial death pathways, we induced oxytosis with glutamate or ferroptosis with erastin in wild-type cells and analyzed the respective effects of the well-established inhibitors ferrostatin-1 and the bid inhibitor bi-6c9 on cell death and mitochondrial paradigms. these results were then compared to the effects of glutamate or erastin in crispr/cas9-bid-knockout cells. bi-6c9 inhibited glutamate-induced morphological changes of ht-22 cells and also prevented cell death as assessed using the mtt assay and annexin v/pi staining. similar results were observed with ferrostatin-1 in the model of erastin-induced ferroptosis. subsequent facs analysis of lipid peroxidation by bodipy staining demonstrated that bi-6c9 abolishes lipid peroxide formation in the erastin model and ferrostatin-1 in the model of oxytosis. facs analysis was further employed for the detection of mitochondrial ros formation. mitosox staining revealed a significantly decreased production of mitochondrial ros by bi-6c9 and ferrostatin-1 in the respective model systems. investigating the crispr/cas9-bid-knockout ht-22 cell line revealed that bid knockout prevented cell death, lipid peroxidation and mitochondrial toxicity in both model systems of cell death, oxytosis and ferroptosis. in conclusion, the present study exposes bid as a pivotal molecular link between the previously separated cell death pathways oxytosis and ferroptosis at the level of mitochondria. parkinson's disease is a common neurodegenerative movement disorder characterized by midbrain dopaminergic neuronal loss in the substantia nigra that has been linked to alpha-synuclein toxicity. however, the molecular mechanisms underlying alphasynuclein-mediated toxicity in human dopaminergic neuronal loss are not well defined. the goal of this study was to investigate the deleterious effects of alpha synuclein in particular mitochondrial toxicity in human dopaminergic cells. therefore, we have generated neuron specific, adeno associated virus type 2 (aav2) expressing cytosolic as well as mitochondrial targeted alpha synuclein and egfp expressing viruses used as respective controls. overexpression of both, the cytosolic and the mitochondrial variants of alpha synuclein severely disrupted the dendritic network, induced loss of cellular atp, enhanced mitochondrial ros production, and was associated with activation of caspases and dopaminergic cell death in a time-dependent manner. in addition, real-time analysis of mitochondrial bioenergetics using the seahorse bioscience system following aav infection elicited a complete damage to mitochondrial respiration capacity in the dopaminergic neurons. our results suggested that mitochondrial targeted expression of alpha synuclein appeared to be more toxic than the cytosolic form of alpha synuclein. in addition, ultrastructural mitochondrial morphological analysis by transmission electron microscopy illustrated a number of deformed cristae in cells expressing the cytosolic alpha synuclein and a complete loss of cristae structure and massively swollen mitochondria following the expression of mitochondrial targeted alpha synuclein in the human dopaminergic neurons. in addition, we found that inhibition of caspases by the broad spectrum caspase inhibitor qvd significantly ameliorated alpha synuclein-induced dopaminergic neuronal death. interestingly, inhibition of caspases preserved neuronal network integrity, atp levels and mitochondrial respiration capacity in both paradigms of cytosolic and mitochondrial alpha synuclein overexpression. overall, our findings show that cytosolic as well as mitochondrial targeted expression of alpha synuclein is detrimental to human dopaminergic neurons, while inhibition of caspases amend alpha synuclein toxicity at the level of mitochondria. thus, caspase inhibitors provide promising therapeutic potential to prevent dopaminergic neuronal death in parkinson's syndromes that are associated with alpha synuclein toxicity. degradation of and adverse effects caused by tattoo and permanent make-up pigments upon sunlight exposure and laser removal have been occasionally reported in the last decades. until now, only the ban of certain azo-pigments has been addressed in the national legislation. the regulation was based on a number of studies showing the cleavage of azo-bonds by ultra violet light and laser-irradiation leading to the formation of carcinogenic aromatic amines. as a result, especially german tattoo ink manufactures switched to the use of more light-fast polycyclic pigments assuming these would be safer for this kind of application when compared to azo-pigments. to assess the potential risks of polycyclic pigments in terms of decomposition in the skin, we compared the photochemical cleavage of the widely used azo-pigment orange 13 and the polycyclic pigment copper phthalocyanine blue. main decomposition products are qualitatively and quantitatively analyzed after q-switched laser irradiation of 1 mg/ml aqueous suspensions and tattooed pig skin. irradiated specimen were extracted with ethyl acetate and analyzed with gas chromatography coupled to mass spectrometric detection (gc/ms) using liquid injection and head-space sampling techniques. we were able to confirm the cleavage of pigment orange 13 at the azo-and other weak bonds in our experimental set-up (fig.1a) . amongst other substances, the carcinogens aniline (max. conc. 1.01 ± 0.12 µg/ml) and 3,3-dichlorobenzidine (max. conc. 0.88 ± 0.18 µg/ml) are formed. despite the lack of such weak bonds, the highly stable porphyrin-like structure of copper phthalocyanine blue is as well decomposed upon laser-irradiation (fig. 1b) . here, 1,2-benzenedicarbonitrile (max. conc. 1.11 ± 0.12 µg/ml) were found as the main decomposition product in all experimental setups. concentrations of cleavage products were generally higher in aqueous suspensions compared to pig skin extracts with both pigments. additionally, the highly toxic gas hydrogen cyanide (max. conc. 35.8 ± 4.32 µg/ml) and the human carcinogen benzene (max. conc. 0.19 ± 0.06 µg/ml) were formed from both pigments, dependent on the laser wavelengths used. cyanide levels of ≥50 µg/ml evolving upon ruby laser irradiation of >1.5 mg/ml aqueous suspensions of phthalocyanine blue were proven to significantly reduce cell viability in human skin cells in vitro. reference 1 schreiver, i., hutzler, c., laux, p., berlien, h. p. & luch, a. formation of highly toxic hydrogen cyanide upon ruby laser irradiation of the tattoo pigment phthalocyanine blue. sci rep 5, 12915 (2015) . understanding the interactions between nanoscaled objects and living cells is of great importance for risk assessment, due to rising application of nanomaterials in foodrelated products. several studies show that silver nanoparticles can reach the intestinal epithelia in nanoform in a human in vitro digestion model. nevertheless, only sparse data concerning the direct quantification of cellular uptake of silver nanoparticles are available. therefore, this study was focused on a systematical quantitative comparison of the cellular uptake of differently coated silver nanoparticles of comparable size. intracellular uptake was determined quantitatively via a transwell tm -system with subsequent elemental analysis (aas) and ion beam microscopy (ibm). silver nanoparticles were coated with poly (acrylic acid) and polyvinylpyrrolidone and characterized extensively by tem, dls, saxs, zetasizer and nanosight. agpure tm as a widely used reference nanoparticle coated with tween 20 and tagat to v was also used for comparison. different intestinal cell models were applied to get closer to the complex in vivo situation: beside the widely used caco-2 model we also investigated particle uptake in a model which considered the enterocyte-covering mucus layer, as well as in a model specialized on particle uptake, the so-called m-cell model. our findings suggest that silver uptake is clearly a particle-and not an ion-related effect. the internalization of silver nanoparticles was enhanced in uptake-specialized m-cells, although no enhanced transport through the cells was observable. furthermore, the mucus did not providing a substantial additional barrier for nanoparticle internalization. rutherford backscattering spectrometry (rbs) via ibm allowed distinguishing between adsorbed an internalized material and the results were in accordance with the transwell tm -data. additionally, ibm investigations via particle-induced x-ray emission (pixe) showed intracellular association of silver with sulfur. the quantification of silver nanoparticle internalization revealed a clear particle-specific and a coating-related uptake. furthermore, a high amount of silver nanoparticles is taken up in cell models of higher complexity. thus, an underestimation of particle effects in vitro might be prevented by considering cell models with greater proximity to the in vivo situation. analyzing iron oxide nanoparticles for drug delivery -innovative investigation tools for nanotoxicology nanoparticles offer promising new possibilities for medical applications including therapy and diagnosis of various diseases. especially nanoparticle systems with magnetic cores provide a broad application spectrum as contrast agents, magnetic transporters, or heat carriers in hyperthermia treatment. for bench to bedside translation of superparamagnetic iron oxide nanoparticles (spions) for medical applications, safety issues have to be clarified. for that, reliable standards must be established on the basis of comprehensively validated physicochemical and biological characterization methods. spions consisting of maghemite and magnetite are usually of brown or black color. due to these special properties, spions and other metal oxide nanoparticles are prone to interfere with classical toxicological assays relying on optical detection of colorimetric, fluorescence or luminescence signals. particularly, nanoparticle concentration and cellular uptake are further influencing factors. consequently, for reliable analysis of nanoparticle mediated effects, alternative robust and interference-free readouts have to be established. based on long lasting experience working with spions, we suggest a combination of complementary methods to analyse nanoparticle-mediated effects: multiparameter analyses in flow cytometry deliver statistically relevant data and link uptake of nanoparticles (side scatter increase) with cellular effects in a high-content style. combination of noninvasive, label-free impedance measurements (xcelligence system) with real-time (fluorescence) microscopy enables us to monitor cellular proliferation and morphology over several days without interference by nanoparticles. additional experiments in multicellular tumor spheroids provide information about tissue infiltration and thus, more closely resemble the in vivo situation. using those complementary methods, several drug-loaded spion systems dedicated for medical applications have been successfully characterized previously. in sum, nanotoxicology is a complex and interdisciplinary challenge, where physicochemical parameters, as well as in vitro and in vivo behavior of nanoparticles have to be considered. to address these basic requirements, we are working on a stringent standardized road of characterization for iron oxide nanoparticles synthesized for medical applications. reference: lyer s, tietze r, unterweger h, zaloga j, singh r, matuszak j, poettler m, friedrich rp, duerr s, cicha i, janko c, alexiou c. nanomedical innovation: the seon concept for an improved cancer therapy with magnetic nanoparticles. nanomedicine (lond). 2015; 10 (21) acrylamide (aa) is an α,β-unsaturated compound, which is categorized as probably carcinogenic to humans [1, 2] . aa is known to arise in foods by heat treatment in the course of the maillard reaction between reducing sugars and amino acids at processing temperatures > 120 °c [3] . dietary aa exposure has mainly been estimated on the basis of dietary recall, assessing consumption of foods with known aa contents. the use of human biomarkers of aa exposure, primarily haemoglobin adducts of aa and its genotoxic metabolite, glycidamide ( ga) in red blood cells, as well as mercapturic acids excreted in the urine, is a promising alternative. such biomarkers are to be validated by exact measurement of aa uptake in duplicates of food as consumed (duplicate diet studies) [4] . we here present results of a nine-day human intervention study with 14 healthy male volunteers. aa contents were determined in duplicates of servings as consumed and kinetics of aa-associated mercapturic acids (aama and gama) monitored in total urine [5] . the study design included washout periods with an aa-minimized diet (21 -41 ng /kg bw), a low aa intake day (0.6 -0.8 µg /kg bw) as well as a high aa intake day (1.3 -1.8 µg /kg bw). after a three-day washout period an aama baseline level of 93 ± 31 nmol/d was determined. low aa intake led to an aama excretion within 24 h of 225 ± 37 nmol/d, high intake to 404 ± 78 nmol/d corresponding to an aama excretion rate of about 30% of the ingested aa dose within 24 h, whereas aama output within 72 h corresponded to 58% of the respective aa intake, the aama baseline after 3 days washout corresponds to a net exposure level of 0.2 -0.3 μg aa/kg bw/d. whether this represents a true baseline level is to be clarified in a follow-up study. in summary, this study provides important quantitative information on kinetics of urinary short-term exposure biomarkers validated by analytically verified dietary aa intake at present day food contamination levels. [1] deutsche forschungsgemeinschaft (dfg), mak-und bat-werte-liste 2013 , 2013 doi: 10.1002 iarc, iarc monographs on the evaluations of carcinogenic risks to humans 1994, 60. [3] tareke et al., j. agric. food chem. 2002, 50, 4998-5006. [4] efsa panel on contaminants in the food chain (contam), efsa journal 2015; 13(6):4104 [321 pp.] . [5] ruenz et al., arch. toxicol. 2015 doi: 10.1007 /s00204-015-1494 heinrich-heine-universität, institut für toxikologie, düsseldorf, germany objective: flavonoids are known to modulate distinct signaling pathways thereby causing different physiological effects. effects of the flavonoids baicalein and myricetin as well as several methylated derivatives were analyzed in the nematode caenorhabditis elegans and in in hct116 colon carcinoma cells and to get insights in molecular mechanisms modulated by these compounds. methods: radical-scavenging activity (teac, dcf), stress resistance (sytox, sodium arsenite), modulation of signaling pathways (nrf2/skn-1, daf16), life span. results: baicalein enhances the resistance of c. elegans against lethal thermal and sodium arsenite stress and dose-dependently prolongs the life span of the nematode (median life span: + 57%). using rna interference we were able to show that the induction of longevity and the enhanced stress-resistance were dependent on skn-1 (homolog to mammalian nrf2), but not daf-16 (homolog to mammalian foxo), another pivotal transcription factor. negletein was the only methylated derivative which was able to enhance the life span of the nematode. in hct116 cells, baicalein activates nrf2; the methylated derivatives oroxylin a and negletein showed a comparable redox-active potential in these cells, but only negletein was able to activate nrf2. the dietary flavonoid myricetin as well as the methylated derivatives laricitrin, syringetin and myricetintrimethylether strongly enhance life span of c. elegans, decreased oxidative stress (dcf) and accumulation of lipofuscin. in contrast to myricetin, the methylated compounds strongly enhanced the resistance against thermal stress. furthermore, treatment with the derivatives induced a much stronger nuclear localization of the daf-16 transcription factor. conclusion: baicalein increases stress-resistance and life span in c. elegans via skn-1 but not daf-16. experiments with methylated baicalein derivatives suggest that the redox-active potential has a minor impact on the nrf2/skn-1 activation since only distinct derivates activate this pathway. in case of myricetin, the methylation increases the stress resistance of the flavonoid. methylation seems to enhance the biofunctionality of the flavonoids. our results may be useful to understand molecular mechanisms of flavonoids and methylated derivatives used as food supplements or pharmacological extracts. the loss of progesterone during menopause is linked to common sleep complaints of the affected women. consequently, a previous study of our laboratory demonstrated sleep promoting effects of oral progesterone replacement in postmenopausal women [1] . the oral administration of progesterone, however, is compromised by individual differences in bioavailability and metabolism of the steroid. we therefore investigated the sleep-eeg effects after intranasal application of progesterone in 12 healthy postmenopausal women (50-70 yrs).in a randomized doubleblind protocol each subject received four treatments, 2 doses of intranasal progesterone (4.5mg mpp22; 9.0 mg mpp22), 10mg of zolpidem and placebo. the 4 conditions consisted of 2 experimental nights (adaptation + examination) separated by at least one week. during each examination sleep eeg was recorded from 23:00 to 07:00. simultaneously blood was collected every 20 min between 22:00 and 07:00 by long catheter for later analysis of the hormones growth hormone (gh), cortisol, melatonin and progesterone. conventional sleep-eeg was statistically evaluated by multivariate analyses of variances (manovas) with repeated measures designs after removal of two outliers, which showed a low sleep efficiency index (sei) after 4.5 and 9.0mg mpp22. univariate f-tests in the manovas pointed to the following results (significant p-values at α=0.05). sei was higher after zolpidem than after the other three treatments. after 9.0mg mpp22 sei was elevated significantly in comparison to placebo. subjects spent more time in nonrem sleep and less time in intermittent wakefulness after 9.0mg mpp22 and after zolpidem than after placebo. total sleep time was elevated and wake after sleep onset (waso) was reduced after 9.0mg mpp22 and after zolpidem. after all active treatments with mpp22 and zolpidem the time spent in sleep stage 2 was higher than after placebo. the amount of slow-wave sleep was higher after zolpidem than after placebo. in addition, the higher dose of mpp22 resulted in an increase of spindle and β frequencies combined with a decrease of δ oscillations during nonrem sleep. in comparison, administration of zolpidem resulted in strong increase of δ, spindle and high β frequencies as well as strong decrease in θ and α frequencies. nocturnal progesterone levels increased after 9.0 mg mpp22. no other changes of hormone secretion were found. our study show sleep promoting effects of 9.0 mg mpp. as expected the sleep promoting effect of zolpidem was confirmed. the spectral signature of intranasal progesterone partly resembled the well-known sleep-eeg alterations induced by gaba active compounds. progestereone levels were elevated after 9.0 mg mpp22. no other endocrine effects were observed. introduction: anticholinergic drugs or drugs with anticholinergic side effects are commonly used for the treatment of various diseases in the elderly population. elderly patients are particularly vulnerable to anticholinergic-related cognitive effects. moreover, there is a relationship between anticholinergic exposure and cognitive impairment. however, there is currently a lack of data on the anticholinergic burden in geriatric patients in germany. it was therefore the aim of this study to evaluate the anticholinergic burden in a large representative cohort of geriatric patients. materials and methods: in this retrospective cohort study, (co)-prescriptions of anticholinergic drugs as well as anti-dementia drugs were evaluated using the discharge medication of geriatric patients between january 2013 and june 2015 from the geriatrics in bavaria-database (gib-dat). anticholinergic drugs were classified according to the anticholinergic cognitive burden (acb) scale in three groups (definite anticholinergics with a score of 2 or 3 and possible anticholinergics with a score of 1). the acb scale was modified by omitting trospium and by adding the three drugs biperiden, metixen and maprotilin, which are used in germany, with a score of 3. a patient's individual score of 3 or higher is considered to be clinically relevant. in total, 130,186 geriatric patients (median age 82 years, 66.3% female, median no. of drugs 9) were evaluated. of these, 41,456 (31.8%) patients took at least one drug with anticholinergic properties. two or more anticholinergic drugs were coprescribed in 10,941 (26.4% of the patients taking anticholinergic drugs) patients. 11,241 (27.1% of the patients taking anticholinergic drugs) patients had a score of 3 or higher. the most common anticholinergic drug combinations involving two definite anticholinergic drugs were amantadine/quetiapine (58), amitriptyline/quetiapine (41) and amitriptyline/carbamazepine (35). 2,885 (7.0%) patients received anticholinergic drugs in combination with anti-dementia drugs. conclusions: one third of patients in a large geriatric population were prescribed at least one anticholinergic drug. one quarter received a co-prescription of anticholinergic drugs. caution is advised prescribing anticholinergic drugs to elderly patients especially with dementia. the antiglaucoma agents brimonidine and timolol are novel substrates of the organic cation transporters oct2 and mate1 expressed in human eye c. neul purpose: glaucoma is a leading cause of visual loss in the world population. lowering intraocular pressure by topical administration of antiglaucoma agents is still the mainstay for glaucoma treatment. 1,2 although many effective drugs exist, the major challenge is their efficient intraocular delivery, which is estimated to amount to 3 the involvement of membrane drug transporters in the intraocular delivery of the widely prescribed antiglaucoma prostanoid latanoprost has been described. 4 however, it is currently unknown whether the cationic drugs brimonidine and timolol, which are also commonly used antiglaucoma agents, are similarly transported by drug transporters and whether these transporters are expressed in human eye. brimonidine is an α 2 -adrenergic agonist, which inhibits the activity of the adenylate cyclase subsequently leading to a reduced production of aqueous humor. timolol is a β-adrenergic receptor antagonist, which blocks β-receptors on the ciliary epithelium also resulting in a reduced aqueous humor production. the aim of the present study was to determine whether brimonidine and timolol are substrates of the organic cation drug transporters oct1 (encoded by slc22a1), oct2 (slc22a2), oct3 (slc22a3) and mate1 (slc47a1). a further aim was to investigate whether these transporters are localized in different human eye substructures. experimental design: transport of brimonidine and timolol was studied using the mammalian cell line hek293 stably expressing the organic cation transporters oct1, oct2, oct3 or mate1. 5 intracellular accumulation of brimonidine and timolol was analyzed by mass spectrometry. immunohistochemistry and immunofluorescence experiments were performed to study the localization of these transporters in different substructures from glaucomatous and non-glaucomatous human eyes. results: uptake experiments revealed that brimonidine is transported by oct2 and mate1 in a time-and concentration-dependent manner, but not by oct1 or oct3. timolol is only transported by mate1, but not by the octs. as shown by immunolocalization studies, the oct2 and mate1 transporter proteins were expressed in all anterior eye substructures of non-glaucomatous and glaucomatous eyes, i.e. the cornea, the conjunctiva and the ciliary body. conclusion: our data demonstrate that oct2 and mate1 may play a role in the ocular disposition of the antiglaucoma drugs brimonidine and timolol and may contribute to interindividual variability of drug concentrations and effects. references: 1. zhang et al., nat rev drug discov. 2012 jun 15; 11(7) :541-59. 2. lavik et al., eye (lond). 2011 may; 25(5):578-86. 3. gaudana et al., pharm res. 2009 may; 26(5):1197 -216. 4. kraft et al., invest ophthalmol vis sci. 2010 51(5):2504 -11. 5. nies et al., plos one. 2011 6(7) :e22163. supported by the robert bosch foundation, stuttgart, germany. immature platelet count or immature platelet fraction as optimal predictor of antiplatelet response to thienopyridine therapy c. stratz 1 , t. nuehrenberg background: previous data suggest that reticulated platelets impact significantly on antiplatelet response to thienopyridines. it is unknown which of the parameters describing reticulated platelets is the optimal predictor of antiplatelet response to thienopyridine therapy. methods: this study is a prespecified subanalysis of the excelsiorload trial that randomized elective patients undergoing coronary stenting to loading with clopidogrel 600mg, prasugrel 30mg or prasugrel 60mg (n=300). adp-induced platelet reactivity was assessed by impedance aggregometry before loading (=intrinsic platelet reactivity) and on day 1 after loading. multiple parameters of reticulated platelets were assessed by an automated whole blood flow cytometer: immature platelet fraction (ipf, proportion of reticulated platelet of the whole platelet pool), highly immature platelet fraction (hipf), absolute immature platelet count (ipc). results: each parameter of reticulated platelets correlated significantly with adpinduced platelet reactivity: ipf (r s =0.18; p=0.002), hipf (r s =0.18; p=0.002), ipc r s =0.26; p<0.001). in a multivariable model including all three parameters, only ipc remained as significant predictor of platelet reactivity (p<0.001). after adjustment to known predictors of on-clopidogrel platelet reactivity including cytochrome p450 2c19 polymorphisms (*2 and *17), age, body mass index, diabetes, smoking and intrinsic platelet reactivity, ipc s21 was the strongest predictor of on-treatment platelet reactivity (partial η 2 =0.045; p<0.001) followed by intrinsic platelet reactivity (partial η 2 =0.034; p < 0.002). these findings prevailed when analyzing subgroups of patients on clopidogrel or on prasugrel. conclusion: immature platelet count is the strongest platelet count derived predictor of antiplatelet response to thienopyridine treatment. given its easy availability together with its even stronger association with on-treatment platelet reactivity when compared to known predictors including the cyp 2c19*2 polymorphism, immature platelet count might become the preferable predictor of antiplatelet response to thienopyridine treatment. cutaneous squamous cell carcinoma (cscc) is the second most common human cancer with continuously rising incidences worldwide. primarily caused by cumulative uvb exposure, cscc accounts for considerable costs for health care systems and poses a deadly risk especially to organ transplant recipients [1] . current chemotherapy needs to be improved, because even the topical treatment for cscc's carcinoma in situ bears limited efficacy and painful adverse effects [2] . however, animal-based approaches in preclinical development contribute to the frequent failure of investigational new drugs in clinical trials [3] . herein, we characterized a human cell-based cscc model, normal reconstructed human skin (rhs) served as control. whereas rhs exhibited low proliferation, the co-culture with cscc increased ki-67 index 23-fold in the cscc model (p£0.001). while the presence of claudin-4 and occludin were distinctly reduced, zonula occludens protein-1 was more wide-spread, and claudin-1 was heterogeneously distributed within the cscc model compared with rhs. this is in accordance to the in vivo situation [4] and likely contributes to the impaired barrier function of the cscc model, as demonstrated for 2.6-fold increased caffeine permeation. finally, the ingenol mebutate effects in the cscc model and rhs closely mimic the anti-tumor effect and the adverse reactions in patients [2] , both linked to the drug's inherent cytotoxicity. in conclusion, the thoroughly characterization of disease models fosters both advanced preclinical drug development and improved cscc treatment. funded by the german government, the berlin-brandenburg research platform bb3r with integrated graduate education was launched in 2014. the aim of this research platform, along with the associated graduate school, is to close substantial knowledge gaps in the fields of the 3rs and to find alternatives to animal experimentation within the next years. a panel of 3r experts has been set up to provide advice and assistance and to raise awareness in society for 3r-related issues. research in bb3r investigates physiological functions on different levels to establish alternative methods for preclinical drug development and basic research. the principal investigators aim at facilitating research collaborations and sustainable research activities in the region berlin-brandenburg and abroad. an integrated bb3r graduate program has been developed to offer structured training to graduate students in a specific, mandatory course program on 3r including modules on ethics and legislation. currently, 14 phd students are qualified for management positions in professional areas related to the 3rs, and three junior research groups are now ready to expand their regional research activities nationwide. furthermore, the concept of a novel lecture series for master students and undergraduates has been designed and awarded the animal welfare research award for berlin-brandenburg in teaching and education. the state government of berlin supports the research platform bb3r and will be funding an additional professorship at the fu berlin to further promote research on alternative testing. finally, co-operations with national and international partners are being built to facilitate the project-based exchange of scientists and joint research. currently, the identification and evaluation of skin sensitizers is mainly restricted to animal testing using the guinea pig maximization test, buehler test or the murine local lymph node assay. recently, an adverse outcome pathway of skin sensitization has been released by the oecd, identifying the key events leading to allergic contact dermatitis. in vitro tests address these key events and two assays are now regulatory adopted (oecd 442c and 442d). the use of the current in chemico and in vitro models is, however, limited since they do not reflect dermal penetration, complete biotransformation and cell cross-talk in an organotypic environment. in this study, we aimed to overcome these limitations by establishing reconstructed skin tissues containing langerhans cells (lcs). in vitro generated immature monocyte-derived (molcs) or mutz-3-derived cells (mutz-lcs) cultivated with keratinocytes on a dermal compartment with fibroblasts form a stratified epidermis after 14 days as indicated by the expression of epidermal differentiation markers. molcs or mutz-lcs were mainly localized in suprabasal layers of the epidermis and distributed homogeneously in accordance with native human skin. topical application of the extreme contact sensitizer 2,4-dinitrochlorobenzene (dncb) induced il-6 and il-8 secretion in skin models with lc-like cells, whereas no change was observed in control rhs lacking immune cells. increased gene expression of cd83 and pd-l1 in the dermal compartment indicated lc maturation. we confirmed the enhanced mobility from epidermal to dermal compartments for mutz-lcs and molcs in the presence of dncb. in summary, we successfully integrated immature and functional lc-like cells into reconstructed human skin. this fosters the development of animal-free test systems for advanced and potentially individualized hazard assessment of skin sensitization. computational methods for prediction of in vitro activity of new chemical structures. background: with a constant increase in the number of new chemicals synthesized every year, it becomes highly important to employ the most reliable and fast in silico screening methods to predict their safety and activity profiles. in recent years, in silico prediction methods received great attention as alternatives to animal experiments for evaluation of various toxicological endpoints, complementing the theme of replace, reduce and refine (3rs). various computational approaches have been proposed for prediction of toxicity of chemicals ranging from quantitative structure activity relationship modeling to molecular similarity based methods and machine-learning methods. within the "toxicology in the 21st century" screening initiative, a crowdsourced platform was established for development and validation of computational models to predict the interference of chemical compounds in nuclear and stress receptor pathways based on a training set containing more than 10,000 compounds tested in high-throughput screening assays. methods: here we present the results of various molecular similarity-based and machine-learning-based methods over an independent evaluation set containing 647 compounds. further, we compare the performance of these methods when applied individually and together. in retrospect we also discuss the reasons behind the superior performance of an ensemble approach, that combines a molecular similarity approach and a naïve bayes machine learning algorithm in achieving best prediction rates in comparison with other individual methods, explaining their intrinsic limitations. results and conclusions: our results suggest that, although prediction methods were optimized individually for each modeled target, an ensemble of similarity and machinelearning approaches provides promising performance indicating its broad applicability in toxicity prediction. charité -universitätsmedizin berlin, structural bioinformatics group, berlin, germany a multitude of drug candidates (approx. 20 %) fail in the late drug development due to toxicity and adverse effects [1] . immunotoxicity can be divided into four types of immune-mediated adverse effects: immunosuppression, immunostimulation, hypersensitivity and autoimmunity. current safety evaluations of drug candidates with respect to immunotoxicity are comprised of in vivo and in vitro assays. here, we present an in silico approach for predicting immunotoxic substances based on immune suppressive and not toxic compounds using the laplacian-modified naϊve baysian model as a supervised machine learning method. therefore, we examined the relations between about 51,000 compounds and 115 cancer cell lines from the national cancer institute's (nci) nci-60 growth inhibition data [2] with focus on five immune cell lines (rpmi-8226, ccrf-cem, . different fingerprints encoding the chemical structures have been evaluated for their predictive power (e. g. extended-connectivity fingerprints, substructure fingerprints) and showed good prediction rates in a retrospective analysis. acting in phase ii metabolism, sulfotransferases (sult) transform endo-and exogenous molecules such as drugs into more hydrophilic entities that are easily excreted from the human body [1] . although serving detoxification, sult-mediated transformation of molecules has also been associated with the formation of chemically reactive metabolites that could promote adverse reactions [2] . the development of a computerbased model that allows prediction of molecules susceptible to metabolism would improve drug development and drug safety [3] , and encourage the reduction of in vivo testing according to the principles of the 3rs (replacement, reduction and refinement). in our study, we developed an in silico model to predict human sult subtype 1e1 activity acting in phase ii metabolism. structure-based molecular modelling of sult activity is challenging due to the broad and overlapping substrate spectrum of sult subtypes. this low substrate specificity can be attributed to the high degree of conformational flexibility of the enzyme, particularly in the active site. therefore, molecular dynamics simulations were performed to address enzyme flexibility and the broad substrate spectrum of sult ( figure 1 ). based on a collection of selected enzyme conformations from molecular dynamics simulations and a dataset of active sult1e1 ligands, ensemble docking was utilized to generate ligand-protein complexes that served as a basis for 3d pharmacophore development. eight specific 3d pharmacophores were created that allow identification of sult1e1 substrates and inhibitors. for further refinement of the computer-based prediction, machine learning models and post-filtering steps were generated that allow classification of predicted hits. the final prediction model was used to screen the drugbank (a database comprising over 6,500 experimental and approved drugs). a major part of the predicted hits could be confirmed from literature. from the remaining hits, a representative selection of molecules was experimentally tested for sult1e1 inhibition or biotransformation. experimental results were in agreement with our computer-based models and revealed previously-unknown biotransformation by or inhibition of sult1e1 for compounds listed in the drugbank. introduction: vascularization of the dermal equivalent of full-thickness skin constructs by endothelial cells is highly desirable, because such constructs closely mimic the architecture of real skin. unfortunately, the realization of a capillary network in skin constructs is still difficult. in our study of full-thickness skin constructs, following the methodologies of küchler et al. (2011) , there were alterations in the epidermal differentiation after endothelialization of the dermal equivalent. the aim of this study was to characterize these changes on a morphological level. material and methods: non-endothelialized constructs (keratinocytes, fibroblasts) were prepared according to küchler et al. (2011) . to obtain endothelialized constructs, the dermal equivalent of the non-endothelialized constructs was enriched with endothelial cells. after two weeks of in vitro culture, the skin constructs were processed for quantitative as well as qualitative assessment by light and electron microscopy. results: both types of skin construct developed all strata, i.e., stratum basale, spinosum, granulosum, corneum of a stratified soft-cornified epidermis, although the two constructs displayed differences in every stratum: significantly more mitoses occurred in the epithelial germ layers of the endothelialized constructs (p=0.013). in addition, significantly more keratohyalin granules were counted within their stratum granulosum (p=0.010). 50% of the shapes of the spinous and the granulosum cells were irregular and these cells were separated by wide intercellular spaces. the typical epidermal lamellar bodies appeared in the endothelialized constructs more often than in the nonendothelialized ones. at the stratum granulosum -stratum corneum interface, no cohesion between the strata was present. background and novelty: during the last decade organ-on-a-chip technologies received increasing attention in the scientific community. the idea of combining different tissue types on a physiological-like system creates completely new options on how substances can be characterized without the use of animal experiments. animal models were used for the investigation of skin sensitization as a standard method until animal testing for cosmetic substances was banned in the e.u. in 2013. by combining skin models with an immunological counterpart, new data will be presented to see if the multi-organ-chip add value to the current need of alternative methods regarding skin sensitization and immunotoxicity testing. experimental approach: the multi-organ-chip platform is designed to combine different human cell and tissue types like 3d-spheroids, barrier models or biopsies in one microfluidic system. a peristaltic on-chip micropump enables circulation of medium, allowing for a constant perfusion between the compartments. first experiments were performed using a dendritic-cell-only approach in the 2-organ-chip. in subsequent cocultivation experiments ex vivo human epidermis and dendritic cells were cultivated each in one culture compartment connected by the microfluidic channels. for analysis we measured the typical activation marker cd86 and the vitality of the dendritic cells by flow cytometry. functionally different sensitizers were selected to investigate their effects in our model. finally a more complex 3d matrices including different immunological cell types to emulate in vivo-like reactions like in the human lymph node were cultivated in the 2-organ-chip. results and discussion: our data show a strong influence of pump pressure and pumping frequency on the activation of dendritic cells. hence, we established an adequate set up by cultivating the dendritic cells in cell culture inserts, preventing cell activation due to shear stress. compared to existing sensitization assays, the main advantage of the perfused 2-organ-chip sensitization assay is the presence of an epidermis equivalent, partially integrating important parameters such as metabolism and skin barrier function. we compared our data with reference cd86-values from the pbmdc (peripheral blood monocyte derived dendritic cells) skin sensitization assay. for identical substances, we observed differences in dendritic cell activation between the pbmdc assay and the 2-organ-chip perfused assay. here we present the first-time cultivation of primary derived immune cells cultivated on our microfluidic system which is a promising enhancement to integrate immunological reactions on further multi-organ combinations. due to growing social and political pressure, the interest in alternatives to animal testing has constantly increased during the past 10 years stimulating the development and validation of new in vitro test systems including reconstructed skin models. additional to toxicological studies and permeability assays, skin models are of high interest for fundamental research to elucidate basic physiological and pathophysiological processes in human skin [1, 2] . as for today, most of the in vitro skin models are grown from primary keratinocytes and fibroblasts that were either isolated from excised human skin or from juvenile foreskin following circumcision. in this project, we aimed for the generation of in vitro skin models using hair folliclederived cells. therefore, different methods to optimize cell isolation and expansion of outer root sheath (ors) cells from human hair follicles were systematically investigated. the best procedure for isolation of ors cells was the direct cell outgrowth on a cell culture insert which was co-cultured with a feeder layer of postmitotic human dermal fibroblasts. following outgrowth, the cells were either further cultivated with feeder cells in specific serum-enriched cell culture medium to obtain hair follicle-derived keratinocytes or using the same culture medium without feeder cells to obtain fibroblasts. afterwards, the generation of hair follicle-derived fibroblasts and keratinocytes was verified via the fibroblast-specific markers vimentin and desmin and the keratinocyte marker cytokeratin (ck) 14 clearly showing that vimentin and desmin are expressed in hair follicle-derived fibroblasts and in dermal fibroblasts. as expected, these cells were negative for ck14, which was abundantly expressed in hair folliclederived keratinocytes. moreover, the expression of collagen type i, iv, tgf-beta, alpha sma and il-1 alpha in hair follicle-derived fibroblasts and dermal fibroblasts showed no significant differences. ultimately, hair follicle-derived keratinocytes and fibroblasts were used to grow full-thickness skin models which were subsequently characterized with regard to epidermal differentiation, skin permeability and skin surface ph. again, no significant differences compared with skin models grown from skin-derived cells were detected showing the potential of using hair follicle-derived cells for generating in vitro skin models. [1] vávrová, k., henkes, d., strüver, k., sochorová, m., školová, b. et al. filaggrin deficiency leads to impaired lipid profile and altered acidification pathways in a 3d skin construct. the journal of investigative dermatology. 134, 746-753 (2014 bundesinstitut für risikobewertung, experimentelle toxikologie und zebet, berlin, germany background: the eu directive 2010/63 has been drawn up with the aim of ultimately replacing animal testing. wherever animal experimentation is necessary, the 3-rprinciple of russel and burch (replace, reduce, refine) has to be observed. the primary goal of the 3-r-principle is to replace animal testing with alternative methods. if no alternative method can be applied, the total number of animals is supposed be reduced. consequently, some animals are used multiple times in the course of an experiment. for example, in imaging studies, rodents are exposed to anesthesia several times in order to control the progress of a disease. however, the directive claims that "the benefit of reusing animals should be balanced against any adverse effects on their welfare, taking into account the lifetime experience of the individual animal". objective: we are looking into whether multiple exposures to anesthesia cause more stress than a single exposure. methods: the most common mouse strain c57/bl6 j and anesthetics isoflurane and the combination of ketamine/xylazine are used. with regard to recent studies, the animals are anesthetized six times for 45 minutes over a period of three weeks. all parameters observed are compared between controls, animals with a single and repeated anesthesia. the interval between the administration of the anesthesias is three to four days. when the animals are under anesthesia, their vital parameters are continuously monitored and afterwards their general condition is examined. the grimace scale is scored 30 and 150 minutes after anesthesia. besides pain, the grimace scale can also assess anxiety, stress and malaise. the display of so-called luxury behaviors like nest building and burrowing behavior serves as an indicator of wellbeing. furthermore, activity, food and water intake are monitored for 24 hours. a behavioral test battery including the free exploratory paradigm, open field, balance beam and rota rod test is performed one, seven and ten days after the last anesthesia. motor coordination and balance are assessed by the balance beam and rota rod. the open field is a test to investigate anxiety-related and exploratory behavior, the free exploratory paradigm estimates trait anxiety. moreover, corticosterone metabolites are measured in feces and fur in order to prove evidence of cumulative stress. results: the first results of our study will be presented at the 82 nd meeting of dgpt. conclusion: we are confident that the results of our study will contribute to the assessment of the severity level caused by multiple exposures to anesthesia and in this way be a benefit for the welfare of laboratory rodents. bb3r is funded by bmbf. in 1959 the 3r-principle was defined by the british scientists william russel and rex burch in their book 'the principles of human experimental techniques'. the 3r refer to replace, reduce, and refine. they set the goals to use alternative non-animal methods (replace), to reduce the number of laboratory animals (reduce) and to minimize the distress of laboratory animals and to refine their welfare (refine). the implementation of the 3r-concept is the overall goal of scientific animal welfare. article 4 of the 'directive 2010/63/eu on the protection of animals used for scientific purposes' state that the research on refinement is as important as on replacement and reduction [1] . according to the current statistics on laboratory animals, the mouse is the most commonly used animal in experiments with approximately 70 % [2] [3] . effective pain treatment is crucial not only for ethical and legal considerations but also to achieve highquality results [4] . pain in mice is only obvious if it is severe or acute pain. however, it is difficult to identify slight or moderate pain. the determination of pain levels and effective dosages of analgesics is therefore challenging. the most commonly used analgesics for postsurgical pain treatment in mice are opioids. however, the recommendations for their use show vast dosage ranges. for example, the recommended dose of buprenorphine ranges from 0.05 to 2.5 mg/kg per bodyweight [5] [6] [7] depending on the pain model used. additionally, putative pharmacogenetic strain differences have to be considered. for example, the analgesic treatment with morphine shows mouse strain specific differences in pain sensitivity [8] . the goal of the project is the refinement of the recommendations for effective dosage of opioid analgesics in laboratory animals for mouse inbred strains by incorporating strain specific differences. regarding the phenotype, we want to identify a putative inbred strain dependent pain threshold and measure the drug level in plasma and brain. additionally the metabolic capacity and mrna expression level will be investigated. genotype identification is based on a data bank analysis used for correlation with phenotypical parameters. [1] rl 2010/63/eu. [2] bmel (2014) . anzahl der für versuche und andere wissenschaftliche zwecke verwendeten wirbeltiere. [3] eu commission (2013) . seventh report on the statistics on the number of animals used for experimental and other scientific purposes in the member states of the european union. [4] carbone l (2011) pain in laboratory animals: the ethical and regulatory imperatives. plos one 6, e21578. [5] gv-solas, a.f. a.d. (2013) . empfehlung zur schmerztherapie bei versuchstieren. [6] carpenter, j.w., t.y. mashima, and d.j. rupiper (2001) . exotic animal formulary, 2nd edition, w.b. saunders co., phila. [7] flecknell, p. (1996) . laboratory animal anaesthesia, 2nd edition, academic press, san diego, ca. introduction: göttingen minipigs™ are frequently used large animal models for orofacial research, for example dental implant surgery. requests from experimental surgeons for detailed anatomical information can not be answered because there is no existing data, especially not age-related. because of unavailable data and the false choice of animal age, surgical interventions fail or lead to enormous post-operative suffering. therefore the aim of this study is the acquisition of detailed anatomical data of the mandibula and other organs and structures without sacrificing pigs for this reason. animals, materials and methods: ct scans of a 64-slice scanner were collected from 18 female minipigs, consisting of 6 animals aged 12 months (group 1, n=6) and 12 animals (group 2; n=12) examined at the age of 17 and 21 months. these minipigs were involved in experiments, approved by the regional office for health and social affairs, berlin. image analysis was performed using vitrea advanced® (vital images). more than 50 parameters concerning teeth, the mandibular body, frame and canal, coronoid process and mandibular condyle were defined and measured. for now, we focused on the development of the mandibular canal and the distance between the dorsal borders of the mandibular canal to the alveolar ridge at the most posterior mental foramen, parameters immensely important for planning interventions when testing new dental implants. results: the measurements by computed tomography showed variations of several parameters between left and right ramus mandibulae and within the different age groups. the volume of the canalis mandibulae increases in time. animals of the same age show significant differences in volume, with a range of up to 65% where the largest volume was 13,4 ml and the lowest 4,7 ml. the distance between the dorsal borders of the mandibular canal to the alveolar ridge decreases between 12 and 17 months of age. comparing 17 and 21 months old minipigs, no significant difference in distance could be observed. from the age of 17 months the position of the mandibular canal in relation to the alveolar ridge remains constant. conclusion: the decrease of the distance between the mandibular canal and the alveolar ridge between 12 and 17 months of age indicates ongoing anatomical changes of this parameter until the age of 17 months. therefore animals should be older than 17 months if included in long-term studies after orofacial experiments, like implant surgery of the mandibula. because of the described individual differences, the authors strongly suggest to support the planning of orofacial interventions by ct imaging or other radiographic techniques. background: laboratory housing conditions are standardized to a high level. under these conditions, the occurrence of stereotypic behaviour (sb) can be observed. stereotypies are commonly known as deviations from normal behaviour that are repetitive, invariant and without any obvious function or aim for the animal [1]. worldwide it is estimated that over 85 million animals perform sb, with the highest prevalence in laboratory animals and the agricultural sector. fvb/n is a typical inbred mouse-strain that shows different types of stereotyped movements. it is known that environmental enrichment decreases the incidence of sb, yet they still occur [2] . since animal's behaviour highly influences its metabolism and immune system, differences in handling, caring and keeping can lead to varying results, even with an identical experimental setup [3] . aim: of the study aim of the study is to observe different life stages of fvb/n-mice and immediately detect the development of sb. observations are linked to various behavioural tests and the characterisation of the metabolic and immunological phenotype. the results will lead to a better understanding of the mechanisms driving the development of sb and clarify its implication to animal welfare or to what extent the performance of stereotypies even reflect emotional suffering. the strain-related behaviour and sb are recorded with computer-assisted programmes. observational periods already begin with the parental generation. as possible indicators for later developing sb, data on reproductive success and maternal care are collected, such as different behavioural tests. the animals are characterized by a specific protocol for detecting the metabolic and immunological phenotype. finally, histological and molecular biological analyses follow. outlook: it is of paramount importance to take good care for the welfare of laboratory animals (3r-refinement). though, the knowledge about the ethological particularities of animals and the motivational base of animals performing sb are not enough to generally avoid stereotypies. therefore the meaning according to the character of sb has to be analysed more intensively to understand the needs of laboratory animals and evolve recommendations for optimizing the breeding and keeping such as for the assessment of possible distress in animals performing sb. objective: thermoregulation is a vital function in both humans and animals with the serotonin (5-ht) system, in particular the 5-ht 1a receptor, playing a major role. activating 5-ht 1a receptors by the 5-ht 1a receptor agonist 8-hydroxy-2-(dipropylamino)tetralin (8-oh-dpat) leads to reduced body temperature. while there is consensus that hypothermia is induced by the stimulation of postsynaptic 5-ht 1a receptors in rats and humans, the regulatory mechanisms in mice are less clear. in our group, within phenotyping a transgenic mouse line permanently overexpressing the 5-ht 1a receptor in serotonergic projection areas, bert et al. (2008, pmid: 18396339) revealed exaggerated 8-oh-dpat-provoked hypothermic response. thus, the objective of the present study was to substantiate the contribution of postsynaptic 5-ht 1a receptors to thermoregulation, more precisely to the hypothermic effect of 8-oh-dpat, in mice. methods: we used radio telemetry technique to monitor the basal body temperature and the hypothermic effect of different doses of 8-oh-dpat (0.1 mg/kg -4 mg/kg i. p.) in male transgenic mice in comparison to nmri wild-type males. additionally, we investigated whether reduction of serotonergic activity by pretreatment with the 5-ht synthesis inhibitor parachlorophenylalanine (pcpa; 100 mg/kg, i. p. on four consecutive days) would alter the effects of 8-oh-dpat on body temperature in transgenic mice postsynaptically overexpressing the 5-ht 1a receptor. results: 5-ht 1a overexpressing mice revealed lower levels of basal body temperature than wild types (transgenic mice: 36.0 °c; nmri wild-type mice: 37.4 °c). in both genotypes, systemic administration of 8-oh-dpat dose-dependently decreased body temperature, being significantly more pronounced in mutant mice (-2.8 °c compared to -1.5 °c in nmri wild types). dose response curves of 8-oh-dpat revealed an ed 50 = 0.4 mg/kg in transgenic and an ed 50 = 0.57 mg/kg in nmri wild-type mice. pcpa pretreatment did not alter the hypothermic response to 8-oh-dpat in mice. the dose-response curves indicate a higher potency of 8-oh-dpat in transgenic mice. the exaggerated hypothermic response to 8-oh-dpat in mutant mice implies that postsynaptic 5-ht 1a receptors could be involved in thermoregulatory function in mice. this assumption is further confirmed by the fact that 8-oh-dpatevoked thermal responses were not influenced by pretreatment with pcpa, most notably in transgenic mice overexpressing 5-ht 1a receptors postsynaptically. genetic variation within g protein-coupled receptors compromises the therapeutic application of drugs targeting these receptors. one of the most intensely studied variation is p.arg389gly in the human beta1-adrenoceptor (adrb1). the adrb1 carrying arginine at position 389 in helix 8 in the proximal carboxy terminus is more frequent among caucasians and is hyperfunctional. yet, the molecular basis for the differences between the beta1-adrenoceptor variants arg389-adrb1 and gly389-adrb1 is poorly understood. despite its hyperfunctionality, we found the arg389-variant of the adrb1 to be hyperphosphorylated upon continuous stimulation with norepinephrine when compared to the gly389-variant. using adrb1 sensors to monitor activation kinetics by fluorescence resonance energy transfer (fret), the arg389-adrb1 exerted faster activation speed and arrestin recruitment than the gly389-variant. both depended on phosphorylation of the receptor as shown by knockdown of g protein-coupled receptor kinases and by fret experiments using phosphorylation-deficient adrb1 mutants. point mutation of single serines and threonines in the carboxy terminus of the adrb1 finally revealed a variant-specific phosphorylation pattern that determines arrestin recruitment. taken together, these findings suggest that differences in receptor phosphorylation determine the differences in activation speed, efficacy and arrestin recruitment of adrb1 variants. opioid drugs are the most potent analgesics, which are used in the clinic; however, by activating the μ-opioid receptor (mor) they also produce several adverse side effects including constipation, antinociceptive tolerance, and physical dependence. there is substantial evidence suggesting that g protein-coupled receptor kinases (grks) and βarrestins play key roles in regulating mor signaling and responsiveness. following phosphorylation by grks, β-arrestins bind to phosphorylated mors, which prevents further interactions between the receptor and g proteins even in the continued presence of agonist resulting in diminished g protein-mediated signaling. we have previously shown that agonist-induced phosphorylation of mor occurs at a conserved 10-residue sequence, 370 trehpstant 379 , in the carboxyl-terminal cytoplasmic tail. morphine induces a selective phosphorylation of serine 375 (s375) in the middle of this sequence that is predominantly catalyzed by g protein-coupled receptor kinase 5 (grk5). by contrast, high-efficacy opioids not only induce phosphorylation of s375 but also drive higher-order phosphorylation on the flanking residues threonine 370 (t370), threonine 376 (t376), and threonine 379 (t379) in a hierarchical phosphorylation cascade that specifically requires grk2/3 isoforms. to investigate this mechanism further, we have developed novel β-galactosidase complementation assays to monitor agonist-dependent recruitment of grk2 and grk3 to activated mors. using this assay, we were able to show that activation of mor by high-efficacy agonists such as damgo results in a robust translocation of grk2/3. in contrast, activation by low-efficacy agonists such as morphine results in a much less pronounced recruitment of grk2/3 isoforms. interestingly, damgo-induced β-arrestin recruitment was strongly inhibited by sirna knock down of grk2 or grk3. conversely, the morphine-induced β-arrestin recruitment was strongly enhanced by overexpression of grk2 or grk3. mutation of s375 to alanine strongly inhibited both grk and β-arrestin recruitment. however, mutation of all 11 carboxyl-terminal serine and threonine residues of mor was required to completely abolish its interaction with arrestins and grks resulting in a complete loss of mor internalization and desensitization. heterotrimeric g proteins are located at the inner leaflet of plasma membranes and are a major primary transducer of cell signaling initiated by g protein-coupled receptors (gpcrs). based on sequence similarity, heterotrimeric g proteins can be subdivided into four main classes, i.e. gi/o, gs, gq/11, and g12/13, which interact with distinct cellular effectors to shape the final cellular response 1 . identification of new selective and cell-permeable g protein inhibitors is of great interest as these may be beneficial in complex pathologies that involve signaling of multiple gpcrs 2 . mechanistically, small molecule g protein inhibitors may, for instance, block nucleotide exchange by inhibiting gdp release (i.e. guanyl nucleotide dissociation inhibitors, gdis) or allow gdp release but block gtp entry by stabilizing the g protein in an empty pocket conformation 3 . here, we set out a new approach to classify g protein inhibitors regarding their mechanism of action in radioligand binding experiments. in particular, we investigated the influence of the specific gα q/11/14 inhibitor fr900359 4 on agonist-radioantagonist binding experiments performed with membranes of cho cells stably expressing the muscarinic m1 acetylcholine receptor (cho-m1). agonistradioantagonist competition under these conditions is biphasic because agonists bind with higher affinity in the ternary complex consisting of agonist, receptor and nucleotidefree g protein compared with a g protein-free receptor 1,5 . we show that fr900359 can be classified as a gdi as it significantly reduced high affinity binding of carbachol in cho-m1 membranes. in contrast to this, bim-46187, a pan g protein inhibitor with a cell-type-dependent preference for gq, did not influence high affinity agonist binding and thus stabilized gq in an empty pocket conformation 3 . interestingly, inhibition of high affinity agonist binding by fr900359 was incomplete in agonist-radioantagonist displacement studies and also when a radioagonist was applied. to fully prevent high affinity agonist binding by fr900359, combined uncoupling of both gi and gs proteins from m1 was required by pre-treatment with pertussis toxin and cholera toxin, respectively. these data demonstrate that not only gq, but also gi, and gs, contribute to the high affinity fraction of m1 receptors. taken together, our findings show that radioligand binding experiments are an attractive approach to classify new g protein inhibitors according to their mechanism of action. universität, würzburg, germany g protein-coupled receptors (gpcrs) are cell surface receptors which, upon a conformational change in the receptor protein induced by an extracellular stimulus, can transduce the signal onto intracellular adaptor proteins such as heterotrimeric g proteins [1] . gpcr-induced cell signaling can be rather complex as several gpcrs may activate multiple different adaptor proteins and can additionally be activated via distinct binding sites, i.e. the orthosteric transmitter binding site and other "allosteric" binding sites [2] . in the present work, we wanted to investigate the influence of an allosteric binding site on receptor activation of muscarinic acetylcholine receptors (machrs). to this end, we employed the orthosteric full agonists acetylcholine and iperoxo as well as several dualsteric compounds consisting of iperoxo linked to an allosteric phthalimide (phth) or naphthalimide (naph) moiety through alkyl chains of different length or through a diamide linker (fri). binding of the allosteric part to the receptor protein may restrict the conformational flexibility of the receptor protein and thus interfere with receptor activation [2] . therefore, application of different linker length may control the signaling outcome. here, we applied the human m1 machr which preferentially activates g proteins of the g q/11 type but can also promiscuously stimulate g s proteins. g q/11 -and g sdependent signaling pathways were analyzed by application of cho cells stably transfected with the human m1 machr in ip1 and camp accumulation assays, respectively. in comparison to the orthosteric building block iperoxo, all dualsteric compounds under investigation showed a decrease in potency for both g q -mediated and g s -mediated signaling. our findings show that the bulkier allosteric naph residue impaired both signaling pathways to a greater extent than the smaller substituent phth. particularly, the compound iper-6-naph completely lost intrinsic activity for both g q/11 and g s activation at the m1 machr. moreover, g s -mediated pathway activation is more sensitive to spatial restriction in the allosteric vestibule than g q -signaling. interestingly, longer linker length led to improved signaling for both pathways (g q and g s ) in both hybrid series. iper-7-phth seems to be an exception as it had a higher intrinsic efficacy for g s -dependent signaling than the other phth hybrids with longer linker chains. strikingly, only iper-fri-phth, which corresponds to iper-8-phth in linker length, but is able to engage increased hydrogen bonding with the receptor protein, acted as a full agonist on m1 machr for both signaling pathways under investigation. taken together, these data strongly suggest that, in comparison to g q/11 -mediated signaling, activation of the g s protein in m1 machr is more sensitive to spatial restriction in the allosteric vestibule. thus, it appears to be possible to control signaling of the m1 machr by allosteric constraint of the receptor's conformational flexibility. for more than three decades 3-(1h-imidazol-4-yl)propylguanidine (sk&f-91,486 (1) [1] ) is known as the prototypic pharmacophore of highly potent histamine h 2 -receptor (h 2 r) agonists of the guanidine class of compounds including, e.g., impromidine and arpromidine. [2] in order to get more insight into the structure-activity relationships of alkylated analogues of sk&f-91,486, we characterized 78 newly synthesized derivatives including several bivalent compounds (e.g., 2) by using different pharmacological in-vitro methods. [3] the potential h 2 r agonists were subjected to a broad screening procedure utilizing radioligand binding assays with membranes of sf9 cells [4] (hh 1,2,3,4 r). compounds were also functionally characterized in the [ 35 s]gtpgs assay (hh 2 r, sf9 cell membranes). [5] selectivity vs. hh 1,3,4 r was determined for selected derivatives also using this technique. organ bath studies (gph 1 r (ileum), gph 2 r (right atrium)) yielded functional data in a more physiological environment. the major part of the new sk&f-91,486 analogues displayed partial or full agonism via hh 2 r and gph 2 r, respectively. the most potent analogue, bivalent thiazole-type bisguanidine 2, was a partial agonist (e max = 88%) and 250-times as potent as histamine vis-à-vis the gph 2 r. attempts to antagonize the positive chronotropic effect of (partial) agonists by preincubation with cimetidine, or by adding a cimetidine bolus at the end of the concentration-response curve, respectively, were successful and furnished pa 2 values for the antagonist (5.87 -6.38) which are in accordance with literature data. however, in the functional in-vitro assay on gph 2 r, the positive chronotropic response evoked by sk&f-91,486 was surprisingly resistant to antagonism by cimetidine and other typical h 2 r antagonists (ranitidine, famotidine), although the compound so far has been unanimously classified by others as a weak partial h 2 r agonist. this behaviour is unique within the large series of sk&f-91,486 analogues studied so far under similar conditions. probably the positive chronotropic effect of the lead compound is -at least in part -the result of a second molecular interaction which has been overlooked so far. [1] parsons, m.e. et al.; agents actions 1975, 5, 464. [2] buschauer, a.; j. med. chem. 1989 , 32, 1963 -1970 [3] pockes, s.; dissertation, univ. regensburg 2015. [4] seifert, r. ; j. pharmacol. exp. ther. 2003, 305 (3) , 1104-1115. the nociceptin/orphanin fq (n/ofq) peptide (nop) receptor is the fourth most recently discovered and least characterized member of the opioid receptor family (mor, kor and dor). nop receptor is widely distributed and modulates several physiological processes by its endogenous ligand nociceptin. the nop receptor is a potential target for the development of ligands with therapeutic use in several pathophysiological states such as chronic and neuropathic pain. consequently, there is increasing interest in understanding the molecular regulation of nop receptor. recently, we generated two phosphosite-specific antibodies directed against the carboxyl-terminal residues serine 351 (s351) and threonine 362 /serine 363 (t362/s363), which enabled us to selectively detect either the s351-or the t362/s363-phosphorylated forms of the receptor. our results show that nociceptin, mcoppb, sch221510 and ro64-6198 induce a stably phosphorylation at s351 and t362/s363 followed by a profound internalization of the receptor. the nociceptin-induced s351 and t362/s363 phosphorylation can be blocked by selective nop receptor antagonists such as j113397 or sb612,111. nnc63-0532, buprenorphine and norbuprenophine failed to induce a phosphorylation at these sites. in the presence of nociceptin, s351 phosphorylation occurred at a faster rate than phosphorylation at t362/s363 indicating that s351 is the primary site of agonistdependent phosphorylation. activation of pkc by phorbol 12-myristate 13-acetate facilitated receptor phosphorylation only at s351 but not at t362/s363, indicating that s351 can also undergo heterologous phosphorylation. using nop-gfp knock in mice, we detected nop receptors in brain, spinal cord and dorsal root ganglia (drg). we were also able to demonstrate a dose-dependent nop receptor phosphorylation at t362/s363 in mouse brain in vivo using western blot and mass spectrometry. in contrast, mcoppb and sch221510 failed to induce phosphorylation in vivo. together, these data provide new insights into the molecular regulation of the nop receptor in vitro and in vivo. several findings indicate that inflammatory diseases are initiated or maintained by an imbalance of receptor baised signaling; the latter referring to the ability of distinct ligands to endue individual receptors with qualitatively different g-protein-and/or b-arrestindependent signaling (1). chemokines and their receptors regulate a wide array of leukocyte functions, including chemotaxis, adhesion, and transendothelial migration, and thus play important roles in regulating inflammation (2) . in man, two cc chemokine receptors ccr2a and ccr2b are present that only differ in their carboxyl terminal portions; the latter known to interact with multi-protein complexes made up of heterotrimeric g proteins (pertussis toxin sensitive and -insensitive) and non-g-protein components, including b-arrestin (2) . interested in differential signaling of ccr2a and ccr2b we comparatively analyzed ligand-induced g-protein-regulated signaling pathways (e.g. activation of phospholipase c isoenzymes and rhogtpase-induced serum response factor [srf] activity) and b-arrestin-regulated pathways (e.g. internalization of receptors and phosphorylation of erk1/2) in the presence of ccl2, ccl8, ccl7, and ccl13. all these chemokines have been shown to interact with human ccr2 receptors. in addition, the structural requirements of the carboxyl terminal portions involved in determining specificity in g-protein-dependent signaling was addressed by using ccr2 receptor mutants. the comparative analysis revealed that differences in ligand-induced activation of g-protein-dependent (pertussis-toxin-sensitive versus pertussis-toxin-insensitive) and/or b-arrestin-dependent signaling exist. for example, activation of ccr2b receptors by ccl2 induced both rho-dependent srf activation and receptor internalization, while ccl8-stimulation resulted in srf but little if any receptor internalization. in contrast, ccr2a-expressing cells showed ccl2dependent srf-activation but any receptor/ligand internalization. analysis of the structural requirements of ccr2 receptors for coupling to g proteins revealed that arginine 313 within the putative 'eighth helix' of the carboxyl-terminal portions of ccr2a and ccr2b is not involved in ga i -mediated induction of erk1/2 and plays a minor role in ccr2b receptor internalization, but is specifically required for the ccr2a/ and ccr2b/ga q -mediated activation of srf. serotonin 5-ht 2c receptors (5-ht 2c r) are functional engaged with gq proteins and expressed in the central nervous system (cns). 5-ht 2c r significantly regulate emotion, feeding, reward or cognition and thus might serve as promising targets for drugs against psychiatric disorders or obesity. due to the technical difficulties in isolating cells from the cns and the hitherto lack of suitable cell lines that would endogenously express 5-ht 2c r, our knowledge about this receptor subtype is rather limited. recently established hypothalamic mhypoa2-10 cells show some characteristics of appetite-regulating hypothalamic neurons of the paraventricular nucleus, where 5-ht 2c r in vivo expression has been detected. thus, we tested mhypoa2-10 cells for putative 5-ht 2c r expression by performing single cell calcium imaging. we observed that serotonin and the specific 5-ht 2c r agonist, way161,503 induced robust calcium transients, which were strongly inhibited by two unrelated 5-ht 2c r-selective antagonist (sb206,553, rs102,221). serotonin and way161,503 also activated a camp response element-dependent reporter gene construct and induced significant phosphorylation of extracellularregulated kinases-1/2 in a sb206,553 and rs102,221 sensitive manner, providing further evidence for functional 5-ht 2c r expression in mhypoa-2/10 cells. intrinsic activity of way161,503 ranged between 0.3 and 0.5 compared to serotonin in all assays, defining way161,503 as a partial 5-ht 2c r agonist. in conclusion, we provide convincing data that hypothalamic mhypoa-2/10 cells endogenously express 5-ht 2c r and thus represent the first cell line to analyse 5-ht 2c r pharmacology, signaling and regulation in its natural environment. optical and electrophysiological methods allow detection and characterization of g i/o -protein coupled receptors j. straub 1 1 walther-straub-institut für pharmakologie und toxikologie, münchen, germany g-protein mediated signaling pathways are essential components of basic cellular functions. of note, g-protein coupled receptors (gpcrs) constitute one of the major drug targets in modern medicine. however, despite their clinical importance, fundamental properties of these receptors remain unknown. in particular, regulation of the major second messenger camp by g s -and g i/o -protein coupled receptors is of special interest. the classical biochemical method to detect receptor-mediated camp level changes uses pre-labeling with 3 h-adenine and calculation of the conversion rate to 3 h-camp. although, this multi-cellular method is highly sensitive and reproducible, it lacks time resolved and spacial assessment of camp formation in single living cells. to measure g s -protein induced increases of intracellular camp levels in single living cells in a time resolved manner, the fret-based camp-sensor epac is commonly used. however, it was unknown whether this sensor might be suitable to detect g i/o -protein mediated decreases of intracellular camp levels as well. in this study, we show that fret-based camp sensors can be deployed to reliably monitor g i/o -protein mediated camp level decreases. fret experiments with adrenergic α 2a or µ 1 opioid receptors in combination with different fret-based camp sensors showed a notable reduction of intracellular camp levels upon receptor activation which could be significantly reduced by selective receptor antagonists. of note, hek293 cells had to be pre-incubated with forskolin in submaximal concentration to increase basal camp levels. our findings suggest that fret based epac sensors are suitable to detect g i/o -protein activation similar to electrophysiological whole-cell measurements with g i/o -protein coupled receptors and trpc5 or heteromeric kir3.1/kir3.2 or kir3.1/kir3.4 channels coexpressing cells. hereby, agonist stimulations caused current increases with characteristic current-voltage relationships. altogether, our findings indicate that both optical and electrophysiological approaches allow time resolved detection and characterization of g i/o -protein coupled receptor activation in single living cells. histamine can exert positive inotropic and chronotropic effects in humans via histamine h 2 -receptors. we have generated and partially characterized transgenic mice (tg) which overexpress the human histamine h 2 -receptor specifically in cardiomyocytes via the α-myosin heavy chain promoter. in these mice, but not in wild type mice (wt), histamine increased heart rate and ejection fraction (ef) measured in vivo by echocardiography under isoflurane anesthesia. to investigate some aspects of the signaling pathway in these mice, we crossed the tg mice with pp2a mice leading to double transgenic mice (h 2 xpp2a = dt). pp2a mice (j biol chem 2004; 279:40827) overexpress the catalytic subunit of protein phosphatase 2a (pp2a) in cardiac myocytes and develop a cardiac hypertrophy. at an age of about 240 days we noted reduced ef in pp2a (33.1 ± 3.5 %, n=16) compared to wt (54.4 ± 4.5 %, n=10) and tg (59.8 ± 2.9 %, n=10). interestingly, in dt the ef (43.9 ± 4.9 %, n=11) was higher than in pp2a at similar heart rates. e´/a´ was elevated in dt compared to wt. relative heart weights were unchanged between these groups. in summary, we demonstrated that pp2a is involved in h 2 -receptor signaling and we tentatively conclude that the h 2 -receptor is able to ameliorate systolic but not diastolic cardiac function of pp2a mice. serotonin (5-ht) can exert positive inotropic and chronotropic effects in humans via 5-ht 4 -receptors. we have generated transgenic mice (tg) which overexpress the human 5-ht 4 -receptor selectively in cardiomyocytes via the α-myosin heavy chain promoter. in these mice, but not in wild type mice (wt), serotonin induced increases in heart rate and ejection fraction. we treated the mice with 30 µg lps (lipopolysaccharide, i.p.; a standard model of sepsis) per g body weight or isotonic sodium chloride solution (as solvent control). echocardiography with isoflurane anesthesia was performed before and 3, 7 and 24 hours after lps treatment. lps led to a time-dependent deterioration of cardiac function in both tg and wt. the deterioration included systolic function (left ventricular ejection fraction=ef) as well as diastolic function (height of a and e waves through the mitral valve: e/a). for instance, ef amounted to 58.8 ± 20.7% seven hours after lps in wt and to 61. [4] [5] [6] [7] [8] [9] [10] [11] [12] p<0 .05 vs pre-lps value). however, 24 hours after lps, diastolic function, measured as e/a, amounted to 1.86 ± 0.43 in p<0 .05 tg vs. wt). moreover, after 24 hours a less pronounced decline in body temperature (probably due to superficial abdominal hyperemia) occurred in tg versus wt. in contrast, while all flow parameters declined after 3 and 7 hours of lps, they were not different between wt and tg. for instance, maximum flow (in mm/s) through the ascending aorta declined from 1158.3 ± 212.2 to 782.5 ± 154.3 in wt and from 1208.4 ± 235.1 to 798.8 ± 170.1 in tg (tg vs. wt, p>0.05, n=10-12; after 7 hours). we tentatively conclude: 5-ht 4 -receptors overexpression protects the heart against sepsis, putatively by interference with the intracellular biochemical pathway of lps in cardiomyocytes. histamine can exert positive inotropic and chronotropic effects in humans via histamine h 2 -receptors. we have generated transgenic mice (tg) which overexpress the human h 2 -receptor specifically in cardiomyocytes via the α-myosin heavy chain promoter. in tg, but not in wild type mice (wt), histamine (ec 50 = 34 nm) or amthamine (ec 50 = 10 nm), a more selective and potent h 2 -receptor agonist, induced positive inotropic effects (pie) and positive chronotropic effects (pce) in isolated left and right atrial preparations, respectively. in order to investigate the signal transduction for the pie in atrium, contractile studies using partially depolarized preparations were performed. therefore, left atrial preparations were equilibrated in the organ bath to 44 mm potassium chloride. thereafter, histamine (100 µm) induced a pie (31.1 ± 10.4 % of control, n=7) in tg but not in wt preparations whereas isoprenaline (10 µm) increased force in both wt and tg. the pie of histamine in potassium treated tg atrium could be blocked by cimetidine. compound 48/80, a releasing agent of endogenous histamine, increased force of contraction in tg left atria to a higher extent than in wt. furthermore, we tested whether analgetics known to release histamine were inotropically active in tg. however, morphine (10 µm) was unable to affect contractility in wt or tg, whereas ketamine and fentanyl increased left atrial contractility in both tg and wt. in summary, we demonstrated an involvement of the l-type calcium channel current in the h 2 -receptor mediated pie in tg atria. we failed to release inotropically active histamine using classical analgetics, arguing that a direct effect also in the human heart is unlikely to occur. the initial step in the homologous desensitization of g-protein-coupled receptors is their phosphorylation by one of the g-protein-coupled receptor kinases (grks). we demonstrate here measurement of the interaction of grk2, a ubiquitously expressed grk, with the μ-opioid receptor (µor) by fret and its dependence on agonist efficacy. hek293t cells transfected with yfp-tagged µor and mturquoise-tagged grk2 as well as non-fluorescent gα i1 , gβ and gγ subunits showed a robust increase in fret upon superfusion with 10 µm [d-ala 2 , n-mephe 4 , gly-ol]-enkephalin (damgo) which was reversible upon agonist washout. the partial agonist morphine (30 µm) also caused a fret increase but the amplitude of the fret signal was reduced to approximately 15-20% of that of the corresponding damgo signal. grk2 binds g-protein βγ (gβγ) subunits, and therefore we aimed to find out how cotransfection of grk2 affected the interaction of gβγ with the µor. however, we could not measure any damgo-induced fret changes between yfp-tagged µor and mturquoise-tagged gβ in the presence of non-fluorescent gα i1 and gγ. this was unexpected because we had previously successfully determined interactions between gβγ and the α 2a -adrenergic receptor or the m 3 muscarinic acetylcholine receptor. this lack of fret was not due to an inability of the tagged gβ to interact with the µor as we could measure damgo-induced fret changes between yfp-tagged gα i1 and gβγ (gβ tagged with mturquoise) in the presence of non-fluorescent µor. moreover, when we attempted to establish an effect of grk2 on the interaction between the µor and gβγ, we could pick up fret between the µor and gβγ. comparison of the on-and off-kinetics of the µor-grk2 interaction with that of the µor-gβγ interaction in the presence of grk2 revealed similar time constants both for the on-and off-kinetics (grk2: k on 0.16 s ). this suggests that, in the absence of grk2, the orientation of the two fluorophores on the µor and gβγ may be unfavorable or the interaction may be too short-lived to produce an appreciable fret signal. in the presence of grk2, however, gβγ changes its position relative to the µor in a way that allows the interaction of the grk2-gβγ complex with the µor to be detected by fret. similarly, we measured fret between gβγ and the α 2a -adrenergic receptor or the m 3 muscarinic acetylcholine receptor in the absence and presence of grk2 and compared the kinetics with the kinetics of grk2 binding and unbinding to these receptors. in both cases, we found that grk2 and gβγ in the presence of grk2 associate and dissociate from these receptors with comparable kinetics. our results suggest that ligand efficacy for µors is already apparent on the level of receptor-grk interaction. institute of pharmacology, university medical center göttingen, ag lutz, göttingen, germany introduction: the monomeric gtpase rhoa is dysregulated in heart disease and in vivo models provide evidence of rhoa signaling being involved in the progression of cardiac fibrosis and hypertrophy. how rhoa is regulated within this context on a cellular level is not defined. objective: the goal of this study was to analyze rhoa activation in adult cardiomyocytes (amcm) from normal and diseased mouse hearts in response to g protein-coupled receptor activation. this project also aimed at providing new insight into the dependence of rhoa localization and activation on the signaling organizing caveolae in neonatal as well as adult cardiomyocytes and engineered heart muscles. methods: cardiomyocytes from sham-operated c57bl/6 mice, from mice subjected to transverse aortic constriction (tac) or from neonatal rats were either directly fixed after isolation or cultured in the presence or absence of methyl-b-cyclodextrin (mβcd). for analyses of rhoa activation cells were treated with endothelin-1 (et-1) for 90 sec. cells were prepared for immunofluorescence analysis or lyzed for immunoblotting. imaging was performed using confocal microscopy. effects of mβcd were further studied in 3d engineered heart muscles (ehm) made from total neonatal rat cardiac cells. the contractile function of ehm was assessed in the organ bath and cells were studied in sections by immunofluorescence analysis. results: in amcm from sham mice active rhoa mainly localizes at the sarcolemma and is augmented in response to et-1 treatment. in tac-amcm the basal level of active rhoa is increased and surprisingly et-1 had no further effect. immunoblot analysis demonstrated that in tac-amcm rhoa expression was per se higher and the major caveolae protein caveolin-3 was reduced. to test the influence of caveolae on rhoa activation and expression, we treated nrcm with mβcd and found the expression of rhoa as well as of rhoa target genes ccn1 and ccn2 to be moderately up-regulated after 24h. in addition, an intensified longitudinal alignment of sarcomeric actin fibers was detectable, which could also be seen in mβcd-treated ehm. however, mβcd had no effect on ehm twitch tension but increased the resting tension compared to control. we further treated amcm with mβcd and found rhoa expression to be increased and its activity less sensitive to et-1 treatment. finally, we could show that the perinuclear localization of cav3 and rhoa was strongly reduced after mβcd treatment. whereas g-protein coupled receptors (gpcrs) have been long believed to signal through cyclic amp only at cell surface, our group has previously shown that gpcrs not only signal at the cell surface but can also continue doing so once internalized together with their ligands, leading to persistent camp production (1). this phenomenon, which we originally described for the thyroid stimulating hormone receptor (tshr) in thyroid cells, has been observed also for other gpcrs (2) (3) (4) . however, the intracellular compartment responsible for such persistent signaling was insufficiently characterized. the aim of this study was to follow by live-cell imaging the internalization and trafficking of tshr, tsh and effector proteins in thyroid cells. mouse primary thyroid cells were transfected with fluorescent-protein tagged tshr, g-proteins, nanobody specific for active g-proteins and/or subcellular markers by electroporation, stimulated with fluorescently labeled tsh and visualized using highly inclined thin illumination (hilo) microscopy. our results suggest that tsh is internalized in complex with its receptor and they traffic retrogradely via the trans golgi network (tgn). while we could not find any evidence of internalized tsh/tshr complexes activating g-proteins in early endosomes, we show that tsh/tshr complexes meet the intracellular pool of gαs in the tgn and activate it, as visualized in real-time by a nanobody specific for active gαs. upon acute brefeldin a-induced golgi collapse, the retrograde trafficking of tsh/tshr via tgn is hindered. bulk tsh stimulations in primary mouse thyroid cells isolated from transgenic mice expressing the camp sensor, epac1-camps, also show a significantly reduced camp production in the presence of brefeldin-a. these data provide evidence that internalized tsh/tshr complexes meet and activate g-proteins at the tgn, which might serve as the main platform of persistent camp signaling after receptor internalization. objective: sphingosine 1-phosphate (s1p) is generated by sphingosine kinase (sk)-1 and -2 and acts mainly as an extracellular ligand at five specific g protein-coupled receptors, denoted s1p 1-5 . after activation, s1p receptors regulate important processes in the progression of renal diseases, such as mesangial cell migration methods and results: here we demonstrate that dexamethasone treatment lowered s1p 1 mrna and protein expression levels in rat mesangial cells measured by taqman® and western blot analyses. this effect was abolished in the presence of the glucocorticoid receptor antagonist ru-486. in addition, in vivo studies showed that dexamethasone downregulated s1p 1 expression in glomeruli isolated from c57bl/6 mice treated with dexamethasone (10 mg/kg body weight). functionally, we identified s1p 1 as a key player mediating s1p-induced mesangial cell migration. using boyden chamber assays, we could show that dexamethasone treatment significantly lowered s1p-induced migration of mesangial cells. this effect was again reversed in the presence of ru-486. conclusion: we suggest that dexamethasone inhibits s1p-induced mesangial cell migration via downregulation of s1p 1 . overall, these results demonstrate that dexamethasone has functional important effects on sphingolipid metabolism and action in renal mesangial cells (koch et al., biol. chem. 2015; 396: 803-12) . the g protein-coupled receptor mrgd is a receptor for angiotensin-(1-7) involving g alphas , camp, and phosphokinase a rationale: angiotensin (ang)-(1-7) has cardiovascular protective effects and is the opponent of the often detrimental ang ii within the renin-angiotensin system. although it is well-accepted that the g-protein coupled receptor mas is a receptor for the heptapeptide, the lack in knowing initial signalling molecules stimulated by ang-(1-7) prevented final verification of ligand/receptor interaction as well as the identification of further hypothesized receptors for the heptapeptide. objective: the study aimed to identify a second messenger stimulated by ang-(1-7) allowing confirmation as well as discovery of the heptapeptide's receptors. we identified camp as the second messenger for ang-(1-7). the heptapeptide elevates camp concentration in primary cells such as endothelial or mesangial cells. using camp as readout in receptor-transfected hek293 cells, we provided final pharmacological proof for mas to be a receptor for ang-(1-7). more important, we identified the g-protein coupled receptor mrgd as a second receptor for ang-(1-7). consequently, the heptapeptide failed to increase camp concentration in primary mesangial cells with genetic deficiency in both mas and mrgd. furthermore, we excluded the ang ii type 2 receptor at2 as a receptor for the heptapeptide, but discovered that the at2 blocker pd123119 can also block the mas and mrgd receptors. conclusions: our results lead to an expansion and partial revision of the reninangiotensin system, by identifying a second receptor for the protective ang-(1-7) but excluding the at2 receptor, and by enforcing the revisit of such publications which concluded at2 function by using pd123119 as a specific at2 blocker. members of the g protein-coupled receptor (gpcr) superfamily are integral membrane proteins that are activated by extracellular ligands and induce cell signaling via g proteins and other adaptor proteins. rhodopsin, the prototypical gpcr that mediates vision, is activated by photons that isomerize its covalent ligand. spectroscopic analyses of the cognate agonist retinal allow a detailed description of rhodopsin dynamics at submillisecond resolution. using rhodopsin as a model, it has been demonstrated that receptor activation, i.e. a switch from the fully inactive to the fully active state, occurs within 1 ms 1 . activation kinetics of other receptors have been studied mainly using fluorescence resonance energy transfer (fret) which allows kinetic studies with high resolution in living cells 2 . in contrast to rhodopsin, activation constants of several gpcrs using agonist superfusion have been determined to range between 30-80 ms 3 . however, it is unknown if all gpcrs with diffusible ligands are really activated on a longer timescale or if ligand diffusion to the binding site is rate limiting. in this study, we intend to overcome ligand diffusion by using photodestruction of caged ligands. we monitor activation-related conformational changes of homodimeric metabotropic glutamate receptor 1 (mglur1) sensors by fret after uncaging of an inert glutamate derivative. 4-methoxy-7-nitroindolinyl-l-glutamate (mni-glutamate) is a caged derivative of glutamate that does not activate mglur1s. upon pre-incubation of hek-tsa cells expressing both cfp-and yfp-tagged mglur1 protomers with mniglutamate, a short uv laser pulse releases active l-glutamate close to the receptor binding site. we demonstrate very rapid mglur1 activation kinetics and this allows us to study the process of signal transduction of this homodimeric gpcrs opioids are still the mainstay of modern pain treatment. most of the clinically established substances primarily exert their effects via the µ-opioid receptor (mor). however, many side effects such as tolerance, constipation and respiratory depression limit their therapeutic use. the efficacy of mor agonists in the treatment of chronic pain is unsatisfactory. in general analgesic effects can be mediated by all four members of the opioid receptor family. the nociception receptor (nop) is the latest member of the opioid receptor family. there is a rapidly growing interest for the development of novel nop and combined mor/nop agonists. the aim of this developement is novel therapeutic agents with improved analgesic characteristics and less classical mor-mediated side effects. here we used buprenorphine as a clinically established opioid which exerts its effect on multiple opioid receptor subtypes. recently, nalfurafine, a potent kappa-opioid receptor (kor) agonist was granted by japanese authorities for the treatment of uremic pruritus. even though kor agonists are known to mediate dysphoria and hallucinations this has not been reported for nalfurafine. rudolf-virchow-zentrum für experimentelle biomedizin, würzburg, germany g protein-coupled receptors (gpcrs) belong to a superfamily of cell surface signaling proteins that mediate many physiological responses to hormones and neurotransmitters. they represent the prime targets for therapeutic drugs in healthcare. however, due to the limited knowledge about the pharmacology of the majority of gpcrs, only few of them are employed as therapeutic target. in our lab, the activation kinetics of the α 2aadrenergic receptor, among others receptors, has been extensively studied in single cell assays (1) (2) . the activation kinetics of the labeled α 2a -adrenergic were monitored by förster resonance energy transfer (fret). the goal of our study is to design a sensor to monitor receptor activation kinetics in high throughput screening assays. for the proof of concept, we used the α 2a -adrenergic receptor as a prototype. to optimize the fret efficiency we exchanged the previous acceptor (yfp) with the halotag technology (3) . additionally, we used halotag in combination with the nanoluc (4) to explore the possibility of using bret as a high-throughput approach to monitor receptor activation kinetics. the fret-based sensor α 2a -halo/cfp showed an increase in fret upon application of the full endogenous agonist norepinephrine with an ec50 value in accordance with the previously published data. this suggests the functionality of the fret-based α 2a -halo/cfp sensor. similar results were obtained with the α 2a -adrenergic receptor bret-based sensor. in contrast to the full agonist norepinephrine, the inverse agonist, yohimbine, decreased the ratio in both, fret as well as bret-based α 2aadrenergic receptor sensors. this suggests the sensitivity of the methods to discriminate among agonist (increased ratio) and antagonist (decreased ratio) induced receptor kinetics. our results show the feasibility of using halotag to monitor receptor activation via fret in a single cells format and halotag, in combination with nanoluc, can be used to monitor receptor activation in high-throughput format. , c. hoffmann the homologous visual arrestins) that has recently been solved by x-ray crystallography. here we investigated both the interaction with gpcrs and β-arrestin conformational changes in real time and in living cells with a series of fret-based β-arrestin2 biosensors. upon stimulation, β2-adrenergic receptors bound β-arrestin2 with a time constant τ = 1.3 ± 0.17 s, indicating that β-arrestin2 binding rapidly terminates their gprotein signaling. we observed a subsequent receptor-mediated conformational change in β-arrestin2 with a τ = 2.2 ± 0.22 s. stimulation of β2-adrenergic vs. m2 muscarinic or ffa4 receptors resulted in different patterns of conformational changes in the various β-arrestin2 sensors and also in downstream kinase signaling, revealing receptor-specificity in β-arrestin2 activation. upon agonist removal, first the interaction (delay = 1.9 ± 0.51 s) and only then the active state of β-arrestin2 (delay = 4.2 ± 0.85 s) were reversed. accordingly, β-arrestin localization at the cell membrane lasted much longer than the direct interaction with β2-adrenergic receptors. our data indicate a rapid, receptorspecific, two step binding and activation process between gpcrs and β-arrestins; they further suggest that β-arrestins remain active following dissociation from receptors, allowing them to remain at the cell surface and presumably signal independently. thus, gpcrs trigger a rapid, receptor-specific activation/deactivation cycle of β-arrestins, which permits their active signaling. patghogenic clostridium difficile produce two large glucosyltransferases, tcda and tcdb, which are the main pathogenicity factors. the cytotoxin tcdb is about 1,000 fold more potent than tcda. tcda and tcdb are a/b structure toxins exhibiting an enzymatically active (a) domain and a binding/translocation domain (b) to deliver the active glucosyltransferase domain into the cytosol of host cells. beside its glucosyltransferase activity by which the substrate proteins mainly of the family of rho gtpases are inhibited, tcdb has additional cytotoxic effects that are independent of rho glucosylation. to investigate the mechanism by which tcdb induces early cell death, we applied chimeras of tcdb from different toxinotypes where different glucosyltransferase domains were combined with different translocation domains. to this end we cloned tcdb from strain vpi10463 (historical strain), strain 1479 (serotype f, variant tcdbf), strain r20291 (hypervirulent strain, ribotype o27), and strain r9385 (hypervirulent strain with tcdbf characteristics). we were able to investigate the impact of the glucosyltransferase domains with different substrate specificity when translocated into the host cell by identical translocation domains. furthermore, we tested different translocation domains to deliver the same glucosyltransferase domain into host cells. we found that the glucosyltransferase domain of tcdbf (strain 1470) is less cytotoxic with respect to early cell death mediated by reactive oxygen species than that from reference strain vpi10463. in addition, the translocation domain also showed significant impact on cytotoxicity, probably by faster intracellular delivery of the gtd. by using glucosyltransferase deficient mutants where the highly conserved dxd-motif was changed to nxn, we were able to show that glucosylation of rho gtpases counteracts the cytotoxic effect, since the mutants were more cytotoxic than wildtype toxins. in conclusion, the cytotoxicity of tcdb mainly depends on the translocation efficiency into the host cell and on the kinetic of glucosylation of their substrate gtpases. thus, sensitivity of target cells towards cytotoxic effect also depends on receptor abundancy and intracellular status of rho gtpases, whereas the cytopathic effect, i.e. cell rounding, is predominatly determined by the substrate specificity. introduction: p63rhogef activates the g protein-coupled receptor (gpcr)-mediated induction of rhoa in different cells. however, its role in cardiac fibroblasts (cf) is not defined yet. thus we studied its localization and function in cf in 2d and 3d culture experiments. methods: neonatal rat cardiac fibroblasts (nrcf) and adult ventricular fibroblasts (amcf) from wild type mice and p63rhogef-knockout mice were adenovirally transduced for 48 to 72 h with recombinant adenoviruses or directly used. for 2d studies the cells were treated with angiotensin ii (ang ii). the location of the involved signaling components, rhoa activation and down-stream effects were studied by confocal microscopy and biochemical analyses. in addition, cf were used to prepare cf-containing engineered connective (ect) or muscle (ehm) tissues. results: we could show that p63rhogef locates at the plasma membrane, adjacent to the golgi apparatus and at the base of primary cilia. in accordance, p63rhogef regulates in response to ang ii the expression and secretion of the connective tissue growth factor (ctgf) in nrcf involving the serum response factor. in ect, p63rhogef increases the stiffness of these tissues and in ehm containing cf expressing gain-and-loss-of-function p63rhogef variants it influences the contractility. interestingly, the increase in ect stiffness was independent of p63rhogef's regulatory function of ctgf, as overexpression of ctgf in cf had no impact on ect properties arguing for a more general role of p63rhogef in auto-and paracrine signaling. moreover, our data on amcf with a genetic deletion of p63rhogef implies that p63rhogef is not only a transducer of gpcr-dependent rhoa activation as its loss led to an increase in rhoa expression accompanied by an increase in rhoa-dependent gene expression suggesting a role of p63rhogef in the feedback regulation of this signaling cascade. conclusion: in summary, our data show that p63rhogef regulates auto-and paracrine signaling in cardiac fibroblasts. the atrophic rhinitis is characterized by a drastic destruction of nasal turbinate bones in different animals. it leads to shortening and twisting of the snout and a growth retardation of young pigs. this bone degradation is induced by pasteuralla multocida toxin (pmt), a toxin produced by p. multocida serogroups a and d. this destructive effect indicates an interaction of pmt with bone cells like osteoclasts and osteoblasts. we demonstrated that pmt stimulates the differentiation of osteoclasts and inhibits the differentiation of osteoblasts in a gq-dependent mechanism. the underlying molecular mechanism of the toxin is the deamidation of an essential glutamine residue in the αsubunits of heterotrimeric g proteins, which results in the constitutive activation of the g protein. until now only the function and the pmt-dependent effects on osteoblasts and osteoclasts were studied in detail, but there is also a third important cell type in bone, the osteocytes. osteocytes are discussed as the regulator of the bone turn-over by interacting with osteoclasts and osteoblasts e.g. via secretion of several osteoclastogenic and osteoblastogenic cytokines. therefore, we studied the effects of pmt on the function of osteocytes in more detail. we utilized an osteocyte like cell line and primary osteocytes isolated from tibiae and femurs from mice. the susceptibility of primary osteocytes and the osteocyte like cell line towards pmt was demonstrated by detection of toxin-induced deamidation of g proteins. we also observed a pmt-induced secretion of different cytokines, like rankl, il-6 and tnf-α, which are known to induce osteoclastogenesis or inhibit osteoblastogenesis. furthermore, we studied the underlying signal transduction pathways and other pmtinduced effects on osteocytes, like morphological changes. in summary, we show that pmt acts on osteocytes by stimulating heterotrimeric g proteins. this might have impact on overall bone metabolism due to modulation of osteoblast and osteoclast activity. pasteurella multocida is an opportunistic pathogen often residing in the nasal pharyngeal space of animals. one virulence factor of p. multocida serogroups a and d is the protein toxin pmt (p. multocida toxin), which is the causative agent of atrophic rhinitis characterized by degradation of nasal turbinate bones in pigs and other animals. on the molecular level, pmt activates distinct members (g q/11 , g 12/13 and g i ,) of heterotrimeric g proteins leading to a modulation of bone metabolism: the toxin stimulates osteoclastogenesis but blocks osteoblastogenesis which results in bone loss. this mechanism of action of pmt might be exploited to counteract the human disease fibrodysplasia ossificans progressiva (fop), a rare and highly disabling disorder of extensive heterotopic bone growth. the underlying cause of fop is a point mutation in the activation domain of acvr1 (r206h), a bmp (bone morphogenic protein) type 1 receptor. this mutation leads to an inflated bmp-signaling and heterotopic osteoblastogenesis. here, we report that c2c12 cells, a mouse myoblast cell line often used as a fop model, are susceptible to pmt intoxication. pmt induces deamidation of g proteins in these cells. furthermore, pmt very efficiently inhibits bmp4-induced osteoblast differentiation in c2c12 cells. this has been shown by measuring alkaline phosphatase expression which is an early marker of osteoblast differentiation. additionally, the impact of pmt on acvr1 r206h induced osteoblastogenesis will be investigated and the involved cellular signaling pathways will be characterized in detail. the data indicate that activation of heterotrimeric g-proteins might be a rationale for pharmacological therapy of fop. p63rhogef is an activator of the monomeric gtpase rhoa and was shown to be expressed in the heart. in cardiac fibroblasts and smooth muscle cells, p63rhogef regulates rhoa in response to angiotensin ii and controls the actin cytoskeleton as well as protein expression and secretion. its role in cardiomyocytes, however, has not been elucidated so far. cardiomyocytes were isolated from neonatal rat hearts (nrcm), wild type mouse hearts (wt-amcm) and homozygous (ko-amcm) p63rhogef knockout mouse hearts. the cells were either directly fixed or adenovirally transduced for 48 h in culture. for activation of the g q/11 -signaling the cells were treated with endothelin-1 (et-1), angiotensin ii (ang ii) or phenylephrine (pe) for 90 s. rhoa activation was assessed by affinity binding or with a specific active-rhoa antibody. other proteins were detected by immunoblot or immunofluorescence analysis with subsequent confocal imaging. in nrcm p63rhogef is involved in the regulation of the et-1-induced rhoa activity and thus increases the expression and secretion of the rhoa target gene ctgf. in accordance, p63rhogef was found to be localized at the sarcolemma as well as in intracellular membrane compartments. the strongest co-localization was detected with the kdel-receptor 3 (kdelr3) which resides in the endoplasmatic reticulum membrane. next, we analyzed rhoa activation in wt and ko-amcm and could show that a loss of p63rhogef led to an increase in basal rhoa activity and an uncoupling from the gpcrs. interestingly, in the ko-amcm caveolin-3 the major component of caveolae, in which several gpcrs are clustered, was reduced in its expression and a shift in localization from transverse to longitudinal membrane tubules was found, arguing for a role of p63rhogef in intracellular protein transport. in accordance, golgi apparatus particles, which were demonstrated to play role in caveolae formation, were reduced in size in ko-amcm. to further address the role of p63rhogef in the transport of membrane proteins, we overexpressed p63rhogef in wt-amcm and could show that this led to an increase in the expression of the kdelr3 and its co-localization with p63rhogef in the perinuclear region and at the sarcolemma. no sarcolemmal localization of kdelr3 was found in control-transduced cells. further, p63rhogef was localized adjacent to golgi apparatus particles which were similar reduced in size as detected in the ko-amcm. finally, we expressed the dominant negative construct p63dn and detected similar changes with respect to kdelr3 localization and golgi structure suggesting that this regulatory function of p63rhogef is not dependent on its activity. conclusion: p63rhogef mediates the activation of rhoa from gpcrs coupled to g q/11 proteins. moreover, it has a function in intracellular transport and distribution of membrane proteins independent of its activity. universität bonn, pharmacology and toxicology section, bonn, germany g protein signaling is a means allowing cells to quickly respond and adapt to environmental changes. four major g protein classes (gs, gi/o, gq/11, g12/13) exist in mammals and these must suffice to convey signals from about 800 g protein-coupled receptors to the cell interior. as such, g proteins receive, interpret, and finally route the gpcr signals to diverse sets of downstream target proteins and thereby permit cells to respond to their ever changing environment. 1 understanding contribution of individual g protein classes or even isoforms to complex signaling networks in living cells requires capacity to activate or inactivate proteins with great precision and selectivity. one approach towards inactivation of g protein function is via chemical inhibition. however, "true specificity" of chemical inhibitors for their associated targets may often be debated. in this study we posit that fr900359, a cyclic depsipeptide isolated from the leaves of ardisia crenata, may clearly be designated as "truly specific" for inhibition of gq signaling. using a broad set of complementary methods based on label-free holistic cell sensing, classical endpoint assays, and bioluminescence resonance energy transferbased g protein biosensors we assign exceptional selectivity to fr for inhibition of gq/11/14 over all other mammalian isoforms ("on-target effects"). in holistic label-free recordings using hek293 cells that lack functional gq/11 alleles by crispr-cas9 genome editing, bona-fide gq stimuli were undetectable. however, reintroduction of gq into the knockout background was required and sufficient to fully restore both, agonist responses and their inhibition by fr. moreover, fr was completely ineffective in cells lacking gq/11 using phenotypic assays that examine basic cellular functions such as cell growth, viability, morphology and expression of housekeeping genes ("off-target effects") 2 . from these results we conclude that fr is of outstanding value as molecular probe to unravel contribution of gq signaling in complex biological processes in vitro, ex vivo and in vivo. just as pertussis toxin, applied world-wide by numerous laboratories to diagnose signaling of gi/o proteins, we anticipate fr to stand out at least equally for investigations into the biological relevance of gq. binary actin adp-ribosylating toxins like c. perfringens iota toxin and c. difficile transferase cdt cause depolymerisation of the cortical actin cytoskeleton, induce the formation of microtubule-based cell membrane protrusions and redirect rab-dependent intracellular traffic (schwan et al. 2009 ). here, we employed the model of toxin-induced protrusions to study the formation of cilia. we found that toxin-induced microtubule-based protrusion formation at the cell membrane depends on recruitment of septins, which are highly conserved, small gtpbinding proteins. similar to toxin-caused protrusions, septins are also recruited to the site of ciliogenesis. inhibition of septins by shrna-based knockdown inhibits ciliogenesis as well toxin-induced protrusion formation. septins are suggested to be involved in exocytotic processes, which are important for ciliogenesis and also for toxin-induced protrusion formation. accordingly, translocation of septins is accompanied by a recruitment of rab proteins and proteins of the exocytotic machinery. the data indicate that septins function as a scaffold at the base of cellular processes like cilia and toxin-induced protrusions, organizing the cross-talk between the actin cytoskeleton and microtubules to regulate the vesicle traffic-and exocytotic machinery. hypervirulent clostridium difficile strains are associated with increased morbidity and mortality. these strains produce the actin-adp-ribosylating clostridium difficile toxin cdt. cdt depolymerizes the actin cytoskeleton, causes formation of microtubule-based protrusions and increases pathogen adherence. septins are essential for cdt-induced protrusion formation. sept2, 6, and 7 accumulate at predetermined protrusion sites and form collar-like structures at the base of protrusions. the septins are a prerequisite for protrusion formation. the inhibitor forchlorfenuron or knock-down of septins inhibit protrusion formation. septins colocalize with active cdc42 and its effector borg which act as up-stream regulators of septin polymerization. microtubules interact with septin structures. precipitation and surface plasmon resonance studies revealed high-affinity binding of septins to microtubule plus end tracking protein eb1 thereby guiding incoming microtubules. the data indicate that cdt hijacks conserved regulatory principles involved in microtubule-membrane interaction, depending on septins, cdc42, borgs and restructuring of the actin cytoskeleton. the zebrafish danio rerio has become an important vertebrate model organism for a wide range of scientific questions [1] . current studies are mainly focused on development, genetics and disease for which the zebrafish is particularly well suited due to its small size, rapid development, short generation time, optical transparency of embryos and larvae as well as conservation in functional domains [1] . hitherto, nothing is known about the composition and endogenous level of different cnmps in various developmental stages and organs of danio rerio. therefore, we used the zebrafish in our study as a vertebrate model to characterize systematically the temporal-and organspecific occurrence(s) of all cnmps including cump in vivo. cyclic nucleotides were quantified by high performance liquid chromatography quadrupole tandem mass spectrometry. we observed specific cnmp patterns in developmental stages and different organs from adult zebrafish, which is in support of the hypothesis of a distinct cnmp signaling code [2] . camp, cgmp and cump were present in tissue samples of both developmental stages (embryos at 24 hours post fertilization, larvae at 5 days post fertilization) and within all harvested organs. remarkably, these three cnmps were the only ones detected in the brain. camp concentration of entrails as well as camp and cgmp concentrations in the brain were similar to those previously described in mouse tissues [3] . ccmp was detected throughout development and was present in all organs except the brain. the identity of ccmp and cump in the zebrafish was confirmed by high performance liquid chromatography quadrupole time-of-flight mass spectrometry (hplc-ms/tof). thus, we unequivocally show for the first time that cump occurs in vertebrates. furthermore, we detected cimp in several developmental stages of the zebrafish, and observed the highest concentrations in testes and heart, but we were unable to unequivocally identify cimp via hplc-ms/tof. in the zebrafish, sac is evolutionarily not conserved and absent, since a search in the ncbi gene data base revealed no entry for sac (also referred to as ac10). therefore, sac can be excluded as cump-and ccmp generator in this system and sgc remains as the only bona fide cump-and ccmp generator in the zebrafish. to test this hypothesis, the effects of no donors, sgc stimulators and sgc activators on cump levels in zebrafish should be examined in future studies. recently, induction of apoptosis in mouse lymphoma cells by ccmp-am has been described [4] . thus, it would be interesting to examine the effect of ccmp-am on zebrafish embryos in future studies as well. [1] seifert, r.: ccmp and cump: emerging second messengers, trends in biochemical sciences 40, 8-15 (2015) . ccmp, 3',5'-cump and 3',5'-cimp were ineffective. to further characterize the action of 3',5'-cgmp on hut-78 cells, we determined apoptosis (propidium iodide/annexin v staining) and proliferation (carboxyfluorescein succinimidylester staining). 3',5'-cgmp significantly increased apoptosis (ec 50 = 75 µm) and inhibited proliferation (ec 50 = 63 µm) of okt3-activated hut-78 cells. interestingly, also 2',3'-cgmp exhibited comparable effects on apoptosis and proliferation with ec 50 values of 92 µm and 75 µm, respectively, while 2',3'-camp, 2',3'-ccmp and 2',3'-cump were ineffective. this indicates that the pro-apoptotic and antiproliferative action of cgmp does not depend on the position of the phosphodiester bond. we also tested 3',5'-cgmp degradation products under the same experimental conditions and found that both 5'-gmp and guanosine increased apoptosis and inhibited proliferation with ec 50 -values between 50 and 100 µm. by contrast, adenosine did not influence cell growth and viability, suggesting that adenosine receptors are not involved in the observed effects. our results suggest that the guanosine moiety is responsible for the pro-apoptotic and antiproliferative effects of 3',5'-cgmp, 2',3'-cgmp, 5'-gmp. it has been reported earlier that guanosine is toxic to jurkat cells, another t cell lymphoma cell line [1]. 3',5'-cgmp may be hydrolyzed by an ekto-phosphodiesterase on the cell surface of hut-78 cells (e.g. enpp1), yielding 5'-gmp, which could be further degraded to guanosine by the 5'-ekto-nukleotidase cd73. a similar pathway may lead from 2',3'-cgmp to guanosine. a previous analysis of phosphodiesterases (pdes) revealed that the dual-specific pde isoforms 3a and 3b as well as the cgmp-selective pde9a also degrade the emerging second messenger cump [1, 2] . we analyzed the enzyme kinetics of pde3b-mediated cump-hydrolysis using recombinant gst-tagged pde3b and a highly sensitive and specific hplc-ms/ms method. our data show that pde3b is a low-affinity enzyme for cump with a cump k m -value of >100 µm. the pde3-selective competitive inhibitor milrinone inhibited pde3b-mediated cump degradation, suggesting that cump binds to the catalytic center. pde3b is highly expressed in adipose tissue [3, 4] . thus, we differentiated murine 3t3-l1-mbx-fibroblasts into adipocytes and analyzed differentiation-dependent alterations of pde3b expression and basal cnmp-concentrations. in both differentiated and undifferentiated 3t3-l1 cells cump and ccmp were detected in addition to the established second messengers camp and cgmp. differentiation to adipocytes reduced camp and cgmp by 66 % and 60 %, respectively, while ccmp was reduced by 78 % and cump even by 85 %. these findings suggest that cump plays a distinct role in adipocyte differentiation. the cump-hydrolyzing pde3b was upregulated ~1000-fold on mrna level after adipocyte differentiation, which may contribute to the observed reduction of basal cump concentrations. we currently investigate the potential biological role of cump in differentiation and lipolysis experiments, analyzing the effects of the membrane-permeant cumpacetoxymethyl ester cump-am. in future experiments, we will also analyze the enzyme kinetics of pde9a-mediated cump hydrolysis. pde9a is the first example of a cgmp-"specific" cump-hydrolyzing pde. background: cgmp and camp are cyclic nucleotide messengers relevant to many physiological and pathophysiological conditions. live-cell imaging with fret-based biosensors is a powerful method to study the spatiotemporal dynamics of cgmp and camp under close-to-native conditions. however, with the existing biosensors it is difficult to resolve potential membrane-associated cgmp microdomains and to monitor cgmp and camp signals in parallel in the same cell. we have generated novel versions of the "green" cfp/yfp-based cytosolic cgmp biosensor, cgi500. they comprise a "green" membrane-targeted version (mcgi500) and a "red" variant (red cgi500) that contains the fluorophores tsapphire and dimer2. methods: the sensors were expressed and characterised in primary vascular smooth muscle cells (vsmcs). intracellular cgmp was elevated in intact vsmcs by application of a nitric oxide donor or natriuretic peptides, and the sensor's sensitivity to each stimulation and its signal-to-noise ratio were determined. to test each sensor's sensitivity and specificity for cgmp versus camp, sensor-expressing cells were permeabilised with β-escin and exposed to defined concentrations of cyclic nucleotides. results: the original cgi500 sensor showed a good signal-to-noise ratio, an ec 50 value of ≈1.1 µm for cgmp, and a high selectivity for cgmp over camp (>100-fold). flincg3, a non-fret-based cgmp sensor, showed similar properties as cgi500. the new membrane-targeted mcgi500 and the new red cgi500 displayed ec 50 values of ≈0.4 µm cgmp and a high selectivity for cgmp over camp (>300-fold). in vsmcs, the red cgi500 showed a better signal-to-noise ratio than the previously described "red" cgmp sensor, red cges-de5. the "green" fret-based camp sensor, epac1-camps, showed a signal-to-noise ratio comparable to that of cgi500, an ec 50 value of ≈3 µm for camp and a selectivity for camp over cgmp of ≈6-fold. finally, imaging of cells expressing both the epac1-camps and the red cgi500 demonstrated the feasibility of combined visualisation of camp and cgmp signals in the same cell. the new cgmp biosensors should be useful for a broad spectrum of applications requiring real-time monitoring of cgmp signals. for example, mcgi500 would be useful to investigate membrane-associated cgmp compartments and red cgi500 to study the crosstalk between cgmp and camp signalling in living cells and tissues. the cgmp-system is a major regulator of blood pressure. cgmp-dependent protein kinases (cgks), located in the smooth muscle layer of vessels, enable cells to dilate and therefore cause a decrease in blood pressure (bp). to the contrary, the reninangiotensin-aldosteron-system (raas) acts as opponent and causes an increase in bp; furthermore, it influences fluid-electrolyte balance. renin, which is secreted from reninproducing cells located in the juxtaglomerular apparatus (jga), is the key regulator enzyme in this system. pharmacological inhibition of the raas, e.g. via ace-inhibitors or at1-receptor-antagonists, is a powerful tool to treat hypertension, but chronically challenges this endocrine system, resulting in an enhancement of renin expression. this is caused by an increased number of renin-expressing cells (the so-called reninrecruitment), which derive from a reversible metaplastic retransformation of extraglomerular and smooth muscle cells of afferent arterioles. next to regulation of renin-function via camp/pka, it has been shown that enos-derived no supports this recruitment via activation of sgc and subsequent generation of cgmp [1] . whether this causes an activation of cgks is not known. these enzymes exist in 3 different isoforms, cgkiα, cgkiβ und cgkii. in contrast to the β-isoform, cgkiα (as well as cgkii) is highly expressed in the jga [2] , [3] . therefore, we analyzed, whether cgkiα also plays a role regarding renin synthesis, secretion or recruitment. to characterize the function of cgkiα in jga-cells, we generated renin-cell specific cgkiα-knockout mice (ren cre-cgki fl/fl) and stimulated renin recruitment via administration of a low salt diet (0.02% na + ) and enalapril (10mg/kg/d) for 3 weeks. we analyzed blood pressure, mrna and renal protein content of renin and cgkiα, plasma renin activity and renin recruitment. furthermore, we activated the cgmp-system in these mice using bay41-8543, a sgcstimulator, and re-analyzed the above-mentioned parameters. our results indicate that cgkiα could be an additional system supporting renin recruitment but is not a crucial pre-requisite. in contrast, the basal renin concentration and activity appears to be downregulated in ren cre-cgki fl/fl-mice, thus, cgki could be an important regulator of renin synthesis. universität regensburg, pharmakologie und toxikologie, regensburg, germany jaw1/lrmp (lymphoid-restricted membrane protein) is a type 2 membrane protein, localised to the cytoplasmic face of the endoplasmic reticulum. it encodes a 539 amino acid protein with a highly conserved coiled-coil domain in the middle third of the protein and a cooh-terminal transmembrane domain [1] , [2] . jaw1 and irag have a limited homology throughout the length of the protein. the coiled-coil domain and the putative transmembrane anchor at the c-terminus of jaw1 and irag share the highest homology [3] , [4] . the coiled coil domains of irag and jaw1 are important for the interaction with ip 3 rs. as already known, irag forms a trimeric complex with cgkiβ and ip 3 r1 and gets phosphorylated by cgkiβ [5] . hence we examined if jaw1 is a new target protein of cgkiβ. the recognition site, where a substrate can be phosphorylated by cgki, is composed of the following amino acids: (k/r)(k/r)x(s/t). in the amino acid sequence of jaw1 possible phosphorylation sites can be found. our in vitro studies with jaw1 and cgki showed that jaw1 gets phosphorylated in a cgmp-dependent manner by cgkiβ. in contrast, jaw1 was not phosphorylated by cgkiα. furthermore, no stable interaction between jaw1 and cgkiβ was detected. to examine the importance of jaw1 in vivo, we generated a conditional knockout mouse. mating with a cmv cre mouse, resulted in an ubiquitous deletion of jaw1. mrna analysis and western blot analysis approved the deletion. the expression pattern revealed high expression in the thymus and weaker expression in the lung, spleen, colon, pancreas and the tongue. as already published by shindo et al., jaw1 was found in sweet, bitter, and umami taste receptor-expressing cells of mouse circumvallate, foliate, and fungiform papillae. we confirmed these results by x-gal staining and mrna analysis. therefore, we decided to analyse if jaw1 influences taste perception. two bottle preference tests did not result in significant differences between wildtype and knockout mice, indicating that taste perception is not altered by jaw1. hence, the function of jaw1 in taste receptor expressing cells has to be further examined in future studies. the cyclic purine nucleotides adenosine 3',5'-cyclic monophosphate (camp) and guanosine 3',5' cyclic monophosphate (cgmp) are well-characterized second messengers. both are generated by nucleotidyl cyclases and degraded by phosphodiesterases. several binding partners of camp and cgmp were already identified and functionally analyzed, e.g. camp-dependent protein kinase (pka) and cgmp-dependent protein kinase (pkg) as well as exchange protein activated by camp 1 and 2, hyperpolarization-activated cyclic nucleotide gated channels and phosphodiesterases. recent data indicate that the cyclic pyrimidine nucleotides cytosine 3',5'-cyclic monophosphate (ccmp) and uridine 3',5'-cyclic monophosphate (cump) also fulfill the criteria of second messengers [1, 2] . the interaction of ccmp with the regulatory subunits of pka (pkariα and pkariiα) has already been shown by using ccmpagarose [3] . additional ccmp-and cump-binding proteins such as calnexin (chaperone), myomegalin (phosphodiesterase-interacting protein) and akap9 (a-kinase anchoring protein) were identified by mass spectrometry analysis. to verify the interaction of ccmp and cump with these potential target proteins, ccmp and cump linked to biotin were used as another approach. the biotin-constructs exhibit lower steric interference than the ccmp-and cump-agarose matrices, which were previously used to confirm the binding of pkariα to ccmp and cump [4] . flag-tagged calnexin, flag-tagged myomegalin and myc-tagged yotiao (smallest splice-variant of akap9) were examined as potential ccmp and cump target proteins. hek293 cells were transiently transfected with the cdna of the respective proteins. the lysates of the protein-overexpressing cells were then incubated with ccmp-and cumpbiotin matrices and bound proteins were purified using strep-tactin® beads (iba). afterwards, the interaction of ccmp and cump with the potential binding partners was analyzed by western-blotting. a pkariα antibody was used as a positive control. analogous experiments were also performed using ccmp-and cump-agaroses. once the interaction between the cyclic pyrimidine nucleotides and the potential binding partners has been confirmed, deletion mutants will be cloned to localize the ccmp-and cump-binding area of the target proteins in further studies. axonal branching is essential for the correct formation of neuronal circuits and enables the simultaneous transmission of information throughout the body. in mice, the bifurcation of axons of sensory neurons at the dorsal root entry zone of the developing spinal cord depends on a cgmp signaling cascade that includes c-type natriuretic peptide (cnp), natriuretic peptide receptor 2 (npr2, also termed gc-b), and cgmpdependent protein kinase iα. in this study it was investigated, if a disturbance of cgmp signaling induced by manipulation of cgmp breakdown or cnp scavenging affects axon bifurcation of murine embryonic dorsal root ganglion (drg) neurons. rt-pcr screens, in situ hybridization, and fret-based cgmp imaging in living neurons revealed phosphodiesterase 2a (pde2a) as the major enzyme for degradation of cnp-induced cgmp in embryonic drg neurons. interestingly, cgmp measurements and dii labeling of pde2a knockout embryos indicated that a strongly elevated concentration of cgmp does not impair sensory axon bifurcation of drg neurons in vivo. the natriuretic peptide receptor 3 (npr3) was found to be expressed in the roof and floor plate of the spinal cord as well as in the dorsal roots of e12.5 embryos. because npr3 binds natriuretic peptides, but does not generate cgmp, it is thought to act as a natriuretic peptide clearance receptor. by scavenging cnp, npr3 could lower the activity of the cnp-npr2-cgmp signaling cascade in drg neurons. in the absence of npr3, the majority of sensory axons showed normal bifurcation, but »13 % of the axons turned only in rostral or caudal direction. this study shows (1) that pde2a is important for the degradation of cgmp in embryonic drg neurons, (2) that the bifurcation of sensory axons in the spinal cord can tolerate high levels of intracellular cgmp in the absence of pde2a, and (3) in the central nervous system, no-dependent cgmp signalling is associated with many different developmental processes and brain functions, and plays an important role in memory consolidation and cognition. to analyse cgmp signals in primary cells, a knock-in mouse was generated which stably and ubiquitously expresses a fret-based cgmp indicator (cgi500). cultured cortical and hippocampal neurons were found to respond to exogenously applied no (gsno). in these cell types, endogenous no is mainly generated by the neuronal no synthase (nnos) isoform which requires a rise in intracellular calcium for activation of the no/cgmp-signalling cascade. here, we show that ampa-type ionotropic glutamate receptors were capable to induce cgmp response in cultured cortical and hippocampal neurons. surprisingly, amparinduced cgmp signals were independent of nmdar activation, as inhibition of nmdars with the nmdar antagonist d-apv (d-2-amino-5-phosphonopentanoic acid) did not block ampar-induced cgmp response. however, cgmp accumulation depends on no synthase activation as the nos inhibitor l-nna (ng-nitro-l-arginine) completely abolished cgmp accumulation. whether ampar-induced nos activation depends on calcium influx via calcium permeable ampars, vgccs (voltage gated calcium channels) or calcium release from intracellular stores will further be investigated in detail. cyclic adenosine monophosphate (camp) is an important and ubiquitous cellular second messenger. a dogma in signaling is that camp is distributed homogenously in the cell and that its concentration changes equally upon stimulation. in contrast, a large body of evidence suggests the existence of concentration gradients (so-called microdomains) of camp. in this regard, phosphodiesterases (pdes), the only enzymes which can degrade camp, have been suspected to be responsible for maintaining those gradients. however, how pdes establish camp gradients is entirely unknown. here, we measure local camp levels in hek cells and cytosolic fractions thereof using the camp fretsensor epac1-camps fused to a phosphodiesterase (pde4a1). we demonstrate the existence of low camp concentrations in close proximity to pdes and show that this gradient is maintained by pde hydrolytic activity. further we establish that camp gradients cannot be maintained solely on the basis of pde activity as the camp turnover is very slow. we provide evidence that pdes are structurally organized in yet unspecified 'microstructures' in which camp diffusion must be considerably slowed down. taken together, we suggest that camp gradients are established by pde hydrolytic activity in cellular regions with slow diffusion of camp. our study sheds light on the organization and maintenance of signaling compartments in cells. the influence of pde hydrolytic activity on camp gradients universität würzburg, pharmakologie und toxikologie, würzburg, germany phosphodiesterases (pdes) are a family of enzymes that degrade cyclic amp (camp) and cyclic gmp (cgmp) to their respective monophosphates. although several pdes have been shown to play an important role in a wide variety of physiological and pathological processes, their complexity and function in cell signalling is only beginning to be understood. it is especially astonishing that eukaryotes express more than 100 different pde isoforms while their single function is to degrade camp, cgmp or both. in recent years, a large body of evidence has suggested that pdes (especially isoforms of the pde families 3, 4 and 5) are key players in establishing signalling compartments. these so-called microdomains are yet unspecified regions in cells where the concentrations of camp and cgmp are higher or lower than in the bulk cytosol. in a companion abstract (bock & lohse) we provide evidence that camp nanodomains, i.e. regions of low camp concentrations, exist in cells in the direct vicinity of pde4. however, the mechanisms by which pdes establish and maintain camp gradients are largely unknown. here we study if establishing camp gradients is a general role of pdes and, moreover, if the size of camp nanodomains mainly depends on pde hydrolytic activity. by fusing an ultra-fast pde2a3 (v max = 120 µmol/min/mg) to a camp fret sensor (epac1-camps) we monitor camp concentrations in direct vicinity of pde2a3. in comparison to pde4, we show that pde2a3, albeit displaying a high camp turnover, only establishes a small camp gradient in both cytosol preparations of transfected hek cells and in living cells. interestingly, this gradient can be increased by deleting the nterminal regulatory domains while maintaining fast camp turnover. biochemical mapping of the camp gradient gives an estimate of the size of nanodomains. taken together, our data suggest that establishing camp gradients does not exclusively depend on pde hydrolytic activity. arglabin is a plant-derived sesquiterpene lactone used for cancer therapy in kazakhstan and russia. signaling pathways targeted by arglabin are poorly understood. we have isolated arglabin by using high performance liquid chromatography from a methanolic extract of artemisia glabella, a plant endemic in kazakhstan. mass spectrometric analyses confirmed the chemical structure and the purity of the isolated arglabin. in j774 macrophages, arglabin strongly induced accumulation of lc3 type ii protein in the absence of inflammasome activators, and also in cells activated with lps and cholesterol crystals. in addition, arglabin induced clustering of lc3-ii at autophagosomal membranes, as evidenced from its punctuated pattern in confocal microscopic images of arglabin-treated macrophages, which is a characteristic sign for autophagy. since autophagy activation leads to increased degradation of nlrp3 and pro-interleukin (il)-1β, we further analyzed whether arglabin inhibits the nlrp3 inflammasome. arglabin reduced expression of nlrp3 and pro-il-1β, inhibited activation of caspase-1, and release of mature il-1β by lps-and cholesterol-crystal-stimulated macrophages consistent with inhibition of the nlrp3 inflammasome. intraperitoneal injection of arglabin into female apoe2.ki mice fed a high fat diet resulted in significantly decreased plasma levels of proinflammatory il-1β. moreover, arglabin markedly reduced mean lesion areas in the sinus and whole aorta in mice. thus, arglabin may represent a new promising drug to treat diseases associated with inflammasome activation, e.g. atherosclerosis. this work was supported in part by a grant from the nouvelle société francophone d'athérosclérose (nsfa). recently, we found that activation of the proteinase-activated receptor (par) 2 stimulates renin release in the isolated perfused kidney model. therefore, we determined in the current experiments the response of plasma renin concentration (prc) to acute intraperitoneal administration of the par2 activating peptide sligrl (100µg/kg), hydralazine (2 mg/kg), isoproterenol (10 mg/kg), losartan (3 mg/kg) and furosemide (40 mg/kg) in conscious wild-type (wt) and par2-deficient mice. prc was measured in plasma obtained by tail vein puncture. renal renin expression was determined by quantitative rt-pcr. renal protein expression was measured by immunohistochemistry. on a control diet (0.6% nacl), plasma renin concentration (in ng angiotensin i per ml per hour) was significantly lower in par2-deficient mice than in wild-type mice (190 ± 35 versus 380 ± 91). renin mrna expression was 50 ± 9% of wt. renin-expressing cells were located at the juxtaglomerular position and renal renin protein expression was lower in par2-deficient mice. as measured by tail-cuff method, systolic blood pressure was not different between par2 (-/-) and wt mice. administration of sligrl increased renin secretion about 6-fold (p<0.01). acute stimulation of renin release by furosemide, isoproterenol, losartan and hydralazine caused significant increases of plasma renin concentration in both par2 (-/-) and wt mice. the absolute changes (delta prc) were similar (3780 ± 770, 2230 ± 400, 2780 ± 70, 1390 ± 460 in wt, and 2320 ± 270, 2530 ± 250, 3010 ± 210, 1230 ± 470 in par2 (-/-)). in conclusion, chronic absence of par2 reduces basal renin expression and renin release. however, par2-deficiency does not alter renin release in response to typical stimuli for renin secretion. therefore, par2 does not appear to be a mandatory and specific requirement for acute regulatory responsiveness. increased myofilament ca 2+ sensitivity could be the underlying cause of diastolic dysfunction. we evaluated acute effects of epigallocatechin-3-gallate (egcg), which has been shown to decrease myofilament ca 2+ sensitivity, on cardiac myocyte contractility and force-ca 2+ relationship of skinned cardiac muscle strips in an hcm mouse model with left ventricular hypertrophy and both systolic and diastolic dysfunction. methods: the hcm mouse model used in this study carries a point mutation in the cardiac myosin-binding protein c gene at the homozygous state (mybpc3-targeted knock-in; ki). we isolated ventricular myocytes from adult ki and wt mice and analyzed sarcomere shortening and ca 2+ transients at 37 °c under 1 hz pacing using the ionoptix system in the absence or presence of egcg (1.8 µm). furthermore, force-ca 2+ relationships of skinned cardiac muscle strips of ki and wt mice were obtained ±egcg (30 µm). results: in baseline settings and absence of fura-2, ki cardiomyocytes displayed higher sarcomere shortening ( type-1 serine/threonine protein phosphatase (pp1) comprises a family of enzymes that dephosphorylate cardiac regulatory proteins, thereby modulating ca 2+ handling and contractility. all pp1 heterodimers possess a catalytic subunit, which is selectively inhibited by inhibitor 2 (i-2). it has been shown by our group that the heart-directed overexpression of a truncated, constitutively active form of i-2 resulted in an improved basal ca 2+ handling and contractility. in contrast, chronic pressure overload by transverse aortic constriction exacerbated the progression of cardiac remodeling and heart failure in transgenic mice. in the present study, we tested whether the overexpression of a full-length form of i-2, regulated by gsk3-dependent phosphorylation at thr 72 , resulted in comparable functional alterations using a model of induced heart failure. for this purpose transgenic (tg) and wild-type (wt) mice were subjected to chronic application of isoprenaline (iso, 30 mg/kg/d) via osmotic minipumps. iso-stimulated mice were compared to mice treated with 0.9% nacl (n=5). after one week of iso administration, cardiac hypertrophy was comparable in tg and wt. ca 2+ transients were measured in isolated, indo-1-loaded myocytes. the peak amplitude of [ca] i was reduced by 39% in tg nacl compared to wt nacl (p<0.05), whereas chronic iso application was associated with comparable effects in tg and wt. [ca] i decay kinetics were comparable in nacl-treated groups but hastened by 25% in tg iso compared to wt iso (p<0.05). consistently, sr ca 2+ load was diminished by 28% in tg nacl compared to wt nacl (p<0.05). chronic iso stimulation led to an unchanged sr ca 2+ content in tg and wt myocytes. biochemical analyses revealed that chronic βadrenergic stimulation was accompanied by a more than 4-fold higher phospholamban phosphorylation at ser 16 in tg (p<0.05). thus, these findings suggest that overexpression of i-2 is able to reduce the progression of heart failure by an improvement of myocyte ca 2+ handling. the ligand-activated farnesoid x receptor (fxr) is a nuclear receptor highly expressed in gastrointestinal and metabolic tissues, such as the duodenum, jejunum, ileum, colon and the liver, but also in lower amounts for instance in macrophages. the endogenous agonists for this receptor are bile acids with the primary bile acid chenodeoxycholic acid as the most active one. activation of fxr regulates the transcription of target genes relevant in bile acid homeostasis, glucose and lipid metabolism, liver protection, inflammation, and cancerogenesis. agonists for fxr have been discussed as possible therapeutic options for the treatment of obesity and the metabolic syndrome. atherosclerosis is the main pathology underlying cardiovasular diseases and often occurs side-by-side with the metabolic syndrome. cholesterol deposition and the formation of cholesterol-loaded foam cells from macrophages lead to the formation of atherosclerotic plaques. this can be prevented by stimulation of cholesterol efflux from macrophages. based on leoligin, a lignan-type secondary plant metabolite, naturally occuring in leontopodium alpinum cass., 168 derivatives were synthesized and subjected to a fxr pharmacophore-based in silico screening. testing of 56 virtual hits in a luciferase-based fxr transactivation assay yielded one compound with promising activity on fxr. moreover, the heterodimer partner of fxr, rxrα, was not activated by this leoligin derivative in a luciferase-based rxrα assay. in addition, this compound was able to increase cholesterol efflux in thp-1 macrophages without affecting cell viability. western blot experiments revealed an increase in atp-binding cassette transporter a1 (abca1) expression in human thp-1 macrophages by this leoligin derivative. the transporters abcg1 and scavenger receptor class b (sr-bi), which also play a key role in macrophage cholesterol efflux, will be investigated. moreover, the effect of the leoligin derivative on liver x receptor activation, the nuclear receptor responsible for upregulation of these transporters, has to be studied. based on this data, further characterization of the molecular mechanism underlying the described effects will provide valuable insights in a possible crosstalk between macrophage cholesterol efflux and fxr activation. background: rho-associated kinases rock1 and rock2 are serine/threonine kinases that are downstream targets of the small gtpases rhoa, rhob, and rhoc. rock1 and rock2 are known to play a pivotal role in the pathogenesis of myocardial fibrosis. however, their specific function in cardiac fibroblasts (cf) remains unclear. remodelling of the diseased heart results in the transition of fibroblasts to a myofibroblast phenotype exemplified by an increased proliferation, migration rate and synthesis of extracellular matrix (ecm) proteins. therefore, we sought to investigate whether rock protein signalling intermediates have an impact on cellular characteristics, intracellular protein expression and mechanical properties in cf and engineered tissues. methods: neonatal cardiac fibroblasts were isolated from wild type rats and downregulation of rock1 and rock2 by 75% was achieved by lentiviral transduction or transfection. wild type fibroblasts were treated with 10 μm fasudil or 3 µm h1152p for general rock inhibition and 3 µm slx-2119 for inhibition of rock2. protein expression and modification was determined by immunoblot analysis, gene expression by qpcr analysis, cf morphology and the localisation of cytoskeletal proteins by immunofluorescence analysis, cell proliferation by automated nuclei counting, cell migration on a planar surface by life cell imaging, and rigidity of engineered tissues by rheological measurement. results: our results show that both rock1 and rock2 influence cf morphology, gene expression, proliferation and migration. the knockdown and inhibition of rocks was associated with changes in cf morphology accompanied by a disorganization of higher-order actin structures including stress fibers and geodesic domes. moreover, the knockdown of rock1 and rock2 in cf increased adhesion velocity, whereas proliferation was attenuated. interestingly, downregulation of rock2, but not of rock1 led to a significantly decreased migration velocity and distance suggesting an isolated principle role for rock2 in cardiac fibroblast migratory behavior. analysis of a three dimensional engineered tissue model composed of cardiac fibroblasts (engineered connective tissue, ect) suggested that rocks are involved in the regulation and turnover of the extracellular matrix (ecm) and thus influence viscoelastic properties of engineered tissues. destructive tensile strength measurement in ect treated with rock inhibitors showed that rigidity was significantly reduced when compared to control tissues. rna sequencing of ect treated with the rock inhibitor h1152p and qpcr analysis of cf with a downregulation of rock1 and rock2 showed that both rocks are involved in the regulation of ecm proteins, such as collagens 4a2, 6a, and 8a1, biglycan, decorin, elastin and its respective degrading enzyme mmp12. conclusion: this study demonstrates that rock signalling controls myofibroblast characteristics of cf via remodelling of the cytoskeleton and the ecm. background: regulation and fine-tuning of gene transcription in cardiomyocytes (cms) is a centerpiece of cardiac development, function, and disease. in order to obtain authentic data, cell type-specific analyses are indispensable. recently, high-purity isolation protocols for cm nuclei were established [bergmann, exp cell res, 2011] and employed for detailed genetic and epigenetic studies on cardiac gene transcription [gilsbach, nat commun, 2014] . however, corresponding protein analyses, which bridge from transcriptional control to cm function are still lacking. therefore, we aimed to map the landscape of nuclear protein expression in newborn and adult mice in order to complement and extend our epigenetic studies. methods and results: cardiac nuclei were isolated from homogenized adult and p1 mouse frozen hearts by sucrose gradient centrifugation. magnetic-assisted cell sorting (macs) with pcm-1 as a nucleus-specific marker was used to enrich cm nuclei to >95%. proteins were extracted from nuclear lysate with 2% sds. quantitative protein data were obtained from silac-based liquid chromatographytandem mass spectrometry (lc-ms/ms) experiments with an ltq orbitrap xl mass spectrometer after in-gel digestion with trypsin. nuclear protein extracts from 3 murine cell lines served as silac (lys8/arg10)-labeled internal standard. finally, protein data are correlated with corresponding mrna data obtained by rna-sequencing. we identified 1041 proteins, 688 of which are annotated to the nucleus. 21%/23% (adult / p1) of nuclear proteins are annotated as dna-binding. 1.5%/1.4% belong to transcription factor complexes; 7.3%/7.5% are able to bind transcription factors. 7.9%/8.3% have chromatin modification functions; 4.3%/4.4% modify histones. nuclearenriched go terms include mrna processing and transport, transcription, nucleosome assembly and protein degradation. 71% of proteins are shared between p1 and adult nuclei. 142 proteins are exclusive to p1, 34 are only found in adult. 93 proteins are enriched (1.3-fold increase in abundance) in p1 hearts, 89 proteins in adult proteins related to heart development, gene silencing, and dna replication are more prominent in or exclusive to newborn mice. adult nuclei strongly express proteins related to regulation of actin fibers and cm function, proteins involved in protein degradation, and chaperones. although high mrna expression increases the chance of protein identification, a significant correlation between mrna and protein level could not be observed on a genome-wide scale. conclusions: we present a comprehensive and specific protein landscape of newborn and adult cm nuclei. young cm nuclei appear as a developing tissue, show the ability for proliferation, and indicate ongoing alterations in gene expression. adult cm nuclei prominently display a focus on regulation of contractile fibers and cm function, as well as chaperones and proteasomal proteins indicative of its arduous function. background: fibrosis is a hallmark of many myocardial pathologies and contributes to distorted organ architecture and function. recent studies have identified premature senescence as regulatory mechanism of tissue fibrosis. however, its relevance in the heart remains to be established. objective: to investigate the role of premature senescence in myocardial fibrosis. methods: murine models of cardiac disease and human heart biopsies were analyzed for characteristics of premature senescence and fibrosis. results: senescence markers p21 cip1/waf1 , senescence-associated ß-galactosidase (sa-ß-gal) and p16 ink4a were increased 2-, 8-and 20-fold (n=5-7; p < 0.01), respectively, in perivascular fibrotic areas after transverse aortic constriction (tac) when compared to sham-treated controls. similar results were observed with cardiomyocytespecific β1-adrenoceptor transgenic mice and human heart biopsies. senescent cells were positive for vimentin (92 ± 0.9%), platelet derived growth factor receptor α (94 ± 0.9%) and α-smooth muscle actin (71 ± 2.3%), specifying myofibroblasts as the predominant cell population undergoing premature senescence in the heart. conclusion: our data provide first evidence for an essential role of premature senescence of myofibroblasts in myocardial fibrosis. it is tempting to speculate that pharmacologic modulation of premature senescence might provide a novel therapeutic target for anti-fibrotic therapies in the heart. introduction: the endocannabinoid system is increasingly studied in cardiac research due to its role in fibrosis, inflammation and cell fate modulation. the deregulation of this system has been implicated in myocardial infarction (mi) and consequent heart failure development. a recent study suggests cannabinoid receptor 1 (cb1) inhibition to improve cardiac function and to reduce adverse remodeling after cardiac stress, but the exact underlying molecular mechanisms of these beneficial effects are still unknown. micrornas (mirnas, mirs) provide a complex layer of post-transcriptional regulation modulating key biological processes such as tissue remodelling in heart failure. the aim of the present study was to explore microrna pathways in the chronic effect of cb1 receptor inhibition after angiotensin-fibrosis induction and left ventricular remodeling. methods and results: adverse cardiac remodelling was induced in mice by chronic administration of angiotensin ii (angii, 1,5 mg/kg/day) with osmotic minipumps for 14 days. treatment with cb1 antagonist, or vehicle was performed every second day during the angii administration period. hemodynamic parameters were measured by echocardiography and cardiac pressure volume catheter and tissue samples were taken for molecular and histological analysis. after two weeks of angii infusion, left ventricular dysfunction was prevented by cb1 antagonist treatment. this was shown by significantly improvements of the myocardial performance index and end-diastolic pressure values. at the tissue level, anti-fibrotic effects of cb1 antagonist treatment was confirmed histologically and by expression analysis of pro-fibrotic genes. these beneficial effects were also observed in cb1 ko mice and in an aging mice model. the particular role of tissue fibroblast in aii-induced cardiac fibrosis was further explored. primary cardiac fibroblasts (cf) from each experimental group were isolated and analysed by next generation deep rna sequencing to identify differentially regulated micrornas. microrna-181a/b family was downregulated by in vivo angii delivery and vice versa upregulated after cb1 antagonist treatment and foxb1 (a direct target of mir-181a family) was differentially regulated, suggesting a possible mechanism of action for the benefits of cb1 receptor inhibition. conclusion: we found that in angii-induced cardiac remodelling, lv function is preserved by chronic cb1 antagonist treatment and that cardiac fibrosis is reduced with concomitant downregulation of fibrogenic genes. also, cf-enriched mir-181a/b family seems to be sensitive to cb1 antagonist treatment, thereby affecting cardiac fibrosis. the current study employs a novel concept regarding chronic cb1 inhibitor treatment and may provide important details and novel targets for anti-fibrotic approaches in heart failure. background: low homoarginine (harg) was recently identified as an emerging biomarker for stroke, myocardial infarction, and heart failure in clinical and epidemiological studies. harg competes with arginine as a substrate for nitric oxide (no) synthase and weakly inhibits arginase. both mechanisms might lead to increased no formation in vivo. the aim of this study was to investigate whether harg effects the development of atherosclerosis as a potential underlying mechanism of cardiovascular diseases. methods: harg-deficient agat-knockout (agat -/-) and wildtype (wt) mice were crossed with apolipoprotein e (apoe) deficient mice and fed with high fat diet (hfd) for three months to induce atherosclerosis. harg plasma concentrations were determined using mass spectrometry. en face preparation of aortae followed by red oil staining of atherosclerotic plaques and quantitative evaluation of plaque areas was performed for female mice. endothelial function of male mice was tested with acetylcholine (ach) and nitroglycerin (ntg) after contraction with prostaglandin f2α. background: the direct oral thrombin inhibitor dabigatran etexilate (dabigatran) is used for the prevention and treatment of venous thromboembolism. obese patients as well as patients with type 2 diabetes mellitus (t2dm) have an increased risk for thrombotic disease and show enhanced thrombin generation. besides its role in blood coagulation, thrombin is known to be involved in many pro-inflammatory processes. in obesity, adipose tissue (at) inflammation plays a crucial role in the development of insulin resistance and t2dm and contributes to atherosclerosis development. the aim of the present study was to analyse the effects of dabigatran on at inflammation in a mouse model of diet-induced obesity in the context of accelerated atherosclerosis. methods: 10-week-old female low-density lipoprotein receptor-deficient (lldr -/-) mice were fed a high-fat diet containing 5 mg/g dabigatran or respective placebo for 20 weeks. results: analysis of visceral at revealed a significant increase in adipocyte size in dabigatran-treated mice, although body weight, fat mass, glucose tolerance, and insulin resistance were unchanged between groups. this effects seemed to be directly mediated by thrombin, as treatment with another thrombin inhibitor (argatroban) also resulted in the development of adipocyte hypertrophy. accordingly, in vitro studies in 3t3-l1 cells revealed an inhibitory effect of thrombin on lipid accumulation in adipocytes. the amount of pro-inflammatory cd11c-positive macrophages (atms) in visceral adipose tissue was significantly reduced, and the secretion of pro-inflammatory il-6 from visceral at was significantly lower in dabigatran-treated animals. in vitro studies using 3t3 l1 cells and primary bone marrow-derived macrophages revealed that the changes in macrophage polarization were not directly mediated by thrombin, but indirectly by a change in the secretion profile of adipocytes. a similar reduction in proinflammatory macrophages as detected in at could also be observed in the aortic wall of dabigatran-treated mice. conclusions: the direct thrombin inhibitor dabigatran inhibits at inflammation and the accumulation of pro-inflammatory macrophages in vat but also the aortic wall of ldlr -/mice. these anti-inflammatory effects of dabigatran might contribute to the known atheroprotective effects of dabigatran. background: sarco/endoplasmic reticulum ca 2+ -atpase (serca2a) and its inhibitor phospholamban (pln) are critical determinants of cardiomyocyte calcium cycling and hence, cardiac contractility. pln exists in an equilibrium between mono-and pentamers. while monomeric pln has been implicated in direct serca2a inhibition, a functional role for the pentamers remains ambiguous. recently it has been shown that pln pentamers modulate pka-dependent phosphorylation of pln monomers in vitro. 1 using transgenic mouse models we now investigated the effects of pln pentamers on pln phosphorylation, myocyte ca 2+ cycling and contractility in cardiac myocytes. methods: phosphorylation patterns of pln were analyzed by western blot using phospho-specific antibodies as well as phosphate affinity sds-page. to assess the phosphorylation at baseline, pln knockout (pln-ko) mice expressing either wild type pln (tgpln) or the solely monomeric pln afa mutant (tgafa) transgene were deeply anesthetized, whereas pln phosphorylation by pka was induced using the betaadrenergic agonist isoproterenol. the consequences on myocyte ca 2+ kinetics were measured in isolated, fura2-loaded and electrically paced (0.5hz) cardiomyocytes as the time to 50% decay of the ca 2+ signal (t 50% ). the time to 50% baseline of sarcomere length (t 50% baseline) characterized the speed of myocyte relaxation. results: under basal conditions, we found stronger phosphorylation of pln pentamers than monomers, pointing at pentamers as the preferred pka target. pln afa monomers showed 3.3-fold stronger phosphorylation signals if pentamers were absent (p<0.0001). consistent with a higher basal phosphorylation of pln afa monomers, measurements of calcium kinetics revealed a faster decay of calcium signals in tgafa compared with tgpln cardiomyocytes (t 50% [ms]: 92±8 and 122±8, respectively, p<0.05). notably, t 50% of pln-ko myocytes was 79±5ms (p= not significant versus tgafa), indicating that the strong basal phosphorylation of monomers leads to near complete inactivation of pln in tgafa. upon stimulation of pka, pln monomer phosphorylation and calcium kinetics of tgpln and tgafa mouse myocytes were indistinguishable, because monomer phosphorylation and the speed of cytosolic ca 2+ clearance strongly increased only in tgpln. acceleration of sarcomere relaxation upon pka stimulation was also more pronounced in tgpln than in tgafa and pln-ko myocytes (increase of t 50% baseline [ms]: 32±8 in tgpln versus 7±4 in tgafa-pln and 5±7 in pln-ko, p<0.05). even high-dose isoproterenol induced phosphorylation of only about half of all protomers of pln pentamers suggesting a high capacity of pentamers to attenuate monomer phosphorylation by acting as a phosphate scavenger. conclusions: our data demonstrate that pln pentamers reduce basal phosphorylation of pln monomers in myocytes. nevertheless, pentamers allow strong phosphorylation of monomers during beta-adrenergic stimulation, thereby extending the range within which pln can modify diastolic ca 2+ kinetics and myocyte relaxation. therapeutic inhibition of micrornas is a promising field in cardiovascular research. vector-based overexpression of an inhibitor construct (e.g. microrna sponge) is one approach to achieve sustained inhibition with potential applicability in humans. yet the strength of expression achieved by the currently available gene therapy vectors (e.g. aavs) in humans remains a limiting factor, therefore inhibitory constructs with increased potency would provide an improvement of this approach and bring it closer to therapeutic application. micrornas are believed to discriminate between potential binding sites, based on additional factors provided by the endogenous untranslated regions at the 3' end of mrnas (3' utrs) and the proteins that are bound to them. aim of this project was to investigate whether selected endogenous 3' utrs can likewise increase the potency of microrna inhibitory constructs. to this end several known targets for a cardiac microrna were selected and their relative potencies of microrna inhibition was compared. to accurately assess changes in the activity of the respective micrornas we constructed dual-fluorescent reporter plasmids and established an automated fluorescent microscopy acquisition and analysis pipeline. among several tested utr constructs we found one which strongly increased the inhibitory potency of the microrna binding site in primary rat cardiac myocytes (nrcms). furthermore a similar effect was obtained when the binding site was exchanged for that of a different microrna and analyzed in the nih-3t3 fibroblast cell line. we therefore conclude that endogenous utr contexts can indeed be successfully applied to increase the potency of vector-based microrna inhibitors. the g-protein-coupled protease-activated receptor-2 (par2) regulates inflammatory responses including monocyte migration and cytokine release. par2 is activated by the coagulation factor-xa or by the tissue-factor (tf)/factor viia complex. the immunomodulatory lipid sphingosine-1-phosphate (s1p) is released from activated platelets and interlinks blood coagulation and inflammation. this study investigates the impact of s1p on the expression of par2, tf and of the anticoagulant protein thrombomodulin (tm) in human monocytes and after pma-induced differentiation into macrophage-like cells. monocytic thp1 and u937 cell lines were used as human monocyte models. primary monocytes were isolated from healthy volunteers using a magnetic bead-based monocyte isolation kit. expression of par2, tf and tm was measured by quantitative real-time pcr and western blotting. differentiation of monocytes into macrophage-like cells was induced by incubation with 50 ng/ml pma (phorbol 12-myristate 13-acetate) over 72 h. calibrated automated thrombin (cat) generation was determined in plateletrich plasma from healthy volunteers. in thp1 and u937 cells s1p induced a time-(1 to 24 h) and concentration-dependent (0.1 to 10 µm) significant upregulation of par2 mrna and total protein expression. par2 total protein was upregulated maximally (about 1.5-fold, n=7) with 1 µm s1p after 16 h incubation. comparable effects were seen in human primary monocytes. in comparison, tf mrna and protein were only marginally elevated in non-differentiated thp1 monocytes and tm was not regulated by s1p. after differentiation of cultured monocytic cells with pma into adhesive macrophage-like cells, incubation with s1p resulted in a time-(1 to 24 h) and concentration-dependent (0.1 to 10 µm) significant upregulation of tf expression within 3 to 6 h of incubation. conversely, par2 total protein expression was reduced by about 50% after 24 h s1p incubation. the expression of tm was again not affected. the generation of thrombin in platelet-rich plasma was determined using pma-differentiated thp1 cells as tf source. timedependent incubation with s1p (1 µm) in differentiated monocytes shortened the time to the onset (lag time) of thrombin generation in plasma from 8.0±1.6 to 6.5±1.4 min and elevated total the thrombin generation capacity from 814±130 to 1286±129 nm. peak thrombin formation was elevated from 66±31 to 130±30 nm/min (control versus s1p for 6h, mean±sd, n=5, respectively). these data suggest that s1p induces an enhanced expression of par2 in undifferentiated human monocytes while tf and tm are not regulated. in differentiated monocytes/macrophages, s1p does upregulate tf expression but attenuates par2 levels. since par2 is involved in regulation cell migration, s1p may stimulate a phenotypic switching from a migratory to a procoagulant phenotype during differentiation of monocytes into macrophages. the pro-inflammatory cytokine interleukin-6 (il-6) plays an important role in vascular inflammation. coagulation factors such as the activated factor-x (fxa) may regulate local inflammatory responses of the vessel wall. in this study we investigated whether fxa regulates il-6 expression and secretion in human vascular smooth muscle cells (smc) as well as in failed thrombosed vein grafts. also, we analysed its possible prothrombotic impact on monocytes. il-6 mrna expression was determined in primary human saphenous vein smc by taqman® real-time pcr. secretion to the cell culture media was measured by elisa. tissue factor (tf) expression in monocytic thp-1 cells was determined by western blot. immunostainings for il-6 and the smc marker smoothelin were performed on paraffin embedded tissue sections from failed thrombosed vein grafts and control veins. incubation of cultured human venous smc with fxa (30 nm) induced a time-dependent (3 -16 h) increase in il-6 mrna expression. maximum expression was observed within 6 h to a 7.2±3.3 fold increase (mean±sd, n=5, p<0.05). incubation with an inhibitor of p38 map kinase (sb203580, 10 µm) or pi3k (ly294002, 10 µm) significantly attenuated fxa-induced il-6 mrna expression (n=5). inhibition of p42/44 mapk, rho kinase or nf-kb had no significant effect. stimulation with fxa for 24h resulted in a markedly increased il-6 secretion into the smc culture media from 0.6±0.2 to 1.1±0.3 ng/ml (p<0.5, n=4). stimulation of thp-1 cells with il-6 (1 ng/ml) induced a time dependent (6 -24 h) increase of up to 2.1±0.6 fold (p<0.05, n=5) in tf protein expression. immunostainings of tissue sample of failed vein grafts revealed enhanced il-6 expression in smc-rich regions in vessel walls compared to non-thrombosed control veins suggesting an elevated il-6 regulation in thrombosed vein grafts in vivo. in conclusion, fxa induced il-6 expression and secretion in venous smc which may be regulated via p38 and pi3k signaling. il-6 enhanced tf expression in thp-1 monocytes and was found in smc-rich regions in failed thrombosed vein grafts. fxa-stimulated il-6 release may be involved in regulating local pro-thrombotic processes during vascular inflammation and possibly vein graft failure. rationale: the transcription factors camp-response element binding protein (creb) and camp-responsive element modulator (crem) bind to camp response elements (cres) and mediate a camp dependent gene regulation. suppression of cre mediated transcription is linked to atrial remodeling in genetic mouse models. inhibition of creb target genes is associated with atrial fibrillation (af) susceptibility in patients. creb and crem affect histone acetylation recruiting the creb-binding protein (cbp/p300). the histone acetyltransferase (hat) activity of cbp facilitates gene transcription by loosening chromatin structure. histone deacetylases (hdacs) catalyze the inverse reaction: histone deacetylation with consecutive gene silencing. mice with heart directed expression of the human cardiac isoform crem-ib∆c-x (tg) show atrial dilatation, morphological and physiological alterations in atria preceding spontaneous-onset af. the hdac inhibitor (hdac i ) valproic acid (vpa) reduced atrial weight and af incidence in tg mice. here we tested the hypothesis, that vpa attenuates the structural remodeling in tg atria by reversing changes in atrial gene regulation due to the transgene. methods and results: tg and wt mice were treated from week 5-16 with vpa (0.71% in drinking water, ad libitum) or vehicle (veh). atrial ultrastructure was studied by electron microscopy (em) (week 7 and 16). veh-treated tg atria showed a progressive dysorganisation of sarcomeres (sm) with less mitochondria and more collagen fibers between cardiomyocytes as compared to veh-treated wt atria. the fraction of sm structure in veh-treated tg atria was significantly reduced as compared to veh-treated wt atria (week 7: tg veh : 33±2%, wt veh : 47±1%; week 16: tg veh : 19±2%, wt veh : 44±1%, p<0.05). vpa led to a more organized ultrastructure and restored, at least partially, the degradation of the sm in the tg atria (tg vpa at week 7: 40±2%, tg vpa at week 16: 34±1%, p<0.05 vs. tg veh ). the structure of wt atria was not affected by vpa. we further analyzed the protein abundance profiles in the groups of all animals (wt veh , wt vpa, tg veh , tg vpa) by using lc-ms/ms. between veh-treated genotypes (tg veh vs. wt veh , p<0.05) 998 proteins were significantly changed while 854 proteins were differentially abundant between vpa-treated groups (tg vpa vs. wt vpa, p<0.05). 109 proteins were regulated by vpa in wt atria (wt vpa vs. wt veh , p<0.05), whereas vpa affected 525 proteins in tg atria (tg vpa vs. tg veh , p<0.05), out of which 43 proteins were common. 295 prominent changed proteins between veh-treated tg and wt atria were significantly regulated by vpa in tg atria in the opposite direction. a functional pathway analysis showed that pathways activated in tg atria such as cardiac fibrosis, mitochondrial dysfunction were inhibited by vpa treatment. conclusion: similar to human af, crem-tg mice present atrial dilatation, ultrastructural changes and impaired conduction and spontaneous af. while vpa had little to no effect in wt mice, valproate improved the tg phenotype by interfering with pathways involved in structural remodeling. this supports the idea that hdac inhibition by vpa antagonizes effects of crem expression in atria. in isolated mouse cardiac preparations, histamine is ineffective regarding inotropic or chronotropic effects, presumably because of lack of receptor protein expression. on the other hand, histamine can exert positive inotropic and chronotropic effects in humans via cardiac histamine h 2 -receptors. hence, we have generated transgenic mice (tg) which overexpress the human h 2 -receptor specifically in cardiomyocytes. in isolated left and right atrial preparations of these mice, we investigated the histamine metabolism on a functional level. preparations of wild type mice (wt) served as control. histamine induced positive inotropic effects (pie) and positive chronotropic effects (pce) in left and right atria of tg mice, respectively, but not in wt. interestingly, the inhibitor of histamine oxidation, aminoguanidine (1 mm), shifted the concentration response curves for the pie of histamine from ec 50 = 110 nm to 37 nm (p<0.05). furthermore, the unspecific inhibitor of mono amine oxidase, tranylcypromine (10 µm), shifted the pie of histamine from ec 50 = 70 nm to 38 nm and increased the efficacy of histamine for the pie (p<0.05). these data indicate that exogenously applied histamine is subject to degradation in the mouse heart by two different pathways namely via diaminoxidase and mono amine oxidase. drugs that inhibit theses enzymes could conceivably alter cardiac function also in the human heart. protein phosphorylation by kinases and dephosphorylation by protein phosphatases has a crucial function in cell signal cascades. it has been shown that cardiomyocyte specific overexpression of serine /threonine protein phosphatases pp1, pp2a, pp2b (calcineurin) and pp5 in mice leads to cardiac hypertrophy and alters cardiac function. to examine the function of another important protein phosphatase in the heart we established a mouse model overexpressing protein phosphatase 2cβ (pp2cβ) under control of the α-myosin heavy chain promoter. cardiac overexpression was demonstrated by western blotting. like other serine/threonine phosphatases, pp2cβ can lead to cardiac hypertrophy. in transgenic mice (tg), relative ventricular weight was increased (4.24 ± 0.14 mg/g) compared to wild type (wt) littermates (3.78 ± 0.20 mg/g; p<0.05) whereas weights of right and left atria were unchanged. therefore, relative heart weight was increased in tg (4.78 ± 0.17 mg/g) vs. wt (4.13 ± 0.22 mg/g; n=8-12; 10-11 months of age; p<0.05). left ventricular function, measured in vivo by echocardiography under isoflurane anesthesia was diminished in tg compared to wt (ejection fraction: 58.33 ± 3.16 % (tg) versus 76.94 ± 0.92 % (wt); n=12-16; 8-11 months; p<0.05 and fractional shortening: 30.84 ± 2.02 % (tg) versus 44.94 ± 0.87 % (wt); n=12-16; 8-11 months; p<0.05). the left ventricle was dilated (systolic diameter: 2.78 ± 0.21 mm (tg) versus 1.84 ± 0.06 mm (wt); n=12-16; 8-11 months; p<0.05; diastolic diameter: 3.97± 0.19 mm (tg) versus 3.33 ± 0.07 mm (wt); n=12-16; 8-11 months; p<0.05). in contrast, atrial function measured as response to β-adrenergic stimulation in isolated left and right atrial preparations was unchanged in tg vs. wt. in summary, our results indicate that pp2cβ overexpression can lead to ventricular dysfunction and hypertrophy. the underlying signal transduction pathways need to be elucidated. the insulin-like growth factor binding protein 5 (igfbp5) -a potential developmental gene is regulated upon cardiac stress m. wölfer background: cardiac remodeling is a complex biological adaptation process of the failing heart accompanied by a re-activation of embryonic gene expression, which so far has unclear pathophysiological relevance. we and others showed that insulin-like growth factor binding protein 5 (igfbp5) is expressed in the early pre-cardiac region in mouse embryos and its up-regulation impairs cardiac progenitor differentiation. igfbp5 functions as an extracellular growth factor binding protein for igf and also has igfindependent activities. the role of this factor in the context of cardiac remodeling is still unknown. the aim of this study was to investigate the relevance of igfbp5 in cardiogenesis and cardiac remodeling and its role as a potential target for ameliorating stress-induced cardiac remodeling methods and results: we investigated the expression of igfbp5 in murine cardiac tissue at different developmental stages by qpcr normalized to tpt1 (tumor protein, translationally-controlled 1). this analysis showed temporal changes of cardiac igfbp5 expression from developing to postnatal hearts, where a high expression was detected in early heart stages, which decreased during cardiac development and became low in the postnatal heart. the analysis of igfbp5 expression in different heart cells showed a very low igfbp5 in adult cardiomyocytes in contrast to a high expression in undifferentiated sca-1 positive cells. in a mouse model with cardiac specific wnt/βcatenin activation, which led to cardiac dysfunction, igfbp5 was found up-regulated (p<0.05). further we found an increased igfbp5 expression after pressure induced cardiac hypertrophy using mice with transverse aortic constriction (tac) (p<0.01). in line with this data, an in vitro model of human heart muscle hypertrophy using engineered cardiac heart muscle (ehm) showed an up-regulation of igfbp5 upon adrenergic activation via norepinephrine stimulation accompanied by a functional deterioration in comparison to untreated controls (p<0.05). all these findings were further supported by rna-sequencing analysis from human aortic stenosis patient samples, where igfbp5 expression was found increased in patients with compensatory hypertrophy and in a higher extent in patients with heart failure in comparison to non-failing heart samples. interestingly, the expression of igfbp5 in angiotensin 2 or norepinephrine stimulated neonatal murine cardiomyocytes, as well as in hearts of mice treated with angiotensin 2, showed the opposite results, namely a reduction in its expression (p<0.01; p<0.05, respectively). summary and conclusion: our results show active igfbp5 transcription in the early developing heart but a low expression in the postnatal heart. a re-activation of expression was found in the process of pathological heart remodeling in mouse and human, in vivo as well as in vitro, indicate the participation of igfbp5 in a conserved manner. we hypothesize that igfbp5 may participate in the developmental gene program becoming activated in the diseased adult heart again. the functional role and regulation of igfbp5 is under investigation. we have recently shown that perivascular adipose tissue (pvat) plays a crucial role in obesity-induced vascular dysfunction. in pvat-free aortas isolated from male c57bl/6j mice fed a high-fat diet (hfd) for 22 weeks, the endothelium-dependent nitric oxide (no)-mediated vasodilator response to acetylcholine remained normal. in contrast, a clear reduction in the vasodilator response to acetylcholine was observed in aortas from obese mice when pvat was left in place. these results suggest that the reason for vascular dysfunction in diet-induced obese mice is a pvat dysfunction rather than an endothelial dysfunction. treatment of hfd mice during the last 4 weeks with crataegus extract ws® 1442 (150 mg/kg/day) completely normalized vascular function in pvatcontaining aorta. the expression of endothelial no synthase (enos) was not changed by ws® 1442, neither in pvat nor in aorta. phosphorylation at serine 1177 is the most important positive modulation of enos activity. hfd-induced obesity was associated with a reduction in enos phosphorylation at serine 1177 in pvat, but not in aorta. ws® 1442 treatment significantly improved enos serine 1177 phosphorylation selectively in pvat but had no effect in aorta. a major upstream kinase for enos serine 1177 phosphorylation is akt. the activity of this kinase is inhibited in the pvat of hfd mice, which was largely reversed by ws® 1442 treatment. in addition, ws® 1442 treatment enhanced the mrna expression of the nad-dependent deacetylase sirtuin-1 (sirt1), known also as a longevity gene. the activity of sirt1 depends, among others, on the intracellular content of its cofactor nad. ws® 1442 treatment led to an upregulation of nicotinamide phosphoribosyltransferase (nampt), a rate-limiting enzyme in the salvage pathway of nad biosynthesis. one of the non-histone substrates of sirt1 is enos. deacetylation of enos at lysine residues 497 and 507 by sirt1 enhances the activity of the enos enzyme. currently, we are studying the effect of ws® 1442 on nad synthesis, sirt1 activity, and enos (de)acetylation. in conclusion, crataegus extract ws® 1442 reverses obesity-induced vascular dysfunction by improving pvat function. the molecular mechanisms may involve enos phosphorylation at serine 1177 and upregulation of sirt1. the raf kinase inhibitor protein (rkip) inhibits g-protein-coupled receptor kinase (grk2) and the raf-erk1/2 pathway. these two functions of rkip could counteract each other. while grk2 inhibition is cardio-protective, inhibition of the pro-survival erk1/2 axis promotes signs of heart failure in patients and experimental models. in view of this ambivalent nature, the function of rkip in vivo is not clear. furthermore, rkip could have a pathophysiological role because heart specimens from patients with heart failure showed rkip up-regulation (ref. 1). to investigate the impact of cardiac rkip upregulation in vivo, we generated transgenic mice with myocardium-specific expression of the human rkip gene (pebp1) under control of the alpha-mhc promoter. two different rkip-transgenic lines with 2.7-fold and 3.4-fold increased cardiac rkip protein level were generated (ref. 2, and jax id number 911819). we investigated the cardiac phenotype and found that tg-rkip mice developed cardiac hypertrophy with a significantly increased heart weight to body weight ratio and a decreased left ventricular ejection fraction relative to non-transgenic fvb controls, as early as 10 weeks of age. histology analysis revealed progressive atrial and ventricular enlargement of tg-rkip hearts. ecg abnormalities, a lower maximum rate of left ventricular pressure rise, and a strongly decreased left ventricular ejection fraction of 32.9±2.3 % (n=6; ±s.d.) were documented at an age of 8 months. down-regulation of the transgenic rkip by lentiviral transduction of an rkip-targeting mirna retarded the cardiac phenotype of tg-rkip mice. thus, dual-specific inhibition of the grk2 and raf-erk1/2 axis by the human rkip gene (pebp1) triggers signs of heart failure in vivo, and the documented upregulation of the cardiac rkip in heart failure patients could aggravate disease pathogenesis. these findings are in contrast to rodent rkip (pebp1), which does not seem to inhibit the raf-erk1/2 axis in vivo but instead confers grk2 inhibitionheterodimerization between the at1 receptor (at1r) for the vasopressor peptide, angiotensin ii, and the b2 receptor (b2r) for the vasodepressor peptide, bradykinin, enhances angiotensin ii-stimulated signalling in cells. in addition, at1r--b2r heterodimerization has a major pathophysiological role and contributes to the angiotensin ii hypersensitivity in women with preeclampsia hypertension. to analyse the vascular function of the at1r--b2r heterodimer in vivo, we generated a transgenic model of at1r--b2r heterodimerization (tg-b2r+) by transgenic expression of the b2r gene (bdkrb2) in the b2r-deficient tg-b2r-/-strain. fluorescence resonance energy transfer (fret) imaging was applied to analyse the interaction between different gprotein-coupled receptors in the aorta of transgenic mice. we report here that fret imaging detected the close interaction between the aortic at1r and b2r at a distance of less than 9 nm in tg-b2r+ mice whereas the at1r--b2r heterodimer was absent in tg-b2r-/-mice. in contrast, fret was not detectable between the endothelin eta receptor (etar) and the b2r in the aorta of tg-b2r+ mice, although immunofluorescence and immunohistology confirmed the aortic (co-)-localization of both, etar and b2r. the efficient at1r--b2r heterodimerization in tg-b2r+ mice was accompanied by an enhanced angiotensin ii at1r-stimulated vasopressor response relative to that of tg-b2-/-mice, which lack the at1r--b2r heterodimer. as a control, the endothelin-1-stimulated vasopressor response mediated by the etar, which did not dimerize with b2r, was not significantly different between tg-b2r+ and tg-b2r-/-mice. together these findings provide strong evidence that at1r--b2r heterodimerization occurs in vivo and enhances the angiotensin ii at1r-stimulated vasopressor response. dysfunction of the cardiac energy substrate metabolism is a characteristic feature of late-stage heart failure. the dysfunctional cardiac substrate metabolism contributes to insufficient energy generation and has limited treatment options. in search for a treatment approach, we investigated whether inhibition of g-protein-coupled receptor kinase 2 (grk2) could confer cardioprotection by targeting the dysfunctional cardiac substrate use. the impaired substrate metabolism of late-stage heart failure was reproduced in a transgenic model with myocardium-specific expression of fatty acid synthase (fasn), which is the major palmitate-synthesizing enzyme. experiments with a seahorse xf24 extracellular flux analyzer revealed that in an adult-like lipogenic milieu, fasn-transgenic cardiomyocytes reproduced the overall depressed substrate use of late-stage heart failure with a switch from fatty acid to predominant glucose utilization. the impaired substrate use was largely retarded by co-expression of a small peptide inhibitor of grk2, grkinh. the grkinh-mediated protection against cardiometabolic remodelling required an intact raf-erk1/2 axis and involved the erk1/2-dependent inactivation of the heart failure-promoting peroxisome proliferatoractivated receptor gamma (pparg) by phosphorylation of serine-273. as a consequence of erk-dependent phosphorylation of pparg on serine-273, the expression of heart failure-related pparg targets such as fatty acid synthase, resistin and adiponectin was decreased. the importance of pparg serine-273 phosphorylation was further shown in transgenic mice with myocardium-specific expression of the phosphorylation-deficient pparg serine-273a mutant, which was resistant to the cardioprotective activity of grkinh. taken together our experiments show that grk2 inhibition could target cardiometabolic remodelling by inhibition of the heart failure-promoting transcription factor pparg. the effect of sodium valproate on the action potential of atrial myocytes of crem ib∆c-x transgenic mice introduction: in mouse, cardiomyocyte directed over-expression of transcription factor crem (camp response element modulator) causes an atrial phenotype characterized by hypertrophy, reduced contractility and increased duration of the monophasic action potential (map). moreover, this animal model (crem ib∆c-x) showed spontaneous atrial fibrillation (af) episodes as early as 5 weeks of age in homozygous mice and 10-12 weeks of age in heterozygous mice (phenotype delayed towards adult stage). previous studies in heterozygous mice targeted hdac2 inhibition by sodium valproate (vpa, an anticonvulsant drug, acting also as inhibitor of hdac class i>ii). vpa treatment delayed significantly the development of atrial hypertrophy and the incidence of af episodes, without affecting cellular hypertrophy. our aim was to investigate the effect of chronic vpa treatment on the electrical activity of atrial myocytes isolated from crem ib∆c-x and wild type (wt) littermate mice. methods and results: atrial myocytes were isolated from 12 weeks old wild type mice (wt) and heterozygous crem ib∆c-x transgenic mice with enlarged atria (tg), treated for 7 weeks with vpa (0.4 mm in the drinking water) vs. water (vehicle control). action potentials (ap) were measured at room temperature using the patch-clamp technique. atrial myocytes of water treated tg mice had the ap amplitude significantly reduced by 7 mv compared to water treated wt, and, in line with previous results for the map, the tg cells depolarized with a slower slope of 90.2±8 v/s (tg: n=33 cells) vs. 109.8±5.1 v/s (wt: n=30, p=0.05). moreover, ap of atrial myocytes isolated from water treated tg mice had longer duration (apd) at 50% (tg: 11.6±1.4 ms, n=35 vs. wt: 6.9±0.5 ms, n=30, p<0.01), at 70% (in ms: 21.2±2.5 vs. 13.6±1, p<0.05) and at 90% (in ms: 46.8±4.5 vs. 34±2.5, p<0.05) repolarization. vpa treatment reduced ap amplitude in wt mice by 6 mv (n=22, p<0.05) vs. water treated wt, without altering the slope of depolarization or the apd. in vpa treated tg mice the apd was reduced (50%: 7.3±0.8 ms, 70%: 13.8±1.5 ms, 90%: 30.9±3.2 ms, n=21, p<0.05 vs. untreated tg), the amplitude was increased by 5 mv (n.s.) and the slope of depolarization was increased by 21% (p=0.16, n.s.). membrane capacitance evaluation, as an estimation of atrial myocyte size, showed that in untreated tg mice the cells were larger than in wt (tg: 104±7.2 pf, n=27 vs. wt: 55±5.4 pf, n=30, p<0.001), in line with the occurrence of cellular hypertrophy in tg atria. chronic vpa treatment did not change the cell size in either genotype (wt-vpa: 64.8±7 pf, n=17 n.s. vs. wt; tg-vpa: 88±7 pf, n=14, p<0.05 vs. wt-vpa; p<0.01 vs. wt; n.s. vs. tg). conclusions: in hypertrophied atrial myocytes of crem-ib∆c-x, ap were characterized by smaller amplitude, slower onset of depolarization and increased duration compared to wt cells. despite having no effect on atrial myocytes size, vpa treatment reduced the duration and showed a tendency to increase the amplitude and the slope of depolarization of the action potential in tg mice to values similar to wt. these data suggest that chronic treatment with vpa restored partially the electrical activity of atrial myocytes and may reverse the electrical remodeling via hdac2 inhibition. (supported by the dfg) of the transcription factor crem (camp response modulator) icer, smicer and crem-ib∆c-x are inducible by β-adrenergic stimulation and code for similar or even identical proteins. thus, these isoforms are able to repress expression of respective target genes in response to camp and might play a role in an arrhythmogenic remodeling during the development of chronic heart diseases. here we test this hypothesis in a mouse model with transgenic expression of crem-ib∆c-x (tg). these mice develop not only spontaneous onset atrial fibrillation but likewise arrhythmogenic alterations in the ventricle. patch clamp experiments revealed an increased na + -ca 2+ exchanger current (i ncx ) and decreased transient outward current (i to ) in tg ventricular cardiomyocytes (vcms) vs. wild-type controls (ctl). these alterations were associated with an increased arrhythmogenicity in tg vcms. action potentials were prolonged in tg vcms vs. ctls leading to an increased proportion of vcms displaying early afterdepolarizations. ca 2+ imaging revealed that the transduction rate of spontaneous sub-threshold ca 2+ -waves into supra-threshold transient-like ca 2+ -events which is mediated by the ncx was increased in tg vcms. at the same time the serca mediated ca 2+ transport rate (r serca ) was enhanced in tg vcms potentially limiting ca 2+ extrusion by the ncx. underlining the in-vivo relevance of our findings ventricular extrasystoles (ves) were augmented in ecgs of tg mice (ves/mouse during 10 -6 m isoproterenol challenge, tg: 3.65*, ctl: 0.4; n=20/condition). the increase in i ncx and r serca and the decrease in i to went along with an increase of ncx1, serca2a and decrease of kchip2 protein levels. however, the respective mrna levels (slc8a1, atp2a2 and kcnip2) were unaltered between groups pointing to a post-transcriptional regulation of these genes. in a mrnasequencing approach we identified the downregulation of precursor mirnas inter alia for mir-369 (fold change in tg: 0.04*) and mir-1 (fold change in tg: 0.13*) (n=10/condition). atp2a2 is a predicted target of mir-369 and mir-1 has recently been shown to regulate ncx1 and i to related potassium channel subunits. (*p<0.05 vs. ctl) our results demonstrate that transgenic expression of crem-ib∆c-x in mouse vcms leads to distinct arrhythmogenic alterations. they further indicate that the repression of micro rnas by short crem repressor isoforms may lead to the upregulation of genes in the context of an arrhythmogenic remodeling. since crem-repressors are inducible by chronic β-adrenergic stimulation our results suggest that the inhibition of credependent transcription contributes to the formation of an arrhythmogenic substrate in chronic heart disease. ( chronic overstimulation of cardiac β-adrenergic receptors (β-ar) is a major trigger for the development and maintenance of cardiac hypertrophy and heart failure. although the camp activated proteinkinase a (pka) is known as a prominent downstream effector of β-ar signaling, its functional contribution to pathological cardiac remodeling is neither well understood nor directly studied so far. to address this issue we used mice carrying a point mutation in the regulatory pka subunit riα (pkariαb), which prevents binding of camp and consequently diminished kinase activity. this dominant negative mutation was controlled by a tamoxifen (tam) inducible αmhc promotor driven cre transgene which allows a selective expression in the ventricular myocardium. the inducible and tissue specific gene expression was analyzed and confirmed by pcr, rt-pcr and immunohistochemistry. furthermore diminished phosphorylation of several pka targets verifies impaired pka activity in tam treated double transgenic animals. hypertrophic response in ventricular pka mutants was studied in genetic, pharmacological and surgical mouse models of heart disease as well. genetically induced heart failure was observed following tam treatment in mice expressing an inducible myocardial-specific cre transgene. this deleterious cardiac phenotype develops independently of the presence of the floxed transgene. 8 days after tam treatment, controls displayed elevated heart weight to bodyweight ratio (hbr) and heart weight to tibia length (htr). hbr shifted from 6.2(mg/g) in untreated control animals to 10.9(mg/g) in tam injected mice. in contrast pka inhibited mutants displayed a minor increased hbr of 7.7 (mg/g). for the pharmacological induction of cardiac hypertrophy we implanted osmotic mini pumps, delivering a combination of isoproterenol and phenylephrine. control animals showed a significantly increased hbr (8.4 mg/g) compared to saline treated animals (6.8mg/g) and pka mutants (7.1 mg/g). paradoxically, all pka inhibited animals displayed a consistent elevation in important hypertrophic markers like anp. surgical constriction of the aortic arch (transverse aortic constriction tac) led to a pressure induced hypertrophic response (hbr: 6.5 vs 7.9 mg/g) followed by a pronounced elevation in several hypertrophic factors such as anp, myh6/7 ratio and myocytes size. in contrast pka mutants displayed an irregular progression of cardiac hypertrophy presented by two groups with either an unchanged (6.9 mg/g) or a strongly elevated hbr (13.5 mg/g). however, additional hypertrophic factors including anp, myh6/7 ratio and myocyte size were significantly increased in both groups. to our knowledge this is the first report, which directly studies the role of ventricular pka activity in cardiac hypertrophy in a genetically altered mouse model. our results suggest that in an early stage of cardiac remodeling pka inhibition alleviates cardiac weight gain but provokes a detrimental shift during further progression, which implicates a protective role of ventricular pka activity in cardiac disease. acetyl-coa carboxylase catalyzes the first step in the biosynthesis of fatty acids in bacterial and eukaryotic cells, i.e. the conversion (carboxylation) of acetyl-coa into malonyl-coa. acc-generated malonyl-coa functions as a substrate for de novo lipogenesis and acts as an inhibitor of mitochondrial β-oxidation of fatty acids. because of its role in lipid metabolism this enzyme has become an interesting target in drug discovery in the field of metabolic diseases and cancer. despite this interest in acc, no attention has as yet been given to the role of acc in endothelial cells. we aimed to investigate the role of acc in two functional key aspects of angiogenesis: endothelial cell proliferation and migration. we used the acc inhibitor soraphen a, a polyketidic natural compound isolated from the myxobacterium sorangium cellulosum, as well as an rnai-based approach to inhibit the function of acc. primary human umbilical vein endothelial cells (huvecs) were used as in vitro model. first, we analyzed the action of soraphen a on cell viability. the compound did neither lower the metabolic activity of huvecs up to a concentration of 100 µm after 24 and 48 h (ctb assay) nor increase in the apoptosis rate after 24, 48, or 72 h up to 100 µm. measuring adenosine triphosphate (atp) levels revealed that 30 µm soraphen a does not alter the atp levels in huvecs after 24 h treatment. in contrast, a 48 h treatment significantly lowered the atp levels by 12 %. also gene silencing of acc1 in huvecs attenuated the atp levels by 11 %. mitochondrial membrane potential (mmp) assays showed decreased mmp levels (10 %) in soraphen a-treated cells after 24 h. interestingly, the compound inhibited the proliferation of endothelial cells with an ic 50 value of 34 µm. cell cycle analysis showed that soraphen a decreases the amount of cells in the g 0 /g 1 phase by 26 % and increases the number of cells in the g 2 /m phase by 50 %. the compound also inhibited the activation of akt (western blot analysis). in a wound healing/scratch assay, 30 µm soraphen a lowered the migration of endothelial cells by 65 %. gene silencing of acc1 in huvecs strongly decreased endothelial migration, whereas a knockdown of acc2 had no influence. furthermore, boyden chamber assays revealed that soraphen a can also lower chemotactic migration by 34 %. since actin rearrangement is necessary for migratory processes, we analyzed the factin cytoskeleton (microscopy) and found that soraphen a decreases the number of filopodia by 60 % but did not influence stress fiber formation. surprisingly, soraphen atreated cells did not exhibit significant alterations in their capacity to form tube-like structures on matrigel. in summary, we could gather first hints that inhibiting acc has an immense impact on the proliferation and migration of primary endothelial cells. the mechanistic basis of this phenomenon will be investigated in future studies by analyzing the lipid profile and the transcriptome of endothelial cells. acknowledgement: this work was supported by the german research foundation (dfg, for 1406, fu 691/9-2) . introduction: statins are among the best examined drugs with excellent efficacy and safety profiles. lowered low-density lipoprotein (ldl) cholesterol goals, new indications for treatment and new knowledge about their pleiotropic effect have promoted a considerable increase in statin use. but as statin use becomes more widespread, awareness of their adverse effects as well as the recognition of statin intolerance problems increase. statin intolerance is a significant problem in the treatment of dyslipidemia, understood as the inability to tolerate a dose of statin required to reduce individual cardiovascular risk sufficiently and could result from different statin-related side effects. muscle-related adverse events, elevation of liver enzymes, cognitive problems and new onset diabetes mellitus have all been described, especially at higher doses. although muscle symptoms are the common side effects observed, excluding other adverse events might underestimate the number of patients with true statin intolerance. these patients represent a target population for the newest lipid lowering drug category i.e. the proprotein convertase subtilisin/kexin type 9 (pcsk9) inhibitors. this work aimed to give an overview of published definitions derived from clinical studies, associations as well as major drug regulatory agencies. we discussed overlaps, differences and limitations in the current definitions. methods: literature based search included pubmed and uptodate publications in english and german language until october 2015. we performed hand searches of the references retrieved and performed an overview. results: a definition of statin intolerance of the european medicines agency (ema) or the us food and drug administration (fda) is not available. in clinical studies, different definitions are chosen and the results are not comparable. also different associations, such as the american heart association (aha), the european atherosclerosis society (eas), the canadian working group or the national lipid association (nla) are not able to agree on one common definition. statin intolerance definitions included different types of muscle symptoms, integration of ck levels and minimal requirements of statin doses. there are currently no validated questionnaires or specific laboratory parameters available. in addition, the term 'myopathy' is often considered as a synonym to statin intolerance. overall, only a few major studies have been conducted with statin intolerant patients so far using inconsistent definitions. discussion and conclusion: there is an unmet need to find a robust and clear definition of statin intolerance as overemphasizing it might hinder appropriate clinical use of this important drug class. thus, further work is required to develop a consensus definition on statin intolerance or a more focused definition regarding statin-associated muscle symptoms only. subsequently, these definitions could be implemented in patient care and their relevance being analyzed and tested in future studies. background: development of cardiac hypertrophy is characterized by reactivation of genes involved in cardiac development. wnt/β-catenin signaling is essential for embryonic cardiac development and is known to be dysregulated in pathological heart remodeling. our previous work suggested a cardiac specific protein complex regulating wnt/b-catenin/tcf transcription in the adult heart. we aim to identify and to characterize this complex in order to find potentially interesting targets for pharmacological therapy preventing maladaptive cardiac remodeling and the onset of heart failure. results: we previously demonstrated that the krüppel-like-factor 15 (klf15) is a βcatenin interaction partner and a cardiac specific nuclear inhibitor of the wnt/β-catenindependent transcription. because klf15 and β-catenin are ubiquitously expressed, we suggest the existence of cardiac specific co-factors responsible for cardiac specificity in this complex. we identified the basic leucine zipper and w2 domain containing protein 2 (bzw2), a phylogenetically conserved protein, as a β-catenin and klf15 interaction partner using yeast-two-hybrid screen. in vitro overexpression experiments and coimmunoprecipitation validated these interactions, which were also confirmed by mass spectrometry. in the developing mouse embryo bzw2 mrna expression is detectable in the heart, neuronal tissue, somites, limbs and branchial arches as shown by whole mount in situ hybridization. in the adulthood expression of bzw2 is confined to the heart, predominantly in cardiomyocytes and in cardiac progenitor cells compared to cardiac fibroblasts (*p<0.05, cm n=4, cfb n=4, cpc n=2), and skeletal muscle. bzw2 was localized in both the cytosol and in the nucleus. mutation analysis showed the importance of the n-terminus of bzw2, containing a putative bzip dna interaction domain, for the nuclear placement of the protein. bzw2 protein expression was significantly increased under cardiac wnt/β-catenin signaling activation in vivo in two mouse models (klf15 knockout (ko) mice **p<0.01 n=3, and in a cardiac specific β-catenin stabilized mouse model, **p<0.01 n=6). a mouse model with constitutively bzw2 loss of function (bzw2 ko) showed cardiac specific upregulation of β-catenin on rna level (***p<0.001, p<0.05, ctrl n=4, bzw2 ko n=5) and on protein level (**p<0.05, ctrl n=7, bzw2 ko n=9). echocardiography analysis in eight-weeks-old bzw2 ko mice showed increased left ventricle wall thickness indicating a hypertrophic phenotype at baseline. we also observed increased levels of bzw2 expression in angiotensin ii treated mice as a model for cardiac hypertrophy (*p<0.05 ctrl n=4, angii n=3) as well as in human samples derived from patients with dilated cardiomyopathy and ischemic cardiomyopathy (*p<0.05 ctrl n=3, dcm n=11, icm n=10). conclusion: these data demonstrated that bzw2 is associated with components of the canonical wnt cascade and suggest its relevance in the constitutive regulation of the wnt/β-catenin components specific in the heart. this study further contribute to the elucidation of the tuning of the wnt-off/-on states aiming to establish a proof-of-concept model for wnt-modulation as a therapeutic strategy in hypertrophic-induced heart failure. objective: sphingosine-1-phosphate (s1p) is involved in the regulation of cell growth, survival, migration and adhesion. it is formed by sphingosine kinases and degraded by phosphatases and s1p lyase [1] . mice that lack s1p lyase are characterized by the accumulation of s1p and sphingosine in their cells and tissues, and by lymphopenia, generalized inflammation, multiple organ damage, and a strongly reduced life span [2] [3] [4] . on the other hand, embryonic fibroblasts from s1p lyase-deficient mice (sgpl1 -/--mefs) are resistant to chemotherapy-induced apoptosis [5] , in part due to an upregulation of multidrug transporters of the atp-binding cassette (abc) transporter family [6] . interestingly, s1p lyase-deficient mice have elevated plasma levels of cholesterol and triglycerides, while suffering from strongly reduced body fat [7] . the aim of the present study was to analyze the link between s1p lyase deficiency and altered cholesterol homeostasis using sgpl1 background: cardiac gene expression changes during cardiac development and under pathophysiological conditions. these alterations in gene expression are regulated by several processes and the exact regulation of gene expression is essential for the proper development and function of the heart. crucial steps in transcription regulation are rna polymerase ii (pol ii) recruitment and changes in pol ii activity. pol ii activity is tightly linked with phosphorylation at serine-2 (p-ser2) of the carboxyterminal domain of pol ii. thus, the aim of the present study was to identify cardiomyocyte-specific genomewide pol ii and p-ser2-pol ii enrichments to get insight into pol ii activity and recruitment in development and disease. methods and results: to get insight into rna polymerase ii dynamics, genome-wide maps of rna polymerase ii occupancy were generated by chromatinimmunoprecipitation in cardiomyocyte nuclei purified from normal neonatal and adult mouse hearts. in addition, cardiomyocyte nuclei were obtained from adult hearts after 4 weeks of pressure overload induced by transverse aortic constriction (tac). cardiomyocyte nuclei were isolated by magnetic beads with an anti-pcm1 antibody. nuclei were used for pol ii chromatin-immunoprecipitation followed by deep sequencing (chip-seq). to test if pol ii marks correlate with nuclear mrna expression in cardiomyocyte nuclei all coding genes were ranked according to their expression level. genes expressed in cardiomyocyte nuclei (> 0.062 fpkm, gene expression rank < 12,653) showed high pol ii enrichment at promoters as well as in genomic regions. many of the gene promoters showed high levels of pol ii accumulation at the transcription start site, as compared to genic regions, which have been associated with pol ii pausing. in contrast, p-ser2-pol ii showed enrichment downstream of the transcription start site. the genomic region of troponin i type 1 (tnni1) which is expressed in cardiac muscle only during development but not in adult cardiomyocytes, was enriched for pol ii in neonatal cardiomyocytes. at the tnni1 gene, pol ii was absent in adult or pressure-overloaded cardiomyocytes. in contrast, pol ii enrichment at the alpha actin 1 (acta1) locus was only present in pressure-overloaded cardiomyocytes. these data are consistent with cellular rna-seq data showing an induction of acta1 after tac. furthermore no pol ii enrichment could be detected in the genomic region of biglycan (bgn), a matrix proteoglycan that is not expressed in cardiomyocytes. this confirms a high purity of cardiomyocyte chromatin. conclusions: this study provides, for the first time, cardiomyocyte-specific landscapes of rna polymerase ii occupancy in heart development and disease. cardiac myocyte maintenance dna methyltransferase 1 is essential for embryonic heart development but is dispensable for cardiac function and remodeling postnatally t. nührenberg background: recent studies have identified dynamic changes in dna methylation in cardiac myocytes during development, postnatal maturation and in disease. however, the enzymes involved in shaping the cardiac myocyte dna methylome are only partially known. here, we explored the role of maintenance dna methyltransferase dnmt1 in cardiac development and in remodeling after chronic left ventricular pressure overload. methods: in mice, deletion of the dnmt1 gene was accomplished by use of two different cre recombinases. crosses of homozygous dnmt1 fl/fl mice with heterozygous dnmt1 fl/+ mice expressing a cre recombinase under control of the atrial myosin light chain gene promoter (myl7-cre) resulted in embryonic deletion of dnmt1 (ko). embryos without myl7-cre served as controls (ctl). embryos were dissected and genotyped at e11.5, e12.5, e13.5 and e14.5. rna-seq and pyrosequencing of genomic dna was performed on e11.5 hearts, histology on e12.5 hearts and electron microscopic imaging on e13.5 hearts. for deletion of dnmt1 in adult mice, homozygous dnmt1 fl/fl mice expressing an inducible cre recombinase (myh6-mcm) were given tamoxifen i.p. over 4 days. homozygous dnmt1 fl/fl mice not carrying myh6-mcm as well as myh6-mcm carrying mice without dnmt1 fl alleles were also injected with tamoxifen and served as controls. cardiac phenotyping including histology, echocardiography and qpcr was carried out without (sham) or with left ventricular pressure overload induced by transverse aortic constriction (tac). results: myl7-cre mediated loss of dnmt1 resulted in progressive embryonic lethality with absence of living ko embryos after e14.5. ko embryos displayed loss of cardiomyocyte gene expression patterns, decreased promotor cpg methylation of aberrantly expressed genes and ultrastructural features of wide-spread cardiac myocyte cell death. in contrast, tamoxifen-induced ablation of dnmt1 in adult mice did not affect survival of ko mice. cardiac phenotyping of adult mice revealed no significant differences between ko and ctl mice under sham and tac conditions. conclusion: dna methyltransferase dnmt1 in embryonic cardiac myocytes is essential for proper heart development. in adult cardiomyocytes, dnmt1 is dispensable for normal cardiac function and for adaptation to chronic cardiac pressure overload. background: recent studies showed that mice with general deletion of the oxidoreductase tet3 involved in dna demethylation are embryonic lethal, the underlying cause remaining unknown. this prompted us to investigate wether embryonic lethality is caused by cardiomyocyte-specific loss of tet3. methods: female mice homozygous for tet3 flox and male mice heterozygous for tet3 flox and heterozygous for myl7-cre were mated and offspring were genotyped after weaning. mice homozygous for tet3 flox and heterozygous for myl7-cre (ko) or homozygous for tet3 flox without myl7-cre (controls) were sacrificed at 12 weeks of age and ventricles were harvested. mrna of the ventricles was isolated and expression of cardiomyocyte-specific genes was evaluated by quantitative real-time pcr. cardiomyocyte-specific genomic dna from ko and control mice was obtained from facs-sorted cardiomyocyte nuclei and bisulfite-converted for analysis of dna methylation by pyrosequencing. results: ko mice showed embryonic lethality of nearly 50 %. born ko mice developed without phenotypic abnormalities (normal heart weight/tibia length ratio) and displayed compensatory upregulation of tet1 and tet2. cardiomyocyte genomic dna of ko mice showed significantly higher methylation levels in the body of the atp2a2 gene and at the binding site of the transcription factor gata4 but not near the promotor and the binding site of the transcription factor tbx5. higher methylation levels were not accompanied by changes in atp2a2 expression. however, both myh7 and nppb were upregulated in ko mice compared to control mice. conclusions: our findings suggest that tet3 is involved in dna demethylation in cardiomyocytes. loss of tet3 expression resulted in embryonic lethality. compensatory upregulation of tet1 and tet2 isoenzymes may contribute to the incomplete penetrance of this phenotype. further studies are ongoing to investigate the functional relevance of tet3 in cardiomyocytes. to identify novel proteins secreted by the myocardium, we have previously conducted a genetic screen, which led to the identification of protease inhibitor 16 (pi16). a recent gwas analysis showed an association of a genetic variant in the pi16 genomic locus (rs1405069) with chemerin plasma levels. here we tested the hypothesis that pi16 determines chemerin plasma levels through regulation of chemerin processing. we generated mice deficient for pi16, which did not display an overt phenotype under basal conditions. plasma levels of chemerin were found significantly lower in pi16-deficient animals compared to littermate controls. to investigate whether pi16 and chemerin interact, we performed co-immunoprecipitation experiments. indeed, we found pi16 to co-precipitate with chemerin from both murine plasma and cell culture supernatants. as chemerin is proteolytically processed and activated, we next asked whether the presence of pi16 would affect the processing of pro-chemerin to its processed forms in native tissue. western blot analysis on cardiac and adipose tissue lysates that detected both the unprocessed precursor and the processed forms of chemerin showed a significant shift towards the processed forms upon genetic deletion of pi16. when we assayed for the activity of the chemerincleaving protease cathepsin k, we found recombinant pi16 to potently inhibit cathepsin k activity. taken together, we propose pi16 to act as a regulator of chemerin processing. the transient receptor potential canonical 6 (trpc6) is a second messenger-gated cation channel, which mediates depolarization and ca 2+ entry. it is known to be activated by diacylglycerol derivatives (dag, 1-oleoyl-1-acetyl-sn-glycerol oag) [1] in a pkc-independent manner and plays important roles in lung and kidney physiology. gain-of-function mutations in the trpc6 gene can cause focal segmental glomerulosclerosis (fsgs), a kidney dysfunction leading to end stage renal disease. [2] thus, the discovery of potent inhibitors of trpc6 may help to develop new therapeutic strategies. urban et al. discovered that larixol, a natural product with a labdane skeleton found in the oleoresin of the european larch (larix decidua), blocks the oag-dependent activation of trpc6. [3] larixyl acetate, another component of the resin showed an even higher potency in trpc6 inhibition (ic 50 = 0.26 µm) and a 12-fold selectivity compared to trpc3. these findings led to the idea that further modifications of the larixol lead structure may reveal even more potent inhibitors. furthermore, changes in selectivity and efficacy of such compounds may also provide a deeper insight about relevant structural elements for channel binding. as larixyl carbamate was assumed to exhibit a higher metabolic stability as larixyl acetate, this compound was already investigated in previous studies. it showed a potent and subtype-selective inhibition of trpc6. hence, the development of further carbamates was a priority objective. as an alternative to the use of different isocyanates for the introduction of a carbamate function at the c1 position of the molecule we found an elegant way via formation of an active ester with carbonyldiimidazole. this precursor allowed the design of several isosteric compounds like larixyl methylcarbamate, larixyl hydrazide and larixyl methylcarbonate, which were all able to block trpc6 with similarly low ic 50 values. the introduction of more bulky side chains appeared to diminish the bioactivity of the compounds, the stereochemistry at the c1 position, however, seems to play no important role for the inhibition of trpc6 currents. larixyl methylcarbamate lead to trpc6 inhibition with an ic 50 value of 0.15 ± 0.06 µm. compared to larixyl carbamate and larixyl methylcarbonate, which are also very potent blockers of trpc6, this compound bears the benefit of high subtype selectivity towards trpc3. even with concentrations up to 50 µm of larixyl methylcarbamate, no complete inhibition of the ca 2+ influx via trpc3 channels could be achieved. this fact distinguishes this larixol derivative as a very promising compound for further studies of trpc6 in health and disease. poisoning by organophosphorus compounds (opc) including pesticides and highly toxic nerve agents is based on irreversible inhibition of acetylcholinesterase (ache) resulting in an excess of acetylcholine causing accumulation. the subsequent overstimulation at nicotinic and muscarinic receptors finally leads to respiratory arrest due to paralysis of the respiratory muscles. therapy focuses on competitive antagonism at muscarinic acetylcholine receptors and reactivation of inhibited ache by bisquarternary pyridinium oximes. thereby, nicotinic malfunction is not directly approached. for that reason, an alternative strategy appears rational using nicotinic acetylcholine receptor (nachr) active substances to counteract the effects of accumulated acetylcholine and thus to restore the loss of function of nachrs. different bispyridinium-non-oxime-compounds (bps) have been demonstrated to be able to serve as target structures for the identification of new positive allosteric modulators of nicotinic receptors. unlike nicotinic agonists, positive allosteric modulators can reinforce the endogenous cholinergic neurotransmission despite of acetylcholine accumulation in the synaptic cleft. to this end, the following electrophysiological in vitro study investigated the effect of twelve diversely substituted bps on human α7 nachr using whole-cell patch clamping under voltage-clamping conditions (-70 mv) performed with planar electrodes in an automatic system (nanion technologies gmbh, munich). cholinergic currents of hα7 nachr that have been expressed in stably transfected cho cells were activated by the agonist nicotine. measurements of the effect of various bps concentrations in the presence of nicotine were performed to establish concentration-response relations. cholinergic inward currents were generated by human α7 nachrs in response to low nicotine concentrations. at high concentrations of the drug the currents were decayed reflecting both, desensitization of the receptors and presumably block of the open channel by high agonist concentrations. four out of twelve bps co-applicated with nicotine showed a concentration-dependent enhancement of peak agonist-evoked currents and, most pronounced, 4-tert-butyl-substitued-bp, also demonstrated a marked elongation of the evoked response. this suggests a positive allosteric effect of these compounds on the nicotinic receptor. however, at high bp concentrations in the presence of agonist, responses were decayed significantly, presumably resulting from an open channel block induced by bps. hence, further compounds have to be synthesized to identify promising candidate compounds for improvement of effective therapy against nerve agent poisoning. the transient receptor potential channels (trp) are a family of tetrameric nonselective cation channels, which are involved in a variety of physiological and pathological processes (1). among the 28 mammalian trp channels, the canonical channel 5 (trpc5) is a ca 2+ -permeable ion channel, which is predominantly expressed in the brain (2) . many aspects of trpc5 function are still elusive although behavioral experiments with trpc5-knock-out mice suggest a role in innate fear-response (3) and some studies indicate a trpc5-mediated down regulation of neurite outgrowth in nerve cells (4, 5) . to elucidate trpc5 function on a cellular level, selective and potent compounds are required to acutely control channel activity. despite extensive research, trpc5 modulators often lack selectivity or exhibit toxicity, limiting their applicability in vivo (6, 7) . thus, there is still a need for identifying novel and efficient trpc5 modulators. we therefore screened a compound library (chembionet) and identified a benzothiadiazine derivative (btd) as a novel, potent, and selective trpc5 activator. hek293 cells heterologously expressing trpc5 upon tetracycline induction (hek trpc5 ) show a btd-induced concentration-dependent activation in both ca 2+ assays (ec 50 = 0.71 µm) and in electrophysiological whole cell patch clamp recordings (ec 50 = 1.1 µm). btd elicits currents with an n-shaped i/v curve, typical for trpc5. the resulting activation is long lasting, reversible and sensitive to clemizole, a recently established trpc5 inhibitor (8) . mtt assays revealed that incubating hek trpc5 cells for 24h with btd concentrations above 1 µm results in a concentration-dependent decrease in viability and cell proliferation, indicating a ca 2+ -mediated cytotoxic effect in consequence of sustained channel activity. non-induced control cells remain unaffected by btd at concentrations up to 10 µm. ca 2+ assays showed no influence of btd on closely related trpc4 channels, as well as trpc3/6/7 at concentrations up to 10 µm. the same applied to more distantly related trpv and trpm channels. besides a homotetrameric organization, trpc5 subunits can also assemble to heteromeric channel complexes with their closest relatives trpc1 and trpc4 (9) . trpc1/5 and trpc4/5 heteromers can also be activated by btd as evident from their typical i/v curves in patch clamp experiments, suggesting a high selectivity of btd for channel complexes bearing at least one trpc5 subunit. transient receptor potential canonical channels 3/6 and 7 are controlled by membrane lipids and highly expressed in neuronal and cardiac tissues. the involvement of these channels in development and (patho)physiology of these tissues is well documented, while our understanding of structure-function relations, specifically in terms of the lipid sensing machinery, in these channel proteins is still incomplete. using a homology model of trpc3, based on the recently available structural information on trpv1, we performed structure-guided mutagenesis and identified a single residue in transmembrane domain 6 (g652), which is conserved within the canonical family of trp channels. single point mutations at position 652 in trpc3 largely eliminated lipid sensitivity. trpc3 g652a expressed in hek293 cells was found resistant not only to activation via the phospholipase c pathway but also to direct administration of diacylglycerols. on the contrary, a synthetic agonist of trpc3/6/7 channels (gsk1702934a) activated wild-type trpc3 and trpc6 channels as well as the respective lipid insensitive mutants (trpc3 g652a, trpc6 g709a ). interestingly, the synthetic activator was found to generate substantially enhanced trpc conductances in cells expressing the lipid-insensitive mutants as compared to wild-type proteins. closer inspection of sensitivity of the wt and mutant proteins to various gsk derivatives argue against a contribution of g652 to gsk recognition by trpc3. our results demonstrate the existence of two different mechanisms of trpc3/6 activation supposedly involving distinct gating movements in the channel complex. we suggest that lipid gating of trpc3/6 involves a hinge-point and/or requires a certain level of flexibility within transmembrane segment s6 provided by g652. lipids and synthetic activators of trpc3/6 may be capable of initiating markedly different structural rearrangement in these channels. objective: organophosphorus compounds (opcs), i.e. nerve agents or pesticides, are highly toxic due to their strong inhibition potency against acetylcholinesterase (ache). inhibited ache results in accumulation of acetylcholine in the synaptic cleft and thus the desensitisation of the nicotinic acetylcholine receptor (nachr) in the postsynaptic membrane is provoked. as the therapeutic efficacy of oximes is limited, e.g. poisoning by soman or tabun, the direct targeting of nachr may be an alternative therapeutic approach. studies with the non-oxime bispyridinium compound (bp) mb327 (1,1'-(propane-1,3-diyl) bis (4-tert-butylpyridinium) di(iodide)) demonstrated a therapeutic effect against soman in vitro and in vivo. consequently, studying the affinity of bps at muscle-type nachrs and functional effects are topics of interest. to identify potential candidates, homologous series of substituted and non-substituted analogues (linker c 1 -c 10 ) of mb327 were investigated by using binding and functional assays. experimental procedures: crude membranes from frozen electric organ of torpedo californica were purified by sucrose-gradient density centrifugation and used in both affinity and functional assays. in competition radio-ligand binding assays, the influence on [³h]epibatidine binding sites of torpedo muscle-type nachr was determined. functional assessments were carried out with a bilayer method to investigate the effect on the cholinergic signal induced by 100 µm carbamoylcholine. results: bispyridinium compounds bearing unsubstituted pyridinium rings and long alkyl linkers (> c 7 ) inhibited the binding of [³h]epibatidine and decreased the cholinergic signal of 100 µm carbamoylcholine in the functional assay. mb327 and several bispyridinium structure analogues (mainly c 2 -c 4 linker) exhibited no regular displacement curves at [ 3 h]epibatidine binding sites and enhanced the carbamoylcholine-induced signal. the results demonstrate that the described affinity and functional screening methods detected some structure-activity-relationships (sar). depending on linker length and substitution pattern, the investigated bispyridinium compounds seemed to interact as positive allosteric modulators. further research is necessary to verify this hypothesis. non oxime bispyridinium compounds with an effect on soman-blocked respiratory muscle function have no effect on normal muscle function the life threatening toxicity of organophosphorus compounds (op), like nerve agents or pesticides, lies in the inhibition of acetylcholinesterase (ache) which causes cholinergic crisis. the accumulated acetylcholine in neuromuscular synapses results in the desensitization of nicotinic acetylcholine receptors (nachr) and paralysis of respiratoric muscles. the 4-tert-butyl-substituted bispyridinium compound mb327 showed therapeutic efficacy in soman and tabun poisoned guinea pigs in vivo. partial restauration of neuromuscular transmission by bispyridinium compounds (bp), e.g. mb327 or mb420, could also observed in soman paralysed respiratory muscles in vitro and was partly attributed to an interaction of bp with nachrs. however, it is unknown, whether these bp might affect normal respiratory muscle function in the absence of cholinergic crisis. therefore this study investigated the effect of bp on physiological rat diaphragm muscle function. force generation of rat diaphragm hemispheres was determined after incubation with increasing bp concentrations (1-300 µm) and compare to sham treatment. the diaphragm hemispheres were stimulated every ten minutes by an indirect electrical field (20, 50, 100 hz). muscle force was analyzed as time-force integral and is expressed as percentage of the individual control values, measured at the outset of the experiment. the muscle force dropped during the experiment. the application of the bispyridinium compounds mb327 (1,1′-(propan-1,3-diyl) bis (4-tertbutylpyridinium) di(iodide)) and mb420 (1,1′-(propan-1,3-diyl) bis (2-ethylpyridinium) di(iodide)) in the tested concentration range (1-300 µm) did not change muscle force production compared to the sham treated muscle. this was equally true for low (20 hz) and high (50 and 100 hz) stimulation frequencies. this study showed that bispyridinium compounds which can partially reverse somaninduced neuromuscular block in rat diaphragms show no effect on respiratory muscle function in absence of the op-induced neuromuscular block. these results suggest that the bp tested in this study interacted with desensitised nachrs only, but do not affect physiological neuromuscular transmission. this effect needs to be investigated with further, promising bp compounds. interaction of recombinant pain-relevant atp-and proton-gated ion channels in an expression system; potentiation of the p2x3 receptor-induced current by the opening of asic3 channels g. stephan 1 , p. illes 1 1 universität leipzig, rudolf-boehm-institut für pharmakologie und toxikologie, leipzig, germany the p2x3 receptor (r) is a ligand-gated cationic channel, which is activated by extracellular atp. the acid sensing ion channel 3 (asic3) belongs to the enac/degenerin family and is gated by extracellular protons. despite their different amino acid sequences both ion channels share the same structure and pore architecture, by i.e. consisting of three identical subunits. besides, they are both located at partially overlapping subpopulations of dorsal root ganglia neurons and are implicated in acidic pain signaling. consequently, their physical interaction in the cell membrane or even the formation of heteromeric receptor channels from p2x3 and asic3 subunits has to be taken into consideration. we transfected rat (r)p2x3r and rasic3 constructs individually or together in a ratio of 1:1 into cho cells. we further used the whole cell patch clamp technique to analyze the current responses either elicited by the application of α,β-methylene-atp (α,β-meatp) or by a decrease in the extracellular ph value. the functionality of the individually transfected p2x3r-and asic3-constructs was verified by recording concentration-response curves for the agonists α,β-meatp and protons, respectively. after co-transfection of both ion channels, a ph-shift from 7.4 to 6.7 caused a rapidly desensitizing current response and a subsequent strong potentiation of the α,β-meatp-induced current. an even larger potentiation was achieved after a decrease of the ph value to 6.5. the opening of asic3 channels failed to facilitate the p2x3r current when 2-guanidine-4-methylquinazoline was used to stimulate a non-proton ligand-sensor of asic3. then, we substituted ca 2+ in the extracellular medium by ba 2+ or decreased the intrapipette concentration of egta, to modify the free intracellular ca 2+ concentration. in cells individually transfected with the receptor-channels, external ba 2+ increased the effect of α,β-meatp but decreased the effect of protons. the lowering of intrapipette egta modified p2x3r-and asic3-specific currents in a similar manner as external ba 2+ . in cells co-transfected with p2x3r/asic3, the ionic manipulations mentioned above abolished the potentiation of the α,β-meatp currents by asic3 activation. taken together, our results suggest that p2x3r and asic3 interact with each other, since the activation of asic3 had a marked impact on the p2x3r specific current response. further experiments are required to clarify the mechanism of this interaction, although it has been shown that extra-and intracellular ca 2+ and the proton sensor of asic3 appear to critically participate in this process. university of duisburg-essen, institute for anatomy, essen, germany background: the sperm acrosome reaction is an all-or-none secretion process, mainly following the conserved principles of calcium-regulated exocytosis in neurons and neurosecretory cells. however, the relationship between the formation of hundreds of fusion pores and the required mobilization of calcium from the lysosome-related acrosomal vesicle has only been partially defined. hence, the second messenger, nicotinic acid adenine dinucleotide phosphate (naadp), known to promote efflux of calcium from lysosome-like acidic compartments, was analyzed for its ability to trigger acrosome reaction in mouse sperm. in addition, the expression of two-pore channel (tpc) proteins, which are primarily localized in lysosome-related acidic organelles and which present potential molecular targets of naadp were examined in mammalian spermatozoa. methodology/ principal findings: our results show that treatment of spermatozoa with naadp resulted in a loss of the acrosomal vesicle, which shows typical properties, described for tpcs: (i) registered responses were not detectable for its chemical analogue nadp, and (ii) where blocked by the naadp antagonist trans-ned-19. in addition, (iii) two narrow bell-shaped dose-response-curves were found, with maxima either in the nanomolar or low micromolar naadp concentration range. performing immungold-electron microscopy with a tpc1 specific antibody, a co-localization with naadp-binding at the acrosomal region was detectable. moreover, quantifying loss of the acrosomal vesicle in tpc1 null sperm upon application of different naadp concentrations, responsiveness to low micromolar naadp concentrations was completely abolished. conclusions/significance: our finding that two convergent naadp-dependent pathways are operative in driving acrosomal exocytosis and that zona pellucida induced acrosomal exocytosis is prevented by trans-ned-19 support the concept that both naadp-gated cascades match local naadp concentrations with the efflux of acrosomal calcium, thereby ensuring reliable and complete fusion of the large acrosomal vesicle. since the acrosome reaction shares the same basic sequence of events typical for the conserved process of calcium regulated exocytosis, such as tethering, docking, priming and final vesicle fusion, the sperm model system may also be useful to comparatively examine whether the same convergence of naadp-dependent pathways is also operative in cellular systems with many secretory vesicles. walther-straub-institut, münchen, germany trpv4 channels are members of the vanilloid family of trp proteins. the channel is nearly ubiquitously expressed and can be found in brain, kidney, skin, heart, blood vessels as well as in the lung. pulmonary expression of trpv4 has been identified in endothelial cells (1), epithelial cells (2) and arterial smooth muscle cells (3) . most interestingly, the channel is known to be involved in the development of several lung diseases such as cough, asthma and pulmonary edema formation, due to its activation by heat, changes in osmolarity and shear stress (reviewed in 4). it is a matter of debate however if trpv4 activation in pulmonary endothelial as well as epithelial cells induces disruption of the barrier and an increased fluid leak into the alveolus as described for trpc6 (5) which is also expressed in both cell types. to analyze the potential role of trpv4 on ischemia-reperfusion-injury(iri)-induced pulmonary edema formation we utilized the isolated perfused mouse lung model. much to our surprise, we detected a significantly enlarged edema formation after 90 minutes of ischemia in trpv4-deficient mice in comparison to wild-type (wt) mice. this effect was observed by constant weight measurements as well as wet-to-dry ratio gain and was not dependent on the initial perfusion rate. most interestingly, edema formation of trpv4/trpc6 double deficient lungs was indistinguishable from wt lungs, indicating antagonizing effects of both channels, because trpc6 deficiency protected lungs from iri-induced edema (5) . moreover, we identified reduced expression levels of aquaporin 5 in trpv4-deficient lung lysates compared to wt lungs. these findings raise the intriguing possibility that trpv4 might be involved in the regulation of aquaporin expression in lung endothelial cells. endosomes and lysosomes are cell organelles involved in transport, breakdown and secretion of proteins, lipids, and other macromolecules. endolysosomal dysfunction can cause storage disorders such as mucolipidoses, sphingolipidoses, or neuronal ceroid lipofuscinoses, but is also implicated in the development of metabolic and neurodegenerative diseases, retinal and pigmentation disorders, trace metal dishomeostasis, infectious diseases, and cancer. endolysosomal ion channels and transporters are highly critical for the tight regulation of the multiple endolysosomal fusion and fission processes including endo-and exocytotic events as well as the regulation of proton and other ionic concentrations in the lumen of endolysosomal vesicles. methods to patch-clamp endolysosomal organelles are continuously improving. yet until now it has not been possible to selectively enlarge endosomal or lysosomal organelles with pharmacological tools for patch-clamp experimentation. we show here by using a combination of two small molecules that we can selectively enlarge early endosomes to a degree sufficient for patch-clamp experimentation. the ability to more selectively patch-clamp intracellular organelles will substantially improve the functional investigation of endolysosomal ion channels under physiological and pathophysiological conditions. the transient receptor potential (trp) channels are a superfamily of non-selective ion channels involved in a variety of physiological processes and in the pathgenesis of many disorders. in the kidney, trp channels have been implicated to be involved in diabetic nephropathy, focal, segmental glomerulosclerosis, polycystic kidney disease, hypomagnesemia with secondary hypercalcemia and idiopathic hypercalcuria. the melastatin-like trp channel subfamily 3 (trpm3) has been shown to be expressed in human kidney 1, 2 . using newly developed anti-trpm3 antibodies, we are able to visualize trpm3 protein in epithelial cells of proximal tubule as well as collecting ducts in the mouse kidneys. therefore, we compared renal function of male, five month old mice lacking trpm3 (trpm3 the atp-gated p2x7 receptor (p2x7r) is a non-selective cation channel widely expressed in epithelia, endothelia, and cells of hematopoietic origin. it plays a central role in cytokine release and studies in p2x7 -/animals indicate its involvement in inflammatory and neurodegenerative diseases. in addition, accumulating data suggests a functional role in neurons and its involvement in neurotransmitter release in the brain. however, despite its importance as a drug target, its precise localization and its molecular and physiological functions remain poorly understood. in particular, the location and function of p2x7r in neurons remain a matter of ongoing debate. to clarify the cellular and subcellular distribution of the p2x7r and to investigate its physiological and pathophysiological role in the brain we generated bac transgenic mouse models in which murine polymorphic variants of egfp-tagged p2x7r are overexpressed under the control of the endogenous p2x7 promoter. the egfp-tagged p2x7rs are efficiently overexpressed in the plasma membrane and can be directly visualized by green fluorescence or indirectly by anti-egfp antibodies. the obtained mouse lines show different expression levels but identical expression patterns with predominant expression in the cerebellum, hippocampus, and thalamus. using cell type-specific markers, p2x7-egfp was identified in almost all microglia and subpopulations of oligodendrocytes and astrocytes in the brain. in the spinal cord, numerous astrocytes in the white matter showed egfp immunoreactivity. so far, no egfp immunostaining was found on map2-and neun-positive cells indicating that under non-pathological conditions, the p2x7 receptor is not expressed in neurons of the cns. interestingly, a higher expression level of cd68 protein was observed in the p2x7r-overexpressing mice. these results suggests that overexpression of p2x7rs alone is sufficient to induce microglia activation, even under non-pathological conditions. since cd68 primarily localizes to lysosomes and endosomes, this further supports a role of the p2x7r in the regulation of phagocytosis. to validate these data, conditional knockout mice are generated. the current status of the project will be presented. the two-pore channels (tpcs) -tpc1 and tpc2 -are located in membranes of intracellular organelles of the endo-lysosomal system. the tpc-protein-monomer contains two homologous domains with six transmembrane α-helices each. a functional tpc probably consists of a dimer of two tpc-proteins resembling an ion channel architecture with the typical four domain organization like voltage gated na + or ca 2+ channels or such as trp channels. due to their biophysical properties, tpc1 and tpc2 are assumed to be involved in the efflux of ca 2+ from intracellular organelles and thereby contribute to fusion/fission processes of endosomes and lysosomes. thus, tpcs are supposed to be important regulators for vesicle trafficking, sorting and degradation/recycling processes. recently, it was shown that virus entry and replication of certain strains of filoviridae -such as ebola -depends on functional tpcs and that either block or genetic inactivation of tpcs reduces virus infectivity. a large family of bacterial protein toxins elicit their effects by modification of intracellular target proteins of host cells. these toxins are taken up by receptor mediated endocytosis and follow different endosomal routes to reach their final cytosolic destination. these toxins principally use two different intracellular routes: the first group uses an entry route via early or late endosomes (short-trip toxins), the second group takes a retrograde route via endosomes, golgi network and the endoplasmic reticulum (long-trip toxins) to get access to the cytosol. translocation of short-trip toxins -such as diphtheria toxin (dt), pasteurella multocida toxin (pmt) and bacillus anthracis lethal factor (pa/lf) -from early and late endosomes into the cytosol is driven by ongoing acidification. long-trip toxins -including cholera toxin (ct) -are retrogradely transported after endocytosis via the golgi apparatus to the endoplasmic reticulum (er). within the er a specific peptide-motif allows the translocation into the cytosol. due to the role of tpc1 for vesicle fusion & fission processes we investigated a potential impact of tpc1 on the uptake of bacterial toxins. first we determined the precise localization of tpc1 in intracellular compartments. to deduce its role for trafficking processes we performed co-localization and correlation studies with a whole set of established markers such as rab-gtpases and pips. second we intoxicated wild-type and tpc1 deficient cell lines with different bacterial protein toxins such as cholera (ct), diphtheria (dt) or pasteurella multocida (pmt) toxin. using cell viability and other intoxication assays we investigated the consequences of tpc1-deletion on bacterial toxin uptake, translocation and cytotoxicity. universität des saarlandes, institut für experimentelle und klinische pharmakologie und toxikologie, homburg/saar, germany trpm3 ion channels are considered to be involved in hormone release from pancreatic islets and the pituitary gland 1,2 . the trpm3 gene encodes a number of different splice variants that differ in their permeation properties and their activity in response to agonists 3, 4 . variations include the presence or absence of five stretches of 10 to 25 amino acid residues within the aminoterminus, long or short pore loops and long or truncated carboxytermini 5 . furthermore, three different aminotermini of human and mouse proteins have been described 5 , but presumably these differences are caused by the activity of different promoters. screening of a mouse pituitary gland cdna library identified 12 variants that differ in exons 8, 13, 15, 17 and 20 . however, only variants bearing the short, ca 2+ permeable pore loop were detected. 3´ rapid amplification of cdna ends (3´race) revealed that trpm3 proteins of the pituitary gland carry truncated c-termini exclusively. 5´race identified five independent regions of transcription initiation within the trpm3 gene implying the presence of five independent promotors. the different transcripts encode four trpm3 amino termini α, β, γ and δ of 1-155 amino acid residues including those described in humans (β,γ). however, the activity of these variants after stimulation with pregnenolone sulfate varied largely just as their frequency in the pituitary with shortened γ-variants being most abundant (~76 %). two-pore channels (tpcs) are a small family of ion-channels found throughout the endolysosomal system of eukaryotic cells. phylogenetically tpcs belong to the voltagegated ion channel superfamily sharing common traits with ca v / na v and trp channels. tpcs show a duplicated architecture with two homologous trans-membrane domains. each domain is build up by six membrane spanning alpha helices linked by short loops. it is very likely that tpcs form dimers maintaining the four-fold symmetry found in other members of the voltage-gated ion channel superfamily. due to their localization in the endolysosomal system tpcs are not accessible to conventional patch clamping. to investigate tpcs electrophysiological properties we use black lipid bilayer measurements. purified channels are integrated into an artificial phospholipid bilayer that separates two chambers enabling us to apply different buffers. upon activation by naadp ions flow through the channel and can thereby pass the diffusion barrier. the movement of charged molecules through tpcs results in currents which can be amplified and recorded. the controlled environment of the lipid bilayer setup allows testing of different ions as well as putative activating and inhibitory substances. one of the major drawbacks of conventional lipid bilayer setups are long preparation times between each measurement. stability of the phospholipid bilayer can pose another issue. often several membranes have to be established before a measurement can be performed making it very time consuming to achieve adequate experiment numbers. new multichannel systems resolve this issue supporting fast formation of bilayers while allowing measurement of up to 16 different bilayers at a time. here we utilize the "orbit" multichannel system with a meca16 chip by ionera to measure tpc1 channel activity. we will present data generated by the "orbit" system and a conventional bilayer setup using different channel constructs and charge carriers. cardiac action potentials are generated and propagated through the coordinated activity of multiple ion channels, including voltage-gated sodium channels (nav1) and potassium channels (like kv1, kv4). the voltage-gated na + channel nav1.5 initiates the cardiac action potential (ap), is essential for rapid depolarization, and is also known to control the ap duration in cardiomyocytes. the voltage-gated k + channel kv4.3 is responsible for the early repolarization of action potentials in human heart. similar to many membrane proteins, nav1.5 and kv4.3 have been found to be regulated by several interacting proteins. the transmembrane β subunit dipeptidyl aminopepidase-like protein (dpp) 10 is known to interact with the kv4.3 channel complex, modulating kinetics and voltage dependence. the overexpression of dpp10 in ventricular cardiomyocytes of rats revealed strong reduction of ap amplitude and significant slowing of ap upstroke velocity and ap duration, which could not be explained by the effects on cardiac kv4 channels. to study the potential influence of dpp10 on nav1.5 channels, we performed whole-cell patch-clamp analysis of transiently transfected cho-k1 cells, expressing scn5a alone or with dpp10. surprisingly, we observed significant effects of dpp10 on nav1.5 channel voltage dependence and kinetics. thus, the co-expression of dpp10 significantly shifted the half-maximal voltage of steady-state activation and steady-stateinactivation to more positive potentials compared to nav1.5 channels alone. in addition we analysed the effects of dpp10 on the kinetics nav1.5 currents. while time to current peak was not affected in cells co-expressing nav1.5 and dpp10 compared to nav1.5 alone, dpp10 slightly accelerated the inactivation. in addition, the time course of recovery from inactivation was clearly accelerated in cells expressing both nav1.5 and dpp10 compared to nav1.5 alone. in summary, we provide first evidence that dpp10 not only interacts with kv4 channels, but also influences nav1.5 channels modulating the depolarization as well as the early repolarization phase of the cardiac ap. therefore, it becomes likely that these ion channels are part of large, multi-protein complexes, and that the pore-forming subunits kv4.3 and nav1.5 behave very differently depending on the expression of its associated proteins like dpp10. cardiac fibroblasts (cf) comprise the most abundant cell type of the mammalian heart and it is known that they contribute to maladaptive cardiac remodeling processes. in response to pressure or volume overload, ischemia-reperfusion injury or myocardial infarction, cardiac fibroblasts proliferate and transdifferentiate into myofibroblasts which produce collagen and pro-hypertrophic cytokines influencing cardiomyocyte function and size. it was shown that β-adrenergic stimulation of cfs with isoproterenol leads to angiotensin ii (at-ii) production and autocrine stimulation of these cells (jaffre et al, 2009 ). activation of phoypholipase c triggered by at-ii leads to formation of inositol trisphosphate (ip 3 ) and subsequent release of calcium from intracellular stores as well as calcium entry across the cell membrane. the focus of our research is the identification of the plasmalemmal channel proteins such as trpc channels mediating this calcium entry, and whether these calcium entry pathways in cfs contribute to pathological remodeling. to date the precise role of calcium entry for these pathological processes is largely unknown. trpc channels are candidates for the analysis of calcium homeostasis in cf. recently, we showed that trpc1/c4-deficient mice are protected from maladaptive cardiac remodeling after neurohumoral stimulation or pressure overload, respectively, which can be explained by a significant reduction of a background ca 2+ -entry (bcge) pathway in cardiomyocytes; this bgce is enhanced by stimulation with agonists such as isoproterenol or angiotensin ii and it critically depends on trpc1 and trpc4 (camacho londoño et al., 2015) . nevertheless, the role of trpc1/c4 for calcium homeostasis in cfs has not been analyzed so far. we established an in vitro model that allows the analysis of calcium release and entry triggered by several (patho)physiological agonists in cultured primary adult cfs from mice. cfs were isolated using langendorff-perfusion and were cultured for maximal 6 days. our results show that there is no difference in 100 nm at-ii induced ca 2+ -release or ca 2+ -entry in trpc1/c4-deficient cf compared to wt. to evaluate whether the lack of trpc1/c4 can be compensated by other trpc channels we currently analyze cfs from trpc hepta ko mice lacking all seven trpc channel proteins concerning at-ii induced ca 2+ -release and ca 2+ -entry and we will also analyze the influence of other agonists on cfs which are known to evoke a longer lasting rise in the [ca 2+ ] i like isoproterenol, 5-ht, thrombin and endothelin-1. cardiovascular and metabolic diseases are currently the primary cause of morbidity and mortality in the western world and are spreading to the rest of the world following globalization. adipose tissue, in particular perivascular adipose tissue (pvat) is recognized as an important player in the development of these diseases. the release of relaxing factor(s) from the pvat has been a matter of interesting and highly spirited debates about its nature, the channels that govern its activities and its role in vascular dysfunction. data from our laboratory indicate that adipose-derived relaxing factor (adrf) is an important player, however the potential channels necessary for its downstream activities are still under study. our recent research primarily focuses on kv7.1 channels, which are known to be expressed in vascular smooth muscle cells. k v 7.1 voltage-gated potassium channels are expressed in vascular smooth muscle cells (vsmc) of diverse arteries, including mesenteric arteries. based on pharmacological evidence using r-l3 (k v 7.1 opener), hmr1556, chromanol-293b (k v 7.1 inhibitors), these channels have been suggested to be involved in the regulation of vascular tone. however, the specificity of these drugs in vivo is uncertain. we used kcnq1-/-mice to determine whether k v 7.1 plays a role in the regulation of arterial tone. we found that r-l3 produces similar concentration-dependent relaxations (ec 50 ~1,4 µm) of wild-type (kcnq1+/+) and kcnq1-/-arteries pre-contracted with either phenylephrine or 60 mm kcl. this relaxation was not affected by 10 µm chromanol-293b, 10 µm hmr1556 or 30 µm xe991 (pan-k v 7 blocker). the anti-contractile effects of pvat were normal in kcnq1-/-arteries. chromanol-293b and hmr1556 did not affect the anti-contractile effects of perivascular adipose tissue (pvat). isolated vsmcs from kcnq1-/-mice exhibited normal peak k v currents. the k v 7.2-5 opener retigabine caused similar relaxations in kcnq1-/-and wild-type vessels. we conclude that k v 7.1 channels are apparently not involved in the control of arterial tone by alpha 1 adrenergic vasoconstrictors and pvat. in addition, r-l3 is an inappropriate pharmacological tool for studying the function of native vascular k v 7.1 channels in mice. introduction: according to international guidelines [1] [2] [3] [4] [5] [6] [7] , both human and animal skin in vitro models have been used and validated to predict percutaneous penetration in humans. excellent correlations have been found for domestic pig as surrogate for human skin [8] [9] . material and methods: tissue the skin samples are descended from female pigs of german landrace (50 kg weight, approx. 4 month). process approved under german welfare law. after narcotization and euthanization the animals were shaved with an electric shaver, washed and dried. microbiological investigation swabs from 10 different skin area (fig. 1) were taken before next preparation step. skin areas were cut, stretched and subcutaneous fatty tissue was carefully removed. the skin was harvested at thickness of 1.000 µm by dermatome. after dermatomization, samples were taken for histological examination. skin disks of 30 mm were punched out from the frozen skin stripes and stored at -20°c. hplc and skin absorption waters corporation hplc containing 2767 sample manager, 2545 binary gradient pump, 2998 pda detector (optional: 3100 electron spray mass spectrometer); column: nucleodur® 100-5 c18 ec 50mm x 4.0 mm id. hanson microette™ vision® diffusion test system (hanson vision® autoplus™ autosampler/autofill™ collector, 6-cell drive system with vertical diffusion cell "standard". the permeation experiment was performed over a period of 48 h at 32°c. the dosage compartments of each cell were filled with approximately 300 µl of the caffeine solution (10 mg/ml). samples of 1 ml are taken after 0, 12, 24, 36 and 48 hours from receptor medium of each cell. aliquots from each vdc are analyzed by hplc in duplicate. microbiology staphylococcus spp. were found explicitly on pig skin surface. bacteria are facultative anaerobe, gram-positive bacteria that are physiologically colonizing the skin, oropharynx and the gastrointestinal tract. histology and skin thickness he staining and mechanical skin thickness determinations confirmed intact dermatomizing process of skin (fig. 2) . thickness was in the order of magnitude between 897 to 1.230 µm and intra variations were less than 10 %. caffeine skin absorption permeability coefficients and lag-phases recorded are in the same order of magnitude of previous work [10], demonstrating intact barrier properties of the membranes after 3 month storage process. until today, intra-assay variations are superior or equal to interregional and inter-animal variations. discussion: well characterized dps1000 provides ready to use research tool for locally and systemic skin investigations. ongoing experiments will cover skin structure analysis by raman spectroscopy, biophotonics and storage impact on skin barrier function. respirable biopersistent granular dusts (gbs) should fulfill the criteria of i.) a negligible solubility in physiological lung fluid that ii.) do not exhibit a specific surface chemistryrelated toxicity at volumetric non-overload conditions in lungs. in 2012, the mak commission derived a new threshold value of 0.3 mg/m 3 for gbs with a density of 1, recognizing that at this concentration a chronic inflammation and increase of the lung cancer risk will not occur. -the objectives of the project were i.) to determine an experimental dissolution value for 'low soluble' gbs using six candidate dusts; ii.) in addition, to measure the inflammatory response in lung lavage fluid and to decide on the criterion 'inert dust'; iii.) to investigate whether nanoscaled dusts could possibly fulfill the criteria to be included in the gbs class. -six micro-and nanoscaled dusts (one of them a well-characterised inert tio 2 dust (microscaled; rutile modification) were compared analysing the solubility in the lung fluid (day 3, 28 and 90) and the lung toxicity after intratracheal instillation in rats (day 3 and 28): tio 2 (rutile, micro), tio 2 (anatase, nano), eu 2 o 3 (micro-nano mixed), baso 4 (micro), zro 2 (micro) and amorphous sio 2 (nano). two doses of 0.5 and 1.5 µl per rat were administered to wistar rats; these volume doses resulted in a non-overload and moderate overload of lungs, respectively. -the differential cell count showed only slight inflammatory cell levels after treatment with tio 2 (rutile) and baso 4 (pmn < 5% after 3 days in the low dose group; < 15% in the high dose group; full recovery after 28 days). in contrast, the tio 2 (anatase) showed a stronger response (pmn > 30% after 3 and 28 days). the rare earth eu 2 o 3 (micro-nano) dust showed the strongest effect (approx. 40% pmn after 3 and 28 days) including a red-coloured lung lavage fluid. µ-zro 2 and amorphous sio 2 showed a strong acute response after 3 days, however, mostly complete recovery after 28 days. the low solubility criterion was met by the following dusts: tio 2 (both) and zro 2 . -similar volumetric lung burdens were deposited in a parallel validation experiment (14-day subacute inhalations). overall the physiological inhalation route confirmed the results obtained in the instillation study, thus suggesting the applicability of the latter as a tool for identification for gbs dusts. however, for nanoscaled dusts an individual toxicological characterization seems to be adequate. polycyclic aromatic hydrocarbons (pah) represent a complex mixture of compounds and occur in considerable amounts as contaminants in the environment and food. some pah have been demonstrated to be carcinogenic and mutagenic. benzo[a]pyrene (bp), the most known and studied member of the potent carcinogenic pah, is classified by iarc as group 1 carcinogen and is present in a wide variety of food items. however, other non-carcinogenic pah such as pyrene (pyr) and fluoranthene (fa) are also found in substantial amounts in the diet and are strongly suspicious to cause interactive effects. reporter gene assays were used for analyzing interactive effects of a ternary mixture including bp, pyr and fa in relative proportions occurring in food on the nuclear receptors aryl hydrocarbon receptor (ahr) and constitutive androstane receptor (car). the observed activations were verified at the gene expression level in the human hepatoma cell line heparg. beside the well characterized ligand bp, 25 µm of pyr and 20 µm fa also activated the ahr, even though to a much lesser extent. no significant higher activation over the level of bp alone was reached when testing different pah mixtures. however, in heparg cells the analysis of cyp1a1 gene expression as a model target gene of ahr showed synergistic effects after pah co-exposure. in addition, the activation of human car was analyzed. pyr and fa are each strong agonists, whereas bp was less potent. for the ternary pah mixture with bp a strong decrease of the induction was observed. this inhibiting effect was verified at the mrna level using the model car target gene cyp2b6 in heparg cells. in conclusion, really occurring mixtures with non-carcinogenic pah can modulate the effects of carcinogenic pah. such effects warrant to be investigated in more detail to enhance our knowledge of interaction of pah mixtures at the molecular level. cytochrome p450 enzymes and transporters are important for the turnover of pharmaceutical compounds. their expression levels and activity influence bioavailability and convey drug-drug interactions. moreover, transporters mediate barrier maintenance of several organs such as blood-brain-barrier and placenta-barrier. overexpression of export transporters in tumors can lead to multiple drug resistance. however, membrane associated proteins are difficult to quantify by conventional bioanalytical methods such as sandwich immunoassays because of their hydrophobicity. antibody -based analysis of cytochrome p450 enzymes and transporters is challenging due to their sequence homology. therefore, we developed a test system for protein quantification which combines the sensitivity of immunoprecipitation and the specificity of mass spectrometry: this method is especially convenient for hydrophobic proteins because denatured samples are analyzed on peptide level. one peptide from each protein, which can be assigned unambiguously, is identified via tandem ms and quantified by means of an isotope labeled reference. prior to ms-read-out, the peptides are enriched by antibodies which recognize a very short c-terminal epitope. these epitopes are selected in such way that they are common in peptides derived from target proteins and therefore allow the analysis of protein groups with few antibodies. the major advantage of this method is that whole cell or tissue lysates -without preparation of microsomal fractions -can be used for quantification by lc-ms. also, samples from different model organisms can be analyzed with the same assay which enhances the comparability of experiments. physiologically based toxicokinetic modeling (pbtk) is an in silico tool to predict compound kinetics based on test substance related properties and physiological parameters of the organism. pbtk is a key element for inverse dosimetry to relate effect concentrations in vitro to external, e.g. oral doses. in our investigations, we use 8 compartment models for the rat including adrenals and testes or ovaries. test substance specific properties taken for pbtk modeling are molecular weight and logp o/w as well as ivis based tissue specific partition coefficients, hepatic clearance, intestinal permeability and plasma protein binding. berkeley madonna software was applied to solve consequent differential equations. here we present the above described model for the 3 test substances bisphenol a (bpa), fenarimol (fen) and ketoconazole (keto). using the lowest effect concentrations (loecs) of bpa, fen and keto from 1) an in vitro yeast based assays with human estrogen and androgen receptor combined with a reporter gene and 2) the interaction of steroidogenesis model calculations were made to relate in vitro concentrations to oral doses in the rat. model calculations, based on in vitro loecs of 10 µm (bpa), 3 µm (fen) and 0.01 µm (keto), for concentrations in target organs resulted in estimated oral loels of 16, 4 and 0.04 mg/kg. when calculations were made for plasma levels oral loels were estimated to be 608, 77 and 16 mg/kg for bpa, fen and keto, respectively when compared to existing in vivo data with endocrine related loels of 375 mg/kg bw day for bpa (1), 50 mg/kg day for fen (2) and 6 mg/kg day for keto (3) , it can be concluded that for the exemplary test substances addressed, ivis related risk assessment approaches based on target tissues seems overpredictive, whereas plasma related loels were closer to the in vivo situation, to study the activation of the nuclear receptor pxr a gal4/uas-based pxr transactivation assay was used. the pxr-mediated induction of cyp3a4 promotor activity was investigated using a pxr-dependent cyp3a4 reporter gene assay. cyp3a4 induction was analyzed at the mrna and protein levels in hepg2 cells using qpcr and western blot. to cover the most frequently occurring pa structures (retronecine, heliotridine and otonecine type as well as monoester, open-chain diester and cyclic diester) the four pa senecionine, heliotrine, echimidine and senkirkine were selected as representative pa for initial analyses. out of the four investigated pa only echimidine activated pxr. accordingly, pxrmediated induction of cyp3a4 promotor activity could only be detected for echimidine. cyp3a4 induction by echimidine was verified at the mrna and protein level in hepg2 wildtype and hepg2 pxr-overexpressing cells. taking heinrich-heine universität düsseldorf, institut für toxikologie, düsseldorf, germany introduction: in higher concentrations, the blood pressure regulating hormone angiotensin ii (ang ii), leads to vasoconstriction, hypertension and oxidative stress by activation of the renin angiotensin system (ras). here we investigate if nadphoxidases are responsible for ras-mediated oxidative stress in kidney and heart. nadph-oxidases (7 isoforms are known, nox 1-5, duox 1 & 2) are membrane-bound enzymes that produce reactive oxygen species (ros) for example during the immune response and cell signaling. material and methods: to clarify the role of nadph-oxidases, wildtype mice and nox 1-, nox 2-and nox 4-deficient mice were equipped with osmotic minipumps, delivering ang ii in a concentration of 600 ng/kg during 28 days to stimulate high blood pressure. kidney and heart were investigated for steady-state ros levels and dna damage (dna single and double strand breaks). results: in wildtype mice, ang ii leads to hypertension, a declined renal function, formation of ros in kidney and heart and to dna single and double strand breaks in the kidney. all nox-knockout mice exhibited ang ii-mediated hypertension and albuminuria. the lack of nox 2 and nox 4 could neither protect from the formation of oxidative stress in the kindey nor from dna double strand breaks in the kidney. initial findings from the nox 1-knockout mouse do not show an increase in dna double strand breaks in the kidney. discussion: contrary to published results of nox 1-knockout mice, we observed a constant rise in blood pressure over the treatment period compared to the control. this can possibly be due to different ang ii doses. in nox 4-knockout mice we observed increased oxidative stress and increased renal dna damage already in untreated control animals, which is in line with reports suggesting a protective effect of nox4. conclusion: separate eliminations of 3 nadph-isoforms did not allow the identification of the enzyme which is responsible for ang ii-induced oxidative stress. a possible explanation is that oxidative stress is caused by more than one nox-isoform or other enzymes like xanthine oxidase or nitrogen synthase take major part in the formation of ros. of mice (rats, cats, dogs, monkey) and men -how to measure kidney biomarkers across species introduction and objectives: there is a need for reliable biomarker assays to detect organ toxicity induced by drug candidates. in the last 50 years about 60 drugs were withdrawn from the market due to liver and/or kidney damage. for the detection of druginduced organ injury, safety-tox studies in rodents, dogs, non-human primates and humans are mandatory in the drug development process. currently, several protein biomarkers for kidney, liver and cardiovascular organ toxicitity are being clinically validated by international consortia like the safer and faster evidence-based translation (safe-t) or the predictive safety consortium (pstc). we are developing mass-spectrometry-based immuoassays suitable for the detection of these markers in animal models to support these efforts method: urinary proteins are proteolytically digested to peptides using trypsin. subsequently synthetic isotope-labelled peptide standards are spiked in at known concentrations. we employ multi-specific antibodies (txp-antibodies) targeting cterminal amino acid motifs for the enrichment of peptides derived from the protein biomarkers. finally, the protein biomarkers are quantified using nanolc-parallel reaction monitoring-ms. the use of our group-specific txp-antibodies allows protein analysis of samples from different species using the same antibody. results and discussion: we established an ms-based immunoassay platform for the analysis of kidney (diki) injury protein biomarkers in urine across 5 species; human, cynomolgus, mouse, rat and dog. we analyzed the potential diki biomarkers aquaporin 2, podocin, synaptopodin, retinol-binding protein 4, clusterin and osteopontin in urine samples from toxicity studies in cynomolgus monkeys, rodents and humans. conclusion: the application of group-specific txp-antibodies and mass spectrometry allows the quantification of biomarkers in urine of all relevant model organisms. the results strongly support the validation of translational drug-induced organ injury protein biomarkers. although effective anticancer-therapeutic regimen are available, they are accompanied by severe adverse effects on normal tissue, for instance chemotherapy induced peripheral neuropathy (cipn) caused by platinum compounds. the pathophysiology of this clinically highly relevant side-effect is still unknown and neither prophylaxis nor specific treatment is available. therefore, further research elucidating the underlying molecular mechanisms of platinating anti-tumor drugs leading to cipn is required as basis for future development of preventive or therapeutic strategies. in general, platinum compounds lead to cell death mainly via dna damage induction (mostly intrastrandcrosslinks) and through interference with the redox homeostasis of cells. here, we introduce and suggest the well-known nematode model organism c. elegans to elucidate mechanisms of neurotoxicity triggered by platinating agents. so far, we determined doses for cis-and oxaliplatin, which have only moderate effects on development, reproduction and body movement (muscular read-out). however, these doses are sufficient to trigger apoptosis in c. elegans and to induce a considerable amount of 1,2-intrastrand crosslinks in dna (measured by south-western blotting). even more important they lead to strong neurotoxicity in a functional read-out (pharyngeal pumping). with regard to redox homeostasis, we determined the oxidative stress resistance showing that e.g. cisplatin sensitizes c. elegans to reactive oxygen species (ros), which could be prevented if worms were co-or pretreated with n-acetylcysteine. furthermore we determined the level of ros in living c. elegans after treatment with platinating agents and also in combination with protective compounds. using the advantages of c. elegans as a genetic model system, we will further clarify the relevance of different defense mechanisms, including dna repair (nucleotide excision repair, base excision repair), detoxification systems (antioxidative stress factors, metallothioneins) as well as drug transporters and signaling proteins. this will be achieved by using rna interference approaches that allow targeting either the whole animal or specific tissues (i.e. neurons) only. first results of this approach will be presented. finally we aim to use this setup to identify neuroprotective compounds that prevent chemotherapy induced peripheral neuropathy induced by platinating anti-tumor drugs. clostridium botulinum c3 exoenyzme (c3) exclusively adp-ribosylates rhoa, b and c leading to reorganization of the actin cytoskeleton and morphological changes. in addition to the enzyme-based inhibition of rho-gtpases, c3 promotes in an enzymeindependent manner axonal and dendritic growth in neurons. as c3 lacks the canonical binding and translocation domains of bacterial protein toxins, cell entry is currently not well understood. based on overlay assays and mass spec analyses the intermediate filament vimentin was identified as the putative membrane receptor for c3. knock down of vimentin by sirna and application of the selective vimentin disruptor acrylamide led to a significantly delayed uptake of c3. moreover, addition of extracellular vimentin to cells induced an enhanced uptake of c3. proof of principle experiments in astrocytes and neurons from vimentin knock out mice showed c3-induced morphological changes (astrocyte stellation and axon growth) to a reduced extent and a significantly delayed uptake of c3 compared to wild type cells. as vimentin knock out did not completely inhibit c3 uptake into cells, an additional uptake mechanism or additional receptor for c3 is likely. nevertheless, our data reveals that c3 employs a specific endocytosis mechanism with involvement of the intermediate filament vimentin to gain access to host cells. the primary target organ of organic hg species-mediated toxicity is the central nervous system (cns). humans are exposed to organic hg mainly in the form of methylmercury (mehg) via the consumption of contaminated fish and other seafood products. in terrestrial food sources hg is mostly found as inorganic hg. thiomersal is a further organic hg compound which is used as a preservative in medical preparations. exposure to organic hg promotes primarily neurological effects. the understanding of transfer mechanisms regarding the cns is an important precondition for an evaluation of hg species-induced neurotoxicity. thus, primary porcine in vitro models of the bloodbrain barrier and the blood-cerebrospinal fluid (csf) barrier were used to investigate effects of mehgcl, thiomersal and hgcl 2 on the barriers as well as transfer properties into and out of the cns in vitro. the results show significant transfer differences of the various incubated species as well as in the different barrier systems. whereas the bloodbrain barrier seems to account for the transfer of organic hg species from the blood side to the brain side, these species are transferred in the contrary direction by the blood-csf barrier. inorganic hgcl 2 was not transferred across both brain barriers towards the brain side but was able to leave the brain side across the blood-brain barrier. additionally, cytotoxic effects of the hg species by themselves as well as the combination of organic and inorganic hg species have been investigated in human astrocytes and human differentiated neurons. differentiated neurons were much more sensitive towards all hg species. organic species exerted stronger cytotoxic effects in both cell types as compared to hgcl 2 . interestingly, a coincubation of organic and inorganic hg species led to an increased cytotoxicity in the astrocytes. this cocytotoxic effect is currently investigated in differentiated neurons. the species-specific differences with respect to both, effects on and transfer across the blood-brain and the blood-csf barrier in vitro as well as toxic effects in brain target cells, clearly emphasizes the necessity for comparative analyses. introduction: the neural crest is a multipotent stem cell population that arises at the neural plate border during early fetal development. neural crest cells (nccs) migrate to target sites in the periphery, where they differentiate into multiple cell types, including melanocytes, cranial bones and peripheral neurons. failure of ncc migration can lead to severe disorders, such as hirschsprung's disease. aim: to test whether toxicants interfere with human ncc migration, a high-throughput migration assay was established. this test system was used to screen an 80 compound library of potential developmental toxicants. methods: nccs were derived from human embryonic stem cells. the cells were allowed to migrate for 24 h before toxicants were added to the cells. migration and viability of the cells were then measured after another 24 h by high-content image analysis and a custom-developed software package. results: the screening library was assembled by the us national toxicology program (ntp) and consisted of different substance classes, e.g. organophosphates, organochlorines, drug-like compounds, pesticides and polycyclic aromatic hydrocarbons (pahs). out of the tested potential developmental toxicants, 26 compounds reduced ncc migration at non-cytotoxic concentrations. hit-confirmation testing confirmed 23 of the compounds as concentration-dependent inhibitors of ncc migration. among the potential developmental toxicants identified here, there were several organophosphates (e.g. chlorpyriphos) and drug-like compounds as well as polybrominated diphenyl ethers (pbdes) and organochlorine pesticides (e.g. ddt and dieldrin), while none of the tested pahs inhibited ncc migration. the negative controls in the screening library, like acetylsalicylic acid, acetaminophen and saccharin, proved to be non-toxic. conclusion/outlook: the newly established test system allows screening of potential developmental toxicants in a high throughput manner for interference with human ncc migration. confirmation in other types of migration assays is ongoing, and selected compounds from amongst the screen hits are undergoing mechanistic evaluation. oxidative stress is regarded as a major trigger for neuronal dysfunction and death in the ageing brain and in multiple neurodegenerative disorders. how oxidative stress mediates neuronal death and whether the associated mechanisms are accessible for therapeutic intervention strategies is not clarified. increasing evidence suggests, however, that oxidative stress triggers molecular mechanisms of regulated necrosis that involve the activation of receptor interacting protein 1 (rip1) independently of death receptor activation. here, we show that erastin-induced ferroptosis which involves inhibition of the glutamate-cystein transporter (xc -), glutathione depletion and lethal formation of reactive oxygen species (ros) 1 , triggers mechanisms of regulated necrosis independent of tnfα-signaling. in hippocampal ht-22 cells erastin promotes activation of rip1 and subsequent rip1-rip3 necrosome formation which has been investigated as a hallmark of regulated necrosis 2 . in fact, silencing of rip1 by sirna or by the rip1 inhibitor necrostatin-1 prevents ferroptosis-induced cell death whereas the ferroptosis inhibitor ferrostatin-1 fails to protect cells against tnfα-induced classical necroptosis, a form of programmed cell death that is mediated by receptor interacting protein-1 (rip1) and rip3 kinases downstream of death receptor activation (e.g. tumor necrosis factor receptor tnfr) 2, 3 . recently, a genome-wide sirna screen linked cylindromatosis (cyld) to rip1/rip3-dependent necroptosis 4 and also in the present paradigm of ferroptosis, cyld depletion promotes neuronal survival and decreases rip1-rip3 complex formation, suggesting a role of cyld in intrinsic pathways of regulated necrosis triggered by oxidative stress. the ns5a inhibitor daclatasvir is used in combination with other antivirals such as the polymerase inhibitor sofosbuvir for treatment of chronic infection with the hepatitis c virus. daclatasvir is embryotoxic and teratogenic in rats and rabbits at exposures at or above the clinical exposure. in contrast, no teratogenic effects were observed in rat and rabbit developmental toxicity studies with ledipasvir, another ns5a inhibitor. we studied these compounds in the embryonic stem cell test (est) alone and in combination with sofosbuvir. the ns5a inhibitors were obtained from selleckchem, the main metabolite of sofosbuvir, psi-6202, was from medchem express. murine embryonic stem cells (es-d3) were obtained from atcc. they were kept in iscove's modified dulbecco's medium (imdm). substances were dissolved in dmso at a final dmso-concentration of 0.1% in the culture medium. a cytotoxicity assay as well as a differentiation assay were performed. after 10 days in culture the cells were evaluated. cytotoxicity was measured by an mtt test. differentiation into contracting myocardial cells was determined using direct phase contrast microscopy. the substances were tested at concentrations between 0.1 and 30 mg/l, which is a broad coverage of the therapeutically relevant concentrations reached in patients. at a concentration of 10 mg daclatasvir / l medium and higher the substance inhibited differentiation of cells. we observed contracting myocytes in 23, 22 and 2 wells out of 24 wells in total at concentrations of 1, 3 and 10 mg/l. at 30 mg/l no differentiation was observed. effects on cell viability were observed at 30 mg/l. unexpectedly, we found a higher potency with ledipasvir. at the low, therapeutically relevant concentration of 1 mg/l this nsa5-inibitor showed a clear impact on differentiation with 6 out of 24 wells affected and no differentiation at higher concentrations. addition of sofosbuvir or its main metabolite psi-6206 at concentrations up to 30 mg/l had no influence on the concentration effect curves established for daclatasvir or ledipasvir. this is the first indication of an embryotoxic potential of ledipasvir. the difference to the results from the routinely performed animal experiments is unknown. possibly, metabolic activity in the maternal organism is responsible for this discrepancy. dimoxystrobin is a european-registered pesticidal active ingredient. biologically it is acting as an inhibitor of the fungal respiratory chain. for the purpose of european registration a full set of toxicological studies has been conducted with dimoxystrobin, including reproduction toxicity studies (according to the most recent oecd tg 416) and developmental toxicity studies (oecd tg 414) in rats and rabbits. dimoxystrobin interferes with the iron transport in rats and mice. this leads to lower serum iron levels and anemia in rats after repeated exposure. this holds true for treated dams and offspring in reproduction toxicity studies. furthermore, offspring effects seen at the high dose of the 2-generation toxicity study were a hypochromic microcytic anemia, impaired body weight development, which only developed postnatally, and reversible cardiomegalies in some 21-days old pups. for all effects clear noaels were determined. in the 2-generation toxicity study no dose adjustment during pregnancy and lactation was performed, which resulted in considerably higher food and compound intakes in dams and offspring during these lifestages. as a result, it seemed, that pups were more severely affected by body weight effects compared to the parental generation. by performing a life-stage specific comparison of body weight and substance intakes, as well as benchmark dose calculations (bmd) for these parameters, it could be demonstrated that the point of departures (pods) and the loaels for direct dimoxystrobin-related effects were comparable for offspring and parents. the heart effects (cardiomegaly), which were reversible, occurred only after direct dimoxystrobinexposure and are considered to be secondary to the detected offspring anemia. both effects (lower body weights and offspring cardiomegalies) only occur postnatally and are not the consequence of in-utero exposure, as no respective effects at higher doses in rat prenatal toxicity studies were seen. two new mechanistic studies (1-generation toxicity study and a 3-week study in young and adult rats, additionally investigating serum iron levels and anemia) confirmed, that pups and young rats were not more sensitive than adult animals to develop anemia or decreased serum iron levels. in 2006, dimoxystrobin was classified with r63 (possible risk of harm to the unborn child) by the ecb, which was the european authority responsible for classification and labeling, before echa in helsinki was formed. the r63 (which has been translated into the ghs classification repr. 2, h361d) was based on offspring body weight and heart effects seen in the 2-generation toxicity study. based on a comprehensive re-evaluation of existing and on new data of dimoxystrobin, the conclusion can be drawn, that a classification for reproduction toxicity is scientifically not justified and should be reconsidered. perfluorooctanesulfonic acid (pfos) and perfluorooctanoic acid (pfoa) are perfluorinated substances (pfas) which are used for the fabrication of surfaces with water-and dirt-repellent properties. due to their reprotoxic properties and their persistence in the environment, the use of pfos was restricted in 2009 and a restriction program for pfoa was initiated in 2013. therefore, industry switches to pfoa and pfos substitutes, which are predominantly pfas with a shorter carbon chain length, or structure-related compounds. in contrast to pfoa and pfos only few toxicological data are available for their substitutes. aim of this study was to examine endocrine effects of the substitutes perfluorohexanesulfonic acid (pfhxs), perfluorobutanesulfonic acid (pfbs), perfluorohexanoic acid (pfhxa), perfluorobutanoic acid (pfba) and 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)propionic acid (genx) in comparison to pfoa and pfos. a hek-293t cell-based dual-luciferase reporter gene assay was used to investigate the potential of these compounds to affect the activity of the human estrogen receptors herα and herβ. the reporter gene assay revealed no activation of herα or herβ by the pfas tested in this study. to investigate the potential inhibition of herα and herβ by pfas, a coincubation with the estrogen receptor agonist 17β-estradiol was performed. none of the tested pfas inhibited herα or herβ activity. however, in the case of herβ an enhancement of 17β-estradiol-stimulated activity was observed. thus, pfas do not directly activate or inhibit the human estrogen receptors but have an impact on herβ activity as they amplify the activation mediated by 17β-estradiol. further studies will be conducted to examine this synergistic effect in more detail. the xeer-reporter cell line: a novel dual-color luciferase reporter assay for simultaneous detection of estrogen and arylhydrocarbon receptor activation p. tarnow consumers are exposed to a multitude of anthropogenic and natural substances capable of activating or inhibiting ligand activated transcription factors, respectively. this in turn can lead to adverse health effects, particularly for substances acting on signalling pathways that are subject to regulatory crosstalk such as xenoestrogens and polycyclic aromatic hydrocarbons (pahs). xenoestrogens are known to activate human estrogen receptors (ers), whereas pahs or dioxins act on the arylhydrocarbon receptor (ahr). importantly, both receptor signalling pathways are interconnected by a complex crosstalk on multiple levels. this ranges from direct protein-protein interactions to competition for common co-factors. however, although this cross-talk has been long known we still lack a deeper understanding of its molecular mechanisms and physiological implications. one reason for this is a lack of tools to visualise and investigate receptor interaction in vivo. based on the breast cancer cell line t47d we thus developed a dualcolour reporter assay which allows time-resolved simultaneous monitoring of the activation of er and ahr in living cells. the assay uses two beetle luciferases emitting luminescence in the red (slr) and the green (eluc) spectrum, respectively. while eluc is expressed under the control of a 6-fold repeated xenobiotic response element (xre) slr is subject to transcription regulation by a 6-fold repeated estrogen response element (ere). both constructs were stably transfected into t47d human breast cancer cells, which endogenously express erα and ahr and are thus ideally suited for monitoring interactions with both receptors. the respective "xeer"-cell line has been successfully subjected to proof of principle studies, using prototypical er-and ahrligands as well as various phytochemicals and xenobiotics. besides e2 and tcdd ligands included various pahs, polychlorinated biphenyls, alpha-and betanaphthoflavone, cosmetic ingredients (butylparaben, benzophenone-2 and 4-mbc), bisphenol a, genistein, resveratrol, diindoylmethane as well as pharmacological antagonists of both receptors. asian women consuming soy rich food throughout life possess lower levels of 17betaestradiol (e2) in plasma (pl) than western women, whose diet is characterized by less soy consumption during early life and possible intake of soy based dietary supplements during adulthood. however, the impact of these soy exposure scenarios on estrogen (biotrans)formation and the consequence thereof in female mammary glands (mg) has not been investigated yet. thus, female august copenhagen irish rats were fed either isoflavone (if) depleted diet (idd, western exposure scenario without if supplement) or if rich diet (ird, asian exposure scenario) until the end of the study at postnatal day (pnd) 81. furthermore, rats fed idd until pnd74 were fed ird for 7 days (idd+ird, western dietary exposure scenario with if supplement). estrous was determined histologically. levels of transcripts were determined by qpcr and e2 and estrone (e1) in pl and mg were quantified by gc-ms/ms. statistical analyses of estrogens were performed by kruskal wallis and unpaired wilcoxon tests and of transcript levels by linear regression models considering the explanatory variables tissue levels of e2 and diet (idd vs ird and idd vs idd+ird). e2 levels in pl and mg did not coincide with those predicted by estrous. furthermore, median levels of e1 and ratios e2/e1 in mg and ratio of e2 levels in pl/mg were not affected by diet. in contrast, diet tended to affect e2 concentrations in pl (p=0.1211) due to an increase in the ird group (p=0.0056) whereas e2 levels in the idd+ird group only tended to be elevated (p=0.0788). in mg, ird and idd+ird increased e2 levels only weakly (p=0.0788 each). likewise, besides significant changes in transcript levels of cyp1a1 and 1a2, putatively decreasing oxidation of e2 to catechols, in the idd+ird group and (not significantly) also in the ird group, no changes in transcript levels putatively affecting e2 levels were observed. moreover, no decrease in levels of transcripts indicative for cellular (oxidative) stress (gclc, tp53, mt1a) was observed in the idd+ird group. e2 mg levels were significantly associated with an increase in transcript levels of areg and pgr, indicating activation of estrogen receptor (er). in contrast, ird was associated with a significant and idd+ird with a not significant decrease in pgr transcript levels. e2 levels but not diet were significantly associated with gata3 transcript levels, indicating tissue differentiation. furthermore, levels of transcripts involved in intercellular communication (egfr, wnt4) were significantly decreased by idd+ird and not significantly by ird and differed from that affected by e2 (increase in gdf15, hgf, igf1r, wnt5a). bmf, a marker transcript for apoptosis was increased by ird, but not affected by e2 and even decreased not significantly by idd+ird. taken together, despite an increase in e2 levels in pl, less er activation was observed after dietary exposure to if. whereas e2 and transcript levels of enzymes involved in e2 (biotrans)formation as well as er activation and cellular communication were affected similarly but to a different extend in both asian and western if exposure scenarios, differences in apoptosis were observed between ird and idd+ird groups. supported by dfg le 1329/10-1. august copenhagen irish (aci) rats with 17β-estradiol (e2)-releasing implants are an accepted model to study the etiology of breast cancer, but neither e2 (biotrans)formation in mammary gland tissues (mg) during tumorigenesis, nor the impact of isoflavones (if) shown to affect tumorigenesis in aci rats, has been investigated, yet. therefore at postnatal day (pnd) 75 and 175, placebo (-e2) or silastic implants containing 4 mg e2 were implanted in female aci rats exposed to either if depleted diet (idd) or if rich diet (ird) since conception until the end of the study at pnd 285. palpable mg tumors (pt) and 1-2 mg per animal without pt were characterized histologically and categorized into normal (-e2 group, n=12), hyperplasia and non-pt and pt with and without solid tumors (+e2 group, n=32). e2, estrone (e1), their hydroxylation products and methylation (meo-) products thereof, as well as conjugates of e1 and e2 in plasmas and mg were analyzed by gc-and uhplc-ms/ms, respectively. levels of 49 transcripts involved in (biotrans)formation of e2 and estrogen receptor (er) activation were determined by taqman®-pcr. without exogenous e2, plasma e2 as well as e1 and (borderline) e2 levels in mg were higher in ird. plasma e2 as well as e1 and e2 levels in mg were lower in the -e2 group than that in the +e2 group. e2 levels as well as e2/e1 and e2 mg/plasma ratios were elevated in pt, accompanied by a significant increase in transcript levels indicative for estrogen receptor activation (areg, pgr) and proliferation (mki67). ird increased e2/e1 ratio in pt and, although ird did not affect er activation (areg, pgr), ird increased differentiation (gata3) in normal and hyperplastic tissues and tended to decrease proliferation in hyperplastic (ccnd1) tissues. levels of e1 and 2-meo-e1 were highest in hyperplastic tissues, accompanied by an increase in transcript levels of hsd17b2 (conversion e2 to e1) and cyp1a1. transcript levels of gstm1 and gstm2 were decreased in the whole +e2 group and of gstt1 and gstt3 in hyperplastic tissues, possibly decreasing inactivation of electrophilic metabolites. accordingly, maximum transcript levels of tp53 and mt1a indicating cellular (oxidative) stress were observed in hyperplastic tissues. ird did neither affect levels of 2-meo-e1 nor cellular stress (gclc, mt1a, tp53). of note, neither 4-meo-e1, nor e1 catechols, nor e2 catechols nor methylation products of the latter were observed in any sample. furthermore, no conjugates of e1 or e2 were detected in plasmas and mammary gland tissues. thus, changes in transcript levels of conjugating enzymes induced by tumorigenesis and by ird were not related with detectable conjugate levels of e1 or e2. taken together, whereas hyperplastic tissues were characterized by maximum oxidative metabolism of e1 and cellular (oxidative) stress, pt exhibited highest e2 levels and er activation. ird increased differentiation and decreased proliferation in normal and hyperplastic tissues but increased e2/e1 ratio in pt. supported by dfg le-1329/10-1. level of 17beta-estradiol (e2) in human breast tissue is considered to affect breast cancer initiation, promotion and progression. although putatively beneficial and adverse effects of soy isoflavones (if) on the human mammary gland, in particular in western women, have been discussed extensively, the influence of if levels on estrogen formation in human mammary gland tissue has not been investigated yet. thus, glandular tissues were dissected from 37 mammoplasty specimen obtained from women (age 18-66 years old) not taking estrogen active drugs. 14 of these women had been exposed to if by their usual diet or by intake of a soy-based dietary supplement for 7 days prior to mammoplasty. information on soy consumption and lifestyle were collected by questionnaire and tissues were characterized histologically. genistein, daidzein their conjugates (n=12) and bacterial metabolites (n=7) as well as the estrogens estrone (e1)-sulfate, e1, e2 and 2-methoxy-e1 were determined by uhplcand gc-ms/ms, respectively and transcript levels of 19 enzymes involved in e2 (biotrans)formation were quantified by taqman®-pcr in glandular tissues. isoflavonoids were categorized into the if parameters aglycones (agl) and conjugates (con) of either genistein, daidzein or sum of both and were further statistically analyzed by spearman`s rank correlation analysis. a positive correlation of e2/e1 ratio with agl(+con) was observed in glandular tissues (r=0.49, p=0.002), accompanied by a significant negative correlation of e1 levels with agl (r=-0.35/p=0.032), possibly due to reduction of 17beta-hydroxysteroid dehydrogenase 2 (conversion of e2 to e1) expression as indicated by a weak negative correlation of transcript levels of 17beta-hydroxysteroid dehydrogenase 2 with agl+con (r=-0.25, p=0.080). further statistical analysis taking into account multiple variables using linear regression models will provide more insights into variables affecting e1/e2 ratio. taken together, estrogen profile in human glandular breast tissue seems to be affected by if levels. supported by dfg le-1329/10-1. allergic contact dermatitis (acd) is a widespread disease often caused by substances in consumables. the eu prohibits the testing of cosmetic ingredients in vivo. this urges the development of reliable in vitro testing strategies. activation of dendritic cells (dcs) represents a key step during sensitization as they are essential for selection and priming of allergen specific effector t cells. in an integrated omics approach we aimed to further elucidate the molecular mechanisms of dc activation using quantitative metabolomics and proteomics. monocytic thp-1 cells were used as a model system and treated with the sensitizer 2,4dinitrochlorobenzene (dncb; 5, 10 and 20 µm) and the irritant sodium dodecyl sulfate (sds; 100 µm). samples were taken after 4, 8 and 24 hours. thp-1 activation was analyzed by measuring the established activation markers cd86 and cd54 after 24 hours. a targeted lc-ms/ms approach was used to analyze 188 metabolites including amino acids and lipids. protein levels were quantified by nano-lc-maldi-ms/ms after stable isotope labeling by amino acids in cell culture (silac). data sets were examined by multivariate analyses for identification of biomarker candidates. regulated metabolites and proteins were subjected to pathway analysis. the data presented might contribute to the further development of suitable in vitro testing methods for chemical-mediated sensitization. drug induced liver injury (dili) is one of the most frequent causes of acute liver injury and a main cause for drug withdrawals. currently there are no reliable models to test the dili potential of new compounds available. kupffer cells (kc) play an important role in hepatic cell stress mediated through chemokines and release of endogenous proteins. kc activation by damaged or stressed hepatocytes can lead to activation of the nf-κb signaling pathway transmitted by reactive oxygen intermediates (roi). we have recently established a liver model composed of primary human hepatocytes (phh) and kc which enables investigation of immune reactions after induction of hepatocyte stress (kegel et al., 2015) . aim of the present study was the kinetic investigation of hepatic cell stress induction and macrophage activation after treatment with subtoxic concentrations of hepatotoxic drugs. primary human hepatocytes (phh) and kc were isolated from human liver resectates using a two-step collagenase perfusion technique. initial kc activation was characterized by the dcf assay and immunofluorescence staining. phh were incubated with different concentrations of acetaminophen (apap) and diclofenac (dic) for different time intervals. cell stress was evaluated by measurement of oxidative stress (dcfassay) and viability (xtt-assay). in order to simulate macrophage activation following hepatocyte damage, kc and macrophages derived from the monocytic cell line thp-1 were incubated with supernatants of phh treated with hepatotoxic compounds. kc and thp-1-macrophage activation were investigated by measuring intracellular formation of roi using the dcf-assay and cell activity using the xtt-assay. the characterization of kc activation revealed a donor and disease dependent kc activation resulting in kc differentiation to pro-and anti-inflammatory macrophages. therefore, kc were substituted by macrophages derived from thp-1 cells. evaluation of hepatic cell stress showed the strongest effect on thp-1-macrophages when phh were incubated with apap or dic for 4 h.treatment of kc and thp-1-macrophages with supernatants of phh challenged for 4 h with hepatotoxic compounds indicates that thp-1-derived macrophages react similar to kc when treated with phh supernatants in terms of cell activity and roi-production. in conclusion, thp-1 derived macrophages might be a suitable alternative to kc concerning macrophage activation. the evaluated kinetic window of 4 h covering hepatic stress induction and immune reaction allows to perform these measurements in a since the use of cerium dioxide nanoparticles is known to be beneficial e.g. in terms of reducing fuel consumption when added to diesel fuel it has become a frequently used nanomaterial. to compensate the concurrent lack of information on its toxicology a 90day nose-only inhalation study was initiated. by comparing the results to a combined chronic inhalation toxicity and carcinogenicity study using the same test items and experimental conditions (basf, ludwigshafen, germany) early indicators for genotoxic and carcinogenic effects should be determined. rats were exposed to 0, 0.1, 0.3, 1 and 3 mg/cm³ ceo 2 as well as 50 mg/m³ baso 4 nanoparticles (6 h/day, 5 days/week, 13 weeks). animal dissections were conducted at five time points (exposure day 1 and 28; recovery day 1, 28 and 90) aiming for endpoints mandatory according to oecd guideline 413. additionally, gene expression analyses in isolated pneumocytes type ii were performed using pathway arrays for inflammation, oxidative stress, genotoxicity, apoptosis and lung cancer. the given results intend the identification of marker genes displaying modulated expression in response to nanoparticle exposure. investigations on ceo 2 and baso 4 retention in the lung are also included in this project. in bronchoalveolar lavage fluid (balf) a time and dose-dependent increase of inflammatory cells has been detected up to the end of exposure. the amount of inflammatory cells decreased during post-exposure; however, in the high dose group a persistent inflammation up to 90 days was detected by balf and histopathology examination. based on our current results effects of ceo 2 nanoparticles on the respiratory system are suggested. its relevance in the context of long term effects such as tumor development needs to be estimated considering all investigations included in this study. the inhalt-90 project is funded by the german federal ministry of education and research (bmbf) -03x0149a. core or coating material? what dictates the uptake and translocation of nanoparticles in vitro? nanoparticles are becoming increasingly important role in consumer-related products. understanding the interactions between nanoscaled objects and living cells is therefore of great importance for risk assessment. in this context, it is generally accepted that nanoparticle size and shape are crucial parameters regarding the potential of nanoparticles to penetrate cell membranes and epithelial barriers. current research in this field additionally focuses on the particle coating material. in order to distinguish between core-and coating-related effects in nanoparticle uptake and translocation behavior, this study investigated two nanoparticles equal in size, coating and charge but different in core material. silver and iron oxide were chosen as core materials to ensure similar nanoparticles characteristics after particle synthesis. nanoparticles were coated with poly (acrylic acid) (pas) and extensively characterized by tem (transmission electron microscopy), saxs (small-angle x-ray scattering), zetasizer tm and nanosight tm . for uptake and transport studies the widely used human intestinal caco-2 model in a transwell tm -system with subsequent elemental analysis (aas) was used. for evaluation and particle visualization transmission electron microscopy (tem) and ion beam microscopy (ibm) were conducted. although similar in size, charge and coating material, the behavior of particles in caco-2 cells was quite different. the internalized amount was comparable, but pas-coated iron oxide nanoparticles were additionally transported through the cells. by contrast, pascoated silver nanoparticles remained in the cells. our findings suggest that the coating material influenced only the uptake of the nanoparticles whereas the translocation was determined by the core material. in summary, a core-dependent effect on nanoparticle translocation was revealed. both the uptake and transport of nanoparticles in and through cells should be considered when discussing nanoparticle fate and safety. nanotechnology is having a great impact not only on basic research but also on many sectors of industry opening the market for numerous new applications ranging from electronics to the health care system. besides their great innovative potential, the large variety of existing synthetic nanomaterials used in the last decade represents a major challenge for scientists and regulators in terms of measuring and assessing the potential hazard caused by the materials or the products themselves. equally, consumers often miss reliable and easy-to-understand information on nanomaterials and nanotechnology and do not know where to get such information. therefore, the international dana 2.0 expert team brings together its expertise and knowledge from different research areas dealing with all aspects of nanosafety research in order to create and provide easy-to-understand, up-to-date and quality-approved nanomaterials' knowledge base on www.nanopartikel.info. this information platform covers the 25 market-relevant nanomaterials focusing on their effects on the safety of humans and the environment.in order to manage and asses the rapidly increasing number of publications related to nanosafety issues, the dana 2.0 project developed a customised methodology «literature criteria checklist», which includes mandatory and desirable assessment criteria covering physico-chemical characterisation, sample preparation and (biological) testing parameters. this checklist facilitates the discrimination between high-and low quality publications and all positively evaluated literature is then fed into the dana knowledge base. accounting for the need to harmonise experimental practices, the dana team also developed a template for standard operation procedures (sop) to support careful scientific practice. validated protocols generated within the german bmbf-funded nanosafety research projects are presented together with results from the swiss ccmx project v.i.g.o. and available for download. another unique feature of the dana knowledge base is the integrated application-based database that provides a unique link between nanomaterials in real applications (e.g. environmental remediation or medical products) and their potential impacts/ toxicological effect(s) that can be easily accessed by the interested visitor. additionally, dana2.0 provides a list of faqs, a link platform with contact data to other information portals and the opportunity to directly pose questions to our experts via e-mail. dana 2.0 is also present on twitter, follow us @nano_info. background: particulate matter of combustion processes enhances cardio-vascular diseases and increases associated mortality rates. around 13% of total pm10 emissions are emitted by wood burners (uba 2006) . how wood combustion aerosols (particles and gasses) can affect human lung cells and how such cellular responses depend on the usage of different wood types and burners is widely unknown. methods: in an exposure chamber imitating the human respiratory tract human alveolar cells (a549) were exposed at an air-liquid-interface (ali) to gasses and particles of wood combustion aerosols. log wood of beech, birch and spruce was burnt in a conventional oven and compared to the combustion of wood pellets in a modern pellet burner. the combustion aerosols were diluted 1:40 and directly delivered to the exposure chamber. after 4h exposure the lung cells were lysed and rna was isolated. in an array based transcription analysis of the whole genome the effects of the aerosol exposures on lung cells was assessed. in parallel, physical and chemical parameters of the combustion aerosols were analyzed. results: the combustion aerosol of wood pellets contained less organic substances than the log wood aerosols, but was higher in its zinc content. genome-wide we found a higher number of regulated genes with combustion of pellets compared to combustion of log wood. the gas phase alone (filtered aerosol) showed comparable gene regulatory activity as the particle-containing total aerosol. aerosol from log wood burning induced mainly genes of the xenobiotic metabolism and cellular signaling. pellet aerosols additionally regulated apoptosis and dna repair processes. conclusions: modern pellet burners reach better combustion efficiencies than conventional log wood ovens, but their emissions seem to stress human lung cells stronger. one reason might be the higher zinc content of wood pellet aerosols. multiwalled carbon nanotubes (mwcnts) may pose as a risk similar to asbestos in causing cancer, notably mesothelioma, which is a malignant tumor originating from mesothelial cells. to identify molecular cues leading to mesothelioma development, we performed genome-wide transcriptome analysis using microarrays in primary human peritoneal mesothelial lp9 cells treated with two different tumor-inducing tailor-made mwcnts (rat model; rittinghausen et al. 2014 part fibre toxicol 11:59), or amosite asbestos at 3 µg/cm2 for 24 h. specifically, we determined how the transcriptomic changes of the highly tumorigenic mwcnt a would differ from another tumor-inducing mwcnt with albeit lesser potency (mwcnt d), long amosite asbestos as positive control, and milled mwcnt a as material control. initial analysis using bioinformatic tools, revealed 3788 significantly differentially regulated genes for mwcnt a, 1680 for mwcnt d, 145 for amosite, and 4 for milled mwcnt. further analyses with ingenuity pathway analysis comparing the two different mwcnt types and amosite, found common as well as exclusive biomarkers. interestingly, we identified many differentially regulated genes implicated in cellular senescence, a growth arrest in response to different stressors including dna damage, disrupted chromatin, and strong mitogenic signals. paradoxically, cellular senescence can represent both tumor suppression and tumor promotion mechanisms. more important, we found differential expression of genes associated with senescence-associated secretory phenotype (sasp) such as inflammatory cytokines, chemokines, proteases, and growth factors, which were manyfolds up-and down-regulated in mwcnt a, compared to mwcnt d and amosite. the mechanisms leading to mesothelioma induction by mwcnts are from far clear, but the key information emerging from the present transcriptomic data, together with our previously identified senescence markers, indicate that cellular senescence has a likely role. nanotechnology offers great advantages for the food industry despite its partly unknown risks, whose enlightenment is the main target of nanotoxicology. due to variability in terms of size, material, shape, surface texture and several endogenous influences, the toxicity of most frequently used and ingested nanomaterials is difficult to estimate. therefore, the aim of this study was the in vitro investigation of toxicological endpoints such as cell viability, dna integrity and the induction of apoptotic processes in human colon carcinoma cells (ht29). for this purpose ht29 cells were exposed for 24 hours with metal nanoparticles (gold, silver) and metal oxide nanoparticles (copper oxide, titanium dioxide, zinc oxide) in concentrations of 2-10 µg/ml. at first the cellular uptake of the nanoparticles by means of an icpms was determined. the influence of cell viability was demonstrated by the trypan blue staining and the mtt assay. the alkaline comet assay gave information about possible dna damages and the use of the repair enzyme formamidopyrimidine dna glycosylase (fpg) additionally allowed the detection of oxidized bases. the induction of programmed cell death was examined using by annexin v fitc assay. the icp-ms data showed a maximum particle content of 1.39 pg per cell for the used concentration range. the metal oxide nanoparticles resulted in a significant reduction of cell viability with a decrease up to 40 % after copper oxide and zinc oxide treatment. for metal particles, only for silver a reduced cell metabolism of about 50 % was detectable by the mtt assay. low genotoxic effects could be determined for silver nanoparticles (tail intensity about 12%; control about 6%), while for titanium dioxide the amount of oxidized bases was additionally increased (tail intensity about 20%; control about 6%) for concentrations above 8 µg/ml. induction of apoptosis was determined for silver particles (up to 24% early apoptotic and 20% late apoptotic cells) as well as for titanium dioxide and zinc oxide (10% each early apoptotic cells), whereby the most significant increase in late apoptotic cells was detected for zinc oxide (up to 90%). the results obtained in our studies indicate a clear particle-dependent influence on cell viability and apoptosis-triggering processes, depending on the used material or the concentration deployed, while only minor changes of dna integrity were detected. the evolution in the field of nanotechnology led to a variety of novel materials at the nanoscale. among them are different carbon materials like buckyballs, graphene nanoplates and carbon nanotubes (cnts). cnts are hollow carbon fibres with either one (sw) or multiple sidewalls (mw). mwcnts usually show a diameter of up to 100nm and can be several micrometres long. because of their nanoscale diameter cnt-uptake can take place directly through the plasma membrane of cells by the so called nanoneedle effect [1] . additionally cnts, like most nanomaterials, show a high surface to volume ratio and, because of their micro scale length, a potentially high loading capacity. these properties make cnts interesting for the potential use as drug delivery carriers (ddcs). mwcnts, produced via chemical vapour deposition with a diameter of 45nm and 16µm length, were used in three different forms, unmodified, acid oxidized (ox_cnt) and ground. cytotoxicity testing was performed in human umbilical vein endothelial cells (huvec). the cells were seeded in 48-well plates and exposed to doses of 1, 5, 10 and 25µg/cm² growth area of the respective cnt type for 24h. the wst-8 assay was applied for testing cell viability and the ldh cytotoxicity assay to identify potential damage to the plasma membrane and to calculate overall cytotoxicity. the results show that an increased oxidation time for the ox_cnts, in a h 2 so 4 /hno 3 mixture, leads to decreased cytotoxicity in huvec, compared to unmodified mwcnts. during the oxidation reactive oxygen groups are formed on the cnt surface [2] . these groups lead to a reduced hydrophobicity of the cnt surface which could be responsible for the decline in cytotoxicity. future investigations will include the toxicological analysis of mwcnts functionalized with polyethylene glycol (cnt-peg). the hydrophilic polymer peg will be covalently bound to the cnt surface and is expected to further reduce the cytotoxic effect. for these investigations different analytical methods will be used. among others, cell cycle analysis, the brdu assay, pathway arrays and qrt-pcr for the investigation of gene expression and cytokines will be measured. these methods will involve a co-culture model of huvec and human umbilical vein smooth muscle cells (huvsmcs) for a better approximation to the cellular in vivo situation. additionally the peg modified mwcnts will be tested for their loading capacity and efficacy with the anticancer drug doxorubicin for a potential use as an intravenous drug delivery carrier in vivo. although aluminium is one of the most common elements in the biosphere, up to now little is known about its impact on human health. aluminium and its chemical derivatives are highly abundant in food, food contact materials and consumer products what leads to an exposition via the gastrointestinal tract (gi tract), the lung and via skin contact. recently, aluminium is hypothized to cohere with cancer and neurodegenerative disorders. lately, due to an increasing attentiveness on this topic, limiting values for food additives have been tightened by the eu commission. however, cellular effects of aluminium and especially aluminium-containing nanomaterials, are in the focus of our research activities, for example in the international solnanotox project. we established an in vitro simulation system of the gi tract, where nanomaterials undergo the different physiological, chemical and proteinbiochemical conditions of saliva, gastric juice and the intestine. the artificially digested nanomaterials, as well as soluble aluminiumchloride as ionic control substance, were subjected to several analytical and biochemical methods to characterize their change of appearance and their cytotoxic effects on intestinal cellular models. we observed the fate of the nanomaterials during typical ph-values of saliva, gastric and intestinal juice with dynamic light scattering measurements and icp-ms in the single particle mode. after observable disappearance at ph 2 the particles recovered in the simulated intestinal fluid. the simulation of the gi tract, mainly the change of ph settings, may lead to a certain chemical activation of aluminium that can increase bioavailability in the intestine after oral uptake of aluminium-containing food products. in vitro assays like ctb, mtt and cellular impedance measurements showed that there were no acute cytotoxic effects measurable after a period up to 48h after incubation, comparable to undigested particles. in contrast, high amounts of aluminium ions showed additional effects on cell viability compared to non-digested aluminium ions. although toxicological potential of al ions to healthy tissue appears to be low, increased hazardous potential cannot be ruled out to pre-damaged tissue and can have a relevance for special consumer groups with for example chronical intestinal inflammation or dietary eating behavior combined with high exposure to al-containing food products. in the eu, there is a strong need for solutions to substitute halogenated flame retardant (hfr) additives, employed in the fabrication of fr thermoplastic and thermoset materials. these materials are used in diverse commercial products, applications and markets, such as electrical/electronic devices, low-voltage wires or household appliances. the phoenix project, funded by the european union 7 th framework program (grant agreement no. 310187), therefore investigates e.g. several tailor-made, nano-layered hybrid particles as alternatives to hfr additives. considering "safer-bydesign" strategies, potential fr nanomaterials (nm) were physico-chemically characterized (e.g. particle size distribution, zeta potential) and screened early in development for their (geno)toxic and pro-inflammatory potential to timely reject nm with high health hazard. as inhalation is the most important exposure route for nm, lungrelevant cells were used as in vitro screening models. to better enable detection and differentiation of the biologic effects of the most promising nm, screening was started with primary rat lung alveolar macrophages (am; cells of first contact for inhaled nm), at a high concentration of 50µg/cm 2 (24h of incubation), using membrane damage (lactate dehydrogenase release assay), direct dna-damage (alkaline comet assay), and il-8 liberation (elisa) as primary endpoints, and quartz dq12 and al 2 o 3 as particulate positive and negative controls, respectively. in this screening system, biologically inert nm could be differentiated from more active ones. thereby, mg(oh) 2 nanoplatelets (mg1; mean lateral size: 1,5-2 µm; mean thickness: 15-20 nm) represented the least, and pristine few layers graphene nanoplatelets (gr1; mean lateral size: 2 µm; mean thickness: 3 nm; graphene layers: 8 ± 0.5) the most biologically active nm. clear concentration dependencies were detected for gr1 in follow-up experiments. mg1 and gr1 were further tested in other lung-relevant cell types, i.e. mrc-5 primary human lung fibroblasts and a549 lung adenocarcinoma epithelial cells. interestingly, mrc-5 cells were less sensitive towards biological effects of gr1, compared to am, whereas a549 cells showed nearly no effect, keeping in mind that lung epithelial cells are the target cells of lung tumor development. to test the hypothesis that the observed cellspecific differences in sensitivity might in part be based on cellular uptake, cells were exposed for 24 h to 12.5 or 25 µg/cm 2 of gr1 on chamber slides. slides were finally stained with dapi and analyzed by dark field microscopy. cells indeed demonstrated differences in uptake capacity and also showed unique pattern of cellular localization of gr1, i.e. with tight perinuclear agglomeration in am, a more scattered cytoplasmic distribution in mrc-5 cells and limited uptake in a549 cells. additionally, biological activity of the diverse nm seemed also to correlate with cellular uptake, as determined by light and dark field microscopy in am and mrc-5 cells. in conclusion, an am-based screening system was able to differentiate biological activity of diverse nm, with morphology, physico-chemical characteristics, and related cellular uptake most likely to be key for nm-and cell type-specific hazard. lung carcinogenicity and putative systemic effects of low-dose life-time inhalation exposure to biopersistent nanoparticles were examined in a chronic inhalation study performed according to oecd test guideline no. 453 with several protocol extensions. female rats (100/group) were exposed to cerium dioxide (nm-212, 0.1; 0.3; 1; 3 mg/m³) for two years; a control group was exposed to clean air. after one year exposure, 42 µg/lung was found in animals exposed to 0.1 mg/m³ and 2.6 mg/lung in animals exposed to 3 mg/m³. histological examination of lungs revealed several adverse and non-adverse effects in the lung. the non-adverse effects comprised accumulation of particle-laden macrophages in alveolar/interstitial areas and in the balt, particle-laden syncytial giant cells in the balt and bronchiolo-alveolar hyperplasia (alveolar bronchiolization). the adverse effects included (mixed) alveolar/interstitial inflammatory cell infiltration, alveolar/interstitial granulomatous inflammation, interstitial fibrosis and alveolar lipoproteinosis. the incidence and severity of the effects were concentration-related. alveolar lipoproteinosis was not observed at low concentrations of 0.1 and 0.3 mg/m³ ceo 2 . neither pre-neoplastic nor neoplastic changes were observed after 12-months exposure. a no observed adverse effect concentration could not be established in this study. the comprehensive histopathological examinations of lungs and other tissues will be finalized in 2017. this project is part of the eu project nanoreg. moreover, german federal ministry for the environment, nature conservation, building and nuclear safety, german federal institute for occupational safety and health, german federal environment agency funded this project. lung carcinogenicity and putative systemic effects of low-dose life-time inhalation exposure to biopersistent nanoparticles were examined in a chronic inhalation study performed according to oecd test guideline no. 453 with several protocol extensions. female rats (100/group) were exposed to cerium dioxide (nm-212, 0.1; 0.3; 1; 3 mg/m³) and barium sulfate (nm-220; 50 mg/m³) for two years; a control group was exposed to clean air. lung burdens and burdens in extrahepatic tissues were measured at various time-point. the two year exposure period was successfully terminated and 50 animals per dose group were examined for organ burden and histopathology. the remaining animals currently are kept exposure-free for maximally 6 additional months. up to two years exposure to both nanoparticles did not lead to body weight reduction compared to control animals. the mortality rates were in an acceptable range. macroscopically evident tumors were not detected after two years. the ceo 2 lung burdens were maximally 3.5 mg/g lung tissue at the highest exposure concentration of 3 mg/m³. in comparison, highest ceo 2 burdens in organs remote to exposure were liver and spleen with maximally roughly 1 x 10 -3 g/g tissue. in brain, maximum ceo 2 levels were 7x10 -6 mg/g lung tissue. baso 4 lung burdens were comparatively low (1 mg/g) within the first 13 weeks of exposure and steeply increased to 6 mg/g lung tissue after one year. the comprehensive histopathological examinations of lungs and other tissues will be finalized in 2017. the european centre for ecotoxicology and toxicology of chemicals (ecetoc) 'nano task force' proposes decision-making framework for the grouping and testing of nanomaterials (df4nano) that consists of 3 tiers to assign nanomaterials to 4 main groups, to perform sub-grouping within the main groups and to determine and refine specific information needs. the df4nanogrouping covers all relevant aspects of a nanomaterial's life cycle and biological pathways, i.e. intrinsic material and systemdependent properties, biopersistence, uptake and biodistribution, cellular and apical toxic effects. use (including manufacture), release and route of exposure are applied as 'qualifiers' within the df4nano to determine if, e.g. nanomaterials cannot be released from a product matrix, which may justify the waiving of testing. the four main groups encompass (1) soluble nanomaterials, (2) biopersistent high aspect ratio nanomaterials, (3) passive nanomaterials, and (4) active nanomaterials. the df4nano aims to group nanomaterials by their specific mode-of-action that results in an apical toxic effect. this is eventually directed by a nanomaterial's intrinsic properties. however, since the exact correlation of intrinsic material properties and apical toxic effect is not yet established, the df4nano uses the 'functionality' of nanomaterials for grouping rather than relying on intrinsic material properties alone. such functionalities include system-dependent material properties (such as dissolution rate in biologically relevant media), bio-physical interactions, in vitro effects and release and exposure. the df4nano is a hazard and risk assessment tool that applies modern toxicology and contributes to the sustainable development of nano-technological products. it ensures that no studies are performed that do not provide crucial data and therefore saves animals and resources. the the grouping decisions of df4nano for 24 nanomaterials were validated against grouping by results of existing in vivo data and demonstrated 23 concordant grouping decisions. serum concentrations in cell culture medium influence the effect of cerium oxide nanoparticles on human lung a549 cells: cytotoxicity, proinflammation, cellular uptake, and particle properties k. burchardt the wide use of cerium oxide (ceo 2 ) nanoparticles (np), e.g. as fuel additive and for industrial and biomedical applications, evoked intense studies to understand the effects those particles might have on living organisms. contradictory results of ceo 2 nanoparticle toxicity have been published as they depend on many variables like shape, size, diluent and others. in our work we used an in vitro model of ceo 2 nanoparticles and lung carcinoma cells to investigate the role of serum content in the cell culture medium on cellular toxicity, particle uptake, proinflammation and particle characteristics. proinflammatory ceo 2 np with an average diameter of 15-30nm and different concentrations were diluted in cell culture medium with different fetal calf serum (fcs) concentrations (0.01, 0.1, 1 and 10%) and were used to expose human lung adenocarcinoma cells (a549) for up to 24 hours. at 100µg/ml ceo 2 np showed little to no toxic effecton growth arrested a549 cells at fcs concentrations of 1% or below, but cell viability was decreased to about 80% in proliferating cells in cultures with 10% fcs. the proinflammatory effect of ceo 2 np was investigated through measurement of il-8 mrna expression after 3 hours and cellular il-8 secretion after 24 hours. the qrt-pcr showed that the expression of il-8 mrna in cells treated with 100 µg/ml ceo 2 np in 10% fcs medium was three times higher than in cells treated with lower fcs concentrations. this finding correlated with the cellular il-8 secretion, which showed a stronger increase by cells treated at 10% fcs. differences in cellular uptake of ceo 2 np was determined by fluorescence-activated cell sorting (facs) after 2 and 24 hours of exposition. after 2 hours, the cells treated with ceo 2 np in 10% fcs medium showed a lower mean granularity (∆gmean) as measure for cellular particle uptake than those with less fcs in medium. after 24 hours all probes showed about the same granularity. to examine the effect the fcs concentrations on ceo 2 np characteristics in cell cultures we used dynamic light scattering (dls) and phase contrast microscopy (pcm). dls measurements revealed an increasing hydrodynamic diameter of the particles with decreasing fcs concentrations (about 1030nm (10% fcs) to 4090nm (0.01% fcs)), which was correlated by an increasing particle agglomeration shown by pcm. our results show that the fcs concentration in cell culture medium has a direct or indirect impact on the cytotoxicity, the proinflammatory effect, the facs parameter for cellular particle uptake as well as on particle properties, which should be taken into account when designing, performing and interpreting in vitro experiments to investigate the toxicity of nanoparticles. toxic and inflammatory effects of shape-engineered titanium dioxide nanoparticles in nr8383 rat alveolar macrophages. knowledge about the contrasting toxicity of nanoparticles (np) of different chemical composition has steadily increased over the past decade. however, available literature often reveals considerable differences in effects within a specific type of nanomaterial. these contrasts have been contributed to different handling and testing protocols as well as to sample-specific differences in physico-chemical properties of np that could affect their mode of interaction with cells. within the nanometrology project setnanometro, the highly controlled generation and characterisation of a large set of shape-engineered tio 2 np allows us to investigate the potential role of subtle shape-and surface structure changes on np toxicity. as inhalation represents the most relevant uptake route of np, the nr8383 rat alveolar macrophage cell line was selected for in vitro toxicological testing. since oxidative stress and inflammation are considered as key biological pathways in nanotoxicity, we evaluated the expression of the oxidative stress marker genes heme oxygenase-1 (ho-1) and g-glutamylcysteine synthetase (g-gcs) as well as the pro-inflammatory genes interleukin (il)-1β, il-6, il-18 and inducible nitric oxide synthase (inos) by qrt-pcr. protein levels of il-1β and tumour necrosis factor-α (tnf-α) were measured by elisa. cytotoxicity testing of the tio 2 np by wst-1 assay overall revealed only minimal toxicity in comparison to sio 2 np which were used as reference material. ho-1 and g-gcs mrna analyses indicated that specific tio 2 np triggered a moderate induction of oxidative stress. il-6 was only induced after sio 2 treatment, whereas il-18 was not affected by any of the tested np. in contrast, various tio 2 np caused a significant induction of il-1β mrna expression. however, no significant induction of il-1β and tnf-α protein secretion was observed for any of the tio 2 np. the results obtained from these and ongoing investigations will be linked to the physico-chemical database as being developed for all tio 2 np within the setnanometro project, with the overall aim to build and model nano-structure activity relationships (nsar) for this widely applied type of nanomaterial. acknowledgements: the setnanometro project is supported by the eu-fp7 programme. specific types of tio 2 particles were obtained by solaronix (switzerland), evonik and cristal. potential of silver and silver nanoparticles to reduce n-acetyltransferase 1 (nat1) activity j. lichter 1 , b. blömeke 1 1 trier university, department of environmental toxicology, trier, germany humans are exposed to various kinds of engineered nanoparticles including silver, which is frequently used in consumer and biomedical product due to its bactericide properties. despite their widespread usage, knowledge about influences on cellular functions is still incomplete. n-acetyltransferase 1 (nat1), an enzyme which is ubiquitously expressed in human tissues, catalyzes the transfer of an acetyl group to its substrates and although its endogenous function is not clear yet, it is well known to be involved in the n-acetylation of arylamines. in addition nat1 enzyme activity is known to modulated by non-substrates including metals and certain nanoparticles, however, the influence of silver on nat1 has not been analyzed yet. to address whether human nat1 is a target of silver nanoparticles and released irons on protein, purified nat1 was exposed to silver ions (agno 3 ) and silver nanoparticles (ag10-cooh, average size 10 nm, carboxyl functionalized), and nat1 enzyme activity was analyzing the n-acetylation of the nat1 substrate para-aminobenzoic acid (paba). therefore, purified nat1 (1 ng/µl) was co-exposed for 20 min to paba (1 mm) and agno 3 or ag10-cooh (0.01, 0.1, 1, 10 and 100 µg/ml each), resulting in a nat1:silver relation of 1:0.01-100 (w/w). both, agno 3 and ag10-cooh inhibited the n-acetylation of paba in a concentration dependent manner. using equal amounts of silver and nat1 (w/w, 1 µg/ml) enzyme activity was reduced about 98±0.2% (agno 3 ) and 82±0.6% (ag10-cooh). the lowest concentration analyzed (0.01 µg/ml) reduced nat1 activity about 24±5% (agno 3 ) and 17±5% (ag10-cooh). fifty percent activity reduction was caused by 0.11 ± 0.01 µg/ml of agno 3 and 0.21 ± 0.04 µg/ml of ag10-cooh, which is 10-fold lower compared to the published ic50 values for other metal oxide nanoparticles (3-15 µg/ml). these data indicate that both chemical silver species are able to modulate paba acetylation. further studies will be performed to clarify whether silver ions and/or silver nanoparticles could affect the specific n-acetylation of arylamines in human cells. colloidal silver has been used in medicine for centuries and nanosilver is present in many consumer-related products. however, despite of intense research in the past few years, the potential of nanosilver to induce effects different from ionic silver in vivo and in vitro, is still under debate. in this study, we compared proteomic effects of nanosilver (agpure™) and ionic silver (silver acetate) in the kidney of male rats after repeated oral delivery in a rat 28-days toxicity study. in order to avoid overt signs of toxicity, silver was dosed moderately in amounts of 60 and 6 mg/kg body weight for nanosilver and corresponding amounts of silver acetate (9.3 and 0.93 mg/kg). accordingly, no pathologic effects, including results from clinical chemistry and hematology, were reported. kidney tissue protein crude extract was separated by 2-d gel electrophoresis and differentially expressed spots were identified by maldi-ms. 374 unique proteins, showing a log 2 ratio of ≤ -0.3 for downregulation and ≥ 0.3 for upregulation were identified in all treatment groups. protein lists were analyzed with ingenuity pathway analysis (ipa). when comparing effects of particulate and ionic treatments, similar alterations were indicated for canonical pathways associated with glycolysis, gluconeogenesis and tricarboxylic acid cycle. regarding inflammatory responses, stronger effects were derived for ionic treatments. for both types of silver exposures, changes of protein expressions were linked to changes of fatty acid metabolism and nrf2-mediated stress. mitochondrial dysfunction was highlighted for both nanosilver treatments only, as well as activation of the insulin receptor. in the top-scored network of the higher dose nanosilver treatment, upregulated 14-3-3 protein zeta (ywhaz) displays a central position. ywhaz, an important regulator of cell cycle and apoptosis, interacts with the insulin receptor and is well known to be envolved in many types of cancer. overall, both forms of silver treatment revealed similar patterns of affected cellular and molecular functions in rat kidney, supporting common and overlapping mechanisms of particulate compared to ionic silver. because of the widespread application of nanomaterials and the fact that for some nanomaterials effects on different organisms where shown, nanomaterials are still in focus of interest. moreover the fate of nps is only partiallyassessed over the lifecycle of products containing nanomaterials. while general toxicological properties of nps are well described in diverse in-vitro and in-vivo experiments, the distribution of these particles during the whole and complex process of waste incineration shows big knowledge gaps. in the "nanoemission"-project the entire route from the residual material via incineration, filtering of the exhaust gas up to a possible release into the environment are considered together with the toxicological evaluation of effects on humans and the environment. in these experiments the influence of thermal waste treatment on the toxicological profile of nanoparticles, contained in the waste, will be described. after a complete characterization of the two types of baso 4 -nps from two different manufacturers by scanning electron microscopy/ energy dispersive x-ray analysis (sem/edx), measurement of the specific surface area (bet) and dynamic light scattering (dls) we investigated the impact of the pure baso 4 -nps on primary cells (normal human bronchial epithelial cells (nhbec) and peripherial lung cells (plc)). both materials show statistically significant cytotoxic effects in the resazurin-assay (decreased viability below 40 % for 1 mg/ml after 72 h). in general the effects of both nps were almost similar. additionally the effects of the baso 4-nps were compared more in detail. uptake of the baso 4 was quantified by icp-ms after 24 h and 72 h as well as the release of ba-ions into the cell culture medium after centrifugal separation. the incubation with baso 4 of plc and nhbec to 0.1 mg/ml over 24 h and 72 h leads to ~200 µg baso 4 /1 mio cells. the uptake is dose dependent but not time dependent. the impact on the secretion of inflammatory cytokines was determined by bead-based multiplex-elisa flow cytometry. tnf-α, il-8 and il-33 could be detected in nhbec and plc after np incubation. the possible induction of apoptosis was measured by flow cytometry as well. first investigations showed no induction of apoptosis for both materials. the impact of both nps on the intracellular glutathione level was measured by hplc and showed a decrease of gsh after 72 h. summing up baso 4 -nps showed toxic effects in primary human lung cell cultures after 72 h for concentrations under 1 mg/ml. c3 exoenzyme from c. botulinum is an adp-ribosyltransferase that inactivates selectively rhoa, b and c by coupling an adp-ribose moiety. rho-gtpases represent a molecular switch integrating different receptor signalling to downstream transcriptional cascades that regulate various cellular processes, such as regulation of actin cytoskeleton, cell proliferation and apoptosis. previous studies with the murine hippocampal cell line ht22 revealed a c3-mediated inhibition of cell proliferation and a prevention of serum-starved cells from apoptosis (rohrbeck et al., 2012) . former results of studies on map kinase signalling indicated c3-induced modulations of downstream signalling modules. therefore, ht22 cells treated with 500 nm c3 for 48 h were applied for screening of the activity of 48 various transcription factors followed by luciferase reporter assays. five transcription factors namely sp1, atf2, e2f-1, cbf, stat6 were identified as significantly regulated in their activity. for validation of identified transcription factors, studies on the protein level of certain target genes were performed. western blot analyses exhibited an enhanced abundance of sp1 target genes p21 and cox-2 as well as a raise in phosphorylation of c-jun. in contrast, the level of apoptosisinducing gadd153, a target gene of atf2, was decreased. our results suggest that c3 is able to modulate the activity of transcription factors whose target genes are involved in the regulation of cell proliferation and apoptosis. via covalent binding of chemical compounds to dna, adducts can be formed. as a consequence, mutations may occur, which represent stages of chemical mutagenesis and carcinogenesis. the isolation of leucocytes represents an essential field of work within dna-adductomics of blood cells, in which adducts serve as markers of exposure for biological monitoring. peripheral mononuclear blood cells (pbmc) comprising monocytes and lymphocytes can be separated from other blood cells like granulocytes, erythrocytes (and dead cells) by isopycnic density gradient centrifugation of buffy coats (bc´s). bc´s are blood concentrates rich in leucocytes and thrombocytes, prepared from whole blood samples thorough removal of plasma and erythrocytes. for the experiments only bc´s from healthy donors were used. while erythrocytes contain no dna, lymphocytes, which constitute a subcategory of leucocytes, carry genetic information. a variety of commercially available fluids with a density of 1.077g/cm 3 , based on different polymers, exists. these materials form the gradient required for the centrifugation. furthermore, there are several methods available, which differ in the ratio blood vs. fluid, duration of the different work up steps, centrifugation parameters and others. the aim of this work was to compare the effectivity of the different fluids and optimize the workflow. for isolation of pbmc the fluid was put in a tube and then covered by a thin layer of blood. after centrifugation, five layers were obtained (figure 1 ). the interphase was removed carefully, washed and the cells were counted. the yield is expressed as percentage of isolated vs. total leucocytes in the bc, which was based on the leucocyte count of the whole blood sample. as separation fluids, ficoll® paque plus (ge healthcare), histopaque® (sigma aldrich) and lsm® (ge healthcare) were tested. no significant differences between the different fluids were observed. although dilution is often recommended in literature, it was found that dilution had no effect on the yield. similarly, using different fluids (rpmi or pbs) for the washing steps did not reveal any differences. increasing centrifugation speed from 400 to 500 xg resulted in higher yields. in general, large variations in yield (46.8% ± 17.1%) among the various bc´s arose. this result is due to the different parameters such as age, storage, leucocyte and erythrocyte counts of the different bc´s used. therefore, the test conditions were optimized using the same batch of bc. the results show that the low priced separation fluids are comparable in performance tothe more expensive ones. by the direct lamination with bc (without dilution) the wastage could be minimized, the yield increased and thus the isolation made more efficient. the franz cell is a well-established model in lots of skin research fields. we adapted the diffusion cell system to additives and contaminants of consumer products which are designated for skin contacts. we were aimed to simulate real exposure as realistic as possible. to meet requirements like longevity, haptic properties and factory costs, different polymers are used as the raw material of choice and modified by a variable number of additives in the majority of commodities. besides additives with a defined function such as plasticizers, stabilizers, colorants and vulcanization accelerators, contaminants as well as decomposition products or so-called non-intentionally added substances (nias) can be part of the material, among them potentially harmful substances. in a first step, polymers of consumer products like flashlights or tools have been characterized concerning additive composition. possible breakdown products were identified by means of gc-ms/ms or pyrolysis-gc-ms. we focused on analytes of toxicological relevance including antioxidants such as n-phenyl-2-naphthylamine (neozon d), which is suspected of causing cancer, and on degradation products like cresol and its derivatives (e.g., mesitol or 2-tert-butyl-4-methylphenol). subsequently, analytes of interest were brought into direct skin contact using porcine, human and artificial skin models in the franz cell chamber assay. the analytes were either followed up layer by layer using tape stripping or examined utilizing cryo sections. for visualization purposes, analytical evaluation has been completed by use of imaging techniques like he staining or atr-ftir (attenuated total reflectance fourier transform infrared) microscopy. the latter was used with intrinsic markers for tissue specific distribution. our project provides evidence for the potential of polymer components to overcome the natural epidermal barrier and, in part, to enter the viable layers of the epidermis. during skin contact with consumer products several substances migrate out of the matrix and penetrate the skin, among them substances which are hazardous to health such as neozon d. depending on its dose, the blister agent sulfur mustard (sm) may lead to painful erythema, blistering with complicated wound healing, pulmonary edema, pulmonary bleeding and temporary blindness. sm is listed as a schedule 1 chemical in the chemical weapons convention and thus its production, stockpiling and use is prohibited. sm still represents a serious threat for civilians and military forces, especially in asymmetric, terroristic and accidental scenarios. after exposure, the highly reactive molecule alkylates nucleophilic sites in endogenous biomacromolecules forming the characteristic and stable hydroxyethylthioehtyl (hete) residue. hence, bioanalytical methods targeting theses adducts in forensic post-exposure verification analysis are of high interest. herein, we present an optimized, accurate and comparably simple method to detect adducts of sm and human serum albumin (hsa) alkylated at its cysteine 34 -residue. since albumin extraction from human plasma is a time-consuming and expensive step of an established procedure, an alternative method for direct proteolysis of human plasma was developed. plasma samples were cleaved directly with pronase resulting in the alkylated dipeptide hete-cysteine-proline (hete-cp) which is detected by micro-liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (µlc-esi hr ms/ms). in order to optimize reproducibility and yield of proteolysis, kinetics were investigated for different kinds of plasma (edta-, citrate-and heparin-) as well as serum. two different mass spectrometers, a triple quadrupole system (4000 qtrap) and a hybrid quadrupole time-of-flight instrument (tt5600 + ), were compared. the latter one proved to be the more selective and sensitive system. the method was successfully applied to in vitro and in vivo samples of real cases of sm poisoning. organophosphorus compounds (op), which were originally intended to be used as pesticides to increase agricultural yields at the onset of the 20 th century, still represent a considerable threat to human health. by irreversible inhibition of acetylcholinesterase op lead to cholinergic crisis due to uncontrolled increase of acetylcholine in the synaptic cleft. finally, sudden death by respiratory failure may result if medical countermeasures are lacking. therefore exceptionally toxic compounds were designed und synthesized as chemical warfare agents (cwa), among which v-type nerve agents, i.e. vx, chinese vx and russian vx, belong to the most toxic artificial substances. recent events like the first gulf war, terrorist attacks in tokyo and the conflict in syria underline the need for ongoing and strict surveillance of cwa prohibition by the chemical weapons convention. unambiguous evidence of such substances (verification analysis) plays an important role with great political and legal impact. a variety of such bioanalytical methods have been established at the bundeswehr institute of pharmacology and toxicology in munich, which is accounted for medical chemical defence in germany. exhibiting quite short half-lives in vivo nerve agents can hardly be detected days or even weeks after exposure. accordingly, there is a great need for additional long-term biomarkers like specific protein adducts. consequently, the current work focuses on examination of adducts between nerve agents and human serum albumin (hsa) as its high abundance and stability in vivo provide relative ease of sampling. after incubation of hsa with v-type nerve agents in vitro the protein was subjected to proteolysis. subsequently resulting peptides were separated using microbore high-performance liquid chromatography (µlc) and detected on-line by modern high-resolution tandemmass spectrometry (hr ms/ms). this allowed unambiguous identification of already known phosphylated tyrosines as well as novel adducts between cysteine-proline dipeptides and the thiol-containing leaving group of v-type nerve agents. simultaneous detection of both biomarkers was realized by a new method, which was applicable even at the very low toxicologically relevant concentrations of v-type agents. therefore, this method represents a valuable and novel supplement of existing methods for verification. fatty acid esters of glycidol (glycidyl esters) are processing contaminants formed as byproducts of industrial deodorizing of plant fats or during other heating processes. following oral intake, glycidyl esters are mainly cleaved to release the reactive glycidol in the gastrointestinal tract. according to the national toxicology program (ntp), glycidol is carcinogenic, genotoxic and teratogenic in rodents. it is classified as probably carcinogenic to humans (iarc group 2a). the exposure assessment of the oral intake of glycidyl esters in humans is difficult, because the current data set for glycidyl ester contents in food is incomplete. we developed a method for the determination of the internal exposure to glycidol by mass spectrometric quantification of a hemoglobin adduct reflecting the total glycidol burden over approximately three months. a modified edman degradation was adapted for the cleavage of the valine residues from the n-termini of hemoglobin by fluoresceinisothiocyanat (fitc) (von stedingk et al. (2011 ) chem res toxicol 24, 1957 resulting in the formation of dihydroxypropyl-valine-fluorescein thiohydantoin (dhp-val-fth). the target analyte is purified with mixed-mode anion-exchange solid-phase extraction and analyzed by lc-ms/ms. a major advantage of the technique is the application to whole blood samples, which renders the time-consuming isolation of erythrocytes unnecessary. we synthesized dhp-d 7 -val-fth as an internal standard for the quantification of the glycidol adduct by lc-ms/ms multiple reaction monitoring. a limit of detection of 5 fmol per injection (5 pmol adduct/g hemoglobin) was achieved. the application of this method will possibly allow future monitoring of the internal exposure of glycidol in human studies. glycidol and 3-monochloropropane-1,2-diol (3-mcpd) are carcinogenic food contaminants, which are present in heat-processed oils and fats mainly in form of fatty acid esters. the risk assessment concerning human consumption of these substances is complicated by various reasons. for example, the data on the occurrence in food stuffs is incomplete. also, the amounts of the proximate carcinogens released from ester hydrolysis in the gastrointestinal tract in humans are not known. monitoring of the internal exposure would be an alternative strategy to support the assessment of possible health risks related to the intake of glycidol and 3-mcpd and their fatty acid esters. for short-term monitoring of the internal exposure, urinary metabolites are suitable biomarkers. we study the potential use of two different substances as descriptors of the oral intake of glycidol and 3-mcpd. the metabolite 2,3-dihydroxy mercapturic acid (dhpma) is generated following glutathione conjugation of both compounds. the second target analyte is 3-mcpd itself, which may also be formed from glycidol in the reaction with hydrochloric acid in the stomach. a method for the quantification of urinary 3-mcpd by gc-ms is currently developed. an lc-ms/ms multiple reaction monitoring technique was devised for the quantification of dhpma in urine samples with the isotope-labeled reference compound 13 c 2 -dhpma. the limit of quantification is 10 µg dhpma/l. related to creatinine, the analyte was detected to be in a relatively small concentration range in urine samples from humans. the average concentration in urine samples (n = 45) of one male volunteer collected over ten days was 154 ± 21 µg dhpma/g creatinine. a meal of a highly contaminated, commercially available frying fat (containing 1.1 mg of glycidol equivalents) did not lead to a visible increase of the urinary concentrations. the considerable background levels of dhpma in urine of humans and also in urine samples of other mammals support the hypothesis that dhpma may be also be formed from an endogenous c 3 -metabolite, as already reported by eckert et al. furfuryl alcohol is a common food contaminant formed by acid-and heat-induced dehydration from pentoses. it induced renal tubule neoplasms in male b6c3f1 mice and nasal neoplasms in male f344/n rats in a study of the national toxicology program (ntp). the neoplastic effects may originate from sulfotransferase (sult)-catalyzed conversion of furfuryl alcohol into the dna reactive and mutagenic 2-sulfoxymethylfuran. the incomplete data set of furfuryl alcohol contents in food does not allow estimating the human exposure. thus, we sought a method for the determination of the internal exposure. recently, the dna adduct of furfuryl alcohol n 2 -[(furan-2-yl)methyl]-2′deoxyguanosine was detected in specimen of human lung tissue. however, human biomonitoring of dna adducts has various disadvantages. for example, dna adducts are removed by various repair systems and human dna samples are usually not accessible in sufficient quantities. we decided to develop a biomarker for the internal exposure to furfuryl alcohol using blood proteins as dosimetric targets. bladder cancer (bc) is a smoking and occupation related disease showing a substantial genetic component. though the prognosis is generally good, a major problem is the frequent relapses affecting about half of the patients. n-acetyltransferase 2 (nat2) is well-known to modulate bc risk in persons heavily exposed to carcinogenic aromatic amines. we aim to investigate the impact of nat2 genotypes, in particular, the ultraslow genotype, on relapse-free time after first diagnosis in 772 bladder cancer cases. we used follow-ups of three case-control studies from lutherstadt wittenberg (n=207), dortmund (n=167) and neuss (n=398). nat2 was genotyped using seven characteristic polymorphisms (rs1801279, rs1041983, rs1801280, rs1799929, rs1799930, rs1208, rs1799931). haplotypes were reconstructed using phase v.2.1.1. we compared slow to rapid acetylators. additionally, we differentiated between the most frequent slow nat2*5b/*5b and *5b/other slow haplotypes as well as between the ultra-slow *6a/*6a and *6a/other slow haplotypes compared to rapid acetylators. chi-square tests used to check the frequency of relapses in ultra-slow, slow and rapid acetylators. genotype differences in relapse-free time up to 5 yr after first diagnosis of bc were analysed using cox proportional hazards models adjusted for age, gender, smoking habits, invasiveness and study group. a total of 371 (49%) patients showed a relapse within the first 5 yr after bc diagnosis. slow acetylators show a higher frequency of relapses than rapid acetylators (51% vs. 46%, p=0.154). this frequency is even higher in ultra-slow acetylators (61%, or=1.81, p=0.019) but not in slow *5b/*5b genotypes (49%, p=0.609). ultra-slow acetylators had a significantly shorter relapse free time within 5 yr after bc diagnosis than rapid acetylators (median 0.66 vs. 0.94 yr, hr=1.57, p=0.009). this trend was not that pronounced in all slow acetylators combined (0.78 yr, hr=1.19, p=0.127) nor in the subgroup of nat2*5b/*5b genotypes (0.79 yr, hr=1.20, p=0.255). the effect of ultraslow nat2 is even more pronounced in smokers (hr=1.78, p=0.003) but absent nonsmokers (hr=0.89, p=0.781). ultra-slow nat2 seems to be associated with a higher recurrence risk and a shorter relapse-free time, especially in smokers. slow nat2 in general seems to have less impact on recurrence. aalborg university, department of biotechnology, chemistry and environmental engineering, aalborg, denmark xenoestrogens with the potential for endocrine disruption like bisphenol a (bpa) may bind to the estrogen receptors (ers) and modulate expression of er target genes mimicking the natural ligand 17β-estradiol (e2). the potential for endocrine adversity is still predominantly assessed in vivo as existing in vitro tests have only limited value for an exposure-based risk assessment. thus, the development of reliable bioassays for the detection of endocrine disruptors is one of the paramount challenges faced by modern toxicology. a targeted metabolomics approach in mcf-7 cells treated with e2 or bpa revealed potential biomarkers for the estrogenic potency of the studied compounds. among them were several phosphatidylcholines and amino acids. we further addressed proline levels that were found to be strongly increased. investigations of proline levels over time showed a clear proliferation correlated concentration dependency after both e2 and bpa stimulation. furthermore, sirna knockdown experiments suggested an influence of the oncogenic transcription factor myc and the dependency of erα activation on the estrogen-mediated proline increase. our study demonstrates metabolomics as a powerful tool for biomarker identification and hypothesis generation. the results could be used further to develop bioassays for the detection of endocrine disruptive chemicals. children are considered to be more sensitive to most chemicals than the general population due to a variety of factors, including dynamic growth and developmental processes as well as physiological, metabolic and behavioral differences [1]. however, only a few data are available on the magnitude of preschool children's exposure to most chemicals present in many consumer products. several of these chemicals are linked to endocrine disrupting effects in animal studies and are suspected to have also adverse effects e.g. on development and function of the reproductive organs as well as on neurological and behavioral development in humans. among of the chemicals that have been a major focus of discussion in the last years are phthalates, dinch, parabens, bisphenol a, and triclosan due to their suspected health effects. therefore we aimed to investigate exposure levels to metabolites of different phthalates and parabens as well as to bisphenol a and triclosan in urine samples collected from preschool children in german day-care centers from north rhine-westphalia (lupe iii; 2011/12). urine specimens from children aged from 20 to 80 months from 23 different day-care centers were analyzed. in total, 253 preschool children were recruited with mean age of 54 months. our study results show that nearly all children (>95 %) of the study population had urine concentrations equal to or above the limit of quantification for five most common phthalates metabolites (mnbp, mibp, 5oh-mehp, 5oxo-mehp, 7oxo-minp), for bisphenol a and methylparaben. triclosan was detected in 16 % of the study population. in general, the median urinary concentrations of the above mentioned phthalate metabolites were about 5-50 µg/l in spot urine samples. the highest amount among the phthalate metabolites was observed for mibp with maximal values of about 1000 µg/l. median urinary concentration for methylparaben and bisphenol a were about 48 µg/l and 2 µg/l respectively. the maximum methylparaben, bisphenol a and triclosan level found were 1770 µg/l, 72.4 µg/l and 55,6 µg/l respectively. in conclusion, our study shows a widespread exposure of young children to various phthalates, parabens and bisphenol a in north rhine-westphalia, germany. a follow-up human biomonitoring study (2014/2015) has finished recruitment and is in the process of analyzing data. polycyclic aromatic hydrocarbons (pahs) represent a large group of organic compounds that are common environmental contaminants. they are formed by incomplete combustion of organic matter such as coal or crude oil and are often known to be carcinogenic, mutagenic and teratogenic. the acute toxicity of pahs is rather low, but because of their stability and lipophilic character those compounds can accumulate in the human body and cause severe chronic effects. additionally pahs may enter the food chain when preserving meat or fish by exposure to smoke. in the european union maximum levels of 2 µg/kg benzo[a]pyrene and 12 µg/kg as the sum of benzo[a]pyrene, benz[a]anthracene, benzo[b]fluoranthene and chrysene in the meat of smoked fish and smoked fishery products are set, respectively. smoked fish is often handmade in small fishery stores in schleswig-holstein, where self caught fish is prepared in smoke houses. this technique implies the danger of pahs to accumulate in smoked fishery products above allowed maximum levels. here, we report our findings of pahs in smoked fishery products bought in local convenience and fishery stores in schleswig-holstein and give a brief overview about actual contaminant levels. hplc with fluorescence detection was used to determine the quality and quantity of several toxic pahs in smoked fishery products made locally. pahs may constitute risks for human health when exposed to hazardous levels and therefore it is important to have knowledge about given contaminant levels. colorectal cancer (crc) is one of the most frequent cancers worldwide and is tightly linked to dietary habits. epidemiological studies provided evidence that the intake of red meat is associated with an increased risk to develop crc [1]. red meat contains high amounts of heme iron, which is thought to play a causal role in tumor formation. the underlying molecular mechanism, however, remains elusive and may involve increased cell proliferation and dna damage induction by heme-iron. in this study, we set out to analyze the genotoxic and cytotoxic effects of heme-iron in human colonic epithelial cell lines. we used hemin (fe iii ) as commercially available heme source, which was compared to inorganic iron chloride (fecl 3 ). first, the time-dependent internalization of hemin and fecl 3 into hct116 cells was determined using icp-ms/ms analysis. treatment of cells with inorganic iron resulted in a maximum of intracellular iron content after 1 h at all doses tested, while hemin particularly at high doses caused an iron accumulation up to 24 h. hemin catalyzed the formation of reactive oxygen species (ros) in a dose-dependent manner in caco-2 and hct116-cells as shown by flow cytometry. consistent with this finding, hemin dose-dependently induced the oxidative dna lesion 8-oxoguanine (8-oxog) as revealed by slot blot analysis and fpg-modified alkaline comet assay. using a pharmacological inhibitor of mutt homologue 1 (mth1), which protects the nucleotide pool by hydrolysis of 8-oxogtp, 8-oxog dna adduct levels in hemin-treated cells were further enhanced. in contrast, inorganic iron hardly affected the cellular ros level and only slightly increased oxidative dna damage. subsequently, a time-and dose-dependent activation of the dna damage response (ddr) by hemin was shown in hct116 and caco-2 cells using western blot analysis, which was followed by a reduction in cell viability at high doses after 72 h. finally, the cytotoxic effects of hemin and inorganic iron were tested using an ex vivo model of intestinal crypt organoids. preliminary results indicate that hemin is highly cytotoxic in organoids, whereas inorganic iron does not impair their viability. taken together, this study demonstrated that hemin induces oxidative stress and dna damage, resulting in the activation of the ddr and subsequent cytotoxicity. in contrast, inorganic iron displayed only modest effects. further in vivo studies using dna-repair deficient and proficient animals will shed light on the contribution of specific dna lesions to hemeassociated colorectal carcinogenesis. red and processed meat consumption is known to be a crucial risk factor in the development of colon cancer, which is one of the most common cancers worldwide. a diet rich in red and processed meat increases n-nitrosation within the colon leading to an increase in endogenously formed nitroso compounds. these compounds are able to alkylate dna of gastrointestinal tract cells, resulting in the formation of dna adducts such as -meg is repaired by mgmt, the potential of this enzyme to repair o 6 -cmg in cells is not well characterized. additionally, adenomas containing a k-ras gc-at transition mutation have lower mgmt levels than adenomas without this mutation. therefore, the aim of this study is to determine the role of mgmt in protecting colorectal cells from genotoxicity by repairing o 6 -cmg adducts. for this purpose, an mgmt-deficient non-transformed human colon epithelial cell line was established by stable transfection via rna interference to inhibit the expression and therefore the activity of mgmt. the transfected cell line was analyzed for complete mgmt gene silencing by activity and expression analyses and cell characterization. results confirmed that there was neither expression nor activity for mgmt in the transfected cells, and the cell characterization data showed that mgmt deficiency did not lead to differences in growth behavior or cell morphology or malignant cell transformation. cytotoxicity experiments performed in the transfected and nontransfected cell lines by treatment with dna alkylating agents suggest that the mgmtdeficient cell line is more sensitive to dna alkylating agents than the non-transfected cell line. these results were also supported by dna damage analysis via comet assay. asarones are secondary plant constituents mainly occurring in acorus calamus l. and guatteria gaumeri. the essential oil of the rhizome of acorus calamus l. is used for flavoring of food and alcoholic beverages. the concentration of β-asarone (ba) in these oils varies between 0.3% and 95%. the bark of guatteria gaumeri, which is rich in αasarone (aa), is used as cholesterol-lowering drug and to treat gallstones in traditional mexican medicine. both, aa and ba are carcinogenic in rodents and mutagenic in the ames test after metabolic activation and thus classified as genotoxic carcinogens. [1, 2] previously, the major metabolites in microsomal incubations with aa and ba were identified and synthesized in our laboratory. side-chain epoxides, the corresponding diols, side-chain alcohols and aldehydes were identified as the major metabolites of aa and ba. [3] the investigation of the mutagenic potency in the ames fluctuation test showed positive results in the salmonella strain ta 100 for aa and ba with metabolic activation and for aa-and ba-epoxide without metabolic activation. while it is known that epoxides are reactive against nucleophiles we set up the hypothesis of dna-adduct formation by epoxides to explain the mutagenic effect. this adducts are currently isolated, characterized by nmr-spectroscopy and used to quantify adducts in cells. at the same time primary rat hepatocytes were incubated with non-cytotoxic concentrations of aa and ba for 24 h. after harvest and lysis of the cells, the dna was isolated by chloroform/phenol extraction and enzymatically hydrolyzed. [4] the residual hydrolysates will be used to identify the dna-adducts formed in cells and to determine the adduct formation rate. preliminary experiments indicate that both, aa and ba also form dna-adducts in intact cells in a concentration-dependent manner. [1] göggmann, schimmer (1983) . mutagenicity testing of α-asarone and commercial calamus drugs with salmonella typhimurium. mutation research. 121, [191] [192] [193] [194] wiseman et al. fatty acid esters of 3-chloro-1, and of 2-chloro-1, are food process contaminants that are formed, e.g., during refinement of vegetable oils. after ingestion, the esters are hydrolyzed in the gut, thereby releasing free 3-mcpd and 2-mcpd. 3-mcpd has been identified by the international agency for research on cancer (iarc) to be possibly carcinogenic to humans (class 2b) and is therefore in the focus of food safety authorities. to elucidate the toxicological properties of these compounds at the molecular level (mode of action) a proteomic study was conducted in which 10 mg/kg b.w./day of either 3-mcpd or 2-mcpd were orally administered to rats over a period of 28 days. total protein was extracted from different tissues of the animals and separated via twodimensional gel electrophoresis (2de). among others, the redox sensor protein dj-1 was identified to be deregulated in liver, kidney, testis, and heart of rats treated either with 3-mcpd or 2-mcpd. up to six different isoforms of dj-1 were identified by 2de-western blot analysis, all of them having the same molecular weight but different pi values, indicating protein modifications of low molecular weight but with a strong impact on the protein charge. treatment of the animals with either 3-mcpd or 2-mcpd predominantly led to a shift of the abundance between two dj-1 isoforms in the rat tissues. this effect was more pronounced with 3-mcpd compared to 2-mcpd. mass spectrometric analysis of these two isoforms identified an oxidation of a conserved cysteine residue (cys106) of dj-1 to a cysteine sulfonic acid to be the protein modification induced by treatment of the rats with either 3-mcpd or 2-mcpd. dj-1 is discussed to participate in a number of biological processes such as proteolysis, protein folding, or redox regulation. oxidation of cys106 was shown to be crucial for the activity of dj-1, and the irreversible oxidation of cys106 to a cysteine sulfonic acid as observed in the present study was shown to result in a loss of protein function and was correlated with diseases related to oxidative stress such as parkinson's disease. thus, the potential impact of 3-mcpd and 2-mcpd on cellular oxidative stress and on associated neurodegenerative diseases has to be considered in the ongoing risk assessment of these food contaminants. pyrrolizidine alkaloids (pa) are secondary plant compounds and widely spread in plant kingdom. humans can therefore be regularly exposed via direct or indirect contamination of food, like herbal teas, honey, wheat or salad. 1,2-unsaturated pa are known for their potentially harmful effects. an acute intoxication may cause venoocclusive disease leading to hepatomegaly, ascites as well as liver hardening resulting in high mortality. on the other hand, chronic pa intoxications due to regular consumption of small amounts of pa are characterized by hepatic necrosis, fibrosis and cirrhosis. an initial whole genome transcriptome study in hepatocytes revealed that molecular pathways related to cancer development, cell cycle regulation and cell death are regulated by the four structurally different pa echimidine, heliotrine, senecionine and senkirkine in short-term exposure (24h). additionally, lipid and bile acid metabolism was affected. however, the uptake of pa by food is more likely to be continuous than singular. therefore, the aim of this study was to investigate molecular effects of longterm exposure (14d) comparatively to short-term exposure (24h) in the hepatoma cell line heparg with the four structurally different pa. in this context we analyzed selected cellular parameters like cytotoxicity and morphology. in contrast to short-term exposure, structure-and concentration-dependent cytotoxicity was found for the long-term exposure (sn>he>em/ski). furthermore, obvious morphological changes such as destructuration and perforation of the heparg cell monolayer were seen after 14d of incubation. based on these findings, a possible induction of apoptosis or necrosis by pa was examined. effects of long-term exposure to pa on gene expression were investigated for a set of genes found to be regulated in the short-term whole genome transcriptome study. the identified regulation of gene expression was confirmed for both terms, with the strongest regulation for cyp7a1 (down-regulation), a gene involved in cholesterol metabolism. therefore, the effects of pa on various parameters related to cholesterol metabolism were analyzed, showing pa effects on cholesterol levels and bile acid secretion. short-term exposure to pa did not affect cell viability. however, repeated doses of pa resulted in severe effects on hepatic cells, concerning viability and morphology. at the mrna level, short-and long-term incubation with pa seem to affect a wide range of signaling pathways. in conclusion, we show for the first time that heparg cells can serve as an in vitro model for hepatotoxicity following chronic intake of pa. shiga toxin-producing e. coli (stec) strains cause a diversity of enteric symptoms in humans, ranging from mild diarrhea to severe diseases such as the hemolytic uremic syndrome (hus). hus is a life threatening disease with microvascular endothelial damage in the gastrointestinal tract and the kidneys, which often leads to haemolytic anemia, thrombocytopenia and acute renal failure. shiga toxin plays a major role for the pathogenesis of hus but the subtilase cytotoxin, which was identified during an hus outbreak in 1998 in stec strains, might contribute as an additional potent enterotoxin. like shiga toxin, subab is an ab 5 protein toxin. the pentameric binding/transport subunit (subb) delivers the enzymatic active subunit (suba), a protease, into the endoplasmic reticulum (er) of human target cells. in the er, suba cleaves the chaperone bip/grp78, which results in cell stress and caspase 3/7dependent cell death. recently, we discovered that higher concentrations of suba (10 mg/ml) causes cytotoxic effects in human epithelial cells (hela, in the absence of subb. the cytotoxic effects were investigated in hela cells in more detail. here, suba binds in a concentration dependent manner to the cell surface and induces dramatic morphological changes as well as caspase-dependent cell death [1] . in contrast to hela and caco-2 cells, cho fibroblasts did not respond to suba. currently, further cell types including macrophages are tested for suba effects and the molecular mechanisms underlying the cytotoxic effects caused by suba and the cell type selectivity are investigated. although there are strong cytotoxic effects caused by suba on some human epithelial cells in vitro, the situation in vivo and the pathogenic relevance of the newly observed suba effect are not known. thermal treatment of fat-containing foodstuff in the presence of salt leads to formation of 3-mcpd and its esters. high amounts of 3-mcpd esters detected in food raised toxicological concern. recent studies revealed that food may also contain significant amounts of structurally related 2-mcpd and its fatty acid esters. toxicological studies indicate genotoxicity in vitro and a carcinogenic potential of 3-mcpd, pointing to kidney and testes as main target organs. 3-mcpd esters were shown to cause similar, but milder effects. few unpublished data exist for 2-mcpd, showing similar organ toxicity compared to 3-mcpd and identifying heart as additional target organ. no such data exist for 2-mcpd diesters. in consequence, further toxicological data were required in order to complete risk assessment for 3-mcpd, 2-mcpd and their esters. hence, an oral 28-days study was performed in male rats in order to fill data gaps and provide essential information for risk assessment. a proteomic analysis was included in order to compare molecular effects induced by 3-mcpd and 2-mcpd and its dipalmitic ester in rat liver, kidney, testes and heart. in order to avoid overt toxicity, moderate doses of 3-mcpd + 2-mcpd (10 mg/kg body weight), and 2-mcpd-dipalmitate (53 + 13.3 mg/kg body weight) were applied. accordingly, no pathologic effects were reported. here, we present proteomic results obtained after analysis of heart tissue. after separation of heart tissue protein crude extract by 2-d gel electrophoresis , differentially expressed spots were identified by maldi-ms. a total of 270 unique proteins deregulated at a log 2 ratio of ≤ -0.5 for downregulation and ≥ 0.5 for upregulation at p ≤ 0.05 were identified. comparing deregulated spots induced by different treatments revealed that a higher number of spots was deregulated by 2-mcpd versus 3-mcpd. dipalmitate treatment even caused more deregulation than 2-mcpd. upregulated cytochrome b-c1 complex subunit rieske (ucri) and downregulated acetyl-coa acetyltransferase (thil) were among the top deregulated proteins after 2-mcpd and 2-mcpd dipalmitate treatment. pronounced upregulation of respiratory chain protein ucri, not deregulated after 3-mcpd treatment, indicates an effect on energy metabolism. downregulation of thil, involved in ketone body metabolism, was only weakly affected after 3-mcpd treatment. protein dj-1 (park7), a multifunctional redoxsensitive chaperone and protease protecting the cell against oxidative stress, was significantly downregulated after treatment with 3-mcpd and the higher dose of the 2-mcpd diester. tropomyosin beta chain (tpm2) was commonly upregulated after 3-mcpd and 2-mcpd treatments, possibly indicating a tgf-beta induction of actin stress fibers. for rat heart, data show that similarities but also some significant differences of 2-mcpd-and 2-mcpd dipalmitate-induced proteomic changes exist compared to 3-mcpd, indicating different mechanisms of toxicity for this structural analogues. oxidation products (oxy) of cholesterol (chol) such as 7α-hydroxy-chol (7α-ho-chol), 7β-hydroxy-chol (7β-ho-chol), 7-keto-chol (7-o-chol), 5,6α-epoxy-chol (α-epoxy-chol) and 5,6β-epoxy-chol (β-epoxy-chol) are formed by autoxidation of chol and are discussed as biomarkers for inflammation and oxidative stress in human tissues to be used in the identification of risk factors for disease. however, oxy-chols are also present in processed foodstuff where 7β-ho-chol (milk) and 7-o-chol (fish, meat, and egg) tend to represent the main oxychols, whereas epoxy-chols, are generally minor constituents. thus, levels of oxychols in tissues may result from both endogenous formation as well as dietary exposure. since quantitative profiles of oxy-chols have not been determined in human adipose tissues yet, levels of oxychols and chol were quantified using gc-ms/ms (internal standards: deuterated oxychols) and gc-fid (internal standard: 5α-cholestan-3β-ol), respectively in breast adipose tissues of 48 healthy women undergoing mammoplasty. furthermore, tissue levels of 25 fatty acids in adipose tissues were determined by gc-fid (internal standard: undecanoic acid) to assess relative levels of pentadecanoic acid, docosahexaenoic acid and elaidic acid, indicative for consumption of dairy products, fish, and processed fats, respectively. all oxychols were detected in all breast adipose tissues. the most abundant oxychol was β-epoxy-chol (median: 0.36 nmol/g tissue, range: 0.06-1.55 nmol/g), followed by α-epoxy-chol > 7-o-chol > 7α-ho-chol> 7β-ho-chol (0.009 nmol/g, range: 0.002-0.11 nmol/g). tissue levels of chol (2.8 micro mol/g, range: 1.88-3.87 micro mol/g) did not correlate (spearman's rank analysis) with that of epoxy-chols and correlated even negatively with that of 7α-ho-, 7β-ho-, and 7-o-chol (r = -0.29, p=0.024-0.044) median oxychol/chol ratios ranged from 0.0119 (5, to 0.0003 (7β-ho-chol). 7-o-chol and 7-ho-chols correlated strongly with each other (r=0.81-0.91, p oxy-chol levels did not correlate with relative amounts of pentadecanoic acid and docosahexaenoic acid, whereas total oxychol, 7β-ho-chol, and β-epoxy-chol levels correlated with relative amounts of elaidic acid (r=0.30, 0.30, and 0.31, respectively, p=0.037, 0.034, 0.028, respectively) . no correlations of oxychol levels, individual or total oxychol/chol ratios with age or bmi were observed. taken together, oxychol profiles in breast adipose tissues were different from that usually present in food but could be affected by dietary habits. classification and labelling of hazardous substances and hazardous consumer products (1) has proven to be a very efficient tool for risk communication. consumer products, such as glue, varnish, or washing and cleansing products need to be classified and labelled if they contain dangerous ingredients that render the mixture hazardous. personal care products, however, need not be classified and labelled if they contain dangerous substances above the thresholds for classification. they are excluded in the clp regulation. what would happen without this exception? when i applied the criteria for classification and labelling to a selection of cosmetic product formulas in a conservative approach, most products would have to be labelled and classified, mainly due to hazardous effects to the eye and to the skin (2) . benefits of personal care products can go along with unwanted properties such as the hazards for the human health, and consumers should be informed about them (3) . risk communication for every day products like personal care products should be clear, easily and quickly understandable. according to the cosmetic regulation (4) the ingredients must be listed on the containers. it must be questioned whether the listing of the ingredients without hazard pictograms on the products could be considered as a clear, easily and quickly understandable risk communication instrument (3). the results show that it is urgent to inform consumers better about the potential dangers of personal care products, because cosmetics need to be applied even with more care than any other consumer product. it is strongly recommended to delete the exception provision in the clp regulation for personal care products. the infochemical effect describes that anthropogenic substances can interfere with the chemical communication and influence organisms so that they perceive their chemical environments differently (1,2). infochemicals play a role in life history, habitat search, food related aspects and survival which shows that disturbed communication (infodisruption) could affect population vulnerability at various decisive points (3). the classical ecotoxicological standard tests do not allow to observe the infochemical effect. systematic analyses are needed to find out more about the relevance of this new chapter in ecotoxicology for natural ecosystems. the first crucial step is to select suitable test substances. repellents (substances used to keep away target organisms, e.g. invertebrates such as midges or fleas via olfaction) enter surface waters mainly indirectly via wastewater discharges from sewage treatment plants or directly by being washed off from the skin and clothes of bathers. there are various indications that repellents which are not toxic for aquatic animals could induce effects like organismic downstream drift of non-target species (downstream dislocation of e.g. crustacean and insect larvae in streams). in a literature study, three repellents were identified to be suitable test compounds for proof of concept of the infochemical effect. deet (cas 134-62-3), icaridine (cas 119515-38-7) and ebaap (cas 52304-36-6) (4). these substances are investigated in new test designs in a subsequent experimental part of the project which are designed to detect and quantify the infochemical effect. persistent, bioaccumulative and toxic (pbt) substances or very persistent and very bioaccumulative (vpvb) substances are compounds with hazardous properties. the non-biodegradability (persistence) and high accumulation in organisms (bioaccumulation) may elicit long-term adverse effects in the environment. persistent and bioaccumulative substances concentrate in the environmental compartments (water, sediment, soil, air) and can be distributed in the food chains. ecotoxicological effects are strengthened by bioaccumulation and appear often in remote areas like marine and polar regions. in the framework of pbt assessment, contrary to the risk assessment, the substances are evaluated regardless of the emission into the environment. an evaluation of medicinal active ingredients under assessment in the german federal environment agency (uba) identified less than 10% as potential pbt candidates. due to data lacks in many cases a definite pbt classification is not possible. the poster presents results of an extensive literature research on the global occurrence of pharmaceuticals in the environment. data on measured environmental occurrences from more than 1,000 international publications have been transferred to a database, with more than 120,000 entries. according to the database, pharmaceuticals have been found in the environment of 71 countries worldwide in all five un regions. most published data are for the compartments surface water and sewage effluent; less information is available for groundwater, manure, soil, and other environmental matrices. more than 600 active pharmaceutical substances (or their metabolites and transformation products) have been detected in the environment. most findings have been published for industrialized countries. monitoring campaigns are increasingly being conducted in developing and emerging countries. these have revealed the global scale of the occurrence of pharmaceuticals in the environment. for example, diclofenac, a non-steroidal inflammatory drug, has been detected in the aquatic environment in 50 countries worldwide. a number of globally marketed pharmaceuticals have been found in both developing and industrialized countries. the available ecotoxicological information indicates that certain pharmaceuticals pose a risk to the environment at measured concentrations. options for cooperative action to address the risk of are also presented. the aim of the research presented was to support the discussion of the proposed emerging policy issue pharmaceuticals in the environment under the strategic approach to international chemicals management (saicm), which is a global initiative of united nation environment programme (unep). the european chemicals' legislation reach aims to protect man and the environment from substances of very high concern (svhc). chemicals with (very) persistent, (very) bioaccumulative and toxic properties (pbt and vpvb compounds), substances that are carcinogenic, mutagenic and toxic to reproduction (cmr compounds), as well as chemicals of comparable concern like endocrine disruptors (eds) may be subject to authorization. the identification of eds is limited as yet, because specific experimental assessments are not required under reach. evidence is currently based on a combination of few experiments, expert judgement and structural analogy with known eds. structural alerts for the identification of potential eds: predictions of properties and effects from chemical structures are based on similarities with either active or inactive substances. structural alerts for the identification of potential estrogen/androgen-ed activities are relevant parts of the structures of compounds that are known to interact with estrogen and androgen receptors as either agonists or antagonists. in addition to the backbones of the chemical structures (pharmacophore) for steric fit to the receptors, modulating factors may be small substructures for local interactions with receptor binding sites and physicochemical properties related to the strength of binding to the receptor. comparison of evidence from in silico, in vitro and in vivo assays for potential eds: identification of potential eds based on findings from mammalian long-term reproduction studies, fish life-cycle tests, receptor assays, and chemical alerts were compared and differences analysed. agreement is limited because in vivo, in vitro and in silico methods address different aspects of potential effects on endocrine systems regarding toxicological targets, modes of action and functional similarity of chemical structures. a combination of toxicological and chemical assays can provide comprehensive and complementary information to support evidence-based identification of potential eds among the chemicals released into the environment. application of structural alerts for the identification of potential eds to the einecs inventory: more than 33000 discrete organic einecs compounds are within the applicability domain of the structural alerts for potential estrogen/androgen-ed activities. among them, 3585 chemicals (ca. 11%) are indicated as potential candidates for endocrine effects based on structural alerts. due to the possibility that these chemicals may interact with estrogen/androgen receptors they should be subject to further investigations regarding their potential for endocrine effects, eventually leading to regulatory actions. within the imi (innovative medicine initiative) project "intelligence-led assessment of pharmaceuticals of the environment " (ipie;http://i-pie.org/), a more intelligent environmental testing and tools for prioritisation of legacy compounds shall be developed. regarding the evaluation of fish toxicity, screening approaches in fish embryos are pursued. while the standard fish embryo toxicity (fet) test is restricted to lethal parameters more relevant as substitutes for acute effects, additional sublethal endpoints may provide expanded applicability of the fet assay for chronic effect assessment in fish. in this respect, the analysis of the metabolome could provide additional insights into biochemical processes elicited by pharmaceutical compounds and could potentially support the extrapolation from fish embryo to chronic fish toxicity as displayed in the standard early life stage (els) test. in the context of ipie a pilot study was performed with the aim to quantify and comparatively assess changes in the metabolic signatures of fish and fish embryos induced by the reference compound amikacin, an aminoglycoside antibiotic, in order to identify metabolic patterns applicable as biomarker profiles that can be linked to apical endpoints in terms of an integrated approach. therefore, toxic effects in fathead minnow embryos and els fish were investigated following a 4 and 32 days exposure, examining conventional endpoints and additionally using a combined direct injection and lc-ms/ms assay for metabolite identification and quantification. metabolic endpoints were found to be at least as sensitive as standard apical endpoints such as growth and mortality as detected in the longer-term fish study. furthermore, multivariate data analysis (pca-x, opls-da) revealed substance induced metabolic perturbations specific for fish and fish embryos, respectively. beyond that, the statistical approach of shared and unique structure (sus) identified some metabolites from the classes of amino acids, biogenic amines and lipids to be similarly changed in both developmental stages related to amikacin treatment, representing shared biomarker candidates. in this first pilot study, the integrated metabolomics approach yielded insights into the molecular consequences of amikacin exposure and provided indications for biomarkers for common effects in fish embryos and fish. due to the different exposure levels in the fet (1 -100 mg/l) and els study (0.0003 -0.03 mg/l), more definitive conclusions regarding the predictivity of metabolic responses in fish embryos for apical endpoints in chronic fish tests are yet not possible. further studies are ongoing with a range of pharmaceutical compounds of different chemical classes which will reveal more substantial information on the applicability of this technology in the prediction of longterm effects in fish. the human cationic amino acid transporter hcat-1 (slc7a1) represents the major uptake route for arginine and other cationic amino acids (such as the essential amino acid lysine) in most mammalian cells. it thus provides these amino acids for protein synthesis as well as for essential metabolic pathways. in endothelial cells, special attention has been given to the role of hcat-1 for supplying arginine to nitric oxide synthase. in spite of its wide distribution, hcat-1 expression is highly regulated both, on the transcriptional and post-transcriptional level. however, the genetic elements involved in transcriptional regulation a largely unknown. here we studied the expressional regulation of hcat-1 in human ea.hy926 endothelial cells. starvation of these cells from cationic amino acids led to a pronounced induction of both, hcat-1 mrna and protein. the mrna induction was almost completely abolished by the transcription elongation inhibitor drb (5,6-dichloro-1-β-dribofuranosylbenzimidazole), suggesting the involvement of transcriptional regulation. we thus aimed at identifying the promoter elements in the hcat-1 gene responsible for this regulation. to our surprise and in contrast to data published by others 1 the chromosomal region up to 5 kb upstream of the first hcat-1 exon exhibited no promoter activity in either endothelial or dld-1 colon carcinoma cells that exhibit a very strong endogenous hcat-1 expression. we could however identify a promoter element within the first intron of the hcat-1 gene. transcriptional activity of this element increased upon amino acid starvation in a similar way as endogenous hcat-1 expression. our results indicate starvation-induced transcriptional regulation of hcat-1 through newly identified promoter regions distinct from those published previously. the transport of a multitude of drug molecules into the cell is mediated by multispecific organic cation transporters (octs), belonging to the solute carrier group (slc). one of these families within this group of membrane transport proteins is the slc47-family consisting of the two multidrug and toxin extrusion transporters mate-1 (slc47a1) and mate2-k (slc47a2). while mate-1 is highly expressed in several different tissues like kidney, liver, skeletal muscle, adrenal glands, testes and heart, mate2-k exclusively occurs in the apical membrane of proximal tubular epithelial cells within the kidney. both transport proteins translocate organic cations in exchange of protons into as well as out of the cell. to define the affinity of both transporters for the anti-diabetic drug metformin and to investigate their interactions with 26 different anti-neoplastic agents comparative transport experiments with the model substrate 1-methyl-4-phenylpyridinium (mpp) have been carried out. therefore stably transfected hek293-cells expressing mate-1 or mate2-k transport proteins generated by portacelltec biosciences gmbh and vector transfected hek293-cells were used. the interaction analyses were carried out by determining the uptake of [ 14 c]-metformin and the inhibition of [ university of basel, basel, switzerland drug transporters play a pivotal role in pharmacokinetics by modulating the cellular entry or efflux of compounds. one transporter facilitating the transport of bile acids, steroid hormones, and statins is the organic anion transporting polypeptide (oatp) 2b1 that is highly expressed in placenta, liver, and small intestine. especially its activity in small intestine and liver is assumed to be basis for specific drug-drug interactions. understanding mechanisms involved in pharmacokinetics is a prerequisite in drug development. to test whether there are species differences in transport activity we compared the expression and function of the human and rat orthologue. first, we determined the transport activity of the known oatp2b1 substrate estrone 3sulfate (e 1 s), and observed a significantly lower k m for mdck-hoatp2b1 compared to .00 ± 10.23 µm, *p<0.05 student´s t-test) whereas there was no difference in v max (mdck-hoatp2b1 119.4 ± 11.3 fmol/min/µg protein; mdck-roatp2b1 102.3 ± 12.8 fmol/min/µg protein). the human oatp2b1 exhibits multiple binding sites for its substrates that may explain specific drug-drug interactions [1] . to identify whether rat oatp2b1 owns the same characteristics, the cellular accumulation of 50 µm e 1 s (low affinity site) or 0.005 µm e 1 s (high affinity site) was determined in presence of specific inhibitors/substrates of oatp2b1. as observed for atorvastatin, a known inhibitor of both affinity sites, the rat transporter failed to exhibit the low affinity site. in detail, while atorvastatin reduced the accumulation of e 1 s in mdck-hoatp2b1 cells (110.0 ± 14.6 fmol/min/µg protein vs. 60.7 ± 7.5 fmol/min/µg protein, *p<0.05 student´s t-test), there was no inhibition of e 1 s accumulation in mdck-roatp2b1 cells (53.2 ± 13.2 fmol/min/µg protein vs. 49.0 ± 17.4 fmol/min/µg protein). additionally, we observed a different membrane localization of both transporters as assessed by immunofluorescent staining showing an apical localization for rat oatp2b1 while human oatp2b1 is localized at the basolateral pole of the cellular model. absolute quantification of mrna (copy number per 1000 ng of total rna) in different tissues of rat revealed a high expression of oatp2b1 in liver (159.6 ± 45.5), a moderate expression in small intestine (14.9 ± 4.0) and colon (57.0 ± 12.3), and a low expression level in kidney (2.3 ± 0.8) . the latter is in accordance with previous findings showing that oatp2b1 is abundant in rat kidney as quantified by absolute proteomics technics [2] . our data demonstrated species differences in localization and activity of the drug transporter. further studies are warranted to proof whether this knowledge will help in future drug development and which molecular cause is responsible for the observed data. background: intestinal drug absorption depends on various factors including aqueous volume, site-specific ph and intestinal motility. moreover, the expression of efflux-and uptake-transporters vary in dependence of the anatomical localization making the gut a complex barrier for drug transfer into the body. in a recent study, site-specific protein and mrna expression levels of 10 drug transporters were determined along the entire length of the human gut. interestingly, discrepancies between mrna expression and protein levels were observed. moreover, there were quantitative differences between small intestine and colon. as a consequence the question arose if this observation could be explained by small non-coding rnas acting as highly tissue-specific posttranscriptional regulators of gene expression. hence, in our current study, we aimed to investigate the impact of mirnas on site-specific transporter expression along the human intestine. methods: total rna was isolated from biopsies obtained from six disease-free organ donors. tissue samples were acquired from the duodenum, the upper and lower jejunum, the upper and lower ileum, and the transversal or descending colon. the expression of 754 mirnas was measured using rt-pcr based low density arrays. expression of all detected mirnas was correlated with transporter protein data recently determined by lc-ms/ms-based targeted proteomics. mirnas and transporter genes showing an inverse pearson's correlation between mirna and protein expression underwent an in-silico search (microcosm targets v.5, targetscan7.0) for putative mirna/mrna interaction. to investigate those interactions in more detail, reporter gene assays and site directed mutagenesis were conducted. results: out of 754 mirnas, 248 were detected in all tissue types. out of ten transporters five showed significant inverse correlations with 10 putatively targeting mirnas (e.g. pept1 and hsa-mir-27a, r= -0.498, p=0.002). reporter gene assays indicated interactions of mir-193a/b with pept1 3'-utr (p = 0.0025 and p = 0.0012) as well as of mir-27a with abcb1 3'-utr (p = 0.0006). the site-specific abundance of intestinal drug transporters is significantly affected by microrna-mediated post-transcriptional regulation under physiological conditions as exemplified for pept1 and abcb1. background: the human uptake transporter nact [gene symbol slc13a5; also known as mindy, the human orthologue of the drosophila indy (i´m not dead yet) transporter] is expressed in liver and brain. nact is important for energy metabolism and brain development and mediates the uptake of tricarboxylic acid (tca) intermediate such as citrate and succinate. reduced expression of this transporter, as studied in knock-out mice, mimics aspects of dietary restriction, promotes longevity and protects against insulin resistance and adiposity. furthermore, mutations in the human slc13a5 gene are associated with epileptic encephalopathy with seizure onset in the first days of life, possibly due to the reduced uptake of tca intermediates into neurons. to gain more insights into the role of nact in drug transport and into structure-function relationships, we studied the inhibition of nact-mediated citrate transport by various drugs and analyzed the effect of known mutations in the slc13a5 gene on nact-mediated transport. methods: drug inhibition studies were performed using hek cells stably expressing human nact with citrate as prototypic substrate. twenty-four drugs were used as potential inhibitors of nact-mediated uptake. the effects of eight mutations, three of them (nactp.g219r, -p.t227m and -p.l488p) associated with epileptic encephalopathy, were analyzed using a transient transfection approach. furthermore, the first computational-based structural model of the nact transporter was established and the impact of all mutations on substrate transport was modeled. results: inhibitions studies demonstrated that only a few drugs (three out of 24 tested) inhibited nact-mediated citrate transport at the tested drug concentration of 100 µm. from these, benzbromarone shows the strongest inhibition with an ic 50 value of 83.2 µm. furthermore, citrate transport was also slightly inhibited by pioglitazone and rosiglitazone. citrate transport of the mutated proteins nactp.g219r, -p.g219e,p.t227m, -p.l420p and -p.l488p was totally abolished and the effect of these mutations could be explained on the basis of the structural model. conclusion: inhibition studies demonstrated that simultaneously administered drugs can inhibit nact-mediated uptake of the prototypic substrate citrate. nact-mediated uptake is abolished by mutations in the slc13a5 gene associated with epileptic encephalopathy. the effect of these mutations can be explained on the basis of the first structural model of this uptake transporter. the atp-binding cassette subfamily b member 1 (abcb1) is a drug efflux pump responsible for the classic multi-drug resistance phenotype in cancer cells. increased activity of abcb1 in cancer cells contributes to protection against a wide range of chemotherapeutic agents. this dramatically decreases therapeutic options and the chance of patient survival. knowledge of the underlying mechanisms for abcb1 deregulation is a critical step for the reversal of this unfavorable condition. of note, phosphatidylinositol-4,5-bisphosphate (pip 2 ) is a known regulator of abcb1. the protein "myristoylated alanine-rich c-kinase substrate" (marcks) is known for its ability to bind and sequester the phospholipid pip 2 . in various forms of colorectal cancer the deregulation of marcks protein goes hand in hand with an increase in malignancy and therapeutic resistance. in this study, we characterized the enigmatic marcks as a modulator of abcb1 activity. we focused on a subgroup of colon cancers, where marcks resides in a hyperphosphorylated state for which the established cell line ht-29 served as a model. we employed various in-vitro methods for the measurement of abcb1 activity, in combination with imaging experiments, assays for cellular viability and classical methods of molecular biology. we combined these approaches with pharmacological inhibition of marcks phosphorylation state or rnai-mediated depletion of marcks. with these interventions we could modulate endogenous abcb1 activity and re-sensitize our cell model against chemotherapeutic agents like 5-fluorouracil which are known to be substrates of abcb1. taken together, our findings suggest a new way how a cancer cell can gain a state of therapeutic resistance. the exploitation of marcks as modulator of abcb1 might be a new approach to target resistant tumors without interfering with the natural function of abcb1 in non-malignant tissue. background: in several large clinical studies low blood concentrations of homoarginine were identified as independent risk marker for stroke, cardiovascular events and mortality. experimental data suggest an active role of homoarginine deficiency in disease development. interference with l-arginine-dependent pathways and signaling has been implicated as a possible mechanism. it was the aim of the present study to identify transport mechanisms involved in the cellular uptake and tissue distribution of homoarginine, which is poorly understood, so far. the experiments focused on cationic amino acid transporters (cats) and possible interactions with known cat substrates. methods: uptake assays were performed using [ 3 h]-labeled homoarginine as substrate and human embryonic kidney (hek293) cells stably overexpressing human cat1 [gene: slc7a1 (solute carrier family 7)], cat2a (slc7a2a) or cat2b (slc7a2b). cells transfected with an empty vector were used as control. unlabeled known catsubstrates l-arginine and asymmetric dimethylarginine (adma) were investigated as inhibitors. results: compared to the uptake into control cells, uptake of homoarginine was significantly higher in hek cells overexpressing cat1 (7-fold), cat2a (1.6-fold) or cat2b (2.2-fold) after 2.5 min using 100 µm substrate (each p<0.001). apparent k m values for cellular net uptake of homoarginine were 174.6 µm for cat1, 3640 µm for cat2a and 522.8 µm for cat2b. homoarginine uptake by the three cats could be inhibited by addition of l-arginine and adma. conclusion: the protective biomarker homoarginine is a substrate of the human cationic amino acid transporters cat1, cat2a and cat2b. compared to other cat substrates, the cat1-and cat2b-mediated homoarginine transport is characterized by relatively high affinity. the uptake of homoarginine by all three cats can be inhibited by l-arginine and adma. taken together these findings make cat-mediated transport of homoarginine a possible target for interactions and pharmacological interventions aimed at homoarginine homeostasis. this project is supported by the doktor robert pfleger-stiftung. background and aim: organic cation transporter 1 (oct1, alternative name slcc22a1) is a polyspecific membrane transporter, which has been suggested to play a role in absorption, distribution and elimination of drugs and toxins. besides endogenous compounds like thiamine (vitamin b1), known oct1 substrates are toxins like mpp + as well as drugs like metformin, o-desmethyltramadol, ranitidine, and sumatriptan. tissue distribution of oct1 has been shown to have strong inter-species differences. in rodents oct1 is strongly expressed in both the sinusoidal membrane of hepatocytes and the basolateral membrane of kidney epithelial cells. human oct1 is strongly expressed on the sinusoidal membrane of hepatocytes, but not in the kidney. furthermore, substrate specific differences have been observed between the human and rodent oct1 orthologs. the aim of this study is to characterize the mechanisms causing substrate specificity between rodent and human oct1 orthologs. methods: overexpression of oct1 orthologs in mouse, rat and human cells was performed by targeted chromosomal integration of t-rex™ 293. the cells were characterized in detail for their ability to transport the model substrates tea + , mpp + , and asp + , the drugs ranitidine, sumatriptan and fenoterol. results: mouse and rat oct1 orthologs showed similar transport activity for all the model substrates and drugs tested. however, significant differences were observed when rodent orthologs were compared to the human oct1. human oct1 showed a 5fold higher v max for the uptake of asp + and 2-fold increase of v max for sumatriptan in comparison to the rodent oct1 orthologs. conversely, human oct1 showed a 4-fold lower v max for the uptake of fenoterol compared to rodent oct1s. there was no difference between rodent and human oct1 in the uptake of ranitidine. these differences were further characterized in detail using chimeric mouse-human oct1 constructs and by comparing the effects of key point mutations in mouse and human oct1 orthologs. conclusions: these data demonstrate strong differences in the substrate specificity between rodent and human oct1 orthologs. therefore oct1 pharmacokinetic data obtained in mouse or rat models could not be directly extrapolated for use in human. furthermore, comparative functional analyses of orthologs may help reveal the mechanisms underlying polyspecificity of oct1. ranitidine is a histamine h 2 -receptor antagonist which is commonly used without prescription for the treatment of pyrosis and gastric ulcers. approximately 30% of the systemic clearance of ranitidine is via hepatic metabolism. ranitidine is known to be a substrate of the hepatic organic cation transporter oct1 [1]. oct1 is expressed on the sinusoidal membrane of human hepatocytes and is highly genetically variable. sixteen major oct1 alleles are known [2] . thereof 12 alleles confer partial or complete loss of oct1 activity. the observed loss of activity was highly substrate specific and should be analyzed substrate by substrate. in this study we analyzed the effects of genetic polymorphisms in oct1 on the uptake of ranitidine. we used hek293 cells stably transfected to overexpress wild type or variant oct1 isoforms. the variant oct1 alleles oct1*1a (met408val), oct1*1c (phe160leu), oct1*1d (pro341leu/met408val), oct1*2 (met420del), oct1*3 (arg61cys), oct1*4 (gly401ser), oct1*5 (gly465arg/met420del), oct1*6 (cys88arg/met420del), oct1*7 (ser14phe), oct1*8 (arg488met), oct1*9 (pro117leu), oct1*10 (ser189leu), oct1*11 (ile449thr), oct1*12 (ser29leu) and oct1*13 (thr245met) were analyzed. we characterized in ranitidine uptake determined k m and v max for the different polymorphic oct1 isoforms. wild type oct1 showed a time and concentration dependent uptake of ranitidine with a k m of 62.91 ± 4.32 µm and a v max of 1125.41 ± 86.12 pmol/min/mg protein. variants oct1*5, oct1*6, oct1*12 and oct1*13 completely lacked ranitidine transport activity. variants oct1*2, oct1*3, oct1*4 and oct1*10 showed v max values decreased by 64, 77, 91 and 50 %, respectively. oct1*8 variant showed an increase of v max by 25 %. there was no significant changes in ranitidine uptake by variants oct1*1a, oct1*1c, oct1*1d, oct1*7, oct1*9 and oct1*11 compared to the wild type. there were no significant differences in the k m values between the wild-type and variants. in conclusion, we confirmed ranitidine as an oct1 substrate and demonstrated that genetic polymorphisms in oct1 can strongly affect ranitidine uptake. the effects of oct1 polymorphisms on ranitidine pharmacokinetics in humans remain to be analyzed. otto-von-guericke university, institute for pharmacology and toxicology, magdeburg, germany serotonergic hallucinogens ( s hgs), such as lysergic acid diethylamide (lsd), induce profound alterations of human consciousness, which are thought to be mediated by activation of 5-ht 2a receptors. with repeated application, the mind-altering effects of most s hgs rapidly are undermined by tolerance. the only exception seems to be dimethyltryptamine (dmt), whose mind-altering effects for reasons unknown even with repeated application do not decrease. assuming that dmt differs from other s hgs in its capacity to regulate 5-ht 2a receptors, we here compare lsd and dmt with regard to processes of 5-ht 2a downregulation. in heat-exposed rats, lsd and dmt induce a marked increase in body core temperature (hyperthermia) accompanied by "defensive hypersalivation". both effects are mimicked by the 5-ht 2 selective agonist dimethoxybromoamphetamine (dob) and can be blocked by the selective 5-ht 2a antagonists ketanserin and mdl100907. after repeated application, the temperatureregulatory effects of lsd are subject to tolerance, whereas the ones of dmt are not. tolerance to lsd (as measured by dob induced [ 35 s]gtpуs binding) is paralleled by a desensitisation of frontocortical 5-ht 2 receptors; for dmt, there is no such decrease. applying techniques of immunocytochemistry, transphosphatidylation, and quantitative real-time pcr to (ha-5-ht 2a transfected) hek293 and (endogenously 5-ht 2a expressing) c6 glioma cells, respectively, we furthermore demonstrate that dmt in contrast to lsd fails to internalise 5-ht 2a receptors, fails to activate the phospholipase d (which is needed for 5-ht 2a internalisation), and fails to inhibit the synthesis of 5-ht 2a receptors. given that dmt unlike lsd turns out to be inactive as to all processes of 5-ht 2a downregulation investigated, our data suggest that the differential tolerance development noted for dmt and lsd indeed might be accounted for by differential regulation of 5-ht 2a receptors. lsd and dmt both have recently regained scientific attention as potential therapeutics in the treatment of depression and/or anxiety disorders. providing mechanistic insights into their action, thus, is of timely clinical relevance. increased gaba release in human neocortex at high intracellular sodium and low extracellular calcium -an anti-seizure mechanism? ] e , this reduction might induce an anti-seizure mechanism by augmenting gat-mediated gaba release, a mechanism absent in rats. aging is complex on the systems as well as on the molecular level. the process of aging is characterized by a progressive loss of physiological functions and accumulation of cellular damage. one hallmark of aging is an impaired protein homeostasis. the imbalance of the quality control of both de novo protein synthesis and protein degradation, therefore, is likely to contribute to the phenotype of aging. we investigated protein turnover rates with the state-of-the art techniques funcat (dieterich et al., 2010) and sunset (schmidt et al., 2009) in aging neuronal cell cultures . using these techniques we show a prominent decrease in protein synthesis and degradation that progressed gradually in aging neuronal cells cultured up to div 60. in order to rejuvenate the protein turnover in aged neuronal cells we applied the selective eukaryotic elongation factor-2 kinase inhibitor a 484954 and the polyamine spermidine and observed protein translation utilizing funcat and sunset. whereas both a 484954 and spermidine had no effect on de novo protein synthesis in juvenile neurons (div 20) , both substances increased the de novo protein synthesis to a juvenile level in aged neuronal cultures (div60). this effect is seen in neuronal somata and dendritic spines. the molecular function of spermidine as an "anti-aging agent" is not defined yet. thus, additional pharmacological interventions are used for further examination of specific molecular spermidine targets. in conclusion, the described experimental setup is used to investigate impaired protein homeostasis as one hallmark of aging. agents with a presumed "anti-aging" effect can be tested for a potential rejuvenating effect on the level of protein homeostasis. a screening approach to test tolerability of multitargeted drug combinations for antiepileptogenesis in mice a large variety of brain insults can induce the development of symptomatic epilepsies, particularly temporal lobe epilepsy. in the latent period after the initial insult multiple molecular, structural, and functional changes proceed in the brain and finally lead to spontaneous recurrent seizures. prevention of these developments, called antiepileptogenesis, in patients at risk is a major unmet clinical need. several drugs underwent clinical trials for epilepsy prevention, but none of the drugs tested was effective. similarly, most previous preclinical attempts to develop antiepileptogenic strategies failed. in the majority of studies, drugs were given as monotherapy. however, epilepsy is a complex network phenomenon, so that it is unlikely that a single drug can halt epileptogenesis. recently, multitargeted approaches were proposed ("network pharmacology") to interfere with epileptogenesis. developing novel combinations of clinically used drugs with diverse mechanisms that are potentially relevant for antiepileptogenesis is a strategy, which would allow a relatively rapid translation into the clinic. we developed an algorithm for testing such drug combinations in a screening approach, modelled after the drug development phases in humans. tolerability of four repeatedly administered drug combinations was evaluated by a behavioral test battery: a, levetiracetam and phenobarbital; b, valproate, losartan, and memantine; c, levetiracetam and topiramate; and d, levetiracetam, parecoxib, and anakinra. as in clinical trials, tolerability was separately evaluated before starting efficacy experiments to identify any adverse effects of the combinations that may critically limit the successful use in preclinical studies on antiepileptogenesis and translation of these preclinical findings to the clinic. based on previous studies, we expected that tolerability would be lower in epileptic mice than in nonepileptic mice. therefore nonepileptic mice were used as a first step, followed by epileptic mice and mice during the latent period shortly after status epilepticus. except combination b, all drug cocktails were relatively well tolerated. in contrast to our expectations, except combination c, no significant differences were determined between nonepileptic and post-status epilepticus animals. as a next step, the rationally chosen drug combinations will be evaluated for antiepileptogenic activity in mouse and rat models of symptomatic epilepsy. major depression is one of the most common mental disorders worldwide, with serious social and economic consequences. there are many different hypotheses concerning the pathophysiology of this disease. the complex brain serotonin system and particularly the serotonin 1a receptors (5-ht 1a r) apparently play a pivotal role in the development of depression. the involvement of an altered, 5-ht 1a r-mediated signalling in adult neurogenesis is also discussed. however, in this context the effects of pre-and postsynaptically located 5-ht 1a rs have not been clarified yet. mice with a permanent overexpression of postsynaptic 5-ht 1a rs (oe mice) represent a unique tool to elucidate the effects of postsynaptic 5-ht 1a rs in adult neurogenesis and depressive-like behaviour. previous studies demonstrated an increased proliferation and survival of newborn cells in the adult dentate gyrus of female oe mice in comparison to controls. in the present study, we investigate the proliferation and survival of adult born cells after chronic treatment (15 days) with the selective 5-ht 1a r agonist 8-oh-dpat (0,5 mg/kg/day) in young adult oe and wt mice. on the last three days of treatment, newly generated cells of oe and wt mice are labelled by injections with bromodeoxyuridine (brdu; 50 mg/kg/day). mice are sacrificed either one day (proliferation) or 21 days (survival) after the last injection. we hypothesise that the data we will present will confirm our previous results, with possibly more pronounced proneurogenic effects and differences in male mice. further immunohistochemical studies post-exercise and behavioural analyses are in progress to identify the relation between chronic postsynaptic 5-ht 1a r stimulation, depressive-like behaviour and hippocampusdependent learning. dystonia is a common movement disorder characterized by intermittent and prolonged muscle contractions resulting in involuntary movements and/or abnormal postures. the lack of knowledge of the pathophysiology of dystonia hampers the development of effective therapeutics. although benzodiazepines can improve dystonic symptoms, tolerance and side effects limit their use. there is evidence for striatal dysfunctions in human dystonia. gabaergic striatal interneurons (in) are important for the regulation of striatal signaling. in the dt sz mutant hamster, a model of paroxysmal dystonia, immunoreactive in were reduced at the age of maximum severity of dystonia (33 days), but not after spontaneous remission (age 90 days). as indicated by unaltered homeoboxprotein nkx 2.1 (cell density, mrna), the age-dependent deficit seems not to be related to a disturbed migration, but to a retarded maturation of in in mutant hamsters. here we further determined the maturation of striatal gabaergic neurons in the dt sz hamster compared to healthy controls. kcc2 and cavii mrna, used as markers for the gaba-switch, were unchanged in 18 and 33 day old mutant hamsters, indicating that there is no general delay in gabaergic maturation. as a retarded maturation seems to be specific for in, we used another marker for gabaergic maturation: the expression of specific gaba a receptor (gaba a r) subunits (mature striatal in express the alpha1 subunit). by stereological determination, we found a 46% decrease in alpha1 subunit expressing neurons. a lower immunoreactive intensity was restricted to the somata of dorsomedial striatal in (32%) of dt sz hamsters, indicating both a reduced density as well as a delayed maturation. these findings prompted us to examine the effects of the αlpha1 gaba a r preferring compound zolpidem in comparison with the benzodiazepine clonazepam. zolpidem (2.0 and 10.0 mg/kg i.p.) only exerted moderate antidystonic effects compared to the benzodiazepine clonazepam (0.5 and 1.0 mg/kg i.p.) in the dt sz hamster. examinations of αlpha2 gaba a r preferring compounds are ongoing. in summary our studies indicate that there is no general defect in striatal gabaergic maturation in the dt sz mutant but a specific alteration of striatal gabaergic interneurons which express αlpha1 gaba a r subunits. changes in αlpha1 gaba a r subunit expression and differences in the antidystonic efficacy of zolpidem and clonazepam indicate that further investigations on the role of gaba a r subunits could lead to new therapeutic approaches for the treatment of dystonia. 2005) . therefore, the hypothesis of the present study was that pregnenolone attenuates the inhibition of synaptic transmission elicited by cannabinoids. methods: 250 µm-thick slices containing the cerebellum and the nucleus accumbens were prepared from the brains of mice and rats. spontaneous and electrically evoked gabaergic inhibitory postsynaptic currents (sipscs and eipscs) and evoked glutamatergic excitatory postsynaptic currents (eepscs) were analyzed in superfused brain slices with patch-clamp electrophysiological techniques. results: a) the synthetic cannabinoids jwh-018 (5 x 10 -6 m) and jwh-210 (5 x 10 -6 m) inhibited the spontaneous gabaergic synaptic input (sipscs) to purkinje cells in mouse cerebellar slices. the inhibition by jwh-018 was not affected by pregnenolone (10 -7 m), the inhibition by jwh-210 was only marginally attenuated. b) the depolarization of the purkinje cells induced suppression of the gabaergic input to purkinje cells (dsi); pregnenolone (10 -7 m) did not affect this endocannabinoid-mediated form of synaptic suppression. c) in rat nucleus accumbens slices, gabaergic and glutamatergic synaptic input to medium spiny neurons was activated by electrical stimulation of axons. ∆ 9 -tetrahydrocannabinol (2 x 10 -5 m) suppressed the gabaergic and glutamatergic synaptic transmission in the nucleus accumbens. these suppressive effects of ∆ 9tetrahydrocannabinol were not changed by pregnenolone (10 -7 m). d) finally, we tested whether we can observe neurosteroid-mediated effects in our brain slice preparations. tetrahydro-deoxycorticosterone (thdoc, 10 -6 m) markedly prolonged the decay time constant (τ) of spontaneous gabaergic postsynaptic currents (sipscs), similarly as in previous experiments (ej cooper et al., j physiol 521: 437-449, 1999) . the results show that inhibition of gabaergic and glutamatergic synaptic transmission by synthetic-, endogenous,-and phyto-cannabinoids is not changed by pregnenolone. therefore, it is unlikely that interference with cannabinoid-induced inhibition of synaptic transmission is the mechanism by which pregnenolone attenuates behavioural and somatic effects of ∆ 9 -tetrahydrocannabinol in vivo. the hypothalamus is one of the key players in the regulation of the energy homeostasis. cold stress leads to an activation of neurons in the paraventricular hypothalamic nucleus (pvn) and increases thermogenesis. the thyrotropin-releasing-hormone (trh) neurons have an important function in this effect. however it is hardly understood which role the trh neurons exactly play and how they are connected to other regions of the brain. we have transduced neurons in the pvn of mice with a recombinant adeno associated virus which contains an activating "designer receptors exclusively activated by designer drugs" (dreadd) system under the control of a shortened trh promotor. two weeks after transduction the animals were injected with clozapine-n-oxide (cno). to analyse the physiological function of this neurons we performed indirect calorimetry, measured rectal temperature and thermogenesis in the brown adipose tissue (bat), analysed drinking feeding behaviour and the home cage activity. after stimulation we measured the expression of genes in bat as well plasma hormone levels of pituitary hormones. propranolol and the specific β3-antagonist sr59230a were used to analyse the relevance of the sympathetic system. to further characterise the transduced neurons and their projections we used immunohistochemistry methods. after stimulation with cno the energy expenditure and body temperature were increased. these effects were mostly driven through an activation of the brown adipose tissue (bat). in dreadd transduced trh-receptor 1 (trh-r1) knockout mice this effects were abolished. in parallel the plasma levels of tsh, the ucp1 mrna level in the bat, the home cage activity as well the food and water intake were increased. after the treatment with propranolol and sr59230a the effects on the thermogenesis were reduced, but the home cage activity was not affected. sr59230a treatment normalised the food intake and increased in parallel the plasma leptin concentrations after cno stimulation. transduced neurons project into the raphe nucleus, the medial part of the thalamus and the spinal cord. with our experiments we could provide strong evidence for a sympathetic connection of the transduced neurons in the pvn to the bat and for the involvement of thr neurons in these effects. therefore, this system is a suitable tool to investigate the metabolic relevance of trh neurons in detail. background & objective: during obesity development, tissue factor signalling contributes coagulation-independently to inflammatory and metabolic dysfunction of adipose tissue. adipogenesis involves proliferation and differentiation of preadipocytes, apoptosis and hypertrophic growth of differentiated adipocytes, angiogenesis and extracellular matrix reorganisation. the coagulant protease thrombin promotes similar processes in various cell types, through activation of protease-activated receptors par-1, par-3 and par-4. in human adipose tissue, par-1 is found in vascular stromal cells and par-4 in preadipocytes and differentiated adipocytes. thrombin stimulates mitogenic kinase signalling and induces inflammatory cytokine and angiogenic growth factor secretion in adipocytes. we have examined the contribution of thrombin receptor activation to adipogenesis processes in 3t3l1 cells. results: differentiation of 3t3l1 preadipocytes with insulin, dexamethasone and isobutylmethylxanthine increases leptin and pparg gene expression and accumulation of triglycerides and oil red o-stained lipids. par-4 is time-dependently upregulated in maturing cells while par-1 expression is detectable but not altered. in preadipocytes, thrombin (3 u/ml) activates the mitogenic kinase erk1/2, promotes cell proliferation and induces gene expression of the maturation markers leptin and pparg and the inflammatory marker tumor necrosis factor alpha (tnfa). repeated stimulation of differentiating adipocytes with thrombin suppresses induction of leptin and pparg and attenuates lipid accumulation, while expression levels of the proliferation marker ki67 and the inflammatory cytokine interleukin (il)-6 are increased compared to differentiated control cells. similar proliferative and anti-adipogenic effects are seen with the selective par4-activating peptide (gypgkf, 100 µm) and cathepsin g, a proteolytic par-4 activator released from neutrophils and mast cells. repeated exposure of maturing 3t3l1 cells to conditioned medium from degranulating mouse peritoneal mast cells (mccm) augments lipid accumulation and il-6 expression. pretreatment of mccm with a par-4 inhibitor further drives lipid accumulation, the induction of il-6 by contrast is suppressed. conclusion: par-4 activation by thrombin or inflammatory cell-derived cathepsin g appears to suppress adipogenesis, possibly by maintaining proliferative capacity and preventing the growth arrest essential for initiating matuation. increased par-4 expression in maturing adipocytes may instead support inflammatory changes, thereby promoting the onset of insulin resistance. the chemokine receptor cxcr4 antagonist amd3100 exerts deleterious effects in endotoxemia in vivo s. seemann 1 , a. lupp the chemokine receptor cxcr4 is a multifunctional receptor which is activated by its natural ligand c-x-c motif chemokine 12 (cxcl12). although a blockade of the cxcr4/cxcl12 axis revealed beneficial outcomes in chronic inflammatory diseases, its importance in acute inflammatory diseases remains contradictious and not well characterized. as cxcr4 seems to be part of the lipopolysaccharide sensing complex, cxcr4 agonists or antagonists may have a positive impact on tlr4 signaling. additionally, cxcr4 is involved in the production of pro-inflammatory cytokines, suggesting the receptor to be a promising target in terms of mitigating the cytokine storm. therefore, we aimed to investigate the impact of a cxcr4 blockade on endotoxemia by applying a sublethal lps dose (5 mg/body weight) in mice. the selective cxcr4 inhibitor amd3100 was administered intraperitoneally shortly after lps treatment to ensure an immediate effect after endotoxemia onset. 24 hours after lps administration, the clinical severity score, the body temperature and the body weight of the animals were determined. afterwards, the mice were sacrificed and serum tnf alpha as well as ifn gamma levels were measured. furthermore, the oxidative stress in the brain, liver, lung and kidney tissue was assessed. in addition, the biotransformation capacity of the liver was evaluated and finally, the expression of gp91 phox as well as of heme oxygenase 1 in the spleen and liver were determined by means of immunohistochemistry. the mice of the amd3100 plus lps treatment group displayed a significantly impaired general condition, a reduced body temperature and a decreased body weight in comparison to the control and to the lps treated animals, respectively. tnf alpha levels were significantly increased by more than 200% or 35% when compared to the control or to the lps group, respectively, whereas ifn gamma levels were elevated by about 11% in comparison to mice which had received lps only. in all investigated organs, but especially in the liver and in the kidney, co-administration of amd3100 and lps caused massive oxidative stress. furthermore, the protein contents and the activities of several cyp enzymes in the liver were significantly reduced. immunohistochemistry revealed gp91 phox to occur above average, whereas heme oxygenase 1 expression was distinctly decreased. our results indicate that a blockade of the cxcr4 in endotoxemia is disadvantageous and even worsens the disease. co-administration of amd3100 and lps impaired the health status of the animals, caused massive oxidative stress and diminished the biotransformation capacity. thus, handling acute systemic inflammation with a cxcr4 antagonist cannot be recommended, hence indicating the activation of cxcr4 to be an attractive treatment option. toll-like receptors (tlrs)recognizemicrobial pathogens and trigger inflammatory immune responses to control infections. in acne vulgaris, activation of tlr2 by propionibacterium acnes contributes to inflammation. although glucocorticoids have immunosuppressive and anti-inflammatory effects, acne can be provoked by systemic or topical treatment. enhanced tlr2 expression by glucocorticoids has been reported in undifferentiated keratinocytes, however, human skin cells of different epidermal and dermal layers have not been investigated. in this study, the modulation of tlr expression by dexamethasone was assessed in monolayer cultures of primary human keratinocytes and dermal fibroblasts, as well as the immortalized keratinocyte cell line hacat. constitutive tlr2, tlr1 and tlr6 mrna and protein expression was confirmed in basal keratinocytes, calcium-induced differentiated keratinocytes, hacat cells and fibroblasts by qpcr and western blotting. dexamethasone induced tlr2 expression in a time-dependent and concentration-dependent manner and reduced tlr1/6 expression in keratinocytes but not in hacat cells or fibroblasts. stimulation with dexamethasone in the presence of the pro-inflammatory cytokines tnfα or il-1β further increased tlr2 mrna levels. gene expression of mapk phosphatase-1 (mkp-1) was also upregulated by dexamethasone. glucocorticoid-induced tlr2 expression was negatively regulated by p38 mapk signalling under inflammatory conditions through mkp-1 induction which functions to deactivate mapks. as expected, dexamethasone inhibited the immune responses linked to tlr2 signalling as demonstrated by reduced il-8, il-1β, mmp-1 and mcp-1 levels. however, the expression of traf6, a critical cytosolic regulator of tlr-and tnf family-mediated signalling, was further upregulated by the tlr2 agonist hklm (heat-killed lysteria monocytogenes) in dexamethasonepretreated basal keratinocytes.conclusively, our results provide novel insights intothe molecular mechanismsof glucocorticoid-mediatedtlr expressionand function in human skin cells. psoriasis is a cutaneous chronic inflammatory disease characterized by increased amounts of il-1 cytokines and t helper 17 (th17) related cytokines in lesional psoriatic skin. treatment with beta-adrenoceptor antagonists is associated with induction or aggravation of psoriasis, however, the underlying mechanism is poorly understood. previously, we could demonstrate a pivotal role for langerhans cells and dermal dendritic cells in antimalarial-provoked psoriasis by maintaining a potent th17 activity. in the present study, we investigated the effect of propranolol on human monocyte-derived langerhans-like cells (molc) and dendritic cells (modc) under inflammatory conditions. in the presence of il-1β, propranolol induced the th17 priming cytokines il-23 and il-6 in a concentration-dependent manner. the increased cytokine release was not mediated by camp suggesting gpcr-independent pathways. in contrast, il-36γ and lps failed to increase il-23 release in molc and modc in the presence of propranolol but further induced secretion of il-1β. autophagy has been linked with the secretion of il-1 family cytokines that are upregulated in chronic inflammatory disorders such as psoriasis. propranolol upregulated the expression levels of the autophagy marker p62 and lc3-i to lc3-ii conversion, induced accumulation of lc3 positive vesicles, as well as expression of il-1 signalling downstream adapter molecule traf6, indicating a late-stage block in autophagy. in summary, our results suggest a prominent role of cutaneous dendritic cell subtypes in psoriasis-like skin inflammation mediated by propranolol and possibly other beta blockers. langerhans cells (lcs) represent a highly specialized subset of epidermal dendritic cells (dcs), yet not fully understood in their function of balancing skin immunity. in the present study, we investigated in vitro generated langerhans-like cells obtained from the human acute myeloid leukaemia cell line mutz-3 (mutz-lcs) to study tlr-and cytokine-dependent activation of epidermal dcs. mutz-lcs revealed high tlr2 expression and responded robustly to tlr2 engagement, confirmed by increased cd83 and cd86 expression, upregulated il-6, il-12p40 and il-23p19 mrna levels and il-8 release. tlr2 activation reduced ccr6 and elevated ccr7 mrna expression and induced migration of mutz-lcs towards ccl21. similar results were obtained by stimulation with pro-inflammatory cytokines tnf-α and il 1β whereas ligands of tlr3 and tlr4 failed to induce a fully mature phenotype. despite limited cytokine gene expression and production for tlr2-activated mutz-lcs, co culture with naive cd4+ t cells led to significantly increased ifn-γ and il-22 levels indicating th1 differentiation independent of il-12. tlr2-mediated effects were blocked by the putative tlr2/1 antagonist cu-cpt22, however, no selectivity for either tlr2/1 or tlr2/6 was observed. computer-aided docking studies confirmed non-selective binding of the tlr2 antagonist. taken together, our results indicate a critical role for tlr2 signalling in mutz-lcs considering the leukemic origin of the generated langerhans-like cells. the stagnation in the development of new antibiotics during the last decades and the concomitant high increase of resistant bacteria emphasize the urgent need for new therapeutic options. antimicrobial peptides are promising agents for the treatment of bacterial infections and recent studies indicate that pep19-2.5, a synthetic antimicrobial and lps-neutralizing peptide (salp), efficiently neutralizes pathogenicity factors of gram-negative and gram-positive bacteria and protects against sepsis. in the present study, we investigated the potential of pep19-2.5 and the structurally related compound pep19-4lf for their therapeutic application in bacterial skin infections. primary human keratinocytes responded to tlr2 (fsl-1) but not tlr4 (lps) activation by increased il-8 production, as determined by elisa. western blot analysis showed that both salps inhibited fsl-1-induced phosphorylation of nf-κb p65 and p38 mapk. furthermore, the peptides significantly reduced il-8 release and gene expression of il-1β, ccl2 (mcp-1) and hbd-2 as assessed by qpcr. no cytotoxicity (mtt test) was observed at salp concentrations below 10 µg/ml. in lps-stimulated monocyte-derived dendritic cells, the peptides blocked il-6 secretion, downregulated expression of the maturation markers cd83 and cd86, as analysed by flow cytometry, and inhibited ccr7-dependent migration capacity. similarly, monocyte-derived langerhans-like cells activated with lps and pro-inflammatory cytokines showed reduced il-6 levels and cd83/cd86 expression in the presence of salps. in addition to acute inflammation, bacterial infections often result in impaired wound healing. since re-epithelialization is a critical step in wound repair, we tested whether pep 19-2.5 affects keratinocyte migration using the scratch wound assay. the peptide markedly promoted cell migration and accelerated artificial wound closure at concentrations as low as 1 ng/ml and was equipotent to tgf-β. conclusively, our data suggest a novel therapeutic target for the treatment of patients with acute and chronic skin infections. recently, we and others have shown that the transcription factor nuclear factor erythroid 2-related factor 2 (nrf2), a major regulator of the cellular antioxidant defence system, is activated by mechanical ventilation. during ventilator-induced lung injury, nrf2 exerts a protective role by interaction with the stretch-induced growth factor amphiregulin. in the current study, we aimed to investigate the role of nrf2 in acid-induced lung injury, a model for aspiration-induced ards. methods: nrf2-deficient (nrf2 -/-) mice and wild type (wt) littermates were tracheotomised and ventilated for 30min (v t =16ml/kg, f=90min -1 , peep=2cmh 2 o, fio 2 =0.3), before 50 µl hydrochloric acid (hcl) with ph=1.5 or ph=1.3 were instilled intratracheally, controls received nacl. mice were then ventilated for further 6h under monitoring of lung mechanics and vital parameters. blood gases as well as proinflammatory mediators, neutrophil recruitment and microvascular permeability were examined to assess lung injury. results: instillation of hcl ph=1.5 induced mild lung injury, indicated by hypoxemia (po 2 /fio 2 ~300mmhg) and continuously increasing lung tissue elastance (stiffness), from which nrf2 -/mice were protected. pulmonary inflammation, characterized by liberation of cytokines, chemokines and oedema formation, was attenuated in nrf2 -/mice. in contrast, hcl ph=1.3 caused more severe lung injury (po 2 /fio 2 ~200mmhg) with a steeper incline in elastance and more severe inflammation in both wt and nrf2 -/mice. conclusion: we conclude that the presence of nrf2 augments mild acid-induced lung injury, but plays no role in more severe injury. these discrepant results will be elucidated in future investigations. uniklinik rwth aachen, institut für pharmakologie und toxikologie, aachen, germany 2 fakultät für maschinenwesen der rwth aachen, werkzeugmaschinenlabor, aachen, germany rationale: reproducibility is key to science. in recent times, the reproducibility of biomedical research has been questioned increasingly. this reproducibility crisis also affects complex animal experiments, which -if not reproducible -might also be regarded as unethical and lose public acceptance. part of the problem is frequently that the provided documentation is not sufficient for reproduction. therefore, in this study we analyzed the potential of conventional quality management tools -used as standard in machine production -as an approach to improve the documentation and ascertain the quality of complex animal experiments. methods: quality management tools were transferred to an experimental animal set up -the mouse intensive care unit (micu) -which we use for lung injury studies. the tools included visualization of the experimental set-up, transfer of the experimental procedures to an event-driven process chain (epc) and statistical process control (spc) of all crucial pulmonary and cardiovascular parameters. data from ventilator-and acidinduced lung injury studies acquired in the micu were analyzed retrospectively. results: schematic visualization of the micu resulted in a chart comprising medical components, hardware, software and generated data types. the customized epc included all important activities and the resulting events for preparation of the mouse and the workplace, the actual animal ventilation experiment and sample-taking. in addition, checklists were provided for these activities and events, to ensure standardization of every work step. lung impedance and cardiac functions from ventilator-and acid-induced lung injury models were analyzed by spc and correlated with events in the epc. the spc proved to be suitable to identify outliers, predict processes and thereby validate the lung injury models. conclusions: conventional quality management tools were successfully adapted to analyze the quality of lung injury experiments in the micu. we suggest that this new approach is suitable to standardize animal testing procedures and increase the reproducibility of animal studies. background: a dysfunctional endothelial l-arginine-nitric oxide (no) pathway is a key pathomechanism of idiopathic pulmonary arterial hypertension (ipah) that can be provoked by hypoxia in cell culture models [1] [2] [3] [4] . the small peptide apelin is involved in the maintenance of pulmonary vascular homeostasis and angiogenesis although its precise mechanism of action is still unclear [5] . asymmetric dimethylarginine (adma) is known to be an endogenous inhibitor of endothelial no synthase and is associated with several cardiovascular diseases [6] . adma is degraded by dimethylarginine dimethylaminohydrolase 1 and 2 (ddah) enzymes [7] . objective: to determine the effect of apelin on the l-arginine/no pathway in human pulmonary microvascular endothelial cells (hpmecs). methods: hpmecs were cultured under normoxic and ph-related hypoxic conditions and treated with apelin. the expression of regulators of the l-arginine/no pathway were analysed using real-time pcr. the effect of apelin on the phosphoinositide-3 kinase (pi3k)/akt signalling pathway was determined using immunoassays and specific inhibitors[lh1] . apelin and adma concentrations were measured in cell culture supernatants using an enzyme-linked immunosorbent assay and a liquid chromatography-tandem mass spectrometry assay. results: treatment with apelin resulted in a reduced expression of the apelin receptor (aplnr) on hpmecs suggesting a negative feedback mechanism. apelin directly influenced the l-arginine/no pathway by increasing the expression of ddah1 and ddah2 enzymes. thus, the concentration of adma was decreased in hpmecs supernatant following treatment with apelin. the effect of apelin could be abrogated by modulation of the pi3k/akt pathway. conclusion: apelin modulates the l-arginine/no pathway and mediates enhanced degradation of adma via an upregulated expression of ddah 1 and 2 enzymes. the pi3k/akt pathway might play a decisive role in regulation of the effect of aplein. an apelin receptor agonist could be a novel and promising therapeutic option for ipah treatment. background and purpose: there is presently no proven pharmacological therapy for the acute respiratory distress syndrome (ards). recently, we and others discovered that the heptapeptide angiotensin (ang)-(1-7) shows significant beneficial effects in preclinical models of acute lung injury (ali). here, we aimed to identify the best time window for ang-(1-7) administration to protect rats from oleic acid (oa) induced ali. experimental approach: the effects of intravenously infused ang-(1-7) were examined over four different time windows before or after induction of ali in male sprague-dawley rats. hemodynamic effects were continuously monitored, and loss of barrier function, inflammation, and lung peptidase activities were measured as experimental endpoints. key results: ang-(1-7) infusion provided best protection from experimental ali when administered by continuous infusion starting 30min after oa infusion till the end of the experiment (30-240min). both pretreatment (-60-0min before oa) and short-term therapy (30-90 min after oa) also had beneficial effects although less pronounced than the effects achieved with the optimal therapy window. starting infusion of ang-(1-7) 90min after oa (late-term infusion) achieved no protective effects on barrier function or hemodynamic alterations, but still reduced myeloperoxidase and angiotensin converting enzyme activity, respectively. conclusions and implications. our findings indicate that early initiation of therapy after ali and continuous drug delivery are most beneficial for optimal therapeutic efficiency of ang-(1-7) treatment in experimental ali, and presumably accordingly, in clinical ards. airway epithelium functions as a physicochemical barrier against dust, air pollutants and other pathogens and plays a critical role in physiological and pathological processes including modulation of the inflammatory response, innate immunity and airway remodeling such as in human asthma, copd and equine recurrent airway obstruction (rao). models of the airway epithelia are, indeed, missing for the horse; thus, we established long-term equine bronchial epithelial cell cultures using the rock inhibitor y-27632 and cell growth and differentiation was characterized. bronchial epithelial cells (ebec) from adult horses were cultured in the presence and absence of 10 µm y-27632 under conventional and air-liquid-interface (ali) culture conditions. cell proliferation and differentiation were analyzed. formation of a functional epithelial barrier was investigated by transepithelial electric resistance (teer) measurement and immunocytochemical staining of the tight-junction-protein zonula occludens-1 (zo-1). under conventional culture, y-27632 induced higher growth rate of primary ebec and increased the passage number up to 5 passages with retained epithelial cell behavior. in the presence of y-27632, ebecs under ali showed higher teer values. expression of zo-1 correlated with the increase in teer, but in y-27632-treated ebec tight-junctionformation was more rapid, indicating accelerated differentiation, as well h/e-staining and scanning electron microscopic imaging showed a higher amount of cilia and microvilli and pas-positive cells. in conclusion, the data suggest that the rock inhibitor y-27632 facilitates long-term culture of equine bronchial epithelial cells which can be used to study airway disease mechanisms and to identify pharmacological targets. leishmaniasis is a neglected disease of tropical and subtropical regions with millions of people at risk of infection with severe consequences including death. current antileishmanial drugs exhibit serious side effects and also development of resistances is rising. this disease is caused by protozoal organisms from the genus leishmania. in their insect vector they exist in the promastigote form, while in the mammalian host they survive as amastigotes inside the phagolysosomes of macrophages. this makes a specific pharmacotherapy complicated. due to the success of artemisinin in malaria therapy, it was of interest whether endoperoxides are also useful to treat leishmaniasis. in a previous study we demonstrated that ascaridole, an endoperoxide from chenopodium ambrosioides, can cure cutaneous leishmaniasis in a mouse model and exhibited ic 50 values for the viability in the low micromolar range [1] . even though in chemical model systems some basic ideas about the mechanism of activation of these endoperoxides exist, in biological systems including leishmania parasites this activation step has never been demonstrated. therefore, we set up experiments to identify primary drug intermediates formed from ascaridole by activation in leishmania tarentolae promastigotes using electron spin resonance spectroscopy in combination with spin trapping methods. ascaridole was activated in a cell-free system by fe 2+ . the radicals were trapped by 2-methyl-2-nitrosopropane (mnp). the resulting esr spectra consisted of the triplet of duplets. spectral simulations revealed coupling parameters of a n = 16.8 g, and a h = 1.8 g. these coupling constants are compatible with iso-propyl radicals as primary intermediates. in the cellular system, consisting of leishmania tarentolae promastigotes, instead of mnp the less cytotoxic 5,5-dimethyl-1pyrroline-n-oxide (dmpo) was used for spin trapping. without addition of fe 2+ a six line esr signal was observed. spectral simulations of the dmpo spin adduct revealed coupling constants of a n = 16.1 and a h = 24.6 g. according to previously published data [2] from other spin trapping experiments, this corresponds to the formation of carboncentered radicals from ascaridole by leishmania parasites. additional experiments using iron chelators and antioxidants as well as a comparison with the endoperoxide artemisinin were performed. in summary, this study for the first time demonstrated the activation of the endoperoxide ascaridole by a protozoal organism to its active intermediate as a prerequisite to understand its mechanism of action. [1] l. monzote, j. pastor, r. scull, and l. gille. antileishmanial activity of essential oil from chenopodium ambrosioides and its main components against experimental cutaneous leishmaniasis in balb/c mice. phytomedicine 21:1048-1052, 2014. nitric oxide (no), produced by the inducible nitric oxide synthase (inos) has many functions in physiological and pathophysiological pathways. after induction of inos expression by cytokines and other agents the enzyme produces high amounts of no in a ca 2+ -independent way. this high no production can have beneficial microbicidal, antiparasital, antiviral and antitumoral effects. in contrast, aberrant inos induction may have detrimental consequences and seems to be part of many diseases such asasthma, arthritis, multiple sclerosis, colitis, psoriasis, neurodegenerative diseases, tumor development, transplant rejection or septic shock. analysis of the human inos-mrna structure revealed the existence of an upstream open reading frame (µorf) and a putative internal ribosome entry site (ires) in the 5' untranslated region (5'utr) in front of the start codon of the inos coding sequence (cds). to analyze the function of the µorf and the putative ires we cloned different egfp and luciferase reporter constructs and transfected them into the human colon carcinoma cell line dld1. using a plasmid construct with the µorf fused with the egfp cds, we could show that the µorf can be translated. however, compared to the positive control plasmid less egfp was produced, which can be explained by a weak kozak sequence of the µorf. blocking the mrna cap-dependent translation by cloning a stem loop structure in front of the inos 5'utr within a luciferase reporter plasmid led to a remarkable loss of luciferase production. thus, the expression of inos seems to be cap-dependent. furthermore, transfection experiments with dld1 cells using constructs coding for a bicistronic renilla-firefly luciferase mrna showed that there is no ires in front of the inos cds. taken together, the inos expression seems to be cap-dependent and without influence of an ires, while the µorf is translatable. therefore we speculate that inos expression is only possible due to a leaky scanning mechanism depending on the weak kozak sequence of the µorf. objectives: vascular oxidative stress is considered a pathophysiologic factor promoting cardiovascular diseases such as coronary artery disease, heart failure, diabetes and hypertension. there are several sources of superoxide in vascular smooth muscle and endothelial cells but whether an impairment of the catalytic function of enos and thus generation of oxidative stress is involved in blood pressure (bp) regulation and/or the development of hypertensive disease states is unknown. methods: we generated a mutant enos in which one of the two essential cysteines required for the coordination with the central zn-ion, correct dimer formation and normal activity is replaced by alanine (c101a-enos). normal enos (enos-tg) or a novel dimer-destabilized c101a-enos described previously (antioxid redox signal. 2015 sep 20; 23(9) :711-23) were introduced in c57bl/6 in an endothelial-specific manner. mice were monitored for enos expression and localization, aortic relaxation, systolic blood pressure, levels of superoxide and several post-translational modifications indicating activity and/or increased vascular oxidative stress. some groups of mice underwent voluntary exercise training for 4 weeks or treatment with sod mimetic tempol. results: c101a-enos-tg showed significantly increased superoxide generation, protein-and enos-tyrosine-nitration, enos-s-glutathionylation, enos 1176/79 phosphorylation and amp kinase (ampkα) phosphorylation at thr172 in aorta, skeletal muscle, left ventricular myocardium and lung as compared to enos-tg and wild type (wt) controls. the localization of enos-c101a-tg was restricted to endothelium as evidenced by immunohistochemically staining for enos and an endothelial-specific marker cd31. exercise training increased phosphorylation of enos at ser 1176/79 and of ampkα at thr 172 in wt but not in c101a-enos-tg. aortic endothelium-dependent and endothelium-independent relaxations were similar in all strains. in striking contrast, c101a-enos-tg displayed normal blood pressure despite higher levels of enos, while enos-tg showed significant hypotension. tempol completely reversed the occurring protein modifications and significantly reduced bp in c101a-enos-tg but not in wt controls. conclusions: by means of a novel transgenic mouse model we demonstrated that vascular oxidative stress generated by endothelial-specific expression of a dimerdestabilized variant of enos selectively prevents bp reducing activity of vascular enos, while having no effect on aortic endothelial-dependent relaxation. these data suggest that oxidative stress in microvascular endothelium may play a role in the development of essential hypertension. the herbal medicinal product myrrhinil-intest ® consists of myrrh, chamomile flower dry extract and coffee charcoal. clinical data prove the effectiveness of this herbal preparation for inflammatory intestinal disorders. to further investigate the anti-inflammatory potential of the single components as part of a multi-target principle, an ethanolic (my) and aqueous (mya) myrrh extract, ethanolic chamomile flower extract (ka) and aqueous coffee charcoal extract (cc), were examined in an in vitro tnbs inflammation model using rat small intestinal preparations. the effect of the plant extracts on tnbs induced inflammatory damage was characterised based on tnfα-gene expression analysis, isometric contraction measurement and histological analysis. furthermore, tnfα-release from lpsstimulated thp-1 cells was determined. budesonide was used as positive control. additionally, microarray gene expression analysis was performed in lps/ifnγ stimulated native human macrophages to determine potential underlying mechanisms. the tnbs-induced overexpression of tnfα-mrna was reduced after ka (0.1 mg/ml) and mya (1 mg/ml) treatment down to 24% and 16% resp.; tnbs-induced loss of contractility and reduction of mucosal layer thickness was inhibited after ka (3 mg/ml) treatment by 26% and 25% resp.; after mya (0.1-1mg/ml) treatment by 17% and 44% resp. lps-induced tnfα release from thp-1 cells was inhibited concentrationdependently by my (ic50 = 60.65 μg/ml; 97% inhibition), ka (ic50 = 439 μg/ml; 71% inhibition) and cc (ic50 = 1886 μg/ml; 44% inhibition). furthermore, ka (200 µg/ml) and cc (500 µg/ml) inhibited the lps/ifnγ-induced expression of genes associated with chemokine signalling up to 100-fold (for cxcl13). the presented study demonstrates further evidence for anti-inflammatory properties of the herbal components which contribute to the reported clinical effectiveness. introduction: the purine nucleoside adenosine, which is involved in a variety of physiological functions, regulates immune and inflammatory responses and acts as a modulator of gut functions. although it is present at low concentrations in the extracellular space, stressful conditions, such as inflammation, can markedly increase its extracellular level up to micromolar range. by activation of different receptor subtypes adenosine is able to induce anti-inflammatory or pro-inflammatory impacts. aim: the current study examined the impact of adenosine a2a receptors (a2ar) and adenosine a2b receptors (a2br) to regulate contractility in untreated and inflamed rat colon preparations using a specific a2ar agonist (cgs 21680) and an a2br antagonist (psb-1115) on acute inflammation in rat colon preparations. further it focused on interactions of the multi-herbal drug stw 5 with a2ar as a possible mechanism of the protective effect of stw 5 in gastrointestinal disorders. methods: inflammation was induced by intraluminal instillation of 2,4,6-trinitrobenzene sulfonic acid (tnbs). contractions were measured isometrically in an organ bath set up. gene expression was determined using rt-pcr. radio ligand binding assays (competition experiments) were carried out with rat brain homogenates. morphological changes were estimated after van gieson staining. results: all four adenosine receptor subtypes were expressed in untreated colon preparations. activation of a1, a2b, and a3 receptor with specific agonists reduced the acetylcholine (ach, 10µm)-induced contractions, while activation of a2br enhanced it. after incubation with tnbs morphological damages in colonic mucosa and muscle walls were detectable followed by reduced ach-contractions. the tnbs-mediated decrease of ach-contractions as well as the morphological damages were partially normalized by co-incubation of tnbs with cgs 21680 (10µm) or with psb 1115 (100µm). the same effects with smaller intensity were found for stw 5 (512 µg/ml) in female but not in male colon preparations. these results are in accordance with ligand binding studies indicating that stw 5 interact with the a2ar. conclusion: anti-inflammatory mechanisms and cell protective actions of stw 5 are partly due to the interaction with adenosine receptors. the results give a clear-cut correlation with symptom improvements in clinical trials and thereby highlight the relevance of stw 5 as a therapeutic approach in ibs. (allescher 2006) . therefore, a multi-target approach is a promising therapeutic strategy, as is exemplified by stw 5(ottillinger et al. 2013) . stw 5 (iberogast®) is a fixed combination of nine plant extracts with iberis amara (stw 6) as one of its components. it is successfully used for treatment of functional dyspepsia and irritable bowel syndrome (ibs). to allow an overview of targets addressed by stw 5 and the role of its components in relation to the different forms and causes of functional gi diseases, an evaluation of the data, which have been gained from more than 150 pharmacological tests, is needed. all data from studies including stw 5 alone, or stw 5 and its components, were retrieved and sorted according to types of study models (human and animal systems, animal disease models, gi-preparations, cell cultures, in vitro-systems) and respective etiologic mechanisms related to fgds and then visualized in the form of 2d histograms (lorkowski et al. 2015) . results: more than 150 pharmacological tests indicated anti-oxidative activity, electrophysiological effects, ulcer protection, anti-inflammatory actions, pro-kinetic and spasmolytic effects as well as reflux and acid reduction. moreover, the analysis indicated that the components of stw 5 contribute differently to the overall effect of stw 5. altogether, the evaluation of the data shows that stw 5 is active in response to multiple etiologic factors involved in fgds, especially functional dyspepsia and irritable bowel syndrome, and to which extent the herbal extract components of the combination are relevant for the different mechanisms of action and their translation to clinical efficacy. conclusion: multi step clustering allows the transformation of complex data sets. it makes the allocation of specific actions to the different components of stw 5 manageable, so also giving support to its clinical use in patients with different symptoms. introduction: stw 5 ii has been recently developed in an effort to reduce the number of active extracts in the mother multi-component herbal preparation, stw 5 (iberogast ® , steigerwald arzneimittelwerk gmbh, darmstadt, germany) without affecting the overall therapeutic efficiency. stw 5 consists of a mixture of 9 standardized extracts: bitter candytuft (iberis amara), lemon balm (melissa officinalis), chamomile (matricaria recutita), caraway fruit (carum carvi), peppermint leaf (mentha piperita), liquorice root (glycyrrhiza glabra), angelica root (angelica archangelica), milk thistle (silybum marianum) and celandine herb (chelidonium majus), whereas stw 5 ii lacks the last 3 components. stw 5 was shown to be effective clinically to treat functional dyspepsia (1) and irritable bowel syndrome (2) and was shown experimentally to be effective to guard against the development of radiation induced intestinal mucositis (3) and in the management of ulcerative colitis (4) . the present study was initiated to show whether stw 5 ii with the reduced component extracts would also be as effective in the latter condition. this was induced in wistar rats by feeding them with 5% dextran sodium sulfate in drinking water for 1 week when lesions were observed in the colon evidenced by histological examination as well as colon shortening and reduction of colon mass index. this was associated with a rise in myeloperoxidase and a fall in reduced glutathione, glutathione peroxidase, and superoxide dismutase in colon homogenates as well as a rise in tnfα in serum. oral administration of stw 5 in doses of 2 and 5 ml/kg or stw 5 ii in a dose of 2 ml/kg for 1 week before and continued during dss feeding tended to normalize all the changes in a fashion comparable to sulfasalazine, used as a reference drug in a dose of 300 mg/kg. conclusions: the modified preparation, stw 5 ii thus proved to be as effective as stw 5, thereby reflecting its potential usefulness in ulcerative colitis possibly by virtue of its anti-inflammatory and anti-oxidant properties. (1) schmulson mj (2008) the emetic pathways include the action of neurotransmitters dopamine, serotonin and substance p in the emetic centers localized in the brainstem, area postrema and vagal nerve afferents. previous in vivo studies in beagle dogs revealed that the plant alkaloid lycorine potentially induce nausea and emesis. though antagonists of the tachykinin receptor 1 (maropitant) and serotonin receptor 3 (ondansetron) prevented lycorinemediated emesis, the molecular mechanism of nausea and vomiting remain still unknown. to study the mechanism of action of the emetic agents, we analyzed the effect of lycorine (direct activation of nk1) and channel opening (activation of 5ht3) on the intracellular calcium homeostasis (using fluorometric ca 2+ analysis) and cell proliferation rates in endogenously nk1 and 5ht3 receptor expressing cell lines as well as in cho and hek cells stably expressing the receptors. neither endogenously receptor expressing nk1 or 5ht3 cells nor receptor overexpressing cells showed calciumflux or calcium mobilization after stimulation with lycorine. furthermore, we are measuring the receptor number and subtypes using radioligand binding studies. it is planned, moreover, to obtain fluorescent labeled constructs of the nk1 receptor to gain insights into the involvement of receptor internalization which might mediate emesis. by characterizing these molecular principles of the nk1 and 5ht3 receptors, we are attempting to obtain more information in predicting drug-induced side effects such as nausea and emesis. the intestinal epithelium is completely renewed every 4-5 days. this process is driven by stem cells, which reside within specialized niches in the intestinal crypts and give rise to several differentiated cell types, including enterocytes, paneth, enteroendocrine, goblet and tuft cells. however, the molecular mechanisms that establish and maintain differentiated cell numbers and proportions remain largely unknown. here, we systematically analyzed the intestinal expression of semaphorins and plexins, which constitute a ligand-receptor system that plays central roles in cell-cell communication in various biological contexts. we identified plexin-b2 and its semaphorin ligands to be highly expressed in intestinal epithelial cells. genetic inactivation of plexin-b2 in intestinal organoids strongly reduced the number of enteroendocrine cells. our data suggest that semaphorin-plexin-b2 signaling promotes differentiation of intestinal epithelial cells towards the enteroendocrine lineage. the gastric epithelium contains several types of differentiated cells, including foveolar cells that produce mucus, parietal cells that secrete gastric acid and intrinsic factor, chief cells that synthesize pepsinogen and gastric lipase, and enteroendocrine cells that release different hormones. these differentiated cell types all originate from multipotent stem cells, yet little is known about how this differentiation process is regulated on a molecular level. the gap protein rasal1 controls the activity of small gtpases of the ras family, and its expression levels have been shown to inversely correlate with progression of stomach cancers. however, functional studies on the physiological role of rasal1 in the gastric epithelium are lacking. here, we established and characterized a mouse line with inactivation of the rasal1 gene. we observed that these mice showed increased numbers of enteroendocrine cells in the gastric mucosa. conditional inactivation of rasal1 in enteroendocrine cells, using a mouse line in which cre expression is driven by the atoh1 promoter, further corroborated that rasal1 expression in enteroendocrine cells determines enteroendocrine cell numbers. these findings identify rasal1 as a regulator of gastric epithelial cell differentiation. ). the present study investigates the impact of two faah inhibitors (arachidonoyl serotonin [aa-5ht], urb597) on a549 lung cancer cell metastasis and invasion. lc-ms analyses revealed increased levels of faah substrates (aea, 2-ag, oea, pea) in cells incubated with either faah inhibitor. in athymic nude mice faah inhibitors were shown to elicit a dosedependent antimetastatic action. in vitro, a concentration-dependent anti-invasive action of either faah inhibitor was demonstrated, accompanied with upregulation of tissue inhibitor of matrix metalloproteinases-1 (timp-1). using sirna approaches, a causal link between the timp-1-upregulating and anti-invasive action of faah inhibitors was confirmed. moreover, knockdown of faah by sirna was shown to confer decreased cancer cell invasiveness and increased timp-1 expression. inhibitor experiments point toward a decisive role of cb 2 and transient receptor potential vanilloid 1 in conferring the anti-invasive effects of faah inhibitors and faah sirna. finally, antimetastatic and anti-invasive effects were confirmed for all faah substrates. collectively, the present study provides first-time proof for a pronounced antimetastatic action of the faah inhibitors aa-5ht and urb597. as underlying mechanism of its anti-invasive properties an upregulation of timp-1 was identified. regenerative activity in tissues of mesenchymal origin depends on the migratory potential of mesenchymal stem cells (mscs). the present study focused on inhibitors of the enzyme fatty acid amide hydrolase (faah), which catalyzes the degradation of endocannabinoids (anandamide, 2-arachidonoylglycerol) and endocannabinoid-like substances (n-oleoylethanolamine, n-palmitoylethanolamine). in boyden chamber assays, the faah inhibitors, urb597 and arachidonoyl serotonin (aa-5ht), were found to increase the migration of human adipose-derived mscs. lc-ms analyses revealed increased levels of all four aforementioned faah substrates in mscs incubated with either faah inhibitor. following addition to mscs, all faah substrates mimicked the promigratory action of faah inhibitors. promigratory effects of faah inhibitors and substrates were causally linked to activation of p42/44 mitogen-activated protein kinase (mapk), as well as to cytosol-to-nucleus translocation of the transcription factor, peroxisome proliferator-activated receptor α (pparα). whereas pparα activation by faah inhibitors and substrates became reversed upon inhibition of p42/44 mapk activation, a blockade of pparα left p42/44 mapk phosphorylation unaltered. collectively, these data demonstrate faah inhibitors and substrates to cause p42/44 mapk phosphorylation, which subsequently activates pparα to confer increased migration of mscs. this novel pathway may be involved in regenerative effects of endocannabinoids whose degradation could be a target of pharmacological intervention by faah inhibitors. background: the hematopoietic disorder chronic myeloid leukemia (cml) is one of the most extensively studied neoplasms. it is caused by translocation between chromosomes 9 and 22 leading to the formation of the philadelphia chromosome and the bcr-abl fusiongene. first-line targeted therapy is still the tyrosine-kinase inhibitor imatinib (im), which led to tremendous success in treatment. however, the amount of therapeutic resistances is increasing, caused either by bcr-abl-dependent mechanisms (e.g. bcr-abl amplification/overexpression, point mutations) or bcr-ablindependent mechanisms. these might be linked to alterations in drug transporter expression or particularly, microrna-expression levels. in our previous study, we analyzed the changes of microrna expression profiles during the development of imresistances in the leukemic cell line k562. an inverse correlation of mir-212 expression and protein levels of the efflux transporter atp-binding cassette transporter g2 (abcg2) was observed in cells resistant to different im-concentrations, pointing to a relation of mir-212 to im-resistance. hence, we investigate in current studies, how the influence of mir-212 on im-sensitivity could be explained. methods: we transfected k562 cells, sensitive treatment-naïve cells and cells resistant to various im-concentrations, either with mir-mimic pre-mir-212 or inhibitory anti-mir-212, challenged them with im and analyzed effects on cell viability, activation of apoptosis and cell death using wst-1-, caspase glo 9-assay and cell counting. in addition, we analyzed changes in abcg2 expression using flow cytometry and qrt-pcr and investigated alterations in im-efflux using hplc and hoechst efflux assay. results: under im-treatment, sensitive k562 showed an effect of mir-212-inhibition using anti-mir-212. this led to a significant promotion of cell survival apparent on the level of respiratory chain function (p<0.01) and cell membrane integrity and reduced capase-9 activity (p<0.05). furthermore, these mirna-effects are dose-dependent as confirmed in concentration row-experiments. regarding transport and abcg2 expression, we found that 2 µm im-resistant k562 do not express higher amounts of abcg2, but showed higher transport rates of im or the abcg2-substrate hoechst 33342. conclusions: overall, these experiments indicate that mir-212 does not only affect abcg2-expression, but also influences cell sensitivity to im in a more direct manner. further analysis will now be performed to reveal the underlying mechanism, how cell sensitivity to im is altered and if these effects occur due to a direct regulation of abcg2. in summary, these findings could be relevant in cml-therapy, overcoming imresistances with a better understanding of mirna-and drug transporter alteration in cml. acknowledgments: we would like to thank all the authors for their contribution to this project. this work was funded by the university hospital schleswig-holstein. oxidized silicon nanoparticles and iron oxide nanoparticles for radiation therapy s. klein radiation therapy often combined with surgery and/or chemotherapy is applied to more than 50 % of patients at some point of their treatment. the cytotoxic effects of ionizing radiation occur from their ability to produce dna double-strand breaks through the formation of free radicals within cells. however, the curative potential of radiotherapy is often limited by intrinsic radio resistance of cancer cells and normal tissue toxicity. to overcome this resistance and enhance the effectiveness of ionizing radiation, radio sensitizers are used in combination with radiotherapy. in our studies we used amino functionalized, oxidized silicon nanoparticles (sinp), superparamagnetic iron oxide nanoparticles (spion) and iron doped silicon nanoparticles (fe(1%)-sinp) to increase the formation of reactive oxygen species (ros) in cells. cancer and tissue cells loaded with the various nanoparticles were irradiated with a single dose of 1-3 gy using a 120 kv x-ray tube. after irradiation, the formation of the different ros species including superoxide, hydroxyl radicals and singlet oxygen was investigated. sinps with sizes around 1 nm can easily cross the cell and nuclear membrane. the positively charged amino functionalized sinps stick in all membranes as well in those of the mitochondria. irradiation of the mitochondria may cause the depolarization of the mitochondrial membrane, which enables the release of cytochrome c and simultaneously, an inhibition of the respiratory chain, which leads to an increased generation of superoxide. amino functionalized sinps, as being embedded in the outer mitochondrial membrane, evidently enhance the depolarizing effect of the x-ray radiation on the mitochondria and therefore increase the concentration of superoxide. [1] oxidized sinps with larger sizes accumulate in the cytoplasm and generate mainly singlet oxygen after irradiation. spions enter the cells via endocytosis, whereas the uncoated spions remain in the vesicles and the citrate coated spions accumulate in the cytoplasm. cells loaded with citrate coated spions show no higher ros concentration than in media-cultured cells. but after irradiation, the ros formation increased drastically. this enhancing effect is explained with the impact of x-rays onto the surface of spions which is due to the destruction of surface structures. the freed spion surface contains easier accessible iron ions. this ions can participate in the fenton and haber-weiss chemistry and thus, catalyze the hydroxyl radical formation. [2] 1 to 5 % iron doped sinp increase the formation of hydroxyl radicals as well as the generation of singlet oxygen after irradiation. chronic pain in response to tissue damage (inflammatory pain) or nerve injury (neuropathic pain) is a major clinical health problem, affecting up to 30% of adults worldwide. currently available treatments are only partially susceptible and are accompanied with therapy limiting side effects. thus it is important to elucidate molecular mechanisms of pain signaling in detail to obtain new insights in potential future therapies. recent data indicate that hydrogen sulfide (h 2 s) contributes to the processing of chronic pain, however pro-as well as antinociceptive effects have been described so far. moreover the sources of h 2 s production in the nociceptive system are only poorly understood. here we investigated the expression of the h 2 s releasing enzyme cystathionine g-lyase (cse) in the nociceptive system and characterized its role in chronic pain signaling using cse deficient mice. paw inflammation and peripheral nerve injury led to upregulation of cse expression in dorsal root ganglia. however, conditional knockout mice lacking cse in sensory neurons as well as global cse knockout mice demonstrated normal pain behaviors in inflammatory and neuropathic pain models as compared to wt littermates. thus, our results suggest that cse is not critically involved in chronic pain signaling in mice and that sources different from cse mediate the pain relevant effects of h 2 s. this work was supported by the deutsche forschungsgemeinschaft (sfb815-a14) and in part by loewe-schwerpunkt "anwendungsorientierte arzneimittelforschung". heinrich-heine-universität, institut für toxikologie, düsseldorf, germany 2 heinrich-heine-universität, urologie, düsseldorf, germany background: cisplatin (cispt) is frequently used in the therapy of advanced stage urothelial cell carcinoma (ucc). yet, inherent and acquired drug resistance limits the clinical use of cispt. here, we comparatively investigated the response of epithelial-like (rt-112) and mesenchymal-like (j-82) uc cells following cispt treatment. methods: upon selection with equitoxic doses of cispt for months, we obtained cispt resistant variants (rt-112 r , j-82 r ). cell viability was measured using the alamar blue assay. cell cycle distribution was analysed by flow cytometry. immunocytochemistry was used to quantify the number of nuclear γh2ax and 53bp1 foci representing dna double strand breaks (dsbs), while western blot was used to unravel the role of dna damage response (ddr) to acquired cispt resistance. qrt-pcr was performed to analyse the mrna expression of genes associated with cispt resistance. j-82 and j-82 r cells were treated with different concentrations of lovastatin and selected ddr inhibitors to elucidate their influence on cell viability. results: untreated rt-112 cells showed an about 2-3-fold higher resistance to cispt than j-82 cells. both cell lines differed in the expression pattern of genes that are associated with cispt resistance. rt-112 r and j-82 r revealed a 2-3-fold increased cispt resistance as compared to the parental cells. during the selection procedure, we observed that acquired cispt resistance goes along with morphological alterations that resemble epithelial mesenchymal transition (emt). cell cycle analysis of rt-112 r cells disclosed a reduced apoptosis and enhanced g2/m arrest following cispt exposure as compared to rt-112 wild-type cells. by contrast, induction of cell death was similar in j-82 and j-82 r cells. notably, j-82 r cells showed a reduced formation of cispt-induced dsbs. correspondingly, the related ddr was diminished in j-82 r as compared to their parental cells. this was not found when ddr was comparatively analysed between rt-112 r and rt-112 cells. data obtained from qrt-pcr analysis indicate that different mechanisms contribute to acquired drug resistance of j-82 r and rt-112 r . unexpectedly, j-82 r and rt-112 r shared the upregulation of xaf-1. treatment of j-82 r cells with statins and protein kinase inhibitors revealed an enhanced sensitivity to pharmacological inhibition of chk-1 and, moreover, re-sensitization to cispt by chk-1 inhibitor. based on the data we suggest that mechanisms of acquired cispt resistance of epithelial and mesenchymal uc cell lines are different with apoptosisrelated mechanisms appear to be more relevant for epithelial-like rt-112 cells and ddr-related mechanisms dominating cispt susceptibility in mesenchymal-like j-82 cells. furthermore, our findings indicate that chk-1 might be an appropriate target to deal with acquired cispt resistance in ucc. in many patients, gastric cancer treatment with conventional cytostatic agents shows only limited clinical response. novel therapeutics, which inhibit rtk signaling by targeting c-met or her family receptors, have demonstrated some efficacy; however, primary resistance of gastric cancer cells against these inhibitors is still a major problem. in the present study we investigated the mechanism of heregulin (hrg)-promoted survival of gastric cancer cells after treatment with c-met inhbitors or sirna-mediated downregulation of c-met. we found that hrg treatment of gastric cancer cells with a c-met amplification partially rescued the cells from the antiproliferative effects of pharmacological c-met inhibition or sirna-mediated downregulation of c-met. moreover, c-met inhibition or downregulation led to an induction of her3 expression on mrna and protein level, whereas other her family receptors were unaffected. downregulation of her3 impaired the hrg-mediated rescue of cell survival upon c-met inhibition. in other tumor entities the chromatin organizer special at-rich sequence-binding protein 1 (satb1) has been described as a regulator of her family receptor expression involved in adaptive responses of tumor cells. thus, we investigated the contribution of satb1 in the upregulation of her3 after c-met inhibition. of note, c-met inhibitors as well as c-met-specific sirnas markedly induced satb1 expression in gastric cancer cells, and the downregulation of satb1 by sirnas completely prevented the induction of her3 upon c-met inhibition. in contrast, her1 or her2 expression levels were not affected by satb1-specific sirnas. the function of satb1 as transcriptional regulator is controlled by its phosphorylation status, which in turn is modulated by pkc activity. thus, we also tested the effect of pkc inhibitors on her3 expression after c-met inhibition. interestingly, the upregulation of her3 in gastric cancer cells was significantly reduced by pkc inhibitors. to summarize, satb1 and pkc are critically involved in the regulation of her3 expression in gastric cancer cells after treatment with c-met inhibitors and the oncogene her3 plays a crucial role for tumor cell survival in this context. thus, inhibition of pkc or satb1 may help to overcome resistance against c-met inhibition in this tumor entitiy. in the rising field of nanomedicine, development of new approaches in diagnosis and treatment of cancer is a challenging task. typically, a nanocarrier is synthesized and linked to functional compounds displaying either diagnostic or therapeutic effects in cancer models. recently, nanomaterials combining both diagnostic and therapeutic properties, so-called 'theranostics', became of primary interest. here we used a human albumin-polyethylene glycol (peg) copolymer (hsa) as a theranostic platform for molecular integration of the chemotherapeutic drug doxorubicin (dox) and the magnet resonance imaging (mri) contrast agent gadolinium (gd) yielding gd-hsa-dox nanoparticles. besides in vitro testing, which demonstrated cytotoxic efficacy of gd-hsa-dox, we used the chorioallantoic membrane (cam) of fertilized chick eggs as a preclinical xenotransplantation model. the cam assay, which in legal terms does not represent an animal experiment, allows testing of compounds in an in vivo setting. this model is particularly helpful to narrow the gap between in vitro and in vivo applications in rodents, because it can help to reduce number of elaborate experiments with typically nude mice, and it reduces or even avoids exposure of those animals to adverse effects and distress. treatment-resistant mda-mb-231 breast cancer cells stably transfected with luciferase were xenotransplanted onto the chorioallantoic membrane. after formation of solid breast cancer xenografts, gd-hsa-dox was injected intravenously and its antiproliferative effect was evaluated by ivis imaging of luciferase activity and by immunohistochemical analysis of the tumor xenografts for the ki-67 proliferation antigen. in comparison to conventional dox, gd-hsa-dox showed increased antiproliferative efficacy and reduced general toxicity in the cam assay. on the basis of these findings, a rodent model was established, where the mda-mb-231 breast cancer cells were orthotopically xenotransplanted into the mammary fat pads of female nmri nu/nu mice. in this model, we further investigated biocompatibility, as well as diagnostic and therapeutic properties of the engineered nanomaterial. after repeated administration of gd-hsa-dox into the tail vein of the animals, biocompatibility of gd-hsa-dox was confirmed by uncompromised liver, kidney and hematopoietic parameters. to warrant diagnostic properties, accumulation of the nanomaterial in tumor tissue is indispensable. by small animal mri of gd, kinetics of intravenously applied gd-hsa-dox in tumor tissue was monitored. an enhancement of the engineered nanomaterial in tumor tissue was detected for up to 47 h after injection indicating successful enrichment of gd-hsa-dox within the tumor tissue, which can be ascribed to the enhanced permeability and retention (epr) effect observed in the microenvironment of many solid tumor tissues. we are currently investigating the antitumor efficacy of gd-has-dox in this mouse model and preliminary data seem to indicate a dose-dependent anticancer effect. supported by the volkswagenstiftung. tubulin-binding agents are the most important anti-tumoral drugs. due to the side effects and the development of resistances, the discovery of new agents is still of importance. recently, pretubulysin (pt), a naturally occurring precursor of the myxobacterial compound tubulysin, was identified as a novel tubulin-binding compound. in the dfg research group for 1406, pt was characterized as anti-tumoral, antiangiogenic and vascular-disrupting compound. moreover, pt was also found to inhibit the formation of metastases in vivo. aim of the present study was to gain first insights into the mechanisms underlying this anti-metastatic effect by investigating the influence of pt on the interaction of endothelial and tumor cells in vitro. pt treatment of primary human endothelial cells (huvecs) strongly increased the adhesion of breast cancer cells (mda-mb-231) on huvecs, but limited their transmigration through the endothelium (transwell assay). based on this data, the gene expression of presumably involved adhesion molecules was determined by qrt-pcr: icam-1, vcam-1, e-selectin, n-cadherin, and galectin-3. moreover, the chemokine system cxcl12/cxcr4 was analyzed. it could be demonstrated that the mrna level of endothelial n-cadherin is upregulated by pt. while total protein expression of ncadherin was enhanced in pt treated huvec, its surface expression was not largely influenced by pt (western blot, flow cytometry). in line with this, blocking endothelial ncadherin by a neutralizing antibody revealed that this protein is not involved in ptevoked tumor cell adhesion. interestingly, pt strongly augmented the mrna and protein expression of cxcl12 in huvecs (qrt-pcr, western blot), whereas its endothelial secretion was not affected by pt (elisa). an autocrine action of cxcl12 could be excluded, since blocking the cxcl12 receptor cxcr4 on endothelial cells with plerixafor did not influence cancer cell adhesion. by microscopic analyses, we observed that pt treatment causes transient gaps in the huvec monolayer, where tumor cells prefer to adhere. since β1-integrins on the tumor cells could mediate interactions between cancer cells and extracellular matrix proteins in the gaps (e.g. collagen), their influence in cell adhesion and transmigration assays was examined. both the pt-evoked increase in cell adhesion and decrease in transmigration was completely abolished when β1-integrins were blocked on mdas by a neutralizing antibody. these results indicate that the anti-metastatic action of pretubulysin might be based on the trapping of tumor cells on the endothelium. whether this effect is also relevant in vivo, will be analyzed in future studies using intravital microscopy. this work was supported by the german research foundation (dfg, for 1406, fu 691/9-2). introduction: tyrosine kinase inhibitors (tkis) for the treatment of non-small cell lung cancer (nsclc) patients harboring activating mutations in the epidermal growth factor receptor have shown prominent success. nevertheless, patients treated with tkis eventually acquire resistance and relapse (1). based on an evolutionary cancer model (2) , weekly high dose-pulsed tki regimens were proposed to delay resistance. using data from nsclc bearing mice treated with erlotinib at different dosing regimens, we developed a semi-mechanistic pharmacokinetic/pharmacodynamic model for erlotinib effects on tumor killing and resistance development. methods: data was available from experiments in xenograft mice bearing nsclc tumors (pc9 and hcc827 cell lines; both erlotinib sensitive) (3). plasma concentrations from two single-dose groups, 30mg/kg and 200mg/kg, were used for pharmacokinetic modeling. relative tumor volume changes in mice randomized to five dosing regimens (15mg/kg daily, 30mg/kg daily, 200mg/kg every 2 days, 200mg/kg every 4 days, or vehicle) was the pharmacodynamic endpoint. a tumor growth inhibition model was developed by testing linear, exponential and logistic models to account for the tumor growth kinetics, as well as fitting an emax model to explain the effect of exposure on killing the sensitive tumor cells, and resistance development. analysis was performed using nonmem 7.3. results: absorption was dose dependent, and a precipitate compartment accounted for dissolution limited absorption for the 200mg/kg dose. a 1-compartment model with first order elimination kinetics described distribution and elimination. to describe tumor volume changes, a tumor was assumed to be a mixture of sensitive and resistant cells (represented by distinct compartments and ordinary differential equations). exponential kinetics best described natural growth (doubling times: 13 and 52 days, for sensitive and fully resistant cells, respectively). a tumor was found to transit through a less sensitive phase before acquiring full resistance. an e max model (less than linear) best described effect on the sensitive cells (ec 50 =0.53μm for both cell lines), and on the partially sensitive transit phase (ec 50 =1.24μm and 3.00μm, for hcc827 and pc9 cell lines, respectively), urging to provide adequate trough erlotinib concentrations for optimal effects. conclusions & future perspectives: an exposure-driven tumor growth inhibition model accounting for the kinetics of resistance development was developed. the model emphasizes the need for establishing an adequate trough erlotinib concentrations to delay disease progression. extracts of the stem bark of ficus platyphylla (fp) have been used in traditional nigerian medicine to treat psychoses, depression, epilepsy, pain and inflammation. previous studies have revealed the analgesic and anti-inflammatory effects of fp in different assays including acetic acid-induced writhing, formalin-induced nociception, and albumin-induced oedema. in this study, we assessed the effects of the standardised extract of fp on hot plate nociceptive threshold and vocalisation threshold in response to electrical stimulation of the tail root in order to confirm its acclaimed analgesic properties. we also investigated the molecular mechanisms underlying these effects, with the focus on opiate receptor binding and the key enzymes of eicosanoid biosynthesis, namely cyclooxygenase (cox) and 5-lipoxygenase (5-lo). fp (i) increased the hot plate nociceptive threshold and vocalisation threshold. the increase in hot plate nociceptive threshold was detectable over a period of 30 min whereas the increase in vocalisation threshold persisted over a period of 90 min. (ii) fp showed an affinity for µ opiate receptors but not for δ or κ opiate receptors, and (iii) fp inhibited the activities of cox-2 and 5-lo but not of cox-1. we provided evidence supporting the use of fp in nigerian folk medicine for the treatment of different types of pain, and identified opioid and non-opioid targets. it is interesting to note that the dual inhibition of cox-2 and 5-lo appears favourable in terms of both efficacy and side effect profile. despite the fact, that the enormous economic burden and individual suffering caused by gastrointestinal infections permanently persists in developing and newly industrialized countries, healthcare systems in first world countries underestimated its significance for a long time. the alarming prevalence of multidrug-resistant gram-negative bacteria, combined with a high epidemic potential of gastrointestinal pathogens, however, demonstrates the urgent need for new antibiotics and antiinfectives worldwide. 2,5 million deaths per year were actually caused by acute diarrheal infections. the most common causative agents of acute diarrheal infections, amongst others, are yersinia enterocolitica, campylobacter jejuni, salmonella spp., shigella spp., escherichia coli, vibrio cholerae, and clostridium difficile. the established treatment based on antibiotics is mostly ineffective or may even have adverse side effects and result in prolonged shedding. in either way, antibiotic treatment also eradicates at least parts of the intestinal microbiome, and thereby disrupts colonization resistance, fosters overgrowth of pathogens and prolongs shedding times. therefore, the development of future drugs should be focused on highly specific antiinfectives, which enable a direct pathogenspecific treatment. one very promising strategy is the inhibition of the biogenesis of outer membrane virulence factors. due to the fact that many decisive virulenceassociated outer membrane proteins (omps) of gram-negative enteropathogens are substrates of the periplasmic chaperone sura exclusively, we developed a new assay format to determine sura in vitro chaperone activity. previous publications by behrens et al., 2001 and buchner et al., 1998 documented an assay to determine sura in vitro chaperone activity with extremely limited sensitivity and minimal detectable concentration, which was not suitable for high throughput screening (hts). we now developed a luciferase-based screening assay. this highly sensitive and robust test system has been validated extensively and now gives reliable output with an appreciable z-factor of > 0,6. in cooperation with the hzi braunschweig (germany) and the hzi saarbrücken (germany), we were able to screen over 7000 purified compounds and over 500 extracts of myxobacteria. during the ongoing screening period, the assay generated four validated primary actives, which corresponds to a positive hit rate of 0,05 %. additionally, we developed an elaborate follow-up strategy to validate positive hits, which includes a well-established mouse infection model. we are looking forward to escalate our screening efforts and would like to use this abstract to invite all scientist who are interested in testing compound/natural extract libraries for an activity against the target structure sura. the potential atypical antipsychotic and dopamine d 2 receptor partial agonist 2bromoterguride antagonizes phencyclidine-and apomorphine-induced prepulse inhibition and novel object recognition deficits in rats e. tarland objectives: schizophrenia is a disabling mental disorder affecting more than 21 million people worldwide. available medical therapies are effective in the treatment of psychosis and other positive symptoms, however come with considerable side effects and often fail to ameliorate cognitive deficits and negative symptoms of the disorder. the dopamine d 2 receptor partial agonist 2-bromoterguride (2-bt) has recently been shown to exhibit antipsychotic effects in rats without causing adverse side effects common to antipsychotic drugs [1]. to determine its atypical character in vivo, the ability of 2-bt to antagonize the disruptive effects of phencyclidine (pcp) and apomorphine on sensory motor gating was determined in the prepulse inhibition paradigm. the effect of 2-bt on cognitive deficits was assessed in the novel object recognition (nor) test after object recognition memory deficits were induced by pcp treatment. method: 10 week old male sprague-dawley rats were injected with 2-bt (0.1 or 0.3mg/kg; i.p.) followed by pcp (1.5mg/kg; s.c.) or apomorphine (0.5mg/kg; s.c.). prepulse inhibition was measured in two sound-proof startle chambers. the attenuating effect of 2-bt (0.1 or 0.3mg/kg; i.p.) on visual learning and memory deficits following subchronic administration of pcp (5.0mg/kg; i.p. twice daily for 7 days) was assessed in the nor task consisting of a 3min acquisition trial and a 3min retention trial separated by a 1h inter-trial interval. clozapine (5.0mg/kg; i.p) or haloperidol (0.1mg/kg; i.p) were used as positive controls. results: the dopamine d 2 receptor partial agonist 2-bt (0.3mg/kg) and the typical antipsychotic haloperidol successfully antagonized apomorphine-induced ppi-deficits. interestingly 2-bt also ameliorated the pcp-induced ppi-deficits to the same extent as the atypical antipsychotic clozapine. preliminary data from the nor test indicate that 2-btreduces subchronic pcp-induced cognitive deficits in novel object recognition analogous to clozapine. the disrupting effects of pcp on ppi are mediated by non-competitive antagonism at nmda sites indirectly influencing a series of neurotransmitter systems. our results indicate that 2-bt mediates actions at multiple neurotransmitter receptors as it successfully ameliorated both the pcp-and apomorphine-induced ppi disruptions in rats, showing an atypical antipsychotic character. furthermore, our preliminary results support the potential atypical antipsychotic effect of 2-bt as it restored performance in the nor test, a test with good predictive validity. due to the previously shown properties and antipsychotic-like effects of 2-bromoterguride [1], this substance may be a promising candidate for treatment of schizophrenic patients. ongoing experiments investigate the potency of 2-bt to improve social deficits following a sub-chronic pcp regime in rats. background and objectives: cannabinoid-1 receptor signaling increases the rewarding effects of food intake and promotes the growth of adipocytes, whereas cb2 possibly opposes these pro-obesity effects by silencing the activated immune cells that are key drivers of the metabolic syndrome. pro-and anti-orexigenic cannabimimetic signaling may become unbalanced with age because of alterations of the immune and endocannabinoid system. methods: to specifically address the role of cb2 for age-associated obesity we analyzed metabolic, cardiovascular, immune and neuronal functions in 1. 2-1.8 year old cb2 -/and control mice, fed with a standard diet and assessed effects of the cb2 agonist, hu308 during high fat diet in 12-16 week old mice. results: the cb2 -/mice were obese with hypertrophy of visceral fat, immune cell polarization towards pro-inflammatory sub-populations in fat and liver and hypertension, as well as increased mortality despite normal blood glucose. they also developed stronger paw inflammation and a premature loss of transient receptor potential responsiveness in primary sensory neurons, a phenomenon typical for small fiber disease. the cb2 agonist hu308 prevented hfd-evoked hypertension, reduced hfdevoked polarization of adipose tissue macrophages towards the m1-like proinflammatory type and reduced hfd-evoked nociceptive hypersensitivity but had no effect on weight gain. conclusion: cb2 agonists may fortify cb2-mediated anti-obesity signaling without the risk of anti-cb1 mediated depression that caused the failure of rimonabant. leishmaniasis is a neglected tropical disease caused by leishmania, eukaryotic protozoal organisms, which infect humans and other mammals. this disease is transmitted by sandflies of the genus phlebotomus. due to global warming the endemic region of these vectors expands further to northwards and threatens south european countries as well. the treatment of leishmaniasis is difficult due to toxicity and resistance development for current drugs. the so far unexplored inhibition of mitochondrial functions in leishmania by natural products or even food ingredients seems to be an interesting alternative. two food ingredients, resveratrol (res) and xanthohumol (xan), were widely studied in mammalian cells but little is known about their actions on protozoal parasites. therefore, we compared the influence of res and xan on the function of leishmanial and mammalian mitochondria. anti-leishmanial activities of xenobiotics were assessed in cell culture of leishmania tarentolae promastigotes (ltp), leishmania amazonensis amastigotes (laa) and compared to peritoneal macrophages from mouse (pmm) using viability assays. furthermore, mechanistic studies regarding mitochondrial functions were conducted in ltp, mitochondrial fractions isolated from ltp and bovine heart submitochondrial particles using oxygen consumption measurements, assays of individual mitochondrial complex activities, membrane potential and superoxide radical formation by photometry, fluorimetry and electron spin resonance spectroscopy. in ltp, xan inhibited the viability more effective than res (ic 50 : xan 23 µm, res 161 µm). likewise, xan and res demonstrated anti-leishmanial activity in laa (ic 50 : xan 7 µm, res 14 µm) while had less influence on the viability of pmm (ic 50 : xan 68 µm, res > 438 µm). in contrast to res, xan strongly inhibited oxygen consumption in leishmania. further studies demonstrated that this is based on the inhibition of the mitochondrial electron transfer complex ii/iii by xan which was less pronounced with res. however, xan also demonstrated inhibitory activity on mammalian mitochondrial complex iii. in addition, xan caused no decrease of the membrane potential in leishmanial mitochondria, while res resulted in mitochondrial uncoupling. neither xan nor res increased mitochondrial superoxide release in ltp. these data show that res, a major polyphenol from red wine, and xan, an ingredient of hop-containing beer, may have selective anti-leishmanial activity. tryptophan hydroxylase (tph) is the rate-limiting enzyme in serotonin (5-ht) biosynthesis. its two existing isoforms are exclusively expressed in the periphery (tph1), or the raphe nuclei of the brainstem (tph2) and the respective 5-ht populations are distinctly separated by the blood-brain barrier, offering the possibility to pharmacologically modulate central and peripheral functions in an independent manner. peripheral 5-ht is mainly produced by tph1-expressing enterochromaffin cells of the gut and taken up into platelets and transported in the blood stream. upon platelet activation, 5-ht is rapidly released and locally induces multiple effects, such as vasoconstriction, cell proliferation or fibrosis and is furthermore involved in the regulation of e.g. vascular tone, gut motility, primary hemostasis, insulin secretion and t-cell-mediated immune response. following the classical early drug development pathway, we developed a fluorescencebased tph activity assay and performed a high-throughput screening of about 37000 small chemical compounds. we discovered a novel class of tph inhibitors, which was thoroughly validated in a variety of in vitro assay setups. combining medicinal chemistry and x-ray crystallography, we further aimed to develop these inhibitors into preclinical drug candidates. to date we were able to generate and patent a series of novel tph inhibitors with optimized affinity and an in vitro ic 50 in the low nanomolar range. this novel class of tph inhibitors could potentially be used to treat a variety of disorders with aberrant peripheral 5-ht signaling, such as gastrointestinal disorders (e.g. irritable bowel syndrome, crohn's disease, various forms of diarrhea), cardiac valve diseases, pulmonary hypertension, chronic respiratory diseases and some neuroendocrine (carcinoid) tumors. primary hepatocellular carcinoma (hcc) is the most frequent type of liver cancer. therapeutic options are rare. beside sorafenib, a tyrosinkinase inhibitor, which is only used in end stage liver cancer, the surgical intervention is the only successful clinical treatment option. hence there is an urgent need to develop new therapeutic strategies and to identify new drugs for therapy of hcc. hcc often arises in fibrotic or cirrhotic liver, which is accompanied by a change of the extracellular matrix (ecm) composition. in addition it was shown that hepatoma cells express different integrins, which interact with ecm and intracellular cell signaling, compared to hepatocytes. snake venoms have gained increased attention, as it was shown that some of their enzymes and peptides directly act on tumor cells and their multicellular arrangement or indirectly by influencing the stroma environment of the tumor. aim of the present study was to investigate the effect of snake venoms on liver cancer related cell lines as well as their specific action on the ecm-integrin axis. the effects of the snake venoms vipera palestinae (vp), calloselasma rhodostoma (cr) and echis sochureki (es) on a cellular level (mtt, ldh release), on cell-cellconnections (caco2 permeability assay) and on cell-matrix-interactions (adherence test) were investigated. cell-matrix interactions were tested with an adhesion assay using collagen i (c-i), collagen iv (c-iv), fibronectin (fn) and laminin (lm) as ecm compounds. in our in vitro models we used hepg2 as a hcc tumor cell line and the fibroblast cell line fi301 as stroma simulation. additionally caco2 cells were used, a colon carcinoma cell line representing colorectal liver metastasis. the toxicity of snake venom on liver cancer related cell lines was determined in the range of 0.01 -100 µg/ml and plotted into dose response curves. the noaels were calculated from these dose response curves: vp: 0.5 µg/ml -cr: 1 µg/ml -es: 5 µg/ml. performance of the caco2-transwell permeability assay revealed no influence of the tested venom concentrations on the integrity of the cellular arrangement. investigations for integrin inhibition revealed that the venom from vp reduced adherence on lm coated plates and the venom of ec reduced adherence on lm and fn coated plates compared to untreated cells. there was no effect on the adherence on any matrix from the venom of cr observable. co-incubation of the snake venoms of vp and es (below or near noael concentrations) with 5-fluorouracil (5fu), which is used as a chemotherapeutic agent, caused a reduction of its ic50 values. the results indicate that components of vp and ec inhibit the formation of cell-matrixinteractions possibly acting as disintegrins. the co-incubation experiments demonstrated a synergistic effect of 5fu and snake venoms. further experiments should enable the isolation of therapeutic active venom compounds, identification of disintegrins and their role in synergistic mechanisms in liver cancer therapy. modulation of the blood-brain barrier with peptidomimetics to improve drug delivery s. dithmer after decades of research, the blood-brain barrier (bbb) still remains a major problem for successful delivery to the brain for the vast majority of drugs. the main component forming the bbb is the brain microvascular endothelium. the paracellular permeation is limited by tight junctions (tjs), a multiprotein complex composed of the members of the claudin family claudin-1, -3, -5, -12. claudin-5 is known to be the key tj protein tightening the bbb. therefore, claudin-5 has been selected as target to modulate the bbb. for this reason, drug enhancer peptides (peptidomimetics) were designed to modulate transiently claudin-5 and, thereby, permeabilize the bbb. by combining biochemical protein/peptide interaction and tissue culture methods, we identified, validated and optimized peptide sequences modulating claudin-5 containing barriers. the claudin-5 targeting peptides decreased the transcellular electrical resistance and increased the permeability through mdck-ii cell monolayers stably expressing yfpclaudin-5 and immortalized brain endothelial cells (bend.3). the peptides decreased the amount of claudin-5 and zo-1 at cell-cell contacts and changed the cell morphology from spindle-shaped to more round-shaped. all tested peptides showed no signs of toxicity on cell cultures and in vivo (intravenous injection). permeability measurements in mice proved enhanced permeation of na-fluorescein (376 da) through the bbb, which was confirmed by magnet resonance imaging of contrast agents (gd-dtpa, 547 da). in summary, we identified new peptides with the potential to enhance cerebral delivery of small molecules through the bbb. treatment of cerebral diseases is limited by the capability of pharmacologically active agents to penetrate the blood-brain barrier (bbb). this paracellularly tight diffusion barrier is formed by brain capillary endothelial cells. the paraendothelial cleft is sealed by tight junctions (tjs), a multiprotein complex. cerebral tjs predominantly consist of claudin-5 (cldn5) which tightens the bbb for molecules <800 da. consequently, cldn5 is a potential target for transient and size-specific modulation of the bbb to improve cns penetration for pharmaceutically active agents. in high throughput screening using a cldn5 assay, the barrier opener 1 (bo1) was identified as a cldn5 modulator. initially, a significant removal of cldn5 from the plasma membrane was shown by confocal microscopy using epithelial and endothelial cell lines. measurement of transcellular electrical resistance and of paracellular permeability using lucifer yellow (mw 521 da) demonstrated the effect of bo1. concentration dependent treatment (50-150 µm) of cell monolayers with bo1 reduced tightness of the tjs between some hours and 24 h. applying 2-hydroxypropyl-ß-cyclodextrin as a solubilizer, opening activityof bo1 became detectable in mice. due to short stability (< 2 h) of bo1 in the bloodplasma repeated administration (1.5 mg/kg i.v.) was required to induce significantly increased permeability of the bbb for na-fluorescein(mw 376 da). the small molecule bo1 is a promising new approach for transient opening of the bbb in vivo. further modification of the stability and solubility of bo1 is necessary to optimize its applicability. the complex of tight junction (tj) proteins is located between opposing epithelial or endothelial cells. tjs restrict the paracellular permeation of ions and other solutes. tricellulin (tric) tightens tricellular tjs (ttjs) and regulates bicellular tj (btj) proteins like claudins and occludin (occl). current data suggest an important role of ttjs at the blood-brain barrier (bbb). a main pharmacological problem is modulation of the bbb to improve drug delivery to the cns. therefore, tricsi has been developed as a peptide taken from tric to open tissue barriers specifically and transiently.initially, a recombinant protein was generated based on a sequence of an extracellular loop of tric, tagged with maltose binding protein. the fusion protein caused down-regulation of tric, internalization of both tric and occl (confocal laser scanning microscopy), and a significant decrease in transcellular electrical resistance (ter) of a human epithelial colorectal adenocarcinoma cell line. then, studies with the synthetic peptide tricsi indicated its capacity of cell barrier openingafter about 16 h of incubation with concentrations varying from 100 to 150 µmaffecting the membrane localization of tricand occl. barrier opening was proven by decreasing ter, increasing permeability coefficient of lucifer yellow (457 da) and fitc-dextran (10 kda); the localization of tric elongated from ttjs towards btjs and cldn1 was weakened at btjs.physiochemical properties of tricsiexamined by circular dichroism spectroscopy suggested ß-strand structure and no helical propensity. taken together, a tric-derived peptide has been identified increasing the paracellular permeability of tissue barriers and redistributing the cellular localization of tj proteins. tricsi is a novel, promising tool to overcome cerebral barriers with the potential to improve drug delivery to the cns. further experiments are needed to better understand the role of tric in tissue barriers as well as to clarify the mode of action of tricsi. introduction: lung transplantation has become an established treatment option for a variety of end-stage lung diseases, but the long-term survival is often disappointing. the leading cause of death is generally chronic rejection which is characterized by inflammation and fibrous obliteration of the small airways, progressively leading to a reduction of the airflow. the mouse heterotopic tracheal transplantation model is widely used as an experimental model to study the development of obliterative airway disease. despite its widespread application, the heterotopic transplantation model does have a number of limitations, as for example the lack of airflow. the present study provides a description of the orthotopic tracheal transplantation mouse model, which shares more similarities with transplant situation in humans, and provides the analysis of airway obliteration via micro ct and histological evaluation. methods: a seven-ring donor trachea from balb/c mice was implanted into the recipient c57bl/6 mice. c57bl/6 mice without transplantation were used as normal controls. donor c57bl/6 mice to recipient c57bl/6 mice were served as the isograft group. 42 days after transplantation, mice were scanned using an in vivo small animal µct (skyscan 1176). tracheal tissue was harvested and fixed in formalin, embedded in paraffin, cut and stained with hematoxylin and eosin (h&e) as well as sirius-red/fast-green. results and conclusions: histologic evaluation showed luminal narrowing with subepithelial inflammatory cell infiltrates and fibrosis, as well as partially damaged and flattened epithelium. the aerated volume of the allogeneic grafts, analyzed by micro ct was significantly reduced compared to the isogenic control grafts and normal controls. non-invasive imaging via micro ct may offer an option for longitudinal monitoring of the progression of obliterative airway disease as well as response to treatment. c. elegans is a well-established model organism to study the aging process as well as effects of various substances in vivo. its lifespan is regulated by multiple signaling pathways (e.g. insulin or mtor signaling), which are well conserved up to humans. the insulin/igf-1 pathway was the first pathway shown to effect ageing in animals. mutations that decrease the activity of daf-2 (igf1r) lead to a significant increase of lifespan accompanied by a decrease of age pigment accumulation in c. elegans. the relevant effector of the insulin/igf-1 pathway is the transcription factor daf-16 (hfoxo3a). inhibition of hmg-coa reductase (enzyme of mevalonate pathway) by statins, which are frequently used as cholesterol-lowering agents in the clinic, has been shown to attenuate protein prenylation and glycosylation. notably, prenylated-, membrane-bound small gtp-binding proteins are important for the regulation of the afore mentioned age-related signaling pathways like the insulin/igf-1 pathway. recently, a cohort study showed that a decreased mortality rate in humans between age 78 -90 correlates with statin treatment, but is independent of total cholesterol levels. as c. elegans harbors the mevalonate pathway, but the branch leading to cholesterol synthesis is missing, it is a well-suited model to study cholesterol -independent effects of statins on aging-associated phenotypes and the underlying molecular mechanisms. here, we show that exposure of c. elegans to statins substantially decelerated the accumulation of age pigments. while the level of age pigments roughly doubled in control animals, there was only a slight increase in the lovastatin group. the use of atorvastatin gave comparable results indicating a more general effect of the inhibition of the hmg-coa reductase. the retarded accumulation of age pigments could be partly phenocopied using an inhibitor of the small gtpase rac1 or using rnai against the hmg-coa reductase. a reduced level of age pigments is prognostic for an elevated mean lifespan (about 20%) in c. elegans. a post reproductive treatment with lovastatin, mimicking the use of statins in patients of advanced age increased the mean lifespan in c. elegans even further. in addition, we could show a mild reduction of fertility and a developmental delay as well as a marked increase in acute thermal stress resistance mediated by lovastatin. besides the reduced accumulation of age pigments and the increased lifespan these are phenotypes which are usually observed under accumulation of daf-16 overactivity. consequently we found an increased nuclear localization of daf-16 in the presence of lovastatin and lovastatin completely failed to reduce age pigments in a daf-16-ko mutant background. rt-qpcr brought jnk-1, a known activator of daf-16, into play as a possible effector induced by statins. this is currently under investigation. in summary, statin exposure induces a longevity phenotype in c. elegans, which might be daf-16 dependent. this findings indicates that a product of the mevalonate pathway might influence the insulin/igf-1 pathway and particularly the transcription factor daf-16. the high-fat diet (hfd)-fed, streptozotocin (stz)-treated rat model is one of the experimentally-induced animal models of diabetes. this model is often used to evaluate the antidiabetic activity of several agents. according to srinivasan et al. (2005) , prolonged exposure of high-fat diet leads to insulin resistance, and the development of diabetes occurs only in insulin-resistant hfd-fed rats following low dose stz, because the hfd-fed rats are already mildly hyperglycemic due to insulin resistance (1). in hfd/stz model, the rats are fed with high-fat diet for 2-4 weeks or for a relatively long time (≥ 3 months) in order to simulate the insulin resistance and/or glucose intolerance. after induction of diabetes with multiple or single low-dose of stz (30-35 mg/kg), some of the diabetic rats receive treatment (2) . in this way, the impact of treatment can be determined by comparing the differences between groups. despite the lack of methodological information concerning the feeding time in some studies, all rats should be allowed to continue to feed on their respective diets until the end of the study. but what would happen if the hfd was switched to normal pellet diet in these diabetic rats? in our experience, the feeding of npd for 4 weeks significantly decreased fbg in diabetic rats compared to hfd-fed diabetic rats (234.40 ± 42.71 mg/dl vs. 464.00 ± 23.88 mg/dl, p < 0.05). although diet regulation could not restore normal blood glucose, such a decrease was unexpected. in addition, the body weights of the npd-fed diabetic rats were significantly lower than the body weights of the hfd-fed diabetic rats (249 ±6.00 gvs. 288.00 ±4.41 g, p < 0.05). there was no significant difference in body weight between nondiabetic control rats and diabetic rats fed npd for 4 weeks. further details can be found in table 1 . diet regulation and weight loss may prevent, control and reverse diabetes. however, at later stages of the disease, it is difficult to improve blood glucose control without medication, because the disease progresses from insulin resistance to insulin deficiency (3) . according to some diabetes researchers, the amount of residual functional betacells mass is an important issue, and another important question is whether hfd/stz rat mimics an early or late stage of type 2 diabetes (4). these preliminary findings suggest the possibility that hfd/stz rat model may simulate the characteristics of early stage more than the final stage of type 2 diabetes, and hyperglicemia in the experimental model can partially reverse with diet regulation. references: 1. srinivasan, k., viswanad, b., asrat, l., kaul, c. l., ramarao, p. (2005) . combination of high-fat diet-fed and low-dose streptozotocin-treated rat: a model for type 2 diabetes and pharmacological screening. pharmacol res 52 (4): 313-320. 2. oztürk z, gurpinar t, vural k, boyacıoglu s, korkmaz m, var a. (2015) . effects of selenium on endothelial dysfunction and metabolic profile in low dose streptozotocin induced diabetic rats fed a high fat diet. biotech histochem 90 (7): 506-515. 3. franz, m. j. (2007) . the dilemma of weight loss in diabetes. diabetes spectr 20 (3) animal models are pivotal for studies of pathogenesis and treatment of movement disorders. dystonia, characterized by sustained or intermittent muscle contractions causing twisting movements/postures, is regarded as a basal ganglia disorder. the pathophysiology is however poorly understood. in mouse models of dyt1 dystonia, which is caused by a gag deletion in tor1a that encodes for the protein torsin a, ex vivo electrophysiological studies have shown an abnormal d2 receptor mediated release of acetylcholine from striatal interneurons. in these models, which do not exhibit a dystonic phenotype, the functional relevance of the increased d2 receptor mediated acetylcholine release has not been examined yet. the aim of present study was to (1) generate more powerful tests to detect behavioural alterations in the dyt1 knock-in mouse and to (2) examine the behavioral effects of the d2 receptor agonist quinpirole. for this purpose, a sequence of cognitive, motoric and sensorimotor tests were performed in this mouse model. only the adhesive removal test that explores sensorimotor connectivity revealed significant impairments in the dyt1 knock-in mice compared to controls. to induce a more characteristic and stronger phenotype, the "rotating beam test" was developed. this motoric test measures motor coordination and balance. interestingly, dyt1 knock-in mice showed significant motor deficits in the rotating beam test. based on these results, the acute effects of quinpirole (0.25 -1 mg/kg i.p.) were tested in dyt1 knock-in and wildtype mice. subsequent to the injections, mice were tested in the open field, the rotating beam test and the adhesive removal test, respectively. in the open field test, dyt1 knock-in mice showed increased thigmotaxis at a dose of 0.5 mg/kg quinpirole. in the rotating beam test, both groups showed a dose-dependently reduced performance. in the adhesive removal test, quinpirole improved the reaction time in dyt1 mice independently of dosage, while no effects were observed in the wildtype littermates. however, in vehicle follow-up (post-drug control), this effect remained consistent in the dyt1 model, suggesting a habituation effect. in conclusion, we generated a new test, i.e., the rotating beam test which improves the detection of mild motor impairments in dyt1 knock-in mice. furthermore, the adhesive removal test revealed sensorimotor dysfunctions in this animal model. these results represent an important step for our ongoing optogenetic examinations on the role of abnormal neuronal plasticity in dyt1 dystonia and for pharmacological studies. the first data on the effects of quinpirole do not indicate a critical role of d2 dysregulated acetylcholine release, but this has to be clarified by ongoing local striatal injections of quinpirole and by pharmacological manipulations of the cholinergic system. renal fibrosis is characterized by decreased nitric oxide (no) bioavailability and pronounced transforming growth factor β (tgfβ) signalling subsequently excessive extracellular matrix (ecm) deposition. here, the effects of the soluble guanylate cyclase (sgc) stimulator bay 41-8543 after unilateral ureter obstruction (uuo) have been studied. kidney fibrosis was induced by unilateral ureter obstruction (uuo) in wild type (wt) and cgki knock-out (cgki ko) mice. starting one day after uuo, the sgc stimulator bay 41-8543 was (4mg/kg/daily) i. p. injected for seven days. biomarkers indicating remodelling processes in the kidney were analysed via mrna expression and protein expression. bay 41-8543 administration influenced the activity of the ecm degrading matrix metalloproteases (mmp2 and mmp9) and their inhibitor timp-1, the expression pattern of extracellular matrix proteins (e.g. collagen and fibronectin) of profibrotic mediators (e.g. connective tissue growth factors (ctgf) and plasminogen-activator inhibitor-1 (pai-1)) and the secretion of cytokines, e.g. il-6. thereby, bay 41-8543 increments the cgmp pool among others via modulation of endothelial no synthase (enos) expression. agents, which enhance no and cyclic guanosine monophosphate (cgmp) ameliorate the progression of fibrotic tissue. however, the molecular mechanism by which cgmp via cgki affects the development of kidney fibrosis has not fully been elucidated. accordingly, the present study investigates the functional role of sgc stimulation in regulating the fibrotic process, the signalling pathway and the underlying mechanisms involved. we hypothesize that the antifibrotic potential of bay 41-8543 might be related to the increased cgmp pool and the inhibition of the mapk and smad signalling pathway. the elucidation of the signalling allows the development of new therapeutic options. infection of mice with listeria monocytogenes (lm) results in a strong t-cell response that is critical for an efficient defense. here, we demonstrate that the adapter protein sly1 (sh3-domain protein expressed inlymphocytes 1) is essential for the generation of a fully functional t-cell response. the lack of sly1 leads to reduced survival rates of infected mice. the increased susceptibility of sly1 ko mice was caused by reduced proliferation of differentiated t cells. ex vivo analyses of isolated sly1 ko t cells displayed a dysregulation of forkhead box protein o1 (foxo1) shuttling after tcr signaling, which resulted in an increased expression of cell cycle inhibiting genes, and therefore, reduced expansion of the t-cell population. foxo1 shuttles to the cytoplasm after phosphorylation in a protein complex including 14-3-3 proteins. interestingly, we observed a similar regulation for the adapter protein sly1, where tcr stimulation results in sly1 phosphorylation and sly1 export to the cytoplasm. moreover, immunoprecipitation analyses revealed a binding of sly1 to 14-3-3 proteins. altogether, this study describes sly1 as an immunoregulatory protein, which is involved in the generation of adaptive immune responses during lm infection, and provides a model of how sly1 regulates t-cell proliferation (schäll et al., eur j immunol. 2015) . the catalytical isoforms p110γ and p110δ of phosphatidylinositol-4,5-bisphosphate 3kinase γ (pi3kγ) and pi3kδ play an important role in the pathogenesis of asthma. two key elements in allergic asthma are increased eosinophil and ige levels. whereas dual pharmacological inhibition of the catalytical subunits p110γ and p110δ reduces asthmaassociated eosinophilic lung infiltration and ameliorates disease symptoms, it has been shown that dual genetic deficiency in pi3kγ and pi3kδ in p110γ ko δ d910a mice increases serum ige and basal eosinophil counts in mucosal tissues and blood. this suggests that long-term inhibition of p110γ and p110δ might exacerbate asthma. here we analysed p110γ/δ -/mice and determined ige and eosinophil counts in a basal state and the immune response to ovalbumin (ova)-induced allergic asthma. we found that serum concentrations of ige, il-5 and eosinophil numbers in blood, spleen and bone marrow were significantly increased in p110γ/δ -/mice in comparison to single knock-out (ko) and wildtype (wt) mice. nevertheless, p110γ/δ -/mice were protected against ovainduced infiltration of eosinophils, neutrophils, b cells and t cells into the lung tissue and the bronchoalveolar space. moreover, p110γ/δ -/mice, but not single ko mice, showed a reduced bronchial hyperresponsiveness as measured with the isolated and perfused lung. we conclude that although the dual deficiency of p110γ and p110δ causes eosinophilia and ige hyperproduction, p110γ/δ -/mice are not prone to develop ovainduced allergic asthma. an increase of plasma extravasation induced by activation of constitutively expressed endothelial bradykinin type 2 receptors (b2) has been shown to contribute to the development of angioedema occurring as a sometimes life-threatening side effect of angiotensin-converting enzyme inhibitors such as enalapril (new engl j med 2015:372; 418-425) . these drugs inhibit the degradation of bradykinin and increase its vascular steady-state concentration. hence, it is reasonable to assume that bradykinin may destabilise the endothelial barrier, i.e. may increase physiologic extravasation. while the commonly used miles assay provides a useful and relatively easy tool to study the effect of permeabilizing mediators in-vivo, it does not distinguish between intravascular and interstitial evan's blue dye. likewise, extravasation can only be quantified at one particular time point per animal, usually 20-30 min. furthermore, evaluation of physiologic extravasation is not possible. in contrast, non-invasive twophoton laser microscopy may allow separating the intravascular from the interstitial compartment and thereby investigations of changes of the physiologic endothelial barrier induced by drugs or transgenes. therefore, we have evaluated this methodology for its suitability to study endothelial permeability in mice in vivo. to establish this, we used two different fluorescent dyes of different molecular weight. a 200,000 kda dextran equipped with a green fluorescent chromophore which cannot leave vascular lumen was injected intravenously to visualize small dermal blood vessels of the mouse ear located approximately 200 µm below the surface. after stabilization of the green fluorescent signal, a 10,000 kda dextran equipped with a red fluorescent chromophore which easily traverses the endothelial barrier was applied by intravenous injection. the red fluorescence permeates into the interstitium during physiologic extravasation and accumulates in the interstitial space. this process can be followed by measuring the decrease of intravascular red fluorescence over various time periods. using this methodology we have studied whether endothelial-specific overexpression of b2 changes physiologic endothelial permeability. this newly developed transgenic mouse line (b2 tg ) was established using a plasmid consisting of pbluescript ii sk+ -vector, the tie-2-promotor, the human b2 cdna, the sv40 poly-a-signal and a tie-2 intron fragment. we observed that b2tg showed a significantly stronger extravasation than their transgene negative littermates as evidenced by the more rapid extravasation of the 10,000 kda dextran at each time point (fig. 1) .we conclude that two-photon laser microscopy is suitable to study endothelial permeability non-invasively in-vivo and that this methodology allows to study the effects of drugs and transgenes on the endothelial barrier under non-inflammatory conditions. furthermore, our results suggest that endothelial-specific overexpression of b2 increases physiologic extravasation. non-allergic angioedema such as angioedema induced by angiotensin converting enzyme inhibitors (acei) develops as a consequence of increased activation of bradykinin receptor type 2 (b2). using a plasmid consisting of pbluescript ii sk+ -vector, the tie-2-promotor, the human b2 cdna, the sv40 poly-a-signal and a tie-2 intron fragment a transgenic mouse line harbouring an endothelial-specific overexpression of b2 was generated and backcrossed to c57bl/6 for more than 10 times (b2 tg ). lung mrna using primers specific for the human or the mouse b2 cdna revealed a 12.5-fold stronger expression of human b2 in b2 tg (n=6), while the expression of murine b2 mrna was unchanged and similar to transgene negative littermates (b2 n ). we have evaluated the specificity of several antibodies directed against b2 and found that a rabbit monoclonal anti b2 antibody appears to be reliable, i.e. there was just a faint staining in lung tissue of b2 -/mice. however, this antibody primarily stains rodent b2 and has only little cross-reactivity to human b2. hence, we were not able to detect a significant increase of b2 protein in tissues of b2 tg . previous experiments have shown that bradykinin induced concentration-dependent constrictions of aortic rings with a maximal effect at 1 µm of bradykinin. the contraction due to bradykinin was completely inhibited by icatibant or diclofenac indicating that it is mediated by endothelial b2 activation and dependent on cyclooxygenase activity. in striking contrast to their transgene negative littermates b2 n , we found a significant icatibant sensitive aortic dilation in b2 tg following preincubation with diclofenac which indicates functional overexpression of b2 in conductance vessels of b2 tg . to evaluate whether this applies to dermal micro vessels we used the miles assay to quantify dermal extravasation of the albumin-bound dye evans blue following intradermal injection of 30 µl of either vehicle, bradykinin, labradimil and histamine (control). increasing concentrations of bradykinin caused a significant increase of extravasation reaching 4.41±0.11 fold at 18.9 nmol bradykinin in c57bl/6 (n=6 each, p<0.0001 vs. vehicle). a similar increase was found in b2 n (4.45±0.25 fold, n=7, p<0.0001 vs. vehicle), while there was a stronger response in b2 tg (5.50±0.16 fold, n=7, p<0.0001 vs. vehicle) which was significantly different to b2 n (p<0.01) and c57bl/6 (p<0.01). in another set of experiments the specific and ace-resistant b2 agonist labradimil (1.89 nmol) was used instead of bradykinin. labradimil increased extravasation by 3.736±0.121 fold in c57bl/6 (n=6 each, p<0.0001 vs. vehicle), by 4.51±0.11 fold in br2 n (n=7 each, p<0.0001 vs. vehicle) and 4.88±0,21 fold in b2 tg (n=6, p<0.0001 vs. vehicle) which was significantly different to c57bl/6 (p<0.01) but not to b2 n (p>0.05). the effects of bradykinin and labradimil were largely blocked by 10 nmol icatibant (i.v.) in c57bl/6, b2 n and b2 tg mice (p<0.0001) and hence mediated by activation of b2. these data suggest that overexpression of b2 in b2 tg is functionally active in endothelial cells of large conductance and small dermal vessels. therefore, b2 tg represents a new animal model suitable for cardiovascular and non-allergic angioedema research. pharmacokinetic pharmacodynamic modeling of irreversible effects: the rituximab example f. keller 1 1 universitätsklinikum, innere 1, nephrologie, ulm, germany background: for pharmacokinetic-pharmacodynamic modeling usually the sigmoid emax model is used as described by the hill equation. however, treatment regimens exist where the effect is only exerted as long as the drug concentration increases whereas decreasing concentrations produce no longer an effect. examples are the pulse anti-cancer therapy such as originally proposed by the devita protocols. methods: here, the new model for irreversible drug action is derived from the time dependent change of the concentration that must be larger than the time-dependent growth of the number of target cells (tumor or bacteria). the irreversible effect can be assumed if the is no further growth of the target cells occurs. de/dt = + dc/dt -dn/dt dn/dt = 0 a solution for the above differential equation can be obtained by use of the integral exponential function iec based on the euler-mascheroni constant (gamma = 0.5772 …). this model of an irreversible effect was applied.to the example of rituximab where the initial effect on cd19+ and cd20+ b-cells completely persists for 6 month. to obtain a numerical solution, the following parameters are needed to be determined: the target concentration ctarget = 100 mcg/ml and the infusion time t = 2 hours. results: it can be shown that a plausible result for rituximab can be estimated only under the condition that a short distribution half-life is assumed of t1/2 = 2 hours (not shorter than the time t of infusion). with the terminal half-life of 460 hours no plausible solution is obtained. under these conditions two observations are made: there is a negative effect both, initially for low concentrations and after cessation of the infusion when concentrations decrease (in reality this means no effect in both cases). the irreversible effect is proportional to the target concentration. the shorter the half-life comes out relative to the infusion time (t1/2 < t) the stronger is the effect (figure) . occasionally there are specific questions occurring on the ruminant xenobiotic metabolism: 1) are the observed metabolites ruminant specific and formed directly in the rumen? 2) are ruminants able to cleave plant specific metabolites like glycosides to the respective aglycon? in the past new additional in vivo goat metabolism studies with at least one animal were performed. the aim of the project was to elucidate an alternative in-vitro method to replace the existing in vivo method in order to address robustly specific questions on xenobiotic metabolism in ruminants for registration of ppp. fresh sheep rumen fluid was incubated in-vitro >7 days by using rumen simulation technology (rusitec). the conservation of the physiological conditions were proven by measurement of ph (~ph 6.6) and redox potential (~-300 mv). the microflora composition and their viability (bacteria, protozoa and fungi) of the rumen were monitored by microscopy, incubation on agar plates and performing several viability tests (e.g. glycosidase-test, nh3 and short fatty acids). all the tests showed that the rusitec is a successful tool to maintain sheep rumen fluid for at least 7 days in -vitro. the metabolic behavior and performance of the rumen fluid was tested by e.g. incubating 14c-triazol derivative metabolites (tdm) like triazol-alanin (ta); triazol-acetic-acid (taa) and triazol-lactic-acid (tla), which are usually formed in plants after application of triazol-containing fungicides. it was shown that ta was cleaved within 72 h to 1.2.4.-triazol, while taa and tla were stable under these conditions. these data are in a good accordance with available in vivo data in cows. moreover glycosides (12c-polydatin, octyl-14c-β-d-glucopyranosid) were cleaved within 1 hour completely. all these data show, that the rumen fluid maintained its metabolic performance by using rusitec. basf identified the rusitec method, which is usually used in different areas (e.g. investigation of methane production in-vitro) as suitable and adapted this method for the purpose of investigation of ruminant xenobiotic metabolism. it was shown that rusitec is a robust method to analyze rumen xenobiotic metabolism and therefore can clearly substitute in vivo animal studies on ruminant metabolism studies beyond oecd503 and contribute significantly to animal welfare (3r: replacement). results: by using the training set, physicochemical (e.g. lipophilicity) and pharmacokinetic characteristics of mtx (e.g. v max for active tubular secretion) were slightly adjusted. using the gfr formula of morris et al. (1982) and including an empiric correction factor, mrd for the training set was 2.49 whereas bias was 2.80 µmol/l. by applying the developed pbpk model to the test set the respective values were mrd=3.92 and bias=1.43 µmol/l. for the covariates "at least one potentially interacting co-medication" and "trimethoprime/sulfamethoxazole" a significant impact on the prediction quality was found. conclusions: using the developed pbpk-model, a good prediction of the pharmacokinetics of hd mtx in severely ill children was found. by including additional factors influencing the prediction of mtx characteristics (e.g. co-medications) an improved prediction of mtx-sl might be reached. in prospective clinical trials, those more complex models should be evaluated and might be helpful to predict hd mtx pharmacokinetics and reduce unwanted side effects. hypericin is a natural polycyclic quinone found in hypericum perforatum. although hypericin reportedly has numerous pharmacological activities, only a limited number of studies have been performed on the absorption and transport characteristics of this compound, presumably, because hypericin is a highly lipophilic compound which is poorly soluble in physiological medium. recently we have shown that quercitrin and isoquercitrin, but not hyerosid, quercetin or rutin increased the uptake of hypericin in caco-2 cells. the major aim of this study was to get a detailed understanding of the exposure and fate of hypericin in the caco-2 cell system under different experimental conditions. the permeation characteristics of hypericin (5 µm) in absence or presence of hypericum extract 145, 62.5 µg/ml) were studied in the absorptive direction. following application of hypericin to the apical side of the monolayer only negligible amounts of the compound were found in the basolateral compartment. the amount of hypericin in the basolateral compartment increased concentration-dependently in the presence of the extract (from 0 to 7.5 %). the majority of hypericin was found after cell extraction (44% in absence and 76% in presence of the extract). the recovery was in the range of 90 %, and significant amounts of hypericin found after cell extraction. fluorescence microscopy and imaging analysis revealed that hypericin is mainly accumulated in the cell membrane. the precise mechanism through which hypericin might overcome the hydrophobic barrier of cell membranes remains to be elucidated. however, our experiments demonstrated that the permeation characteristics of hypericin significantly improved in presence of the extract. background: the combination of gamithromycin (gam), a novel drug with the big advantage of a once weekly administration, and rifampicin (rif) is used in the treatment of lower airway disease in foals. both are effective in the therapy of infections with rhodococcus equi, a gram-positive coccobacillus bacterium, which is known to survive and reproduce within alveolar macrophages. macrolides are combined with rif to prevent resistance developing with single agent therapy. both drug classes reach high concentrations in the lung, penetrate into phagocytes and kill intracellular pathogens. methods: a controlled, single-and multiple dose study with four-periods was conducted in 10 healthy foals (5 ♂ and 5 ♀, age: 42-63 days, body weight: 100-177 kg) which were treated once with rif alone (10 mg/kg s.i.d., p.o., a) followed by the administration of gam (6 mg/kg once weekly, i.v., b) for 3 weeks. study period 3 ("rif-gam acute", c) includes the administration of gam and rif for 7 days with an administration interruption after the first rif dose for blood sampling. for the last study period ("rif-gam chronic", d) both gam and rif were coadministered for 2 weeks. all periods were completed with blood sampling for pharmacokinetic analysis for 48 ( . rif is also accumulated in the lung, but to a much lower degree than gam (elf/c 24 h : 1.2 ± 0.5; balc/c 24 h : 2.01 ± 1.24). conclusion: pharmacokinetic data of the present study provides surprising results. in previous studies coadministration of clarithromycin and rif show a dramatic decreased plasma exposure of the macrolide, whereas the balc/c 24 h -ratio was unaffected (peters et al. 2011) . in contrast, systemic exposure of gam increase significantly in case of the combined therapy and the balc/c 24 h -ratio was nearly halved. both macrolides have in common, that they are intensively accumulated in the lung (elf << balc). at the moment there is further research required (e.g. in vitro studies) for a better understanding of the very interesting in vivo data. 1 1 institut für klinische pharmakologie, göttingen, germany background and aim: desvenlafaxine is a selective serotonin and norepinephrin reuptake inhibitor, which is approved in the usa (but not in europe) for treatment of major depressive disorder. desvenlafaxine is the major active metabolite of the antidepressant venlafaxine. desvenlafaxin is produced by o-desmethylation via cyp2d6. direct administration of desvenlafaxine should bypass the variability in venlafaxine pharmacokinetics caused by the highly polymorphic cyp2d6. however, desvenlafaxine is less lipophilic than venlafaxine and may require carrier-mediated transport to penetrate cell membranes. based on our in vitro data, desvenlafaxine is a substrate of the hepatic organic cation transporter oct1 and common genetic polymorphisms abolished desvenlafaxine cellular uptake. about 9% of caucasians are compound heterozygous carriers of loss-of-function oct1 polymorphisms. therefore, oct1 polymorphisms may cause substantial inter-individual variability in the hepatic uptake and plasma concentrations of desvenlafaxine. in this study we evaluated the influence of genetically-determined loss of oct1 function on the pharmacokinetics and pharmacodynamics of desvenlafaxine. primary aim was dependence of desvenlafaxine plasma concentrations (represented by auc as primary endpoint) on the number of active oct1 alleles. methods: 50 mg desvenlafaxine (pristiq®) was orally administered to 41 healthy subjects preselected according to their oct1 genotypes. oct1*1 allele was regarded full active, oct1*2 to *6 alleles were regarded loss of function. plasma concentrations of desvenlafaxine and its main metabolite didesmethylvenlafaxine were quantified in plasma sampled up to 60 hours after administration using lc-ms/ms. pupillographic measurements were performed as possible surrogate markers for desvenlafaxine pharmacodynamics. results out of the 41 subjects 14 carried two active, 13 one active, and 14 zero active oct1 alleles. age, height and weight were 26.9 ± 6.4 years (mean ± standard deviation), 1.75 ± 0.11 m and 70.9 ± 12.1 kg with no significant differences among the oct1 genotypes. there were strong variations in the pharmacokinetics of desvenlafaxine and its metabolite didesmethylvenlafaxine. the auc 0-infinity of desvenlafaxine varied between 52.8 and 282.2 min*mg/l and auc 0-infinity of didesmethylvenlafaxine between 3.5 and 30.7 mg*min/l. however, neither desvenlafaxine nor didesmethylvenlafaxine pharmacokinetics significantly differed among the three oct1 genotypes. concerning pharmacodynamics of desvenlafaxine, pupil diameters at maximal constriction after a standardized light exposure were on average 14% greater around the time of t max than before administration. in line with the pharmacokinetic results there were no significant differences in maximal constriction or other pupillographic parameters among the oct1 genotype groups. conclusions: our results suggest that oct1 genotype does not affect the pharmacokinetics of desvenlafaxine and therefore no dose adjustment in respect to oct1 genotype should be considered. other factors like renal transporters or polymorphic glucuronidation may explain the great variability in desvenlafaxine pharmacokinetics. background: cardiovascular disorders and medication are highly prevalent in elderly (1). due to age related changes in the body, the elderly are particularly vulnerable to side effects and adverse drug reactions. some psychotropic drugs are linked with reports of cardiac side effects. additionally, some cardiac drugs may also cause psychiatric symptoms. of these, angiotensin converting enzyme inhibitors, beta blockers, methyldopa and calcium channel antagonists can induce or exacerbate symptoms of depression (2) . the aim of this study was to provide information on the concomitant use of cardiovascular drugs among elderly patients who took psychotropic medication. methods: we conducted a single-center, retrospective study between september 2013 and december 2013 using the medical records of elderly patients (≥65 years of age) admitted to basin sitesi polyclinic, izmir ataturk research hospital, turkey. demographic characteristics of patients, diagnoses, prescription drugs were evaluated, and spss 16.0 statistical software was used for data analysis. number, percent, mean and standard deviation were used as descriptive statistics. results: a total of 541 elderly patients with psychiatric disorders were identified. one in four patients receiving psychotropic medication took at least one cardiovascular agent concomitantly (n=135). median age was 72 (min:65, max:98), 84 patients were female (62.2%). according to medical records of 135 patients, the most commonly used drugs were escitalopram, sertraline, mirtazapine, quetiapine, mianserin and risperidone. the proportion of the concomitantly use of cardiovascular drugs was higher among the patients who took more than one psychotropic drug (69.6% vs. %30.4) compared to patients taking psychotropic monotherapy. a higher percentage of women used diuretics (65.4% vs. 33.3%) and angiotensin receptor blockers (36.9% vs. 23.5%) concomitantly with psychotropic drugs when compared to men. the proportion of men using angiotensin-converting enzyme inhibitors and lipid-modifying agents was higher than women (58.8% vs. 38%, 68.6% vs. 20.2%, respectively). conclusions: the world's population is ageing rapidly. according to world health organization, over 20% of elderly suffer from a psychiatric or neurological disorder. our data showed that use of cardiovascular drugs among elderly patients with psychiatric disorders was extensive. the effects and interactions of these drugs should be discussed and carefully evaluated before starting treatment in the elderly. further studies focusing on drug use in elderly will increase the success in geriatric pharmacotherapy. since the adoption of the ich e14 guideline [1], the thorough qt (tqt) study has become a standard element of clinical drug development. however, with the iq/csrc study [2] the ability to detect qtc-prolongations of about 10 ms in a phase i setting has been demonstrated. as a consequence, regulatory agencies have begun to grant waivers for a tqt study based on negative qt findings obtained from first-in-man studies. a concentration-response model is the key tool that gives sufficient power to an analysis based on data from single or multiple ascending dose studies. this power has been investigated in subsampling studies that simulate situations comparable to those encountered in first-in-man studies [3, 4] . other topics to be addressed are the assurance of sufficient quality of the ecgs obtained, in particular in doses that cause adverse reactions, and a replacement for the active control that is part of a tqt study. if a model based statistical analysis is used for confirmatory inference, it must be specified in advance. this pre-specification includes tests to ascertain that the model assumptions are met and alternative methods to be used in case they are violated. in particular linearity and the absence of a hysteresis, i.e. a delay between the drug concentration and the observed qt effect need to be tested. this is an area of active research. in this contribution, i will share current experience from a statistical perspective, both based on real data and on simulated studies. i will also discuss critical points in the design of first-in-man studies that are intended to be used to obtain a waiver for a tqt study. human platelets express the g-protein-coupled angiotensin receptor-like-1 (apj) receptor. apj is activated by apelin, which is produced as pre-apelin and cleaved into several bioactive peptides such as apelin-12, -13 and -17. apelin and apj are expressed in a variety of tissues such as the heart, the vessel wall, several tumor types, and in platelets. to date, there is no description or a suggested function of the apelin/apj system in platelets to date. here, we investigate apj expression and function in human platelets. apelin and apj expression were determined in platelet-rich plasma from healthy donors by immunofluorescence, western blotting and flow cytometry. in a pilot study apelin and platelet apj expression were analyzed in 23 patients with nstemi, 4 stemi patients and 14 controls. here, platelet aggregation was analyzed by light transmission aggregometry (lta); platelet cd62 and apj expression by flow cytometry and circulating plasma apelin by elisa. in resting human platelets, apj receptor expression was observed predominantly in the outer cell membrane, as determined by immunofluorescence staining and flow cytometry. activation with a selective thrombin receptor-activating peptide (ap1) resulted in decreased apj protein levels determined by western blotting in platelet lysates compared to untreated controls. preincubation of platelets with different apelin isoforms for 30 sec to 5 min reduced platelet aggregation in lta studies by up to 20 % for apelin-17. this effect was inhibited by preincubation of the platelets with the enos inhibitor l-name (300 µm), suggesting the involvement of a no-dependent mechanism. in patients with myocardial infarction the expression of platelet apj was significantly reduced compared to the control group (56,84% ± 9,28 % in mi versus 100 % ± 19,35 %in controls; p = 0.029). this reduction in apj expression on platelets was accompanied by decreased plasma levels of apelin-17 in patients with mi (14.95 ± 0.6 pg/ml versus 16.98 ± 0.6 pg/ml; p = 0.035). interestingly, the decreased apj expression on platelets in mi patients significantly correlated inversely with the troponin t plasma levels (r = -0.46; p = 0.03). this may suggest an association of apj expression with lower plasma levels of troponin t and possibly tissue damage. in conclusion, our study shows for the first time the expression of apj and a possible function in human platelets. apj may act as an endogenous inhibitor of platelet aggregation in response to certain apelin isoforms, predominantly apelin-17. upon platelet activation, apj is internalized and surface expression is reduced by about 50 %. in mi patients, plasma levels of apelin-17 and platelet apj expression were reduced. this correlated inversely with troponin t levels. reduced circulating apelin-17 levels and platelet apj expression may be associated or partly account for platelet hyperactivity in mi patients. anticholinergic drug use, m 1 receptor affinity and dementia risk-a pharmacoepidemiological analysis using claims data f. thome background: dementia is characterized by cumulative cognitive decline and progressive inability for independent living. the lack of suitable therapies for terminating the progression of this disease underlines the importance of the detection of risk factors. anticholinergic drugs have been shown to enhance cognitive decline in the elderly. the classification of anticholinergic drugs according to their anticholinergic burden, however, is inconsistent. since cholinergic transmission is mainly mediated by the m 1 muscarinic acetylcholine receptor in the brain, we classified anticholinergic drugs from anticholinergic risk lists according to their affinity for the m 1 receptor subtype and calculated the risk for the onset of incident dementia. methods: our analyses are based on claims data of the public health insurance fund aok. data include information of inpatient and outpatient diagnoses and treatment from 2004 to 2011. inclusion criteria comprised the initial absence of dementia and age of 75 years or older in 2004. anticholinergic drugs were taken from three anticholinergic risk lists. the pdsp-database and literature search were used to define k i -values for the substances. hazard ratios were calculated using time-dependent cox regression including covariates like age, gender, and several comorbidities. results and conclusion: anticholinergic drug exposure increases the risk for dementia. we found that anticholinergics with small k i -values are at a higher risk than those with greater k i -values. furthermore, conventional risk factors for dementia (e.g. age, depression, stroke) could be confirmed. in conclusion, the intake of anticholinergic drugs increases the risk for incident dementia in the elderly. taking into account the m 1 receptor affinity may be a contribution in determining the anticholinergic load in view of the risk for incident dementia. safety signal detection in a large german statutory health insurance databasefirst results of a feasibility assessment f. andersohn 1,2 , s. schmiedl 3, 4 , k. janhsen 3, 5 , p. thuermann 3, 4 , j. walker background: during the last years, approaches to routinely screen health care databases based on electronic medical records or claims to identify drug safety signals were proposed. to evaluate the performance of such methods, reference sets of index drugs have been compiled consisting of (1) drugs with a known association to a certain adverse event (=positive controls) and (2) drugs without any evidence to cause this adverse event (=negative controls). the best possible signal detection method would identify a safety signal for 100% of the positive control drugs, and for none of the negative controls. ryan et al. 2013 developed drug reference sets for four adverse events of interest (acute myocardial infarction=ami, acute kidney injury =aki, acute liver injury=ali, and upper gastrointestinal bleeding=ugib) and have shown feasibility of using these reference sets in us health care databases. if the use of these us specific drug lists for evaluation of signal detection methods is also feasible within german health care databases, is unknown. aims: to evaluate if the drug reference sets developed by ryan et al. (drug saf. 2013 ;36 suppl 1:s33-47) could be used for testing signal detection methods in a large german statutory health insurance database. methods: data source was the health risk institute (hri) database, an anonymized healthcare database with longitudinal health insurance data from approximately six million germans. new users (initiators) of index drugs in 2010 to 2013 were identified and followed-up for one year from their first prescription. exposed person-time to the respective index drug was assessed to estimate for which of these drugs an increase in risk of 50% (relative risk 1.5) compared to the background incidence of the respective adverse event (ami, aki, ali or ugib) could be identified with 80% statistical power. results: from a total of 182 index drugs in the reference sets of ryan et al., 142 (78.0 %) were also available on the german drug market and were used by at least one insurant in the hri database during the study period. a total of 5,485,722 index drug initiators were included in the analysis. for a total of 16 index drugs, a relative risk of 1.5 could be detected with 80% power. the numbers of index drugs for each of the outcomes of interest were: ami (3 positive controls; 6 negative controls); aki (3 positive controls; 1 negative control); ugib (2 positive controls; 1 negative control). as the background incidence of ali was low, no positive or negative control with sufficient power was identified for this outcome. conclusions: using the set of reference drugs proposed by ryan et al., the number of drug-event pairs with 80% power to detect a relative risk of 1.5 was low, despite the magnitude of the database used. this may be attributable to differences of drug exposure in germany and the us. hence, an adaptation of the drug list to the german drug market and consumption data might be relevant for future evaluations of signal detection methods using german databases. correlation of sativex™ doses to steady state concentrations of 11-nor-9-carboxyadministration of the oromucosal spray sativex™ represents a therapy option for treatment of spasticity in patients with multiple sclerosis. sativex™ is an extract containing equal amounts of the cannabis-derived cannabinoids ∆ 9tetrahydrocannabinol (thc) and cannabidiol (cbd). in cases of cannabis abuse a long elimination half life of some thc metabolites is known. therefore, in patients receiving sativex™, the long elimination half life of these metabolites should allow a drug monitoring under conditions of steady state. due to the fact that immunologically based methods for thc determination are very common in medical chemistry, a monitoring might be simply performed even in patients under sativex™ therapy. in a preliminary observational study 11-nor-9-carboxy-∆ 9 -thc (thc-cooh) concentrations were measured with a commercial immunoassay in urine samples of 16 patients with multiple sclerosis obtaining sativex™. in addition, thc-cooh, thc, cbd as well as the hydroxy metabolites of thc and cbd were measured by gc/ms in urine and blood samples. using this analytical technique, only an excessive dosing (as compared to the declaration by the patient) can be detected. as a result of this approach, thc-cooh concentrations determined by the immunoassay were found not to correlate to the daily applicated amount of sativex™ as indicated by the patients (spearman rang order test: p > 0.05). two patients mentioned not to have taken sativex™ on the day the samples had been taken, and one patient predicated an additional cannabis abuse. in three patients the immunological thc-cooh determination was negative or nearly negative. interpretation of the data is hampered by the fact that an incorrect declaration of sativex™ applications by the patients cannot be excluded. introduction: learning analytics seeks to enhance the learning process through systematic measurement and analysis of learning related data to provide informative feedback for students and lecturers. however, which parameters have the best predictive power for academic performance remains to be elucidated. objective: to analyze the potential of different learning analytics parameters to predict exam performance in undergraduate medical education of pharmacology. methods and results: hypertext preprocessor (php) as server-side scripting language was used to develop a learning analytics platform linked to a my structured query language (mysql) database for storage and analysis of data (www.tumanalytics.de). the database consisted of 440 lecturer-authored multiple choice questions that were made available to a cohort of undergraduate medical students enrolled in a pharmacology course (winter term 2014/15) at technische universität münchen (tum). the course consisted of a 28-day teaching period, followed by a 12-day self-study period and a final written exam. students' assessment data of tumanalytics was collected during the self-study period and correlated to the individual exam results in a pseudonymized manner. a total of 224 out of 393 (57%) students participated in the study. the coefficient of multiple correlation (r) was calculated for different parameters in relation to exam results as a measure of predictive power. of different parameters investigated, the total score and the score of the first attempt in tumanalytics had the highest positive correlation with exam performance (abb. 1). no sex-specific differences were observed. summary and conclusion: in this study we systematically investigated the potential of different learning analytics parameters to predict learning outcome and exam performance. total score and score of the first attempt were identified as parameters with the highest predictive power. in conclusion, our study underscores the potential of learning analytics as valuable feedback source in undergraduate medical education of pharmacology. in educational settings, tests (e.g., written or oral exams) are usually considered devices of assessment. however, a recent and intriguing line of evidence from basic cognitive psychological research suggests that tests may not only help to assess what students know, but may also help to improve the learning and long-term retention of information. the goal of the present study was to apply such test-enhanced learning to pharmacological teaching. after the last lecture of a pharmacology class (n=194 3 rd -year medical students, n=213 4 th -year medical students: basic or clinical pharmacology, respectively), one week prior to the final exam, students were given the opportunity to voluntarily participate in online exam. because pilot work from previous semesters had revealed relatively low levels of participation in such formative exams, students were offered bonus points for (successful) participation as an incentive. the online exam consisted of 60 items (i.e., selected pieces of information from the lectures and seminars) and was provided on the e-learning platform ilias. twenty of the 60 items were presented as statements for restudy, 20 items were tested using single-choice questions, and 20 items were tested using short-answer questions. randomly a third of the students were assigned to different sets of questions. the summative final written exam for each group consisted of 30 single-choice questions, 15 questions of which had not been used before (as a standard of comparison). the remaining 15 questions of the final exam were taken from the previous online exam, but were slightly reworded to avoid ceiling effects. each of 5 of these reworded 15 questions from the final exam corresponded to restudy items, single-choice items, and short-answer items from the online exam, respectively. the main points of interest were (i) whether the re-processing (rewording and asking for transfer of knowledge) of information in the online exam affected participants' performance on the final exam, and (ii) whether any effect depended on the specific type of re-processing (restudy vs. single-choice test vs. shortanswer test). if previous findings from basic cognitive psychological research on testenhanced learning can be generalized to more applied settings and educationally more relevant materials such as pharmacological information, students' performance in the final exam should be better for questions corresponding to previously tested items than for questions corresponding to previously restudied items. moreover, if more difficult tests lead to more test-enhanced learning than less difficult tests, as is suggested by recent findings from cognitive psychological research, performance for questions corresponding to (supposedly more difficult) short-answer items should even exceed that for questions corresponding to (supposedly less difficult) single-choice items. the present findings bear direct implications for educational practice. safe and rational prescribing is one of future physicians' key skills [1] . in order to address the persisting prescribing deficiencies [2] , we set out to develop a learning tool for pharmacotherapies of the most important diseases worldwide. the format, scope, information architecture, and functionalities of the app were identified through assessment of existing apps, literature analysis, app simulation-based student surveys, and expert advice. a fully functional offline app format for smartphones was selected based on the trends in using digital technologies for educational purposes [3] and on the unreliable internet and power availability in many learning settings. a relational database based on semantic relationships was chosen to minimize information redundancy and to enable the retrieval of drug-related information in the context of mechanisms, contra-and indications, adverse drug reactions, interactions, and common prescribing situations. the usability was optimized using a simulation of the app evaluated by medical students from germany and tanzania, and by experts. a list of 67 indications was assembled beginning with disease burden data for the seven who world bank regions. each disease accounts for at least 0.3% of life years lost due to premature death or lived with ill-health or disability (daly) in at least one region. the list was further complemented according to expert recommendations. therapeutic recommendations are based on current guidelines, considering cheaper treatment alternatives provided in the who list of essential medicines. a novel dual-scale classification system lists drug mechanisms according to the affected physiological process and to the resulting therapeutic effect [4] . contraindications, adverse drug reactions, and interactions were compiled using drug monographs of the european medical agency, the us food and drug administration, and health canada. unexpectedly, we found significant differences among these sources in respect of adverse drug reactions. this necessitated the ongoing verification through surveying general practitioners and specialists in internal medicine. during the dgpt meeting we will present the results of testing of the cardiovascular section comprising 8 indications, 28 drugs representing 25 mechanisms, and up to 120 adverse drug reactions. the european certified pharmacologists (eucp) programme was lauched in july 2014 by the federation of european pharmacological societies with the intention to identify experts in the field of pharmacology whose competency profile, in addition to their personal specialised scientific expertise, covers expert knowledge in all major fields of the discipline. seventeen ephar member societies have declared their active participation in the eucp programme so far (austria, croatia, czech republic, finland, france, germany, greece, hungary, italy, the netherlands, norway, poland, portugal, serbia, slovenia, spain, turkey) . eacpt, the european association of clinical pharmacology and therapeutics, has also recently decided to participate in the eucp programme. national programmes must meet all requirements of the eucp guidelines including a clear catalogue of criteria with respect to knowledge, practical awareness and skills, as well as general rules including rules for final assessment of candidates. such programmes may be based on existing diplomas or training schemes or may consist of a set of rules how applicants may submit credentials for their expertise with respect to the eucp criteria. so far, three ephar member societies have submitted a national eucp program: austria, italy and the netherlands. the programmes differ in structure and reflect the flexibility of the eucp programme with respect to the respective national conditions. while the italian programme is based on a catalogue of criteria where applicants have to certify and document their expertise on the basis of this catalogue and the dutch program is based on a structured phd training course, the eucp scheme submitted by the austrian pharmacological society aphar is based on the legally regulated diploma medical specialist (facharzt) in pharmacology and toxicology (aphar also plans to submit separate regulations for specialists in clinical pharmacology and for nonmedically qualified pharmacologists). the aphar eucp scheme has been approved by the eucp committee in november 2015 and its regulations are available on the eucp website (www.eucp-certification.org). the differently structured programs of the italian and dutch pharmacological societies will also be available at this website, once approved by the eucp committee and may thus serve as 'case studies' for other ephar and eacpt member societies wishing to take part in the eucp programme. introduction: the outstanding importance of pharmacovigilance (pv) for the safe use of medicines has increasingly been recognised during recent years. the multidisciplinary character of pv requires know-how in topics as different as pharmacology, clinical medicine, pharmacoepidemiology, information technology, pharmaceutical manufacturing, legal aspects, public health policies, and medical traditions in different regions of the world. in this complex situation there is a growing need for pv capacity building, in particular by professional training through high quality pv courses with different focuses and different levels of detailing. against this background, the world health organization (who) and the international society of pharmacovigilance (isop) have co-operated to create a curriculum for teaching pv which can be used for a wide range of audiences and in very different settings and situations. the purpose was to provide an inventory and systematically structured overview of pv including recent developments of topics like pharmacogenomics, consumer reporting of adrs, risk management and who-led international projects, and to propose a range of tasks for practical training. we made use of several relevant already existing packages of pv topics and concepts of pv teaching from national and international institutions. we also drew from extensive printed material as well as comprehensive reviews, textbooks and guidelines developed by international organisations which are often available online. results: the curriculum includes a main component consisting of a major part for theoretical lecture-based training and a minor component with suggestions for hands-on exercises. the theoretical part has a three-level hierarchical and modular structure with evenly divided tiers. there are 15 chapters. each of them is divided into four sections and each section into four to six sub-sections. the practical part consists of twelve times three or four proposals for practical tasks which are related to the theoretical lectures. since its launch in 2014 1 it has successfully been used in several international courses. currently a pilot project is under way to explore its use for 'crowd sourcing': it is placed on the isop homepage with a programme allowing for institutions or persons experienced in pv teaching to upload any relevant presentations they may have with a link to related chapters, sections or subsections. these presentations will be offered for downloading by interested users. the curriculum provides a comprehensive coverage of almost all areas of pv. the structure and content allows almost every kind of focusing on specific issues and going into depth, while maintaining the overall context. it offers opportunities of tailoring courses specifically to the needs of different audiences and can be applied to various forms of training, such as broad, comprehensive and intensive courses, short overviews or focuses on specific narrow topics in perspective. according to the reach regulation (ec) no. 1907/2006 chemicals produced, marketed or used within the european union have to undergo a registration process, wherein the registrants have to provide information on hazard and potential risks presented by the substances. however, the standard information requirements defined in annexes vii to x of the regulation might be waived or adapted by the registrants if adequate documentation and justification according to criteria specified in annexes vii to xi are provided. to evaluate the data availability in registration dossiers of high tonnage substances (above 1000 tpa) and their compliance with the reach regulation, the federal institute for risk assessment (bfr) in cooperation with the federal environment agency (uba) developed a systematic web-based scheme. in total, 1932 dossiers were checked for selected human health and environmental endpoints such as repeated dose toxicity, genetic toxicity and ecotoxicity. a remarkable high rate, 43% to 82 % depending on the endpoint, of the evaluated dossiers included waiving or adaptations from the standard information requirements. therefore, those dossiers were not concluded, but categorised as 'complex' (springer et al., 2015) . the use of waiving and adaptations in 'complex' endpoints were part of a follow-up project. herein, it was evaluated whether the given justifications were in accordance with the criteria set out in the respective reach annexes. the results will show the frequency and pattern of waiving/adaptation approaches for the human health as well as the environmental endpoints. besides this general overview, specific problems regarding the application of the reach regulation were identified and their significance with regard to remaining data gaps will be discussed. the german commission for the investigation of health hazards of chemical compounds in the work area has re-evaluated dimethylformamide, and classified it in the carcinogen category 4. this category is for chemicals with carcinogenic potential for which a non-genotoxic mode of action is of prime importance and genotoxic effects play no or at most a minor part provided the mak and bat values are observed. under these conditions no contribution to human cancer risk is expected. dmf was identified as a substance of very high concern by european commission. the amount of dmf manufactured and/or imported into the eu is, in the range of 10000-100 000 t/y. n,n-dimethylformamide is a hepatotoxin in humans and rats. the carcinogenicity studies in both mouse and rat were conducted with test material of an acceptable purity and physical form. the critical study involved administration of dmf via inhalation, which is relevant to human exposure. there is conclusive evidence that dmf induces significant increases of hepatocellular carcinomas in rats after exposure to 800 ml/m³ and in mice in response to 200 ml/m³ and higher. several in vitro and in vivo studies have indicated that dmf is not genotoxic. the results of the long-term studies reveal that the tumors develop in the liver only after chronic toxic inflammatory and degenerative changes have developed in this organ. the commission concluded that the tumors are a result of chronic liver damage, occurring at high exposure concentrations. the available evidence therefore suggests that there is a threshold dose for the carcinogenic effects of dmf. accordingly, dmf was classified in carcinogen category 4 with a mak-value of 5 ml/m³, an exposure concentration which does not induces liver toxicity and as a consequence is not associated with an increased cancer risk. today, a large majority of people is constantly exposed to electromagnetic radiation. many studies have been performed to investigate whether this type of radiation has a potential to affect biological systems at low intensity levels. even though no complete consensus has been reached so far in this issue, most of the investigations do not indicate a harmful potential of this radiation. two questions remain open until today, i. e. long-term effects and specific effects on children. it has been demonstrated that in comparison to adults, children absorb far higher doses of mobile phone radiation in the skull, particularly in the bone marrow, where hematopoiesis takes place. these absorptions occasionally exceed the recommended safety limits. the aim of this study was to elucidate, whether cells of the hematopoietic system can be affected by different forms of mobile phone radiation. as biological systems, two cell types were investigated, hl-60 cells as an established cell line, and human hematopoietic stem cells. the radiation was modulated according to the two major technologies, gsm (900 mhz) and umts (1950 mhz). additionally, lte (2.535 mhz) modulation was applied because this technology is used worldwide already but has not been studied sufficiently. cells were exposed for a short and a long period and with different intensities ranging from 0 to 4 w/kg. studied endpoints included oxidative stress, differentiation, dna repair, cell cycle, dna damage, histone acetylation, and apoptosis. appropriate negative and positive controls were included and three independent replicate experiments were performed. exposure to radiofrequency radiation did not induce any alterations of cell functions, measured as oxidative stress and cell cycle. cell death in the form of apoptosis was not observed. primary dna damage was not induced and dna repair capacity for nucleotide excision repair was not changed. epigenetic effects (measured as histone acetylation) were not observed. finally, differentiation was not affected. the effect of treatment with various chemicals as positive controls was different in the two cell types. all in all, mobile phone radiation did not induce effects on human hematopoietic cells. in 2014 who published guidance on evaluating and expressing uncertainties in human health hazard characterisation (hc). in this approach, the outcome of hc is expressed as an interval or distribution rather than a "traditional" deterministic point estimate, such as a reference dose (rfd), thereby communicating potential uncertainties more clearly. risk management protection goals, such as the acceptable magnitude of effect (m) and incidence (i) in the population, are made explicit quantitatively along with the confidence with which they are achieved, e.g. by an rfd. specifically, the goal of this approach to hc is to estimate the "hd m i" , i.e. the "true" human dose associated with m and i (e.g. body weight decreased by ≥ 10% (m) in 5% (i) of the population). if uncertainties are expressed by providing estimates of the hd m i as confidence intervals or uncertainty distributions, both the "degree of uncertainty" (ratio of upper and lower limit of the interval/relevant distribution segment) and the "coverage" (statistical confidence) associated with a given rfd value can be characterised. alternatively, one may start from a chosen coverage and calculate the associated "probabilistic rfd". uncertainty in each hc aspect, e.g. inter-/intraspecies or time extrapolation, can be characterised by a "generic default uncertainty distribution" which has been derived from historical data, but may be replaced by a substance-or effect-specific distribution (analogous to a chemical-specific adjustment factor in the "traditional" deterministic approach), where available. the uncertainty distributions for the individual hc aspects can then be combined into an overall uncertainty interval/distribution in (1) a simple non-probabilistic way, (2) a more refined "approximate probabilistic analysis" (aproba, a free spreadsheet tool for easy implementation also by non-statisticians is available from the who website), or (3) a fully probabilistic monte carlo analysis. the hc aspects contributing most to overall uncertainty are also identified and may be prioritised for refinement in a next assessment tier. the who approach uses a tiered strategy which may start with evaluating the uncertainties in the outcome of a "traditional" deterministic hc. in this way it represents a unified framework integrating deterministic and probabilistic hc methodologies. moreover, the concept can be easily combined with exposure uncertainty assessment. risk managers may use the additional information in better weighing potential health effects against other interests. when they consider the overall uncertainty larger than desirable in view of the problem formulation, they may decide to ask for a more refined (higher tier) assessment. if all possibilities for refinement are exhausted, the new approach can also aid in the selection of new data which might need to be generated. due to a constant improvement in analytical methods an increasing number of substances are found in drinking water. the joint project toxbox aimed for the development of a reliable test battery, allowing for a rapid evaluation of single substances in water. eleven partners either from the research (nine) or the business sector (2) formed the project. the attention was focused on genotoxic, neurotoxic and endocrine effects, which are considered to be of most concern to the consumer. by the end of the project a set of guidelines is published that describes the analytical methods in detail. the project was based on a theoretical concept, called "health-related indicator value" (hriv), which was developed by the german federal environment agency (uba) for the assessment of substances with incomplete toxicological data. depending on the type of effect a hriv between 0.1 and 3.0 µg/laws derived for the substance which had to be evaluated. during the years an increasing amount of substances as well as an increase in finds was observed in drinking water. this called for the creation of an evaluation scheme that offers rapid and at the same time reliable evaluations of chemicals for which there are no data available. the concept is in accordance with tox21, which envisages the trustworthy evaluation of relevant endpoints by two or three in vitro assays. in the context of toxbox this was provided for the endpoints genotoxicity, neurotoxicity and endocrine effects. in all cases a hierarchic strategy is applied that enables a first assessment via relatively simple test assays and only when these test give a hint towards an effective more elaborate techniques are applied for a final assessment. the ames test and micronucleus assay in combination with the umu test will form the panel for genotoxicity testing. neurotoxicity will be assessed by comparing necrotic and apoptotic effects as well as the development of reactive oxygen species in human nervous cell with human liver cells. additionally neuron specific assay like the neurite outgrowth test are performed. this is complemented by measuring the activity of acetyl choline esterase activity and the development of the side line organ in zebra fish (danio rerio). the test battery for endocrine effects consists of hormone specific reporter gene s81 assays in addition to the h295r assay. when necessary a reproduction assay in the mud snail potamopyrgus antipodarum is carried out. during the project some 60 substances were evaluated. this allowed for the development of a reliable test strategy. currently the guidelines for performing the required tests are in the making. metabolomics has gained increasing interest over the last years with numerous possible applications ranging from strain optimization for industrial production over drug discovery to improved toxicity testing. however, regulatory acceptance of this promising approach is still not reached, mostly because standardization and evaluation of reproducibility are still mostly lacking. the metamap®tox database has been developed by basf over the last ten years containing toxicity and metabolome profiles of more than 750 different compounds. to ensure maximum reliability, data was gained from plasma samples of highly standardized 4 week rat studies. animal maintenance and treatment, sampling and work-up of plasma, measurement of the metabolome as well as data interpretation and storage were standardized including thorough documentation, the compliance with sops and safe data storage. data from more than 80 control groups with each 10 males and females were analyzed to assess variability. an in depth analysis of this showed a high stability and robustness of the metabolome over a period of ten years. after artificially splitting the groups of 10 control groups into groups of five animals and comparing the number of statistically significantly regulated (false positive) metabolites, the peak of the distribution curve was located to the left of the exact (gaussian) center, but tailed off to the right more than expected under the normal distribution. from this analysis we were able to calculate density distributions (relative ratio and standard deviation) for the control values of each metabolite, which can serve as a historical control displaying the range of changes which can be expected as normal. during the course of our project we have used more than ten exact repeats to show reproducibility and reliability of the metabolome analysis (kamp et al., 2012) . comparing these exact repeats at different levels of statistical significance, we noted that at a level of statistical significance of approximately p = 0.1, the best balance between matches (metabolites regulated in the same direction) and mismatches (metabolites regulated in opposite directions) was obtained. the high quality standards applied as well as the examination of control data increase the robustness of this approach, going also hand in hand with improved data quality. this significantly facilitates decision making based on the gained data. due to these improvements a new level of transparency is reached, which might allow inclusion of metabolome data in a regulatory environment. hydroxycitric acid (hca) is a fruit acid naturally occurring in fruits of the tropical plant garcinia cambogia. a number of dietary supplements intended for weight loss contain hca, which is added in form of g. cambogia extracts. the composition of these extracts is often not clearly specified. health concerns about safety of the hca-containing supplements have been raised, based on results from animal studies, which observed toxic effects on the testis and on spermatogenesis after administration of preparations containing high hca doses. in the current risk assessment, the possible health risks associated with consumption of hca-containing dietary supplements (hca doses of approximately 1000 to 3000 mg per day) were evaluated based on relevant animal and human studies with the focus on testicular toxicity as a critical endpoint. in several published animal studies, repeated (short-term or subchronic) ingestion of certain hca-preparations (g. cambogia-extract or ca 2+ -hca salt) induced testicular atrophy (i. e. atrophy of seminiferous tubules, degenerative changes of sertoli cells at histological examination) and impaired spermatogenesis (i. e. decreased sperm counts) in male rats at high doses (noael and loael of 389 and 778 mg hca/kg body weight & day, respectively). animal studies with other hca-preparations (ca 2+/ k + -hca salt) found no such effects at the highest hca-doses tested (noael: 610,8 mg hca/kg bwt & day). human intervention studies which addressed the safety of hca in healthy test persons reported no substancespecific adverse effects after ingestion of hca doses up to 3000 mg per day over the period of up to 12 weeks. however, the question of possible adverse effects of hca on the human testes was not adequately addressed in studies with human volunteers. in a single clinical study with 24 male test subjects, no significant changes in endocrinologically relevant parameters such as serum inhibin b or fsh were observed after consumption of 3000 mg of hca for 12 weeks. however, no investigations of direct parameters that might inform on potential effects on spermatogenesis, such as sperm quality and sperm count, were conducted in this study. considering the serious adverse effects on the testes observed in several animal studies as well as in view of lack of the adequate human data on the safety of the long-term use of hca-preparations, it is concluded that knowledge gaps and substantial uncertainties exist regarding the safety with respect to human health of high amounts of hca found in commercially available food supplements, particularly with regard to the human male reproductive system. a critical look on the passing-bablok-regression b. mayer 1 1 universität ulm, institut für epidemiologie und medizinische biometrie, ulm, germany background: the passing-bablok (pb)-regression is a commonly used approach to prove the equality of different analytical methods when studying quantitative laboratory data. it is based on the assumption that the measurements of two methods are linearly related. if then one method is regressed onto the other and the respective confidence intervals of the intercept and the slope include 0 and 1, respectively, it is assumed to have a proof of methods equality. however, this conclusion is problematic in respect of an essential principle of statistical hypothesis testing. methods: in this talk the general idea behind the pb-regression is discussed critically. although the method makes use of confidence intervals a decision is made, which is why it is important to discuss how the results of a statistical hypothesis test have to be interpreted. moreover, alternative statistical approaches to investigate agreement in biometrical practice are pointed out by means of a practical example and their advantages and limitations are addressed. results: all approaches applied to a sample data set led to the same conclusions. demonstrating methods equality though necessitates an a-priori definition of an appropriate equivalence margin. conclusion: the pb-regression may give useful advice when comparing two measurement methods towards equality. however, its results are statistically inconclusive, since the pb-method does not follow the principle of equivalence testing. alternative measures of agreement should be applied instead to ensure results which are not attackable and serve as a statistical proof. insulin is an important parameter both in toxicology (toxicity to the endocrine pancreas) and pharmacology (models of diabetes and metabolic syndrome). currently available elisa and ria methodologies for insulin often require up to 100 µl plasma or serum for a single measurement. in order to meet the general trend to include more relevant parameters in animal studies and restrictions through animal welfare requirements to limit the volume of interim blood draws we explored the rat/mouse insulin singleplex assay of meso scale discovery (msd) as an alternate assay consuming only 10 µl serum or plasma or less for a single measurement. the assay is a sandwich immunoassay, whereby insulin in the sample binds to the capture antibody immobilized on the working electrode surface at the bottom of each well and recruitment of the labeled detection antibody (anti-insulin labeled with electrochemiluminescent compound, msd sulfo-tag™ label) by bound analyte completes the sandwich. voltage applied to the plate electrodes then causes the label bound to the electrode surface to emit light the intensity of which is a quantitative measure of insulin. the msd insulin assay was characterized by a robust calibration and only small variations within repeated measurements. the assay presented a broad dynamic range and differences in insulin levels of normal and rats suffering from metabolic syndrome could readily be demonstrated. furthermore, the high sensitivity may even allow the use of smaller sample volumes. these features render this assay an attractive alternative for the measurement of insulin. the lack of corresponding quality control samples for internal quality control may be considered as a relative drawback. however, the cross reactivity of the assay with human insulin provides the opportunity to use qcs designed for human assays and to possibly participate in ring trials for human insulin for external quality control if needed. surfactants are main constituents of different consumer products, e.g. detergents or cosmetic cleansing products. since surfactants show an intrinsic skin irritation potential, dilutions are used in the final products to avoid adverse effects like irritant contact dermatitis from product use. in addition, mixtures of different surfactants are typically formulated, as it is a long-standing experience that those mixtures exhibit much lower acute irritation potential than expected from the mere summation of their individual irritation potential, an effect coined surfactant antagonism. only few studies were performed to gain a more fundamental understanding of the effect, and it's mechanistic basis remains unclear. however, a thorough understanding of the surfactant antagonism is not only of value for the formulation of products that are considered 'mild to the skin'. it is also important for the classification of products according to the clp regulation in cases when data of the mixtures is missing, because summation of the ingredients' irritating effects usually results in over-classification as skin irritant. due to the progress in the development of alternatives to animal testing, different in vitro methods have become available to determine skin irritating properties of substances. methods like the oecd tg 431 and 439 especially aim at deriving a classification for skin irritation/corrosion effects according to the clp regulation. however, even though these methods became the preferred test methods for skin irritation testing, to our knowledge hitherto isolated investigations on the surfactant antagonism were only performed either by human patch test studies or by non-standard in vitro assays. in this study, the irritation potential of binary mixtures of sodium dodecylsulfate (sds), linear alkylbezene sulfate (las), cocamidopropyl betaine (cabp) and alkylpolyglucosid (apg) compared to the single compounds was investigated using open source reconstructed epidermis (os-rep) models. combinations of sds or las with cabp and apg, respectively, resulted in a clear decrease of the irritation potential compared to the irritation exerted by the single surfactants, even though the total surfactant concentration was higher in the mixtures. in addition, the effect of surfactant antagonism was also observed in a mixture of cabp and apg. the reduced irritation potential of mixed surfactants came along with both reduced skin penetration of fluorescein and reduced release of ldh. since no surfactant antagonism is observed in monolayer cultures of keratinocytes that were exposed to mixtures of surfactants, it is assumed that keratinocytes in the viable parts of the reconstructed epidermis are promptly damaged by the surfactants once the model's barrier is destroyed. hence, surfactant antagonism appears to be primarily driven by the mixture's lower ability to damage the skin model's barrier. the micronucleus (mn) test is a reliable method for the detection of cytogenetic damage in proliferating cells. in recent years, substantial progress has been made on automated, thus faster and more objective scoring of mn test samples, i.e., methods based on flow cytometry. the aim of the present study was to use the adherently growing human bladder cancer cell line rt4 to carry out a comparison between traditional (fluorescence microscopy) and automated (flow cytometry) mn scoring. for this purpose, different substances which are known to be positive controls were used. rt4 cells were either continuously incubated for 72 h (approximately 1.5 cell cycles duration) with methyl methanesulfonate (mms; 50-200 µm), benzo[a]pyrene (b[a]p; 0.1-1 µm), vincristine (3-20 nm) , and colcemid (10-20 nm) or cells were irradiated with xrays (0.5-2 sv) and then cultured for 72 h. for standard mn scoring, cells were harvested, subjected to hypotonic treatment, fixed with methanol/acetic acid, placed on glass slides, stained with acridine orange and observed by fluorescence microscopy. for the flow cytometric method, harvested cells were stained in two sequential steps. intact cells were subjected to ethidium monazide bromide followed by photoactivation (75 w, 20 min) to label dead or dying cells. then, cells were lysed and stained with sytox green for a pan-dna labelling and analyzed on a flow cytometer. both, chemically-and radiation-induced treatment led to a dose-dependent induction of mn when evaluated by fluorescence microscopy. when the flow cytometry-based method was applied, clearly positive results including a dose-dependent induction of mn, however, were obtained only for 3 out of the 5 treatments (vincristine, colcemid and x-rays); whereas, treatment with mms and b[a]p led to only minor increases in relative mn frequencies (≤2-fold), even at the maximum concentrations. in summary, flow cytometry-based mn scoring has been successfully applied in rt4 cells. however, our initial results suggest that flow cytometry-based mn scoring is less sensitive than microscopic scoring when rt4 cells are used. so far, only few adherently growing cell lines have been applied to flow cytometry-based mn scoring. further substances (positive and negative controls) and possibly other adherent cell lines need to be tested to expand our knowledge on the effectiveness of automated mn scoring in vitro and compared to traditional approaches. background: the platinating agent cisplatin is commonly used in the therapy of various types of solid tumors, especially urogenital cancers. its anticancer efficacy largely depends on the formation of bivalent dna intrastrand crosslinks, which impair dna replication and transcription. these crosslinks stimulate mechanisms of the dna damage response (ddr), thereby triggering checkpoint activation, gene expression and cell death. the clinically most relevant adverse effect associated with cisplatin treatment is nephrotoxicity, which mainly results from damaged tubular cells. here, we analyzed the influence of the hmg-coa-reductase inhibitor lovastatin on the cisplatin-induced geno-and cytotoxicity in the rat renal proximal tubular epithelial cell line nrk-52e. methods: cell viability was determined by using the alamar blue assay, as well as by electrical impedance measurements via the icelligence system. alterations in cell cycle progression were assayed by flow cytometric analysis. the formation of pt-(gpg) intrastrand crosslinks was determined via southwestern blot. the amount of dna double-strand breaks (dsbs) was quantified by measuring the level of s139 phosphorylated h2ax (γh2ax) via immunocytochemistry as well as by western blot. additionally, neutral and alkaline comet assays were performed to determine the amount of dna single-and dna double-strand breaks. mechanisms of the ddr were analyzed by western blot as well as by quantitative real-time pcr. results: the data show that pretreatment of nrk-52e cells with a subtoxic dose of lovastatin reduced the cytotoxicity evoked by high doses of cisplatin by protection from cisplatin-stimulated apoptotic cell death. moreover, lovastatin had extensive inhibitory effects on cisplatin-induced ddr, as reflected on the level of p-atm, p-p53, p-chk1, p-chk2 and p-kap1. furthermore, activation of mitogen-activated kinases (mapks) was also reduced. the lovastatin-mediated mitigation of cisplatin-induced ddr was independent of the initial formation of dsbs as well as of pt-(gpg) intrastrand crosslinks. lovastatin protects nrk-52e cells from cisplatin-induced cytotoxicity by interfering with proapoptotic mechanisms of the ddr independently from initial dna damage formation. with respect to the clinic, the data indicate that lovastatin might be useful to mitigate cisplatin-induced nephrotoxicity. the influence of oxidant tert-butylhydrochinone (tbhq) on endothelial cell migration in wrn-deficient cells k. laarmann 1 , g. fritz 1 1 institut für toxikologie, düsseldorf, germany introduction: wrn is a dna helicase and possesses a 3´-5´exonuclease and atpase activity as well as a single strand annealing activity. it is involved in dna repair, by interacting with proteins of base excision repair (ber) and nucleotide excision repair (ner). defects of wrn are marked by genome instability which, in turn, is caused by defects in dna damage repair. patients with a mutation in the wrn gene show premature aging and early mortality. the latter is mainly caused by arteriosclerosis. furthermore, wrn participates in the regulation of genotoxic stress responses stimulated by reactive oxygen species (ros) and alkylating agents. the aim of this study was (i) to investigate whether endothelial cell migration and adhesion were effected by sub-toxic (ic 10 ) and moderate toxic (ic 40 ) concentrations of the oxidant tertbutylhydrochinone (tbhq) and (ii) whether wrn influences migration and adhesion in the presence or absence of tbhq. methods: endothelial-like ea.hy926 cells were treated with different concentrations of the redox cycling and thus ros producing oxidant tbhq. viability was measured by the alamar blue assay. ic 10 and ic 40 were determined after 24h permanent treatment. to investigate the influence of wrn on endothelial cell migration and adhesion, a wrn knock-down was performed in ea.hy926 cells using rna interference. to measure migration, a confluent cell monolayer was scratched using a pipet tip, 24h after permanent tbhq treatment. pictures were taken at the time points 0h, 4h and 8h after performing the scratch. the non-closed area was measured. in a second part, adhesion of the calcein-labeled colon adenocarcinoma ht-29 cell line on the ea.hy926 monolayer was investigated. wrn-deficient or non-deficient cells were treated with 100 µm and 160 µm tbhq or with tnfα. results: for ea.hy926 cells, 100 µm and 160 µm tbhq were determined as ic 10 and ic 40 , respectively. performing the migration assay, ea.hy926 cells showed 75% gap closure, whereas wrn-deficient cells showed a closure of only 35% after 8h. the gap was closed of 65% and 55% after 100 µm and 160 µm tbhq treatment. in wrndeficient cells no remarkable effect on migration was observed after 100 µm tbhq treatment, whereas the treatment with 160 µm tbhq showed a slight decrease in migration of about 10% compared to wrn-deficient cells. no effect on adhesion was observed after tnfα treatment. after 160 µm tbhq treatment a slight increase of adhesion was detected in ea.hy926 cells. the influence of moderate tbhq concentration on adhesion was reduced in the absence of wrn. conclusion: wrn influences endothelial cell migration. in contrast to wild-type ea.hy926 cells, no significant effect of tbhq was observed on migration of wrndeficient cells. furthermore, the moderate toxic concentration of tbhq showed slightly increased ht-29 adhesion to ea.hy926, which was not found in wrn-deficient cells. outlook: in forthcoming studies we analyse the effect of alkylating agents on migration and adhesion. data will be presented and discussed. the aim of the present work was to compare the sensitivity of leukemia cell lines (hl60, jurkat and tk6) and hematopoietic stem cells with regard to the response to genotoxic agents. chromosomal damage was analyzed by evaluation of the micronucleus frequency. furthermore, changes in the proliferation index and the frequencies of apoptotic and mitotic cells were assessed. several cytostatic drugs with different mechanisms of action were used as genotoxic agents. doxorubicin was used as an intercalator, radical producer and topoisomerase ii inhibitor. also, the effects of vinblastine, a mitosis-inhibiting drug and of methyl methanesulfonate, which forms dna-adducts and stalls replication forks, were analyzed. in general, a difference in sensitivity between the different substances was observed. with regard to the formation of micronuclei after treatment with doxorubicin, jurkat and tk6 cells showed similar increasing trends, whereas hl60 cells showed a much higher increase in micronucleus frequency. a clear decrease in proliferation and the frequency of mitotic cells was observed at the highest concentration (100 nm doxorubicin) investigated, and only a slight increase in the number of apoptotic cells could be shown. the biggest differences in formation of micronuclei could be detected after treatment with vinblastine. hl60 cells showed only a slight increase of micronuclei, but the effect on jurkat cells was stronger. the highest micronucleus frequency after vinblastine treatment was detectable for the tk6 cells. the results for the highest investigated concentrations (31.6 nm and 100 nm vinblastine) showed a significant reduction of the proliferation index. this effect is reflected by the increasing numbers of apoptotic cells in all cell lines. the results for methyl methanesulfonate demonstrated only a small increase in micronucleus formation for the jurkat cells, but higher values for the tk6 cells. in contrast the hl60 cells did not lead to a concentration-dependent effect with methyl methanesulfonate. these results are complemented by preliminary findings in hematopoietic stem cells at selected compound concentrations. the different results between the leukemia cell lines and the stem cells might possibly originate from the different p53 status of hl60 (null), jurkat (multiple mutations), tk6 (wild type) and hematopoietic stem cells (wild type). this difference might also cause differences in cell cycle control or repair mechanisms, and needs further investigations. hyperinsulinemia is thought to enhance cancer risk. a possible mechanism is induction of oxidative stress and dna damage by insulin, here, the effect of a combination of metformin with insulin was investigated in vitro and in vivo. the rational for this were reported antioxidative properties of metformin and the aim to gain further insights into mechanisms responsible for protecting the genome from insulin mediated oxidative stress and damage. comet assay, micronucleus frequency test and a mammalian gene mutation assay were used to evaluate the dna damage produced by insulin alone or in combination with metformin. for analysis of antioxidant activity, oxidative stress and mitochondrial disturbances, the cell-free frap assay, the superoxide-sensitive dye dihydroethidium and the mitochondrial membrane potential-sensitive dye jc-1 were applied. accumulation of p53 and pakt were analysed. as an in vivo model, hyperinsulinemic zucker diabetic fatty rats, additionally exposed to insulin during a hyperinsulinemic euglycemic clamp, were treated with metformin. in the rat kidney samples, dhe staining, p53 and pakt analysis, and quantification of the oxidized dna base 8-oxodg was performed. metformin did not show intrinsic antioxidant activity in the cell free assay, but protected cultured cells from insulin mediated oxidative stress, dna damage and mutation. treatment of the rats with metformin protected their kidneys from oxidative stress and genomic damage induced by hyperinsulinemia. metformin may protect patients from genomic damage induced by elevated insulin levels. this may support efforts to reduce the elevated cancer risk that is associated with hyperinsulinemia. the human skin is the primary barrier against environmental and chemical impacts. as such it shields us against a plethora of xenobiotics such as potentially carcinogenic polycyclic aromatic hydrocarbons (pahs). at the same time it is the second most densely populated organ, harbouring more than 1000 bacterial species and population densities of up to 10 7 cfu per cm 2 . yet little is known about this microbiome's potential to metabolise and toxify pahs such as benzo[a]pyrene (b[a]p). previous work at the bfr showed that degradation of b[a]p and other pahs is a universal feature of the skin's microbiome (sowada et al., 2014) . the corresponding metabolites only partly overlap with those known from eukaryotic metabolism and possess cytotoxic as well as genotoxic properties. excretion of these metabolites will lead to exposure times of 20-30 hours or longer for full and partial metabolisers, respectively. while in vitro studies show the corresponding substances to exert their effects synergistically, an assessment of their potential impact on human carcinogenesis is pending. one obvious mode of action would be direct genotoxicity. however, another option is interference with uv-damage repair. ultraviolet radiation (uvr) from sunlight is regarded the main causative factor for the induction of skin cancer. it induces two of the most abundant mutagenic and cytotoxic dna lesions, that is cyclobutane-pyrimidine dimers (cpds) and 6-4 photoproducts (6-4pps). these lesions are repaired primarily by nucleotide excision repair (ner), a system that is also responsible for the removal of pah-derived dna adducts. we therefore wanted to know whether and to what extent bacterial b[a]p metabolites have the capacity to interfere with ner, potentially contributing to uv-induced dna-damage. to investigate this selected genotoxic metabolites were examined for their potential to affect the dna repair capacity of skin cells (hacat). following treatment with uva/b and bacterial b[a]p-metabolites the skin's repair capacity was assessed using a modified comet-assay. ionizing radiation (ir) is a well-established model to induce dna double-strand breaks (dna-dsbs), but it also generates a broad range of other dna lesions including dna single-strand breaks as well as oxidative dna base modifications. furthermore, ir is able to modify membrane components and triggers the activation of epidermal growth factor receptor. a more specific dsb-inducer is cytolethal distending toxin (cdt), which is produced by a variety of gram-negative bacteria and harbours an intrinsic dnase-like endonuclease activity [1] . dsbs are potent cytotoxic lesions and promote genomic instability, e.g. by formation of chromosomal aberrations. a cellular mechanism to prevent genomic instability and maintain cell homeostasis could be autophagy. this process is highly regulated involving the lysosomal degradation of damaged organelles and proteins. here, we study autophagy induction following dsb generation in human colorectal cancer cells as well as in primary human colonic epithelial cells (hcec) and analyzed regulatory mechanisms. first, the autophagy-specific marker lc3b was shown to increase in a dose-and time-dependent manner after treatment with both cdt and ir as assessed by confocal immunofluorescence microscopy and western blot analysis in hct116. similar results were obtained in sw48 and hcec cells via western blot. these findings are in agreement with the enhanced formation of autophagosomes and the dose-dependent decrease of the autophagy substrate p62 as observed by flow cytometry and western blot analysis in hct116, sw48 and hcec. cdt-and irinduced autophagy rates in hct116 increased over time correlating well with the dsb induction. importantly, a dnasei-defective mutant of cdt did neither cause dsbs nor induce autophagy. additionally, the time-dependent accumulation of the lysosomal associated membrane protein 1 (lamp-1) was observed by confocal immunofluorescence microscopy. dsb-induced autophagy was blocked by chemical inhibitors. next, we showed that both ir and, to a lesser degree, cdt induce the phosphorylation of akt at ser473. pharmacological inhibition of akt in hct116 cells enhanced the cdt-and ir-induced autophagy shown by accumulation of lc3b and lamp1 after 48 h and increased autophagosome formation. upregulation of dsbinduced autophagy by akt inhibition resulted in a decreased cytotoxicity after 72 h and significantly lower apoptosis/necrosis rates after 48 h, which were determined by mts cell viability assay and annexin-v/pi staining. ongoing studies will evaluate the impact of other dna damage response pathways and the potential protective role of autophagy against genomic instability. mustard agents are potent dna alkylating agents. among them, the bi-functional agent sulfur mustard (sm) was used as a chemical warfare agent due to its vesicant properties. although the use of sm in warfare has been banned in most countries of the world, its use in terroristic attacks or asymmetrical conflicts, such as the syrian civil war, still represents a realistic and significant threat. on the other hand, especially nitrogen mustards, such as cyclophosphamide or melphalan, have been used as chemotherapeutic agents due to their cytostatic properties. thus, mustard-induced dna damage, in particular dna crosslinks, can trigger complex pathological states, as it is observed in sulfur mustard exposed victims, but on the other hand also lead to the chemotherapeutic effects of clinically-used nitrogen mustards. mass spectrometric monitoring and quantitation of mustard-induced dna adducts can help to unambiguously identify and verify sm-exposed victims and to monitor the efficiency, as well as potential side-effects of mustard-based chemotherapy. up to now, the verification of mustard-induced nucleic acid damage is mainly based on immunohistochemical methods, which have several drawbacks such as limited specificity, sensitivity, and low dynamic range of quantitation. with this project, we aim to develop a (hplc/uplc)-ms/ms-based platform for the quantitation of the most common mustard-induced dna adducts including bis(n7-guanine-ethyl) sulfide dna crosslinks. up to date, we established methods for the quantitation of the several common dna adducts induced by the mono-functional sulfur-mustard derivative 2chloroethyl ethyl sulfide ("half mustard", cees). for that reason purification protocols, chromatographic conditions and mass spectrometric settings were developed to detect n7-ethylthioethyl-2´desoxyguanosine (n7-ete-dg) and n3-ethylthioethyl-2´desoxyadenosine (n3-ete-da) and their thermal hydrolysis products n7ethylthioethyl-guanine (n7-ete-gua) and n3-ethylthioethyl-adenine (n3-ete-ade), respectively, and the sensitivity was compared to immunohistochemical methods. additional non-radioactive isotope-labelled standards are being synthesized, which will be spiked into samples to account for technical variability during sample work-up and to improve ms-based quantitation. this procedure requires minimal cellular material and therefore should be transferred to quantitation of dna adducts in human blood samples. this will allow to monitor dna adducts as biomarkers of exposure in potential smexposed victims as well as in mustard-based chemotherapy. this method also sets a basis to investigate specific mustard-induced dna repair mechanisms and their cellular consequences. the γh2ax assay vs. comet assay for genotoxicity testing universitätsmedizin mainz, institut für toxikologie, mainz, germany dna damage leads to activation of the cellular dna damage response (ddr). this signalling network results in activation of various dna repair proteins and chromatin structure modulators. a frequent manifestation of ddr is the phosphorylation of histone 2ax (gh2ax), which can be visualised as gh2ax foci by immunocytochemistry. in the present study, we tried to assess if gh2ax is a reliable biomarker for detecting the cellular response to dna damage. we selected 14 well-characterised genotoxic compounds and compared them with 10 non-genotoxic chemicals in the wellcharacterised cho cell system. we measured quantitatively γh2ax by manual and automatic scoring of γh2ax foci, and by flow cytometry counting of γh2ax positive cells. the cytotoxicity dose-response was determined by the mtt cell proliferation/viability assay. we show that a) all genotoxic agents were able to induce dose-dependently γh2ax in the cytotoxic range whereas no induction was observed after treatment with non-genotoxicants; b) manual scoring of γh2ax foci and automated scoring gave similar results, with the automated scoring being faster and more reproducible; c) data obtained by foci counting and facs analysis of γh2ax positive cells showed a significant correlation. further we compared dna damage induced by 4 selected genotoxins at the time-points using the alkaline and neutral comet assay. significant correlation with the alkaline and neutral comet assay was observed for some but not all genotoxins and, predominantly, at earlier time points. we suggest that comet assays detect mainly primary dna damage, whereas γh2ax assay detects a specific response to dna damage which can persist longer. the γh2ax foci and flow-cytometry assays allow for a rapid and reliable determination of genetic damage in mammalian cells and can be used as additional genotoxicity assays. available in vitro methods to investigate the genotoxic potential of drugs fall short of throughput, specificity and mode of action information. a set of mechanistic biomarkers for clastogenic, aneugenic or apoptotic effects may help to overcome these limitations. thus, a staining assay amenable to flow cytometric analysis is being developed by litron laboratories, rochester, ny, supported by international collaborators. the experimental design of this assay consists of 3 stages. the objective of this work is the evaluation of this assay in the laboratories of bayer pharma ag. the biomarkers covered by the assay are associated with dna damage response pathways that have potential for class discrimination (clastogen/aneugen/cytotoxicant) of in vitro genotoxicants: dna double strand breaks (γh2ax), nuclear division (phospho-h3, dna content), apoptosis (cleaved parp). based on the pilot work at litron laboratories, tk6 cells were introduced to the genetic toxicology of bayer pharma ag. cells were exposed for 4 and 24 hrs in triplicates on a 96 microwell plate to one reference clastogen (etoposide, eto), aneugen (vinblastine, vb) and cytotoxicant (carbonyl cyanide 3-chlorophenylhydrazone, cccp). after staining, the samples were analyzed with the flow cytometer bd accuri c6 (bd biosciences, heidelberg, germany). the reference substances yielded the responses expected from the pilot study at litron laboratories: vb showed distinct increases of phospho-h3 events at 4 and 24 hrs and polyploidy at 24 hrs time point. eto induced a clear increase of yh2ax with a simultaneous reduction of phospho-h3 at 4 and 24 hrs. finally, cccp caused a reduction of phospho-h3 events, increased cleaved parp events and did not influence γh2ax. moreover, benchmarking experiments under pilot work conditions were performed with high content imaging analysis. we compared yh2ax and phsopho-h3 pilot study results as well as cleaved parp with caspase 3/7. in addition, the tunel assay (click-it ® tunel alexa fluor, thermofisher) was executed to benchmark cleaved parp. the benchmarking results support the selected biomarkers of the multiplexed assay. in stage 2, additional reference compounds (three aneugens/clastogens/cytotoxicants) were investigated. so far, the chosen biomarkers of dna damage response appear useful for class discrimination and provide additional information to existing genotoxicity tests. cell-cell contacts are involved in keeping a physiological balance between proliferation, differentiation and apoptosis. far less is known about the role of cell-cell-contacts in regulating necrosis, for instance in response to oxidative stress. previous findings of our group demonstrated that, in contrast to semi-confluent proliferating cultures, confluent murine fibroblasts (nih3t3, mef) and human keratinocytes (hacat) are protected against necrosis induced by tert-butyl hydroperoxide (t-booh). comparison of confluent cells (g0/g1 = ~70 %) and semi-confluent cultures, similarly arrested in the g0/g1 phase by serum-starvation or the mek inhibitor u0126, ascertained that the resistance against t-booh is mediated by cell-cell contacts and not by cell cycle arrest. we further revealed that confluent cultures are protected against t-booh-induced dna double strand breaks as assessed by the neutral comet assay and against mitochondrial damage detected by flow cytometric analysis of dioc6 staining. to better understand the protective role of cell-cell-contacts in ros-mediated necrosis, we started characterizing the signaling cascade induced by t-booh in semi-confluent proliferating cultures. in accordance with the observed formation of dna double strand breaks in response to t-booh, we detected phosphorylation of the checkpoint kinase chk2. however, inhibition of atm, the kinase responsible for chk2 activation, did not influence t-booh-induced cell death. interestingly, first experiments gave a hint for the participation of rip1, since the chemical rip1 kinase inhibitor necrostatin-1 (nec-1) blocked cell death up to averagely 50 %, what is described as a specific marker for regulated necrosis. in line with this observation, t-booh-induced cell death could not be blocked by the pan-caspase inhibitor z-vad-fmk strongly indicating that caspase activity is not required. moreover, parp-1 and p53 are probably not involved. deeper analyses could give evidence that nec-1 did not block formation of dna double strand breaks nor mitochondrial damage indicating that the kinase blocked by nec-1, possibly rip1, acts downstream of dna double strand breaks and / or mitochondrial damage. in the end, we could identify a crucial role of ca 2+ signaling for t-booh-mediated toxicity. as the calcium chelator bapta-am was able to completely block not only cell death, but also mitochondrial damage and dna double-strand break formation, there is a strong need for further investigations of the possible interplay between regulated necrosis and calcium, regulated by cell-cell contacts among oxidative stress. the work was supported by the hoffmann-klose-stiftung, the promotionsförderung rheinland-pfalz, the johannes gutenberg-university and the university medical center of the johannes gutenberg-university. the mammalian target of rapamycin (mtor) forms two multiprotein complexes (mtorc1 and mtorc2) and influences cell growth, proliferation, survival and metabolism. constitutively activated mtor was found to be deregulated in several cancer types, which makes it an interesting target for therapeutic cancer strategies. rapamycin is able to inhibit mtor and its downstream targets and is currently studied for its anticancer properties in clinical trials. despite previous evidence, there are studies that show an adverse effect in cancer treatment causing tumour growth, evolving the question of the effectiveness of the drug in cancer treatment. therefore, we examined the transformational potential of rapamycin in a balb/c cell transformation assay (cta) as well as markers of proliferation and protein synthesis. the balb/c 3t3 cell transformation assay mimics different stages of in vivo carcinogenicity (initiation, promotion, post-promotion phase) and is a promising alternative to rodent bioassays. balb/c fibroblasts are treated for 3 days with the tumour initiator mca (3-methylcholanthrene) followed by 13 days with the promotor tpa (12-o-tetradecanoylphorbol-13-acetate). upon treatment with these chemicals cells are transformed into morphologically aberrant foci and can be visualized after six weeks by giemsa staining. it is possible to apply additional substances during the whole assay or in several phases of transformation and evaluate the colony formation. furthermore, our improved protocol allows additional westernblot or immunofluorescence analysis. the influence on cell proliferation of different concentrations of rapamycin was investigated by cell counting (living and dead) to choose a suitable concentration for the cta. performances of balb/c ctas with 10 nm rapamycin showed, contrary to expectations, an increase in cell transformation. by administration of rapamycin only in the promotion phase we could detect an increase in colony formation, whereas a treatment with rapamycin in the post-promotion phase with already established foci, seemed to reveal its therapeutic properties. to better understand the role of mtor in our cell transformation system we used another mtor inhibitor called osi-027. surprisingly, an incubation with 3 µm osi-027 led to a decrease in colony formation. we are now able to investigate the underlying mechanism with westernblot and immunofluorescence analysis and can compare regulations of downstream targets like the marker of protein synthesis p-s6. our investigations revealed different cell transformation outcomes by comparing the two known mtor inhibitors rapamycin and osi-027, which need to be further evaluated. in the ongoing project we want to detect differences between rapamycin and osi-027 by protein analysis and identify key proteins, which are involved in this opposed colony formation of the balb/c cells. these results can be helpful to better understand mtor inhibition in matters of tumour therapy. introduction: over the past 50 years, the biguanide compound metformin has been widely prescribed as an insulin sensitizer in type 2 diabetes mellitus. interestingly, recent meta-analyses of epidemiological studies have shown that metformin might be involved in risk reduction of carcinogenesis. in vitro studies have described amp-activated protein kinase (ampk)-dependent, by inhibition of the respiratory chain complex i, as well as ampk-independent actions of metformin. however, the detailed molecular mechanisms by which metformin affects cell proliferation and carcinogenesis have not been well identified up until now. method: to evaluate the protective potential of metformin, balb/c 3t3 cell transformation assays were performed. this valid toxicological method is an alternative to in vivo carcinogenic testing and mimics the different stages of cell transformation during carcinogenesis. in detail, mouse fibroblasts are treated with metformin and/or the tumour initiator 3-methylcholanthrene (mca) and the tumour promotor 12-otetradecanoylphorbol-13-acetate (tpa). in the first experiment several metformin concentrations (0.1-1 mm metformin) were applied answering the question of an effective metformin concentration. next, metformin treatment during the different phases of carcinogenesis (initiation, promotion, post-promotion phase) was done determining the most effective phase for an intervention, i.e. chemopreventive or chemotherapeutical properties of metformin. additionally, the effect of metformin on the energy metabolism of the cells was analysed using various methods like immunoblot and oxygen measurement by clark electrode. results/discussion: analysis of different metformin concentrations revealed a concentration-dependent effect of metformin. in detail, decreased colony forming potential of balb/c cells was most prominent using 1 mm metformin. this effect was not caused by growth inhibition of metformin itself since 1 mm metformin showed no growth inhibitory properties in a cellular growth pretrial. interestingly, the 2 phase cell transformation assay showed that the metformin effect is more pronounce in the postpromotion phase than in the initiation and promotion phase pointing to a chemotherapeutical potential. investigating several energy metabolism parameters, the results indicate that metformin may affect cell respiration as well as energy-dependent mechanistic markers like ampk. the presented results support rather the idea of the chemotherapeutic potential of metformin than a chemopreventive, using 1 mm metformin. the initial analysis of energy metabolism markers discovered interesting starting points for further investigations. johannes gutenberg university, institute of toxicology, 55131 mainz, germany nvp, widely used e. g. as a monomer for polyvinylpyrrolidones (pvp) with applications in food technology or cosmetics is a known hepatocarcinogen in rats after inhalative exposure to 5, 10, and 20 ppm for 2 years. nvp is tested in a battery of genotoxicity assays (e.g. ames, hprt, mouse lymphoma, uds, chromosome aberration, cell transformation, micronucleus test (mnt) in mice bone marrow) [1]) that all yielded negative results. however, nvp induces cell proliferation in liver (loaec: 0.5 ppm) after whole body exposure to vapor [2] . to confirm the absence of genotoxicity in the context of a potentially non-genotoxic mode of action, a five day whole body inhalation study to nvp vapor with concentrations of 0, 5, 10, 20 ppm was conducted in wistar rats (six animals per gender and group, ethyl methanesulfonate 200 mg/kg bw p.o. as positive control). genotoxicity was investigated by the mnt in bone marrow and the comet assay (± fpg) in liver and lung. further investigated endpoints related to possible non-hepatogenotoxic moa were: enzyme induction (erod, prod, brod), oxidative stress (gsh-, gssg-, non-protein sulfhydryl group level), and peroxisome proliferation (cyp4a, cyanide-insensitive palmitoyl-coa-oxidase). at carcinogenic inhalative doses, the results of this study proved the absence of genotoxicity in lung, liver and bone marrow as neither the tail intensity in the comet assay nor the number of micronuclei in the mnt was increased compared to the controls. however, also the non-genotoxic parameters (cyp-enzyme activity, glutathione levels, cyanide-insensitive-palmitoyl-coa-oxidase) were not affected by nvp-treatment. as potential metabolic activation cannot be excluded and may essentially contribute to the understanding of the carcinogenic mechanism, in vitro investigations in rat liver systems (subcellular fractions, hepatocytes, precision cut liver slices (pcls)) were performed additionally. up to now, 2-pyrrolidone is the only identified in vitro metabolite. as these results cannot mimic the in vivo situation of two described, ring-and vinylmajority containing unidentified metabolites [3] detailed investigations on metabolism may be a future perspective to approach the overall understanding of the carcinogenic mechanism of nvp. introduction: in ischemic conditions such as wound healing and myocardial infarction, new vessels are generated by vasculogenesis and angiogenesis. these processes are stimulated by the signalling peptide vascular endothelial growth factor which therefore has been proposed as a promising compound for the treatment of ischemic conditions. however, results of respective clinical studies have not been fully convincing yet. here, we investigated principles underlying the selforganization of newly formed vessels to functionally adequate microvascular networks indispensable for proper tissue substrate supply. intravital microscopy of the chick chorioallantoic membrane (cam), a non animal model as defined by the american national institutes of health's office for protection from research risks, was used to study peripheral expansion of existing arteriolar and venular trees by recruiting segments of the dense polygonal capillary mesh. this process we call "emerging angiogenesis". methods: white leghorn chicken eggs were put into incubators on embryonic day 0 (e0) at 37.5°c and 82% humidity. on e3, the eggs were cracked open and transferred into petri dishes. on e10, cam microcirculation was recorded using time-lapse intravital videomicroscopy at discrete time points for up to 48 hours. to improve the visibility of the capillary mesh, videorecordings were processed offline by generating coefficient of variation images of pixel grey values over time. changes of network topology during the observation time were investigated. results: in the cam, a sequence of specific events leading to extension of existing vessel trees was observed: in a capillary mesh region near terminal branches of existing vessel trees, homogeneous flow distribution is transferred to inhomogeneous flow distribution: preferred flow pathways through the mesh evolve carrying most of the blood. over time, these flow pathways exhibit diameter increase, straighten and connect the mesh to arteriolar and venular trees. in contrast, less perfused parallel mesh flow pathways and transversal mesh segments exhibit progressive decrease of flow and diameter resulting in vessel regression. as a result, hierarchical vessel tree structures are extended into the mesh region. while newly generated tree extensions are located above the mesh at the beginning, they sink to a lower level at later stages until they are finally covered by a reconstituted mesh network. the cam ex ovo model is well suited for studying emerging angiogenesis. vessel tree extension occurs via parallel processes of vessel maturation and capillary mesh segment regression. at later stages, newly formed vessel tree branches sink and the capillary mesh is reconstituted above. in the next step of our project, we will implement these phenomena in a computer simulation and use theoretical modeling to further investigate and better understand principles underlying microvascular network maturation. this will allow us to derive effective therapeutic strategies which could be tested in the cam model. chemicals are able to induce cancer in a wide range of organs. therefore, it is very important to investigate the toxic properties of chemical substances, especially their carcinogenic potential. in this context the number of animal experiments will drastically increase in the future. in order to avoid the use of expensive and time consuming animal experiments for long-term carcinogenic studies, the development of an in vitro system to test the carcinogenic potential of a high number of chemicals in a highly reproducible manner within a short period of time is imperative. by combination of the well-established balb/c cell transformation method with the soft agar colony formation assay, we developed a high-throughput in vitro system to identify effects of chemicals on cell transformation for the first time. balb/c mouse fibroblasts are treated with 3-methylcholanthrene as a tumour initiator and 12-o-tetradecanoylphorbol-13-acetate as promotor for several days, whereby foci of transformed cells are developed. after the promotion phase of the common balb/c cell transformation assay, cells are transferred into soft agar to further monitor the anchorage independent growth of transformed cells only. the established soft agar transformation assay reproduces the foci growth of previous experiments and is performed in 96-well plate format. hence, we can analyse the carcinogenic potential of several chemical substances in parallel and are also searching for alternative endpoint analysis, e.g. the usage of fluorescing cells stably expressing irfp, instead of the former time-consuming microscopic assessment. the here presented new technique is a high-throughput and low priced alternative for the evaluation of the carcinogenic potential of chemical substances in a short period of time without animal testing. the effort to develop new or refine established in vitro test systems rises due to animal welfare, scientific and/or regulatory reasons (e.g. the animal testing ban concerning the risk assessment of cosmetic product ingredients in march 2013). this progress, among others, leads to an increased performance of cell-based assays. the majority of model cell lines are routinely cultured using medium supplemented with fetal bovine serum (fbs) in amounts between 5-10%. the application of serum-substitutes will provide a reduction of the animal number needed, which corresponds to the guiding principles of the three r's (3r), described by russel and burch in 1959. in addition, chemically defined serum-substitutes have the potential to reduce the inter-experimental variability of test conditions caused by the inherent differences in chemical composition across fbs batches 1 , resulting in a refinement of in vitro testing. in this study, human tk6 cells were gradually adapted to serum-free conditions, where they show comparable growth gradients at the exponential phase. for cells under serum-free conditions a mean doubling time of 19.3 (± 1.7) h was observed while fbs supplemented cells showed a doubling time of 13.5 (± 0.8) several non-animal test methods addressing key events in the sensitization process have passed formal validation and oecd (draft) test guidelines are available. one of these methods is the direct peptide reactivity assay (dpra) assessing the ability of a chemical to bind to proteins to form a complete antigen (oecd tg 442c). the test is used to obtain a yes/no answer on whether the substance has a protein-binding potential. for a complete risk assessment, however, an estimation of a chemical's potency is also needed. in this study we examined if an assessment of potency could be achieved by 1) determining reactivity class cut offs based on published data on 199 substances for the dpra performed according to oecd 442c to predict un ghs sensitizer classes, 2) a variant of the dpra assessing reaction kinetics (time and concentration) for 12 substances or 3) an extended protocol testing several test substance concentrations for 50 reference substances and estimating the concentration of a test substance that is needed to cause a peptide depletion of 6.38% (ec6.38%). results of the first approach indicated that cut offs to differentiate the un ghs sensitizer classes 1a and 1b could indeed be defined. secondly, evaluating the reaction time based assay in which several time points between 5 min and 24 hours were assessed, it was found that not all reactions followed ideal kinetics. hence further investigations are needed to eventually derive a reaction time based prediction model. the results of the 3 rd approach (the standard protocol of the dpra was amended by testing three concentrations i.e. 1, 10, and 100 mm) indicated that potency classes could be assigned using the ec6.38% value to assess potency. in summary, using quantitative information derived from the dpra in particular using ec6.38% value may support the assessment of the skin sensitizing potency. identification of pre-and pro-haptens with non-animal test methods for skin sensitization since pro-haptens may be metabolically activated in the skin, information on xenobiotic metabolizing enzyme (xme) activities in cell lines used for testing of sensitization in vitro is of special interest. metabolic activity of e.g. n-acetyltransferase 1 (nat1) and esterase in the keratinocyte (keratinosens tm and lusens) and dendritic cell-like cells (u937 and thp-1) was previously demonstrated. aldehyde dehydrogenase (aldh) activities were found in keratinosens tm and lusens cells. activities of the investigated cytochrome p450-dependent alkylresorufin o-dealkylases, flavin-containing monooxygenase, alcohol dehydrogenase as well as udp glucuronosyl transferase activities were below detection in all investigated cell lines. a set of 27 putative pre-and pro-haptens (no obvious structural alert for peptide reactivity but positive in vivo) was routinely tested using the above mentioned cell lines as well as in the direct peptide reactivity assay (dpra). 18 of the compounds were unexpectedly positive in the dpra und further analyzed by lc/ms techniques to clarify the reaction mechanism leading to true positive results in this assay. oxidation products like dipeptide formations or the oxidation of the peptide-based sulfhydryl group led to positive results for benzo[a]pyren or 5-amino-2-methylphenol, respectively. in contrast, covalent peptide adducts were identified for 12 putative pre-haptens, indicating the dpra to be suitable for compounds requiring abiotic oxidation to get activated. for some dpra negatives, the keratinocyte and dendritic cell based assays provided true positive results. a combination of dpra, keratinosens tm and h-clat within a '2 out of 3' prediction model provided a high sensitivity of 81% for the set of the pre-/pro-haptens. the sensitivity of this combination of non-animal test methods in the '2 out of 3' prediction model in a set of 95 direct haptens was comparable (sensitivity = 87% when compared to llna skin sensitization testing is mandatory for all substances produced or marketed in volumes larger than 1 tonne per year under the european reach legislation. with reach supporting in vivo testing only "as a last resort" and the marketing ban for finished cosmetic products with ingredients tested in animals, attention has been given to developing integrated testing strategies combining in vitro, in silico and in chemico methods. key challenges are which tests to select and how to combine non-animal methods into testing strategies. this study suggests a bayesian value of information (voi) approach for developing non-animal testing strategies, which consider information gains from testing, but also expected payoffs from adopting regulatory decisions on the use of a substance, and testing costs. the 'value' of testing is defined as the expected social net benefit from decision-making on the use of chemicals with additional, but uncertain information from testing. the voi is calculated for a set of individual nonanimal methods including dpra, oecd qsar toolbox, are-nrf2 luciferase method covered by keratinosens and lusens, and hclat, seven battery combinations of these methods, and 86 two-test and 360 three-tests sequential strategies consisting of nonanimal methods. their voi is compared to the voi of the local lymph node assay (llna) as the animal test. we find that battery and sequential combinations of nonanimal methods reveal a higher voi than the llna. in particular, for small prior beliefs (i.e. a chemicals is, prior to testing, assumed to be a non-sensitiser), a battery of dpra + lusens reveals the highest voi. if there are strong beliefs that a chemical is a sensitizer, a sequential combination of the battery dpra + lusens, followed by keratinosens + hclat at the second stage and by the oecd qsar toolbox at the third stage performs best. for given specifications of expected payoffs the voi of the nonanimal strategy significantly outperformed the voi of the llna, for the entire range of prior beliefs. this underlines strong economic potential of non-animal methods for skin sensitization assessment. a chemical series to predict the proarrhythmic potential of drugs with low solubility for which no reliable purkinje fiber results could be obtained. these validation results showed that this cardiosafety in silico model can successfully be applied in r&d to predict the proarrhythmic potential of drug candidates within the model ad. introduction: the use of p-phenylenediamine (ppd) and derivatives (tab. 1) in oxidative consumer hair dye products is considered as key in hair dye allergic contact dermatitis [1] [2] [3] [4] . in recent supplement, 2-methoxymethyl-ppd (me+) shows significantly reduced sensitizing properties [5, 6] . since overcoming the skin barrier is a prerequisite for sensitization, numerous in vitro an in vivo studies on skin penetration of ppd and derivatives have been performed. the aim of the present study is the in silico prediction of the penetration of ppds, because such computations may help in understanding the processes involved in sensitization. for the first time, software dskin [7] is challenged to simulate this class of compounds. in silico results are retrospectively compared to previously published experimental data and may assist in future tailoring of in vitro experiments. material and methods: the permeabilities, lag-times and the time-dependent accumulated amounts of ppds were computed using dskin. input parameters for the latter were a concentration of 1 mg/ml (1%), finite dosing and 30 min in use incubation periods. molecular structures were optimized ab initio and the condensed fukui functions (ff) were estimated from mulliken population analyses [8] and electrostatic potentials using gamess [9] . results: initial results agree with experimental results using ppd in white petrolatum, demonstrating the applicability of dskin to ppds. the four ppds exhibit only small differences in permeabilities in silico (tab. 1). toluene-2,5-diamine shows a higher accumulated mass due to increased lipophilicity (fig. 1) . in general, the ff were very similar for all ppds and indicated that the n atoms would be the preferred targets for radical and electrophilic attack. discussion and outlook: in silico methods may be used to model the permeation of ppds despite their low molecular weight and low lipophilicity. the low amounts of ppds under in use conditions result from oxidative conditions. computed me+ permeation was not different to other ppds, therefore other properties account for the reduced sensitization potential. the very similar ff values hint at similar reaction pathways. furthermore, ppd and its derivatives are prone to n-acetylation in living skin resulting in metabolites exhibiting higher molecular weight and greater lipophilicity than the parent compounds. the effects of n-acetylation and reactions of ppd and its derivatives with histidine and cysteine residues are subject of upcoming computations. dermal absorption is an important factor in regulatory science regarding the registration of chemicals, agrochemicals and cosmetics. the issue has gained importance since it has been realized that the skin is not completely impenetrable for chemical substances. [1] the different ways to assess dermal absorption range from qsar models to complex in vivo studies including a complete toxicokinetic examination. the choice of method depends on the question that has to be answered as different systems give different results: absorption as % of applied dose in in vivo studies or permeability coefficient and lag time in infinite dose in vitro studies [1] . ideally both data would be available. since the oecd has adopted a guideline for assessing dermal penetration in vitro in 2004 the number of in vitro studies is rising continuously. depending on the chosen method results may vary in reliability and in acceptance by regulatory authorities. the skinab database [ba1] contains data for about 600 substances on dermal absorption which has been found through the echemportal [3] and extended with data from the edetox database. for selected substances with a broad spectrum of data available further analysis has now been started. 165 chemicals have been investigated in a comparable test system; from these 79 were shown to have a low dermal absorption of less than 1% and 51 compounds showed a high absorption rate of more than 50%. for the assessment of dermal exposure either the absorbed dose in percent or the flux can be measured. data analysis showed that only for 15 substances both is available: flux data from in vitro studies and absorption data from in vivo studies. this data could be used to clarify which parameter would be most useful for exposure assessment regarding dermal exposure. seven substances in the dataset were conspicuous for their range of absorption rates in different studies: less than 1% to more than 50%. an in depth analysis revealed the complex influence that different exposure parameters have on the results of dermal absorption studies. for some chemicals the influence of exposure time on increasing absorption values could be clearly demonstrated. beside other factors such as the chosen vehicle, and the (non-)occlusion of the site of exposure especially the choice species introduced a high variability; this holds even for the most common laboratory animals t. a review published by jung in 2015 [5] which comes to the conclusion that i.e. hairless species are usually not a good model to predict dermal absorption in humans. [1]who (2006) ehc 235 dermal absorption [2] scholz et al (2014) naunyn-schmiedeberg´s arch pharmacol 3 (suppl 1):s86 [3] www.echemportal.org [4] http://edetox.ncl.ac.uk [5] jung et al (2015) in-silico methods have evolved to indispensable tools in various areas of life sciences. several stages in drug development including hit identification and lead optimization, for instance, highly benefit from an accurate estimation of binding free energies associated with biological host-guest systems. as a consequence, the need for laboratory experiments including in-vivo experiments and animal testing is considerably reduced. another field profiting from free energy calculations is human as well as ecotoxicology. upon the development and risk assessment of new chemicals, transformation products arising from biotic or abiotic degradation of the parent substance have usually been neglected. however, since few years, the risk assessment of new chemicals often includes transformation products probably causing more harm than the parent substance itself. such studies as well are mostly carried out on the basis of in-vitro and in-vivo tests. moreover, many metabolites can be detected but neither enriched nor synthesized in amount sufficient for toxicological evaluations. at this stage, computational methods come into play. using classical molecular dynamics simulations in combination with an empirical linear prediction model, we have investigated several metabolites of the drugs sulfamethoxazole and carbamazepine and prioritized them according to their estimated binding affinities to potential biological target proteins. consequently, a couple of metabolites were identified that bind to one or more human cytochrome p450 variants and the bacterial enzyme dihydropteroate synthase, respectively, which are known to be sensitive to the two drugs. the investigations were carried out in the framework of the bb3r poject funded by the german government through bmbf. instituto superiore di sanità, environment and primary prevention dept., rome, italien introduction: in vitro methods have been increasingly used to characterize pharmacological and toxicological properties of substances. to address the problem of nominal versus actual concentrations, in vitro biokinetic studies were recently undertaken (truisi et al., toxicol lett 233:172-86, 2015) . we use those data as input into a physiologically based human kinetic model (pbhkm) to model the in vivo doses leading to the in vitro measured concentrations. methods: a pbhkm was used to simulate the concentration time profile of ibuprofen in the hepatic vein after oral administration. the details of the model and the physiological parameters used have been described elsewhere (abraham et al., arch. toxicology 79: 63-73, 2004) . we modelled the concentration time profile exploring the dose which would lead to a concentration at 1 hour and at 24 hour as similar as possible to the concentration measured in the supernatant of human freshly prepared cell cultures after dosing the culture with ibuprofen. we parametrized the pbhkm with the parameters which have been estimated from the in vitro kinetic studies (clearance between 3 and 15 µm 3 /sec (truisi et al., 2015) and an absorption of 100% and an absorption rate of 1/h (cristofoletti and dressman, j pharm sci 103: 3263-75, 2014). results: the data of the in vitro study with 100 µm ibuprofen could well be modelled. when assuming a clearance of 15 µm 3 /sec the dose of 1480 mg resulted in an 1 hour concentration of 66.5 µm in the hepatic vein of pbhkm equal to 133.0 nmol/well (volume of the well = 2 ml) in the in vitro study in which the measured concentration was 138.75 nmol/well. the concentration at 24 hours of 8.7 µm (equal to 17.4 nmol/well) corresponded with the in vitro concentration (16.5 nmol/well). the modelling approach was less successful with in vitro dosing of 1000 µm. the 10 fold higher dose of 14800 mg lead to nearly double the concentration at 1 hour than measured in vitro. with a dose of 8500 mg/kg an approximation was feasible resulting in 396.7 µm in the hepatic vein at 1 hour which is equal to 793.4 nmol/well whereas the measured concentration in vitro was 782.85 nmol/well. even with a clearance value as low as the 2.5 percentile (3 µm 3 /sec), the concentration at 24 hours was modelled to be lower than the in vitro measured value (in vivo model: 98.7 µm which corresponds to 197.4 nmol/well; measured in vitro concentration: 979.2 nmol/well). discussion: this is the first attempt to use kinetic data obtained in vitro to feed in in a pbhkm for reverse dosimetry finding the dose which corresponds in vivo to the in vitro situation. in the case presented here, the in vitro dose assumed to be low in vitro (100 µm) corresponds to a dose of 1480 mg (note: the highest approved daily dose is 2,400 mg). for the high in vitro dose modelling was successful only for the concentration 1 hour after dosing and a dose of 8,500 mg. conclusions: in vitro kinetic parameters, such as clearance, can successfully be used for parametrizing a pbhkm. it is of utmost importance for the relevance of in vitro finding to assure that the concentrations used in vitro can be obtained with relevant in vivo doses. in this case, the in vitro concentrations were within (low dose) and 3.5 fold above (high dose) the in vivo relevant therapeutic concentration range. introduction: a variety of drug residues have been detected in sewage plant run-offs, rivers and lakes, but also in groundwater and tap water samples. studies have yet to identify a risk for human health from these contaminants, but adverse health effects have been reported for various species, including fish and birds. it has recently been suggested that for a comprehensive risk assessment toxicologists should also consider transformation products (tps) of such water contaminants that may arise from abiotic and biotic (metabolic) reactions. with aciclovir (acv), a well-known antiviral drug, as the parent drug we tried an in-silico approach to identify tps that might be of interest due to some mutagenic or carcinogenic toxicophores. methods: from a literature and database search we picked up 12 acv-tps. predicted acute toxicities and mutagenic / carcinogenic properties for these tps were derived from an expert system analysis using the lazar portal (http://lazar.in-silico.ch/) as front end. results: two of the identified acv-tps could not be handled by lazar because of insufficient training data in one out of eight queried categories. the highest score (4 positive out of 8 possible genotoxicity categories) was assigned to 9 of the 12 tps, including acv itself. this is a rather low score when compared to other water-borne drug residues, e.g. carbamazepine. cofa, an imidazole derivative of acv seen in advanced oxidation processes, had shown antiproliferative effects in several ecotoxicologic screening assays, e.g. [1], but was unremarkable in our tests. additionally, a computer-based simulation of the respective tps interacting with human cyp isozymes did not support concerns that these tps may pose a risk for human health. conclusions: our in-silico analyses of 12 acv-tps did not provide evidence for any adverse health effects in the micromolar concentration range. further studies are needed to clarify if the biological activity of some acv-tps in ecotoxicological assays may eventually affect yet unidentified biological targets in the human body. sulfur mustard (sm) is a chemical warfare agent which was first used in world war i, but has found use in several conflicts afterwards. although sm is prohibited by geneva protocol, terroristic attacks cannot be ruled out. latest news give rise to concern that is may be in the possession of sm and is willing to deploy it. even 200 years after the initial synthesis of sm its mode of action is not fully unraveled. thus, no antidote does exist. however, chemosensing ion channels have been shown to be activated by highly toxic chemicals and might represent a specific therapeutic target. previous studies have shown that the sm-surrogate cees (mono-functional alkylating agent) is able to activate transient receptor potential ankyrin 1 (trpa1) channels that are known to affect mapk cell signaling. mapk-pathways, especially perk1/2, are known to increase protein biosynthesis through activation of transcription factors binding to the serum response element (sre). it is unknown whether alkylating agents have also impact on mapk signaling mediated through trpa1 activation. our results demonstrate that aitc resulted in phosphorylation of the mapk perk1/2 and increased protein biosynthesis of sre-regulated genes in hek293 cells overexpressing htrpa1. cees increased perk1/2 levels already after 2.5 min which could be prevented by the trpa1 blocker ap18. activation of target genes through perk1/2 signaling was also evident, but less pronounced compared to aitc. our results demonstrate that alkylating agents have impact on cell signaling through trpa1 channel activation. thus, trpa1 might represent a promising target for counteracting sm toxicity. sulfur mustard (sm) is a chemical warfare agent that provokes severe inflammation and blistering upon exposure to the skin accompanied by disturbed wound healing. the potential use of sm in terroristic assaults amplified the interest in understanding the underlying cellular and molecular pathomechanisms in order to improve therapeutical intervention. autophagy is a highly conserved catabolic pathway in eukaryotes that ensures the degradation and recycling of cellular components through the lysosomal machinery. autophagy is important for cell survival in physiological and pathological stress situations. emerging knowledge indicates that imbalanced regulation of autophagy disturbs basal cell functions including proliferation, differentiation and migration, thus contributing to the pathophysiology of various diseases. after penetration into skin cells, sm alkylates and thereby modifies nucleic acids and proteins thus forming aggregates of dysfunctional proteins destined for autophagic disposal. in our studies, we analyzed the influence of sm on protein expression (western blotting) of autophagy-related (atg) genes as well as proliferation (wst-1) of primary normal human keratinocytes (nhek) and primary normal human dermal fibroblasts (nhdf). preliminary results demonstrate that sm strongly dysregulates the biosynthesis of atg proteins that may contribute to the diminished cell migration and proliferation under these conditions. our findings suggest that sm affects autophagy in correlation with an impairment of physiological functions in keratinocytes and fibroblasts that are essentially required for normal tissue regeneration. thus, application of pharmacological modulators of autophagy might be useful in the treatment of the delayed wound healing in skin upon exposure to sm. exposure of the respiratory tract to airborne particles is a major risk to human health. due to the ubiquitous application of these particles in the field of pharmacy, industry and in daily life, there is a strong necessity to investigate the toxic properties and the underlying pathomechanisms of these inhalable substances. in addition, the eu chemicals regulation requires not only that all substances placed on the market have to undergo a toxicological characterization, including the identification of potential toxic inhalation hazards (reach), but also that animal testing shall be undertaken only as a last resort ("3rs" principle) and the promotion of the development of alternative methods. thus, the development, establishment and validation of alternative in vitrobased test systems for the assessment of pulmonary toxicity are in the focus of current research. until now, most of the available in vitro cell culture models are limited to some extent as in those studies the exposure is either done under submerged conditions, not resembling the exposure conditions in vivo, or a homogeneous particle distribution is not guaranteed. the cultex ® radial flow system (rfs) is a specially designed in vitro modular exposure system that overcomes these limitations. it enables the homogenous exposure of human lung epithelial cells at the air-liquid interface (ali), thereby mimicking the physiological conditions of the alveolae. however, further optimizations are needed for the enhancement of the cultex® methodology. aim of this study was first the optimization of the test methodology in general (i.e. focus on clean air controls of the human lung epithelial cell line a549), and second the improvement of cultivation conditions. parameters such as handling of the cultex® device (proper closing and opening operation of the cultex® rfs module; improved washing conditions and media supply), treatment of the incubator controls, adjustment of clean air pressure and flow rates, and integration of two additional filters were sequentially adjusted in order to enhance the methodical setup. our results show that the test parameters for clean air exposure of the a549 cells were successfully optimized resulting in more accurate and robust data. cultivation conditions were improved by changing from closed-wall cell culture inserts to open-wall cell culture inserts. the openwall inserts turned out to be more suitable for exposure experiments as they provided a better medium supply and preserved humidity. deductive, the change of the cell culture inserts was identified as the deciding factor for the improvement of cell morphology. hence, we have successfully optimized the cultex ® rfs methodology for clean air exposure of a549 cells. human primary hepatocytes represent the gold standard in in vitro liver research. due to their low availability and high costs, alternative liver cell models with comparable morphological and biochemical characteristics have come into focus. the human hepatocarcinoma cell line hepg2 is often used as a model for liver toxicity studies. however, under two-dimensional (2d) cultivation conditions the expression of xenobiotic-metabolizing enzymes and typical liver markers is very low. cultivation for 21 days in a three-dimensional (3d) matrigel culture system has been reported to strongly increase the metabolic competency of hepg2 cells. in our present study we extended previous studies and compared hepg2 cell cultivation in three different 3d culture systems: collagen, matrigel and alvetex culture system. cell morphology, albumin secretion, cytochrome p450 monooxygenase (cyp) enzyme activities, as well as expression of xenobiotic-metabolizing and liver-specific enzymes were analyzed after 3, 7, 14, and 21 days of cultivation. our results show that the previously reported increase of metabolic competency of hepg2 cells is not primarily the result of 3d culture but a consequence of the duration of cultivation. hepg2 cells grown for 21 days in 2d monolayer exhibit comparable biochemical characteristics, cyp activities and gene expression patterns as all 3d culture systems used in our study. however, cyp activities did not reach the level of heparg cells. in conclusion, the increase of metabolic competence of the hepatocarcinoma cell line hepg2 is not due to 3d cultivation but rather a result of prolonged cultivation time. in vitro assessment of the neurotoxic potential of arsenolipids arsenolipids are organic, lipid-soluble arsenic compounds, which occur mainly in marine organisms. major human exposure routes are fatty fish including herring or fish oil-based food supplements. about 55 different arsenolipids have been identified so far. thereby, arsenic-containing hydrocarbons (ashc) and arsenic-containing fatty acids (asfa) represent two subgroups of the arsenolipids [1]. our in vitro studies have demonstrated high cellular bioavailability and a high cytotoxic potential of ashcs in human liver and bladder cells [2] , whereas asfas were less toxic [3] . a substantial transfer across an intestinal barrier model (caco-2) indicated that ashcs are highly intestinal available. in comparison, asfas showed lower intestinal bioavailability and underwent a presystemic metabolism [4] . moreover, in drosophila melanogaster ashcs exerted late developmental toxicity and accumulated in the fruit fly's brain. these results suggest that ashcs might pass the blood-brain-barrier due to their amphiphilic structure [5] . in order to assess the neurotoxic potential we currently investigate the toxicity of several arsenolipids in differentiated, human neurons (luhmes). after 48 h incubation with ashcs or asfas, cell number (hoechst) as well as cellular dehydrogenase activity (resazurin) were measured, with the latter endpoint turning out to be more sensitive. ashcs showed substantial cytotoxic effects (ic 50 ~ 7-12.5 µm) in a concentration range comparable to that of arsenite (ic 50 ~ 7.5 µm), whereas asfas were less cytotoxic (ic 50 > 100 µm). after incubation with ashcs the cellular arsenic concentrations increased 10-20fold as compared to incubation with arsenite. further studies indicated that one possible toxic mode of action of arsenolipids could be a disruption of the cellular energy level. therefore, the mitochondrial membrane potential was investigated after incubation with the arsenic compounds in differentiated neurons. whereas arsenite did not exert an impact, ashcs reduced the mitochondrial membrane potential significantly. this might be due to interactions of the amphiphilic ashcs with mitochondrial membranes. currently we investigate the impact of the arsenolipids on neurite outgrowth as a developmental toxicity endpoint. standard treatment of poisoning by organophosphorus compounds (op; e.g. nerve agents and pesticides) consists of co-administration of atropine and an oxime-based reactivator of inhibited cholinesterases. due to lack of efficacy of clinically used oximes against various op-inhibited human acetylcholinesterase (ache) (e.g. soman) research started focusing on new therapeutic approaches. several research groups conducted in silico screenings [1, 2] in order to identify new non-oxime reactivators, presenting amodiaquine as a promising candidate for paraoxon-inhibited hache. for decades, antimalarial drugs like amodiaquine and chloroquine have been closely investigated regarding their side effects, thereby discovering interaction with cholinesterases, which could pose a new potential therapeutic benefit for inhibited cholinesterases. therefore, in this study interactions between antimalarial agents in presence or absence of ops were examined spectrophotometrically by a modified ellman assay. reversible inhibition of cholinesterases was observed with both antimalarial agents. amodiaquine had higher inhibitory potency for hache than human butyrylcholinesterase (bche), being confirmed by ic 50 values of 0.67 ± 0.02 µm for hache and 81.28 ± 0.04 µm for hbche. ic 50 values with chloroquine were 28.37 ± 0.02 µm for hache and 55.62 ± 0.02 µm for hbche, thus representing a weaker inhibition of hache than amodiaquine. furthermore, reactivation of paraoxon-(pxe), sarin-(gb), cyclosarin-(gf), and vxinhibited hache and hbche by amodiaquine and chloroquine was determined. after 60 minutes, only paraoxon-inhibited hache (50%) and cyclosarin-inhibited hbche (10%) were reactivated by 500 µm chloroquine. on the contrary, 10 µm amodiaquine reactivated all tested ops after 60 minutes in the following order: pxe > vx > gf > gb. in contrast, with hbche the highest reactivation was generated with 100 µm amodiaquine in the following order: vx > gb > gf > pxe. due to the high reversible inhibitory potency of amodiaquine, an increased concentration does not result in a higher reactivation of op-inhibited hache. in summary, our results show that amodiaquine is a reactivator of op-inhibited cholinesterases. in the future, non-oxime reactivators that are structurally-related to amodiaquine should be further investigated. [1] bhattacharjee, a.k., marek, e., le, h.t., gordon, r.k., eur. j. med. chem., 2012, 49, 229-238. [2] katz, f.s., pecic, s., tran, t.h., trakht, i., schneider, l., zhu, z., ton-that, l., luzac, m., zlatanic, v., damera, s., macdonald, j., landry, d.w., tong, l., stojanovic, m.n., chembiochem., 2015 , 16, 2205 -2215 bundesinstitut für risikobewertung, lebensmittelsicherheit, berlin, germany development of mammary gland tumors is connected to a deregulation of breast epithelial cell differentiation, a complex process which cannot be reproduced in vitro under standard cell culture conditions. however, cultivation of cells in a tissue-like environment in an in vitro three dimensional (3d) model can mimic general architecture, function and differentiation of mammary bulks. in this project, a 3d model was used consisting of the permanent breast epithelial cell lines mcf10a (er -, estrogen receptor negative) and mcf12a (er + , estrogen receptor negative) grown in matrigel tm , which mimics the complex extracellular matrix in vivo. the 3d culture of mcf10a and mcf12a cells in matrigel tm results in the formation of growth-arrested, polarized spheroids with a lumen (acini-like organoids). in order to perform a semi-quantitative estimation on the influence of substances on the differentiation of the breast cells for the identification of non-genotoxic carcinogens a scoring method was developed. this scoring method provides information about substance-induced morphological changes of the spheroids during differentiation based on the following parameters: size of the spheroids, the formation of the lumen, and the degree of polarization. furthermore, the model allows distinguishing between erdependent (mcf12a) and er-independent (mcf10a and mcf12a) effects. the 3d in vitro model is a useful tool for toxicologists to study substance effects on differentiation processes. the system will be used to examine the potential of e.g. food contaminants such as phthalates or perfluorinated substances (pfas) to disrupt the differentiation process of breast epithelial cells and will therefore serve as a valuable in vitro tool to assess their carcinogenic potential. inflammatory episodes occur erratically throughout life and are likely to play a critical role in the alteration of the individual susceptibility of a person to idiosyncratic druginduced liver injury (idili), a particular severe form of drug-induced liver injury (dili). in concordance with the inflammatory stress hypothesis, modest inflammatory stress can lower the threshold for hepatotoxicity and make an individual susceptible to develop liver injury during exposure to therapeutic doses of a drug. in order to evaluate the role of immune cells and its secreted factors during drug therapy, we established an in vitro test battery consisting of two cell culture systems in presence or absence of proinflammatory factors (lps, tnfα): (a) the monoculture of human hepatoma (hepg2) cells and (b) co-culture systems of human monocytic or macrophage-like (thp-1) and hepg2 cells. with these different test settings we aimed to identify whether the introduction of inflammatory immune cells and/or pro-inflammatory factors could increase the sensitivity of liver cells towards idili compounds. three reference substance pairs were tested, namely troglitazone -rosiglitazone, trovafloxacin -levofloxacin, and diclofenac -acetylsalicylic acid, each of them being composed of a compound that is known to induce idili and a partner compound of the same substance class that does not induce idili. first, all compounds were tested for cytotoxicity towards the single cell systems using the wst-assay. co-culture experiments with hepg2 and thp-1 monocytes or macrophages as well as co-exposure experiments with lps or tnfα were then done at about 20% cytotoxicity of the respective substance in the most sensitive cell type. subsequently the results were compared to the experiments in the monoculture of hepg2. we observed that every idili compound showed a significant increase in cytotoxicity in a minimum of one exposure combination while this effect was not observed with the corresponding non-dili partner compound. in conclusion, a combination of different culture systems and co-exposures with proinflammatory factors is needed for a valid differentiation between non-dili and idili compounds. this test battery could provide a useful tool for the prediction of inflammation-associated idiosyncratic drug-induced hepatotoxicity. furthermore, our results support the inflammatory stress hypothesis and points to an involvement of proinflammatory factors in the development of idili. extensive animal models of carcinogenicity ensure a safe usage of chemicals. to elucidate fundamental molecular mechanisms of carcinogenicity these methods are expensive, time consuming and above all too complex. in contrast, most in vitro methods are rather simple and detect only selected endpoints, like dna damage, mutations or changes in proliferation. the balb/c cell transformation assay is a validated toxicological method to identify potential tumour initiators and promotors. first, balb/c mouse fibroblasts form a monolayer culture and get contact-inhibited after reaching confluence. upon treatment with a tumour initiator (3-methylcholanthrene) and promotor (12-o-tetradecanoylphorbol-13-acetate) transformed cells do not stop proliferation and grow as morphologically aberrant foci over the monolayer of normal cells. after fixation with methanol at day 42, morphological aberrant foci can be visualized with giemsa staining. because the balb/c assay mimics different stages of the malignant cell transformation process (initiation, promotion and post-promotion phase) and detects with the colony formation a late endpoint of carcinogenicity we improved this method for mechanistic cancer research. using the example of insulin-signalling pathway we can show that (1) several substances have a different impact on the transformation process, (2) it is possible to identify for each substance the phase with the greatest effectiveness and (3) we can detect additional endpoints to elucidate the mechanistic mode of action. therefore we used several compounds (linsitinib, metformin, rapamycin, …) to manipulate the insulin-signalling pathway on different levels (insr, ampk, mtor, …) and analysed a number of characteristic endpoints of carcinogenesis. changes on protein level and signalling (westernblot, immunofluorescence, flow cytometry) or parameters of energy metabolism (oxygen consumption, glucose or atp measurement) are measurable and enable new insights into the process of cancer origin. summing up, the balb/c 3t3 assay proves to be a cheap and short-time alternative to rodent bioassays. although this method does not mimic the whole in vivo neoplastic process, it can be used to provide essential information regarding key proteins and their signalling, during the different stages of transformation. is there hope to correctly classify severe ocular irritant agrochemical formulations using in vitro methods: a proof of concept using the isolated chicken eye test, two modified bcop protocols and an epiocular™ et50 protocol while some in vitro methods addressing ocular irritancy have gained regulatory acceptance, to date the draize rabbit eye test (oecd tg 405) is the only world-wide regulatory accepted test for the determination of the full range of eye irritation potential. further although several in vitro methods for the severe eye irritation have gained regulatory acceptance, agrochemical formulations are nor explicitly included nor excluded from the applicability domain to predict severe ocular irritant formulations. systematic analyses are only available for e.g. the hen's egg test-chorioallantoic membrane (het-cam), and bovine corneal opacity and permeability (bcop, oecd tg 437) assays both showing that the used protocols do not provide sufficient sensitivity to reliably predict severe ocular irritating formulations. the purpose of this study was to evaluate whether the regulatory accepted isolated chicken eye (ice, oecd tg 438) test including corneal histopathology (as suggested for evaluation of the depth of injury), as well two modified protocols of the bcop and/or an et50 (exposure time reducing viability of treated tissue to 50%) protocol using the reconstructed cornea model epiocular™ are useful to predict severe ocular irritant agrochemical formulations. a proof of concept comprising the testing of ten to twelve agrochemical formulations with available in vivo data in each assay was conducted. in summary, based on the ice evaluation described in oecd tg 438, one of the five severe ocular irritant formulations (un ghs cat 1) was predicted correctly. using both modified protocol versions of the bcop the result for one of the four tested un ghs cat 1 formulations was just above the un ghs cat 1 classification border for using one of the modified protocols. lastly and most promising, the epiocular™ et50 predicted four of five tested un ghs formulations correctly with the fifths being close to the classification border. additional agrochemical formulations will be tested to further evaluated the epiocular™ et50 protocol to identify severe ocular irritant agrochemical formulations. drug-induced pancreatic toxicity comprises effects on the exocrine and/or the endocrine pancreas, which both can have serious clinical implications, e.g. acute pancreatitis or diabetes mellitus. adverse effects on the pancreas are occasionally observed during drug discovery and development and often prohibit further development. hence, there is a need for reliable in vitro models to early on identify the pancreas-toxic potential of drug candidates. permanent cell lines and primary cells have many shortcomings, e.g. loss of cell-to-cell and cell-to-matrix relationships or changes in cell physiology due to the isolation procedure. pancreas tissue slices are a potential alternative, circumventing most of these limitations. their preparation is rather elaborate which may explain its rare use. so far, pancreas tissue slices have predominantly been used to address physiological or pharmacological questions, although they might also serve as valuable in vitro model for toxicological applications. therefore, this work aimed to establish and characterize rat pancreas tissue slices as in vitro model for studying drug-induced pancreatic toxicity. results will be compared to the responses of the permanent endocrine (ins-1e) and exocrine (ar42j) pancreatic cell lines to evaluate a potential added value. rat pancreas tissue slices were prepared by a protocol adapted from marciniak et al. (nat protoc, 2014 . 9(12): p. 2809 . briefly, pancreas was infused and embedded with agarose. tissue sections of app. 200 µm were prepared using a vibratome and maintained in cell culture medium for up to 6 days. cell viability was determined by daily measurement of lactate dehydrogenase (ldh) in medium supernatants and by microscopic evaluation following fixation in 10 % formalin and h&e staining. functional integrity of acinar and beta cells were assessed by cell-type specific secretory responses (i.e. insulin, amylase, lipase) to physiological stimuli. moreover, the effects of the pancreas toxins streptozotocin (stz), alloxan (all), and the cholecystokinin (cck) analogue cerulein on the viability and functional integrity of tissue slices were compared to the respective responses of the cell lines. we were able to establish an optimized isolation and cultivation procedure for rat pancreas tissue slices applying minor modifications to the original protocol. cell viability declined over the cultivation period. stimulation of the cell lines with glucose or cerulein increased secretion of insulin (ins-1e cells) or amylase/lipase (ar42j cells), respectively. the pancreas slices responded to both stimuli, demonstrating functional integrity of endocrine and exocrine cells. treatment of ins-1e islet cells with the betacell toxicants all or stz only slightly affected islet cell viability, whereas treatment of ar42j acinar cells with cerulein at supraphysiological concentrations had no effect. this set of experiments is currently completed by investigating the effects of all, stz and cerulein on the viability of acinar and islet cells in pancreas slices. our preliminary data demonstrate feasibility to prepare and cultivate rat pancreas tissue slices over a period of 6 days thereby maintaining functional integrity to some extent. coculture of human monocytes with the keratinocyte cell line hacat in serumcontaining medium leads to higher sensitivity to weak contact allergens: an improvement for the loose-fit coculture-based sensitization assay (lcsa) a. sonnenburg 1 , j. the loose-fit coculture-based sensitization assay (lcsa) has proved reliable for the in vitro detection of contact sensitizers in the past. however, the use of primary human keratinocytes has some disadvantages. to facilitate high throughput screening of chemicals, we replaced primary keratinocytes from the original assay setup (setup a) by the human keratinocyte cell line hacat. these cells were cocultured with monocytederived dendritic cells in serum-free medium (setup b) or fetal calf serum (fcs)containing medium (setup c). upregulation of the dendritic cell maturation marker cd86 assessed by flow cytometry served as endpoint. we have tested four substances known as sensitizers and four non-sensitizers in both new setups as well as in the original setup with primary cells. three out of four sensitizers (2,4-dinitrochlorobenzene, 2-mercaptobenzothiazole, and coumarin) , and three out of four non-sensitizers (glycerol, monochlorobenzene, and salicylic acid) were correctly assessed under all culture conditions. the weak sensitizing potency of resorcinol was only detected by setup b with fcs supplemented medium. a false positive reaction to caprylic (octanoic) acid in all three setups confirms earlier results from our laboratory that some fatty acids are able to induce cd86 on dendritic cells in vitro. culture in fcs supplemented medium led to generation of dendritic cells showing a more pronounced upregulation of cd86 after application of substances with rather high sensitization potency compared to dendritic cells which are formed under serum-free conditions. therefore, we characterized dendritic cells from setups b and c by flow cytometric measurement of additional dendritic cell surface markers. dendritic cells from the original setup a had been characterized extensively before (schreiner et al., toxicology 2008; 249:146-1529) . dendritic cells generated in fcs supplemented medium were cd1a+/cd1c+, whereas dendritic cells from serum free culture conditions were cd1a−/cd1c− regardless whether cocultured with primary human keratinocytes or hacat. populations with cd1a+/cd1c+ dendritic cells in coculture seem to show a higher sensitivity to weak sensitizers, which proved beneficial for the identification of resorcinol. in conclusion, modification of the lcsa protocol led to an increased sensitivity of the assay. due to ethical and social reasons, in vitro assays are being developed to replace animal tests for addressing e.g. toxicological questions. for the induction of skin sensitization by chemicals, resulting in tolerance or allergic contact dermatitis after repeated exposure, prerequisites are the induction of inflammatory responses in keratinocytes supporting maturation of dendritic cells (dc), which is needed for the t cell response. although related in vitro assays consisting of one single cell type have good hazard prediction capacities, they have limitations in predicting sensitization potency. one drawback could be the lack of communication between keratinocytes and dc. with respect to the activation of keratinocytes and maturation of dc, intercellular communication between these two cells may include the release of danger molecules such as cytokines, damage-associated molecules such as atp, and metabolized chemicals. beside this, microrna (mirna), among them those that can regulate dc activation or maturation, can be differentially expressed upon stimulation but can also be transferred between cells. for skin sensitizers, we reported already that cross talk between hacat keratinocytes and thp-1 cells, as model for dc, enhanced cyp1 enzyme activity in hacat cells exposed to benzo[a]pyrene (b[a]p) and eugenol, belonging to a subgroup of chemicals (prohaptens) whose sensitizing potential depend on prior metabolic activation e.g. via cytochrome p450 (cyp) enzymes. furthermore, coculture clearly increased the upregulation of the cell surface molecule cd86 on thp-1 cells after incubation with these prohaptens and also several other skin sensitizers. in this study we further elucidate the cross talk between thp-1 cells and hacat cells by analyzing the impact of hacat cells on the expression of mirnas in thp-1 cells by microarray technology. we identified 6 differentially expressed mirnas in cocultured thp-1 cells compared to monocultured thp-1 cells irrespective of the treatment (medium, 0.2% dmso as solvent control, b[a]p). in the presence of dmso and b[a]p (after 48h) 8 additional mirnas are differentially expressed. up to now it is not clear whether the cross talk between hacat and thp-1 cells comprises the exchange of mirna between the cocultured cells or whether it influences the expression of these mirna in thp-1 cells, or both. given that one mirna has several gene targets these results illustrate that the cross talk between thp-1 and hacat cells also impacts on the mirnome. walther-straub-institut der lmu-münchen, münchen, germany transient receptor potential (trp) proteins represent a large superfamily of nonselective cation channels sensing toxic stimuli in the human body. trpa1 expresses a high number of aminoterminal ankyrin repeats and is the only member of the trpa family. channel monomers form homotetramers in the plasma membrane with six transmembrane segments (tm) and a pore forming loop between tm5 and 6. trpa1 has been extensively described in sensory nerve endings as an important cellular detector for toxic stimuli and as an oxygen sensor (reviewed in 1). although recently two reports identified trpa1 in pulmonary epithelial and endothelial cells (2, 3) , its expression in non-neuronal tissues is still a matter of debate. after isolation and identification of different murine lung cells we were able to identify murine trpa1 protein in primary endothelial cells, pneumocytes type ii (atii) and fibroblasts by using specific antibodies in a western blot analysis, but not in cells from trpa1-deficient mice. atii cells were identified by specific cell markers such as surfactant protein c and were further differentiated to ati cells characterized by their specific expression of podoplanin. quantitative trp expression patterns will now be evaluated by quantitative reverse transcription (rt)-pcr as well as utilizing nanostring ® technology in different lung cells. to characterize trpa1 on a cellular level we cultured a hek293 cell line stably expressing trpa1 (4) . allylisothiocyanate (aitc) a specific activator as well as hypoxia and hyperoxia was able to induce ca 2+ -influx in this cell line, which was blocked by the specific inhibitor a96079. in the future, we will utilize the isolated perfused lung model (5) to quantify toxin-induced edema formation in ex vivo lungs from wt and trpa1-deficient mice after exposure to potential toxic inhalation hazards (tih see 6) to challenge the hypothesis of trpa1 as an important toxin sensor in the lung. by this strategy we hope to understand trpa1 function in lung cells and to evaluate trpa1 proteins as potential pharmacological targets for a specific therapeutic intervention during toxin-induced edema formation. metabolism by the intestinal microbiota is likely to contribute essentially to the plasma metabolite profile of the mammalian host organism and it requires adequate identification of effects of the microbiome on the endogenous plasma metabolite patterns. the current investigations present insights in the mammalian-microbiome cometabolism of endogenous metabolites. antibiotics have a profound effect on the micro-organism composition of the microbiome and hence on the mammalian-microbiome co-metabolism. the consequences, however, on the functionality of the microbiome (defined as the production of metabolites absorbed by the host) and which of these changes are related to the microbiome are not well understood. to identify plasma metabolites related to microbiome changes due to antibiotic treatment, we have employed a metabolomics approach. to this purpose broadspectrum antibiotics belonging to the class of aminoglycosides (streptomycin, neomycin, gentamicin), fluoroquinolones (moxifloxacin, levofloxacin) and tetracyclines (doxycycline, tetracycline) were administered orally for 28 days to male rats including blood sampling for metabolic profiling after 7, 14 and 28 days. fluoroquinolones and tetracyclines can be absorbed from the gut whereas aminoglycosides cannot. to distinguish between metabolite changes caused by systemic toxicity of the antibiotics and microbiome related changes, the metabolites identified in the metabolome pattern were compared to a list of metabolites known to be produced by the gastro-intestinal micro-organisms. beside changes mainly concerning amino acids and carbohydrates, hippuric acid and indole-3-acetic acid were identified as key metabolites being affected by antibiotic treatment. for each class the following gut metabolites were found to be unique: indole-3-propionic acid for aminoglycosides, taurine for fluoroquinolones, 3indoxylsulfate, uracil and allantoin for tetracyclines. for each class of antibiotics specific and selective metabolome patterns could be established. the results suggest that plasma based metabolic profiling (metabolomics) could be a suitable tool to investigate the effect of antibiotics on the functionality of the microbiome and to obtain insight in the mammalian-microbiome co-metabolism of endogenous metabolites. drug-induced liver injury (dili) is still a major reason for termination of clinical trials and thus is an important concern in drug development. identification and prediction of dili in the clinic and in preclinical safety testing still relies on the classical clinical chemistry panel and histopathology with known limitations in sensitivity and specificity. in the last years bile acids (bas) have been studied as potential biomarkers to better characterize drug-induced liver injury with promising results (ellinger-ziegelbauer et al., 2011; luo, schomaker, houle, aubrecht, & colangelo, 2014; yamazaki et al., 2013) . to evaluate whether a targeted bile acid profiling via lc-ms/ms in plasma and liver tissue can improve assessment of liver injury, methapyrilene (mpy) a known hepatotoxin, or corresponding vehicle, was administered daily to male wistar rats at a low (30 mg/kg) and a high (80 mg/kg) dose. rats were sacrificed following 3, 7, or 14 consecutive daily doses, or after recovery 10 days following 14 consecutive administrations of mpy or vehicle. in addition to bile acids which were determined both in plasma and tissue, conventional preclinical safety endpoints (histopathology and clinical chemistry) assessment and gene expression profiling was performed in liver to obtain mechanistic information about potential changes in regulation of bile acid levels. conventional findings included periportal necrosis, inflammation and biliary hyperplasia, and increased liver enzyme activity and bilirubin levels during the treatment phase. the bile acid pattern showed increased levels of conjugated and unconjugated bile acids in low dose and high dose groups compared to the controls after administration of methapyrilene. furthermore, although liver enzyme activity and bilirubin levels in serum were decreased again in the recovery groups, suggesting recovering liver injury, bile acid concentrations remained elevated with no signs of recovery. analysis of transcriptomics data revealed decreased levels of mrna encoding α-methylacyl-coa racemase (amacr) 4 and 15 days after dosing, a gene responsible for bile acid synthesis. membrane transport systems for bile acids like sodium/taurocholate co-transporting polypeptide (ntcp) and organic anion transporting polypeptide 1 (oatp1) expression were down regulated as well, indicating that the increased bile acid concentrations in plasma and tissue could be attributable to reduced uptake by the hepatocyte. in summary the data suggest that targeted bile acid profiling could be used as potential biomarkers to enhance assessment of drug-induced liver injury. photorhabdus asymbiotica is an entomopathogen and emerging human pathogen causing soft tissue infections in humans. photorhabdus asymbiotica produces the bacterial protein toxin patox, which is cytotoxic for various cell lines and kills insect larvae. previous studies have established that patox harbors two enzymatic active domains, a glycosyltransferase and a deamidase domain. the glycosyltransferase domain inactivates host gtpases of the rho family by glcnacylation of a tyrosine residue in the effector binding loop, which results in the disassembly of the actin cytoskeleton. the deamidase domain deamidates a crucial glutamine residue in heterotrimeric gα i and gα q/11 proteins, which renders the g proteins constitutive active. sequence and structural homology analyses of patox revealed a third domain (patox p ) resembling peptidases of the c58 protease family. patox p contains the conserved catalytic triade (c/h/d) of papain-like cysteine proteases and shares sequence similarity with effectors from yersinia pestis (yersinia outer protein yopt) and pseudomonas syringae (avirulence protein avrpphb). transient expression of patox p in hela cells induces cell rounding and indicates a cytotoxic potential of patox p . incubation of patox p with linearized bovine serum albumin (bsa) results in cleavage products of bsa assuming proteolytic activity of patox p . mutation of the catalytic cysteine in patox p prevents cleavage of bsa and blocks cytotoxicity. we were not able to observe autocatalytic cleavage of patox constructs under various conditions. the intracellular activity of the protease domain is most likely involved in the pathogenicity of patox. vitamin d metabolism -involved in triazole fungicide toxicity? a. lehmann 1 1 background: in a 28-day rat feeding study with the azole fungicides cyproconazole (c), epoxiconazole (e), propiconazole (p), tebuconazole (t), prochloraz (pz) as well as combinations c+e and c+e+pz, a reduction of vitamin d (vitd) receptor mrna levels was reproducibly observed in adrenals for c, e and p. transcription of various enzymes related to vitd homeostasis (including cyp2r1, gc, cyp3a, ugt1a) in liver was also affected, while initial indications for modulation of renal cyp24a1 and renal and hepatic cyp27b1 could not be confirmed. a possible induction of parathormone (pth) was noted for the high dose of c, but statistical significance could not be shown. we have now performed supporting analyses for serum vitd levels, measured additional transcript levels and will provide a framework for the interpretation of the findings. methods: male wistar rats (n=5 for single substances, n=10 for combinations) were treated for 28 days at dose levels tested based on noaels from 90-day subchronic feeding studies and ranged from noael/100 to noaelx10. quantitative rt-pcr analyses were performed on organ samples obtained at sacrifice. serum vitd levels were determined using the total (25-oh) vitamin d elisa (drg instruments gmbh, marburg, germany). results: the elisa established for diagnostic analysis of human serum and plasma samples could be applied to rat serum. vitd levels in control animals (n=30) were 64.7±8.3 ng/ml (min/max: 48/78 ng/ml), i.e. in the range of values reported previously for rats. for the high dose of c (1000 ppm in food, n=5), there was a statistically nonsignificant reduction of vitd levels to 71.3±17.6% of the concurrent control (n=5). however, for 4 of 5 animals of this group, measured vitd level were below the range observed in pooled controls (n=30). an according follow-up is ongoing. qrt-pcr analysis of adrenal tissue showed deregulation of apoptosis related genes (p21 for c, e and pz; cdk1 and gadd45a for e; cdkn1c for c), which is in agreement with an involvement of vitd in the autocrine/paracrine regulation of cell proliferation. conclusion: reduction of circulating vitd levels would be plausible as a result of induction of hepatic cyp3a1/2 and ugt1a. however, this could not be confirmed by elisa as a general mechanism for all azole fungicides under investigation. only for rats fed with 1000ppm cyproconazole, there were indications for a moderate reduction of 25-oh vitamin d, which would correlate with the previously reported moderate increase in serum pth for this group. hansen's disease during pregnancy and lactation: two babies born to a mother using antileprosy drugs z. ozturk hansen's disease, also known as leprosy, during pregnancy has been rarely reported in europe and united states. early diagnosis is important, and medication can decrease the risk of those living with leprosy patients from acquiring the disease. this report presents a case of multidrug antileprosy therapy during pregnancy and lactation. a 26-year-old multiparous woman with a known case of multibacillary leprosy presented with unplanned pregnancy. her pregnancy was discovered in the 9th week, and she has been taking a multidrug therapy (dapsone 100 mg/day, rifampicin 600 mg/month, clofazimine 50 mg /day and clofazimine 300 mg/month) for the past 8 months. diagnosis of leprosy was established in her previous pregnancy. the patient was informed about the risks of drugs used in pregnancy. the treatment was continued unchanged during pregnancy. a detailed fetal ultrasonography was offered to scan the development of the fetus at about 20 weeks. in the 8th, 22nd, 28th weeks of pregnancy, prenatal sonographic examinations revealed normal fetal growth and amniotic fluid volume. at 28 weeks pregnant, she was diagnosed with gestational diabetes. diabetes did not cause any symptoms during pregnancy, and it was controlled with a reduced-calorie diet in a week. the patient delivered a healthy baby girl by vaginal birth in the 39th week of gestation without perinatal complications. the baby was also healthy (apgar 8-9,3300 g,51 cm), and its growth and development were normal during a 6-month follow-up period. the patient decided to breastfeed while taking medication. she had a previous experience with use of anti-leprosy drugs while breastfeeding, her other child was 15 months old and healthy. as well as in the first child, skin discoloration was observed in newborn due to clofazimine during lactation. after 3 months, she stopped breastfeeding, and the infant's skin changes were reversed. for pregnant women and practitioners, treatment of leprosy in pregnancy can be complicated. physical and neurological damage may be irreversible even if cured. multidrug therapy consisting dapsone, rifampicin and clofazimine is highly effective for people with leprosy and considered safe, both for the mother and the child. antileprosy drugs are excreted into human milk but there is no report of adverse effects except for skin discolouration of the infant due to clofazimine. therefore, multidrug therapy for leprosy patients should be continued unchanged during pregnancy and lactation. methods: individuals included in the analysis were participants of the berlin initiative study (bis). bis is a population-based prospective cohort study initiated in 2009 in berlin, germany, to evaluate kidney function in people ≥ 70 years. medication was assessed through personal interviews and coded using the anatomical therapeutic chemical classification system. for estimation of glomerular filtration rate (egfr) we used the ckd-epicr equation. predictor analysis was conducted via logistic regression. results: figure 1 illustrates the percentage of drug use for the three noacs and phenprocoumon, the most common vitamin k antagonist in germany, over the course of four years. table 1 shows the characteristics of patients for each oral anticoagulant group during the four-year follow-up visit (from january 2014 until april 2015). the probability of dabigatran use rose with increasing age (+12%), and the probability of phenprocoumon use rose in case of egfr < 60 ml/min/1.73m 2 (+54%) or male sex (+82%). discussion: our data show that also in the elderly noac use increased over the past years. characteristics such as age, sex or kidney function had an impact on the choice of oral anticoagulation. objective: orthostatic hypotension (oh) is an important factor in determining cardiovascular mortality especially in older age. different factors were discussed to influence oh. arterial stiffness, medication and frailty were demonstrated as modifying factors of oh. the aim of this study was to assess prevalence of and influencing factors on oh in nursing home residents (nhr) in germany. methods: systolic (sbp) and diastolic (dbp) blood pressure as well as pulse pressure (pp) and pulse wave velocity (pwv) as markers of arterial stiffness were measured in nhr aged ≥ 65 years in 12 nursing homes in berlin, germany. measurements were first performed in the sitting position and then repeated after standing up. oh was defined as a sbp decrease of > 20 mmhg and/or dbp decrease of > 10 mmhg within 3 min after standing up. hypertension was defined as the presence of diagnosis arterial hypertension, the prescription of at least one antihypertensive drug, or mean sbp values > 139 mmhg and/or mean dbp >89 mmhg. information about antihypertensive medication was received from interviews and medical records. frailty was determined by geriatric assessments, e.g. "timed up and go test" (tug) or barthel scale. results: oh testing could be performed with 96 nhr (mean age = 84.5 ± 7.3 years). in total, 15 subjects (15.6%) had oh. the mean change in sbp from sitting to standing was 19.2 ± 15 mmhg (range +8.5 to -52.5 mmhg) in patients with oh and 1.5 ±10.9 mmhg (range +44.5 to -17 mmhg) in patients without oh. mean sbp was significantly higher (143.6 ± 17.1 mmhg) in people with oh than in those without (131.5 ± 20.1mmhg). all of the nhr with oh were hypertensive compared to 89% of the nhr without oh. sex, mean age, pwv and pp was not significantly different between individuals with or without oh (p>0.05). medication data was available for 89 patients. all individuals with oh and 60 nhr without oh (80%) had antihypertensive medication. more than 2 different antihypertensive drugs were present in 11 patients with oh (78.5%) and in 43 patients without oh (57.3%). the intake of beta-blockers had no impact on oh development. geriatric assessments did not differ significantly between the oh group and the non-oh group. more than 75% of patients in both groups reached 80 points as maximum in barthel scale defining a need for assistance and tug analyses demonstrated that around 50% of patients with oh as well as patients without oh needed more than 19 sec showing a motor slowing. conclusion: we found a relatively low prevalence of oh in our very old patient cohort and the overall bp control was good. similar to earlier publications mean sbp was significantly higher in nhr with oh. all of the other investigated factors were not associated with the occurrence of oh. the small cohort size might have limited the detection of cardiovascular, epidemiological or geriatric associations. in addition, important confounding factors such as the inability to stand of some nhr and the lack of standardized fraility assessments must be addressed. impact of reticulated platelets on the initial antiplatelet response to thienopyridine loading in patients undergoing elective coronary intervention c. stratz 1 , t. nuehrenberg are known to be involved in cell metabolism pathways and therefore ccrcc is supposed to be a metabolic disease. in order to facilitate a better understanding of cancer metabolism and to support tumor classification on the metabolite level we have developed a novel analytical approach for comprehensive metabolomic profiling of small molecules and lipids in kidney tissue. the method was established and validated based on porcine tissue and, as proof of concept, applied to a small cohort of human normal and ccrcc tissue samples for molecular tissue differentiation. methods: five fresh frozen ccrcc samples and corresponding normal tissue were used for cancer-specific metabolomic profiling and were derived from patients who underwent partial or radical nephrectomy. metabolites and lipids were recovered from tissue samples by a two-step extraction protocol. tissue homogenization and extraction of polar metabolites was performed in methanol/water (aqueous extract) by a beadbeating approach. lipids were recovered by consecutive extraction of the pellet with methanol/methyl tert-butyl ether (organic extract). metabolites in aqueous extracts were separated by hydrophilic liquid interaction chromatography whereas compounds in organic extracts were separated by reversed phase chromatography prior high resolution mass spectrometry. results: reproducibility of tissue extraction and metabolite analysis was assessed by the analysis of multiple individually prepared porcine kidney samples. more than 1000 metabolic features including amino acids, nucleotides, small organic acids, phospholipids, sphingolipids, glycerolipids and fatty acids could be reproducible (cv ≤ 30 %) analyzed with the novel non-targeted metabolomics approach. the validated protocol was applied for metabolomic profiling of kidney tissue derived from ccrcc patients. based on unsupervised multivariate statistics, a clear differentiation between cancerous and normal tissue for the small metabolites profile as well as for the lipid profile could be observed. a first subset of differentially regulated metabolites responsible for tissue differentiation could be tentatively identified. conclusion: metabolomic profiling of kidney tissue extracts enables differentiation between ccrcc and normal kidney tissue samples based on the lipid and small molecule metabolomic profiles. further studies on larger and independent sample groups are necessary to confirm and validate our preliminary findings. in summary, the presented approach provides a first basis for comprehensive metabolomics studies in human kidney tissue and thus offers great potential for the metabolic characterization of ccrcc with important prognostic and therapeutic implications in the future. introduction: clomiphene (clom) citrate as mixture of trans-and cis-isomer (60:40) is the first line therapy for the treatment of infertility caused by the polycystic ovary syndrome. treatment schedule includes dose escalation from 50 mg/d clom citrate to up to 150 mg/d in case of non-ovulation. however, therapy outcome is variable and approximately 10 -30% of patients do not benefit from clom treatment. the pro-drug clom is bioactivated via 4-hydroxylation of trans-clom by the highly polymorphic cytochrome p450 (cyp) 2d6 leading to the major active metabolite trans-4hydroxyclomiphene (trans-4-oh-clom) [1] . recently, we identified a less active trans-3-oh-clom which is also formed by cyp2d6. besides the formation of the active metabolites, their plasma concentrations are influenced by their clearance e.g. via glucuronidation and sulfation. here we investigated the glucuronidation and sulfation of both hydroxyl-metabolites. methods: isoforms of udp-glucuronosyl-transferase (ugt) and sulfotransferase (sult) responsible for conjugation of oh-clom were identified using commercially available supersomes. glucuronidation and sulfation kinetics were determined in pooled human liver microsomes. conjugated clom metabolites were quantified in plasma and urine samples obtained from healthy female volunteers who received a single dose of 100 mg clom citrate. results: incubations with human liver microsomes revealed an almost 60-fold higher glucuronidation rate for trans-3-oh-clom, which is exclusively catalyzed by ugt2b7, compared to the more potent trans-4-oh-clom. for the latter a pattern of multiple ugts was identified. in contrast, the intrinsic clearance of trans-4-oh-clom to its sulfate is 16-fold higher compared to 3-oh-clom. for both metabolites a participation of sult1a1 and sult1e1 was identified. these results were in line with previous studies, which identified the same sults [2] and ugts [3] responsible for the conjugation of the structurally related trans-4-hydroxytamoxifen. in addition, in vivo data from plasma and urine samples confirmed the reverse regioselective glucuronidation and sulfation of trans-3-oh-clom and trans-4-oh-clom. overall, concentrations of clomglucuronides were significantly higher than those of sulfates. highest concentrations in plasma and urine samples were measured for trans-clom-3-o-glucuronide. conclusion: our results suggest a new metabolic route via trans-3-oh-clom which appears to be a potential inactivation pathway of clom. 1 1 institut für pharmakologie und toxikologie der bundeswehr, münchen, germany for decades the biological effect of sm has been investigated. it is well known how sm interacts and destroys cells. unfortunately, it is still unknown if and how a cell can become resistant against sm. within the here described experiments we investigated a new approach adapting cells to the presence of sm. over a time period of nearly three years the cells were cultivated in presence of sm with increasing concentrations. before starting the initial sm sensitivity was investigated. at the beginning cells were cultivated with a concentration of 0.07 µm sm (ic 10 ). today the cells are able to tolerate a concentration of 7.2 µm sm (ic 90 ), which reflects to a concentration of which 90% of the original cells would have died. to determine cellular characteristics, the resistant cells were compared with wildtype cells. the following cell characteristics were investigated: proliferation, apoptosis, clonogenicity, size of nuclei and cytoplasm, cell-cell contacts, dna adducts formation, secretome, screening of mirna expression, next generation sequencing, vital observation and scratch assay, nad(p) + /nad(p)h, h 2 o 2 , glutathione, ca 2+ -influx, mdrchannels, resistance to other alkylating agents and the reversibility of the resistance. the resistant cells demonstrate smaller nuclei and cytoplasm, less dna adducts, a higher clonogenicity as well as proliferation and less apoptosis. the secretome analysis showed an up-regulation of anti-apoptotic acting cytokines timp and ang and the proproliferative acting cytokines timp and pdgf-aa. in contrast, immunologically active cytokines were down-regulated. concerning cell-cell contacts no differences were seen. in the mirna screening 49 significant up-regulated and 20 significant down-regulated mirnas have been observed. noteworthy was the regulation of various members of 11 different families. during vital observation and in a scratch-assay the resistant cells were show to have disadvantages. the observed resistance was not unique for sm but also towards other alkylating agents and cytostatic drugs. by analyzing the reversibility cells stayed resistant over more than 35 weeks. in conclusion, many aspects investigated in this study have an influence on the sm resistance, pointing out that it is a combination of various effects that are involved to switch on resistance. more likely, there are many aspects working together. the present results are an important step in the characterization of the sm-resistant cell line and further studies may be able to directly use these as a start for target identification in antidote or prophylactic agent discovery. the arylhydrocarbon receptor (ahr) is localized in a cytosolic complex that contains several co-chaperones and associated factors. the protein is shifted into the nucleus in response to endogenous and xenobiotic ligands. however, a transient nuclear transport does also occur in the absence of any ligands, while the predominant cytoplasmic compartmentalization is maintained by parallel export. we have analyzed the interplay between this basal nucleo-cytoplasmic shuttling and ligand induced transport in hepg2 cells, using a yfp-tagged fusion protein that is capable to respond to ligands and to trigger the induction of cyp1a1 expression. basal import was assessed in cells that had been treated with leptomycin b (lmb), an inhibitor of crm1-mediated nuclear export. interestingly, the apparent ahr import rate in lmb-treated cells was comparable with nuclear import as trigged by xenobiotic (b-naphthoflavone) or endogenous (kynurenine) ligands. this observation was confirmed for endogenous ahr in hepg2 cells, since both ligands and lmb showed comparable effects on nuclear compartmentalization. however, the basal nuclear import rate in lmb-treated cells was strongly increased by ahr ligands. ligand-induced nuclear transport was therefore confirmed as an import step in receptor activation. interestingly, lmb did also accelerate nuclear import of ahr after pretreatment of cells with ahr ligands. these data suggest that nuclear export of the ahr is maintained in the presence of ligands. receptor activation might therefore comprise several rounds of shuttling, thereby involving both accelerated import and continued export of the ahr protein fraction that has not already undergone interactions with arnt or dna. we suggest that nuclear export provides an additional kinetic control of ahr activation and function. mitochondrial toxicology: rescuing mitochondria in wilson disease avoids acute liver failure h. zischka 1 1 institut für molekulare toxikologie und pharmakologie, ag zischka, neuherberg, germany in wilson disease (wd) functional loss mutations in the hepatocyte atp7b gene cause dramatic copper overload leading to acute liver failure, posing an unmet therapeutic issue. we find that the pathology of severe wd cases is mirrored in lpp (-/-) rats carrying a functional loss atp7b mutation. this is especially apparent in the hepatocyte mitochondrial compartment. a progressive copper deposition increasingly harms the lifesustaining mitochondrial membrane integrity. thus, depleting this devastating mitochondrial copper burden is a core requirement for a treatment strategy against acute liver failure in this wd animal model. preparation for the master degree program in toxicology started in 2006 as a cooperation of charité universitätsmedizin berlin with the university of potsdam and other institutions of the region. first enrollment of students was done in 2008. the program was accredited in 2011 by the central evaluation and accreditation agency. it offers a modern curriculum encompassing a wide variety of scientific aspects with an interdisciplinary character. this training program in toxicology is organized in modules and ends with the degree "master of science" (m.sc.). the goal of this program in toxicology is to teach the basis of the interactions between substances at toxic concentrations and living organisms, as well as the molecular mechanism of the adverse effects of chemicals. the understanding of the mechanism of a toxic action is an important prerequisite for the scientifically based evaluation of a hazard associated with a substance. furthermore, only with the knowledge of the mechanism of action and a deduction of structure activity relationships it is possible to predict toxic effects of new substances. this knowledge should enable students to perform a risk evaluation of chemicals or to predict the adverse effects of chemicals with the aim that human beings and the environment can be protected from harmful consequences of chemical exposure. the program allocates 30 places per year to an average of 60 applicants. most applicants have a basic training in the fields biology, chemistry, pharmacy, veterinary medicine and nutritional sciences. about 75% of the students are female. the majority of them have a bachelor's degree before starting the master program, other degrees are diploma and state examination as pharmacists or physicians. ninety percent of the students pass the final examination consisting of the master's thesis and disputation at the end of the four semesters. afterwards, most of the graduates aim to obtain a phd degree. the program is well established in the education of toxicologists in germany. respiratory injury due to chlorine developed from consumer products. still an issue in germany u. stedtler 1 , m. hermanns-clausen 1 1 uniklinikum freiburg, vergiftungs-informations-zentrale, freiburg, germany objective: in the last decades strong effords have been took to improve product safety, especially in products intended for domestic use. hypochlorite-containing cleaners may develop chlorine gas when acidified e.g. by adding an acid sanitary cleaner. usually these cleaners contain sodium hydroxide or other strong alkalines to avoid this reaction. we analysed reports to our poisons center concerning inhalation exposure to chlorine developed from hypochlorite-containing mixtures. method: retrospective search in the case database of the poisons center. human inhalative exposures to chlorine released from mixing hypochlorite as well as human inhalative hypochlorite exposure alone were analysed. frequency and symptoms were compared. results: from 2010 to 2015 in total 85 cases of human exposures to chlorine developed from mixtures of hypochlorite and acids (0.8 of 1000 cases) were registered. in 55 cases the exposure was due to mixtures of products intended for domestic use. 94 % of the exposed patients reported symptoms. only in two cases the symptoms were not considered to be caused by the inhalation accident. most frequent symptoms reported were (percent of symptomatic patients): cough (45 %), dyspnea ( 33 %), irritated upper airway (26 %), abdominal discomfort (pain, nausea, vomiting) (21 %), thoracic pain (20 %), irritated eyes (11 %), dizziness (8%), and bronchospasm (6 %). further symptoms were malaise, headache, irritated nose, sweating, muscle pain, and others. in 12 patients (14 %) the symptoms were graded as moderate severe. main symptoms in this group were dyspnoea ( 83% ), cough, and irritated airway. one third of the patients experienced bronchial obstruction. all symptomatic patients developed symptoms while exposed or shortly after exposure. there were no severe or fatal cases (especially no lung edema) and all symptoms were expected to resolve completely. because hypochlorite containing procucts sontanously release "chlorine-like" smelling gases, we additionally analysed inhalation exposures to hypochlorite solutions alone in the same period. there were 42 patients in the same period exposed to hypochlorite evaporation alone. 36 of them (86 %) had symptoms of which in 30 cases these were considered to be caused or possibly be caused by the hypochlorite. most frequent symptoms were irritated upper airway (33 %), nausea or vomiting (30 %), cough (23 %), irritated eyes (20 %). dyspneoa was less fequent than in the mixture group (10 %). all symptoms were considered mild. there was no bronchospasm or thoracic discomfort. conclusion: respiratory injuries by chlorine from hypochlorite-containing solutions still occur despite clear warning on the label. the majority of cases was due to products for domestic use. symptoms develop shortly after exposure. the γh2ax assay for genotoxic and nongenotoxic agents: comparison of h2ax phosphorylation with cell death response perturbation of mitosis through inhibition of histone acetyltransferases: the key to ochratoxin a toxicity and carcinogenicity? regulation of chromatin by histone modifications transcriptomic alterations induced by ochratoxin a in rat and human renal proximal tubular in vitro models and comparison to a rat in vivo model in vitro gene expression data supporting a dna non-reactive genotoxic mechanism for ochratoxin a fragment ion patchwork quantification for measuring site-specific acetylation degrees combinatorial patterns of histone acetylations and methylations in the human genome inroads to predict in vivo toxicology -an introduction to the etox project value of shared preclinical safety studies -the etox database acknowledgements: support of the bfr through grant 1322-530 is gratefully acknowledged. acknowledgement: supported by the robert bosch foundation, stuttgart, germany. [1] mürdter t, et al. hum mol genet, 2011, 21:1145-54 [2] nishiyama t. et al., biochemical pharmacology, 2002, 63:1817 -1830 [3] sun d. et al., drug metabolism and disposition, 2007, 35:2006 background: infections are a major problem in patients with burn diseases (bd). due to severe injuries of their total body surface area (tbsa), burn patients have altered pharmacokinetic characteristics. therefore, insufficient plasma concentrations may be achieved, when standard dosing schedules are applied for antibiotics such as piperacillin. for time-dependent antibiotics, the duration how long drug concentration exceeds the minimal inhibition concentration (mic) is crucial for their antibacterial effects. since pseudomonas spp. is the main problematic pathophysiological bacterium for bd patients. the aim of the present study was to monitor the plasma concentrations of piperacillin during piperacillin/tazobactam treatment in bd patients. patients from intensive care units (icu) served as controls. methods: 10 bd patients (5/5 m/f, 43.4±5.3y, tbsa 40 .9±5.9%) and 5 patients (74.4±3.9y) from the icu were included in this observational study. blood samples were taken within the 3 rd interval of the 8h-lasting dosing period of piperacillin/tazobactam (4/0.5g within 0.5h) at 1, 4 and 7.5h after the end of infusion. total and free piperacillin concentrations were determined in plasma using hplc-uv after deproteinisation with acetonitrile and by ultrafiltration, respectively. pharmacokinetic parameters and dosing simulations were calculated by tdmx (www.tdmx.eu). free plasma concentrations of piperacillin exceeding at least 1xmic but preferably 4xmic over the whole dosing interval were considered to be sufficient for antibiotic efficacy (mic 16 mg/l for pseudomonas spp.,www.eucast.org). results: the pharmacokinetic parameters of total piperacillin, calculated for each bd or icu patient using the concentrations at 1, 4, and 7.5h, were as follows: c max 69.6±7.9 vs.116.3±10.4 mg/l, p<0.05, half-life 1.8±0.3 vs. 2.3±0.3h, p>0.05, clearance 12.6±1.9 vs. 6.5±0.4 l/h, p<0.05, volume of distribution 27.8±2.0 vs. 20.9±1.3l, p<0.05. free concentrations (which were included in tdmx calculations) were 87±2 vs. 81±2% (p<0.05) of total concentrations. duration per day while concentrations exceeded 1xmic (15.6±1.9 vs. 22.0±1.1h, p<0.05) or 4xmic (5.4±1.3 vs. 9 .4±0.7h, p<0.05) were lower in bd than in icu patients. moreover, tdmx simulations predicted that the duration per day for 4xmic could be enhanced to 16.2±1.5h if the piperacillin amount will be increased to 4x8 g/d and the infusion duration to 3h. pharmacokinetic parameters have, however, to be determined in a pilot study with bd patients to ensure predicted values. conclusions: standard dosage regimens for piperacillin/tazobactam could result in suboptimal plasma concentrations of piperacillin in bd patients as well as in icu patients. drug monitoring and tdmx simulation of kinetic parameters may easily help to improve piperacillin treatment in bd patients. background: high dose methotrexate (hd mtx), defined as >1000mg mtx/m 2 bodysurface-area (bsa) is used in children to treat a variety of malignant diseases since the 1950s. clinicians observe relevant rates of severe unwanted side effects. identifying patients having an increased risk for toxicity due to altered mtx pharmacokinetics is urgently needed. we aim to develop and evaluate a physiology-based pharmacokinetic (pbpk) model for hd mtx in children using pk-sim® (bayer technology services gmbh, leverkusen, germany) with a special emphasize on relevant covariates. methods: in this non-interventional observational study, children receiving hd mtx intravenously at two major german pediatric oncology departments during the years 2004-2009 were included if at least one mtx serum level (mtx-sl) was determined during clinical routine. 29 patients aged 2-18 years (male = 19, female = 10) with following diagnoses were included: acute lymphoblastic leukemia, non-hodgkin lymphoma, burkitt lymphoma, brain stem glioma and glioblastoma multiforme. in total, 103 mtx treatment cycles corresponding to 300 mtx-sl were used in this study. patients were randomized into two patient sets (training set and test set). based on literature data, mtx pbpk-models were developed and slightly adapted taking into account mean relative deviation (mrd) and bias of predicted versus observed mtx-sl of the training set. the pbpk model with the lowest mrd and bias was chosen and finally evaluated using the test set. the impact of the covariates urine ph <6.5, trimethoprime/sulfamethoxazole, proton-pump-inhibitors, non-steroidal anti-inflammatory drugs and ß-lactam antibiotics on the prediction quality was assessed using the mann-whitney u test. ochratoxin a (ota) is a wide-spread food contaminant and one of the most potent renal carcinogens [1] . recent data by our group demonstrate that ota inhibits histone acetyltransferases (hats), thereby causing a global reduction of lysine acetylation of histones and non-histone proteins [2] . based on these findings and the importance of specific histone acetylation marks in regulating gene transcription [3] , we speculated that repression of gene expression as the predominant transcriptional response to ota [4, 5] may be linked to loss of histone acetylation. in this study we therefore used a novel mass spectrometry approach, which is based on chemical acetylation of unmodified lysine residues of histones using 13 c-labeled acetic anhydride and subsequent calculation of the degree of acetylation based on the measured intensities of heavy and light acetylated isotopologues [6] , to identify and quantify site-specific alterations in histone acetylation in human kidney epithelial (hk-2) cells treated with ota. our results demonstrate ota-mediated loss of acetylation at almost all important lysine residues at histones h2a, h2b, h3 and h4. we further selected acetylation at histone h3 lysine 9 (h3k9), a well-known euchromatic hallmark that is elevated at promoter regions of transcriptionally active genes [7] and which was reduced from ~ 3% in controls to < 0.1% in response to ota, to establish a link between loss of h3k9 acetylation and expression of genes consistently shown to be down-regulated in response to ota [4, 5] . using chromatin immunoprecipitation followed by quantitative real-time pcr (chip-qpcr), we observed ota-mediated loss of h3k9 acetylation at promoter regions of the selected genes (% of controls: amigo2: 45%, clasp2: 60%, ctnnd1: 54%). overall, these data provide first evidence for a mechanistic link between h3k9 hypoacetylation as a consequence of ota-mediated inhibition of hats and repression of gene expression by ota. a new paradigm to assess the proarrhythmic potential of drugs is proposed by the cipa (comprehensive in vitro pro-arrhythmia assay) initiative combining a suite of a priori in vitro assays (7 most important ion channels for cardiac activity) coupled to in silico reconstructions of cellular cardiac action potential (ap). the etox consortium has developed a multiscale simulation in silico model based on o'hara/rudy incorporating the principles of this new paradigm. the core model simulates the effects of drugs on a virtual cardiac tissue composed by different types of cardiomyocytes. the input of this model, the blockade of a set of 3 ion channels (ikr/herg, iks, ical), can be obtained experimentally or predicted using advanced 3d-qsar models. the system predicts the % change of the qt interval at different drug concentrations in order to facilitate risk assessment. this in silico model was validated using purkinje fiber assay results (input: ap prolongation and arrhythmogenic risk assessed by early after-depolarisation occurrence) from 500 in-house drug candidates. the validation showed that predictivity is highly dependent on the model's applicability domain (ad): for some chemical series the proarrhythmic potential could not be identified, for others, however, most of the positive drugs were correctly predicted with sensitivities up to 80-90% (average prediction accuracy was 70%). retraining of this model with additional internal data should help to improve the model ad and predictivity. it is important to note that ap prolongation was correctly predicted for many proarrhythmic drugs with only low (> 30 µm) in vitro herg inhibition. furthermore, the model showed high additional benefit for read-across within bayer pharma ag, investigational toxicology, berlin, germany etox [1] [2] started in 2010 and is a public-private partnership project within the european innovative medicines initiative (imi) [3] . the etox project is building a toxicology database relevant to pharmaceutical development and to elaborate innovative strategies and software tools. the overall goal is to better predict the toxicological profiles of new chemical entities in early stages of the drug development pipeline based on existing in vivo study results contributed by the participating efpia * companies in the consortium. the etox database is a relational database with a specifically designed schema to store complex and comprehensive preclinical safety data like the study design, toxicokinetics, adme data, clinical chemistry, hematology, gross necropsy, histopathological findings and general toxic effects. in addition relevant data from public sources has been included into the database. the primary focus for data collection are systemic toxicity (up to 4 week) repeated dose studies, mostly in rodent. overall more than 7000 study reports for approximately 1400 investigated compounds. in order to optimize the usage and mapping of data from different sources the development of common ontologies was a key task within the project. this timeconsuming step was necessary to make a high quality read-across analysis possible and valuable. therefore the ontobrowser [4] tool was developed to curate and harmonize the verbatim terms to standardize terms which are used within the etox database. until now more than 13 million verbatim terms were curated. additionally to the toxicology database, a web-based user interface called etoxsys was developed to allow the retrieving of toxicity information, as well as the prediction of toxic endpoints for chemical compounds. due to the complex search capabilities, the database can be queried for structural similarity, similar target classes and specific toxicological endpoints. approximately 150 prediction models based on public data are available and first models based on in vivo data are in development. the etox database therefore represents a valuable tool for early animal-free assessment of drug candidates [5] . * european federation of pharmaceutical industries and associations cell lines background: consumers are constantly exposed to chemical mixtures e. g. to multiple residues of different pesticides via the diet. this raises questions concerning potential cumulative effects, especially for substances causing toxicity by a common mode of action. since substances are tested for regulatory purposes on an individual basis at generally high dose levels, there is only limited data available on potential mixture effects especially in the low dose range. with more than 400 active substances approved for being used in pesticides and over 100000 chemicals registered under reach there are more possible combinations than one could test with classical animal experiments. the development of in vitro tools for assessment of mixture effects consequently is of tremendous importance. methods: as a first step in the development of such in vitro tools we used a group of fungicides, (tri-)azoles, as model substances in a set of different cell lines from known target tissues, basically liver (human: hepg2, heparg, rat: h4iie) and adrenal gland (human: h295r). concentrations were taken from measured tissue concentrations in vivo to ensure that used concentrations of the (tri-) azoles reflect realistic effect levels. the cell lines were exposed with the triazoles cyproconazole and epoxiconazole as well as with the azole prochloraz as individual substances and in binary or ternary combinations of these substances at three dose levels and three different time periods. the effects of the substances were subsequently analysed by transcriptomics and metabolomics. a support vector machine will be utilized to integrate the data from the different sources to gain a complete picture of affected adverse outcome pathways and mechanistic information about the applied fungicides. first results indicate combination effects of the substances also at the omics level depending on the specific endpoint and the concentration used. some of these are comparable to effects found with similar methods in a standard toxicity test, a 28-day feeding study in the rat, thus raising hope for the development of in vitro methods suitable to detect combination effects. background: plant protection and biocide products are chemical mixtures, which contain one or more active substances as well as several co-formulants (e.g. solvents, wetting agents, thickener or preservatives). nevertheless, to this day extensive toxicological testing is performed only with the individual active substances, while the plant protection products are only evaluated for acute toxicity, ie, a single dose group experiment with rats is performed as well as testing for skin-and eye-irritation. current pesticides regulation foresees testing of potential harmful mixture effects but only when adequate methods are available making the development of such methods a high priority. several published studies both in vitro and in vivo have shown fortified toxic effects of plant protection products compared to individual active substances. methods: here we present effects of plant protection products as a whole as compared to the individual active substances or co-formulants in a set of human cell lines of hepatic and renal origin (hepg2, heparg, hek293). cytoxicity has been analysed by wst-1 and nru assay as well as gene expression of several marker genes involved in xenobiotic metabolism. additionally reporter gene assays have been conducted for nuclear receptors such as ahr and car. results: while some active substances showed lower toxicity as compared to the respective products, this cannot be confirmed as a general rule for all endpoints for all of the analysed fungicides or herbicides containing active substances such as epoxiconazole, cyproconazole, azoxystrobin or glyphosate. chemical compounds may induce skin sensitization in humans, resulting in tolerance or allergic contact dermatitis after repeated exposure. mechanistically, the activation of dendritic cells is one of the prerequisites for the induction of skin sensitization. a subgroup of sensitizing chemicals, prohaptens, need metabolic activation, e.g. via cytochrome p450 (cyp) enzymes. thus, xenobiotic metabolism may crucially impact on a chemical's potential for the induction of skin sensitization by activation, but also deactivation of reactive molecules via conjugation, which determines the concentration and the chemical species available for protein haptenation and cell activation. we established a coculture model consisting of hacat keratinocytes and thp-1 as surrogate dendritic cells for the detection of sensitizing chemicals and found enhanced cyp1 enzyme activity in hacat cells exposed to benzo[a]pyrene (b[a]p) and eugenol as well as clearly increased expression of cell surface molecule cd86 on thp-1 cells after incubation with these prohaptens (hennen et al., 2011) . here, we studied the impact of intercellular cross talk on activation and conjugation capacities in more detail. treatment of thp-1 with b[a]p and eugenol in coculture with hacat cells augmented cyp1a1 and/or cyp1b1 mrna levels, while this was not found for thp-1 monoculture. augmentation of cyp1a1 mrna needed continuous presence of hacat cells. in coculture, levels of 3-oh-b[a]p as exemplary cyp-dependent metabolite were increased compared to single cultures. in contrast to this, total glutathione contents as well as n-acetyltransferase 1 enzyme activities in both cell types were not modulated in coculture, furthermore the capacity for sulfation/glucuronidation of 3-oh-b[a]p was maintained in coculture. additionally, the decrease of the total glutathione content in thp-1 cells by 2,4-dinitrochlorobenzene (dncb) was much less pronounced when exposed in coculture with hacat cells, showing that hacat cells provide additional targets for cysteine-reactive chemicals such as dncb, diminishing the total amount of chemicals available for thp-1 cells.overall, results indicate that the cross talk between keratinocytes and antigenpresenting cells enhances their capacities for metabolic activation of chemicals, while hacat cells also provide supplementary capacities for phase ii reactions. references: hennen j et al. cross talk between keratinocytes and dendritic cells: impact on the prediction of sen-sitization. toxicol sci 2011 123:501-510.toxicology -toxic pathway analysis/aop 395 background: reticulated platelets are associated with impaired antiplatelet response to thienopyridine treatment. this interaction might be caused by intrinsic properties of reticulated platelets or a decreased drug exposure due to high platelet turnover reflected by reticulated platelets as surrogate. we investigated the impact of reticulated platelets on antiplatelet response to thienopyridines and if this effect is linked to platelet turnover. methods: this study randomized elective patients to loading with clopidogrel 600mg or prasugrel 60mg (n=200). adp-induced platelet reactivity was assessed by impedance aggregometry 30 to 120 minutes and day 1after loading but before intake of the next dose of thienopyridines. immature platelet count (ipc) was assessed as marker of reticulated platelets by whole blood flow cytometry. results: platelet reactivity increased with rising tertiles of ipc (figure) . this effect was more pronounced in patients on clopidogrel as compared to patients on prasugrel. overall, ipc correlated well with on-treatment platelet reactivity at 120min (r=0.21; p<0.001). this correlation did not change over time indicating an effect independent of platelet turnover (comparison of correlations 120min/day 1: p=0.57 for clopidogrel, p=0.76 for prasugrel). conclusion: a high immature platelet count is associated with impaired response to thienopyridine loading. this effect is independent of platelet turnover indicating a relation to intrinsic properties of reticulated platelets. introduction: one of the biggest drawbacks of protein-based therapeutics with intracellular targets is their inability to enter the cytosol. targeted toxins are known to be used in drug delivery. aim of the study was to target epidermal growth factor (egf) receptor overexpressed on pancreatic carcinoma using a novel well-defined targeted toxin consisting of egf fused to the toxic plant ribosome-inactivating protein dianthin and a glycosidic triterpenoid (so1861) as efficacy enhancer. methods: the enzymatic activity of dianthin-egf was verified by an adenine release assay. the kinetics of cytotoxicity were evaluated in pancreatic adenocarcinoma bxpc-3 and miapaca-2 cells in comparison to the non-target cell line nih3t3 with an impedance-based real time cell analyzer (xcelligence) and final cytotoxicity analyses with conventional end-point mtt assays. acute toxic of dianthin-egf was studied in male balb/c mice. a xenograft solid tumor model was developed in male nude mice by injecting bxpc-3 cells into the dorsal part subcutaneously. dianthin-egf was administered at the vicinity of the tumor and so1861 by subcutaneous injection at the neck. after the tumor reached a diameter of 2 to 3 mm in size 6 treatments were given in total. tumor volumes and body weight shifts were observed twice weekly to determine the potency of dianthin-egf when given alone and in combination with so1861 in comparison to placebo. immunohistochemical detection of egf receptor was performed according to the manufacturers's advice (dako, glostrup, denmark, k1492). complete blood count analysis was done by labor 28 gmbh, berlin. results: the adenine release mediated by dianthin-egf was 47.8 pmol adenine/pmol toxin/h. the in vitro efficacy of the targeted toxin was proven by an ic50 value of approximately 1 nm for egf receptor expressing miapaca-2 and bxpc-3 cells as compared to 100 nm for non-target nih3t3 cells. real time measurement of the cytotoxicity showed a dose-dependent decrease in cell viability from 10 pm to 1 µm. toxicity studies in balb/c mice revealed 0.4 µg/mouse to be non-toxic and maximum tolerated dose (mtd) whereas 40 µg caused moribundity accompanied with white ocular discharge. efficacy studies were performed for a period of 28 days. the combination therapy showed that the average tumor volume measured by a digital vernier caliper was found to be 80% less than for placebo whereas single therapy using dianthin-egf alone caused a further increase in tumor volume which was although yet 50% less when compared to placebo. immunohistochemistry slides showed egf receptor expression in each of all untreated xenograft tumors, which further confirms the presence of egf receptor overexpression in the target bxpc-3 cell line. enlarged spleen was only observed in untreated xenografts. no significant change in various blood parameters (rbc counts, wbc counts, hgb, hct, mcv, mch and mchc) were observed on hematological analysis except for the platelet (plt) counts in comparison to healthy male nude mice. conclusion: combination therapy with so1861 proves to be a promising approach for the targeted delivery of toxins instead of single therapy administering targeted toxin alone. the strategy is specific for egf receptor overexpressing tumors such as pancreatic cancer. introduction: moringa oleifera (mo) is a popular herbal supplement used for treatment and management of diverse diseases in sub-saharan africa. its intake among individuals infected with hiv/aids has increased recently due to the purported immune boosting property. limited information, however, is available regarding its potential to cause interactions with commonly prescribed medications that are substrates of cyp3a4 and p-glycoprotein. methods: the methanol extract and four fractions of mo were tested on recombinant cyp3a4 at different concentrations with and without nadph to determine the ic 50 shift reduction. the crude methanol extract of mo was incubated with testosterone (tst) and cryopreserved hepatocytes to evaluate its influence on clearance of tst. effect of mo on the efflux transporter, p-glycoprotein was investigated by incubating the methanol extract with mdr1 -mdckii cells. virtual screening was conducted to predict physicochemical properties, bioavailability and interaction potential of phytochemical compounds unique to mo using combination of molinspiration version 2014.11 and admetsar. results: fractions (f1-f3) indicated ic 50 shift reduction ≥5 post-incubation with and without nadph. mo showed moderate interaction (auc i /auc = 2.46) with tst in cryopreserved hepatocytes. also, mo mildly inhibited the transport of digoxin (ic 50 = 35.45 µg/ml) across mdr1 -mdckii cells. niaziminin indicated 85.57% bioavailablity via the human intestinal membrane with 61% chance of inhibiting cyp3a4. βsitostenone showed strong p-gp inhibition (83.27%) with 100% absorption via the intestine. conclusions: mo has the potential to inhibit the metabolism or excretion of other medications that are eliminated by cyp3a4 or p-glycoprotein, respectively, if adequate amounts of the active constituents such as niaziminin and β-sitostenone enter the circulation. background: herb-induced liver injury (hili) has attracted attention in the past years due to an increasing number of publications reporting cases of hepatotoxicity associated with use of phytotherapeutics. here, we present data on hili from the berlin case-control surveillance study fakos. methods: fakos was initiated in 2000 to study serious toxicity of drugs including hepatotoxicity. potential cases of liver injury were ascertained in more than 180 departments of all 51 berlin hospitals from october 2002 until december 2011. through a standardised face-to-face interview and review of medical charts information on all previous intakes of drugs or herbals, on co-morbidities, and demographic data was ascertained. inclusion criteria were an elevation of alanine aminotransferase or aspartate aminotransferase threefold above the upper limit of normal or an elevation of total bilirubin higher than 2 mg/dl. excluded were patients with underlying liver disease (e.g., alcoholic fatty liver disease). drug or herbal aetiology was assessed based on the updated council for international organizations of medical sciences (cioms) scale. results: of all 198 cases of hepatotoxicity included into the fakos study, herbs were involved in ten cases (5.1%). demographic, clinical, and laboratory characteristics of these ten cases are illustrated in table 1 . among the six patients with available liver biopsy results, five patients showed signs of necrosis, either disseminated or predominantly near the central vein. portal inflammation was more common than lobular inflammation, and the infiltrates contained mostly lymphocytes, neutrophil or eosinophil granulocytes. herbal aetiology was judged two times as probable (ayurvedic herb in patient 1, pelargonium sidoides in patient 6), and eight times as possible (valeriana in patients 3, 4, 8, 9, 10 , mentha piperita in patient 5, hypericum perforatum in patient 2, eucalyptus globulus in patient 7). in nine cases other non-herbal drugs were also suspected as potentially hepatotoxic (exception: patient 6). seven cases occurred in the ambulatory setting requiring hospitalisation, three cases occurred during hospital stay. discussion: this case series provides further information on laboratory and clinical aspects of hili. it corroborates known risks for valeriana and ayurveda treatment, and suggests that further herbals rarely or never associated with liver injury before such as pelargonium sidoides, hypericum perforatum or mentha piperita could also exhibit a hepatotoxic potential. clinical routine often requires to evaluate the cause of a newly occurring adverse event. if this event is regarded to be iatrogen, further information of the association between the drugs in the current medication list and the adverse event is needed. this information should ideally reflect the true risk and allow ranking of the drugs according to this risk to identify which drug to discontinue first. we discuss the summary of product characteristics (spc), the sider side effect resource and openvigil 2 as possible sources of information. spcs are becoming more and more a vindicative charter for pharmaceutical companies that contain misleading information which is not based on evidence (ref. 1). since it relies on the spcs, sider inherits these shortcomings and flags warnings that result from confounding factors (ref. 1, fig. 1 ). furthermore, if any rates are given, they are not easily comparable since they stem from different studies. pharmacovigilance data are biased by the very nature of the data and the collection method. however, once confounders are eliminated, pharmacovigilance offers better information om how to rank the drugs than spcs/sider. we present decision-guiding information obtained by sider and by openvigil 2 for one of our patients ( fig. 2 & 3 ) and discuss how this information was used to modify the therapy. institut für naturheilkunde und klinische pharmakologie, universität ulm, ulm, germany background: differences (polymorphisms) in target genes or genes encoding drug transport proteins or drug metabolizing enzymes may be responsible, among other factors, for observed variation in patients' response to medications. pharmacogenetics aims at identification of patients at higher, genetically determined, risk of adverse drug effects or ineffective medication, to modify dosage or switch to alternative therapy. there is, however, a lack of awareness of pharmacogenetic-based clinical practise guidelines. methods: a systematic literature review was conducted which focused on published guidelines on genotype-based (germ-line genetic variants) dosage modification or selection of drugs. we serched the medline and the pharmacogenomics knowledgebase (pharmgkb) databases. prescribing information was also screened for pharmacogenetic guidance. results: the systematic review revealed recommendations for 61 drugs (table) that enable the translation of genetic test results into actionable prescribing decisions. for 20% of these drugs the respective german drug labels recommend or even require pharmacogenetic testing (table, 3rd column). although pharmacogenetic testing is recommended, the prescribing information not always provides guidance on how to adjust the drug dosage based on the pharmacogenetic test result. compared with the german or european drug labels, the fda drug labels povide more detailed information on pharmacogenetic dose modifications. conclusions: academic working groups have a front-runner role in the development of prescribing recommendations based on genetic markers. to date, drug labels rarely contain detailed guidelines how available genetic test results should be used to adjust drug dosage. because pharmacogenetics has a growing role during drug development and pre-prescription genotyping will become more widespread, it is expected that specific pharmacogenetic guidance for the treating physicians will become increasingly important. bisphenol a (bpa) is a high production volume compound mainly used as a monomer to make polymers for various applications, including food-contact applications. people are exposed to low levels of bpa because very small amounts of bpa may migrate from the food packaging into foods or beverages. however, other potential sources of exposure, such as dermal contact have also been identified (efsa, 2015) . a substance evaluation process (corap) was initiated for bpa by the european chemicals agency (echa). as part of the safety evaluation of bpa, a study was required by echa to assess absorption and metabolism of bpa following dermal exposure to human skin. an in vitro study with human skin was requested according to oecd tg 428 under consideration of the scientific committee on consumer safety (sccs) criteria for the in vitro assessment of dermal absorption. to investigate potential dermal bpa metabolism fresh human skin was used. abdominal skin was obtained fresh from surgery from 4 different donors. split-thickness human skin membranes were mounted into flow-through diffusion cells (n=4 per dose and donor) and the receptor fluid was pumped underneath the skin at a constant flow rate. the skin surface temperature was maintained at 32°c throughout the experiment and electrical resistance barrier integrity testing was performed at the start (0 h) and end of the experiment (24 h). four test preparations at final bpa concentrations of 2.4, 12, 60, and 300mg/l were investigated. the highest concentration was chosen based on the maximum solubility of bpa in water and the lowest concenration was chosen based upon the specific activity of the radiolabelled [ 14 c]-bpa that could be used for mass balance. percutaneous absorption was assessed by collecting receptor fluid (tissue culture medium (dmem), containing ethanol (ca 1%, v/v), uridine 5'-diphosphoglucuronic acid (udpga, 2 mm) and 3'-phosphoadenosine-5'-phosphosulfate (paps, 40 µm)), at multiple time points througout the experiment. at termination the skin was removed from the cells and the stratum corneum was removed with 20 successive tape strips. the exposed epidermis was separated from the dermis using a scalpel. metabolism was investigated for the highest concentration (300 mg bpa/l) only, using a hplc with in-line radiodetection and confirmed bpa-glucuronide (bpa-g) and bpa-sulfate (bpa-s) standards for comparison. no metabolism was observed in any of the epidermis samples, however some metabolism is observed in dermis and receptor fluid samples. metabolites were identified with retention consistent with bpa-g and bpa-s, and also some more polar components. the mean total absorbed dose (receptor fluid + receptor chamber wash + receptor rinse) was between 1.7 and 3.6% of the applied dose and the mean dermal delivery (epidermis + dermis + total absorbed dose) was between 16 and 20% of the applied dose, with the majority of the radioactivity associated with epidermis samples compared to dermis and receptor fluid samples. a linear dose-response relationship is observed over the whole concentration range. anastrozole is a well-known non-steroidal aromatase-inhibiting drug approved for the second-line treatment of breast cancer after surgery and for treating postmenopausal women. treatment with the only available dosage form, anastrozole film-coated tablets for oral administration, is frequently associated with concentration-dependent unwanted side effects like hot flashes, fatigue, joint pain, joint stiffness, vaginal dryness, hair loss, skin rash, nausea, diarrhea and headache. in order to minimize the local gastrointestinal as well as systemic side effects, a system for transdermal anastrozole delivery has recently been developed. in this study, we describe the first experimental in vivo application of a transdermal therapeutic system (tts) to beagle dogs and, as a necessary prerequisite for the analysis of the time course of anastrozole release and uptake, a simple, sensitive and accurate lc-ms method for quantifying anastrozole in plasma. the detection of fragment ions at m/z 225 and 237 instead of the molecule ions (m/z 294 and 306) generated from the elevated collision energy, and the use of a deuterated internal standard resulted in increased relative abundances and improved signal-to-noise ratios.the lower limit of quantification and the limit of detection were 1.4 ng/ml and 0.5 ng/ml, respectively. the developed method was successfully applied in a pharmacokinetic study of anastrozole plasma levels in beagle dogs, measuring percutaneous drug absorption from an experimental, newly designed glycerol-based patch / tts. a distinct time course was observed, with an initial linear increase over 24 hours and a plateau thereafter. this offers promising strategies for the transdermal application of anastrozole with improved pharmacokinetics. background: the monocarboxylate transporter 4 (mct4), encoded by the slc16a3 gene, mediates h + -coupled transport of lactate across the plasma membrane. for cells with high glycolytic activity lactate export is of major importance for the maintenance of the glycolytic metabolism and for the prevention of intracellular acidification. in glycolytic tumor cells, the acidic extracellular environment resulting from export of lactate and h + , furthermore promotes anti-apoptotic effects and metastasis. clear cell renal cell carcinoma (ccrcc) is the most common subtype of renal cell carcinoma (rcc) and is characterized by a metabolic shift towards enhanced aerobic glycolysis and hence, increased lactate production. mct4 and its epigenetic regulation by slc16a3 promoter methylation has previously been identified as prognostic marker for ccrcc outcome and as target for ccrcc treatment. since metastatic ccrcc is associated with poor overall survival and represents a major challenge for treatment, mct4/slc16a3 might represent a promising prognostic marker and a target for therapeutic intervention also for metastatic disease. methods: mct4 protein expression was analysed in 130 paraffin embedded tissue samples of distant metastases derived from different organs by immunohistochemical staining of tissue microarrays. protein expression was evaluated semi-quantitatively using tissue studio v.3.6 (definiens ag). dna methylation in the slc16a3 promoter, specifically at the previously identified cpg site with prognostic potential in primary ccrcc, was analysed in 82 paraffin embedded metastasis samples by maldi tof-ms. mct4 protein expression data and dna methylation at the specific cpg site in the slc16a3 promoter were correlated with clinicopathological parameters and outcome data. results: distant metastases of primary ccrcc showed high mct4 protein expression irrespective of the affected organ. the most frequently affected organs like lung or bone, with approximately 28% and 14% in our cohort respectively, showed similar expression levels as less frequent metastatic sites such as thyroid gland or spleen. accordingly, dna methylation at the identified cpg site in the slc16a3 promoter was low in metastatic tissue in all investigated organ sites. an association of low promoter dna methylation level at the previously identified prognostic cpg site in metastases with poor tumor-specific survival of the patients was observed. conclusion: from these results we hypothesize that dna methylation at specific cpg sites in the 5'-regulatory region of mct4 may not only serve as a predictor for patient outcome and as potential novel target for therapeutic intervention in primary, but also for metastatic disease. tamoxifen is used to treat pre-and postmenopausal women with estrogen-receptor (er) positive breast cancer. as a prodrug, tamoxifen undergoes extensive hepatic metabolism resulting in a complex mixture of metabolites with estrogenic and antiestrogenic effects. while endoxifen and (z) 4-hydroxytamoxifen are the most potent antiestrogenic metabolites, bisphenol and both isomers (e) and (z) of metabolite e are the most potent compounds with estrogenic properties at the er. the mixed antagonist/agonist pharmacodynamic effects of the selective estrogen receptor modulator tamoxifen at the er have been mainly attributed to tissue specific action of er coregulators, yet little is known about agonistic metabolites contributing to its estrogenic actions. the aim of the present study was to clarify whether there is a genetic component for interindividual differences in the formation and clinical effect of agonistic tamoxifen metabolites. a genome-wide association study (gwas) was conducted on steady-state agonist plasma levels in 390 postmenopausal breast cancer patients of european origin who were treated with 20 mg/day of tamoxifen for at least 6 months. plasma concentrations of estrogenic metabolites bisphenol, (e), and (z) metabolite e were quantified using a recently established lc-ms/ms method 1 . promising snps for an association between genotype and either plasma metabolite concentration or clinical outcome were confirmed for their relevance in an independent patient cohort of 313 premenopausal breast cancer patients mainly of european descent 2, 3 . twelve snps close to or above genome-wide significance (p <5e-08) were found to be associated with allele-dependent variable (e) or (z) metabolite e plasma levels, while no genomic hit was found for the tamoxifen metabolite bisphenol. here, positive intergenic or genic regions mapped to chromosomes 1, 2 and 16 for (e) metabolite e and to chromosomes 15 and 18 for (z) metabolite e. upon genotyping of the validation cohort, two genetic loci with minor allele frequencies < 5% were confirmed as putative candidates: rs662106 was associated with a 21-39% variant allele-dependent increase of (e) and (z) metabolite e isomers (p< 0.05), and rs3731872, mapping to a gene encoding zinc finger protein znf124, was associated with increased risk of reccurrence or death (hr carriers 2.6, 95% ci: 1.3 -3.4; p < 0.005). these findings suggest the existence of genetic loci that may contribute to the formation and clinical effect of estrogenic tamoxifen metabolites and therefore could explain therapeutic failure of tamoxifen and/or the occurrence of adverse events during treatment. introduction: metabolomic monitoring of endogenous biomarkers is of increasing importance for the assessment of drug safety and efficacy during clinical drug development. myrcludex b, a novel lipopetide-based entry inhibitor for the therapy of hepatitis b and d, exerts its function through inhibition of the hepatic bile acid transporter na + -taurocholate cotransporting polypeptide (ntcp). in order to assess a myrcludex binduced metabolomic response in humans, lc/ms-based monitoring of endogenous metabolites was performed in blood and urine samples from healthy individuals before and during treatment with myrcludex b. methods: plasma and urine samples were collected from healthy volunteers participating in clinical phase i trials to evaluate safety, tolerability, and pharmacokinetics of single doses of the ntcp inhibitor myrcludex b. using quadrupole time-of-flight mass spectrometry coupled to reversed-phase chromatography (lc-qtof-ms) a set of 15 known ntcp substrates (bile acids) was quantified by targeted metabolomics. protein precipitation was performed in the presence of deuterium-labeled internal standards (istds) which allowed absolute bile acid (ba) quantification in low amounts of plasma. ba profiling in urine was performed after dilution with methanol/water (1:1) in the presence of istds. both methods were validated according to fda guidance and applied to monitor the effect of myrcludex b treatment on human bile acid homeostasis. results: dynamic quantification in plasma and urine was achieved in the range from 7.8 nm to 10000 nm depending on the ba species analyzed. intraday-and interday accuracy and precision were in the 15% tolerance range for all analytes in all matrices. matrix effects were between 39-104% (plasma) and 31-95% (urine), apparent recoveries in plasma were above 97%. basal plasma ba level (mean ± sd) in fasting healthy subjects were 667 ± 574 nm (unconjugated bas), 935 ± 629 nm (glycine-conjugated bas) and 104 ± 62 nm (taurine-conjugated bas). urinary ba level (nmol/g creatinine) were 193 ± 225 nm (unconjugated bas), 89 ± 29 nm (glycine-conjugated bas) and 6 ± 2 nm (taurine-conjugated bas). myrcludex-induced ntcp inhibition resulted in significantly elevated amounts of conjugated ba species demonstrating a spillover of ntcp substrates into the systemic circulation. furthermore, higher urinary ba level were observed during treatment indicating accelerated elimination of excessive bas from the body. conclusion: lc/ms-based monitoring of endogenous biomarkers has been successfully established and applied to study the effect of myrcludex b treatment on human ba metabolism. the results obtained by our assay demonstrate that a myrcludex-induced ntcp inhibition drastically affects human ba homeostasis. this observation provides valuable insights into the drug´s mode of action and will be indispensable for the assessment of side effects and dose-finding processes during future clinical trials. further studies are required to assess a possible role of ba modification (e.g. sulfation) in the process of ba detoxification during myrcludex treatment. key: cord-006230-xta38e7j authors: nan title: deutsche gesellschaft für experimentelle und klinische pharmakologie und toxikologie e.v. date: 2012-02-22 journal: naunyn schmiedebergs arch pharmacol doi: 10.1007/s00210-012-0736-0 sha: doc_id: 6230 cord_uid: xta38e7j nan nucleoside diphosphate kinases (ndpks) are multifunctional enzymes involved in a variety of cellular processes including cancer metastasis and heart diseases. the plasma membrane content of the three major ndpk isoforms ndpk a, b and c is increased in human heart failure. we have previously shown that the ndpk b isoform regulates camp levels and cardiac contractility through a receptor-independent gprotein activation involving direct g protein β subunit phosphorylation. the precise role of ndpk c in the heart is unknown and was the object of this study. ndpk c function was assessed in neonatal (nrcm) and adult (arcm) rat cardiomyocytes with real-time pcr, immunoblotting, and quantification of camp content. heart failure was induced by chronic treatment with isoproterenol (iso, 2.4 mg/kg/d 4 days) via minipumps. chronic iso increased mrna levels of ndpk c by 9.4±1.9-fold and its protein levels by 2.1±0.11-fold. immunoprecipitation of the g protein β subunit resulted in coimmunoprecipitation of ndpk c and the stimulatory gαs subunit: iso enhanced this interaction. upon iso stimulation, ndpk c translocated from the cytosol to the plasma membrane within 3 hours in both nrcms and arcms. adenoviral overexpression of ndpk c in nrcms caused a 1.5-fold increase in basal and iso induced camp synthesis, whereas sirna mediated knockdown of endogenous ndpk c decreased camp levels by ~50%. our results establish ndpk c as a novel and critical regulator of camp synthesis and gs signaling in the heart. the up-regulation of ndpk c and the increased responsiveness to iso in failing hearts point to ndpk c as a potential counterregulatory factor in the onset of heart failure. uptake and metabolism of methylated myricetin derivatives: studies in cell culture and c. elegans ackermann d. 1, 2 , büchter c. secondary plant compounds like flavonoids that are ubiquitary present in fruits and vegetables are believed to exert health protective effects in terms of lowering the incidence of widespread diseases such as cardiovascular diseases and cancer. besides their antioxidative effects, flavonoids may also modulate cell signaling pathways and thereby performing their disease-protective actions. though this class of dietary polyphenols has become increasingly popular as dietary supplements, only little is known about their metabolic fate in vivo. therefore, we investigated the absorption and metabolism of several flavonoids such as myricetin and its methylated derivatives laricitrin, syringetin, and myricetin-3',4',5'-trimethylether in the human colon carcinoma cell line hct116 and the human hepatoma cell line hepg2 as well as the model organism caenorhabditis elegans. all flavonoids were rapidly taken up by both cell lines as shown by hplc analyses. the intracellular amount of myricetin and laricitrin did not increase with time and was only half of that of syringetin and myricetin-3',4',5'-trimethylether. interestingly, no metabolites of these flavonoids could be detected which might at least in part be due to their low intracellular amounts. absorption as well as intracellular distribution was also evidenced by using the fluorescent dye "naturstoff reagent a" nsra) . fluorescence microscopy indicated a predominant cytosolic distribution of the employed flavonoids. in the model organism caenorhabditis elegans, the flavonoids were exclusively distributed in the intestine as visualized by nsra. the antioxidative capacity of the four flavonoids (measured by using the cell free teac assay and the h 2dcf-da assay in hct116 cells) decreased with increasing methyl groups in the b-ring. myricetin-3',4',5'-trimethylether (three methyl groups) was the least effective radical scavenging flavonoid in both the cell free system and in hct116 cells compared to myricetin (no methyl groups). in conclusion, for exerting their biological effects, uptake and distribution as well as metabolism of flavonoids in certain organs such as liver and gut are important and more research in that field is warranted. munich heart alliance, münchen, germany signaling through g protein-coupled receptors is affected by receptor polymorphisms, yet the molecular basis for the functional differences of individual receptor variants is unclear. to investigate the impact of the frequent gly389arg variant of the β1-adrenergic receptor (β1ar) on receptor conformation we used β1ar-sensors capable of fluorescence resonance energy transfer (fret). these sensors retained the pharmacological and functional characteristics of the native receptors. upon stimulation of the sensors we determined the activation characteristics of the polymorphic receptors in real time and in living cells. we found the β1ar variants to behave similar upon a single stimulation with an agonist, but to differentially respond with a change of their activation kinetics during subsequent stimulations. while the arg389-β1ar did not show altered activation kinetics after prestimulation, the gly389-β1ar became slower compared to the initial stimulation suggesting that β1ars possess a memory of previous activation. we then permeabilized β1ar-sensor-expressing cells with saponin to remove soluble cytosolic factors. upon permeabilization the β1ar variants did not display receptor memory, suggesting that the β1ar memory depended on the interaction of the receptors with soluble cytosolic factors upon their initial activation including the phosphorylation of agonist-bound receptors by protein kinase a or g protein-coupled receptor kinases. our findings suggest an intrinsic, polymorphism-specific property of βars that alters activation kinetics upon continued stimulation and that might account for individual drug responses. micro-rna replacement therapy: nanoparticle-mediated in vivo delivery of mirna-145 or mirna-33a exerts antitumor effects in colon carcinoma xenograft mouse models weirauch u. 1 micro-rnas (mirnas) control the expression of various genes, and under pathological conditions several mirnas are up-or downregulated. previous in vitro studies have established a pro-apoptotic and anti-proliferative role of mir-145, which shows decreased levels in colon carcinoma. in contrast, while mir-33a is only weakly expressed in several tumors as well, its role in cancer has not been analysed so far. in this study, we demonstrate the tumor-relevance of mir-33a and identify the protooncogenic kinase pim-1 as a target of mir-33a. pim-1 harbours a highly conserved mir-33a binding site within its 3'-utr, and seed mutagenesis of this target sequence abolishes the mir-33a-mediated downregulation of pim-1. the knockdown of pim-1 by rnai or mirna transfection inhibits proliferation in leukemia and in colon carcinoma cells by decelerating cell cycle progression, thus establishing a tumor inhibitory function of mir-33a. we furthermore introduce polyethylenimines (peis) for the therapeutic application of mirnas in vivo, which is critically dependent on the development of appropriate delivery tools. peis are able to form non-covalent complexes with mirnas, leading to mirna protection after systemic application in combination with an attractive biodistribution profile and the efficient uptake in target organs/cells. therapeutic effects of pei-mediated mirna delivery were demonstrated in subcutaneous colon carcinoma xenograft mouse models. the in vivo application of mirna-145 through systemic or local injection of pei/mirna complexes resulted in efficient mirna delivery and in antitumor effects, based on the concomitant repression of erk5. likewise, tumor growth inhibition was observed upon treatment of tumor-bearing mice with pei-complexed mir-33a. this is due to the mir-33a-mediated downregulation of pim-1 expression and resembles the pim-1 knockdown through rnai / pim-1 sirnas. taken together, in tumor xenograft mouse models we establish mirna replacement therapy through the pei-complexation of mirnas as a novel therapeutic strategy and demonstrate that mir-145 and mir-33a may be promising mirnas in colon carcinoma therapy. md 288, a hybrid of chloroquine and primaquine, is a potential drug against infectious diseases such as malaria. since one moiety of the hybrid, the known antimalarial drug chloroquine, is a known intercalator, the potential of md 288 to intercalate into dna was determined. due to the ability of intercalators to cause frame shift mutations, the mutagenic potential of md 288 was also investigated. the potential of md 288 to intercalate into dna was investigated by means of fluorescence based micro plate assay using ethidium bromide (eb) and isolated calf thymus double stranded dna. as a positive control chloroquine was used. the potential of md 288 to cause gene mutations was determined using the hypoxanthine-guanine phosphoribosyltransferase (hprt) test in chinese hamster v79 lung fibroblasts (v79 cells). v79 cells were treated with 0.4 µm, 0.8 µm and 1.5 µm md 288 or the positive control, the direct mutagen 4-nitroquinoline-n-oxide (nqo, 1 µm) for 24 h. on day 6, mutants exhibiting loss of hprt function were selected with 6-thioguanine . whereas at 1 µm chloroquine, a 38% decrease in fluorescence intensity of eb (indicating dna intercalation) was observed, 10 µm md 288 were needed to observe a similar decrease (28%) in fluorescence intensity of eb. therefore, md 288 is 10fold less potent to intercalate into dna than its moiety chloroquine. the frequency of spontaneous 6-tg resistant mutants per 10 6 colony-forming cells was 9 ± 2. as expected, 1 µm nqo caused a significant increase in the mutant frequency (mf, 168 ± 9) . in contrast, mf was not significantly affected by treatment with md 288 at both noncytotoxic (0.4 µm: 5 ± 4) and cytotoxic (0.8 µm: 9 ± 7) concentrations. in conclusion, md 288 is a less potent intercalator than the known antimalarial drug chloroquine. furthermore, md 288 does not cause gene mutations in the hprt test. since current studies show that various metabolites of md 288 are formed in vitro, their mutagenic potential is currently under investigation as well. -conducting channels but depends critically on the membrane potential. trp channels form cation entry channels thereby either contributing to ca 2+ entry or depolarisation. recently, we showed that trpm4 acts as a ca 2+ -activated non-selective cation channel and critically determines the driving force for ca 2+ influx in mast cells following fcεri-stimulation (1) . in addition to trpm4 we also identified the expression of other trp transcripts in bone marrow derived mast cells (bmmc) including those encoding trpc2, trpc3, trpc5, trpc6 and trpm7. in peritoneal mast cells (pmc), rt-pcr indicated expression of trpc1, trpc4, trpc5, trpc6, trpm2, trpm4 and trpm5. to identify the functional role of those trp channel proteins for mast cell activation we analysed ca 2+ signaling using microfluorimetry in bmmcs and pmcs after stimulation with substances known to activate trpc6 channels in other cell systems such as the diacylglycerol analogue oag, the hyperforin analogue hyp-9 and flufenamic acid (ffa), but could not evoke a rise in the [ca 2+ ]i in both pmc and bmmc. sphingosine 1phosphate and lysophosphatidylcholine, which were reported to activate trpc5 channels, induced only minor rise in [ca 2+ ]i in bmmcs, respectively. here, we will present our analysis of ca 2+ signaling following stimulation of the fcεri receptor and application of secretagogues that are supposed to affect ca 2+ -dependent mast cell activation such as adenosine, endothelin-1, substance p and compound 48/80 in bmmcs and pmcs derived from mouse lines with inactivation of trpc1, trpc3, trpc4, trpc5 or trpc6 since specific antagonists are still lacking for these trp channels. the α2a-ar is the main ar in the central nervous system and it plays a crucial role in regulating norepinephrine (ne) release from nerve terminals via presynaptic feedback inhibition. it is also associated with a number of physiological effects, including hypotension, pain perception, sedation and modulation of mood. ne, once released in the synaptic space, binds to α2a-ars and induces a rearrangement of the receptors from the inactive state into an active conformation. this allows the binding and activation of the cognate gi-protein and, hence, the transduction of the transmembrane signal to the downstream effectors. generally, α2a-ar activation has been deduced from the stimulation of a receptor-mediated biological response that could be easily followed experimentally. however, most of these approaches do not employ living cells and are normally applied under equilibrium conditions that need prolonged incubation periods incompatible with the physiological temporal dynamics of ne. here, we monitored the ne-mediated α2a-ar and gi-protein activation by using a fluorescence resonance energy transfer (fret)-based approach in living cells. to examine the effects of increasing concentrations of ne on the speed and extent of α2a-ar activation with very high temporal resolution, we took advantage of the previously described α2a-ar flash/cfp sensor [1] . the results indicate that in our system the efficacy of ne in eliciting α2a-ar flash/cfp activation increases in a time-dependent way and reaches the maximum with a half-life of ~ 70 ms. the ec50 values decrease in an exponential manner and arrive at ~ 2 µm with a half-life of ~ 330 ms. next, we analyzed the ability of increasing concentrations of ne to trigger a downstream intracellular response after α2a-ar stimulation by monitoring the kinetics and amplitude of gi activation in living cells. we applied the previously well characterized gi cfp/yfp sensor [2] . the results show that both the efficacy and the potency of ne in inducing gi activation reach the steady state slower compared to receptor activation (half-life ~ 700 ms and ~ 3,000 ms respectively). in conclusion, we were able to monitor ne-mediated events occurring in the millisecond time scale and reaching the equilibrium in a time interval compatible with physiological conditions. sphingosine-1-phosphate (s1p) is an immune modulator produced by sphingosine kinase 1 (sphk1) and sphingosine kinase 2 (sphk2) and de-phosphorylated or degraded irreversibly by s1p phosphatases and a lyase, respectively. we recently showed that tlr4-induced il-12p70 is selectively counter regulated by sphk1, s1pr1 and its extracellular ligand s1p. on the other hand, spiegel et al. have demonstrated that specific, sphk1-dependent, binding of s1p to traf2 enhances the tnf-alpha signaling. therefore we were interested whether the tlr/tir and tnf-alpha-signaling pathways are interfered with each other and are modulated by s1p. in a first approach we focused our investigations on sphk1 effects on both traf2 and traf6 stimulatory signals and cytokines produced downstream. experimentally, with gm-csf expanded, bone marrow-derived dcs we first desensitized the lps-tlr4 or cpg-tlr9 signal by a defined time period of costimulation with tnf-alpha. the initial results showed a partial decrease of il-12p70 secretion in tnf-alpha-co-stimulated dcs in contrast to lps stimulation alone. this might indicate that traf2 activated via tnf-alpha interacted with the traf6 pathway to reduce il-12p70. further series with dcs derived from sphk1-deficient mice confirmed our former results that il-12p70 in contrast to other cytokines is specifically sensitive to sphk1-s1p feedback, but did not change the effects of tnf-alpha on wt dcs il-12p70 release. in comparison, cpg-tlr9-induced il-12p70 release reached only 20% of lps-induced il-12p70 levels and was less sensitive to tnf-alpha costimulation. however, sphk1-deficiency strongly augmented cpg-dependent il-12p70 production. in ongoing experiments we started to analyze the details of traf2/rip1 and traf6/tak1 activation by ubiquitination blots in wt, sphk1-and s1plyase-deficient dcs. in conclusion, we hope to unravel possible mechanisms of the observed differential effects of s1p and its enzymes on inflammation and cancer-relevant cytokines. identification of the kh type splicing regulatory protein (ksrp) as a new important mediator of the anti-inflammatory effects of resveratrol art j. 1 , besche v. 2 , bros m. 2 , li h. 1 , handler n. 3 , bauer f. 3 , erker t. 3 , behnke f. 4 , mönch b. 5 , förstermann u. 1 , dirsch v. m. 6 , werz o. 5 , kleinert h. 1 , pautz a. university of vienna department of pharmacognosy, althanstr. 14, 1090 wien, austria resveratrol, a polyphenol derived from different plants, possesses multiple pharmacological functions such as anti-oxidative, anti-diabetic, cardioprotective, anticancer, neuroprotective and anti-inflammatory properties. many of these effects have been attributed to its anti-oxidative activity but resveratrol also modulates signal transduction pathways like the p38 mapk pathway or the activity of different transcription factors like nf-κb redox-independently. moreover, the histone deacetylase sirtuin 1 (sirt1) is an important mediator of resveratrol effects. nevertheless the direct molecular target of resveratrol remains unclear. in target fishing experiments we identified the rna-binding protein ksrp as direct resveratrol binding partner. ksrp is an rna-binding protein that controls proinflammatory gene expression on the post-transcriptional level by modulation of mrna stability. moreover, it is involved in the biogenesis of mirnas. resveratrol treatment of human dld-1 cells resulted in a decreased mrna expression of a number of well known ksrp target mrnas and enhanced mirna-155 function. downregulation of ksrp expression by sirna prevented the mrna destabilizing effect of resveratrol. as the activity of ksrp is mainly regulated on the post-translational level by phosphorylation of different serine and threonine residues we analyzed whether resveratrol changes ksrp activity by altering the phosphorylation of the protein. indeed, our immunoprecipitation experiments demonstrated that resveratrol reduces the p38 mapk-mediated phosphorylation of threonine residues in the ksrp protein and thus leads to an increase of ksrp activity. interestingly, resveratrol does not block p38 mapk activation or activity. in addition we have evidence that sirt1 is not involved in the resveratrol mediated activation of ksrp. so we believe that activation of ksrp by resveratrol is the major mechanism mediating the anti-inflammatory effects of resveratrol. in vitro testing of oecd reference nanomaterials (nm-series) in rat precision cut lung slices aumann a. 1 the oecd has defined reference nanomaterials (nm) to be tested in different endpoints concerning human health and environmental safety (1) in order to evaluate if the toxicity of nanomaterials can be linked to their physico-chemical properties. for nanomaterials, inhalation presents the major exposure route of concern and can be assessed using acute inhalation toxicity studies in rodents. however, these in vivo studies are resource intensive and animal consuming. the oecd working party on nanomaterials has named several alternative methods as being of particular interest for testing of nanomaterials; among them is the precision-cut lung slices model (pcls) to estimate respiratory toxicity. we have tested all 16 nm in pcls measuring cytotoxicity, apoptosis, oxidative stress and inflammatory response of the tissues as well as observing them histological. for in vitro exposure of pcls the test material was dispersed in medium. since it is the nature of these materials to change their surface characteristics and agglomeration state in different environments, a standardized dispersion method (nanocare) using bovine serum albumin as a stabilizing agent, was used. particle size-distributions of the nanomaterial dispersions were characterized via analytical ultracentrifugation and found the nanomaterials well dispersed. silver and zinc oxide but none of the other nm showed cytotoxicity to the lung tissue in the tested concentrations. however, differences in cytokine profiles among the nm were observed and showed several correlations to the results obtained in in vivo inhalation or instillation studies. universität des saarlandes institut für molekulare zellbiologie, gebäude 61, 66421 homburg, germany tmem2 proteins show similarities in their primary sequence to motifs that are conserved amongst various members of the trp protein family. based on hydropathy analysis these proteins exhibit 6 to 10 membrane spanning domains. in contrast to trp channels there is no evidence that these proteins form ion channels in the plasma membrane following overexpression of their cdna in hek293 cells. tmem2 -/mice are viable and show no obvious signs of disease, but exhibit increased pancreatic amylase and lipase plasma levels. microfluorimetric measurements using fura-2 revealed that the elevation of the cytosolic ca 2+ concentration after stimulation with carbachol and the cholecystokinin analogue caerulein is unchanged in tmem2-deficient acinar cells. tmem2 is expressed in several cell types including pancreatic acinar cells, cardiac myocytes, cardiac fibroblasts, but their subcellular localization is still unkown. we generated several constructs encoding tmem2 fusion proteins with fluorescence protein tags by fusing eyfp, mcherry and tagrfp-t to the n-and c-terminus of the protein, respectively. based on western blot experiments and expression in hek293 cells the tmem2-eyfp construct was most suitable for further colocalisation analysis and generation of viral vectors including adenovirus and semliki forrest virus. in contrast to the prediction by the psort ii algorithm tmem2-eyfp could not yet be identified in the plasma membrane of fibroblasts, cardiac myocytes or acinar cells but showed a vesicular subcellular localization pattern. we localized tmem2-eyfp in acidic compartments and predominantly in lysosomes (pearson coefficient (pcc) 0,79 ± 0,03, n= 4 using lysotracker ® dye). analysis of subcellular localization with independent tmem2 fusion constructs and additional vesicular markers will be presented as a framework to get insights towards the cellular function of tmem2 and to reveal the mechanisms underlying increased amylase release from acinar cells of tmem2 -/mice. the small molecule bcl-2/mcl-1 inhibitor tw-37 shows single-agent cytotoxicity in neuroblastoma cell lines bachmann h. s. 1 , akdeli n. high-risk neuroblastoma (nb) remains a therapeutic challenge in paediatric oncology. pro-survival bcl-2 family proteins critically regulate apoptosis, and may represent important therapeutic targets in nb. primary nb tumours heterogeneously express mcl-1 or bcl-2, with high expression correlating to high risk phenotype. co-expression can be detected in approximately 10% and is correlated to reduced survival. recent studies with two inhibitors that predominantly target bcl-2 and other proteins, but not or to a lesser extend mcl-1, elucidated the importance of mcl-1 inhibition for cytotoxicity in nb. tw-37 is a small molecule inhibitor that showed almost equal affinity to bcl-2 and mcl-1. to explore the effect of combined bcl-2/mcl-1 inhibition on neuroblastoma cells, four cell lines (sk-n-as, imr-5, sy5y and kelly) were treated with tw-37 and changes in growth properties were determined. furthermore, nude mice with kelly (human neuroblastoma cell line) xenografts were treated with tw-37. using sirna, we investigated the functional relevance of mcl-1 and bcl-2 in kelly cells. for in vitro cell viability we observed ic50 values of 0.59 ± 0.39 µmol/l. on treatment with 1 µmol/l dose of tw-37, all neuroblastoma cell lines analyzed showed significantly reduced proliferation and increased apoptosis rates. bcl-2 as well as mcl-1 knockdown induced apoptosis in kelly cells. interestingly, tw-37 was able to reduce, but not to abrogate growth of kelly neuroblastoma xenografts in nude mice. in conclusion, combined inhibition of bcl-2 and mcl-1 using tw-37 exhibits strong single-agent antitumor activity on human neuroblastoma cells in vitro, but limited single-agent activity in vivo. therefore, inhibition of bcl-2/mcl-1 may represent an interesting therapeutic strategy, most likely in combination with conventional chemotherapy and other specific inhibitors. localization and functional characterization of membrane transporters for sulfated steroid hormones in the human testis bakhaus k. 1 , wapelhorst b. circulating sulfated steroid hormones like estrone sulfate (e1s) or dehydroepiandrosterone sulfate (dheas) are delivered to the testis via membrane uptake carriers such as the sodium-dependent organic anion transporter (soat). inside the cell these sulfated steroids can be metabolized to active steroid hormones by the catalytic activity of the steroid sulfatase (sts), which shows high enzymatic activity in the testis ("sulfatase pathway"). in addition to soat, other candidate carriers like the organic solute carrier protein 1 (oscp1) and the organic anion transporting polypeptides oatp6a1 and oatp1c1 are predominantly expressed in the human testis and demonstrate transport activity for sulfated steroids. we aimed to evaluate the cellular expression of soat and the other steroid sulfate carriers and their co-localization with the steroid sulfatase (sts) in human testis. furthermore we want to perform functional transport studies with the steroid sulfate carriers in stably transfected hek293 cells. we detected soat by rt-pcr and western blot analysis in the human testis. single cell analysis and in situ hybridization revealed pachytene primary spermatocytes to express the soat mrna. soat expression in specimens showing maturation arrest at the level of early round spermatids seems to be severely reduced or absent. sts mrna was detected by rt-pcr in testis homogenates. preliminary immunohistochemical data showed that sts may be expressed in germ cells and interstitial leydig cells. hek293 cells stably expressing the soat carrier protein showed significant transport activity for dheas. this was demonstrated by using a radiolabeled [ 3 the hepato-intestinal induction of the detoxifying enzymes cyp3a4 and cyp3a5 by the xenosensing pregnane x receptor (pxr) constitutes a key adaptive response to oral drugs and dietary xenobiotics. in contrast to cyp3a4, cyp3a5 is additionally expressed in several, mostly steroidogenic organs, which creates potential for induction-driven disturbances of the steroid homeostasis. using cell lines and mice transgenic for a cyp3a5 promoter we demonstrate that the cyp3a5 expression in these organs is noninducible and independent from pxr. instead, it is enabled by the loss of a suppressing yin yang 1 (yy1)-binding site from the cyp3a5 promoter which occurred in haplorrhine primates. this yy1 site is conserved in cyp3a4, but its inhibitory effect can be offset by pxr acting on response elements such as xrem. taken together, the loss of yy1 binding site from promoters of the cyp3a5 gene lineage during primate evolution may have enabled the utilization of cyp3a5 both in the adaptive hepato-intestinal response to xenobiotics and as a constitutively expressed gene in other organs. our results thus constitute a first description of uncoupling induction from constitutive expression for a major detoxifying enzyme. they also suggest an explanation for the considerable tissue expression differences between cyp3a5 and cyp3a4. serum albumin adducts as biomarkers for systemic bioavailability of active metabolites of various glucosinolates in animal models and humans barknowitz g. 1 , engst w. glucosinolates (gls) are natural pesticides of brassicales, which comprise many important food and feed plants. upon physical damage to the plant, the enzyme myrosinase can convert gls to reactive metabolites (e.g. isothiocyanates). the same reaction can also be catalyzed by enzymes of the intestinal microbiota. modification of sensor proteins (e.g. keap-1) by some gls metabolites leads to adaptive responses, resulting in enhanced detoxification of reactive metabolites and other protective reactions. at least in experimental models, this mechanism can be exploited for chemoprevention of carcinogenesis induced by various chemical carcinogens. however, own research revealed that certain reactive gls metabolites can covalently bind to dna in vitro and in vivo, involving possible genotoxic and carcinogenic risks. animal models are useful for studying beneficial and adverse effects of gls. however, it has to be taken into account that the toxicokinetics of gls may differ between rodent and humans and that the active metabolites are short lived and thus difficult to detect and quantify. likewise, exposure of humans to gls and their breakdown products may enormously vary depending on (i) food preferences, (ii) cultivars, growth conditions and preparation of plants consumed and (iii) variations in human xenobiotic metabolizing system and composition of intestinal microbiota. in order to estimate individual levels of systemic exposure to reactive gls metabolites, blood protein adducts may be useful. we have developed lc-ms/ms methods for quantifying serum albumin adducts formed by glucoraphanin, glucotropaeolin and neoglucobrassicin. the method involves digestion of the protein to amino acids and the usage of isotope-labelled amino acid adducts as internal standards. serum albumin adducts were detected in mouse models after feeding broccoli and pak choi respectively, as well as after administration of purified gls and breakdown products. likewise, gls adducts were detected in human blood plasma after consumption of broccoli or cress. this work was financially supported by the bundesministerium für bildung und forschung (grant 0315370d) . barlow s. harrington house, 8 harrington road, brighton, bn1 6re, great britain values for cancer endpoints for the application of the ttc approach have been derived by linear extrapolation of results from animal carcinogenicity studies to calculate "virtually safe doses" (vsds). a vsd is defined as an exposure that represents an estimated upper bound increase in risk of 1 in a million of developing cancer during a lifetime. in 1995, from consideration of a range of vsds for carcinogens, the us food and drug administration (fda) proposed and adopted a value of 0.5 micrograms/kg of diet (0.5 ppb) as a threshold of regulation to be used for substances present in food contact materials. the fda considered that if exposure to a substance in the diet was below this value, consumers would be protected "with reasonable certainty of no harm" and no toxicological data on the substance need be submitted. the value of 0.5 ppb is equivalent to 1.5 micrograms/person per day, assuming that 1500 g of food and 1500 g of fluids diet might be consumed daily. this value was subsequently incorporated into the ttc approach to be used for assessment of substances without a structural alert for genotoxicity. in 2004, kroes and colleagues further explored cancer as an endpoint and recommended a lower ttc value of 0.15 micrograms/person per day for substances with a structural alert for genotoxicity and exclusion from the ttc approach of certain groups of high potency carcinogens (with vsds below this value). in this presentation, the data underpinning these ttc values will be discussed from the perspective of their reliability for risk assessment of substances with low exposures, for which there are no toxicity data, but which may in fact be genotoxic or non-genotoxic carcinogens. stable conjugates which are recognized by the immune system. in the current investigations, the ability of chemical pre-treatment to interfere with antibody-protein binding has been investigated using ovalbumin (ova) as the model protein and naturally occurring anti-ova igg from healthy human donors. preparation of conjugates: ova (1 mg/ml) was dissolved in sodium borate buffer (0.1m, ph 9.4) . for chemical treatment various amounts (50-200 mg) of 1-fluoro-2,4dinitrobenzene (dnfb; sensitizer) or 2,4-dichloro-1-nitrobenzene(dcnb; non-sensitizer) was added and stirred for 2 hrs at room temperature. unbound compound was removed by consecutive dialysis against phosphate buffered saline (pbs) and distilled water. inhibition elisa: plates were coated with 100 µg/ml ova and blocked with 10% fcs in pbs. ova samples (native or conjugated; 0.6 -10 µg/ml) were pre-incubated with polyclonal antibodies (ab) from pooled normal human serum for 30min and then added to the plates. human anti-ova igg abs were detected by colorimetric analysis using ortho-phenylendiamine as a substrate. the concentration of soluble native ova or conjugate required to displace 50% of ab to plate-bound ova (ic50) was calculated (minimum of n=3 independent experiments). treatment of ova with the chemical sensitizer and protein reactive dnfb resulted in increased ic50 value, whereas mock treatment resulted in comparable ic50 values to native ova. treatment with dcnb showed that the presence of a chemical per se (and possible denaturation) was not sufficient to alter the ic50 value; conjugation of the compound to the protein was required. the analysis is not test chemical specific (specific ab against compound-protein conjugates are not required) and the colorimetric analysis is unaffected by absorbance of the compound itself. thus, this method may have utility for the identification of chemical sensitizers which are directly protein reactive. 2´-deoxy-camp in human cell lines: another second messenger? beckert c. 1 , hinz c. we have shown that 2´-deoxy-camp (dcamp) is synthesized by recombinant human soluble adenylyl cyclase (sac). here, we report that dcamp can be detected and quantified by liquid chromatography coupled to mass spectrometry (lc-ms) in various human cell lines. in most cells a ratio of camp : dcamp of ~10 was observed. as a remarkable exception, in hl-60 promyelocytic leukemia cells, the dcamp concentration exceeded the camp concentration more than 3-fold. a differential regulation of camp versus dcamp was determined upon replacement of the incubation medium (proliferating condition with serum / serum-free resting condition). for example, camp was dramatically reduced in hek293 cells after 24 hours under resting conditions whereas dcamp was significantly increased. in cellular subfractions of hek293 cells ac assays (mn 2+ /forskolin-or mn 2+ /bicarbonate-stimulated) with either atp or datp as substrate revealed that comparable amounts of camp and dcamp accumulated. in addition to sac, membranous acs such as ac v were capable of forming dcamp with vmax and km values for datp comparable to those for atp. we also analyzed the substrate-specificity for several human phosphodiesterases. pde3 and pde4 hydrolyzed dcamp more effectively than camp. taken together, these data point to a putative second messenger role of dcamp in human cells. we are currently investigating the regulatory role(s) of camp and dcamp in apoptosis of hek293 cells. as a novel tool for these studies, we will use the cellpermeant dcamp-acetoxymethylester which penetrates the plasma membrane and releases dcamp intracellularly. the biogenic amine histamine is recognized by target cells via four different histamine receptors subtypes (h1r -h4r), which all belong to the family of g-protein-coupled seven-transmembrane receptors. histamine plays a crucial role in allergic reactions such as rhinitis or conjunctivitis and also in allergic asthma. previously, we showed an interaction of the effects of antagonists at the h1r and h4r in a mouse model of allergic asthma. however, not much is known about the signaling pathway activated by murine h1r and h4r. in order to analyze these signaling pathways, we established a cellular model using transfected hek 293 cells which stably express recombinant mh1r or mh4r. proper expression of the receptors was verified by western blot analysis and flow cytometry. in functional assays we demonstrated that histamine stimulation results in the increase of intracellular ca 2+ concentration ([ca 2+ ]i) in cells expressing either of both, mh1r (pec50 = 8.2) or mh4r (pec50 = 6.9). as a second readout, we analyzed the modulation of forskolin-induced camp-accumulation. in mh1r-expressing cells the intracellular camp concentration was increased by stimulation with histamine, while in mh4r-expressing cells forskolin-induced camp accumulation was reduced. the histamine-induced effects in h1r-expressing cells were blocked by the h1r antagonist mepyramine ([ca 2+ ]i: pkb = 8.6) and those in the h4r-expressing cells by the h4r antagonist jnj7777120 ([ca 2+ ]i: pkb = 8.8) or by pertussis toxin, which selectively blocks receptor gi-protein coupling. jnj7777120, which behaves as a partial mh4r agonist in the steady-state gtpase assay using membranes of infected sf9 cells, was without effect on [ca 2+ ]i and forskolin-induced camp-accumulation in the mh4r-expressing cells. currently, we are investigating mitogen-activated protein (map)-kinase pathways activated by the h1r and the h4r. using a phospho-map-kinase array, histamine dependent phosphorylation of erk1/2, p38, jnk, creb, pkb (akt), and mkk 3/6 were detected in cells expressing either of both, mh1r and mh4r. in summary, the hek 293 cell lines stably expressing selective histamine receptors are very useful tools to investigate hxr signaling pathways in-vitro. enhanced fibroblast motility in the absence of the β3 regulatory subunit of voltage-activated calcium channels belkacemi a. 1 cavβ subunits of voltage-activated ca 2+ channels are required for trafficking the poreforming cavα1 subunit to the plasma membrane and modulate the kinetics of its current. mouse embryonic fibroblasts (mefs), acutely isolated cardiac fibroblasts (cfs) and nih 3t3 fibroblasts do express cavβ2 and cavβ3 subunits, but we could not detect any voltage-activated ca 2+ influx. whereas in mouse cardiomyocytes or hek 293 cells coexpressing cavβ3 and cavα1 subunits a dihydropyridin-sensitive voltage-activated ca 2+ influx was readily detectable. apparently, cavβ subunits serve functions in fibroblasts unrelated to voltage-activated ca 2+ influx. among the proteins potentially interacting with cavβ3 are the inositol 1,4,5-trisphosphate receptors (ip3rs) [1, 2] . we therefore coexpressed mouse cavβ3 and mouse ip3r type 1, 2 or 3 in cos-7 cells and found coimmunoprecipitation of ip3rs using an antibody for cavβ3 and vice versa. to study the release of ca 2+ from ip3-sensitive stores we performed fura-2 measurements on fibroblasts isolated from wild type and cavβ3-deficient mice either in the presence of thapsigargin or after stimulation of gq-coupled receptors by par-1, lpa or bradykinin. receptor-activated ca 2+ release was more pronounced in β3-deficient mefs and cfs, whereas thapsigargin-induced ca 2+ release was the same in cells from both genotypes. in addition, ip3 production measured by a radioreceptor assay was already increased in β3-deficient cells under basal conditions. fibroblasts are migrating cells and involved in various physiological and pathophysiological processes. we therefore started in vitro assays for proliferation, migration and angiogenesis as well as in vivo assays for skin wound healing. angiogenesis and proliferation were apparently not different in both genotypes but migration (measured as transwell migration and in scratch assays) and wound healing were affected in different ways. fluorescent staining of cytoskeleton and quantification of the f-actin/g-actin ratio show similar results in both genotypes, suggesting that the increased migration rates and wound repair in β3 knockout may result, in part, from the increased amount of ip3-releasable ca 2+ . [1] berggren, yang, murakami, et al., removal of ca channel β3 subunit enhances caoscillation frequency and insulin exocytosis. cell 119, (2004) 273-284. [2] müller, haupt, bildl, et al., quantitative proteomics of the cav2 channel nanoenvironments in the mammalian brain. pnas 107, (2010) 14950-14957. bender-sigel j., closs e. i. universitätsmedizin mainz institut für pharmakologie, obere zahlbacher straße 67, 55101 mainz, germany human cationic amino acid transporters (hcat) are a family of multimembrane spanning proteins that mediate the transport of cationic amino acids through the plasma membrane. our earlier results have demonstrated that activation of either protein kinase c (pkc) by pma or cdc42 by egf leads to an internalization of these transporters. in addition, in a recent collaboration with the group of alexander sorkin (university of colorado denver) we found that ubiquitination and clathrin-dependent endocytosis are necessary for the down regulation of hcat-1-mediated arginine transport by pma (vina-vilaseca et al, j biol chem 2011 286:8697) . this mechanism requires nedd4 e3 ligases, but hcats do not contain a ppxy motif to bind the ligases, suggesting that an adaptor protein takes part in this process. however, an involvement of the adaptor protein beta-arrestin in this mechanism could be excluded. using sirna against pkc alpha we now show that pkc alpha is the major isoform that induces the reduction of arginine transport in human u373 glioblastoma cells overexpressing hcat-2a-egfp. in addition, sirna-mediated knock down of cdc42 prevented the decrease of hcat-2amediated transport induced by pma. taken together pkc seems to negatively influence the constitutive cycling of cats by activation the ubiquitination machinery and clathrinmediated endocytosis. cdc42 is part of this pathway. converging of the classical mitochondria-related pathway in parkinson and nuclear dna-repair signaling? scherr a. -l. 1 parkinson disease is the second most neurological disorder worldwide. despite the fact that most cases are idiopathic and only few can be traced back to specific genes, general progression between both tracks of the disease is comparable. the variety of clinical symptoms in motor control like tremor, rigor and postural problems all originate from loss of dopaminergic neurons in the substantia nigra pars compacta of the brain. several proteins mutated in pd are involved in surveillance pathways, monitoring functionality and integrity of proteins and organelles either by the proteasome degradation machinery or by clearance of mitochondria via autophagy (mitophagy). disturbed calcium-and redox-homeostasis seems to play a major role in susceptibility to cell death signals in dopaminergic neurons, but if this is a preceding or successive event in cell death related to pd progression is not known. on the other hand, experimentally elicited parkinsonism by oxidative stress inducers like paraquat or rotenone and mptp (inhibitors of mitochondrial complex i) lead to damage in nuclear dna and activation of the dna repair protein poly(adp-ribose) polymerase 1. by consuming substantial amounts of its substrate nad + , this enzyme can drastically decrease energy levels and disturb the redox balance within a cell, also sensitizing it to stress induced cell death. inhibition of poly(adp-ribose) polymerase 1 has been proven to be beneficial to some extent in cell culture models as well as in experimental pd in mice. our research focuses on a putative crosstalk mechanism we recently discovered between the two pathways of experimentally induced cell death in culture models, i.e. mitochondrial signaling and parp1-dependent poly(adp-ribosyl)ation. both converge on two mitochondrial chaperones, mortalin and trap1. whereas mutations in mortalin have been reported recently to be responsible for some parkinson disease cases in humans, trap1 is a specific target of the kinase pink1 (pten induced putative kinase), which is often mutated in autosomal-recessive forms of the disorder. pink1 is a central regulator of the mitophagy process, tagging mitochondria with dissipated membrane potential for destruction. we could show now that both chaperones bind to short-chain poly(adp-ribose) specifically synthesized by poly(adp-ribose) polymerase 1. we will present our most recent findings about regulation of these two chaperones after application of parkinson-inducing toxins. aldrich). the effect was reversible within ten minutes when cells were re-incubated in regular cell culture medium. stimulatory effects were not due to osmolarity or cell stress due to medium exchange. analysis of different components of both media (table 1) revealed that bicarbonate stimulates accumulation of ccmp and cump besides cgmp and camp in a time-and concentration-dependent manner. bicarbonate is known to activate soluble adenylyl cyclase (sac) and particulate guanylyl cyclase g (pgc-g), regulating sugar metabolism, sperm motility and olfaction by synthesis of camp and cgmp, respectively. in order to identify a responsible cyclase for ccmp and cump generation after bicarbonate treatment, we are currently analyzing transiently and stably transfected hek293 cells overexpressing various known adenylyl and guanylyl cyclases (sac, mac1, mac3, mac9, soluble guanylyl cyclase, pgc-g, pgc-a, and pgc-d) for their pyrimidinyl and purinyl cyclase activity in vivo and their regulation by bicarbonate. in addition, cell fractions will be analyzed for the detection of specific cyclase compartments. question: long term ventricular pacing, especially at the right ventricle (rv), results in left ventricular (lv) failure. there are several lines of evidence that disturbed ca 2+ homeostasis is involved in the pathophysiology of human heart failure. in this study we examined if ventricular pacing affects the na + -and ca 2+ -channels and the expression of ca 2+ -handling proteins and investigated if there is a differential effect between right ventricular free wall (rvfw) pacing and left ventricular apex (lva) pacing. methods: after av-node ablation 14 minipigs underwent ventricular pacing at 120 beats/min (ddd mode) for one year. 7 minipigs were paced from the rvfw and 7 minipigs from the lva, respectively. 7 minipigs with normal sinus-rhythm served as control group. patch-clamp-experiments were studied to measure na + -and ca 2+ currents. western-blots were carried out to investigate the expression of the ca 2+handling proteins l-type ca 2+ -channel, serca2 and phospholamban. results: both rvfw-and lva-pacing led to significant decreased ca 2+ -currentdensities in cardiomyocytes of the lv compared to the control group. the plateau phase of the action potential was significantly shortened after ventricular pacing in relation to control minipigs. furthermore cardiomyocytes of rvfw-and lva-paced minipigs had significant lower na + -current-densities than control minipigs. the action potential amplitude was significantly decreased after rvfw-and lva-pacing whereas the diastolic potential remained unchanged. the expression of the l-type ca 2+ -channel was significantly reduced after ventricular pacing, regardless of the pacing site. in contrast rvfw-and lva-paced minipigs showed significant increased serca2-expression. the expression of phospholamban remained unchanged after rvfw-and lva-pacing compared to control minipigs. conclusion: in a chronic animal model ventricular pacing leads to remodeling of ionchannels and ca 2+ -handling-protein-expression, regardless of the pacing site. investigation on metabolic competence of dermal systems: native human skin, in vitro skin models and keratinocytes blatz v. 1 the implementation of reconstructed human skin equivalents (rhes) as an alternative method for dermal toxicity testing became very prominent in the last decades. their advantages are e. g. the human cell origin and an organ-like 3d structure. already regulatory accepted methodologies are widely in use for testing the skin corrosion (oecd 431) and irritation (oecd 439) within rhes. but there are still some questions open, one of them the metabolic competence of such dermal systems. in this context, enzyme activities of oxidizing (cyp; fmo; adh; aldh) and conjugating enzymes (nat; ugt) were investigated in subcellular fractions of in vitro systems such as keratinocytes and rhes (epidermis model epiderm tm (mattek), full-thickness skin models epiderm tm ft (mattek) and phenion ® ft (henkel ag)) and compared to those of native human skin. activities of cyp 1a, 2b and 3a isoenzymes were measured fluorometrically by oxidative desalkylation of alkoxyresorufines. fmo 1/3 activities were evaluated by hplc/fld detection of n-oxygenated product of benzydamine [1] . adh and aldh activities were investigated by photometrical detection of nadh generation during ethanol (adh) [2] or propanal (aldh) oxidation [3] . nat1 activity was followed by hplc/uv detection of acetylated p-aminobenzoic acid. ugt1 activity was quantified fluorimetrically by glucuronidation of methylumbelliferone [1] . during the course of this study the following results were observed: (loq = limit of quantification) since the metabolic competence of rhes is confirmed, these in vitro systems are estimated as suitable for further toxicity tests (e. g. genotoxicity by comet assay), where metabolic activation of substances may play a crucial role. however, for the data assessment, the determined metabolic profiles should be taken into account. we acknowledge bmbf funding this project (0315226d). signalling pathway indicates that in b104 cells the adenine receptor couples to a gqprotein followed by activation of phospholipase c pathway. these findings represent a new signalling pathway of the adenine receptor and allow the assumption that different adenine receptor subtypes exist in the rat brain. in the scope of the project lexukon ("foodborne exposure to environmental contaminants -data analysis to support and standardise exposure assessments based on nvs ii") exposure to the heavy metals cadmium (cd), lead (pb) and mercury (hg) via food consumption has been assessed for the german adult population based on the national nutrition study ii ( the updated intake assessments show that especially foods regularly consumed such as vegetables and grain contribute mainly to exposure of cd that is about 1.5 µg/kg body weight (bw) per week for average consumers over all food groups. this corresponds to 58% of the tolerable weekly intake (twi) of 2.5 µg/kg bw for cd defined by the european food safety authority (efsa) in 2009. beverages and vegetables are the food groups most relevant for exposure to pb. about 3.7 µg pb/kg bw is taken up by average consumers that is below the benchmark dose for renal toxic effects (4.41 µg/kg bw) defined by efsa for the weekly pb intake. for hg the intake amounts for all population groups examined were significantly below the toxicological reference values. for average consumers the weekly intake of hg is 0.49 µg/kg bw that is primarily taken up by eating fish and fishery products. however, individual population groups and high consumers reach and/or exceed the toxicological reference values for the daily intake amounts for cd and pb. high consumers almost reach the twi for the cd with 94%. for pb a weekly intake of 5 µg/kg bw was estimated for high consumers that exceeds the benchmark dose for renal toxic effects for the weekly pb intake. the results show that data collection should also focus on highly consumed and not only on highly contaminated foods. further, uncertainties in concentration levels should be reduced e.g. by lowering and standardizing the analytical limits. it's recommended to consider further measures in view of the reduction of contents of environmental contaminants in foods. however, other sources can also contribute to the intake of the mentioned heavy metals (e.g. smoking). major cell biological processes are regulated by rho-gtpases, actin-mediated processes in particular. amongst others, rho-gtpases are stimulated by the receptormediated activation of gα12/13 and gαq via specific rhogefs. the p63rhogef is activated by gαq and plays a major role in the acute response of vascular smooth muscle cells to angiotensin ii treatment. the aim of the present study was to establish a fret-assay between gαq-cfp and venus-p63rhogef and characterize the dynamics of p63rhogef-gαq-interaction in single living cells. the fusion of p63rhogef with venus resulted in a functional gαq-regulated p63rhogef-protein as determined by means of rho-luziferase-assays. whereas no specific fret signal was observed between the two interaction partners in the absence of receptor stimulation, a robust and rapid fret signal developed in response to stimulation of histaminergic h1and cholinergic m3-recptors. the onset of this signal after rapid application of agonist paralleled gαq activation kinetics. similar to the kinetics of gαq-protein deactivation the dissociation of p63rhogef and gαq after withdrawal of agonist was slow (tens of seconds). the specificity of the fret signal between gαq-cfp and venus-p63rhogef was verified by introducing point mutations rendering p63rhogef unable to bind to active gαq. furtehrmore we observed a robust acceleration of the dissociation of p63rhogef and gαq upon cotransfection of rgs2, suggesting a very short lifetime of the p63rhogef-gαq-complex or the ability of rgs2 to bind to p63rhogef-associated gαq. taken together, fret-based imaging of the interactions between p63rhogef and gαq revealed fast interaction kinetics closely resembling g-protein activation kinetics, both of which can be regulated by rgs2. toxicity of silver nanoparticles in intestinal cells boehmert l. 1 the rapid development of nanotechnology has been accompanied by an increased concern for the safety of nanomaterials. especially silver nanoparticles are used in many manufacturer identified consumer products including silver coated food contact materials or hydrosol silver supplements. these products lead to an intentional or unintentional oral uptake of silver nanoparticles and hence to a contact with the intestinal barrier. the human cell line caco-2 is a well established model system in studying effects on human enterocytes. although these cells are colon carcinoma cells and exhibit typical features of cancer cells when they are kept sub confluent, these cells have the capability to differentiate into polarized cells with morphological and biochemical properties of small enterocyte cells. we investigated the effects of silver nanoparticles on these colon carcinoma (proliferating) and small intestinal epithelium like (differentiated) caco-2 cells. the silver nanoparticles agpure were commercially available from rent-a-scientist gmbh. the behaviour of these silver nanoparticles in cell culture medium were characterised using asymmetric flow-fild flow fractionation (a4f), small ankle x-ray scattering (saxs) and dynamic light scattering (dls). we investigated the particle toxicity on both cell states using cell titer blue assay, xcelligence impedance measurements, annexin-v and caspase measurements, diclorofluorescein assay and antioxidant pre incubated cells. the agpure stock solution is an aqueous suspension of silver particles with a metal core radius of 7.2 nm stabilised with tween-20 und polyoxyethylenglycerol trioleate. agpure silver nanoparticles were toxic for proliferating as well as differentiated caco-2 cells in a time and concentration depending manner. the presence of foetal calf serum in the incubation medium has a minor influence on the toxicity. prior to cell death, morphological abnormal adherence characteristics and morphological changes in the cells were observed using microscopy and quantified by xcelligence impedance measurement. it is concluded that cell death is caused by an oxidative stress related mechanism rather than apoptosis. the release of sphingosine-1-phosphate from human platelets during acute coronary syndrome is attenuated by aspirin böhm a. 1 , polzin a. in addition to atherosclerosis ttp knock out mice develop more cardiovascular dysfunctions. in tail vein bleeding assays we monitored a significant difference in the bleeding times of ttp deficient mice in comparison to wildtype mice, triggered by a stronger granulopoeisis. our results leed us to the assumption that the chonic inflammation seems to be more improtant for the development of cardiovascular diseases in ra patients than the traditional risk factors. differentially expressed cardiac genes in a mouse model with heart-specific overexpression of pp2a bollmann p., makarova e. a., gergs u., neumann j. institute for pharmacology and toxicology medical faculty, magdeburger str. 4, 06112 halle (saale) , germany in transgenic (tg) mice with cardiac myocyte-specific overexpression of the catalytic subunit of protein phosphatase 2a (pp2a) reduced cardiac protein phosphorylation, cardiac hypertrophy and impaired cardiac contractility were noted compared to wild type (wt) littermates. the hearts of tg mice also suffered from ventricular dilatation and a diminished response to β-adrenergic stimulation. analyses of mrnas expressed in tg and wt hearts (n=3) using affimetrix mouse genome microarray chips resulted in several candidate genes possibly differentially regulated. in this study, we focussed on verifying the mrna data of selected genes important for stress response and signal transduction on protein level in cardiac homogenates by western blotting. hearts from wt littermates were used as control. compared to wt heat shock protein 25 (hsp25) and calcium calmodulin dependent protein kinase type ii (camkii) mrnas were upregulated in tg but only hsp25 protein was increased (p<0.05, n=7-8) but not camkii (p>0.05). protein phosphatase type 5 (pp5) and superoxide dismutase (sod) were downregulated on mrna level in tg but on protein level this could be found only for sod (p<0.05, . in contrast, pp5 protein was upregulated (p<0.05, in tg compared to wt. for comparison the regulatory a-subunit of pp2a and hsp90 were studied. both genes were unchanged on mrna level in tg: western blotting revealed the same results for the corresponding proteins. in summary, mrna expression data could only partially be confirmed on protein level elucidating the importance of western blotting studies. these data indicate that increased pp2a activity is associated with modified gene expression in tg hearts possibly affecting stress response and regulation of cell signalling. (supported by the deutsche forschungsgemeinschaft) center for regenerative therapies, technische universtität, dresden, germany cell therapy in the form of beta cell replacement to cure diabetes has been practiced for decades without become a routine clinical therapy. more widespread clinical application is hindered by the scarcity of suitable organ donors, a dramatic loss of transplanted cells within the first days post-transplant, the requirement of long term immunosuppression to maintain graft survival, and despite this, a loss of graft function from a recurrence of autoimmunity in some patients. research is currently dedicated to overcome each of these limitations. additional beta cell sources investigated include embryonic stem cell derived insulin producing cells, human insulin producing cells lines, and xenogeneic beta cells. parallel to these efforts are the development of encapsulation devices to protect these sources from immune and inflammation mediated destruction, and transplantation into new sites such as the muscle and bone marrow to infuse beta cells. additional therapy to reduce immune suppression includes the infusion of t regulatory cells to control autoimmune and alloimmune response, and cytokine and chemokine receptor directed compounds aimed at blocking early inflammation or autoimmunity. these efforts are likely to lead to an expansion of clinical activity to replace beta cells in diabetes, and to novel pharmaceutical therapies that may be more generally applicable in patients with diabetes. pdgf-bb induces the h2s producing enzyme cystathionine-γ-lyase via a rosdependent mechanism in rat renal mesangial cells boosen m. 1 , hassan m. there is increasing evidence that hydrogen sulfide (h2s) that is endogenously produced in several cell types serves as a potent gasotransmitter in a wide variety of physiological processes involving vascular homeostasis and inflammation. in the present study we investigate the expression and the regulation of the hydrogen sulfide synthesizing enzyme cystathionine γ-lyase (cse) in cultured rat renal mesangial cells. as demonstrated by qpcr and western blot experiments, mesangial cells showed a marked time-and dose-dependent upregulation of cse mrna and protein levels after treatment with platelet-derived growth factor (pdgf-bb). the cse upregulation by pdgf-bb is accompanied by a marked increase in reactive oxygen species (ros) formation. interestingly, co-administration of the ros scavenger n-acetylcysteine, the glutathione peroxidase mimetic ebselen and the nadph oxidase inhibitor diphenylen iodonium chloride (dpi) drastically reduced pdgf-induced cse expression, indicating a role for endogenously produced ros in mediating regulation of cse. as demonstrated by electrophoretic mobility shift (emsa) experiments pdgf-bb induces binding of the redox-sensitive transcription factor nf-e2-related factor 2 (nrf2) to a consensus antioxidant response element and this effect was also diminished by co-administration of antioxidants (dpi, nac, ebselen) . furthermore, lps/ifnγ-as well as pdgf-bb-induced cse upregulation was nearly completely abolished in nrf2 -/spleen macrophages and mesangial cells, respectively. as a consequence of the elevated cse levels we could demonstrate increased h2s levels and a higher cse enzyme activity in mesangial cells after stimulation with pdgf-bb by using the colorimetric methylenblue method and a cse activity assay. importantly, in a rat model of anti-thy-1-induced proliferative glomerulonephritis we observed a marked upregulation of cse protein during the course of the disease paralleled by a stabilization of nrf2 protein. from our data, we hypothesize that pdgf-bb-mediated regulation of cse via a redox-mediated activation of nrf2 may constitute a protective mechanism during glomerular inflammatory disease. rac1 knockout protects from acute hepatic damage following doxorubicin treatment bopp a., wartlick f., fritz g. heinrich-heine-universität institut für toxikologie, universitätsstr. 1, 40225 düsseldorf, germany rac1 belongs to the best characterized members of the ras-homologous (rho) family of small gtpases, which are key regulators of the actin cytoskeleton. furthermore, rac1 is part of the activation of the nadph oxidase, which produces reactive oxygen species and regulates the activity of stress kinases (e.g. sapk/jnk) and transcription factors such as nf-κb and ap1. anticancer drugs cause dna damage, which in turn stimulates the dna damage response (ddr) regulating dna repair, cell cycle progression and, in case of non-repairable dna damage, triggers apoptosis. so far, a role of rac1 in the ddr has not been reported. based on its exceptional function as a regulator of transcription and because of its recently found ability to translocate to the nucleus, we hypothesize that rac1 may be involved in the ddr. to study the in vivo function of rac1 we used an inducible cre-based knockout mouse model (rac1 flox/flox/mxcre ). mice were treated with different doses of doxorubicin for different periods of time. we monitored gh2ax foci formation as a marker of dna strand breaks, used the masson-goldner staining for the detection of collagen accumulation, analyzed phosphorylated histone 3 as a marker of mitotic events and performed a tunel assay to detect apoptotic cells. in the absence of rac1 the basal mrna expression of pro-fibrotic ctgf was decreased. collagen levels were increased and mmp1 mrna expression was reduced in the liver of rac -/animals as compared with rac1 proficient animals. in addition we found more apoptotic cells in rac1 -/mice. 96 hours after treatment with the anthracycline derivative doxorubicin the number of gh2ax foci in rac1 -/animals was reduced in comparison to rac1 +/+ animals. we also found lower level of ctgf mrna expression and reduced amount of collagen in rac1 -/mice. none of these protective effects resulting from rac1 deficiency could be detected after administration of three consecutive doxorubicin injections over a time period of 21 days. there were no significant differences in the number of gh2ax foci or collagen accumulation. the mrna expression of ctgf was even higher in rac1 -/animals. furthermore the number of mitotic events was almost two times higher in the rac1 -/mice compared to the rac1 +/+ mice. summarizing, our findings show that impaired hepatic expression of rac1 protein is hepatoprotective against acute damage following doxorubicin exposure, but does not protect against doxorubicin-induced subacute toxicity. in vitro cytotoxicity of tbhq (tert-butyl-hydroquinone) braeuning a., vetter s., schwarz m. institut für experimentelle und klinische pharmakologie und toxikologie toxikologie, wilhelmstrasse 56, 72074 tübingen, germany at high concentrations, tert.-butyl-hydroquinone (tbhq), a phenolic antioxidant frequently used as a food preservative, exerts cytotoxic effects, which are closely linked to its ability to form reactive oxygen species as a consequence of redox cycling processes. here we describe that treatment of murine 3t3 cells with tbhq in 96-well culture plates induces the death of untreated cells in neighboring wells on the same plate. the mechanisms underlying that effect were investigated. death of the seemingly untreated neighboring cells was caused by a more toxic and volatile tbhq oxidation product which was formed in a non-enzymatic process involving metal ions and oxygen. the unexpected perturbation of cytotoxicity testing by the volatile tbhq metabolite shows that not only metabolic processes, but also non-enzymatic mechanisms have to be considered as important parameters for in vitro assays. furthermore, our data show that even cells several wells distant from the site of treatment do not necessarily constitute proper "untreated" controls when cells are treated with tbhq, e.g. in assays aimed to analyze the activity of the tbhq-inducible nrf2 pathway. s14 056 angiotensin ii causes oxidative stress and dna damage in mouse kidneys via the angiotensin ii type 1 receptor brand s. 1 , amann k. 2 , schupp n. 1 1 universität würzburg institut für pharmakologie und toxikologie, versbacherstr. 9, 97078 würzburg, germany 2 universität erlangen-nürnberg institut für pathologie, krankenhausstraße 8, 91054 erlangen, germany angiotensin ii (ang ii), the reactive peptide of the renin-angiotensin-system, causes vasoconstriction and, in higher levels hypertension, which is connected with an increased cancer risk in the kidney. treatment of male c57bl/6 mice with ang ii results in the formation of superoxide radicals and dna damage in the kidney as well as in the heart. to answer the question if the dna damage is caused by hypertension or by elevated ang ii concentrations, mice were treated with different compounds: the angiotensin-converting-enzyme blocker ramipril, the ang ii receptor blocker ramipril, the ang ii receptor candesartan, the antioxidant tempol and the vasodilator hydralazine. the effect on blood pressure and renal function of ang ii-treated c57bl/6 mice was examined. treatment with ang ii led to a significant increase in blood pressure. candesartan and hydralazine led to a decrease, whereas intervention with ramipril and tempol had no effect. equal conditions could be found by examining renal function regarding the excretion of urinary albumin, which was ameliorated by candesartan and hydralazine. in addition, histopathological changes were investigated. there was significant glomerular damage and tubulointerstitial damage in ang ii-treated animals compared to control animals, which was significantly improved by candesartan and tempol. hydralazine and ramipril mitigated the observed renal damage but were less effective than candesartan. furthermore, the ang ii-induced formation of superoxide radicals in the kidney and the heart was slightly affected by all interventions. genomic damage, in the form of double strand breaks was prevented by the ang ii receptor antagonist candesartan and the antioxidant tempol. to sum up, the results from this study show that ang ii induces the elevation of markers of kidney failure and dna damage, which is prevented by substances lowering blood pressure like candesartan, showing the receptor responsibility for the induction of dna damage. actually by substances not lowering blood pressure like tempol, the oxidative stress and dna damage was ameliorated, showing the involvement of reactive oxygen species. optimization of the balb/c-3t3 cell transformation assay by coupling a drug metabolizing system brauneis m. d., steinberg p. stiftung tierärztliche hochschule hannover institut für lebensmitteltoxikologie und chemische analytik, bischofsholer damm 15, 30173 hannover, germany the analysis of the carcinogenic potential of chemicals plays an important role in toxicology. up to now the acquisition of such data requires a large amount of animal experiments. the aim of this study is to reduce the number of experimental animals being used by further optimizing the balb/c-3t3 cell transformation assay, an already well-established in vitro method. this method, which is also well suited for high throughput screening applications, allows a quantitative analysis of the aforementioned carcinogenic potential. the incubation of balb/c-3t3 cells (murine embryonic fibroblasts) with mutagenic compounds leads to a loss of contact inhibition between these cells, which results in the development of so-called foci. these foci can be distinguished by characteristic changes in cell growth behaviour, a result of the treatment with carcinogenic compounds, and their number is therefore directly related to the genotoxic potential of the latter. a major disadvantage of the "classic" balb/c-3t3 cell transformation assay is that a number of compounds initially require a metabolic transformation to gain their full genotoxic potential. hence, without prior metabolic transformation many chemicals are not detected as carcinogenic in the abovementioned test system. to overcome this drawback the balb/c-3t3 cell transformation assay has been coupled to a drug metabolizing system, in this case the so-called liver s9. in a first step the well-known genotoxic agents benzo[a]pyrene, aflatoxin b 1 and nnitrosodimethylamine were tested in this assay. all three compounds led to a concentration-dependent increase in the number of foci formed, whereby this concentration-dependent increase was observed in a non-cytotoxic concentration range. in a next step the balb/c-3t3 cell transformation assay will be coupled to further drug metabolizing systems as well as to the soft agar assay. this study is being financially supported by the stiftung set and the doerenkamp-zbinden foundation. adenylyl cyclases (acs) synthesize the second messenger camp. the family of acs consists of nine membranous and one soluble isoforms with ac5 and ac6 being the predominantly expressed isoforms in the heart. in the heart, acs integrate β-adrenergic (β-ar) signaling as the main physiological mechanism to improve cardiac performance. although ac5 and ac6 share high sequence homology, opposing effects on cardioprotection have been reported, where disruption of ac5, as well as overexpression of ac6 both exerting beneficial effects in heart failure. prospective pharmacological treatment of heart failure on the level of ac is under investigation. our study explored the impact of ac5 ko on ac-activities in the heart at a functional level. complementary, mrna expression studies of the β-ar-g-protein-ac signaling cascade were performed to detect possible compensatory alterations. hearts from 16-20 week old homozygote ac5 knockout and wild-type male littermates were examined in this study. ac activities where measured in cardiac membrane preparations from left ventricles. ac activities were assessed under β-ar and g-protein (g s) stimulation by isoproterenol, guanosine 5'-triphosphate (gtp) and 5'-o-(3thiotriphosphate) (gtpγs) as well as for direct activation by forskolin. relative mrna expressions for ac1-9, gs-, gi-a and β1-, β2-ar where measured by quantitative realtime pcr. surprisingly, assessment of basal, β-ar and g-protein-mediated ac-stimulation as well as direct activation by forskolin revealed no changes in ac activities. besides from detection of the ac5 knockout, mrna expressions analysis of ac1-9, gs-, gi-a and β1-, β2-ar did not detect any compensatory alteration. these findings suggest that proximal adrenergic signaling in the heart does not necessarily require ac5. whether physiological integration of beta adrenergic signaling in the heart is mediated by both isoforms ac5 and ac6, or can be attributed to one main isoform remains to be elucidated. melanocortin-promoted pka activation decreases ampk activity via erk-1/2 and lkb-1 in hypothalamic gt1-7 cells breit a., ellen d., gudermann t. goethestrasse 33, 80336 münchen, germany α-melanocyte stimulating hormone (α-msh)-induced activation of the melanocortin-4 receptor (mc4r) in hypothalamic neurons increases energy expenditure and inhibits food intake. intrahypothalamic injection of melanocortins decreased food intake due to the inhibition of amp-activated protein kinase (ampk) that has recently been reported to enhance food intake in rodents. until now, it is not clear if α-msh affects ampk via direct intracellular signaling cascades or if the release of paracrine factors is involved. herein, we used a murine, hypothalamic cell line (gt1-7 cells) and monitored ampk phosphorylation at thr 172 which has been suggested to increase ampk activity. we found that α-msh dephosphorylated ampk at thr 172 and consequently decreased phosphorylation of the established ampk substrate acetyl-coa-carboxylase at ser 79 . inhibitory effects of α-msh on ampk were blocked by specific inhibitors of protein kinase a (pka) or extracellular-regulated kinases-1/2 (erk-1/2), pointing to an important role of both kinases in this process. since α-msh-induced activation of erk-1/2 was blunted by pka inhibitors, we propose that erk-1/2 serves as a link between pka and ampk in gt1-7 cells. furthermore, down-regulation of liver kinase b-1 (lkb-1), but not inhibition of calcium-calmodulin-dependent kinase kinase-β or transforming growth-factor-beta-activated kinase-1 decreased basal phosphorylation of ampk and its dephosphorylation induced by α-msh. thus, we propose that α-msh inhibits ampk activity via a linear pathway including pka, erk-1/2 and lkb-1 in gt1-7 cells. given the importance of the melanocortin system in the formation of adipositas detailed knowledge about this pathway might help to develop drugs targeting obesity. autism spectrum disorder (asd) is a complex neurodevelopmental disorder with dysfunction of social interaction and communication. a hitherto unknown complexgenetic principle of origin probably underlies asd. so far, more than 100 candidate genes were identified in literature. the patients affected with the monogenic timothy syndrome show multiorgan dysfunction including lethal arrhythmias, immune deficiency, skeleton-dysplasia, syndactylia and autism. this single gene disorder serves as a model disease for asd, giving insights in a possible pathophysiology. here, a point mutation in a highly-conserved region of the pore-forming subunit of the voltage-dependent calcium channel (ca v) cav1.2 gene (cacna1c) results in incomplete inactivation of the l-type calcium currents (splawski et al., cell 2004; 119:19-31) . functionally similar biophysical effects can be induced by structural variation β1-and β2-subunits of the voltagedependent calcium channels (herzig et al., faseb j. 2007; 21:1527-38; jangsangthong et al., pflugers arch. 2010; 459:399-411) . supported by findings in a meta-analysis of linkage data of asd patients (trikalinos et al., mol psychiatry. 2006; 11:29-36) , we are investigating a function-based candidate gene hypothesis linking the β2 subunit gene (cacnb2) with asd. we performed a case control study sequencing all exons and flanking intronic regions of cacnb2 in 155 patients with asd. we found three rare missense mutations in asd patients, but not in 259 unaffected controls. all three mutations occur at highly conserved positions and might alter protein function; additionally results one amino acid substitution highly probable in a post-translational modification by phosphorylation. so far, we characterized two of these mutations and also a phosphorylation-mimicking mutant in electrophysiological studies. all variants show a decelerated and incomplete time-dependent inactivation of the co-transfected cav1.2 subunit. furthermore, two variants exhibit a significant increased slope factor of voltage-dependent steady-state inactivation. we here present mutations in the β2 subunit gene of asd patients that result in a retardation of inactivation behavior, thus phenocopying the monogenic timothy syndrome mutations of cav1.2. β2 subunit mutations may influence neuronal function or development in some asd patients. jangsangthong, w., kuzmenkina, e., khan, i.f., matthes, j., hullin, r., and herzig, s. (2010) . inactivation of l-type calcium channels is determined by the length of the n terminus of mutant beta (1) subunits. pflugers arch 459, 399-411. herzig, s., khan, i.f., grundemann, d., matthes, j., ludwig, a., michels, g., hoppe, u.c., chaudhuri, d., schwartz, a., yue, d.t., et al. (2007) . mechanism of ca(v)1.2 channel modulation by the amino terminus of cardiac beta2-subunits. faseb j 21, 1527-1538. splawski, i., timothy, k.w., sharpe, l.m., decher, n., kumar, p., bloise, r., napolitano, c., schwartz, p.j., joseph, r.m., condouris, k., et al. (2004) . ca(v)1.2 calcium channel dysfunction causes a multisystem disorder including arrhythmia and autism. cell 119, 19-31. trikalinos, t.a., karvouni, a., zintzaras, e., ylisaukko-oja, t., peltonen, l., jarvela, i., and ioannidis, j.p. (2006) . a heterogeneity-based genome search meta-analysis for autism-spectrum disorders. mol psychiatry 11, [29] [30] [31] [32] [33] [34] [35] [36] background: brain serotonin (5-ht) has been implicated in the regulation of food-intake. the ingestive effects of 5-ht are mediated by a number of different receptor subtypes under which the 5-ht1a-receptor plays a central role. former in vivo studies have shown an increased intake of food, elicted by 5-ht-receptor agonists. the aim of this behavioural pharmacologic project was to determine if the hyperphagic effect is mediated by presynaptic 5-ht1a autoreceptors in the raphe nuclei or by postsynaptic 5-ht1a heteroreceptors in serotonergic terminal structures. methods: the effect of the 5-ht1a receptor agonist 8-oh-dpat (0.1, 0.5 or 1.0mg/kg) was investigated on feeding behaviour in non-food-deprived young-adult and adult nmri and transgenic l35 mice. l35 mice are characterized by an overexpression of postsynaptic 5-ht1a receptors. results: the administration of the 5-ht1a receptor agonist induced hyperphagia in all groups of mice, except for the adult transgenic mice which showed no drug effect. conclusion: the results confirm a key role of the 5-ht1a receptor in food intake. further, we make the assumption that the hyperphagic effect of 8-oh-dpat is mediated by presynaptic 5-ht1a autoreceptors in the raphe nuclei which decreases 5-ht function in the central nervous system. it can be speculated that the aberrant feeding behaviour of the adult transgenic mice refers to a possible opposite role of the postsynaptic 5-ht1a receptors. these receptors might affect the release of neuropeptides in the hypothalamus. the efflux transporter abcc2 (mrp2) expressed at different compartment barriers is important for the elimination of various endogenous and exogenous compounds. with some evidence inflammatory processes regulate abcc2 expression and cause changes of absorption, distribution and clearance of a number of xenobiotics. the investigation of the influence of interleukin (il) 1β on abcc2 mrna and protein expression in various cell lines representing specific tissues is the aim of our study. a further aim is to characterize the signaling pathways regulating abcc2 expression while inflammation. three different cell lines a) hepg2 cells (liver tissue) and b) caco2 (colon tissue) both without naïve il1β expression and c) skhep1 cells representing physiological liver tissue with naive il1β expression, were stimulated with different concentrations of il1β (range 10 pg/ml to 10 ng/ml). over a period of 48h samples were taken at defined time points. abcc2 mrna and protein expression were quantified by qrt-pcr and western blot analysis, respectively. by using small molecule kinase inhibitors for signal transduction proteins (p38 mapk, akt, erk1 and jnk) we analysed the signal transduction pathways associated with il1β-mediated transcriptional abcc2 regulation. on abcc2 mrna level an up-regulation in caco2 cells (1, 35 fold) and hepg2 cells (3, 6 fold) within the first hour after stimulation with 1 ng/ml il1β was shown. in contrast skhep1 cells demonstrated a decreased abcc2 mrna expression (0,59-0,62 fold) in comparison to unstimulated controls. the abcc2 protein expression exhibited a time and il1β dependent regulation as well. the analysis for the signal transduction showed for p38mapk a moderat time dependet down regulated phosphorylation (15%) in hepg2 cells whereas it showed no effect in caco2 cells. concluding, the expression of abcc2 is regulated moderately by il1b in a concentration and time-dependent manner. interestingly, the effects are strongly tissue-dependent concerning abcc2 expression and signal transduction pathways and show partly contradictory results. the regulation of the different signaling pathways is currently subject of ongoing investigations. introduction: despite the remarkable success of imatinib treatment of chronic myeloid leukemia (cml), therapy resistance emerged as a major clinical problem. the aim of this study was to identify micrornas, which may serve as biomarkers for therapy response or predict pathways involved in pharmacoresistance of imatinib treatment. methods: blood was collected from 21 cml-patients, ten of whom responded to imatinib therapy. after rna extraction from leukocytes, we performed a taqman low-density array screen to determine the expression of 667 micrornas. statistical analysis using the 2 -∆ct method was performed. micrornas showing a p-value<0.01 and a fold change>2 were considered to be significantly differently expressed. in addition, by using microrna target prediction databases (targetscan 1 , mirdb 2 , pictar 3 , microcosm 4 , diana microt 5 ), selected putative target genes were further functionally investigated by the david bioinformatics database 6 . results: comparing treatment-naïve responders and non-responders four micrornas were identified to be deregulated that were predicted to target 97 genes, especially transcription regulators (21%). pathway analysis showed that six of the predicted genes are relevant in cancer pathways, four of which play a role in cml (smad4, nras, rb1, raf1). when comparing patients' expression profiles before and under treatment, seven micrornas were identified to be deregulated in responders and five micrornas in nonresponders. ninety-nine targets of the latter include transcription regulators (19%), but also cellular transporters (18%, especially uptake transporters of the slc-family). most target genes are involved in mapk signalling or endocytosis pathways. conclusion: analysis of microrna expression profiles revealed four micrornas involved in imatinib-response and 12 micrornas deregulated during imatinib treatment. predicted target genes code mainly for transcription factors as well as oncogenes relevant for cml and are involved in transporter expression and endocytotic processes. dissociations in the effects of beta2-adrenergic receptor agonists on camp formation and superoxide production in human neutrophils brunskole i. 1 , buschauer a. activation of the β2-adrenergic receptor (β2ar), a classically gs-coupled receptor, in neutrophil granulocytes results in an inhibition of inflammatory responses [1] , which could be further therapeutically exploited. the aim of the present study was to evaluate the effects of various β2ar ligands on cyclic adenosine 3',5'-monophosphate accumulation (camp assay) and n-formyl-l-methionyl-l-leucyl-l-phenylalanine (fmlp)induced superoxide anion production (o2 •assay) in isolated human neutrophils, which are a physiologically relevant native test system. camp concentration in neutrophils was determined by hplc/tandem mass spectrometry, and o2 •formation was assessed by monitoring the superoxide dismutase-inhibitable reduction of ferricytochrome c. (-)-isoproterenol, (-)-adrenaline, salbutamol and dobutamine were more potent in inhibiting fmlp-induced o2 •production than in stimulating camp accumulation. (-)-ephedrine and dichloroisoproterenol were devoid of any agonistic activity in the camp assay, but could partially inhibit fmlp-induced o2 •production at higher concentrations. moreover, (-)-adrenaline and dobutamine were equi-efficacious in both assays whereas the efficacy of salbutamol was more than two fold higher in the o2 •assay. this suggests that salbutamol is able to stabilize a different receptor conformation than the other two ligands. thus, ligand-directed signaling via β2ar can also occur in human neutrophils. in addition, differences between the data from neutrophils and recombinant test systems [2, 3] were noticed, pointing to the problem of insufficient comparability of effects in recombinant and native test systems. the investigation of β2ar antagonists on neutrophil granulocytes is subject of ongoing work, in order to find out whether pkb values of β2ar antagonists in the camp assay and the o2 •assay are different. such differences were previously reported for β2ar antagonists in other test systems [4] . moreover, studies with protein kinase a inhibitors should give deeper insight into the signaling events in neutrophils that result in inhibition of fmlp-stimulated o2 •production and clarify how camp increase interferes with this events. agonist-selective internalization of the human 5-ht2a receptor buchborn t., kahl e., höllt v., koch t. otto-von-guericke-universität magdeburg institut für pharmakologie und toxikologie, leipziger straße 44, 39120 magdeburg, germany the serotonin 2a (5-ht2a) receptor is a g protein coupled receptor and the molecular target of lsd-like hallucinogens. downregulation of 5-ht2a receptors is an adaptive process considered relevant for the therapeutic action of diverse serotonergic antidepressants, such as ssris. since the antidepressant targeting of 5-ht2a receptors, however, is largely restricted to indirect agonists and/or antagonists, little is known about the mechanisms and implications of their regulation by direct agonists. in the present study we, therefore, investigated the capacity of various agonists to regulate the human ha-tagged 5-ht2a receptor by internalization. using immunocytochemical techniques in stably transfected hek293 cells, we show that agonists differ in their capacity to internalize the receptor. serotonin, quipazine and doi are the agonists most efficaciously internalizing the receptor, dmt and methysergide, on the other hand, hardly internalize; other agonists like psilocin, ergotamine and lsd induce low to intermediate internalization. the specificity of the agonistic effect was demonstrated by the 5-ht2a selective antagonist ketanserin, which blocked the agonist-induced internalization. in additional experiments, we show that the internalized 5-ht2a receptors colocalize with a488-labelled transferrin receptors, and that the internalization can be blocked by high molar sucrose; these results are indicative of a clathrin associated sequestration of 5-ht2a receptors in recycling endosomes. also, we demonstrate that the proteinkinase c activator pma efficaciously induces 5-ht2a internalization in the absence of an agonist, and that the doi-induced internalization can be blocked by the proteinkinase inhibitor staurosporine. we, thus, confirm previous findings that the activation of proteinkinases seems to be necessitated for the 5-ht2a internalization to occur. overall, we conclude that the internalization of the human 5-ht2a receptor is agonist-selective, and employs a proteinkinase (possibly pkc) dependent, clathrincoated endosome associated pathway. as there is recent evidence that the regulation of 5-ht2(a) receptors by agonists might have antidepressant (-like) properties, knowledge about the agonist-selective processing of 5-ht2a receptors could help to identify agonists most promising for future (pre-)clinical research. non-clinical safety assessment of homeopathic medicinal products: criteria for establishing a first safe dilution buchholzer m. -l., werner c., knoess w. bfarm bundesinstitut für arzneimittel und medizinprodukte zulassung 4, kurt-georg-kiesinger-allee 3, 53175 bonn, germany like all human medicinal products the homeopathic medicinal products for human use must demonstrate adequate safety. in general, they are regulated according to the analogue non-clinical safety principles (points to consider on non-clinical safety of homeopathic medicinal products of botanical, mineral and chemical origin, adoption by hma 2007). one particular approach is the recently introduced concept of a first safe dilution (fsd; introduction to the list of first safe dilutions, adoption by hma 2010) . this contribution summarizes the first experiences in establishing fsds of a selection of given homeopathic preparations by bfarm. for a given preparation the major toxicological concern and available data set is identified. this determines the safety assessment route: food regulation, permitted daily exposure (pde), threshold of toxicological concern (ttc) or lowest human recommended dose (lhrd/100). finally the acceptable amount/tolerable daily intake is derived and the respective fsd is calculated. for example the draft evaluation for reserpinum (ph. eur. method 4.1.1) and for atropine (ph. eur. method 3.1.1 or 4.1.1) based on lhrd leads in each case to a suggested fsd of d7 related to 10 g of preparation. furthermore, the draft evaluation for potassium iodide (ph. eur. method 3.1.1 or 4.1.1) based on food legislation emerged a proposed fsd of d6 related to 10 g of preparation. the concept of fsd combines a scientific and at the same time pragmatic approach in differentiated risk assessment of homeopathic medicinal products. impact of myricetin and its methylated derivatives laricitrin, syringetin and myricetin-3`,4`,5`-trimethylether in c. elegans büchter c. 1 , ackermann d. polyphenolic compounds ubiquitously present in herbal food are discussed to contribute to the health beneficial effects of a diet rich in vegetables and fruits. additional to a strong antioxidative activity of various flavonoids, most of these substances display a variety of other pharmacological properties. we investigated the flavonoid myricetin found in several species of berries, as well as the methylated derivatives laricitrin, syringetin and myricetin-3`,4`,5`-trimethylether. in this study caenorhabditis elegans was used as a model to explore the impact of myricetin and its methylated derivatives in vivo and to investigate molecular modes of action. myricetin (100 µm) caused an increase in mean and median adult lifespan of c. elegans. this longevity effect was associated with a decrease of the aging marker lipofuscin as well as a decrease in ros induction, measured by using the h 2dcf-da assay. however, myrictin failed to improve heat stress resistance, an attribute often associated with longevity in c. elegans. the methylated myricetin derivatives (100 µm) showed a decrease in lipofuscin accumulation and ros induction and they further improved the heat stress resistance. in order to elucidate the basis of the life prolonging action of myricetin, we investigated its influence on factors known to have important functions in stress response and the regulation of aging, namely the foxo homologue daf-16, the nad + -dependent protein deacetylase sir-2.1 and the heat-shock transcription factor hsf-1, respectively. lifespan extension by myricetin disappeared in daf-16 and sir-2.1 loss of function mutant strains, showing the effect is at least partially dependent on these signaling molecules. by using a hsf-1 loss of function mutant strain of c. elegans, it was further shown that the life prolonging effect of myricetin is independent of hsf-1. in conclusion, our results indicate that the life prolonging effect of myricetin is at least in part dependent on daf-16 and sir-2.1, probably due to a modified expression of target genes. stimulatory and inhibitory control of phospholipase c-gamma 2 bühler a., walliser c., becker l., gierschik p. universität ulm institut für pharmakologie und toxikologie, albert-einstein-allee 11, 89081 ulm, germany activation of phospholipase c-γ2 (plcγ2) upon b cell antigen receptor (bcr) stimulation has been implicated to be a critical step in the bcr-mediated calcium signaling. therefore it is important to understand the mechanisms of how the activity of plcγ2 is stimulated and inhibited. the mammalian plcs are divided into six subfamilies, designated β, γ, δ, ε, ζ, and η. within the plcγ subfamily, the two plcγ isoforms share a number of features that are distinct from those of the other plc subfamilies. the most striking difference is the insertion of additional domains between the catalytic subdomains x and y. this specific array (sa) contains a second, split pleckstrin homology (spph) domain, consisting of two halves separated by two src homology 2 (sh2) domains and one src homology 3 (sh3) domain. there is abundant evidence in the literature that plcs are autoinhibited in their basal state by structural elements within their x/y linker, pointing to a conserved role of the x/y linker in autoinhibitory regulation of plc isozymes. data from our group show that plcγ 2 is also regulated by autoinhibitory elements within its specific array (walliser et al., 2008; everett et al., 2011) . our recent data demonstrated that plcγ2 is negatively regulated by its sa. specifically, within the sh domain tandem, the c-terminal sh2 (sh2c) and the sh3 domain in combination, but not either one alone, cause the strongest autoinhibitory control of plcγ2. plcγ2 has been shown to be phosphorylated at tyrosine residues 753 and 759 upon bcr stimulation (kim et al., 2004) . both tyrosines are located in the linker between the sh2c and the sh3 domain, which we have shown to be the major elements involved in autoinhibitory regulation of plcγ2. interestingly, a novel phosphorylation site in plcγ2 was found in non-small cell lung cancer (nsclc) tissue which is located at tyrosine residue 733 (rikova et al., 2007) . in this work, we demonstrate, for the first time, the activation of plcγ2 by phosphomimetic mutations in these three positions and the functional interplay of the three tyrosine phosphorylation targets. most interestingly, mimicking phosphorylation of tyr733 is critical to fully activate the enzyme. the results not only point to a crucial role of plcγ2 in pulmonary tumorigenesis, but also prompt and stimulate the search for the protein kinase involved in phosphorylating plcγ2 at tyr733. molecular characterization of hepatotoxic effects of perfluorooctanoic acid (pfoa) buhrke t., scharmach e., lampen a. bundesinstitut für risikobewertung (bfr) lebensmittelsicherheit, max-dohrn-str. 8-10, 10589 berlin, germany perfluorooctanoic acid (pfoa) is an industrial chemical that is used for the fabrication of numerous products with oil-, dirt-and water-repellent properties. pfoa is resistant to chemical, thermal and biological degradation and has become a global contaminant of soil, water, air and food in the meantime. the toxicological data of pfoa give cause for concern as the substance was shown to damage the liver of rodents and to impair embryo development. currently, the hazard potential of pfoa for humans is controversially discussed. in this study the human liver cell line hepg2 was employed to analyse the hepatotoxic effects of pfoa on the cellular and on the molecular level. pfoa was shown to stimulate cellular proliferation at concentrations in a range between 5 µm and 25 µm. at concentrations higher than 25 µm the substance was cytotoxic to the cells (ic 50 47µm). cytotoxicity was not due to apoptotic mechanisms as no increase of caspase activity was detected up to a level of 100 µm pfoa. on the molecular level pfoa is known to act as an agonist of the peroxisome proliferator-activated receptor alpha (pparα), and the observed hepatotoxic effects in rodents are associated with pfoa-mediated pparα activation. here we show that pfoa has the capacity also to activate the human isoform of pparα. additional human nuclear receptors were tested for activation by pfoa, and pparγ as well as the pregnane x receptor (pxr) were shown to be activated at high concentrations of pfoa whereas pparδ and the liver x receptor alpha (lxrα) were insensitive to activation by pfoa. notably, we observed a significant inhibition of the activity of the hepatocyte nuclear factor 4α (hnf4α) by incubating the cells already with moderate concentrations of pfoa at a level of about 1 µm. these findings indicate that additional, pparα-independent mechanisms may contribute to the observed hepatotoxicity of pfoa. the elucidation of novel modes of action of pfoa is relevant for the ongoing risk assessment of the substance. s17 070 human breast stem cells as a toxicological model for endocrine disruptors, such as soy isoflavones stempin s. 1 , bumke scheer m. (kao et al., 1995) . two daughter cell lines were developed from m13sv1 after x-ray irradiation (m13sv1 r2) and an additional transfection with a mutated erbb2 oncogene (m13sv1 r2-n1), resulting in high and low tumorigenicity respectively and showing a change in estrogen response after growth in minimal media (wang et al., 2010) . isolated isoflavones are currently widely used in the treatment of postmenopausal symptoms of women. according to the stem cell theory of carcinogenesis, breast stem cells are the ideal target for the proposed research. however, the epidemiological data to the effect of isoflavone intake on breast cancer is contradictory. therefore, we want to develop a toxicological model using these human breast stem cell lines to test the effect of endocrine disruptors, such as soy isoflavones in a human relevant model. in the present study we analyzed the effect of the phytoestrogen genistein, the most intensively studied soy protein, on the differentiation of the 3 hbec lines. the expression of different luminal epithelial cell markers, estrogen receptors and stem cell markers was measured on the mrna level by quantitative real time pcr. the analysis of several of these markers was also performed on the protein level using western blot. additionally, a broad number of genes related to breast cancer and estrogen receptordependent signal transduction were studied using a commercial pcr-array. in parallel we are also analyzing the changes on the protein level using 2d gel electrophoresis. we want to use this panel of different markers to establish a toxicological model that can be used in the future to analyze a wide range of different endocrine disruptors. kao cy, nomata k, oakley cs, welsch cw, chang cc. (1995) bronchial asthma is a common inflammatory disease of the airways whose occurrence has increased dramatically over the past decades. histamine plays an important role in mediating the inflammatory response leading to characteristic symptoms like wheezing, coughing, chest tightness, and shortness of breath. since antagonizing the histamine h1-receptor (h1r) shows no ameliorating effects on asthmatic symptoms, h4r antagonists may be new drugs for asthma therapy. in addition to the h3r-and h4rselective antagonist thioperamide, the selective h4r antagonist 1-[(5-chloro-1h-indol-2yl)carbonyl]-4-methylperazine (jnj7777120) is used in pharmacodynamic studies. a correct interpretation of the collected data requires the detailed knowledge of the pharmacokinetics of the applied substances. for this reason, we developed a fast and robust method based on high performance liquid chromatography coupled to tandem mass spectrometry (hplc-ms/ms) which allows the simultaneous quantitation of thioperamide and jnj7777120 as well as the selective h3r antagonist 1-({4[3-(piperidin-1-yl) propoxy]phenyl}methyl)piperidine (jnj5207852) in murine plasma and lung tissue. the treatment of plasma samples based on protein precipitation performed with a mixture of methanol and 0.2 m znso4 using 30 µl of plasma. analyte extraction from lung tissue was achieved by treating 150 -200 mg of tissue with a mixture of ethanol and water followed by rigorous mixing using a fastprep-system. ten µl of the extracted samples were transferred to a synergy polar-rp 80a mercury column (10 x 2mm; 4µm) connected to a polar-rp security guard. chromatographic separation was performed via a gradient using an acetate buffer (ph 5) and methanol at a flow rate of 0.4 ml/min. the analytical run-time was 5 minutes. for plasma samples the assay was linear over a concentration range of 0.078 -40 µg/ml for jnj7777120 and jnj5207852, and 0.313 -40 µg/ml for thioperamide, respectively. in tissues, thioperamide could be quantified in a concentration range of 0.008 -2 µg/sample, jnj7777120 and jnj5207852 in a range of 0.004 -2 µg/sample. our results show that the developed hplc-ms/ms method is suitable for the quantitation of all tested histamine-receptor ligands in murine plasma and lung tissue. the functionality of the heart greatly depends on strict homeostasis and interplay of a range of signalling cascades. deregulation of either one is always harmful and eventually detrimental for life. some of the most relevant signals in the adult heart are triggered by the stimulation of g protein-coupled receptors such as adrenergic or angiotensin receptors. those in turn are modulated by a small subset of kinases, the g protein-coupled receptor kinases (grks). interestingly, grks, which for the longest time were believed to regulate only g protein-coupled receptors were shown to modulate also non-receptor-mediated signalling pathways. by now it is well documented that grk5 plays important roles in both the physiological as well pathological setting of the adult heart. in spite of the important functions of grks in the adult heart, it must be assumed that grk5, one of the two main cardiac grks, may also be involved in signal modulation in the course of heart development. deregulation of grk5-dependent pathways may very well be causal for impaired cardiac development up to congenital heart disease. in fact, grk5 is already expressed during embryogenesis and we can detect it in the developing heart. however, grk5 has not been studied yet for a potential function during embryonic development in general or heart formation in particular. we have established zebrafish as a very time and cost efficient vertebrate model to investigate the role of grk5 on cardiac signalling and development. tools for grk5 specific loss-as well as gain-of-function analyses have been developed in our lab. we revealed an unexpected role of grk5 in the development of left-right asymmetry in zebrafish. clinically, this has been associated with disorders such as heterotaxy and other syndromes linked to ciliary dysfunction. many of those disorders are known to affect proper heart development resulting for example in septum defects or in detrimental translocation of the outflow tract. precisely, depletion of the close homolog of human grk5 in zebrafish mirrors the human syndrome called heterotaxy by displaying randomized placement of inner organs, aberrant heart looping and disrupted valve formation in the heart. in addition, loss of zebrafish grk5 results in a lower heart rate as well as dilatation of the embryonic heart at later stages of development. therefore, we believe that grk5 may potentially serve as a candidate gene for congenital heart disease. identification of a hcn3 interacting protein in mouse brain cao-ehlker x., hammelmann v., zong x., fenske s., biel m. department of pharmacy, center for drug research ludwig-maximilians-universität, munich center for integrated protein science cipsm, butenandtstr. [5] [6] [7] [8] [9] [10] [11] [12] [13] 81377 münchen, germany hyperpolarization activated cyclic nucleotide-gated cation channels (hcn) pass a depolarizing current (ih) that is involved in cardiac pacemaking and the control of numerous basic functions in neuronal circuits. the four hcn channel types (hcn1-4) display specific expression pattern in brain suggesting that each channel fulfills a distinct physiological function. while hcn1, hcn2 and hcn4 channels have been studied in quite some detail there is only little information on the particular role of the hcn3 channel. as an important step towards achieving a better understanding of hcn3 function we set out in this study to identify proteins that are assembled with hcn3 in brain tissue. to this end we performed a yeast two hybrid screen with a mouse cdna library using the hcn3 c-terminal domain as bait. several proteins were obtained and confirmed for interaction with hcn3 using heterologous coexpression in hek293 cells. here, we provide an in-depth analysis of the functional interaction between hcn3 and one of the identified interacting proteins (hip3.1). we show that hip3.1 physically binds to hcn3 channels in vitro and in vivo. still several open issues remain to be clarified i.e. the precise function of tpc1 and its tissue-specific and subcellular distribution. therefore we established a mouse model with a general deletion of tpcn1 and generated a series of tpc1 antibodies. using these tools we investigate the closer molecular and vesicular environment by different biochemical approaches i.e. affinity purification from native tissue derived from wild-type and as a control from knock-out mice, density gradient based vesicle separation, fluorescence activated organelle sorting (faos), total internal reflection fluorescence (tirf) and confocal microscopy. so far, we confirmed the tpc1 knock-out model by our self-generated tpc1 antibodies. tpc1 knockout mice are viable and do not show any obvious deficits. to isolate tpc1 containing vesicles or protein complexes, tissue or cell culture derived material was prepurified by sucrose density gradient centrifugation. for a subsequent mass spectrometric analysis this preparation was taken as a source material for coimmunoprecipitation or faos respectively. in another approach the migration pattern of tpc1 containing endosomes on linear density gradients was compared with a series of endolysosomal markers i.e. different rab-, er-, golgi-and lysosomal antibodies. potentially interesting markers were then in turn analyzed for their co-localization with tpc1 by confocal microscopy/tirf. by combining these results with that from mass spectrometric analysis of faos samples we collect data to get detailed information on the precise endolysosomal distribution pattern of tpc1. foxos are involved in a wide spectrum of cellular functions, including cell proliferation, apoptosis and regulation of oxidative stress. in order to identify novel target genes of foxo transcription factors and to achieve further insight into their role in cancer cells, dna microarray analysis was performed using wild type mcf-7 breast cancer cells and mcf-7 cells overexpressing foxo. we found that several genes involved in the tnf receptor/nf-κb pathway were differentially regulated. one of the genes that was identified to be up-regulated in foxo4 overexpressing cells was a20 a negative regulator of nf-κb signaling pathway. at both mrna and protein level foxo4-dependent up-regulation of this ubiquitin modifying enzyme was confirmed. to determine whether a20 is a direct target of foxo4, a luciferase reporter containing a 1.2 kb of the a20 promoter was co-transfected with different amounts of foxo4 wild-type expression construct. foxo4 induced a dosedependent increase in a20 promoter activity, supporting the assumption that a20 is a direct transcriptional target of foxo4. overexpression of foxo4 led to decreased activity of nf-kb signaling pathway as confirmed by reporter gene and nf-kb specific elisa assays. in addition, mcf-7 cells can be sentisized to tnf-α mediated cytotoxicity which is assocciated with a dimineshed activation of nf-κb. altogether, we identified a20, an ubiquitin modifying enzyme, as a novel foxo4 target gene. our data implicate that sustained foxo4 expression may be involved in regulation of tnf receptor/nf-κb pathway and leading to reduced cell survival. trpm7 is a bi-functional protein consisting of a transient receptor potential ion channel segment linked to an α-type protein kinase domain. trpm7 is essential for motility, proliferation and cell growth. up-regulation of trpm7 function is involved in anoxic neuronal death, cardiac fibrosis and tumor cell proliferation. recently, we have demonstrated that the recombinant trpm7 channel is inhibited by the known modulators of sk1-3 channels such as antimalarial plant alkaloid quinine, cyppa, dequalinium, ns8593, ska31, ucl 1684. the most potent of these compounds, ns8593 (ic50 1.6 µm), interferes with the regulation of trpm7 by cytosolic mg 2+ . here we show that ns8593 (10 µm) fully and reversibly inhibits native trpm7-like currents in hek 293 cells, freshly isolated smooth muscle cells, primary podocytes and ventricular myocytes. furthermore, we examined whether targeting of the native trpm7 currents by ns8593 would impact cellular processes known to be affected by a genetic inactivation of trpm7. we found that ns8593 (10-30 µm) suppressed motility of hek 293 cells without a detectable effect on cell viability. taken together, our findings indicate that ns8593 is a potent and reversible inhibitor of endogenous trpm7 currents and may be a good candidate drug for pharmacological targeting of trpm7. sulfur mustard (2,2'-dichlorodiethylsulfide; sm) is a highly toxic and mutagenic warfare agent classified as a weapon of mass destruction. as soon as sm was first used as a warfare agent, research aimed at the development of an effective antidote was launched. early studies with first-generation inhibitors of poly(adp-ribose) polymerases (parp) have revealed promising therapeutic potential in sm-induced skin injury, but the underlying mechanism remains elusive. the current renaissance of parp inhibitors in cancer chemotherapy has revived the discussion on their use for treatment of sm injury. thus we established a comprehensive study aiming the elucidation of the role of parp in sm pathology based on model substance 2-chloroethyl ethyl sulfide (cees), which is not classified as warfare agent. we have recently demonstrated that parp becomes rapidly activated in living human keratinocytes (hacat) after treatment with cees. the maximal parp activity was observed 10 minutes after treatment with 3 mm cees. the activation was transient and dose dependent. to our knowledge this is the first demonstration of parp activation after treatment with mustards in the context of live cells. an important question is how parp-1 becomes activated upon treatment with mustards. parp-1 is a first-line protein involved in the cellular response to dna strand breaks. however, mustards do not directly induce large numbers of such lesions. one possibility is that parp-1 is activated by dna breaks incorporated as base excision repair (ber) or nucleotide excision repair (ner) intermediates. thus, we performed knockdown experiments of ape1 and ercc1, i.e. endonucleases involved in ber and ner, respectively. the reduction of ape1 expression had no effect on parp activity. surprisingly, the knockdown of ercc1 almost completely abolished the cellular par production after cees treatment. the functional consequence of the errc1-parp cross-talk with regards to adduct removal is under investigation. however, our present data indicate that parp activity is not obligatory for the survival of cells upon cees treatment, as revealed by the lack of effect of the potent parp inhibitor abt-888. expression and activity of g protein coupled receptor kinase 2 (grk2) are elevated in several conditions of compromised heart function. although grk2 inhibition has been characterized as a promising therapeutic strategy in heart failure, a specific grk2inhibitor is not available. raf kinase inhibitor protein (rkip) inhibits raf1 but it also acts as a physiological inhibitor of grk2 upon phosphorylation by pkc at serine153. a detailed understanding of the rkip/grk2 interaction may help to identify inhibitory compounds for grk2. since phosphorylation often induces homo-oligomerization of proteins, we investigated whether this could be implicated in switching rkip from a raf1-into a grk2-inhibitor. co-immunoprecipitation assays showed that rkip self-association was substantially increased after pkc-mediated phosphorylation of rkip. rkip mutants either lacking or mimicking s153 phosphorylation confirmed that this phosphorylation is indeed a prerequisite for rkip/rkip association. cross-linking experiments with myc-tagged rkip in living cells or with purified rkip revealed that rkip phosphorylation by pkc promotes rkip dimers -not oligomers. to test whether dimerization is a critical step for the association of rkip with grk2, we generated a peptide to inhibit rkip dimerization. intriguingly, the peptide did not only prevent rkip dimerization but also attenuated rkip/grk2 association. this implicates, that dimerization of rkip is essential to bind grk2. to determine whether rkip dimers consequently inhibit grk2 activity, we established rkip mutants with high tendency to form dimers. subsequent functional analyses demonstrated that enhanced dimerization of rkip indeed translates into increased grk2 inhibition. we conclude that pkc-mediated phosphorylation of rkip is important for dimerization and that these dimers are essential for grk2 binding and inhibition. our results reveal new insights in the molecular mechanism of rkip/grk2 interaction and will help to develop specific grk2 inhibitors. expression and function of trpm3 ion channels in epithelial mdck2 cells dembla s., meiser j., philipp s. university of saarland institute for experimental and clinical pharmacology and toxicology, kirrberger str. 1, 66421 homburg, germany trpm3 proteins build ca 2+ permeable cation channels [1] activated by steroids [2] and sensitive to increased temperatures [3] . trpm3 channels are expressed in pancreatic ßcells as well as neurons of the dorsal root ganglion, where they act as mediators of insulin release [2] or as nociceptors of noxious heat, respectively [3] . however, northern blots and in situ hybridization experiments revealed that trpm3 is also expressed in epithelial cells of the choroid plexus and the ciliary body [1] as well as in the kidney. pcr analysis of different epithelial cell lines indicated that trpm3 is also expressed in madin-darby canine kidney2 (mdck) cells. quantitative analysis of trpm3 expression by qrt-pcr revealed a ~ 5 fold upregulation in mdck2 cells grown in confluency compared to well separated, proliferating cells. in contrast the level of expression of trpm7, a related ion channel described as regulator of proliferation in other cell types, remained constant. hek293 cells overexpressing trpm3 channels did not proliferate in the presence of the trpm3 agonist pregnenolone sulphate. however, as indicated by impedance analysis, the proliferation of mdck2 cells in the presence pregs was only slightly affected. when we analysed the transepithelial resistance (ter) of mdck2 epithelial cells in transwells as a measure for the formation of tight junctions, we found that the ter of cells grown in the presence of pregs was reduced. interestingly, ca 2+ imaging experiments using fura2 revealed that pregnenolone sulphate induces ca 2+ entry in well separated mdck2 cells but not in cells growing in confluency. we hypothesize that trpm3 might act as a regulator of cell proliferation and/or the formation of tight junctions in mdck2 cells. inhibition of grk2 by rkip improves cardiac contractility and structure in a transgenic mouse model of heart failure denzinger s., schmitt j. p., lohse m. j., lorenz k. institut für pharmakologie und toxikologie pharmakologie, versbacher str. 9, 97078 würzburg, germany the raf kinase inhibitor protein (rkip) has been identified as a physiological inhibitor of g-protein coupled receptor kinase 2 (grk2). grk2 initiates g protein coupled receptor (gpcr) desensitization. since expression and activity of grk2 are upregulated in human heart failure, it has been proposed that grk2 inhibition may resensitize badrenergic receptor activity in heart failure patients. in this study, we evaluated chronic grk2 inhibition by rkip as a potential strategy to improve cardiac function in heart failure. to analyse the effect of rkip on heart failure, rkip transgenic mice were crossed with mice carrying a mutation in phospholamban (pln r9c ). pln r9c causes severe heart failure and premature death in humans and transgenic mice. cardiac function was significantly improved in the presence of rkip as shown by left ventricular catheterization and echocardiography. expression of heart failure marker genes anf and bnp was indistinguishable between wild-type mice and mice co-expressing rkip and pln r9c . in line with these findings, the life span of double transgenic mice was significantly prolonged compared to pln r9c transgenic mice. slow calcium transport into the sarcoplasmatic reticulum was characterised as cause for dilatated cardiomyopathy of pln r9c transgenic mice. since western blot analyses of rkip transgenic heart lysates showed increased phosphorylation of important regulators of cardiomyocyte relaxation, we analysed calcium transients and contractility of isolated cardiomyocytes as possible mechanism of the rkip mediated rescue. in the presence of rkip, calcium reuptake into the sarcoplasmatic reticulum was accelerated and cardiomyocyte relaxation improved. furthermore, coexpression of rkip significantly attenuated pathological cardiac remodelling. interstitual fibrosis and apoptotic cells were quantified in histological sections after sirius red-and tunel-staining. this study revealed a protective function of rkip in a genetic mouse model of human dilated cardiomyopathy by improving cardiac contractility and attenuating interstitial fibrosis and apoptosis. a detailed understanding of this rescue may help to find a new therapeutic strategy to improve cardiac contractility in heart failure. gαi-proteins comprise a group of three highly related members characterized by specific expression patterns. based on previous work of gi-mediated signaling pathways in cardiomyocytes and platelets, we checked gαi expression in mouse heart and platelets. the analysis revealed the presence of gαi2 and gαi3 with gαi2 as the predominant isoform. gene-targeted mice lacking either gαi2 or gαi3 were analyzed to unravel the physiological role of gαi-proteins in the cardiovascular system. extraordinarily prolonged bleeding times in gαi2-deficient animals were an obvious phenomenon. detailed analysis using isolated platelets gαi2-deficient mice exhibited reduced platelet activation and attenuated aggregation in response to stimulation by various agonists accompanied by reduced thrombus formation and diminished stability on a collagen-coated surface. employing in vivo injury/thrombosis models revealed abrogated thrombus formation selectively in gαi2-deficient mice. comparable results were obtained in experiments using mice with megakaryocyte/platelet-specific gαi2deficiency. to assess the pathophysiological consequences of platelet gαi2 function, we challenged these mice in experimental models of myocardial and cerebral ischemia. the results clearly show that platelet-gαi2-deficient mice were protected from both, myocardial and cerebral ischemia. in contrast, conventional gαi2-deficient mice subjected to the heart ischemia/reperfusion model exhibited a significantly increased susceptibility to ischemic injury as compared to wild type controls. in contrast, gαi3deficient mice were strongly protected from injury. thus we suggest that gαi2 and gαi3 play distinct roles in major cardiovascular disorders pointing to specific, non-redundant functions of these two highly related gαi isoforms. the cgmp signaling pathway is activated by nitric oxide (no), natriuretic peptides (anp, bnp & cnp), and cgmp-elevating drugs. it regulates several physiological functions such as smooth muscle relaxation, platelet inhibition, and cell growth and differentiation. recent studies indicate that cgmp signaling might also play a role in tumorigenesis, but the cellular and molecular mechanisms of cgmp's potential pro-and/or anti-tumor activities are not well understood. this study has examined the expression and function of components of the cgmp pathway in melanoma cells of murine and human origin. we have found that mouse b16 melanoma cells specifically express the alpha isoform of the cgmp-dependent protein kinase type i (cgkialpha) but not the beta isoform. treatment of intact cells with the membrane-permeable cgmp analog 8-br-cgmp induced the phosphorylation of cgki substrates, vasodilator stimulated phosphoprotein and phosphodiesterase 5. anp and cnp, ligands of the membrane-bound guanylyl cyclase gc-a and gc-b, respectively, did also activate the endogenous cgmp/cgki pathway. however, b16 melanoma cells did not respond to dea-no, which stimulates no-sensitive soluble guanylyl cyclases. interestingly, activation of cgmp/cgkialpha signal transduction was associated with an increase in erk1/2 and p38 phosphorylation, growth and migration of b16 melanoma cells. similar results were obtained with wm1205 human melanoma cells. in conclusion, we have identified a gc-a/gc-b/cgmp/cgkialpha pathway in melanoma cells, which stimulates tumor cell growth and migration in vitro. pharmacologic inhibition of cgmp signaling may offer a promising strategy for the treatment of melanoma. an increasing body of evidence supports important roles for voltage-gated calcium channels in idiopathic generalized epilepsies (iges), however which calcium channels participate in ige pathogenesis and how has yet to be fully understood. recently, it has been proposed that cav2.3 (r-type) and t-type calcium channels jointly contribute to oscillatory bursting in the reticular thalamus (rt) 1 , which is associated with absence epilepsy. cav3.2 is one of the two t-type calcium channels known to be expressed in the rt 2 . it has been demonstrated that ablation of either cav2.3 or cav3.2 reduces susceptibility to experimentally induced epilepsy 3;4 and in addition that both channels share several pharmacological properties [5] [6] [7] . to gain further insight into interacting mechanisms of these two channels in epilepsy, we tested cav2.3(-|-), cav3.2(-|-) and cav3.2(-|-)xcav2.3(+|-) mice side-by-side in the kainic acid model of epilepsy. we provide first in vivo data supporting a synergistic mode of action for cav2.3 and cav3.2 calcium channels in epileptogenesis. the deubiquitinase cyld regulates mechanisms of rip1/rip3-dependent necroptosis in neuronal cells diemert s. 1 , krieg s. vivo model of cerebral ischemia, we found, that cyld -/-mice exhibit significantly reduced infarction volume compared to control littermates. overall, these data reveal a role for cyld in rip1/3-dependent mechanisms of necroptosis in a model of glutamate toxicity in neuronal cells and further suggests cyld-mediated mechanisms of neuronal cell death as a potential therapeutic target after acute brain injury in vivo. cyanamide-mediated inhibition of n-acetyltransferase 1 dierolf d., bonifas j., blömeke b. university trier department of environmental toxicology, universitätsring 15, bldg. n, 54286 trier, germany cyanamide has been used for decades for medical purposes in the treatment of alcoholism and for agricultural purposes as a plant growth regulator and bud-breaking agent. its therapeutic effect is mediated by reversible inhibition of aldehyde dehydrogenase and it was reported to be metabolised in vivo mainly via coenzyme a dependent n-acetylation by n-acetyltransferases. reported to be a substrate for n-acetyltransferases, cyanamide has a different molecular structure to arylamines and hydrazines, the preferred substrates for nacetyltransferases. therefore a more detailed investigation of its interrelations with nacetyltransferases was performed. we analysed nat1 enzyme activities after incubation of thp-1 cells with cyanamide for 24h, and found that the metabolic conversion of the classic substrate para-aminobenzoic acid was significantly reduced at physiologically relevant concentrations. in detail a significant dose-and time-dependent nat1 protein inhibition was observed for 100 and 1000 µm cyanamide using over-expressed human recombinant nat1 (insect cell cytosol containing recombinant human nat1*4). however, no inhibition was found in the presence of recombinant nat2*4. as we also provide evidence that cyanamide is not metabolised via coenzyme a dependent nacetylation in vitro by human nat1 or nat2 cytosol, by thp-1 cells or by human liver cytosol, we can conclude that this inhibition is not based on substrate-dependent downregulation of nat1. further mechanistic and kinetic studies indicated that cyanamide reacts with the active site cysteine residue of nat1, leading to its rapid inhibition. since the presence of the reduction agent dithiothreitol did not reverse the results it could be that it is possibly not caused by oxidative processes. in sum these data indicate that cyanamide is able to interact with cysteine residues of human nat1, which causes its inhibition but not by a substrate-dependent mode of action. taken together our results show, that cyanamide is not n-acetylated by human nats, but might modulate nat1 dependent detoxification and activation of arylamines. dissecting the signal transduction pathway of acute hypoxic vasoconstriction (hpv) in precapillary pulmonary arterial smooth muscle cells ( low levels of oxygen in the pulmonary airways induce acute hypoxic pulmonary arterial vasoconstriction (acute hpv) redirecting blood flow to normoxic areas of the lung to assure optimal uptake of oxygen during ventilation. acute hpv lasting several minutes occurs predominantly in the precapillary region of the pulmonary vascular tree [1] . therefore, precapillary pulmonary arterial smooth muscle cells (pasmc) have been suggested as sensor as well as effector cells and trpc6 a member of the classical transient receptor potential (trpc) ion channel family was identified to be essential for the initiation of ca 2+ influx and the subsequent contraction of pasmc [2] . however, the underlying oxygen sensor and the exact signal transduction pathway(s) in pasmc have not been fully elucidated yet. by using gene-deficient mouse models as well as downregulation of potential candidate proteins by specific small interfering rnas (sirnas), we aim to dissect signaling cascades of trpc6 channel activation in acute hpv. for pasmc isolation and culture from mice we use a technique based on magnetic separation of intrapulmonary arteries originally developed in rats [3] . trpc-expression in freshly isolated and passaged pasmc cultured in low (5%) and high fetal bovine serum (25%) was analyzed. interestingly higher passage numbers resulted in a significant down-regulation of trpc1 and trpc6 the most predominantly expressed channel monomers in pasmc, while different serum concentrations resulted in no significant changes in their expression rates. sirnas were designed, transfected and successfully tested in hek293 cells and pasmc. initial results of the dissection of the signal transduction pathway activating trpc6 and inducing acute hpv in pasmc will be presented. references [1] staub, n. c. (1985) . site of hypoxic pulmonary vasoconstriction, chest 88, 240s-245s. [2] weissmann, n. et al. (2006) . classical transient receptor potential channel 6 (trpc6) is essential for hypoxic pulmonary vasoconstriction and alveolar gas exchange, proc. natl. acad. sci. u.s.a. 103, 19093-19098 trpv5 is a highly selective calcium channel expressed in various tissues amongst others in placenta. the channel may be involved in transcellular calcium transport in epithelial tissues thereby playing some role in calcium homeostasis of the body. in the placenta trpv5 is assumed to contribute to the maternal-fetal calcium transport. most probably trpv5 is part of a multiprotein channel complex but most of the components of this complex are unknown so far. our aim is to find interaction partners of the trpv5 protein in the placenta that might contribute to the regulation of the trpv5 protein function. therefore we expressed the intracellularly located n-and c-terminal parts of trpv5 (aa 1-330 and 571-723) as trpv5-gst (glutathione-s-transferase)-fusion proteins and used the purified recombinant proteins for pulling down proteins from human placenta cell extracts. the proteins pulled down by this approach were analysed by mass spectrometry. we identified several potential trpv5 interacting proteins which were not associated with the gst protein only. one of the proteins which was highly enriched with the n-terminal part of the trpv5 protein is calpain 6. in contrast to the classical calpains (calcium activated cystein proteases), calpain 6 is unique in that it lacks the cysteine residue in the active site. calpain 6 is mainly expressed during embryogenesis and is reported to be involved in cytoskeleton stabilisation but with unknown function in placenta. the interaction between trpv5 and calpain 6 was confirmed in reciprocal pulldown experiments and the trpv5 binding region for calpain 6 was narrowed down by using overlapping n-terminal trpv5-gst fusion proteins. after injection of trpv5 crna into xenopus laevis oocytes calcium uptake into the oocytes was measured; this uptake was largely reduced after co-injection of the calpain 6 crna. in further experiments we want to study potential regulatory effects of the trpv5 protein on the calpain 6 function in cell culture models. comparative studies on the effects of the human carcinogen inorganic arsenite and its recently identified thiolated metabolite thio-dma v on human urothelial cells ebert f., leffers l., unterberg m., schwerdtle t. universität münster institut für lebensmittelchemie, corrensstr. 45, 48149 münster, germany it has been demonstrated that chronic ingestion of 50-200 µg/day inorganic arsenic is associated with an increased risk for cancers of the skin, the lung and the bladder, but until now the underlying toxic modes of action are still under debate. in this context, in the last five years one main focus has been given to the role of human inorganic arsenic metabolism and nowadays it is generally accepted that human biomethylation contributes to inorganic arsenic induced carcinogenicity. due to further improvements in arsenic speciation techniques recently a new thiolated arsenite metabolite, the thio analogue of the well known metabolite dimethylarsinic acid (dma v ), the so called thiodimethylarsinic acid (thio-dma v ), has been discovered in human biological samples. after synthesizing and analytically characterizing this metabolite (bartel et al. 2011 , j toxicol. 2011 we investigated its toxic effects in direct comparison with ias iii in human urothelial cells. thereby cell cycle studies revealed a g2/m-and s-phase arrest as well as subg1 peak formation in case of thio-dma v . moreover, thio-dma v induced apoptosis (subg1, caspase 3 activity) at lower concentrations and earlier time points as compared to ias iii . most likely this is partly due to the higher cellular bioavailability of thio-dma v (aas/icp-ms). regarding genotoxicity, a generation of dna single strand breaks (alkaline unwinding technique) as well as an increased formation of reactive oxygen species (ros, dcfh-da-fluorescence) occurred only at high cytotoxic concentrations. however, thio-dma v strongly increased h2o2 induced ros formation at very low nanomolar concentrations, which might result in cogenotoxic effects. since our earlier studies have shown a strong inhibition of h2o2 induced poly(adp-ribosyl)ation especially by trivalent methylated arsenic metabolites, actual studies investigate the impact of thio-dma v on cellular poly(adp-ribosyl)ation, parp-1 gene expression, protein level and cellular cleavage, which might hopefully give further hints regarding the mode of action behind thio-dma v induced apoptosis. mitochondrial dysfunction in models of alzheimer´s disease eckert a. universitäre psychiatrische kliniken basel neurobiology laboratory, wilhelm klein strasse 27, 4012 basel, switzerland the histopathological characteristics of alzheimer's disease (ad) are amyloid-ß (aß) containing plaques and neurofibrillary tangles (nfts) as well as neuronal and synaptic loss. until today, the underlying mechanisms of the interplay of plaques and tangles remained unresolved. there is increasing evidence that mitochondrial dysfunction might be a possible link, as revealed by studies in several app and tau transgenic mouse models. recently, we examined mitochondrial function in a novel triple transgenic mouse model (pr5/app/ps2) -triplead mice -that combines both pathologic features of the disease in brain. using comparative, quantitative proteomics (itraq) and mass spectroscopy we found a massive deregulation of 24 proteins, of which one-third were mitochondrial proteins mainly related to complexes i and iv of the oxidative phosphorylation system (oxphos). remarkably, deregulation of complex i was related to tau, whereas deregulation of complex iv was aß dependent, both at the protein and activity levels. the triplead mice showed synergistic effects of aß and tau already at the age of 8 months, resulting in a depolarized mitochondrial membrane potential. at 12 months, the strongest defects on oxphos, synthesis of atp and reactive oxygen species were exhibited in the triplead mice, again emphasizing synergistic, ageassociated effects of aß and tau in impairing mitochondria. evidences from ad post-mortem brain as well as cellular and animal ad models indicate that aß and tau protein trigger mitochondrial dysfunction through a number of pathways, such as impairment of oxidative phosphorylation, elevation of reactive oxygen species production, alteration of mitochondrial dynamics, and interaction with mitochondrial proteins. moreover, recent reports indicate that aß may also interact directly with intracellular proteins such as the mitochondrial enzyme abad (aß binding alcohol dehydrogenase) in executing its toxic effects. mitochondrial dysfunction occurs early in ad, and aß's toxicity seems to be in part mediated by inhibition of abad. in total, a vicious cycle as well as several vicious circles within the cycle, each accelerating the other, can be drawn emphasizing the synergistic deterioration of mitochondria by tau and aß. olesoxime is a novel mitochondrial-targeted compound that is orally active and crosses the blood brain barrier. the cholesterol-oxime targets proteins of the outer mitochondrial membrane and represents a promising drug candidate for neurodegenerative diseases 1 . we evaluated olexoxime's neuroprotective effects against mitochondrial dysfunction in an animal model for alzheimer`s disease (ad). dissociated brain cells (dbc) and mitochondria were isolated from brains of c57/bj6-thy1-appsl (ad-mice) mice that were fed with 600 mg olesoxime/kg feed for 3 months. drug plasma levels reached approx. 600 ng/ml. respiration of isolated mitochondria were significantly diminished in ad-mice due to reduced complex i, i+ii and cox activities. consequently, mitochondrial membrane potential (mmp) was significantly reduced in dbc from ad-mice. olesoxime normalized respiration chain complex activities and the mpp. to further evaluate the beneficial effects of olesoxime on complex i activity, we challenged dbc with rotenone ex vivo and observed that olesoxime treatment was protective. to further clarify the mode of action, we analyzed the ability of olesoxime to prevent opening of the mitochondrial permeability transition pore (mptp) in vitro using energized brain mitochondria by measuring ca 2+ -and atractyloside (atr) induced swelling. the opening of mptp precedes apoptosis and can be induced by mitochondrial dysfunction due to calcium overload, oxidative stress, elevated phosphate concentrations or adenine nucleotide depletion. olesoxime prevented ca 2+ -as well as atr induced mitochondrial swelling. atr inhibits the adenine nucleotide translocase (ant) that requires appropriate membrane properties to mediate mitochondrial permeability transition (mpt). since cholesterol (cho) and its derivates represent potent modulators of membrane viscosity, we related the effects of cho and olesoxime on mptp opening to membrane properties. both, cho and olesoxime reduced the flexibility of membrane acyl-chains in energized mitochondria and prevented atr induced mptp opening. however, cho didn`t prevent ca 2+ -induced mptp opening, indicating a different mode of action for olesoxime. our data confirm olesoxime as drug candidate against mitochondrial dysfunction, which is considered to play a pivotal role in neurodegenerative diseases. the work was supported by the european union under the 7th framework program for rtd -project mitotarget -grant agreement health-f2-2008-223388. several inflammatory glomerular kidney diseases are accompanied with a massive production of reactive oxygen species (ros) that may attack the glomerular filtration barrier by affecting podocyte function and may contribute to apoptotic or necrotic cell death of mesangial cells. otherwise, ros also trigger fine-tuned signaling processes that may result in cell proliferation or cell migration. to define such redox-driven signaling devices, we performed a non hypothesis-driven proteomic approach, to identify homo-or heteromeric protein complexes induced by ros. to this end, protein lysates of human podocytes were treated with or without hydrogen peroxide (250 µm) for 10 min. thereafter, the cell lysates were subjected to diagonal 2d gel electrophoresis and putative redox-affected proteins were analyzed by ms/ms-analysis. by this approach, we could identify a series of proteins that form interprotein-disulfide bonds in a redoxdependent manner. one of those proteins could be characterized as the regulatory subunit of protein kinase a (r-subunit of pka), which belongs to the family of serine/threonine kinases. to evaluate whether ros is capable to activate pka also in a more physiological setting, we treated rat mesangial cells with pdgf-bb to induce ros formation and we could demonstrate that pdgf-bb induces dimerization of r-subunits in a redox-dependent manner. to demonstrate whether pdgf-bb induces pkadependent pathways, we analyzed the effects of pdgf-bb on phosphorylation of serine 157 of vasodilater stimulated protein (vasp) a classical target of pka. in fact, pdgf-bb induced vasp phosphorylation independently of intracellular camp levels. moreover, elevating camp levels via activation of adenylate cyclase with forskolin did not change the dimerization state of r-subunits. pdgf-bb-induced dimerization of the r-subunits and subsequent phosphorylation of vasp was blocked by diphenyljodonium (dpi), indicating activation of a nadph oxidase is essential for pka activity. taken together, we demonstrate a redox-dependent activation of pka by pdgf-bb and this may hint also for a probably protective role of ros in rat mesangial cells. testing the potential sensitizing capacity of chemicals is currently done by using the murine local lymph node assay (llna). animal welfare and eu cosmetics directive demands alternative methods to animal tests. the purpose of this study was to establish an in vitro assay for the prediction of skin sensitizers. based on the finding that the majority of skin sensitizers are electrophilic or have the potential to be metabolized to electrophilic substances, it is assumed that they can activate the nrf2-keap1-antioxidant response element (are) regulatory pathway. here, we report the results obtained from the lusens assay that detects electrophilic chemicals using the nrf2 pathway. the cell line lusens was derived from immortalized keratinocyte hacat cells and carries a luciferase reporter gene under the control of an are-element from the rat nadph quinone reductase nq1. the lusens assay was in house validated with a panel of 54 chemicals and cosmetic ingredients including the 22 performance standard substances of the local lymph node assay. the predictivity of this assay was compared to the predictivity of the murine llna and to human patch test data and can be considered as reliable screening approach (accuracy of 83% compared to human data). however, in order to cope with the complex multi-step mechanism of skin sensitization, an integrated approach of in vitro assays mimicking several steps was designed; thereof, the are-dependent gene activation represents one module. time-resolved fluorescence ligand-binding assays for parathyroid hormone receptors emami-nemini a. 1 ligand-binding studies represent essential tools for pharmacological research on g protein-coupled receptors. in recent years, time-resolved fluorescence gained significant relevance as readout for ligand binding studies. however, ligand-binding assays for parathyroid hormone receptors (pthrs) utilizing fluorescent parathyroid hormone (pth) were missing. therefore, we generated various fluorescent pth analogues which exhibit properties of native pth in terms of affinity, potency and internalization. for the purposes of academic and commercial research, we utilized labeled pth to set up three time-resolved fluorescence assay formats: (i) classical separation binding assay, based on time-resolved fluorescence and suitable for native receptors; (ii) homogeneous timeresolved fluorescence resonance energy transfer (htrf) based on tag-lite technology for high through-put screening; (iii) htrf based on antibodies, a synergistic approach using htrf with minimized receptor modification. this work will facilitate the development of new drugs directed to the pthr as well as fundamental research on the pthr. anandamide production in eosinophilic granulocytes is independent of il-5 and eotaxin stimulation engeli s., reinke j., zörner a., tsikas d., jordan j. medizinische hochschule hannover institut für klinische pharmakologie, carl-neuberg-straße 1, 30625 hannover, germany introduction: some animal and in vitro studies suggest that endocannabinoids exert anti-inflammatory effects. specifically, inhaled anandamide reduced the obstructive effect of leukotrien d4 in airways, and a specific cannabinoid receptor agonist significantly reduced pulmonary inflammation in guinea pigs. we have recently shown that segmental bronchial allergen challenge is associated with significant increases of anandamide concentrations in bronchoalveolar fluid of patients with asthma. the concomitant increase in eosinophilic counts, eotaxin and il-5 concentrations in bronchoalveolar fluid led us to hypothesize that anandamide is produced by eosinophilic granulocytes in response to chemotactic stimuli. peripheral eosinophilic granulocytes were isolated from whole blood by means of percoll gradient centrifugation and magnetic separation employing cd16antibodies conjugated to magnetic beads. isolated cells were counted and anandamide measurements were typically performed in whole cell lysates of 1.5x10 6 eosinophils. we stimulated eosinophils with varying concentrations of il-5, eotaxin-1 (ccl11), eotaxin-2 (ccl24), and eotaxin-3 (ccl26). to prevent anandamide degradation, a specific fatty acid amide hydrolase (faah) inhibitor (oloxa) was employed. anandamide concentrations and faah activity were determined by stable isotope dilution using lc-ms/ms protocols. results: first, we confirmed the ability of eosinophilc granulocytes to synthesize anandamide. however, cellular anandamide content could only be measured when faah was effectively blocked with oloxa, and strong faah activity was demonstrated in eosinophils. with oloxa, typical anandamide concentrations were in the range of 2-4 pm/1.5x10 6 eosinophils. neither il5 (50-500 pg/ml), nor any of the eotaxins (50 ng/ml either alone or in varying combinations) did stimulate anandamide production after 30min of incubation. our results suggest that chemotactic molecules like eotaxin and il-5 are not responsible for increased anandamide formation in eosinophils during allergen challenge. in a next step, the effects of anandamide on eosinophils and bronchial epithelial cells need to be determined. the suprisingly high faah activity in eosinophils may point to an alternative pathway facilitating prostaglandin and leukotriene synthesis by production of arachidonic acid. screening methodology for estimatation of dermal absorption in vitro fabian e., goth c., guth k., mehling a., van ravenzwaay b., landsiedel r. basf se experimentelle toxikologie, carl-bosch-strasse 38, 67056 ludwigshafen, germany dermal absorption is used in the evaluation of the effectiveness of pharmaceutical or cosmetic formulations, but often dermal absorption is a critical parameter in risk assessment of pesticides or chemicals. therefore, knowledge of dermal absorption is e.g. helpful in formulation development. skin absorption is routinely measured in vivo or in vitro following oecd tg 427 or 428. however, these tests are complex, time consuming and expensive. therefore, a method was developed to allow simple and rapid screening. the experiment uses dermatomized skin in modified franz type diffusion cells. 10 µl of test substance preparation are applied to the skin preparation. after 6h, the skin is washed and the amount of penetrated substance is quantified. the receptor fluid and the washing solutions are optimized for subsequent analyses by lc-ms. we performed dermal absorption screenings in parallel to our routine guideline studies and demonstrated a good correlation of the results of both study types: the total recovery found in the screening studies is somewhat lower than in the corresponding guideline studies but is always in an acceptable range above 80%. the efficacy of the skin washing procedure is lower than under routine conditions, most probably due to the change to an lc-ms-compatible washing solution. overall, the dermal absorption screening is an easy, fast and cost-effective screening method for the estimation of dermal absorption of a wide variety of test substances and formulations. the p53 tumor suppressor protein is frequently inactivated in human cancers by diverse mechanisms. owing to its fundamental role in the maintenance of genomic stability and cancer prevention, p53 is an attractive target in cancer therapy and several approaches were pursued to restore p53 function in tumor cells. polyamidoamine (pamam) dendrons are positively charged molecules with a systematically branched structure that interact with the negatively charged cell membrane, inducing cellular uptake via endocytosis. in the present study, biotin-labeled p53 protein was attached to a dendronized streptavidin nanocarrier to facilitate its internalization into different tumor cell lines. first, biotin-substituted pamam dendrons were conjugated with streptavidin to allow formation of the dendronized streptavidin nanocarrier. the nanocarrier displayed uptake into hela cervix carcinoma and a549 lung adenocarcinoma cells without detectable cytotoxicity. biotin-labeled p53 was then conjugated to the dendronized streptavidin, preserving its specific dna-binding in vitro. immunoblot analysis revealed efficient internalization of biotin-p53 into hela cells in the presence of dendronized streptavidin. in line with this finding, specific cellular uptake of biotin-p53 was observed by confocal microscopy, which showed a cytoplasmic and peri-nuclear localization in hela, a549 and saos osteosarcoma cells. the internalized biotin-p53 also partially co-localized with early endosomes. importantly, the delivery of biotin-p53 into p53-deficient saos cells resulted in impaired cell viability and upregulation of caspase 3/7 activity, demonstrating its biological functionality. this study intriguingly demonstrate the efficient delivery of functional biotin-p53 into different tumor cell lines using the novel streptavidin nanocarrier, which can be further modified to allow cell-type specific targeting and combined with cytotoxic drugs such as doxorubicin. identification of novel ahr target genes in rat liver oval cells faust d. 1 , vondracek j. the aryl hydrocarbon receptor (ahr) is a transcription factor involved in physiological processes, but also mediates most, if not all, toxic responses to 2,3,7,8tetrachlorodibenzo-p-dioxin (tcdd), some polycyclic aromatic hydrocarbons (pahs) and dioxin-like polychlorinated biphenyls (pcbs), such as pcb126. activation of the ahr by these ligands leads to its dimerization with arnt and transcriptional activation of several phase i and ii metabolising enzymes. while it is generally accepted that many pahs are thereby transformed to genotoxic metabolites, this classical signalling pathway so far failed to explain the tumour promoting effects of the nongenotoxic compounds tcdd and pcb126. thus, there is an urgent need to define genetic programmes orchestrated by ahr to unravel its role in physiology and toxicology. we have recently shown that treatment of rat liver oval cells with tcdd or pcb126 leads to a release from contact-inhibition involving activation of the ahr, elevation of jund protein levels and transcriptional activation of cyclin a (1, 2) . loss of contact-inhibition is one hallmark of tumour promotion. to better understand ahr-driven pathways we identified the transcriptional programme using high density microarrays in response to pcb126. already 6 h after treatment, 69 genes were found to be upregulated and 76 genes downregulated indicating that these are direct ahr-dependent target genes. david analysis revealed that these genes are involved, for instance, in drug and lipid metabolism, cancer pathways, tgf-b signalling and cell-cell communication. ten of the 69 genes were selected for confirmation by semi-quantitative rt-pcr. using the ahr inhibitor ch-223191 and sirna directed against ahr and arnt, we further demonstrated that ahr-and arnt-function is required for transcriptional activation of the selected genes. finally, we identified the transcription factor foxq1as a novel ahr target protein in rat liver oval cells. although the function of foxq1 is poorly understood, it has been shown very recently that foxq1 is overexpressed in colorectal cancer and is involved in epithelial-mesenchymal transition in breast cancer cells. its function in ahrmediated tumour promotion, however, remains to be determined. the practical relevance of histamine h1 and h3 receptors in the brain can be easily deduced since h1 receptor antagonists of the first generation have a sedative effect and an inverse h3 agonist, pitolisant (close to its introduction to the market), is active against excessive daytime sleepiness associated with narcolepsy. in this context the question arises whether also h2 and h4 receptors possess a practical relevance in the brain. to this end, we examined whether the electrically evoked 3 h-noradrenaline release in superfused human cerebral cortex slices is affected by agonists at the above receptors. the h2 agonist impromidine 10 µm failed to affect noradrenaline release in human cortex slices although it facilitated noradrenaline release in guinea-pig cortex slices; the maximum extent of facilitation was 50 %, the pec50 was 6.9 and the pa2 of the h2 antagonist ranitidine against impromidine was 7.2. with respect to h4 receptors there is some controversy in the literature whether they occur in the brain at all. however, we were able to detect h4 receptor mrna in the human and mouse cortex by the reverse transcriptase polymerase chain reaction. in cortex slices of either species, noradrenaline release was not affected by the h4 agonist 4-methylhistamine 10 -30 µm but inhibited by histamine 10 µm via h3 receptors by 28 and 55 %, respectively. in mouse cortex membranes, 4-methylhistamine 30 µm also failed to affect 35 s-gtpγs binding although r-α-methylhistamine 3 µm, acting via h3 receptors, increased it by 20 %. in conclusion, h2 receptors facilitating noradrenaline release are detectable in the isolated guinea-pig but not human cortex. despite the presence of h4 mrna in the brain, functional readouts of this receptor, i.e. modulation of noradrenaline release (humans, mice) and modulation of 35 s-gtpγs binding (mice), could not be shown. murine cx40 promoter activity is dependent on the transcription factor creb fels b., nunes f., schmitz w., müller f. u. westfälische wilhelms-universität münster institut für pharmakologie und toxikologie, domagkstraße 12, 48149 münster, germany connexin 40 (cx40) is a gap junction protein expressed in atrial myocytes and the ventricular conduction system, mediating the electrical intercellular communication in the myocardium. alterations in cx40 function were linked to the pathophysiology of atrial fibrillation and heart failure. in the heart, camp dependent gene transcription is regulated by members of the creb/crem/atf family which bind to camp responsive elements (cres). similar to the human cx40 gene promoter, the murine promoter contains one cre. cardiomyocyte-specific overexpression of a crem-isoform (crem-ib∆c-x) led to cx40 down-regulation, suggesting that creb related transcription factors are involved in cx40 gene regulation. in order to study the functional role of creb in the regulation of the cx40 promoter we have generated a luciferase based murine cx40 promoter reporter gene construct and monitored its activity in a permanent cell line upon overexpression of constitutive-active creb (cacreb) and a non-phosphorylatable dominant-negative creb (dncreb) isoform. surprisingly, overexpression of cacreb and dncreb both lead to a reduction of cx40 promoter activity (cacreb 46% ± 5% vs control 100% ± 3%; p<0.05 vs control , n=15), dncreb 45% ± 2% vs control 100% ± 3%; p<0.05 vs control, n=18). the activity of the murine connexin 40 promoter is modulated by creb. both cacreb and dncreb led to cx40 down-regulation, which could be explained by induction of inhibitory transcription factors creb/crem/atf1 transcription factor family, which in turn could suppress cx40 promoter activity. (supported by the dfg) results: fxa increased par-2 mrna, protein and cell-surface expression and augmented par-2-mediated mitogenesis. par-1 expression was not influenced. the regulatory action of fxa on par-2 was concentration-dependent and mimicked by a par-2 selective activating peptide. the thrombin inhibitor argatroban or par-1 gene silencing did not influence fxa-stimulated par-2 expression. fxa increased oxidative stress and expression of the nadph oxidase subunit nox-1 in smc. nox-1 gene silencing prevented fxa-stimulated par-2 regulation, as did ebselen and catalase. exogenous hydrogen peroxide increased par-2 expression and mitogenic activity. fxa induced nuclear translocation and par-2 dna binding of nuclear factor kb (nfkb). inhibition of nfkb prevented fxa-stimulated par-2 expression. in separate studies, fxa promoted par-2 mrna stabilisation through increased human antigen r (hur)/par-2 mrna binding and cytoplasmic shuttling. hur gene silencing abolished fxa-stimulated par-2 expression. conclusion: expression and mitogenic activity of vascular par-2, but not par-1, is upregulated by fxa. this action involves transcriptional and post-transcriptional mechanisms mediated through nox-1-containing nadph oxidase and its downstream effectors hydrogen peroxide, nfkb and the mrna stabilising protein hur. continued generation of fxa by the mural thrombus, and the autoregulatory feedback control of par-2 may maintain the inflammatory and proliferative state of the injured vessel, thereby promoting vascular remodeling. the mrna stabilising factor hur is a critical regulator of human proteaseactivated receptor 4 aim: we recently reported that functional expression of par-4 thrombin receptors is induced in human saphenous vein smc exposed to high glucose. this effect could be attributed in part to transcriptional mechanisms mediated through nfkb but the contribution of post-transcriptional effects such as mrna stabilisation is not known. this study explored the potential role of the mrna stabilising factor human antigen r (hur) in the regulation of par-4. methods: human saphenous vein smc were serum-deprived prior to study. gene expression was determined by realtime pcr, protein expression by western blotting. gene silencing utilized commercially available sirna. hur binding to par-4 mrna was determined by immunoprecipitation ("pull-down") pcr. results: high glucose (25 mm vs 5.5 mm) slowed par-4 mrna degradation in the presence of actinomycin d. par-1 mrna decay was not affected. hur binding to par-4 mrna and nucleo-cytosolic shuttling was enhanced by high glucose, total hur protein expression was not affected. hur sirna abolished the high glucose-stimulated induction of par-4 mrna. hydrogen peroxide (h 2o2) also induced cytosolic hur shuttling and increased par-4 mrna and total protein expression. the role of endogenously generated h2o2 in the regulatory effect of high glucose on par-4 expression was investigated with the nadph oxidase inhibitors apocynin/diphenyliodonium (to prevent h2o2 generation) and cell-permeant catalase (to degrade cellular h2o2). both approaches prevented the stimulatory effect of high glucose on par-4 expression. cyclic amp has been reported to suppress hur activation and in the present study, the cyclic amp stimuli forskolin and cicaprost (prostacyclin analog) suppressed basal hur shuttling and par-4 transcript stability. cicaprost also attenuated high glucose-induced hur binding to par-4 mrna and as a consequence normalised par-4 expression and inflammatory signalling in high glucosetreated cell. conclusion: the regulation of par-4 thrombin receptors in human vascular smc is critically dependent on the mrna stabilising actions of hur. through activation of hur, high glucose and other hur stimuli such as ang ii and exogenous h2o2, increase par-4 expression, while cyclic amp agonists such as prostacyclin oppose this effect. such interactions could potentially represent a fine-tuning mechanism to control par-4 expression and ultimately also the mitogenic and inflammatory actions of thrombin in the vessel wall. nucleoside diphosphate kinase b (ndpk b) is a member of a family of ubiquitously expressed enzymes required for nucleoside triphosphate synthesis. thus, they are involved in the regulation of a variety of cellular processes, e. g. g protein mediated signal transduction. however, whether ndpk b has a specific role in the regulation of angiogenic processes in endothelial cells is unknown. therefore, we studied the function of ndpk b in the vasculature in a developmental, an ischemia-induced and an in vitro model of angiogenesis. firstly, depletion of ndpk b expression was achieved by morpholino-mediated knockdown in zebrafish embryos in which the developing vasculature can be visualized by egfp expression in the endothelium. 72h post fertilization, ndpk b knockdown larvae showed a dramatic inhibition of intersegmental and dorsal longitudinal anastomatic vessel formation compared to control injected fish. this phenotype could be rescued by early re-expression of ndpk b. secondly, ischemia driven angiogenesis was studied in ndpk b-depleted mice and wildtype littermates after excision of the left femoral artery. hind limb blood flow was assessed by laser doppler perfusion imaging immediately before and after ligation (day 0) and on postoperative days 3, 7, 14, 21, 28, and 35 . a significant reduction of recovery was observed in the ndpk b depleted mice at days 3 and 7. thirdly, in vitro-sprouting angiogenesis was analyzed in human umbilical vein endothelial cell (huvec) spheroids with and without sirna-mediated ndpk b knockdown. vascular endothelial growth factor (vegf)induced sprouting was significantly attenuated by ndpk b knock down by more than 50% in comparison with control transfected huvec. we conclude from these results that ndpk b is an essential regulator of angiogenesis. the loss of ndpk b may specifically interfere with the vegf-induced migration and proliferation during endothelial sprouting. ethylene oxide in blood of ethylene-exposed volunteers ethylene (et) is a commercially important high volume industrial chemical. inhaled and endogenous et is metabolized in mammals to ethylene oxide (eo), which is carcinogenic in rats and mice. until now, no data on the oxidation of et in et-exposed humans has been published. in the present study, we investigated the formation of eo in four male adult volunteers exposed for 4 hours to constant atmospheric et concentrations of 5, 20, or 50 ppm by means of a breathing mask. during exposure, et concentrations were measured in inhaled and exhaled air by gc/fid and eo concentrations in venous blood by gc/ms. rates of et metabolism were obtained from the product of the differences in the et concentrations with the pulmonary ventilation. in each subject, linear correlations were found between the et exposure concentration and the rate of et metabolism or the eo concentration in blood. mean rate of et metabolism was 5.5 ± 1.4 nmol/h/ppm/kg body weight. steady-state concentrations of eo in blood differed by a factor of 2 between the volunteers. these inter-individual differences likely reflect the polymorphism of glutathione s-transferase theta, the main eo metabolizing enzyme in human liver. mean eo concentration in blood at steady state was 1.5 ± 0.08 nmol/l blood per ppm of et. the data will be used for validating a physiological toxicokinetic model which will describe the et related eo tissue burdens in rodents and humans. the model predictions will support risk evaluations of et. financially supported by the lower olefins sector group of cefic. in vitro effect of stw11 on human dendritic cells fink c. 1 , bonaterra g. a. extracts of echinacea (purple coneflower) are used in the prevention and therapy of infectious diseases. the medicinal product stw 11 contains the extract from purple coneflower, and in addition, extracts of monkshood, venom of honey bee and bushmaster snake in homeopathic dilutions. previous studies showed a stimulation of the cellular and humoral immune response. dendritic cells (dcs) are antigen presenting cells that act at the interface of the innate and adaptive branches of the immune system. during stages of dc differentiation, the ability to internalize antigens varies and decreases during maturation. in this study, we determined the influence of stw11 on the expression of maturation related genes (cd1a, cd83), cytokines (tnfα, il-4, il-12), chemokines (ccr7), major histocompatibility complex ii(mhc-ii) and toll-like receptors (tlr2, tlr4). in mature (mdc) and immature dc (idc) using real-time rt-pcr. peripheral blood mononuclear cells (pbmcs) were isolated from buffy coats of human volunteers by densitygradient (ficoll ® ) and seeded in 6 well plates. non-adherent cells were eliminated. to induce idc development, 50 ng/ml il-4 and 50 ng/ml granulocyte macrophagecolony stimulating factor (gm-csf) were added. at day 6, maturation was induced by addition of lipopolysaccharide (lps) at a final concentration of 1µg/ml and cultured for additional 3 days. after incubation with different concentrations (0. in idc, compared to control, we found a significantly increased expression of cd83 (2.1-2.6 fold) and tnfα (2.6-3.3-fold) genes after treatment with 0.1-5% stw11, respectively, but no effect was found on the expression of cd1a, il-4, il12, adam19, cd11c, cd40,tlr2, tlr4, mhc-ii and ccr7. in summary, these data demonstrate a stimulatory effect of stw 11 in idc and especially in mdc, concerning an increase of various genes related to maturation (cd83), immunomodulation (tnfα, cd40), adhesion (ccr7) and antigen presentation (mhc-ii) and are in accordance with the therapeutic use in infectious diseases. waterproofing sprays are widely used consumer products containing for example fluorinated polymers or silicon based compounds dissolved in alcohols or volatile petroleum distillates. there have been repeated reports on cases of severe acute respiratory disorders especially when using products that newly entered the market. it is hypothesized that impairment of the pulmonary surfactant by deposition of inhaled respirable particles of the active compound is one of the main causes of the acute lung injury. since the inhalation toxicity cannot be predicted a priori based on the physical and chemical properties of the formulation, proper test strategies are required to ensure consumer safety. we propose to combine screening tests addressing both, exposure and acute lung toxicity. the exposure potential of the spray product is characterized by determining the release fraction of the active compound in the respirable particle size range under conditions relevant for the product application. this is carried out by spraying defined quantities of the product into a control volume and measuring the concentration of health related size fractions. this procedure takes into account spray ageing, especially size reduction of the droplets due to solvent evaporation. the isolated perfused lung is used as a model for testing acute toxicity. ventilated rat lungs are exposed to aged aerosols with proper particle size of approximately 1 µm mmad generated from the liquid spray formulation. lung compliance and lung resistance are continuously monitored during exposure. dose dependent deviations from the normal values (without exposure) are used as read-out parameters. using the combined procedure, different sprays could be ranked according to their realistic exposure risk and, most importantly, sprays with known lung toxicity could be uniquely distinguished from those that have been shown to be safe. in its current stage of development the simple test method is recommended for screening of substances only. induction of oxidative damage in calf thymus dna by the fusarium mycotoxin zearalenone after metabolic activation with liver microsomes fleck s. c., pfeiffer e., metzler m. kit -institute of applied biosciences chair of food chemistry, adenauerring 20a, 76131 karlsruhe, germany zearalenone (zen) is an estrogenic mycotoxin produced by fusarium species. the adverse effects of zen and its reductive metabolite zearalenol (zel) are often compared to those of 17-beta-estradiol (e2) and estrone (e1). these endogenous estrogens are associated with an increased risk for cancer, which may be mediated by two mechanisms, i.e. (i) hormonal activity and (ii) genotoxic effects by p450-catalyzed metabolic activation to catechols (wang et al., chem res toxicol 23, 1365 . like e2 and e1, zen and zel exhibit marked estrogenicity and also undergo aromatic hydroxylation to catechol metabolites (pfeiffer et al., mol nutr food res 53, 1123 . the aim of the present study was to examine the formation of catechol metabolites of zen by liver microsomes of various species and their potential for redox cycling. catechol metabolites are frequently associated with the generation of reactive oxygen species and subsequent oxidative damage of dna, for which 8-oxo-7,8-dihydro-2'deoxyguanosine (8-oxo-dg) is a common biomarker. the propensity of the catechol metabolites of zen and zel to cause the formation of 8-oxo-dg in isolated calf thymus dna was determined using a lc-esi-ms/ms method. to this end, zen was incubated with microsomes from human, rat, mouse, bovine and porcine liver as well as with human cyp1a2, and the incubations were extracted with ethyl acetate. the extract was analyzed with lc-ms and then added to a solution of calf thymus dna in the presence of copper(ii) ions and nadph. the formation of 8-oxo-dg could be demonstrated with each extract. the levels of 8-oxo-dg correlated directly with the extent of catechol formation, which increased from steer to swine to human to mouse to rat microsomes. 15-hydroxylated zen/zel, which is the major catechol, was more reactive than 13-hydroxylated zen/zel to form 8-oxo-dg. in conclusion, our study has shown that the catechol metabolites of zen are highly reactive and give rise to oxidative dna damage in vitro. in addition, recent research from our laboratory revealed that the catechols of zen are less efficiently inactivated by catechol-o-methyl transferase than the catechols of e2 and e1. the genotoxic potential of zen may constitute another biological activity in addition to the well-known estrogenicity. supported by deutsche forschungsgemeinschaft (grant me 574/32-1) and "food and health" of kit. thrombin regulates expression of sphingosine kinase-1 (sphk-1) in human vascular smooth muscle cells -inhibition by dabigatran reduces vascular sphk-1 expression and atherosclerotic burden in vivo flößer a. 1 results: thrombin induced a time-and concentration-dependent (1-100 nmol/l) increase in sphk-1 mrna and protein expression in human saphenous vein smc, n=6-7. this was mimicked by a synthetic par-1 ligand. inhibition of sphk-1 attenuated thrombin-induced smc proliferation but not smc migration (n=5). the regulatory action of thrombin on sphk-1 expression was suppressed by sirna against the mrna stabilisiserhur. in thrombin-stimulated smc, hur binding to sphk-1 mrna and subsequent nucleo-cytosolic shuttling was enhanced. accordingly, thrombin induced sphk-1 mrna stabilisation in smc in the presence of actinomycin d. in apoe-deficient mice, long-term treatment with the direct thrombin inhibitor dabigatran significantly reduced aortic sphk-1 expression by 50% (n=5) and plaque size by 35% compared to control animals (n=10). conclusions: thrombin induces sphk-1 expression and s1p synthesis in vascular smc via the mrna stabilising protein hur. this leads to increased smc proliferation. inhibition of thrombin by dabigatran treatment in vivo attenuates progression of plaques possibly by reducing sphk-1 expression. mycotoxin contamination and cytotoxicity of grain mill products typical grain mill products from north-rhine westphalia, i.e. grains, flour, wholemeal flour and bran (from wheat, rye and spelt) as well as typical by-products (outsourced fractions) from the milling process were analysed for their mycotoxin content by lc-ms/ms. the cytotoxicity of sample extracts with known mycotoxin composition was then assessed in v79 cell cultures by means of the neutral red uptake assay, in parallel with pure reference mycotoxin mixtures. extracts from flour and wholemeal flour samples with low levels of deoxynivalenol and enniatin b (from not detectable to 0.4 µg/g don and 0.5 µg/g ennb) induced no measurable cytotoxicity. on the other hand, although mycotoxin contamination levels were also rather low in bran, these samples induced strong cytotoxicity: extracts of bran derived from rye and spelt were more cytotoxic than those of wheat bran. the cytotoxic effects of the bran samples cannot be related to their mycotoxin content as comparable concentrations of pure mycotoxins and mycotoxin combinations tested in parallel were not cytotoxic. by-products from certain stages of the milling process (sorting and waste fractions) were found to contain mycotoxins at rather high levels: enniatin b was detected in nearly all samples, and also t-2 toxin, ht-2 toxin, ergotamine, ergocornin and deoxynivalenol were present. waste sample extracts with notable mycotoxin levels (up to 6 µg/g don, 7 µg/g ennb, 16 µg/g ergotamine, 50 ng/g ht-2 toxin) exert pronounced cytotoxicity in v79 cells. the cytotoxicity of these samples was somewhat stronger than expected when compared with mixtures of reference mycotoxins tested in parallel. in summary, the tested flour and wholemeal flour extracts contained only low levels of mycotoxins and were not cytotoxic. in contrast, bran samples showed cytotoxicity which cannot be explained solely by their mycotoxin content. this unexpected observation in real samples and combination effects of mycotoxin mixtures require further studies. the ahr is a ligand-activated transcription factor that mediates the toxicity of dioxins and related compounds. upon ligand binding the ahr translocates into the nucleus and dimerizes with arnt to modulate gene expression, e.g. of cyp1a1. recently, we have shown that uvb irradiation of human keratinocytes results in activation of the ahr and associated egfr signaling leading to an enhanced expression of cyp1a1 and proinflammatory cox-2, respectively. the initial step is the uvb induced intracellular formation of the tryptophan photoproduct 6-formylindolo [3,2b] carbazole (ficz), a high affinity ahr ligand. thus, the ficz activated ahr is an important mediator of the dna damage independent part of the uvb response. our current study aims to identify further aspects of ahr mediated uvb responses. therefore, we analysed changes in protein expression, proliferation and apoptosis in ahr+/+ and ahr-/-keratinocytes (nctc 2544) by western blot, flow cytometry and brdu incorporation. uvb exposure of nctc cells led to a dose-dependent increase in apoptosis. compared to ahr+/+ cells, ahr-/-cultures exhibited an increased amount of apoptotic cells. this finding was confirmed in irradiated ahr+/+ cells, pretreated with the ahr antagonist 3'methoxy-4'-nitroflavone. moreover, the proliferation of sham as well as uvb irradiated ahr-/-cells was significantly decreased. in ahr-/-cells we found a reduced expression of checkpoint kinase 1 (chk1), an important cell cycle regulator that arrests the cell in g2/m upon dna damage. interestingly, uvb exposure led to a higher net phosphorylation of chk1 in ahr-/-cells, indicating that this pathway is responsible for the observed ahr-dependent differences in proliferation and apoptosis. further expression analyses of chk1 client proteins emphasize our hypothesis. in conclusion our study identifies the ahr as an anti-apoptotic player in uvb irradiated human nctc cells. therefore, we propose that the ahr is a suitable target to prevent uvb induced skin diseases. synthetic progestins exert divergent effects on thrombosis in a murine model of atherothrombosis background: medroxyprogesterone acetate (mpa), a synthetic progestin often used in postmenopausal hormone replacement therapy, has previously been described to be pro-thrombotic in a murine model of accelerated atherosclerosis. however, nothing is so far known about effects of progestins with receptor profiles different from mpa (i.e. agonism or antagonism of mineralocorticoid-or androgen-receptors), such as drospirenone, levonorgestrel and norethisterone acetate. methods: apo -/mice were bilaterally ovariectomized (ovx) and substituted subcutaneously with mpa, drospirenone, levonorgestrel and norethisterone acetate as well as the respective placebo pellets for 90 days on a western-type diet. subsequently, thrombosis was induced by photochemical injury to the right carotid artery using rose bengal and a green light laser. results: compared to placebo, animals substituted with mpa showed significantly shortened times to occlusion of the right carotid artery (placebo mpa: 50.0 ± 5.8 min. vs. mpa: 33.4 ± 4.2 min., n = 6 -9, p < 0.05). in contrast, drospirenone, levonorgestrel or norethisterone acetate did not alter thrombotic responses. however, at least drospirenone (placebo drospirenone: 53.7 ± 5.4 min. vs. drospirenone: 47.5 ± 4.8 min., n = 7 -8) and levonorgestrel (placebo levonorgestrel: 47.0 ± 3.2 min. vs. levonorgestrel: 41.8 ± 2.1 min., n = 6) showed a trend towards shorter times to stable occlusion. furthermore, analysis of aortic gene expression revealed that in aortas of mpa-treated mice expression of matrix-metalloproteinase 9 (mmp-9) was induced as compared to placebo-treated mice. conclusion: mpa, a progestin with glucocorticoid effects, exerts a pro-thrombotic effect that is either progesterone-or glucocorticoidreceptor-dependent while progestins with receptor profiles different from mpa do not show a significant pro-thrombotic effect. furthermore, the pro-thrombotic effect exerted by mpa may be associated with increased expression of mmp-9, a metalloproteinase being known to destabilize atherosclerotic plaques and make them more prone to rupture. rapid screening for mitochondrial toxicity in vitro using an oxygen-sensitive phosphorescent probe freyberger a. bayer healthcare bph gdd ged toxikologie -p & cp, aprather weg 18, 42096 wuppertal, germany impaired mitochondrial function has been implicated with disease, aging, and druginduced toxicities. analyzing mitochondrial respiration (mr) rates is one of the most informative ways to assess mitochondrial function as it provides information on the the bioenergetic capacity of a tissue, however, previously measurements using polarography (clark electrode) were cumbersome with only low throughput. in this work we explored luxcel's water-soluble phosphorescent oxygen-sensitive probe mitoxpress tm for the assessment of mitochondrial toxicity in freshly isolated male rat liver mitochondria (rlm) in a 96-well plate format using glutamate/malate (20 mm/0.5 mm) and succinate (25 mm) as respiratory substrates. inhibition of mitochondrial complexes i to iv, adenosine triphosphate synthetase and the adenosine diphosphate (adp) / adenosine triphosphate (atp) antiporter by rotenone, thenoyltrifluoracetone (ttfa), antimycin a, potassium cyanide, oligomycin, and atractyloside was readily detected in adp-stimulated rlm. use of the two different substrates in parallel allowed to discriminate complex i inhibition by rotenone from complex ii inhibition by ttfa, whereas downstream of these complexes inhibition by the other model inhibitors occurred independent of the substrate used. decoupling of mr from oxidative phosphorylation by carbonylcyanid-p-trifluormethoxyphenylhydrazone (fccp) was detected best in the absence of adp. compared to polarographic measurement, the use of an oxygen-sensitive probe is superior with regard to assay cycle time and sample throughput and offers new opportunities to characterize and screen for mitochondrial toxicity, but also to support studies on mitochondria-mediated modes of action of new chemical entities. the murine local lymph node assay (llna) and the guinea pig maximization test (gpmt) have been used to study the sensitization potential of a series of unsaturated compounds by kreiling et al. (2008) . we have examined the same substances in the loose-fit coculture-based sensitization assay (lcsa), developed by our working group (schreiner et al., 2007) . eight unsaturated compounds [oleic acid (oa), linoleic acid (la), linolenic acid (lna), undecylenic acid (ua), fumaric acid (fa), maleic acid (ma), squalene (sq), 1-octyn-3-ol (oc)] and succinic acid (sua) were investigated using a coculture of keratinocytes and dendritic cell-related cells (dcrc). sensitization potential was quantified by flow cytometry measuring the increase of cd86 expressed on dcrc (ec50 = half maximal effective concentration). a pronounced induction of cd86 at low concentrations was seen with la, lna and oa (ec50: 3, 5 and 5 µmol/l, respectively). ua exhibited an intermediate response (ec50: 307 µmol/l). with oc and ma, we observed effects at higher concentrations only (ec50: 1340 and 2293 µmol/l). no significant increase of cd86 was observed with fa, sua and sq. because of poor solubility, sq could not be studied adequately. induction of cd86 was generally observed at concentrations which did not cause a major impairment of cell viability. our results show a high degree of concordance with those obtained by the gpmt, except for oa. in comparison with the results of the llna, those compounds which showed a strong effect in the llna (oa, la, lna) also induced an increase of cd86 at low concentrations, whereas those with low stimulation indices in the llna induced no significant increase of cd86 (fa, sua) or only at higher concentrations (ua). we observed a discrepancy between the tests with ma and oc, causing a strong stimulation of the murine lymph nodes, while the expression of cd86 was increased at high concentrations only. we assume that ma and oc might be false-positives in the llna, because they were also negative in the gpmt. background: the first step in elimination of many cationic drugs is their uptake from the blood into hepatocytes and renal proximal tubular cells by the organic cation transporter 1 (oct1) and oct2, respectively. the pivotal role of octs in the excretion of cationic drugs raises the possibility of drug-drug interactions in which one drug reduces octmediated elimination of a second drug. although many psychoactive drugs are cationic at ph 7.4 and some of these have already been recognized as oct inhibitors, a systematic screen of this class of compounds is missing. methods: we screened a drug library of 50 most frequently prescribed psychoactive drugs (inpatient prescriptions in germany, at least 4 million ddd each) for their inhibitory interaction with oct1 and oct2. human embryonic kidney (hek) cells stably overexpressing oct1 or oct2 and the prototypical oct substrate 1-methyl-4phenylpyridinium (mpp+) were used as a test system. cells transfected with the empty vector were used as controls. results: at 20 µm, 59% and 51%, respectively, of the tested compounds significantly decreased oct1-and oct2-mediated uptake of mpp+. the most potent inhibitors (inhibition >75%) of oct1 were chlorprothixen and clomipramine, whereas olanzapine, clomipramine and doxepin were the most potent inhibitors of oct2. in contrast, neither at 20 µm nor at 200 µm carbamazepin, haloperidol, lithium, moclobemide and valproic acid did significantly inhibit mpp+ uptake into hek-oct1 or hek-oct2 cells. there was a significant correlation between the degree of oct1 and oct2 inhibition (p conclusions: our results demonstrate that inhibition of oct function by psychoactive drugs has to be considered as a potential mechanism underlying drug-drug interactions. considering estimated peak sinusoidal concentrations e.g., of chlorprothixen and clomipramine between 30 and 80 µm in humans, inhibitory interactions of these compounds with hepatic oct1 have to be taken in account. our data will help to create a chemoinformatic model to predict potential oct-dependent interactions of psychoactive drugs with the hepatic or renal elimination of coadministered drugs. this project is supported by the german federal ministry of education and research (bmbf), project grant no. 01 ex1015b. cardiac gene expression is altered during the development of hypertrophy and heart failure compared to the healthy heart. the molecular mechanisms controlling gene expression in cardiac failure are only partially known. dna methylation is one epigenetic mechanism that regulates long-term changes in gene-expression. to elucidate whether dna methylation is altered during the development and progression of chronic heart failure, genome-wide dna methylation profiles were determined in myocardial biopsies from control patients and patients with cardiac hypertrophy or failure. cardiac biopsies were obtained from patients with aortic aneurysm who served as control and did not show clinical signs of chronic heart disease (ef: 58±3 %, n=3) and from patients with aortic stenosis. the latter group was subdivided according to the ejection fraction into hypertrophic (ef: 63±7 %, n=3) and failing patients (ef: 28±1 %, n=3). after bisulfite conversion of extracted dna, the methylation status of genomic dna was quantified using the infinium® humanmethylation450 beadchip (illumina). this microarray allows analysis of more than 485,000 methylation-sites throughout the whole genome at single-base-pair resolution. these experiments identified 1280 cpg sites in hypertrophic samples and 1365 cpg sites in failing samples which were differentially methylated compared to control specimens (delta >15%; p<0.05). 523 cpg sites were significantly altered in both aortic stenosis groups compared with control hearts. from these cpgs, 496 sites were altered concordantly in hypertrophic and failing samples. analysis of regions harbouring distinct cpg densities revealed that most changes occured in shelf regions of cpg islands whereas the methylation status in the cpg islands was more stable. further analysis showed that differences in methylation were most frequent in gene body, enhancer and 3`utr regions. specifically 9 probes spanning a cpg-island at the promotor region of the muscle-specific serine kinase 1 (srpk3) showed diminished cpg-methylation in hypertrophic (-11.5±0.02%) and failing (-11±0.02%) as compared to control biopsies. remarkably, no alterations of dna-methylation were observed in loci of classic marker genes of chronic heart failure like nppa, serca, ctgf, myh6 or myh7. these results indicate that dna methylation is specifically altered in chronic heart disease but does not affect classic marker genes of chronic heart failure. gliomas are the most abundant type of primary brain tumor in the central nervous system in adults. the current standard of glioblastoma multiforme (gbm) therapy is surgery followed by radiotherapy and chemotherapy. however the morbidity and mortality of gbm remain very high and the median survival period is only 15 months even with treatment. therefore it is important to identify novel drugs to reduce gbm cell proliferation. purine-analogues (pa) are well known for their anti-proliferative effects on eukaryotic cells. in this study novel pa were synthesized and the library of substance-derivatives was tested using different gbm cell lines namely ln18, u87-mg and gl261. the effect on proliferation and viability was assessed by using brdu and resazurin assays. using these in vitro methods we were able to identify several compounds with cytotoxic and anti-proliferative effects in vitro showing ic50 values in the deeper µm range. cytotoxicity of selected compounds was further analyzed by assessment of caspase 3 and propidium iodide based cell cycle facs analysis to discriminate between apoptosis and cell cycle arrest. based on these data purine-derivatives might inhibit proliferation and induce apoptosis in glioma cells. as a result we hypothesize that these compounds could be potentially interesting for the drug-development of gbm therapy and therefore a clue for chemical modifications. further studies are required to identify the exact underlying mechanism of action of the tested purine-analogues. the biological role of adenosine receptors in brown adipose tissue gnad t. 1 brown adipose tissue (bat) is responsible for basal and inducible energy expenditure in mammals. bat contains large amounts of mitochondria and is highly vascularized. bat lipolysis and thermogenesis are stimulated by sympathetic neurons. importantly, recent findings indicate that adult humans possess metabolically active bat 1 . here, we analyzed the expression and function of adenosine receptors in bat. adenosine receptors (ador) are members of the superfamily of g protein-coupled receptors. there are four subtypes of adors in humans referred to as adora1, a2a, a2b and a3. they are widely expressed in tissues and mediate a variety of cellular functions, mostly due to their regulation of camp levels within cells. interestingly, it has been shown that adenosine can either inhibit or stimulate lipolysis in white adipocytes through adora1 or a2a, respectively 2 . however, the role of adenosine in the differentiation of brown preadipocytes to adipocytes and in bat function is not clear. to analyze the role of adors in bat, we use preadipocytes isolated from bat of newborn mice and subjected them to a differentiation protocol. 3 abundance of adora1, a2a, a2b and a3 mrna was measured using qpcr. all four receptor subtypes are present in preadipocytes with adora2b being the most abundant. adora1, adora2a and adora3 are significantly transcriptionally upregulated -albeit at varying degree -during differentiation. adora1 is upregulated 4.4 fold (+/-0.3 fold) and 11 fold (+/-0.49 fold) at day 4 and at day 7, respectively, as compared to preadipocytes (n=5). adora2a is 23 fold (+/-1.96 fold) upregulated at day 4 and 57 fold upregulated (+/-2.48 fold) at day 7, respectively (n=4). adora3 was found upregulated 3.6 fold (+/-0.46 fold) at day 7 (n=4). in contrast to this, ador2b was downregulated to 0.87 fold (+/-0.09 fold) at day 4 and to 0.69 fold (+/-0.04) day 7 compared to preadipocytes (n=5). to investigate the functional role of ador in bati cells, we analyzed lipolysis in mature cells after acute treatment with specific agonists and antagonists. we observed that adora2a activation by cgs21680 significantly increased lipolysis by 88% (+/-0.33%) compared to untreated control. moreover, adora1 antagonist psb36 increased lipolysis by 35% (+/-0.05%) (n=4). in conclusion, ador are highly regulated during brown fat cell differentiation. lipolysis of mature brown fat cells is significantly increased by adora2a agonist or adora1 antagonist, respectively. munich heart alliance, münchen, germany activation of the sympathetic nervous system and the subsequent activation of βadrenergic receptors (βars) through catecholamines represents the strongest mechanism to increase cardiac function. however, long-term activation of cardiac βars is clearly detrimental and β-blockers have been introduced as an effective treatment modality in cardiac failure. despite their central role in cardiac physiology and disease, our knowledge about the intracellular mechanism of βar stimulation is confined to a few targets and is likely incomplete. here, we report a functional proteomics approach to directly assess the entire phosphoproteome of βar-stimulated mouse hearts in vivo. to identify proteins that are phosphorylated in response to β-adrenergic stimulation in vivo, we treated mice with isoproterenol or, as a control, with propranolol. after lysis of hearts and tryptic digest, phosphopeptides were enriched by tio 2 or immobilized metal ion affinity chromatography (imac). subsequent analysis of eluated peptides by tandem mass spectrometry (ms/ms) mapped several phosphopeptides to cardiac proteins, among which known mediators of βar signaling such as phospholamban, troponin i and myosin binding protein c. we then employed multiple reaction monitoring (mrm) as a quantitative approach to assess changes of phosphorylation after βar stimulation. using this combination of ms approaches, we identified 39 peptides with pka consensus phosphosites that were more abundantly detected under βar stimulation. among those, we found myozenin-2 (myoz2) and g protein signaling modulator 1 (gpsm1, also termed ags3) as proteins previously not related to βar signaling. we validated the βar-dependence of phosphorylation at these sites in isolated cardiomyocytes by in vivo labelling or phosphoepitope-specific antibodies. current efforts aim at the functional characterization of these novel candidate mediators of βar signaling in the heart. taken together, we report the β-adrenergic phosphoproteome of the mammalian heart in vivo. we have identified several new targets of βar signaling that may represent essential factors in cardiac physiology and disease. background: drug measurement in autopsy material is normally used to investigate the cause of death. in our study it was possible to measure concentrations of drugs that were part of a regular treatment without connection to the cause of death. metamizole is used as an analgetic and spasmolytic agent. the active metabolite maa (4-methyl-aminoantipyrin) is metabolized by the liver and eliminated by the kidney. hepatic and renal dysfunction can therefore influence maa clearance. methods: maa concentrations were measured in different samples of the autopsy material (heart blood, venous blood, urine, liver, kidney and brain) using an hplc-ms/ms method. information about the dosage and time of drug application as well as information about existing renal or hepatic disorders were taken from the corresponding patient records. because of the low number of cases an explorative single-case study was necessary. results: 10 cases with oral intake of metamizole in a customary continuous dosage could be indentified. the maa distribution into body liquids and organs depended on the time between last oral intake and death. in two cases without renal or hepatic diseases maa blood levels were below 10 µg/ml. five cases with combined renal and hepatic disorders showed either increased blood levels of 40-50 µg/ml or prolonged maa elimination half-life of up to 12 hours. in one case with manifest hepatic insufficiency an maa concentration of more than 200 µg/ml was measured in venous blood. two cases with renal insufficiency alone had maa venous blood levels of less than 10 µg/ml. (pet) . pet detects the positron emission of neutron-deficient radioactive nuclides and allows their external localization in vivo. fet, a modified amino acid, is not incorporated in proteins but accumulates in glioblastomas. one pathway responsible for its accumulation is the preferential transport into the tumor cells, probably via amino acid transporters. we investigated in more detail (a) which individual, cloned amino acid transporters accept fet as substrate and (b) which transporter is responsible for the major fet transport into glioblastoma cells. studies with xenopus laevis oocytes, expressing individual human amino acid transporters, revealed that system l, y + l and b 0+ amino acid transporters recognize fet as substrate (lat1 and 2, y + lat2, and b 0+ at, respectively). in contrast, y + lat1 and atb 0,+ did not transport fet. rna expression studies using qrt/pcr revealed that lat1 is the dominant amino acid transporter in all glioblastoma cells investigated (ln229/u373/u87mg/u251/a172/t98g). a strong lat1 expression was also shown on the protein level. to find out whether lat1 is the main transporter responsible for fet accumulation, we first studied transport of the parent amino acid l-tyrosine in ln229 glioblastoma cells. [ 3 h] tyr uptake was completely na + -independent and inhibited by leu, phe and trp, but not by arg, pro or ser. sirna-mediated down-regulation of lat1 in ln229 cells led to a concomitant decrease of lat1 mrna and tyr transport (down to 3% and 20%, respectively). these results indicate that tyr is exclusively transported by lat1 in ln229 cells. we are currently performing transport studies using [ 18 f]fet to investigate whether fet transport is also exclusively mediated by lat1 in glioblastoma cells. a further question is if lat1, a sodium-independent transporter, can be responsible for the accumulation of fet observed in glioblastoma cells. if true, other amino acid derivatives that are lat1 substrates might also proof useful in cancer diagnosis. telmisartan reduces adipose tissue inflammation and biglycan accumulation in diabetogenic ldl-receptor knockout mice grandoch m., nagy n., fischer j. w. institut für pharmakologie und klinische pharmakologie, universitätsklinikum der heinrich-heine-universität düsseldorf, moorenstraße 5, 40225 düsseldorf, germany in addition to lowering blood pressure some of the angiotensin ii at1 receptor antagonists (arb) such as telmisartan have additional beneficial effects on the onset of type 2 diabetes mellitus and obesity. this was contributed mainly to peroxisome proliferator activated receptor (ppar)γ modulating activity. hyaluronan (ha), a high molecular weight polysaccharide and the small leucine rich proteoglycans, decorin and biglycan, are known to be involved in atheroprogression. mechanistically these matrix components contribute to inflammatory processes via toll-like receptor-signalling and are supposed to modulate lipid retention. the aim of this study was to elucidate the effects of telmisartan in comparison to valsartan, an arb without pparγ activity, on extracellular matrix remodelling and inflammation in atherosclerosis and the interrelationship with adipose tissue inflammation using the ldlr-/-model of accelerated atherosclerosis. male ldlr-/-mice were fed either a diabetogenic diet alone or in combination with telmisartan (10 mg/kg), valsartan (25 mg/kg) or valsartan (50 mg/kg) from 8 weeks of age for 17 weeks. all treatment groups except of the lower valsartan dose showed significant effects on reducing the aortic plaque score. the content of ha, collagen and decorin in the aortic root were not changed. however, telmisartan reduced the content of biglycan in the aortic root significantly in contrast to valsartan. in addition, a trend towards decreased mac2-positive macrophages in abdominal adipose tissue was detectable after telmisartan treatment as well as a strong reduction in the adipose tissue mrna expression of biglycan. finally, telmisartan reduced the expression of hyaluronan catabolizing enzymes potentially leading to an increase of high molecular weight ha in the adipose tissue, which is thought to be homeostatic and antiinflammatory. in summary, the results of this study underline the pronounced anti-inflammatory capacity of telmisartan on atherosclerosis and adipose tissue inflammation in comparison to valsartan and strongly suggest that biglycan might be an additional target of telmisartan not only concerning matrix composition of atherosclerotic lesions but also concerning the structure of adipose tissue and metabolic effects of the compound. human primary malignant cancer cells derived from peritoneal effusions of a patient with colorectal carcinoma, as assessed by comet assay. the primary cancer cells were more efficient in dsb repair than ht-29 cells, and their doxorubicin ic50 was four times higher. comparative protein expression levels showed that the primary cells had less rad 51 and 52 as well as less topoiiα, while ku70 and 80 levels were similar. another very interesting protein is the mrn (mre11-rad50-nbs1) complex that initializes the phosphorylation of atm and thereby starts the signalling cascade. the newly described mrn-atm pathway inhibitor mirin interrupts mrn activity by inhibiting the exonuclease activity of mre11. the toxicity of mirin in ht-29 cells was measured using a luminescence-based assay detecting the amount of atp, which is correlated with cellular viability. mirin did not show any toxic effects up to a concentration of 100 µm and incubation times of 24 hours, indicating that mirin can be used under these conditions without detrimental effects. we are currently investigating the effect of mirin on the toxicity of topoiiα inhibitors. the inhibition of dna repair may be a valuable strategy to enhance the effect of dnadamaging anticancer drugs. since tumours (even of the same entity) are not only heterogeneous, but also polyclonal, a broad selection of response modifiers of anticancer drugs would be helpful to individually enhance chemotherapeutic effectiveness. evidence has been provided that diet and environmental factors directly influence epigenetic mechanisms associated with cancer development in humans. epigenetics play an important role in the control of gene expression. epigenetic mechanisms comprise modulation in dna methylation, histone modification and non-coding rna. several polyphenols have been reported to possess histondeacetylase (hdac) inhibitory properties [1] . histone deacetylation is generally linked to transcription repression. furthermore, hdac belongs to the group of small ubiquitin-related modifier (sumo) substrate proteins. sumoylation of hdac is associated with a modulation of its biological activity [2] . little is known so far about the mechanism by which hdac sumoylation mediates inhibition of gene transcription. we addressed the question whether sumo e1 and hdac 1 expression and whether potential hdac-sumoylation will be affected by polyphenols such as chlorogenic acid, genistein and (-)epigallocatechin-3-gallate (egcg). chlorogenic acid, genistein and egcg decreased sumo e1 protein level in the human colon carcinoma cell line ht29 after 24h of incubation measured with western blot analysis. egcg exhibited the most pronounced effect at concentrations ≥ 50 µm. hdac 1 expression was also affected by these polyphenols. the direct impact of polyphenols on the hdac sumoylation is detected by co-immunoprecipitation experiments with the respective antibodies against hdac-1 and sumo e1. these experiments are still under investigation. in conclusion, chlorogenic acid, genistein and (-)-epigallocatechin-3-gallate influenced the sumo and hdac expression in vitro. in further studies the direct impact on subtract-sumoylation will be investigated. these studies contribute to a better understanding of potential chemopreventive effects of dietary polyphenols on specific epigenetic alterations may provide chemopreventive strategies for reducing cancer risk. the no/cgmp cascade is thought to be essential for penile erection. within the smooth muscle of corpus cavernosum, nitric oxide activates the no-sensitive guanylyl cyclase (no-gc) which raises the intracellular concentration of cgmp. this second messenger activates the cgmp-dependent protein kinase i (pkgi) and subsequent phosphorylation of target proteins leads to relaxation of cavernosal smooth muscle. knock out of key enzymes of the no/cgmp cascade has led to discrepant results: the deletion of pkgi in the mouse has been shown to lead to erectile dysfunction whereas mice lacking neuronal no synthase are fertile. to investigate the role of the no receptor in fertility we have generated mice lacking no-gc (gcko), a bottleneck enzyme of the no/cgmp cascade. we have shown that lack of no-gc resulted in arterial hypertension concomitant with a totally abolished no responsiveness of vascular and gastrointestinal smooth muscle. in addition, we generated a mouse line in which no-gc was specifically deleted in smooth muscle cells (sm-gcko). using these ko strains we here examined the role of no/cgmp signaling with regards to the smooth muscle relaxation of corpus cavernosum. no failed to affect corpus cavernosum from gcko in organ bath experiments: neither exogenously produced no by no donors nor endogenous no release from neurons induced by electrical field stimulation led to relaxation. similar results were observed in the corpus cavernosum of sm-gcko mice. to our surprise, the gcko animals were fertile and produced offspring albeit at a reduced rate compared to wt animals. our data show that interruption of no/cgmp signaling results in complete absence of no-induced relaxation of penile corpus cavernosum in mice and reduces the ability to produce offspring but does not abolish fertility. novel modes of invasive cell motility regulated by the formin class of actin nucleators khan j., grosse r. philipps-universität marburg, pharmakologisches institut, karl-von-frisch-str. 1, 35034 marburg, germany pathological invasive cell migration essentially reqires actin polymerization. formins are the largest group of rho-gtpase effectors involved in actin nucleation and assembly as well as microtubule dynamics. here we studied the role of formins in cytoskeletal regulation during homotypic cancer cell invasion. we identified the actin-dependent steps and structures involved for this process. using live cell analysis we characterize the distinct actin dynamics controlled by formin-like 2 and rho function. the specific involvement of this signaling module will be discussed. formin-driven nuclear actin assembly controls mal/srf activity baarlink c., wang h. polymerization of actin in the cytoplasm is tightly linked to transcriptional activation of the srf cofactor mal (also known as mrtf-a) through release of actin/mal interactions and subsequent nuclear accumulation of mal. formins directly promote assembly of actin filaments thereby efficiently regulating mal-dependent transcription for cell shape, adhesion and motility. here we show that formins assemble f-actin and promote mal activation inside the mammalian nucleus. the rho-gtpase effector mdia2 rapidly enters the nucleus in a signal-dependent fashion and an active mdia confined to the nucleus potently promotes release of g-actin from mal to specifically activate srf. live cell imaging reveals formin-mediated nuclear actin dynamics. moreover, using actin assembly assays we find that inhibition of endogenous mdia formins controls f-actin turnover in isolated nuclear extracts. thus, formin activity is dynamically compartmentalized to the mammalian nucleus to potently regulate actindependent mrtf function. in women the placenta becomes the main source of maternal estrogens during pregnancy. placental estrogen biosynthesis is located in the syncytiotrophoblast, a syncytium that builds the main part of the placental barrier and limits the transfer of substances between the fetal and maternal compartment. since the human placenta is unable to convert cholesterol into 17-oh-pregnenolone, the placenta tissue highly depends on the supply of c-19 steroids for their conversion into c-18 estrogens. in contrast to lipophilic unconjugated steroids that penetrate the cell membrane passively via diffusion, circulating sulfated steroid hormones are delivered to the placenta via carrier-mediated transport, followed by their reactivation via the catalytic activity of the steroid sulfatase (sts). dheas of maternal and fetal origin contributes about equally to the placental formation of estrone (e 1) and estradiol (e2), while 16αoh-dheas supplied by the fetus contributes to over 90% of placental estriol (e3) synthesis. soat, a member of the slc10 family with highest expression in hormone-responsive tissues such as testis, placenta, and mammary gland has been shown to transport the sulfoconjugated steroid hormones dehydroepiandrosterone sulfate (dheas), estrone sulfate (e1s), and pregnenolone sulfate (pregs) [1] . aim of this project is to investigate the role of soat for placental estrogen synthesis by means of the choriocarcinoma cell line jeg-3 as in vitro model for the human syncytiotrophoblast. therefore, we characterized a jeg-3 cell line that transformed dhea into e2 and 16αoh-dhea into e3. by qrt-pcr we found expression of sts and aromatase, both essential for estrogen synthesis in these cells. upon transient transfection of soat the carrier was located in the cell membrane of transfected jeg-3 cells. currently we investigate the transformation of dheas of these soat-jeg-3 cells by lc-ms-ms. we could demonstrate transport of 16αoh-dheas for stably transfected soat-hek293 cells. in situ hybridization and immunohistochemistry showed coloured syncytiotrophoblasts and vascular endothelial cells in late term placenta. in conclusion, soat-mediated transport of sulfated steroids could play a pivotal role for placental estrogen synthesis from sulfated steroid hormones. developing non-animal test systems for evaluation of toxicity was important in the past and will remain essential in the future. here we present a toxicity test using the chicken yolk sac area vasculosa (cav) of fertilized white leghorn chicken eggs [1, 2] and compare it to hen's egg test on chorioallantoic membrane (het-cam) [3] for polymer toxicity testing. fertilized chicken eggs were incubated and after 72 h explanted shell less into sterile petri dishes. test substances were applied on the cav and the appearance of different effects (vascular lysis, haemorrhage, aggregation of blood components, lethality) was determined by light microscopy after 1 -48 h (fig.1 ). these effects were combined to a cav test score based on the irritation score calculation used for het-cam evaluation. different polymers like poly(ethylene glycol) (peg; neutral), poly(ethylene imine) (pei; cationic) and dextran sulphate (ds; anionic), as well as guideline-conform (recommended het-cam protocol from the interagency coordination committee on the validation of alternative methods) negative (0.9 % nacl) and positive controls (1 % sodium dodecyl sulphate (sds) and 0.1 n naoh) were investigated. additionally ld 50 values for different cationic polymers have been determined. within the selected incubation times (1 -48 h) , effects such as vessel lysis and blood component aggregation could be detected. additionally to het-cam, lethality as well as recovery of the cav could be observed. differences between neutral, positively and negatively charged polymers were obtained. pei showed strong vessel lysis and aggregation of blood components whereas ds and peg showed none of these effects. lethality was found to increase from peg < ds < pei and is concentration and time dependent. the results demonstrate that differences, regarding the toxicity of the used polymers, can be shown with this test. these findings in the cav test can be well correlated with already existing data. in summary, the cav test provides same data (testing control substances) and more information (recovery and lethality) compared to het-cam and could be a suitable model for toxicity testing of polymers. risk characterisation of chemicals consists of three steps (1) hazard identification and characterisation, based on substance-specific toxicological hazard data, (2) estimates of the level of exposure toward the substance and (3) the comparison between the toxicologically safe level and the exposure level. in contrast to the classical risk assessment approach, the threshold of toxicological concern (ttc) approach is developed as a tool to assess the risk of substances without toxicity data. its application requires (1) information on human exposure, for which it is essential that exposure is fully captured and (2) knowledge of the chemical structure to assess whether the chemical is not excluded from the application of the ttc concept. instead of chemical specific no observed (adverse) effect levels (noels/noaels), the ttc approach utilises knowledge on the empirical distribution of several hundreds of noels/noaels, originally 613, based on toxicological testing in animals (munroe et al., 1996) . with the basis on noaels, the ttc concept builds on the fundamental principle of toxicology, that toxicity is a function of dose and that a dose exists, below which no adverse effects of the substance can be detected. it is assumed that exposures below this level will not result in health risks. three separate ttcs were derived (munroe et al., 1996) by classifying the chemicals into three toxicity classes using a decision tree based on a series of 33 questions related to chemical structure, and on natural occurrence in food and in the body (cramer et al., 1978) . the ttc values are derived from empirical distribution of the noels/noaels in the class taking the 95th percentiles and dividing them by the default uncertainty factor of 100. it is assumed that the probability is very low that the unknown noael of a not tested chemical will be lower than the value of the 95th percentile in the distribution of the known noels/noaels. hence, at exposures below the ttc values, the probability of adverse effects on human health is considered to be very low. introduction: kibra, mainly expressed in kidney and brain tissue, is involved in brain development and memory formation as a postsynaptic scaffold protein. in podocytes, kibra is proposed to regulate cell motility as a linker between components of the cytoskeleton and polarity protein complexes (duning et al, jasn 2008) . furthermore, kibra has been identified as key regulator of the hippo pathway, which is involved in organ size control and tumorigenesis. in the current study, we focused on the identification of kibra gene expression regulation and functional promoter characterization. methods: serial promoter deletion constructs were generated by cloning 3627 bp of the 5'flanking region of kibra into the pgl3-vector system. deletion constructs were transiently transfected into human neuroblastoma cells (sh-sy5y) and immortalized human kidney epithelial (ihke) cells. potential transcription factors (tfs) were investigated in cotransfection experiments. transcriptional start sites (tss) were determined by rapid amplification of 5'cdna ends (5'race) . tss utilization between cell lines was assessed by semiquantitative pcr. transcriptional activity (ta) of the kibra promoter p1 was separated by ~1060 bp into two distinct regions, promoter p1a and p1b. deletion constructs harbouring promoter p1b were transcriptionally active only in ihke cells. 5'race revealed two alternative tss in both cell lines upstream of the annotated tss (nm_015238). exclusively in ihke cells, two additional tss were detected in intron 1, resulting in two alternative exons. deletion constructs harbouring the putative regulatory regions (p2 and p3) of both exons were transcriptionally active only in ihke cells. overexpression of full length tcf7l2 (transcription factor 7-like 2 [t-cell specific, hmgbox]) resulted in a ~3-fold increase of promoter p1a and intron promoter p2 ta. kibra gene expression is driven by a complex alternative promoter system comprising the constitutional promoter p1 and three alternative promoters p1b, p2 and p3. the tss utilization is cell type-specific. subsequent usage of an alternative translation start site within exon 3 could result in truncated kibra protein isoforms. tcf7l2 is involved in the differential kibra gene expression regulation. resulting kibra protein isoform and their cellular function will be assessed in further studies. skin absorption in vitro based on the study of human/animal skin ex vivo or reconstructed human epidermis, respectively, is an alternative method which is accepted by the oecd. guideline tg 428 and a corresponding technical guidance document (gd 28) give technical guidance how to perform valid experiments 1, 2 . the requirements include integrity evaluation tests for the skin samples. different tests are proposed to ensure an exclusively use of undamaged skin. to decide which test suites best to our routine test strategy, we investigated the correlation between integrity test results and absorption profiles of various penetrants (logp range: -0.07 -6.2). finite dose experiments using rat and human skin were performed with 14 c-labeled testosterone, caffeine, mcpa (4-chloro-2-methylphenoxyacetic acid) and its 2-ethylhexyl-ester mcpa-2ehe. for each experiment at least three of the five following integrity tests were conducted: transepidermal electrical resistance (teer), transepidermal water loss (tewl), transepidermal tritiated water flux (³h2o), transepidermal absorption of methylene blue (blue) , transepidermal absorption and flux of a ³h-labeled internal standard (istd); ³h-testosterone or ³h-mannitol was used as istd. the applied radioactivity of the ³h-istd was selected to show no analytical interference with the 14 c-penetrants. teer, tewl and ³h2o represent pre-study, istd concurrent and blue post-study tests. calculated maximal permeability constants (kp) and absorbed doses (ad) of the penetrants were compared to the results of the integrity tests. individual linear regression analysis was used to evaluate the correlation the correlations varied over a wide range for all five methods and four penetrants. the best correlations in average were achieved with the istd. no inverse correlations were obtained for the istd, but partly for tewl, teer, ³h2o and blue. in conclusion, the istd represents best the achieved absorption profiles of the test compounds and is based on that the most suitable integrity test for our dermal absorption studies. we will further confirm its effectiveness and generate a sufficient historical database in order to include the istd in our routine test protocol. investigation of mirna expression and dna methylation in focal and non-focal brain tissue of therapy-resistant epilepsy patients haenisch s. 1 background: resistance to anticonvulsants affects one third of all epilepsy patients. limited bioavailability of the drug at the target site caused by increased expression of efflux transporters on the blood brain barrier or alterations of target genes are potential mechanisms for therapy resistance. however, these mechanisms alone cannot completely explain the observed resistance and it is likely that multifactorial alterations lead to pharmacoresistance. there is increasing evidence that expression of micrornas probably caused by dna modifications is deregulated in many neuronal diseases. we hypothesize that mirna regulation of target genes is involved in drug resistance in epilepsy. methods: hippocampal focal and cortical non-focal brain tissue samples from 13 patients diagnosed with mts (mesial temporal sclerosis) who underwent neurosurgery have been screened for mirna expression using taqman low density arrays. in silico approaches for both a hypothesis-based (efflux-transporter and target gene) as well as a hypothesis-free approach were used to identify potential phenotype-relevant target genes. using the program r (bioconductor) a mann-whitney-u test was performed to compare mirna expression between brain regions. pyrosequencing was performed to investigate methylation status 5'-upstream of dna regions encoding for selected candidate mirnas. results: out of 754 mirnas, 150 were detected in both tissue types. the expression of one mirna was 7.2 fold higher (q=0.01) and another was 3.8 fold lower (q=0.01) in the hippocampus relative to the cortex. evidence could be found that down-regulation of the latter is possibly caused by hypermethylation of 5'-flanking region of its encoding dna locus. bioinformatic analysis has identified eight genes important for neuronal regulation and signal transmission (e.g. sox11, mecp2, bsn), as well as one abc effluxtransporter, as potential targets for these differentially regulated mirnas. conclusion: differential regulation of two mirnas could contribute to an altered function of several genes resulting in an imbalance between neuronal excitation and inhibition that is independent from mechanisms presently targeted by anticonvulsants. this work was supported by a fellowship from dfg and nih grant gm61390. recently, it has been reported that human b cells express and secrete the cytotoxic protease granzyme b (grb) after the combined stimulation of the il-21-and the b cell receptors. grb produced by b cells is enzymatically active and b cells deliver grb to sensitive cancer cell lines, thereby inducing apoptosis. to date, there is little experimental evidence on the mechanisms involved in grb expression, or its function in b cell biology. as experimental transgenic murine systems should enable us insights into these issues, we assayed for grb in c57bl/6 b cells using an extensive array of physiologically relevant stimuli, but were unable to detect either grb expression or its proteolytic activity, even when antigen specific transgenic b cell receptors were cross-linked. similar results were also obtained with b cells from dba/2, cba or balb/c mice. in vivo, infection with either influenza virus or murine γ-herpesvirus induced the expected expression of grb in cytotoxic t lymphocytes, but not in b cell populations. we also investigated a possible role of grb on the humoral immune response to np-klh, but grb-deficient mice produced normal amounts of antibody with typical affinity maturation and heightened secondary response, demonstrating conclusively the redundancy of grb for antibody responses. our results highlight the complex evolutionary differences that have shaped the immune systems of mice and humans and demonstrate the need to develop novel in vivo systems to study human humoral immune responses. investigations of the cholinergic neurotransmitter system in dyt1 mice hamann m. 1 early-onset torsion dystonia is an autosomal dominant inherited movement disorder associated with the dyt1 gene defect with deletion of a glutamic acid residue in the protein torsina. despite the gene defect, the pathophysiology is poorly understood. animal models can help to understand the underlying mechanisms and thereby to develop new therapeutic strategies. sharma et al. (2005, j. neurosci. 25 [22] , 5351-5355) initially described a transgenic mouse model (dyt1 mice) with overexpression of mutant torsina. previous studies in these mice pointed to alterations in the cholinergic system. to investigate the functional relevance of these in-vitro findings, we carried out pharmacological in-vivo experiments and determined the density of striatal cholinergic interneurons as well as the expression of choline acetyltransferase in different brain regions. the acute intraperitoneal administration of the cholinomimetic drug pilocarpine (75, 100 and 125 mg/kg) as well as a long-term treatment over 21 days (100 mg/kg/d) did not induce pronounced effects in dyt1 mice compared to wildtype controls. the higher incidence of epileptic seizures in dyt1 mice compared to controls after repeated local striatal applications of pilocarpine (25 and 50 µg/0.5 µl/hemisphere) let presume an altered synaptic plasticity in dyt1 mice. the immunohistochemical investigations revealed a moderately reduced density of striatal cholinergic interneurons in the dorsomedial subregion of dyt1 mice compared to wildtype controls, while significant differences in other striatal subregions were not detected. western blot analysis did not show clear differences in the expression of choline acetyltransferase between dyt1 and wildtype control mice. these results indicate that the cholinergic system seems not to play a key role in this line of dyt1 mice. ongoing receptor autoradiographic analysis of binding to different muscarinic receptors subtypes have to further clarify the existence of possible alterations within the cholinergic system of these dyt1 mice. inhibitors direct against cell cycle-regulatory kinases are being tested in clinical trials as anti-proliferative agents. thus, the atp-competitive kinase inhibitor pd332991 which inhibits cdk4 and cdk6 is currently tested in patients with solid tumors such as glioma. we found that pd332991 suppressed il-1-induced expression of il-8 suggesting that cdk4 or cdk6 may have unknown anti-inflammatory properties. to study the effects of cdk6 on the il-1-signaling network, we established a bidirectional doxycyline-inducible system to express a constitutively active mutant of cdk6, cdk6 s178p, in asynchronized hela cells. cdk6-expressing cells were identified by gfp which was expressed from the same promoter, isolated by laser-microdissection and analysed for mrna expression using a down-scaled rt-qpcr assay. compared to the uninduced state, cdk6 s178p enhanced il-1-induced il-8 and il-6 mrna expression. moreover, shrna-mediated suppression of endogenous cdk6 confirmed a role of this kinase in regulation of maximal il-1-induced gene expression of il-8. these data also revealed that the contribution of cdk6 to inflammatory gene expression is highest in g1, when activity of endogenous cdk6 is activated by d-type cyclins. these findings were corroborated in hela cells expressing fluorescent ubiquitin-dependent cell cycle indicator (fucci) proteins. hela-fucci cells from g1, g1/s, g2 or mitotic states were isolated by laser-microdissection and analyzed by rt-qpcr for tnf-inducible gene expression. stable knockdown of cdk6 in hela fucci or inhibition by pd332991 suppressed inducible il-8 expression. microarray experiments identified many additional genes that required active cdk6 for maximal il-1-or tnf-inducible gene expression. we also found that cdk6 co-immunoprecipitated with p65 nf-κb, colocalized with p65 in the nucleus and was recruited together with the p65 subunit to the proximal il-8 promoter as assessed by chip and re-chip experiments. collectively, these results suggest an unexpected control of inflammatory gene expression through a classical cell cycle regulatory pathway. these results also imply that pharmacological targeting of cdks may have effects and side-effects on the immune system in addition to inhibition of cell cycle progression. trp channels form a heterogeneous family of calcium-permeable channels, which play major roles in physiological functions ranging from sensory reception to cellular signal transduction. members of the trpc subfamily (classic transient receptor potential channels) are downstream targets of hormone receptors. of particular interest is the biological role of trpc6 channels. they are directly activated by diacylglycerol due to phospholipase c-driven signalling pathways which are involved in smooth muscle contractility, neuronal plasticity, keratinocyte differentiation and renal function. their impact in renal function became evident from analyzing patients suffering from familial forms of focal segmental glomerolusclerosis (fsgs) which could be linked to trpc6 mutations. since the first descriptions at least 17 different pathogenic mutations have been identified in humans to cause fsgs. in order to study the underlying pathophysiological mechanisms of trpc6 mutations, we have analysed all mutated trpc6 channels known to date heterologously expressed cells. one set of mutations showed a gain-of-function phenotype which has been previously suggested to cause an increased intracellular calcium load and subsequent cell death. hence, gain of function mutations fit to the current paradigm of fsgs pathophysiology. however, another set of mutations found in the patients showed a loss-of-function phenotype. our results enable a change in the current paradigm for the role of trpc6 in renal pathophysiology and may provide a basis for our understanding of the pathophysiology of loss-of-function mutations in familial focal segmental glomerolusclerosis. karlsruher institut für technologie (kit) institut für angewandte biowissenschaften, abteilung lebensmittelchemie und toxikologie, adenauerring 20a, 76131 karlsruhe, germany risk assessment for genotoxic carcinogens is an important challenge in toxicology. even though manifold attempts have been made to substitute carcinogens and to reduce exposures, their complete elimination appears to be not possible. thus, low concentrations of known or suspected genotoxic carcinogens are present at workplaces, in the environment and in food. in order to deal with this situation and to set priorities for risk management, different concepts have been established such as the alara principle (as low as reasonably achievable) and the margin of exposure (moe), based on the ratio between concentrations being carcinogenic in experimental animals and the actual exposure of humans for example via foodstuff. while usually linear doseresponse-relationships have been used as default assumption, analytical methods are now available to assess the induction and repair of dna lesions on low exposure conditions, including environmental background exposure, and to relate the extent of exposure-induced dna lesions to endogenous dna damage. this may be an important prerequisite to establish health-based limit values for selected genotoxic carcinogens. within this workshop, different examples will be discussed and research need will be identified. dendritic cells from h4r-deficient mice lose their ability to properly stimulate t lymphocytes hartwig c., seifert r., neumann d. mhh pharmakologie, carl-neuberg str. 1, 30625 hannover, germany the incidence of allergic airway diseases is increasing throughout the world, especially in western countries. although histamine (ha) is found at high concentrations in asthmatic lungs, a role for ha in bronchial asthma is still a neglected topic in clinical research. in particular, the capacity of ha to modulate the underlying immune reaction is far from being understood. the histamine h4-receptor (h4r) is involved in acute inflammation and th2 cytokine production. consequently, we intended to analyze the role of h4r in a murine th2 lymphocyte transfer-based model of asthma. specifically the ability of h4r expressed on dendritic cells (dcs) to modulate t cell function was analyzed. ova-specific cd4 + t cells were polarized in vitro under th2-favoring conditions with ova peptide-pulsed dcs, obtained either from wild-type or h4r -/mice. analysis of the polarized t cells after in vitro restimulation revealed a marked decrease of il-4 production in t cells polarized in the presence of h4r -/-dcs compared to those polarized in the presence of wild-type dcs. thus, on dcs, the h4r is essential for proper stimulation of spleen t cells and for directing their polarization towards a th2 phenotype. the transfer of in vitro polarized t cells into recipient mice and subsequent provocation elicited an asthma-like disease. the h4r on dcs not only affects in vitro polarization of t cells, but also the in vivo function of the obtained polarized t cells. a parameter indicating allergic inflammation is the enhanced influx of inflammatory immune cells into the lung tissue, mainly driven by eosinophils, which are virtually absent in non-asthmatics. when analyzing the number of eosinophils, a dramatic difference due to the polarizing conditions of t cells occurs. in bal fluids of mice that received t cells polarized in the presence of wild-type dcs, about 40% eosinophils were detected. in contrast, the transfer of t cells polarized in the presence of h4r -/-dcs yielded only about 10-20% eosinophils in bal fluids. in summery, the h4r on dcs plays an important role for t cell polarization and consequently affects the allergic reaction during sensitization. since the lack of the h4r on dcs reduced their ability to stimulate proper th2 polarization of cd4 + t cells, we conclude that ha via the h4r significantly affects the manifestation of asthmatic inflammation. antioxidant polyphenols and their effects on nrf2 (skn-1) signalling in a cell culture system and the model organism c. elegans havermann s., wätjen w. heinrich-heine-universität düsseldorf institut für toxikologie, p.o. box 101007, 40225 düsseldorf, germany oxidative stress has been connected with a variety of diseases, (e.g. alzheimer`s and parkinson´s disease), cancer and ageing over the last years. certain polyphenols were shown to have an antioxidant capacity as well as being able to activate the protective nrf2 signalling pathway. compared to direct radical scavengers modulators have the advantage of building up a permanent defense against oxidative insults whereas scavengers do not protect any more after consumption or may even cause stress due to redox cycling. we have employed cell culture based assays (dcf, western blot, gfp reporters) to analyse the effects of polyphenols. further we tested the coumpounds in vivo in the nematode c. elegans where skn-1 is the nrf2 homologue. baicalein and caffeic acid phenethylester (cape) protected cells and the nematode from ros accumulation after application of stress (shown by dcf assay). activation of nrf2 signalling is correlated with translocation of the transcription factor into the nucleus. in both systems nrf2::gfp accumulation in the nuclei could be observed after incubation with baicalein (fluorescence microscopy). but while cape is a potent activator of nrf2 in cells, it has no effect on skn-1 localisation. further the effect on the nrf2 protein amount was investigated by western blot analysis. the expression of target genes can be investigated by differing means: while pcr methods and western blotting are standard for in vitro studies, the vast number of available gfp reporter strains offers opportunities for research using c. elegans. we have performed congruent assays in a cell culture system and the model organism c. elegans to compare antioxidative capacity and effects of polyphenols on nrf2 signalling. therefore, depending on the substance tested, c. elegans is a suitable model system to investigate effects of natural compounds in an organism. being associated with adverse health effects, the human exposure to dehp is subject to concern. quantifying the population's exposure and determining the contributions of different exposure routes is a key task of environmental health risk assessment. the study presented comprises a review of the available data on dehp levels in foods, consumer products, and house dust. extensive survey data, e.g. from the current national nutrition survey ii and the eu rapex system were processed for modeling the exposure by the oral, inhalative and dermal path of the population in germany. the study also included analytical analyses of dehp levels in selected foods and consumer goods (incl. migration rates for mouthing). probabilistic techniques allowed elucidating the exposure's variation and the relevance of different routes. mean exposure estimates for german children and adults to dehp are 36 and 26 µg/(kg d), resp. for children, food accounts for 36% of the total exposure, followed by mouthing (30%) and house dust (19%). the adult exposure is almost entirely (82%) due to food. as dietary exposure is a result from concentration and consumption, foods exhibiting high contamination e.g. butter (8%) and dressings (mayonnaise) (14 %) as well as highly consumed foods e.g. bread and bakery (16%) and vegetables (6,8 %) contributed significantly. the mean estimate of children's dehp exposure via mouthing revealed 0,9 µg/(kg d). high exposures were estimated (95th percentile) up to 10,8 µg/(kg d). on average people in germany are exposed to dehp below the current tdi of 50 µg/(kg d). however, individual exposures exceeding the tdi still cannot be excluded. current data on dehp and other plasticizers in foods are scarce, which warrants broader monitoring. our findings highly facilitate further exposure modeling focusing on dehp substitutes and risks of combined exposure. this study was funded by the federal ministry for the environment, nature conservation and nuclear safety in the frame of the environmental research plan (umweltforschungsplan, förderkennzeichen (ufoplan) 3707 61 201). on the basis of the available measurements of dehp, the exposure assessment has been focused on 37 food categories characterising a selection of the most important food groups covering all major food classes of the german population. based on an extensive literature survey, the analysis considered the available data of dehp measurements in food, as well as the official german food control data taken from the national food consumption survey. the high amount of considered data allowed the consideration of several exposure assessment tiers (deterministic and probabilistic by using monte carlo simulation). a quantitative evaluation of the uncertainties of the estimate of the 37 food categories groups has been performed by means of a sensitivity analysis by using the methodology proposed within the 2008 who ipcs guidance document of characterising and communication uncertainty in exposure analysis. qualitative uncertainty analysis (tier 1) was applied to determine the most important sources of uncertainty, i.e. concentration of dehp in all food categories. the probabilistic monte carlo simulation (tier 2) was then used to rank the cumulative probability distributions of the exposure assessments of 37 food categories on the basis of the food categories that appear to dominate. sensitivity analyses were applied to prove the impact correlation of food groups for uncertainties. by a scenario based concept, the aggregation of the 37 food groups to 10 groups has been evaluated, as well as the sensitivities by characterising particular scenarios. for this purpose, particular "meals" have been described as fixed combined scenarios and. the aggregation leads to a considerable higher exposure estimate which can be explained by the combination of high contaminated foods with others of high consumption. the evaluation confirms the considerable role of possibly high contaminated foods e.g. fats, or mayonnaise. the evaluation shows that quantitative probabilistic sensitivity analysis is a suitable and pragmatic tool for uncertainty analysis in exposure assessment. the transcription factor camp response element (cre)-binding protein (creb) plays a critical role in regulating gene expression in response to activation of the campdependent signaling pathway, which is implicated in the pathophysiology of heart failure. we observed creb knock-out cardiomyocytes to be larger than wildtype cardiomyocytes (cell area in µm 2 ; mean±sem; creb-ko 4871±258 vs. wt 3396±171; n=68/69 cells, n=4 mice, p<0.01 vs. ctr.). the nuclear factor of activated t-cells c3, nfatc3, is another transcription factor involved in the development of heart failure and also a known positive regulator of hypertrophy. hence, we investigated whether inhibition of the cre-dependent transcriptional activation has an impact on the nfatc3 signaling pathway. we first studied the effects of an overexpression of a dominant negative creb mutant (dncreb) or of nfatc3 on the activity of a nfat-dependent model promoter in a permanent cell line. overexpression of dncreb evoked an 8.0±1.5 fold increase of the nfat model promoter activity (n=36; n=6 transfections), but had no impact on kv4.2 promoter activity which is known to be regulated by nfatc3 (1.2±0.1 fold; n=18; n=3; p<0.05 vs. ctr.). nfatc3 overexpression led to a 4.5±0.7 fold increase of the nfat-dependent model promoter activity (n=12; n=2) and to an inhibition of kv4.2 promoter activity (0.8±0.1 fold; n=18; n=3; p<0.05 vs. ctr.). we conclude that creb is a negative regulator of nfat-mediated gene transcription and that activation of nfatc3 might contribute to the observed hypertrophy of creb-ko cardiomyocytes. neuroleptika der perazin-klasse sind potente modulatoren des p2x7-rezeptors -perazine-type neuroleptic drugs are potent modulators of p2x7 receptors hempel c., nörenberg w., urban n., sobottka h., schaefer m. universität leipzig rudolf-boehm-institut für pharmakologie und toxikologie, härtelstraße 16-18, 04107 leipzig, germany p2x7 receptors belong to a family of atp-gated, non-selective cation channels, which play an important role in immune cell activation, inflammatory hyperalgesia and neuropathic pain. they differ from other p2x family members by the low atp affinity, and by the ability to form or recruit dilated pores in the sustained presence of atp. owing to its involvement in many diseased states, p2x7 is a promising target for pharmacological intervention. accordingly, p2x7 blockers are currently tested in phase ii clinical trials. in an attempt to identify p2x7-modulating properties of approved drugs or natural compounds, we performed a medium-throughput screen, using an appropriate compound library (spectrum collection) and a stably transfected hek293hp2x7 cell line. with ic50 values of 1-2 µm, the tricyclic antipsychotics prochlorperazine and trifluoperazine showed a high potency and efficacy to block the atp (1 mm)-triggered increases in the intracellular ca 2+ concentration ([ca 2+ ]i) that was mediated by human p2x7 (hp2x7). the closely related phenothiazine-class neuroleptic drugs, such as chlorpromazine or triflupromazine did not have an appreciable effect on hp2x7mediated ca 2+ influx. whole-cell inward currents, measured at -60 mv, were blocked by more than 60% by 3-10 µm prochlorperazine. the inhibitory effects of perazines developed within about 200 ms, hinting to a direct mode of action by binding to the p2x7 protein. prochlorperazine added intracellularly via the patch pipette did not substitute for the extracellularly applied drug, indicating that its binding site is accessible from the extracellular side. in addition, both compounds blocked yo-pro-1 uptake when preincubated before p2x7 stimulation with 1 mm atp or when applied subsequent to the agonist. interestingly, when added to a hek293 cell line expressing the rat p2x7, perazines potentiated the atp-induced increase in [ca 2+ ]i. measurements in human monocyte-derived macrophages confirmed the ability of prochlorperazine and trifluoperazine to inhibit atp-evoked increases in [ca 2+ ]i, changes in yo-pro-1 permeability and whole cell currents. taken together, we conclude that perazine-type neuroleptics impede on p2x7 activity in a species-specific manner, presumably by binding to an extracellularly accessible binding site of recombinant or natively expressed p2x7. similarly, pre-treatment with lov also lowered dox-induced stabilisation of p53 and phosphorylation of chek1 and sapk/jnk. while lov had no influence on ir-induced initial dna damage formation in huvec and rat cardiomyoblasts (h9c2), it decreased dox-and eto-induced phosphorylation of histone h2ax, which is a surrogate marker of dna-double strand breaks. this indicates that lov specifically protects against the genotoxicity of topoisomerase type ii poisons. in an acute and subacute balb/c mouse model lov protected from ir-induced toxicity. this effect rested on inhibition of pro-inflammatory and pro-fibrotic processes as measured via quantification of mrna levels of il6, ctgf and tnfα. the same was true for dox-induced toxicity, i.e. heart and liver damage. similar to the in vitro experiments, dox-induced hepatic dna-damage was attenuated by lov treatment. overall, liver and heart toxicity were reduced by lov as mirrored by the serum levels of gldh/gpt and ctn-i, respectively. both in liver and in heart we observed collagen rich perivascular areas following dox treatment. under situation of lov-co-treatment these areas occurred more rarely and were less pronounced, pointing to a lowered level of fibrosis. pcr-array-based mrna analyses showed inhibitory effects of lov on dox-triggered expression of genes involved in oxidative stress response, drug transport, dna repair, cell cycle progression and cell death. for instance, up-regulation of p21, wee1, cjun/fos and hmox-1 following dox administration was attenuated by lov. altogether, we suggest that including lov in current cancer therapeutic regimen might widen the therapeutic window of anticancer therapeutics by lowering normal tissue damage. the p values. in addition, array data underwent cluster analysis for identification of substantial differences of gene regulation among the three different types of biopsies. results: of particular interest in our study was the expression of genes coding for metabolism and transport proteins. therefore 42 genes from the 156 significant differentially regulated genes, were selected for the qrt-pcr analysis. genes coding for abcb1 and abcg8 transport proteins showed higher expression in the jejunal tissue one year after surgery compared to the duodenal tissue (fold change 1.80 and 1.73). moreover, cyp7a1 mrna involved in metabolic processes is higher expressed in postoperative jejunum than in the jejunum tissue taken during the surgery (fold change 1.9). in conclusion roux-en-y gastric bypass operation leeds a change of mucosal gene expression profile in the jejunum during one year. there was also a significant differential gene expression between the original duodenum and jejunum one year after surgery. these results give strong evidence that jejunum not exposed to pancreatic but only to gastric fluids may change its gene regulation. background. numerous genome-wide association studies (gwas) identified polymorphisms located in transporter genes such as slc2a9, abcg2, npt1, and urat1 as predicitive for the serum levels of urate 1 . these genes encode membrane proteins expressed in the apical membrane of human kidney proximal tubule cells and are assumed to facilitate tubular exchange of urate 2, 3, 4, 5 . importantly several single nucleotide polymorphisms (snp) located in vicinity of slc2a9 have been identified as highly associated with serum urate levels. little is known about the transcriptional regulation of slc2a9. therefore, the aim of our study was to investigate which sequences in the slc2a9 gene harbour ciselements and regulate its gene expression. we also asked whether intronic snps influence gene expression at the transcriptional level. methods and results. performing dual luciferase reporter gene assays we found gene regulating modules in the slc2a9 gene. dna from human kidney samples was then genotyped for rs6449237 being part of this region. next total slc2a9 mrna-expression levels of the samples were determined using real-time quantitative rt-pcr assay. male samples with two minor alleles of snp rs6449237 showed lower slc2a9 mrna levels than samples with the wild type alleles. the effect was not seen in females. reporter gene constructs with either minor or major allele of rs6449237 were then used in luciferase assays, however showing no significant difference in activity. furthermore mrna-expression levels of other urate transporter genes were determined in kidney samples. after linear regression a positive correlation of mrna-expression of slc2a9, urat1, npt1, and oat10, respectively was observed. conclusion. our data suggest that the slc2a9 snps rs6449237 and rs74794351 might influence slc2a9 mrna-level without controlling the transcriptional activity. it needs to be elucidated whether those snps alter mrna stability. however, the mrna coexpression of slc2a9 and other urate transporter might be attributed to a common gene regulating pathway of an "transportosome" controlling urate homeostasis. sulfotransferases mediate the bioactivation of methyleugenol to a reactive sulfate ester binding to dna in vitro and in vivo herrmann k. 1 methyleugenol (me) is a secondary metabolite occurring in many herbs and spices. although me is hepatocarcinogenic in rodents, standard genotoxicity tests were negative. this may be due to the lack of critical activating enzymes responsible for the terminal bioactivation of me to a genotoxicant. me is initially hydroxylated by cytochrome p450 enzymes yielding 1´-hydroxymethyleugenol (1´-ohme). this alcohol can be further activated by sulfotransferases (sults) to an electrophilic sulfate ester that can be easily attacked by dna. the dna adducts formed could lead to mutation and further carcinogenicity observed in animals. the aim of the present study was to clarify whether individual human (h) and murine sult forms are involved in the activation of me to a genotoxicant. in order to identify critical sults, mutagenicity tests including bacteria expressing different sult forms were conducted. (±)-1´-ohme (separated into its enantiomers) served as test compound. we could show that hsult1a1, standing out due to its high expression level in many tissues, can efficiently activate both enantiomers even at low concentrations. furthermore, dna adduct formation in hsult1a1-proficient and sult-deficient bacteria was examined after incubation with 7 µm of (+)-or (-)-1´-ohme. for selective detection and quantification of me-derived 2´-deoxyadenosine (da) and 2´-deoxyguanosine (dg) adducts we developed a sensitive tandem mass spectrometry method including stable isotope dilution analysis. adduct formation was only observed in bacteria expressing hsult1a1. the concentration dependence of adduct formation in hsult1a1-proficient bacteria was examined for (+)-1´-ohme. both adducts turned out to be concentrationdependent. to check the extent and organ specificity of adduct formation in vivo we administered 54 mg/kg bw (±)-1´-ohme (i.p.) to mice carrying the hsult1a1/1a2 gene cluster. mice getting only the vehicle served as controls. animals were sacrificed and dna from eight organs was extracted. by means of tandem mass spectrometry adducts were measured and quantified. da and dg adduct formation was observed in all tissues studied, but not in untreated animals. furthermore, adduct levels were higher than in experiments using wild-type mice. altogether, we herein could show that sulfo conjugation leads to bioactivation of me to a dna-binding intermediate in vitro and in vivo. this work was financially supported by bundesinstitut für risikobewertung. objective: the soluble adenylyl cyclase (sac) activates the na + /k + -atpase in renal epithelial collecting duct cells. nuclear sac constitutes a functional complex with camp response element binding protein (creb), suggesting a more general role of sac in overall gene regulation. we determined the chromatin binding capacities of sac at cre sequences and its influence on genes, which play a role in aldosterone signalling. furthermore, we functionally characterised expression relevant promoter portions of sac and the influence of aldosterone and camp mediated signalling pathways on sac gene regulation. design and methods: in vascular endothelial cells (ea.hy926) and in human kidney cell lines (hek293t; ihke), we performed chromation immunoprecipitation (chip) assay with antibodies against sac and creb. we conducted transfection with a cre luciferase reporter vector and sac promoter constructs, following treatment with sac inhibitors and aldosterone. total rna of ea.hy926 cells, which were treated with sac inhibitors and aldosterone, was isolated and subsequently analysed by real-time pcr for expression of genes involved in aldosterone signalling. in vivo binding of sac at cre motifs was shown using cre consensus sequences in chip experiments. specific pharmacological inhibition of sac led to a significant decrease of transcriptional activity of the cre control vector in endothelial and kidney cell lines. furthermore, we were able to show the different effects of sac on the expression of downstream targets of aldosterone signalling, e.g. mineralocorticoid receptor and na + /k + -atpase alpha1 and beta1 and sac itself. regulation of sac itself is mediated by two different promoter portions, which are influenced by aldosterone and inhibition of sac and differentially accessed in kidney and endothelial cells. sac has transcriptional trans-acting properties as it interacts with cre sites and potentially influences the expression of genes, which play a role in aldosterone signalling. transcription of sac is regulated via aldosterone and camp. the location of promoter ta is cell type-and stimulation-specific. the role of the sodium-calcium exchanger (ncx1) in cardiac pacemaking herrmann s., stieber j., ludwig a. friedrich-alexander-universität erlangen-nürnberg institut für experimentelle und klinische pharmakologie und toxikologie, fahrstrasse 17, 91054 erlangen, germany the mammalian heart is driven by the sinoatrial node, the primary cardiac pacemaker. the unique feature of sinoatrial node (sn) cells is the ability to generate a spontaneous diastolic depolarization that periodically initiates action potentials which set the heart rhythm. the molecular origin of this cardiac pacemaker activity is still a matter of debate. recent findings point to a coordinated interplay between intracellular ca 2+ -cycling processes and plasma membrane-localized ion channels which determines the origin, periodicity and rate modulation of pacemaker potentials. in this study, we investigated the contribution of the cardiac sodium-calcium exchanger (ncx1) to pacemaking. ncx1 is a key sarcolemmal protein for the maintenance of calcium homeostasis in the heart. it was speculated that the membrane depolarizing current incx, whose activity is dependent on intracellular ca 2+ -fluctuations, represents a main determinant of the spontaneous diastolic depolarization. we used an inducible and sinoatrial node-specific cre-transgene to delete ncx1 in the murine pacemaker system. the successful creation of a cardiac pacemaking and conduction system specific ncx1 knockout (cpncx1ko) was demonstrated by transcript quantification as well as immunofluorescence experiments. telemetric ecg recordings of cpncx1ko displayed a distinct cardiac phenotype. mutant animals were deeply bradycardic and lost their capability of maintaining a stable heart beat as demonstrated by various ecg abnormalities like sn arrhythmia, sn pauses, av block and ventricular tachycardia. analysis of the spontaneous activity of isolated sn preparations showed a slower and arrhythmic contraction rate of the mutant tissues strips confirming that the bradycardia and arrhythmia induced by deletion of ncx1 results from a slower and arrhythmic intrinsic pacemaker activity. a battery of experiments using different heart rate lowering as well as increasing drugs revealed an altered heart rate modulation in cpncx1ko animals as compared to controls. in conclusion, these initial results establish ncx1 as a major contributor to cardiac pacemaking. a wide variety of contaminants are ingested through food, among them the procarcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene (bp) which is resorbed and partially metabolized in the enterocytes of the small intestine. previous in vitro studies revealed that bp phenols are excreted as phase ii metabolites including bp glucuronides and bp sulfates. this export is mediated by the breast cancer resistance protein (bcrp/abcg2). the ultimate carcinogenic phase i bp metabolite anti-bp-7,8dihydrodiol-9,10-epoxide (bpde) can be detoxified by glutathione conjugate formation catalyzed by various glutathione s-transferases. in the present study, differentiated human intestinal caco-2 cells were used as a model for the human small intestine to investigate the detoxification of bpde and the subsequent transport of the stereoisomeric glutathione conjugates in the presence of an inhibitor (acivicin) of the glutathione-cleaving enzyme gamma-glutamyl transpeptidase (ggt) at the surface of the cells. the results indicate that the glutathione conjugates of bpde are formed and excreted mainly to the apical and to a minor extent to the basolateral side of the polarized caco-2 monolayer. to stimulate the transport rate several inducers known to enhance gene expression of xenobiotic-metabolizing enzymes as well as transport proteins were used (quercetin, oltipraz, butyrate). however, solely oltipraz substantially increased the efflux of bpde glutathione conjugates after inhibition of ggt. inhibition studies revealed that the multidrug resistance-associated proteins (mrps/abccs) are involved in the transport of the bpde glutathione conjugates. stable abcc1, abcc2 and abcc3 knockdown cell lines were generated allowing to demonstrate that abcc1 mediates the basolateral, abcc2 the apical excretion of the bpde glutathione conjugates. in conclusion, the ultimate carcinogen bpde is detoxified via glutathione conjugation and subsequently excreted by caco-2 cells in both apical and basolateral directions. .this finding is equivalent to a transport into the feces as well as blood system in the in vivo situation. signaling via irag regulates store operated calcium entry (soce) in aortic vsmc hieke b., hüttner j., schlossmann j. university of regensburg department of pharmacology and toxicology, universitätsstr. 31, 93053 regensburg, germany the mechanisms involved in the activation of store operated calcium entry (soce) through depletion of intracellular stores and their regulation are not yet fully understood. we examined the effect of inositoltriphosphate-receptor associated cgmp-kinase substrate (irag) on soce. aortic vascular smooth muscle cells (vsmc) from wild type (wt) and irag-knock-out (ko) mice were loaded with the calcium indicator fura 2-am and soce was measured as a change in the intracellular calcium concentration. in experiments with vsmc from wt mice soce was attenuated by the application of 8-br-cgmp. this effect was not observed in vsmc isolated from irag-ko mice. these differences in the strength of the soce-signal were abolished by the replacement of extracellular sodium with n-methyl-d-glucamine. the observed sodium dependence of the soce regulation via irag suggests, that an alternated sodium conductance might be responsible to some extent for the differences detected in wt and irag-ko vsmc. as a change in sodium conductance might result in a changed membrane potential we tried to track these changes with the flipr membrane potential assay kit while executing the soce protocol with and without 8-br-cgmp. no significant differences in membrane potential could be detected in the various stages of soce. in conclusion, our results indicate that irag exhibits a dual action on calcium regulation as it inhibits not only the intracellular calcium release but also the extracellular calcium influx through soce. induction of the icer promoter in vascular smooth muscle cells hildebrandt i. 1 tokyo metropolitan institute of gerontology, tokyo japan several transcription factor isoforms are encoded by the crem (camp response element modulator) gene. one prominent isoform is the inducible camp early repressor (icer), which acts as a transcriptional repressor on so called camp responsive elements (cres) in its target gene promoters. the icer mrna expression is regulated by an intronic promoter of the crem gene. in vascular smooth muscle cells (vsmcs) crem/icer is involved in the regulation of cell proliferation and apoptosis with physiological consequences in vivo. for instance crem-knockout mice, in which none of the known isoforms can be expressed, exhibit an increased neointima formation after carotid ligation as well as an increased atherosclerotic plaque formation after high fat diet on an apoe background. these observations were associated with an increased proliferation rate in isolated crem deficient vsmcs. on this background we wanted to clarify the specific role of icer isoforms in the vasculature. in first experiments we examined the inducibility of icer in primary vsmcs and smooth muscle cell lines. reporter luciferase assays showed that the activity of the icer promoter is induced at the maximum of fourteen fold after 4 hours of stimulation with forskolin (fsk) in immortalized rat vsmcs (13.7 ± 0.93; n=12 from 3 isolations). in a7r5 rat smooth muscle cells the icer promoter showed a maximum stimulation of 3.2 ± 0.16 fold after two hours of fsk stimulation (n=20 from 4 isolations). these pilot experiments showed that the icer promoter is inducible in vsmcs by camp dependent pathways. further experiments have to be carried out to elucidate the role of icer in the vascular system for example by stimulation of primary vsmcs and analysis of icer knockout mice. (supported by the dfg) overexpression of transmembrane channel-like proteins (tmcs) uncouples receptor-mediated calcium mobilisation hill k., urban n., straub i., schaefer m. universität leipzig -universitätsmedizin rudolf boehm-institut für pharmakologie und toxikologie, härtelstr. 16-18, 04107 leipzig, germany the family of transmembrane channel-like proteins (tmcs) consist of 8 members (tmc1-tmc8) all tmc genes are predicted to encode transmembrane proteins with at least six membrane-spanning helices. mutations of tmc1 cause deafness in human and mice whereas tmc6 and tmc8 (also referred to as ever 1 and 2) are linked to epidermodysplasia verruciformis (ev), a skin disorder, which is characterised by an enhanced susceptibility towards cutanous infections by human papillomaviruses. the cell biological and physiological functions of tmc proteins still remain elusive. we have overexpressed tmc6 and tmc8 in hek293 cells to get insights into their physiological function. all tmcs were located within the endoplasmic reticulum (er) after overexpression of yfp or cfp-tagged constructs. ratiometric calcium imaging revealed that after overexpression of tmc6 or tmc8, stimulation of gq-coupled receptors with carbachol and atp resulted in a greatly reduced amount of calcium release from the er. moreover, challenging tmc6-or tmc8-expressing cells with the serca pump inhibitor thapsigargin was also not followed by a release of er-based calcium within the cell. to test whether the lack of calcium release was caused by a reduced calcium content within the er, we investigated calcium dynamics within the er using an er-targeted fret-based calcium indicator (d1er cameleon). the experiments revealed that the amount of calcium within the er was reduced upon overexpression of tmc6 or tmc8. recently, it has been reported that tmc6 and tmc8 might influence intracellular zinc distribution in human keratinocytes. we could confirm the presence of tmc6 and tmc8 mrna in a human keratinocytes cell line (hacat). upon overexpression of tmc8, hacat cells revealed the same phenotype as described above for the hek293 cells with an uncoupling of the receptor-mediated calcium mobilisation due to a depletion of the er calcium store. the mechanism by which overexpression of tmc proteins causes a reduced calcium concentration within the er remains unclear. considering that the presumed topology of the tmc proteins distantly resembles those of other ion channel superfamilies such as anoctamins, one might speculate that a conductance through the tmc protein itself leads to a calcium leak from the er. terahertz radiation is defined as radiation between 0.1 thz and 10 thz. a number of applications are currently being developed using radiation in this frequency range. these applications will lead to exposure of the general public, making it very important to study potential effects on biological systems. historically, only a few studies on effects caused by terahertz radiation have been conducted because of the lack of suitable generators and detectors. during the last decade, a number of studies on effects caused by radiation with frequencies around 100 ghz have been published. the present study investigated the genotoxic potential of terahertz radiation at three different frequencies, 0.106 thz, 0.380 thz and 2.520 thz. two skin cell types were used, primary human dermal fibroblasts (hdf) and a keratinocyte cell line (hacat). the cells were irradiated applying different exposure times and different power intensities. two genotoxicity tests were applied: the comet assay quantifies dna strand breaks as well as alkali-labile sites whereas the micronucleus test quantifies chromosomal damage. all experiments were performed and evaluated under blinded conditions as three independent replicate experiments. positive (mms-treated) and negative (untreated, sham-exposed) controls were included. in the comet assay no dna damage was observed as a consequence of the exposure under all experimental conditions. the same was true for the chromosomal damage investigated with the micronucleus test. the latter finding was particularly interesting for the experiments at 0.106 thz, because this type of radiation had been reported to cause mitotic disturbances. therefore these experiments were extended, applying higher power intensities and longer exposure periods. also with these modifications, no genomic damage was observed in the form of micronucleus formation. all in all, terahertz radiation did not induce genomic damage under the applied experimental conditions. this result is in line with published findings on genotoxicity of low-frequency terahertz radiation around 0.1 thz. the question, why the reported mitotic disturbances do not lead to manifest genomic damage remains open and requires further research. introduction: tea flavonoids derived from camomile and green tea such as apigenin and epigallocatechin gallate (egcg) can inhibit intestinal neoplasia. recurrences of adenomas and cancers were reduced in patients with resected colorectal cancer by treatment with tea bioflavonoids after tumor operation [1] . to clarify the biomolecular pathway for suppression of neoplasia we investigated the anti-inflammatory effect of a nutritional supplement flavo natin® (fn) which had been used in the clinical study on tertiary tumor prevention and of egcg in a colon tumor cell line. the aim of our study was to investigate if tea flavonoids are capable to suppress the inflammatory markers produced by tumor cells after cytokine stimulation. method: we studied the cytotoxicity of fn in the colon cancer cell line t-84 by resazurin fluorescence and compared it with the placebo supplement. additionally, the t-84 cells were incubated with fn, egcg or placebo and stimulated with tnf-alpha, if-gamma and il-1-beta. after the cytokine stimulation the mrna expression of ip-10, il-8 and tnf-alpha was measured by quantitative real-time pcr (qrt-pcr). results: stimulation of t-84 cells increased the expression of ip-10 (gamma-interferon inducible protein 10), tnf-alpha and il-8. by preincubation with fn at 10 µm the mrna expression of ip-10 was strongly reduced (log2-ratio -14). the tnf-alpha mrna was also but less decreased by fn. egcg displayed an inhibition pattern similar to fn. placebo did not influence the mrna expression of the chemokines and tnf-alpha. discussion & conclusion: clinically useful dietary tea bioflavonoids inhibit the expression of inflammatory genes in a colon cancer cell line. down-regulation of inflammatory gene products could be achieved in vivo by botanicals without clinically relevant side effects. [1] h. hoensch, b. groh, l. edler, w. kirch (2008) . prospective cohort comparison of flavonoid treatment in patients with resected colorectal cancer to prevent recurrence. world j gastroenterol, 14, 2187-2193. the cxcr7 c-terminal domain mediates efficient cxcl12 uptake and degradation hoffmann f., müller w., schütz d., schulz s., stumm r. universitätsklinikum jena institut für pharmakologie und toxikologie, drackendorfer str. 1, 07747 jena, germany cxcl12-signaling mediated by the g protein-coupled cxcr4 receptor plays a key role during embryonic development and disease states including cancer and inflammation. the second cxcl12-receptor cxcr7 modulates cxcl12/cxcr4-signaling by acting as a cxcl12-scavenger. given the distinct functions of cxcr4 and cxcr7, we hypothesized that trafficking and receptor stability are differently regulated by the distinct c-terminal domains. here, we examined epitope-tagged wild type and c-terminal mutant receptors expressed in human embryonic kidney cells (hek293) with respect to trafficking, stability, 125 i-cxcl12 radioligand degradation, and g protein-coupling. we found that the 24 c-terminal residues of cxcr7 were sufficient for cxcr7 to undergo rapid spontaneous internalization. replacement of the cxcr7 c-terminal domain with that of cxcr4 (cxcr7-4tail mutant) abolished spontaneous internalization but permitted ligand-induced internalization in conjunction with c-terminal phosphorylation. conversely, replacement of the cxcr4 c-terminal domain by that of cxcr7 caused ligand-independent internalization of cxcr4. receptor-mediated 125 i-cxcl12-uptake, release of 125 i-cxcl12-degradation products, and degradation of the receptor protein itself were accelerated with receptors bearing the cxcr7 c-terminus. while the cxcr7 c-terminus was sufficient to abolish g protein coupling in the cxcr4-7tail mutant, replacement of the cxcr7 c-terminus, cxcr7 second intracellular loop or both domains with the corresponding cxcr4 domain did not generate a g protein-coupled cxcr7 chimera. taken together, we provide evidence that the cxcr7 c-terminal domain influences the ligand-uptake/degradation rate, g protein-coupling, and stability of the receptor. this suggests that heterologous regulatory pathways targeting the cxcr7-c-terminal domain may effectively control cxcr7 functions. soluble guanylyl cyclase is a key mediator of brown adipocyte differentiation hoffmann l. s. 1 brown adipose tissue (bat) uses energy to produce heat by inducible thermogenesis. recent studies show that active bat is present in adults and involved in human energy balance, suggesting that the energy consuming property of bat might be exploited to fight obesity and related diseases. the no/cgmp signaling pathway is a key player in diverse physiological processes. recently, we have shown in bat that cgmp signaling is connected with insulin signaling and abrogation of cgmp signaling leads to impaired bat differentiation and function (haas, b. et al., sci signal, 2009 ). here we investigated the role of the cgmp generating enzyme soluble guanylate cyclase (sgc) in bat differentiation in vitro. mesenchymal stem cells isolated from bat of newborn sgcβ 1 -/mice and wt littermates were differentiated in vitro into brown adipocytes in the presence or absence of cgmp. abundance of sgc isoforms was determined by qrt-pcr and western blotting. bat differentiation was assessed by redo staining of accumulated intracellular lipids, measurement of triglyceride (tg) content, determination of expression of bat marker proteins pparγ, c/ebpα, ap2 and bat marker genes ucp1, pgc1α, cidea. the α2 and β1 isoforms of sgc were highly expressed in bat whereas α1 sgc could not be detected. redo staining of wt brown adipocytes showed basal differentiation which was increased upon addition of 8-pcpt-cgmp. in contrast, staining was lower in sgcβ1 -/cells compared to wt under control conditions and increased in the presence of cgmp. tg measurement showed that sgcβ1 -/brown adipocytes contain approximately 50% less lipids than wt cells under basal conditions. addition of cgmp doubled tg content in both genotypes. similar results were observed for marker protein expression. deletion of sgc resulted in 56-40% decrease in c/ebpα, pparγ and ap2 expression compared to wt. again, cgmp roughly doubled protein expression in sgcβ1 -/and wt cells compared to control. under basal conditions, bat marker gene expression was decreased by approximately 80% in sgcβ1 -/cells compared to wt cells. this decrease was prevented by addition of cgmp. these results show that sgc deletion leads to dysfunctional bat differentiation and emphasize the central role of cgmp signaling in bat differentiation. further investigation of sgc/cgmp signaling in bat might reveal new drugable targets bringing bat-dependent pharmacological therapy to treat obesity and related disease into closer reach. comparative inhalation toxicity of carbon-nanomaterials (multi-wall carbon nanotubes, graphene and carbon black) hofmann t. 1 carbon black is a spherical carbon anomaterial whereas multi-wall carbon nanotubes (mwcnt) are cylindrical and graphene is a laminar allotrope of carbon. processing and handling as well as abrasion processes can set free inhalable cnt particles. results of rodent studies collectively show that regardless of the process by which cnts were synthesized and the types and amounts of metals they contained, cnts were capable of producing inflammation, epithelioid granulomas, fibrosis, biochemical and or toxicological changes in the lungs (lam et al. 2004 , muller et al. 2005 , ma-hock 2009 , pauluhn 2010 . graphene possess similar physical properties as cnt but may different toxicological property. we performed short-term inhalation studies in rats to compare the toxic potency of four different cnt, two graphenes and one carbon black. the materials are characterized thoroughly according to the oecd list. the four mwcnt caused morphological changes as descriped above. several biochemical and cytological parameters in the broncho-alveolar lavage fluid were strongly increased consistent with the histological findings. two mwcnt exhibited a higher toxic potency than two other mwcnts and findings caused by one graphene typ were even less severe. the graphene with lower surface area as well as low surface area carbon black did not cause any adverse effects up to 10 mg/m 3 . the short-term inhalation studies were able to descriminate different toxic potencies of carbon-based nanomaterials and is hence used for the selection of less toxic materials for further product development as well as to define and prioritize higher-tier toxicological testing of nanomaterials. 165 synthesis and analytical assessment of possible dna adducts formed after activation of the tobacco alkaloid myosmine högg c., zwickenpflug w., gudermann t. walther-straub-institut abt.: toxikologie, nußbaumstraße 26, 80336 münchen, germany myosmine represents one of the minor tobacco alkaloids and its effective uptake from smokeless tobacco or tobacco smoke, as well as by consumption of food is not understood in detail. myosmine can be activated by peroxidation and n-nitrosation yielding 4-hydroxy-1-(3-pyridyl)-1-butanone (hpb) which is well known as reactive intermediate during activation of a variety of tobacco specific n-nitrosamines (tsna). therefore, myosmine may be a potential candidate for possible mutagenic or carcinogenic risk to human health. furthermore, myosmine n-nitrosation yields the tobacco specific nitrosamine n-nitrosonornicotine (nnn), which is classified as carcinogenic to humans. considerable efforts have been undertaken, especially in organic synthesis, to verify and elucidate the significance of the hpb precursor the pyridyloxobutyl (pob) intermediate and its dna-adducts, which were analysed only in animal experiments till now. the formation of 3-pyridylmethanol was observed under myosmine peroxidation and identified as a metabolite in rat urine after application of myosmine to rats. the formation of the 3-pyridylmethanol intermediate, might provide for the reactive electrophilic picolinium ion which could interact with dna. these possible adducts might be of special interest to elucidate the role of myosmine concerning its uptake by smokers and passive-smokers in contrast to non smokers ingesting the substance by consumption of food. dna adducts may help to obtain more information about possible risk assessment of myosmine and to differentiate between the activation from the other nicotinoids and tsna. the specific dna adducts, 5-methyl-1,3-bispyridin-3-ylmethyl-1h-pyrimidin-2,4-dion and 3-(3''-picolyl)thymidine have been synthesised as reference substances. the former was prepared by addition of diisopropylazodicarboxylate (diad) to a mixture of 3-pyridylmethanol, thymine and triphenylphosphine. the reaction product was identified using nmr ( 1 h, 13 c, cosy, hmqc, hmbc). for synthesis of 3-(3''-picolyl)thymidine the hydroxyl groups of thymidine were initially acetylated using acetic acid anhydride and 4dimethylaminopyridine (dmap). the existence of this thymidine adduct was confirmed using nmr. this adduct was labelled with 5-([4,6,-dichlorotriazin-2-yl]amino)fluorescin (dtaf) to enhance the sensitivity using hplc-fluorescence chromatography and used as reference substances for analysis of dna samples from biological tissue. supported by dfg grant (ty 81/1-1). cancer and cardiovascular diseases such as atherosclerosis are the most important causes of death in western societies. common to both diseases is a deregulation of cell death, with significant contribution of inflammatory processes. enhanced oxidative stress plays a dominant role in such events as it forms a vicious cycle with inflammation and controls multiple forms of cell demise. therefore, anti-oxidative enzyme systems gained considerable interest since control of reactive oxygen species (ros) has the capacity to regulate cell death in either direction. the human enzyme family of paraoxonases consists of three members, known as pon1, pon2 and pon3. while pon1 is found predominantly in the circulation, pon2 and pon3 are intracellular enzymes with established anti-oxidative functions. it has been shown that both pon2 and pon3 are protective against atherosclerosis. underlying mechanisms of their protective and antioxidative functions however remained elusive. here we demonstrate that both enzymes locate to the endoplasmic reticulum (er) and mitochondria where they fulfill vital functions in the control of ros generation. in particular, pon2 and pon3 were shown to interact with coenzyme q10 which diminishes mitochondrial ros formation. as a consequence, these enzymes reduce execution of mitochondrial apoptosis, such as cardiolipin peroxidation, cytochrome c release and caspase activation. moreover, pon2 and pon3 reduced er stress-triggered cell death, i.e. by diminishing jnk signaling and chop expression. while these results elucidate their protective role in cardiovascular diseases, it also establishes a relevant function in survival of tumor cells. in accordance, we demonstrate that both enzymes are frequently found overexpressed in various tumors. in cancer cell culture studies, overexpression of both enzymes granted considerable resistance against chemotherapeutics. in turn, knock-down of pon2 caused spontaneous apoptosis of several cancer cell lines. finally, our analyses also revealed that pon2-knockout mice show severe alterations of the hematopoetic stem cell compartment, which implies a significant role in leukemias where these enzymes are frequently found overexpressed. together, our results propose pon2 and pon3 as new putative anti-tumor candidates and demonstrate the efficacy of interventions targeting cellular redox-balance. steigerwald arzneimittelwerk gmbh wissenschaftliche abteilung, havelstr. 5, 64295 darmstadt, germany stw 5 (iberogast ® ), a multi-component herbal drug, is successfully used in the therapy of functional dyspepsia and irritable bowel syndrome (ibs). previous studies revealed effects of stw 5 on disturbed motility and inflammatory processes. although the antiinflammatory properties of stw 5 are well examined, the contribution each of the individual herbal constituents to the anti-inflammatory effect remains unclear. therefore, we studied the effects of stw 5 and its components on inflammation-induced cell death and on the release of the pro-inflammatory cytokine tnf-α. the aim of these investigations was to analyse additive or synergistic effects of the components. the experiments were carried out on caco-2 cells after stimulation with lps (10 ng/ml) for 2 hours. cytotoxicity was evaluated using a commercially available ldh (lactate dehydrogenase)-assay. furthermore, the release of tnf-α after lps (100ng/ml) stimulation of differentiated thp-1 cells was measured using a commercially available elisa. stw 5 (62.6 -500.5 µg/ml) reduced lps (10 ng/ml)-induced cell death in a concentration-dependent manner with a maximum inhibition of 50.0 %. the herbal components in equivalent concentrations contributed to the inhibitory effect of stw 5 to different extents. the maximum inhibition differed in a wide range between the components. stw 5 (500.5 µg/ml) reduced significantly the release of tnf-α by 87 % in lps (100 ng/ml)-stimulated differentiated thp-1 cells while having no effect in untreated cells. in concentrations equivalent to stw 5 caraway, milk thistle, lemon balm and greater celandine had no effect on the lps-induced increase in tnf-α release. bitter candytuft, peppermint, chamomile, liquorice and angelica reduced the tnf-α release, though less pronounced as compared to stw 5, indicating a possible synergistic effect. our results indicate a multi-target effect of stw 5. the anti-inflammatory effect may be due to a reduction of the cytotoxic effect on intestinal mucosa cells and to an inhibition of the release of the pro-inflammatory cytokine tnf-α from immune cells. the individual herbal components seem to contribute by different mechanisms of action to the overall effect of stw 5. immune cell-induced local steroidogenesis in the lung: implications for asthmatic disease and therapeutic intervention hostettler n. 1 , brunner t. glucocorticoids are steroid hormones with potent anti-inflammatory properties. synthetic glucocorticoids are frequently used for the therapeutic treatment of inflammatory disorders, such as asthma. endogenous glucocorticoids are predominantly produced in the adrenal glands in response to emotional, physical and immunological stress. recent years, however, revealed several alternative sources of these immunoregulatory steroid hormones. thus, we have found that the intestinal epithelium is a rich source of glucocorticoids and intestinal glucocorticoids contribute to the maintenance of local immune homeostasis (1) . as the intestinal and the pulmonary epithelium have much in common, i.e. barrier functions and transport of nutrients, resp. gases, we wondered whether the lung mucosa is also capable of synthesizing immunoregulatory glucocorticoids in response to immune cell activation. the murine lung was found to expresses all enzymes required for the synthesis of corticosterone from cholesterol. while most enzymes where expressed in a constitutive manner, cyp11a1, encoding p450ssc, was strongly induced in response to immunological tress. treatment of mice with t cell-activating anti-cd3 antibody of macrophage-activating lipopolysaccharides induced strong local glucocorticoid synthesis, which was effectively blocked by the corticosterone synthesis inhibitor metyrapone, indicating that glucocorticoids measured were produced bona fide in the lung tissue. surprisingly, allergen-induced allergic airway inflammation failed to trigger local glucocorticoid synthesis despite the massive infiltration of neutrophils, eosinophils and t cells. in contrast to that in the intestinal epithelium local glucocorticoid synthesis in the lung was found to be dependent on the presence of adrenal glands as adrenalectomy abolished pulmonary steroidogenesis. in line with the notion that the lung metabolizes steroid precursors we found that ex vivo cultured lung tissue metabolized 3 hduring the last decade small rna molecules has been identified as important regulators for gene expression. these micrornas (mirna) are single-stranded transcripts, which are expressed in many cell types, where they modulate rna stability and translation and, therefore, controlling various cellular mechanism and tissue development. against this background, in the present study the mirna expression and potential target genes were studied in the human placenta as a tissue demonstrating various developmental changes in a limited period of time. taqman®array microrna cards for profiling of 365 mirnas in placentas of different gestation times revealed a significant expression of 277 mirnas by comparing placentas of early gestation (<10.week), preterm (<30.week) and term (>37.week). when comparing the analyzed groups, 106 of these mirnas expose a continuous up-(including mir-20a, mir-379) or downregulation (including mir-9, mir-200a). while comparing placentas of early gestation to term placentas, 30% (e.g. mir-9, mir-429) were up-and 70% (e.g. mir-126, mir-373) were downregulated. by focusing on the latter group with early preterm placentas the ratio is opposed. here, 60% of mirnas showed higher (e.g. mir-107) and 40% lower expression (e.g. mir-627, mir-191). with emphasize on these mirnas, a computational prediction algorithm using the mirò database predicted a potential interaction between corticotropin-releasing hormone (crh) and the mirnas mir-9 and mir-429. specific expression analyses validate an inverse expression between mirnas and target with a reduced expression of mir-9 (93%) and mir-429 (94%) in term placentas, while crh is upregulated 114fold. this interaction was verified on functional level by reporter gene assay. a significantly suppressed luciferase activity of the reporter plasmid containing the 3´-utr sequence complementary to crh was exhibit for the predicted mir-9 binding site. here, the mirna-mrna-interaction reduces the luciferase activity by ~40%, whereas the decreased luciferase activity for the predicted mir-429 binding site is not significant. the results demonstrate a gestation dependent expression of placental mirnas, which may help to explain gestational changes in gene expression and highlight the potential of mirnas as biomarker for pregnancy-related pathologies. since homo sapiens era wound treatment by early civilizations was based on the use of local flora. scilla indica knuth liliaceae (s.i) is a perennial herb with a pear shaped, tunicated bulb, bearing fibrous roots, white flowers and leaves on the stem resembling u. maritima. it is used as cardiac tonic, against inflammation, ulcers and sinus diseases. traditionally the powdered bulb is used topically for warts treatment, while roasted and crushed bulbs are applied to corns of the feet soles. the plant contains steroid glycosides (bufadienolides 1-3%), glucoscillarene a, proscillaridine a, scillarene a, scillcyanoside ,scilliglaucoside ,mucilage and alkyresorcinol derivatives exerting skin healing properties similar to wheat bran products. the study is focused on the investigation of the restoration quality of a skin dorsal incision wound in wistar rats in three groups, control (a1) and experimental (a2,a3 ) of rats weighing 350-450g local application of dichloromethane extract of s.i in vehicle olive oil preparations of 50 and 100mg were used. animals were anaesthetized ( ether pro narcosi ), trauma 2 cm long and 2mm deep was performed by lancet on depilated dorsal area skin until the muscular aponeurosis and were treated for 4 days with 60λ of each preparation. group a3 showed increased remodelling of trauma area (limited width). granulomatous tissue was more pronounced in length, width and surface in group a2. the macroscopic ulcus dimensions were better in group a3 while the microscopic view were similar in a2 and a3 and better in comparison to control a1. a tendency to trauma remodelling was observed, without statistical significance (t =0.3) in recent years, it has been suggested that nanoparticles generated from combustion processes (e.g. diesel engine exhaust particles), may contribute to the pathogenesis of neurodegenerative diseases such as alzheimer's disease (ad). the aim of our current study was to investigate the effects of subchronic exposure to diesel engine exhaust (dee) in the 5xfad mouse model, which is characterised by progressive behavioural deficits as well as amyloid plaque formation and neuron loss. ten weeks old female 5xfad mice and their nontransgenic littermates were exposed by whole body inhalation to diluted dee (~1 mg particles/m 3 ) or clean air (controls) for 3 or 13 weeks (5 days/week and 6 hour/day). subsequently, all animals were subjected to a series of behavioural tests. at ten days post-exposure, mice were sacrificed to investigate lungs and brain tissues for pathological and biochemical and molecular-biological changes. in line with the expectations, the 5xfad mice displayed typically age-dependent behavioural deficits and amyloid plaque formation in cortex and hippocampus. a significant dee exposure-related effect was observed for the string suspension test, representing a measure of motor coordination/grip strength. dee exposure was also associated with mrna expression changes of markers of inflammation and oxidative stress in specific brain regions, including the olfactory bulb. histopathology of plaqueload in cortex and hippocampus (from a limited number of animals investigated so far) did not reveal clear evidence for increased plaque formation due to the dee exposures. further research is needed to evaluate the effects of long term exposure to nanoparticles in the central nervous system. this work is supported by funds from the research committee of the medical faculty of the university of düsseldorf (9772-365), the dfg graduate school grk1033 and the rivm -centre for environmental health, bilthoven, netherlands. mono-glucosylation of (h/k/n)ras by clostridium sordellii lethal toxin (tcsl) blocks critical survival pathways, including the pi3k/akt, the ralgef/ral, or the raf/erk, and results in apoptosis. in this study, ras glucosylation is presented to result in expression of the cell death-regulating small gtpase rhob based on transcriptional activitation. rhob expression depends on k-ras inhibition, as sirna-mediated knock-down of specifically k-ras (neither h-ras nor n-ras) provokes rhob expression. rhob expression further depends on inhibition of pi3k/akt, as activation of pi3k/akt using a pi3k activator prevents rhob expression downstream of inactivated ras. newly synthesized rhob is gtp-loaded and rapidly degraded in a proteasome-and a caspase-dependent manner, providing first evidence for caspase-dependent degradation of a rho family protein. although often characterised as a pro-apoptotic protein, rhob suppresses caspase-3 activation in tcsl-treated fibroblasts. conclusions: 1. rapid and efficacious ras inactivation by tcsl turns out to be particularly useful in the characterisation of ras inactivation-induced rhob expression as an immediate-early gene response. 2. the finding on the cytoprotective activity of rhob in tcsl-treated cells re-enforces the concept that rhob exhibits cytoprotective rather than pro-apoptotic activity on cellular background of inactive ras. the activity of the rhob promoter is suppressed by rhoa (through a not yet identified pathway or ras (through the pi3k/akt pathway. ras glucosylation by tcsl results in de-suppression of the rhob promoter and rhob expression. the calcium-binding protein annexin a4 (anxa4) is involved in diverse cellular processes including e.g. vesicular transport, ion channel regulation and transcriptional regulation. upon ca2+ binding anxa4 undergoes conformational changes, which lead to oligomerization of anxa4 to homotrimers and cause an increased affinity for membrane phospholipids, which in turn provokes the translocation from the cytosol and the nucleoplasm to plasma and nuclear membranes. in order to examine the effect of anxa4 on cre-and creb-(camp response element-binding protein) dependent transcription, a series of transient transfections using a luciferase reporter gene driven by the creb target promoter of the inducible camp early repressor (icer) was carried out. when the luciferase reporter construct was co-transfected with anxa4, there was significant reduction of basal (dmso) luciferase activity ( the implantation of drug eluting stents (des) after coronary artery intervention was an important step treating coronary artery disease. indeed, cytotoxic compounds like sirolimus used on des are responsible for reduced in-stent restenosis in comparison with bare metal stents. pimecrolimus, a potent anti-inflammatory drug has also been investigated for its efficacy on des. preclinical studies in pigs revealed promising antiproliferative effects of pimecrolimus on neointima formation. however, in humans, pimecrolimus coated stents exerted adverse effects. we hypothesize that compared to the highly active sirolimus, pimecrolimus may influence additional cellular processes leading to the worse outcome. in order to identify those processes we conducted in vitro studies in human coronary artery endothelial cells (hcaec) and smooth muscle cells (hcasmc). brdu in vitro assays of hcaec treated with pimecrolimus examined an ic50 value of 6.787 µm [5.745 to 8 .017] which is much higher than ic50 values of sirolimus described in literature [0.1nm to 1nm]. genechip array analysis comparing gene expression in pimecrolimus and sirolimus treated hcaec and hcasmc showed significant induction of several genes involved in interferon signaling. in detail, the expression of ifnβ activated genes like irf9 and ifitm1 was up regulated in cells treated with pimecrolimus while no or oppositional effects were observed with sirolimus. gene regulatory effects were validated by real time pcr. incubation with ifnβ itself showed similar effects in up regulation of genes involved in interferon signaling. furthermore, we were able to demonstrate a significant increase of ifnβ secretion in hcasmc and hcaec treated with pimecrolimus. however, comparison of ifnβ and pimecrolimus on proliferation of hcaec and hcasmc revealed different cellular responses. while ifnβ significantly decreased hcasmc and increased hcaec proliferation, treatment with pimecrolimus lead to anti-proliferative effects on both cell types. in conclusion, pimecrolimus activates pathways involved in interferon signaling but exerts different pharmacological effects, compared to the endogenous compound suggesting that infβ secretion is not the major factor contributing to the difference in pimecrolimus function. identification of novel phospho acceptor sites of the mu opioid receptor regulating receptor internalization illing s., just s., doll c., schulz s. uniklinikum der fsu jena pharmakologie und toxikologie, drackendorfer straße 1, 07747 jena, germany the opioid alkaloid morphine is among the most potent clinically used analgesics. however, the clinical utility of morphine to treat chronic pain is limited by its rapid development of tolerance and dependence. the stimulation of the mu opioid receptor (mor) with damgo or morphine results in different patterns of receptor phosphorylation and trafficking. so far, the three major phosphorylation sites of the mu opioid receptor namely serine 363 (s363) threonine 370 (t370) and serine 375 (s375) have been identified using phosphosite-specific antibodies. however, mutations of these three residues to alanine (s363a/t370a/s375a) did not prevent agonist-dependent internalization. in the present study, we have constructed a series of phosphorylationdeficient mutants showing that mutation of at least six residues (s363a/t370a/s375a/t376a/t379a/t383a) was required to completely block agonistdriven endocytosis. consequently, we generated phosphosite-specific antibodies to t376 and t379 which enabled us to provide direct evidence for agonist-dependent phosphorylation of these sites. our analysis of time-and dose-dependent phosphorylation of mor revealed that s375 was the primary phosphorylation site, whereas t370, t376 and t379 were secondary sites. moreover, the partial agonist morphine induced only the phosphorylation of s375 but not of t370, t376 of t379. our results show, that mor phosphorylation occurs in a hierarchical and agonist-selective manner directly regulating receptor sequestration. assessment of mda effects from toxicity studies with regard to endocrine modulation jäger r. 1 methylene dianiline (mda; cas no. 101-77-9) is on the usepa list 2 for endocrine disruption screen testing. in a yeast androgen screening (yas) assay (in vitro assay on receptor binding or blocking) mda revealed a significant anti-androgenic activity at a concentration of 0.01 mm. to assess the biological relevance of this observation under in vivo conditions the existing data from animal toxicity studies with mda were reviewed for indications of possible endocrine modulating (em) effects (weight-of-evidence approach). in addition, literature was searched for relevant studies on mda and structural analogues using the american chemical society scifinder client-server software. structural analogues indicated several drivers for em activity, notably the diphenolic ring structure. however, in vitro receptor binding assays showed mda had no androgenic or anti-estrogenic activitiy. one report described a weak estrogenic binding at 0.2 mm mda, while another described no effect at similar concentrations and a rat estrogen receptor binding study found no effect at 0.2 mm. overall it is concluded that mda does not bind to the estrogen receptor. endocrine related effects of mda were investigated in several species and dose routes in unvalidated research-type protocols. guideline subchronic and chronic toxicity studies with rodents revealed neither adverse organ weight changes nor histopathological alterations of sex organs. the main systemic effects from the oral 13-week studies were body weight reduction and histopathological changes of the thyroid and bile duct (lowest loael: 7.5 mg/kg bw). mda was carcinogenic to rats and mice (thyroid and liver) after oral administration over 2 years. non-neoplastic effects in the thyroid (rats and mice) were observed (lowest loael for systemic toxicity: 9 mg/kg bw). mda inhibits iodide oxidation which with concomitant decreased thyroid hormone formation is known to induce thyroid tumors. in summary, in vitro screening tests revealed no consistent endocrine related effects of mda. in vivo, there was an effect on the thyroid gland, possibly by enzyme inhibition. there were no histopathological changes of gonads and accessory sex organs and no evidence of sex hormone related em. the evidence from the full dataset on mda does not indicate androgenic or estrogenic effects. overall, based on a weight of evidence assessment there are insufficient alerts for em activity to suggest further testing should be done. the gtpase arfrp1 controls the assembly of apoa-i to and the lipidation of chylomicrons in the golgi of intestinal epithelium jaschke a. 1 background: the uptake and processing of dietary lipid by the small intestine is a multi-step process that involves luminal digestion, cellular uptake of fatty acids by the mucosa, and subsequent synthesis and export of chylomicrons. the gtpase adpribosylation factor related protein 1 (arfrp1) is a member of the arf-family and controls the arf-like 1 (arl1)-mediated golgi recruitment of grip domain proteins which in turn bind several rab-gtpases. the aim of the study was to define the role of arfrp1 in intestinal nutrient absorption. methods: for the generation of intestine-specific null mutants arfrp1 flox/flox mice were crossed with mice expressing the cre recombinase under the control of the intestinespecific villin promoter (vil-cre) and arfrp1 expression was suppressed by sirna in caco2-cells. the phenotype in respect to lipid absorption and chylomicron production in the intestinal epithelium and in caco2-cells, respectively, was analyzed. results: arfrp vil-/mice were viable but showed an early postnatal growth retardation (mean body weight of arfrp1 vil-/was 43.3±5% lower than that of control mice at the age of 28 days) arfrp1 vil-/mice displayed reduced triglycerides, free fatty acids and glucose plasma levels indicating that the growth retardation is the result of a malabsorption. uptake of glucose and amino acids were unaffected by the deletion of arfrp1. in contrast, lipid uptake as elucidated by oral fat tolerance tests was impaired in arfrp1 vil-/mice. arfrp1 vil-/enterocytes as well as arfrp1 mrna depleted caco-2 cells absorbed fatty acids normally but secreted chylomicrons with a markedly reduced triglyceride content. in addition, while the release of apolipoprotein a-i (apoa-i) was dramatically decreased apoa-i accumulated in the arfrp1 vil-/epithelium and was predominantly colocalized with rab2. our results demonstrate that the growth retardation of arfrp vil-/mice is a consequence of impaired intestinal fatty acid absorption. we suggest that arfrp1 is required for the assembly of aopa-i to the chylomicrons and for the further lipidation of chylomicrons in the golgi of intestinal epithelial cells. this finally leads to an secretion of chylomicrons with a markedly reduced triglyceride content. rhoa influences adhesion and spreading of cardiac fibroblasts via complex regulation of cytoskeletal proteins jatho a. 1 the monomeric gtpase rhoa is thought to be involved in the pathology of heart diseases, however, its role in cardiac cells is not well defined. therefore we intended to analyze the effect of rhoa in cardiac fibroblasts by using a lentivirus-based knockdown approach. by doing so, we could show that the knockdown of this gtpase by about 50% resulted in a massive change in cell morphology, but displayed no effect on cell viability. the appearance of the cells in the 2d-culture changed from a fibroblast-typical stretched morphology with intracellular stress fibers to a more epithelial-like cell morphology. by morphometrical analyses we demonstrate that fibroblasts with reduced rhoa expression display an increase in cell surface by 2.2-fold and in perimeter by 1.5fold. moreover, the depletion of rhoa significantly influences the adhesion velocity, as within the first hour after cell detachment about 20% of the rhoa knockdown cells reattach, but only 5% of the respective control cells. based on these findings, we investigated the distribution and composition of different cytoskeletal proteins by immunofluorescence stainings and immunoblot analysis. we found, that the amount of b-actin is not reduced in rhoa knockdown cells, however, the distribution is markedly changed. in these cells internal star-shape bundles of actin could be found instead of the commonly appearing stress fibers in control cells. in contrast, the cortical actin fibers, mainly consisting of g-actin, were not affected. in addition, smooth muscle-actin, which is characteristic for myofibroblasts, was clearly reduced in rhoa knockdown cells compared to control cells by 33%. this reduction might be responsible for the more relaxed cell shape. in summary, the knockdown of rhoa influences cell adhesion and the morphological characteristics of cardiac fibroblasts, without obviously affecting cell proliferation and viability. using site-specific fluorescence labeling to study uptake of pasteurella multocida toxin into eucaryotic cells jehle d., aktories k., orth j. institut für experimentelle und klinische pharmakologie und toxikologie abteilung 1, albertstraße 25, 79104 freiburg, germany pasteurella multocida is an opportunistic pathogenic bacterium living in the nasal pharyngeal space of animals. p. multocida is of particular importance in the livestock management of pigs. under special conditions infection of pigs with p. multocida leads to an atrophic rhinitis, which is characterized by the atrophy of nasal turbinate bones accompanied by a shortening and twisting of the snout. the causative agent of the atrophic rhinitis was found to be the bacterial protein toxin pmt (pasteurella multocida toxin). after entering the cell the 146-kda toxin activates various signal transduction pathways by stimulating heterotrimeric g proteins of the gαq, gα13 and gαi family. the underlying mechanism of the activation of heterotrimeric g proteins is a deamidation of an essential glutamine residue leading to a constitutive activation of the g protein. the uptake of pmt into cells is not comprehensively understood. therefore, we utilized a recently described technique, called "sortagging" (popp mw et al. nat chem biol. 2007; 3: 707) , to specifically couple fluorescence tags to the n-or c-terminus of pmt. the enzyme sortase a (srta) from staphylococcus aureus attaches proteins to the bacterial cell wall. the substrates are recognized by an lpxtg motif. srta cleaves the peptide bond after the threonine and adds a glycine-containing nucleophile. we introduced these motifs into pmt to express srta-recognized toxin and coupled the toxin with fluorescence tags, respectively. fluorescently labeled pmt was used to study the uptake of the toxin into eucaryotic cells by laser scanning microscopy. munich heart alliance, münchen, germany cells within the myocardium communicate by secreted factors and this has been suggested to contribute to cardiac remodeling. to identify novel factors secreted by the myocardium, we have previously reported a genetic yeast screen which led to the identification of protease inhibitor 16 (pi16). here we report the generation of a mouse line where pi16 can be deleted globally or conditionally using the cre/loxp system. after electroporation of the pi16 floxneo targeting vector in embryonic stem cells and injection into murine blastocysts we gained a mouse line that carried the targeted modification of the pi16 allele. global pi16 deficiency (pi16 lox/lox ) per se did not lead to a cardiac phenoytpe (hw/bw (mg/g): pi16 +/+ = 5.4 ± 0.2 (n = 6), pi16 lox/lox = 5.9 ± 0.7 (n = 4), p > 0.05; fibrosis (%): pi16 +/+ = 3.3 ± 0.2 (n = 7), pi16 lox/lox = 3.9 ± 0.8 (n = 5), p > 0.05; fractional shortening (%): pi16 +/+ = 29.8 ± 0.7 (n = 7), pi16 lox/lox = 26.4 ± 2.5 (n=5), p > 0.05). in addition we carried out an immunohistochemical analysis of pi16 expression using pi16-deficient mice as negative controls. pi16 localized to cardiac fibroblasts, to the epididymis and the trachea. in the failing heart we detected accumulation of pi16 preferentially in fibrotic areas. we are currently applying cardiac stress models to gain a broader understanding of the function of pi16 and its potential as a therapeutic target molecule. enantioselective determination of r-and s-hyoscyamine in mammalian plasma and urine samples john atropine (atr) is the racemic mixture of the tropane alkaloids s-and r-hyoscyamine (hyo). s-hyo acts as a competitive acetylcholine antagonist at the muscarinic receptors (eutomer) inducing mydriasis, excitations, hallucinations, coma and ultimately death, whereas r-hyo does not (distomer). atr is used for clinical intervention of poisoning with organophosphorus (op) pesticides or nerve agents. despite well known differences in pharmacological behavior, individual pharmacokinetics of both atr enantiomers have rarely been addressed in the literature [1, 2, 3] . therefore, we initially developed a nonchiral liquid-chromatography-electrospray tandem mass spectrometric method (lc-esi ms/ms) that allows quantification of atr and additional natural and synthetic tropane alkaloids from plasma after simple deproteinization [4] . to discriminate both atr enantiomers the sample preparation step was expanded by an enzymatic pretreatment. samples were incubated either with diluted human serum (not containing atropinesterase, atre, procedure a) or with diluted rabbit serum (procedure b). rabbit serum contains atre (ec 3.1.1.10) which is suitable for stereospecific hydrolysis of shyo into tropine and tropic acid while r-hyo remains unaffected. after sample precipitation, hyoscyamines were quantified by the lc-esi ms/ms method. following procedure a the concentration of total hyo and following procedure b remaining r-hyo were determined. s-hyo was calculated by the difference between these concentrations [3] . the impact of potential matrix ingredients that may appear in samples from oppoisoned patients under atr therapy were evaluated (oximes, op agents, carbamates) [3] . the assay was applied to diverse toxicological and pharmacological samples. i) measurement of natural s-hyo in an extract of atropa belladonna leaves as well as in plasma and urine of a female patient who was poisoned after ingestion of such leaves revealed that no biotransformation to r-hyo occurred. ii) analysis of plasma obtained from an op-poisoned female patient under atr therapy revealed faster elimination of shyo when compared to r-hyo [3] . iii) in contrast, no enantioselective differences were obvious in healthy anaesthetized swine after intravenous injection of atr [5] . data indicate that the enzymatic enantioselective procedure represents a useful tool to characterize in vivo behavior of r-and s-hyo allowing to reveal individual kinetics. failing hearts are unable to adequately supply the body with blood and oxygen. common therapeutic strategies interfere with cardiac remodelling, reduce cardiac preand afterload or aim at direct improvement of cardiac contractility. cardiac contractility is mainly controlled by β1-adrenergic receptors. resensitization of b-adrenergic receptors by inhibition of g-protein coupled receptor kinase 2 (grk2) is discussed as a potential strategy to treat heart failure. we characterized raf kinase inhibitor protein (rkip) as a physiological inhibitor of grk2 and found rkip to increase contractility of neonatal cardiomyocytes. the present study evaluated the role of rkip in heart failure. we assessed its effects on cardiac function in pressure overload induced heart failure and determined the expression patterns of rkip in failing hearts of humans and mice. transverse aortic contriction (tac) was performed on 7-week-old c57/bl6 mice to induce heart failure by pressure overload of left ventricles. after three weeks of tac, echocardiography showed distinct signs of decreased cardiac function in wild-type mice. fractional shortening was reduced and left ventricular diameters were increased. histological analyses revealed increased interstitial fibrosis, caspase-and tunelassays indicated myocyte apoptosis. western blot analysis showed significant upregulation of rkip expression in failing hearts compared to non-banded control hearts. interestingly, this upregulation of rkip expression was also detected in failing human hearts and in samples of patients with aortic valve stenosis but not in healthy control samples. to assess the effects of rkip overexpression on heart failure, we analysed heart function and structure of rkip transgenic mice and wild-type mice 3 weeks after tac. while left ventricular hypertrophy was increased to similar extents in wild-type and rkip trangenic mice, rkip mice did neither develop dilatation of the left ventricle nor a decrease in fractional shortening. in contrast to wild-type mice, the expression of the heart failure markers bnp and anp was not upregulated in banded rkip transgenic mice after 3 weeks of tac. taken together, cardiac overexpression of rkip prevented left ventricular dilatation and loss of contractile function in a mouse model of pressure overload induced heart failure. therefore, increased rkip expression may be an interesting target to prevent detrimental effects from increased left ventricular pressure. the main focus of this study was the chromatographic separation. the most frequently prescribed antibiotic drugs clarithromycin, erythromycin, clindamycin, cefuroxim, doxycyclin, amoxicillin, levofloxacin, and ciprofloxacin were selected for the screening method. method: a relatively short operation time and a sufficient separation were reached by column, eluent, and gradient optimization with poplc (phase optimized liquid chromatography). in the first step columns with the five stationary phases c18 eps 2, c18 sh 2, phenyl 2, cn 2, and c30 were used to determine the retention times of the drugs in an isocratic mode. the stationary phases, the column length and the retention times were fed in the poplc software and the optimal column was calculated. this column contained different stationary phases and was compared with customary columns. results: using the optimal column a sufficient chromatographic separation of the eight antibiotic drugs was reached. that was not possible with the customary columns. with the optimal column the time of measurement was too long. using a mobile phase gradient the measuring time could be reduced. discussion: with lc-ms/ms a complete chromatographic separation of all analytes is not necessary. but when measuring many transitions in a biological matrix two problems should be excluded: ion suppression and a too small number of measurement points per peak. especially when using positive and negative ionization in the ms a good separation is mostly necessary. to determine only the eight antibiotic drugs an optimized column is not necessary, but for a screening method with more than twenty drugs the poplc system is very helpful. we have investigated the uptake mechanism of cdt and in particular the intracellular membrane translocation of cdta. our data indicate that cdt requires acidification of the endosomal lumen for translocation of cdta across endosomal membranes into the cytosol. bafilomycin a1, an inhibitor of endosomal acidification protects vero (african green monkey kidney) cells from intoxication with cdt. consistently, translocation of cdta was observed when the acidic conditions of the endosomal lumen were mimicked at the cytoplasmic membrane of intact cells. next, we tested whether host cell factors are involved in membrane translocation of the toxin. radicicol, a specific pharmacological inhibitor of the chaperone heat shock protein hsp90 as well as cyclosporine a, an inhibitor of cyclophilins delayed the intoxication of cells with cdt but not with toxins a and b [1] . this result was confirmed by analyzing the adp-ribosylation status of actin from such cells in the presence or absence of the inhibitors. in addition, we excluded that the inhibitors of hsp90 and cyclophilins have any effect on receptor binding, endocytosis or enzyme activity of cdt. the data strongly suggest that the participation of hsp90 and cyclophilin is crucial for translocation of cdta into the cytosol. comparable results were obtained for the related binary iota toxin of c. perfringens. in vitro purified immobilized hsp90 and cyclophilin a specifically bound to the enzyme components of cdt and iota toxins. in conclusion, the results imply a common hsp90/cyclophilin a-dependent translocation mechanism for the family of binary actin-adp-ribosylating toxins. our current investigations focus on the participation of fk506-binding proteins (fkbps), another group of peptidyl-prolyl cis/trans isomerases in the membrane translocation step of these toxins. [1] kaiser, e., kroll, c., ernst, k., schwan, c., popoff, m. r., fischer, g., buchner, j., aktories, k. and barth, h. (2011) . complex multicellular in vitro coculture models represent a promising tool regarding e. g. cellular interactions with nanoparticles, since they more closely mimic the cellular composition of the body. therefore, we used our developed coculture model of the alveolar-capillary barrier composed of lung epithelial cells (nci h441) on top and microvascular endothelial cells (iso-has-1) on the bottom side of a filter-membrane to study nanoparticle cytotoxicity and cellular uptake. with a coculture period of about 10 days the cells achieve a more differentiated and polarized state and develop a tight barrier, which can be measured via ter (transepithelial electrical resistance). regarding cellular uptake of fluorescently labeled amorphous silica nanoparticles (asnps, 30 nm) the coculture took up much less asnps than conventional monocultures. besides, we could not verify a specific uptake mechanism (e. g. clathrin-, caveolae-mediated endocytosis) via immunofluorescence staining of the cells. however, we detected asnps incorporated in flotillin-1 and -2 labelled vesicles. former studies concerning cytotoxicity (lactate dehydrogenase assay) of amorphous silica nanoparticles (asnps, 30 nm) revealed that our coculture behaved much more robustly compared to conventional monocultures. however, regarding inflammatory responses (e. g. sicam, il-8 increase) the coculture responded more sensitively than conventional monocultures. in a further development we added a third cell type, the alveolar macrophage (am), to our coculture. since ams embody the front-line of alveolar defense against inhaled pathogens and particles, they play a central role in regulating lung immunity. as model we applied the human acute monocytic leukemia cell line, thp-1 (prestimulated with 8 nm pma for 4d) apically to the epithelial monolayer of the coculture. our preliminary studies concerning inflammatory responses of the tripleculture (h441/iso-has-1 with thp-1) revealed a higher sensitivity of the triple-culture compared to the double coculture. the triple-culture responded with an increased il-8 release upon lps or tnf-a stimulation. in conclusion, this triple-culture model offers a promising prospect to mimic more closely realistic cell interactions with nanoparticles in the distal lung. ethanol is a component of many herbal fluid preparations [1] , since it is an excellent extraction solvent for the phytochemical components of herbal drugs and contributes to the stability of these medicines. toxicological and pharmacokinetic evaluations [2] have shown that the small amounts of ethanol applied with therapeutic doses are safe even in children. despite that these medicines have been used safely since many decades, they have occasionally been subject of discussion in the public, triggered by the increasing problem of recreational abuse of alcoholic beverages by children and young persons [3, 4] . therefore, there is a growing need of a systematic evaluation of pharmacovigilance data on these medicines. for evaluating the experience gained from the therapeutic use of these medicines, 16 pro-and retrospective studies with 10 herbal medicinal products containing ethanol at doses of 40 to 240 mg per single dose, depending on the age group, have been analyzed, covering 49 816 patients of 0-12 years of age. in these studies, altogether 15 adverse drug effects have been described, none of which was attributable to the ethanol content of the medicines. in a survey of the worldwide use of these and other 6 herbal medicinal products it was shown that during the past few years, more than 764 mio daily doses have been sold, corresponding to more than 33 mio of patients (data obtained from manufacturers; figures available partly from 1993 onwards, partly from 2003/4 onwards). from the packages sold in germany in the years between 2005 and 2009, 48.1 mio were attributable to self-medication, 10.8 mio to prescriptions reimbursed by health insurance (ims, frankfurt). as non prescription medicines are reimbursed in germany only in children, at least the latter part of the prescriptions can be attributed mainly to children. all of these medicines are registered or licensed by regulatory authorities. adverse effects are covered by the pharmacovigilance system, and no adverse effects attributable to the ethanol content have been reported. this set of data supports the conclusion drawn from the experience of a safe use over decades, i.e. that the ethanol content of herbal medicinal products does not give any causes for concern regarding their safety even in children. dedication: this contribution is dedicated to prof. dr. hilke winterhoff, institute for pharmacology and toxicology, university of münster, who died on 9 may 2010. she has initiated this work. prucalopride was introduced as a new selective 5-ht4 receptor agonist that is approved for treating obstipation. whereas one could expect -due to the fact that 5-ht4 receptors are functionally expressed in the human heart -in clinical trials prucalopride did not show cardiac effects. this is quite surprising because other 5-ht4 receptor agonists have been withdrawn from the market just for that reason. in this study we used prucalopride for in vitro studies with atrial preparations from transgenic (tg) mice with cardiac myocyte-specific overexpression of the human 5-ht4a receptor. isolated electrically driven (1 hz) left and spontaneous beating right atria of tg mice were compared with those of wild type (wt) littermates. moreover, we used isolated electrically driven (1 hz) human right atrial preparations from patients undergoing cardiac surgery. finally gr113808, a 5-ht4 receptor antagonist, was added. prucalopride exhibited a dose dependent positive inotropic effect in left atria and a positive chronotropic effect in right atria of tg mice with a logec50 of -6.8 and -7.1, respectively (p<0.05 vs. wt, n=3). in human atrial tissue prucalopride also acts as an agonist, leading to an inotropic effect. all effects could be antagonized by 10 µm gr113808. we could demonstrate that prucalopride acts as an agonist at the 5-ht4 receptor in our transgenic mice model and also in human right atrial preparations. these findings suggest that tachycardia and arrhythmias are possible side effects, which should be carefully looked for. the involvement of psychological factors, especially stress, are known to play an important role in functional gastrointestinal diseases (1, 2) probably by affecting the brain-gut axis. based on the good correlation between stress & functional dyspepsia (fd), many animal models for fd have been developed where animals are subjected to various kinds of psychological stress either during the neonatal period or in adulthood. this stress was found to induce gastric motor dysfunction resembling symptoms of fd. two models for stressinduced fd were performed in order to choose the more adequate one for studying sensitivity changes of the fundus to various mediators as well as changes in some relevant hormone levels in the blood for subsequently testing the efficacy of treatment with stw 5 (a 9 component herbal preparation of proven clinical efficacy in fd and in irritable bowel syndrome (3, 4) ). in one model, maternal separation (5) was performed on weanling rats starting from postnatal day 2 for 3 h each day for 3 weeks. rats were then allowed to mature to an adult age. the other model was that of restraint stress (rs) (6, 7) . adult animals were restrained for 90 min/ day for 1 week. the animals of both models were eventually sacrificed, the stomach fundus was isolated and its sensitivity in vitro to carbachol, potassium chloride, serotonin and adrenaline was tested. blood samples were taken to assess levels of ghrelin, corticosterone releasing factor (crf) and corticosterone. the sensitivity of fundus strips from restrained rats towards the agents tested, partly representing autonomic responsiveness, was more depressed than those from maternally separated ones. levels of ghrelin, crf and corticosterone were also more elevated in the rs model. that model was therefore chosen to test the efficacy of stw 5 in restoring the deranged parameters. a group of animals received stw 5 orally once daily starting treatment 1 week before exposing them to rs and continuing treatment for a further week during subjection to rs. stw 5 was effective in normalizing the depressed stomach fundus responses exhibited by animals subjected to rs and to normalize to a large extent the deranged blood levels of ghrelin, crf and corticosterone. the findings lend further evidence to the role of the brain-gut axis in fd and gives supportive evidence for the first time for the clinical usefulness of stw 5 in this condition. there is good evidence that oxidative damage to dna leads to down-regulation of transcription of affected genes and epigenetic gene silencing in a mechanism dependent on the 8-oxoguanine dna glycosylase (ogg1), which generates harmful repair intermediates [1, 2] . we have recently shown that the magnitude of inhibition of transcription of an egfp reporter gene by single 8-oxo-7,8-dihydro-deoxyguanosine (8-oxodg) varies between the opposing dna strands of the gene [3] . we now have addressed the question, to which extent the transcription inhibitory potential of 8-oxodg depends on its position in the gene and on the dna microsequence surrounding the modified nucleobase. to investigate the effect of position, we produced plasmid vectors containing single 8-oxodg or dg (underlined) in the second position of an agc trinucleotide. measurements of egfp expression in transfected mammalian host cells showed that a single 8-oxodg caused a strong inhibition of gene transcription. the magnitude of this effect for 8-oxodg situated in the transcribed dna strand of the gene was the same as in the non-transcribed dna strand. similarly, there was no quantitative difference between the effects of 8-oxodg present in the 5′-versus 3′-utr of the gene. the results thus indicate that inhibition of transcription by this base modification does not depend on the position in the gene. further comparisons were done between the effects of 8-oxodg nucleobases localised in the same dna strand but within different sequence contexts. gene expression analyses in the repair proficient host cells showed that the degree of inhibition of transcription caused by single 8-oxodg was dependent on the neighbouring nucleotides. among three tested sequence motifs, the minimal effect on the gene transcription was found to correlate with a significantly less efficient base excision by the purified human ogg1 protein. the results thus support the initiatory role of ogg1 in the mechanism of transcriptional repression. in addition, the finding of the effect of dna sequence on the base excision activity of ogg1 suggests that repair rates of single base modifications in genome could also be heterogeneous. allgayer j., kitsera n., epe b., khobta a. johannes gutenberg university of mainz institute of pharmacy and biochemistry, staudingerweg 5, 55128 mainz, germany interference of dna base modifications with gene transcription is an important biological consequence of genotoxic damage [1] . an efficient method for incorporation of a single 8-oxo-7,8-dihydroguanine (8-oxog) at a defined position in the egfp gene in a plasmid vector was recently developed in our lab. the method relies on the availability of adjacent sites for a sequence-specific nicking endonuclease, which allow the insertion of a synthetic oligonucleotide containing 8-oxog in a chosen position. we further showed that this single lesion inhibits transcription after excision by ogg1 [2] . in order to determine to which extend the observed effect depends on the position of the modified base in the gene, we constructed several new plasmid vectors which allow incorporation of the same dna oligonucleotide containing 8-oxog or g in different positions in different dna-strands. dna sequence cassettes were designed to contain a 5′-cattgcttcgctagcacg nucleotide sequence in different orientations, which was flanked by two unidirectional bsrdi recognition sites (5′-gcaatgnn). adapters containing the restriction sites for directional cloning into the 5′-or the 3′-utr of the plasmid-borne egfp gene were added. the produced plasmid vectors thus allow the insertion of a single 8-oxog in the same sequence context into opposing dna strands in the 5'utr and in the 3'utr. keratinocytes are the major cell type of epidermis and are responsible for the formation of an outer barrier, the statum corneum, to protect an organism against harmful environmental influences. for generation of this barrier, keratinocytes pass through a complex differentiation program that is accompanied by synthesis of lipids, like cholesterol and ceramides. finally, the differentiation of keratinocytes leads to apoptosis. another function of keratinocytes is to sense environmental factors, some of which are decoded by members of the transient receptor potential (trp) ion channel family. trpv3, for example, is predominantly expressed in keratinocytes, and decodes different chemical and physical stimuli like the terpenoid-derived ligands camphor and thymol or temperatures above 33°c. less is known about the influence of cholesterol on trpv3 signalling. we modified the cholesterol content of hek293 cells stably transfected with trpv3 and performed flipr-based calcium measurements. these experiments revealed that cholesterol enrichment robustly potentiates trpv3 by sensitizing it to lower agonist concentrations. we verified these results with whole-cell patch-clamp measurements. in contrast, trpv2, another heat-sensing channel, was not affected by cholesterol modification. since former studies showed a defective formation of epidermal barrier in trpv3 -/mice, our results imply that a cholesterol-regulated trpv3 signalling may contribute to the progression of differentiation or initiation of apoptosis of keratinocytes. ischemia-reperfusion injury causes severe problems in the early period after lung transplantation. since transient receptor potential (trp) channels are important regulators of vascular permeability and tone, we investigated the influence of a trpc6 blocker on pulmonary function after simulated transplantation, using an ex vivo model of isolated perfused and ventilated rabbit lungs. to this end, heart-lung blocks were excised and mounted in an artificial thorax chamber. negative pleural pressure ventilation was initiated, and lungs were perfused with albumin-containing tyrode-solution (100 ml min -1 ). after equilibration in a stable perfusion and ventilation mode for 30 min, lungs were flush-perfused with perfadex ® solution, followed by an ischemic storage for 4 h on ice. subsequently, ventilation and perfusion were re-initiated to simulate a transplantation situation. in the oxygenated group, pulmonary artery po2 was adjusted to 120 mmhg, in the deoxygenated group, the perfusate inflow was gassed with nitrogen to achieve a pulmonary artery po2 of 50 mmhg. both transplantation conditions were conducted in the absence or in the presence of the trpc6 blocker larixol acetate (5 µm). hemodynamic and ventilatory parameters were continuously monitored. the weight of deoxygenated lungs steadily increased during 2 h of reperfusion from 22.1 ± 1.32 g to 35.4 ± 4.23 g. this weight gain was inhibited by supplementation of the trpc6 blocker (from 21.4 ± 0.91 g to 27.4 ± 2.42 g 2 h after reperfusion). in contrast, oxygenated lungs showed no marked weight gain after reestablishment of perfusion (from 17.5 ± 1.51 g to 21.5 ± 2.29 g 2 h after reperfusion), and the trpc6 blocker had no additional effect (initial 19.5 ± 1.28 g, 2 h reperfusion 21.3 ± 1.22 g). we conclude that a trpc6-blocking compound to the lung perfusate during simulated transplantation counteracts the endothelial permeability increase and the resulting post transplant weight gain. the results indicate a role for trpc6 in the regulation of pulmonary vessel permeability and support the concept that perfusion of donor lungs with trpc6 blockers may prevent edema formation caused by ischemiareperfusion injury shortly after lung transplantation. regulation of human inducible nitric oxide synthase (inos) expression by noncoding rnas (ncrnas) kleinert h., schmitz k., koch k., hahn s., pautz a. universitätsmedizin der johannes gutenberg-universität mainz institut für pharmakologie, obere zahlbacher str. 67, 55101 mainz, germany the transcriptome analyses of human cells showed that additionally to the 30.000 protein coding sequences the human dna codes for much more (450.000 ?) non-coding rnas 1 . beside the ribosomal, and transfer rnas (rrna and trna) involved in the mechanism of translation there are also short (snrna, sorna, mirna) and long noncoding rnas (ncrnas) implicated in regulation of gene expression (splicing, translation, chromatin packaging etc.). matsui et al. described regulation of il-1β-induced inos expression by an antisense rna (as-3-utr-rna) in rat hepatocytes 2 . a promoter located on the antisense strand 3' to the last exon (exon 27) of the rat inos gene drives the expression of an as-rna complementary to the 3'-utr of the rat inos mrna. using different sense primers with homology to the 3'-utr sequences of the human inos gene for specific rt-pcr reactions (detecting only an as-rna) we were not able to detect such an as-3-utr-rna in human cells. in addition, transient transfection analyses using constructs containing a 2.7 kb fragment of the 3'-flanking genomic sequences (as used by matsui et al. in the rat system) of the human inos gene in front of a luciferase reportergene into dld-1 cells revealed no promoter activity of these sequences. korneev et al. described the expression of a 2 kb anti-inos-ncrna in different brain tumors and showed reciprocal expression to the inos mrna in human embryonic stem cells 3 . analyzing the expression of this anti-inos-ncrna in cytokine-treated dld-1 cells also showed a basal expression of this as-rna and an enhancement of the expression by cytokine-treatment. downregulation of the anti-inos-ncrna by sirnas reduced whereas overexpression enhanced cm-induced inos expression in human dld-1 cells. this indicates that this anti-inos-ncrna regulates cytokine-induced inos expression in a positive manner. rna 14, 2030 rna 14, -2037 rna 14, (2008 . using directed evolution to improve functional expression of class b g-protein coupled receptors klenk c., scott d. j., plückthun a. universität zürich institut für biochemie, winterthurerstrasse 190, 8057 zürich, switzerland the class b of g-protein coupled receptors (gpcrs) comprises 15 peptide hormonebinding receptors which regulate important endocrine and neuroendocrine functions of the human body. several of these receptors are implicated in the pathogenesis of severe diseases such as diabetes, osteoporosis, growth disorders and depression, which makes them attractive targets for drug therapy. to develop new compounds targeting these receptors a detailed understanding of the molecular structure is required which has not been succeeded to date. structural studies of proteins by x-ray crystallography or nmr spectroscopy generally require large and homogenous quantities of protein. for gpcrs this prerequisite is difficult to achieve as the vast majority of gpcrs exhibits low endogenous expression and is very unstable in solution. therefore, improved expression conditions are necessary for the efficient characterization of new gpcr structures. here we present a method to optimize class b gpcrs for improved heterologous expression and increased thermostability by means of directed evolution. libraries of class b gpcrs were obtained by random mutagenesis and were expressed in a heterologous expression system in which functional gpcr is targeted to the inner membrane of e. coli. mutants that display increased receptor expression levels and ligand binding were selected by flow cytometry using fluorescently labeled ligands. repetitive cycles of randomization and selection allow to gradually increasing the level of protein expression and stability. with this evolutionary approach key residues within the receptor sequence can be identified rapidly that are responsible for improved biophysical properties without affecting the pharmacological features of the receptor. such gpcr mutants will become a valuable tool on the way to express high quantities of stable receptor protein for subsequent structural studies. pasireotide (som230) is currently under clinical evaluation as a successor compound to octreotide for the treatment of acromegaly, cushing's disease and carcinoid tumors. whereas octreotide acts primarily via the sst2 somatostatin receptor, pasireotide was designed to exhibit octreotide-like sst2 activity combined with enhanced binding to other somatostatin receptor subtypes. somatostatin and octreotide stimulate the complete phosphorylation of at least six carboxyl-terminal serine and threonine residues namely s341, s343, t353, t354, t356 and t359, which in turn leads to a robust endocytosis of the sst2 receptor. surprisingly, pasireotide failed to phosphorylate the four threonine residues and induced only a detectable phosphorylation of s341 and s343. somatoprim and ke108 are recently developed somatostatin analogs capable of binding to four of five somatostatin receptor subtypes. here, we performed an in vitro study comparing the effects of pasireotide, somatoprim and ke108 on sst2 somatostatin receptor binding, phosphorylation, internalization and signaling. further somatostatin, octreotide, pasireotide, somatoprim and ke108 were tested for functional selectivity at sst5 receptor mutants, which possess a carboxyl-terminal sst2tail. this approach allows detection of receptor activation by phospho-specific sst2 antibodies. compared to octreotide, somatoprim activates sst2 but has a higher activity on sst5. ke108 and pasireotide are partial agonists at the sst2 receptor. pasireotide, ke108 and somatoprim show comparable effects on sst5 receptor. however, none of these new pan-somatostatin analogs behaves like natural somatostatin on the sst5 receptor. cadmium modulates ahr-associated gene expression in the rat intestine after oral exposure kluxen f. m. 1, 2 , höfer n. cadmium has been shown to mimic steroid estrogen effects in vivo and in vitro. we have recently identified cross-talk of estrogen receptor (er) and aryl hydrocarbon receptor (ahr) in the rat uterus where 17beta-estradiol (e2) and cdcl2 modulate ahrassociated genes via er after i.p. injection in a similar fashion (kluxen et al., arch toxicol doi 10 .1007/s00204-011-0787-x). however, the predominant route of exposure to cadmium in non-smokers is via diet. moreover, uterus expresses mainly the receptor subtype eralpha, whilst small intestine and colon express mainly erbeta. thus, we now investigated by real-time rt-pcr the effects of cadmium (2mg/kg b.wt cdcl2 (cd2)) or steroid estrogen (0.1 mg/kg b.wt. 17alpha-ethinylestradiol (ee2)) on ahrassociated gene expression (i.e., ahr, cyp1a1, gsta2, nqo1) in the small intestine of rats after oral exposure (3 days gavage). the animals were also co-treated with cd2 and pure anti-estrogen (2.5 mg/kg b.wt zk191703 (zk)) or ee2, to asseess whether ermediated processes are involved. we also measured cyp1a1 mrna expression in two estrogen receptor negative colon cancer cell lines (ht-29 and caco2) treated for 5 days with cdcl2 (1µm) and e2 (0.01µm). the dose-dependency of cadmium induced ahr target gene modulation was studied in a second animal experiment, with administration of cadmium in drinking water for 28 days (0.4-9 mg/kg b.wt cdcl2 equivalent to 5, 50, and 150 ppm) and ee2 (0.08 mg/kg b.wt) as steroid reference. in summary we present two major results: the metalloestrogen cadmium modulates dose-dependently the ahr-associated gene expression in the intestine after oral exposure. yet, since the cadmium induced modulation of ahr target genes was not antagonized by anti-estrogen in the small intestine in vivo and was also found to occur in er-negative colon cells in vitro, we propose that er-independent mechanisms might play a role in this effect. meg) is the most cytotoxic lesion. if not repaired by o 6 -methylguanine-dna methyltransferase (mgmt), o 6 meg/t mismatch is recognized by the mismatch repair system (mmr) that performs futile repair cycles. during this process secondary lesions (i.e. dna single-strand brakes) are formed, which block dna replication in the next replication cycle, leading to dna double-strand breaks (dsbs). these dsbs eventually signal to apoptosis and other genotoxic endpoints. here, we wished to address the question whether autophagy is part of the cellular response triggered by o 6 meg. we also assessed whether autophagy influences apoptosis induced by tmz in glioma cells. we show that tmz induces autophagy in u-87 mg and ln-229 glioma cell lines. the maximum amount of autophagy was observed several days (96 h) after tmz treatment and mgmt proficient cells did not display significant autophagy. thus, the data show that mgmt protects against tmz induced autophagy, pointing to o 6 meg as the critical lesion responsible for induction of autophagy. using colon cancer cell lines proficient and deficient in mmr, we show that mmr is required for tmz-induced autophagy. because dsbs, which emerge during the processing of o 6 meg, are repaired preferably by homologous recombination (hr) ln-229 cells stably down-regulated for hr were tested for autophagy induction. the data indicate that dsbs are involved in tmz-induced autophagy. because autophagy following tmz treatment occurs earlier than apoptosis we hypothesize that autophagy protects glioma cells against apoptosis. using an early stage autophagy inhibitor 3-methyladenin we have shown, that autophagy inhibition sensitized glioma cells to tmz-induced apoptosis. taken together our data point out that tmz induces autophagy in glioma cells and that autophagy protects glioma cells against tmz-induced apoptosis. o 6 meg is the lesion responsible for autophagy induction. furthermore, the data also shows that mmr and dsbs are involved in the induction of autophagy after tmz treatment. work was supported by bmbf (02nuk016). retinitis pigmentosa (rp) is a severe human retinal disease characterized by a progressive degeneration of rod photoreceptors and a secondary loss of cone function. in most cases, rp finally leads to legal blindness. mutations in the regulatory subunit of the rod cyclic nucleotide-gated (cng) channel (cngb1a) have been found in patients suffering from rp. we used cngb1-deficient (cngb1 -/-) mice to establish a gene replacement therapy as a potential treatment for rp by means of recombinant adenoassociated viral (raav) vectors. the packaging limitations of raav vectors required a capacity-optimized vector of the large cngb1a cdna (approx. 4 kb). therefore, we replaced regulatory elements within the expression cassette and used a short mouse rhodopsin promoter element for rod-specific expression. after injection of therapeutic raavs (serotype 8) into the subretinal space of 2-week-old cngb1 -/mice, we assessed the restoration of vision by analyzing i) protein expression and localization, ii) retinal function and morphology and iii) vision guided behavior. we found that treated cngb1 -/mice expressed full-length cngb1a and cnga1, which were previously downregulated. both proteins co-localized in rod outer segments and formed regular cng channel complexes in the treated area of the cngb1 -/retina. using electroretinography (erg) we observed a distinct rescue of rod-driven responses. moreover, cngb1 replacement significantly preserved outer segment morphology and delayed retinal degeneration. finally, treated cngb1 -/mice performed significantly better than untreated mice in a modified water maze task designed to test for rod-mediated, vision-guided behavior. in summary, this work provides a proof-of-concept for the treatment of rod channelopathyassociated retinitis pigmentosa by raav-mediated gene replacement. most endocrine disruptors interact with hormone receptors or steroid biosynthesis and metabolism, thereby modifying the physiological function of endogenous hormones. here, we present an alternative testing paradigm for detection of endocrine modes of actions that replace and reduce animal testing through refinement. receptor mediated endocrine effects were assessed using the yeast based receptor mediated transcriptional activation yes/yas assays and effects on steroid hormone biosynthesis were assessed using the human cell line h295r in the steroidogenesis assay. in our testing paradigm we propose to complement the in vitro assays with a single in vivo repeated dose study in which plasma samples are analyzed for their metabolome profile. the combination of these methods does not only contribute to refinement and reduction of animal testing, but also has significantly increased the efficient allocation of resources and allows for a sound assessment of the endocrine disruption potential of compounds. thus, this proposal constitutes a potentially attractive alternative to epa's endocrine disruptor screening program. data on 14 reference substances for which the in vitro yes/yas and steroidogenesis assays and the in vivo metabolome analysis were performed to assess their putative endocrine mode of action is presented here. the bovine corneal opacity and permeability (bcop) test has been adopted by oecd for the identification of corrosive and severe ocular irritants (ghs category 1) for single component substances and multi-component formulations. eye irritation tests (eit) using human reconstructed tissue models (such as epiocular) have been described to predict ocular non-irritants (ghs no category). thus the ultimate repaltement of the draize rabbit eye irritation test (oecd tg 405) by a combined or tiered testing strategy could be possible. the purpose of this study was to evaluate whether the bcop with additional corneal histology together with the eit could be used to predict eye irritancy of agrochemical formulations according to different classification schemes including un ghs and epa systems. we have performed the bcop (plus histology) and the eit of 50 agrochemical formulations for which in vivo eye irritation data were already available (for registration purposes). using the oecd tg guideline evaluation scheme for opacity and permeability in the bcop was not predictive for the agrochemical formulations assessed here, while corneal histology grades and the epiocular tissue viabilities were useful predictors of eye irritancy potencies and could be applied for the different classification schemes. the nanomaterials offers extraordinary opportunities. the nano-structure can change the physical and chemical properties, and often also alters the biological effects. hence the toxicity of a nanomaterial can differ from its larger-scale material; but as of today, no new quality of a general nano-specific toxic effect has been observed. therefore the established testing methods are generally suitable. it is, however, difficult to apply the nanomaterials to in vitro test systems, since it is the nature of these materials to change their surface properties and agglomeration stateand the uptake and distribution in the body may differ from their larger-scale materials. while the methods for topical effects may be used for nanomaterials without further modification, the in vitro methods for genotoxicity testing require the dispersion in culture media. the use of reproducible and well-documented dispersion protocols andthe characterization of its particle size distribution is de rigueur . [1] . for many nanomaterials published genotoxicity studies did not give a consistent picture [2] and therefore there are rather effects of individual nanomaterials than nano-genotoxicity per se. modern toxicology is based on the insight into toxic pathways. for nanomaterials a testing strategy will include testing for their primary effects (which might be only a handful: particle effects, catalyzing the formation of reactive molecules and ion release) and their uptake, distribution and clearance. the use of dermal penetration studies in vitro for the risk assessment of sunscreen nanomaterials has been demonstrated [3] . in vitro methods for specific effects (such as inflammation, pulmonary toxicity, sensitization) are currently awaiting validation (for both chemicals/molecules as well as nanomaterials). in the meantime alternative short-term in vivo studies with optimized biological readouts can deliver information on the toxic pathways as well as the biokinetics and dose-response relation of nanomaterials in the body. a short-term inhalation test for nanomaterials has already been used successfully [4] . testing strategies based on those methods engage less animals and provides more significant data than classical testing. moreover data from these methods will serve as a benchmark and a validation for the in vitro models under evaluation. nanoparticles (np) are increasingly used in various field of industry which necessitates evaluation of their safety. also in the food industry, nps have gained strong interest, for example as food additives or to improve food packing. however, the potential risks of ingested np have been rarely investigated. inhalation studies have revealed that inflammation and oxidative stress may represent unifying mechanism for the induction of adverse health effects of toxic np. in the present study, a co-incubation model of human polymorphonuclear neutrohils (pmns) and caco-2 human intestinal epithelial cells was used as a model to address potential genotoxic effect of np during intestinal inflammation. oxidative dna damage induction (measured by the fpg-modified comet assay) was induced in the caco-2 cells by activated pmn and this effect increased with increasing pmn to caco-2 cell ratio. the crucial involvement of the phagocyte nadph oxidase complex could be demonstrated using treatment of caco-2 cells with bone marrow-derived pmn from nadph oxidase deficient mice. dna damage by pmn as well as h 2o2 was increased in buthionine sulphoximine (bso) pre-treated caco-2 cells, illustrating the importance of the cellular glutathione (gsh) status in these target cells. gsh depletion in caco-2 cells could also be shown upon treatment with various types of np. our data suggests that ingested np may increase the susceptibility of the colon mucosa to genetic damage during the occurrence of intestinal inflammation. the ingestion of seafood contaminated by acute toxic doses of the marine toxin okadaic acid (oa) is responsible for diarrhetic shellfish poisoning. it is recently known that both the rat and the human hepatic cytochrome p450 monooxygenases (cyp) are able to metabolize this toxin. currently, there is a lack of data about the toxicity and mode of action of oa after xenobiotic metabolism. the aim of our study was the measurement of the toxicity and oxidative stress status in hepg2 cells incubated with oa in the absence and presence of s9 mix. pure oa, as well as oa pre-activated with liver homogenisates (s9 mix) were used to treat human hepg2 cells that have nearly undetectable levels of functional cyp but express phase ii enzymes. the experiments were performed with both human and rat s9 fraction plus cofactors of phase i enzymes. the cell viability was measured after 4 h using mtt-test and xcelligence real time cell monitoring system. furthermore, levels of intracellular reactive oxygen species (ros) were detected by 2´,7´-dichlorofluorescein diacetate and additionally by measuring the intracellular glutathione content. in the presence of both human and rat s9 mix oa showed a higher toxicity than the parental substance. oa pre-incubated in rat s9 mix was toxic at 75 nm oa. strong effects could be observed when oa was pre-activated with human s9-mix at a concentration of 50 nm oa. pure oa was non-toxic in that concentration range. we could also detect an increase of oxidative stress in hepg2 cells treated with oa in the presence of all investigated s9-mix. these results suggest that oa is activated after oxidative xenobiotic metabolism into metabolites which possess a higher cytotoxic activity and increase the amount of intracellular ros in hepg2 cells. ballast water treatment -emerging health risks werschkun b., banerji s., krätke r. bundesinstitut für risikobewertung, max-dohrn-strasse 10, 10589 berlin, germany the introduction of invasive marine species into new environments by ships' ballast water, via ships' hulls and other vectors has severe impacts on the oceans. in 2004 the international maritime organisation (imo) launched the international convention for the control and management of ships ballast water and sediments which requires ballast water to be treated in order to eliminate alien aquatic species. ballast water treatment may include mechanical, physical or chemical measures. any ballast water management system using active substances needs imo approval. therefore identification of active substances, relevant chemicals and submission of specified datasets on their physical, chemical and toxicological properties is required in order to assess the safety for the aquatic environment and for human health. the bfr is the german federal agency responsible for health risk assessment and has been involved in more than twenty assessment and approval processes so far. the majority of imo approved systems are based on oxidative principles such as chlorination and ozonation. these methods can generate disinfection by-products (dbps), which are a mixed group mostly of halogenated organic substances like trihalomethanes, haloacetic acids and haloacetonitriles. the formation of dbps is well known from the disinfection of drinking water. some dbps are regulated under drinking water directives because of their long-term toxicity but many are unregulated and have unknown toxicological properties. the formation of dbps may vary significantly depending on the treatment system as well as on environmental parameters like temperature, ph and composition of the organic matter within the aquatic environment. in sea water, sources for dbp formation besides ballast water treatment are aquaculture and the cooling systems of coastal power plants. in order to address possible health and environmental risks from dbps formed during ballast water treatment a conference on emerging risks from ballast water treatment was held at bfr in october 2011. here we summarise the main conference findings and identify areas for future research. two presentations corroborated that significant amounts of dbps can be formed in sea water and a presentation on the toxicological properties of dbps pointed out that many have genotoxic properties. accordingly, the determination of dbp species and generated concentrations under different ballast water treatment conditions was seen as a mayor task. different approaches for health and environmental risk assessments were also discussed. appropriate human exposure scenarios and methods for exposure assessment, taking into account common approaches used in risk assessment were presented during the conference. a suitable approach based on derived pec-values for exposure quantification was proposed in order to improve the procedure available for risk assessment of chemical agents used for ballast water treatment. agonist-selective signaling of µ-opioid receptors in t lymphocytes kraus j., börner c., lanciotti s., koch t., höllt v. inst. für pharmakologie und toxikologie, leipzigerstr. 44, 39120 magdeburg, germany opioids are the most potent analgesics and irreplaceable for the treatment of severe pain. in addition to their central effects, opioids modulate a great variety of immune effector cell functions, which may result in unwanted side effects during opioid treatment. the effects of most of the commonly used opioids are mediated by µ-opioid receptors, which belong to the superfamily of g protein coupled receptors. recent data support the concept that g protein coupled receptors function as dynamic entities, which may occupy multiple conformations and activate multiple signaling pathways in a ligand-dependent manner. consequently, different ligands activating the same receptor may have different cellular effects, which has been termed "agonistselective signaling". little is known about agonist-selective signaling of µ-opioid receptors in immune effector cells. in a first attempt to understand if and why such different profiles among different opioids occur we investigated effects of different opioids in human jurkat t cells. we report that the µ-opioid receptor ligands fentanyl, methadone, loperamide and betaendorphin induce internalization of a µ-opioid receptor-green fluorescent reporter construct, whereas morphine and buprenorphine did not induce internalization. the internalization was dependent on p38 mapk and phospholipase d2. in line with this, we observed marked phosphorylation of p38 mapk and activation of phospholipase d2 induced by the internalizing opioids, but no or little such activity by morphine and buprenorphine. as a physiological result, fentanyl, methadone, loperamide and betaendorphin treatment of primary human t cells and jurkat t cells resulted in a strong, up to 100 fold induction of il-4, which was dependent on p38. in contrast, morphine and buprenorphine only showed a weak, approximately one order of magnitude lower induction of il-4. by inducing il-4 opioids significantly modulate the t helper cell balance into the type 2 direction, which influences various immune responses, e. g. the antiviral, t helper cell type 1-mediated response. considering the vital necessity of opioid use in humans, it is an intriguing goal to identify analgetically feasible opioids that have little or no immunosuppressive or -modulatory effects. modulation of cgmp signals by phosphodiesterases in smooth muscle cells krawutschke c., koesling d., russwurm m. ruhr-universität bochum pharmakologie und toxikologie, universitätsstrasse 150, 44780 bochum, germany within the cardiovascular system, cgmp mediates smooth muscle relaxation and inhibition of platelet aggregation. cgmp is formed by particulate guanylyl cyclases and nitric oxide-sensitive guanylyl cyclases, that are activated by natriuretic peptides or nitric oxide (no), respectively. besides the cgmp-forming enzymes, the cgmp-degrading phosphodiesterases strongly determine amplitude and shape of cgmp signals. in vascular smooth muscle cells, three phosphodiesterases are considered to be responsible for cgmp degradation: pde5, the cgmp-specific phosphodiesterase is activated directly by cgmp binding to its gaf domains; this activation if further stabilized by cgmp-dependent protein kinase-mediated phosphorylation. pde1, the ca2+/calmodulin-stimulated pde constitutes the majority of cgmp-hydrolyzing activity in smooth muscle cells at least in the presence of high intracellular ca2+ concentrations. and lastly pde3, the cgmp-inhibited pde displays some cgmp-degrading activity, although cgmp binding to its catalytic domain is primarily thought to inhibit the campdegrading activity of pde3. cgmp signals measurable in radioimmunoassays require stimulation with cgmpincreasing vasodilator concentrations that are orders of magnitudes higher than those required for relaxation. thus, we developed fluorescent sensors for real-time measurement of cgmp signals in single cells. by using these indicators, we analyzed the contribution of different cyclic nucleotide-degrading phosphodiesterases to the modulation of cgmp signals elicited by physiologically relevant vasodilator concentrations. hyaluronan (ha) is a major component of extracellular matrices and is thought to control cellular phenotypes such as proliferation and migration. therefore, ha synthesis may play an important role in the pathophysiology of atherosclerosis. there are three major ha-synthase isoenzymes (has1-3). the has3 gene is alternatively spliced. has3 transcript variant 2 (has3v2) encodes the smallest has isoenzyme which has a different c-terminus and contains only two transmembrane domains compared to has3 transcript variant 1. the aim of the present study was to investigate whether has3v2 is expressed by vascular cells, how it is regulated and where it is localized in cells and whether it indeed synthesizes ha. has3v2 mrna expression was monitored by quantitative real-time rt-pcr. protein expression was determined by western blotting using a polyclonal antibody that was raised in rabbit. an n-terminal eyfp-has3v2 fusion protein and a ddk-tagged has3v2 construct were expressed for subcellular localization studies and co-immunoprecipitation (co-ip). endogenous has3v2 mrna was expressed in both vascular smooth muscle cells (vsmc) and endothelial cells. furthermore, western blotting revealed has3v2 protein expression in vsmc and platelets. in vsmc has3v2 mrna expression was strongly up-regulated in response to interleukin-1β (il-1β, 10 ng/ml) whereas stimulation with interleukin-10 (10 ng/ml), platelet-derived growth factor-bb (10 ng/ml), transforming growth factor β (10 ng/ml), tumor necrosis factor α (10 ng/ml) and interferon-γ (10 ng/ml) had no effect. transfection of hek cells with eyfp-has3v2 fusion protein revealed localization to the endoplasmic reticulum but not to the plasma membrane. furthermore, co-ip experiments showed that tagged has3v2 proteins were precipitated together suggesting formation of multimeric has3v2 complexes. transfection of has3v2 did not cause increased secretion of ha into the cell culture supernatant in hek cells. in conclusion, has3v2 is present in vascular cells and responds to inflammatory cytokines such as il-1ß. because of the intracellular localization and the lack of ha secretion in has3v2 transfected cells, has3v2 may serve intracellular functions apart from ha synthesis. the tubulin antagonist pretubulysin shows strong vascular-disrupting properties in vitro several epidemiological studies indicate a correlation of human exposure to ultrafine particulate air pollution caused by incomplete combustion processes and an increase in the incidence of pulmonary immune diseases like asthma. as a possible mechanism behind this pathological phenomenon, the adjuvant effect of lung inflammation induced by poorly soluble environmental particles has been hypothesised. the aim of our study was to investigate the causal link between carbon nanoparticle-induced lung inflammation and modulations of immune cell populations during processes leading to sensitization, and allergic immune responses of the airways. therefore mice were treated with ovalbumin (ova) alone or in combination with carbon nanoparticles (cnp) by pharyngeal aspiration. the induction of inflammation and the immune adjuvant activity were studied in the lungs and lung draining peribronchial lymph nodes (pbln) at the level of sensitization, and at the level of the immune response. ova-specific ige antibodies were measured in blood serum, and the development of allergic airway inflammation was studied after ova challenge. results at the level of sensitization showed that cnp-induced immediate airway inflammation had immune adjuvant activity resulting in an increase of specific cell populations in pbln and in a stimulation of asthma-specific th2 cytokines. a specific reduction of the neutrophilic lung inflammation by application of the compatible solute ectoine significantly reduced the adjuvant effects of cnp. in ova-sensitized mice, application of cnp 12 hours prior to allergen challenge, led to a significant increase in inflammatory cell infiltrate and respective cytokines in broncho-alveolar lavages. coapplication of 1 mm ectoine together with cnp reduced the particle-induced effects. our data show a link between neutrophilic lung inflammation and adjuvant effects of cnp. a specific reduction of neutrophils by the application of ectoine attenuated this np induced adjuvant effect, indicating that particle-induced lung inflammation rather than the direct interaction of nanoparticles with immune cells is the critical step in environmentally modulated pulmonary immune diseases like asthma. introduction: drug-eluting stents (des) are commonly used in the treatment of acute artery occlusion. however, even if released cytotoxic drugs reduced neointimal proliferation significantly there is still the risk of in-stent thrombosis. it is presumed that this is due to reduced reendothelialization. it has been suggested that coating the stent with biomolecules may provide a new approach to circumvent the lack of healing of the endothelial layer. one approach would be the use of biomolecular signals, such as (poly)peptides and growth factors. rgd and redv are peptide motifs, known to enhance cell attachment and spreading. aim: the aim of our study was to evaluate the efficacy of proteins, derived from elastin-like proteins (elp) and artificial modified by incorporating with the amino acid motifs rgd,redv and p15, in terms of endothelial healing on stents and other cardiovascular devices. results: in this work, we generated vectors encoding for different biopolymers consisting of various bioactive signal molecule sequences. the peptides, e.g. based on the elastinlike matrix (vpgig)2-vpgkg-(vpgig)2, were synthesized using heterologous expression. after optimizing culture conditions and extraction procedures their biological activity was assessed using human umbilical vein endothelial cells (huvec). elastin like proteins with differently incorporated bioactive signals (redv, rgd and a small p15 peptide) were linked covalently via carbodiimide coupling to poly(l-lactide) (plla) films. huvec growth was determined on these modified surfaces using the brdu assay (cell proliferation) and resazurin assay (cell viability). the chemically modified plla surfaces conferred higher cell viability after 1 h adhesion (60%) and an enhanced proliferation (63%, 1 h adhesion, 24 h cultivation) in comparison to the unmodified plla. these results indicate that the synthesized elp incorporated with amino acid motifs promote an accelerated endothelialization of the biodegradable stent material plla. discussion: in summary, we were able to generate elastin-like proteins modified by bioactive sequences. those sequences enhanced endothelial cell proliferation and adhesion. further studies are warranted to determine the activity on smooth muscle cells of these peptides. (1) the failing heart is characterized by excessive extracellular matrix production by myofibroblasts (myocfs) causing fibrosis and myocardial stiffening. myocfs represent phenotypically transformed cardiac fibroblasts (cfs) and are characterized by the expression of contractile proteins like a-smooth muscle actin (α-sma) and enhanced secretion of growth factors (e. g. ctgf). identification of intracellular enzymes that modulate this transformation process is desired to therapeutically modulate pro-fibrotic progression in heart failure. we show that pde2a, a phosphodiesterase isoform, that is able to hydrolyse cgmp and camp, is markedly upregulated in failing hearts from patients with end-stage heart failure (2-3-fold, p<0.05, n=8). notably, pde2a protein abundance is 4-fold higher in myocfs compared to cardiomyocytes from neonatal rat hearts (p<0.05, n=7). to this end we tested whether pde2a modulates the transformation of cfs isolated from neonatal rat hearts to myocfs. indeed, as assessed by immunoblotting and fluorescent microscopy (α-sma, phalloidin, dapi), adenoviral pde2a overexpression induced α-sma expression (3.2-fold p<0.05, n≥8) and to a lower extent ctgf synthesis (1.5-fold, p<0.05, n≥5). mechanistically, pde2a showed preferential subsarcolemmal localisation with diminished total cgmp levels (-56%, p<0.05, n≥5). consistently, parallel stimulation with atrial natriuretic peptide (anp), a selective activator of membrane-bound guanylyl cyclase, normalized ctgf synthesis indicating that pde2a controls cgmp in a discrete subdomain near the plasma membrane. moreover pde2a overexpression diminished the protein levels of vasodilator-stimulated phosphoprotein, a membrane cytoskeletal component (-60%, p<0.05, n≥5). these data implicate pde2a-dependent subsarcolemmal cgmp regulation in myofibroblast formation and potentially cardiac fibrosis. therefore, targeting pde2a may lead to regression of the fibrotic remodeling associated with heart failure. several anorganic nanoparticles (np) causedhigher inhalation toxicity than the corresponding coarse particles (oberdoerster et al. 2005) . we examined an organic pigment and a polymer dispersion each as nanomaterial and as the chemical identical coarse material in short-term inhalation studies in malerats. the polymer was an anionic acrylic ester copolymer containing free carboxylic groups. three different particle sizes were synthesized by varying polymerization conditions: 12, 80 or 250 nm. although polymeric acrylic ester was reported to be irritating to the respiratory tract at 3 mg/m3, all three tested polymers -including the np (12 and 80 nm) -did not cause any changes in lavage fluid and in histopathology at 10 mg/m3. the organic pigment was a poorly soluble pyrrol with an intense orange color. the np (10 to 50 nm width and 30 to 400 nm length) and the coarse pigments (70 to 200 nm width and 0.3 to 3 µm length) are both needle-like. they were tested at 1, 3, 10 and 30 mg/m3 for the np and 3, 10 and 30 mg/m3 for the coarse materials. mild and partly reversible morphological changes were observed in lung and lymph nodes at the highest concentrations, but the more pronounced effect were found in rats exposed to the coarse material. likewise there was an increase of lavage parameters in rats exposed to thecoarse material but not to the np. these data demonstrate that inhalation of finer np is not necessarily associated with higher toxicity compared to the coarse material. the results were obtained with two organic particles of rather different size and composition but are in contrast to the more severe effects seen with several anorganic np when compared to the corresponding coarse particles. within the nanocare project a standard short-term inhalation test to examine the toxicity of inhaled aerosols from nanomaterials has been developed. the inhalation toxicity of nano-andpigmentary materials was studied: baso4, zno, ceo2, al-doped ceo2, zro2, amorphous silica, surface-coated amorphous silica, titania, carbon black and three multi-wall carbon nano tubes, all with complete phys-chem-characterization as planned for the oecd sponsorship program. quartz dust tio2 and zno were tested as pigmentary materials. rats were exposed nose-only to three concentrations of one of these materials, 6 h a day for five consecutive days. positive controls were exposed to quartz dust or pigmentary zno, negative controls to clean air. a wide range of endpoints for pulmonary toxicity were evaluated immediately after the last exposure and after 3 days and 3 weeks after the last exposure. among these parameters, polymorphnuclear granulocyte count in bronchoalveolar lavage fluid is the most sensitive early parameter indicating inflammation process in lung, while histological examination reveals the type and localization of inflammation. among these substances, we identified baso4 as having the lowest toxicity. all mwcnts were most potent in producing progressive inflammation in the lung; granulomas in lung and lung associated lymph nodes were observed without indication for fibrosis. the noaecs of the 16 substances ranged between < 0.1 and >50 mg/m 3 . generally the material was only found in the lung (surface and macrophages) and in the draining lymph nodes. surface modified amorphous silica was also found in the spleen. the data demonstrate that the method is able to differentiate the toxic potential of different nanomaterials and to indicate regression or progression of the effects effects. moreover the lung burden and potential translocation to other tissues was detecable. comparing the material properties and effects of the 16 materials, no general relationship between the toxicity and either particle size, specific surface area or aerosol particle number concentration was found. hence we must not expect to find a gerneral "nanotoxicology" or a unifying dosimetry for all nanomaterials. we must rather be prepared to test individual nanomaterials for their effects. and to develop grouping concepts not only based on material properties but also on biopersistence, biokinetics and biological effects. part of this studes has been sponsored by bmbf (nanocare). endpoint-centric search for toxicological information and data to support the information retrieval for regulatory programs landsiedel r. 1 , wächter t. the eu reach regulation no 1907/2006 requires industry to ensure the safety of chemical use and manufacturing. all substances manufactured or imported in quantities above one tonne per year must be registered. information requirements for the dossiers increase with increasing tonnage or once hazards are suspected. searching for substance specific literature and the compilation of hazard data for safety assessments are highly challenging procedures. the novel web-based search engine go3r, accessible free of charge at www.go3r.org, has been created to allow quickly finding relevant hazard information and data. go3r provides an endpoint-centered literature search to all scientists and regulatory authorities seeking for toxicological information. furthermore, go3r specifically highlights information on animal testing alternatives. search results are presented automatically linked to an "intelligent table of contents" which enables the user to sort the literature listed in pubmed or the toxicology data network (toxnet) in a fast and comprehensive manner. retrieved documents are automatically organized in categories relating to the iuclid 5 chapters. hereby, the user can browse directly through the entire 21 million documents without even having to start the search with an initial query. the semantically enriched platform supports the user during query formulation, allows for bibliographic analysis, and specifically highlights information related to the replacement, reduction, and refinement of animal experiments. search results in go3r are shown in an dynamic table of contents (left) making them browsable for the contained information on animal testing alternatives and toxicologogical endpoints. towards a differentiation therapy of acute myelogenous leukemia with histamine h2-receptor agonists laue s., burhenne h., seifert r. hannover medical school institute of pharmacology, carl-neuberg-str. 1, 30625 hannover, germany acute myelogenous leukemia (aml) is a devastating malignancy characterized by a differentiation block of myeloid progenitor cells. recently, histamine dihydrochloride in combination with interleukin 2 has been approved as orphan drug for the consolidation treatment of aml (1) . it is assumed that histamine exerts its effects by activating the histamine h2-receptor (h2r) in human neutrophils, resulting in improved anti-tumor function of t killer cells by inhibiting nadph oxidase-catalyzed superoxide formation. previous studies had shown that histamine also induces myeloid differentiation (2) . considering the fact that all-trans-retinoic acids constitutes a powerful differentiation therapy of acute promyelocytic leukaemia, a specific subtype of aml (3), we initiated a study to explore the possibility that h2r-mediated myeloid differentiation provides an alternative or complementary strategy to treat leukemias associated with differentiation blocks. as model system, we used hl-60 cells. in hl-60 cells, histamine and various h2-receptor agonists induced concentrationdependent increases in camp levels. interestingly, ligands differentially increased cytosolic calcium concentration and extracellular receptor kinase (erk) pathways, indicative for ligand-specific h2r conformations. h2r activation resulted in myeloid differentiation as assessed by enhanced formyl peptide receptor-mediated increases in cytosolic calcium concentration. h2r agonists showed no signs of cytotoxicity. intriguingly, following h2r activation, the majority of the formed camp was exported into the extracellular space via multi-drug resistance protein (mrp) 4, indicating that export is a more important pathway for signal termination than cleavage of camp by phosphodiesterases. despite effective camp export, even a short-term exposure (30 minutes) of cells was sufficient to induce expression of functionally active formyl peptide receptors. these data indicate that in contrast to previously held dogma, induction of myeloid differentiation does not require continuous presence of a camp signal. from a therapeutic point of view this is very important since "spike" therapy with campincreasing substances may be sufficient to induce a therapeutic effect in aml, thereby also reducing toxic side effects. currently, we are systematically exploring the effects of h2r agonists on signal transduction pathways and differentiation in various myeloid cell types to identify highly efficacious compounds. introduction. activation of gαq/11 protein-coupled receptors of postsynaptic neurons can elicit the production of endogenous cannabinoids (endocannabinoids), which in turn inhibit transmitter release from axon terminals by activating presynaptic cb1 receptors. the aim of the present experiments was to study the mechanism of the endocannabinoid production. specifically, we wanted to clarify the role of ca 2+ release from intracellular stores in triggering endocannabinoid production. methods. patch-clamp-and ca 2+ imaging experiments were performed on purkinje cells in mouse cerebellar brain slices. glutamatergic excitatory postsynaptic currents (eepscs) were elicited by electrical stimulation of parallel fibers. the gαq/11 proteincoupled metabotropic glutamate receptor 1 (mglur1) was activated by superfusion of (rs)-3,5-dihydroxyphenylglycine (dhpg) or -more physiologically -by burst stimulation of the parallel fibers. results. both dhpg superfusion and burst stimulation of parallel fibers elicited an increase in intracellular ca 2+ concentration in the postsynaptic purkinje cells. dhpg superfusion and burst stimulation suppressed eepscs, and this suppression was abolished in the presence of the mglur1 antagonist cpccoet. the suppression of the eepscs was also sensitive to the cb1 receptor antagonist rimonabant, pointing to involvement of endocannabinoids and cb1 receptors. the suppression of the eepscs was attenuated after depletion of the endoplasmic reticulum ca 2+ stores by thapsigargin, cyclopiazonic acid and ip3. the results indicate that after activation of the gαq/11 protein-coupled metabotropic glutamate receptor 1 (mglur1) of the postsynaptic neuron ca 2+ is released from the endoplasmic reticulum. this ca 2+ release significantly contributes to the production of endocannabinoids. the endocannabinoids diffuse in the synaptic cleft retrogradely to the terminals of afferent axons and inhibit transmitter release there through presynaptic cb1 receptors. the guanine nucleotide exchange factor dock9 controls reelin dependent cdc42effects on radial migration pichler m. 1 the regulation of blood glucose levels is under tight control of a complex system including hormone and neurotransmitter signalling. many of these cellular signalling pathways are initiated by binding of the ligand to a g-protein coupled receptor (gpcr), e.g. noradrenaline inhibits insulin secretion upon binding to a gi-coupled receptor. upon gpcr activation the heterotrimeric g-protein is activated and both the α-subunit and βγdimers are released and interact with their specific target proteins. by the usage of bordetella pertussis toxin (ptx) as a common gαi inhibitor gαi-dependent signalling pathways are interrupted which leads to increased insulin secretion, and significantly improves glucose tolerance. since the gαi-isoform specific roles in the regulation of glucose homeostasis are still debated we studied the glycemic control in gαi2-deficient mice. surprisingly and in contrast to the ptx data, glucose tolerance was unchanged in the gαi2-deficient mice compared to wild type controls. however, the plasma insulin levels were significantly reduced upon glucose challenge. these findings point to disturbed islets function and improved peripheral insulin sensitivity. analysing gαi2deficient islets we show that islet size and number of nuclei are reduced. nevertheless, in vitro insulin secretion is improved at low (3 mm) and high (16 mm) glucose concentrations and can be further stimulated upon ptx-treatment. these data indicate that gαi2 proteins influence islet development and inhibit insulin secretion. in addition, these findings support our hypothesis that gαi2-deletion influences peripheral insulin sensitivity. therefore, we investigated glucose homeostasis and pakt-levels after two hours feeding ad libitum in gαi2-deficient mice. under feeding conditions no differences in plasma insulin levels were visible although blood glucose levels were significantly reduced in gαi2-targeted mice. pakt-levels of liver and skeletal muscle were unaltered, whereas akt phosphorylation in white adipose tissue was significantly increased, indicating improved glucose uptake of adipocytes. in conclusion, gαi2 is a negative regulator of both insulin secretion and peripheral insulin sensitivity and important for the maintenance of glucose homeostasis. 11689, 1992) in a radioligand binding test and to determine their functional effects with a membrane potential test using the dye r7260 (molecular probes, 0.125 mg/ml, excitation 505 nm, emission 530 nm). the affinity of compounds in radioligand binding was slightly higher in sur2b than in sur2a-type channels, but the enantiomeric ratio in sur2a channels matched that one determined for the sur2b-type indicating some conformity of the binding pockets of sur2a and sur2b-proteins. surprisingly, however, the membrane potential tests revealed that the (3r,4s)-enantiomer acted as agonist (a) whereas the (3s,4r)-enantiomer acted as antagonist (b): (3r,4s)-bms-191095 induced membrane hyperpolarisation whereas (3s,4r)-bms-191095 repolarised cells prestimulated with submaximally effective concentrations of diazoxide. concluding, bms-191095 is not selective for sur2a as compared to sur2b-type k atp channels. its enantiomers activate and block sur2-type katp channels in a stereospecific manner. thieno-thiadiazine derivatives with full agonistic activity at sur2b-type katp channels act as partial agonists at cardiac sur2a-subtypes oldenhage c., grittner d., schmidt c., lemoine h. heinrich-heine universität, inst. für lasermedizin, mol. wirkstoff-forschung, universitätsstr. 1, 40225 düsseldorf, germany new potassium channel openers (kco) of the thieno-thiadiazine(ttd)-type initially developed as agonists for the sur1-type katp channels (nielsen et al., j med chem 45: 4171, 2002) were characterized in sur2b-type katp channels as agonists and antagonists, if r contains a quaternary (methyl-cycloalkyl) and a tertiary (r = cycloalkyl) carbon, respectively (lemoine et al., this journal 375, r45, 2007) . to investigate the selectivity of ttd-derivatives for myocardial katp channels the membrane potential actions of compounds were tested in hek 293(kir6.2/sur2a)-cells and compared to hek 293(kir6.1/sur2b)-cells as a model for smooth muscle-type katp channels. membrane potential was measured by fluorescence (excitation 505 nm, emission 530 nm) using 0.125 mg/ml of the dye r7260 (molecular probes). standard-kco induced hyperpolarisation with ~10-fold smaller potency (pec50) in sur2a. ttd-compounds with ch3-cycloalkyl residues not only lost potency but also intrinsic activity for channel activation (emax) in sur2a. possibly, this loss of emax would be much greater in native heart cells with a normal channel density. in contrast, ttd-compounds with cycloalkyl residues acted as antagonists of cells pre-hyperpolarized with diazoxide with similar affinity in sur2a and sur2b-type katp channels. concluding, selectivity of kco for katp channel-subtypes cannot only be achieved by a different affinity but also by a selective stimulation of the channel of interest. small-conductance calcium activated potassium (kcnn/sk/kca2) channels maintain neuronal calcium homeostasis, shape synaptic functions and prevent excitotoxic neuronal death. so far, little is known about the function of kca2 channels in nonneuronal cells. the aim of this study was to investigate the expression of kca2 channels in microglial cells and their potential function in microglial activation and maintenance. expression of kca2 channel subtypes in microglial cells was assessed by mrna analysis, western blots and immunocytochemistry. lipopolysaccharide (lps)-induced microglial proliferation was evaluated by the xcelligence impedance-based system and mtt assays, and immunogenic activation of microglia was determined by measuring cytokines and nitric oxide (no) release into the cell culture medium. the kca2.2 and kca2.3 channel activator cyppa (25 µm) and specific inhibitory peptides (50 µm) were applied to distinguish effects mediated by the kca2 channel subtypes. all kca2 channel subtypes were detected on mrna and protein levels in resting and in lps-activated microglial cells. xcelligence real-time measurements and mtt assays demonstrated that lps (200 ng/ml) induced microglial proliferation. the kca2.2/kca2.3 channel activator cyppa reduced lps-induced microglial proliferation in a concentration-dependent manner. specific peptide inhibitors of kca2.3 channels, but not of kca2.2 channels, reversed the cyppa-effects on lps-induced microglial proliferation. cyppa alone did not alter the production of tnf-alpha or il-6, but strongly reduced the lps-dependent cytokine production. interestingly, chelation of extracellular calcium by edta induced differential cytokine kinetics by decreasing lps-dependent il-6 production while tnf-a production was not affected. moreover, using inhibitory sk3/kca2.3 channel peptides, we demonstrated that sk3/kca2.3 channels modulate lps-induced cytokine il-6 production in a calcium-dependent manner, while the tnf-a release was independent of extracellular calcium. in summary, the present study revealed that kca2.3 channel stimulation reversed microglial activation. thus, kca2.3 channels may serve as a therapeutic target for reducing microglial activity and related inflammatory responses in cns diseases. (3r,4s)-(3s,4r)intracellular amyloid beta (aß) oligomers and extracellular aß plaques are key players in the progression of sporadic alzheimer disease (ad). still, the molecular signals triggering aß production are largely unclear. we asked whether mitochondria-derived reactive oxygen species (ros) are sufficient to increase aß generation and thereby initiate a vicious cycle further impairing mitochondrial function. complex i and iii dysfunction were induced in a cell model using the respiratory inhibitors rotenone and antimycin resulting in mitochondrial dysfunction and enhanced ros levels. both treatments lead to elevated levels of aß. presence of an antioxidant rescued mitochondrial function and reduced formation of aß demonstrating that the observed effects depended on ros. conversely, cells overproducing aß showed impairment of mitochondrial function such as comprised mitochondrial respiration, strongly altered morphology, and reduced intracellular mobility of mitochondria. again, the capability of these cells to generate aß was partly reduced by an antioxidant indicating that aß formation was also ros-dependent. moreover, mice with a genetic defect in complex i, or ad mice treated with a complex i inhibitor, showed enhanced aß levels in vivo. several lines of evidence show that mitochondria-derived ros result in enhanced amyloidogenic amyloid precursor protein processing, and that aß itself leads to mitochondrial dysfunction and increased ros levels. we propose that starting from mitochondrial dysfunction a vicious cycle is triggered that contributes to the pathogenesis of sporadic ad. comparison of methods to derive health-based guidance or limit values for chemicals licht o., voss j. -u., mangelsdorf i. fraunhofer item chemikalienbewertung, nikolai-fuchs-str. 1, 30625 hannover, germany health-based guidance or limit values are derived for chemicals to compare measured or estimated exposure concentrations with these values. if the exposure is below the limit value, adverse effect for human health can be regarded as negligible, e.g. the exposure is expected to be tolerable. in germany such values have been derived since years for chemicals that can be found in soil, water and air as well as human blood and urine (biomonitoring). in a research project sponsored by the german umweltbundesamt several methods used by the agency are compared to the method laid in reach guidance document r.8 to derive a derived no effect level (dnel). the aim was to identify possibilities for standardization as well as to figure out specific elements in individual methods. in addition to extrapolation factors the public availability of guidance and specific derivations as well as procedures for consensus on the limit value were evaluated. the comparison of extrapolation factors revealed that, although they are named differently such as extrapolation, safety or assessment factors, they are used in a comparable manner. factors for interspecies and intraspecies extrapolation are presented in more detail. the standard factor for such extrapolation is 10 in most cases. in the reach guidance this factor consists of a part for allometric scaling and remaining differences. other factors are only defined in some methods, like a factor for extrapolation from loael to noael, data quality or data gaps. a factor for data quality is not laid down in the basic scheme for setting of indoor air guidance values, but is used in some of the recent derivations of limit values. also the who guidelines for drinkingwater quality use comparable factors to account for adequacy of studies or database and nature and severity of effect. a transparent and documented derivation is necessary for acceptance of the value. the derivation methods as well as the evaluation document on a specific substance are available through publications or the internet in nearly all cases. for the dnel only the numeric value is available at the echa website, but not any information on starting point and extrapolation factors. although all guide or limit values are derived in a comparable way, differences, however, exist in some details. in most cases detailed explanation is lacking when deviating from standard or default assumptions. often such deviation is based on expert judgement. hepatocellular carcinoma (hcc) is the fifth most common cancer in the world and has a poor prognosis with limited therapeutic options. up to now, no curative systemic therapy exists emphasizing the high clinical importance of new therapies for hcc. therefore, the identification and characterization of novel drugable targets is a relevant goal. cyclin-dependent kinase 5 (cdk5) is well characterized for its function in cns development and disease. recently, few reports indicate functions of cdk5 in cancer. cdk5 was shown to regulate tumor growth, and our group discovered that cdk5 regulates angiogenesis. since hcc is a highly vascularized tumor and anti-angiogenic treatment (sorafenib) has shown some therapeutic benefit, we hypothesize that cdk5 is an interesting target for hcc therapy. the aim of this study was to characterize the function of cdk5 in hcc. histology of tissue micro arrays indicates an increased expression of cdk5 in human hcc tissue in comparison to healthy liver tissue of the same patient. to investigate the function of cdk5 in hcc, we analyzed the impact of both pharmacological inhibition of cdk5 and specific downregulation of cdk5 with rna interference on hcc cells. pharmacological inhibition of cdk5 with the small molecule roscovitine (r-roscovitine, seliciclib) decreased proliferation and clonogenic survival, induced g2/m cell cycle arrest and cell death, and reduced motility of huh7 and hepg2 cells. transient downregulation (sirna) and stable knockdown (shrna) of cdk5 also reduced proliferation, clonogenic survival, migration and invasion of huh7 cells. in a subcutaneous hcc xenograft model, treatment with roscovitine reduced tumor growth and angiogenesis, indicated by decreased tumor weight and volume, and reduced vessel density. moreover, cotreatment of hcc cells with roscovitine and tumor necrosis factor related apoptosis inducing ligand (trail) resulted in an over-additive additive effect on the induction of apoptosis. this coincided with reduced phosphorylation and activity of the anti-apoptotic transcription factor stat3 at ser727 that is directly phosphorylated by cdk5, and tyr705. in line with this, the expression of the antiapoptotic protein mcl-1 is reduced by inhibition of cdk5. our results point to an important function of cdk5 in hcc and suggest cdk5 as an interesting pharmacologically druggable target for hcc therapy. delivery of mono-biotinylated rnasea into macrophages with streptavidinconjugated clostridium botulinum c3 toxin lillich m. 1 , chen x. clostridium botulinum produces the adp-ribosyltransferase c3, which modifies and thereby inactivates exclusively the small gtp binding proteins rho-a,-b and -c. recently, we discovered a specific endocytotic internalization of c3 toxin in macrophages and myeloid leukaemia cells, but not in epithelial cells [1] . thus, c3 toxin provides a tool to target cells of the monocyte/macrophage lineage, which are involved in various diseases and are of great clinical interest. we used a biochemical crosslinking approach to design a delivery system based on an enzymatic inactive c3bot mutant (c3mut) and streptavidin. the c3 portion mediates uptake of the transporter into monocytes/macrophages and streptavidin allows for binding of biotinylated cargo molecules to the transporter. in vitro, the generated c3mut-streptavidin bioconjugate showed specific and concentration dependent binding to biotinylated oligonucleotides as demonstrated by electrophoretic mobility shift assay. cell fractionation experiments indicated an uptake of the bioconjugate into the cytosol of j774a.1 macrophages. in the next step, mono-biotinylated bovine pancreatic ribonuclease a (rnasea) was used as a model cargo for the delivery of macromolecules by the bioconjugate. rnasea is a highly stable, well studied protein which catalyzes the degradation of rna. mono-biotinylated rnasea interacts in a specific and concentration dependent manner with the c3mut-streptavidin bioconjugate in vitro as analysed with dot blot technique. the c3mut-streptavidin bioconjugate efficiently mediates the internalization of biotinylated rnasea into j774a.1 macrophages as analyzed with laser scanning microscopy in fixed cells. this finding was also confirmed by live cell imaging. furthermore, cell fractionation showed a cytosolic delivery of biotinylated rnasea in the presence of c3mut-streptavidin. as expected we could not observe a cytotoxic effect of biotinylated wild-type rnasea on j774a.1 macrophages, which is attributable to the presence of ribonuclease inhibitor protein in mammalian cells. in summary, the c3mut-streptavidin bioconjugate mediates the efficient internalization of biotinylated (macro)molecules into macrophage like cells, and therefore represents a useful tool for the transduction of exogenous molecules into macrophages. in addition, cytotoxic rnasea mutants are available and will be used in further studies. organometal compounds such as cisplatin or the second generation complexes carboplatin and oxaliplatin have become more and more important as antitumor agents. nevertheless there is still an increasing demand for novel metal-based compounds. this is necessary due to severe side effects and the occurence of resistent tumour cells. in this context we investigated the cytotoxic effects of imidazole-based phosphane gold(i) complexes as potential agents for cancer treatment. initially we have used the mtt-assay to examine the toxic potential of the gold complexes in h4iie rat hepatoma cells. in this context cw60 (a diphosphane ligand with azoyl substituents r2p(ch2)2pr2, r= thiazol-2-yl) turned out to be the compound with the highest cytotoxic potential with an ic50 value of 6,5mm (24h incubation). further investigations revealed that cw60 induced an apoptotic cell death in h4iie demonstrated by the activation of caspase 3/7 (48h incubation with 10mm cw60). in addition the induction of apoptosis was confirmed by the dna ladder formation (24h incubation with 5mm cw60). in connection with the molecular mechanisms of apoptosis induction we used the comet assay to analyse the generation of dna strand breaks as well as the dcf-assay to detect the formation of reactive oxygen species. however neither dna strand breaks nor increased levels of reactive oxygen species were detected after 1h of incubation. furthermore we analysed if the compound influences intracellular signalling pathways such as the jnk pathway and the pi3k/akt but after 24h of incubation neither pakt nor jnk were influenced. the imidazole based phosphane gold (i) complex cw60 shows strong toxic effects in h4iie cells and turned out to be a promising compound as a potential agent for cancer treatment. the high and inappropriate intake of loop diuretics in hypertensive elderly reported in former studies has again been confirmed. remembering that inappropriate intake of loop diuretics can lead to exsiccosis and electrolyte loss especially in elderly, better medical education has to follow these alarming results to improve the pattern of diuretic prescription. furthermore, our results lead us to assume a high estimated number of unreported cases of torasemide use in uncomplicated arterial hypertension in elderly. this loop diuretic agent shows a longer duration of action compared with furosemide (elimination half-life: 3-4 hrs vs. 1 hr) and is effective in decreasing blood pressure in subdiuretic doses. it must be pointed out that loop diuretics are still frequently inadequately prescribed because current guidelines recommend loop diuretics only in complicated arterial hypertension. the role of cgmp/cgki signaling and trpc channels in regulation of vascular tone loga f., domes k., hofmann f., wegener j. pharmakologie und toxikologie for 923, biedersteiner str 27, 80802 münchen, germany signaling by intracellular cgmp and cgmp-dependent protein kinase i (cgki) is the major pathway in vascular smooth muscle, by which endothelial no regulates vascular tone. recent evidence suggests that trpc channels are targets of cgki in smooth muscle and mediate, at least partially, the relaxant effects of cgmp. we tested this concept by investigating the role of cgmp/cgki signaling on vascular tone and peripheral resistance using cgki-, trpc6-, and trpc3-, and trpc3/c6-double knock-out mice. we found larger contractile responses to α-adrenergic stimulation in intact aorta from cgki-, trpc6-, and trpc3/c6-double knock-out mice as compared to aorta from ctr and trpc3-knock-out mice indicating a functional link between cgki and trpc6 channels. no differences were found if the vasodilator tone, provided by the no generation in the vascular endothelium, was inhibited by l-name. likewise, no differences were observed in the increase in peripheral resistance by α-adrenergic stimulation using the hind limb perfusion system. activation of cgki by 8-br-cgmp diminished aortic tone and peripheral resistance to a similar extent in control, trpc6-, trpc3-, and trpc3/c6-double knock-out mice. no effect of 8-br-cgmp was observed in preparations from cgki -/mice. to test the co-localization of cgki and trpc channels, we performed immunocytochemistry on isolated smooth muscle and endothelial cells from aorta of ctr, trpc3-, and trpc6-knockout mice. trpc3 could be detected in both smooth muscle and endothelial cells whereas trpc6 was only detected in endothelial cells. the results suggest that absence of cgki or trpc6 impairs the vasodilator tone induced by endothelial no production but that cgki and trpc6 channels are not functionally coupled in vascular smooth muscle. we thank profs birnbaumer (nih) and freichel (homburg) for providing us with trpc6 -/-, and trpc3 -/mice and prof. flockerzi (homburg) for the antibodies against the trpc channels. whole genome microarray analysis of the effects of tcdd and pcb 153 in human hepatic cell models lohr c. 1 , neser s. after the treatment with tcdd, however, a total of 281 genes were more than 2-fold up regulated in hepg2 e.g. cytochrome p450 1a1 (cyp1a1) (32-fold) a sensitive marker for ahr activation. additional up regulated genes in hepg2 were; arylhydrocarbon receptor repressor (ahrr) 15-fold, aldehyde dehydrogenase 3 a1 (aldh3a1) 11-fold, and cytochrome p450 1b1 (cyp1b1) 10-fold. 44 genes were more than 2-fold down regulated in hepg2 cells e.g. -proprotein convertase subtilisin/kexin type 9. markedly different findings were obtained in hheps, i.e., 117 genes were up regulated, the highest up regulated gene was cyp1b1 with a 95-fold increase in gene expression, followed by cyp1a1 (41-fold) and aldh3a1 (21-fold). only a small group of genes were significantly down regulated (17 in total), e.g., solute carrier family 2 (facilitated glucose transporter). comparing both human cell types, there was an unexpected small overlap of genes being up or down regulated. interestingly, in both cell types, only 30 in common genes were up regulated, including cyp1a1, cyp1a2, cyp1b1 and aldh1a3. only platelet-derived growth factor receptor, beta polypeptide, was down-regulated in both hepg2 and hheps. in conclusion, our data indicate pronounced differences in the patterns of tcdd-regulated genes between hepg2 cells and hheps. detection of redox modified proteins in nociceptive processing lorenz j. e. 1 , kallenborn-gerhardt w. recent data indicate that redox modifications of proteins induced by reactive oxygen species (ros) contribute to sensitization of pain pathways during persistent pain. however, little is known about the targets of ros in pain processing, because the relatively unstable nature of many reversible protein oxidation states hampers the reliable detection and identification of modified proteins. here, we used the quantitative thiol trapping technique termed oxicat to identify proteins which are redox modified during nociceptive processing. we investigated spinal cords of untreated mice, after zymosan injection into a hindpaw (inflammatory pain model) and after spared nerve injury (neuropathic pain model). we identified several proteins with marked changes in their redox states after nociceptive stimulation. our results show that the oxicat method is an efficient method to detect redox modifications in proteins and that redox modifications seem to play a role in pain processing. supported by the deutsche forschungsgemeinschaft (sfb 815/a14). additive antinociceptive effects of a combination of vitamin c and vitamin e after peripheral nerve injury lu r., kallenborn-gerhardt w., geisslinger g., schmidtko a. pharmazentrum frankfurt/zafes institute of clinical pharmacology, goethe university, frankfurt am main, germany accumulating evidence indicates that increased generation of reactive oxygen species (ros) contributes to the development of exaggerated pain hypersensitivity during persistent pain. in the present study, we investigated the antinociceptive efficacy of the antioxidants vitamin c and vitamin e in mouse models of inflammatory and neuropathic pain. we show that systemic administration of a combination of vitamins c and e inhibited the early behavioral responses to formalin injection and the neuropathic pain behavior after peripheral nerve injury, but not the inflammatory pain behavior induced by complete freund's adjuvant. in contrast, vitamin c or vitamin e given alone failed to affect the nociceptive behavior in all tested models. the attenuated neuropathic pain behavior induced by the vitamin c and e combination was paralleled by a reduced p38 phosphorylation in the spinal cord and in dorsal root ganglia, and was also observed after intrathecal injection of the vitamins. moreover, the vitamin c and e combination ameliorated the allodynia induced by an intrathecally delivered ros donor. our results suggest that administration of vitamins c and e in combination may exert synergistic antinociceptive effects, and further indicate that ros essentially contribute to nociceptive processing in special pain states. -206, -213 and -214) replaced by leucine residues. both amino acids are comparable in terms of hydrophobicity, volume and the preference for forming α-helices, but only methionine is oxidizable to a sulfoxide, in contrast to leucine. in the present study we examined the protein-protein interaction (ppi) of recombinant ac 1, expressed in sf9 insect cell membranes, with cam, cam-206, -213, -214 and -215 by measuring the catalytic activity of ac 1. cam-mutants show a 3-4-fold lower potency than cam, but they are more efficacious than cam. most prominently, cam-215 was 133 % more efficacious than cam. such striking differences between cam and cam-mutants have not yet been observed for other mammalian effector proteins. as a result of the exchange of all methionine against leucine residues in cam-215, it is more hydrophobic than cam and this leads to a better ppi with ac 1. in future studies we will examine the effects of cam inhibitors, antidepressants and antipsychotics on cam/ac 1 interaction. furthermore we will analyze the effects of oxidized cam and cam-mutants on the catalytic activity of ac 1. because oxidative stress is of great importance in aging, it is important to know more about the abovenamed interaction in view to the demographic change. taken together, our data point to a unique cam/ac 1 interaction that may be selectively targeted by small molecules. in particular, enhancers of these interaction could be useful to improve memory and learning. gender differences in fat distribution and diabetes prevalence in nzo mouse lubura m., scherneck s., zucker a., schürmann a. deutsches institut für ernährungsforschung experimentelle diabetologie, arthur-scheunert-allee 114-116, 14558 potsdam, germany background: excessive fat accumulation in visceral but not subcutaneous fat depots as well as ectopic fat storage in liver, skeletal muscle and pancreas are associated with an increased risk for the development of type 2 diabetes in humans. in this study we aimed to examine the influence of early fat distribution on onset of type 2 diabetes in mice. methods: nzo mice are regarded as insulin resistant model in which only males become diabetic. we used male and female mice fed with high-fat and standard diet. we determined fat distribution by computed tomography for three times and conducted oral glucose tolerance tests on two different time points. besides we assessed body weight and blood glucose levels on weekly basis. results: contrary to previous findings, we observed that not only male nzo mice on high-fat diet develop diabetes. blood glucose levels at the 16 th week of age and total pancreatic insulin content indicated diabetes prevalence of 68% in males and 25% in females these results lead to the conclusion that high-fat diet counteracts protective action of estrogens against diabetes. inversely to the findings in humans, female mice tend to store more fat in abdominal region than males. there was no relationship between early accumulation of fat in abdominal region and onset of type 2 diabetes. however, visceral fat was associated with liver fat in males as well as in females. furthermore, at the age of ten weeks hepatic fat content correlated with blood glucose levels (r² = 0.69) indicating that the early hepatosteatosis is a predictor for hyperglycemia. however, there was no correlation between hepatic insulin sensitivity (indicated by quantitative insulin sensitivity index-quicki) and amounts of hepatic fat we conclude that early hepatosteatosis does not predict for glucose intolerance in nzo mice. in the nzo mouse, the amount of liver fat but not the early fat distribution predicts for the later onset of type 2 diabetes. further experiments are needed to examine the gender dependent differences in the diabetes prevalence of this mouse strain. with a prevalence of about 20-30% non-alcoholic fatty liver disease (nafld) represents the most common liver disorder in europe. nafld manifestation ranges from steatosis through steatohepatitis (nash) to fibrosis and cirrhosis, followed in some cases by liver failure and hepatocellular carcinoma. fatty degeneration of liver cells, increased oxidative stress with concomitant lipid peroxidation and an induction of pro-inflammatory cytokines are proposed as possible causes for developing inflammation and fibrosis, but the exact pathogenesis of the progression of nafld into nash is still unknown. thus, besides life style modifications and weight reduction interventions, no established pharmacological therapy exists so far. to gain further insights into the pathogenesis of nafld and nash and to develop new therapeutic strategies, appropriate animal models are essential. thus, in the present study three different dietary animal models for nafld were evaluated and compared to the biochemical and metabolic alterations seen with nafld and nash in man. male adult lewis rats were given standard food or one of three different diets: fatty liver diet [fld] , methionine/choline deficient diet [mcd] or methionine/choline deficient plus high fat diet [mcd+hf] . after 1, 2, 4, 6 or 12 weeks of treatment, animals were sacrificed and body and liver weights, laboratory parameters (asat, alat) as well as histopathological changes in the livers and different parameters indicating oxidative stress or representing the biotransformation capacity of the livers were analyzed. with fld and mcd+hf a normal body weight gain was observed, whereas with mcd body weight gain was strongly impaired. liver weights were mainly increased after mcd+hf. elevation of asat and alat values and hepatic steatosis were more pronounced after mcd and mcd+hf than after fld. all three diets caused an increase in the oxidative stress in liver tissue, but especially with mcd a tremendous elevation in the hepatic levels of lipid peroxidation products was seen. with regard to liver biotransformation capacity, with all three diets mainly an induction of the cytochrome p450 2e1 and 4a1 isoforms expression and activity was observed, which was most pronounced after mcd and mcd+hf. in summary, the changes induced by mcd or mcd+hf most closely resemble the alterations described in literature for nafld in man and thus should be preferred over fld in future investigations on nafld and nash. ep3 receptors for prostaglandin e2 convey stimulatory and inhibitory effects. e.g., their stimulatory effect leads to vasoconstriction in the human pulmonary artery and their inhibitory activity to reduction of neurotransmitter release from neuron endings. the aim of our study was (1) the pharmacological characterization of ep3 receptors in human pulmonary arteries and (2) the examination of the involvement of these receptors in the regulation of the neurogenic tachycardia in pithed rats. l-826266 served as the ep3 antagonist. experiments were performed in human pulmonary arterial rings isolated from patients undergoing lobectomy during resection of lung carcinoma and in pithed and vagotomised rats. the ep1/ep3 agonist sulprostone (1 nm -100 mm) concentrationdependently contracted human pulmonary artery rings (pec50 and emax; 6.89±0.12 and 106.5±5.2%, relative to the contraction induced by kcl 60 mm). the concentrationresponse curve of sulprostone was not affected by the ep1 antagonist sc 19920 (10 µm) but shifted to the right by l-826266 (10 µm) (apparent pa2 6.22). extending the exposure time to l-826266 from 0.5 to 3 h increased its antagonistic potency to 7.39 (schild plot-based pa2; concentrations 0.1, 1 and 10 µm). in pithed rats electrical stimulation (0.66 hz, 1 ms, 50 v for 5 s) of the preganglionic sympathetic nerve fibers or intravenous isoprenaline (0.15 nmol/kg) increased heart rate (hr) by 55 beats/min. sulprostone (10 -1000 nmol/kg) did not affect the isoprenaline-induced increase in hr but inhibited the neurogenic tachycardia dose-dependently, maximally by 80%. l-826266 (3 µmol/kg) diminished the inhibitory effect of sulprostone 1000 nmol/kg on the neurogenic tachycardia by 20%. in conclusion, ep3 receptors (1) located postsynaptically strongly contract human pulmonary arteries and (2) located presynaptically on sympathetic nerve fibres supplying the heart of rats strongly inhibit the neurogenic tachycardia. -5-bromo-n[3-(5voltage-gated ca 2+ channels of the central nervous system control a multitude of ca 2+ dependent processes such as neurotransmitter release, neuronal excitability, neurite outgrowth, synaptogenesis, plasticity and neuronal survival. the cav2.1 ca 2+ channelalso known as p/q-type channel -belongs to the subfamily of high voltage activated ca 2+ -channels. ca 2+ influx via cav2.1 ca 2+ channels located at presynaptic nerve terminals triggers vesicle fusion and transmitter release at brain synapses and at the neuromuscular junction. thus, cav2.1 ca 2+ channels play a crucial role in synaptic transmission. the global cav2.1 knock-out phenotype is characterized by severe ataxia, dystonia and lethality during the first postnatal weeks and is therefore an unsuitable model to analyze the importance of cav2.1 ca 2+ channels for learning and memory. therefore, we crossed a floxed cav2.1 mouse line with nex-cre transgenic mice to establish a viable, forebrain specific knock-out mouse line (fbko-mice). results from western blot analysis confirmed an efficient knock out of cav2.1 in hippocampal and cortical preparations, whereas the expression level in the cerebellum was not altered. to investigate the specific role of cav2.1 channels in hippocampus and neocortex dependent behavior, we performed tests for motor functions and sensory abilities and in particular learning and memory tasks. mice with a forebrain specific cav2.1 knock-out show significant deficits in spatial learning & reference memory and a significant reduced recognition memory as revealed by the morris water maze and an object recognition task. the fbko-mice exhibit no obvious locomotor deficits during behavioral tasks in the open field test and elevated plus maze. some fbko-mice demonstrate episodes of seizures in the morris water maze and during different rotarod tasks. to assess motor-function of fbko-mice in a stress reduced environment, we performed home cage based running-wheel motor-learning tasks. in summary, the diverse phenotypes of the forebrain specific knock-out mouse line emphasize the critical importance of cav2.1 for learning and memory. helicobacter hepaticus-infected rag2 -/mice emulate many aspects of human inflammatory bowel disease (ibd), including the development of colitis and colon cancer [erdman et al., 2009 , pnas 106: 1027 -1032 . toward the goal of elucidating mechanisms of inflammation-induced carcinogenesis and developing biomarkers of inflammation, we undertook a comprehensive analysis of macromolecular damage products during disease progression in h. hepaticus-infected rag2 -/mice. infected mice developed severe colitis and hepatitis, accompanied by infiltration of myeloperoxidase-positive neutrophils and f4/80-positive macrophages, by 10 wks postinfection (pi), progressing into colon carcinoma by 20 wks pi. qpcr array-based gene expression profiling revealed that pathophysiological changes were associated with characteristic alterations in the expression of genes related to inflammation, dna repair, and oxidative stress response. to study inflammation-related macromolecular damage, colon and liver tissues were analyzed by isotope-dilution chromatography-coupled mass spectrometry to quantify a battery of 16 different dna, rna and protein damage products thought to represent the full spectrum of inflammation-related chemistries. our data revealed a significant predominance of chlorinated dna-, rna-, and protein damage products by 20 weeks pi. in contrast, levels of damage products arising from oxidation, nitration and nitrosation changed only modestly or remained unchanged. our analyses also revealed higher levels of damage products in rna than in dna and demonstrated organ-specific differences of oxidative damage products, such as 8-oxo-dg and its oxidation products spiroiminodihydantoin and guanidinohydantoin. collectively, these results suggest that neutrophil and myeloperoxidase-induced chlorination chemistry may serve as a biomarker of ibd and may play important roles in the pathophysiology of ibd and colitis-associated cancer. characterization of a membrane protein expressed in mouse heart and brain mannebach s. 1 recently, a novel membrane protein in drosophila was shown to be localized in presynaptic vesicles. it appears to mediate a ca influx after vesicle fusion with the plasma membrane. disruption of the corresponding gene leads to endocytic defects in drosophila [1] . apparently, this protein plays a role in exo-and endocytosis and could serve as a ca channel supplying ca required for endocytosis. we have identified a protein in mouse, c90rf7, which shares 26,3% amino acid sequence identity with the drosophila protein. it covers 171 amino acid residues. using rt-pcr the full length transcripts could be identified in brain, kidney, pancreas, heart, spleen, thymus and mast cells. coexpression of c90rf7 and the ca v2.2 channel in xenopus oocytes reduced the amount of the α1b and cavβ3 subunits of the ca 2+ channel in the plasma membrane but did not affect the gating properties of the cav2.2 channel. expression of c90rf7 alone did not yield any channel activity. we therefore started to produce recombinant protein using the his-sumo-prokaryotic expression vector. the protein was efficiently expressed as his-sumo-c9orf7-fusion in e.coli (yield 2mg at 1mg/ml). we are currently preparing the c9orf7 part of the his-sumo-c9orf7fusion protein by ulp1-protease digestion followed by various chromatographic steps. the purified recombinant protein will be used to immunize rabbits to get antibodies. in parallel we generated antisera by immunizing rabbits with peptide fragments derived from the c9orf7 sequence. we could not identify any homologues of c9orf7 in the mouse genome and to analyze its function we are currently generating c9orf7 deficient mouse lines by gene targeting. we have chosen a strategy for conditionally inactivation of the gene with the option to study the cellular localization of c9orf7 by expression of the bgalactosidase gene under the control of the endogenous c9orf7 promoter. by southern blot analysis we´ve already identified 210 homologous recombinant embryonic stem cell clones out of 300 analyzed ones and we will proceed with blastocyst injection to get chimeric mice and finally mice carrying the introduced mutations in the c90rf gene. parps are involved in various biological processes such as regulation of dna repair, cell cycle progression, and cell death. consequently, several parp inhibitors are currently in clinical development as chemo-and radiosensitizers as well as monotherapeutic agents following the concept of synthetic lethality. pharmacological and toxicological studies call for an accurate analysis of parp activity in terms of a detailed knowledge of the structure of par and a reliable method for its quantification. we have developed a sensitive, precise, and accurate bioanalytical method based on liquid chromatography coupled to electrospray tandem mass spectrometry (lc/ms-ms) to characterize and quantify par with femtmol sensitivity: par is extracted from cells and hydrolysed to specific monomeric units, i.e., ribosyladenosine, which is characteristic for linear par, diribosyladenosine, which is characteristic for branching points, and adenosine, which represents the terminal part of the polymer. using this method, we are currently analyzing par levels in different cell lines and in primary human peripheral blood mononuclear cells (pbmcs) both under physiological conditions as well as upon genotoxic stress and in the presence of potent parp inhibitors. we expect that after completing method validation this assay will be useful for a wide range of applications in pharmacology and toxicology. gene mutagenic potential and metabolite profile of 17β-estradiol in cultured v79 cells expressing human cytochrome p450 1a1 martínez jaramillo d., lehmann l. university of wuerzburg, institute of pharmacy and food chemistry section of food chemistry, am hubland, 97074 wuerzburg, germany oxidative metabolism of the female sex hormone 17β-estradiol (e2) is considered to play a major role in the initiation of hormone-induced carcinogenesis. in extrahepatic tissues, e2 undergoes metabolic activation by cytochrome p450-dependent monooxygenase (cyp) isozyme 1a1 to 2-hydroxy-(2-ho) and to a lesser extent to 4-ho-e2. if not conjugated, these catecholestrogens (ce) can further oxidize to electrophilic quinones (q), which may react with dna and induce thereby mutations. conjugation of these ce in extrahepatic tissues is mainly catalyzed by catechol-omethyltransferase. in order to identify possible mutagenic metabolites (i) the induction of gene mutations by e2 was determined in male chinese hamster lung fibroblasts (v79 cells) expressing human (h) cyp1a1 and (ii) the metabolite profile of e2 in these cells was analyzed via gas chromatography/mass spectrometry after solid phase extraction of the cell suspension in the culture medium. (i) gene mutations were assessed using the hypoxanthine-guanine phosphoribosyltransferase assay. the promutagen benzo[a]pyrene (bap) served as positive control requiring metabolic activation by hcyp1a1 and dimethylsulfoxide as solvent control. v79 hcyp1a1 were treated with 100 nm e2 for 3 weeks and the resulting 6-thioguanine (6-tg) resistant mutants selected at weeks (w) 2 and 3. the frequency of spontaneous 6-tg resistant mutants per 10 6 colony-forming cells ranged from 5 ± 1 (w2) to 9 ± 4 (w3). as expected, 0.25 µm bap induced a significant increase in mutant frequency (mf, 266 ± 13, w2 and 132 ± 46, w3) . treatment with 100 nm e2 resulted in a 3-fold (17 ± 2, w2) and a 2-fold (23 ± 4, w3) increase in mf, suggesting slight mutagenic activity. in culture medium of v79 hcyp1a1 treated with 100 nm e2, 2-ho-e2, 2-methoxy-(meo)-e2, 3-o-methyl-2ho-e2 and 4-meo-e2 (suggesting intracellular formation of 4-ho-e2) were detected. while 4-meo-e2 concentration remained constant over the exposure period, the concentration of the other metabolites increased in a timedependent manner. the maximum concentration increase was reached at w3 for methylcatechols and at w2 for 2-ho-e2, correlating with the maximum increase in mf, observed after 2 weeks as well. in conclusion, e2 possessed a slight mutagenic potential after hcyp1a1-mediated activation to 2-, 4-ho-e2 and their corresponding methylcatechols. cumulative effects of three triazole fungicides in a broad dose range in vitro rieke s., kneuer c., bumke scheer m., lampen a., hirsch-ernst k., marx-stoelting p. bundesinstitut für risikobewertung chemikaliensicherheit, max-dohrn-str., 10589 berlin, germany consumers are exposed to multiple residues of different pesticides via the diet. this raises questions concerning potential cumulative effects, especially for substances causing toxicity by a common mode of action. the aim of this work was to investigate potential combination effects of the three triazole fungicides epoxiconazol, tebuconazol and flusilazol for selected parameters in a broad dose range in vitro. parameters investigated were cytotoxicity, hormone synthesis (17-β estradiol, progesterone and β-hcg), expression of a panel of androgen-or estrogen-responsive genes in the human placental choriocarcinoma cell-line jeg-3 and transactivation via estrogen receptors α and β in stably-transfected hek 293 cells. the ability to inhibit steroidogenesis was analysed by measuring the concentrations of 17β-estradiol and progesterone in cell culture supernatants of jeg-3 cells. additionally, the placental peptide hormone β-hcg was measured. while no change in β-hcg and 17β-estradiol concentrations were observed, all triazoles induced a dose-dependent decrease in progesterone concentration and a cumulative effect was observed implying dose additivity at individual doses of >1.7 µg triazole/ml. significant activation of erβ by the three triazoles, especially by flusilazol, was observed at 5 µg triazole/ml and combined exposure showed additive effects, while no significant activation of erα was observed. based on the data, our findings suggest dose-additivity of triazole pesticides with the same mode of action for selected parameters in vitro. no significant effects were observed at lower doses [1ng -1µg triazole/ml] neither for substances applied individually nor in combination. transient receptor potential channels as mediators of catecholamine release mathar i. 1 trp proteins form cation channels that are regulated through strikingly diverse mechanisms. recently, genetic association studies identified many trp genes including trpm4 as risk factors for disease states such as arrhythmias, hypertension and cardiomyopathy. the melastatin trp channels trpm4 and trpm5 have distinct properties within the trp channel family; they form non-selective cation channels activated by intracellular calcium ions and are expressed in heart, aortic endothelial cells, kidney and adrenal gland. disruption of the trpm4 gene in mice leads to increased basal blood pressure without evidence for impairment of endothelium-or smooth muscle-dependent regulation of contractility of peripheral resistance vessels, the renin angiotensin aldosterone system, basal cardiac output or body fluid homeostasis. instead, trpm4-deficient chromaffin cells exhibit increased acetylcholine-induced exocytosis of catecholamines which is associated with elevated level of epinephrine in the plasma and its metabolites in the urine. this indicates that trpm4 serves as an inhibitory regulator of exocytotic catecholamine release, at least in chromaffin cells. whether catecholamine release is also regulated by trpm4 in other cells of the sympathetic nervous system such as perivascular neurons still needs to be clarified as well as the molecular mechanism underlying how trpm4 regulates catecholamine release. besides trpm4 we recently identified transcripts encoding additional trp channels including trpc5 and trpc6 in chromaffin cells isolated by laser capture microdissection but their functional role in these cells is still unknown. measurements of the time course of the intracellular calcium concentration before and during acetylcholine stimulation (10µm) of catecholamine release as well as the analysis of the number of released vesicles in chromaffin cells relvealed no changes in trpc1/c5/c6 triple knock out mice compared to wildtype controls. although it seems that these trpc proteins are not directly involved in catecholamine release from chromaffin cells induced by acetylcholine application in our hitherto existing experiments, their contribution to the modulation of catecholamine release by agonists of gq-coupled receptors still needs to be analysed. aims: sulfonylureas (sus) are among the most widely used oral hypoglycaemic drugs that stimulate insulin secretion. in addition, sus have pleiotropic effects on other tissues. regarding the effects of sus on adipocytes conflicting findings were reported. we have now investigated the actions of glimepiride and glibenclamide (=glyburide) in primary human adipocytes. methods: primary cultured human white pre-adipocytes were differentiated in vitro according to a standard protocol. lipid accumulation was assessed by oil red o staining and determination of triglyceride content; gene expression was measured by real-time pcr and western blotting. results: we initially characterized the genes regulated during human preadipocyte differentiation by a global microarray analysis. treatment with glimepiride and glibenclamide caused a strong accumulation of lipid droplets and an increase in triglyceride content. genes involved in lipid metabolism were induced, chemokine expression was decreased. interestingly, the effects of sus were over all qualitatively and quantitatively similar to pioglitazone. in direct comparison glibenclamide was more potent than glimepiride in respect to the induction of fabp4 (ec 50 0.32 vs. 2.8 µm), an important adipocyte marker gene. su-induced differentiation was virtually completely blocked by the pparγ-antagonist t0070907 but not affected by diazoxide, indicating pparγ activation by sus. repaglinide, causing insulin liberation like sus but being structurally different, had no effect on adipocytes. conclusions: in primary human pre-adipocytes, glibenclamide and glimepiride strongly induced differentiation, apparently by activating pparγ . thus, sus but not repaglinide may be used to influence insulin resistance beyond their effect on insulin liberation. the role of at1a and at1b receptors as mechanosensors in myogenic vasoconstriction blodow s. 1 , schneider h. arterial myogenic tone denotes the intrinsic property of vascular smooth muscle cells to constrict in response to an elevated intraluminal blood pressure. this physiological reaction is more distinct in small resistance arteries than in large conduit arteries. understanding the underlying mechanisms should provide useful information for the treatment of diseases like anaphylactic shock and systemic hypertension in which this reaction is altered. whereas the underlying signaling cascade has been extensively studied, the molecular identity of the mechanosensory elements still remains elusive. recent studies at the cellular level suggest a sensory function for a subgroup of gprotein coupled receptors (gpcrs) coupling to gq/11-proteins. by determining mrnaexpression levels of selected gpcrs in consecutive pairs of resistance and conduit vessels, we could identify a subset of gq/11-coupled receptors such as angiotensin ii at1b, vasopressin v1a, endothelin eta and etb and α1a adrenoceptor significantly enriched in resistance vessels. by pharmacological blocking of those highly expressed gpcrs by different antagonists and inverse agonists, we evaluated their influence on the formation or the intensity of myogenic tone, as measured in isolated murine mesenteric arteries ex vivo. while blocking of v1a receptor and α2a and α2ab adrenoceptors showed no differences of myogenic tone, blocking of at1a and at1b receptors by losartan and candesartan, eta receptor by bq123 and α1a adrenoceptor by prazosin caused significant reductions of the vascular response. analyzing the myogenic response of at1a -/mice with and without additional blocking of at1b receptors by candesartan suggested that especially at1b receptors play a dominant role for mechanosensitivity in mice. this was further supported by investigating the myogenic response of at1b -/mice. these findings suggest that mechanosensitive gq/11-protein coupled receptors, especially at1b receptors, play a dominant role for the development of myogenic vasoconstriction. trpm3 ion channels are activated by steroidal compounds and noxious heat and are considered to be involved in insulin secretion and pain perception. the expression of the trpm3 gene generates a variety of different transcripts which arise by alternative splicing and the use of different promoters [1] . they encode a substantial variety of isoformes and so far we have identified more than 20 distinct trpm3 proteins in mouse and rat each varying in exons 1, 2, 8, 13, 15, 17 and 24 . these variants differ enormously in their biophysical properties. for example splicing within exon 24 affects the channel pore and causes significant changes of the ionic selectivity of trpm3 channels [2] , whereas splicing of 54 nucleotides encoded by exon 13 leads to dormant trpm3 proteins. however, the frequency of these different isoformes in trpm3 expressing tissues is completely unknown. to get insight into the significance of the different trpm3 isoformes we investigated the abundance of alternative trpm3 transcripts in different tissues and cell types by reverse transcription quantitative pcr (rt-qpcr). we found that the frequency of splicing within exon 13 ranges from 5 up to 20 % in different cell types and tissues. furthermore we analyzed the trpm3 transcriptome in the choroid plexus of the brain and the pituitary gland, tissues in which trpm3 transcripts are most abundant. for that purpose we sequenced more than 120 clones, each. corresponding to our rt-qpcr result, we found a significant number of transcripts lacking exon 13. in cells of the choroid plexus nearly all (124 /126 clones) carried the short ca 2+ permeable pore. furthermore, we identified seven variants spliced in exon 20 encoding truncated trpm3 proteins. however, the composition of the trpm3 transcriptome in the choroid plexus and pituitary gland differed enormously, indicating the importance of alternative splicing for trpm3 function in different tissues. the concept of "thresholds of toxicological concern" (ttc) defines tolerable dietary intakes for chemicals without toxicity data and is widely applied to chemicals present in food in low concentrations such as flavorings. based on a statistical evaluation of the results of many toxicity studies and considerations of chemical structures, the ttc concept derives a maximum daily oral intake without concern of 1800, 540 or 90 µg/person/day for non-genotoxic chemicals depending on the allocation to so-called cramer classes i, ii or iii. for substances with a structural alert for genotoxicity a ttc value of 0.15 µg/person/day might be used. recently, it has been investigated, whether the ttc values, which were derived based on mostly chronic oral dietary rodent studies would cover all relevant toxicities (neurotoxic, repeated dose, reproductive and developmental, immune effects and endocrine-related effects). several authors using different specific databases have confirmed that the ttc values derived using cramer classes are also covering immunotoxic, neurotoxic, reproductive and developmental effects. a respective decision tree is going to be presented, also considering substances or substance classes which shall be excluded from the ttc approach. there are several areas in which the ttc concept is already used, or a ttc approach is considered useful, to assess low-level human exposures, or help in prioritizing toxicological testing; as for example the assessment of plant metabolites and degradates of pesticide active substances, feed and food additives, chemicals with a low exposure profile under reach, residues, metabolites and impurities in plants, chemicals, plant protection products or pharmaceuticals. if no structural alert for genotoxicity is given or standard genotoxicity tests are negative the cramer class iii value of 90 µg/person/day, which corresponds to a dose of 1.5 µg/kg bw is considered to represent a chronic tolerable daily intake of the test substance. examples for current and future uses of the ttc concept in regulatory toxicology are presented. objective: hyaluronan (ha), synthesized by three ha-synthases (has1, -2, -3), is a prominent matrix component of atherosclerotic lesions. the aim of the present study was to identify the has isoenzyme that is associated with ha-matrix remodeling in inflammatory regions of atherosclerotic plaques. furthermore the underlying regulatory pathways were determined and functional aspects of this regulation in vascular smooth muscle cell (vsmc) were addressed. methods and results: during atherosclerosis in apoe deficient mice the peak of macrophage invasion at 14 weeks coincided with ha deposition and induction of has3 in aortic root plaques. in human symptomatic carotid artery plaques has3 was by far the most prominent has isoenzyme as determined by quantitative real time rtpcr. in vitro, in human vascular smooth muscle cell (vsmc) has3 was specifically induced via activation of nfkb by interleukin-1β (il-1β) and tumor necrosis factor alpha (tnfa) as shown by chip assay and utilization of nfkb inhibitor bay 11-7082. has3 was also upregulated in a co-culture system by activated macrophages via paracrine release of tnfa and il-1β as verified by neutralizing antibodies. in human atherosclerotic lesions nfkb positive vsmc were frequently detected in close proximity with ha and f4/80 positive macrophages as shown by immunohistochemistry. to study the effects of has3 mediated ha synthesis in human coronary vsmc, lentiviral overexpression and knockdown of human has3 were employed. overexpression of has3 resulted in increased migration and proliferation whereas knock down had the opposite effect. the effects of has3 were mediated by both pi3k signaling and mapk signaling via hyaluronan receptors cd44 and rhamm. conclusion: the present results suggest that has3-dependent ha synthesis is induced in human vsmc by inflammatory cytokines released from activated macrophages. moreover, has3-mediated ha production induced phenotypic activation of vsmc. pulmonary inflammation and airway remodeling are major features of chronic obstructive lung disease (copd). in addition, pulmonary hypertension is a common comorbidity, which is associated with a poor prognosis of the disease. recent studies in a guinea pig model of allergic asthma have shown that increased arginase activity, which converts larginine into l-ornithine and urea and competes with nitric oxide synthases for the common substrate, contributes to allergen-induced airway inflammation, hyperresponsiveness and remodeling. there is evidence that cigarette smoke and lipopolysaccharide (lps), both involved in the pathogenesis of copd, increase the expression of arginase, however, its role in the pathogenesis of copd is currently unknown. this study aimed to investigate the role of arginase in pulmonary inflammation and remodeling, using a guinea pig model of lps-induced copd. to this aim, guinea pigs were instilled intranasally with lps or saline twice weekly for 12 weeks and were pretreated by inhalation of the arginase inhibitor (2)s-amino-boronohexanoic acid (abh) or pbs. repeated lps exposure increased lung arginase activity, resulting in increased lornithine/l-arginine and l-ornithine/l-citrulline ratio's. both ratio's were reversed by abh treatment. repeated lps exposure also induced increased il-8 levels, neutrophils, goblet cells, hydroxyproline and airway collagen content in the lung, which were all abrogated by abh. moreover, repeated lps exposure increased right ventricular mass, indicative of pulmonary hypertension, which was similarly prevented by abh. in conclusion, increased arginase activity contributes to pulmonary inflammation, airway remodeling and right ventricular hypertrophy in a guinea pig model of copd, indicating that arginase inhibitors may have therapeutic potential in the treatment of this disease. (supported by msd). behavioral abnormalities in hcn3-deficient mice michalakis s., schöll-weidinger m., mader r., cao-ehlker x., fenske s., wahl-schott c., biel m. center for integrated protein science munich (cipsm) department of pharmacy -center for drug research, ludwig-maximilians-universität münchen, butenandtstr. 5-13, 81377 münchen, germany hcn3 encodes a hyperpolarization-activated and cyclic nucleotide-gated channel, which is expressed in various brain regions including thalamic, hypothalamic and habenular nuclei as well as brain stem and olfactory bulb. in this study we performed a comparative analysis of hcn3 -/and hcn3 +/+ mice using a battery of behavioral tests and telemetric biopotential measurements to evaluate a potential role of hcn3 in central nervous system function. in general, the knockout mice showed normal motor function as assessed by the rotarod and open field tests. telemetric home cage activity and core body temperature measurements confirmed a normal circadian behavior, but revealed a lower basal activity that concurred with decreased body temperature during the light phase and the light-dark transition phase. hippocampus-dependent spatial learning was normal. by contrast, hcn3 knockout mice showed more immobility than control mice on day two of the porsolt forced swimming test, which could reflect increased depressionlike behavior. however, center exploration in the open field test as well as performance in the light-dark transition and the elevated-plus maze tests was normal in hcn3 -/mice. this suggests that general anxiety was not changed in the knockout mice. in addition, hcn3 knockout mice were less active on the second day of the open field test, which supports a habituation phenotype. finally, hcn3 -/mice had higher burying scores in the marble-burying test, which is a test for certain aspects of obsessive compulsive disorder in rodents. taken together, genetic deletion of hcn3 in mice results in distinct behavioral abnormalities related to behavioral despair and expression of repetitive behaviors in response to mild stressors. mielke h. 1 , gundert-remy u. alcohol consumption when breast feeding is discussed controversially. some groups recommend breast pumping before alcohol consumption and feeding the stored milk instead of breast feeding after drinking alcohol. this study was performed to simulate the blood concentration in the breastfed baby and to assess the health impact. method: we established a physiologically based kinetic model. its parameters were calculated (partition coefficients tissue/blood ; schmitt, 2008) silva et al.,1993) . we simulated 1. the alcohol concentration in a breastfed neonate and a 3-month-old suckling infant after the nursing mother had consumed alcohol,2. the alcohol concentration in utero/fetal compartment during pregnancy assuming the identical alcohol consumption of the pregnant woman 3. the alcohol concentration during infant´s treatment of bloating by an approved herbal drug containing alcohol. results: peak maternal alcohol concentration was 0.59 ‰ after consuming 0.25 l of wine, peak concentration was 0.0033 ‰ in the newborn, 0.0038 ‰ in the 3-month-old infant and 0.38 ‰ in the utero/fetal compartment. the peak concentration after herbal drug treatment was 0.015‰ in the neonate and 0.015‰ in the 3-month-old infant, respectively. we discuss the results of the simulations and compare it with doses and published concentrations measured in experimental animals or in vitro studies. conclusions: we conclude that the recommendation "1 to 2 glasses of wine on occasion" (agence nationale d'accréditation et d'évaluation en santé, assante 2002) is in accordance with the simulation results presented here whereas stricter rules are not scientifically sound. (2002) http://www.has-sante.fr/portail/upload/docs/application/pdf/ breastfeeding_guidelines.pdf da silva et al. (1993) adenylyl cyclases (ac) mediate physiological responses in virtually all cells, where their regulation through receptors and g proteins results in the modulation of camp. in the present study we focused on the kinetics of interactions between the alpha-subunit from inhibitory g protein type 1 (gαi1) and adenylyl cyclase type v (ac5). these proteins were labeled with cfp and yfp, respectively. the dynamics of their interactions was monitored by means of high temporal resolution fret imaging in hek cells expressing unlabeled a2a-receptor and gβg subunits. to activate the signaling pathway, we applied agonist using a rapid superfusion device. application of norepinephrine resulted in the development of a fret signal, indicating interaction between gai1-cfp and yfp-ac5. after withdrawal of agonist the fret signal recovered with a remarkably slow time course compared to the deactivation kinetics of gi proteins reported previously (bünemann et al. 2003) . to further analyze the properties of the dissociation between gai1 and ac5 we measured in parallel the offset kinetics of the interaction between gai1-yfp and gβg-cfp (gi1-fret) after agonist withdrawal under comparable conditions. in addition we tested to what degree the coexpression of rgs4 accelerated the deactivation of gi proteins and the dissociation of gai1-cfp from yfp-ac5. these experiments revealed that in the absence of rgs4 the dissociation of gai1 from ac5 after agonist withdrawal takes about 3 times longer than the deactivation of gi proteins. in the presence of rgs4 this difference is even larger due to the pronounced acceleration of g protein deactivation. the dissociation of gai1 from ac5 was only marginally accelerated by rgs4. these observations lead us to hypothesize, that ac5 might trap activated g protein-subunits and thereby affect the g protein cycle by shifting the equilibrium towards activated g proteins. if this hypothesis is true, it should result in a left-shifted dose response curve compared to g protein activation dose response. in support of this hypothesis we found that the concentration response curve for gai1-ac5 interaction was several-fold leftward-shifted compared to the concentration-response curve of gi-protein activation under very similar conditions. influencing the dynamics of the g protein cycle by effectors may represent a novel and powerful mechanism for finetuning the sensitivity of receptor evoked responses in an effector-specific manner. obesity, the excessive accumulation of white adipose tissue (wat), has reached pandemic dimensions. the factors that determine fat mass are not fully understood, but adipocyte hypertrophy and adipokine secretion are thought to be important. in present study, we investigated the role of the cyclic gmp (cgmp) signaling pathway focusing on cgmp-dependent protein kinase i (pkgi) in white adipocytes. pkgi is expressed in wat, preadipocytes and differentiated adipocytes as demonstrated by real-time pcr, western blot and immunochemistry. differentiation of pkgifl/fl preadipocytes, using an optimized protocol, resulted in an enhanced lipid accumulation as evidenced by oil red o staining. deletion of pkgi in pkgifl/fl adipocytes infected with a cre lentivirus (lv-cre, pkgi0/0) exhibited reduced differentiation. analysis of the triglyceride (tg) content revealed a significant decrease of tg levels by 65% ± 1% in pkgi0/0 as compared to pkgifl/fl adipocytes. western blot analysis of white adipocytes showed a significant decrease of c/ebpalpha (19% ± 4.8%), ppargamma (66% ± 2.9%) and ap2 (37% ± 10.6%) expression in pkgi0/0 cells as compared to pkgifl/fl. treatment of 3t3-l1 cells with cgmp resulted in increased lipid accumulation and enhanced expression of fat marker genes. lentiviral overexpression of pkgi further increased differentiation. importantly, pkgi significantly induced mitochondrial biogenesis in 3t3-l1 cells. concomitant activation of pkgi in 3t3-l1 preadipocytes and treatment with the demethylating agent 5-aza-deoxycytidine significantly increased expression of uncoupling protein-1 (ucp-1) -a unique protein of brown fat cells. we found rhoa as major target of pkgi signaling with increased phosphorylation of rhoa at ser-188 in pkgi overexpressing cells. moreover, pkgi-dependent phosphorylation counteracts the effects of rhoa on insulin signaling as well as adipokine expression. taken together, pkgi is a key player in white adipocyte differentiation that regulates cell size and has an anti-inflammatory effect. pkgi decreases the secretion of proinflammatory adipokines via inhibition of rhoa signaling. in addition, activation of pkgi can establish a brown fat cell like phenotype during white adipocyte differentiation if the ucp-1 promoter is accessible. the rag gtpases, raga, ragb, ragc, and ragd form a subfamily gtpases of the ras-related superfamiliy. rag proteins are characterized by a modified ras-like gtpbinding domain and a unique c-terminal region lacking a lipid modification motif. interestingly, rag proteins have been proposed to function as heterodimeric complexes consisting of raga or ragb associated with ragc or ragd. rag gtpases have been implicated in the control of mammalian target of rapamycin (mtor) function, in particular in regulation of the nutrient-stimulated and/or hormone-regulated mtor activity. the protein kinase mtor is found as the catalytic subunit of two larger protein complexes referred to as mtor complex 1 and 2, mtorc1 and 2. under amino-acidrich conditions, activated mtorc1 promotes protein synthesis and inhibits autophagy, while under starvation autophagy inhibition is released. increasing evidence suggests that activation of rag gtpases contributes to mtorc1 function. thus, rag proteins were found to be associated with a protein complex termed ragulator, a major regulatory protein of mtorc1 function and guanine nucleotide exchange of rag gtpases within the rag-ragulator-complex were described to promote mtorc1 translocation to its functional lysosomal compartment. however, the guanine nucleotide exchange properties of rag proteins are poorly characterized, and it is currently unknown, how amino acids promote rag proteins to facilitate the formation of the active, raptor-binding state of the rag heterodimers. to characterize the guanine nucleotide exchange properties of the rag gtpases as momomers or heterodimers in more detail, recombinant rag proteins were expressed in bacteria and purified from this source to near homogeneity. first, the parameters of gdp/gtp exchange of each of these proteins were compared using the non-hydrolysable gtp analogon gtpgs. the results showed that the rag isoforms are distinct in their guanine nucleotide exchange activities. in particular, nucleotide exchange on raga and ragc, but not on ragb and ragd, was only observed at low concentrations of gdp and mgcl 2 in extraction and assay buffers, i.e. conditions favoring the gdp/gtp exchange. these findings may indicate that guanine nucleotide exchange on raga and ragc is controlled by guanine nucleotide exchange factors and suggest specific functions of the individual rag gtpases within individual rag heterodimers. in in-vitro studies on rat and canine mast cells and human mast cell leukemia cells hmc1.2 bz at micromolar concentrations inhibited mediator release which appeared to be related to an inhibition of the intracellular camp pathway. in order to identify potential targets on/in mast cells at which bz may cause an inhibitory effect on mast cell activation, the 1,4-bz flunitrazepam (flu), clonazepam (clo) and 4chlorodiazepam (4-cd) were selected because of their different affinity and selectivity to/for the gaba-a-receptor and the translocator protein (tspo): flu and clo bind with nanomolar affinity to gaba-a receptors, whereas 4-cd is a selective ligand at tspo with nanomolar affinity to tspo but only micromolar affinity to gaba-a receptors. flu also possesses nanomolar affinity to tspo, whereas clo has no or only micromolar affinity to tspo. after incubation of hmc1.2 cells with 4-cd, flu and clo for 1, 6 and 24 hours up to 712 genes were significantly differently expressed in a substance-specific and timedependent manner. comparison of the genes differently expressed at 6 hours revealed that the expression of 217 genes was regulated by both flu and clo but only 8 genes were regulated by both 4-cd and flu suggesting that flu and clo induce gene expression by acting at a target site different from that of 4-cd. the difference between the gene regulation by flu and clo on the one hand and that of 4-cd on the other hand is also reflected in pathway analysis. since it was conceivable that the beneficial effects of the 1,4-bz could be mediated by the recognition sites targeted by the 2,3-bz, i.e. the gria2-encoded ionotropic glutamate receptor ampa2, we investigated by quantitative pcr whether hmc1.2 cells express gria2, tspo, the genes encoding the subunits of the gaba-a receptor and the gaba-forming enzyme glutamic acid decarboxylase. tspo, gabra3, gabrb3, gabre and gabrd were moderately expressed. in addition, there was a week or very week expression of gabra2, gabra4, gabrb2, gabrg3 and gabrr2. expression of gria2 was not detectable. taken together, it cannot be decided yet from our data whether the inhibitory effect of benzodiazepines on mast cell activation is due to an action at tspo or at gaba-a receptors of a novel subunit composition. monien b. h., glatt h. german institute of human nutrition (dife) department of nutritional toxicology, arthur-scheunert-allee 114-116, 14558 nuthetal, germany 5-hydroxymethylfurfural (hmf) and furfuryl alcohol (ffa) are common constituents of foodstuffs in which they are formed by heat-and acid catalyzed reactions from carbohydrates. hmf and ffa have been reported to induce the formation of hepatocellular adenomas in female mice and renal tubule neoplasms in male mice, respectively. we studied whether the carcinogenic effect of these hydroxymethylsubstituted furans may originate from sulfotransferase (sult)-catalyzed formation of electrophilic esters. hmf was inactive in in vitro mutagenicity tests using standard activating systems. in contrast, it was mutagenic in v79 cells genetically engineered for expression of human sult1a1 suggesting that hmf is converted into the reactive 5sulfooxymethylfurfural (smf). following incubation of mutagenic smf with porcine liver dna in vitro, specific methylfurfural adducts were detected using liquid chromatography tandem mass spectrometry (lc-ms/ms), i.e., n 6 -((5-formylfuran-2-yl)methyl)-2'deoxyadenosine (n 6 -ffmda) and n 2 -((5-formylfuran-2-yl)methyl)-2'-deoxyguanosie (n 2 -ffmdg). these adducts were also detected in dna from v79-sult1a1 cells incubated with hmf. in order to determine sulfo conjugation of hmf in mice in vivo, we conducted pharmacokinetic measurements showing that about 500 ppm of the hmf dose was converted to smf and reached the circulation. like hmf, ffa was negative in the standard ames test and various other in vitro genotoxicity tests. we showed that ffa is mutagenic in salmonella typhimurium ta100 engineered for expression of human sult1a1. the putative mutagen 2-sulfooxymethylfuran was synthesized and incubated with porcine liver dna, in which various nucleoside adducts were found. the main adducts, -mfda were detected in dna of ffa-exposed salmonella strain ta100-sult1a1 and in dna of liver, lung and kidney of fvb/n mice that had received about 390 mg ffa/kg body weight per day via the drinking water for 28 days. in summary, both furan derivatives form mutagenic sulfate esters in vitro and in vivo. in the future, we will use genetically engineered mice to characterize the role of single murine and human sult forms in the bioactivation of the furan derivatives and the contribution to tumor induction. background: micrornas are small non-coding rnas that can negatively regulate gene expression on a post-transcriptional level and have been shown to interact with epigenetic mechanisms like dna methylation. mecp2 (methyl cpg binding protein 2) is a protein that binds methylated dna cpgs in the promoter region of genes and can thus regulate their expression. otherwise, mecp2 is known to be a target gene for several micrornas including the cluster mir-132/212 in the brain. recently, our group could show that mecp2 expression is downregulated in human heart failure suggesting that mecp2 might be involved in cardiac pathogenesis. the aim of this project is to study the upstream regulation of mecp2 by the cluster mir-132/212 in the heart during cardiac hypertrophy in-vitro and in-vivo. methods and results: to test whether hypertrophic stimuli can induce mir-132/212 expression, we treated cultured nrcms with 40 µm norepinephrine for 72 hours. this induced cardiomyocyte hypertrophy and expression of the hypertrophy marker nppa, but also of mir-132 (1.79 ± 0.14-fold of untreated cells, p<0.01) and of mir-212-3p (1.73 ± 0.26-fold of untreated cells, p<0.05) and downregulated mecp2 mrna and protein levels (0.80 ± 0.04-fold of untreated cells, p<0.05). to check whether mecp2 downregulation also occurs by direct mir-132/212 activation we increased levels of mir-132 and mir-212 in cardiac myocytes by transfecting precursor mir-132 and mir-212-3p molecules. again, we observed nrcm hypertrophy, nppa mrna upregulation and mecp2 mrna and protein downregulation (0.75 ± 0.05-fold of control, p<0.05) after mir-132 overexpression. similar results were obtained by overexpression of mir-212-3p. to test the effects of adrenoceptor activation on the mir-132/212-mecp2 axis in-vivo, wild-type mice received isoprenaline and phenylephrine via osmotic minipumps (30 mg/kg/day each). after 7 days, cardiac ventricles were analyzed. nppa gene expression (2.81 ± 0.32 -fold of control animals), mir-132 and mir-212-3p levels (3.67 ± 0.5 and 2.62 ± 0.4 -fold of control animals, p<0.01 and p<0.05, respectively) were increased while mecp2 protein levels decreased to 77% (p<0.05) conclusion: these results suggest that in-vitro and in-vivo adrenoceptor stimulation leads to the activation of mir-132/212 expression and to downregulation of mecp2 in cardiac myocytes in-vitro and in-vivo. leopold-franzens-universität, innsbruck, austria at-1 receptor antagonists block the angiotensin ii-enhancing effect on noradrenaline release from sympathetic neurons. in a cell-free assay the binding affinity of the at-1 receptor antagonists telmisartan and valsartan to the gamma peroxisome proliferatoractivated receptor (pparγ) is close to that of the pparγ selective agonists thiazolidinediones (tzds). we tested whether the tzds rosiglitazone and pioglitazone would also modify the prejunctional facilitatory effect of angiotensin ii. left ventricular slices of rats were incubated with tritiated noradrenaline, perifused and electrically stimulated. the negative logarithm of the drug concentration that caused a 30% increase of control (pec30%) was calculated. angiotensin ii caused a concentration-dependent increase of tritium overflow induced by electrical stimulation [pec30%=8.6±0.2 (mean±sem, n=18); maximum increase=110±8%]. neither rosiglitazone nor pioglitazone (0.3-3 µm) had a direct effect. the concentrationresponse to angiotensin ii in the presence of fixed concentrations of rosiglitazone was shifted to the left with increase of the maximum (pec30%=8.8±0. 2, 9. 2±0.2 and 9.3±0.3; maximum increase=118±14%, 146±13% and 148±16%, in the presence of 0.3, 1 and 3 µm of rosiglitazone, respectively, n=4-6, each). in contrast, pioglitazone in concentrations up to 3 µm had no effect on the release-enhancing effects of angiotensin ii. results show that rosiglitazone but not pioglitazone potentiates the noradrenalinerelease enhancing effect of angiotensin ii. this action might contribute to the risk for myocardial infarction from rosiglitazone use but not from pioglitazone use. deleted in liver cancer 1 (dlc1) is a tumor suppressor whose allele is lost in 50% of liver, breast, lung and 70% of colon cancers. despite its significance, the molecular mechanisms that drive cancerous transformation upon dlc1 loss remain unclear. we found that the transcriptional coactivators megakaryoblastic leukemia 1 and 2 (mkl1/2) are constitutively localized to the nucleus in hepatocellular and mammary carcinoma cells that lack dlc1. moreover, dlc1 loss and mkl1 nuclear localization correlated in primary human hepatocellular carcinoma. nuclear accumulation of mkl1 in dlc1-deficient cancer cells was accomplished by activation of the rhoa/actin signaling pathway and concomitant impairment of erk-mediated mkl1 phosphorylation. dlc1 loss led to constitutive activation of the mkl-dependent, tumor-relevant target genes ctgf, cyr61, myl9 and myh9. furthermore, we identified a novel target gene, integrin a5, with a key role in cell migration and metastasis, that exhibited a dlc1-and mkldependent regulation. depletion of mkl1/2 suppressed not only cell migration, but also cell proliferation and anchorage-independent cell growth induced by dlc1 loss. our data provide insight into the mechanism by which dlc1 loss initiates tumorigenesis. as mkl1 and 2 have a key role in this process, this pathway may provide promising pharmacological targets for cancer therapy. universität bonn, pharma-zentrum bonn pharmazeutisches institut, pharm. chemie i, an der immenburg, 53121 bonn, germany membrane receptors activated by purine and/or pyrimidine nucleotides ("p2 receptors") are widely distributed in the body and constitute novel (potential) drug targets. they are subdivided into g protein-coupled p2y receptors (p2y1, 2, 4, 6, 11, 12, 13, 14) , and homo-or heterotrimeric ligand-gated ion channel or p2x receptors (subunits: p2x1-7). we have been interested in the identification and development of potent and subtype-selective ligands -as tool compounds and potential drugs -for the various p2y and p2x receptor subtypes. our strategy involves (i) establishment of a proprietary compound library consisting of synthetic small molecules and natural products; (ii) development of screening assays suitable for medium throughput screening; (iii) careful analysis of structure-activity relationships at each target and systematic optimization of the lead structures; (iv) pharmacological evaluation of selected compounds. this approach has led to new biological tools for several targets, including p2y and p2x receptors [1] [2] [3] [4] [5] [6] . fine particles in particulate matter (pm) are effective vehicles to transport toxicants into the lung; depending on their size, smaller particles may reach the bronchiolar or alveolar space. in recent years the pm fraction pm2.5 has especially been correlated with both pulmonary and cardiovascular diseases. in order to better characterize pm emission and distribution of environmental tobacco smoke (ets) from cigarettes (reference cigarette (rc), brand cigarette (bc)) we have developed an ets emitter to simulate human smoking emission and measured pm2.5 concentration in a telephone booth (1,75 m3 volume) as an example for small indoor spaces like cars. fine particulate matter was measured using an aerosol spectrometer with 6 sec time resolution; laser scatter allowed a size resolution from 0,25 µm to 32 µm. for the pm2.5 concentration the following values were calculated: cumulative pm2.5 concentration as auc-pm2.5 (µg/m3/sec), peak pm2.5 concentration as cmax-pm2.5 (µg/m3) and average pm2.5 concentration cmean-pm (µg/m3). in closed door condition both cigarettes produced particulate auc-pm2.5 values of 59 000 ± 15 000 µg/m3/sec (rc) to 85 000 ± 31 000 µg/m3/sec (bc after myocardial infarction (mi) inflammatory cells and cardiac fibroblasts (cf) determine the remodeling response. interleukin-6 (il-6) is induced in the ischemic myocardium and is known to stimulate the differentiation of fibroblasts to myofibroblasts. hyaluronan (ha) is an extracellular matrix component synthesized by ha-synthase isoenzymes (has 1-3) and is also known to control fibroblast phenotypes. however, it is presently unknown whether il-6 participates in the remodeling of the ha-matrix or whether the ha-matrix modulates the responses to il-6. therefore, the aim of the present study was to elucidate whether il-6 regulates the expression and function of ha-matrix in cfs. cells were isolated from c57bl/6j mice and used during passage 2-3 for experiments. cfs were stimulated with il-6 or hyper-il-6 which is a fusion protein of il-6 and soluble il-6 receptor (sil-6r). after 10 and 30 min signal transducer and activator of transcription 3 (stat3) was phosphorylated in response to hyper-il-6 but not in response to il-6. rt-pcr revealed rapid upregulation of has 1 (5.94 ± 2.49 fold of unstimulated control, 1h) in response to hyper-il-6. has2 was induced to a lesser degree (2.04 ± 0.55 fold of unstimulated control, 1h) whereas has3 was not responsive (1.49 ± 0.42 fold of unstimulated control, 1h). in contrast, il-6 had no effect on transcript levels of has isoenzymes. in turn, expression of has1 and has2 in response to hyper-il-6 was inhibited by ag490, which indicates the involvement of stat signaling. interestingly, despite induction of has1 and has2 the amount of secreted ha as determined by an elisa-like assay was not affected by hyper-il-6. this may indicate that il-6 regulates the cell surface associated ha-matrix of cfs. in conclusion, the present data demonstrate that cardiac fibroblasts respond to il-6 trans-signaling (hyper-il-6) via the soluble il-6r and subsequent stat3 signaling with increased ha-synthesis. the fact that il-6 had no significant effect suggests that the expression of the non-signaling membrane-bound il-6 α-receptor (il-6r) in cultured murine cardiac fibroblasts is not sufficient to induce has 1 and -2 gene expression. therefore, il-6 trans-signaling mediated by il-6 and the circulating sil-6r might be necessary to mediate the il-6-induced has expression in vivo. mrgprd receptor endogenously expressed in dorsal root ganglia: evidence for an activation by 3-aminoisobutyric acid müller s., hoffmann k., von kügelgen i. universität bonn institut für pharmakologie und toxikologie, sigmund-freud-straße 25, 53127 bonn, germany the gpcr mrgprd (mrgd) is highly expressed in small diameter dorsal root ganglion (drg) neurons and has been implicated to play a role in nociception. the receptor was previously shown to respond to β-alanine. in the present study we searched for agonistic activity of structural analogues of β-alanine. for further characterization of the receptor we used fura-2 fluorimetry, a nfat luciferase reportergene assay and the determination of the inhibition of forskolin-induced camp production ([ 3 h]-camp affinity assay). first, we confirmed the activation of the receptor by β-alanine and gaba. in reportergene experiments we then identified 3-dlaminoisobutyric acid as an agonist, with similar potency but weaker affinity when compared to β-alanine (ec50 165 µm). fura-2 fluorimetry showed an increase in intracellular ca 2+ levels by 3-dl-aminoisobutyric acid (300 µm). moreover, 3-dlaminoisobutyric acid reduced the forskolin-induced camp production by up to 65 % (ec50 195 µm). in addition to 3-dl-aminoisobutyric acid, we identified 3-dl-aminobutyric acid as a weak agonist acting at the mrgprd. other closely related substances failed to show significant responses. next to the agonists we further characterized antagonists inhibiting the response to βalanine mediated by mrgprd. chlorpromazine shifted the concentration-response curve of β-alanine to the right with an apparent pkb of 4.9 (nfat assay), thioridazine with an apparent pkb of 5.4 (nfat assay) and 5.3 (camp assay) and rimcazole with an apparent pkb of 5.2 (nfat assay) and 5.2 (camp assay). in conclusion we show for the first time that 3-dl-aminoisobutyric acid is an agonist at the mrgprd and that the structure-activity relationship of agonists at mrgprd is very close. the sdf-1-chemokine receptor cxcr4 plays a key role during embryogenesis and regulates functions of immune and stem cells in adult life. furthermore, cxcr4 is involved in disease states including inflammation and cancer. it is well established that sdf-1-stimulated cxcr4 receptors activate gi protein-dependent signal transduction pathways and undergo c-terminal phosphorylation and internalization. because the cxcr4 c-terminal domain contains 15 serine and 3 threonine residues, it is incompletely understood which of the potential phosphorylation sites contribute to homologous and heterologous regulation of cxcr4. here, we analyzed the phosphorylation pattern of cxcr4 at 3 c-terminal sites after stimulation of the receptor with sdf-1 and after pma-induced activation of the pkc pathway as a model for heterologous receptor phosphorylation. using phospho-specific antibodies against s324/325, s338/339 and s346/347 in immunoblot analyses, we showed that the 3 sites were phosphorylated after stimulation with sdf-1 or pma. stimulation with egf or forskolin did not induce phosphorylation at these sites. sdf-1-induced phosphorylation at s324/325, s338/339 and s346/347 was reversible after wash out of the ligand. time course analyses revealed that phosphorylation occurred first at s346/347 and then at s324/325 and s338/339. taken together, these results indicate that the c-terminus of cxcr4 is phosphorylated at multiple sites by homologous and heterologous pathways and that phosphorylation at the different sites may be hierarchically organized. human milk represents the best form of nutrition for infants early in life. however, it can also contain toxic contaminants that may adversely affect infant's development. the nephrotoxin ochratoxin a (ota) is present in human milk (tab. 1 in [1] ), but information on transfer from maternal blood to milk is scarce: published data [2] indicate that levels of ota in milk are roughly one tenth (0.1) of those in blood. but, the efficiency of the ota-transfer at various stages of breastfeeding may vary since studies in animals revealed that transfer of ota is apparently time-and dose-dependent. thus, the aim of this study was to assess the ota transfer from blood to milk at different stages of breastfeeding in humans. in a small chilean cohort, 18 lactating women were asked to provide blood and milk on the same day. these samples were collected on four different occasions within the first months after delivery and analyzed using hplc with fluorescence and/or tandem mass spectrometric detection [1] . the transfer of ota from blood to milk was quantitatively assessed by measuring the milk to plasma ratio (m/p). the average ota level in blood plasma was 235 ± 129 ng/l, and no major variations were observed over time (p = 0.19). on the other hand, ota levels found in colostrum (85 ± 60 ng/l) were higher than in mature milk (p < 0.05). in line with these data, higher m/p ratios (table) were obtained with samples collected in the first six days after delivery. this study showed that the transfer of ota from blood to milk was more efficient with colostrum (m/p 0.43 ± 0.26) than with mature milk. thus, a higher exposure to ota can be expected for neonates than for infants at later stages of breastfeeding. moreover, the lactating women have lower average ota levels in plasma than non lactating women from chile [3] , indicative of milk as additional excretion route. acknowledgement: this work has been supported by a stipend from conicyt/daad to km. exposure of infants to ochratoxin a (ota) deserves particular attention since ota is nephrotoxic, and one of the most potent rodent renal carcinogen studied to date [1] . moreover, infants may be more vulnerable to the toxic effects of contaminants than adults. ota-levels in plasma of infants are indicative of an early exposure in life [2] . but blood sampling is an invasive method not readily applicable for breastfed infants. thus, the aim of this study was to implement a non invasive biomonitoring method to assess ota-exposure in this group. to assess the exposure to ota, breast milk and infants' urine specimens were collected, from two different cohorts: chile (n= 28) and turkey (n=10, only urine). analysis of the samples was performed using enzymatic hydrolysis prior to extraction and hplc-ms/ms [3] . the magnitude of infants' exposure was assessed by calculating the ota-daily intake with human milk and relating it also to urinary ota levels. calculations of the daily intake with human milk [4] showed that infants may be exposed to ota at high levels, exceeding the tolerable daily intake (tdi) of 5 ng/kg-bw/day set for adults [1] . in both cohorts, most of the urine samples tested positive for ota (chile 72%, turkey 80%). ota levels observed in urine samples from the turkish infants (range: 116 -1,361 ng/l) were 10 fold higher than levels found in chilean samples (range positive samples: 17 -320 ng/l). further analysis of phase ii metabolites in urine confirm the excretion of ota as conjugate (glucuronide) in highly exposed infants. in conclusion, ota exposure of infants early in life was documented. given that otaintake by several infants exceeded the tdi for adults, further biomonitoring in this vulnerable group is advised including also suitable biomarkers of effect. a mixture of (e)-and (z)-clomiphene citrate is the first line therapy of female infertility. however, up to 30% of patients do not respond. (e)-clomiphene is structurally closely related to another selective estrogen receptor modulator, tamoxifen which is frequently used for the treatment of hormone receptor-positive breast cancer. like tamoxifen, clomiphene is extensively metabolised by the cytochrome p450 system. using the estrogen receptor response assay (e)-4-hydroxyclomiphene and (e)-4-hydroxy-n-desethylclomiphene (ec50: 2.5 and 1.4 nm, respectively) turned out to be 100 times more active at the er compared to the parent drug isomers and de-ethylated metabolites. using recombinant expressed human cyp isoforms and inhibitory antibodies, cyp2d6 revealed to be the major isoenzyme involved in the formation of 4-hydroxlated metabolites. n-deethylation was catalysed by cyps 3a4/5, 2d6, 2c19 and 2c8. rates of 4-hydroxylation in microsomes from 30 human liver donors correlated with the number of functional cyp2d6 genes. these in vitro results were confirmed in a pharmacokinetic study with female healthy volunteers receiving a single dose of clomiphene. in carriers of two non-functional cyp2d6 genes (poor metabolizers) cmax of (e)-4-hydroxyclomiphene and 4-hydroxy-ndesethylclomiphene was 8 and 12 times lower, respectively, when compared with subjects with at least one fully functional cyp2d6 allele. in contrast, half-life of (e)-clomiphene and (e)-n-desethylclomiphene was 10 and 50-fold higher, respectively, in poor metabolizers. our data provide first evidence of a pharmacogenetic rational for the variability in the response to clomiphene treatment. among the tested compounds, compound 1 proved to be the most active derivative, showing a significant toxicity at a concentration of 2,5 µm. compounds 2 and 3 showed significant toxic effects at a concentration of 5 µm. the compound 4 showed no toxicity up to a concentration of 100 µm. all derivatives 1, 2 and 3 have a ec50 between 10 and 15 µm. we further proved the induction of apoptosis by apo-one assay (caspase 3/7 activity) and life/dead-assay (fluorescence microscopy). in conclusion, these gold complexes exhibit an example of interesting potential candidates for future anticancer pharmaceuticals due to relatively high cytotoxicity. gene regulating effects in mouse liver subsequent to treatment with selected dioxin-like compounds and pcb 153 using whole genome microarray analysis neser s. 1 , lohr c. 1 , van ede k. i. 2 , andresen k. 3 interaction with the aryl hydrocarbon receptor (ahr), with 2,3,7,8-tetrachlorodibenzo-pdioxin (tcdd) being the most potent congener amongst the ahr agonists. recent risk assessments have employed the toxic equivalency factor (tef) concept. the current eu-project systeq aims at developing, validating, and implementing human systemic tefs as indicators of toxicity for dl-compounds. at present, the best known parameter of ahr mediated effects is the induction of cytochrome p450 isoenzymes (cyps), i.e., cyp1a1, 1a2, and 1b1. one of the major objectives of the systeq project is the identification of novel quantifiable biomarkers. in a three day study, female c57bl/6 mice were treated with single doses of six dl-congeners (tcdd, pcb 118, pcb 126, and pcb 156) , and the 'non-dioxin-like' (ndl) pcb 153. quality tested (agilent ® bioanalyzer) mrna isolated from livers was analyzed using the agilent ® mouse whole genome array (4x44k) system. the quantity of genes affected (≥ 2fold) was highly heterogeneous amongst the dl-compounds. whereas tcdd-treatment upregulated 89 genes, and down-regulated 66, 4-pncdf-treatment had impact on 3208 (up), and 2396 (down). treatment with pcb 118 led to marginal numbers of 16 up and 6 down-regulated genes. with 64 (up), and 38 (down) genes shared, the most extensive overlap occurred between tcdd-and 1-pncdd-treatment. no overlap was found due to treatments with the ndl pcb 153 (21 up, 1 down) and tcdd. when comparing the effects of all dl-congeners, minor numbers of genes of 19 up, and 5 down-regulated remain, most of them being related to drug metabolism. while pcb 118 regulated only genes involved in drug metabolism, omission of pcb 118-regulated genes resulted in consistently 51 (up), and 24 (down) regulated among dl-compounds. in conclusion, our findings suggest that the pattern of gene regulation in mouse liver elicited by pcb 153 was strictly different from tcdd, while a very limited coincidence of genes was found even among dl-compounds. comparison of these 'core' genes with data from human models is required with respect to determination of novel biomarkers. introduction: proper use of antibiotics is essential with regard to effective treatment of bacterial infections. providing adequate information for patients can contribute to achieve this aim. materials and methods: data was collected from the relational database of the drug information service at dresden university of technology. the patients, who used the service, were interviewed concerning socio-demographic characteristics, reason for enquiry, number and kind of drugs taken, and diseases. possible contact paths were phone, e-mail or letter. in the present evaluation, all enquiries from the years 2009 and 2010 were analyzed descriptively focussing primarily on systemic antibiotics as reason for the enquiry. results: in the evaluated period, 4454 enquiries were registered in total. in 4.7% of those enquiries systemic antibiotics were named with a total number of 283 drugs. 50.9% of those antibiotics were found to be the direct reason for the enquiry. most common information requested by patients corresponded to adverse drug reactions (50.7%), diagnosis/treatment (35.4%), drug application (29.2%) and drug-druginteractions (19.4%). the majority of the requesting patients (61.5%) was born before 1970. a correlation between incidence of enquiries especially concerning antibiotics and quarterly statistics could not be detected. conclusion: mainly patients aged 40 years or more seem to need or search for further information about antibiotic medication. advice is required especially regarding adverse drug reactions and diagnosis or treatment. in order to this, the advisory service can help patients to lose their insecurity and to gain more confidence in handling antibiotic drugs. colon cancer is one of the most frequent cancers in the industrialized nations. epidemiological studies show a correlation between highly processed meat and the development of colorectal tumours. it is assumed that the risk of developing colorectal cancer, among various different factors, is related to the uptake of toxic substances contained in food such as heterocyclic aromatic amines that arise during the processing of fish and meat. phip is the most abundantly formed heterocyclic compound, and therefore has the biggest impact. in a previous study, we measured the absorption of phip in different intestinal segments of the rat. in the present study we focussed on the potential mechanisms by which phip is reabsorbed. the unidirectional phip transport from the mucosal to the serosal compartment (j ms) and in the opposite direction (jsm) was examined using the ussing chamber technique and 14 c-phip as a radiotracer. the proximal jejunum and distal colon of male fischer 344 rats in short-circuit current chambers was clamped, so that mucosal and serosal compartments were built. the phip flux rates were determined at defined intervals over 120 min. the experimental conditions were selected in such a way that negative net flux rates (jnet = jms-jsm) were indicative of an active secretion. both intestinal segments showed large differences. while in the jejunum jms and jsm of phip were not significantly different, there was an active secretion in the colon. in a next step the transport proteins involved in this process should be examined. introduction: human organic anion transporter 2, oat2 (slc22a7), is abundantly expressed in kidney and liver and mediates the sodium-independent uptake of clinically relevant drugs like 5-fluorouracil, paclitaxel, bumetanide, tetracycline, and zidovudine. while immunohistochemical studies have localized human oat2 to the basolateral membrane of kidney proximal tubules, its hepatic localization is currently unknown. we, therefore, firstly determined oat2 localization in human liver. because interindividual variability of oat2 expression may affect hepatobiliary drug uptake and elimination, we next systematically investigated the influence of genetic and non-genetic factors on hepatic oat2 expression. methods: an expression profile of oat2 for 20 human tissues was determined by realtime quantitative polymerase chain reaction (taqman). oat2 mrna expression was analyzed in well-characterized human liver samples from 150 caucasians that were accompanied by detailed demographic and clinical data. oat2 was localized in human liver cryosections using a commercial rabbit polyclonal antibody and hepatic oat2 protein levels were determined. resequencing, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and genome-wide single nucleotide polymorphism microarray technology served to genotype variants in the slc22a7 gene region. results: oat2 mrna was expressed in several human tissues, including liver. moreover, a new alternatively spliced variant of oat2 was identified in human liver. hepatic expression of full-length oat2 mrna and oat2 protein varied 37-fold and 41fold, respectively. oat2 mrna and protein levels did not correlate with each other. oat2 was localized to the sinusoidal membrane of human hepatocytes. no novel variants in the 9 exons, the 5'-flanking region, or the 3'-untranslated region of the slc22a7 gene were identified. univariate analysis showed that oat2 mrna is reduced in patients diagnosed for cholestasis (p=0.0012) and is affected by genetic variants. whereas the influence of genetic variants on hepatic oat2 expression appears to be limited, cholestasis significantly contributes to the variable interindividual oat2 expression. this indicates consequences for hepatic drug elimination of and response to oat2 drug substrates such as paclitaxel or tetracycline. the life threatening toxicity of organophosphorus (op) nerve agents is caused by the inhibition of the acetylcholine esterase (ache). oximes were shown to be potent reactivators of inhibited ache, but in poisoning by some compounds, e.g. soman, they have only a small therapeutic effect. for such cases, an alternative new strategy may be the intervention at nicotinic acetylcholine receptors (nachr). previous studies with the bispyridinium non-oxime mb327 demonstrated therapeutic effects against soman in vitro and in vivo which was partly attributed to its direct interaction with nachrs [1] . we investigated the interaction of mb327 and several structure analogous at the orthosteric binding site of human α7 nachr (hα7 nachrs), a subtype which appears to be widespread in the human body, and compared the results with data obtained from torpedo-nachrs, which show a high degree of homology with human muscle-type nachrs. interaction of compounds with the orthosteric binding site of hα7 nachrs were investigated with radioligand binding experiments performed as high-throughput method [2] . membrane preparations of gh 4c1 cells stably expressed hα7 nachrs were incubated with the nachr agonist [³h] epibatidine and appropriate concentrations of the unlabelled competitors e.g. bispyridinium compounds. after incubation, bound and free [³h] epibatidine were separated by rapid vacuum filtration. ki values of the competing compounds were calculated with nonlinear regression. three bispyridinium compounds, mb442, mb456 and mb770 exhibited ki values at micromolar concentrations while three other compounds, mb327, mb583 and the pharmacological inactive mb424 (negative control) did not show any interaction with the orthosteric binding site of hα7 nachrs. with torpedo-nachrs, ki values were in similar orders of magnitude -except mb442 which indicated significant subtype selectivity. interestingly, the affinity of monomeric pyridinium derivates did not correlate with their bispyridinium structure analogues. obviously, no correlation between the affinity to the orthosteric binding site and the functional improvement of neuromuscular transmission exists, although species-related differences cannot be excluded. in this study, we analysed the cytotoxic and clastogenic effects of the anticancer drug nimustine (acnu) in cells deficient in repair proteins involved either in homologous recombination (hr) or non-homologous end-joining (nhej). we show that hr mutants are extremely sensitive to acnu as measured by the induction of apoptosis and colony formation as well as the induction of chromosomal aberrations. the nhej mutants were slightly sensitive to acnu and differed in their sensitivity, with the ku80 mutants being moderately sensitive and the dna-pkcs mutant resistant, comparable to the wild-type (wt). cell death was mostly executed via the caspase-dependent apoptotic pathway with involvement of caspase-3 and -7, and necrosis was also induced. further, we investigated the kinetics of dna double-strand break (dsb) formation that resulted from the repair of acnu-induced interstrand cross-links by means of γh2ax and 53bp1 foci analysis in wt and mutant cells. cells mutant in hr did not repair dsb and went into the apoptotic or necrotic pathway, whereas wt cells were able to repair most of the dsb. cells deficient in ku80 formed at early times after acnu treatment less γh2ax and 53bp1 foci compared to the corresponding wt, which might be due to a reduced capacity of recognising dsb. at later times after treatment, ku80 mutant cells show foci levels similar to the wt indicating restitution of h2ax phosphorylation. we also analysed whether dsb formation after acnu treatment was replication-dependent using synchronised cells. we determined the formation of γh2ax and other dsb marker in wt cells that passed through the first cell cycle after demecolcine synchronization. the level of γh2ax foci increased significantly in the s-phase and remained at a high level during g2 where a fraction of cells remained arrested. rad51, atm, mdc-1 and rpa-2 foci were also formed and shown to co-localize with γh2ax. these foci were ameliorated significantly in s-and g2-phase, which was similar to the time course of γh2ax foci formation. in western blots, we confirmed a higher phosphorylation level of atm and chk2 and less phosphorylation of chk1 in hr mutants. the data indicate that acnuinduced dna cross-links give rise to cyto-and genotoxicity via the formation of dsbs that activate the cellular dna damage response. the endocannabinoid system has been established as a mediator of numerous central and peripheral biological functions. cannabinoids have emerged as attractive alternatives or supplements to therapy with opioids for chronic pain states. however, in human the activation of cannabinoid receptors is associated with side effects. for clinical exploitation of the analgesics properties of cannabinoids, a major challenge is to devise strategies that reduce or abolish their adverse effects on cognitive, affective and motor functions without attenuating their analgesics effect. in animal studies, the anti-nociceptive efficacy of cannabinoids has been unequivocally demonstrated in several models of inflammatory and neuropathic pain. however, there are marked inconsistencies between different reports with respect to the locus of these pain-protective effects. we are working towards establishing the contribution of cb1 receptors expressed on the peripheral terminals of nociceptors to cannabinoid-induced analgesia. using cb1 globally knock-out animal as background, we induce the expression of cb1 specifically in nociceptive neurons localized in the peripheral nervous system and test the analgesic effects of cannabinoid systemical delivery in these mice. our results support the development of peripherally acting cb1 analgesic agonist with reduced central side effects. furthermore, we are utilizing proteomics approach to identify protein complexes that interact with cb1 receptor which hold promise in understanding cannabinoid signaling in health and disease. most chemoattractants, including chemokines, complement c5a, fmlp, and leukotriene b4 are signaling through heterotrimeric g proteins of the pertussis toxin (ptx)-sensitive gi family. the functional inactivation of all gαi proteins with ptx leads to a fulminant decompensation of the immune system, whereas the constitutive inactivation of a single gαi coding gene results in mild phenotypes in mice. we are mostly interested in the nonneuronally found gαi2 and gαi3 isoforms and their redundant and specific roles in immune function and infection. for this purpose cellular in vivo and ex vivo models and in vivo infection model with listeria monocytogenes are being used. macrophages were isolated from the peritoneal cavity of wild type (wt) and gαi-deficient mice 4 days after i.p. injection of 4% thioglycolate that induces peritonitis in vivo. we confirmed previous observations that in gαi2-deficient mice the migration of macrophages into the peritoneal cavity was reduced after induction of peritonitis. regarding the expression levels of gαi and gβ isoforms in the lavage samples, the predominant gαi isoform gαi2 was upregulated in gαi3-deficient macrophages. vice versa gαi3 was upregulated in gαi2-deficient macrophages. concerning gβ isoforms, both gβ1 and gβ2 were strongly reduced in the gαi2-deficient macrophages which resulted in a reduced total amount of gβ. surprisingly, the gαi3-deficient macrophages showed reduced gβ1 protein levels only which caused a change in the gβ2/ gβ1 quotient in favour of gβ2. we are currently establishing an in vivo infection model with l. monocytogenes in gαi2and gαi3-deficient mice. our previous in vitro infection studies in mice embryonic fibroblasts provided us with information about possible distinct roles of these two isoforms as far as the uptake of l. monocytogenes in the cells is concerned. challenging the immune system of gαi-deficient mice with this pathogenic organism will give us new insights into the systemic immune response in these mice upon bacterial infection. our data indicate that we may surmount the redundancy between these two isoforms and focus on their distinct and specific roles in pathogen defense. fret-based β-arrestin2 biosensors reveal conformational changes upon binding to the β2-adrenergic receptor in real time and living cells nuber s., zabel u., ziegler n., hoffmann c., lohse m. j. institut für pharmakologie und toxikologie pharmakologie, versbacherstr.9, 97078 würzburg, germany β-arrestins are multifunctional adapter proteins that regulate seven transmembranespanning receptor (7tmr) signaling and initiate also alternative signaling pathways. studies have shown that β-arrestins undergo conformational changes upon receptor stimulation, which are thought to be necessary for its downstream actions. to investigate these conformational changes in living cells we constructed fret based biosensors of β-arrestin2, in which cfp was fused to the c-terminus and the flashbinding motif (ccpgcc) was inserted to different positions within the n-or c-domain of β-arrestin2. upon β2-adrenergic receptor (β2ar) stimulation we observed a decrease of the intramolecular fret signal between cfp and flash at the n-domain (β-arrestin2flash2), indicating a conformational change moving the c-terminus and the ndomain of β-arrestin2 relative to each other. kinetic analysis revealed that this conformational change immediately follows β-arrestin2/β2ar interaction on a timescale of seconds. a β2ar mutant that was previously shown not to interact with β-arrestin2 was utilized as control and did not induce a conformational change in the β-arrestin2 molecule. our data provide evidence that β-arrestin2 changes it`s conformation upon binding to the activated β2ar in living cells. the β-arrestin2flash2 sensor could serve as universal biosensor for gpcr activation. studies on the physiological role of annexin a4 in the heart nunes f. 1 the calcium binding protein annexin a4 has been examined in the context of heart failure in the past. annexin a4 expression level was found to be elevated in ventricles of human failing heart in comparison to expression levels in non-failing ventricles. furthermore the intracellular localization pattern in atrial cardiomyocytes was found to be altered in the failing human heart (moravec and matteo, cardiovasc res 2000). in order to gain insight into the possible physiological significance of these findings we utilized an annexin a4 gene trap model (gt) in which the annexin a4 protein content was not detectable in ventricles and atria. measurements of sarcomere shortening and calcium transient kinetics in isolated ventricular cardiomyocytes revealed a prolonged calcium transient decay at stimulation frequencies of 0.5 hz, 1hz and 2 hz as well as an increased sarcomere shortening at 1 hz and 2 hz in anxa4 gene trap animals in comparison to wild type (wt) ( the effects of the β-adrenoreceptor agonist isoprenaline (iso) on the shortening of ventricular cardiomyocytes was increased in gt as compared to wt (0,2±0.013 vs. 0.17±0.012, *=p<0.05 vs. wt; n=14-18/5). western blot analyses indicated that the expression of the sarcoplasmic reticulum (sr) ca 2+ -atpase (serca2a) and the phosphorylation status of its regulator protein phospholamban (plb) did not differ between groups (n=7). however, co-immunoprecipitation experiments suggest, that anxa4 is able to interact with hax1, which acts as a repressor of serca2a (n=4). we performed force measurements in isolated and electrically stimulated left atria in response to rising isoprenaline concentrations (10 -9 m-10 -5 m). the positiv inotropic effect of isoprenaline was significantly increased in gt atria (rel. force at 10 -4 m iso [%]: wt: 433±76; gt: 699±63 *= p<0.05 vs wt; n=8-10). in conclusion, annexin a4 contributes to the regulation of cardiomyocyte contractility. the anxa4 up-regulation might therefore contribute to diminished cardiac performance in heart failure. matteo rg, moravec cs. immunolocalization of annexins iv, v and vi in thefailing and non-failing human heart. cardiovasc res. 2000 mar;45 (4) background: pregnane x receptor (pxr) is considered the most important sensor of natural and anthropogenic xenobiotics in vertebrates. in contrast, the amphibian ortholog is involved in neural development and irresponsive to xenobiotics. instead, the xenopus laevis constitutive androstane receptor (car) was recently found to possess pxr-like properties, featuring low basal activity and a pronounced ligand spectrum. thus a structural and functional characterisation of x. laevis car may provide further insights into human car basal and ligand-induced activity. methods: the time-point of origin of car genes was determined by macrosynteny analyses of car, pxr, and vdr (vitamin d receptor) gene loci, which form the nr1i subfamily of nuclear receptors. based on a 3-dimensional protein model of xenopus laevis car, docking studies with structurally diverse agonists were conducted. proteinligand-interactions as well as sequence comparisons were performed in order to select amino acids to be mutated towards human car. the organ response to car activators was determined in xenopus laevis using rna microarrays. results: car emerged together with pxr and vdr from an ancestral nr1i gene in early vertebrates via two whole-genome duplications. this was followed by losses of car from the fish lineage and of pxr from sauropsida (reptiles and birds). amino acids important for ligand binding were identified. structural features responsible for the pronounced basal activity in human constitutive androstane receptor are not present in x. laevis car. in human pxr the inter-helical loop in front of helix 3 is part of the ligandbinding pocket and supposed to be responsible for the wide substrate spectrum. in amphibian car this inter-helical loop plays no role in ligand binding. car agonists resulted in a pronounced induction of antimicrobial peptides in the ovary. conclusions: car emerged already in early vertebrates and it is conserved in land vertebrates, whereas xenosensing pxr is found only in the fishes and mammals. we provide a comprehensive modeling and mutational analysis of this first reported amphibian xenosensor. the induction of antimicrobial peptides by car activators suggests a link between xenosensing and innate immunity. the latter one may play a previously unrecognized role in the amphibian reproduction. background: retigabine belongs to a novel class of potent anticonvulsant drugs and is currently being investigated in clinical routine. the therapeutic range of retigabine serum concentration is unknown. a therapeutic drug monitoring (tdm) is used for most other anticonvulsant drugs. the aim of this study was to develop a method for the determination of retigabine in serum of patients and to compare the effect and the side effects of retigabine with the blood levels of the drug. method: a hplc method with tandem mass spectrometric detection for the sensitive determination of retigabine was developed. solid-phase extraction (spe) of 250 µl serum with oasis hlb cartridges allowed a reliable quantification down to 5 ng/ml. in order to develop an assay with high sample throughput and to obtain maximum response for the analytes we required the shortest possible retention time. to implement the determination of retigabine in a second step in the routine tdm of anticonvulsant drugs the corresponding hplc method was selected: a purospher rp18 column (55 mm x 2 mm; 4 µm, merck) and a mobile phase with a steep acetonitrile gradient. results: the great advantage of having analytes with different molecular masses and similar retention times in combination with ms/ms detection enabled us to aim at a minimum separation that might remove some salts or matrix components that can suppress or interfere with the analyses from the target components, while maintaining good sample throughput. the method was validated. the assay is precise, accurate, fast, sensitive, and selective. discussion: the developed method is suitable for therapeutic drug monitoring of retigabine. the correlation of the serum concentration and the effect of the drug and thus the necessity of tdm have to be tested. targeting inflammatory t lymphocytes with conditional chemokine receptor antagonist expression for a tissue-specific therapy of chronic inflammatory disorders ogrissek n., giegold o., pfeilschifter j., radeke h. h. uniklinikum der goethe-universität pharmazentrum / zafes, theodor-stern-kai 7, 60590 frankfurt am main, germany chemokines and their receptors are known to be involved in the pathogenesis of chronic inflammation and autoimmune diseases. several approaches tried to use chemokine receptor antagonists as therapeutics to reduce exagerrated immune response, however, due to compensation and systemic side effects clinical trials often failed. in previous experiments our group identified three promising antagonists. cxcl11(4-79) has antagonistic function for cxcr3, cxcl12(p2g2) is able to inhibit cxcr4 and the herpesvirus 8 encoded protein vmip-ii interferes with ccr1, -2 and -5 as well as with cxcr3, -4 and cx3cr1. their expression and secretion was confirmed in pichia pastoris and antagonistic function has been proven by a reduction of t cell migration. the aim of this project is to develop a cell-based therapy for chronic inflammation with a treatment that is based on the collective effect of cxcl11(4-79), cxcl12(p2g2) and vmip-ii. with targeting of stable transduced memory t cells these antagonists should be conditional expressed and secreted directly in the centre of inflammation, resulting in inhibition of further inflammatory t cell accumulation. to realize this project we first cloned constitutive lentiviral constructs containing these antagonists and optimized transduction of t cells, such as the ova-specific memory th-1 cell clone if12 with the potential to initiate antigen specific nephritis in scid mice. next we investigated expression and secretion of cxcl11(4-79), cxcl12(p2g2) and vmip-ii with pcr, western blot and elisa. at the moment we want to measure the inhibition efficiency of t cell migration in vitro with chemotaxis and flow chamber assays. construction of an inducible lentiviral vector plasmid to ensure expression of the antagonists only upon t cell activation, is also part of our current work. finally we would like to test the chemokine receptor antagonists in vivo in two relevant mouse models of type-1-diabetes and contact dermatitis. small heterodimer partner 1 (shp-1) is a member of the superfamily of nuclear receptors (nrs). in contrast to other nrs this orphan receptor lacks the dna binding domain. however, shp-1 is known to inhibit activity of several nrs by direct proteinprotein interaction. importantly several of the interacting nrs have been shown to directly regulate shp-1 expression, suggesting that shp-1 is involved in negative feedback loops of various metabolic pathways, such as cholesterol-, bile acid-and drug metabolism and glucose homeostasis. recently binding sites for nrs were identified in the promoter region of shp-1, including hnf4α, lrh1, lxr, fxr, srebp1c and pparγ. the aim of our study was to identify single nucleotide polymorphisms (snps) in the promoter region of shp-1 and to determine their impact on the transactivation of shp-1. 120 dna samples from subjects of the population based cohort study of health in pomerania were analyzed by sanger sequencing, thereby we identified four snps namely -594t>c (rs71636795), -413g>c, -423 c>t (rs78182695) and del-195ctga (rs145613139). subsequently those polymorphisms were tested for their functional consequence performing cell based reporter gene assays testing all above mentioned modulators (lrh1, lxr, fxr, srebp1c and pparγ) of shp-1 expression. only the transactivation by hnf4α was decreased in the presence of the -423 c>t polymorphism to 69% and the -413g>c polymorphism to 75%. in conclusion we described snps with impact on transactivation. it will be aim of future studies to determine the potential impact on physiological processes or disease development. autosomal recessive polycystic kidney disease (arpkd) is a rare genetic disease, afflicting about 1 in 20.000 individuals. arpkd is characterized by cystic fusiform dilatations of the renal collecting ducts leading to massive enlargement of the kidneys and ultimately loss of renal function. in addition, the patients suffer from congenital hepatic fibrosis (chf), possibly leading to portal hypertension and liver enlargement. so far, there is no cure for arpkd. therapy is focussing on controlling the disease symptoms [1] . mutations in the pkhd1 gene cause arpkd. more than 300 different mutations in this gene have been reported, all leading to the same phenotype, though there are differences regarding the severity of the disease [2] . in animal models of autosomal dominant polycystic kidney disease (adpkd) as well as arpkd elevated levels of camp were shown [1] [2] [3] . in isolated kidney cells camp stimulates cl-secretion and activates the b-raf /mek/erk pathway. these both are important factors for cyst development and disease progression [2, 3] . intracellular camp regulation is based on conversion of atp to camp by adenylyl cyclases (acs) and degradation by phosphodiesterases . referring to this, we asked the question if there are differences in the activation and expression pattern of acs in pck rats, an animal model of arpkd [4] and in sprague dawley rats. therefore, we examined membranes in a radioactive ac activity assay using various stimulatory compounds, e.g. forskolin, a direct ac activator, or hormones like glucagon and vasopressin to characterize acs. furthermore, we examined ac isoform expression on the mrna level via rt-pcr. we observed that in pck rats ac activity was decreased in general in comparison to sprague dawley rats. in future experiments we are aiming to obtain further knowledge about the influence various hormones exhibit on pck rat acs and to biochemically characterize acs. the major pathogenicity factors tcda and tcdb from clostridium difficile monoglucosylate and thereby inactivate small gtp-binding proteins of the rho subfamily after entering host cells via receptor-mediated endocytosis. although the intracellular mode of action of the toxins is well understood, far less is known about binding structure and internalization pathway of tcda and tcdb. since antibodies directed against the c-terminal located clostridial repetitive oligopeptides (crops) are able to neutralize toxin cytotoxicity the crop domain is acknowledged to mediate receptor binding. however, we recently demonstrated that crop deletion mutants of tcda (tcda 1-1874 ) and tcdb (tcdb 1-1852 ) enter host cells and exhibit full cytopathic potency though lacking the proposed receptor binding domain. we therefore refute the accepted opinion of a solely crop-mediated toxin uptake and re-evaluate the role of the crops in toxin endocytosis. tcda 1-1874 and tcdb 1-1852 induced time and concentration dependent cell rounding and rac1-glucosylation. however, depending on the cell line, truncated toxins exhibit up to 10-fold reduced potency towards host cells compared to the respective full length toxin. the observed difference in toxin potency might reflect the recognition of different receptor structures or the use of various endocytotic routes. interestingly, pre-incubation of cells with the isolated crop domain enhances binding as well as cytotoxicity of subsequent applied truncated tcda indicating that the crops primarily determine toxin uptake. in fact, competition experiments revealed that tcda and tcda 1-1874 predominantly use different receptor structures corroborating the notion of alternative internalization processes utilized by tcda. different routes for cellular uptake might enable the toxins to enter a broader repertoire of cell types leading to the observed multifarious pathogenesis of c. difficile. thus, characterization of alternative endocytotic pathways used by the c. difficile toxins might therefore be the basis to investigate the opportunity of toxin uptake inhibition as therapeutic option. in neurodegenerative diseases, such as alzheimer´s disease and parkinson´s disease, mitochondrial pathways of apoptosis are considered as major features of the underlying neuronal cell death. such mitochondrial mechanisms of apoptosis are mediated by the bh3-only protein bid, a member of the bcl-2 family that triggers mitochondrial permeabilization and the subsequent release of death-promoting proteins into the cytosol. the pivotal role of bid in apoptotic cascades of neuronal cells has been shown in our previous studies showing a neuroprotective effect of bid sirna and small molecule bid inhibitors such as bi6c9 in vitro. in vivo, however, the available bidinhibitors failed to protect brain tissue likely because the compounds were not bioavailable or did not cross the blood brain barrier. therefore, chemical modifications of bi-6c9 were generated resulting in new structures and molecules with different pharmacophors. the aim of the present study is to identify novel potent bid inhibitors available for applications in model systems of brain damage in vivo. for the first screening of 80 compounds we used a model of glutamate toxicity in immortalized mouse hippocampal neurons (ht-22 cells). in this model system, glutamate induces a decrease of intracellular glutathione levels resulting in lipoxygenase activity and enhanced formation of toxic reactive oxygen species (ros). to investigate the compounds' ability to prevent glutamate induced cell death, we first analyzed the cell viability by the mtt assay. in addition, we examined the cell survival by using real time monitoring of cell impedance (xcelligence system) to determine the neuroprotective potency of the new structures. using these assays, we identified 10 novel molecules that significantly prevented glutamate-induced toxicity in ht-22 cells. further we were able to express and to purify recombinant bid in a high amount. in the ongoing study the purified bid protein will be used for co-crystallization with the identified neuroprotective structures for further optimization of novel bid inhibitors for therapeutic applications in experimental models of neurodegenerative diseases in vivo. polymorphic enzymes, urinary bladder cancer risk and structural change in the local industry ovsiannikov d. 1 , selinski s. in the 1990s, an uncommonly high percentage of glutathione s-transferase m1 (gstm1) negative bladder cancer cases (70 %) was reported in the greater dortmund area (golka et al., 1997) . the question arose whether this uncommonly high percentage of gstm1 negative bladder cancer cases was due to environmental and occupational exposure decades ago. thus, 15 years later, another study on bladder cancer was performed in the same area after the coal, iron and steel industries had finally closed in the 1990s. in total 196 bladder cancer patients from the st.-josefs-hospital dortmund-hörde and 235 controls with benign urological diseases were investigated by a questionnaire and genotyped for gstm1, gstt1 and the n-acetyltransferase 2 (nat2) tag snp rs1495741. the frequency of the gstm1 negative genotype was 52 % in bladder cancer cases and thus much lower, compared to a previous study performed from 1992-95 in the same area (70%). nat2 genotypes were distributed equally among cases and controls (63% slow acetylators). less gstt1 negative genotypes were present in cases (17%; controls 20%). apoptosis inducing factor (aif) has been identified as a key factor in intrinsic pathways of caspase-independent neuronal death in model systems of acute brain injury and neurodegenerative diseases, such as alzheimer's disease and parkinson's disease. aif is a mitochondrial intermembrane flavoprotein with the capacity to translocate to the nucleus where it induces chromatin condensation and large-scale dna fragmentation. previous studies revealed that aif deficiency leads to protective effects in different models of neuronal death in vitro and in vivo. however, aif also plays an important physiological role for the integrity and function of the mitochondrial respiratory chain. thus, aif deficiency may significantly alter mitochondrial functions and metabolic homeostasis thereby preconditioning the cells to tolerate subsequent stress stimuli. the present study addresses this hypothesis and investigates whether neuroprotection by aif depletion was attributed to a preconditioning effect, i.e. protecting mitochondrial function and integrity. as model system we use glutamate induced oxytosis in immortalized mouse hippocampal ht-22 neurons. silencing of aif expression by sirna (20nm) protected mitochondrial morphology and integrity against glutamate induced damage. microscopy analysis of the mitochondrial morphology revealed that aif sirna prevented mitochondrial fission. furthermore, facs analysis confirmed that mitochondrial membrane potential was stable in cells with aif silencing. this protection of mitochondrial morphology and integrity by aif depletion was associated with preserved atp levels and inhibition of cell death as detected by an mtt assay. pronounced formation of lipidperoxides as another indicator of mitochondrial damage was also attenuated in cells preconditioned by aif sirna. these protective effects of aif sirna were highly similar to effects obtained with low doses of rotenone (20nm), which was applied as an inhibitor of complex i and mediated comparable preconditioning effects in the ht-22 cells. overall, these findings support the conclusion that aif depletion mediates a preconditioning effect protecting neuronal cells from a subsequent glutamate toxicity. in order to link these preconditioning effects to complex i functions, protein expression and functional analysis of complex i are being analysed to identify the molecular mechanisms of aif dependent control of neuronal life and death. -dependent inactivation and display very slow voltage-dependent inactivation. both properties are of crucial importance in ribbon synapses of retinal photoreceptors and bipolar cells, where sustained ca 2+ influx through cav1.4 channels is required to couple slowly graded changes of the membrane potential with tonic glutamate release. mutations in the gene coding for cav1.4 cause severe impairment of retinal circuitry function and have been linked to congenital stationary night blindness type 2a (csnb2), aland island eye disease (aied) and cone-rod dystrophy type 3 (cordx3). the clinical phenotypes of these eye diseases vary substantially regarding the ratio of rod to cone functional impairment. the reasons for this variability are not known. to gain more insights into the pathophysiology caused by loss of cav1.4 function we analyzed the visual phenotype of cav1.4-deficient mice. to this end, we combined immunohistochemistry, electroretinography (erg) and vision-dependent behavioral testing. immunohistochemical analysis using synaptic and postsynaptic markers revealed severe synaptic defects in cav1.4-deficient mice. heterozygous cav1.4 mice showed mosaic synaptic defects most probably caused by random x-chromosomal inactivation of the healthy allele. electroretinography revealed a loss of scotopic and photopic photoreceptor function. this loss of retinal network function resulted in impaired performance of cav1.4 knockout mice in a water maze-based behavioral test of rod and cone function. in conclusion, loss of cav1.4 channels strongly impairs rod and cone retinal function and vision in mice. lsr is the host cell receptor for clostridial iota-like toxins papatheodorou p., aktories k. albert-ludwigs-universität freiburg institut für experimentelle und klinische pharmakologie und toxikologie, albertstr. 25, 79104 freiburg, germany the human enteric pathogen clostridium difficile is the most serious cause of antibioticassociated diarrhea and pseudomembranous colitis. hypervirulent strains of the pathogen, associated with more severe disease and increased death rates, produce the binary actin-adp-ribosylating toxin cdt (c. difficile transferase) in addition to the rho glucosylating toxins a and b. cdt is member of the family of clostridial iota-like toxins, including c. spiroforme toxin (cst) and the eponym c. perfringens iota toxin. the toxins induce depolymerization of the actin cytoskeleton and the formation of microtubulebased cell protrusions that increase adherence and colonization of clostridia. using a haploid genetic screen, we identify the lipolysis-stimulated lipoprotein receptor (lsr) as the target molecule for entry of cdt into host cells. in addition, we present evidence that lsr is shared as a cell entry point by all members of the iota-like toxin family. identification of the toxin receptor provides a most valuable basis for antitoxin strategies. bisphenol a (bpa) is a chemical of high interest due to its endocrine activity. controversy exists concerning the blood concentration due to normal exposures. some authors claimed to have measured concentrations in the ng/ml range which is in contrast to kinetic properties of bpa. bpa is excreted in the urine as glucuronide and sulfate metabolites. recently, data on the in vitro metabolism of bpa by recombinant udpglucuronyltransferase 2b15 enzymes (ugt2b15) revealed that ugt2b15.2 and ugt2b15.5 had markedly lower intrinsic clearance as compared to ugt2b15.1 (hanioka et al., 2011) . using the in vitro metabolism data, we scaled the kmand vmaxvalues in an established human physiologically based toxicokinetic (pbtk) model (mielke and gundert-remy, 2009, mielke et al., 2011) to the values of the variants. for oral doses at relevant exposure levels, the maximum blood concentration (cmax) for the ugt2b15.2 variant (v2) was 5 fold and those of the ugt2b15.5 variant (v5) was 7 fold higher than that of the ugt15.1 variant. with dermal exposure at a relevant exposure level, the cmax values were 1.4 (v2) and 1.6 fold (v5) of ugt15.1 variant. a combined exposure of oral and dermal exposure, an exposure scenario, which occurs in daily life, resulted in 2.4 fold (v2) and 3.2 fold (v5) higher cmax values as compared to ugt15.1 variant. the values for the area under the blood concentration time curve (auc) were for a relevant oral dose 5.7 fold (v2) and 8.6 fold (v5), for relevant dermal exposure 1.4 fold (v2) and 1.6 fold (v5), and for combined exposure 1.9 fold (v2) and 2.5 fold (v5) of ugt15.1 variant. from the results we conclude: (1) polymorphism of udpglucuronyltransferase (2b15.2 and 2b15.5) has an impact on the blood concentrations which, however, is less than 10 fold for cmax and for auc. the effect is more pronounced for oral as compared to dermal or combined exposure. (2) polymorphism of metabolism does not explain the blood/plasma concentrations in the ng/ml range measured by some authors. hanioka n, oka h, nagaoka k, ikushiro s, narimatsu s. effect of udpglucuronosyltransferase 2b15 polymorphism on bisphenol a glucuronidation. arch toxicol. 2011, 85(11) :1373-81. mielke h, gundert-remy u. bisphenol a levels in blood depend on age and exposure. toxicol lett. 2009 8; 190(1) :32-40. mielke h, partosch f, gundertthe heart responds to maladaptive pro-hypertrophic stimuli by stimulating intrinsic signals that contrast and dampen the onset and development of hypertrophy. cyclic guanosine monophosphate (cgmp) and its downstream effector cgmp kinase i (cgki) have been suggested to be an important anti-hypertrophic signaling pathway (1) . intracellular levels of cgmp can be raised by the action of nitric oxide (no) and natriuretic peptides (anf, bnf), or by inhibiting cgmp-degrading phosphodiesterases (pde). a growing body of evidence suggests that the pde5 specific inhibitor sildenafil (sil) prevents and reverses hypertrophy and chamber remodelling in the heart of mice subjected to thoracic aorta constriction (tac) by elevating cgmp levels and cgki activation (1) . in contrast, using a mouse model that lacks cgki expression in every cell type except smooth muscle cells (βres mice; see ref. 2), we recently showed that the absence of this kinase does not alter the onset of hypertrophy induced by tac or isoproterenol infusion (2) . sil is believed to increase cardiac cgmp levels, although it is unclear, if its target (pde5) is expressed in cm (2). sil may act on other pdes, such as pde1c which is abundant in cms. it is also unclear if sil effects are mediated by other cardiac cell types, in particular by cardiofibroblast. to answer these questions, we are currently investigating whether sil is able to prevent hormone induced cardiac hypertrophy in the absence of cgki in cm. preliminary results on βres mice show that even in the case of chronic angii infusion, lack of cgki in cm does not alter the induction of hypertrophic response, at least in the initial phase (7days of angii infusion at 2mg/kg/day). interestingly, βres mice showed impaired cardiac function, as indicated by decreased fractional shortening. sil was able to partially block the onset of cardiac hypertrophy in wt animals, but not in βres mice, indicating a requirement of cgki in this process. in particular, sil was able to block the transcription of pro-fibrotic genes such as tgfβ, ctgf, collageni and fibronectin. the overexpression of the somatostatin receptors sst2 and sst5 in neuroendocrine tumors provides the molecular basis for therapeutic application of the stable somatostatin analogs octreotide and pasireotide. whereas the phosphorylation of the carboxyl-terminal region of the sst2 receptor has been studied in detail, little is known about the agonist-induced regulation of the human sst5 receptor. here, we have generated phosphosite-specific antibodies for the carboxyl-terminal threonines 333 (t333) and 347 (t347), which enabled us to selectively detect either the t333-or the t347-phosphorylated form of sst5. we show that agonist-mediated phosphorylation occurs at t333, whereas t347 is constitutively phosphorylated in the absence of agonist. we further demonstrate that the pan-somatostatin analog pasireotide and the sst5-selective ligand l-817,818 but not octreotide or ke108 were able to promote a clearly detectable t333 phosphorylation. however, none of these compounds was able to stimulate t333 phosphorylation and sst5 internalization to the same extent as the natural somatostatin. agonist-induced t333 phosphorylation was dose-dependent and selectively mediated by g protein-coupled receptor kinase 2 (grk2). like that observed for the sst2 receptor, phosphorylation of sst5 occurred within seconds. however, unlike that seen for the sst2 receptor, dephosphorylation and recycling of sst5 were complete within minutes. we also identify protein phosphatase 1g (pp1g) as sst5 receptor phosphatase. together, we provide direct evidence for agonist-selective phosphorylation of carboxyl-terminal t333. in addition, we identify grk2-mediated phosphorylation and pp1g-mediated dephosphorylation of t333 as key regulators of rapid internalization and recycling of the human sst5 receptor. termination of signaling of activated g protein-coupled receptors (gpcrs) is essential for maintenance of cellular homeostasis. although the regulation of agonist-induced phosphorylation has been studied in detail for many gpcrs, the molecular mechanisms and functional consequences of receptor dephosphorylation are far from understood. recent studies have shown that phosphatase inhibitors, such as okadaic acid and calyculin a, can block the dephosphorylation of a number of gpcrs including the ß2 adrenergic receptor, d1 dopamine receptor, parathyroid hormone receptor 1, thromboxane a receptor and the vasopressin receptor 1. however, a specific phosphatase has not been identified so far. in present studies, we have examined the mechanism and function of receptor dephosphorylation using the sst2a somatostatin receptor and the µ-opioid receptor (mor) as models. within those analyses, we have identified protein phosphatase 1beta (pp1ß) as the phosphatase for the cluster of phosphorylated threonines (353ttetqrt359) within the sst2a somatostatin receptor carboxylterminus using sirna knock down screeening. those phosphorylation sites mediate ß-arrestin binding. we have also identified protein phosphatase 1gamma (pp1γ) as mor phosphatase that catalyzed t370 and s375 dephosphorylation at or near the plasma membrane within minutes after agonist removal. here, we show the different activated phosphatases with functional selective mutants. we examined tailswap mutants which specify the different phosphatase activities. therefore we produced a mor-rsst2a chimera with the rsst2a c-terminal tail and a rsst2a-mor chimera with a mor c-terminal tail. detoxification by conjugation of glutathione? formations of dna adducts of patulin activated by glutathione pfenning c., lehmann l. university wuerzburg, institute of pharmacy and food chemistry section of food chemistry, am hubland, 97074 wuerzburg, germany as a frequent contaminant in apple juice, the mycotoxin patulin (pat) has shown mutagenic potential in cultured mammalian cells at concentrations which are equivalent to those found in marketable foods. this fact is in contrast with the assumption that conjugation to the major intracellular nucleophile glutathione (gsh) leads to detoxification of the electrophile pat. although pat reacts readily with gsh, previous studies showed that co-incubation of pat with model thiols and amine compounds increased the reactivity of pat towards amines forming mixed-type adducts. thus, we hypothesise that the potential to react with dna bases after being activated by gsh might contribute to the mutagenicity of pat. adduct formation of dna bases (adenine, guanine or cytosine) with pat in the presence and absence of gsh was studied under neutral conditions. liquid chromatography coupled with electrospray ionization tandem mass spectrometry was applied for identification and structure elucidation of putative adducts. besides published as well as hitherto unknown pat-gsh adducts, several pat-dna base adducts were formed both in presence and absence of gsh. in addition, with each of the three dna bases one product exhibiting a mass to charge ratio and fragmentation pattern suggesting a mixed thiol-amine adduct was detected. based on the fragment ions of adducts formed with gsh and chemically modified derivatives, we postulate a cyclic structure of the pat-gsh-dna base adducts, resulting from the reaction of the α-amino group of the glutamic acid residue with the c7-carbonyl function of pat. the exocyclic amino group of the dna base is linked to c1 of the pat backbone by an amid bond. thus, the present study demonstrates the reactivity of pat towards dna bases and the participation of gsh in adduct formation. the postulated structures of dna adducts could be used as biomarkers for the determination of the internal exposure to pat in humans. prescribing information detailed in the summary of product characteristics (spc) forms the officially approved basis for safe prescribing of drugs. in a project funded by the german federal ministry of education and research (bmbf) we aimed to derive an internationally valid data set for safe prescribing of psychiatric drugs and therefore analyzed and compared the content of internationally available prescribing information. a team of pharmacists and clinical pharmacologists performed an in-depth comparison of the german, swiss, british and us-american spcs of 10 top prescribed psychiatric drugs. for 7 drugs (of identical pharmaceutical form) the spcs from the same manufacturer were available for all countries, whereas for three drugs spcs were only available from different companies. in these cases the most recent prescribing information from each country was included in the comparison. in 40 spcs 2220 individual data points (55.5±17.4 per individual spc) were compared. between countries the timeliness of prescribing information for an individual drug varied by a median of 18.5 (range: 6-134) months. the respective spcs covered on average 71.4±30.3% (range: 12.5-100%) of all mentioned indications and 70.1±24.4% (range 15.4-100%) of all mentioned contraindications. the warnings and precautions section of an individual spc covered on average 59.5±17.1% (range: 12.5-93.3%) of all mentioned warnings and precautions for that drug. the variation observed was only marginally improved when restricting the analysis to the 28 spcs of the 7 drugs available in all four countries from the same manufacturer. across countries, the summary of product characteristics of individual psychiatric drugs show substantial variation in crucial prescribing information. as different manufacturers are unlikely to explain much of the observed variation, these data argue for a better international cooperation and standardization of the content of summary of product characteristics. this project is supported by the german federal ministry of education and research (bmbf), project grant no. 01 ex1015b. protein kinase ck2 (former name 'casein kinase 2') is a highly conserved serine/threonine kinase, which acts as a component of regulatory networks implicated in many cellular processes but is also linked to various types of human cancer [1, 2] . elevated ck2 activity has been associated with aggressive tumor behavior and results in growth advantage, enhanced survival and dynamic adaption to stress of cancer cells [3] . ck2 is a heterotetramer consisting of two catalytic subunits (ck2α) attached to a dimer of regulatory subunits (ck2β) [4] . ck2β stabilizes ck2α against denaturation and modulates the substrate specificity of the catalytic subunit [5] . due to the relatively small and hydrophobic ck2α-ck2β interface (832 å²) [4] , low molecular weight inhibitors are able to interfere with the ck2 subunit interaction and thus affect the kinase activity [6] . such inhibitors might exhibit an increased specificity in comparison to those compounds interacting with the conserved atp binding site [3] . we have developed an elisa-based ck2α-ck2β binding assay using recombinant human ck2 subunits. different blocking reagents were analyzed to minimize nonspecific binding. the optimized binding assay was then applied to screen for inhibitors of the ck2 subunit interaction. primary hits were further characterized by determination of the parameters ic50 and ki as well as by comparing the results from the binding assay with literature-known or recently obtained crystal structures. numerous epidemiological studies have shown associations between exposure to ultra fine particles and an increase in cardiovascular diseases such as atherosclerosis, coronary heart disease and myocardial infarction. ultra-fine particles have an aerodynamic diameter of < 0.1 µm and are highly diverse with impurities of transition metals and organic compounds (e.g. polycyclic aromatic hydrocarbons). posttranslational modification (ptm) of proteins, particularly phosphorylation, is a key element in the regulation of cell function and any disturbance can lead to multiple diseases. the present study focused on the proteomic-based identification of phosphorylated proteins to understand the mechanism behind ultrafine particle exposure and cardiovascular disease development. as one of the major sources for ufp emissions are diesel exhaust, therefore to mimic the diesel particles, carbon black (cb) and benzo[a]pyrene loaded carbon black (cb+) were used in the present study. cells of the endothelial cell line ea.hy926 were exposed for 14 days to 100 ng/ml cb and cb+. phosphoprotein extraction of whole cell lysates was carried out by the method developed in our lab 1 . the obtained proteins were then separated by two-dimensional gel-electrophoresis followed by maldi-tof-ms (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) analysis of differently expressed proteins. to further validate the results invasive potential of cells were monitored by plating exposed cells for 24 hrs on top of matrigel-coated inserts. differential expressions of 20 phosphoproteins were found in cb-treated cells while an altered expression of 33 phosphoproteins was observed in cb+-treated cells. the maldi-tof analysis revealed proteins involved in the regulation of the endothelial permeability and the cellular plasticity such as vimentin, actin and transitional endoplasmic reticulum atpase. further, the invasion assay supported these results as the cb-exposed cells showed a high invasive potential as compared to control. [1] pink m. et. al. , precipitation by lanthanum ions: a straightforward approach to isolate phosphoproteins, j.protomics, 75, 2011, [375] [376] [377] [378] [379] [380] [381] [382] [383] 302 application of the ttc approach in cosmetics platzek t. bundesinstitut für risikobewertung, max-dohrn-straße 8-10, 10589 berlin, germany regulatory toxicologists in europe have been discussing the ttc approach since more than a decade, e.g. the previous scientific committee on food in 1996. since then, the concept was further developed and is now applied in the eu for the assessment of flavouring substances by efsa. two committees are discussing possible applications: the efsa scientific committee prepared an opinion exploring options for the application in food and feed, e.g. for impurities of food additives, thermal reaction products, food contact materials, contaminants etc. in addition, an eu non-food expert committee consisting of members of sccs, scher and schenir discussed the ttc concept in general as well as additional possible fields of application with the focus on cosmetics and an opinion was already published for public consultation. the proposal to apply the ttc approach also for cosmetic ingredients was introduced by a paper published in 2007 by a colipa (the european cosmetics association) supported working group. major aspects to be considered are the following: 1. applicability domain. the chemical space of the ttc dataset (> 600 compounds) has to be compared with that of cosmetic ingredients (> 10 000 compounds). route to route extrapolation. since no adequate dermal toxicity database is available both data on oral intake used in the ttc approach and on dermal exposure to cosmetic ingredients have to be transfomed to internal exposure figures. gastrointestinal and dermal bioavailability as well as route specific differences in metabolism have to be integrated. exposure. the reliability of exposure estimation is the second pillar of the ttc approach. compared to food, data on exposure to substances in cosmetics and consumer products is scarce. a pragmatic step forward is the comparison of ttcs and noael-derived safe exposure levels for cosmetic ingredients. this work was already done with substances in food contact materials and chemicals from the elincs list. for cosmetic ingredients a similar european project is ongoing. further refinement of the ttc approach is needed taking into account the up-to-date toxicological knowledge. with cosmetics specific problems may arise in praxi: according to the new eu cosmetic legislation the safety of cosmetic products available on the market has to be assessed by the manufacturer or importer and also assessors with limited toxicological experience may apply the ttc approach, e.g. by running the toxtree software. plöttner s., marczynski b., käfferlein h. u., welge p., groth h., engelhardt b., schmitz k., erkes a., brüning t. institut für prävention und arbeitsmedizin der deutschen gesetzlichen unfallversicherung -institut der ruhr-universität bochum (ipa) toxikologie, bürkle-dela-camp-platz 1, 44789 bochum, germany polycyclic aromatic hydrocarbons (pahs) comprise several hundred compounds with different carcinogenic potentials, typically occurring in complex mixtures. due to a lack of data risk estimations for pah mixtures are usually based on those for benzo[a]pyrene (b[a]p). the aim of our present study was to explore the suitability of a permanent human lung cell line as tool for future studies on genotoxicity of pah mixtures. in this pilot study we investigated the time-and concentration-dependent generation of specific anti-benzo[a]pyrene -7,8-diol-9 ,10-epoxide (anti-bpde)-dna adducts as well as cytochrome p450 (cyp) 1a1/1b1 enzyme activities after b[a]p-incubation in vitro. we used metabolically competent a549 lung carcinoma cells which display several characteristics of alveolar epithelial type ii cells. after 24 h and 48 h incubations with different b[a]p-concentrations cytotoxic effects were assessed with the neutral red assay and cyp1a1/1b1 activities using luminescent tests. the formation of specific anti-bpde-dna adducts was determined by hplc with fluorescence detection. a time-and concentration-dependent formation of anti-bpde-dna adducts was observed with maximum rates of 340.5 ± 39.1 and 599.6 ± 132.4 anti-bpde/10 8 nucleotides after incubation with 1 µm (24 h) and 3 µm b[a]p (48 h), respectively. however, the mean adduct rates decreased at higher b[a]p-concentrations. the reduction was more pronounced after 24 h than after 48 h. increased cyp1a1/1b1 activities were observed at > 0.1 -1 µm (24 h) and > 0.1 -3 µm (48 h). a clear decrease of enzyme activities was observed at higher concentrations for both incubation times. in the neutral red assay no more than 10% cytotoxicity in relation to the negative control were found after 24 h incubation with ≥ 10 µm b[a]p and after 48 h with ≥ 1 µm b[a]p. overall, incubation of a549 cells with b[a]p resulted in a time-and concentration-dependent increase of cyp-activities and anti-bpde-dna adducts. this clearly shows that a549 cells are able to generate mutagenic dna-adducts. thus, the in vitro model used in the present work appears suitable for genotoxicity studies of individual pahs and pah mixtures, and therefore may be a useful tool for research on syncarcinogenesis. the β subunit of the cav1.2 channel complex has been shown to play a key role in cav1.2 channel trafficking and channel characteristics like opening probability. furthermore, the last exon of the cacnb2 gene coding for cavβ2, exon 14, contains several potential phosphorylation sites, e.g. for protein kinase a or ca 2+ /calmodulin dependent camkii. pka-dependent phosphorylation mediates β-adrenergic stimulation of cav1.2. potential phosphorylation sites are ser478 and ser479 (in the β2 subunit in rat, perez-reyes et al. 1992 a ) and ser1928 in the c-terminus of the poreforming α1c subunit (lemke et al. 2008 b ) . in cardiomyocytes camkii regulates ca 2+ release and reuptake from and into the sr and is involved in the facilitation of the calcium channel. potential interaction sites between the cav1.2 channel complex and camkii are thr498 in the β2 subunit (in rat, grueter et al. 2006 c ) and ser1512 and ser1570 in the cterminus of the α1c subunit (blaich et al. 2010 d ) . however, the exact pathways remained widely unclear up to now. to clarify these mechanisms and to identify the relevant phosphorylation sites for pka and camkii we established a mouse line carrying a stop codon in exon 14 after aa pro501. this mutation prevented translation of the cavβ2 c-terminus containing the corresponding potential phosphorylation sites mentioned above (cavβ2stop mouse). these mice were viable, showed unaltered expression of the truncated of cavβ protein and unchanged ecg and echocardiography. electophysiological analysis of isolated cardiomyocytes showed no differences in current density, the effect of isoproterenol, the time course of inactivation and the facilitation property when compared to cells isolated from littermate controls. for further investigations we bred the cavβ2stop mice with s1928a mice (lemke et al. 2008 b ) lacking the pka phosphorylation site s1928 in the α1c subunit (s1928aβ2stop mouse) or with sf mice (blaich et al. 2010 d ) lacking the camkii phosphorylation sites s1512 and s1570 in the α1c c-terminus (sfβ2stop). both mouse lines were viable and showed unchanged echocardiography recordings compared to their control littermates and unaltered ecg for s1928aβ2stop mice. electrophysiological investigations on cardiomyocytes of s1928aβ2stop mice showed unchanged β-adrenergic stimulation with isoproterenol compared to littermate controls. these results suggest that β adrenergic regulation of the cardiac cav1.2 channel is not mediated by these phosphorylation sites. introduction. chronic atrial fibrillation (caf) is marked by increased fibrosis which contributes to the perpetuation of the disease. in addition to the role of fibrosis in structural remodeling of cardiac tissue, fibroblasts can couple with cardiomyocytes via gap junction thereby altering the electrophysiological properties of the later and potentially participating in atrial electrical dysfunction. in order to understand the importance of fibroblasts in the pathophysiology of caf, we compared the electrical properties of atrial fibroblasts isolated from patients in sinus rhythm (sr) and caf. methods. fibroblasts were isolated by outgrowth culture from right atrial biopsies and cultivated in medium containing 10% fetal calf serum. we used whole-cell patch clamp techniques to investigate ion currents and membrane potential. results. sr and caf fibroblasts showed similar capacitance (sr: 43.6 ± 4.6 pf, n=33; caf: 54.7 ± 5.1 pf, n = 17) and membrane potential (sr: -21.0 ± 4.3 mv, n = 14; caf: -27. 4 ± 4.8 mv, n = 16) . in both groups, we observed fast activating outward currents with a mean threshold at -20 mv. interestingly, current amplitude was significantly larger in sr than caf cells (sr: 23.8 ± 4.2 pa/pf, n = 15; caf: 6.1 ± 1.0, n = 6; p < 0.05). when maintained in culture for 3-5 weeks, cells from both groups developed na + currents. surprisingly, the fraction of caf cells displaying such currents was larger than the sr counterpart (caf: 38%; sr: 15%). furthermore, na + current amplitude was significantly larger in caf fibroblasts (sr: 6.1 ± 2.0 pa/pf, n = 5; caf: 17.4 ± 4.4 pa/pf, n = 6; p < 0.05). na + currents were not altered by 100 nm tetrodotoxin (ttx), but 10 µm ttx reduced current amplitude to 42% of control, suggesting that the channel involved is the cardiac ttx-resistant isoform nav1.5. conclusion. in the context of caf, fibroblasts undergo electrophysiological changes which need to be thoroughly described. understanding whether those changes contribute to the af substrate might provide new therapeutic targets for the treatment of caf. the initial recruitment of gi to the alpha2a-adrenergic receptor is affected by gprotein-coupled receptor kinases and arrestins prokopets o. s., krasel c., 35043 marburg, germany most g-protein-coupled receptors undergo homologous desensitization after agonist stimulation. in this process, agonist-activated receptors are phosphorylated by gprotein-coupled receptor kinases (grks), followed by binding of arrestins to the still agonist-occupied, phosphorylated receptors. it is assumed that arrestin competes with heterotrimeric g-proteins for the receptor molecule and thereby causes desensitization of g-protein-mediated responses. we tested this idea by investigating the effect of grks and arrestins on the recruitment of g-proteins by the alpha2a-adrenergic receptor. hek293t cells were transfected with yfp-tagged alpha2a-adrenergic receptor and the three subunits of gi1. the g(beta) subunit was cfp-tagged. upon stimulation with noradrenaline, a very rapid, robust increase in fret between the receptor and the g(beta) subunit was observed, confirming previous observations that the alpha2aadrenergic receptor recruits gi with a half-life of around 150 milliseconds. when arrestin3 and grk2 were also co-transfected, the half-life of gi recruitment was substantially delayed, increasing to around 2 seconds. there seemed to be no effect of grk2+arrestin3 cotransfection on a second stimulation; neither the kinetics nor the extent were altered compared to the first stimulation. interestingly, grk2 alone seemed to cause a similar but slightly less pronounced delay of g(beta) recruitment to the alpha2a-adrenergic receptor. in corresponding experiments, we measured the recruitment of arrestin3-cfp to yfp-tagged alpha2a-adrenergic receptors in the presence of grk2. upon stimulation with noradrenaline, we observed a robust increase in fret between the receptor and arrestin3, confirming previous observations that the alpha2a-adrenergic receptor recruits arrestin3. co-transfection of the three subunits of gi1 had no effect on the kinetics of arrestin3 binding to the alpha2a-adrenergic receptors. based on previous results with other receptors, an attenuation of gi recruiting to the alpha2a-adrenergic receptor is quite unexpected. our data suggest that the relation between receptors, g-proteins, grks and arrestins may be more complex than previously postulated. introduction: human urinary bladder expresses mrna of the three known badrenoceptor (b-ar) subtypes (b1, b2, b3) in detrusor and urothelium. we have shown previously that only b3-ar is involved in human detrusor relaxation. to investigate the urothelium-induced modulation of b-ar-mediated relaxation, we have examined systematically whether other b-ar subtypes are involved. human detrusor tissue samples were obtained from patients undergoing radical cystectomy for the treatment of bladder cancer. detrusor strips were studied with and without an intact mucosa layer. muscle strips were precontracted with 1 µm carbachol and relaxation was studied in response to the b-ar agonist ne. a-ar mediated processes were blocked with the a-ar antagonists phentolamine and prazosin. selective b-ars antagonists were used to investigate b-ar mediated relaxation. at the end of each experiment 10 µm forskolin was used to determine maximum camp-mediated relaxation. the presence of intact urothelium reduces potency but not effectivity of ne indicating involvement of urothelium-mediated processes not only during detrusor contraction but also during relaxation. ne-mediated detrusor relaxation is mediated through b3-ar in the absence of urothelium. but in intact detrusor strips b2-ars seem to have an additional inhibitory effect. affinity of the selective b3-ar antagonist l748,337 is unchanged, therefore the intact urothelium does not interact with the function of b3-ars. aldosterone causes oxidative stress and dna damage independent of blood pressure in vivo queisser n., schupp n. universität würzburg institut für toxikologie, versbacherstr. 9, 97078 würzburg, germany background: an inappropriate increase of the mineralocorticoid aldosterone (ald) can be induced by a stimulated renin-angiotensin-aldosterone system. epidemiological studies exploring the connection between hypertension and cancer found higher cancer mortality and an increased risk to develop kidney cancer in hypertensive individuals. we recently showed that ald produces oxidative stress, activates transcription factor nf-kb and is genotoxic in kidney tubule cells. objectives: this study investigated the capacity of ald to induce oxidative/nitrosative stress, dna damage, dna repair, apoptosis, cell proliferation and the activation of nf-κb in rat kidneys. methods: mineralocorticoid-dependent hypertension was induced by ald/salt in sprague dawley rats. dna damage and oxidative/nitrosative stress markers were detected immunohistochemically. results: ald/salt treatment caused increased blood pressure compared to untreated rats. tempol, an antioxidant, and hydralazine, a vasodilator acting independent of the renin-angiotensin-aldosterone system, could lower the blood pressure, while the mineralocorticoid receptor (mr) antagonist spironolactone was administered in a subtherapeutical dose not lowering the blood pressure. ald/salt treatment caused oxidative and nitrosative stress, structural dna damage, double strand breaks, dna repair and nf-κb activation. spironolactone decreased these markers significantly. tempol was also able to reduce these markers, while hydralazine had no effect. ald/salttreated kidneys showed a tendency to lower apoptosis and to increased cell proliferation compared to control rat kidneys. discussion: this study provides a first hint of blood pressure-independent effects of ald. the mr and the production of ros seem to be crucial for the damaging effects of ald. an aberrant or long-term activation of nf-κb by persistently high ald levels could support resistance to apoptosis and the survival of cells with damaged dna, and increase cell proliferation. these actions could contribute to the increased cancer incidence in hypertension by initiating carcinogenesis. grant support by the dfg is gratefully acknowledged. creb regulating transcriptional coactivator 1 (crtc1) is a transcriptional coactivator of the transcription factor creb. we have recently shown its expression in cardiomyocytes and its activation by beta-adrenergic signaling. beta-adrenergic signaling contributes to the pathogenesis of cardiac hypertrophy, leading to heart failure, as evidenced by the therapeutic success of the beta-adrenoceptor antagonists. in order to investigate if crtc1 is involved in this process, we investigated the expression of crtc1 in hypertrophied myocardium from mice and humans. methods: protein lysates from mouse and human samples were investigated for crtc1 protein expression. we distinguished between an acquired and an inherited form of cardiac hypertrophy. acquired cardiac hypertrophy is an adaptation of the heart to an increased cardiac workload and can be found in patients with an aortic valve stenosis. in mice this kind of hypertrophy can be evoked by transverse aortic constriction (tac). the inherited form of cardiac hypertrophy is caused by mutations in genes coding for proteins of the sarcomeric apparatus and is referred to hypertrophic cardiomyopathy (hcm). as a model for hcm, transgenic mice with a mutation in the mybpc3 gene, coding for the sarcomeric protein cardiac myosin-binding protein c (cmybp-c), were used. these transgenic mice were characterized by a hypertrophied heart. in case of human samples we distinguished between patients with an acquired form of hypertrophy due to an aortic valve stenosis, and patients with a hypertrophic obstructive cardiomyopathy (hocm), a special form of hcm. results: in the tac mice and in the myppc3 mutant mice the expression of crtc1 was significantly higher than in the controls (2.1-and 1.9-fold, respectively; n=3). in both forms of human hypertrophy, we found a significantly 5-fold upregulation of crtc1 protein level in comparison to human samples from non-failing myocardium or samples from patients with a dilatative or ischemic cardiomyopathy (n=7-10 for each group). conclusions: crtc1 is upregulated in cardiac hypertrophy with acquired or geneticbased origin. the evaluation of the role of crtc1 in the heart may help to elucidate the role of beta-adrenergic signaling in the development of cardiac hypertrophy. microarray gene expression profiling reveals up-regulation of the cardiac lipid metabolic process at the onset of heart failure fu x., abd alla j., quitterer u. eth zürich molekulare pharmakologie, winterthurerstrasse 190, 8057 zürich, switzerland atherosclerosis and chronic pressure overload are major cardiovascular risk factors for the development of heart failure in patients. to mimic those risk factors in experimental models we used atherosclerosis-prone apolipoprotein e (apoe)-deficient mice, and chronic pressure overload was imposed by abdominal aortic constriction (aac). cardiac function was monitored by echocardiography. severe atherosclerosis in aged apoedeficient mice or chronic pressure overload induced signs of heart failure as evidenced by a significantly reduced cardiac ejection fraction (<30%). to investigate pathomechanisms underlying the development of heart failure, microarray gene expression analysis and qt-rt-pcr were performed of failing heart tissue relative to age-matched controls. gene ontology analysis of the microarray data revealed that the onset of heart failure, in two different experimental models, was characterized by a strong up-regulation (≥2-fold) of the cardiac lipid metabolic process and lipid overload. lipid metabolism genes were involved in lipid synthesis, storage and oxidation. the major palmitate-synthesizing enzyme, fatty acid synthase, was causally related to the development of cardiac dysfunction by enhancing cardiomyocyte apoptosis. taken together the data support that the onset of experimental heart failure is characterized by a dysfunction of the cardiac lipid metabolism promoting cardiomyocyte death. at1-receptor blockers (arbs) are established for the treatment of high blood pressure and new onset of diabetes is reduced by arbs. in the past years evidence increased that body weight may also be lessened particularly in rats and mice. however, less data are available whether arbs still reduce weight, when treatment was initiated not until animals became obese by diet. prior to drug treatment, spontaneously hypertensive rats were fed for 6 months with chow but also with a cafeteria diet (cd) to develop obesity. controls received only chow (conchow). cd-fed shr were treated for 3 months with telmisartan (tel 8mg/kg/d) or amlodipine (aml, 12 mg/kg/d), whereas controls received vehicle (concd). systolic blood pressure (sbp), feeding behaviour, body weight, abdominal fat mass (by mrt) and energy expenditure (by indirect calorimetry) was monitored. leptin sensitivity was assessed by measuring energy intake and expenditure after repetitive injections (s.c.) of leptin. insulin sensitivity was functionally determined by glucose and insulin tolerance tests. due to cd feeding body weight was increased after 6 months by more than 60 g. tel normalized sbp whereas it remained >200 mmhg in concd and conchow. tel additionally reduced cd-induced increase of body weight and abdominal fat mass. food intake was diminished during the first 4 weeks, but raised beyond control levels during the last 4 weeks of treatment. the shift of the respiratory index to lower levels indicated improved energy expenditure. in response to exogenous leptin, the food intake of concd was higher compared to conchow, indicating a leptin resistance. this assumption is further supported by high triglyceride concentrations of concd. after tel, leptininduced food intake was reduced and energy expenditure was increased compared to concd, indicating that leptin sensitivity was at least partially restored. accordingly, triglycerides were reduced. compared to concd, the insulin sensitivity was improved by tel since maximal increases in plasma concentrations of glucose and insulin in response to glucose challenge were reduced, but glucose response to insulin challenge was diminished. even though reduction in blood pressure was almost similar between tel and aml, metabolic and antiobese efficacies of aml were markedly attenuated. we conclude that telmisartan reveals wide efficacies in improving all symptoms of the metabolic syndrome. the pleiotropic effects are not related to the hypotensive action of tel. a β2-adrenoceptor -camp mediated, immediate stimulation of β2-adrenoceptor gene expression in human lung fibroblasts is opposed by a delayed up-regulation of inhibitory factors racké k., lamyel f., kämpfer n., schütz i., warnken m. univ. bonn dept. pharmacol. &toxicol., 53105 bonn, germany based on their bronchodilatory effects, β2-adrenoceptor agonists constitute essential elements in the treatment of bronchial asthma and copd. however, treatment with long-acting β2-adrenoceptor agonists has been associated with possible worsening of airway hyper-reactivity, possibly because of loss of β-adrenoceptor function. therefore, the molecular regulation of β2-adrenoceptor expression was addressed here. mrc-5 human lung fibroblasts were cultured for up to 48 h in absence or presence of test substances, followed by β2-adrenoceptor mrna determination by qpcr. β2-adrenoceptor mrna decreased with a half-life of 25 min after inhibition of mrna synthesis with actinomycin d (30 µm), but increased by 333±85%, 502±52% and 640±165% (means±sem) within 1.5, 4 and 6.5 h, resp. after inhibition of protein synthesis by cycloheximide (30 µm). the β2-adrenoceptor agonists formoterol and olodaterol (1-100 nm) induced a rapid increase in β2-adrenoceptor mrna (maximally within 1 h by 100±19% and 110±19% at 10 nm, resp.). however, after 4 h exposure to 10 nm formoterol or olodaterol a reduction in β2-adrenoceptor mrna by 59±8% and 58±6%, resp., was observed. both, the stimulatory and inhibitory effects of β2adrenoceptor agonists were mimicked by forskolin (10 µm, increase by 88±14% and inhibition by 49±4%) and cholera toxin (5 ng/ml, increase by 76±12% and inhibition by 77±7%). the formoterol-induced up-regulation of β2-adrenoceptor mrna was blocked by actinomycin d, but not by cycloheximide. moreover, in presence of cycloheximide, β2adrenoceptor agonist induced inhibition was converted into a marked stimulation. in conclusion, expression of β2-adrenoceptors in human lung fibroblasts is highly regulated at transcriptional level. the observations with cycloheximide indicate that the β2-adrenoceptor gene is under strong inhibitory control of short-living, not yet identified suppressors. although both, the time-dependent up-and down-regulation of the β2adrenoceptor gene expression by β2-adrenoceptor activation appears to be mediated via adenylyl cyclase -camp signalling, only the stimulatory effect appears to be a direct action on the β2-adrenoceptor gene. et-1 appears to be involved in the pathogenesis not only of pulmonary hypertension, but also in fibrotic remodeling associated with chronic obstructive airway diseases. since human lung fibroblasts (hlf) are a source of et-1 and have been shown to be controlled by muscarinic receptors and β-adrenoceptors, a possible muscarinic and βadrenergic modulation of et-1 expression in hlf was explored. mrc-5 hlf were cultured for up to 24 h in absence or presence of test substances, followed by prepro-et-1 (ppet-1) mrna determination by qpcr. the muscarinic agonist oxotremorine (10 µm) induced an increase in ppet-1 mrna by 180%, an effect prevented by 10 nm tiotropium. the β2-adrenoceptor agonist olodaterol (up to 100 nm) caused a reduction of ppet-1 mrna expression by 45%. the effect of 10 nm olodaterol was prevented by ici 118,551 (1 µm), but not affect by cgp 20712 (3 µm) . the pka agonist 6-bnz-camp (500 µm) caused a reduction in ppet-1 mrna expression by 65%, whereas the epac agonist 8-cpt-2'-o-me-camp (100 µm) caused only a marginal inhibition by 22%. olodaterol (10 nm) strongly opposed the stimulatory effect of 10 µm oxotremorine. an increase in ppet-1 mrna expression by 185% caused by 0.3 ng/ml tgf-β was effectively opposed by 10 and 100 nm olodaterol, resulting in an inhibition comparable to that in absence of tgf-β. however, the increase in ppet-1 mrna caused by a maximally effective concentration of tgf-β (1 ng/ml, increase by 620%) was not significantly affected by 10 or 100 nm olodaterol. likewise, the pkaagonist 6-bnz-camp (500 µm) opposed the increase in ppet-1 mrna expression caused by 0.3 ng/ml tgf-β, but not that caused by 1 ng/ml tgf-β. tgf-β caused, with an ic 50 of 0.3 ng/ml, a marked down-regulation of β2-adrenoceptor mrna expression, maximally by 90% within 6 h. et-1 expression in hlf is stimulated by muscarinic receptors and inhibited by β2adrenoceptors. the effect of β2-adrenoceptors may be mediated via pka. et-1 expression in hlf is markedly up-regulated by tgf-β, but only effects of sub-maximally effective concentrations of tgf-β are opposed by the β2-adrenoceptor -pka pathway, in part because of tgf-β-induced down-regulation of β2-adrenoceptors. since et-1 can promote pro-fibrotic features in hlf, inhibition of et-1 expression could contribute to long-term beneficial effects of long-acting β2-adrenoceptor agonists such as olodaterol and long-acting muscarinic antagonists such as tiotropium. cardiac hypertrophy leads to up-regulation of dipeptidyl aminopeptidase-like proteins in human and rat radicke s., hutschenreuther a., schaefer m. universität leipzig rudolf-boehm-institut für pharmakologie und toxikologie, härtelstr. cardiac hypertrophy is a major risk factor for heart failure and associated morbidity and mortality. functional down-regulation of k + currents is a prominent feature of cells isolated from failing ventricles. a marked decrease in the transient outward potassium current ito has been shown in various models. changes in the k + channel expression differ depending on the species, and the mechanism of induction of heart failure. to study the regulation of ito channel subunits we compared the hypertrophic responses in human ventricular tissues from failing hearts with doxorubicin-induced hypertrophy in rats and in h9c2 embryonic rat cardiac cells. specifically, we quantified mrna expression of the pore-forming subunits kv4.3 and kv4.2, the cytosolic β-subunit kchip2 and the transmembrane subunits dpp6 and dpp10 using rt-pcr. treatment with doxorubicin (2 µm) induced hypertrophy and increased the mrna expression of the hypertrophy marker genes anf, bnp and beta-mhc in h9c2 cardiac myoblasts. while kv4.3 was detected in h9c2 cells and hearts from human and rat, kv4.2 mrna was only expressed in adult rats. during hypertrophy kv4.3 was downregulated in human tissue as well as in doxorubicin-treated h9c2 cells compared to the controls. in rat hearts kv4.2 expression was increased after doxorubicin treatment. interestingly, kv4.2 was also found to be up-regulated in rat heart tissues and h9c2 cells after treatment with doxorubicin. as previously shown, kchip2 mrna expression was significantly reduced in tissues of failing hearts. in contrast, kchip2 mrna was upregulated in hypertrophic rat hearts and h9c2 cells. the expression of dpp6 and dpp10 was observed only in human hearts. but mrna levels of both were significantly increased in failing tissues. dpp6 and dpp10 were not expressed in the adult rat heart or h9c2 cells, whereas in rats with doxorubicin-induced cardiac hypertrophy and in doxorubicin-treated h9c2 cells, the mrna of dpp6 and dpp10 was up-regulated. h9c2 cells showed almost identical hypertrophic responses to those observed in human ventricles and rat hearts. this finding validates h9c2 cells as a model for in vitro studies of cardiac hypertrophy. in further studies we will investigate the consequences of a knock-down of dpp6 and dpp10 in doxorubicin-induced hypertrophic h9c2 cells. in preliminary experiments specific short-hairpin rna, targeting dpp6 and dpp10, has been designed and tested in heterologous expression systems. the nonsynonymous c.521t>c germline genetic variation in the liver-specific organic anion transporter slco1b1 is associated with methotrexate pharmacokinetics in pediatric acute lymphoblastic leukemia radtke s. 1 background: methotrexate (mtx) plasma concentration is related to its clinical effect. transport proteins, such as abcc2, slco19a1, and slco1b1, have been implicated in the disposition of mtx. here we investigated whether common reduced-function variants in abcc2, slco19a1, and slco1b1 contribute to the interindividual variability in methotrexate pharmacokinetics in children with acute lymphoblastic leukemia (all). we analyzed mtx pharmacokinetics (mtx plasma concentration at the end of infusion c24h, mtx auc24-48h, and mtx clearance cl) in an unselected population of 419 children with all from the all-bfm 2000 trial (clinicaltrials.gov: nct00430118) who received 1676 courses of mtx at 5 g/m 2 as 24 h infusions. the contribution of genes (genetic component, rgc) to the interindividual variability in mtx pharmacokinetics was estimated according to the method of kalow et al. (1998) . abcc2 c.-24c>t (rs717620), slco19a1 c.80g>a (p.his27arg, rs1051266), slco1b1 c.521t>c (p.val174ala, rs4149056) and slco1b1 388a>g (p.asn130asp, rs2306283) genotypes were analyzed by taqman polymerase chain reaction. there was substantial interpatient variability in average (± sd) mtx c24h (50.95 ± 24.15 µmol/l), auc24-48h (57.44 ± 37.52 h*mg/l), and cl (390.72 ± 223.95 ml/min/m²). the rgc values of c24h, auc24-48h, and cl ranged from 0.61-0.71 suggesting that variation in mtx pharmacokinetics has a substantial genetic component. after adjustment for age and sex by multiple regression, the slco1b1 c.521t>c snp was significantly associated with c24h (p<0.001), auc24-48h (p<0.001), and cl (p=0.011) of mtx. compared with the wildtype genotype, in patients with the tc genotype c24h and auc24-48h increased by 18% (p=0.009) and 28% (p=0.003), respectively, whereas cl significantly decreased by 15% (p=0.012). pharmacokinetic variables significantly changed with increasing number of variant c.521t>c alleles (p<0.02, jonckheere-terpstra), suggesting a per allele effect consistent with a co-dominant model of association. in contrast, the abcc2 c.-24c>t, slco19a1 c.80g>a, and slco1b1 388a>g polymorphisms did not show an association with mtx pharmacokinetics. conclusions: the nonsynonymous c.521t>c polymorphism in slco1b1 contributes to the variability of mtx pharmacokinetics in this study of high-dose mtx in pediatric all. this project is supported by the johannes und frieda marohn-stiftung. the antitumorigenic mechanism of the selective cyclooxygenase-2 (cox-2) inhibitor celecoxib is still a matter of debate. using human lung cancer cell lines (a549, h358, h460), the present study investigates the contribution of cox-2 and peroxisome proliferator activated receptor γ (pparγ) to apoptosis elicited by celecoxib. celecoxib was found to cause apoptotic cell death in a concentration-dependent manner (10 -50 µm), whereas structurally-related cox-2 inhibitors (etoricoxib, rofecoxib, valdecoxib) were inactive in this respect. apoptotic cell death by celecoxib was suppressed by preincubation of tumor cells with the selective cox-2 inhibitor ns-398, the pparγ antagonist gw-9662 and by sirna targeting cox-2 or pparγ. celecoxib-induced apoptosis was paralleled by a time-and concentration-dependent upregulation of cox-2 and pparγ at both mrna and protein level. using an established cox-2 activity assay monitoring immediate conversion of exogenously added arachidonic acid to the respective prostaglandins (pgs), ns-398 was shown to suppress celecoxib-induced cox-2 activity when added prior to arachidonic acid. among the cox-2-dependent pgs analyzed, pgd 2 and its dehydration product 15-deoxy-∆ 12,14 -pgj2 were found to induce cytosol-to-nucleus translocation of pparγ as well as pparγ-dependent apoptosis. celecoxib-elicited translocation of pparγ was inhibited by preincubation of cells with ns-398 which itself did not alter celecoxib-induced total pparγ protein expression. finally, a cox-2-and pparγ-dependent proapoptotic mechanism of celecoxib was confirmed in primary tumor cells obtained from brain metastases of two lung cancer patients. together, our data demonstrate a proapoptotic mechanism of celecoxib involving initial upregulation of cox-2 and pparγ and a subsequent nuclear translocation of pparγ by cox-2-dependent pgs. uncoagulated ppp contained 40-60 nm thrombin throughout. fxa was initially measured in clots at 10 nm. this level declined over time while clot-conditioned pbs accumulated fxa. exposure of human aortic smc to clots or native unclotted ppp for 24h only marginally influenced smc apoptosis but increased mitogenesis over 15-fold. this was reduced by all 4 inhibitors. clot-stimulated induction of tnfα and interleukin-6 mrna was also attenuated by the inhibitors. denatured ppp (no protease activity) increased smc mitogenesis to a level seen in smc exposed to clot and combined hirudin + dx9065a, reflecting the well-known mitogenic actions of serum alone. conclusion: coagulation of human plasma generates nanomolar amounts of thrombin and fxa, sufficient to stimulate the proliferative and inflammatory properties of adjacent smc. our observations validate the use of purified thrombin and fxa at nanomolar concentrations for in vitro studies, and support the individual and coagulationindependent roles of these proteases in cell proliferation and inflammation. antithrombotic therapy with argatroban or rivaroxaban may limit the cellular effects of clot-derived thrombin and fxa, while normal anti-platelet therapy would not. this aspect should be considered in the clinical use of these agents, specifically in healing processes after vessel injury. rhogef17 mediates cgmp/cgk induced adherence and relaxation of vascular smooth muscle cells rauch j. 1 , stephan-schnatz k. the guanine nucleotide exchange factor rhogef17 is the only gef known so far to be directly activated by cgmp-dependent kinase. it is expressed in various types of smooth muscle cells and has been shown to play a role in the regulation of cell integrity. in a previous investigation we showed that the knockdown of rhogef17 by a shrna approach caused a loss of actin stress fibers and a subsequent change of smooth muscle cell morphology that finally resulted in cell rounding. we now provide evidence that the expression level of rhogef17 influences the re-attachment of cultured rat aortic smooth muscle cells (rasmc) to a surface after detachment. although rhogef17 depleted rasmc were still able to adhere and spread, their cell surface area remained considerably smaller than that of control cells in the first 24 hours after seeding. cell counting revealed that 6 to 12 hours after seeding the percentage of adherent cells was significantly lower in the rhogef17 knockdown group compared to the control group. these data indicate a delay in attachment. interestingly, the knockdown of rhogef17 was paralleled by a loss in rhoa and cadherine expression. as rhogef17 mediated a cgmp-induced activation of the small gtpase rhoa in rasmc, we studied the effect of a stable cgmp analogon (8-pcpt-cgmp) on the adhesion process. in accordance with previously published data, cgmp treatment accelerated the attachment of rasmc to the surface within the first 12 hours. in contrast, the adhesion of rasmc after rhogef17 knockdown was no longer stimulated by cgmp. as these data indicate that rhogef17 mediates cgmp-dependent signalling in a physiological process we wondered whether this protein might also play a role in the regulation of cgmpdependent relaxation of vascular smooth muscle cells. thus, we performed a collagenbased contraction assay with rhogef17 depleted rasmc. while the contraction in response to serum was not affected by the depletion of rhogef17, we observed a slight decrease in basal contractility. interestingly, cgmp was not able to counteract the serum-driven contraction of rhogef17 knockdown cells. there was no cgmp-induced relaxation in these cells. we conclude that rhogef17 is involved in the cell adhesion of vascular smooth muscle cells and likely promotes the expression of specific proteins. its activation is required to mediate cgmp-induced signalling in terms of vascular smooth muscle cell adherence and relaxation. human eosinophil and neutrophil granulocytes are cells of the innate immune system. they both express formyl peptide receptors (fpr) und histamine h2 receptors (h2r). activation of fpr leads to a release of reactive oxygen species (ros). h2r activation results in an increase of intracellular 3'-5'-cyclic adenosine monophosphate (camp) concentration and inhibition of fpr-mediated ros release via adenylyl cyclase activation. in this study we compared the effects of various h2r ligands on camp accumulation and formyl peptide n-formyl-l-methionyl-l-leucyl-l-phenylalanine (fmlp)induced ros release in isolated eosinophils and neutrophils. camp concentration was determined by hplc/tandem mass spectrometry, and ros release was assessed by monitoring superoxide dismutase-inhibitable reduction of ferricytochrome c. in eosinophils, histamine, amthamine and 5-methylhistamine exhibited similar potencies and efficacies with regard to camp accumulation and inhibtion of ros release. in marked contrast, in human neutrophils, we observed dissociations in potencies and efficacies of ligands at increasing camp accumulation and inhibition of ros production. our data suggest that in human eosinophils, but not neutrophils, camp mediates inhibtion of ros production. in a broader context our data provide a compelling example of the context-dependency of the pharmacological properties of g-proteincoupled-receptors. specifically, one has to be cautious when extrapolating experimental observations from one cell type to another, even when they are very closely related to each other. comonomers and monomers are used as dental restorative materials (e.g. in dental composites). unconverted compounds can be released from dental composites and can enter the body in humans. comonomers can induce various side effects in humans. this study was evaluated to qualify and to quantify eluted compounds from various dental composites. following composites were tested (producer in parentheses): els extra low shrinkage (saremco), synergy duo shade (coltène), grandio (voco), tetric evo ceram (vivadent), venus (kulzer), gradia (g.c.), and premise (kerr).polymerized composites (100 mg) were incubated in gc vials with 1 ml dest. water or 1 ml methanol, each at 37 °c for 72 hours. aliquots were taken, and eluted compounds were analyzed with the method of gas chromatography/mass spectrometry (gc-ms) and liquid chromatography/mass spectrometry (lc-ms).from all composites 18 different compounds were found. following comonomers were quantified (µg/ml; mean ± s.d.; n=4)( fig. 1 ).following range of the eluted and detected comonomers from dental composites was found (dest. water; decreasing elution): venus > gradia > synergy duo shade > tetric evo ceram premise > grandio > els extra low shrinkage. * n.d. = not detectable (below limit of detection). triphenylstibane was detected in tetric evo ceram (5 ± 2 µg/ml). reimann c., lupp a., schulz s. institut für pharmakologie und toxikologie universitätsklinikum jena, drackendorfer str. objective: cxcr4 is a plasma membrane chemokine receptor which is involved e.g. in organogenesis, hematopoesis and inflammation. in the adult organism cxcr4 is physiologically expressed on various cell types, in particular on lymphocytes. with respect to neoplastic tissues, in the current literature an over-expression of cxcr4 is described in different types of tumors, especially in breast and prostate cancer. additionally, an involvement of cxcr4 in tumor metastasis is discussed. subsequently, detection of cxcr4 expression in a given human tumor sample would provide a valuable predictive information on disease prognosis and possible therapeutic intervention. however, previous attempts to localize cxcr4 using poorly characterized mouse monoclonal or rabbit polyclonal antibodies have yielded predominant nuclear and occasional cytoplasmic staining, but did not result in the identification of cell surface receptors. thus, the aim of the present study was to reassess the cxcr4 expression in a panel of formalin-fixed and paraffin-embedded human normal and neoplastic tissue samples by means of immunohistochemistry using the well characterized novel rabbit monoclonal anti-cxcr4 antibody umb-2. methods: in comparison to negative and positive control samples (cxcr4-knockout and wild-type mouse embryos) the extent of staining in the different normal and neoplastic tissue specimens was scored from zero (no expression) to three (high expression). results: cxcr4 was found to be expressed in all neoplastic tissue entities analyzed. in many cases, the receptor was predominantly localized at the plasma membrane of the tumor cells. however, in all cxcr4-expressing tumor entities a huge interindividual variability both in the percentage of positive cells and in the intensity of staining was noted which strongly differed also between the various types of cancer. the most intense (score three) staining was found in the samples of (small cell) lung cancer, ovarian cancer and of pheochromocytoma. additionally, lymphatic organs such as lymph nodes, spleens and tonsils were cxcr4 positive, with mainly b cells displaying a distinct staining of the plasma membrane. the rabbit monoclonal antibody umb-2 may prove to be of great value in the assessment of cxcr4 expression in different human tumor entities and of the mechanisms underlying the formation of metastases, thus helping to find new targets and strategies in cancer therapy. link between β2-adrenoceptor-mediated inhibition of formyl-peptide-induced o2 β2-adrenoceptor (adrb2)-agonists are in daily use for asthma therapy. although most cases of asthma are controlled by standard medication, a subpopulation of asthmatics remains difficult to treat. the adrb2 gene contains a total of 49 polymorphisms. this variability could cause part of the ~70 % genetically-determined differences in therapy response. genetic and corresponding functional data on adrb2 can help to understand the complex disease and, in cases of severe asthma, optimize therapy with adrb2agonists for each individual. (ortega et al. 2007; chung et al. 2011) our present study connects sequence data with pharmacological data of prototypical adrb2-ligands, namely, (r)-isoproterenol, (r,r)-and (s,s)-fenoterol, (r)-and (s)salbutamol and (r,r)-formoterol. as a pathophysiologically relevant cell type, we analysed human neutrophils from peripheral blood of healthy volunteers. formyl-peptide-induced o2 .production and its inhibition by the agonists are examined in a 96-well cytochrome-c assay. characteristic pharmacological values (pic50, emax) are obtained for each individual. the data-set for each individual is supplemented by a differential blood cell analysis and an asthma-related questionnaire. most importantly, each volunteer's adrb2-sequence variant is determined by sanger-sequencing. complete determination of a 1,490 bp sequence, including the entire adrb2 exon and part of the flanking 5'-and 3'-untranslated regions, allows mapping of the most common, but also of new or rare polymorphisms. first data demonstrate the inter-day and intra-individual robustness of the functional data. in the next step, we will link sequence variants and functional differences within a population of sixty volunteers with sufficient statistical power. collectively, this study represents a straightforward approach to link functional and genetic data of a clinically relevant receptor. cyclic nucleotide phosphodiesterases (pdes) are classified into eleven families and are essential for second messenger metabolism in human cells. 1 recently, we have shown that several human pdes possess a much broader substrate-specificity than previously assumed, being capable of hydrolyzing not only the purine nucleotides cyclic adenosine 3',5'-monophosphate (camp) and cyclic guanosine 3',5'-monophosphate (cgmp), but also pyrimidine nucleotides such as cyclic uridine 3',5'-monophosphate (cump). 2 these data were obtained using a highly sensitive hplc mass spectrometric assay which is quite expensive and whose technical requirements are available only in few laboratories. in our present study we developed a fluorimetric pde activity assay using 2'-o-(n'methylanthraniloyl) (mant)-substituted cyclic purine and pyrimidine nucleotides that can be used more broadly in the scientific community. human pde3a is important for the regulation of platelet aggregation, oozyte maturation, vascular smooth muscle relaxation and contractility of cardiac myocytes. 1 moreover, this pde shows a broad substrate-specificity, hydrolyzing camp, cgmp, cump and cyclic inosine 3',5'-monophosphate (cimp). 2 using this enzyme, here, we demonstrate that various mant-substituted cyclic nucleotides are substrates of pde3a and undergo a significant change in fluorescence whilst being hydrolyzed, thus allowing a quantitative analysis of catalysis via fluorescence detection. in fact, not only native cump but also mant-cump is a substrate of pde3a. this finding is consistent with data published by hardman and sutherland 3 who described a cump-degrading pde activity in homogenates from beef and dog heart, leading to the assumption that the pde activity described there could be attributed to pde3a. as cump has furthermore been proven to be present in mammalian cells 4 , to differentially activate camp-and cgmp-dependent protein kinases 5 and to be synthesized from utp by mammalian soluble guanylyl cyclase 6 , our present study supports the hypothesis that this cyclic nucleotide could play an important role in cell metabolism. the newly established fluorescence assay with mant-cump facilitates future studies on pde3a and the assumed second messenger function of cump. rhoa is reportedly involved in stat-dependent transcription. however, the pathway connecting the gtpase and stat signaling has not been characterized. we made use of bacterial toxins, which directly activate rho gtpases to analyse this pathway. cytotoxic necrotizing factors (cnfs) are produced by pathogenic escherichia coli strains and by yersinia pseudotuberculosis. they activate small gtpases of the rho family by deamidation of a glutamine, which is crucial for gtp hydrolysis. we show that rhoa activation leads to phosphorylation and activation of stat3 and identify signal proteins involved in this pathway. rhoa-dependent stat3 stimulation requires rock and junkinase activation as well as ap1-induced protein synthesis. the secretion of one or more factor/s activate/s the jak-stat pathway in an auto/paracrine manner. we identify ccl1/i-309 as an essential cytokine, which is produced and secreted upon rhoa activation and which is able to activate stat3-dependent signaling pathways. the knowledge about the connection between rhoa and stat signaling is crucial for understanding several deseases, especially cancer. acid sphingomyelinase-deficient mice are protected from the lethal cardiovascular effects in tnf-induced septic shock reiss l. k. 1 christian-albrechts universität institut für immunologie, michealisstrasse 5, 24105 kiel, germany introduction: the cytokine tumor necrosis factor (tnf) is a mediator of septic shock. sepsis is a major cause of acute respiratory distress syndrome (ards), a heterogeneous lung disease with a mortality of about 50%. the present study was designed to investigate the effects of high systemic tnf-levels on the lung and on the systemic circulation in wildtype and acid sphingomyelinase-deficient (asm -/-) mice. the enzyme acid sphingomyelinase generates the signaling molecule ceramide that plays a critical role in edema formation and vasodilatation. material and methods: asm -/and wildtype mice were ventilated mechanically at vt=8ml/kg and f=180min -1 with fio2=0.3 and peep=2cmh2o while lung mechanics were followed. half of the mice received 50µg of murine tnf intravenously. blood pressure was stabilized by intra-arterial fluid support and body temperature was kept at 37°c to prevent lethal shock and to allow investigation of blood gases, lung histopathology, pro-inflammatory mediators and microvascular permeability 6 hours after tnf application. results: tnf induced septic shock in wildtype mice, as indicated by metabolic acidosis, high serum levels of the sepsis marker procalcitonin, decreasing blood pressure and reflex tachycardia. interestingly, asm -/mice were protected from the tnf-induced cardiovascular effects and mortality. in the present study, circulating tnf failed to cause lung injury. lung mechanics stayed stable during ventilation in all groups and also pulmonary histopathology, cytokine levels and microvascular permeability were unaffected. conclusion: circulating tnf alone is not sufficient to cause acute lung injury. we conclude that the cardiovascular effects in tnf-induced septic shock are partly mediated by acid sphingomyelinase. cyclophilin a sirna provides mitoprotection and prevents aif-dependent neuronal cell death reuther c. 1 cyclophilin a (cypa) is a peptidyl-prolyl-cis-trans isomerase which is localized in the cytosol. recent data suggested that neuronal cell death involved cytosolic cypa translocation to the nucleus, where it formed a pro-apoptotic complex with apoptosis inducing factor (aif). this cypa-aif complex induced caspase-independent chromatin condensation, dna degradation and cell death in various paradigms of apoptosis. on the basis of these data, the selective inhibition of the aif-cypa complex was proposed as a potential strategy to prevent aif-dependent cell death in neurons. therefore, the aim of this study was to determine effects of cypa silencing in a model of glutamate toxicity in immortalized hippocampal ht22 neurons. first, we addressed the interaction of aif and cypa by immunoprecipitation and their translocation to the nucleus by immunohistochemistry and confocal fluorescence microscopy. after exposure of ht-22 cells to glutamate the translocation of aif and cyp a occurred prior to cell death. cypa sirna attenuated glutamate-induced cell death as detected by the mtt-assay, impedance measurements (xcelligence system), and by facs analysis after annexin v/ propidium iodide staining. most intriguingly, cypa sirna also preserved the mitochondrial membrane potential as shown by facs analysis after tmre staining. further, confocal microscopy showed that cypa silencing prevented mitomorphology alterations and blocked the release of mitochondrial aif to the nucleus. the inhibition of the aif translocation to the nucleus was also shown by western blot analysis. in summary this study demonstrates that silencing of cypa prevents mitochondrial disruption and attenuates glutamate toxicity in vitro. thus, cypa is a promising target for mitoprotection as a basis for novel strategies of neuroprotection. up to now the question is unresolved how the ingested dose influences the absorption and metabolism of chlorogenic acids (cga) from food. so far no studies have been performed on the impact of the dose on cga absorption, circulation and excretion. recently we performed a dose-response study in a randomized, double-blinded, crossover design with five ileostomy subjects. in three trials the volunteers consumed after a two day polyphenol free diet coffee with varying cga content (high 4525 µmol; medium 2219 µmol; low 1053 µmol). the cga concentrations in plasma, ileal effluent and urine were subsequently identified and quantified by hplc-esi-ms, hplc-esi-ms/ms and hplc-dad. the results showed that the consumption of higher cga concentrations lead to a faster ileal excretion measured in the ileal effluents. this corresponded to the renal excretion of 8.0 ± 4.9% (high), 12.1 ± 6.7% (medium) and 14.6 ± 6.8% (low) of total cga and metabolites. we found that cgas with a caffeic acid moiety are predominantly sulphated and those with a ferulic acid moiety are predominantly conjugated via glucuronidation prior renal excretion. furthermore, in the ileal effluents, sulphation of both structural units dominated. in plasma samples (after enzymatic deconjugation) the auc values were determined by the major cga classes in coffee, the caffeoylquinic acids: 551.5 ± 93.8 nm*h -1 (high); 299.3 ± 79.6 nm*h -1 (medium) and 222.8 ± 91.3 nm*h -1 (low). no major differences in the metabolic pattern were observed. additionally, we were able to identify new metabolites of cga in urine and ileal fluids. we conclude that the consumption of high concentrations of cga via coffee might influence the gastrointestinal transit time and consequently affect cga bioavailability. this study was supported by the nestlé research centre (lausanne, switzerland). interaction of antagonists with the atp binding pocket at the human p2x3 ion channel helms n., riedel t., illes p. rudolf-boehm-institut für pharmakologie und toxikologie, härtelstraße 16-18, 04107 leipzig, germany the homomeric p2x3 receptor (p2x3r) is a rapidly activating and desensitizing cation channel, gated by extracellular atp. it consists of three homomeric subunits. this representative of the p2x receptor family is highly expressed on sensory afferent neurons and plays a significant role in chronic pain, bladder reflexes and taste sensation. therefore, the development of selective antagonists for p2x3 receptors and knowledge about the binding of these antagonists are of great significance for future pain therapy and therapy of urge incontinence. to simulate the shape of the rapidly desensitizing agonist-induced current responses via p2x3 receptors, we created a specific markov model to describe the binding of agonists and competitive antagonists. this model can be used to prove the competitive character of inhibition and to calculate the association and dissociation constants of the antagonists. furthermore we use this model to fit current responses at p2x3 wild type receptors and their mutants to α,βmethylene atp in the presence of different antagonists. whole-cell patch-clamp recordings were performed on hek 293 cells, heterologously expressing the human p2x3 receptor, to determine the concentration-response relationship of different antagonists. by applying increasing concentrations, differences of antagonist potency could be observed at the wild type receptor. afterwards, we chose amino acid residues for replacement by alanine, which seem to be important for agonist binding and should be so for competitive antagonist binding as well, based on our homology model, developed from the zebrafish p2x4r crystal structure and previous mutagenesis studies. we intend to identify those amino acids which are important for competitive antagonist binding by monitoring the altered antagonist potency on the mutated receptor when compared with the wild-type receptor. analysis of the p2x3 agonist binding site by double mutant cycles riedel t., wiese s., illes p. rudolf-boehm-institut für pharmakologie und toxikologie, härtelstraße 16-18, 04107 leipzig, germany purinergic p2x receptors belong to the family of ligand-gated ion channels. they are non-selective cation channels, activated by extracellular atp. one of the seven members of the p2x receptor family, the p2x3 receptor, is localized at the plasma membrane of sensory neurons and is involved in pain perception. therefore, this receptor is a possible target for new drugs in pain treatment. the development of such drugs can be supported by an exact knowledge of the receptor structure and function. there are many hints to the atp binding site, but the interaction of the p2x receptor with its agonists and antagonists remains still unknown. in this study, we investigated the effects of single alanine substitutions of amino acid residues in the supposed atp binding site of the homomeric human p2x3 receptor on the effect of nucleotide analogues. the mutant receptors were expressed in hek293 cells and the nucleotide effects were measured by means of the whole-cell patch-clamp method. modifications in the receptor binding site changed the concentration-response dependency as well as the current kinetics during fast pulsed agonist applications. based on this fact, we were able to distinguish binding from gating, conductance, and desensitisation, using a markov model that describes the complete channel behaviour by a matrix of rate constants. the results were also checked for consistency with a structural hp2x3 model that we developed from the known zebra fish p2x4 crystal structure in the closed state. voltage-dependent modulation of alpha2a adrenergic receptor signaling rinne a., birk a., bünemann m. philipps-universität marburg institut für pharmakologie und klinische pharmazie, karlvon-frisch str. 1, 35034 marburg, germany g protein-coupled receptors (gpcrs) are proteins that regulate numerous signaling pathways by activation of intracellular g proteins. gpcrs are activated by extracellular stimuli, such as light, hormones and neurotransmitters. recent evidence suggests that some gpcrs exhibit voltage-sensitivity leading to a modulation of their activity by the membrane potential (vm). we used a fret-based biosensor of the α2a adrenergic receptor to analyze receptor activation at defined membrane potentials in hek 293 cells by means of voltage-clamp recording. the biosensor was stimulated either with the partial agonist clonidine or with the full agonist norepinephrine (ne) and receptor activation was measured as decrease in the ratio of acceptor-/donor-fluorescence. receptor stimulation by ne was inhibited at depolarizing membrane potentials but enhanced by hyperpolarization. inhibition of ne activated receptors was strong at low concentrations (500 nm: 60 % inhibition) but almost absent at saturating agonist concentrations (100 µm: 9 % inhibition). both agonist-induced and hyperpolarizationinduced receptor activation exhibited a similar monoexponential time course and speed of activation was primarily dependent on agonist concentration for both activation modes. the latter indicates that depolarization lowers the apparent affinity of the ne receptor interaction and thus causes receptor deactivation by means of ne release. application of clonidine (1 µm, vm=-90 mv) resulted in a fret response that was inhibited by 40 % at +60 mv. in contrast to ne, strong receptor inhibition at +60 mv was present even at super-saturating concentrations of clonidine (100 µm), suggesting that voltage alters the equilibrium between active and inactive conformations of the receptor. voltage-dependence of the a2a adrenergic receptor also modulated downstream receptor signaling: g protein activation or the recruitment of arrestins, which we determined in fret assays that directly detect gαi protein activation or receptor-arrestin interactions, were both substantially inhibited at vm = +60 mv. therefore we conclude that negative membrane potentials promote active conformations of the a2a adrenergic receptor, increase affinity of full agonists and enhance receptor signaling. in conclusion the present data show that scc cells extrude more ha, possibly related to increased levels of has3, in comparison to keratinocytes. increased amounts of ha appear to be essential for the uvb induced tumourgenesis of sccs in mice. this effect might be related to the pro-proliferative property of high molecular weight ha. furthermore biological active ha fragments derived from ha degradation by hyaluronidases (hyal1,2) are thought to be pro-angiogenetic, anti-apoptotic and proinflammatory, thus possibly also promoting tumour growth and malignancy. estradiol induced paracrine release of egf from keratinocytes protects the dermal hyaluronan/versican matrix during photoaging röck k. 1 , meusch m. hyaluronan (ha) and versican are key components of the dermis and are responsive to uvb induced remodeling. the aim of the present study was to investigate the molecular mechanisms of estrogen (e2) mediated effects on ha-rich ecm during actinic aging. 10 weeks of uvb irradiation (3 x 1 med (80 mj/cm 2 ), weekly) of hairless skh-1 mice caused a marked decline of dermal ha, which was aggravated by ovariectomy (ovx). subcutaneous substitution of estrogen (e2) by means of controlled release pellets abolished these effects confirming the stimulatory role of e2. the increase of dermal ha correlated with induction of ha synthase has3 by e2. in addition the ha-binding proteoglycan versican was induced by uvb and further increased by e2. however in cultured skin fibroblasts e2 reduced the expression of versican and had no effect on has3. therefore, direct upregulation of has3 and versican in fiborblasts by e2 was excluded. however, e2 increased the expression of egf in uvb irradiated skin in vivo and in keratinocytes in vitro. egf in turn upregulated the expression of has3 and versican in dermal fibroblasts. furthermore the supernatants of estradiol treated keratinocytes led to the same effects in dermal fibroblasts, which could be abolished by previous treatment of the supernatant with neutralizing egf antibody or treatment of the fibroblasts with egf receptor blocker erlotinib. functionally, dermal ha and versican induction by e2 correlated positively with proliferation and negatively with accumulation of inflammatory macrophages in the dermis. collectively these data suggest that e2 treatment increases the amount of dermal ha and versican via paracrine release of egf which may be implicated in the pro-proliferative and anti-inflammatory effects of e2 during photoaging. differential pro-and eukaryotic toxicity of silver released from nanocomposite surfaces increases the therapeutic window of silver in antibacterial treatments röhl c. 1 , hrkac t. silver has been used since ancient times as antimicrobial agent. recently, silver gained new attention due to its higher effectiveness in its nanoform. this led to new developments of silver nanomaterials, e.g., for medical devices and consumer products. though, it is generally assumed that silver is less toxic for eukaryotes than for prokaryotes, concern is raised, if nanosilver at the same time might also increase mammalian cytotoxicity. in our study we examined the toxicity of silver released from nanocomposite surfaces with that of silver from agno3 solutions for adherent bacteria and mammalian cells. therefore, we established an in vitro reference system which enabled us to compare the therapeutic window between prokaryotic toxicity and eukaryotic integrity in both exposure settings. we focussed especially on the comparability of the bacterial and mammalian cell systems and the development of characterized ag/tio2 nanocomposite coatings with well-defined silver filling factors and silver surface release, which could be varied over a wide concentration range. as reference cells the e. coli sar 18 strain and human dermal fibroblasts, which are of special relevance in the context of medical devices like implants or wound dressings, were chosen. bactericidal effects were determined by direct growth visualization of the gfp-producing e. coli strain by epifluorescence microscopy. mammalian cell growth and toxicity was determined by the mtt assay, protein measurements and phase contrast microscopy. the ag/tio2 samples were prepared by sputter co-deposition from two separate magnetron sources. the silver surface concentration release was determined by xps and the silver release by icp-ms. in solution a concentration-dependent constant silver concentration could be determined between 2 and at least 72 hours at the surface. while lowest bactericidal and cytotoxic concentrations of ag + from agno3 solutions with 0.64 and 0.95 mg/cm 2 , respectively, differed only slightly, the therapeutic window increased significantly if ag + was released from the nanocomposite surface. while the toxicity on the fibroblasts was unchanged the bactericidal potency increased at least one order of magnitude. taken together, it can be concluded that local exposure factors i) can be modulated by silver nanocomposites and ii) play an important role for the differential toxicity of surface silver on bacteria and mammalian cells. charite -universitätsmedizin institut für integrative neuroanatomie, funktionelle zellbiologie, philippstr. 2, 10115 berlin, germany c3 exoenzyme (c3bot) a clostridial adp-ribosyltransferase does not possess a cellbinding/-translocation domain. nevertheless, c3 is able to efficiently enter intact cells, including neuronal cells but the mechanism of uptake is not yet understood. in the present work, binding of c3bot to the hippocampus-derived ht22 cell line was characterized by means of binding and blot overlay assays as well as mass spectrometry analysis to identify binding partners of c3bot. the binding assays established that c3bot bound in a concentration-dependent manner to ht22 cells. in the overlay assay we detected one clear band of 55 kda. to elucidate whether glycosylation is important for the c3bot-protein interaction, ht22 cells were incubated with glycosidase f resulting in a decreased binding of c3 to the 55 kda band. to explore the involvement of phosphorylation in the binding of c3 to the putative binding protein, blot was pre-treated with cip (calf intestinal phosphatase) before overlay with c3bot. pretreatment greatly reduced the c3bot-protein interaction. moreover, inhibition of dephosphorylation by vanadate before in intact cells showed an increased level of c3botprotein interaction in the following overlay. thus, interactions between c3bot and ht22 cell proteins may require phosphorylation. to further characterize the 55 kda band as binding target of c3bot, the 55 kda band was digested with trypsin and then subjected to lc-orbitrap mass spectrometry analysis. from this 55 kda single gel band 141 proteins were identified. further analysis of the identified proteins will provide a possible interaction partner of c3bot. in sum, protein overlay assays revealed that phosphorylation and glycosylation are critical for efficient c3bot-protein interaction. s76 335 solvent effects on enzyme kinetics in vitro rokitta d., pfeiffer k., gerwin h., streich c., fuhr u. uniklinik köln institut für pharmakologie, gleueler str. 24, 50931 köln, germany kinetic parameters provide essential quantitative information for characterisation of drug metabolising enzymes. such enzymes are located in an a partially queous environment, but to solve potential lipophilic substrates for in vitro measurements organic solvents are regularly needed. to preserve the enzymes from denaturation and other solvent related effects, the concentration of these solvents must be kept low. data on nature and extent of such solvent effects is sparse. in this study, we investigated the effects of methanol, ethanol, acetonitrile and dimethylsulfoxide (1% to 4%) on the assessment of k m, vmax and clint with regard to the 1-hydroxylation of midazolam via cyp3a4 and the cyp1a2 catalyzed metabolism of caffeine to paraxanthine in vitro. the presence of acetonitrile showed the highest vmax value for paraxanthine formation but the lowest values for 1-hydroxymidazolam formation. the km value for midazolam showed no systematic effects of organic solvents, while for caffeine km was up to eightfold lower for solvent free samples compared to solvent containing samples. the present example suggests that the presence of organic solvents may considerably influence enzyme kinetic parameters beyond a mere change in apparent activity. these effects are differing between enzyme-substrate systems and solvents. it remains to be determined to which extent such effects compromise in vitro -in vivo extrapolations, and which solvents are most appropriate. atrial remodeling and arrhythmia induced by the transcription factor er81 rommel c. 1 , rösner s. introduction: the transcription factor er81 (ets related 81) belongs to the large family of ets-transcription factors that are involved in developmental processes and in the pathogenesis of cancer. er81 is activated by gq-and gs-coupled receptors leading to a phosphorylation of the transcription factor by map-kinases and protein kinase a, respectively. cardiac er81 mrna expression is increased in failing human hearts. however mechanical unloading by a left ventricular assist device leads to normalization of er81 expression. thus, the aim of the present study was to investigate the cardiac function of er81 in genetically modified mouse models. we previously generated transgenic mice overexpressing er81 under control of the cardiomyocyte-specific α-myosin heavy chain gene (αmhc) promoter by pronuclear injection and established independent transgenic lines. electrocardiography (ecg) was assessed in mice at day 5 after birth (p5) and in adult mice (3 months) during isoflurane anesthesia and by ecg telemetry in awake mice, respectively. ecg analysis revealed no differences between the genotypes at day 5 after birth. however, we found a decreased heart rate, a replacement of regular p-waves by an undulating baseline and frequent supraventricular extrasystoles in adult er81 αmhc transgenic mice. next, isometric contractile force measurements on isolated left atria were carried out in organ baths. while wt left atria responded to increasing concentrations of isoprenaline, nkh477 and calcium with an increase in contractility, the maximal positive inotropic responses to these substances were severely blunted in er81 αmhc atria. we performed western blots to identify potential aberrations of calcium handling and regulatory proteins. phosphorylation of serine 16 of phospholamban (pln) was reduced in er81 αmhc mice. in addition, protein phosphatase 1 (pp1) expression was significantly increased in er81 αmhc mice, which is consistent with the increased dephosphorylation of phospholamban. furthermore, we found a decreased expression of calsequestrin and serca2a protein in er81 αmhc atria. electron microscopy revealed the significant structural remodeling of er81 αmhc atria at 3 months of age. conclusion: increased cardiac expression of the ets-transcription factor er81 leads to structural and electrical remodeling of the atria. thus, er81 may play an important role in the pathogenesis of cardiac arrhythmias in chronic heart failure. signal transduction pathway of atp and utp in neonatal rat cardiac myocytes rothkirch d., gergs u., neumann j. institute for pharmacology and toxicology medical faculty, magdeburger str. 4, 06097 halle (saale), germany extracellular atp and utp can be released from the heart during pathological conditions such as ischemia or hypoxia. in humans, atp and utp levels are increased during myocardial infarction. atp and utp can act via p2-purinoceptors which are further divided in p2x1-7 and p2y1-14-receptors. as previously shown atp and utp can induce inotropic effects in cardiac preparations of mice and man. for rat articular chondrocytes and human intestinal cells it has been demonstrated that the mapk cascade can be activated by atp and utp. therefore, the cardiac effects of atp and utp on force of contraction probably occur via the mapk pathway. to investigate the signal transduction pathway involved, we studied the effects of atp and utp on mapk phosphorylation in isolated neonatal rat cardiac myocytes using phosphorylation-specific antibodies. 100 µm atp as well as utp transiently increased phosphorylation of erk 1/2 and p38 mapk with a maximum effect at 5 to 10 minutes after application of atp and utp in neonatal cardiac myocytes (n=3 preparations each). the maximum phosphorylation of p38 increased with atp to 284% ± 68% (p<0.05) at 10 minutes and with utp up to 204% ± 21% (p<0.05) at 5 minutes. the phosphorylation with erk 1/2 mapk increased with atp to 234% ± 46.5% (p<0.05) at 5 minutes and to 337% ± 27% (p<0.05) with utp at 10 minutes of basal values, respectively. after 20 minutes, predrug values of mapk phosphorylation were reached again. in summary, we noted an atp-and utp-induced phosphorylation of erk 1/2 and p38 mapk in isolated neonatal rat cardiac myocytes. the involved receptor subtype(s) and the link between mapk phosphorylation and inotropic effect of atp and utp need to be elucidated. hameel: use of elearning in teaching pharmacology and toxicology -the halle experience rulf k. 1 , gergs u. introduction: during the past three years, our faculty has started to integrate items of elearning into the standard curriculum of a classical medical school: the "hallesches medizinisches elearning -hameel". our hypothesis was that these new elearning tools would improve the willingness of students to spend more time into learning and this would lead to an improved outcome (in multiple choice tests). methods: hence, we offered medical students (5 th or 10 th semester) additional learning environments. the courses for students (experimental pharmacology and toxicology or clinical pharmacology) were existed of a weekly lecture and in addition tutorials (problem-based-learning style, paper cases) or classical seminars. furthermore, we offered the possibility to use an online multiple choice quiz (involving 8 -12 previously used tests) and/or an online module on heart failure each week. we used the learning management system ilias software in combination with the content management system stud.ip. all students were subjected to an introductory test (to assess knowledge prior to our teaching section and allowing us to exclude a conceivable bias due to previous knowledge, involving basic items from prior teaching opportunities), a mid-term test and a final test to assess gain of knowledge. a maximum of 60 points could be obtained as a sum of both tests. results: in the means 40% of students used the new elearning tools (quizzes, heart failure module). however, there was no association between the use of self-assessment quizzes and examination results. the usage of the online quizzes increased in the periods before the exams. however, usage of the heart failure module was accompanied by significantly increased scores in exams. moreover, in a formalized evaluation system, students positively commented on our elearning efforts. conclusions: while usage of our quizzes did not improve test marks, another more sophisticated clinically oriented elearning module seemed to be improving test outcomes marginally. targeting of erk thr188 phosphorylation attenuates cardiac hypertrophy but preserves the anti-apoptotic effects of erk1/2 ruppert c., vidal m., lohse m. j., lorenz k. institut für pharmakologie und toxikologie pharmakologie, versbacher str. 9, 97078 würzburg, germany background and aims: the extracellular regulated kinases 1 and 2 (erk1/2) play an important role in cardiac hypertrophy and cell survival. erk1/2 are phosphorylated at the so-called tey motif, which in turn activates erk1/2. hypertrophic stimuli lead to an additional autophosphorylation threonine188 (thr188). this autophosphorylation of erk1/2 stimulates activation of nuclear erk targets, which are known to induce hypertrophy. the aim of this study is to investigate whether specific targeting of erk thr188 phosphorylation affects both erk functions -erk mediated hypertrophy and cardioprotective cell survival. methods and results: for the analysis of cardiomyocyte hypertrophy in vitro, we stimulated cardiomyocytes with phenylephrine and measured the incorporation of tritiated isoleucine. cardiac hypertrophy was assessed by echocardiography before and after transverse aortic constriction (tac). for analysis of cell survival, caspase activity and dna fragmentation was determined upon hydrogen peroxide stimulation in vitro and in response to tac in vivo. to differentiate between inhibition of erk1/2 activity and prevention of erk thr188 phosphorylation, we either inhibited erk activity with pd98059 or overexpressed a mutant of erk2, which cannot be phosphorylated at thr188 phosphorylation. while inhibition of overall erk activity with pd98059 attenuated cell survival and hypertrophy in vitro, specific targeting of erk thr188 phosphorylation by overexpression of the phosphorylation deficient mutant (erk2 t188a ) attentuated phenylephrine induced hypertrophy, but preserved the anti-apoptotic effects of erk. cardiac overexpression of erk2 t188a significantly reduced tac-induced hypertrophy compared to wild-type erk2 overexpressing mice. in line with the in vitro experiments, erk thr188 inhibition only prevented hypertrophy in the tac model without promoting apoptosis. conclusions: these results show that blockade of erk thr188 phosphorylation attenuates cardiomyocyte hypertrophy but preserves anti-apoptotic effects of erk1/2. therefore, specific targeting of erk thr188 phosphorylation might be a promising strategy for the treatment of pathological hypertrophy. intracellular camp levels are determined by interplay of camp formation by adenylyl cyclases and camp degradation by phosphodiesterases (pde). eleven families of pdes are known. one of the most recently identified pdes is pde10, a pde in principle capable of hydrolysing camp as well as cgmp. pde10 contains a tandem of so called gaf domains in its n-terminal regulatory domain that mediate activation by camp. because current knowledge about the tissue distribution of pde10 was mostly based on the analysis of mrna distribution, we generated antisera against pde10 to analyze tissue distribution of the protein level. using these antibodies, we found a prominent occurrence of the enzyme in testis and in brain, where it was confined to the striatum. thus, pde10 displays a comparably restricted tissue distribution which is in contrast to that of many other pdes. low camp levels in so called medium spiny neurons of the striatum have been implicated in schizophrenia. furthermore, studies using the nonspecific pde10 inhibitor papaverine as well as specific pde10 inhibitors suggest pde10 as a target for the treatment of schizophrenia. here we set out to analyze the contribution of pde10 to camp degradation in striatum, to identify the physiological pathways pde10 is involved in and to clarify the functional impact of the proposed phosphorylation of the enzyme. identification of cgmp-dependent kinase i substrate complexes salb k., schlossmann j. universität regensburg lehrstuhl pharmakologie, universitätsstrasse 31, 93053 regensburg, germany the cgmp-dependent kinases (cgks) are components of the no/cgmp/cgk-signalling pathway and have a great physiological importance in a multitude of tissues and organs such as smooth muscles and platelets. two isoforms of the cgki and the cgkii are known. cgkiα and cgkiβ differ only in their first ~ 100 amino acids which constitute the leucine zipper and the autoinhibitory domains. the n-terminal leucine zipper domains mediate homodimerization of the kinase and the interaction with diverse substrate proteins. since cgkiα and cgkiβ express different n-termini they interact with different substrates. the cgkiβ isoform is assembled in a macrocomplex at the endoplasmic reticulum (er) with the intracellular calcium release channel inositoltrisphosphate receptor i (insp 3r-i) and the inositol-trisphosphate receptor associated cgmp kinase substrate (irag). we investigated, whether irag also interacts with the insp3r-ii and the insp3r-iii in murine platelets and tissues. additionally, we analyzed the interaction between the 52 amino acid peptide phospholamban (plb), which is also located at the er and regulates the er calcium reuptake by the sarco/endoplasmic reticulum ca 2+ -atpase (serca), and the two cgki isoforms. we performed cgmp-agarose experiments with murine wt and irag-ko platelets to examine the irag-insp3r interactions. the insp3r-ii isoform was neither bound to cgmp-agarose nor detected in the anti-irag immunoprecipitate. on the other hand, insp3r-iii from wt but not from irag-ko platelets was bound to cgmp-agarose. hence, insp3r-iii interacts directly with irag but not with cgkiβ in murine platelets. however, in colon smooth muscle lysate, insp3r-iii not only interacted with the irag protein but was also detected in the anti-cgkiα-immunoprecipitate. phospholamban from wt and irag-ko platelets was also bound to cgmp-agarose. subsequent immunoprecipitation experiments with the respective antibodies against the two cgki isoforms revealed that plb interacted both with cgkiβ and cgkiα. these results were supported by analysis of colon smooth muscle tissue from wt and irag-ko mice. in conclusion, irag interacts with insp3r-i and insp3r-iii but not with insp3r-ii in murine platelets and colon smooth muscle tissue. moreover, phospholamban is an interacting partner of both the cgkiα and the cgkiβ isoform. the human immunodeficiency virus type 1 enhancer binding protein 1 (hivep1) is regulated by proinflammatory stimuli and statins salomon a. 1, 2 , schmitz b. objective: hivep1 binds nf-ĸb and other proinflammatory consensus sequences, and is suggested to be involved in inflammatory processes. we recently identified two tagging snps, one positioned 90 kb upstream (rs169713) and another in exon 4 (rs2228220) of the hivep1 gene, to be replicatively associated with venous thrombosis in gwas and follow-up studies (ajhg, 2010; plos one, 2011) . methods: total rna isolation was performed after treatment of vascular endothelial cells (ea.hy926) with proinflammatory cytokines or statins (24h). serial hivep1 promoter deletion constructs were cloned into the pgl3-basic vector, a potential enhancer fragment, harbouring rs169713c/t, into the pgl3-promoter vector. in ea.hy926 cells and thp1 monocytes, reporter gene assays were performed by transient transfection and overexpression of transcription factors. chip and bandshift assays were performed to identify candidate transcription factors. results: in ea.hy926 cells, endogenous hivep1 expression was increased by proinflammatory cytokines tnfα and il-1β. simvastatin (1.2 and 2.4 µm) and atorvastatin (9 µm) -but not pravastatin or aspirin -both dose-dependently decreased basal and tnfα-stimulated hivep1 expression. the construct harbouring rs169713t exerted significantly higher transcriptional activity (ta) compared to rs169713c (p<0.001). for an intronic modulator, reporter gene assays demonstrated a regulatory effect on hivep1 expression in ea.hy926 and thp1 cells. cotransfection of sp1 and egr1 led to an increase in ta, while wt1 exclusively upregulated ta of constructs comprising the intronic modulator. chip and bandshift assays combined with specific antibody detection revealed binding of sp1 to the 5'-flanking region and the intronic modulator of hivep1. conclusion: increased hivep1 expression during inflammatory conditions can be repressed by simvastatin and atorvastatin, and not by pravastatin or aspirin. basal hivep1 expression is regulated by sp1 combined in a transcription factor module with egr1 and wt1 under basal and/or inflammatory conditions. the rs169713 site harbours potential activational capacity for hivep1 gene transcription and may communicate with the sp1/egr1/wt1 module. to date, the treatment of various movement disorders of the central nervous system is still insufficient. in most cases this is due to the sparse knowledge of the pathophysiology. l-dopa-induced dyskinesias (lid) represent a severe complication of long-time pharmacotherapy in parkinson's disease that deserves novel therapeutics. an increased activity of striatal projection neurons, which express kv7.2/3 channels, seems to be involved in the pathophysiology of these spontaneous involuntary dystonic and choreatic movements. previous studies demonstrated an antidyskinetic effect of the kv7.2-7.5 channel opener retigabine after acute and chronic treatment in a rat model of lid. in order to clarify if this effect was based on the modulation of kv7.2/3 channels, we examined the acute effects of the preferred kv7.2/3 channel opener ica 27243 on lid in this animal model. four weeks post 6-ohda lesioning of the left forebrain bundle, dyskinesia was induced by chronic treatment with 10 mg/kg l-dopa and 15 mg/kg benserazide for 20 days. three subtypes of dyskinesia (limb, axial and orolingual) were rated according to a score system from 0 to 4 over 180 min. for drug testing, ica 27243 (5, 10 and 15 mg/kg) was administered intraperitoneal additionally to l-dopa (or vehicle). effects of drug action in comparison to vehicle controls were detected by adding up the severity scores of each observation time. additionally, effects on parkinsonian symptoms were examined 20 min after drug administration using the block and the stepping test. ica 24273 reduced the severity of dyskinesia significantly at all doses while no negative impact on the antiparkinsonian effect of l-dopa was observed. whereas the antidyskinetic effect was restricted to the first 20 min after the application of 5 mg, it lasted up to 110 min in rats treated with 10 mg ica 27243. a higher dose of 15 mg did not further enhance the antidyskinetic effect. the results of our study suggest that the antidyskinetic effect of the k v7 channel opener retigabine was based on its action on striatal kv7.2/3 channels. in line with the results of previous studies with retigabine, this action does not seem to interfere with the antiparkinsonian effect of l-dopa. this study was supported by the micheal j. fox foundation. background: skeletal muscle toxicity is the major side effect of hmg-coa-reductase inhibitors (statins) and can be simulated in engineered skeletal muscle. statins are known to exert "pleiotropic" effects, e.g. reducing endothelial dysfunction by inducing no synthases and no production. the role of no synthases in skeletal muscle under statin treatment is largely unknown. interestingly, some skeletal muscle pathologies (e.g. duchenne muscular dystrophy) may be exacerbated by increased inos activity. here we tested whether or not statin-induced skeletal muscle toxicity would be associated with enhanced no synthesis. we generated engineered skeletal muscle (esm) from rat skeletal muscle cells, matrigel and collagen. esms displayed typical skeletal muscle properties (differentiated muscle fibres, tetanic contractions). under baseline conditions esm expressed enos most abundantly, followed by inos and nnos (n=4-5). myotoxic cerivastatin (0.01, 0.1, 1 µm for 5 days) caused a concentration-dependent decrease of contractile force (p<0,05, n=17-20) paralleled by an increase in inos transcript (mean±sem: 0.01 µm 4±0.9-fold, n=3 p<0.05; 0.1 µm 9.3±2.8-fold, n=3 p<0.05) and protein (0.01 µm 5.1±2-fold, n=4 p<0.05; 0.1 µm 7.8±0.8-fold, n=3 p<0.05). mevalonic acid fully prevented the inos increase suggesting that the induction is hmg-coa reductase-dependent. to test whether inos may contribute to the decrease in contractile force we co-treated esm with 1400w, a specific inos inhibitor. we applied 5 µm of 1400w, a concentration found to potently reduce lipopolysaccharide (lps)induced no-production in cultured myotubes. however, we did not observe a rescue effect (n=9-15). also, l-name (10 mm), an unspecific nos inhibitor, did not improve contractile function, instead we observed increased myotoxictiy (n=6-13, p<0.05). to further investigate the role of no for muscle function we treated the esms with increasing concentrations of the no-donor snp. only high concentrations of snp (10 µm) caused a reduction of contractile force. combined treatment with cerivastatin and 0.1 µm snp showed a tendency towards improved force development in esm. conclusions: statins increase inos activity in our skeletal muscle model (esm). however, this does not seem to functionally contribute to myopathy in esm. increased production of no may in fact be a protective measure. esm may help to dissect clinically relevant functional changes in statin myotoxicity. characterization of primary skin fibroblasts of patients with 3m syndrome and mutations in the cul7 gene meyer k., hieber m., engelhardt s., sarikas a. technische universität münchen institut für pharmakologie und toxikologie, biedersteiner str. 29, 80802 münchen, germany 3m syndrome is an autosomal-recessive disorder characterized by pre-and postnatal growth retardation (< -4 sd), facial dysmorphism and skeletal anomalies. the majority of patients harbor missense mutations of the cul7 (76%) or obsl1 (16%) gene, respectively. cul7 constitutes an e3 ubiquitin ligase that is involved in the regulation of the insulin-like growth factor 1 (igf-1) signaling pathway via ubiquitin mediated degradation of insulin receptor substrate 1 (irs-1). to investigate the role of cul7 mediated irs-1 degradation in the pathogenesis of 3m syndrome. primary skin fibroblasts of seven 3m syndrome patients (six with cul7 mutations, one with a obsl1 mutation) and control fibroblasts were analyzed for proliferation rate (cell counter), cell cycle profile (facs), cell morphology and cellular senescence (histochemistry), irs-1 protein concentrations and activation of the igf-1 signaling pathway (western blot). the proliferation rate of 3m patient fibroblasts was significantly increased when compared to control cells. in contrast, irs-1 protein levels and activation of the pi3k/akt and erk mapk pathway were only increased in a subset of 3m cells that carried cul7 mutations, but not in cells from a patient with the obsl1 mutation. no significant differences in cell cycle profile, cell morphology or cellular senescence were observed in 3m patient fibroblasts when compared to control cells. to determine the pathogenetic contribution of increased irs-1 levels to the observed phenotype, human imr90 fibroblasts were stably transfected with retroviral vectors encoding irs-1. despite 20-fold overexpression of irs-1 compared to empty vector controls, no significant effect of igf-1 stimulation on proliferation rate or pi3k/akt and erk mapk signaling was observed. skin fibroblasts of 3m patients with cul7 mutations displayed an increased proliferation rate and enhanced activation of the igf-1 signaling pathways. despite accumulation of irs-1 in fibroblasts from a subset of 3m patients with cul7 mutations, no pathomechanistic role for irs-1 could be demonstrated. collectively, our data indicate that a dysregulated igf-1 signaling may contribute to the pathogenesis of 3m syndrome, yet in an irs-1 independent manner. pharmacases.de -a student-centered elearning project of clinical pharmacology zollner b., berg c., gros n., muß n., oestreicher d., engelhardt s., sarikas a. technische universität münchen institut für pharmakologie und toxikologie, biedersteiner str. 29, 80802 münchen, germany pharmacases.de is a novel e-learning website of clinical pharmacology that presents clinically relevant aspects of pharmacology and toxicology in an interactive and multimedial manner. the aim of the project pharmacases.de was to develop an innovative concept for creating high quality elearning content that i) integrates and promotes the theoretical and cooperative skills of final year medical students and ii) is easily adoptable by cooperating institutes and hospitals. a peer-teaching concept was developed in which final year medical students with the elective pharmacology (pj wahlfach pharmakologie) independently researched and wrote elearning lessions ("pharmacases"). subject-specific expertise was acquired by consulting elective students of other disciplines. at present (11/2011) , this "peer network" consists of elective students of nine cooperating institutions (pathology, microbiology, radiology, cardiology, psychiatry, dermatology, neurology, ophthalmology, pediatrics) at the technische universität münchen. the average time for the generation of one elearning lession by the peer network was 10 days. to date, the website consists of 49 pharmacases that are available to all students online (http://www.pharmacases.de). the website also contains a discussion forum and evaluation form for direct feedback. on average, pharmacases.de has 1000 visitors per month with the following evaluation results: "excellent": 76%, "good": 15% and "satisfactory": 9% (n=33). the didactic concept of pharmacases.de enabled the efficient generation of high quality elearning content in a student-centered and interdisciplinary manner. the peer-teaching approach supports the collaborative skills of final year medical students and facilitates the transfer of theoretical pharmacological knowledge into clinical practice. improved glucose tolerance, less chronic adipose tissue inflammation and reduced adipose tissue mass in mice with adipocyte-specific loss of tak1 sassmann a., offermanns s., wettschureck n. max-planck-institut für herz-und lungenforschung pharmakologie, ludwigstr. 43, 61231 bad nauheim, germany tgf-β activated kinase 1 (tak1) is known to be involved in numerous inflammatory processes by linking receptors for inflammatory stimuli like lps, interleukin-1 or tnfa to ikk, p38 and jnk activation. chronic inflammation of white adipose tissue is one of the major causes for the development of insulin resistance and impaired glucose tolerance in states of obesity. to investigate the role of tak1 in white adipose tissue, we crossed the tamoxifen-inducible white adipocyte-specific cre mouse line adipoqcreer t2 with animals carrying floxed alleles of the tak1 gene. adipoqcreer ; tak1 fl/fl animals and cre negative control littermates are viable and fertile and do not show any developmental defects. after tamoxifen induction and high fat diet feeding adipocytespecific tak1 knockout mice show improved glucose tolerance and lower fasting insulin levels compared to control animals. in line with this, serum levels of the adipose tissuespecific hormone resistin are reduced in adipocyte-specific tak1 knockout mice. these findings are accompanied by a lower state of chronic inflammation of adipose tissue as indicated by a dramatic reduction of adipose tissue macrophage number and lower serum levels of tnfα and interleukin-6. stimuli like tnfα, interleukins and tgf-β released from macrophages and adipocytes are known to promote obesity-related adipose tissue inflammation. when stimulated with these substances tak1 deficient adipocytes show reduced activation of jnk and p38 which both play an important role in the development of insulin resistance. interestingly, we observe a lean phenotype in adipocyte-specific tak1 knockout mice when fed a high fat diet which reflects a reduction of white adipose tissue mass. currently we are investigating the molecular mechanisms underlying the reduced adiposity and lower state of chronic inflammation in adipose tissue. growth of small cell lung cancer (sclc) cells is regulated via the autocrine stimulation of g protein coupled receptors (gpcrs), i. e., neuropeptide and muscarinic acetyl choline (ach) receptors. the activation of gq/11 and calcium-dependent gpcr pathways results in the stimulation of erk signaling which is necessary for the mitogenic effects of neuropeptides or ach on sclc cells. in contrast, the role of calcium-independent gpcr signaling and its interplay with gq/11-regulated pathways in sclc cells are less well defined. the aim of our studies was to characterize the molecular make-up and the interaction of these pathways, and to delineate the phenotypic effects of calciumdependent and -independent signaling cascades in sclc cells. using a panel of sclc cell lines, we found that the stimulation of neuropeptide receptors led to an increase of calcium which was independent of extracellular calcium and could be prevented by depleting internal calcium stores. this calcium increase was sufficient to activate the tyrosine kinase pyk2 and subsequently the erk1/2 cascade. the role of pyk2 for the growth of sclc cells was further supported by the fact that inhibition of pyk2 using a sirna approach or a novel specific inhibitor, pf431396, exerted pronounced cytotoxic effects on sclc cells, whereas non-sclc cells were less sensitive. interestingly, the inhibition of g 12/13 signaling by sirna-mediated g(alpha)12 or g(alpha)13 knockdown also markedly reduced the growth of sclc in vitro or in subcutaneous tumor xenografts, and increased the sensitivity of sclc cells towards certain cytostatics. to further define the role of calcium-dependent signaling via pyk2 versus the role of calcium-independent signaling via g12/13, we tested the effect of pyk2 inhibition in cells with impaired g12/13 signaling. notably, pyk2 and g12/13 double inhibition led to an even increased proliferation. thus, we propose that dysbalanced g protein signaling favoring either pyk2 activation or g12/13-dependent cascades inhibits the growth of sclc cells, whereas the parallel inhibition of both pathways restores again the balance and the growth capacity in this tumor entity. dendritic cells (dcs) are essential for the initial immune response and for the defence against inhalated pulmonary toxins and carcinogens in lung. to differentiate dcs, the cell line thp-1 were used for 7 days and stimulated with various cytokines (il-4, gm-csf, tnf-a, ionomycin). the dcs were characterized by flow cytometry with different typical dendritic cell markers (for example cd11c, cd209, cd83) and by immunfluorescence compared to monocytes. the bronchial tract contains up to 800 dcs per mm² and therefore we established a triple culture model to mimic the situation in vivo. the triple culture consists out of primary human epithelial cells from small bronchi (hbec) and lung fibroblasts which are cultured under air-liquid conditions on filter membranes for 4 weeks and dcs which were added after the differentiation phase of the bronchial cells. during the cultivation time the hbec formed an epithelial layer expressing both tight and adherens junctions. they also produced mucus, formed functional cilia with a beat frequency of between 16 to 20 hz and the transepithelial resistance values were stable between 600 to 800 ω·cm². pathomechanisms of pulmonary toxicity in vivo are difficult to investigate, so the tripleculture model is the basis for investigations of the toxic effects at cellular level. lungtoxic substances such as organophosphates are usually absorbed through inhalation. organophosphates are dangerous nerve agents for the human organism. at high concentrations organophosphates damage in the coculture without dcs the cell-cell contacts of the epithelial layer. in the triple culture dcs firstly respond to inhaled organophosphates and seem to compensate effects on the other cells. in summary, it is very important to understand the pathogenic mechanisms of lung injury in relation to the role of dendritic cells in lung. they could play an essential role in therapy against damage of organophosphates in the lung. co-purification of arf gtpase-activating protein git1 and cavb3 schalkowsky p., wissenbach u., fecher-trost c., flockerzi v. universität des saarlandes institut für experimentelle und klinische pharmakologie und toxikologie, kirrbergerstraße, 66421 homburg, germany high-voltage activated ca channels are assembled from pore-forming α1 subunits and two distinct types of auxiliary subunits, cavβ1-β4 and, maybe, α2δ1-δ4. by a cavβ3-specific antibody based affinity chromatography the cavβ3 protein was highly enriched from rat brain microsomal membranes. proteins associated with cavβ3 were identified by mass spectrometry (lc-esi-ms/ms) and include α1-subunits, α2δ-subunits and β-subunits. in addition to these expected interacting proteins additional proteins were co-purified with the cavβ3 protein, including the g protein-coupled receptor kinase-interactor 1 (git1). the 770aa git1 is a ubiquitously expressed multidomain protein which may serve as a scaffold to bring together molecules to form signaling modules controlling, for example, vesicle trafficking, cytoskeletal organization and cell migration. in rat brain lysates the git1 and cavβ3 proteins were co-immunoprecipitated by the antibodies for cavβ3 and git, respectively. we cloned the git1 cdna from mouse brain and co-expressed it with the cavβ3 subunit in hek cells. like in brain lysates the git protein was retained by cavβ3 precipitated by the antibody for cavβ3 and cavβ3 was retained by the git1protein precipitated by the antibody for git1. both proteins, cavβ3 and git1 are endogenously co-expressed in mouse embryonic fibroblasts (mef). we could not observe potassium-induced voltage-activated ca influx in these acutely prepared cells. accordingly, mefs can be used as a model system to study the impact of cavβ3-git1 interaction in the absence of functional cav channels. in addition, using mefs from cavβ3-deficient mice enables us to control the impact of cavβ3 on git1 function. vice versa down-regulation of git1 by specific sirnas might allow to control the impact of git1 on cavβ3 function. as read-outs we use cell migration assays and monitor receptor-dependent and receptor-independent calcium signaling in these cells. effects of sphingosine-1-phosphate and fty720 on epidermal hyperproliferation and inflammation in an imiquimod induced mouse model of psoriasis the sphingolipid sphingosine 1-phosphate (s1p) is a mediator that modulates various physiological functions of skin cells. s1p has distinct direct effects on keratinocytes as it diminishes proliferation and induces differentiation which is a classical goal of psoriasis therapy. furthermore, s1p modulates the function of various immune cells, mainly to an anti-inflammatory direction. thus, the strategy of targeting immune cells with locally acting s1p was explored in an experimental animal model of psoriasis vulgaris, the recently established imiquimod induced psoriasis mouse model and in the mouse tail test. topical administration of imiquimod onto back and ear skin led to a distinct inflammatory response characterized by epidermal hyperproliferation, scaling and redness which was scored with a modified pasi (psoriasis area and severity index). the positive control diflorasone diacetate and s1p, but not fty720 reduced the epidermal hyperproliferation by topical administration onto ear skin, indicating a mode of action for s1p via the s1p2 receptor, which is not activated by fty720. there was also a moderate reduction of inflammatory cell influx and edema formation in ear skin by s1p treatment, which was even more pronounced by treatment with diflorasone diacetate. the pasi determined on back skin was, however, only significantly reduced by diflorasone diacetate. the discrepancy between outcome on ear and back skin remains elusive. in the mouse tail assay, the influence of s1p in stratum granulosum formation (orthokeratosis) was tested compared to the positive control calcipotriol. whereas topical administration of calcipotriol led to the expected significant increase of stratum granulosum in mouse tail epidermis, s1p lacked such an effect, indicating a different mode of action in epidermal differentiation. taken together, these results imply that topical administration of s1p might be a new option for the treatment of mild to moderate psoriasis lesions. inhalation of toxicants such as sulphur mustard (sm), an alkylating chemical warfare agent, cause pulmonary complications like respiratory failure, pulmonary edema and secondary pneumonia. in order to investigate pathomechanisms of pulmonary toxicity, an in vitro alveolar-capillary co-culture model has been established recently by our group. in this model the human lung adenocarcinoma epithelial cell line (h441) is mimicking the epithelial site of the alveoli while the human hemangiosarcoma cell line (iso-has) represents the endothelial site. acute respiratory injuries are accompanied by disruption of the alveolar-capillary barrier that can be detected by the use of biochemical markers (e.g. ldh) and electrochemical indicators (e.g. transepithelial resistance). sm-mediated pulmonary injury is characterized by the increased secretion of proinflammatory mediators (e.g. il-6). a shortcoming of this model is the missing inflammatory component in the lung. aim of the present project is the addition of macrophages to the established co-culture model to improve the model and to investigate the relevance of inflammatory processes in toxic lung injury. the effect of sm on this triple-culture model is characterized with special regard to the interaction of epithelial cells and macrophages. the human acute monocytic leukemia cell line (thp-1) was stimulated to allow differentiation into macrophages. validation of the cellular differentiation was checked by specific clusters of differentiation (e.g. cd206) using flow cytometric analysis. after successful differentiation into macrophages, these inflammatory cells were added to the co-culture model before and after exposure with sm, respectively. the cytotoxicity of sm on the triple-culture model was evaluated by xtt assays and ter measurements. furthermore, immunohistochemical staining of tight junction proteins (e.g. zo-1) and of adherens junction proteins (e.g. e-cadherin) was conducted to enhance the knowledge of the function of the intercellular junction in injured and rejuvenated regions as well as the interaction of epithelial cells and macrophages. for the contact allergen para-phenylenediamine (ppd) we showed that concentrations above 50 µm are accompanied with inhibition of nat1 activity in human keratinocytes [1] . in the following we investigated the impact of ppd on nat1 activity in antigenpresenting cells using dendritic cell-like cells, namely the monocytic thp-1 cells. measured nat1 activity of thp-1 was comparable to those found in primary keratinocytes. a 24h treatment of thp-1 cells with physiologically relevant concentrations of ppd (10-200 µm) led to a 47% reduction of nat1 activity. comparable results were found for mono-acetylated ppd (mappd) whereas di-acetylated ppd demonstrated no inhibition. time-dependent studies found a significant decrease in enzyme activity already 8h after application of ppd or mappd while nat1 mrna levels were not modified. these results are indicative for a substrate-dependent inhibition. further investigations concentrated on the restoration of nat1 activity after treatment with ppd or mappd. here we found that n-acetylation capacities were restored after 24h cultivation of the treated cells in fresh medium. independent of the enzymatic activity, certain compounds are known to oxidise the catalytic cysteine or form adducts with the nat1 protein. therefore we studied whether ppd and/or oxidised ppd including the trimer bandrowski´s base interact additionally with recombinant nat1 protein itself in the absence of acetyl-coenzyme a. we found that all compounds but mappd bind to nat1 protein after 2h. the greatest inhibition was found for oxidised ppd (up to 50%). due to the greater inhibition by oxidized ppd we propose that oxidation products interact with the protein whereas ppd itself modulates nat1 enzyme activity in a substrate-dependent mode of action. overall we demonstrated that ppd can inhibit nat1 in two different ways. the work was partially financed by federal office of public health (foph), switzerland and stiftung zur förderung begabter studierender und des wissenschaftlichen nachwuchses objective: fabry's disease is a rare progressive multisystem disorder resulting from deficiency of the lysosomal enzyme alpha-galactosidase a (gla, ec 3.2.1.22). we hypothesize that genetic gla variants, especially those in its promoter region are of pathophysiological relevance for the development and progression of fabry's disease phenotypes. this study focuses on the characterization of the gla promoter, identification of functional genetic variants and impact of transcription factor eb (tfeb), a regulator of lysosomal genes. we screened 4011 bp of the 5'-flanking region of gla in 60 patients with fabry's disease and 60 controls for genetic variants. serial promoter deletion constructs for reporter gene assays were designed and identified genetic variants were introduced by site-directed mutagenesis. constructs were transiently transfected into immortalized human kidney epithelial (ihke) cells and human vascular endothelial cells (ea.hy926) to determine transcriptional promoter activity (ta). sequencing of patients' dna revealed five genetic variants in the 5'flanking region of gla, significantly more frequent in fabry's patients compared to control group (rs2071225; rs3027580; rs3027579; rs59647857; rs3027575; all minor alleles p<0.0027). we identified two regions, a proximal one between -110 and -425 and a distal region between 1106 and -1421 with significant ta, in both cell lines. cotransfection with tfeb activated ta of both regions significantly up to 5.3-fold (p<0.001). in ihke cells, insertion of the minor t allele (rs2071225) significantly enhanced basal ta of the proximal promoter region (p=0.0006), while insertion decreased basal ta (p<0.0001) of the distal promoter portion. the combined insertion of the minor c alleles (rs3027580; rs3027579), which were in complete linkage disequilibrium, significantly increased basal ta of the distal promoter region (p=0.0037). our results indicate that three genetic variants, overrepresented in fabry's patients, are located within transcriptionally active regions, possibly altering tf binding sites and therefore, affecting gla expression. future analysis will assess the impact of gla promoter variants and gla regulation by tfeb with respect to fabry's phenotypes. multiple sclerosis (ms) and its animal counterpart experimental autoimmune encephalomyelitis (eae) have a major inflammatory component that drives and orchestrates both diseases. ceramides (cer) are known as mediators of inflammatory processes, but until now their role in ms was not elucidated. we measured the ceramide levels in the cerebrospinal fluid of ms patients and control patients using lc-ms/ms. interestingly, the c16:0-cer levels were 1.9 fold increased in ms patients. this translates into the finding that c16:0-cer levels were also significantly elevated in the lumbar spinal cord of eae mice. the raised c16:0-cer levels in the lumbar spinal cord were caused by a transiently increased expression of ceramide synthase (cers) 6 in macrophages. nitric oxide (no) and tumor necrosis factor alpha (tnf-α) secreted by interferon gamma (infγ ) induced macrophages play an essential role in the development of ms. astonishingly, rnai experiments reveal that cers6 and its product c16:0-cer are mediators of inf-γ induced no/tnf-α release in raw macrophages. moreover, treatment of eae mice with l-cycloserine prevented the increase of c16:0-cer and of inos/tnf-α expression and caused a remission of the disease. in summary, cers6 plays a critical role in the initial phase of ms, most likely by regulating the no and tnf-α synthesis. this let us speculate, that a substance designed to inhibit cers6 and therefore to limit the inflammatory effects of c16:0-cer may represent a new drug in ms therapy. role of cgmp-dependent protein kinase i for kidney fibrosis schinner e. 1 cgmp is synthesized via nitric oxide-or natriuretic peptide-stimulated guanylyl cyclases and exhibits pleiotropic regulatory functions also in the kidney. hence, the integration of cgmp signaling via cgmp-dependent protein kinases (cgk) might play a critical role for renal physiology. both isozymes were detected in arterioles, mesangium and within the cortical interstitium. in contrast to cgkiα, the β isoform was not detected in the juxtaglomerular apparatus and medullary fibroblasts. here, we focused on the function of cgki in the renal interstitium emphasizing a functional differentiation of both isoforms. the interstitium exists mainly of fibroblasts playing a prominent role in the interstitial fibrosis. accordingly, cgki could also be involved in this pathophysiological process. therefore, we studied whether cgki influences renal fibrosis which was induced by unilateral ureter obstruction (uuo). at first we analysed the role of the no/cgmp signaling by application of cgmp increasing yc1 or isdn. thereby we detected antifibrotic effects of these substances. subsequently we tested whether these effects are mediated by cgki by using mutant mice. on the one hand we examined αsm-rescue mice (expressing cgkiα only in smooth muscle under the control of the sm22 promotor with a cgki-ko background) and cgki-ko mice (expressing no cgki). on the other hand we used tgtg mice expressing more cgkiα in smooth muscle than wt mice (transgenic cgkiα under the control of the sm22 promotor). due to the steeply increased use of nanomaterials for commercial and industrial applications, toxicological assessment of their potential harmful effects is urgently needed. moreover, the continuous development of novel materials requires the implementation of hazard-predicting models to prevent potential health effects resulting from human exposure. in the present study, we studied the toxic potential of a set of nanoparticles (np) with varying physicochemical properties in human a549 lung epithelial cells, hepg2 liver epithelial cells and hk-2 proximal tubule epithelial cells. the used nanomaterials incorporated five tio 2 samples, two zno samples (i.e. uncoated and coated), two multi-walled carbon nanotube (mw-cnt) samples and a nanoparticulate ag sample. cells were treated with np at doses ranging from 0.3 to 80 µg/cm 2 for cytotoxicity and from 06 to 40 µg/cm 2 for genotoxicity. dna damage was evaluated using the alkaline comet assay while concurrent cytotoxicity was determined by the wst-1 assay. marked contrasts in cytotoxic and dna damaging properties were observed among the different materials. the overall strongest responses were observed with the uncoated zno-np sample and with ag-np, although effects were found to depend on the cell type. notably, the dna damaging effect of ag-np could at least partly be attributed to its dispersant. present results form part of a growing data set which are generated in the framework of the eu fp7 project enpra (fp7-nmp) to establish dose-response relationships and a mathematical model to predict the hazard of nanoparticles. increased spontaneous hprt mutant frequency in v79 cells expressing human cytochrome p450 1b1 schlechtweg a., esch h. , martínez jaramillo d., lehmann l. university of wuerzburg/institute of pharmacy and food chemistry section of food chemistry, am hubland, 97074 wuerzburg, germany the hypoxanthine-guanine phosphoribosyltransferase (hprt) assay in chinese hamster v79 lung fibroblasts (v79 cells) represents a widely-used mammalian test system to detect gene mutations. since v79 cells do not express any cytochrome-p450dependent monooxygenase (cyp) isozymes, usually an activating system has to be added. therefore, v79 cells expressing human (h) cyp isozymes have been commercialized. to test these v79 cells for their use in the hprt test, v79 h1a1 and h1b1 cells were characterized regarding (i) spontaneous frequency of 6-thioguanineresistant clones per 10 6 clonable cells (smf), (ii) the stability of which over 4 weeks (w), and (iii) the mutational spectrum (ms) of cdna from mutant clones. ms of cdna was determined by isolation of total rna, reverse transcription/amplification of the coding region by polymerase chain reaction and sanger sequencing of the amplification product. activity of cyp isozymes was verified by ethoxyresorufin-o-deethylase (erod) assay. (i)/(ii) whereas the smf of v79 cells (w2:13±4; w4:2±1) and v79 h1a1 (w2:17±4; w4:3±0) only varied within the range of historical controls, smf of v79 h1b1 increased continuously over time (w2: 36±6; w4: 68±13). (iii) although the smf of v79 and v79 h1a1 were similar, the mutational spectrum of v79 cells was characterized by as many transversions as transitions and deletions of exon 4 or exon 4+5, whereas the mutational spectrum of v79 h1a1 was characterized exclusively by transversions and deletion of exon 7+8. surprisingly, with 57 out of 59 cdnas derived from v79 h1b1 mutant clones, no amplification product was detected. first results indicate that there is at least one gene mutation in the untranslated region before and behind the coding region precluding amplification with the original primers. (ii)to reduce the smf of v79 h1b1, cells with wildtype hprt activity were cloned and one clone with an erod activity which did not differ significantly from the original cell population was further characterized. initially, smf of the clone varied between 0.7±0.6 and 1.2±0.0. yet its smf was unstable reaching up to 104±6. in conclusion, the mutational spectrum differed between the v79 cell lines. furthermore, h1b1 expression seemed to enhance smf in v79 cells. even though a temporary reduction of the smf by cloning was possible, smf of v79 h1b1 cells was unstable. we wanted to investigate the possible antithrombotic effects and elucidate the chemical identity of the active principles involved in inhibitory effects against adp-induced aggregation of human platelets by wild garlic, allium ursinum l. method: bioassay-guided isolation procedure was used followed by spectrometric identification of pure active compounds. for the bioassay, blood was taken from healthy human volunteers and platelet rich plasma (prp) was prepared for turbidimetric platelet aggregation tests. prp, stimulated with 20µm adp, was treated with extracts of different polarities, fractions and isolated single compounds from allium ursinum. the extracts were investigated by thin layer chromatography, hplc, mass spectroscopy, esi-ms and 1d/2d 1h/13c-nmr spectroscopic techniques. for references the adt-antagonist mes-amp was used. result: fresh allium ursinum leaves were extracted with ethanol, which was the potent form that effectively inhibited adp-induced aggregation of human platelets. this ethanolic extract was subjected to liquid-liquid partition. whilst the aqueous phase containing the moiety of cysteine sulphoxide and thiosulphinate derivatives showed only weak activity on platelet aggregation, the ethyl acetate and especially the chloroform partitions showed highest aggregation inhibiting potency. thus, in our bioassay effects of alliins/allicins could be neglected. the chloroform phase, possessing the strongest activity, was separated into 28 fractions by gradient elution open cc on silica gel. the most active fractions 11-17 were separated again yielding 10 subfractions. this afforded 1,2-di-o-α-linolenoyl-3-o-β-d-galactopyranosyl-sn-glycerol and β-sitosterol-3-o-β-dglucopyranoside, the structures of which were determined by esi-ms and 1d/2d 1h/13c-nmr spectroscopic techniques. furthermore, the diminutive amounts of volatile oil of a.ursinum leaves obtained by steam distillation according to ph.eur. could be evaluated as a third aggregation inhibiting principle. conclusions: at the first time two active, non-sulphur-containing constituents of wild garlic, namely a galactolipid and a phytosterol, could be identified exhibiting inhibitory action on adp-induced aggregation in human blood platelets. as a major constituent, the galactolipid 1,2-di-o-α-linolenoyl-3-o-β-d-galactopyranosyl-sn-glycerol, not yet found in allium spec., appears as a new, highly useful marker substance for a.ursinum drugs, or their pharmaceutical preparations. in recent years, public attention focused more and more on risk factors which may impair sperm quality and thereby human reproduction. in this context, for example pesticides, alcohol, cigarettes, and even mobile phones are discussed. a variety of parameters exists including sperm counts as well as sperm motility, which are considered to be two of the most important parameters to evaluate sperm quality in animal models with the final aim to assess human risk. in recent years computer assisted sperm analysis (casa) devices mostly replaced the formerly used manual counting and manual motility assessment. however, although casa offers multiple opportunities and can allow for an objective and more detailed evaluation, several pitfalls exist which can alter the results profoundly and consequently compromise the quality of the data and ultimately the validity of a study. the aim of the present study was to establish and validate the casa device tox ivos sperm analyzer from hamilton thorne and thereby to gain detailed knowledge about the practical advantages but also intricacies which may alter the obtained results. in this regard healthy adult male rats (10-12 weeks old) were used. ultrasonic sound resistant sperm heads were isolated from the testis and in addition, sperms were isolated from the cauda epididymis. testicular sperm head counts and sperm motility were assessed using different isolation procedures and/or instrument settings. results different instrument settings modulate both -sperm motility and testicular sperm counts. in this regard, a wide range of results including slight changes as well as false positive/negative results were obtained. in addition, the modification of the isolation procedure can lead to variable results especially for sperm motility. conclusion isolation procedures as well as instrument settings can alter the results. consequently, in an experimental setting, potential adverse effects can be confounded with methodologically mediated apparent findings exerted via inappropriate use of the device -depending on the respective conditions in the test laboratory. this study demonstrates the relevance of standardization of testing conditions adopted for computer assisted sperm analysis and the need for a robust validation prior to use in experimental settings. orai and stim proteins have been identified as central components of the highly ca 2+ selective, store-operated current in immune cells (icrac). the molecular basis of selective orai-mediated activation of the calcineurin/nfat pathway and the crosstalk with other channel and scaffold molecules of the trpc family are still incompletely understood. using patch clamp recordings complemented by fluorescence and tirf microscopy we investigated interactions between orai1 and trpc3 in plasma membrane microdomains of rbl-2h3 mast cells. orai1-mediated crac currents, activated by passive store depletion, were found significantly reduced by over-expression of trpc3. this negative impact of trpc3 on icrac was independent of channel function as the trpc3 pore dead mutant (e630k) inhibited icrac to a similar extent as wild type trpc3. importantly, despite a reduction in icrac, nfat translocation in trpc3 overexpressing rbl cells remained unchanged, or was even slightly promoted. store depletion-induced nfat translocation in rbl cells was as well unaffected by trpc3e630k but substantially reduced by trpc3 mutants with either i) eliminated fkbp12/calcineurin binding (p704q) or ii) deficiency in pkc phosphorylation (s712a). moreover, inhibition of pkc phosphorylation by (gfx109203x; 3 µm) strongly suppressed nfat signaling. we suggest trpc3 as a scaffold that links orai-mediated ca 2+ -entry to nfat/calcineurin signaling within plasma membrane microdomains. neurally-induced bronchoconstriction in human and guinea pig precision-cut lung slices schlepütz m. 1 , rieg a. d. introduction: precision-cut lung slices (pcls) are well suited to study peripheral airway responses in different species. airway tone is under close control of the autonomic nervous system and dysregulation may contribute to airway hyperresponsiveness as observed in human lung diseases such as asthma. hence, the aim of the present study was to characterize neurally induced bronchoconstriction (bc) in guinea pigs (gp) and to compare the results with those in human pcls. methods: pcls were prepared from gp or human lung tissue. nerve endings in pcls were activated by electric field stimulation (efs) or capsaicin addition. cholinergic nerve responses were proven by atropine. capsaicin was used to show excitatory nonadrenergic non-cholinergic (enanc) responses. ruthenium red or skf96365 were used to confirm transient receptor potential (trp) channel contributions upon enanc activation. results: gp and human pcls were both sensitive to efs and airways contracted to 39±26% of the initial airway area (%-iaa) and 63±21%-iaa, respectively. in frequency response curves half maximal response was found at 7.0±1.2 hz for guinea pig pcls and 8.1±0.8 hz for human pcls. efs-induced bc was inhibited by atropine in both species. capsaicin contracted gp to 18±15%-iaa. 60% of human pcls were responsive to capsaicin and airways contracted to 72±10%-iaa, respectively. in gp ruthenium red and skf96365 blocked capsaicin-as well as efs-induced bc. conclusion: gp and human pcls contain atropine sensitive cholinergic and capsaicin sensitive enanc nerve endings. since gp pcls were sensitive to trp channel inhibitors, the involvement of those channels can be characterized with respect to lung diseases. in conclusion, gp pcls resemble the human distal lung innervation and represent a useful model to study neural airway pharmacology. the erk1/2-pathway is involved in pkc-induced nox4 up-regulation schlufter f., xia n., förstermann u., li h. universitätsmedizin mainz institut für pharmakologie, obere zahlbacher straße 67, 55131 mainz, germany nadph oxidases (nox) are major producers of reactive oxygen species in the vascular wall and nox4 is the most abundant nox isoform in human endothelial cells. we have previously shown that treatment of human ea.hy 926 endothelial cells with phorbol 12myristate 13-acetate (pma) for 48 h leads to an up-regulation of nox4 expression. this effect of pma is mediated by protein kinase cα, because it is preventable by the pkc inhibitor gö 6983 and by pkcα-sirna. the present study is aimed to investigate the signal transduction cascade downstream of pkcα. pma-induced nox4 up-regulation can be attenuated by pd 98,059 (an erk1/2 inhibitor), but not by sp 600125 (a jnk inhibitor), indicating in the involvement of erk1/2. consistently, pma treatment leads to a sustained activation of erk1/2, and sirnamediated knockdown of erk1/2 markedly reduces the pma-induced nox4 up-regulation. h89, an inhibitor of the mitogen-and stress-activated protein kinases (msks) has no effect on the pma-stimulated nox4 expression, indicating that msks are not the target molecules of erk1/2 in this scenario. on the contrary, knockdown of the transcription factor elk-1 by sirna significantly reduces the pma-induced nox4 up-regulation. in conclusion, erk1/2 and elk-1 are involved in the pkcα-induced nox4 up-regulation. determination of spontaneous mutation frequencies in normal human mammary gland tissue using the random mutation capture technique schmalbach k., lehmann l. university of wuerzburg section of food chemistry, am hubland, 97074 wuerzburg, germany annually, over 57,000 women develop breast cancer in germany. the accumulation of genetic mutations in mammary gland tissue during lifetime may be reasonable for developing breast cancer. in particular mutations in tumor suppressor genes, e.g. p53, seem to play an important role in developing cancer. up to now, lack of a method sensitive enough to determine the expected very low spontaneous mutation frequency (smf) in normal mammary gland tissue precluded the investigation of the role of spontaneous mutations acquired in the p53 gene in epidemiological studies. the only test with the potential to determine low smfs was the random mutation capture (rmc) assay, a genotype selective method which detects mutants that render the mutational sequence non-cleavable by the taqi restriction enzyme after accumulation of the target sequence. therefore, the suitability of the rmc assay to determine smf in p53 gene in normal human mammary gland tissue was evaluated. thus, the rmc assay was optimized concerning (i) dna isolation, (ii) pcr conditions, and (iii) amount of mammary gland tissue. (i) genomic dna from normal human mammary gland tissue, obtained from healthy women who underwent mamma reduction surgery for cosmetic reasons, was isolated using an extended proteinase k digestion prior to chloroform extraction. (ii) the target sequence in intron 6 of p53 gene was captured by hybridization with a complementary uracil-containing dna-probe synthesized via polymerase chain reaction (pcr), followed by magnetic separation from the remaining genomic dna. the copy number of the target sequence was quantified by competitive pcr. the number of mutants was detected after cleavage of the target dna with taqi by means of pcr with a primer set flanking the restriction site. (iii) with 2 g of normal mammary gland tissue a smf of 2.2±1.4x10 -7 per base pair was determined indicating the rmc assay suitable for smf determination. in conclusion, the smf in the p53 gene in normal human mammary gland tissue was determined for the first time, enabling the future investigation of factors influencing the smf during breast cancer development. cigarette smoke-induced release of pro-inflammatory cytokines including interleukin-8 (il-8) from inflammatory as well as structural cells in the airways, including airway smooth muscle (asm) cells, may contribute to the development of chronic obstructive pulmonary disease (copd). despite the wide use of pharmacological treatment aimed at increasing intracellular levels of the endogenous suppressor cyclic amp (camp), little is known on its exact mechanism of action. we report here that next to the β2-agonist fenoterol, direct and specific activation of either exchange protein directly activated by camp (epac) or protein kinase a (pka) reduced cigarette smoke extract (cse)-induced il-8 mrna expression and protein release by human asm cells. cse-induced iκbαdegradation and p65 nuclear translocation, processes that were primarily reversed by epac activation. further, cse increased extracellular signal-regulated kinase (erk) phosphorylation, which was selectively reduced by pka activation. cse decreased epac1 expression, but did not affect epac2 and pka expression. importantly, epac1 expression was also reduced in lung tissue from copd patients. in conclusion, epac and pka decrease cse-induced il-8 release by human asm cells via inhibition of nf-κb and erk, respectively, pointing at these camp effectors as potential targets for antiinflammatory therapy in copd. however, cigarette smoke exposure may reduce antiinflammatory effects of camp elevating agents via down-regulation of epac1. polycyclic aromatic hydrocarbons (key marker substance benzo[a]pyrene (bap)) have been assumed to play a role in the development of bladder cancer. the objective of the present study was to unravel cellular and in particular cytoskeletal response to bap. to follow the sequential steps of chemical carcinogenesis the differential proteomic profile was analyzed at early and late time points. the study was carried out in a superficial human bladder cancer cell line (rt4) exposed to 0.5 µm bap, a subacute concentration based on results of proliferation (brdu) and dna damage (tunel) tests. cells of a human bladder cancer cell line (rt4) were exposed to 0.5 µm bap for 24 h (n=5), 4 wk (n=7) and 8 wk (n=3). proteins of whole cell lysate were separated by twodimensional electrophoresis (fig. 1) . differentially expressed proteins were identified by matrix-assisted laser desorption/ionization-time of flight analysis. cortactin, actin and tubulin were immunohistochemical stained. changes in migration and colony forming ability were assessed by scratch wound-healing assay and soft-agar colony formation. results: by using several databases (uniprot, reactome, panther) the identified proteins were categorized into different functional classes such as mrna processing, translation, protein metabolic process, or several areas associated with the organization of the cytoskeleton. 45 % of the differentially expressed proteins after 24 h of treatment are involved in the processing of pre-mrna (20 %) and protein metabolism (25 %). this pattern changed after 8 wk of treatment. then, 48 % of the proteins affected the cytoskeleton whereas still 26 % were categorized to protein metabolism and only 7 % to pre-mrna processing. in the immunhistochemical staining, the treated cells appeared after 8 wk of exposure larger and rounder predominantly due to the modifications of the actin cytoskeleton. merged images of actin and cortactin revealed that both proteins colocalized in invadopodiae. after 8 wk, a higher number of treated cells moved toward the centre of the wound and they formed more soft-agar colonies compared to vehicle conditions, suggesting a transformation of the cells. in conclusion, bap exposure causes in an early phase an activation of the spliceosome which can led to an epithelial-tomesenchymal transition. two coordinators of a cell-type-specific splicing program, epithelial splicing regulatory proteins 1 and 2, are currently being validate by pcr. fused master gel : representative 2-de gel of rt4 cells exposed to 0.5 µm bap for 8 wk. protein spots which were differentially expressed compared to control and identified were marked. cannabinoids stimulate mesenchymal stem cell migration via a mitogen-activated protein kinase pathway schmuhl e. 1 , ramer r. mesenchymal stem cells (mscs) are known to be involved in various regenerative processes such as cardiac, ocular, skin and bone tissue healing. however, little is known about the pharmacotherapeutical options aiming at tissue healing steps such as the mobilization and homing of mscs. here, we show that cannabidiol (cbd), a nonpsychoactive cannabinoid, stimulates the migration of human adipose-derived mscs in both boyden chamber and in vitro scratch wound assays. in boyden chambers cbd (0.01 -3 µm) was shown to promote cell migration in a time-and concentration dependent manner. this promigratory action was inhibited by am-630 (cb2 receptor antagonist) and by o-1602 (g protein-coupled receptor [gpr] 55 agonist). moreover, cbd activated the mitogen-activated protein kinase (mapk) pathway as evidenced by increased phosphorylation of extracellular signal-regulated kinase (erk) 1/2. blockade of erk activation by pd98059 prevented cbd-stimulated msc migration, whereas inhibition of p38 mapk by sb203580 was inactive in this respect. furthermore, am-630 and o-1602 were found to attenuate cbd-induced erk activation. an erk-dependent promigratory action was likewise demonstrated for the phytocannabinoid ∆ 9 tetrahydrocannabinol and for the hydrolysis-stable anandamide analogue r(+)methanandamide. we conclude that cbd promotes msc migration via receptordependent erk activation, possibly contributing to tissue healing. the duffy antigen receptor for chemokines (darc) binds promiscuously many inflammatory chemokines without showing intracellular signal transduction. it is mainly expressed on endothelial cells of postcapillary venules and on red blood cells, where it acts as a transendothelial transporter of chemokines and as a chemokine sink, respectively. surprisingly, as shown for human and mouse brain, darc is also expressed at high density in the cerebellum. however, nothing is known about the function of darc in this location. we addressed this question by subjecting c57bl/6 wildtype and darc-deficient mice to a series of behavior experiments including morris water maze-, elevated plus maze-, rotarod-and actometer tests. while the results from the water maze experiments are ambiguous, elevated plus maze trials show a strong aversion of darc -/mice to walk to the end of the open arm, which is consistent with anxiety-like behavior. moreover, darc -/mice show greatly reduced locomotor activity, which is at least partly caused by episodes of reduced mobility occurring more frequently than in the corresponding wildtype controls (elevated plus maze, actometer). finally, darc -/mice spend a significantly reduced time on the rotating rod compared to c57bl/6 wildtype controls, which may indicate an impaired cerebellar function. we conclude that darc in fact modulates brain function. surprisingly, this appears to be happening under homeostatic conditions, although darc binds for the most part to inflammatory chemokines. it remains to be elucidated, how this effect can be caused by a non-signaling chemokine receptor. it may be an indirect consequence of altered brain chemokine concentrations or of as yet unknown signaling pathways activated by darc. transporter gene expression in human head-neck squamous cell carcinoma and epigenetic regulation mechanisms schnepf r. 1 hals-nasen-ohren-klinik, kopf-und halschirurgie, friedrich-alexander-universität erlangen-nürnberg, waldstraße 1, 91054 erlangen, germany background: membrane transporters may affect the disposition and thereby treatment efficiency of anticancer drugs in human head-neck squamous cell carcinoma (hnscc). the gene expression profile of transporters in hnscc, however, is unknown and was evaluated in this study. moreover, we evaluated mechanisms by which transporters are regulated in hnscc. we focused on the role of the nuclear pregnane x receptors (pxr, nr1i2) and epigenetic mechanisms. methods and results: real-time rt-pcr revealed a significantly increased mrna expression of slco1a2 and slco1b3 and a significantly decreased expression of transporters such as slco2b1, slco2a1 and abcc3 in human hnscc tissue samples compared to adjacent normal mucosa. moreover, an association between slco2b1 mrna levels in tumor tissues and five-year survival of hnscc patients was observed (χ 2 =6.59; p=0.010; n=34). bisulfite sequencing revealed that promoter cpg islands of abcc3 and slco2a1 were not methylated and thus these genes were not epigenetically silenced in hnscc tissues. in the hnscc-derived umscc-1 and scc-15 cell lines, transcript expression of transporters (e.g., abcc3, slco2a1; p<0.001) and pxr (nr1i2; p<0.001) was markedly induced by the dna methyltransferase inhibitor decitabine. cotreatment with the prototypical pxr activator rifampicin significantly reversed decitabine-induced abcc3 and slco2a1 expression. conclusions: transporter expression profiles significantly differed between hnscc and normal mucosa and expression levels of slco2b1 may serve as a marker for prognosis. modulation of the epigenome with the anticancer drug decitabine substantially affects transporter expression in umscc-1 and scc-15 cells, suggesting epigenetic regulation mechanisms. moreover, interactions between epigenetic and nuclear receptor-mediated mechanisms in transporter regulation occur. this work was in part supported by the johannes und frieda marohn foundation. the role of hcn2 in neuropathic and inflammatory pain schnorr s. 1 , eberhardt m. the pacemaker current ih is carried by hyperpolarization-activated cyclic nucleotidegated cation channels (hcn1-4) and contributes to cellular excitability in the heart and the nervous system. hcn1 and hcn2 are the two most abundant hcn subunits in peripheral sensory neurons with hcn2 being the prevalent isoform in nociceptive small sized c-fibre dorsal root ganglion (drg) neurons. we examined the role of hcn2 for peripheral sensitization and spontaneous neuronal activity in neuropathic and inflammatory pain. we generated a conditional deletion of hcn2 by using a nociceptor specific cre-transgene driven by the nav1.8 promoter. the nociceptor-specific knockout of hcn2 in drg neurons (nosphcn2ko) was confirmed by quantitative rt-pcr and western blot. immunohistochemical staining revealed that the deletion of hcn2 was mainly restricted to small sized drg neurons. the conditional loss of hcn2 resulted in a significant reduction of ih positive small diameter drg neurons pointing to a central role of this isoform to the hcn current in nociceptive neurons. behavioral studies showed that the lack of hcn2 did not influence basal pain responses but led to a significant reduction in mechanosensation in both neuropathic and inflammatory pain models. however, thermosensation of the mutants was only decreased in neuropathic pain conditions. in wild-type animals, intraperitoneal, intraplantar and even intrathecal injection of the hcn channel blocker zd7288 nearly eliminated tactile allodynia caused by inflammation in contrast to thermal hyperalgesia which remained unaffected. in contrast, pain thresholds in nosphcn2ko mice did not significantly increase after pharmacological block of ih. additionally, experiments revealed that the inflammatory condition induced an upregulation of hcn2 protein in the spinal dorsal horn compared to saline injected mice. our results suggest that hcn2 might be a new target in the treatment of neuropathic and inflammatory pain. the proper functioning of the central, as well as the peripheral nervous systems is of outstanding importance to the survival and well-being of humans. yet, the integrity of neuronal systems is constantly challenged by a plethora of environmental and occupational toxins. some of these toxins preferentially target neural cells. these neurotoxins can exert their devastating effects by very different modes of action. neurotoxins may induce apoptosis or necrosis of neurons, or interfere with axon growth and elongation. these processes can be identified by specialized in vitro tests. furthermore, neurotoxins have been described to alter glial function which may compromise the viability of surrounding neurons. as another important mode of action, several neurotoxins act on neurotransmitter receptors, thereby altering signal propagation within neuronal networks. countless natural and synthetic substances have been characterized for their effects on neurotransmitter receptors and today can be used for detailed studies of receptor function. however, environmental toxins of anthropogenic origin and occupational toxins that both represent constant sources for human exposure are still poorly studied with respect to their effects on neurotransmitter receptors. thus, the need for a better understanding of the susceptibility of neurotransmitter systems for toxic effects exerted by these substances is of outstanding importance for the protection of human health. here, we introduce an imaging-based approach for the screening of the effects of potential and known neurotoxins on neurotransmitter receptors of intact cells in vitro. different neuronal cells were tested for their sensitivities for classical neurotransmitters using life-cell imaging experiments. in more detail, we examined the proportion of responding cells and determined the ec50 values for the most prominent neurotransmitters in cell lines widely used for in vitro neurotoxicity studies on the one hand, namely sh-sy5y and lumes cells, and primary mouse neurons on the other hand. with these data at hand, we are now able to identify and characterize the effects of neurotoxins on receptor function in chronic, as well as acute exposition paradigms. the use of an in vitro imaging-based physiological test system is at the interface between non-functional in vitro approaches and in vivo toxicity tests, thus, giving mechanistic insight into neurotoxic processes without requiring animal experiments. apomorphine acts on trpa1 channels scholze a., schaefer m., hill k. universität leipzig -universitätsmedizin rudolf boehm-institut für pharmakologie und toxikologie, härtelstr. 16-18, 04107 leipzig, germany apomorphine is a non-narcotic derivative of morphine which acts as a dopamine agonist and is clinically used to treat "off-states" in patients suffering from parkinson´s disease. adverse effects of apomorphine treatment include dopaminergic effects such as nausea, but also ulceration and pain at the injection site. we wanted to test whether an activation of trp (transient receptor potential) channels in sensory neurones contributes to the perception of pain after apomorphine injection. while the warm/heat receptors trpv1, trpv2, trpv3, and trpv4 and the cold receptor trpm8 were insensitive towards apomorphine treatment, trpa1 could concentration-dependently be modulated by apomorphine. low micromolar apomorphine concentrations potently activated heterologously expressed trpa1 channels in a stably transfected cell line (hek293-trpa1), as well as natively expressed trpa1 in cultured dorsal root ganglion neurones. on the other hand, when using higher concentrations of apomorphine, we observed inhibition of trpa1 activity. previous studies have shown that subcutaneously administered apomorphine produces a biphasic dose response relationship in rats, inducing hyperalgesia at low doses whereas high doses of the substance cause antinociception. from our studies we conclude that such in vivo effects of apomorphine are presumably mediated by activation/inhibition of trpa1 expressed in sensory neurones agonist binding to a g protein-coupled receptor (gpcr) induces a conformational change of the receptor protein, which results in the activation of receptor-associated heterotrimeric g proteins [1] . in radioligand binding studies, conducted to investigate ligand binding to specific gpcrs, receptors are usually probed with radioantagonists. as in other gpcrs [2] , agonists of the muscarinic m2 receptor exhibit biphasic kinetics and biphasic competition curves with radioantagonists, indicating a more complex situation probably caused by g protein interactions. here, we present a detailed study of the binding of agonists to muscarinic m2 receptors including the novel super-high affinity agonist iperoxo and a differential chemical knockout of g proteins. in addition to membrane homogenates living cells were employed. we demonstrate that the high affinity fraction in biphasic curves does not differ between selected full agonist and is sensitive to pertussis toxin, thus indicating that this receptor population is associated with gi proteins. however, despite promiscuous signalling properties of m2 receptors, the low affinity fraction is not associated with any other g protein, since low affinity binding is insensitive to high concentrations of guanylnucleotides and cholera toxin. moreover, high affinity agonist binding appears solely in membrane homogenates but not in experiments conducted with living cells, probably due to their high intracellular concentration of guanylnucleotides. taken together the chemical knock-out of g proteins revealed that the high affinity binding of agonists in membrane homogenates is associated with the interaction of the muscarinic m2 receptor with gi proteins. the low affinity binding cannot be related to another g protein, although the muscarinic m2 receptor exhibits promiscuous g protein signalling properties. interestingly data obtained with living cells do not reveal any high affinity binding of agonists. prolonged stress leads to a dysregulation of the hypothalamus-pituitary-adrenal (hpa)axis and may affect the sensitivity of pain perception. however, it is not yet known whether the alterations of hpa-axis and increased pain sensitivity are related. to create a long lasting stressful situation, male wistar rats were exposed to a restraint-stress for 1 h daily over a period of two weeks. the effect of stress on the hpa-axis was determined by adrenal morphology and stress hormone levels, the influence on mechanical pain sensitivity was evaluated by the randall-selitto paw pressure test. on day 15 the animals exhibited a significant mechanical hyperalgesia. they also showed increased acth and corticosterone plasma levels and an enlarged zona fasciculata of the adrenal gland, indicating a dysregulation of the hpa-axis. for testing the correlation of hpa-axis dysregulation and hyperalgesia a persistent increase in plasma corticosterone in wistar rats was generated by the administration of corticosterone via the drinking water for two weeks. these animals also showed an increased mechanical nociceptive sensitivity with an accompanied decrease of the adrenal glands and reduced acth levels. the results show that chronic stress leads to a dysfunction of the hpa-axis with an accompanied mechanical hyperalgesia which can be mimicked by oral administration of corticosterone. thus, this in-vivo test system may provide a new animal-friendly pharmacological model for stress-related pain disorders. the alternaria mycotoxins aoh and ame induce cyp1a1 and apoptosis in murine hepatoma cells dependent on the aryl hydrocarbon receptor mycotoxins are secondary metabolites of fungi including the genus alternaria (black mold). alternaria fungi are known to infest different types of foodstuffs and produce diverse toxins amongst them the mycotoxins alternariol (aoh) and alternariol methyl ether (ame) which are potential carcinogens. as planar compounds, aoh and ame are preferentially metabolized by cytochrome p450 (cyp) 1a1 and 1a2. the most prominent regulator of cyp1a1 is the dimeric transcription factor complex ahr/arnt, which is activated by planar ligands. therefore we studied the activation of ahr/arnt by aoh and ame and monitored cyp1a1 induction in murine hepatoma cells (hepa-1c1c7). indeed, aoh and ame enhanced the levels of cyp1a1 in hepa-1c1c7 cells but not in cells with inactivated ahr (hepa-1c1c12) or arnt (hepa-1c1c4). furthermore, we studied the cytotoxicity of aoh and ame. by using a fluorescence-based microscopic readout we measured effects on cell counts, apoptosis, senescence and micronuclei formation. both aoh and ame reduce the cell number and the cell nuclei show drastic morphological changes e.g. enlargement after aoh treatment or micronuclei formation. the observed effects where, except for the induction of apoptosis, independent of ahr/arnt. in summary, aoh and ame activate the ahr/arnt pathway to induce cyp1a1 expression and apoptosis. however, the predominant cytotoxic effect of aoh and ame in hepatoma cells is a profound reduction in cell numbers, which is independent of the ahr/arnt pathway. special purpose databases are the first place for researchers in the life sciences to obtain expert curated data. naturally, such resources are limited in terms of timeliness and comprehensiveness. the literature database pubmed alone lists more than 20,000,000 scientific abstracts, and 700,000 are newly added every year. the protein sequence database uniprotkb stores over 10,500,000 sequences, a hundred times more than ten years ago. turning these data into meaningful information and making it accessible to both humans and computers have become an essential part of biological discovery and biomedical research. text mining techniques have proven useful to extract the missing links in areas such as drug-target and drug-disease prediction, the extraction of mutation-phenotype associations, or the prediction of protein-protein and protein-ligand interactions. by systematically extracting information from available literature, reports or patents, text mining techniques can help to refine existing categorical knowledge stored in databases, and hence will support drug repositioning, the discovery of novel cancer biomarkers, or help to understand causes of hereditary diseases. in the area of regulatory toxicology we developed go3r, the first knowledge-based search engine for alternative methods to animal experiments. the system not only helps retrieving information on the availability of alternative methods that allows for replacing, reducing or refining animal experiments, but also provides an endpoint-centered literature search to all scientists and regulatory authorities seeking for toxicological information and data. the up-to-date taxonomicstructured "table of contents" provided by go3r allows for search in the literature listed in pubmed or the toxicology data network (toxnet) in a fast and comprehensive manner. the semantically enriched platform supports the user during the query formulation, allows for bibliographic analysis, and reveals existing relations to replacement, reduction, and refinement of animal experiments. impaired cardiac excitation-contraction-coupling in mice with complete inactivation of the crem gene schulte j. s., tekook m., schmitz w., müller f. u. westfälische wilhelms-universität institut für pharmakologie und toxikologie, domagkstraße 12, 48149 münster, germany the structurally related transcription factors camp response element binding protein (creb) and camp response element modulator (crem) mediate a regulation of gene transcription in response to camp and are expressed in the heart. mice with complete inactivation of crem display impaired cardiac contraction and relaxation, decreased expression of serca and down-regulation of β1-adrenoceptors. to elucidate the underlying functional mechanisms on the cellular level we here investigated cellular electrophysiology and ca 2+ -cycling in ventricular cardiomyocytes from crem ko mice (ko). adult ventricular cardiomyocytes were isolated from ko and wildtype (wt) mice (age 16-20 weeks) and subsequently used for experiments within 6 hours after isolation. action potentials (aps) were recorded from ventricular cardiomyocytes (perforated patch, whole cell current clamp inactivation of crem seems to have no consequences for ap duration and possibly associated ion channels but leads to impairment of ca 2+ cycling and sarcomere shortening under basal conditions well explaining the previous findings in vivo. our results show that crem is essential for a regular excitation-contraction coupling in the mouse heart. (supported by the izkf münster) new mechanistic insights in no/cgmp actions in the vasculature schulte k., koesling d., universitätsstr. 150, 44781 bochum, germany hypertension, a major risk factor for cardiovascular diseases, is associated with vascular changes resulting in increased vascular contractility und vascular peripheral resistance. a prominent factor in the development and maintenance of hypertension is the reninangiotensin-aldostrerone system. angiotensin ii (ang ii) is the main mediator of this system and a powerful vasoconstrictor. ang ii increases the intracellular ca 2+ concentration thereby activating myosin light chain (mlc) kinase, which enhances mlc phosphorylation and subsequent vascular contraction. opposite to angii-induced vascular contraction, no/cgmp pathway promotes vascular relaxation by decreasing ca 2+ concentration and lowering mlc phosphorylation. responsible for mlc dephosphorylation is the mlc phosphatase (mlcp), whose activity is regulated by different phosphorylations. phosphorylation of mlcp via rhoa-activated rho-kinase enhances phosphatase activity while phosphorylation via the cgmp-dependent protein kinase has been proposed to decrease enzymatic activity. to investigate the interplay of angii with the no/cgmp pathway, we treated wild-type and ko mice lacking the cgmp forming no receptor, no-gc1, with angiotensinii (1.44 mg / kg bw / d) for two weeks. in addition to various cardiovascular parameters, physiological changes in vascular reactivity of aortic rings of angii-treated wt and no-gc1 ko mice were assessed in organ bath experiments and correlated with biochemical parameters as the phosphorylation state of mlc, mlcp and rho-kinase activities examined by immunoblot analysis. analysis of cgmp levels revealed that angii treatment decreased cgmp in wt mice to levels comparable to those of the ko mice which were unaltered by the treatment. our study will provide further mechanistic insights in the molecular interactions between constrictor and dilator stimuli in the vasculature. nanomaterials are already used today and offer even greater use and benefits in the future. the progress of nanotechnology must be accompanied by investigations of their potential harmful effects. for airborne nanomaterials, lung toxicity is a major concern and obviously the particle size is discussed as a critical property directing adverse effects. while standard toxicological test methods are generally capable of detecting the toxic effects, the choice of relevant methods for nanomaterials is still discussed. we have investigated two genotoxic endpoints -alkaline comet assay in lung tissue and micronucleation in polychromatic erythrocytes of the bone marrow -in a combined study 72 hours after a single instillation of 18 µg gold nanoparticles (np) into the trachea of male adult wistar rats. the administration of three test materials differing only in their primary particle size (2-, 20-and 200-nm) did not lead to relevant dna damage in the mentioned tests. the measurement of clinical pathology parameters in bronchoalveolar lavage fluid (balf) and blood indicated neither relevant local reactions in the animals' lungs nor adverse systemic effects. minor histopathology findings occurred in the lung of the animals exposed to 20-nm and 200-nm sized nanomaterials. in conclusion, under the conditions of this study the different sized gold np tested were non-genotoxic and showed no systemic and local adverse effects at the given dose. platelet dense granule secretion mediates platelet-dependent enhancement of tumor cell transmigration and formation of metastases schumacher d., strilic b., wettschureck n., offermanns s. mpi für herz-und lungenforschung offermanns, ludwigstr. 43, 61231 bad nauheim, germany tumor cell metastasis to distant organs is the primary cause of mortality in cancer patients. tumor cells leave the primary tumor, intravasate, survive in the circulation and extravasate through the endothelial cell layer to grow in the target organ. it has long been known that blood platelets play an important role in tumor cell survival and dissemination, but the mechanism by which platelets promote metastasis remained unclear. given that platelets are found closely associated with tumor cells shortly after vascular arrest, we explored whether platelets can facilitate the transmigration of tumor cells through the endothelium and thereby promote extravasation of tumor cells into the organ parenchyma. the ability of various mouse and human tumor cells like lewis-lung carcinoma cells (llc1), b16f10 melanoma cells or human neuroblastoma cells (sh-sy5y) to transmigrate through an endothelial cell layer was strongly enhanced by seeding tumor cells together with mouse or human platelets onto the endothelial cell layer. this indicates that platelets facilitate tumor cell transmigration in vitro. we found that platelet granule secretion is involved in this process as supernatant from platelets incubated with tumor cells but not from resting platelets was sufficient to enhance tumor cell transmigration. additionally, no platelet-mediated increase of tumor cell transmigration was observed in dense granule secretion-defective platelets of munc13-4 deficient mice. thus, dense granule secretion is required for platelet-dependent tumor cell extravasation in vitro. while the growth and weight of primary tumors after subcutaneous injection of llc1 and b16 cells was indistinguishable between wild-type mice and animals lacking munc13-4, the number of metastases in the lung was strongly reduced in munc13-4-deficient animals. the strong decrease in formation of metastases in munc13-4 deficient mice was also observed after i.v. injection of llc1 and b16f10 cells. thus, platelet dense granule secretion plays a critical role in tumor cell metastasis by enhancing tumor cell transmigration through the endothelial cell layer. formation of dna adducts in mouse tissues by the brassica ingredient 1methoxy-3-indolylmethyl glucosinolate and its break-down product 1-methoxy-3indolylmethyl alcohol schumacher f. 1 , engst w. glucosinolates are secondary metabolites present at substantial levels in cruciferous vegetables, such as broccoli and cabbage. after injury of plant tissue they are activated by the enzyme myrosinase to form various electrophilic degradation products like isothiocyanates. we previously showed that 1-methoxy-3-indolylmethyl (1-mim) glucosinolate (or neoglucobrassicin) is a potent genotoxicant in bacterial and mammalian cells after activation by myrosinase. the induction of mutations could be correlated with the formation of dna adducts [1] . we have identified and synthesized the major dna adducts n 2 -(1-mim)-dg and n 6 -(1-mim)-da. moreover, we developed a highly sensitive uplc-esi-ms/ms method for selective mrm quantification of these adducts using stable-isotopic labeled adducts as internal standards. while the plant enzyme myrosinase is probably almost completely inactivated after cooking the vegetables, the glucosinolates reach the gut mostly intact due to their good heat and ph stability. enzymes of individual intestinal bacteria are able to cleave the glycosidic bond of the glucosinolates, which leads to the formation of reactive metabolites within the gut lumen. we were able to detect significant levels of n 2 -(1-mim)-dg and n 6 -(1-mim)-da in a dose-dependent manner in the large intestine of mice treated orally with isolated 1-mim glucosinolate. the peak levels of n 2 -(1-mim)-dg and n 6 -(1-mim)-da in the murine large intestine were reached 8 h after a single administration of 600 µmol 1-mim glucosinolate/ kg body weight. the oral application of the relatively stable metabolite 1-mim alcohol to mice led to the formation of identical dna adducts. this benzylic alcohol can be activated by sulfotransferases to an electrophilic sulfo conjugate. in contrast to the intact glucosinolate the orally administered 1-mim alcohol generated significant levels of n 2 -(1-mim)-dg and n 6 -(1-mim)-da not only in the large intestine but also in other tissues, such as the liver, of mice. [1] h. glatt, c. baasanjav-gerber, f. schumacher, b. h. monien, m. schreiner, h. frank, a. seidel, w. engst, chem.-biol. interact., 192 (2011) human pregnane x receptor genotype of the donor but not of the recipient is a risk factor for delayed graft function after renal transplantation schwab m. 1, 2 , schaeffeler e. delayed graft function (dgf) is an important immediate complication in renal transplantation significantly contributing to decreased long-term allograft survival. in addition to donor-and recipient-related risk factors altered renal excretion of xenobiotics by membrane transporters may influence dgf as well. using recipients' and donors' dna, we assessed the impact of genetic variants on dgf for the transporter proteins, pglycoprotein (abcb1) and multidrug resistance protein 2 (abcc2), and the nuclear pregnane x receptor (pxr/nr1i2), regulating the transcription of drug metabolizing enzymes and membrane transporters. in our local cohort of transplant patients (n=178) dgf occurred in 27.5 %. logistic regression analysis using four different genetic models (i.e. co-dominant, dominant, recessive and log additive) indicates that only the donor's pxr rs2276707 8055tt genotype was significantly associated with dgf (recessive model: or, 9.0; 95%ci, p=0.004 unadjusted) , even after correction for multiple testing (p=0.035 holm-adjusted). when we performed multivariate analysis including genetic and 16 clinical co-variates (i.e. age, gender, hla mismatches, panelreactive antibodies, immunosuppression using cni, t cell-depleting agents, anti-il-2 receptor antibody and steroids, cold or warm ischemia time, living vs deceased donors or graft loss) again dgf was significantly associated only with the pxr rs2276707 tt genotype of the donor (recessive model: or, 16.08; 95% ci, 2.0-129.46; p=0.0046 unadjusted) which held true after correction for multiple testing (p=0.04). for abcc2 variants only the donor rs17222723 3563t>a genotype correlated with dgf by univariate (or, 4.65; 95%ci, p=0.005 unadjusted) as well as by multivariate analysis (or, 5.5; 95%ci, p=0 .01; table 4 ) but not after correction for multiple testing (p=0.12). for variants in the abcb1 gene no significant associations with dgf were detected for both the donor's and the recipient's genotype. in summary, our findings suggest for the first time that pxr may be a risk gene for the development of dgf independently from previously identified risk factors. supported by the robert-bosch foundation, stuttgart, germany, the bmbf grant 03is2061c (berlin, germany) and by the ferdinand eisenberger grant of the german society of urology (id krs1/fe-10). formation, morphology and structural requirements for formation of microtubule protrusions by clostridium difficile toxin cdt schwan c., kruppke a. s., nölke t., aktories k. institut für experimentelle und klinische pharmakologie und toxikologie i, albertstr. 25, 79104 freiburg, germany clostridium difficile is an anaerobe, gram-positive pathogen. it causes antibioticassociated diarrhoea and pseudomembranous colitis by production of the rho gtpaseglucosylating toxins a and b. recently emerging hypervirulent clostridium difficile strains additionally produce the binary adp-ribosyltransferase toxin cdt (clostridium difficile transferase). cdt is taken up via receptor-mediated endocytosis after binding to the lipolysis stimulated lipoprotein receptor (papatheodorou et al., pnas 2011) . in the cytosol, cdt adp-ribosylates actin at arg 177, thereby actin polymerization is blocked, resulting in disruption of the f-actin network. cdt and other binary actin-adp-ribosylating toxins induce redistribution of microtubules in the cell interior and formation of long (>150 µm) microtubule-based protrusions at the surface of intestinal epithelial cells which increase bacterial adherence (schwan et al., plos pathog 2009 ). the clostridial actin-adp-ribosyltransferases influence the dynamicity of microtubules and their capture at the cell cortex by indirectly affecting different microtubule regulating proteins like clasp2 and acf7. besides the influence of cdt on microtubule regulatory proteins, the formation of protrusions depends on plasma membrane composition. depletion of cholesterol, the breakdown of sphingomyelin or inhibition of sphingolipid-synthesis reduce the formation of microtubule-based protrusions. surprisingly, most of the cdt-induced processes contain membrane-tubules derived from the endoplasmatic reticulum (er). the remodeling of the er is microtubule dependent and is mainly mediated by stim1 that usually functions as a calcium sensor in the er and activates the store operated orai1 calcium ion channels in the plasma membrane. the data suggest that toxin-induced changes of the microtubule system including alterations of the er, may affect trafficking and er-dependent signalling. bilobalide is a neuroprotective constituent of ginkgo biloba with an unknown mechanism of action. in the present study, we first used microdialysis in mice to evaluate changes in the extracellular fluid of the brain during and after stroke. microdialysis probes were implanted into the striatum of cd-1 mice, and dialysates were obtained while a monofilament was inserted for 60 min via the common carotid artery (cca) to block perfusion through the middle cerebral artery (mca). while glucose levels dropped immediately upon middle cerebral artery occlusion (mcao), glutamate concentrations in the microdialysates -as measured with a cma 600 analyzer -rose extensively during ischemia to more than 2000% of baseline level. both glucose and glutamate levels recovered rapidly when mcao was terminated after 60 min. when bilobalide (10 µm) was perfused into the striatum through the microdialysis probe during mcao, glucose levels dropped but the neurotoxic rise of glutamate was significantly attenuated and reached only 500% of baseline level (p<0.01). in the following experiments, we investigated the activity of mitochondria in ischemic brain. ischemia was induced by mcao, and ischemic as well as "healthy" tissue from the opposite hemisphere was obtained. mitochondria were isolated and mitochondrial respiration was monitored using the oroboros ® oxygraph. significant deficits of respiration were observed after ischemia. in the healthy hemisphere, the respiratory states (leak i+ii, complex i+ii+iv, oxidative phosphorylation (oxphos) and electron transport system (ets) capacity) showed a decrease of oxygen consumption to 60-70% of sham-operated mice. in the ischemic hemisphere, several values were lower at 40% of sham-operated mice (leak i+ii, complex ii+iv,oxphos and ets) whereas complex i showed a remarkably low respiratory capacity of 16% of baseline. direct addition of bilobalide (10 µm) to post-ischemic mitochondria caused a 2-fold increase of complex i activity in vitro. pretreatment of mice with bilobalide (10 mg/kg i.p.) one hour before mcao caused a significant, 3-fold improvement of complex i respiration when measured ex vivo. these data clearly indicate that bilobalide targets mitochondrial processes within the respiratory chain, preserving complex i function during ischemia. this action likely explains its neuroprotextive activity in vivo. unreacted resin monomers such as 2-hydroxyethyl methacrylate (hema) are environmental stressors released from dental composites after incomplete polymerization. the production of reactive oxygen species (ros) is a major response of cells to monomer exposure. moreover, adverse effects of monomers including delayed cell differentiation or mineralization processes, dna damage or apoptosis are associated with increased ros production. the intracellular redox homeostasis is controlled by the major non-enzymatic antioxidant glutathione (gsh), and antioxidant enzymes. here, we hypothesized that cells exposed to hema responded by a differential expression of antioxidant enzymes such as superoxide dismutase (sod-1), catalase (cat) or glutathione peroxidase (gpx1/2). raw246.7 mouse macrophages were exposed to hema (0-8mm) for 24h, and protein expression was analyzed by western blotting. to study the influence of intracellular gsh on enzyme expression, gsh synthesis was reduced by the inhibitor buthionine sulfoximine (50µm bso), or enhanced by 2-oxothiazolidine-4-carboxylate (5mm otc) and n-acetyl cysteine (10mm nac). expression of sod-1 found in untreated cultures was decreased in the presence of hema and even further reduced by bso. in contrast, nac counteracted hemainduced inhibition of sod-1 expression. cat expression was not detected in untreated cells, however, the enhanced expression of cat in cells exposed to hema indicated the decomposition of abundant levels of hydrogen peroxide. the minor influence of bso or otc showed that expression of cat was independent of gsh levels while a decrease of hema-induced cat expression in the presence of nac indicated reduced oxidative stress. gpx1/2 was expressed in untreated cultures, and its down-regulation by bso indicated that this enzyme was primarily responsible for h2o2 decomposition. the inhibitory effect of hema on gpx1/2 expression was enhanced by bso but counteracted by otc or nac. these findings indicate that h2o2 is the predominant reactive oxygen species generated in the presence of dental resin monomers like hema. abundant h2o2 production leads to the activation of cat expression and a feed-back inhibition of sod-1 expression. the hema-induced reduction of gpx1/2 expression is most likely a consequence of reduced gsh levels because of the formation of glutathione disulfide (gssg) or by gsh-hema adducts. the life-threatening effects of certain organophosphorus compounds such as soman or fenamiphos cannot be antagonized adequately by the treatment with atropine and oximes. alternative approaches are necessary. since the adequate restoration of disturbed muscle function is considered to be life-saving, a model is needed for screening of potentially therapeutic substances. an established model for the development of such new therapies is the diaphragm preparation. however, this model requires a large number of animals and experimental available time frame is limited to some hours. here, the organotypic nerve-muscle co-culture may be an appropriate alternative, because a large number of specimens with low numbers of animals and a long period of investigation over several days is possible. in the present study, the restoration of vx paralysed muscle function with obidoxime was investigated by using both models. slices of spinal cord and muscle tissue were dissected from mice embryos (e 14-15) , fixed to coverslips and incubated in roller tubes for about 2-4 weeks. spontaneous muscle activity was recorded by video microscopic techniques and was quantified offline. muscle force production in mice diaphragm preparations was elicited by indirect field stimulation technique in a 12 chamber organ bath and quantified as time-force diagrams (auc) that were expressed as relative changes of the muscle force compared to the control data. application of the nerve agent vx (0.75 µm) resulted in a strong reduction of muscle activity in the co-culture and of muscle force production in the diaphragm muscle. after obidoxime (10 µm) was added spontaneous muscle activity in the co culture recovered from 0.45 ± 0.24 hz to 1.67 ± 0.24 hz (control 1.79 + 0.22 hz) . muscle force remained stable over the next days. the vx-blocked muscle force of diaphragm was restored to 69.9 ± 21.3 % by obidoxime compared to control. muscle force production after indirect stimulation was stable for 6 hours only. our results suggest that the organotypic nerve-muscle co-cultures may be an appropriate tool for the screening of new therapeutic approaches in restoration of blocked neuromuscular transmission. moreover, the model offers an additional advantage as long-term experiments may be performed and pre-and postsynaptic effects may be assessed directly. additionally, the number of experimental animals could be reduced. the modulation of gene expression by the transcription factor crem (camp responsiveelement modulator) represents a fundamental mechanism of gene control in response to elevation of intracellular camp levels. in vascular smooth muscle cells (vsmcs) crem is involved in the regulation of cell proliferation and apoptosis supporting its relevance for vascular proliferative diseases. mice with a global inactivation of crem (cko) showed a significant increase in neointima formation after ligation of the carotid artery and an increase of atherosclerotic plaque formation after high fat diet on an apoe knockout background compared to wildtype controls (wt). on the cellular level a crem deficiency was associated with a 1.3 fold increased proliferation rate of primary vsmcs after stimulation with the platelet-derived growth factor (pdgf; n=10 from 6 isolations). microarray analysis and subsequent realtime-rt-pcr validation revealed that the alpha-type platelet-derived growth factor receptor (pdgfra) the regulator of g-protein signaling 5 (rgs5) and peptidylprolyl isomerase a (ppia) were 1.5-2.5 fold upregulated in pdgf treated cko vsmcs compared to wt vsmcs (n=6). transcripts of rgs5 (2.6 fold, ) and ppia (1.8 fold, were also upregulated in the carotid artery of cko mice in comparison to wt mice (n=10-12). we conclude that crem deficiency is associated with transcriptional changes of rgs5, pdgfra, ppia, which might explain the increased proliferation rate in cko vsmcs and the increased responsiveness to pathophysiological conditions. (supported by the izkf münster). the role of non-catalytic p101 and p87 subunits in regulating phosphoinositide 3kinase γ by gβγ and h-ras shymanets a. 1 phosphoinositide 3-kinase γ (pi3kγ) controls a plethora of cellular responses. pi3kγ, a heterodimer formed by non-catalytic p101 or p87 and catalytic p110γ subunits, is regulated by gβγ and ras. earlier we speculated that p101 binds to gβγ to translocate cytosolic pi3kγ to the plasma membrane, enabling direct activation of p110γ (brock et al., j. cell biol. 2003) . however, the p87 subunit does not function as gβγ adapter (kurig et al., pnas 2009) . since the impact of each non-catalytic subunit in regulating p110γ by gβγ and ras still remains elusive, we studied their role in detail. gβ1γ2 variants harbouring mutations in positions involved in interaction with gα subunit were purified from sf9 cells and tested for their ability to activate pi3kγ. we observed that p101, but not p87, was able to rescue the stimulatory activity of gβ1γ2 mutants incapable to activate p110γ (shymanets et al., biochem. j. doi:10 .1042/bj20111664). to further study the functional impact of the non-catalytic subunits on pi3kγ regulation, we have designed phospholipid vesicles containing similar amounts of recombinant pi3kγ variants. although p87/p110γ exhibited stronger sensitivity to gβ1γ2 than p110γ, the activity of vesicles-associated p101/p110γ was significantly higher as compared to vesicles-associated p87/p110γ or p110γ in the absence and in the presence of gβ1γ2. to study an effect of ras proteins on pi3kγ variants, recombinantly expressed h-ras was purified from sf9 cells. the posttranslational processing and lipidation of the protein was verified by mass spectrometry analysis. the impact of h-ras on regulation of p87/p110γ and p101/p110γ differed, which may explain integration of pi3kγ variants in different signalling pathways. taken together, p87 and p101 subunits implement discrete functions in respect to (i) membrane recruitment of pi3kγ and (ii) regulation of enzymatic activity by gβγ and h-ras. preparation of consolidated exposure scenarios for mixtures under reach sica m., dorn s., mostert v. dr. knoell consult gmbh, marie-curie-str. 8, 51377 leverkusen, germany under reach, formulators of mixtures need to include substance-related information into extended safety data sheets (esds), if mixtures are classified as dangerous according to the dangerous preparation directive (directive 1999/45/ec). one way to add information on substances into esds of mixtures is to generate exposure scenarios (ess) for mixtures. in order to fulfil this task, two approaches have been developed for the identification of the risk-determining substances (lead substances) in the mixtures: the critical component approach (cca) relies on dnels and pnecs for all substances, their concentrations in the mixtures as well as substance and use-specific availability parameters (echa, 2008) . in contrast, the dpd+ method is based on the legislation for classification of preparations (directive 1999/45/ec). the dpd+ method defines a lead substance for each route of human exposure and for the aquatic environment (cefic, 2010) . however, each of these methods has certain limitations. the aim of the present work is to improve the preparation of consolidated ess for a number of mixtures and provide information about their safe use. to this end, we first adopted information on risk management measures (rmms) and operational conditions (ocs) of the lead substances using the dpd+ methodology. at the same time, we considered the specific conditions of use of the mixtures (e.g. spraying, brush painting). we then conducted risk assessments by deriving dnels for the mixtures and using exposure modelling tools recommended under reach (e.g., ecetoc tra, riskofderm, art). we compared the outcome of these assessments with results obtained from the application of the dpd+ methodology. the work presents the results of application of dpd+ approach and the cca and indicates possible improvements for the risk assessment of mixtures. to check for seasonal and weather dependent influences in the prescription rate of drugs used to treat cardiovascular and respiratory diseases (atc codes c and r) a survey covering all prescriptions of a specimen german general regional health insurance (aok plus, data for saxony, largest health insurance service, approx. 50 % of all saxonian citizens are inscribed to this service) for the years 2005 to 2007 was analysed on a monthly basis. the number of prescriptions for cardiovascular drugs changed approximately +/-20 % around the mean for the different month without a clear seasonal pattern. for respiratory drugs only the systemic anticholinergics and drugs used to treat obstructive lung disease displayed a distinct seasonal pattern with a 50 -70 % above average prescription figure during spring time (february to may) and a 25 to 40 % trough in late summer/autumn (july to october). the data have to be analysed for further cofounders (e.g. influenza prevalence, environmental conditions etc.) to fully understand the fluctuations observed. the prescription rate for cardiovascular drugs and respiratory drugs seems to be influenced by multiple factors aside seasonal influences. pasteurella multocida toxin prevents osteoblast differentiation by activation of heterotrimeric g proteins siegert p., aktories k., orth j. institut für experimentelle und klinische pharmakologie und toxikologie abt. i, albertstr. 25, 79104 freiburg i. br., germany pasteurella multocida toxin (pmt) is a major virulence factor of pasteurella multocida causing pasteurellosis in man and animals and is responsible for atrophic rhinitis in pigs. the toxin modulates various signaling pathways by acting on the heterotrimeric g proteins gαq, gα12/13 and gαi. pmt activates gq to increase inositol phosphate production via phospholipase cβ and alteration of gene expression via the jak/stat pathway. the toxin also activates rhoa via gαq and gα12/13 family proteins. we showed that the underlying mechanism of the activation of heterotrimeric g proteins is a deamidation of an essential glutamine residue leading to a constitutive activation of the g protein. because pmt is the causative agent to induce progressive atrophic rhinitis in pigs, which is characterized by loss of nasal turbinate bones leading to a twisting and/or shortening of the snout, we studied the effect of pmt on bone cells. here we studied the effect of the toxin on osteoblast differentiation in st-2 cells and in primary osteoblasts from rat calvaria. st-2 cells are stromal derived cells, which can be differentiated into osteoblasts or adipocytes. the toxin inhibits the differentiation of st-2 cells into osteoblasts studied by determination of specific osteoblast markers. additionally, pmt represses the induction of transcription factors essential for osteoblast differentiation. moreover, the principal pathways activated by pmt to induce these effects were investigated. ventilator-induced lung injury (vili) is a serious problem in intensive care medicine. its mechanisms are only incompletely understood, although it is widely accepted that ventilation-induced inflammation (biotrauma) makes an important contribution. the isolated perfused mouse lung (ipl) is a valuable tool to investigate the mechanisms of vili. several studies have shown considerable differences between various mouse strains with respect to lung mechanics and inflammatory responses. therefore, we hypothesized that the pulmonary responses to mechanical ventilation differ between c57bl/6 and balb/c mice. in addition, this study introduces the novel half lung technique that allows to obtain lung tissue from the same mouse at two different time points. isolated perfused mouse lungs from c57bl/6 or balb/c were subjected to high (25cmh 2o) or low pressure (8cmh2o) ventilation for 240 minutes. after 180 minutes the left lung was removed and used for western blot analysis. the right lung was ventilated for another 60 minutes. by the end of experiment the right lung was removed and qrt-pcr performed. during the whole experiment perfusate sample were taken from the venous catheter and used for protein quantification by elisa. it was possible to remove half of the lung and to further ventilate the other half without acute changes in lung mechanics. in both strains high pressure ventilation elicited a significantly higher cytokine release than low pressure ventilation. c57bl/6 mice showed higher tnf, il-1β and amphiregulin levels after high pressure ventilation, whereas balb/c exhibited increased production of cxc chemokines (cxcl-1, cxcl-2) and il-6. kinase activities (jnk, akt, erk1/2, p38 map kinase) were increased in high pressure ventilated animals, but were strain independent. the novel half lung technique builds on the well established ipl method. it permits to separately analyze the left and the right lung at different time points during continual ventilation. this method reduces animal numbers by 50% and allows statistical within subject analysis. using this method, the present study showed that inflammatory response to mechanical ventilation differ between c57bl/6 and balb/c. these findings show that the biotrauma response in mice depends on the strain that is studied. macrophages play an important role as an integral part of the first line of immune defense. two different macrophage populations have been described. m1 macrophages produce proinflammatory cytokines and are involved in inflammatory processes. by contrast, m2 macrophages release anti-inflammatory cytokines and extracellular matrix components. they can enhance wound repair and angiogenesis, but they can also promote tumor progression. recently, industrial nanoparticles have raised concerns because of their putative toxic effects. on the other hand, specifically designed nanoparticles can be used as clinical diagnostics and as drug carriers for pharmacotherapy. thus, investigations on the interactions of engineered nanoparticles with living cells and organisms are of great importance. macrophages as phagocytosing cells scavenge nanoparticles circulating in the bloodstream. therefore, we analyzed how nanoparticles with different surface functionalization might affect functions of human macrophages. monocytes were isolated from buffy coats and differentiated to macrophages with macrophage colony-stimulating factor. carboxy-(ps-cooh) and amino-(ps-nh 2) functionalized polystyrene nanoparticles were produced by the miniemulsion polymerization process and the average particle size, the polydispersity index and their zeta potential were determined by dynamic light scattering. the macrophages were cultured in the presence or absence of different concentrations of ps-cooh and ps-nh2 nanoparticles for up to 6 days. analysis of cell viability revealed that ps-nh2 but not ps-cooh concentration-and time-dependently reduced the macrophage viability. by annexin v/propidium iodide double staining we could show that ps-nh2 trigger apoptosis in macrophages. we further polarized macrophages to either m1 or m2 using ifn-γ and lps or il-4, respectively. these macrophage populations were characterized by their expression of extracellular markers by flow cytometry and their production of cytokines by elisa. the effects of functionalized polysterene nanoparticles on the cytokine production and surface marker expression of m1 and m2 macrophages were analyzed. our data indicate that surface functionalization is a critical parameter in the nanoparticle-induced toxicity in human macrophages. this work was supported by the dfg spp1313. loos c., lunov o., syrovets t., simmet t. ulm university institute of pharmacology of natural products & clinical pharmacology, helmholtzstr. 20, 89081 ulm, germany nanoparticles are currently used for various medical applications including imaging, diagnosis and drug delivery. due to particle size and surface area, their fundamental properties differ significantly from those of corresponding bulk materials. nanoparticles circulating in the blood are mainly sequestrated by the reticuloendothelial system that consists predominantly of phagocytic macrophages. macrophages express a variety of cellular receptors for sensing and internalizing particular material like viruses, microorganisms, and foreign particulate matter including nanoparticles. therefore, a detailed understanding of the intracellular fate and processing of the nanoparticles by macrophages is indispensable for controlled biomedical applications of nanoparticles. introducing distinct surface modifications, one might control nanoparticle uptake by different cell types and thereby target specific tissues and cellular compartments. tumor cell lines are frequently used as models for primary cells to analyze the effect of nanoparticles on cells. here we show that carboxy-(ps-cooh) and aminofunctionalized (ps-nh 2) polystyrene nanoparticles of ~100 nm in diameter are internalized by human macrophages, thp-1 monocytic leukemia cells, and by pmadifferentiated thp-1 cells via different mechanisms. in buffer, macrophages and thp-1 rapidly internalize both types of nanoparticles, yet, the carboxy-functionalized particles were taken up to a higher extent. the uptake of both nanoparticles was drastically reduced in media containing serum. using pharmacological and antisense in vitro knockdown approaches, we showed that the specific interaction between cd64 receptors and the particles determines the macrophage uptake of particles by phagocytosis, whereas particle internalization by thp-1 cells occurred via dynamin iidependent endocytosis. by contrast, pma-differentiated thp-1 cells took up the particles via macropinocytosis. in line with the in vitro data, more intravenously applied ps-cooh particles accumulated in liver tissue, whereas ps-nh 2 were preferentially targeted to tumor tissue. these data show that the amount of particle internalization, the uptake mechanisms, and kinetics differ significantly among primary cells and model tumor cells, whether differentiated or not, and that they are further critically dependent on the particle opsonisation by serum proteins. this work was supported by the dfg spp1313. specifically designed and functionalized nanoparticles hold great promise for a variety of biomedical applications. to ensure their safe application, such particles require a rigorous analysis of their effects on cell functions. here we demonstrate that aminofunctionalized polystyrene nanoparticles (ps-nh2) of ~100 nm in diameter in contrast to carboxy-(ps-cooh) and nonfunctionalized (ps) particles induce an nlrp3 inflammasome activation and the subsequent release of il-1β in human macrophages. amino-functionalized ps nanoparticles induced time-dependent lysosomal destabilization followed by release of lysosomal enzymes. this resulted in mitochondrial damage and formation of reactive oxygen species. accumulation of mitochondrial reactive oxygen species was accompanied by oxidation of thioredoxin, a protein playing a central role in maintaining the cellular redox balance. upon oxidation, thioredoxin dissociated from the thioredoxin-interacting protein (txnip). liberated txnip, in turn, interacted with the nlrp3 protein resulting in a conformational change of the pyrin domain of the nlrp3 protein as predicted by molecular modeling. txnip interaction with nlrp3 led to assembly of the nlrp3 inflammasome complex, to caspase-1 activation, and release of il-1β. using an in vitro knockdown approach, we showed that ps-nh 2 induced activation exclusively of nlrp3, whereas other inflammasomes remained unaffected. treatment of macrophages with n-acetyl-l-cysteine, a scavenger of reactive oxygen species, abolished both, the caspase-1 activation and the subsequent release of il-1β caused by ps-nh2 nanoparticles. these data reveal a novel mechanism of the nlrp3 activation induced by amino-functionalized nanoparticles and provide a strategy as to how such an effect can be functionally antagonized by supplementation with a radical scavenger. this work was supported by the dfg spp1313. the semi-permeable barrier of the endothelial cell lining of the blood vessels has important synthetic and metabolic functions including transport of cells and biomolecules, regulation of vascular smooth muscle tone, and control of hemostasis. plasmin is a serine protease, which is generated from its zymogen plasminogen under physiological and pathological conditions. small amounts of plasmin are produced in the context of contact activation during inflammation. consistently, increased generation of plasmin has been reported during atherosclerosis. we have shown previously that plasmin, in addition to its role in fibrinolysis, could induce proinflammatory activation of various cells including monocytes, macrophages, and dendritic cells. therefore, we analyzed how plasmin might affect the functions of endothelial cells, which could be relevant during inflammation and atherosclerosis. using flow cytometry, western immunoblotting and fluorescent microscopy, we show that endothelial cells of different origin express the plasmin receptor complex composed of annexin a2 and s100a10. addition of plasmin to human umbilical vein endothelial cells (huvec) induced timeand concentration-dependent cytotoxic effects in the cells. in addition, within 30 min plasmin triggered a rapid and prolonged expression of free radical oxygen species (ros) in endothelial cells as analyzed by microscopy and fluorometry using the rossensitive dye carboxy-h 2dcfda. the ros production in endothelial cells was accompanied by cell detachment. fluorometric and western blot analysis of caspase 3 activation in the cells treated with plasmin showed that plasmin induced apoptotic cell death in endothelial cells, which was evident already several hours after exposure to plasmin. thus, plasmin might induce production of ros in endothelial cells, their detachment and apoptosis, events which might be relevant for the development of atherosclerosis. this work was supported by the dfg. sesquiterpene lactones (stl) comprise a large group of secondary plant metabolites that constitute the active principle of a number of traditional anti-inflammatory phytomedicines. specifically helenalin and parthenolide have recently gained considerable attention as lead compounds or putative therapeutics for the treatment of inflammation and possibly cancer. both compounds have been shown to interfere with the signal transduction through inhibition of the nuclear factor κb (nf-κb). whereas the inhibitory effects of the stl on nf-κb are undisputed, their molecular mechanism of action remains a matter of debate. surface plasmon resonance (spr) analysis allows label-free measurement of molecular interactions. yet, analysis of the interaction of immobilized recombinant proteins with small molecular ligands remains a technically challenging task. in the present study we used spr technology to investigate the molecular interaction of the stl helenalin with putative intracellular target proteins such as the nf-κb protein p65/rela, the catalytic subunits of the ikk complex, namely ikkα and ikkβ, and the intracellular antioxidant glutathione (gsh). at physiological ph 7.4, helenalin interacts with rela (k d = 4.8 µm), yet it failed to bind either ikkα or ikkβ. hence, when dna with nf-κb binding consensus sequence was immobilized on sensor chips, the binding of rela was inhibited by helenalin with an ic50 of 5.0 µm. moreover, we provided several lines of evidence that stl may modify rela on cysteine 38 by a michael-type addition. this interaction was confirmed by molecular docking that identified the best matching interaction between rela and helenalin with predicted hydrogen bonding interactions between helenalin and residues arg35, lys37, gly44 and ile118 of rela. consistent with our hypothesis that helenalin interacts with sulfhydryl groups at ph 8.0, helenalin was also able to interact with reduced, but not oxidized, glutathione with a kd of 24 µm, though no significant interaction was observed at ph 7.4. thus, we showed that the sesquiterpene lactone helenalin interacts with the nf-κb protein rela but not with ikkα or ikkβ. moreover, at physiological ph, helenalin does not interact with glutathione to any significant extent. direct interaction of helenalin with rela leading to inhibition of rela-dna binding and transactivation might present the molecular mechanisms underlying the anti-inflammatory effects of stls. although nanosized materials are quickly taken up by macrophages, our understanding of the involved processes is still rather limited. therefore, we analyzed the uptake of diagnostically used carboxydextran-coated iron oxide nanoparticles of two different sizes, superparamagnetic iron oxide nanoparticles of 60 nm (spio) and ultrasmall superparamagnetic iron oxide nanoparticles of 20 nm (uspio), by human macrophages. by pharmacological and in vitro knockdown approaches, the principal uptake mechanism of macrophages for both particles was identified as clathrin-mediated, scavenger receptor a-dependent endocytosis. further, we created a mathematical model of the nanoparticle uptake by macrophages that permitted determination of key parameters of endocytotic process, such as the uptake rate, the mean uptake time, the number of particles taken up by a cell, and the correlation between the number of internalized particles and their extracellular concentration. the model also provided information on the individual and collective wrapping time of the nanoparticles and described the relation between biophysical parameters such as cytoskeletal forces, membrane elasticity, and the uptake time. finally, we gained information on the minimal linear spacing between simultaneously acting neighboring endocytotic pits that contain single nanoparticles and govern the collective uptake process. the calculated parameters were further confirmed experimentally using spinning disc confocal microscopy. thus, the new model provides important insights into the biophysical processes involved in endocytosis of nanoparticles by human macrophages. this work was supported by the dfg spp1313. prostaglandins (pg) are hormones which are formed during inflammatory processes from arachidonic acid by cyclooxygenases and prostaglandin synthases [4] . in the subsequent metabolism, in which the five-membered ring is dehydrated, α,β-unsaturated carbonyl compounds are generated [2, 3] . these come along with mercapto groups of amino acids in a michael addition reaction associated with activation of cellular enzyme cascades [1] that potentially contribute to their possessed antiinflammatory, antineoplastic and antiviral effects [5] . however little is known so far about possible adverse health effects.we addressed the question whether selected cyclopentenone prostaglandins (cypg) exhibit potential mutagenic and genotoxic properties in the hamster lung fibroblast cell line v79. induction of dna damage was investigated by single cell gel electrophoresis assay (scge). the impact of cypg on cellular redox status was detected by total glutathione (tgsh) assay. the induction of micronuclei and apoptosis was determined by staining with 4',6-diamidino-2-phenylindole (dapi). furthermore the hypo-xanthine-guanine phosphoribosyltransferase (hprt) assay was used for mutagenicity testing. , followed by prostaglandin a2 (pga2), showed the most distinctive genotoxicity, i.e., induction of micronuclei, and apoptotic effects. furthermore, the 15dpgj2 and pga2 -induced significant decrease in the tgsh level in v79 may contribute to the observed increase in oxidative dna-damage. however, none of the tested cypg exhibited mutagenic properties in the hprt assay. in conclusion, a potential in vitro genotoxicity of cypg has been observed which may be involved in carcinogenesis associated with chronic inflammation. parabens and methylisothiazolinone are used as preservatives in personal care products. sensitization to parabens and methylisothiazolinone is relatively rare considering their wide use in cosmetics, but only few quantitative or clinical data exist. therefore, we have tested methyl-, ethyl-, propyl-, isopropyl-, butyl-, isobutyl-, pentyl-, phenyl-and benzylparaben , and methylisothiazolinone in the loose-fit coculture-based sensitization assay (lcsa) developed by our working group. the coculture of primary human keratinocytes and allogenic dendritic cell-related cells (dc-rc) in this assay emulates the in vivo situation of the human skin. sensitization potential of the test substances was determined by flow cytometric analysis of the dc-rc maturation marker cd86. determination of the concentration required to cause a half-maximal increase in cd86-expression (ec 50) allowed a quantitative evaluation. the irritative potential of the substances was assessed by 7-aad (7-amino-actinomycin d)-staining. the concentration required to devitalize 50 % of the examined cells compared to a zero control was termed ec50%. parabens exhibited weak (methyl-, ethyl-, propyl-and isopropylparaben) or strong (butyl-, isobutyl-, pentyl-and benzylparaben) sensitizing potential, phenylparaben was found to be a moderate sensitizer, with ec50-values ranging from 16.67 µmol/l (pentylparaben) to 325.36 µmol/l (methylparaben). due to a pronounced cytotoxicity (ec50% = 70.94 µmol/l), we could not estimate an ec50-value for methylisothiazolinone. sensitization potential of parabens correlated with side chain length. parabens showed no (methyl-and ethylparaben) or weak irritative potential (propyl-, isopropyl-, butyl-, isobutyl-, phenyl-and benzylparaben) , only pentylparaben was rated to be irritative. apart from phenyl-and benzylparaben, irritative potential also correlated with side chain length but did not correlate strictly with the sensitization potential. overall, we were able to demonstrate and compare the sensitizing potential of parabens in this in vitro test. it was weak for methyl-and propylparaben, the most commonly used parabens. furthermore, we showed an irritative potential for most of the perservatives. thus the lcsa is a useful in vitro test to compare the sensitizing potential of xenobiotics. phosphorylation of the neurodegeneration-related septin 4 by protein kinase dyrk1a at serine 107 affects protein stability soppa u. 1 septins are gtp-binding proteins forming heterooligomeric complexes and filaments by interactions of the 13 family members. these complexes have important functions by building scaffolds for proteins involved in cell cycle or cell polarity but their subcellular distribution as well as their regulation remain largely unclear. septin 4 (sept4) was found in neurodegeneration related protein aggregates and is associated with migration of cortical neurons. here we first describe a potential mechanism for regulation of sept4 by phosphorylation via dual specificity tyrosine-phosphorylation-regulated kinase 1a (dyrk1a). dyrk1a is overexpressed in down syndrome and supposed to be involved in neurodevelopment and neurodegeneration. by site directed mutagenesis of flag tagged mouse sept4 and overexpression in hela cells we identified serine 107 as the major phosphorylation site of dyrk1a and generated a phosphospecific antibody. transient coexpression of sept4 and dyrk1a in hela cells increased phosphorylation of serine 107 by 50% in relation to basal phosphorylation. in contrast, cotransfection of the kinase deficient dyrk1a mutants k188r and d287n did not increase serine 107 phosphorylation. moreover we could show, that inhibition of kinase activity by the dyrk1a inhibitor harmine reduced phosphorylation of exogenous sept4 at serine 107 about 25% in hela cells. furthermore, down regulation of dyrk1a by rna interference lead to decreased phosphorylated serine 107. these results indicate that endogenous dyrk1a contributes to sept4 phosphorylation in hela cells. finally we analyzed protein stability of wild type sept4 compared to the phosphorylation resistant s107a mutant in hela cells by inhibition of translation with cycloheximide. we found that in living cells the sept4 s107a mutant is more stable than wild type sept4. in summary, our results suggest phosphorylation at serine 107 by dyrk1a as a novel mechanism to regulate sept4 stability and indicate a possible link of these proteins in cellular processes. is formaldehyde a good example for a "genotoxic carcinogen" with a threshold mode of action? speit g. institut für humangenetik, universität ulm, 89069 ulm, germany formaldehyde (fa) induces toxic and genotoxic effects in directly exposed cells (site of contact). several studies in which fa was administered to rats by inhalation showed evidence of tumor induction in nasal epithelium. there is also some epidemiological evidence that fa causes nasopharyngeal cancer in humans. although fa is a known mutagen, it is still a matter of discussion whether carcinogenesis is primarily mediated via a mutagenic mode of action. there is evidence that cytotoxicity and induced proliferation are the main causes for tumor formation. however, a decisive role of mutagenesis cannot be excluded and a mutagenic mode of action has to be considered for risk estimation. the basic assumption is that mutagens have a non-threshold mode of action. a threshold mode of action for a chemical is likely when a substance with a known mutagenic potential does not induce mutations at low concentrations due to a specific type of reaction with the genetic material and / or physiological protective mechanisms. because fa is a directly acting dna-reactive substance, a threshold mode of action may only be considered because of physiological protective mechanisms. after inhalation, pre-lesion protection occurs by unspecific binding to mucus, cellular proteins and glutathione. furthermore, fa is efficiently inactivated by enzymatic pathways. if fa reaches and damages the nuclear dna, dna repair mechanisms act as efficient postlesion protection mechanisms. in vivo inhalation studies with rats indicated that fa induces primary dna damage in the nasal epithelium but increased mutation frequencies were not measured. data are now available to show the relative distribution of endogenous versus exogenous dna adducts in different locations of the nose and other organs. considering the fa concentrations present in every living cell and the background levels of endogenous dna adducts, appropriate risk assessment and the identification of practical thresholds for fa-induced genotoxicity become feasible. fraud and misconduct in clinical trials steffen c. formerly federal institute for drugs and medical devices clinical trials unit, kurt-georg-kiesinger-allee 3, 53175 bonn, germany plagiarism in scientific publications has been the subject of public ("guttenplag") and scientific debate. plagiarism violates, however, "only" the intellectual property of its authors. in clinical trials, misconduct may also endager the health of patients. the suppression or falsification of data in clinical trials may mislead patients and doctors to use worthless treatments. clinicians may be provoked to repeat these trials, thereby wasting time and money. in clinical trials, misconduct includes everything from suppression or their repeated publication, the "correction" of unwanted results to the complete invention of the data, their intentional or negligent misinterpretation leading to the publication of biased conclusions from otherwise correctly performed clinical studies. the peer review system cannot protect against fraud, as few reviewers will have the means and the time to reevaluate original data. they will have to rely on these data, the calculations and statistics that are presented to them. some kinds of fraudulent behavior, such as double publications, can be found in the review process, but this is also time-consuming and depends on a helpful librarian. other kind of fraud, as the suppression of patient data in clinical trials (the deletion of "non-responders", according to cinderella: the good ones go into the pot, the bad ones go into your crop) can only be detected by the national competent authorities performing an inspection according to good clinical practice. even if the results of such an inspection become public as in the case of ukrain (gansauge et al. 2002) , there is no institution that will further analyze and publish the misconduct or initiate the retraction of the incriminated paper. a german agency similar to the us office of scientific integrity could foster good clinical practice in germany. although it is sometimes very difficult to decide whether improper results arise from fraud or error, a bias for the source of funding is obvious. trials funded by for-profit organizations were significantly more likely to recommend the experimental drug as treatment of choice (als-nielsen et al. 2003) . medicinal products with disputed efficacy such as orally applied enzymes for systemic action, bacterial preparations for irritable bowel syndrome and food supplements are to be reviewed with special attention. further examples from the author's experience will be presented. ]i favors further kca opening, kca may establish a feed-forward regulation of ca 2+ influx. in the present study we analyzed whether kca channels of sk4 and bk type play a role in sustaining ca 2+ oscillations at g1-to s-phase transition of primary mouse tumor cells and in human breast cancer cells, aiming towards a better understanding how kca modulate tumor cell proliferation. methods: kca expression was quantified by qpcr in human breast cancer biopsies, transgenic mmtv/c-neu + mouse mammary tumors and primary tumor cells derived thereof. the identity of the tumor cells was verified by gliolan staining. proliferation of the primary mammary tumor cells in the presence or absence of bk and sk4 modulators was tested using a real time cell monitoring system. the cellular dna content as a measure for cell ploidity was determined by propidium iodide staining and flow cytometry. changes in [ca 2+ ]i oscillations and peak amplitude were determined using ca 2+ indicator fura-2am. results: sk4, but not bk, expression is detectable in human and mouse breast cancer biopsies and in primary tumor cells derived from the mmtv/c-neu + mouse model. sk4 inhibition by tram-34 dose-dependently (0,1 to 10 µm) inhibits the growth of primary mammary tumor cells probably by a g1 cell cycle arrest. ca 2+ oscillations in proliferating mmtv/c-neu + tumor cells were ablated upon pharmacologic inhibition of sk4 channels. -dependent cell cycle progression is dependent on sk4 activity. blocking sk4 disrupts a feed-forward loop that coordinates ca 2+ influx via trp or crac channels in tumor cells. the consequences of sk4 inhibition in mammary tumors in vivo will be discussed. synthesis of a triphenylphosphonium substituted derivative of 5-hydroxymethyl-5-methylpyrroline n-oxide stolze k. 1 esr combined with spin trapping is a well-known analytical approach to detect free radicals formed in various biological systems, e.g. superoxide, hydroxyl and a series of carbon-centered free radicals, which are involved in oxidative stress. our aim was to modify the established spin trap 5,5-dimethyl-pyrroline n-oxide (dmpo) with a functional side chain, which can be used further as anchor for moieties enabling the spin trap to penetrate mitochondrial membranes, such as the positively charged triphenylphosphonium substituent. several synthetic routes were tested to introduce a 4-carboxybutyltriphenylphosphonium-substituent to the spin trap 5-hydroxymethyl-5-methylpyrroline noxide (hmmpo). while the activation of the carboxy group via the corresponding chloride was not successful, the use of a mixed anhydride with acetic acid appeared to be a promising way, although the reaction is considerably slower. preliminary spin trapping experiments have been performed with model systems generating superoxide, hydroxyl-, and carbon-centered radicals. othman e. m., stopper h. universität würzburg toxikologie, versbacher str. 9, 97078 würzburg, germany type 2 diabetes mellitus (dm2) is a growing health problem affecting more than 150 million people worldwide. it is associated with severe acute and chronic complications that negatively influence both the quality of life and survival of affected individuals. epidemiological studies clearly indicate that the risk of several types of cancer (including pancreas, liver, breast, colorectal, urinary tract and female reproductive organs) is increased in diabetic patients. diabetic patients are exposed to oxidative stress which plays a pivotal role in the pathogenesis of both micro-and macro-vascular complications. this is due to a decreased antioxidant capacity and chronic exposure to increased levels of reactive oxygen species (ros). since the insulin resistance in dm2 leads to hyperinsulinemia we studied the cellular consequences of the elevated insulin level and showed that it generates superoxide anions (o 2-) and dna damage by a nadph oxidase dependent mechanism in cultured cells. in addition, we found elevated genomic damage in the lymphocytes of diabetic patients as well as oxidative stress and genomic damage in kidneys of diabetic rats. this effect of insulin may contribute to the pathogenesis and progression of dm2 complications including the elevated cancer risk. the classical transient receptor potential (trpc) channel subfamily is regarded as nonselective, calcium permeable cation channels involved in a wide range of physiological events that require calcium signaling. until now, the specific roles of trpc channels in neuronal function are still elusive. given that trpc1 is able to form receptor-operated heterotetrameric channel complexes with other trpc channel subunits, we investigated the role of trpc1 for receptor-operated calcium influx in the heterologous expression system as well as in neurons. for this electrophysiological whole-cell measurements, fluorimetric calcium measurements, mn 2+ quenching and qpcr analysis were applied. furthermore, the effect of trpc1 knock-down on neuronal migration was monitored performing scratch assays, videomicroscopy and g-actin/f-actin assays. employing these techniques, we found that recombinant trpc1 was not able to function as a homomeric channel. instead, trpc1 subunits formed functional receptor-operated heteromeric channel complexes with trpc3, 4, 5, 6, and 7. heteromers containing trpc1 subunits showed significantly decreased calcium permeation in heterologous cell systems. mutation of amino acids in the putative pore forming region of trpc1 further reduced calcium permeability. in gnrh neurons endogenously expressing trpc1, 2, 5, and 6, downregulation of trpc1 by shrna resulted in increased basal cytosolic calcium concentrations and elevated calcium permeability. trpc1 was not involved in store-operated cation influx in gnrh neurons. moreover, trpc1 suppressed the migration of gnrh neurons without affecting cell proliferation. these findings suggest a novel regulatory mechanism relying on the expression of trpc1 and the subsequent formation of heteromeric trpc channel complexes with reduced calcium permeability, thereby fine-tuning neuronal migration. the transient receptor potential melastatin-3 (trpm3) is a calcium permeable nonselective cation channel that can be activated by the neurosteroid pregnenolonesulfate (pregs) or heat. trpm3 is expressed in various tissues, including insulin-secreting βcells and a subset of sensory neurons from dorsal root (drg) and trigeminal ganglia. the ability of pregs to evoke trpm3-like currents in pancreatic β-cells and to induce insulin secretion indicated its involvement in blood glucose regulation. however, trpm3 -/mice show so far no metabolic deficits but further investigations are recommended to evaluate its function in insulin secretion. further studies showed that trpm3 is a nociceptor channel involved in sensing heat and inflammatory thermal hyperalgesia. we performed a calcium-based screening of a compound library (spectrum collection) that identified several natural compounds as trpm3 blockers. the most potent blockers were the citrus fruit flavonoids hesperetin and naringenin as well as ononetin, a chalcon from ononis spinosa. the ic50 values of the substances are in the low micromoles ranges. electrophysiological whole cell measurements as well as calcium measurements confirmed the potency of the trpm3 blockers. furthermore, we could show that these blockers are effective on endogenous trpm3 in drg neurons from mice and isolated β-cells. by drinking grapefruit juice naringenin could be consumed in concentrations that are sufficiently high enough to block trpm3 activity in vivo. in sensory neurons, trpm3 may exert similar functions as trpv1. thus, trpm3 blocker could bear a therapeutic potential for analgesic treatment. xtt-based cell viability assay was used to determine the half-maximum effect concentration (ec50) for the investigated composite components in hgf. following concentrations of substances were used to determine the induced double strand dna breaks (dsbs): 1 /25× ec50, 1 /10× ec50 , 1 /3× ec50, and 1× ec50. each experiment was performed at least four times. hgf were incubated with various concentrations of substances for a period of 6 hours. induced dna double-strand breaks (dsbs) were tested by the γh2ax focus assay, which is a direct marker for dsbs using anti γh2ax antibodies. for quantitative γh2ax analysis foci in cell nucleus were counted by eye down using a fluorescence microscope. each experiment was performed at least four times. in the xtt test following ec50 values of substances were found (mmol/l;mean +/-sem): tmp(eo)9ta a, b 0.087 ± 0.011; 1,6-hddma b, c 4.500 ± 0.700; etma a, c ; 12.000 ± 1.100; a significantly (p < 0.05) differently to 1,6-hddma, b significantly (p < 0.05) differently to etma, c significantly (p < 0.05) differently to tmp(eo)9ta. after six hours of exposure with tmp(eo)9ta at 0.00348 mm there were induced 0.55 γ-h2ax foci-formations in hgfs, at 0.00870 mm 0.67 foci, at 0.02900 mm 0.86 foci and at 0.08700 mm 0.97 foci. after exposure with 1,6-hddma at 0.180 mm there were induced 0.45 γ-h2ax foci, at 0.450 mm 0.64 foci, at 1.500 mm 0.94 foci and at 4.500 mm 1.32 foci. after exposure with etma at 0.48 mm there were induced 0.43 γ-h2ax foci, at 1.2 mm 0.50 foci, at 4.0 mm 0.61 foci and at 12 mm 0.71 foci. the negative controls dmso and medium cultures displayed 0.31 -0.34 γ-h2ax foci/cell. it was found that the induction of foci/cell were concentration-dependet for all xenobiotics in the order of: 1,6-hddma > tmp(eo)9ta > etma. these results show that dental composite components can induce dsbs in primary oral cells and therefore these substances demonstrate a genotoxic potential. effects of antioxidants on the dna-toxicity of dental (co)monomers in human gingival fibroblasts styllou p. 1 , scherthan h. unreacted (co)monomers can be released from restorative dental materials and may show biologic activity after ingestion in the human organism. in previous studies the mutagenic/carcinogenic effect of dental monomers/co-monomers (e.g. methacrylates) on the human dna was demonstrated. in this study the effects of the antioxidants vitamin c and n-acetylcysteine on the dna toxicity of the (co)monomers triethylenglycol-dimethacrylate (tegdma) and 2-hydroxyethyl methacrylate (hema) was investigated. the induction of dna double-strand breaks with (co)monomers alone and in combination with antioxidants was investigated in human gingival fibroblasts (hgf). hgf were incubated with substances without or with antioxidants for a period of 6 hours. induced dna double-strand breaks (dsbs) were tested by the γh2ax focus assay, which is a direct marker for dsbs using anti γh2ax antibodies. for quantitative analysis of the γ-h2ax test, foci were counted by the same investigator by eye down the fluorescence microscope. each experiment was performed at least four times. the halfmaximum effect concentration ec 50 (mmol/l) of triethylenglykol dimethacrylat (tegdma) and 2-hydroxyethyl methacrylat (hema) was taken from of a previous study after using xtt-based cell viability assay. tegdma induced significantly (p < 0.05) higher dsbs compared to hema (1.91 ± 0.04 vs 1.66 ± 0.02). the mean number of cells scored and the standard deviation (sd) were calculated. when cells were exposed to tegdma in combination with the antioxidant vitamin c an increase of dsbs was observed (2.02 ± 0.06), compared to tegdma alone. when cells were exposed to hema in combination with vitamin c an increase of dsbs was observed (1.89 ± 0.07), compared to hema alone. when cells were exposed to tegdma in combination with the antioxidant nacetylcysteine a decrease of dsbs was observed (1.64 ± 0.04), compared to tegdma alone. when cells were exposed to hema in combination with n-acetylcysteine a decrease of dsbs was observed (0.76 ± 0.02), compared to hema alone. these results show that dental (co)monomers can induce dsbs in primary oral cells. it also shows for the first time that the genotoxic potential may be reduced by the addition of the antioxidant n-acetylcysteine. purpose: we aimed to investigate the role of superoxide and peroxynitrite generated by genetically destabilized enos for the development of endothelial dysfunction and vascular remodelling. methods: a mutant of bovine enos in which cys 101 was replaced by ala (c101a) resulting in destabilization of enos has been generated (enos-c101a). transgenic mice carrying c101a were generated on a c57bl/6 background using the endotheliumspecific tie-2 promoter. by breeding these mice with enos knockouts (enos-ko), mice that express enos-c101a (enos-ko/enos-c101a-tg) exclusively in the endothelium were obtained. unilateral common carotid artery ligation experiments were performed in c57bl/6, enos-ko, and enos-ko/ enos-c101a-tg to study a role of destabilized enos for vascular lesion formation. results: western blot analysis confirmed the expression of enos in enos-ko/enos-c101a-tg in aorta (37.1±8.4%, n=9), skeletal muscle (45.4±5.3%, n=10) and myocardium (17.4±4.9%, n=7) and revealed an increased phosphorylation of enos on ser1176/79 (470±47%) as compared to c57bl/6 (p<0.05, n=8). endothelium-specific overexpression of destabilized enos induced a large increase in superoxide and peroxynitrite formation in the aorta and the heart of enos-ko/enos-c101a-tg (p<0.05, n=5-8), which was abolished by nos-inhibitor l-nitroarginine (l-na) suggesting enos-c101a as a source of elevated radical generation. endothelium-specific introduction of enos-c101a at ~ 35% of c57bl/6 level almost completely restored aortic endotheliumdependent relaxation. experiments with l-na, soluble guanylyl cyclase inhibitor odq, peg-catalase and no-scavenger fe(detc)2 indicated that endothelium-dependent relaxation in enos-ko/enos-c101a-tg is nos-and cgmp-dependent and nomediated. four weeks after the carotid artery ligation, neointima formation, media thickening and luminal narrowing were observed in the ligated arteries of all studied genotypes (p<0.05, n=4-7). consistent with vasoprotective roles of enos, neointima formation was accelerated in enos-ko (n=4-7, p<0.05). despite significantly higher vascular levels of nitrotyrosine and peroxynitrite, neointima formation in enos-ko/enos-c101a-tg was substantially lower then in enos-ko and tended to be similar to c57bl/6. conclusions: increased vascular superoxide and peroxynitrite formation caused by destabilization of enos does not induce endothelial dysfunction in healthy mice and has negligible effect on neointima formation. fenton reactivity as a determining parameter for the interaction of manganese oxide nanoparticles with lung epithelial cells sydlik u. 1 , bieschke c. nanoparticles consisting of manganese oxide have been suggested for several innovative technological approaches, including the use in nanomedicine and diagnostics. therefore, the interaction of such nanoparticles with human target cells is of particular interest for the success of nanomedical approaches but also with regard to unintended side effects. to address this problem, we tested different kinds of manganese nanoparticles (mnnp) in an in vitro system which we earlier evaluated for proliferative, apoptotic, and pro-inflammatory endpoints induced by carbon nanoparticles (cnp). mnnp were synthesized by hydrothermal treatment of manganese salt solutions. the particles were subsequently characterized by scanning electron microscopy and dynamic light scattering. biological and toxic effects of the generated particles were studied in comparison to carbon nanoparticles (cnp) in experiments with rat and human lung epithelial cells (rle-6tn and 16hbe14o-). cytotoxicity was determined as measures of membrane damage (lactate dehydrogenase release) and metabolic activity (water soluble tetrazolium conversion). the oxidative capacity of the particles as well as the generation of intracellular oxidative stress was monitored using dichlorofluorescein diacetate in cell free experiments and flow cytometry assays (facs), respectively. the particle-specific phosphorylation of src family kinases (sfk) and mitogen activated protein kinases erk1/2 were investigated using western blot techniques. after physico-chemical characterization, a set of three mnnp consisting of mn3o4 or mno2 with significant differences in size and shape were selected. according to the different oxidation stages of manganese, the particles showed significant differences in fenton reactivity in the cell free system. these data did not reflect the capacity of the particles to induce intracellular oxidative stress. the characteristic to trigger membranedependent signaling processes, however, was correlated to the intrinsic oxidative capacity of mnnp than to the ability to induce intracellular ros. furthermore, the metabolic activity (wst) was negatively correlated with intracellular ros, indicating a link between mitochondrial activity and ros generation. none of the particles had effects on the membrane integrity of the cells. the data demonstrate that mnnp, unlike other poorly soluble nanoparticles (e.g. cnp), mainly trigger adverse health effects through ros production via the fenton reaction. acute ozone induced airway inflammation does not effect resting human sympathetic nerve traffic tank j. 1 numerous mediators released in inflammatory and neuropathic pain states activate gprotein-coupled receptors (gpcrs) and modulate nociception via activation of gs, gi/o, g12/13, or gq/11 g proteins. each of the g protein-coupled receptor pathways is involved in nociceptive modulation and pain processing, but the relative contribution of the individual signaling pathways in vivo has not yet been worked out. the gq/11 signaling branch is of particular interest in pain research because it leads to the activation of phospholipase c, protein kinase c, and the release of calcium from intracellular stores. using a conditional gene-targeting approach we generated double-deficient mice lacking gaq and ga11 selectively in nociceptors to investigate the contribution of the entire gq/11signaling pathway in nociceptors towards the regulation of pain. we observed that mice lacking gq/11 in nociceptive neurons show normal development of the nociceptive circuitry. the nociceptor-specific loss of gq/11 results in reduced pain hypersensitivity following paw inflammation or spared nerve injury. surprisingly, our behavioral and electrophysiological experiments also indicated defects in basal mechanical sensitivity in gq/11 deficient mice, suggesting a novel function for gq/11 in tonic modulation of acute nociception. patch-clamp recordings revealed changes in voltagedependent tetrodotoxin-resistant and tetrodotoxin-sensitive sodium channels in nociceptors upon a loss of gq/11, whereas potassium currents remained unchanged. our results indicate that the functional role of the gq/11 branch of g-protein signaling in nociceptors in vivo not only spans sensitization mechanisms in pathological pain states, but is also operational in tonic modulation of basal nociception and acute pain. provocation of arrhythmic events in single primary isolated adult mouse ventricular cardiomyocytes tekook m., fehrmann e., schulte j. s., schmitz w., müller f. u. westfälische wilhelms-universität institut für pharmakologie und toxikologie, domagkstraße 12, 48149 münster, germany ap duration and ca 2+ cycling are altered in cardiomyocytes of different genetic mouse models. here, we systematically tested various protocols to study the inducibility of arrhythmic events in mouse cardiomyocytes. adult ventricular cardiomyocytes were isolated from wildtype (wt) mice by enzymatic digestion and subsequently tested to trigger arrhythmic events within 6 hours after isolation. in patch clamp experiments (perforated patch, whole cell current clamp) aps were stimulated for 1 second (5hz; 10hz; 5hz + s2-stimulus after 50-80ms) followed by a 4s rest period. the resting-membrane-potential (rmp) was observed over 30 cycles. we observed rmp-fluctuations of different length (amplitude <5 mv; % of 13 observed cardiomyocytes, mean events/cell; <1s: 100%, 17.1; <3s: 92%, 11.5; >3s: 69%, 5.8), spontaneous depolarizations (>5 mv; 92%, 5.1) and spontaneous aps (62%, 1.9). intracellular ca 2+ transients and sarcomere shortening were measured after loading cardiomyocytes with indo-1/am. after preconditioning (10 min/1 hz) cells were measured under basal and continuous isoprenaline (10 -6 m) stimulation (iso). a 4 min pacing period was followed by a 1 min interval of no pacing. pacing frequency was reduced after each cycle (1 hz, 0.5 hz, 0.25 arrhythmic events were provocable with stimulation/rest protocols both by field stimulation and direct stimulation via patch pipette. however, low stimulation frequencies seem to lead to distinct destabilization of cardiomyocytes probably due to ca 2+ overload. we conclude that the tested stimulation protocols are able to provoke arrhythmic events even in wt single adult mouse ventricular cardiomyocytes and may serve as a tool to test for the relevance of potential proarrhythmic substrates in mouse models. ruhr-universität bochum pharmakologie ma nord1, bochum, germany cgmp is a second messenger involved in many (patho-)physiological processes such as smooth muscle relaxation, platelet inhibition, and the development and plasticity of the nervous system. however, it is not fully understood how cgmp regulates these and other processes on a mechanistic level. in particular, the existence and functional relevance of global and local cgmp signaling domains is not clear. recently, highly specific genetically-encoded optical biosensors for cgmp have been developed. these cgmp indicators are either based on fluorescence resonance energy transfer (fret), with cgmp-binding domains sandwiched between fluorescent proteins with overlapping spectra, or they consist of a single fluorescent protein fused to cgmp-binding domains. with these cgmp indicators, the spatiotemporal dynamics of cgmp signals, which result from the interplay between cgmp-producing guanylyl cyclases, cgmp-binding effectors, and cgmp-degrading phosphodiesterases (pdes), can be monitored in living cells. here, we report the generation of transgenic mice expressing the fret-based cgmp indicators cgi500 and cgi6000 with apparent cgmp affinities of 500 nm and 6000 nm, respectively. one mouse line expresses cgi6000 driven by a cmv promoter in neural cells. fret experiments were performed with isolated cerebellar granule neurons, hippocampal neurons, and astrocytes. we observed nitric oxide (no)-induced cgmp transients and analyzed the capability of other agents (natriuretic peptides, glutamate) to induce cgmp responses. in another mouse line, the sm22alpha promoter directs cgi500 expression specifically to smooth muscle cells (smcs). fret experiments have been performed with smcs isolated from aorta, bladder and colon, as well as with intact vessels in the retina and cremaster muscle of transgenic animals. in primary smcs we studied responses to no, atrial and c-type natriuretic peptide (anp,cnp). in different smc types we observed differences in the overall ability to react to these stimuli and in the kinetics of the induced cgmp transients. we also studied the effects of pde inhibitors on the no-, anp-, and cnp-induced cgmp signals. importantly, we were able to detect cgmp transients upon no stimulation in intact vessels of the retina and cremaster. we conclude that the cgi transgenic mouse lines are valuable tools to visualize cgmp signals in living cells in vitro and, possibly, also in vivo in the intact animal under physiological and pathophysiological conditions. current research data dealing with pharmacotherapy of α-ama intoxication shows a particularly high variability regarding the protective effect of silibinin. the aim of this study was therefore to evaluate the influence of the frequently used clinical antidotes benzylpenicillin, silibinin and their combination in human hepatocyte culture intoxicated with α-ama. cytotoxicity and apoptosis testing were performed after two and five days of simultaneously exposure to α-ama and/ or tested antidotes. to quantify apoptosis, necrosis and cell viability, we used cell death detection elisa plus®, toxilight® bioluminescence assay and cell proliferation kit ii (xtt). furthermore, we analysed the ways of apoptosis by using immunohistochemistry (differential detection of caspase 3, 8 and 9, activated caspase 3, and aif). exposure of hepatocytes to α-ama at concentrations of 0,2 µm, 0,5 µm and 1 µm resulted in disorder of cell cultures, apoptosis and reduction in cell viability compared with unexposed hepatocytes. in hepatocyte cultures treated with benzylpenicillin at concentrations of 30 µm and 1mm, silibinin at 50 µm and 100 µm and a combination of both (30 µm benzylpenicillin and 50 µm silibinin, 1mm benzylpenicillin and 100 µm silibinin), toxilight® values in the supernatant and xtt values were not significantly different from untreated cultures. simultaneous exposure to α-ama (at all tested concentrations) and benzylpenicillin, silibinin or combination of both showed higher cell viability and lower values of necrosis compared to the cultures exposed to α-ama alone (exept 50 µm silibinin at 0,2 µm α-ama); however, in both groups dosed with benzylpenicillin the highest hepatocyte viabilitiy was observed. this protective effect was particularly revealed at high α-ama concentrations (0,5 µm and 1 µm). in conclusion, our data suggest that benzylpenicillin in monotherapy is more effective than in combination with silibinin or silibinin alone. glucocorticoids (gcs) are important hormones in the regulation of metabolic homeostasis. synthetic gcs, such as dexamethasone (dex), play a fundamental role in the treatment of inflammatory diseases. there are numerous side effects of a dex therapy, e.g. the development of hypertension. in the pathogenesis of hypertension oxidative stress is a crucial factor. glucocorticoid-induced hypertension has been shown to be associated with an imbalance between nitric oxide (no) and superoxide. however, the source of this elevated superoxide production is unknown. we hypothesize that an uncoupling of the no synthase (enos), a key mediator of vascular homeostasis, may contribute to dex-induced oxidative stress. incubation of human endothelial cells (ea.hy 926) with dexamethasone led to a decrease in enos expression at mrna and protein levels. this effect of dex was timeand concentration-dependent. since the major cause of enos uncoupling is a deficiency of its co-factor tetrahydrobiopterin (bh 4), we analyzed the amount of bh4 in ea.hy 926 by hplc. a concentration-dependent reduction of bh4 and also bh2 (dihydrobiopterin) could be demonstrated in response to treatment with dexamethasone. bh4 can be synthesized endogenously by two different pathways -the de novo pathway (from gtp with gtp cyclohydrolase i, gch1, acting as the rate-limiting enzyme) and the salvage pathway (conversion of sepiapterin to bh4 involving dihydrofolate reductase, dhfr). treatment of ea.hy 926 cells with dex decreased mrna and protein expression of both gch1 and dhfr. because bh4 is the major "coupling switch", an enos uncoupling is likely to occur in dex-treated cells. in summary, we showed that dex treatment led to a reduced availability of the important co-factor bh4 which could lead to enos uncoupling. the uncoupled enos may possibly contribute to glucocorticoid-induced vascular oxidative stress. the cellular oncoprotein c-fos is a major component of the heterodimeric transcription factor ap-1 and has been commonly found over-expressed in tumors and cancer cells of different origin. previous work showed that mouse cells lacking the immediate-early gene c-fos are hypersensitive to ultraviolet (uvc) light. here we demonstrate that in human telomerase-immortalized vh10tert foreskin fibroblasts (behaving like primary cells) and sv40-immortalized gm637 fibroblasts, uvc-triggered induction of c-fos protein is a delayed and long-lasting event. sustained up-regulation of c-fos went along with transcriptional stimulation of the nucleotide excision repair (ner) gene xpf, carrying an ap-1 binding site in the promoter. c-fos mrna was induced in a biphasic manner. an immediate c-fos mrna expression (30-90 min after exposure) was not translated into the protein, the second wave of transcription (4-24h after uvc exposure) resulted in c-fos protein expression, 18-48h post-uv. the stress-activated/mitogen-activated protein kinases (jnk, p38k and erks) were immediately induced upon uvc exposure and stayed active for at least 24h. inhibitor experiments revealed that c-fos was phosphorylated by erks and jnk. the activation of c-fos preceded re-synthesis and the induction of xpf mrna, which was observed 24-40h post-uvc, resulting in the increased expression of the xpf protein. cells over-expressing c-fos showed an accelerated induction of xpf mrna, and consequently a faster repair of cyclobutane pyrimidine dimers (cpds). sirna-mediated silencing of c-fos (transient c-fos knockdown) resulted in abrogated uvc-triggered induction of xpf, attenuated repair of cpds and increased apoptosis. finally, we observed that the removal of cpds but not of photoproducts was significantly faster when cells were pre-exposed to a low uvc dose, indicative of an adaptive response to dna damage. the work was financed by deutsche forschungsgemeinschaft (dfg ch 665/2-1). the addition of clopidogrel to aspirin reduces ischemic events in patients with acute coronary syndrome and in those undergoing percutaneous coronary intervention (pci). however, recurrent ischemic event occurrence during dual antiplatelet therapy remains a major concern. variability in the pharmacodynamic response to clopidogrel is well recognized, and patients with higher platelet reactivity while receiving clopidogrel are at increased risk of ischemic cardiovascular events. clopidogrel is an inactive prodrug requiring biotransformation to form the platelet inhibiting metabolite. interindividual differences in clopidogrel metabolism are the major source of variability in antiplatelet response. polymorphically expressed cytochrome p450 (cyp) enzymes play a critical role in the metabolism of clopidogrel. these findings gave rise to the concept of personalized antiplatelet therapy -i.e. individual platelet function testing and correction of insufficient platelet inhibition to reduce ischemic events in patients with high on-clopidogrel platelet reactivity (hcpr). gravitas was the first study to test this concept by comparing double-dose clopidogrel to standard-dose clopidogrel in patients with hcpr. gravitas failed to correct hcpr consistently in the study arm, which coupled with a low overall event rate precluded demonstrating a substantial benefit from improved platelet inhibition. the trigger-pci trial tested the effectiveness of the more potent thienopyridine prasugrel versus clopidogrel in patients with hcpr after elective pci with implantation of drug-eluting stents (des). switching from clopidogrel to prasugrel in patients with hcpr afforded effective platelet inhibition. however, given the low rate of adverse ischemic effects using contemporary des after pci in stable ischemic heart disease, the clinical utility of this strategy could not be demonstrated and the study was terminated prematurely for futility. multiple studies have shown that both heterozygotes and homozygotes for loss-offunction cyp2c19 alleles have higher rates of adverse cardiovascular events as compared with noncarriers on approved maintenance dosing of clopidogrel (75mg qd), albeit carriage of cyp2c19 loss-of-function alleles accounted for only a minor proportion of the variability in on-clopidogrel platelet reactivity. results of ongoing studies with antiplatelet treatment stratified by cyp2c19 genotyping are awaited to assess the clinical benefit of this approach. the organic cation transporter novel type 2 (octn2/slc22a5) represents a high affinity uptake system for carnitine. besides metabolic disease like severe system carnitine deficiency, genetic variants within the slc22a5 gene have been associated with inflammatory diseases like colitis ulcerosa. against this background, we characterized octn2 expression in peripheral blood cells thereby identifying its expression in all cell types. in the present work we studied octn2 expression in monocytes and thp-1 cells as an in vitro model for this cell type. in addition we examined transcriptional regulation of the carnitine transporter in lps activated thp-1 and investigated the effect of carnitine and its analog mildronate on the respective cytokine response. octn2 expression was characterized on monocytes and thp-1 cells on mrna and protein level. transporter mrna expression could be shown in both cell types by realtime pcr. however, the protein expression was analyzed by western blot, flow cytometry and immunofluorescence microscopy demonstrating octn2 specific signals as well as a localization in the plasma membrane. following thp-1 cells were activated using lps (10ng/ml) for up to 6h, thereby indicating the expected cytokine response as demonstrated by increased tnfα (24fold induction) and il-1β (37fold induction) mrna levels. in addition, octn2 expression was analyzed identifying an initial reduction of around 60% compared to untreated cells. in parallel activated thp-1 cells were coincubated with increasing concentrations of the octn2 substrate carnitine or its analog resulting in reduced cytokine release as shown by elisa for tnfα. here, the tnfα effect was diminished by 64% in the presence of 50mm carnitine. this effect does not rely on a direct neutralization of lps by carnitine since it was also present in cells only preincubated with carnitine. in the present work we could show that thp-1 cells represent a useful model to study octn2 expression and function. in addition, we demonstrate immunosuppressive effects of octn2 substrates like carnitine. further experiments will be necessary to identify the underlying mechanism of this observation. castor oil has been used for more than 3000 years for its laxative effects and also to induce labor in pregnant women. despite its wide-spread use, the mechanism of action remained unknown. the active metabolite of castor oil is ricinoleic acid which is released from castor oil by intestinal lipases. we have found that exposure of meg-01 cells to ricinoleic acid caused an increase in [ca 2+ ]i, an effect which was dose-dependent and abolished by pretreatment of cells with pertussis toxin, suggesting the involvement of a g-protein coupled receptor. to search for a putative receptor, we determined ricinoleic acid-induced [ca 2+ ]i increases in cells transfected with a sirna library directed against human gpcrs. in this way, we identified prostaglandin e2 receptors ep3 and ep4 as mediators of ricinoleic acid-induced effects. to test if ep3 and ep4 receptors mediate pharmacological effects of castor oil in vivo, we analyzed laxative effects induced by castor oil in wild-type (wt) mice, ep3-deficient (ep3 -/-) or ep4-deficient mice (ep4 -/-). while ep4 -/mice responded similarly to the wt mice, ep3 -/animals were totally insensitive to castor oil-induced laxation. moreover, mice lacking the ep3 receptor only in the smooth muscle cells did not respond to castor oil, in contrast to mice which lack ep3 receptor only in epithelial cells of the intestinal mucosa. similarly, ricinoleic acidinduced contractions of isolated ileal segments were absent in segments lacking ep3, consistent with a preferential expression of the ep3 receptor in the longitudinal muscle layer of the intestine. also, ricinoleic acid-induced contractions of isolated uteri were dependent on the expression of ep3 receptor in the myometrium. these findings identify the cellular and molecular mechanism underlying the effects of castor oil and indicate a role of the ep3 receptor as a pharmacological target to induce laxative effects. introduction: patients seek health information from various sources. they are facing the challenge to differentiate between reliable and untrustworthy sources and at the same time identify the best drug therapy for them. furthermore generalised health information confuses more than they benefit or rather unsettle. patients are not necessarily qualified to assess the evidence of statements properly. there is thus a need for providing competent drug information, which is offered by the independent drug information service at the institute of clinical pharmacology in dresden, germany. for the present descriptive evaluation we selected 5 drugs (arimidex ® , cipralex ® pentalong ® , onbrez ® and pradaxa ® ), that were affected by new referenceprice formation, generic registration, warnings or directions in 2011. in specified time frames we assessed the increase in and the cause of enquiries. deductively we draw conclusions for a perspicuous presentation of patient information. since generic registrations of the aromatase inhibitor arimidex ® enquiries on side effects of this drug were stable, but 17 additional consultations were held on generic changeovers. the antidepressant cipralex ® as well as the long-acting β-agonist onbrez ® were assigned to reference-price groups, which resulted in an 8-times (cipralex ® : 5 → 40) and 5-times (onbrez ® : 2 → 10), respectively, increase in enquiries. main aspect was to give background information on reference prices and point out therapeutic alternatives (cipralex ® : 34 of 40; onbrez ® : 10 of 10). an additional amount of 22 conversations were carried on the fictive registered drug pentalong ® after health insurance companies advised practitioners to avoid recourse by not prescribing this organic nitrate. notable insecurity was aroused by media reporting on lethal bleeding after taking pradaxa ® for anticoagulation. every tenth enquiry in the evaluation period was focussing on these instigative reports (21 of 227). patients are confronted by current changes, but often do not get enough background information from their health care providers to become acquainted with the tidings. health seekers may find eligible data from media coverage. however the individual assessment as well as the risk-benefit-relation may not be feasible for them. the drug information service for patients is a convenient helpline to reduce lack of knowledge and uncertainties and therefore support shared decision making. insulin effects on hyaluronan production -a possible link between diabetes and cancer? twarock s., fischer j. w. institut für pharmakologie und klinische pharmakologie, universitätsklinikum der heinrich-heine-universität düsseldorf, moorenstraße 5, 40225 düsseldorf, germany background: epidemiological studies have shown an elevated incidence of certain tumor entities in diabetes type 1 and type 2 patients. to reveal the underlying mechanisms we focused on the effects of increased glucose uptake in cancer cells with respect to matrix production. abundant production of hyaluronic acid (ha) in the vicinity of gastrointestinal cancer cells is a hallmark in tumor development. esophageal cancer is a rare but severe kind of gastrointestinal cancer which is differentiated in adenocarcinoma and squamous cell carcinoma (scc) of the esophagus. we studied the effects of increased glucose levels on ha production in an scc cell line (osc1). in starving and full media, elevated glucose concentrations increased the production of ha secreted to the medium in 24h as measured by an ha-binding protein linked assay (starved, 0g/l glucose: 100±4.7%; 1g/l: 185.7±44.8%; 4g/l: 362.6±58.3%; full medium 100±15.2%; 155.3±32.3%; 422.7±32.0%). surprisingly, total ha concentrations were about 2.2-2.8 fold higher under starved conditions. to investigate whether this effect might be due to insulin actions, starved cells were treated with 10 mg/l insulin for 24h. we observed a dosedependent decrease in ha production following insulin treatment (control vs insulin, 1g/l glucose: 100±6.5% vs 67.83±4.4%; 4g/l: 100±2.2% vs 71.8±6.6%). this finding might suggest that insulin directs glucose usage to the glycolytic pathway thereby diminishing ha synthesis. a premise to this assumption is the ability of osc1 cells for insulin independent glucose uptake. to verify this thesis, mrna expression levels of insulinindependent and insulin-dependent glucose transporters (glut1, glut4) were analyzed by qrt-pcr. the relative abundance was 24.32±3.49 in favor of glut1 indicating the presence of insulin-independent glucose transport. conclusion: in osc1 the absence of insulin actions caused increased ha production which might be due to diminished insulin driven glycolysis, thus leading to the use of early glucose metabolites for ha production instead of energy gain. this finding could be important in the context of diabetes type 2, where insulin actions are also diminished because of insulin resistance. since increased ha production is of critical importance for cancer growth and spread, the cellular shift in glucose usage from glucose catabolism to ha anabolism could therefore indicate a possible link between diabetes type 2 and cancer progression. schwarz m., unterberger e. universtität tübingen, institut für klinische und experimentelle pharmakologie und toxikologie abteilung toxikologie, wilhelmstraße 56, 72074 tübingen, germany chemical hepatocarcinogenesis is a multi-stage process triggered by an intitiating mutation in a gene encoding an important cell-regulatory protein. tumour initiation may be caused by genotoxic substances which directly interact with the dna, causing mutations. cells carrying permanent mutations experience clonal expansion which may be accelerated by exposure of the experimental animals to tumour promoters during the following step of tumour promotion. it has been shown that substances which constantly activate certain nuclear receptors act as tumour promoters in rodent liver, such as the model tumour promoter phenobarbital which, amongst others, activates constitutive androstane receptor (car). since these tumour promoters do not seem to directly interact with dna causing mutations they can be regarded as non-genotoxic carcinogens. however, the molecular mechanisms of non-genotoxic carcinogenesis are still widely unknown which also poses a major problem in preclinical drug-development. the aim of the marcar (biomarkers and molecular tumour classification for nongenotoxic carcinogenesis) project is to establish early biomarkers for non-genotoxic carcinogenesis by creating a comprehensive molecular profile of tumours generated by a regimen including model tumour promoters such as phenobarbital. the ultimate aim is to differentiate spontaneous liver tumours from tumours generated by non-genotoxic carcinogens. this molecular profile includes mutational analyses, immunostaining for known tumour-specific markers, phospho-proteome analyses, genome wide and promoter-specific dna methylation analyses, as well as mirna analyses. mutation analyses were carried out with mouse and rat tissue from phenobarbital promoted liver tumours to identify mutations which phenobarbital provides a growth advantage for. furthermore, real time pcr measurements show that the expression of a particular non-coding rna and mirna precursor is up-regulated in tissue isolated from phenobarbital promoted mouse liver tumours. additional in-situ-hybridisation experiments demonstrated the localisation of this transcript in ctnnb1-mutated tumours. larch-derived diterpenes are potent and selective trpc6 blockers urban n. 1 , kübler w. the transient receptor potential channel trpc6 is a poorly ca 2+ -selective cation channel that is activated by the membrane-resident second messenger diacylglycerol (dag). consistent with the major sites of trpc6 expression, its activation has been implicated in pulmonary and renal diseases, such as pulmonary hypertension, lung edema, chronic obstructive lung disease, allergic airway disease, and focal segmental glomerulosclerosis. amongst various plant extracts, conifer oils and resins are traditionally used to treat pulmonary ailments. therefore, we reasoned that they may contain constituents with a biological activity to modulate trpc6 activity. the true turpentines, oils and resins of various coniferous genera were tested with respect to a possible inhibition of dag-or receptor-induced activation of trpc6 and trpc3. indeed, turpentines and resins, but not coniphere oils blocked trpc6 and trpc3 in a concentration-dependent manner. interestingly, the larch-derived turpentine exerted a trpc6-prevalent inhibition. we identified larixol and its mono-and diacetates as the specific compounds that are contained in larch resin and give rise to a trpc6-selective block. larixol acetates displayed an ic50 towards the dag-or receptor-stimulated trpc6 activity of about 0.3-1 µm, but did not strongly inhibit a number of other trp channels, including trpv1, trpm2, trpm3, trpm8, or trpa1. selectivity for trpc6 compared to its closest relative, trpc3, was about 30-fold. unlike conipherous oils, which contain toxic pinenes, the resin constituent larixol ant its acetates exerted no significant cellular toxicity at concentrations that are required to block trpc6. electrophysiological analysis confirmed the highly potent block, which was voltageindependent and reversible. in a murine hypoxia-induced pulmonary vasoconstriction (hpv) model, larixol acetate abrogated the euler-liljestrand mechanism and, thus, mimicked the phenotype of trpc6 -/mice. we conclude that trpc6 blockers and, more specifically, larixol-related derivatives may provide novel therapeutic strategies to treat or prevent pulmonary diseases. dioxin is an environmental contaminant, believed to affect basic biological equilibria such as calcium and iron homeostasis. however, the molecular mechanisms underlying these effects are still largely unknown. this strongly hampers the estimation of the hazard to humans associated with dioxin exposure and necessitates further studies aimed at the clarification of these mechanisms. it has been suggested that nearly all biological and biochemical processes are mediated by protein complexes. the most commonly used technology for monitoring changes in the expression of complex protein mixtures is still 2d gel electrophoresis, but this method suffers from poor expression of low or moderately abundant proteins. blue native page and subcellular fractionation form an ideal partnership when it comes to enrichment and analysis of intracellular organelles and low abundant multiprotein complexes. the aim of the study is to identify and characterize multiprotein complexes by blue native page to elucidate the network of protein-protein interactions that regulate protein function after dioxin exposure. sample preparation and subcellular fractionation rt4 cells were cultured in mccoy's 5a medium. cells at confluence were harvested and fractionated into cytosolic, membrane/organelle and nuclear fraction by using the proteoextract subcellular proteome extraction kit. first dimension (bn-page) 50 mg of protein sample was mixed with 5% of coomassie blue g-250 (cbb g-250) and loaded in each lane of 4-15% polyacrylamide native gradient gels. the lanes from the first dimension were cut into individual strips and were placed into a 12% sds gel. the gels were stained with coomassie and the spots were picked up for mass spectrometry. bn/sds-page combined with ms led to the identification of proteins involved in the regulation of both calcium and iron homeostasis in dioxin-exposed cells. these results demonstrate for the first time that dioxin exposure simultaneously affects calcium and iron metabolism. since important iron and calcium requirement changes occur during the regulation of cell growth, the protein expression changes observed in our study may be associated with dioxin-dependent cell-fate decisions. the murine protease inhibitor serpina3n inhibits mechanical allodynia in a model of neuropathic pain vicuna l. 1, 2 , simonetti m. several chronic diseases are accompanied by strong, long-lasting pain. a majority of chronic pain diseases are not well understood yet and cannot be controlled by conventional analgesics or non-pharmacological approaches. therefore, there is a major need to develop novel therapeutic principles. using a genetic screen, we identified serpina3n, a serine protease inhibitor, which is homologous to human a1-antichymotrypsin, to be a determinant of low neuropathic pain. we found that serpina3n is expressed in the dorsal root ganglia (drg) and spinal cord and that it is upregulated in these tissues in mice developing neuropathic pain. importantly, we observed that spinal delivery of recombinant serpina3n inhibits mechanical allodynia in a mouse model of neuropathic pain. we identified a novel serine protease substrate for serpina3n, which is upregulated in the spinal cord in mice undergoing neuropathic pain ('enzyme e'). recombinant enzyme e delivered intrathecally to the spinal cord of mice elicited rapid and long-lasting allodynia, which was fully blocked by concomitant administration of serpina3n. our results suggest that serine protease-serpin signaling modulates spinal neuronal and glial cell networks involved in processing pain and that activity-induced spinal release of serpina3n constitutes an endogenous defence mechanism against establishing chronic pain hypersensitivity. these data have important implications for the pathophysiology of pathological pain and potentially hold therapeutic relevance. gβγ subunits are involved in β-adrenergic receptor induced cardiac hypertrophy vidal m., lohse m. j., lorenz k. institut für pharmakologie und toxikologie pharmakologie, versbacher str. 9, 97078 würzburg, germany introduction. activated β1-adrenergic receptors and their g protein gαs induce the development of cardiac hypertrophy. however, the hypertropic effects of direct activation of downstream effectors, such as adenylyl cyclase, camp or pka, are controversely discussed. recently, a hypertrophic pathway involving a g protein βγ subunit induced phosphorylation of the mitogenic kinases erk1/2 at threonine 188 (erk thr188phosphorylation) has been described to mediate erk-induced hypertrophy. this study aims to investigate whether erk thr188 -phosphorylation is involved in cardiac hypertrophy triggered by β-adrenergic receptors. -phosphorylation was detected in hek cells overexpressing β1-receptors, murine hearts and neonatal rat cardiomyocytes after isoprenaline treatment. we performed [ 3 h]-isoleucine incorporation assays to assess cardiomyocyte hypertrophy in vitro. neonatal rat cardiomyocytes (nrcms) overexpressing wild-type erk2 showed a significant increase in [ 3 h]-isoleucine incorporation after isoprenaline treatment. in contrast, nrcms transfected with erk thr188 -phosphorylation deficient mutants (erk2 t188a and erk2 t188s ) or pretreatment with the erk inhibitor, pd98059, significantly attenuated cardiomyocyte hypertrophy. for in vivo studies, isoprenaline was given subcutaneously for 14 days to wild-type mice and transgenic mice overexpressing either wild-type erk2 t188t or erk2 t188s . echocardiography and histological analyses revealed that erk t188s mice developed less left ventricular hypertrophy than control mice. hypertrophic target proteins of erk (e.g. elk1) are located in the nucleus. western blot and confocal microscopy analyses showed that overexpressed erk2 t188a or erk2 t188s are retained in the cytosol and prevented elk1-phosphorylation after isoprenaline stimulation. co-immunopreciptation assays in hek cells and nrcms underlined the direct involvement of g protein βγ/erk interaction upon isoprenaline stimulation. in line with this finding, direct activation of adenylyl cyclase by forskolin did not lead to gβγ induced erk thr188 -phosphorylation. conclusion. taken together, gβγ-subunits participate in β1-adrenergic receptor mediated hypertrophy by enhancing erk thr188 -phosphorylation. these findings add important insight to the molecular signaling of g proteins in cardiac hypertrophy. the protein tyrosine kinase src and its role upon alpha-toxin stimulation of human platelets vogel k. 1 , burke m. introduction: alpha-toxin, a 34 kda calcium pore forming exotoxin, is a major virulence factor in the pathogenesis of staphylococcus aureus infections. alpha-toxin affects human blood cells such as platelets and induces aggregation that is accompanied by multiple changes in platelet protein tyrosine phosphorylation and dephosphorylation (1). in the present paper, we focused our interest on the protein tyrosine kinase src, the most abundant member of the src-family kinases present in platelets (2) . by the use of various inhibitors, we studied src and its role in α-toxin-induced platelet aggregation. methods: isolated human platelets from healthy volunteers were stimulated with α-toxin in the presence or absence of the src-family member inhibitors pp1, pp2 or su6654 (referred as src inhibitors). src and autophosphorylation of src were analyzed by sds-page and western blotting using specific antibodies against src and tyr-416-phospho-src from calbiochem and cell signaling, respectively (3). furthermore, calpeptin, an inhibitor of the calcium-dependent protease calpain, was used. platelet aggregation was measured by the method of born. staphylococcal α-toxin induced platelet aggregation in a concentration-dependent manner (0.18 -3.0 µg/ml of toxin). pre-incubation with 3 src inhibitors (pp1, pp2 or su6654) reduced α-toxin-induced platelet aggregation by about 50%. similar inhibitory effects have been observed by the use of calpeptin that acts as an inhibitor of the src degrading protease calpain. with respect to src itself, a-toxin induced autophosphorylation at tyr-416 followed by a fast and complete dephosphorylation within 10 min. while calpeptin modified the time course of dephosphorylation, only little effect of the src inhibitors has been seen on tyr-416 phosphorylation/dephosphorylation. the typical calpain-dependent degradation of src can be blocked by calpeptin (1µm), but also by depletion of extracellular calcium indicating that it is a calcium-dependent process. conclusion: taken together, our data demonstrate that α-toxin of staphylococcus aureus induces platelet aggregation accompanied by src degradation and autophosphorylation at tyr-416 typically observed in activated platelets. inhibition of the cellular tyrosine kinase src as well as the protease calpeptin reduces aggregation indicating an important role of src and/or other src-family members in α-toxin-induced platelet stimulation. the five subtypes of muscarinic acetylcholine receptors belong to the superfamily of gprotein coupled receptors. the even-numbered subtypes m2 and m4 prefer coupling to gi proteins, whereas the odd-numbered receptors m1, m3 and m5 prefer coupling to gq proteins. with respect to ligand binding and m2 receptor activation, the conserved epitope trp 7.35 at the beginning of tm7 displays remarkable functional features. it is located at the junction between the orthosteric and the allosteric binding site of the m2 receptor [1] . in the inactive m2 receptor, it provides subtype-independent baseline affinity for allosteric antagonists [1] . in the active receptor, m2 trp 7.35 affords binding affinity for the full agonist acetylcholine and intrinsic efficacy for the partial agonist pilocarpine [2] . to study the role of trp 7.35 for m3 receptor activation, agonist-induced formation of dmyo-inositol-monophosphate was measured in cho-cells transfected with the respective human receptor-cdna. surface receptor expression measured by radioligand binding was similar in hm3 wild-type-cells and hm3 trp 7.35→ala-cells, amounting to 0.07 and 0.11x10 6 receptors per cell, respectively. the intrinsic efficacy of acetylcholine was not influenced at m3 trp 7.35→ala relative to m3 wild-type, whereas potency was reduced about tenfold. these findings resemble those made previously in m2 and the corresponding mutant. in the case of pilocarpine, replacement of trp 7.35 by alanine in m3 did not reduce intrinsic efficacy. this finding is in contrast to m2, where the corresponding mutation induced a loss of pilocarpine's intrinsic efficacy. the potency of pilocarpine was diminished about tenfold at the m3 trp 7.35→ala mutant relative to m3 wild-type. this finding is also in contrast to m2, at which pilocarpine's potency was not sensitive to the trp 7.35→ala mutation. taken together, the diverging sensitivity of pilocarpine to the trp 7.35→ala mutation between the m3 and the m2 receptor suggests that the role of this epitope for receptor function may differ between even-and odd-numbered muscarinic acetylcholine receptors. in vivo experiments for inhalation toxicity are time and animal consuming. thus several in vitro methods aim to replace or reduce and refine the in vivo experiments. human 3dtissue models are commercially available reconstructed from different donors (normal, smokers, chronic obstructive pulmonary diseases), which show a normal human bronchiole tissue that reveals a pseudostratified epithelial structure, numerous microvilli and cilia on the apical surface of the cultures. the presence of tight junctions and mucus secretion has also been confirmed comparable to the in vivo situation. these 3d-models are cultured on a porous membrane as air-liquid interface. test substances can be applied apically, either as solution or with an aerosol-inducer. in our in house validation to test the strengths, handling and reproducibility of such 3dmodel systems as well as determining the correlation between in vivo inhalation data, we have assessed the epiairway tm model from mattek, usa. a set of 20 substances were selected with known in vivo toxicity data and mode of action. the substances were tested in the epiairway model an in parallel, in 3t3 and a549 cell lines to assess putative unspecific cytotoxic effects of the test substances. a comparison of toxicity data from the 3d-model and the in vivo data revealed, that the model is only predictive of respiratory toxicity in vivo for a subset of substances with specific modes of action. the epiairway tm model has proven to be robust, showing high reproducibility between pre-and main-tests as well as in the concurrent controls but it will need a strict definition of its applicability domain or further development of the test protocol to achieve a wider applicability. remodeling of intracellular ca 2+ handling and cyclic amp-dependent signaling in atrial myocytes from patients with chronic atrial fibrillation. voigt n. 1 background: in atrial myocytes ca 2+ entry through l-type ca 2+ channels (ica,l) triggers a larger ca 2+ release (ca 2+ transient,cat) from the sarcoplasmic reticulum activating contractile myofilaments. reduced ica,l is a hallmark of atrial remodeling in chronic atrial fibrillation (caf) and is supposed to contribute to action potential shortening and contractile dysfunction. however, the coupling efficiency between ica,l and cat and its regulation by camp-dependent signaling in caf patients are unexplored. methods: ica,l (voltage-clamp) and cat (fluo-3) were measured simultaneously in rightatrial myocytes from sinus-rhythm (ctl) or caf patients. a saturating concentration of the non-selective β-adrenoceptor (ar) agonist isoprenaline (iso, 1µm) and the nonselective phosphodiasterase (pde) inhibitor 3-isobutyl-1-methylxanthine (ibmx, 10µm) were used to increase cellular camp content. camp content was assessed with immunoassay. results: in caf amplitudes of ica,l (3.3±0.3pa/pf, n=12/6 [myocytes/patients] vs 6.2±0.8 pa/pf, n=15/10, p<0.01) and cat (182.2±26.8nm vs 307.5±30.9nm, p<0.01) were lower than in ctl myocytes, whereas diastolic [ca 2+ ]i levels were unchanged (caf, 312.3±56.7nm; ctl, 305.3±43.6nm). the coupling efficiency between ica,l and cat was similar in ctl and caf. application of iso increased ica,l amplitude to 10.4±2.0pa/pf (n=7/4) in caf and to 14.3±1.7pa/pf (n=9/7) in ctl. the corresponding cats increased to 493.0±132.3nm in caf and to 545.0±79.0nm in ctl. although the amplitudes of ica,l and cat also increased after pde inhibition with ibmx, the magnitude of these increases was smaller than the iso-induced enhancements. both iso and ibmx had no effect on diastolic [ca 2+ ]i and coupling efficiency. however, the relative iso-induced increases in ica,l (caf, +202.3±47.3% vs ctl, +98.4±16.1%, p<0.05) and cat (caf, +215.4±57.8% vs ctl, +101.9±20.3%, p=0.06) were significantly higher in caf compared to ctl myocytes and a similar tendency was found for ibmx. basal camp levels were higher in caf compared to ctl (caf, 9.9±1.5pmol/mg, n=6 vs ctl, 5.0±0.6pmol/mg, n=7, p<0.05), pointing to an increased camp-dependent signaling in caf patients. conclusions: these data point to remodeling of camp-dependent signaling in caf patients which likely contributes to the stronger relative increases of ica,l and cat amplitudes after β-ar stimulation and pde inhibition. remodeling of camp-dependent signaling might be a novel contributor to af pathophysiology. direct visualisation of g-protein-coupled receptors and heterotrimeric g-proteins using single-molecule microscopy wagner j. 1 g-protein-coupled receptors (gpcrs) form the largest family of membrane-bound receptors and mediate the effects of several extracellular stimuli. although the basic mechanisms of gpcr signalling have been extensively studied, a full characterization of the involved protein-protein interaction is still missing, largely due to technical limitations. in this study, we developed new methods for labelling gpcrs and g-protein subunits based on snap-and clip-tags and visualise them with single-molecule sensitivity. the snap-tag is a mutant of the dna repair protein o 6 -alkylguanine-dna alkyltransferase that reacts with fluorescent benzylguanine derivatives, whereas the clip-tag is reacting specifically with o 2 -benzylcytosine derivatives. these tags allow labelling proteins directly in living cells with very high specificity and low background. snap/clip-tagged receptors and g-proteins were covalently labelled with small organic fluorophores and visualised by total internal reflection fluorescence microscopy, which allows to selectively illuminate only fluorescent molecules located on or immediately underneath the cell surface. particles were automatically analysed with previously published as well as newly developed algorithms. the results indicated that both receptors and gproteins, although diffusing with high speed on the cell surface (diffusion coefficients: receptors ~ 0.05 mm 2 /s, g-proteins ~ 0.1mm 2 /s), can be visualised and correctly tracked. a variable fraction of receptors and g-proteins are immobile or show hop movements, possibly suggesting their interaction with cytoskeletal or other membranebound proteins. our data also suggest the feasibility of performing two-colour analyses with snap-and clip-tagged proteins aimed at directly visualizing transient interactions between receptors and g-proteins or among g-protein subunits. in-vitro screening systems are particularly well suited to preclinical toxicology testing at an early stage of drug development as they have the advantage of being fast and requiring only a small amount of test substance. the demands for in-vitro screening assays for systemic toxicity are multiple and include the need of organ specific cell systems, the use of optimal cell numbers, cell passages and incubation times. even minimal changes in the conditions of the test system may lead to significant changes of the biological system. therefore a reliable normalization compensating biological variability is crucial prior to any interpretation of results generated from a biological system. basf has developed an in-vitro metabolite profiling assay and a subsequently tuned normalization strategy allowing the prediction of specific organ toxicity. the in-vitro assay consist of exposing cells lines to test substances and to determine the metabolite profile using chromatography coupled to mass spectrometry systems. herein, we compare five different normalization strategies referring to their suitability in the application to in-vitro metabolite profiling data. the strategies comprise statistical approaches, approaches referring to reference values from each individual sample or samples generated in dependent batches. best results were achieved by an individual strategy using a new reference value correlating well over a large range of cell counts previously used for generating corresponding cell extracts. statistical analysis revealed the normalization based on the new reference value greatly improved the quality of the results compared to non-normalized samples as well as to all remaining strategies. generation and application of this new reference value and the corresponding normalization strategy will be presented the first time. validation will be featured on the basis of extracts of the human hepatocellular carcinoma cell line hep g2. molecular mechanisms of the inhibitory function of rhoh in phospholipase cmediated signalling walliser c., löschmann y., ziegler v., kühne e., schilling p., rasonabe z., bühler a., vatter p., gierschik p. universitätsklinikum ulm institut für pharmakologie und toxikologie, albert-einstein-allee 11, 89081 ulm, germany rho gtpases are a subfamily of ras gtpases regulating diverse signalling pathways, for example those regulating the reorganisation of the actin cytoskeleton. among them, rac2 and rhoh show an expression restricted to the hematopoietic lineage. rhoh is constitutively active, because it carries mutations in two positions (s13 and n62) known to be important for gtp hydrolysis. hence, rhoh is controlled on the level of protein expression and, possibly, by tyrosine phosphorylation. rhoh has been implicated in human malignancies, since the gene is subject to somatic hypermutation in its noncoding regions and to translocation to the gene encoding laz3/bcl6 or to other genes in human b-cell lymphomas. furthermore, rhoh is overexpressed in primary human chronic lymphocytic leukemia (cll) cells. these findings suggested that rhoh is involved in the initiation and/or progression of cll. we previously showed that rhoh acts as a potent inhibitor of both rac2-mediated phospholipase c-β 2 (plcβ2) and plcγ2 activation in intact cells. the aim of this study was to elucidate the molecular mechanisms of the inhibitory effect of rhoh on plc activity. the results showed that rhoh directly inhibited the activity of constitutively active variants of plcγ2, plcβ2, and plcδ1, but that it had little or no effect on the activity of plcγ1 and plcε. the amino acid residues s13 and n62, likely to be the cause for the gtpase-deficiency of rhoh, are not required for the inhibitory function of rhoh. furthermore, the switch-i and switch-ii regions of rhoh are not necessary for the inhibitory effect of rhoh, since rhoh mutants carrying switch-i or switch-ii regions of rac2 caused inhibitory effects on rac2-mediated plcβ2 and plcγ2 stimulation indistinguishable from wild-type. interestingly, rhoh seems to interact with regions of plcγ2 distinct from those which are necessary for rac2 interaction, as the split pleckstrin homology domain of plcγ2, which is essential for its interaction with activated rac2, is dispensable for the inhibitory effect of rhoh. in summary, our results indicate, that rhoh acts as a plc-isozyme-specific negative regulator of the activity of plcβ2 and plcγ2, both of which are specifically expressed in hematopoietic cells. these findings suggest a novel mechanism of plc isozyme regulation by rhoh. the results also suggest that rhoh plays an important role in b cell maturation, function, and leukemogenesis by modulating b-cell-receptor-mediated plcγ2 activation. effect of rac1 inhibition on doxorubicin mediated cell response wartlick f., fritz g. heinrich-heine-universität düsseldorf institut für toxikologie, universitätsstrasse 1, 40225 düsseldorf, germany background: the small gtpase rac1 is a well characterized member of the rashomologous (rho) family. rac1 is not only a key regulator of the actin cytoskeleton but also regulates the activity of nadph oxidase, stress kinases and transcription factors (e.g. nf-κb, ap1). furthermore, rac1 can translocate into the nucleus and interacts with topoisomerase type ii (topo ii). yet the general nuclear function of rac1 is still unclear. here, we address the question how rac1 influences the genotoxicity of the topo ii poisons doxorubicin and etoposide. methods: to study the function of rac1, human hepatoma cells were pretreated with the rac1-inhibitor eht 1864 before they were exposed to doxorubicin, etoposide or, for control, ionizing radiation (ir). to check the influence of rac1 inhibition on the outcome of genotoxin treatments, cell viability and cellular stress response were analyzed by the wst-assay, western blot (wb), co-immunoprecipitation experiments, facs-analysis and the alkaline comet-assay. results: as compared to the control, cells that have been pretreated with the rac1 inhibitor showed a higher viability, less phosphorylation of h2ax (s139) and a reduced dna damage formation (measured by alkaline comet-assay) after treatment with doxorubicin and etoposide but not after treatment with ir. furthermore inhibition of rac1 resulted in a reduced phosphorylation of topo iiα (s1106) and an increased interaction of topo iiα with hsp90 in doxorubicin treated cells. the data indicate that inhibition of rac1 protects human hepatoma cells against topo ii poisons due to interference with topo iiα function. the presence of drugs or other potential toxic substances in milk has enormous toxicological and nutritional consequences for consumers of dairy products. the atpbinding cassette (abc) transport protein breast cancer resistance protein (bcrp; abcg2) is expressed in alveolar epithelial cells of the mammary gland in cows, sheeps and goats. bcrp is known to play a major role in the active secretion of a variety of xenobiotics into human milk. so far there is little information about the transport activity and substrate specificity of dairy bcrp. therefore we aimed to establish a mdck cell in vitro model expressing bcrp of dairy animals. bcrp mrna was isolated from bovine, caprine and ovine mammary gland. full-length clones were generated using race (rapid amplification of cdna ends) pcr. the final full-length bovine, ovine and caprine abcg2 cdna-clone sequences were submitted to the ncbi genebank (eu570105, gq141082 and gq241418). stable transfection of bcrp in mdck cells was performed and the subcellular localization of bcrp at the apical plasma membrane was identified by confocal laser scanning microscopy. bcrp-mediated transport of the substrate hoechst 33342 was measured and the selectivity was determined by the bcrp inhibitor ko143. inhibition studies using hoechst 33342 identified various drugs including the antibiotic enrofloxacin or anthelmintic agents like oxfendazole as substrates of bovine, caprine and ovine bcrp. to further characterize bcrp carrier activity, bidirectional transport studies were performed with transwell® filter inserts that allow studying drug transport between an apical and basolateral compartment. cell monolayer integrity was checked by measuring teer values as well as by measuring the paracellular flux marker atenolol by lc/ms. bidirectional transport studies with enrofloxacin were performed to characterize the bcrp transporter activity. our results may contribute to increase the understanding of carrier associated drug transport into the milk of dairy cattle and therefore enlarge consumer protection. acrolein and acrylamide: excretion of mercapturic acids after consumption of potato chips watzek n., scherbl d., berger f., feld j., eisenbrand g., richling e. technische universität kaiserslautern fachbereich chemie; fachrichtung lebensmittelchemie & toxikologie, erwin-schrödinger-str. 52, 67663 kaiserslautern, germany acrolein (ac) and acrylamide (aa) may be formed from food constituents during heating of food. ac is supposed to be generated via heat induced formation from glycerides/glycerol, aa is known to arise during the maillard reaction from asparagine and reducing carbohydrates. ac also has also been suggested to be formed by endogenous metabolism as a side product of carbohydrate and/or amino acid turnover or by oxidative desamination of polyamines [1] . as an α,b-unsaturated aldehyde, ac forms 1,4-michael-adducts with biomolecular nucleophiles, such as sulfhydryl and amino groups. in the organism, ac and aa are preferentially conjugated to glutathione and are excreted as mercapturic acids (ma), n-acetyl-s-(3-hydroxypropyl)-cysteine , n-acetyl-s-(carboxyethyl)-cysteine (cema), (n-acetyl-s-(2-carbamoylethyl)-cysteine (aama), and (n-acetyl-s-(2-hydroxy-2-carbamoylethyl)-cysteine (gama). data on human exposure to ac and its occurrence in the diet are scarce. in general, contents in heat treated foods are considered to be in the low ppb range (µg/kg) [2] . nevertheless, in a pilot study in humans urinary 3-hpma excretion of 14 non-smokers was reported to be about three fold higher, as compared to aama [3] . in the present human intervention study we monitored the excretion of mas in five healthy volunteers (male) after ingestion of commercially available potato chips (175 g), equivalent to an uptake of 44 µg aa (absolute amount), together with an as yet unknown amount of acrolein [4] . urinary ma contents were monitored by hplc-ms/ms following solid phase clean-up of urine for up to 24 h after test meal uptake. the results demonstrated kinetics of 3-hpma and cema excretion in human urine to be clearly related to ingestion of the potato chip meal. on the basis of auc values, total excretion of 3-hpma plus cema exceeded that of aama plus gama by a factor of about four. the results confirm earlier findings on urinary mas, suggesting markedly higher human exposure to dietary ac / potential ac precursors than to aa. it is an as yet unresolved question, whether and to what extent concomitant substantial ac exposure may influence toxicology of such dietary heat-induced toxicants. [1] stevens, j.f. and maier, c.s. (2008) molecular nutrition & food research 52; 7 [2] osorio, v. m. and de lourdes cardeal, z., (2011) journal of chromatography a 1218; 3332 [3] schettgen, t., musiol, a., and kraus, t., (2008) rapid communications in mass spectrometry 22; 2629 [4] ewert, a., granvogl, m., and schieberle, p., (2011) lebensmittelchemikertag 2011 450 protective effects of increased nad + levels in human peripheral blood mononuclear cells exposed to dna damaging agents weidele k., beneke s., bürkle a. university of konstanz molecular toxicology group, department of biology, jacob-burckhardt-str.31, 78457 konstanz, germany the dna damage-activated enzyme poly(adp-ribose) polymerase 1 (parp-1) acts as a nick sensor and modifies target proteins by covalent attachment of poly(adp-ribose) [par] using nad + as substrate. the intracellular levels of par and nad + are important parameters for biological responses to genotoxic stress and influence diverse cellular functions including dna repair or maintenance of genomic stability. notably, loss of genomic stability is a hallmark of both carcinogenesis and the ageing process. here we analysed the impact of elevated nad + levels in human blood peripheral mononuclear cells (pbmc) with regard to (i) poly(adp-ribose) formation, (ii) cell death, (iii) initial dna damage and subsequent repair, as well as the influence on (iv) genomic stability under genotoxic stress. after ex vivo supplementation of pbmc with low concentration of nad + precursor nicotinic acid (na) intracellular nad + level significantly increased up to 2 fold in unstimulated [1] and 1.5 fold in mitogen-stimulated cells. after dna damage infliction, parp activity was dramatically increased in supplemented cells, necrotic cell death was reduced and dna strand break repair was significantly affected. furthermore the frequency of micronuclei decreased significantly after irradiation damage, emphasizing the fundamental role of adequate nad + levels in maintaining genomic integrity. the cyclic purine nucleotides adenosine 3':5' monophosphate (camp) and guanosine 3':5' monophosphate (cgmp) are well-examined second messengers with many proven biological functions. in a recent study, using a highly sensitive and specific mass spectrometry method, we have shown that cyclic 3':5' cytidine monophosphate (ccmp), a pyrimidine nucleotide, is naturally occurring in several mammalian cells [1] . ccmp activates both camp-and cgmp-dependent protein kinases with low potency [2] but the physiological function of ccmp is still very poorly understood. in an effort to delineate the function of ccmp, we analyzed expression of the early response gene egr1. we chose this gene because it is regulated by numerous stimuli including camp [3] . in our first study, we showed that dibutyryl-ccmp and ccmp failed to increase egr1 gene expression levels after stimulation of kb cells under various experimental conditions using real-time pcr (taqman®). we have now changed the experimental set-up using hela cells and the new ccmp analogue, ccmp-acetoxymethyl ester (ccmp-am), still focusing on gene expression of egr1. esterification of the negatively charged cyclic phosphate of ccmp allows better transport of the nucleotide across the cell membrane, thus augmenting possible intracellular effects. hela cells were stimulated in cell culture medium with extracellularly applied ccmp-am (3, 10, 33, 100 µm) over 15 to 240 min 24 h after seeding. analysis of real time pcr (taqman®) experiments, using β-actin as a housekeeping gene, showed a significant increase of egr1 expression in a time and concentration dependent manner. these effects were specific for stimulation with ccmp-am but not the control phosphate trisacetoxymethyl ester. hela cells were also cultured in serum free resting medium (mcdb 153, sigma) that induces growth arrest, one to eight hours prior to stimulation. here, even higher egr1 expression levels through ccmp-am stimulation could be seen. these results suggest that ccmp could function as a second messenger just as camp and cgmp do. studies are in progress to further examine the mechanisms of the ccmp-am effects on egr1 expression in hela cells. methyl-cpg-binding protein 2 (mecp2) recognizes methylated dna, it is involved in chromatin remodeling and it acts as a transcriptional repressor or activator. we have previously shown that expression of mecp2 is diminished in murine and human heart failure. prevention of mecp2 downregulation in transgenic mouse models aggravated cardiac hallmarks of heart failure. in patients with rett syndrome, which is caused by mutations in the mecp2 gene, mitochondrial function was found to be altered in the central nervous system. as the impact of mecp2 on mitochondrial function in the heart is unknown, the aim of the present study was to characterize the significance of mecp2 of cardiac mitochondria in mouse models with cardiac myocyte-specific expression or ablation of mecp2. in order to investigate the cardiac function of mecp2, two genetically modified mouse models were previously generated, including mice with inducible transgenic expression of mecp2 in cardiac myocytes under control of the tetracycline-system (mecp2-tg) and mice with targeted ablation of mecp2 in myocytes (mecp2 mlccre ). these mice were analyzed under basal conditions and after chronic transverse aortic constriction (tac). at baseline, cardiac-specific overexpression of mecp2 did not cause any difference in cardiac function as compared to control mice using millar catheterization. isolated interfibrillar mitochondria showed a decrease in citrate synthase activity. after chronic pressure overload, the decrease in cardiac mecp2 expression could be completely prevented by the mecp2 transgene. cardiac contractility and relaxation were significantly decreased in mecp2-tg animals. upon electron microscopical investigation, transgenic mecp2 expression was associated with a significant reduction of interfibrillar mitochondria, clustering of mitochondria in the perinuclear region and smaller mitochondrial cross sections as compared with control specimens. in contrast, cardiac myocyte-specific ablation of mecp2 caused a rightward shift in the size distribution of mitochondria as compared with mecp2-tg hearts. epigenetic processes, including the recognition of dna methylation by mecp2, may play an important role in the control of mitochondrial gene expression, structure, subcellular localization and function in the heart. thus, precise control of mecp2 expression and function is essential to prevent deterioration of metabolic function during chronic heart failure. normalisation of blood pressure does not prevent angiotensin ii-induced dna damage in kidney and heart of ren2 rats weissenberger s., hey v., lau d., schupp n. universität würzburg institut für pharmakologie und toxikologie, versbacher strass 9, 97078 würzburg, germany increased activity of the renin angiotensin system (ras) with enhanced levels of angiotensin ii (angii) leads to oxidative stress with endothelial dysfunction, hypertension and atherosclerosis. epidemiological studies revealed a higher cancer mortality and an increased kidney cancer incidence in hypertensive patients. we could show in vitro and in vivo that angii causes structural dna damage dose-dependently in kidney cells and in the kidney. elevated angii levels therefore might contribute to carcinogenesis of the kidney. in a model of high angii organ levels, the transgenic ren2 rat, carrying an additional renin gene, dna damage in the kidney was analysed in animals of 13 and 32 weeks. untreated ren2 rats exhibit increased blood pressure from the age of 8 weeks on. therefore, the line is kept on angiotensin i converting enzyme inhibitor therapy, which normalizes blood pressure and kidney function to values of control sprague dawley rats. despite this normalized blood pressure of the ren2 animals, a significant higher superoxide production could be observed in kidneys already in 13 week old animals. also a higher frequency of structural dna damage and double strand breaks could be detected in the comet assay and with an antibody against the double strand break marker γ-h2ax in kidneys. further, fittingly, an increased dna repair activity exists in kidneys of ren2 rats compared to control rats. as another organ affected by hypertension the heart of the ren2 animals was analysed for oxidative dna damage. although only a marginal increase of superoxide production could be found, also in the heart a significant higher frequency of dna double strand breaks and cells positive for dna repair activity could be observed. our data let us conclude that normalization of blood pressure in a state of activated ras is not sufficient to prevent angii-induced genotoxicity. this further implies that also patients with treated hypertension still might suffer from endorgan-damaging effects of elevated angii levels. the d541a pore mutation leads to complete inactivation of trpv6 channels in epididymis weißgerber p. 1 , kriebs u. replacement of aspartate residue 541 by alanine (d541a) in the pore of trpv6 channels in mice disrupts ca 2+ absorption by the epididymal epithelium resulting in abnormally high ca 2+ concentrations in epididymal luminal fluid and in a dramatic but incomplete loss of sperm motility and fertilisation capacity raising the possibility of residual activity of channels formed by trpv6 d541a proteins (sci signal 4, ra27, 2011) . it is known from other cation channels that introducing pore mutations even if they largely affect their conductivity and permeability can evoke considerably different phenotypes compared to the deletion of the corresponding protein. to gain insights whether the trpv6 d541a pore mutant still contributes to residual channel activity and/or channelindependent functions in vivo, we compared important fertility-parameters between trpv6 -/and trpv6 d541a/d541a mice: the fertilization rate observed in permanent matings, the in vivo fertilization rate as judged by the rate of embryos isolated from plug positive females of matings with males homozygous for either trpv6 mutation as well as the motility, in vitro fertilization capacity and viability of sperm were reduced to the same extent in both genotypes. also, no differences were observed in copulatory behavior between trpv6 -/and trpv6 d541a/d541a males. the profound reduction in ca 2+ uptake by the epididymal epithelium was identical in trpv6 -/and trpv6 d541a/d541a males. this direct comparison of these parameters indicate that deleting trpv6 does not further aggravate the phenotype observed in trpv6 d541a/d541a mice, and -in our opinion -allows the conclusion that the d541a pore mutant of the trpv6 protein leads to complete inactivation of the trpv6 channel activity or channel-independent scaffolding functions in epididymal epithelium. characterization of a naturally occurring c-terminal mutation (n996i) on herg channel function in hek293 cells sellmaier v. 1 , moretti a. long-qt-syndromes (lqts) are acquired or inherited disorders which predispose patients to cardiac arrhythmias and sudden death. in affected individuals, the electrocardiogram shows a prolongation in the qt interval, due to an unstable repolarization of the action potential. acquired forms of lqts are often the result of treatment with medications that block cardiac potassium channels, such as class iii antiarrhythmic drugs or antihistamines. inherited lqts are caused by mutations in cardiac ion channels. congenital long qt syndrome 2 (lqt2) is caused by loss-offunction mutations in the human ether-á-go-go-related gene herg (also known as kcnh2 or kv11.1). herg encodes the pore-forming α-subunit of the rapid delayed rectifier potassium current i kr, whose physiological role is to repolarize the late phase of cardiac action potentials. herg channel α-subunits exist as 2 isoforms (1a and 1b) that are identical except for structurally divergent n termini. native cardiac ikr channels are tetraheteromers containing 2 of each α-subunit types. a loss-of-function can be due to either defects in a) channel opening (gating), b) ion permeation or c) protein maturation and trafficking. we have identified a so far uncharacterized dominant missense mutation in the herg1 gene (n996i) in a patient with lqt. both herg1a and herg1b subunits were cloned from a human heart cdna library and the specific n996i mutation introduced by site directed mutagenesis. hek 293 cells were transiently transfected with equal amounts of mutated herg1a and wild type (wt) herg1b cdnas and the resulting potassium current compared to herg1a/b wt. whole-cell patch-clamp analysis showed similar current densities for wt versus mutated channels. also the voltage-dependence of activation was unchanged with a halfmaximal activation at -27 mv for wt and -29 mv for the mutated channel assembly. differences were found for the deactivation and inactivation kinetics. the deactivation was faster in the mutated channels with t fast = 62 ms and tslow = 480 ms versus tfast = 90 ms and tfast = 620 ms in wt channels, determined from the tail current at -40 mv after a 5-second pulse to + 50 mv. the half-maximal steady-state inactivation of the tail current was shifted by 20 mv to the depolarizing direction in the mutated channel compared to the wt. a defect in channel gating appears to be the most likely explanation. background: abcc2 is adjacent to p-glycoprotein the most important efflux transporter for various endogenous and exogenous compounds and is expressed at several compartment barriers. by increasing evidence it is shown that the abcc2 polymorphisms are of clinical significance. the aim of our study is to analyze the epigenetic regulation of distinct abcc2 haplotypes by the influence of micrornas. methods: abcc2 cdna clones containing five distinct haplotypes were generated by site-directed mutagenesis, cloned into pires-zsgreen expression vectors and transfected into different cell lines for further functional analysis. one modified vector set contained a short 3'utr sequence of abcc2 whereas the other contained the full length (fl) sequence. mirnas potentially interacting with abcc2 were identified form an mirna array after rifampicin stimulation of hepg2 cells. results: we could demonstrate that there is no difference in the basal protein expression level comparing the two vector types (fl 3'utr vs. mod. 3'utr) concerning the -24c/1249g/3972c (cgc) abcc2 wt in hepg2 cells. using the fl vector construct, the expression level of cac haplotype protein was increased (136,15% ± 20%). transfection assays with the mir-397, which was identified as a candidate mirna targeting abcc2 mrna, and the two different vector constructs harboring the cac or the cgc (wt) haplotype, confirmed that mir-379 was able to down regulate the abcc2 protein expression. there was no significance in downregulation for one abcc2 haplotype, respectively (modified 3'utr: cgc 22-34%; cac 20-40%, fl 3'utr: cgc 20-25%; cac 17-44% down regulation compared to mir-negative control). discussion: differences of abcc2 protein expression level are due to the epigenetic regulation of abcc2 haplotypes. to further characterize the effect of mir-379 on tcg, tgt and cgt abcc2 haplotypes, transfection assays are currently performed using cell cultures as well human primary leukocyte cultures. sex differences affect the pathophysiology and pharmacology of leukotriene biosynthesis werz o. friedrich-schiller-university jena, institute of pharmacy, philosophenweg 14, 07743 jena, germany inflammatory diseases affect more females than males. thus, women suffer more often from asthma, rheumatoide arthritis, alzheimer´s disease, and many autoimmune diseases than men. of interest, sex differences also exist in drug responses, with respect to both pharmacodynamics and pharmacokinetics. we have recently discovered a sex bias in the biosynthesis of pro-inflammatory leukotrienes (lts) due to testosterone, which may represent a molecular basis for gender differences in inflammation and asthma. interestingly, testosterone downregulates lt biosynthesis and also causes a sex bias in the efficiency of lt synthesis inhibitors, which demands for the clinical evaluation of a gender-tailored therapy with anti-lts. we found that certain inhibitors of lt biosynthesis were more efficiently in females than in males, and that androgens are responsible for these gender differences. in fact, the flap inhibitor mk886 effectively reduced ltb 4 pleural levels in female but not in male rats treated with carrageenan, and mk886 increased survival only of female mice in the lt-related disease model of paf-induced lethal shock. administration of testosterone to female mice abolished the protective effects of mk886. in view of the current active development of lt synthesis inhibitors as therapeutics in respiratory and cardiovascular diseases, our data prompt for consideration of gender issues in the development and use of such drugs, in order to optimize medical therapy both for men and women. considering the complexity of deposition and kinetics of air-borne nanomaterials in the lung, potential pulmonary toxicity of biopersistent nanomaterials should be evaluated by inhalation studies. those studies demand special equipment and large quantities of test material. intratracheal instillation appears as a simple and low substance-consuming alternative, although bolus dosing and the more central distribution of the particles in the lung are a well known trade-off. we compared the inflammatory response of the lung to amorphous silica (as) after instillation and inhalation. for inhalation the established short-term protocol for nanomaterials was employed (ma-hock et al. inhalation toxicology, 21:102, 2009 ): male wistar rats were exposed to the test items for 6 h/day on five consecutive days. the lungs were evaluated by analysis of bronchoalveolar lavage fluid (balf) and by histopathology three days and three weeks after the end of the exposure. in a parallel study, rats were intratracheally instilled and equally evaluated three days after instillation. assuming a deposition rate of 10%, the instilled dose corresponded to the aerosol concentration of 10 mg/m 3 used for inhalation. results show that inhalation and instillation of nominally equal mounts of amourphous silica elicit different results in the lung with inhalative treatment being less harmfull. this difference may be due to the bolus effect inevitable linked to instillation. instillation stuldies with amorphous silica may, therefore, be of limited value with respect to doseresponse assessment. sunscreen products containing uv filters protect consumers from the harmful effects of uv exposure. pigmentary grades of metal oxides like zno result in an opaque whiteness as a result of scattering visible light, whereasnanoparticles result in transparent products for better consumer acceptance and thus improved protection of human skin against uv-induced damage. in addition scatter uv light is most efficiently reflected at a nanosize of 60-120 nm. in the last 2 years the toxicological properties of nanozno in comparison with pigmentary zno were examined, the results of these comprehensive studies are presented. all tests were performed according to oecd guidelines, which were modified, especially in regard of substance preparation where appropriate. nanosized zno showed no acute toxicity after dermal application, in the bcop assay as well as in the epiderm assay it showed no corrosion / irritation potential. nanosized zno does not penetrate the intact as well as the sunburned skin. a dermal absorption test in rats (oecd 427) with 65 zn-labelled test item as well as penetration tests in weanling pigs after uv radiation did not show a penetration of the zno nanoparticles through the skin. genotoxicity was tested in vitro in the ames test, in the chromosomal aberration test in v79 cells, both showing negative results whereas the mouse lymphoma mutation test / l5178y/tk+/-cells was positive. in vivo no mutagenic effect was detected in two mouse micronucleus tests, on with intraperitoneally application and another after repeated inhalation. nanosized zno was tested in 5-days, 14-days and 90-days inhalation studies, in all studies the predominant effects were reversible local inflammatory changes in the nasal cavity and lungs, with a noaec of 1.5 mg/m3 in the 90-day study. in a prenatal developmental toxicity study according to oecd tg 414, with repeated inhalation exposure to female wistar rats from gestation day 6 through 19, maternal toxicity was observed by increase lung weights and inflammations in the lungs. but no substance-related effects on reproductive parameters (conception rate, corpora lutea, implantation sites, preimplantation loss, postimplantation loss, resorptions, dead fetuses) and no increase in external and soft tissue malformations and variations could be detected. the overall result of all the toxicological studies with nanosized and pigmentary zno is that the toxicological profile of both is very similar. studies were sponsored partly by cefci lri and partly by basf se. use of reach registration data for improving thresholds of toxicological concern (ttc) wieneke n., dorn s., jakupoglu c., schäfer c., sica m., wiegand c., mostert v. dr. knoell consult gmbh, marie-curie-str. 8, 51377 leverkusen, germany the threshold of toxicological concern (ttc) concept is utilised to identify human exposures that are so low that in-depth toxicological investigations are expendable. this is called "exposure-based waiving". exposure-based waiving serves to focus available resources on substances with relevant human exposure potential. important work into establishing ttc values has been published by munro et al. (1996) . the initial report used a database of 613 organic substances compiled from publicly available sources. in total, 2941 noels were collected in this fashion. the munro concept used the cramer classification to categorise substances according to their hazard potential. we broadened the ttc database by including noaels published on the echa website as per 3 november 2011, containing data for more than 3900 registrations. only nongaseous mono-constituent substances with oral noaels were included in the ttc database. organophosphates and genotoxic substances were excluded from the database as well as noaels obtained for surrogate substances. noaels for all systemic endpoints (general toxicity, developmental toxicity, fertility, neoplasia) were taken into account. where appropriate, default assessment factors of up to 6 were used to establish chronic noaels for each substance. for every eligible substance, we collected the published clp category for acute oral toxicity as a potential predictor of overall hazard potential. this gives rise to five categories of acute oral toxicity. a ttc is calculated from the 5 th percentile of noaels in each of these categories using the reach rules for establishing dnels for workers and the general population. this poster presents the preliminary results for more than 1000 substances. the results indicate that the ttc concept becomes more robust when using the very broad echa database. it also suggests that acute oral toxicity categories can be used as a predictor for the overall hazard potential of a substance. comparison of different in-vitro models for inhalation toxicology with respect to the effects of cigarette smoke total particulate matter wiese j. 1 , schumann b. b-and l-moc can be cultivated up to 70 days without loss of viability, as determined by resazurin-assay. viability of cell cultures was determined by mtt-assay after incubation with increasing doses of tpm. exposure of h322 to tpm (30 mg/l) reduced viability to 95% or 83% after 24 or 72h, respectively. in a549 viability was 67% after 72h with tpm (30 mg/l). the same dose of tpm lead to a decrease in viability to 82% (24h) or 25% (72h) in nhbec and to 74% (24h) or 28% (72h) in plc. as a marker of oxidative stress the level of intra-cellular glutathione (gsh) was determined by hplc. in both the tumor cell lines analysed gsh-level was increased by tpm (5 mg/l). in h322 the induction was 1.6 and 1.2 fold after 24 or 72h, respectively. while in a549 it was 1.3 (24h) and 1.4 fold (72h). in nhec and plc tpm (5 mg/l) did not have a significant effect on gsh-levels after 72h. in n-moc tpm (5 mg/l) also did not modulate gsh after 24h, but it diminished gsh-level by 0.5 fold upon prolonged contact (72h). in b-moc gsh concentrations also decreased to 0.6 or 0.8 fold the level of controls after 24 or 72h incubation. the results presented show that in-vitro models of varying complexity and origin within the respiratory tract clearly differ in their response to tpm, which was used a model inhalative toxicant. the tumor cell lines used seem to be better adapted to chemical stress, while the models closer to the in-vivo situation are more vulnerable. the nongenomic effects of the mineralocorticoid receptor in transgenic mouse heart winter s. 1 , schreier b. within the renin-angiotensin-aldosterone system (raas), the mineralocorticoid receptor (mr) is important for the regulation of fluid and electrolyte balance in the kidney, salivary glands, sweat glands and colon. however, survival of patients with severe heart failure is increased when mr blockage is combined with standard therapy suggesting aldosterone, the mr ligand, as a key factor in the development of cardiovascular diseases, but the mechanism is not yet fully understood. in recent years, evidence accumulated that besides its function as a hormone-activated transcription factor the mr also functions via nongenomic pathways. to investigate the function of the nongenomic effects of the mr in cardiovascular dysfunction, we generated a transgenic (tg) mouse model expressing a truncated human mr lacking the dna-binding site (hmr def ) under control of the cardiac specific α myosin heavy chain promoter (αmhc), a model for nongenomic effects of the mr in the heart. in this mouse model no enhanced mortality could be observed. body weight (bw), heart weight and relative heart weight were not different compared to wild type (wt) while left atrial weight/bw was increased by 20 % (wt 0,25 ± 0,01 mg/g vs. tg 0,30 ± 0,02 mg/g, p<0.05, n=12). compared to wt mice neither surface electrocardiographic experiments nor echocardiographic experiments revealed modified parameters for tg mice under basal (i.e. unstimulated) conditons as well as under β-adrenergic stimulation by isoproterenol (iso, 100 µl 1 mm iso intraperitoneally applied). to uncover the role of aldosterone in the development of cardiovascular diseases treatment with aldosterone and high-salt diet (1%) was performed. after 28 days cardiac function and heart dimensions were analyzed, surface electrocardiographie uncovered increased p duration (16 ± 0.5 ms vs. 14 ± 0.7 ms, p<0.05) and qtc interval (61 ± 2 ms vs. 50 ± 3 ms, p<0.05) in tg (n=7) compared to wt (n=5) animals. these findings probably indicate more sensitive conduction pathways to aldosterone in tg mice. oligomerization is important for regulation of phospholamban activity wittmann t., lohse m. j., schmitt j. p. institut für pharmakologie und toxikologie, versbacher straße 9, 97078 würzburg, germany phospholamban (pln) is a heart specific protein located in the membrane of the sarcoplasmic reticulum. it inhibits the ca 2+ -atpase serca2a, thereby decelerating cytosolic ca 2+ clearance during diastole of the cardiac cycle. upon phosphorylation the inhibitory activity of pln on myocyte ca 2+ transport is attenuated. further, it is believed that phosphorylation favors the formation of (rather inactive) pentamers and that pln pentamers itselves were an inferior substrate for phosphorylation compared to monomers. this would suggest an important role of pln oligomerization in the regulation of pln activity. to prove this hypothesis, we are investigating the patterns and kinetics of pln phosphorylation in the context of alterations in pln structure. the introduction of specific point mutations into the transmembrane region of pln yielded mutants that are purely monomeric (l37a, i40a and c41f) or favor pentamer formation (i45a, v49a). transfected hek293 cells expressing these mutants or wildtype pln were stimulated with forskolin to induce pln phosphorylation before lysis of cells and western blot analysis using antibodies directed against phosphorylated pln. surprisingly, phosphorylation was increased for both monomeric and pentameric pln after stimulation with 0.25µm forskolin for less than one minute. at increasing forskolin concentrations phosphorylation signals increased in parallel for monomers and pentamers. for measurement of phosphorylation kinetics stimulation of cells with 2.5µm forskolin was stopped at different time points. we found phosphorylation of both pln monomers and pentamers within seconds of stimulation. differences in phosphorylation patterns became more pronounced when assays were performed at low temperature (14°c). intriguingly, preliminary analyses suggest that pka dependent phosphorylation occurs first in pentamers and that phosphorylation of monomers may catch up only after pentamer phosphorylation is almost complete. our data suggest that both pln pentamers and monomers are suitable substrates for pka dependent pln phosphorylation. unlike the prevalent assumption, kinetics of pentamer phosphorylation seem to be at least as fast as that of pln monomers in transfected hek293 cells suggesting an important role of pln pentamers in the regulation of pln activity. regulation of cardiac contractility by nucleoside diphosphate kinases in zebrafish wolf n. m. 1, 2 , abu-taha i. in the heart, nucleoside diphosphate kinases (ndpks) can interact with heterotrimeric g proteins, thus regulating camp synthesis in a receptor independent manner and thereby influencing contractility in cardiomyocytes. we further investigated the interaction of ndpk isoforms with heterotrimeric g proteins in the heart in vivo and in vitro using zebrafish embryos and embryonic fibroblast from ndpk a/b double knockout mice (ndpk a/b ko mefs). in zebrafish the morpholino-mediated knockdown of ndpk a did not lead to an obvious phenotype, although the total ndpk activity was reduced. depletion of ndpk b caused a cardiac phenotype characterized by severely impaired atrial and ventricular contractility and insufficient blood flow. the depletion of ndpk b was associated with a significant decrease of protein levels of the heterotrimeric g protein subunits gβγ, gα s and gαi. the knockdown of ndpk c led to a more restricted cardiac phenotype with markedly reduced pumping function of the ventricle, while the atrium was unaffected. in accordance to the reduced cardiac pumping function, camp levels were significantly diminished in the ndpk b and ndpk c morphants. similar findings were obtained in ndpk a/b ko mefs. the absence of ndpk a and b resulted in a decrease of the plasma membrane content of gβ and gαs and a significant reduction in camp synthesis. the protein expression of the isoform ndpk c was also significantly reduced in the ndpk a/b ko mefs. the re-expression of ndpk b but not ndpk a rescued the basal camp production and the membrane content of g proteins. interestingly, the overexpression of ndpk c led to a 5-fold enhancement of the camp level and a significant increase of the membrane content of gβ and gα, and thus rescued the knockout phenotype. our data indicate, that the ndpk isoforms b and c are essential for cardiac contractility, most likely by forming a signaling complex at the plasma membrane including ndpk b, ndpk c and heterotrimeric g proteins. the isoform ndpk c, with its n-terminal hydrophobic region, might serve as a membrane anchor for the ndpk/g protein complex. induction of apoptosis via pka-dependent and pka-independent pathways by cyclic purine and pyrimidine nucleotides in mouse lymphoma cell lines wolter s. 1 camp is a second messenger that plays an important role in intracellular signal transduction of various hormones and neurotransmitters. a major function of camp in eukaryotes is the activation of camp-dependent protein kinase a (pka). pka is involved in the control of a variety of cellular processes. pka exists as an inactive tetramer of a dimeric regulatory (r2) and two catalytic (c) subunits that releases the active c-subunits upon binding of camp. stimulation of the mouse t-lymphoma cell line s49 wild-type (wt) with dibutyryl (db)-camp induces apoptosis by an intrinsic, mitochondria-dependent mechanism. apoptosis induced by db-camp occurs via a pka-dependent mechanism, since s49 kincells lacking the catalytic subunit of pka are resistant to db-camp-mediated cell death. db-camp is cleaved by esterases into the biologically active compound n 6 -mb-camp and into 2'-o-mb-camp. other cyclic nucleotides (cnmps) in addition to camp, like ccmp and cump can also function as second messengers and activate pka and cgmp-dependent protein kinase (pkg) 1 . therefore, we investigated the effects of a series of membrane-permeable analogues of camp, cgmp, ccmp and cump in s49 wt und s49 kincells on apoptosis. stimulation with db-ccmp or db-cgmp induced neither apoptosis in s49 wt nor in s49 kincells. interestingly, we observed apoptosis in s49 wt and s49 kin cells after incubation with membrane-permeable nucleotide acetoxymethyl ester (am)-analogues of cgmp, ccmp, cump and also camp. induction of apoptosis occurs via pkadependent and also pka-independent pathways. a potential role of pkg and of the exchange protein activated by camp (epac) in the induction of apoptosis is unsolved and will be explored by specific pkg-and epac-activators in this system. investigations are on the way to identify the targets, the involved signal transduction pathways and the mechanisms of pro-apoptotic actions mediated by cnmp-ams. (1) wolter s, golombek m, seifert r (2011) differential activation of camp-and cgmpdependent protein kinases by cyclic purine and pyrimidine nucleotides. biochem biophys res commun. in press apoptosis in s49 cells : induction of apoptosis in s49 wt and in s49 cells lacking the catalytic subunit of pka (s49 kin-) with a) db-cnmps and b) cnmp-ams for 72h. nebivolol reduces vascular inflammation in spontaneously hypertensive rats wu z., xia n., förstermann u., li h. institut für pharmakologie, obere zahlbacher str. 67, 55131 mainz, germany nebivolol is a third generation β1 receptor blocker with additional effects on endothelial nitric oxide production. the aim of the present study is to investigate the antiinflammatory effects of nebivolol in vivo. 48 spontaneously hypertensive rats (shr) were divided into two groups: control or nebivolol treatment group. nebivolol treatment (5 mg/kg/day for 10 days) significantly reduced the blood pressure and the heart rate in shr. the drug had no effect on coagulation. aorta from nebivolol-treated rats showed significantly improved endothelial function. nebivolol did not change the expression levels of aortic nf-kb, but significantly reduced its dna binding activity. furthermore, nebivolol decreased the expression of adhesion molecules (e.g. icam-1, vcam-1) and pro-inflammatory cytokines (e.g. il-6). in conclusion, nebivolol reduces vascular inflammation in experimental hypertension. it inhibits nf-kb activity, decreases the expression of adhesion molecules and pro-inflammatory cytokine, and improves endothelial function. characterization of the cellular activity of pde4 inhibitors using two novel pde4 reporter cell lines wunder f., quednau r., barg m., tersteegen a. bayer pharma ag lead discovery wuppertal, aprather weg 18a, 42096 wuppertal, germany cyclic nucleotide-specific phosphodiesterases (pdes) play an essential role in cellular signal transduction by regulating the intracellular levels of camp and cgmp and, therefore, are important pharmacological targets. we report here the generation and pharmacological characterization of two novel pde4 reporter cell lines. plasmid constructs encoding human pde4b1 or pde4d3 were stably co-transfected with the beta1-adrenoceptor in a parental camp reporter cell line expressing a cyclic nucleotide-gated (cng) cation channel, acting as the biosensor for intracellular camp. in this reporter cell line, camp levels can be monitored in real-time via aequorin luminescence stimulated by calcium influx through the cng channel. by using different pde4 and non-pde4 inhibitors, we could show that our novel pde4b1 and pde4d3 reporter cell lines specifically monitor pde4 inhibition with high sensitivity. pde4-selective inhibitors alone did not increase basal luminescence levels in this experimental setting. however, these inhibitors induced concentration-dependent luminescence signals in combination with the adrenoceptor agonist isoproterenol. in contrast, in a stable beta1-adrenoceptor reporter cell line with no recombinant pde4 expression, pde4 inhibitors had no effect on isoproterenol-stimulated luminescence signals. we compared the cellular activity of different pde4 inhibitors with the in vitro inhibition of full-length and truncated (catalytic domain) pde4d3 from cell lysates. two different groups of pde4 inhibitors could be identified. the first group, including the allosteric inhibitors pmnpq and d159153, showed high cellular activity and much better inhibition of full-length versus truncated pde4d3. the second inhibitor group, including classical competitive inhibitors like roflumilast, cilomilast and piclamilast, showed comparably lower cellular activity and similar inhibitory activity on full-length and truncated pde4d3. the results imply that these novel pde4 reporter cell lines are well-suited for the characterization of the cellular activity of pde4 inhibitors and may also support a better understanding of the complex pde4 pharmacology. plexin-b2 is required for kidney regeneration after acute renal failure xia j. 1 , gröne h acute renal failure is a common clinical problem with unsatisfactory therapeutic options and high mortality in humans. therefore, unraveling the mechanisms that promote kidney regeneration and repair may provide new therapeutic strategies for acute renal injury. plexin-b2 belongs to a family of transmembrane receptors which mediate the cellular effects of semaphorins. while plexins have first been described in the context of axon guidance, several recent studies have established them as key regulators of organogenesis, the immune system and cancer. we have recently found that plexin-b2 is highly expressed in the adult kidney, particularly in tubular epithelial cells which are most sensitive to acute ischemic injury. to study the role of plexin-b2 during kidney regeneration we generated mice lacking plexin-b2 specifically in tubular epithelial cells. under physiological conditions, these mice displayed normal kidney morphology and function. in contrast, following ischemia/reperfusion injury, plexin-b2 conditional knockout mice exhibited severely impaired kidney regeneration. while the renal function of control mice fully recovered within 3 weeks after injury, plexin-b2 knockout mice had strongly elevated serum creatinine and urea levels associated with increased morbidity and mortality. this was accompanied by hyperproliferation of tubular epithelial cells and obstruction of tubular lumina. we conclude that plexin-b2 is required for regeneration after acute ischemic renal injury and that pharmacological interventions activating plexin-b2 might represent a new therapeutic strategy in acute renal failure. the nadph oxidase enzyme complex consists of two membrane-bound catalytic subunits (a nox protein and p22phox) and several cytosolic regulatory components including p47phox, p67phox, p40phox and the small gtpase rac1. we have previously shown that treatment of apolipoprotein e knockout mice with resveratrol led to a downregulation of nox2 and nox4 in the heart. our recent data demonstrated that resveratrol also reduced the enzymatic activity of cardiac nadph oxidase. because activation of nadph oxidase enzyme complex is induced by translocation of the regulatory subunits, we studied whether the reduced enzymatic activity is due to an inhibition of such a translocation. indeed, resveratrol treatment prevented rac1 membrane translocation from cytosol. resveratrol is known as an activator and expression enhancer of the longevity gene sirtuin 1 (sirt1). we then wanted to find out whether the effect of resveratrol on rac1 was mediated by sirt1. sirt1 is a histone/protein deacetylase. in vitro incubation of rac1 with sirt1 led to a reduction of lysine acetylation. deacetylation of rac1 on lysine 166 could be identified by mass spectrometry analyses. the lysine 166 lies within the p67phox-binding region of rac1. consistently, in vitro incubation of rac1 with sirt1 markedly reduced its binding activity to p67phox. in conclusion, we provide evidence that rac1 is a direct target molecule of sirt1. sirt1 deacetylates rac1 on lysine 166 and thereby inhibits its interaction with p67phox. this is a novel mechanism of nadph oxidase inhibition by sirt1/resveratrol. mutational analysis of the effects of the cardioprotective drug dexrazoxane on topoisomerase ii beta in vitro yan t., deng s., frensch i., gödtel-armbrust u., wojnowski l. universitätsmedizin der johannes gutenberg-universität mainz institut für pharmakologie, obere zahlbacher straße 67, 55101 mainz, germany dexrazoxane (drz, icrf-187) is the only approved drug shown to protect against anthracycline-induced heart failure. the protection is usually ascribed to the ironchelation by drz, which is thought to reduce anthracycline-induced oxidative stress. however, similarly to anthracyclines, drz is also an inhibitor of the anthracycline target topoisomerase ii (top2). we hypothesized that the cardioprotective effects of drz are mediated by the prevention of the anthracycline-induced dna damage mediated by top2b, the dominant cardiac top2 isoform. this was investigated using top2b mutants resistant to drz, which were expressed in cells depleted of wild-type top2 isoforms. top2b-mediated double-strand dna breaks were assessed as γ-h2ax. the levesl of dsb generated by the top2b mutants in response to the anthracycline doxorubicine (dox) was indistinguishable from that mediated by a wild-type top2b. preincubation with drz depleted wild-type top2b and this was accompanied by a decrease in the dna damage following a subsequent exposure to dox. in contrast, neither top2b depletion nor the reduction of dsb by drz was seen in drz-resistant top2b mutants. furthermore, the cardially ineffective drz analog icrf-161, capable of iron chelation but not of top2 binding, affected neither the stability of top2b, nor the dox-induced dna damage mediated by this enzyme. these results indicate that drz may exert its cardioprotective effects by reducing the dna damage mediated by doxpoisoned top2b rather than by iron chelation. they also suggest a cardioprotective function of top2b, which is currently under investigation using cardiomyocyte-specific top2b mouse knockouts. aminoglycosides are important antibiotics in the treatment of life-threatening infections, especially those caused by gram-negative bacteria. their nephrotoxic and ototoxic potential is well-known, but little is known about the effects of aminoglycosides on the male reproductive system. we studied the effects of four aminoglycosides on sertolicells in vitro. rat sertoli-cells from the cell line serw3 were cultivated for 3, 6, and 9 days in dmem supplemented with three different concentrations of amikacin, streptomycin (30 mg/l, 100 mg/l, 300 mg/l), gentamicin or tobramycin (10 mg/l, 30 mg/l, 100 mg/l). we determined the expression of two junctional proteins (connexin 43, ncadherin) and one protein of the cytoskeleton (vimentin) by western blot. cells were solubilized in lysis buffer. lysates were separated by sds-page and electroblotted on a pvdf-membrane. after incubation with primary antibodies overnight and horseradish peroxidase-conjugated secondary antibody the visualization was achieved by a chemiluminescence-detection system. in addition, proteins were detected by immunohistochemistry. after three days in culture amikacin caused the most pronounced effect. at the lowest concentration tested (30 mg/l) connexin 43 and n-cadherin were reduced to 55±19% and 92±10% of the controls (n=6). no change was recognized for vimentin (102±16%). effects obtained with streptomycin were less pronounced for these these proteins (68±16%, 96±7%, and 102±13%, respectively). similar, but less pronounced effects were observed with gentamicin and tobramycin at a concentration of 10 mg/l (connexin 43: 88±15% and 81±15%; n-cadherin: 95±18% and 103±11%; vimentin: 81±12% and 102±19%) and 30 mg/l (connexin-43: 84±19% and 78±18%; n-cadherin: 98±19% and 103±13%; vimentin: 102±8% and 115±15%). after incubation for 6 and 9 days the effects occurred in the same range. the substances showed no influence on the viability of serw3 sertoli-cells up to 300 mg/l in the mtt assay. by immunohistochemistry we showed that the localisation of the proteins -connexin 43 and n-cadherin at the cell membrane and vimentin in the cytoplasm -was not influenced by the aminoglycosides. large conductance calcium-and voltage-gated potassium (bk) channels play an important role in controlling membrane potential and calcium influx, and are strongly modulated by protein kinases at multiple sites. the stress-regulated exon (strex) adds to the bk channel c terminus a cysteine-rich insert of 59 amino acids that inverts the channel regulation by protein kinase a (pka) from excitatory to inhibitory. here we investigated the mechanisms by which the strex insert influences bk channel regulation by protein kinase c (pkc). activity of bk channels without strex insert (bk-zero), transiently expressed in hek cells, was inhibited by pkc in inside-out membrane patches (~ 50% inhibition). bk channels with strex insert (bk-strex), however, were insensitive to pkc. phosphomimetic mutation of a pkc phosphorylation site (s700e) in bk-strex, resulted in a ~50% reduction of basal channel activity, whereas the s700a mutant retained normal activity. to examine whether palmitoylation, and thus association of the strex domain with the plasma membrane, prevents pkc inhibition of bk channel gating, palmitoylation was abolished by either site-directed mutagenesis (c12:13a) or by pharmacological inhibition of palmitoyl transferases with 2-bromopalmitate (2-bp). both, mutation and pretreatment with 2-bp resulted in the expression of bk-strex channels which were sensitive to pkc (~ 50% inhibition of channel activity). no inhibitory pkc effect was observed in patches of the bk-strex s700a channel mutant pretreated with 2-bp. in a clonal rat somatomammotroph pituitary cell line (gh3b6), in which pcr products without (zero) and with the 174 bp strex exon could be identified, the pkc activator pma blocked channel activity by ~25 %. this inhibition was increased to over 50% when gh3b6 cells were pretreated with 2-bp, indicating that both channel isoforms were functionally active. in summary, the present study demonstrates that palmitoylation of strex prevents bk channel regulation by pkc, which is mediated by phosphorylation of ser700, probably by steric hindrance. our results provide further evidence for a cross-talk between palmitoylation and phosphorylation as a crucial mechanism underlying the dynamic regulation of ion channels. human pleural mesothelial met-5a cells are a limited in vitro model system in determining potential asbestos-like genotoxic effects of multiwall carbon nanotubes ziemann c. 1 , reamon-büttner s. multiwall carbon nanotubes (mwcnt) are nanomaterials with important technological impact. depending on their diameter, length, and biopersistence, however, some mwcnt seem to exhibit a fiber-like cytotoxic and genotoxic potential, similar to asbestos. thus, a project funded by the german federal ministry of education and research (bmbf contract no. 03x0109a) focuses on potential adverse biological effects of different types of mwcnt to enlarge the knowledge base about toxicity determining parameters. this project comprises both in vitro (rat) and in vivo endpoints with long amosite asbestos as a positive control. as mesothelial cells are target cells for adverse effects of asbestos, in particular mesothelioma development, the human sv40transformed, non-malignant pleural mesothelial cell line met-5a was chosen as the main in vitro model in this project. in the present study part, met-5a cells were characterized concerning their usefulness as an in vitro model to study potential asbestos-like cytotoxic and genotoxic effects of different mwcnt varieties. using an mwcnt-optimized lactate dehydrogenase liberation assay and proliferation parameters derived from cell counts, concentration-dependent cytotoxicity of long amosite asbestos (2, 10, and 20 µg/cm 2 ) was demonstrated in met-5a cells. cells also showed asbestosinduced increase in dna-strand breaks and oxidative dna-damage in the hogg1modified comet assay. thus, met-5a cells were responsive to asbestos treatment. owing to asbestos potential to induce aneugenic effects and spindle fiber damage, micronucleus induction, determination of numerical chromosome aberration, and altered meta-, ana-, and telophase morphology were planned as in vitro endpoints. met-5a cells were thus initially characterized in this regard and were found to exhibit highly variable chromosome numbers with lower than 10% cells exhibiting a normal diploid chromosome set, an up to twentyfold higher spontaneous micronucleus frequency, as compared to polychromatic bone marrow erythrocytes in rodents, and a profound number of aberrant meta-, ana-and telophases with bridges, lagging chromosomes and multipolar divisions. in conclusion, met-5a cells are of only limited value as an in vitro model system to study potential asbestos-like effects of mwcnt and also biopersistent fibers. the cells are indeed responsive to asbestos, but unfortunately demonstrate marked genomic instability and thus limited significance concerning genotoxic effects. waixenicin a inhibits cell proliferation through magnesium-dependent block of trpm7 channels zierler s. 1 transient receptor potential melastatin 7 (trpm7) channels represent the major magnesium-uptake mechanism in mammalian cells and are key regulators of cell growth and proliferation. they are abundantly expressed in a variety of human carcinoma cells controlling survival, growth and migration. these characteristics are the basis for recent interest in the channel as a target for cancer therapeutics. we screened a chemical library of marine organism-derived extracts and identified waixenicin a from the soft coral sarcothelia edmondsoni as a strong inhibitor of overexpressed and native trpm7. waixenicin a activity was cytosolic and potentiated by intracellular free magnesium (mg 2+ ) concentration. mutating a mg 2+ -sensitive site on the trpm7 kinase domain reduced the potency of the compound, whereas kinase deletion enhanced its efficacy independent of mg 2+ . waixenicin a failed to inhibit the closely homologous trpm6 channel and did not significantly affect trpm2, trpm4, and crac (calcium release activated calcium) channels. therefore, waixenicin a represents the first potent and relatively specific inhibitor of trpm7 ion channels. consistent with trpm7 inhibition, the compound blocked cell proliferation in human jurkat t-cells and rat basophilic leukemia cells. based on the compound's ability to inhibit cell proliferation through mg 2+ -dependent block of trpm7, waixenicin a or structural analogs may have cancer-specific therapeutic potential, particularly since certain cancers accumulate cytosolic mg 2+ . release of metals from different sections of domestic drinking water installations zietz b. p., richter k., laß j., suchenwirth r., huppmann r. governmental institute of public health of lower saxony division of environmental medicine and environmental epidemiology, roesebeckstraße 4-6, 30449 hanover, germany different metals were used as important piping materials in the drinking water supply for a long time. due to corrosion metals can leach into the tap water. of special importance is the toxic element lead. however other heavy metals in drinking water such as copper, nickel and cadmium can also give reason for health concerns. in this study it was investigated in which amount relevant metals were released from different parts of domestic installations into the cold water. for the spatial allocation of the emission sources a sequential water sampling protocol was used after three hours of stagnation time representing the first 5 litre of the water column. after stagnation ten sample volumes were collected in series. existing facilities of domestic installations constructed with different plumbing materials were examined predominantly from residential buildings. the elements al, as, cd, cr, cu, fe, mg, mn, ni, pb, sb, se, u and zn were detected by means of icp-ms. in total 16 water pipe strands of 11 domestic installation systems were examined. they comprised 379 single water samples and 5306 single parameters. depending upon the type of plumbing different courses and concentration ranges of the elements could be measured in the tap water samples. terminal taps or installation parts were frequently responsible for a release of nickel and in several cases of cadmium. the concentration courses of the element zinc proved as a good indicator for the allocation of the emission source to a brass containing section of the installation (zinc as an alloy component of brass). one can conclude that an investigation by means of a sequential water sampling protocol and multi-element detection can be a valuable non-destructive method for drinking water-hygienic investigations of domestic installations. novel interaction partners of the murine trpc4 protein zimmermann j., beck a., flockerzi v. universität des saarlandes experimentelle und klinische pharmakologie und toxikologie, kirrberger str. 1, 66421 homburg, germany in this work novel interaction partners of the murine protein transient receptor potential canonical 4 (mtrpc4) were identified. the trpc4 protein is the major subunit of a cation channel, residing in the plasma membrane. it comprises six trans-membrane domains and cytosolic amino and carboxyl termini. two major splice variants of the trpc4 gene exist, trpc4a (974 aa) and trpc4b (890 aa), trpc4b lacks aa 781 to 864 of the trpc4a variant. both trpc4 variants are co-expressed in endothelial cells, intestinal smooth muscle and brain. to identify trpc4-interacting proteins a yeast two-hybrid system, cytotrap®, which allows identification of protein-protein interactions within the cytosol was used. a premade mouse brain cdna library was screened by the cytosolic amino and carboxyl terminal parts of mouse trpc4a (aa 1 to 324; aa 622 to 974). for the carboxyl terminal part fourteen proteins were identified. to independently prove the interaction, the fulllength cdnas of all fourteen proteins were cloned, fused to a flag-tag and coexpressed with trpc4 in hek 293 cells. co-immunoprecipitations were performed for all candidates using both the anti-flag-antibody and the antibody for trpc4. in addition, changes of cytosolic calcium were monitored and trpc4 currents were recorded in hek 293 cells expressing the candidate cdnas and stably expressing the trpc4a or trpc4b and the muscarinic receptor type 2 cdnas. the tarbp2 protein, one of the candidates shown to interact with trpc4, changed calcium influx when coexpressed with trpc4. in order to identify the domains of trpc4 responsible for its interaction with the tarbp2 protein, six trpc4-gst-fusion proteins covering the carboxyl terminal 353 aa of trpc4a were expressed in e. coli and used for pull-down experiments. by this approach two domains of trpc4 could be identified to interact with tarbp2. one of these domains is well conserved within the trpc5 protein, corresponding to the result, that trpc5 and tarbp2 effectively co-immunoprecipitate, too. the tarbp2 protein has been shown to be a component of the risc loading complex, also known as the micro-rna loading complex which is composed of dicer1, eif2c2/ago2 and tarbp2 (chendrimada et al. 2005) . by its interaction it may link trpc4 to pre-microrna processing. increased levels of angiotensin ii provoke dna damage and have influence on dna repair in mouse kidneys zimnol a., brand s., schupp n. universität würzburg institut für toxikologie, versbacherstr. 9, 97078 würzburg, germany the renin-angiotensin system (ras) plays a crucial role concerning the blood pressure, electrolyte balance and cardiovascular homeostasis. angiotensin ii (ang ii), the active hormone of the ras, in higher concentrations leads to vasoconstriction, oxidative stress and hypertension. hypertensive patients have an increased risk to develop cancer, especially kidney cancer. we have shown in vitro and in vivo, that ang ii is capable to cause an elevation of blood pressure as well as dna damage dose-dependently. to investigate whether the high blood pressure or the enhanced levels of ang ii are responsible for dna damage, male c57bl/6-mice were equipped with osmotic minipumps, delivering ang ii in a concentration of 600ng/kg · min during 28 days. additionally they were treated with ramipril, an angiotensin-converting-enzyme blocker, with the ang ii receptor antagonist candesartan, the vasodilator hydralazine, and the antioxidant tempol. dna damage was analysed with the comet assay. we measured the base excision repair (ber)-related dna repair in the kidney with a comet-based in vitro repair assay. furthermore, the distribution and expression of the ang ii-type 1 (at1) receptor in the kidney was analyzed by immunohistochemistry. treatment with ang ii led to a significant increase of blood pressure, whereas the medication with candesartan decreased the systolic blood pressure. the intervention with hydralazine lowered the blood pressure only for a short time. the other substances had no effect at all on the blood pressure. genomic damage, quantified with the comet assay, was augmented by ang ii and improved by all interventions, particularly by candesartan and tempol. beyond that, ang ii showed a tendency to reduce dna repair. treatment with candesartan, hydralazine and tempol increased the repair capacity. furthermore, ang ii tended to result in a downregulation of the at1 receptor in kidney tubule cells. candesartan and ramipril, especially were able to augment the expression of the at1 receptor, whereas hydralazine achieved the opposite. these results demonstrate that ang ii leads to dna damage in the kidney independent of blood pressure. apparently elevated levels of ang ii affect dna repair and expression of at1 receptor. to confirm these findings we are going to examine more precisely the manifestation of other enzymes, which are implicated in dna repair. regulation of hcn channel activity by cyclic cytidine 3´, 5´-monophosphate zong x., krause s., chen c. -c., gruner c., cao-ehlker x., fenske s., wahl-schott c., biel m. lmu münchen, department pharmazie, pharmakologie für naturwissenschaften center for integrated protein science munich (cipsm), butenandtstr. [5] [6] [7] [8] [9] [10] [11] [12] [13] 81377 münchen, germany hyperpolarization-activated cyclic nucleotide-gated (hcn) channels play a key role in controlling cardiac pacemaker activity and are essential for normal function of neuronal circuits. hcn channels are principally gated by voltage but are coactivated by the cyclic nucleotides camp and cgmp which directly bind to a c-terminal binding domain. recently, cyclic cmp (ccmp) was shown to be present in various cell lines and tissues at concentrations that are comparable to cellular cgmp levels. moreover, there is recent evidence that ccmp can activate camp-and cgmp-dependent protein kinases in vivo. here, we examined whether ccmp exerts effects on hcn channels. to this end, we recorded hcn channel-mediated currents (i h) in hek 293 cells that stably express hcn1, hcn2, hcn3 or hcn4, respectively. currents were measured either with a standard patch-clamp setup or by employing the planar patch-clamp technology. in hcn2 and hcn4 channels, ccmp shifted the membrane potential for half maximal activation (v0.5) to more positive values. in addition, ccmp accelerated activation while it slowed down deactivation kinetics. the ec50 for ccmp was 30 µm which is about 5 times higher than the ec50 of cgmp. cyclic cmp is a partial agonist of hcn channels since it activates only 65 % of the maximal current obtained with camp or cgmp. to identify in vivo effects of ccmp we recorded ih of murine sinoatrial node (san) cells in the presence and absence of 1 mm ccmp. like for heterologously expressed hcn channels, ccmp shifted v0.5 of ih by about +5 mv. importantly, the steepness of the diastolic depolarization of san pacemaker potentials which is mainly determined by the amplitude of ih was profoundly increased by ccmp, compared to control conditions. as a consequence of the upregulation of ih the frequency of san pacemaker potentials was increased by about 20 % in the presence of ccmp. our results suggest that ccmp is a physiological regulator of hcn channel activity. a 3d actinic keratosis like construct for assessment of innovative tumour therapeutics zoschke c. 1 the incidence of actinic keratosis (ak) has increased dramatically in the last decades and it is considered the most frequent carcinoma in situ today. especially immunosuppressed patients are at high risk to develop invasive squamous cell carcinoma (scc) [1] which asks for most efficient and well-tolerated ak therapy. yet, current measures do not fit with these demands. nucleotide analogues, recently identified by molecular modelling [2, 3] , outperformed the current standard for the therapy of actinic keratosis, 5-fluoruracil, when tested in the tumour cell line scc25, in normal human keratinocytes and fibroblasts [4] . as next step in pre-clinical drug assessment, we aimed to characterise the effect of the most selective nucleotide analogue oxbu in reconstructed human tumour skin. based on the 3d construct of scc developed by höller and co-workers [5] for start we introduced several adaptations with respect to keratinocyte, fibroblast and scc12 seeding to grow an aklike construct with scc12 cells forming nests in particular in the epidermis. in addition, first experiments with the oxbu on the ak like constructs showed promising results for an efficient treatment of actinic keratosis. efficacy was derived from immunohistology (marker for proliferation: ki-67, marker for scc: cytokeratin-10, axl, marker for invasion: mmp2, marker for apoptosis: caspase-7, nuclei were stained with dapi) as well as the effects on the secretion of cytokeratin-18 and its caspase-induced cleavage product into the culture medium following drug exposure for up to 7 days. efficacy of a 0.05% oxbu solution proved close to or even better when compared to both a 0.1% 5fluorouracil and 0.025% aphidicolin solution. the former being the gold standard of current ak therapy, the latter is a frequently used inhibitor of human polymerase alpha and delta, however, failed to be introduced into clinical use. -in fact, in monolayer cultures aphidicolin proved most toxic for normal human keratinocytes which was not true with oxbu [4] . -therefore, these 3d tumour constructs offer a new approach to pre-clinical drug assessment and may be added to other 3d models of skin diseases currently gaining increased interest as test platforms. ima910, a multi-peptide cancer vaccine for advanced colorectal cancer, induces multiple cd8+ and cd4+ t-cell responses associated with improved survival walter s. 1 , kuttruff s. ima910 is a novel peptide-based vaccine consisting of 10 hla-a*02 and 3 hla-dr binding synthetic peptides that were identified based on natural presentation on human colorectal cancer (crc) samples. ima910 was characterized in a phase i/ii trial in advanced/metastatic crc patients being at least clinically stable after 12 weeks of first-line oxaliplatin-based therapy. all patients received a single application of low-dose cyclophosphamide for immunomodulation. this was followed by repeated ima910 vaccinations in combination with low-dose gm-csf (cohort 1; n=66) or ima910 / gm-csf plus topically applied imiquimod (cohort 2; n=26). before and post vaccination, patients were analyzed by hla-multimer assay and intracellular cytokine (ics) assay for cd8 + t-cell responses and by ics assay for cd4 + tcell responses. as immune status biomarkers, 6 phenotypically defined myeloid derived suppressor cell populations (mdsc1-6) were analyzed prior to immunotherapy. tumor status of patients was monitored repeatedly by ct/mri according to recist and corresponding tumor scans were reviewed centrally. clinical assessment included disease control rate (dcr), time to progression (ttp), progression-free survival (pfs) and overall survival (os). ima910 overall was immunogenic in 73/81 (90%) evaluable patients, with 43% and 65% of patients mounting multiple cd8 + and cd4 + t-cell responses, respectively. patients that received the immunomodulator imiquimod presented with significantly more multiple cd8 + cell responses as detected by ics (p=0.016). multiple cd8 + and multiple cd4 + responses were individually associated with significantly better clinical outcome. the association was most pronounced for patients with both multiple cd8 + and multiple cd4 + responses. these patients had significantly higher dcr at 6 months (p=0.002), improved ttp (p=0.006) and improved pfs (p=0.009) than other patients. most importantly, a trend for prolonged os was also observed in these patients (p=0.088, hazard ratio 0.53). in the study population, levels of 5 different mdsc phenotypes were significantly increased as compared to age/gender matched controls. high mdsc levels were associated with fewer immune responses and for mdsc4 and mdsc5 high frequencies were associated with shorter os (p=0.007 and p=0.019, respectively). to summarize, both hla-a*02 and hla-dr restricted peptides in ima910 were immunogenic. a significantly better clinical outcome of multi-tumap responders in comparison to patients with one/no tumap response strongly indicates clinical activity of ima910. acrylamide (aa), a genotoxic carcinogen (iarc class 2a) is formed in food by thermal treatment from different precursors. after oral ingestion, aa is metabolically epoxidized in the liver by cyp450 2e1 into glycidamide (ga). ga binds to dna, forming covalent adducts, primarily at n7 of guanine (n7-ga-gua). both, aa and ga undergo conjugation to glutathione (gsh) to be excreted via urine as mercapturic acids (ma), namely nacetyl-s-(2-carbamoylethyl)-cysteine (aama), and n-acetyl-s-(2-hydroxy-2carbamoylethyl)-cysteine (gama). in a dose response study, encompassing the dosage range from human dietary exposure levels up to 10 mg/kg bw, female sprague dawley (sd) rats on a diet devoid of detectable aa content were gavaged with single doses of aa. formation of urinary mas and of n7-ga-gua dna adducts in liver, kidney and lung was measured 16 h after application, a time point where cmax of n7-ga-gua was reached. the untreated control group was found to excrete about 0.8 nmol (aama plus gama) in the urine (16 h), indicating a background of endogenous aa formation. compared to untreated control, the lowest dosage of 0.1 µg aa/kg bw neither resulted in significantly enhanced ma excretion, nor in a detectable n7-ga-gua adduct levels in any organ tested (limit of detection, lod, 0.2 adducts/10 8 nucleotides). at the tenfold higher dose (1 µg/kg bw), adducts were found in kidney (about 1 adduct/10 8 nucleotides) and lung (< 1 adduct/10 8 nucleotides), but not in liver. at 10 and 100 µg/kg bw, adducts were found in all three organs, at levels not significantly different to those found at 1 µg aa/kg bw (about 1-2 adducts/10 8 nucleotides). the results of this in vivo study and of further recent research on aa toxicology will be discussed with respect to risk assessment. exposure of rats to single doses of aa in the range of human dietary exposure (0.1-10 µg/kg bw ) leads to n7-ga-gua adduct levels in the tissues monitored obviously not exceeding the range of steady state background dna lesions associated with endogenous/exogenous exposure to various genotoxic electrophiles. thus, the question of significant impact on human background dna damage resulting from exposure to a given genotoxic carcinogen, and on potentially ensuing biological consequences may become a highly relevant issue in risk assessment. pharmaco-economic impact of price, volume and demographic development böcking w., kirch w. institut für klinische pharmakologie, medizinische fakultät der tu dresden, fiedlerstr. 27, 01307 dresden health insurance costs in germany have grown by 3% p.a. over the last ten years and amount to approx. 280 bn eur in 2009. while costs for stationary treatment as the largest cost category have been intensely analyzed over the past years, pharmaceutical expenses have been analyzed in less detail, mostly focusing on the statutory health insurance side, even though pharmaceutical expenses have grown almost twice as much as costs for ambulant treatments. therefore, the question was asked how pharmaceutical expenses in a large german private health insurance company are allocated with respect to age and indication groups, and how those have developed from 2007 to 2011. the data of a private health insurance company with more than 600.000 customers was split into price and volume effects per age group to understand if price or volume drives the cost development. additionally, the two largest indication groups are analyzed in detail. as a result, both price and volume effects drive an overall cost increase. these effects are even stronger in older age groups. this cost increase is not sustainable for the german health insurance system over a longer period of time and will even further increase due to the ageing of the german population. a novel animal replacement system for the detection of endocrine disruptive capabilities in sexual development scheider j. 1, 2 , winter p. alternatives to animal testing for prediction of local toxicity and genotoxicity have been recently established. however, currently these methods are not suitable for measuring endocrine effects in developing organs such as e.g. embryonic gonads. here we present a phenotypic anchoring of a comprehensive study on sex-specific gene expression analysis accompanied by histological analysis of endocrine disruption in chicken embryo gonads, having the potential for an animal replacing system for endocrine disruptive toxicologic and ecotoxicologic examinations of chemicals. chicken embryos were inoculated with different amounts of tributyltin (tbt) and bisphenol-a (bpa). embryos were incubated and their gonads analyzed histologically 2 d prior to hatching. from identically treated embryos right and left testes and ovaries were separated and genome-wide transcription profiles generated using supertag digital gene expression (st-dge, supersage) profiling. male and female gonadal tissues both revealed histological aberrations in response to tbt and bpa. female gonads became masculinized in response to tbt and, viceversa, bpa-treated male gonads underwent feminization whereas in female gonads clearly visible structural aberrations occurred. in both chemicals mortality increased especially in the most affected sex (tbt: females, bpa: males). the expression profiles of more than 60 million mrnas revealed massive effects of both chemicals, tbt and bpa, on important cellular signaling pathways. gene expression differences were most pronounced in the phenotypically most affected sex. our results demonstrate that endocrine disruptive chemicals exert their effects on several levels including but not restricted to known hormone-based pathways. together with an ongoing study of gene expression differences in very young life stages and different chemicals these data will form the base for a blow-by-blow analysis of sexspecific gene expression of embryonic development. the project builds on already existing and further to generate data with the aim of the development of an in vitro method for testing chemicals at chicken eggs for 1) replacement of tests on juveniles and (sub-) adult rodents, 2) stages with impossibility of sensation of pain in the individuals, 3) highly sensitive prospects of modes of action of chemicals, which 4) might show consequences in the next generations. 3 channels are critical for oscillatory burst discharges in the reticular thalamus and absence epilepsy differential distribution of three members of a gene family encoding low voltage-activated (t-type) calcium channels hippocampal seizure resistance and reduced neuronal excitotoxicity in mice lacking the cav 2.3 e/r-type voltage-gated calcium channel transcriptional upregulation of cav3.2 mediates epileptogenesis in the pilocarpine model of epilepsy structure and functional expression of a member of the low voltage-activated calcium channel family a molecular determinant of nickel inhibition in cav3.2 t-type calcium channels histidine residues in the is3-is4 loop are critical for nickel-sensitive inhibition of the cav2.3 calcium channel substrate recognition and translocation by polyspecific organic cation transporters proton pump inhibitors inhibit metformin uptake by organic cation transporters (octs) structural determinants of inhibitor interaction with the human organic cation transporter oct2 (slc22a2) functional characterization of the human organic cation transporter 2 variant p.270ala>ser extra-adrenal glucocorticoid synthesis in the intestinal epithelium: more than a drop in the ocean? local glucocorticoid production in the mouse lung is induced by immune cell stimulation biomimetic materials in tissue engineering biomaterials offer cancer research the third dimension synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering injectable self-assembling peptide nanofibers create intramyocardial microenvironments for endothelial cells directed growth of fibroblasts into three dimensional micropatterned geometries via selfassembling scaffolds novel pcl-based honeycomb scaffolds as drug delivery systems for rhbmp-2 tissue engineering spatio-temporal vegf and pdgf delivery patterns blood vessel formation and maturation presentation of rgds epitopes on self-assembled nanofibers of branched peptide amphiphiles controlling mammalian cell interactions on patterned polyelectrolyte multilayer surfaces langmuir avintegrins as receptors for tumor targeting by circulating ligands heparin binding nanostructures to promote growth of blood vessels tirrell endothelial cell adhesion to the fibronectin cs5 domain in artificial extracellular matrix proteins design and bioproduction of a recombinant multi(bio)functional elastin-like protein polymer containing cell adhesion sequences for tissue engineering purposes stimuli-responsive thin coatings using elastin-like polymers for epac as a novel effector of airway smooth muscle relaxation ) camp inhibits airway smooth muscle phenotype modulation functional roles of epac and pka in human airway smooth muscle phenotype plasticity assessment of the sensitizing and irritative potential of preservatives by loose-fit coculture-based sensitization assay (lcsa) sonnenburg a nsc-631570 (ukrain) in the palliative treatment of pancreatic cancer. results of a phase ii trial association of funding and conclusions in randomized drug trials: a reflection of treatment effect or adverse events a general method for the covalent labeling of fusion proteins with small molecules in vivo robust single-particle tracking in live-cell time-lapse sequences correlation of structural class with no-observed-effect levels: a proposal for establishing a threshold of concern trbp recruits the dicer complex to ago2 for microrna processing and gene silencing index a 009, 019, 035 011, 014 003, 044 objective: hypertension and arterial stiffness is influenced by environmental and genetic factors. high plasma sodium concentration leads to mechanical stiffening of endothelial cells resulting in endothelial dysfunction and elevated blood pressure. here we investigated whether endothelial cell stiffness of ex vivo preparations of human arteries is linked to plasma sodium concentrations and functional genetic variants of the mineralocorticoid receptor (nr3c2), rs2070951 modulating blood pressure, renin, and aldosterone levels, and rs5534, which alters a mirna binding site. design and methods: twenty patients were enrolled after a vein stripping procedure and collateral arterial blood vessels were prepared for atomic force microscopy (afm). plasma sodium concentration was routinely determined and dna for genotyping was extracted from edta blood samples. sodium levels >140 mmol/l were defined as 'high'. after application of 5 µm amiloride, a specific blocker of the endothelial sodium channel (enac) changes in endothelial cell stiffness, were defined as 'weak' (≤10%), or 'strong' (>10%). statistical analyzes were performed by anova. results: in ex vivo artery preparations of patients with high sodium levels (n=12), mechanical stiffness of endothelial cells was tend to increase (∆ amiloride) (p=0.06). both nr3c2 variants were associated with a change >10% in endothelial stiffness after amiloride treatment. the rs2070951 c allele was significantly associated with a strong amiloride response (p=0.024), while the rs5534 a allele only showed a trend towards stronger amiloride effects (p=0.06). conclusion: our findings indicate that high plasma sodium concentration results in an increased endothelial amiloride response and thus influencing mechanical stiffness, modulated by functional nr3c2 variants. our novel approach linking patients' sodium levels and genetic status to endothelial stiffness by afm will be further evaluated in larger clinical settings. protein expression changes in bap-exposed human bladder cancer cells from spliceosome activation towards redistribution of the cytoskeleton after long-term exposure to subacute concentration schmitz-spanke s., pink m., jeske e., stempelmann k., rehn s., verma n., rettenmeier a. w. universitätsklinikum essen institut für hygiene und arbeitsmedizin, hufelandstr. 55, 45122 essen, germany deregulation of the β-catenin signaling pathway plays an important role in the development of hepatocellular tumors. activating mutations in ctnnb1 (encoding β-catenin) are frequently observed in murine and human liver tumors (e.g. human hepatoblastomas). activation of β-catenin signaling induces an overexpression of several cytochrome p450 (cyp) enzymes, including cyp2e1. cytotoxicity of acetaminophen (aap) is based on its cyp2e1-catalyzed metabolism to the electrophilic compound n-acetyl-p-benzo-quinone imine, which forms covalent adducts with cellular macromolecules if depletion of glutathione occurs. treatment with aap should therefore lead to a selective damage of cyp2e1-overexpressing ctnnb1mutated hepatoma cells. mice were injected with a single dose of the liver carcinogen n-nitrosodiethylamine (den) and subsequently treated with the tumor promoter phenobarbital to select for ctnnb1-mutated tumors. administration of a single dose of aap (300 mg/kg of body weight) followed the tumor promotion protocol. two days after treatment immunohistological analysis of the livers showed about 90% necrotic tissue in the larger tumors which were positive for glutamine synthetase (gs), a marker for ctnnb1-mutated tumor cells. by contrast, gs-negative tumors remained unaffected. at later time points we observed regeneration processes with infiltration of the necrotic tissue by inflammatory cells followed by fibrotic cells. proliferation of normal hepatocytes surrounding the damaged areas could also be observed. however, repopulation of parts of the former tumor areas by remaining gs-positive tumor cells was also detected. these results suggest that treatment with aap might serve as a future therapeutic possibility to selectively poison cyp2e1-overexpressing hepatoma. release of 5,6-epoxyeicosatrienoic acid (5,6-eet) upon neuronal activity induces trpa1-dependent mechanical pain hypersensitivity sisignano m. 1 , epoxyeicosatrienoic acids (eets) are cyp-epoxygenase (cyp450) derived metabolites of arachidonic acid (aa) which act as endogenous signaling molecules in multiple biological systems. we investigated the specific contribution of 5,6-eet to transient-receptor potential-(trp)-channel activation in nociceptor neurons, and its consequence for nociceptive processing. we found that during capsaicin-induced nociception 5,6-eet-levels increased in the drg and it is released from activated sensory neurons in vitro. 5,6-eet potently induced a calcium flux [10 nm] in cultured drg-neurons which was completely abolished when trpa1 was deleted or inhibited. in spinal cord slices 5,6-eet dose-dependently enhanced the frequency, but not the amplitude of spontaneous excitatory postsynaptic currents (sepsc) in lamina ii neurons that also respond to mustard oil (aitc), indicating a presynaptic mechanism. furthermore, 5,6-eet-induced enhancement of sepsc frequency was abolished in trpa1 null mice, suggesting that 5,6-eet pre-synaptically facilitates spinal cord synaptic transmission via trpa1. finally, intrathecal injection of 5,6-eet caused mechanical hyperagesia in wild type but not trpa1 null mice. we conclude that 5,6-eet is synthesized upon acute activation of nociceptors and leads to mechanical hypersensitivity via trpa1 at central afferent terminals in the spinal cord. sisnaiske j. 1 , hardelauf h. introduction: neurite outgrowth and plasticity of neuronal networks are essential processes e.g. during brain development and learning. thus, morphological readouts of neuronal connectivity are thought to be important endpoints to assess neurotoxic effects of environmental chemicals as well as when discovering new drugs. to analyze neurite outgrowth and connectivity level rapidly and easily in vitro we developed the network formation assay (nfa) (pct/ep2010/002811). this platform requires a spatially standardized hexagonal array for culturing neuronal networks with no need to fix or stain the cells to visualize neuritic processes. methods: to demonstrate the feasibility of the nfa we performed experiments in which we disrupted mature neurite networks or inhibited generating networks of human sh-sy5y cells with different concentrations of acrylamide (acr). we also observed the counteracting effects of brain-derived neurotrophic factor (bdnf) and calpeptin in these systems. to create the hexagonal array we used a poly(dimethylsulfoxide) bilayer stencil comprising through holes for adhesion spots and interconnecting tracks. plasma stencilling a peg-coated glass substrate produces adhesive nodes for the neurons and micron-scale-tracks for guiding neurite outgrowth and connectivity. results: in both systems, the developing and mature network, we found not only a concentration dependant effect of acr and bdnf but also a time dependant effect with a limited capability of the developing system to regenerate, even in the presence of acr. the co-treatment of the cells showed that inhibition of calpains by calpeptin might reduce the effect of intracellular elevated ca2+, a known neurotoxic mechanism of acr. moreover, the neurothrophin bdnf acts via trkb receptors on pathways stimulating neurite outgrowth and thereby counteracting the adverse effect of acr. conclusion: with the nfa we provide a rapid and simple way to analyze neurite outgrowth and connection formation in real time. by spatially standardizing the array we provide assay coordinates to streamline the analysis process and bring it towards high throughput testing. furthermore preliminary data showed that modification of the surface with biomolecules allows cell adhesion of other neuronal celltypes (e.g. primary mouse neurons) and extracellular matrix proteins (e.g. laminin) stimulate neurite outgrowth via integrins. transcriptional regulation of nox4 by histone deacetylases siuda d. 1, 2 , zechner u. 3 , prawitt d. 4 nox4 is a member of the nadph oxidase family, which represents a major source of reactive oxygen species (ros) in the vascular wall. nox4-mediated ros production mainly depends on the expression levels of the enzyme. the present study is aimed to investigate the regulation mechanisms of nox4 transcription by histone deacetylase (hdac). in cultured human ea.hy 926 endothelial cells, treatment with the pan-hdac inhibitors (scriptaid, trichostatin a, tsa, and suberoylanilide hydroxamic acid, saha) leads to a drastic decrease in nox4 mrna expression. a similar down-regulation of nox4 mrna expression can be achieved with sirna-mediated knockdown of hdac3. hdac inhibition in endothelial cells is associated with enhanced histone acetylation in the human nox4 promoter region, with no significant changes in dna methylation. consistently, scriptaid-treated cells show increased chromatin accessibility in nox4 promoter. in addition, we provide evidence that c-jun plays an important role in controlling nox4 transcription. knockdown of c-jun with sirna leads to a marked downregulation of nox4 mrna expression. in response to scriptaid treatment, the binding to c-jun to the nox4 promoter region is reduced despite the open chromatin structure. in parallel, the binding of polymerase iia to the nox4 promoter is significantly inhibited as well, which may explain the reduction in nox4 transcription. in conclusion, hdac inhibition decreases nox4 transcription in human endothelial cells by preventing the binding of transcription factor(s) and polymerase(s) to the nox4 promoter. this is very likely because of a hyperacetylation-mediated steric inhibition. cyclopentenone prostaglandins induce oxidative dna-damage in hamster lung fibroblast v79 cells solecki g. m. 1 key: cord-015021-pol2qm74 authors: nan title: third international congress on the immune consequences of trauma, shock and sepsis —mechanisms and therapeutic approaches date: 1994 journal: intensive care med doi: 10.1007/bf02258437 sha: doc_id: 15021 cord_uid: pol2qm74 nan this issue of the journal contains the abstracts for the third international congress on the immune consequences of trauma, shock and sepsis -mechanisms and therapeutic approaches. we hope that the information contained in this special issue will stimulate you to participate in the congress, to contribute to the knowledge being developed in this field and to use this information to help you in providing better care for your patients. we thank the editors and the editorial board and publishers of the journal for their interest and support in preparation of this special issue. we also, on behalf of the scientific committee, welcome you to the third international congress in munich on 2-5 march 1994. when, in the mid-1980s, we thought of having a worldwide congress, we hoped to bring together investigators to discuss this theme. the explosion of knowledge occurring around that time provided an excellent background against which the first conference in 1988 provided stateof-the-art information and consensus on factors involved in injury and sepsis. in 1991, the second congress was held at the time of another resurgence of research, study and information on injured and operated patients. it seemed then that there would be a lull in the development of new information and therapy, and that another state-of-the art conference might not be necessary until 1995 or 1996. however, the explosion in molecular biology has continued. the wonderful world of cytokines has gone from ill to il-6 to il-11, il-12 and il-13 and beyond. the vast amount of information about mediators and their importance in disease is impressive. this has all suggested a magic bullet that might be used to alter or block inflammatory responses. this has not happened, however, and the question is "why not"? our science is powerful, but our therapy is still weak. what are the issues, then, in 1994, to be dealt with at this symposium and congress? (1) proposals for new terminology. there have been a number of proposals for new terminology and new classifications of injury, sepsis, inflammation and various other problems related to human illness. the question is whether this is the way to go. will this contribute to better clinical trials, information basis and better research? the pros and cons of this development will be reviewed by those making the proposals and those questioning the need for and wisdom of this effort. (2) magic bullets: the prospect of a magic bullet to deal with inflammation in injury and infection seemed highly promising earlier. many preclinical trials and a lot of animal research suggested the possibility of a great breakthrough in clinical care. what has become, then, of all the expensive and extensive multi-institution randomized, placebo-controlled, double-blind clinical trials of agents that block mediators and endotoxin. many such studies have yielded equivocal, marginal or negative results. the reasons for this and the future of clinical research will be the subject of presentations and discussions to set the stage for further work. (3) should future clinical trials be based on new classifications of illness such as mods, sirs, apache iii, sap ii, mrm, etc., or should trials be dedicated to specific diseases -urinary tract infections, pneumonia, trauma patients, cardiac surgery and other specific problems, rather than generalized problems of sepsis, the sepsis syndrome and other classifications? in other words, should we now begin to have clinical trials on specific diseases with causes that are known and can be attacked? the causality of disease becomes an important consideration in this regard. (4) a multitude of potential therapeutic agents has been proposed on the basis of animal studies. how should we decide which of them should be brought to clinical trial? the possibilities are endless as we develop new clinical information about the mechanisms and pathogenesis of human disease. (5) information on the pathogenic mechanism of disease states and of injury continued to emerge in an explosive fashion, and in light of our gathering knowledge we can look forward to working out a cohesive system of response to injury. (6) additional information will be provided in plenary sections, many symposia and free communication sessions and posters, which will update the participants on a variety of relevant topics presented by many of the leading in-iv vestigators in these fields. topics will range from molecular mechanisms, such as signal transduction, through the explosive growth of information on the role of cytokines and pathophysiology, to practical considerations in the design of immunomodulatory therapeutic regimens. these merely touch on a few areas, from the basic to the clinical, which will be the subjects of those symposia. all this information will fit into the jigsaw of this exciting area and its stimulus to further research study. this promises to provide an exciting, educational programme with experts and participants from all over the world. we hope it will set the stage for many years to come and will increase our understanding of trauma, shock and sepsis and help us to provide better therapy for those of our patients who are affected by such problems. a. the clinical syndrome of mods versus mof will be reviewed in detail by those who have made these proposals. b. an extensive review of the design and interpretation of clinical trials in patients with shock and injury will be provided. the reasons why so many clinical studies in the recent past have been negative will be reviewed. the therapeutic strategies that are being developed for the treatment or prevention of mods or mof will be the subject of another panel discussion by experts who have been involved in and contributed to this area. a consensus conference or controversy conference will be presented about various aspects of mods or mof, including the benefits of supernormal oxygen delivery, bacterial translocation, parenteral nutrition, the immune response and other aspects. the successes and failures of completed clinical trials will be presented by those who are involved in these clinical trials, with a refreshing review of the problems related to that injury. there will be late news about studies just being completed at present or after the beginning of 1994 and where they stand. c. the mechanisms and biochemical profiles of specific organ dysfunction or failure will be reviewed. what are the definitions? what are the mechanisms? how can organ dysfunction and/or failure be defined? an extensive review of the biological mechanisms involved in production of injury by mediators will be presented. a session will be devoted to how future ongoing trials might be better designed and what can be done about the studies recently completed, many of which are negative. d. the immunological or inflammatory pathways resulting in organ injury will be reviewed in detail in presentations and a panel discussion. we look forward to welcoming you to an exciting and rewarding conference, which undoubtedly possesses the potential to become a landmark event and major reference point for any scientific discussion about the complex of host defense dysfunctions following trauma, shock and sepsis. studies over the past 25 years have established that the contact system, which forms bradykin/~, is gax important mediator in hypotensive septicemia. in addition to hradyk{nln, another product of the contact system, kailikrein, can mediate inflammation by virtue of its chemotaetic mad neutrophj/activating properties. using functional and immunochemical tech~2ques, we have demonstrated activation of the contact system in the adult respiratory distress syndrome in typhoid fever and clin/cal sepsis. we have also been able to inhibit the hypotension but not the disseminated intravaseular coagulation in a model of primate sepsis by the use of a monoclonal antibody directed agsi~st factor xii, the initiating protein of the contact system, in volunteers given e. coil endotoxin, who did not develop hypotension, we were also able to demonstrate activation of the contact system with a rise of alpha-2macrogiebulin-kalllkrein complex. we have also examined, j~ an i~tensive care situation, patients with sirs. we found that serial measuremezzts of the contact system were useful in eva~u~ting prognosis+ these studies suggest that inhibition of kalllkrein a~d l e r bradykinin actions might be useful i~ obviating many .of the features seen in sepsis and septic shock. dextran sulfate (dxs) activates the contact system and, in vivo, produces transient hypotension. in order to better define the mechanisms underlying the dxs-induced hypotension, we investigated the effects of either the plasma kallikrein inhibitor, des-pro2-iarg]5]aprotinin (bay 4620) or the b2 kinin antagonist, hoe 140 on the hypotensive response to dxs. in the first study, anesthetized miniature pigs (5 pigs/group, randomly assigned) were given one of the following treatment protocols: 1) dxs (5 mg/kg), 2-5) dxs plus bay 4620 (45, 90, 180, or 360 rag), or 6) saline. dxs alone produced a profound but transient systemic arterial hypotension with a corresponding reduction in plasma kinin-containing kininogen. circulating kinin levels, complement fragment c3adesarg and fibrin mom)mer were all increased. bay 4620 produced a dose-dependent delay or attenuation in these effects with the highest dose completely blocking dxs-induced hypotension and elevations of kinin, c3adesarg and fibrin monomer levels. thus, the effects of dxs are solely dependent on contact system activation and this activation is sensitive to bay 4620. llowev~:r, contact system activation is known to produce changes in a variety of vasoactive mediators, all of which can affect blood pressure. in a second study, two groups of pigs (3/group) were given either dxs alone (2 mg/kg) or dxs 10 minutes after a bolus injection of hoe 140 (30 #g/kg). dxs alone produced transient hypotenmon. this response was completely blocked by hoe 140 pretreatment. both groups had identical reductions in kinin-containing kininogen. we conclude that dxs-induced hypotension is produced by activation of the contact system which results in the production of bradykinin. liberation of bradykinin is both necessary and sufficient to produce all of the hemodynamic changes observed. dr. matthias siebeck, department of surgery, university of munich, klinikum lnnenstadt, nussbaumstrasse 20, d-80336 munich, germany in experimental animals exposed to i.v. injection of endotoxin accumulation of leukocytes in various organs as lungs and the liver is a prominent feature. as a part of these morphological changes damages of endothelial ceils are regularly seen. this process, which is a part of endothelial-cellular interaction, leeds to exposure of the sub-endothelial basement membran. the basement membran is known f6r its capacity to activate the contact system of plasma. during this cascade activation, coagulation factor xii is converted to the active factor xii. this activation might produce increased plasma kallikrein activities and thereby give release of the vasoactive substance bradykinin. using a porcine model we have noticed that endotoxin infusion (0,01 mg/kg) induces elevated plasma kailikrein activities within two hours after the start of the infusion. this enzyme activity remained increased during the next hours and reached value of up to 50 u/1. in patients with sepsis we also have observed elevated plasma kallikrein activities with enzyme activities up to 200 u/1. in order to further elucidate the significance of these elevated enzyme activities, we prepared human plasma kallikrein and injected it intravenously in anaesthetized pigs (1). when very small plasma kailikrein activities (0,3 u/kg bodyweight) were given intravenously a 50% decrease in arterial blood pressure was seen in the animals. in the patients with sepsis also decreases in prekallikrein values and functional plasma kallikrein inhibition are frequently seen. furthermore, degradation of high molecular weight kioinogen is found in these patients indicating formation of bradykinin. these experimental and clinical studies underline that contact activation in sepsis might results in the release of very powerful mediator substances which can be of pathophysiological importance in this disease. a number of pathological disorders as reperfusion injury, bone marrow transplantation, polytrauma and septic shock are associated with capillary leakage. as the activation of the complement system and the contact phase play a major role in these diseases we investigated whether cl-lnhibitor (c1-inh), which inactivates cl-esterase, kallikrein and clotting factors xii and xl, could abolish vascular leakage. a capillary leakage was induced in rats by the administration of interleukin-2 (5 x 106 iu/kg). the increased vascular permeability was monitored for one hour as the extravasation of fitc marked rat serum albumin from a mesenterial vessel by a video-image processing system. ci-inh (berinert®, behringwerke) given as a single i.v. bolus in concentrations of 100, 250 or 500 u/kg dose-dependently prevented the capillary leakage. carrageenaninduced inflammation in the rat leads to vascutar leakage and to edematous swelling of the paw. ci-inh in this model leads to a dose-dependent decrease in paw edema formation. finally, we investigated the effect of ci-inh (infusion (100-125 u/kg x h) on a lps-induced shock in the rat by combination therapy with the antithrombotlc agents antithrombin ill (kybernin®) or rec. hirudin (both substances from behringwerke). in this animal model mortality was 90 % in the untreated control. both antithrombotic agents decreased mortality rates by inhibiting formation of dic; a further significant improvement of survival was achieved by the treatment with ci-inh. thus+ it could be concluded that c1-inh has a beneficial effect in diseases associated with a vascular leakage. iclb and laboratory for experimental and clinical immunology, university of amsterdam, the netherlands; 2thrombosis research center, temple university, penn., usa; 3oklahoma medical research foundation,. ok. city, usa. to evaluate the contribution of the contact system to activation of other mediator systems in an experimental model of sepsis, we investigated the effect of mab c6b7 which inhibits activation of factor xli, on activation of complement and fibrinolytic cascades and activation of neutrophils in baboons suffering from a lethal sepsis. activation of the complement system was assessed by measuring circulating levels of c3b/c and c4b/c, and a significant reduction was observed in 5 animals that had received a lethal dose of e. coli together with mab c687 (treatment group), compared to 4 animals that had received a lethal dose of e. coil only (control group). activation of the fibrinolytic system as reflected by circulating plasmin-=2antiplasmin complexes and tissue plasminogen activator, and activation of neutrophils, assessed by measuring circulating elastase-=l-antitrypsin complexes, was also significantly less in the treatment group. we conclude that activation of the contact system protein factor xll during the inflammatory response to a lethal dose of e. coil in this baboon model, modulates directly or indirectly activation of the complement and fibrinolytic systems and that of neutrophils. in a prospective study, plasma levels of c3a, c3, and c5a were measured in 30 patients from an internal intensive care unit. 20 patients were clinically septic defined by the criteria of bone et al.(l) . the remaining 10 patients were critically ill but didn't fulfill the clinical criteria of sepsis. from both groups of patients blood samples were taken over a l0 days period. during the first 3 days blood samples were drawn every 8 h, on day 4-6 every 12 h and the last 4 days once daily. mean plasma concentrations of c3a within the first 32 h after clinical onset of sepsis were 1019 + 618 pg/ml, whereas non-septic-patients exhibited mean values of only 595 +_ 312 p_g/m/. c3 levels were lower for septic-patients (840 + 480 lag/ml) than for non-septic-patients (1340 _+ 770 lag/ml). the most profound difference between both groups was found, when the c3a/c3 ratio was compared (1.84 + 2.28 for septic-patients and 0.4 _+_ 0.18 for the control group). no significant differences between both patient groups were observed in c5a plasma levels (5.9 + 2.1 ng/ml in septic-patients vs. 5.6 _+ 1.5 ng/ml in control patients). in 13 of 20 cases of clinically defined sepsis causative organisms like bacteria, protozoa or fungi could be cultured from blood, bronchoalveolar lavages and/or section materials. application of the complement parameters to survivors (n=9) and non-survivors (n=l 1) within the septic-group revealed, that the c3a/c3 ratio could also be used as a prognostic parameter for clinical outcome. the possibility of rapid and easy measurement of c3a and c3 in only 20-25 minutes (2) and the significant difference of the c3ajc3 ratio between the septic and non-septic group renders this parameter a good candidate for early diagnosis of sepsis in the intensive care unit. hirudin, a single polypeptide chain composed of 65 amino acids with 6 cysteine residues (mr 7000 daitons), is the most potent and specific thrombin inhibitor, which is now available as a genetically engineered product (rec. hirudin -hbw 023, behringwerke; marburg). the aim of our study was to establish a rabbit model of tissue factor (tf) induced activation of the extrinsic pathway of coagulation and to evaluate the therapeutic efficacy of rec. hirudin. coagulation was induced in female nzw rabbits by infusion of 0.5 p.g/kgxh thromboplastin for 7 hours. development of disseminated clotting was manifested by a decrease of fibrinogen and platelets to 27.0 % and 33,0 % respectively, and by an increase of fibrin monomers from 13.2 to > 85.0 ~tg/ml. we administered rec. hirudin to rabbits in 3 different concentrations (0.5, 1.0 and 2.0 mg/kg); treatment started simultaneously with the infusion as an i.v. bolus. rec. hirudin significantly prevented the decrease of fibrinogen, platelets and the increase of fibrin monomers. this effect was dose dependent and long lasting, even 7 hours after the administration of rec. hirudin, clotting was still significantly reduced. as could be drawn from the plasma levels, rec. hirudin had been cleared from plasma at this time. in a post-treatment study we administered rec. hirudin (0.5, 1.0 and 2.0 mg/kg i.v. bolus) as late as 2 hours after the start of tf infusion. at this time there was already a prominent activation of coagulation. even in this post-treatment regimen rec. hirudin significantly prevented disseminated clotting. hence, it was concluded, that rec. hirudin by inkihiting thrombin could be effective in the prevention of coagulation disorders including disseminated intravascular clotting (dic) induced by a septic disease. research laboratories of behringwerke ag, 35001 marburg, germany $3 novel protease inhibitory activities of the second domain of urinary trypsin inhibitor (r-020) and its effect on sepns-lnduced organ injury in rat atsuo murata 1, hitoshi toda 1, ken'ichi uda 1, hidewaki nakagawa 1 , takesada mori 1, hideaki morishita 2, tom yamakawa 2, jiro hirese 2, atsushi ni~ 2, nariaki matsuura 3 1osaka university medical school, osaka, 2mochida pharmaceutical co. ltd. tokyo, 3wakayama medical schoof, wakayama, japan inhibitory-activities of the second kuntz-type inhibitor domain of human urinary trypsin inhibitor (uti) and its effect on sepsis-induced organ injury in rat were investigated by using the recombinant protein. uti is a glycoprotein with a structure in which 2 kunitz-type inhibitor domains are linked in a row. we isolated the gene encoding the second kunitz-type inhibitor domain of uti, and then constructed expression plasmids by ligating it to the e. coli phoa signal peptide gene. these plasmids expressed the second domain in e. coil strain je5505 which lacks the membrane lipoprotein. the recombinant second domain (r-020) innb[ted trypsin, plasmin, neutrophil elastase and chymotrypsin. in addition it inhibited blood coagulation factor xa and plasma kallikrein in a concentration dependent and competitive manner. the in vivo effect of the recombinant r-020 was investigated in a rat model of septic shock induced by cecal ligation and puncture. the administration of r-020 significantly improved the survival rate of the rats and attenuated the pathological changes of lung and iiver. we found out the novel protease inhibitory activities of the second domain of uti and its protective effects on sepsis-induced organ injury. macrophages are known to secrete lysosomal proteinases,mainly cathepsin b and cathepsin l, and also ~-proteinase inhibitor (pi),related to acute phase proteins.disturbances of proteinases/ proteinase inhibitors correlates with inflammatory process,leading sometimes to noncontrol "pathglogical" proteolysis (jochum et ai.,1990) . the cathepsin l-like and cathepsin b-like activity were measured in serum of 42 patients with chronic bronchitis (14 -with obstructive, 28 -with nonobstructive bronchitis),acute bronchitis (15) and 35 healthy persons.simultaneously the level of~pi was determined in the same groups.cysteine proteinases were measured with help of fluorogenic substrates,as was presented earlier (korolenko et ai.,1991) , ~pi with help of immune enzyme method. it was shown increase of cathepsin l-like and cathepsin b-like activities during aggravation of chronic bronchitis comparatively to the controls (2-4 fold) .after treatment there was a tendency to normalization of indices,but the increase was about 30-40% more than the control values.~pi level in this group was also increased (two-fold),in patients with acute bronchitis -2-4-times more comparatively to the control.it is possible to conclude that chronic bronchitis induced increased secretion both cysteine proteinases and d{pi into blood. some peculiarities of ratio were noted in patients with emphysema. endotoxins are microbial products derived from the outer cell membrane of gram negative bacteria. the active component of endotoxin is lipopolysaccharide (lps), a complex macromolecule consisting of polysaccharide covalently bound to a unique lipid, termed lipid a. now recognized to embody the endotoxic principle of lps, lipid a consists of a/31-6 diglucosamine backbone, both ester and amide linked fatty acids, some of which are acyloxyacylated, and charged constituents such as phosphate, phosphorylethanolamine and 4 amino arbinose lps, exerts its biological effects in vivo by noncytotoxic interactions with a variety of host inflammatory mediator cells, primarily the mononuclear phagocyte and the endothelial cell, although other host cells also participate. these interactions are modulated by lps-specific binding proteins found in plasma, including lps-binding protein (lbp) scd14 and perhaps other proteins as well. specific receptors for lps have been identified on mammalian cells which mediate signal transduction via multiple pathways. lps-activated host cells are stimulated to secrete or express multiple proinflammatory mediators, including tnf-a, illa, il-1/3, ifn-a, il-6, il-8, il-10, paf, pge, ltb 4 and procoagulant activity. the overproduction of these proinfiammatory mediators results in the manifestations of endotoxemia, observed experimentally as fever, hypotension, disseminated intravascular coagulation and death. modulation of activity of these mediators protects animals against lethality. similar pathways are thought to be operative in gram negative sepsis, and control studies with human volunteers support such conclusions. immunotherapeutic approaches in clinical gram negative sepsis have, to date, been less successful. in vitro experiments and studies in animal models have recently shown that several proteinaceous bacterial exotoxins can evoke cytotoxic effects that ultimately lead to cardiovascular collapse and shock. since the possible relevance of bacterial exotoxins in the pathogenesis of septic shock has received very little attention in the past, an attempt will be made here to provide a brief overview of this generaily neglected topic. protein toxins act intracellularly or they dz~nage the integrity and function of the plasma membrane. major representatives of the former group are the adenosine diphosphate (adp)-ribosylating toxins, e.g. cholera and cholera-like toxins, diphtheria toxin), and the neurotoxins. most medically relevant toxins of this category have been studied in great detail. although often responsible for severe and sometimes fatal disease, their association with septic shock is rare. in contrast, experimental evidence is accumulating for a role of membrane<lamaging toxins in the pathogenesis of inflammatory lesions. formation of transmembrane pores is probably the most widespread mechanism via which such physical membrane perturbation can occur (1,2), the present discussion will be restricted to this category of toxins. the author shall first discuss basic properties of pore-forming proteins, and consider the factors that direct their attack to specific targets. thereafter, attention will be drawn to diverse functional consequences that may follow membrane damage so that a general concept of how pore-forming toxins may contribute towards the development of shock will evolve. i. bhakdi, s. and tranum-jensen. j. 1987 gram positive bacteria produce exotoxins that, at least in part, exhibit the unusual properties of superantigens. superantigens (sag) activate v/3 selectively t cells to produce lymphokines including i1-2 and tnf/lymphotoxin. systemic application of the sag staphylococcal enterotoxin b (seb) to d-galactosamine sensitized mice causes lethal shock within hours. seb reactive v/38 + t cells accumulate within 90-120 min mrna for the ii-2, i1-4, tnfai~ and ifn-3,. parallel in time high concentrations of bloodborne i1-2 and tnf are noted. anti tnf t~//3 neutralizing mab protect mice against seb induced t cell dependent lethal shock thus indicating a key role of tnf in effecting lethal toxicity. within 12-18 h distinct levels of tolerance to seb become discernable on ligand reactive v/38 t ceils, dependent of the dose of seb used. these levels include anergy, t cell receptor downregulation, loss of cd2, cd4, cd8 accessory molecules and programmed cell death. m.freudenberg, y.yaegashi, p.nielsen, c.galanos. the sensitivity towards endotoxin (lps) is genetically determined, however it may be increased under different experimental conditions. these include treatment with hepatotoxic agents such as d-galactosamine, and with live (infection) or killed bacteria. it is well established now that d-galactosamine sensitizes to lps by increasing the susceptibility of the host to toxic activity of lps-induced tnf~. sensitization by bacteria, especially by gram-negative once is of especial interest because it implies that infecting microorganisms not only produce lps, but als0 sensitize the host to its lethal activity. sensitization to lps by bacteria proceeds in all lps-sensitive (lps n) mouse strains that have been investigated so far. it also proceeds in lps-resistant (lps d) c3h/hej mice. the absence of sensitization to lps in lps d c57bl/10sccr mice was identified as being due to their inability to produce interferon-7 (ifn?). administration of exogenous ifn? to these mice conferred sensitivity to lps. conversely, administration of anti-ifn? to lpsn; mice inhibited the development of sensitization'by bacteria. it may be concluded therefore that ifn7 is the mediator of sensitization to lps by bacterial infection. the ifn? defect of c57bl/10sccr mice concerns only the ifn7 response to bacteria, since the response to the t-cell mitogen con a and to cd3 monoclonal antibodies was not impaired. the ifn?producing cells themselves were shown to be intact. on closer investigation the impaired ifn? response was identified as the result of a defective accessory function of macrophages. macrophages of c57bl/10sccr mice are incapable of producing interferon-~ (ifn~) which we could show to be obligatory for the production of ifn? in response to bacteria. although antibiotics are required to clear bacteremia, they do not reverse evolving shock, because endotoxin free in the bloodstream or exposed on the surface of circulating bacteria continues to activate the production of inflammatory mediators, furthermore, antibiotics have been shown to induce release of endotoxin from gram-negative bacteria during the treatment of severe infection. in an in-vitro study, using live escherichia coil, we have observed the release of endotoxin upon treatment with several antibiotics like cefuroxim, imipenem, ceftazidime and aztreonam. incubation with imipenem or ceftazidime induced an early lyeis and a mild rise in endotoxin levels, whereas incubation with cefuroxime or aztreonam resulted in the formation of filaments with a late but dramatic rise in endotoxin levels. in a second study we studied the influence of different antibiotics on the tnf-= production of e.coli stimulated human monocytes. we found again great differences in the production of this important cytokine among the different antibiotics according to their capacity to induce liberation of endotoxin. these differences in the amount of endotoxin release are probably caused by penicillin-binding-proteins (pbp) specificities of the antibiotics with resultant differential morphological changes, in a rat sepsis model treatment with imipenem or ceftazidime resulted in a transient increase in il-6 levels, whereas treatment with aztreonam resulted in progressively increasing levels of il-6 and a significant increase in mortality. the increased mortality in pbp-3 specific aztreonam treated rats might have been the result of filament formation in the abdominal cavity of the septic rats. the endotoxin sequestered at local sites may account for the precipitation of gram-negative shock. finally, we've also demonstrated that in patients under treatment for septic shock endotoxin release can be related to the administration of antibiotics. carbapenem-and cephalosporin-lnduced release of lps from smooth and rough pseudomonas aeruginosa: in-vivo relevance j. jackson and h. kropp, merck research laboratories, rahway, nj b-lactam (cell-wall active) antibiotics are generally deemed the class of antibiotics most responsible for the release of free endotoxin (lps) from gram-negative bacteria due to their cell lytic actions although all antibiotic classes studied thus far induce release of endotoxin. we have recently shown in-vitro that antibiotics within the b-lactam class (i.e. imipenem , meropenem and ceftazidime , with equivalent mics) differ in their propensities to release excessive amounts of lps from both smooth-(s-) and rough-(r-) lps laboratory and clinical and antibiotic-sensitive and -resistant p. aeruginosa isolates and that lps release is independent of cell lysis. additionally, variations in the induction of endotoxin release is antibiotic concentration dependant and correlate with antibiotic penicillin-binding protein (pbp) affinities which result in morphological changes. these studies also show that lps differences measured via chromogenic lps (c-lal) assay correlate with lps lethality in lps-sensitive mice. release of lps was compared to changes in cfus, cell mass, morphology and antibiotic pbp-specificity. corresponding in-vivo studies in cd1 mice against s-lps p. aeruginosa isolates show that, despite equivalent in-vitro protection, antibiotic efficacy in mice differ as much as 100-to lo00-fold. to establish a possible in-vivo role for antibiotic-induced release of free lps we compared antibiotic efficacy results in mice challenged with virulent parent s-lps and its relatively avirulent r-lps mutant (lacking only its 0 side chain) p. aeruginosa isolates. results show the relevance of quantity and physiochemical composition (bioreactivity) of free lps to virulence and in-vivo antibiotic efficacy. in other in-vivo studies chemical agents that destroy cells (m~) which mediate lps lethality in-vivo were used to pretreat mice prior to antibiotic therapy and bacterial challenge with wild type pseudomonas. we conclude that circumstances in which antibiotic-induced filamentation occurs are also conditions that yield excessive lps release, the in-vivo effects of which are shown through the requirement of larger amounts of antibiotic to afford equivalent protection. bacterial endotoxins have been reported to play a significant role in the pathogenesis of gram negative sepsis by their interaction with, and stimulation of, host inflammatory mediator cells to produce cytokines, biologically active lipid intermediates, and procoagulant activity (tfa). although both microbe-associated and free endotoxin are capable of stimulating mediator production, studies from our laboratory suggest that free endotoxin may be the more potent stimulus for tnf and tfa. further, our studies suggest that the majority of the proinflammatory activity released from antibiotictreated e. coli coisolates with lps by two different fractionation techniques. to assess whether actions of endotoxin directly contribute to lethality in gram negative sepsis, an experimental model was developed in which mice were made hypersusceptihle to the lethal effects of endotoxin by treatment with d-galactosamine (dgaln). challenge of cf-1 dgaln-treated mice with e. coli olll:b4 resulted in an lds0 of approximately 2.5 x 104 cfu. resolution of the peritonitis and bacteremia by treatment with cell wall-active antibiotics resulted in an increase in the ldso. ceftazidime and imipenem, which differ in their mechanism of action and in their capacity to effect endotoxin release in vitro, also differed in their in vivo capacity to provide chemotherapeutic protection (3 fold vs 8 fold respectively, p< .01). parallel studies with endotoxin hyporesponsive c3h/hej mice indicate significantly greater levels of protection with imipenem (> 80 fold vs saline controls). collectively these data suggest that endotoxin may contribute directly to the pathogenesis of experimental gram negative sepsis. bacterial lipopolysaccharides (lps) are the endotoxins of gram-negative bacteria and represent their major surface antigens. lps is made up of three chemically, biologically and genetically disctinct regions, i.e, the o-chain, the core region and the lipid a moiety whereby the latter represents the endotoxic center. it is our current understanding that lps is responsible for many of the pathophysiological events observed during gramnegative infections and that one of the major mechanisms leading to shock and death is the lps-induced activation of macrophages resulting in the production and release of lipid and peptide mediators, among which tumor necrosis factor seems to be the most important. therefore, in the fight against the lethal outcome of gram-negative infections, modern strategies, in addition to antibiotic treatment, aim at i) the neutralization of tumor necrosis factor, ii) the inhibition of the production of tumor necrosis factor or iii) the neutralization of the activation potential of lps for macrophages by monoclonal, preferably human antibodies. the latter approach, to be effective against a broad spectrum of gram-negatives, must be directed against common structures of lps (lipid a and core region). the molecular basis of this approach and the controversy in this field will be discussed. passive immunotherapy has been used since 1893, when von behring described the administration of immune horse serum to treat a patient with diphteria infection. even if this therapy was sometimes successful in bacterial infections, it has been largely replaced by antibiotics. however, antibiotics have their limitations, especially in critically-ill patients. to improve outcome, adjunctive therapies such as immunotherapy with polyclonal and monoclonal antibodies particularly against endotoxin are again considered. the role of humoral immunity in host defenses against bacterial infections is weu known. for instance, tile importance of antibodies in the defense against gramnegative infections has been established clinically by studies relating the outcome of patients with gram-negative bacteremia to tilers of antibodies directed at the offending pathogens at the onset ofbacteremia (mccabe 1972; pollack 1979) . ever since we know the role of endotoxins in the pathophysiology of sepsis, antibodies against the s-and r-lps have also been detected in sepsis patients. the aim of the administration of iv/g to the sepsis patient is as follows: 1 ) enhancing of opsonization and phagocytosis(antibactericidai activity) 2) synergistic effects with [3-1actam antibiotics 3) neutralization of endotoxin, the main pathogenic mediator of gram-negative sepsis 4) modulation and/or inhibition of cytokine release the enhancement of opsonic-and phagocytic-activity especially with igg via fc and c3 receptors has been well documented. monoclonal antiendotoxin antibodies, proven in clinical studies, do not appear to neutralize endotoxin in vitro and are not reproducibly protective in animal models of sepsis. also they can not suppress endotoxin-induced tnf-~, il-6 release in mice (baumgartner 1990, corriveau and danner 1993) . in conlrast, recent studies of a polyclonal immunoglobulin preparation, containing high levels of antibodies against gram-negative bacteria and their o-antigen of lps in igg, igm and iga classes (pentaglobin®) provide evidence to neutralize endotoxin. this effect is demonstrated in vitro (berger 1990 (berger ,1993 , in animal models (stephan 1985 , berger 1993 and also in prospective, randomized, controlled clinical trials (schedel 1991 , poynton 1992 , behre 1992 . furthermore mortali b' was reduced statistically in patients with septic shock and endotoxemia by using this preparation, as has been demonstrated by sehedel. anti-core lps monoolonal antibodies: binding specificity and biological properties f.e. di padova, r. barclay, e.th. rietschel. bacterial lps and cytokines are responsible for the pathological processes of gram-sepsis and are suitable targets for therapeutic interventions. chemical characterization and structural analysis of different lps have revealed common features. the inner core region of lps shows a high degree of similarity among e. coli, salmonella and shigella. among a large number of broadly cross-reactive murine anti-core lps mab one of these igg2ak) has been selected and chimerized into a human igglk (sdz 219-800). in elisa and in immunoblots on purified lps both sdz 219-800 and wni 222-5 show a strong reactivity with all smooth lps from e. coli and salmonella. reactivity with all the known complete core structures from e. coli and salmonella (ra) is evident. reactivity with re structures or free lipid a is not observed. this mab cressreacts with all clinical e. coli isolates from blood, urine and feces and with other enterobacteriaceae. sdz 219-800 and wni 222-5 have biological activity as they inhibit the lal assay and the secretion of monokines (il-6 and tnf) by mouse and human macrophages. moreover, sdz 219-800 and wni 222-5 inhibit the release of il-6 and tnf in vivo. in vivo sdz 219-800 as well as wni 222-5 neutralize the pyrogenic activity of e. coli lps and protect mice from lethality in d-gain-sensitized mice. the possibility to use wni 222-5 as a capture antibodies in the immunolimulus assay opens the possibility to differentiate the origin of the lps in patients with endotoxemia. franco di padova, sandoz pharma ag, ch4002 basel, $chweiz $7 presentation of lps to cd14 by lps binding protein peter s. tobias, julie gegner, katrin soldau, lois kline, loren hatlen, douglas mintz, and richard j. ulevitch. the activation of myeloid cells by lipopolysaccharides (lps) has been shown to require the serum glycoprotein lps binding protein (lbp) and binding of lps to membrane bound cd14 (mcd14). other cells such as human umbilical vein endothelial cells (huvec), smooth muscle cells, and some epithelial cells, which do not express mcd14 but nevertheless respond to lps in the presence of serum, have receptors for complexes of lps with the soluble form of cd14 (scd14). these complexes of lps with scd14 are only formed efficiently in the presence of lbp. we have begun to characterise the mechanisms by which lbp enables lps to bind to cd14, either soluble or membrane bound. with the use of fluorophore and radiolabelled reagents we have developed procedures for quantitative measurement of the association of lps with lbp and of lps-lbp complexes with cd14. these results show that the delivery of lps to scd14 is catalysed by lbp, i.e., lbp is not included with the lps-scd14 complex. in contrast, on the surface of cells, lbp does not dissociate from the cells after lps binds to mcd14. the kinetics, equilibria and stoichiometry of these reactions will be discussed in the context of models for cellular activation by lps and cellular uptake of lps. supported by nltt grants gm37696, ai32021, ai15136, gm08172, and assistance from the pharmaceutical research institute of johnson and johnson. the scripps research institute, imm-12, 10666 n. torrey pines rd. la jolla, ca usa 92037. modulation of endotoxin-induced cytokine production by lps partial structures h.-d. flad, h. loppnow, t. mattern, and a.j. ulmer department of immunology and cell biology, forschungsinstitut borstel, d-23845 borstel lipid a constitutes the active moiety of endotoxin (lps) of gramnegative bacteria. it activates mononuclear phagocytes to produce cytokines, such as tnf, i1_-1, and il-6, which are the major mediators of the endotoxic effect of lps in vivo. lipid a precursor la (synthetic compound 406) does not induce cytokines, but is able to specifically antagonize lps-or lipid a-induced mediator production in human mononuclear cells, vascular endothelial cells, and smooth muscle cells. furthermore, we present evidence for the first time that t-lymphocytes proliferate in response to lps and express mrna for interleukin-2 and interferon-~ and that these responses are also antagonized by synthetic lipid a precursor la. when comparing the agonistic and antagonistic activity of lipid a and different partial structures at the functional and binding level, the number and length of the fatty acids and the number of phosphoryl groups were pound to be of crucial importance. unexpectedly, lipid a precursor la, although biologically inactive, turned out to be both the most potent antagonist and competitor in inhibiting the binding of lps. taken together, our results provide evidence for a model in which lipid a partial structures compete with lps for specific cell surface receptor(s). in this sense, biologically inactive lipid a analogues may be good candidates as therapeutic agents for the prevention of gram-negative septic shock. two mammalian lipid a-binding proteins have been identified that are believed to have important roles in mediating the host response to endotoxin: lipopolysaccharide-binding protein (lbp) and bactericidal/ permeability-increasing protein (bpi). human lbp shares a 45% amino acid sequence identity with human bpi. despite the sequence homology, the two lipid a-binding proteins have very different functional activities. lbp is an acute phase serum protein that markedly potentiates the proinfiammatory host response to gram-negative infection by a mechanism which involves binding of the lbp-lps complex to cd14 receptors on monocytes, neutrophils and endothelial cells. in contrast, bpi is a neutrophil granule protein with potent bactericidal and lps-neutralizing activities. the divergent functional properties of these two lps-bindlng proteins can be explained by the inability of bpi-lps complexes to bind to cell-surface cd14 receptors. a recombinant protein (rbpi23), corresponding to the amino terminal 23kd fragment of human bpi, has been shown to retain the potent biological activities of the hdlo protein and may represent a novel therapeutic agent for the treatment of gram-negative infections, sepsis and endotoxemia. for therapeutic effectiveness in many clinical situations, rbpi23 will have to successfully compete with relatively high serum levels of lbp (5-60 ~g/mi) for binding to endotoxin and gram-negative bacteria. to evaluate this issue, experiments were conducted to compare the relative binding affinities of rbpi23 and human recombinant lbp (rlbp) for lipid a. the binding of both proteins to iipid a was specific and saturable with apparent kd's of 2.6 nm for rbpi23 and 58 nm for rlbp. in a competition assay format rbpi23 was approximately 75-fold more potent than rlbp in inhibiting the binding of nsi-rlbp to lipid a. these results demonstrate that rbpi23 has a significantly higher affinity for endotoxin than does rlbp and may explain the potent inhibitory activity of low concentrations of rbpi23 in a variety of in vitro functional assays for lps activation of cells despite the presence of high lbp levels. for example, rbpi23 at 0.2 ~tg/mi was able to totally inhibit lps-induced tnf release from monocytes despite a 100-fold weight excess of rlbp over rbpi23. and for heparin binding. three separate domains which inhibit the lal reaction to lps and bind to heparin were identified in amino acid regions 17-45, 65-99 and 142-169 . a single synthetic peptide (85-99) was bactericidal. these results suggest that rbpi23 contains three separate functional domains which may contribute to its high affmity interaction with gram-negative bacteria and heparin. the individual activity of each domain and the cooperative interaction among domains provide the basis for developing rbpi23 analogues with increased biologic efficacy. a considerable body of experimental data has accumulated implicating tumour necrosis factor (tnf) as a principal mediator of the pathophysiological features of septic shock. these data prompted the development of clinical strategies designed to limit excess (inappropriate) tnf production. monoclonoal antibodies (mobs) were developed and a phase ii dose escalation trial in 80 patients confirmed that the mab was safe, and suggested that it was having a beneficial effect on certain parameters. preliminary results of a large phase iii study indicated that (a) the mob was safe; (b) that it was of no discernible benefit in non-shocked patients; (c) that it reduced mortality in shocked patients, especially during the first 3 days. an alternative strategy was to take advantage of the high binding affinity of soluble receptors for tnf (stnfr). stnfr-iggfc constructs were made for both the p55 and p75 receptors. both were effective in animal models of lps challenge, but when a clinical trial was done with the p75 stnfr-fc there was unexpected mortality in the treated arm. using an animal model of live e.coli sepsis, we have shown that this may have been due to the release of bound tnf from the construct. plasma enhances while bpi inhibits lps-induced cytokine production from peripheral blood mononuclear cells (pbmc). pseudomonas species produce cytokine-inducing substances which are different from lps as indicated by the fact that polymyxin b blocks only 40% of the cytokine-inducing activity of these pyrogens. we now tested the effect of plasma and bpi on the il-1[3-inducing activity of pseudomonas maltophilia -derived pyrogens (pmp). bacteria were cultured to the log phase and filtered (300kd) to obtain prop. dilutions of pmp or lps were added to pbmc alone or to pbmc in 5% plasma +/-bpi (500 ng/ml). pbmc were incubated for 24 hours at 37°c and total il-i~ was measured by ria. results: il-i[~ in ng/ml (n=7, mear~+sem, *p<0.05 vs control). 0 control 0.1_+0 + bpi 0.1+0 5% plas. 0.2_+0 + bpi 0.2_+0 pmp (ng/ml) lps (ng/ml) 60 600 3000 1 10 3.2_+1 8.0_+1 16.3_+3 1.2_+. 2 5.2+.6 0.2_+.1 3.3_+.9" 13_+3" 0.1_+0" 0.1_+0" 2.2_+. 5 5.7+1 14_+2 5_+.6* 14_+2" 0.6+. 4" 5.3+1 15-+2 0.1-+0" 0.5_+.3" cba, c57bl/6, balb/c, akr, dba, swiss mice, guinea pigs, rabbits have been used in research work. the toxicity, immunogenicity, mitogenic and immunomodulating activity of lps have been studied. the possibility of reduction of the toxic activity of lps on macroorganism by bioglycansimmunomodulators obtained from sea invertebrates anymals (crenomytilus grayanus, stromhus gigas) have been investigated too. lps has been shown to induce specific antibody response of laboratory animals. cba mice are high responsive to lps. lps stimulates humoral immune response of mice to tdependent and t-independent antigens and suppresses intensity of the delayed hypersensitivity. the small doses of lps stimulate functional activity of macrophages, the large doses of lps -decrease one and show the cytotoxic effect. the bioglycans enhance the resistance of mice to the lethal effect of lads and provide protection 59-75% of mice. one opens possibility to use of bioglicans for reduction of toxinemia in generalizated forms of pseudotuberculosis. thus, lps from y.pseudotuberculosis is immunogen and immunomodulator wich has influence on humoral and cellular factors of immunity and plays the important role in immunopathogenesis of infection. endotoxaemia is implicated in the pathophysiology of obstructive jaundice. the lirnulus lysate (lal) assay is the gold standard method for measuring endotoxin concentrations, but inherent biochemical and technical problems limit the usefulness of this assay. the endocab elisa is a novel assay which measures endogenous antibody (igg) to the inner core region of circulating endotoxins (acga). objectives we evaluated the significance of endotoxaemia in biliary obstruction using the endocab assay and subsequently the specificity of the humoral response to endotoxin compared with an exogenous antigenic challenge [tetamls toxoid (tt) ]. materials and methods in experiment i three groups of male wistar rats (250-300g) were studied [no operation (n=39) , sham operation (n=40), and bile duct ligation for 21 days (bdl)(n=50)]. plasma was collected and assayed for bilirubin, endntoxin(lal) and acga(endocab). in experiment ii 30 rats were actively immunised with tetanus toxoid ('it) and then randomised to have no op(n=8), sham op(n=8) or bdl(n=i4). blood was taken at this time (to) and 21 days later(t20 at sacrifice for acga concentrationslendocab] and igg produced to tt(ttab) [elisa] . antibody concentrations are expressed as % increase from control values.results in bdl rats, acga concentrations were significantly increased compared with controlslp<0.001, mann-whitney]. endotoxin concentrations were sporadically elevated in the jaundiced rats but the rise was not significant. in experiment [i there was no difference between the acga or ttab concentrations in the fllree groups at to, bdl rats had a significant rise in acga concentrations by t21 [p<0,001,paired t-test] and humoral response to tt was significantly impaired in bdl rats compared with control groupslp<0.05, paired ttest data plasma endotoxin was measured by means of an endotoxinspecific endospecy test after pretreatment of the plasma with a new perchloric acid method that we developed. the normal value of plasma endotoxin is less than 9.8 pglml. polymyxin b was administered at a dose of 12,500 u every 6 hours. plasma endotoxin rapidly decreased to the normal range in 40 of the 42 patients. body temperature fall significantly. apache ii scores were also significantly improved. tumor necrosis factor-o~ and interleukin 6 decreased in survivors, while in high values tended to persist in patients died. no side effects were observed in any of the patients. in conclusion, intramuscular injection of minute of polymyxin b was useful in the treatment of endotoxemia. 19-1 uchimaru, morioka 020, japan. l e v a n t g r a m n e g a t i v organisms. m e t h o d s : u n d e r general anesthesia, 12 n o r w e g i a n b r e d landrace pigs (17-30 kg) of either sex, 6 pr group, u n d e r w e n t t r a c h e o s t o m y a n d w e r e v e n t i l a t e d on a 50/50 air a n d o x y g e n m i x t u r e a i m e d at m a i n t a i n i n g a n o r m a l p h a n d a isocapnic level. ventilation w a s not readjusted d u r i n g the observation period. the anesthesia w a s k e t a m i n e 0.6 m g / k g h a n d d i a z e p a m 2.4 m g / k g h i n t r a v e n o u s l y . h e m o d y n a m i c m o n i t o r i n g of m e a n aorta, p u l m o n a r y artery, central v e n o u s a n d p u l m o n a r y capillary w e d g e pressures w a s p e r f o r m e d w i t h a 5f s w a n -g a n z catheter a n d an aorta catheter. a continous infusion of r i n g e r ' s acetate ( 1 0 m l / k g h ) w a s g i v e n intravenously. w h e n stabilised, the a n i m a l s w e r e g i v e n 2.5 x l09 cfu of e colt intraperitoneally as a bolus in 100ml saline, the a n t i b o d y g r o u p received in a d d i t i o n 5 m g / k g e5 a n t i e n d o t o x i n i n t r a v e n o u s l y over 1 h o u r via a n infusion p u m p at the start of the observation period. the a n i m a l s w e r e observed for 6 hours. results : a t 5 a n d 6 hours, the o x y g e n c o n s u m p t i o n increased by 30 % in the a n t i b o d y treated g r o u p w h e r e a s there w a s a significant fall of 30 % in the sepsis group. in the a n t i b o d y group, the arterial p h a n d the cardiac index were also significantly h i g h e r at the s a m e p o i n t s in time. there w a s no significant difference in arterial po2. in severe bacterial infections it would be beneficial to neutralize the plasma endotoxin content with complex forming compounds. the phenothiazines are able to form complexes with endoto×in and the existence of these complexes were already shown in differential speetrophotometry and animal experiments, however, the mechanism of partial neutralization was not clarified. therefore some representative phenothiazines and structurally related compounds were tested for anti-endotoxin activity. the endotoxin neutralizinb effects of several benzophenothiazines were investigated in differential speotrophotemetry, tnf induction and in the conventional limulus test. in animal experiments some beneficial effect of complex forming compounds was found. the benzophenothiazines were not able to inactivate the biological effect of endotoxin in the limulus test. the recent findings indicates that a multifocal effect can be responsible for "anti-endotoxin action in vivo". effects of tnf inducing effect of endotoxin in leukocytes and bypotensiv action in experimental animals were reduced by some phenothiazine derivatives. monophosphoril lipid a was without effect. of microbiology, albert szemt-gydrbyi medical university, odm t~r lo, h-6720 szeged~ hunbary 46 involvement of streptococcus pyogenes erythrogenic toxins in the induction oflstreptococcal toxic shock syndrome 3 heide mgller-alou~* , joseph e. alouf , die [er gerlach , ~atherine fitting., and jean-marc ca~aillon . unit6 des toxines microbiennes and "unit6 d'immuno-allergie, institut pasteur, 28, rue du docteur roux -75015 paris (france) ; institut f~r experimentelle mikrobiologie, jena (germany). superantigen erythrogenic toxin a (eta) is thought to be involved in toxic shock syndrome in humans by inducing massive release of cytokines by patient immune cells. the cytokineinducing capacity of eta w~:s £:ompa~ed to that of lps, a gram-negative bacterial cell wall component. eta elicited weak production of il-1 d and ~, tnf ~ and il-6 in purified human monocytes whereas lps stimulated the production of high amounts of these cytokines. in the presence of t cells, eta elicited the production of significant amounts of il-i~, il-i~, il-6 and il-8. however, the most preponderant cytokine was tnf~, which peaked at i0 ng/ml after stimulation with i0 ~g eta. comparable amounts of tnfd (ca 4 ng) were induced by 0.i ~g eta and 0.i ~g lp$. in contrast to lps, eta was a strong inducer of tnf~ which was produced only in marginal amounts by lps. these results suggest that the septic shock induced by gramnegative bacteria (lps) and by gram-positive bacteria {extracellular superantigens) follows different pathogenic pathways. lps-induced shock is mainly mediated by monocytes and monocyte-produced cytokines (il-i and tnf). the eta-induced shock is mediated by t-cells or depends on t cell help for the production of monocyte-liberated cytokines. production of t cell cytokines such as tnf~ and interferon 7 in addition to the other cytokines contribute very likely to the severity of the toxic-shock resulting from s. auzeus and s. pyogenes infections in humans. the present study was utidertakc~l to cvalu~tlc the effect of soluble chemically modified giucan during septic shock. carboxylnethyl-b-i,3-glucan (ram 120000) was injected twice 48 and 24 h before the shock i.v. in a dose of 50 ing/kg. shock was induced in u~?esthetizcd (sodikm~. l)mntobarbital) rats by i.v. injection of endotoxin of escherichia colli 0127 bs, 5 mg/kg. aiiofcmg pretreated ruts survived during first 24haher ¢ndotoxine, while in controi shock group the lethality was 90%. the concentration of ~col)terin in serum was significantly elevated 24 hafterthc second cmginjection (appare~tly 80 % if compare with the control rats), but didu't chartged 60 rain and 1 s0 rain after endotoxin injectjom cardiac output in cmogroup was higher a* the i20 and 180 min after endotoxine onset ( 8i % trod 72~, respectively of initial level) than in the control shock group (64 % and 52% at the same time). pretreatment of rals with soh~ble giucan w~ts associated with beneficial effects o~ the hepatic c~ergy $ia[tls after 3 h after challenge of endotoxiae: the tissue level of lactale was ahnost twice lower than in the control ruts, me~mthne the tissue atf in cmg pretreated group was higher at 40 %. twice injected macrophage stimuhttor soluble glucan can prevent the endotoxic shock, and extremely ir~creased survival rate after endotoxine injection. the national committee of surgical infections of the spanish association of surgeons have produced a computer program for the collection and analysis of information on surgical infections. the program is suitable for ibm compatible hard disk personal computers and works through the ms-dos system. the main menu is called up on the screen when the operating disk has been installed; it reads as follows: i. new record; 2. modify records; 3. erase records; 4. searches; 5. reports; 6. configure; o. ouit. if you ask fdr a new record the screen will prompt you to enter the number of case, record number, hospital, age and sex. the next screen will come up and the words "topographic diagnosis" will flash. a menu of 34 areas or organs will be displayed. then, the words "type of pathology" (inflammatory, neoplastic, traumatic and other). days of postoperative period. type of surgery (programmed and emergency). type of operation (clean, clean contaminated, contaminated and dirty). duration of surgery. this is followed by "order of operation" and the "type of anaesthesia (general, regional or local). you are then required to supply the "diagnostic code of who" (icd9) and the "procedure code of who. analytic and concurrent illnesses (total proteins, albumin, haemoglobin, haematocrit, leucocytes, red corpuscles, glucose and bilirubin). the next screen asks for "risk factors" (obesity, uraemia, neoplasia, malnutrition, urinary catheter, distant infection, artificial valve, immunosuppressive drugs, over 65 years and anergy. this is followed by a screen headed "postoperative complications". "evolution" (the questions asked are drainage, systemic antibiotics, and on each ocasion a choice of 31 antibiotics is displayed), local antiseptics, reoperation, etc. under "microhiology" is a choice of 37 organisms and the chance of identifyin 3 organisms. finally, "sepsis score". our recent work had shown that renshen-fuzi-chaihu mixture could increase the survival rate in experimented study. the purpose of this study was to determine the effect of combined administration of renshen-fuzi-chaihu mixtuer and antibitics (sa) in patients with septic shock. the result showed that, in sa group (40 cases), the total effective rate was 87,5%, in the contral group (combined administration of gentamycin and dexamethasone, 25 cases) the total effective rate was 84%. however the obviously effective rate in sa group 70% was significantly higher than in contral group 44% (p<o.os). sa group appeared to be more obvious than the contral group in raising arterial pressure, recovering consciousness and pulse, improving cold lilmbs and reducing fever (p<o.o5). it was found that sa could inhibit the pathogeneses changes of septic shock such as the decreases of superotide dismutase (sod) and glutathione peroxidase (gsh-px) in erythrocytes and the increases of plasma free hemoglobin (phb), plasma malondialdehyde (mda) and 13-glucuronidase (13-gc) occured in septic shock. sa had stronger effect than gd in inhibiting the changes mentioned above. hence, these results suggest that sa has beneficial effect in the treatment of septic shock. sa could diminish the decrease of the antioxdase activities and inhibit lipid peroxidization and stabilize cellmembrance. this may be one of the principal mechanisms of the beneficial effect of sa in the treatment of septic shock. donna l. lehman, heather a. childers, irshad h. chaudry and alfred ayala. the thymus is thought to be a privileged sight for tcell generation. here the t-lymphocytes are either selected for their potential to recognize and react competently to foreign antigens or de-selected, due to their potential to react to auto-antigens, de-selected. tcells with this latter capacity are thought to be deselected through a process referred to as programmed cell death or apoptosis. while it is known that thymic apoptosis is elevated by a variety of stressers associated with infection and inflammation, little or no information is available concerning its presence in polymicrobial sepsis. it was, therefore, the aim of this study to determine whether thymocytes exhibit increased apoptosis during sepsis. to assess this, thymocytes were harvested from c3h/hen mice at i and 24 h following cecal ligation and puncture (clp; to induce sepsis) or sham-clp (sham). thymic yields were assessed and the extent of apetosis determined by staining the cells with propidium iodide (a dna intercalating dye) for facs analysis or by examining the extracted dna electrophoretically for the endogenous endonuclease activity the results (n=6/grp) indicate that at i h post-clp there was no marked changes in any of these parameters. however, at 24 h the thymic cell yield from clp mice was significantly decreased (sham:3.6!0.2 x l06 vs clp:0.3i0.1 x 106* cells; *,p<0.05), the % of cells apoptotic by facs analysis increased from 1.7i0.1% in sham to 8.7±3.2%* in clp, and the septic mouse genomic dna exhibited dna degradation these results indicate that clp, but not simple laparotomy, induces marked thymic apetosis, which may contribute to the lymphocytic immune deficiency seen in these mice the purpose of this retrospective study was to evaluate effectiveness of irrigation to treat septic knee joints from longterm results. from 1982 to 1992 31 patients with septic knee joints have been treated. treatment in acute cases started with asp#at[on of the effusion to evaluate for cultures. without awaiting the result treatment was continued with immediate joint irrigation under arthroscopic control. three redon tubes ( ch 16 ) were introduced for continuous irrigation and for intermittent distention of the joint cavity. in case of chronic joint infection additionally all foreign and synthetic material was removed. systemic antibiotics were given. patients could be re -examined with a mean follow-up of 6 years. re-examination showed that immediate arthroscopic lavage started at the onset of septic sings had best results (less signs of osteoarthr[tis, less synovitis, less pain while loading and less range of motion loss ). 21 patients (80%) out of 26 patients with s tre_=.tsd acute se.~tic knee joint had excellent functional recovery, whereas all 5 patients with chronic septic knee joints showed poor results. additionally, in 3 of these 5 patients with chronic septic knee joint after irrigation a further procedure was necessary due to recurrent septic knee joint. the concept that bacteria can translocate across the intestinal mucosa under a variety of circumstances is well established. however, due to the inherent limitations of in vivo studies, the cellular mechanisms underlying the process of bacterial translocation (bt) across the epithelial barrier are poorly understood. this is especially true in those circumstances in which there is no merphologic evidence of mucosal injury. consequently, we have utilized an in vitro cell culture model system to investigate the cellular events involved in the translocation process. in this model system, a polarized monolayer of intestinal cells (caco-2 cell line) is grown on a 3 micron filter in a two compartment system. after tight junction integrity is established, bacteria are placed in the apical surface chamber and bt across the caco-2 monolayer is measured by culturing the basal chamber. the results of our studies utilizing the caco-2 system indicates that; l) several strains of non-pathogenic bacteria will translocate across the caco-2 monolayer in a time-dependent and dose-dependent fashion. however, the incidence and magnitude of bt are highest for those strains of bacteria (gram-negative enterics) that are most commonly cultured in vivo. 2) caco-2 cells are actively involved in the transcellular passage of bacteria across the caco-2 monolayer, since inhibition of caco-2 microtubule or microfilament function decreases the magnitude of st. 3) lectin but not integrin receptors are involved in bacterial translocation of e. col~ across the caco-2 cell monolayer. 4) caco-2 cells have the ability to ingest and kill certain strains of bacteria. the mechanisms involved in caco-2 bactericidal activity appear similar to that of pmns to the extent that both are oxidant but not nitric oxide mediated. these in vitro studies suggest that caco-2 cells can function as "nonprofessional" phagocytes and that both bacteria and enterocytes are involved in the translecation phenomenon. bacterial translocation due to gut barrier dysfunction is considered to be a major cause of septic complications and multiple organ failure following trauma, burn injury, hypoxia, shock and shock-like states. the invasion of bacteria into the circulation from the gastrointestinal tract or even from other sources, however, should not necessarily result in systemic infection or organ cohinisation, as long as the humoral and cellular defense mechanisms are not impaired. thus, a reduced res clearance capacity and hepatic clearance function of bacteria and toxins can be observed under the same conditions which enable bacterial translocation from the gut. in order to evaluate the influence of circulatory, metabolic and humorul disorders on the elimination of bacteria out of the blood circulation, a series of experiments were performed in anesthetized and ventilated rabbits which received defined numbers (1.3 x 108 colony-furming units) of escherichia coli as a bolus injection. in unaffected control animals the intravenously injected bacteria are eliminated about 99.9% within the first 15 minutes and are totaiy cleared within 90 minutes. 57% offue total cfu counted in the cultures of the organ homogenates were found in liver and spleen, whereas only very low numbers could be detected in the lungs and kidneys. systemic injury of the animals by hemorrhagic shock, endotoxinemia, hypoxia, coagulation disorder, systemic vasoconstriction by norepinephrine and other types of injury reduced the elimination rate of bacteria and changed the pattern and degree of their dissemination and tissue distribution. the bacterial clearance was delayed mostly in rabbits subjected to hypoxemic hypoxia in which about 100 cfu of viable e.eoli per ml blood could still be counted 3 hours following their injection. the number of viable bacteria was some ten powers higher in organs of hypoxic rabbits in comparison to control ardmais, and an excessive colonisation of the lungs and kidneys with 25% respectively 11% of the total cfu of the cultared organ homoganates was observed. unilateral iscbemia of the kidney prior to the intravenous injection of e.coli only moderately delayed the bacterial clearance, bowever, caused an intense eohinisation of the affected reperfused organ. in contrast to this, inhalation injury did not promote the accumulation of bacteria in the lungs. conclusion: shock, systemic and local affections reduce the clearance of bacteria from the blood circulation and enable their dissemination and accumulation in vital organs. the degree of this reduction and the organ pattern of the bacterial distribution varies with the type of injury. thus, these factors seem to be essentially involved whether or not bacterial translocation and multiple organ failure will follow the invasion of bacteria into the circulation as in the ease of gut barrier dysfunction. movement of enteric baaterla from gut to extralumeaal sites (bacterial tra~sloeation) m~y play a role ~. nraltipl~ orga~ failure. traumatic stress (shock, hffectiort, and im~lammation) and severe illness are common alateeedents. tnt~st~aal lleus is e common proximme causal factor. we have exanlined the hypothesis that bacterial ttanslecation from the gut in expe~me~tal paaerestitis is due to bacterial overgrowth as a cuas~ucnee of a deere~ae in intestinal transit. ne~rotit_.~g pancreatitls fbliowlng bile duct iigation in the opossum is associated with colonization of the pa,se ~re, aa by gut derived organisms or salmonella of biljary origin in infected animals (previously reported). to ti~rther define the mechanism of transloemdon in panereati* inflammation, pancteatitis was induced hy iigatlon of t~e lower bile duet distal to the pancreatic duets m the rat. intestinal transit, enteric bacteriology, and tromsloeatien of bacteria to mesanterie lymph nodes, blood stream and splanelmie organs wen deletmhaed at 24 (n~ 7), 4g (n ~ 10), and 96 (n~ 10) hours with the fallowing results: ly~testinai,..transfi". geometric mean of fluorescent marker -25 % at 24 hour~ with return to 51% of gut leugth (similar to ~m) at 96 hour~. bacterial overgrowt_hff -increase in total bsctetia ~ad gram negative rod.~ at 2,; hours with return to sham control at 96 hours (9 log cfu/g veraus 6 log cfu/g ia ileum). translocation to mesenteric nodes -71, 90, and 30 percent at 24, 48, and 9fi hours compared to 49b in sham controls (n=l of 26). conclusion -bacterial translocation following panetoatieobiliary duct ljgation induced panereatitls ha the rat is tighlly linked to intestinal trausit and bacterial overgrowth within the gut lumen. speclrjlation -the ilens in in/~arm~ation may be related to activation of eytokiaes and mediated by nitric oxide. preliminary evidence for this notion will be discussed. in a series of studies we investigated: a) the time course of bacterial translocation (bt), b) the kinetics of lps and cytokine appearance (tnf, il-6, soluble tnf receptor [stnf-r] ) in the circulation, and c) the role of lps and/or tnf with regard to haemorrhagic shock (hs) related pathophysiologie alterations and mortality in baboons and/or rats. method: baboons were subjected to femur fracture, soft tissue trauma (acute experiment), and hs (40 mmhg mean arterial pressure: map for 2-4 h terminated at an accumulated oxygen debt of 180-200 ml/kg followed by resuscitation). a chronic model of hs was set up by administration of zymosan activated plasma (zap) before and after hs but without trauma. hs was induced in rats by bleeding the animals to map of 30-35 mmhg 90-180 rain followed by resuscitation. results and conclusion: in rats, lps increased in the systemic circulation, plasma tnf levels were found to be significantly elevated at 90 min and were still markedly increased at 150 rain postshock. plasma tnf, il-6, and stnf-r levels increased already during the shock period in baboons, while tnf was not detectable. our data suggest that haemorrhagic shock may lead to early bt in the intestinal wall (at 30 min following resuscitation in rats subjected to hs for 90 min, similarly at 3 h after the end of oxygen debt period in baboons ) and transient access of gut-derived lps into the circulation (levels became significant at 120 min, while in baboons higher levels up to 300 pg/ml were found at the end of shock and 1 h after reinfusion, partially accompanied by bacteremia). in addition, since the treatment of rats with anti lps measures or anti tnf therapy significantly improved the 48-hour survival rate it can be assumed that endogenous lps and lps-induced mediator(s), in particular tnf, may lead to organ injury and mortality following haemorrhagic shock. alteration of the mesenteric blood flow and the consequent ischemia / reperfusion injury to the intestine appear to be one of the earliest events in the systemic inflammatory response following severe thermal injuries. the causative relationship between the subsequent disruption of the intestinal integrity and the potentiation of the translocation of indigenous bacteria and/or endotoxins to remote body organs and the systemic circulation has been deeumented in several studies. among other effects, endotoxemia solely or acting synergistically with thermal injuries has been shown to promote bacterial translocation. as these sequences continue without adequate intervention, multiple organ failure and eventually death may become inevitable. hence, the crucial need of treatment modalities with the capacity of modulating the intestinal blood flow and preventing the manifestations of these pathological incidents. although the underlying mechanisms of this process have not yet been fully elucidated, there is accumulating evidence that various interacting vasomediators are involved. the actions of these mediators can probably be modulated by either nonspecific or selective agents. among the nonspecific agents, the composition, volume and timing of the resuscitation fluids appear to play an important role in this process. nitropmsside, a nonspecific vasodilator, has been shown to prevent the decrease of the postbum mesentefic blood flow when selectively infused in the mesenteric artery. the nonspecificity of various vasodialators with their possible undesirable systemic effects emphasizes the importance of the identification and modulation of selective vasomniiators. thromboxane a2 (txa2) and angiotensin ii (a-ii) appear to be the primary mediators of the postburn vasoconstriction. both mediators were found to be elevated following thermal injuries. in a previous study, pretreatment with oky-046, a thromboxane synthetese inhibitor was found to attenuate the postbarn mesenteric vasoconstriction. in a bum/sepsis porcine model the efficacy of dup753, an a-11 receptor antagonist as a posthum treatment modality was evaluated. treatment with dup753 prevented the early posthum and the late posteodotoxin elevation in the mesenteric vascular resistance and subsequently abrogated the fall in the mesenteric blood flow. the incidence of burn & endotoxin induced bacterial translocation was significantly reduced by dup753 from 86% to 29% (p<0.05, fisher's exact test). the indigenous flora of the gastrointestinal tract exerts a potent influence on the developmental maturation of normal systemic immunologic responsiveness. moreover, both oral and intraportal administration of experimental antigen is associated with a systemic state of immunologic hyporesponsiveness, known as oral tolerance. since trauma and critical illness are associated with changes in the flora of the gut and with altered systemic immune reactivity, we have hypothesized that the interactions of an altered gut flora with immune tissues in the gut and liver play a role in the pathogenesis of the immunologic derangements of critical illness. prolonged feeding of two clinically relevant killed organisms-pseudomonas and candida-to a rat results in significant impairment of cutaneous delayed hypersensitivity (dh) reactivity. conversely, measures directed against gram negative bacterial overgrowth in experimental peritonitis result in partial restoration of impaired dh reactivity. to ascertain the role of the liver in the pathogenesis of these alterations, we studied the differential immunologic sequelae of portal versus systemic (ivc) infusion of killed bacteria. experimental infusion of live or killed gram negative organisms into the portal vein produces dh suppression in rive, and profound suppression of mitogen-driven lymphocyte proliferation in vitro; systemic administration of the same organism has no effect on these phenomena. suppression is tgf3-dependent, and mediated by a soluble factor released by fixed tissue macrophages of the lung and spleen. the suppressive influence appears to be mediated at the level of interactions between il-2 and its high affinity receptor. release of this factor can be induced by a second circulating factor present in the plasma of portally-, but not systemically-infused animals two hours after bacterial administration. these studies suggest that the release of inducible immunoregulatory factors from the liver, and possibly from the gut, may play a role in the pathogenesis of the profound derangements of systemic immune homeostasis that accompany trauma and critical illness. acad.hospital st. radboud,postbus 9101,6500 lib nijmegen introduction. the central question being tested in this study was whether liposome encapsulated dichloromethylene-diphosphonate (ci2mdp) mediated elimination of liver and splenic maorophages would influence zymosan induced systemic toxicity (st) and bacterial lranslocation (bt). materia|s and methods. 90 male swiss (icr) mice were used weighing 21-25 g elimination of liver and spleen macrophages was accomplished by intravenous injection of 200/11 suspension of ci2mdp-liposome suspension. control mice received an i.v. injection with 200 pt phosphate-buffered saline (pbs) . two days later all mice were challenged with an i.p. injection of zymosan suspended in paraffin (z) at a dosage of either 01 mg/g, 0.5 mg/g or 1.0 mg/g body weight (n=8 in each group), signs of st and bacterial translocation bt were measured 24 hours later the st was assessed by blindly grading the degree of lethargy, conjunctivitis, diarrhea, and ruffled fur of each mouse on a four point scale. bt to the mesenteric lymph nodes (mln) and systemic organs (liver, spleen, lung and blood) was tested by culturing on blood and macconkey's agar at the time of sacrifice sections of the distal ileum were taken for histology. an additional two control groups (n=6 in each group) were tested to verify that neither paraffin nor pbs or ci2mdpqiposome suspension alone induced bt or st. furthermore survival over a 12-day period was assessed in a control and macrophage-depleted group with a dose of 1d mg/g z. results. neither the paraffin nor ci2mdp-tiposome suspension induced bt or st the incidence of bacterial translocation to the mln was similar between macrophage depleted and control groups. however, macrophage depletion was associated with a significantly higher incidence of systemic spread of bacteria. data expressed as positive tissues/total tissues tested: 11/32 vs. 1/32 at 0.1 mg/g zymosan (p~o.01); 19132 vs. 10132 at 05 mg/g zymosan (p~q05); 23/32 vs. 11/32 st 1.0 mg/g zymosan (p~o.o1).the magnitude of bt however did not show any differences. although systemic bt was greater in the macrophage depleted groups, the systemic toxic response was significantly less at doses of 0.5 mg/g zymosan (3.14 -+ 0.69 vs. 7.13 -+ 3, p~o.01) and 1.0 mg/g zymosan (4.38 -+2.50 vs. 11.25 -+ 2.5, p<_.o.01) . the 12 day mortality after 1.0 mg/g zymosan was 27% in the control group and 0% in the macrophage depleted group (p=o.05). histology did not show any differences between the control and macrophage depleted groups. conclusion. the lethal and toxic effects of zymosan in this model appear to be more related to the excessive activation of macrophages than to the systemic spread of bacteria. bacterial translocation (bt) in hemorrhagic shock was studied using a porcine model. in this study three groups were randomized: one control (n=3) and two experimental (n=6 each). a portal vein catheter was placed and remained in situ 48 hrs. a femoral artery and vein catheter were in place 12 hrs. the arterial eaanula was used for monitoring mean arterial pressure (map) in all animals and to bleed the experimental animals and the venous cannula to repelfuse with ringer's lactate (rl) or autologous blood (ab). baseline samples and values were taken in this period. control animals were observed for a sham shock period of 6.5 hrs., given 2 more hours of anesthesia, and studied for further 48 hrs. 40% of the estimated blood volume was withdrawn from each experimental animal over 30 min. these animals were kept in shock for 6.5 hrs. at the end of the shock one group was perfused with rl and the other with ab. two hours later they were extubated and further studied for 48 hrs. samples from portal blood for cultures and measurement of leukotriene b4 (ltb4), prostaglandin e2 (pge2), and nitric oxide (no) metabolites were taken from all animals at intervals beyond baseline up to 48 hrs. after 48 hrs. under general anesthesia a second laparotomy was performed and samples for culture from mesenteric lymph nodes (min), liver and spleen were taken; as well as samples for histologic study with light and scanning electron microscopy were taken from jejunum, ileum, and colon. animals were then euthanized. results: significant shock was induced as indicated by map and arterial blood gases. significant bt to min was observed in all groups. no significant bt was observed in cultures from liver and spleen in any of the groups. increased values for ltb4, pge2 and no metabolites were found during the period of shock. these results suggest that the isehemic phenomenon triggers a significant inflammatory response, and that bt to min may be an epiphenomenon without apparent significant influence on the inflammatory response. objective: to study the potential effects and mechanisms of action of systemic administration of monoclonal antibodies anti-endotoxin (ha-1a) in a animal model of gut-origin sepsis. materials and methods: exoeriment a. balb/c mice were transfused with allogeneic blood (from c3hb-iej mice). five days post-transfusion the animals were gavaged with lxl09 viable escherichia coil and randomized into 3 groups (n=22/group) to receive a sham burn (sb group) or a 20% thermal injury immediately followed by the systemic administration, via a tail vein, of monoclonal antibodies (6 mg/kg) (ha-1a group) or a 20% burn injury and administration of equal volume of sterile saline (c group). all 3 groups were resuscitated by intraperitoneal injection of 0.3 ml of saline. the animal survival rate was observed for 10 days post-burn. experiment b, transfusion and burn procedures were identical to experiment a but the mice (n=8/group) were gavaged with 109 viable e.coli labeled with mmndium oxine. four hours after burn the mesenteric lymph nodes (mln), liver, lungs and blood were harvested to determine plasma endotoxin levels and the magnitude of transtocation of labeled bacteria as measured by the residual radioactivity (dpm) in the organs. circulating endotoxin levels were determined by limulus colorimetric assay. diamine 0xidae(dao) is an enzyme existing in the mucosa of small intestines, but its activity is low in the plasma. it is evoked in the blood with the intravenous injection of much heparin. it is well known that da0 declines under the chronic mueosal injury when the small intestines are cut off or the mucosae become atrophied. aim of this paper is to confirm that da0 goes up to the measureable level by the intravenous injection of a small amounts of low molecular heparin(lmh objectives: the aims of this study is to investigate the inhibitor" effect of the prolease inhibitor, urinastatin, on endotoxin induced bacterial transleeation in mice. materials and methods:lcr mice were divided in six groups as the fotlows, group i: control mice receiving intraperitoneal injections (ip) of saline 24.5 and 24 hr prior to sacrifice, group ih treated mice receiving ip of utinastatin 24.5 hr and endotoxin (5mg/kg) 24hr prior to sacrifice,group ilk sham4reated mice receiving ip of saline 24.5 hr and endotoxin (5mg/kg) 24hr prior to sacrifice,group iv: saline control mice receiving ip twice during 30 rain intervals,group v: mice received ip of utinasmtin, and 30 min later they were received ip of endotoxin (5mg/kg), group vi: mice received ip of saline,and 30 min later they received ip of endotoxin (5mg/kg). in group l,ii and ill ,the mice were killed by cervical dislocation.using stetile techniques,a middle-line incision was made and the mesenteric lymph nodes were harvested in order to culture on selective agars for quantitation of bacterial trauslocation. in group iv, v and vi, survival was recorded for 3 days. unnastatin pretreatment could rednce mortality significantly. first surgey, shiga university of medical science, seta ,ohtsu 520-21,japan increased gut permeability correlates with inflammatory response in multiple trauma aptients. pape, h.-c. dwenger, a. grotz, m. regel, g. purpose: disturbances of intestinal permeability have been advocated as a pathogenetic factor of posttranmatic multisystem organ failure (mof). a close relationship with impairment of the stationary reticulocndothelial system (res) and a systemic inflammatory response (sir) was discussed. these tactors were investigated prospectively in patients with multiple trauma showing posttraumatie complications (multiple organ failure, mof). methods: 31 polytranmatized patients were studied prospectively. according to a mof-score (goris) those with posttrattmatic complications were selected (>7 points at 2 days), others were excluded. every second day gut permeability according to the ratio of urine concentrations of lactulose and mannitol (l/m) was evaluated (enteral application). at parallel time points res clearance capacity (k-value, invasion constant, normal range 0.6 -0.8 mind) was studied after i.v. injection of 100 mbq 99rotehuman albumin. liver perfusion was calculated from these data, total serum bilirubin (/zmol/l) was documented. serum elastase (#g/l) levels were determined enzymatieally. results 86.0 148+ 239+ liver perfusion did not ehangu, bilirubin showed progressive worsening indicating mof. a positive correlation was present between l/m and k (r=0.88) and between l/m and ela (r=0.71). conclusions: there is a positive correlation between the time pattern of intestinal permeability dysfunction and res hyperactivity as well as between intestinal permeability and the systemic intlammatory response (elastase levels). the results speak in favor of an interaction between intestinal and extraintestinal inflammatory systems, which in eombiuation are likely to be responsible for post~anmafic complications. endotoxemia, il-6 release and consecutive acute phase reaction are observed as a host response to surgical trauma. as well vasodilative prostaglandins (pg) and thromboxane (tx) are released after abdominal meaenteric traction (mt). the following hypotension and acute hypoxeraja are duo to prostacyelin (pgiz) arm can be avoided by perioperative cyclooxygenase inhibition. we therefore focused on the effect of pg and tx liberated following mt on the induction of endotoxemia. methods: in a prospective, randomized double-blinded protocol 50 patients, who were scheduled for major abdominal surgery (pancreatic or infrarenal abdominal surgery), were studied. ibuprofen (400 mg i.v.) or a placebo equivalent was administered 15 minutes before skin incision. mt was applied in a uniform fashion. baseline values were obtained before induction of anesthesia. further measurements followed before the incision of the peri[onenm (tl) and 5, 30, 90, 180 min, . the plasma concentrations (,pc) of 6-keto-pgft,, txb: and-ki-12-pgf ~ (stable metabolites of pgi2, txa2 and pge~) were determined by ria. we measured endotoxin pc by limulus-amoebocyte-lysate test and il-6 levels by elisa. data are given as mean+sem (* p< 0.05 placebo vs. [ibuprofen] ). results: endotoxin plasma levels increased before incision of the peritoneum tl both in the ibuprofen pretreated and in the placebo group. peak pc were observed 180 minutes after mt. endotoxin pc were significantly higher in the ibuprufen treated group (t5 0.16+0.02e[0.32+0.06] eu/ml). il-6 pc demonstrated an increase continuously from t4 to t7 (t7 115+12 [91 + 15] ng/l) in both groups. after intentional abdominal mesenteric traction we observed a marked increase of 6-keto-pgf~,, pc up to 6h after mt in untreated patients with a peak of 2450*[60] ng/1 at tl. also txb: and kh2pge 2 pc showed a considerabe increase up to 6h after mt in the placebo group. in ibuprofen pretreated patients the pg and tx pc remained within the normal range. discussion: our data clearly indicate a significant endotoxemia and il-6 release following major surgical trauma which is not initiated either by prostaglandin or thromboxane release. moreover endotoxemia is accentuated by ibuprofen pretreatment. therefore we hypothesize that in major abdominal surgery prostacyclin release-after mt may play a crucial physiological role in maintaining splanclmic microcirculation and thus preserving gut mucosal barrier function. objectives of the study it has been shown recently that parenteral and certain euteral diets promote the translocation of gut flora to the mesenteric lymph nodes (mln) and systemic organs, a process termed bacterial translocation (bt). in chow fed rats bt usually does not occur without further promoting factors. the goals of the present study were to determine whether the provision of defined amounts of standard lab chow during iv-tpn administration wotfld redane the incidence of bt, materials und methods male spf spragnle-dawley rats were divided into 6 groups. group 1 received standard laboratory chow feeding ad lib. in group 2 a central venous catheter was placed, ligated and secured by a spring coil tether attached to a swivel allowing free movement in the housing cage and chow was fed ad lib. in group 3 50% of the calculated daily required calory intake (drci) (307/kcal/kg) was given by iv-tpn (28% glucose, 4,25% amino acids) and 50% by limited chow administration. groups 4 and 5 received 70% and 90% of the drci by i.v. tpn and 30% and 10% respectively by chow feeding. group 6 received iv-tpn only. after 7 days the rats were sacrificed and the mln, liver, spleen and cecum removed aseptically, homogenized and cultured for bt samples of distal ileum were taken for light microscopy. the group with the least amount of chow shown to be protective against bt received the amount of non-fermentable fiber of that chow regimen during iv-tpn feeding and bt was studied. 4 5,5+0,9 4 12 23,9 -2,3 16 ,7 2/12 63+11 125~1 "7,7-+0,7 6,7-+1 5 12 24~6 -3,5 67 8/12 241+~243 170+163 8_+0,7 7+1,1 6 18 25, 5 -4 88,8 16118 2061-+3224 2211+4015 8,4~0,7 8,1-+1~1 conclusions: the administration of 30% of drci by chow feeding during iv-tpn significantly reduced the incidence of bt and maintained gut barrier function. the addition of the respective amount of dietary fiber of this group did not prevent iv-tpn-indueed bt. dr. med. m naruhn., dep. of general surgery, eberhard-karls-university, hoppe-seyler-str. 3 previous experimental studies have suggested that a disturbed ecology of the enteric bacterial population might contribute to the development of bacterial translocation from the gut in acute liver failure (alf). in the present study, the effect of oral administration of lactobacillus reuteri r2lc and oat fiber on bacterial overgrowth and translocation was investigated in rats with acute liver failure induced by subtotal (90 %) liver resection. the oatmeal soup base was anaerobically inoculated with lactobacillae and fermented for 15 hours, after which the animals were fed with either fermented or unfermented oatmeal or saline daily for 6 days prior to the operation. bacterial translocation to mesenteric lymph nodes (mln) and the systemic circulation was determined, as well as the intestinal bacterial flora and enterocyte protein content. the incidence of bacterial translocstion to the systemic circulation was nit in rats subjected to sham operation and saline treatment and 17 % in animals subjected to 90 % bepatectomy and lreatment with fermented oatmeal, while 80-90 % and 34-50 %, respectively, in rats subjected to hepatectomy and treatment with either saline or unfermented oatmeal. only one rat with fermented oatmeal demonstrated bacterial growth in mln (p < 0.05 vs hepatectomy and treatment with saline or unfermented oatmeal). the enterocyte protein content significantly decreased (p < 0.01) in salinetreated animals following 90 % hepatectomy, while there was no significant difference between bepatectomized animals with oral administration of fermented or unfermented oatmeal. the number of anaerobic bacteria, gram-negative anaerobes and lactobacillus significantly decreased and the number of e.cnli increased in the distal small intestine and colon in hepatectomized animals with enteral saline or unfermented oatmeal as compared with animals subjected to sham operation or bepatectomy with fermented oatmeal. our results thus show that the occurrence of bacterial translocatiou from the gut in 90 % hepatectomy-induced alf could be prevented by enteral administration of fermented oatmeal, maybe partly due to a positive effect on the enteric bacterial ecology. 190_+20" 16+_3" 11.7" data=mean_+sd, * stats anova p<0.05 vs control. l+air and lap groups, both exposed to exogenous i.ps shnwm:t m significant increase (p<.05) in lps gut translocation compared to control and l+co2. this correlated with a significant increase in peritoneal inflammatory responses (o2-,tnf) above that of the control and l+co2 groups, while mac-1 and cr3 opsonized phagocytosis were significantly impaired. the absence of significant differences between l+air and lap groups indicates that lps rather than wound factors is the principle mediator. thus, lps plays a significant role in regulating peritoneal responses in the early post-operative period dept of surgery, rcsi, beaumont hospital, dublin 9, ireland brlke e, berger d, staneseu a, buttenschsn k, vasilescu c, seidelmann m, beger hg in 32 patients undergoing a colonoscopy, endotoxin, endotoxin neutralizing capacity (enc), thromboxane b o (stabile metabolite of tbmomboxane ~), 6-keto-prostaglansin, leueotriene c4, interleukin 6 and the incidence of bacteremia were determined before and then every five minutes during the procedure. twenty-one of 32 patients showed a significant increase of endotoxin plasma levels during colonoscopy (p=0.003), whereas only one patient had a positive blood culture with bacteria obviously derived from the gastro-intestinal tract. the enc decreased significantly five minutes after the beginning of eolonoscopy and was diminished further thereafter. the baseline values were reached after 24 hours. ~hromboxane b o levels also increased after five min. from 89 to 376 pgyml peaking at 30 min. with 1332 pg/ml. 6-keto-prostaglandin,leucotriene c 4, ii-6 and crp remained unchanged. a control group of i0 volunteers who were not subjected to endoscopy, were prepared for eolonoscopy by orthograde lavage. the blood sampling procedure remained identical. no differences were seen in all described parameters for the controls. these data show that the gut barrier can be compromised by mininml invasive procedures, at least, concerning bacterial products. living bacteria, on the contrary, do not pass the gastro-intestinal wall. endotoxin, when determined by enc, is more sensitive than the conventional limulus-amebocyte-lysate test. no acute-phase reaction was induceri by the observed endotoxin translocation. it can be speculated from the dramatically enhanced thromboxane b 2 levels, together with its hemodynamie effects, that the thromboxane release may support translocation of bacterial products. sepsis is common after hemorrhagic shock. this study aims to demonstrate that hemorrhagic shock alone can promote translocation of gut bacteria from intestinal tract to its regional nodes and subsequently to blood. one hundred twenty mice, divided into 4 groups were subjected to 30, 60 and 120 minutes of 20%, 30% and 40% of hemorrhagic shock. on the specified time, blood cultures were taken and mice were sacrificed. the intestinal tract were histologically examined for any changes which allows translocation and its regional nodes were quantitatively cultured for translocated bacteria. there was a direct relationship between duration and degree of hemorrhagic shock and incidence of translocation (p 0.05). there was a high incidence of gut bacterial translocation to the mesenteric and mesocolic nodes in all degrees of shock (p 0.05). bacterial growth in the regional intestinal nodes increased and blood cultures were positive in direct proportion to degree and duration of shock. histologic evaluation of segments of git showed submucosal congestion to allow bacteria normally contained within the gut to cause systemic infections. translocation of gut bacteria in untreated hemorrhagic shock is clearly shown in this study on animal models. in this study, guotobiotic rats with 5 known species of bacteria were subjected to total parenteral nutrition(tpn) and subsequent hemorrhagic shock. the purpose of the study was to observe the impairment of gut barrier function following tpn and hemorrhagic shock and to study the mechanism of enterogenic infection induced by tpn and shock.the results were as follows: 1.long term(8-12 days) tpn induced impairment of gut barrier function, evidenced by atrophy of intestinal mucosa, significant decrease in diamine oxidase activity of intestinal mucosa and blood, and marked microecologic imbalance of the intestinal mucosa flora with dorminant growth of aerobes and relative decrease in anaerobes. the degree of mucosal damage were proportional to the duration of tpn. 2.in tpn+shock groups, failure of gut barrier function was found. ri,~ere were further damage in the mucosa, with a large number of gramnegative organisms invading mucosa and submucosa and a significant decrease in dao activity as compared with each relative tpn groups. these changes were significantly correlated with enhanced bacterial translocation, elevation of lps and mda levels in the plasma. these findings suggested that long term standard tpn impaired the gut barrier function, precipitating posttraumatic gut barrier failure. thus infec. fion following shock might be oi'iginated from the gut and it was obviously related to the impaired gut defence resulted from antecedent tpn. the determination of plasma dao activity might provide a valuable tool for the ear. ly diagnosis of gut injut;y during tpn and after trauma. in our earlier studies we have investigated the dynamics of granuloayte infiltration of the ischemic/reperfused s~all intestine (g. illy~s, j. hamar int. j. exp. 9athol. 73. 161. 1992.) . there was a increasing infiltration of the mucosa c111m~nating at the 3d to 4th hours of reperfusion. in the present series we have studied sc~e of the conseqn/ences and the possible role of this cellular reaction. 45 ~in isehemia was followed by a 4 hour reperfusion in the anesthetized rat. arterial ~/ad mesenteric venous blood samples were collected at 5 m_in, i, 2~ 3, and 4 hours of reperfusion. elastase and lactate concentrations were determined and hamoculture was carried out from the blood samples, and tissue pieces from the heart, lung, liver and kidney were collected for histological analyses at the above mentioned times of reperfusion. all blood samples were free of cell bacteria. staphylococci appeared only occasionally at the 4th hour in the arterial blood .and at the 3d and 4th hours in the venous blood, respectively. arterial and venous elastase activities were high throughout the reperfusion, venous concentrations being higher at all times. lactate concentrations of the arterial and mesenteric venous blood samples increased during shock. ~ranuloeyte infiltration of all organs studied appeared during the 2d hour and it increased at later times of reperfusion. it is concluded that heavy infiltration of the intestinal mucosa can block bacterial translocation in most of the cases during reperfusion. granulocytes activated either by the reperfused area or by the released cytokines infiltrate other organs contributing by this way to the mesenteric shock s!rndrc~e. intestinal motility plays an important role for maintaining nutrient transport and absorption and for balancing the enteric bacterial population. disturbances of intestinal motility may be one of the earliest notable changes in intestinal function. in the present study, we aimed at determining early alterations in intestinal transit time following ischemia-reperfusion injury induced by occlusion of the superior mesenteric artery in the rat. intestinal ischemia was induced for 20 and 30 minutes by applying a microvascular clip on the superior mesenteric artery followed by reperfusion 2, 4 and 6 hours after clip removal. intestinal transit time was measured by the propulsion of a radiolabelled solution (cr51). light microscopy was performed on intestinal samples. macroscopical pathological changes were not observed. however, microscopically, mucosal epithelial oedema, degeneration or slight ulceration occurred in rats 6 hours after reperfusion in ischemia-20 rain group and 4 and 6 hours after reperfusion in the ischemia-40 rain group. delayed small intestinal transit time was seen from 2 hours and on after intestinal ischemia for both 20 and 40 rain ischemia followed by reperfusion. the distribution of radioactivity demonstrated that most radioactivity was accumulated in the first two segments following intestinal ischemia and reperfusion, significantly differing from what was seen in animals subjected to sham operation (p < 0.01). the distribution of radioactivity in segments 4 and 5 in the group with repeffusion 6 hours after intestinal iscbemia for 20 rain was significantly higher than that noted in the group with repeffusion 6 hours after intestinal ischemia for 40 min (p < 0.01). q'he results indicate that a delayed intestinal transit time may be one of the earliest pathophysiological alterations noted, associated with duration of gut ischemia, and a potential factor for the development of bacterial overgrowth, gut barrier failure and bacterial translocation, in hypovolemic conditions. bacterial infections still constitute a major cause of morbidity and mortality in patients with acute liver failure. the present study aimed at evaluating the effect of ethylhydroxyethyl cellulose (ehec) on bacterial translocation following surgically induced acute liver failure. acute liver failure was induced by subtotal hepatectomy (90 %) in the rat. water-soluble ehec was administered orally 1 and 12 hours prior to hepatectomy. the incidence of bacterial translocation from the gut to mesenteric lymph nodes (mlns) and systemic and portal circulation was evaluated and the number of isolated bacteria from these samples and from intestinal content were determined. intestinal transit time, bacterial adherence onto the intestinal surface, intestinal mucosal mass, bacterial growth and dna synthesis, bacterial surface characteristics (hydrobiology: hydrophobicity, hydrophilicity and neutrality; surface charges: positive, negative and neutral) were also determined. hepatectomized animals showed a 80-100 % translocation rate to mlns or blood 2 and 4 hours after operation, while only 0-1% of rats subjected to sham operation or animals with 90 % hepatectomy and pre-treatment with ehec (p < 0.01). bacterial overgrowth, increased bacterial adherence onto the intestinal surface as well as decreased intestinal mucosal masses were observed in animals with subtotal liver resection alone, alterations that were prevented by enteral ehec treatment. a delay in intestinal 2-hour transit time occurred in both groups with subtotal liver resection, with or without enteral ehec. ehec inhibited bacterial growth and dna synthesis, and altered bacterial surface properties following 1hour incubation with bacteria. in conclusion, the findings in the present study imply that ehec alters enterobacterial capacities for metabolism, proliferation and invasion by effects on e.g. bacterial surface characteristics. furthermore, ehec seems to possess a trophic action on the intestine, rather than exerting its effect by enhancing intestinal motility. department of surgery, lund university hospital, s-221 85 lund, sweden disturbances in intracellular calcium signalling can potentially result in impairments of cellular responses vital to the functional integrity of both immune and non-immune cells, and thus contribute to a decrease in host resistance against infection and to multiple organ system failure during sepsis. studies in our laboratory have focused on assessments of intracellular ca 2÷ regulation and ca~+-depended cellular responses in the liver, skeletal muscle and splenic tlymphocytes harvested from rats subjected to gram-negative intraabdominal sepsis. cytosolic ca 2+ concentration, [ca2*]i, and ca 2+ fluxes were measured by the use of fluorescent ca 2+ chelating dyes (fura-2 or indo-1) and 45 ca respectively. to assess sepsis-related changes in ca 2+ dependent cellular responses, we measured the acute phase protein response in the liver, the regulation of protein and sugar metabolism in the skeletal muscle, and the proliferation response in the splenic tlymphocytes. altered ca2+ i signalling with sepsis was correlated with an exaggerated inappropriate acute phase protein response (100% ¢) in the liver, and a blunted insulin mediated sugar utilization (60% 4) and increased proteolysis (50% ~) in the skeletal muscle. in t-lymphocytes, a decrease in mitogen induced elevation of [ca2+]i by 35-40% was correlated with a significant depression in their proliferative capacity. these studies clearly suggest that altered calcium signalling is correlated with disturbances in cellular responses in both immune and non-immune cells during sepsis. the altered cellular responses adversely effect the outcome of the septic injury. (supported by nih grant gm 32288). alfred ayala, ping wang and irshad h. chaudry. changes in macrophage capacity to respond to foreign pathogens are thought to be central to the developing immunosuppression associated with traumatic injury. in this respect, the suppression seen in m~ functions following hen (a common component of traumatic injury) may be mediated by the direct or indirect inhibition of their capacity to perceive external stimuli (e.g., opsonized & non-opsonized bacteria, and their cellular components, etc.} due to the breakdown of the receptormediated signal transduction system. results of a number of studies by our laboratory and others indicate that this inability to respond to external stimuli is in part due to the loss of cell surface receptors. decreases have been documented for not only la antigen, but also c3b, fc, and tnf receptors following hem in mice. furthermore, studies which have examined second messenger generation in these cells indicate that m~ derived from the peritoneum and spleen exhibit a decreased capacity to mobilize ca +2 from intracellular stores. this protein kinase dependent process of [ca+2] i mobilization appears to be linked to the inability to synthesize inositol triphosphate. of interest, the depression in ca +2 signal generation appears to be inversely related to presence of elevated levels of camp in m~ from hen mice. we have reported that m~ priming agents, such as ifn-7 (which exhibits salutary effects on m~ function following hem), appear to restore cell signal transductive capacity while reducing the levels of camp. nonetheless, the extent to which depressed receptor signal transduction in hem, is due to receptor loss~dysfunction or elevated antagonistic second messenger levels remains to be determined. conclusions: significant impairment of calcium signaling occurs at all time-points prior to and following pha stimulation in trauma patients. tcell activation failure can, in part, be explained by the inadequacy of this essential intracellular second messenger system. restoration of immunocompetence following trauma will have to address strategies to better assess and restore this vital step in the activation sequence leading to proliferation during the antigen recognition process. patrick a. bseuerle institute 01 biochemistry, albert-ludwigs-university, hermann-herder-str. 7, d-79104 freiburg, germany the active form of the transcriptional activator nf-~b is a heteredimer composed of a 50 and 65 kda polypeptide. in this form, nf-'<b can bind with high affinity to decameric sequence motifs (consensus: 5"-gggpunnf'ypycc-3") found in enhancers and promoters of many genes activated upon a wide variety of pathogenic conditions, including viral and bactedai infections, acute phase response, oxidative stress and uv and gamma irradation. clinically important target genes for nf-k:b encode inflammatory cytokines (il-1b, tnf, [l-6, il-8, gm-csf etc), cell adhesion molecules (icam-1, elam-1, vcam-1), acute phase proteins and immunoregulatory ceil surface receptors. in most cell types, nf-~:b is sequestered in the cytoplasm and, therefore, unable to initiate transcription of these genes. the cytoplasmic form of nf-~:b contains one additionai subunit, referred to as h,;:b. i~b can reversibly suppress dna binding and nuclear uptake of nf-~b by virtue of its association with p65. upon stimulation of cells, ikb is proteolytically destroyed, allowing nf-kb to enter the nucleus and activate genes upon binding to transcriptional enhancers. we have demonstrated that various antioxidative compounds inhibit the activation of nf-~b in response to many inducers. moreover, nf-~:b was shown to be activated upon treatment of cell cultures with p.m-amounts of hydrogen peroxide. these data suggested that nf-~b prinicipaliy is an oxidative stress-responsive transcnption factor. the notion is supported by numerous reports on the induction of oxidative stress by many nf-nb-inducing agents. nf-~b activition can likewise be suppressed by protease inhibitors. these drugs apparently inhibit a yet unidentified protease responsible for the inducible decay of inb. we propose to use nf-nb inhibifors as novel drugs with a very broad application under acute phases el inflammation, injury and infection. northern blot experiments revealed that expression of the recently discovered lipopolysaccharide binding protein (lbp), a 58/60 kd glycoprotein, in the liver rises substantially during the acute phase response. experiments with an in-vivo acute phase model using new-zealand-white rabbits and also in-vitro experiments employing stimulated hep-g2 cells showed that lbp transcripts could be induced 10 to 100 ford within 24 hours after induction with cytokines. by using a newly established nonradioactive nuclear run-on technique we show that induction of lbp during the acute phase is transcriptionally regulated. furthermore we screened a human genomic library and were able to clone the lbp gone, containing the promoter 1.5 kb upstream. several liver-specific and "acute-phase-typical" potential binding sites were found fn the promoter region and reporter gone assays were set up. several caat-boxes, the il-6 responsive elements hstf-hsp 70.5 and h-apf-1 rs as well as the transcription factor hnf-5 and the glucocrticoid-responsive elements oct-2-d and gcre were found, which correlates with the induction pattern of lbp mrna in hep-g2 cells by il-6, il-1 and dexamethasone. pcr-based analysis of the exon-intron pattern of the lbp gene revealed similarities with the genes of two other lps binding proteins, namely bpi and cetp. further analysis of this novel acute phase protein, that also has an important role in endotoxin recognition and immunomodulation will help in understanding the complex acute phase mechanism and potentially lead the way to new therapeutic strategies. lps activation of nucle?/r fa-ctor:~b " (nf:~bj"in cd14.transfected fi'broblasts does not appear to require tyrosine kinase activity d. t. golenbock, r. delude, r. savedra, s. vogel and m. j. fenton lipopolysaccharide (lps) is the major constituent of the outer leaflet of gram-negative bacteria. during the course of serious infection, shock may occur as a result of the interaction of lps with macrophage lps receptors. cd14, a 55 kda glycosyl phosphatidylinositol (gpi)-linked protein, functions as an lps receptor. a critical issue in lps activation concerns the mechanism by which cd14, which has no transmernbrane domain, transduces a sig,nal after lps binding. evidence exists which suggests that cd14 may signal cells after lps binding by interacting with an lps signal transducer with tyrosine kinase (tk) activity. wild-type chinese hamster ovary fibroblasts (cho) normally do not respond to lps, but can be activated following transfection with cd14 (cho/cd14). stimulation of cho/cdi4 with as little as 1 ng of lps/ml resulted in the release of 3h-arachidonic acid (3h-20:4) into the culture supernatant. in addition to 3h-20:4 release, we examined lps-imiuced activation (transl0cation) of the transcription factor nf-~b. similar to lps-induced 3h-20:4 release, these responses were observed only in cho cells lransfected with cd14 closely resembling the same response in the marine macrophage cell line raw 264.7. maximum nf-~cb expression occurred at 30 minutes. in contrast to lps-induced 3h-20:4 release, dexamethasone, cycloheximide, and actinomycin d had no effect on activation of nf-r,.b in cho/cd14, suggesting that nf-r,b activation begins early after lps binding to cd14 and does not require transcription or translation. we then examined cells for lps-induced tk activity by western blotting cellular ly~ates with anti-phcsphot-'rosin:~ anl',bodie~" although tk activity was seen in lps-stimulated raw cells and phorbol ester stimulated cho/cd14, lps-induced tk activity in cho/cd14 was not observed. although the tk inhibitor herbimycin inhibited the release of 3h-20:4 in cho/cd14 and raw cells, neither herbimycin nor genestein effectively blocked nf-r,b activation in either cell type. these results suggest that tk activity is involved in a portion of the signaling pathway that is distal to initial signal transduction. the absence of observable lps-induced phosphotyrosine residues in cho/cd14 cells as well as the failure of tk antagonists to inhibit lps-induced nf-~cb activation suggest that the proposed cd14-related lps signal transducer in cho/cd14 is not a tymsine ldnase. dimished cardiac inotropic function and response to 13-adrenergic receptor (13-ar) stimulation are frequently seen in septic patients, these cardiac defects are also seen in animal models of entoxemia. to determine the characteristics of the decreased transmembrane signal associated with the decreased beta adrenergic response we measured the force and rate of contraction of atria harvested from sprague-dawley rats exposed to endotoxin (0:2 gm/kg). to stimulate various components of the transmembrane signal cascade of 15-ar was isoproterenol (i3-ar), acetylcholine (m2ach receptor), naf(gs and gi proteins), forskolin (adenylate cyclase), and calcium (intracellular contracttile signal) were used. to eliminate the transmembrane control of calcium and test the intrinsic contractile response, hypothermia (22°c) was used the results showed alterations in the inotropic function with no significant difference in chronotropic response. the inotropic reponse for the endotoxin exposed atria and controls is shown below these results show that there is a transmembrane signal defect both for the i~-ar signal and for ca +. these data indicate that the level of the defect appears to be at the g proteins. ischemia-reperfusion of the rat intestine produces an injmry both to the intestine and to distal organs, notably the lungs and liver. prior studies have shown that 1 h of intestinal ischemia leads to a nemtrophil independent reperfusion injury of the gut. thus, rats rendered neutropenic by pl~l antiserum have been shown to express a severe local gut injury when subjected to ischemia and reperfusion. this gut injury is postulated to he mediated at least in part by the complement system. the soluble (s) cri receptor which binds to c3b and cdb was given to rats at a dose of 12 mg/kg by intravenous bolus, 15 min before reperfusion of the ischemic gut. a control group was given pbs buffer. at 4 h of rmperfmsion the animals were sacrificed. pbs treatment led to a significant activation of both classical and alternate complement pathways while scri inhibited both pathways (fall to zero of chbo ). intestinal mucosal injury score, assessed by histological grading was reduced by scri (2.49±0.22 to 1.73_+0.17,p<0.05) . intestinal neutrophil sequestration estimated by assay of myeleperoxidase was also reduced (0.05 ± 0.01 to 0.02 ± 0.01 u/g). c3 was detected in the gut hy i~unohistochemistry. remote organ injury was modified that is lung permeability (ration of concentration of radiolabelled albumin in broncho-alveolar lavage fluid to that in blood) was reduced (11.3±1.4 x i0 3 to 3.9±0.8 x 103, p<o.05). however, neutrophil sequestration by the lungs was unaffected. this remote protection is thougth to be related to prevention of ic3b deposition on imng andotheli~ which activates adherent neutrophils. these findings support the hypothesis of complement mediated local and remote injury. we postulate that complement fragments may be deposited either as a result of the ischemia induced loss of membrane bound complement regulatory proteins, such as decay accelerating factor or membrane cofactor protein, which allow complement fragments to accumulate on the endothelial cell surface. alternatively, increased expression of p-selectin on the endothelial cell surface may, by means of its complement binding domains, act as a lattice for complement deposition. indeed, p-selectin has been detected on the surface of endothelial cells ans platelet-neutrophil aggregates recovered from the intestine of these animals. ischemia (4hr) followed by reperfusion (dhr) of rat hind limbs resuits in local (muscle) injury as well as in remote (lung) injury, which is characterized by increased vascular permeability (leakage of j2sl labeled albumin) and neutrophil accumulation (tissue content of myeloperoxidase, mpo). within 60 minutes of reperfusion following ischemia, tumor necrosis factor-o~ (tnfc 0, interleukin-i (1l-i) and il-6 plasma levels increased significantly, reaching maximum levels after 2 hours ofreperfusion. polyclonal antibodies to tnfet and 1l-i provided significant protection against muscle and lung injury. muscle and lung vascular permeability decreased in anti-tnfct treated rats by 41% and 74% respectively and mpo was reduced by 72% and 83%. antml-1 yielded a protection in muscle and lung vascular permeability of 24.7°,4 and 52.4% respectively whereas muscle mpo was reduced by 46.4% and lung mpo by 25.9°,4. these results were confirmed by the use of soluble tnf~ receptor and il-i receptor antagonist (il-ira). when icam-i expression in vivo was examined using 125mabeled antmcam-i, constititive expression of icam-1 was evident only in lung but not in muscle. during reperfusion icam-1 expression increased by 98.2%. treatment with anti-tnfcx or il-ira reduced icam-i upregulation by 73.9°,4 and 74.8% respectively. frozen sections of normal rat lung revealed no evidence of e-selectin expression, whereas lung sections obtained after ischemia and reperfusion immunhistochemically demonstrated expression of e-sdectin. lungs from rats pretreated with anti-tnfct were negative for e-selectin staining. our data indicate a role for cytokines in the upregulation of adhesion molecules in ischemia/reperfusion injury. adherence of neutrophils to the coronary endothelium is associated with significant myocardial injury following 90 min of ischemia (mi) and 4.5 h of reperfusion (r). p-selectin is expressed by activated endothelial ceils and platelets within 10 min and tethers polymorphonuclear leukocytes (pmns) to the endothelium. we examined the cardioprotective effects of a monoclonal antibody (mab) designated pb1.3 to p-selectin in a feline model of mi+r. pb 1.3 (1 mg/kg) administered 80 rain post-occlusion (10 min prior to reperfusion) significantly attenuated myocardial necrosis compared to a non-blocking mab (nbp1.6) for p-selectin (15 + 3 vs 33 + 5% of area-at-risk, p<0.01). endothelial dependent relaxation to acetylcholine was also significantly preserved in ischemic-reperfused coronary arteries isolated from cats treated with pb 1.3 compared to nbp1.6 (67 + 6 vs 11 + 3, p<0.01). furthermore, pmn adherence to ischemic-reperfused coronary endothelium was significantly attenuated in pb1.3 treated cats (64 + 7 vs 136 + 12 pmns/mm z, p<0.01). also, normal coronary endothelium activated with thrombin (2 u/mt) had increased pmn adherence under conditions of shear stress, an effect which was significantly (p<0.01) blocked by pb1.3, but not by nbp1.6. furthermore, p-selectin is markedly upregulated 10-20 min post-reperfusion in coronary arterial and venous endothelinm. similar results were obtained in a rat model of mesenteric ischemia/reperfusion, including upregulation of p-selectin on mesenteric vascular endothelium. these results demonstrate that tethering of neutrophils to endethelium by p-selectin is an important early consequence of reperfusien injury, and a specific monoclonal antibody to p-selectin exerts significant endothelial preservation and cardioprotection in ischemia and reperfusion states. as recent interest has increased in the role of extracellular matrix proteins in inflammation, we examined the role and relationship of laminin and fibronectin to the processes of cell infiltration in a rat model of heart isografts. changes occurring in this system are on a nonimmunological basis and may be an effect of the initial surgical manipulation and the warm and cold ischemic times of transplantation. lewis (lew) donor hearts were transplanted into lew recipients, naive lew acted as naive controls (nc). grafts were harvested 2, 4, 8, 16, 24, 48, 72, 120 and 168h after transplantation (n=6/time point) . snap frozen sections of organs were stained for cd-4 (w3/25), cd-8 (ox8), t-lymphecytes (tl) (ox-19), macrophages (ed1, ed2), neutrophils (pmn) (mom), icam-i (iap29), laminin and fibrinogen. results were evaluated visually by counting cells/field of view (cf) for w3/25, ox8, ox19, ed1, ed2 and mom, staining for icam1, lain and fib was assessed visually using a scale (ve=0-10). as early as 2h after transplantation, fibrinogen and laminin expression increased in i, peaking 8h after transplantation (ve= 4.5; nc: ve=i.5) . at 16 and 24h in i, on both vessels and interstitial cells laminin and fibrinogen expression had declined to baseline (ve=i.5) but rose again at 48h, peaking at 72h (ve=4) and remaining elevated thereafter (ve=3). the high expression at 8h correlated well with the maximal pmn infiltration at that time (cf=6) in i returning to baseline at 24h (cf=0.5) and thereafter. the second peak at 72h correlated with the onset of macrophage and t-lymphocyte infiltration. thus, laminin and fibrinogen seem to guide leucocyte infiltration following graft ischemia during the time of reperfusion. background. reperfusion of ischemic kidneys with xenogeneic blood leads to activation of endothelial cells and circulating leucocytes with the final consequence of microvascular thrombosis. in concordant systems, the mechanisms of rejection can be studied due to cross-reactivity of mediators with anti-human monoclona~ antibodies. aim of the study. to obtain information about the kinetics of proinflammatory cytokines and production of soluble adhesion molecules in the acute phase of reperfusion, eight kidneys from rhesus monkeys were perfused ex-vivo with human blood (group b/o) for one hour in a closed system. blood levels of il-lb, il-6, tnf-a, soluble icam and e-selectin were measured in elisa-technique under steady-state conditions. results. cytokine levels rose significantly within the 60minute interval (il-lb: 6,1__+2,6 to 161,1__+98,5 pg/ml; il-6:30.24-7,7 to 274,2__+75,8 pg/ml; tnf-a: 544,2__+363,6 to 1651,0+25,7 pg/ml; p<0.05). immediately after the beginning of reperfusion, soluble icam-1 and selectin levels were abnormally high and constantly rising throughout the observation period, reaching significance at 60 minutes. discussion. high levels of proinflammatory cytokines may lead to an induction of adhesion molecules, thus upregulating the leukocyte-endothelial interaction in an complement-independent mechanism. specific pretreatment with monoclonal antibodies against icam-1, lfa-1 or other soluble mediators may be useful in downregulating hyperacute rejection in transspecies transplantation. prolonged ischemia has been associated with later dysfunction of solid organs as it may cause upregulation of adhesion molecules, production of cytokines on vascular endothelium and migration of host cells into the graft. these early events may lead to irreversible organ injury. this study evaluates the effects of global ischemia on early immunohistological changes in cardiac grafts transplanted in rats. isografted hearts (lewis->lewis) were were divided into ischemic and non-ischemic groups (n=30/group). all donor hearts were flushed immediately with cold saline. non-ischemic hearts were then transplanted within 30 rain, ischemic hearts were stored in cold ringer's solution for 3 hours before revascularization. representative grafts were removed after 12. 24, 72hrs, 10 and 100 days, and evaluated immunohistologically (cells/field of view=c/f). restitution of ventricular activity was significantly delayed in ischemic grafts (5 vs 1 rain). after 12 hrs, all ischemic grafts exhibited an extensive interstitial edema, declining slowly thereafter. at the same time, numbers of pmn peaked (3 vs 1 c/f in non-ischemic grafts), whereas edl+macrophages (9 vs 2 c/f) and tnfe expression peaked by 24hrs. by 72 hrs t-lymphocytes began to enter ischemic myocardium and icam-1 was moderately increased. after 10 days cellular infiltration had returned to baseline, and no differences were seen among both groups after 100 days. global myocardial ischemia inhibits initial graft function, and engenders a brisk inflammatory reponse, primarily pmn and macrophages, with increased mhc class ii and cytokine expression. leukocyte -endothelial interactions are the result of endothelial activation, leukocyte activation or combination of both, which are accompanied by nee-expression, upregulation or shedding of adhesion molecules (selectins, inlegrins). such interactions differ with regard to the stimulus (e.g. thrombin or histamine for p-selectin, endotoxin or tnf/il-1 for e-selectin), the time course of response (minutes versus hours) and the localisation in different organs. recently assays are available for circulating soluble fragments of the cell bound adhesion molecules e.g. se-seleetin was found to be increased in plasma concurrent with high circulating endoloxin and cytokine levels. the importance of adhesion molecules for the sepsis event is evident, while effectiveness of anti-adhesion inolecu]e therapy is controversial e.g. beneficial anti-e-selectin therapy in baboon bacleremia but deleterious effects of amti-cd18 treatment in the same model. in other species similar controversial results with anti-cd18 therapy in sepsis were reported. steven l. kunkel,theodore standiford* and robert m. stricter. the migration of leukocytes to the lung during endotoxemia is dependent upon the coordinated expression of lung vascular adhesion molecules and the subsequent production of appropriate leukocyte chemotactic proteins. in experimental animals, neutrophils accumulate within the lung soon after the administration of endotoxin, while mononuclear cell infiltration occurs in a more distal manner. a kinetic analysis of lung leukocyte levels revealed a 3-fold increase in neutrophil numbers associated with dispersed lullg tissues 2 hours after lps treatment, while macrophage levels increased by 4-fold at the 24 hour time point. thus, the recruitment of different leukocyte populations to the lungs during endotoxemia is likely directed by different mechanisms. recent studies have identified a supergene family of small inducible chemotactic cytokines (chemukines) which possesses chemotactic and activating properties for neutrophils. the prototype of this family is interleukin-8 (il-8). interestingly, a related supergene family has been identified which possesses activity for recruiting mononuclear cells. examples of this group of inflammatory chemukines are monocyte chemotactic protein-i (mcp-i) and macrophage inflammatory protein-i alpha (mip-i). in initial in viva studies we examined whether mip-i was expressed systemically or in a compartmentalized fashion post lps challenge. assessment of plasma cytokine levels revealed maximal tnf levels occurred i hour post lps administration, returning to baseline by 4 hours, while mip-i levels were maximal at 2 hours (2,5 ng/ml), with a second peak at 24 hours after lps challenge. interestingly, aqueous extracts of liver homogenates from lps treated animals demonstrated no mip-i levels, while aqueous extracts of lung revealed a 4-fold increase in mip-i levels over control lungs. immunohistochemical analysis of the lungs from 24 hour lps treated animals demonstrated the alveolar macrophage was a rich source of mip-i protein. cell-associated mip-i was also expressed by blood monocytes adherent to the pulmonary vascular endotheliun, however the expression of monocyte-mip-i was observed by 2 hours post lps administration. immunohistochemical analysis also demonstrated that mip-i antigen is associated with the extracellular matrix on the interstitial side of the endothelium. this suggests that the extracellular matrix, which is produced during inflammation, can bind mip-i and this may serve as a depot for the prolonged presence of nip-1. in additional studies we have demonstrated that the intratracheal instillation of rmui [ip-l(loong) activation of polymorphonuclear leukocytes by inflammatory stimuli may contribute to the development of multiple organ failure in septic patients. thereby pmnl are proposed to avidly adhere to vascular endothelium causing damage by the subsequent release of toxic agents. as cellular adhesion is primarily mediated by 62-integrins and lselectins, the present study compares the expression of these adhesionmolecules on pmnl in septic patients and healthy volunteers. methods: expression of 62-integrins and l-selectins on pmnl was measured in whole blood by flow cytometry using the monoclonal antibodies ib4 and dreg 200, baseline values were determined immediatley after drawing blood. in addition cells were incubated 45 min at 37°c to allow for spontaneous regulation of adhesion molecules. 55 blood specimens from 8 septic patients were obtained during the course of their illness. control values were determined in 12 healthy volunteers. results: baseline expression of 62-integrins and l-selectins was not signifcantly different in septic and in healthy subjects. in contrast, there was a significant upregulation of g2-integrins and shedding of l-selectins of pmnl in septic patients (sp) compared to healthy volunteers (hv). the local or systemic production of inflammatory cytokines, such as tumor necrosis factor alpha (tnfc~), can serve to modulate multiple aspects of neutrophil function. the ability of neutrophils to leave the circulation and migrate to areas of infection is one essential component of host defense. l-selectin, a leucocyte-associated adhesion molecule, is responsible for the initial reversible contact between neutrophils and endothelium and the subsequent roiling action of neutrophils along the vessel wall. in contrast to other adhesion molecules, l-selectin expression is rapidly down-regulated after neutrophil activation. the loss of l-seleclin may thus be a critical determinant of how neutrophils become unbound from their endothelial attachments and enabled to proceed towards an underlying extravascular area of infection. we hypothesize that the shedding of l-selectin is a strictly controlled process, occurring primarily at localized sites of inflammation, which may be modulated by tnf~, a flow cytometric method of staining neutrophhs by monoclonal antibodies in whole blood is described whereby the kinetics of l-selectin shedding may be followed in real time. the dose response and time course of in-vitro l-selectin shedding by neutrophils from normal human subjects was assayed after exposure to n-formyl-methionylleucyl-phenylalanine (fmlp) and tnfc~. either singly or in combination, our results show that l-selectin shedding can be reliably followed over time. a significant percentage of cells shed l-selectin after exposure to 2000 pg/ml tnfc~ or 1000nm fmlp (but not at 400pg/ml tnfc~ or 200nm fmlp). greater numbers of cells were able to shed their l-selectin when fmlp and tnf~x were presented in combination rather than alone. high levels of tnfc~ did not appear to alter the threshold concentration of fmlp required to induce shedding, we conclude that the extent and rapidity of l-selectin shedding may be modified by different combinations of ligands and that shedding, by vidue of the high concentrations of cytokines or chemotactic factors required, is a process localized to sites of infection or inflammation. we prospectively studied 23 patients with severe sepsis syndrome; group a : septic shock with or without adult respiratory distress syndrome lards) (n =7, bacteremia = 6); group b : sepsis syndrome without septic shock (n = 16, bacteremia = 9). serial plasma samples obtained on day 0, 1, 3, 6, 10 and 14, were assayed using elisas method (british biotechnology), normal control levels of soluble icam-1 and e-selectin, obtained from 11 healthy volunteers, were respectively 205 ± 19.4 ng/ml and 45 ± 5.7 ng/ml (mean _+ se), acute lung injury was quantified dally on a tour-point score system (murray, am rev respir dis,1989) . compared to control mean values, initial levels of groups a and b were significantly higher for icam-1 (p < 10 -4) and e-selectin (p < 10-3). comparisons of group a and [] (* = p<0.05; ** = p<0.00t) soluble icam-1 levels of group a enhanced significantly (p<0.05) during the first 24 hours, and a sustained high levels was of bad prognosis (25% of survivors at day 6). the evolution of soluble icam-1 and e-selectin levels were significantly correlated with murray's score (spearman test : p <0.001). conclusion: these results suggest that endothelial adhesion molecules are released into the plasma of patients with severe sepsis syndrome. soluble icam-1 and e-selectin are correlated with endothelial lung damage, and loam-1 seems to be a better indicator of the severity of endothelial injury. introductory remarks to anti-adhesion molecule strategies as a therapeutic modality ch wortel, repligen corporation, one kendall square, building 700, cambridge, ma 02139, usa. the development of antimicrobial therapy represented a major breakthrough in the struggle against disease. it strengthened the notion that disease could be overcome by eliminating foreign invaders threatening the host. this paradigm has proven to be very successful, the threat of many infectious diseases has significantly changed, some have even been eradicated. nevertheless, sepsis has remained a severe condition, increasing in incidence while mortality remained very high. more recently, it has become increasingly clear that besides the nature and treatment of an exogenous agent, the reaction of the host defense itself plays a pivotal role in the outcome of the event. endogenous mediators, such as tnf, il-i, il-6 and il-8, govem many of the actions of the host defense system. while the expression of these cytokines more often than not benefit the host, (over)-expression can cause severe damage. based on this hypothesis,anticytokine strategies, such as those targeted against tnf or il-1, have been evaluated for the treatment of sepsis. results of these early studies have not yet indicated success in improving the outcome of the disease. it has been difficult to define a patient population where a benefit could be reproducibly shown. furthermore, it has been documented that synergy between cytokines occurs, but detailed knowledge of the cytokine network is not yet available. it is conceivable, that neutralization of one cytokine prompts the induction of another which will evoke the intended response in the host. recent data obtained in human endotoxemic volunteer models seem to confirm this. if this turns out to be the case, neutralizing a single cytokine may not be a successful approach. cytokines in tum, induce various adhesion molecules, such as icam-1. such molecules regulate for instance the neutrophil-endothelial cell interactions, which are thought to play an important role in the pathogenesis of systemic organ injury. the potential for monoclonal antibodies to adhesion proteins to reduce vascular and tissue damage has been studied in a large number of experimental models. protective effects have been observed in a wide variety of inflammatory, immune, and ischemia-reperfusion injuries. thus, altering the host response by modulating the function of adhesion molecules may attenuate the inadvertent injury caused by inappropriate behavior of host defense cells. targeting cellular surface interactions has been added to the efforts to change the outcome of disease. modulation oftheseprocesses seems very promising, but may temporarily leave the host without effective defense mechanism. great care therefore, must be exerted when studying this powerful two-edged sword in a clinical setting. our knowledge of the role of adhesion molecules in the intlammatory response has increased rapidly due to the availability of new reagents and mice geneticly deficient in adhesion molecules. these molecules are important in interactions of leukocytes with endothelial cells, other leukocytes, platelets, and epithelial cells. when these molecules are engaged, they can also play a role in activating leukocytes and their effector functions. in the venules of the systemic circulation, adhesion often occurs through a series of sequential interactions. initial interactions are mediated by members of the selectin family to loosely associate the leukocytes with the endothelium and are followed by firm adhesion requiring members of the integrin and immunoglobulin family. later interactions with endothelium may require pecam. adhesion molecules are usually required for leukocyte emigration in response to extravascular stimuli and for neutrophil-mediated endothelial cell injury. they are critical for host response in many diseases including infections. however, when the inflammatory response results in damage to host tissues, patients may benefit from blocking the leukocyte response. anti-adhesion molecule agents are an important potential antiinflammatory therapy. the focus of anti-adhesion therapy may be at any step of the sequence. diseases where anti-adhesion molecule therapy may benefit patients include ischemia/reperfusion injury in many organs, ards and mof, and transplantation, both to protect the donor organ from ischemia/reperfusion injury and to inhibit graft vs host disease. many strategies have been considered and include: 1) blocking the ability of adhesion molecules to recognize their ligand using antibodies that have been humanized or soluble receptors linked to igg to prolong their circulating halflife, 2) blocking the ligands for adhesion molecules using soluble adhesion molecules, peptide analogues, or oligosaccharides, and 3) blocking the production of the adhesion molecule using anti-sense oligonucleotides. because the synthesis of adhesion molecules is usually regulated by cytokines, inhibiting the action of cytokines is another potential site for interrupting the adhesion process. although important issues of safety must be evaluated, the potential for modulating the inflammatory response make this an exciting area of improvement in health care delivery. claire m. doerschuk, m.d.; riley hospital for children, room 2600; 702 barnhill drive; indianapolis, in 46202 usa. modulation of neutrophil-endothelial cell adhesion with anti-cdl i/cd18 monoclonal antibodies as a therapeutic modality. ch wortel, repligen corporation, one kendall square, building 700, cambridge, ma 02139, usa. the central role of inflammatory cells in the pathogenesis of lung and systemic organ injury is well recognized. binding of neutrophils to endothelial cells and migration into the parenchyma are largely regulated by complementary adhesion molecules. the leukocyte integrins are glycoproteins expressed on the neutrophil surface and in the cytoplasmic granules. integrins consist of a common beta2 or cluster differentiation (cd)18 chain covalently linked to one of three different alpha chains (cdlla, cdllb, cdilc) and exist on the cell surface as three distinct heterodimers. cdlla/cd18 is expressed on all leukocytes, whereas cd 11 b/cd 18 and cd 11 c/cd 1.8 are restricted to cells of myeloid origin. cd i 1/ cd 18 interacts with intracellular adhesion molecule-1 (icam-i), its ligand on endothelial cells. the potential for monoclonal antibodies to adhesion proteins to reduce vascular and tissue damage has been studied in a large number of experimental models. protective effects with anti-cd18 antibodies have been observed in a wide variety of inflammatory, immune, and isehemia-reperfusion injuries, such as arthritis, burns, endotoxic shock, bacterial meningitis, autoimmune diabetes, nerve degenemrion, allograft rejection, allergic asthma, acute lung inflammation, skin lesions, and ischemia-reperfusion models of the intestine, myocardium, lung, skeletal muscle, and central nervous system. protective effects have also been observed in animals resuscitated following hemorrhagic shock. blockage of cd18, however, would affect all leukocytes, as would antibodies to cdlla/cdi8. targeting cdllb/cd18 would affect cells of the myeloid lineage only, which could prove to be beneficial. cd11b/cd18 is not only involved in transendothelial migration, but is also implicated in adherencedependent formation of reactive oxygen species. blocking cd1 lb/cd18 may therefore not only reduce the numbe r of leukocytes accumulating in the tissue, but also attenuate the oxidant stress of infiltrated neutrophils. anti-cd 11 b treatment has been used effectively to reduce tissue injury initiated by ischemia-reperfusion, complement activation and endotoxemia. altering the host response by modulating the function of adhesion molecules may attenuate the inadvertent injury caused by leukocytes, but may also temporarily leave the host without effective defense machinery. overall, animal studies suggest that it may be safe to inhibit neutrophil adhesion for a limited period of rime. these observations will have to be confirmed in carefully designed clinical trials. c, arbobydrams are ubiquizom constir~uts of cell sv.rfaees, and possess many c~xssfies ttm~ m~,e ~em ide~. canaidates for r~ognifioa mole~ule& in m~y systems whe,~ cer udhesioa ~lays a critical ro~ car~hydram l:~dtag ~otegas have been shown to b~ad tocell surfa~ earbohydzaxes ~nd pzrl~pate in cell-ceil lumtaefion& such sys,.ems include ~rti~za~io=, deveaopmeat, l~thoge~-hcet reeog--ition ~d i~zmmadon_ in particular, tb.z recent di%~ve~ of lhe selec~ and th~ impo.~a~c~ in teukccy~udo~lelium adh~ion has -~f~m av.c~on ok l~in m~ted cell adhe~on. s~vere/poten~s/cs.rbohydr~ l~ga~s hrve ~e~l ~u~ilied for ~he s~lcc~ins. the,~ c~u be broadly di,,sded la~o ~wo m'oups -sibyl l~wis x m~ mh~.~l oligo~chadd~s, ~d sf/~ ca~ohydmma, all ~:~ ~l~dns bind m siflyl l~wis x (sie$ o!igos~ccb.e.rkms, zlthou~ w~ differing avi~re~. 'we have i~¢n~ed the functional g~oups 0a s~ex ~n~ med/a~ ~he b~u ~di~g of ~h~ c~b0hydmm = e-se/sedm we have used ~hat iv.formation to sya~esize sle ~ '~mt0gs r.he, t focus on replacing slslic ~sd ~nd fuc0s¢ wi~ simpler, more stable strunt~es. a[~ou~a ~ proeer~ is ongoing, we hve been ~ucee,.~ful a~ rep~aein8 t.ke si~ic a~id. residue wi~ std.fzte. ~ce~ or la~c amd groupa we t'we ex2aninad &e ten, bunion of ezed~ hydroxyl group of the fizeose residue ~ billding of e-, l-~nd p-selees..u. we have also found m~fi~fio~ of the reducing end ~¢.cha'i~ ~z increase mtagovsst activity. the, m¢ond. group of figs,rids a.r eontzin su~a~ 0u a ea.rbohydr~t¢ support,, und seem to bi~.d to t~e sele~ti~s wi~ dlf:ferem characteristics c0.an does sle:, s=h compounds are m2ogniz~d by l-selects. md p-selectia, bur., in genera/, not e, selecti~ these dam may mdicam r.hat l-and p-s~ ¢at~ h~d via o, second ~te thaz operates lu~.ead of, or in conjunction with ~tc sle" b~ding ~iite. dam rela~&~g to ±e, se two types of ,ml~ liga~ds have beam t~ed to desig~ potential the2~peutics for i~fi~anmat0ry disease. lr:rng maimai models of acute lung lu3ury we can demo~trate that eompmmds that inhibit seleetiu birding ~ ~i~o hzve ber~ficial effects when uc~d in rive. progressive microvascular damage in the tissue adjacent to a cutaneous burn injury results in extension of burn size. the role of leukocytes in the pathogenesis of microvascular injury was investigated by inhibition of their adherence to the microvascular endothelium using monoclonal antibodies directed to leukocyte cdi 8 or its endothelial ligaud, intercellular adhesion molecule-1 (icam-1, cd54). a model of thermal injury was developed using new zealand white rabbits. two sets of three full-thickness burns separated by two 5 x 30-mm zones were produced by applying brass probes heated to 100°c to the animals' backs for 30 sec. cutaneous blood flow determinations carried out with a laser doppler blood flowmeter were obtained for 72 hours. there were five experimental groups: controls given saline alone; animals given monoelonal antibody to the cd 18 r 15.7 prior to burn injury (pre-r 15.7); animals given r 15.7 30 min after burn injury (post-r 15.7); animals given a monoclonal antibody to icam-i, r 6.5 prior to burn (pre-r 6.5); and animals given the r 6.5 30 min postburn injury (post-r 6.5). blood flow in the marginal "zone of stasis" between burn contact sites was significantly higher in the antibody-treated animals. administration of the antibodies 30 min after injury was as effective as preburn administration in preserving blood flow. at 72 hr post-burn all antibody -treated animals had blood flow in the areas at risk for progression (i.e., the zone of stasis) at or above baseline levels while the control animals had levels equal to 34.7 _+ 12% of baseline (p < 0.05 by analysis of variance and mann-whitney u test). these results indicate that leukocytes play an important role in the pathogenesis of burn wound progression, and that this progression can be attenuated by moduiating adherence to endothelial cells. a wealth of information now supports the hypothesis that inhibition of cell adhesive mechanisms will nter the course of immunologicand inflammatory processes. what remains unclear is whether inhibition of specific mechanisms wfl[ be of therapeutic benefit in any specific human disease. current data derived from animal models are not inconsistent with the hope of therapeutic benefit, but techniques for inhibition (e.g., antibodies, antisense oligonucleotides, inhibitory peptides, inhibitory carbohydrates, smaii synthetic inhibitors, etc), tissue and species differences in the relative contributions of adhesion molecules to the inflammatory process, and the cascade model of adhesive interactions are all confounding issues, making predictions of therapeutic benefit in any specific human disease process very difficult. additional concerns involve the potential roles of adhesive mechanisms in host resistance to infection. as human therapeutictdals are initiated, more exact information on the roles qf specific adhesion molecules in human disease should emerge. inhibition of leukocyte adherence to endothelial cells can represent a novel therapeutic approach to septic shock. we performed a pilot study to evaluate the safety and tolerability to cy-1787, a monoclonal antibody against human e-selectin, in patients with septic shock. septic shock was defined by clinical signs of sepsis, a documented source of infection, and fluid-resistant hypotension requiring the use of vasopressors. eleven patients entered the study, but 2 patients who died during the first 24 hours were excluded, as this was part of the protocol. cy-1787 was administered as a single intravenous bolus of 0.1 mg/kg (n=3), 0.33 mg/kg (n=3) or i mg/kg (n=3) mg/kg. the antibody was well tolerated. none of the 9 patients died during the 28 day follow-up period. organ failure was assessed for 5 organs (cns, lungs, liver, kidneys and coagulation). the mean number of organs failing, which was initially 2.7 ± 1.2, decreased to 0.2 ± 0.4 at the end of the study (p<o.o01). these results are therefore encouraging. administration of an antibody to e-selectin in patients with septic shock requires further study. the importance of cytotoxic t lymphocytes (ctl) in the host defense mechanism against viral as well as parasitic and bacterial diseases is becoming increasingly appreciated. the design of vaccines to generate cytotoxic t ceils from nonviable or subunit constructs, however, poses challenges due to the unique characteristics of antigen processing and presentation by class i major histocompatibility complex (mhc) molecules. since the final antigen recognized by ctl are short peptides capable of high affinity binding to class i mhc molecules, we have designed strategies: a) to define the structural characteristics of peptides that are required for their high affinity binding to mhc; and b) to formulate these peptides into immunogenic vaccines that are capable of generating cytotoxic t lymphocytes. the strategy and results to accomplish these two goals will be discussed. amnon altman and erich gulbins ras proteins represent essential intermediates in receptor-mediated growth and differentiation pathways. they couple signals initiated by receptor or nonreceptor protein tyrosine kinases (ptks) at the cell surface to cytoplasmic targets which coordinate signal transduciion to the nucleus. ras also participates in t lymphocyte activation initiated by the antigen-specific t cell receptor (tcr)/cd3 complex, which leads to lymphokine production and proliferation. receptor ligation causes activation of associated ptks of the src and syk families as the earliest identifiable event in this pathway. the importance of ras in t cell activation is indicated by the findings that an oncogenic ras protein activates the interleukin-2 (il-2) gene promoter; conversely, a transdominant inhibitory ras mutant inhibits this event and the activation of map kinases. the rate-limiting step in ras activation is the exchange of bound gdp for gtp, which is catalysed by guanine nucleotide exchange factors (gefs). we identified the sh2/sh3 domain-containing vav protein, which is selectively expressed in hematopoietic cells, as a ras-specific gef coupled to several hematopoietic receptors. vav can be activated by two pathways, i.e., by ptk-mediated phosphorylation or by binding of diglyceride second messengers to a cysteine-rich, protein kinase c (pkc)homologous vav domain. these pathways are independent as demonstrated by structure/function analysis and the use of pharmacological inhibitors. the two activation pathways may enable vav to integrate signals from distinct hematopoietic cell receptors, and couple them to ras. t cells express another, ubiquitous ras gef, i.e., sos, which is known to be coupled to activated tpk receptors. t cell activation leads to membrane association of a signaling complex consisting of sos and two other signaling proteins, grb-2 and shc, via direct binding of the shc sh2 domain to the tyrosine-phosphorylated chain of the tcr/cd3 complex. thus, multiple receptors, ptks and ras activators participate in signaling pathways that lead to hematopoieitc cell growth and differentiation. the interactions and function of these various signal transducers will be reviewed. phagocytes. immunity is mediated by specific t lymphocytes, cytokines produced by these specific t cells activate antimicrobial capacities in mononuclear phagocytes, thus contributing to protection. other immune mechanisms also participate in the acquisition of resistance. on the other hand, t cells are also crucial for many pathologic sequelae of intracellular bacterial infections. although ifn-y is central to macrophage activation, synergistic and antagonistic effects with other cytokines occur. the cd/3 t cells, representing the major t cell population in experimental mice, are the main mediators of antibacterial immunity; an auxiliary role for y/~ t cells appears likely. the c~//3 t cell population further segregates into cd4 t cells which recognize microbial peptides in the context of gene products of the major histocompatibility complex (mhc) and cd8 t cells which see their peptide in the context of mhc class i. the -y/~ t cells are generally double-negative. knock-out mice in which the genes for various t cell receptor chains, for mhc class i or mhc class ii molecules, for cytokines or cytokine receptors had been deleted, have provided extremely helpful tools for analyzing the role of t cell subsets and cytokines in antimicrobial immunity. using these mutant mice, the role of distinct t cell subsets and cytokines in bacterial elimination and granuloma formation was analyzed in detail. these studies underline the central role of a//3 t cells and ifn-y in antibacterial immunity and, furthermore, show a distinct function of 7/$ t cells. moreover, these studies show that the relative contribution of cd8 or cd4 t cells to antimicrobial immunity varies in response to different pathogens. apoptosis is an active form of cell death in which the cell participates in its own demise, providing the biochemical pathways that trigger and mediate the complex events of the process. although this phenomenon has been studied for many years, it is only recently that significant progress has been made towards the elucidation of the molecular mechanisms that control apoptosis. once such mechanism came as something of a surprise: in a number of eases, apoptosis can be promoted by the action of the c-myc protein, a protooncogene product that had previously been associated only with cell proliferation (and presumably, survival) . expression of c.myc in cells can cause them to enter an apoptotic pathway or proliferate, mad this "decision" depends upon additional signals, such as growth factors that inhibit apoptosis and allow the ceils to proliferate. thus, c-myc directs cells out of rest and the choice of proliferation or death depends upon the presence or absence of survival signals. other survival signals are generated by oncogene products that can cooperate with my¢, including bcl-2 and abl there are implications of this interplay between apoptotie and anti-apoptorlc signals in cell iransformation and the resistance of some tumor ceils to therapeutic agents capable of inducing apoptosis. oncogene interactions therefore control the life or death of transformed ceils but also have important roles in normal, development processes. in the immune system, developing t ceils are "screened" for potential self-reactivity by a process of negative selection, in which activation via the t cell receptor (tcr) induces apoptosis. this process can be mimicked in t cell hybridomas, and appears to depend upon the expression of the c-mye protein. evidence that this represents a requirement for the myc/max heterodimer will be presented. as above, these cells are presented with the "choice" to proliferate or die and this depends not only upon myc but also other signals. thus, expression of functional v-an kinase in these ceils can render them resistant to the activation of apoptosis via tcr ligation, surprisingly, bcl-2 does not appear to be capable of blocking this form of apoptosis, and the reasons for this will provide new insights into the development of the immune system and the process of apoptosis. although the oncogene products discussed here probably do not directly participate in the biochemical process of apoptosis, they represent molecular controls on this complex mechanism. as such, they gave us new ways to look at the life and death of a cell recent attention has focused on macrophage release of cytokines, such as il-1, il-6 and tnf as important mediators of septic shock. however, serum cytokine levels have correlated poorly with outcome of septic shock. the purpose of this study was to compare the functional status of macrophages to serum cytokine levels in early sepsis by an analysis of splenic macrophage protein synthesis during abdominal sepsis. macrophage total protein distribution and biosynthetic activity was assessed by large format 2-d page, autoradiography of 35s-labeled proteins and computer assisted densitometric analysis. sepsis was induced in sprague dawley mts by cecal ligation and puncture (clp). untreated and sham operated rats served as controls. splenic macrophages were harvested at 3 and 24 hrs. following clp. serum il-6 levels were determined at 3,12, and 24 hours by the 7 tdi bioassay. by 12 hrs. 100% of the clp mls exhibited multi-microbiaj bacteremia. control or sham operated rats did not show bacteremia. clp resulted in a significant 286% elevation (p < 0.05) in serum il-6 levels at 12 hrs. there was no difference in il-6 at 3 and 24 hrs. when compared to the control groups. 2d page studies examined splenic macrophage total protein distribution and biosynthetic activity at 3 and 24 hrs. post-clp. at 3 hrs. macrophages from clp rats showed a substantial decrease in total protein pattern (353 protein spots vs 503 protein spots) when compared to non-septic control macrophages. a decrease in the number of lower molecular weight proteins (< 40 kd) was observed. at 24 hrs. post-clp, splenic macrophages from clp rats showed a substantial increase in the synthesis of low molecular weight (< 40 kd) proteins, when compared to non-septic controls. we conclude that: l) fulminate abdominal sepsis induces an early (3 hrs.) staining with cd14 and cd16 mabs will identify two monocyte subpopulations in human blood: a major population of regular monocytes, which strongly expresses the cd14 antigen (cd14++) and a minor population with weak expression of cd14 and expression of the cd16 antigen lcd14+/ cd16+ cells). in healthy control donors(n=35) the latter cells account for 45+22 cells/mm 3 and 9%+5% of the monocytes. in sepsis patients, the cd]4+/cd16+ cells can become a major population with more than 50% of all monocytes in 3 of 18 patients and with more than 500 cells/mm 3 in 4 of 18 cases. there was no correlation of cd14+/ cd16+ cells to any clinical parameter except for cd14+/cd16+ percentage and body temperature (p= 0.013). the cd14++ monocytes showed a substanttial decrease in cd14 antigen density in 9 of 11 patients. three-color-if shows that the cd14+/ cd16+ cells in sepsis patients when compared with the cd14++ monocytes exhibit a higher level of class ii antigen and a lower level of cdiib and cd33 antigens, consistent with a more mature nature of the cd14+/cd16+ cells. levels of il-6 were increased in sepsis patients: 3 of 5 patients with high numbers of cdi4+/cd16+ cells ( 200/mm 3) had high levels of il-6 (250u/ml). these data suggest that septicemia may lead to substantial changes in blood monocyte composition and this may be related to elevated levels of cytokines such as il-6. human peritoneal macrophages were exposed to increasing doses of lipopolysaccharide (lps) or a synthetic lipid a analogue (mrl 953) and production of the cytokines il-lb, il-6, tnf-a and g-csf was assessed at the protein-and mrnalevel. cells were also stimulated with low doses of lps and mrl 953 to study their "adaptation" to a secondary challenge with high doses of lps. tnf-a production after stimulation with lps could be almost completely relieved by preincubation of cells with low doses of lps and similarly, il-6 production was suppressed. surprisingly, however, "adapted" cells were able to synthesize even larger quantities of of g-csf and il-lb when exposed to a secondary lps challenge. the changes in tnf-a, il-6 and g-csf protein synthesis could a/so be detected on the mrna level, whereas transcript levels for il-lb remained unaltered as assessed by northern blot analysis. high doses of the synthetic lipid a analogue mrl 953 were also able to "adapt" macrophages for a secondary lps challenge by downregulating tnfa-and il-6-production, while priming secretion of g-csfand il-lb as well. taken together, here we describe for the first time the discordant adaptation of human peritoneal macrophages to a secondary lps stimulus in vitro. these findings appear to bear ramifications for the in-vivo endotoxin response during inflammation and gramnegative septicemia. shock states with inadequate cellular oxygen delivery may contribute to macrophage (mo) activation or dysregulation. in this study we compared the effect of transient hypoxia and endotoxin pretreatment (lps 1) on mo mediator release with a second endotoxin stimulus (lps2). methods: in vitro cultures of marine peritoneal exudate mo were exposed to 2 hr. of hypoxic or normoxic conditions, then incubated 22 hr under identical normoxic conditions _+ 10 ng/ml of lps 1 pretreatment. during the final 24 hours all m~l were exposed to a range of lps 2 concentrations. m~i supernatants were assayed for tnf, il-1, il-6, pge2, nitric oxide (no), and superoxide release. results: lps 1 markedly inhibited mo tnf release by lps 2 but hypoxia had no effect on lps 2-triggered tnf release. hypoxia increased mo il-6 production in the absence of lps 1, but inhibited lps 1 augmentation seen under normoxic conditions. pretreatment with lps 1 increased no production from lps 2 under normoxic conditions, but bypoxiainhibited this effect. pretreatment with lps1 signifcantly augmented mo release of il-i and pge 2, but hypoxia had no significant effect (data not shown) superoxide production was increased by lps 1 under normoxic conditions, but hypoxia significantly inhibited superoxide release (not shown a. lepape, c. docile, f. arpin, m. c. gutowski, v. banssillon and j. bienvenu. i aim of the study. increased levels of tnf are inconsistently correlated with the severity of sepsis. one possible explanation is a decrease ability of ceils to produce eytokines. the objectives of this study were m compare at different levels of of clinical severity the responsiveness of cytokine producing cells. this was evaluated by the response to a stimulation by lps as well as to the inhibitory effect of ptx. the technic used is a whole blood ex-vivo model, as described by desch et al. (1) . h materials and methods. different groups of icu patients, postoperative (n=12), sepsis (n=12), septick shock (n=13), and healthy control (n=10), were studied. blood was drawn in endotoxio free heparinized robes. stimulation was achieved by addition nf75 pl oflps (10ng/ml) to 675 id of whole blood in presence of various concentrations of ptx (0, 10 "5, 10 -4, 10 "3, i0"2m). after 5 hrs incubation at 37°c, the tubes were centrifuged and supematants frozen at -80°c. tnfa and/l6 were measured by elisa (medgenix r and immunotech r respectively). monocytes were quantified on a technicon h6000. the results are expressed as median values. non parametric tests are used for testing differences between groups. hi results, tnftx and 11,6, corrected for monocytes count, are given as % of their initial value, the baseline before stimulation or inhibition being 100%, 1) stimulation by lps. the control group is much more stimulated (>21000% for il6, >8000% for tnfa). blood samples taken postoperatively and in patients with simple sepsis are significantly less stimulated (>1500% for il6, >600% for tnfa ). the lowest stimulation was observed in patients with septic shock (median = 168%), some patients being not stimulated at all. 2)effects of ptx.the inhibitory effect of ptx on tnftx production is effective in all groups at 10 -3 m (reduction to less than '¼ of the median values), and is almost complete at 10" 2 m. the septic shock group has a decreased sensitivity to ptx. il6 production exhibits a lesser reduction at 10 -3 m (~ 'a to ½ of the median values), further increased at 10 -2 m. the septic shock group is again less sensitive to ptx. iv conclusion: the reduced ability of circulating monocytes to produce cytokines during severe infections is confirmed here. ptx is able to reduce significantly tnfc~ at 10 -3 m and the inhibition is nearly complete at 10 -2 m. surprisingly, there is a lesser, but significant suppressive action of ptx on il6, not found in experiments using purified monocytes. one possible explination could be the interplay between cytokines production. (1) lymphokine research (1989) cdna sequencing constitutes a powerful method of measuring steady-state mrna levels for all genes transcribed in a given cell or tissue at a particular stage of differentiation. by comparing transcript abundance both prior to and following differentiation, individual genes can be identified whose transcription is regulated both positively and negatively. in order to examine monocyte activation, the human monocyte line thp-1 was induced with phorbol ester (48 h) and activated for 4 h with lipopolysaccharide (lps) after which polya + rna was purified. the rna from control and lps-treated cells were each used to construct a cdna library under identical conditions, and all resulting clones were selected for cdna sequence analysis. each clone sequence was evaluated by matching with both genbank and our own gene databases. very different patterns of gene expression were seen in the two libraries, the latter reflecting very high levels of known inflammatory mediators such as il-1 and tnf. a second set of libraries were made from umbilical vein endothelial cells (huvec), both with and without lps stimulation, and were analyzed in a similar fashion. the effects of lps induction on specific gene transcription in both cell types will be discussed. t. tadros, md, th wobbes, me) phd, rja goris, md phd to investigate whether the preactivation of regional macrophuges by liposomes containing muramyl tripeptide (mtp-pe) can counteract the detrimental effect of blood transfusions on both anastomotic repair and host susceptibility to infections. methods eighty lewis rats received lmg/kg of either empty or mtp-pe encapsulated liposomes, intraperitoneally (ip). twenty-four hours thereafter, the animals underwent resection and anastomosis of both ileum and colon, and received 3 ml of either saline or blood from brown norway donors,iv. the animals were killed 3 or 7 days after surgery and examined for septic complications and anastomotic repair. the average anastomotic strength, as assessed by bursting pressure (+sd), was significantly diminished in the transfused animals, as compared to the non-transfused animals (ileum;day 3; 44-+8 vs 99+7, p<0.001). transfused animals pretreated with mtp-pe encapsulated liposomes showed a significant improvement of their anastomotic bursting pressure (93+9, p<0.001 vs transfusion). pretreatment with mtp-pe encapsulated liposomes decreased significantly the incidence of anastomotic abscesses in transfused animals ( from 90% in ileum on day 3 to 20%, p<0.001). conclusions preactivation of regional macrophges by intraperitoneal administration of mtp-pe encapsulated liposomes prevents the detrimental effects of transfusions on anastomotic repair and reduces the incidence of intraabdominal sepsis. academic hospital nijmegen, dept of general surgery, pb 910 i, 6500 hb nijmegen, the netherlands. leukemia cell line, teip-1. robin s. wa, gner*, perry v. halushka "~, and james a. cook*, departments of physiology , pharmacology "l" and medicine "t, medical university of south carolina, charleston, s.c. 29425. adherence of monoeytes to endothelium and extracella/ar matrix proteins is essential for accumulation at sites of inflammation. txa2, an arachidonic acid metabolite, inhibits human monocyte chemotactic responses suggesting that txa 2 may alter monocyte adhesiveness. we selected the thp-1 cell line, a human monocytic leukemia cell line to further investigate the effect of txa 2 on adhesion. we tested the hypothesis that txa 2 alters lpsinduced adhesion of thp-1 cells and that txa 2 exerts its effect on adhesion via a camp dependent mechanism. thp-i cells were exposed to s. enteritidis endotoxin (lp.g/ml) _+ the cyelooxygenase inhibitor lndomethacin (in), the txa 2 mimetic i-bop (0.11.tm,) or txa 2 receptor antagonists bms180291 and l657925 (10~m). cells were allowed to adhere for 24 hours and adherent protein/well was determined. lps-induced a significant (p<0.05;n=7) increase in adherence of thp-1 cells (basal, 2.4+0.85gg protein/well; lps, 20.4+_6.0p.g protein/well). the amino acid glutamine is an essential compound for synthesis of purine and pyrimidine basis and therefore necessary for rna-and dna synthesis. in human plasma the concentration of glutamine is between 0.5 -0.6 mm, and is reduced in septic patients up to 80 % (0.1 -0.2 mm). monocytes play a central part in the inunune system and it was of interest, whether glutamine is involved in the modulation of cell surface markers and phagocytosis of these cells. human peripheral blood mononuclear ceils were obtained from 500 ml heparinized blood of apparently healthy donors by ficoll-paque density gradient and isolated by counterflow elutriation. the puritiy was more than 90 %. subsequently cells were cultured in phenolred-free rpmi 1640 medium with various concentrations of glutamine (0.05, 0.1, 0.2, 0.3, 0.6, 1, 2 mm) in teflon-fluorinated ethylene propylene bottles to exclude cell adhesion and possible cell activation. aider seven days culture, cell viabilty was determined by trepan blue exclusion and varied between 70 and 90 %, independent of glutamine concentrations. cell surface markers were detected by flow cytometry, noaspecifie phagoeytosis was measured with latex beads and specific phagocytosis with opsonizied e.eoli using a facscan. lower concentrations of glutamine decreased the expression of hla-dr and icam-1/cd54 on monocytes in a dose-dependent manner. the receptor for fc'/rucd64 as well as the receptors for complement cr3/cdllb and cr4/cdllc were down-regulated. cr1/cd35 which is only slightly expressed on monocytes was not influenced. furthermore, no effects on the expression of cdi4, the receptor for transferrin cd71 and fc'friii/cd16 were seen. our data indicate, that lower concentrations of glutamme influence the phenotype of monocytes. we are now interested to study whether glutmnine influences non-specific phagocytosis, or whether specific phagocytosis correlates with the decreased expression of fc'/r and complement receptors. we investigated immunologically more than 300 patients who were admitted to icu because septic syndrom during the last four years. patients were immunologically followed up 2-3 times per week until release from icu. the expression of hla-dr antigen on monocytes turned out to be the best prognostic parameter. the persistence (> 5 days) of low hla-dr expression (< 30%) predicts fatal outcome (mortality > 85%). the altered phenotype was associated with a functional deactivation of monocytes (diminished apc, ros formation, cytokine secretion). we called this phenomenon "immunoparalysis". ifn-gamma and gm-csf were able to restore the altered phenotype and function in vitro. however, addition of autologous plasma from septic patients with "immunoparalysis" to these cultures prevented the cytokine-induced restitution. the inhibitory activity could not be removed by dialysis. therefore, we started a study to prove the therapeutic efficacy of plasmapheresis. indeed, [7 of 33 patients recovered from "immunoparalysis" following repeated plasmapheres; 16 of them survived (46 %). 7 patients recovered temporarely and 9 patients did not respond (all 16 died). the survival rate in the control group of septic patients with persistent "immunoparalysis" was 4 of 38 (11%; p<0,01). in summary, plasmapheresis in association with immune monitoring may be an alternative strategy to improve survival rate in severe sepsis. taurolidine, a synthetic taurine-formaldehyde derivative has antiadherent, bactericidal and anti-lps properties functioning primarily through binding of the lipid a region of the lps molecule. the active derivative of taurolidine, taurine, modulates calcium channel activity, critical to the initiation of a number of immunostimulatory pathways. we hypothesised that taurolidine may have direct immunostimulatory activity. the aim of this study was to investigate the immune effects of taurolidine on peritoneal macrophage (pmo) function and then determine the role of taurine in this response. study 1: in vivo stimulation:cd-1 mice (n=35) were randomized to receive taurolidine (200mg/kg bw/i.p.) or saline cor~trol. peritoneai cells were harvested after 24 hours and were assessed for pm0 function [superoxide anion generation (o2-), nitric oxide (no), tumor necrosis factor (tnf), fc/cr3-mediated phagocytic function (phago) study 2: control pm0 were harvested and cultured in vitro with taurine (1.0mg/ml for 10 hrs), after which time they were assayed for 0 2-and tnf release. in vivo stimulation with taurolidine taurolidine has specific immunological effects on m0. release of the inflammatory mediators 0 2-and tnf, and fc/cr3-mediated phagocytosis were significantly increased, while release of the endothelial relaxing factor no was significantly reduced. in addition, the amino acid taurine, which is released as a byproduct of taurolidines breakdown has an immunostimulatory effect on pmo and may be the active moeity of the compound tanrolidine. in sepsis, a number of mediators which affect vasomotor tone and cardiovascular function are produced. inasmuch as sepsis causes decrease in systemic vascular resistance (svr), attention is usually focussed on vasodilators such as lactate, tumor necrosis factor, interleukin-i & 2, and nitric oxide. but injury and inflammation als0 cause production of several vasoconstrictors whose effect may not be evident in changed svr, but may significantly affect organ blood flow or function in the paracrine environment. endothelin (et) is a 21 amino acid peptide vasoconstrictor produced by ischemic or injured endothelial cells (ec's). et is also a potent constrictor for renal mesangial and coronary vessels, an endocrine regulator, and a negative cardiac inotrope. systemic et levels increase significantly in hypoperfusion and ischemia. while et is principally produced by ec's, we asked if human monocytes might also produce et and thereby regulate vasomotor tone in areas of inflammation. monocytes from healthy donors were separated on ficoll, resuspended in rpmi 1640 +5% fetal calf serum and stimulated with i0 ug/ml endotoxin (lps). et was measured by radioimmunoassay. lps-stimulated monocytes produced 87 ! 6 fm of et/106 cells (vs. unstimulated controls of <25). this calculates to 15-30% of the amount of et observed in patients with low cardiac output, sepsis or ischemia. we conclude that et is a cytokine produced by both ec's and monocytes with potent effects on numerous cells and organs in the critically ill. wuppertal 5, germany we and other authors showed that fatal outcome in septic disease is associated with a decreased capacity of peripheral blood monocytes for the in vitro production of proinflammatory cytokines, especially tnf-alpha. we found that this monocytic deactivation is completed by a persistent and marked decrease of hla-dr expression on monocytes (< 30 % hla-dr+ monocytes) and a diminished antigen presenting activity whereas the capacity to form the antiinflammatory il-1 receptor antagonist remains high. in order to evaluate the in vivo situation and to determine at which level tnfproduction/secretion is altered we assessed the tnf-alpha mrna expression in freshly isolated peripheral blood mononuclear cells (pbmnc) from septic patients. tnf-mrna was onty rarely detected by semiqaantitative polymerase chain reaction in pbmnc's from septic patients with monocyte deactivation. meanwhile, it was found in almost all pbmncs from septic patients without monocytic deactivation. we wondered, whether il-i0, which ,is known to depress monocytic proinflammatoly response and mhc class ii expression, could be one of the mediators in fatal sepsis. in fact, we found that il-10 message in pbmncs of septic patients peaked in the beginning phase of monocytic deactivation. in further investigations we found that tnf-administration can induce monocytic deactivation in a murine model/n vivo and provoke il-10 message in human pbmncs in vitro. these results support our hypothesis that an excessive delivery of proinflammatory cytokines in a first phase can induce an overwheiming inhibitory feedback, mediated by immuninhibitory mediators like il-l0, which leads to often fatal monocytic deactivation in a second phase. interferon-gamma which is known to counteract il-10 production and the effects of il-10 on monocytes restores the function and phenotype of monocytes from septic patients with monoq, te deactivation in vitro and could be a possible therapeutic agent in otherwise fatal sepsis. our laboratory previously reported that lps dependent macrophagederived tnf-a production can be enhanced by pretreatment with lps at substimulatory lps priming doses coincident with a suppression of lps dependent nitric oxide (no) production (zhang and morrison, j. exp. med 17:511, 1993) . in order to extend the characterization of these lps priming effects in mouse macrophages, we examined the capacity of substimulatory lps to modify lps dependent il-6 production. macrophages were obtained from peritoneal exudate of thioglycollate treated c3heb/fej mice and cultured in rpmi 1640 medium containing 10% fetal bovine serum. macrophages were pretreated with various subthreshold stimulatory concentrations of lps (olll:b4) for 6 hours, washed three times, and then stimulated with the effective stimulatory concentration of lps for 18 hours. the amount of il-6 in the supernatant was measured by il-6 dependent cell line (b9 and 7td1) proliferation assay. il-6 was produced by macrophages at lower threshold doses of lps than those required for tnf-o~ or no production. subthreshold doses of lps modulated il-6 production in a biphasic manner characterized by an initial suppression and then potentiation. higher doses resulted in secretion of il-6 during the initial incubation with lps and subsequent desensitization. il-6, like tnf-~ and no, is, therefore, also affected by lps pretreatment. moreover, tnf-a and il-6 shared the similar potentiational pathway, but differed by the fact that only il-6 was inhibited. (supported by r37 ai23447 and po1 a54474.) department of microbiology, molecular genetics and immunology and the cancer center, 1000 wahl east, university of kansas medical center, kansas city, ks 66160-7832. korolenko t.,urazgaliev k.,and arkhipov s. the role of macrophage (mph) stimulation in mechanism of protective effect of new immunomodulators yeast polysaccharides -heteropolysaccharide cryelan and homopolysaccharide mannan rhodexman (both produced by petersburg chem.-pharm. inst.) was studied. in vitro according to nst test incubation of murine peritoneal mphs with cryelan or rhodexman, 150 ~g/ml,30 min was followed by increase of potencial microbicidic activity of mphs. in vivo mph stimulation by immunomodulators studied included increase rate of carbon particles phagocytosis during single i.v. or i.p. mode of administration to mice 1-14 days after (peak at 2nd day for i.v. and 5th day for i.p. mode of administration of the same dose of 5 mg/100 g b.w.).the preliminary injection of cryelan (5 mg/100 g, 24 or 48 h before) to mice with acute cold stress (-10 ° c, 1 h) revealed protective effect restorating the value of depressed phagocytosis up to the normal level;the positive effect on ultrastructure of hepatocytes was noted also.there was no changes of plasma corticosterone level between group with acute cold stress and mice with cryelan + acute cold stress (several fold increase comparatively to the control mice).as was suggested, the mechanism of protection can include mph stimulation and secretion of some acute phase proteins responsible for positive effect of immunomodulators. new yeast polysaccharides cryelan and rhodexman can be used for macrophage stimulation,especially in pathological states. immunomodulators were shown to increase production and secretion of lysosomal enzymes (like zymosan). secreted enzymes,especially cysteine proteinasescathepsins b and l -involve in the process of inflammation;however, excessive release of these enzymes may lead to noncontrolled proteolysis followed by tissue degradation (assfalg-machleidt et al.,1990) .the effect of zymosan,bcg and new immunomodulator carboxymethylglucan (cmg), second fraction on secretion of lysosomal enzymes by murine peritoneal macrophages was studied. zymosan increased the secretion of n-acetyl-~-d-glucosaminidase and ~-galactosidase into the culture medium (3-4 fold); bcg possessed similar effect.cmg in the same concentrations (50 /~g/ml) increased release of these enzymes only saightly (1.6 times).it's known that zymosan-induced secretion reflects the enzyme release from formed lysosomes (warren,1989) .it was suggested that cmg activated macrophages via interaction with scavenger-receptors,followed by weak secretion of lysosomal enzymes and as a result decrease of tissue damage. in vivo zymosan induced stimulation of mononuclear system of phagocytes followed by increase of cysteine proteinases activity in liver at the 5th day. in the same time in blood n-acetyl-~-d-glucosaminidase and n-acetyl-~-d-galactosidase activity increased 2-5 fold. it was concluded that in drug design it's possible to select such immunomodulators,e.g. cmg,which can activate mononuclear system of phagocytes and do not damage tissue. endothelin-i (et-i) is produced by injured/ ischemic endothelium, mobilizes intracellular ca ++ and is a potent vasoconstrictor. it is also a ca ++ agonist for anterior pituitary or renal mesangial cells and monocytes. et-i causes monocytes to produce interleukin-l, 6, 8, prostaglandin e2, and substances which trigger neutrophil superoxide production. et-i levels increase in shock and et may play a role in activating leukocytes post shock causing reperfusion injury. but blood flow experiments suggest splanchnic circulation changes more profoundly in shock than peripheral circulation. we therefore asked if et-4 (or vic), the et which predominates in splanchnic vessels, had any effect on monocyte cytokine production. human monocytes from health~ blood donors were separated on ficoll. 0.5 x 10ucells/ ml in rpmi 1640 + 10% fcs were incubated i0 min., 6 & 24 hrs. with 10 -8 m et-i, 10 -8 m vic or i0 ug/ml of lps. supernatants were assayed by elisa. we have shown that low dose endotoxin pretreatment (lps 1 ) for 24 hrs markedly inhibits the macrophage (mo) release of tumor necrosis factor (tnf) and increases interleukin-1 (il-i) in response to a subsequent endotoxin stimulus (lps2). in this study we examined the kinetics of lps 1 inhibition of tnf and augmentation ofil-1. methods: murine peritoneal exudate mo from balbc mice were exposed in vitro to medium or 10 ng/ml of lps1 for intervals of 0 to 24 hours. culture medium was then replaced with 0, 10 or 1000 ng/ml of lps2 for 24 hrs. tnf and il-1 in mo supernatants were measured by specific bioassays. during sepsis endotoxin (lps) activates macrophages (mo) to release mediators such as tumor necrosis factor (tnf), interleukin-6 (il-6), interleukin-i (il-i) and prostaglandin e2 (pge2). we showed that preexposure to lps (lps 1) alters the response of murine m~i to subsequent lps stimulation (lps2). we hypothesized that in vitro cytokine release by lps2 in human monocytes (mo) is also be altered by preexposure to lpsi. methods: human peripheral blood mo were obtained from healthy volunteers (n=8), cultured in vitro 24 hrs, then pretreated 24 hr _+ lps 1-cultures were then stimulated with lps 2 and mediators in mo supernatant measured: tnf, il-i, and il-6 by specific bioassays, pge2 by immunoassay kit. serum cytokine levels (specific elisa kits) were compared to in vitro supernatant levels. data is expressed as % control_+sem, lps2= 1000 ng/mh the table shows that all mediators were increased, in the absence of lps 1. pretreatment with lps 1 resulted in complete inhibition of lps2-triggered tnf release. in contrast, lps 1 significantly increased mo secretion of il-1, il-6 and pge 2 (data not shown). serum cytokine levels were as follows: tnf 407_+21, il-i 241+208, and il-6 8.0-+2.5 ng/ml. these serum levels were low, showed an extremely wide variation, and did not correlate with in vitro lps2-triggered mediator production. conclusion: human monoeyte mediator production is differentially regulated by preexposure to lps 1. provocative in vitro testing of monocytes may ultimately be clinically useful to identify prior in vivo lps exposure or mo macrophages release numerous secretory products involved in host defense and inflammation. activated macrophages with cytokines produced have been implicated in tissue damage in sepsis and multiple organ dysfunction. aimed to elucidate the organ-association phenomena,this study is to compare peritoneal macrophage(pm),alveolar macrophage(am), and kupffer cells(kc) during sepsis in terms of cellular protein contents as symbol of activation by flow cytometry analysis. sepsis were produced by cecal ligatien and perforation (clp) in wistar rats weighing 210-290g.pm were obtained by peritoneal lavage,am by bronchial lavage and kc by incubating the collegenase digested liver with pronase-e. leukocytes have been implicated as a mediator of the microvascular dysfunction associated with reperfasion of ischemic tissues. a role for ieukocytes is largely based on observations that rendering animals anutropenic with anti-neutrophil serum or preventing leukocyte adhesion with monoclonal antibodies attenuates the increased fluid and protein leakage from the vaseulature that is normally observed in postischemic tissues. we have recently undertaken studies designed to determine the relationship between leukocyte-endothelial cell adhesion and albumin leakage ia rat mesenterlc venules exposed ~o ischemia-reperfusion (i/r). leukocyte adherence and emigration as well as albumin extravasafion were monitored in single postcapillary venules using iatravital fluorescence microscopy, lschemia was induced by complete occ!usion of the superior mesenteric artery and ~dl parameters were monitored at various intervals following reperfusion. the magnitude of the leukocyte adherence and emigration, and albumin leakage elicited by i/r was positively con-elated with the duration of ischemia. the albumin leakage response was also highly correlated with the number of adherent and emigrated leukocytes. monoclonal antibodies against the adhesion glycoproteins cd18, cdllb, icam-1 and l-selectin, but not p-or e-selecdn, reduced i/r-induced leukocyte adherence and emigration as well as albumin leakage. phauoidln, an f-aetin stabilizer, largely prevented the emigration (but not adherence) of leukocytes and greatly reduced, the raicrovascular protein leakage. plateletleukocyte aggregates were formed in postischemic vemdes; the number of aggregates was reduced by antibodies against p-selecdh, cdilb, cd18, and icam-1, but not e-selectin or lselectin. a significant fraction of the mast ceils surrounding the posteapillary venules degranulated in response to ischemia/repeffusion, but mast cell stabilizers did not afford protection against the albumin leakage elicited by i/r. these results indicate that reperfusloninduced albumin leakage is tightly coupled to the adherence and emigration of leukocytes in posteapillary venules. this adhesiomdependent injury response is primarily mediated by cdllb/cdi8 on activated neutrophils and icam-1 on venular endothellum, and appears to require l-selecda dependent leukocyte rolling. mast cell degranulation does not appear to conwibate to the vascular pathology associated with i/r. m.d. rod=iek, boston, ma, usa the polymorphonuclear neutrophil (pmn) has long been known to pa~tlcipats in the inflammatory rebpons~ as a phagocyte and killer of invading organisms, but little attention has been given to its potential as a participant in the in~une interaction of lymphocytes and macrophages. we and others have shown that the pmn may have i~m~/nomcdulatory effects both in vitro and in vlvo. more recently it has been proven that the pmn can make mrna for and secrete the proinflammatory oytokines illa, il-ib, tnfs, il-6 and il-8 as does the other major circulating phagocyte, the monocyte/macrophags. furthermore it has been shown to make the potentially autoregulatory oytokines gcsf and gmcsf. these functional capabilities suggest that the pmn is not an wend cell ~, but one which has a potential role in regulation cf ~he immune response and that this potential ~cle should no longer be ignored when considering the immune abnormalities existing in patients following majo~ injury or surgery. we have investigated the proinflaznmatory oytokine secretion patter~ by pmn in patients following major ~hermal or tra~matic injury and in volunteers fellowinq endotoxemia. ?ollowing major injury there is variable pmn secretion of these cytokines when stimulated in vlero. following endotoxemia in a group of human volunteers pmn showed a hypo=esponsivenesa to lps 4 hrs following endotoxin infusion followed at 24 hre by an overshoot. pretreatment with steroids modulated this overshoot phenomenon, suggesting that receptors for steroids are involved in the regulation of cytokin® secretlon by fmn. these results sugges~ that the pmn, the most numerous cell in the circulation and the first to respond to an ins~l~ may be a so~rce of the prolnflammatory cytokine cascade following injury that has been recognized as significant in the process which often leads to multiple o;gan failure, the immunosuppresslon which occurs following major thermal injury may predispose these individuals to infection and sepsis, which remain a significant cause of morbidity and mortality. included among the many immune aheratlons are the p2 integrln (cdlla, b,c/cd18) dependent activities of adhesion, chemotaxls, diapodesls, and phagocytosls. our investigations indicate that, following major thermal injuries, the expression of the [~2 integrlns, but not cd14, is significantly decreased on neutrophlls (pmns). it remains unclear if pmns from thermally injured patients respond normally to lps, the effects of treatment in vitro with lps and f-met-leu-phe (fmlp) on the expression of cdtlb was examlned on pmns from the peripheral blood of healthy volunteers and non-septic burn patients (>30~; total body surface area, >ls~ full thickness), the pmns were incubated with lps (]ng-10p.g/ml) or f'mlp (10 "1 1 to 10"6m) et 37oc for 30 mln, in 1~; human ab serum, the expression of the 132 ]ntegrins was detected using monoclonat antibodies and flow cytometry. lps and f'mlp resulted in a slight increase (3 fold) in the expression of cd11b on pmns from burned patients compared to an 8 and 14 fold increase, respectively, on pmns from healthy individuals. this inability of lps or fmlp to increase cd11 b expression was not due to the amount of lps bound to the two cell populations. because the same defect is seen after either lps or fmlp stimulation, it is speculated that the defect must be in the amount of preformed cd1 ] b or its transport to the plasma membrane. platelet-activating factor (paf) and neutrophils have been implicated in the patbophysiology of ischemia-repeffusion injury, in addition, paf stimulates neutrophi[ (pmn) oxidative metabolism in vitro. the present study examined the potential role of paf in repeffusion injury in an in viva rabbit model. eight anesthetized rabbi~s underwent retroperitoneal exposure of the infrarenal abdominal aorta after percutaneous insertion of a catheter through the jugular vein into the infrahepatic inferior vena cava. doppler flow probes were placed around the abdominal aorta and the right common femoral artery to assess flow through these vessels. an occlusive ligature was placed around the abdominal aorta (superior to the flow probe) at t = 0 and total occlusion of blood flow to the lower extremities was maintained for g0 mins., after which the ligature was released allowing for reperfusion of the ischemic lower limbs. effluent blood from the ischemic hind-limbs was collected through the ivc catheter at the times indicated below and assayed for paf by a direct radioimmunoassay. in addition, neutrophil h202 production was determined by a previously described 2'7'-dichlorofluorescein flowcytametric assay. 185 _+ 53 amean _+ s.e.m, pg/ml blood; brelative fluoresenee (% of baseline); caortic and femoral artery flow (% of baseline); *p < 0.01 vs. baseline; 1"p < 0.05 vs. baseline. a significant elevation of paf was observed in ischemic hind-limb effluent blood at 15 min. after release of the aortic ligature during the repeffusion phase, as compared to baseline levels. in addition, pmn h20 2 production was increased by 2.3-fold above baseline values by 1 hour after ligature release during the reperfusion phase. both of these elevations were transient and returned toward baseline by 2 hours post-isehemia. tatar occlusion of hind-limb flow was achieved as evidenced by the absence of aortic or femorat flow at 90 rain. post-ischemia, however after release 01 the ligature a significant reactive hyperemia was observed by 15 mln. into the rapeffusion phase. histolog[c examination of reper[used gastrocnemius muscle revealed moderate pmn infiltration into the interstitium. in conclusion, these data indicate that paf is released into the circulation during repeffusion, and is likely involved as a mediator in the observed pmn oxidative burst activity, thereby contributing to reperfusien injury. following thermal injury and infection granulocyte function ts abnormal. to elucidate the mechanism by which thermal injury and infection affect the granulocyte's ability to polymerize and depolymedze actin, we serially measured f-actin levels in granulocytes from 14 burned patients (mean age 42,1 +_ 17.7 years, mean burn size 39.8% _+ 22.2%) during the first s weeks post injury. six of the patients had infections during the course of the study, (septicemia, wound invasion and pneumonia). actin levels in granulocytes from eleven healthy volunteers (mean age 34 years) were measured repeatedly and served as controls. lysecl white blood cell preparations were brought to 370c and incubated with n-formyl-met-leu-phe (stim) or with dulbecco's phosphate unbuffered sellne (unstim). the cells were concomitantly stained and fixed with formaldehyde, lysoleclthln and fiuoresceln phafioidin. actin depolymedzation (depol) was measured by incubating stimulated cells at 0°c before the stain-fixative was added. baseline (base) f-actln levels were assessed by adding stsln-fixatlve to icecold unstimulated cells. fluorescence was estimated in a facscan and expressed as ilnesr mean channel fluorescence_+ sem (mcf). figure 1 displays granulecyle fectln levels in infected and uninfected patients as compared to controls. f-actln levels were consistently lower in control cells than in those from burned or burn-infected patients under all measured conditions. granulocytes from infected burned patients demonstrated a significant decrement in their ability to depofymerlze f.actin compared to both uninfected burned patients and controls, while there were no significant differences between infected and 1,~ uninfected patients in the baseline, unstlmuleted and stimulated conditions. those results indicate la5 that grsnulocytas from burned and bum-infected patients contain higher levels of polymerized actln than ~ ,2s control cells. in order to study tumor necrosis factor (tnf) receptor sensitivity in septic critically ill patients we investigated 20 blood samples of such people in reaction of leucocyte migration inhibition. migration of their polymorphonuclear leucocytes (pmns) was studied with stimulation with human recombinant tnf in concentration of 166.7 u/ml (recommended by manufacturer is the range of 10-200 o/ml) and without such. ten healthy blood donors formed control group. the results obtained showed diminished pmn reactivity to tnf in patients (migration inhibition was absent) oscaring with significantly increased migration ability of their pmns (107.2% of that in control group). at the same time normal pmns in control group did show migration changes upon tnf stimulation. considering all the above we come to a conclusion that externally added tnf fails to activate pmns in critically ill patients more than they are by their endogenous tnf. moreover, this tnf no longer serves a positive chemotactic factor for such pmns. these findings may suggest that in critically ill septic patients reactivity of pmns to tnf is deeply altered. tnf receptors of pmns are either exhausted as such by excessive stimulation with endogenous tnf or further transmission of their message is impossible due to "fatigue" of the cell's activation mechanisms. we express our gratitude to reanal factory of laboratory chemicals for generously providing us with a tnf com~rcial sample. ~-sanguis medical, ekaterineburg russia; s-urals med.lnst. activated neutrophils infiltrating the local site of inflammation following trauma release high amounts of destructive lysosomal enzymes into the extracellular space. cytokines were discussed to be involved in regulation of this early process. the task of this investigation was to evaluate the possible regulatory role of interleukin-6 (il-6) and its potential immunosupressive opponent, the transforming growth factor-&, in regulation of neutrophil degranulation. we analysed the concentration of the al-proteinase-inhibitor complex of the lysosomal elastase as marker for the degranulation of neutrophils as well as the levels of il-6 and tgf-61 in the plasma probes of patients undergoing multiple trauma and severe surgeries. the time courses of il-6 and elastase were found to be highly correlated, wheras the concentrations of the cytokine tgf-e~ were found to be not significantly altered in comparison to the control group. this close temporal correlationship was confirmed by investigation of fluids derived from sites of inflammation. interstingly, the inhibitory potential (~zcproteinase inhibitor, antithrombin iii) was dramatically reduced in the early inflammatory phase. to prove this in vivo findings, the effects of il-6 and tgf-i~ 1 on the degranulation of isolated human neutrophils of healthy donors was investigated in vitro. pathological high concentrations of rhll-6 up to 104u/ml (as detected in fluids derived from local inflammatory site) were found to be capable to induce a significant release of lysosomal elastase in a concentration-dependent manner, whereas the degranulation of neutrophils was uneffected by tgf-61. in conclusion, these data suggest a contribution of il-6 in regulation of neutrophil activation at sides of inflammation. the immunosuppressive cytokine tgf-i&~ seems to have no direct regulatory effect beside its described chemotactic function on neutrephils. postirradiation chan~es of adhesive properties arid supercoiled nucleoid dna structure of blood leukocytes were studied in macaca nemestrina andrats. the dynamics of membrane chan~es after nonlethal irradiation of rats demonstrated the temporary increase of the leukocyte adherence at 24 h followed by return of this parameter to normal levels at 48 h. after lethal irradiation of both animal species the increase in adhesive leukooytes fraction was detected as early as at 3 h. this hi~her index persisted until the end of experiments (5 days). the early (3-5h) temporary loosin~ of supercoiled dna structure was demonstrated in the leukocytes of nonlethally irradiated animals. this phenomenon seems to be connected with the lymphocyte fraction chan~es. this process was not dependent on altered adhesive properties of leukocyte membranes. the membrane chan~es of leukocytes preceded decondensation of supercoiled dna after lethal irradiation of animals, in this case loosin~ of supercoiled dna pro-~ressively increased at 24 h and at the later terms of postirradiation period. the systemic inflammatory response syndrome (sirs) involves many inanunological reactions of the host including acfivatinn of inflammatory mediator cascades and depression of cellular reactivity in t-lymphecytes (1). there are reports of nentrophil dysfunction in inflammatory disorders of the skin (2), are there dysfunctions concerning the unspecific host defense in sirs, as well? in this study, we examined the reactivity of neutrophil granolocytes from patients suffering from sirs. twenty-one patients (apache ii-score 21±5) with diagnosis of sirs entered the study. granulocytes were prepared as reported previously (2) . in parallel, granulocytes from 20 healthy individuals were tested. two granulocyte functians were studied in vitro: 1. migration of the ceils in a boyden chamber through a filter matrix following stimulation with 5 different receptor dependent stimuli (c5a, intefleukin-8, platelet-activating-factor, leukotrien b4, fmlp). 2. release of 13glucuronidase following stimulation with the aforementioned activators. the results demonstrate, that the release of 13-glucuronidase in patients suffering from sirs was comparable to the enzyme release of granulocytes prepared from healthy individuals. each stimulant induced release of p-glucuronidase in a characteristic dose dependent fashion. all granulocyte preparations from the healthy donors showed a positive chemotaxis response in the migration-assay. in contrast, only ten out of twenty-one patients had granulocytes migrating after stimulation. the two groups of patients displaying reactive or non-reactive granulocytes differed clinically: the nonreactive group consisted of patients with multiple organ failure (10/11) and nonsurvivors (8/11), whereas 8/10 patients in the reactive group survived. thus, the in vitro chemotaxis of granulocytes is impaired in a subgroup of patients with sirs. this defect of the non-specific host defense may contribute to poor prognosis and outcome of these patients. dermatol. 85: 194-198, 1985 klinik ffir an~isthesiologie und operative intensivmedizin der cau kiel, schwanenweg 21, 24105 kiel, germany. objectives of the study: major emphasis has been given to the analysis of interactions of antibiotics with microorganisms. effects of antibiotics on cells of primary host defense mechanisms, such as the neutrophils, are less well known. therefore, attention has been focused on clindamycin, a member of the lincoseamide family. materials and methods: the effect of clindamycin (i -i00 ~g/ml) on 8 granulocyte functions (healthy volunteers) such as random migration, chemotaxis (agarose method), ingestion (radiometric assay), superoxide (cytochrom c reduction) and hydrogen peroxide production (phenol red oxidation), lucigenin-and luminol-amplified chemiluminescence (luminometry) and degranulation (turbidometry with micrococcus lysodeicticus) were investigated in vitro. results: motility and degranulation were inhibited, ingestion of saccharomyces cerevisiae, zymosan-induced lucigenin-and luminol-amplified chemiluminescence, superoxide and hydrogen peroxide production were stimulated in a dose dependent fashion. conclusion: clindamycin has granulocyte function modulating properties. recognition of immunomodulating effects of antibiotics may have therapeutic significance, especially in patients with long-term antibiotic therapy or immune deficiencies. the intense muscle activity (ea) of rats resulted in increase of neutrophil influx in muscles during the recovery. we investigated neutrophil proteinases involvement in neutral proteinases balance of skeletal muscles by na. the rats were submited to swim with the load (8% of body mass) till exhaustion. immediately after na the neutrophil antiserum was injected i.p. to rats of experimental group. saline was injected to control animals° injections were repeated in 12 h of the recovery and cytosol proteolytic activity (ph 7.4; fitc-casein) was determined. isolated soleus muscles were incubated also in vitro and proteolytic activity of incubation media was measured. it was found that there was 2-3 -fold proteinases activity increase in cytosols of all investigated muscles (soleus, white and red portions of quadriceps) of control animals by 6 h of the recovery (the comparison was done with the sedentary rats). in 12 h cytosol proteolytic activity decreased and then increased again by 24 h of the fast. antiserum injections resulted in relible decrease of the proteolytic activities at every investigated time. when incubating m. soleus in vitro the activities of proteinases in incubation media turned out reliably less if soleus muscles were isolated from the animals to which antiserum was injected. the conclusion is that neutrophil proteinases can be involved in the balance of rat skeletal muscle neutral proteinases after ~a. a lot and new clinical problems complicating the outcome of polytrauma, burn and septic patients in surgical intensive care units, have arisen as the care improvement prolonged the patient's survival: a progressive degradation of organ and system functions often develops, usually making its first clinical appearance by ards, followed by the other organ failure (mof) and sepsis symptoms. the clinical picture is polymorphic, the end result of a complex systemic pathophysiological reaction trigg~ed off by trauma consequences (tissues disruption, hypo~xygenatiun and necrosis). nowadays there is not a preventi~ or specific therapy to lower the mortality rate (70-80%) and-'mdy-a~ early, aggressive surgical approach .-evacuating haematomas, stopping bleeding, toileting all septic, necrotic foci and restoring anatomic continuity-, seems to be of some help this complex clinical entity has not an univocal denomination yet. the proper labelling of an illness should come from the full understanding of its pathopysiology and suggest the proper treatment choice. clinical and experimental studies demonstrated that pathophysiologic mechanisms involved in the past-traumatic illness, share the same anatomo-pathological elemem: the interstitial edema, due to a generalised endothelial micro circulatory injury. this alteration, as constantly seen in polytrauma patients, develops in a few hours after trauma as a consequence of the deregulation of the homoeostatic and immune mechanisms. in fact the overproduced oxygen free radicals and r~ombinam cytokines (il1,tnf), together with the complement degradation fragments, the proteolytic enzymes and many other mediators are all strongly h~l ~ ,_he e,,j,yheha! ceils. our~osect, atim~,-bnsed on examination of autopsical specimens from 85 polytraanm patients, showed that such endothelial damage, supporting the interstitial edema, is widely and simultaneensly distributed, ensues shortly arer trauma and shows its effects in different organs at different times, only because each apparatus has different fimctienal reserves: the lung is the first organ to fail just because its ah, celocapillary membrane is one of the most delicate bodily structure, and its function is irroplace~le. we think it will be of a great help, in planning a preventive therapy, to chose a denomination focusing the physician's attention on the earl)" generalized endothelial injury and its effects, as in trauma patients it is present -even if latenflysince the first few hours. we would like to see the generalised endothelial microcircolatory injury properly highlighted when considering the best definition and the optimal nomenclature for the post-traumatic s3mdrome. the presence of interleukin (il)-8 in bronchoalveolar lavage fluid of critically ill patients correlates clinically with the development of the adult respiratory distress syndrome lards), and inhibition of il-8 in animal models can attenuate lung injury. collectively, evidence to date suggests that il-8 attracts and activates neutrophiis (pmn), which are then responsible for the capillary leak of ards. however, an alternative explanation is that il-8 is directly toxic to the endothelial cell (ec). in this study, we have hypothesized that il-8 can disrupt endothelial integrity independent of pmn. meth ods: human umbilical vein (huv) ec monolayers were cultured to confluency on collagen-coated micropore filters. to assess ec integrity, 1251.albumi n leak was quantitated by measuring the 1251 counts which crossed the monolayer, using a gamma counter. il-8 (lpg/ml) was incubated in the culture medium with 1251.albumi n for 21 hrs. the il-8 dose was not cytotoxic. to determine the involvement of protein synthesis in this process, selected monolayers were pretreated with cycloheximide (ch) prior to 11.-8 addition. statistical analysis was performed using anovmfisher plsd. we have previously shown that platelet activating factor (paf) enhances cdt8 expression and primes pmn's for subsequent 02generation. both are important steps in pmn mediated injury and are assumed to occur in concert. following major trauma non-specific pmn inflammation is activated, however, unbridled systemic pmn activity needs to be minimized. since circulating catecholamines are high early post-injury, we hypothesised that they downregu/ate cd 18 expression and pmn priming via the [3 adrenergic signal transduction pathway. methods: normal human pmns were primed with paf (10ng/ml for 5 min) or pre-treated with 10-5m of isoproterenol (i) or forskoklin (f) for 5 rain and then primed with paf. cd18 expression was measured by flow cytometry (fig.l) and 02-generation in response to 10-rm fmlp was determined as sod inhibitable reduction of cytochrome c ( fig.2 holler** and georg w. bornkamm* lymphocyte-endothelial interactions are crucial for various immune responses, including cytokine driven inflammatory processes. protein kinase c (pkc)-inhibitors on the other hand are discussed as potential cytokine antagonists. in the present study we investigated the influence of the pkc-inhibitor gf109203x on cytokine-and endotoxin induced expression of intercellular adhesion molecule 1 (icam-1) and on adhesion of lymphocytes to cytokine activated endothelial cells. we found that tumor necrosis factor alpha (tnfo0-and lipopolysaccharide (lps)-induced icam-1 expression on human endothelioma celts (eahy926) were unaffected by the pkc-inhibitor and thus appeared to be independent of pkc activation. in contrast, gf109203x significantly reduced icam-1 expression induced by interferon-y (ifn-?) and interleukin-1 (il-1). the functional relevance of these findings was evaluated in an adhesion assay using human umbilical vene endothelial cells (huvec) and peripheral blood mononuclear cells (pbmc). in fact, the ifn-? and il-1 induced adhesion of pbmc to cytokine treated huvec could be downregulated by the pkc-inhibitor, whereas tnfc~-and lps-mediated adhesion was not influenced. additionally, the il-1 driven icam-1 expression on eahy926 cells as well as the il-1 induced adhesion of pbmc to huvec was found to be tnf-dependent, since both effects could be inhibited by an anti-tnf monoclonal antibody (195f) . these in vitro data further support the idea of examining pkc-inhibitors, such as gf109203x, for their biological relevance in cytokine related dysregulations. seiffge, d., bissinger, t., laux, v., during inflammation there are some key processes, which occur in the microcirculation: the release of mediators from various cell types, the migration of inflammatory cells towards a chemotactic stimulus in the tissue, the expression of adhesion molecules on different cells, and the extravasation of plasma proteins. the aim of the present study was to elucidate the mediator induced interaction of leukocyte adhesion and plasma leakage in postcapillary venules. using an analogous video-image analysing system we have studied the effect of different mediators on leukocyte adhesion and macromolecular permeability in the mesentery of the rat. the increase in permeability was measured as changes in optical density. we found that topical administration of leneotriene b 4 (ltb4, 5x10 "6 tool/l) or intravenous injection of interleuldn-2 (il-2, 5-106 iu/kg b.w.) and lipopolysaccharide (lps, 15 mg/kg b.w.) resulted in a significant extravasation of fitc-labelled rat serum albumin (fitc-rsa) in venules but not in arterioles. we could correlate the changes in vascular permeability with a locally increased number of rolling and sticking leukocytes in venules. both effects were dose dependently inhibited by different drugs. pentoxlfylline inhibits lps-indueed fitc-rsa extravasation and leukocyte adhesion at a dose of 3 mg/kg b.w., superoxid-dismutase (sod, 10.000 iu/kg b.w.) was able to decrease the ltb 4 effect, and the immuumodulating drug leflunomide (hwa 486) exerted inhibitory effects on il-2-induced permeability at a dose of 3 mg/kg b.w.i.v. the obtained results demonstrate that lps, ltb 4 or il-2 induced extravasation of fitc-rsa is mediated by activated leukocytes and can be deminished following administration of different drugs. platelet-endothelial cell adhesion molecule-i (pecam-i), a member of the immunoglobulin superfamily, is constitutively expressed at high levels on the endothelial cell surface. in vitro data have suggested that pecam-i functions as a vascular adhesion molecule, specifically in neutrophil transmigration across the endothelium. this current work is the first demonstrating the in vivo role of pecam-1 in neutrophil migration. blocking antibodies to human pecam-1, in which the antibodies are crossreactive with rat pecam-1, were able to block the movement of neutrophils into the rat lungs after igg immune complex deposition. furthermore, when human foreskin was transplanted into mice with severe combined immunodeficiency and the site injected with tnf-alpha, anti-pecam-i blocked neutrophil emigration into the dermal interstitium. it has already been established that neutrophil recruitment is dependent upon selectin mediated rolling, followed by firm adherence that is icam-1/ integrin mediated. these data suggest, for the first time, that a third endothelial adhesion molecule (pecam-i) is involved in the coordinated recruitment of neutrophils in vivo. to test whether trauma causes generalized activation or priming of pmns, cdi8 adherence receptors were measured with iinmunomonitoring in whole blood after lps stimulation ex vivo. anesthetized (fentanyl) mongrel pigs (15-25 kg) were subjected to 40% arterial hemorrhage + soft tissue injury and after liar, resuscitated with all the shed blood + supplemental fluid. blood was collected at 24hr intervals from unanesthetized animals with indwelling catheters, pmns were counted, and lps was added (0,1,5,10 i.tg/ml) ex vivo. after 3hr incubation at 22-24°c, %cd18 (+) pmns were determined with fitc-ib4 and flow cytometry from mean channel fluorescence histograms. 49±8 # p<0.05 vs baseline * p<0.05 vs sham $p<0.05 vs no anesthesia these observations provide direct evidence for time-dependent changes in pmn priming following major injury because cd18 expression was depressed for at ]east 24hr after trauma relative to sham but by 72hr, was enhanced, relative to sham, and because fentanyl anesthesia at 72hr had a greater effect on cd18 expression in trauma vs sham. neutrophil (pmn) adhesion to vascular endothelial cells (•c) is a key element in the inflammatory response and tissue injury. inflammatory mediators such as lps (exogenous) and tnf (endogenous) can promote pmn-ec interaction which is believed to be responsible for capillary leakage and subsequent organ injury. however, the mechanism of this injury remains unclear.we hypothesised that the mechanism of tissue injury is due to ec necrosis with release of toxic products and that activated pmn are responsible. human pmn were obtained from healthy donors, separated by density gradient, and activated with lps (50ng/ml), tnf(10ng/ml), and lps/tnf(25ng/10ng/ml). cultures of the human ec tine(ecv-304) were used as surrogates of the microvasculature, were exposed to either lps, tnf, lps/tnf and pmn activated with lps, tnf, lps/tnf and incubated for 6, 12, 18, and 24hrs. ec necrosis was assessed by a 51cr release cytotoxicity assay. pmn activation was assessed by cd1 lb receptor expression and respiratory burst activity 6hr 0_+0 1.7-+ 1 0-+0 3.2_+ 1 0_+0 4.4_+ 1 0_+0 2.3_+0.9 12hr 0+0 3.24_3 0_+0 9.3_+3 0_+0 11_+3" 0+_0 12+--2.1" lghr 04-0 0.9_+2 0+_0 284-12" o:fo 25,12" 0~0 30+-9.5* 24hr 0_+0 4.14-4 0-+0 33+_3* 0_+0 30_+9* 0_+0 32_+10" data = ec % necrosis mean_+sd stats: student's t-test with significance (*) set at p<0.05 vs control. ( our previous studies have indicated that despite the increased cardiac output and maintenance of tissue perfusion, hepatoceliular dysfunction occurs during early sepsis. nonetheless, it remains unknown whether vascular endothelial cell function (i.e., the release of endothelium-derived relaxing factor/nitric oxide) is depressed under such conditions and, if so, whether endothelial cell dysfunction also occurs at the microcirculatory level. to determine this, rats were subjected to sepsis by cecal ligation and puncture (clp), following which these and corresponding shams received 3 ml/100g bw normal saline. at 5 hr after clp (hyperdynamic sepsis) or sham operation, the thoracic aorta was isolated, cut into rings, and placed in organ chambers. norepinephrine (ne, 2xi0 -7 m) was used to achieve near-maximal contraction. responses for an endothelium-dependeut vasodilator, acetylcholine (ach, via nitric oxide), were determined. in additional studies, the small gut was isolated at 5 hr post-clp. after pre-contraction of blood vessels in the isolated gut with 5xl04 m ne, vascular responses to ach (5x10 6 m) and an endotheliumindependent vasodiiator, nitroglycerine (ntg, 5xl0 -7 m), were determined. total vascular resistance (tvr, mmhg/mi/min/100g) was then calculated as pressure/ perfusinn rate. ach-induced relaxation (%, n=6/group) in the aortic rings were: ach lxl0 i~s, st-in ~ ~ significantly at 5 hr post-clp (i.e., increased *p(o 05 vs. sham; n-4 per group. tvr) in the absence of any changes in ntginduced relaxation (fig. a) . thus, the vascular endothelial cell dysfunction observed in the aorta in early sepsis also occurs at the microcirculatory level. introduction: the cytokine-mediated adherence of leulcooytes to vascular endothelium is considered as an early step in the cascade of pathologic reactions culminating in the "systemic inflammatory response syndrome" (sirs); the purpose of this study was to evaluate the influence of interleakin-1 on leukooyteendothelial cell-interactions and microoirculation in the liver after hemorrhagic shock by means of intravital microscopy. methods: in anesthetized female sprdrats co.w. 200-230g) shook was induced by fractionated withdrawl of arterial blood within 5 rain and maintained for 1 h (map at 40 mm hg, cardiac output 50 % of baseline). rats were adequately resuscitated with 60% of shed blood and twice the volume in ringer's solution additionally. following 5 h of reperfusinn (map >100 mm hg, co >120% of baseline) the microcirculation in liver lobules was examined by intravital fluorescence microscopy after labelling of leukocytes. continuous administration of il-lra (synergen, boulder, colorado, 5mg/kg/h) was started at different time points in a randomized and blinded manner. the animals in group p (n=6) received the il-lra as pretreatment beginning 5 min prior to shock induction. in the group t (n=6) the application of il-lm started at the beginning of the reperfusion period with a bolus injection of 5 mg/kg and was followed by continuons administration of 5 mg/kg/h. the control group c (n=4) received equal volumes in nac10,9%, the sham-operated group s (n=4) was not exposed to shock. results: macrohemodynamics were comparable in all shook groups. the increased percentage of permanendy adherent leukocytes after hemorrhagic shook (s: 9,1% +1,1%; c: 42,1% _+5,4%) was significantly reduced by pretreatment or treatment with il-lra (p: 16,9% -+1,9%; p<0.001, t: 28,8% -+3,6%, p<0.01, anova). temporary adhesion of leukocytes was unaffected by application of il-lra. liver microcirculation measured by volumetric blood flow in liver sinusoids and sinusoidal diameters was impaired after hemorrhagic shock in all groups and was not affected (c: 31993iam3/s +40971um3/s, p: 32768llm3/s +4445}am3/s, t: 326211ams/s -+2998 lam3/s, s: 390201am3/s -+39041am3/s). di.seu~sinn: the results demonstrate that permanent adherence of leukocytes to endothelium is in part regulated by il-1. pathological adherence could be reduced by application of illra, even given at die time of resuscitation. the effect of ll-lm on permanent adhesion is a specific event and might be caused by reduced expression of specific receptors on sinusoidal endothelial cens and leukocytes. objectives of the study. the adhesion of activated neutrophils (pmn) to endothelial ceils (ec) and the concomitant production of reactive oxygen metabolites (rom) initiates organ damage after trauma, sepsis, shock and organ reperfusion. aien of this study was to investigate the effect on adhesion and rom production of the highly water-soluble, membrane-permeable and physiological ascorbic acid (asc). materials and methods. adhesion of pmn to nylon fiber (cell count) and simultaneous rom production (chemiluminescence-cl-response) were measured up to 6 retool/1 asc as well as adhesion, rom production and ec damage (lllln-release from labeled ec) of endotoxin-activated pmn to cultered ec moanlayers. in an in vivo animal model (sheep with lung lymph fistulas) the effect of asc (1 g/kg bw bolus, followed by 0.2 g/ kg-h infusion) on the endotoxin-induced (0.5 ixg/kg bw) neutropenia (cell count), lung capillary permeability damage (lung lymph protein clearance) and rom production of neutrophils (zymosan-induced cl response) was measured. results. asc scavenged rom dose-dependently during adhesion of pmn to nylon fiber (p<0.0005 at 6 mmol/l asc), adhesion itself was unchanged. during the activated pmn/ec interaction asc scavenged rom (p<0.025 at 6 mmol/l asc) and reduced the adhesion dose-dependently (p<0.0005 at 6 mmol/l asc); ec damage was also reduced (p<0.0005 at 6 retool/1 asc). in the in rive model asc increased the endotoxin-induced blood pmn decrease (p<0.05), decreased the protein clearance (p<0.05) as well as the zymosan-induced rom production (p<0.03), indicating the asc-mediated reduction of adhesion, rom production and lung tissue damage processes. conclusions. by in vitro and in rive experiments ascorbic acid reduced the adhesion-and rom production-initiated tissue damage. therefore, i.v. administration of ascorbic acid is recommended for oxidative stress-associated states after trauma, sepsis, shock and organ reperfusion. for neut rophi l-accumulat ion and activation. we investigated the influence of or to the activation and the expression of lecam-i and cdiib,cdi8 on neutrophils and lymphocytes. methods: from blood samples (n=5) all white blood cells (wbc) and neutrophils (nc) were isolated and cultured. or were produced via the xanthine oxidase/hypoxanthine system. after 0,5, 15,30,60 and 120 minutes a giemsa-staining to determine the granulation of neutrophils (n: normal, r : reduced ) and a facs-analysis with monoclonal antibodies detecting cdiib,cdi8 and lecam-i was performed. results: under the influence of or a degranulation of neutrophils starting at 15min was observed in wbc-cultures (n/r: 0min 91/9, 15min 76/24, 30min 68/32, 60min 52/48, 120min 49/51). these data were confirmed in the dot-plots of facs-analysis. only in wbc-cultures or induced a significant increase of lecam-i expression on neutrophils up to 30min followed by a decrease to normal values at 60min. lecam-i on lymphocytes disappeared totally during the observed period. cdllb,cdl8-expression was not altered. conclusion:increased lecam-i expression on neutrophils due to or could enhance the 'rolling' of neutrophils along the endothelium which is a prerequisite for neutrophil sticking and migration. further or are able to activate neutrophils without endothelium. these changes seem to be mediated by other wbc. introduction. multiple organ failure (mof) has been hypothesized to be the result of an excessive uncontrolled autedestructive inflammatory response. since the complement system is an important mediator and initiator of the inflammatory response, interruption of this cascade could theoretically lead to an attenuation of mof. in order to test this hypothesis we evaluated the response of c5-delicient mice in a model of zymesan indt~ed mof. materials and methods. 25 c5-deficient b2d10/oid and 25 c5-sufficient b2d10/new mice were used in this study. on day 0 all mice received an intraperitoneal injection with zymosan suspended in paraffin in a dose of 1 mg/g body weight. between day 0 and 12, biological parameters (temperature, body weight and clinical condition) were measured daily and mortality was monitored. clinical condition was assessed by blindly grading the degree of lethargy, conjunctivitis, diarrhea, and ruffled fur of each mouse on a two point scale (maximum score=4). on day 12 all surviving mice were sacrificed and relative organ weights of lungs, liver, spleen and kidneys (relative organ weight= (organ weight/body weight)x100) wore calculated. earlier experiments with our model have shown a good correlation between histological organ damage and relative organ weights. statistical analysis of biological parameter was performed using the koziol curve analysis. analysis was divided in an acute phase (day 0-4) and a late phase (day 8-12). relative organ weights were analyzed using wilcoxon's test and mortality rate using fischor's exact test. results. all zymosan injected mice showed a typical triphesic illness. deterioration of the clinical condition as indicated by the symptom score and the decrease in temperature and body weight in the acute phase were all significantly lass severe in c5 deficient mice (all p<0.005). in the late phase no differences could be noticed in the courses of biological parameters. overall mortality was 2/25 (8%) in c5 deficient mice and 8/25 (32%) jn c5 sufficient mice (p=0.049), a difference mainly due to a difference in the acute phase. organ damage assessed as the relative organ weights did not show any statistical differences for any organ between both strains. conclusion. complement factor c5 appears to play an important role in the acute hyperdynamic septic response in this model but deficiency of c5 could not prevent organ damage in the late mof phase. this suggests that other factors could be more important in the development of the inflammatory response leading to mof. proinflammatory cytokines are thought to play a critical role in the pathophysiology of multiple organ failure (mof). in mice, zymosan-lnduced generalized inflammation (ztgi) leads to mof. therefore we performed a sequential study into plasma levels of, and macrophage production capacity for, four cytokines during the development of mof in the zigi model. male young-adult c57bl/6 mice received zymosan (1 mg/g body weight) intraperitoneally. groups of 14 animals were killed after 3, 6, 8, 18 and 24h and subsequently at each day until day 12. plasma was collected and peritoneal macrophages were isolated and cultured overnight with or without lipopolysaccharide (lps). interleukin 1-ct, and -6 (il-lc~,~,), and tumour necrosis factor-o~ (tnf-c 0 were measured in plasma and culture fluid by means of a ria (detection limit 0.1 ng/ml). interleukin-6 (il~) levels were assayed using the b9 hybddoma cell proliferation assay. zymosan induces a three-phase disease in mice. after an acute phase the animals recover. around day 7, they start to develop clinical signs which resemble mof. plasma tnf-~ peaked within 3h after zymosan injection and disappeared within 8h. from day 8 onwards, tnf levels started to rise again. plasma il-6 behaved almost similarly in the acute phase, but in the mof phase plasma il-6 remained low. no circulating il-16 could be detected at any time point. macrophage lps-stimulated production of il-lcq il-11~ and tnf--c~ was suppressed immediately after zymosan injection. production of il-16 and tnf-~ was normalized within 6h, while production of il-lc~ remained lower than that in macrophages from untreated control mice. only at day 6 did production of il-i~ reach control values. il-16 production was higher than control values from day 3 onwards. il6 production was similar to that of ili-il the production of tnf-ct was strongly elevated between days 1 and 6 and again during days 10 to 12. the development of mof-like symptoms during zlgi in mice is accompanied by increased plasma levels of tnf-ct without enhanced il-16 or il-6. also, the ability of macrophages to produce excessive amounts of il-16 and tnf--~, as well as the suppressed capacity to produce il-lcq could be important mechanisms in the pathophysiology of mof. when conjugated to an asialoglycoprotein, dna and oligonucleotides are specifically taken up by the hepatocytes via the asialoglyccprotein receptor which is unique to the liver. human asialoglycoprotein (~1-acid, asgp) was derivatized with low molecular weight poly(l)lysine(pll) and complexed with 5 antisense dna's (as) complementary to the 5' region of the il-6 gpl30 receptor. the antisense were 5'-agtttagggatgagg-3' (asl), 5'-atcttcatcttctgaat-3' (as2), 5'-aagtgaatgattaaaacact-3' (as3), 5'-aaacctttataggcg-3' (as4), and 5'-cgttctacaactgcaacgt-3' (as5). using hepg2, the biological effects of these antisense complexes on the high affinity il-6 receptor were evaluated by scatchard analysis, cellular proliferation, and acute phase protein expression by radioimmunoprecipitation and two dimensional gel electrophoresis. scatchard analysis demonstrated that high affinity receptor expression was inhibited by incubation of cells with asgp-pll-asi for 24 h. underivatized asl was less effective and the complex, asgp-pll-as2, had minimal effects on high affinity binding. when the cells were treated with the conjugates and stimulated with il-6 (i00 units) asgp-pll-asi alone showed a dose dependent (0.i-0.4 ~m) inhibition of 3ss fibrinogen synthesis. two dimensional gel electrophoresis showed that expression of other acute phase proteins was also blocked. these results indicate that the targeted delivery of antisense molecules via conjugates recognized by the asialoglycoprotein receptor can block the cytokine stimulated acute phase protein response in hepatocytes, this approach may be relevant to the therapeutic management of patients with severe injury and sepsis. it has been established that immune cells are able to express neuropeptide genes and to release products that were considered to be of neuroendocrine origin. we have shown that proenkephalin (penk), a neuropeptide encoding gene, is expressed in lymphoid cells in culture. to study the physiological significance of these observations we have used the model of experimental endotoxemia. in this model, a disease state is induced by bacterial lipopolysaccharide (lps), that activates the immune system, the adrenocortical axis and the nervous system. we found that the expression of penkmrna is markedly enhanced in vivo immediately after lps injection both in the adrenal glands and in the lymph nodes. in situ hybridization analysis combined with immunohisto-chemistry indicated that the induced penk expression is confined to macrephages within the lymph nodes and chromaffin cells in the adrenal medulla. furthermore, this expression in lymph nodes is modulated by ligands of the adrenergic system. our results strongly support the notion that immune derived opioids participate in the bidirectional communication between the nervous and immune systems. of neurology hadassah university hospital, jerusalem 91120, israel. objectives of the study: multiple-organ-failure is recognized as the most severe, and often lethal, complication after multiple trauma. however there is no adeqate animal model available. our goal was to develop an animal model, in which reproducable irreversible failure of parenchymal organs is achieved in the late phase after insults in the early phase (trauma). materials and methods: l0 female merino-sheep were included (mean weight: 30kg). day 0: hemorrhagic shock (mean arterial pressure (map) 50mmhg for 2 hrs.), closed femoral nailing (ao-technique), day 1-5: bolusinjection of endotoxin (et) (0,75~tg/kgbw) und zymosan-activated plasma (zap) (20ml) every 12hrs., day 6-10: observation. bronchoalveolar lavage (bal): day 1, 6, 10. the course of representative parameters of organ function was documented: cocardiac output (i/min), svr -systemic vascular resistence (dyn ~ s cm-5), pap -putm.art.pressure (mmhg), pap2 -arterial oxygen pressure (mmhg), bill -bilirubin (;xmov1), crci -creatinin clearence (ml/min) statistics: data as means+sem, *significant from baseline (wileoxon test; p<0005) results: baseline day 4 day 6 day 8 day 10 heart: co 4,96_+0,32 5,33_+0,36 5,4_+0,2 7,30_+0,77* 7,98_+0,66* svr1520_+134 1395+141 1403_+89 1119+_122" 1089+-91" lung: pap 17,0_-20,7 19,6_+1,6" 22,0+-1,2"25,8+2,0" 28,8+-1,7' pap2 103,1+1,5101,5+-5,7 97,3_+4,7 91,9+-5,6 89,8+_3,9* liver: bill 2, 94_+0, 344, 82_+1, 34' 5, 13_+0, 68'6, 13_+0, 7" 7, 19_+0, 91" kidney:crcl 86, 5+_30, 9 109, [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] 4 74, 7_+10, 868, 8+14, 1 53, 1+17, 6 histologic specimens showed all signs of fulminant mof. combination of hemorrhagic shock, femoral nailing, et und zap (insults in the early phase) lead to an irreversible organ failure in the late phase. prostaglandin e (pge) levels are elevated by trauma, shock or sepsis and can profoundly affect the immune response. pge2 is produced by many cell types including fibroblasts, macrophages, monoeytes, follicular dendritic cells, and epithelial cells and is induced by il-i, bacterial lps, components of the complement cascade, tnf, il-6 and crosslinking of surface fc receptors for igg, iga and ige. our research has shown that pge inhibits b cell activation (specifically enlargement, class ii ~c and fc~ rii expression), proliferation, igm and igg3 responses, t cell proliferation, and il-2 synthesis in the mouse model. in contrast, pge greatly promotes class switching to ige,the isotype responsible for type i allergic hypersensitivity. thus, our model mirrors th~ general immunosuppression and elevated ige titers of the trauma or sepsis patient. pge increases the number of cells secreting ige and iggl, acts on surface igm positive b cells, synergizes with il-4 and lp$ to induce preswitch germline transcripts, and induces more rapid expression of mature vdj~ mp~a than in eontro~ pge intracellular signalling occurs through cyclic adenosine monophosphate (camp) levels and can be mimicked by camp-inducing agents and blocl~ed by an inhibitor of campdependent protein kinase a. pge action requires de novo protein synthesis and candidate pge-inducible regulatory proteins have been identified by 2d gel eleetrophoresis. thus, pge inhibits a number of immune mechanisms while promoting ige production. a deeper understanding of pge immune regulation may lead to more effective treatment of immune perturbations as sequelae of trauma, shock or sepsis. during infrarenai aortic surgery mesetueric traction (re.t.) results in prostacyclin (pgi:) release and consecutively in hemodynamic disturbances (decreased systemic vascular resisteace, mean arterial pressure; increased cardiac output, heartrate). these symptomes are bypassed by cyclooxygenase inhibition. hemodynamic symptoms vanish after 30-45 rain even without cyclooxygenase inhibition although pgi2 levels remain elevated. to study the endocrine vasopressor system in a prospective double blinded protocol, we investigated 26 patients undergoing major abdominal surgery as compared to 26 ibuprofen (400 rag, i.v.) pretreated (ibu) patients. the surgeon applied m.t. in a uniform fashion. we chose a general anesthesia combined with a supplemental thoracic epidural anesthesia. at the points in time 5, 15, 30, 45, 90, 180, 360 , 1440 rain after and before (to) mesentzrie traction we determined the plasma concentrations (pc) of 6-keto-pgf~o~pr~, epinephrine, norepinephrine, dopamine, renin, aldosterone, adh and cortisol. pc of 6-k-pgf~,tp~, peaked 5 minutes after m.t. (2274_+284, ibu: 102_+12, to: 60+i ng/l) and declined monotonously over 6 h (203 +_42, ibu: 84_+ 12 ng/1). catecholamine pc "s did not exceed the reference range during the observation period. reninpc peaked after 45 rain (66_+23, ibu: 18+3, to: 16-+2/~u/ml); aldosteronc also presented a maximum after 45 rain (110 + 12, ibu: 66-+ 15, to: 56 +-7 pg/ml), whereas cortisol demonstrated irrespectively of circadian rhythms a maximum 6 h after m.t. (26+_1, ibu: 24-+3, to: 10+_1 ~g/1). adh pc peaked 5 min after m.t. (71+7, ibu: 20-+6, to: 5+_1 pg/ml) and showed analogously to 6-k-pgft~j~ pc a monotone decline over the observation period. our data demonstrate a counteractive reaction to pgiz mediated vasodilation via adh secretion. the second regulative is the renin-angiotensin-aldosterone system (raas), which is activated 45 min after m.t., the aldosterone pc does not paratlel the cortisol pc, which peaked post operafionem in both groups, probably due to the end of anaesthesia. a regulative release of catecholamines could not be documented. the activation of adh and raas after mt is not a hormonal response primaryly related to surgical trauma and/or stress but a counterregulation to systemic vasoditafion induced by prostacyclin. although adh and raas support systemic circulation, angiotensin and vasopressin may compromise local organ blood flow (e.g. splancimic vascular bed). insfitut f. klin. chemic, anaesthesiologie ~, chirurgie l*, univ. ulm, 89070 elm, expression of c-fos protein in rat brain following occlusion of superior mesenterie artery. takanobu there is general agreement that neurologic abnormalities are seen in sepsis. the aim of this study is to examine what effect does the brain receive in case of sma occlusion by immunohistochemistry using antibody to c-fos, an immediate early gene, which is recently recognized as a genetic marker of activated neurons. moreover, we investigated the correlation between c-fos induction in the brain and plasma endotoxiu level. 20 rats of them received sma clipping and others wee used as control. control and treated rats at 2, 4, 6, 8 hours were perfused and fixed. the brain were sectioned at 20 pm and stained by abc method using c-fos antibody. plasma endotoxin level of 5 rats were measured at 0, 2, 4, 6, 8 hours after the treatment by chromogenic limulus method. immunohistochemical study showed scarcely no immunoreactivity in control rat brain. in treated rat brain, the significant expression of c-los was detected in specific nuclei including the habenula, some hypothalamie nuclei, amygdala, locus ceruleus and nucleus tractus solitarii. such immunoreactivities were increased in time curse, which well corresponded plasma endotoxin levels. the mean plasma endotoxin level of 0, 2,4, 6, 8 hours after the treatment were 6.48 ± 5.57, 15.0 _-4-4.37, 10.0 _+ 4.18, 14.4 ± 3.52 and 58.4 ± 28 .6 pg/ml, respectively. the results indicate that limbic and hypothalamic-brainstem systems are involved in sma occlusion, and suggest that such neuronal actival.jon may precede the elevation of plasma endotoxin icy.el. systemic vascular resistance and increased cardiac output accompanied presumingly by a increased pulmonary shunt (qs/qt). this response is induced by prostacyclin (pgi2). we examined oxygen transport after traction on the mesentery root and the transpulmonary prostacyclin levels in a prospective placebo controlled study with intravenous ibuprofen. methods: with approval of the human [nvestigadon review board we studied 52 patients in a prospective, randomized double-blinded protocol who were scheduled for major abdominal surgery. ibuprofen (400 mg i.v.) or a placebo equivalent was administered 15 minutes before skin incision. pulmonary artery thermodilution and radial artery catheters were placed after induction of anesthesia. mt was applied in a uniform fashion. baseline values preceded the incision of the peritoneum (to). fulther assessments followed 5, 15, 30, 45, . tile plasma concentrations (pc) of 6-keto-pgft, (stable metabolite of pgi2) were determined in arterial and mixed venous blood by radioimmunoassay. at all points in time we measured arterial and mixed venous blood gases. qs/qt was calculated by standard formula. data are given as median (p < 0.05 placebo vs. [ibuprofen] [117] mmhg (*p< 0.0i). these changes were accompanied by a marked increase of 6-keto-pgf~ pc up to 180 rain after mt in arterial and mixed venous blood of untreated patients with a peak of 2450*[60] ng/l tl (*p< 0.00ol). there was no difference between arterial and mixed venous pc. ibuprofen pretreated patients (n=zr) demonstrated stabile qs/qt and pao2 while 6-keto-pgf~ pc remained within the normal range. discussion: our data clearly indicate that mesenteric traction response includes a critical rise in qs/qt followed by significant decrease of paov stable oxygen transport determinants following cyclooxygenase inhibition signify an action mediated by prostacyclin. an indicative transpulmonary gradient for 6-keto-pgft~ was not detectable. a splanchnic vascular source for pgi2 release seems to be likely, but could not be proved by our current data. department of anesthesiology, cliu. chemistry * and surgery*; university clinics uim, prittwitzstral]e 43, 89075 ulm, germany it is unclear whether injuries like bums, in general, directly result in alterations of cell-mediated immunity that, in turn, promote endotoxic and bacterial translocation or, alternatively, whether these conditions allow increased bacterial invasion that, in turn, inhibits cmi. aim: to determine whether infectious challenge, as clp alone or combined with ti causes further immune abnormalities in the days following clp. study plan: on day 1, two groups of n=36 8 week old aj mice were subjected to either a 20% scold burn (ti), or were untreated (c) n=18. on day 10, 18 mice (ti+clp) and 24 mice (clp) were subjected to clp. the two other groups (ti and c) were untreated. at days 11, 12 and 13 after thermal injury splenocytes (sp) were harvested and cultured with cona for an assay of il-2 and adherent splenocytes (as) were cultured with lps for il-1, tnf, il-6 and pge2. results: either ti + clp or clp alone result in significantly decreased secretion of all cytokines tested. in the ti group almost every cytokine production determined was elevated in comparison to ti + clp and prosmcyclin (pgi2) has been implicated in the pathophysiology of septic shock. however, pgi~'s role in the inflammatory response to sepsis is not well-defined. the purpose of this study was to identify which acute septic events are mediated by pgi 2 during graded bacteremia. methods: eleven ~nesrhetized, hemodynamically monitored adult swine were infused iv with aeromonas h. (109/ml) at rates increased incrementally from 0.2 to 4.0 mi/kg/hr over 4 hours. animals were studied in two groups: septic control (sc), graded bacteremia only (n=6); pga (n=5), graded bacteremta plus anti-pgiz antibody, 50 ml/hr iv, beginning at 2 hours. mean systemic (map) and pulmonary arterial (pap) pressures and arterial po2, mmhg, cardiac index (ci), l/min/m2, oxygen delivery index (do2i) and consumption index (vozi), ml/min/m2, and oxygen extraction (er), %, )latelet aggregometry (plt), %max., plasma pg6-keto f1 alpha ; in the first instance~ peak values of lt ~4 after i~ hrs post infarction were 6 times higher than in the controls and excess leucocyte infiltration was noted at the infarction zone. in second instance two levels of lt b4 led to weak infiltration of the infarction zone by leucocytes. a. mo~e~o, in~.~p~siolo~,d~t.e~.cardiolo~,bogotsolets4, ~ev252024, ukrmne systemic lesion$of erythron in traumatic disease and possibilities of their regulation by opioid peptides. redkin y. v., fominih s. g. using clinical (121 patients) and experimental material(128 rats and 74 dogs) we revealed general regularities of erythron lesions after hard mechanical trauma of various genesis as well as some mechanisms of development of posttraumatic anemia and possibilities of its correction with preparations of opioid peptides. the condition of central and peripheral compartments of erythron was studied with unified morphologic, immunogematological, biochemical and radiological methods. it was revealed that irrespective of the experimental animal species (dogs, rats) or in clinical experiments (patients) and irrespective of the injuring factor type (skeletal trauma, craniocerebral trauma, loss of blood) in erythron can be observed one-directed unspecific reaction realized by the considerable lowering of hemoglobin concentration, erythrocytes number and hematocrit. in the initial period (1-7 days) in the system of erythron prevail processes of distraction and elimination of er~zthrocytes relatively to the general production of stimulated erythropoiesis. the primary alterating factor is the prolonged intensification of peroxydation of membrane iipids of erythrocytes with simultaneous lowering of reserves of reduced glutathione. the distraction of erythrocytes is supported by the developing phenomena of autoallergization of organism that becomes apparent by the appearance of sensitized t cells and antierythrocyte antibodies. the intensified production of erythropoietin rules to the realization of he program of fetal and terminal (reserved) erythropoiesis. failure of erythropoiesis function is supported by disturbances of the processes of the injuring of cell metabolic apparatus. using of dalargin (15 microgram per kilogram of body mass intrap'eritoneally within 5 days after the trauma) showed the precise pharmacotherapeutic effect revealed by the diminishing of anemia of experimental rats, more . fiberbronohoscopic procedures are known to produce "peep-like" effects and to increase pulmonary artery (pa) resistance [2] . peep can affect rv function by reducing preload and ejection fraction (ef) [3] . since changes of rv function during bronchoscopy in septic patients are not reported, we measured rv parameters before, during and after fiberoptic bronchoalveolar lavage (bal). method: this 34-year-old patient (apache-ii:24) developed a hyperdynanlic septic state due to staphylococcus aureus (blood culture). we inserted a "fast response" thermistor pa-catheter (baxter-edwards) to evaluate rv performance [4] . the therapeutic procedure included volume replacement, vasopressors (dopamine 5, dobutamine 3 gg/kg/min. iv) and analgosedatior/. before bronchoscopy (olympus bf-30, od=6 mm) the patient received pancmonium for muscle relaxation. ventilation was not changed during the procedure (endotracheal tube: id=8 ram, bennett 7200a, pressure controlled mode, pm~x=25 mbar, peep=8 mbar, i:e=i:i, fio2=1.0). we measured rv enddiastolic volume (edv), stroke volume (sv), ef, heart rate (hr), cardiac index (ci) and mean pa pressure (mpap gerlach h, gerlach m, clauss m, falke kj renal hypoxia and/or ischemia initiates the development of a deteriorated medullary perfusion based on fibrin deposition in the peritubular capillaries, vasoconstriction, and perivascular edema, which is followed by a swelling of the tubular epithelial ceils, intraluminal tubular obstruction, and a backleak of fluid through the injured tubules into the renal interstitium, finally leading to an acute tubular necrosis (atn) [1 ], clinically diagnosed as acute renal failure (arf). one important pathway for induction of enhanced vascular procoagulant activity and permeability is based on the synthesis and expression of macrophage-derived cytokines, which bind to specific endothelial cell surface receptors. we recently described the identification and purification .of a new 55,000 dalton polypeptide, which is synthesized and expressed by murine macrophages after stimulation with lipopolysaccharide, and exerts procoagulant activity on cultured endothelial cells [2] . in the presented study, we demonstrate that the new polypeptid is also synthesized by macrophages under hypoxic conditions. the protein binds to specific receptors, which are expressed by endothelial cells dependent on the environmental oxygen tension. animal studies were performed after approval by the local committee for animal safety; the animals were anesthetized, treated and supervised in accordance with the guidelines of this committee. in contrast to other authors, who performed long-term hypoxia experiments in awake animals, we preferred to implement the studies under anesthesia for ethical reasons, although regulatory functions for ventilation might be influenced. animal studies demonstrated that the intravenous injection of the polypeptide initiates fibrin formation in the peritubular vessels. keeping the animals under hypoxic conditions induces similar effects, which are reduced by a rabbit-antiserum against the new protein. in conclusion, the new polypeptide obviously contributes to the pathogenesis of acute renal failure by tubular necrosis during and after hypoxic events. the use of verapamil as cardioprotective agents for management of patients with acute ischemic/reperfused heart is based on the assumption that the increased intracellular ca+ level is a key factor in causing cell death. our in vitro study was designed to focus on effects of verapamil on the metabolic potential of cardiac slices after reversible ischemia in rats. the material consisted of two main groups : group a (non ischemia/reperfusion group) and group b (ischemia/reperfusion group), each is subdivided into two subgroups (a and b). each subgroup included 10 rat hearts. group aa is the control group, group ab is verapami] added group. group ba is ischemia group without verapamil. group bb is verapamil added group. ischemic cardiac slices were obtained from rats subjected to 15 min. haemorrhage to induce reversible global ischemia. both nonischemic and ischemic cardiac slices were placed in well oxygenated krebs ringer phosphate buffer containing 150 mg% glucose & 5 gm% bovine albumin and incubated in dubnoff shaking water bath for 60min at 37°c the results revealed that there was an enhancement in release of free fatty acids (ffa) (240%) and lactate (163%) and in glucose uptake (160%) in group ba as compared with group aa. these metabolic alternations produced by ischemic cardiac slices were reversed by verapamil addition (200 ml%) but in group ab verpamil did not alter the release of ffa & lactate from non-ischemic cardiac slices, whereas it inhibited glucose uptake from these slices by 67%. the improvement of the metabolic intervention of ischemic myocardium indicates that verapamil may be of importance in reducing the extent and severity of acute myocardial ischemic injury in acute haemorrhage. severe endothelial dysfunction occurs following injury to carotid arteries which is characterized by a decreased ability of these arteries to dilate when challenged with ach or a23187, but not with a direct vasodilator (nano2). this failure to relax to ach and a23187 reflects an inability of endothelium to generate edrf, but relaxation recovers gradually to control values by 2 weeks. exogenous no donors (e.g., c87-3754 or spm-5185), accelerate the recovery of the injured endothelium in rat carotid arteries. intravenous infusion of an no donor (30 p.g/day) with an implanted osmotic pump significantly accelerated the recovery of regenerated endothelium to produce edrf at 7 days. rat carotid artery rings relaxed only 17 + 5% and 15 + 5% to 10 gm ach in vehicle treated rats and in inactive no donor treated rats respectively 7 days following injury compared with 67 + 6% in no donor rats (p<0.001). relaxation to 100 gm nan02 was normal in all groups indicating that the differences in relaxation were not the result of damage to vascular smooth muscle. contraction to l-name (1 mm) was markedly reduced by injury, but was protected by no donors (p<0.01). thus, exogenous no donors enhance the ability of the endothelium to regenerate and to release edrf in response to endothelium-dependent vasodilators. this may be due to an anti-proliferative and anti-mitogenic effect of no on vascular smooth muscle cells, allowing the endothelium to regenerate without intimal thickening. no also has been shown to inhibit platelet aggregation, and to attenuate neutrophil adherence and activation. the superoxide scavenging effect of no is not the basis for these effects since hsod is inactive in preserving endothelial function in injured arteries. thus, no exerts a variety of cytoprotective effects which may be of importance in protecting against vascular injury. much evidence has now accumulated to show that the excess production of the vasodilator nitric oxide (no) in sepsis is an important contributor to the hypotension and multiorgan failure characteristic of this condition. various cytokines play an important role in this process through their ability to induce the production of one of the enzymes responsible for no synthesis, the inducible no synthase (inos). we have studied the effects of cytokines on the induction of this enzyme both in vitro using vascular smooth muscle cells, and in a murine model of gram-negative sepsis. tn smooth muscle ceils, the cytokines il-1, ifnq', and tnf-oc show strong synergy with one another in the production of inos. in order to define the molecular basis for this synergic effect, we have linked the promoter of the inos gene to a "reporter" gene, chloramphenicol acetyl transferase (cat), and transfected these constructs into vascular smooth muscle cells. assays of cat activity reflect the activity of the promoter in this system, and by generating sets of deletion mutants of the promoter sequence we have been able to define the area within the promoter which mediates the synergic effect of these cytokines. in addition to stimufatory effects on inos production, certain cytokines are able to down-regulate the production of inos in vascular smooth muscle cells, and the effects of these counterregulatory cytokines will be discussed. the interaction of these cytokine effects in the whole organism has been studied in a murine model of gramnegative sepsis. widespread induction of inos occurs in this model as assayed by enzyme activity and through use of specific antisera to inos. neutralizing antibodies to tnf-~ and tfn-y are both able to prevent death in this model, but it is only the anti-ifn-y which attenuates the induction of inos assayed in the liver. clearly there is some redundancy in the effects of cytokines on the production of inos in sepsis, and greater understanding of the most important factors in inos production is required in order to target anti-cytokine therapy most appropriately. effects of nitric oxide on hepatocyte metabolism in inflammation. j. stadler, department of surgery, tu mqnchen, frg hepatocellular nitric oxide (no) synthesis is induced by proinflammatory mediators such as tumor necrosis factor, interleukin-1 and interferon gamma or by bacterial toxins such as lipopolysaccharide. stimulation of the hepatocytes (hc) with a combination of these agents leads to an output of no in quantities which are not seen in any other celltype. it has been demonstrated by various investigators that important effects of these cytokines and bacterial toxins on hc metabolism can be attributed to the action of no. in contrast to other celltypes hc seem to be relatively resistant to suppression of basic metabolic functions such as energy metabolism by no. therefore, cell damage has not been described to a significant extent following exposure to no. however, no does inhibit total protein synthesis. the exact biochemical mechanism of this phenomenon has not been uncovered yet, but it has been demonstrated for some specific proteins that their production is inhibited at a posttransscriptional level. as in many other celltypes cgmp generation is elevated in hc by no through activation of the soluble guanylate cyclase. cyclic gmp may possibly exert a plethora of metabolic functions, but it is interesting to note that most of the cgmp seems to be transported out of the cell. some very specific effects of no on hc metabolism include the inhibition of the glyceraldehyde-3-phosphate dehydrogenase (gapdh) and the cytochrome p450 (cyp) enzymes. inhibition of gapdh activity is mediated through nitrosylation of critical domains of the enzymes by no which enhances auto-adpribosylation. this effect on gapdh activity might be responsible for the inhibition of gluconeogenesis by no, which has been described recently. finally, no-mediated inhibition of cyps may help to explain the suppression of hiotransformation processes which is a characteristic featur,'~ r ~ "~flamed liver. nitric oxide (no) is an endogenous inhibitor of polymorphonuclear leukocyte (pmn) adhesion which limits pmn-endothelial cell interactions under normal conditions. we have previously demonstrated that following ischemia, no production by the vascular endothelinm is dramatically reduced. accordingly, we investigated the effects of no-donors on pmn accumulation and tissue injury following hemorrhagic shock and ischemia. hemorrhagic shock was induced in anesthetized rats by bleeding to 35 mmhg for 3 hours followed by reperfusion. segments of superior mesenteric artery (sma) were isolated and suspended in organ baths. in rats receiving saline sma relaxation to acetylcholine (ach, 100 nm) was reduced by 80% compared to control sma segments (p<0.01) while relaxation to sodium nitrite (100 gm) was unaffected. in addition, mesenteric tissue pmn accumulation as determined by myeloperoxidase (mpo) activity was significantly elevated compared to controls (p<0.0l). interestingly, treatment with the no-donating agent, s-nitroso-n-acetylpenicillamine (snap) significantly preserved sma relaxation (p<0.01), attenuated mesenteric mpo (p<0.05) activity, and significantly improved survival compared to saline vehicle. in anesthetized, open-chest dogs we investigated the cardioprotective actions of a novel no-donor, spm-5185 (schwarz pharma), following regional myocardial ischemia (1 hour) and reperfusion (4.5 hours) . treatment with spm-5185 (500 rim) significantly reduced myocardial necrosis by 70% (p<0.01) compared to an no-deficient analog of spm-5185, spm-5267. furthermore, mpo activity within the ischemic-reperfused zone was also significantly (p<0.01) reduced following treatment with spm-5185 compared to spm-5267 (1.6 + 0.5 vs. 3.8 + 0.3 u/100 mg tissue). these data strongly suggest that no is a potent inhibitor of pmn-mediated tissue injury following hemorrhagic shock as well as in acute myocardial ischemia-reperfusion injury. overproduction of nitdc oxide (no') may contribute to sepsis-induced hypotension. during septic shock, excess no" is produced by an isoform of nitric oxide synthase (nos) which is induced by inflammatory mediators. nonselective nos inhibitors have been proposed as a new therapeutic approach to treating hypotension in septic shock. we studied the differential hemodynamic effects of n~-methyi-l-arginine (l-nma), a nos inhibitor, in normal canines versus those challenged with endotexin (lps) and compared the activity of this drug across the venous, pulmonary and systemic vascular beds. awake canines were challenged with lps (0 mg/kg, n=15:8 mg/kg, n=21 ; or 16 mg/kg, n=30) and treated with l-nma (0, 1,2, 4,10 mg/kg/hr) for 22 hours following a 0, 10, or40 mg/kg loading dose. animals were resuscitated with iv ringers solution (10 ml/kg/hr). hemodynamic data were collected at 0, 2, 6, 10, 14, and 22 hours using intravascular catheters and radionuclide heart scans and analyzed by anova. in both normal and endotoxemic animals, l-nma at all doses studied similarly increased mean arterial pressure (p=0.07), and systemic vascular resistance index (p=0.ol) and decreased cardiac index (p=0.05) and oxygen delivery index (p=0.01). in contrast, the effect of l-nma on mean pulmonary artery pressure, central venous pressure, pulmonary capillary wedge pressure, and pulmonary vascular resistance index was greater in lps-challenged canines compared to normal animals (p<0.05), but this differential effect on the venous and pulmonary circulation occurred, >6 hours after lps challenge. l-nma did not significantly increase survival rates or times at any of the doses studied (1, 2, 4, or 10 mg/kg/h) in either the low (8 mg/kg) or high dose (16 mg/kg) lps-challenge groups. a nonsignificant (p>0.2) trend toward a beneficial effect on survival ol low dose l-nma (1 mg/kg/h) in animals given the 8 mg/kg lps-cha[lenge was not enhanced by increasing the lethality of the model or by administering higher l-nma doses. at the highest l-nma dose used in this study (10 mg/kg/h), survival time decreased significantly for both the low and high dose lps-challenge animals (p<0.05). this increased mortality was not explained by changes in plasma concentrations of either lps or tnfc~. thus, l-nma did not have a greater effect on the systemic arterial circulation in endotoxemic compared to normal canines. however, in the venous and pulmonary vascular beds, the effect of l-nma increased with time after endotoxin-challenge these data suggest the induction of nos activity by endotoxin in canines may be relatively greater in venous and pulmonary vessels compared to systernic arteries. l-nma, a nonselective nos inhibitor, did not decrease mortality in endoloxemic canines and the highest dose studied was harmful. pulmonary hypertension (ph) and arterial hypoxemia are characteristic features of the adult respiratory distress syndrome (ards). reducing pulmonary vascular pressures may promote the resolution of pulmonary edema. intravenously infused vasodilators lower ph in ards, but, as a result of their general vasodilatatory effects, systemic mean arterial pressure may also decrease. furthermore, blood flow may be increased to non-ventilated or poorly ventilated lung areas resulting in a rise of intrapulmonary shunt, thus causing a further fall in pad 2. recently, short term inhalation of low concentrations of the gas nitric oxide (no), an endogenous endothelium derived relaxing factor, which is rapidly inactivated in blood by hemoglobin, was reported to decrease ph without causing systemic vasodilation in sheep [1]. similar changes have been observed in patients with severe ards during repeated short term inhalation of no (18 and 36 ppm), which rapidly and selectively decreased the mean pulmonary artery pressure (pap) and, in contrast to intravenously infused prostacyclin, induced a remarkable increase of pad2 [2] . this improvement in oxygenation was caused by a redistribution in blood flow away from intrapulmonary shunt areas to normal ventilated lung regions. continuous no inhalation (5-20 ppm) consistently lowered the pap and augmented the pao2/f.o 2 for up to 53 days. no negative side effects were observed during the whole time span examined. in particular methemoglobin levels always remained below 1.5%. following these investigations, it could be shown that these effects may also occur using concentrations in the parts per billion range [3] , which may reduce possible toxic side effects. however, in the same study it was demonstrated that the dose-response curves for pa02 and pap have different patterns. whereas pap presented a continuous dose-dependent downward tendency with an eds o of approximately 2-3 ppm, the improvement of oxygenation had a maximum at 10 ppm and, at higher doses, drifted back towards the baseline data. the ed~o was estimated at approximately 100 ppb, i.e. more than ten times lower than for the reduction of pap. in conclusion, inhalation of no by patients with severe ards may result in persistent and reproducible decreases in pap associated with an evident improvement in pad2, thus allowing reduction of the f.o 2. no inhalation should be performed using low concentrations which are less toxic, although any possible risks still have to be considered carefully. dose-response studies for the individual patients are recommended urgently. finally, controlled randomized studies are required to demonstrate that additional no inhalation is able to reduce mortality of ards. inhibition of the activity of glyceraldehyd-3-phosphate dehydrogenase (gapdh), an enzyme of the glycolysis/gluconeogenetic pathway, through adp-ribosylation is promoted by nitric oxide (no). since no is produced in the septic liver and hypoglycemia is a major problem of late sepsis, it was investigated whether no interferes with gluconeogenesis of hepatocytes. hepatocytes (hc) were isolated from sprague-dawley rats using a collagenase perfusion technique and differential centrifugation. exogenous no was applied by incubation with the no-donors s-nitrosyl-acetylpenicillamine and sodium-nitroprusside. endogenous no synthesis was induced by incubation with cytokines (tnfcq il-1, ifnj and lipopolysacchafide (lps). 24 hrs later the incubation medium was changed to a solution containing lactate, ornithine, lysine, ammoniumchloride and glucagon for optimal conditions of gluconeogenesis. after 3 more hrs glucose and nitrite levels were determined spectrophotometrically. gapdh activity was measured by the nadh-dependent conversion of 1,3-diphosphoglycerate to glyceraldehyde-3-phosphate. incubation of hc with no-donors led to a concentrationdependent inhibition of gluconeogenesis and gapdh activity. however, gapdh activity was about 5 times more sensitive to the inhibitory effect of exogenous no. incubation of hc with cytokines and lps induced nq synthesis as measured by an increase in nitrite concentrations. endogenously produced no suppressed gluconeogenesis by 49_+3%. in contrast to exogenously applied no, the effect of endogenous no synthesis was less on gapdh activity resulting in an inhibition of only 32_+12%. in conclusion, exogenous and endogenous no inhibited gluconeogenesis as well as gapdh activity. however, there was no correlation between the extent of inhibition of these two parameters of hepatocellular glucose metabolism. we have shown that inhibition of hepatocyte (hep) synthesis of nitric oxide (no) potentiates cell injury in a model of acetaminopheninduced oxidative stress and the extent of damage was paralleled by depletion of reduced glutathione (gsh) stores. to clarify the role of no in modulating the redox state of hep, we studied the effect of inhibition of cytokine-mediated no production on hep gsh stores, in a system of isolated rat hep in primary culture, no synthesis was induced (stim) by exposure to il-1, tnf, ifn, and lps for 16 hours. 20, 60, 120 and 200 ~m of n-monomethyi-l-arginine (nmma), a specific inhibitor of no synthesis, was added. cells incubated in media alone served as controls (cont). the no metabolite (no2); aspartate aminotransferase (ast), an indicator of cell injury; and gsh were assayed. (data presented as mean + sem; n=4.) gsh (nmovma orotein) ..~ (nmol/ma orotein) cont 14.1 + 0.3 32 + 4.0# stim 13.4 + 0.6 139 + 12 stim+2o tzm nmma 12.7 + 1.1 88 + 5.0# stim+60 ~m nmma 9.9_..+ 0.4* 68 + 4.5# stim+120 pm nmma 7.6 + 0.6* 57 + 3.0# stim+200 )lm nmma 5.0 + 0.5* 37 + 2.5# anova 0,0002 0.0001 (* p < 0.05 versus stim, # p < 0.01 versus stim; anova with neuman-keuls) gsh in cont+120 i~m l-nmma was equivalent to that of cont (13.3 vs.14.1). ast release was equivalent in all treatment groups. these data show that inhibition of hep synthesis of no depletes intracellular stores of reduced gsh. we conclude that hepatocyte no production modulates cellular gsh homeostasis and as a result, may be hepatoprotective in oxidative injury. nitric oxide (no) is a modulator of immune response and may be involved in the changes in immune reactivity after major trauma and operations. we investigated no-generation in rat and mice spleen cells (sc) after partial hepatectomy (ph). c57bl/6 mice and lew rats underwent a 50% and 70% ph, respectively. sc were prepared 1-6 days after ph and plated at 1 to 2 x 10ecells per well. after 20 h incubation at 37°c, no-production was measured as nitrite levels (griess reagent). normal mouse sc did not produce no, neither basal nor in response to lps or con a starting at the second day after ph, we found a substantial production of no. in rats, also sc from control animals were able to generate no; both basal and stimulated no-generation were further enhanced after ph (table, values expressed as mean --se). after shame operation, there was only a modest elevation of noproduction in rat and mouse sc. in first experiments we could demonstrate no-production also in phagocytes from a patient 3 days aider liver partial resection (1.4 nmol nitrite/106 cells) enhanced no-production in macrophages may contribute to the changes of immune reactivity after partial hepatectomy. nitric oxide (no) is recognized as an important mediator in endotoxemia and sepsis. increased synthesis of no has been demonstrated in septic humans and animals, and no inhibitors have been used in the treatment of septic shock. recent reports have, however, suggested that this form of therapy may cause serious organ damage. in the present investigation circulatory and metabolic changes in the liver were studied during treatment with the no-synthase inhibitor n-nitro-l-arginine-methyl ester (l-name) in endotoxemia. methods: juvenile pigs were randomized to one of the following treatment groups: 1) encletoxin and l-name, 2) endotoxin, 3) naci and l-name, 4) nach preliminary results from groups 1 (n=7) and 2 (n=8) are presented. catheters for pressure measurement were introduced into the aorta, hepatic and portal veins and ultrasonic transit time flow probes were placed on the hepatic artery and portal vein. a catheter was introduced into the pulmonary artery. endotoxin (1.7 gg/kg/h) was given as a continous portal infusion over the entire observation period of 8 hrs. l-name (25 mg/kg) was given as a bolus after 3 hrs. of endotoxemia. results: endotoxin transiently reduced portal vein flow (pvf) by 25%* and hepatic artery flow (hal e) by 45%*, while l-name caused a further and lasting reduction in flow (pvf 78%, haf 90%)*. transhepatic (portal-hepatic vein) vascular resistance increased to 3 times baseline value during endotoxemia while l-name caused a further marked increase in resistance to 12 times initial value. portal oxygen saturation (so2) decreased by 60%* during endotoxemia. l-name caused a reduction in portal so2 by 87%*. arterial so2 was unchanged in both groups. hepatic oxygen uptake was not changed by endotoxin, but was markedly reduced after addition of l-name. endotoxin caused a 27% reduction in cardiac output (co). the addition of l-name reduced co by a total of 71%*. *: p < 0.05. conclusion: is the present model of endotoxemia treatment with the nitric oxide synthase inhibitor l-name markedly reduced liver perfusion and portal oxygen supply. this might explain the increased liver damage reported in previous studies using no-inhibitors. the increase in transhepatic resistance found after l-name treatment will tend to cause pooling of blood in the splanchnic veins, resulting in reduced filling of the heart and thus contribute to the observed reduction in cardiac output. institute for surgical research, rikshospitalet, the national hospital, university of oslo, 0027 oslo, norway. we have investigated the role of tumour necrosis factor (tnf) and interleukin-i (il-i) in the induction of nitric oxide synthase (nos) by bacterial endotoxin (lipopolysaccharide; lps; 2 mg kg -i i.v.) in vivo. in anaesthetized rats, pretreatment with a monoclonal antibody for tnf (tnfab; 20 mg kg -i s.c., at 16 h prior to lps) or with an il-i receptor antagonist (il-ira; 16 mg/kg bolus and 2.4 mg/kg/h infusion) ameliorated the fall in mean arterial blood pressure (map) at 90-180 min after lps. for instance, endotoxaemia for 180 min resulted in a fall in map from 114-+6 (control) to 79-+4 mmhg (p<0.01; n=8). in contrast, animals pretreated with tnfab or il-ira prior to lps injection maintained significantly higher map at 180 min when compared to lps-control: 118-+3 mmeg (n=5) and 103-+7 mmhg (n=5), respectively (p<0.01). three hours of endotoxaemia significantly reduced the contractile effects of noradrenaline (na) in the thoracic aorta ex vivo. the hyporeactivity to na was partially restored by in vitro treatment of the vessels with ng-nitro-l-arginine methyl ester (l-name, 20 min, 3x10 -4 m). pretreatment of rats with tnfab or il-ira significantly (p<0.05) prevented the lps-induced hyporeactivity of rat aortic rings ex vivo. l-name did not alter or only slightly enhanced the contractions of aortic rings obtained from tnfab or il-ira treated lps-rats, respectively. at 180 min after lps there was an induction of calcium-independent nos activity in the lung (5.14-+0.57 pmol citrulline/mg/min, n=8), which was attenuated by tnfab and !l-ira by 37-+6% and 46-+6%, respectively (n=5; p<0.05). thus, the production of both tnf and il-i contributes to the induction of nos by lps in vivo. the protective effect of agents which inhibit the release or action of tnf or il-i in shock may be, in part, due to inhibition of nos induction. neal garrison, md objective: sepsis is often accompanied by organ dysfunction, in part due to impaired microvascular perfusion. recently, nitric oxide (no) has been described as an important mediator of the hemodynamic changes of sepsis, and no synthase (no-s) inhibitors have been advocated for treatment of septic shock, but their visceral microcirculatory effects are inadequately characterized. we postulated that no-s inhibition would exacerbate the impaired organ perfusion of sepsis. methods: six groups ofdecerebrate rats were studied. bacteremia was induced with 109 live e. coli, which consistently increased cardiac output 15-20% above baseline (bl). the no-s inhibitor nm-nitro-larginine methyl ester (l-name,1 mg/kg iv), prevented this increase and elevated map by 25-30%. in the first 3 groups, total hepatic blood flow (thbf, ml/min by time transit flowmetry) and microvascular perfusion (mi-ibf, ¼ bl by laser doppler flux) were measured. in the other 3 groups, in vivo videomicroscopy was used to observe renal microvascular responses (ila=interlobular artery, aff=afferent arteriole, eff=efferent arteriole; % bl for all). results: data are 60 rains after e. cob. n=5-7/group. * p<0.05 vs bl by remanova and § p<0.05 vs e. coli alone by anova. ec+l-name 11-+2 -57_+10" § -45_+4* § -89_+3* § -33+5* -20+5* § conclusions: l-name administration in controls decreased renal blood flow, indicating no contributes to basal renal tone. bacteremia decreased mtlbf but not thbf, and mi-ibf was further impaired by no-s inhibition. e. coli caused renal preglomemlar, but not postglomerular constriction and reduced flow. l-name exacerbated these e. coli-induced alterations and caused eff constriction. these data indicate that no-s inhibition exacerbates bacteremia-induced impairment of renal and hepatic blood flow, suggesting that no is an importam compensatory dilator mechanism in these organs during sepsis. irf (iron responsive factor) is the central regulatory protein of intracellular iron metabolism able to bind to responsive rna elements (ires) present atthe 5'untranslated region (utr) of ferritin mrna and 3'utr of transferrin receptor mrna. binding of irf to ires results in repression of ferritin mrna translation and increased stability of transferrin receptor mrna leading to enhancement of transferrin receptor translation. we describe here that either tetrahydrobiopterin dependent stimulation as well as cytokine (ifn-~)/lipopolysaccharidemediated induction of nitric oxide synthase activates irf, which is due to direct interaction of nitric oxide with the iron-sulphur-cluster of irf. this was shown by gene expression studies using a plasmid containing a ferritin ire and a cat indicator box which was transfected into k562 myelomonocytic cells, which were shown to have a constitutive form of nitric oxide synthase (nos). furthermore, the increased binding of 1re to irf due to irf activation of irf by nitric oxide was demonstrated by gel shift assays. irf activity was much more increased in cellular extracts from murine macrophages (j774) where a cytokine inducible form of nos has been characterized earlier as compared with irf activity in k562 cells, where nos was stimulated by increasing the availability of the essential nos cofactor 5,6,7,8-tetrahydrobiopterin. we then demonstrated that activation of irf by nitric oxide is accompanied by alterations in ferritin translation as checked by metabolic labeling and immunoprecipitation. these results suggest a reasonable mechanism for the regulation of iron disturbances under chronic inflammatory disorders, characterized by increased concentration of immune activation parameters like ifn-5or neopterin and low serum iron and hemoglobin concentrations. taken nitric oxide, no, the putative endothelial derived relaxant factor, edrf, has been shown to be a potent inhibitor ofplatelet aggregation in vitro. in vivo evidence however, is scarce. accumulation of platelets in the lungs has been shown to occur during extracorporeal circulation. the aim of the present study was to investigate the effect of inhaled no on this reaction. materials and methods: the animals were divided into two groups, each consisting of 7 pigs. platelets were selectively labelled with luln-oxine. dialysis was instituted via catheters in the femoral vessels. in group 1, no, 50 ppm, was added to the inhaled gas from the start of dialysis. in group 2 no was not given. the activity over the lungs was followed dynamically with a gamma camera. central hemodynamics was monitored via a swan -ganz catheter. results: the activity was significantly lower in group 1, from 2 minutes after start of dialysis and onwards, indicating diminished accumulation of platelets in the lungs. parallel to this the hemodynamic response in terms of increased pulmonary artery pressure and pulmonary vascular resistance was blunted in this group conclusion: inhaled no in this model seems to affect pulmonary platelet sequestration. an associated attenuation of the changes in central hemodynamics was also seen. previous studies from our laboratory have demonstrated that vascular contractility decreased in endothelium-intact blood vessel rings in early and late stages of sepsis. although endothelium removal in early sepsis restored vascular contraction, the depressed smooth muscle contractility observed in late sepsis was not restored by endothelium removal. this indicates that impairment of smooth muscleper se may be responsible for such dysfunction in late sepsis. the aim of this study, therefore, was to determine whether or not smooth muscle-derived nitric oxide (no) plays a role in producing vascular smooth muscle dysfunction during late stages of sepsis. to study this, rats (250-300g, n=4-5/group) were subjected to sepsis by cecal ligation and puncture (clp). septic and shamoperated rats then received 3 rrd/100g bw normal saline. the animals were killed at 10, 20, or 35 h post-clp (10 h post-clp=early sepsis; 20-35 h post-clp=late sepsis), and thoracic aortic rings were prepared for contraction studies using organ chambers. the complete removal of endothelial cells was tested by the absence of any significant acetylcholine-induced vascular relaxation. contractile responses to norepinephrine (ne, 10 9 to 10 -5 m) were determined in the aortic rings without intact endothelium. ng-monomethyl-l-arginine (l-nmma, 300/~m, an inhibitor of no synthase) was then added to the organ chamber and ne-induced peak contraction was determined before and after the addition of l-nmma. the peak contraction (rag/rag tissue, mean_+sem) is shown below: the results indicate that the addition of l-nmma did not significantly affect ne-lnduced peak contraction in endothelium-denuded vessel rings at 10 and 20 h after clp. in contrast, l-nmma administration produces an 18% increase (p<0.05) in peak contraction during late sepsis. therefore, the vascular smooth muscle contractile dysfunction observed at 35 h post-clp is partially due to smooth muscle-derived no over-production. thus, unlike macrophages in which inducible nitric oxide synthase (inos) is observed in early sepsis, the inos in vascular smooth muscle appears prominent only in the late stages of sepsis. in three cases of human septic shock in which ng-monomethyi-l-arginine, (l-nmma) a nitric-oxide-synthase-inhibitor was applied, we isolated three completely different types of pathogens: candida, pseudomonas aeruginose and multiresistant coagulase-negative staphylococci. this observation suggests that endotoxin alone is not the main factor triggering hypotension in septic shock by the nitric oxide pathway. in a 68-years-old woman in severe septic shock due to a candida and pseudomonas aeruginosa infection complicated by adult-respiratorydistress-syndrome conditions deteriorated despite adequate conventional therapy. in this trial, effects of l-nmma on cytokin-levels were investigated. the study-protocol was approved by the ethical committee of the department of surgery. after two boll of 200 mg of l-nmma, a continuous infusion was installed (0.05 mg/minute and kg body weight l-nmma). as expected mean arterial blood pressure rose (62 to 134 mmhg}, heart rate stayed stable (124 + 4 b/rain), systemic vascular resistance increased (225 to 354 dyne.sec/cm5), cardiac output decreased (17 to 15.2 l/rain), and cardiac index declined (9.94 to 8.63 l/min/m2}. before and after 90 minutes while the infusion of l-nmma, blood samples for immunological measurements were taken and processed together. pulmonary-shunt-volume was observed before the application of l-nmma, after one hour and after 140 matutes. neopterine increased from 16.51 to 32.55 ng/ml, tumour-necrosis-factor-a increased from 24.16 to 36.61 pg/ml and intedeukin-6 increased from 61.9 to 98.2 pg/ml. immunoglobulines a, g, and m (3.2 to 2.9, 8.1 to 7.6, 1.1 to 0.9 g/i), complement factor c-3c and c-4 (0.54 to 0.50, 0.22 to 0.23 g/i), alpha-l-antitrypsine (3.86 to 3.83 g/i), c-reactive-protein (111.1 to 105.6 rag/i), interleukin-1 (0 pg/ml) and soluble interleukin-2 (2128 to 1983 units/ml) did not change significantly. pulmonary-shuntvolume decreased from 54.1% to 35.4% within one hour and to 36.6% after 140 minutes. in septic shock blocking nitric oxide as an intervention at the end of a not ~,et ful!y understood cascade might have important influences on pulmonary-shunt-volume and inter-cell-communication. department of surgery, pharmacy* and immunology**, university hospital of zurich, r~imistrasse 100, 8091 zurich, switzerland we previously reported that hypoferremic cba mice had an increased resistance to salmonella infection, and that injection of ammonium ferric citrate (afc) to these mice led to enhanced infection (ganthier et at. 1986. microbiol.immuno130:425) . because nitric oxide (no) is involved in the antimicrobial activity of routine macmphages towards various inttacellular pathogens, we investigated the influence of iron on the bactericidal activity of cba mouse macrophages towards s.typhimurium and on the production and activity of reactive nitrogen intermediates (rni). peritoneal macrophages hum cba mice were cultured in the presence (or not) of afc 50 ,um, ifn-,/250 u/ml, lps 1 fig/m/, ngmonomethyl-l--arginine (mmla) 2ram. nitrite (no2-) content of the supematants was determined by a standard griess reaction, and h202 release was measured by the peroxidese dependant oxidation of phenol red. for intracellular killing, macrophages monolayers were infected, and, at various intervals, lysed by triton x-100, and surviving bacteria enumerated by colony counting on agar. for in vivo experiments, mice were infected ip with 0.5 ml of a suspension of 5.10 ~" s.typhimurium, strain c5, and injected with aminoguanidine (ag) 1 mg/ml in saline. our results show that the rn[ inhibitor ag strongly accelerates the mortality of infected mice, the survival rate decreasing from 60% in the control group to 20% in the treated group, 10 days after challenge. correlatively the rni inhibitor mmla induces in vitro a decrease in the rate of bacterial killing, fxom 78% to 57%, in macrophages triggered with ifn-? + lps. the cultivation of macrophages in the presence of afc leads to a decreased no 2-accumulation, 4.7 nmole/well v.s. 21 nmole/well. conversely h202 production is enhanced from 09 nmole/well up to 2,33 nmole/well. nevertheless, macrophages cultivated in the presence of afc exhibit an increased tale of intracellular killing, 77% in iron exposed macrophages v.s, 68% in control macrophages. when triggered with ifn-~, alone, macrophages have a reduced antibacterial activity (57% v.s. 68%) whereas the addition of afc to these macrophagas restores an elevated (72%) rate of killing. in conclusion, the results show that bactericidal activity of cba macrophages towards s.typhimurium depends on the production of no by these macrophages ; but they also demonstrate that no is not the only reactive species involved in the intracellular kil/ing of s.thyphimurium ; indeed afc which strongly inhibits rni production, stimulates h20 2 release by these macrophages and increase their bactericidal activity in vitro. nevertheless afc may promote bacterial growth in vivo. crssa. unit4 de microbiologie. bp 87. 38702 la tronche cedex france. henning jahr, ulrike noack, karin braun the large amounts of no produced by the inducible no synthase in rat macrophages have direct antimicrobial effects, but inhibit the activation of the lymphocyte-dependent host defense system. the aim of this study was to investigate if complement activation influences no-generation. spleen cells from lew rats were incubated at 37°in tcm-199/5% fcs, with or without additional rat serum. after 20 h, nitrite (end product from no metabolism) was measured by oriess reagent. in rat spleen cell preparations, most of the no is produced by macrophages. complement activation in vivo was carried out by i.v. injections of 240 u cobra venom factor/kg b.w. at days 0 and 2. significantly higher (p<o.o1) lps-stimulated no-generation was found in spleen cells prepared at day 4 (13.0 4-1.3 nmol nitrite/106 cells, n=6) compared to spleen cells from non-treated animals (3.6 ± 1.3 nmol, n=6) complement activation was effective also in vitro. when incubated in 25% zymosan-activated rat serum, both basal and lps-stimulated noproduction were enhanced in spleen cells (table, values increased production of nitric oxide (no) is associated with sepsis, however, the effect of no inhibition on survival during sepsis is unclear. we examined the effect of no inhibition on host's ability to eliminate bacteria and survival in mice sepsis model. sixty female balb/c mice were injected with e.coli (10qbody) into peritoneal cavity. the treated group received n-ultro-l-arginine-methyl-ester (l-name), an inhibitor of nos, one hour before bacterial challenge. thirty mice were observed for survival after e.coli challenge and the other mice were sacrificed at 4 hours. blood was obtained by cardiac puncture and peritoneal lavage fluid (plf), liver and lung were harvested. the number of peritoneal exudative cell (pec) was counted and colony forming units (cfld of bacteria in the tissues, blood, and plf were also determined. in addition, pec was cultured in vitro with or without lipopolysaecharide (lps). tnf levels in the blood, plf, and supematants of cultured pec were measured by l929 bioassay. survival rate (%) qrql/,p n 8 hr 24 hr 4 dav control (normal saline 0.1ml/body i.p.) 10 100 50 40 n10 (l-name 10mg/kg i. administration of l-name before e.coli injection increased the number of bacteria in plf and tnf level in plasma, the result suggests that nos inhibitor may depress the host's ability to kill the injected bacteria and that it may induce greater systemic tnf production. we conclude that nos inhibitor is detrimental to animals in sepsis. the hypothesis that sod prevents reperfusion injury to the liver by protecting the endogenous endothelium derived vascular relaxing factor (no) from being inactivated by oxygen free radicals should be investigated. male wistar rats were subjected to 60 min of partial liver ischemia (70%) and 30 min of consecutive reperfusion in situ. some rats were pretreated with the no synthase inhibitor nitro-l-arginin (nla: i0 mg/kg). sod, when used, was given as mn-containing preparation at a dose of 6000 u. i.v. i0 min prior to ischemia. results (sod/ nla+sod/ ni~,, respectively): bile flow (~i/g/min) : 0.93±0.11"#/ 0.52±0.08/ 0.48±0.05. alt (u/2) • 171±40~#/ 477±173/ 506±182. gldh (u/l): 5.2±1.2e#/ 10.7±4.2#/ 24.3±9.1. in absence of nla, sod enhanced postischemic bile flow and reduced leakage of alt and gldh into the plasma, while the effects of sod disappeared, when endogenous 140~synthesis was inhibited by nla. nla had no ~ffect on untreated animals submitted to the same protocol. these results may suggest, that oxygen free radicals interfe~:e with the endogenous vasodilator no and that the benefit of sod can be attribuated in large part to a protection of no during reperfusion. nitric oxide (no) reduces pulmonary vascular resistance and increases oxygenation by inhalation in ards patients [1] . animal studies on the endogeneous no production proved that no is exhaled after synthesis in the respiratory tract [2] . with approval by the hospital ethics committee, we studied healthy volunteers and ventilated patients by measurement of inand exhaled no by no/no2-chemiluminescence in segments of the upper respiratory tract. we found that no is synthesized predominantly in the nose and in the upper nasopharynx, leading to inspiratory tracheal no concentrations of 0.07-0.13 parts per million in non-smoking volunteers; data during mask ventilation per nose were as follows: non 0.089 0.0422 0.0721 0.0311,2 parts per million (ppm) no, mean, n = 10; ~ p < 0.05 (t-test) between smokers and non-smokers; 2 p < 0.05 (t-test) between in-and expiration. about 50% of no is resorbe.d by the lung, and the exhaled no -in contrast to former presumptions -is the remaining part of the autoinhaled no. intubated and ventilated patients are deprived of no autoinhalation, and the exhaled no in the trachea decreases more than 95% {o.043 ppm before intubation vs. 0.003 ppm after intubation, ~, < 0.01), whereas the nasal no concentration increases by cumulation. hence, low-dose no inhalation in ards patients might be considered as a no replacement, especially since the tracheal no concentrations we measured in volunteers are similar to those which could be demonstrated to be effective for ards in former studies [3] . the data demonstrate that no is synthesized in the nose, and inhaled and resorbed by the lung. this phenomenon of no autoinhalation, which is reduced in smokers and in ventilated patients, does possibly contribute to the regulation of the ventilation-perfusion ratio in the lung. kikuchi 1, yoshihiro inoue 1, masao yoshida 2 1 critical care and emergency center, and 2department of bacteriology, iwate medical university. nitric oxide (no) is produced by macrophages and vascular endothelial cells. it is thought to be the true form of endotheliumderived relaxing factor, and to be involved in the fall of blood pressure during septic shock. we have reported previously that endotoxin (lps), tnf-c~ and il-2 contribute to the occurrence of septic shock (circ shock 38:264; . the present study investigated the in vitro effects of lps, tnf-o~, il-2 and ifn-'i on the production of no by macrophages. peritoneal macrophages were cultured with lps, il-2, tnf-~ and ifn-¥. in culture with lps, no was produced in a dose-dependent manner, and the production was suppressed by a no production inhibitor, l-nmma. ifn-y, il-2 and tnf-c~ used alone did not promote the production of no, but helped lps to facilitate no production. a combination of il-2 and tnf-c( enhanced lps-facilitated no production to a greater extent than il-2 or tnf-c~ alone. the above results suggest that these cytokines are involved in endotoxin shock through the mechanisms that facilitate no production. 19-1 uchimaru, morioka 020. japan. t. yolk 1,2, i. ioannidis 1, w.j. kox 2 and h. de groot 1 objectives: nitric oxide (no.) is known to play an important role in neurotransmission, vasoregulation, and bactericidal as well as tumoricidal cellular defense systems. by means of no-donating agents we studied the mechanism of no-mediated cytotoxicity against rat liver microvascular endothelial cells. materials and methods: 3-morphoiinosydnonimine-n-ethylcarbamide (sin-l) and sodium nitroprusside (snp) were used as no. donators, ldh release and trypan blue exclusion were applied to indicate cell viability. results: sin-1 (5mm), which releases both no. and o2-., caused a time-dependent cytoreduction of 60% and 80% after 3 and 6 hrs, respectively. this effect was not inhibited by superoxide dismutase (sod), which removes 02--to form h202 and 02. in contrast, the addition of catalase (cat), which removes h202 to form h20 and 02, diminished the cytotoxic effect ot sin-1 to 30%, equivalent to high concentrations of snp (20mm), which solely releases no.. in addition, the amount of h202 formed by 5mm sin-1 equaled the amount ot enzymaticany produced h202 by 5mu/l glucose oxidase (god). whereas god in this concentration and snp in low concentration (5mm) alone were not toxic to endothelial cells, the combination caused an 80% reduction of viability, comparable to the effect observed with sin-1 (5mm) alone. moreover, toxicity mediated by high concentrations of snp (20mm and 50ram) was reduced by 20% under hypoxic conditions indicating synergism with no-and 02. conclusions: we conclude, that no.-induced toxicity against endothelial cells is enhanced slightly in the presence of oxygen and is not due to an interaction with 02-. however, toxicity increases dramatically in the presence of h202. this may possibly occur either by a chemical formation of more potent reactive hydroxyl radicals or by independent actions on different cellular targets. although it is becoming increasingly clear that nitric oxide (no) is associated with otxan dysfunctions in septic patients, little is known as to die kinetics of no production in these patients to clarify these kinetics, we have investegated serum arid monocyte associated no in septic patients. five patients who had undergone gastrointestinal surgeries mid were complicated postoperatively with severe sepsis served as the subjects in this study (sgroup), using twelve healthy volunteers as a control group (c group). serum no2 and no3 levels were measul*d sliectrophotometricallymonocyte cultures were done for 24 itrs with or without lps+ifn-t stimulation mid no2 and no3 levels in the supernat,'utts were also measured spectrophotometrically. furthermore,using an no selective electrode,time course of the lps+ifn-t induced no production by monocytes was studied. l) serum no2 levels in s group were slightly lfigher than dmse in c group,bnt there were no significoatt differences between the two. 2)both no2 levels in the supematmlts of monocytes cultured with lps+ifn-t mid no2, no3 levels ill lhe supernatants of monocytes cultured without lps+ifn-y were significantly higher in s group. further,the time required for the no production by enltnred monoeytes was siglfificantly shorter also in s groap. conclusions l)in severe septic patients, monocyte no productions wei~ not only primed, but also activated concomitantly, suggesting that these monocyte activations m'e strongly associated with organ dysfunctions in sepsis. 2)based on the restdts of serum no nteasuremeuts, it can be deduced that no p~'oduced by monocytes in septic patients affects tissues and org~s ioeoregiomflly. objectives of the study: nitric oxide (no) is an important messenger which accounts for a wide spectrum of physiological and pathophysiological processes, e.g. mediating vasodilation in endoloxin shock investigating the signal lransducfion pathways involved in the regulation of the inducible nitric oxide synthase (inos), we report the involvement of phosphatidylcholinespecific phospholipase c (pc-plc) and proteinkinase c (pkc). materials and methods: no-production was induced in the murine macrophage cell line j774 with lipopolysaccharide (lps) [lljg/ml] and interferon ;f (ifny) [200u/ml]. after 18 hours of incubation no-release was determined as nitrite (no2) by using a colorimetric assay based on the griess-reaetion. results: preincubation of cells with the pkc-inhibitors staurosporine (stp), chelerythrine, bisindolylmaleimide (bim) and d609, that recently has been identified as a selective inhibitor of pc-plc, diminished significantly lpsand ]fnt-induced no2-production (p<o,001). although we observed no toxic effect on cell viability, no2-production was related to protein concentrations to exclude any inhibitory effect on celt growth. celts preincubated with stp [lo0nm] released 20% less no 2 in response to lps and ifn,[ compared to control cells cultured without inhibitor. chelerythrine [30}jm] and bim [ioijm] reduced the no2-formation by 50% and 85%, respectively. interestingly, the most potent inhibitor of no-production turned out to be the xanthate d609 cells pretreated with d609 [301~g/mt] released 96% less no 2 than stimulated controls the inhibitory effect on no 2production decreased the later the inhibitore were added after stimulation; e.g. bim added 6 and 12 hours after stimulation reduced no2-production only by 50% and 12%, respectively. accordingly, inqs activity present in stimulated cell lysates supplemented with l-arginine and co-factors was not suppressed by the inhibitors tested. conclusions: our findings suggest, that the induction of inos but not its activity is a pkc and particularly pc-plc dependent process. dr. k tschaikowsky, institut for anaesthesiologie, universital erlangen--nqrnberg, krankenhausstr 12, 91054 erlangen interleukin-1 (il-1) affects nearly every cell type by increasing the expression of a variety of genes associated with the promotion of inflammatory processes. these include upregulation of cyclooxygenase and nitric acid synthases. the il-1ri transmits a signal(s) through the cytoplasmic domain which is structurally related to the fruit fly toll protein. we have recently cloned the promotor most proximal to the 5' utr of the human il-1ri gene. the genomic sequences reveal a lack of tata and caat boxes but rather a considerable (75%) gc rich region. the translation of the type i il-1r appears under a significant suppression and may limit the biological activities of il-1 by low level of expression. on the other hand, the type ii il-1r, lacking a significant cytoplasmic domain, does not transmit a signal and acts as a decoy receptor preventing the binding to the type i receptor. there are several approaches to reducing il-1 activity; inhibition of il-1 converting enzyme which cleaves pro-il-l[3, anti-il-1ri, the il-i receptor antagonist (il-1ra) and soluble (extracellular) il-1ri and il-1rii. il-1ra is structurally related to il-1 and binds at 4°c to the il-1ri with near equal affinity as that of bona fide il-i; however, il-1ra does not transduce a signal. il-1ra has been given to humans in pharmacologic levels (mean plasma levels of 30gg/ml) without evidence of agonist activity. in addition, there has been no affect on normal homeostatic parameters, suggesting that il-i does not play a role in health. in healthy humans given intravenous endotoxin, il-ira reduced endotoxin-associated neutrophilia by 50% and completely reversed endoloxin-iuduced immunosuppression, il-tra is produced naturally and is elevated in the circulation in several diseases. in a variety of diseases, plasma levels of il-1ra are in 100-fold molar excess to those of il-i [3. it is unclear whether these endogenous levels of il-1ra are sufficient to block il-1 activity in disease. a single injection of ]l-6 or ifna in humans does not induce il-1 but rather high levels of il-1ra (10ng/ml). prof.dr.l.a. aarden interleukin 6 (il6) is a multifunctionai cytokine with a central role in host defense mechanisms and consequently in inflammation. il6 is thought to be involved in the pathogenesis of various diseases. therefore, specific inhibitors could have therapeutic value. a human-mouse chimeric monoclonal antibody to il6 has been applied in a phasei clinical trial in patients with multiple myeloma and in primate models of sepsis. no toxicity was observed and the most remarkable outcome of this study was the build up in the circulation of il6-anti-il6 complexes, suggesting that plasma il6 measurements represent a gross underestimation of il6 production in the patient. until now no natural antagonists of il6 have been described. the biological activities of il6 results from binding to a lowaffinity il6-receptor (il6r). the il6/il6r complex induces disulfide-linked homodimerization of the signal transducing receptor component, gp130. studies on the structure-function relation of il6 revealed that disruption of the gpl30-interaction site on il6 gives rise to a mutant il6 that, by virtue of its intact binding to the il6r, acts as a potent antagonist on human hepatocytes and on human b cells. in vivo studies using this molecule will be initiated in the near future. the evolution of infla~ation involves a dynamic series of cellular events controlled by specific signals which are important to the initiation, maintenance, and final resolution of the inflammatory response. the various phases of inflammation are influenced via the expression and subsequent regulation of a number of key mediators which play a predominant role during each particular stage of the developing lesion. for example, the initial elicitation of blood born leukocytes to on inflamed area is a complex system that is dependent upon the expression of early response cytokineso these proximal mediators, including both interleukin-i (il-i) and tumor necroisis factor (tnf), are important in the initiation phase by activating both immune and non-immune cells and establishing a series of cytokine networks, thus, the above proximal mediators can stimulate endothelial cells, epithelial cells, smooth muscle cells, and fibroblasts to generate more distal cytokines. these latter cytokines include interleukin-8 (il-8), macrophage inflammatory protein-i (mip-i), and monocyte cbemoattractant protein-i (mcp-i). the importance of the above chemukines can be found in the role they play in leukocyte elicitation, as well as wound repair. numerous investigations have shown the diverse cellular sources of il-8 and the ability of il-8 to elicit neutrophils in vitro at pm to nm concentrations, however, recent studies have sho%~ an additional role for il-8 during the resolution phase of the inflammatory process. using corneal pocket animal models of angiogenesis/neovascularization, il-8 was found to be a potent angiogenic factor at concentrations ranging from 2-40ng/ml. sequential fluorescein angiograms demonstrated that il-8 induced neovascularization by 14 days, which regressed by 6 weeks. in further analyses, both conditioned media recovered from either inflamed human heumatoid synovial tissue macrophages or lps-stimulated peripheral blood monocytes were found to possess potent angiogenic activity, which was effectively blocked by neutralizing antibodies directed against human il-8. in addition, an il-8 antisense nligomunclentide blocked the expression of a functionally active angiogenic factor from lps treated monocytes. the above data demonstrate that il-8 has multifumctional activities during the initiation, maintenance, and resolution of a local inflammatory response. inhibits specific t-cell responses to soluble protein antigens and alloantigens. the proliferative responses of th0, thl and th2 cd4 ÷ t-cell clones and cytokine production by these t-cell subsets are equally well inhibited. the inhibitory effects of il-10 are indirect and related to inhibition of antigen-presenting capacity and accessory function of the antigen-presenting cells (apc). however, il-10 also directly inhibits t-cell proli/eration induced by cross]inked anti-cd3 or anti-cd2 mabs (e.g. in the absence of professional apc), by specifically inhibiting il-2 gene transcription and il-2 protein production. il-10 also inhibits the production of the pro-inflammatory cytokines il-l-a, ~, il-6, and tnf-a and the chemokines il-8 and mip-i-u, which are strong chemo attractants for neutrophils and macrophage, respectively. in contrast, il-10 enhances the production of the il-1 receptor antagonist, a cytokine with anti-inflammatory activity. il-l0 produced by monocytes downregulates class ii mhc expression and il-10 production by these cells in an autoregulatory fashion. collectively, these in vitro data indicate that il-10 is a powerful suppressor factor for immune-and inflammatory responses and therefore may have potential clinical utility as an anti-inflammatory agent. this notion is supported by recent studies which indicated that il-10 also prevents lethal lps-induced toxic shock in mice. five of the chamekines (hip-i~,era~tes. ip-io and il 8) also chemoattraet t lymphocytes, while others act on mast cells. we have studied the effaces of these chemokines on t cell subsets. il 8 preferentially chemoattracts uns~imulated t cells. raises ~ttracte resting as well as activated cd4 and cd8 memory t cells. ip-10 chemoattracts only preactivated (not resting) cd4 or cd8 t cells. hip-l~ preferentially chemoattracts gd8 t calla, while hip-18 more readily attracts the cd4 t call subset. the 8 chemoklnes and ip-io were also sho~rn to promote the adherence of ~he same subsets of anti-cd3 activated t cells to ili activated human umbilical vein endothelial cells (hitvec). this was not associated with a~y i~crease in the number of lymphocyte adhesion molecules, bu~ could be inhibited by neutralizing monoclonal antibodies re lfa-i and vla-4. these chemokines also rapidly increased ~ha adhesion of resting t l~phoeytes to plates coated wi~h 8 integrlns (i-cam or v-cam) or matrix proteins such as f~bronectln. this induction of adhesion began wiehln 30', peaked by 1 hr and was completed in 3 hr. this suggests that chemoklnee rapldly increase the binding affinity of adhesion prote~ns on t cells. we also recently observed ~hat mcaf/mcpi and rantes chemoattract unatimulated mast calls. furthermore, igs actlvared mast calls were chemoattraeted by mcaf, ra~tes, pf4 and mip.i~. however. ~he chemoklnee did not induce mast cells ~o release histamines. presumably, chemokines as regulators of the adhesion, migration and homing of all kinds of leukocytes participate in many types of inflammatory diseases. natural killer cell stimulatory factor or interleukin-12 (il-12) is an heterodimerie cytokine formed by two covalently linked glycosylated chains of 35kd and 40 kd. il-12 was originally purified from the supermatant fluids of ebv-transformed human b ly~phobiastoid cell lines. however, in addition to b cells, phagocytic cells, i.e. monocytes, macrophages, and neutrophils, have been identified as the major physiological producers of il-12. phagocytic cells produce il-12 in response to bacteria, bacterial products such as lps, and intracellular parasites. the cell types on which il-12 exert biological activity are t and nk cells. the major direct biological function of il-12 on these cell types are the induction of cytokine production, primarily ifn-y, the enhancement of a generation of cytotoxic cells, and a mitogenic effect on antlgen-activated lymphocytes. in part thromgh its ability to induce ifn-y production, il-12, produced in response to infections, represents an important functional link between effector cells of innate resistance, phagocytic cells and nk cells, and effector cells of adaptiye resistance, t and b lymphocytes. the ability of il-12 to induce ifn-y production from circulating. nk cells and at least a proportion of t cells determines a rapid, antigen-independet production of ifn-y during infections. ifn-y acts as a potent enhancer of phagocytic and bacteriocidal activity of phagocytic cells, participating early in infection to activate some of the inflammatory mechanisms that represent the first innate resistance against infection. this antigem-independet activation of phagocytic cells that involves, in addition to il-12, other monocyte derived cytokines such as tnf and il-l, has been shown to be important in experimental animals for the resistance against infection by microorganisms such as limteria monocytogens and toxoplasma gondii. in addition to this role, early in infection in the antigen-independet phagocytic cell activation, il-12 has a major effect in the ensuing antigenspecific admptive immune response by facilitating the generation of t helper type 1 (th-i) response and suppressing a th-2 response. the presence of il-12 during antigen stimulation in vitro using human peripheral blood lymphocytes, or both in vivo and in vitro in experimental animals, induces generation of th-i cells producing ifn-y and il-2, and inhibits the generation of th-2 cells producing il-4. il-12 induces a direct differentiative effect on th-cells, priming them for high ifn-y production. ~is priming effect is stable and maintained even when t cell clones are cultured for extended periods in the absence of il-12, however, il-12 needs to be present during the first few days after stimulation with antigen or mitogen and established th clones are resistant to the priming effect of ifn-y. unlike the generation of high ifn-y producing clones, the il-12-mediated inhibition of il-4 producing cells in probably due to selective mechanisms, which may include a selective mitegenic effect of il-12 for thl cells and an inhibitory effect of ifn-y on th2 cell proliferation. in several experimental systems, it has been shown that the enhancing effect of il-12 on th-i responses is dependent on production of if~-y and on the presence of nk cells, possibly needed for the early production of ifn-y. the importance of phagocytic cells and nk cells in determining the characteristics of the th response during antigenic stimulation indicated that the mechanisms of innate resistance have a determining influence on the subsequent antigen-specific immune response: il-12 appears to have a key role in mediating the interaction between phagocytic cells. nk cells, and t lympocytes in these proccessem. trinchieri, g. 1993, interleukin-12 and its role in the generation of t e 1 cells, imm~ol. on peripheral blood monocyte/macrophages, il-13 behaves like il-4 in a variety of assays, and shows both similar and contrasting, activties with either il-10 or ifn-y. il-13 reduces inflammatory monokine and il-10 production. it reduces the pyrogen-induced expression of tissue factor (herbert et el, 1993, febs lett. 328, 268) . it mpregulates manmose receptor and ~c class ii expression, while reducing cdi4 expression. il-13 produces morphological changes (extensive cell processes, aggregation, giant cell formation) which have previously been observed with il-4. it inhibits niv-i production (montaner et el, 1993, i. exp. mad. 178, 743) , and interferes with hiv-induced cell fusion. the activities of il-13 and il-4 differ on t-ly•ocyte and large granular lymphocyte population. in particular il-13 does not exhibit the suppressive effects of il-4 on thl-type-helper functions (ifny production) and cytotoxic functions (perforin production). data will be presented showing that il-4 and il-l3 share a common receptor on a nu~er of cell types, but not on t-lympocytes (see also zmrawski st el. 1993, embo j.7. 2663-2670), explaining both thai common and divergent activities. the levels of il-13~a in activated peripheral blood lymphocytes are greater than or equivalent to those of il-4 mp~ja and the two ml{nas are expressed by different t lympocyte population: in particular, t8 cytotoxic lymphocytes express markedly higher levels of il-13~rna. in vivo, il-13 may thus be contributing, significantly to some of the activites previously ascribed to il-4 il-10. recently, we cloned the human cdna encoding interleukin-13 (il-13). human il-13 is a non-glycosylated protein of 132 aa with a molecular mass (mr) of 10 kd and has biological activities on monocytes and b cells, il-13, like il-4, strongly enhanced the expression of class ii mhc antigens on monocytes and induced expression of cd23 (fc~rii). furthermore, il-13, like il-4 and il~10, inhibited the production of pro-inflammatory cytokines il-1, ]l-6, tnfo~ and chemokines miplc~ and tl-8 by lps activated monocytes. both il-13 and il-4 enhanced the production of il-lra by monocytes. in contrast, il-13 lacked the t cell stimulating activities of il-4. the responses of monocytes to il-13 and il-4 were not additive or synergistic, supporting the hypothesis that il-13 and il-4 receptors are complex and share a common component which is important for signal transduction, taken together, these data indicate that il-13, il~4 and il-10 have important anti-inflammatory activities on human monocytes. in addition, these results indicate that il-13 is unique in that it can modify inflammatory reactions without stimulating (as does il-4) or inhibiting (as does il-10) t cell responses. transforming growth factor beta i (tgf-fll) belongs to a family of proteins that have potent immunoregulatory properties ranging ftom effects on the growth and dfflerentiafion of primitive stem cells to the differentiated functions of immune system effector cells. tgf-i~i is synthesized as a large secretory precursor polypeptide of 390 amino acids that is inactive until processed to a disulfide linked homodimeric molecule of 1t2 amino acids each. recently it has been found that tgf-[~i can have autocrine as well as paracrine effects on the immune system indicating that immune cells can activate the inactive secreted form of tgi~-~i. in addition, tgf-i~i has differential intraceuular effects on cell surface receptor modulation, tyrosine phosphorylation and cytokine gene transcription as well as cell mediated cytotoxicity. the systemic administration of tgf-131 has proven beneficial in a variety of animal models such as septic shock, allograft rejection and experimental forms of autoimmunity including collagen induced arthritis and experimental autoimmune encephalomyelitis. moreover the increased levels of tgf-ill and other tgf-13 isoforms found in several disease states associated with immunosuppression such as different forms of malignancy, chronic degenerative diseases and aids implicate the involvement of tgt~-i~ in the pathogenesis of some diseases. hopefully, well designed clinical trials will define the potential roles of the tgf-[3 family of proteins in the treatment of diseases of man. patient development of systemic inflammatory response syndrome (sis) after severe trauma is accompanied by a wide range of immune aberrations and altered cytokine production. in particular, t lymphocyte proliferation and ifn, t production is suppressed while monocyte (mo) tnfc~, il-6, pge2 and tgf~ production is massively increased concomitant to decreased antigen presenting capacity. recently, two new cytokines, il-10 and il-12, have been characterized as critical in regulation of mo tnfa and il-6, as well as in induction of t lymphocyte proliferation and ifn 7 production. in this study, peripheral blood leukocytes (pbl) from severe trauma patients (injury severity score >35) were analyzed for their il-10 levels, their in vitro proliferation to mitogen (pha) and their response after il-12 addition. since il-10 produced either by mo or by t lymphocytes can depress m~ antigen presenting capacity, inhibit t cell ifn,/production and directly diminish t cell proliferation, it might be suggested that immunosuppressed patients' mo and/or t lymphocytes would have increased il-10 levels. increased patient il-10 production might also be resulting from the high levels of tnfa a known stimulator of il-10. conversely, since il-12 augments mo antigenpresenting capacity, thl induction and proliferation, post-trauma leukocytes might be il-12 deficient. pbl of trauma patients were compared to normals' pbl, either unstimulated or ptta induced, and their levels of il-10 found to be dramatically and significantly reduced. patients' isolated m~, either stimulated with the bacterial cell wall analogue, mdp, or unstimulated, also had depressed il-10 production concomitant to elevated tnfa production when compared to normals' mo. mechanisms for the depressed patients' mo il-10 were explored. increases in tgf[3 may have partially contributed to the patients' depressed il-10 level, but elevated pge2 had no effect. addition of il-12 to patients' pbl significantly increased their mitogen responses. these data imply that sis is characterized by disruption in the interactions between mci and t lymphocytes so that patients' m~i produce excesses of some mediators (tnfa, il-6, pge2) and a dearth of other monokines (il-12, il-io). t lymphocytes are not activated and, therefore, unable to function in both immune defense and monocyte regulation. it is known that lge receptor-mediated or ca-ionophore-induced activation of mouse bone marrow-derived mast cells (13mmc) may result in the production of different cytokines including the interleukins (il) 3, 4, 5, and 6 as well as gm-csf and tnf-a. in the present study we analyzed the effects of exogeneously applied pro-inflammatory cytokines (il-1, 1l-6, tnf-c 0 as well as various mast cell growth factors (il-3, il-4, il-9, il-10, ngf, kl (kit ligand)) on cytokine production in primary mouse bmmc using a standard activation protocol (lxl06 bmmc/ml; ll.um ionomycin; 24-48h). the actixdties of bmmc supernatants were assessed in specific biological (il-3, il-4 il-6, 1l-9) and/or elisa assays (il-5, il-9). here we show that homogeneous populations of bmmc (>97%alcian blue+/safranln-; in vitro age: 4 weeks) generated in the presence of recombinant (r) rail-3 from normal balb/c mice produced modest amounts of 1l-6 and low or undetectable levels of il-3, -5, and -9 after induction with lp.m ionomycin only. however, a dramatic increase (5-to 10-fold) of these cytokine activities was noted, when in addition to ionomycin also human (11) rll-la was provided during the induction period. this il-1 effect was dose dependent with a maximgm at 2-10 u/ml hrll-la and specific, as pre-incubation (lh) of bmmc with 20 ng/ml hrll-1 receptor antagonist abolished the action of 2 u/ml hrll-lcc similar effects were noted with hrll-lg or rurll-lb (lng/ml, respectively), but not with rhll-6 or rmtnf-~. both mrll-10 and hrll-10 substantially enhanced ionomycin-induced 1l-6 production of bmmc in the absence or presence of il-1. il-10 significantly enhanced il-6 and il-9 production while decreasing il-3 activities to abont 50-90% of control levels, when il-i0 was provided in the presence of il-l/ionomycin. a monoclonal anti-nfil-t0 antibody (ascites 1:200) abrogated the effects of mrll-10. other mast cell-active cy~okines (]1,-3, il-4, 1l-9, ngf, or kl) added to ionomycia-or 1l-1/ionomycin-treated bmmc had no major effects on cytokine production. il-1 and il-i0 did not induce significant cytokine release in the absence of ionomycin suggesting tlmt cadependent signalling was required. at doses of 10"6m, dexamethasone, corticosterone, or hydrocortisone almost completely abolished ionomycin/il-1/ll-10induced cytokine production. the inducer cocktails per se did not interfere with the cytokine bio-assays. in case of il-9 inducibility of this cytokine in bmmc was confirmed at the mrna level by northern blot analysis. hence our data show that activated mast cells are a source of il-9 previously recognized as a product of th2type lymphocytes only. moreover, our study reveals novel functional roles for i-l-i, il-10, and ghicecorticoids in the regulation of cytoldne production in mast ceils. accumulating data suggests that cytokines, peptides involved in regulation of both physiological and pathological immunological responses, predominantly are produced at the local site of antigen stimulation. a new method was used to detect cytokine-producing cells in haman tissue at the protein level. single-cell production of 17 different httman cytokines, ilia, ill [3, illra, il2, il3, il4, il5, il6, ils, ill0, gm-csf, tnfa, ifn 7 and tgf[31.3, was identified by indirect immunohistochemical staining procedures and use of carefully selected cytokine-specific mab's. frozen sections were fixed with 4 % paraformaldehyde and permeabilized by 0.1% saponin treatment, eluting cholesterol from the membranes. the intracellular presence of all cytokines except ill, illra (late) and tfg[31_3, could be demonstrated by a characteristic perinuclear configuration in producer cells. in addition, the immunoreactivity extended over a large extracellular area encompassing the producer cell. a localization of the cytokine to the golgi-organelle was established by use of two culour staining including a haman golgi complex specific mab. this staining pattern was only evident in producer cells because injection of recombinant human cytgkines into the tissue caused a membraneous and extracellular staining pattern. both the extra-and the intracellular types of staining reaction could, however, be blocked by preincubating the cytokine specific mab with pure human interleukins. oxygen radicals (or) directly induce lipid peroxidation, indirectly they trigger adhesion and activation of pmn leukocytes. we investigated whether or also lead to a release of acute-phase response cytokins such as tnf-alpha, il-i beta or il-6 in whole blood cultures to maintain the induced inflammatory reaction. methods: blood samples from healthy volunteers (n=10) were incubated at 37°c. or were produced by the xanthine oxidase (xo)/ hypoxanthine (hx) system. after 0,30,60,120,240 and 360 minutes plasma levels of tnf-alpha, il-i beta and il-6 were determined with elisa kits. results: under the influence of or tnf-alpha plasma levels increased from 0,5 pg/ml at 0min to 14pg/ml, 236pg/ml, 343pg/ml after 60, 120 and 240 min. il-ibeta (0,3pg/ml, 0,6pg/ml, 1,5pg/ml, 81pg/ml and 169pg/ml after 0,60,120,240 and 360min) and il-6 (0,2pg/ml, l,lpg/ml, 4,1pg/ml, 58pg/ml and 72,9pg/ml after 0,60,120,240 and 360min) plasma levels were increased 60min later than tnf-alpha. summary: these data suggest that or do not only play an important role in initial accumulation and activation of pmn leukocytes but also lead to a stimulation of monocytes to produce the acute phase reaction cytokins tnf-alpha, il-i beta and il-6 to maintain and strengthen the inflammatory reaction. department of general surgery, steinhsvelstr.9, 89070 ulm, germany jan k. horn md, greg a. hamon md, robert h. mulloy md, greg chen bs, rebecca chow bs, and christof birkenmaier md. transforming growth factor-i~l (tgf-131) is released from inflammatory ceils following injury and in sepsis. in vitro experiments have confirmed that low concentrations of tgf-131 (0.1-1.0 ng/ml) are chemoattractive for monocytes, whereas higher levels of tgf-131 (>5.0 ng/ml) potentiate production of the immunedepressive prostaglandin e 2. other investigators have shown that tgf-]31 can cause the appearance of cd16 (fc immunoglobulin receptor) on monocytes exposed to 10 ng/ml of tgf-[~i for 24 hours. monocytes also express on their surface a glycoprotein that binds complexes of lipopolysaceharide (lps) and lpsbinding protein (lbp). such binding is associated with generation of proinflammatory cytokines such as tumor necrosis factor alpha. we have shown that cd14 is depressed in septic patients and therefore we hypothesized that tgf-131 could account for the down-regulation of cd14 observed in these individuals. we incubated normal human monocytes with platelet-derived tgf-[31 for 2 and 24 hours at 37°c and examined ceils for cd14 and cd16 expression using flow cytometry after immunnfluoreseent staining with appropriate monoclonal antibodies. monocytes were selected on the by usual criteria for size and granularity. non-viable ceils were excluded with the use of propidium iodide. two populations of monocytes could be found afcer incubation at 37°c alone. one displaying high density of cd14 had increased fluorescence over the homogeneous expression of cd14 in cells maintained at 4°c (baseline). the other population displayed decreased cd14 expression relative to the baseline cells. tgf-i~i (10-50 ng/ml) caused a shift of ceils from the high density into the low density cd14 population. this trend was observed within 2 hours of incubation and was complete by 24 hours. we observed a net decrease in cd14 expression 0f32% for all subjects studied (p<0.05 vs controls). phorbol myristate acetate (100 ng/ml) also caused down-regulation of cd14 to a similar degree as tfg-i~i. we also confirmed that monocytes could be induced to express cd16 after incubation with tgf-131 (10 ng/ml) for 24 hours. these studies demonstrate that monocytes incubated with immunodepressive levels of regulation of cd14 by tgf-131 deplete their surface expression of cd14 while generating cd16. this down-regulation of cd14 by tgf-131 correlates with our clinical observations of lower cd14 expression on monocytes obtained from septic patients. for over 25 years, activated t lymphocytes have been considered to be the cellular source of mif. we recently isolated and cloned the murine homolog of mif after identifying the specific secretion of this protein by lpsstimulated pituitary cells in vitro and in vivo. however, further experiments showed that mif protein is detectable both in t-cell deficient (nude) and hypophyseetomized mice, suggesting that yet additional cell types may produce mif in vivo. since monocytes/macrophages are a major source of the cytokines that appear in response to lps administration, we examined the possibility that mif also is expressed in cells of the monocyte/macrophage lineage. we found that mif is expressed constitutively in the murine macrophage-line raw264.7 and in thioglycollate-elicited peritoneal macrophages. significant amounts of mif mrna (rt-pcr) and protein (western blotting) were observed in cell lysates. in raw 264.7 cells, mif secretion was induced by as little as 10 pg/ml of lps (e.coli 011 l:b4), peaked at 1 ng/ml, but was not detectable at lps concentrations >1 txg/ml. similar data were obtained with elicited macrophages, but higher lps concentrations were required, unless the cells had been preincubated with ifn 7. production of mif by lps-stimulated (l ng/ml) macrophages peaked at 12 hr. expression ofmif mrna and tnf mrna by lps-stimulated raw 264.7 macrophages was investigated by rt-pcr. as expected tnf mrna expression increased over the range of lps concentrations (1 pg/ml to 1 p_g/ml). in contrast, levels of mif mrna correlated inversely with lps concentration. by competitive pcr, mif mrna was observed to increase approximately 2-fold after lps induction (100 pg/ml). mif secretion also was induced by tnfoc (1 ng/ml) and ifn? (10 iu/ml), but not by il-113 and il-6 (up to 10 ng/ml). lps and ifn 7 had additive effects in inducing mif secretion. in separate experiments, macrophages stimulated with recombinant mouse mif (1 gg/ml) were found to secrete bioactive tnf~ (>750 pg/ml by l929 cytotoxicity). we conclude that the macrophage is an important albeit overlooked cellular source of mif in vivo. mif secretion is induced by lps, tnfc~ and ifn?. mif also stimulates macrophages to secrete tnf. taken together with previous observations that anti-mif antibody protects against lethal endotoxemia, these data implicate mif as a critical mediator of inflammation and septic shock. inflammation is characterized by an exacerbation of proinflammatory cytokine production. cytokines such as il-4, il-10, and tgf8, have been identified as anti-inflammatory mediators thanks to their ability to down regulate the production of il-1, il-6, il-8, tnfc~ by activated monocytes / macrophages. however, other cells, including polymorphonuclear cells (pmn) do contribute to the release of pro-inflammatory cytokines. we investigated the capacity of the so-called anti-inflammatory cytokines to control the release of il-8 by activated neutrophils. human pmn were purified following glucose-dextran sedimentation and ficoli-hypaque centrifugation. the cells were cultured at 37°c for 24h in the absence or presence of lipopolysaccharide (lps) or tnfa. il-8 release was measured in the supernatants using a specific elisa. among tested cytokines, il-10 was the most efficient inhibitor of il-8 production by lps-activated pmn. il-4 was also active, whereas no down regulation was noticed with tgfp~i. when tnfa was used as a triggering agent, none of the cytokine could prevent il-8 production. northern analysis are under investigation to precise the level of the il-4-and il-10-induced inhibition of il-8 production by pmn. our data illustrate that il-10 and il-4 possess the capacity to down regulate the production of il-8 by both monocytes and pmn, whereas tgfb has a more limited inhibitory activity. ciliary neurotrophic factor (cntf), a member of the il-6 superfamily, has recently been shown to promote axonal growth and neuronal healing. cntf production is also increased during neuronal and muscle damage, associated with soft tissue injury or trauma. we postulated that production of cntf may explain the loss of skeletal muscm protein that occurs in inflammation. 40 female, wistar (175-200 gm) rats received either 250 or 25 pg/kg bw s.c. injections of recombinant rat cntf for seven days, or received sham injections and were freely-fed. additional animals were pretreated with 10 mg/kg ibuprofen lp prior to 250 pg/kg bw cntf. rats treated with 250 ,ug/kg bw cntf lost 6.8_+0.4 gms bw as compared to freely-fed controls which gained 17.0_+3.1 gms (p<o.001 by anova and lsd), and ibuprofen treatment had no effect on cntf-induced weight loss (-5.8_+0.7). animals pair fed to the high dose cntf gained body weight (10.2-+3.5), although weight gain was less than seen in freely-fed controls. rats treated with 25 pg/kg bw had reduced body weight gain (+7.8+_4.4 gin). food intake was also reduced by 29% in 250 pg/kg cntf (from 23.7+_0.7 gm/day to 16.8_+0.6 gm/day; p<o.05) and was also unaffected by prior ibuprofen treatment (18.4+-0.6 gm/day). despite the loss in body weight noted in the high dose cntf animals, there was no appreciable hepatic acute phase response, since liver weight (8.6+0,1 gms vs. 9.4_+0.3 gins), total protein (54.5+-2.9 gms/l vs 44.8+-3.6 gins/l) and albumin (29.0+1.5 gms/l vs. 26.0+_1.6 gms/l) were not different between cntf and freely fed animals. we conclude that cntf causes anorexia, and increases body weight loss in excess of the associated anorexia. unlike the cachexia associated with il-1 and tnf infusions, which is associated with an acute phase response, cntf acts predominantly on food intake and carcass weight through a prostaglandin-independent mechanism and has only minimal acute phase inducing properties, n. joseph espat, md. university of florida, department of surgery, j-box 100286, gainesville, fl. 32610. of cytokines by neurotrophic factors, the neuroendocrine system and phenotiazines. cytokines, including tnf and il-1, have a pathogenetic role in various diseases related to infection and inflammation. the action and/or production of cytokines can be regulated by drugs or endogenous mechanisms that involve the cetral nervous system (cns) we found that the antipsychotic chlorpromazine (cpz) potently inhibits tnf production and protects mice against endotoxic shock. cpz also inhibits brain tnf production in mice intracerebrally injected with endotoxin, whereas cpz analogs that do not cross the blood-brain barrier inhibit only perypheral, not brain, tnf. tnf production and susceptibility to the toxicity of endotoxin, tnf and il-1 are increased in adrenalectomized mice, indicating that endogenous corticosterone (cs) controls cytokine production and toxicity, in a feedback fashion since endotoxin and cytokines increase serum cs. increased tnf production was also observed in mice where the hypothalamus-pituitary-adrenal axis was blocked by chronic administration of dexamethasone, following a two-day washout. administration of cytokines acting through the gp130 signal transd,uction protein, including il-6 and ciliary neurotrophic factor, cntf potentiated il-l-induced elevation of cs. il-6 and cntf also induced hepatic acute-phase proteins. thus il-6 and cntf might have antiinflammatory activity, by potentiating the feedback responses of the neuroendocrine axis. these data indicate how complex can be the interregulatory relationships between the brain and the immune system, how they can be used to pharmacomodulate cytokine action and how their disregulation might exacerbate il-1-and tnf-mediated pathologies. lipopolysaccharide (lps) constitutes a well established activator of monocytes/ macrophages (mizi) and murine b lymphocytes. the hydrophobic part of lps, termed lipid a, represents the endotoxic principle of lps. we now demonstrate that lps is also capable of inducing proliferation of human t cells at a dose of 10 to 1000 pg/ml. the specificity of this stimulation was analysed by the following experiments: i) the synthetic hexaacyl escherichia coiltype lipid a (compound 506) also stimulated human t cells; ii) the synthetic tetraacyl lipid a precursor (compound 406) or the 1phosphono-oxyethyl analogue pe-4, structures known to antagonize lps-induced monokine production in human mz, also inhibited lpsinduced t-cell proliferation. the t-cell proliferation expressed the following features: i) cd4 ÷-as welt as cd8 ÷ t cells proliferate in response to lps, ii) limiting dilution analyses reveal that the frequency of responding cells was between 1/500 to 1/1000 cells; iii) purified t cells alone cannot be stimulated by lps, but need the help of monocytes. this help cannot be replaced by b cells or fixed monocytes. furthermore, for activation a direct cell-to-cell contact between t cells and monocyte is neccessary; iv) t cells stimulated with lps express mrna for il-2 and ifnf, but not for il-4 or il-5 (determined by pcr). in supernatants, ifnf was detectable (elisa); v) lps-activated cd4 ÷ as well as cd8 + t cells express cd25, the high affinity il-2 receptor. our results indicate that in contrast to previous reports human t cells respond to lps with il-2 receptor expression, lymphokine production, and proliferation. the reactive cells appear to belong to the t,1 cell type, the finding that human t cells can be activated by lps is of important relevance for the understanding of the pathomechanisms of infections by gramnegative bacteria. this may lead to new strategies for the therapy of sepsis. we have recently shown that human eosinophils produce mrna for interleukin (il)-8 when stimulated with calcium ionophore (eur. j. immunol. 23 (1993), 956). we now present evidence that human eosinophils contain preformed il-8 protein although they do not express mrna for il-8 as measured by rt-pcr. il-8 protein expression was determined using specific anti-human il-8 monoclonal antibodies by western blotting, facs analysis and immunohistochemistry. moreover, preformed il-8 was re/eased into supernatant by 99% pure eosinophils after priming with gm-csf or il-5 and subsequent 25-min stimulation with paf, rantes, ionomycin or pma, however not with il-1 or il-2, as measured by elisa. this observation implies that activation of protein kinase c and/or intracellular calcium mobilization are necessary events for il-8 release in human eosinophils. the determined amounts of preformed il-8 protein was higher in patients with asthma compared to normal individuals suggesting a role for eosinonhi! derived il-8 in asthma. since the eosinopnit is the ,,r~:=z~mmant cell in the asthmatic airways, we determined il-8 concentrations in bronchoaiveolar lavage (bal. ~ids from normal !i~.~ijvtduals and asthmatic patients. indeed, il-8 concentrations in bals from both groups were similar to those seen in the supernatants released by activated eosinophils. the role for il-8 in asthma remains to be determined. based on a well established rat model named activity-induced ulceration (asu) or activity based anorexia (aba), we have developed a rat model for chronic stress. male rats were exposed to a feeding schedule in order to reduce body weight by 20-30% within 14 days. while one group of rats was kept sedentary, the other had access to a running wheel. food restricted rats allowed to exercise developed highly increased activity as compared to ad libitum fed rats in wheels (ca. 15 versus 2 kin/day). they developed elevated plasma corticosterone levels (30_+5gg/dl) at their circadian peak, increased adrenal weight and decreased thymus and spleen weight as compared to control rats and weight matched sedentary rats, establishing this paradigm as a model of chronic stress. no differences in plasma acth were seen among the various groups, suggesting increased sensitivity of the adrenal glands to acth. the effect of chronic stress accompanied by hypercorticism on baseline and endotoxin-induced cytokine levels was studied in this model. under baseline conditions,splenic il-1 a was suppressed in both food-restricted groups. this was more pronounced in rats with access to a running wheel. il-6 and tnf activity in plasma was not affected. after administration of 1001.tg/kg lipopolysaccharide (lps), tnf activity in plasma was suppressed by food restriction but there were no differences between sedentary or exercising rats. in addition, no differences were found among groups for corticosterone, spleen il-lc~ and plasma il-6 activity. we conclude, that the food restriction-induced hyperactive rat model is a useful model for studies on the effects of chronic stress. in this model, under basal conditions il-le~ in tissues may be subject to downregulation by glucocorticoids, whereas after lps-stimulation of cytokine production only tnf activity is regulated by fast acting feedback loop between cytokines and the hypothalamo-pituitary-adrenal axis (hpa-axis). this is supported by strong correlations between plasma tnf activity and corticosterone in stressed rats. cytokine antagonists modulate cytokine actions and it appears that a balance between production of cytokines and cytokine antagonists is important to control of host responses. recent observations show that during myocardial infarction there is an increase in circulating cytokines such as tumor necrosis factor (tnf) and interleukin 6 (il-6). we tested the idea that compensatory increases in cytokine antagonists likewise occur and investigated changes in plasma concentrations of tnf and interleukin i (il-1) and those of their specific antagonists interleukin-1 receptor antagonist (il-1 ra) and soluble tumor necrosis factor receptor type i (s tnf r-i). we also measured plasma concentrations of ~-melanocyte-stimulating hormone (~-msh), an endogenous neuropeptide that occurs in pituitary, brain, skin, gut and other tissues. when administered to laboratory animals, this peptide has potent antiinflammatory and antipyretic activity, perhaps by mimicking an endogenous modulatory influence on cytokine actions. samples were obtained from 20 patients with acute myocardial infarction at presentation in the coronary unit, every three hours for 24 hours, and daily thereafter until discharge. we found elevations in the plasma cytokines il-1 and tnf only in a proportion of subjects whereas cytokine antagonists c~-msh, il-lra, and stnfr were elevated in all patients, o~-msh was elevated at presentation but it decreased progressively in successive tests, reaching normal values within 24 hr whereas stnfr and 1l-lra peaked at 3-6 hr or later. there were significant correlations between plasma concentrations of cytokine antagonists and enzymes released from injured myocardium, including creatine kinase (ck), aspartate serum transferase (ast), and lactate dehydrogenase (ldh). the data show that cytokine antagonists increase in the circulation of patients with acute myocardial infarction likely to modulate prninflammatory effects of cytokines. objects of the study: interleukin-6 (il-6) is a multifunctionai cytokine known to be involved in important homeostatic processes. il-6 levels are increased in many pathological situations, and correlates with disease activity and patient outcome. to exert its effect il-6 binds to its receptor on the membrane of its target cell. this receptor is also present in a soluble form in plasma and in urine. in this study we quantitatively measured the availability of soluble il-6 receptor (sll-6r) in biological fluids in healthy volunteers. materials and methods: blood, urine, cerebrospinal fluid (csf), and synovial fluid (sf) are obtained from healthy volunteers, sil-6r and il-6 are measured using elisa's. both elisa's are tested for interference of sil-6r mid il-6. the effect of sll-6r in the il-6 bio-assay (b9) is tested. results: baseline levels are (mean + sd): 76.6 -+ 19.3 ng/ml in serum, 3.7 -+ 1.3 ng/ml in urine, 1.64 _+ 0.4 ng/ml in csf and 11.6 + 3.3 ng/ml in sf. there is no interference of sll-6r in the il-6 elisa or bio-assay and no interference of 1l-6 in the sil-6r elisa. conclusions: in all investigated biological fluids sll-6r can be found. these data provide baseline values for investigations concerning pathological situations. both elisa's described are useful tools to do these investigations. trauma-associated immune aberrations coincide with augmented release of immunoregulatory and proinflammatory cytokines. the present study examines the effect of thermal trauma on the capacity for specific (receptormediated) response of lymphoid cells to interleukin 1 (il 1) and to turnout necrosis factor ~x (tnfc0. methods. bum patients (n=24, 25->75 % total body surface area) were studied weekly up to 42 days post-injury. the limulus amoebocyte lysate (lal) test was used to measure plasma endotoxin levels. the percentage of il 1~-and tnfcz-binding t(cd 5) lymphocytes was assessed by flow cytometry analysis. levels of il 1 receptor antagonist (il lra) in patients' plasma and cultures of peripheral blood ceils (pbc) were determined by immunoassay. results. plasma endotoxin concentrations were significantly (p<0.05) increased up to 3 weeks post-bum (means 3.5+1 in non-surviving and 2.6+ 0.6 u/ml in surviving patients vs < 1u/ml in the control). within 2 weeks of bum, the percentage oft ceils expressing receptors for tnfa and il 1[~ constitutively was elevated (by 10-15 fold). in contrast, the capacity for de novo receptor expression by activated pbc was reduced. serum levels of il ira were significantly increased (range 2.5-40x10 j pg/ml vs <0.2x10 j pg/ml in the control). in all patients, high concentrations of il lm were released spontaneously in unstimulated cultures of adherent ceils (range 20-30x10 -3 pg/ml vs 4-8x10j pg/ml in the control). however, its secretion was decreased in lps-stimulated parallel preparations. conclusions. in the bum patient, susceptibility to the immunoregulatory effect of tnfcz and tl 1~ may be modulated by infection-related products. alterations in the capacity for receptor expression and secretion of 1l lra may affect il 1-regulated biological responses including specific immune reactions. while studies suggest that il-10 is an important lymphokine involved in cell-mediated immunity, little is known about this mediator's role in hem-induced immunesuppression. our aims, therefore, were to determine: i) if il-10 contributes to depressed t-cell responses seen following hem; and 2) how other agents, known to play a role in hem, effect il-10 release. to study this, c3h/hen mice were bled to and maintained at a map of 35 mmhg for 1 h and then adequately resuscitated. mice were killed 2 h post-hem to obtain splenic t-cells (nylon-wool purified). il-10's immunosuppressant role was demonstrated by the ability of monoclenal antibody (mab) to il-10 to markedly improve the t-cell proliferative response [2.5 #g the marked increase in capacity of t-cells from hem mice to produce il-10 was significantly reduced by treatment with either ibu or mabs. since ibu, tgf-~, as well as il-6 are all reported to directly/indirectly influence prostanoid synthesis, this implies that eicosanoids play a major role in inducing il-10 release by t-cells following hem which depresses t-cell function. the mechanisms underlying immunosuppression induced by thermal injury and alcohol ingestion are in part due to cytokine dysregulatinn. il-10 down-regulates production of eytokines by maerophages and may be an important regulator of the initiation of the immune response. il-10 has also been demonstrated to inhibit the production of no by macrophages. this study examined the alterations in eytokine production and effect of inhibition of no production on immunologic function in a routine thermal injury model. methods: balb/c mice (n=40) were randomized to 4 groups: saline-sham(ns-sham), alcohol-sham(etoh-sham), ns-bum, etoh-bum. animals received 20% etoh or ns daily for 14 days by gavage. a 30% full thickness bum was induced 4 hrs after the last dose of etoh or ns. animals were resuscitated, then sacrificed 4 days post bum. splenic lymphocytes were cultured for 5 days with lps, and lps with two concentrations of n-monomethyl-l-arginine, a nitric oxide inhibitor (l-nmma 2.5ug/ml, 10ug/ml). splenocyte production of il-10, interferon-gamma, il-2, pge2 were measured, and lymphocyte proliferative response examined. results: il-10 production was significantly suppressed in thermal injury. exogenous l-nmma normalized the suppression of 11.-10 in a dose-dependent manner, indicating nitric oxide may modulate il-10 and interferon-gamma production in thermal injury. il-10 production is normal in etoh-burn animals. conclusion: il-10 and interferon-gamma production is altered in this murine thermal injury model, and may contribute to this injury-induced immunosuppression. inhibition of no synthesis normalizes il-10 production and should be investigated further as an immanomodalator in thermal injury. surgery, infection and inflammation results in the production of pro-inflammatory cytokines which mediate metabolic and immunologic host responses. the aim of this study was to characterise the elaboration of cytokine release following a variety of surgical procedures. twenty one patients undergoing elective intermediate, hip, knee and major gastrointestinal surgery were studied. levels of interleukin-1 (i1-1), interleukin-6 (i1-6), the interleukin-1 receptor antagonist (i1-1ra) and the acute phase c-reactive protein (crp) were measured in bloods drawn 0, 1, 2, 4, 6, 12, 24 and 48 hours following operation. a portion of the results are shown (mean -+ sem). 135+37 1738-+843 145_+24 one and two factor anova; *p<0.0002, #p<0.0001, §p<0.0009, ¶p<0.0014, for differences between groups i1-1 was not detected at any time point. both ii-ira and i1-6 increased after surgery. maximum responses occurred following major git and hip surgery, minimal responses were seen after intermediate and knee surgery. ii-ira levels increased within two hours and remained elevated for 24 hours; the b-ira increase was a thousand fold greater than the rise in i1-6 levels. i1-6 levels increased up to 24 hours after surgery. crp levels reflected maximum ii-ira and i1-6 levels (r2=.676, p<0.0001 and r2=.282, p<0.04 respectively). high ii-1ra and i1-6 levels reflect major surgery, however the ii-ira response is more rapid and of greater magnitude. the strong i1-1ra correlation with crp may indicate that this regulatory cytokine is itself a mediator of host responses to surgery. dept. of surgery, meath/adelaide hospitals, heytesbury st., dublin 8, ireland. change of il-6 and soluble il-6 receptor levels after surgery s. hisano, k. sakamoto, s. mita, t. ishiko, m. ogawa [objectives] under surgical stress, il-6 plays a main role in producing acute phase proteins and contributes to host defense mechanism. soluble il-6 receptor (sll-6r) is considered to be agonistic to il-6, unlike other soluble type receptors of cytokines. here we measured il-6 and sll-6r levels in the serum and drain fluid from surgical field in order to investigate the changes of il-6 and sll-6r after surgery and their origins. [materials and methods] serum and drain fluid samples from 21 cases (6 of esophagectomy and 15 of gastrectomy ) were serially collected before and after surgery. il-6 and sll-6r levels were measured by elisa. [results] (1) serum il-6 : all cases reached the maximum level on pod-l, more precisely 3-6 hours after operation. (2) il-6 in the drain : maximal il-6 levels in the drain were recognized 3-6 hours after operation, at almost the same time as serum il-6. furthermore the il-6 values in the drain were much higher, about 100 times, than those in serum. (3) sll-6r in the serum : all cases reached minimum levels 3-12 hours after operation and recovered to the preoperative levels a few days later (decrease ratio : 43.9+5.6~,, range : 9-84~'). (4) sll-6r in the drain : sll-6r levels in the drain showed almost the same value and change as serum sll-6r. [conclusions] (1) il-6 is produced from the cells gathering around operative fields whereas sll-6r is considered to be produced in the cells which do not gather around the operative fields. (2) there may be a mechanism that down-regulates sll-6r in the early stage of surgery. [objectives] il-6 plays an important role in host defense in the early stage after surgery. in the present study, we examined changes in il-6 concentration after major thoracoabdominal surgery and elucidated the effect of surgical trauma and factors influencing postoperative elevation of serum il-6. [materials and methods] thirty-eight patients undergoing elective surgery of the thoracoabdomen were classified into 6 groups according to the location of the operation. bloods and drain fluids were serially obtained and samples were frozen until measured, keukocytes were simultaneously collected for northern blot analysis. concentration of il-6 was measured by elisa and il-6 mrna was detected by northern blotting after total rna was extracted by the acid guanidium phenol chloroform method. [results] (1) serum il-6 levels reached the maximum concentration on the 1st postoperative day in all patients. (2) the il-6 peak was significantly correlated with surgical trauma as defined by the operation length and the volume of blood loss during operation (r=0.554, p<0.01, r=0.427, p<0.01, respectively). (3) the peak concentration of serum il-6 in patients undergoing esophagectomy was significantly higher than in those undergoing pancreaticoduodenectomy (p<0.05), despite a similar degree of surgical trauma. (4) peak 1l-6 concentration observed in a patient who underwent esophagectomy was about 100 fold greater in the drain fluid of thorax than in the peripheral blood. (5) il-6 mrna was demonstrated in leukocytes from thoracic and abdominal exudate at 6, 24 and 48 hours after surgery. in contrast, il-6 mrna could not be detected in leukocytes from the peripheral blood. [conclusion] il-6 is mainly produced in the operative field and subsequently enter the peripheral blood to induce cytokinemia. the operation length, volume of blood loss and thoracotomy are factors influencing the concentration of cytokine in the blood. zaragoza spain age may be an important factor influencing the function of immunocompeteut cells releasing cytokines after both accidental and surgical trauma the aim of the present paper is to ascertain if patients (pts) over 60 years old show a different serum level cytokine pattern than pts under 60 after a standard surgical procedure considered as a "medium strength trauma". patients and methods: 33 pts(22 females 11 males)with gallstone disease were perspectively studied, pts were allotted in two groups: gr.a:17 pts under 60 years(mean age:49.5+-7)gr.b:16 pts over 60 years(mean age:65.5_+5). all pts underwent cholecystectomy and cholangiography. 6 pts in gr.a and 7 pts in gr. b underwent common duct exploration. 1 spbintercctomy was performed in each group. on the day of surgery (pre) and on the 1st and 7th postoperative day(leo, 7po) : percentages of cd19, cd5, cd4, cd8 and cd16 cells we measured by means of flow cytometry using moab. and levels of il-1, il-4, il-6 and tnf "in vivo" by elisa using moab. results: ere: cd16% was 5.3_+3 in gr.a and 7. 5 objectives of the study. after surgery for esophageal cancer multiple organ damage has been reported to be caused by polymorphonuclear leukocyte (pmn)-mediated injury. we measured serum granulocyte colony-stimulating factor (g-csf) and interleukin 8 (il-8) levels to determine a role of g-csf and il-8 in pmn function after surgery for esophageal cancer. materials and methods. peripheral pmn counts, peripheral pmn chemiluminescence, serum g-csf levels, and serum il-8 levels were measured before and after surgery in 12 patients with esophageal cancer (ec), and 12 patients of gastric cancer (gc). esophagectomy with thoracotomy and laparotomy were performed for patients with ec, while subtotal gastrectomy with laparotomy were performed for patients with gc. results. peripheral pmn counts (p<0.01) and peripheral pmn chemiluminescence (p<0.05) of patients with ec were significantly decreased compared to those of patients with gc at 4 and 8 hours after surgery. serum g-csf levels of patients with ec were significantly (p<0.01) increased compared to those of patients with gc at 4 and 8 hours after surgery. serum il-8 levels of patients with ec were significantly (p<0.01) increased compared to those of patients with gc at 4, 8 and 24 hours after surgery. significant inverse correlations (p<0.0l) between peripheral pmn count and serum g-csf and il-8 levels were seen at 4 hours after surgery. conclusion. these results suggest that many circulating pmns, which are excessively activated by g-csf and il-8, may adhere to the endotherial cells and then migrate into the tissues, and cause multiple organ damage after surgery for esophageal cancer. immunnogical changes in patients with severe brain trauma receive increasing attention since morbidity and mortality ere still high. interleukin-6 (il-6) was previously detected in the cerebrospinal fluid (csf) during different pathologies of the nervous system (1, 2, 3). in our study we monitored il-6 and nerve growth factor (ngf) production in the csf after human brain trauma. since astrocytes within the brain constitute one of the major cell type contributing to the inflammatory response through the release of cytokines and other factors after injury, we investigated the functional relationship of il-6 and ngf on a single cell niveau using cultured astrocytes. methods csf was obtained from patients with severe brain injury (glasgow coma score (gcs) <8 and ct abnormatities or gcs <8 over 6 hours) after implantation of intraventricular icp monitoring device for therapeutic purpose and collected over 24 hours csf and serum. il-6 and ngf were assayed by elisa. astrocytes were isolated from neonatal mouse brain as described (4) . ngf production by cultured astrocytes was measured by elisa in the presence of csf, il-6 and il-6 antibody. astrocyte migration was tested in a chemstaxis chamber. results 17 head trauma patients were included in this study (approved by the university hospital medical ethics board) and the csf was obtained through intraventricular catheters. high levels of il-6 were detected in the csf of these patients when compared to serum during the first days after brain trauma. furthermore ngf could be found inside the intracerebral compartment. csf containing high levels of il-6 could stimulate ngf production in cultured astrocytes. this effect could be [nhibited partially by il-6 antibodies, purified il-6 exposed to cultured astrocytes in vitro, stimulated the migratory activity of these cells in a dose response fashion. il-6 was found in the csf of brain injured patients, suggesting a role for this cytokine in the pathophysiology of brain injury. since astrocytes are involved in maintaining the homeostasis of the brain, we further investigated the possible role o1 il-6 on astrocyte functions, il-6 promoted ngf production in vivo and in vitro, thus contributing to neuronal cell survival and regeneration. furthermore il-6 stimulated astrocyte migration in a dose response fashion, potentially contributing to astrocytosis following brain injury and inflammation, these results show that il-6 represents a key cytokine in traumatic human brain injury with possible systemic effects, which are at preserlt under investigation. we studied a) the role of tnf and b) the therapeutic effect of a mab to tnf with regard to haemorrhagic shock (hs) related ,pathophysiologic alterations and mortality in rats. method: a prolonged hs was induced by bleeding to a blood pressure of 30-35 mmhg for 180 pin followed by reinfusion of shed blood (sb) and resuscitation with two times of sb volume of ringer's lactate over 50 rain. 15 animals received a bolus dose (20 mg/kg) of tnf mab (celltech, berkshire, uk) at 15 min after resuscitation (tn3). the control group (n = 15) was treated similar to the tn3 group but received ringer's lactate (con). results: at 180 min the prolonged hs resulted in a metabolic acidosis indicated by a significant decrease of blood ph (7.16 + 0.04), hco3-(16.3 ___ 2.0 mm), and base excess (-12.5 + 1.9 ram) values with pco2 (47.9 + 7.7 mmhg) and po2 (136.2 + 20.1 mmhg) in the tn3 with no difference to the con group. immediately after resuscitation (230 min) plasma endotoxin levels were found to be increased in both groups (48.8 + 73.3 in tn3 vs 30.1 _ 25.7 pg/ml in con group) . prior to the treatment with tnf mab (230 min) there was also no difference between plasma tnf levels of the two groups (42.1 + 60.7 in tn3 vs 60 + 141.8 pg/ml in con group). treatment with the tnf mab at 65 rain post-hs improved the 48hour survival rate to 73.3 % as compared to 26.7 % in the control group. macropathologic evaluations revealed frequency of intestinal bleeding in oniy 2 animals in the tn3 vs 11 in the con group. no bleeding in the kidneys was found in the tn3 but in 4 rats in the con group. the significant increase in lung wet weight observed in non-survivors in the con (n = 11) was prevented in animals which died in the tn3 (n = 4) group ((9.3 +_ 2.4 vs 6.3 +_ 1.0 g/kg). conclusion: our data suggest that tnf formation induced by hs in rats is an important mediator for pathophysiologic alterations leading to multi organ failure and lethality. antibodies to tnf might be a useful agent in the treatment of haemorrhagic shock related disorders. 55-+8 n=ll*$ 15-+5 n=7 78_+22 n=5* * p<0.05 vs baseline :~p<0.05 no anesthesia vs anesthesia thus 1) tnf production increased 2-3 fold by 48-72 hrs following trauma in unstimulated blood, but was reduced or not changed after lps stimulation, so circulating leukocytes are probably not an important source of tnf post trauma; 2) anticd18 had no obvious effect on tnf production in unstimulated or lps stimulated blood, relative to vehicle, which suggests that the protective mechanism of anticd18 does not involve tnf suppression; 3) fentanyl anesthesia at 72 hrs following trauma unexpectedly decreased lps-evoked tnf production, which suggests that anesthesia alone can influence an inflammatory response. proinflamrnato~ cytokines have been shown to play a signific~t role in the pathogenesis of sepsis, which is a very common occurrence in born injury. tnfa is infrequently detected in the blood of burned patients, the ability to detect the shed receptors of stnfg has not been determined. serial serum mmples from burn patients were collected from the time of admission until death from septic shock. these samples were analyzed using an enzyme-linked immunosorbent assay (elisa) for stnfr, l-ira, tnf-a, and il-ib. the patients ranged in age from 26 to 72 yeas of age. the percentages of bum ranged from 40% -90%. cytokine concenlrntions vmled from patient to padent irrespective of bum size. tnfa levels were consistentiy in the range of30pgjml -150pg/ml. peaks in the tnfa values were above 90pg/ml and were also associated with a peak in the stnfr levels. these levels began at <10,000pghnl within the in,st 6 ins of injury and gradually increased with time. clinically. ti~ appearance of eytoklnes was independent of positive wound, blood, or respiratory cultures however peak values in tnfa and stnfr were ~ialed with a fluid requirnmenl levels of il-i ra were also elevated independent of clinical findings as well as extent of injury. in pl5 there is a significant corresponding peak in il-trn (>40~8/ml) at the same time as t/~:a and stnfr levels. we aimed to characterise the pattern of secretion of interleukin-1 beta 0l-ii3), intefleukin-6 (il-6) and tumour necrosis factor alpha (tnfa) in multiply injured patients and to relate these results to their clinical condition and outcome. two hourly blood samples were taken from ten patients from the time of injury until 48 hours. cytokine levels were measured using sandwich enzyme-linked immunosorbent assays (elisas). injury severity scores (iss) were calculated and haemorrhage was assessed from the blood transfusion requirement over the 48 hours. patients' ages ranged from 17 to 86 years. iss varied from 9 to 50 and transfusion requirement from 0 to 14 units. five patients died after the study period. ]1,-6 was raised in 9/10 patients (max level 12,500 pg/ml) but was unrelated to condition or outcome. 5/8 showed a rise in il-1b (max level 90 pg/ml) which was negatively correlated to iss (i=-0.789, p<0.02). tnfa was raised in 2/10 (max level 95 pg/ml). peak tnfc~ was positively correlated with iss (1=0.676, p<0.05) and haemorrhage (i=0.602 but p<0.5). il-ib and tnfa production was mutually exclusive. there was no common cytokine profile for these patients. unlike elective surgery there was no correlation between peak 11,-6 and severity of injury: tissue damage may not be the stimulus for the cytokine response to multiple injury. periods of ischemia or hypoxia produce endothelial damage in peripheral organs. tumor necrosis factor-alpha (tnf) plays a central role for regulation of endothelial physiology during septic events, taking influence on vascular permeability and coagulant activity [1] . animal experiments demonstrated a synergism between hypoxia and septic shock on letality, leading to the hypothesis that low oxygen tension leads to enhanced sensitivity of target cells for tnf [2] . radioligand binding studies with ~261odid-tnf on cultured human endothelial cells were performed after incubation in several environmental oxygen tensions (pc2) for 12 hours. data were achieved by nonlinear regression of an idealized saturation curve according to the equation: b = n " k./(1 + k,); b = totally bound tnf; k,: association constant (concentration for half-maximal binding); n: number of binding sites per cell. p_o0o (mm h¢i): _k, (nm}: n (molecules/cell): 130 -150 1.83 ± 0.15 2600 _+ 500 55 -70 1.27 ± 0.15 3600 + 500 25 -35 0,41 ± 0.13 4800 -+ 500 10-15 0.18 + 0.07 5900 -+ 300 presented are calculated values on the idealized curve + 95 % percentiles. hypoxia induces enhanced binding of tnf to specific receptors on the endothelial cell surface in a time-and dose-dependent manner by a mechanism, which is not dependent on oxygen radicals, as shown by additional protocols with radical-scavenging drugs. with respect to former findings about a correlation between growth and tnf receptor affinity [3] , these data lead to the hypothesis that enhanced tnf binding during hypoxia is due to a biochemical conversion of the receptor protein from the low affinity to the high affinity state, possibly by posttranslational phosphorylation of the binding protein by intracel)ular kinases. the proposed involvement of tnf-dependent pathways in pathogenesis of organ dysfunction and multiple organ failure after hypoxia/ischemia may provide a basis for understanding the initiation of hypoxic vascular injury, as manifested by increased permeability and prothrombotic tendency, and, thus, merits further attention. the levels of activity of circulating cytokines (ill, il-6 and tnf-alpha) which are believed to play important regulatory role in response to trauma are determined (by hioassays and respective anti-cytokine antibodies) in mice and rats subjected to scald injury ion c,10 see, °0v bsa, ld 50) and (80 c, 30 see, 20 ~ b ~^)~ , respectively. biphasic increase of cytokine activity was noted in mice: initial increase of il-i and il-6, 1-3 hr following injury and of try activity 6hr after scald, followed by elevated levels of il-i and il-6 at 12hr, with tendency of decrease of activity at later time points. increased activity of tnf was noted 24 hr following injury, in rats, initial, short-lived increase of il-i and tnf activity was detected lhr following injury, folowed by increase on days i and 3 postburn. il-6 increase peaked 3-12 hr after scalding and levels remained elevated 3-5 days following injury. similar kinetics of appearance of proinflammatory cytokines (il-i and tnf-alpha) both in lethal and ncnlethal injury concomitant with differential profile of circulating il-6 activity (early,short-lived increase and later slow decrease of activity in lethal burn injury) with late persistent high levels of activity in nonlethai injury demonstrated in the present study highlight the need for investigation the relationship of these cytokines in burn-injury induced inflammation. zikica jovicic,lnstitute for medical research, mma,crnotravska 17,11002 belgrade~yu. asadullah k (1), woiciechowsky c (2), liebenthai c (1), doecke wd (1), volk hd (1), vogel s (2), v. baehr r (1); depts. of med. immunology (1) and neurosurgery (2) , medical school (char#d), humboldt university berlin, frg in patients after polytrauma or major abdominal surgery a hyperinflammatory phase seems to be followed by the development of a phase of monocyte inactivation. the latter is charaeterised by a decrease of monocytic hla-dr expression and a shift to anti-inflammatory cytokine production. as shown, by us and others, this phenomenon indicates severe immunodepression with a high risk of infection. however, the mechanisms leading to monocyte inactivation in the above mentioned syndromes may be multiple. to elucidate the influence of a selective, sterile trauma to the central nervous system (cns) on immune reactivity the neurosurgieal patient is an interesting model. initially, 30 patients who developed a systemic inflammatory response syndrome following neurosurgery were analysed. in all of them a marked decrease of monocytic hla-dr expression was observed soon after the operation. these results suggest that neurosurgery alone can induce immunodepression and lead us to conduct a prospective study, in which we closely monitored l0 patients undergoing neurosurgery from the first preoperative day until at least day 6 after the operation. hla-dr expression was decreased hi all patients to various extent only hours after surgery. in one patient only we found a persistently reduced hla-dr expression and this was the only patient to develop sepsis syndrome. this suggests that a prolonged, postoperatively decreased hla-dr expression is predictive of infection following cns trauma. in order to assess, whether a decrease of hla-dr expression was associated with a preceding inflammatory response, local cytokine release in the cns was compared with systemic cytokine release. for this purpose, paired samples of earebrospinal fluid (csf) from a vantricle drainage and peripheral blood plasma were obtained. in the csf extremely elevated futerleakin (il)-6 levels, peaking already a few hours after the operation were found. in plasma, by eontrast, il-6( and tnf-alpha) was detectable not until days later and only if infection was present. the antiinflammatory ili-ra, on the other hand, was also present in csf but peaked after il-6 and was detectable in peripheral plasma too. we believe there is an association between the inflammatory response in the cns and the following depression of hla-dr expression on peripheral blood monocytes. our results suggest that even a sterile cns-trauma by itself may contribute to general immunodepressinn leading to septic complications. the aim of this study was to evaluate the effect of haemorrhagic shock (hs) a) on total capacity of the host, and b) the circulating blood cells to produce tnf immediately after bleeding. in vivo studies: baboons were subjected to a limited oxygen deficit (180-200 ml/kg) hypotension phase (mean arterial pressure = map of 35-40 mmhg for 2-4 hours followed by adequate resuscitation). rats subjected to hs (map of 30-35 mmhg for 90 rain followed by reinfusion of shed blood and fluid resuscitation) were challenged with endotoxin (1 ~g/kg i.v.) at the end of shock (rhs group). the control group (rco) received the same dose of endotoxin as rhs group but without prior bleeding. in vitro studies: whole blood (wb) obtained from both baboons and rats before and at the end of hs were incubated with endotoxin (100 ng/ml) for 2 hrs at 37 °c. results: at 90 min post-lps challenge we found significantly higher plasma tnf levels in rats that were subjected to hs prior to the endotoxin challenge as compared to the control group (956 _+ 585 vs 276 + 199 pg/ml) . after hs the tpc was significantly decreased in in vitro stimulated cbc of both rats (12 + 4 post-hs vs 164 + 89 ng tnf/ml pre-hs) and baboons (8 ± 16 post-hs vs 233 ± 188 pg tnf/ml pre-hs). in contrast, the il-6 productive capacity was increased in baboons cbc (not yet analysed in rats) stimulated at the end of hs (224 ± 106 pre-vs 1545 ±_ 1254 pg il-6/ml post-hs). conclusion: from our data we suggest that despite of down regulation of the cbc to produce tnf the overall tpc is enhanced at the early stage of i-is. with regard to the related literature (chaudry's group) it can be assumed that among the macrophage/monocyte populations, as the main source only the kupffer cells (kc) exhibit enhanced tnf production capacity following haemorrhage. the mechanisms of down/up regulation of cytokine response of cbc and/or kc following hs remain to be examined. d. eg~er, s. geuenich °, c. dertzlin~er °, e. schmitt*, r. mailhammer, h ehrenreich #, p. drrmer, and l. h01mer gsf-instimt fox experimentelle h~znatologie, °medizinische kliulk iii, klinikum groghadern, munich, *institut for immunologic, johannes gutenberg universit/it, malnz, and #psychiatrische k/in& der georg-aagust-universi~t, grttingen, germany. it has been shown previously (ehranreich et al., 1992, new biol. 4: 147 ) that mouse bone marrow-derived mast cells (bmmc) synthesize and secrete endothelin-1 (et-i) and express eta-type endothelin receptors (eta). so far, however, no functions of et-1/et a in bmmc have been described. in the present study we investigated the effect of exogeneously administered et-1 on the release of histamine, serotonin, and leukotriene c 4 (ltc4) by primary mouse bmmc (in vitro age: 4 weeks) caltured with different recombinant mttrine cytokines (interleukin 3 (il-3) and/or kit ligand (kl) in the presence or absence of il4) for two weeks prior to activation. et-1 (5x10 -8 to lxl0 -6 m) induced an extremely rapid (_<lmin) and dose-dependent release of histamine and serotonin (up to 20% and 16% of total mediator content, respectively, comparable to ige/ag-indueed mediator release) from bmmc cultured with il-3 and il-4. only when cultured in the additional presence of il-4 rather than in medium containing kl or kl plus il-3 alone, bmmc released histamine and serotoaln upon challenge with et-1.furthermore, et-1 stimulated ltc 4 biosynthesis up to 4-fold in bmmc cultured in the presence of il-4. in contrast, the peptide was without major effect on ltc4 biosynthesis in bmmc culturd without il-4. the release of histamine and sereotonin was not altered if bmmc were preincubated for i h with il-4 prior to activation with et-i, suggesting that a differentiation process rather than a functional priming effect had been initiated by ]l-4. et-1 (10"6m) -induced histamine and serotouln release from bmmc was inhibited by an eta-specific antagonist (cyclic id-asp-pro-d-val-leu-d-tel ) in a dose-dependent manner, with a complete inhibition observed at an antagonist concentration of 10 -8 m. crude peritoneal cells (containing 2-3% serosal mast cells) obtained from normal balb/c-mice released 87% histamine upon challenge with et-1 (10 -6 m; 20 rain). taken together, these resu/ts demonstrate that et-1 can directly function as a histamine and serotohin secretagogue and as a stimulator of ltc 4 production in mast cells. il-4 appears to be involved in the differentiation of immature mast cell prec~trsors towards an et-l-reactive functional phenotype. when inflammatory reactions are advanced, type ii phospholipase a2 (type ii pla2) is produced in high levels by various cells at the site of inflammation. cytokines such as tnf-a and il-i activate type ii pla2 and promote the production of eicosanoid, which is one of the mechanisms by which they enhance the inflammatory reaction. in the present study, the blood levels of type ii pea2, endotoxin, tnf-c~, il-6 and il-8 were determined in patients with sepsis to examine the relationship between these levels and the patients' pathological conditions. the levels of type ii pla2 and tnf-a in these patients were 218.3 ± 179.9 ng/ml and 98.5 ± 92.1 pg/ml, respectively, the two parameters being significantly correlated (r = 0.6460, p = 0.0001 ). there were also a significant correlation between the level of type ii pla2 an il-6 (r = 0.4262, p = 0.0188). there was no significant correlation between the level of type ii pla2 and il-8. these in vivo findings suggest that tnf-a may be involved in the production of type ii pla2. a. filzmaver, g. reisbach, e. schmitt °, r. mailhammer, and l. hiittner. gsf-iustitut ff~r experimentelle h~imatologie, munich, and °iustitut fttr immunelone , johannes gutenberg universit~t, mainz, germany the product of c-kit, a transmembrane tyrosin ldnase receptor, and a corresponding kit ligand (kl) are critically involved in the control of different hemopoietic cell lineages including the proliferation, maturation, migration, and function of mast cells. it has been reported previously that interleukin (il)-4 down-regulates kit receptors in human primary myeloid leukemic cells and in a human mast celt line (sinaber et al. 1991, j. immunol. 147: 4224) . transforming growth factor (tgf) gl, was also found to down-modulate c-kit mrna and kit receptor proteins in human aml blasts, a mechanism probabely contributing to the anti-proliferative effect of tgfih on kl-stimulated aml ceils in vitro (de vos et at. 1993, cancer res. 53: 3638) . in porcine endothelial cells transfected with a retroviral construct carrying the human (hu) e-kit gena, exogenous hu kl transiently down-regulated kit receptors (blume-jeusen et al. 1991, embo j 10: 4121) . in the light of these previous findings we analyzed the effects of these exogenously applied cytokines (il-4, tgfgl, kl) on kit receptor expression and kl-mediated proliferation and chemotactic migration of primary mouse bone marrow-derived mast cells (bmmc) (in vitro age: 4 weeks). our results show that in bmmc cultures initiated with recombinant (r) marine (mu) il-3 the additional presence of rmull-4 for 3 or 14 days did not change the expression of kit protein (assessed by facs analysis with the anti-mouse c-kit antibody ack-2) nor kl-mediated proliferation or chemotaxis. in contrast, il-4 syeergistically increased kl-stimulated growth of bmmc in a short-term proliferation assay (mtt-tes0. pre-incubation of bmmc with rhutgfl~ 1 (5.0, 1.0, or 0.1 ng/ml) for 24h had no effect on kit receptor expression, although tgffil at concentrations of 0.5 -5.0 ng/ml completely suppressed the proliferative action of kl on these ceils. similar anti-proliferative effects of tgfg 1 were observed in various factor-dependent mast cell lines stimulated with different mast cell growth factors (il-3, 1i,-4, il-9, and/or il-10). moreover, pre-incuhation (24h) of bmmc with tgf-gl (>500 pg/ml) significantly enhanced spontaneous undirected cell movement (chemokinesis) and synergistically increased il-3-or kl-induced chemetaxis. when bmmc were preancuhated with rmukl (200 ng/ml) for 1, 3. or 5 days, a transient down-modulation of kit receptors with a maximum effect on day 1 was demonstrated by facs analysis and correlated well with a decreased chemotactic response of these cells. in conclusion our results show that neither il-4 nor tgfi31 affect expression of kit receptors in primary murine bmmc. it is reasonable to suggest that c-kit expression is controlled in a cell type-specific manner.interestingly, tgfgl is obviously able to dissect the proliferative from the migrational signal transducted by kl in these cells. objectives of the study: antisense strategies using dna-otigonucleofides (odn) to modulate the cytokine response are presently under investigation. odn are thought to act very specifically with little or no relevant negative side effects. we now report that odn unspeeifically protect wehi 164 cells from tnf-mediated cytolysis. material and methods: wehi 164 subclone 13 ceils (5 x 105), that are highly sensitive to the cytolytic activity of tnf, were grown on 96-well culture plates in rpm11640 medium. after 24 hours, phosphorothioate(ps)and partially ps-modified-odn as well as phesphodiester-odn (20-29bp) were added (0.1, 1 and 10 pm). four hours after incubation with odn, ce(i lysis was induced by recombinant murina tnf. after 18 hours the plates were washed and stained with crystal violet cell lysis was determined by reading the absorbance (abs) at 590 nm. results: wehi 164 ceils incubated with tnf (1-8ng/ml) were completely lysed after 18 hours (0% abs). interestingly, wehi cells incubated with tnf and odn resisted complete lysis, eg cells incubated with 2.5ng/ml tnf and 11jm odn showed still 30% of the absorbance observed in control ceils without tnf (100% abs). the protective effect of odn started at 0.1pm, reached a maximum at 1,um, and diminished at 101jm. with increasing amounts of tnf the protective effect of qdn decreased and no protection was detectable at 8ng tnf per ml conclusions: dna-oligonucleotides were found to unspecifically inhibit tnf-induced cytolysis. we hypothesize, that this protective effect of qdn results from an inhibition of the binding of tnf to its receptor, or from interference of odn with the subsequent signal transduction mechanisms. as a consequence, to discriminate the specific effect of odn in biologic systems, several control odn should be used. secondly, whether dna released by degradation of tumor cells or leukocytes can significantly impair tumor-and immune-defense mechanisms merits further investigation dr. med. michael meisner, institut for anaesthesiologie der universitat erlangen-nqmberg, krankenhausstral~e 12, d-91054 erlangen. in this study we investigated the involvement of serine protease and free radical generation in the systemic release of tumor necrosis factor-alpha (tnf) and interieukin i(il-1), in the sepsis model of lipopolysaccharide (lps, 5mg/kg i.p.) induced hepatitis in galactosamine (gain, 18rag/mouse, i.p.) sensitized mice. treatment of gain-sensitized mice with lps (gain/lps) led to dramatic increase in serum cytokine (tnf and il-i) ievels and transaminase activity at 3 hr and 8 hr respectively. pretreatment of serine protease inhibitor, c~jantitrypsin (a j-at, 50mg/kg i.p.), 30 rains prior to gain/lps treatment, fully protected the animals against the hepatotoxic challenge with significantly reduced serum tnf and il-1 levels. in order to block and scavenge superoxide generation, the mice were pretreated with xanthine oxidase inhibitor, allopurinol (al, 2 x 100mg/kg i.p.) and pyran polymer-conjugated superoxide dismutase (sod, 2 x 200 unit/mouse i.v) r6spectively. pretreatment with al and sod (24 and 1 hr prior to gain/lps) prevented gain/lps hepatitis and blocked lps induced released of tnf and il-1 into serum of the mice. the protective agents like cq-at or al/sod did not protect the mice against th~ hpp~totoxi£ ch~llpn-e indllee4 b'~ th~ recombinant mmlse tnf-o' (0.3 ~/rno~e j.p.) ~d oi~lps 1~ caln-.~dlfa%aed mlce. it-l cett~aged la tnf (x/gain treated mjde was not detectable in animals pretreated with oq-at or al/sod. our study suggests that a serine protease sensitive to cq-antitrypsin is responsible in regulating tnf release, possibly by proteolytic cleavage of a tnf-precursor or membrane bound tnf. in addition our evidence suggest that the balance of extracellular protease/antiprotease activity may be regulated by free radical generation, possible superoxide anion, resulting in inactivation of the antiprotease. il-1 release may be subsequent to tnf release. objective: during sepsis one can observe a dramatically impaired production of proinflammatory cytokines like the tumor necrosis factor alpha (tnf-a), interleukin i-alpha (il-la), intedeukin i-beta (il-i&) and interferon gamma (if~) upon in vitro stimulation of circulating cells. however there is also evidence of a decreased ability to produce cytokines in other immuno-deficient states. in this study we compared the capacity to secrete proinflammatory cytokines upon in vitro stimulation of patients in severe sepsis and patients with malignant tumors. methods: heparinized blood samples of ten patients (62+ 14 years) in severe sepsis (sepsis score > 15 according to e}ebute and stoner) were drawn at onset of disease, from fifteen patients with solid growing carcinoma (59+19 years) blood was drawn at diagnosis prior to any therapy. controls were obtained from fifteen healthy volunteers. 50 pl of whole blood were incubated either with 450/4 of a standard medium or with 400 pl of a standard medium and 50 pl of phytohemagglutinin (pha) a potent mitogen. after an incubation period of 24 hours plasma concentrations of tnf-a, il-la, il-16 and if-~ were determined by elisa. comments: our results suggest that down-regulation of cytokine secretion or of cell responsiveness to non-specific mitogens during sepsis has occurred. we observe a similar phenomenon for the group of carcinoma patients vs control significant for stimulated tnf-a and stimulated if-t. sustained immunological interactions between tumorcells and cytokine producing cells could effect responsiveness of the latter, a general increased immuno-tolerant state in patients with carcinoma has to be discussed. however we found significant differences between sepsis and cancer concerning the in vitro capacity of responsable cells to produce il-la and il-i#. the dramatically decrease of the ability to produce il-i upon in vitro stimulation could be more sensitive for a septic state than stimulated tnf-a or if-3,. objective: tumor necrosis factor alpha (tnf-a) has been implicated as a central mediator of sepsis and its sequelae. increased systemic levels of this cytoklne seem to be correlated with severity of sepsis and outcome. however mechanism of action and metabolism of tnf-g are not fully understood. in most studies blood samples for tnf-a determinations are obtained either by peripheral venipuncture, a central venous catheter or by an indwelling arterial catheter. very often blood samples are taken in different manners within the same study. in this study we measured circulating tnf-a and the amount of tnf-a released upon in vitro stimulation in arterial and central venous blood. methods: heparlnized arterial and central venous blood samples of ten patients (6 males, 4 females, mean age 62+_14) with severe sepsis (sepsis score > 15, elebute and stoner} were drawn on day 1,3,6,8, and 14 of disease. blood was immediately placed on ice and processed within 1 hour. 50 pl of whole blood were incubated with 450 pl rpmi-medium supplemented with antibiotics and l-glutamlne or with 400 pl of rpmi-medium and 50 pl phytohemagglutinin (pha) a potent mitogen. after an incubation period of 24 hours samples were centrifuged and plasma was harvested and stored at -30 ° celsius before assessment of tnf-a concentration by elisa. statistical analysis was performed with the paired student-t-test. results: we found a significant difference (p < 0,05) for circulating mean arterial tnf-a concentration (237 pg/ml _+ 34 sem} and central venous tnf-a (203 pg/ml +_ 29 sem). upon in vitro stimulation there was also a significant difference (p < 0,002) between released arterial tnf-~' {746 pg/ml _+ 88 sem) and venous tnf-a (637 pg/ml +_ 76 semi. conclusions: these results are difficult to interprete but could reflect the influence of pao 2 and sao 2 on tnf a release. it could also be the result of different concentrations of tnf-o release influencing factors like for example endotoxin, interferon-f or prostaglandin. a possible pulmonary and/or a hepatic metabolism of tnf-n and tnf-a producing cells cannot be ruled out. however for better interpretations of tnf-a release in septic states it is necessary to use either arterial or venous blood samples. early inflammatory processes following trauma and/or infections were found to be associated with the secretion of high amounts of proinflammatory cytokines. besides intedeukin-t (il-1), tumor necrosis factor-a (tnf-c 0 and interleukin-8 (il-8) the multifunctional cytokine intedeukin-6 (il-6) was described to be a central regulatory element of the primary cellular and humeral defence reaction. the previously described close temporal correlation of pathologically elevated il-6-concentrations and the extracellulary release of lysosomal enzymes from activated pelymorphnuclear neutrophils suggests, that il-6 may be a potential substrate of these preteases. the serine preteases elastase (ec 3.4.21.37 ) and cathepsin g (ec 3.4.21.20) derived from the azurophilic granules were assumed to be mainly involved in unspecific proteolysis at sites of inflammation by cleavage of structural as well as soluble proteins at random sites, if the inhibitory potential is decreased. the possible proteolytic activity of elastase and cathepsin g toward the proinflammatory cytokine interleukin-6 (il-6) was investigated. the addition of purified neutrephil elastase and cathepsin g to recombinant human il-6 leads to a rapid sequential degradation in vitro. at least two intermediate products could be detected by silver staining and western blotting following protein separation under reducing conditions. the serine protease inhibitor g-anitrypsin was shown to prevent the proteolytical degradation of intedeukin-6. furthermore the loss of the biological activity of both, recombinant and natural human il-6, was demonstrated by determination of the capacity of protease-treated il-6 to stimulate hybddoma growth (7td1 bioassay). these data suggest a possible downregulation of pathologically elevated il-6 levels by proteolytic activity of extracellulary released enzymes at sites of inflammation. the aim of the study was to compare circulating levels of three cytokines -il-1, il-6, 11_-8 -between critically ill subjects who developed gram-negative sepsis and who did not. materials and methods: the patient population consisted of 39 patients admitted to an intensive cars unit, with different underlying diseases. sepsis diagnosis was given according to pre-estabilished cdteda. nineteen cases were enrolled in sepsis group, twenty in control group. serum sampling was collected in sterile tubes at study entry and every three days until study dismissal. serum concentrations of il-1, 11_-6 and il-8 were measured using commercially available test kits, based on the dual immunometric sandwich principle. results: the causative patogens of sepsis were: pseudomonas aeruginosa, acinetobacter, eseherichia co~i, serratia marceseens, proteus mirobilis and citrobacter freundl the time of observation was equal to 12 days, for a total of four tests performed (to, tl, t2, t3). i1.-1 was not detected in any samples. the serological profiles of the two cytokines 11.-6 and 11_-8 were similar; augmented levels were found at study entry and throughout the observation period, peaking at t3 and decreasing at t4. however, in patients with sepsis, il-6 and 11_-8 concentrations were significantly higher in respect to control group. conclusion: our observations shown that in icu patients increased il-6 and il-8 release may be induced by cdtical illness; however, in subjects in which sepsis occurred, il-6 and il-8 production appears more significantly elevated, suggesting a role of il-6 and 11_-8 in the pathophysiology of sepsis. the fact that ii. objective: to check whether continuous veno-venous haemofiltration (cvvh) could remove the cytokines, namely tumour necrosis factor alpha (tnfc 0 and interleukin 6 (il-6) from the circulation of critically ill patients with sepsis ad multiple organ failure (mof). setting: the intensive therapy unit of the medical school teaching hospital. patients: nine critically ill patients with sepsis and mof treated with cvvh. methods: blood samples were collected before the cvvh had been started. then, blood and ultrafiltrate samples were collected simultaneously after 4 hours and every 24 hour. tnfct and il-6 levels were measured using the bioassays with cell lines wehi-164 ci13 and 7td1, respectively. other data were recorded from the patient notes and intensive therapy unit charts. results: no measurable concentrations of tnfct were detected in either blood or ultrafiltrate samples. il-6 was found in all the patients' plasma samples and five patients' (55.5%) ultrafiltrate samples. the il-6 blood level ranged from 17.4 to 3061.7 u/ml (mean 518.2, sd 686.8). the il-6 level in positive ultrafiltrate samples ranged from 21.0 to 1150.2 u/ml (mean 252.2, sd 401.8). conclusions: our preliminary results suggest that il-6 is present in bloodstream of septic patients. we assume we could not detect tnfa in any sample because we usually started observations when septic state had developed. cvvh could extract cytokines from the circulating blood. it remains under discussion, whether that extraction may be beneficial to patients with mof. the pattern of some significant cytokines tnf, il-1 and il-6 and their pharmacomodulation were evaluated in an experimental model of polimicrobial sepsis induced in cd-1 mice by cecal ligation and puncture (clp) in order to understand their roles. this model of sepsis, which resembles the clinical situation of bowel perforation, was also compared with that induced by administration of pure endotoxin (lps). tnf was detectable in serum and tissues during the first 4h with a peak 2h after clp at a significantly lower level than after lps. il-1 was measurable in serum only after 24h, significantly increased in spleen and liver after 4 and 8h and in mesenteric lymphonodes from 8 to 24h after clp compared with shammice. il-6 was significantly increased in serum throughout the first 16h after clp. pretreatment with dexamethasone (dex), ibuprofen (ibu) and nitro-l-arginine (n-arg) significantly reduced the survival time while chlorpromazine (cpz) and tnf did not affect it. only the antibiotics and pentoxifylline (ptx) significantly increased the survival in clp. however cpz and dex protected from lps-mor~ality. in conclusion, by inhibiting tnf with dex, cpz, ptx a reduced, unchanged and increased survival time was observed and by increasing tnf with ibu and tnf administration the survival was decreased or unchanged respectively suggesting that the modulation of this cytokine does not seem to play a significant role in clp unlike lps_ moreover the negative effects of ibu and n-arg suggest an important and protective role by prostaglandins and no in clp. to gain more insigths on the contribution of tnf~, il-i~ and if 7 to lps toxicity, we explored the time-course of the cytokine production in ealb/c mice given different doses, from the lethal (= 3 ld50) to the sublethal (= 1/3 ld50) of three different lps (e.coli oiii:b4 and 026:b6; p.aeruginosa r261) endowed with different degree of toxicity cytokines were measured in serum and organs with specific elisas up to i0 h after lps administration. results demonstrate that i) circulating and organ levels of tnf~ do not reflect lps toxicity. in fact, the lethal dose of lps 026:b6 induced as much tnf~ as the sublethal dose of lps 0111:b4; furthermore, lps r261, whose cytokine inducing capability is far lower than that of lps from e.coli, induced higher tnf~ levels at the sublethal than at the lethal dose. in addition, policlonal anti tnf ab, that were able to protect mice from e.coli lps induced mortality, failed in mice treated with lps r261 2) circulating il-i~ levels are generally low and increase significantly only in muribond animals. on the contrary, in spleen and lung very high levels of il-i~ are persistent from i to 8 h post lps administration moreover, the treatment with 6 mgr of neutralizing policlonal anti il-i~ ab, did not modify survival in lps challenged mice. 3) circulating and organ levels of if 7 are proportional to the dose and degree of toxicity of all the administered lps even if lps r261 was again a less efficient cytokine inducer than lps from e.coli. csa is an immunos~ppressive drug, able to inhibit gene expression for many cytokines, including if 7. to study the effect of cytokines modulation on lps toxicity, csa was administered to mice twice at the oral dose of i00 mg/kg before the challenge with lps. mice were monitored in terms of mortality and tnf~, il-i~ and if 7 production. together with the total ablation of if7, the strong reduction of tnfu and unmodified il-i~ levels, a significant increase of lps toxicity was also observed. these results suggest the hypothesis that the numerous factors that jointly mediate lps toxic effects, can also be protective, the final outcome depending on their relative ratio rather than on the absolute amount interleukin-1 (il-1) mediates the septic shock syndrome and affects intestinal secretion in vitro. we studied the intestinal production of il-t and its effects on diarrhea during endotoxic shock. cd-1 mice were randomized to 15 mg/kg e.coli 0111:b4 lps or saline infusion (i.p. or i.v.). diarrhea invariably occurred following lps infusion. mice were sacrificed at 0, 30', lh, 2.5h, 4h, 6h, 12h, and 24h (3 mice/group/time-point). the small bowel was compressed and the intestinal contents were weighed and expressed per g sb weight. the small (sb) and large bowels (lb) were eventually frozen, weighed, and homogenized for either cytosolic protein or total rna. il-i~ (cell-associated agonist) was measured with a radioimmunoassay specific for mouse il-l~ (detection limit 100 pg/ml) and expressed as ng/g weight + sem (lowest detectable amount 1 ng/gwt). northern analysis of total rna and in sfu hybridization of paraformaldehyde-fixed frozen tissue were done with [0~-32p]-iabeled mouse il-lc~ cdna probes. only sb had il-i~ constitutively present (6.2 + 1.6 ng/gwt). lps i.p. or i.v. induced elevation of il-lc¢ in both organs in a biphasic pattern; lps i.v. induced 3-fold more il-i~ than lps i.p. following lps i.p., il-i~ in sb was 19.2 + 0.6 ng/gwt at lh, reached maximal levels at 2.5h (31.8 -+ 0.5 ng/gw-i) and returned to baseline at 24h. saline controls maintained their constitutive il-i~ levels. sb had 2fold more il-1 ¢ than lb and identical kinetics, but lb showed a clearer doseresponse. northern analysis of sb-total rna showed induction of il-i~ mrna by lps in correlation with il-lc¢ kinetics. il-i~ mrna producing cells were mononuclear cells in the lamina propda and epithelial cells at the bottom of the crypts of ueberkuhn. mucus and fluid were increased in the small bowel post-lps in correlation with intestinal il-lc~ kinetics (r2=0.9). separate mice were pretreated with saline i.p. orthe il-1 receptor antagonist (irap, 30 mg/kg bolus i.p.) and were challenged 20 rain later with 1.5 mg/kg lps i.p. or saline i.p. specific blockade of il-1 by irap decreased intestinal secretion at 4h and 6h post-lps challenge (p<_. 0.05, student's-t-test). these data indicate that local (intrinsic) intestinal il-i~ mediates sepsis-induced intestinal changes. inflammatory cytokines initiate the host response to endotoxemia, causing severe physiological and hemodynamic changes which may lead to septic shock. among the regulatory systems that play an important rote in controlling host inflammatory responses is the pituitary. it has been known for many years for example, that hypophysectomized animals are extremely sensitive to lps lethality. while investigating the possibility that protective, pituitary mediators might explain this phenomenon, we identified the cytoldne mif to be a specific secretory product produced by pituitary cells in vitro and in vivo after lps challenge. analysis of serum mif levels in control, t-cell deficient (nude), and hypophysectomized mice revealed that pituitary-derived mif contributes significantly to the rise in serum mif that occurs after lps administration. of note, pituitary mif content (0.05% of total pituitary protein) and peak serum mif levels (80-340 ng/ml) were determined to be within the range observed for other pituitary hormones that are released after pituitary stimulation. to investigate a possible beneficial role for mif in septic shock, we co-injected mice with purified, recombinant murine mif (rmif) together with lps (15 mg/kg). surprisingly, rmif markedly potentiated lps lethality compared to control mice that were injected with lps alone (85% vs. 35%, p = 0.003). to confirm these results, mice were treated with anti-rmif antibody prior to injection of a high dose of lps (17.5 mg/kg). anti-rmif antibody fully protected mice against lps lethality, increasing survival from 50% to 100% (p = 0.0004). serum levels of tnf,~, the first cytokinc that appears in the circulation after lps challenge, were reduced by 38.0 _+ 9.5% in anti-rmif-treated mice. we conclude that pituitary derived mif contributes significantly to circulating mif in the post-acute response in endotoxemia and may act in concert with other pituitary mediators to regulate both pro-and antiinflammatory effects. moreover, mif may play a critical regulatory role in the systemic host response in septic shock. our results suggest that anti-rmif antibody might be of potential therapeutic use in the treatment of septic shock. although anti-interleukin-1 (il-1) antibodies and il-1 receptor antagonist have been shown to improve survival in animal models of endotoxemia and abrogate the lethal effects of tnf, the presence of il-1 in the serum does not correlate well with outcome. we hypothesized that this may be because il-1 acts mainly in a paracrine fashion and is metabolized before it diffuses into the circulation. methods: we measured the il-i~ mrna expression with the differential reverse transcription polymerase chain reaction (rt-pcr) using g-actin as internal standard in the peritoneal macrophages and lung tissue in normal controls and mice after cecal ligation and puncture (clp). clp resembles human intra-abdominal sepsis in that it is characterized by very slight elevations of serum il-1 levels. results: il-lg mrna levels after clp are expressed as % of normal (mean+sem, n=5 in several experimental models of infection exacerbation of disease was observed, when infected animals were depleted of tuajor necrosis factor (tnf). after sublethal cecal ligation and puncture (clp) leading to peritonitis and sepsis the survival of mice also critically depends on tnf as demonstrated in earlier studies, when clp-treated mice injected with anti-tnf antibody died, whereas mice injected with a control antibody survived after clp (echtenacher et al. 1990, j. inununol. 145: 3762) . from a panel of different cell types (macrophages, neutrophils, t lymphocytes, natural killer cells, mast cells) able to produce tnf upon activation~ the mast cell is apparantly the only one capable of storing in cytoplasmic granules preformed tnf-ct which is rapidly released following challenge. in the present study-we analyzed serum tnf after lps injections as well as the outcome of clp in severely mast cell deficient mutant mice (wav v) as compared to syngeaeic wild-type littermates (+/+). we proposed that concentrations and/or kinetics of serum tnf should be different between wavv mutants and wild-type mice, if mast cell-derived tnf significantly contributes to the rise in serum tnf levels following systemic stimulation with endotoxin. although similar levels of increased tnf were detected in the sera of both genotypes after 1 and 2 hours of lps injection (100 btg/ 0.5 ml / mouse i. p.), mast ceil-deficient mice indeed showed decreased serum tnf levels 30 iron after injection amounting to only 12 to 25% of the concentrations observed in the corresponding sera of normal wildtype mice. in the clp model of septic peritonitis we found that mast celldeficient mutant mice were dramatically more sensitive to clp than syngeneic normal mice resulting in 92% mortality in w/w v versus 8% mortality in +/+ mice 2.5 days after initiation of clp. further experiments with w/w v mutants selectively reconstituted with cultured bone marrow-derived mast cells from normal syngeneic wild-type mice and the use of an antibody specifically blocking the action of tnf tn vivo should clarify a potential protective function of mast cells in this model of septic peritonitis. interleukin-10 (il-10) inhibits cytokine production, including tumor necrosis factor (tnf), by lipopolysaccharide (lps)-aetivated maerophages. we recently observed that lps injection (e.coli 055:b5, 100 gg ip) into balb/c mice induces the rapid release of circulating il-10 (17±9 u/ml at 90 min). blocking endogenous il-10 using monocional antibody (jes5-2a5, 2 mg, 2h before lps) resulted in a massive increase in tnf production (107±47 in lps+anti-il-10 treated mice vs 6±2 ng/ml in lps alone, p<0.05, n=4 to 5 mice per group) and an enhanced lps-induccd lethality (53% vs 7% in anti-il-10+lps or lps alone respectively, p=0.01, n=15 mice per group). irrelevant igg1 rat monoclonal antibody (lo-dnp) did not influence neither tnf production nor lethality associated with endotoxin shock. this led us to study the production of il-10 during human septicemia. plasma samples were obtained from 65 patients with gramnegative (gns, n=22) or gram-positive septicemia (gps, n=43) and from 20 healthy volunteers. among these patients, 17 suffered from septic shock at the time of sampling. il-10 levels were measured by elisa (detection limit: i 1 pghrd). we found that 35 patients (54%) had increased il-10 plasma levels (range 12 to 2740 pg/nd). patients with gps had il-10 levels similar to the ones observed in gns (median: 12 vs 17.5 pg/m, respectively). patients with septic shock had higher il-10 values (median: 58 pg/ml) than septicemic patients without shock (11 pg/ml, p=0.002). no il-10 was detected in plasma from healthy volunteers. we conclude that il-10 is produced daring human septicemia. our experimental data suggest that il-10 might be involved in the control of the inflammatory response induced by bacterial products. dr arnand marchant, immunology department, hopital erasme, 808 route de lennik, 1070 brussels, belgium. to provide information about the role of tnf in sepsis and mods we measured tnf and stnfr-i levels in septic patients and investigated if there is a relation between plasma concentration of these molecules and the severity of sepsis evaluated by two scores (apache 1i and sss). patients and melhods: 20 septic patients fullfilling sepsis criteria of american college of chest physician and society of critical care medicine were studied. tnf-cc and stnfr-i (60kda) were measured by enzyme immuneassays (norms1 values = 13+8 pg/ml and 1.06_+0a ng/ml respectively). results: the mean tnf and stnfr-i values for each patient (mean+sd) were 93+64 pg/ml and 11.1+5.5 ng/ml respectively. these values are approximately seven and ten times greater than those observed in normal healthy volunteers (p<0.001). mean tnf concentrations for each patient were significantly greater in non survivors (112+52 vs 64_+74 pg/ml p<0.05); stnfr-i levels also were greater in this group, but the difference was not statistically significant (12.6+5.2 vs 8.8_+5.5 ng/ml). plasma tnf and stnfr-i concentrations were significantly correlated (r = 0.65 p<0.005). mean tnf levels were significantly correlated with apache ii (r = 0.49 p<0.05) and sss (r = 0.54 p<o.01); stnfr-i levels were likewise correlated with both scores (r = 0.80 p<0.001 and r = 0.76 p<0.001 respectively). tnf and stnfqgi levels increase with the number of dysfuctional organs; in particular tnf and stnfr-i increase when hemodynamics deteriorate and kidney failure ensues. conclusions: the correlations between tnf and stnfr-i levels and sepsis severity scores suggests that tnf is involved in sepsis and may influence the occurrence of mods and finally clinical outcome. tnf and stnfr-i are in fact especially increased when mods occurs. in particular their concentrations are elevated when cardiovascular system and kidney failure ensue, suggesting that tnf can have a direct role in hemodynamic derangements caused by sepsis and that kidneys are involved in tnf and stnfr-i excretion. lif is a pleiotropic cytokine that has been implicated in the pathogenesis of sepsis in animal models. we conducted a prospective study in 33 septic patients to correlate lif plasma levels with patient's clinical and biological data (among them il-6). plasma was drawn daily from day one to .ten and lif presence was determined with a specific elisa and with the dai,a bioassay (sensitivity of 100 pg/ml and 1 ngml respectively). risk factor associated wftt~ with elevated lif plasma level was determined by an univariate analysis. a multivariate stepwise logistic regression analysis was subsequently performed with a bmdp statistical software. lif was detected with the elisa and with the bioassay in 11 and 2 out of the 33 patients, respectively. lif peak plasma levels were statistically associated with high creatmin levels, temperature and il-6. multivariate regression analysis showed that high serum creatinin level was the major independent risk factor associated with elevated lifplasma levels. this correlation was also found for il-6. peak plasma levels of both lif and il-6 correlated inversely with patients survival duration. cox's analysis for plasma levels of lif >480 pg/ml yelded a hazard ratio of [exp (1.86)=6.44]. our study indicates that lif levels were associated with clinical and biological parameters of illness severity and significantly increased (cut-off value 480 pg/mi) in patients with fatal outcome. current consensus exists about the central role of tumor necrosis factor (tnf) alpha in initiating the systemic inflammatory response syndrome (sirs). a correlation with sirs has inconsistently been found. tnf effects its pleiotropic reactions upon two distinct cellular receptors. soluble extracel]ular fragments of the human 55 kda tnf receptor (stnfri) and the 75 kda receptor (stnfrii) are detectable in the circulation. the kinetics of these endogenously produced tnf-inhibitors were measured to evaluate their role in patients with sirs. fourteen patients of an operative icu were included with the diagnossis of sirs (mean apache ii score: 20 points). serial blood samples were obtained within 12 h after diagnosis of sirs, every 6 hrs for the first 48 hrs and every 12 hrs thereafter until patients died or recovered. soluble tnfri and stnfrii were assayed by an enzymed-linked immunological binding assay. soluble tnfri and ii could be detected in all samples with a significantly higher level (p<o,o01) compared to healthy controls (normal value: tnfrh 1,5 ng/ml; tnfrii: 2,3 ng/ml). the serum concentration of tnfri (14,1 ng/ml) as well as tnfrii (18,2 ng/ml) were significantly higher in nonsurvivors (n=6) compared to survivors (8,6 and 9,5 ng/ml; n=8) at the onset of sirs and during the course of the inflammation response (p<0,05). a positive correlation exists between both receptors (r-0,57; p < 0,01 ). increasing levels and maximal values of stnfri (59 ng/ml) and i1 (82 ng/mi) were detected only in patients who died. the serum concentrations of patients who recovered did not change and are remaining at the initial level. tnf alpha bioactivity was detected in 10 out of 14 patients. no significant correlation exists between the concentration of both receptors and tnf alpha as well as the apache ii score at the onset of sirs. elevated serum concentration of stnfri and 11 are found in patients with sirs. these naturally occuring antagonists reflect the inflammatory process. the measurement of soluble tnf receptor levels may be useful in monitorina sirs and in the prediction of outcome. 11-2 is given to patients with advanced melanoma or renal carcinoma. however, this therapy is accompanied by severe side effects, including the vascular leak syndrome (vls) that closely resembles septic shock. to investigate the involvement of cytokines and phospholipase a z in the pathogenesis of the vls we collected serial plasma samples from patients during the first 24 hours after administration of il-2. in these patients we monitored temperature, blood pressure and heart rate and assessed levels of the cytokines tnf, il-6 and il-8 using immuno assays. in these plasma samples we also measured phospholipase a 2 activity using an enzyme activity assay, since this enzyme is beleived to play a major role in the generation of lipid mediators. we demonstrate that during il-2 therapy the cytokines tnf, il-6, and il-8 are produced and that the release of these cytokines is followed by an induction of phospholipase az activity in the plasma of these patients, we further discuss the role of these mediators in the development of the vascular leak syndrome observed in patients receiving il-2. objectives of the study: sepsis caused by candida albicans (ca) is no longer a clinical rarity but a common consequence of immunosuppression and surgery, and is associated with high mortality. tumor necrosis faetor-c¢ (tnf-<~), interlcukin-lg (il-lg) and interleukin-6 (il-6) are thought to play an important role in the pathogenesis of the sepsis syndrome. we therefore studied the in vitro production of tnf-<x, il-ib and il-6 by human peripheral blood mononuclear cells (pbmc) stimulated by ca compared to stimulation by bacterial lipopolysaccharide (lps). materials and methods: pbmc were isolated from venous blood of 5 healthy volunteers and cultured at a concentration of 2.5 • 10 ~ eells/ml in culture medium with a dose-range of either heat-killed ca or lps. supernatants were harvested after 24 hours. using elisa, tnf-c~, il-ib and il-6 concentrations were measured. ca was isolated from a patient with ca sepsis and cultured under sterile conditions. results: ca and lps stimulate the production of tnf-~t, il-11] and il-6 by human pbmc. we observed a dose-dependent synthesis of these three cytokines in human pbmc. tnf-c¢, il-ib and il-6 in ng/ml as mean _+ standard error. ca/ml 0.0 1. (2) we constructed a single-chain fv-fragment (sc-fv) from the variable domains of a neutralizing antibody to human tumornecrosisfactor-alpha (tnf) because sc-fv should have some advantages in vivo. the sc-fv has a similar affinity (3,8x10-9m) as the fab-fragment (2,7x10-9m) but bind the antigen with a eightfold lower avidity compared to the parental mab (4,8x10-10). the parental mab as well as its fragments can compete the binding of rhutnf to both tnf-receptors. this was shown by competitive binding to the cell line hl-60 and to immobilized rtnf-receptors. in the nutralization assay on the cell line, l 929, the sc-fv can neutralize tnf but in comparison to the mab or its fabfragment, the capacity was three-fold lower as expected from the molar ratio of the dissociation constants. the in rive-neutralizing capacity was tested in a mice receiving toxic dose of rhutnf. the sc-fv showed a 15-fold lower neutralizing capacity in comparison with the fab-fragment. this could be explained in different manners: i) partial instability of scfv at 370c, ii) shorter half-life because sc-fv but not fab-fragments are eliminated via kidney, iii) elimination of tnf-immunocomplexes may be improved by opsonization mechanisms which requires some structures of the constant region (as in fab or mab). in summary, despite the encouraging in vitro experiments, the in vivo data suggest that sc-fv are not the right strategy for neutralizing tnf in vivo. tnf production in endotoxin non-responder mice, effect of a glucocorticoid antagonist g. iazgtr jr, e. duda, j. ol~th, e. husztik, g. iazar glucocorticoids inhibit lipopolysaccharide (lps)-induced tnf production and tnf can stimulate pituitary adrenocorticotropin secretion, resulting in the release of corticosterone. earlier studies (1) reveales that the glucocorticoid antagonist ru 38486, nufepristone, sensitizes both endotoxin responder and nonresponder, c3h/hej, mice to endotoxin lethality. we have also shown that the glucocorticoid antagonist ru 38486 sensitizes endotoxin-tolerant (endotoxin-pretreated) and normal mice to the lethal effect of septic and endotoxin shock. on this basis, we set out to study the influence of ru 38486 on tnf production induced by endotoxin in endotoxin responder and non-responder mice. one and 2 hrs following lps injection, ru 38486 significantly increased the tnf concentration in the serum, liver and spleen of treated animals as compared with the control and methylprednisolonetreated groups. in tissue cultures, ru 38486 induced the tnf synthesis of myeloid cells and increased the tnf production of genetically modified hela cells, which synthesize tnf constitutively. normal and tumor cell cultures exhibited an increased sensitivity toward tnf in the presence of ru 38486. however, the same dose of lps did not induce tnf production in the serum, liver and spleen of endotoxin non-responder mice and this was not influenced by concomitant ru 38486 treatment. these studies suggest that ru 38486 aggravates lps lethality via factors other than tnf in endotoxin non-responder, c3h/hej mice. the cytokine response to a novel exnacellular mitogenic factor, mf, produced by the group a streptococci (gas), and to the streptococcal pyrogenic exotoxins (spe) a and b, was determined by in vitro stimulation of peripheral blood mononuclear cells (pbmc) obtained from healthy blood donors. the induction and kinetics of 14 different cytokines was studied at single-cell level using cytokine specific monoclonal antibodies and intracellular immunofluorescent staining. all three mitogens induced a massive ifny and tnfi3 response in 10-16% of the pbmc after 48-96 hours of cell stimulation. in contrast, il2 and tnfc~ containing cells was only detected in 1-3% of the pbmc. the induction ofil3, il4, il5, and ill0, was weak or completely absent. thus, the response indicates activation of th1 cells. however, illc~, illl3, illra and il8, were consistently found and gradually produced, peaking at 24 hems in approximately 5-8% of the pbmc. mf induced an equally extensive cytokine as well as proliferative inducing capacity, as did spea and speb, which suggests that also mf is a superantigen. a marked inter-individual variation could be noted between lymphocytes isolated from different individuals, both in the toxin induced proliferation and cytokine induction. this may be one explanation for the varying clinical severity noted in patients after infection with clonal gas strains. introduction -tumour necrosis factor (tnf) is released by mononuclear cells in response to inflammation and sepsis. it has been implicated as a possible mediator in inflammatory bowel disease (ibd) but its pathogenic role remains uncertain. this study assessed the relationship between tnfct and disease activity by measuring plasma and faecal tnf concelnmtions in an experimcntai model of ibd. methodologo' -colitis was induced in male wistar rats by intmcolonic instillation of 30rag 2,4,6-trinitrobenzenesulphunic acid in 0.5ml 50% ethanol (n=12). control animals received an isovohlmetric instillation of normal saline (n=12). faecal pellets were collected before induction of colitis (day 0) and throughout the experiment. after 8 days the colon was excised, ueighed and the colon macroscopic score (cms) assessed plasma tnf (ptnf) samples were assayed by bioassay and elisa. faecal samples were vortcxed in water and the supernatant removed for estimation of protein and faecal tnf (itnf) content (elisa). there is a significant increase in itnf on day 4 -#p=0 02 alld the total ftnf measured during the study correlatcs with the cms oll day 8 -p=0.04 there is no correlation between ftnf and ptnf. conclusions -therc is evidence of coloific tnf excretion by macroscopically normal animals and following induction of colitis there is increased faecal tnf which correlates with disease actmty. there is no significant elevation in bioactive or imnmnoreactive plasma tnf in colitie animals this data would suggest that faecal tnf is a marker of colonic inflamrnatiort and serial tnf assessment inay be useful as an indicator &severity in ibd. to study the effect of in-vivo hypoxia/reoxygenation on cytokine elaboration by routine peritoneal macrophages. materials and methods: adult male cba mice (20-25g) were primed intraperitoneauy with either lipopolysaccharide (lps) 10ug]anlmal or saline. five days later, the animals were subjected to hypoxia (8%02) for 160, followed by 20 of normoxia. harvested peritoneal macrophages were plated in-vitro, culture supernatants were collected at 20, 40, and 60 and assayed for tumor necrosis factor (tnf), prostaglandin e2 (pge2) and nitric oxide (no). control animals underwent the same procedures, but were maintained in normoxia (21%o2 disease 5, berlin, germany the precise mechanisms of reactivation of latent human cytomegalovirus (cmv) infection are still unknown. apart from the permissive effect of diminished cellular immunity there is evidence of a cytokine mediated cmv reactivation as it is known for other systemic virus infections (e.g. hiv). we found that tumor necrosis factor-alpha (tnf) -in contrast to all other cytokines tested -can stimulate the activity of the cmv-ie-enhancer/promoter region in the human monocytic cell line, hl 60. we wondered whether our in vitro results were actually reflected by the incidence of cmv reactivation in diseases which go together with high tnfalpha levels. cellular immunodeficiency as well as at least temporarily increased tnf levels are known to occur in septic disease. we tested for the presence of cmv infection in peripheral blood mononuclear cells (pbmnc) by means of 24h centrifugation culture, immunocytological detection of different cmv antigens (apaap-technique), and pcr technique (cmv-ie dna). in striking contrast to healthy controls almost all examined septic patients were positive, irrespective of the method for cmv-ddtection applied, including the less sensitive immunocytology. follow-up studies showed that cmv-antigen positive pbmnc ceased to be detectable in patients with good outcome once septicaemia had been overcome. to further back up our hypothesis, we looked for a coincidence of enhanced tnf-alpha serum levels and cmv antigenaemia (immunocytology) in some other diseases which are known to have either enhanced tnf serum levels or increased incidence of active cmv infection and found the majority of patients with b-cell chronic lymphocytic leukemia, liver cirrhosis, and common variable immunodeficiency to be positive for these two parameters. we now wondered whether, apart from cmv reactivation, tnf-alpha could also cause cellular immunodeficiency this way promoting cmv replication and spreading and eventually causing cmv disease. in summary, we were able to show that a diminished cellular immune response results from/n vitro or in vivo methylprednisolone (mps) is used to suppress both inflammatory and immune responses. il-1 receptor antagonist (il-lra) and tnf binding protein (tnfbp) are naturally occuring anti-cytokine proteins, possibly modulating inflammatory and immunological responses. to define mps modulation of cytokine and anticytokine responses, we examined effects of mps on il-lra production and tnfbp generation as well as il-i~ and tnfa production by human peripheral blood mononuclear cells (pbmc) stimulated with lipopolysaccharide (lps). pbmc were purified from the blood of healthy volunteers, then incubated with lps (10ng/ml) supplemented with different concentrations of mps (0, 500pg/ml, 50mg/ml). after 24hrs incubation, the supernatants were collected for rias of secreted cytokines. the cell lysates obtained by 3 cycles of freeze-thaw, were also collected for r ias of cell-associated cytokines. both the supernatant for secreted cytokines and the cell lysates for the cell-associated cytokines were subjected to rias for il-i~, tnfa, il-1 ra, and tnfbp-i (tnfsrp55). il-la, il-1 ra, and tnfbp were produced mainly as cell-associated forms in response to lps, whereas most tnfa was secreted into the supernatant of lps-stimulated pbmc. mps supplement resulted in reduction of il-la, il-lra, and tnfa production. il-la production was reduced up to 5.4+1.6%(50mg/ml) by mps in a dose dependent fashion, whereas about 50% decrease in tnfc, and il-lra production was observed at both high and low dose mps supplement. however, total tnfbp generation was not reduced significantly by mps supplement. furthermore, tnfbp secretion was increased 12 times more in the supernatant of lps-stimulated pbmc with 500gg/ml of mps. these data suggest that mps could effectively block tnf activity by both reducing its production and up-regulating tnfbp generation and that mps works as an anti-inflammatory drug as well as an immunosuppressant by modulating cytokine and anticytokine responses. in severe gram-negative sepsis, excess of proinflammatory cytokines is associated with increased morbidity and the mortality. we have found that immunocytes possess functional 13-adrenergic receptors (~-ar) which, when activated, down regulate the lipopolysaccharide (lps) induced gene expression and subsequent secretion of tnf-~ in vitro. in the present study, we used a lethal endotoxic model in mice to test the hypothesis that 13-ar stimulation will down regulate the gene expression thus decreasing the circulatory levels of proinftammatory cytokines and improve the survivals in severe endotoxicosis. the animals (iso group) were injected 3 times with !3-ar agohist isoproterenol (0.5 mg/kg i.m. and 0.5 mg/kg s.c.) 4, 2 and 0 hours prior to a lethal dose of lps injection (25 mg/kg i.p.). control group (ctl) received same volume of saline at the same times before the lps insult. at the appropriate time points after lps injection animals were sacrificed and venous blood was collected from the inferior vena cava. the plasma levels of tnf-cc and il-loc were determined by elisa. in the mortality study, each grou the data showed that isoproterenol significantly decreased the mortality and the circulatory levels of tnf-c~ and il-lcc (fig. 1) . these data suggest that 13-ar activation of immunocytes down regulates cytokine gene expression, decreasing circulatory eytokine levels thus reducing endotoxicosis mortality. these results provide a new pharmacological approach to reduce the excess of proinflammatory cytokines and mortality in sepsis. introduction: cytokines released from monocytic cells are thought to play an important role in the pathophysiology of sepsis. during the last few years several investigations have been performed aimed at modulating this mediator response. in the present investigation using a full blood model we have studied the effect of a standard immune globulin and an antitipopolysaccharide (lps) immune globulin preparation and a monoclonal anti-lps antibody in modulating the endotoxin induced cytokine response. methods: citrated blood from healthy human donors was incubated at 37 °c in rotating polystyrene tubes. samples were taken before addition of 1 x 103 ng/l e. col± endotoxin and after 2 hours. plasma was assayed for the cytokines minor necrosis factor alpha (tnf-~) and interleukin-6 (il-6) using elisa methods. the eodotoxin concentration was assayed by lal and end-point chromogenic substrate technique. the blood was preincubated with standard immune globuline (human igg from pooled plasma, gammagardr, baxter) or with an anti-lps immune glubuline (human anti-lps igg obtained by screening of blood donors, novo nordisk) in the concentrations of 4mg/ml or 8 mg/ml or with the monoclonal lipid a igm antibody ha-1a for ten minutes before the endotoxin was added. results: two hours after endotoxin addition markedly elevated tnf-~ and il-6 values were found.in the plasma. cytokine values at 2 hours: tnf-ec 1689 pg/ml (mean) and 1l-6 1123 pg/ml (mean). preincubalion with 8 mg/ml standard immune globulins reduced il-6 values by 49 % (mean) while there were no effects on tnf-c~ values at 2 hours. a slight reduction in il-6 values was also found with the lower concentration, 4mg/ml, but this was not statistically significant. preincubation with anti-lps immune globulins had no effect on il-6 nor on tnf-cc values. preincubation with the monoclottal lipid a igm antibody ha-1a in variable concentrations had no effect on cytokine release in this model. in this full blood in vitro model standard immune globuline reduced endotoxin induced 1l-6 release while tnf-cc values were unchanged.the anti-lps immune glubuline preparation and the monoclonal lipid a igm antibody ha-1a had no effect on the il-6 or tnf-ct release. the effect of anti-tnf treatment in severe haemorrhagic/traumatic shock was investigated in a conscious rabbit model. the jugular vein (for administration of substances and blood withdrawal) and right femoral artery (for measurement of bp) were cannulated and an aortic thermistor probe inserted into the left; femoral artery for the measurement of cardiac output (co). the following day, upto 40% of the estimated blood volume was withdrawn over 80-40 rain, to reduce bp to 40-50mmttg and co by 2/8 mad withheld for 60 rain. shed blood (60%) was then reinfused followed by lactate solution to give a total potential dreulat%ag fluid volume of 120%. zymoeen activated plasma (0,36gg/ml) was given 10 rain prior to haemorrhage and during blood r~n%eion, all ~,~ir, ak were killed at 72h and o~gane removed for histology. rabbita received either monoclonal antirabbit tnf (r6, rat igg1, 8mgkg i.e. n=7) or emine (nffi6), 60 rain prior to haemorrhage. the cardiovascular and biochemical changes during the haemorrhage and hypotensive phase (is. haemorrhagic stimulus) were similar in both groups. upon reinfusion, haematccrit and white cell count remained sigutfia~utly (p<0.05) higher in saline compared to r6-treated animals, indicating a relative haemoconcentration (i,e. reduced circulating volume) in saline animals. the lettkocytosis persisted in the saline animals upto 72h. plasma glucose, lactate and asparteto .m~-o transferase all rose similarly in both gorups but plasma ¢rea~!~+-e levels were markedly (p<0.05) higher in ealine ve r6 animals at 48 and 72h indicative of kidney damage, using a macro and micro. pathological scoring system, major organ damage was seen in the lung, liver and kidney which was significantly (p<0.05) attenuated in r6 treated animals. in the hmg and liver, this was primarily due to a reduced bleeding and pmn infiltrate and to an additional maintenance of tubular integri~:y in the kidney. hence, in tlais model anti-tnf treatment markedly reduced the biochemical and histological indices of severe ,, ~gan damage during liaemorrhage and ranerfn~ion. thermal injuries and systemic bacterial sepsis produce a wasting diathesis with anorexia and marked loss of lean tissue and body fat. endogenously produced cytokines, including interleukin-1 (il-1 ), are thought to mediate many beneficial as well as pathologic host changes that occur during burn injury and infection. to evaluate whether bluckade of il-1 in vivo would affect survival and attenuate the anorexia and cachex[a associated with a thermal injury and chronic fulminant gram negative infection, 24 sprague-dawley rats were studied. anesthetized rats were divided in 3 groups, implanted with alzet r minipumps and randomized to receive the following treatment for 14 days:i: 25 gg/kgbw/min of il-lcc; ii: 25 ~tg/kgbw/min of il-1 receptor antagonist (il-ira); iii: buffered vehicle. the animals were subjected to a 30% bsa, full thickness scald burn and the wounds then infected with 10 + cfu/ml pseudomonas aeruginosa. eight additional rats were anesthetized, but were not burned, and served as healthy controls. . il-hx reduced food intake transiently but was otherwise without effect however, il-lra significantly attenuated weight loss over the first 8 days and improved food intake between days 5 and 8. we conclude that blockade of il-1 after a thermal injury with superimposed infection does not adversly affect the progression of the disease, but does attenuate the anorexia and weight loss associated with this model. these data indicate that il-i receptor blockade may offer a means to minimize the morbidity associated with catabolic illness. tumor necrosis factor (tnf) and its downstream induced mediators have pivotal roles in the pathogenesis of sepsis and septic shock. the suppressive effect of pentoxyfillin (ptx) on tnf production may be of clinical importance. when peripheral white blood ceils were stimulated with either lps or staphylococcus aureus, pentoxyfillin inhibited tnf production. in contrast, no inhibitory effect was observed on the induction of il-6. ptx inhibited not only the tnf production of monocytes but also the tnf secretion of both granulocytes, and unseparated whole blood. the facs analysis revealed that the expression of cd14 antigen on monocytes was not influenced by pentoxyfillin. the in vitro tnf and il-6 producing capacity in septic patients were higher than in the control group, but decreased on subsequent days especially in fatal cases. administration of ptx (200 mg/day) in septic patients resulted in tnf and il-6 productions similar to those found in control donors. it also subsequently led to an improvement of the clinical status. a further improvement in apache score could be achieved by administration of pentaglobin ° (biotest)( 5ml/kg/day ). the prevention of lpsinduced in vitro tnf and il-6 production by pentaglobin ° could be demonstrated in in vitro experiments involving the use of whole blood rather than purified lymphocytes, very probably because of the presence of lps binding protein in the plasma. the level of soluble icam-1 in sera of septic patients was significantly higher (800-1200 ng/ml) than in normal individuals (50-150 ng/ml), but it decreased following the ptx and pentaglobin ° therapy. it is presumed that ptx and pentaglobin ° can antagonize eytokine production at different levels, resulting synergistic action being beneficial in the treatment of sepsis. albert szent-gy6rgyi medical university, institute of microbiology, szeged, hungary h-6720 ddm t. 10. e~i~im~/tii, septic sh0~ '. tnf alfa/pge, and somatostatln lands ji., alvarez j., gram m., llanos k., alcalde j., sanchez ja., balibr~d jl. taurine, a semi-essential sulphur-containing t:~-amian acid, promotes neutrophil phagocytic activit3' against yeasts and enhances intracellular killing of e.coli. paradoxically it protects against hypochlorous acidmediated biomembmne oxidatio~ and abrogates the pyrogenic effects of intracerebral endotoxin. it is therefore possible that taurine mediates some of these effects by modulating the cytokine cascade. aim to assess the cytekine responses in a rat model of intraperitoneal sepsis, pretreated with supplenmntary taurine, by measuring tnf and il-6 concentrations. methodology female wistar rats (wt 160-200g) (n-6 per group) were prefed with 2% tanrine, 3.33% glyciuc ( in tap water) or tap water (tw) for 15 days prior to caecal ligation aud puncture (clp). normothermia was maintained intraoperatively and the animals received 0.9% saline subcutaneously (3 mls/100g) post-procedure. animals were venosected by direct cardiac puncture at 6 or 18 hours post-operation and the blood placed in endotoxin-free tubes. centrifilgation and storage of plasma at -70°c was completed within 2 hours. tnf and il-6 were measured using wehi and b9 bioassays respectively. results (means ± sem) bioactivc tnf concentrations were not significantly different between clp or sham groups. however plasma il-6 estimation revealed a statistically significant reduction at 6 hours in tanrine-treated animals compared to glycino and tw controls ( objective: to evaluate the effects of allogeneic blood transfusion, thermal injury and bacterial garage on interteukin 4 (il-4), tumor necrosis factor alpha (tnf) production and host mortality and to study if the administration of thymopentth (thy) could affect these events. materials and methods: balb/c mice were transfused with allogeneic blood (from c3h/hej mice) and treated daily with thy (i mg/kg)(t+thy) . control animals were transfused but not treated (t). after 5 days systemic blood was harvested to determine the circulating levels of il4 and tnf. a second group was treated with thy for 5 days and then received a gavage wide lxl09 escherichia coli and a 20% burn injury (bg+thy). one hour post-bum, blood was collected to measure cytokins. control animals received the same treatment but thy (bg). a third group was transfused, treated with thy for 5 days and then received gavage and burn (tbg+thy). one hour post-burn, blood was collected to measure cytokins. control animals received the same treatment but thy (tbg). all treated and control groups had 8 mice/group. in separate groups (n=20/group), receiving the same treatments, mortality was observed for 10 days post-burn or transfusion. the production and release of oxygen radicals by phagocytic leukomytee is one of the most relevant and important functions of these substances in biology. when neutrophilic granulocytes or certain macrophages phagocytize they produce super oxide and hydrogen peroxide and form secondary products of these activated species. all of these substances are released in to the phagosome. it has been appreciated since 1977 that the amount of oxygen consumed by phagocytes in producing free radicals is prodigious. what has not been totally appreciated is the significant contribution this 02 consumption contributes to total body oxygen consumption. our understanding of increased oxygen consumption following sepsis is compounded by a paradox of increased cardiac index and a narrow av02 difference. it was mclean in a study reported in 1967 that showed an increase in cardiac index and a narrowing of the avo z difference in humans following sepsis. other studies documented similar changes in humans and it has recently been speculated that increasing o~ delivery might have a favorable impact on outcome. at least three different explanations have been put forward as to why there are possible alterations in energy metabolism during sepsis. these include decreases in oxygen supply, peripheral shunts and direct action o~ cellular mediators on o~ delivery, a provocative review by hodgkiss and nark in 1992 shewed that in experimental animals sepsis did not cause any evidence of bioenergatic failure in muscle i liver, heart or brain tissue. they further showed that the increase in serum lactate is mot necessarily due to cellular hypoxia. they conclude that cellular hypoxia and defects in energy producing metabolites are not the cause of metabolic abnormalities in sepsis. we set out on a series of experiments to try to explain the paradox in cellular metabolism and whether or not white cells might contribute to total body oxygen consumption during sepsis. the first set of experiments involved growing rat cardiac myocytes on tissue culture. pyruvate was added to the medium as substrata and physiologic levels of peroxide were also added simultaneously, carbon 14 labeled co~ production was measured as was nucleotides and degradation products from the cell. the peroxide induced a significant inhibition of pyruvate dehydrogenase. in addition, there was a loss of cellular atp and a corresponding rise in the release of inosine and adenine from the cell. we concluded from this first sst of experiments that physiologic levels of peroxide are a potent inhibitor of pyruvate dehydrogenase in isolated cardiac mitochendria as well as the cultured myocyte. we further showed that pyruvate dehydrogenase inhibition blocks the aerobic oxidation of glucose and leads te adenine nucleotide metabolism with adenosine and inosine release from the cell. other enzymes within the tca cycle did not appear to be specifically blocked by peroxide. this would explain the increase in lactate and the ability of the cell to continue to make high energy phosphate by entry of metabolites in to other entry points of the tca cycle, the second set of experimbnts was designed to estimate the magnitude of whole body reactive oxygen generation via nadph oxidase following granuloeyte activation invivo. using a guinea pig model baseline oxygen consumption was determined. ~iaphenyl iodinium (dpi) was used as a specific inhibitor of granulocyte nadph oxidase. animals were divided into two groups; those that received dpi and those that served as control, all animals were then injected with phorbcl myristate acetate (pma) intravenously. pma is a potent stimulus for granulocyte activation and subsequent reactive oxygen generation. whole body oxygen consumption measurements were then repeated after the p~la injection. in addition, arterial blood gas amalysis was performed, circulating neutrophils were collected and assayed for their ability to generate hydrogen peroxide and the r~ght lung was removed for wet and dry weight measurement. total body oxygen consumption increased by 25 percent after pma injection. arterial oxygen tension fell significantly in the control animal. neutrophil hydrogen peroxide generation rate doubled and lung water in the untreated animals increased by 25 percent. in this animal model we could show that granulocyte activation substantially increases whole body oxygen consumption to approximately 120 percent of control values. the nadph oxidase inhibitor, dpi, decreases granulocyte hydrogen peroxide generation invivo and prevents the pulmonary injury induced by pma. using this animal modal it is possible to transpose this to the human septic state. if we assume there are 5,000 neutrophils per cubic millimeter of blood, a 70kg man would have ~1.75xi0 u circulating neutrophils. when activated ixl0 ~ neutrophils generate one nanemole of hydrogen peroxide per minute. t~erefore, the circulating neutrophil pool could generate -1.75xi0 nanomoles of hydrogen peroxide per minute. this corresponds to an oxygen consumption of -4.2ml/ojmin by the circulating neutrophil pool alone. this does not take into account the production of oxygen by macrophages or the amount of oxygen consumed to produce nitric oxide. manipulation of oxygen delivery in human sepsis may or may not be beneficial. irshad h. chaudry, ping wang, and alfred ayala life threatening blood loss, due to soft tissue and bony injuries, is a frequent complication in trauma victims. although numerous organs and cells are adversely affected by hemorrhage, the focus here will be to discuss whether or not there are any differential alterations in splenic and hepatic immune functions. in particular, m~ antigen presentation (ap) and associated processes will be described since one of the important functions of the m6 is to activate resting t-cell lymphocytes by processing and presenting foreign antigens in a mhc class ii-restricted fashion. studies have shown that splenic m6 and kupffer cell (kc) ap was significantly depressed following hemorrhagic shock and remained so for at least 96 h alter resuscitation. the presentation of antigenic fragments associated with mhc class ii antigen itself was not decreased following hemorrhage, but the catabolism of antigens in antigen-presenting cells was decreased. this is likely due to a significant loss of cellular atp. although m6 ap was depressed in splenic, as well as kc, only kc showed an enhanced capacity to produce inflammatory cytokines, il-1, il-6, and tnf. this difference in response between kc and splenic md may be due to differences in the microenvironment in which these cell populations are found, as well as potential differences in the effects that hemorrhage has on the environment from which these cells are obtained. splenic m6 from hemorrhage-resuscitated mice exhibited a significantly reduced cytotoxic response, whereas kc showed an enhanced cytotuxic capacity. the increased kc cytotoxicity is probably mediated through the increased expression of cell-associated tnf and the release of reactive nitrogen. the enhanced production of reactive nitrogen may play an important role in the clearance of microbes translocated from the gut following hemorrhage. however, excessive release of reactive nitrogen by kc may have deleterious effects on the adjacent hepatncytes. such an effect could, in part, explain the reduction in active hepatocellular function, which is seen following hemorrhage-resuscitation. thus, hemorrhage induces differential effects on splenic and kc m6 populations; it decreases splenic m6 but increases kc capacity to mount a cytotoxic response. the depressed splenic md and kc ap was, however, significantly improved by posttreatment of animals with atp-mgcia, chloroquine, diltiazem or interferon-'/. these agents also decreased the susceptibility to sepsis following hemorrhage. thus, the use of atp-mgci~, dfltiazem, chloroquine or interferon-7 offers new therapeutic modalities in the treatment of immunosuppression and for decreasing the susceptibility to sepsis, which is observed following severe blood loss. the production of macrophages from bone marrow precursor cells is a dynamic and highly regulated process. the differentiation of myeloid lineage-restricted progenitor cells is initially controlled by interleukin-3, which drives the progenitors along a maturation pathway that leads to their response to specific lineagerestricted colony-stimulating factors(csfs) we have previously hypothesized that thermal injury can initiate a self perpetuating cycle of production of defective immune cells: thermal injury can initially affect circulating immune cells. then these cells, by unbalanced production of colony stimulating factors and other mediators, can cause a derangement of hematopoiesis resulting in an abnormal production profile of tnf, il-i, and il-6 and cytotoxic activity of bone marrow derived cells. the results of our studies so far have supported parts of this hypothesis. il-3 and il-3+m-csf treatments increased the production of tnf in burn 4 day macrophages and progenitor cells compared to normal cells. il-3+m-csf+il-10 increased the production of il-6 by burn progenitor cells compared to normal macrophagee. preliminary results for the ratios of tnf/~ actin mrna indicate that il-3+m-csf increased the mrna for tnf in the 4-day macrophages derived from burned animals compared to macrophages derived from normal animals. ratios of il-6/~ actin mrna indicated that il-3, il-3+m-csf, and m-csf increased the il-6 ~rna in progenitor cells from burn animals compared to progenitor cells from normal animals. these preliminary results support our hypothesis that bone marrow derived macrophages and progenitor cells from burned animals act differently compared to cells from unburned animals regarding the production of inflanunatory cytokines. more specifically, the results suggest that the different cell types are regulated in different ways by cytokines, and cells from burned animals are regulated differently than cells from unburned animals. it i,~ to ~ssume that the translneatinn is due to a ixx~h,v functioning ~astroiutestlnal surface pro|ection system, which leads to an unacceptably high load ¢ln the imrsunc dcfcncc system, partlcul~ly the local system in the gastrointestinal wall, leading tu exh~ustiou of ihi~ system. tlae g&~lrointesliual i]'ac( can be regal'deal a.s a t~2servojf of appr 200 m ~ separating 10 i~ eucayotic ceils of the hosl ttom l0 ~4 bacterial culls. in the human body the the myriad of cndngan¢nts micrix)rganistns, exogenous itl,~ttdittg micro~ganisms altd taxies m-'e sepia"areal li'otll lhe rest of the body by a simple epithclhd layer. the barrier function is heavily tlcpcnding tm =~ proleclive layer cmtsisting ie. surl~:emts, nluclts, probiotic bacteria, and ,~ub~tances with strong antioxid~lt, antipfotease al~d other i~rotective eft'ecru, the surfaet~ml htycr consists at tile besl if1 i(.) bimolecular layers, fl is thus very iltil~, the epilhelial layer is renewed every 24 hours , and tile tanuwer of the mucus is ~so fa,~t, all attdphy of die epithelial layei induced by g&sttointestinal st~'vation does also include the mu~usprndocing cells, gnhlet cells, which leaduhg lu slmnage aad luwer quality of gi mucus, altered viscoelas,jc properties and reduced protection, ]~'obioiic bactcrla keeps the ppm's low, produce nutrients for the intestinal mllcosa, eliminates tuxia~ and stimulates the local immune system, the gl surface is conslanlly under hc wv wearing by ingested bacteria and their enzymes, ingested substaucas, chefftie,'ds at~d toxins. 'll~e n~tective flora is reduced in disease, by stress and by cm'clcss antibiotic therapy. wc have made attempts tn recondition the i,,.astroiniesli!l~d. tnucosa by rcstlpply of surfactants or sttrfactanfliko glyc~lipids, gels with pseudomuein [until.oil, and probiotl¢ laetobaeilli. by supply of glycolipids we have bt:cn able m prevent and heal intlammatory and tlleerallve injuries (and io p)'cveut microbe transhteatkln) in uppe~' and lower (.31 u'acl, this c~al be fu~'ther augmentented by shnultaueos supply el' a p.~uedomucin,likc gel like pectin. ()at contain~ one hundred times more ot membrane lipid$ th~a =nay other food, much of fiber, ~spceially watcrsnlnhlc betaglucaal~, at~d has an close to ideal amino acid cornpo~ition, <')at, termented by species-specific lactobacilli, has a strol~ ability to prevent local inflammatory and ulcerative chunges,transh)cadnn and sepsis in experimeut~d attimals mid to pl~ven~ revert mof in ba,nan.~.lt seems us, that it the mucos~lnucus/probiotic bacterial filnclinns cat'= 1%' restated by mucosa reconditioning, even in severely sick iudividuals) the hx:~d iuuuuue ~y,~teme will be capable tu pr~)tec! the ix~y ag~lls~ sepsis ~nd mof. the impaired immune response associated with multiple system organ failure (msof) has been most widely studied following surgery for inflammatory disease and trauma. the majority of late deaths following trauma are due to infection and msof. not all patients with multiple organ failure have concomitant infection present, yet most have been septic at some time in their hospital course. their generalized inflammatory condition often persists whether or not organisms have been recovered. immune failure probably precedes msof with or without overt infection and indeed may perpetuate such widespread sepsis. macrophage tumor necrosis factor-alpha (tnf) production is thought to represent an important pathogenic mechanism by which this is mediated. cecal ligation and puncture (clp) is a clinically relevant murine model of intra-abdominal infection and sepsis. serum tnf and endotoxin levels, and peritoneal macrophage tnf production are only slightly elevated in this model even in endotoxin sensitive animals. mortality in this model, therefore, does not appear to be attributed to overwhelming endotoxemia and/or tnf production. it has therefore been postulated that tnf and other cytokines may act in a paracrine fashion to cause local injury. tnf messenger rna (mrna) expression was therefore measured and found to be increased in the lung and liver following clp. a different pattern of expression was seen after endotoxin administration, characterized by a more rapid rise and fall, and enhanced tnf mrna expression in the spleen as well. route of spread of infection as well as the type of stimulus may determine the organ specific cytokine response. these data support the concept of locally produced tnf as a contributing factor in tissue damage and multiple organ failure during sepsis. the estimation of histamine's role in sirs changed over the years based on increased knowledge in shock pathogenesis, evidence later found to be faulty, and the discovery of other, more fashionable mediators. over the decades, the prevailing view has been that histamine is a noxious mediator contributing to the fatal outcome of shock. the analysis of experimental data on exogenously administered, endogenously released and/or newly formed histamine in septic/endotoxic shock suggests that histamine contributes to the early cardiovascular changes via hi-receptor vasoconstriction thus excerbating (directly and indirectly) the effects of catecholamines. in the later phase of septic shock (low out-put, high resistance states), however, it can be assumed that histamine exerts strong vasodilator and positive inotropic effects via h2-receptors. these effects are considered beneficial in counteracting or attenuating the effects of sympathetic responses of catecholamines and other mediators. thus, a blockade of the early reaction with hi-antagonists and a stimulation of the h2-receptors by h2-agonists are worthwhile therapeutic approaches. shock produc~s ~ij alterations, an encrgy crisis and eventual c~ll damage, however, shock in itse|f do~ not lead freqoenfly to r~mote organ damage. in animal ~xpcrhnents and clinical expariez:ces in korea and vietnam, shock i~r s¢ was not associated with lung damage, renal failure or ards. the same is true with g.l bleeding. however, when shock is due to trauma or associated with tlssus injmy, then organ malfunction m~d damage is frequent. shock is bclleved to increase the frequency of infection, but clinical evidcnc~ for this is scant. hemonhsgio shock akme is an unusual o~orrenee dinica[ly. immune dysfolletion dons occur witl~ experhnental hypovolemic shock and in pailcnts with hypotcnsion or after receiving blood ~ansfosions. tbc most significant factor affecting reoorrcnce of resented ca,lorectal liver mclastasis was hypotensivc episodes during operation. blood transfosions depress immune function, improve transplant ~raft survival and increase ttll'rmr rec~r'tcnos ill co[eli and head and nc~k cancers. in experimental hemorrhagic shock, we found decreased response of [ymphocytes to mitogens, el:clad mixed lymphocyte reactilln and i[,-2 decreased. othe~.x hsvc found decreased macrophage and t and b ¢¢lt function, deci~ased re,ease of/l~i, increased poe v depressed macrophagc antigen presanlatiota, increased ien y, decreased il-2, 3 and 5, and shared mscrophags funclion with loss nr f and c 3b r~ccpto~s. this is inhibited by chlornqoine, ibuprofen, dihiazem, and atp. shock may activate ncutrophils contributing to ilappitlg in cspillarics, no refluw, increased adhesion, cytotoxicity and organ damage. this may be related to decreased clearance of bacteria after shock. t~lls shock, hypotcnsion and decreased microcirculatory blued flow initiate or set the stage for immune dysfmtction. they, along with tissue injury trigger inflammatory cascades. this also contributes to immunologic dysfonctitm, which is associated with increased lufoction. thus altered bh,od flow and mediator cascades are bolh involved, the question now is can wc rapidly improve microcirculatory blood flow, and dg'masa inflsrmnatory fosponss, which is also necessary for survival the effe~ of ~nterferon ~amma @n wour~ healing was ewaluated ~n a routine mo~el. ~mma ~n~erferon we8 g~ven in doses of ~37.5 to ~2,500 u~%$ synchronous with ereatio~ of a pa~splnal woumd then daily for is ~lays. ~mteet~on ~amma impaired tensile strength a% all ~oses relative to control. m0zphology suggested ~reas~ ¢oll~gen deposition and r~du~-~ neovag~ulaeit¥ ~n t~eat~4 animals, interferon ~amma was a2so ~valuated in a murine mo~el of cecal llgatlon and punneure. one hundred to 22,s00 uni~ vere ~iv.n following ~nju~ an~ than daily. interferon qamma was associated with inco:ea~ed mortalit~ and earlier death a~/a~n ~n a dose dependant manner (p< 0,05). afmlnistra~ion the proinfiammatory cytokines tumor necrosis factor-c~ (tnf-c0, interleukin-16 (il-ii3), interleukin-6 (il-6), and interleukin-8 (il-8) have been implicated in the pathogenesis of the multiple organ dysfunction syndrome (mods) following shock and trauma. these mediators which are released in high concentrations by activated macrophages, endothelial cells, and connective tissue cells cause a systemic inflammatory response syndrome (sirs), thus leading to a shock-like state and tissue destruction. recently, a number of proteins have been characterized in in vitro and in vivo studies demonstrating potent anti-inflammatory properties. these latter cytokines include interleukin-4 (il-4), interleukin-10 (il=10), transforming growth factor-6 (tgf-j3), and the newly sequenced interleukin-13 (il-i3). they are considered antiinflammatory, since in vitro studies using purified macrophage cultures or whole blood assays revealed an inhibitory effect of these antmnfiammatory cytokines on ~he synthesis and release of tnf-cl and il-u3. in addition, they reduced the severity of disease and reduce plasma levels of tnf-c~ and il-ii3 when administered to animals with infection or inflammation. furthermore, naturally occurring substances have been described that specifically neutralize the bioactivity of il-1b and tnf-a. the il-1 receptor antagonist (il-i ra) is related structurally to il-i and binds to the same cell-surface receptors, but does not stimulate the cell. in animal models ore. cob-induced shock, inflammatory bowel disease, and peritonitis, injection of il-lra significantly reduced the severity of disease. the cytotoxicity of tnf-c~ can be inhibited through two types of soluble tnf receptors (stnfr) type i (p55) and type ii (p75). these stnfrs are proteolytic cleavage products of the cell-bound tnfreceptors. when given to baboons to combat e. coil-induced shock, soluble receptm's to the tnf type [ receptor decreased the severi~ of the shock-like state. it has been well accepted that shock and severe injury result in an increased release of proinflammatory cytokines with a high incidence of sirs and mods. our studies investigating plasma levels of anti-inflammatory mediators (il-4, il-lra, stnfrs) in patients after severe injury further suggest that shock and injury lead to an activation of anti-inflammatory processes with altered plasma levels of these mediators. however, while plasma levels of il-4 and 1l-lra were significantly increased in trauma patients compared to healthy volunteers, stnfrs did not differ from control samples. thus, shock and severe injury result in different activation patterns of anti-inflammatory processes. m,l. rodrick, boston, b~usa following severe thermal injury significant suppression of the immune response has been demonstrsfed. these changes are associated with increased susceptibility not only to co,on human pathogens, but to organisms not normally pathogenic in the uncompromlsed host. early observations included prolonged graft rejection and skin test anergy in patients following burn injury. work from our laboratory and that of numerous others has shown that the~e is a profound £mmunoeuppresslon following severe thermal injury. i~mune deficiency has been identified in these patients hyz ii lack cf t nell responses to mitogens, 2) early decreases in total t and helper t cells in the peripheral bloo~ 3} lack of response to tetanus toxoid i~uniza~io~ in spite of increased non-speoifi~ i~k~unoglobulin praduction and 4) severe and prolongei deficiency of interleukin-2 (il-2) prcductlon by peripheral blood mononuclear cells (pbmo). this il-2 deficiency could be an underlyin~ cause of all the afo~ementloned immune defects by failing to activate both helper and suppressor t cell functions. mo~a recent work has focused on the molecular mechanisms of this il-2 deficiency end shown that the defect lies post-transcrlptionally since mrna for il-2 is also depressed following thermal injury. we have also investigated the potential of a defect in ii-2 receptor status on peripheral blood mononuclear uolls from patients fqllowing burn injury and in a murine model. in both oases no decrease in il-2r was found either by phenotypic markers or by funational assays. another hallmark of thermal injury is hyperao~ivatlon of proinflammatory ~ytok~ne secretion, which may play a significant role in multiple organ failure following burn injury. therapy o~ major burn injury may reasonably be aimed, therefore, at up-regulating t cell ~unction and down-regulating hyperactive secretion of proieflaam~ntory cytokines. the majority of patients dying of bums succumb to sepsis which coincides with dysfunction in the specific and non-specific host defences. generally, concepts relating to the mechanism(s) of specific immune failure in thermal trauma imply that inhibition of t (cd4+) cell responses is imposed by the injury or infection-related factors. however, the same factors elicit strong biological reactions within the lymphoid compartment. recent evidence indicates that systemic activation is inherent in the burn patient. to establish the relation of molecular and cellular events to the development of post-bum anergy we examined the state of and the capacity for t cell activation with regard to: 1) occurrence of activation-induced cell death (aicd), 2) activation-related phenotypic and functional_ diversity in the pool of peripheral cd4+ t cells. initially, (up to 7 days post-bum), peripheral blood lympliocytes from severely injured (> 35% total body surface area) patients exhibited high levels of constitutive expression of surface receptor for ]l 2 (cd 25) and spontaneous blastogenesis. the presence of activation-related t cellproducts in bum plasma was also apparent. subsequent impairment of the t cell receptor (tcr)-regulated t cell responses in vitro was accompanied by significantly increased dna fragmentation that is associated with cell death by the mode of apoptosis. using molecular markers we established that flesh peripheral blood ceils from immunosuppressed patients also contain large numbers of apoptotic cells. fluctuations in the number of viable (pi-) peripheral blood lymphocytes involved primarily cd4+/cd45ro+ (memory) subset of t ceils. the above observations suggest that thermal trauma-associated t cell anergy develops through aicd, a phenomenon commonly associated with the tolerogenic activity of bacterial superantigens. persistence of staphylococcal infections in the burn patient may support this assumption. response following trauma jane shelby, ph.d. the immune system is integrated with other physiologic systems, and is exquisitely sensitive to changes in nervous and endocrine systems changes following traumatic stress challenge. the immune, nervous and endocrine systems interact via both direct and indirect pathways which utilize neuro and endocrine hormones, neurotransmitters, neurepeptides and immune cell products. it is now known that the immune system may be affected by all of the neuroendocrine products produced during a stress response, with evidence for innervation of iymphoid organs, lymphoid cell receptors for neuroendocdne products, and leukocyte production of chemicals which are virtually identical to certain neuroendocdne peptides (acth, endorphins). trauma induced alterations in the equilibrium of various neuropeptides and neuroendocdne hormones have a significant impact on immune response potential, affecting control of proliferation, differentiation and function of immune cells. for example, the neurohormone melatonin is thought to be a natural antagonist to counteract glucocorticeid associated immunosuppression resulting from stressful challenges, such as surgery and trauma, plasma melatonin levels are known to be significantly reduced in burn patients. the administration of exogenous me[atonin improved cellular immune response following burn injury in an animal model. melatonin was also shown to have in vivo cytokine regulatory activity, increasing the potential for il-2 secretion and downregulating excessive il-6 and ifn~ in burn injured, stress susceptible mice. the regulatory interactions between the immune, nervous and endocrine systems provide mechanistic pathways for trauma associated immune dysfunction. increased knowledge of these interactions will enhance the potential for the design of novei clinical interventions to improve immune response and decrease the risk for infection in trauma and surgical patients. . animals receiving e were given a single dose daily of either 2.4 g/kg of e in a 20% solution by garage (ge), or 1.25 g/kg of sterile ive in saline. four hours following the last dose, bum animals were subjected to a 30% body surface area bum injury to their dorsum. twentyfour hours following injury, the animals were sacrificed and spleen cells were harvested for assessment of lymphocyte function. splenocytes were prepared by mincing the spleen, followed by incubation on glass petri dishes to remove adherent macrophages. non-adherent cells were then tested for proliferative response to t-cell mitogen concanavalin a (con a) and b-cell mitogen lipopolysaccharide (lps). data were analyzed by anova. results: chronic alcohol exposure and burn injury independently inhibit lymphocyte response to con a but not to lps. the combination of e plus bum injury, however, pmfouedly decreases this response to both con a and lps as outlined in the this data clearly identifies the synergistic impairment of immune function produced by ethanol and bum injury. it is furthermore apparent that ibis effect is gut mediated and that gastrointestinal exposure to alcohol is necessary to produce this effect. further studies will work to identify cellular and subcellular mechanisms to explain this effect. in experimental animal studies and investigations on human volunteers endotoxin infusion is mgulary accompanied by the release of the cytokine tumor necrosis factor a (tnf-~) determined by elisa technique. in patients with menigococcal sepsis also elevated tnf-a values have been found using a functional assay. we have studied the role of tnf-et in surgical icu patients with sepsis. using functional technique, we were not able to detect tnf-~ activities in the patient plasmas. when this cytokine, however, was determined by immunochemicai technique (el1sa) elevated tnf-e~ values where frequently oberserved. in order to further elucidate these observations, we studied shedding of tnf receptors in the patients. in these studies, we noticed that shedding of tnf receptors oecured regulary in the patients. at the time of diagnosis, soluble tnf receptor p55 and p75 were both 2-3 fold higher than values found in plasma samples obtained prior to die diagnosis of sepsis. we also observed that the sepsis patients revealed higher maximum values of p55 and p75 during the icu stay compared to values found in surgical icu patients without sepsis. these observations indicate that soluble tnf receptors are available in sufficient amounts to bind tnf-ot which is released in surgical patients developing sepsis. this mechanism may explain why functional tnf-c~ was not detected in the patients. institute for surgical research, rikshospitalet, the national hospital, university of oslo, 0027 oslo, norway. decker, d., sch6ndorf, m., bidlingrnaier, f., hirner, a., yon rfcker, a. the advantage oflaparoscopic cholecystectomy over conventional open surgical approaches in the treatment of symptomatic cholelithiasis has been shown convincingly by clinical studies. in order to facilitate comparisons of different surgical approaches, we evaluated the cell biological characteristics of tissue trauma by measuring changes in various cell surface markers on leukocytes and eytokines in plasma as a possible means to assess tissue trauma in choleeystectomy. patients recruited into our study had experienced at least one typical bifiary colic, had ultrasound-proven cholelithiasis (stages 0-ii according to me sherry), were 35-55 years old, and presented for elective choleeysteetomy. patients could choose between laparoscopic and conventional eholeeystectomy after being informed about the advantages and disadvantages of each procedure. cell surface markers on leukoeytes were determined using whole blood techniques with the help of commercially available fluorescent monocloml antibodies and flow cytometry. shed cell surface markers in plasma and cytoldnes were measured with the help of sandwich-elisa kits. blood samples were drawn 24 h before surgery, immediately before incision (after anaesthesia), 2 h and 24 h after incision. seventeen cell surface markers were examined on different cell populations and cellular subsets in 18 laparoscopic and 15 open-surgery patients. three soluble cell surface markers and six cytokines were monitored. by statistical analyses (multivariate regression analysis, student's t test, wilcoxommann-whituey's rank sum test) the six markers/cytekines that best distinguished open surgical from laparoscopic procedurea were determined. these were 1. the interleuldn-2 receptor and im soluble form (cd25/scd25); 2. the activation antigen fd-1 and its soluble form (cd30/scd30), a member of the nerve-growth-factor receptor family; 3. the cd45ro epitope which characterizes t memory ceils; 4. the trausferrin receptor cd71; 5. the soluble adhesion molecule icam-1; and 6. the cytokines interieukin-6 and interleuldn-8. on the basis of these results, a tissue trauma activation (tta) index was calculated by combining the marker/cytoldne measurements by simple multiplication. anaesthesia and pre-ineision maneuvers did not significantly change cell marker or cytokine levels in either surgical approach as compared to 24 h before surgery. 2 h after incision the tra index in open cholecystectomy showed a distinct 22-37 fold increase, whereas in laparoseopic surgery a mere 2-3 fold increase was noted. 24 h after incision, the tra-index returned to near pre-surgery levels. in conclusion, our results demonstrate that changes in cell surface markers and cytokines can help evaluate the magnitude of tissue trauma in diffei'ent surgical approaches. the relationship between lymphocyte subpopulation changes after thermal injury and the increased susceptibility of burned patients to infection is unclear. in this study, we have attempted to correlate such subpopulation changes with the presence of infection in burned patients. peripberal blood from 19 patients was monitored for lymphocyte subpopulation changes three times weekly for three weeks postburn and weekly thereafter for three additional weeks. mean bum size was 44.7% (range 24%-84%) of total body surface and mean age was 38 years. infection was diagnosed by carefully defined clinical and laboratory criteria and its presence or absence noted each time blood was drawn. samples taken when patients had wound infection, bacteremia, or pneumonia were compared with samples taken in the absence of systemic infection. whole blood samples were stained with four monoclonal antibodies, the red blood cells lysed and the leukocytes fixed and analyzed by flow cytometry. for each patient sample, the proportion of lymphocytes falling within the light scatter gates was determined as the percentage of cells negative for cd14 and most strongly positive for cd45. this percentage was used to correct each sample for the presence of debris or nonlymphocytic cells. the proportion of cd4 and cd8 positive cells was slightly greatc~ in the samples from infected patients, while the proportion of b cells (cd20+) was unchanged and nk (cd16+) cells were decreased by ahnos[ 50% compared to sampie~ li'om uuiuleclcd patients. the percentage of cells positive for cdilb (c~ integrin) decreased sharply and cd4ro (memory cells) decreased slightly in samples from infected patients while the expression of the lymphocyte homing receptor and cd29 were unchanged. cd25 (il2 receptor) and cd69 (early activation marker) were significantly increased in the samples from the infected patients while hladr was unchanged. these changes in lymphocyte phenotype correlate with the presence of infection. if they closely precede or occur during the early development of infection they may be valuable clues to the mechanism of susceptibility following thermal injury. trauma patients are subjected to an immediate massive impact on their host defense integrity due to the combined effect of tissue trauma, shock and endotoxemia. cytoldnes are playing a crucial role within the course of an impaired cell mediated immune response (cmi) resulting from a disruption of intact m%/tcell interaction. the current study was undertaken to further elucidate the mechanisms of dysfimctional cmi following major burn and mechanical trauma -via comparative analysis of mrna expression and protein release. the major regulatory levels for different cytokines were determined in mitogen, respectively lps stimulated peripheral blood mononuclear cell (pbmc) cultures of trauma patients on consecutive days (13) t, 3, 5, 7 and 10 post injury. we analyzed the cumulative data for interleukin-1 beta (il-i[3), il-2, il-8 as well as tumor necrosis factor alpha (tnf-~) and saw a considerable impairment of the protein release in the stimulated pbmc cultures until d5 post-trauma and recovery thereafter. *p < 0.05, ** p < 0.01 vs control comparing the autoradiographies of the specific cytokine mrna expression with the protein release in the supernatants, we saw a good correlation between mrna signal intensity and protein synthesis for il-8 and 11,-2, suggesting that for these cytokines the main regulatory mechanisms are located at the pre-/transcriptional level. for the other cytokines investigated one has to suppose posttranseriptional mechanisms. the analysis of our data clearly indicates a severe impairment of forward regulatory immune mechanisms following trauma. most likely the regulatory mechanisms, that are involved are greatly different among the cytokines investigated. it may be concluded, that depressed cmi responses post-trauma are partly due to an impaired pro-inflammatory cytokine production. the severity of the injury (iss) correlated with the development at multiple organ failure (mof-score; r=0.612). the levels of mediators and markers of the inflammatory response were generally higher in the more severely injured group (iss>30, n=16). i1-6, 11-8, g-csf, fpa, and c3a -levels differed significantly (p<0.005) between the iss-groups (>-< iss 30) at the time of admission, whereas on day 3 tnfa, c3a, 11-6, and ealpi showed significant differences. beyond the first week, major differences were restricted to pge 2 and c3a. the formation of two groups with respect to later multiple organ failure (mof < 3; mof > 2 n= 14) yielded similar results. leukocyte-facs analysis revealed significant differences mainly in the cd14 (monocytes), cd3/cd25 (i1-2r + t-cells), and cd29/cd4 (th 2calls) populations. summarizing our findings we were able to detect some alterations in the surface antigens of immunocompetent cells. the inflammato d response, however, seemed to be more pronounced and correlates wi~ the further clinical course. using an experimental bum model in rodents, we have demonstrated that administration of a full thickness, scald burn involving 20% or more of the total body surface area (tbsa) elicits systemic responses which are characterized by numerous alterations in t-ceu function (i.e., lymphokine production and contact hypersensitivity (ch) responses) plus an enhanced susceptibility to bacterial infection. in the present study we questioned whether the apparent systemic effects mediated by large burns would be elicited as site-specific alterations in immune function following administration of small area burn trauma (5% tbsa). following a 20% tbsa burn, ch responses to contact sensitizing antigens were found to be altered. the depression in ch responses could be induced independent of the site used for topical skin sensitization. following a 5% tbsa thermal injury, development of ch responses were affected in a site-specific manner. immunization of 5% tbsa thermally injured mice in a site near the position of the burn resulted in depressed responsiveness, whereas immunization through a contralateral site resulted in responses that displayed both the intensity and kinetics of a ch response equivalent to sham-bumed mice. similar systemic and site-limited changes in lymphokine production were observed with 20% and 5% tbsa thermal injuries, respectively. a 20% tbsa injury affected the lymphokine producing potential of all cells regardless of which lymphoid tissue the cells were isolated from. the effect of a 5% tbsa burn was significant but site-specific. thus, ceils from lymph nodes receiving drainage from thermally injured tissue were specifically affected, whereas lymphokine production by cells from lymphoid organs receiving drainage from unaffected skin was normal. it was concluded that modulation of lymphokine production and cellular immune responses may be a normal consequence of burntrauma regardless of the size of the burn. changes in immune competence can be mediated either regionally or systemically in direct proportion to the area of skin exposed to the burn injury. this work is supported by phs grant gm46899 and the office of navy research n00014-92-j-1612. division of cell biology and immunology, department of pathology, university of utah school of medicine, salt lake city, ut 84132. post spleneetomy septic sequelae may be fatal, but the mechanisms remain unclear. the objectives ef this study were to assess the mortality from concomitant splen-'etomy and ]~eritoneal bacterial challenge and to elucidate the local cetkdar responses. 50 cd-1 mice were randomised to receive laparotomy and sham splenectomy (l) or splenectomy (s) with simultaneous ca'-cal ligation and "):mcture and the survival patterns assessed. subsequently, 150 cd-1 mice were randomised into control (c), l or s groups and peritoneal cells studied at 24 hours for bacterial phagocytosis and killi:~g, superoxide (02-) and tumour necrosis factor (tnf) production and macrophage activation vsing mac-i(cd-11b) receptor in~.ensity expressed es mean channel of fluorescence (mcf). these resides indicate that sf!enectomy predisposes to nrortal~ty from bacterial sepsis ia the early pos~ operative period compared to sham operated animals. failure ~f p'.acrophages to kill bacteria in the splenectomv group '~:cured in t?~e absence of impairment of oxygen freeradical or tnf pred:~ctien. the macrovh~ge ac!ivotion marker mac-1 was significantly reduced in both l and s groups and impaired phagocytosis of bacteria oceured in both operative groups compared to controls. laparotomy a!one reduces macrophage activity in terms of surface re:eptor mac-1 expression and !ingestive capacity. splenectomy however s~gnificantiy ~mpairs r-acrophage-wediated l~,acterial killing and this qefect rttav co~tribut~ sig~ifjcav'ly to th-~ dissemination of local infection and to n':ortalit). depts of haem~ tology & surgery, beaumont hosoital, dub!in 9,eire. introduction: loss of cell membrane integrity appears to be a common pathway of injury to tissues subjected to high-voltage electrical shock. the cell membrane is the most heat labile structure in the cell, and is also the most vulnerable to externally-imposed electrical forces. skeletal muscle and nerve cells are particularly susceptible to electroporation by clinically relevant electric fields. restoration of membrane integrity is essential for cell survival in victims of electrical shock. we have studied the effect of non-ionic triblock copolymers ( poloxamer class) on the transport properties of isolated rat skeletal muscle cells following electroporation-induced membrane disruption. 1-3 mm long adult skeletal muscle fibers were isolated by enzymatic digestion from the rat flexor digitorium brevus and maintained under standard culture conditions. they were loaded with the calcein-am dye and placed in a 37,c chamber for recording by real-time video confocal microscopy. the cells were subjected to 4 msec, 150 v/era, 1 a field pulses with a low duty cycle to allow thermal relaxation. peak temperature rise was 0,2.c. the uye content of the cell was monitored in real time. experiments were carried out in calcium-free phosphate buffered saline, with 1 mm mg%. experiments were repeated with 4 mm neutral dextran ( the aim of the present paper is to ascertain if thuracotomy induces a different pattern of variations of cytokines, immunocompetent cells and antibodies from laparotomy in the early postoperative period. 60 patients (34 males 26 females,mean age: 59.5_+5) 30 with gallstone disease and 30 with non neoplastic pulmonary disease were studied. none of these patients received blood transfusion, biological response modifiers, radiotherapy or surgery for at least 6 months before being included in our study. anaesthetic procedures were similar in all patients and none were matnourished. on the day of surgery and on the 1st and 7th postoperative days (pre, lpo, 7po) percentages of cd19, cd5, cd4, cds, cdi6 were measured by means of flow cytometry using moab., and levels of ig a, lgg, igm, ige. by nephelometry cytokine levels in peripheral blood(il-1, il-2, il-4, il-6, tnf) were measured in 10 pts. of each group by means of elisa using moab. _r. esults:variations of il-2 and il-4 were not s.s.. il-6 increased but differences between groups were not statistically significant (s.s). il-i decreased on 1po and increased on 7po in both groups but were only s.s. in the th.g., and therefore, the differences between groups were s.s (p<0.05).tnf decreased in the l.g. and increased in the th.g. on the 1po, the difference was s.s(p<0.05); on 7po, tnf decreased in the l.g. and decreased in the th.g. but these variations were not s.s. cell percentages decreased an lpo and increased on 7po, except for %cd19 cell that increased on lpo and decreased on 7po ,in both groups of pts. differences were not s.s. ig a, igm decreased and ige increased in both groups (p< 0.0i), but differences between them were not s.s. in contrast, igg decreased on 1po (p<0.01) and increased on 7po in both groups, but the decrease iu the th.g. was greater than in the l.g. twenty male children,aged from six months to 3 years,admitted for elective inguinal operation were studied. the operations were performed under balanced combined anaesthesia (fentanyl,thiopemtone,vecuronium, 70% nitrous oxide in oxygen) and blood samples were collected before flunitrazepam premedication,after anaesthesia,4 and 24 hours after anaesthesia. cells from the wound were collected with cellstick sponge which was removed from the wound 4 or 24 hours after anaesthesia. the study was approved by the local ethical committee. the percentage of neutrophils was increased and that of lymphocytes was decreased in perpheral blood after the operation.the values in the wound were close to the values found in peripheral blood. the percentage of t-lymphocytes (cd3) and helper-t-cells (cd4) decreased in peripheral blood being lower in the wound than in peripheral blood after the operation. the percentage of t-eytotoxic cells (cd8) also decreased in peripheral blood and was similar to that in the wound. b-lymphocyte (cd19) percentage was increased in pe~pheral blood after the operation and was higher than in the wound. the percentage of activated t-cells (cd3+hla-dr-positive cells) in peripheral blood increased while that of natural killer cells (cd3+cd16+leu19-pos) was increased just after anaesthesia being decreased at g and 24 hours after the operation. spontaneous lymphocyte proliferative responses didn't change while phytohemagglutinin a and concavalin a induced responses were decreased in peripheral blood samples 4 hours after the operation with recovery at 24 hours.pokeweed mitogen induced lymphocyte proliferative responses were decreased at 4 hours (p<o.01) and 24 hours after the operation we previously reported that plasma ige increased after traumatic injury. in this study, we measured plasma ige ievels in 40 trauma patients on hospital admission and 3x weekly in the intensive care unit to evaluate ige kinetics. patient characteristics were: mean age 36-+ 16 years (33 m, 9 f), mean injury severity score (iss) 26+ 10, sepsis (19 patients), sepsis syndrome (9 patients), ards (4 patients), renal failure (5 patients). ige increased in 28 patients (70 %). the mean increase was 527 ng/ml (range 0 to 7362 ng/ml; 95% ci was 160 to 895 ng/ml). increase in plasma ige was significantly greater in patients with sepsis syndrome (p=0.021) and renal failure (p<0.001). although mean plasma ige was higher with ards, pneumonia, hepatic dysfunction, and shock, these differences were not significant (p>0.05). plasma ige increase was not related to severity of injury by iss score (p =0.42). the mean day to highest ige was 9.1-+6.5. the day sepsis was first observed preceded the day of highest ige by 4.2+6.6 days. there was a significant association between the day of sepsis onset and the day of highest ige (p=0.018). eight of nine patients with sepsis syndrome had > 100% increase in plasma ige from admission. one patient's ige levels were normal (20-100 ng/ml) for 24 days and then increased to 7300 ng/ml over the next 12 days, after onset of sepsis syndrome. changes in ige plasma levels may reflect the action of cytokines, such as il-4, which concurrently regulate production of ige and il-1 receptor antagonist in a response to sepsis. sepsis remains a leading cause of late mortality in trauma and hs. although hs-induced bacterial translocation is supposed to be the major cause of sepsis and mof, depression of the res increases susceptibility to infection after injury. the purposes of this study were: a) to evaluate the res in the lung, spleen and liver after hs and subsequent hypertonic saline (hsl) treatment, and b) to document the patterns of phagocytic activity in these organs during 24 hrs. adult male wistar rats (250+_23 gin) were submitted to hs (sbp 40 tort) and after t hr (shock i hr) and 24 hrs (shock 24 hrs) hsl (nac17.5%, 3.5 ml/kg) treatment, 1131 e. coli (i 131) was injected into the portal vein 10 ~tci (n_>12). twenty minutes later, the lungs, spleen and liver were harvested and scintilographic counts obtained. data is depicted as mean_%+sem * p<0.05, ~" p<0.001 and statistical analysis was performed by analysis of variance and wilcoxon tests. one hr after treatment, lung 1131 uptake was increased and liver and spleen 1131 uptake were reduced compared to sham. twenty four hrs after treatment, all organs, except lung uptake, returned to normal values. radioautographic histological analysis revealed radiolabeled particles inside phagocytic cells of all organs. we conclude that pulmonary phagocytic activity increases after 1 hr of hs hsl reatment, diminishing by 24 hrs although still above normal values. in contrast, res suppression occurs in liver and spleen after 1 hr hs hsl treatment, returning to normal values by 24 hrs. these results may explain lung complications and immunosuppression after trauma. infusion of endotoxin as well as major surgery is followed by lymphopenia in peripheral blood. the purpose of this study was to investigate to which tissues the lymphocytes are redistributed in response to endotoxaemia and major surgery. in addition changes in lymphocyte subpopulations and expression of mecii was measured. lymphocytes were isolated from peripheral blood of 30 rabbits, labelled with 111indium-tropolene and reinjected intravenously into the rabbits, i0 rabbits received an infusion of escherichia coli endotoxin 2 ~g/kg, while i0 rabbits were subjected to a major sham operation and i0 rabbits served as a control group. the redistribution of lymphocytes were imaged with af gamma camera, and calculated with an interfaces computer before, and 2, 4 and 6 hours after major surgery or infusion of endotoxin or saline. interleukin-l~ and serum cortisol were measured. in addition we followed cd2, cd3, cdlla/b, cdis, cd20, cd44, mhcii and cd4/cd8 ratio. following endotoxaemia interleukin-lf~ increased significantly, following endotoxaemia as well as major surgery serum cortisol increased significantly. following major surgery as well as endotoxaemia there was significant lomphocytepenia in peripheral blood with a decreased cd4/cd8 ratio while the cd3 positive subpopulation increased. in addition there was a decrease in the expression of mhcii on the lymphocytes peripheral blood. the radioactivity of the lymphatic tissue in and around the intestine increased to 129% of initial values following endotoxaemia and to 125% following major surgery. the results indicate that endotoxaemia as well as major surgery induces redistribution of lymphocytes from peripheral blood to lymphatic tissue. among the lymphocytes staying in peripheral blood there was a decreased expression of mhcii and a relative decrease in cd4 cells compared to cd8 positive lymphocytes. in order to analyze the effects of immune suppressive substances on expression of mrna of interleukin-2(il-2) and interleukin-2 reeeptor(il-2r), this study was carried out. twenty male rabbits with comminuted fracture were used in the study. ten ml blood were taken at 0, i, 2, 4, 6 days after injury. the sera were tested for the effects on lymphocyte blastogenesis and induction of il-2 stimulated by concanavalin a(con a): the sera from the rabbits 2 days after injury were analyzed with sds-page gel eleetrophoresis, and divided into three groups by ultrafiltration (ufpi ttk, 30kd,milipore; centricon-10,10kd,amicon), that are less than 10kd, between i0 and 30kd, and more than 30kd. each group of the substances also was tested for the expression of il-2 and il-2r by the dot blot hybridization. the results showed that: i) all sera from the rabbits after injury had significant suppression on lymphocyte proliferation and secretion of il-2 by the con a-stimulated splenocyte in mice; 2) the sera from the rabbits 2 days after injury had more profound suppression than other injured sera; 3) there was a marked band at about 10kd in sera from the rabbits 2 days after injury, but nothing at the same position in normal sera analyzed with electrophoresis; 4) the substance with molecular weight of about iokd had more obvious suppressive action on expression of mrna of il-2 and il-2r than other groups substances, of which molecular weights are more than 10kd. it is concluded that: i) the sera from the injured rabbits can reduce immune response; 2) there is kind of substance, of which molecular weight is about 10kd, it is probable the main factor involved in the pathogenesie of postinjury suppression immune; 3} the substance can depress the expression of mrna of both il-2 and il-2r. research institute of surgery daping, chongqing, 630042 p. r. china acute ethanol uptake prior to injury modulates monocyte tnfo~, production and mononuclear cell apoptosis. g. szabo, b. verma, p. mandrekar, d. catalano monocytes (mo) have been shown to contribute to immunosuppression after both major injury and alcohol consumption. we reported that acute ethanol exposure of m(3 results in decreased antigen presentation, induces tgf-13 and pge2 while inhibiting inflammatory monokine production. we also showed that post-trauma immunosuppression is mediated by hyper-elevated mo tnfc~ and il-6. consequently, here we investigated rnonokine production in trauma patients (n=10) who had elevated (>o.lmg/dl) or had no blood alcohol level (n=t0) at the time of emergency room admission. none of the patients had chronic alcohol use history. met tnfc~ production from trauma patients with prior alcohol uptake was undetectable during days 0-3 post-injury in contrast to patients without alcohol exposure. furthermore, decreased tnf~x levels were found in alcoholic patients' mci after mdp or ifny + mdp induction. however, mcl tnfc~ levels during the 4-8 days post injury period became higher in alcoholic trauma patients. furthermore, over 9 days post-injury, alcoholic trauma patients showed significantly elevated mci tnfo~ production after adherence isolation, mdp, or ifn+mdp stimulation compared to patients without alcohol. these results suggest that acute ethanol uptake prior to injury decreases tnf(x inducibility in the early post-trauma period, but these patients' mo produce hyper-elevated tnfa levels later post-injury, thereby prolonging their cytokine shock risk. tnf ng/ml 0-3 days post-injury 9 days post injury stimulus ale. pt. pt 9.3 11.9 5.6 4.0 immunosuppression might also be increased by the elevated apoptotic activity found in trauma patients' mononuclear ceils, which was even greater in alcoholic trauma patients' cells. in non-alcoholic trauma patients' preactivated mo, in vitro acute ethanol (25-150mm) exposure resulted in a significant down-regulation of tnfc~ (p<0.001) and il-6 (p<0.01) production. in contrast, in vitro ethanol exposure increased the production of inhibitory monokine, tgfi]. these results provide both in vivo and in vitro evidence for the effect of acute ethanol exposure increasing immunosuppression and cytokine shock. the 'systemic inflammatory response syndrome' (sirs) with consecutive septic multi-organ dysfunction represents the major cause of late death following major mechanical and burn trauma. systemic hyperinflammation and concurrent depression of cell mediated immune response (cmi) render the traumatized host anergic, resulting in profound susceptibility to opportunistic infection. monooytes/macrophages (mo) play a central role within the host defense system in developing and manifesting states of injury, shock and sepsis. the mechanistic scrutiny of the synthesis patterns of crucial cccytokines appears to be a helpful tool to further analyse mo behaviour in the compromised individual. the objective of this study was to further dissect the characteristics of cytokine regulation in pbmc under stressful conditions, via analysis of the expression of cd14+ receptor, the proinflammatory mediator il-6, the macrophage activating factor ifn-5,, and neopterin (npt) a metabolite of activated mo. we investigated pbmc's on consecutive days 1, 3, 5, 7 and 10 after mechanical trauma of 9 and after bum trauma of 5 patients (mean age 38~17 years; mean iss 34±2 pts). in trauma patients we saw a massive increase of pha induced neopterin synthesis compared to controls. however, when discriminating the npt levels in the supernatants for the amount of mo stimulated, the npt output of the individual cell was lower compared to mo of nontraumatized individuals. interestingly there was a contrary coarse in the cumulative protein release patterns of il-6 and ifn-7 in mechanical versus burn trauma patients. wheras in burn patients ifn-y was decreased significantly (16+5 u/ml) compared to controls (58+6 u/ml) as well as mechanical trauma (59+12 u/ml). il-6 showed a significant suppression following mechanical trauma (662+58 u/ml) vs control (1266+91 u/ml) and bum patients. the rt~,na signal intensity for beth eytokines was in concurrence with the protein release in more than 85% of the individual patients investigated. from these data we can conclude that the inadequate low npt synthesis predominantly in bum patients appears to be a sign of cellular immaturity and is probably partly due to low t-cell ifno t signals. in addition we could state that the quality of trauma is apparently responsible for the different synthesis patterns of ]l-6 and ifn-q,. it has been postulated that bacterial invasion or endotoxemia are necessary for cytokine production following burn injury. we studied the organ distribution and kinetics pattern of il-fc~ (cell-associated il-1 agonist) in eutrophic rats subjected to either 20% tbsa cutaneous scald injury (bi), muscle scald injury of equivalent 20% tbsa (mbi), sham muscle bum (resection of skin only, up to 20% tbsa) (smbi), and sham cutaneous burn (sbi), followed by saline resuscitation (15 mukg i.p.). separate rats were infused with 15 mg/kg e.coli 0111:b4 lps or saline lv. unmanipulated rats were baseline normal controls. liver, lung, spleen, ileum, thymus, kidney, skin, and plasma were harvested at various time-points within the first 24h. tissues were frozen, weighed, homogenized, the homogenates centrifuged and the supernates assayed with a radioimmunoassay specific for rat il-l(z (detection limit 150 pg/rnl). il-lc~ was expressed as ng/g weight + sem (lowest detectable amount 1.5 ng/gwt). il-lo~ was constitutively present only in the skin (102 + 16.4 ng/gwt). cutaneous burn and sham cutaneous bum induced biphasic elevations of il-lcc in the liver and lung only, with maximal levels at 2.5h (in the liver, bi = 16.5 _+ 6.2 ng/gwt, sbi = 1.7 + 0.1 ng/gwt, p _< 0.05; in the lung, bi = 10.3 + 1.3 ng/gwt, sbi = 1.9 + 0.8 ng/gwt, p -< 0.002). of note, both bi and sbi rats had detectable il-i~ in the liver at timepoint 0 already (2 min real-time). these levels increased in parallel until 30 min and became eventually different by 1 log at 1 -2.5h. all other organs as well as plasma were below detection limits. muscle burn injury and sham muscle burn (skin resection) induced similar elevations of il-10~ in the liver at lh, indistinguishable from each other and from cutaneous burn. in contrast, lps challenge induced dramatic elevation of il-t~ in all organs tested except for the kidney; the spleen was the most responsive organ to lps-induced il-lo~ production. these data indicate that thermal or mechanical injuries induce very early and organ specific production of il-1 c~ in vivo by mechanisms other than endotoxemia. injury-induced complement and platelet activation may be involved as well as the neuro-endocrine axis, which may explain the low levels of il-lo~ induction observed in all rats at the very early time-points. trauma services, massachusetts general hospital, and department of surgery, harvard medical school. fruit, st, boston, ma 02114. j. f. schmand *#, a. ayala* and i. h. chaudry* studies indicate that i.v. infusion of the colloid hes in normal animals does not adversely affect non-specific immunity. it remains unknown, however, if lies affects cell mediated, specific immune functions after trauma and hemorrhage (hem). to study this, non-heparinized c3h/hen mice underwent midline laparotomy to induce trauma and were then bled to and maintained at a bp of 40 mmi-ig for 60 rain. the animals were then resuscitated with either 4 times (x) the shed blood vohune as lactated ringer's solution (lrs) or 2x lrs + lx 6% lies. sham mice were neither hemorrhaged nor resuscitated. at 2 or 24 hours post hem serum, peritoneal (pm~) and splenic macrophages (sm~) were obtained. bioassayes were employed to assess the levels of ii-l, il-6 ( alternatively pmqb showed no differences in il-1 release between all groups at 2 and 24h, while sm~ from the lrs + hen group showed a depression at 24h. tnf production by pm~ was depressed in all groups at 2h and remained so in the lrs + hes group at 24h. sm~b showed decreased tnf release values in both hem groups at 2 and 24h. in summary, the levels of inflammatory cytokines (particularly the values of circulating il-6) after trauma/hem are positively influenced by the administration of hes. this might be due to a protective effect on pmqb and sm~, but also on other cytokine producing cells, e.g. kupffer ceils. we conclude that hes is not only a safe, but also beneficial agent in the resuscitation of patients atler trauma/bemorrhagic shock. this study investigated endotoxemia and consecutlve immune response in 39 patients with multiple trauma (median injury severity score = 20,5). blood samples.were collected shortly after injury and after 0,5, 1, 3, s and l0 days. endotoxin was measured with limulus-amebocyte lysate test and the specific antibody content (sac) against endotoxins of the classes igg, igm and lga by elisa-technique. five antigens were used: lipopolysaccaride (lps) of e.coli (ec), lipid a of e.coli (la), lps of pseudomonas aerog. (pa), lps of vibrin cholerae (vc) and cx-hemolysin of staphylococcus anreus (oth). a nephelometer indicated the total concentrations of igg, igm and iga. differences were checked with wilcoxon-test and p<0,0s was considered significant. cross-reactivity was calculated with rank correlation coefficients. results: endotoxemia peaked shortly after injury (0-3h) at 0,425 eki/ml (median), decreased thereafter to 0,04 eh/ml at day s and remained on this level. sac oflgmclass increased to all endotoxins and peaked at day 3 revealing the lfighest level to la followed by pa (= 80% of la-sac), ec (= 65% of la-sac) and vc (= 40% of la-sac). lga antibodies increased as well but only slightly and not significant (exception: sac to la was elevated significantly at day 5). igg antibodies increased similar to iga class only slightly and again only sac to la was significantly higher at day 5 and 10. however sac to (xh of all ig-classes remained continuously on the same level troughout the observation time. correlation analysis revealed strong cross-reactivity (r>0,5; p<0,01) most often between antibodies of igm-elass (73%) followed by igaclass (40%) and lgg class (2%]. conclusions: multiple trauma is associated with temporary endotoxemia. endotoxins probably translocated from the gut cause specific increase of anti endotoxin antibodies in blood of the igm-class. endotoxins cause no increase of antibodies to gramposilave bacteria. igm antibodies are most unspecific. during cardio-pulmonary bypass, as well as postoperatively, high levels of endotoxin, interleukin-6 (ii-6) and c-reactive protein (crp) were measured in 30 patients. i0 female and 20 male, ageing from 30 to 73 with a median age of 59. blood sampling was done preoperatively, immediately after induction of anaesthesia, after thoracotomy, after cannulation of the aorta and right atrium after the first half of the reperfusion phase, after closure of the thorax, 2 and 4 hours after the operation and then every morning until the 5th postoperative day. blood was drawn into heparinized tubes (i0 iu/ml) which were free of endotoxin. crp levels were determined through the use of the behring nephelometer. 11-6 levels were measured by using commercially-available elisa test. the endotoxin level was determined by a chromogenic modification of the limulus amebocyte test. the statistical analysis was done using the wilcoxon ranks test and correlation analysis. a significant increase {p 0.01) in endotoxin plasma occurred during surgery, culminating in a peak (median value of 0.795 eu/m!) during reperfusicn. plasma levels of endotoxin continued to be slightly raised till the 5th day after surgery, whereas those of interleukin-6 rose at the end of the operation and were at their highest 4 hours later (median value of 217.5 pg/ml). crp levels were also high postoperatively with a median value of 114 mg/l, and were markedly raised on day 2 (191 mg/l). a definite, statistically significant correlation between the plasma levels of endotoxin and 11-6 during the operation was establisthed (p 0.05), leading us to conclude that the endotoxin liberated during cardiac surgery acts as the main trigger in the releasing of 11-6, and thus induces the postoperative acute phase reaction. there was no evidence of a correlation between crp and endotoxin or 11-6 plasma levels. impaired immune function is well described following trauma and hemorrhagic shock (hs). prior studies have utilized peripheral blood or spleen cells to index immune function following hs. however, changes in mucosal immunity are not weii characterized in this setting. gut origin sepsis is thought to be an important cause of organ failure and death following trauma. a rodent model was utilized to allow comparison of mucosal-associated immune function vs, systemic compartments after hs. fischer rates underwent hs (map 35±5mm hg) for 60 minutes followed by resuscitation with shed blood and lr. sham animals were instrumented only. rat tears were collected at 24 and 72 hours following hs for quantitation of slga by ria. animals were sacrificed at 24 hours and spleen (spl), peripheral lymph nodes (pln), and mesenteric lymph nodes (mln) harvested for cell population analysis using flow cytometry and mitogen stimulation analysis. cell marker expression analysis revealed no changes in t or b ceil populations following hs. mitogen mucosal immune function appears relatively spared following hs. the mechanism(s) for this variability in immune function requires further investigation. we have found that transplantation of bone marrow in a hind-limb graft to syngeneic lethally irradiated recipient is followed not only by rapid repopulafion but also overpopulation of bone marrow cavities. the question arises whether this unexpected phenomenon could be the result of stimulation of stem cells by factors (cytokines) released from surgical wound at the site of anastomosis of graft with recipient. aim of the study was to investigate which tissues damaged during the procedure of limb transplantation may be a potential source of humoral factors accelerating in vivo bone marrow proliferation. methods. experiments were carried out on lew rats in 5 groups. in group i, the hind limb was transplanted orthotopically to a syngeneic recipient; in group ii, sham operation was performed; in group iii, a four-cm long cutaneous wound was made on the dorsum; in group iv, limb skin was harvested, fragmented and implanted into peritoneal cavity; in group v, bm from femur and tibia was implanted intraperitoneally. bm, lymphoid tissues and blood were sampled 10 and 30 days later for cell concentration and phenotype evaluation. results. the yield of nucleated cells from tibia was on day 10 in the control 29.3 + 3.2, in group 1 58.1 +2.0, in group ii 47.1 + 1.6, in group iii 50.8+2.0, in group iv 48.5_+2.0, in group v 37.4_+4.9x10(6). the evident increase in bmc yield in all groups continued until day 30. increase in weight and total cell count of spleen and mesenteric lymph nodes in all but group iii was also found. no differences in percentage of maturing erythroid cells, but higher of mature myeloid cells and lower of lymphocytes were observed. conclusions. trauma of skin, muscles, and bone brought about an increase in bone marrow cellularity and acceleration of maturation of myeloid lineage. transplantation of bm ceils alone did not produce this effect. transplantation of bm in limb graft is a good model for studies of natural factors reaulatin~ bm hemormesis. this study sought to determine a relationship, if any, between the degree of hypochclesterolemia upon trauma patients' admission and their subsequent outcome. all blunt and penetrating trauma patients admitted to a level i facility from 1987 through 1991, and who had serum cholesterol assayed during the first 24 hrs were retrospectively studied for development of death or significant organ dysfunction. the mantel-kaenzel chisquared test was used to determine significance of data at the p< 0.05 level. results: 2909 trauma patients were admitted during the four-year period who had serum cholesterol assays performed in the first 24 hrs. 24 patients had cholesterol levels less than 50 mg/dl; 8 of these (33.3%) died, 3 (12.5%) developed ards, 5 (20.8%) developed acute renal failure, and 8 (33.3%) developed multisystem organ dysfunction; hypocholesterolemia in these patients was not due to liver injury or massive fluid administration. the risk of death was 5 times greater and risk of multi-organ failure 3 times greater in this group than in those with a normal serum cholesterol (>if0 mg/dl; 1820 patients; p<0.05). conclusions: admission serum cholesterol level in the trauma patient serves as a powerful marker for those at risk of subsequent organ failure or death. hypocholesterolemia in this setting may result from organ hypoperfusion and humeral mediator release. lung tissue contains many immunocompetent cells. resection, therefore, is expected to activate extensively inflammatory mediators such as pmn-elastase, pmstanoids and pteridines. in a prospective clinical study we compared 35 patients (pts) undergoing either thomcotomy with or without lung tissue msectioh and tboracoscopic lung resection concerning activation of inflammatory response. material & methods: group a pts (n=8) had thoraantomy but no lung tissue injury; group b pts (n=ls) had thoracotomy and lung tissue resection due to benign diseases; group c (n=9) represents group b tissue resection but using a thomcoscopic procedure. the following parameters were determined pre-, peri-, and postoperatively: elastase and crp as indicators of activation of pmn-leukocytes and injury severity; prostacyclin (pgi 2) and thromboxane (txa~) as parameters of lung endothelial response; prostaglandin f2~ (pgf~) and pgm representing pulmonaly metabolic activity; pge a and neopterin as proof of macmphage activation. statistics were performed using analysis of variance for repeated measures. results: group b pts revealed postoperatively an increase in crp (p<0.001) indicating a higher injury severity in comparison to the thoracoscopic procedure (c). both, controls (a) and group c pts did not show pmn-activation, whereas group b demonstrated a reversible increase in elastase. surgical trauma caused in all groups a release of pgi z and txa 2 which was more pronounced in c (p<0.05) and most in b (p<0.01). similar results were found for pge~ and pgf2=. there was no activation of maerophages since neopterin did not increase. apparently, metabolic lung function was not impaired because there was no marked rise in pgm except in b (p<0.05 vs. c). discussion: our results demonstrate that lung tissue injury aggravates the mediator release induced by thoracic traum. these mediators among others are able to increase capillary pressure and hence lung edema formation. impairment of lung function, however, seems dependent on the extent of the liberation. therefore, the maximal release reactions occured in group b and c after lung tissue resection, whereas the controls showed the highest levels immediately after the incision. we conclude that thoracoscopic procedures are superior in reducing the resection trauma per se and hence might prevent severe mediamr-induced (pulmonary/systemic) sequelae. in a prospective study we investigated 45 patients using radiochemical method according to sch~dlich (s) and photometric method according to hoffmann (h). serum of severly traumatized patients was withdrawn directly after admission at our emergency room and in narrow time intervals during first 24 hours after trauma. follow up control samples were taken daily until day ten. whereas no elevated pla-ca was found during first 24 hours, a peak was regularly observed around day four. there was high correlation between pla-ca and iss (r=0.71, p<o.o001) except patients suffering from thoracic trauma, thoracic trauma {tt) provoked similar gons-score-and pla-ca-values compared to multiple trauma (mt) despite significantly lower iss: pla-ca (h) mt 32.9 + 6.4 tt 28,7 +_ 9,5 n.s. pla-ca(s) mt 11.7 +_ 2.6 tt8.3 _+ 2.6 n.s. goris mt 3.5 4-0.7 tt3.6 _+ 1.3 n.s. iss mt 35 4-3 tt21 4-3 p<0.01 we found delayed elevation of pla-ca after severe trauma, however detection of delayed pla-ca in the serum of traumatized patients cannot give reliable information about the exact role of pla in the inflammatory reaction after trauma as latest results show that pla is secreted early but bound at lipophilic sites of vascular membranes. hern~mdez m j, amiguet ja, monreal ji, vid~n jr, jim6nez f j, l6pez-vivanco g acute phase reactant proteins increment in serum of patients with traumatisms and inflammation has been previously reported. alpha-1 antitrypsin (aat), alpha-2 macroglobulin (amg) and ceruloplasmin (cp) are evaluated in the following of 33 patients, 27 males and 6 females (mean age: 43 years), with compound fractures. 57 % of them were located in tibia and fibula. intercurrent infection developed in 4 patients and internal fixation rejection in one. measurements were made by radial immunodiffusion on days 0, 3, 10, 17, 24, 31 and 38. a control group of 30 healthy volunteers was also evaluated. aat and cp showed increased values from day 3 which kept elevated untill the end of the study. amg elevated from day 24. when infection developed, aat and cp values were even higher whereas amg values decreased. fixation rejection caused aat, amg and cp increment. aat and cp increment in non complicated fractures might be due to accompanying inflamation, by means of cytokines release and aprps hepatic synthesis induction. therefore, increment is higher when infection or rejection develop. amg behaviour at early stages of evolution is secondary to hiperconsumption, considering mean life shortening after proteases ligation. amg modifications in fixation rejection remain obscure. aprps increment modulates inflammation and bony callus formation by means their antyprotease and antioxidant properties. clinically, aprps might be useful parameters in determining complications onset in fractures evolution. hemorrhagic shock (hs) is a common complication of trauma, as well as some major surgical procedures. the alteration of the immune system by hs is thought to contribute to the increased septic morbidity and mortality seen in these patients. multiple mediators are released following hs. il-1, il-6, tnf, pge 2 and tgf-b have all been implicated in these immune system changes. in vitro, pge 2 causes many of the immune system changes following hs, yet the time course of pge 2 release has not been well deliniated. the aim of this study was to determine the time course of pge 2 release by peritoneal and splenic macrophages following hs. c3h/hen mice were bled to a mean bp of 35+5 mm hg for 60 minutes. the animals were resuscitated with the shed blood and normal saline. control animals were cannalated, but blood was not withdrawn. the animals were sacrificed at 2, 12, 24, 48, and 72 hours following hs. peritoneal and splenic macrophages were harvested from each animal and cultured with lps for 24 hours. the supernatants were collected and frozen until assayed. pge 2 was assayed using an elisa (cayman chemical). results are shown below for the peritoneal macrophages: pge 2 release by peritoneal macrophages was significantly elevated at 24, 48 and 72 hours over control. it was maximal at 48 hours, but by 72 hours it had started to decline. splenic macrophage pge2 release was much more variable. (data not shown.) there was less consistency between time points and no clear trend was seen. in conclusion, pge 2 is significantly elevated at 24 hours following hs, and reaches a peak at 48 hours. pge 2 release may be responsible for the immune system changes seen in the first few days following hs. splenic and peritoneal macrophages respond differently in their release of pge 2 following hs; which may be due to differences in their milieu. there is an obvious association between the altered immunocompetence of patients following traumatic injury of various types and the increased riskto develop complications. the present investigation was undertaken in order to analyze the value of both neopterin (n) and c-reactive protein (crp) in monitoring disease course in patients with multiple trauma. we were interested to compare nconcentrations as marker of t-cell/macrophage activation with crp levels as indicator of the inflammatory acute-phase response daily serum concentrations(n and crp) were measured in 12 patients (9 male, 3 female, mean age 31.42), admitted to our icu following serious trauma (mean iss=34) and in 10 control subjects. the clinical course of each patient was evaluated with apacheii score according to knaus. mean stay in icu was 9.25 (range 5-13). 5 patients didn't develop organ failure(nof-group), 4 patients survived, developing mof (s,mof-group), 3 patients died with mof (ns, mofgroup). three patients(one s and two ns) showed signs of septicemia and septic shock. the serum samples were measured for neopterin (immu-test-ria, henning-berlin) and for crp (immunoturbidimetric method) -at the university hospital for internal medicine, innsbruck, austria. the highest crp levels were measured 48h after trauma, but they were not accompanied by significant increase in n-values. we found significantly elevated n-concentrations from the days 5-6. n and crp levels showed a parallel release peaks on the days 6-7 and the values discriminated significantly among the three outcome groups(the second crp peak is lower in comparison to the first posttrauma peak). by the ns-group we found constant and parallel increasing crp and n levels, reaching extremly high values 24-48h and preceding clinical signs of mof and sepsis. the values increased dramatically before death. the serum levels by all 10 control subjects were upper the normal limit for n and crp.. the parallel occured peak changes in n and crp concentrations showed a significant correlation with clinical status, using a disease activity score (apac h eli). our findings suggest that both-cellular and humoral mediated immune mechanisms are activated after multiple trauma and that simultaneous determination of n-crp may be of advantage as diagnostic and prognostic parameter in polytrauma-patient monitoring panel. it is apparent, that measurement of very high levels may be of value as a srong indicator in developing of mof or septic complications and thus offer the possibility for earlier therapeutic intervention. the modification of t-cell/macrophage and acute-phase responses may be potentially useful in the treatment of icu patients, resuscitated after severe trauma, and suggest that n-crp may be relevant indicator for testing experimental immunomodulating therapies. although several in vitro studies suggested the catalytic action of iron in oxygen free radical generation, few data are available concerning iron-metabolism following ischemia-reperfusion injury and hemorrhage in vivo. aim of the present studies was to evaluate iron metabolism following mild and severe hemorrhage in the presence and absence of intraperituneal hematoma. methods. male lewis rats (300 g) divided in 4 groups (observation time 120 hrs): a: mild hemorrhage by sequential blood sampling (0.9 ml each)(0, 2, 4, 6, 24, 48, 72, 96, 120 hrs). b: a with hemoperitoneum (hp)(2 ml/100g), c: hemorrhagic shock: blood withdrawal of 3 ml/100g over 30 min and resuscitation with ringer's lactate (2 x shed blood) and isogenic donor blood; d: c with hp. parameters: serum iron ([g/dl], ferrozin-method); 59fe labeling of erythrocytas (application of 1 ci 59fe i.a. into a donor rat, 5 days later bleeding of the rat and injection intraperitoneally in experimental rat; plasma transferrin ([g/ml], ria). t-tests and mann whitney. data are expressed as mean + sem. results. compared to baseline, mild and severe hemorrhage caused a significant increase in serum iron at 24 and 48 hrs, respectively (212+30 vs. 248+35; 185+_28 vs. 255+_22). hp reduced this increase (209+_17 vs. 177+21; 194+25 vs. 197+-48, p<.05). i-ip significantly increased hemoglobin between 24 to 96 hrs in mild (12.1+_.8 vs. 14.2+.7) and severe hemorrhage (10.3+.6 vs. 13.1+.6). in mild and severe hemorrhage comparable maximal resorption (59fe labeled erythrocytes in circulating blood) of intraperitoneal hematoma occured at 24 hrs (mild: 318+_71 vs. severe: 364+-114 counts/rain). hp increased survival-rate following hemorrhagic shock (no hp: 10/17; hp: 10/13). in mild hemorrhage no alterations of plasma transferrin concentrations. in shock group without hp, there was at 96 hrs a significant higher transferrin level than with hp alone (3.2+_.5 vs. 2.23 + .2). in conclusion, hemoperitoneum with hemorrhage prevents increase in plasma iron levels by increasing hemoglobin through resorption of erythrocytes and thereby suppressing iron mobilization from endogenous iron resources, e.g. liver. this suggests that intraperitoneal hematoma not necessarily promotes iron-mediated oxygen free radical production, especially since survival in these groups was improved. increased transfarrin levels in group c at the end of the experiment suggest increased iron turnover which was not observed in shock groups associated with hemoperitoneum. primary immune response to thymus-dependent (srbc) and thymus-independent (vi-polysaccharide) antigens was determined during 30 days after cct. antigenic challenge was made on day i, 5, 15 or 30. number of spleen plaque forming cells (pfc) and serum haemagglutinin (ha) titre were studied on day 5 after immunization. cct stimulated immune response to t-dependent, but not to t-independent antigen. enhanced response was noted when srbs were given in posttraumatic day i, it was higher than preceding one after injection on day 5 and reached a maximum when antigen was presented on day 15 after trauma (pfc number 2.4 times exceeded the level of immunized, but nontraumatized rats). the rise of pfc number in spleen correlated with increased ha level. thymectomy had been performed 30 days before cct completely abolished posttraumatio stimulation of immune response to t-dependent antigen. standard thoracotomy also enhanced humoral response when srbc were administered on day 15 after the operation. thymus trauma modeled by pincett squeezing of its right lobe and performed at the same time with thoracotomy fully abrogated as well as thymectomy stimulation of immune reaction. thus, different experimental chest traumas are able to increase antibody production via thymus-dependent mechanisms. endothelin is a 21 amino acid peptide produced by ischemic or injured endothelial cells. in vitro and in animal studies, et is also a potent constrictor for renal mesangial and coronary vessels, a negative cardiac inotrope and an endocrine regulator. our lab has shown that et is an agonist for monocyte production of interleukins i, 6 & 8, prostaglandin e 2 (pge2) , and substances which trigger superoxide production. systemic et levels increase in hypoperfused and septic patients, and et may be yet another important cytokine in critical illness. but while et has been shown to be a potent vasoconstrictor in healthy dogs, systemic vascular resistance does not increase in critically ill patients with high et levels. (we hypothesize that this might be due to pgez-mediated vasodilation predominating over et-mediated vasoconstriction.) we next asked what happened to systemic et levels in patients with major burns (>20%.) ten hemodynamically stable patients resuscitated by a modified parkland formula to a urine output >30 cc's per hour had et levels drawn on admission, at i, 6 12, 24, and 48 hrs. et levels were measured by radioimmunoassay. mean levels were elevated at 205±28 pg/ml at all time points versus levels in healthy controls of 39±9. in summary, systemic et levels increase significantly in patients with major burns. et may be yet another cytokine playing a significant role in the immune, inflammatory and multiorgan dysfunction observed with major burns. restoration processes in an organism after ischemic damage are realized through ~n~lammatory mechanisms~ the intensity of which is significantly defined by blood levels of neuropeptides. myocardial infarction (mi) was chosen for studyin 9 these processes since it eradicates the influence of infectious factc~rs. 32 dogs~ in whom mi underwent different forms o¢ healer, g; bhn~ed ~h~t during the acute phase of the disease there was a characteristic rise of ne!~ropeptides in the blood. these neuropeptides had nociceptive and antinociceptive effects. particularly substance p and -endorphins triggered off the development of compensatory and adaptive mechanisms and defined the intensity of inflammatory reaction at the zone of ischem~t: damage-notable fall in substance p levels after an ~nitial increase, while the ~-endorphins stayed high was an important condition for non complicated healing of mi. on the other hand high levels of substance p with low ~-endorphin concentrations lead to increased infiltration o~ neutrophils into the infarction zone and weakened the activity of synthetic processes~ thereby leading to left ventricular aneurysm. at the same time low intitial levels of substance p slowed down the development of necrotic processes which lead to delay in refunctioning of the heart and complicated the healing process. thus, regulation of the levels of neuropeptides in the blood in trauma forms a perspective method of its treatment. of laparascopic versus open choleocystectomy c. schinkel, s. zimmer, v. lange, d. fuchs, e. faist the impairment of immune function due to surgical trauma may be followed by deleterious septic sequelae. compared to open abdominal surgical procedures (lap), laparaseopic surgery (lsc) is associated with a decrease in hospital stay and in accelerated patient recover. the aim of the study was to evaluate the sensitivity of the immune sermn parameters of il-6, saa and neopterin, the percentage of cd14+ cells, the in-vitro il-2 synthesis after mitogen stimulation and lymphocyte proliferation, in order to purposefully discriminate differences in the severity of trauma. we investigated the blood of 27 patients with cholecystolithiasis undergoing either laparascopic (16) or open (i 1) cholecystectomy on consecutive perioperative days -1, 1, 2 and 3. there was no significant difference between the two groups concerning age and sex. patients with clinical signs of acute cholecystitis were excluded from the study. operation time and hospital stay were obviously longer in lap patients (67 versus 145 minutes, 5 versus 20 days) compared to the lsc group. concerning the unspecific acute phase reaction we could show no difference in the increment of senun amyoid a (saa) synthesis in the lsc group (d-i 40 + 1 lng/ml, d1 362 + 43ng/ml) versus lap group (d-1 61 + 19ng/ml, d1 457 + 45ng/ml), while in serum il-6 levels we saw a less steep increment in the lsc group (7-fold from d-1 to d 1) compared to the lap group (10-fold from d-1 to d 1). the analysis of cd14+ receptor expression and serum neopterin did not reveal any difference between the groups. lymphocyte function showed an impairment of proliferation to antigen stimulation in lap (d -1:24.000 + 3.330 cpm, d 1:17.300 + 3.000 cpm) compared to the lsc group (d -h 22.500 + 4.200 cpm, d h 26.000 + 2.900 cpm). in both groups il-2 synthesis was decreased post-operatively. our data indicate that laparascopic cholecystectomy reusults in a less distinct unspecific acute phase reaction post-trauma compared to that following lap. neopterin serum levels and cd14 receptor expression show that these parameters apparently are less useful markers to detect differences of surgical trauma severity while it appears that the impact of lap is reflected most impressively on the lymphocyte compartment. trauma alters the host resistance of organism and is accompained by appearence of excgenic and endogenic proteins in the body. to understand the molecular mechanisms of host resistans disorders in trauma, as a first step, the genetic regulatory mechanisms of immune response after antigen injection has been studed. the appearence of specific protein factors (13-19 and 25 kda), in the nucleus of rat splenic and brain cells, accordingly, was shown after immunization with sheep erythrocytes. the stimulatory effect of these factors on the il-2 mrna and il-2 production was detected. the nucleotide sequences of the human il-2 gene regulatory region bounding by the splenic nuclear proteins were determined between +39-141 b.p. the il-2 trans-factors shows the affinity to splenic and thymic lymphocytes in vitro. thus, the antigen causes the appearence of specific protein factors in the cells,which act on the gene level,stimulate il-2 production and the host resistance. these results cause the next step of experiments using the same model, but after trauma. these investigations will let us verify the hypothesis that the protein il-2 gene trans-factors may play a definite role in the decrease of the cell immune responce after trauma. confronted with the routine procedure of prophylactic treatment of candidates for surgery in a rural african hospital, we initiated studies on the fre'quency of post-surgical malaria. in tanzania 195 non-pregnant patients from rural areas were followed. of preoperative patients 10% had a parasitaemia and those maintaining it showed no increase or complaints. nine percent of patients without detectable parasitaemia before surgery came down afterwards and one-third had malaria-like complaints. spinal and general anaesthesia were equally applied in these last patients. in burkina faso we studied 120 patients of which 16% had a parasitaemia on admission and 6% had postoperative malaria. half of the surgical patients came from rural areas, whilst only 14% of those with malaria lived in the city (with much less exposure and immunity). 57% underwent major surgery and 43% minor. bloodtransfusions (4% with parasites) never evoked a parasitaemia in recipients. post-surgical malaria is thus a reality in about 10% of the adult cases, both in east and west africa. surgery evokes a cascade of factors, varying from cortison to interleukines and acute phase proteins; immune responses may temporarily be suppressed. clinical attacks of malaria in otherwise immunes could be evoked by one of these factors. though malaria can easily be cured, the differential diagnosis is difficult because of post-surgery fevers; we found that 16% was treated without justified indication. the involvement of "student-doctors" a. this study examines glucose uptake and hexose monophosphate (i~ip) shunt activity in normal human peripheral lymphocytes and polymorphonuclear leukocytes (pmn). glucose uptake was determined by measurir,g the uptake of tritiated deoxyglucose, a non-metabolized glucose analogue. adsorption of co2 derived from [i-14c] glucose was used to determine knp shunt activity. in vitro assays were carried out in hormone concentrations approximating normal and elevated trauma blood levels. (normal -cortisol 0.12 ~g/ml, glucagon 80 #g/m1, epinephrine 20 ~g/ml, insulin 18 t~u/ml; traumaeortisol 0.40 ~g/ml, glucagon 500 /*g/ml, epinephrine 420~g/ml, insulin 36 ~ij/ml. analysis of twenty subjects showed a reduction of 207° ~mp shunt activity by lymphoeytes and a ]3% reduction in glucose uptake by p~n in normal vs. trauma hontc,nes p < 0.05. lymphocyte glucose uptake was also reduced by trauma hormones p~0.05. it ha~ be.ea~ suggested thgt idiopatno pulmonary fibrous (y.pf) [s a consequence of severe alveolar epithelial injury and is associated with an nveolar irnammamry reactio~ and the presence 0f.neutr0phils. there~bre, neutr0pk~ chemoattra~ant~ are probably important in the genegs oft.he infial lesions of ipf. the obse,"wson that stimulated macrophages are or~n histologically promin~t in fibmfio [-~gs ~.nd am capable of p~oducmg a v~dery 0f flbrogenic pep'ides also a~gues for their role ~n the pathogenic prc~e~ oflpf. the observation that stimume~ maerophages ere often histologica[iy prominent in fibrotio lungs and ~re ~pable of producing a varie~, offibroge.~e peptide~ also argues for tkek role in the pathogenic process, therefore, we ha-~e tested the potentn for iater!eukln-8 (i1..-8) and mo~tocyte chemotacde pop, de 1 (x¢cp-1) to induce neutro~hil ~d mononuclear phagocyte accumuhdon in lungs of pafient~ with pulmonary .~r~idosis and i~f. brenet~o.alveolar lavabo (bal) fluids from 12 ipf and 15 sar~qidosis patient were conexntratea by reversed-phase chromatography, ~d ii.4 arid mcp-i asso.~ed by ells& ehemotaxis mad enzyme-reieasing ~ssas's on msnocyte~ and neatrophiis. elisa revealed significenfly elevated b al-eoneentrations o£mcp-i (20.1 ng]mg aibumm) in purisms with p~monary sarcoidodis artd in ipf (41.8 ng!mg) in comparises to 1.1 normal individuals (4.24 ng/mg) and to 15 patients w~th obreic bronentis (cb) (~, 16 rig/rag). similarly, chemota*dc ac~a~' for monocles (mcp-1 e.qu/va]ent) was strongly increased in sareoidosis (86.03 ngjmg) as well as ~n 12f pag,nts (54.47 ng/mg). norra.al indlvidu~s and cb patiants hzd a 7. or 3-fold lower ~cn%i~y, re~peefively. patients with ipf and sarcoidosi~ also h~l eievated il-8 ievei~ (15.5 and 26.0 rig/rag, respe~veiy; nomzls: 2.14 rig/rag; cb: 4.23 ng/mg) mad nvatropmi ohemotax~ (60.25 ~'~d 49.68 nnmg, res!z~ztiveiy; aormals: 0.25 ng,'mg; cb: 1l06 ngmg). these data suggest that increased ievels of born mcp.1 ~d il-8 may be oharacted~tie for ~arcoidosis or ipf_ it appears iikely that both ehernoattraetants ~ontribute to the influx ofmonocytes and neutrophils into the pulmonary alveoius and interstit~um in these dlsea~es. we have recently shown that the combined administration of noninjurious doses of lps and paf in the rat produce ards-like lung injury characterized by neutrophil adhesion to lung capillary venules, neutrophil accumulation in lung parenchyma, pulmonary edema, and increased protein and neutrophil count in bal fluid. this new paradigm of lung injury was associated with elevated serum tnfc~ and pretreatment with anti tnfa mab dose-dependently prevented these responses. also, the combined administration of lps and paf induced lung mrna levels of tnfe~ (5 fold vs. lps or paf alone), ll-lg (80 fold), kc (60 fold) and il-6. taken together, these data suggest that this new paradigm of lung injury is cytokinemediated and that lps/paf in vivo can functionally couple to the activation of gone expression of a multi-cytokine network system, all of which may be involved in the pathogenesis of ards. materials and methods. the sheep model included hemorrhagic shock and closed femoral nailing at day 0, 12hourly injections of e. coli endotoxin and zymosan-activated autologous plasma at clays 1-5 and further observation and measurements at days 6-10. from venous blood and bronchoalveolar lavage(bal)fluid of ten merino sheep (mean weight 30 kg) neutrophil counts (10e6 pmn/ml blood or epithelial lining fluid-elf-), the elf/ plasma ratio of albumin (r), and the zymosan-induced (stim) and non-induced (spont) chemiluminescence response (cl) of blood (10e6 cpm/250,000 pmn), and of blood-and bal-isolated pmn (10e6 cpm/25,000 pmn) were measured. for statistical calculations the wilcoxon test was used. data of the changes in polymorphonucleur leukocyte (pivinl) metabolism have been suggested to play a pivotal part in the post-traumatic systemic inflammatory response syndrome. the underlying cellular mechanisms which control this response are not yet completely understood. since the 'ca 2+ second messenger'-system has been shown to be involved in regulation of pmnl-'respiratory burst', we investigated changes in pmnl-ca z÷ regulation in relation to oxygen free radical mediated injury. methods. in 12 polytranmatized patients (mean injury severity score = 32) arterial and venous blood samples during 15 days. daily evaluation of horowitz-quotiant (po2/fio2), plasma lactate (mg/dl) and body temperature ( results. body temperature peaked at day 6 and 7 (day 0: 36+.4; day 7: 39.8+.4). plasma lactate was significantly increased at day l (36+2) and day 7 (16.2+2). hurowitz-quotient (day 0: 344+43) was low at day 4 (199+19) and day 7 to 10 (178+25)(p<.05). at day 7 a substantial rise in venous pmnl-superoxide production (day 5: 1.9+_.45, day 7: 3.3+.7, day 9: 1.72+_.8), oecured with significant increase in plasma lipid peroxidation (day 1:0.6+0.2 ; day 7: 1.3+0.2). pivin~-myeloperoxidase activity was high at day 2 (2.3+--.5) and then continuously declined (day 7: 1.3+.3). plasma antiexidant activity (glutathione pemxidase) was reduced by 34% at day 7 (day 1: 5.3+.3; day 4: 4.4+_.2; day 7: 3.5+.2). whereas basal ca 2+ concentration remained unchanged (day 4: 68+_17, day 7: 68+_15), fmlp-stimulated cytosolic ca 2+ mobilization increased at day 7 (day 4: 457+89, day 7: 11084410, day 9: 670+262). conclusion. the present study in polytraumatized patients shows, that seven days after injury the agonist-induced pmnl ca 2+ mobilization is significantly enhanced. at the same time, pmnl-oxygen free radical release and phagocytotic activity, systemic fever response and lactate concentrations were maximal. these observations were accompanied by post-tranmatic respiratory failure and in some patients by clinical signs of multiple organ failure. preliminary data from an ongoing study using hes-and dextran-infusions in these patients show attenuation of this inflammatory response. stefan rose, m.d., trauma surgery, univ. of saarland, 66421 homburg/saar 1donnelly sc, 1haslett c, 1dransfield i, 2robertson ce, 3grant is, 4carter c, 4ross ja, 5tedder tf. 1dept's of respiratory medicine, 2accident & emergency, 3intensive care, 4surgery, university of edinburgh, scotland and 5dept. tumor immunology, dana farber cancer institute, boston. the selectins are a family of adhesion molecules (l-selectin, e-selectin, pselectin), all of whom are implicated in inflammatory cell transendothelial migration. they, as a family can be proteolytieally cleaved from their parent cell and exist in a soluble form within the circulation. ards is a disease state in whic neutrophils and neutrophil transendotheliat migration have been implicated. in this study we wished to investigate whether the levels of these circulating soluble receptors from patients at-risk of ards at initial hospital presentation, correlated with subsequent ards progression. eighty-two patients were enrolled (pancreatitis (n=19), perforated bowel (n=12), and multiple trauma (n=51)), of whom 14 progressed to ards. assays for soluble l,p & e-selectin were performed on collected plasma samples via a sandwich elisa. (ns = not significant, **** = p<o.0001) we have found a highly significant inverse correlation between a low circulating soluble l-selectin (and not soluble e & p-selectin) and subsequent ards. these results further our understanding of neutrophilendothelial interactions in the early stages of ards pathogenesis and offer the promise of sl-selectin in combination with other markers of inflammatory events being used to identify individuals at particularly high risk of ards progression on initial hospital presentation. it has been suggested that polymorphonuclear leukocytes aggregate in the lung capillaries of patients developing the adult respiratory distress syndrome (ards) and account for the endothelial damage while releasing toxic metabo-iites such as o.a. free oxygen radicals. among many antioxidants which have been investigated in in vitro systems and in animal models, n-acetylcysteine (nac) has been established to be useful as a free radical scavenger in vivo. to assess the influence of n-acetylcysteine (nac) on the activation of neutrophils in patients undergoing cardiopulmonary bypass (cpb) surgery with extracorporeal circulation (ecc), we evaluated the plasma levels of elastase and myeloperoxidase (mpo) throughout the operation and up to 24 hours after surgery. four hours after surgery also a bronchoalveolar lavage was performed. a spectrophotometric method was used for the detection of plasma elastase activity and mpo levels were measured using a radioimmunoassay. we found that pretreatment with intravenous nac prevented the increase in elastase activity (632 _+ 231 pmol/i vs 1666 _+ 187; p -0.027), but not the release of neutrophil related mediators (2.20 _+ 0.56 ng/i vs 1.80 _+ 0.17; p=0.63). besides, nac had the capability to prevent the enhancement of neutrophil numbers in lavage fluid as is normally seen during cpb (1.4 + 0.8 % vs 25.0 _+ 14.3; p =0.04). although not significant, nac prevented also the increase in elastase levels in lavage fluid (19.0 + 18.5 pmol/i vs 172.7 + 101.4; p = 0.12). it is concluded that nac is able to protect against the inactivation of a 1-proteinase inhibitor, the main inhibitor of elastase activity, and that nac interferes with adhesion and migration of neutrophils. therefore nac may be useful in the prevention of tissue damage. this study is supported by inpharzam belgium. sodium nitroprnsside (snp) has previously been shown to attenuate the neutrophil induced lung injury associated with limb ischaemia and repeffusion. glyceryl trinitrate (gtn), already widely used in aortic surgery, also increases nitric oxide but has a less marked splanchnic "steal" effect. we examined the effect of gtn on lung injury, neutrophil infiltration and activation in a model of aortic occlusion (30 rain) and repeffusion (120 min). sprague dawley rats were randomized into: control (n=i 1); ischaemia repeffusion (ir) (n=12); and ir treated with gtn (2ug/kg/min) during repeffusion (n=10). myeloperoxidase activity (mpo) measured pulmonary neutrophil influx. pulmonary endothelial permeability was measured by wet to dry weight ratio (w/d), broncboalveolar lavage protein (bal) and neutrophil counts (bal pmn). neutrophil superoxide release (mean channel fluorescence mcf) was measured by flow cytometry in a further ir v gtn experiment (n=6 per group). control 573_+t02"* 992_+125 579_+54** bal pmn/mm 3 36l+-126 3"219+1087# 820_~221 *p<0.05, #p<0.02 v control/gtn;**p<0.01 v ir. students t test the significant increase in mpo activity produced by ir was attenuated by gtn. the increase in pulmonary microvascular leakage after reperfusion was also prevented by gtn. neutrophil superoxide release increased significantly from 9.4+0.76 to 13.9_+1.92 mcf after 40 minutes repeffusion (p<.01). this increase in superoxide was prevented in the gtn treated group. these results indicate that gtn inhibits neutropbil superoxide release during limb reperfusion, thus preventing pulmonary vascular leakage and neutrophil infiltration. these data suggest that the beneficial effects of gtn in aortic surgery may be due in part to a protective effect onpulmonary microvasculature. department of surgery, beaumont hospital, dublin 9, ireland. lipton adult respiratory distress syndrome (ards) is marked by increased vascular permeability of the lung to white blood ceils (wbc). indeed, influx of wbc into the lung is believed to be the central mechanism in acute lung injury of ards. evidence is developing that the neuropeptide c~-melanocyte stimulating hormone (c~-msh), a proopiomelanocortin derivative, inhibits inflammatory reactions in animai models of inflammatory responses in humans. to test the influence of a-msh on experimental ards, the peptide was administered to rats after intratraeheal infusion of endotoxin (s. typhosa, 500 #g in 0.25 ml saline). three groups (n=10 each) received: intratracheai infusion of endotoxin plus ip injections of o~-msh (100 ~tg in 0.2 ml saline) at 0, 2, and 4 hr post-endotoxin treatment; intratracheal endotoxin infusion plus saline injections; control intratracheal saline infusion plus saline injections. the bal data showed overall differences among groups (f2,29=6.2, p<.003), o~-msh treatment inhibited endotoxin-induced wbc migration into the pulmonary tree (p<.05). circulating wbc counts were markedly lower in both groups given endoroxin than in the control group (p<.01), but there was no difference in wbc count between these two endotoxin-treated groups. the results indicate that c~-msh can reduce wbc migration in experimental lung injury. in further experiments we tested the hypothesis that c~-msh, administered alone or in combination with an inhibitor of gram-negative bacterial growth, increases survival in a model of peritonitis/endotoxemia/septie shock. cecal ligation and puncture were performed under sodium pemobarbital anesthesia (75 mg/kg, i.p.) in female balb/c mice (16-20g) that were then randomly assigned (15-17 mice per group) to receive at time 0 and 3 hr later: control saline thjectioas (i.p.), injections of ~-msh (100 ~tg), gentamicin sulfate (i0 mg/kg), or both ~-msh and gentamicin. one ml of normal saline was given s.c. to each animal for fluid replacement. both ix-msh and gentamicin treatments administered singly improved survival; when given in combination survival was even greater these findings suggest that the anticytokine ot -msh molecule might be useful for treatment of systemic inflammation, perhaps particularly when used in combination with agents that inhibit bacterial growth. previous studies showed a close relationship between xanthine oxidase activation, membrane damage and postischemic cardio-respiratory failure following aortic clamping and reperfusion. aim of the present study was to evaluate alterations of polymorphonuclear leukocyte (pmnl)-metabolism and signal lransduction systems during ischemia/reperfusion induced by aortic clamping in man. methods, in 14 patients (3 female, 59+4 years) with elective reconstruction of infrarenal aortic anearysms, arterial (a) and venous (v) blood samples were obtained before clamping and declamping, and 60 min after declamping. arterial pla 2 concentrations were higher than venous (113+32 vs. 71+30). while cytosolic basal pmnl ca 2+ concenlxation was not altered at the end of ischemia (v: 52.3+8; a: 42_+6) and during reperfusion (v: 60+8; a: 69+12), fmlp-stimulated cytosolic ca 2+ mobilization showed a significant arterial increase as compared to venous (1652_+601 vs. 638+85, p < .05). these presented data indicate that the reperfusion syndrome after infrarenal aortic clamping is characterized by 1) pulmonary membrane damage possibly due to pmnl-degranulation, 2) activation of pmnl-superoxide radical production with release of activated pmnl in arterial circulation, and 3) a significant arterial increase of pmnl cytosolic ca 2+ mobilization after fmlp-stimulation parallel to pmnl-activation. in conclusion, ischemia/reperfusion in man induces pulmonary damage and activation of pmnl superoxide production possibly due to an altered pmnl-ca 2+ messenger system by inflammatory mediators (e,g. ii-8, tnf, paf). the septic lung diseases (i.e. ards) generally show distinct alveolar damage and surfactant disturbances. it is thought that alveolar macrophages are involved in specific release products like h202 and cytokines like tnf. in this pathway the type ii alveolar epithelial cell (p2 cell) is not only an important target, but as recently shown it is also capable of producing h202. the aim of this study was to investigate the influence of endotoxin on h202 release of p2 cells. we obtained p2 cells from lungs of female wistar rats and investigated the effect of lipopolysaccharid (lps) on h202 release of fresh isolated cells and on cells cultured for 2 days in a fibronectin matrix. furthermore we conditioned isolated alveolar macrophages for 16 hours with 1 p.g/ml lps and tested the conditioned medium (mcm) on cultured p2 cells. p2 cells 5 h ex vivo were _> 70 % pure, _> 95 % vital and had an basal h202 release of 64.5 _+ 1.8 nmol h202 per hour and million cells. adding 1 p.g/ml lps to the incubation medium the h202 release decreases slightly but significantly to 54.2 _+ 2.3 nmol. adding 0.3 p.g/ml phorbol myristate acetate (pma) to the basal incubation medium the h202 release increased 3-fold to 148.3_+ 26 nmol. pma induced h202 release decreased to 128.3 + 41.5 nmol after addition of 1 p.g/ml lps. after 2 culture days the p2 cells were _> 98 % pure and showed a pma inducible h202 release of 174.8 _+ 9.9 nmol addition of 1 p.g/ml lps had the inverse effect as on freshly isolated cells as it increased the h202 release up to 216.2 _+ 2.4 nmol. addition of mcm to cultured p2 cells increases pma-stimulated h202 release to 223.3 +_ 10.7 nmol. the release decreased to 191.9 _+ 2.4 nmol when an murine anti-tnf-alpha antibody was added. vitality of cultured cells was > 96 % in all experiments. the results show that lps has an direct effect on p2 cells cultured on fibronectin. we conclude that the observed additional stimulatory effects of mcm seems to depend on tnf-alpha. the induction of h202 release of p2 cells could be important for generating internal oxidative stress in p2 cells before external oxygen radicals exceed. the produced h202 did not necessarily damage p2 ceils, but it can effect surfactant metabolism, especially when extracellular h202 release of alveolar macrophages following an immune response is increasing. introduction: primary stabilization of femoral shaft fractures in patients with multiple trauma is beneficial. however, in patients with associated lung contusion we have found an increased incidence of ards, apparently associated with primary reamed femnral nailing (rfn). previous animal studies revealed, that perioperative disturbances of lung ftmetion appear to be related to the reaming procedure, ix~ssibly due to pulmonary embolizafion of bone marrow fat. in a prospective clinical analysis we compared effects of intrameduuary nailing with and withont reaming on parameters known to be related to ards-pathoganesis. in order to gain further insight into the role of endotoxin and cytokines in the pathogenesis of the adult respiratory distress syndrome (ards), we enrolled 23 patients with severe lung injury after sepsis (17) or polytrauma (6) and obtained multiple blood samples (14 days) for endotoxin, tumor necrosis factor e (tnfa), interleukin 18 (il-18) and interleukin 6 (il-6) determination. to evaluate the cytokine releasing capacity of the blood, plasma concentrations of tnfe, il-l8 and il-6 were also determined after the "in vitro" stimulation of the whole blood samples with lipopolysaccharide (lps, 0.1 ng/ml) for 2 hours at 370 c (stimulated values). the difference among stimulated cytokines levels and the basal plasma concentrations were defined as "delta values", an expression of the cytokine releasing capacity of the blood. the pao2/fiao2 quotient was used as an index of the severity of lung injury (sli). the endotoxin plasma level was significantly higher in patients with sli < 200 (0.288 ± 0.038 eu/ml, mean values ± sem) versus the patients with a sli > 200 (0.175 ± 0.023 eu/ml, p<o.01). the basal plasma concentration of ll-6 also correlates with the sli index (p<o.o01). the delta tnfe and delta il-6 values were s~gnificantly decreased in patients with a severe lung injury. compared with the survivors (n:11), the deceased patients (n=12) showed higher endotoxin level, a reduced tnfa and il-6 releasing capacity of the blood and higher basal il-6 levels. we can conclude that endotoxemia, high il-6 plasma level and a decreased capacity of the blood to release tnfa an il-6 under endotoxin stimulation associates with the severity of lung injury. [objectives] experimental and clinical findings implicate the involvement of inflammatory cytokines in the pathogenesis of the decreased oxygenation in the lung after major surgery, though the mechanism remains unclear. in the present study, we elucidated the involvement of il-8 in the pathogenesis of lung injury defined by deterioration of pulmonary oxygenation after esophagectomy. [materials and methods] seventeen patients underwent subtotal esophagectomy through a right thoracotomy. in all patients, tbe alveolar-arterial oxygen tension difference (aado2) was measured everyday and bronchoalveolar lavage fluid (balf) samples were obtained from a single segment ($4 or $5) of the right and left lung through a bronchoscope on days l, 3, and 5 days after surgery. they were then examined for cell analysis, measurement of cytokines by elisa, and measurement of neutrnphil elastase (ne) by elisa. [results] aado2 unexceptionally deteriorated postoperatively and the mean aado2 in 17 patients reached the maximum (mean ± sem; 372 _+ 23 mmhg) on the 3rd postoperative day. the mean concentrations of il-8, ne and neutrophil count in balf also increased postoperatively and reached their maximum level (2137 + 618 pg/ml, 1603 -+ 493 gg/l, and 1561 ± 477 x 103 cells/ml, respectively) on the 3rd postoperative day. these increases in balf were recognized in both the right and left lung. a highly significant correlation was observed between il-8 and ne levels in balf (r=0.82, p=0.0001). also, significant correlations between aado2 and the neutropbil count, and the concentration of ne or 1l-8 were observed. in contrast to il-8, neither il-6, il-113 nor tnfa was detected in balf. [conclusion] major surgical trauma such as esophagectomy may induce recruitment of neutrophils to the lung and cause lung injury. il-8 increased in the lung is an important mediator of neutrophil recruitment and activation in the lung. phorbol myristate acetate (pma) is commonly used to cause edema and other tissue damages in the lung. lung injuries induced by the administration of pma has been shown to be mediated mainly by neutrophils. neutrophils recruited to the lower respiratory tract may damage lung tissues by releasing reactive oxygen species, neutral proteases and lysosomal enzymes. the present study was conducted to investigate whether c~tocopherol, entrapped in dipalmitoylphosphatidylcholine liposomes and delivered directly to the lung, could counteract some of the pma-induced lung injuries. plain liposomes or c~tocopherol-containing liposomes (8 mg c~-tocopherol/kgbody wt.) were instilled into the lungs of rats 24 h prior to pma exposure (25 pg/kg, intratracheally) and treated rats were killed 3 h later. lungs of control animals exposed to pma developed increases in lung weight and lipid peroxidation as well as decreases in lung angiotensin converting enzyme (ace) and alkaline phosphatase (akp) activities. pma treatment also caused an increase in myeloperoxidase (mpo) activity in the lung, suggestive of neutrophi] infiltration. pretreatment of pma-treated rats with plain liposomes had no effect on pma-induced injuries. in contrast, pretreatment of rats with liposomal c~-tocopherol, 24 h prior to pma administration, resulted in a significant elevation of pulmonary a-tocopherol concentration, accompanied by a concomitant reduction in mpo activity and reversal of pmainduced changes in lung edema, lipid peroxidation, ace and akp activities. these results appear to demonstrate that the intratracheal administration of a liposome-associated lipophilic antioxidant, such as a-tocopherol, can significantly ameliorate the toxic effects of reactive oxygen species, released from pmastimulated pulmonary target cells and infiltrating neutrophils. jk wojcik, g palasiewicz, w roszkowski immunology department,institute of tuberculosis and lung diseases~ warszawa, poland, m~larhospital, eskilstuna, sweden. introduction:the critical point in neutrophil migration to the alveolar space in the course of ards is the attachment between giyeoproteins of neotrophil membranes(sialyl lewis x carbohydrate epitope containing u-l-fucose) and lectin domains of endothelial p and e selectins (gmp-140,elam-i). a potential beneficial therapeutic strategy may be designep to suppress neutrophil recruitment to the lungs by preventing their rolling and attachment to the pulmonary endothelial cells. objeclive:to investigate if tz-l-fueose prevent experiments pulmonary hypertension in ards as effective as metylpredniselone. methods:only healthy rabbits (n-24) weighing 5.d-5.5 kg oaselinepao>11 kpa and mean pulmonary arterial pressure (mpap)<i.5 kpa were included in this study. anaesthesia was induced with thiopental 20-25 mg/kg bw and "blind" jntubation was performed. a 5f swan ganz catheter was introduced via the left jugular vein into the pulmonary artery. pma, as was used in our experiments activates neutrophils and produces acute respiratory failure with pulmonary hypertension and pulmonary edema. eight rabbits serving as control animals received pma 25 ug/kg bw iv only. sixteen other animals were divided into two groups receiving either ~-l-fucose 100 mg iv or metylprednisolone 150 mg iv 15 min before pma admininstration. results:after pma administration notable physiological changes including marked increase in mpap(2.0-+o.~6 kpe3 ..lere found. the increase of npap was observed to be completely blocked in both groups of animals which received ~-l-fucose or metylprednisolone. conclusion:in aur rabbit model ards cz-l-fucose pretreatment prevents pulmonary hypertension after pma administration. the effect is comparable with high dose metylprednisolone. the possible explanation is that g-l-fueose molecules block iectin domains of gnp-140 or elam-i preventing the adhesion of the neutrophils to the pulmonary endotheliom. bartens c, elmadfa i, steltzer h, fischer m, druml w introduction: various micronutrients and vitamins are particulary effective in modulating immune functions and host defense. the nutritive ant±oxidants vitamin c, e and b-carotene, as well as selenium lower the burden of reactive free radicals and protect the structural integrity of cells and tissues of the immune system, as well as other systems in the body. certain cells of the immune system like polymorphonuclear leucocytes (pmn's) use free radicals as a weapon to destroy invading pathogens. these reactive molecules, when released into the surrounding area, mediate lipid peroxidation and tissue injury. there is growing evidence that pmn's are activated in vivo by complement or immune complexes in disorders such as ards. the respiratory host defense mechanisms of patients with adult respiratory distress syndrome (ards) may be defective. the aim of this study was ta evaluate the status of the free radical scavengers and their activity in ards and to investigate the respiratory burst of ards patients. methods: seven ards patients (3 sepsis, 4 trauma) with an fie 2 of 0.59+0.(36, a peep of 8.50±0.99, and a murray score of 2.83 ±0.18 were included in this study. 15 healthy adults served as controls. superoxide anion production in granulocytes was measured photometrically, and the hydrogen peroxides by fluorimetric assay. vitamin a, e, and g-carotene concentrations were assessed by hplc, and plasma vitamin c photometrically. plasma selenium was determined by dectrothermal aas. plasma lipid peroxidation pruducts, expressed as malondialdehyde (mda), were determined by thiobarbituric acid assay and hplc. results: mda-plasma (/xmol/l) 0.74~: 0.46* 0.37 ± 0.16 *= <0.01, **=p<0.001 condusion: ards patients showed significantly decreased plasma concentrations of vitamin c, e and g-carotene, as well as selenium. these nutritive ant±oxidants are consumed during increased oxidative stress. mda, a final product of lipid peroxidation, is increased. however, a difference in rates of release of hydrogen peroxides and superoxide-anion of pmn could not be detected. therefore, oxygen radical accumulation is more likely a result of excessive inspiratory oxygen concentrations (f.io2), pro-oxidant drugs, and a high settlement of pmn in the lungs, and not an increased release of free radicals of the single pmn. kd glycoprotein secreted by the alveolar type ii cells. recent study showed that sp-a exists in the sera from normal healthy adults and that the significantly elevated serum sp-a levels were observed in the patients with interstitial pneumonia and pulmonary alveolar proteinosis. these findings means there may be a mechanism that the sp-a produced in the alveoli escapes into the bloodstream. here we examined the serum sp-a levels in the patients with critical illness including sepsis and ards. ]clinical subjects & methods] a total of 99 serum samples were collected from 39 patients hospitalized in the division of critical care medicine(icu), sappom medical college hospital. serum sp-a levels were measured by an enzyme-linked immunosorbent assay using monoclonal antibodies to human sp-a. ]results] there was no significant difference between the septic (n=12) and non-septic (n=27) patients. patients with ards (n=10, 52.0+4.85ng/ml) and with respiratory disease other than ards (n=12, 49.6+-5.35ng/ml) showed significantly higher levels of serum sp-a than those with non-respiratory disease (n=17, 27.4_+4.86ng/ml). as to the ards patients, it was likely that the serum sp-a levels were getting higher in their clinical courses. however, there was no significant correlation between the serum sp-a levels and the pa%/fio 2 ratio. ]discussion] at the present time, the exact mechanism of the escape of sp-a into the bloodstream remain to be unclear. our results may suggested that there was some relation between this leakage of sp-a and the alveolar-capillary permeability because of the higher sp-a levels observed in the ards patients. however it is also likely that higher serum sp-a level represents the proliferation of alveolar type ii cells as the regeneration of damaged lung. further studies are now being done. neutrophils contain a number of enzymes within intracellular granules,potentially damagingto lung tissue.release of these enzymes into the lung tissue from the sequestred activated neutrophils is felt to play significant role in lung injury in the course of aros. using 6-hours acute animal model of aros the activity of cathepsins,beta-glucuronidase and acid phosphatase was determined in the lung tissue. results of this research were compared with neutrophil proteases activity in serum and broncho-alveolar lavage.also hemodynamic,respiratory and biochemical investigations were performed before beginning of experiment,and in a two-hours time intervals of its duration.after 6 hours was determined total and free activity of cathepsins,beta-glucuronidase and acid phosphatase in full homogenate of the lung tissue,mitochondriallysosomal fraction and the final supernatant. in the course of our experiment we observed an increase (30.i-49.6%) both total and free activity of neutrophil proteases in all fractions of the lung tissue.generally accepted current pharmacological treatment of aros only attenuated this increase but never caused reversion to the level before beginning of our investigations. the presence of aros was histologically proved.an activity of acid phosphatase in serum and the lung tissue fractions was compared with histochemical pictures of the lung. results of our research indicate that neutrophil proteases are a very important mediators in aros and they are a cause of the lung injury. in addition neutrophil-derived proteases can amplify the inflamatory process by enzymatitally activating of many other mediators and they also may to increase an oxidant induced injury by imparing of antioxidant defenses. oepartment of general surgery, sk~odowskiej 24a 15-276 ~ia~ystok, poland, tel/fax +48 85 619742. in vitro studies demonstrated that paf activates polymorplionuclear leukocyte (pmn) 5-1ipoxygenase, but the generation of leukotrienes (lts) is critically dependent on exogenous arachidunic acid (aa) disposal (f. grimminger et at., mol. pharmacol. 41:757, 1992) . we investigated the features of paf-evoked lt synthesis in a model of pulmonary microvascular leukostasis, in the presence and absence of intravascular n-3-and n-6 -prescursor fatty acid supply. human neutrophils were infused into isolated, ventilated and perfused rabbit lungs to achieve microvascular sequestration of ligand-responsive leukecytes. leukotrienes (lt) and hydroxyeicosatetra(penta)enoic acids (het(p)e) were quantified by itplc techniques. intravascular application of paf (5 um) or arachidonic acid (aa;ioum) provoked the generation of limited quantities of 4-series lts and 5-hete (total sum of 5-1ipoxygenade products rd. 7 and rd. 27 pmol/ml.; both in pmn-preloaded and non-preloaded lungs). combined administration of pat r and aa caused amplification of 5-1ipoxygenase product formation with predominance of cysteinyl-lt synthesis both in lungs without (total sum rd. 67 pmol/ml) and, much more strikingly, with pro-application of neutrophils (total sum rd. 308 pmofml). eicosapentaeooic acid (10 um) elicited exclusive generation of 5-series lts and 5-hepe (total sum rd. 82 pmol/mi). dual stimulation with paf and epa provoked amplification of epa-derived 5-1ipoxygenase product formation, again with predominance of cysteinyl-lts, in lungs without (total sum rd. 224 pmul/ml) and in particular in those with preceding microvascular pmn entrapment (total sum rd. 545 pmol/ml). we conclude that the paf-evoked 5-1ipoxygenase product formation in the neutrophil-harbouring lung capillary bed is critically dependent on intravascular precursor fatty acid supply, with epa representing the preferred substrate as compared to aa. pmn-related transcellular eicosanoid synthesis is suggested to underly the predominant generation of cysteinyl-lts. these findings may be relevant for lipid mediator profiles provoked by infusion of n-3-versus n-6enriched lipid emulsions in diseases with pmn-related inflammators events. adult respiratory distress syndrome (ards) involves damage to the alveolar epithelium and microvascular endothelium. products released from neutrophils (pmn) are thought to be among the chief causes of this damage, while the role of mononuclear cells (mnc) is uncertain. we studied patients at risk of or with acute ards. we measured the concentration of myeloperoxidase (mpo) and elastase (el) within circulating pmn as an index of pmn activation, elastin degradation products (edp) in the plasma as an index of tissue damage, as well as the cytotoxici~y of pmn and mnc to endothelial cells (ec) in vitro. cells and plasma were prepared from venous blood of healthy controls; or systemic arterial blood of patients on ventilators in intensive care but not at risk of ards; at risk of ards because of sepsis; at risk because of multiple transfusion/major trauma; or with acute ards. lung injury, multi-organ failure and apache h scores were recorded. to measure cytotoxicity, human umbilical vein ec were labelled with cr-51, incubated with either pmn or mnc for 18 hours, and the supernatant counted. the adherence of the pmn and the mnc to the ec was also measured. standard assays were used to measure mpo and el in the pmn, mad edp in the plasma. the mnc cytotoxicity did not differ between the groups. the pmn cytotoxicity was higher in the transfusion/trauma patients compared to each other group, which did not differ from each other. there was no correlation between cytotoxicity and adherence for any group. the mpo and the el concentrations were significantly decreased in the pmn from each of the patient groups, including the patient controls, compared to healthy controls. the edp were only increased in patients at risk of or with ards. thus pmn degrannlation occurred in patients not at risk of ards, but tissue damage as measured by increased edp only occurred in patients with or at risk of ards, suggesting that factors additional to pmn activation and degranulation are required for the progression to ards. there was no correlation between the parameters measured and the indices of disease activity, or outcome. these measures of pmn and mnc activation and function are not of value as prognostic indicators in patients with or at risk of ards. the liver is made up of several different cell types which subserve distinct functions in vivo. in addition to the different functions of the distinct cell types, it is now evident that even within the population of hepatocytes substantial functional heterogeneity exists. this heterogeneity occurs in a very organized fashion in the normal liver based upon the position of the hepatocyte in the acinus. although it has been proposed that the acinar heterogeneity of hepatocytes arises from migration of immature hepatocytes from the periportal region to the perivenous region where they become senescent and die, many of the heterogeneous responses can be accutely reversed by reversal of the oxygen gradient via retrograde perfusion. under normal conditions in which the acinus is exposed to unidirectional flow, the periportal hepatocytes are exposed to relatively high levels of oxygen, substrates and hormones. as these substances are extracted along the aeinus their concentration decreases leaving relatively low concentrations for the perivenous hepatocytes. in the normal liver, the heterogeneity of response is attenuated by extensive communication via gap junctions. the major liver gap junctions are made up of hexamers of connexin 32 (cx32) which forms channels between cells capable of pas.~ing any molecule smaller than 1000 d. coupling is sufficient to allow diffusion of molecules such as atp or camp from one hepatocyte into > 20 adjacent hepatocytes within 60 seconds. during stress conditions such as endotoxemia or zymozan inflammation, expression of cx32 is markedly decreased while the secondary gap junction protein cx26 is either unchanged or even increased. while cx 26 readily effects electrical coupling, molecules > 350 d pass only very slowly. this would result in restriciton of transmission of moecules the size of atp or camp. since inhibition of gap junctions also attentuates metabolic response to hormone or nerve stimulation, it is evident that modulation of hepatocyte hetereogeneity by gap junctions must be considered in determining the mechanisms of metabolic alterations during stress. already minor haemorrhage decreases portal venous blood supply to the fiver and the reduction in portal blood flow becomes more pronounced with more profound btood loss. severe hacmorrhagic hypovolemia also reduces hepatic arterial blood supply which, however, is maintained over a vide range of haemorthage. the net effect of blood loss is a reduction in liver oxygee supply and this reduction is in proportion to the vulume iossed. however, oxygen supply to the liver exceeds the demands of the normal liver and this is the ca~ stilt following reduction of 30 % of blood volume. the situation in sepsis is more complicated. po~l venous supply to the liver is redur.~i fairly early following normovolemic sepsis while hepatic arterial blood supply is maintained at le,~t initialiy, oxygen saturation might be maintained in arterial blood but may also be slightly reduced during sepsis, oxygen saturation of portal venous blood is significantly reduced during sepsis due to increased extraction of the intestines. therefore oxygea delivery to the liver during sepsis becomes sigalfkzntly reduced. at the s,~ne time and for mai.v.ly unknown reasons the need for oxygen becomes significantly increased in the ~-~ptic liver. as a consequence liver oxygen consumption becomes flow dependent and the liver is likely to suffer from ischemia during septic conditions. $94 although liver failure is well recognized in sepsis, it is generally thought to be a late complication following pulmonary and renal failure. jaundice, hypoglycemia, encephalopathy and bleeding secondary to low levels of liver-synthesizing clotting factors are, however, signs of rather severe end-stage hepatic failure. furthermore, elevated liver enzymes (sgot and sgpt) represent hepatucyte damage and not hepatocellular dysfunction. in view of this, a more sensitive indicator of hepatic function is desirable in order to detect early hepatic abnormality. in this respect, indocyanine green (icg) is a tricarbocyanine dye that possesses several properties which makes it particularly valuable inthe assessment ofhepatic function. this dye is bound m albumin and is cleared exclusively by the liver through an energydependent membrane transport process and is nontoxic at lower doses. we propose that maximal velocity (vm~,) of icg clearance is a valuable measure of active hepatocellular function, since the total concentration of functioning receptors is directly proportional to vm~. we have utilized a fiber optic catheter and an in vivo hemoreflectometar to continuously measure the administered icg in vivo and consequently determine its clearance without the need of blood sampling. using this technique, we have found that in the early stages of sepsis (i.e., 2 and 5 h following cecal ligation and puncture), the vm~ and kinetic constant (k=) of icg clearance was significantly depressed. it should be noted that at this stage of sepsis, there was no elevation in serum enzyme levels. furthermore, hepatic blood flow and cardiac output increased at the above mentioned time points. thus, the extremely early depression in active hepatocellular function in sepsis, despite the increased hepatic blood flow and cardiac output, may form the basis for cellular dysfunctions leading to multiple organ failure during sepsis. additional studies indicated that following hemorrhage, active hepatocellular function was markedly depressed. this returned to prehemorrhage levels after ringers lactate resuscitation, however, this function was not maintained and decreased significantly after fluid resuscitation. nevertheless, the depressed active hepatocelinlar function following hemorrhage was markedly improved by post-treatment of animals with either atp-mgci2, peutoxifylline or diltiazem. thus, the use of icg clearance provides an early sensitive indicator of hepatic abnormality during sepsis and following hemorrhage and this method should be used, not only experimentally, but also in the clinical arena for the early detection of hepatocellular abnormality. although multiple organ dysfunction syndrome (mods) remains a major cause of mortality and morbidity in intensive care units, very little is known about the mechanisms that precipitate its development. since an episode of inadequate tissue oxygenation is considered to be the trigger for mods, we have proposed that a primary localized injury such as ischemia/reperfusion may be sufficient to cause a change of gene expression of remote and apparently unaffected organs. such modulation of remote organ gene expression may decrease the organ's tolerance to a subsequent stress contributing to the development of mofs. to test this hypothesis, rats were subjected to hepatic regional ischemia by clamping the blood flow (hepatic artery and portal venous inflow) of the left and median liver lobes. intestinal congestion was prevented by allowing flow through the smaller right and caudate lobes. after 60 minutes of ischemia, the clamp was removed and the blood flow restored. the animals were allowed to recover for 1, 4 and 24 hours. kidneys were removed, total rna was isolated and poly(a) ÷ selected by affinity chromatography on oligo(dt) columns. message was in vitro translated using rabbit reticulocyte iysates in the presence of radioactive amino acids. the gene products (radiolabeled polypeptides) were fractionated by two dimensional gel electrophoresis, and visualized by fluorography. analyses of the two dimensional fluorograms indicate that there is a dramatic change in the electrophoretic pattern of in vitro translated products in samples corresponding to kidneys obtained after 60 minutes of hepatic ischemia and 24 hours of reperfusion with respect to kidney samples obtained after sham operation or from control rats. the latter were not subjected to any surgical manipulation. these studies suggest that the gene expression of the kidneys is specifically modified after a remote organ injury (hepatic ischemia/reperfusion). we speculate that this change of gene expression in kidneys after an indirect injury may be part of the early events leading to the development of mods. a priming event, e.g. local ischemia, in combination with a second insult, e.g. sepsis, may amplify a host's response and lead to multiple organ failure. to better understand the mechanisms involved in the pathophysiology, male fischer rats were subjected to 20 min of hepatic ischemia followed by reperfusion (rp) and injection of 0.5 mg/kg salmonella enteritidis endotoxin (et) at 30 min of rp. et injection potentiated the postischemic liver injury as indicated by histopathology and an increase of plasma alt activities from 870+122 u/l (i/rp only) to 3900+470 u/l at 4h rp. inhibition of kupffer cells (kc) with gadolinium chloride (7 mg/kg) attenuated liver injury in this model by 70%, however, monoclonal antibodies (cl26, wt3) directed against adhesion molecules (132 integrins, cd18) on neutrophils had no effect on the injury despite the substantial accumulation of neutrophils in the liver at that time (467+52 pmns/50 hpf; baseline: 13+3). isolation of kc and neutrophils from the postischemic liver indicated a 10-fold increase of the spontaneous superoxide formation only in the kc fractions [3.85+0.68 nmol o2-/h/10%elts (kc2); 6.82_+0.92 (kca) ] at 4h rp compared to control cells. in addition, stimulation with phorbol ester or opsonized zymosan revealed a substantial priming of kc for reactive oxygen formation. in contrast to the short-term experiments (4h), the antibody wt3 (4 mg/kg) attenuated liver injury by 90% at 24 h of rp and improved survival. conclusion: liver injury during the early rp phase is mediated mainly by kc generating excessive amounts of reactive oxygen while neutrophils are primarily responsible for organ damage during the later rp period. (es-06091 and gm-42957) tumor necrosis factors (tnf) are cytokines which are cytotoxic towards some tumors in vivo and certain tumor lines in vitro. moreover, these polypeptides are powerful immunomodulators and have been found to be distal mediators in several models of septic shock and septic organ failure. one of the best-characterized experimental systems is the hepatitis caused by lps or tnf in galactosamine (galn)-sensitized mice. here we describe a cell culture system, in which the direct toxicity of tnf towards mouse hepatocytes was examined. the toxicity of tnf, as determined by ldh-release or formazan-formation, was dose-and time-dependent. the threshold of toxicity was 10 ng/ml, which corresponds to serum concentrations found in mice after lpsinjection. toxicity was only observed in hepatocytes sensitized with transcriptional inhibiters such as galn, actinomycin d (actd) or cxamanitin. sensitization was neither observed with different translational inhibitors nor with various other metabolic inlaibitors or toxins. inhibitors of protein synthesis or protein processing such as cycloheximide, puromycin, tunicamycin and ricin protected actdsensitized hepatocytes from tnf-induced cytotoxicity. tnf induced apoptotic changes and dna-fragmentation in sensitized hepatocytes which is in line with the above findings that cell death is dependent on protein synthesis. thus tnf may be a trigger of programmed cell death during inflammatory organ damage. with the purpose of studying the role of complement activation in tissue injury after ischaemia and reperfusion we blocked the complement cascade in a model of rat liver isehaemia and reperfusion, either by administration of soluble human complement receptor type 1 (scri), 25 mg/kg iv after vascular occlusion (n=8) or by depleting the complement system using cobra venom factor (cvf), 0.5 mg im, 24 and 6 hours before ischaemia (n=10). non-ischaemic rats (n=8) and ischaemic non-treated rats (n=15) were used as controls. the experimental procedure consists of the temporary interruption of arterial and portal blood flow to the left lateral and medial lobes of the liver during 45 minutes, followed by reperfusion, recording the liver blood flow and haemoglobin saturation with a laser doppler flowmeter and photometer during one hour after declamping; alt levels were assayed and immunoperoxidase stainings for c3 and c9 were performed. there were statistically significant differences between the experimental ~roups and the untreated ischaemic control group in terms of post-isehaemic blood flow (p<0.001) and haemoglobin saturation (p<0.001). c3 and c9 were present in the endothelium of the ischaemic control group. no deposits of c3 or c9 were found in the cvf group. few c3 and no c9 were found in scri treated rats. these results show that the effect of reperfusion injury in the rat liver is ameliorated either by depleting complement with cvf or by regulating complement activation with scri. hepatic dysfunction, a major cause of mortality following hemorrhagic shock, has not yet been well characterized. the present study was designed to assess the effects of liver blood flow and cytokine levels on hepatic function following resuscitation from severe hemorrhagic shock in normal and cin-hotic rats. methods: aftor pentobarbltal anesthesia, 23 control and 38 cirrhotic sprague-dawley rats were subjected to severe hemorrhage to reduce their systolic blood pressure to 50 + 5 mm hg. this level of hypotension was maintained until the skeletal muscle transmembrane potential (era) depolarized by 25%.; the animals were then resuscitated with ringer's lactate solution in three times the volume of the shed blood. serial blood samples for tumor necrosis factor (tnf) determination (a modified flow-cytomeuic wehi cell bioassay) were obtained at baseline, during hemorrhage and following resuscitation. liver blood flow measurements by low dose galactose clearance (glc) and functional bepatocyte mass (fhm; defared as galactose elimination capacity [gec] from the zero order portion of the plasma disappearance curve following an intravenous galactose bolus [500 mg/kg], divided by liver weight) were measured before shock and after resuscitation. results: higher survival rates (p < .05) were observed in control as compared with cirrhotic rats. shock produced a significant reduction in gec (to < 0.01); fhm (19 < .01); and liver blood flow (p < 0.01) in normal and cirrhotic rats. decreases in gec and fi-im were greater (p < 0.05) in cirrhotic rots. tnf levels were higher (p < 0.05) in cirrhotic rats at baseline and during induction of shock. pre gap junctions provide pathways for metabolic signals between cells. in the liver, the majority of gap junctions are composed of connexin 32 (cx32) polypeptide subunits, and are regulated by gluconeogenic hormones. since sepsis and other inflammatory states alter hepatic glucoregulatory control, we have evaluated the contribution of gap junctional conductance to the metabolic dysregulation in the liver. an acute inflammation was induced in rats by injection with e. coli endotoxin (lps lmg/kg). northern blot/hybridization analysis of total rna isolated from livers after endotoxin injection show a decrease in the steady state transcript levels of cx32 to 18% of sham controls. immunostaining of liver sections using anti-cx32 revealed punctate fluorescent staining on the plasma membrane at regions of call-cell contact in saline injected animals, whereas, staining was only observed in cytoplasmic vesicles 6 hrs after animals were treated with lps, suggesting the internalization of cx32 without replacement on the cell surface. the staining was quantitated and expressed as % of pixels above threshold. at 6hr post injection 3.6% ofpixels exceeded threshold, compared to 16.2% in sham controls. functional gap junctional communication was assessed by dye coupling using lucifer yellow in an isolated perfused liver under intravital fluorescence microscopy. dye diffusion was markedly decreased 6hr after endotoxin injection. this suggests that decreased metabolic coupling after lps injection results from decreased gap junction abundance. the present data suggest that metabolic dysregulation during sepsis may arise in part from changes in intercellular communication caused by a decrease in gap junctional expression and communication. given the marked metabolic heterogeneity of hepatocytes with respect to acinar location, metabolic signaling via gap junctions most likely serves to moderate this heterogeneity, contributing to a coordinated metabolic response. altered cellular ca 2÷ regulation might be a critical step in organ dysfunction during sepsis and ischemia/reperfusion events. the aim of the present study was to evaluate hepato-ceuular ca 2÷ regulation in isehemiah'eperfusion after hemorrhage and to assess effectiveness of tnfc~-monoclonal antibody (tnfo~-moab). methods. male sprague-dawley rats (250g, n>_6/group; pentobarbital 50 mg/kg) with hemorrhage for 60 rain at 40 mm hg. reperfusion by ringer's lactate (2 x maximal bleed out/60 min) and 60% of citrated shed blood. tnfcz-moab (tn3, ceutech, 20 mg/kg in 0.9% nac1) infused during flrst 5 min of reperfusion. at baseline, end of ischemia and 60 min of reperfusion, hepatecyte isolation by liver collagenase perfusion. " 45 hepatocyte incubation (30 mg w.w./ml) with caci 2 (0.05 2+ 2+ 2+ mbq/ml) for 60 rain (ca influx [slope, 1/mini; ca uptake [nmol ca /mg protein]) w/ and w/o epinephrine (epi, 100 nm). hepatecyte resuspension in radioisotope-free medium and farther incubation (exchangeable ca 2+ (ca2+ex) [nmol ca2+/mg protein]; ca 2+ membrane flux [nmol ca2+/mg protein'min]). during incubation, aliquots (100 ~tl) were centrifuged through oil/lanthanum gradient and acivity measured by scintillation counting. statistics: anova. mean + sem. results. hepatocyte ca2+ex and membrane ca 2+ flux were significantly increased at both, the end of ischemia (14.3+.9; 0.41+.05) and reperfusion (16.4+.9; 0.50+.2), as compared to sham-operated animals (7.6+_.5; 0.09+.02)(19<.05 ). tnfc~-moab treatment significantly prevented reperfusion-induced increase of ca2+ex (10+.8) and membrane ca 2+ flux (0.17+.03)(p<.01). fast ca 2+ influx was significantly increased by epinephrine in hepatecytes from sham-operated rats (0.23+.03 vs. epi: 0.52+.1, p< .05). this hormone effect was not observed in isehemia (0.47+.05, epi: 0.6!-_.2) or reperfusion (untreated: 0.13+.06, epi: 0.07+.01; tnft~-moab: 0.26_+.04, epi: 0.33+.07). conclusion. the present study clearly demonstrated hepato-cellular ca 2+ overload in ischemia and reperfusion as a result of hemorrhagic shock. analysis of membrane ca 2+ fluxes and hormone ca 2+ mobilization suggests disturbances of membrane ca 2+ transport mechanisms, e.g. through ca2+-atpases. reperfusion-induced oxygen free radical generation which affect exchange kinetics of cellular ca 2+ buffering compartments might also be operative. prevention by tnfct-moab indicates the pivotal role of tnf as an early inflammatory mediator of hepatocellular alterations in signal transduetion mechanisms and cellular homeostasis. although the precise mechanism has not yet been elucidated, bacterial translocation and endotoxin absorption have been frequently shown after burn, and have been postulated to be one of the underlying processes of sepsis. the purpose of the current study is to define the hemodynamic response of the liver to endotoxin release in burns, in correlation to bacterial translocation. twelve female minipigs, weighing 20-28 kg, underwent a laparotomy & transition time ultrasonic flow probes were positioned on the portal vein, the common hepatic artery, and the superior mesenteric artery. 6.5 fr catheters were inserted in the superior mesenteric vein and the left hepatic vein. a jejunostomy was also performed. after five days all animals were anaesthetized and randomized to receive 40% of tbs a third degree burn. eighteen hours after burn. 100 gg/kg e. coli lps was intravenously administered over 30 rain. ali animals were studied for additional 24 hours and then sacrificed. several recent data suggest that in severe injuries, such as shock state, the gradual activation of kupffer cells and the excessive release of destructive and immunosuppresive products from macrophages may contribute to the development of "multiple organ failure". in in vivo experiments in mice, the effect of kupffer cell phagocytosis blockade on the correlation between the tissue distribution of lps, endotoxin sensitivity and lps-induced tnf production was investigated. to depress the activity of the kupffer cells, gadolinium chloride (gdc13) or carrageenan was used. th~e studies indicate the dissociation of tissue localisation of cr jllabelled endotoxin and endotoxin lethalithy. both gdc13 and carrageenan depressed kupffer cell activity, but endotoxin sensitivity was enhanced only by carragenan treatment. however, there was a close correlation between the sensitivity to lps and lps-induced tnf production as measured in the serum, since lpsinduced tnf production was enhanced only by carrageenan treatment. on the other hand, gdc13 pretreatment significantly increased tnf production in the spleen. these results support our earlier findings that gdc13-indueed kupffer cell phagocytosis blockade leads to activation of the spleen, and may explain some of the immunological effects of gdc13. inositol(l, 4, 5) triphosphate (ip3) has been proposed as a second messenger for calcium mobilization. the addition of ip3 at low concentration has been shown to cause calcium release from intracellular microsomal store in rat hepatocytes. the effects of sepsis on the ip3 binding from microsomal fraction of rat hepatocytes during sepsis were investigated. sepsis was induced by cecal ligation & puncture (clp). control rats were sham-operated. three microsomal fractions (rough, intermediate and smooth) were isolated from rat liver. study of ip3 receptor binding was performed with tridium label ip3. the results shewed that the ip3 binding was significantly depressed by 40-47% (p<0.05) during late sepsis (18 hrs after clp), but not in early sepsis (9 hrs after clp). the ip3 binding depression during late sepsis was most significant on rough and intermediate endoplasmic reticulum (p<0.05), but not on smooth subfraction. since ip3 binding plays an important role in the regulation of intracellular calcium homeostasis in hepatocytes, an impairment in the calcium release due to depressed ip3 binding on smooth and intermediate endoplasmic reticulum during late sepsis may have a pathophysiological significance in contributing to the development of altered hepatic metabolism during septic shock. septic organ failure is currently recognized as an overactivation of the nonspecific immune system by bacterial stimuli giving rise to proinflammatory mediators. little is known about the mechanisms of the resulting cellular injury. here, a synergism is described between tnf as a major mediator of septic organ injury released by macrophages and hydrogen peroxide (h202) as a representative of reactive oxygen species as formed by e.g. neutrophils. rat hepatocytes are only slightly sensitive to either agent alone. when treated with a conbination of tnf and h#09 a stronq synergistic toxicity was found, especially w~e~ tnf-treatment preceeded challenge with h~o~. we have recently described a coculture model bfzrat liver macrophaqes and hepatocytes where lps induces hepatocyte cell death partially mediated by macrophage tnf release. when h202 was also employed in fhis more complex cellular system a similar synergism was found: the ecc~ of lps was 62 consecutive patients with liver cirrhosis admitted to the department of surgery over a 1 year period from january to december 1992 were studied for their complement profiles in relation to other parameters of liver function, the aim of the study was to determine if a direct correlation existed between low complement levels and end stage liver cirrhosis. cirrhotic patients were divided into child's a, b and c categories using child's classification. complement levels (c3, c4) were measured and functional assay for complement (ch50) were performed in each of these groupings in addition to normal blood donor controls. these results show that the qualitative c3, c4 and the functional chs0 complement assays have good predictive values in assessing deteriorating liver function• in particular, the functional assay for complement (ch50) showed marked impairment in child's c patients (p<0.000001) confirming the impaired immunological status of these patients. sera from this group of patients (child's c) were titrated with pig red blood cells (rbcs) in a haemolytic assay. the results showed that there were significantly less haemolysis of pig rbcs in these patients (p=0.0177) as compared to the controls. this findings strongly support an impaired immunological status in child's c liver cirrhosis and may explain the high incidence of sepsis as a terminal event in these patients. aim:kupffer cells(kc) have an importamt play to cause hepatocellular injury in sepsis, because these cells release many kinds of substances. we reported that oxygem radicals released by kcs stimulated by lipopolysaccharide (lps) caused hepatocellular injury. aim of this study is to investigate the relationship between imtracellmlar calcium(ca) concentration of cultured rat kcs stimulated by lps and release of oxygen radicals, and effect of prostaglandin e~ (pge~) on imtracellular ca concentration. production of acute phase proteins (c-reactive protein, crp, transferrin, tf) and £erritin (f) in rat hepatocytes (hps) and its dependence on extracellular matrix components were studied. hps isolated from the liver by collagenase perfusion were cultured at ~o5 per 0.5 ml medium fi2+dmem (1:1) with 10% fetal calf serum for 2 days on uncoated or type i collagen coated plastic surface or in the presence of dextrane sulphate in the medium. hps were stimulated by conditioned medium (gm) from i~ia-p or e. coli lps preineubated human blood mononuclear cells. production of crp, tf and f by hps was detected by elisa. it was found that both cms decreased tf synthesis in hps by 15-50% (p<o.os). the level of f synthesis didn't change or only slightly decreased. the addition of cms led to the enhancement of crp synthesis more than 2.5 times. the most reproducible results were obtained when plastics had been coated with collagen. dextran sulphate markedly increased crp synthesis. thus using this model of an acute phase reaction in vitro we found the enhancement of crp synthesis and the decrease of tf synthesis. our data correspond with nublished materials on the acute phase in vivo and point to relevance of the proposed model. metabolism of hepatocytes under traumatic illness has not been sufficiently investigated. therefore we have applied absorption and fluorescent cytophotometry techniques to study the content of rna, glycogen and its fractions in hepatocytes of patients with severe mechanical trauma, both with and without endointoxication at various stages. for these purposes slides were prepared from the repeated aspiration biopsy material according to special techniques (kudryavtseva et al., 1983) . slides were stained by gallocyanin-chromalum to measure rna or underwent fluorescent pas-reaction to measure glycogen and its fractions (kudryavtseva et al., 1970 (kudryavtseva et al., ,1974 the ability of 02 metabolites derived from the xanthine-xanthine oxidase system to inhibit mitochondrial function was examined using freshly isolated rat liver mitochondria. under uncoupled conditions, mitochondria exposed to free radicals exhibited a significant decrease in 02 consumption supported by nad+-iinked substrates, but showed almost no change in 02 consumption in the presence of succinate and ascorbate. the activity of electron transfer complex i (nadh oxidase and nadh-cytochrome c oxidoreductase) and the energy-dependent reduction of nad+ by succinate were unaltered by oxidative stress. exposure to free radicals also had an uncoupling effect at all three coupling sites. the degree of mitochondrial swelling was closely correlated with the inhibition of state 3 oxidation of site i substrates and with the increase in state 4 oxidation of succinate. the immunosuppressive agent cyclosporin a (cya) completely prevented the mitochondrial damage induced by oxygen free radicals (swelling, ca2+ release, sucrose trapping, uncoupling, and selective inhibition of the mitochondrial respiration of site i substrates). the same protective effect was found when ca2+ cycling was prevented, either by chelating ca 2+ with egta or by inhibiting ca2+ re-uptake with ruthenium red. these findings suggest that the deleterious effect of free radicals on mitochondria in the present experimental system was triggered by the cya-sensitive and ca2÷-dependent membrane transition, and not by direct impairment of the mitochondrial inner membrane enzymes. correspondence should be addressed to: naoshi takeyama the aim of this study was to clarify the critical role of reduced glutathione (gsh) on the progression of lipopolysaccharide (lps)induced liver damage of mouse. the conditions of gsh-depletion and -richness were produced by intraperitoneal administration of buthionine sulfoximine (bso), a specific inhibitor of gsh synthetase, and of gsh-monoethyl ester (gsh-me), respectively. gsh-me, which was esterified the caboxyl group of the glycine residue, is readily transported into cells and is hydrolyzed intracellularly; this leads to greatly increased the cytosolic and mitochondrial levels of gsh. bso and gsh-me were administered intraperitoneally 3 mmol/kg and 10 mmollkg, respectively, and lps (2 mg/kg) given 1 h later. hepatocytes were isolated by in situ perfusion of the liver with collagenase 6 h after lps injection. the degree of hydrogen peroxide (h202) synthesis and mitochondrial membrane potential were measured by flow cytometry using fluorescence probe dichrolofluorescein diacetate and tetrametyl rhodamine ethyl ester, respectively. the degree of mitochondria membrane permeability transition (mmp) was assessed by [014]sucrose entrapment. we found that lps treatment results in a decrease in hepatic gsh level and mitochondrial membrane potential, and an increase in h202 synthesis and mmp. lps administration to bsq-pretreated mouse worsened at[ these parameters. in contrast, gsh-me pretreatment ameliorates. all together, gsh may acts an important role in cellular defence against lps-mediated liver damage, and mmp may result in the decrease in mitochondrial membrane potential. it has been shown, up to now, that disturbances of enzymatic processes in the cell organella are the basic factor of the changes taking place during endotoxin shock it has been observed that lysosomes are biochemically changed as result of the effect of lipopotysacharides of gram negative endotoxic bacteria. the purpose of the experiment was: 1.evaluation nfthe course of the ees due to the biochemic changes 2.determination of the influence of the ees on the activity of lysosomal enzymes in liver cell 3.deterrmnatian of the influence of h and d on the activity of some lysosomal enzymes in liver cell during ees material and method: examinations ware carried out on 30 dogs. the shock was evoked by i.v. administration of endotoxin e.coli. the dogs were divided into 5 groups: icontrols, i[ -dogs with untreated shock, iii-dogs in shock treated with h, ivdogs in shock treated with d, v -dogs in shock treated with h and d. the following parameters were determined: 1.activity of acid phosphatase in the venous blood serum. 2-6.total and free activity of acid phosphatase and catepsins in the full liver cell homogenate, in mitochondrial-and-lysosomal fraction, and in the superuatunt (all after 4 hours of the experiment). conclusions 1. essential increase of activity of lysosomal enzymes was observed in the ees. 2. increased activity of lysosomal hydrolases in the liver cell homogenate corresponds, as a result of the endotoxin effect, with increased enzymatic activity in the peripherial blood. extensive investigations from our laboratory of the pathophysiology of hepatic no-flow ischemia (45 min) -reperfusion injury demonstrated substantial kupffer cell activation with reactive oxygen formation during the first few hours of reperfusion as indicated by increases in plasma glutathione disulfide (gssg) levels in vivo (am. j. physiol. 260: g355, 1991) and enhanced superoxide formation by kupffer cells (kc) isolated from the postischemic livers (free radic. res. commun. 15:277, 1991) . the early kc-induced injury initiated the later, primarily neutrophilmediated reperfusion injury (faseb j. 4: 3355, 1990 ). to test whether or not similar mechanisms are operative during hepatic ischemia induced by hemorrhagic shock, male fischer rats (230-250 g) were subjected to 60 rain of hemorrhage (mean arterial pressure 40 mm hg) followed by resuscitation (rs) (reinfusion of shed blood). hepatic atp levels declined from 2.3+0.2 txmol/g to 0.24_+0.08 (60 min shock) indicating severe ischemia, and recovered to 1.04--0.2 at 90 min rs. to test for potential oxidant stress during rs, plasma gssg conc. were measured. although gssg levels increased by 120% compared to baseline levels (0.69!-_0.31 i.tm), there was no significant difference to shamoperated controls. spontaneous superoxide formation of isolated kc was 0.32+0.05 nmol o2-/min/10ecells (kc2) and 0.51+0.11 (kc3) in controls and did not change significantly after shock and 90 rain rs. addition of phorbol ester or opsonized zymosan to isolated kc did not indicate a priming of these cells. conclusion: in striking contrast to our previous findings with hepatic no-flow ischemia, there is no evidence for a significant kc activation after 60 min of hemorrhagic shock and up to 90 rain of resuscitation. objectives-this study investigates morphometric changes in kc nltrastmcture which might account for this diminution in phagacytosis. materials and methods -three groups of wistar rats were studied: control, sham-operated [sham] and bile duct ligated [edl] . after 3 weeks animals were sacrificed and livers were "perfusion fixed" with 3% glntaraldehyde and processed for transmission electron microscopy and image analysis. results -in the the bdl group kupffer cell numbers were greatly increased. biliary ductular proliferation was evident and sinusoidal spaces were compromised. eleven nuclear and cytoplasmic parameters were measured and statistically significant results are summarised in the results: ascorbic acid levels were slightly elevated after the hs and returned to basal values after 120 rain. glutathione concentrations were increased significantly after the hs and the gsh levels kept rising continuously to as much as 4-fold of basal levels at the end of 120 min. mda showed no significant variation before and after the hemorrhagic shock. plasma concentrations of ratine, teucine, isoleucine, phenylalanine, proline, threonine and serine showed significant increase over basal levels during the whole ischemic period. glutamic acid was slightly elevated at the onset of hs and remained the same for the rest of ischemic period. hydroxyproline was significantly below the basal value at the mid-point of ischemic period. alanine, glycine, histidine and aspartate did not change significantly throughout experimental time course. conclusions: (1) oxidative stress might not be severe in the systemic hemorrhagic shock for pigs since no significant variations were observed for ascorbic acid and mda (2) the increase of gsh might be that it was released from hepatocytes when the animal was under systemic ischemia. (3) celzain amino acids might be acting as transmitters in the event of ischemia. however, fm"lher investigations are needed to clarify the detailed mechanism in regard with antioxidants and amino acids. dr. fang-ku p'eng taichung veterans general hospital talchung, taiwan 407, r.o.c. jia shi yong, huang zhi oiang and men xian jun. in patients underi~ent major hepatic surgery,obstructive jaundice has been implicated with fnoreased susceptibility to sepsis and liver failure. jaundice was said to he associated with derangement of iamune function and result in expression of pr,:.inflar...aatory cytoki~es that cause hepatooellular damage. this study is ~.o investigate the effects ,.,t lip,~polysaccharide(lps)rats. jaundice were produced in healthy ~istar rats of either sex weighing 180-220 m~ by common hi l~ duet l igation(bl)l).seven days post l igation, rats were given lps(236.gg.10)0.7rag/kg intraperitoneally. at the end ,:,f l,gand 5 hours after lp$ ehanllenge, liver were removed and prepared for forzen sections immediately. i)emonstration of inf in liver parenchyna were performed by method of abu i~munohistochemistry using r~onoolonal antibody against tnf, leve of mrna for tnf ~ere measured by in-situ hybridization using biotin-labeled edna probe. results sho'~ed that tnf stained granules appeared in kup~'fer eell~ and polymorphonuclear leukocytes(pnnl,but not in hepatoeytes nor endothelial eel is. they ~'ere located within cytoplasms and peaked at 3-5 hours after lps ohal lenge. there was vocomitant tnfmrna expression but peaked earlier 1 hour post lps challenge. tnfmrna vcere detected notably in necrotic area and barely in bdl rats ~ithout lps challenge. it is therefore postulated that production of tnf by inflammatory effeetor cells-kupffer cell and phn in site, in the obstructive jaundice rats during sepsis may atribute to the hepatocellular damage. it also provide evidence for the beneficial effects of early release of biliary obstruction. in the present study, an animal model of a0c in wlstar rats was developed by tigatlng the common bite duct and injecting d.3mt solution of e. goli % b, (g×10' efm/mt) into the bite duct. the mortality rate was 40~ at 48h after aoc. at 6, 12, 24, 48h after aoc, kcs were isotated and cultured atone or cocuttured with hepatocytes to evaluate the modulation effect of kgs on hepatoeyte protein synthesis. the results showed that tactodehydrogenase leakage was increased progressively as the injured k~ function and structure developed. tnf and il-i tn the supernatant were detected with the peak values at 12h after aoc. at 48h after aoc, ~-teueine incorporation was obviousty decreased in the normal hepatocytes eocuttured with stimulated kcs compared to the uormai hepatocyte cultured group (p<8.01), but it was slgnificantiy increased in the group cocultured with normal kcs. in the a0c group, 'h-teuclne ineorporatien was decreased progressively in the injured hepatoeytes cocuttured with stimulatedkgs and it was markedly elevated when cocuttured wlthnormat kcs at 48h after a0c (p<0. og). it is concluded that the impaired hepatocyte proterin synthesis was directly mediated hy the stimulated kc function during aoc. such a mechanism possibly may be involved in the patho0ensis of hepatic dysfunction and m0f following h06. " the project supported by nsfc. in the past few years it has become evident that cytokines are implicated in various pathophysiological mechanisms. therefore measurement of cytokines and their potential inhibitors in biological fluids may be helpful in diagnosing and monitoring of diseases. however, dependent on the type of assay used investigations performed in different laboratories have often yielded conflicting results. in order to assess reliability and reproducibility of cytokine measurements two comparative studies for the determination of cytokines in biological fluids were launched by several investigators interested in this field: one national austrian ~tudy and one european study in the context of the european workshop for rheumatnlogy research. serum and synovial fluid samples were distributed by the organisers, and participants had to measure one or several of the following cytokines in all samples: il-1, il-2, 1l-6, i58, tnf-alpha, ifn-gamma, gm-csf, and the soluble receptors for il-2 and tnf; both commercially available immunoassays and home-made assays were allowed. so far, the main conclusions emerging from these preliminary studies are: (1) the same immurzoassay (elisa) yielded comparable results in different laboratories; (2) immunoassays from different manufactureres usually measured the highest concentrations in the same samples, yielded similar relative values but disparate absolute values; (3) assays for soluble receptors yielded less discrepant results; (4) some assays seem to be more suitable for measuring cytokines in biological fluids than others. the mean retrieval of exogenous recombinant human cytokines was approximately 50% for most cytokines tested. however, it must be borne in mind that natural and recombinant cytokines may behave differently in immunoassays. this raises the question as to the necessity of setting up international standards for all cytokines. possible interferences by serum components such as (lipo)proteins, complement, rheumatoid factors, etc., which may either decrease assay sensitivity or yield false positive results, must also be considered. in addition, natural cytokine binding proteins (e.g. soluble receptors) might interfere with cytokine determination in immunoassays. the problem of immunoassays versus bioassays has not yet been approached, but is without any doubt worth indepth investigation. thus, these studies are a promising first approach to deal with the difficulties of cytokine analysis in biological samples, and it is hoped that they will help to overcome some of the major methodological problems in the near future. tumor necrosis factor (tnf) is a pleiotropic cytokine which plays a key role in a number of immunologically mediated disorders like septicemia or cachexia. tnf receptors are found on the surface of a variety of cells, where they provide molecular signals for the cytokine effect. there exist two types of soluble tnf receptors (stnf-rs), tnf-r p55 and tnf-r p75. these stnf-rs can compete with the cell surface receptors and block their activity. the expression on and shedding from the cell surface of stnf-rs is enhanced by interferon gamma. increased concentrations of stnf-rs can be found in patients with infectious as well as malignant disorders. in patients undergoing immune stimulatory therapy with interleukin-2 and interferon alpha a simultaneous increase of tnf and its soluble receptors can be seen. in patients with hiv infection a strong correlation exists between the levels of stnf-rs and markers of immune activation like neopterin or beta-2-microglobulin. likewise patients with hematological disorders show elevated stnf-r levels. patients with weight loss have higher concentrations than patients with stable weight. the final value of stnf-rs within the complex network of cytokines is not yet clear. however, there exist striking parallels between infectious and malignant disorders pointing to a key role of endogenous immune activation. biochemicatly, d-erythro-neopterin derives from guanosine triphosphate. in vitro studies show that large amounts of neopterin are produced by human monocytes/macrophages stimulated with interferon-j (ifn-j). neopterin is biologically stable, and its halflife in the blood seems to solely depend on renal excretion. specimen for neopterin determination can be stored without special precautions. increased concentrations of neopterin have been described in body fluids of patients suffering from diseases which are apparently associated with an activated cellular immune response and with enhanced endogenous formation of ifn-~', e.g,, 1 ) increasing neopterin levels predict rejection episodes in allograft recipients 2) virus infections induce increased neopterin concentrations, and the degree of neopterin elevation predicts prognosis in these patients which is particularly true in hiv-1 infection 3) in autoimmune disorders such as systemic lupus erythematosus or rheumatoid arthritis neopterin levels reflect activity of the disease 4) increased neopterin concentrations in patients suffering from certain types of cancer indicate poor prognosis. significant associations between changes of neopterin and decrease of hemoglobin as well as the development of weight loss and cachexia, e.g., in cancer patients and in patients with hiv-1 infection, support the concept that endogenously formed cytokines such as ifn-~" and tumor necrosis factor-~( are involved in the pathogenesis of such symptoms. in conclusion, neopterin monitoring is a sensitive tool to detect the activation status of the cell-mediated immune system in vivo. repair and healing or perpetuation of acute inflammation is characterized by a complex network of interacting cellular and humoral defence mechanisms. of the numerous inflammatory mediators/effectors investigated hitherto, the proteolydc lysosomal enzyme pmn elastase has turned out to be highly destructive when released extracellularly thus contributing considm:ably to organ dysfunctions in severe posttraumatic and postoperative courses. besides various cytokines, elastase in complex with its main antagonist c¢ 1-proteinase inhibitor (a 1pi) is also supposed to induce the shedding of intercellular adhesion molecules from inflammatory cells (pmns, monocytes/macrophages, endothelial cells). these soluble adhesion molecules may modulate the inflammatory process in many different ways, e.g. via acting as chemotaxins, blocking neutrophil activation or competing with the membrane-bound forms in cell-cell adhesion. thus, measuring circulating or local levels of elastase-a 1pi and soluble adhesion molecules (icam, e-selectin,-pselectin, l-selectin) may be a helpful tool in judging the severity of an acute inflammatory process. in several clinical studies on surgical patients suffering from multiple trauma and/or sepsis we could demonstrate that the measurement of these parameters in consecutive plasma samples and local body fluids was highly valuable for early diagnosis and prognosis of multiple organ failure. prof. dr. m./ochum, institut fdr klinischechemie und biochemie, lmu miinchen, nufibaumstrage 20, 80336 m0nchen, germany procalaitonin (proct) a 116 aa peptide is dramatically increased in the blood of patients with various sepsis and infections. the increase begins as soon as 3 hours after the andoto:edn release and readied maximal level with a 200 to 80b-fold increase 24 h after li~ edrmrdstration. prcc, alcitorfin response was temporally different from these of tnf~ and ii-6, that reached peak levels at 2 h and 3 h respectively. at the opposite of eytokine (tnfcz, ild, j, is), proct is a very stable molecule. the half iife of proct in ~he blood is surprising if we consider the half llfe (about nine mirtute) of mature ¢alcitonin (co. we have found that it is due to the binding of proct with a protein on the n-termktal part. thin complex of about 80 000 daltons is unable to cross the kidney and the c_n9 barriers and the half life is at least elghteen hours. this long half llfe is very useful in the evaluation of a sepsis. a very sensitive and specific sandwich in'ununoassay has been developped. the results cart be delivered in two hours. at time, we know that in the healthy adults, the values are less thau 0.1ng/ml. it is also clear that proct is not increased ~n patients wlth inflammatory dleeasea without lnfectlon. for instance, proct is not signlffcat3vely increased in purpura rhumatom, arthritis, crohn's disease, rectocolitis, also in various cancer patients with inflammatory components. am other interesting finding, is the absence of increase or a very low increase in the viral infections. at the opp0site, in all the patients with bacterial sepsis or in the malaria pat/ants, progt was dramatically increased, from i00 to 10 000 fold. p~oct can be useful to the follow up of patients with sepsis. actually, we don't know the origin of this proct production, in the united states, will also be discussed. the septest portion of this study will eventually enroll over 500 patients tentatively diagnosed as septic. the results of septest will be compared with commonly accepted symptoms and criteria associated with sepsis in order to determine the clinical utility of this endotoxin assay. preclinical results of patients and normals be presented as well as data on the time course of endotoxemia and clinical outcome of selected septic patients. preparation of dna libraries clifford w. schweinfest, ph.d. a fundamental tool of the molecular biologist today is the ability to work with purified dnas representing genes of interest with respect to the investigator's field of study. originally applied almost 20 years ago to the genetic dissection of simple organisms such as viruses and bacteria, molecfllar cloning was rapidly applied to study the more complex genetics of the mammalian cell. today, virtually any study of cell physiology which depends upon specific gene expression is approachable using the tools of the molecular biologist. therefore, stress induced gene expression (such as the heat shock gene, hsp70) or the regulation of the nitric oxide synthetase gene or the induction of cytokines and growth factors as a result of trauma or injury lend themselves to investigation at the level of the gene. during these workshops, we will discuss the means by which cdnas and genes are isolated, amplified and identified. we will discuss the strategies and methods for cloning cdnas and genomic dnas, for organizing such cloned libraries, for finding specific genes and for characterizing those genes. this speaker will focus on the techniques and considerations involved in the synthesis of cdna libraries. topics include rna isolation, mrna quality, reverse transcription, choice of cloning vectors and library quality. genomic dna library synthesis will also be discussed with respect to the vector/host systems available and applications which are specific to genomic dna. finally, polymerase chain reaction (pcr) will be discussed briefly with regard to its impact on dna cloning. the identification of the genes or gene products that have a role in cellular processes is fundamental to our understanding of genetic control and methodologies to achieve this aim are currently available. all levels of the various signal transduction pathways can b~ molecularly analyzed to define the mechanism by which critical steps in each pathway are achieved. the questions that are unresolved in each area of investigation serve to direct the scientist towards analyses of gene products or gene regulation of the gene itself. thus, selection of the type of library (genomic or cdna) to be used is dependent upon the specific goals of each investigator. to facilitate resolution of this question, an overview of the uses for the different types of libraries will be presented. at least four methodologies are currently available for screening a library, which are dependent upon specific interaction between nucleic acids, nucleic acids and proteins, antibodies and antigens or between different proteins. the use and advantages of each of these methodologies will be discussed along with a description of probe preparation. sequence methodologies required to begin characterization of gene clones will be presented. hopefully, the participants in the workshop will help define areas of emphasis and allow time for directed interaction to discuss specific applications based upon their own experimental systems. alterations of physiological conditions, such as those occurring after shock and sepsis, induce changes in gene expression of cells composing the major organ systems. the challenge is to detect and characterize these changes in geae expression and relate them to the injury. the development of cloning techniques has emerged as the methodology of choice. changes in gene expression are usually characterized by variations in the concentration of cellular messages because synthesis of the final product "the polypoptide" is usually proportional to the concentration of mrna. due to the rapid regulation of gene expression the cellular concentration of messages changes very quickly. this is commonly referred to as steady-state levels, a balance between transcription and degradation. steady-state levels of mrna can be detected in a single cell (in situ hybridization) or in total rna isolated from cells or organs and separated in agarose gels (northern blots). although analysis of mrna steady-state levels are very useful, they do not provide information about the mechanisms that regulate gene expression. changes in gene expression primarily occur at the level of transcription; characterization of the rna species being synthesized at a given time is performed by the nuclear run-off technique. when there is no a priori information about the gene that may be regulated after a particular situation such as sepsis, the total pool of messages present in cells at given time can be characterized by a combination of in vitro translation of the cellular messages and fractionation of the in vitro translated products by two dimensional gel electrophoresis. such approach permits the visualization of the general pattern of gene expression, a "finger print", of the physiologic state. in summary, researchers now have access to a great variety of techniques to identify and characterize genes expressed in response to a physiological condition that may be utilized as "molecular tools" to study the organism's responses to shock and sepsis. the acute phase proteins are a set of plasma proteins with greatly increased plasma concentrations during acute inflammations and after tissue trauma, e.g. after major surgery. the proteins counteract the source of injury, facilitate clearance of infectious particles by phagocytes, assist immune responses and initiate wound healing. the corresponding genes are expressed in liver hepatocytes and regulated by the inflammatory mediators interleukin 1 and interleukin 6 (ill, il6). class1 acute phase genes are mainly controlled by ill and by combinations of ill plus il6; class2 genes mainly by ii,6. the human c-reactive protein(crp), serum amyloid a(saa) and complement c3 genes will be discussed as examples of class1 genes, and rat c~2 macroglobulin as a prototypical class2 gene. both classes of genes use different types of ¢ytokine response elements in their promoter-proximal transcription control regions; class1 genes use the transcription factor nf-il6 or c/ebp as the key factor to mediate cytokine responsiveness; class2 genes use a different, so far unidentified factor to achieve responsiveness to il6. ongoing efforts to purify this factor, called the il6-response factor (il6-rf), from rat liver nuclear protein extracts will be described. the il6-rf apparently mediates the effects of il6 in a broad range of cell types, including b lymphocytes and other hematopoietic cells. it is therefore likely to be of equal general importance for gene regulation by il6 as nf-il6. the importance of transcription factors as potential targets for novel bioactive substances and possibly therapeutic agents will be discussed with the help of several recent examples. new methods for altering the germ lines of mice provide powerful tools for understanding the roles of individual genes in normal and pathological processes, and for reproducing specific genetic mutations that result in congenital disease. three approaches will be discussed. conventional transgeulc mice are frequently constructed using artificial transgenes that express genes from a promoter other than the normal gene promoter. this paradigm is used to produce very high levels of a gene product, for example, with a viral promoter, or to attempt to regulate gene expression using an inducible promoter. alternatively, normal promoter sequences can be hooked to a gene whose expression is lethal to the cell (e.g., diptheria toxin a chain) to ablate all cells expressing that gene. another approach uses an intact gene with its normal regulatory sequences. here, an elevated level of the gene product is delivered from the correct cells in the correct tissues at the correct developmental stages or in response to natural signals. this approach has been refered to as "genetic pharmacology," and provides a precise delivery of the gene product to the target. however, to be most effective, the transgene should contain arl regulatory sequences as welt as the entire genomic segment containing gene coding regions. the introduction of conventional transgenes is limited to roughly 50 kilobases, while genes containing all regulatory sequences frequently stretch over dozens or even hundreds of kilobases. to overcome this problem, procedures have been developed recently to introduce yeast artificial chromosomes (yacs) into mouse embryonic stem (es) cells. yacs can include over 2 mb of human dna, large enough to encompass the largest known human genes. es cells are also useful for targeted gene deletions and mutations. homologous recombination can be targeted to any known gene in es cells. when these modified cells are injected into a mouse blastocyst, they can be incorporated into all tissues of the resulting mouse, including germ cells, so subsequent generations will pass the genetic modification as an inherited trait. assessment of the effects of a null allele on development can provide valuable insights into its normal role. ultimately, these technologies may be adapted to human cells -not for embryonic intervention, but to provide modified stem cells for various tissues (e.g., gut, eye, hematopoietic system) which could then be applied to somatic therapies. with major illness/injury that respirswy faihne fi'equently followed sepsis, tilney el al nse, d the term "soqueutial syste~ failure" for patients with renal failure after solmir of ruptured abdominal aortic ~aeurysms, i then reviewed our experiences after inju~ and oporstion and suggested that multiple, progsesslve or sequential systems or organ failure was a syndrome to be reckoned with in the, 70's. eiseman et al then wrote about "multiple organ failure", especially with liver failure. polk el m found that renmte orgau failure often signalled occult anita-abdominal infection. fry et al ampbasized the role of uncontrolled infection in organ failure, border et ai described metabolic alterations with mof and used the term multiplesystems organ feilmo (msof). other early conltibutots to our knowledge of mof include faist, trunkey and miller, marshall and dimic&, cassone, catlleo, meakins and marshall, (}otis et at., mater and many others. mof or msof were used eommoifly. cerra coined the terms "post-trsumatic septic sfndromc" and "hyper metabolism otgau failure complex", and border et al, used lhe ex3~ression "gut origin seplic states" to illdioate important eurrem aspects of abe gt.'nerlc problem of organ failure and death, other terms include acute organ-system failure, multiple organ injury syndrome, post-traumatic multi-system organ failure nod post-~aumatic organ system infection s~dmmc (fi~sis), i bdieve the latin "maltiple organ failure" is clean, neat and says it all, now there is a proposal for a new set of torminolugios -mods and sirs. will they be helpful in the study and esro of patients? our speakers in this session will review thcsa proposals and the evidence as we strlvo for eoasensns. it has been well demonstrated that the administration of pro-inflammatory cytokines, and especially tumor necrosis factor (tnf) can result in a picture similar to septic shock with secondary multiple organ failure. it is also clear that tissue hypoxia can lead to organ damage. we believe that it is the interaction between the two phenomenons that can lead to mof. on the one hand, the inflammatory response is amplified in the presence of hypoxia. on the other hand, the release of the mediators of sepsis can increase tbe oxygen demand ef the tissues, alter their oxygen extraction and decrease myocardial contractility, and the combination of these elements accounts for the development of septic shock. this hypothesis is supported by two lines of evidence. first, mof is usually preceded by an episode of acute circulatory failure (even though frank circulatory shock may not be evident). second, recent experimental and clinical studies indicated that immunotherapy (especially antibodies to tnf) is particularly effective in the most severe forms of sepsis, associated with circulatory shock. hence, an effective way to prevent mof may be a combination of aggressive cardiovascular management and immunotherapy. development of the multiple organ dysfunction syndrome (mods) in the critically ill follows an heterogeneous group of stimuli including infection, sterile inflammmion, extensive tissue injury, ischemia, intoxication with a variety of substances, and immune system activation. whether the resulting physiologic abnormalities reflect a common pathologic process, or are simply a non-specific manifestation of tissue injury is unknown. moreover, the lack of clearly-defined functional criteria for the characterization of the syndrome on the one hand, and of reliable biochemical markers of its evolution on the other, has made attempts to define its epidemiology and natural history difficult. using a numeric scoring system to quanfimte the clinical severity of mods, we demonstrated that, although mods is more common in patients who have significant infection at the time of icu admission, a much stronger correlation is evident between the severity of mods and the subsequent development of nosocomial icu-acquired infection. furthermore the severity of mods correlates strongly with the severity of the clinical septic response, and with the degree of immunologic compromise evident on delayed type hypersensitivity skin testing. to determine the relative importance of the initial stimulus, and the host response to that stimulus, to the subsequent emergence of mods, we undertook a matched cohort study of 109 critically ill patients admitted to the icu with infection, matched by age, sex, and apache ii score to 109 uninfected controls. organ dysfunction scores were higher in patients admitted with infection, but were also significantly associated with the expression of a septic response at the time of icu admission, independent of the presence of infection. by stepwise regression analysis, the magnitude of the septic response was the most powerful predictor of the severity of mods in both infected and uninfected patients. these data suggest a model of mods in which a stimulus (eg infection) and the host response to that stimulus (eg the septic response) result in a state of altered systemic homeostasis (manifested in this example by immunologic dysfunction). moreover they suggest that the principle determinant of the emergence of mods is the response of the host, rather than the nature of the stimulus, and are consistent with the hypothesis that a stereotypie systemic response to an heterogeneous group of injurious stimuli underlies the pathogenesis of mods. arthur e. baue, m.d, ever since the attorapt 10 build the tower ef babel, as desclibcd in the old testament of the biblc, it has been difficult to got agreement o;x common definitions or language, although mof has served well, there will always he now classifications proposed with good reasons for them, thus mods and sirs are not the final oxpressinns in the lexico~aphic sweepstakes. attcmpts to develop standard animal shock and scpsis models (hemorrhage, endotoxin, foes[ pellets, cecal ligation and puncture, e. colt hlf0sioi0 to evaluate therapy have net been suoca.ssf~. clinical syndromes of mof, sepsis, sepsis syndrome and athers have not been aeacpte.d by all. our problem is that we arc tryittg tu d¢ffino a non-emily. mof or mods or sirs is net a disease or oven a syndrome. it is a series of complications of injury, disease and intensiva care which loads to death for many patients in intensive care units, it is not a fixed entity, but an ewilving picture. we have lumped (ugethet for therapy and definition a number of diseases and problems according to clinical manifestatieos rather than the eause of ihe problem, the search for unified mechanisms has not bean successful. will we benefit oleo from a more precise classification and definition of a non-outjty, a hodgu-podge of human disorders which have charsmeristic.s and manifestations of tissue injury, inflammation and infactian? re.hi clinical trials of potential "magle ballets" suggest that we should go in a different direction--instead of lumpors, we should be splitters, to seek effective therapy, we must fucus on specific disorders such as trauma, specific infections, inflammatory disease and chronic or acute organ damage (copd -renal failure, etc.), i will say more aboet this later ha the me.ethag. thcre is cue potentially important pert of the mods proposal, and that would be do~mcntation of organ dysfunction before fsiiure occurs and before tile need for external supporl dovelope. this could lead to hatter methods of preventing organ failure. preveution is ultimately the only answer to organ failure, m mof, mods and sirs. have wo reached a consensus?--only that we arc dealing wilh a diffl~x:lt probtolr looking for solutlons. acute lung injury (ali) and its most severe form, acute respiratory distress syndrome (ards) characterized by severe hypoxemia, diffuse infiltration of both lungs, a reduction of compliance and a wedgepresure lower than 18 mm hare related to direct or indirect injury. when aliresults from direct injury (toxic agents, lung contusion, pneumonia), the lesions of epithelial barrier and the activation of lung macrophages are the first events leading to the release of inflammatory mediators wh:ch are responsible for alteration of the membranar permeability and for invasion of the alveoli by fluids, proteins and cells. an inflammatory reaction develops, with injury to endothelial cells, adhesion of polymorpbonuclear leucocytes (pmn) and the release in blood stream of intlammatory mediators dispersing the inflammatory reaction to other organs and tissues. the failure of lung function also results into hypoxia in other organs, leading to tissular ischemia, triggering an inflammatory reaction leading to organ dysfunction. frequent complications of direct lung injury are septic pneumonia with endotoxin release, phagocytes activation and cytokine production disseminating inflammation. by this way, direct lung injury can be at the origin of the multiple organ dysfunction syndrome (mods), the frequence of which having been estimated to 40% and is increased when all has occured in patients alreadypresenting an organ dysfunction (immunodepresslon, cancer...). when ali results from redirect or extrathoraclc injury (trauma, infections, hypovolemic shock, acute pancreatitis...), inflammatory mediators and micro-aggregates released in particular organs or tissues reach the lung via the blood and lymph streams, are filtered in the organ or trapped in the lung capillaries, and are responsible for a local inflammatory reactaon (endothelial cell activation, pmn trapping and adhesion, platelet aggregation, release of mediators). sepsis acts by the way of endotoxiu and sequential eytokines release (tnf, ill,...) while trauma with important blood loss acts especially by the way of hypoperfusionreperfusion phenomena leading to a disseminatedinflammatory reaction and to a possible bacterial transloeation when intestinal hypopeffnsion is present. in these conditions of indirect injury, all (or ards) is a iocal early (often the first) manifestation of a general phenomenon of altered membrane permeability and inflammatory reaction, easily and rapidly recognized by its clin~cai signs. the iung is one of the numerous organs participating to the mods, in these conditions, the mortality rate is high, reaching more than 80% when sepsis is present or when at least 3 organs are implicated in mods. by direct or redirect injury, lung is so a target organ for mods and injured lung can be the cause of mods or simply a participant to this syndrome. jorge cervts-navarro main determinants for brain dysfunction are breakdown of the blood-brainbarrier and raise of intracranial pressure (icp), in second place decrease of systemic blood pressure, disorders of blood coagulation and respiratory function. the blood-brain-barrier of cerebral blood vessels can be opened by stroke, cerebral trauma, inflammation, convulsions, acute hypertension and changes in the blood osmolarity. the consequence is brain oedema, increase of lop and decrease of the perfusion pressure. a close correlation between intraventricular pressure and the fate of patients with severe brain injuries has been established. regional cbf values of 16 to 18 ml/100g/min are reflected in isoelectric eeg, and values about t5ml/100g/min, result in loss of somatosensory evoked potentials. for ionic pump failure the threshold has been determined by values of about 10 ml/100g/min and is reflected in massive release of intracellular potassium. in stroke and in brain trauma when the oedematogenous condition is initially localized the tissue pressure variance within and between the hemispheres lead to tentorial and falciform herniations are the common cause of death even before the critical threshold of intracranial pressure is reached. the increase of the icp over the level of the perfusion pressure leads to the arrest of the cbf. if this persists {or periods exceeding 6 seconds of global brain ischemia loss of conciousness and developing seizures appear. if it exceeds 10 min irreversible injuries of the brain appear. following cerebral ischemia and after severe head injury a tendency to increased coagulation can be detected. vascular occlusion may develop either as a result of local thrombus formation or from emboli caused by circulating platelet aggregates. the formation of microthrombi is triggered by the plateletactivating factor a mediator in central nervous injury also responsible for aggravation of posttraumatic brain edema. acute hemorrhagic leucoencephalitis and brain purpura are known to arise as complications of acute viral infections and following gram-negative septicaemia. institute of neuropathology, free university of berlin, hindenburgdamm 30, 12200 berlin, germany undoubtedly, attempts to quantify as objectively as possible the degree of dysfunction of the various organs is very valuable. however, cardiovascular failure has a peculiar place in the context of sepsis. the development of acute circulatory failure (shock) is of paramount importance as a cause rather than as a consequence of organ failure. it is therefore essential to clearly separate patients with and without circulatory failure. once this has been done, attempts to list the cardiovascular system among the other systems have been unconvincing. in a number of studies, cardiovascular complications are assimilated to a low systemic vascular resistance (svr), but since svr is basically the ratio of blood pressure over cardiac output, and since hypotension is already included in the definition of shock, then a low svr is basically equivalent to a high cardiac output, which is a characteristic rather than an ominous complication of severe sepsis. development of a low cardiac output unresponsive to fluids is usually a terminal event. it is not advisable to list other problems such as severe arrhythmias, myocardial infarction or tamponade as complications of severe sepsis. host defense dysfunction following trauma, shock and sepsis e. faist, g. f. babcock * major accidental, burn and operative injury often precipitates a profound multicentric immunologic depression that is believed to predispose patients to become anergic and thus to develop towards a higb probability of infection. anergy or a lack of immunologic reactivity stands for a total elimination of skin test reactivity and recall antigen responses in the inununocompromised patient. anergy following overwhelming trauma or major septic conditions results from a massive impairment of cell mediated immunity (cmi) together with a whole body malignant inflammationiike state. we and others have demonstrated clearly that the skeration of cmi following trauma is mainly due to the disruption of intact monocyte (moo) / t-cell interaction. within this phenomenon we see a shift of the cell ratio in the compartment of peripheral blood mononuclear cells (pbmc) with a considerable increase of prostaglandin e 2 synthesizing m~ and a simultaneous decrease of functionally competent cd3+ and cd4+ lymphocytes. t-cell dysfunction in states of profound stress is characterized by impaired synthesis of two crucial lymphokines -interleukin-2 (il-2) and gamma-interferon (y-ifn). the inability to produce adequate amounts of il-2, most likely attributable to alterations in the transmission of signals from the cell membrane to the nucleus, results in incomplete proliferative t-cell responses to antigenic stimuli, while a lack of ?-ifn results in inadequate m~ antigen processing and presentation. the role of the two functionally distinct t-helper subsets, t-helper-1 (thl) secreting principally il-2 and ?-ifn and t-helper-2 (th2) acting principally in inducing antibody formation with a secretion of il-4, il-5, il-10 and tnf-[3 has still to be established within the context of major trauma. antibody formation to common protein antigens is impaired, the predominant finding is a shift from igm to igg synthesis. although excessive secretion of prninflammatory cytokines (il-u3, tnf-c~, il-6, il-8) has been considered to be deleterious for the host in states of major inflammation and infection, in-vitro and whole blood investigation of me function has clearly revealed that there is initially a marked suppression of moo from septic patients to synthesize and secrete proinflammatory cytokines to endotoxin. this probably will result in immunodeficiency of traumatized as well as septic patients, since these cytokines in low concentrations are involved in the upregulation of the essential humoral and cellular immune functions. abnormalities of pmn function noted after serious injury have been interpreted by some investigators as signs of unresponsiveness of this cell population, others have demonstrated signs of pmn byperactivation. latest findings revealed that there is a clear pmn dysfunction in circulating blood: 70% of all pmn's in this compartment show downregulated or non existent ~-integrins within 72 hours posttrauma -these cells do not phagocytose, have a reduced respiratory burst and appear to be completely degranulated. restoration of these cell functions within 7 days post-trauma is significantly correlated to survival of traumatized patients. other reports however stress increased expression of cr3+ receptors (cdll/cd18 complex) on circulating pmn's, increasing the ability of these cells to adhere to intercellular adhesion molecules, thus contributing to play a major role in inducing the pulmonary capillary leakage. our knowledge about the perturbation of host defenses associated with serious injury and sepsis is continuously increasing and represents the precondition for the design of effective therapeutic measures to prevent and counteract the resistance to invading microorganisms. dept. of surgery, klinikum grosshadern, ludwig-maximilians-university, munich, germany. * dept. of surgery, university of cincinnati, cincinnati, ohio, usa lg thijs~ ce n~ck sepsis is the most common cause of acute disseminated intravascular coagulation (dic) and coagulation disorders as well as abnormalities of fibrinolysis are common in septic patients. some clinical and experimental studies indicate that dic is a pathogenetic factor for the development of multiple organ failure. endotoxin and various mediators (tnf, il-i) enhance tissue factor and decrease expression of thrombomodulin on endothelial cells in vitro. also, tpa activity is suppressed and release of pai-i is stimulated. in such a way the endothelial surface becomes procoagulant whereas fibrinolysis is inhibited. following administration of endotoxin to normal volunteers levels of prothrombin fragments (fi+2) and thrombin-antithrombin (tat) complexes increase, indicating thrombin generation. also the fibrinolytic system is activated as evidenced by a rise of levels of tpa and plasmin-antiplasmin (pap) complexes but this is counteracted by a subsequent release of pai-i. in severely ill septic patients levels of tat complexes are increased and concentrations of the natural inhibitors of coagulation (at iii, protein c) are usually decreased. also, tpa levels snd pap complexes as well as concentrations of pai-i are elevated. the severity of many of these alterations is related to mortality. thus, both coagulation and fibrinolysis are activated in sepsis, but not in a balanced way, thereby promoting coagulation. one of the hallmsxk pathological alterations associated with sepsis is the development of microvascular thrombosis, an entity ~lmre~erizod by microvas~tlar plugbing with leukoeyies (predominately nentrophils) and fibrin deposits. preranably, ischemia remlt~g fibm ve~-'ttlac ow,.luwion contributes sisni.ficantly to end orgen darnaje and consequent dysfatlctlon. p, er~t srldies haw focused pmdominately on the role of adhesion molecules in the pathogenesis of neu~ophil sequestration during infection and s~sis. this preparation will review the mechanisms underlying intravak-'v.ler i~brin deposition and its contribution to organ injury. the normal endothelial cell sure is generally conddered 1o be anticos~am. this state is maintained by two major anticoag~lam molecules on the cell so,ace: hepax'an su,lfale and thmmbomodulin. studies performed over the past decade have do~mentod a general shii~ towards a procoa~qtlant staw during gram negative hffeodon, aft e~| mediated mainly by changes in the endothalial cell surface. antt-tf antibodies have been shown to be pro~ective during l~'ml endotexemia. in summary, ~erova~'~ler fibrin deposition, in ~rt mediated by ~rcgulation of cellular tf, is a consistemt patholo~cal flndin~ during sepsis and contributes to organ injury. strategies designed to abrogate this event may n~nimjze the magnitude of erg~ dysftm~on. n, v, christou md phd, department of surgery, megill university, montreal, quebec, canada interactions between immune calls and the endothelium vie adhesion molecules serve to modulate immune cell traffic between the blood strum and the extrmvascular space, these families of molecules acting in a cascade and closely integrated with the oytoklna network, blood coagulation and the complement system, are responsible for the homing of leukocytes to areas of inflammation and the realrculatlon of tymphocytes. there are three types of immune ceils that must exit the blood stream into the extravascular space: lymphocytes ; polymorphonuclear leukocytes; and non-lymphocyta mononuclaar cells (monooytes, basophlls, aoslncphils). b lymphocytes exit the blood stream and recircu}ate mostly via the high endothelial venule (hev) in lymphatic tissue, rarely, in chronic }nfectlon=, they may recirqulate vie hev in non-lymphoi:t tissue. t-lymphocyte recircutation on the other hand can occur via hev in lymphoid tissue, as well as, the endothalium of the skin, returning to peripheral lymph nodes via the lymphatic channels. lymphocyte recirculation is the mechanism which allows ¢ontaot between the immune repertoire and pathogens, and homing delivers these cells to the appropriate environment for activation and for effeotor function, pmns exit the blood stream through endotbelium in close proximity to the site of inflammation after appropriate endothelial membrane receptor expression. they must reach the site of pathogen tnveslon quickly in order to be effective. their exudation to the inflammatory site can be modulated by their intravascular pdmlng, which results from appropriate receptor expression. because endotoxin leak from the gut has been postulated as an important trigger mechanism for the systemic inflammatory response syndrome seen after serious injury, the immunologic and metabolic effects of bacterial endotoxin infusion into normal volunteers can be expected to shed considerable light on the validity of this hypothesis. we and ethers have used tbis model to show that endotoxin causes a temporary paralysis in the ability of circulating monocytes to prudce the proinflammatory cytokines interleukin-i (il-i) and tnf-a. endotoxin, however, causes increased production of pge2 by the same cell population. endotoxin infusion causes significant suppression of circulating t-cell numbers and a profound suppression in the ability of the circulating t-cell population to proliferate and to produce interle~in-2 (il-2) upon in vitro stimulation. after endotoxin circulating t-cells are also more sensitive to the inhibitory action of exogenous pge2. administration of cyclooxygenase inhibitors at the time of endotoxin infusion largely abrogates the effect of endetexin on t-cell proliferation and il-2 production. endotoxin infusion also causes the release of increased levels of circulating catabolic hormones including cortisol. administration of cortisol alone or cortisol along with endetowin to volunteers has allowed dissection of cortisol effects from those of endotoxin itself. cortisol infusion alone causes a diminution in the circulating t-cell population but the remaining ceils continue to produce il-2 after appropriate stimulation. cortisol infused along with endotoxin blocks the overshoot in monocyte proinflammatory cytokine production seen 24 -48 hours after endetoxin infusion. at present, one may conclude that administration of sublethal doses of bacterial endetoxin to human volusteers produces alterations in cytokime production and tlymphocyte activation which are both similar and dissimilar to those seen following serious injury. hemodynamic support is based first on intravenous fluids and second on vasoactive agents. colloids are usually preferred to crystalloids, for their more rapid hemodynamic effects and their lesser passage in the interstitial space. in the presence of hypotension, the pharmacological profile of dopamine makes it the first agent of choice. it is only when high doses of dopamine are insufficient to restore a sufficient tissue perfusion pressure that norepinephrine should be added. even when cardiac output is not decreased, a dobutamine infusion is often indicated to counteract the myocardial depression, prevent vasoconstriction, and increase oxygen delivery to the tissues. the benefit derived from the administration of "renal" doses of dopamine is doubtful, but stimulation of dopaminergic receptors may increase blood flow also to the gut. in any case, none of these catecholamines has been shown to significantly improve the oxygen extraction capabilities of the tissues. agents with antiinflammatory properties but also with some vasodilating properties, such as n-acetylcysteine, prostaglandin el or pentoxifylline represent more attractive options to increase oxygen extraction. cardiovascular support should aim not only at the restoration of a sufficient arterial pressure but also at the maintenance of an optimal oxygen delivery to the cells. it should thus be guided also by measurements of cardiac output, mixed venous oxygen saturation and oxygen extraction, and indexes of cellular hypoxia, such as blood lactate levels, gastric intramucosal ph or vanearterial pc02 gradients. supranorma[ 02 delivery contributes to the prevention of tissue hypoxia tissue hypoxia is considered to be the common final pathway in the pathogenesis of multipte systems organ failure. it may directly contribute to cellular injury and organ dysfunction as well as enhance further mediator activation and release by a positive feed back mechanism. prevention of tissue hypoxia, is therefore, one of the most important aims in the supportive therapy of critically ill patients, especially in those with sepsis and septic shock. mismatch of cellular o= supply and demand in these patients may resuit from the impairment of the cardiocircu[atory system on all levels. 1. myocardial depression with a drop in 02 delivery (do~) 2. redistribution of blood flow between the different organ systems 3. inadequate nutritive blood flow due to tissue edema and endothelial damage (gic, leucocyte swelling etc.) these pathological changes may go along with increased metabolic needs. cellular and organ 02 supply may therefore be inadequately matched with cellular 02 needs in patients with normal and even supral normal doz. hyperdynamic circulation is often present in adequately volume resusucitated patients. it is most likely that one of the bodys main compensatory mechanisms would maintain cellular o= supply in the face of increased metabolic needs and impaired oz extraction capabillities. this pathophysiology may explain the findings in several clinical investigations that patients with normal or even supranormal doz can have signs of inadequate regional tissue oxygenation. it is also consistent with the results of different studies which demonstrated that the achievement of supranormal doz levels, either spontanuously or due to adequate supportive therapy, resuits in better patient outcome. any attempt to prevent or treat tissue hypoxia in these patients, however, has to take into account the effects of therapy not only on global dgz but especially on regional and microcirculatory blood flow. r. rossaint, d. pappert, k. lewandowski, h. gedach, k. slama, k.j. falke klinik for anaesthesiologie und operative intensivmedizin, ukrv, freie universit §t berlin, germany important contributory factors to the high mortality of severe acute respiratory distress syndrome lards) are the aggressive therapies required, such as mechanical ventilation with high inspiratory oxygen concentrations and airway pressures. as specific therapies for reducing or preventing the general inflammatory reaction of the lung resulting in severe hypoxemia is unknown, today's therapy is limited to procedures which predominantly support or maintain pulmonary function, i.e. pressure limited ventilation with peep and permissive hypercapnea, avoiding or treatment of fluid overloading, and positional maneuvers. extracorporeal lung assist combined with low frequency positive pressure ventilation has been recommended, when conventional therapy fails. today, extracorporeal gas exchange may be carried out using a system internally coated with covalently bound heparin. this allows for a lower level of systemic heparinzation reducing the risk of severe hemorrhagic complications of extracorporeal respiratory support. from april 1989 to august 1993 89 patients, aged from 15 months to 60 years, were transferred to our intensive care unit (icu) for treatment of severe ards. all fulfilled at least the slow entry criteria of the u.s. national ecmo study. due to hypoxemia 13 patients required veno-venous extracorporeal membrane oxygenation (ecmo) immediately after transfer to our icu. the other 76 patients were first treated with pressure controlled mechanical ventilation, permissive hypercapnea, prone position, and dehydration, if necessary. in 23 out of these 76 patients, in which the gas exchange did not improve in the following days, veno-venous ecmo with heparin-coated systems was additionally performed. heparin was given to achieve a partial thrombin time between 40 -55 s and an activated clotting time between 120 -150 s. if surgery was performed during ecmo, heparin infusion was interrupted. no severe bleeding complications related to anticoagulation for the extracorporeal bypass could be observed, however, in some patients, after three weeks of ecmo thrombi in the drainage cannula were observed. a problem of of membrane leakage shortened the life span of the membrane lungs to a mean of about four 4 days. in the conventionally treated group 87% survived and in the additionally with ecmo treated group 58% survived, representing a 75% survival rate in patients whose risk of death is considered more than 50%. on the basis of these experiences, we conclude that the described strategy, including veno-venous ecmo with heparin-coeted systems, may improve the survival in patients suffering from severe ards. abnormally high skeletal muscle po 2 in septic patients and its normalization during recovery: evidence against tissue hypoxia but for impaired oxygen metabolism of skeletal muscle? p. boekstegers, k. werdan department of medicine i, gro6hadern university hospital, munich, germany a pathological oxygen uptake supply dependence was suggested to indicate tissue hypoxia in sepsis-a hypothesis which is discussed controversially. because the detection of reduced oxygen transport to tissue and &tissue hypoxia would have important implications for therapy, skeletal muscle oxygenation was studied in patients with sepsis. intermittent and continuous determination of skeletal muscle po 2 by polarographic needle or catheter probes provided no evidence for skeletal muscle hypoxia even in severe stage of sepsis (boekstegers et al., crit care med 1994) . in contrast, mean skeletal muscle po 2 was found to be abnormally high in patients with sepsis (48 _+ 10,4 mmhg, n=39) compared to patients with limited infection (28, 4 + 4, 7 mmhg, p<0.001, n=16) , to patients with cardiogenic shock (22,3 + 6,2 mmhg, p<0.001, n=ls) and to healthy subjects (33,3 _+ 8,1 mmhg, p<0.001, n=10). furthermore, serial measurements in patients with sepsis showed that skeletal muscle po 2 was higher (44,1 _+ 10,3 mmhg) on days with severe sepsis than on days with intermediate state (30,1 _+ 8 mmhg) and than on days without sepsis (26, 8 _+ 5, i mmhg) . thus, a decrease of abnormally high skeletal muscle po 2 was related to improvement of sepsis. this was also demonstrated by comparing the decrease of abnormally high skeletal muscle po 2 with the decrease of apache ii score or elebute score as well as interleukin-6 serum levels in a separate study (boekstegers et el., shock, in press). in summary, our data provide no evidence for skeletal muscle hypoxia in patients with sepsis but support the assumption &impaired oxygen metabolism in severe sepsis. neutropnlic emigration from the vascular space into the extracellular matrix is important for the response to tissue injury or contamination by providing a large recruitable pool of tissue-based phagocytes. the consequences of neutrophil exposure to intravaseular chemoattractants, likely relevant for il-8 and c5a in sepsis, trauma, and in burn injury, are suppression of neutrophil attachment to endothelial cells and matrix proteins. it is probable that proteoglycan-bound attractants are of considerable biologic relevance, and may result in enhancement of responses to subsequently encountered cytokines. in the presence of connective tissue proteins expressing integrin-specific binding ligands, neutrophils respond to tnf-cq gm-csf and other cytokines by producing large amounts of hydrogen peroxide. this response is likely important in the digestion of contaminated or injured tissue by facilitating activity of neutrnphil granular proteases and inactivating plasma anti-proteases. this matrix-dependent oxidative response is important in defining potential novel targets for therapeutic intervention. the response appears to involve signalling by integrin-linked tyrosine kinases. the net effect of matrix protein injury through this mechanism is enhancement of the fibrotic response which underlies much of the prolonged ventilator dependence of patients with the adult respiratory distress syndrome. this adherence dependent response has also been implicated in direct hepatocyte injury, and may represent an early component of the syndrome of multisystem organ failure. study purpose: in this patient (pat.) population with a considerable sepsis morbidity and mortality, parameters proposed for sepsis assessment (elebute sepsis score [ele], sirs) were investigated with regard to their use in the early classification of pop. pat. at risk for developing sepsis. patients and methods: in a previous observational study on 110 consecutive pat. after elective open-heart surgery, we had determined ele daily for at least 3 pop. days (in pat. with a longer stay: until icu discharge). after publication of the newly proposed sirs definition, these criteria were retrospectively calculated and compared to ele. results: on the total of n =515 patient-days, both ele and sirs showed significant (p<0.0001) correlations with parameters reflecting the general and sepsis-related severity of the pop. clinical course. compared to sirs, the ele correlations were better (icu mortality: 0.59 vs. 0.32; icu treatment days: 0,51 vs. 0.32; days on mechanical ventilation: 0,54 vs. 0.32; days with vasopressor requirement: 0.59 vs. 0.33; number of microbiological findings: 0.44 vs. 0.29), the optimal classification criteria for the mainly sepsis-related outcome gave maximal youden indices of 0.87 for ele (ele >_12 on >_2 days, accuracy: 94%) compared to 0.42 for sirs (sirs present on >3 days, accuracy: 67%). accordingly, ele roc curve areas for both overall (0.92) as well as sepsis-related prognostic evaluation (0.99) were significantly (p<0,05) larger compared to sirs (0.79 and 0.86, resp.), this higher overall accuracy of the ele criterion was primary due to a more valid assessment already on the first and second pop. day, where sirs still had a high false positive classification rate (46% and 53%, compared to 7% and 1%, resp.). conclusion: in the early postoperative course after cardiac surgery, the sirs definition displayed a high false-positive classification rate (low specificity) for subsequent sepsis-related mortality compared to better classification results obtained by the elebute sepsis score. from the departments of medicine i and of "cardiac surgery, grosshadern university hospital, marchioninistr. 15, d-81377 munich, frg. correlation between physiological and immunological parameters in critically ill septic patients. ma rogy, h oldenburg, r trousdale, s coyle, l moldawer, sf lowry a relationship between physiological parameters of severe sepsis and immunological function has not been established. in an effort to assess such a relationship we prospectively evaluated nine severely ill septic patients. physiological risk was assessed by the apache iii score 1, while one component of immunologic function was evaluated by peripheral blood mononuclear cells (pbmc) eytokine production after in vitro lps stimulation 2. four of the nine patients died. apache iii scores at 72 h were lower in survivors (s) than in non-survivors (ns), (51-+13 vs 111-+1 5 p<0.05), while apache iii scores at admission were not significant different between s and ns (82-+13 vs 95-+13). down regulation of cytokine production by pbmc upon lps stimulation was a transient event in s. while s demonstrated an 3 fold increase of tnf-a bioactivity with[r~ 72 hours, ns did not demonstrate any increase at all. a similar pattern was demonstrated for il-1[3 and il-6 immunoactivity. tnf was measured by wehi bioactivity, il-1[~ and il-6 immunoactivity were determined by elisa. the sensitivity was 35 pg/ml for tnf, 30 pg/ml for il-ll3 and 35 pg/ml for il-6, respectively. in conclusion, both physiological as well as immunological functions of severe critically ill septic patients demonstrate predictive value for ultimate survival. while patients biological status seems to be more predictable by apache iii at day 3, p<0.05, the pattern of cytokine production by pbmc upon lps stimulation over the first 72h might be a reliable predictor as well. introduction: therapy of sepsis and its sequelae depends largely on its early recognition. many studies have investigated the change of certain mediators during sepsis and their potential to predict multiple organ failure and outcome. it was the objective of this study to investigate whether the onset of sepsis can be predicted by alterations of levels of interleukin-6 (il-6), tumour-necrosis-factor (tnf), pmn-elastase and c-reactive protein (crp). materials and methods: over a one year period, 40 polytraumatized patients were prospectively studied (mean age 42y, 71% male, iss 28). serum and edta-plasma samples were taken in 12h intervalls until the patient left the icu. il-6, tnf, elastase, and crp were determined immunologically. sepsis was defined according to the criteria of 'systemic sepsis' (veterans" administration study, 1987) with at least 4 of 7 clinical signs: (1) tachycar-dia> 100/min, (2) temperature >38,9°c, (3) blood pressure < 100mmhg, (4) mechanical ventilation, (5) leukocytosis > 15.000 /ml, (6) thrombocytopenia < 100.000/ml and (7) presence of an obvious septic focus. clinical parameters, sepsis severity and serum levels were documented on a daily basis, beginning on day 2 after trauma. results: 21 of 40 patients developed a systemic sepsis (52.5%), and 5 died. all mediator levels were elevated under septic conditions. the clinical severity of sepsis correlated well with the respective levels of mediators. in patients, who developed a sepsis the following day, il-6 (510 vs. 195 ng/l; p=0.029), crp (160 vs. 121 mg/l; p=0.035) and tnf (32 vs. 15 ng/l; p=0.045) were significantly increased as compared to those patients who remained non-septic. elastase levels were considerably elevated but did not reach the level of significance. we conclude that il-6, tnf and crp appear to be sensitive markers for prediction of septic complications in polytraumatized patients. objectives of the study: the assessment of liver function in polytraumatized patients who are at risk of developing mof is too inaccurate and late by using conventional biochemical parameters. methats: the injury severity of the patients (n=26) was determined by the injury severity score (iss). lidocaine is given at a dose of 1 mg/kgbw over 2 rain. i.v. and is metabolized in the liver by a cytochrome p-450 mechanism to monoethylglycinexylidide (megx). the metabolite is measured by a fluorescence polarization immunoassay. serial determinations of the test were performed between the 1 ~t and the 11 ~ day after trauma and were compared with other liver function tests (bilimbin, gldh, alt, ast). the systemic inflammatory response syndrome (sirs) is still a challenge concerning early diagnosis, therapy and prognosis. therefore, evaluation of inflammatory and disease activity becomes more important. c-reactive protein (crp) is a well established acute phase protein in chronic inflammatory diseases. recent reports suggest an induction of crp by interteukin-6 (il-6), a cytokine involved in the mediator cascade of sirs. on the other hand, tumornecmsisfactor alpha (tnfcx) is a very early released mediator in sirs removed very rapidly from circulation. in addition, soluble tnf receptors (stnfr~5, stnfr75) are released into circulation in the acute phase response. this study examines the kinetics of five acute phase proteins (crp, il-6, tnfot, stnfr55 , stnfr75 ) in patients suffering from sirs. eighteen patients entered the study after diagnosis of sirs. blood samples were drawn every six hours during the first two days and every twelve hours thereafter. crp was measured in an routine turbimetric assay. il-6 was detected in an biological assay using the/l-6 dependent 13-cell line 1313/29. detection of tnfc~ was performed in an elisa system using a monoclonal antibody" for tnfo~. soluble tnf receptors were also measured by elisa. crp levels were elevated (>10 mg/l) in all patients and at all time points. crp values did neither differ significantly in patients with (143±33 mg/l) or without (129a:39) multiple organ failure (mof) nor in survivors (133±41) or non-survivors (147:t:44). in contrast, 1l-6 was elevated in patients wilh mof (mean 740 pg/ml, range 0-8900 pg/ml). il-6 levels correlated especially with lung dysfunction. tnf(x levels were consistently elevated in patients with mof. crp, il-6 and tnfoc did not correlate with each other. in contrast, levels for both stnfr showed a positive correlation (r=0.7). patients could be divided into two groups by values for stnfr~5 and stnfr75: the group with higher soluble tnf receptor levels showed increasing values combined with a poor prognosis. the group with lower levels of soluble tnf receptor consisted of patients surviving mof or without mof. in conclusion, crp does not monitor the course of sirs adequately. in contrast, il-6 correlates with mof and episodes of high disease activity. high stnfr levels may indicate poor prognosis. klinik f0r an/isthesiologie and operative intensivmedizin der cau kiel, schwanenweg 21, 24105 kiei, germany. ch. waydhas, md; d. nast-kolb, ivid; m. jochum, phi); l. schweiberer, mi) objective: to evaluate the irfflarranatory response after different types of orthopedic operations and compare them with the systemic effects of accidental trauma of varying severity. patients: in 135 consecutive patients with multiple injuries (iss 40.6) the inflammatory response to trauma was prospectively studied. the patients were divided into 4 groups according to their iss points. additionally, the alterations after 79 secondary operations (>24 hr) were determined (msteosynthesis of the femur (n=28), pelvic girdle (n=ll) and spine (n=8), facial reconstruction (n=13), smaller osteosynthesis (n=13) and others (n=6)). methods: specific and unspecific parameters of the inflammatory response were determined in the trauma patients every 6 h, beginning on admission of the patient to the emergency room for a period of 48 hr, and in the operative patients on the morning of the operation, at the end of the procedure and every 6 hr during the first two days. results: lactate, neutrophil elastase, heart rate, po2/fio2-ratio, and other parameters discriminated significantly between the 4 injury severity groups during the first 48 hr (kruskal-wallis-test, p<.05). the degree of postoperative changes differed significantly (kmskal-wallis-test, p<.05) between the 5 types of operations for lactate, heart rate, po2/fio2-ratio, nitrogen excretion and showed a strong discriminating tendency for neutrophil elastase and c-reactive protein. the extent of changes were highest after operations of the pelvic girdle, followed by procedures on the femur, spine, smaller bones, and the facial region. the postoperative changes after osteosynthesis of the femur or pelvis were comparable to the alterations noticed after smaller (iss 1 to 24) or moderate (iss 25 to 40) accidental trauma for neutrophil elastuse, heart rate, po2/fio2-ratio and parameters of the coagulation system. conclusions: there is a considerable inflammatory response to operative procedures that varies with the type of surgery. large operations cause changes in the body homeostasis that resemble those after multiple injuries. it remains to be established whether the inflammatory sequelae of surgical trauma are additive to the changes caused by accidental trauma. objective of the study: we retrospectively compared characteristics of elderly patients (~65 years) and yeunger patients admitted to a surgical {sicu) and a medical intensive care unit (micu). we further studied the relations between advancing age, chronic disease, sepsis, organ system failure (osf) and mortality in the elderly group. material and methods: during a 1-year period, 538 patients were consecutively admitted into the 8icu; and during a 2-year period, 487 patients were consecutively admitted to t~mich. criteria for chronic disease, sepsis, osfsi.e. cardiovascular (cf), pulmonary (pf), renal (rf), neurological (nf), haematological (hf), hepatic (lf), and gastrointestinal failure (gf)-were derived from the literature. results: patients from the sicu and~cu were similar in age, number of osf, and length of stay. however, when compared to sicu patients, micu patients had more cf (p<o.o1), sepsis (p<o.o01), and mortality rate (27% vs. ii~, p<o.ool). in contrast, sicu patients had more hp and nf, and prior chronic disease (all, p<o.o01). of the total group of 1,025 patients, there were 406 (40%) patients 65 years of age or older. elderly and younger patients had comparable length of stay in the icj, but elderly patients had significantly more chronic disease, sepsis, and number of osf (all, p<o.o02). all osfs, except hf and nf, were more ~o~mon i~ mdeljl, eompard to you-ger patients. ~he mortality is also higher in elderly patients (29% vs. 11% in younger patients). after multiple logistic regression, only number of osf increased mortality by a factor of 2.48 (95% confidence limits, 1.97-3.12), age did not increased [odds ratio 1.05 (i.01i.i0)], while admission to the sicu reduced risk of death by a factor of 0.15 (0.08-0.30). conclusions: based on these results, we conclude that age does not have any impact on icu outcome in the elderly. the only prognostic factor is the extent of acute disease, as measured by the number of osf. hence, prevention of oaf may influence outcome in elderly patients with critical illness. the lower risk of death in sicupatients may be explained by the large proportion of postoperative elective patients with relative mild chronic disease, and the lower incidence of sepsis. department of surgery, free university hospital, de boelelaan 1117, 1081 ~v amsterdam, the netherlands. septic shock and low output syndrome h.miyakawa, t.noguchi, s.hattori, a. mizutani, m. mori and n.honda objectives: cytckines are thought to cause the acute phase reactions under several critical conditions. we had investigated the relationships between cytokines network which included il-6,1l-8,acth and cortisol,and the outcome of the patients with septic shock or low output syndrome (los). materials and methods: in the septic group and los group,nine and four patients were enrolled respectively. furthermore, the patients in the septic group were divided into two groups,survival (n=5) and non-survival group (n=4).tn these patients,ll-6,1l-8,acth and cortisol had been measured every day after diagnosis of septic shock or los.the total number of measurements were 12 points in survival septic group, l l points in non-survival septic group and 10 points in los group.statistical analysis was used student's t-test to compare the mean of each group,and the changes were reported as clinicat significant if p<0.05. results: il-8 levels in non-survival septic group were higher than in both survival septic and los group.ll-6 levels in non-survival septic group were more significant than in both survival and los group.otherwise,there were no differences with respect to acth and cortisol among three groups. conclusions: we assumed that los would make several organs dysfunction as well as septic shock. but our results showed septic shock, especially non-survival,had different cytokins network mechanism for organs failure from los. lasson ,~, g/3ransson j, jijnsson k. eady diagnosis of complications after trauma is imperative to decrease morbidity and mortality. for this purpose an easy, cheap and fast diagnostic method is needed. the aim of this study was to analyse if complications after surgical trauma of various extent could be diagnosed earlier using preoperative or daily postoperative measurements of various plasma proteins than by using climcal signs of complications. material and methods: two acute phase proteins (c-reactive protein and antichymotrypsin), lbur main plasma protease inhibitors (alpha-2-maeroglobulin, c 1estemse inhibitor, antithrombin ill and alpha-2-antiplasmin) and one collagen precursor (procollagen iii peptide) were measured preoperatively and then once dally for 7 days postoperatively. for the plasma protease inhibitors immunorcactive as well as functional levels were measured. haemoglbin, albumin and the white blood cell count were also measured. measurements were done preoperatively in 256 patients and continued postoperatively in 78 patients, 70 % of the patients were operated on for malignancy. resulls: preoperative plasma c-reactive protein levels were higher in patients developing postoperative complications. this discrimination increased when plasma was sequentially analysed postoperatively, when a remaining high level ora second increase in plasma levels depicted a complication 1-3 days earlier than clinical signs. high levels were seen both in patients developing local as well as in patients developing general complications. there was no clear correlation between plasma protease inhibitory levels and complications, neither pre-nor postoperatively. procollagen iii paptide levels were slightly (but not significantly) higher postoperatively in patients developing complications. contusions: the acute phase response, especially concerning c-reactive protein, predicts postoperative complications better than both plasma protease inhibitors or collagen precursors. preoperative plasma c-reactive protein levels indicate the risk of postoperative complications, while daily postoperative measurements more readily depicts a complication, usually preceeding the clinical signs of complication by 1-3 days. dept. of surgery m'alm6 general hospital, s-2 i4 01 malmr, sweden. mof is a major threat to the extensive burned patients and associated with a high mortality rate. the role of endotoxemia in the pathogenesis of mof in burned patients remains controversial. in this study, we examined the relationship of plasma endotoxin levels to development of mof, and outcome in patients with thermal injury. methods: a prospective cohort study of 17 patients admitted with burns covering more than 70 per cent of body surface area was undertaken. circulating endotoxin concentrations were measured by modified limulus amebocyte lysate assay in serial samples of plasma. results: 7 out of 17 burned patients developed mof according to multiple criteria. the plasma endotoxin concentrations of patients with mof were 0.512--1.127 eu/ml, significantly higher than that of 10 patients without mof (0.216-0.553 eu/ml) on 3, 14, 21 and 28 days postburn (p< 0.05--0.01). significantly more incidence of positive endotoxin test (>_ 0.120 eu/ml) was found in patients who developed mof as compared to that of non-mof during the observation period (p<0.05). as the mean endotoxin levels increased, the prevalence of mof and death also increased (see table below), persistent endotoxemia carried a poor prognosis. conclusions: the present investigation provide further evidence that endotoxemia in severely burned patients commonly occur. cimulating endotoxin has also been found to be strongly associated with development of mof and mortality following major burn injury. multiple hemostatic changes occur in sepsis mad multiple organ failure (mof). to evaluate the role of platelcts in patients with sepsis and mof, we examined changes in surface glyeoproteins on circulating platelets of t4 patients with suspected sepsis and mof. the severity of sepsis and mof was assessed by eiebute and apache 1i scoring system, respectively.using flow cytometric techniques and platelets specific monoclonal antibodies, platelet surface expression of fibrinogen receptor on gpiib-iiia, ofvon willebrand receptor gpib, and of granula glycoproteins (thrombospondin, gmp-140, and gp53) was measured. receptor density of gpiib-illa mad gpib on circulating platelets was not affected by sepsis or mof. in septic patients surface expression of activated fibrinogen receptor (libs1 expression) was significantly elevated (p<0.05) and correlated well with severity of disease (f0.597). no significant change in surface expression ofthrombospondin, gmp-140 or gp53 was noted in septic patients. in contrast, degranulation ofgraanle glycoproteins was significantly elevated in mof (!0<0.05) that correlated well with severity of mof (gmp-140, r=0.611; thrombospondin, r=0.643).we speculate, that platelets in sepsis circulate in a hyperaggregable (fibrinogen receptor activation ) but still reversible state that results in increased risk of microthrombotic events. in the course of the disease, irreversible platelet degranulation might occur and may play an important role in development of mof. abdominal sepsis is still associated with high morbidity and mortality. the present study aimed at evaluating patients with abdominal sepsis treated at our surgical intensive care unit during a 10-year period with the aim of identifying potential prognostic factors, bacteriological cultures, diagnostic procedures, treatment and outcome. during the period 1983-1992 i66 patients with abdominal sepsis were treated at the icu at our university hospital. 60 patients were women and 106 men with a mean age of 64 (17-101) years. in 74 cases, the abdominal sepsis occurred as a postoperative complication. the patients were scored according to apache ii and bacteriological cultures and the occurrence of organ failure were noted. the patients were hospitalized in median for 40 (-273) days out of which 7 (-178) in the intensive care unit. 46 out of 166 patients (28 %) died in median after 18 (1-194) days. the primary cause of mortality was multiple organ failure (38/46; 83 %). apache ii scoring could not predict a fatal outcome. abdominal bacterial cultures were dominated by bacteria of enteric origin (83 %) and in 60 % cultures grew multiple bacteria. 134 patients bad organ failure and 64 multiple organ failure. 29/166 patients (17 %) had abdominal sepsis due to diffuse peritonitis despite a morphologically intact gastrointestinal tract and the absence of localized abscess formation. mortality in this group was significantly higher as was the percentage of positive blood cultures and the occurrence of multiple organ failure. abdominal sepsis is still associated with a high mortality, predominantly caused by multiple organ failure. abdominal culture findings are dominated by bacteria of enteric origin. in about 1/5 of patients with severe abdominal sepsis a diffuse peritonitis with intact gastrointestinal tract without localized abscess formation was found. in this group the mortality was increased as well as the risk of developing multiple organ failure. during the period from january 1985 to september 1993 56 patients, mean age 24+4 years were referred to our department of resuscitologywith the diagnosis of eclampsia. all the patients were delivered by cesarian section and were mechanically ventilated for 7.3_+5.7 days. diagnosis of sepsis was confirmed in 6 cases by clinical and microbiological methods. patients were divided in two groups: lnon septic patients, 2-patients with sepsis, the control group consisted of 20 patients after cesarian section without symptoms of eclampsia or infection. we determined plasma concentrations of immunoglobulins a,g,m(a,g,m), complement factors (c3,c4), alphal-antitrypsin (aat), trausferrin (trf) and albumin (alb) using beckman (usa) analyzer,protein concentration, using kone (finland) analyzer. a(mg/dl) g(mg/dl) m(mg/dl) c3(mg/dl) c4(mg/dl) k 203+-31 971+47 205_+21 134+12 30+-3 1 154-+29" 568-+48* 142_+24" 81-+14' 17_+4" 2 102+_21'* 420-+30** 96-+21"* 67-+10"* 11_+3" in a prospective study we investigated serum of 12 severly traumatized patients withdrawn directly after admission at our hospital (tr i). follow up controls were taken daily until day ten after trauma (tr ii). two control groups were performed: serum of healthy volunteers (co, n =24) was investigated as. well as serum of patients undergoing elective herniotomy (n=12) 24 hours before (op i) and 24 hours after operation (op ii). serum bactericidal index (sbi) was determined using a hemolytic e.coli strain 08:k87:h19. 200/11 suspension with a final concentration of 250 -400 cfu were incubated with l oopl serum. after overnight incubation sbi was calculated according a special formula. results: co 74.8 _+ 6.3 opi 77.8 _+ 6.4 opii 68.2 _+ 7.7* tri 38.4 _+ 9.1"* trii 57.6 + 8.9** (*:p< 0.05; **:p<o.01) sbi represents a simple and reproducible method to detect immunobiological alterations after trauma and elective surgery. patients undergoing elective surgery showed slight but significant diminuation of sbi 24h after operation. deterioration of sbi was observed in serum of traumatized patients directly withdrawn after admission at our hospital. ten days after trauma significant improvement of sbi was found. further investigations have to be done to proof sensitivity and prospectivity of this simple but reliable test. a sepsis like syndrome (sls) occurs in i0 % of our patients following cardiac surgery. sls is characterized by a severe decrease in systemic vascular resistance requiring the use of vasopressors to maintain adequate hemodynamics in the presence of negative blood cultures. in order to determine whether preoperative factors predict the occurrence sls we retrospectively studied 62 patients with sls (group i: age 61.1 ~ 10.8 years); they were compared to 52 control patients (group 2: age 58 ± 14.3 years). treatment of sls consisted of aggressive fluid replacement and norepinephrine infusion. rydrocortisone (200 mg/24 hrs) was given on the first day. antibiotics were switched prophylactic to cover gramnegative bacteria in 48 pts. preoperatively pts in group 1 revealed a significantly lower cardiac index (2.5 ± 1 i/min/m 2) compared to group 2 (3.1 ± 1.2 i/min/m2; p < .02) higher left atrial pressure (group i: 17.8 ~ 8.1 mmhg; group 2: 13.1 ~ 6.1 mmhg; p < .005) and lower ejection fraction (group i: 57.9 z 16.3 %; group 2 65.2 ± 10.9 %; p < .03). bypass time, minimum and mean blood pressure on bypass and aortic clamp time were not different between the groups. immediately postoperatively (pop), the white blood count was elevated (group i: 15,700 ± 8,200/ui; group 2: 10,200 ~ 4,400/ui; p < .0005) and core temperature 12 hours pop was higher (group i: 38.3 7.6 °c; group 2: 37.6 ~ 8.4 °c; p < .004). serum lactate was already elevated on arrival on the icu pop (group i: 5,0 z 1.9 mmol/l, group 2: 2.2 ~ 0.9 mmol/l) but dropped within 24 hours (p < .0005). icu stay (group i: 3.7 ! 4.3; group 2: 1.6 ± 1.9 days; p < 0.001) and mortality over 3 months (group i: 19.1 %; group 2: 3.1 %; p < .005) were significant higher in group i. sls is observed more frequently in patients with preoperative cardiac congestion and results in longer icu stay and higher mortality. although intraoperative hemodynamics are similar in both groups, the white blood count and serum lactate levels suggest an intraoperative cause for sls. further investigations will focus on possible intraoperative causes of sls. the objectives of the study were to estimate the problem of stress ulcers in critically ill patients and to compare ranitidine and omeprazole to determine their efficacy in the prophylaxis of stress ulcers. 33 patients who were admitted to the intensive care units between january and july '92 with polytrauma, sepsis or those requiring ventilatory support for more than 48 hours were selected for the study. the patients were randomized into 3 groups to receive ranitidine, omeprazole or a placebo. the changes in gastric ph were monitored 4 hourly and the patients underwent an upper gi endoscopy 48 hours after the start of the study to look for stress ulcers and hemorrhage. the drugs were withdrawn one week after the start of the study. of the 33 patients selected for the study, 14 had undergone cardiac surgery, 6 had polytrauma and 13 had sepsis. the change in average pre and post drug gastric ph was +1.1 in the omeprazole group, +0.5 in the ranitidine group and -).9 in the placebo group (p < 0.05). endoscopic evidence of stress ulcers was seen in 62% of patients not on prophylaxis, 18.1% of those on omeprazole and 54.5% of those on ranitidine. while all the patients in the placebo group who had stress ulcers showed endoscopic evidence of hemorrhage, 82% of the patients with ulcers in the ranitidine group and none of the patients with ulcers in the omeprazole group showed hemorrhage (p < 0.01). in critically ill patients the incidence of stress ulceration is high. an alkaline gastric ph is protective against stress ulcers. omeprazole when used for prophylaxis against stress ulcers in critically ill patients or those on short term ventilation will reduce the incidence of formation and hemorrhage from stress ulcers. the early and accurate diagnosis of sepsis is of key importance for critically ill patients survival. many studies have shown that tumor necrosis factor ~x (tnf ~) and interleukin-6 (il-6) are mediators of the inflammatory response in sepsis. bacterial antigens interact also with antigen specific t cell receptors and the activation of t ceils takes place. activated t cells release soluble interteuldn-2 receptors (sil-2r). the aim of this study was to evaluate: the significance oftnf c~, il-6 and sll-2r in the diagnosis of bacterial sepsis and to evaluate the correlation of clinical and laboratory parameters with serum levels of tnf ~x, il-6 and sll-2r. we examined 19 patients with clinical signs of sepsis treated in the intensive care units of university medical centre ljubljana. venous blood was drawn from each patient within 24 hours after the first clinical signs of sepsis. clinical data (body temperature, blood pressure), laboratory dam (peripheral white blood cell count with differential, platelet count, c-reactive protein), and quantitative blood cultures were recorded. serum levels of tnf c~, 11,-6 and sil-2r were measured in 19 septic patients and in 20 adult healthy controls. tnf ct, il-6 and sil-2r serum levels were measured by commercially available ria and elisa (amersharn and eurogenetics) kits. the differences in serttm levels of tnf ~, il-6 and sil-2r between healthy control and patients and correlation between clinical and laboratory parameters were calculated. we found that only sll-2r serum levels were significantly (p<0.005, student t test) increased in patients in comparison with healthy controls. of all indicators of sepsis that we compared, only hypotension was significantly correlated with tnf ~x levels and leukocytosis with ]1,-6 levels. we conclude that only the increased serum sil-2r levels were predictive of bacterial sepsis within 24 hours after the first septic signs. tnf c~ and il-6 levels were not significantly increased at that time in lifts small group of patients. in order to create high precision prognostic system for patients with sepsis and septic shock we have made prospective and retrospective analis of 200 cases of sepsis. patients were included in this analis according bone's criterions of sepsis. comparing with apache-h, we have excluded such phisiologic variables as:temperature,na+ ,k+ ,}it, ph,which had a little influence on prognos of illness. we have added the other, more important variables: biilirubin, plateles,pti. we took into account: the seat arrangement, methods of respiratory support, comorbid conditions as: caneer, diabets,obersity. in general new score has 5 headings: -physiologic variables, -age, -seat arrangement, -chronic comorbid conditions, -methods of respiratory support. roc -analis have showed a greater precision of our score, compared with apache-ii score. there is significant difference between the roc-curve of apache-ii scare. the k.p.tit~v.,i.e.gurmanchuk. objectives of the study. sepsis is a severe complication of the local infection, th e fever and the systemic inflammatory response syndrome may take place in such a case as manifistations of the inflammation induced by bacteria. the systemic fever observed in infection is known to develop due to the action of certain cytoldnes. other systemic manffistation of inflammation are the production of acute phase reactants, acute phase proteins, the activation of the complement system. ivratertals and methods. we observed 35 children with light and 45 children with severe course(l-3 class of serionuess) of the sepsis. the ftmetional activity of the complement system (ch50, level of c 1-c5, factors b told d, c3a), level of c-reactive protein (crp} and tumor necrosis factor (tnf) were investigated, results. the level of crp was increased in the children with more severe class of the septic process in both group (60%-1 st and ioo%-2 nd}( with light and hard eours of disese) of patients. parameters of the complement system -the activity of cl, c4, c3 components, activity of b and d factors-were increased in the seram of children with light course of sepsis. in children with severe course of sepsis these parameters were signffieantty decreased, especially in the group of chiidrellwith higer level of crp. we found the negative correlation of the tnp-alfa concentration with the functional activity of classical pathway components (cl~3) and the positive onewith the funfional activity of the alternative pathway factors b-and d of the complement system. conclusions. thus, in children with different course of sepsis certain changes in levels and functional activity of an acute phase o[ inflammation proteins -crp and the complement system ones -&ire characteristic. the relationship between the tnf-alfa level and the activity of classical and alternative pathways proteins indicates on its role in the complement proteins genes expression. schr6der j, braun j, rennekampff ha, schr6der dw various alterations of the immune response are known in trauma, but the course will not be complicated by multiple organ dysfunction syndrome (mods) in all patients. phenotyping of cells offers a rapid system of evaluation certain aspects of the immune response. different subsets of lymphocytes and neutrophils were determined to evaluate their prognostic role in developement of mods. in 16 trauma patients with an injury severity score (iss) > 20 (mean iss = 32; mean age 41 years) lymphocyte and neutrophil phenotypes cd 3 (t-cells), cd 4 (t-helper cells), cd 8 (t-suppressor cells), ratio cd 4/cd 8, cd 11b (receptor for cr 3) and cd 16 (fcriii) were measured on day 1,2,3,5,7 and 10 post trauma. the expression of class ii histocompatibility antigen (hladr) on monocytes (hladr+ cd 14) and il 2-receptors on t-helper cells (cd 25/cd 41 were determined as well. the percentage of cells was monitored by immunofluorescence using monoclonal antibodies and three color cytometry. the percentage of hladr+ cd 14 were significantly lower an day 2,5,7 and 10 in patients who developed mods (p<0,05) compared to patients without mods and a healthy control (p<o,o01). the expression of il 2receptors on cd 4 was significantly different only on day 2. no differences could be measured in lymphocyte phenotypes cd 3, 4, 8 and cd 4/cd 8-ratio as well as in expression of cd 11b and cd 16 on neutrophils. class ii histocompatibility antigen (hladr) on monocytes offers the best ability to reflect cellular immunosuppression in trauma. this parameter allows the best differentiation of patients with and without mods. we retrospectively studied the relative importance of age, prior chronic disease, sepsis and organ system failure (osf) to determine outcome in critically ill patients with acute renal failure (arf). arf was defined as serum creatinine > 280/zmol/i, a twofold creatinine rise in prior renal insufficiency or the need of acute renal replacement therapy. definitions for prior chronic disease and other osfs -i.e. cardiovascular (cf), pulmonary (pf), neurological (nf), haematological (hf), hepatic (lf), and gastrointestinal failure (gf)-were derived from the literature and described previously. of the 1025 consecutively admitted patients to a surgical and a medical intensive care unit during 1 -ye0r period, 136 (13%) had arf. arf mortality was 52%. ninety-eight percent had other osf. overall, cf, pf, gf, and nf was significantly more common in nonsurvivors than in survivors (all, p<o,02). although both the number of osf before and after arf was higher in nonsurvivors than in survivors (p<0.02), the number of osf before was higher compared to after arf in both groups of patients (p<0.001). furthermore, age, cf and pf before the ontset of arf, nf thereafter, and renal replacement therapy were related to mortality (all, p<0.03). however, prior chronic disease was not related to mortality and chronic renal insufficiency was inversely related to outcome (p<0.002). after multiple logistic regression, advancing age, cardiopulrnonary failure and sepsis before arf, and nf after arf remained significant. these results show the high prevalence of arf in critically ill patients and that arf rarely occurs alone. the prognosis of arf in critically ill patients is poor, especially if associated with advancing age, additional osf, particularly cardiopulmonary failure and sepsis before arf, and nf after arf. hence, prevention of cardiopulmonary and sepsis before the outset of arf may influence outcome in arf in patients with critical illness. the search cominnes for a magic bullet ta help critically ill petienta, bat recent elini ',.ml 1rials of agents that showed ixomise in animal studies raise questions about such trials. expcrlmemai uvidan~ :in anlmats and human volanteet~ for concepts, mechanisms and treatment of inju~ ur illness can be substentlal, l~rauasive and exciting but the positive effects of potential therapy suggested by such animal and ctinieal research may be difficult to prove in sick patients. efficacy of a new txeatment can be difficult to prove became elinie.al situations, hi spi~e of important biologic phenomena, are v~iable and conrplex. there is redundancy and overlap of mediators ~d problems of timing of therapy. a majt~r cause of this dilemtns is not with the trials or hypotheses, but rather the promem of the cause or the causability of the human dise~.se that is trebled. this is/hu "causa nets" or '*specific cause" as described by stehbens, when prevention or tre.atmcnt is based em the sauna nero, extinetiem of the disease is ix~ssible as with saul/ pox. only elimination of the cause wl12 cure the disease. careful animal studit~s evaluate single pcrturbmiuns for survival or other changes. an ldso preparation may be infiuancad aignificantly by a ~mall clangs which may be insignific~lt ht pstiants, most human experiments ate carried out in normal volunleers with a single porlurbation, such as a low-level, non-lethal dose of a substance. such studies are huportant in defining mechanisms attd mediators of disease, in our world of injm'ed, operated, infected and inflslned patients, there are many diseases, mof is not one of lhem. it isn't even a syndrume, it is the final pathway for a number of diseaaes~ each of which has a cause. mecormock believes thal a "multi-causal" etiology is a euphemism for ignoranc# or a synonym fo~ unknown etiology. admission iv sn icu, a high apache seer% the sepsis syndrome, mof, muds, or sirs and having predictors indicating potential mortality, are not diseases. the inmpors are getting negative results in trials of agents on eiek or septic icu patients and aru now becoming splitters, dafining subgaoups at higher risk for death. such an approach may produce positive results if the diseases are identified and the treatmeul is specific for them, a major challenge in deterraintng the efficacy of new therapies for sepsis nnd shock is identifying patients whose e#temtc inflammatory response is appropriate from those in whom the mediator| are contributing to end organ system damage end death, traditional and recently revised categorical patient descriptions such as sepsis syndrome, sepsis syndrome with shock, multiple organ system failure, or the recently proposed systemic inflammatory response syndrome (sirs) may not be sufilcient since they identify individuals at varying levels of risk for short term mortality, the efficacy of new lmmunotherapy may be directly related to the patient's severity end risk of mortality at entry to the study. we recently reported (jama 1993;270:1z33-1~41) a comprehensive risk assessment approach for patients with sepsis syndrome, a common eategarlcal description used as an entry eriterlon for many trials. by screening s database of 58,737 recent admissions to 107 hospitals in the united states and western europe, we identified 1,195 patients who met sepsis syndrome criteria but were not enrolled tn clinical trinh, we then developed a survival model end severlty assessment method to estimate their 2g.day mortality risk. k..ente physiologic abnormalities were the most important prognostic factors (82% of total chl square) influencing outcome. the specific disease resulting in ice admission and the days the patient spent in the hospital and icu before qualification were independent risks, as were age and a clinical history of cirrhosis. the predictive model was cross-validated within the original 1,195 patients with a receiver d_perator characteristics (roc) area of 0.76 and in two independent databases, the first independent database contained 295 hospitalized patients with gram-negative infection meeting criteria for sepsis syndrome (roc=0.80); in the second, the 302 placebo patients withtn the recently completed phase ill e~aluatlan of il-1ra (rocffi0.76). in all databases the risk model was able to accurately risk stratify patients throughout the range of risks that varled from a very low 1% to a very hlglt 99%. by drawing on the ready avatlabillty of large clinically aeuurate, current databases and using the power of modern statistical techniques to model the bodfs response to sepsis, we are able to produce very aeettrate pre-treatment risk estimates, these prospectively defined and continuous risk estimates reduce reliance on multiple subgroups and data dredging that can compromise a stady's integrity. they cart provide a nnlform method for patient description across clinical trials of different attempts at immmlotherapy. risk calculations can also be useful in developing entry criteria for phase ii evaluations in order to initially test efficacy on a limited number of high risk patients, fonowing sueeessinl completion of a definitive trial, the risk estimates can be used to develop specific |ndlcattons for s drug's use and can monitor the impact of both new and multiple compounds on patient outrome, when dealing with therapeutic trials for the treatment of sepsis, there are 2 ways of checking the comparability of the groups (placebo and treated groups) -to use several description items such as age, sex, preexisting disease, number and nature of organ failures, physiology score, such as saps il (1) -to use a model for risk of death either from a score (apache ii, lii, (2) saps ii) or directly (mpm ii system (3)) before using the model for risk of death in septic patients, is it mandatory to validate it (by calibration, i.e. goodness of fit, and discrin'dnation, i.e., roc curves) first on a data base using the same definition for sepsis as in the trial and secondly on the placebo group of the trial ? or is it better to use a specific model for each trial ? the general models usually do uot fit for septic patients. there are 2 methods to make them fit : either to add variables to the original score (model for sepsis using the apache iii system (4)) or to customize the model. for saps ii the customization is applied to the equation regression, using the same saps ii score. for mpm 1i each component of the model can be customized. several remarks : only one model is published to be applied at the icu entry ( mpm ii 0) before any therapy. 2 -most of the published scores are calculated from the lsl icu day. applying these scores to the let day of sepsis could have a very different significance. 3 -is the accp classification of sepsis useful at the bedside to select oatients ? ( the pathophysiology of sepsis involves a dynamic interaction between the host and the infecting microorganism(s). a multitude of biochemical and hemodynamic interactions occur which function in concert to determine the ultimate outcome of the septic episode. the complexities of the septic process preclude outcome analysis by in vitro systems or computer models, animal studies have become indispensable in evaluating new therapeutic agents in the treatment of sepsis. these studies provide valuable information on safety, pharmacokinetics, relative efficacy and dosing strategies for potential therapeutic agents in the treatment of sepsis. there are three basic types of animal models: (1) toxicity models with injection of bacterial endotoxin or exotoxins; (2) live or killed bacterial challenge models; (3) actual infection models. all models employ some form of external manipulation under controlled conditions to induce sepsis and to analyze potential therapeutic efficacy. these experimental requirements may limit the ability of animal models to accurately predict the clinical value of new agents in human sepsis. four major end points are analyzed in the various animal models: (1) hemodynamic parameters; (2) modulation of host-derived inflammatory mediators; (3) resolution of end organ injury; or (4) lethality. each animal model has certain advantages and limitations. species-specific differences in physiologic and immunologic features make it difficult to directly extrapolate the results of animal experiments into clinical medicine. while no ideal animal model exists, consistent benefit of a new immunotherapeutic agent in multiple animal systems provides the most compelling preclinical evidence of its therapeutic potential in septic patients. consensus-assisted development of a study protocol on sepsis: an important difference from previous randomized trials there have been several, and perhaps too many metaanalyses of cliuical trials on surgical infections, but their results have only rarely been incorporated into the design of new randomized trials in sepsis. metaanalysis is retrospective. it seems more important to analyse and to criticize the design of tria/s before they are ongoing. incorporation of that into development of a study protocol is the aim of the following procedure: the time-schedule of about two years should include further cell culture and animal experiments after the first successful studies showing effectiveness. exhaustive evidence for a dose-dependent cost-banefit and near complete explanations of the pathomechanism should not be awaited for development of the protocol of the clinical study. however, a discussion forum of experts should be performed in a recently published, almost perfect trial in order to see the frontiers of the present research in cell and molecular biology, clinical pharmacology, statistics and decision making. this should be followed by a metaanalysis of at least 10 study designs on sepsis with methods of formalized medical decision making (decision tree). clinical algorithms -developed by objective methods including nominal group processes -should be developed to standardize any concomitant treatment in the centers considered for a multinentre trial. the latter is usually necessary in the field of sepsis. finally, a pilot study should be performed to demonstrate not only feasability, but to learn about all possible types of interventions which modify endpoints as response variables. especially mortality is not free from such effects. the time for the individual phases of this consensus-assisted process of protocol development have to be shortened by their temporal hiterloeldug. due to the fast discovery of new chemical factors in sirs it wilt otherwise be impossible to come up with results of randomized trials which are fascinating enough to be incorporated in daily routine treatment. severity scoring systems are intended to provide a population-based estimate for the risk of an unwanted outcome resulting from some disease process. in the area of infections and the "septic" response, this has routinely been defined as death. for anti-infective trials, there has been a reasonable correlation of severity (apache ll) with outcome measured as persistent or recurrent infection, but this is not as robust as the association with mortality. it is expected that non-lethal failure would not immediately correlate with disturbed physiology: the basis for antiinfective failure depends on a variety of factors not measured in severity scoring, such as type of infecting organisms, density and susceptibility to administered antimicrobia[s, and anatomic features of infection (e.g., pancreatic). severity scoring is important in differentiating these "categorical" variables from continuous (physiologic) variables. there are, additionally, a series of problems related to the cause of death in relation to severity scoring. in those tdals in which cause of death is analyzed, high severity scores did not routinely predict death as an immediate sequelae of sepsis/septic shock. many of the predicted deaths occurred remotely of progressive background diseases. other important problems with severity scoring have to do with lead-time bias: hew long patients were ill before entry into the study. statistical techniques for condensing various categorical and continuous variables, as well as factoring in information requiring the pharmacodynamics of agents which affect outcome, are being developed to further refine deviation from predictive equations as an outcome measure. stepwise logistic regression analysis has identified the presence of shock, intm-abdominal or unknown source of infection, hospital acquired infection, age greater than 70 years, neutropenia and inappropriate antimicmbial therapy as independent variables associated with increased mortality in sepsis. maldistribution or" these variables is a concern in targe randomized clinical studies. it should be noted that only the selection of antimicrobiai therapy could be addre.~'sed and altered at the time of enrollment in a sepsis clinics] trial. inappropriate antimicrobial therapy has been noted in 41%-66% of septic patients. well designed, randomized sepsis trials do not assure the absence of imbalance between treatment and control groups with regard to inappropriate antibiotic therapy, especially with subgroup analysis. statistical manipulation to correct for any imbalance is appropriate but avoiding imbalance is ideal. selection of antibiotics in septic patients requires such considerations as clinical setting (history, physical examination and underlying disease), appropriate laboratory tests (such as gram stain), identification of source and associated bacterial flora, antibiotic susceptibility patterns for that hospital and likelihood of resistant organisms. determination of inappropriate antibiotic therapy is made retrospectively after culture results are available. in some circunmtances the organism and antibiotic sensitivities cannot be predicted, while in others it can. leibovici et al identified hospital acquired infection, antibiotic treatment before a bacteremic episode, endotracheal intubetion and thermal trauma as independent variables which predicted isolation of a multiresistant organism. the same authors developed a predictive index for resistant organisms, which when compared to prescriptions of attending physicians, improved antibiotic selection in critically ill patients. including a standard antibiotic regimen for all septic patients in a clinical trial is impractical. there is too much interhopsital variability in flora and antibiotic susceptibility. in addition, the aggressive broad spectrum coverage necessary for some patients is inappropriate for others. a protocol which guides a prescribing physician through a logical chain of reasoning might improve antibiotic selection in critically ill patients and could be included in the design of a clinical trial in sepsis. although this approach might minimize potential for maldistribution of inappropriate antimierobial therapy, it would likely create multiple problem areas. will the patient's physicians agree to the protocol choice? what is the availability of speciftc antibiotics at a participating hospital? what is the aplpiieability of study results in typical clinical situations? if there is agreement that thin is an appropriate antibiotic protocol, should it not be used in all seriously ill septic patients in the hospital? in other conditions such as ards, the success of protocol therapy is dependent upon measurable goals such as pao 2 and highest plateau pressure, while measurable goals are not as clear in choosing antibiotics. based on the above confounding issues, a complex and extensive algorithm covering many potential possibilities of error and specific antibiotics seems impractical; however, an algorithm focusing on three to four major common errors in antibiotic selection and dealing with classes of antibiotics is plausible for inclusion in sepsis clinical trials. in this session we will attempt to outline the methodology of such a future study, emphasizing the immense difficulties involved in its proper conduction including: design, selection of patients, surgical considerations, supportive treatment, the "human factor" and ~nd points. only a close cooperation between a few dedicated surgeons, obsessively studying a large number of well selected and stratified patients over years, could make such a study possible. algorithmis have frequently been mistaken for the graphic format in which they are presented. however, an algorithm is neither a flow chart nor a list of instructions; it is a step-by-step approach to solving a problem. likewise, a clinical algorithm is a step-by-step approach to solving a clinical problem. cas are deterministic, which does not mean that they are rigid. they are practical rather than mathematical algorithms, that tan be used only with clinical judgment, and must be tailored to each patient. there are a number of types of, or uses for, cas that can improve sepsis management, including: diagnostic or severity scorm cas, a management ca for managing sepsis constructed according to recently published standards for use in teaching research protocol algorithms that must be used to collect data or guide implementation of a clinical trial aimed at validating one or more dicisions in the management ca; finally, protocol-style cas drawn from the management ca and should be validated using matching outcome studies. organisational problems peculiar to multicentre trials the organisation of any randomised clinical trial is fraught with difficulties, and that of a multicentre trial particularly so. there are problems associated not only with the principles on which the investigation is based but also with the detailed organisation and analysis. are all the participants truly unbiased about the relative merits of the regimens being studied? is like being compared with like -are all the end points and the standards for measuring them clearly defined so that they cannot be misunderstood? are all eligible patients being studied, and their results reported? is truly informed consent being sought that will satisfy not only the patient's right to choose what happens to him, but also the law of the land? and, finally, how can the participants be sure that the trial will be stopped at the right time, to ensure that no treatment is given to any patient that might be harmful? these questions may seem insoluble, but until we tackle them we shall never be certain. dr.m. ev~s, scar~mu~ hospital, scarborough, nonhyo~s~ y0126ql, u.k. increasing knowledge of the pathogenesis of sepsis and septic shock has led to the development of many new therapies and technologies capable of preventing, blocking or even reversing the deleterious cycle of events. recent results of subsequent multicenter trials testing some novel therapeutic drugs, however, did not confirm the promising sometimes overwhelming effects obtained experimentally. it is postulated that this is largely due to inappropriate design and conduct of multicenter trials as currently performed. one of the shortcomings emphasized in this paper is the "treatment mix" problem. multicenter trials in sepsis are performed because no single center is able to recruit the necessary number of eligible subjects within a reasonable time. each single center (icu) has its own individual policy which cannot be standardized for the trial (treatment mix). even if we ascribe that each icu did the "best" for their patients, it is highly probable that the outcome differs between the icus. this translates to the effect of the new therapy under investigation. ]t is therefore worthwhile to consider that icus must first demonstrate a certain standard based on the patients' outcome (audit) before becoming accepted as a participating center. this prerequisite ensures comparable quality between participating centers, led to the reduction of "noise level" and increases the probability of positive study results. the riyadh-icu program based on standard mortality ratios is recommended as an audit instrument. during the last decade most clinical trials of potential therapeutic agents in systemic sepsis have failed to demonstrate benefit from the drug under study. although in many instances it is entirely possible that the agent in question does not have clinical efficacy, an equally plausible explanation for the multitude of negative results is that the studies were improperly designed and thus could not show therapeutic benefit, if it existed. problems which have been identified in these various trials include inappropriate or unrealistic definitions of response to the drug; inadequate sample size, with attendant insufficient statistical power to demonstrate a difference, if it existed; insufficient standardization of customary therapeutic maneuvers in septic patients; imprecise criteria for patient entry into the study, which creates unacceptable inhomogeneity between control and treatment groups and invites unjustified post-study subgroup analysis; the lack of a formal sequential monitoring procedure for interim data analysis, which could potentially affect patient safety; and a primary analysis of study results that excludes protocol deviants and is not according to intent-to-treat. these and other problems have impacted upon the validity of the various trials conducted to date and may well have prevented the resolution of controversies surrounding the use of certain agents in the treatment of septic patients. the complexity of bacterially-induced septic shock involves a cascade of events that evolve over time, beginning with the proliferation of offending organisms and followed by the release of toxic mediators from the bacteria. endotoxins [e.g. lipopolysaccharides) from gram-negative bacteria initiate septic shock by precipitating a systemic inflammatory response syndrome (sirs) through release of a broad spectrum of hostderived mediators. over the last century, hundreds of these endogenous mediators have been proposed to serve as pathophysiological substances in this "alphabet soup" of cytokines, eieosanoids, biogenic amines, kinins, peptides, ions and hormones. an evaluation of the literature on septic shock leaves the investigator with a sense that, although many pieces of the septic shock puzzle have been presented, no clue was provided to indicate how these pieces fit together, in an attempt to assess objectively the causal role of individual mediators, we recently completed a collective meta-analysis of 24 mediators that were individually reviewed and subjected to koch-dale analysis of causality by distinguished authorities in the field (1). in summary, our attempt analyze available evidence to support a cause-and-effect relationship for putative mediators of septic shock revealed that only eight of the 24 mediators analyzed could be strongly inferred as playing a causal role in septic shock. evidence supported the involvement of endotoxln, endorphins, complement, interleukins, tumor necrosis factor, eieosanoids, platelet activating factor and calcium. other mediators either lacked adequate supportive evidence or were not conclusively involved. posttraumatic outcome may be reduced by an amplified stress response mediated by neural and humoral stimuli. the classical hormonal catabolic response is predominantly mediated directly or indirectly by neural stimuli, while most changes in inflammatory responses such as leukocytosis, acute phase proteins and changes immunofunction are mediated by humoral mediators. clinical experience from controlled studies suggest afferent neural blockade and modification of stress response to improve organ function and postoperative outcome, and limited experimental studies also suggest neural blockade to be of beneficial value in shock states. of special value may be the improved gastro-intestinal motility after neural (visceral) blockade. no outcome studies are available from patients with major truma, multiple organ failure or sepsis, conditions where humorai mediators may be more important to modify than neurally mediated responses. future studies should investigate the clinical outcome effect of combined neural and humoral mediated blockade, as well as the potential role of an initial neural blockade in elective trauma (first hit) to improve the metabolic scene and outcome if a postoperative complication (second hit) should occur. neutrophil elastase is the predominant protease of the human neutrophil. this enzyme, and a variety of other enzymes, including neutrophil collagenase, are released by activated neutrophils in the setting of high concentrations of reactive oxygen metabolites. in addition, in this extracellular environment, the normal antiprotease defense mechanisms of the organism may have been severely inactivated oxidatively especially along capillary endothelial cells. accordingly, release of neutrophil elastase which mediates inflammation and degrades most extracellular matrix proteins, including elastin, fibronectin and many forms of collagen, may contribute to tissue injury and organ failure in a variety of diseases ranging in severity and acuity from pulmonary emphysema to the adult respiratory distress syndrome (ards). as a result, significant efforts by the pharmaceutical industry have been directed not only toward the development of human neutrophil elastase inhibitors, but also toward their characterization in a variety of animal models of neutrophil mediated diseases, and their evaluation as potential therapeutics for the treatment of a variety of human clinical conditions. data from animal models of organ injury and failure along with data collected from a series of patients at risk for ards and with ards will be presented and discussed as a rationale for inhibition of neutrophil elastase. parameters that need to be monitored to obtain success in future clinical studies will also be presented in this context. have turned out to initiate and maintain organ dysfunctions in severe posttraumatic and postoperative courses. such proteolysis-induced disorders are especially rendered possible by the concurrently arising consumption of inhibitory regulators (e.g. a vproteinase inhibitor=a 1pi, antithrombin iii=at iii) via cofnplex formation and/of proteolytic/oxidative inactivation. besides the well-known destructive potency against protein substrates, proteinases such as elastase or thrombin (active or in complex with the serpin inhibitors ~ 1pi or at iii) are also supposed to increase the inflammatory reaction by inducing cytokine release or shedding of soiuble adhesion molecules from various inflammatory cells (pmns, monocytes/macrophages, endothelial cells). those cell-derived mediators may provoke further cell activation through an autocrine/paracrine loop thus increasing significantly the proteinase burden at the inflammatory focus. therefore, this noxious circle might be interrupted by a convenient proteinase inhibitor therapy to ameliorate the inflammatory process. to verify this hypothesis, high dosis of at iii (mean plasma levels up to 140 %) were applied in first controlled randomized studies on surgical patients suffering from multiple trauma or severe sepsis. in control patients we could clearly demonstrate the sequential appearance of proteinase/serpin-complexes (fa 1pi, tat), cytokines (il-8, il-6), and soluble intercellular adh6sion molecules (icam, elan, p-selectin) in the circulation. these factors turned out to be a helpful tool for early diagnosis and prognosis of forthcoming organ dysfunctions. interestingly, m at iii-treated patients a significantly reduced release/production of inflammatory mediators became obvious concomitant with the improved clinical course in comparision to an increasing deterioration in control patients. soluble mediators of inflammation are cytokines (e.g. il-1, il-6 and tnfe) as well as breakdown products of complement proteins (e.g. c5a and terminal complement complex). therefore, attenuation of both, release and generation of these synergistically functioning mediators together with stimulation of release of antagonists including soluble forms of receptors are potentially beneficial in all kind of inflammatory diseases. intravenous immunoglobulins (ivigs) are known to have an anti-inflammatory effect in humans (nih consensus conference on wig, 1990) . the mechanism of action becomes more and more clear. in single cells stimulated by anti-cd3 mab wig was able to down-regulate production of il-2, il-10, ifn% and tnfj~ significantly. igg coated solid-phase stimulates release from monocytes of interleukin-1 receptor antagonist (il-lra) but not of il-1; this observation is very much in contrast to the effect of endotoxin in the same in vitro system. furthermore, naturally occurring anti-cytokine antibodies can be demonstrated in apparently healthy individuals and in wig prepared from plasma pools. some of these antibodies can be detected by antigen/antibody reactions in fluid phase (anti-ll-le, anti-il-6), some only by solid-phase detection systems (anti-ll-8, anti-ifn% anti-tnfc 0. infusion of wig to patients suffering from inflammatory disorders have resulted in a decrease in il-6 in circulation. during infusion wig might induce an acute but transient rise of tnfo~, il-6, and il-8. self limitiation of this process might be due to demonstrable instant release of il-1 ra and soluble tnfo~ receptors. in vitro ivig has also been shown to attenuate complement activation via the classical pathway. furthermore, acid haemolysis of erythrocytes in paroxysmal nocturnal haemoglobinuria could partially be prevented by ivig. ivig was able to attenuate forssman shock in guinea pigs. we have seen a patient with congenitally elevated turnover of alternative pathway of complement who repeatedly showed an increase of haemolytic titres after administration of ivig. thus, wigs apparently have multiple potencies including attenuation of soluble mediators of inflammation and might therefore be of benefit in trauma, shock, and sepsis. significance of sinusoidal liuing cells and of humoral factors on hepatic function following sepsis and major surgery hirata k, katsuramaki t, yamaguchi h, mukaiya m, yamashiro k. regeneration of the liver after hepatic necrosis or partial removal has been extensively studied, but uncontrolled mechanisms to irreversible hepatic failure following sepsis or major surgery of liver or with hepatic dysfunction have so far not been well documented. we suggested the importance of the intrasinusoidal aggregation between sinusoidal lining cells and intrasinusoidal running ceils in the mechanisms of hepatocyte dysfunction. thus, the purposes of this study was to investigate the changes in each sinusoidal lining cell during this process and the effect of cytokine on hepatocyte under various oxygenation. [ materials and methods ] ( 1 ) clinical study : thirty two patients with histologically proved malignant tumor underwent major hepatectomy were devided in two groups, i_e. the cirrhotic liver group and the non-cirrhotic [aver group. most of the former were the patients with hepatocellular carcinoma and most of the latter were the patients with metastatic carcinoma of colorectal cancer. five patients were complicated septic conditions with hepatic failure. following operation in each patient, a verous blood sample was taken for measurement of serum il-1, il-6, tnf and c-reactive protein concentration. and hepatic biopsies were sometimes undergone in patients with anomalous hepatic function. (2) experimental study : kupffer ceils, sinsoidal endothelial cells and hepatocytes were separated from mouse ( c3hhen ) liver. and also polymorphonuclear cells and platelets were purified from blood and peritoneal macrophages were from peritoneal cavity of mouse. co-cultures between or among these cells with iipopolysaccharide were performed. cytokine concentrations in media and hepatocytic function were compared. [ results and conclusion ] our findings in above experiments demonstrated the cleanly different concept for the mechanism of hepatocyte dysfunction following sepsis or major surgery. namely, the effect of hepatoeytie function by cytokines or superioxide were significantly affected under low oxygenation, but not significant under good oxygenation. hirata m.d.,nishi 17,hokkaido,japan $120 470 immune complexes as .mediators of sepsis and immune dyafumcffon laa k horn, chxistof birkenmaier, and greg h2mon departmen~ of surgery, san frandsco general hospital, urdversr 3, of calif(~rrda, san frandsco. immune complexes (ic) a;:e commonly found circulating in parians during infection and sepsis; and following traumm, bums, and massive l~ensfusion. clearance of 1c occurs by phago~'tmsis of esll-bou_nd or opsonized p~tlcles. monocyte activation du~jng phmgocytos/s could lead ~o release of cytokilies ieletexiot~ to the hosh to examine tb/s phenomenon, we formed insoluble ic by precipitating iow-endotoxin bovine serun~ albumin (bsa) with rabbit ant/-bsa igg. we have ~town that these ic are ingested by monocytes and concomitantly stimv/ate re~pizatory bu~t activity measuxed by assay of in%racellul~ peroxldaffon. we have shown that ic ingesffon produces signlqcant el,era,lore in the monoey~e phenotype. spedacally, cdi4 is down-regu/ated, along wl~ cd 32 and cd64. thus responsiveness to lipopolysacchmdde (128) a~xd i=m~unoglobulin fe st~mu/mtlon is reduced. in cert,+rest, the complement rote.p tot cdc42 is u~-regulamd, indicaling mcremsed re~ponalx=~ness to opsonized pat,rotes, we examined monocyte superna~n~s for production of hzmor necrosis factor alpha (tnfg) following ic stimulation and showed that ic are capable of provoking ~elaase of hrmaunl)reac~ve tni~. j[c also increase mono=yte produchon of prnstaglandin e2. in summary, ~/~ese results demonstrate lhmt ic are capable of indudng production of ¢ytokines and the imrau-nosuppressive prostaglandin pge2. ic also allez ~he surface expression of important receptors involved in actlvaffon by lp~ and phagocyiosis. thus, ic aze competent for indud/on of the mepl/e s!0otlse. by examining m0n0cyte phenotypms, it may be possible to d~mrmkne extent ohn'u~u~e complex activation and predict immune depression iu criffcally ill pa~ents. a great deal of experimental work has focused on preventing and/or treating sepsis and organ failure following injury. many of these strategies have tried to attack mediator substances released during the systemic inflammatory response following injury. sugermann has demonstrated the improvement of pulmonary function, but not eappillary leak using [buprofen in the porcine model. in a clinical trial, faist demonstrated the effectiveness of indomethacine in amelierating the post trauma of cellular amenity using in vitro parameters. morbidity and mortality, however, were not different between control and treatment groups. baue und chaudry have suggested that atp magnesium chloride may have protective effect in renal failure um kupfer cell dysfunction seen after shock. pentoxiphyllin has been found to be effective in improving tissue oxygenation and oxygen consumption following hemorrhage, and there may be promise for this drug in the ischemia reperfusion injury. monoclonal antibodies against endothxin and inflammatory mediators seemed promising at first glance; however, preliminiary clinical data of the treatment of sepsis with either ha-ia or e5 monoelonoal antibodies against bacterial cell wall have been disappointing. initial studies with il-i receptor antagonist also appeared encouraging, but no difference was seen in the most recent trial. currently studies are ongoing with monoclonal antibodies to tumor necrosis factor which may have more efficacy given the fact that it is a macrophage mediator released by endotoxin. although we seem closer to understanding and preventing the problems of sepsis and organg failure after trauma, we must continue to appreciate the complex interrelationships and delicate balances inherent in the host defense system. overzealous attempts at restoring immunity in the absence of understanding pathophysiology may be just as dangerous at post-traumatic immlmospremsion. severe acute pancreatitis was caused by different etiologic factors, but once pancreatic tissues were infected, patients show a similar pattern of aggravation and develop organ failure. remarkable elevation of serum il-6 was unexceptionally observed in patients with severe acute pancreatitis. the serum levels were more than 500 pg/ml (normal: less than 4 pg/ml) during the first one or two days after onset. there was a significant correlation between the peak serum il-6 level and the number of organs showing failure (r=0.883). tnf-a production by isolated peritoneal macrophages following lipopolysaccharide (lps) stimulation was significantly increased in cerulein-induced pancreatitis rats. serum tnf-a activity was elevated following intraperitoneal administration of lps as the septic challenge both in pancreatitis rats and in control rats, being significantly higher in the former (p<0.05). histological findings and liver function tests revealed that lps induced more severe liver damage in pancreatitis rats than in control rats. these results indicate that increased tnf-a production by peritoneal macrophages in acute pancreatitis augmented lps-induced liver injury. organ failure in severe acute pancreatitis could be caused, at least in part, by increased cytokine production. it is clear now, that in mods, organ injury is not directly due to exogenous factors, such as bacteria or toxins, but instead is largely a consequence of the host's own endogenously produced mediators. there is an extensive body of literature on the inhibition of mediator production, release and action in different cascade systems by glucocorticoids. they can serve as a host protection against overactive defense reactions if used adequately. a new understanding of the regulation of cytokine synthesis, especially tnf, may lead to an insight of the conflicting findings between the benefits of glucocorticoid therapy in animals and its current failure in recent clinical trials. glucocorticoid shares its role with other novel therapeutic approaches also shown to be successful only in experimental settings. a mete-analysis of steroids in sepsis, however, revealed a beneficial effect in the subgroup of patients with gram-negative infections. there is now a series of arguments to reevaluate the indication for steroid thera-py and/or prophylaxis in mods and sepsis. honjo i-1-1, kumamoto 860, japan in septic shock j. briegel, m. halter, h. forst, w. kellermaun, m. bittl, k. peter imrodnetlea and methods: hydrocortisone (hc) at an infusion rate similar to the maximal secretory rate of cortisol in man improves hemodynamics in human septic shock (1). we hypothesized that the hc-induced hypercortisolemia prevents a harmful overshoot of immune reactions. to test this hypothesis we prospectively investigated 12 patients admitted to the icu in refractory septic shock. after resuscitation (mechanical ventilation, fluid resuscitation, titration of vasopressors, and initiation of antimicrobial therapy) hc was started with a loading dose of 100 mg/ 30 rain, followed by a continuous infusion (10 mggh). with hemodynamic improvement (dopamine < 2 gg/kg/min) the infusion rate nfhc was tapered over a period of 3 days. phospbolipase a 2 (pla2), c-reactive protein (crp), and elastase-proteinase inhibitor complex (e-pi) were deierminated daily. results: according to our previous results hemodynamics improved during hc infusien. after 4 days dopamine requirements decreased to less than 2 gg&g/min in all patients. this corresponded to an attenuation of the systemic inflammatory response syndrome (sirs) indicated by the abrogation of fever and the decrease in heart rate and inflammatory mediators. pla 2 activity and e-pi concentrations decreased to near normal values and crp concentrations decreased as well. after cessation of hc infusion (between day 5 and 7) a reinforcement nfthe sirs was observed despite cortisol levels within the normal range. this was first indicated by e-pi, followed by fever, increases in heart rate, crp, pla2, and finally by the relapse of septic shock in four patients on days 7,8 9, and 1o. a second infusion of hc again resulted in shock reversal and in a decrease in pla2, crp, and e-pi in the patients under study. discussion: there is growing evidence that the endogenous steroid hc and proinflammatory cytokines integrate a complex immunoregulatory feec~ack circuit. the elaboration of tnf, il-16, il-6, and il-8 is attenuated by-100 rag hc prior to endotoxin challenge in humans (2, 3) . cytokines like tnf, il-16, il-6, and il-8 are potent proinflammatory signals for acute reactants (--, clip), neutrophil degranulation (--, e-pi), and pla 2 synthesis and secretion. our data provide evidence that hc at an infusion rate similar to the maximal secretory rate of cortisol in man attenuates the systemic inflammatory response in human septic shock. to evaluate the feasibility of a nigh-dose regimen of antithrombin iii (at iii) treatment in patients with multiple injuries regarding blood levels of antithrombin iii and undesired side effcts and to analyse effects of the initibitortherapy on the systemic inflammatory response. patients and methods: 20 patients with an iss nf more than 28 points who could be treated with at iii administration within 180 rain after trauma were randomized to receive either at iii or placebo. at iii was given to reach an antithrombin iii inhibitory capacity in plasma of 140% by using the following calculation: at iii to administer = (140% -at iii measured) x body weight. at iii was given every six hours for a period nf48 hours and on the 4th day. patients were followed up throughout their stay in the icu but at least 14 days. in this abstract the data of the first 10 patients are included. results: the patients' median injury severity score was 41 points with a median age of 42 years. the patients were equally distributed between verum and placebo groups with respact to age , iss, prehospital treatment, blood pressure, hemoglobin and at nmevel on admission, and time from the accident until the test solution was given. the patients of the at iii group received 18,100 units of the inhibitor on average during the first 4 days. with our regimen, the at iii levels could be kept between 125% and 140% of normal, whereas control patients an at hi concentration of 40% to 60% was observed. patients with at ni treatment required less red blood cells bur received the same amount of ffp and fluids. there were no differences with respect to platelet count, partial thromboplastin time, l~rothrombin time and prothrombim. we did not observe bleeding disorders or any other side effect with peak concetrations of up to 206% of normal. white blood count, pmn--elastase, interleukin 6 and 8 and c-reactive protein tended to be lower in the at iii group. there were no differences with respect to renal and hepatic function parameters but the duration od mechanical ventilation was shorter in the at ii1 group (8.5 vs. 15.8 days) conclusions: we conclude that our treatment protocol (a) was able to restore at iil levels to the desired range of 140%, (b) did not lead to coagulation disorders, (c) did not increase transfusion requirements, (d) did not reveal other undesired side effects, and (e) showed a tendency towards reduced posttranmatic inflammation and mechanical ventilation requirements department of trauma surgery, 45122 essen, germany antithrombin-lll (at-ill) acts as an inhibitor of thrombin, further proteases and the alternative pathway of the complement system. inhibiting thrombin, at-ill works as a functional antagonist of paf~ therefore, in state of hypevolemic-traumatic shock, continuous supranormal serum -and tissue -concentrations of at-ill may be efficient to reduce the local posttraumatic reactions resulting from complement and pmn-activation. 20 patients with severe multiple trauma (7 treatment [at-ill] --13 controls [c]) were investigated. study cdteda were age > 16 and < 65 years, injury severity (iss) > 30 points and glasgow-coma-scale > 8 points; randomization and treatment has to be started within 6 hours after trauma. permission for the clinical study was given by the local ethic committee. bradykinin (bk) and related kinins are potent inflammatory peptides which possess the ability to induce, vasodilation, increased vascular permeability and hyperalgesia. cp-0127, a novel homodimer bk antagonist has previously been shown to increase survival in rat and rabbit models of lethal endotoxin shock and is now in clinical trials for sepsis. we have now evaluated the effect of cp-0127 in other models of inflammation. male rats were precannulated with a catheter in the carotid artery. 24h later bk was injected ia and the pain score ranked from 0 (no responses) to 5 (vocalization). cp-0127 at 3.6 umoles/kg completely inhibited the pain responses for a period of 2.5 -3h. cp-0127 at 3.6 umoles/kg s.c. was also found to inhibit the increase in paw volume and hyperalgesia induced in rats over a 3-4h period by an intraplantar injection of 0.1% carrageenan. the abdominal constriction response 1o an intraperitoneal injection of kaolin was inhibited in a dose-dependent manner by cp-0127. when 50 ul of 0.5% formalin was injected into the paw of a mouse a characteristic licking response was observed which was biphasic in nature. cp-0127 significantly inhibited both the first (0-5min) and second (15-30 min) phase responses. ]n a rat burn model, where the hind paw is immersed in water at 55°c for 15 sec the increase in paw volume was significantly reduced by pretreatment with cp-0127, 3.6 umoles/kg s.c. finally cerebrai edema was induced in rats by applying cold (-78°c for 10 sec) to the dural surface following a craniectomy. cp-0127 at 3.6 umoles/kg s.c. produced a significant reduction in the amount of edema compared with sham controls 24 h later. these data suggest that bk is an important mediator of inflammation and hyperalgesia and that the bradykinin antagonist, cp-0127, may be useful in the treatment of such inflammatory, hyperalgesic disorders. partial hepatectomy in humans is associated with a considerable morbidity due to hemodynamic and metabolic derangements, which increase the risk for organ failure and mortality. we hypothesized that endotoxemia may play a pivotal role in these complications. we therefore, investigated whether peri-operative infusion of rbpi23, a recombinant protein of the human neutrophil bpi with bactericidal and endotoxin-binding capacity, could prevent postoperative derangements following partial hepatectomy. male wistar rats (230-250 g.) received a 70% liver resection (phx) or a sham operation (sh), and a continuous intravenous infusion of either 0.2 mg/kg/hr rbpi23 (phx-bpi, n=8; sh-bpi, n=7) or the (iso-electric, iso-kd) control protein thaumatin (phx-con, n=8; sh-con, n-8). various parameters were measured 4 h after the resection or sham operation. mean arterial pressure, cardiac output and heart rate were significantly decreased in phx-con rats compared with sh rats, which effects were not observed in phx rats treated with rbpi23. blood ph was significantly decreased in the phx-eon group, whereas the leucocyte count, hematocrite and il-6 levels were significantly increased compared to sham levels. in the phx-bpi group, these parameters were restored to near sham levels. in vitro experiments with rat plasma and human mononuclear cells (mncs) revealed that plasma of phx-con rats is highly capable of activating mncs, accompanied by the release of cytokines. this activation is attenuated with phx-bpi plasma. in vitro added acd14 or polymyxin b was able to reduce the activation by phx-con rat plasma to the levels of phx-bpi rats thus, these data suggest that systemic endctoxemia, possibly of gut origin, is a major cause of postoperative hemodynamic and metabolic derangements following phx and that rbpizz can prevent these changes. more recently we reported a transient appearance of both endotoxin and tnf in the circulation of rats subjected to the haemorrhagic shock (hs) already at 90 -120 rain. similar to bpi, recombinant bpi was found to bind lps and inhibit tnf formation in vitro. the aim of this study was to investigate the effects of rbpi21 (kindly provided by xoma corporation, berkeley, ca) against haemorrhage related endotoxemia and mortality in rats. method: a prolonged hs was induced by blood withdrawal to a mean arterial pressure of 30-35 mmhg for 180 rain followed by reinfusion of shed blood (sb) and resuscitation with two times of sb volume of ringer's lactate over 50 rain. rbplg. 1 was administered at a total dose of 5 mg/kg i.v. (2.0 mg/kg at the16-eginning followed by two doses of 1.5 mg/kg each at end of shock and the end of resuscitation). the control group was treated similar to the bpi group but received thaumatin as a protein control preparation at the same dose as rbpi21. results: imrffe?diately after resuscitation (230 min) the detected plasma endotoxin levels in the control group (mean = 61, range = 0-120 pg/ml) were almost neutralized by rbpi21 treatment (mean = 13, range = 0-53 pg/ml) . plasma tnf levyis were not significantly influenced by rbpi treatment at the two time points 230 and 320 min of experiment (means: 65 and 51 in bpi vs 49, 65 pg/ml in the control group). the 48-hour survival rate was improved from 6/16 (37.5 %) in the control to 11/16 (69 %). conclusion: these data suggest that haemorrhagic shock may lead to bacterial translocation and/or transient endotoxemia with concomitant cytokine formation that may play an important role in the pathogenesis after shock and trauma, rbpi21 might be a useful therapeutic agent against endogenous bacterfal/endotoxin related disorders in hemorrhagic shock. morbidity and mortality after hypoxia of the vital organs had been correlated to the production of oxygen radicle which is mediated by xanthine oxidase activity, in this study we have evaluated the survival rate after allopurinol. 42 rabbits weighed 900 + 200 grams divided into two groups. group i included 22 tabbits were treated with allopurinol 80 mg/kg for seven days before induction of haemorrhage. group ii as a control included 20 rabbits. all rabbits were subjected to 25% arterial blood loss through the central ear artery for one hour then resusciatation was done by the heparinized withdrawn blood through a marginal ear vein. during the experiment blood pressure and heart rate were monitored through the central ear artery. also uric acid, lactic acid, glutathione activity were estimated. animal survival was followed for 10 days. postmortem vital organ histochemistry and histopathology examinations were done. in group i the survival after three days was 10 out of 22 while in group ii it was two out of 20. our conc|usion, allopurinol had increased the survival in aiiopurinol pretreated rabbits which may indicate the value of allopurinol premedication for patient prepared for elective bloody surgical intervention . h2 receptor antagonists are commonly used for stress ulcer prophylaxis, but their actions on the septic response are largely unknown, in an experimental model, 26 pigs were first anesthetized, then injured with 100 joules of energy to the posterior thigh, then hemorrhaged 30-40% of their blood volume. after i hr of shock, all the shed blood plus 2x the hemorrhage volume as lactated ringers was infused. following resuscitation, ranitidine (1.5 mg/kg iv twice daily) or saline placebo was begun. the treatment group was randomly assigned in a blinded fashion. after 72 hrs, a septic challenge was administered (15 bg/kg of e. coil endotoxin (lps)). serial gastroscopy, gastric ph, hemodynamics, abg's, physiologic dead space ventilation, leukocyte counts, and tumor necrosis factor (tnf) levels were recorded for 180 min. baseline values and units were cardiac index 102 _+ 9 ml/min/kg (ci), arterial po2 228 + 11 mmhg(pao2), base excess 6.5 -+ 1 meq (be), physiologic dead space fraction 23 +_ 4% (pds), and tnf 0.4 + 0.1 units/ml. baseline gastric ph was 3.9 -+ 0.4 and 4.8 _+ 0.5 in the placebo and ranitidine groups, respectively. the gastritis following hemorrhage was marginally attenuated in the ranitidine group. following lps infusion the following were obtained: ci pao2* be* gastric* pds* peak* 120 rain 60 rain 120 rain ph 120 min tnf ranitidine 167_+17 118_+15 -4.4±1. bum injury results in hypermetabolism, fever and nitrogen wasting. endotoxin (lps) has been proposed to mediate these effects, either directly or via activation of macrophages to produce cytokines such as interleukin-6 (ii-6). this study was designed to clarify the role of lps and 11-6 in the metabolic response to bum injury. twenty-five burn patients (49 -+ 17%; 16 + 15% ft bsa burn; 32 _+ 16 years old) were studied serially for three weeks post bum. patients underwent partitional calorimetry to assess metabolic rate and compartmented heat loss. nitrogen was assayed using chemiluminescence. lps and i1-6 were measured with limulus amebocyte lysate assay and elisa. patients were excluded if they suffered smoke inhalation, showed any sign of sepsis or failed to rapidly meet their nutritional needs via the enteral route. ten patients received intravenous polymixin b (5,000 u/kg/day to bind lps). these patients did not differ for the remainder. all patients were hypermetabolic and febrile in proportion to the size of their bum wound but were not endotoxemic (3.1 +_ 5.6 pg/ml; normal <40 pg/ml). i1-6 did demonstrate a significant correlation with cole temperature (tr~ = 37.6 + 0.21ogi1-6, p=0.04) and with nitrogen excretion (nou t = -5.2 4-0.091ogi1-6 + 0.14tr, p=0.01). administration of polymixin b had no effect on metabolic rate, temperature or i1-6 levels but did reduce nitrogen excretion resulting in more positive nitrogen balance (0.t grn/day vs. -0.1 gm/day, p=0.04). although bum injury does not produce an obligatory endotoxemia, i1-6 does appear to play a role in the fever and nitrogen wasting seen with such injuries. the effect ofpolymixin b on nitrogen excretion suggests that lps may play a role either locally or in the portal system. introduction: there is substantial evidence that release of inflammatory mediators by activated kupffer cells contribute to the course of a systemic inflammatory process, e.g. after shock or lrauma. besides the systemic effects of mediators such as tnf, paf or interleukines, local actions on hepatic microvasculature and hepatic inflammatory response have to be considered. our aim was to assess the role of tnf and paf by blocking their effects using anti-tnf monoclonal antibody, pentoxifylline and a paf antagonist. methnds: in anesthetized sprd-rats, hemorrhagic shock was induced by withdrawl of arterial blood within 5 rain and shock state was hold for 1 h at a map of 40 mm hg (cardiac output of 50 %). following adequate resuscitation with 60% of shed blood and twice of this volume as ringer's solntion, animals recovered to map >1130 mm hg and co >120%. hepatic microcirculation and sinusoidal leukocyte-endothelium interactions were examined by intravital epi-fluorescence microscopy at 1, 3, or 5 hours after resuscitation. in a blinded fashion, a rat-specific monoclonal anti-tnf antibody [2 mg/kg, celltech, uk) , pentoxffylline (ptx, 50 mg/kg, hoechst, d), and a paf antagonist (web 2086, boehringer, ingh., d) were given either as pretreatment or at the time of resuscitation (n=6-8 group bolla. k*., duchateau, j., hajos, gy., mbzes, t., hern~di, f. prevention of temporary/secondary immune deficiencies or reduction of their severity and/or duration as well as the reduction of the perifocal inflammatory processes belong to the rational targets of posttraumatic/pedsurgical medication. such a targeted medication can result in less frequently occurring nosocomial infections, and in reducing the duration of the intensive care and convalescence period. the results of in vitro studies performed with the amino acid sequence 32-35 of thymopoietin, i.e., with thymocartin in whole blood and peripheral mono-nuclear celi(pbnc) cultures clearly show some characteristic effects of this immunomodulator. preincubation with the tetrapeptide significantly (p<o.ol) and concentration-dependent reduced the endotoxin(lps)-induced tnfalfa production from 95,7 to about 20 pglml, but did not abolish it. the inhibition of the lps(0.1 ug/ml)-induced tnf production occured already in presence of 0,1 pg peptide/ml and peaked at the concentration of 0,01 ng/ml. higher concentrations did not increase this effect. in contrast to the other two small thymic peptides (tp-3 and tp-5), thymocartin did not enhanced the pwm-induced pge 2 production in pbmc cultures up to a concentration of 1 ng/ml, inclusive. the selection of thymocartin (tp-4) for perisurgical medication and/or for treatment of trauma-induced temporary immune depression has been strongly supported also by targeted animal experiments. thus, mortality of about 90% registered in mice infected with pseudomonas aeruginosa after bum injury could be reduced with the tetrapeptide treatment to a level which did not differ significantly from that of the infected non-burned controls. moreover, observations gathered first of all with the inflammatory model of air poach/croton oil grenuloma (selye) clearly showed, that a very definite, antiexudative effect can be achieved with this thymic peptide. as to this effect, thymocartin has been confirmed, e.g., to be equipotent with locally administered fluocinolone acetonide and/or s.c. injected prednisolone. the interim results of two explorative double-blind clinical studies (involving more than 260 patients after polytrauma and hip-fracture untill now, respectively) correspond to the expectations. the treatment significantly reduced the occurence of nosocomial infections, the days spend in intensive care and on antibiotics as well as it shortened the bed-out time and reduced the additional medication. objective of this prospective randomized study was to further delineate the mechanisms leading to these alterations and to investigate the effects of immunomodulatory intervention. patients and methods: 20 patients (pts) formed control group a. 20 group b pts received 3x50 mg indomethacin (indo) from the first (dl) to the fourth postoperative day (d5) intravenously to block prostaglandin e2induced suppression of cmi response as well as 50 mg thymopentin (tp-5) subcutaneously preop., on d2 and d4 to augment cmi reactivity. in vitro investigations preoperatively, on dl and d7 included measurements of the percentage of cd4+ t-helper (th) cells, pbytohemagglutinin (pha)induced synthesis of interleukin (il)-2 as one cytokine primarily produced by thl-cells, and pha-induced synthesis of il-6 as one cytokine primarily produced by th2-cells. delayed type hypersensitivity (dth) skin response to an antigen skin test battery and specific antibody (ab) production following tetanus vaccination preoperatively and on d7 were used as in vivo tests for thl-and th2-induced immune response respectively. results: postoperatively, group a pts showed a persistent significant reduction of cd4+ th cells, il-2/il-6 ratio, and dth skin response as compared to baseline values (bl), while ab production increased (p<0.05 vs.bl). in group b pts no change in cd4+ th cells, il-2/il-6 ratio, or dth skin response was observed (p<0.05 vs.group a), and postoperative ab production increased significantly (n.s. vs. group a). conclusions: 1.these results clearly indicate a significant suppression of th1 induced cmi response, while th2 induced response remains normal following ohs. 2.these alterations may gain clinical significance whenever a postoperative infection requires a th1 response and may explain the susceptibility to opportunistic microorganisms observed in pts after ohs. 3 the aim of this study was to find out whether the establishment of administration of thymostimulin (tp-i) in jaundiced and anergic rats would return the rats to the state of immunocompetence. material and methods. male wistar rats weighing 250-300 g were injected with haemocyanin (klh). all rats with a positive response were included in the study and we evalu ated the t cell subpopulations and il-2. they underwent laparotomy and the common duct was ligated and divided. those rats whic become jaundiced and anergic one week after the operation, were divided in two groups: control group ( objective of study. the goal of this study was to prove the necessity of thymus derived immunomodulators treatment in acute period of infantine sepsis, materials and methods. immune status was investigated in 11o infants. clinical conditions of children were defined by syndrome of multiple polyorgan]c insufficiency. the heaviness nf condition was estimated as the sum of points according to efforts needed to restore of basic living functions -respiration, hemodynamjcs, hemocoagulatlon, metabolism, functions of liver and kidneys. it was expressed in form of clinic condition classes from 1 to 4. results.it was found that in 100% of infants with 3-4 classes of heaviness immunode[iciency took place simultaneously with ii and iii stages of hemocoagulattve syndrome. amounts of t cells and t helpers were decreased more then 2 times, ratio th/ts was reduced to 1.5 or lower. concentration of lgg and igawas reduced almost a hull in comparison to control. phagocytic and metabolic activities nf neutrophils were depressed. complement system state was changed: the functional activity of the alternative pathway factors b, d and the level or c3a subcomponent were increased but the level of c2, c3,, c4 and c5 components of the classic pathway were decreased. during the development of coagnlopathy the direct correlation between chso, levels of plasminogen and antithrombine iii was found. in greater extent the function of immune system was disodered in accordance with 2 point metabolic violations and hemocoagulative disturbances considered as disseminated inmtravaseular clotting syndrome on ii or ili stages. conclusions. the rehabilitation of the immune system functions was promoted by taetivine and thymaline in doses of i-3 mcg/kg per day and 2 mg/kg per day accordingly during a week in children with 3 -4 classes el heaviness. aiter 11-14 days tactivine had advantage. when eoagulopathy took place thynaaljne promoted restoration of (1-3)-~-d-glucan (bgc) is a major cell wall compornent of pathogenic fungi and shows many immunopharmacological activities on experimental animals. lps is also derived from gram-negative bacteria and have in~ttnomodulating activities on immune systems positively or negatively. because of difficulty to cure contaminating lps from bgc preparations, it is very hard to evaluate the exact activities of bgc. in this study, we compared the immunological activities of lps ans highly purified bgc on murine system. materials and methods: c3h/hen or c3h/hej mice, 6 to 12 wkolds of both sexes were used through experiments. highly purified (1-3 )-{5-d-glucan (gurdran; bgc)was courteous gift from seikagaku kogyo co. tokyo, japan and resolved in endotoxin-free ultra-pure water. escherichia cell lps was purchased from sigma chemicals, usa. mitogenic activities of bgc or lps on splenic cells were measured by mit dye assay. in vitro activation of peritoneal exudate macrophage (pen) have moniotored by phagocytosis and intraphagocytic killing of pseudomonas aeruginosa (z~b-139. induction of cytokine productions from pem or spleen cells induced by bgc or lps were detected by cytotoxic assay. results and (bnclnsions: i ) in vitro cultures of pem with bgc have induced enhanced phagocytic and bactecidal activities in c3h/hen and c3h/hej mice, whereas lps only induced such activities in c3h/hen mice. most effective dose of bgc was 10 ug/ml. 2) bgc-dependent proliferative response of c3h/hen splenic cells was not detected under the culture condition used. 3) induction of tnf~ prodction was apparently observed in bgc-stimulated c3h/hej pd4 cultures, but not in lps stimulated one. these findings indicate that activation mechanisms of pr94 and splenic cells by bgc are different from that by lps. to investigate the bsc-induced p~4 activation, intracellular signaling pathways of p~4 by bgc-stimulation are under consideration. yamagami k, uedono y, kawakami k, kitazawa y and tanaka t according to the latest reports of use of il-2 in a burned-sepsis model, the dose was too high to adjust for human therapy. adoptive transfer of l~ymphokine-activated killer (lak) cells has been demonstrated as a means of enhancing therapy in malignant tumors. we therefore attempt to use lak therapy on burned-infected mice instead of il-2. under anesthesia, 8-weekold male c3h-hej mice were subjected to a 20% scald or sham burn and then resuscitated. sham burned mice served as controls. scald burn mice were subjected to peritonitis by intraperitoneal innoculation of 105 organisms of p. aeruginosa 24 hr after burn. burned-infected-mice were divided into two groups. one group had no further treatment, and the other group received lak cells (2x105) intraperitonelly after septic challenge. the mice were scored for survival daily. on day 7 after burn, using moabs dual-color flow cytometry, we studied lymphocyte expression in mice with use of blood and spleen. simultaneousely we studied the alteration of il-1 and il-6 in their serum using immunoassy kits. sham burned animals had no mortality. the mortality of the lak-treated mice was significantly reduced when compared with that of the burned-infected mice. the most consistent changes observed were depressions in percentages of thyl, 2 positive cells and a remarkable depression of nk cells in spleen. after lak cell treatment, those two percentages of cells significantly increased. all the data of assay of ill and [l 6 were not detected on sham burn mice serum. due to burn and septic challenge the levels of illand il 6 (n=6) were raised to 45.9_+17.4 and 3866_+1045 pg/ml, while the levels in lak treated mice were reduced to 4.23_+2.37 and 437_+110 pg/ml, respectively. the results reported here demonstrate that lak therapy is able to improve cell-mediated immunity in burned-infected mice. this is associated with a significantly improved survival rate, since ill has been demonstrated to mediate immune and inflammatory responses. also a cause effect relationship between elevated endetoxin levels and increases of [l6 has been recently established. the aim of our present study was to investigate the effect of parenteral indomethacin on the immune system of patients under major operations. our study included 20 operated patients, 10 of which received 100mg indomethacin before and 1,2 and 3 days after surgery (group a) and 10 patients received no antiinflammatory therapy (group b). we measured total t-cells helper (cd4), suppressor (cd8), nk-cells and interleukin-1 and tnf production by pha or endodoxin (lps) stimulated peripheral blood mononuclear cells at day 0 and 1, 3 and 7 days after operation. in group a we detected a significant (p<0.05) increase in cd4/cd8 ratio on day 3 while no differences were ~oted in nk-cells or cytokine production in both groups (table 1) . it seems, therefore, that parenterai indomethacin may have a benefitial effect on the immune system of patients under major operations by increasing the t-helper /t-suppressor cell ratio while having no effect on the production of cytokines by peripheral blood mononuclear cells. this study was undertaken td dete~nnine the role of prostaglandin synthesis inhibitor on survival post traulna sepsis. analysis of the effects of prostaglandin synthesis inhibitor on survival of four groups of 15 mice each were made after laparatomy and intravenous administration of live e.coli. post trauma sepsis, group a received no treatment; group b received antibiotics im; group c received prostaglandin synthesis inhibitor im and group d received both antibiotics and prostaglandin synthesis inhibitor im. survival time was longest in group treated with prostaglandin synthesis inhibitor and antibiotics post trauma sepsis. the singular use of either antibiotic or prostaglandin synthesis inhibitor did not alter the outcome on survival. mortality rate was lowest in the group treated with combined prostaglandin synthesis inhibitor and antibiotics. use of this combination showed a reduction in bacterial growth in peritoneal and blood samples, mild morphologic changes secondary to sepsis in the heart, lungs, liver, kidney and stomach and marked enhancement of mean level of activity. the study indicates that addition of prostaglandin synthesis inhibitor in treatment of trauma-sepsis has clear beneficial effects. interferon-? (ifn-y) is a potent immunostimulant which has been shown to be detrimental when administered during septic shock. blockade of this cytokine however is beneficial during the acute septic response. the aim of this study was to evaluate the cellular effects of this cytokine and its blocking monoclonal antibody (moab) in lethal sepsis following injury. 100 female cd-1 mice were randomized into two groups: septic=lps vs injury=hind limb amputation (hla vs igg. nstats= anova=p<0.05 vs lgg ifn-y was detrimental when given to control septic animals. however its blocking moab was protective (p<0.05). conversely ifn-y was proteetive in injured septic animals compared with anti-ifn-? (p<0.05). these findings demonstrate that the immune stimulant ifn-? has therapeutic potential in the immunosuppressed injured host predisposed to sepsis, while blockade of this cytokine is required for protection in the septic host with a normal immune response. dept of surgery, rcsi, beaumont hospital, dublin 9, ireland. oxidants play a role in physiology. the potential noxious oxidant biochemistry is restrained by chemically distinct antioxidants. these antioxidants, which may reside in different cellular compartments, can act synergistically. in normal physiology an oxidant/antioxidant bai&nce exists. unbalance in favour of the oxidants is called oxidative stress. oxidative stress has been implicated in many pathologies including lung diseases(e.g, doxorubicin induced cardiotoxicity), ischemia/reperfnsion damage and central nervous system trauma. transition metals such as iron are involved in the early events leading to oxidative damage in these pathologies. this has been underlined by the finding that postischemic cns pathology closely resembles that caused by a chemical oxidative insult (e.g. iron microinjection). moreover, a protective effect of oxygen-radical scavening agents or compounds that inhibit lipid peroxidation (antioxidants) in brain ischemia/trauma is apparent. some known drugs (e.g. anti-arrhythmics or histamine h2-antagonists may act as non-specific antioxidants. howe~er, recently, interesting new membrancespecific antioxidants (e.g. 21-aminosterdids) have been designed. subt&e effects on iron redox chemistry contributes to the potent antioxidant activity of these 21aminosteroids. since a few years, one area that greeted enthusimastlcally in clinical medicine is that of reoxygenatlon injury or ischemia-repeffusion, a phenomenon accepted to make a majo:" contribution to organ dysfunction in trauma, shock and sepsis through the formation o( oxygenated free radical species. however, thei detection in vivo always remains a difficult challenge. efforts have been made recently to directly establish the formation of free radicals in biological samples as complex as blood, plasma or tissue with the use of electron spin resonance (esr) spectroscopy. using spin trapping agents, the presence of reactive carbon-and oxygen-centered radicals has been demonstrated in animals blood submitted to heart, renal and intestinal ischemia-repeffusion or to liver transplantation. recently, the in vivo formation of free radicals in the cerebra; extracellnlar fluid during cerebral isehemia-repeffusion has been assessed by the spin trapping technique coupled to brain microdialysis. esr investigations have also been conducted to detect endotoxin-induced free radical generation in rat or baboon liver and oxygen free radicals (ofr), produced both at intra-and extra-cellular levels, seem to play a major role in the pathophysiology of tissue injury and organ dysfunction in sepsis, through induction of lipid pemxidation in cell membranes. oxidative damage can result both to an ofr-overpmduction or to a depletion of endogenous antioxidant defenses, which are represented by scavenger enzymes (e.g. sod), hydrosoluble and liposoluble antioxidants (e.g. ascorbate, vitamin e), and other mechanisms such as metal-ion sequestration. in animal models, oxidative damage has been proved by detection of increased levels of lipoperoxidative products, and a depletion of antioxidant defenses was found both in plasma and tissues. in the few clinical studies a similar results have been reported, and oxidative damage appears to correlate with dic, with organ dysfunction and with red blood cell deformability. in a group of critically ill septic patients we found increased levels of conjugated dienes (cd) (bioproducts of lipoperoxidation) and decreased plasma levels of alpha-tocopherol and coenzyme qlo (coqi0)(endogenous liposoluble antioxidants). a relationship between cd and uric acid (p<0.004) was found, may be related to xantine-dehydmgenase to xantine-oxidase conversion. antioxidant depletion is due both to overconsumption and to a nutritional deficiency, even if a reduced synthesis can also be present. in fact one more relationship between coqi0 and plasma cholesterol (p<0.0005) demonstrates the commun hepatic biosynthetic defect. is there any role for antioxidative therapy in sepsis? can it achives a significant improvement in the septic course, organ dysfunction, and final outcome? several antioxidant drugs, have been evaluated in experimental and clinical sepsis: scavenger enzymes, natural antioxidants, alloporinol, 21-aminosteroids or lazaroids, have been proved to reduce lipopemxidative damage, to protect organ dysfunction, and in some cases to improve survival. several questions must be addressed in evaluating safety and utility of antioxidative therapy. more specific and standardized methods must be applied to detect and quantify the lipoperoxidative damage. antioxidant drags must act selectively on ofr-production, without interference with other than that are beneficial for the organism. antioxidants must be able to achive in adequate amount the tissue or cellular compartment where their action is required. many antioxidants have also a pro-oxidative effect which can he detrimental in some conditions. the timing of administration, the dosage used and the association wih others antioxidant drugs must be defined. nutrition plays an important role as antioxidative therapy, since it supplies all compounds needed to maintain adequately levels of endogenous antioxidant or to support their synthesis. thiobarbituric acid reactive substances (tbars) concentration in serum has been determined in a large population of healthy subjects and in patients suffering acute hepatitis and chronic cases of hepatitis c, as well as several other processes. the correlation with different physiological and clinical parameters in these populations is also presented. treatment with interferon of the chronic active hepatitis c patients, 5x10 u three times a week during two months, led in those patients whose sgpt activity normalized in serum, to a concomitant decrease in serum tbars content. the possible theoretical involvement of peroxidation and antioxidants in this beneficial effect of i~terferon in hepatitis c patients is discussed. the results presented confirm the value of tbars as laboratory test in the management of disease and as a useful tool for the study of pathogenic and/or therapeutic mechanisms. 4-hydroxyalkenals are among the different substances measured by the tbars assay. these compounds show several biological effects that have been demonstrated mainly in different experimental models. we have studied the capacity of different tissues to conjugate these compounds patients surviving the initial subarachnoid hemorrhage (sah) from a ruptured in~raeranial aneurysm are prone to develop cerebra] ischemia from vasospasm which is associated with significant morbidity and mortality. among the many treatments that have been prol~osed to treat this condition, none has been shown to be satisfactorily effective. recently, a new drug, namely the 21-aminosteroid tirilazadmesylats, has been studied in a large, prospective, multicentor trial for its effectiveness to improve the outcome of patients with aneurysma! said. the study was conducted in europe and australia in 41 neurosurgical centers and included 1023 patients. all patients received presently accepted standard treatment, including nimodipine. patients were randomized to four different groups: placebo and 8 groups of tirilazad in escalating dosage (0.6 -6 mg/kg/day). the clinical outcome was compared for these 4 groups and cerebral a~giography was performed in 75 % of patients on day 7 -11 follovang sah, to study the effect 0f'the dflf~ off cerebral vasospasm. the study reached its planned endpoint 6 months ago. preliminary results ~how a significant improvement with regardto mortality in those patients receiving the highest tirilazad dose as compared to placebo and the two lower dose groups. additional beneficial effects were seen in the 6 rag/day groups with regard to the occurence of symptomatic vasospasm and good clinical outcome. although the final analysis of all data is still pending, these results suggest that tirilazad-mesylate could represent a major improvement for the treatment of patients with sail. experimentally lipidperoxidation products (lpo) are increased in acute pancreatitis (ap) due to oxygen radicals (or). the purpose of this clinical study was to determine the antioxidant status and lpo in patients suffering from ap and to evaluate the effect of antioxidant treatment with sodium selenite (se) thus improving the function of the se dependant glutathione peroxidase which is the major detoxifying system for or. materials and methods: in z0 patients sufferin s from severe ap (c-reactlve protein >120 me/l) we determined on day 1 and day 8 after admission the lpo ma!ondialdehyd (mda), conjugated dishes (cd), reduced (gsr) and oxidized (gssg) glutathione, the vitamins a,c,e and se. moreover the patients were evaluated clinically using the ranson and the apache ii score. i0 patients were randomly treated with 600 ug/day of se for 8 days. results: all patients suffered from a severe depletion of antioxidants,especially a low concentration of se (only 1/3 of normal). thereby the increase in lpo correlated with the clinical course. during se treatment lpo decreased and the levels of antioxidant vitamins improved. se had no influence on leth-slity the lenl or the chan in rs or ap ii. background: since reperfusion injury occurs when oxygen is reintroduced into ischemic tissue, the ideal timing for administration of therapeutic compounds aimed at ameliorating oxygen radical mediated injury is at the time of initial fluid resuscitation. currently used colloid or crystalloid preparations do not provide optimal, or even significant, anti-oxidant protection. systemic iron chelation affords protection against the iron catalyzed components of oxygen and lipid radical mediated tissue injury. the conjugate resulting from chemical attachment of the clinically approved iron chelator, deferoxamine (dfo, desferal ®, ciba), to hydroxyethyl starch (hes) represents a novel approach to colloid based fluid resuscitation. hes-dfo contains 85% hes and 15% chemically bound dfo. the polymer-drug conjugate has a lower molecular weight than that of hes in order to allow more rapid excretion. results: preclinical and initial clinical trials indicate that hes-dfo is well tolerated, even at high doses. in animal studies, fluid resuscitation with hes-dfo does not significantly improve central hemodynamic recovery beyond that observed with hes, but hes-dfo seems to afford better protection of microcirculation in organs at risk (lung, liver and gut), possibly by decreasing neutrophil sequestration. in a burn model, total fluid requirements are lower and oxygen utilization higher in hes-dfo treated animals compared to hes controls, suggesting decreased vascular leak and improved tissue perfusion. conclusion: hes-dfo represents a means by which potent antioxidant protection can be administered at resuscitation. iron has been suggested to play a pivotal role in oxygen flee radical mediated tissue injury. in vitro experiments indicated its critical role as a katalyst in hydroxyl free radical generation 0fenton-reaction). since iron chelator deferoxamine administered in shock alone demonstrated severe side effects, a hydroxyethylstarch (hes)daferoxamine (dfo)-conjugute was used to modulate oxygen free radical injury during the ischemia/reperfi~ion syndrome induced by hemorrhagic shock. methods. female lewis rats (190-215 g, n>6; pentobarbital anesthesia 50 mgjkg), in hemorrhagic shock ( the aim of the study was to elucidate (1) whether the generation of or would affect lung and kidneys as primary shock organs in the very early phase of sepsis and (2) whether dfo-hes could prevent this tissue damage. methods: in 67 rats sepsis was induced by cecal ligation puncture (clp) peritonitis. the animals were randomly assessed to 2 groups: one group was treated with 3 ml dfo-hes (100 mg/kg iv), the other rats received solely 3 ml of the carrier starch solution. 30, 60, 120, and 240 min after induction of sepsis respectively, the animals were sacrificed, the organs collected, and tissue contents of glutathione (gsh), malondialdehyde (mda), myeloperoxidase (mpo) and conjugated dienes (cd) determined. plasma samples were obtained for analyses of endotoxin (chromogenic lal test). blood pressure (map) was measured via a carotid artery catheter. results: clp caused sepsis with high (> 1.84 eu/ml) endotoxin levels. map in both groups decreased slightly but significantly during sepsis regardless any treatment. in the lungs mpo concentration was increased (p<0.05) in the lies group already 30 min after sepsis induction. concomitantly, tissue gsh level decreased and lipid peroxidation was pronounced as shown by elevated mda and cd levels. dfo-hes diminished tissue pmn accumulation and mpo concentration. moreover, at each time point lung mda and cd levels were lower (p<0.02). histomorphological examination showed marked micro-atelectases, destruction of the alveolar septa, and splicing of the basal membranes in the lies group. in contrast, in dfo-hes treated rats the alveoli remained well-ventiiated and only some enlarged reticular fibers without splicing were observed. almost similar results were found for the kidneys. mpo levels differed neither within nor between both groups. the slight decrease in gsh levels seen after 60 min in the dfo-hes group seems to demonstrate an oxidative stress to a lesser degree. the most impressive effect of iron chelation, however, was revealed by the lipid peroxidation products. at each time point, mda and cd levels were lower (p<0.02) compared to the hes group. light and electron microscopic examination disclosed tubulotoxic and mitochondriat damages while dfo-hes lxeatment prevented that alterations. conclusion: both the biochemical and histological results of this study reveal an early and remarkable generation of or in peritonitis-induced sepsis. thereby, these or obviously cause pulmonary and renal tissue damages, intravenous application of dfo-hes may, however, benefit by preventing early lipid peroxidation of the tissue. the proteolytic irreversible conversion of xanthine dehydrogenase (xd) to xanthine oxidase (xo) is triggered by calcium flux. the aim of our study is to clarify ~he link between intracellular ca2+ levels and xo activity determined by uric acid release, and to evaluate the efficacy of verapamil, on the generation of hydrogen peroxide associated with reperfusion by assaying lactate & pyruva~e release and the levels of cytosolic free nad /nadh ratio. experimental protocol consisted of :(a) non ischemic/reperfused experiment in which normal cardiac slices of rats were perfusated with oxygenated kreb's ringer phosphate buffer containing glucose (150mg%) and bovine albumine (5gm%) for 60min at 37°c.it composed of 3 groups, group aa (control group), and groups ab & ac (perfusate supplemented with verapamil in the dose of loo&200 mi% respectively). (b) ischemic reperfused experiment in which ischemic cardiac slices were obtained from rats subjected to 15 min ~aemorrhage.lt was also divided into two groups; group ba and bb (verapam~/ 200mi% added to perfusate}. verapamil stimulated uric acid release from normal rat cardiac slices were 267% in group ab and 767% in group ac(dose related). rates of uric acid release is enhanced by verapamil in group bb. moreover, rates of uric acid release in groups ac & bb are insignificant. in verapmil added groups (group ab, ac & bb), increase uric acid release is associated with an enhancement in pyrurate release and with increase levels of cytosolic free nad+/nadh ratio, although it is not evident ~ ischemic group (group ba).it is concluded that the conversion of xd to xo is calcium independent. eicosanoids like thromboxane a2, leukotriene b4 and leukotriene c4 are known as promoters of initial inflammatory reactions. we investigated whether oxygen radicals (or) are able to induce a release of these eicosanoids in whole blood. blood from healthy volunteers was incubated with xanthine oxidase/hypoxanthine to generate oxygen radicals. after 0,5,15,30 and 60 minutes plasma levels of thromboxane b2 (txb2), leukotriene b4 (ltb4) and leukotriene c4 (ltc4) were determined via elisa technique. another volunteer had taken 1000mg aspirin one day before taking the blood sample (no7). results: txb2 plasma levels increased from 14pg/ml at 0min to 144pg/ml, 350pg/ml, 370pg/ml and 660pg/ml at 5,15,30 and 60min (p<0,01) . ltb4 and ltc4 plasma levels showed an increase during the first few minutes (ltb4: 0min: llpg/ml,5min: 87pg/ml; ltc4: 0min: 23pg/ml, 5min: 97pg/ml (p<0,05)) followed by a decrease to normal values at 15min. in the sample no7 the cyclooxigenase-pathway was completely inhibited, the txb2 plasma-levels did not alter at all, whereas ltb4 and ltc4-plasma levels weren't affected. opallogeneic blood transfusion jane shelby, ph.d., and edward w, nelson, m.d, there have been numerous investigations dudng the last two decades examining the effect of surgery, anesthesia, blood loss and transfusion on vadous immune parameters in humans and animal models. there appears to be concurrence among several well controlled studies that transfusion of whole blood (containing leukocytes), has regulatory effects on immune ceil function which include decreased cell mediated immune response, and inhibition of il-2 secretion. these effects occur following transfusion alone and in con.cart with the distinct immune effects of surgery, trauma and anesthesla, the clinical consequences of this immune modulation by transfusion include decreased allogeneic response to transplanted organs, which has been exploited clinicelly in renal transplant patients. additionally, there is evidence for a strong association with increased risk for infection in transfused patients following surgical procedures. aiiogeneio blood transfusions have been shown to inhibit cellular anti.bacterial mechanisms, causing increased susceptibility to bacterial pathogens, in humans and in animal models. there is also concern that allog~neic transfusion may adversely affect cancer patients, resulting in decreased disease-free survival. several stategies have been proposed to minimize the adverse effects of blood transfusion. there is evidence that the risk of immune mediated infectious complications associated with transfusion may be greatly minimized wlth the use of autologous blood and leukocyte free allogeneic blood.products in surgical and trauma patients, it also appears that the inhibition of cellular immune response and il-2 productiorl following atlogeneic blood transfusion may be mediated by increased prostaglandin e2 secretion, and that immune response may be preserved in allogeneio whole blood transfused subjects receiving c3lc~oxygenase inhibitors such as ibuprofen. among these are various alterations in immune function. efforts have therefore been made to utilize alternatives to homologous transfusions. these include the use of autologous predonation, supplemental iron therapy, and recombinant human erythropoietin. although initially considered innocuous, these therapies are now recognized to have potential deliterious immune sequelae. erythropoietin, by its ability to lower serum iron levels, can impair both lymphocyte and nk cell activity. autologous donation impairs nk cell function. finally, supplemental iron therapy can stimulate bacterial growth and increase the rate of infectious complications. this talk will present a discussion of these factors as well as a weighting of their importance. r.l rutan, rn;bsn, shriners burns institute and the university of texas medical branch, galveston tx, usa the serious sequelae of homologous blood transfusions have resulted in vigorous efforts at identifying alternate therapies for correcting red blood cell (rbc) deficits. erythropoietin (epo) was hypothesized to exist in the early 20th century, however the protein was not isolaled until 1977. the human gene was identified and cloned in 1983, which permitted the production of epo through recombinant techniques. the earliest clinical trials were performed in anemic end-stage renal failure palients on hemodialysis. treated patients experienced increases in erythropoiesis with normalization of hematocrit and hemoglobin levels, cessation of lrans-fusion requirements and improvement in general wellbeing. these studies, however, identified side effects of epo treatment such as hypertension, seizures and ee deficiency. volunteer trials have established that the hypertension is not a direct pressor effect but rather the result of abnormally rapid increases in red cell mass in the face of the incompetent volume-controlling mechanisms of the end stage renal failure patient. lower doses of epo and the subsequent gradual increases in red cell mass are associated with significantly lower incidences of hypertensive complications of epo therapy. likewise, seizure activity is not the result of a direct epileptogenie effect but parallels the incidence of hyper-tensive-related sequelae during high.dose epo treatment. in cross-over designed studies, pre-existing iron deficiency has been demonstrated to decrease or negate stimulated erythropoiesis but effective-hess can be restored with appropriate fe supplementation. exogenous epo is effective whether given by iv or sq routes and dose response curves do not vary with route of administration. increases in rbc mass are directly related to the dose of epo, both in amount and frequency of administration although there is a 3-5 day time lag between the first epo dose and laboratory indications of its action (i.e. increase in the number of reticulceytes in peripheral wood). epo is currently labelled for use in the treatment of anemias associated with end-stage renal disease and aids. however, its use in the surgical population has been explored because of its unique direct dose-response, epo has been used to effectively increase the blood harvest amounls in autologous pre-donation, significantly increase hematocrils in children following thermal trauma and successfully increase red blood cell mass following essential surgical procedures in patients with religious aversion to transfusion. by blood transfusion in colorectal cancer surgery mm heiss md, ch delanoff md, r stets md, j hofinann, e faist md, kw jauch md, fw schildberg md allogeneic blood transfusions are associated with an increased risk for postoperative infections in colorectal surgery when compared with autologous blood transfusions. attribution of this effect to immunomodulation was suspected in our previous study (lancet 1993; 342:1328-33) . task of the recent investigations was to analyze which specific effector systems were affected in-vivo by this transfusion-associated modulation. for global in-viva assessment of cell-mediated immunity (cmi) multiple recall skin-reactions were applied prior and post-operative. the specific humoral immune mechanisms were investigated by applying tetanus-toxoid one day preoperatively and deterimnating the quantitative igg-response. for indication of macrophage stimulation in-vivo tnf-levels were determinated by bioassay. dth-responses were significantly suppressed (p<0.05) in patients receiving allogeneic blood (n=36) or operated without blood transfusions (n=17). dthresponses were not suppressed and tendentiously increased in patients with autologous blood transfusions (n=24). in contrast, specific igg-levels increased sigmficantly (p<0.05) in patients receiving allogeneie blood (from 1.6 + 4.4 to 9.7 _+ 18.4 ie/ml) whereas in patients receiving autologous blood a smaller increase (from 1.9 + 4.1 to 4.0 + 6.7; p=0.068) was observed. tnflevels demonstrated a similar pattern with a higher increase in patients receiving allogeneic transfusions (l 1.4 + 12.0 to 28.5 + 11.4 u/ml) compared to those patients with autologous blood (8.7 + 12.5 to 17.1 + 23.0). in conclusion these data indicate that allogeneic blood transfusions lead to a remarkable macrophage/rhs stimulation. this is corroborated by the boostered humoral igg-response which was initiated before onset of surgical trauma and blood transfusion. concerning cmi this caused a substancial suppression probably due to a stimulated secretion of immunosuppressive monokines. objective: firstly, to analyse the concentrations of the cytokines tumor necrosis factor (tnc), interleukin-1 (il-i), interleukin-6 (il-6) and coagulatioo/fibrinolysis parameters in postoperatively retrieved blood from a surgical area, secondly to characterize the correspanding cytokine patters in the patients and thirdly to study cytokine concentrations in the initial portion of drainage blood from a surgical area. materials and methods: blood retrieval was performed in a closed-loop system without anticoagulant during 4-6 hours after surgery in 10 patients undergoing arthroplasty (9 hips and 1 knee). 7kf, il-1, it-6, thrembin-antithrombin complexes (tac) and antithrombin (at) ~ere determined in shed blood. patient plasma tn v, il-i and il-6 concentrations ~ere analysed at the beginnlqg and end of the 4-6 hour blood retrieval period. in a separate study (5 hip arthroplasties) f~f, il-i and il-6 ~ere determined in the initial portion of drainage blood. cytekine analyses ~re performed usiog ipmuooassays. an omidolytic method was used for at determinaf.ion and tac was analysed by elisa. n~n-poram~tric tests was used for the statistical comparison. results: the patient plasma il-6 coocemtratiems rose from a median value of 0 to 116 pg/ml, p<o.01, during the blood ret.rieval period. in c was detectable in 4 patients, range:15-50 pg/ml. il-i was not detectable in the patients. in retrieved blood tag was >200 mg/ml in all samples (ref:<4.1 mg/ml) and at was 0. 16-0.51 units/ml (ref:o.80-1.20) . the il-6 concentrations in retrieved blood was >1000 pg/ml in all samples. tn v or il-i was not detectable. in the separate study, (n=5), characterlzing eytokine content in the initial portiere of drainage blood, in= (range: 11-277 pg/ml) and il-i (range:5-125 pg/ml) ~re present in all samples but ii-6 (range:o-25 pg/ml) was detectable in o.qly one semple. conclusion: theses findings indicate that hypereoagulability and hic~ ccrcentratioos are present in retrieved blood. the cytokine pattern in the initial portion of blood from a surgical area differed from these observed in retrieved blood and in the systemic circulation. to identify the role of both autologous and homologous blood on postoperative infections in elective cancer surgery. materials and methods: 159 patients with colo-rectal cancer submitted to curative elective surgery were prospectively studied. on hospital admission the following nutritional measurements were assessed: serum level of albumin, cholinesterase, delayed hypersensivity response , total lymphocyte count and weight loss, as were age and sex, duration of operation , operative blood loss, amount and type of blood given, pathological dukes' stage of the disease and the attending surgeon were also recorded. results : eighty-four patients (52.8%) were perioperatively transfused. thirty-six (42.8%) patients were given autologous blood , while 48 (57.2%) received homologous blood. no patients received both autologous and homologous blood. twenty eight (17.9%) patients developed postoperative infections. non transfused patients had a 9.3 % infection rate , those receiving autologous blood had a 13.9 % infection rate, whi]e in the homologous blood group the infection rate was 33.3% (p < 0.001). univariate analysis showed that infections were significantly related to operative blood loss (p<0.01), length of operation (p<0.05) blood transfusion (p<0.05) and attending surgeon (p<0.05) . multivariate analysis identified homologous blood transfusion as the only variable related to the occurrence of postoperative infections , while the other variables failed to reach statistical significance. blood transfusion (bt) remains an essential life-saving treatment for surgical patients. however, besides the beneficial short-term impacts, negative longer-term effects are observed, which include various alterations in the immune responsiveness. in surgical patients these alterations may contribute to the increased risk for infections and cancer recurrence. since relatively few data demonstrate immunologic changes occurring in other lymphoid compartments than blood after bt, we studied the effect of et on the frequency and responsiveness of immune cells in bone marrow (bm), spleen (spl) and blood (b) in a rat model. normovalemic, 2 month old rats were transfused intravenously with syngeneic heparinized venous blood (3x2ml, every other day), and 3, 7 and 14 days after the last transfusion bm cells ( leh is an experimental oxygen-carrying resuscitation fluid. since leh is cleared from the circulation primarily by the mps, its effect on the development of sepsis and the nature of its relationship with the mps remain a major concern. preliminary in vivo data from our laboratory failed to show any leh effect on the hemodynamic and hematologic responses to endotoxin lipopolysaccharide (lps) in the rat. in contrast, leh exacerbated the lps-induced tnfa production and early mortality. the exacerbation of early mortality by leh was attenuated by pretreatment with the tnfu synthesis inhibitor rolipram. ex vivo, peritoneal macrophages from rats treated with leh and lps have shown increased il-lg mrna signal as compared to lps alone. also, leh increased tnftx production by peritoneal macrophages in response to lps stimulation in vitro. additionally, recent pilot studies indicate that leh attenuates pma-induced superoxide production from rat peritoneal macrophages and that leh augments fmlp-induced migration of human monocytes. taken together, these data strongly support possible interactions of leh with the mps and therefore the nature of such interactions should be further explored. over the last decade, we have developed liposome encapsulated hemoglobin (leh) as an artificial oxygen carrying fluid, or blood substitute. our efforts have focused on studies to define the safety and efficacy of this resuscitative solutions. leh consists of distearoyl phosphatidylcholine, cholesterol, dimyristoyl phosphatidylglyeerol, and alpha tocopherol in a 10:9:0.9:0.1 mole ratio and can encapsulate hemoglobins of different origin (bovine, human, recombinant human). leh is fabricated using hydrodynamic shear to create an average particle size of 0.3 microns. leh can be lyophilized using disaccharides and stabilized in the dry state and easily reconstituted before administration. histopathology and clinical chemistries indicate that leh rapidly accumulates in tissue resident macrophages in small animals injected in the tail vein, principai1y in the liver and spleen. the consequences of accumulation in the reticuloendothelial system are manifest by transient increases in liver transaminases (ast, alt), bilirubin, and bun over 24-48 hours with no change in biliary function (ggt, ap) . clearance through the liver and spleen is observed over the course of 1-2-weeks. more recent attention has been focused on secondary consequences of leh administration especially with regard to inflammatory eytokines. leh does not elicit expression of tumor necrosis factor in vivo and in isolated macrophage cultures, but does result in a transient increase in serum il-6. we have also examined the interaction of leh with lps in vitro macrophage culture to further understand how this blood substitute may effect the immune system. we have labeled leh with technetium-99m (99mtc) to study the biodistribution of leh non-invasively in anesthetized rabbits. rabbits were infused with a 25% topload of leh (200 mg of phospholipid, 2.5 g of hemoglobin per kg of body weight) and imaged continuously with a gamma camera. at 20 hours, images were again acquired. animals were then sacrificed and tissue counts obtained, images revealed an initial rapid uptake bythe liver, 8% at 30 minutes and 15% by 2 hours. the spleen accumulated activity at a slower rate, 3% at 30 minutes and 7% at 2 hours. at 20 hours, autopsy biodistribution studies revealed that approximately 42.6% of the dose is in the blood pool, 15.4% in liver, 18.1% in spleen, 3.2% in lungs, 2.4% in muscle and 1.6% in urine, with trace levels in kidney, brain and heart (<1 °/o). in a hypovolemic model, rats were 10% or 50% exchange transfused with 99mtc-leh. in the 10% exchange model, 99mtc-leh was rapidly taken up by the liver and spleen with minimal activity in the circulation at 20 hours. with the 50% exchange, 50% of the leh was in circulation at 20 hours. the interaction of leh with platelets labeled with indium-111 was also studied. after infusion of leh, the labeled platelets rapidly moved from the circulation to the lungs and liver. over the next 30 minutes, the platelets gradually returned to circulation. this effect was not seen with iiposomes of the same lipid composition but containing no hemoglobin. non-invasive imaging is proving to be a very useful tool for the investigation of leh. the need for a safe, efficacious and commercially viable blood substitute is unequivocal. of the several strategies pursued to invent an adequate blood substitute, liposome entrapped hemoglobin (leh) has been already established as a leading possibility. major advances in liposome technology have already resulted in liposome preparations compatible with clinical use for drug delivery. recent technological advances made by the u.s. naval research laboratories resulted in the capacity to entrap hemoglobin into liposomes in a way which secludes hemoglobin from interacting freely with biological systems. the leh produced has already been tested in in vivo systems and was foun.d to be well tolerated. moreover, the leh originally produced as a solution can be transformed into a lyophilized form which can be reconstituted and delivered as a fresh solution. while important milestones in leh development for a practical blood substitute have been achieved, several issues remain to be explored. most notably, the long term consequences of leh on host defense mechanisms and, in particular, immune cell function. in addition, it is important to understand more fully the metabolic fate and repercussions of leh delivered at clinically relevant dose/schedule regimens. finally, while leh is a highly promising strategy for a blood substitute, the present formulations consist of human hemoglobin derived from human blood, to improve the safety profile, a recombinant preparation for liposome entrapment will be much desired, aa-ginine, a semi-essendai dietary amino acid, possesses several unique and potentially pharmacologic properties. argirdun is a potent secretagogue for pituitary growth hormone and prolacfin and for pancreatic insulin and glueagon; it modulates host protein metabolism by increasing nkmgen retention and enhancing wound collagen synthesis. it also is a potent t call function regulator. ait of these effects coupled with its relative lack of toxicity and safety make it an a~antive nulritionai pharmacologic agem (t). rodents fed supplemeutal arginine exhibit increased thymsc weight which is due to increased numbers of thymic lymphocytes present in the gland. thymic lymphocytes from animals fed supplemental ar~e demonstrate increased blastogenesis in response to coma. and pha (2) . peripheral blood lymphocytes from humans given supplemental arginine also have heightened mitogunic responses to mitogen or antigens (3) . in postsurgery padents supplemental arginine abrogates or diminishes the deleterious effects of trauma on lymphocyte responsiveness and restores peripheral blood lymphocyte responses much faster than observed in controls. overall host immunity is also enhanced by arginine. allograft rejection is enhanced and septic animals survive longer when given supplemental arginine (4) . tumor bearing urginine-supplemented animals have decreased tumor growth and enhanced survival (i). lastly, asgmine can induce t cell maturation and t cell mediated responses in athyrnic nude mice. arginine also has remarkable effects on host nitrogen metabolism post-injury. in increases nitrogen retention in healthy human volunteers and in surgical patients. this beneficial effect on overall nitrogen metabolism is accompanied by a unique effect on the healing wound. supp]emental arginine increases wound collagen synthesis which also translates into increased wound breaking strength (5) . arginine has no effect ou epithelialization. douglas w. wilmom, m.d. boston, ma gintamine is the most abundant amino acid in the body, but it has long been considered a nonessential amino aeid because it is synthesized in many tissues. fohov~g st,~'vation~ injury or infection, skeletal muscle pmteln inoresses its net tale of degradation and releases amino acids into the blunds~mm at an aocelerared rate. app~o)~mately one-third of the amino nitmgea is ghitamine, which is metabolized by the kidney where it parth:~pates in acid-base homeostasis, is the primly ~ for lymphocytes, mac~optmgcs and untexocyms, and contm'butcs to the synthesis of giumth~une. olmamine degrades slowly while in ~olu~ou, especially at usual room teml~mtums. because giulamine was considered nonessential, it has beer absent r'om nil intravenous and most gluts.mine should be considered a cendittona]ly essential nutrient for individuals with serious ilinesses, uspccially those confoanded by infcctinn and inflammation. over the uc~:t 5-7 years, glutamine will be incorgorated into most feeding formulas designed for patients with critical illness. 526 o]~ga-3 pufa there continues to much interest in the application of the 0mega-3 pufa in clinical nutrition. the basic principle has been that the 0mega-3 pufa will displace arachidunic acid and result in a decrease in eic0san0id production. in addition these changes in pufa will after the physical characteristics of the membrane including flujdity, receptor function and transmembrane signals. animal studies have shown that there is omega-3 incorporation with continuou~ enteral feeding both in control and endotoxic animals within 3 days. this includes the liver, spleen, circulating and alveolar marc0phages and the lung. this incorporation resuls in significant changes in the eicosan0id production including pgf 2 and ket0-pgflalpha. there is improvement in the cardio-vascular reep0nse of these animals with ~ecreamed lactic acidosis and improved cardiac contractility. as well there is improved immune function with improved t cell response to mit0gens. the ~ of a mumber of pharmacological agents blocking cicosanoid production can enhance the cell effects of 0mega-3 pufa. clinical studies using short term entsral nutrition with 0mega-3 either alone or with other enteral supplements in a number of clinical settings have shown significant 0mesa-3 incorporation and decreased eicosan0id production. these positive results must be discussed with the additional evidence that long term omega-3 supplementation decrease eic0san0id production but als0 induce a state of immune suppression that is capable of increasing transplant sunvival. these 10ng te~ inune effects may benefit clinical conditions including rheumatoid arthritis and cr0hn' disease early enteral nutrition instituted i~mediately afte~ injury will decrease the entry of bacteria into the intestinal wall and decrease the number of bacteria that translocate into the portal blood. these reductions are associated with & decreased catabolic response, decreased plasma cortisnl levels, end decreased vma excretion in the urine and prevention of mueosal atrophy. sdecific nutrients also affect the transloeation process. addition of arginlne to the diet significantly improves the ability to kill translocated organisms. however. translooetion across the gastrointestinal barrier is not affected. in contrast, glutamine diminishes the rate of translooation across the imtestinal barrier and also improves killing of the beetarla that do translooate. the omega 3 fatty acids in the form of fish oil slightly decrease the rate of translocation but more significantly increase the ability of the animal to kill translo~ated organisms, all three dietary additives, i.e. argini~e, glu=amine and fish nil. significantly improve survival, hut adding glyoine or medium chain triglyeeridem do not, combinations of srginine and glutamlns, glutamine and fish oil, and fish ell end arginine each improve survival, and to a greater degree than a combination of all three. these studies add further evidence that translocation is an important determinant of survival after injury, early feeding with immunonutrlent enriched dices will improve survival and dsarease transloeation to varying degrees, depending upon the nutrients provided. objectives: we studied effects of supplementing a commercial enteral diet, impact r (imp, sander nutr lnc), with fiber (imp/fib) or alanyl-glutamine (imp/ag, exogenous glutamine (gln) 14 gms/l) on influencing the incidence of bt to mesenteric lymph nodes (mln) in burned mice. fiber has been shown to improve gi integrity under certain stress/treatment conditions. the dipeptide ag is a water-stable source of gln, which is a specific fuel for many cells including enterocytes. traumacal (trcal), a high-protein, high-fat enteral diet (mead johnson iuc), was also studied, as well as rodent chow (harlan teklad inc), which contains very high protein & fiber. methods: anesthetized cf-1 mice aged 8-12 wks received 32% tbsa fullthickness dorsal burns & were resuscitated with 2 cc ip saline. diets were allowed ad lib; caloric intakes were comparable in all gps except fasted gp (fast 24 hrs, chow 24 hrs). at 48 hrs postburn mln were sterily removed, homogenized and plated on heart brain infusion agar; cfu/g mln tissue were determined. bt was analyzed by fishers exact test, cfu/g by anova-bonferroni. * p<0.01, ** p<0.05 compared to imp and burn-fast gps. background. infectious complications following trauma, major operation, or critical illness adversely affect hospital cost and length of stay (los). some key nutrients have been shown to possess immune enhancing properties. this multicenter trial was conducted to determine if early administration of an enteral formula supplemented with arginine, dietary nucleotides and fish oil can decrease los and infectious complications in icu patients. methods. this was a prospective, randomized, double-blind study of 326 adult icu patients who required enteral feeding for > 7 days. patients entered the study within 48 hr of the event, were stratified by age and disease, and were randomized to receive either the supplemented formula (impact®) or the conventional formula (osmolite ® hn). feedings were initiated at full strength and advanced to at least 60 ml/hr by 96 hr after event. results. both groups tolerated administration of formula well. for patients fed > 7 days, the median los was 25% shorter (p=o.ol) for the--supplemented group (21 days) compared to the conventional group (28 days). the incidence of most infectious complications was lower in the supplemented group, but this difference reached significance only for urinary tract infections (p=o.o05). the supplemented group had a significantly shorter los from onset of infectious complication until discharge for patients with pneumonia (19 vs. 27 days) and skin/soft tissue infection (19 vs. 37 days). conclusions. administration of the supplemented formula was safe and well tolerated. when fed > 7 days, it reduced the incidence of most infectious complications, and significantly reduced los. materials and methods: twenty-seven patients were randomised into 3 groups ( n= 9 each) to receive either a standard enteral formula, the same formula enriched with arginine, rna and omega 3 fatty acids (enriched group) or isonitrogen, isocaloric parenteral nutrition. early enteral nutrition was started within 12 hours following surgery (10 ml/hour). it was progressively increased reaching a full regimen on day 4. on hospital admission and on post-operative day 1 and 8, the following parameters were assessed: serum level of transferrin , albumin , prealbumin, retiool binding protein (rbp), cholinesterase. delayed hypersensitivity response, igg, igm, iga, lymphocyte subsets and monocyte phagocytosis ability were evaluated on admission and on post-operative day 1, 4, 8. the three groups were comparable for sex, age, cancer stage, type and duration of surgery, intra-operative blood loss and amount of blood transfused . in all groups a significant drop in all the nutritional and immunological parameters was observed on postoperative day 1. comparing post-operative day 1 versus day 8 a significant increase of prealbumin (p<0.02) and rbp (p<0.005) was found only in the enriched group. with respect to immunological variables an increased phagocytosis ability (p<0.001) and a significant recovery in delayed hypersensitivity response (p< 0.005) was observed only in the enriched group. conclusions : these data are suggestive for a more effective post-operative recovery of both. nutritional and immunological status in cancer patients fed with enriched enteral formula. gastrointestinal intolerance was equivalent (18% in each group) and laboratory screening confirmed that both diets were safe. when analyzing clinical outcome for all patients, there were no significant differences in septic complications (immun-aid = 41% vs vivonex ten = 35%), mean mof score (immun-aid = l.b vs vivonex ten = 3.6), or mortality (immun-aid 0% vs vivonex ten = 6%) . kowever, when analyzing the subgroup of patients with severe injury (iss or ati _> 25), patients receiving immun-aid appeared to have fewer septic complications (33% vs 50%) and their mean mof was significantly lower (1.8 _+ 0.7 vs 5.8 + 1.7, p = 0.05, student's t-test) . these preliminary data indicate that immun-aid is tolerated well when aggressively delivered immediately postinjury. the ultimate affect on clinical outcome appears ~avorable for immun-aid, but needs to be confirmed in larger patient groups. kemp?n, m., neumann, h.a., he i[michh b: as both increased, normal and reduced phagocytic capabilities of polymorphonuclear leukocytes (pmn) and monocytes in acute batterial infections have been reported, the role of phagocytes in patients with severe sepsis is less clear.we examined pmn and monocytes from 10 patients in septic shock and 17 heailhy votunteers for phagocytic function. phagocytosis was determined by flow cytometry (facscan) and was measured by the ability of pmn and monocytes to phagocytose e.coli marked with fluorescent antibodies. a septic shock was defined by the presence of a ~ource of i, nfoctiqn with a known bacteriology, distinct signs of a systemic response and defined minimum scores in 3 icu scoring systems indicating the presence of a multiple organ failure. additionally we examined how phagocytosis is influenced when a new enteral diet formulation containing substrates suggested to improve immune function or arginine, one of its major compononts, is added in vitro in defined concentrations and incubated for 10 minutes. pmn (p{o,05) and monocytes (p<o,01) of the septic patients showed a significantly enhanced phagocytosis compared tolhe phagocytes of the healthy group. the incubation with the enteral diet in vitro was followed by a higher dose-related rate of phagocytosis, especially in the healthy group, that was not statistically significant. after addition of l-argintne we atso observed an iqcrease in phagocytosis, that was lower than in the group with the complete diet. using a modern immunefluorescence-cytometric essay we founfl an improved phagocytic function in septic shosk. there are signs for an influence of nutrient substrates on phagocytosis which seems to be complex and should be further investigated. arg and gln were lower in the 49%-bcaa vs 16%-bcaa group; arg was related directly to arg dose and inversely to bcaa dose (p<o.o01 for all). clearances of bcaa increased with increasing bcaa dose (p<o.o01); arg and gln clearances were directly related to the bcaa clearances (rz=o.65, p<o.o0i). relationships between arg, gln and other aa seem to be ruled by patterns involving specific intracellular aa transport systems. regulation of arg and gln clearances through bcaa administration may be related to an increased flux of aa into synthetic processes. intestinal segments from rats immunized by infection with the nematode trichinella spiruus undergo intestinal anaphylaxis or type i hypersensitivity when challenged in vitro with parasite antigen. immunized rats exposed to t-radiation (7gy) and sustained on rat chow,fail to fully express type i hypersensitivity up to 14 days post irradiation (dpi). we hypothesized that enteral nutritional support immediately following irradiation may be effective in preserving mucosal immune responsiveness or in reducing the recovery time after radiation-induced injury. rats were infected with 3x103 t.spiralis larvae. thirty to fifty days later rats received 7gy t-radiation from a co source as total abdominal irradiation (tai). immediately following tai, rats were fed an elemental amino acid diet (eaad) and at various dpi sacrificed and tested in ussing chambers for local anaphylaxis, expressed as antigen-induced ci secretion. the normal ci secretory response to antigenic challenge which is suppressed after irradiation,was restored by 3 dpi when rats were placed on the eaad. antigen-induced c[ secretion is dependent on the interaction o~ antigen with specific ige antibodies on the surface o5 mucosal mast cells and their subsequent degranulation. mast cell-derived 5-ht,histamine and pg, together effect ci secretion.in irradiated rats on rat chow,the failure to respond to antigenic challenge appears to be associated with the destruction of mast cells. they are undetectable with standard staining procedures and mucosal histamine levels are drastically reduced. also,el secretion can be induced by direct stimulation of epithelium with cr secrctagogues and circulating ige levels are normal. despite a more rapid recovery of immune responsiveness in irradiated rats receiving eaad,mast cells were not restored. we conclude that the eaad contributes to an adaptation that supports the expression of local anaphylaxis in the absence of mast cells. total parenteral nutrition (pn) and bowel rest may influence the host response to infection. this study examined the different host response to infection in parenterally and enterally fed animals. forty-three rats were divided into pn group and total enteral nutrition (en) group. they received identically constituted isocaloric isonitxogenous diets for seven days. animals were then challenged intraperitoneauy with 3xl(y escherichia coli and survival were observed. in other experiments, they were sacrificed before and two hours after bacterial challenge. peritoneal cavity was lavaged with 10ml saline and peritoneal exudative cells (pec) were harvested and cultured in vitro for 24 hours with and without lipopolysaccharide (lps). colony f(m, ning units of bacteria (cfu-b) in the peritoncal lavaged fluid (plf) were determined and tnf in the plf, serum and pec culture supernal,ants were measured by l929 bioassay. twenty-fonr hour survival rate in en group was significantly greater than that in pn group (60% vs 0%). the data suggest that local immune responses of pn-treated animals may be depressed with consequent disturbance in the clearance of bacteria. this may lead to a greater systemic cytokine response and resulting in a poor survival after bacterial challenge in pn group. the effects of intragastfic tube feeding of diets enriched with m-6 polyunsaturated fatty acids (pufa) from safflower oil (so) or (o-3 pufa from menhaden oil (mo) on eicosanoid production, and onthe membrane phospholipid fatty acid composition were investigated in a severe acute septic shock model. the percent calories from fat maintained at 30% in the diets was adjusted to provide 10% each of saturated, monounsaturated, and pufa using olive oil and palm oil as filler fats. after feeding for 5 days, lipopolysaccharide (lps, 9mg/100g) was injected through the tail vein. the percent of animals surviving in the mo group was 67% (14/21), and did not differ significantly compared to 57% (12/21) in the so group. the plasma concentrations of tnf ct for the groups of animals fed so (1.2+3.0 ng/ml) and those fed mo (1.8+1.1) did not differ significantly. the levels of prostaglandin(pg)e2, 6-keto-pgflc~ and tumor necrosis factor-ct (tnf-c 0 were determined 90 min after lps injection. the fatty composition (relative mole%) of the liver was determined before (lps-) and 3h after the lps administration (lps+). the data (mean_+sd; n=6) are as follows. group these data indicate that a marked reduction in the plasma levels of poe2 and 6-keto-pgflct for rats fed mo diet was associated with a reduction in aa which was displaced by epa in the membrane phospholipids. further, in these rats, we observed that there was a 2.8% reduction in the percent of epa following lps challenge compared to the basal levels. however, for the group of rats maintained on so diet, the liver membrane phospholipid fatty acid composition before and after lps administration was unaltered. these finding suggest that although a reduction in aa was associated with a decrease in the production of pro inflammatory eicosanoids after 5 days of continuous mo feeding, there was no marked effect on the survival following lps challenge. animal models have been used to examine the effects of various nutrients and nutrient combinations upon the immune system. we studied the effects of standard and specialized enteral formulations on the response of mice to infectious challenge with listeria monocytogenes, influenza a or candida albicans in order to test the efficacy of specialized ingredients. cf-1 outhred female mice (12-15 g) were fed purina #5002 chow, commercially available osmolite hn ®, perative ® or impact*. mortality and tissue burden of pathogen were examined during the 21 d period following induced infection. the results indicate that there were no differences between the groups fed the liquid formulas with regards to mean survival time or % survivors in these models of infection. examination of spleens in the groups challenged with l. monocytogenes and kidneys in the groups challenged with c. albicans revealed no differences in tissue burden of pathogenic organisms. alterations in the length of pre-feeding from 0 to 8 days prior to infection did not affect these results. these data demonstrate that, contrary to previous reports, the use of specialized nutrients did not improve resistance to disease in mice challenged with l. monocytogenese, influenza a or c. albicans as compared with mice fed osmolite hn. animals fed standard chow tended to be more resistant to infectious disease than those fed the liquid formulas perhaps due to the form of diet or complexity of ingredients. the results indicate that these animal models of infectious challenge may be of limited use to distinguish effects of modified nutrient composition of enteral formulas. laboratories the beneficial effects of sesame oil (ses) have been attributed to the presence ofsesamin, a non-fat constituent which is claimed to iultibit a-5 desaturase activity. we investigated the effects of ad libidum feeding of ses enriched diet on fatty acid composition of phospholipids from plasma, liver, on eicosanoid production, and on the protective effects in mice after cecal ligation and puncture (clp) and compared with safflower oil (so) diets. olive and palm otis were added as filler fats to the so diet to maintain the ratios of saturated, and unsaturated fatty acids similar to ses diet. thus, while the total fat remained at 15wt%, the only difference between the two diets is the presence ofsesamin in ses diet. the animals were fed for 3 weeks. the incorporation of dihomo-y-linolealc acid (dgla: 20:3 o)-6) in the phospholipids from plasma and from the liver for the group of mice fed so diet ranged between 1-3% compared to the undetectable levels for those fed so diet. there were no significant differences in the incorporation of other fatty acids either in plasma or in the cell membranes between the two groups. following an intraperitoneal administration oflps (10p.g/kg body wt), the plasma levels of both pge2, and txb2, were significantly decreased (p<0.02) by nearly 50% for the group of animals maintained on ses diet compared to those maintained on so diet. these findings suggest that sesamin inhibited the release of arachidonic acid (aa) which upon accumulation is retroconverted to dgla which could reflect a modest increase in the content of dgla. failure to decrease the formation of aa could mean that the effects of sesamin on chain elongation process may be after aa is formed, and that sesamin may not inhibit a-5 desaturase activity. on the other hand, our data suggest that sesamin present in sesame oil inhibited the activity of cyclooxygenase which converts aa to its metabolites), or alternatively could block the activity of phosphalipase a 2 (pla2) (which releases aa from membrane phospholipids) as is evident from a marked decrease in eicosanoid production. the percentage of animals surviving after cecal ligation and puncture in rite group of mice fed ses diet was 65% (13/20) which was markedly higher (p<0.01) compared to only 20% (4/20) for the so group. increase in survival in the group of mice fed ses diet was associated with nearly 50% reduction in the production of tnf in response to lps i.p. challenge, for ses group compared to so fed animals. our data suggest that sesamin, present in sesame oil, decreased the production tnf~, inhibited the activity ofpla 2 and consequently offered a significant protection against clp. thus sesamin or sesame oil appear to be novel antiinflammatory agents which are present naturally in many edible products. [dept. surgery, ~e. deasoness hosp.,harvard medical school, boston, usa] stress causes alterations in the homeostasis of an organism with major changes in various organs, lifespans of cells, protein turnovers, metabolic pathways of activation or stimulation, energy mobilization, etc. punctual changes have been described in several organs and various cell types of the nervous, endocrine and immune systems. many stored or newly synthesized proteins are released to the bloodstream to be transported to target organs or to be disposed of. as stress alters protein synthesis and requirements in the targets, the bloodstream is a useful system for monitoring the relevant changes. we present here the pattern of newly synthesized or released serum proteins in c57bl/6 mice that have been stressed by immobilization. the proteins have been labelled with 3ssmethionine in vivo and the study was performed by two dimensional gel electrophoresis based on the method originally described by o'farrel. the electrophoretic run was carried out in an isodalt apparatus. a molecular dynamics laser scanner and the kepler image analysis system (lsb, rockville, usa) were used for modelling the radiofluorographic images. the radiofluorographic images of the gels from serum samples obtained from mice injected with i mci of 3ss-methionine reveal about 440 protein spots, from which a small fraction -about 20are altered in both qualitative and quantitative manner. the major changes are in the range of 25 to 60.000 d. the analysis displays a highly reproducible pattern, where clear differences between stressed and non-stressed conditions are shown. differences between patterns attributed to chronic vs. acute stress will be presented. patients with severe trauma or major surgery are known to acquire alterations in neutrophil functions which contribute to their enhanced susceptibility towards microbial infections. we analyzed neutrophil functions from fourty patients admitted for major surgery 6 days and 1 days preoperatively and on the ist and 6th postoperative days (study-days i, 2, 3, and 4) in a randomized placebo-controlled double-blind study. patients were divided into two groups, one ~eceived 6 days preoperatively an enteral nutrition enriched with omega-3 fatty acids, arginiee, and rna (impact), the other an isocaloric, not enriched control nutrition (placebo). meutrophils were isolated from the peripheral blood and stimulated with the ca-ionophor a23187 (7.3 pm). the capacity of the respective neutrophils to generate leukotrienes was analyzed by rp-hplc. neutrophils from impact group generated significantly higher amounts of ltb5 (mean values 6.6 ng/l x i~ cells) compared to the placebo group (2.6 ng) at study-day 2 [p, 0.01). in contrast, the capacity of neutrophils to produce ltb4 was not significantly different in both patients populations at study-days 2 and 3. the omega-oxidation of ltb4 to its biologically less active metabolites oh-ltb4 and cooh-ltb4 was not significantly different in both groups. however, neutrophils from group f patients generate more ltc4 at study-day 2 (p(0/5). the capacity of the patients'neutrophils to generate reactive oxygen radicals upon stimulation with fmlp, pma or the caionopbore a23187 exhibited patient dependent differences but no correlations to the repective nutritional supplementation as was measured by luminol dependent ohemiluminescense= these data indicate that an enteral nutrition enriched in omega-3 fatty acids lead to alterations in distinct parameters of neutrophil functions. clinical patient characteristics and mean caloric intake were similar between the two groups. both formula were tolerated well and the incidence of diarrhea was low. the number of infectious and wound complications as well as the apache ii score was evaluated for the two study groups. although the number of complications in the group supplemented with arginine, rna and omega-3 fatty acids was lower no significant difference in the occurance of infectious and wound complications has been observed between the two groups. however, the apache ii score indicated a significantly improved clinical outcome in patients with supplemented diet. in conclusion, early enteral nutrition with an arginine, omega-3 fatty acids and rna supplemented diet helps to recover more rapidly after surgical total parenteral nutrition (tpn} is associated with intestinal atrophy and dysfunction attributed to the absence of the non-essential amino acid glutamine in current parenteral nutrition solutions. in this animal study with 18 male wistar-rats we tested the hypothesis that glutamine-dipeptide supplementation during tpn for 10 days would restore small bowel atrophy and tpn induced dysfunction. a conventional tpn solution (250 kcai/kg bw) was compared to an isocaloric and isonitrogenous tpn (0,5 g n/animal/d) supplemented with alanin-glutamin (ala-gin) dipeptide (0,15 g n derived from ala-gin =30% gin-n). an enteral fed cqntrol group was included. mucosal thickness, villous height and architecture, absorption of water and glucose as well as dissacharidase activity of maltase and alkaline phosphatase were evaluated. total parenteral nutrition in rats causes villous atrophy, a significantly reduced absorption rate and a decreased activity of villous enzymes compared to an enteral fed control (p<o,01). addition of ala-gin to parenteral nutrition solutions prevents intestinal atrophy and restores functional alterations with a significantly increased rate of water and glucose absorption as well as a higher dissacharidase activity compared to the standard tpn group (p<0,01). there was no significant difference found between the enteral fed control and the dipeptide supplemented animals concerning intestinal structure and absorption, the enzyme activity was significantly decreased in the ala-gin supplemented group compared to the enteral fed control (p<0,01). in this rat model supplementation of parentera] nutrition solutions with ala-gin dipeptide restores tpn induced intestinal atrophy and dysfunction. boston. ~ usa injury, infection and major o1~'ations s~nulate a variety of factors whi~ initiate the body's response to th~ st~..sses. 'i"hese reactions are m~liated by a cbmage ia the neta'al hormonal, eac.ranmemt, by the elaboration of eytokines and tl~ougb the stimu/ation of other inflammatory mediators. this eatabolio setting zesulis in body protein loss, which g-taduai1y erodes both fimetianal and stm~ tissue. wiu3_b the normally nom'ished individual can withslzod "d~ response for a brief period of time, ircolonged eatabollsm l~ associated with ~,nmunosuppmssion, poor wound healing, and proioagcd convalescence. surgeons have rej2ed on intraveaous or eater~ feedings to ~everse this process. a va~ety of data now indicates that it is extremely di~edt to replete protein-containing tissue d~g the eataholie state of illness; the umutt response to hypercaloise feedings in this setting is the incxeased accretion of body fat md water. administration of gxow~ ho~moae ~cesults i.a n shift of the body's oxidative machinery to fat rather than eazbohydmte utiq~afic~; ¢oncomitaatiy, protein synthesis is stimulated. this metabolic state may have clinical utility in p~e.n.ts who need to heal large wotmds, in those who need to gain strength in order to be w~ed 5ore veatilatory support, or those patients who ~ve mulfiorgan failure and nee~ to ealaaace protein syatsesis to in.suze recovery. 235als are now in progress to dcterm~e the benefit of growth hormone in these and other disease state.s, rn the fature, the increased use of growth hormone, will occur;, this and other growth factors will serve to su~2po~ the host and shorten convalesceace following catabolic diseases. our objective was to study effects of pre-trauma growth hormone (gh) treatment on liver and skeletal muscle metabolism in a trauma model in piglets. twenty-four piglets were randomized to three treatment groups; one group was pretreated with gh 24 iu daily three days before the experiment (gh-3), another group treated with gh 24 ie at the start of the surgical procedure (gh-1) and one group served as untreated controls (con). they underwent a standardized surgical procedure to place sampling cathethers in the portal, hepatic and femoral veins and doppler flow probes around the portal vein, the hepatic and the femoral arteries. all pigs recieved a primed constant infusion of u-14c-glutamine. substrate fluxes were measured one (lh) and five (5b) hours after termination of the surgery. nitrogen excretion was significantly decreased in the gh treated animals (gh-3:30.8+4.2 mg/kg, gh-i: 37.6+_.5.8 mg/kg, con: 55.4+-3.1 mg/kg, p=0.004). in the hindieg, there was reduced net glutamine efflux due to decreased absolute glutamine release (gh-3:0.9 + 0.8gmol/min at lh and -2.4+1.4 in.tool/rain at 5h, gh-i: -3.4+1.5 i.tmol/min at lh and -1.4+1.2 pmol/min at 5h, con: -8.7+_2.8g mol/min at lh and -11.6+3.4/amol/min at 5h, p=0.005, p=0.006), whereas the absolute uptake of glutamine in hindleg was unchanged (p=0.84). glucose uptake in the hindleg was decreased (p=0.0006). net glutamine uptake in liver was attenuated (p=0.03 at 5h), whereas net glutamate release from liver was increased (p=0.005). thus, pre-trauma treatment with gh improved nitrogen economy, attenuated net glutamine loss from hindieg due to decreased absolute release, and attenuated hindleg glucose uptake. liver net glutanaine uptake was decerased and net glutamate release increased. patients with major abdominal surgery exhibit a considerable catabolic response with negative nitrogen balance and protein breakdown, which may have detrimental effects on outcome. we investigated the effects recombinant human growth hormone on substrate metabolism in parenterally fed patients. 48 metabolic healthy patients were randomized to receive either placebo or hgh in a dosage of 0,075; 0,15 or 0,3 i.u. per kg bw. substrate oxidation rate, energy expenditure as well as short life proteins were measured daily. six patients were excluded from analysis as drop outs, due to surgical complications. we could find a dose-dependend effect of growth hormone on resting energy expenditure, carbohydrate oxidation, fat and proteine oxidation. with increasing growth hormone resting energy expenditure increased from 20.9 calories per kg body weight to 26.6 calories. this was mainly due to an increased carbohydrate and fat oxidation, while proteine oxidation decreased. this was in accordance with a decreased urea.production rate and an improvement in cumulative nitrogene balance, which increased from minus 12.7 to plus 8.5 g n / 7 days. in consequence short life proteins were also increased. the only negative side effect was an increased blood glucose concentration in some of the patients, making insuline administration mandatory in 3 patients. our results are in accordance with other studies showing improvement of nitrogen balance, maintainance of lean body mass and muscular strength. if growth hormone administration may have any impact on morbidity, mortality and length of hospital stay in these patients it should be evaluated in a larger number of patients. in 30 patient after liver transplantation (oltx) the effect of recombinant human growth hormone (rhgh) on protein metabolism and hormonal changes was studied. patients and methods: the underlying disease in all patients was end stage liver cirrhosis, non of the patients included in the study was suffering from malignancies. the patients were randomly allocated to receive either 81u of rhgh bid (sc) or no alternative medication. the study period lasted 14 days. from daily 24 hrs udne collection urinary nitrogen loss and daily loss of urea were determined. body compostion analysis (bca, bioimpedance, akern-ltaly) was performed preoperativly and at the postoperative days 7 and 14 to evaluate changes in body water and body cell mass (bcm). the effect on resting energy expenditure (ree) was analysed using indirekt calorimetry wrhin the same intervalls (metabolic monitor, datex). blood levels of gh, igf1 and igf2, igfbp3 were measured daily during the study period, on day 3 a 24 hour profile of gh was.performed. samples were collected each 20 minutes. results: cumulative udnary nitrogen and urea loss were not signifcantly different for the whole study period but for the first 4 study days. bia indicated for the rhgh-group a loss of bcm to a lesser extent than for the controls (n.s.), changes in bodywater where similar for both groups. ree changes (kcal/kg bcm) were not significantly different for the two groups. blood levels of gh and igf1 differed significantly between the groups for the whole study period, but not igf2 and igfbp-3. the circadian rhythm of endogenous gh-excretion had not been altered by gh, but absolute levels of gh were increased. conclusion: during the first 4 days after oltx we could proof the protein sparing effect of gh treatment. although the differences between the two groups were not significantly different for the whole study period the results showed on all days a less catabolic situation for the rhgh treated patients. to possibly improve protein balance after major abdominal surgery we studied the effects of gh on protein metabolism after ltx. excluding malignant diseases, 29 candidates for ltx were randomized to either postoperatively receive the usual treatment (co=control group, n=14, age range 25-59 yr, bmi 23.9+5.7 kg/m 2) or for 14 days additional gh (2x8 i.u.s.c.) (gh group, n=14, 21-59 yr, 21.8+2.2 kg/m2). metabolic studies (calorimetry and lsc-leucine infusion) were performed 5 (test a) and 11 days (test b) after surgery. tracer analysis was done by gas chromatography-mass spectrometry. during the 2-week study clinical parameters did not differ significantly between groups; however, there was a tendency for increased glucose levels, insulin need, and energy expenditure at the time of test b in the gh group. leucine oxidation (x+se) during test a;b was 117+10; 111+15 (co) and 110+7; 100±12 (gh) pmol/kg ffm/h (n.s.). protein breakdown was 134±16; 131+30 (co) and 156+14; 178+30 (gh) pmol/kg ffm/h (n.s.). nonoxidative-leuine disposal was 119±9; 127+16 (co) and 139±11; 177+20 (gh) pmol/kg ffm/h (p<0.05 for the rise in the gh group). tracer data were supported by cumulative urea nitrogen excretion (days 1-4: gh 52+3 vs co 66±7 g; p<0.05). we conclude that after major abdominal surgery gh lowers nitrogen excretion by increasing total body protein synthesis. it is not known which organs contribute to this effect. in several investigations it has been shown that the protein saving effect of recombinant human growth hormone (rhgh) in the postoperative state is accompanied by raised igf-1 levels in plasma. it was concluded that the anabolic effects of rhgh were probably, at least in part, mediated by igf-1. this study was aimed at detecting the efficacy of igf-i on modulation of the protein metabolism in the catabolic state. patients and methodes: 38 patients (both sexes, age: 40-75 years, bmi 17-30 kg/m 2) with major upper gastrointestinal surgery (gastrectomy or partial gastrectomy) received 80 ug rhlgf-1/kg body weight/twice daily or placebo during 5 treatment days beginning on the first postoperative day in a double-blind, randomized study. all patients received hypocaloric and hyponitrogenous total parenteral nutrition (3g chng, o.6g aa/kg) troughout the whole study period. cumulated nitrogen balance, 3-methyl-histidin exretion in urine as well as serum-igf-1 levels have been determined. the two tailed wilcoxon test was used for statistical analysis results: first results will be presented previous studies have suggested that growth hormone (gh) potentiates various activities of leukecytes. however, it is not clear whether gh can improve survival of animals with gram-negative sepsis. we investigated the effects of gh on host response to infection in septic mice model. methods balb]c mice were randomized to 3 groups (n=10/gronp): gh-high (4+8mg/kg/day), gh-iow (0a8mg/kg/day) or control (saline). following subcutaneous treatment (every 8 hours for 6 days) with gh or saline, mice were challenged i.p. with e.coli (lxl08/body). survival rate was recorded every 2 hours. in the next experiment, gh-high and control animals were sacrificed 4 hours after bacterial challenge. peritoneal cavity was lavaged with 3ml saline and the peritoneal exudative cells (pec) were harvested and counted. colony forming units (cfu) of bacteria in the peritoneal lavaged fluid (plf), liver, lung and blood were determined. pec were cultured in vitro for 24 hours with lipopolymccharide (10p.g/ml in normotensive adults growth hormone (gh) rises when clonidine challenge is performed. recently evidence was shown for gh to accelerate wound healing. while gh secretion patterns are inconstant the gh-dependant insulin like growth factor (igf-1) and the insulin like growth factor binding protein (igfbp-3) reflect the integrated gh secretion over days. since we administer clonidine to patients with acute alcohol abuse we determined plasma levels of igf-1 and igfbp-3. materials and methods: 21 patients sheduled for esophagus resection were investigated once they had given written informed consent. 8 patients showed acute alcohol abuse and were treated with clonidine postoperatively• 13 patients with no history of acute alcohol abuse served as controls. besides liver enzymes igf-1 and igfbp-3 were determined. results: both groups had a comparable hepatic capacity of synthesis with decreased cholinesterase levels as to be expected after a major operation. regression analysis of igf-1 and igfbp-3 levels showed no differences between the groups. discussion: igf-1 and igfbp-3 plasma levels are not increased in normotensive patients who are treated with clonidine for prevention of postoperative alcohol withdrawl syndrom. further studies are required to discriminate whether our results are due to impaired postoperative liver function or if clonidine has not any impact on the gh-igf-axis in patients with alcohol abuse. recent studies have revealed that igf-i has several immanoregulatory roles. however, little is known about its effects on septic animals. we tested whether igf-i could increase the survival o f mice challenged intraperitoneally (i.p.) with e. coli. m~thqd$ balb/c mice received either high dose igf-i (n=10, 24mg/kg/day), low dose igf-i (n=10, 2.4mg/kg/day) or saline (11=10) every eight hours subcutaneously for six days. animals were then challenged i.p. with lx108 e. coli. survival was observed every two hours. in the other experiment, mice that received high dose igf-i or saline were sacrificed four hours after bacterial challenge. peritoneal cavity was lavaged with 3ml saline and the peritoneal exudative ceils (pec) were harvested and counted. colony forming units (cfu) of bacteria in the peritoneal lavaged fluid (plf), liver, lung and blood were determined. in addition, pec were cultured/n vitro for 24 hours with lipopolysaccharide (10).tg/ml). tumor necrosis factor (tnf) in the supernatants of pec culture, plf and serum were measured by l929 bioassay. results igf-i improved the survival rate at a dose of 24mg/kg/day. severe chronic neutropenia (scn) is a disorder characterized by severe neutropenia secondary to either a maturational arrest of myelopoiesis at the level of promyelocytes (kostmann-syndrome; scn) or regular cyclic fluctuations in the number of blood neutrophils with a median absolute neutrophil count (anc) of less than 500/~tl (cyclic neutropenia). patients suffering from scn have an impaired acute inflammatory response to injury and an increased susceptibility to infections, i.e. bacterial or fungal infections, resulting in chronic oral inflammation, severe pneumonia, abscess formation and bacteraemia. we have treated 36 patients (32 scn, 4 cyclic neutropenia) with r-methug-csf (filgrastim). thirty of 32 patients with scn and all 4 cyclic neutropenia patients responded to this treatment with an increase of the median anc to greater than 1000/~tl. the dose of r-methug-csf needed to achieve and maintain the response varied between 0.8 and 120 ~tg/kg/d. long-term treatment did not exhaust myelopoiesis: the anc remained stable after up to 6 years of treatment and antibodies to r-methug-csf were not detected. the increase of anc was associated with dramatic clinical responses: significant reduction of severe bacterial infections, reduction of intravenous antibiotic treatment, and reduction of hospitalizations. no severe bacterial infections occurred in any of the r-rnethug-csf responders during longterm treatment. severe adverse events, most likely due to the underlying disease, included the development of mds/leukemia in two patients, and osteopenia/osteoporosis in 12 patients. these results demonstrate the beneficial effects of r-methug-csf treatment in severe chronic neutropenia patients. the newborn is predisposed to mortality during infections due in large part to developmentally immature host defenses. recently cytokines have been identified that regulate the development of these defenses. one such cytokine is specific for neutrophils and is termed granulocyte colony stimulating factor (g-csf). in the studies to be discussed, we have attempted to impact hematopoiesis in the fetus using exogenous rhg-csf delivered through the pregnant dam. our rationale was that an enhanced myeloid system may result from such in utero treatment leading to, perhaps, enhanced protection to microbial insult. rhg-csf crossed the placenta and reached peak fetal serum concentrations 4 hours after administration. these levels were 1000-fold lower than the levels detected in the adult dams serum. white blood cell counts were elevated approximately 2-4-fold over control newborn blood. this increase was due to circulating numbers of neutrophils. the marrows from these pups were hyperplastic consisting of predominately immature and mature neutrophilic cells. no changes were seen in the thymus or livers. pups born to mothers treated with rhg-csf since day 15 of gestation (parturition in rodents occurs on approximately day 21) survived a lethal challenge of group b-13 hemolytic streptococcus. in contrast their untreated yet infected counterparts did not survive. recent clinical developments have led to trials of rhg-csf in certain neutropenic states including cyclic and chronic neutropenia. we have identified several of these patients who were pregnant during treatment with rhg-csf. biologically and anfigenically identifiable rhg-csf was detected at increased levels in the cord serum of these neonates. we will discuss our findings with these patients in light of our animal model of neonatal myeloid development. we have previously demonstrated decreased g-csf production and g-csf mrna expression from activated mononuclear cells from newboms compared to adults (cairo, pediatr res 31:574, 1992) . we have also recently observed that administration of a single dose of rhg-cse to one-day-old newborn rats induced significant neatrophilia, while administration for 7 days induced neulrophilia and increased the bone marrow (bm) neutrophil storage and progenitor pools (cairo, blood 76:1788 , 1990 cairo, pediatr res 27:612, 1990) . we embarked on a phase i trial exploring the safety, pharmacokinetics, and biological efficacy of g-csf in newborns with presumed sepsis. 42 infants <72 hrs old with a diagnosis of presumed sepsis were stratified into three groups: very preterm (26-30 wk), preterm (31-35 wk), and term (>36 wk) and randomized to receive either a placebo or 1, 5, and 10 gg/kg/qd or 5 and 10 p.g/kg/bid of rhg-csf infused by pump over 1 hour for 3 consecutive days. cbcs were obtained at 2, 6, 24, 48, 72 and 96 hrs. tibial bone marrow aspirations were performed 72 hrs after study entry and differential counts and cfu-gm pools were determined. c3bi expression was determined at 0 and 24 hrs after rhg-csf, and g-csf pharmacokinetics were performed after the first dose of rhg-csf utilizing a sandwich elisa. a significant increase in the anc was observed at 48, 72 and 96 hrs following administration of both 5 and 10 ~tg/kg/d of rhg-csf. the maximum increase in the anc occurred 72 hrs after 5 and 10 ~tg/kg/d (397 -116%) (p<0.01) and (621% -+ 200%) (p<0.001), respectively. there was a significant dose-dapendeat increase in the bm neutrophil storage pool (18 _+ 4% vs. 62 + 6%) (p<0.01) (placebo vs. 10 ~tg/kg/d). there was no significant difference in the nantrophil proliferative pool. an increase in cfu-gm and cfu-gemm was seen at all doses tested, compared to placebo (53.5 _+ 8.6 vs. 87 -+ 15) (colonies/l(p cells/plate). c3bi expression was significantly increased 24 hrs after 10 bg/kg/d of rhg-csf (233 + 81% vs. 83 +-26%) (p<0.012). peak serum g-csf levels occurred at 2 hrs and were dosedependent. the half-life of rhg-cse was 4.44 + 0.4 hrs. most importantly, there was no observed toxicity from g-csf in all patients studied. 31 of 42 patients were on ventilators prior to administration of rhg-csf and there was no increase in pulmonary toxicity. these preliminary data suggest that rhg-csf is well tolerated at all gestational ages in newborns with presumed sepsis. a multi-center phase ii/iii randomized double-blindad placebo controlled trial is required to determine the efficacy of rhg-csf in this clinical setting. we investigated the effects of recombinant canine granulocyte-colony stimulating factor (g-csf) on survival, cardiopulmonary function, serum endotoxin levels and tumor necrosis factor (tnf) levels in a canine model of lethal bacterial septic shock (clinical research.41:240, 1993) . methods: awake 2 ylo beagles had serial cardiopulmonary and laboratory studies before and for up to 10 days after intraperitoneal placement of an e. celi infected clot. nine days before and daily until 3 days after clot placement, animals received high (n=17) or low dose (n=17) g-csf or protein control (n=20) subcutaneously. results: survival in high dose g-csf animals (14/17) was significantly improved compared to low dose (10117) and controls (12120) (p<0.04 wilcoxon). high dose g-csf also improved cardiovascular function evidenced by a higher mean left ventricular ejection fraction (day 1 after clot, p<0.001) and mean arterial pressure (day 2, p<0,02) compared to low dose and controls. high dose rcg-csf increased (p<0.001) peripheral neutrophil numbers both before and after clot implantation (2 hours to 4 days) compared to low dose and controls. in addition, high dose rcg-csf produced a more rapid (p<0.0001) rise (day 2) and fall (day 4) in alveolar neutrophils determined by bronchoalveolar lavage compared to low dose and controls. lastly, high dose rcg-csf decreased serum endotoxin (2 to 8h, p<0.002) and tumor necrosis factor (tnf, 2h, p<0.02) levels compared to low dose and controls. discussion: these data suggest that therapy with g-csf sufficient to increase peripheral neutrophil numbers during peritonitis and septic shock may augment host defense and endotoxin clearance, reduce cytokine levels (tnf) and improve cardiovascular function and survival. the use of g-csf in sepsis prophylaxis in neutropenic patients is well established and has been ascribed to accelerated recovery in granulccyte counts. here, an additional sepsis-prophylactic property could be demonstrated in healthy volunteers: eleven volunteers were employed in a sinqle-btind, controlled study and were given 480 uq g-csf or saline placebo via subcutaneous injection. blood was withdrawn immediately before and 20 or 44 hours later. lps-inducible tnf, il-1, stnf-r p75 and il-lra were assessed in the supernatant of whole blood incubations stimulated with 10 ug/ml lps from salmonella abortus equi. similarly to previous animal studies, lps-inducible tnf was attenuated by about 50% 20 hrs. after treatment. the same was true of il-lb. in contrast, lps-inducible stnf-r p75 which was indetectable in blood incubations from untreated donors increased dramatically 44 hrs. after g-csf treatment. il-lra found after lps challenge was increased tenfold by g-csf treatment. it is concluded that g-csf treatment switches peripheral leukocytes to an antiinflammatery state characterized by an attenuation of il-i and tnf releasing capacity and an augmentation of the release of cytokine antagonists. this findinq minht offer a novel concept in septic shock prophylaxis. objective.the aim of the study was to investigate the effect of recombinant human g-csf (rhg-csf) on survival, bone marrow neutrophil myelopoiesis, neutrophil counts, levels of bacteria and some important sepsis mediators in a model of rat abdominal sepsis. lethal peritonitis was induced with a 4 mm coecal perforation (cp) in male wistar rats. rhg-csf was administered as 10 /.tg/kg iv every 12 h, first dose at sepsis induction. bone marrow neutrophi] progenitors were determined as blast colonies, cfu-gm and cfu-g. neutrophils and bacteria were determined in peripheral blood and peritoneal fluid. lps, tnf, endothelin 21 and lactate were measured in blood from femoral vein. mortality rates were registered with g-csf treatment starting either 1 or 7 days before or 4 hours after cp. results. mortality was reduced from 90% to about 50% with rhg-csf intervention and there was no difference between the pretreatment and treatment groups. bone marrow blast colonies were not influenced while neutrophil myelopoiesis was augmented at the stages of cfu-gm and cfu-g. neutrophils in blood and peritoneal cavity were enhanced and numbers of bacteria in the same compartments were substantially reduced. circulating lps, tnf, endothelin 21 and lactate were attenuated the first 24 hours after cp. neutrophil myelopoiesis is augmented with increased number of neutrophils in blood and peritoneal cavity, resulting in enhanced clearance of pathogens. lps, tnf, endothelin 21 and lactate are suppressed the first 24 hours during sepsis course. a. wendel, j. barsig, g. tiegs gm-csf stimulates the proliferation and differentiation of granulocytic and monocytic progenitor cells. in addition the hemopoietic cytokine activates the inflammatory response in mature leukocytes. the priming effect of gm-csf towards lipopolysaccharide (lps)-induced cytokine production in vitro has been described, but little is known about proinflammatory gm-csf effects in vivo. we detected gm-csf in plasma of lps-challenged mice with kinetics similar to tnf, reaching peak levels 2h after lps administration. gm-csf pretreatment (50 ~tg/kg i.v.) enhanced mortality in mice challenged by a sublethal dose of lps. plasma levels of tumor necrosis factor (tnf) and interleukin-6 (il-6) were significantly enhanced. a monoclonal antibody, which neutralizes gm-csf bioactivity, rendered mice less sensitive towards lethal lps-challenge. tnf-and il-6-tevels were reduced in these mice compared to control animals without antibody treatment. in addition, severalfold potentiation of lps-induced cytokine release by gm-csf was observed in vitro in murine bone marrow cell cultures. these data demonstrate the proinflammatory capacity of gm-csf and suggest that the hemopoietic cytokine plays also a role as an endogenous modulator of lps toxicity. immune dysfunction, developing in the wake of multiple trauma, overwhelming infection and other forms of critical surgical illnes% is associated with increased infections, morbidity and mortality. the mechanisms responsible for alterations in immune regulation are incompletely understood but monocyte appear to play a central role. polymorphonuclear leukocytes (pmn) are known to play a central role in the inflammatory response of the host toward invading microrganisms. reports of defects in all the aspeots of pmn function have been accumulated in recent years. the possible role of gm-csf in modifing the state of immuno suppression detected in severe intraabdominal infected pt~. inspite of surgical appropriate procedures and in reducing the expected mortality is investigated. the safety of rh-gm-csf administration in sepsis is also evaluated. a double blind randomized study is proposed. this study include 60 icu patients who do not exhibit signs of shock and/or ards, with clinical signs and symptoms of abdominal infection. immunodepressed patients-aids, chronic chemotherapy or chronic steroid administration do not partecipate to the study. patients will receive rgm-csf (l~g/kg/day) or placebo in 24 hs. continuous infusion for 15 days. safetyandefyieacy will be assessed till to day 30. the apache ii score is adopted for risk stratification of patients because it is reliable and validated, objective and composed of information that is indipendent of diagnostic criteria. patient's entry criteria is apache ii > 16 (score 16 corresponds to expected mortality rate of 38%).in this protocol the surgeons report the judgement of the efficacy of surgical procedure to remove or not the focus of infection. objectives: infections and subsequent septic responses remain the leading cause of death among surgical intensive care (sicu) patients despite tmprovetaunts in supportive care and brond-epectrum antibiotics. usually invading bacteria are efficiently cleared by neutrophil granulocytes. however, during sepsis various neatrophil dysfunctions have been demonstrated, leading to impaired host defense. granulocyte colony-stimulating factor (g-csf) induces a sustained increase in circulating neutrophils and enhances various noutrophil functions. it was the purpose of the present study, to evaluate the safety and efficacy of g-csf (filgrastim) in sicu patients at risk of sepsis. materiel a.d methods: the study was designed as an open-label phase-ll study of filgrastim. ten consecutive slcu patients, with a therapeutic interveotion score greater than 30, were included in the study. filgrastim was given by daily continuous intravenous infusion for 7 days or discharge from the sicu. apache ll-score, multiple-organ-failure (mof) score, definitions of infections, sepsis, systemic inflammatory response syndrome (sirs), and acute respiratory failure were applied daily. a response to filgrastinl th_erapy was defined as an improvement in disease severity quantified by a decrease of > 4 apache 1i score points on day 4 after onset of treatment. results: none of the 10 patients developed a sepsis or mof later on and no patient died during hospitalization. specific postoperative complications occured in one patient ~jth a leekage of the oesophagou-gastric anastomosis after oesophageus resection. at study entry the leucocytes amounted to 10.110 + 2.000/~tl (mean + sem) and reached a level of 29.280 +_ 5.810/tal at day 6 after onset offilgrastim therapy. the apache ii score initally was 14 + 1.57 (mean + sem) and as an indicator of filgrastim response a decrease of 5 points ~dthin 4 days oceured in 7 out ot 10 patients. filgrastim was well tolerated, side effects were not noted. growth of solid tumors might be modulated by the activity of inflammatory and/or immune effector cells of undefined specificity. in this study patients undergoing surgical treatment for gastric (n= 41) or colorectal (n= 47) cancers were evaluated for endogenous serum levels of granulocyte colony-stimulatingfactor (g-csf) during a pre-and postoperative time period. from the same blood specimens mononuelcar cells (mnc) were prepared. the release of ifn-%, and il-2, which are secreted by thl cells, were stimulated in vitro by pha during a cell culture period up to 48 hours. the patients were further classified for their immunreactivity by responses in dth skin testing to seven different antigens (e.g. tetanus toxoid, ppd, diphtheria toxin, trichophyton, streptococcus, candida and proteus antigens). dth testing has been repeated in each patient two remarkable results were obtained. the serum levels of endogenous g-cse showed a biphasic increase with maximum values of 900 pg/ml (preoperative < 70 pg/ml) on day 1 and day 3 to 5 after surgical treatment. similar patterns of g-csf production were found in both groups of patients with gastric or colorectal cancers. high serum levels of g-csf were significantly (p < 0,01) correlated with infectious complications in patients whh gastric cancer (n= 17/22). secondly patients could be arranged into two groups according to an anergic (n= 25) or normergi¢ (n = 63) responsiveness in dth testing. the frequency of anergi¢ responsiveness was similar in both patients with gastric (n= 11/41) or colorectal (n= 14/47) cancers. interestingly we found a significant correlation (p < 0,01) between low serum levels of g-csf and anergy during the postoperative period in both groups. stimulation of mncs from anergic patients (n= 30) within the pre-and postoperative period resulted in reduced mean values (about 50%) for ifn-ff release (preoperative means llo0 pg/nfl), if compared to patients with normergic dth (n= 63, preoperative means 1960 pg/ml). similar, but less significant results were obtained for il-2 secretion. our results confirm a correlation between infectious complications and g-csf in the postoperative period, however elevated levels were also found in some patients without any signs of infections. more interestingly there might be an association between cytokine (c~csf, ifn-% and il-2) release and dth, which is known to be mediated by activated thl calls. to recognize anergic dth as a possible higher risk in the postoperative outcome of cancer patients extended periods of observation are needed. objectives of the study effects of recombinant huraan granulocyte colony-stimulating factor(rhc-csf)a galnst severe septic infections were investigated by its single use or by its corn b{nation with cephera antibiotlcs.we examined its effects on the mortality,and circulating blood neutrophyis counts and functlons,such as phagocytic activity and h 2 02 production using the rat severe septic model. rats were subcutaneously administsrd rhc~csf(s0orl0o ~ g/k~ body wt)after on set of peritonitis brought about by cecal ]igation and one puncture withe2-gaug e needle once a day for three days.in addjtlon,cefmetazol na(cmz)(50m$/k9 bo dy wt)was injected intrarnustularly to the rats tv~ce a day for three days. cirehlatlng blood neutrophyls counts were determoned electronically with a hem ocytometer,and blood smears stained with may~runwaldm.qlemsa~taln. neutrophyls functions in vltro,such as phagocytic activity and h 20 2 producti on using the rat severe septic model was analyzvd by automated flow cytometri c single cell-analysis methods. the reortallty rate after 2 weeks was significantly decreased by administratlon of rh~-csf(p<0,01).ln addjtion,a combination therapy of rhg-csf wlte cephern ant~biotics(cmz)showed a significantly survive] advantage and the rate had b een reached 57.1%. nextly,treatn%ent wlth rhg-csf(s0 ~ $/k9 body wt)increased the nuzaber of the peripheral blood neutrophjls slgn[fieantly(p<0.01). iv~oreover,functions of neutrophlis which were phagocytic activity and h2 02 p roduction were remarkably enhanced by admlnlstratlon of rhg-cs~(50 ~ 9/ks b ody wt) (p<0.(15). these findings suggest that combination therapy of rhcrcsf with cephern antib iotlcs(cmz)is an efficient regime against severe infectlons.and the increased ne utrophils counts and enhanced neutrophiis functions were played a important ro le about the survival advantage. granulocyte macrophage colony-stimulating factor (gm-csf) is a haematopoietic growth factor active on neutrophils and macrophages. leukopenia often occurs following renal transplantation and can be associated with infection and/or the myelosuppressive effect of azathioprine. aim: we report the use of gm-csf in 17 renal allograft recipients with leukopenia. nonglycosylated recombinant gm-csf was obtained from e. coli transvected by human gm-csf gene. m~terial ~,nd methods : written informed consent was obtained from all patients. patients were suffering from toxic neutropenia (neutrophils < 500/mm 3) with medullar hypocellularity on bone marrow aspiration, or leukopenia (neutrophils < 1000/ram 3) with cytomegalovirus infection requiring ganciclovir administtation. gm-csf was given subcutaneously at a dally dose of 2 to 5 mcg/kg/day, according to renal function. results : in all cases, neutrophil counts returned to normal levels within 1 to 4 days. in most of them, spectacular correction was observed within 24 hours, with a single injection. adverse events due to gm-csf at this dose were mild and easily managed (2 cases of bone pain treated with paracetamol). one acute rejection episode was observed after correction of leukopenia. conclusion : on the basis of this study, it appears that gm-csf at a dose below 5 mcg/kg/day is an effective treatment for renal transplant recipients with leukopenia associated with cmv infection or toxic neutropenia. department of nephrology, 161, rue de s~vres, hopital necker, 75743 paris, france. changes in serum g-csf and il-6 after surgical intervention hitoshi toda 1, atsuo murata 1, hidewaki nakagawa 1 , takesada mori 1, nariaki matsuura 2 1osaka university medical school, osaka, 2wakayama medical school, wakayama, japan we measured serum immunoreactive interleukin 6 (il-6) and granulocyte colony-stimulating factor (g-csf) levels of the patients undergoing major thoraco-abdominal surgery for esophageal cancer. serum samples were collected from eight patients on the day before surgery, at the time of operation, and thereafter at suitable intervals for one week. il-6 and g-csf were measured by means of enzyme linked immunoassay. the normal range of serum ]l-6 was less than 2 pg/ml and g-csf less than 4 pg/ml. values between groups were compared with linear regression analysis. both serum g-csf and il-6 levels reached their maximal levels at the first postoperative day and decreased thereafter. the correlation between g-csf (y) and il-6 (x) was y=3.28x+6.16 (r=0.786, n=86, p<0.01 ), showing a significant correlation. in the case who suffered from aspiration pneumonia and ards at the second postoperative day, the peak level of il-6 was 50 pg/ml and g-csf 69 pg/ml respectively. the estimated value of g-csf was 171 pg/mi by the regression equation. this means the real g-cse level was less than half of the estimated value. it suggests that low responsiveness of g-csf is one of the reason of immunodeficient state after the major surgery, neutrophils from injured patients ingest and kill bacteria less efficiently as compared to those of healthy individuals, probably reflecting the suppression in respiratoly burst which occurs after severe trauma. one of the main mechanisms of killing bacteria by neutrophil granulocytes is production of oxygen radicals (respiratory burst). granulocyte colony-stimulating factor (g-csf), a 20kilodalton cytokine, leads to a sustained, dose-dependent increase in circulating neutrophils. thus, it was investigated whether filgrastim (recombinant human granulocyte colony-stimulating factor, rhg-csf) therapy fits for prophylaxis of sepsis in postoperative/posttraumatic patients, and whether, besides an expected increase in neutrophil count, filgrastim would also augment neutrophil function. material and methods: this study was designed as an open label, prospective phase ii study of filgrastim and performed in a surgical intensive care unit (sicu) (university hospital). 10 postoperative/post-traumatic patients with a therapeutic intervention scoring system (tiss) score greater than 30 were treated with filgrastim (0.5 -1 l.tg/kg/day) for prophylaxis of sepsis on 7 days or until discharge from the sicu. production of oxygen radicals can be quantified by analysis of fmlp-and zymosan-induced chemiluminescence. neutrophil oxygen radical production was tested by fmlp-and zymosan-induced chemiluminescence by the polymorphonuclear cells (pmn) of these patients in multiple blood samples over a period of up to 14 days. results: none of the patients treated with filgrastim for prophylaxis of sepsis developed sepsis. in vitro fmlp-induced (10 -7 reel/l) neutrophil oxygen radical production was significantly increased under therapy with filgrastim by a maximum of 797% +-570% (217% -1826%) compared to pretreatment values of 100%. tapering of filgrastim resulted in a reduction of fmlp-induced neutrophil oxygen radical production within 48 hours. in contrast, zymosan-induced neutrophil oxygen radical production was not affected by filgrastim treatment. conclusions: besides its quantitative effect on neutrophil counts enhanced neutrophil function, documented here as increased fmlp-induced oxygen radical production, may account for the beneficial effect of filgrastim for prophylaxis of sepsis in posttraumatic/post-operative patients. granulocyte colony stimulating factor (g-csf) and granulocytemacrophage colony stimulating factor (gm-csf) have been recently introduced in the treatment of chemotherapy-induced neutropenia. effects of these csfs on cellular immune system were evaluated in 38 neutropenic gynecological cancer patients during chemotherapy. g-csf and gm-csf were equally able to induce a rapid recovery of white cell count within one or two days. g-csf treatment resulted in a significantly higher concentration of leukocytes measured in the peripheral blood although by gm-csf a sufficient effect was achieved (p<0.05). before initiation of csf treatment urinary neopterin was similar in both groups of patients (283+/-38 and 267+/-40 lamol/mol creatinine for gm-csf and g-csf respectively expressed as mean +/-one sd). in g-csf treated patient only a marginal induction of neopterin was observed. on day 4 the mean value was about 40% above the basal level (p<0.05). on the other hand gm-csf treated patients were characterized by a pronounced increase in urinary neopterin levels. in comparison with the basal level a more than 2 fold induction was noted and the difference between g-csf and gm-csf was highly significant (p<0.01). this effect was confirmed in vitro by investigating the effects of these csfs on interferon-gamma mediated pathways in thp-1 human myelomonocytic cells. results suggest activation of immune effector cells by gm-csf which may help the organism to overcome infections. however, activated macrophages produce several growth factors which may increase malignant proliferation, and augmented neopterin production as sign of macrophage activation has also been associated with poor prognosis m several malignancies. more data are therefore necessary to clarify whether csf mediated immune activation is beneficial or deleterious for cancer patients but considering our results caution in applying csfs in oncology seems advised. from a historical perspective, the development of humoral immunity to bacterial endotoxin has assumed a prominent position in the spectrum of therapeutic approaches which have been explored for the treatment of gram negative septic shock. predicated upon the fact that rough strains of bacteria manifest lps containing exclusively conserved structural features common to lps from all gram negatives, specific antibodies were elicited which conveyed cross protective immunity in experimental models of bacteremia and endotoxemia. such studies culminated in a well-conducted, randomized, double-blind placebo-controlled clinical trial using passively administered human polyclonal antiserum to treat patients with suspected gram negative sepsis. the efficacy of treatment established in that trial spurred efforts to develop monoclonai reagents which, to date, have not been uniformly successful in reproducing those earlier studies with polyclonai antibodies. nevertheless, the numerous successes which have been documented in experimental models of endotoxemia continue to foster promise for this immunotherapeutie approach. several recent studies with human polyclonalimrnunoglobulin preparations containing antibodies reactive with lps and lipid a have yielded promising results in treatment of patients with sepsis. in addition, the recent development of an antiidiotypic monoclonal antibody which reflects an internal image of a kdo specific monoclonal antibody has provided an alternative experimental approach to generate anti-lps antibody. immunization of mice with the antiidiotype provides significant protection against subsequent lps lethality consistent with the development of circulating immunoglobulin specific for lps. thus, the use of polyclonal immunoglobulins contrives to provide an alternative and potentially cost effective method for the treatment of endotoxin shock. supported by r37 a123447 and pot ca54474. john holaday, anne fortier, shawn green, glenn swartz, john madsen, carol naey, and jan dijkstra entremed, inc.. rockville, md, 20850. at the time of diagnosis, the signs and symptoms of septic shock are an indication that the systemic inflammatory response is well underway; thus, it has been argued that the endotoxin "cat is out of the bag", and that subsequent passive immunization may be too late to achieve therapeutic benefit. our approach has been to evaluate active immunization as a prophylax~s against sepsis. mice were inoculated twice (two weeks apart) with liposomes containing dmpc[i.8], dmpg[0.2], cholesterol [1.5] , and monophosphoryl lipid a [20-200 gg/txmole phospholipid] by several routes (i.p., i.m.), and serum was collected 10-11 days after each inoculation. after a single injection, highest tilers of ab were produced in mice inoculated i.p., but mice inoculated by all routes produced anti-lipid a ab. following the second injection. ab levels were roughly equivalent in mice inoculated by all routes, regardless of lipid a concentration. mice vaccinated i.p. with liposomes containing 0, 20 or 200 gg lipid a were treated with cyclophosphamide to produce neutroperda and then challenged with e. cole in an infection model of gram negative sepsis. the lds0 for control (liposomes with no lipid a) mice was 3x10 bacteria; ld50 for mice vaccinated with 20p.g was 24x103 (8-fold increase in resistance) and with 200~tg was 48x103 bacteria (16-laid increase in resistance). mice vaccinated as before were also treated with actinomyein d to increase sensitivity to lps (salmonella minnesota) challenge in an endotoxemia model of grain negative sepsis. the ld50 for control (liposomes with no lipid a) mice was 30 ng lps; the ld50 for 20gg lipid a was 70rig lps (2-fold increase in resistance) and for 2001xg was 570ng lps (19-fold increase in resistance). mice were similarly vaccinated and challenged with an aggressive gram negative pathogen, francfsella tularensis. the ld50 of franciseua in normal mice or mice inoculated with liposomes without lipid a was 20-40 bacteria. in contrast, mice vaccinated with liposomal lipid a (200ggl survived challenges as high as 30,000 bacteria, (3 logs of protection). the impressive protective capacity of this vaccine did not correlate with ab liter in any of the sepsis models, nor did it correlate with classic nonspeeific events, such as macrophage activation. maerophages harvested from the peritoneum of mice vaccinated and protected against sequelae of gram negative infections did not spontaneously kill the bacteria in vitro, but could be activated by ifn-y for antimicrobial activity equivalent to that of macrophages from unt#eated mice. research is underway to defme the protective mechanism(s) activated by this liposomal-lipid a vaccine. intervention by monophosphoryl lipid a in septic shock jon a. rudbach, ribi immunochem research, inc., hamilton, montana, usa monophosphoryl lipid a (mla), the clinical form of which is called mpl®-immunostimulant, has been tested extensively as an intervenient material in septic shock. mla is protective when given to experimental animals prior to a live microbial challenge or challenge with lethal doses of microbial products or certain cytokines. this is shown with gram negative and gram positive bacteria, gram negative bacterial endotoxins, and gram positive bacterial exotoxins. furthermore, animals treated with a regimen of mla which results in a refractory state to a lethal dose of gram negative bacterial endotoxin concomitantly display increased resistance to a live bacterial challenge. thus, both endotoxin tolerance and nonspeciflc resistance to infection can be manifested simultaneously. also, prophylactic doses of mla do not interfere with other therapies given subsequently; an additive or a synergistic protective effect can be demonstrated with certain combinatorial treatment regimens, such as mla followed by antiendotoxin monoclonal antibodies. the preclinical studies were extended to human trials wherein the safety of agonistic doses of mla was verified. furthermore, when mla was administered to human volunteers 24 hr before challenge with a pharmacologically active dose of reference endotoxin, febrile, cardiac, tnf, il-6, and il-8 responses were all decreased significantly as compared with the responses of subjects pretreated with a control solution and challenged with endotoxin. human trials with mla are being extended into patient cohorts which have high probabilities of developing septic shock; this will expand the safety base and establish clinical efficacy for mpl®-immunostimulant. a considerable body of in vitro evidence supports the concept that the effects of lps on cells of the immune/inflammatory systems are controlled by interactions of lps with cd14. to evaluate if blocking lps-cd14 interactions has potential as a therapeutic in septic shock we have evaluated the effect of anti-cdi4 monoclonal antibody (mab) on lps-induced cytokine production and physiologic changes in an experimental model of endotoxin shock performed in cynomolgus monkeys. a novel model has been established where animals were treated with interferongamma for three days prior to infusion of highly purified lps over an eight hour period. in this model lps challenge resulted in marked release of eytokines in the blood, substantial hemodynamic changes, release of liver enzymes and alteration in lung permeability observed over a 24 hour period. to evaluate the effect of treatment with anti-cd14 mab, animals were given either nothing, an isotype control or anti-cd14 mab (5mg/kg) 30 rains, prior to the beginning of the lps infusion. evaluation of physiologic changes including mean arterial blood pressure and cardiac output, quantitative analysis of eytoldne levels including tnfct, il-113,1i,-6, il-8 and il-10, and liver enzymes during a 24 hour period revealed that treatment with anti-cd14 mab markedly attenuated all parameters of injury including decreased mean arterial blood pressure, increased cytnkine levels and the release of liver enzymes observed in animals given the isotype control mab or those not treated. administration of anti-cd14 mab to interferon-gamma treated animals not challenged with lps did not induce any detectable physiologic changes or increases in cytoldnes. these studies suggest that strategies to block lps-cd14 interactions will have utility in diseases such as septic shock or ards where lps plays a central role in initiating injury. preclinical studies with recombinant bactericidal/permeability increasing proteins (rbpi and rbpi23). p.w. "frown, dept. of preclinical science, xoma corporation, berkeley, california, usa. bactericidal/permeability increasing protein (bpi), from neutrophils, binds to and neutralizes lipopolysaccharide (lps); it also specifically kills gram-negative bacteria (gnb). these properties, which reside in the n-terminal half of the molecule, indicate potential therapeutic application in the treatment of gram-negative sepsis. the gene for human bpi has been cloned and recombinant holoprotein (rbpi) and a 23 kd n-terminal fragment (rbpi;3) have been produced in sufficient quantities for preclinical studies. both rbpi and rbpi23 bind to lipid a and neutralize the biological activities of lps derived from a variety of organisms, rbpi23 has equivalent antibacterial activity to bpi against rough gnb but is up to 30x more potent than bpi vs. serum-resistant and smooth gnb. rbpi and rbpi23 compete with lps-binding protein (lbp) for binding to lps under physiological conditions. consequently, both rbpi and rbpi23 block the cd14-dependent lpsinduced synthesis of the cytokines tnf, il-1, el-6 and il-8 in vitro. rbpi23 has also been shown to inhibit the lps-induced synthesis of reactive 02 metabolites, endothelial adhesion molecules and the procoagulant molecule tissue factor. in animals, rbpi has been reported to increase survival of endotoxin-challenged rats and mice, to inhibit the dermal schwartzman reaction in rabbits and to increase survival of neutropenic rats with pseudomonas bacteremia, rbpi23 increases survival and decreases cytokine production in endotoxin challenged mice and rats. it normalizes lps-induced changes in hemodynamic, pulmonary and/or metabolic parameters in lps-induced rats, rabbits and pigs. treatment with rbpi23 also increases survival and decreases cytokine production in bacterial challenge models in rats and mice. rbpi23 was not toxic to rats after 10 daily consecutive i.v. doses of 10 mg/kg. this combination of properties indicate that recombinant bpi may be useful in the treatment of sepsis. phase i/ii clinical trials of rbpi23 have begun. the discovery of lps binding protein (lbp) and subsequent identification of cd14 as a receptor for lps or lps-lbp complexes has resulted in a new understanding o£ how lps responsive ceils are stimulated. cd14 is found either as a glycosylphosphatidyl-inositol (gpi)-anehored membrane glycoprotein (mcd14) of myeloid cells or as a soluble serum protein (scd14) lacking the gpi-anchor. binding of lps to mcd 14 triggers cell activation while binding of lps-scd14 complexes to cells such as endothelial or epithelial cells that normally do not express mcd14 activates these cells. these pathways are shown in schematic form below. 4 ~di mcd14 plays a crucial role in presentation of lps to additional membrane components that make up a functional lps receptor. an immediate consequence of engagement of this functional receptor is protein tyrosine phosphorylation. the molecular mechanisms leading to these events will be discussed. understanding of these pathways will lead to the development of new therapeutic approaches to controlling host responses to lps. pretreatmen t posttreatment (before or after tnf peak) d) with different antibody dosages: 15 mg/kg ---0.1 mg/kg pretreatment with anti-tnfab prevented death in most model situations (except peritonitis), but also posttreatment up to 4h after sepsis induction was successful in the few studies performed. there is additional evidence that low-dose tnfab is partially effective. especially baboon anti-tnfab studies provided many insights into the pathophysiological sequences of sepsis induction, due to crossreactivity with human reagents. those events include the cytokine sequence with tnf-dependent il-i, il-6, or il-8, but also il-lra or stnf receptor release. granulocyte as well as endothelial cell activation were shown to be partly tnf related, and the procoagulatory response was influenced by anti-tnf treatment. from many animal studies the concept that tnf plays a pivotal role in sepsis is clearly evident and therefore anti-tnf therapy is a major candidate tbr clinical studies. the beneficial or harmful effects of tnf-mediated inflammatory responses depend on the clinical context. decreasing exaggerated tnf-mediated inflammatory responses may be useful in some patients with organ failure. tnfr:fc (immunex, seattle, wa) is a recombinant human protein composed of two identical extracellular p80 tnf receptors linked by the fc region of iggl. it neutralizes tnf with an affinity for tnf<z of 10-10 m and has a t1/2 of • 63h in normal humans. methods: 18 normal volunteers were randomized to receive either tnfr:fc vehicle (n=6), or low dose (ld) tnfr:fc (10 mg/m 2 iv, n=6), or high dose (hd) tnfr:fc (60 mg/m 2 iv, n=6) infused over 30 rain prior to endotoxin (e) (escherichia coil 0:113; 4 ng/kg iv) administration. plasma cytokines were measured using elisa and tnf bioactiv'dy. systemic hemodynamics were evaluated with indwelling arterial and pulmonary artery catheters and radionuclide heart scans and compared before and after fluid loading in subjects given vehicle with hd tnfr:fc. results: endotoxin-related symptoms were unchanged after ld or hd. peak temperature occurred at a later time point (4-5h) in subjects given tnfr:fc compared to vehicle (3h) (p=.004). at 3h, 5h (post fluid load ), and 8h, subjects given vehicle developed increased cardiac index and heart rate and decreased systemic vascular resistance index (all p=.0001 ). hd tnfr:fc did not alter this hyperdynamic cardiovascular response. ld and hd inhibited the e-induced teukopenta at i h post endotoxin (p<.03) and later leukocytosis (p<0.05). peak tnf bioactivity in subjects given vehicle was 154 + 44 pg/ml and <10 pg/ml in the ld or hd tnfr:fc groups (p<.001). two immunoassays for tnf were performed because the detection of receptor bound tnf varies with elisa. tnf (biosource) was decreased in ld vs vehicle or hd (p=.0001) whereas tnf (r&d systems) was increased after hd or ld vs vehicle (p<.001). decreased levels of il-8 (p=.00t), granulocyte colony stimulating factor (p=.03), and il-1 receptor antagonist (p=.001) were found after ld or hd vs vehicle. il-6 levels were lower after ld vs vehicle or hd (p=.006). il-10 levels were similar among subjects given vehicle, ld, and hd tnfr:fc. conclusions: ld and hd tnfr:fc delayed the febrile response to e but did not alter the early hyperdynamic cardiac response. this suggests that mediators other than tnf play an important role in the initial cardiovascular response to e in humans. tnfr:fc attenuated the release of some e and tnf-induced cytokines. ti~e antiinflammatory responses of tnfr:fc may be useful in some patients with disease processes resulting from excessive tnf-mediated inflammatory responses. the biosynthesis of tnf within macrophages is regulated both at the transcriptional and the translational levels. the 5' flanking region of the tnf gene contains several transcriptional control elements, including two kb enhancers similar to those of immunoglobulin genes and a "y box" similar to that of the mhc class ii promoter. these elements are critical for the enhancement of transcription noted after stimulation of macrophages with lps. despite these and other elements, transcriptional regulation alone accounts neither for the absence of tnf protein during normal homeostasis nor the profound increasein tnf production following stimulation with lps or other secretagogues. the translation of tnf mrna into protein is also tightly regulated via mechanisms involving the octameric "ttatttat" motif present in the 3'untranslated region of tnf, human inducible no synthase, and several other cytokine genes. this region not only destabilizes mrna, but also confers a translational blockade so that tnf protein is not normally synthesized despite leaky basal transcription. lps stimulation releases translational blockade and, together with enhanced trancriptional rate, accounts for significantly increased tnf secretion. opportunities for inhibition of tnf production are available at each of these two levels. agents which increase intracellular camp (pentoxif¥1ine, amrinone) inhibit accumulation of tnf mrna; in contrast, corticosteroids act primarily at the level of translation, inhibiting lps induced translational activation. understanding the regulation of tnf biosynthesis may assist in clinical strategies designed to regulate tnf secretion. the serine protease thrombin is formed by enzymatic conversion from its proenzyme form after coagulation activation and may enhance the inflammatory response in various ways. in the last step of the coagulation cascade, thrombin cleaves its substrate fibrinogen, thus forming fibrin. the fibrin clot may prevent phagocytosis of bacteria and promote local bacterial growth. thrombin can also stimulate a specific th.rombin receptor that exists on a variety of cells. for example, minute amounts of thrombin ma), stimulate platelets to develop tissue factor and boost the activation of more thrombin, thus initiating a positive feedback loop. other cellular effects of thrombin receptor stimulation include increase in intracellular calcium of platelets and monocytes, contraction of smooth muscle cells, increase in endothelial permeability, thromboxane generation by neutrophils and lymphocytes, pulmonary vasoconstriction, and enhanced cytokine production. animal experiments have been conducted with a variety of thrombin inhibitors including heparin, antithrombin ili, hiradin, and hirudin-like peptides. an alternative approach is the inhibition of the coagulation cascade at an earlier point in the cascades, or administration of the anticoagulant enzyme protein c work from our own group in a porcine model of lipopolysaccharide-(lps)-induced shock with disseminated intravascular coagulation has shown that prelreatment with recombinant desulfatohirudin (r-h) or with a preformed complex of human donor antithrombin iii with heparin or post treatment with r-h are able to block the degradation of fibrinogen and the formation of fibrin monomer. in addition, after pretreatment with r-h it was found that lps-induced lung injury was reduced. since both natural hirudin as well as r-h were well tolerated by healthy volunteers, adminislration of r-h may be a promising therapeutic approach in patients with sepsis and sirs. corticosteroids have been used in the management of various shock syndroms including sepsis. the data concerning the effects of this therapy is, however, conflicting and in large prospective studies, the use of early administration of high doses of corticosteroids have been found to give no beneficial effects in patients with sepsis. we have particularly been engaged in studies on possible effects of corticosteroids on the plasma cascade systems and on the mediator release from leukocytes. in in vitro studies, high doses of methylprednisolone were found to facilitate endotoxin induced generation of plasma kallikrein. furthermore, plasma prekallikrein and kallikrein inhibitor values decreased together with formation of kallikrein-c1 inhibitor complexes (1). in this in vitro plasma model we also found that methylpredoisolone alone in doses of 1 mg/ml induced contact activation with the formation of kallikrein-c1 inhibitor complexes. methylprednisolone was also found to have an additive effect on endotoxine induced decreases in kallikrein inhibitor functions. in the same experimental model on endotoxemia methylprednisoione was found to give an additive effect on activation of the initial part of the complement cascade (2) . activation of the terminal part of complement, however, was inhibited by the same dose of methylprednisolone. addition of methylprednisolone alone to plasma was also found to activate the initial part of complement. using a full blood model, we studied the effect of methylpredoisolone in modulating the endotoxin induced cytokine response. when 103 ng/l e. coli endotoxin was added to citrated blood from healthy human donors preinkubation with 20 gg/ml methylprednisolone significantly reduced the release of tumor necrosis factor et (79 %) and interleukin-6 (63 % ) at two hours after exposure to endotoxin. in conclusion, these studies show that corticosteroids might facilitate contact activation of plasma and reduce the function of major protease inhibitors. furthermore, we also observed that this drag in in vitro conditions facilitated activation of complement. on the other hand, corticosteroids were found to reduce endotoxin induced cytokine release from leukocytes in whole blood. these studies also showed a dose dependence of the effects of corticosteroids on endotoxin induced cellular and cascade system activation. different predisposing conditions like extensive trauma, infection or pancreatitis result in a remarkably similar inflammatory response. although thls inflammatory response primarily aims at the removal of devitalized tissues, destruction of bacteria and reestablishment of the integrity of host tissues, it may potentially become unregulated and generalized and thereby producing deleterious effects to vital organs or the body" as a whole. cytokines play an important role in the development of the systemic inflammatory response. most of their biological effects, however, are not directly mediated but exerted through other mediator systems. when the inflammatory response results in the formation of capillary leak syndrome and refractory hypotension, the unregulated activation of complement and contact systems appears to be the cause. both systems are almost exclusively regulated by c1-1nhibitor. am unbalanced degradation of the inhibitor protein was found to be associated with the onset of capillary leakage in various conditions such as bone marrow transplantation, severe burns, and septic shock. these observations suggest a relative deficiency of biologically active c3-1nhibitor in these septic shock like syndromes. therapeutic intervention studies were initiated using high doses of c1-1nhibitor concentrate. first results are promising, as in most patients the administration of the drug was followed by a quick and marked improvement of the patient's hemodynamics. whether the administration of c1-1nhibitor alone or in combination with other drugs is also sufficient to improve long-time survival has to be investigated in double-blind, placebocontrolled studies. salutary effect of human soluble complement receptor type-1 in models of adult respiratory distress syndrome g. feuerstein, m. dimartino, and *r. rabinovici, smithkline beecham pharmaceuticals, king of prussia, pa, usa, and *jefferson medical college, philadelphia, pa, usa adult respiratory distress syndrome (ards) is a severe pulmonary insufficiency syndrome associated with diverse diseases and injuries. ards is believed to be the consequence of a massive acute inflammatory reaction consisting of activated neutrophils infiltration into the lung. neutrophil activation is likely the result of multiple factors of which complement is believed to play a major role. a potent complement regulatory system in the form of the complement receptor type-1 (cr-1) exists on many cells where protection against complement injury is provided by inhibition of both the alternative and classical pathways for c3/c5 convertase formation. we have recently purified the human recombinant cr-1 in a truncated form (brl55730, tp-io, scr-1) and tested this soluble protein for protective effects in several models of ards. specifically, we have used the following rat models: 1) endotoxin-induced ards in paf primed animals; 2) 11.-2induced ards; 3) thermal injury (30% body surface)-induced ards; 4) acid aspiration-induced ards. ards-like features included edema (increase in lung wet weight and water content), neutrophil accumulation (histological inspection and myeloperoxidase assay) and increase in cells and protein in the bronchoalveolar lavage (bad. scr-1, 1-25 mg/kg, i,v., given as prophylactic (and in some cases, therapeutic) treatment significantly inhibited edema formation and leukocyte infiltration and, in some cases (e.g., endotoxin/paf or il-2), virtually completely. these comprehensive studies strongly support the therapeutic potential of scr-1 in ards due to diverse injuries. the xanthine derivative pentoxifylline (ptx) and several analogs of ptx have been evaluated for their protective effect in various animal models of septic shock. data from these in vivo studies demonstrate that (1) ptx suppresses lps-induced tnf release from activated macrophages; (2) ptx prevents tnf-induced pmn activation; (3) ptx attenuates lps-or tnfinduced acute lung injury as evidenced by prevention of increased pulmonary vascular permeability and detoriation of gas exchange; (4) ptx exerts its .protective effects even when administered following initiation of sepsis. the sum of these actions suggest that administration of ptx in sepsis may have therapeutic potential. hans hoffmann md, dept. of surgery, klinikum grosshadern, ludwig-maximilians-universit~.t, marchioninjstrasse 15, d-81377 m0nchen, germany. pentoxifylline [pof] and related xanthins are drugs of known haemorrheologieal activity and widely used clinically. recently, new pharmacological aspects of these established drugs could be demonstrated. it was found that pof selectively inhibits the formation of tnf but not il-6 most likely by causing accumulation of intraceliular camp via inhibiting phosphodiestersse activity, and, secondly, by inducing prostacyclin in different cell types. furthermore, pof is able to counteract tnf-mediated adherence of neutrophils to vascular endothelium and to inhibit fmlp-indueed respiratory burst in neutrophils. we and others have, therefore, studied its effect in different animal models. it was found that pof protected from increased pulmonary vascular permeability and sequestration of neutrophils into the lung in different animal models of acute lung injury. moreover, in pigs with induced faecal peritonitis the drug improved haemodyrmmle variables as well as pulmonary compliance and reduced the number of pulmonary neutrophila and lymphocytes. consequently, as a general outcome, pof could be found to improve survival in different models of haemorrhagie and endotoxie shock. the protective effect of pof was dose-dependent and observed even when pof was given up to 4 hours after endotoxin. in order to evaluate these pharmacological effects of pof in humans, we studied pof under controlled conditions of endotoxlnemia in volunteers. we found that pof selectively inhibits the lps-indueed endogenous formation of tnf without affecting il-6 and il-8. moreover, pof counteracts the lps-indueed initial leukocytopenia by interfering with the adherence of neutrophils in the microvasculature. meanwhile the inhibition of endogenous formation of tnf by pof could be demonstrated under different clinical conditions of acute and chronic cytokine release syndromes, {okt3 first-dose reaction, cancer-or tuberculosis-induced cachexla}. it appears worthwile and promising to investigate wether pof exhibits beneficial effects also in septic patients. objectives: to determine the specific role of paf in both lethal primate bacteremia and nonlethal human endotoxemia. materials and methods: two double-blinded placebo controlled studies were performed. first, ten baboons (14.5 +.7 kg), were randomized to a control group receiving 101° cfu/kg of intravenously administered e. co/i (n=5) and a treatment group (n =5), pretreated with .1 mg/kg of paf receptor antagonist ro24-4736 two hours prior to e. co/i infusion. in a second study, ten healthy male volunteers were randomized to a control group receiving 4 ng/kg of intravenously administered endotoxin (lps) (n=5) and a treatment group (n=5) pretreated with 10 mg of ro24-4736 eighteen hours prior to lps administration. physiologic parameters and cytokine levels were measured continuously in both studies for 24 hours. results: baboons pretreated with the paf antagonist had improved oxygenation (96 + 11 mm hg v 59 -+ 6 in controls, p < 0.05) and creatinine clearance 12 hours post e.coli (39.8 + 23.4 ml/min v 0.1 _+ 0.1 in controls, p < 0.05). similarly, volunteers pretreated with the paf antagonist experienced fewer symptoms, including rigors and myalgias at 1 hour post lps. this was in concert with a diminished peak cortisol (668 +_ 107 mmot/l v 959 +-159 mmol/l in controls, p < 0.05), epinephrine secretion (1057 + 165 nmol/l v 2029 + 431 nmol/l in controls, p < 0.05). hemodynamics and circulating tnfa levels (data not shown) were unaffected in either study by prior paf antagonism. concluslons: paf influences some components of the multi-organ failure syndrome in lethal gram negative sepsis and the counter-regulatory hormonal system in human endotoxemia through tnf-independent mechanisms. paf antagonists may serve as adjuncts to cytokine blockade in the treatment of gram negative sepsis. the mediator role of platelet-activating factor in gramnegative septic shock pierre g. braquet, david hosford and matyas koltai institut henri beaufour, le plessis robinson, france considerable evidence has been accumulated to support the concept that a paf/cytokine autogenerated feedback network is responsible for priming and amplification of inflammatory mediator release in septic shock. cytokines, such as tnf~x, il-1 and other interleukins interact with paf which then amplifies cytokine production, therefore, paf may be the principle mediator in endotoxin shock. a single factor identified as tnf is suggested to play a pivotal role in the fatal phase of septic shock. presumably excess tnf release is the result of amplification process primarily triggered by paf. one may assume that the high tnf concentration in the blood of 'non survivors' is the consequence rather than the cause of cell death. the dubious results of clinical trials with anti-tnf antibodies and il-1ra, a naturally occurring il-1 antagonist protein, have risen debate around the exceptional role of cytokines in the pathomechanism of septic shock or systemic inflammatory response syndrome (sirs) induced by gramnegative microorganisms. there are similar problems with the interpretation of the results obtained in clinical trials of monoclonal igm antibodies against e. coli endotoxin, ha-1a and e5. future studies will answer the question as to the effectivity of inefficiency of this treatment. perhaps when the plasma concentrations of lps and cytokines exceed a critical level, treatment fails to save the patients life. with regard the causal role of lps in septic shock, the putative role of bacterial porins may have to be taken into consideration. the marked release of paf induced by endotoxin leading to excess txa2 production may be responsible for the microvascular failure and death in septic shock. antagonists of paf markedly protect against the release of txa2, and may interrupt the vicious circle of amplification process. no produced by the inducible no synthase plays a pivotal role in causing vascular paralysis in lps-induced sirs. to explore the relationship of paf to no synthesis will provide better understanding of the pathomechanism of septic shock. several paf antagonists, including bn 52021, have undergone phase ii and phase iii clinical trials. the strategy in the treatment of sirs, however, remains multifactorial, since little is known about the sirs caused by microorganisms other than gram-negative bacteria. evidence has begun to accumulate which suggests that in sepsis excess production of lps and cytokines can lead to uncontrolled production of inos and that this might in part explain the severe unresponsive hypotension seen in some septic patients. a number of strategies have been explored in an attempt to limit excess no production. a favourite target has been competitive inhibition of nos by arginine analogues such as l-nmma. in animals, some investigators have shown that the haemodynamic effects of lps challenge can be attenuated by l-nmma, but there is a narrow toxic-therapeutic ratio. in a model of live e. cell sepsis, we have been unable to reproduce these findings. localisation of no production by immunohistochemistry suggests that no production is compartmentalised, and more subtle methods of regulation may be required if this approach is to have clinical valuc. anti-adhesion molecule strategies ch wortel, repligen corporation, one kendall square, building 700, cambridge, ma 02139, usa. the neutrophil has been implicated as a pivotal player in the pathogenesis of multiple organ dysfunction. an important first step in the process of neutrophilmediated organ injury involves the binding of neutrophils to endothelial ceils. this process is largely regulated by complementary adhesion molecules, some of which are present constitutively on the cell surface and others that can be upregulated in response to chemotactic and pro-inflammatory stimuli. several different adhesion molecules have been described. l-selec tin is expressed on the plasma membrane of neutrophils and is shed from the surface early in the inflammatory process. it is therefore thought to mediate the initial interactions with endothelial cells. e-selectin and p-selectin are endothelial adhesion molecules. e-selectin is not constitutively expressed but is upregulated by inflammatory mediators, particularly il-1 and tnf. p-selectin is present in the weibel-palade bodies within the endothelial cytoplasm and can be upregulated rapidly in response to thrombin, histamine, and oxidants. all selectins recognize oligosaccharides, particularly sialylated lewis x antigen. this carbohydrate moiety is expressed on many proteins, including the selectins themselves. the leukocyte integrins are glycoproteins expressed on the neutrophil surface and in the cytoplasmic granules. integrins consist of a common beta2 or cluster differentiation (cd)i 8 chain covalently linked to one of three different alpha chains (cd11 a, cd1 lb, cd1 lc) and exist on the cell surface as three distinct heterodimers. cd1 l a/cdi 8 is expressed on all leukocytes, whereas cd1 l b/ cd18 and cd1 lc/cd18 are restricted to cells of myeloid origin. cd1 i/cd18 interacts with intracellular adhesion molecule-1 (icam-1), its ligand on endothelial cells. icam-1 is a member of the immunoglobulin family of adhesion molecules. it is constitutively expressed on endothelial cells and can be upregulated by cytokines. the potential for monoclonal antibodies to adhesion proteins to reduce vascular and tissue damage has been studied in alarge number of experimental models. protective effects have been observed in a wide variety of inflammatory, immune, and ischemia-reperfusion injuries such as arthritis, bums, endotoxic shock, bacterial meningitis, autoimmune diabetes, nerve degeneration, allograft rejection, allergic asthma, acute lung inflammation, skin lesions, and ischemia-reperfusion models of the intestine, myocardium, lung, skeletal muscle, and central nervous system. protective effects have also been observed in animals resuscitated following hemorrhagic shock. since modulating the function of adhesion molecules may temporarily leave the host without effective defense machinery, safety evaluations are of utmost importance in evaluating this therapeutic approach. treatment of gram-negative septic shock with an immunoglobulin preparation: a prospective, randomized clinical trial ingolf schedel, ursula dreikhausen; birgit nentwig; marion hockenschnteder, dirk rauthmann, salim balikcioglu, rolf coldewey, helmuth deicher, md endotoxin may play a major role in the pathogenesls of muitiorgan failure in the context of the toxic process in septicemia induced by gram-negative pathogens. the hypothesis which has led to a prospective clinical study consisted in the idea of inhibiting the biological effects ofendotoxin during the early phase of septic process, and thereby attampting to attain a positive effect on the clinical course of the disease. -objecave: to evaluate the effectiveness ofa polyclonal immunoglobulln (ig) preparation containing igg, lgm, and iga as an adjunctive therapy for septic shock. -desigru prospective, randomized clinical trial. patients: fifty-five patients w~th septic shock were randomly allocated to two groups according to criteria of septic shock. -intervention: one group of patients (n = 27) received a commercially available immunoglobulin preparation (containing high titers of antibodies specific for determinants to bacteria1 endotoxin) during the first 3 days after inciosion in the study. the other randomized group (n = 28) did not receive any immunogiobulin preparation. -measuremems and main resmts: during the period of <6 wks after the beginning of clinically apparent septic shock, death related to the septic process occurred in one (4%) of 27 patients who received immunoglobulln. by comparison, nine (32%) of 28 control group patients died during this period (p <. 01 ). within the first 48 hrs after onset of the clinically apparent septie process, significaptly increased activity of circulating endotoxin and simultaneously decreased specifie igg serum titers to lipid a were detected in the group of nonsurvivors. the rationale for the use of polyvalent intravenous immunogiobulins (ivig) to treat severe infections stems from in vitro and animal studies documenting that high-dose igg enhance opsonization and phagocytosis of pathogenic bacteria. moreover, recent clinical studies have shown that in septic patients the phagocytic function of circulating polymorphonuclear neutrophils (pmn) is defective; the degree of such impairment correlates to the severity of the septic state, the beneficial effect of administering high dose igo in septic patients can be two-fold: i) ivig provides large quantities of antibodies against invading pathogens; the igg bound to the pathogens can opsonize their phagocytosis; 2) the opsonization of phagocytosis by exogenously administred polyvalent igo would be of special importance in those patients who present a significant defect of their phagocytic function, when they are challanged by a large bacterial invasion. a prospective, randomized, double-blind study was carried out comparing the administration of ivig (sandoglobulin@) vs, paeebo (human albumin) in severely septic surgical patients, only patients with gepis score >_20 (meaning a mortality risk >70%) were accepted into this protocol. patients were randomized to receive 0.4 g/kg of ivig or placebo on day 0 (when they reached sepsis score>20), repeated on day +1 and +5. at the beginning of icu treatment, the two groups of patients were similar for severity of sepsis, age, concomitant disease, type of surgical procedures, antra and perioperative procedures, antibiotic administration. the results of the study indicated a significantly reduced mortality in patients with severe surgical sepsis treated with ivig as compared to placebo control patients (mortality: 38% vs, 67% respectively; p< 0,05). in conclusion, the results of our study in patients with severe surgical sepsis were the following: 1) ivig plus multimodal treatment of sepsis, including antibiotics, reduce mortality significantly', 2) the reduction of mortality seems to be due to a decreased incidence of lethal septic shock. despite substantial clinical research, the avallable data regarding the effectiveness of supplemental immunoglobulin (ig) treatment in sepsis in adult patients do not yet allow definitive conclusions. in view of the persistently high sepsis mortality there is a need to continue clinical investxqations regarding supplemental sepsis treatmen~ in general, as well as concerning ig administration in particular. we present and discuss the protocol of the ongoing ,,score-based-immuneglobulin therapy of sepsis (sbits)" study. the protocol (theoret surg 8(1993)61-83) of this multicenter, randomized, prospective and double-blind trfal relies on the results of an observational trial on i.v. igg treatment in 163 patients with sepsis and septic shock (infection 19~1991)216-227), carried out as a prerequisite for the present trial. using microcomputer-based bedside routine score monitoring, we regard quantitative measures of severity of disease and sepsis: only patients with a certain degree of both severity of disease (apache ii score 20-35) and severity of sepsis (elebute sepsis score 12-27) will be included. by observing these previously validated inclusion criteria, this trial snould iqentify a priori and include patients with potentially optimal response to therapy, consisting o~ either placebo (0.i % albumin) or polyglobin n" -12 ml (0.6g)/kg on day 0 and 6ml (0.3 g)/kg on day i. with an anticipatedpopulation size of 800 patients the study should comply with the statlstical requirements (estimated mortality: 30%, with a 33 % reduction in 28-day mortality in the treatment groupl to prove or disprove the question of igg effectiveness in sepsis in terms of improved prognosis. up to november 1993, more than 460 patients had been included; patient enrollment will be finished in 1994. previous studies have demonstrated rhll-i ra, a naturally occurring antagonist of il-1, increases survival in animal models of andotoxemia and eschehchia coli bacteremia and attenuates the decrease in mean arterial pressure resulting from challenge with both gram-negative and gram-positive bacteria. previously, in 99 patients, rhll-lra was demonstrated to increase survival in patients with sepsis syndrome and septic shock in a dose-dependent manner. methods: a randomized, double-blind, placebo-controlled, malticenter, clinical trial enrolled 893 patients at 63 academic medical centers in europe aad north america. eligible patients received either placebo (vehicle) or rhil-lra (anakinra) 1.0 or 2.0 mg/kg/hr by continuous intravenous infusion for 72 hours. the presence of organ dysfunction (i.e., ards, dic, renal, and hepatic) at study entry was determined prospectively by a clinical evaluation committee using definitions which were developed a-priori. survival time was evaluated over 28 days utilizing a linear dose-response model, assuming a log-normal distribution. results: 563 patients had one or more sepsis-induced organ dysfunction(s) at study entry. a dose-related increase in survival time was observed with rhll-lra compared to placebo in patients with ards, dic, and renal dysfunction (p --< 0.04 endotoxin infusion releases platelet-activating factor (paf), a potent phospholipid mediator which leads to an autocatalytic amplification of cytokine release. bn 52021 (ginkgolide b), a natural paf receptor antagonist, has provided significant protection against sepsis in different animal models• a randomized, placebo-controlled, double blind, multicenter trial on efficacy (mortality at d28) and tolerance of bn 52021 (2 iv infusion of 120 mg x 2/day over 4 days) in severe sepsis has enrolled 262 pts. the 28day mortality rate was 51% for the placebo group and 42% for the bn 52021 group (p = .17). the efficacy of bn 52021 was greater in pts with gram-negative sepsis: the 28-day mortality rate was 57% for the placebo group and 33% for the bn 52021 group (p = .01). bn 52021 also reduced mortality among pts with gram-negative septic shock (mortality was 65% for placebo vs 37% for bn 52021; p = .01). using statistical adjusments for pronostic factors, the relative risk of death of the bn 52021 group was 0.61 (0.34 -1.08, 95% confidence interval; p = .09). this risk corresponds to an adjusted reduction in mortality of 39% for pts receiving bn 52021. no differences in mortality rates were found between the placebo and the bn 52021 groups in the absence of gram-negative sepsis• there were no differences in adverse events between the placebo and the bn 52021 groups. bn 52021 is a safe and promising treatment for patients with severe gram-negative sepsis. a confirming study, focused on gram negative sepsis, is in progress. v~3lliam a. kanus m.d. and the rhll-lra it has been traditional within the field of infection and sepsis to think in terms of specific indications for drugs based on the type of infecting organisms, advances in antibiotic therapy now control or ltnflt the growth of bacteria. the majority of deaths are now caused by either an initial overwhelming response to infection or subsequent multiple organ system failure attributed, in part, to the effects of intrinsic biologic responses of the host. type of organism, therefore, may not be as critical as determining the exact severity of the host's severity or risk of death from infection. we also know that both the relative benefit of a new treatment across groups and its absolute benefit for an individual patient will vary with their risk in a predictable fashion. we recently iuve~iguted the relationship between one measure of host response, the acute risk of death as prospectively estimated by u comprehensive risk mode[ for 28-day mortality (jamb. 1993; 270:12,33-1241) , by its retrospective application to the results from the phase in evaluation of recombinant human intcrlenkin-1 receptor antagonist (rhll. ira). we found that there was a significant interaction between the patient's predicted risk of mortality at the time of entry to the study and the ability of rhil-lra to prolong survival time (x2 = 7.6, p [] 0.02, log.normal) for all 893 patients in the trial• survival benefit began st approximately 24% baseline risk of 28-day mortality. for the $80 patients with a predicted risk > 24%, there was a 22% reduction (p=0,00$ log normal). when we examined the variation in patients above and below the 24% risk level with hazard functions, i.e., their daily risk of death during the study period, we found that placebo patients with < 24% risk had lltile acute daffy risk during the hlltial two days follawh~g study entry and this risk was little affected by rhil-lra, in contrast, patients with > 24% risk had high daily mortality risks during the tuttlal two days that high dose rhtl-lro substantially reduced. these results are compatible with our current understanding of outcome from sepsis and the proposed mechanism of action o£ immunotherapy, the earliest deaths from sop sis are secondary to an immediate inflammatory response followed closely by deaths secondary to multiple organ system failure, later deaths (after 14 days) are not as closely related to the acute effeete of the inflammatory cascade. because of the timing and action of most proposed tmmunotherapy, they may be capable of preventing mortality primarily in these initial two phases. in this study, an independent predicted risk of mortality reflected this mortality pattern ned illustrated the potential benefit of immtmotherapy. use of a predicted risk of mortality in the design and analysis of clinical trials could improve our understanding of the clinical benefit of these new therapeutic approaches. the systemic inflammatory response syndrome (sirs) is a term recently proposed to describe patients with systemic inflammatory responses to insults such as infections (sepsis), trauma, burns, pancreatitis, and other initiating events. patients with sirs may have similar activation of inflammatory mediators and similar outcomes independent of the initiating event. these outcomes include organ dysfunction and failure, shock, and death. challenges to the successful conduct of clinical trials in sirs include the complexity of illness in these patients and the important--but limited--clinical benefits of novel compounds that may be limited to selected patient subsets. addressing these challenges will require new tools and approaches. these will include more sensitive and appropriate endpoints, and the use of methods such as baseline risk adjustment, to allow detection of drug risk interactions not captured adequately by categorical definitions, such as sepsis syndrome. on the basis of supportive preclinical and phase i safety studies, we have initiated phase ii clinical trials of a novel bradykinin antagonist, cp-0127, in four sirs subcategofies: sepsis, multiple trauma, burns, and pancreatitis. each of these studies is designed to measure the effect of cp-0127 on mortality, organ dysfunction and failure, and activation of mediators. in addition to investigating rates of organ failure using standard definitions--a new endpoint--a continuous summary measure of organ dysfunction (the acute physiology score of apache tm iii) is being used to quantify the degree of organ dysfunction and the speed and pattern of recovery of physiologic stability. in the sepsis study, another new approach--a study specific risk model based on the apache ill database--has been developed which will be used to assign a pre-treatment baseline risk to each patient enrolled. the primary outcome variable will be risk adjusted survival time to 28 days. this type of risk-adjusted analysis may allow for more efficient and powerful trials and more accurate and useful indications for use. study purpose: in post-cardiac surgical patients (pat.) at risk for sepsis, the efficacy of early i.v. immunoglobulin (ig) treatment was compared to a matching historical control (con.) population. postoperative risk assessment: using apache ii scores lap) (first postoperative [pop.] day) in a pilot study phase, we were able to differentiate between the large population (95.2%) of pop. low-risk pat. (ap< 19; mortality: 1%) and the small groups of pop. pat. at risk lap= 19-23) and high risk lap_24) with a significantly higher mortality (14% and 76%, mainly due to sepsis). subsequently, among 1341 consecutive pop. pat. we prospectively identified and treated these pat. iq treatment reqimens: first study period (n =41): (gg (psomaglobin n a, tropon biologische pr~parate, cologne, frg, day 1:8 ml/kg, day 2:4 ml/kg). second study period (n=25): iggma (pentaglobin r, biotest, dreieich, frg, 5 ml/kg on days 1 to 3). results: ig pat. and con. were comparable in demographic data, operation characteristics and baseline disease severity lap and elebute sepsis scores). in contrast to con. (risk: n=21, high-risk: n-21), the ig pat. showed a marked improvement in disease severity (fall in ap), especially in the high-risk group (igg, n=26: p<o.05; iggma, n=13: p: 0.08). in this group, ig led to higher (p<0.05) response rates, defined as an ap score decrease ->7 within four days (igg: 54%, iggma: 62%; con.: 19%), and reduction in mortality (igg: 46%, iggma: 46%; con.: 76%), statistically significant (p<0.05) for ig treatment as a whole (igg and iggma). conclusion: given the good comparability of the study groups, our results indicate, despite the non-randomized design, that early supplemental ig treatment can improve disease severity and may improve prognosis in prospectively apache ii score-identified high-risk patients after cardiac surgery. objective. elevated plasma levels of endothelin (et) have been demonstrated in both experimental and human sepsis. et has been proposed as a sepsis mediator leading to vasoconstriction with tissue hypoperfusion and organ failure. the aim of the study was to determine the effects of sepsis treatment with volume resuscitation, antibiotics and the anti-lps monoclonal antibody es® on big et and active, 21 aminoacids et (et 21) in rat abdominal sepsis. methods. lethal peritonitis was induced with a 4 mm coecal perforation (cp) in male wistar rats. plasma levels of big et and et 21 were determined with amersham tm endothelin rias 4, 8 and 12 h after sepsis induction. experimental groups: 1. cp control, 2. volume replacement (vr); 0,9% saline 10 ml/kg/h continous iv infusion started after 2 h, 3. antibiotic; imipenem 40 mg/kg iv after 2 h, 4. e5®; 10 mg/kg iv after 2 h, 5. vr + imipenem + es® after 2 h. results. high concentrations of both big et and et 21 could be demonstrated after 2 h and lasting for 12 h after cp. neither volume replacement nor imipenem did influence the elevated plasma et. e5® significantly reduced et 21 both 4, 8 and 12 h after sepsis induction, but did not reduce big et. when es® was combined with vr and imipenem, reduction of et 21 was the same as for e5® alone. these results strongly suggest that bacteria and hypovolemia per se are not decisive stimuli for et production during sepsis. e5® reduces circulating lps and tnf which is the probable mechanism of the suppressed et 21 synthesis. the unaltered big et fraction after e5® treatment indicates conversion of big et to et 21 as the site of action responsible for reduced et 21. conclusion. lethal peritonitis in the rat is followed by elevated plasma levels of big et and et 21. e5® anti-lps antibody significantly reduces plasma et 21 while volume resuscitation and antibiotics failed to do the same. es® did not reduce plasma big et. pmx treatment on severe endotoxemia with multiple organ failure was safety and effect in prognosis, and sepsis related parameters. it was certified that reduction of plasma endotoxin was effective in severe endotoxemia. a. lechleuthner,s. aymaz, g. grass, c. stosch, s. dimmeler, m. nagelschmidt, e. neugebauer. ii. dept. surgery, university of cologne, germany. introduction: the cardiovascular therapy of hypodynarnic shock states is a challenging problem. in clinical as well as experimental studies beneficial functions of a new hg-agonist bu-e-75 in congestive heart failure has been demonstrated 03aumann, 1989). therefore, we investigated the effect of bu-e-75 in hypodynamic shock in pigs. materials and methods: pigs (deutsches hausschwein, pitrain, [20] [21] [22] [23] [24] [25] were anesthesized with fentanyl/dormicum, ventilated (n20:o 2 = 2:1) and cardiovascular parameters were monitored with a complete icu-eqnipment. the hypodynamic model was established in a pilot study (4 animals) to evaluate the effective concentration of bue-75 in healthy and endotoxin (lps)-treated animals. endotoxic shock was induced by continous infusion of 10 ~g lps/kgkg/h (055:b5, fa. difco). the hypodynamic state was defined as a decrease of cardiac output by 30 % of steady state levels. a wedge pressure of 10-12 mmhg was kept constant by volume resucitation during the experiment. in a subsequent randomized controlled trial (rtc) 3 groups with 6 animals per group were studied. the groups were treated as follows: group i, lps and 0,9 % nac1; group ii, lps and bu-e-75 (100 #g/kgkg/h); group iii, famotidine (h2-blocker) pretreatment (1 mg/kgkg), lps and bu-e-75. results: the pilot study in healthy pigs revealed, that bu-e-75 had positive inotropic effects. these effects were inhibited by the h 2antagonist famotidin. bu-e-75 however had no beneficial effects in the hypodynamic phase of endotoxic shock in the rct. cardiac index (ci) and the oxygen delivery (do2) were not significantly influenced by bu-e-75 application (group i versus group ii). bu-e-75 did not ameliorate the negative inotropic effect measuring left ventricular stroke work (lvsw) in hypodynamic shock phases. on the contrary, bu-e-75 led to a further significant decrease of lvsw (p < 0,05). famotidin pretreatment did not affect the response (group iii versus group ii). conclusion: in hypodynamic shock states the h2-agonism seemed to have no beneficial effect under these experimental conditions. receptor down regulation or changes of signal transduction under septic conditions may be responsible. cellular studies may help to identify these mechanisms. objectives. antithrombin iii inactivation of proccagulant proteases is so far the only inhibitory therapeutic approach to disseminated intravascutar coagulation (dic). we therefore set out to investigate whether cll substitution reduces coagulation activation in an endotoxin induced rabbit dic model. materials and methods. 29 male rabbits chbb:hm(spf) were randomty assigned to one of the following groups. group k : naci 0.9% (control without endotoxin, n=10). group e : endotoxin 120 tjg kg "1 bolus i.v. + naci 0.9% (control with endotoxin, n=10). group c : endotoxin 120 pg kg -1 bolus i.v. + cll 400 u kg -1 bolus + 400 u kg "1 4h "~ i,v. (treatment group, n=9). all animals were anesthetized and mechanically ventilated. blood samples were drawn prior to endotoxin administration (m1) and after 120 (m2) and 240 rain. (m3). thereafter, lung and liver tissue samples were taken intravitatly in a standardized fashion for h&e microscopic fibrin quantification using a triple score (fibs). from all blood samples the prothrombin time (pt), activated partial thromboplastin time (aptt), fibrin monomers (fm), and d-dimers (dd) were measured. for statistical significance of differences between the groups anovas and the wilcoxon test (fibs) were performed. results. fibs for lung/liver were significantly different (p<0.05) between group e (lung 180, liver 159) and c (lung 89, liver 0) (group k : lung 11, liver 0). , a synthetic serine proteinase inhibitor, has an anticoagulant activity in the absence of" antithrobim iii. gabexate has been reported to be useful in the treatment of disseminated intravascular coaguiation due to neoplastic diseases. in this study, we investigated gabexate therapy for the treatment of dic due to sepsis in the postoperative critical patients. materials and methods: from july 1992 to june 1993, 15 patients in the surgical intensive care unit met the criteria of dic or pre-dic. eleven were male and four were female with the mean age of 66.7 years. all these patients suffered from some complication of operations which led to the development of sepsis. foy was administered at the rate of 2mg/kg/hr untii the coagulation profile retumed to normal or the patient died. the coagulation parameters were monitored before and on the 1st, 3rd, 5th and 7th day. results: fourteen of these fifteen patients died despite transient improvement of the coagulation parameters in five patients. these patients suffered from sepsis resulting from surgical complications which could not be well controlled. the only survival was a case of recurrent intrahepatic duct stone with biliary tract infection complicated with sepsis and dic. after choledocholithotomy and the use of foy, the patient recovered gradually. conclusion: dic is a late manifestation of sepsis in the critical surgical patients. the most important thing is to eradicate the cause of sepsis. if the underlying septic focus cannot be controlled, dic will persist despite the use of gabexate mesilate. emergency surgery, taipei veterans general hospital, taipei, taiwan. there are 2 main types of bradykinin (bk) receptor, namely bk~ and bk z. the bk 2 receptor is constitutive. the bk 1 receptor is also constitutive but in the majority of cases is inducible and involved in chronic inflammatory syndromes such as sepsis, hyperalgesia and airways hyperreactivty in animals. the mechanism(s) involved in the upregulation of the bk 1 receptor is unclear, however a variety of agents including lps, e coil and ill are particularly efficacious in vitro and in vivo. ill and bradykinin acting at their respective receptors are believed to be involved in sirs/sepsis. we have investigated the effect of antagonists at ill (antril), bk 2 (bradycor [cp-0127]),bk~ (cp-0298) and bkz/bk 1 (cp-0364) receptors on the de novo generation of bk~ receptors (reflected by hypotensive responses to a bk 1 agonist) in the lps-treated (10ug iv) rabbit. in lps treated rabbits hypotensive responses to bk~ but not bk 2 agonists increased with time and at time 210min appeared maximally induced. constant iv infusions of cp-0127 blocked bk 2 but not bk~ and cp-0298 bk~ but not bk 2 responses. cp-0364,cp-0127+cp-0298 and antril+cp-0364 blocked both bk 2 and bk~ responses. antril alone had no effect on bk 2 or bk~ responses. within 30-60 min after stopping the infusions of antagonists the responses to bk~ and bk z agonists were the same as those in nonantagonist infused rabbits. these results indicate, at least in the lps-treated rabbit, that neither bk2,bk ~ or ill receptors alone or in combination, are involved in the de novo generation of bk 1 receptors. in vitro studies demonstrated that beth bradycor and cp-0364 (but not antril) were antagonists at both bk z and bk~ receptors. if both bk z and bk 1 receptors are significantly involved in chronic inflammatory situations in man such as sirs/sepsis then the rationale for the use of compounds such as bradycor or cp-0364 is clear. infection is a major cause of or contributor for morbidity and mortality in liver transplant recipients. effectiveness of prophylactic and therapeutic protocols is important for the success of liver transplantation ( olt ). sdd is used as prophylaxis for reduction of infection caused by gram negative or fungal microorganisms. between september 1988 and july 1993 400 olt's in 368 patients were performed at our department. the actuarial 1-year patient survival is 90 %. infection prophylaxis is started with sdd and ciprofloxacin once the patient is accepted as an olt candidate. perioperatively metronidazol, tobramycin and cefotaxim, postoperatively cotrimoxazol are prescribed additionally. the table shows pneumonia, peritonitis, major wound and urinary tract infection are common nosocomial infections following severe injury. in a series of severely injured patients from the university of louisville hospital, pneumonia was the most common infection followed by peritonitis, intra-abdominal abscess formation and burn wound infection. pneumonia is actually the leading cause of death from nosocomial infection. these are defined as occurring from 48 to 72 hours after hospital admission. this definition has important implications for antibiotic therapy because the likely pathogens and their respective sensitivities are different for community acquired pneumonia. the diagnosis of nosocomial pneumonia is difficult following major injury as many patients will have pre-existing fever, leukocytosis, tachypnea, and chest x-ray changes. reliance on sputum gram stain and culture is important and best obtained by a bronchoalveolar lavage or protected specimen brush during bronchoscopy. predisposing risk factors include severe head injury, emergent intubation and shock, and such patients have been shown to benefit by early tracheostomy. staph aureus has been the most common pathogen isolated from the sputum and the remainder gram-negative organisms with pseudomonas aeruginosa, and klebsiella pneumonia predominating. bacteria recovered by site as well as by intensive care unit is published in the six month antibiogram which also includes recent antibiotic sensitivities. this aids in empiric antibiotic selection against such nosocomial organisms. in a series of severely injured patients (iss -30), mean temp. was 101.4f, leukocytosis was 16k, pan2 was 87, fin2 was .47, and peep was 5.1 at the time of diagnosis (ards excluded). there was marked reduction in class ii histocompatibility antigen (hla-dr) density on peripheral and bal monocyte/macrophages which recovered over time with resolution of pneumonia. immune suppression occurred prior to development of pneumonia, was especially localized to the infected tissue, but recovered with clinical improvement. specific immune modulation targeted to pulmonary white cells may hasten clinical recovery and minimize pulmonary dysfunction. -clinical experience j. tnllemar amphntericin b remains the drug of choice for many systemic fungal infections. its advantages include a broad spectrum of activity and intravenous administration. the major disadvantages of amphoterlcin b is its severe side-effects, especially the nephrotoxicity. to decrease the toxic side..cffccts various liposomal amphoteficin b formulations have been produced. it was found that these liposemal formulations were as effective as amphotericin b but in contrast had a low incidence of toxicity. at present there are three ~different variations of lipid formulations under assessment: amphotericin b lipid complex (ablc), amphotericin b coloidal dispersion (abcd) or true liposomes. the ablc has a ribbon like structure. it has been shown to have a reduced toxicity and an efficacy ranging from being as effective to four times less effective that conventional amphotericin b. regarding abcd the particles have a disk-like structure with a diameter of around t00 am and a thickness of 4nm. the ami-fungal efficacy is 2-4 times less than that of conventional amphotedcin b. both ablc and abcd are presently investigated in phase ii/iii studies in the us. ambiseme is currently the only commefieally available true lipesome. ambiseme is a spherical small unilamellar lipesome with a diameter less than 100 nm with a mutina ld50 of > 175 mg/kg. it has been used in dosages up to 6 mg/kg/day in compassionate based studies with good tolerability. the mycological efficacy range from a 83% response rate for invasive candida infections to 41% response rate for aspergillosis. ambisomc have been evaluated as anti-fungal prophylaxis in randomized trials in bone marrow (bmt) and liver transplant (ltx) recipients. it was well tolerated. in bmt recipients the incidence of proven fungal infections was 8% among placebo treated patients compared to 3% for the ambisome treated patients (ns). in ltx recipients ambisome prophylaxis was effective, significantly reducing the incidence of deep fungal infections from 16% to 0% ill placebo and ambisome treated patients respectively (p<0.01). prospective randomized trials comparing these various amphotericin b preparations with conventional amphotericin b is needed to determine their future place in the therapeutical arsenal. two patlentgroups ere particularly at risk to develop serious cmv disease: cmv seronegative transplant recipients of seroposltlva donors and those patlants treated for rejection with anti t-ceil preparations, we have evaluated the value of prophylactic anti-cmv immunoglobulin (cytotect", biotest pbarma gmbh, dreieich, frg) administration in high risk heart and kidney transplant recipients, in a double blind placebo controlled study 40 kidney transplant recipients, treated for biopsy proved re)action with rabbit atg, received globullntplacebo infusions. the preparatlons were given i,v, in a dose of 100 mg/kg at day 0,7,14,35,56 and 77 after the initiation of anti = rejection therapy, passive immunization completely prevented cmv related death, although it did not reduce th~ incidence of cmv isolation, viraemia or disease, this effect was mainly observed in cmv saronegativa recipients of a serop0sitive donorktdney. seroposltive recipients did not benefit from treatment and seronegatlve recipients of a seronegetlye donor were not et risk for cmv infection at e!l. in a open study the incidence of cmv infection and disease was evaluated in 143 consecutive i~eart sllograft recipients. sixty-five patients were cmv seronagatlve and they all received passive immunlzation according to the dosage schedule used in the kidney patients, but starting on the day of transplantation, this scheme resulted in median snti-cmv igg titers of 1200 elisa units during 3 months. cmv infection occurred in 21/65 ~eronegetlve and in 40/81 seropositive recipients (n,s,), in ssronegetive donor-recipients pairs the incidence was significantly lower (3/34] , the passively immunized seronegstive recipients of e seroposltlve donorheart showed comparable incidence of cmv infection f18t29) vs the seropositive recipients. primary infection more often resulted in disease than secondary infection (11121 v8 10/40), but no difference in incidence of disease (11165 vs 10/81 ) or severity in symptoms was noted between the immunoglobulln treated serone(]ative patients and the seropositiva recipients. apparently passive immunization induces anti-cmv immunity which crossly resembles naturally acquired resistance. abdulkadirov k.,chebotkevich v., moiseev s. the incidence of infection is still high in patients underwent bmt. this complication is the major cause of mortality if it is not recognized and treated promptly and properly. our data showed that from 21 patients with different types of leucemia after autologous and allogenzc bmt 19 had the episodes of fever. in the ma i ority of these episodes the bacterial etiolog$ gram negative bacflli and gram positive cocci) can be proved. on the other hand, in 62% of the fever cases we detected also viral respiratory (corona-, adeno-, rs-and other) infection. our previous investigations showed that even in healthy persons the viral infection has influence on antibacterial immunity, in the cases of model experimental reaction in volunteers we found the decrease of delayed hypersensitivity 10-14 days after intranasal inoculation of influenza virus a (h3n2-3) to bacterial (staphylococcal, streptococcal and pneumococcal) and ~iycoplasma pneumoniae antigens in the leucocyte migration inhibition test. these results showed that respiratory viruses may be the important pathogenic factor in the development of bacterial infection in posttransplanted period. we consider the constant control of latent and visual respiratory viral infection in bmt patients to be very important. ficcb the ~ter£~li of the nation~l institute of trad/~atoloqy in budapest 1.00 consecutive cases of revision hip grafting were carried out arthroplasties wlth hemoloquous bone between the years 1990 and 1993. in the same period of time 732 pri~ total hlp replacen~nts were performed under i4entieal technical conditions. the average septic rate for the 'total hip althroplasties was less than 1%. in the selected i00 cases the septic rate was 4% indicating the role of bone grafting° homografts were prepared by deep freezing~ it .is recognized that the cells of the hl~grafts become destroyed by the ium~unological, response of the host~ and the patients develop ~ti-hl~, ar~tib'o~ies. the dead ~trix, however, has a bone-inducing capacity that stimulates host osteoblasts to recolonize the *i~/trix which serves as scaffolding. the sequence of events favours the infections. for this reason, beside preventive perioperative systemic ant/biotic treatment, local ~ntibioties were also applied in the form of antibiotic-//npregnated cement. the role of age and the .immune status of the patients .is discussed.. the purpose of this study is to evaluate the rate of toxemia in patients with acute panereatitis and to find this coudition to the activation of cascade systems that are encountered in the subsequent complications of the disease. we studied a series of 45 patients with acute pancreatitis, the severeness of which was evaluated by the ranson's criteria and the apach-ii scoring system. all of them were considered to have severe acute puncreatitis. the determination of toxemia was made using the limulus test (lal test). we also determined the levels of the third (c3) and fourth (c4) complement components as weu as the coagulation factors, iibrinolysis faeters and kimns by serial measurements. the severity of the disease was serially determined by the apach-ii scoring system. it was found that complement activation ( which was also assessed using a graphically illustrated method by a aggregometer ) was followed by an increase of morbitity and mortality .we also detected that toxemia (positive lal-test) was closely correlated with complement activation and more of the ranson's criteria. a clear relation existed between the number of ranson's signs and the enmplieations' rate (1"= -0.766, p < 0.001). the documentation of toxemia and the complement activation cannot predict the kind and the severity of complications. the study of coagulation, fibrinolysis and kinms systems didn't reveal any results with statistical significance. necrotizing pancreatitis still represents a life-threatenthg disease. infectious complications dominate among the causes of death. differences in the individual immune response could possibly explain different clinical courses even in patients with comparable pancreatic morphology. to explore the inflammatory response in acute pancreatitis, the following investigation was performed. methods: peripheral-venous blood was withdrawn on admission and furthermore twice weekly in as yet 36 patients with acute pancreatitis and tested for the parameters mentioned below. in parallel, polymorphounciear granaiocytes were isolated using density gradient centrifugation and assessed for superoxide anion and hydroxyl radical producing capacity using electron spin resonance techniques. results: total leukocyte cotmt and total lymphocyte count did neither reflect the clinical course nor predict complications. this comes tree also for serum igg, igm, iga, c3, c4, crp, alpha-l-antitrypsin and neopterth as well as for plasma il-la, il-ib, il-1ra, il-2, il-2r, il-6r, tnf-ct, tnf-~r (p75) and icam-1. in contrast, pmn-elastase, il-6 and il-8 closely correlated to the clinical course. isolated pmn's in vitro capacity to produce oxygen radicals depended on the respective radical species and was slightly elevated (superoxide anions) or decreased (hydroxyl radicals), respectively. patients with a cd4+/cd8+ ratio below i were seen at risk of developing septic complications. in contrast, a percentage of monocytes of 30 % or more among total mononuclear cells indicated an uncomplicated course, in general. conclusions: the immune status of the individual patient may significantly influence the course of acute pancreatitis. the cytokine pattern in peripheral blood is very complex and most parameters are of little use for the clinician. the pmn-elastase, il-6 and il-8, however, closely correlate to the clinical course and may prove valuable for follow-up. the cd4+/cd8+ ratio was found the best predictor of septic complications, but it failed in non-septic patients. a percentage of 30 % or more of monocytes among total mononuclear ceils indicated a rather mild course. the reduced ability of the pmns to produce hydroxyl radicals may help to explain the frequent development of septic complications in severe necmtizing pancreatitis. peroxidation of membrane lipids contributes to ceil injury in pancreatitis. overwhelming release of toxic metabolites by infiltrating neutrophils is regarded a major pathogenetic factor, too. as yet little is known about the mechanisms by which oxidative stress and leukocytes damage pancreatic cells. the present study examines (i) the susceptibility of pancreatic acinar cells to attacks by oxidants and leukocytes and (2) the potential of antioxidants to prevent such damage in order to better understand the cellular mechanisms of pancreatic injury in inflammatory states. methods: freshly isolated rat pancreatic acinar ceils were exposed to a model system of oxidative stress consisting of 20 mu/ml xanthine oxidase (xod), 500 mm hypoxanthine (hx), 50 mm fec13 and 75 mm edta. in a second set of experiments, acinar cells were exposed to excess autologous neutrophils or neutrophils obtained from patients with acute pancreatitis. neutrophils were stimulated by zymosan a, pma, and il-8. cell viability was assessed by both cellular uptake of trypan blue (tb) and by release of ldh. results: the xod/hx system caused a time-dependent acinar cell injury. this injury was effectively prevented by catalase (cat) and gfutathione peroxidase (gpx). in comrast, superoxide dismutase (sod) enhanced cell injury. addition of both sod and cat abolished the damage seen with sod alone. the non-enzymatic scavengers mannitol, dmso, dmtu and the iron chelator deferoxamine were not protective and at a higher concentration even accelerated cell decline. the newly developed antioxidants of the lazaroid type effectively prevented oxidative acinar cell damage. stimulated neutrophils, both autologous and heterologous, did not damage healthy acinar cells but had even protective effects. conclusion: pancreatic acinar ceils are very susceptible to oxidative injury. a combination of catalase and sod prevented cell damage effectively. sod when given alone may rather damage than protect aelnar cells when h202 is generated in concentrations overwhelming the capacity of endogenous catalase. therapeutic approaches to pancreatic disease using antioxidants should, therefore, include combinations of protective substances. the lazaroids seem to be candidates for clinical use as antioxidants in pancreatitis. the results argue against direct toxic effects of stimulated neutrophils to pancreatic acinar cells. are ch~act~z~ by the presence of a polymicrobial flora, the pmtotyi~ cffthese inf~ons is secend~,y bacterial pedtonitlw, whereby a pathololoeal process in the ~trointesfimd tract r~ful~ in tim disrup~on ofi~ inteffrlty and ¢ollseqtlent sptl]nge of inte~.i,o~.l gontents into the peritoneal c~iry. the ensuing infection invariably contains a mixtm~ of gt~m negative enteric bacilli, gram positive b~eria and anaerobe& experimental and clinical =t~ies have de~ed the eantrlbution of each of th¢~ components to ti~ ovemu virulence of these in~ons, gram negative enteri~ such as f.veher~chla coil ere endowed with a virulent l~l~x~lyse~haride ptill~ly t~sponsible for lethality, by contrast, bacteroldes sl~cles, which rarely c~se death, prornot~ abscess fonllation, a uniqm~ capsul~ polyseccluu'ide, particularly on b.j~ogiljs slrai~, oontributes to tjtis erect, several mecltanims have bccn pml~ed whereby or~ microorganism mi~t interact with its microbial ~net to augment the overall virulence of a r~xed im~edan. these include: l) provision of nutrients by one apexes which stimulates the growth of its ~opathoge& 2) inhibition of host deletes by one of the migroorganisms so that the other microbes might persist and exert their virulence, 3) the trant~ of vim.©n~e traits between ~renr~a.,dsms and 4) the ~.mizatian d the mi~oe~vironmental con~tion$ by one d the baetez'isl pa#, so that the other might persist. exampl~ for each of these m~banisms imv~ been provided by experimental ttudies i~stigating e.co!l-b.p~flls synergistic in~ra~ons. byproducts ofg.coli metabolim l~¢ovide essential short ebath fatty acids £~ optimal b,frosili~ ga'owth. fm-ther, oxygen ¢ons~tmption by kcelt lowers oxygen tension end redox potantial to levels eomlucive to b#a#lts gro~h. coawr~ely, b,~agtlis rolea~s proteases and fatty acids wl~¢h impair pl'tsgocy~¢ ~lt rmctlon tnd permit f-..¢oli proliferation and expression of its intrinsic virulent. in summaxy, interactions among the separate microbial cemponents of mixed infections heighten the overall virttienee of these lafectiot~, this knowledge provides ~r rationale for targetting of antibiotic therapy against the knowa eantributors of these synergistic pro~¢sses, intraabdominal abscess formation and the macrophage william g. cheadle, m.d., department of surgery, university of louisville school of medicine, louisville, ky 40292 inflammation of the peritoneal cavity following bacterial contamination has been classified into primary, secondary and tertiary, the last two relating to bacteria originating from the gastrointestinal lumen. the natural history of such infection is either resolution without clinical sequelae, which is uncommon, abscess formation, or generalized peritonitis, which occurs as a result of failure of peritoneal host defenses. early clearance of microorganisms by peritoneal fluid circulation and filtration througti subdiaphragmatic lymphatics into the thoracic duct and systemic circulation occurs as well. simultaneously peritoneal macrophages and the omentum approach the area of inflammation and lead to neutrophil influx and abscess formation adjacent to the affected viscus. we have found a shift in peritoneal macrophage function from antigen presentation to proinflarnmatory cytokine production that occurs early after experimental peritonitis produced by cecal ligation and puncture. this is also reflected by reduced class ii histocompatibility antigen expression on peripheral blood mononuclear cells and peritoneal macrophages. this is accempauied by an influx of both neutrophils and macrophages into the peritoneum and subsequent abscess formation. interestingly, there is little serum endotoxin or tnf seen in this model despite tnf mrna expression in peritoneal macrophages. we believe this model is more clinically relevant than other models of endotoxemia or bacteremia in which different patterns of cytokine expression are seen. newer agents aimed at reduction of systemic manifestations of sepsis originating from intra-abdominal infection such as monoclonal antibodies against cytokines or il-1 receptor antagonists may need to be directed against remote organ macrophage populations while preserving peritoneal macrophage function. inflammation is a complex process involving microcirculatory changes, extravasation of fluid and a cellular influx in the affected body area. in our communication, we will only consider the regulation of the cellular infiltrate which plays a major role in the defense of the peritoneum against microbial invasion. until recently, it was thought that the influx of leukocytes in the abdomen was induced by bacterial products, local humeral factors and secretions of resident macrophages. there is now increasing evidence that this view is too simplistic. many other cell types present in the abdominal cavity or composing the peritoneal membrane (mast-cells, mesothelial cells, fibroblasts) are able to release or secrete vasoactive or chemotactic substances such as histamine, prostagtandines, or cytokines. they are most likely to play a role in the regulation of intraperitoneal inflammatory reactions. the emigration of leukocytes towards the abdominal cavity is also modulated by a previous contact with gram negative bacteria. in the rat, this intriguing phenomenon is long lasting, cannot be transferred by serum and seems independent from t lymphocytes. the clinical relevance of these various regulating mechanisms has still to be determined. kinnaert paul, h6pital erasme, route de lennik 808, 1070 bruxelles belgium generalized response in secondary peritonitis the clinical course of an intraabdominal infection may depend on a variety of variables including the capacity of host defense mechanisms and the degree of the inflammatory response. if local defense mechanisms fail to restrict the inflammation to the abdominal cavity a generalized inflammatory reponse will result. in a first stage generalized signs of a local inflammation become detectable whereas the second stage comprises the overwhelming systemic inflammatory response. the extent of this systemic response determines the outcome. sometimes it may appear to be unrelated to the severity of the intraperitoneal findings. the activation of plasma systems and cellular elements leads to a fast release of cytokines, inflammatory mediators and other substances. these parameters precisely reflect the degree of the generalized response. inflammation of the peritoneum causes significant morbidity. objektives: to test the hypothesis that peritoneal mesothelial cells play a role in regulating inflammatory responses within the peritoneal cavity, we examined neutrophil-chemotactic activity (interleukin 8) and monocyte-chemotactic cytokine (mcp) release by sytokine-etimulated mesothelial cells. confluent human peritoneal mesothelial cells were exposed to varying concentrations of phorbolmyristate-acetate (pma) and the cytokines tumorneerosis factor a (tnf a) and interleukin i~ (il-i~). the supernatant was examined for il-8 by elisa and for mcp by investigating the ehemotactic activity for isolated human monocytes. mesothelial cells express low levels of il 8 and monocyte chemotactic activity when cultured. these activies were significantly increased (2-fold) after stimulation with either tnf a or il-i~. additionally macrophage inflammatory protein was detected. these observations provide a probably important mechanism whereby peritoneal mesothelial cells respond to imflammatory stimuli released during peritonitis and how leucocyte recruitment by liberation of chemotactic cytokines is regulated. the perioperative course of lps, tnfa and il-6 in 19 patients with bacteriologic proven abdominal infection (intraabdominal abscess 10, diffuse peritonitis 4, pancreatic necrosis 2, pancreatic abscess 3) was followed prospectively and evaluated for possible correlation with septic state and organ function. methods: patients were studied in a 6 to 10 hours period during their first surgical intervention because of intraabdominal infection. all were monitored for their cardiovascular, respiratory, hepatic and renal function. plasma samples for lps. tnfa and il-6 determination were drawn preoperatively, intraoperatively, and until 2h postoperatively in regular intervals (min 7/pat), results: preoperative apache ii was 12 in median (rain 4, max 25). 10 patients fulfilled the criteria of sirs. 4 of them were in septic shock.there was a significant correlation between preoperative tnfa and apache ii (p=0,000 i, spearman coefficient). preoperative cardiovascular (systol. rr<90 mmhg) and respiratory (pao2<75mm hg) dysfunction were associated with significantly elevated tnfa (cardial: p=0,000 i, wilcoxon; pulmonal: p=0,0003) and il-6 (cardial: p=0,2; pulmonal: p=0.0001) overall, lps, tnfa and il-6 values varied considerably during the observation period. however, tnfa was markedly higher in patients with sirs and septic shock (group a: n= i 0, mean 182 pg/ml) than in those who did not fulfill these criteria (group b; n=9, mean 24 pg/ml; p=0,00 i, wilcoxon). il-6 was significantly higher in group a (mean 2169 pg/ml) than in group b (mean 191 pg/ml; p=0,0o i wilcoxon). conclusion: perioperative tnfa and il-6 were shown to correlate significantly with preoperative organ function, apache ii and the severity of sepsis. these results could help to define patients that might benefit from further therapeutic strategies, e.g. antibody administration. department of surgery, university vienna, akh wien, wahringer gurtel 18-20, 1090 wien. aim of the study: the purpose of this pilot study was to establish and to prove a standardized reproducible animal model of intraperitoneal sepsis induced by e.coli-endotoxinaemia in lew.lw-rats in order to investigate early immunoserological responses to find a mediator based evaluating system of peritonitis sepsis. materials and methods: in 30 lew. lw-rats, diffuse peritonitis was induced by intraperitoneal injection of a mixture of e.coli (khu +) and autogenous haemoglobin solution. in the control animal group (n= 12) an intraperitoneally injection of physiological saline solution was done. blood samples were obtained by heart puncture after 6 hours. stastistieal calculations were performed on a personal computer with the spss programm vers. 4.01 (correlation with pearson's r, mann-whitney-u-test, descriptives statistics, discriminant analysis). results: in contrast to the sham treated rats, the peritonitis animals showed significant differences in the concentrations of endotoxin, interferon-gamma (wn-y), the pteridin derivate biopterin and serum pla2-activities [endotoxin range from 0.064 eu/i, sd=0.19 to 102.38 eu/1, sd-145.4 (p < 0 0001), ifn-¥ levels, range from 147.9 pg/ml, sd-141.6, to 4591 pg/ml, sd=6541 (p < 0.0001), circulating pla2-activities range from 116.2, sd=45.4 to 185.4 u/1, sd=105.4 (p < 0.01) and biopterin range from 54.4 nmol/l sd=17.2 to 127.8 nmol/l, sd=76.8 (p < 0.001)]. for the peritonitis group we found strong correlations between the degree of endotoxinaemia to elevated levels of ifn-'~ (rp = 0.72, p < 0.0001) and bioptefin synthesis (rv= 0.82, p < 0.0001). the increase of ifn-t levels was correlated to the regulatory synthesis of biopterin (r = 0 73 p < . .. p • , . 0.0001) and to the pla2-actwtues (rp = 0.50, p < 0.005). the biopterin synthes~s correlates slightly with the pla2-actn,ities (rp= :0.38; p < 0.05). using the para, meters of endotoxin, ifn-y levels, biopterin and the pla~ -activities only, the statistical procedure of the linear discriminant analysis makes it possible, to distinguish between non-septic animals and septic animals correctly at a rate of 87 %. anaerobes were found in 53.4%, anaerobes were isolated in 22.8%. there were aerobic and anaerobic associations in 12.1% and microflora was not found in 11.6% of the cases. express method of anaerobes discovering let to receive information on 1 -3 days early than in generally accepted nethods. intraaotal transfusion of oxygenate blood and laser irradiation of blood reduces the duration of anaerobic sow, disminishes intoxication and accelerate the patients recovery. patients with abdominal sepsis are subject to long periods of hospitalization and high associated morbidity and mortality rates. this category of patients is thus consuming extensive facilities and costs. as the age-related outcome of abdominal sepsis is not fully known, the aim of the present study was to investigate abdominal sepsis in the elderly. out of 166 patients with abdominal sepsis treated at the surgical intensive care unit during a 10-year period, 72 (43 %) had an age of 70 years or more. 28 were women and 44 were men, a sex distribution not differing with patients younger than 70 years. the patients were scored according to apache ii and septic severity score (sss) upon arrival to the intensive care unit. bacterial cultures, the occurrence of organ failure, hospitalization and outcome was noted. in median two operations were performed for both "younger and elderly" patients. the median time of hospitalization in the elderly was 29 (-183) days including in median 10 days in the icu. figures in patients less than 70 years of age were comparable (34 (-293) days out of which in median 10 days in the icu). apache ii and sss-scores did not significantly differ (14.2 vs 13 and 23.3 vs 26.5, respectively), between the groups. neither did the incidence of organ failure differ (57/73 vs 77/94). however, the incidence of multiple organ failure was significantly lower in elderly patients (21/72 vs 42/94 (p < 0.001)). the mortality rate, however, did not differ between the groups (21/72 vs 26/94). in conclusion, severe abdominal sepsis in the elderly was not associated with an increase in mortality, incidence of organ failure or hospital stay. with the help of light transmissional scanning electron microscopy morphology of erythrosytes of peripheric blood was studied in patients with different stages of diffuse peritonitis before and after intravascu!ar irradiation of blood with heliun-neon laser. peritoneal morphology was investigated in patients who died from peritonitis, it was established that in all phases of peritonitis occured stomatocytoric and echinocytoric transformation of erythrocytes which progressed simultaneously with increase of intoxication. it combined with strongly pronounced vessels variability of microcirculatory peritoneal bed which displaied by erythrocytes aggregation, stasis and microtrombogenesis. in intravascular laser irradiation of blood number of erythrocytes which underwent to stomatocytoric and echinooytorie transformation was lower than in patients without laser irradiation. it indicated that the intravascular irradiation of blood with helium-neon laser can prevent development of severe alterations of rheological property of blood and consequently variability of microcirlatory peritoneal bed in patients with diffuse peritonitis. abdominal sepsis is still associated with high morbidity and mortality rates, frequenfly caused by multiple organ failure. it has been reported that changes in capillary permeability play a role in the pathogenesis of multiple organ failure. the present study aimed at evaluating the influence of intraabdominal sepsis induced by cekal ligation and puncture on capillary permeability in multiple organs and tissues. 96 adult male sprague-dawley rats were subjected to laparotomy with separation of the cekum (sham operation) or induction of intraabdominal sepsis by cekal ligation and puneatre (n--48 in each group). at 3, 6, 12, 24, 48 and 72 hours (n=6/timepoint), the animals were evaluated concerning mortality and capillary permeability as determined by the passage of :25i-labelled albumin from capillaries to the peritoneum, the proximal and distal small intestine, cekum, colon, spleen, kidneys, lungs. the mortality rate in rats with intraabdominal sepsis was 33 % both at 48 and 72 hours. capillary permeability in the peritoneum, cekum, colon and kidneys significantly increased from 6 hours and on in rats with intraabdominal sepsis. in septic animals, capillary permeability in the lungs and spleen increased from 12 hours and on and in the proximal and distal small intestine from 24 hours and on. different types of alterations in capillary permeability seem to appear: 1) a temporary short increase e.g. in the proximal small intestine and spleen; 2) a temporary longer increase e.g. in the colon and kidneys; 3) a persisting increase e.g. in the peritoneum, cekum, distal small intestine and lungs. we conclude that experimentally induced intraabdominal sepsis induces early alterations in capillary permeability in multiple organs and tissues. such changes may contribute to explain the development of sepsis-induced multiple organ failure. despite a number of significant advances in the care of burn and non-burn traumatic injury, infection and sepsis remain major causes of morbidity and mortality. the severe immunosuppresslon often seen in patients with severe trauma or large burns may predispose these patients to life threatening infections. included among the many immune alterations are changes in the functional capabilities of neutrophlls (pmns). we have examined the expression of the p 2 integrins (cd1 l a, b,c/cd18), and the fc'?r (cd16, cd32, and cd64), as well as several functional parameters, on pmns from thermal and non-thermal traumatic injury, pmns were obtained from patients sustaining severe trauma (initial apache ii score >10) or thermal injury (>30~ total body surface area, 15% full thickness), and healthy controls. the expression of cd11 b and c and to a lesser degree cdi 1 a was significantly reduced on pmns. the expression of cd16 and cd32 but not cd64 was also significantly reduced. pmns displaying this reduction in receptor expression have a significantly reduced ability to phagocytose bacteria and undergo the oxidative metabolic burst response. thermal and traumatic injury result in global reduction in the expression of 132 integrins and for which may lead to decreased functional capabilities, these abnormalities may in turn account at least in part for the increased rate of infection in these patlems, institute, dept. of surgery, 2~1 8ethesda ave, cincinnalt, oh, usa, 45267-0s5b, antibiotic-phagocytic cell interactions: their effect on endotoxin release. c g c-emmet1, dep[baeteriolog.z, univer_sitv of glasgow, scotlan~_d increasingly it is recognised that pathogenic bacteria are capable of surviving intracellularly within phagocytic cells in addition to their capacity to produce disease whilst in the extracellular milieu. as well as providing protection from certain antibiotics which fail to penetrate the phagocyte, such intraceltular bacteria may be transported from the initial site of infection to a distant more vulnerable body site wherein they may proliferate. it is also known that some antibiotics are capable of becoming concentrated within phagocytic cells mid displaying bioactivity therein. such bioactivity might be responsible for the release of endotoxia #orn gram-negative bacteria which when liberated from the celt could ~gger the cytokine cascade. anfib,.'otic-induced damage to the ultrastructure of bacteria can also occur when the target bacteria are exposed to low (sub-mic) concentrations of certain drugs. such bacteria may present quite altered surface components m host-defense cells as well as releasing biologically active ceil wall components such as endotoxin. the nature of these interactions at the cellular level as well as the consequences for the host will be discussed. new jersey medical school: umd, newark, nj 07107 a technique of physiologic state classification has been developed based on the m~itlvariable analysis of patient derived data sets of seventeen physiologic variables. these multivariable data sets obtained from critically ill patients requiring intensive care, were aormallsed by the mean and the standard deviation of recoverin~ trauma patients who were not critically ill, the resulting normalized seventeen variable sets were then clustered. seven independent data groupings were developed. the normal stress response hyperdynamic state seen post-trauma and in compensated sepsis (a stets)/ metabolic insufficiency seen in septic decompsnsation (b stste}; early (c,) and late (e2) respiratory insufficiency associated with ards; cardlogenlc dscompensation (n state); post-trauma hyvolemla without shock (r stats). the stats closest to a new patient's values allows patient classifi0atlon with regard to his previous physiologic state. classifying observations f~om patients who lived or died who fell into these physiologic states enables a probability of death (p death) to be obtalned. utilizing this criteria for the staging of severity in 44 recent trauma patients the physiologic states accurately and significantly predicted the likelihood that the patient had an increased circulating level of the eytoklnes tnf and il-1. the probability of death (p death) as well as the cytoklne levels appear to be a function of the physiologic b state with the highest levels being seen in the b state of metabolic insufficiency and the c~ state of oombined respiratory and metabolic insqffioienoy characteristic of septlc ards. the increase in the magnltude of metabolic abnormalities associated with the transition from non-sepsls to septic a, septic b, or septic c z states was associated with an increasing probability of death (p denth)(mean a state =.28, mean b state = .57, mean ~ state = .61). the accuraay of this estimate was prospectively analyzed in this group of 44 m~itlple patients of whom 72% had sepsis and 36% had ssptlo ards. the 22 survivors had a mean p death of 0.39 and the 22 deaths had a mean p death of 0.63. the severity of post-trauma sepsis can be quantified by probability analysis and stra~ifie~ by physiologic state. serologic tests have not been extensively tes'~ed in surgical patients but seem to be of limited value. we use nystatin as the main form of chemoprophyhxis. patients "~'ith signs of infection who do not rapidly improve with antibacterial therapy are candidates for anti-funsal therapy, amphoteradn b remains the first llne of therapy although combination therapy '~'ith flueonazole is use;l with increasing freque~;c)', the recovery of c~dida from an antra-abdominal site represents a challenging problem, anti~ngal therapy in such patients depends on the underlying disease, the nature of the infected material and overall patient risk. role of neural stimuli and pain principles and practice of anesthesiology effect of combined prednisolone, epidural analgesia and indomethacin on the systemic response after colonic surgery arginine: biochemistry, physiology and therapeutic irnplications immunosfimulatory effects of arginine in normal and injured rats arginine stimulates lymphocyte immune response in heahhy humans rote of arginine in trauma, sepsis and immunity arginine enhances wound healing in humans if labrecque t, gv campion t, and the rhll-lra phase i//sepsis syndrome study group the cleveland clinic foundation a murine-anti-human tnf-monoclonal antibody known as cb6 was the first anti-tnf mab which was studied in a phase ii multinational trial in the treatment of patients with severe sepsis.this was an open-label, dose-escalation trial consisting of 80 patients who were enrolled into one of four treatment groups: (1) 0.1 mg/kg of anti-tnf mab, (2) 1.0 mg/kg, (3) 10 mg/kg or (4) 1.0 mg/kg at study entry and the second dose 24 hours later. the small sample size in each group (n=20) precludes detailed statistical inference in this study. nonetheless, a considerable amount of useful information was obtained from this investigation. irst, this study demonstrated the clinical feasibility of specific anticytoldne therapy in septic patients. second, the measurement systemic levels of tnf proved to be an elusive target; interleukin-6 may prove to be a more useful indicator of cytokine activation. third, immunologic reactions including tnf: anti-tnf mab immune complexes and human anti-routine antibodies were frequently found in these patients. despite their apparent lack of overt toxicity in this study, these immunologic reactions may complicate this form of anticytokine therapy. additionally, the potential benefits of anti-tnf mab therapy occur within the first 48 hours of therapeutic administration in these septic patients. infecting organisms differ in their potential to induce tnf in vitro and these differences correlate with circulating tnf levels observed in septic patients. rapid methods to define those patients most likely to respond to anticytokine therapy are needed to determine the ultimate therapeutic potential of these agents in clinical medicine. wherry, j., abraham e., wunderink r., silverman h., perl t., nasraway s., levy h., bone r., wenzel r., balk r., allred r., pennington j. and the tnfa mab sepsis study group.tnfa mab (bay x1351) is a murine monoclonal antibody raised against human tumor necrosis factor. tnf~ mab has been shown to reduce morbidity and mortality in animal models of septic shock and has been safely administered to septic and non septic patients.to evaluate the efficacy and safety of tnf~ mab in patients with sepsis syndrome, a prospective, multicentered, double-blind, placebo-controlled trial was conducted in 31 hospitals in north america. patients were prospectively stratified into shock or nonshock groups and then randomized to receive a single intravenous infusion either of 15 mg/kg tnf~ mab, 7.5 mg/kg tnf~ mab or placebo (0.25% human albumin).patients received standard aggressive medical/surgical care during the 28 day post dosing period.the three treatment arms were well balanced with respect to demographics, apache ii score and other parameters. for all infused sepsis syndrome patients, those who received tnf~ mab had slightly reduced 28 day all cause mortality compared to placebo. among shock patients there was a more pronounced trend towards efficacy at day 28 post dosing with lower mortality rates in both active treatment arms. among nonshock patients tn~ mab did not appear beneficial. the initial clinical experience with a chimeric anti-tnf monoclonal antibody, ca2, was undertaken in septic patients. the objectives of the study were to determine the safety, pharmacokinetics and effects on cytokine levels of ca2. as a single infusion or in combination with ha-1a in septic patients. the study was conducted with the intent to progress to an efficacy trial based on the information collected.the trial was conducted in three stages. stage 1 was an open label trial in which 4 groups of 5 patients each with the clinical diagnosis of sepsis received ascending doses of ca2 (0.1, 1, 5, 10 mg/kg). stage 2 was a randomized, double blind study in which patients received a single dose of ha-1a (100 mg) and placebo or one of 3 doses of ca2 (1,5,10 mg/kg). stage 3 was a randomized, double blind study in which patients received a single dose of placebo or one of 3 doses of ca2 (0.1, 1, 10 mg/kg). in addition to usual laboratory tests, the following assays were performed: chimeric anti-tnf concentration, anti-chimeric antibody, endotoxin, tnf, il-1, and il-6 levels.a total of 141 patients were enrolled from 13 clinical sites (20 in stage 1, 60 in stage 2 and 61 in stage 3). primary analyses were performed on patients in stage 1 and 3. there were 65 patients who received ca2 exclusively and 16 patients received placebo. administration of ca2 was well tolerated at doses up to 10 mg/kg. no patient discontinued treatment due to adverse events. human anti-chimeric antibody responses were positive in 61% (30/49) of evaluated patients. mean cma × and auc increased proportionally with increasing doses of ca2. the mean half-life was -70 hrs (58-96 hrs). a dose related decrease in tnf concentration was observed 1 hr post infusion of ca2. tnf is considered to be one of the central endogenous mediators for the inili'ation of the pathophysiological changes in patients with sepsis and septic shock. high tnf levels were demonstrated to correlate with patient outcome. blocking or neutralising tnf with specific antibodies was effective in preventing death in some animal modets of sepsis. in a placebo controlled prospective randomized study we tested the mur~ne derived antibody mak 195f. it is a f(ab')2 fragment. the fragment rather the complete antibody was selected in order to reduce the potential immunogenicity and to facilitate tissue penetration. 122 patients with severe sepsis or septic shdck were enrolied in the study, three different doses of mak 195f or placebo were administered (0,1; 0,3 and i mg/kg) over a perid of 72 hours in random order. the patients were evaluated for side effects, hemodynamics, organ dysfunction, cytokines (il 6, il 8 and tnf), and outcome. at this time only an interim analysis of 60 patients is available i indicating that mak 195f in all 3 dosage groups resulted in a decrease in il 6. this contrasted to a further in crease of il 6 in the placebo patients. no serious side effects have been reported so far. a more detailed analysis on all 122 patients in the study will be presented and discussed.$152 s154 staubach,k.h., otto, v., kooistra,a,, rosenfeid,j.a., bruch, h.p., univ. lfibeek, germany once endotoxinemia occurs in sepsis a vieieus cycle with translocation of et can be established. increasing the clearance capacity for et would therapeutically be the ulimate aim. we developed a new et on-line adsorption (ad) system in whole blood by means of polymyxin b (pb) coupled eovalently to a matrix (acrylic particles) via a 13 atom-chain spacer. the detoxification capacity was 150 ug[et/ml column material. the biocompatbility resulted in 90~ platelet recovery. the column contained 7 ml of admaterial and was sterilized by high steam autoclave, anticoagulation was achieved by heparine 1.000 iu/h in the inflowline after bolus injection of 5.000 iu. hp was performed on 8 pigs at a rate of 50 ml/min by means of a roller-pump until the animals succumbed (h). 8 animals served as controls (c). serum et levels rose from 3.40 pg/ml to 74,9 pg/ml after 5 hours in the c and from 2.77 pg/ml only to 28 pg/ml in the h group after 7 hours whieh was highly significant. survival time could be extrended from 216 to 313 min. results are listed in the following l. blinzler, p. zaar, m. leier, r. b(2rger, d. heuser clinic of anaesthesiology , city hospital nuremberg, germany sepsis and multiple organ failure (mof) are still related with poor prognosis inspire of pharmacological and technical progress. impressed by revealing reports about blood purification the continuous veno-venous hemofiltration (cvvh) was used as supporting treatment beside the critical cam basic therapy of mof. from 1989 to 1991 78 consecutive patients were treated by cwh. mof was caused by hemolrhagic-traumatic noxa in 21°,4 and by septic-toxic event in 79%. all patients required mechanical ventilation (fio2>0, 5) . 86°4 showed hyperdynamic shock. 85% had renal and 53% hepatic failure. medium appache ii score amounted to 29,8 points. cvvh was performed in postdilution mode with a polyamide membrane (fh 66) and high volume exchange (50 l/die). anticoagulation was done with heparin. hemofiltration in mof was installed, when critical cam basic therapy including adequate respiratory and hemodynamic management, pamnteral nutrition, antibiotic treatment, etc., failed to stabilize organ functions. during consequent application of cvvh most of these patients showed improvement of their clinical course. pulmonary stabilization was seen in 71%, hemodynamic in 68% and renal in 66% of the cases. 42% of the patients survived and were discharged from hospital. 10 of 45 non-survivors (58%) died because of fatal mof within 24h after admission to icu. patients with early application of cvvh in mof showed a better survival rate.mediators of mof, i.e. products of the complement cascade measured in blood and nitrafiltrate by elisa, were partially removed by cvvh. the testing ultrafiltrate by hplc demonstrated decreasing spikes ofpolypeptides during hemofiltration. mof seems to be generated by cascade-activation of immune competent cells and plasmatic mediators (e.g. bmdykinin, eicosanoides, cytokines, anaphylatoxins, etc.). therapeutic approaches aim to inactivate or eliminate single substances. cwh with high-flux membranes in combination with high-volume exchange allows elimination of many mediators with different molecular weight and therefore may contribute to improve the prognosis of mof. other significant advantages of this teqalnique like adequate nutrition, optimized fluid balance and control of body temperature should not be negicctod. introductioni pseudomonas (p) aeruginosa has to be considered an important pathogen of nosocomial pneumonia and septic organ failure. the lung seems to be the predominant target organ for the pore-forming p. aeruginosa cytotoxin, thus inducing microvascular injury. with respect to therapeutical consequences, the potential protective effects of paf-antagonist (web 2086), cyelooxygenase inhibitor (diclofenac) and specific and unspecific antibodies on cytotoxin-induced pulmonary vascular reaction and mediator release were studied in the isolated perfused rabbit lung. methods: cytotoxin (6p_g/ml) was administered into the perfusion fluid in all groups, either in the absence of inhibitors (n=6), or after pretreatment with web 2086 (5xl0-gm, n=6), or diclofenac (10#g/ml, n-6). furthermore, the application of specific antitoxin (mg/ml, n=6) was tested in comparison with the unspecific immunoglobulins (venimmun®, behring, 7.5 mg/ml) (n=6) and the combination of immunogiobulins, web 2086 and diclofenac (n=6). six experiments without toxin served as controls. the arterial pressure mad the weight gain as an indicator of edema formation were continuously monitored during the three hour peffusion phase. arachidonic-ucid metabolites, as well as lactate dehydrogenase (ldh) and k + concentrations were determined at 30 rain intervals. results: cytotoxin caused a gradual increase in pulmonary arterial pressure, reaching a maximum value of 2.5 times higher than the control, starting after 1 min and a delayed onset of edema formation resulting in a mean weight gain of 20 g after 120 min. this was paralleled by a significant increase in prostacyclin generation and a continuous release of k + and ldh. thromboxane synthesis exceeded about 10 times that of controls in the toxin treated lungs. pretreatment with web 2086 or diclofenac significantly attenuated the pressure response and edema formation evoked by cytotoxin. the addition of the unspecific immunognbulin preparation alone induced a transient pressure increase within the first minutes, but mean values remained below those of the cytotoxin group in the continuing observation period. mmost complete inhibition of the pressure reaction, the edema formation and the metabolic alterations was achieved mainly by the combination of immunoglobulin, web 2086 and diclofenac and to lesser extend by the specific toxin antibody. conclusion: the current results point towards the crucial role of paf and aa-metabolites as mediators of cytotoxin induced microvascular injury. the systemic or local application of cytotoxin antibodies or even unspecific immunoglobolins in combination with paf-antagonist and diclofenac appears to be a promising therapeutic approach in the case of infection with cytotoxin-preducing strains. cytokines have long been shown to be of particular importance in the metabolic derangements occurring in lps-induced shock. recent studies strongly imply the involvement of platelet aggregating factor (paf) in the pathogenesis of gram-negative bacterial sepsis. an autocatalytic feedback network has been postulated to exist between paf and tumor necrosis factor (tnf), a key cytokine involved in septic metabolic cascade, leading to an uncontrolled amplification of inflammatory mediator release. we have previously shown that st 899 (4-n,n,n trimethylammonium-(r)-3-isovaleroyloxy-butanoic acid z-3-(5-chlorphtalidiliden) ethyl ester bromide) was quite effective in inhibiting the "in vitro" binding of 3h-paf (ki=7.5x10 -8 m) to rabbit platelets. the present study shows that pretreatment of c57bl/6 mice with st 899, administered by different routes, dose-dependently and significantly reduces the lethality induced by endotoxin (e.coli 055:b5 injected at 30 mg/kg intraperitoneally). very interestingly, st 899 administered at the same doses as above (i.e. 1.25, 2.5, and 5 mg/kg body weight) results to be significantly effective in reducing the endotoxin-induced release of serum tnf. the reported dual activity of st 899 (i.e. paf antagonism and decreased circulating tnf levels) may turn out to be greatly beneficial, in combination with current therapies, in the treatment of diseases that involve overproduction of tnf and paf such as septic shock. introduction: recently, we reported that prophylactic whole body hyperthermia (42.5°c) induces heat shock protein ('asp) 72 and increases smvival 2-4 fold in a mouse endotoxin model (am. j. physiol. in press). other investigators reported that prophylactic pharmacologic induction of hsp-72 by sodium arsenite improves survival in a rat sepsis model (abstract a95 am. rev. resp. dis. vol. 147, 1993) . the effects of heat are complex and in addition to formation of lisp-72 include release of cytokines, changes in cellular ph etc. thus, the protective mechanisms of heat may differ from those due to pharmacologically induced . the purpose of this study was to compare the protection of heat vs the protection of pharmacologically induced hsp-72 in a mouse endotoxin model to determine if different protective mechanisms were likely to be involved.. i%'lethods: both sodium arsenite (10 mg/kg) and ethanol (400 ~1 of 39% ethanol) caused marked induction of hsp-72 in lung, gut, kidney, and liver, which was comparable to heat-induced hsp-72. female nd4 mice weighing 22-25 gms were pretreated with arsenite or alcohol 8 hours prior to challenge with escherichia coli endotoxin (-ld 80) and survival was compared to control mice. results: survival at 24 hrs. for arsenite treated and alcohol treated mice was 96% and 88% respectively and was statistically different from the 40% survival for control mice. (p<0.01) (n=25 mice per group). however, at 7 days post endotoxin, there were no differences in survival in the 3 groups, i.e., ~ 4% survival for all 3 groups. in contrast, the protective effect of hyperthermia remains present at 7 days, i.e., ~ 70% survival vs 20% survival control. conclusion: the protective effect of heat is probably due to other factors such as the effect of hyperthermia to release il-lc~ and is not due solely to hsp-72 formation. it was the aim of the study to examine whether bacteria play a causative role in the pathogenesis of anastomotic insufficiency following gastrectomy in man.the study was carried out in form of a prospective, randemised, double-blind, multicenter trial. primary endpoints were the rate of anastomotic insufficiencies, infectious-and uncomplicated postoperative courses. all pat. received a periop, i.v. prophylaxis with cefotaxim. identical numbered vial either contained placebo or polymyxin b, tobramycin, vancomycin and amphotericin b . the vials were administered 4x per day from the day be ~ fore the operation until the 7th postop, day. insufficiencies were detected by gastrographin swallow and recorded by x-ray on day 7 postop.. evaluation was carried out on an "intention to treat'basis. statistical analysis was done with the pearson's chi square and fisher's exact tests~ results: interim analysis was carried out in 3/93 after 137 pat. had been recruited. along with a significant reduction of s.aureus and enterobacteria there was a reduction in the rate of anastomotic insufficiency of the esophago-jejunostomy from 10.6 % in the placebo-group to 3.5 % in the treatment group. the difference was not yet significant. the rate of nosocomial infections (e.g. respiratory tract infection and uti) were significantly reduced from 33.3 % in the placebo-group to 16.4 % in the treatment-group (p ~ 0.0281;fisher's exact test). in march 1994 final results with more than 200 patients will be presented for the first time. (= po2 <80 mm hg, b s-creatinin >2 mg%). respiratory insufficiency was the most frequent systemic complication followed by sepsis and respiratory insufficiency. etiology of pancreatitis and initial serum increase of pancreatic enzymes predicted neither complications nor outcome. only 2 of 14 deaths occurred during the 1st week, all other deaths occurred late (after 4-12 weeks), generally as the consequence of septic complications and multi-organ failure. high levels of crp were correlated with a compliacted course and a fatal outcome. although same cytokines (e.g. 11--6) were found increased in severe disease, the predictive value of these markers was not better than the combination of ctinical scores (ranson, imrie, apache ii) with gt or crp. conclusions: intensive care medicine can often control the inital shock situation in severe pancreatitis. thus. only 15% of deaths today occur eady in the course of the disease, whereas this percentage varied between 40-70% just 10 years ago. nowadays, most deaths are caused by late septic complications and multi-organ failure. ranson-and ct-scores as well as serum crp predict a course with systemic complications; they are less helpful for prediction of sepsis and late mortality. it is doubtful whether measurements of cytokines will help to better predict the late outcome. as yet, only careful and continuous monitoring of patients (e.g. by apache scores) may help to early identify those who develop septic complications and multi-organ failure. the classic description of severe acute pancreatitis has hinged upon the release of large volumes of activated enzymes into the peritoneal cavity and thertce the lymphatics and blood stream. these activated enzymes escape from the pancreas due to disruption of cells with associated ischaemia and occasional infarction of tissue. for 20 to 25 years it has been postulated that the bocly's defence system to activated pancreatic enzymes required supplementation iu the form of anti-protease support either in the vascular space or in the peritoneal cavity. all controlled studies have shown that this is either impracftcal or unnecessary.hore recently release of a large number of cytokines from monocytes, macrophages and neutrophils have been considered to be harmful to the body and various agent~ which oppose the action of tnf alpha, paf and similar cytokines are being examined in experimental anim~is and certain clinical trials, it has clearly been shown that higher levels of cytokines are released in the patients with objectively graded severe acute pancreatitis than in those with milder disease. we now seem to be moving into an exciting phase of potentially beneficial therapy in acute pancreatitis which has had no specific effective therapy through studies utilising aprotinin, gabexate mesilate and fresh frozen plasma. inflammation cascades may play a role in the pathogenesis of acute pancreatitis. to evaluate the status of the cellular immune system we examined serum concentrations of immune activation markers in 24 patients with acute pancreatitis (14 males, 10 females; median age: 56 years, range: 32-81 years). concentrations of neopterin, serum soluble tumor necrosis factor receptor (stnf-r) and serum soluble intercellular adhesion molecule type 1 (slcam-1) were determined using immunoassays (henning, bender, t cell sciences). 13/14 had increased concentrations of stnf-r compared to the 75th percentile obtained in healthy controls (>4.2 ng/ml), and 9/24 patients had increased neopterin (> 8.7 nmol/i), 9/24 presented with elevated slcam-1 (>440 u/i). all patients with increased neopterin also had increased stnf-r, 4 patients had concentrations of all three markers outside the normal range. there existed a significant correlation between neopterin and stnf-r (rs = 0.728, p < 0.001 ). weak associations between age and stnf-r (rs=0.435, p=o.05) or neopterin (rs=0.456, p = 0.044) were also found. our results demonstrate activation of the cell-mediated immune system taking place in a sub-group of patients with acute pancreatitis. the finding of increased neopterin and stnf-r levels implies that activated monocytes/macrophages are involved in the pathogenesis of the disease. further data are necessary to evaluate potential associations between changes of marker concent-rations and the course of the disease. pancreatic injury after heart surgery was reported as soon as 1970 ( 1,2) and characterized by increased serum or urine amylase levels (in about 33% of patients) in the 5 fi~t postoperafi.'ve days. this pancreatic injury, which sometimes led to acute pancreatitis, was atreaay at~buted to inappropriate perfusion of this organ. in the 198ffs, 3 studies were published dealing with pancreatic suffering alter heart surgery, in large series of patients, concluding ~n~at panc~a~c injury (with a low incidence of pancreatifis) is more common than previously recognized and is a potential source of complication after camliac surgery (3, 4, 5) . in a recent study (6), evidence of pancreatic cellular injury was found in 80 out of 300 patients undergoing cardiac surgery, with 22 out of these 80 patients presenting abdominal signs or symptoms and 3 developing severe pancreafitis. this injury was associated w~th preoperative renal insufficiency, valve surgery, ~..stoperalive hytxxension, calcium administered periopuratively and length of bypass. we studied 300 patients submitted to cardiopulmunary bypass (cpb) for heart surgery and used the measurement of un:~sin, pancreatic iso-amylase and lipase in plasma for biochemical characterization of pancreatic cellular injury. blood samples were obtained before surgery, directly aller surgery (return to inte~ve care unit), 8 hours alter surgery and in the folfowing days alter surgery (days 1, 2, 3, 4 and 8). computed tomography scan of pancreas was performed in patients presenting hi~ levels of amylase on day 1. we measured abnormal levels of trypsin and pancteatic iso-amylase in 30% of patients and observed 2 simultaneous releases of these 2 enzymes, the fi,'st one in the 24 hours after surgery and the second more intense from day 4 and pa~icularly on day 8 after smgery. this second release was concomitant with abnormal levels of llpase. these biochemical observations were accompanied by radiological and clinical signs of pancreatic injury in about 2% of our patients : pancrealic abnormalities were revealed by scan in 7 patients and acute pancreatitis in i patient. more pronounced pancreatic suffering was observed in patients undergoing valve replacement than in patients undergoing coronam-anrtic bypass grafm~g. analysis of trypsin and pare're, tic 1so-amylase are sw.cific of pancreatic cellular injury and their simultaneous ir~rease in plasma alter cpb in our padents confirms the presence of an exocrine pancreatic injury. the presence of a simultaneous peak of lipase mcaezse~ the specificity of overt pancreatic injtu 7 diagnosis. the precise cause of th/s injury could he related to hypoperfnsion leading to ischemic injury of foe splancbnic area, pancreas being largely sensible to hypoperfnsion (7). this hypoperfosion could he responsible for the ftmt release of pancrealac enzymes observed in our patients and would contribute to the deterioration of other organs leading to an inflammatory reaction developing in the following days and responsible for the second release of pancreatic enzymes observed in our patients. patients with necrotizing pancreatitis show a heigh rate of pulmonary, renal and septic complications, whereas the course in acute interstitial pancreatitis is generally very mild. we have prospectively analysed the value of endotoxin, interleukin-6(il-6) and transferrin in compare with c-reactive protein(crp) for the early assessment of the severity of acute pancreatitis. patients aud methods: the values of endotoxin(measured by limulus-lysate-test), ii-6(elisa), transferrin and crp (nephelometry) were analysed daily along the first i0 days of hospitalisation by 28 patients with acute pancreatitis admitted to our hospital from 4/1991 to 4/1993 . it was judged whether the patients have either interstitial (aip) (n=9) or necrotizing (anp) (n=lg) pancreatitis. 5 patients with anp have died during the course of pancreatitis (mortality=17.5%). results: 1-severity o~ pancreatitis: signifcant differences (p<o.05, mann-whitney test) could be calculated between aip and anp for endotoxin, il-6, transferrin and crp during the i0 days of monitoring. 2-mortality: except of il-6 on days 2, 3, 4 and 5 after ad-mission there were no significant differences between the values of analysed parameters in patients who survived and those values in patients who died. 3-organ failure: the patients with pulmonary, renal or multiorgan failure have significantly heigher values of endotoxin, il-6 and crp along the i0 days of monitoring in compare with those patients without an organ failure. conclusions: endotoxin, il-6 and transferrin, an important endotoxin carrier, are promising parameters to differentiate between the severe and mild form of pancreatitis comparable with crp. this parameters may be helpfull in the early clinical assessment of patients with acute panereatitis. the determination of cytokines may elucidate the pathophysiologic mechanisms that produce early systemic complications in acute interstitial (i) or necrotizing (n) pancreatitis (ap). the appearence of respiratory insufficiency (ri) is used to appraise the early systemic complications and is compared to the results of cytokine blood levels. in a prospective clinical trial from 10/92 -8/93, 23 patients with ap were recruited and blood samples taken for cytokine determination with commercial elisa-kits. the differentiation between i (n=12) and n (n=l 1) ap was made through ct-scan in all and laparotomy for drainage and necrosectomy in 13 patients. the etiology of ap was gallstones in 9, alcohol abuse in 7, hyperlipidemia in 2 and other or unknown causes in 5 patients.five of 11 patients with nap died early (n=l) or of late septic complications. none died of iap.the peak of cytnkine level in the first three days of hospitalisation was used for calculation. ri was diagnosed in the first week of hospitalisation, if oxygenmask or iutubation for at least 3 days was nescessary to treat arterial hypoxemia. for statistical analysis u-test of wilcoxon, mann and whitney was applicated.the il-6 concentration in blood reached up to 2600pg/ml in the first days depending on the severity of ap and dropped to almost zero in the next days, independently of the clinical course. the differentiation of i-versus nap, using a cutoff-line of 600 pg/ml was correct in 20 patients (87%, sensitivity (se): 82%= specificity (sp): 91%, (z:0,001). high levels of il-6 over 500 pg/ml correlated well with the prognosis ifri in the first week (accuracy: 87%, se: 80%, sp: 100%, c~: 0,001). the blood levels of il-8 reached a maximum of 1381 pg/ml in the first days, depending on the severity of ap, and showed a correlation to the clinical course in the following days. the peak of 1l,-8 blood levels indicated correctly the severity of ap in 18 out of 23 patients using a cut offtevel of 200 pglml (accuracy: 78%, se: 82%, sp: 75%, ~: 0,01). there was no correlation between cytokine blood levels and mortality, also there seemed to be no correlation between the levels of il-6 and il-8. in the bloodsamples of five patients with i-or nap no c~-tnf was detectable.the blood levels of il-6, and to a lesser extent of [l-8, can predict the severity and early systemic complications of ap in men. the excessive rise of cytokines can be explained by the stimulation of immunological cells ( macrophages, lymphocytes and endothelial cells) in the course of ap. objectives: in an opossum model, ligation of the sphincter of oddi or separate ligation of the bile and the pancreatic duct together leads to severe haemorrhagic pancreatffis while single ligation of the pancreatic duct causes only an exocdne atrophy. since bo has been discussed to be a contdbutor t¢, res dysfunction, this may depdve the organism of a potent defensive mechanism thus promoting the course of pancreatitis. the present study wa,,; carded out tc develop a new method for measuring dynamic liver res func.. tion and to test the hypothesis that bo causes a res dysfunction. material & methods: 18 opossums were randomly placed in three groups. all received a central venous line. after midline laparotomy, a specially designe0 tourniquet was placed around the common hepatic duct above its junction with the pancreatic duct (group a, n=6), around the pancreatic duct (group b, n=6) or around both ducts (group c, n=6). after one week, the tourniquets were closed. using a scintillation camera, the liver uptake of nanocoll, a 99mtcalbumin colloid with a diameter of 25 nm, was measured before, 2,5 and 6 days after the obstruction. the curves were analysed by a computer and two parameters (r, s) were calculated using natural logarithmic regression. as a common measure for res function, the corrected granulopexic index (a) wa,,; determined, after day 6, the pancreas of each opossum was searched for necrosis by morphometdc methods. results: all animals survived till day 6. the opossums in groups a & 8 had a macro-and microscopic normal pancreas, while those in group c showed a severe hemorrhagic pancreatitis. the granuinpexic index showed a significan~ change to the worse starting at day 5 in group a & b (p<o,05) and at day 2 it~ group c (p<0,ol). at day 6, res function in group c was significantly worse than that in group a & b (p<0,01). the regression parameter r showed no significant change in groups a & b, but a highly significant decrease in group c starting at day 2 (p<0.005), thus showing a dysfunction of the hepatic res. conclusions: obstruction of the hepatic or pancreatic duct alone does no{ result in immediate dysfunction of the hepatic res, while obstruction of beth ducts does. because only ligation of both ducts is followed by a severe hereof.. rhagic pancreatitis, res dysfunction could be an important factor in the pathogenesis of pancreatitis and resulting organ failure.logarithmic regressinrl has proven to be a valuable tool to analyse dynamic hepatic res function. (b6) in lewis rats weighing 220-250g. there were five groups: group a (n=14) were untreated controls; group b (n=7) were given isotonic saline intraperitoneally and observed for 48 hours; group c (n=7) 2 ml x 108 e. coli intraperitoneally and observed for 48 hours; group d (n=7) were given 4 ml x 108 e. coli and observed for 48 hours; group e (n=7) 4 ml x i08 e. coli and observed for 24 hours, the rats were than killed, the spleens were removed and heparinised blood taken for immunological tests. total t-ceils, helper t-ceils, suppressor t-cells, monocytes, and the interleukin 2 receptor were determined by monoclonal antibodies, indirect immunfluorescence, and flow cytometry (facs analyser 11). statistical analysis was by the t test on rank transformed data.in group b (isotonic saline) the total t-ceils increased in the blood (p=0,003) and spleen (p=0,0001 )com pared with the untreated rats (group a), in the groups with peritonitis (c, d, and e) we found highly significant rises in suppressor t-cells in the blood and spleen (both p<0,0001 ) compared with the non-infected control groups a and b. this increase was significantly higher in group d than in groups c and e. as well as other alterations, we found a uniform increase in the number of t suppressor ceils in our model of acute peritonitis that was both time and dose dependent, department of surgery, university vienna, akh wien, w~hringer gurtel 18-20, 1090 wien. d~'na~'aics of som~ l~mnunologic indices in children with abdo;~tinal shock v.z.i~ioskalenko, s.f.vesiol$,t.v.p~bina serum (iga,:~i,g, circulating imaune co!~plexes, co:apleaentary serum activity, antimesothelialvisceral and parietal antibodies) and cellular (i-l~q~hocytes, ~s/th,b-lyaphoeytes,i nlaunoleukosis to aesothelial allergens; spontaneous blast cell transformation to phytohemaaglutinin and .nesoth<-~lial ~,togen; phagooftic neutrophil activity) im:~une indices v.'ere studied in 60 children operated v:ith symi~to'as of abdo~ainal shock. ,'~!-~ of them had appendicnlar(26-1ocalized jo-diffuse, 9-~eneralized) and 5-hemoperitoni-~is (dull abdominal trau;na with injayy of 'the spleen-4, the spleen and liver-q), flo patients tjere operated on for co~iplete iieus (~-intussnsc.sption, 5-com:lissural ileus). 2he follo~'~ing serum factors: cireula:;ing im~aune complexes and ~).esothelial antibodies are the aost i~portant ~ar~ers of the abdominal shock course. :yith the progress of the process the indices increase irresponsive of t.l~e cha±.aeter of pathology. in case of ileus regress and hemoperitonotis an abrupt drop in ~he titer of anti~aesothelial antibodies is noted. in bacterial peritonitis a high ~iter of anti:aesothelial parietal antibodies in great dilutions ("+++","++++"-q ; 720) persisted even in the period of clinical recovery. cellular factors cham'acterize dfnaaics of the abdominal shock coulees less specifically llast cell transfor~aation and phagocyue neut~ophil activit 2 can inderectly characterize transition of shock f~orn reversible into irreversible phases. the past so years have brought a number of major advances in burn care. the initial advance was the discovery that burn victims required large volumes of resuscitation fluid in the initial 24 hours to maintain hsmodynamic stability, this was followed hy the dsvelopmsnt of topical antimicrohlal agents to prevent end treat infections. next, was the reporting that early excision of burn wounds, with autografting of the excised wounds was possible.finally, was the identification of the importance of aggrsssive nutritional support for the hypermstabolic burn patient.these advances have markedly increaesd survival rates for given burn mimes, s~ch that burns which would previously have been considered to be aniformly fetal, are now readily survivable.thsre remain two unsolved problems in burn care, which are limiting survivability cf burn patients.the first ie ths treatment of inhalation injury. ths sscond is how to obtain coverage of ths maaaivsly burned patient, who has limited skin graft donor sites available.this ssssion will focus on the new treatments being developed to obtain permanent coverage of burn wounds.these include the use of various tcplcal growth £aotore which can speed the rate of reepithelialization of wounds, both the beneficial and dstrimental effects of using cultured karatinooyte preparations, to obtain in excess of one squats meter of a pseudoskin from a single fifteen square centimeter biopsy of unburned skin, will be discussed, finally, the used for artificlal dermal preparations will be discussed. integumentary wound healing consists of simultaneous epithelializalion and contraction. not long ago, it was thought that healing proceeds at an optimum fixed rate and could only be delayed. the recent explosion in molecular biology and recombinant gene techniques have produced information on the biological activity of compounds previously believed to be biologically inert. many current attempts to modulate the rate of wound healing center on the topical application of various growth factors produced by integumentary cells or agents designed to modulate growth factor expression or response. epidermal growth factor (egf), plateiet derived growth factor (pdgf), transforming growth factor (tgf) and fibroblast growth factor (fgf) have been demonstrated, both in vitro and in vivo, to stimulate the proliferation and diferentiation of their designated cells types. egf and pdgf have been clearly demonstraled to increase the rate of wound closure in partial and full thickness wounds. pdgf also has demonstrated efficacy in the closure of recalcitrant pressure sores, whereas tgf increases the tensile strength of incisiona[ wounds and fgf stimulates angiogenesis. it appears that epidermal keratinocyte migration during wound healing depends on integrin-mediated interactions with compounds and ceils extracellular matrix. additionally, the activities of extracellular glycoproteins such as fibronectin, fibrinogen, laminin and thrombospondin are coordinated by multiple integrins with specific distributions and binding affinities. currently, agents which incorporate specific protein sequences essential for the interaction of the glycoproteins with the cells of the extracellular matrix are being investigated. it is has been clearly established that the rate and nature of wound healing can be influenced by the application of various agents and that sufficient quantities of these agents can be manufactured. the future challenge is to develop techniques to now the objective evaluation of the clinical efficacy of these various agents and to employ the appropriate agents in a cost-effective manner. transplantation; and 4) the immune status/manipulation of the host.multiple interactive variables will determine the effect of employing allogeneic cells and tissue in wound coverage design and a batter understanding of the dynamics of the wound-graft microenvironment will facilitate the dove opment of clinicauy beneficial graft composites. cadaver allograft skin (cas) is the "gold standard" for wound coverage, but has limitations including varied availability & quality, immunogenicity leading to rejection, & potential for disease transmission. our development of a temporary living skin replacement (tlsr) addresses these issues; the tlsr consists of highly screened human neonatal fibroblasts (hf) cultured in biobrane a, a bilayer dressing consisting of nylon mesh laminated to a thin silicone membrane which controls water-vapor transmission. studies in our lab indicated that "fresw ~ tlsr grafts reproducibly close full-thickness wounds in mice & perform better than bb controls. we studied wound adherence & structural/molecular properties of fresh & cryopreserved (cryo) tlsr. methods: tlsrs were prepared by seeding 4000/co 2 hf in bb nylon mesh in dmem. hf quickly attached to the nylon mesh & were allowed to proliferate 3-6 wks. fibroblast mitochondrial activity was assayed by mtt assay. matrix proteins were identified by immunostaining, mrnas for specific growth factors were identified by reverse-transcription polymerase chain reaction amplification, & quantitated by gel scans. bb structure was studied by scanning electron microscopy & stretching measurements pro/post cryo. 80 grafts were cryo'd in 10% dmso, quick-thawed & sutured onto 2x2 cm excised full-thickness dorsal wounds on athymic mice. 40 "fresh" grafts were similarly placed. controls grafts were cas and aeellular bb. adherence was quantitated by a device which peeled grafts from wounds at intervals following placement, using a serve motor and a strain gauge. results: postcryo, storage and subsequent thawing tile tlsrs maintained >60% cell viability by the mtt assay, indicating continued mitochondrial activity, and bb structure & stretchability were maintained. multiple matrix proteins secreted and deposited in the bb nylon mesh (types l/iii collagen, decorin, fibroneetin) were identified by specific immunostaining. growth factor mrnas in the tlsrs (afgf, bfgf, kgf, tgf~,p~,) were present in 10-10,000x higher levels in fresh/cryo tlsrs than in adult hcs. grafts adhered to wounds on mice through 40 days of followup. histologic exams on days 6-40 showed excellent vascular ingrowth and minimal inflammation. adherence of tlsrs to wounds was >cas adherence. burn wound coverage in the massively burned patient remains a difficult problem. although cultured keratinocytes have been utilized for burn wound coverage, their impact on the patient with burns greater than 80% total body surface area has not been spectacular, with poor graft take and unstable epithelium.current investigations have been directed toward dermal replacement beneath either very thin split-thickness autografts (stag) or utilizing cultured keratinocytes. current products include: collagen dermal replacement with thin stag (burke, et al). collagen dermal replacement with cultured keratinocytes and fibroblasts (boyce, et ai). allograft dermis with cultured keratinocytes (cnno, et al). allograft dermis with thin stag (life cell). polyglactin acid mesh and neonatal human fibroblasts with thin stag (hansbrnngh, et al).investigations regarding culture media, use of growth factors, topical nutrients and antibiotics, and melanocytes for pigmentation as well as safety and efficacy are needed before any of the current products become viable options for coverage of the massively burned patient. the~ is a growing world-wide problem with the ujc of cadaver tissues and ocgans bae, au~ of the tren~m~s~km of dilemma such a; cmutzfeldt.jukob disease and iiiv as we1] as ready availability of urdform lis~ue~. on dec~mt~r 14, 1993, the fda assumed control of as1 tissue bar~s in the uldtod st=tea in an attempt to bflng ~s difficult problem of dise~s~ transmission under ¢onlrol. in europe, ~om¢ of the governments are consldofll~ a c~mplcte bat) on the use of cadaverlc fissu~s such as ddn, 'this |ncroam in regulation of cadavefle ~s,quct will incmar¢ the difficulty of obtain~g and dlslflbulmg them. however, thc nc~ for these tissues contlnue~ m incrcaso, we will discuss ~'l¢ solulion to this important pmbl~n: tissue engineering. tlssu~ engineering is an in~rdisdpllnary field that applies pdnclplc~ of angin~edng and die life sclcnce~ reward the development of ~olok~¢al sub~dtute,~ ih= mslom, maintain, or improve tissue function, "13ssuc ongln~cdng can provide ~ho nccassary tlssuoa for wound repair ~d ibe assuranoe fl'~t the lissuos are d.ls¢~¢ free. in addition, a ds~uo-cng~ne~n~l wound covering will bo u~lvemally acceptable and evntlublc as "off g~o shell", consis~t products, them are several approaches to restating thls function in a large wound, 'l'nosc i~elud~ tmmcdiete long term coverage, short t=nn coverage, uandtl~el coverage and compost= dssu¢ coverage, "flssuo onglncrcd wound coverings that meet those vaflous ne,.cds will he r~vlowod.cllni~:sl and experimental d~la in venous ulcer, dlabctl¢ ulcers, prossur~ ulcers and bum wounds wgj be mvlcw~, a~ welt as new approacl~s u~ csrtilag¢, bone, liver and bone marrow it~suos. c oomplon, k nadirs, w press, g wetland, j fallen iv, shrtners burns institute and massachusetts general hospital, boston, ma~schusetts, usa the clinical "take" rate o? cultured epithelial autografts (cea) has been observed to increase with transplantation to allodermls, but the reasons for the improved clinical performance have not yet been defined. the aim of this study was to determine the biological impact of normal human dermis on cea differentiation and maturation, biopsies of cea transplanted to engrafted and de-opldermlzed human homograft dermis have been compared to nopsles of cea transplanted to granulation tissue in tullthickness burn wound beds on the same patient, each patient serving as hls or her own control. paired test and control biopstes from six patients have acquired from as early as one week postgrafting to as late as 3 years postgrafting (one patient) and analyzed histopathologlcally, ultrastructurally and immunoh[stochemloally, results demonstrate more rapid normalization of differentiation markers (e,g., involucfln, fllaggrln, cytokeratln profiles) in the cea transplanted to allodermls compared to their corresponding controls by in all patients, the proliferation rate within the basal layer ot the epidermis as determined by ki-67 (proliferation-associated antigen) is seen to norh~altze more quickly in the cea transplanted to allodermls in every case, persistence of allodermal matrix can be dooumented in all patients by elastic tlssue-trichrome stain, allowing visualization of the dermal elastin network. the popu;atlon densities ot intraepldarmal langerhans cells are conslstently and signlflcantly higher in cea transplanted to ,allodermls, possibly reflectlng an immunologlcal reaction to the underlying allogenlc tissue. overall, these preliminary results indicate that transplantation to a normal human dermal matrix accelerates the maturation of cea-deflved epidermis, wound closure continues to be a major problem in patients who have sustained a major thermal injury, cultured epidermal autografts (cea) have been utilized extensively since 1984 when galllco et el reported theh'use in two brothers with greater than 90% total body surface area burn. unfortunately, cea take rate varies widely and the resultant skin coverage is often fragile and the cosmetic results are less than optimal however the overall take rate and durability of the coverase can be markedly improved by using nn allodermls base as the recipient bed. a review of cea applications performed by physicians using cultured outologens epithelium obtained from blusurfaoe teclmology, inc. shows a marked discrepancy in the results obtained utilizing different methods of wound bed preparation. tgf-b is an important modulator coordinating complex physiological events associated with growth and development. it is assumed that tgf-b is also involved in the well-coordinated process of cutaneous wound healing by regulating proliferation, differentiation, chemotaxis and matrix deposition. the purpose of our study was to analyze the spatial and temporal pattern of tgf-b expression during granulation tissue formation in patients with accidanutl surgical trauma (monotraumata mid polytraumata) and bum wounds. after debridement (day 0), the full thickness wounds were covered with epigard, a synthetic dressing until day 10. after this time the granulated wounds were closed by transplantation of mesh graft. biopsies of the wound center were taken from 30 patients at the beginning of surgical treatment (day 0) and after 3, 6, and 10 days. cryosections were stained with antibodies against tgf-fi s using the apaap technique and -for standard histology -with hematoxylin-eosin. for identification of the cell type expressing tgf-6, double staining immunofluorescence experiments were conducted using antibodies specific for monocytes/macrophages, polymorphoanclear neutropkils and fibroblasts. the results showed a characteristic pattern of tgf-t~ distribution during wound development. tgf-fi appearence was mainly cell-associated znd the absolute and relative number of cells that were positive increased with lime. infiltrating cells and developing blood vessels were most prominently stained; epithelial and t-cells showed no immuno-reactivity. a delay of emergence for tgf-b during the time course could be seen in one patient group. this might reflect various regulation patterns depending on the type and severity of injury.(1) pharmatec gmbh, frankfurt (2) institut fiir immonologie and serologic, heidelberg ( immune cells extravasating specifically in skin recognize and eliminate the invading antigens (bacteria, viruses, etc.) either in situ or transport them to regional lymph nodes. they also participate in the process of skin wound healing. cells which traffic through the skin can be harvested from efferent lymph drained from a given area of skin. the type of migrating cells changes after trauma, heating and infection. we have developed a method for collection of human afferent lymph in lower limbs. the method allows obtaining immune cells from normal and injured skin and their characterization. aim of the study was to characterize skin immune cells in situ and in skin lymph with use of immunohistological methods (staining, facs). results. group 1, cells migrating through skin: 75+4% t lymphocytes (cd3), 6+3% langerhans and dendritic cells (cdla, hla dr, s100), 41+9% cd4, 18+6% cd8, no b cells (cd22, 23), 74% cd45r0 (memory cells), 6+3% il2r. approximately 90% cells possessed cdlla and 18 antigens. cd1 lc was expressed only on large cells. the frequency of all phenotypes was different from the blood populations. group 2, cells in skin: langerhans cells were found only in epidermis, cd3,4 and 8, cd45r0, rb, ila/18 cells around venules, cd68 (macrophages) uniformly dispersed, no il2r and b cells. hla dr positive were endothelial and some dispersed mononuclear cells. group 3, one, three and thirty days after surgical wound (simple varicous vein extirpation): high density of epidermal langerhans cells, hla dr positive keratinocytes and all endothelial ceils, few il2r cells, perivenular infiltrates of cd68, 45r0 but less cd3 cells, high density of cdlla/18 cells. classic staining of isolated and in situ located ccl!s with mgg or he did not allow to follow kinetics of changes. conclusions. this study presents the first in the literature quantitative data of immune cell traffic through normal and injured human skin. in the controlled release of biological response modifiers for soft tissue regeneration. alan s. rudolph, helmut speilberg, mariam monshipouri, and florence rollwagen, and barry j. spargo. we have employed lipid microstructures as controlled release vehicles for the delivery of growth factors in wound repair. traditional liposomes as well as novel lipid based microcylinders have been examined for their in vitro kinetics of the release of transforming growth factor beta (tgf-b). in vitro reiease has been examined by setting up models with examine the physical release of iodinated tgf-b as well as a cell based bioassay (based on the ht3 bioassay). the hollow lipid microcylinders (50 microns in length and i micron in diameter) show an initial burst (5-10 ng) followed be zero order kinetics which result in the release of approximately i ng tgf/day. this release behavior can be modified by temperature based on the phase behavior of the lipid bilayer which comprises the microcylinder.we have also examined the cellular response to lipid microcylinders applied in vivo. the lipid microcylinders are mixed in agarose and implanted as a composite hydrogel block under the flank of a mouse. the blocks are removed 1, 3, and 5 days following implant and the cells analyzed by facs sorter analysis. the observed pattern of ceil recruitment to the blocks mimics that seen in a local inflammatory response. cell surface phenotype studies included the determination of cd3 and cd4, mac-l, and ig bearing cells. we have also begun to examine the change in cell surface phenotype and kinetics of recruitment following the inclusion of tgf-beta in the lipid microcylinders.center for biomolecular science and engineering, code 6900, naval research laboratory, washington, dc. 20375-5348. expression pattern of heat shock proteins in acute, good healing and chronic human wound tissue. abstract: wound healing is a complex biologic process that is well characterized at the histological level, but its molecular regulation is poorly understood. after clot formation, inflammatory cells are rapidly drawn into the wound, followed by migration of fibroblasts and epithelial cells that divide and repopulate the wound area. during the last decade peptide growth factors and cytokine are thought to play a key role in initiating and sustaining the phase of tissue repair. these factors which are released from different cells appear to initiate the cascade of events that lead to healing. different studys described the rapid activation of a family of proteins,named heat shock proteins (hsp) in differnt tissue that were exposed to various forms of stress (heat, toxic agents, mechanical). in this context hsp's have the ability to regulate protein folding and assembly, to transport proteins across cytoplasm and membranes, to disrupt protein complexes, to stabilize, degrade and regulate the synthesis of proteins and to take part in dna replication and repair. we now attempted to find out if hsp-gene activation is also involved in injury and wound healing, which likewise resemble a stress situation for cells. therefore we collected tissue samples during operation and single biopsies from chronic wounds (decubitus for example) and granulation tissue. after rna preparation from these samples we used rna-pcr and nothern analysis to study the expression of objectives of the study chronic, non-healing cutaneous tflcers are a challenging clinical and socioeconomic problem. several animal studies have shown that cytukines (e.g. egf, pdgf, fgf, tgfb) accelerate the healing process and tissue repair in general. results from first clinical trials indicate a promising value of cytokines in the treatment of chronic non-healing diabetic and venous ulcers. recent reports in the literature indicate that the biological activity of the solution of platlet derived wound healing formula (pdwt~) released from c~-granules (mainly pdgf & tgfi~) is greater than the activity of the recombiant single factors like e.g. pdgf-bb (robson, lancet 1992) . the aim of our study was to determine whether a correlation exits between the concentration of tgfi~ & pdgf and the time course of wound healing. materials and methods pdwhf was prepared from 200ml of auto]ogous patient blood and diluted with a special buffer to a final concentration of 30 ng/ml g-thromboglobulin. the concentrations of pdgf and tgfg were determined by elisa-tests developed in our laboratory. 22 patients with chronic non-healing ulcers have been evaluated alter treatment by topical application of pdwhf. pdfg and tgff~ concentrations of the topical solution were measured and two patient groups formed for analysis the time course of wound healing was regularly and meticulously documented and evaluated by photography and casting. the time from initiation of treatment instil o wound volume reduction to 50 go of the origional size (t50%) was noted• results: healing of extensive burn wounds can be accelerated by grafting cultured autologous or allogeneic keratinocytes. the stimulation of granulation tissue formation and reepithelialization is presumably based on growth factors and cytokines released by keratinocytes. we wanted to prove this hypothesis by investigating the bfgf expression during wound development, bfgf is mainly described as an angiogenic protein with mitogenic activity on various mesodermal and ectodermal cell types pointing to its stimulating potential in wound heating. in the present study we compared the pattern of human bfgf m-rna expression and the localization of bfgf protein during the first days of wound healing. biopsies were taken from 5 juvenile human bum patients, immediately after wound debridemerit mad on day 8 after transplantation of cultured allografts. biopsies were snap frozen and cryosected. the pattern of bfgf expression was assessed by in situ hybridization of the bfgf m-rna with a digoxigenin-labelled antisense-rna and the parallel detection of the mature protein with an anfi-bfgf monoclonal antibody. our study revealed typical patterns of bfgf-m-rna-expression and intense bfgfprotein deposition during granulation tissue formation and reepithelialjzation of healing bum wounds. 'it, is known that major thermal injuries cause early impairment of wound healing followed by decreased influx of granuiocytes st. the site of injury. the role of granuiocytes in the process of wound healing is not ~"~ "" elucidated, it is now assumed that they are not merely phagocytic cells but active participants in ~n~*'1~.,.,a+~o~: processes secreting_ a number of various cvt-;kines, in order to investigate the effect of there is accumulating evidence that neuropeptides could be involved in the pathogenesis of several inflammatory reactions. vasocactive intestinal polypeptide (vip) and substance p (sp) have been detected by immunohistochemistry in normal as well as inflammed skin mostly in perivascular and periglandular location. both vip and sp are involved in vasodilatation, mast cell degranulation and irnmunomodulation.we determined the influence of sp and vip on the proliferation of lymphocytes in patients with psoriasis and healthy individuals. peripheral blood t-lymphocytes of 6 psoriatics and 6 healthy controls were isolated by density gradient centrifugation and passage over nylon wool. cell enrichment was controlled by facs analysis, lx105 t-lymphocytes were then incubated alone or in coculture with 2x104 irradiated autologous lymphocytes in culture medium containing 10 -7 mol/i sp or vip. cell proliferation was measured semiquanfitatively by tdr uptake in a betacounter. significance was tested by the wilcoxon signed-rank test.our results show that sp and vip exert only an effect on unstirnulated t-cells. in healthy individuals but not in patients with psoriasis sp increases significantly proliferation of t-cells. vip, however stimulates significantly the blastogenesis of t-lymphocytes only in psoriatics.our results confirm the psychoneuroimmunologic component in inflammatory reactions and vip and sp could be partially implicated in their pathogenetic mechanisms. moreover psoriatic lymphocytes show an altered reaction to sp and vip. this might be due to a preexisting (genetic?) or more likely to an epiphenomenal receptor defect. the adhesive interactions between endothelial cells and circulating ~enkocytes in shock and innammatory vondltions is mediated by several distinct families of ce11-surface determinants. of particular importance are the leukocyte integrins cdib / cdlla-c. in this study monoclonal antibodies to two of the u chains (cdlla & cdiib) and the common [~ chain (cdib) have been used to investigate leukocyte-dependent and leukocyte-independent plasma leakage in tee skin of rabbite. plasma leakage was measured as the local accumulation of t2si-hsa over a 30 rain period, the chemotac~c peptide imlp (0.1. 30ng) and bradykinin were used to induce cell.dependent and cell-5ndependent leakage respectively, the antibodies used were 6.5e (cdis), nri85 (cdlla) and antibody 198 (cdllb). ]ntradermal in~ections of bradyklnin and ~dlp both caused a dose dependent increase in plasma extravasatien (15.~. ffi 2.1p.l to 54.4 z b.bttl and 16.4 ,-1.8~ to 59.8 z 6.71d respectively. 6.5e (0.0072-2.4mf,/k~ iv) caused a dose dependent inhibition of imlp-induced but not bradyldnin.inducecl plasma exudation. at 0.24mk/kg, the plasma leakage was completely inhibited, antibody nr185 produced similar results, treatment with antibody 198 did not cause inhibition o£ plasma leakage due to either tnedi~tor. in vitro, the irmnune system ex~nination in persons with bone, chest and abdominal traumatic injury (i group . 22 patients without infectious coz~lications and 2 group -53 patients with wound infections development) was carried out. to restore found immunity disorders and host defense to infection 23 patients of the 2 group were treated with thymalin-the biologically active peptides prepared from bovine thymus. the examination on t~e i-3 days after injury revealed a considerable decrease of lymphocytes, ed3",$d4 ~ and cd8 cells amo~it in the blood, cd4 /cd8 ratio and indexes of let~ocyte migration inhibition test in both groups of patients. the imm~lity disorders recovered to norm on the 7-10 days in pateents of+the i group. but stable ~eple$ion of cd3 and cd4 cells amount, lower cd4 /cd8 ratio and indexes of leukocyte migration inhibition test in patients of the 2 group were observed~ besides that, these persons showed higher cd19cells amount and ig level in the blood. after thymalin therapy valid ii~rovement of inun~e status was discovered. also good clinical effect of immunotherapy and best wo~id healing observed in 80% of cases. these results allow us to propose that the thymus involution and the reduction of cell-mediated immunity responsiveness with disturbances of immu_uoregulatio~ on the level of restriction of activated cd4 tho cells play the most important role in the pathogenesis of wound infections development in persons with traumatic injury.dept. of immunology, military-nedical academy, lebedeva str. 6, 194175, st.petersburg, russia a severe impairment of neutrophil (pmn) function often occurs following severe thermal or non-thermal traumatic injury. our laboratory has previously reported that following severe burn or non-burn traumatic injury the expression of the p2 integrlns (cd11a,b,c/cd18) and the fw receptors (cd16, and cd32) were significantly decreased on pmns, in this study, the effects of gm and g-csf on the expression of the f~ r and the ~2 integrln family on pmns were examined, pmns were obtained from severe trauma (initial apache ii score ;z 10) or thermal injury (>30~; total body surface area, >15~ full thickness) and incubated /n v/tro with gm or g-csf. the j32 integrins or fcyr were detected with monoclonal antibodies and flow cytometry. gm end g-csf induced a sllght increase in the percentage of pmns expressing cd1 lb, cd16, and cd18 while gm bur not c-csf induced an increase in the percentage expressing cdi 1 a, cd1 lc, and cd32, gm-csf and to a lesser extent g-csf induced an increase in the density (1,5 fold) of the ~2 integrlns on pmns from normal, burn, and trauma patients, these data suggest that cytoklne modulation with csfs could have a role clinically in certain situations. institute, dept. of surgery, 231 bethesda ave, cincinnati, oh, usa, 45267-0558. funl~al infections after solid organ transplantatlon(sot) lewis flint, md and ed,~-afd e. etheredge, me) dept. of surgery tullrte univ. school of medicine new orleans. louisiana infections contribute to increased gra5 loss and mortaliw following sot. pr~isposing facton include diabetes, hepatitis, leukopenia, cc.¢xistem infection, and intense, especially triple drug, immunosuppression. funga] infections occur ~s isolated conditions in 6% and in association with bacterial infection(l 1%), viral infection(3*/.), and combined infections(it%), candida sp. is the most common fungus recovered but aspecgillus, coccidiodies, cryptococcus, histoplasma, mueor~ ghizopus, tinea, and toruiop~is s?. also are pathogens. clinical syndromes vary among orga.aizms or may be variable with a single p~tthogen, for ~ample, with aggressive immunosuppression, candlda my be localized esophagitis or cystitis or systemically iavaslve with an associated high mortality. aspergilius presents ~ a diffuse pneumonia while cryptococcus causes pulmonary and centrad nervons sy'stem infection, clinical examination, ct scanning and aggressive sampling for c'ultures a.s wall as serologic tests contribute to diagnosis. empiric the~py is ind',cated where there is a high level of suspicion. preventlon of ca.adlda izfection is ~ci~itated by early remov-a.1 of central }ants, ca~hetess and stents as well as by the use of oral nystatin. amphotericin ]~ remains the drug of choice for treatment of in.save fungd infection, surgical resection of infectious loci in the lung and brain is indicated in selected patients. the main problems of diagnosis in lower respirator-), tract infection are the differentation of infection from colonization or contamination, and the isolation of a reliable and true pathogen. expectorated sputum may be unreliable in pneumonia, because of contamination by oropharyngeal flora. although blood cultures may be negative, they provide a precise diagnosis and should be obtained in all pneumonias. other more invasive procedures are transtracheal needle aspiration, fibrobronchoscopic techniques including protected specimen brush and bronchoalveolar lavage with quantitative culturing and cytological analysis, transthoracic needle aspiration, thoracoscopy -guided biopsy and open lung biopsy. recently m. e1-ebiary, a. torres et al, 1993 reported quantitative cultures of endotracheal aspirates for the diagnosis of ventilator-associated pneumonia offering reliable results in these patients and should be further investigated. any invasive procedure in a severely ill patient should be carefully directed weighing the risks as well as the benefits, whilst taking the underlying diseases and expected survival into consideration. -current therapeutic approach is based mainly on monotherapy with broad spectrum antibiotics. combination therapy is apparently indicated only in p. aeruginosa infections and severe s. aureus pneumonia. graft infection can lead to fulminant graft failure or rapid progressive cirrhosis. for prevention of graft infection immunoprophylaxis, i,e. administration of human polyclonal anti hbs hypedmmunoglobutin (hig), starting in the anhepatic phase during operation, has proved to be at least partially succesful when performed on a long term basis.from a total of 415 olt in adult patients 100 olt were performed for hbsag positive liver disease (cirrhosis n=86, fulminant liver failure n=8, retransplantation n=6) in 94 pat. all pat. received 10.000 u hig in the anhepatic phase and 2.000 u/per day for the first week. a small group of 9 pat. received hig only for i week (short term immunoprophylaxis), in all other pat. hig is administered on a long term basis to keep anti hbs serum levels above 100 uii or until graft infection occurs (long term immunoprophylaxis);one-year survival rates are 100% in pat. who were transplanted for fulminant hepatitis, 96% in pat. with cirrhosis and long term prophylaxis, and 78% ir~ pat. with short term prophylaxis. all fatalities were related to hbv graft infection. the total rate of graft infection was 100% under short term prophylaxis and was independent from preoperative hbv dna status, under long term prophylaxis graft infection occurad in 28% in pat, negative for hbv dna. in hbv dna positive pat. infection rate was 56%, the total rate of reinfection for all pat. with long term prophylaxis was 36%the results of liver transplantation in hbsag positive pat. are comparable to other indications, graft infection with hepatitis b virus ist the major risk factor for these patients. under long term therapy with hig the rate of graft infection can be significantly reduced. the crucial cellular element for mods-mof: monocyi'f_./m acrophaoe ronald v. meier, m,d., f.a,c,s. the severely :injured or crldcally ill surgical patient is at high risk for immune dysfunction. a major consequence of this immune dysfunction is multiple organ dysfunction and failure leading to death, the underlying etiology is now recognized to be an uncontrolled, unfocused, disseminated activation of the host normally protective inflammatory. ,, cascades.. the resultant "mahgnant' systemic" inflan'a'natlon produces d~ffuso multiple organ bystander injury !eading to progressive organ dysfunction and failure. systemic malignant inflammation involves diffuse actlvatton of all components of the humoral and cellular inflammatory host response. of these various components, the macropha~e is the crucial central cellular element. the tissue fixed macrophage is ideally located diffusely throughout the various organs injured to orchestrate the inflammatory process. the macrophage is long-lived and highly metabolic, the macrophage regulates both the extent and the dissemination of the inflammatory processes. the macrophage is an exu'emely active c¢11 capable of producing and releasing not only directly eytotoxlc agents, s irnil~, to the neutrophil, including oxidants and numerous proteases out also the multitude of other cytokines and initiators of the interacting inflammatory cascades. the macrophage is the central source for ehemotactic agents (il-8, ltb4, c5a) for neutrophils and other inflammatory cells, production of vasoaetive arachidonie acid metabolites (tx, pgi2, poe, lt's), complement components (c3a, csa), thrombotic agents (pca, tx), metabolic and physiologic modulators (il,1, il-6 or tnf), and immunosuppressivc agents (poe2, il-10). these products of the macrophage are highly effective in enhancing and augmenting the inflammatory response. disseminated activation otthe macrophage is critical to the induction of the long-term diffuse activation of inflammation necessary to induce multiple organ injury and failure. our ability to elucidate the molecular mechanisms that control the macrophage will lead to our ability to conu'ol the maerophage response and prevent mods-mof.flarborview medical center, 325-9th ave za-16, seattle, wa 98104 usa key: cord-023026-2r84ndzv authors: nan title: posters date: 2013-06-14 journal: glia doi: 10.1002/glia.22530 sha: doc_id: 23026 cord_uid: 2r84ndzv nan university of colorado school of medicine, aurora, united states oligodendrocyte progenitor cells (opcs) migrate long distances before differentiating and myelinating axons. it is well established that both short and long-range soluble guidance factors, as well as contact with the surrounding extracellular matrix (ecm) and neighboring cells, can modulate opc migration and differentiation. however, the influence of cell-matrix interactions on opc responses to guidance molecules is less clear. previous in vitro studies of opc migration in response to semaphorins and netrin-1 yielded inconsistent results, but these studies were performed on a variety of ecm substrates, i.e., explants, collagen, poly-d-lysine. in the current study, we systematically studied opc migration in response to guidance factors on several different ecm substrates, to better understand the influence of ecm interactions on opc responses to chemotropic molecules. understanding the regulation of opc migration and differentiation has important implications for the treatment of demyelinating diseases such as multiple sclerosis (ms). for example, semaphorins 3a and 3f are expressed in active, but not chronic, ms lesions, and in demyelination/remyelination studies in animal models, semaphorins influence opc migration into lesions, affecting the rate at which remyelination occurs. in addition, aberrant expression of ecm ligands occurs in and around ms lesions. we performed live imaging experiments of opc migration on different ecm substrates in response to gradients of chemotropic molecules. these studies are combined with ihc, co-ip, western blot and rt-pcr analyses to determine the effects of ecm substrates on opc migration and signaling pathway responses to chemotropic molecules. our preliminarily results suggest that ecm substrate can regulate both the direction and rate of opc migration. semaphorin 3a is repulsive to opcs on laminin 1, while on fibronectin opc migration remains uniform in the presence of semaphorin 3a. since laminin 1 is the ligand for a6b1 integrin heterodimers, while opc migration on fibronectin is mediated by avb3 integrins, the repulsive effect of semaphorin 3a appears likely to function through b1 integrin. thus, signaling through the neuropilin-plexin receptor complex that underlies the repulsive effects of semaphorin 3a on opc migration may be modulated by interactions with b1 integrins. x. bai university of saarland, homburg, germany secondary injury processes after acute brain trauma involve activation of different cell types like astrocytes, oligodendrocytes and microglia cells. a complex and yet not completely understood sequence of cellular responses initiate functional recovery after the neurodegeneration process. by using double-transgenic mice gcpb (gfap-egfp 3 plp-dsred1) mice, we were able to identify a particular type of activated glia expressing astro-and oligodendroglial properties simultaneously (ao cells) that transiently appeared after three types of acute cortical injuries (stab wound injury, pial vessel disruption and middle cerebral artery occlusion). ao cells could be labeled by oligodendrocyte precursor cell (opc) markers olig2 and sox10, but not for markers of mature astrocytes or neurons. two-photon live imaging of gcpb mice revealed that ao cells originated from plp-dsred-positive oligodendrocyte lineage cells. electrophysiological inspection of ao cells in acutely isolated brain slices revealed the expression of delayed rectifying, voltage gated k 1 currents, a typical property of ng2 glia. therefore, we classified ao cells as opcs. to follow the fate of ao cells which disappeared about 15 days after the injury, we generated gfap-n-cre 3 plp-c-cre 3 rosa26eyfp triple transgenic (crec) mice. under non-injury conditions, few recombined cells were observed. however, numerous recombined cells appeared after 1 week of stab wound injury adjacent to the lesion site, and lasted over 8 weeks. we found that 55-70% of recombined cells were gfap-positive, 18-29% of them were pdgfra-positive and 19-30% were positive for the oligodendrocyte marker gstp. in line with that, about 46% of newly generated gfap-positive astrocytes were observed from ng2-creert2 x rosa26tdtomato mice after 1 week of stab wound injury. these results provide a strong evidence for opcs giving rise to astrocytes after acute cortical injuries. to further understand the molecular mechanism, we performed intra-cerebral injection of bone morphogenetic protein 4 (bmp4, known to promote astrocyte, but blocking oligodendrocyte differentiation) into gcpb and crec mice. bmp4, but not leukemia inhibitory factor (lif), significantly increased the number of ao cells as well as astrocytes in the respective mice. in conclusion, ng2/opcs display strong potential to differentiate into astrocytes after acute cortical injury. for instance, sox9 and sox10 have a crucial role in opc specification and differentiation, respectively. using microarray analysis of oligodendrocyte lineage cells, we previously identified sox17 as a major regulator of oligodendrocyte development (sohn et al., 2006) . in opc cultures, sox17 expression was maximal at developmental corresponding to cell cycle exit and onset of differentiation. these findings, suggest that sox17 has critical functions in the controlling of opc cell cycle exit and differentiation. in order to decipher the functional role of sox17 in oligodendrocyte development, we generated a tet-sox17:sox10 rtta double transgenic mouse line. this mouse strain allows a sox17 overexpression specifically in sox101 cells in a doxycycline dependant manner (tet-on system). in the cns of dox-treated transgenic animals, we showed that sox17 overexpression was specifically targeted in sox101 oligodendroglial cells. in the developing cns, sox17 overexpression was targeted in pdgfra1/nkx2.21 opcs and in olig21/cc11 differentiated oligodendrocytes. to assess the effect of sox17 gain-of-function on oligodendrocyte development and myelination, we analysed opc proliferation, apoptosis and differentiation, after doxycycline induction from e12.5 to p15. in the embryonic spinal cord, we showed that overexpression of sox17 did not modify cell proliferation or the number of pdgfra1 opcs. these data strongly suggest that sox17 is not involved in early steps of oligodendrocyte development. interestingly, we found that sox17 gain-of-function drastically reduces the density of cc11 mature oligodendrocytes at postnatal stages, correlating with a delay in myelination in transgenic mouse spinal cords. altogether, our data reveal critical functions of sox17 in oligodendrocyte lineage progression and myelination. supports: arsep and nmss (usa). m.f is a phd fellow of the french mesr (ed3c). m. rocha, p. fernandes, a.c. manhães, p.c. barradas, f. tenorio universidade do estado rio de janeiro, instituto de biologia, rio de janeiro, brazil nutrient restriction during the perinatal period exerts a profound influence on brain development. previous studies using an undernutrition protocol (0% protein diet) during the first half of the lactation period in rats showed that the offspring presented an altered feeding pattern, which reflected a metabolic imprinting effect on feeding behavior. there is a delay in the pattern of npy expression in the arcuateparaventricular (arc-pvn) pathway, reflecting an effect on the development of the hypothalamic circuitry, leading to metabolic imprinting. here we studied the effects of undernutrition on glial differentiation using the same model. we used rats from 5 to 30 postnatal (p) days of age whose dams were either fed a 0% protein diet (dg) or a normoprotein diet (cg) from p1 to p10. we assessed ki67, vimentin, gfap, ed-1 and cnpase distribution in hypothalamic nuclei using immunohistochemistry. our results showed impairment in cell proliferation, more evident in the pvn and in the lateral hypothalamus (lh), where dg animals showed a lower number of ki-67positive (ki671) cells at p5 when compared to cg. however, at p10 and p15, the number of ki671 cells was significantly increased in dg. from p20 onwards, staining intensity remained relatively stable and similar in all groups. dg animals showed a delay in astroglial differentiation, presenting a decrease in gfap expression and a peak of vimentin expression at p10 and p15 accompanied by an increase in vimentin/ki671 cells. an increase in the number of ed1 positive cells next to the third ventricle was also shown at the same ages in dg. cnpase staining was very faint in most nuclei and did not show any obvious difference in dg animals. our results suggest that glial differentiation is delayed in dg, which may contribute to the changes in hypothalamic circuitry that causes metabolic imprinting. recent studies have demonstrated that neural precursor cells (npcs) residing in the adult subventricular zone also exhibit the capacity to generate new oligodendrocytes following experimental demyelination. the relative capacities of these two cell types to contribute to oligodendrogenesis in this context remain largely unexplored. we have adopted transgenic approaches that enable either the specific labeling of npcs or oligodendrocytes and the interrogation of their subsequent fate within the corpus callosi of mice subjected to central demyelination induced by cuprizone. adult nestin-creer t2 : r26r-eyfp mice were administered tamoxifen (0.3g/kg/day) for 4 days by oral gavage, inducing the permanent expression of yellow fluorescent protein (yfp) in npcs and their progeny. mice were subsequently fed 0.2% cuprizone for 6 weeks followed by 6 weeks recovery on normal food to enable the analysis of remyelination. expression of yfp and other cellular markers was examined immunohistochemically to determine the fate and migratory potential of npcs in the remyelinating corpus callosum (cc). rostrocaudal analyses of cc in the cuprizonechallenged mice demonstrated that approximately 60% of yfp 1 npcs commit to an oligodendroglial fate. there was robust recruitment of npc-derived oligodendroglial cells, with a 14-fold increase in yfp 1 sox10 1 cells compared to unchallenged mice. most of these cells were mature oligodendrocytes (yfp 1 cc1 1 ) and their density in the remyelinating cc was highest adjacent to the dorsolateral corner of svz and decreased proportionally with distance in the mediolateral axis. on the other hand, fate mapping of opcs using pdgfracreer t2 : r26r-eyfp mice revealed that oligodendrocytes derived from parenchymal opcs were distributed in a converse manner to svz-derived oligodendrocytes. in addition, we found that, independent of the cellular source, many oligodendrocytes repopulated the corpus callosum as linear arrays of clonally derived cells, in a manner that recapitulated the postnatal development of these structures. these results demonstrate that lineage determination and maturation of oligodendroglia from npcs occurs efficiently in situ. we also reveal that remyelination occurs in a coordinated way, most likely secondary to interactions between a restricted pool of axons and a sentinel opc induced to proliferate in situ. play an important role in developmental oligodendrogenesis and myelination and mounting evidence from rodent models of demyelination suggest that they also promote remyelination. the therapeutic application of thyroid hormone to demyelinating disorders is limited by the potential for cardiac side effects mediated by thyroid hormone receptor (thr) a signaling. here we report that gc-1, a thyromimetic with selective thrb1 action promotes in vitro oligodendrogenesis from both rodent and human oligodendrocyte progenitor cells. in addition, we used pdgfar-creer;rosa26-eyfp double-transgenic mice to examine the effect of gc-1 on the fate of oligodendrocyte progenitor cells and find that treatment with gc-1 during developmental myelination promotes oligodendrogenesis in vivo. these results indicate that a b receptor selective thyromimetic can enhance ol differentiation in vitro and during developmental myelination and warrants further study in demyelinating models. the olfactory epithelial layer contains multipotent horizontal basal cells (hbcs) that differentiate into olfactory sensory neurons. we generated olfactory sphere (os) cells in cultures that were derived from adult rat olfactory mucosa. fluorescence-activated cell sorting and immunofluorescence analyses showed that os cells were derived from hbcs. os cells underwent neuronal and glial differentiation in vitro. to examine multipotent differentiation in vivo, os cells were transplanted into injured rat spinal cords. the transplanted cells integrated into host tissue and underwent glial differentiation. our data showed the stem cell properties of hbcs. oligodendrocyte progenitor cells (opcs) comprise the main cycling cell population of the cns parenchyma during the early postnatal period and at adult stages. however, the molecular mechanisms implicated in opc divisions are still by large obscure. with the aims to unveil i) whether opc divisions exploit the same molecular machinery of neuronal precursors, and ii) whether distinct opc subsets exist with different cell division machineries, we focused on a mutant mouse where citron-kinase, a crucial regulator of cytokinesis in neuronal precursors, is germinally ablated (cit-k ko). these mice have severe cns developmental abnormalities, reduction of selected neuronal populations due to apoptosis triggered by defective divisions, display ataxia, epileptic seizures and die by the third postnatal week. interestingly, they were reported to have myelin defects, suggesting that the cit-ko affects oligodendroglial cells. notably, already early after birth we found a 2-fold decrease in opc density in both the cortical grey (gm) and white matter (wm), compared to wild-type mice (wt). here, most opcs were hypertrophic and multinucleated, indicating a cytokinesis failure after nuclear division. at later ages, opcs progressively disappeared from the cortical gm, while in both wm and ventral areas of the telencephalon (i.e. striatum, hypothalamus), uni-and multi-nucleated opcs could be detected, although at lower densities compared to wt. these data suggests opc heterogeneity in both cit-k requirement for cytokinesis and susceptibility to death. however, even where normal uninucleated opcs persisted, we could not find pre-myelinating or myelinating cells, in line with additional differentiation defects. to discriminate the contribution of cell-autonomous vs. environmental factors in differentiation impairment, we performed homochronic grafts of wt fluorescently tagged (gfp1) cells into perinatal cit-ko mice. strikingly, gfp1 opcs invaded the whole cit-k ko brain and actively divided. however, they hardly ever differentiated into more mature cells at difference with cells grafted in the wt, indicating that altered environmental signals contribute to myelination defects in cit-k ko. in conclusion, our results show that cit-k is involved also in glial cell divisions and suggest distinct cit-k requirement and proneness to death of dorsal and ventral opcs. additionally, cit-k ablation results in pervasive nervous tissue alterations affecting opc maturation. further studies will clarify the molecular mechanisms underlying these alterations. knowledge of how these cells act in repair is lacking, partly due to a limited understanding of their behaviour in normal developmental processes. radial glia cells are a unique population of neural progenitors that have recently been suggested to function in neurogenesis in the cerebral cortex. however, many questions still remain regarding their specific role in the developing spinal cord. using an organotypic spinal cord slice culture model and two-photon microscopy we directly visualized radial glial cell behaviour in the developing spinal cord. 400 mm slices of rat embryonic spinal cord (e12-e16) were cultured in order to preserve the 3d cytoarchitecture of the cellular environment. using electroporation, slices were transfected with a brain-lipid binding protein (blbp) driven gfp expression plasmid, which specifically labels endogenous radial glial cells. slice cultures were then imaged using two-photon time-lapse microscopy, allowing for high spatially and temporally resolved 4d imaging. we were able to follow individual gfp expressing radial glial cells over time (24 hours to 6 days) and characterized precise morphological changes. at early developmental ages, gfp expressing cells initially exhibited a radial morphology with the soma located in the ventricular zone and processes extending out to the pial surface. over time, many cells developed a multipolar morphology and migrated away from the lumen, primarily using somal translocation. using immunohistochemistry we identified populations of transfected cells which also expressed gfap, representing the differentiation of blbp-expressing radial glia cells into astrocytes. we also found that radial glial cells differentiate into oligodendrocytes (olig2 and cnpase positive) but found little evidence for gfp co-localization with neuronal markers (neun, dcx, biiitubulin). using this model we can directly observe complex developmental behaviours of specific cell populations and elucidate the relevant genetic regulation and molecular mechanisms that govern spinal cord development. by understanding these intricate events, we will gain insight into how a simple tube of undifferentiated neuroepithelial cells generates different cell populations that will eventually develop into the adult spinal cord. methods: we used the new cre-expressing mouse strain cx 3 cr1 creer to express a diphtheria toxin receptor (dtr) specifically on microglia/macrophages. in these mice, administration of tamoxifen leads to cre-mediated excision of a loxp flanked transcriptional stop element in both macrophages and microglia, thus allowing for the expression of the dtr as well as an yfp reporter. due to their fast turnover macrophages are replaced by unaffected precursors whereas microglia persist in the modified stage. these mice were used for in vivo and ex vivo analysis of microglia by facs and histology. results: our optimized protocol with two tamoxifen injections at the age of 2 weeks resulted in 80-90% of reporter-positive microglia in adulthood, whereas all peripheral macrophages were reporter-negative. with additional three successive dt injections we could achieve a microglia ablation efficiency of 80%. interestingly, histological as well as facs analysis revealed 50% of microglia repopulation already 4 days after depletion. two weeks after depletion microglia numbers were even higher compared to unaffected controls. on day 6, we found a cd45.2 high ly6c 1 population, indicating replenishment by peripheral monocytes. in bm chimera experiments, where peripheral cells were labeled with a congenic marker, we obtained striking evidence showing that almost all of the repopulating cells are of peripheral origin. conclusions: after depletion microglia were rapidly repopulated, which emphasizes their critical role in brain homeostasis and function. even though ms has an onset in young patients, it persists throughout life and progresses along adulthood and aging. therefore, mscs from aged individuals should be tested for their pro-oligodendrogenic activity before moving into the clinical trials. this is insofar of clinical relevance, since the brain's capacity for self-repair and remyelination strongly declines with aging. thus, the aim of this study was to determine whether mscs from aged individuals maintain their known prooligodendrogenic activity. mscs were obtained from bone marrow of young (2 months) as well as old (17-20 months) rats and analyzed their stem cell properties as well as their pro-oligodendrogenic activity. as expected, mscs derived from aged bone marrow presented diminished stem cell properties and differentiation capacity. to test the msc prooligodendrogenic activity, npcs were incubated with msc conditioned medium. surprisingly, soluble factors derived from old rats mscs maintain their ability to induce an increase of mbp-expressing oligodendrocytes on npcs. moreover, aged mscs conserve their oligodendrogenic activity also on npcs derived from old donors (20 months). these results indicate that mscs keep their oligodendrogenic activity regardless of age and, therefore, this study provides a rationale for clinical trials of autologous msc transplantation in ms therapy. 6 munich cluster for systems neurology (synergy), munich, germany astrocytes fulfill essential functions under physiological conditions and are involved in various beneficial and adverse functions after brain injury (reviewed by sofroniew 2009). in order to improve functional recovery after injury, it is essential to delineate the beneficial from adverse effects. however, little is yet to know which extent distinct sets of astrocytes perform distinct functions or whether all astrocytes behave in a uniform manner. live imaging of astrocytes after stab wound injury in the adult cerebral cortex in vivo revealed a striking heterogeneity of astrocyte behaviour with distinct subsets extending long processes towards the site of injury, others proliferating and yet other subtypes undergoing only hypertrophy. most strikingly, the vast majority of astrocytes proliferating were located with their somata directly at blood vessels (capillaries or post-capillary vessels). this close proximity was confirmed at ultrastructural level and identified as juxtavascular positions, sometimes adjacent to pericytes located in the perivascular space. this novel population of reactive astrocytes at juxtavascular positions is of particular interest, as the capacity of astrocytes to reactivate proliferation ( cell stem cell, in press). therefore we examined whether this population of juxtavascular astrocytes would also preferentially proliferate in even more severe injury conditions, such as 1 hour of middle cerebral artery occlusion (mcao). the proliferation of juxtavasular astrocyte was analyzed immunohistochemically in fixed brain sections co-stained for s100b/gfap/ki67/cd31 at different time points after injury. at 3 days post injury, 18% of all astrocytes were actively dividing (ki671). strikingly the majority of these was again located at juxtavascular positions (68%), suggesting a general bias of astrocytes at this position to proliferate after injury, while astrocytes further distant from blood vessels virtually fail to divide. we shall further present data from conditional knock-out mice with reduced proliferative activity of juxtavascular astrocytes in order to elucidate the function of this highly specific astrocyte population and their proliferative reaction after injury. a.d. rivera, a. butt university of portsmouth, portsmouth, united kingdom the generation of oligodendrocytes (ols) from their precursors (opcs) occurs throughout adult life and is particularly important following demyelinating insults, such as occurs in multiple sclerosis (ms). glycogen synthase kinase 3-b (gsk3b) acts as a constitutively active negative regulator of multiple pathways that regulate proliferation, survival and differentiation of opcs. lithium is a potent inhibitor of gsk-3 and is used for the treatment of bipolar disorder, but in addition is neuroprotective in a wide range of diseases, including stroke, amyotrophic lateral sclerosis, and alzheimer's disease. lithium inhibits gsk-3 by binding directly to the enzyme's magnesium-sensitive site and indirectly by enhancing its phosphorylation. notably, by inhibiting gsk3b, lithium acts as a wnt mimetic to promote b-catenin-dependent transcriptional events, including enhancement of genes involved in apoptotic inhibition. here, we have examined the effects of lithium on oligodendrocytes in the adult mouse optic nerve, a typical cns white matter tract; all procedures were in accordance with the animals scientific procedures act (1986) . transgenic mice aged postnatal day (p) in the developing and adult cns multipotent neural stem cells reside in distinct niches. specific carbohydrates and glycoproteins are expressed in these niche microenvironments that are important regulators of cell fate determination and stem cell maintenance. in previous studies we described lewisx (lex) glycan as a specific marker of neural stem precursor cells (nspcs) in developing brain and showed it to be involved in nspcs migration and proliferation. using lex glycosylation as a biomarker we aimed to identify the lex carrier glycoproteins which could have a functional relevance for cns development. in addition to already known lex carriers we have found lrp1 as new major lex carrier and for the first time showed it's expression in nspcs and developing brain. lrp1 is essential for the normal neuronal function in adult cns, whereas the role of lrp1 in development is still unclear, mainly due to the lethality of the lrp1 knock-out in mice. in the current study we investigated the basic properties of lrp1 knock-out nspcs created by means of cre-loxp mediated recombination. the elimination of lrp1 in vitro was induced by the addition of cell permeable cre-recombinase to nspcs derived from embryonic brain of lrp1flox/flox mice. the functional status of lrp1-deficient cells was subsequently studied using proliferation, migration and differentiation assays. lrp1 knock-out cells maintained as neurospheres, retained the ability to migrate and differentiate. interestingly, lrp1 knock-out nspcs generated 3-times less opcs in comparison to lrp1wt/wt cells. this suggests that lrp1 is involved in the control of the oligodendroglial differentiation. astrocytes are multifaceted cells in the adult central nervous system and react to pathology of the adult brain with a finely graded continuum of morphological and behavioral changes. given that astrocytes in healthy adult brain rarely divide outside the stem cell niches, but activate both proliferation as well as a stem cell potential after injury (for review see robel et al., 2011) , it is important to understand their exact mode of cell division. indeed, a defining hallmark of a stem cell is the capacity to self-renew -often by asymmetric cell division. therefore we set out to image reactive astrocyte divisions in vitro by developing a primary culture system for reactive astrocytes obtained from the somatosensory cortex of adult mouse at 5 days after stab wound injury. continuous live imaging and single cell tracking (costa et al., 2008 (costa et al., , 2011 allowed us observing the mode of cell division mode as well the cell fate of their immediate progeny. results of our study reveal (i) that reactive astrocytes can undergo multiple rounds of cell division, (ii) the shh signaling has a profound influence on this behavior, (iii) reactive astrocytes divide with distinct modes of cell division, which can be influenced by the local microenvironment. taken together, this new culture system allows close examination of signals on the mode and multitude of reactive astrocyte cell divisions and provides a great tool to identify signals derived from the extracellular matrix, other cell types or as diffusible signals. interestingly, even without injury temporary depletion of proliferating opcs leads to behavioral deficits which will be described. to identify the molecular mechanisms by which ng2 1 cells may react and influence other cells after brain injury, we isolated sox10-gfp 1 cells by facs either from the intact or the lesioned cerebral cortex at three days after stab wound injury. comparative genome-wide expression profiling revealed exciting candidates for opc function in the physiological and pathological brain. oligodendrocytes are the myelin forming cells of the central nervous system (cns) that enable fast salutatory signalling transduction and mediate trophic support and protection of axons. oligodendrocytes originate from a population of precursors cells (oligodendrocyte precursor cells, opcs) that are formed at distinct developmental stages. the biological process leading to opc differentiation involves a cascade of molecular events that eventually constitute the complex and multifaceted cellular program that determines the generation of mature oligodendrocytes. to identify the earliest detectable transcriptional correlates of differentiation we used a microarray approach investigating changes in gene expression occurring in primary rat opcs within the first 12 hours after purification. amongst the most regulated factors that were detected was hairless (hr), a nuclear receptor co-repressor that has been associated with thyroid hormone signalling in the neonatal cns. validation of the microarray results by rt-qpcr, western blot and immunochemistry confirmed a rapid increase of hr expression during opc differentiation in media containing thyroid hormone. in the absence of thyroid hormone, hr expression was suppressed and associated with impaired opc differentiation. similarly, rnai mediated gene silencing of hr induced an impairment of opc differentiation demonstrating an important functional role of hr in the differentiation program. thyroid hormones regulate hr expression via the thyroid hormone receptors thra and thrb as rnai-mediated gene silencing of each of the receptors resulted in a decrease in hr expression. hr is known to negatively regulate rar-related orphan receptor alpha (rora) and vitamin d receptor (vdr). however, rnai-mediated gene silencing of rora and vdr did not change the course of opc differentiation. interestingly, an interaction between hr and histone de-acetylases (hdacs), of which hdac1 and 2 are known regulators of opc differentiation, has been noted in the past. using an in situ protein-protein interaction assay we were able to demonstrate a direct interaction of hr with hdac1 and hdac2 in differentiating opcs. moreover, chip qpcr analysis revealed hr-hdac1 recruitment to the opc differentiation inhibitor hes5. taken together these data show that thyroid hormone promotes opc differentiation via hr by modulating hdac1 and 2 function. as iodine deficiency results in hypothyriodism our results give an example of a developmental disease that is triggered by environmental factors (the absence of iodine) causing epigenetic dysregulation. c. stagni, a.d. rivera, a. butt university of portsmouth, portsmouth, united kingdom astrocytes respond to all forms of cns insults by reactive astrogliosis, which involves changes in their molecular expression and morphology, and in most severe cases glial scar formation. reactive astrogliosis is potentially neuroprotective, but can also act as chemical and physical barrier to axon growth and repair. however, mechanisms underlying this process are still poorly understood. the enzyme glycogen synthase kinase 3-b (gsk-3b) is a key regulator of many signalling pathways involved in cell proliferation, survival and differentiation, including wnt signalling. lithium is a potent inhibitor of gsk-3 and acts as a wnt mimetic to promote b-catenin-dependent transcriptional events. wnt signalling is altered following cns injury and in a range of neurodegenerative diseases, such as stroke, amyotrophic lateral sclerosis, and alzheimer's disease. notably, lithium is neuroprotective in these same diseases. here, we have examined the effects of wnt3a and lithium on astrocytes of the adult mouse optic nerve, a typical cns white matter tract; all procedures were in accordance with the animals scientific procedures act (1986). we used transgenic mice aged postnatal day (p) 35 inwhich gfap drives the expression of egfp. optic nerves (ons) were isolated with the retina intact and maintained in organotypic culture for 3 days in vitro (div), in control medium, or medium containing lithium chloride, or wnt3a. both agents resulted in a significant increase in the number of astrocytes, compared to controls (p < 0.05, unrelated t-tests), but lithium and wnts3a had markedly divergent effects on astrocyte morphology. in the presence of lithium, astrocytes became highly polarised, extending long thin processes that traversed the nerve perpendicularly to the long axis. in contrast, wnt3a treatment resulted in the generation of morphologically simple astrocytes, with short, fine branching processes that extended radially from a centrally located cell body. genome wide microarray analysis indicated wnt3a and lithium differentially altered chondroitin sulphate proteoglycans (cspgs), a family of extracellular matrix molecules highly expressed at cns injury sites. further experiments are required to define the molecular phenotypes of the novel astrocytes generated by these treatments, but the results provide evidence that wnt signalling regulates astrogenesis in situ and is therefore a potential molecular target to modulate astrogliosis. the contrasting effects of lithium suggest it is acting via other pathways, which require further study. studies of mouse schwann cell culture. the proliferation of scs is regulated by axonal signals and several growth factors. growth factors that are essential for rodent sc survival in culture include heregulin-b1 and forskolin. in the case of rat scs, these factors are also sufficient to allow a robust proliferation. in contrast, the proliferation of mousederived scs in culture requires the presence of other growth factors. we studied effect of several factors known to be expressed in scs after nerve injury on mouse sc proliferation. these included fibroblast growth factor type 2 (fgf), pituitary extract (pe), epidermal growth factor, platelet-derived growth factor bb, and transforming growth factor b1. we found that heregulin-b1 and forskolin are essential for mouse sc to survive in culture, fgf and pe were the best combination of growth factors promoting the mouse sc proliferation, and that neuronal axons provide effective mitogenic environment for mouse scs. the expression of the large myelin-associated glycoprotein in pns was studied in scs cultured alone and in cocultures of scs and drgns. when schwann cells are alone without axons, expression of the cytoplasmic domain of l-mag is seen in the nucleus of schwann cells. when schwann cells make contact with neuronal axons, the location of the expression shifts out of the nucleus to the perinuclear and cytoplasmic regions; the extracellular domain is not visible. until the schwann cells initiate the myelination programme. these results support the hypothesis that l-mag might have role in the regulation of the myelination programme in the pns. it has been postulated that trophic support provided by either stem cells or precursors is actuallyone of the most beneficial effects in the cell-based therapies. to address this issue, a comparison between the neuroprotective properties of opcs and the stem cells derived from warton jelly was carried out in the co-culture system with ischemically injured organotypic hippocampal slices. those two cell types were selected in respect of the advancement in their differentiation process. while the mesenchymal stem cells residing in warthon jelly are still pluripotent, the opcs are already the glia-committed precursors. to evaluate their neuroprotective properties, we set -up an ex vivo co-culture system of either stem cells or opcs isolated from neonatal rat brain with organotypic hippocampal slices subjected to a short oxygenglucose deprivation (ogd). the cell death, as well as the number of the newborn cells has been quantified in slices co-cultured for one week. a microarray analysis of a broad spectrum of trophic factors and cytokines expressed by opcs was performed for the purpose of finding the factor(s) contributing to the observed effect. three of them were selected for the subsequent blocking experiments with specificl antibodies to verify their influence on the injured tissue. the results obtained in the study prove that both cell types secrete soluble factors and are potent in exerting the neuroprotective effect in a paracrine-like manner, promoting cell survival and proliferation in damaged hippocampal slices. in conclusion, the observed advantageous qualities of stem cells and oligodendroglia-biased progenitors significantly contribute to anticipating a clinical improvement in cell replacement therapies. ng2 is a type i transmembrane glycoprotein and also known as chondroitin sulphate proteoglycan 4 (cspg4). in the central nervous system ng2-expressing cells have been identified as a novel type of glia with a strong potential to generate oligodendrocytes in the developing white matter. for temporally-controlled gene targeting of ng2 glia in vivo, we generated a mouse line in which the open reading frame of the tamoxifen-inducible form of cre recombinase (creert2) was inserted into the ng2 locus by homologous recombination. here, we investigated the differentiation potential of ng2 glia at different developmental stages of the forebrain. cre recombinase activity was induced at embryonic day 17.5, postnatal day 0 (p0), p3, p8, p30 and p60 by intraperitoneal tamoxifen injections into novel tgh(ng2-creert2) mice crossbred to rosa26-tdtomato and rosa26-eyfp reporter lines that helped to identify ng2 glia and its progeny. recombination and cell identification were determined 10 to 60 days after the first tamoxifen injection. induction of recombination at embryonic stages revealed that embryonic ng21 cells mainly generated more ng2 glia and oligodendrocytes, however, a significant number of astrocytes could be detected as well. recombined cells were predominantly restricted to the ventral brain. in contrast, after tamoxifen injections at p0 and p3 recombined cells were widely found in all brain regions, thereby suggesting the presence of a distinct subpopulation of ng21 cells after birth. interestingly, only very few recombined astrocytes could be found, which are probably derived from few remaining embryonic ng2 glia. starting from p8, ng2 glia stopped generating astrocytes, with the progeny largely restricted to the oligodendrocyte lineage. however, consistently, we found recombined neun1 cells with the morphology of neurons in the ventral cortex after administration of tamoxifen at p8. even more of such cells were present in the whole cortex when cre activity was induced in adult mice. the morphology, the presence of neun immunoreactivity and our electrophysiological characterization that demonstrated bona fide action potentials clearly identify these cells as functional neurons. our results suggest that ng21 cells display a broad differentiation potential that appears to be highly age-dependent during development. brdu-assay) and the number of surviving cells (by counting dapi-stained nuclei). after we had observed, that the choice of the basal medium (neurobasal, rpmi or dmem) exerts a pronounced effect on opc proliferation we here investigated the effect of cell density on opc proliferation. starting from a small seeding density (10.000 cells/cm 2 ) a tenfold increase in seeding density only resulted in a fourfold augmentation of the density of surviving cells after 7 days in culture. using plating densities of 5.000; 20.000 and 80.000 cells/cm 2 we observed, that an increase in seeding density led to a decrease in the proliferation rate, as tested with a brduassay and monitored after 3 days in culture. similarly, the percentage of a2b5-positive, immature, cells was lower, the higher the cell density. we then tested whether this finding is due to proliferation inhibiting factors secreted in cultures with high seeding densities and whether the remaining microglia population could be involved. to this aim we seeded opcs at low density (5.000 cells/cm 2 ) and cultivated them with the medium supernatant of cultures with high density (80.000 cells/ cm 2 ). furthermore we seeded opcs in low density (5.000 cells/cm 2 ) and co-cultivated them with an added density of microglia as counted in cultures of high density (80.000 cells/cm 2 ). the resulting cell counts after 3 days showed, that the percentage of brdu positive cells after cultivation with the supernatant was significantly reduced compared with control cultures, although the number of a2b5-positive cells was not influenced by this treatment. the addition of microglia to the cultures lead to a strong reduction of surviving cells, even though the percentage of brdu and a2b5 positive cells were not altered. so apoptosis, necrosis or phagocytosis might be responsible for the reduction of surviving cells. our findings suggest that the reduction of surviving opcs in high density cultures is due to secreted factors inhibiting opc proliferation and that microglia could be involved in this effect. in neuropathological studies, it is important to detect microglial cells; however, a specific microglia marker has yet to be determined. [methods] in this study, we examined and compared the usefulness of various microglia markers, including human glucose transporter-5 (glut-5), mhc class ii, cd68, and iba-1. [results] all of the microglia markers consistently stained for microglia in paraffin sections fixed with formalin. cd68 staining of microglia in the gray matter was not as well defined as the other microglia markers. moreover, cd68 did not clearly stain the microglial processes. with regard to mhc class ii, it was difficult to observe the microglial processes in the white matter. in contrast, both glut-5 and iba-1 clearly stained for microglia in the gray and white matter, and provided detailed staining of microglial processes. although it has previously been shown that perivascular cells are negative for glut-5, we found perivascular macrophages to be glut-5 positive. [conclusions] nevertheless, glut-5 and iba-1 may be considered as markers suitable for routine histopathological staining procedures. neurons and glia of the enteric nervous system (ens) originate from a pool of sox101 progenitors. in addition to being a scaffold for enteric neurons, enteric glial cells (egcs) have been suggested to function in mucosal integrity, neuroprotection, adult neurogenesis, neuro-immune interactions and synaptic transmission. currently, diverse glial populations are known to reside within the ens. however, whether, specific functions are carried out by specialized glial subtypes, with characteristic morphologies and molecular markers and within specific locations in the gut remains elusive. to address this question we used a repertoire of genetic tools, immunostaining and imaging techniques. for high single-cell resolution study of egcs, we mosaic analysis with double markers (madm) in conjunction with sox10-cre. we analyzed adult gut tissue to characterize the different glial populations based on morphology, position in the ens and their relationship with the intestinal epithelial and vascular system. moreover, we used reporter mice (sox10-icreer t2 ; r26r-yfp and gfap-icreer t2 ; r26r-yfp) to quantify the expression of three glial markers -gfap, s100b and sox10 in myenteric plexus (mp) preparations of adult mice. our investigation on madm-mp preparations, revealed three subtypes of enteric glia in the mp of adult mice. type i and type ii glia, present in the primary plexus of mouse ens have been previously defined. in addition, we characterized a third subtype in the extraganglionic space of the mp of adult mice, at present termed as type iii-extraganglionic glia. furthermore, our analyses using 3d-imaging, on madm-gut sections allowed us to characterize the relationship of enteric glia located within the mucosa, with the intestinal epithelium and vascular system of the villi. our marker expression analysis showed that while the majority of glial cells co-expressed gfap and s100b, a large fraction of glia within the myenteric ganglia was positive for either s100b ($30%) or gfap ($10%). we also found that s100b 1 /gfapglia were abundant outside the ganglia. most glial cells co-expressed sox10 with gfap and s100b yet a significant proportion ($10%) of mainly extra-ganglionic glia (type iii) expressed only sox10. surprisingly, $5% of the cells that were s100band sox10displayed high gfap expression. use of gfap-icreer t2 ; r26r-yfp mice showed that about $30% of yfp-labelled cells did not express gfap, implying dynamic regulation of gfap expression. overall, our high resolution studies reveal extensive heterogeneity of enteric glial cells in terms of morphology, position and expression of molecular markers suggesting differential functional roles. our future experiments will address whether different physiological roles of egcs can be assigned to specific subtypes. university of washington, seattle, united states m€ uller glia are the major type of glia in the retina, spanning its entire thickness. it is known that bmp signaling promotes astroglial differentiation in cortex, but its role in m€ uller glial development in the retina has not been studied. the purpose of this study was to investigate the role of bmp-smad1/5/8 signaling in m€ uller glial development. c57bl/6 mouse retinas were collected at various ages between postnatal day 0 (p0) and p21 in order to analyze the expression and activation of smad1/5/8. smad1/5/8 was transiently but robustly activated in the inner nuclear layer, including developing m€ uller glia between p5-p8. the timing of this transient activation of smad1/5/8 corresponds with the timing of m€ uller glial differentiation and maturation. to test the hypothesis that activation of bmp-smad1/5/8 signaling is essential for m€ uller glial development, retinas were explanted at p3 or p6 and treated with bmp4 or a bmp inhibitor dorsomorphin (dm) for 5 days in order to activate or inhibit smad1/5/8, respectively. when p3 and p6 retinal explants were treated with dm for 5 days in vitro, there was a significant reduction in m€ uller glial markers (cralbp, gs, sox9, and sox2). smad1/5/8 activation and cralbp expression were also significantly attenuated in vivo 1 day after intraocular injection of a bmp inhibitor dm at p5. our data suggest that activation of bmp-smad1/5/8 signaling is necessary for the development of m€ uller glia. this work was supported by 1r01ey021482 to tar. huazhong university of science and technology, wuhan, china rho-associated kinase (rock) has been identified as an important regulator of proliferation and cell cycle progression in a number of cell types. although its effects on astrocyte proliferation have not been well characterized, rock has been reported to play important roles in gap junction formation, morphology, and migration of astrocytes. in the present study, our aim was to investigate the effect of rock inhibition by [(1)-(r)trans-4-(1-aminoethyl)-n-(4-pyridyl) cyclohexanecarboxamide dihydrochloride] (y27632) on proliferation and dna synthesis in cultured astrocytes from rat spinal cord and the possible mechanism involved. western blots showed that treatment of astrocytes with y27632 increased their expression of cyclin d1, cdk4, and cyclin e, thereby causing cell cycle progression. furthermore, y27632-induced astrocyte proliferation was mediated through the extracellular-signalregulated kinase signaling cascade. these results indicate the importance of rock in astrocyte proliferation. here we used cell transplantation experiments to determine to which extent this differential behavior is regulated by environmental signals differing between the wm and gm, or intrinsic fate determinants restricted to one or the other cell population. these experiments indicate that the wm promotes differentiation of opcs to mature oligodendrocytes, as the rate of differentiation of gm cells increased when exposed to this environment. interestingly, cells derived from the wm retained their higher differentiation rate also in a less supportive environment like the gm. these results reveal not only important environmental differences for opc maturation, but also support the concept of intrinsic heterogeneity among adult opcs, persisting even when exposed to more inhibitory environments. thus, this work provides the basis to identify molecular pathways regulated by distinct niche/environmental signals and involved in the heterogeneity of adult opcs. multiple sclerosis (ms) is a chronic inflammatory and neurodegenerative demyelinating disease of the central nervous system (cns) characterized by inflammation, which leads to formation of demyelinating areas due to loss of oligodendrocytes, astrogliosis and, finally, axonal degeneration. different studies have suggested that 3 0 -5 0 -cyclic adenosine monophosphate (camp) levels might play an important role in neuroprotection and neuroinflammatory response so the modulation of this nucleotide intracellular levels might control the neuroinflammatory pathological process and, consequently, to delay the progression of ms. intracellular camp levels depend on its synthesis by adenylyl cyclases, and on its degradation by cyclic nucleotide 3 0 ,5 0 -phosphodiesterases (pdes). specific inhibitors for the different isoforms of pdes family, particularly camp-specific pde, emerge as putative treatments of this kind of disease. pde7 has emerged as a new therapeutic target, not only for a variety of immunological and immunodeficiency conditions to alleviate chronic inflammation, but also for several neurodegenerative disorders, including ms, in which both immune system and cns are implicated. in the present work, we have detected the expression of pde7 in oligodendrocyte precursor cells (opcs) isolated from cerebral cortex of p0 and p15 mice and from adult human samples derived from neurosurgery of epilepsy. opcs are abundant in the mice and human cns during development but they also represent 5-7% of the total number of cells in the adult cns, where they serve as a source of new oligodendrocytes to replace those which die. however, in ms these endogenous opcs are not able to effectively replace dead oligodendrocytes. in vitro, we have demonstrated that two newly-developed pde7 selective inhibitors (tc 3.6 and vp 1.15) favour opc survival and differentiation towards mature oligodendrocytes, being both more effective than the commercial pde7 inhibitor brl-50481. erk intracellular pathway has been identified as a key in these cellular processes related with the camp/pka/creb pathway. moreover, we have observed that both specific inhibitors (vp1.15 and tc3.6) increase the differentiation of human opcs. these findings, combined with the already known anti-inflammatory effect carried out by these pde7 inhibitors, point them as potential therapeutic agents to treat ms. the knowledge of factors that affect the biology of murine opcs and even more, human opcs, are critical for possible remyelination in this kind of pathologies. their role in cns remyelination has not been fully investigated. previous studies using genetic fate mapping techniques revealed their recruitment into injured spinal cord and differentiation into astrocytes and oligodendrocytes. in this study, we used aninducible cre-floxed-stop-rosa26-yfp mediated lineage tracing strategy to characterize the fate of foxj1 expressing ependymal cells during remyelination of toxin-induced demyelination. we found that in unlesioned spinal cord, foxj1 labelled ependymal cells. however, it also labeled a population of cells in the spinal roots with immnochemical features of fibroblasts. following lysolecithin-induced demyelination in the dorsal funiculus, there were few yfp expressing cells in the lesion area at 5 and 14 days post lesion (dpl) indicating that the ependymal cells were not recruited in demyelinated area. however, in ventral white matter lesions, which were adjacent to damaged nerve roots, many cells were detected expressing yfp. the distribution of yfp positive cells overlapped that of remyelinating schwann cells and a number of yfp expressing cells colocalised with mature schwann cell marker periaxin. these data revealed a foxj1 expressing population in peripheral nerve which migrates into demyelinated lesions andcontributes to cns remyelinaiton. the generation of myelinating cells from multipotential neural stem cells in the cns requires the initiation of specific gene expression programs in oligodendrocytes (ols). we reasoned that micrornas (mirnas) could play an important role in this process by regulating genes crucial for ol development. here we identified mir-7a as one of the highly enriched mirnas in oligodendrocyte precursor cells (opcs), overexpression of which in either neural progenitor cells (npcs) or embryonic mouse cortex promoted the generation of ol lineage cells. blocking the function of mir-7a in differentiating npcs led to a reduction in ol number and an expansion of neuronal populations simultaneously. we also found that overexpression of this mirna in purified opc cultures promoted cell proliferation and inhibited further maturation. in addition, mir-7a might exert the effects just mentioned partially by directly repressing proneuronal differentiation factors including pax6 and neurod4, or prool genes involved in oligodendrocyte maturation. these results suggest that mirna pathway is essential in determining cell fate commitment for ols and thus providing a new strategy for modulating this process in ol loss diseases. a. guzman de la fuente 1 , j. huang 1 new sheaths can be regenerated following the recruitment of adult neural progenitor cells (opc) into areas of injury and their differentiation into myelinating oligodendrocytes. although this regenerative process (called remyelination) occurs efficiently in the early stages of multiple sclerosis, its efficiency gradually declines leaving axons demyelinated and vulnerable to degeneration. several signalling pathways are involved in regulating opc differentiation and subsequent myelination. understanding their mechanisms is necessary to design regenerative therapies to overcome remyelination failure. we have identified rxrc as a key positive regulator of opc differentiation and remyelination (huang et al., 2011). rxrc is a nuclear receptor forms a heterodimer with other nuclear receptors to activate its downstream signalling cascades. here we describe rxrc binding partners expressed in both opc and oligodendrocytes, and the binding of vdr, rarb and pparc to rxrc within oligodendrocyte lineage cells by coimmunoprecipitation. vdr-rxrc heterodimer is necessary for the oligodendrocyte differentiation. treatment of oligodendrocytes with vdr antagonist inhibits opc differentiation and abrogates the effect of 9-cis retinoic acid, an rxrc agonist, in opc differentiation. our data suggests a model in which rxrc is an essential anchor for different nuclear receptors, including vdr, with a shifting and complex rxrc signaling in oligodendrocyte lineage cells. cleavage of type i membrane proteins by a-and c-secretases to yield biologically active fragments is best exemplified for the evolutionarily conserved protein notch. here we show that the protein ng2, a type i membrane protein which has homologues in mouse (ng2) and drosophila (kontiki) and is expressed by multiple immature cell types including glia, is processed in a similar fashion to notch. in both mouse and drosophila this generates a major 290 kd shedded ectodomain, a c-terminal fragment (ctf) and a small intracellular domain (icd). the ng2 icd is present after overexpression in cells of biochemical isolates of nuclear fractions, and can be visualized in the nucleus by immunofluorescence of cultured murine glial cells. in glial nuclei of developing drosophila larva, a striking intranuclear staining of the kontiki icd is observed. levels of expression of ng2 mrna and protein in mouse glial cells are regulated by the neurotransmitter glutamate and overexpression of cleaved fragments modulates protein expression in murine glial cells. these results demonstrate that cleavage of ng2 takes place in mouse and drosophila glia and that this generates fragments with signaling properties. modulation of ng2 expression by glutamate provides a way for neuronal activity to regulate ng2 signalling. our goal is to understand the glial signaling that controls inflammatory responses in the central nervous system (cns). this glial response can play both a detrimental and beneficial role. toll like receptor (tlr) signaling is implicated in responses to pathogens or endogenous signals. tlr signaling mediates immune response by inducing cytokines, including type i interferons (ifn). ifn-beta, a member of the type i ifn family, is a first-line therapeutic for multiple sclerosis. type i ifns signal through a common receptor, ifnar. the aim of the present study was to investigate the in vivo response of microglia and astrocytes to cns administration of tlr ligand/agonist, and to examine whether this response involves type i ifn signaling. mice were administered tlr ligands/agonists by injection to the cisterna magna. we analyzed leukocyte entry to the cns by flow cytometry, and their localization by immunostaining. the induction of ifn-beta was examined by in vivo imaging of an ifn-beta reporter mouse. astrocytes and microglia were sorted by facs and gene expression was measured using quantitative real time-pcr. injection of ligands/agonists for tlr2, 3 and 4 resulted in infiltration of cd451 leukocytes after 18 hrs, most strongly by tlr2. immunostaining showed parenchymal localization of infiltrating cells in cerebellum. facs sorted astrocytes expressed equivalent levels of tlr3 mrna to microglia but lower levels of tlr2 or 4. astrocytes were induced by tlr3 signaling to express interferon regulatory factor 7, which regulates the induction of type i ifn. the induction of ifnbeta in cns in response to tlr3 signaling was verified in ifn-beta reporter mice. tlr2, 3 and 4 signaling led to increased levels of mrna for glial cxcl10. together these results suggest the involvement of type i ifn signaling. however, unlike cxcl10 gene expression, that was dependent on ifnar signaling, tlr2-induced leukocyte infiltration was not affected in ifnar deficient mice. these studies point to a role for tlr signaling in the innate glial response that regulates cns inflammation. microglial phagocytosis plays a key role in neuroprotective and neurodegenerative responses of the innate immune system in the brain. here we investigated the regulatory function of the signaling protein phosphoinositide 3-kinasec (pi3kc) in phagocytosis of bacteria and zymosan particles by mouse brain microglia in vitro and in vivo. using genetic and pharmacological approaches our data revealed pi3kc as an essential mediator of microglial phagocytosis. unexpectedly, microglia expressing lipid kinase deficient mutant pi3kc exhibited similar phagocytosis as wild-type cells. detailed analysis disclosed pi3kc dependent stimulation of camp phosphodiesterase as a crucial mediator of phagocytosis. both stimulation of protein kinase a and epac were shown to convey inhibitory effects of camp on phagocytosis. together our findings indicate pi3kc-dependent suppression of camp signaling as an indispensible regulatory element of microglial phagocytosis. v. kovaleva, e. berezhnaya, m. rudkovskii, a. uzdensky southern federal university, rostov-on-don, russian federation photodynamic therapy (pdt) is currently used in oncology, particularly, in treatment of brain tumors. the possible role of no-mediated signaling in photodynamic injury and protection of neurons and surrounding glial cells (gc) was studied. crayfish stretch receptor consisting of a single neuron enveloped by gc was photosensitized with alumophthalocyanine photosens (10 nm, 30 min incubation) and irradiated with laser diode (670 nm, 0.4 w/cm2). application of no generators notably 10 mm sodium nitroprusside and 10 mm nonoate reduced pdtinduced necrosis of gc and showed the same tendency in neuronal necrosis. nonoate significantly increased pdt-induced apoptosis of gc. inhibitor of neuronal no-synthase l-name (1mm) significantly increased the percentage of necrotic glial cells after pdt but did not influence neuronal necrosis. this confirmed the anti-necrotic effect of no in glial cells and involvement of neuronal no synthase in protection of glial cells. 1 mm l-name and 1 mm l-nna (another inhibitor of neuronal no-synthase) as well as 50 lm s-methhylisothioharnstoff sulfat (inhibitor of inducible no synthase) protected gc from pdtinduced apoptosis. therefore no may be involved in pdt-induced apoptosis of glial cells. inhibition of no-activated protein kinase g with 10 lm kt5823 decreased the percentage of necrotic glial cells but not neurons. therefore protein kinase g appeared to be involved in pdtinduced necrosis of gc, possibly independently on no. kt5823 also increased the level of apoptosis of gc indicating the anti-apoptotic role of protein kinase g. thus no is involved in regulation of pdt-induced necrosis of neurons and glial cells as well as in apoptosis of glial cells. g. tyzack, t. cymes, n. lau, a. lakatos university of cambridge, cambridge, united kingdom background and question. emerging evidence suggests that signalling by damaged neurones leads to reactive astrocyte response that may have a fundamental impact on synaptic reorganisation. soluble factors, such as cytokines, have been described to induce such astrocyte activation. however, rapid contact dependent signalling between damaged neurones and astrocytes is also a possibility. in light of recent findings of ephb1 upregulation on injured neurons closely surrounded by reactive astrocytes, we hypothesized that ephb1 can signal to ephrin-b1 expressing astrocytes. methods. to test this in simplified in vitro assays, purified astrocyte cultures were treated with clustered-ephb1-fc, and the characteristic features of glial activation, such as cytoskeletal protein expression, stat3 phosphorylation, and p-stat3 nuclear transfer were monitored. results. we have demonstrated that 1) astrocytes express ephrin-b1, 2) ephb1 triggers a two-fold increase in gfap and ezrin expression, 3) ephb1 induces both stat3 phosphorylation and nuclear transfer by a three-fold value, and 4) all effects were significantly diminished by knocking down ephrin-b1 in astrocytes. conclusions. our data show that ephb1 induces astrocyte activation by triggering ephrinb1-mediated reverse signalling via stat3 activation. this work therefore suggests a potential novel route in neuron-astrocyte communication after injury. a further characterisation of this signalling pathway may provide a better understanding of mechanisms underlying glial activation and its role in plasticity. n. kramann 1 , r. pf€ ortner 1 , u.-k. hanisch 1 , k. hagemeier 2 , t. kuhlmann 2 , w. br€ uck 1 , c. wegner 1 1 university medical center g€ ottingen, department of neuropathology, g€ ottingen, germany 2 university hospital m€ unster, institute of neuropathology, m€ unster, germany introduction: laquinimod (laq) is an oral well-tolerated molecule that has been shown to reduce brain atrophy, disability progression and relapse rate in patients with relapsing-remitting multiple sclerosis. recent experimental data indicate that laq minimizes demyelination, inflammation and axonal damage in mice with cuprizone challenge. aim: since astrocytic nfjb activation plays a crucial role in cuprizone-induced demyelination, we investigated the effects of laq on cns cells in vitro and in vivo. methods: in vitro experiments used primary cells to test effects of laq on oligodendroglial survival as well as on cytokine secretion and nfjb activation in astrocytes and microglia. primary mouse astrocytes and microglia were pre-treated with 0, 0.25 and 2.5 mm laq and stimulated by pro-inflammatory cytokines. a dual-luciferase reporter assay was used to measure nfjb activity. for in vivo experiments, 10-week-old male c57bl/6j mice were challenged with 0.25% cuprizone and treated daily with laq (25 mg/ kg) or water. to assess astrocytic and microglial nfkb activation in vivo, nuclear translocation of nfjb p65 was assessed in astrocytes and microglia by double immunofluorescence with antibodies against p65 and iba1 or gfap. results: in vitro, astrocytic but not microglial nfjb activation was markedly reduced by laq as evidenced by nfjb reporter assay. in astrocytes, pre-treatment with 0.25 and 2.5 mm laq significantly reduced the induced nfjb activity after tnfa stimulation compared to stimulated controls. pre-treatment with 2.5 mm laq also significantly reduced nfjb activation after stimulation with the combination of il-1b and ifnc compared to untreated stimulated controls. oligodendroglial viability and survival were not affected by laq treatment. to confirm the in vivo relevance of these findings, we also examined astrocytic and microglial p65 translocation in mice with and without laq treatment after cuprizone challenge. the proportion of astrocytes with nuclear p65 immunoreactivity was significantly reduced in laqtreated mice (14.0% 6 0.9%) compared to untreated controls (25.8% 6 1.1%). microglia did not display marked translocation of nfjb p65 after cuprizone challenge. conclusion: our data indicate that laq prevents cuprizoneinduced demyelination by attenuating astrocytic nfjb activation. in vitro, laq reduced the astrocytic nfjb activation by up to 46% as evidenced by nfjb reporter assay. similar quantitative findings were obtained in vivo when laq treatment also led to a 46% reduction of astrocytes with nfjb activation, as evidenced by nuclear translocation of p65. these findings suggest that targeting the astrocytic nfjb pathway might have therapeutic effects in demyelinating cns disorders. . we asked whether brain-derived neurotrophic factor (bdnf), known to be highly expressed in a circadian manner in scn, might be involved in the mechanisms governing these astroglial rearrangements. using an analog-sensitive kinase allele murine model (trkb f616a ) and semi-quantitative electron microscopy, we found that the pharmacological blockade of the tropomyosin-related kinase receptor type b (trkb), the high affinity receptor of bdnf, abolished the day/night changes in glial coverage of vip dendrites. the bdnf/trkb signaling pathway therefore exerts a permissive role on the ultrastructural rearrangements that occur in scn across the day/night cycle. in contrast, the extent of glial coverage of non-vip dendrites was not different at daytime and nighttime in trkb f616a mice submitted to trkb inactivation or not receiving any pharmacological treatment. considering the well-established role of bdnf in the clock's photic synchronization process, these dataprovide strong support to the view that the daily astroglial rearrangements in scn could be involved in the photic entrainment of circadian rhythms. above all, they highly suggest that they are necessary for the modulatory action of bdnf on the scn responses to light. features of this form of neurodegenerative ataxia, including a striking reduction in lifespan when polyq atrophins are expressed in the glia. a sensible hypothesis is that affected glia will act non-autonomously on neurons as in other neurodegenerative pathologies. this is an emerging field that yields new and important therapeutic possibilities. we now wish to elucidate the mechanisms through which degenerating glia impairs organism functions, and identify genes involved cell-autonomously and non-autonomously in generating this aspect of the pathology. first, we investigated the impact of glial polyq atrophin on lifespan. using different glia marker, we have demonstrated that the expression of polyq atrophin in glia, or specifically in astrocyte-like cortex glia, has a dramatic effect on drosophila lifespan while brood brain barrier glia do not have any phenotype. we will then investigate the effect of other subtypes of glia looking at lifespan and motility to identify the relevance of each subtype in drpla. second, to identify new regulators of gliopathology we will conduct unbiased genetic screens for mutations in neurons that modify the lifespan of drpla flies, expressing polyq atrophin within the glia. our studies will be paramount in deciphering the complex glia-neurons interactions in ataxias and may be beneficial for several ataxias. they will open up new avenues for therapies aimed at targeting glianeuron interactions during disease progression. in alzheimer's disease, microglial clearance of beta-amyloid (ab) is considered neuroprotective. whether ab uptake regulates microglial cell responsiveness, and whether macrophages participate, remains unclear. here we investigated whether in vivo phagocytosis of endogenously-produced ab disturbs the turnover of microglia and macrophages by changing rates of cellular proliferation and apoptosis. using app swe /ps1 de9 tg mice, we show that plaque-associated microglia and cd11b 1 cd45 high macrophages bind and ingest endogenously-produced ab in vivo. plaqueassociated microglia and cd45 1 leukocyte-like cells were also observed in brains from patients with alzheimer's disease. phagocytic microglia and macrophages had a reduced rate of proliferation compared to abcells. instead, high proportions of apoptotic annexin v 1 microglia and macrophages were found in aged app/ps1 tg mice. phagocytic microglia and macrophages had greater caspase activity and increased p53 expression after in vivo uptake of ab, than cells that did not phagocytose ab. in vitro, we found that ab uptake induced caspase activation in microglia, whereas blocking ab uptake triggered apoptosis-induced cell death in macrophages. our data demonstrate that ab uptake impairs microglial/ macrophage viability and responsiveness. amyloid clearance may fail as phagocytic microglia and macrophages undergo ab-induced apoptosis. f. michetti, s. ceccariglia, a. d'altocolle, f. pizzolante, m. barba, a. delf a, c. gangitano universit a cattolica del s. cuore, rome, italy trimethyltin (tmt) is a neurotoxicant producing neuronal degeneration in the mammalian central nervous system , especially in the hippocampus (for reviews, 1,2). magnetic resonance imaging (mri) investigation in tmt-treated rats has evidenced dilation of lateral ventricles, possibly correlated to alterations in blood brain barrier permeability. we have investigated in the hippocampus and cortex of rats the expression of aquaporin 4 (aqp4), a glial water channel protein which is regarded to play a role in brain oedematous conditions, to explore the molecular mechanisms involved in the phenomenon. tmt-treated aqp4 expression was tested both by real-time pcr and western blotting analysis in hippocampus and cortex homogenates. to confirm molecular results and visualize the aqp4 cell distribution, double-label immunofluorescence for aqp4 and gfap was performed. real-time pcr and western blotting data show a significant upregulation of aqp4 starting from 14 days of tmt treatment in the hippocampus and, at a lower degree, in the cortex. accordingly, the immunofluorescence shows an intense astrogliosis and aqp4 immunoreactivity diffusely pronounced in the hippocampal and cortex areas starting from 14 days after intoxication. aqp4 immunolabelling was localized in astrocytic end-feet encircling the blood vessels, as expected. the study of the rhodamine b fluorescent tracer, intraperitoneally administered, also revealed an intense vascular reaction, characterized by hypertrophic vessels with abnormal course and dimensions in the brain of tmt-treated rats, indicating a vascular involvement in the tmt-induced neurodegenerative processes. aqp4 over-expression and the concurrent astrogliosis occurring in the brain of tmt-treated rats might be candidated to play a role in alterations of vascular permeability and brain oedema formation evidenced by mri studies. amyotrophic lateral sclerosis (als) is a neurodegenerative disease affecting predominantly motoneurons but with a particular pathological role of non-neuronal cells. previous research suggested that increased concentration of extracellular potassium could lead to motoneuron dysfunction. the na 1 /k 1 -atpase should regulate the homeostasis of potassium ions and thus help maintain regular neuronal activity. this homeostasis may be regulated by na 1 /k 1 -atpase in both neurons and astrocytes. in order to reveal the possible role of the na 1 /k 1 -atpase in als pathology, we explored the expression of the alpha1 catalytic subunit of na 1 /k 1 -atpase in neurons and astrocytes from different brain regions of the sod1 g93a rat model of als. labeling immunofluorescently the alpha1 isoform of the na 1 /k 1 -atpase revealed a decreased signal in the trigeminal nucleus and cerebellum of the als rat. double immunofluorecence with neun for neurons showed that the reduced alpha1 was on the neuronal surface in the trigeminal nucleus. in addition, the alpha1 ring-like staining of cerebellar granule neurons was lower in als as compared to the wild type rat. in comparison to neurons, astrocytes from the wild type rat were characterized with a lower expression of alpha1. in the examined brain regions we did not observe a prominent difference in alpha1 expression between astrocytes of wild type and als rats. based on the findings of this study the prominent reduction of na 1 /k 1 -atpase level observed in neurons and not in astrocytes could contribute to further understaning of impaired ion homeostasis affecting proper neuronal physiology in als. the role of glial na 1 /k 1 -atpase needs to be further studied with markers of other alpha subunit isoforms. although commonly used as nutritional supplements, excessive intake of bcaas might favour the establishment of neurotoxic conditions as indicated by the severe neurological symptoms characterising inherited disorders of bcaa catabolism such as maple syrup urine disease (msud). recent evidence indicates that bcaas induce excitotoxicity through mechanisms that require the presence of astrocytes. in the present study, we evaluated the effects of bcaas on microglia, the main immune cells of the brain. as an experimental model we used primary microglial cells harvested from mixed glial cultures that had been kept in normal or high bcaa medium (h-bcaa). we show that h-bcaa microglial cells exhibit a peculiar phenotype characterized by a partial skewing toward the m2 state, with enhanced il-10 expression and phagocytic activity but also increased free radical generation and decreased neuroprotective functions. we suggest that such an intermediate m1/m2 phenotype might result in a less efficient microglial response, which would promote the establishment of a low grade chronic inflammation and increase the likelihood of neurodegeneration. although based on in vitro evidence, our study adds on to an increasing literature indicating that the increasing use of dietary integrators might deserve consideration for the possible drawbacks. in addition to excitotoxicity, the altered immune profile of microglia might represent a further mechanism by which bcaas might turn into toxicants and facilitate neurodegeneration. the proteolipid protein gene (plp1), located on the x-chromosome, undergoes alternative splicing to produce the most abundant proteins of central nervous system myelin: plp and dm20. related to the extent of myelin defect, mutations in the plp1 gene in humans are associated with a spectrum of x-linked disorders, from the severe pelizaeus-merzbacher disease (pmd), to the mild form spastic paraplegia type 2 (spg2). phenotype-genotype correlation exists, large duplications of plp1 gene are predominantly found in pmd and null mutations in spg2. plp is almost 100% conserved across mammalian species, and the large spectrum of phenotype severity is also found plp transgenic mice. experiments on these models have contributed to our understanding in the pathophysiology of plp related disorders. in one hand, mice with extra copies of the plp1 gene (1plptg mice) have neurological symptoms and cns pathology similar to those found in pmd patients while mice with plp1 gene inactivation (plp null mice) share similarities with spg2 patients. then, these two mouse models represent very useful tools to evaluate the efficacy of a therapeutic strategy for the severe and the mild form of the plp related disorders on the neuropathological and behavioral aspects. olesoxime (tro19622), a cholesterol-like small molecule, has been shown to rescue motor neurons from cell death and to promote axonal regeneration both in vitro and in vivo. more recently, olesoxime has also been shown to promote central remyelination by accelerating oligodendrocyte maturation both in vitro and in vivo. altogether, these results strongly suggest that olesoxime may be a potential drug candidate for the treatment of white matter neurodegenerative disorders and notably to plp related disorders. here we treated 1plptg mice with olesoxime for 2 months starting at birth, as well as plp null mice from 3 to 12 months of age (presymptomatic treatment) or from 9 to 15 months of age (symptomatic treatment). behavioral and neuropathological analysis revealed that olesoxime failed to correct the development of symptoms in mice modeling the severe form of plp related disorders. on the contrary, treatment of plp null mice with olesoxime resulted in a 3-month delay in the onset of abnormal behaviors related to anxiety and in the slowing of central nerve conduction. these beneficial effects are observed only when the drug is administered before disease onset. treatment with olesoxime also reduces the effect of aging on rotarod performance decline in both wt and plp null old mice. altogether these results suggest that olesoxime could be of interest as a treatment to delay the onset of symptoms in plp related disorders provided that patients suffer from a mild form of the disease and are treated before the establishment of symptoms. objectives: glial cells play an important role in amyloid-beta (aß) clearance, however little is known about the cellular routes involved in internalization of different aß aggregation forms by adult human microglia and astrocytes. therefore, we evaluated the uptake mechanism of aß 1-42 by primary human adult microglia and astrocytes isolated from brain tissue of non-demented control and ad cases. methods: cells were preincubated for 1 hour with different inhibitors, before exposure to fluorescence labelled (fam) oligomeric and fibrillar forms of synthetic aß. the number of amyloid positive cells was quantified by flow cytometry. inhibitors of various pathways tested, include cytochalasin b (inhibits actin polymerization; general endocytosis inhibitor), nocadozole (inhibits tubulin depolymerization; general endocytosis inhibitor) and fucoidan (scavenger receptor inhibitor). results: cytochalasin b inhibited aß fibril uptake (90% reduction, p < 0.01) and to a lesser extent oligomer (50% reduction, p < 0.05) uptake in microglia, whereas no effect was seen on aß uptake by astrocytes. in contrast, nocodazole was found to inhibit aß fibril uptake (54% reduction, p < 0.05) and oligomer (77% reduction, p < 0.01) uptake in astrocytes, whereas no effect was seen on aß uptake by the microglia. interestingly, fucoidan prevented aß uptake in both astrocytes (oligomers:86% (p < 0.01); fibrils: 79% (p 5 0.01) and microglia (oligomers:67% (p < 0.001); fibrils: 79% (p < 0.05)). conclusions: these data indicate that both human astrocytes and microglia use scavenger receptors to internalize aß oligomers and fibrils. whether different types of scavenger receptors are involved in aß uptake by astrocytes and microglia will be further investigated. tel-aviv university, tel aviv, israel alzheimer's disease (ad) accounts for the majority of cases of dementia worldwide, affecting over 50% of the population over 85. the disease is characterized by a progressive memory loss, cognitive deterioration, behavioral disorders, deposit of beta-amyloid (ab) peptides, neurofibrillary tangles, reactive astrocytosis and activation of microglia cells. mutations in the genes that encode app (amyloid precursor protein) and components of the proteases that generate amyloid beta cause familial forms of ad. microglia, the resident immune cells of the central nervous system (cns) were suggested to play both beneficial and harmful roles in the course of the disease. therefore, modulating the microglial response is being considered as a promising approach for the treatment of this disease. we have recently shown using in vitro and in vivo systems that the ectoenzyme cd38, regulates microglial activation. in view of the significant role of activated microglia in ad and the important role played by cd38 in microglial activation, we hypothesized that cd38 deficiency would affect the course of the disease. in order to test this hypothesis, we implicated the app swe ps1de9 transgenic mouse model for ad, and compared the cognitive performance (as evident by behavioral studies) of aged app swe ps1de9 mice, to app swe ps1de9 cd38 -/mice. in addition, abload, accumulation of microglia and astrocytes was assessed in the brains of these mice. our preliminary results show that cd38 deficiency has a neuroprotective role in this ad mouse model as indicated by improvement in cognitive performance and dramatic reduction in ab load. oxidative stress induced by reactive oxygen species (ros) is associated with various pathological conditions, including aging, neurodegenerative disorders, traumatic and ischemic insults. the mechanism by which the gap junction protein connexin43 (cx43) contributes to oxidative stress-induced cell death or the "rescue" of the dying cells is still unclear. cx43 is highly expressed in astrocytes. we have previously shown that astrocytic cx43 is critical for neuroprotection during ischemic insults in vivo. to investigate the protective effect of cx43 in oxidative stress pathways, we induced oxidative stress in wild-type and cx43 knockout astrocytes using hydrogen peroxide (h 2 o 2 ). cx43 knockout astrocytes exhibited increased h 2 o 2 -induced cell death as measured by the loss of membrane integrity using the dye rhodamine b dextran, supporting a cell protective effect of cx43; this effect was not observed in wild-type astrocytes. to examine the involvement of cx43 in h 2 o 2 -induced cell death, we studied the expression and distribution of cx43 in response to h 2 o 2 in wild-type astrocytes. h 2 o 2 treatment altered the expression level and phosphorylation of cx43. this was accompanied with changes in cx43 distribution and gap junction plaque formation. hemichannel activity was measured using dye uptake. h 2 o 2 (0.7 mm) increased dye uptake, an effect that was blocked by gap26 (syntethic mimetic peptide, 200 mm) or carbenoxolone (100 mm), two connexin hemichannels blockers. however, h 2 o 2 treatment did not result in any measureable gap junction activity in cx43 knockout cells as measured by a scrape-loading dye transfer assay. these findings suggests that gap junction activity contributes to cx43-mediated protection in response to ros. one of the receptors found on microglia is the triggering receptor expressed on myeloid cells 2 (trem2), which signals intracellularly via an adapter molecule referred to as tyro protein tyrosine kinase-binding protein (tyrobp, known also as dap12). in humans, loss of function of either trem2 or tyrobp leads to nasu-hakola disease, which is characterized by neuroinflammation and bone cysts. by using a trem2 knock-out (ko) mouse line, we provided further insights on the trem2 function in neurodegenerative diseases. first, we observed mild signs of dopaminergic neurodegeneration and a slight increase in microglial iba1-immunoreactivity in different brain regions of 3 months trem2 ko mice compared with the wild type (wt) control animals. however, the transcription levels of inflammatory cytokines (tnfa, il1b) or inducible nitric oxide synthase (inos) were unaffected in mice of different ages (3, 9 or 12 months). to shorten the time required for neurodegeneration to occur, we have injected the mice intraperitoneally with lipopolysaccharides (lps) on four consecutive days and analyzed the dopaminergic neurodegeneration in the substantia nigra after a three weeks period. while the quantification of dopaminergic neurons in the substantia nigra showed a slight decrease in number for wt animals, a higher loss was recorded in the trem2 ko mice after systemic lps challenge. concomitantly, significant increase of microglia activation was detected in the lpstreated trem2 ko mice compared with lps-treated wt animals. our results are pointing towards a neuroprotective effect of the microglial trem2 receptor under chronic and systemic inflammatory conditions. increasing evidence suggest that brain energy metabolism is severely altered in multiple sclerosis patients, which may contribute to the process of neurodegeneration. imaging studies reveal a disturbed cerebral perfusion and glucose uptake and metabolism in ms patients. moreover mitochondrial dysfunction is recognized to play a crucial role in ms pathology. to date little is known about the distribution of glucose transporters (gluts), key molecules for the cellular uptake of glucose, in human brain and their role in the pathogenesis of ms. therefore, the aim of this study was to provide a complete overview of the distribution and expression levels of glucose transporters in control and ms brain. our data so far show that mrna levels of glut1, 3 and 4 as well as their regulators hypoxia inducible factor (hif)-1a, hif-2a and peroxisome proliferator-activated receptor gamma coactivator (pgc)-1a, are increased in normal appearing white matter (nawm) of ms patients compared to controls. immunohistochemical analysis revealed that brain endothelial cells not only express glut1 but also glut3 and glut4. glut 1, 3 and 4 are present in resting microglia and highly expressed in activated microglia and macrophages in active ms lesions. interestingly reactive astrocytes expressed increased levels of glut1 and glut4 compared to control, in addition glut3 positive astrocyes were observed in ms brain. axons expressed high levels of glut3 in active ms lesions, which was evidently reduced in chronic lesions. strikingly, all glucose transporters studied were down regulated in chronic ms lesions. together, these data emphasize the extent to which glucose metabolism appears to be affected in all stages of ms. a better understanding of these metabolic changes, especially in the nawm and chronic stage of the disease, is essential to develop strategies to improve neuronal survival and to gain more insight in the mechanisms underlying lesion formation. pericytes are vascular mural cells which are enriched within the capillary basement membranes of the brain. recent studies have provided evidence that pericytes regulate the blood-brain barrier (bbb) and modulate cerebral blood flow. neurovascular dysfunction and bbb damage are hallmarks of alzheimer's disease (ad), but the role of pericytes in ad pathogenesis is still unclear. we have examined post-mortem samples of middle frontal gyrus in collaboration with the university of cambridge after ethical approval by the local irb. ten cases with neither history of neurological disorder nor evidence of neuropathology were compared with 10 ad cases provided by the neurological foundation of the new zealand human brain bank. we performed immunostainings for laminin and plateletderived growth factor receptor (pdgfr)-b which mark vessels and pericytes, respectively. we also stained for ab 1-42 for evidence of amyloid pathology. quantification of pericyte and vessel density was performed using stereological methods, which are considered as gold standard in morphometric quantification. the spaceballs method was used to measure the density of capillaries. pericyte density was assessed using the optical fractionator. the age of the subjects did not differ between the control and ad groups (76,2 6 4,0 vs. 75,4 6 5,5 years). the cortex of ad cases contained more capillary vessels than controls (mean 6 sd: 308 6 40 vs. 365 6 41 mm/mm 3 ; p < 0,01; fig. 1a ). the capillary density increased in correlation to the total plaque load (r 5 0,58; p < 0,001). in contrast, we could not observe a significant difference in the number of pericytes per mm capillary length between control and ad cases (8,56 6 1,21 vs. 8,17 6 0,94 cells/mm; p 5 0,44; fig. 1b ). relative to tissue volume, the cortex of ad individuals showed a tendency to contain more pericytes than controls (2624 6 406 vs. 2985 6 588 cells/mm 3 ; p 5 0,13; fig. 1c ). this is the first study using state-of-the-art techniques to investigate the relationship between capillaries and pericytes in ad brains. impaired clearance of b amyloid across the pericyte-regulated bbb and deficient microvascular regulation of blood flow may contribute causally to ad pathogenesis. in consequence, loss of pericytes could contribute to disease progression. however, our results show that ad cortical pathology is characterized by an increase in the density of capillary vessels without deficit in pericyte coverage. thus, they do not substantiate the notion that pericyte loss is a significative feature of ad. m. noack, j. leyk, c. richter-landsberg carl von ossietzky universit€ at oldenburg, oldenburg, germany histone deacetylase 6 (hdac6), a member of the class ii hdacs, is a unique cytoplasmic a-tubulin deacetylase that interacts with the microtubule (mt)-associated protein tau. in healthy human brain six tau isoforms are expressed generated by alternative mrna splicing. these contain either three (3r-tau) or four (4r-tau) microtubule binding repeats regulating mt assembly and stability. tau hyperphosphorylation impairs its mt-binding activity. tau deposits in nerve cells and glia are the characteristic hallmark of a number of neurodegenerative diseases termed tauopathies. in progressive supranuclear palsy and corticobasal degeneration, pathogenic glial cell inclusions are observable in oligodendrocytes, the myelin forming cells of the cns. hdac6 plays an important role in the degradation and accumulation of misfolded proteins by promoting transport of aggregated proteins to the aggresome, localized at the microtubule-organizing center where protein aggregates are deposited and processed by autophagy. the present study was undertaken to elucidate the functional significance of hdac6 in oligodendrocytes and its role in pathological protein aggregate formation. towards this cultured rat brain oligodendrocytes and oln-t40 cells, an oligodendroglial cell line expressing the longest human tau isoform, were used. treatment of primary oligodendrocytes with tubastatin a (tst), a selective inhibitor of the tubulin deacetylation activity of hdac6, caused a significant increase in the level of acetylated tubulin, as determined by indirect immunofluorescence and immunoblot procedure. mts appeared more bundled and stabilized. inhibition of hdac6 attenuated the phosphorylation of tau at the phf-1-epitope (serine 396/ 404) and its enhancement at the 12e8-epitope (serine 262, located within the microtubule binding repeat region 1). furthermore, altered protein levels of tau isoforms containing either three or four mt binding repeats were observed. while 3r-isoforms were reduced, 4r-isoforms were increased in primary oligodendrocytes suggesting an impact on microtubule binding ability. hdac6 knockdown in oln-t40 cells revealed no alteration in tau phosphorylation at phf-1-epitope, but also augmented phosphorylation at the 12e8-epitope. cells were morphologically damaged and mt organization was disturbed. the data indicate that hdca6 modulates tau expression and phosphorylation, its inhibition leads to the accumulation of 4r-tau which is also prominent in tauopathies with oligodendroglial pathology. to test the hypothesis, that these cells are part of a possible compensatory mechanism to cope with the 6-ohda-induced loss of dopaminergic neurons, we studied the expression of tyrosine hydroxylase (th), the rate-limiting enzyme in the catecholamine synthesis pathway, in the cortex of 6-ohda-lesioned animals. performing immuncytochemistry at 4d after the lesion we demonstrated a 21-fold increase in the number of th-positive (th1somata in rat cortex following 6-ohda injection) compared to sham-lesioned animals. th1somata in the parietal cortex were restricted to the layers ii-v with most of the cells being bipolar. combining th immuncytochemistry with classical nissl stain yielded complete congruency. a small fraction of th1cells co-expressed calretinin thus pointing to an interneuron affiliation. virtually none of the th1cells expressed other markers of interneurons (calcium binding proteins, neuropeptides) or the glial markers gfap and nestin. in contrast, we found a co-localization of th with markers of glial progenitor cells (sox2 and s100b) and with psa-ncam, which has been shown to be expressed in immature, but not recently generated cortical neurons in cortical layer ii of the cat (varea et al.; front. neurosci. 5: 17 2011). taken together, these findings indicate that 6-ohda lesions might induce a glial de-differentiation followed by a fate re-direction towards neuronal committed cells. future studies have to be performed to confirm this and to find out if the th1cells are capable of dopamine synthesis. objective: using the mouse optic nerve (mon) wm model, we tested whether hydrogen in drinking water reduced functional wm ischemic injury, and if it reduced cellular lipid peroxidation and mitochondrial dysfunction including mitochondrial and nuclear dna oxidation. methods: functional integrity of mon was determined by quantitatively monitoring the area of mon compound action potential (cap) before, during and after a standardized 60 min period of oxygen and glucose deprivation (ogd), the ischemia equivalent ex vivo. nuclear 8-oxoguanine (nu8-oxog) was used as a marker of oxidative dna damage. results: a 60 min period of ogd caused prompt loss of the cap, followed by an average 20% recovery. in mice that had received hydrogen-containing drinking water for 7-10 days, the cap area did not disappear during ischemia and recovered to a significantly greater extent during reperfusion (normal oxygen and glucose levels). immunostaining of axonal neurofilament by smi-31 showed significant protection in mons from mice drinking hydrogen-water. accumulation of nu8-oxog was observed mainly in oligodendrocytes after ogd. the levels of 8-oxog after ogd were significantly reduced in optic nerves from hydrogen-water drinking mice. the 'protection' induced by 7-10 days of hydrogen-water drinking lasted several days. conclusions: our results show that several days of hydrogen exposure reduced the extent of cns wm irreversible injury associated with ogd. the importance of these observations is that oligodendrocytes may play an important role in the protective effect against ischemic injury. these observations raise intriguing therapeutic options. amyloid-b (ab) deposits in the brain extracellular space (ecs) and neurofibrillary tangles are typical features of alzheimer's disease (ad). both affections are expressed in triple transgenic (3xtg-ad) mice, making them a useful model for studying the pathophysiology of ad. in these mice, significant changes in astroglial morphology appear in the limbic brain structures [1, 2] , which may affect the diffusion of neuroactive substances through the ecs. using the real-time iontophoretic method, we determined the ecs volume fraction a (a 5 ecs volume / total tissue volume) and the geometrical factor tortuosity k (k 2 5 free / apparent diffusion coefficient) in the ca1 region of the hippocampus, dentate gyrus (dg) and prelimbic cortex (plc) in brain slices obtained from 10-month-old (10m) and 20month-old (20m) 3xtg-ad mice and age-matched wild-type (wt) controls. to study potential diffusion anisotropy, the measurements were performed along 3 orthogonal axes: medio-lateral, rostro-caudal and ventro-dorsal. astroglial morphology and the presence of ab were assessed using immunohistochemical staining for gfap and ab. we found no anisotropy in the ca1 region, dg, or plc, thus the data for each structure along the three orthogonal axes were pooled. the ecs diffusion parameters measured in the ca1 are shown in table 1. in the ca1 of 10m mice, there was no significant difference in the ecs diffusion parameters between wt and 3xtg-ad mice. in 20m mice, a decreased by 9% in wt but increased by 27% in 3xtg-ad mice in comparison with the values found in the younger animals. during aging, there was also a significant decrease in k in wt but not in the 3xtg-ad mice. a similar pattern of changes in the ecs diffusion parameters during aging was also observed in the dg and plc. unlike in the ca1, we found significant differences in young animals: a was lower in the dg and k was higher in the dg and plc of 3xtg-ad mice than in wt mice. in 20m 3xtg-ad mice, ab deposits accompanied with astroglial atrophy or hypertrophy were observed. we suggest that the amyloid load in 3xtg-ad mice and the subsequent prevailing astroglial atrophy affect the normal aging process by inducing an increase in the ecs volume during aging instead of the normal decrease. this leads to a larger ecs space in aged 3xtg-ad mice than in age-matched wt mice, which may alter the efficacy of extrasynaptic as well as synaptic transmission and contribute to the impaired cognitive functions in ad. the study was supported by the grants: gacr p304/11/0184, gacr p304/12/g069. significant differences between 10m and 20m mice of the same group are marked with *, differences between wt and 3xtg-ad mice of the same age are marked with 1. sequence or any treatment. however growing evidences are in favor of the involvement, besides neurons, of several partners such as glia and muscles. to better characterize the time course of pathological events in an animal model that recapitulates human als symptoms, we investigated functional and cellular characteristics of hsod1 g93a mice. we have evaluated locomotor function of hsod1 g93a mice through dynamic walking patterns and spontaneous motor activity analysis. we detected early functional deficits that redefine symptoms onset at 60 days of age, i.e. 20 days earlier than previously described. moreover, sequential combination of these approaches allows monitoring of motor activity up to disease end stage. to tentatively correlate early functional deficit with cellular alterations we have used flow cytometry and immunohistochemistry approaches to characterize neuromuscular junctions, astrocytes and microglia. we show that (1) decrease in neuromuscular junction's number correlates with motor impairment, (2) astrocytes number is not altered at pre-and early-symptomatic ages but intraspinal repartition is modified at symptoms onset, and (3) microglia modifications precede disease onset. at pre-symptomatic age, we show a decrease in microglia number whereas at onset of the disease two distinct microglia sub-populations emerge. we are now establishing the transcriptomic profile of microglia over the course of the disease. in conclusion, precise motor analysis updates the onset of the disease in hsod1 g93a mice and allows locomotor monitoring until the end stage of the disease. early functional deficits coincide with alterations of neuromuscular junctions. importantly, we identify different sets of changes in microglia before disease onset as well as at early-symptomatic stage. this finding not only brings a new sequence of cellular events in the natural history of the disease, but it may also provide clues in the search for biomarkers of the disease, and potential therapeutic targets. in order to gain more knowledge about the disease pathophysiology, two mouse models for vwm are developed in our lab. co-cultures between astrocytes (wt and vwm) and wt oligodendrocyte progenitor cells (opcs) are done to investigate the effect of astrocytes on opc maturation. furthermore, induced pluripotent stem cells (ipscs) from both vwm and wt mice are compared in their glial differentiation efficiency in vitro and in vivo. the astrocyte-opc co-cultures show that vwm astrocytes inhibit opc maturation. this suggest that astrocytes might have a crucial role in the development of the oligodendrocyte and myelin pathology in vwm. currently our research focuses on rescuing the maturation inhibition in vitro, which can aid the development of stem cell therapy for vwm. particularly in response to b-amyloid. using the appps1 alzheimer's disease mouse model, we observed the production of the common interleukin (il)212 and il-23 subunit p40 by microglia. genetic ablation of various il-12/il-23 signaling molecules, of which deficiency in p40 had the strongest effect, resulted in a drastic decrease in cerebral amyloid burden. although deletion of il-12/il-23 signaling from the radiation-resistant glial compartment of the brain was most efficient in mitigating cerebral amyloidosis, peripheral administration of a neutralizing p40-specific antibody likewise resulted in reduction of cerebral b-amyloid in appps1 mice. furthermore, intracerebroventricular delivery of antibodies to p40 significantly reduced soluble ab species and reversed cognitive deficits in aged appps1 mice. our results suggest that inhibition of il-12/il-23 signaling reduces cerebral amyloidosis and cognitive dysfunction, and may pose a novel potential pharmacological target to combat alzheimer's disease. charit e -universit€ atsmedizin berlin, berlin, germany question: microglia are attracted to and surround b-amyloid deposits, the main hallmark of alzheimer's disease (ad), suggesting a role for these cells in disease pathogenesis. however, recent in vivo data using the cd11b-hsvtk system for microglial depletion suggests that resident microglia are not sufficiently capable of restricting and clearing bamyloid plaques. to underpin the cd11b-hsvtk system and shed more light into the biological mechanistics of microglial depletion mediated by ganciclovir (gcv), we aim to intravitally monitor the depletion process. methods: we are using cd11b-hsvtk transgenic mice crossed to fractalkine-gfp 1/mice harboring green fluorescent microglia. a chronic cranial window is implanted onto the head of the offsprings followed by depletion of microglia and subsequent in vivo two-photon (2p) imaging. as 2p imaging is able to visualize the upper 300 mm of the cortex, a new topical depletion approach of cd11b-hsvtk 1 microglia was established. here, gcv is applied directly onto the brain surface in the area of the cranial window through a catheter. results: manipulation of the brain skull by installation of the cranial window and placing a catheter for topic gcv application resulted in strong depletion of microglia up to 85% in fractalkine-gfp 1/mice crossed to cd11b-hsvtk mice. conclusions: taken together, we established a minimal invasive technique for visualization of the microglia depletion process in cd11b-hsvtk mice using intravital 2p microscopy. these tools will allow the underpinning of the cd11b-hsvtk system as well as the study of relevant biological questions involving resident and peripheral derived microglia in ad. (icrea) , barcelona, spain x-linked adrenoleukodystrophy (x-ald) is a metabolic genetic disorder of the central nervous system characterized by demyelination in brain and/or axonopathy in the spinal cords, adrenal insufficiency and accumulation of very long-chain fatty acids (vlcfa) in plasma and tissues. the disease is caused by inactivation of the abcd1 transporter with the consequence production of oxidative stress mediators and damage. here we aimed to determine: i) the existence of endoplasmic reticulum (er) stress and the ensuing unfolded-protein response (upr) in brains and fibroblasts from x-ald patients, and in the x-ald mouse model (the abcd1 null mouse); and ii) whether the early and rampant oxidative stress formerly identified accounts for the er stress. indeed, here we report signs of er stress and upr response in human and mouse tissues. a common feature is the activation of atf6 and lower amounts of ire1, two er transducers constituting the core signature of upr in x-ald. chaperone grp78 and grp94 expression, by contrast, differ between variants and stages of the disease. we highlight how chaperones are down-regulated in x-ald mice at 12 months of age, pointing to selective depletion of upr-related factors overtime. treatment of the mouse model with a combination of antioxidants reversed the initial activation of transducers and chaperones, indicating that oxidative stress caused er stress. altogether, we postulate that oxidative-stress elicited er stress occurs in x-ald, and that a defective upr may contribute to axonopathy progression. thus, potentiation of the upr may be a therapeutic avenue in x-ald. representing opc-like cells with the potential to differentiate upon growth factor withdrawal and addition of triiodothyronine (t3) into premyelinating oligodendrocytes. upon differentiation, significant fewer cg4_wt-a-syn cells express the myelin basic protein (mbp; see figure a) as a marker for terminal differentiation and additionally, mrna levels were remarkably reduced compared to control cells (see figure b ). as mrna levels of the myelin protein proteolipid protein 1 (plp1) as well as the major myelin-gene regulatory factor (mrf) are also reduced in differentiated cg4_wt-a-syn cells (see figure b) we hypothesized that the process of differentiation is delayed upon a-syn expression. using various differentiation-promoting agents targeting distinct maturation-associated pathways we aimed to dissect the a-synmediated effect on differentiation in more detail and evaluate potential disease-modifying approaches for this devastating neurodegenerative disorder. methods: two nutritionally distinct groups of male newborn ratswell-nourished (w; n 5 26) and malnourished (m; n 5 33) were treated from the 5th to the 24th day of postnatal life with daily intraperitoneal injections of saline (sal) or cona (sigma-aldrich) at doses of 1 mg/kg (group w-l1 and m-l1) or 10 mg/kg (groups w-l10 and m-l10). two groups of "naive" (non-injected) pups were used as additional controls. when the pups reached adult age (90-120 days), under ip anesthesia (1 g/kg urethane 1 40 mg/kg chloralose) they were submitted to the csd recording (ecog and slow potential change) in two points of the parietal cortical surface. csd was triggered every 20 minutes by applying 2% kcl for 1 min at a point in the frontal cortex. this study was approved by the ethics committee of our university, case no.: 23076.031272/2010-53. results: compared to w-sal controls (mean 6 sd velocity in mm/ min 5 3.41 6 0.10), m-sal rats presented significantly higher velocities (4.26 6 0.16; p < 0.05). in both nutritional conditions l1 and l10 treated groups presented significantly lower csd velocities (3.12 6 0.14 and 2.82 6 0.18 for the w-l1 and w-l10, respectively, and 3.74 6 0.13 and 3.25 6 0.16 for the m-l1 and m-l10). this effect was dose-dependent, and was greater in the m-condition. the na€ ıve-and salgroups had similar csd velocities. conclusions: the lectin cona administered early in life to rats decelerated csd in a dose-dependent manner at adulthood, suggesting a long-lasting effect. the largest relative reduction was observed in the m group, suggesting modulation of the effect by the nutritional status. the involvement of glial cells in this effect is discussed. the endocannabinoid system is of growing interest as a therapeutic target in neurological diseases. the two major endocannabinoids 2arachidonoylglycerol (2-ag) and n-arachidonoyl ethanolamine (anandamide, aea), are the endogenous ligands for the cannabinoid receptors 1 (cb 1 ) and 2 (cb 2 ). cb 1 is mainly expressed in the brain and associated with neuroprotective effects, whereas cb 2 is primarily found in hematopoietic cells and associated with anti-inflammatory effects. in neurodegenerative diseases, e. g. amyotrophic lateral sclerosis (als) and parkinson's disease (pd), an increased cb 2 expression on microglia has been reported and is therefore an interesting therapeutic target. furthermore, an increase of endocannabinoid levels occurs in the affected neuronal tissues. stimulating this disease-related increase in endocannabinoids might thus be of therapeutic interest. in our project, we evaluated the novel, highly selective inhibitor kml29 of an enzyme central to the degradation of the endocannabinoid 2-ag, the monoacylglycerol lipase [1] . an in vitro monoacylglycerol lipase inhibitor screening assay for the comparison of the utilized inhibitor with others was performed. additionally, behavioral experiments including body temperature measurement, pain threshold measurement and motor activity analysis in healthy control mice were performed. furthermore, endocannabinoid system baseline levels of healthy control mice vs. sod1 (superoxide dismutase 1) mice as an als mouse model were compared at the gene expression and protein level. in vitro inhibition of the monoacylglycerol lipase by the recently published compound kml29 resulted in lower ic 50 values and therefore indicated a higher potency of kml29 when compared to the other monoacylglycerol lipase inhibitors jzl184 and cpc12 applied in the assay. in vivo administration of the reported monoacylglycerol lipase inhibitor kml29 resulted in significant decrease in body temperature, increase in pain threshold in b6sjlf1/j mice as well as in impairments in the nocturnal motor activity of c57bl/6j mice when compared to vehicle-treated mice. in summary, our data show a comparatively high potency in vitro and significant dose-dependent effects on physiological parameters in vivo of the monoacylglycerol lipase inhibitor kml29 and therefore lay the foundation for a future therapeutic study targeting neuroinflammation in mouse models of als and pd. glaz/apod protects against oxidative stress and promotes axon regeneration after injury, but its mechanism of action is unknown. we hypothesize that glaz/apod modulates membrane dynamics and oxidation. to contrast this hypothesis we study glaz protective role on the polyq-based spinocerebellar ataxia type i (sca1) model in drosophila. human polyq-ataxin1 expression in fly photoreceptors triggers glaz up-regulation. overexpressing glaz in photoreceptors or in retinal support cells rescues neurodegeneration. when expressed in retinal support cells, the glaz rescue is dependent on lipocalin-receptor expression by photoreceptor neurons, and the glaz-gfp fusion protein co-localizes with photoreceptor markers, suggesting the existence of receptor-mediated endocytosis of glaz. upon neurodegeneration, oxidative stress and induction of autophagy coexist. to test whether the glaz beneficial effects are mediated by the modulation of autophagy we quantified atg8a expression and monitored the accumulation of ubiquitinated proteins and p62. overexpression of glaz reduces atg8a induction, but concurrently reduces the accumulation of ubiquitinated proteins and p62. upon autophagy stimulation by rapamycin treatment, the levels of sca1-dependent accumulation of ubiquitinated proteins and p62 are further reduced by glaz. in addition, glaz decreases the sca1-dependent induction of gsts1, a sca1 genetic modifier contributing to the clearance of lipid peroxides. our data support that glaz enters the degenerating neurons by a receptor-mediated mechanism and promotes the resolution of autophagy, increasing its flow and helping to clear polyq-induced protein aggregates. moreover, the beneficial effects of glaz are linked to lipid peroxide clearance, either directly or through receptor-mediated modulation of protective gene networks. micinn(bfu2011-23978); jcyl(va180a11-2); fund. rodr ıguez-pascual. we investigated the effect of three different intensities of running exercises on the survival of sod1 g93a mice. at the early-symptomatic stage (p60), males were isolated and randomly assigned to 5 conditions: 2 sedentary groups ("sedentary" and "sedentary treadmill" placed on the inert treadmill), and 3 different training intensity groups (5 cm/s, 10 cm/s and 21 cm/s; 15 min/day, 5 days/week). we first demonstrated that an appropriate "control" of the environment is of the utmost importance since comparison of the two sedentary groups evidenced an 11.6% increase in survival in the "sedentary treadmill" group. moreover, we showed by immunohistochemistry that this increased lifespan is accompanied with motoneurons survival and increased glial reactivity in the spinal cord. in a second step, we showed that when compared with the proper control, all three runningbased training did not modify lifespan of the animals, but resulted in glial and motoneuronal changes. conclusions/significance: we demonstrate that increase in survival induced by a slight daily modification of the environment is associated with motoneurons preservation and strong glial modifications in the lumbar spinal cord of sod1 g93a . using the appropriate control, we then demonstrate that all running intensities have no effect on the survival of als mice but induce cellular modifications. our results highlight the critical importance of the control of the environment in als studies and may explain discrepancy in the literature regarding the effect of exercise in als. alzheimer's disease is the most prevalent neurodegenerative disorder, characterized by occurrence of senile plaques, neurofibrillary tangles and aberrant function of classical neurotransmitters and peptide messengers, such as neuropeptides and growth factors. in the last years a role of astrocytes in mediating and amplifying the progression of neurodegenerative disorders has been proposed. moreover, a growing body of evidence has shown that astrocytes can release non-peptide and peptide transmitters to influence neuronal development, function and plasticity. here we analyzed the impact of the main component of senile plaques, the amyloid-b (ab), on two key components of peptide vesicles, carboxypeptidase e (cpe) and secretogranin iii (sgiii), in astrocytes in vitro and in vivo. we consistently located cpe and sgiii proteins in cultured and in situ astrocytes. traffic and secretion of astroglial cpe and sgiii were analyzed under basal and stimulated conditions in primary cultures. we found that exposure of astrocytes to ab1-42 markedly impairs expression, traffic and secretion of glial cpe and sgiii. protein levels were decreased in the media, but increased into the cells, by ab1-42 incubations. the effect of ab on cpe and sgiii in glial cells was also investigated in vivo using the amyloid-forming transgenic mice appswe/ps1de9. an aberrant accumulation of cpe and sgiii was found in numerous activated-astrocytes surrounding senile plaques through all the cns of aged transgenic mice. taken together, the present study shows that ab dramatically alters expression, traffic and secretion of cpe and sgiii. because cpe and sgiii are essential in the process and targeting of neuropeptides and growth factors, an involvement of the astroglial secretory pathway in the pathology of alzheimer's disease is suggested. glia cytokine that is produced subsequent to h-i, that collaborates with other cytokines to stimulate the production of astrocytes from subventricular zone glial progenitors. the specific goal of this study was to evaluate gliogenesis after h-i when the tgfb1 receptor alk5 is antagonized in the vanucci p6 h-i rat model. by q-pcr, the relative amount of tgfb1 mrna peaked 7 days after h-i. therefore, sb-505124, and alk5 antagonist, was given intraperitoneally (i.p.) 7 days after the injury to a group of rats while another group received pbs. animals were sacrificed at different time points and brain samples processed for western blot analysis. validating the importance of alk-5 signaling, sb505124 reduced the levels of phosphorylated smad 2/3 that increased after h-i. in another study, sb-50512 was administered i.p. from p10 to p15 and animals sacrificed at p20 for immunohistochemistry for gfap, iba-1, gstpi and mbp. gfap and iba-1 positive cells were dramatically increased in the injured striatum and corpus callosum after h-i and mbp staining was decreased. by contrast, both the extent of injury and the degree of reactive gliosis was decreased by sb-505124 treatment. altogether, our results indicate that sb-505124 inhibits alk5 signaling in the damaged brain. furthermore, sb-505124 administration decreases the extent of microgliosis and astrogliosis while preserving myelination in the damaged brain after neonatal h-i. supported by nih r01 hd052064 and a grant from the leducq foundation awarded to swl. [2] . despite efforts to improve air quality, dep persists as a problem and the share of diesel engined passenger cars in western europe is increasing steadily. honey bees are an important pollinator of food crops and wild flowering plants. they not only contribute to food security by providing pollination services of an enormous economic value but also play an important ecological role. over the last five years, bee keepers worldwide have reported a decline in honey bee populations. the reasons for this decline remain unknown, but it is thought to be multi-factorial [3] . honey bee colonies are confronted with a number of stressors, for example infection with the mite varroa destructor or exposure to pesticides. to forage successfully honey bees rely on their learning and memory abilities. our research aims to establish whether dep might be a factor contributing to declines in honey bee populations through impairment of healthy cns function. methods: we have investigated the impact of dep exposure on the learning abilities of the honey bee using the proboscis extension reflex and classical conditioning. western blotting and histology were used to determine regional expression levels of stress proteins in the brain in response to an acute diesel exhaust exposure. we are examining the morphological appearance of glial cells to investigate the impact of dep in the honey bee cns of dep exposed, and control, forager bees. results and conclusions: we hypothesize that dep acts as a stressor in the brain of the honey bee causing changes in glial cells that are detectable using morphological and molecular techniques -similar to microglial activation or immunological responses of astroglia in the mammalian brain. this work will further our understanding of how dep acts on the brain and how it might impact on the health of the animal. current results indicate that diesel exhaust pollution impacts on the neurobiology of the honey bee and this may contribute to decline by impairing cns functions. hepatic encephalopathy (he) is a serious neurological disorder caused by liver failure. the prime candidate responsible for he pathology is an increased ammonium concentration; and following acute liver failure, ammonium can reach levels of up to 5 mm in the brain. astrocytes are a major target of ammonium toxicity and earlier studies suggested that this toxicity includes a disturbance in intracellular calcium signaling. in the present study, we analyzed the effect of acute hyperammonia on the intracellular calcium concentration of astrocytes in tissue slices of mouse brain, using quantitative intracellular calcium imaging with the indicator dye fura-2. astrocytes were identified by staining with the vital dye sr101; cerebellar bergmann glial cells were identified based on their morphology. in all brain regions studied, the hippocampal ca1 area, somatosensoric cortex, and cerebellar cortex, bath perfusion with 5 mm ammonium for 30 min induced a small, but persistent elevation in intracellular calcium, averaging about 50 nm. in addition, a fast and transient increase by on average 100 nm that lasted about 10 minutes was seen in a small percentage of astrocytes in hippocampal and cortical slices at the onset of ammonium perfusion. this transient increase was never observed in cerebellar bergmann glial cells. both the transient, as well as the plateau phase of ammonium-induced calcium changes were unaltered in the presence of ttx, indicating that they are independent from action potential generation by neurons. furthermore, ammonium-induced calcium increases largely persisted upon removal of extracellular calcium, indicating that they are caused by release from intracellular stores. taken together, our experiments demonstrate that ammonium evokes complex changes in the intracellular calcium concentration of astrocytes in the intact tissue, which differ both between cells in one preparation as well as between different brain regions. because of the central role of astrocyte calcium in gliotransmission, the observed ammonium-induced calcium dysbalance might result in disturbed neuronglia interaction and contribute to the pathology of he. it has been shown in a number of animal models that microglia in the degenerating brain are primed to show exaggerated cytokine responses to subsequent stimulation with toll-like receptors agonists such as lps and poly i:c. it is not clear whether the degenerating brain shows similarly exaggerated responses to pro-inflammatory cytokines. in the current study we hypothesised that glial cells in the hippocampus of animals with chronic neurodegenerative disease (me7 prion disease) would display abnormal responses to central cytokine challenges. in normal animals it has previously been established that intracerebral cytokine challenges elicit specific pathways, i.e. il-1b a cxcl-1 a neutrophil recruitment or tnfa a ccl-2 a monocyte recruitment. unilateral 1ll doses of tnfa (300ng/ll), il-1b (10ng/ll) or saline were administered intrahippocampally via pulled glass microcapillary, in normal (nbh) and me7 mice. these animals were terminally perfused for formalin fixation and paraffin embedding at 2, 24 and 72 hours post challenge. at 2 hours post challenge me7 microglia produced il-1b following either il-1b or tnfa challenge while nbh microglia did not. furthermore, there was very robust nuclear localisation of the nfkb subunit p65 in the astrocyte population and this was associated with very marked astrocytic synthesis of the chemokines cxcl-1 and ccl-2 in response to both cytokine challenges in me7 animals. conversely, very limited expression of these chemokines was apparent in nbh animals similarly challenged. thus astrocytes are the primary chemokine synthesizing brain cell and in the prion-diseased brain are they primed to produce exaggerated chemokine responses to acute stimulation with pro-inflammatory cytokines. this abnormal pattern of chemokine expression is predicted to have consequences for leukocyte infiltration and the ramifications of an altered cellular infiltration pathway for the degenerating brain will be discussed. these findings suggested that the presence of mutated htt in astrocytes alters glial glutamate transport capacity early in the disease process and may contribute to excitotoxicity. to decipher whether astrocytic mutated htt expression was directly associated with msns dysfunction, we investigated the functional properties of individual msns in proximity of mutated htt expressing astrocytes in acute slices. results from whole-cell patch clamp technique showed that msns surrounded (0 -100 microns distance) by astrocytes expressing mutated htt vs. astrocytes expressing normal htt did not differ with regard to passive membrane properties (input resistance, resting membrane potential), action potential firing rates nor spontaneous excitatory postsynaptic current properties (averaged amplitude and frequency). thus, astrocytic expression of mutated htt does not lead to readily apparent functional consequences in msns in these conditions. our data show that 11 month old tg mice displayed a significant increase of ab-plaques in the brain, compared to wt mice. furthermore, we show that the a1c subunit colocalizes with 90% of the abpositive plaques. the cellular expression of a1c was predominantly found in activated gfap1 astroglia around plaques. qpcr profiles of the hippocampus revealed expression of the majority of calcium channel subunits, which was stable during aging. the auxiliary beta4 subunit was expressed in these mice but did not co-localize with a1c1 astroglia around plaques. in cultures of primary astrocytes, ab(42) slightly enhanced the a1c mrna expression after 3 days. we are currently underway to characterize this expression in more detail. in summary, our data provide evidence for a largely stable expression of most ltcc subunits in alzheimer tg mice during aging. finally, the expression of a1c but not beta4 is upregulated in activated astrocytes located around ab-plaques and ab(42) may directly induce astroglial ca v 1.2 calcium channels. this study was supported by the sonderforschungsbereich sfb f4405-b19 and f4406-b19 of the austrian science funds. increasing evidence indicate that both physical and cognitive stimulation induce plastic changes in the brain, such as increase in synaptic and spine densities or neurogenesis, and counteract b amyloid (ab) pathology recovering cognitive function. in the present study we, for the first time, demonstrate the effects of psychostimulation on glial fibrillary acid protein (gfap) architecture in the dentate gyrus (dg) of 3xtg-ad, a well-established mouse model of ad. starting from 3 months of age, 3xtg-ad and their corresponding controls (non-tg) were housed either in the presence of a running wheel (run) or in the enriched environment (enr) for 9 months. as reported previously (olabarria et al., 2010), in standard housing, 3xtg-ad animals showed marked atrophy of gfap-positive profiles, evidenced by significant reduction in gfap surface area, by 39%, and volume, by 42%, compared with non-tg mice. however, physical and cognitive stimulation affected astrocyte morphology by increasing their cytoskeleton surface area and volume, both in non-tg and in 3xtg-ad mice. the surface area of gfap-ir astrocytes increased by 145% and 154% in 3xtg-ad mice housed in run and enr, respectively, compared with transgenic mice kept in standard housing. in addition, the cell volume of gfap-ir astrocytes was higher by 169% and 199% in 3xtg-ad mice housed in run and enr, respectively. the hypertrophy was also evidenced by an increase in the surface area and the volume of astroglial somata and processes in both non-tg and 3xtg-ad mice. further correlation analysis between 3xtg-ad and their corresponding control mice housed under the same conditions, revealed that run and enr not only reversed the genotype-induced astrocytic atrophy, but even induced a hypertrophy in 3xtg-ad mice, with gfap positive profiles rising to the levels of non-tg mice. thus our study indicates that long-term exercise and enriched environment restore and improve astroglial plasticity in the context of adlike pathology, which may recover cognitive functions by compensating disease-associated degeneration of neuronal-glial network. methods: intraperitoneal administration of wa at a dosage of 4mg/ kg of body weight was initiated from postnatal day 40 (p40) till end stage in sod1g93a mice, from 9 months till end stage in sod1g37r mice and from 6 months of age in tdp43a315t mice. results: wa was able to improve the survival by more than a week in sod1g93a and by two weeks in sod1g37r familial mouse model. in addition wa administration also conferred significant neuroprotection in tdp43a315t transgenic mice model. beneficial effects of wa in sod1g93a mice model was accompanied by reduction in loss of motor neurons and also by reduction in level of misfolded sod1 protein in the spinal cord as detected by immunoprecipitation with antibody specific to misfolded sod1. wa was found to be an inducer of heat shock protein 27 (hsp-27), which could possibly explain reduced level of misfolded sod1 and increased neuroprotection in wa treated mice. moreover real-time imaging with the use of biophotonic sod1g93a transgenic mice carrying luc (luciferase) and gfp (green fluorescent protein) reporter genes under the control of the murine gap-43 promoter revealed that wa was able to reduce neuronal stress at post symptomatic stage. conclusion: taken together, our results suggest that wa may represent a promising therapeutic drug for treatment of als. the temporal lobe epilepsy (tle) is the most common form of epilepsy that originates from the hippocampus and then propagates to other limbic areas such as the amygdala and entorhinal cortex. the pathological feature associated with tle is hippocampal sclerosis which is characterized by atrophy, induration, gliosis and loss of neurons in ca1, ca3 and the dentate hilar regions. some animal models, albeit do not exactly match the complex etiologies identified in humans, are found to recapitulate most of the pathological features observed in tle. there is evidence that administration of kainic acid can cause seizures in the ca3 region of the hippocampus that can lead to loss of neurons and astrogliosis characteristic of tle, but the underlying mechanisms associated with the degeneration of neurons remain unclear. since lysosomal enzymes, cathepsins b and d, can have important roles in the loss of neurons in a variety of experimental conditions, we evaluated their potential roles along with the insulin-like growth factor-ii (igf-ii) receptor, which is involved in the intracellular transport of these enzymes, in the kainic acid treated rats. our results clearly showed that systemic administration of kainic acid evoked severe loss of neurons along with hypertrophy of astrocytes and microglia in the hippocampal region of the adult rat brain. the levels and expression of cathepsins b and d as well as igf-ii receptor increased progressively with time in the hippocampus of kainic acid treated rats compared to control rats. the activity of both cathepsins b and d was also found to be enhanced in the hippocampus of treated rats. our double labelling studies revealed that expression of both cathepsins were initially increased in the pyramidal neurons and then decreased with time. this was accompanied by the expression of immunoreactive igf-ii receptors as well as cathepsins b and d in a subset of gfap-labelled activated astrocytes and iba1-labelled microglia in kainic acid treated rats. these results, taken together, suggest that enhanced levels/expression and activity of lysosomal enzymes may have a role in the loss of neurons observed in kainic acid treated rats. in addition, increased ng2 glia immunoreactivity and myelin loss were found specifically in the gray matter of patients' motor cortex and spinal cord ventral horn. furthermore, oligodendroglial specific monocarboxylate transporter 1 expression is decreased not only in als mouse models but also in patients. given that oligodendrocytes not only facilitate saltatory conduction of action potentials by myelinating axons, but also support neuronal functions metabolically through monocarboxylate transporter 1 (mct1), the injury to oligodendroglia in als may have pathogenic consequences. methods. to further investigate whether oligodendrocytes play a role in als development, we selectively removed mutant human sod1 (g37r) from postnatal ng21 cells in pdgfar-creer;loxsod1(g37r) mice. results. the administration of 4-hydorxytamoxifen (4ht) caused significant decrease in the expression of mutant human sod1 transgene in ng2 glia as determined by quantitative pcr analysis. we found that excision of mutant hsod1 from ng2 glia dramatically delayed disease onset and early disease, and significantly prolonged animal survival. in addition, at disease onset stage, activated astroglial and microglial responses were delayed in the animals received 4ht treatment. moreover, the removal of mutant sod1 helped to preserve mct1 expression at disease onset. conclusions. these data indicate that expression of mutant sod1 in ng21 cells and their oligodendrocyte progeny has a deleterious effect on motor neuron survival, and suggest that a key negative consequence of mutant sod1 expression in oligodendrocytes is to diminish their capacity to provide metabolic support to neurons. recent studies suggest that innate immunity might be also involved in the pathogenesis of neurodegenerative diseases, cerebral ischemia and brain injury. although the recent works demonstrated that adaptive immune system is involved in the motor neuron disease process, the role of innate immune system in motor neuron disease was not fully investigated. to assess the contribution of innate immunity in the pathogenesis of amyotrophic lateral sclerosis (als), the gene expression profile of lumbar spinal cord from symptomatic mutant sod1 mice was obtained by microarray approach and subsequent pathway analysis indicated the involvement of innate immune pathway. next, to test the role of innate immunity in the pathogenesis of als, sod1 g93a als model mice were mated with myd88 and trif (tir domain-containing adaptor inducing ifnb) deficient mice. myd88 and trif are the essential adaptor proteins for toll-like receptor mediated signaling pathway. as compared with sod1 g93a mice, myd88/trif double-deficient and trif-deficient sod1 g93a mice exhibited the substantially shorter survival times with accelerated disease progression. the disease duration was shortened by 50% in trif-deficient sod1 g93a mice. in contrast, elimination of myd88 in sod1 g93a mice showed marginal effect in survival time. in addition, the expression levels of pro-inflammatory chemokines, ccl5 and cxcl-10 were significantly suppressed in the spinal cord of trifdeficient sod1 g93a mice, as compared with sod1 g93a mice. to determine the cell type in which trif-dependent pathway contributes to the production of these chemokines, we examined the expression of poster abstracts s75 glia chemokines in lps-stimulated primary microglia or astrocyte derived from trif-deficient or myd88-deficient mice. trif-dependent induction of these chemokines was observed only in lps-stimulated microglia. moreover, we found that infiltration of t-lymphocytes and other immune cells were significantly decreased in the spinal cord of symptomatic trif-deficient sod1 g93a mice. these results suggest that the basal level of trif-dependent innate immune activation of microglia is beneficial to slow disease progression of als models through the maintenance of pro-inflammatory chemokines and the infiltration of the immune cells to the spinal cords. the detailed analyses to clarify the role of these infiltrating immune cells are underway. alzheimer's disease (ad), the most common age-dependent neurodegenerative disorder, causes a chronically progressive decline in cognitive functions. there is growing evidence that glial changes are early involved in this pathology. pdapp mouse, a well-defined model of ad, accumulates toxic soluble and deposited ab, derived from proteolytic processing of the amyloid precursor protein (app) and develops adlike synaptic deficits and cognitive impairment. on the other hand, autophagy has been associated to the neurodegenerative process, in particular with the clearance of aggregation-prone proteins like ab. the amyloid plaques, mainly located in cortex and hippocampus, are closely surrounded by reactive and hypertrophic gfap1 astrocytes. the aim of this work was to evaluate the potential astrocyte autophagic activity during the progression of ad in pdapp mice from 5 to 20 months (m) of age. lc3ii/i ratio was studied by western blot. amyloid deposit load, stained with congo red, exhibited a continuing rise according to age in transgenic mice. the markers gfap and lc3 were analyzed by immunofluorescence and confocal microscopy on hippocampal sections showing an increasing colocalization that reached the top at 14 m, where 52.5 6 9.9% of plaque associated gfap cells were lc31. at 20 m, this proportion was lower. conversely, the subpopulation of astrocytes located far from ab deposits were gfap1/lc3-, besides a decreased cell volume compared with control mice astrocytes. additionally, the density of astroglial cells in the stratum radiatum diminished with aging (5 compared to 14 m, total gfap1 cells, p < 0.05) along with higher plaque load, in a more prominently manner than in control mice. our results clearly show that 1) astroglia is strongly affected during ad progression: two different morphologic subpopulations -close to or far from deposits-are distinguished in the hippocampus. aging correlates with a glial density reduction; 2) a gradual increase of autophagic activity in plaque associated-astrocytes is verified by the first time, suggesting a contribution to ab clearance until 14 m in pdapp mice, with a significant decay after this age. it also shows widespread neuron loss and gliosis in the brain. interestingly, in cstb-/-mice pronounced microglial activation has been detected in selected brain areas already in presymptomatic mice, preceding astrocytosis and neuronal death. in this study, we examine the microglial contribution to the neuronal dysfunction and death in epm1. we aim to characterize the functional properties of cstb-/-microglia and their effects on the survival of cstb-/-neurons. our results show that the cstb mrna level in cultured wild type mouse microglia analyzed by real time quantitative pcr is high in relation to primary astrocytes or neurons. a gene-expression profiling of microglia from cstb-/-mice and from wild type mice has been obtained by using the affymetrix mouse exon 1.0 st array and reveals a down-regulation of interferon-regulated pathways in cstb-/-microglia. the stimulation of primary wild type mouse microglia with the endotoxin lipopolysaccharide (lps) up-regulates cstb mrna expression measured by real time quantitative pcr. cstb-/-microglia show an altered inflammatory response to the stimulation with lps by increased secretion of chemokines demonstrated by cytokine array analyses. moreover, the release of nitric oxide from cstb-/-microglia measured by griess assay is elevated. our data indicate an altered response of primary cstb-/-microglia to inflammatory stimuli, which may contribute to neurodegeneration in epm1. the data provide a basis for further detailed studies on the pathophysiology and therapy of this devastating disease. activation of astrocytes and microglia surrounding amyloid extracellular plaques in the brain is a hallmark of alzheimer's disease (ad). increasing evidence suggests that chronic neuroinflammation and elevated levels of several cytokines play important roles in ad development. beside astro-and microgliosis, demyelination and oligodendrogenesis can be observed in human patients as well as in murine models of ad. oligodendrocyte progenitor cells, also termed ng2 cells because they express the neuron/glial antigen 2 (ng2) proteoglycan, comprise about 5% of total cell number of the adult brain and are the main proliferating cells present. ng2 cells receive gabaergic and glutamatergic synaptic input and can secrete pro-and antiinflammatory substances upon activation by microglia-derived cytokines. overall, ng2 cells are highly reactive cells and one of the first cells which respond to changes in the cns. however, their role during ad pathology is still uncompletely characterized. using immunohistochemistry, we have analyzed their expression pattern in a transgenic murine model of ad (coexpressing a mutated form of the amyloid precursor protein (app) and the presenilin1(ps1) gene). we focused on the spatial arrangement of ng2 cells in the hippocampus of transgenic mice at 3, 7 and 10 months of age and age-matched controls (n 5 4/ time point). our goal is to provide an anatomical and structural basis for elucidating the role of ng2 cells in ad. the first results indicate an uniform distribution of ng2 cells in the hippocampus of app/ps1 mice, with an accumulation at the sites of plaque deposits in close association with microglia. ng2 cells are normaly absent from the pyramidal and granular cell layers. however, when plaques deposits were found at these principal cell layers, ng2 cells could be seen surrounding them, suggesting an active migration. changes in ng2 cell phenotype were noted from 3 months of age on. like microglia, ng2 cells assume an activated morphology, as in a more "bushy" appearance. in numerous plaques, ng2 immunoreactivity was strongly increased, mostly in the intermingled processes that encircle the core of the plaques. whether these activated ng2 cells proliferate and differentiate into oligodendrocytes or communicate with activated microglia in the plaque microenvironment remains to be investigated. further, levels of the glutamatergic ampa receptor expression will be evaluated. ultimately we aim at understanding how ng2 cells and microglia interact in ad, which could be usefull in the development of novel therapies. accumulating evidence supports an increasing role of astrocytes in the initiation or progression of a variety of neuropathological conditions. in alzheimer's disease (ad), numerous data indicate that astrocyte properties are modified with potential deleterious effects on neurons. accordingly, we have described changes in the expression of astroglial connexins, the gap junction channel and hemichannel forming proteins, in the vicinity of amyloid-b (ab) plaques in brains from ad patients and murine models of ad. also ab peptide was shown to trigger astroglial cx43 hemichannel activation in culture and in acute slices leading to neuronal degeneration. hence, we have investigated hemichannel function of astroglial connexins in 8-9 months old app swe /ps1 de9 mice that exhibit ab plaques in the cortex and hippocampus. ethidium bromide (etbr) uptake performed in acute brain hemisphere slices was used as an index of hemichannel activation and was quantified in astrocytes identified by gfap immunostaining. in app swe /ps1 de9 mice, compared to wildtype mice of the same age, an increased uptake of etbr was detected in the overall population of astrocytes. this uptake was blocked by carbenoxolone and lanthanum ions, indicating connexin hemichannel involvement. such activation may be linked to the increase in resting astroglial [ca 21 ] i described in this mouse model. interestingly, etbr uptake was higher in reactive astrocytes contacting ab plaques than in non-reactive astrocytes located far from (! 50 mm) these deposits. we are currently investigating their respective pharmacological profiles. moreover, in app swe /ps1 de9 mice knock-out for cx30 generated in our facility, etbr uptake was comparable to that observed in app swe /ps1 de9 mice, suggesting that cx43 is the major contributor to the hemichannel activity observed. altogether, these results indicate that neuroglial interactions could be affected by astroglial hemichannel activation and could account for neuronal alterations or death observed in neurodegenerative diseases. since it is well established that amyloid plaques are associated with activated astrocytes we asked whether concentrations of the astrocyte-derived proteins glial fibrillary acidic protein (gfap) and s100b in human csf might serve as additional biomarkers. to functionally link the role of astrocytes to mechanisms involved in memory formation, we asked whether the amount of gfap-positive astrocytes correlates to the level of ltp in the hippocampus of aged ad transgenic mice. we used elisa kits for the quantitative analysis of gfap (ibl-international, hamburg, germany) and s100b (ibl). in a mixed disease cohort (n 5 52 diseased patients, n 5 16 controls), we correlated standard biomarkers (abeta and tau-protein) and gliaderived proteins. furthermore we compared mean levels between groups of ad patients (n 5 36) and healthy control patients (n 5 35) and correlated levels of astroglial proteins in the csf and cognitive performance (mmst values). in transgenic (app/ps1 transgenic mice), aged (10-15 months) mice (n 5 10), we performed ltp experiments at the schaffer collateral synapses in the ca1 region of the hippocampus. the number of gfap-positive astrocytes was quantified contralaterally. results: in the mixed cohort, we found a significant correlation of t-tau and gfap levels in csf (r 5 0.261, pbetween the s100b levels and the patients cognitive performance as measured by the mmst values (r 5 -0,42) as well as between the gfap levels and the mmst values (r 5 -0,38). when correlating the number of astrocytes in the ca1 region to the level of ltp, 30 min after induction, we found a significant correlation of the number of astrocytes per mm 3 to the level of ltp (r 5 0,648, p conclusions: the astrocyte-derived proteins gfap and s100b can be reliably detected in human csf. measurement of astroglial biomarkers might allow an improved pathobiological staging of ad, also since astrocytes appear to play a functional role in memory formation in the context of an ad-like pathology in mice. retinitis pigmentosa is a group of inherited disorders affecting photoreceptors or retinal pigment epithelium (rpe) that leads to progressive loss of vision. the pde6b rd1 mouse model is a very well-known animal model for retinal degeneration, in which rods carry a mutation in b subunit of rod cgmp-phosphodiesterease. glial cell line-derived neurotrophic factor (gdnf) has already been shown to rescue morphology as well as function of rod cells in pde6b rd1 mouse. this effect was indirect, through stimulation of m€ uller glial cells (rmg). to better understand the neuroprotective effect of gdnf, primary retinal rmg were stimulated in vitro with gdnf and the secreted proteome was analyzed by proteome profiler arrays. among others, cyr61/ccn1, a member of ccn family, was found strongly induced upon rmg stimulation with gdnf. when applied directly to medium, cyr61 significantly reduced photoreceptor death in organotypic ex vivo cultures of pde6b rd1 retinas. in order to identify the target cells of cyr61 we treated, arpe19 and mio-m1 cell lines with cyr61, and observed an increase in phosphorylation of akt and erk1/2 signaling molecules. these results suggest that stimulation of rpe and rmg cells may have a protective influence on photoreceptor survival in organotypic ex vivo conditions. we postulate cyr61 as a novel potential candidate for future therapeutic approaches in neurodegenerative retinal disorders. since astrocytes react to multiple physiological and pathological stimuli in the microenvironment of the brain, they could be mediators for gene x environment interactions in the pathogenesis of psychiatric disorders. altered gene/protein expression and regressive changes have been reported for astrocytes in post-mortem studies of psychiatric disorders, i.e. schizophrenia (scz), bipolar disorder (bp) and autism spectrum disorders (asd). by contrast, no genetic link to astrocytes has emerged from the extensive genomic analysis of psychiatric disorders. genetic findings mostly relate to neurons for disorders rooted in neurodevelopment (asd, scz). it is timely to perform a formal analysis of genomic information emerging for psychiatric disorders for a contribution of genes expressed by astrocytes. methods: we generated a meta-list of genes highly expressed in rodent astrocytes using published gene lists and literature mining. datasets were prepared for candidate genes and genes from gwas in attention deficit/hyperactivity disorder (adhd), asd, bp and scz, by using literature and databases (szgene, sfari base, dbgap). datasets for copy number variations (cnvs) associated with neurodevelopmental delay were also assembled. then the overlap between the meta-list of genes highly expressed in astrocytes and gene datasets for psychiatric disease was determined. overlapping genes were pooled and subjected to david bioinformatics analysis. results: the meta-list of genes highly expressed in astrocytes contained n 5 2,680 genes (13.4% of the genome). datasets for risk genes in psychiatric disorders showed random overlap with the meta-list (adhd 18%; asd 13%; bp 12%; scz 13%). cnvs related to neurodevelopment were not enriched for astrocytic genes (8%). when the overlapping genes were pooled from all disorders, a small set of shared astrocytic genes emerged (n 5 23). cntnap2, nrxn1 and sdc2 were linked by david analysis (p 5 0.013). conclusions: this correlative analysis makes it unlikely that genes highly expressed in astrocytes make a major contribution to the genetic risk in four major psychiatric disorders. no genetic link was found between astrocytes and neurodevelopmental delay. changes in gene/ protein expression and pathology in astrocytes in the adult brain in scz and bp may be explained by chronic neuronal dysfunction, chronic medication or acute changes. the neuronal ceroid lipofuscinoses (ncls, batten disease) are a group of autosomal recessively inherited lysosomal storage disorders affecting children and young adults, each of which is caused by a mutation in a different gene. a common feature across all ncls is the early, localised glial activation that occurs long before the onset of neuron loss. this glial activation appears to be an accurate predictor of which neuron populations are vulnerable and will subsequently die. both astrocytes and microglia express the ncl gene products and it is likely that normal glial cell function may be compromised. given the close functional relationship between neurons and glial cells it is possible that glial activation and/or dysfunction may affect neuronal health. we have begun to explore the biology of astrocytes and microglia in the juvenile form of ncl using primary cultures from cln3 -/mice. defects in both astrocytes and microglia, including altered protein secretion profiles and impaired morphological and proliferative responses were apparent. co-culturing with mutant microglia and astrocytes influenced the survival and morphology of wild type neurons, with more profound effects upon cln3 -/neurons. intriguingly, these effects were largely reversed by substituting wild type glia, which rescue mutant neurons. a pilot study has provided similar evidence for glial dysfunction in infantile ncl, including and altered protein secretion and response to stimulation. these changes appear to be different from those observed in juvenile ncl and we are in the process of exploring whether the glial cell phenotypes discovered so far, and their impact upon neurons are specific to the different forms of the disease. the accumulation of aggregated proteins in nerve cells and glia underlie the pathogenesis of many neurodegenerative diseases. glial pathology is characteristically observed in tauopathies, such as corticobasal degeneration and progressive supranuclear palsy, and alexander disease, a primary disease of astrocytes caused by mutations in the gfap (glial fibrillary acidic protein) gene. irreversibly damaged proteins can be degraded by the proteasomal system. for proteasomal degradation they are covalently linked to ubiquitin in a three-step enzymatic pathway (e1, ubiquitin-activating enzyme; e2, ubiquitin-conjugating enzyme; and e3 ubiquitin ligases). the polyubiquitin chain needs to be removed before the translocation of the substrates to the lumen of the proteasome. this is achieved by deubiquitinating enzymes (dubs), which oppose the functions of e3-ligases and assist in targeting substrates to specific pathways. to assess whether impairment of dubs may contribute to age-related neurodegenerative diseases and the regulation of cell death and survival, we have subjected primary cultures of rat brain astrocytes to pr-619, a dub inhibitor with broad specificity. immunoblot analysis demonstrates that after a 18h treatment, pr-619 caused the induction of heat shock proteins, hsp70 and hsp32, and an increase in p62, which is considered a cargo receptor for ubiquitinated proteins and a common constituent of ubiquitinated protein inclusions. as analyzed by indirect immunofluorescence, protein aggregates positively stained by antibodies against ubiquitin and p62 assembled in the perinuclear region at the microtubule organizing center (mtoc). these aggregates were surrounded by a cage-like network of gfap intermediate filaments, thus resembling aggresomes. using mitotracker and lysotracker staining procedures, the fluorescent images demonstrate that after dub inhibition lysosomes and mitochondria are recruited to the mtoc. this process was dependent on an intact microtubule network, since it was interrupted after treatment with nocodazole, a microtubule destabilization drug. furthermore, pr-619 caused mitochondrial fragmentation which may be a stress response to enable the removal of damaged mitochondria by mitophagy. in conclusion, dub inhibition impairs the cellular architecture, leads to protein aggregate formation and mitochondrial alterations in astrocytes. our data sustain the hypothesis that an imbalance in dub activities may contribute to the pathogenesis of neurodegenerative diseases. shown that the collapsin response mediator protein 2 (crmp-2), which play a significant physiological role in neuronal cell bodies and axons within the cns, is phosphorylated during the neurodegenerative phase of the inflammatory disease. methods: we investigated the limitation of axonal degeneration by transducing retinal ganglion neurons with a phosphorylation mutant of crmp-2 utilising an intraocular adeno-associated virus 2 (aav2) delivery system in eae-induced mice. the other group of mice injected with aav2 consisting of the green flourescent protein reporter only (aav2-gfp) with eae was used as a control (n 5 8 per each group). results: we showed substantial preservation of axons of the optic nerve in the eae-induced mice injected with the aav2 carrying the crmp-2 phospho-mutant compared ($10-fold difference) with those mice injected with aav2-gfp. the aav2-gfp transduced axons showed significant degeneration during the peak stage of eae. conclusion: our data suggest that phosphorylation of crmp-2 may be a central mechanism that governs axonal degeneration during inflammatory demyelination of the cns (as occurs in ms) and inhibition of phosphorylation of crmp-2 may be of therapeutic potential for the progressive phase of the disease in ms patients. y. shinozaki, s. koizumi univ. of yamanashi, yamanashi, japan atp, a major gliotransmitter integrating neuron-glia networks, is known to be released or leaked from injured cells. in the cns, atp dramatically changes glial phenotypesand could affect pathogenesis of brain disorders. however, whether such glial responses facilitate or rather inhibit the diseases is still under debate. to address this issue,we studied the neuroprotective role of atp/purinergic signaling using in vivo stab injury model on mouse cerebral cortex. without injury or the contralateral side of the injured brain exhibited no fluoro-jade(fj)-positive neuronal death, cd45 1 infiltrating leukocytes or gfap 1 reactive astrocytes. three days after injury, fj signals, cd45 1 cells and reactive astrocytes were increased in the ipsilateral side of the brain. the cd45 1 cells were enclosed by reactive astrocytes-formed glial scar. administration of an atp-degrading enzyme apyrase, a broad p2 receptor antagonist ppads, and a p2y 1 receptor antagonist mrs2179 significantly reduced the stabincreased fj signals. immunohistochemical analysis revealed p2y 1 receptor was expressed in gfap 1 astrocytes. p2y 1 receptor knockout (p2y 1 ko) mice also exhibited reduced fj signals and infiltration of cd45 1 cells. they exhibited higher level of gfap expression, more remarkable hypertrophy of astrocytes and tighter glial scar formation than wild type mice did. the accelerated glial scar formation reduced cd45 1 cell number and inhibition of astrocytes by fluorocitrate increased cd45 1 cell number. although glial scar formation was accelerated, no enhanced proliferation was observed. we then analyzed the mechanism of enhanced glial scar formation and neuroprotection using in vitro scratch-wound model. in cultured astrocytes, the scratch induced a transient increase in extracellular atp, which was followed by decrease in extracellular atp mainly due to an increase in ectoatpase activity. suppression of purinergic signaling by either apyrase, ppads or mrs2179 enhanced scratchevoked astrocytic migration rather than proliferation. neither activation nor inhibition of p2y 1 receptors in cultured cortical neurons affected the scratch-induced damages. our data suggest that the decrease in atp/ p2y 1 receptor-mediated signal should be a trigger that transforms astrocytes into migratory phenotype, which forms tighter glial scar structure thereby suppressing neuronal death. background: oligodendrocyte damage and loss are key features of multiple sclerosis (ms) pathology and oligodendrocytes appear to be particularly vulnerable to ros. in vitro studies showed that ros induce cell death and prevent the differentiation of oligodendrocyte precursor cells (opcs) into mature myelin-producing oligodendrocytes. hence, a potential therapeutic strategy to protect these cells from ros-mediated damage is urgently needed. here we investigated the efficacy of several compounds that are able to boost antioxidant enzyme production, including monomethyl fumarate (mmf), tert-butylhydroquinone (tbhq), sulforaphane (sfn) and protandim. these compounds are thought to exert their protective function via activation of the nuclear-factor-e2-related factor-2 (nrf2) transcriptional pathway, which is involved in the production of antioxidant enzymes necessary for oxidative stress defense. methods: primary rat oligodendrocytes were treated with different concentrations of mmf, tbhq, sfn and protandim. expression of antioxidant enzymes were analyzed by pcr and western blot analyses. to study the beneficial effects of the different nrf2 activators, oligodendrocytes were first incubated with nrf2 activators and subsequently exposed to various concentrations of hydrogen peroxide. oligodendrocyte cell survival was measured by a live/dead cell viability assay. results: sfn, mmf and protandim are well-tolerated and induce nrf2driven antioxidant enzyme production in oligodendrocytes. protandim was the most potent compound with regard to antioxidant enzyme induction and protected oligodendrocytes against ros-induced cytotoxicity. conclusions: our findings indicate that several nrf2 activators are able to induce antioxidant enzyme production in oligodendrocytes. interestingly, protandim, a dietary supplement consisting of herbal ingredients, was the most potent protector of primary rat oligodendrocytes. in future experiments we will determine whether protandim can also promote the differentiation of opcs under oxidative stress and test the clinical efficacy of protandim in an experimental animal model for ms. the arising of the "tripartite synapse" concept and the discovery of the astrocytic excitability have been highlighting astrocytes as active elements concerning information flow in the brain. however, the impact of such remarkable features in complex brain function is still under-explored mostly due to the difficulty of studying neuron-astrocyte interactions in vivo. the aim of this work was to use an in vivo model of astrocytic dysfunction and investigate the impact of this treatment in complex cognitive functions. the rationale consisted in studying behavioral performance that relies on the prefrontal cortex, such as behavioral flexibility and working memory in an animal model of astrocytic dysfunction. for that purpose, we used a pharmacological model in which wistar-han rats were subjected to bilateral intracranial injections of aminoadipate in the prelimbic portion of the medial prefrontal cortex to cause astrocyte depletion specifically in this region, mimicking pathological states such as depression, in which marked decreases of gfappositive cells are observed. this animal model was tested for its cognitive abilities by the attentional set-shifting task (asst) and water maze-based tests. a clear impairment of cognitive function was observed in the animals treated with aminoadipate. a detailed morphological and histological analysis showed that along with the astrocytic lesion, neurons were also affected at the site of lesion. this seems to contribute to the cognitive decline observed in this model and may explain similar observations under pathological states. a. sternotte, e. hermans universit e catholique de louvain, brussels, belgium amyotrophic lateral sclerosis (als) is an adult disease characterized by a selective loss of motor neurons, resulting in progressive paralysis and death within 3-5 years. in several inherited cases, the disease is caused by point mutations in the gene encoding for superoxide dismutase 1 (sod1) which promotes its aggregation, leading to biochemical damages, including mitochondrial dysfunction. alteration of mitochondrial membrane potential and/or respiratory chain leads to atp depletion and these metabolic dysfunctions could contribute to motor neuron death in patients and animal models of als. in motor-neurons, the combination of altered axonal transport with mitochondrial dysfunction likely leads to considerable decrease in atp levels at distant neuromuscular junctions. commonly know as the fuel gauge of mammalian cells, amp-activated protein kinase (ampk) is a key enzyme in the control of cellular atp and energy homeostasis. in this study, the implication of ampk in the progression of als was specifically investigated by measuring the expression and activity of this enzyme in the spinal cord of mice overexpressing the mutated human sod1 (hsod1 g93a ), a commonly used experimental model of als. no consistent modification of the enzyme was detected in spinal cord samples throughout the progression of als. indeed, such experiment did not discriminate between cell types present in the tissue. hence, a more detailed characterization of ampk in cultured astrocytes derived from als animals revealed a robust induction of the enzyme activity, which correlates with decreased atp levels in these cells. in a second part of our study, we have investigated the consequences of ablating ampk in the mouse model of als by breeding ampk knock out mice with hsod1 g93a mice. while we did not detect any difference in the lifespan of ampk(-/-)/hsod1 g93a mice as compared to hsod1 g93a mice, we observed substantial alterations in the progression of the disease in selected behavioural tests. in particular, analysis of the gait (catwalk) which enables early detection of muscle weakness revealed that the onset was delayed by up to 30 days while the progression of the disease after onset was accelerated. further studies will be conducted to establish the importance of atp-in the disease, and to validate this enzyme as a putative pharmacological target in als and in other neurodegenerative diseases involving mitochondrial dysfunction. institute of neuroscience, sibs, cas, shanghai, china ng2 glia is the fourth type of neuroglia in vertebrate nervous system, which also known as oligodendrocyte progenitor cell (opc) in a developmental point of view. in adult brain, ng2 glia has a small cell body with highly branched extensions and represents a new type of glial cell differing from other glial cell types such as astrocyte, microglia and mature oligodendrocytes. published studies by others have shown that ng2 glia is able to promptly respond to brain injury and activated in several neurodegenerative disease animal models including 6-ohdainduced rat parkinson's disease model. however, the pattern and function of these activated ng2 glia remain largely unknown. here, we performed a time-course study of ng2 glia activation in mptp mouse model of parkinson's disease using immunohistochemistry. it was found that ng2 glia was activated as early as 24 hours after the last mptp injection in the substantia nigra (sn) of mptp-treated mice. this temporal character is very similar to those of microglia, indicating ng2 glia respond to mptp challenges very quickly. the activation of ng2 glia became stronger over time and reached the peak at about 5 days after the final mptp injection. then, the extent of ng2 glia activation declined gradually and diminished 10 days after mptp injection. interestingly, we found that prominent ng2 glia activation could only be observed in the substantia nigra. the dorsolateral striatum which is the projection target of nigral dopaminergic neurons, however, was devoid of ng2 glia activation in all the time-points examined. in summary, we characterized the ng2 glia activation in mptp mouse model of parkinson's disease. these data suggest that activated ng2 glia may play a role in the degenerative process of dopaminergic neurons in pd. hallmarks of cns inflammation, including microglial and astrocyte activation, are a feature of post-mortem tissue from amyotrophic lateral sclerosis (als) patients and in transgenic mice overexpressing mutant superoxide dismutase-1 (sod1 g93a ). administration of glucocorticoids does not significantly alter disease progression, but this may reflect poor cns delivery. here, we sought to discover whether cns-targeted liposomally-packaged glucocorticoid would inhibit the cns inflammatory response and reduce motor neuron loss. sod1 g93a mice were treated with saline, free methylprednisolone (mp, 4mg/kg/week) or glutathione pegylated liposomal mp (2b3-201, 4mg/kg/week) and compared to saline treated wild-type animals. animals were treated weekly with intravenous injections for 9 weeks from 60 days of age. animal weights and motor behaviour were monitored during this period. at the end of the experimental paradigm (116 days) mice were imaged using t 2 -weighted mri for brainstem pathology, and brain and spinal cord tissue was collected for histological analysis. all sod1 g93a animals showed a significant decrease in motor performance compared to baseline from $100 days. sod1 g93a animals showed a significant increase in signal intensity on t 2 weighted mr images, which may reflect the combination of neuronal vacuolation and glial activation in these motor nuclei. treatment with 2b3-201, but not free mp, significantly reduced t 2 hyperintensity, which correlated with significantly reduced histopathological manifestations in the brainstem motor nuclei, but not the spinal cord. interestingly, there was a significant reduction in astrogliosis but not microglial activation following treatment with 2b3-201. the results of this work indicated that the cns-targeted anti-inflammatory agent 2b3-201 has therapeutic potential in als. the toxic effects of new antiretroviral drugs on the central nervous system (cns) are unclear. because these drugs penetrate the brain even at low concentrations, it becomes crucial to determine the doses which can be toxic for the cns resident cells. moreover, after the recent introduction into clinical practice, it is unclear whether the efficacy of the antiretroviral drugs of new generation may also derive from their ability to exert extravirological effects on factors responsible for the development of hiv brain injury, e.g. matrix metalloproteinases (mmps). objective: to investigate on the toxicity of four different antiretroviral drugs and their ability to modulate the expression of gelatinase b (mmp-9) in astrocyte cultures. methods: primary cultures of rat astrocytes were activated by exposure to 10 mg/ml lipopolysaccaride (lps) (positive control) and simultaneously treated for 20 h with increasing doses (1-5-10-25. 50 mm) of: efavirenz (efv); darunavir (drv); maraviroc (mvc) or raltegravir (ral). mmp-9 mrna expression was assessed by rt-pcr. quantitative determination of mmp-9 expression was done by computerized scanning densitometry. single drug toxicity was assessed by the mtt test. each drug was considered toxic at the concentration able to induce a percentage of cell survival above 60%. results: the treatment with antiretroviral drugs inhibited mmp-9 mrna expression in lps-activated astrocytes in a dose-dependent manner. in particular, a statistically significant inhibition of mmp-9 expression was observed when astrocytes were treated with 25mm efv (54% of inhibition) or with 50 mm ral (43% of inhibition). as assessed by the mtt test, the toxicity of the antiretrovirals ranges from 10 and 50 mm. in particular, efv was toxic for astrocytes at the concentration of 25 mm, mvc at 10 mm, while drv and ral were toxic at the concentration of 50mm. conclusions: the present results indicate that efv and ral directly inhibit mmp-9 expression in lps-activated astrocytes with mechanisms that are independent from their antiviral activity. the toxic doses of antiretrovirals are much higher than those found in the csf of hiv-positive patients. our results highlight some beneficial/deleterious extra-viral effects of the antiretroviral drugs that may be useful to improve the development of new therapeutic strategies for the management of hiv infection. we used a parahippocampal kainate injection mouse model to induce mtle-hs and to study early expression patterns of kir4.1 and apq4 after se. mice were sacrificed 24 h, 72 h and 7d after kainate injection. immunhistochemistry of the hippocampus was performed to assess kir4.1 and aqp4 expression, and gfap staining was used for identification of astrocytes. gfap expression was increased compared to saline injected controls, suggesting that kainate injection induces astrogliosis. comparison of kir4.1 and aqp4 staining showed a similar change in the expression pattern. our data indicate a drop in kir4.1 and aqp4 expression within 24 hrs after se followed by an upregulation within the first week. altered expression of kir4.1 and aqp4 could contribute to dysregulation of potassium and water homeostasis and play a role in the early phase of epileptogenesis. c. l€ o€ ov, a. erlandsson uppsala university, neuroscience/neurosurgery, uppsala, sweden we have previously shown that astrocytes effectively engulf dead cells after trauma both in vitro and in vivo, but that they store the ingested material rather than degrade it. compared to macrophages, which degrade engulfed, dead cells within hours, our data show that astrocytes store the ingested material for weeks before the degradation is completed. to further study the routes of degradation and the possibilities to speed up this process we have used a cell culture model where uvtreated, dead cells are added to stem cell-derived astrocytes. by labeling the dead cells with the ph-sensitive dye phrodo prior to the engulfment, we demonstrate that the ph in the astrocytic phago-lysosomes is higher than in professional phagocytes. interestingly, lamp1 and lamp2, which are involved in the phago-lysosome fusion, are both highly expressed in the astrocytes, particularly around the ingested material. dendritic cells are known to express the inhibitory protein rab27a that slow down the degradation in order to preserve antigen for presentation. rab27a prolongs the actin coating around the phagosomes, which physically inhibit the phago-lysosome fusion, and interacts with nox-2, which leads to an increased consumption of protons. western blot analysis shows a high expression of rab27a in our cell cultures which may explain the slow degradation. moreover, dead cells in astrocytes are surrounded by actin rings for a long period of time after the ingestion. to counteract this, the astrocytes were treated with 0.1 or 1 mm of the actin inhibitor latrunculin b. the number of actin rings were significantly lower in the cultures treated with 1 mm of latrunculin b compared to controls, but the intensity of phrodo was unaltered indicating that the prolonged degradation may be due to a higher ph in the lysosomes rather than a delayed actin coating. next we will investigate the effect of rab27a expression on the degradation process by performing sirna experiments. one of the promising strategies for the treatment of spinal cord injury is the transplantation of glial cells obtained from the olfactory system; olfactory ensheathing cells (oecs). effective proliferation and migration of oecs are essential for optimizing clinical applications and there is a need for identification of small molecules that regulate glial cell biology. curcumin is a natural polyphenol compound found in the spice turmeric, which is known for its neuro-protective properties and has been reported to have an effect on neurogenesis and nerve regeneration. however, the effect of curcumin on oecs has not been determined. we have examined the effect of curcumin on oecs at the cellular level using fluorescence and timelapse microscopy. oecs were purified from s100b-dsred transgenic mice in which oecs express the fluorescent protein dsred. different treatments: (i) control medium, (ii) curcumin (0.5 to 20 mm), (iii) g5 commercial growth factor, or (iv) a combination of g5 and curcumin were used to determine the effect of curcumin on oecs proliferation and migration. cell proliferation was quantified at two different times by cell counting and mts proliferation assay. timelapse microscopy was used to visualize changes in cell morphology and cell migration. cell branching, number and area of lamellipodia and speed of migration per cell were measured for each treatment. we found that lower concentrations of curcumin (0.5 and 1 mm) increase oec proliferation. as well, combination of g5 and curcumin showed the highest proliferative effect on oecs suggesting a possible synergistic relation between g5 and curcumin. live cell imaging analysis showed that curcumin increased the number and area of lamellipodia resulting in faster migration of oecs. these results suggest that curcumin can regulate the proliferation, morphology and migration of oecs which could improve the therapeutic use of oecs for spinal cord injury repair. methods: the dams were divided in four groups and had received 50 ml of nonalcoholic or alcoholic beer solution -control, vehicle (nonalcoholic solution), etoh 5% (nonalcoholic solution 1 5%v.v ethanol) or etoh 10% (nonalcoholic solution 1 10%v.v ethanol) during gestational day 1 up to weaning (postnatal day 22). male offspring (30 -35 days old) were sacrificed and hippocampal acute slices were prepared. we analyzed gfap content, s100b and glutamate uptake as astrocyte parameters. we tested the animals in the plus-maze discriminative avoidance task to check anxiety and memory. results: gfap content decreased after pnee for etoh 5% and etoh 10% treatment (f 3,59 5 17.75, p < 0.0001). interestingly, we found that only the etoh 10% treatment showed an increase of hippocampal s100b content (t 5 2.38, p 5 0.02) and in the csf (f 3,18 5 3.249, p 5 0.0462), but no difference in secretion. glutamate uptake was significantly decreased for etoh 10% group (f 3,20 5 4.069, p 5 0.0208). to perform the plus-maze discriminative avoidance task we selected pups from control, vehicle and etoh 10% treatment on pnd 30. two-way anova revealed significant effects of treatment (f 2,46 5 4.32, p 5 0.019), arm type (f 1,46 5 103.9, p < 0.0001) and treatment x arm type interaction (f 2,46 5 6.66, p < 0.01). in the test session, only a significant prenatal treatment group effect (f 2,46 5 6.44 p < 0.01). bonferroni's post-hoc test revealed that only control group presented significantly less time in the enclosed aversive arm than nonaversive one. conclusions: in this study we showed that pnee with moderate doses could alter the structure and functional astrocytes balance. thus, high levels of s100b are indicated in some neurodegenerative disorders. taken this data together we could demonstrate that moderate ethanol exposure can be harmful to fetal brain. aging has been associated to neuroinflammation in the central nervous system, however it is not known whether microglial changes induced by aging are affected by early effects, such as litter size and sedentary life style. in addition, the lateral septum has been recognized by its complementary role in the memory processing for tasks of object recognition for identity and spatial location. in the present report, we investigated whether aging cognitive decline and microglial morphological changes in the lateral septal region are influenced by changing litter size early in life and by a sedentary life style. to assess these questions, wistar rats suckled in litters of either six or 12 pups per dam were raised sedentarily in groups of 2 up to 3, from the 21 st post-natal day onwards. at 4 (young adult) or 23 (aged) months-old, half of the sedentary rats underwent progressive daily treadmill exercise for five weeks, while the others remained sedentary. after performing the tests of recognition for spatial localization and object identity all of the animals were sacrificed and their brains were processed for selective microglia/macrophages immunolabeling with anti-iba-1 antibodies. a representative sample of the immunolabeled cells in the lateral septum was analyzed after three-dimensional reconstruction with neurolucida software (microbright field inc.) and morphological features of each cell were quantified by neuroexplorer (microbright field inc.). it was found that sedentary life style of wistar rats maintained in standard laboratory cages is associated with spatial memory deficits in both mature and aged subjects no matter the litter size, and that exercise decreased these effects in aged subjects raised in small but not in large litters. on the other hand, all sedentary aged rats, despite of the litter size, presented impairment of object recognition memory for identity, and exercise decreased this effect in animals from both large and small litter size groups. microglial morphological analysis revealed that cell soma area, perimeter and branches volume seem to be more intensely affected by aging and that these changes are mainly associated with animals raised in large litters. furthermore, it was observed important shrinkage and thickening of the microglial branches in aged individuals on a higher proportion in the sedentary group suckled in large litters. taken together the results suggest that litter size and sedentary life style may affect object recognition and spatial memories in both adult and aged rats and that exercise minimizes these effects on animals suckled in small but not larger litters. in addition, we suggest that these changes are related to distinct effects on the soma and branching patterns of septal microglia from young and aged subjects. huntington disease (hd) is a dominant inherited neurodegenerative disorder caused by an unstable expansion of a cag repeat within the gene encoding for huntingtin protein (htt). this mutation induces formation of a poly-glutamine tract in the mutant htt (mhtt) protein that prones its aggregation into the cells. hd is characterized by an initial and massive degeneration of the medium spiny neurons in the striatum with later neuronal loss in cortex, globus pallidus, and other structures leading to motor and cognitive symptoms. alterations of energy metabolism contribute to hd pathogenesis but the mechanisms responsible for these alterations are not well understood. a recent pet study provided evidence for a selective impairment of striatal glycolytic and not oxidative metabolism of pre-symptomatic hd patients. in the brain, glycolysis is predominantly an astrocytic metabolic process, whereas oxidative metabolism is primarily neuronal raising the question that metabolic defects in hd could originates from astrocytes. the purpose of our study was to characterize (1) the metabolic alterations in vivo in a transgenic mouse model of hd (bachd mice) and (2) the respective roles of astrocytes and neurons in metabolic defects occurring in hd using an in vitro strategy. bachd mice express the human full length htt protein with 97 glutamine repetitions under the human htt promoter into a bacterial artificial chromosome. bachd mice present a slow disease progression with motor deficits occurring at 6 months and progressive neuronal aggregates from 12 months, reflecting human pathology. first, we performed [14c]-2-deoxyglucose autoradiography experiments in vivo to assess energy metabolism in 14 months bachd (n 5 6) and wt (n 5 6) mice. we performed a 3d analysis of the whole brain glucose uptake without any regional a priori. using statistical parametric mapping (spm), a voxel-wise statistical analysis approach, we showed that compared to age-matched wt mice, 14 months bachd mice present hypometabolism in the striatum, like pre symptomatic hd patients, and also in the hippocampus. hypermetabolism was also detected in the hypothalamus. second, we performed an in vitro study to dissect out the cellular mechanisms responsible for these metabolic changes. by using cell insert, we realized neurons/astrocytes co-culture without any physical contact. this culture system allowed us to grow neurons in presence of wt or bachd astrocytes. we showed that both bachd and wt neurons have a decreased [14c]-2-deoxyglucose uptake when cultured in presence of bachd astrocytes but not wt astrocytes. these results collectively suggest that energy defects in bachd mice are due to noncell autonomous mechanisms by which astrocytes expressing mhtt induce neuronal metabolic dysfunction. naag and naa are abundant metabolites in the brain which can be utilised by oligodendrocytes to make myelin. but elevated levels of naag and naa are associated with myelin loss in the leukodystophies canavan and pelizaeus-merzbacher-like disease, whereas lower levels of naa are observed in brain tissue of ms patients. currently, the physiological function of these two metabolites as well as their role in white matter pathology is unknown. naag and naa can also act as signalling molecules as they can affect glutamate receptors. oligodendrocyte precursor cells (opcs) express nmda receptors and activation of these receptors may be important for their differentiation and efficient myelination. in disease opcs are recruited to demyelinating lesions where they proliferate and differentiate into new myelinating oligodendrocytes. we therefore investigated the effects of both physiological and pathological concentrations of naa and naag on opc biology in vitro. we used calcium imaging to see whether naag or naa can induce intracellular calcium changes in opcs as they are known to act on neuronal nmda receptors. 1mm naa or 1mm naag evoked intracellular calcium changes in opcs (df[340/380] 44.6 6 0.7 3 10 23 and 9.0 6 3 3 10 23 respectively), but only one third of the cells responded which corresponds to the proportion of nmda responsive cells in the cultures. however, not all opcs that responded to nmda did also respond to naag or naa. therefore, it seems that the naa and naag evoked calcium raise is via an nmda receptor independent pathway. naag and naa evoked a small but significant increase in intracellular calcium in opcs, which even after prolonged application of pathological levels of naa and naag did not affect the cells' viability as propidium iodide staining of opcs showed no significant difference in viability. over 90% of the opcs survived 4hrs application of 1mm naa and 1mm naag. naag and naa are produced and released by neurons but it is not known how opcs take them up from the environment and how it affects them. we therefore addressed which possible transporters opcs express and the effect naa and naag have on opcs' proliferation and differentiation. it is unlikely that naa and naag even at high concentrations have a detrimental effect on opcs. perhaps these high concentrations of naa and naag found in canavan and pelizaeus-merzbacher-like disease indicate rather opcs' deficiency to transport and utilise naa and naag for energy and myelin formation. vanishing white matter (vwm) disease is a severe leukoencephalopathy, classically starting in childhood. the disease is characterized by neurological symptoms, including uncoordinated movements and balance disturbances which are slowly progressive. episodes of rapid deterioration are triggered by febrile infections and minor head trauma, which can lead to coma. a treatment is unavailable and eventually all patients die within a few years upon diagnosis. the disease is caused by mutations in any of the five genes encoding the subunits of the eukaryotic translation initiation factor 2b (eif2b). although this protein has an important function in all cells, mainly the cells in the brain white matter (astrocytes and oligodendrocytes) are affected by these mutations. pathologically the brain shows cystic degeneration with meager gliosis, dysmorphic astrocytes, lack of myelin and an increased number of oligodendrocyte and astrocyte progenitor cells. we have developed two mouse models for vwm to further unravel the disease mechanisms at molecular level and to develop and test possible treatments. the 2b4 mouse line is homozygous for a mutation in the eif2b4 gene, encoding the eif2bd subunit, and the 2b5 mouse line is homozygous for a mutation in the eif2b5 gene, encoding the eif2be subunit. both mutations have been described in human patients and are associated with a severe form of vwm. preliminary data show that these mice models recapitulate the clinical and neuropathological phenotype observed in vwm patients. pathologically, the central nervous system white matter is vacuolated and shows lack of myelin while astrocytes are dysmorphic and immature. these transgenic mice appear to be a representative model for vwm. microglial cell lines were differentiated from ips cells and represent in vitro models for human microglia. rt-pcr and flow cytometry analysis of ipsdm showed gene transcription and protein expression of cd33 respectively. the protein expression was increased by breaking the cis-interacting sialic acids on the microglial glycocalyx using sialidases. cd33-fc fusion protein was designed and purified via the fc tag to study binding patterns of cd33 to various cell types expressing different glycocalyx structures. additionally, ipsdm exhibiting either an over expression or knockdown of cd33 will be used to determine protein functions. furthermore, preliminary immunohistochemical analysis of control brain tissue compared to ad patients showed increased cd33 protein expression in cd68 positive cells, in the latter. in summary, data show that cd33 is expressed by human microglial cell line enabling further characteristic and functional analysis of the protein. furthermore, preliminary analysis on primary brain tissue corroborates the association of cd33 -expressed on microglia -to lateonset ad. the neurotrophins are essential for peripheral nervous system development and myelination. we have previously demonstrated that the neurotrophin bdnf exerts contrasting influences upon myelinationacting through neuronal p75 ntr to enhance myelination, but inhibiting it via neuronal trkb. recently we have generated a small peptide called cyclo-dpakkr that structurally mimics the region of bdnf that binds p75 ntr . here we aim to investigate whether utilising cyclo-dpakkr to selectively target p75 ntr is an approach that could exert a unified promyelinating response. like bdnf, cyclo-dpakkr promoted myelination of ngf-dependent neurons in vitro, an effect dependent on the neuronal expression of p75 ntr . importantly, cyclo-dpakkr significantly enhanced the myelination of bdnf-dependent neurons in vitro, whereas bdnf inhibited it. local injection of cyclo-dpakkr adjacent to the neonatal sciatic nerve in vivo significantly enhanced myelin protein expression and increased the number of myelinated axons. we found that injection of cyclo-dpakkr also significantly upregulated the expression of neuregulin 1 type-iii, a key factor required to induce peripheral myelination. thus, our data demonstrate that cyclo-dpakkr promotes peripheral myelination in vitro and in vivo. to investigate whether cyclo-dpakkr promoted the remyelination of peripheral neurons, we utilized the ean, a rodent model of peripheral demyelinating neuropathy. our data show that administration of cyclo-dpakkr significantly delayed the disease onset and reduced clinical severity in ean. this improved disease phenotype is supported by reduced demyelination, as cyclo-dpakkr substantially reduced the extent of myelin damage in myelinated peripheral nerves subjected to ean. collectively, our data demonstrate that using cyclo-dpakkr to selectively target p75 ntr promotes peripheral myelin development, and is effective at ameliorating ean disease and protecting against myelin damage. our findings suggest that selective targeting of p75 ntr is a strategy worthy of further investigation for the treatment of peripheral demyelinating diseases introduction huntington's disease (hd) is a neurodegenerative disease characterised by huntingtin protein misfolding and the intra-cellular accumulation of mutant huntingtin (mhtt). accumulation of mhtt is most pronounced in neurons and is thought to underpin neuronal dysfunction. oligodendrocytes have been shown to accumulate mhtt aggregates, which may contribute to cellular dysfunction in these cells. evidence from diffusion tensor mri (dt-mri) studies shows that white-matter changes are amongst the earliest pre-symptomatic changes observed in hd. these findings have been taken to implicate axonal and or glial aberrations prior to disease onset. methods we have used a variety of techniques, which include, emulsion in situ hybridisation, whole brain sub-fractionation, immunohistochemistry, meso scale cytokine assay, and western blots. results our investigations using the r6/2 mouse model show that mhtt aggregates result in a specific down-regulation of the small heat shock protein (shsp) hspb5 (alpha-b-crystallin). localisation of hspb5 by whole brain sub-fractionation shows that hspb5 is enriched in the myelin fraction. emulsion in situ hybridisation further shows localises hspb5 in oligodendrocytes. detergent extraction of myelin fractions show differential hspb5 partitioning between soluble and insoluble fractions. cumulatively, these findings suggest that hspb5 is constitutively expressed in oligodendrocytes and is present in two distinct pools. recent studies implicate hspb5 as being a negative downregulator of inflammation. as such down-regulation of the shsp in r6/ 2 mice may promote increased oligodendrocyte vulnerability to mhtt cytotoxicity. to investigate the role of hspb5 as a negative regulator of inflammation, we obtained transgenic hspb5 knockout mice. the knockout mice are viable and show very little phenotypic difference against littermate controls. we have analysed the inflammatory profiles of these mice using the meso scale. to validate our findings in the r6/2 mice, we are currently investigating human tissue from distinct vonsattel stages of disease, to see if hspb5 downregulation is also observed in the human disease. conclusion as hspb5 has several critical cellular roles, its downregulation may compromise oligodendrocyte structure and function, which ultimately has negative consequences for neurons. it is therefore worthwhile to investigate it's specific role in the cns. the myelin sheath is attached to the axon through multiprotein complexes that form the axon-glial interactions. they consist of cell adhesion molecules and voltage gated ion channels and divide the axon into distinct domains: the node of ranvier, the paranode, the juxtaparanode and the internode. this molecular organization is crucial since recent studies identified distinct perinodal proteins as autoantigens in ms patients: nodal and paranodal neurofascin isoforms and juxtaparanodal tag-1/contactin-2. tag-1 is a cell adhesion molecule expressed both by axons and glial cells. in the adult nervous system, tag-1 organizes the juxtaparanodal domain of the fiber, where it interacts with caspr2 and the potassium channels (vgkcs). question: in this study we have analyzed the alterations of the juxtaparanodal proteins caspr2, tag-1 and vgkcs in relation to paranodal caspr and nodal sodium channels upon the onset and progression of experimental autoimmune encephalomyelitis (eae) and in the cuprizone model of toxic demyelination. methods: we immunohistochemically and biochemically analyzed both models of cns demyelination. results-conclusions: our results show a higher paranodal susceptibility to disruption compared to juxtaparanodes in eae. as eae progresses there is subsequent compensatory efforts for myelinated fiber restoration that include paranodal protein re-clustering and increased heminodal formation, without subsequent juxtaparanodal protein aggregation. in contrast, juxtaparanodal organization was observed only when remyelination occured in the cuprizone model of toxic demyelination. supported by: imbb-forth scholarship, manassaki scholarship (university of crete), the project "myelintag" (593) is implemented under the "aristeia" action of the "operational programme edu-cation and lifelong learning" and is co-funded by the european social fund (esf) and national resources. multiple sclerosis (ms) is a chronic disease of the human central nervous system that is characterized by focal lesions with inflammation, infiltration of immune cells, demyelination and axonal damage. activation of cannabinoid cb 1 /cb 2 receptors is considered a potential therapeutic strategy for the treatment of ms, based on the evidence that exogenous cannabinoid agonists exert neuroprotective and immunosuppressive effects in experimental models of the disease. nevertheless, the therapeutic use of synthetic and/or plant-derived agonists acting on brain cannabinoid receptors is limited by the possible adverse responses related to memory and learning impairment. an alternative approach that could avoid this limitation consists of enhancing the concentration of the main endocannabinoids (aea, 2-ag) by increasing their synthesis or decreasing their degradation. the main objective of this study was to analyze the effects of jzl184, a selective inhibitor of 2-ag hydrolyzing enzyme monoacylglycerol lipase (magl), in the chronic eae model of ms. mice were treated daily with jzl184 (8 mg/ kg and 32 mg/kg) or vehicle from onset of the motor symptoms to the end of the experiment. comparison of the motor score curves indicated that both doses of jzl184 ameliorated the deficits observed in vehicletreated mice during the disease course. nevertheless, the beneficial effect of the 32 mg/kg dose was no longer evident in mice scored at 40 dpi. chronic treatment with 8 mg/kg jzl184 reduced the conduction latency of the corticospinal tract measured at the end of the experiment, as well as the number and size of inflammatory lesions in spinal cord, whereas chronic administration of the high dose of the magl inhibitor had no effect on these parameters. importantly, treatment with the 32 mg/kg dose of jzl184 reduced the coupling ability of cannabinoid receptors to g i/o proteins, measured by [ 35 s]gtpcs autoradiography, in all the brain areas analyzed. finally, incubation with jzl184 prevented cytotoxicity by activation of ampa-type glutamate receptors in oligodendrocyte precursors in vitro. this protective effect of the jzl184 was blocked by the cb 1 receptor antagonist am281. our findings point to the involvement of cb 1 receptors in the in vivo therapeutic effects of jzl184, and suggest that chronic administration of magl inhibitors may be a promising strategy for the treatment demyelinating disorders in which oligodendrocyte excitotoxicity plays an important role as mechanism of white matter damage. under disease conditions, connexins can also release these signaling molecules into the extracellular milieu through hemichannels. since astrocytes are involved in the disease progression of als, we are exploring if abnormal gj activity can serve as a potential mechanism for the reported astrocyte-mediated toxicity. we used the sod1 g93a mutation as a als mouse model to address the role of connexins in als. we observed that at the end stage of the disease, spinal cords of sod1 g93a mice exhibited a significant increase in expression of connexin 43 protein compared to their spinal cords of control mice. this increase in cx43 co-localizes primarily to astrocytes in the gray matter of the spinal cord. importantly, human post-mortem tissue from als patients compared to control patients exhibited a significant increase in cx43 rna and protein levels in the motor cortex and a trend for increase in cx43 in the cervical spinal cord. this is an important finding in pursuing cx43 as a potential candidate contributing to astrocyte-mediated toxicity in als. in addition, when astrocytes were isolated from wt and sod1 g93a mice, endogenous levels of cx43 rna and protein were elevated in sod1 g93a mice compared to the wt derived astrocytes. we are currently investigating if functional properties of astrocytes from sod1 g93a mice are altered in addition to biochemical changes. in vitro studies using cx43 specific blockers to assess neuroprotection are also being conducted in co-cultures of neurons and astrocytes. these findings will be novel and an important step towards harnessing therapeutic strategies for als. the pdz protein omi/htra2 is localized in the intermembrane space of mitochondria. it is involved in mitochondrial homeostasis and may act as a chaperone. mutations in the serine protease domain of omi/ htra2 (mnd2 mice), leads to massive loss of striatal neurons and neuromuscular disorders in mice confirming the protective function within mitochondria. upon cellular stress, omi/htra2 is translocated from mitochondria into the cytosol when the mitochondrial outer membrane is permeabilised. in the cytosol, omi/htra2 blocks the action of the inhibitors of apoptosis proteins (iaps) by competitive binding or by degradation via the protease activity. this leads to caspase activation and apoptosis induction. omi/htra2 also regulates apoptosis in a caspaseindependent manner via its protease activity; recent studies have defined multiple substrates of the serine protease. in a yeast twohybrid screen we identified the ng2 proteoglycan as a binding partner of omi/htra2. ng2 is expressed by oligodendrocyte progenitor cells (opc). in the presence of hydrogen peroxide, which is a model of cellular oxidative stress, omi/htra2 is released from mitochondria and can bind to ng2 in opcs. binding of ng2 to omi/htra2 via the pdz domain may thus be a way to sequester or regulate the serine protease. oligodendrocyte-lineage cells are known to be particularly sensitive to cellular stress and white matter diseases of new borns is a major pathology in situations where premature infants are exposed to unphysiological oxygen concentrations. it has been recently shown that glioma cells often express the ng2 protein which may play a protective role. our results indicate that the interaction between omi and ng2 may help protect opcs from cellular stress. microglia are the immunocompetent cells of the cns that continuously survey the extracellular milieu for foreign antigens and play an instrumental role in brain inflammation. in contrast, astrocytes extend highly ramified processes between neurons and synapses and play a vital role in regulating brain homeostasis, synaptic function and plasticity. interestingly, both microglia and astrocytes adopt a specialized 'activated' or 'reactive' state when exposed to adverse brain conditions. the changes in their molecular profiles include the release a diversity of proteins such as pro-and anti-inflammatory cytokines and extracellular matrix molecules that could impact each other's behavior and regulate the properties of nearby neurons. in alzheimer's disease (ad), activated microglia and reactive astrocytes are detected around plaques in mouse models of ad and human ad tissues. however, exactly how these glial types contribute to the onset or progression of ad still remains an open question. to gain a better understanding of the bidirectional communication and response of microglia and astrocytes in the development of ad-like symptomatology, we investigated the properties of these cells types in the crnd8 transgenic ad mouse model by applying confocal imaging and western blot analysis. crnd8 is an early-onset model of ad-like symptomatology showing ab deposits present at 2 months of age and neuritic pathology by 5 months. our results show complex spatio-temporal changes in the interaction of microglia and astrocytes around ab plaques during the progression of the disease in both cortex and hippocampus. we found that subtle rearrangements of microglial morphology and astrocyte reactivity were detected as early as 1 month when ab plaque deposition is absent. by 2-3 months, low numbers of activated microglia and reactive astrocytes begin to surround small ab deposits. the establishment of complex 'reactive glial domains (rgds)' can be observed at 4 months, when amoeboid microglia fully encompass large ab plaques and become surrounded by reactive astrocytes forming an elaborate outer shell-like structure. intriguingly, after 6 months, the domains become progressively disrupted due to the reorganization and recruitment of additional microglia and astrocytes. we also observed gradated inflammatory phenotypes of glial cells. these are first restricted to rgds in the early and middle stages of ad but spread locally in late stages when disorganized rgds are observed. interestingly, neuronal dystrophy was correlated with the severity of the inflammation. we conclude that communication between microglia and astrocytes may be a key process in the ability of the brain to surmount a reparative response to early neurodegenerative conditions but may be disrupted or become overwhelmed in later stages of ad. supported by alzheimer society of canada (db) and by cihr (kkm and rq). poster topic 04 extracellular matrix and cell adhesion molecules opcs exist in mature rodent and human central nervous system (cns; around 5-7% of the total cells) and constitute an interesting source regenerative therapies in demyelinating diseases, like multiple sclerosis (ms). different molecules are involved in opc morphofunctional development during embryogenesis and postnatal stages, and some of them are upregulated in ms lesions, suggesting their involvement in the pathogenesis of the disease. this is de case of anosmin-1, an extracellular matrix glycoprotein coded by the kal1 gene and responsible for the x-linked form of kallmann syndrome. the best known mechanism of action of anosmin-1 seems to be mediated through the interaction of this protein with fibroblast growth factor receptor 1 (fgfr1) and the modulation of the activation of this receptor by fgf2. this protein participates in the adhesion, migration and differentiation of various cell types in the cns; among others, anosmin-1 promotes the adhesion of neurons, neurite outgrowth, axonal guidance and branching of different cns projection neurons, as well as having a role in the migration of different types of neuronal precursors, immortalized gnrh-producing neurons and embryonic opcs. in addition, previous results of our group also suggest a role of anosmin-1 in demyelinated lesions from ms patients and point to the feasible pharmacological and genetic manipulation of the fgf2/fgfr-1/anosmin-1 system on endogenous and/or exogenous opcs in demyelinating lesions. in the present work we studied the functional implications of anosmin-1 and fgf-2 on neurobiology (cell death, proliferation, motility, migration) of postnatal/adult murine opcs isolated from cerebral cortex (p0, p15, p60) and of opcs isolated from adult human biopsies, using both in vivo (immunohistochemistry) and in vitro (chemotaxis chambers, migration, brdu uptake, tunel, immunocytochemistry) techniques. these results would be useful for the design of effective neuroreparative therapies in ms and other demyelinating diseases. funded we generated ipsc lines from fibroblasts isolated from pitx3-gfp knock-in mice which allowed facs-purification of mature midbrain da neurons upon in vitro differentiation based on the expression of the mature da marker pitx3. subsequently, mouse pitx3-gfp knock-in ips cells were transduced with lentiviral vectors containing genes encoding for hl1 and for stx, the enzyme that add psa onto ncam. next they were differentiated in vitro into da neurons. the effects of hl1 and psa-ncam on differentiation, survival and outgrowth of ipsc-derived da neuron were analyzed in vitro and in vivo. indeed, we were able to obtain a pure suspension of mature ipscderived da neurons upon in vitro differentiation and fac sorting. transfection of pitx3-gfp knock-in ips cells with hl1 or stx did not affect the pluripotent potential of these cells and did not interfere with the process of dopaminergic differentiation itself. we continued to establish the beneficial effect of l1 and psa-ncam expression on survival and outgrowth of ipsc-derived da neurons in vitro and after grafting in the striatum of parkinsonian rats. vascular endothelial growth factor (vegf) is a major regulator of neurovascular remodeling following brain injury. while much have been learnt about vegf functions on target endothelial cells, little is known about its intracellular trafficking, mode of secretion and matrix interactions in "source" cells, i.e. mainly reactive astrocytes. here, we generated a vegf::gfp fusion protein to follow the distribution of vegf165 during its trafficking in primary astrocytes and cos7 cells. we found that vegf::gfp forms dimers and gets glycosylated similarly to wild type, and as expected, the molecule follows the endoplasmic reticulum-golgi pathway. however, its post-golgi trafficking suggests a unique route with features that do not conform the classical constitutive secretory pathway: the secretion of vegf165 occurs even at 19 c and shows a ca21-and pkc-induced increase. we investigated the distribution of vegf in polarized primary astrocytes in an in vitro scratch wound assay. in these cells, vegf::gfp appeared to follow a vectorial distribution and accumulated behind the leading edge. the accumulation appeared to be on the extracellular surface, moreover, ultrastructural immunogold labeling revealed that extracellular vegf::gfp remains associated with discrete areas of the cell membrane and is accumulated in caveolae as well as in microvesicular shedding elements. this particular localization corresponds to fibrillar adhesions (fb), where vegf::gfp is co-localized with fibronectin. we also observed that vegf::gfp is targeted to adherens junctions between astrocytes, however, at these sites vegf::gfp was not co-localized with fibronectin. finally, we show in frap experiments that integrin turnover is decreased in vegf associated fbs, which raises the possibility of an autocrine/paracrine regulation of astrocytic functions. together, these findings have strong implications for understanding focal coordination of angiogenesis and vascular remodeling by astroglia derived vegf. after invading the embryonic cns, microglia migrate extensively to occupy their final positions. however, the cellular and molecular mechanisms of microglial migration are unknown. therefore, this study aims to elucidate the microglial migration behavior, cellular interaction partners and ecm-integrin interactions essential for migration in the developing neocortex. methods: immunohistochemical stainings and immunogold labeling for transmission electron microscopy were carried out on fixed brain tissue of c57bl/6 cx3cr1 1/egfp mice embryos (embryonic day (e) 10.5-17.5). multicolor facs analyses were performed on homogenized age-pooled cx3cr1 1/egfp embryonic cortici. microglial migration was recorded in cx3cr1 1/egfp -hgfap-cfp acute brain slices using multiphoton time-lapse excitation and analyzed using mtrackj in imagej. results: laminin (lm) is expressed as punctuate dots near the pia and in the ventricular zone at e10.5 and disappears at e15.5 to remain only in blood vessels and the pia. fibronectin (fn) is present as aggregates from e10.5 until e17.5. both ecm proteins follow the course of radial cells. preliminary data indicate a transient upregulation of the lm receptor (lmr) on e15.5 and e16.5. in addition, the percentage of microglia expressing the fn receptor (fnr) tends to decrease during development. ex vivo time-lapse recordings show that embryonic microglia are dynamic cells that migrate in random patterns. these cells seem to make brief contacts with the processes of radial glia. conclusions: embryonic microglia express laminin and fibronectin receptors and possibly change the expression level during development. they are dynamic cells that migrate in random patterns, and possibly make occasional contact with radial glia. knowledge about ecm-integrin interactions during microglial migration could reveal targets for intervention in pathological settings, such as multiple sclerosis and alzheimer's disease. multiple sclerosis (ms) is a chronic demyelinating disease of the central nervous system in which astroglial cells become hypertrophic, produce various extracellular matrix (ecm) proteins which become aggregated when secreted. these aggregated ecm proteins contribute to a non-permissive environment for repair. tissue transglutaminase (tg2) expression is enhanced in astrocytes in ms lesions. this enzyme is well-known for protein cross-linking capacities. moreover, tg2 can act as a co-receptor for b-integrins to bind to ecm proteins, thereby mediating cell adhesion processes. in this study we questioned whether astrocyte-derived tg2 affects ecm protein production and/or aggregation, and if astrocyte-derived tg2 mediates cell adhesion onto various ecm proteins. primary rat astrocytes were transduced with an empty lentiviral vector (mock) or with a lentiviral vector expressing human wildtype tg2. alternatively, primary rat astrocytes were transduced with a lentiviral vector expressing scrambled shrna or tg2 shrna to knockdown endogenous rat tg2. the rat primary astrocytes overexpressing human tg2 showed an increased production of fibronectin and collagen v. conversely, knocking-down tg2 showed a decrease in fibronectin and collagen production. interestingly, overexpression of tg2 resulted in enhanced aggregation of extracellular laminin. also, human astrocytoma cells (u373) were transduced with an empty lentiviral vector (mock) or with a lentiviral vector expressing human wildtype tg2 and allowed to adhere onto ecmcoated wells. astrocytoma cells overexpressing human tg2 showed increased adhesion onto laminin-, and fibronectin-coated wells. we conclude that astrocyte-derived tg2 affects production/aggregation of fibronectin and laminin. moreover, the astrocyte-derived tg2mediated elevated production/aggregation of fibronectin and laminin coincides with more adherence of astrocytes onto these ecm proteins. we hypothesize that tg2-mediated enhanced production/aggregation of fibronectin and laminin is related to increased astrocyte adhesion which together could contribute to the non-permissive environment for repair in the central nervous system of ms patients. a. tripathi, z. parikh, p. pillai the m.s. university of baroda, vadodara, india background: oligodendrocytes originate as progenitor cells (opcs) in discrete areas of the developing brain which undergoes a complex and precisely timed program of proliferation, migration, differentiation, and myelination in cns. during migration it interacts with its surrounding environment through integrins. purpose: to evaluate the interactions between integrins and growth factors (gfrs) that promote the molecular events such as proliferation, cytoskeletal reorganization and migration in oligodendrocytes leading to oligodendrocyte mediated myelination. methods: cortical cells were prepared from p0 rat cerebral cortex following standard protocols. immunocytochemistry (icc), western blot (wb), immunoprecipitation (ip), migration assay and real-time rt-pcr analysis were performed. for dissecting out the roles of different intracellular signalling proteins in the regulation of events downstream of receptor activation, use of pharmacological inhibitors was carried out. results: tnfa, pdgfa and estradiol significantly increased cell proliferation and cell processes. data also suggest differential effects of extracellular matrix (ecm) on signalling component activation. significant increase in erk expression was found following gfrs-integrin interactions. ecm proteins bind to integrin family members and mediate bidirectional signalling between the cellular interior and the external environment through the activation of focal adhesion complexes. conclusion: integrin switching, oligodendrocyte proliferation, cytoskeletal reorganization and differentiation profile is highly influenced by ecm and gfrs. integrin governs the signalling cascades underlying oligodendrocyte differentiation and myelination. this study was performed in strict accordance with the recommendations for the care and use of laboratory animals. all the protocols were duly approved by the institutional ethical committee, the m.s. university of baroda. topography, roughness and mechanical properties of micro environment are crucial parameters influencing cell adhesion/motility, morphology and mechanics as well as the development of cells e.g. stem/progenitor cells, and neuronal cells [1] [2] [3] [4] [5] . atomic force microscopy is a powerful tool not only to study the morphology in terms of high resolution imaging and roughness measurements, but also to map mechanical and adhesive properties of the sample/cells and tissues. combining these remarkable abilities with advanced optical microscopy allows for extensive characterization of biomaterials and tissue slices [6] [7] [8] . however, commercially existing technical solutions are either time consuming, or only limited practicable for soft, sticky, or fragile samples. therefore, we developed a new force curve based afm mode -quantitative imaging (qi tm ). the novel qi tip movement algorithm prevents lateral forces and controls the vertical forces for nondestructive imaging. to demonstrate the performance and flexibility qi tm mode we investigated topography, adhesion properties, and young's modulus local distribution of living dorsal root ganglion cells. a specialized platform -cellhesion v r -has been developed to run single cell force spectroscopy (scfs) with a need for long-range cellsurface and cell-cell binding experiments with up to 100 microns pulling length. using single cell force spectroscopy (scfc), cell-cell adhesion can be quantified [e.g. 3], and the contribution of different components e.g. from the extra cellular matrix, can be assessed [9] . a new technical solution will be demonstrated if a cantilever attached cell can be transferred through the liquid-air interface. this approach allows to measure the adhesion of the same individual single cell on different materials within different dishes as it will presented for cho cell adhesion on plastic as well as albumin coated surfaces. we want to present the progress in automatic data processing and display to investigate topography, young's modulus and local adhesion phenomena on transparent and non-transparent biomaterials like titanium, peptide functionalized surface or cell layers, and tissue slices. l. vargova 1 , m. cicanic 1,2 , e. sykova 1,2 1 charles university, 2nd faculty of medicine, prague, czech republic 2 institute of experimental medicine, v.v.i., department of neuroscience, prague, czech republic bral-2 is a link protein that is localized in distinct brain regions, where it stabilizes the perineuronal nets of the extracellular matrix (ecm). our previous studies showed that qualitative and quantitative ecm changes, might evoke changes in the diffusion properties of the extracellular space (ecs), thus affecting the movement of neuroactive substances in the cns. in the current study, we determined by the real-time iontophoretic method the extracellular space volume fraction a (a 5 ecs volume / total tissue volume) and the geometrical factor tortuosity k (k 2 5 free / apparent diffusion coefficient) in coronal brain slices obtained from the sensorimotor cortex and the ventral posteromedial thalamic nucleus of sixteen-month-old bral-2 deficient mice (bral-2 -/-), age-matched bral-2 1/1 controls and young adult (5-month-old) bral-2 1/1 controls. the diffusion parameters in the cortex of young adult bral-2 1/1 mice were: a 5 0.21 6 0.01 (mean 6 s. e. m.) and k 5 1.48 6 0.02, n 5 6, n 5 5 (n -number of slices, n -number of animals). in the thalamus, there was a smaller a and a larger k (0.16 6 0.01 and 1.53 6 0.01, respectively, n 5 26, n 5 10). in young adult mice, we found no significant differences in the ecs diffusion parameters between bral2 positive and negative mice in either the cortex or the thalamus. in the cortex of aged bral-21/1 mice we found a smaller a (0.18 6 0.01, n 5 10, n 5 7) than in the cortex of young adult mice. during aging, there was no significant difference in the thalamic diffusion properties in wild-type animals, but in the thalamus of aged bral-2 -/-mice, a significantly decreased (0.13 6 0.01, n 5 28, n 5 11) in comparison to the younger bral2-/-animals as well as to age-matched controls. immunohistochemical analysis of the ecm in the ventral posteromedial thalamic nucleus revealed no positivity for bral2 protein in either group of bral2-/-mice. in the youger mice, bral deficiency was associated with a small attenuation in the positivity of brevican and another link protein, ctrl1; however, in aged bral2-/-animals, brevican and ctrl1 positivity was increased in comparison with the age-matched controls. in contrast to the cortex, we found no decrease in a in the thalamus of aged wt animals, suggesting that the process of aging may differ between the cortex and the thalamus. a decrease in the ecs volume fraction in the thalamus of bral-2 -/-aged mice may result from a disruption of the perineuronal nets and rearrangements of the molecular assembly, which seem to differ in young adult and aged animals. here we analyzed the global transcriptome of cultured astroglial cells incubated with activators of camp pathways. a bulk of astroglial transcripts were differentially regulated by camp signaling. camp analogs strongly upregulated genes involved in typical functions of mature astrocytes, such as homeostatic control, metabolic and structural support to neurons, antioxidant defense and communication, whereas they downregulated a considerable number of proliferating and immaturity-related transcripts. moreover, genes typically activated in reactive cells, such as immunological mediators and scar components, were repressed by camp. gene set enrichment analysis and evaluation in situ of gene expression in astrocytes in different states showed that camp signaling conferred a mature and in vivo-like transcriptional profile to cultured astrocytes. these results indicate that camp signaling is a key pathway restricting developmental and activation features of astrocytes and promoting their maturation. a positive modulation of camp signaling is suggested to suppress the mechanisms of activation driven by pathological situations and to promote the physiologically normal state of differentiated astrocytes. question: microglia, the innate immune system of the central nervous system, are crucial to maintain homeostasis by cleaning debris and attacking stressors. in the ageing brain, increased numbers of microglia with ameboid morphology and increased inflammatory cytokine levels are reported. the altered microglial phenotype in the ageing brain is characterized by hyperresponsive to immune stimuli, which is called immune priming. immune priming could be a potential cause of age-associated neurodegeneration. the aim of this study is to characterize the immune pathways and biological processes that are altered in ageing-induced immune primed microglia and to find genes potential drivers of this process. design/methods: the effect of ageing on microglia has been addressed in naturally aged mice (24 months old) and in ercc1 transgenic mouse model in which accelerated ageing is induced by dna damage accumulation. from each of these models a pure population of microglia was isolated. microarray technology was used to obtain information about the genome wide gene expression profile. weighted gene co-expression network analysis (wgcna), uses correlational structures to find clusters of genes to make optimal use of a dataset in biological interpretation. results: using weighted gene co-expression network analysis, a very robust microglial immune priming gene module was discovered. in this module, there is a significant enrichment of the complement system, antigen presentation, interferon type-1 signaling and phagocytosis-machinery. the average expression of this module is stronger up-regulated in ercc1 transgenic than in normally physiologically aged mice, supporting the notion that ercc1 is characterized by excessive immune priming. conclusion: microglial priming which occurs in normal ageing or in accelerated ageing models is marked by increased expression of several immune signaling pathways. methods: we investigated three single nucleotide polymorphisms (snps) (rs1042713 (arg16gly), rs1042714 (gln27gly) and rs1042711) and five haplotypes in adrb2 to explore whether these polymorphisms were associated with ms progression in 436 well phenotyped ms patients. genotyping was done using illumina bead microarray and missing genotypes were imputed using beagle. results: a trend towards a decreased frequency of rs1042714c allele (27gln) was in people with progressive ms than in people with relapsing remitting ms. this was seen in both sp-ms (p (uncorrected) 5 0.0072) and in pp-ms (v 2 5 6.85, p (uncorrected) 5 0.0325). however, those p values did not reach statistical significance after correction for multiple testing. neither haplotypes nor snps were significantly associated with altered msss, age of onset, time between fist and second relapse, processing speed (sdt) or brain atrophy. conclusion: we found no evidence that polymorphisms in adrb2 influence severity in multiple sclerosis. here we examine the expression of these brain-specific pgc-1a isoforms in neurons and glia cells. we characterize the abundance of the different isoforms of pgc-1a in primary murine cell cultures of neurons, glia cells, including oligodendrocytes, astrocytes and microglia under control conditions and metabolic stress. we show that pgc-1a is differentially expressed in the different cell types, e.g. neurons and oligodendrocytes express much higher levels of the novel brain-specific isoforms of pgc-1a. the different expression levels might reflect the different metabolic needs of the different cell types. s. marques, g. castelo-branco karolinska institutet, stockholm, sweden oligodendrocytes (ol) are neuroepithelium-derived cells that insulate neuronal axons through myelin-containing membranes, essential for axonal integrity and impulse transmission. defects in this process are present in several diseases, including the highly debilitating multiple sclerosis (ms). the process of remyelination can be promoted by ol precursor cells (opc) endogenously and occurs during a short window of time at earlier stages of ms, ultimately failing as the disease evolve. the understanding of development and differentiation regulation of ol is essential to clarify the reasons behind the defective myelination during pathologies and to open the pave to new remyelinating cell therapies. opcs start to be specified early during embryogenesis but regardless of the origin, their terminal differentiation and functional maturation occurs only at post-natal stages. in this project, we are characterizing the transcriptome of opcs during development, which will allow us to better characterize the heterogeneity of these cells and point out what might be the key factors involved in transition between states. recent studies suggest that non-coding rnas can interact with proteins complexes containing chromatin modifying enzymes and transcription factors, regulating their activity, acting as scaffolds and/or recruiting them to specific loci in the genome. therefore, we are investigating their role in opcs by rna interference and how they might modulate the function of key transcription factors. neural stem cells (nscs) represent a potentially valuable resource when considering repair strategies for cns damage. unlike their embryonic counterparts, adult neural stem cells (anscs) in the two neurogenic niches have an astrocyte-like identity, raising the question of what regulates this ansc state. some early postnatal astrocytes in the parenchyma also retain some nsc-like characteristics and in vitro display self-renewal and multipotency, though these properties are lost following in vivo maturation. interestingly, following injury, adult astrocytes can become reactive and reacquire nsc-like properties, whilst others can generate new neurons through forced expression of specific transcription factors. we are aiming to elucidate the gene regulatory networks underlying specific nsc and astrocyte states and transitions between them using transcriptomic and epigenomic tools alongside computational methods in different nsc and astrocyte populations. we will describe our latest findings from an in vitro system that aims to model immature versus mature astrocytes derived from a common progenitor. from microarray data, we have identified candidate genes and pathways involved in regulating astrocyte potential versus differentiation, including a role for specific pro-inflammatory signalling. we will also describe the associated epigenetic signatures that accompany cell state and discuss the implications of our latest findings. microglia are the cns immune-related cells and associated with the pathogenesis of several types of neurological diseases. following peripheral nerve injury (pni), spinal microglia become activated and have crucial roles in generating neuropathic pain. we have previously shown that interferon regulatory factor-8 (irf8), upregulated in spinal microglia after pni, regulates gene expressions that switch to a reactive phenotype. here we identified irf5 as a crucial factor of reactive microglia that works cooperatively with irf8. we found that pni increased expression of irf5 in the spinal cord, the expression of which was restricted to microglia. forced expression of irf8 in cultured microglia markedly increased expression of irf5 in a manner that depended on its ability to bind dna. furthermore, irf8-deficient mice failed to increase the expression of irf5 in spinal microglia after pni, indicating irf8-dependent expression of irf5 in microglia in vivo. interestingly, upregulation of p2x4 receptor (p2x4r; an atp-gated channel crucial for producing neuropathic pain) by forced expression of irf8 in cultured microglia was markedly suppressed by knockdown of irf5 expression. in a model of neuropathic pain, suppressing upregulated irf5 protein by spinal administration of sirna targeting irf5 alleviated pain hypersensitivity. altogether, the irf8-irf5 axis drives a program of gene expression that promotes p2x4r hi microglia gating neuropathic pain. astrocytes are the most abundant glial cell type. they express a range of neurotransmitter receptors localized along their processes that cover synapses and constitute "microdomains" for signalling pathways. to investigate astroglial purinergic p2y1 and glutamatergic nmda receptors we generated knockout mice in which gene recombination of "floxed" receptors was controlled by an astrocyte-specific and tamoxifen-inducible cre recombinase (glast-creert2). here, we present data on the dna recombination efficiencies of glast-creert2 x floxed p2y1 mice and glast-creert2 x floxed glun1 mice in different brain regions (cerebellum, hippocampus, brainstem, optic nerve and cortex). recombination is determined by quantitative real-time pcr of genomic dna using primers across the loxp sequences flanking exon 1 of the p2ry1 gene and exon 11-21 of the grin1 gene, respectively. dna recombination was induced in young adult mice by intraperitoneal tamoxifen injection for three consecutive days and analyzed three weeks later. primers were designed such that reduction of the pcr signal reveals increasing recombination (i.e. gene excision and knockout) compared to control. the degree of reduction in the investigated brain regions is expected to be similar in both mouse lines; allowing conclusions (1) about the reliability of the glast-creert2/loxp system in general (i.e. recombination efficiency at different chromosomal locations), and (2) gives a percentage of glastpositive cells (predominantly astrocytes) in the given brain area. first data demonstrate 30% recombination in the cortex, which is well in line with the percentage of astrocytes in this brain region. these results could be confirmed by using a reporter mouse line expressing the red fluorescent protein tdtomato (r26-tdtomato) and using immunohistochemistry with astrocyte markers. in conclusion, the combination of quantitative real-time pcr analysis of gene recombination and cell-specific cre mouse lines represents a reliable approach to reveal the percentage of a given cell type in a given tissue region. the transcription factor creb is protective in injury and neurodegeneration, but the contribution of neuronal vs astrocytes is unknown. we have generated a mouse with targeted over-expression of a constitutively active creb (vp16-creb) in astrocytes by crossing teto-vp16 and tta-gfap mice. vp16-creb/gfap mice show expression of vp16 in astrocytes -as detected by immunohistochemistry -restricted to cerebellum, hippocampus and outer layers of cortex. we determined the role of astrocytic creb in the outcome of brain injury by subjecting wild type (wt) and vp16-creb/gfap (tg) mice to a focal cryolesion (c) in the frontal cortex. at 3 days post-lesion, wt mice presented a necrotic region filled with macrophages (labeled with lectin) surrounded by a rim of tissue with robust gliosis (labeled with gfap). tg mice showed reduced macrophage infiltration as compared with wt mice. a dna array analysis of the damaged zone (agilent) has revealed differentially expressed genes in paired comparisons. wtc/wt: 13,984; wtc/tgc: 5,821; tgc-t:913; tg/wt:0 (statistical linear model in limma., p values computed by empirical bayes moderated t-statistics at 0.05). there were 159 significantly enriched kegg pathways regulated by the cryolesion, according to gsea and hypergeometric tests set at p < 0.05. vp16-creb had an effect on 127 of the pathways. upon increasing the analysis stringency by setting the cut-off at p < 0.0001, the number of significantly enriched kegg pathways regulated by vp16-creb was reduced to 16. we selected 10 of these pathways related to 5 functions: energy metabolism, inflammation, structural reorganization, genomic stability and apoptosis, which we define as the core signature of the vp16-creb-elicited change. the differential expression of representative genes related to this signature was validated by real-time pcr. thus, vp16-creb rescued the decreased expression of enzymes regulating energy metabolism, while decreasing the expression of pathways related to apoptosis, extracellular matrix and inflammation. the latter is consistent with the decreased macrophage infiltration detected by immunohistochemistry in tg mice. we posit that vp16-creb protects the brain against bionergetic failure and secondary damage due to macrophage infiltration, while rendering the tissue more conducive to neurite regeneration by reducing extracellular matrix components. the study supports the notion of astrocytes as therapeutic targets in brain injury. furthermore, ca 21 /cn/nfat dependent mmp3 expression was confirmed in pure astrocyte cultures derived from neural stem cells (ast-nsc), demonstrating that the induced mmp3 expression occurs in astrocytes, and not microglial cells. in an in vivo stab-wound model of brain injury, mmp3 expression was detected in nfatc3-positive scarforming astrocytes. since [ca 21 ] i increase is an early event in most brain injuries, these data support an important role for ca 21 /cn/ nfat-induced astrocyte mmp3 expression in the early neuroinflammatory response. understanding the molecular pathways involved in this regulation could provide novel therapeutic targets and approaches to promoting recovery of the injured brain. the posterior hypothalamus (ph) is crucial for maintaining wakefulness whereas the anterior hypothalamus (ah) constitutes a sleep-promoting region. consequently, the energy needs of ah and ph should probably change throughout the sleep-wake cycle. the glycogen mobilization altogether with the astrocyte-neuron lactate shuttle (anls) likely constitutes two major mechanisms by which astrocytes ensure the neurometabolic coupling. therefore, we hypothesized that astrocytes of ah and ph may display differences in genes expression involved in these mechanisms during the sleep-wake cycle. to investigate this, we measured the gene expression levels in astrocytes obtained from transgenic mice expressing the fluorescent protein egfp under the control of the astrocyte-specific gene gfap promoter. mice were sacrificed at four time, zt0, zt6, zt12 and zt18 (zt0 corresponding to light-on). after brain dissection, "punches" of ah and ph were micro-dissected from slices. cell suspensions were obtained from a pool of 3 samples by enzymatic digestion and cell trituration. then, gfp-positive cells, which were highly enriched in astrocytes, were sorted by facs. total rna was extracted from these cells and gene expression levels were assayed by qrt-pcr and normalized by cyclophylin mrna levels. seven genes related to anls were tested: the alpha 2 sub-unit of the na-k atpase (atp1a2), the two sub-unit of the lactate dehydrogenase (ldha and ldhb), the glutamate transporters (glast and glt1) and two monocarboxylate transporters (mct1 and mct4). levels of mrna encoding genes related to glycogen metabolism such as ptg, the glycogen synthase (gs) and the glycogen phosphorylase (gphos) were also measured results showed that the levels of mrna encoding ldha, mct1 and mct4 expressed significant circadian variations which can reach 200% for mct4 at zt0, compared to zt6 levels in ah. the glt1 mrna levels as well as mct1 and mct4 mrna levels also displayed significant circadian variations in ph. levels of expression of genes encoding the gphos, gs and ptg did not display major regional circadian variation. this study shows that astrocytes displayed some transcriptional regulation in hypothalamic areas involved in the sleep-wake cycle. the difference in the pattern of expression of anls related genes in ah and ph, such as glt1, suggests that neuro-metabolic coupling capacity of each structure might be different and might play a functional role in the sleep-wake regulation. , are a heterogeneous cell population that is despite its functional importance still not well defined. while astrocytes are no longer just considered the supporting cells in the cns and are recognized to play a significant functional role in e.g. synapse formation, regulation of the blood brain barrier and synaptic transmission, little is known whether specific subtypes of astrocytes fulfill these functional tasks differently. studies in our laboratory described brain region specific differences in the expression of astrocytic glutamate transporter 1 (glt-1) with significantly increased levels of glt-1 in the spinal cord compared to brain. the mechanisms of glutamate transporter regulation are not well defined, and to date little is known about the factors that are responsible for regulating protein expression and transporter activity and whether this regulation is specific for certain subtypes of astrocytes. to study glt-1 regulation in more detail, we generated a family of transgenic mice using increasing lengths of the 5 0 non-coding region of the glt-1 gene controlling expression of tdtomato. these mice were crossed with full length bac-glt-1-egfp mice to compare astrocytic expression of the reporter genes. interestingly, only a subpopulation of gfp-positive astrocytes was found to have strong tdtomato fluorescence signal, when we crossed an 8.3 kb eaat2 promoter fragment mouse with the bac-glt-1 mouse, suggesting the existence of astroglial subtypes that are differentially regulated for eaat2 expression. such differences might account for variance in local glutamate-dependent excitotoxicity to motor neurons. to this end, we purified both tdtomato1/gfp1 and tdtomato-/gfp1 astrocytes from multiple cns regions by fluorescence-assisted cell sorting (facs) and analyzed their transcriptomic profiles using microarray analysis. a list of candidate genes, mainly transcription factors and cell membrane molecules, was complied from this analysis and validated by both quantitative rt-pcr and immunofluorescence. pax6 and kir4.1 were found to be highly expressed in astrocytes, consistent with previous findings. more importantly, kir4.1 mrna was noted to be selectively enriched in cortical tdtomato1/gfp1 cells, compared to gfp1 only cells. it suggests kir4.1 may serve as an astroglial subtype marker. . we have hypothesized that microglial c/ebpb is a potential target to attenuate the neurotoxic effects of neuroinflammation. in order to test this hypothesis in vivo, c/ebpb knockout mice are not suitable because they show multiple phenotypic alterations as a result of the widespread expression of c/ ebpb. mice with specific c/ebpb deletion in microglia would be a better model. unlike gfap promoter for astrocytes, there is not a gold standard promoter for cre expression in microglia. cd11b, cx3cr1 or lysozime m (lysm), among other promoters, have been used to this end. we have generated lysm-cre/c/ebpb fl/fl mice with two main aims: 1) to validate the use of lysm as a suitable promoter for microglial gene deletion by cre-lox recombination 2) to analyze the effects of the absence of microglial c/ebpb in vitro and in vivo lysm-cre/c/ebpb fl/fl mice which were fertile and viable and did not show the female infertility and high perinatal mortality described in c/ ebpb knockout mice. western blot experiments revealed the presence of c/ebpb in protein extracts from wild-type but not from lysm-cre/c/ ebpb fl/fl microglial cultures. immunocytochemistry experiments confirmed this observation. thus, c/ebpb immunoreactivity was present in all cells in wild-type microglial cultures and it was absent in all microglial cells in lysm-cre/c/ebpb fl/fl microglial cultures. the microglial specificity of lysm-cre recombination was demonstrated in primary mixed glial cultures. whereas in wild-type primary mixed glial cultures c/ebpb immunoreactivity was observed both in astrocytes and microglia, in lysm-cre/c/ebpb fl/fl mixed glial cultures it was detected in astrocytes, but never in microglia,. to assess the functional outcome of microglial c/ebpb deficiency, we analyzed lps1ifncinduced no production and we observed a marked decrease in lysm-cre/c/ebpb fl/fl microglial cultures. this decrease was also observed in mixed glial cultures in fitting with the microglial origin of no production in this model. in summary, these findings show that lysm-cre promoter causes recombination in 100% of microglial cells in primary culture and it is therefore suitable for microglial-specific gene deletion in vitro. experiments are in progress to determine the degree and specifity of microglial c/ebpb deletion in vivo in these mice. supported by grants pi10/378 and pi12/00709, instituto de salud carlos iii, spain. to study the functional role of cpeb3, transgenic mice overexpressing cpeb3 in astrocytes were generated. cpeb3 overexpression without the endogenous 3 0 untranslated region (utr) led to the downregulation of astrocytic connexins (cx43 & cx30). consistently, in the hippocampus of cpeb3 overexpressing mice, intercellular gap junction coupling was strongly impaired. cpeb3 overexpression also led to downregulation of glutamate transporter 1 (glt-1) and glutamine synthetase (gs), key players involved in extracellular glutamate clearance. we have now raised mice overexpressing cpeb3 with the endogenous 3 0 utr. in these mice, we observed the downregulation of cx43, cx30, glt-1 and gs. since interastrocytic coupling and astrocytic glt-1 and gs activities are downregulated in epilepsy patients with hippocampal sclerosis, we hypothesize that upregulation of cpebs in astrocytes may contribute to the pathogenesis of epilepsy by causing astrocyte dysfunction. to study the influence of the p53 gene in the structural and quantitative characteristics of astrocytes in the mice retina methods: adult mice of the c57bl/6 strain (12 months old) were distributed into two groups: 1) mice with two extra copies of p53 ("super p53"; n 5 6) and 2) wild-type p53 age-matched control, as the control group (wt; n 5 6). retinas were immunohistochemically processed with gfap. gfap1 astrocytes were quantified. results: in comparison con wt: i) retinal-astrocyte distribution followed established patterns; however, morphological changes were seen through the retinas in relation to p53 availability: astrocytes were more robust and secondary processes were more evident; ii) astroglial density was significantly higher in the sp53 retinas, both in the whole-retina (p < 0,01 student's t-test) and in the intermediate and peripheral concentric areas of the retina (p < 0.05 student's t-test). conclusion: the astrocyte changes in the sp53 retinas might improve the resistance of the retinal cells against oxidative stress and its downstream signalling pathways. however, neuronal damage caused due to excessive microglial activation is a well known phenomenon in neurodegenerative diseases. mirnas are novel regulators of gene function, controlling several biological processes. recent reports show altered mirna expression in immune-mediated pathologies, suggesting that mirnas have roles in modulating immune responses. thus, the present study was initiated to identify micrornas and their target mrnas which can modulate microglial inflammatory responses. a pilot study was designed to understand the role of mirna-200b in modulating microglia-mediated immune response as this was found to be localized in the microglia. recently, mirna 200b has been shown to target several proteins including c-jun, the substrate of jnk mapk (mitogen-activated protein kinase) which mediates the release of proinflammatory cytokines in activated microglia. qrt-pcr revealed the decrease of mir-200b expression level in activated bv2 microglia. loss-of-function and gain-of-function studies confirmed c-jun to be the target of mir-200b in microglia. overexpression of mir-200b in activated microglia resulted in a decrease in c-jun and jnk expression and activity, thereby dysregulating the mapk-jnk pathway and proinflammatory cytokines. further, treatment of neuronal cells, mn9d with conditioned medium obtained from activated microglial cells, resulted in increased inflammatorymediated cell death upon knockdown of mir-200b. overexpression of mirna-200b also reduced the phagocytic ability in activated microglia. taken together, these results demonstrate the important role of mir-200b in modulating the mapk pathway via c-jun which in turn affects different aspects of the inflammatory process accompanying microglia activation including cytokine response, no production, phagocytosis and neuronal cell death. thus, mir-200b may prove to be a useful target for developing therapeutic strategies to control microglial mediated inflammation in neurodegenerative diseases. further in order to understand the global mirna changes contributing to microglial activation, a global mirna microarray was carried out using control, lps-activated and amyloid b-activated primary microglia. this screen identified several families of mirnas differentially expressed between the different treatment groups enabling us to analyze the mirna signature in activated microglia. national university of singapore, singapore, singapore an inflammatory process in the brain as a result of an injury or infection is mediated by the microglia, the prime immune effector cells of the central nervous system (cns). microglia have also been shown to display chronic inflammatory responses leading to neurodegenerative disorders. small ubiquitin-like modifier-1 (sumo-1) is a post-translational protein modifier, which has been shown to be associated with nuclear factor kappa b (nfjb)-mediated inflammatory pathway. in this study, we showed the expression of sumo-1 in microglia and characterized the roles of sumo-1 in activated microglia. we found that sumo-1 is specifically expressed in the amoeboid microglial cells of the early postnatal rat brain. secondly, lipopolysaccharide-mediated activation of microglia led to a significant increase in the expression as well the nuclear translocation of sumo-1 in microglia in vitro. in order to establish a function for sumo-1 in activated microglia, we sought to silence its expression using sirna-mediated gene knockdown. sumo-1 knockdown in lps-treated bv-2 microglia in vitro subsequently showed the decreased nuclear translocation of nfjb and expression of tumour necrosis factor-alpha (tnf-a) level. further, we performed an in silico screening to identify the targets of sumo-1 in the nfjb pathway. our study thus, suggests that sumo-1 regulates nfjb translocation into the nucleus for the transcription of pro-inflammatory genes including tnf-a in activated microglia. microglia are the main resident immunological cells the cns. in the healthy brain, microglial cells are in a surveillance state whereas upon rupture of cns homeostasis they enter activated states. in ischemic stroke, activated microglia is one of the most important cellular components of post-stroke neuroinflammation, which mainly occurs in and around the area of the infarct. microglia activation is characterized by morphological alterations, functional and transcriptional remodeling, which account for the acquisition of immune phenotypes. purinergic receptors are known to play important roles in microglia activation, and p2x4r have been suggested as potential therapeutic target to limit microglia-mediated inflammatory responses associated with brain diseases. in the present study, we have developed an experimental approach based on laser micro-capture to isolate microglia from the penumbra area at different post-lesion times (from 4h to 7 days post-lesion). the repertoire of genes expressed by microglial cells was determined through cdna microarray analysis. with this approach we have been able to determine clusters of genes that are co-regulated thus revealing the time course of microglia activation in this model. in addition, we have compared the transcriptional remodeling of microglia in wild-type and p2x4-deficient mice. our results suggest that wild-type and p2x4r ko mice present specific transcriptional profiles, which only partially overlapped. thus our data suggest that p2x4r play significant roles in the regulation of microglial cells functions. z. chen 1 , m. ghosh 2 , r. sattler 1 , m. robinson 2 , j. rothstein 1 1 johns hopkins university, baltimore, united states 2 university of pennsylvania, philadelphia, united states question: astrocytes, the most abundant cell type in the central nervous system (cns), are a heterogeneous cell population that is despite its functional importance still not well defined. while astrocytes are no longer just considered the supporting cells in the cns and are recognized to play a significant functional role in e.g. synapse formation, regulation of the blood brain barrier and synaptic transmission, little is known whether specific subtypes of astrocytes fulfill these functional tasks differently. studies in our laboratory described brain region specific differences in the expression of astrocytic glutamate transporter 1 (glt-1) with significantly increased levels of glt-1 in the spinal cord compared to brain. the mechanisms of glutamate transporter regulation are not well defined, and to date little is known about the factors that are responsible for regulating protein expression and transporter activity and whether this regulation is specific for certain subtypes of astrocytes. methods: to study glt-1 regulation in more detail, we generated a family of transgenic mice using increasing lengths of the 5 0 non-coding region of the glt-1 gene controlling expression of tdtomato. these mice were crossed with full length bac-glt-1-egfp mice to compare astrocytic expression of the reporter genes. results: interestingly, only a subpopulation of gfp-positive astrocytes was found to have strong tdtomato fluorescence signal, when we crossed an 8.3 kb eaat2 promoter fragment mouse with the bac-glt-1 mouse, suggesting the existence of astroglial subtypes that are differentially regulated for eaat2 expression. such differences might account for variance in local glutamate-dependent excitotoxicity to motor neurons. to this end, we purified both tdtomato1/gfp1 and tdtomato-/gfp1 astrocytes from multiple cns regions by fluorescence-assisted cell sorting (facs) and analyzed their transcriptomic profiles using microarray analysis. a list of candidate genes, mainly transcription factors and cell membrane molecules, was complied from this analysis and validated by both quantitative rt-pcr and immunofluorescence. pax6 and kir4.1 were found to be highly expressed in astrocytes, consistent with previous findings. more importantly, kir4.1 mrna was noted to be selectively enriched in cortical tdtomato1/ gfp1 cells, compared to gfp1 only cells. it suggests kir4.1 may serve as an astroglial subtype marker. conclusions: our data clearly suggest the existence of molecular subtypes of astrocytes. factor expressed by activated astrocytes and microglia. studies with non-glial cells have shown that c/ebpd is able to regulate the expression of proinflammatory genes. however, the role of c/ebpd in neuroinflammation has not been established. we have here analyzed the effects of c/ebpd absence on glial activation in vitro and in vivo. in primary mixed glial and microglial-enriched cultures the expression of the pro-inflammatory genes nos-2, cox-2 and il-6 induced by lps1ifnc, but not by lps alone, was attenuated in the absence of c/ebpd. chip experiments revealed binding of c/ebpd to the promoters of these genes in activated, but not in control, mixed glial cultures. in neuronal-microglial co-cultures the neurotoxicity elicited by lps1ifnc-activated microglia was completely abolished when microglial cells were devoid of c/ebpd suggesting that this transcription factor plays a key regulatory role of the expression of potentially detrimental proinflammatory genes by activated microglia. to analyze the role of c/ebpd in neuroinflammation in vivo mice were treated systemically with lps (100 ug/mouse; i.p.). this treatment induced a marked increase in c/ebpd mrna and protein brain levels. double immunofluorescence experiments showed the presence of c/ebpd in astrocytes and microglia in lps-treated mouse brain. in c/ ebpd -/-mice, systemic lps-induced brain expression of nos-2, tnfa, il-1b and il-6 was attenuated. finally, in mouse experimental autoimmune encephalitis (eae) c/ebpd was upregulated in the spinal cord and the severity of eae symptoms was reduced in c/ebpd -/-mice. altogether these findings demonstrate that c/ebpd plays a major role in the regulation of proinflammatory gene expression in neuroinflammation and point to microglial c/ebpd inhibition as a novel strategy to reduce the detrimental effects of neuroinflammation. supported by pi10/378 and pi12/709 from instituto de salud carlos iii, spain gene targeting strategies have become a powerful technology for elucidating mammalian gene function. the recently generated knockout (ko)-first strategy produces a knockout at the rna processing level and also allows for the generation of conditional ko alleles by combining flp/frt and cre/loxp systems, thereby providing high flexibility in gene manipulation. however, this multipurpose ko-first cassette might produce hypomorphic rather than complete knockouts if the rna processing module is bypassed. moreover, the generation of a conditional phenotype is also dependent on specific activity of cre recombinase. here, we report the use of an efficient molecular biological approach to test pannexin1 (panx1) mrna expression in global and conditional panx1 ko mice derived from the ko-first mouse line, panx1 tm1a(komp)wtsi . using qrt-pcr, we demonstrate that tissues from wild-type mice show a range of panx1 mrna expression levels, with highest expression in trigeminal ganglia, bladder and spleen. unexpectedly, we found that in mice homozygous for the ko-first allele, panx1 mrna expression is not abolished but reduced by 70% compared to that of wild-type tissues. thus, panx1 ko-first mice present a hypomorphic phenotype. crosses of panx1 ko-first with flp deleter mice generated panx1 f/f mice. further crosses of the later mice with mgfap-cre or nfh-cre mice were used to generate astrocyte-and neuronal-specific panx1 deletions, respectively. a high incidence of ectopic cre expression was found in offspring of both types of conditional panx1 ko mice. our study demonstrates that panx1 expression levels in the global and conditional panx1 ko mice derived from ko-first mouse lines must be carefully characterized to ensure modulation of panx1 gene expression. the precise quantitation of panx1 expression and its relation to function is expected to provide a foundation for future efforts aimed at deciphering the role of panx1 under physiological and pathological conditions. agarwal, e. hughes, d. bergles johns hopkins university, baltimore, united states astrocytes elevate intracellular ca21 in response to stimulation of metabotropic receptors. these ca21 transients are linked to processes as diverse as synaptic plasticity and functional hyperemia, but the relevance of this signaling remains uncertain. although most studies of ca21 signaling in astrocytes have focused on somatic events, anatomical and functional studies indicate that ca21 signaling in astrocytes may be compartmentalized into functionally isolated "microdomains." to facilitate analysis of ca21 signaling, we generated transgenic mice in which the cytosolic and membrane anchored variant of genetically encoded calcium indicator gcamp3, referred as cgcamp3 and mgcamp3 respectively, can be expressed conditionally in a cell specific manner. a ubiquitous cag promoter is used to control cgcamp3 and mgcamp3 expression, and a "stopper" sequence flanked by loxp sites was placed upstream of the coding sequence, preventing expression until cre excises this dna. these constructs were targeted to rosa26 locus to ensure widespread expression. to evaluate whether cgcamp3 and mgcamp3 were expressed at levels sufficient to resolve ca21 transients, we bred r26-lsl-cgcamp3 and r26-lsl-mgcamp3 mice to glast-creer mice. double transgenic offspring were injected with tamoxifen at 3 weeks of age and analyzed 2-3 weeks later. histology revealed that cgcamp3 and mgcamp3 were expressed in astrocytes throughout the brain. astrocytes in acute cortical slices exhibited large amplitude, spontaneous increases in both cgcamp3 and mgcamp3 fluorescence. the signal to noise ratio of the fluorescence intensity was greatly improved in astyrocytes expressing mgcamp3, partially due to the ability of mgcamp3 to better resolve near membrane ca21 flux. ca21 transients were typically restricted to small regions of an individual astrocyte, consistent with ca21 elevations within microdomains. spontaneous ca21 transients were occasionally observed in the soma of astrocytes expressing cgcamp3 but not mgcamp3, and were not coincident with activity in the processes, suggesting that these regions are functionally uncoupled. to determine if gcamp3 expression is sufficient to visualize ca21 signaling in vivo, we made cranial windows over the somatosensory cortex and performed 2-photon imaging. microdomain ca21 transients were prominent in cortical astrocytes, indicating that these mice provide new opportunities for understanding the significance of this form of signaling. in the trigeminal ganglion (tg), satellite glial cells (sgcs) surround neurons and are able to release substances that may affect sensory transmission through the tg and contribute to mechanisms underlying craniofacial pain. the aims of this study were 1) to investigate whether sgcs could be provoked to release glu in vitro and 2) to study the in vivo response properties of trigeminal afferent fibres upon artificial elevation of glu concentration in the tg. methods: confocal microscopy was used to assess glu content in and the expression of excitatory amino acid transporter 1 (eaat1) and eaat2 by sgcs of the tg. glu release was investigated in vitro, where sgcs were isolated from the tg of 9 adult male sprague-dawley (sd) rats and treated with control medium or medium containing 10 mm kcl together with 0, 0.1, 1, 10 mm of the eaat1/2 inhibitor tfb-tboa. in vivo electrophysiology experiments were conducted in 6 additional anaesthetized adult male sd rats to determine the effect of repeated intraganglionic injections of glu (500 mm, 3 ml 3 2, 30 min apart) on the response properties of 6 tg neurons that innervated either the temporalis or masseter muscle. cumulative afferent discharge was calculated as the sum of action potentials (ap) 10 min post injection subtracted from the sum of ap 10 min prior to injection. an electronic von-frey hair was used to measure afferent mechanical threshold (mt) at 1 min intervals for 10 min prior to the first and again 20 min after the second microinjection of glu. results: most sgcs were found to be glu positive and express eaat1 and eaat2. treatment with 10 mm kcl resulted in a $2-fold increase in glu concentration, compared to control (10.6 6 1.1 vs. 4.8 6 0.1 mm, respectively; p 5 0.038). treatment with 1 mm tfb-tboa significantly reduced the glu concentration to 5.8 6 1.4 mm (p 5 0.047), which was further lowered to 3.0 6 0.8 mm when 10 mm was applied (p 5 0.001). cumulative afferent discharge evoked in tg neurons by the second injection of glu (20 6 9 ap) was not significantly different from that evoked by the initial injection (21 6 10 ap; p 5 0.92). glu injections significantly reduced muscle afferent mt (baseline: 35 6 19 g) by 30 6 3% (p 5 0.03). these results indicate that glu can be released from sgcs through eaats, and that increased glu concentrations in the tg excite neurons and induce a state of peripheral sensitization. this may hypothetically be a mechanism, whereby activated tg sgcs could contribute to e.g. migraine headache. in an accompanying poster, we present data demonstrating that in cultured neurons l-lactate induces plasticity-related genes expression through a nmda-dependent mechanism (i.e. arc, zif268 and c-fos). here, we describe the effect of l-lactate on neuronal excitability by performing electrophysiological recordings of cultured cortical neurons. we observed that application of l-lactate (10 mm) triggers an inward current with an amplitude of 20.55 1/-0.1 na and slow kinetics, with a peak reached at 189 1/-55s (called ilac). the membrane current decreases gradually to reach a plateau, with a mean inward current of 20.13 1/-0.04 na persisting up to 780s (13 minutes) after addition of l-lactate (called ipyr). in order to assess l-lactate specificity on the generation of these currents l-pyruvate, another monocarboxylate, as well as d-lactate, the non-metabolized enantiomer of l-lactate) were tested. in contrast to l-lactate, l-pyruvate (10 mm) induced a low-amplitude sustained current (20.07 1/-0.03 na) similar to the late-phase slow current evoked by l-lactate (i.e ipyr), but no current similar to ilac. dlactate on its side did not elicit any specific current. pharmacological characterization of ilac and ipyr currents demonstrate that ilac is generated by nmda receptors (as it is blocked by mk801) and relies on active nmda receptors (as it is blocked by either glutamate or glycine binding sites inhibitors). in contrast, generation of ipyr by both l-lactate and lpyruvate is prevented by diazoxide, a katp opener, whereas ilac was unaffected by the treatment. in order to determine the importance of katp for plasticity-related gene expression, the katp closer glibenclamide (200 um) was applied to neurons and mrna gene expression of arc and zif268 quantified. results obtained demonstrate that arc and zif268 mrna expression are unaffected by glibenclamide. as a whole this set of data demonstrates that l-lactate generates two types of inward currents in neurons i.e. nmda-dependent (ilac) or katp-dependent (ipyr). moreover, they highlight the nmda-dependent ilac current as the key mediator, in contrast to katp, of l-lactate-induced plasticity gene expression. this is further sustained by the observation that l-pyruvate does not reproduce the effect of l-lactate on gene expression (see accompanying poster). this set of results provides novel insights into the mechanisms of action of l-lactate as an inducer of plasticity gene expression, and an important signaling molecule for neuronal plasticity. the reliance on drg neuron co-culture for sclcs fate commitment stands as a major hurdle for the therapeutic application of this finding. thus, we aim to unravel the mechanisms underlying sclcs fate commitment in order to develop a drg neuron-free condition for generating fate committed schwann cells. we hypothesised that the switch is brought about by the membrane bound neuregulin (nrg), nrg 1 type iii, but not the other soluble isoforms of neuregulin. as our results show that sclcs treated with soluble neuregulin did not show significant changes in morphology nor marker expression when compared with untreated sclcs. nrg 1 type iii expression was observed on purified drg neurons by immunocytochemistry, western blot analysis as well as reverse transcription pcr for the mrna. mammalian expression constructs for nrg 1 type iii were made by transfection into human embryonic kidney cells, hek 293t, and mouse embryonic fibroblasts (mef) with the aim of generating surrogate cell types for co-culture with sclcs to pursue cell-specific effects of nrg 1 type iii on differentiation of sclcs. the findings promise a way for generating fate committed schwann cells for autologous transplantation for recovery after nerve injury. (2) the co 2 /hco 3 buffer system. the latter being an open buffer system, because biomembranes are usually permeable to co 2 , and, in addition, most cells express hco 3 transporting membrane proteins. the enzyme family of carbonic anhydrases (cas) catalyses the reversible reaction from co 2 and h 2 o to hco 3 and h 1 , and thereby modulates the intracellular h 1 buffer dynamics.we have performed calibrated in situ live-cell imaging with the proton-sensitive fluophore bcecf in glial cells and neurons of acute cerebellar slices of wild-type and knockout mice. the studies were used to quantify the intracellular buffer capacity and to dissect the contribution of the intracellular caii and the extracelluar caiv by comparing intracellular h 1 shifts in glial cells and neurons from wild-type mice and mice deficient in their caii or caiv gene. we found that only one proton in 400000 is unbound and thereby chemically active, and that about 50% of this buffer capacity is mediated by the co 2 /hco 3 buffer system and the other 50% by intrinsic buffers. the rate of co 2 -induced change in intracellular h 1 concentration was increased by intracellular caii in both glial cells and neurons. the extracellular caiv on the other hand affected primarily neurons, but also showed unexpected intracellular activity (schneider et al. 2013, pnas 110). the rates of acidification and alkalinisation in wild-type and knockout mice were significantly reduced in the presence of the ca blocker 6-ethoxy-2-benzothiazolsulfonamid (10 mm). we confirmed ca protein expression by western blot and could also show that the expression of the remaining ca is not upregulated. these results provide new insights into cellular proton-coupled processes in acute neural tissue, which shows some new complex roles of carbonic anhydrases in shaping proton dynamics in cells and tissues. objective: it is becoming increasingly evident that inflammatory mechanisms promote neuronal hyper-synchronization and epileptogenesis following brain insults. our recent studies identified serum albumin as a key player in the inflammatory cascade leading to epilepsy, at least partially through the activation of tgf-b signaling pathway in astrocytes. in this study we set out to explore the mechanisms by which tgf-b1 induces glial activation and epileptogenesis. methods: primary cultures of microglia and astrocytes were treated with tgf-b1 and gene expression analysis along with protein quantification were performed by quantitative pcr and elisa. electrophysiological response to il-6 was obtained in acute brain slices (field potential recordings) and in-vivo (sub-dural corticoencephalography) following to a continuous administration of il-6 into the lateral ventricle for 7 days. results: in glia cultures tgf-b1 induced early and rapid up-regulation of il-6 at both mrna and protein levels whereas the upregulation of other pro-inflammatory cytokines such as il-1b and tnf-a was milder and at a later time point. notably, smad2/3-dependent tgf-b1 signaling induced the expression of il-6 primarily in astrocytes and to a significantly lesser extent in microglia. il-6 was sufficient to induce epileptiform activity in acute brain slices and recurrent seizures within 3-4 days a following continuous intracerebroventricular injection of il-6. conclusions: we thus suggest astrocytic release of il-6 as a potential key mechanism in epileptogenesis. okayama univ., dept. of brain science, okayama, japan dopamine transporter (dat) is expressed not only in catecholaminergic neurons but also in astrocytes. we previously showed that repeated l-dopa treatment markedly induced expression of dat and apparent dopamine (da)-immunoreactivity in the reactive astrocytes in the striatum of animal models of parkinson's disease. it is thought that astrocytes can uptake both l-dopa and da via neutral amino acid transporter lat and dat, respectively. therefore, uptake and metabolism of l-dopa and da in the striatal astrocytes may influence their availability in dopaminergic system of parkinsonian patients. to clarify uptake and metabolism of l-dopa and da in striatal astrocytes, the contents of l-dopa, da and their metabolites were measured after the l-dopa/da treatment using primary cultured astrocytes. first, we revealed the expression of neutral amino acid transporter lat and aromatic amino acid decarboxylase (aadc) in the striatal astrocytes. the level of l-dopa in astrocytes was markedly increased after 4-hr l-dopa exposure, but da was not detected in the astrocytes 4 or 8 hr after the treatment. on the other hand, the da treatment for 4 hr increased levels of da and its metabolites, especially dopac. these results indicate that uptaken da into astrocytes is rapidly metabolized and that uptaken l-dopa never be converted to da in astrocytes. furthermore, level of uptaken l-dopa in cultured striatal astrocytes was rapidly decreased after removing extracellular l-dopa. taken together, the present results suggest that striatal astrocytes act as a reservoir of l-dopa to uptake or release l-dopa depending on the extracellular l-dopa concentration, but have less ability to convert l-dopa to dopamine. s. limmer, c. kl€ ambt universit€ at m€ unster, m€ unster, germany neuronal function consumes a large amount of energy and thus the brain requires a constant but regulated supply of metabolites. in invertebrates, such as drosophila, the nervous system is floating in the hemolymph and all metabolites that enter the brain have to be transported across the blood brain barrier (bbb). as in primitive vertebrates, the drosophila bbb is generated by glia. a layer of so-called subperineurial glial cells completely encapsulates the nervous system and thus, all metabolites reach the neurons en route through glia. the main energy supply of the inner organs of drosophila is provided by trehalose that is found in high concentrations in the hemolymph. trehalose is a nonreducing disaccharide, in which two dglucose units are linked via a a,a-1,1-glycosidic bond. the uptake of trehalose is mediated by two trehalose transporters, which are members of the slc2a gene family. the subperineurial glial cells then either secrete c 6 sugars to the neurons -or alternatively supply energy to neurons by secreting c 3 metabolites as described for the bee retina. to discriminate between these possibilities we have followed a number of different approaches. the relevance of trehalose transporters in the different cell types has been determined by mutant analysis and rnai studies. furthermore, we have studied the role of all other putative sugar and monocarboxylate transporters in glial cells by rnai knockdown. to determine whether c 6 or c 3 carbohydrates are secreted by glial cells we suppressed the expression of core enzymes regulating glycolysis or components of the respiratory chain in either glial cells or neurons. moreover, we have ablated mitochondria specifically in glia or in neurons through expression of a restriction enzyme targeted to mitochondria. we will summarize our results and discuss approaches towards identifying neuronal signals that control glial carbohydrate secretion during energy homeostasis of the brain. microglia have long been known to respond to virtually any insult to the central nervous system. they quickly migrate to the site of the insult, and play many important roles, such as barricading the injury site, phagocytosing debris, and releasing cytokines. the role of microglia in the normal brain, however, has only recently begun to be appreciated. with the advent of in vivo imaging, microglia have been shown to be constantly surveying their environment with rapid extensions and retractions of their processes. subsequent studies have shown that these 'resting' microglia (now known as 'surveying microglia') are indeed active throughout development and adulthood. during development, microglia have been shown to be involved in synaptic monitoring and pruning as well as synaptic stripping after injury. thus far, many studies have focused on the role of microglia at the synapse. however, we report here, for the first time, that in the cortex of normal adult rodents, a small percentage of microglia are specifically associated with the axon initial segment (ais). the ais is characterized by a high density of voltage-gated ion channels and plays an important role in initiation of the action potential and maintenance of neuronal polarity. this interaction seems to be limited to the 'surveying' phenotype of microglia and much less frequent in 'activated' microglia. this overlap of processes appears early in development and continues throughout adulthood although the function of microglia at the ais is still currently unknown. x. liu 1 , j.-m. petit 2,3 , p.j. magistretti 2,3 , c. giaume 1 1 cirb, collège de france, paris, france 2 lndc, brain mind institute, epfl, lausanne, switzerland 3 centre de neurosciences psychiatriques, chuv, prilly, switzerland astrocytes act as modulators of neuronal activity through mechanisms such as neurotransmitter uptake, 'gliotransmitters' release, and metabolic supply. recently, astrocytes were shown to modulate sleep by vesicular release of atp. besides the cellular mechanism, we are interested in the network behavior of astrocytes in sleep-wake cycle. indeed, astrocytes form networks of communicating cells via gap junction channels constituted by connexins (cxs). recently, we have demonstrated that astroglial networking is regulated by neuronal activity and that neuronal activity is affected by deletion of the two main astroglial cxs (cx43, cx30). to investigate how astroglial networks behave in sleep-wake cycle, we used pharmacological treatments and sleep deprivation to manipulate sleep-wake status of mice and determined whether astroglial networks would change in response. astroglial networks in acute cortical slices were revealed by "dye coupling", i.e. by loading of a patch-clamped astrocyte with sulforhodamine b that diffuses into neighboring astrocytes through gap junction channels. also, cxs expression at mrna and protein levels was measured as an index of connections among astrocytes. the results are as follows. first, i.p. injection of modafinil, a potent wakefulness-promoting drug, increased both mrna and protein expression of cx 30 in the mouse cortex. superfusion of modafinil also enhanced dye coupling among astrocytes in acute coronal slices of the mouse somatosensory cortex. this effect was abolished by ttx treatment, which demonstrates neuronal activity is necessary for the effect of modafinil. in contrast, c-hydroxybutyric acid (ghb), a sleep-promoting agent, and propofol, a general anesthetic, decreased astroglial coupling in cortical slices. interestingly, the effect of ghb was not affected by ttx, suggesting a different pathway of action from modafinil. next, we used a "gentle" sleep deprivation model to sleep deprive mice for 6 hours from the onset of the light period. as a result, mrna of cx30, but not cx43, was increased in both the cortex and hippocampus. finally, dye coupling in cortical astrocytes was enhanced in mice after sleep deprivation compared to mice after normal sleep. this increase in dye coupling, however, was not observed in slices from cx30 knock out mice after the same sleep deprivation treatment. altogether these results indicate that astroglial networks are bidirectionally regulated by perturbations in sleep-wake cycle and that cx 30 is the major cx sensitive to such perturbations. in neurons, synaptic and extrasynaptic gaba a receptors (gaba a rs) differ in their subunit composition, conferring them distinct functional and pharmacological properties. oligodendrocyte precursor cells, also called ng2 cells, are contacted by bona fide neuronal gabaergic synapses. however, we recently showed a synaptic to extrasynaptic switch in the mode of transmission between gabaergic interneurons and ng2 cells during postnatal development of the somatosensory cortex. we therefore hypothesized that the postnatal switch of gabaergic transmission in ng2 cells is accompanied by changes in the expression of gaba a r subunits. to test for this hypothesis, we stimulated neuronal fibers to evoke gaba a r-mediated responses in ng2 cells recorded in acute slices of the somatosensory cortex of ng2-dsred transgenic mice. the effect of zolpidem and a5ia on evoked gabaergic responses reveals the predominance of functional a1-and a5-containing gaba a receptors, respectively, at interneuron-ng2 cell synapses early in development. however, the expression level of a5 decreases when responses rely exclusively in extrasynaptic transmission at more mature developmental stages. more importantly, specific pharmacology for the c2 subunit, a crucial molecular component for the clustering of gaba a receptors at postsynaptic sites, demonstrated a down-regulation of this subunit in ng2 cells prior to the complete loss of gabaergic synaptic activity. in keeping with the synaptic nature of the c2 subunit in neurons, this molecular change in ng2 cells correlates with the switch from synaptic to extrasynaptic transmission. to corroborate these functional data, we performed single cell multiplex rt-pcr of a1-5, b1-3, c1-3 and d subunit mrnas in ng2 cells of the second and fourth postnatal weeks. despite the large heterogeneity of subunit mrna expression at both developmental stages, we found that the number of cells expressing c2 mrnas dramatically decreases in the fourth postnatal week. in conclusion, the expression loss of the c2 subunit is an important molecular determinant impacting the change of transmission modes between interneurons and ng2 cells during cortical development. financial support: frc, anr blanche, arsep, dfg (sfb/tr3) and eu (fp7-202167 neuroglia). university of rochester, medical center, rochester, united states synaptic plasticity is a critical process throughout the lifespan for achieving and maintaining normal and efficient operation of the nervous system. while much is known about the functional and structural changes that occur at the synapse during synaptic plasticity, the mechanisms implementing these changes are poorly understood. despite being classically characterized as immune cells, microglia have recently been shown to play a role in normal brain function by restructuring and removing synapses. given the novelty of this role, few details are known about how microglia behave to implement such a role during periods of plasticity. to characterize the changes in microglial behavior during ocular dominance plasticity in the developing visual cortex, we examined microglial morphology in fixed brain sections as well as in vivo using two-photon microscopy. for analysis of microglia in fixed sections, c57/bl6 mice were monocularly deprived for either 12 hours, 1, 2, 4, or 7 days during the visual critical period (p23-p30). fixed coronal sections were stained with iba1, a specific marker for microglia, imaged on a confocal microscope and analyzed using imagej. microglial density decreased rapidly (within 12 hours) and remained low for the 7 day deprivation period. characterization of microglial morphology revealed an increase in the complexity of the microglial process arbor after 12 hours, which steadily decreased back to baseline by 7 days. this suggests that microglia do undergo a rapid change in morphology during plastic events that is reminiscent but distinct from the traditional activation pattern observed during pathological events. for analysis of microglia in vivo, heterozygous cx3cr1/gfp (jung 2000) mice were monocularly deprived directly preceding the first imaging session, the binocular segment of primary visual cortex was imaged, and the same cortical area was re-imaged 2 and 4 days later. while no change in microglial density was observed, analysis of microglial process motility showed a decrease in motility after periods of monocular deprivation. these results, combined with the observed morphological changes, show that microglia undergo behavioral changes after a period of monocular deprivation that are inconsistent with pathological activation, suggesting that microglia take on different roles and activities during normal brain function. g. baltazar, j. oliveira, t. roxo, c. fonseca faculty of health sciences, cics-ubi, covilhã, portugal neuroinflammation is a pathological hallmark in patients and experimental models of parkinson's disease. both present the classical features of inflammation, with evidence of an uncontrolled process. moreover, microglia may become activated early in the disease process and remain primed, responding strongly to subsequent stimuli, and thereby enhancing inflammation-induced oxidative stress and cytokinedependent toxicity in vulnerable neuronal populations. we have previously demonstrated that glial cell line-derived neurotrophic factor (gdnf), released by ventral midbrain astrocyte cultures, potently prevents microglial activation induced by a pro-inflammatory agent. therefore, gdnf is an important mediator in the astrocytemicroglia crosstalk keeping microglia in a resting state. however, in the brain, microglia and astrocytes are not alone and other cell types, e.g. neurons, may interfere and influence this astrocytic control of inflammation. therefore, it is important to investigate if the presence of neurons alters gdnf-mediated astrocytic control of microglial activity. in this work we aimed at investigating whether the presence of neurons, injured or not, changes the ability of astrocyte-derived gdnf to prevent microglial activation. for that purpose, we used primary cultures of ventral midbrain astrocytes and microglia, as well as neuronastrocyte mixed cultures from the same brain region. neuron-astrocyte cocultures were exposed to vehicle (control) or to the da neurotoxin mpp 1 . the media conditioned by control and mpp 1 -challenged neuron-astrocyte mixed cultures, or by astrocyte cultures, was collected and transferred to ventral midbrain microglia cultures, which were then exposed to the pro-inflammatory agent lipopolysaccharide. the effect of conditioned media obtained from the different cell culture systems on microglial activity was determined by analyzing the production of nitric oxide by the griess reaction and of the phagocytic activity by determining the engulfment of fluorescent microspheres by fluorescence microscopy. university of lausanne, lausanne, switzerland lactate is produced by astrocytes and used by neurons as metabolic substrate during activity. recent evidence indicates that lactate may not only serve as energy substrate but also as modulator of glutamatergic or gabaergic neurotransmission. we tested this hypothesis using primary cultures of cortical neurons obtained from wild type and gad67-gfp knock-in mice (c57bl/6). neuronal excitability was monitored by electrophysiological recordings and calcium imaging using the fluorescent probe fluo-4 am. spontaneous action potentials and rhythmic calcium transients were present in more than 50% of cultured neurons, which were either gabaergic or glutamatergic cells. in the presence of 5mm glucose, l-lactate application reversibly diminished calcium transient frequency in a concentration-dependent manner in both cell types (glutamatergic neurons ic50 4.23 6 1.9mm and gabaergic neurons ic50 4.18 6 2.8mm). to test whether lactate effects were dependent on metabolism, we applied the closely related substrate pyruvate (5mm) or switched to different glucose concentrations (0.5 or 10mm). none of these conditions altered the calcium transient frequency. in contrast, the application of d-lactate, thought not to be metabolized by neurons, decreased the spiking activity in the same concentration range as l-lactate (ic50 4.58 6 1.2mm). we determined that d-lactate was taken up by neurons, however more than two-fold less efficiently than l-lactate. these results suggest that the mechanisms of lactate sensitivity are not purely metabolic. since d-lactate has the same potency as l-lactate but it is less internalized, it is likely that lactate acts as an extracellular ligand that triggers the reduction of neuronal activity. this hypothesis is currently under investigation. this modulating effect of lactate represents a novel role for the compound that has to be taken into account in the context of the interactions between astrocytes and neurons. ben-gurion university, ber-sheva, israel microglia integrate within the neural tissue with a distinct ramified morphology through which they scan the surrounding neuronal network, a process which appears to contribute to the integrity, maintenance and functioning of the brain. since microglia are long-lived, they are subjected to senescence processes which may severely compromise their function with age. here we used a digital tool for the quantitative morphometric characterization of fine cortical microglia structures in mice. we thus followed the morphological changes microglia underwent with aging and with the progression of alzheimer's-like disease. compared with microglia in young mice, microglia in old mice are less ramified and possess less branches and fine processes, a phenomenon which was associated with increased expression of pro-inflammatory genes. notably, a similar microglial pathology appeared 6-12 months earlier in mouse models of alzheimer's disease (ad). we thus demonstrate that in addition to promoting neurotoxic inflammation, amyloid plaques attract the microglia and modify their structure. this, in turn, causes a severe microglial process deficiency, which possibly results in compromised neuronal function and repair. blood-borne glucose is the main energy substrate for the brain under physiological conditions. however, the question remains unresolved if the two main cell types in the brain -neurons and astrocytes -consume glucose upon their individual needs or if glucose is mainly taken up by one cell group and glycolytically degraded to energy-rich substrates (e.g. lactate), which are then shuttled to the other cell group. a novel genetically encoded fret glucose sensor (bittner et al., 2010, 2011) together with two-photon microscopy now provides for the first time the opportunity to determine intracellular glucose concentrations in single cells in the intact organism. cortical microinjections of an adenoviral vector (aav9 and aav6) coding for the sensor flii 12 pglu600md6 with the promoter sgfap for astrocytes and synapsin for neurons were performed in mice and chronic windows were implanted above the somatosensory cortex. after two weeks, specific expression in astrocytes and neurons was observed. the ratiometric fret signal in both cell types increased after intraperitoneal glucose injection and decreased after insulin application demonstrating the functionality of the construct in vivo. the astrocytic sensor displayed a large linear range at blood glucose levels ranging from 3 to 20 mmol/l. in a second set of experiments, we examined the dynamic behavior of cellular glucose concentrations upon increased brain activity. during electrical hindpaw stimulation 21% of examined astrocytes showed a 0.5% decrease of the fret signal, whereas neuronal glucose levels did not exhibit activity-dependent fluctuations. to be able to draw conclusions about metabolic rates of substrate oxidation we recently started with experiments where cellular glucose uptake was transiently inhibited using different blockers of glucose transport. drugs were applied systemically and intracortically. we could demonstrate this principle on first measurements on astrocytes. glucose fret sensors allow measurements of the dynamics of glucose concentrations of single neurons and astrocytes in the intact organism. measurements of cellular glucose transients during transport blockade hold the potential to compare glycolytic rates of neurons and astrocytes. this tool may substantially contribute to uncover the complex organization of brain energy metabolism. neuron-glial communication in the cns is fundamentally important for many brain processes including synaptic transmission and plasticity. perisynaptic astrocytic processes (pap) contact excitatory synapses, forming tripartite structures with neurons. in the hippocampus, the morphology of pap has been shown to remodel rapidly and continuously but the mechanisms and roles of this form of structural plasticity remain unknown. this study investigated the physiological mechanisms driving pap movements and their role during long-term potentiation (ltp). pap and spines contacts were labeled by viral gene delivery of farnesylated fluorescent proteins in hippocampal slice cultures and imaged by confocal microscopy. electron-microscopy of infected slices confirmed that pap-synapse morphology is preserved in organotypic cultures. pap movements adjacent to dendritic spines were evaluated with an index of motility (mi). increasing neuronal activity by schaffer collaterals (sc) stimulation elevated pap mi and this was prevented by both ttx and mglur inhibition, suggesting that neuronal activity modulates pap movements. we next investigated whether intracellular calcium (ca 21 i ) in astrocytes influenced pap motility. bapta-am bulk-loading specifically chelated ca 21 i in astrocytes and reduced pap movements. when exogenous gq-coupled receptors mrga1 or mrgc11 were specifically targeted to astrocytes, their agonist fmrfa induced ca 21 i increases and accelerated pap motility. moreover, fmrfa delivery at the synaptic level by two-photons flash photolysis was sufficient to elevate pap mi. we then investigated pap dynamics in relation to ltp. application of theta-burst sc stimulation initially increased pap motility, but then resulted 30min later in a decrease of pap motility. interestingly, the reduction of pap movements correlated with both spine enlargement and increased pap's spine coverage. to assess the consequences of these changes, we then specifically triggered elevation of pap movements at the synaptic level by flash photolysis. spine stability analysis 24 hour after uncaging revealed that spines in contact with activated paps were more stable than others. this study suggests that excitatory synapses control the motility of surrounding paps through the triggering of neurotransmitter-evoked astrocytic ca 21 i elevations. increased pap motility seems to be necessary to elevate their coverage of the synapse during ltp, leading to higher synapse stability. astrocyte reactivity occurs in response to many pathological brain situations. reactive astrocytes display morphological and functional changes that could result in concentration variations of some brain metabolites localized both in astrocytes and in neurons. such changes could be detected by magnetic resonance spectroscopy (mrs), allowing non-invasive monitoring of astrocyte reactivity and thereby neuronal dysfunction in vivo. one of the major peak detected by mrs in the brain corresponds to neuronal metabolite n-acetyl-aspartate (naa). although its function is unclear, it is considered as a biomarker for neurodegeneration and a decrease in its concentration is interpreted as neuronal death or dysfunction. however, there is not clear evidence that other cell types, specifically astroglia, could not contribute to change in naa levels in the brain. in this study, we used a rat model of astrocyte activation by stereotaxic injections of lentiviral vectors encoding for either the cytokine ciliary neurotrophic factor (lenti-cntf) into the right striatum or betagalactosidase (lenti-lacz) into the left striatum, as a control. mrs data were acquired on a 7t magnet from two voxels placed over each injected striatum. a diffusion-weighted laser sequence with echo time te 5 40 ms was used. concentrations were measured using lcmodel software for total n-acetyl-aspartate (naa 1 naag), myo-inositol (ins), total choline (cho), glutamate and taurine relative to total creatine. lenti-cntf injection promotes a sustained, extensive and selective activation of astrocytes, as evidenced by overexpression of gfap and vimentin and cellular hypertrophy (fig.1) . importantly, neurons displayed unaltered morphological, molecular and electrophysiological features, as described previously (escartin et al., 2006; beurrier et al., 2010). mrs evidences changes in metabolite concentration: in the lenti-cntf injected striatum, levels of the astrocyte metabolites ins and cho were increased, whereas levels of the neuronal metabolite naa and glutamate were decreased. increased levels of ins and cho are generally found associated with astrocyte reactivity, however, the decrease in naa is unexpected given that neurons are not dysfunctional with cntf. to understand how astrocyte reactivity can influence naa levels, we are currently characterizing the molecular changes occurring in the naa metabolism using quantitative pcr, biochemistry, histology and hplc. our data suggest that reactive astrocytes alone result in alterations of metabolite concentrations detectable by nmr. although the mechanisms by which activated astrocytes regulate neuronal metabolism remain to be elucidated, our work challenges the mrs dogma that decreased neuronal metabolites can be used as a biomarker for neurodegeneration. microglial phagocytosis is a vital phenomenon for the clearance of damaged and death cells or infectious agents in a context of brain injury or infection, respectively. in addition, microglia can boost synaptic plasticity by the phagocytosis of axon terminals and dendritic spines. therefore, it is crucial to better understand the mechanisms involved in microglia clearance in order to devise new strategies to promote an efficient brain repair. recently, we showed that histamine modulates microglia motility and cytokines release. in this work, we aimed to investigate the role of this molecule and its receptors in microgliainduced inflammation by evaluating microglial phagocytic activity and reactive oxygen species (ros) production. for that purpose, an iggopsonized latex bead assay was performed in n9 murine microglial cell lines exposed to lipopolysaccharide (lps) or histamine 1, 10 and 100 mm. we showed that histamine significantly stimulated phagocytosis of opsonized latex beads via h1 receptor activation. this effect was accompanied by the rearrangement of cytoskeleton analyzed by phaloidin and acetylated tubulin expression and cellular distribution. the phagocytic activity was significantly reduced after pretreatment with apocynin, a putative napdh oxidase (nox) inhibitor. all nox isoforms were expressed by microglial cells, but only the expression of nox1 was increased by treatment of histamine. rac1, an important subunit for the activation of nox1, was also activated by histamine. in addition, histamine induced ros production via h1 and h4 receptor activation. in conclusion, histamine plays an important role in the regulation of microglial phagocytosis, which is mediated by nox1 and rac1 activation. multiple sclerosis (ms) is a chronic inflammatory disease of the central nervous system characterized by demyelination and axonal damage, involving both white (wm) and gray matter (gm) areas. furthermore, cortical pathology correlates with ms progression and cognitive dysfunction. we previously found altered expression of gap junction (gj) proteins connexin32 (cx32) and cx47 in oligodendrocytes and cx43 in astrocytes in wm lesions and normal appearing wm (nawm) in brain samples from ms patients, and in a mouse model of experimental autoimmune encephalomyelitis (eae), implicating uncoupling of myelinating cells as an important aspect of ms pathology. here we studied cx32 and cx47 and their astrocytic partners cx30 and cx43 in and around cortical lesions and the normal appearing cortex (nacx) in ms samples and non-ms controls. we examined their expression by real-time pcr, quantitative immunoblot and immunohistochemichal analysis. compared to non-ms controls, expression levels of cx32 and cx47 mrna were reduced within lesions whereas cx30 and cx43 were increased. in nacx, all four connexins were upregulated with cx47 being significantly increased. immunoblot analysis of ms cortex revealed reduced cx32 and increased cx43 levels, while cx30 and cx47 showed no significant change. immunohistochemical analysis showed severely reduced cx47 gj plaques within lesions with gradual increase toward nacx. in contrast to nawm, cx47 gj plaques were not increased in nacx. both astrocytic gj proteins were increased in ms, but in distinct patterns: cx30 gj plaque counts were higher within lesions and perilesional areas. cx43 was increased in ms cortex compared to non-ms controls, but in contrast to nawm, cx43 gj numbers were higher in nacx than in lesions. similar alterations were found in the eae mice, in which cx32 and cx47 gj counts were reduced whereas cx43 and cx30 gjs were increased. our results indicate that oligodentrocytic as well as astrocytic gjs are affected not only in ms wm but also in the gm. loss of oligodendrocyte gjs and in parallel increase of astrocytic gjs reflecting astrogliosis occurs throughout the ms brain and may negatively affect the prospects of repair and remyelination, worsening disease progression. acknowledgements: this project was funded by the cyprus research promotion foundation (grants access/0308/11 and health/bios/ 0308/01 to kak) and by cyprus telethon (2010-14 grant to kak). post-mortem human brain tissues were kindly provided by the uk multiple sclerosis society tissue bank, imperial college london, funded by the uk multiple sclerosis society. the mainly astroglialocated enzyme glutamine synthetase is required to synthesize glutamine from the re-uptake of either glutamate or gaba. thereby, this enzyme significantly contributes to the control of brain glutamate and gaba levels. there is good evidence that both neurotransmitter systems are dysregulated in depression. hence, we decided to study the cellular expression of this enzyme in subjects with major depression and bipolar disorder. material and methods: we investigated the numerical density of glutamine synthetase immunoreactive astroglial cells in different brain areas (prefrontal cortex, anterior insula) of 14 subjects with major depression, 15 subjects with bipolar disorder and 19 matched control cases. results and discussion: we found that compared with controls in cases with major depression there was a significant reduction of the density of immunostained glial cells in all brain regions studied, whereas bipolar cases showed a normal expression of the enzyme. our findings point to a profound disturbance of the glutamateglutamine-gaba cycle in major depression, which is in good agreement with neuroimaging observations and molecular biologic data of others. m.-c. marx, d. billups, b. billups university of cambridge, cambridge, united kingdom perisynaptic astrocytes are thought to play a key role in the recycling pathway of glutamate by supplying the precursor glutamine to the presynaptic terminal. using fluorescent ph i measurements of astrocytes and direct patch-clamp recordings from the calyx of held synapse in rat brain slices, we have investigated the transport mechanisms that may mediate this release of glutamine from astrocytes in situ and assessed the role of glutamine in maintaining glutamatergic neurotransmission. glutamine transporter currents were elicited by puff application of 10 mm glutamine from a nearby pipette. for imaging astrocytic ph i , hpts was included in the patch pipette. system a (sa) transporter function was probed using the substrate inhibitors meaib, alanine and serine, while system n (sn) function was isolated using its unique ability to transport li 1 in place of na 1 . histidine was used as a substrate inhibitor of both sa and sn. glutamine-induced membrane currents i gln (220.0 6 2.4 pa, n 5 21) were blocked by bath applied alanine (10 mm) and meaib (20 mm), as well as substitution of external na 1 with li 1 . fluorescent ph i measurement revealed an alkalinisation during puff application of glutamine. while bath application of histidine (20 mm) abolished the ph change, meaib and serine (20 mm) only attenuated it. most of the ph change (63 6 11%; n 5 7) was insensitive to the substitution of na 1 with li 1 . in presynaptic terminals, puff application of glutamate did not induce a membrane current indicating a lack of direct glutamate uptake; however, puff application of glutamine resulted in a i gln which was eliminated by the removal of external na 1 and by meaib. miniature epscs recorded from postsynaptic neurons (238.8 6 3.5 pa) were inhibited by meaib (228.6 6 3.4 pa; n 5 5) following turnover of the presynaptic glutamate pool. single epscs were unaffected by meaib indicating no direct effect on postsynaptic ampa receptors. together, these data identify two glutamine transport systems within astrocytes. properties of the glial i gln indicate expression of sa while ph imaging reveals a non-electrogenic glutamine transporter with properties identifying it as sn. sn is known to be readily reversible under physiological conditions and we propose that this mediates the astrocytic glutamine release mechanism within the glutamate-glutamine cycle. furthermore, system a mediates glutamine transport in glutamatergic nerve terminals and is important in maintaining the supply of glutamate for excitatory neurotransmission during periods of high frequency stimulation. a. briens, m. schwalm, f. docagne, d. vivien inserm u919, caen, france astrocytes are key players in the brain, cooperating with neurons to modulate crucial processes. in particular, they are able to uptake and release neurotransmitters and neuromodulators to control their synaptic level. in our study, we hypothesize that astrocytes can regulate some effects of the serine protease plasmin in the cns through a mechanism of uptake. conversion of the zymogen plasminogen to the serine protease plasmin by the activators (tpa or upa) is the basis of fibrinolysis in the vasculature. but cerebral functions for the plasminogen activator/plasmin system have recently arisen. it has been shown that plasmin was essential to activate the brain derived neurotrophic factor (bdnf) in its mature form, promoting long-term hippocampal plasticity. in alzheimer's disease, plasmin plays a protective role by participating in beta-amyloid clearance. plasmin can also lead to neuronal injury by degrading extra-cellular matrix proteins. in animal models of multiple sclerosis, plasmin can also help removing intraparenchymal deposits of fibrin, thus limiting axonal damage. these data suggest that a system of regulation of plasmin effects in the brain is necessary. in this study, we showed that astrocytes can actively and specifically uptake plasminogen and plasmin in a time-dependent and a dose-dependent manner. this mechanism involves clathrin pits formation in astrocytes and the lysine-binding site of plasminogen/plasmin. plasminogen and plasmin, following endocytosis are found in early endosomes, late endosomes, lysosomes and recycling endosomes. finally, we noticed that astrocytes uptake plasmin more efficiently than plasminogen. this study brings out a mechanism of glial uptake of plasminogen and plasmin. this could control their extra-cellular levels and regulate their effects within the brain. further investigations should determine whether this mechanism can modulate plasminogen activation and synaptic plasmin activity. besides, plasminogen activators (tpa) and cerebral substrates of plasmin (pro-bdnf) are also taken up by astrocytes. a link could thus exist between these processes to regulate the amount of synaptic mature bdnf. this would reveal a cross-talk between neurons, releasing precursors in the synapse (pro-bdnf and plasminogen) and astrocytes, regulating their activation and their clearance. understanding and modulating plasmin uptake could be very important in pathological conditions where the plasminogen/plasmin system is involved, such as alzheimer's disease, multiple sclerosis or stroke. healthy brain aging is characterized by neuronal loss and cognitive decline being inflammation a major causative factor. microglial activation is considered to be a major driver for the neuropathological findings in alzheimer's disease (ad). we evaluated whether young vs. aged microglia responded differently to ab soluble oligomers (abo) and to conditioned media from abo-treated hippocampal neurons. microglia from 1 day cd1 pups were incubated for 24 h at 2 (young) and 16 (old) days in vitro (div) with abo (ab1-42 at 50 and 1000 nm). in parallel, e16 mice hippocampal neurons were treated with abo at 4 (young) and 18 (old) div for 24 h, and the incubation media used as conditioned media to treat microglia, at respective div, for further 24h. cell migration (boyden chamber), extracellular content in glutamate (commercial kit), and activity of matrix metalloproteinases (mmp)22 and 29 (gelatin zymography) were assessed. an increased migration towards abo and atp was observed in young microglia but almost undetectable in aged cells (p < 0.01). interestingly, microglia exposure to conditioned media from 1000 nm abotreated neurons markedly decreased both young and old microglia migration, while treatment with media from 50 nm abo-treated neurons enhanced migration, mainly in the young microglia (p < 0.05). concerning glutamate, aged cells released less than young ones upon abo treatment (p < 0.01). in addition, when exposed to neuronal conditioned media, only the young microglia were able to exert a neuroprotective role by reducing the media glutamate content. curiously, while abo promoted a microglia release of mmp-9, independently of cell age, it only induced mmp-2 secretion in old cells in an abo concentrationdependent manner (p < 0.05 for 1000 nm abo). in contrast, conditioned media from abo-treated neurons elicited mmp-2 release only from young microglia. together, our data point to a loss of microglia function towards abo with ageing and are unique in suggesting that senescent microglia may release high levels of mmp-2 in ad. in addition, our results indicate that abo-treated neurons enhance the release of mmp-2 even by young microglia. whether this finding may contribute to enhance inflammatory stress and the onset of ad will be the aim of further studies. human immunodefeciency virus-1 (hiv-1) productively infects microglia within the central nervous system (cns). viral replication within microglia and the resulting immune response and neurotoxicity leads to significant cognitive impairments not limited to dementia, neurocognitive abnormalities, and memory deficits. combination antiretroviral therapy reduces the cognitive impairments associated with hiv-1, but the regulatory hiv-1 tat protein is still produced in the cns during treatment, even in the absence of productive infection. our laboratory analyzes the effect of tat on the murine cns by stereotactically injected into cortex, which mimics in part the neuroinflammation and neurodegeneration characteristic of hiv-1 associated neurocognitive disorders (hands). additionally, in vitro tat-treated bv-2 microglial cells exhibit activation associated with increased cytokine expression and phagocytosis. mixed lineage kinase 3 (mlk3) has recently been implicated in the tat-mediated activation of microglia. mlk3 activity is increased by the presence of tat protein, which results in microglial activation and increased production of inflammatory cytokines. conversely, the brain penetrable, small molecule, new chemical entity urmc-099 inhibits the mlk3 pathway. we hypothesize that urmc-099 attenuates tat-induced microglial activation as measured by microglia process length, phagocytosis assay and cytokine expression. in vitro application of urmc-099 decreased tatinduced production of ccl2 and il-6, microglia process length, and phagocytosis of charged and uncharged beads in bv-2 microglia cells. these data, in aggregate, strongly support a role for the mlk3 pathway in neuroinflammation and suggest that inhibition of this pathway with urmc-099 may have significant anti-inflammatory effects in an inflammatory cns milieu. in conclusion, this work will advance our understanding of mlk3 activity and may provide a viable drug therapy for hands. the contributions of glial cells to the modulation of adult synaptic plasticity are becoming increasingly more appreciated. astrocytes in the supraoptic nucleus (son) of the hypothalamus demonstrate prominent temporary structural changes during physiological events such as dehydration and lactation. such structural remodeling is characterized by a reduction in the astrocytic coverage of oxytocin neurons and their synapses. these astrocyte morphological changes also contribute to the observed functional changes in synaptic activity during dehydration and lactation. in order to elucidate the underlying mechanisms regulating the astrocytic contributions to synaptic plasticity we have established an astrocyte protein expression profile for structural changes in the son during lactation and hyperosmotic challenge. for this, son from virgin, lactating, and hyperosmotic rats were collected from acute hypothalamic slices and analyzed by mass spectrometry to identify proteins differentially regulated by the changes in synaptic structure induced by lactation and hyper-osmolarity. our mass spectrometry data analysis has placed particular focus on regulation of astrocyte-specific proteins and the roles of these proteins in molecular pathways known to be involved in son plasticity, such as cytoskeleton remodeling, cell adhesion, and cell signaling. gene ontology analysis revealed over representation of pathways known to be highly active in astrocytes including metabolism and antioxidant activity. promising target proteins for future functional studies will be discussed. here we elucidate a novel merlin isoform 2 (merlin-iso2)-specific function in maintaining axonal integrity and propose that reduced axonal nf2 gene dosage leads to nf2-associated polyneuropathy. we identify a merlin-iso2-dependent complex corresponding to activation of the gtpase rhoa, enabling downstream rho-associated kinase to promote neurofilament heavy chain phosphorylation. in vivo, specifically merlin-iso2 knockout mice exhibit impaired locomotor capacities, delayed sensory reactions, and electrophysiological signs of axonal neuropathy. sciatic nerves from these mice and sural nerve biopsies from nf2 patients show reduced nf-h phosphorylation, decreased interfilament spacings and irregular-shaped axons. a. catenaccio, p. silva, f. court pontificia universidad cat olica de chile, santiago, chile axonal degeneration is an active process involved in a variety of neurodegenerative conditions triggered by diverse stimuli. this degenerative process can cause permanent loss of function, so it represents a focus for neuroprotective strategies. we have recently shown that axonal degeneration triggered by distinct mechanical and toxic damage is dependent on the activation of the mitochondrial permeability transition pore (mptp), which probably represents a central execution program of axonal degeneration, upon which several pathways converge (barrientos et. al., journal of neuroscience 2011). nevertheless, the participation of other cellular types in this degenerative process has been largely overlooked. after axonal damage in the peripheral nervous system (pns), schwann cells enveloping axons have been regarded as responsible for clearing degenerating axonal debris and to mount an inflammatory response for tissue clearance by invading macrophages. nevertheless, a possible function of glial cells in modulating axonal degeneration has not been addressed so far. here we explored the possibility that glial cells actively participates not only in the clearance of degenerating axons, but directly in the axonal degeneration program during wallerian degeneration. we found that axonal injury activates an early response of schwann cells that results in the fragmentation of their associated axons by actin-rich cytoplasmic domains of schwann cells known as schmidt-lanterman incisures (sli). this first step of schwann cell fragmentation of axons is dependent on the erk signaling pathway, wich control the dedifferentiation program initiated in schwann cells after nerve injury (napoli et. al., neuron 2012). in the axonal compartment, injury triggers a parallel cell autonomous degenerating program dependent on mitochondrial mptp activation, which is cell non-autonomously assisted by schwann cells through the activation of a cell extrinsic pathway of axonal destruction by tnf-alpha secretion. we showed for first that time that axonal degeneration in the pns proceeds by axonal autonomous mechanisms, as well as cell non-autonomous ones dependent on scs. therefore, effective targets for neuroprotection should consider not only the axonal compartment, but also the associated glial cell. glutamate is the major excitatory neurotransmitter in the brain. its concentration is the synaptic area is highly regulated in order to maintain point-to-point transmission and to prevent excitotoxicity associated with chronic activation of glutamate receptors. as there is no endogenous extracellular enzyme to degrade glutamate, the clearance of glutamate is ensured by transporters located on the surface of astrocytes. among these surface transporters glt-1 ensures almost 90% of glutamate uptake at synapses. it is widely accepted that many receptors and transporters on the surface of neurons and glia display surface trafficking properties and are not simply static proteins. the basic surface trafficking properties of glutamate receptors at the synapse have been studied in depth and have allowed us to understand many important trafficking events that occur during synaptic plasticity. the action of glt-1 is fundamentally important in synaptic communication. surface trafficking of this transporter may play a pivotal role in the spatial and temporal properties of glutamate diffusion at the synapse. however, it is still unknown whether glt-1 is dynamically regulated on the surface of astrocytes. thus we set out to uncover the surface trafficking of glt-1 in astrocytes by using a combination of high-resolution imaging, such as single particle (quantum dot [qd]) tracking, molecular biology and electrophysiological approaches. here we have demonstrated that glt-1 is indeed subject to surface trafficking on astrocytes. using pharmacological approaches we have shown that this diffusion is subject to regulation by the activity of the transporter itself as well as neuronal activity. furthermore, we have demonstrated that the speed of glt-1 diffusion is greatly reduced at excitatory synapses. this leads us to the conclusion that glt-1 surface diffusion is highly regulated at the synapse where it can effectively carry out its role as the principal glutamate transporter at the synapse. finally, to elucidate the physiological role of glt-1 surface diffusion, we have carried out electrophysiological experiments in which glt-1 trafficking is effectively reduced using a cross-link (x-link) protocol. we observed that a reduction in the surface trafficking of glt-1 resulted in an increase of neuronal excitability. thus glt-1 surface trafficking on astrocytes has an implicit role in regulating basal neuronal activity through glutamate uptake at the synapse. o. chever, e. dossi, n. rouach collège de france, paris, france astrocytes play crucial roles in brain physiology by dynamic interactions with neurons. they form plastic and extensive networks mediated by gap junction channels. it has recently been shown that astroglial networks limit neuronal network activity. however, it is currently unclear how astroglial networks influence neuronal excitability and population activity. to investigate how astroglial assembly regulates neuronal network activity, we performed electrophysiological recordings in hippocampal slices (ca3 and ca1 areas) from mice with disconnected astrocytes, in which both astroglial gap junction forming proteins, connexin 30 (cx30) and connexin 43 (cx43), are knocked out (gfap-cre cx30 -/-cx43 fl/fl , dko). synchronized excitatory discharges were recorded in an acute pharmacological model of epileptic-like activity. in this model, spontaneous bursting discharges are characterized by a sharp and large amplitude shift in field potential, generated by a major depolarization and firing of all putative neurons. pyramidal cells depolarized up to 40 mv during seconds and such depolarization is followed by a long-lasting undershoot of the membrane potential. we found that the frequency of bursting activity in dko hippocampal slices is drastically increased. however, the duration of neuronal depolarizing bursts is severely reduced. furthermore, pyramidal neurons are more depolarized due to an increase of synaptic bombardment. this suggests that increased synaptic background promoting resting membrane potential depolarization facilitates triggering of neuronal bursts, but compromises the strength of synchronized events. to investigate local dynamics of bursts generation, we performed multielectrodes arrays recordings in the ca3 area of the hippocampus. in slices from dko mice, bursting activity in the ca3 region is generated within a more restricted area, indicating that the number of neurons to be recruited is highly decreased. altogether, these results indicate that gap junction-mediated astroglial networks strengthen the coordination of neurons during synchronized events. acutely loaded hypoxia increases ventilation, and increased ventilation does not immediately return to the pre-hypoxic level, but persists for a while after resuming room air inhalation. this phenomenon is referred to as short term potentiation or plasticity of breathing, and is thought to contribute to the stabilization of ventilation. although extensive studies have been conducted to clarify the cellular mechanism of respiratory plasticity focusing on neurons and synapses, its precise mechanism remains unknown. on the basis of the recent discovery that astrocytes are actively involved in neural plasticity in various brain regions, we hypothesized that the post-hypoxic potentiation of breathing is mediated by astrocytes. we used unanaesthetized unrestrained adult mice. ventilatory parameters (respiratory frequency, tidal volume and minute ventilation) were measured by whole body plethysmography. to test the hypoxic response, mice breathed room air, hypoxic gas (7% o 2 , 93% n 2 ), and again room air. we also analyzed the hypercapnic response that was conducted under a hyperoxic condition to eliminate the influence of the carotid body. to test the hypercapnic response, mice breathed pure o 2 , hyperoxic hypercapnic gas (95% o 2 , 5% co 2 ), and again pure o 2 . after recording of the hypoxic and hypercapnic responses with their recovery time courses, arundic acid (ono-2506) was injected intraperitoneally. arundic acid is a compound that suppresses the function of astrocytes through the inhibition of s100b production in astrocytes. arundic acid did not affect ventilatory augmentation induced by either hypoxia or hypercapnia. however, arundic acid suppressed the short term potentiation induced by hypoxia (but not by hypercapnia) in a dose-dependent fashion. high dose arundic acid completely eliminated hypoxia-induced short term potentiation. these results suggest that the post-hypoxic potentiation of breathing is mediated by astrocytes with the involvement of s100b. the mechanism of hypercapnia-induced potentiation is different from that of hypoxia. we propose a model to explain the cellular mechanism of post-hypoxic potentiation: hypoxia stimulates the respiratory neuronal network in the brainstem through the excitation of the carotid body. neurotransmitters spilled from excited neuronal synapses activate nearby astrocytes. the activation of astrocytes is sustained, and these astrocytes persistently excite respiratory neuronal network by releasing gliotransmitters. monosynaptic c-fibre evoked excitatory postsynaptic currents (epscs) were recorded in lamina i neurons using whole-cell patchclamp in rat transverse spinal cord dorsal-root slice preparations. spontaneous epscs were recorded simultaneously. in all experiments bicuculline and strychnine were added to the bath solution to block gaba a -and glycine-mediated synaptic currents. the superfusion of spinal cord slices in vitro with fkn results in a rapid facilitation of evoked epscs in spinal lamina i neurons receiving monosynaptic input from primary afferent c-fibres. both the microglial cell inhibitor minocycline and a fkn neutralising antibody, completely prevented fkn-induced changes in synaptic transmission. in addition, the inclusion of the ca 21 chelator bapta or the nmda receptor antagonist mk801 in the pipette solution completely prevented fkn-induced enhancement of synaptic strength, suggesting a post-synaptic ca 21 -dependent mechanism of ltp induction. analysis of paired-pulse ratio (ppr) indicated that fkn-induced facilitation of synaptic strength is accompanied by a decrease in ppr suggesting a pre-synaptic mechanism of ltp expression. in support of this finding, fkn increases the number of spontaneous epscs recorded from lamina i neurons. these data indicate that stimulation of microglial cells in order to induce a pro-nociceptive activity state is sufficient for facilitation of synaptic strength within the dorsal horn. both pre-and post-synaptic mechanisms contribute to fkn-induced facilitation of synaptic strength. this work was funded by a wellcome trust flexible travel fellowship to akc. the organization and connectivity of the developing neocortex remains largely unresolved. the establishment of neuronal microcircuits is a complex process that partly depends on the diversity of neuronal cell types and their ultimate role in the mature brain. this complexity is increased by the existence of bona fide synaptic connections between neurons and oligodendrocyte precursor cells, also called ng2 cells, which properties and function remains largely unknown. to unravel the physiological characteristics of gabaergic synapses between interneurons and ng2 cells, we performed paired recordings in the developing somatosensory cortex. experiments were carried out during the second postnatal week in vgat-venus;ng2-dsred double transgenic mice that allowed us to identify interneurons (venus 1 ) and opcs (dsred 1 ) in acute slices. our results demonstrated that ng2 cells are synaptically contacted by fast-spiking (fsi) and non-fast-spiking (nfsi) interneurons. however, a predominant input was received by fsi, a class of interneuron that constitutes only a minor population in the second postnatal week. on the contrary, only a small proportion of the abundant nfsi were connected to ng2 cells. all the connections showed paired-pulse depression, low probability of response, and preliminary analyses suggest that each interneuron form a single synaptic contact onto ng2 cells. paired recordings, extensively used to examine the synaptic properties of neuronal microcircuits, may open a new perspective to understand in detail the mechanisms of gaba release and signaling between interneurons and ng2 cells in the healthy and pathological developing brain. first, we characterized a microglial cell line obtained from cd1 mouse cortex (n9), in order to clarify the mechanisms involved in the activation pattern of these cells. for that, we incubated n9 microglia with 300 ng/ml of lipopolysaccharide (lps) for 24 h. morphology (anti-iba1 staining), phagocytic ability (fluorescent latex beads) and chemotaxis to atp (boyden chamber) were evaluated. we observed that n9 microglia is ramified in control conditions and after lps treatment they change to an amoeboid morphology, which is accompanied by increased phagocytosis and decreased migratory ability, indicating that lps induces activation of n9 cell line. next, we investigated the role of the released factors from a mn-like cell line expressing human sod1 with g93a mutation (nsc-34/ hsod1g93a) on microglia functions. nsc-34 expressing human sod1 wt were used as control. thus, n9 microglia were incubated for 4 and 24 h with nsc-34 conditioned media that had been collected at 1 and 4 days of differentiation (div) and reactivity tests were performed as above. although after 4 h of incubation no significant changes were observed, data evidenced that incubation for 24 h with nsc-34/ hsod1g93a conditioned media collected at 4 div changed the bipolar morphology of the n9 microglia into an active amoeboid state, decreased phagocytic ability (22%, p < 0.05) and induced apoptosis (50%) (fig.1) . moreover, microglia have shown to not be attracted by the released factors from degenerating mn, as determined by the chemotaxis assay. together, our results evidence that released factors from nsc-34/ hsod1g93a degenerating cells although inducing microglia morphological activation trigger a reduction in cell phagocytic ability and loss of viability, suggesting a role for microglia along als progression. organotypic explants culture has a pivotal role in studying the complex structure of neuronal tissue. although embryonic tissue explants and slices are used to obtain long term culture, most applications such as drug testing require adult tissue for reliable results. in this study, we show that nanostructured metal-oxide arrays can be employed to yield long-term organotypic culture of adult neuronal tissue explants as demonstrated for the adult guinea pig retina and adult murine brain slices. even after 14 days of culture, the thickness and the layered structure of the retina were well maintained. quantitatively, the number of nuclei within the inner and outer nuclear layers was comparable to freshly isolated retinae and no significant change in the densities of the nuclei within the layers was observed. the outer plexiform layer, as the site of synaptic connection between photoreceptors and neurons, was still well preserved. moreover, the thickness of the photoreceptor layer was conserved, indicating that the inner and the outer segments of the photoreceptors survived well during two weeks culture. this is hardly observed in long-term cultures since the outer segments were cultured without retinal pigment epithelium. concerning the adult murine brain slices from neo-cortex, after 9 days of culture, the neurofilaments and cell nuclei were still well preserved and the neuronal network displayed the typical arrangement of long axons. however, preservation of the tissue depends strongly on the geometry of the nanostructured array surface such as roughness and spacing, whereby organotypic brain slice culture requires different geometries than retina culture. since organotypic culture of adult tissue could only be obtained for about 7 days with conventional techniques, our new substrate material could be the breakthrough for in vitro studies on tissue regeneration, neuroplasticity, as well as drug testing. the intrinsic optical signal (ios) is widely used for mapping neuronal activity in afferent activated brain areas. ios evoked by afferent stimulation in vitro is generally attributed to glial cell swelling via activation of na 1 /k 1 /clcotransporter that follows postsynaptic activation. despite the phenomenon is known for decades, the relative contribution of different cell types and the underlying molecular mechanisms have not been disclosed in detail. our goal was to explore the glial and neuronal correlates underlying in vitro ios genesis. we characterized ios initiated by schaffer collateral stimulation in the rat hippocampal slice using simultaneous high-frequency imaging and field potential recordings. we used a 464-element photodiode-array device (pda) that enables ios detection with 0.6 ms time resolution, making it achievable to align optical and electrophysiological signals. ios was primarily observed in the proximal region of the stratum radiatum and stratum pyramidale of the hippocampus. ios was decreased by blockade of neuronal activity by voltage-gated na 1 channel inhibitor tetrodotoxin and was significantly enhanced by suppressing inhibitory signaling with the gaba a antagonist picrotoxin. we found that ios was predominantly initiated by postsynaptic glutamate receptor activation and progressed by the activation of astroglial glutamate transporters and mg 21 -independent astroglial nmda receptors. in agreement with previous studies, furosemide, the blocker of both the neuronal k 1 / clcotransporter kcc2 and the glial na 1 /k 1 /clcotransporter nkcc1 decreased the ios amplitude. to decide which cotransporter is responsible for this effect of furosemide, we applied selective blockers of nkcc1 and kcc2, bumetanide and (1)[r]-dioa, respectively. we evidenced, that nkcc1 did not contribute to the ios generation, in contrast to previous suggestions. enhancement and slight inhibition of ios through anion and volume-regulated anion channels, respectively, were also depicted. major players of ios mechanisms disclosed by high-frequency ios imaging imply that spatiotemporal ios reflects glutamatergic neuronal activation and astroglial response, as observed within the hippocampus. our model may help to better interpret in vivo ios and support diagnosis in the future. question: a poor x3 polyunsaturated fatty acids (x3 pufa) status, favored by the low x3/x6 ratio in western diets, seems to contribute to cognitive decline in the elderly, but mechanistic evidence is lacking. we therefore explored the impact of x3 status on the evolution of glutamatergic transmission, astroglial regulation and neurogenesis in the hippocampus during ageing in rats. these processes are involved in memory formation and their dysregulation participates to the agerelated brain damage leading to cognitive decline. methods: we have compared 6 groups of rats aged 6 to 22 months fed x3-deficient, x3/x6-balanced, or x3 (fish oil) supplemented diets: young x3 balanced (yb), deficient (yd) or supplemented (ys), and old x3 balanced (ob), deficient (od) or supplemented (os) rats. we have evaluated synaptic efficacy and plasticity (electrophysiological recording), astroglial regulations (glutamate uptake and gfap expression), neuronal markers (glutamate transporters and receptors), neurogenesis (proliferation of neuronal precursors in the sub granular zone), and analyzed brain fatty acids composition. results: dietary modulation of x3 intakes efficiently modified the incorporation of docosahexaenoic acid (dha, the main x3 in cell membranes) in brain (250% deficient vs balanced, 110% supplemented vs balanced). ageing induced a 35% reduction of synaptic efficacy due to decreased pre-synaptic glutamate release, and a 30% decrease in the astroglial glutamate uptake associated to a marked astrogliosis (1100% gfap). x3 deficiency further decreased these hallmarks of ageing (od vs ob rats: 235% synaptic efficacy, 215% glutamate uptake, 130% gfap). on the opposite, x3 supplementation increased synaptic efficacy (125% os vs od) and seems to abolish astrogliosis (os vs ys : no change in gfap). neurogenesis was altered by x3 deficiency but not by supplementation. conclusion: our results characterize some specific age-related alterations of the glutamatergic synapse in the hippocampus that are aggravated by a dietary deficit in x3 and attenuated by x3 supplementation. methods: sgcs were isolated from the trigeminal ganglion (tg) of adult male wistar rats (n 5 9). animals were deeply anaesthetized and both tg were extracted and digested first in 5 mg/ml collagenase for 15 min and then in 0.125% trypsin containing dnase for 10 min. in initial experiments, isolated sgcs were treated with control medium or medium containing 16.5, 33, or 66 mm cis for 8h. in the subsequent experiments, sgcs were pretreated for 24h with control medium or medium containing 100 mm ibu or 1 mm skf. afterwards, sgcs were stimulated for 8 hours with medium containing 66 mm cis. pge 2 concentration was determined by elisa. results: cis caused a concentration-dependent increase in pge 2 release from sgcs. treatment with 33 mm cis significantly increased pge 2 concentration to 354.8 6 87.3 pg/ml, compared with control 211.3 6 39.0 pg/ml (p 5 0.018). pge 2 concentration was further increased to 464.1 6 77.0 pg/ml (p 5 0.002) following 66 mm cis. pretreatment with ibu significantly lowered the pge 2 concentration compared with cis-stimulated sgcs (197. 5 6 85.2 pg/ml vs. 415.8 6 143.8 pg/ml, respectively; p 5 0.05). a more pronounced inhibitory effect was observed following skf pretreatment, where it reduced the pge 2 concentration to 19.7 6 1.8 pg/ml (p 5 0.003, compared with cis-stimulated sgcs). conclusion: these findings suggest that cis contributes to an inflammatory response in the tg by provoking release of pge 2 from sgcs, which may lead to development or maintenance of peripheral sensitization involved in cipn. the data also suggest that treatment with agents that target pge 2 release cascade may prove beneficial for preventing development or maintenance of cipn. functional alterations in synaptic contacts have often been described as a hallmark of major depressive disorder (mdd). antidepressants (ads) have been shown to restore neuronal circuits through an enhancement of synaptogenesis in some regions of the brain. nevertheless, the underlying mechanisms are still unclear. glia cells have been since long acknowledged as active partners of neurons in orchestrating molecular signals crucial for the proper arrangement of neuronal circuits in the developing and adult brain. therefore, understanding how both neurons and astrocytes respond to antidepressants (ad) is of high interest. using rat c6 glioma cells and primary cultures of astrocytes and neurons, we showed a time-dependent cell-autonomous modulation of the erk/mapk signalling pathway after acute treatment with various classes of ads in both cell types. specifically, glia cells responded to ads with the simultaneous and fast activation (after 10 min treatment) of both erk1/2, in contrast to treatment with antipsychotics or mood stabilizers. this activation induced 48 hrs later an increased release of gdnf, a factor involved in synapse formation and axonal wiring, which was mapk-dependent. on the contrary, hippocampal neurons showed a reduction in erk activity that correlated with neuronal activity inhibition after short term (10 min) ads administration, as demonstrated by quantification of c-fos expression after kcl stimulation. interestingly, this inhibitory effect was reversible after long term (48 hrs) ads treatment. to further identify whether gdnf released from astrocytes treated with ads might be responsible for long term changes in neuronal synapses, we examined how ads influenced synaptic densities in neurons alone or co-cultured with astrocytes. strikingly, we found that the number of synapses was reduced at 48hrs after ad treatments, but only in the presence of intact astrocytes and not in cultures of neurons alone or neurons treated with astrocyte-conditioned media. this effect was reversed after 120 hrs treatment and rescued by the soluble form of the specific gdnf receptor gfralpha1, but not by gdnf alone. moreover, live imaging of astrocytes showed dramatic morphologic changes of their processes in response to ads that might underlie the observed synaptic remodelling. our analysis of changes occurring at the neuronglia interface upon ad treatments might elucidate novel mechanisms of ads action that may open the avenue for unravelling the role of astrocytes in psychiatric diseases and implement pharmacologic treatment regimens for mdd patients. here we extend these findings showing that the neurotrophin nt-3, which also belongs to the putative factors secreted from glial cells after t3-stimulation, also increases the navd in neuron enriched cultures. the effects of nt-3 and fgf-2 are, however, not additive. antibodies against nt-3 potentiated the effects of fgf-2, suggesting that by converging signal cascades nt-3 could reduce the effect of fgf-2. in neuron-glia mixed cultures antibodies against nt-3 enhanced the t3-induced up-regulation of navd, suggesting, that a co-secretion of nt-3 from glial cells could limit the effects of fgf-2. in a second series of experiments we investigated, whether the well-known effect of t3 on the expression of na 1 /k 1 -atpases, thought to contribute to the effect of thyroid hormone on metabolism, is also influenced by glial cells. we found, that the effect of t3 on 3 [h]ouabain-binding and on the expression of na 1 / k 1 -atpase alpha2 subunits detected by western-blots was highest in neuron-mixed cultures, suggesting that a neuron-glia interaction is also involved in the t3 effects on na 1 /k 1 -atpase expression. in the healthy brain microglia engage in bi-directional interactions with neurons and other brain cells, which is crucial for maintaining normal physiology and cognitive function of the brain. disturbances in homeostasis and cell-cell communication may predispose to age-related dysfunction and neurodegenerative disease. for decades it has been speculated that microglia exhibit phenotypic diversity allowing specialisation to a variety of brain microenvironments. previous observations of regional variations in microglial densities and morphologies support the existence of microglial heterogeneity. however it is clear that a more comprehensive understanding, in particular of the molecular basis of microglial phenotypic and functional diversity on a global scale, is required. the limitation of recent gene expression studies is the use of mixed whole brain or dissected mixed cell extracts which preclude the detection of cell-specific gene expression profiles. thus, our aim was to validate an efficient extraction process for microglia, ideally minimising the impact on the resting state. the method developed is based on existing procedures and was refined to ensure consistency and to maximise cell yield and purity. the extraction of highly pure microglia as a single cell type was achieved by a two-step isolation technique using density-gradient and magnetic bead separation. purity between 90-95% was verified by flow cytometry and immunohistochemistry. quantitative pcr also demonstrated expression of specific microglial genes indicating that isolated cells are microglia. cytokine assays suggest that no or only minimal activation of cells was induced by the isolation. hence, the microglia retained their ability to respond to immunogenic stimuli and produce il-1b as a key inflammatory cytokine. overall, the purification procedure was shown to achieve consistent high purities and retain crucial functional properties. thus, the extracted microglia are likely to adequately represent the real in vivo state and enable the study of microglial phenotypes and functionality in the healthy and ageing whole brain and accordingly brain regions. early diversity experiments showed region-specific microglia phenotypes in the brain of healthy and immune challenged animals as well as differences between unchallenged and challenged animals. question: activation of nmda receptors increase the respiratory frequency in mammals. astrocytes are in intimate contact with neurons, specially in glutamatergic synapses, and are able to sense neuronal activity, react to, and even influence nearby neurons. furthermore, they can sense changes in h 1 or pco 2 , and in response to these stimuli, they can release molecules like atp and d-serine, which will activate neuronal circuits. then d-serine, an agonist of the glycine site of the nmda receptor, can affect the respiratory rhythm during hypercapnia. our aim was to study the probable role of d-serine in the modulation of the respiratory rhythm in mice neonates. methods: fictive respiration was recorded with suction electrodes from c4-c5 ventral roots in "en bloc" (brainstem-spinal cord) preparations from p0-p3 cf1 mice. superfusion was done with artificial cerebrospinal fluid equilibrated with o2:co2 5 95%: 5%, (ph 7.4,28 c) containing different d-serine concentrations (0.1-100 mm) in presence or absence of d-amino acid oxidase (daao), which degrades d-serine. in addition, hypercarbic acidosis (switching equilibration from 5 to 10% co2, changing ph from 7.4 to 7.2) was done in absence or presence of daao. results: application of d-serine increased the frequency of the respiratory rhythm in a concentration-dependent way. application of dao attenuated the effects of d-serine application. likewise, daao reduced slightly the effect of hypercarbia on the respiratory rhythm. conclusions: our preliminary experiments indicate that d-serine is a potent agent increasing the respiratory rhythm frequency in in vitro neonatal preparations, and likely, in association with atp, is mediating the respiratory response to hypercarbia. there, we showed that mac-2 positive microglia accumulate within the motoneuron area and phagocyted apoptotic bodies at the onset of motoneuron developmental cell death (from embryonic day 12.5-e12.5). motoneuron developmental cell death and microglia accumulation in the motoneuron area increased at e13.5, decreased at e14.5 and disappeared at e15.5. since it was supposed that microglia promote developmental cell death in the developing central nervous system, at least in vitro, we determined to what extent it was also the case in the embryonic spinal cord in vivo. to address this issue, we analysed in vivo the progression of developmental neuronal death in the spinal cord of transgenic mouse embryos lacking microglia (pu.1-ko mice). as opposed to what is observed in the sc of wild type mouse embryo, we found that activated caspase-3 staining was detectable before e12.5 in pu.1-ko mouse embryos. in pu.1-ko mouse embryos, activated caspase-3 staining was not restricted to the motoneuron area, as several positive cells are also present in the dorsal region of the spinal cord and in the progenitor zone. at e13.5, activated caspase-3 staining was 10 times greater in the spinal cord ventral area of pu.1-ko mouse embryos when compared to wild type littermates. moreover, and opposed to what occurs in wild type animals, caspase-3 staining in the motoneuron area of pu.1-ko mouse embryos increased at e14.5 and persisted at e15.5, being likely partly due to the accumulation of apoptotic bodies in the absence of microglia. our results suggest that, in addition to their phagocytic role, embryonic microglia are likely to protect neuron from death at the onset of developmental cell death and to promote survival of progenitor-like cells in the embryonic sc in vivo. accordingly, microglia could have an opposite effect on cell survival in the embryonic sc when compared to postnatal snc developmental stages and pathological conditions in the adult. we recently demonstrated a key role of the a-secretase tace, also known as adam17, in peripheral nervous system (pns) myelination by tace-mediated cleavage and subsequent inhibition of neuregulin1 (nrg1) type iii activity. unlike the pns in which nrg1 type iii is an essential instructive signal for myelination, oligodendrocyte (ol) development and myelination in the central nervous system (cns) are likely controlled by several growth factors some of which undergo cleavage by secretases. to study the role of tace on opc development, immunopanned a2b51 opcs were cultured in proliferating or differentiating medium and treated with a soluble form of tace (rhtace). when ols were scored according to their morphology, we observed enhanced opc differentiation upon addition of rhtace to proliferating opcs. addition of rhtace to differentiating opcs also increased the number of ols with a complex morphology and already presenting membrane sheets, suggesting that tace might promote opc differentiation. to further investigate the role of neuronal tace in cns, we used an in vitro ol myelinating coculture system. when cocultured with tace null drg neurons, rat wild type opcs never myelinate, suggesting that neuronal tace might control opc differentiation. in agreement, preliminary ultrastructural analyses of transgenic mice lacking tace in cns neurons showed hypomyelination and signs of myelin degeneration. accordingly, conditional transgenic mice lacking tace in ol are normally myelinated. these studies suggest that in the cns the a-secretase tace promotes opc differentiation and might regulate cns myelination. a better understanding of the role of tace will provide novel insights into the mechanism regulating cns myelination. in the excitatory network glutamate is released in the synaptic cleft during synaptic activity. glutamate clearance by astrocytes is mainly performed by na 1 -dependent glutamate transporters. the na 1 load during glutamate uptake evokes an activation of the na 1 /k 1 atpase, which dramatically increases energy demands in astrocytes. astrocytes are also involved in the regulation of extracellular potassium (k e 1 ) which significantly increases during the repolarization of excitatory neurons. in this study we evaluated how these fundamental functions of astrocytes coexist and if they interact. we investigated the impact of altering k e 1 concentrations on glutamate transporter activity measuring intracellular sodium (na i 1 ) using the na 1 sensitive fluorescent dye asante sodium green loaded in cultured astrocytes. we observed that glutamate uptake caused a reversible rise in na i 1 , which was tightly modulated in amplitude, slope and recovery rate by k e 1 levels. in order to evaluate the impact of physiologically relevant k e 1 fluctuations on the kinetics of the glutamate transporter the na 1 /k 1 atpase was inhibited. therefore we found that low k e 1 evoked a significantly faster influx of na 1 , whereas high k e 1 markedly slowed down glutamate capture, indicating that k e 1 directly modulates the kinetics of glutamate transporters. we then investigated the energy demands of astrocytes caused by k e 1 alterations. atp hydrolysis was indirectly measured through the changes in intracellular free mg 21 using the mg 21 sensitive fluorescent probe magnesium green. we found that low k e 1 led to higher energy demands of astrocytes during glutamate uptake. overall these results indicate that k e 1 acts as a negative feedback on glutamate uptake in astrocytes. the physiological consequences of these findings have to be considered in the context of k 1 buffering and of maintenance of neurotransmitter and energy homeostasis. recovery was significantly improved by nf fractions, as well as tubulin (tub), added after lpc removal: compared to cultures treated with lpc alone the number of olp (a2b51 cells), and their proliferation increased significantly, as well as the number of differentiated (cnp1) and mature (mbp1) ol. moreover, nf and tub protected ol from lpc toxicity when added at the time of lpc treatment: they increased olp proliferation, as well as the number of cnp1 and mbp1 ol significantly, compared to cultures treated only with lpc. on the contrary irrelevant proteins (actin, and skin proteins) were ineffective, demonstrating the specificity of the cytoskeleton proteins effects. importantly, nf and tub increase olp survival and proliferation when challenged with lpc, without delaying differentiation and maturation. we hypothesize that release of nf and tub following axonal damage in ms could participate in the regulation of remyelination through this process. this putative phenomenon might be complexified by alterations of axon protein expression, or of proteolysis, known to occur in ms models. purpose: microglial proliferation is commonly accepted as a hallmark of glial activation. an automatic method that allows assessing the number of microglial cells to analyze the proliferative microglial behavior in a laser-induced ocular hypertension (oht) model has been developed. methods: adult albino swiss mice were organized in two groups: naive (age-matched control; n 5 6) and lasered (n 5 6). retinal wholemounts were immunostained with anti-iba1 and images of iba-11 microglias were recorded with a fluorescence microscope. new algorithms of segmentation and control of distances were developed in matlab to obtain the number of iba-1 microglial cells. automatic results were compared with those obtain by direct human observation of the samples. results: the automatic method detected the number of iba-11 cells in the inner and outer plexiform layers of the retina in both, na€ ıve and oht-retinas. the number of cells present in the samples was not an obstacle for the program to run properly. the time required for counting iba-11 cells decreased from the human guide to the program based counting method from days to one hour. results show a strong correlation between both automatic and manual method (pearson correlation test, r 5 0.979; p 5 0.000 and r 5 0.942; p 5 0.000 for outer and inner plexiform layer respectively) indicating the consistency of the automatic counting. conclusions: a new, reliable and quick algorithm was developed with matlab to obtain the number of iba-11 microglial cells and also cellular density maps through retinal wholemounts in both na€ ıve and other proliferative conditions (i.e. glaucoma). due to this new automatic method, a bigger set of images or samples could be included in future studies to analyze the behavior of microglial cells. we are interested to understand whether scs in the peripheral nerves of mammals express functional ionotropic glutamate receptors, and whether scs use these receptors for communication with axons. to start addressing this question, we established a preparation of mouse live sciatic nerve slices employing "know-how" of live brain slices technique. using this preparation, we performed whole-cell patch-clamp recordings of scs at different stages of development (embryonic and early postnatal). we included a fluorescent dye into the pipette solution in order to perform post-recording morphological analysis of single scs. first, we recorded current responses of scs to a series of depolarizing voltage steps, aiming to test whether all scs in the nerve are electrophysiologically akin. we found that mouse scs differ in their passive membrane properties and expression of voltage-gated k1 channels: depending on the age, 3 to 4 cell types could be distinguished. post-recording morphological characterization of the recorded cells showed that scs in the mouse sciatic nerve differ in their size, as well as in the number and length of their processes. next, we used fast pressureinduced application of glutamate to investigate whether scs possess ionotropic glutamate receptors, and whether electrophysiologically distinct sc types differ in their expression of glutamate receptors. application of 1 mm glutamate caused an inward current in at least two types of scs, and preliminary analysis revealed a peak current amplitude in the range of 14-250 pa (n 5 18). glutamate-induced currents in scs were blocked by gyki53655, indicating that mouse scs carry ionotropic glutamate receptors of ampa/kainate type. currently we are studying which subunits of ampa receptors are present in scs, and how these receptors get activated in situ. although cx32 is expressed by myelinating schwann cells in the peripheral nerves, patients with cmt1x develop relatively early axonal degeneration, which correlates best with chronic disability. we found in previous studies of gjb1-null mice, a model of cmt1x, that the diameter of myelinated axons was progressively reduced at the age of 2 months compared to wild type (wt) littermates, resulting from progressive dephosphorylation of axonal neurofilaments and increased packing density. fast axonal transport was slower in distal axons of mutant compared to wt animals, with impaired mobility of synaptic vesicleassociated proteins and accumulation of beta-amyloid precursor protein and tau. how cx32-formed gjs are involved in the regulation of axonal cytoskeleton dynamics remains unclear. methods-here we investigated the signaling mechanisms regulating axonal cytoskeleton focusing on major kinases and phosphatases involved in neurofilament phosphorylation by immunohistochemistry and immunoblot. in addition we examined the localization of axonal mitochondrial, which also depend on axonal transport. we focused on nerve pathology in 2 month-old gjb1-null mice, when demyelination and inflammation is minimal, in order to identify the direct effects of gj loss on the axon. results-our results show that erk1/2 kinase and pp2a phosphatase were localized in non-compact myelin areas, including the schmidt-lantermann incisures and paranodes, where gjs are normally formed by cx32. another kinase, cdk5, was more diffusely localized along the axon. biochemical analysis showed altered amounts of erk1/ 2, pp2a and cdk5 as well as their upstream activators in gjb1-null nerves. the pp2a inhibitor phapi was also localized in non-compact myelin areas and significantly increased in gjb1-null nerves. assessment of axonal mitochondria by porin immunostaining showed significantly increased density in gjb1-null nerves, consistent with slower axonal transport. conclusion-our findings clarify some mechanisms of early axonal pathology that are independent of demyelination in this mouse model of cmt1x. several components of the signaling pathway regulating axonal cytoskeleton and axonal transport appear to be functionally related to gjs at the non-compact myelin sheath. loss of myelin gjs results in impaired regulation of axonal cytoskeleton, energy supply, and axonal transport. we have recently discovered that oligodendrocytes secrete exosomes containing a distinct set of proteins as well as mrna and microrna. intriguingly, oligodendroglial exosome release is stimulated by the neurotransmitter glutamate indicating that neuronal electrical activity controls glial exosome release. in this study we examined the role of exosomes in neuron-glia communication and its implications in glial support. results: to analyze the transfer of oligodendroglial exosomes to neurons, we exposed cultured cortical neurons to fluorescently labeled oligodendroglial exosomes. indeed, cultured cortical neurons internalized and accumulated oligodendroglial exosomes in the neuronal cell soma in a time-dependent manner. addition of endocytosis inhibitors or expression of dominant negative dynamin interfered with neuronal exosome internalization indicating that exosome uptake is mediated by clathrin-dependent endocytosis. furthermore, neuronal internalization of exosomes resulted in functional retrieval of exosomal cargo in vitro and in vivo upon stereotactic injection of exosomes. to investigate the influence of oligodendroglial exosomes on neuronal gene expression we performed a microarray screen of neuronal mrnas after exosome exposure. interesting candidates were further validated by qrt-pcr. conclusion: taken together, our results represent a proof of principle of exosome transmission from oligodendrocytes to neurons suggesting a new route of horizontal transfer in the cns. astrocytes are essential for the regulation of neuronal excitability, synaptic plasticity, and the vascular coupling of brain metabolism. unlike neurons, they are electrically silent, but produce a complex repertoire of ca 21 events that coordinate their major functions. however, the canonical cellular mechanism for ca 21 integration in astrocytes is unknown. using cultured astocytes and astrocytes in brain slices expressing ca 21 sensor, gcamp2, we report a candidate principle for ca 21 integration in single astrocytes. analysis of the spatiotemporal dynamics of ca 21 signaling in cultured astrocytes demonstrated that ca 21 event distribution can be described by a power law. we show that metabotropic glutamate receptor activation or changes in extracellular ca 21 regulate the power law exponent. these findings demonstrate scale-free ca 21 dynamics in astrocytes that can provide a potential computational theory for brain operation and metabolism. the biogenesis of the axon-myelin unit is controlled by reciprocal interactions between oligodendrocytes and neurons, which continue throughout life. oligodendrocytes secrete endosome-derived microvesicles termed exosomes, implicated in intercellular communication. these exosomes carry a specific set of proteins as well as rna species. here, we show that the neurotransmitter glutamate stimulates exosome secretion from oligodendrocytes by provoking ca 21 entry through oligodendroglial nmda and ampa receptors. furthermore, active neurons evoke exosome release from oligodendrocytes, indicating that neuronal activity controls oligodendroglial exosome release. in turn, neurons internalize oligodendroglial exosomes and functionally retrieve the exosomal cargo. thus, oligodendrocytes may influence the neuronal metabolism by an exosome-dependent transfer of bioactive substances to neurons. axonal degeneration resulting from lack of glial support occurs in plp and cnp deficient mice. intriguingly, both proteins are components of oligodendroglial exosomes. a proteomic approach revealed that amount and composition of exosomes derived from plp -/and cnp -/oligodendrocytes are altered, supporting the hypothesis that disturbed intercellular transfer of substances by exosomes may contribute to axonal degeneration in these mouse models. a. shahraz, j. kopatz, h. neumann university of bonn, bonn, germany microglial cells are the resident macrophages of the brain, which are derived from the embryonic haematopoiesis of the yolk sac. recently, we established a protocol for in vitro differentiation of pluripotent stem cells into microglial precursors by mimicking the embryonic haematopoiesis in a neural microenvironment. we now succeeded to produce microglia from human induced pluripotent stem (ips) cells. these human microglial cells were responsive to amyloid-b by increasing reactive oxygen species (ros) production. in addition, we produced primitive neural stem cells (pnsc) from ips cells that differentiated with suitable growth factors towards neurons. in human microglia-neuron co-culture, amyloid-b treatment resulted in shorter neural branches length compare to the control group. furthermore, results showed that the inhibitory human lineage specific receptor sialic acid binding immunoglobulin-like lectin-11 (siglec-11) was expressed on human microglial lines. we are now studying the neuroprotective role of siglec-11 in the amyloid-b-mediated microglia-neuron co-culture model. by expressing the human-lineage specific receptors, these microglial cells will be a suitable model to investigate these receptors in a human microglianeurons co-culture system.x purpose: organotypic retinal culture is a useful tool to perform preclinical drug testing, in which cellular interactions as well as tissue threedimensionality are preserved. the present study aimed to provide a detailed characterization of the morphologic changes of macro and microglial cells in this culture system. methods: retinas were isolated from 7 days old crl: cd(sd) rats with the retinal pigment epithelium attached as described previously (arango-gonzalez et al. 2010). explants were cultured for 4, 10 and 14 days (div4, div10 and div14). glial cell populations were characterized by immunostaining cryosections and wholemounts of age-matched control and cultured retinas using specific antibodies against gfap, vimentin and cd11-b. results: in div4 and div10 cultures, gfap-positive astrocytes were more robust and not homogeneously distributed as observed in vivo: in some retinal areas the astrocytic network was thicker and in other regions astrocytes were sparsely distributed. at div14 only few thinned gfap-positive astrocytes remained. gfap immunoreactivity of m€ uller cells was up-regulated in culture. however, no major difference was observed in vimentin staining between both, in vivo and in vitro retinas. m€ uller cells extended processes from the inner to the outer limiting membrane were observed for all examined retinas. microglial cells stained with cd11-b at div4 and div10 had somas more robust than in vivo and cell processes were thicker and retracted. same cells at div14 exhibited mainly a rounded morphology. conclusions: progressive morphological changes were observed in glial cell populations in retina explants. these changes include reactive macrogliosis, rearranged astrocytic distribution and microglial activation. since glial response is a hallmark of several retinal diseases, our organotypic retinal culture is a valuable resource for future investigations in retinal degenerative processes and therapy. it is widely recognized that grouped housing in enriched environment (enr) impacts the animals' brain structure and function. for instance, rats reared in enr exhibit enhanced neurogenesis in the dentate gyrus, improved hippocampus-dependent spatial memory task performance, and spine density increases of ca3 and ca1 pyramidal neurons. we found that that enr housing for four weeks after weaning induces an enhancement in gamma band local field potential oscillation power and an increase in spine density in the right ca1 stratum radiatum of long-evans rats. the mean spine size, as assessed by serial measurements of postsynaptic density areas, did not change substantially by laterality or rearing condition. one standing question is how glial processes are organized in enr treated rats. unlike cerebellar parallel fiber synapses, the astrocytic coverage of a hippocampal synapse varies from synapse to synapse. our preliminary electron microscopic observation suggests that right ca1 str. radiatum excitatory synapses tend to have larger degrees of astrocytic coverage in enr treated rats than singly caged rats.we are currently making morphometric measurements to quantitatively assess the peri-synaptic glial changes. it is becoming more and more evident that proper functioning of the neuron-astrocyte-microglia triad is fundamental for the functional organization of the brain, and thus it is of the utmost importance to better characterize their interactions in physiological and pathological processes. we recently demonstrated, in rat models of normal brain aging and lps-induced acute inflammation, that astrocytes and microglia actively collaborate in the clearance of apoptotic neurons and neuronal debris associated with programmed cell death. here we studied the interactions between neurons, microglia and astrocytes within the ca1 region of the hippocampus after bilateral common carotid artery occlusion (bccao) in the rat, a valid model of chronic cerebral hypoperfusion which leads to persistent ischemic conditions and ultimately to neuronal death. male wistar rats were subjected to permanent bccao. a group of rats was infused into the jugular vein with dipyridamole (7 days, 4 mg/kg/day by an osmotic minipump). sham-operated animals were used as controls. three months after bccao, immunohistochemical studies were performed on brain coronal slices, focussing on the hippocampal ca1 region. using an antibody against glial fibrillary acidic protein (gfap) no astrogliosis was detected. we found a significant increase in the number of total microglia, visualized using the iba1 antibody, in bccao-treated rats in comparison to the sham group (1 18%, p < 0.01, one way anova and newman-keuls) and this effect was completely reverted by dipyridamole (p < 0.01, one way anova and newman-keuls). as exemplified in figure 1 , in the ca1 and str. radiatum of bccao-treated rats, many neurons (anti-neun antibody, red) showing signs of degeneration were closely apposed to and infiltrated by astrocyte branches (anti-gfap antibody, green) which appeared to be bisecting the cell body into cellular debris, and microglia cells (anti-iba1, antibody, blue) were actively phagocytosing the damaged neurons. this finding is consistent with the scavenging activity of microglia upon dying neurons or debris, a possible mechanism that prevents further injury to neighboring neurons. it will be interesting to investigate which intercellular communication mechanisms allow the recruitment and activation of different glial cells in a well-organized reciprocal interaction to scavenge the damaged neurons and to verify the mechanisms of the protective effects of dipyridamole found in this chronic cerebral ischemic model. astrocytes are glial cells which provide important metabolic support to neurons, actively tune synaptic activity and influence brain microcirculation [1] . one of the key processes which sustain astrocyte communication with neighbouring cells is regulated exocytosis mediating the release of gliotransmitters (peptides, amino acids, atp), delivery of membrane transporters, channels and other molecules to the plasma membrane. the exocytic gliotransmitter release is thought to be associated with snare complexes. these consist of four a-helices where one of these is contributed by syntaxin-1, one by synaptobrevin-2 (sb2) and two by snap-23 in astrocytes. out of these, sb2 is a trans-membrane protein present on the secretory vesicle [2] . however, the subvesicular nano-architecture and the number of sb2 molecules per vesicle are unclear. moreover, it is also unknown how many sb2 molecules are involved in the fusion between the vesicle and the plasma membrane in astrocytes. to study single vesicles and their association with sb2 in live astrocytes, we generated a ph-sensitive indicator yellow synapto-phluorin (ysph) as a marker for sb2 and as a functional readout for monitoring the properties of fusion pores, which are formed following the merger between the vesicle and plasma membranes. in an acidic environment ysph is non-fluorescent and becomes fluorescent upon alkalinisation, permitting the study of fusion of vesicles with the membrane during gliotransmitter release [3] . our preliminary results, obtained by confocal fluorescence microscopy (with the resolution limit of about 200 nm) and by a super-resolution microscopic technique structured illumination microscopy (sim; with the resolution limit of about 120 nm) show that the new probe (i.e. ysph) efficiently reports the fusion pore establishment in astrocytes and the configuration of sb2 molecules in a vesicle. the number of schwann cells is fitted to the axonal length in the peripheral nerves. this setting is lost when tumorigenic stimuli induce uncontrolled schwann cell proliferation, generating tumours such us neurofibromas and schwannomas. schwann cells proliferate as well during wallerian degeneration. in both cases proliferation is finally arrested. we show that in neurofibroma, the induction of jmjd3 removes trimethyl groups on lysine-27 of histone-h3 and epigenetically activates the ink4a/arf-locus, forcing schwann cells towards replicative senescence. remarkably, loss of function of this mechanism allows unrestricted proliferation, inducing malignant transformation of neurofibromas. interestingly, our data suggest that in injured nerves, schwann cells epigenetically activate the same locus and go as well into the senescence program. indeed, when this pathway is genetically blocked, cell proliferation results increased after nerve injury. we postulate that the ink4a/arf-locus is expressed as part of a physiological response that prevents uncontrolled proliferation of the de-differentiated schwann cells generated during nerve regeneration, a response that is also activated to avoid overproliferation after tumorigenic stimuli in the peripheral nervous system. university of rochester medical center, rochester, united states synaptic plasticity is critical for normal neurodevelopment and proper circuit function in the adult nervous system. recent evidence suggests that microglia, classically studied in neuroinflammation, play critical roles in neurodevelopment and synaptic plasticity. however, the mechanisms driving these roles are not well understood. to start elucidating the molecular players involved in microglial communication with neurons during plasticity, we decided to first explore the role of purinergic signaling in this process. while purinergic signaling has been implicated in microglial behaviors, studies have primarily focused on neuroinflammatory roles. however, noninflamed microglia highly express the purinergic receptor, p2y12, which has known functions in microglial chemotaxis in early inflammation and can stimulate the release of cytokines implicated in synaptic plasticity. thus, we posited that purinergic signaling could contribute to the microglial motility that underlies synapse surveillance in the non-inflamed brain. this in turn may be critical for microglial roles in synaptic refinement during development. to explore this possibility, we took advantage of the p2y12 knock-out (ko) mouse and examined the morphology and motility of microglia in the visual cortex as compared to microglia in wildtype mice. first, we used immunohistochemistry for the microglia specific ionized calcium-binding adapter (iba-1) protein to stain microglia in fixed sections. we then used confocal imaging and scholl analysis to investigate the complexity of the microglial processes. we also used 2-photon microscopy to monitor microglial motility in p2y12 ko mice by crossing them with cx3cr1-gfp knock-in mice that allow visualization of microglia including fine processes in vivo. our preliminary evidence suggests that p2y12 disruption alters basal microglial morphology and behavior making microglia less complex and altering their motility patterns. we are now investigating whether microglia-synapse interactions, as well as functional plasticity elicited by visual experience are altered in p2y12 ko mice. our studies will expand our understanding of emerging roles for microglia in neurodevelopment and will pioneer new non-pathological roles for microglial purinergic signaling. a. dvorzhak, a. wojtowicz, r. grantyn charit e -university medicine, berlin, germany in neurodegenerative diseases, the afflicted brain is both an important object of study and an opportunity to characterize a given cellular interaction from a pathophysiological perspective. this dual approach is particularly advantageous when human disease is based on a monogenetic defect and an appropriate animal model becomes available for detailed investigation, as in case of z_q175_ki (q175), a new knock-in mouse expressing a mutant form of murine huntingtin. electrophysiological recordings of gabaergic unitary ipscs from striatal output neurons (sons) in sagittal slices from wild-type and homozygous q175 challenged the current viewpoint that gabaergic transmission is enhanced in the hd striatum. quantal analysis in combination with high frequency stimulation and paired pulse tests revealed that synaptic gaba release is in fact tonically suppressed resulting in disinhibition of striatal output activity (dvorzhak et al j physiol 2013). the underlying mechanism involves a retrograde endocannabinoid signalling pathway linking postsynaptic mglur5 with presynaptic cb1 and gaba release. in addition to this deficit, z-q175_ki homozygotes exhibited a significant reduction of tonic inhibition via extrasynaptic gaba(a) receptors. using pharmacological tools to alter the ratio of ambient glutamate and gaba concentrations made clear that the hd-related depression of synaptic and extrasynaptic gabaergic actions depends on the state of both the astrocytic glutamate transporter glt-1 and the gaba transporter gat-3. in support of h eja and colleagues, our imaging experiments suggest that in normal striatal astrocytes gat-3 is in a releasing mode and driven by the activity of glt-1. however, the latter activity is much lower in the hd striatum. sbfi recordings of glt-1-related na transients and patch clamp recordings of glt-1 transporter currents revealed a marked reduction (less than 50% of wt level) in sr-labelled astrocytes from symptomatic hd mice. together, our results suggest that the deficiency of astrocytic glutamate uptake might represent a pathophysiological key mechanism underlying the observed disinhibition in the striatum of mice affected by huntington's disease. nodes of ranvier are highly enriched in voltage-gated sodium channels (nav), allowing rapid action potential propagation on myelinated axons. cell adhesion molecules (neurofascin-186 (nfasc186) and contactin) and scaffold proteins (ankyring and bivspectrin), which are also enriched at the nodes, have a critical role in assembly and/or stabilization of na v clusters. oligodendrocytes have been described to induce nav channel clustering at nodes in the central nervous system (cns); however, the nature of the signal(s) involved in nodal formation remains mostly unknown in the cns. to gain insight into cns node assembly, we developed hippocampal neuronal cultures from e18 rat embryos. during the first week, as expected, na v channel accumulation was detected at the axon initial segment (ais), co-localized with ankyring and nfasc186, whereas expression along the axon was diffuse and hardly visible. in contrast, at 14 days in vitro (div), regularly spaced clusters of na v channels, ankyring and nfasc186 were detected along the axon. the percentage of hippocampal axons with clusters increased from 4.1 6 3.1% at 14 div, to 15.8 6 3.5% and 17.167.1% at 17 and 21 div, respectively (mean 6 sd). importantly, these clusters were formed in the absence of myelin and no accumulation of the paranodal protein caspr was detected at this stage. to study the role of extrinsic versus intrinsic pro-aggregating factors, we analyzed nearly pure hippocampal neuronal cultures. these purified neurons still formed ais, while only few axons formed na v clusters. interestingly, when these purified neurons were co-cultured with oligodendrocytes or with conditioned medium from oligodendrocytes, the percentage of neurons with nascent nodes was greatly enhanced. taken together, these results confirm that some glial soluble factor(s) might be involved in na v channels clustering, as previously shown for retinal neurons (kaplan et al., 2001). furthermore, nascent nodes were detected on gabaergic neurons, but not on glutamatergic hippocampal neurons, thus arguing also for the role of intrinsic neuronal factors in their establishment. using hippocampal sections from gad67-gfp mice, we showed that some gabaergic interneurons were myelinated in vivo. moreover, we observed that nascent node formation prior to myelination also takes place in vivo, strengthening physiological relevance. the electrophysiological properties of neurons with nascent nodes will be studied. microglia in the degenerating or aging brain are primed by aspects of pathology to produce exaggerated pro-inflammatory responses to subsequent central and systemic inflammatory insults. however, the mechanisms by which microglia become primed, rather than adopting an m1 phenotype, are not clear. triggers for priming may include altered expression of complement, recognition of amyloid or neuronal/synaptic debris for phagocytosis, decreased engagement of microglial regulatory molecules such as cd200r, trem2 or fractalkine receptor. there is also evidence that loss of basal neurotransmitter tone may contribute to this state. microglia are known to express the nicotinic cholinergic a 7 receptor, which has been shown to influence macrophage and microglial reactivity. in the current study we performed limited lesions of the basal forebrain cholinergic system using the murine-p75-saporin immunotoxin (mu-p75-sap), to investigate the degree to which loss of cholinergic tone predisposes the microglia to subsequent inflammatory stimulation and to assess the consequences of this for cognitive function in the vulnerable brain. intracerebroventricular injection of mu-p75-sap (0.08 lg) depleted cholinergic neurons in the basal forebrain and decreased cholinergic innervation of the hippocampus, but left performance on hippocampal-dependent reference and working memory tasks relatively intact. however, decreased cholinergic innervation of the hippocampus conferred increased susceptibility to cognitive deficits induced by systemic lps (100 lg/kg) 40 days after lesioning, but microglia were not primed 40 days after 20% lesions. to investigate the regional, temporal and neurochemical basis of this we induced more severe lesions of the basal forebrain (using 1.2 lg mu-p-75 sap) and assessed microglial priming at 14 or 40 days post-lesion. these data demonstrate that microglia are primed by low dose challenge at 14 but not 40 days and, when more robust lesions are induced, priming remains even at 40 days. these responses are observed at the site of injury (medial septum) but more profoundly at the site of denervation, the hippocampus. indicating that neuronal injury and, particularly, loss of cholinergic tone have a profound effect on the reactivity of the microglia to subsequent inflammatory challenge. these data expand our knowledge of microglial priming and have clear implications for the interaction of cholinergic tone and inflammation in cognitive dysfunction such as dementia and delirium in which cholinergic dysfunction is implicated. ). however, glucose may also be metabolized to glycogen or oxidized to co 2 , so it is not obvious that the stimulation of glut1 and hexokinase leads to lactate release. the objective of this work was to investigate whether lactate may be released by cultured mouse astrocytes within the time frame of the interstitial lactate rise observed in vivo. a first approach with an enzymatic assay showed a significant increase in extracellular lactate after 1 minute of exposure to 12 mm k 1 or 50 lm glutamate. to obtain better time resolution we developed a "lactate sniffer", a hek293 cell that expresses the fret lactate nanosensor laconic (san mart ın et al., plos one 2013). seeded on top of an astrocytic monolayer, the sniffer cell responded within 10 seconds of exposure to elevated k 1 12 mm and a similar response was elicited by 3 mm ba 21 . the response to k 1 and ba 21 supports a role for the na 1 /bicarbonate co-transporter nbce1, previously shown to mediate the stimulation of astrocytic glycolysis (ruminot et al., j. neurosci., 2011). the sniffer cell detected an equally fast increase in extracellular lactate when the culture was exposed to 50 lm glutamate. previous work had shown that glutamate does not stimulate glucose consumption in the short-term (bittner et al., j. neurosci. 2011), and therefore the rapid release of lactate came as a surprise. this phenomenon may relate to glycogen mobilization or perhaps to inhibition of astrocytic oxygen consumption (azarias et al., j. neurosci. 2011). k 1 , ba 21 and glutamate did not produce detectable effects on the sniffer cell in the absence of astrocytes. the rapid release of lactate by astrocytes exposed to k 1 and glutamate supports a role for these cells in the fast increase in interstitial lactate concentration that accompanies synaptic activity. ] i ) are regularly occurring as a result of uptake of glutamate from the synaptic space. glutamate uptake occurs via the glutamate/na 1 co-transporters glast and glt-1, where one glutamate is accompanied by 3 na 1 and 1 h 1 in exchange for 1 k 1 . the salt pump, na,k-atpase (nka), is responsible for the maintenance of the trans-membrane na 1 gradient by exporting 3 na 1 and importing 2 k 1 for each atp. nka is dose-dependently inhibited by ouabain. astrocytes express two isoforms of the catalytic nka a subunit, the ubiquitous a1 and in the cns the glia-specific a2. the isoforms differ with regard to na 1 affinity, which is lower for a2 and with regard to ouabain affinity, which is higher for a2 than for a1. although a2 mutations give rise to neurological symptoms -familial hemiplegic migraine type 2, the relative roles of the a1 and a2 isoforms in astrocytes are still incompletely understood. we hypothesized that the low na affinity of a2 makes it more suitable to cope with transient large increases in na i . to test this, we compared the effect of 200 mm glutamate for 10 min on real time changes of [na 1 ] i in primary astrocytes, which, following transient transfection, predominantly expressed either the a1 or the a2 isoform. in a1 cells glutamate caused a significantly larger increase in [na 1 ] i and longer recovery time to base line [na 1 ] i than in cells expressing a2. glutamate uptake dependence on either a isoform was determined by aspartate uptake in astrocytes expressing endogenous a1 and a2. in the presence of ouabain concentrations that selectively inhibit a2, we found a modest (1.9mm) increase in [na 1 ] i , accompanied by a disproportionately large decrease (24%) in glutamate uptake. it has been suggested that astrocyte glutamate transporters are clustered in microdomains where they may interact with nka. in gst pull down assays on rat brain we found that both glt-1 and glast interacted with the 1st intracellular loop of both a1 and a2, but the interaction was significantly stronger for the a2 isoform. no interaction was found with other segments of the a molecule. the data indicate that the nka a2 isoform, which has a more restricted expression than a1, plays a specific role for the interaction with the glutamate transporters and for sodium homeostasis in astrocytes. this specificity may be attributed to low sodium affinity of a2 and to the relatively high capacity of a2 to interact with the astrocyte glutamate transporters. ozone, a major component of air pollution, has considerable impact on public health. besides its well described inflammatory and dysfunction effects on the respiratory tract, there is accumulating evidence indicating that ozone exposure also affects brain functions. however, the mechanisms through which ozone exerts toxic effects on the cns remain poorly understood. we previously showed that in addition to lung inflammation,ozone exposure caused a neuronal activation in the dorsolateral regions of the rat nucleus tractus solitarius (nts, a sensory nucleus involved in visceral information processing) overlapping terminal fields of lung primary afferent running in the vagus nerves to investigate this hypothesis, we used electron microscopy and immunoblot techniques. in ozone-exposed animals, the astrocytic coverage of nts glutamatergic synapses is increased while the astrocyte volume fraction and the astrocyte membrane densities are not significantly increased. moreover, the expression of specific astrocyte markers gfap, s100beta and ezrin did not change in ozone-exposed animals. altogether, our results indicate that ozone inhalation induces glial plasticity, which is restricted to the peri-synaptic coverage. . we recorded pp-gc synaptic activity after eae induction via adoptive transfert (at-eae) and found that mepsc frequency was increased compared to control animals. moreover synaptic alteration in at-eae mice depended on astrocytic tnfr1 because the effect was absent in tnfr1-/-mice but reappeared upon tnfr1 re-expression in astrocytes. nr2b receptors are also necessary because in vivo ifenprodil injection prevented synaptic alteration. overall, our study reveals a specific mechanism responsible for hippocampal synaptic dysfunction possibly relevant for cognitive impairment in ms. research supported by snsf grant 31003a-140999 and nccr "synapsy" to av. we patched ng2 cells in the hippocampal ca1 region of ng2-dsred mice (8-20 days old) and investigated in current clamp mode how sodium and potassium channels affected synaptic depolarizations. epsps and ipsps were evoked from a resting membrane potential of 285mv by injecting mock epsc or ipsc waveforms derived from miniature currents. epsps which depolarized ng2 cells to more than 245 mv were increasingly shortened by vgcs. at 216 6 1 mv, corresponding to a quantal content of 640, the half width of epsps was shortened to 47 6 3% while the amplitude was hardly altered. application of ttx, 4-ap and tea showed that most of the observed shaping of epsps was primarily due to activation of a-type k1 current rectifying the depolarizing input. na1 currents only slightly ($10%) amplified epsp input, but this effect was outcompeted by the dampening effect contributed by a-type current ($15%). tea was almost without effect on epsps. due to their slower time course ipsps were very differently shaped by vgcs: while recruitment of vgcs by mock ipsps started at a similar threshold voltage, the maximal suppression of ipsp amplitude was much stronger. when depolarizing ng2 cells to 235 6 1 mv, corresponding to a quantal content of 235, ipsp amplitudes were suppressed to 52 6 4% and the duration of ipsps was not shortened but increased to 193 6 5%. a-type k1 current was the primary contributor for rectifying as well. interestingly, if a-type k1 current was blocked, ipsps with a quantal content of 105 unmasked a small ttx-sensitive spikelike depolarization of 20 6 2 mv (duration: 7 6 1 ms). for comparison, while synaptic responses to simultaneous release of 100 glutamatergic vesicles were hardly altered by vgcs, the synaptic response of 100 gabaergic vesicles was substantially low-pass filtered by a-type potassium currents. altogether, our results suggest that vgcs in ng2 cells normalize the synaptic strength of excitatory and inhibitory inputs and support their differentiation by ng2 cells through further emphasizing their pre-existing kinetic differences. to explore the involvement of this pathway in pain and analyze its impact separately in sensory neurons and sgcs, we tested pharmacological inhibitors and transgenic mice in an orofacial pain model. transient inflammation in the submandibular region of mice was induced using complete freund's adjuvant (cfa) and tactile sensitivity was quantified using von frey filaments. although inflammation was resolved by 28 days after injection, tactile hypersensitivity persisted in wildtype mice for at least 53 days, consistent with chronic post-inflammatory pain. tactile hypersensitivity in mice was reversed by systemic injection of the panx1/gap junction blockers mefloquine or carbenoxolone at 7 days (peak inflammation) and at 28 days after cfa-injection, suggesting the contribution of these channels to tactile hypersensitivity. in dissociated trigeminal ganglia (tg), which innervate the submandibular skin, bzatp-induced yopro uptake into neurons and glia was prevented by mefloquine, demonstrating the functional presence of the p2x 7 r-panx1 complex in tg. moreover, tg of cfa-injected mice showed more atp release and immunostaining of tg revealed higher expression of panx1 at 1 week after cfa injection compared to controls. development of hypersensitivity was prevented in p2x 7 r-null and in panx1-null mice, and was attenuated in mice with glia-specific deletion of panx1 (gfapcre-panx1f/f) but not with neuron-specific deletion (mnfhcre-panx1f/f), emphasizing the importance of glial panx1 signaling in pain. because p2x 7 r and panx1 both have a key role in the immune response by activating the inflammasome, we are currently dissecting the relative importance of neuron-glial communication compared to inflammatory responses in the development and maintenance of orofacial pain. overall, our results show that the p2x 7 r-panx1 complex likely plays a major role in signaling events contributing to tactile hypersensitivity. synaptic plasticity is central to the process of learning and memory and is accompanied by strengthening of existing synapses and/or formation of new synapses. numerous studies have investigated the molecular mechanisms of learning and memory with neurons as the primary interest. given the tight coupling between synaptic activity and brain metabolism, it seems reasonable to assume that synaptic plasticity changes occurring at the synaptic level may also occur at the metabolic level in order to keep up with the augmented demand at the synapse. in this study we investigated the importance of genes involved in brain energy metabolism, and in particular those involved in neuronglia metabolic coupling during fear-motivated inhibitory avoidance learning. using ( 14 c) 2-deoxyglucose (2-dg) technique we first defined the brain areas that are involved in this learning process. context-dependent avoidance behavior was tested in c57bl/6 mice using the step-through inhibitory avoidance paradigm (ia). regional brain metabolic activity, as measured by the 2dg uptake, was quantified in several brain regions. brain metabolic mapping revealed increased glucose utilization in hippocampus, amygdala [(basolateral complex (bla) and the central nucleus (cea)], and anterior cingulate cortex and mammillary bodies. quantitative mrna levels were assessed in dorsal hippocampal tissue at different time points following ia learning. results obtained demonstrate that learning modulates the expression pattern of genes involved in brain energy metabolism, i.e. glycogen synthesis and degradation, pyruvate metabolism, the pentose phosphate shunt and the astrocyte-neuron lactate shuttle (anls) in a time dependent manner. we found a late-phase of enhanced dorsal hippocampal gene expression, particularly for anls related genes, including monocarboxylate transporter 1 (mct1). furthermore, we found that mice deficient in mct1 transporter display impaired long term memory in the inhibitory avoidance and spatial learning in morris water maze. these observations indicate that metabolic adaptation shown by neuron-glia metabolic coupling might be a relevant phenomenon following learning in conjunction with synaptic plasticity. in order to systematically as well as functionally analyse rmg reactivity to retinal degeneration and thus explore the rmg-derived signalling molecules in depth, we aim at comprehensively profiling proteomewide cellular responses to stimuli. we thus developed a proteomics approach screening specifically for cell surface and membrane proteins as well as secreted protein expression profiles and in addition monitor quantitative changes induced by prototype inflammatory inducer lipopolysaccharide lps. this workflow utilises sugar residues of cell surface proteins on intact rmg which are mildly oxidized and then covalently coupled to biotin. biotinylated proteins are affinity purified and glycosylated peptides are specifically released by pngasef followed by protein identification and quantification by label-free lc-msms. this workflow resulted in the identification of more than 500 proteins on cell surfaces complemented by more than 700 proteins identified in the rmg secretome. bioinformatic analysis allocates 75% of the cell surface proteins to be truly membrane or extracellular matrix proteins. this comprehensive cell surfaceome includes transmembrane receptors, transporters, adhesion molecules, signalling molecules and proteases, including 18 cd markers, 18 integrins, 41 solute carriers, two ephrins, five ephrin receptors and six plexins. treatment of cells with lps results in a highly reproducible significant shift of cell surface proteome with upregulation of 36 proteins and downregulation of 13 proteins. among those lps-induced cell surface expression changes are proteins that suggest an active role of these glial cells in inflammatory processes. the specific changes will be discussed in detail. cell surface biotinylation on glycosyl-residues in combination with label-free lc-msms is a sensitive and reproducible method to profile cell surface proteomics and sheds light on the biological properties of cells. background: neuronal activity alters calcium ion (ca 21 ) dynamics in astrocytes, but the physiologic relevance of these changes is controversial. to examine this issue further, we generated an inducible transgenic mouse model in which the expression of an inositol 1,4,5-trisphosphate absorbent, "ip 3 sponge", attenuates astrocytic ca 21 signaling. results: attenuated ca 21 activity correlated with reduced astrocytic coverage of asymmetric synapses in the hippocampal ca1 region in these animals. the decreased astrocytic 'protection' of the synapses facilitated glutamate 'spillover', which was reflected by prolonged glutamate transporter currents in stratum radiatum astrocytes and enhanced n-methyl-d-aspartate receptor currents in ca1 pyramidal neurons in response to burst stimulation. these mice also exhibited behavioral impairments in spatial reference memory and remote contextual fear memory, in which hippocampal circuits are involved. conclusions: our findings suggest that ip 3 -mediated astrocytic ca 21 signaling correlates with the formation of functional tripartite synapses in the hippocampus. they are the only non-neuronal cell type in the brain that receives synaptic input from neurons. ng2-cells exhibit a variety of ca 21 -signalling pathways, which might be triggered by pre-synaptic neuronal activity. however, no global increase in the intracellular ca 21 -concentration ([ca 21 ] i ) was detected in ng2-cells employing the minimal stimulation technique at neuron-glial synapses. therefore we assume that post-synaptic ca 21 -microdomains might be activated under physiological conditions. these ca 21 -signals are locally restricted to the plasma membrane. membrane bound ca 21 -sensors with a high signal-to-noise ratio, such as lck-gcamp3, are best suited for optimal visualisation of ca 21 -microdomains. here, we set out to express lck-gcamp3 in ng2cells together with the cytosolic reporter dye dsred. we generated transgenic mice which express a bidirectional promoter encoding both proteins under the control of a tet-off system. this strategy allows an exclusive expression of the transgenes only in the presence of a tet-responsive transcriptional activator (tta). the construct was successfully cloned and expressed in hek293 cells. as expected, lck-gcamp3 was located only at the inner plasma membrane while dsred was expressed all-over the cytosol. functionality of the ca 21 -sensor was tested in vitro. elevation of [ca 21 ] i reliably increased the fluorescence intensity of lck-gcamp3. after zygote injection seven positive founder animals could be identified and will be crossbred with a ng2-tta mouse line. taken together, the presented construct appears suitable for functional imaging of ca 21 -microdomains in post-synaptic ng2-cells. according to the "lactate shuttle" hypothesis, glycogen stored in astrocytes can be converted to l-lactate and exported to neurones as preferred energy substrate. we use optogenetics to investigate signalling between astrocytes and noradrenergic (naergic) neurones of the locus coeruleus (lc). to selectively activate g s -protein-mediated signalling in astrocytes, we generated a viral vector to express a chimera of rhodopsin and b 2 -adrenoceptor (optob 2 ar). we confirmed that this construct activates camp-mediated signalling in astrocytes. in cultured astrocytes, excitation of optob2ar resulted in acidification as detected using snarf-5 indicator, and this acidification could be blocked by pre-incubation with an inhibitor of glycogen breakdown 1,4-dideoxy-1,4-imino-d-arabinitol (dab), suggesting that the acidification was due to build-up of l-lactate. fast scan cyclic voltammetry (fcv) was used to measure noradrenaline (na) release in organotypic slices cut at the level of the lc in which astrocytes were expressing optob 2 ar. light stimulation of astrocytes powerfully induced release of na which could be prevented by pre-incubation with dab or application of d-lactate. bath application of l-lactate (!100 mm) in absence of optogenetic stimulation triggered the release of na. in patch clamp experiments, l-lactate depolarised lc neurones and evoked vigorous firing of action potentials. these experiments suggest that activation of g s -protein-coupled receptors in astrocytes could potentiate the release of na from lc neurones via l-lactate. the cellular and molecular mechanisms of this signalling mechanism are currently under investigation. supported by the british heart foundation. rgcs. in addition to this, the organization of astrocytes in the retina of a rat model of glaucoma was found to be significantly different to those of a healthy retina. these alterations in glial cell morphology may reflect changes in their relationship with rgcs and could signify profound changes in their secretome, which may influence rgc survival. in this study we investigated labeling of astrocytes by sr101 in acute slices from the ventro-lateral medulla and the hippocampus of transgenic mice expressing egfp under the control of an astrocyte-specific promoter. while sr101 efficiently labeled egfp-expressing astrocytes in hippocampus, we found that sr101-staining was very weak in the ventro-lateral medulla and rather unspecific. thus, sr101 is not a reliable marker for brainstem astrocytes. although carbenoxolone decreased the labeling of astrocytes in the hippocampus significantly, mefloquine which blocks pannexin and connexin hemichannels, was unable to prevent sr101 uptake in hippocampal astrocytes. time-lapse 2-photon imaging revealed that in hippocampus both astrocytes and neurons showed temporary sr101-loading. in astrocytes the rise of sr101 fluorescence was slower as compared to egfp-negative cells and sr101 was quickly removed from non-astrocytic cells during washout, while it was retained in astrocytes. in brainstem astrocytes, however, only a very weak and transient sr101-labeling was observed. to test if sr101 is actively removed from astrocytes in the brainstem, we applied mk-571 to block the multi-drug resistance transporters mrp-1. we did not observe any increase of sr101-labeling in brainstem astrocytes. in contrast, astrocytic sr101 labeling was significantly reduced by substrates of organic anion transport, probenecid, estron-3sulfate and dehydroepiandrosterone sulfate suggesting that sr101 is actively transported into hippocampal astrocytes by an organic anion transporting polypeptide (oatp). additionally, the data suggest that astrocytes modulate extracellular concentration of neurosteroids in the hippocampus. as an alternative approach we have used scanning electron microscopy (sem) to obtain high-resolution back scattered images (bsi) of serial ultrathin sections over a wide-area. in tem the size of the specimen is limited to less than 1 mm 2 , whereas in sem it can be expanded to 20-30 mm 2 , allowing us to mount and examine more than 100 serial ultrathin sections on the same stage, collecting 3d information about many nodal structures simultaneously. here we demonstrate a variety of nodal architectures assembled from bsi-sem (su8010,hitachi). in our preliminary study using this technique, the pattern of cellular processes approaching the perinodal axolemma is quite different among axons even in only four nodes examined in optic nerve. in the present study, we analyzed perinodal elements at 25 whole nodes in rat optic nerves and reconfirmed this variety of perinodal glial elements include the followings: 1) perinodal space with extracellular matrix (20-100%); 2) closely abutting astrocytic processes (0-80%) ; 3) unidentified glial cell, presumable ng21 glial processes (0-50%), but could not find 4) pre-synapse-like pseudo-neuronal processes, which was observed at the perinode in the cns grey matter (cerebral cortex) in the previous study. additionally, we found unexpected structures, small teardrop-like protrusion(s) of axolemma at 18 out of 25 nodes examined (72%). in 16 nodes out of these 18 nodes (88.9%), glial elements encircled or densely contacted with the teardrop protrusions. a typical case of 3d reconstructed image is shown in the figure. nodal axon (ax, red), unidentified glial process (yellow; ug, presumable ng2 cell) and teardrop-like protrusion (asterisks) are demonstrated. outer part of the protrusion from the position indicated two arrows is encircled by the process of ug cell. based on these observations, we propose that some glial cells, including astrocytes and/or ng2 cells, might contribute to remove wasted membranous elements from the axon at the node of ranvier, which is a new type of neuron-glial interaction. to understand what makes this ssc network more synchronous and excitable, we performed a morphological study of neurons and astrocytes by estimating densities of neun and s100-positive cells respectively. although we found similar densities of neurons and astrocytes between gaers and non-epileptic controls, the thickness of the ssc was 30% smaller in gaers. western blotting quantification of gfap, another astrocytic marker, was significantly higher at p15-p17, as compared with non-epileptic control. moreover, gfap-immunolabeling suggested that these astrocytes were reactive. we hypothesized that these modifications are linked with an alteration of astrocytic and/or neuronal physiological calcium excitability that could lead to the occurrence of epileptic seizures. to better evaluate the role of astrocytes and neurons networks during the development of ssc between p15 and p25, we used two-photon microscopy imaging coupled with eeg recording in animal under anesthesia and neuroleptanalgesia. we first analyzed neuropile, representing ascendant projection of deeper cortical layers, astrocytes and neurons calcium activities. preliminary results obtained from animals under isoflurane anesthesia showed similar neuropile activities. astrocytic and neuronal activities were too weak to highlight differences between gaers and non-epileptic control. this is likely due to the mode of anesthesia known to strongly decrease astrocyte calcium signaling and neuronal synchronization. current experiments are therefore performed using neuroleptanalgesia know to allow the occurrence of swd. the expected data should lead to a better understanding of neuron-astrocyte interactions during brain development and the mechanism underlying epileptogenesis in a genetic model of epilepsy. purpose: to study the effects of laser-induced ocular hypertension (oht) in the astrocytes of contralateral eyes two weeks after lasering. methods: adult swiss mice were divided into two groups: na€ ıve (n 5 6) and lasered (n 5 6). retinal whole-mounts were immunostained with antibodies against gfap. the gfap-labelled retinal area (gfap-ra) and the number of astrocytes were quantified. results: in comparison with na€ ıve: i) astrocytes were more robust in contralateral eyes. in oht-eyes, the astrocyte population was not homogeneous, given that astrocytes displaying only primary processes coexisted with astrocytes in which primary and secondary processes could be recognized, the former having less intense gfap-ir (p<0.001). the mean percentage of astrocytes in which only primary processes could be detected was 37.8%; ii) gfap-ra was increased in contralateral (p<0.05) and decreased in oht-eyes (p <0.001); iii) the mean intensity of gfap-ir was higher in oht-eyes (p<0.01), and the percentage of the retinal area occupied by gfap1 cells with higher intensity levels was increased in contralateral (p 5 0.05) and in oht-eyes (p<0.01); iv) the astrocyte number did not differ significantly among the eyes analyzed. however, in oht-eyes the number of astrocytes in which primary and secondary processes could be observed were decreased (p<0.01). conclusions: two weeks of laser-induced oht caused changes in the gfap-labelled retinal area but not in astrocyte number in both, contralateral and oht-eyes. on the basis of the astroglial changes detected in the present work, the use of the contralateral eye as an internal control in experimental model unilateral oht should be reconsidered. astrocytes show a high level of functional and morphological heterogeneity and are involved in many aspects of neural function, e.g., formation of the blood-brain barrier, regulation of ion homeostasis, synaptogenesis, and synaptic plasticity. despite the diversity of this cell type, the various astrocyte subpopulations are not well characterized, which is mainly due to the lack of markers that would allow for classification of astrocyte subsets. our study aimed at phenotyping of astrocyte subpopulations based on the expression of cell surface markers, which are associated with certain functions. we used two novel monoclonal antibodies, acsa-1 and acsa-2 (acsa: astrocyte cell surface antigen), directed against extracellular epitopes of astrocyte-specific cell surface markers to identify astrocyte subtypes. the acsa-1 antibody was generated by immunization of glast1 knockout mice and specifically detects the astrocyte-specific l-glutamate/l-aspartate transporter glast (eaat1, slc1a3), whereas the acsa-2 antibody results from an immunization of rats with astrocytes isolated from gfap-egfp transgenic mice. a mass spectrometric approach for the ligand-based identification of the acsa-2 antigen pointed to a number of candidates, which have to be validated by further experiments. the antibodies acsa-1 and acsa-2 were carefully analyzed by costaining experiments with commonly used intracellular astrocyte markers. we found that both antibodies specifically detect astrocytes in the developing and adult central nervous system. in contrast to antibodies against intracellular markers, such as gfap, s100ß, or glutamine synthetase, acsa-1 and acsa-2 can be used to detect and isolate living astrocytes, which enables further analysis and culture of the separated cells. flow cytometric analysis of acsa-1 and acsa-2 antigen expression on cells from different brain regions, as well as immunohistochemical analysis, revealed differences in the expression patterns. this allowed us to define different astrocyte subtypes especially in the cerebellum and olfactory bulb of the neonatal mouse brain. future work will focus on the identification of additional astrocytespecific cell surface proteins for a comprehensive classification of astrocyte subtypes based on cell surface marker expression or expression patterns. the reciprocal communication between neuronal and glial cells represents the key component of the immunosurveillance system of the brain. it has been proposed that the neuro-glial interaction is impaired in human alzheimer's disease. in this study we focus on neuronal "on" and "off" signalling molecules in the transgenic rat model for alzheimer's disease. using enzyme-linked immunosorbent assay we determined the level of cd47, cx3cl1 ("off" signalling molecules) and mmp3, trem2 ("on" signalling molecules) in brain homogenates. our results demonstrated significantly up-regulated level of cd47 (p < 0,001) in our ad rat transgenic animal model. on the other hand, quantification of mmp3 level revealed significant decrease of mmp3 expression (p < 0,001) in tested transgenic animals. previous studies showed that cd47 and mmp3 are predominantly expressed on neuronal cells in cns where cd47 functions normally as a marker of "self" to protect intact body component or "don't eat me" molecule which protect cd47-expresing cells from phagocytosis. our results indicate that overexpression of cd47 on neuronal cells expressing pathologically modified truncated tau protein can be used as a neuroprotective mechanism potentiating spread and accumulation of pathological modified tau protein in neurons affected by ad pathology which in final stage support progression of the developing disease. in conclusion, we showed, that pathologically modified tau protein can modulate specific "on and off" signalling molecules and thus affects neuron-glia interaction in ad. field-excitatory post-synaptic potentials (fepsp) were recorded from the ca1 area of hippocampal slices prepared from wistar rats (3-5 weeks old) as before (fontinha et al., 2008) . after obtaining a stable value of the fepsp slope for at least 30 min, ltp was induced by delivery of 3 trains of 3 pulses at 100hz, each train being separated by 200 ms. ltp magnitude (% increase of fepsp) was evaluated 50-60 min after induction. ltp magnitude in control conditions was 16 6 5.9%, whereas in a second independent pathway of the same slices but in the presence of bdnf (20ng/ml) it was 39 6 4.7% (n 5 7; p <0.05). ltp was completely abolished when hippocampal slices were superfused with the gliotoxin fluorocitrate (fc, 200 mm), which selectively reduces the astrocytic metabolism decreasing intracellular ca 21 signalling and the release of gliotransmitters. interestingly, in presence of fc, the facilitatory action bdnf (20 ng/ml) upon ltp was completely prevented. noteworthy, when fc (200 mm) treated slices were superfused with the selective adenosine a 2a receptor agonist, cgs21680 (30nm), the facilitatory action of bdnf (20 ng/ml) upon ltp was rescued (n 5 5; p < 0.05), so that dltp caused by bdnf (ltp in the presence of bdnf -ltp in the absence of bdnf in two independent pathways of the same slices) in fc 1 cgs 21680 treated slices (19%) was similar to that observed in control slices (23% in the absence of fc and cgs 21680). the results suggest that the facilitatory action of bdnf upon ltp is controlled by astrocytes, and that this role of astrocytes results from their contribution to the extracellular accumulation of adenosine allowing a 2a receptor activation to gate plasticity actions of bdnf. several studies demonstrated the ability of astrocytes to sense, respond to and regulate neuronal function. among the many functions of glial proteins, glutamate (glu) and gaba transporters play important roles in balancing excitatory and inhibitory signals in the brain. here we show that astrocytes regulate the tonic inhibition of neurons by the concerted action of glu and gaba transporters, thereby protecting both neurons and glial cells from overactivation. we demonstrate that the uptake of glutamate triggers the reverse function of glial gat-2/3 transporters by elevating the intracellular na 1 concentration in astrocytes. the released gaba significantly contributes to the tonic inhibition of neurons in a network activity-dependent manner. we also describe the source of the releasing gaba that is synthesized by an alternative pathway from polyamines. moreover, in the low-[mg 21 ] model of epilepsy, we show that blockade of the glial glu/gaba exchange mechanism increases the duration of seizure-like events and also results in increased activity of astrocytes, demonstrating the neuroprotective impact of the mechanism. finally, we show that the released glial gaba modulates the power of gamma range oscillation in vivo, suggesting that the glu/gaba exchange mechanism is also functioning in the intact hippocampus under physiological conditions. revealing this novel molecular mechanism by which astrocytes provide an adjustable, in situ negative feedback on the excitability of neurons is expected to broaden our understanding about the regulation of neuronal activity by astrocytes and may open up new targets for the treatments of pathological conditions, such as epilepsy or ischemia. chronic stress is well recognised to decrease the number of gfap1 astrocytes within the prefrontal cortex (pfc). recent research, however, has suggested that our understanding of how stress alters astrocytes may be incomplete. specifically, chronic stress has been shown to induce a unique form of microglial remodelling, but it is not yet clear whether astrocytes also undergo similar structural modifications. such alterations may be significant given the role of astrocytes in modulating synaptic function. accordingly, in the current study we have examined changes in astrocyte morphology following exposure to chronic stress in adult rats, using three-dimensional digital reconstructions of astrocytes. our analysis indicated that chronic stress produced profound atrophy of astrocyte process length, branching and volume. we additionally examined changes in astrocyte-specific s100b, which is both a putative astrocyte marker, and a protein whose expression is associated with astrocyte distress. while we found that s100b levels were increased by stress, this increase was not correlated with atrophy. we further established that while chronic stress was associated with a decrease in astrocyte numbers when gfap labelling was used as a marker, we could find no evidence of a decrease in the total number of cells, based on nissl staining, or in the number of s100b1 cells. this finding suggests that chronic stress may not actually reduce astrocyte numbers and may instead selectively decrease gfap expression. together, these results provide a significantly more elaborate picture of how chronic stress alters the pfc. the drosophila nervous system is ensheathed by several types of glial cells, one of which, namely the subperineurial glia, forms pleated septate junctions (psj) to build a tight blood-brain barrier (bbb). hence, nutrients from the surrounding hemolymph must cross these glial cells to reach the neurons. the amount of nutrients entering the nervous system is likely to be tightly controlled through neuron-glia communication to ensure optimal metabolic supply while avoiding disturbance of the extracellular homeostasis. we aim to elucidate signals involved in this regulatory interaction. to this end, an rnai-based screen in glia of adult flies is performed using the gal4/gal80 ts system. we analyze the intake of blue-dyed food by photometric measurement. since impairment of the metabolic supply of the brain should severely affect the organism, compensatory changes in feeding behavior are expected. for instance, knockdown of nutrient transporters specifically in glia would then induce excessive feeding in normally nurtured individuals. the first set of rnai lines screened includes genes that have previously been found to be essential in glia for survival or locomotion of the animal. furthermore it comprises metabolic enzymes, all carbohydrate transporters, neuropeptides and their corresponding receptors. candidate genes will be further characterized regarding their role in controlling the energy homeostasis of the nervous system. the discovery that activation of non-neuronal cns microglia plays a causal role in spinal processing of nociceptive signaling has shed new light on the processes underlying neuropathic pain facilitation. however, there remains much uncertainty as to the necessary contribution of microglia to enhanced pain states. we aim to define the particular role of microglia for the initiation of neuropathic pain and by answering this question also learn if the function of peripheral myeloid cells is distinct or redundant in this process. methods: to specifically investigate spinal microglia and peripheral macrophages in the pathogenesis of neuropathic pain, we model chronic pain by performing partial ligature of the sciatic nerve in cd11b-hsvtk mice engrafted with gfp bone marrow (gfp>cd11b-hsvtk). cd11b-hsvtk 1/mice allow the exchange with peripherally-derived, gfp 1 macrophages upon central depletion of endogenous cd11b 1 microglia. following this depletion/ repopulation paradigm, behavioral analyses of mechanical and thermal allodynia are conducted. results: we established a selective tool to exchange cns parenchymal microglia with peripheral gfp 1 myeloid cells. in chronic pain tests for mechanical and thermal hyperalgesia, gfp>cd11b-hsvtk 1/mice show considerable decreases in paw withdrawal thresholds in response to mechanical, but not thermal stimuli ipsilateral to the injury, indicating distinct roles of microglia and macrophages in the facilitation of thermal hyperalgesia. conclusions: we identified a differential contribution of resident spinal microglia vs. peripheral myeloid cells in the development of neuropathic pain in gfp>cd11b-hsvtk chimeras. future studies aim to examine the exact mechanisms underlying the distinction between these two populations. a deeper understanding of the processes involved in the compartmentation of brain energy metabolism is of major interest. not only to enwrap pathomechanisms of brain diseases associated with metabolic deficits but also for a more accurate interpretation of functional neuroimaging signals which often use metabolic signals as surrogate marker for brain activity (e.g. fdg pet). large amount of knowledge in the field has been obtained from small animal neuroimaging studies. but due to their invasiveness they often need anesthesia. anesthetics heavily alter physiological read-outs. the aim of this study was 1.) to test if metabolic imaging using two-photon microscopy (2pm) and genetically encoded glucose sensors is feasible in the awake, head-fixed mouse and 2.) to examine the effects of isoflurane anesthesia on the signals. in two mice, a recombinant adeno-associated virus as a carrier for the genetically encoded glucose sensor flii 12 pglu600md6 was injected into the whisker barrel cortex. by using an astrocyte-specific promoter (short gfap) astrocytic expression was ensured. in addition, a chronic window and a head post were implanted. behavioral training started a few days after surgery. animals had to learn to tolerate head fixation without spontaneous motor activity. within a single trial animals were trained not to move for 10 seconds (imaging period) to receive a water reward. animals were subjected to water restriction during the behavioral training. animals were trained for three weeks before 2pm imaging started. animals learned to tolerate head fixation up to 90 minutes allowing acquisition of up to 400 trials per session. upon oral glucose administration (per single water reward $1 mg glucose was administered) fret signal started to increase within 5 minutes and went up to a maximum of 8%. surprisingly, isoflurane anesthesia led to an even higher increase of the signal (mean increase of 12%) compared to the awake state. in some experiments the barrel cortex was activated by vibrotactile single whisker deflections generated by a piezo bending actuator. fret signal did not show a difference between baseline and stimulated condition (1.133 6 0.3 vs. 1.136 6 0.29). in conclusion, the presented experiments demonstrate for the first time the feasibility of awake 2pm imaging for metabolic measurements on a single cell level. the signal increase upon peroral glucose administration unambiguously supports the functionality of the sensor. isoflurane exhibits a large effect on intracellular astrocytic glucose concentration distinctly revealing the need for experiments in awake animals for unbiased results. future experiments will now be expanded to neuronal glucose sensors to enable comparisons between astrocytes and neurons. our previous studies indicate that neuronal electrical activity controls glial exosome release resulting in subsequent neuronal uptake and functional retrieval of the exosomal content. proteomic analysis of oligodendroglial exosomes revealed a list of candidates with potential neurotrophic action in neurons. to elucidate the impact of oligodendroglial exosomes on the neuronal metabolism, we analyzed the metabolic activity of neurons after co-culture with oligodendrocytes or direct treatment with exosomes. methods: neurons were subjected to different stress paradigms such as oxidative stress, hypoxia, and nutrient deprivation. neuronal vitality was assessed by mtt assay and the mitochondrial membrane potential was visualized by staining with mitocapture. results: neurons grown under optimal conditions were not affected by the presence of exosomes. intriguingly, when neurons were subjected to stress (oxidative stress, nutrient deprivation, oxygen-glucose deprivation) their metabolic activity was significantly increased in the presence of exosomes. when challenged with oxidative stress prior to exosome treatment, neurons were not able to recover. mitocapture staining demonstrated that oligodendroglial exosomes prevent the breakdown of the mitochondrial membrane in nutrient-deprived neurons. conclusions: our results indicate that exosome supply is protective for neurons but is unlikely to support their recovery. we suggest that oligodendroglial exosomes carry neuroprotective substances, which protect neurons from stress. interestingly, this effect of lactate on synaptic plasticity mechanisms was not fully mimicked by glucose, suggesting that the effect was not only related to supporting the potential increase in energy demands related to plasticity, but that l-lactate could act as a signaling molecule for the regulation of expression of plasticity-related genes. we have followed up on these observations and show that l-lactate significantly stimulates mrna expression of key immediate early genes (iegs), in a time-and concentration-dependent manner, in cultured neurons. following one hour of treatment with 20 mm l-lactate, arc, zif268 and c-fos mrna levels were increased by 5.2, 3.7 and 8.2 folds, respectively. in addition, the increased iegs mrna expression levels induced by l-lactate are correlated at the protein level with respectively 5.5, 4.0 and 3.2 fold increase compared to control values at the same time point. these effects are specific for llactate, since d-lactate (non-metabolized enantiomer of l-lactate), l-pyruvate and d-glucose (at equicaloric concentrations) have no effect on gene expression. interestingly, we observed that, in similar culture conditions, l-lactate-induced iegs expression is only observed in neurons but not in astrocytes, implying a cell specific effect. characterization of the underlying molecular mechanisms of l-lactate action on iegs expression demonstrate the involvement of nmda receptors. finally, we obtained evidence that such regulatory mechanisms of ieg expression also operate in vivo as direct intracortical injections of l-lactate (10 mm) into the somatosensory-motor cortex areas resulted, within 1 hour of application, in a significant stimulation of arc, zif268 and c-fos expression (by 61 6 13%, 46 6 8% and 60 6 12%, respectively) as compared to d-lactate (10 mm)-injected contralaterally areas. taken together these findings demonstrate that l-lactate acts as a direct signaling molecule which regulates neuronal plasticity-related iegs expression both in vitro and in vivo. interestingly, the underlying mechanism of action of l-lactate involves nmda receptors.this set of observations therefore reveals a novel signaling role of lactate which may represent a likely mechanism to account for the role of astrocyticderived l-lactate on ltm formation. neuronal activity in the brain is associated with a transient increase in the extracellular k 1 concentration. the excess k 1 is removed from the extracellular space, primarily by the surrounding glial cells, leading to an intracellular accumulation of k 1 via mechanisms not fully identified and/or quantified. post-stimulus recovery of [k 1 ] o has been proposed to be dependent on kir4.1-mediated spatial buffering and/or to na 1 /k 1 -atpase activity. to resolve the molecular mechanisms involved in k 1 clearance, we initially determined the contribution from the different k 1 -transporting mechanisms present in primary culture of rat astrocytes and their k 0.5 for k 1 . the na 1 /k 1 /2clcotransporter, nkcc1, increased its activity within a physiological concentration range of k 1 , while the na 1 / k 1 -atpase saturated at much lower concentrations. consequently, a concentration-dependent switch occurs between the two mechanisms of k 1 uptake in cultured astrocytes. in addition, nkcc1 was capable of mediating robust k 1 -induced astrocytic cell swelling. thus, nkcc1 could potentially act as a molecular mechanism responsible for clearance of the stimulus-evoked rise in [k 1 ] o and the associated shrinkage of the extracellular space. to determine the contribution of each of the three molecular mechanisms to k 1 -clearance in native brain tissue, we used rat hippocampal brain slices and employed ion-sensitive microelectrodes in association with high-frequency electrical stimulation as well as focal appliances of k 1 by ionophoresis. inhibition of kir4.1 (100 um bacl 2 ) or nkcc1 (10 um bumetanide) failed to show a significant effect on the rate of k 1 removal from the extracellular space. in contrast, inhibition of the a2 and a3 isoforms of the na 1 /k 1 -atpase significantly delayed post-stimulus recovery of [k 1 ] o and this delay was further potentiated by additional inhibition of the a1 isoform. the na 1 /k 1 atpase emerged as the primary factor responsible for stimulus-evoked k 1 -clearance with no evidence in favor of kir4.1 and nkcc1 involvement. further characterization of the different na 1 /k 1 -atpase isoforms, by heterologous expression in xenopus laevis oocytes, revealed that the a1 isoform reached its maximal activity already at resting k 1 concentrations, hinting at a "housekeeping" function. the a2 subunit displayed voltage-sensitivity and increased turnover rate along physiologically relevant increments in extracellular k 1 concentration. these a2-related features may render this astrocyte-specific subunit variant specifically geared for post-stimulus recovery of [ as a model of ischemic injury, and sham-operated mice were used as controls. we isolated gfp 1 cells from the dorsal part of the lv of sham-operated-and post-ischemic brains and employed a neurosphereforming assay in order to estimate their ability to proliferate and selfrenew. furthermore, we followed their immunocytochemical and electrophysiological properties during in vitro differentiation and compared the differentiation potential of gfp 1 cells isolated from the controls to those isolated from post-ischemic brains. the gfp 1 cells isolated from the dorsal part of the lv of controls formed neurospheres and differentiated only into a glial phenotype. the gfp 1 cells isolated from the dorsal part of the lv of post-ischemic brains were able to form neurospheres as well; however, besides their differentiation into a glial phenotype, they also gave rise to cells with the properties of neuronal precursors. we also performed in situ immunohistochemical/electrophysiological analyses of gfp 1 cells in the adult brain of controls and those after mcao. in situ analyses revealed that gfp 1 cells expressed the phenotype of adult nscs or neuroblasts in controls or following ischemia and that their number was increased in both hemispheres following mcao. compared to controls, we found that the number of gfp/doublecortin-positive cells significantly increased in the dorsal part of the lv as well as the number of gfp 1 cells in the olfactory bulb, where they probably differentiated into calretinin 1 interneurons. our results indicate that gfp 1 cells with an active mdach1 gene in the dorsal part of the lv play an important role in the increased production of neuroblasts after injury and that this process is also enhanced in the contralateral hemisphere. collectively, our results reveal the involvement of the mdach1 gene in adult neurogenesis. cells expressing this gene exhibit the properties of adult nscs or neuroblasts and respond to mcao by enhanced neurogenesis. experimental cerebral ischemia leads to activation of microglia and astrocytes, which subsequently release pro-and anti-inflammatory factors. furthermore, during the earliest periods of neuronal damage, large quantities of dopamine are released, which may exacerbate the vulnerability of the lesioned tissue. on the other hand, delayed treatment with levodopa has been proven beneficial in stroke patients, suggesting a more complex effect of dopamine. recent studies on astrocytic involvement in neuroinflammation have shown a remarkable modulation of the inflammatory response over the dopamine d2 receptor (d2r). after stroke, reactive astrocytes in the astroglial scar were found to be immunopositive for d2r. in this study, we report the expression of d2r in activated microglia, both after experimental stroke in vivo and after oxygen-glucose deprivation (ogd), an in vitro model of ischemia. using immunohistological stains, we analyzed the expression of d2r in iba1 positive cells 1, 3, 5, 7, and 14 days after middle cerebral artery occlusion (mcao; n 5 2-4 mice/time point). for each animal, >4 pictures were taken from the ischemic core as well as from the contralateral, non-lesioned cortex from 3 sections. a total of >300 (control) and >500 (ischemic core) iba11 cells were analyzed for each time point. in the ischemic core, $25% of cells immunopositive for iba1 expressed d2r in contrast to <1% in control areas. a strong depletion of d2r immunoreactivity was observed in the ischemic striatum, accompanied by an increase in d2r expression in the adjoining cortex and an elevated presence and activation of microglia. for in vitro experiments, we used fluorescence activated cell sorting to isolate cd45 low /cd111 microglial cells. no significant d2r expression at mrna level could be detected by rt-pcr in this population, while we were able to detect d2r in non-microglial cells. interestingly, cultured primary mouse microglia expressed low quantities of d2r, as revealed by western blotting and rt-pcr, suggesting that culturing is sufficient to induce d2r in microglial cells. upon in vitro activation of primary microglia through 60 minutes of ogd, we found a 1.5 fold increase in d2r expression after 24 hours (n 5 4). furthermore, treatment of primary microglia with the selective d2r agonist pramipexole showed a dose-dependent tendency to increase lipopolysaccharideinduced tnfa release (n 5 5). our studies indicate that activated microglia express a functional d2r, which may contribute to the pro-inflammatory reaction of microglia after ischemia. white matter (wm) is injured in most strokes and axonal injury and dysfunction contribute to disability associated with clinical deficits. in young wm, the damage from ischemic injury involves the sequence of energy depletion (ionic pathway), excessive glutamate release (excitotoxicity), generation of reactive oxygen species and oxidative stress (oxidative pathway). in older wm the injury is mediated by ca21-independent excitotoxicity due to an earlier and more robust glutamate release. because excitotoxicity leads to oxidative stress in wm we investigated whether blocking nitric oxide synthase (nos) activity before or after a period of oxygen glucose deprivation (ogd) promoted axon function in an age-dependent manner. acutely isolated optic nerves from young and old (1 and 12 month) swiss webster (sw) or c57bl/6 (bl6) mice were used to ascertain quantitative measurements of wm function and structure. to support a biological basis for nos inhibitor nitro-l-arginine methyl ester (l-name) action in themonpreparation, we evaluated the expression and localization of brain nos (bnos) using immunohistochemistry in conjunction with confocal imaging. the expression of bnos co-localized with gfap (1) astrocyte nuclei, cytoplasm, end-feet as well as nf-200 (1) axons. the pattern of bnos expression paralleled astrocyte morphology with age and became more punctate in appearance. evoked compound action potentials (caps) recovered to 21.8 6 2.8% (n 5 18) after 60 min of oxygen glucose deprivation (ogd) in young bl6 mons. pretreatment of mons with l-name (200 mm) promotedcaprecovery to 69.4 6 11.3% (n 5 8) compared to ogd while caps recovered to 39.9 6 4.4% (n 5 11) when l-name was applied after the end of ogd. pretreatment of mons from 1 or 12 month old sw with l-name improvedcaprecovery to 49.6 6 3.7% (n 5 8, vs ogd 21.3 6 3.7%, n 5 12) or to 51.1 6 9.3% (n 5 7, vs ogd 5.7 61.7%, n 5 8) respectively. l-name application after the end of ogd failed to promote aging axon function (8.8 6 3.5%, n 5 6). changes in nos activity help unveil agedependent oxidative injury mechanisms in ischemic white matter. question: cerebral endothelial cells have been reported to exert a protective effect against brain damage. does transplantation of healthy endothelial cells have a beneficial effect on the outcome of ischemic brain damage? methods: we injected endothelin-1 (et-1) into the rat internal capsule to induce lacunar infarction. seven days after et-1 injection, microvascular endothelial cells (mvecs) prepared from adult rat cerebral cortices were transplanted into the internal capsule. meningeal cells prepared from adult rat cerebra or 0.2% bovine serum albumin-hank's balanced salt solution were injected as controls. two weeks later, the footprint test and histochemical analysis were performed. results: we found that mvec transplantation improved the behavioral outcome based on recovery of hind-limb rotation angle (p <0.01) and induced remyelination (p<0.01) compared with the control groups. also the inflammatory response was repressed by mvec transplantation, judging from fewer ed-1-positive activated microglial cells in the mvec-transplanted group than in the other groups. conclusions: elucidation of the mechanisms by which mvecs ameliorate ischemic damage of the white matter may provide important information for the development of effective therapies for white matter ischemia. objective: though lactate is a known neuroprotective agent, its mechanism of action is not known. we hypothesized that lactate can provide neuroprotection by activating trek channels. the effect of lactate on the electrophysiology of trek channels was studied both in an in vitro brain slice model, after blocking the activity of other ion channels and in a heterologous expression system. results: whole cell patch clamp experiments, on ca1 stratum radiatum astrocytes, in acute hippocampal slices, significantly increased trek channel activity by 23.3 6 6.4% and hyperpolarized their resting membrane potential by 5 6 0.6 mv, on bath application of 30 mm lactate. comparison of rectification index at negative potentials between control and 30 mm lactate treatment showed no significant difference, suggesting that the conductance activated by lactate is outward rectifying. lactate-evoked increase in trek channel activity was reversibly inhibited by 200 mm quinine, a potent inhibitor of trek channels. further, lactate was unable to increase trek channel activity after disruption of intracellular lactate uptake with 2 mm a-cyano-4-hydroxy cinnamate, a blocker of monocarboxylate transporter. this was confirmed by inside-out patch clamp experiments on human trek1 channel expressed in hek293 cells. conclusion: the experimental observations suggest uptake of lactate by astrocytes to activate trek channels intracellularly. regenerative responses occurring after different types of postnatal brain injury often involve expansion of an endogenous neural progenitor cell pool, but the molecular mechanisms underlying this process are still poorly understood. we studied expansion of the glial progenitor cell pool in a mouse model of neonatal hypoxia (hx) that reproduces the hallmarks of perinatal brain injury in infants, including diffused white matter injury (dwmi). we have previously shown that hx causes proliferation of wm oligodendrocyte progenitor cells (opcs), and that this process is regulated by the cyclin-dependent kinase 2 (cdk2) pathway. our analysis in wm in vivo and in cultured cells demonstrated: i) higher expression of cdk2, cycline, prb806/811 and e2f1 in wm opcs after hx; ii) activation of the cdk2 pathway after hx, and iii) reduced hx-induced opc proliferation in cdk2-null mutant mice. here, we studied the upstream molecular mechanisms that regulate activity of the cdk2 signaling pathway resulting in opc proliferation after hx. we show that sirtuin 1 (sirt1) histone deacetylase activity is a major regulator of cdk2 signaling and of the regenerative response of opcs after hx. under hypoxic conditions, sirt1 is upregulated in wm, and interacts with both rb and cdk2. patterns of posttranslational modifications of cdk2 or sirt1 proteins after silencing either of these genes in cultured cells from normoxic and hypoxic wm indicate that cdk2 is a substrate for deacetylation by sirt1, and that sirt1 is phosphorylated by cdk2. also, sirt1 causes rb deacetylation resulting in dissociation of e2f1 from the rb/e2f1 complex, which ultimately leads to higher opc proliferation. knockdown of sirt1 in hypoxic wm cell cultures suppresses opc proliferation. finally, inhibition of sirt1 activity by sirtinol accelerates opc differentiation to mature oligodendrocytes, and reduces the number of opcs. we are currently analyzing the effects of hx on opc proliferation and differentiation in the wm of sirt1-null mice. our results provide evidence that sirt1 is an essential regulator of the cdk2-dependent regenerative response observed in wm opcs after hx, and that inhibition of sirt1 activity facilitates oligodendrocyte regeneration from opcs, which might ultimately promote wm recovery after hx. supported by nih r01ns045702, p01ns062686 and iddrc p30hd40677. gonadal steroid hormones reveal a potential neuroprotective role in acute brain ischemia. in rat in vivo studies using the transient occlusion of the middle cerebral artery as ischemic stroke model, we have shown that 17ß-estradiol and progesterone reduce the infarct area by more than 70% given 1h after the onset of stroke, prevent neuronal death and behavioral deficits. an intriguing aspect of this study was that the application of steroid hormones reduced the number of microglia and microglia-related markers in the penumbra during the first 24 h after the onset of stroke. besides microglia, penumbral astroglia coevally appeared as cellular target for steroid hormones by reducing the expression of proinflammatory-and molecules necessary for the attraction and activation of microglia and lymphocytes. this suggests that protective steroid hormones influence glia cell cross-talk and activity during early damaging conditions in the lesioned brain site. by using an in vitro hypoxic approach, we have now demonstrated that microglia express steroid hormone receptors and directly respond to ischemic conditions and steroid hormone treatment by changing expression and secretion of inflammatory compounds, trans-signaling factors and by modulating phagocytotic activity. this clearly shows that microglia is an important direct target for steroid hormones but also indirectly influenced via cross-talk by adjacent astrocytes at the damaged brain site. this generally points at an extensive bidirectional crosstalk between both glial cell types to convey steroid-mediated neuroprotection in ischemic brain tissue. in summary, the balance of inflammatory astrogliamicroglia interactions within the injured brain tissue during an early phase of ischemic destruction is a pivotal step for tissue repair. cannabinoids are considered as key regulators in the pathology of various diseases, including ischemic stroke. we previously demonstrated that post-ischemic treatment of two naturally occurring phenylpropanoids, trans-and cis-hinokiresinols (hrs) significantly reduced ischemic injury in rats subjected to middle cerebral artery occlusion (mcao) . in studies to screen their cellular targets, we found that hrs selectively bind to both type 1 and type 2 cannabinoid receptors (cb1r and cb2r). in the cb1r reporter gene assay, both hrs demonstrated antagonistic activity. in cb2r-overexpressing u 2 os cell lines, both hrs increased forskolininduced camp accumulation, suggesting their inverse agonism. since camp induces expression of heme oxyagenase-1 (ho-1), a well-known antioxidant stress protein, we further investigated the effect of hinokiresinols on ho-1 expression in mixed cortical neuron/glia cultures. trans-hr increased astroglial expression of ho-1 greater than cis-hr did. a selective ho-1 inhibitor, tin protoporphyrin ix significantly reduces the neuroprotective effect of trans-hr in cortical cultures exposed to oxygenglucose deprivation. in addition, both hrs reduced the number of ed-1immunopositive cells (i.e., active microglia and macrophages) in ischemic lesions. also, both hrs significantly reduced microglial migration induced by an endocannabinoid, 2-arachidonoylglycerol. the present study suggests that hrs reduce ischemic injury via regulation of glial cbrs. , the main anaplerotic enzyme in the brain, is predominantly located in astrocytes. in the adult brain, ischemia is known to reduce pyruvate carboxylation. moreover, reperfusion after ischemia leads to production of reactive oxygen species (ros). the pentose phosphate pathway (ppp) is, by making nadph necessary for the regeneration of reduced glutathione, a major pathway in the protection against ros. however, astrocytic and neuronal metabolic function in the neonatal brain after hypoxic-ischemic brain injury (hi) remains to be explored. methods: hi was induced in 7-day-old rats by unilateral severing of the carotid artery followed by 2 hours recovery and subsequent exposure to hypoxia (8%o 2 ) for 90 min. 30 min after end of hypoxia (the early reperfusion phase), rats were injected with [1,2-13 c]glucose and decapitated 30 min later. one group was sham-operated, and not exposed to hypoxia, thus reflecting normal metabolism in the neonate. extracts of ipsilateral hemispheres from hi and sham animals were analysed with 1 h-and 13 c-nmr spectroscopy. results: the sham animals had similar glutamine content, but lower amounts of 13 c labelled glutamine compared to corresponding values from adult rats, reflecting a generally lower rate of glucose metabolism in the neonatal astrocytes. however, approximately equal amounts of 13 c labelled isotopomers of glutamine were derived from pc and pyruvate dehydrogenase (pdh), resulting in a relatively high pc/pdh-ratio compared the same ratio in adults. following hi and reperfusion, a reduction in glucose metabolism and mitochondrial metabolism was seen by increased glucose and reduced incorporation of 13 c labelling in glutamate, glutamine and aspartate via pc and pdh. however, the pc/pdh-ratio in glutamine was similar in hi and sham. total mean values of glutamate, glutamine and aspartate were decreased, but only the latter to a significant degree. labelling in lactate via the ppp was reduced following hi. conclusion: the low labelling of lactate via the ppp indicates that the flux through the ppp was in fact reduced in the early reperfusion phase after hi. moreover, mitochondrial metabolism of pyruvate from glucose was reduced in both astrocytes and neurons. impaired anaplerosis in astrocytes was confirmed by lower amounts of aspartate. however, the proportionally similar decrease in 13 c labelling in glutamate and glutamine via pc, and the maintained pc/pdh-ratio implies that astrocytes continue to provide metabolic support for the neurons during the early reperfusion phase. ret receptor tyrosine kinase is the signaling component of the receptor complex for the family ligands of the glial cell line-derived neurotrophic factor (gdnf). ret is involved in the development of enteric nervous system, of sympathetic, parasympathetic, motor and sensory neurons and it is necessary for the postnatal maintenance of dopaminergic neurons. ret expression has been as well demonstrated on microglia and several evidence indicate that gdnf regulates not only neuronal survival and maturation but also certain functions of microglia in the brain. here we demonstrated that isolectin ib4, commonly used as a microglial marker in the brain, binds to the glycosylated extracellular domain of ret both on the surface of living nih3t3 fibroblasts cells stably transfected with ret than in adult rat brain sections as revealed by immunoblotting. further, confocal immunofluorescence analysis demonstrated a clear overlap staining between pret and ib4 labeling in primary microglia cultures as well as in adult rat sections obtained from control or postischemic brain after permanent middle artery occlusion (pmcao). interestingly, ib4 staining identified activated or amoeboid retexpressing microglia under ischemic conditions. collectively, our data indicate ret receptor as one of the ib4-reactive glycoconjugate accounting for the ib4 stain in microglia under physiological and ischemic conditions. astrocytes can adapt to lower oxygen tensions by enhancing their glycolytic rate of energy production. in this case, adequate expression of monocarboxylate transporters (mcts) may be essential to sustain prominent lactate efflux and prevent glycolysis inhibition. here we show that oxygen level is an important factor controlling mct4 expression in primary cultures of mouse cortical astrocytes. indeed, while mct4 is clearly expressed by astrocytes in vivo, its basal in vitro expression in primary cultures of mouse cortical astrocytes is undetectable under classical culture conditions (95% air, 5%co 2 ). exposing astrocytes to more physiological brain oxygen tensions (hypoxic chamber flushed with 1%o 2 /5%co 2 /94%n 2 , leading to a concentration of dissolved oxygen in the culture medium of 40-60mmhg) restored mct4 expression. mct4 mrna expression became detectable after 12 hours under lower oxygen tension and was still present after 10 days with a maximum at 36 hours of incubation. this effect was paralleled by the appearance of the mct4 protein which reached a plateau between 48 hours and 96 hours, with a further enhancement at 7 and 10 days of incubation. this induction was specific for mct4 since mct1 expression was not altered. lactate release was significantly enhanced after 48 hours of incubation and further increased up to 10 days compared to astrocytes maintained under atmospheric conditions. treatment with dimethyloxalylglycine (dmog), a compound that mimics hypoxia by stabilizing the hypoxia-inducible factor 1 alpha (hif-1a) , produced a similar effect. both mct4 mrna and protein induction reached their maxima when astrocytes were treated with a concentration of dmog ranging between 1 to 2.5mm, which is also the range for greatest hif-1a expression. again, no effect was detectable on mct1 expression. transfecting astrocyte cultures with a sirna against hif-1a significantly reduced the effect of lower oxygen tension on mct4 expression. our results suggest that 1) under physiological conditions, changes in local oxygenation might regulate the capacity of astrocytes to supply lactate to neighboring cells via notably alteration in mct4 expression 2) under pathological conditions, mct4 may be upregulated as a consequence of hypoxia and contribute to neuroprotection by favouring the release and accumulation of lactate in the extracellular space. indeed, this process might give rise to the beneficial effect of lactate for neurons observed during the recovery from hypoxic/ischemic conditions in vitro and in vivo. early onset dementia and. the loss-of-function of trem2 or its coreceptor dap12 is responsible for the recessively inherited nasu-hakola disease (also known as plosl). the brains of plosl affected patients show strong microglial activation in the cerebral white matter. reports demonstrate that trem2-mediated phagocytic function of microglia is required for debris clearance and cns tissue homeostasis. perinatal brain injury is the underlying etiology for a host of developmental disabilities that includes spastic motor deficits and cognitive, behavioral and learning difficulties. hence, our aim is to characterize the expression of trem2 in the control of neuroinflammation following hypoxia/ ischemia (hi) in the newborn brain. we performed hi brain damage in postnatal day 7 (p7) c57/bl6 mice by permanent left carotid occlusion and litters were exposed to 8% of oxygen balanced with nitrogen for 50 minutes in a hypoxic chamber with controlled humidity and temperature maintained at 37 c. pups were then returned to their dam until sacrifice. the age-matched controls and samples from 3 hours to 7 days after hypoxia were collected and processed for immunohistochemistry. in control animals, trem2 expression was observed in the corpus callosum and in the subventricular zone at p7 that almost disappeared at p10. following hi, an increase in trem2 staining was observed in the corpus callosum, hippocampus, caudate-putamen, fimbria, cortex and thalamus in the ipsilateral (il) hemisphere, following a similar regional pattern of microglia activation. the increased expression of trem2 was observed from 24 hours to 7 days post-hypoxia. trem2 colocalized mainly with microglia markers, such as iba-1 and cd68. oligodendrocyte expression of trem2 was also analyzed. in conclusion, hi produced an increase in trem2 expression on the il damaged regions. these results suggest that the modulation of trem2 might be a possible target for damage control in neonatal brain. increasing evidence shows that astrocytes play a critical role in neuronal protection during an ischemic insult. in the vertebrate retina, most of astrocytic functions are executed by m€ uller glial cells, the major macroglial cell type in this tissue. the aim of this study was to evaluate the role of m€ uller glia in the onset and progression of retinal ganglion cell (rgc) degeneration after acute ischemic injury in the mouse eye in vivo. here, we used the selective gliotoxin fluorocitrate (fc) in order to transiently impair m€ uller glial metabolism at different time points during and after induction of an acute retinal ischemia/reperfusion by means of elevated intraocular pressure (iop). the effect of fc (3 mm) on metabolism was determined by measuring retinal glutamine levels, aconitase activity and mitochondrial membrane depolarization after intravitreal injection of the gliotoxin. retinal ischemia was unilaterally induced by elevating iop for 45 min. fc was intravitreally injected 4 h before onset of ischemia and after 6, 12 or 24h of reperfusion. saline was injected in the contralateral eye and served as control. surviving rgcs were quantified by means of confocal scanning microscopy and automated cell counting-based analysis on retinal wholemounts 7 days after lesion. fc treatment reduced glutamine levels to 35-38% as compared to control 4-6 h after injection. inhibition was completely reversed 24 h after injection. aconitase activity was reduced to $30% from control levels 4 h after fc injection. mitochondrial membrane potential was reduced by 50-70% as indicated by reduced intensity of the specific stain mitotracker red cmxros. one week after ischemia, only 40% of rgcs survived as compared to unlesioned controls. impairment of m€ uller glial metabolism during ischemia further reduced rgcs by 15% as compared to ischemia alone. fc treatment did not significantly modify rgcs survival 6 and 12 h after lesion. results support the notion for a critical neuroprotective role of m€ uller glial cells during ischemia. in the acute phase following ischemia, however, m€ uller glia inhibition does not further affect rgcs survival. further research is required to establish the time point for this switch in m€ uller glial function and the underlying mechanisms. extract has been widely investigated in animal models and clinical studies for various diseases including ischemic stroke. its major metabolite, cordycepin, has been reported to act as a selective a3 adenosine receptor agonist and to protect neurons against ischemic injury. however, their underlying mechanisms remain unclear. in the present study, we report that the standardized c. militaris extract, wib-801c, which contains cordycepin 8% of total dry weight of the extract, markedly reduced ischemic injury by inhibiting post-ischemic inflammatory responses. postischemic treatment with wib-801c (orally administered twice at 3 and 8 h after onset of mcao) significantly reduced infarct size and edema in rats subjected to transient middle cerebral artery occlusion (mcao, 1.5 h) and subsequent reperfusion (22 h). wib-801c also significantly improved integrity of glial cells in ischemic lesions, as evidenced by reduced white matter degeneration and loss of blood-brain barrier. importantly, wib-801c significantly ameliorated neurological deficits in not only transient but also in permanent stroke models. moreover, wib-801csignificantly improved long-term survival of mcao rats over 30 days. as we previously reported with selective a3 receptor agonists introduction: cd200 & cd200r are immune inhibitory molecules that have been shown to be involved in inducing immune tolerance and in contributing to immune privileged status of the cns. the developing brain exhibits distinct morphological as well as physiological characteristics determining a peculiar response to injury showing an aggravated susceptibility to excitotoxicity and pro-inflammatory cytokines, along with an exacerbated inflammatory response. previous studies from our group have described the expression of cd200-cd200r in brain during development showing a distinct pattern of expression in the cortex and the hippocampus. hence, the aim of this study is phenotypic characterization of cd200r1 microglia/macrophages and cd2001 neuronal cells following hypoxia/ischemia (h/i) in neonatal mice brain. wild-type c57/bl6 mice postnatal (p) day 1,3,5,7,10,14,21 and adult were used for developmental studies. p7 mice was used for h/i injury that was administered using vannucci model modified for neonatal mice (8% o 2 , 55 min) and samples were collected 3h, 12h, 24h, 48h, 72h & 7 days after hypoxia for immunofluorescence staining. results: phenotypic characterization of cd200r1 microglia/macrophages showed them to express markers of pro-or anti-inflammatory phenotype in a temporal fashion post lesion. they expressed m2 phenotype as observed by colocalization with cd2061 cells throughout the time points studied but a subpopulation of cd200r1 cells exhibited m1 phenotype as exhibited by mhcii1, cd861 cells from 24-48 hrs post lesion. calretinin (cr) used for the characterization of cd2001 neurons showed that most cr1 neurons were cd2001 especially the interneurons in the innermolecular layer of hippocampus, layer i of the neocortex and the hilus at most age groups studied. after h/i, cd200 immunolabeling was increased in the hippocampal fissure until 7 days post-lesion. this followed a change in cd200r1 microglial cells described above. to conclude, a balance between m1/m2 phenotype of cd200r1 microglia/macrophages and expression of cd200 by cr1 cells are involved in evolution of h/i induced brain injury in neonatal mice. objective: the cytokine interleukin-1 (il-1) and its naturally occurring receptor antagonist (il-1ra) play a key role in determining neuronal cell death and survival in focal cerebral ischemia. the objective of this study was to determine the cellular production of il-1/il-1ra, and to test the neuroprotective potential of post-surgically injected il-1ra overexpressing bone marrow (bm) cells in a mouse model of focal cerebral ischemia. materials and methods: c57bl/6 mice were injected i.v. with bm cells isolated from sil-1ra overexpressing mice, 30 min after permanent middle cerebral artery occlusion (pmcao). physiological parameters and behaviour were recorded post-surgically, infarct sizes were estimated, and microglial-leukocyte expression of il-1/il-1ra was analyzed by flowcytometry, in situ hybridization and immunohistochemistry. results: we identify microglia, and not recruited leukocytes as the major producers of il-1ra after pmcao in mice, and we show by using il-1ra knock out mice that microglial-produced il-1ra is neuroprotective. we report that the sil-1ra produced by the post-surgically injected bm cells, potentiates the neuroprotective effect of microglialderived il-1ra, at both 24 h and 5 days, which is consistent with behavioural improvement at 5 days, and detection of recruited bm cells in the ischemic area 1.5 h after pmcao. interestingly, we also find that the sil-1ra overexpressing bm cells stimulate microglial production of il-1ra 6 h after pmcao, at which time both il-1a and il-1b is upregulated. conclusion: our results provide proof of principle that increasing the production of il-1ra by recruited bm cells can counteract the effect of il-1a/b, increase neuronal survival and improve motor function alone or through induction of microglial-produced il-1ra after pmcao in mice. bogomoletz institute of physiology, kyiv, ukraine short-term oxygen-glucose deprivation (ogd) is known to activate excitatory glutamatergic synapses, induce long-term potentiation and result in neuronal and synaptical ultrastructural remodelling in organitypic cultured hippocampal slices (ochs). in this work we studied changes in glial synaptic coverage following 30 min ogd episode using 3d reconstruction of ochs confocal and electron microscopic images. propidium iodide (pi) and mitotracker orange cmtmros (mto) were used to estimate cell viability and their mitochondrial potential. astroglial and microglial cells were identified using immunohistochemical staining with anti-gfap and anti-iba-1 antibodies. the study demonstrated that astroglial and microglial cells retain viability (there was no significant increase in the amount of pi-labbelled cells). 3d reconstruction revealed significant increase in excitatory synapses glial coverage as early as 1 hour following ogd accompanied by a significant increase in number of astroglial and microglial processes. the deprivation also resulted in gradual increase of mitochondrial potential from 4 hr to 24 hr in both astroglial and microglial cells. it was previously shown that pyramidal neurons under the same conditions lose mitochondrial potential and became pi-positive after 4 and 24 reoxygenetion, indicating damage of cytoplasmic membrane, the effect prevented by application of nmda receptor antagonist d-ap5. thus, unlike neurons astroglial and microglial cells are ogd-resistant and demonstrate marked mitochondrial activation -a process that might be involved in maintaining neuronal function during oxygen-glucose deficiency. using a microfluidic high throughput qpcr platform, we measured the expression of 47 selected genes encoding astrocytic and polydendrocytic markers, ion channels, transporters and receptors that participate in maintaining k 1 and glutamate homeostasis in each of 292 individual gfap/egfp-positive glial cells. in this study we show how the expression of these selected genes changes during development (postnatal days 10, 20, 30 and 50) as well as 3, 7 and 14 days after middle cerebral artery occlusion (mcao). self-organizing maps and principal component analyses divided the cells according to their similarity in gene expression into three subpopulations of astrocytes present within the first 10-50 days of postnatal development (p10-p50). the first subpopulation comprises immature glia mainly from p10, characterized by the high transcriptional activity of all of the studied genes, including polydendrocytic markers. the second subpopulation is dominated by cells from p20 and displayed low transcript levels of all of the studied genes. the third subpopulation represents mature astrocytes mainly from p30 and p50. three, seven and fourteen days after ischemia (d3, d7, d14), additional astrocytic subpopulations appear: resting astroglia (mainly from p50 and d3), transcriptionally active early reactive astroglia (mainly from d7) expressing the mrna of polydendrocytic markers, and, presumably, permanent reactive astroglia (solely from d14). following focal cerebral ischemia, reactive astrocytes undergo pronounced changes in their expression of aquaporins, nonspecific cationic and potassium channels, glutamate receptors and markers of reactive astrocytes and polydendrocytes. from the data obtained from single cell pcr analysis, we calculated the spearman correlation coefficients between pairs of genes, which revealed interesting positive gene expression correlations with polydendrocytic markers (gria2-4, grik1-5, grin3a, kcnj16, kcnk2 and kcnk10 genes). this study was further supplemented by an immunohistochemical analysis of nmda receptor subunits and pdgfar, which confirmed that changes in gene expressions correlate with immunodetected proteins. ga cr 13-02154s, astf 110-2011, gauk 604212. reactive astrogliosis is often regarded as universal type of astroglial response to various forms of injuries. among its most characteristic features one may include: glial fibrillary acidic protein (gfap) up-regulation, cellular hypertrophy and proliferation and gliotic scar formation. an important element of glial cells response is their ability of de-differentiation, which is related with their re-gaining of immunological and molecular properties characteristic for earlier developmental forms. the origin of cells proliferating in response to various types of brain injury is still a subject of debate. among others it is related to the pathomechanism of lesion, affected brain structure and developmental stage of the nervous system. aim: in our study we aimed to assess the differences of de-differentiation and proliferation potential of astroglia localized in the cerebral cortex and striatum to the transient focal brain ischemia. material and methods: transient focal brain ischemia was evoked in 20 adult male wistar rats by placement of the monofilament surgical thread into the internal carotid artery and occlusion of the middle cerebral artery for 1h. the postoperative survival period was up to 6 weeks. immunocytochemical double-staining for astrocytic marker (gfap), developmental (pax6) and proliferative (ki-67) markers was performed and subsequent qualitative and quantitative study was done by means of confocal microscopy. results: double-labeled gfap/pax6 and gfap/ki67 reactive astroglial cells were revealed in the cerebral cortex and striatum of the ischemic region, starting from 24h after initiating the ischemia. the apparent difference in the intensity of morphological changes, quantity of de-differentiating and proliferating astroglial cells was observed between ischemic cerebral cortex and striatum. intensity of astroglial response and proliferative potential were much higher in the striatum than in the cerebral cortex. summary and conclusion: apparent difference in proliferative response between the cerebral cortex and striatum may be consequence of metabolic and molecular differences between astroglial cells populating two mentioned above structures. it may be also consequence of uneven decrease of cerebral blood flow and resulting different metabolic conditions influencing astroglial function in various fragments of ischemic area. reassuming, the differentiated potential of astroglial proliferative response to ischemic changes in different brain regions must be taken into account while analyzing clinical consequences of cerebral infarcts of different localization. triggering receptor expressed on myeloid cells-2 (trem2) is a microglial surface receptor involved in phagocytosis. clearance of apoptotic debris after stroke represents an important mechanism to re-attain tissue homeostasis and thereby ensure functional recovery. the role of trem2 following stroke is currently unclear. as an experimental stroke model, the middle cerebral artery of mice was occluded for 30 minutes with a range of reperfusion times (duration of reperfusion: 6 h/12 h/24 h/2 d/7 d/28 d). quantitative pcr (qpcr) revealed a greatly increased transcription of trem2 after stroke ($10fold). we subsequently analyzed the expression of proinflammatory cytokines, chemokines and their receptors in trem2knockout (trem2-ko) mice via qpcr. microglial activation (cd68, iba1) and cd3-positive t-cell invasion were analyzed via qpcr and immunohistochemistry. functional consequences of trem2 knockout were assessed by infarct volumetry. the acute inflammatory response (12 h reperfusion) was very similar between trem2-ko mice and their littermate controls. however, in the sub-acute phase (7 d reperfusion) following stroke, trem2-ko mice showed a decreased transcription of pro-inflammatory cytokines tnfa, il-1a and il-1b, associated with a reduced microglial activity (cd68, iba1). furthermore, trem2-ko mice showed a reduced transcription of chemokines ccl2 (mcp1), ccl3 (mip1a) and the chemokine receptor cx3cr1, followed by a diminished invasion of cd3-positive t-cells. no effect on the lesion size was observed. conclusions although we initially expected an exaggerated pro-inflammatory response following ablation of trem2, our data support a contradictory scenario that the sub-acute inflammatory reaction after stroke is attenuated in trem2-ko mice. we therefore conclude that trem2 appears to sustain a distinct inflammatory response after stroke. metabotropic glutamate receptors (mglurs) are seven transmembrane domain g-protein-coupled receptors for l-glutamate, the main excitatory neurotransmitter in the mammalian brain. in addition, glutamatemediated excitotoxicity plays a central role in mediating neuron and oligodendrocyte death during ischemic episodes. in vitro, developing oligodendrocytes (ols) in particular have been shown to be highly vulnerable to excitotoxicity and oxidative stress. notably, activation of group 1 mglurs has been shown to attenuate ol excitotoxicity in vitro and in brain slices has been shown to reduce ischemia-mediated impairment of astrocytes. here, we have examined the functional expression of mglurs and their role in acute ischemic injury in developing cns white matter of the mouse optic nerve. calcium imaging experiments were performed in optic nerves from p9-13 wild-type mice. optic nerves were isolated intact, loaded with fluo-4 am and visualized using a zeiss lsm 5 pascal confocal microscope; images were collected in optical z-sections and changes in fluorescence intensity were measured. the application of mglur agonists acpd and dhpg showed that group i mglur mediate a rise in glial [ca 21 ] i . the action of the group i mglur antagonist aida versus acpd indicated that the rise in [ca 21 ] i also involves group ii (and possibly group iii) mglur. immunohistochemistry confirmed glial expression of group i subunit mglur5 and group ii subunits mglur2 and mglur3 in the optic nerve, although further analysis of cellular localization is required. to examine the role of mglurs in white matter ischemia, optic nerves from p8-9 mice in which fluorescent reporters are driven by astrocyte (gfap-egfp) or oligodendrocyte (sox10-egfp) genes were isolated intact into artificial cerebrospinal fluid (acsf) and subjected to oxygen glucose deprivation (ogd) for 1 hour. activation of group i or group ii mglur with the agonists acpd, dhpg or ly379268 protected astrocytes and oligodendrocytes from ischemic cell death. further experiments are required to determine the mechanisms involved, but these findings demonstrate functional expression of mglur in optic nerve glia and indicate that activation of mglur is a possible therapeutic strategy to protect against glial damage in response to ischemia. the cerebral cortex is one of the most often affected areas after stroke, but there are no studies comparing the outcome in different cortical regions after focal ischemia. the aim of this investigation was to evaluate the patterns of glial activation, tissue degeneration and neuronal loss in different survival times following cortical ischemia. focal ischemia was induced by stereotaxic microinjections of endothelin-1 (et-1) into the somatosensory, motor and association cortices of adult rats (n 5 45). the control animals were injected with the same volume of sterile saline (n 5 27). the animals were perfused at 1, 3 and 7 days after ischemia. gross histopathology was evaluated using cresyl violet staining. 20 lm sections were submitted to immunohistochemistry for astrocytes (anti-gfap), activated microglia/macrophages (anti-ed1) and microglia (anti-iba1 and ed1). tissue loss and glial activation were more intense in the somatosensory cortex than in other cortical areas. the motor cortex was the second more affected area. the association cortex was the less damaged area, which was confirmed by quantitative analysis (anova-tukey). the results suggest that an ischemic lesion of the same intensity induces a differential pattern of tissue loss and neuroinflammation, depending on the cortical area, and that the primary sensory and motor areas are more susceptible to ischemia than association areas. tlr4 is required for ipc-induced neuroprotection. in order to elucidate the mechanisms by which microglial tlr4 contributes to ipc, we carried out cell-targeted genomic analyses specifically on microglia exposed to either ischemic conditions in vitro or ipc in vivo. for our in vitro model, we exposed wt and tlr4 -/mouse primary microglia to hypoxic/hypoglycemic conditions for 24 hours. for our in vivo model, we carried out transient middle cerebral artery occlusion (mcao) or sham surgery as our ipc pulse on wt and tlr4 -/adult male mice. three days later mice were sacrificed and microglia acutely isolated from ipsilateral cortex using magnetic affinity chromatography cell separation and ex vivo flow cytometry. microarray analysis was carried out on rna from both in vitro and in vivo microglia. results from both datasets identified robust expression of type 1 and/or type 3 interferon (ifn)-stimulated genes (isgs) as the predominant transcriptomal feature of ischemia-exposed microglia. in vitro, we found that 25 out of the 60 (40%) individual genes that were significantly up-regulated >2fold by hypoxia/hypoglycemia in wt, but not tlr4 -/-, microglia were isgs including mx1, oas2, irf7 and ccl5. promoter analysis demonstrated that the top four most active transcription factors were isgf3, irf2, irf1 and irf7; all ifn regulatory factors (irfs). a similar pattern emerged from the in vivo dataset with multiple irfs again among the most activated transcription factors in sorted microglia from ipcexposed mice. however, unlike the isg response in vitro, the isg response from the in vivo dataset was not tlr4-dependent. the ifn family of cytokines is recognized as a key component of the innate immune response to infection and other types of injury. the role of microglial ifn-signaling in ipc is unknown. these results suggest that broad activation of isgs may be a key feature of the microglial response to ischemic conditions. astrocytes respond to central nervous system (cns) injury by the formation of a glial scar that develops within few days after insult and is characterized by astrocyte proliferation and cellular hypertrophy. reactive astrocytes change their immunohistochemical profile, such as increasing expression of gfap, nestin and vimentin; however, they also markedly alter the expression of ion channels, receptors and transporters, which participate in the maintenance of ionic-, water-and neurotransmitter homeostasis, such as k 1 channels, aquaporins or glutamate transporters. here, we aimed to characterize the gene expression profile and functional properties of reactive astrocytes 5 weeks after global cerebral ischemia (gci) or focal cerebral ischemia (fci) with a specific focus on k 1 channels or non-specific cationic channels. gci was induced in adult rats by bilateral, 15-minute common carotid artery occlusion combined with low oxygen, while fci was induced in adult egfp/gfap mice by permanent middle cerebral artery occlusion. using the patch-clamp, we investigated the membrane properties of astrocytes in situ 5 weeks after gci in the rat hippocampal ca1 region and 5 weeks after fci in the mouse cortex. regardless of the type of ischemic injury, astrocytes from both cns regions depolarized their membrane potential by $14 mv 5 weeks after ischemia. when compared to astrocytes from the non-injured ca1 region or the cortex, the post-ischemic astrocytes displayed large hyperpolarizationactivated time-and voltage-dependent, non-inactivating inward currents, the current density of which increased 3-fold in response to gci or fci. in addition, these non-specific cationic channels were sensitive to zd7288 and to a low extracellular na 1 concentration, suggesting that they may belong to the family of hyperpolarization-activated cyclic nucleotide-gated (hcn) channels. we have taken advantage of fluorescently labeled astrocytes in egfp/gfap mice and isolated egfp 1 cells by facs from non-injured as well as ischemic regions and performed gene expression profiling using single-cell rt-qpcr. our data revealed that cortical astrocytes after fci express, besides the increased expression of gfap, gfapd, aqp1 and 9, extremely high levels of hcn1-3 transcripts that encode hcn 1-3 channels. moreover, our immunohistochemical analyses confirmed the presence of hcn1-3 in reactive astrocytes, especially hcn1, 2 and 3 channels. in summary, our results show that reactive astrocytes from post-ischemic tissue express hcn channels and that their activity might significantly contribute to na 1 influx, possibly maintaining atpase function and/or contributing to intracellular na 1 -dependent events, such as glutamate transporter or na 1 /ca 21 exchanger reversal. ga cr 13-02154s, p304/12/g069 and gauk 383711. the optimal operation of electrogenic astrocytic transporters and exchangers for some well-defined astrocyte brain homeostatic functions depends on the presence of k 1 channels in the cell membranes and the hyperpolarized membrane potential. our previous study showed that astrocytes functionally express two-pore domain k 1 channel trek-1, which helps to set the negative resting membrane potential. however, the roles of trek-1 on astrocytic function under normal and ischemic conditions remain unclear. in this study, we investigated the effects of trek-1 activity on astrocytic function and neuronal death under ischemic conditions. in an in vitro hypoxia model with astrocytes culture, trek-1 immunoreactivity was up-regulated after hypoxia. suppression of trek-1 activity inhibited the glutamate clearance capability, enhanced the inflammatory secretion of astrocytes derived s100b and led to increased neuronal apoptosis after ischemic insult. after middle cerebral artery occlusion induced focal ischemia in rats, the trek-1 expression was significantly increased which correlated with reactive astrogliosis. application of lineonic acid, a trek-1 agonist which could potentiate the astrocytic conductance and hyperpolarization membrane potential, up-regulated the expression of astrocytic glutamate transporter glt-1, inhibited the micro-glial inflammatory response and attenuated neuronal damage. our results suggest that trek-1 activity is involved in astrocytic function and neuronal survival. this would provide evidence showing astrocytic trek-1 involvement in ischemia pathology which may serve as a potential therapeutic target in stroke. oligodendrocytes are the myelinating cells of the central nervous system (cns). they are responsible for ensheathing myelin layers around axons, which contributes to improve conduction of neuronal impulses and additionally provides axonal support. oligodendrocytes are generated by oligodendrocyte precursor cells (opcs), a self-renewing and proliferating adult stem/precursor cell population in the cns. this process occurs not only during developmental myelination but also mediates cns remyelination, a regenerative process that restores myelin sheaths to demyelinated axons. although energy metabolism in the oligodendrocyte lineage is thought to be very active to support the lipid production specific to this cell type, it is still poorly characterized. as a first step towards a detailed characterization of the energy metabolism associated with specific lineage stages we investigated how glucose -the main cerebral energy substrate -is metabolized at an immature opc stage and in differentiated oligodendrocytes. primary cultures of opcs and oligodendrocytes were incubated with medium containing [1,6-13 c]glucose for 4h and metabolites in both cell extracts and culture medium were collected and analysed for excess % 13 c enrichment using gas-chromatography-mass spectrometry (gc-ms) as well as for total amounts using hplc. the significant 13 c-enrichment in citrate, malate, and aspartate in cell extracts indicate that oligodendrocytes oxidize glucose-derived metabolites extensively in the tricarboxylic acid (tca) cycle. moreover, they release 13 c-labelled aspartate and glutamate to the extracellular medium, although a significant consumption of aspartate, glutamate and glutamine from the medium was also observed. enrichment of extracellular lactate and increased alanine synthesis in oligodendrocytes as compared to opcs suggests that the rate of aerobic glycolysis is increased in immature as compared to differentiated cells. in conclusion, these data confirm that both opcs and oligodendrocytes are highly metabolically active and, in addition to extensively oxidizing glucose, synthesize and release distinct metabolites. we anticipate that further studies on this subject will contribute to a better understanding of the physiological role of oligodendrocytes in the cns and the role of glycolysis and mitochondrial metabolism during their maturation. schwann cells in the peripheral and oligodendrocytes in the central nervous system require a tremendous amount of lipids to be capable of producing all cell membranes forming the myelin structure. furthermore, when compared to other cells, myelin membrane presents an extremely high lipid content ($80% of dry weight) coupled to an unique lipid composition. the importance of myelin lipid composition towards myelin formation and maintenance is highlighted by the frequent appearance of myelin defects in lipid-metabolism human disorders. one of the main represented class of lipids in myelin are phospholipids, of which fatty acids (fas) represents the building blocks. most cells preferentially uptake circulating free fas. however, cells under increased membrane production, e.g. highly proliferating cells, can endogenously produce them through the enzyme fatty acid synthase (fasn), which synthesize palmitate. in these cells, fasn is transcriptionally augmented via the mtorc1 pathway. thus, we hypothesized that due to their increased lipid demand myelinating cells would require fasn activity towards myelin production. the functional role of fasn in the metabolic control of myelin membrane lipid composition and myelination is unidentified. thus, we addressed it experimentally by conditionally deleting fasn in myelinating cells in vivo. we show that endogenous fas biosynthesis in myelinating cells is essential to maintain myelin membrane lipid homeostasis. furthermore, maintaining a correct lipid homeostasis is critical for myelination. thus, we analyzed whether fasn is required to allow palmitoylation of myelin proteins and their targeting to the myelin membrane. in addition, we show that interfering with myelin fas homeostasis results in the activation of systemic compensating metabolic loops promoting fas uptake, although not sufficient to preserve efficient myelination. taken together our data demonstrate that in myelinating cells fas homeostasis is under the synergistic control of endogenous metabolic fas production via fasn and systemic functional regulation of free fas uptake. most importantly, maintaining a correct homeostasis of fa-derived lipids in myelin is critical for myelination. homeostasis and signaling, respectively (1). in mammals, two cytosolic and two mainly mitochondrial located glutaredoxins have been identified (2). we already described an essential role for the formation of a functional neuronal network during embryonic development of one of these proteins, the vertebrate specific glutaredoxin 2 (3). here, we will focus on the contribution of glutaredoxin 2 on basic cellular functions of oligodendrocytes, the myelinating cells of the central nervous system, under physiological and pathophysiological situations, e.g. multiple sclerosis, which is characterized by inflammatory axonal demyelination (4). oligodendrocyte progenitors and mature oligodendrocytes are very sensitive against oxidative stress induced by release of nitric oxide by activated microglia (5) . using a2b5 positive glia restricted progenitor cells as well as organotypic cerebellar slice cultures, we found that increased levels of glutaredoxin 2 protect against cell death induced by either activated microglia or s-nitrosoglutathione, a physiological nitric oxide donor. under physiological conditions elevated levels of glutaredoxin 2 increased migration of a2b5 positive cells nearly three fold. western blot analyses, quantitative real time pcr, as well as immunocytochemistry revealed that glutaredoxin 2 inhibited further differentiation of ng2 positive oligodendrocyte progenitor cells. in summary, our data indicate the importance of specific thiol redox signaling during cellular protection and function in this type of glia cells. current multiple sclerosis therapeutics act as immunomodulators and/ or immunosuppressors. this strategy is largely ineffective at preventing progression of the disease. significant benefits to multiple sclerosis patients might be seen with a therapeutic agent which induces remyelination, however, to date no such agents have been developed. using in vitro and in vivo models we have identified nefiracetam as a potential remyelinating therapeutic. to study remyelination in vitro, hippocampal organotypic cultures were exposed to lysophospatidylcholine (lpc) for 18hrs to induce demyelination before being allowed to recover. cultures were treated with nefiracetam for 24hrs of this recovery period. in all cases immunofluorescent labelling of myelin basic protein (mbp) was used as a measure of myelin. nefiracetam was shown to increase mbp expression, relative to untreated controls, accelerating recovery from lpc. two different models were used to investigate the efficacy of nefiracetam in vivo; an inflammatory model, experimental autoimmune encephalomyelitis (eae), and a toxin induced model of demyelination, achieved through the use of cuprizone chow. nefiracetam treatment did not alleviate the symptoms of eae. however, nefiracetam did restore the amount of myelin in cuprizone exposed animals, compared to saline-treated controls. in combination, these observations suggest that nefiracetam is capable of accelerating remyelination through a mechanism of action which is independent of immunomodulation. we conclude that nefiracetam encourages remyelination both in vitro and in vivo. these pro-myelin properties identify nefiracetam as a potential therapeutic for demyelinating disorders. university of naples "federico", neuroscience, naples, italy changes in intracellular [ca 21 ] i levels have been shown to influence the developmental processes that accompany the transition of human oligodendrocyte precursor cells (opcs) into mature myelinating oligodendrocytes and are required for the initiation of myelination and remyelination processes. in the present study, we explored whether calcium signals mediated by the selective sodium calcium exchanger (ncx) family members ncx1, ncx2, and ncx3, play a role in oligodendrocyte maturation. functional studies, as well as mrna and protein expression analyses, revealed that ncx1 and ncx3, but not ncx2, were divergently modulated during opc differentiation into oligodendrocyte phenotype. in fact, while ncx1 was down-regulated, ncx3 was strongly up-regulated during the oligodendrocyte development. the importance of calcium signaling mediated by ncx3 during oligodendrocyte maturation was supported by several findings. indeed, whereas the knocking down of the ncx3 isoform in opcs prevented the up-regulation of the myelin protein markers cnpase and mbp, its overexpression induced an up-regulation of cnpase and mbp. furthermore, ncx3 knock-out mice exhibited not only a reduced size of spinal cord but also a marked hypomyelination, as revealed by the decrease in mbp expression and by the accompanying increase in opcs number. collectively, our findings indicate that calcium signaling mediated by ncx3 plays a crucial role in oligodendrocyte maturation and myelin formation. to explore the role of kif13b in myelination in vivo, we generated conditional knock-out mice with kif13b specific ablation in either schwann cells or oligodendrocytes. we found that kif13bfl/fl p0cre mouse nerves are hypomyelinated with reduced myelin thickness, thus confirming in vitro data. in the pns, axonal neuregulin (nrg) 1 type iii is one of the main signaling promoting myelination and remyelination after injury. nrg1 type iii binds to erbb2/erbb3 receptors in schwann cells, which signal through a plethora of downstream signaling pathways also including the pi3k/akt complex. the pi3k/akt signaling pathway is attenuated by the pten phosphatase, which dephosphorylates ptdins(3,4,5)p 3 (s. goebbels et al., 2010). dlg1 is believed to interact with pten and potentiate its ability to dephosphorylate ptdins(3,4,5)p 3 , thus negatively modulating the akt-mtor pathway (l. cotter et al, 2010). as kif13b interacts with dlg1 in schwann cells, we investigated the pi3k/akt pathway in kif13b-null nerves and surprisingly, we found that dlg1 expression levels are increased whereas akt phosphorylation is decreased in mutant nerves. our findings suggest that kif13b negatively regulates dlg1 activity and acts as a promoter of myelination in the pns. on the contrary, when kif13b is lost specifically in oligodendrocytes, myelin sheaths are thicker. at the molecular level, the hypermyelination is consistent with increased akt phosphorylation. we are currently investigating the molecular mechanisms at the basis of the kif13b/dlg1 interaction in both schwann cells and oligodendrocytes. we have identified the nootropic nefiracetam as a potential novel remyelinating therapeutic using in vivo and in vitro models of demyelination. in this study we investigate the mechanism of action of nefiracetam in the context of remyelination as regulated by wnt/b-catenin signalling and spontaneous activity. to investigate whether nefiracetam acts by modulating wnt signalling, organotypic hippocampal cultures were exposed to nefiracetam or the wnt/b-catenin inhibitor cardamonin. both increased immunofluorescent staining for the opc marker ng2. myelin formation, as assessed by mbp staining, was enhanced by nefiracetam but was not affected by cardamonin. thus, modifying wnt signalling appears to impact opc development without effecting differentiation, suggesting the pro-myelinating effects of nefiracetam involve an additional mechanism. we next investigated the effect of nefircatam on opc differentiation using purified opc cultures. nefiracetam increased the ol:opc ratio, as assessed by immunofluorescent staining for mbp and ng2 respectively, suggesting nefiracetam is capable of promoting ol maturation through a direct action on opcs. finally, we investigated the effect of nefiracetam on spontaneous activity. organotypic hippocampal cultures were exposed to lysophospatidylcholine (lpc) for 18hrs to induce demyelination before being allowed to recover. cultures were treated with nefiracetam for 24hrs of the recovery period. spontaneous calcium activity was then measured using fluo4-am. cultures not exposed to lpc displayed low levels of activity which were unaffected by nefiracetam. lpc exposure caused an increase in activity which was enhanced by nefiracetam. additionally, in these cultures, lpc decreased mbp staining compared to control while nefiracetam post-lpc caused a return to control levels. these data suggest increased activity may be an adaptive response related to myelin repair which is enhanced by nefiracetam. together, these findings suggest a complex mechanism of action for nefiracetam as a remyelinating agent involving modulation of wnt signalling, spontaneous activity and opc differentiation. in an assay in which cortical oligodendrocytes ensheath dorsal root ganglion cells 3 we found that nrg and nmda receptors interact to regulate myelination. without nrg, blocking nmda receptors had no effect on myelination. adding nrg increased myelination at all timepoints analysed (2-5 weeks), suggesting that nrg did not merely accelerate myelination. strikingly, nrg led to the majority of myelination becoming dependent on activation of nmda receptors and action potential activity. nrg's effect was associated with a 4-fold increase in nmda receptor current in oligodendrocyte lineage cells. thus, nrg switches myelination from a default programme, which is independent of neuronal activity, to a mechanism that is regulated by glutamate released from active axons. the effects of nrg were associated with an increase in the phosphorylation of akt and the transcription factor creb, but not associated with changes in the phosphorylation of erk. these data reveal a function for oligodendrocyte nmda receptors. the absence of nrg in multiple sclerosis lesions, and enhanced remyelination by added nrg, suggest a role for neuregulin/nmda receptor dependent remyelination after pathology. low-density lipoprotein receptor-related proteins (lrps) are endocytic receptors that internalize several ligands and are involved in lipoprotein homeostasis in adult brain. lrp-2, also known as megalin, is involved in the development of the central nervous system (cns) and in the transport of molecules across the blood brain barrier (bbb). in a previous work, our group revealed that megalin is selectively involved in sonic hedgehog-mediated effects on oligodendrocyte precursor cell migration and proliferation during the development of the cns. although there are some reports describing the presence of lrps in acute lesions of multiple sclerosis (ms) patients and pointing to an important role of shh in the pathogenesis of this disease, there are not data about the expression of megalin in ms tissue. in the present study, we analyse the distribution and the cellular characterisation of megalin in samples of human brain from ms patients to elucidate the role of this receptor in the pathogenesis of demyelination. changes in megalin distribution parallel the different ms histopathological lesions: we detected megalin in active demyelinating lesions and in the periplaque of chronic-active lesions, areas where remyelination spontaneously occurs. on the contrary, megalin was absent in the demyelinated region of chronic lesions. in addition, we observed the up-regulation of megalin in a subpopulation of perivascular astrocytes within the normal appearing grey matter (nagm) of ms patients. moreover, megalin was up-regulated in blood vessels within active lesions, in the periplaque of chronic-active lesions and in the nagm. in parallel, we also analysed the bbb profile to determine structural and functional alterations. altogether, we suggest that megalin is an important receptor expressed in diverse areas of the cns of ms patients representing different aspects of ms development: i) a potential target to improve remyelination; ii) an indicator of a functional rather than a structural disruption of the bbb. therapeutic strategies to modulate specific lrps would be useful to effectively treat ms. bdnf is known to promote central nervous system myelination both in vitro and in vivo. adopting in vitro myelination assays, we identified that bdnf acts through oligodendroglial-expressed trkb receptors to promote myelination. this was verified in vivo, as deletion of trkb in mature oligodendrocytes (in trkb fl/fl mbp-cre mice) resulted in a hypomyelinating phenotype during development. question: here we investigate the consequence of trkb deletion in oligodendrocyte progenitor cells (in trkb fl/fl cnpase-cre mice) and the signalling pathways downstream of trkb that promote myelination. methods and results: trkb fl/fl cnpase-cre mice were born in mendelian ratios and were phenotypically indistinguishable from littermate control mice. surprisingly, analysis of myelinated axonal tracts in the spinal cord and optic nerve revealed normal myelin development. in addition, the number of cells in the oligodendroglial lineage was the same as control mice during postnatal development. these data indicate that the timing of trkb deletion in the oligodendroglial lineage is critical, as deletion in opcs results in no significant phenotype, whereas deletion in mature oligodendrocytes results in a hypomyelinating phenotype. these data suggest that opcs are able to compensate for the loss of trkb and myelinate normally in the absence of bdnf signalling. our in vitro data have shown that the promyelinating influence of bdnf strongly correlates with erk1/2 activation. here we show that overexpression of erk2 in opcs exerts a more significant promyelinating effect than erk1. as erk1/2 are known to exert some of their effects through direct transcription factor phosphorylation, we have undertaken an in silico analysis of myelin-specific transcription factors and identified several that contain potential erk1/2 binding domains and phosphorylation sites. co-immunoprecipitation experiments show an interaction between erk1/2 and these transcription factors. investigation into the nature of these interactions and their functional consequences are ongoing. conclusions: this work suggests a novel role for erk1/2 signalling within oligodendrocytes that regulates cns myelination, possibly via activation of myelin-specific transcription factors. as erk1/2 is activated by multiple receptors, other receptors could potentially compensate for the loss of trkb in opcs, resulting in a normally myelinated cns in vivo. myelin is vulnerable to impairment by genetic, toxic, and immunological triggers, but the molecular basis for many aspects of myelin pathology has remained unknown. to identify molecules required for the structural integrity of central nervous system myelin we have systematically analyzed its protein composition by quantitative proteome analysis. based on the high abundance of filament-forming septins in myelin, we hypothesized that septin filaments may constitute a cytoskeletal membrane cortex in myelin. we find that septin filaments localize to the non-compact adaxonal myelin compartment, which is considered relevant for the metabolic maintenance and the turnover of myelin. for functional analysis, we generated mice lacking the most abundant septin of myelin, sept8, from myelinating glia. our results indicate that sept2, sept4, sept7 and sept9 multimerize with sept8 and that this interaction is essential for their incorporation into myelin. myelin lacking septin filaments display pathogenic outfoldings emerging from adaxonal myelin. we propose that this phenotype reflects that myelin septins provide long-term structural integrity to the adaxonal compartment of cns myelin. here, we used primary opc cultures to investigate the mechanisms underlying gpr17 endogenous regulation. this is quite important since gpr17 is upregulated at injury sites in both a demyelinating in vivo model (boda et al., glia, 2011) and in multiple sclerosis (ms) patients (chen et al., nat neurosci 2009), which, could, in turn, cause defective myelination. in line with these findings, we have recently shown that gpr17 forced over-expression at late differentiation stages, obtained by transfecting cultured opcs with a gfp-gpr17 fusion vector, indeed impairs terminal cell maturation. this suggests that the receptor needs to be down-regulated/ desensitized to allow opc terminal maturation. physiologically, gpr17 downregulation may occur through agonist-induced receptor phosphorylation via g-protein coupled receptor kinases (grks) (daniele et al., j pharm exp ther, 2011; frantangeli et al., j biol chem, 2013), which are, in turn, controlled by mtor kinases, and are altered in ms. our in vitro data show that rapamycin, an inhibitor of mtor kinase, reduces grk2 levels, with parallel increases in gpr17 expression and strong impairment of opc maturation. globally, these data suggest that dysregulation of these interconnected pathways leading to aberrant gpr17 overexpression may prematurely block opc maturation. analysis of gpr17 in vivo in ms models will confirm if the receptor is dysregulated during disease development and will help finding ways to re-establish myelin repair in demyelinating diseases. loss of central nervous system myelin, and the failure of remyelination by oligodendrocytes, contributes to the functional impairment that characterizes neurodegenerative disorders and demyelinating diseases such as multiple sclerosis (ms). since incomplete remyelination will irreversibly damage axonal connections, treatments effectively promoting (re)myelination are pivotal in precluding progression of disease. although the reasons for remyelination failure are likely multifactorial, one of the major impediments to successful remyelination is the altered lesion microenvironment. our previous findings suggest that fibronectin aggregates, as an environmental factor, contribute to remyelination failure. here, we aim at elucidating whether exogenous gangliosides, i.e., cell surface lipids with a potential to modulate fibronectin-cell surface interactions, could overcome fibronectin-mediated inhibition of myelination. of the gangliosides tested, only addition of gd1a, perturbed adherence of oligodendrocyte progenitor cells to fibronectin, but not to poly-l-lysine, without affecting cell viability. gd1a slightly increased growth factor-mediated proliferation on fibronectin, but in the presence of the lipid migration of oligodendrocyte progenitor cells on fibronectin was reduced. furthermore, addition of gd1a increased myelin-like membrane formation on (aggregated) fibronectin, whereas gm1 reduced oligodendrocyte differentiation, as reflected by a decrease in the number of mbp positive cells. most interestingly, gd1a counteracted the myelination inhibiting effect of aggregated fibronectin in co-cultures of dorsal root ganglion neurons and oligodendrocytes. also, gd1a enhanced remyelination in demyelinated cerebellar slice cultures that contained fibronectin aggregates. taken together, gd1a is able to overcome the inhibiting effect of aggregated fibronectin in remyelination. given the persistent presence of these aggregates in ms lesions, gd1a might therefore act as a potentially novel molecular tool to selectively modulate the impeding signaling environment, apparently detrimental to remyelination. its mechanism of action and exploration of gd1a's potential in the treatment of ms, are currently investigated. in the peripheral nervous system (pns) it is secreted by schwann cells and fibroblasts and its expression is strongly induced upon injury. we are interested in the function of apod in the molecular events that take place after a pns injury. we have studied in vivo the process of wallerian degeneration following sciatic nerve crush and we have assayed ex vivo the phagocytosis of labelled myelin from wt and apod-ko mice by flow cytometry. the analysis of cellular processes and protein or mrna expression of a group of signalling molecules induced by injury shows that the lack of apod results in an exacerbated mcp-1 and tnfa-dependent macrophage recruitment. at the injury site, free aa decreases in the wt. lack of apod results in higher basal levels and a stronger injurytriggered depletion of aa. control by apod of the availability of aa to produce the lipid mediators involved in macrophage recruitment is therefore a key mechanism that conditions both wallerian degeneration and injury resolution later on. on the other hand, the phagocytosis of apod-ko cns myelin is less efficient than the phagocytosis of wt myelin. these results indicate that there are also genotype-dependent differences in myelin composition and/or in the interaction between myelin and macrophages. an electron microscopy analysis of crude myelin preparations reveals that the cns myelin from apod-ko mice has abnormal periodicity and shows defective myelin compaction. lipid analysis of apod-ko myelin shows altered phospholipid composition, particularly in phosphoinositid species. a study of the function of apod in the myelination process is currently underway, our results demonstrate that apod function is relevant for both, myelin membrane properties that influence myelin-macrophage interactions, and the control of the lipid-mediated signalling events controlling the extent of macrophage recruitment. adhesion g protein-coupled receptors (ad-gpcrs) are a subgroup of gpcrs that facilitate cell-cell and cell-matrix interaction. structurally, they differ from other gpcrs by the presence of an extremely larger extracellular n-terminal region that is separated from the 7-transmembrane region by a gpcr autoproteolysis-inducing (gain) domain that in most ad-gpcrs facilitates an auto-catalytic process to produce an nand c-terminal fragments during the maturation process. gpr56 is a member of the ad-gpcr family and its mutations are responsible for a human brain malformation known as bilateral frontoparietal polymicrogyria (bfpp). individuals with bfpp present with changes in the white matter tracts in the form of hypomyelination, in addition to cortical malformation. here, we show that gpr56 knockout mice and zebrafish exhibit a reduction of myelin-specific protein expression, fewer myelinated axons and decreased number of mature oligodendrocytes in the central nervous system (cns). during cortical development, gpr56 functions together with its ligand collagen iii to regulate neuronal migration and cortical lamination. however, it is not clear whether collagen iii is also the ligand of gpr56 during cns myelination. gpr56 has a very long and poorly characterized n-terminal fragment, allowing for the possibility of multiple binding partners that mediate cell-extracellular matrix interactions. indeed, gpr56 also binds to tissue transglutaminase (tg2) in melanoma cells. we have begun to study the possible role of collagen iii and tg2 in oligodendrocyte development and cns myelination. the likelihood of these two proteins as being the ligand(s) of gpr56 during oligodendroglial development will be discussed as will our ongoing work to define the role of gpr56 in cns myelination. proper control of cell cycle, proliferation and differentiation is fundamental to regulate oligodendrocyte (ol) development and recruitment of oligodendrocyte precursors (opc) after brain damage. failure in this process results in severe dysmyelinating disorders and defective brain repair. the molecular machinery that couples differentiation to proliferation withdrawal plays therefore a critical role in brain development and repair and is regulated by multiple extracellular cues including extracellular matrix (ecm) and growth factor derived signals. however, how these signals are integrated into ol to control cell cycle progression and differentiation is only partially understood. we are investigating the role of jun activating binding protein 1 (jab1), an intracellular signalling molecule, shuttling between nucleus and cytoplasm, involved in the control of cell proliferation, survival and gene transcription. recent evidence showed that jab1 function is regulated by ecm and growth factors. we demonstrated that jab1 is expressed in glial cells in peripheral and central nervous system (cns), and conditional inactivation of jab1 in schwann cells results in dysmyelinating neuropathy. here we present preliminary data showing that jab1 plays a role in cns development. mice with conditional inactivation of jab1 in ol, by cnp-cre tansgene, show cns hypomyelination, including corpus callosum, optic nerve and spinal cord. concomitant fiber degeneration was observed. our preliminary data suggest that jab1 expression in ol plays a role in cns myelination and possibility axonal survival. c. gonsior, j. trotter university of mainz, mainz, germany myelin basic protein (mbp), a major component of central nervous system (cns) myelin, is essential for oligodendroglial function and myelination in the cns and its expression is tightly regulated in time and space. the mrna of mbp is sorted to cytoplasmic rna granules and transported up to the distal processes of oligodendrocytes in a translationally silenced state. we and others could show that, dependent on axonal signals, activation of the non-receptor tyrosine kinase fyn leads to a release of translational inhibition allowing synthesis of mbp protein at the axon-glial contact site. a key factor in this pathway is the fyn target and rna-binding protein heterogeneous nuclear ribonucleoprotein (hnrnp) a2, that recognizes the a2 response element (a2re) in the mbp 3 0 utr and orchestrates the dynamics of the mbp mrna granule. moreover, we identified hnrnp f as an additional target of fyn kinase present in these ribonucleoprotein complexes and contributing to posttranscriptional regulation of mbp. cell stress conditions, as present in various disease states such as multiple sclerosis, may result in the formation of cytoplasmic rna-containing stress granules (sgs), potentially influencing the fate of myelin transcripts including their translation.mbp mrna was shown to be sorted to these cytoplasmic foci. we here identified the hnrnps a2 and f to be associated with oligodendroglial sgs. de-regulation of such factors involved in mbp mrna metabolism could affect stress dependent synthesis of mbp and thus (re-)myelination and myelin maintenance. we performed the experiments in biozzi abh mice known to develop a chronic relapsing-remitting form of eae following immunization with myelin oligodendrocyte glycoprotein. our findings suggest that in demyelinating lesions, myelin layers are pulled off from the myelin sheath and form bulb-like structures that we call "myelinosomes". we can detect the formation of such structures in acute and in chronic stages in the animal model, as well in biopsies from multiple sclerosis patients. we are currently investigating the molecular interactions that underlie the formation of myelinosomes to gather further insight into the mechanisms that underlie immue-mediated demyelination. adenosine is a potent neuromodulator which can be released from most cells and its extracellular concentration increases dramatically after brain injury. it acts on four g protein-coupled receptors, a 1 , a 2a , a 2b and a 3, and their activation is involved in myelination as well as in apoptosis linked to neurodegenerative diseases. in this study, we initially found that rat oligodendrocytes in vitro express all four adenosine receptor subtypes, adenosine transporters ent1 and ent2 and the adenosine degrading enzymes adenosine deaminase and adenosine kinase. activation of adenosine receptors caused mitochondrial dysfunction and oligodendroglial apoptosis. notably, selective activation of adenosine receptors revealed that a 3 receptors are the main subtype mediating oligodendrocyte death. thus, a 3 receptor agonist 2-ci-ib-meca caused a robust increase in ros levels, mitochondrial membrane depolarization and caspase-dependent apoptosis. in turn, selective a 3 receptor activation induced mapk modifications compatible with cellular damage, which was prevented by the selective antagonist mrs1220. the modulation of g protein and adenylate cyclase suggested that 2-ci-ib-meca acts via camp-pka. moreover, selective activation of a 3 receptor triggered intracellular calcium mobilization via plc pathway. finally, we used cerebellar organotypic slices and optic nerve ex vivo to investigate adenosinemediated oligodendroglial death in more integral preparations. consistent with data in dissociated cultures, a 3 receptor activation induced a significant damage in the optic nerve as well as in the cerebellum white matter, an effect that was prevented by caffeine, an adenosine receptor blocker. together, these results indicate that adenosine can trigger oligodendrocyte death via activation of a 3 receptors, and suggest that this mechanism may contribute to the etiology of demyelinating diseases. question-one of the commonest forms of inherited neuropathy, xlinked charcot marie-tooth disease (cmt1x) leads to progressive distal muscle weakness and sensory loss. the disease is caused by a large number of mutations in the gjb1 gene encoding the gap junction protein connexin32 (cx32). cx32 is expressed by schwann cells in peripheral nerves and forms important channels of communication between the layers of the non-compact myelin sheath and is necessary for the homeostasis and survival of the myelinating cells and the axon. transgenic disease models generated in our lab as well as clinical-genetic analysis of large number of patients have demonstrated that most cmt1x mutations result in loss of cx32 function and failure to form gap junctions. cx32 ko mice offer a well characterized and relevant model of the disease to study future therapies for cmt1x, for which gene replacement may be an effective treatment strategy. methods-for this purpose, we have generated a novel 3 rd generation lentiviral vector to allow gene delivery to peripheral nerves. in this vector, the cx32 open reading frame is placed under the control of cell-specific promoter for schwann cells, the rat mpz/p0 promoter. the expression cassette also includes a green fluorescent protein (egfp) as a marker. we have successfully produced lentiviral vector particles and delivered them to the sciatic nerves of 2-month old wild type (wt) and cx32 ko mice, immediately distal to the sciatic notch. the expression of egfp on wt background and of cx32 on ko background was determined by western blot analysis and immunohistochemistry of teased nerve fibers at different time points after injection. results-our initial studies in wt mice (n 5 15) show that more than 60% of schwann cells from different nerve fascicles expressed egfp as far as 5 mm distal to the injection site. expression started a week after the intraneural injection and remained stable for up to 3 months. when the lentiviral construct was injected into the sciatic nerves of cx32 ko mice (n 5 5), virally delivered cx32 was expressed and was properly localized at the non-compact myelin areas including the paranodal regions and incisures of myelinated fibers, where gap junctions are normally formed. poster abstracts s139 glia conclusions-thus, we have generated lentiviral vector that leads to efficient gene expression specifically in schwann cells. these results indicate that gene replacement therapy is feasible and may lead to correction of the gap junction loss in myelinating cells of the pns. perinatal inflammation is intensively discussed to have a long-term impact on various cell populations and on the development of autoimmune diseases such as multiple sclerosis (ms) in adulthood. in order to determine long-term effects of perinatal inflammation on the course of demyelination in adulthood we mimic a bacterial inflammation by exposure to lipopolysaccharide (lps) of either pregnant or newborn mice. demyelination as a "second hit" was induced by feeding adult mice with the oligodendrocyte toxin cuprizone (bis-cyclohexanone oxaldihydrazone). a single prenatal lps injection at e13.5 did not affect the course of demyelination in adulthood. in contrast, serial postnatal lps injections lead to a delayed demyelination and a reduced number of activated microglia in the corpus callosum, suggesting a long-lasting effect of perinatal inflammation on the function of microglia. surprisingly, mice exposed to lps after birth showed an enhancement of early remyelination accompanied by an increased number of newly differentiated oligodendrocytes. furthermore, independent of cuprizone administration the perinatal lps injections seem to induce a long-term impact on the blood-brain barrier (bbb) by decreasing the number of claudin-5 positive vessels. in summary, perinatal inflammation mimicked by the administration of lps has long-lasting effects on the bbb, microglia activation, and remyelination. question: epigenetic control is crucial for the differentiation of a variety of cells including oligodendrocytes, the myelinating glial cells of the central nervous system. however, studies about the implication of epigenetic factors in peripheral nervous system maturation are just emerging and we were wondering whether methylation of histones is involved in this process. methods: we describe the function of ezh2 in primary schwann cells and drg cocultures using immunohistochemistry, transfection and lentiviral transduction, shrna, qrt-pcr analysis, morphological analysis, western blotting and chromatin immunoprecipitation. results: here, we demonstrate for the first time the impact of a histone methyltransferase, encoded by the enhancer of zeste homolog 2 (ezh2) gene, on schwann cell differentiation. in sciatic nerves, ezh2 expression was found in schwann cells and to peak perinatally. suppression of ezh2 expression in cultured primary rat schwann cells reduced the length of cell processes. these morphological changes were accompanied by widespread alterations in the gene expression pattern, including downregulation of myelin genes and induction of p57kip2, which we have recently identified as an intrinsic inhibitory regulator of schwann cell maturation. in addition, we show that ezh2 suppression in dorsal root ganglion cocultures interferes with in vitro myelination. chromatin immunoprecipitation analysis revealed binding of ezh2 at the p57kip2 promoter and reduction of histone h3k27 trimethylation upon gene suppression. ezh2 suppression-dependent effects on morphology and myelin genes could be reversed by concomitant suppression of p57kip2, indicating that p57kip2 is a downstream effector of ezh2. furthermore, we describe hes5 as transcriptional repressor of myelin genes in schwann cells, which was induced upon ezh2 suppression and downregulated in p57kip2-suppressed schwann cells. conclusions: we have identified a molecular link between histone methylation and control of schwann cell differentiation and demonstrate that this epigenetic mechanism is crucial for glial differentiation to proceed. previous studies, performed primarily in cell culture, suggest that electrical activity may regulate multiple stages of oligodendrocyte development including proliferation, migration, initial wrapping and myelination. in this study, we have examined the requirement for electrical activity in oligodendrocyte development in vivo using zebrafish as a model system. results: we find that electrical activity is not required for proliferation, migration, or the initiation of axon wrapping. rather, our data indicate a specific role for electrical activity in oligodendrocyte differentiation by regulating a subset of myelin genes, including myelin basic protein (mbp). silencing electrical activity with tetrodotoxin reduced mrna expression of mbp, as assessed by rna in situ hybridization and quantitative rt-pcr. in contrast, myelin protein zero, claudin k, claudin 11a, ugt8, and 36k levels were not regulated by electrical activity. ongoing experiments are aimed to uncover the mechanism of activity-dependent mbp mrna expression during oligodendrocyte differentiation. conclusions: taken together, these data indicate that electrical activity can modulate myelin gene expression in vivo but do not support the notion that electrical activity drives key axon-glia messengers that are required for the initial stages of myelination. (fancy et al., 2011) . eight-week-old lgals3-/-and wild type (wt) mice were fed a diet containing 0.2% cpz w/w during 6 weeks, after which cpz was withdrawn in order to evaluate remyelination 2 weeks after. according to our results, cpz-induced demyelination in lgals3-/mice showed an exacerbated astrocytic and microglial response as compared to wt littermates. electron microscopy showed a significant lack of myelinated axons in lgals3-/-mice as compared to controls. furthermore, the few myelinated axons present were nearly 50% less myelinated than those of controls and were found to be collapsed. remarkably, the remyelination process seemed to be faster in lgals3-/-mice than in wt. remyelinated lgals3-/-mice showed a higher myelin basic protein (mbp) recovery rate as compared to their controls. flow cytometry assays showed a sharper microglial response in lgals3-/-mice, which was supported by an exacerbated number of cd11b1 and cd451 cells. however, electron microscopy images from remyelinated lgals3-/-animals showed, again, collapsed axons with a defective myelin wrap, as compared to wt mice showing normal axons without any relevant myelin wrap disruption. behavioral performance observed during cpz treatment recovery correlates with alterations in the morphological studies, which show that neither lgals3-/-nor wt mice reach basal myelination levels. we have shown that new oligodendrocytes (ols) continue to be generated in healthy adult mice after 8 months of age, even in the optic nerves, which are considered to be completely myelinated already [1] . this raises the question of whether the adult-born ols and myelin are engaged in remodeling existing myelin (e.g. intercalating among the existing myelin sheaths), or replacing ols that die in use. to ask whether ols die and are replaced during healthy adulthood we generated transgenic mice that express icreer t2 under the transcriptional control of the myelin basic protein promoter (mbp6-icreer t2 ). the $6kb mbp upstream fragment that we used is reported to direct transcription to ols but not to either ol precursors (ops) or schwann cells [2] . we confirmed that our mbp transgene targets cre recombination specifically to differentiated ols by crossing to ros26-yfp conditional reporters; when tamoxifen was administered to double-transgenic offspring, essentially all yfp 1 cells in the cns were also cc1 1 . when mbp6-icreer t2 is crossed to tau-mgfp conditional reporter mice (mgfp is membrane-tethered) we can visualize differentiated ols and their myelin internodes in white and grey matter, allowing us to follow the fates of myelinating ols for extended periods of time after tamoxifen labeling. following tamoxifen administration to mbp6-icre t2 : tau-mgfp mice on postnatal day 60 (p60) or p120, a large proportion of ols became gfp-labelled in the corpus callosum, optic nerve and spinal cord. examining these mice at increasing times post-tamoxifen will reveal if and how the number and fraction of mgfp-labeled ols/ internodes changes with age -whether there is net loss or gain of ols or whether new ol synthesis is balanced by the rate of ol death (i.e. there is ol turnover). our preliminary results indicate that new ol synthesis exceeds ol death, so the function of adult ol genesis is not simply to replace dying ols. this study aims to identify protein networks that participate in peripheral myelin formation and maintenance. we use a cell culture system, where c57bl/6j mouse embryos (e13.5) are used for the isolation of dorsal root ganglia neuron explants. explants are cultured in matrigel, and endogenous schwann cells appear in the culture within a few days. myelin formation is induced by adding myelination-promoting substances to the culture medium. immunocytochemistry is used to evaluate the myelination efficiency and the developmental timetable of myelination in culture. electron microscopy is used to study structural features of myelin in culture and to compare its ultrastructural features with those of myelin in situ. rna microarray analysis is performed to study gene expression changes during myelin development in culture. the rna expression data indicates the changes in the rna expression levels, but it does not give information about the amount of final protein product, their posttranslational modifications or stability. we combine the rna expression analysis with a proteomics approach. proteins are extracted for 2-dimensional electrophoresis analysis at the same time points, and protein spots exhibiting time-dependent changes in their expression levels are analyzed by mass spectrometry. we show that mouse dorsal root ganglion explant cultures produce substantial amounts of mature myelin in 3 weeks after the induction of myelination. ultrastructural analysis shows that myelin in our cell culture system has the same structural characteristics as those observed in peripheral nerves in situ. rna microarray analysis reveals changes in myelin-, nervous system-and schwann cell-related genes. the most significant changes in gene expression occur at the induction of myelination. at the protein level, peaks of specific proteins can be observed at the first and the last time points, which complement the results obtained using microarray data. we have developed a reproducible and efficient method to study molecular changes in myelination at the proteomic and genomic levels. this approach will be exploited further for the characterization of the molecular networks involved in the myelination process. results: our findings show that primary rat schwann cells respond to ivig stimulation with altered cell morphologies accompanied by an accelerated growth of cellular protrusions in early stages of the differentiation process. stimulation with ivig was also found to reduce cellular proliferation rates without affecting cell survival. furthermore, we observed that ivig treatment transiently boosts myelin gene expression of immature cells, whereas myelin gene expression of differentiating schwann cells is promoted on a long-term scale. importantly, myelin gene expression responses cannot be detected upon application of igg1 control antibodies (herceptin, synagis, avastin). in addition, we could demonstrate that primary rat schwann cells express the cd64 fc receptor and that in differentiating schwann cells cd64 levels were significantly increased. on the other hand, we could also reveal a specific binding of the human ivig on the schwann cell surface. conclusions: we therefore conclude that schwann cells are not only able to respond to but also specifically bind immunoglobulins and that ivig stimulation can promote their differentiation. here, we demonstrate that cnp deficient mice exhibit alterations in mechanical and thermal sensation. histological analyses of cnp deficient mutants reveal loss of sensory axons when quantified in the dorsal root and the predominantly sensory saphenous nerve. in contrast ventral roots remain unaltered. moreover, electron microscopical assessments of saphenous nerves display a hypermyelination of small to mid-sized caliber axons and an overrepresentation of the smallest fibers. the myelin pathology of sensory axons is reflected by alterations of sensory nerve conduction velocities. thus, our data demonstrate that, contrary to findings in the central nervous system, cnp has an impact on myelination and is especially important for the integrity of small to mid-sized caliber axons within the sensory peripheral nervous system. to unravel the pathogenetic mechanism of the s63del mutation, we generated transgenic mice that express p0s63del protein and manifest a demyelinating neuropathy similar to cmt1b. p0s63del is a misfolded protein retained in the er, where it activates a chronic unfolded protein response (upr). the upr is a cellular response to er stress, aimed to reduce the load of abnormal proteins by attenuating protein translation, increasing the er folding capacity and stimulating protein degradation. among the degradation strategies upregulated by the upr is the er-associated-degradation (erad) pathway. in erad, proteins destined for degradation are retrotranslocated from the er to the cytosol, ubiquitylated and degraded by the proteasome. microarray analysis of s63del nerves revealed that several erad genes, such as derlins, are strongly upregulated and derlin-3 expression returns towards normal in association with the amelioration of demyelination produced by ablation of chop, indicating a possible involvement of this pathway in cmt1b neuropathy. derlins elicit particular interest for exerting a protective role in several er stress-mediated diseases. in s63del nerves, derlins co-immunoprecipitate with p0s63del protein and, finally, derlin-1 overexpression reduces the level of er stress in cos7 cells expressing p0s63del protein, suggesting a positive role in the modulation of p0s63del protein retrotranslocation. these observations indicate that derlins may be part of the schwann cell adaptive response that attempts to cope with chronic er stress by modulating p0s63del clearance. we are now in the process of investigating the role of derlins, in the pathophysiology of pns myelination using in vitro, ex vivo and in vivo approaches. during the myelination process, the oligodendrocyte extends processes to and wraps around multiple axons of different diameter, keeping the number of wraps proportional to the axon diameter. this is one unique example of how cells in the central nervous system establish asymmetry. transport of mrna and local regulation of protein synthesis represent one mechanism by which such cellular asymmetry can be generated and the different requirements for myelin sheath at each axo-glia interaction can be controlled. the mrna of a key myelin sheath protein, myelin basic protein (mbp), is known to be transported into the oligodendrocyte processes, and a tight temporal and spatial control of mbp mrna translation is thought to be required for normal myelination. the molecular basis for the tight control of mrna transport and translation is the assembly of large mrna-protein complexes. prior work has identified a number of proteins that interact with the mbp mrna, including hnrnp-a2, hnrnp-k and hnrnp-e1. however, it is currently unknown how these protein functions together to prevent translation during transport. it is also unknown how targeting of the mbp mrna to the myelin sheets occurs. to delineate the precise role of the individual binding protein in regulating mbp mrna translation, we have identified regulatory elements within the 3 0 utr of the mbp mrna involved in translational inhibition, and we have identified binding sites in the mrna for hnrnp-k and hnrnp-e1. furthermore, we have analyzed the effect of sirna-mediated knockdown and overexpression of these proteins in primary oligodendrocytes. together, our results allow us to propose a model, in which the individual mrna binding proteins are assigned distinct roles for correct spatial and temporal expression of mbp. remarkably, this manipulation was sufficient to achieve both the expansion of oligodendrocyte precursor cells (opc) and the de novo myelination of a large fraction of enlarged parallel fiber axons in the cerebellar molecular layer, independent of axonal neuregulin-1 expression. by laser-guided microdissection and gene expression profiling of cerebellar gc, we identified neuronal factors that promote opc proliferation and myelin synthesis in vitro. these findings suggest that oligodendrocytes and their precursors respond to multiple 'instructive' signals expressed by cns neurons in vivo, but only on axons with diameters >0.2 lm that became 'permissive' for myelination. l. marziali, p. franco, j. pasquini university of buenos aires, caba, argentina promyelinating effects of both apo-transferrin (atf) and thyroid hormone (th) on rat brain are well documented. th effects are mediated by nuclear receptors which act as transcription factors and regulate different cell processes. previous results from our laboratory showed that th promotes the commitment of oligodendrocyte progenitors (opcs) to mature myelinating oligodendrocytes (olg). on the other hand, atf is able to promote the commitment of neural stem cells (nsc) to cells of the oligodendroglial linage and favors olg maturation after an intracranial injection. our previous demonstration that th administration is able to up-regulate tf mrna expression leads us to hypothesize that both factors converge in the control of oligodendrogenesis. to test if the combined effects of both atf and th are required for proper myelination in the rat brain, studies were done at p10 and p20 for each experimental condition. a hyperthyroid state was rendered by daily subcutaneous th administration from the day of birth (hyper). hypothyroidism was achieved by giving propylthiouracil to the mother from gestational day 18 to the end of the experiment (hypo). half of the hypo pups received an intracranial injection of atf at postnatal day 3 (hypo1atf). a set of euthyroid animals received an intracranial injection of atf at postnatal day 3 (atf). control groups received an intracranial injection or subcutaneous saline solution (c). at p10, hyper animals showed an up-regulation of 55% in tf mrna expression and 87% in protein levels as compared to controls. hypo showed a decrease of 25% in tf mrna levels. this effect was not affected by exogenous administration of atf. at p20, hyper showed no differences in the expression of tf mrna or protein levels as compared to controls. hypo showed a decrease of 25% in tf mrna and a 40% decrease in protein levels. this effect was not affected by exogenous administration of atf. no differences were observed in the expression of insulin growth factor i (igf-i) or igf-i receptor between groups at p10 or p20. immunohistochemical analyses were performed at p20 in all groups usingspecific markers anti caii, anti mbp, anti rip and pdgfra. our conclusion is that, in a hypothyroid state, tf is not able to produce olg and myelin maturation at the corpus callosum, which indicates that th is necessary for atf action. however, the finding that hyperthyroid animals showed a significant increase in tf mrna strongly suggests that tf could be involved in th effects. new experiments are carrying out in order to knock down the tf gen to probe this last piece of evidence. h. yigit, l. meyer, c. gonsior, p. hoch-kraft, j. trotter johannes gutenberg university, mainz, germany myelination is the prerequisite for fast saltatory conductance of action potentials. in the central nervous system, the myelin sheath is produced by oligodendrocytes which extend processes and wrap the axons. this structure is subsequently compacted by the help of certain proteins. of major importance here is the myelin basic protein (mbp) which interacts with the cytoplasmic leaflet of the plasma membrane. mbp is a highly basic protein and binds to the negatively charged plasma membrane with high affinity. it has 6 isoforms in mice produced by alternative splicing. the mostly studied 4 isoforms are 14 kd, 17 kd, 18,5 kd and 21,5 kd. interestingly, exon-ii containing 17 kd and 21,5 kd isoforms are actively transported into the nucleus while 14 kd and 18,5 kd isoforms are rather localized to the distal part of the plasma membrane. transport to the nucleus depends on temperature and energy. recently, it was reported that exon-ii contains a non-canonical py nuclear localization signal. as a cytoplasmic protein, mbp is essential for proper myelination. furthemore mbp is supposed to have multiple additional functions, e.g. in calcium homeostasis and cytoskeleton dynamics. it was also shown that the expression level of mbp exon-ii containing is changed in schizophrenia patients. selectively transport of exon-ii containing isoforms to the nucleus raises the question of its nuclear functions which have not been elucidated yet. this study aims to unravel the role of exon-ii containing mbps in the nucleus. gene expression profiling of the microdissected svz regions revealed that wnt-responsive genes were enriched in the postnatal dorsal svz compared to the lateral svz. we performed inhibition of gsk3b by intraventricular infusion of ara to initiate wnt-signalling. several approaches confirmed activation of the wnt-signalling pathway in the dorsal svz as well as within ops. thus, gene expression profiles of wnt-target genes of the dorsal svz indicated that axin2, lef1, fzdl1 and tcf4, but not those other pathways, were dramatically up-regulated. furthermore, nuclear b-catenin was transiently up-regulated in the dorsal svz including in a large population of sox10-egfp expressing cells following pharmacological wnt-activation. gene expression profiling and immunostainings revealed that the densities of nscs, cycling progenitors and subsequent op differentiation in the dorsal svz were augmented. in parallel, we genetically manipulated wnt-signalling in stem and progenitor cells of the dorsal wall of the lateral ventricle. targeted dorsal svz electroporation of cre-gfp plasmids in floxed stabilised b-catenin (gain-of-function) and floxed b-catenin (loss-of-function) transgenic mouse lines were performed and resulted in a phenocopy of the results observed following ara-intraventricular infusion. in summary, our findings illustrate that wnt-signalling endogenously persists in the dorsal svz after birth. pharmacological as well as genetic interventions reveal an important role of this signalling pathway in the regulation of op genesis during the period of myelination. in the cerebellum the generation of neurons and glia and the relationships between these two lineages are poorly understood. all the cerebellar neuronal phenotypes derive from two distinct embryonic germinal neuroepithelia that are restricted regarding the neuronal cell fate: the rombic lip (rl) gives rise to the glutamatergic neurons, whereas the ventricular zone (vz) generates all gabaergic neurons. in particular, different types of inhibitory interneurons are generated during the postnatal life from a common pool of progenitors expressing the transcription factor pax2. on the other hand, the glial development is relatively unexplored. part of the oligodendrocytes derives from extracerebellar sources, whereas some of them derive from endogenous precursors. astrocytes, instead, may derive from progenitors that delaminate from the vz and continue to divide in the prospective white matter (pwm) up to postnatal life. given the vn origin of both astrocytes and interneurons, we wondered whether a lineage relationship exists between these two cell populations and whether they share the same bipotent progenitor. in order to address these issues, we performed a genetic fate mapping study of glastcreert2 mice. we showed that both cerebellar gabaergic interneurons and astrocytes derive from proliferating glast 1 precursors. in particular, we found that these progenitors are able to produce interneurons at earlier developmental phases, whereas after p6 they appear restricted to the glial lineage. proliferating glast 1 cells that may produce interneurons comprise bergmann glia and pwm progenitors. however, when recombination was specifically induced in bergmann glia, we found that these cells were not neurogenic. therefore, we concluded that glast 1 progenitors reside in the pwm. to get further insight in the behaviour of these precursors, we injected in the pwm a gfp-containing lentiviral vector with a specific tropism for astroglial cells. some days after, infected cells generated pax2 1 -interneurons that were able to mature into interneurons of different cortical layers. moreover, in vitro and in vivo clonal analysis of pwm derivatives indicates that the pwm is neurogenic and both astrocytes and interneurons may derive from a common progenitor. evidence is accumulating that neurogenesis in the subventricular zone (svz) is boosted after trauma or ischemia, also through the interaction with surrounding parenchyma or niche cells. nevertheless, the number of newborn neurons that survive and integrate in the damaged areas is negligible, suggesting a non-permissive environment. thus, understanding the complex signaling network guiding neuroblast generation/survival could help identifying strategies to limit negative inputs and promote regeneration. extracellular nucleotides (ents) are among the hypothetical modulators of svz cell functions, especially under pathological conditions where their concentrations raise several folds and they contribute to reactive astrogliosis. few literature data have recently pointed for a role of the p2y 1 receptor subtype (one of the 8 known g protein-coupled nucleotide receptors) in controlling the proliferation and differentiative potential of svz cells. thus, we tested the ability of adpbs, a stable p2y 1 agonist, to modulate stem cell properties in the adult brain, with a focus on the possible modulatory effects exerted by reactive astrocytes. the administration of adpbs in the lateral ventricle of adult mice led to reactive astrogliosis in the surrounding brain parenchima, and to a massive reaction of gfapexpressing precursors and astrocytes in the svz. also proliferation was increased, paralleled by a significant expansion of the population of mash11 transit-amplifying cells and of doublecortin1 neuroblasts. thanks to the conditional glast::creert2 yfp mouse model, we also demonstrated that adpbs promoted the proliferation of glast-expressing progenitors in the neurogenic niche, and sustained their progression towards the generation of rapidly dividing transit-amplifying cells. in vitro the nucleotide analog increased the proliferation of svz cells grown as neurospheres, and their differentiation towards neurons, fully confirming in vivo data. interestingly, a significant enhancement in neurosphere generation was detected when svz cells were initially grown in the supernatant of astrocytes exposed to adpbs, and then shifted to normal medium. this suggests that adpbs stimulates the release of yet-to-be identified astrocytic mediator(s) whose removal from the culture medium boosted proliferation of svz cells. our results further strengthen the notion that the purinergic system is a key regulator of the neurogenic potential of svz cells, both directly and through the involvement of reactive astrocytes. cambridge stem cell institute, cambridge, united kingdom oligodendrocytes, one of the principal glial cell-types of the central nervous system, are critical for neuronal function and represent the primary target of many neurological diseases, including multiple sclerosis and leukodystrophies. despite the recent progress in stem cell research and the possibility to generate various neural cell types in vitro, the derivation of oligodendrocytes from human pluripotent stem cells remains inefficient and unreliable. we employed a new experimental approach termed "direct cellular reprogrammng" which was already succesfully applied for the generation of different types of neurons and neural stem cells. aided by bioinformatic filtering we have generated a pool of seventeen candidate reprogramming factors that could potentially convert fibroblasts directly into oligodendrocyte lineage cells. combinatorial testing of the candidate factors in overexpression experiments has revealed a combination of just two transcription factors that was sufficient to convert mouse embryonic fibroblasts directly into o4-expressing oligodendrocyte precursors. supplementation of additional factors and multicistronic gene delivery strategies render the reprogramming process more efficient and widen the applicability of this protocol. cellular reprogramming offers a new tool for the generation of oligodendrocyte precursors. this approach, that circumvents the protracted specification and differentiation process inherent to the oligodendrocyte lineage will foster the quest for a culture system of human oligodendrocyte lineage cells. transplanted are derived from progenitors within the intestine. to gain further insight in possible stem cell niches the compelling evidence that mouse enteric glia can also be neuronal precursors was related to human tissue. human colon from infants with hirschsprung's disease was investigated with a panel of glial and stem cell markers. tissue samples from infants with hirschsprung's disease were investigated with glial and stem cell markers along the gut axis. the tissue samples from ganglionic, aganglionic and transient segments were immuonstained either for s100/nestin, s100/p75, gfap/nestin and gfap/p75. in all samples investigated, nestin positive ganglia could be found, even in the distal parts with a severe hypo-or aganglionosis. beside nestin positive cells in all segments, there were also different expression pattern glial markers within the ganglia, indicating that distinct phenotypes of glia cells could be found. neural and glial precursor cells are present in the ganglionic as well as in the hypoganglionic segments of hirschsprung's colon, suggesting that these cells might be suitable for ncsc generation and further transplantation. the subventricular zone (svz) of the lateral ventricle, the largest adult stem cell niche, has a striking pinwheel organization specific to regions of adult neurogenesis. the pinwheel's core contains the apical endings of b1 cells and in its periphery two types of ependymal cells: multiciliated (e1) and biciliated (e2) cells. previous studies showed that svz cells are reactivated in response to demyelination. however, the mechanisms inducing svz reactivation in response to inflammatory demyelination such as in multiple sclerosis (ms) are poorly understood. we hypothesize that b1 and e, which are in direct contact with csf through cilia, may play a crucial role in initiation of svz reactivation. in the present study, we examined the svz reactivation in mice in which we targeted an ms-like pathology to the forebrain white matter (t-eae). animals were immunized with subclinical doses of myelin oligodendrocyte protein and after 20 days, received a stereotaxic injection of a cytokine cocktail consisting of tumour necrosis factor (tnfa) and interferon (ifn c) into the corpus callosum. to obtain an en face view of the ventricular surface, we dissected whole mounts of lateral ventricle of control and eae animals at 3, 7, 14 and 28 days post-eae induction (dpi). we investigated by immunohistochemistry whether and how the pinwheel architecture of the svz is modified and ependymal cells are reactivated during disease course of eae. in svz niche of t-eae animals we found that the number of b1 cells increased and peaked at 7 dpi compared to control. the number of e2 cells and pinwheels decreased and then reached control level at 7 dpi. while the number of e1 cells remained steady, their surface area increased at 3dpi and then returned to normal size over time. in addition, the number of their ciliary basal bodies increased during eae time courses, and e1 cells highly expressed gfap in eae animals compared to controls. thus, the findings of our ongoing research suggest that neuroinflammation induces some changes in cell number and morphology of svz niche as well as reactivation of ependymal cells, which altogether may contribute to svz reactivation mechanisms. further study will provide detailed information on the homotypic and heterotypic interactions between pinwheel cells in response to t-eae. to test whether hypothalamic tanycytes act as bona fide neural stem/ progenitor cells in vivo, we analysed their immumoprofile and proliferative potential, and lineage-traced them in vivo. for this, we exploited our earlier findings that in the adult hypothalamus, fibroblast growth factor 10 (fgf10) is expressed exclusively by tanycytes (hajihosseini et al. 2008 mcn 37: 857-68) , a radial glial-cell like population that lines the floor and ventral walls of the third ventricle. using a line of fgf10-lacz reporter mice, we found that fgf101 beta tanycytes express a panel of neural/stem progenitor markers such as nestin, musashi1, blbp and sox2. fgf101 tanycytes also incorporated brdu under cumulative brdu-labelling paradigms, and formed neurospheres in vitro. to directly test the potential of fgf101ve tanycytes, we lineage-traced them at p28 and p70 by activating the constitutive expression of the marker gene, tomato-dsred, in these cells and their descendants. this was achieved by applying tamoxifen to fgf10-creer-t2::rosa26r-tomato double transgenic mice. after short survival time-points, tomato was detected exclusively in tanycytes, whilst with longer survival time-point, tomato1 cells emerged in the neighbouring parenhcyma. the majority of these were neun1 neurons with fine arborizations. we noted that the newly-generated neurons become associated mainly with hypothalamic nuclei that regulate appetite and energy-balance. moreover, they respond to appetite/energy balance regulating signals such as acute food deprivation and leptin administration. our findings establish fgf101 beta-tanycytes as a putative population of neural stem/ progenitor cells that generate appetite/energy balance regulating neurons in the postnatal and adult hypothalamus. we used an inducible transgenic mouse line (hgfap-creert2) to conditionally label and fate map putative gfap(1) nscs populations in the adult svz and spinal cord (sc), and compare their self-renewal and multipotential properties. we evidence that a population of gfap 1 cells that share the morphology and antigenic properties of svz-nscs reside in the dorsal and ventral aspects of the central canal (cc) throughout the spinal cord. these cells are non-proliferative in the intact spinal cord, but incorporate edu, an s-phase marker following spinal cord injury. multipotent yfp-expressing neurospheres (i.e. which derive from recombined gfap-expressing cells) were successfully obtained from both the intact and injured spinal cord, confirming their early gfap origin. notably, only spheres isolated from the injured spinal cord revealed long-term self-renewal properties resembling those of svz neurospheres. altogether, this work highlights discrepancies in the identity of nscs that reside in distinct regions of the adult cns. our observations however reconcile divergent views on the location and identity of spinal cord-nscs by showing a population of multipotent gfap 1 progenitors with limited self-renewal properties co-existing alongside with multipotent, self-renewing ependymal cells within the spinal cord central canal. during development neural precursors (nps) both divide, to expand the cell population, and produce many different kinds of neurons and glia. this balance appears to be regulated by par complex proteins, which polarize neural precursors and can thereby direct daughter cells for different fates. how par complex proteins are regulated to appropriately polarize nps remains unknown. in recent years, regulation of gene function by micrornas has emerged as an important mechanism during development. using bioinformatics we identified the polarity gene pard3 as a candidate target for microrna219 (mir-219). mir-219 is specifically expressed in the developing central nervous system (cns) of vertebrates and mir-219-deficient zebrafish embryos have a deficit of oligodendrocytes, the myelinating glial cells of the cns. because a disruption in polarity could affect the types of cell divisions that nps undergo, thus altering the balance of cell types that arise, we hypothesize that neural precursor maintenance is regulated by modulation of polarity cues through mir-219. by using an in vitro reporter assay we found that mir-219 can downregulate expression of a luciferase gene fused to the pard3 3 0 utr. reduction of pard3 function in zebrafish embryos suppressed the oligodendrocyte phenotype resulting from loss of mir-219 function and injection of a morpholino oligonucleotide designed to block binding of mir-219 to its pard3 target sequence phenocopied mir-219 knock down. together, these data provide strong evidence that pard3 is a functionally relevant in vivo target of mir-219. to further investigate the role of mir-219 function we tested expression of np, radial glial and neuronal markers in mir-219-deficient embryos. the number of cells expressing sox2, a marker of nps and neural stem cells, was significantly increased upon mir-219 knockdown. concomitantly, mir-219-deficient embryos had a dramatic deficit of radial glia and late born neurons. these data provide evidence for a new mechanism of np regulation, in which mir-219 regulates pard3 levels, thereby regulating the transition of dividing neural precursors to differentiated neurons and glia. in demyelinating diseases such as multiple sclerosis myelin repair activities based on recruitment, activation and differentiation of resident progenitor and stem cells can be observed. however, the overall degree of successful remyelination is limited. it is therefore of considerable interest to understand oligodendroglial precursor cell (opc) homeostasis and maturation processes in order to develop remyelination therapies. mesenchymal stem cells (msc) were shown to exert positive immunomodulatory effects, to reduce demyelination, to increase neuroprotection and to promote adult neural stem cell differentiation towards the oligodendroglial lineage. we here addressed whether msc secreted factors can influence primary opcs in a myelin non-permissive environment. methods: to this end we analyzed cellular morphologies, expression and regulation of key genes/proteins involved in oligodendroglial cell fate and maturation upon incubation with mesenchymal stem cell conditioned medium. results: this demonstrated that msc derived soluble factors promote and accelerate oligodendroglial differentiation, even under astrocytic endorsing conditions. accelerated maturation featured elevated levels of 2 0 , 3 0 -cyclic nucleotide 3 0 -phosphodiesterase and myelin basic protein expression, reduced glial fibrillary acidic protein expression and was accompanied by downregulation of prominent inhibitory differentiation factors such as id2 and id4. conclusions: we thus conclude that besides the previously established immunomodulatory and neuroprotective roles of mscs these cells can also positively influence oligodendrogenesis in the adult central nervous system. in this study, we evaluated the effects of epo and the epo splicing variant (vepo) on murine neurogenesis ex vivo. for this purpose, we analyzed the survival of cultured neural stem cells (nscs) either exposed transiently to recombinant protein, or transduced with lentiviral vectors to achieve stable protein expression. in comparison to non-exposed controls, recombinant epo and vepo enhanced survival of nscs ex vivo (epo: 150 6 25%; vepo: 250 6 60% survival). nscs transduced with pcl20-mscv-epo-and pcl20-mscv-vepo showed also higher viability ex vivo compared to nontransduced nscs (epo: 182 6 4% vepo: 131 6 4% survival). furthermore, pcl20-mscv-epo, but not pcl20-mscv-vepo, increased the proportion of differentiated neurons when compared to non-transduced controls. thus, stable expressed vepo may have rather an effect on nsc survival than on nsc differentiation. our results demonstrate that vepos are neuroprotective in vitro. vepo may serve as therapeutic protein for treatment of neurodegenerative disease associated with impaired neurogenesis such as huntington disease. in the subependymal zone (sez) cytogenic niche of the adult mouse brain neural stem cells drive the continuous generation of new cells, mostly of olfactory bulb interneurons, but also of cells of the oligodendroglial lineage. previous reports have shown that newly-born cells exit the sez and migrate to the adjacent corpus callosum (cc) where they differentiate into oligodendroglial cells. however, the real contribution of these niche-derived oligodendrocyte progenitor cells and oligodendrocytes (nopcs and noligos) to the oligodendroglial population of the cc has not been assessed so far. in this study we used the hgfap-cre ert2 3 rosa26-eyfp double transgenic mice in order to label specifically the progeny of sez-located adult neural stem cells. we found that cells expressing markers of opcs, such as pdgfra and olig2, are constantly generated in the sez and migrate to the proximal fraction of the cc. interestingly though, these cells do not remain in the cc for more than 15 days with their contribution to the total population of oligodedroglial cells remaining stably below 2% even in the ageing brain. moreover, immunostaining for markers of cell cycle revealed that a higher fraction of nopcs undergoes mitosis, when compared with local opcs; hence, they constitute almost 25% of proliferating opcs of the cc. notably, during their transient presence in the cc, nopcs express markers of maturing oligodendrocytes and are incorporated in established local cell structures. when the cc is challenged with a focal demyelinating insult the local and the niche-derived oligodendroglial machineries exhibit differential cell kinetics, nopcs increase their contribution to the total pool of oligodendroglial cells; however, their presence remains transient. in the human and mouse retina m€ uller glia (mg) are well known to undergo gliosis in all major types of retinal diseases -which sometimes may even lead to scar formation due to proliferative gliosis. some studies suggest that in the mouse retina mg derived neuronal regeneration can be stimulated, but only to a very limited extent. here, we started to find out, if conditional immortalization might stimulate mg derived proliferative gliosis and /or neuronal regeneration. others and we reported that in the juvenile mouse retina, after retinogenesis is finished, some m€ uller glia shift from a quiescent differentiated state into a proliferative state upon damage of retinal explants ex vivo. we now observed that this process is differentially inducible by mitogens like egf (epidermal growth factor). edu (s-phase marker) pulse chase experiments revealed a tremendous increase in mg proliferation within the first two days, a peak of edu positive cells at day 4 (14 122 sem, n 5 4) and a massive decrease until day 6 (4 120.4 sem, n 5 4) per 400 mm of a central retina section. next, we used transgenic mice with tightly and temporally controlled expression of the proto-oncogene sv40 large t-antigen (csv40lt). it is well reported that csv40lt binds several proteins including the tumor suppressor p53 and retinoblastoma and bypasses cell cycle checkpoints. induction of the csv40lt for 6 days ex vivo led to an overall increase in proliferation compared to control. the number of edu1 cells was 8.5 fold increased (csv40lt: 23 126 sem, n 5 4; control: 3 120.16 sem, n 5 4; p our results so far suggest that induction of csv40lt not only overcomes the proliferative restriction of m€ uller glia but also maintains its progeny in the cell cycle over extended period of time. surprisingly, major parts of the generated cell progeny formed gliotic cell clusters, which were all located within the boundaries of the retina. further, we present data of successful m€ uller glia isolation at high purity by fac-sorting. in our current and future work we study the m€ uller glia and its derived progeny to find out the underlying mechanisms that enable neuronal regeneration and prevent gliotic scar formation. in mammals, regeneration capacity of the central nervous system (cns) is considered too low to recover the neurological functions after traumatic injury. in fishes and amphibians, however, the spontaneous regeneration is vigorous enough to complete the anatomical reconstruction and functional recovery even after severe damage of cns, in which the ependymal cells play the central role to exhibit proliferation, epithelial-mesodermal transition, cell migration, support of regenerating axons and neurogenesis to contribute to recovery from injury. these positive roles of the ependymal cells observed in fish and amphibian damaged cns may be good candidates for treating mammalian cns injury. in this study, we attempted to control the activity of the ependymal cells in the adult rodent spinal cord by virus-mediated gene transfer for imitating the reactions that are observed in the ependymal cells of fish and amphibian damaged cns. we introduced the gene known to be expressed in both the ependymal cells and neural stem cells (nscs) and found the proliferation of ependymal cells. in this case, the ependymal cells expressed glial fibrillary acific protein suggesting that this gene regulates proliferation and differentiation into astrocytes. in addition, introduction of another gene, also known to be expressed in the ependymal cells and nscs, caused cell proliferation without differentiation tendency. these findings indicate that the cell fate of the ependymal cells in the adult rodent spinal cord can be modulated by artificial intervention. future studies will clarify whether cell fate modification in the ependymal cells can contribute to anatomical reconstruction and functional recovery in the adult mammalian damaged spinal cord. methods neural stem cells are isolated from different parts of the human gastrointestinal tract, and also from different compartments (muscle, submucous and mucous layer) using enzymatical digestion approaches. generated neurospheres were screened for neuronal, glial, neural stem cell and pluripotency markers, prior and after differentiation. additional experiments were performed where neurospheres were either co-cultured with tissue blocks from different parts of the brain, or transplanted into brain slices from the rat. results neurospheres could be generated from all areas investigated and differentiated on glas coverslips. nestin and musashi-1 could be found in all neurospheres. moreover sox-10, nanog and oct-4 could be found in the differentiating cultures. after differentiation tubulin and pgp 9.5 positive neurons could be found. transplantation experiments showed that cells from the transplanted spheres migrated into the brain slices and differentiated into neurons or glial cells. neurospheres co-cultured with tissue blocks from cortex, cerebellum or hippocampus showed a significant higher outgrowth than neurospheres cultured without any brain tissue. conclusions in general, the ens is a suitable tissue for the isolation of neural stem cells in the individual patient and might so be an appropriate source for autologous neural stem cells. neurogenesis. at the end of embryogenesis, oligodendrocytes and astrocytes appear during the process of gliogenesis. although in the last decades diverse extrinsic and intrinsic factors involved in these developmental processes were identified, our understanding of detailed mechanisms is still comparatively low. in order to increase the knowledge about the cellular and molecular mechanisms that underlie central nervous system development, transfection of nscs showed to be a promising method to understand the function of specific genes and proteins. to overcome the well-known difficulties to transfect cells of the central nervous system, we improved the electroporation conditions to achieve high efficiency transfection and survival rates for embryonic and adult nscs isolated from mouse forebrain structures. one day after electroporation transfection and viability rates of around 80% were achieved in murine nscs (bertram et al. j neurosci meth 2012). due to our interest in differentiation and maturation of oligodendrocytes we are currently improving the transfection conditions of oligodendrocyte precursor cells (opcs), since a sufficient protocol for the transfection of opcs derived from neonatal rat cortices is currently not established. the opcs are isolated from rat cortical tissue by a shaking protocol and transfected with the 4d-nucleofector (lonza). when we used the same pulse protocol that successfully works on nscs, oligodendrocytes were sensitive to the transfection process, and many cells died or did not undergo normal differentiation. this demonstrates that other pulses are needed to electroporate opcs. in cooperation with lonza we tested several recommended pulses to increase the transfection and survival rates of opcs. a dramatic increase of the viability rate has already been achieved. in further experiments the pulse protocol will be improved to obtain transfection rates above 20%. by establishing this transfection protocol we want to accomplish transfection and viability rates comparable to murine nscs. this improved protocol for the transfection of sensitive opcs will allow for reliable studies of gene function by over-expression or knockdown experiments, and will enhance our understanding of cell biological mechanisms important for oligodendrocyte development. recent studies demonstrate that astroglial cells isolated from non-neurogenic brain regions have the potential to be reprogrammed into functional neurons through forced expression of transcription factors known to instruct neurogenesis. based on our previous studies on the potential of the neurogenic gene cend1 in directing neural stem/precursor cells (nsc) to exit the cell cycle and acquire a neuronal phenotype, in parallel with evidence demonstrating activation of cend1 expression by genes of the neurogenin family, we explored the combined effect of cend1 and neurogenin-2 on their reprogramming potential on postnatal cortical astrocytes. to this end, forced expression of either cend1, neurogenin-2 or both, resulted in an important increase of two morphologically distinct subpopulations of gfapcells with elongated morphology, that strongly expressed the radial glial marker glutamate transporter glt-1. further characterization revealed that a subpopulation of these cells differentiates towards the neuronal lineage, as they were exhibiting a differentiated neuronal morphology and expressed b-iii tubulin, as well as neuronal subtype-specific markers, including gaba and th. further analysis using long time live cell imaging and brdu cumulative labeling revealed that the majority of cend1-transduced astrocytes undergo 1-2 cell divisions before differentiating to gabaergic neurons, whereas most ngn2 1 astrocytes directly transdifferentiate giving rise to th 1 neurons. additionally, only in the doubletransduced cultures, cend1 1 /ngn2 1 astrocytes seemed to take a step back forming colonies of small round glast 1 /nestin 1 cells, detected 24h following transduction. a day later, these colonies grew as three-dimensional spheres of high proliferative potential attached to the culture dish. when 'astrospheres' were isolated and cultured under nsc conditions, they grew as neurospheres and were propagated for over ten passages. importantly, as soon as 'astrospheres' were cultured in the absence of growth factors, they differentiated into neurons, astrocytes and oligodendrocytes, implying that they possess neural stem cell properties. at the moment we are performing electrophysiology recordings to assess the functionality of cend1derived neurons in vitro, while studies are in progress to explore the potential role of cend1 and ngn2 on astrocytic reprogramming in vivo following cortical brain injury. average membrane potential (v m ) was 277 mv and input resistance (ir) was 92 mx, while dcx 1 or map2 1 cells expressing fast activating outwardly rectifying k 1 currents (ka), and delayed outwardly rectifying k 1 currents (kdr) were defined by a v m of 272 mv and high values of ir (1943 mx). ng2 1 cells with a complex current pattern expressed inwardly rectifying k 1 (kir) currents, in addition to kdr and ka currents, and their v m was 276 mv and ir was 383 mx. the electrophysiological analysis revealed that the inhibition of the wnt signaling pathway significantly increased the incidence of cells with a complex current pattern, while the number of cells with the marked expression of outwardly rectifying k 1 currents declined. wnt signaling inhibition also resulted in increased kir and decreased ka current amplitudes. 4oht-treated cells were also hyperpolarized when compared to controls. immunocytochemistry using antibodies against map2 and dcx showed that neuronal progenitors in 4oht-treated cultures were less developed, having fewer and less branched processes. in summary, our data imply that the canonical wnt pathway in the neonatal svz promotes nsc differentiation into cells with neuronal characteristics. gacr p303/12/0855; p304/12/g069. adult neurogenesis plays a critical role in the overall health of, and repair processes occurring within the nervous system. while a great deal of information has been gained about the elements of the neurogenic niche and the factors that mediate neurogenesis, many unanswered questions remain. specifically, studies suggest that microglia have a significant impact on adult neurogenesis, but no unifying theory exists to explain their exact role in this process. in order to determine if microglia play a pivotal role in adult hippocampal neurogenesis and to examine the mechanisms through which microglia exert their effects on the hippocampal neurogenic niche, we employed a transgenic mouse model that allows for the selective ablation of microglia in the central nervous system (cns), the cd11b-herpes simplex virus thymidine kinase (cd11b-hsvtk) mouse. using this system, we removed microglia from the adult hippocampal neurogenic niche and evaluated the effects of this manipulation on neurogenesis and hippocampal homeostasis, including immunohistochemical analyses of stem cell survival and proliferation, as well as analyses of neuroblast development, migration and electrophysiological activity. taken together, these studies help to provide a better understanding of the factors governing adult neurogenesis and further our knowledge about the increasingly diverse role of microglia in the cns. the current assumption is that ependymal damage, caused by viral infections may result in reactive gliosis in the subventricular zone (svz), leading to sgns. however, as the svz is currently recognized as one of the neurogenic niches in the adult human brain, and svz astrocytes have been identified as neural stem cells (nscs), the assumption of sgns being reactive astrocytes has to be reconsidered. therefore, we have immunophenotyped the cell types present in these structures in the brains of individuals of various ages. in addition, we assessed nodule incidence in the svz in cases infected with hiv, where cells in the svz are likely to be to be affected. we found sgns in almost 90% of all cases, independent of age or hiv infection. we determined that cells in the sgns express adult nsc markers, rather than markers for reactive astrocytes. we hypothesized that direct contact of the nscs with cerebrospinal fluid (csf) may induce nodule formation. indeed, we could show that human ventricular csf stimulated the proliferation of human nscs in culture. from our data, we conclude that sgns most likely are formed upon ependymal damage in response to exposure to csf and represent local expansions of the svz. . in experimental murine model of demyelination, the repair capacity is robust and sufficient, even if it might decrease with aging. one major therapeutic goal in ms is to favour endogenous myelin repair, to prevent neurodegeneration and disability progression. using a transcriptomic approach on pure populations of opcs, we try to identify mechanisms specifically involved in opcs activation. we set up a method to isolate a purified population of opcs, by fluorescent-activated cell sorting from pdgfar::gfp mouse brains. we created the gene expression profile of neonatal opcs, adult opcs and adult oligodendrocytes (ols) in control conditions and of adult opcs in cuprizone-induced demyelinating conditions. we analyzed the transcriptomic profile of adult opcs, neo-natal opcs and ols, and identified more than 1000 differentially expressed genes. after demyelination, we identified more than 2000 adult-opc-specific genes that are either up-or down-regulated compared to control conditions. preliminary analysis suggests that adult opcs are more "mature" than neonatal opcs and partially revert to a more immature profile after demyelination. bio-informatic analysis is ongoing, with the perspective of identifying key candidates for myelin repair capacity, then develop functional experiments to validate their involvement in remyelination. in parallel, we plan to compare the transcriptomic profile of adult opcs from aged versus young mice, to get insight into age-related decrease of repair efficacy. question: after damage, astrocytes are activated and exert specific roles for central nervous system (cns) repair. among these, astrocytes within the injury site may represent a novel source of cells with stem cell potential. in this study, we used sedimentation field-flow fractionation (sdfff) method to rapidly sort well preserved astrocyte subpopulations and we identified stem cell properties within one of these subpopulations. methods: cells were collected from newborn rat cortex, separated mechanically and subjected to sdfff. cell fractions obtained were analyzed by immunocytofluorescence using antibodies against specific epitopes: glial fibrillary acidic protein (gfap), o4, b iii tubulin, and cd68, labelling respectively astrocytes, oligodendrocytes, neurons, and microglial cells. then, proteomic analysis was performed on astrocyte subpopulations and their behaviour in culture was analyzed, particularly under culture conditions usually allowing neurosphere development. results: three main fractions (f1, f2, and f3) were isolated and compared with the total eluted population (tp). after one week in vitro, tp and f2 derived cell cultures contained respectively 74.5% and 11.9% of gfap expressing astrocytes while this percentage was extremely high in f1 and f3 derived cell cultures (95.6% and 98.0% respectively). f3 derived cells displayed higher migratory capacities than those of f1. proteomic analysis of f1 and f3 derived cells indicated a high expression of vimentin in f3 derived cells. in the presence of epidermal growth factor (egf), f3 derived cells formed neurospheres containing cells expressing gfap and nestin (figure) that, when placed in appropriate conditions, were able to generate the three major cell types of the cns (neurons, astrocytes and oligendrocytes). moreover, colony-forming cell assay in a collagen-containing semi-solid matrix allowed in the presence of egf the formation of large colonies. in addition, 7 days after cortical lesion in adult rats, cells presenting similar stem properties were obtained after sdfff cell sorting. conclusions. our study demonstrates that sdfff enables the isolation of almost pure astrocyte subpopulations after one week in vitro. in addition, after sdfff cell sorting, we isolated an astrocyte subpopulation expressing vimentin and displaying the self-renewal and multipotency properties of neural stem cells. our data support that sdfff is an efficient tool to explore the different astrocyte subpopulations of the cns, particularly those involved in cns repair. experimental studies have shown that loss of myelin results in axonal loss and disability. so, finding of an expandable and autologous source of myelin-forming cells to enhance remyelination is required. induced pluripotent stem cell-derived neural progenitor cells (ipsc-npcs) have been developed recently. the remyelination potential and safety of these cells still remained to be well-addressed. the main goal of this study is to characterize mouse ips-npcs in vitro and in vivo after transplantation. we used embryonic mouse neural progenitor cells (mnpcs) as control and characterized mouse ips-npcs for their expression of major markers of immature or mature cells by immunostaining. rt-pcr was performed to analyze expression of nestin, olig2, b3-tubulin, olig1, sox10, and nkx2.2 mrnas. furthermore, to investigate the fate of these cells in vivo, we injected the lysolecithin to induce focal demyelination in the spinal cord of adult nude or shiverer mice. we transplanted cells in the lesion site 2 days after demyelination. animals were sacrificed 2 days, 1, 2, 6 and 8 weeks post transplantation to assess survival and differentiation of the grafted cells. our preliminary results showed that mips-npcs similar to mnpcs were nestin1, ki671, olig21, b3-tubulin1 but o4-and map5-. mips-npcs, were more proliferative than mnpc. moreover, mips-npc expressed all mrna transcripts except sox10. a first line of mips-npcs, was transplanted in the newborn shiverer:rag forebrain or the demyelinated spinal cord of immunosuppressed shiverer mice with a constant failure in terms of cell survival beyond 2 days of transplantation, highlighting the fragility of some of the reprogrammed cell lines. cells of a second line of mips-npcs were transplanted in nude mice to be sacrificed at 1, 2 and 6 weeks. an additional serie of transplantation was performed in the demyelinated shi:rag spinal cord. data at 1 and 2 weeks indicate excellent survival and integration of the mips-npc at both time points. some of the grafted cells expressed gfap, olig2 and ki67 (few only) and so far, no tumors were observed at these timepoints. immunohistochemistry with other markers and analysis of 6 weeks post transplantation animals, are in progress. our preliminary results show that although mips-npcs have very similar immature phenotype of mnpcs in vitro, they may have different survival capacities in vivo. future studies will reveal whether transplanted cells which showed short-term survival after grafting are capable of differentiation into myelin-forming cells. . however these putative stem cells have only been partly characterized in vitro and their therapeutic potential for cns repair has not been addressed. we cultured adult mouse drg cells in sphere-forming conditions. in parallel, we derived spheres from adult spinal cord of the same animals and compared their properties. this last tissue contains well-described stem/progenitor cells whose repair capacities have already been proven for spinal cord injury but not for primary demyelination. in vitro, regardless of their origins, all tissues generated self-renewable spheres up to 10 passages. rt-pcr and immunocytochemistry revealed that sphere-forming cells actively proliferate and express neural stem cells markers. drg spheres were multipotent: as they differentiated into schwann cells, neurons and myofibroblasts, whereas spinal cord spheres differentiated into astrocytes, neurons and oligodendrocytes. we then compared the integration, differentiation potential and remyelination capacity of primary sphereforming cells isolated from actin-gfp transgenic mice after their engraftment in two distinct environments: the adult nude lpc-demyelinated spinal cord and the developing newborn shiverer forebrain. both cell types survived but with distinct integration and differentiation patterns. in the mbp deficient mutant, spinal cord cells exhibit a substantial tropism toward white matter tracts and differentiated mainly into myelinating oligodendrocytes while drg cells were largely distributed in the parenchyma in close association with blood vessels and differentiated in vascular smooth muscle cells. surprisingly, after focal demyelination, in the adult spinal cord, drg cells migrated extensively towards the lesion and differentiated into remyelinating schwann cells whereas adult spinal cord cells migrated less efficiently, and gave rise to oligodendrocytes and a large population of astrocytes. our study reveals that both cell types present different but promising potential for cns remyelination. funding by ela foudation, dim nerf, arsep foundation. ischemia-induced progenitor cell proliferation is a prominent example of the adult mammalian brain's ability to regenerate injured tissue resulting from pathophysiological processes. a key locus of regeneration is the neurogenic niche located in the subgranular zone of the dentate gyrus (sgz). within the sgz, radial glia-like progenitor cells generate neural precursor cells which migrate into the granule cell layer (gcl) of the dentate gyrus, mature, and integrate into the hippocampal synaptic network. in order to better understand and ultimately exploit the cell signaling mechanisms that regulate ischemia-induced proliferation in the sgz, we tested the role of the p42/44 mitogen-activated protein kinase (mapk) cascade effector ribosomal s6 kinase (rsk) in this process. using the endothelin-1 ischemia model in c57bl/6 mice, we report that intrahippocampal cerebral ischemia triggered rsk activation in sgz progenitor cells. further, microinjection of a rsk inhibitor significantly reduced ischemia-induced sgz progenitor cell proliferation. using the neurosphere assay, we also show that sgz-and subventricular zone (svz)-derived adult neural stem cells (nsc) exhibit a significant reduction in proliferation in the presence of rsk and mapk inhibitors. taken together, these data indicate that rsk functions as a cell-autonomous regulator of ischemia-induced progenitor cell proliferation. here we demonstrated that the inpcs not only possess npc-specific marker genes, but also have qualitites of primary brain-derived npcs (wt-npcs), including tripotent differentiation potential, mature neuron differentiation capability and synapse formation. moreover, the mature neurons derived from inpcs exhibit significant physiological properties, such as potassium channel activity and generation of action potential-like spikes. importantly, besides survival, the transplanted inpcs exhibit the migratory property responding to inflammatory stimulation in vivo. these results suggest that directly reprogrammed inpcs closely resemble wt-npcs, which may suggest an alternative strategy to overcome the restricted proliferative and lineage potential of induced neurons (incs) and broaden applications of cell therapy in the treatment of neurodegenerative disorders. the pathogenesis of neurodegenerative disorders, including human immunodeficiency virus (hiv)21 associated neurocognitive disorders (hand), is exacerbated by an imbalance between metalloproteinases (mmps) and their inhibitors, tissue inhibitors of metalloproteinases (timps). as the timps exhibit diverse non-classical functions including anti-apoptotic effects, the induction of timp-1 in neuroinflammatory conditions likely serves multiple roles in addition to modulating mmp activity. our work demonstrates differential timp-1 expression in acute versus chronic activation of astrocytes and hiv-1 associated dementia (had) brain tissues. the timp-1 promoter harbors five consensus ccaat boxes. ccaat enhancer binding protein (c/ebp)b levels were elevated in brain specimens from hiv-1 patients and this transcription factor regulated astrocyte timp-1 expression. hiv-relevant stimuli increased c/ebpb expression in human astrocytes and localized the factor to the nucleus. overexpressing c/ebpb in astrocytes increased timp-1 promoter activity, mrna and protein expression, while knockdown of c/ebpb decreased timp-1 mrna and protein expression. erk1/2 activation is critical for il-1b-mediated astrocyte timp-1 expression and p38k activation contributes to il-1b-induced astrocyte timp-1 and c/ebpb expression. these data suggest that erk1/2 signals downstream of c/ ebpb to facilitate il-1b-induced astrocyte timp-1 expression. the role of astrocyte-timp-1 as a neurotrophic factor was examined. interestingly, cotreatment with timp-1 protected neurons from apoptosis and reversed neuronal morphological changes induced by staurosporine (sts) and hiv-1 ada virus. further, the anti-apoptotic effect was specific to timp-1 and partially independent of mmp-inhibition. additionally, timp-1 modulated the bcl-2 family of proteins and inhibited opening of mitochondrial permeability transition pores induced by hiv-1 or sts. together, these findings describe a novel function, regulatory mechanism and direct role of astrocyte-timp-1 in neuroprotection suggesting its therapeutic potential in had and possibly in other neurodegenerative diseases. mounting evidence and our pervious study has also confirmed that the axon outgrowth inhibition receptor ngr was also expressed on microglia, and regulated cell adhesion and migration behavior in vitro. however, microglia has been proposed to play a pivotal role in the neuroinflammation through expressing a range of proinflammatory enzymes and proinflammatory cytokines under pathological stimulus. these findings initiate the possibility of nogo/ngr signal on mediating neural inflammation processes during cns injury beside neurite outgrowth inhibition. in the present study, we found that nogo-p4, a 25-aa core inhibitory peptide sequence of nogo-66, was able to induce microglia expressing cox-2, inos, as well as releasing proinflammatory cytokines including il-1b, no and pge2, which could been reversed by nep1-40 or pi-plc treatment. microglia stimulated with nogo-p4 increased the phosphorylation lever of signal transducer and activator of transcription 3 (stat3). the activation of stat3 further mediated the promotion of nogo-p4 on microglia expression proinflammatory factors. furthermore, administration nep1-40 was capable to attenuate the inflammation response in a mouse spinal cord compression injury model by reducing the expression of several proinflammatory mediators, as well as attenuating the activation of stat3. taken together, our results demonstrated, for the first time, that nogo-66 may directly take part in cns inflammation process by influencing microglia expressing proinflammatory factors, which was related to ngr /stat3 signal pathway. these results imply that the interaction of nogo-66 with ngr expressed on microglia may participate in diverse cns diseases related to neural inflammation, including trauma, stroke, and neurodegeneration diseases, etc. our results indicate that ccl-1 is one of the key molecules in pathogenesis and ccl-1/ccr-8 signaling system can be a potential target for drug development in the treatment for neuropathic pain. demyelination plays a central role in the pathophysiology of multiple sclerosis (ms) not only because it disrupts nerve conduction, but also because of effects that compromise axonal survival. there is a growing consensus that any effective treatment for ms must include a component that restores or enhances remyelination by endogenous oligodendrocyte progenitor cells (opc). however achieving this goal requires a far better understanding of why remyelination fails in the first place. we now report that fgf9 is selectively up regulated in demyelinating ms lesions and acts to inhibit (re)myelination in vitro. to explore the mechanism involved we compared the activity of fgf9 to that of fgf2, another member of the fgf family with well characterised effects on opc differentiation and myelination. fgf2 acts directly on opc to stimulate proliferation, while at the same restricting their differentiation into mature oligodendrocytes. in contrast, fgf9 is neither an opc mitogen, nor is it able to inhibit opc differentiation directly. however, opc differentiation and myelination in myelinating cultures were inhibited by supernatants harvested from fgf9 treated astrocytes, even in the presence of an excess of fgf9 neutralising antibody. microarray studies were performed to identify astrocyte derived factors responsible for this inhibitory effect. this approach identified several potential candidates that were significantly up regulated by fgf9 in astrocytes. these included lif, a member of the gp130/il6 cytokine family that is generally described as having pro-myelinating effects, although our own studies indicate when present in excess relative to other gp130/il6 family members it will inhibit myelination in vitro. our experiments demonstrate that fgf9-mediated signal transduction in astrocytes disrupts their ability to maintain a growth factor milieu that can support myelination. been shown that inflammation is primarily mediated by macrophages that are recruited to the tissue. similarly, a recent study demonstrated reactive microgliosis in the hypothalamus of mice fed a high-fat diet. in the cns, however, the relevance of microglial activation in response to a high fat diet remains unclear. we aim to determine if microglia activation occurring in response to overnutrition is a cause or consequence of dysregulation of energy homeostasis and obesity. to determine if the absence of microglia (and therefore microglia-mediated inflammation) may influence the metabolic response to a high fat diet, we used a transgenic mouse model, the cd11b-hsvtk mouse (tk), which allows for an inducible, specific ablation of microglia in response to treatment with the nucleoside analog, gancilovir (gcv). mice were treated with gcv for 4 weeks during which time they were maintained on a diet high in fat (60%) or a respective control diet. body weight, food intake, locomotor activity and energy expenditure were measured and compared to wildtype mice. analyses revealed alterations in the metabolic phenotype of mice fed both hfd and control diet in the absence of microglia cells. our findings point towards a physiological role of microglia in energy homeostasis, the study of which will be the focus of further experiments. interleukin-10 (il-10), a cytokine classically related with anti-inflammatory and protective functions in the central nervous system (cns), has been reported to be produced by astrocytes and microglia in different neurodegenerative and neuroinflammatory conditions. in order to study the specific role of il-10 in the cns, we have created a new transgenic mouse with astrocyte-targeted production of il-10 (gfap-il10tg). the aim of this study was to evaluate if production of this cytokine in the cns has any effect on the phenotype of glial cells. for this purpose, coronal cryostat free-floating sections from the brain of both adult transgenic mice and their corresponding wild-type (wt) littermates, were processed for the study of astrocytes using gfap immunohistochemistry and microglia using antibodies against iba1 and several markers commonly related to the activated phenotype of these microglial cells, such as cd16/32 (fc receptor), f4/80, cd11b, cd206, cd150 and mhc-ii. our results showed that astrocyte-targeted production of il-10 induces an increase in the area covered by gfap immunolabelling. microglial cells in these gfap-il10tg animals displayed also morphological changes in specific areas of the brain, including the hippocampus, cortex and cerebellum, characterized by coarser processes and a notable enlargement of the cell body. distinctively, in the hippocampus, microglial cells adopted an elongated morphology parallel to the dendrites of pyramidal neurons. in addition, in the aforementioned areas, microglia exhibited a statistically significant increase in iba1, cd16/32, f4/80 and cd11b markers and "de novo" expression of cd150, whereas no detectable levels of either cd206 or mhc-ii was found. in conclusion, these results indicate that in specific areas, glial cells are influenced by the presence of astrocyte-targeted il-10. further studies are necessary to evaluate whether after injury these transgenic animals will present changes in the microglial and astrocyte activation that can influence the evolution of the lesion. networks. brain-resident macrophages, microglial cells were considered ''resting,'' but it has recently become evident that they actively sense neuronal microenvironment with their motile and ramified processes. they develop specific activities (cytotoxic, neuroprotective or phagocytic) depending on brain molecular context. for example, they contribute directly to the development of neuronal networks by eliminating or maintaining synapses in an activity-dependent manner. morphofunctional plasticity of microglial cells is finely tuned in order to keep brain homeostasis and to ensure such developmental activity. like macrophages at the periphery, microglial cells indeed adopt m1 or m2 phenotypes, distinguishable through their pattern of cytokine expression and of specific cell surface markers, a m1 phenotype being more pro-inflammatory while a m2 phenotype is more related to phagocytosis. linoleic acid (la, 12:2n-6) and alpha-linolenic acid (ala, 18:3n-3) are essential fatty acids that cannot be synthesized de novo by mammals and have to be provided through the diet. they are precursors of arachidonic acid (aa; 20:4n-6) and docosahexaenoic acid (dha; 22:6n-3) respectively which are key structural components of brain cell membrane phospholipids and precursors of bioactive lipid messengers involved in the regulation of inflammation and microglial activity. most aa and dha accumulate in the brain during the perinatal period via placenta and milk. we previously demonstrated that depleting the diet in n-3 pufas from the first day of gestation alters some neuronal functions of the offspring at adulthood. we here hypothesized that this is due to an alteration of microglial mrophofunctional activity in the developing brain. to test this hypothesis, mice were submitted to a diet deficient or not in ala throughout gestation and lactation. microglial morphology, phenotypes and functions were determined in the brain of pups at post-natal day 21 (pnd21). we show that dha levels is decreased in membrane phospholipids of microglia. in addition, microglial cells from mice fed with an ala deficient diet present a drastic increase of the cd36 marker expression combined to a dysregulated cytokine/chemokine expression profile and an altered phagocytic activity. all together, our results show that dietary n-3 pufas deficiency is likely to impair brain innate immune system activity at pnd21. the outcome of such microglial impairment on brain function is discussed. in recent years, one of the most promising therapeutic means for ad was thought to be immunization with specific antibodies against ab. several therapeutic antibodies were developed, however, most of the clinical trials were canceled due to unexpected deaths and neuroinflammatory responses in some patients. the causes and mechanisms of such responses are currently only partially understood. we have recently raised monoclonal antibodies against oligomeric forms of ab and were investigating whether these antibodies would prevent the neurotoxicity of ab oligomers in primary neuronal-glial cerebellar granule cell (cgc) cultures. antibodies were not directly toxic to cgcs when applied at concentrations relevant to concentrations reported to be in blood serum. however, surprisingly, the antibodies when in complexes with ab oligomers or firbrils dramatically increased the neurotoxicity of ab. similar effects were also observed with antibodies to other oligomeric proteins: hamster polyomavirus major capsid protein vp1, human metapneumovirus nucleocapsid protein and measles virus nucleocapsid protein strongly potentiated the neurotoxicity of their antigens. the neurotoxicity of antibody-oligomeric antigen complexes was abolished by removal of the fc region from the monoclonal antibodies or by the removal of microglia from cultures, and was accompanied by inflammatory activation and proliferation of the microglia in culture. in conclusion, we find that immune complexes formed by ab oligomers or other oligomeric antigens and their specific monoclonal antibodies can cause neuronal death in primary neuronal-glial cultures via fc-dependent microglial activation. the results suggest that therapies resulting in antibodies to oligomeric ab or oligomeric brain virus proteins should be used with caution or with suppression of microglial activation. additionally, if endogenous antibodies contribute to neuronal loss in ad or viral encephalitis, suppression of microglial activation may be therapeutic. antidepressants have often been recommended for potential treatment of chronic pain, especially for neuropathic pain caused by nerve damage. it is known that nerve injury-induced activation of microglia and astroglia can be a cause for the development of neuropathic pain. recent data indicate that abnormal neuronal activity mediated by glia activation may influence the level of neuroimmune signaling molecules in chronic pain. however, it remains unclear whether the mechanism underlying antidepressant effects is related to glia activation. this study investigated the potential pain-relieving properties of milnacipran and venlafaxine, selective serotonin/noradrenaline reuptake inhibitors (snri), in neuropathic pain and attempted to resolve how these antidepressants affect the factors synthesized by the activated glia in neuropathy. chronic constriction injury (cci) was performed in wistar rats by loose ligation of the sciatic nerve. after a single intraperitoneal (i.p.) injection of antidepressants, the responses to mechanical and thermal stimuli in neuropathic rats were evaluated 7 days after cci by the von frey test and the cold plate test, respectively. to confirm the influence of antidepressants on microglia and astroglia, western blot analysis of iba-1 (microglia marker) and gfap (astroglia marker) in the spinal cord and dorsal root ganglions (drg) was performed. the experiments were carried out according to the iasp and local bioethics committee rules. our findings confirmed that the administration of milnacipran (20 mg/kg) and venlafaxine (10 mg/kg) elicited antiallodynic and antihyperalgesic effects in cci-exposed rats. western blot analysis showed that milnacipran elevated the level of iba-1 protein in the spinal cord (but not in drg) in cci-exposed rats. venlafaxine decreased the cci-induced iba-1 protein level in the spinal cord and in drg. we observed no changes in gfap in the spinal cord and drg after milnacipran and venlafaxine administration. these results provide a substantial evidence that both used antidepressants belonging to snri attenuated allodynia and hyperalgesia in an animal model of neuropathic pain, although their effect on microglial cells and astrocytes was different, namely, milnacipran increased the microglia activation, in contrast to venlafaxine. this effect can be explained by comparing the effects of these drugs during their repeated application, and relevant experiments are currently in progress. myeloid cells can respond to intra-and extracellular danger/stress signals by inflammasome-mediated activation. this process consists of two steps: toll-like receptor-induced production of pro-il-1b followed by inflammasome-mediated cleavage and secretion of bioactive il-1b. inflammasome-mediated activation is strictly regulated by expression of regulatory proteins and by inhibitory processes like autophagy. recent studies describe that microglia are markedly different from other myeloid cells. they originate from a separate progenitor, are chronically exposed to the neural microenvironment and their activation may be regulated differently. the objective of this study was to determine the expression profile of inflammasome components in primary microglia and to compare inflammasome-mediated microglial activation and its regulation with other myeloid cells. primary microglia, bone marrow-and blood-derived macrophages (bmdms) of adult rhesus macaques of the same donor were profiled for all nod-like receptors (nlrs), inflammasome-associated adaptor proteins, caspases, and regulatory proteins by qpcr. inflammasome function and kinetics were assessed by exposing lps-primed microglia to atp, silica or msu (danger-associated molecular patterns: damps) followed by measuring the transcription of pro-il-1b-encoding mrna and the secretion of il-1b. primary microglia expressed nlrs (nalp1-3, nod1/3/4, aim2, ipaf, naip), adaptor proteins (asc), caspases (1/3-5/7/8), and regulatory proteins (a20), and the expression profile closely resembled that of bmdms. priming of microglia with lps induced high levels of pro-il-1b-encoding mrna, but il-1b protein was only secreted in response to subsequent stimulation with various damps. interestingly, in lpsprimed bmdms the window for inflammasome-mediated activation was 4-8 hours, while lps-primed microglia remained sensitive for inflammasome-mediated activation for at least 20 hours. in conclusion, primary microglia express multiple inflammasome components closely resembling the expression profile of bmdms and they can be induced to form functional inflammasomes. importantly, primed microglia remain sensitive to inflammasome-mediated activation for much longer than other macrophages. we will present data on whether this reflects a difference in negative regulation of the inflammasome, or differences in autophagy or apoptosis sensitivity. objective: the susceptibility of the aging brain to neurodegenerative diseases may in part be attributed to the intrinsic senescence of the microglia. cellular aging or senescence is linked to telomere shortening/dysfunction. evidence is accumulating that aging microglia show morphological changes, referred to as microglial dystrophy, which may be accompanied by accumulation of the iron-binding protein, ferritin. here, we investigated the effect of telomere shortening on microglial morphology, numbers, and activation state in telomerase deficient (terc -/ -) mice, a model of premature aging due to shortened telomeres. methods: male terc 1/1 mice and 3 rd generation terc -/mice, which show a clear aging phenotype, were perfusion-fixed with 4% paraformaldehyde. brains were processed into 60 lm thick horizontal sections. every fourth section was used for stereological estimation of cd11b 1 microglia in the molecular layer of the dentate gyrus by the optical fractionator method. sections were also stained for the microglial markers iba-1 to monitor microglial morphology, cd45 and cd68 to study microglial activation, and ferritin to study microglial aging. further sections were stained for the apoptosis marker cleaved poly (adp-ribose) polymerase (cparp). results: unlike resting microglia in littermate terc 1/1 mice, which display thin, finely branched processes, microglia in terc -/mice exhibited partial retraction and hypertrophy of processes. microglial cd45 and cd68 immunoreactivity were similar in terc 1/1 and terc -/mice. absolute numbers of mac-1 1 microglia were significantly reduced in young and old terc -/versus terc 1/1 mice. an increased microglial density in old terc -/versus terc 1/1 mice could be attributed to a volume reduction of the dentate gyrus. we also observed an increased number of cparp 1 cells in old terc -/versus terc 1/1 mice. this increase was most prominent in the sub-granular zone, an active site of neurogenesis, but also occurred in the molecular layer of the dentate gyrus and might reflect microglial apoptosis. we observed no difference in ferritin staining in terc -/versus terc 1/1 mice. conclusion: our findings suggest that telomere shortening impairs microglial capacity for self-renewal. surprisingly, changes in microglial morphology in terc -/mice occurred without differences in cd45 and cd68 expression. ongoing studies will show if increased numbers of microglia in terc -/mice co-express cparp or ferritin as evidence of increased microglial aging. tetrahydrocannabivarin (d9thcv) and cannabidivarin (cbdv). we have developed a series of new cannabinoid quinones, among them the cbg quinone (vce-003) that shows pparg and cb2 receptors agonism. in addition we have found that vce-003 activates the nrf2/are pathway in neuronal cell lines. in the present study, we investigated the therapeutic potential of vce-003 in the experimental autoimmune encephalomyelitis (eae) model of multiple sclerosis (ms) by immunization con mog 33-55 . vce-003 (5mg/kg ip, daily) was administered to susceptible c57/bl6 mice at the onset of symptomatology. clinical score and weights of mice were daily recorded until the day of sacrifice (28 days post-immunization). vce-003 treatment delayed the onset of disease and ameliorated the symptomatology. histological analysis of spinal cord of eae mice treated with vce-003 showed decreased microglia reactivity and reduced cellular infiltrates, in particular cd4 1 t lymphocytes. double labeling with neurofilament and the myelin protein, rip indicated that vce-003 diminished the axonal damage. demyelination was evaluated by luxol fast blue labeling. changes in the expression of several cytokines, adhesion molecules and nrf2-dependent genes were determined by qrt-pcr. the implication of pparg and cb2 receptors in the beneficial effects of vce-003 in the eae model of ms is being investigated by using specific receptors antagonists. taken together our results support the potential of vce-003 for the treatment of ms and other chronic inflammatory diseases. we found that the lipid molecule lactosylceramide (laccer), is upregulated in ms patients. to better understand the role of laccer in ms, we used an animal model of spms, and found that laccer synthase (b4galt6) is upregulated during disease progression by astrocytes, and that its inhibition halts the progressive phase of the disease, attenuating cytokine and chemokine production by the astrocytes, and reducing the recruitment of inflammatory ly6c high monocytes to the cns. moreover, it also skewed the microglia and monocytes towards a m2 phenotype, and shifted the astrocyte phenotype to support re-myelination and axonal growth. in vitro experiments, using primary cells demonstrated that the inhibition of laccer synthesis directly inhibits cytokines and chemokines production in astrocytes. importantly, similar results were also obtained with human astrocytes, reinforcing the biological importance of our findings. in summary, we identified a novel lipid-signaling pathway that promotes the activation of local cns immunity and neurodegeneration in experimental spms and is a potential therapeutic target for spms. converging clinical studies report an increased prevalence of comorbid neuropsychiatric symptoms, particularly major depressive disorders, in a number of conditions (e.g., aging, obesity) and diseases (e.g., atherosclerosis, congestive heart failure, rheumatoid arthritis) sharing inflammation as a common denominator. by using an experimental approach in mice exposed to innate immune system (iss) stimulation, we demonstrated that induction of depressive-like behavior is mediated by cytokine-induced brain activation of a tryptophan-catabolizing enzyme, the indoleamine 2,3-dioxygenase (ido). in the metabolic syndrome (mets), a widely accepted concept that identifies a cluster of individual risk factors for type 2 diabetes and cardiovascular disease (including obesity, metabolic dysregulations and basal low-grade inflammation), neuropsychiatric symptoms emerge as significant factors for aggravation of the disease and related outcomes. recently, we demonstrated in a model of mets, the diabetic and obese db/db mice that cognitive alterations and increased anxiety-like behavior are related to hippocampal inflammation. on the other hand, depressive-like behavior is not affected by basal inflammation displayed by db/db mice. given the data linking increased depressive-like behavior and ido activation by cytokines in conditions of iss stimulation, the question arises as to whether depressive-like behavior is increased in those conditions in db/db mice. to answer this question, we measured in db/db mice and in their healthy db/1 littermates the effect of a lipopolysaccharide (lps) challenge (5 mg/mouse, ip) on behavioral reactivity in a depression test (the forced swim test, fst), plasma levels and hippocampus expression of inflammatory cytokines and related neuronal targets, and brain ido activity. plasma levels and hippocampus expression of il-1b, il-6, tnfa and il-10 are similarly increased 2h after lps in both db/1 and db/db mice. as expected, brain ido activity and duration of immobility in the fst are increased in lps-treated db/1 mice 24h after lps. on the contrary, induction of brain ido activity is significantly blunted in db/ db mice compared to their db/1 counterparts and no increase of depressive-like behavior is observed. moreover, some data suggest an impairment of the neuroimmune interactions in db/db mice. we need now to understand how obesity and related neurobiological alterations impair ido activation by cytokines and their consequences in terms of vulnerability to infections in mets. in this study, we tested it in a collagenase-induced mouse intracerebral hemorrhage (ich) model using tlr2 knock-out (ko) mice. to induce ich, collagenase or blood was injected into the right caudate putamen in 8-10 week old male mice. tlr2 expression was upregulated in the ipsilateral hemorrhagic tissues of the collagenase-injected mice. brain injury volume and neurological deficits following ich were reduced in the tlr2 ko mice as compared to the wild type (wt) mice. heterologous blood-transfer experiments show that tlr2 signaling in the brain-resident cells, but not leukocytes, contributes to the injury. in our study to elucidate underlying mechanisms, we found that damage in the blood-brain barrier (bbb) integrity following the ich was attenuated in the tlr2 ko mice compared to the wt mice, which may be due to reduced mmp-9 activation in brain astrocyte. the reduced bbb damages accompanies with reduced neutrophil infiltration and proinflammatory gene expression the injured brain parenchyma, which may account for the attenuated brain damage in the tlr2 ko mice after ich. conclusively, these data demonstrate that tlr2 contributes to brain injury following ich by compromising bbb through activating mmp-9 in brain. the pathological relevance of this autoantibody response is unknown, but it is generally assumed that it exacerbates demyelination via activation of complement and/or cell meditated effector mechanisms. however in a majority of cases the mog-specific autoantibody titre is far lower than that required to induce widespread demyelination and exacerbate disease severity in animal models of ms. to explore the possibility mog-specific antibodies may play other more subtle roles in disease pathogenesis we investigated possible effects in myelinating cultures derived from embryonic rat spinal cord; a model system that allows us to explore antibody-dependent effects in the absence of exogenous complement and effector cells. myelinating cultures were treated continuously with monoclonal antibodies specific for either mog (clone z2, igg2a), sulphatide (clone o4, igm), proteolipid protein (plp) (clone o10, igm), or an appropriate isotype control from 18 days in vitro onwards. in the absence of an exogenous source of complement none of these antibodies induced demyelination, but by 24 div had all had a significant inhibitory effect on myelination compared to cultures treated with appropriate isotype control antibodies. to investigate possible mechanisms contributing to this inhibitory effect qpcr arrays were used to determine if this complement-independent effect on myelination was associated changes in expression of immune mediators. unexpectedly we found recognition of antigens exposed at the surface of the myelin sheath induced a rapid increase in expression of three chemokines known to be involved in recruitment of effector t cells into the cns. within 24 hours of adding antibody to the cultures expression of ccl5, ccl20 and cxcl11 increased by at least three orders of magnitude and then declined to baseline over the following five days despite continuous presence of antibody. this transient increase in mrna transcripts for these cytokines resulted in sustained protein synthesis and secretion of biological active products as demonstrated by analysis of culture supernatants. these results challenge the traditional view that myelin-specific autoantibodies contribute to the pathogenesis of diseases such as ms and adem by virtue of their ability to initiate immune-mediated demyelination. we now demonstrate that even in the absence of recruited immune effector mechanisms myelin-specific autoantibodies not only inhibit myelination but trigger secretion of chemokines predicted to trigger or exacerbate inflammation within the cns. the blood-brain barrier (bbb), comprising specialized brain endothelial cells, is crucial in maintaining a controlled environment within the central nervous system (cns) to safeguard normal neuronal function. in multiple sclerosis (ms) the function of the bbb is disturbed, leading to uncontrolled influx of metabolites and immune cells and neuroinflammation. therefore, restoring the function of the bbb may be a novel therapeutic tool to halt disease progression or new lesion formation. we previously showed the importance of the vitamin a metabolite retinoic acid (ra) in bbb function during cns development 1 , where fetal astrocyte-derived ra is needed to induce bbb function during cns embryogenesis. considering the possible involvement of developmental pathways that could regenerate the disrupted bbb, we investigated the possible role for ra in the protection or reinstatement of disrupted bbb function. we show that ra counteracts the deleterious effects of inflammatory cytokines such as tumour necrosis factor (tnf)-a and interferon (ifn)c on bbb function in vitro. moreover, ra diminishes the induction of inflammation-related genes in human brain endothelial cells, and induces general immune quiescence in resting brain endothelium. our preliminary data as well as a recently described study in the cuprizone animal model for demyelination suggest that ra synthesis in the cns is increased under inflammatory conditions. the impact of cns-derived ra on the inflamed bbb, as well as the cellular source is currently under investigation. insight into the ra-synthetic pathway and its protective effect on the bbb may lead to the development of novel therapies which are aimed to restore bbb function and reduce the inflammatory cascade in ms lesions. tissue transglutaminase (tg2) is a multifunctional enzyme whose expression and activity is enhanced during inflammatory processes. we previously observed that the expression of tg2 is increased in monocytes in lesions in post-mortem material of ms patients. moreover, tg2 activity and expression is enhanced in chronic-relapsing experimental autoimmune encephalomyelitis (cr-eae) in rats. in this same ms model, pharmacological inhibition of tg2 activity dramatically reduced clinical symptoms and attenuated the influx of monocytes. in the present study we question whether tg2 plays a role in mouse eae, as transgenic mice have to be used for subsequent in vivo imaging of leukocytes/monocytes during eae. methods and results: eae was induced in tg2 -/mice and littermate wildtype mice (c57bl/6) using mog 35-55 . during the early phase of disease (day 14-15), the clinical symptoms observed were significantly less severe in tg2 -/compared to the wildtype mice. moreover, the maximal clinical score was significantly lower in the knockout mice. there was no difference in the day of onset of disease symptoms between the two groups. secondly, we aimed at visualizing leukocytes/monocytes in the spinal cord of lysm-gfp and cd11c-yfp mice suffering from mog 35-55induced eae using intravital video microscopy. a permanent window was fixed on top of the spinal cord of mice to allow reimaging of the same field of view in the same mouse over the disease course. in a pilot experiment we observed numerous gfp positive cells in the white matter around the imaged blood vessel in the spinal cord of a lysm-gfp eae mouse. although equally present, this was less prominent in a cd11c-yfp mouse suffering from eae. subsequent immunohistochemical analysis revealed that various cell types had infiltrated the spinal of cord of both mice. outlook: to further address the role of tg2 in the infiltration of leukocytes/monocytes into the spinal cord of eae mice in vivo, specific inhibitors for tg2 activity will be administered to the transgenic mice and leukocytes/monocytes will be visualized using intravital video microscopy. z. muneer 1 , c. wiesinger 1 , g. regelsberger 2 , j. berger 1 , s. forss-petter 1 1 medical universty of vienna, center for brain research, vienna, austria 2 medical university of vienna, institute of neurology, vienna, austria x-linked adrenoleukodystrophy (x-ald) is the most frequently occurring peroxisomal neurodegenerative disorder and is often associated with cerebral demyelination and inflammation. x-ald is caused by mutations in the atp-binding cassette sub-family d member 1 (abcd1) gene encoding adrenoleukodystrophy protein (aldp), which is a transporter for coa esters of very long-chain fatty acids (vlcfa) across the peroxisomal membrane. adrenoleukodystrophy related protein (aldrp), another member of the abcd sub-family, encoded by abcd2, is the closest homologue of abcd1. upon over-expression, abcd2 has been shown to compensate for abcd1 deficiency in vitro and in vivo. several lines of evidence imply an important role of microglia/macrophages in the disease progression. here we used mouse peritoneal macrophages (mpmu) to correlate the gene expression levels of candidate modifiers with x-ald associated defects, like accumulation of vlcfa and decreased peroxisomal b-oxidation, in abcd1 and abcd2 single ko mutants as well as in abcd1/abcd2 double deficient (doko) mice. by quantitative rt-pcr analysis, in mpmu from wild type mice the abcd2 mrna was present at about half the level of the abcd1 mrna. abcd1 ko mpmu showed elevated accumulation of vlcfa (c26:0) compared to the mpmu from wild type mice as measured by gc-ms. the vlcfa levels of abcd2 ko mpmu were similar to wild type amounts. interestingly, there was much higher accumulation of vlcfa in the mpmu from doko mice. there were no differences in the mrna expression level of the elovl1 gene, encoding the enzyme catalyzing the elongation step of vlcfa biosynthesis. peroxisomal boxidation activity was decreased in abcd1 ko mpmu compared to wild type level. this defect was strongly enhanced in mpmu from abcd1/ abcd2 doko mice. these results show that the increased accumulation of vlcfa in mpmu from doko mice as compared to abcd1 ko mice was due to a more severe defect in peroxisomal b-oxidation in doko mpmu. we conclude that a substantial expression of abcd2 mrna prevents a more severe metabolic phenotype in abcd1 deficient mouse peritoneal macrophages. this study also supports the validity of abcd2 up-regulation as a potential therapeutic target in x-ald. j. claude, b. linnartz-gerlach, h. neumann reconstructive neurobiology, bonn, germany microglia have innate immune receptors recognizing pathogens and disease-associated molecular patterns but also molecules that could sense the intact tissue. a subfamily of these receptors is the inhibitory signaling sialic acid-binding immunoglobulin-like lectin (siglec) group including siglec-e that has an immunoreceptor tyrosine-based inhibitory motif (itim) in the cytoplasmic tail to suppress activatory microglial signals. in this study, we used primary and stem cell-derived microglia that were modified by lentiviral vectors. here we show that siglec-e is expressed on microglia and is up-regulated following interferon-c (ifnc) treatment. we performed lentiviral knock-down and overexpression of siglec-e. lentiviral overexpression of siglec-e decreased, while knock-down increased the phagocytosis of neural debris and its associated reactive oxygen burst. the extracellular domain of siglec-e linked to a fc-part of immunoglobulin bound to the sialic acid residues of the neuronal glycocalyx. therefore, we co-cultured these modified microglia with primary hippocampal neurons. overexpression and knock-down of siglec-e showed an increase and decrease in relative neurite-length, respectively. the neuroprotective effect of siglec-e was abrogated after removal of the sialic acid residues on the neuronal glycocalyx. treatment with the anti-oxidant trolox abolished the neurotoxic effect of the siglec-e knock-down on neurite length. in summary, our data suggests an immunomodulatory function of siglec-e on microglia which leads to a neuroprotective phenotype by decreasing the production of reactive oxygen species and a reduced phagocytosis rate of neural debris. a. nadjar, c. madore, a. sere, a. aubert, s. lay e university of bordeaux 2, bordeaux, france many epidemiological studies have linked maternal exposure to infections during pregnancy to later development of cognitive disorders in the descendants. these alterations are likely to originate from a generalized neuroinflammation in the fetal brain following activation of the maternal immune system. this neuroinflammatory response relies on microglial cells activation. these latter normally contribute to the development of neuronal networks especially by eliminating/maintaining synapses, a phenomenon also known as synaptic stripping, in optimal conditions of brain development. neonatal inflammation may alter the synaptic stripping capacity of microglial cells. thus, targeting this persistent inflammatory microglial activation could represent an original and promising strategy to improve cognitive performances in the descendants. omega-3 are known as immunomodulators and target microglial cells. we thus hypothesized that enriching the diet with omega-3 from the first day of gestation may impair the development of neurological deficits at adulthood, through limitation of microglial inflammatory response in favor of its synaptic stripping activity. to characterize the beneficial effects of omega-3 on microglial activity, pregnant mice were fed with an omega-3-deficient diet, or a balanced omega-3/ omega-6 diet or supplemented with omega-3 and received a peripheral injection of either lps or saline at e18 of gestation. using the golgi method, we first evaluated the morphology of dendrites and quantified the dendritic spines density as a measure of neuronal networks maturation in the hippocampus. we found that a prenatal exposure to lps increased the number of immature spines and this was reversed by an omega-3 supplemented diet. to correlate these data with alterations of microglia-neurons interactions, we took advantage of the cx3cr1-gfp mice and injected them with a neuronspecific lentivirus containing dsred protein, in the hippocampus, 7 days prior to two-photon experiments. we focused on microglia general dynamic and microglia-neuron physical interactions and found an alteration in microglial behavior and microglia-neurons interactions in pups from lps-treated females. these alterations were reversed by an omega-3 supplemented diet. overall, our data show that alterations of microglia-neurons interactions in the developing brain may explain the neurological disorders observed in the descendants of females with prenatal inflammation. these deleterious effects may be prevented by nutritional strategies. , which is characterized by a relapsing phase with inflammatory cell infiltrates and a remitting period, where patients partially recover. among the different cell types involved in the necessary immunomodulation to allow the relapsing-to-remitting transition, the role of myeloid-derived suppressor cells (mdscs) gains importance. mdscs form a heterogenic population of immature myeloid cells that is able to suppress the inflammatory response. this cells act, among other mechanisms, through arginase-i (arg-i) activity on tcells. a previous study of our group showed that arg-i 1 -mdscs transiently enter the spinal cord of eae mice and takes part in the immune response control by inducing t cell apoptosis around the peak of the clinical score. therefore, changes in the mdsc population during eae should modify the evolution of the disease. the retinoid acid family molecules are used for the treatment of different leukaemia due to their role as mdsc differentiation factors into diverse cell populations, abolishing t cell immunosuppression. am80, a synthetic analogue of the retinoic acid with a higher bioavailability and less side effects than the natural ones, has controversial actions on eae depending on its administration period. in this work, we administered am80 specifically in the critical moment for immune modulation (around the maximum clinical score). drug administration affected mdsc population and clearly worsened the eae course. our results point to endogenous strengthening of mdsc population as a new and promising therapeutic strategy to treat ms by speeding up the transition from the relapsing to the remitting period. sildenafil induces cgmp accumulation by pde5 inhibition. we have demonstrated that sildenafil decreases microgliosis and astrogliosis and proinflammatory cytokines expression in a demyelination model. however, little is known about mechanisms of sildenafil neuroprotection. since nfjb plays an important role in the regulation of glial activation, we examine the hypothesis that nfkb is part of the mechanism underlying the sildenafil neuroprotective effects. five male mice (c57bl/6), 6 weeks-old, were used per group. the groups received for four weeks: 0.2% cuprizone (cpz) mixed into the chow; cpz into the chow plus sildenafil (viagra v r , pfizer, 25 mg/kg) in the drinking water, starting concomitantly (sild-t0) or fifteen days (sild-t15) after initiation of cpz; controls received pure chow/water. cerebella were processed for western blotting and immunofluorescence (if). results showed that cpz increased gfap expression compared to control (astrocytes activation). sild-t0 (but not sild-t15) significantly decreased gfap compared to cpz group. in controls, microglia showed resting phenotype, with thin and branched processes positive to iba1/nfjbp65 (inactive fraction) double labeling. cpz treatment decreased inactive-nfjb expression, indicating that this transcription factor was activated. in agreement, ikba (nfjb inactivating protein) was also decreased. if showed increase of iba-1 expression, indicating microglia activation. sild-t0 strongly increased the nfjbp65 and its inhibitory protein, ikba, suggesting inactivation of nfjb. if showed decreasing in iba-1 labeling, suggesting inactive microglia. the delayed treatment (sild-t15) did not decrease iba-1 or increase inactive-nfjb and ikba expression. in conclusion, treatment with sildenafil concomitant with cpz exposure prevents micro-and astrogliosis in mice, possibly through ikba-nfjb signaling pathway. sildenafil may be suitable to improve neuroinflammatory/neurodegenerative diseases treatment. financial support: cnpq, capes, facepe, fapesp. mt-1&2 levels have been found to be increased in several human neurodegenerative diseases including alzheimer's disease (ad). moreover, mt112 are also upregulated in different ad mouse models, for example tg2576 mice, which show a significant up-regulation of these proteins in the vicinity of the amyloid plaques. in the present study we generated a double transgenic mouse line that develops ad-like pathology in addition to having an overexpresion of mt1, in order to determine the role of mts in different aspects of ad pathology. the results show that the overexpression of mt1 does affect the mortality rate of the tg2576 and control mice in a gender-dependent manner and partially reverses the behavioural phenotype of young (4-5 months) tg2576 mice, reducing the exploratory activity and improving the learning process; in contrast, mt1 does not cause relevant changes in deambulations and anxiety. on the other hand, the amyloid cascade and neuroinflammation is increased in the hippocampus of old (15-16 months) apptgmt mice, but the lower level of gliosis in the hippocampus of young male apptgmt mice suggests that the overexpression of mt1 is capable of reducing inflamatory response well before amyloid plaques are formed but not afterwards. further molecular and immunohistochemistry analyses are underway to give further insight into the role of mts in amyloidosis and neuroinflammation in this ad mouse line. exposure to prenatal inflammation is a risk factor for neurodevelopmental and neurobehavioural abnormalities that manifest in later life in disorders such as autism, schizophrenia and seizure development. the amygdaloid complex is composed of more than 10 nuclei with subdivisions that have different cytoarchitectonic, chemoarchitectonic, and projection characteristics and that are responsible for the processing of higher functions such as memory consolidation and emotional conditioning. it is also known that the basolateral nucleus of the amygdala is implicated in seizure propagation and initiation. seizure onset later in life may be linked to abnormal development of the amygdalaoid complex. neuropeptide y (npy) and its receptors are known to be involved in a number of important functions such as feeding behaviours, anxiety and seizure modulation. npy y1 and y2 receptors are the most abundant in the brain and are expressed at different stages of development. during foetal development different brain regions are populated by neurones and glia at different times and the interface between mother and foetus plays a crucial role in the development process. lipopolysaccharide (lps) induced maternal inflammation is a known animal model for studying the inflammatory effects on the developing foetus. in this study we are investigating the effects of an acute prenatal systemic insult using the lps model on amygdala morphology and structure. embryonic day 12 (e12) c57bl6j pregnant mice receive an intraperionteal injection of lps (50 mg/kg) or 0.9% sterile saline. embryos at e12, e16, e18 and pups at p1, p7 p14 and p40 are taken and used to study the developing hippocampus and amygdala. for each age and treatment group foetal brains, maternal brains, maternal serum and placentas are collected. incidence of preterm birth, resorption rate, litter size and weights are also assessed. immunohistochemistry, western blot and rt-pcr are then used to assess the expression of neuronal and glial markers in the amygdala and surrounding limbic structures. specifically alterations in expression, activation and distribution of npy and its receptors, microglia and astrocytes in the developing amygdala and hippocampus at different stages following lps treatment are being investigated. it is hoped that this study will further enhance our understanding of how the maternal environment influences brain development and, if disturbed at a critical time in the development of the amygdala, may predispose individuals to amygdala related disorders in later life. in vivo imaging of transgenic fluorescent mice in the cns by twophoton laser-scanning microscopy (2p-lsm) has become a powerful tool in neuroscience. for in vivo imaging of the cortex a craniotomy of the skull has to be made. in addition to anaesthesia and analgesia treatment, we occasionally apply anti-inflammatory drugs to prevent immunological activity that can obscure the images. as anti-inflammatory drug we used carprofen ((rs)22-(6-chloro-9h-carbazol-2-yl)propanoic acid), a known cyclooxygenase-2 (cox-2) inhibitor. adult cx3cr1egfp mice in which microglia are labeled by expression of the green fluorescent protein egfp were treated with a single dose of carprofen or with vehicle 12 hours before the craniotomy. in all mice we exposed the right somato-sensory cortex, and quantified the microglial response to a laser-induced micro-lesion. this micro-lesion was caused by increasing the power of the laser for 1 second in the middle of the region of interest. results -conclusions unexpectedly, we observed that carprofen reduced the microglial process motility significantly. the speed with which the processes approached the lesion dropped from 0.5 mm per minute in the untreated mice to 0.2 mm per minute in the treated mice. a molecular understanding of microglia response in inflammatory processes and how anti-inflammatory drugs modify normal microglia response will provide a strong impact in developing treatment strategies for diseases with strong inflammatory components. resting and activated (lipopolysaccharide, lps) cultured cstb-/-and control microglia were analyzed for cytokine production, chemotaxis and phagocytosis. microglia extracted directly from the mouse brains were studied for expression of mhc ii and m1-m2 polarization using flow cytometry. mouse brain cortex (p14) was analysed for m1-m2 microglial phenotypes and the presence of other immunological cells from the periphery. moreover, we have checked myeloid cell population in bone marrow and spleen of p14 animals as well as cytokines' level in blood serum. our results from cultured microglia show that secretion of proinflammatory chemokines is elevated by cstb-/-microglia. also, cstb-/microglia are chemotactically more active compared to the controls whereas their phagocytic activity is decreased. cstb-deficient microglia show decreased expression of mhc ii on the cell surface indicating reduced antigen presentation. activated cstb-/-microglia directly extracted from the brain has predominantly proinflammatory m1 phenotype. cstb-/-mice display inflammatory changes in the peripheral tissues at their early postnatal period. we have registered high concentrations of proinflammatory chemokines and cytokines in blood serum of p14 cstb-/-pups, but no difference in amount of neutrophils and lymphocytes between brains of wild-type and knockout mice. also, in bone marrow and spleen amount of granulocytes is enhanced in cstb-/mice during early postnatal period as well as level of granulocyte macrophage colony-stimulating factor in their blood serum. our results suggest a role for cystatin b in regulation of immune response and that cstb-deficiency is associated with early inflammatory processes both in the brain and the peripheral tissues. therefore, we consider that epm1 should be treated as a disorder combining neuropathological and immunological features. the contribution of glial cells in the pathophysiology of epilepsy is increasingly valued. furthermore, clinical and experimental evidence suggests a direct relationship between epileptic activity and cns inflammation, which is characterized by accumulation, activation and proliferation of microglia and astrocytes. concomitant glia-mediated mechanisms of action of several aeds have been proposed. however, their direct effects on glial cells, especially microglia, have jet been hardly investigated. we aimed to investigate the influence of commonly used aeds on the glial viability and microglial in-/ activation state in a physiological and inflammatory modified in-vitro astroglia/ microglia coculture model. methods:primary astrocytic cultures were prepared from brains of postnatal (p0-p2) wistar rats and cocultured with a physiological amount of 5% (m5), as well as 30% (m30) microglia in order to mimic inflammatory conditions. cocultures were treated for 24 hours with valproic acid (vpa), carbamazepine (cbz), phenytoin (phe) and gabapentine (gbt) with a concentration of 10, 25, 50 and 100 mg/ml. viability and proliferation was measured using the tetrazolium (mtt) assay. the microglial activation state was determined by immunocytochemical labeling using a monoclonal antibody to the ed1 marker. results:m5 and m30 cocultures showed a dose-dependent, significant reduction in glial viability after incubation with phe and cbz. furthermore, low doses of vpa led to highly significant microglial activation in m5 cocultures. however, cbz significantly reduced the amount of activated microglial cells and increased the total number of inactivated microglia in the inflammatory modified m30 cocultures. conclusion: cns inflammation is characterized by a disturbance of glial cell functions. strong microglial activation, a typical hallmark of inflammation, was induced by vpa in m5 and continued in m30 cocultures. with regard to the direct relation between cns inflammation and seizures, vpa seems to be unsuitable to reduce inflammatory condition. the reverse effect was achieved after cbz. we noticed significant microglial inactivation, after incubation of the m30 cocultures. as it has been demonstrated for levetiracetam before, we assume a beneficial therapeutic effect of anti-epileptic drugs (aed) with an antiinflammatory glial potential in epileptic patients with persistent inflammation. microglial cell activation due to homeostatic imbalances of external and/or internal etiology implies, among others, secretion of proinflammatory cytokines. the fact that microglial activation, inflammation and neurodegeneration often coexist, suggests that proinflammatory cytokines might induce and/or enhance neuronal damage. we investigated the neurotoxic impact of microglial activation in organotypic hippocampal slice cultures by exposing them to the toll-like receptor 4 ligand, lipopolysaccharide (lps), for 72 hours. microglial activation was characterized by unbiased estimation of their number (stereology) and quantification of soma and branching morphology (neurolucida v r -based cell reconstructions). moreover, proinflammatory cytokine and nitrite levels in the culture supernatant were estimated by elisa and griess. the neurotoxic impact was assessed by morphology (nissl staining, fluoro-jade v r b) and extracellular electrophysiological recordings of spontaneous and evoked neuronal activity in the hippocampal ca3 subregion. lps exposure induced microglial population expansion, morphological changes, such as process thickening and somatic enlargement, as well as substantial secretion of proinflammatory cytokines (tnfa, il6) and nitrite accumulation in the culture medium. however, these changes did not coincide with neurodegeneration and were associated with only minor effects on neuronal function, as demonstrated by the amplitude of evoked neuronal responses and short-term plasticity properties. we conclude that, in contrast to what has been observed in vivo, chronic microglial activation by lps is not sufficient to drive neuronal death and dysfunction in organotypic hippocampal slice cultures. the neonatal brain is particularly susceptible to oxidative stress. our group has previously shown that following hypoxic-ischemic injury, hydrogen peroxide (h 2 o 2 ) levels rise significantly particularly in the neonatal brain and are sustained for up to 24 hours. this rapidly accumulated h 2 o 2 is detrimental in the iron-rich immature brain as it can lead to the generation of dangerous free radicals that can cause extensive injury. to date, there is limited literature on the effects of increased h 2 o 2 levels, particularly on microglial cells, which have been extensively indicated in the mediation of ensuing injury. here we describe the effects of a continuous exposure of microglia to h 2 o 2 , as generated using the glucose oxidase-catalase (gox-cat) system. this system allows us to generate and continuously maintain for up to 24h pathophysiological levels of h 2 o 2 >1 um. microglial cultures were derived from the p1 mouse brain and exposed to either bolus concentrations of h 2 o 2 [1-100 mm] or varying concentrations of gox-cat for varying lengths of time. conditioned medium was collected from cells at 4, 18 and 24h of treatment and analyzed for secreted cytokine levels using the 25-plex cytokine microbead array kit. treated cell extracts were processed for protein and fixed cells were labeled with m1 and m2a phenotype markers. continuous exposure to very low levels of h 2 o 2 can produce 10-20-fold higher (over control) pro-inflammatory cytokine protein levels of il5, il6, il12p40, il12p70, il15, il17 and ifng and at least a 100-fold higher level of il1a, il1b, il7 and tnfa by 18h. anti-inflammatory cytokine (il4, il10 and il13) and chemokine (rantes, g-csf and gm-csf) protein levels were also increased by 18h. interestingly, no prominent cytokine responses were seen with bolus treatment at any of the time points studied. low, continuous h 2 o 2 promoted a predominantly m2a microglial phenotype by 24h. we conclude that studying continuous exposure effects will help delineate the various specific effects h 2 o 2 can have on microglial cells. these specific effects can then be used to clarify when and how microglial cell responses can be beneficial or detrimental following injury in the immature brain. we have previously shown that natural (15-deoxy-d 12,14 prostaglandin j 2 , 15d) and synthetic (pioglitazone) agonists of peroxisome proliferator activated receptor-c (ppar) potentiate intrinsic cellular mechanisms protecting oligodendrocyte (ol) progenitors (ops) from oxidative insults and promote their differentiation to ols. in addition, ppar-c agonists potentiate mitochondrial activities, as the mitochondrial respiratory chain activity and the regulation of cytoplasmic ca21 waves, which are known to be crucial for ol differentiation. in the present study we sought to investigate whether ppar-c agonists can protect ol cultures from conditions causing mitochondrial stress. first, to specifically induce a mitochondrial impairment, we used the complex i inhibitor rotenone. as expected rotenone, at concentration not affecting cell viability, significantly inhibited ol differentiation, as indicated by the reduced number of cells expressing specific markers of differentiation (o 4 and o 1 ). in ppar-c agonist-treated ols the inhibitory effects of rotenone were significantly attenuated, suggesting a protective effect of the agonists against the mitochondrial toxin. we next examined a condition mimicking inflammatory stress by challenging op cultures with tnf-a, an inflammatory cytokine known to retard the differentiating program of ops. in parallel with the expected reduction of the percentage of o 4 and o 1 positive cells, the cytokine induced a significant reduction of mitochondrial membrane potential (mmp), suggesting an impairment of the mitochondrial functions. the simultaneous treatment with tnf-a and ppar-c agonists (15d or pioglitazone) significantly reverted both tnf-a dependent reduction of ol differentiation and mmp. at the molecular level, we found that in op cultures, ppar-c agonists increased the expression of the uncoupling protein-2 (ucp-2), a mitochondrial protein known to contribute to the protection of mitochondria against oxidative stress. these findings suggest that ppar-c agonists protect ols and promote myelination through several mechanisms, including those involving mitochondrial functions. our studies support the therapeutic potential of ppar-c agonists in brain diseases in which mitochondrial alteration, oxidative stress and demyelination occur and point to the need to better understand the role of ppar-c and its agonists in ol biology. it is characterized by lesions of demyelination and inflammation, which can be classified according to the activation state of the microglia/macrophages. well before any myelin and blood-brain barrier damage clusters of activated microglia are noticeable. these clusters are considered to be the first stage of lesion formation and therefore called 'pre-active lesions. however, they do not always develop into demyelinating lesions. here we postulate that stressed oligodendrocytes play an crucial role in pre-active lesion formation, by producing factors that will attract and activate microglia to either a more pro-inflammatory (m1) or antiinflammatory (m2) activation status. this can contribute to the amount of damage to the environment and further lesion formation. this study sets out to identify the best method to mimic this early lesion formation in vitro. first, human primary isolated microglia are stimulated to either a m1 or m2 phenotype and characterized by marker expression and cytokine profile. oligodendrocytes are isolated out of the human brain. medium of (stressed) oligodendrocytes will be used as attractant as well as activator for microglia. by immunohistochemical staining the activation status of microglia in pre-active lesions will be determined. preliminary data reveal that human microglia are able to be skewed towards a pro-and anti-inflammatory phenotype. first experiments of primary human oligodendrocyte cultures, showed pure oligodendrocytes. thus far our data support our hypothesis, however further research is required to make our conclusions more robust. question: although cell transplantation is increasingly suggested to be beneficial for the treatment of various neurodegenerative diseases, the therapeutic application of such intervention is currently hindered by the limited knowledge regarding central nervous system (cns) transplantation immunology. in this study, we aimed to investigate the early post transplantation innate immune events following grafting of autologous mesenchymal stromal cells (msc) in the cns of immune competent mice. methods and results: first, the survival of grafted luciferase/egfpexpressing msc (msc-luc/egfp) was demonstrated to be stable from on day 3 post implantation using in vivo bioluminescence imaging (bli), which was further confirmed by quantitative histological analysis of msc-luc/egfp graft survival. additional histological analyses at week 1 and week 2 post grafting revealed the appearance of (i) graftsurrounding/-invading iba11 microglia and (ii) graft-surrounding gfap1 astrocytes, as compared to day 0 post grafting. while the density of graft-surrounding astrocytes and microglia did not change between week 1 and week 2 post grafting, the density of graft-invading microglia significantly decreased between week 1 and week 2 post implantation. however, despite the observed decrease in microglial density within the graft site, additional phenotypic analysis of graftinvading microglia, based on cd11b-and mhcii-expression, revealed > 50% of graft-invading microglia at week 2 post implantation to display an activated status. although microglial expression of cd11b and mhcii is already suggestive for a pro-inflammatory m1-oriented phenotype, the latter was further confirmed by: (i) the expression of nos2 by microglia within the graft site, and (ii) the absence of arginase 1-expression, an enzyme known to suppress no activity in m2oriented microglia, on graft-surrounding and -invading microglia. conclusions: in summary, we here provide a detailed phenotypic analysis of post transplantation innate immune events in the cns of mice, and warrant that such intervention is associated with an m1-oriented microglia response and severe astrogliosis. it is well known that cell surface immune receptors play a critical role in regulating immune and inflammatory processes in the central nervous system (cns). cd300f is a novel immunoreceptor that dampens inflammatory reactions in experimental autoimmune encephalitis (eae) and diverse allergy models. we have analyzed the function of cd300f immunoreceptor in an nmda excitotoxicity model and in a traumatic brain injury model (tbi). for this purpose, we determined the pattern of expression of both cd300f and its putative ligand(s) in the cns, as well as the neuroprotective role of cd300f after both acute brain injuries. first, we used a human and rat cd300f-ig soluble protein to show by confocal microscopy the presence of endogenous ligand(s) for this receptor mainly in cns white matter oligodendrocytes and on the surface of oligodendrocytes, fibrous astrocytes and neurons in vitro. interestingly, when we analyzed the in vitro expression pattern of rcd300f in brain cells by q-pcr and immunohistochemistry, in addition to the expected expression in microglial cells, we detected expression of cd300f in oligodendrocytes and neurons. in vivo, after a tbi, cd300f is expressed at early timepoints (3d) in small round macrophages, and at later time-points (14d) in neurons of the penumbra. q-pcr studies showed significant upregulation of the mrna for rcd300f at 7 and 14d after tbi. interestingly, a delayed (4 hours after the lesion) intraparenchymal injections of a non-viral modular recombinant gene therapy vector termed nlsct overexpressing hcd300f induced a decrease in the lesion volume 3d after nmda injection or tbi when compared to control gfp over-expression. in addition, the over-expression of hcd300f decreased the long-term sensitive functional deficits observed by the "sticky tape" test. in order to validate these results with the endogenous molecule, we cloned the rat ortholog of cd300f protein. on-going positron emission tomography (pet) studies using 2-[ 18 f]-fluoro-2-desoxiglucosa ( 18 f-fdg) are being used to evaluate the long-term functional recovery after tbi. the overexpression of rcd300f receptor had a comparable neuroprotective effect as the human molecule after nmda injection. these data suggest that cd300f may be important in neuron-glia inflammatory and trophic interactions. in addition, our results suggest that delayed cd300f overexpression by means of a modular recombinant gene therapy may constitute an interesting therapeutic strategy for acute brain injuries. depending on the type of signals produced after an inflammatory event, microglia develops specific activities, including cytotoxic, neuroprotective or phagocytic activities in order to come back to brain homeostasis. as described for macrophages, microglia can adopt m1 (proinflammatory) or m2 (phagocytic) phenotypes distinguishable through their pattern of factors expression and specific cell surface markers. interestingly, polyunsaturated fatty acids (pufas) are precursors of bioactive lipid messengers involved in the regulation of inflammation and in particular, docosahexanoic acid (dha, 22:6n-3) an n-3 pufa, presents anti-inflammatory properties. the aim of our study was thus to investigate the effect of an increase of n-3 pufas on the inflammatory response and cognitive abilities after a peripheral immune challenge. to increase n-3 pufas, we took advantage of transgenic mice carrying the fat-1 gene from the roundworm caenorhabditis elegans. this fat-1 transgenic mouse is capable of producing n-3 fatty acids from the n-6 type endogenously, eliminating confounding factors of the diet. this conversion leads to abundant n-3 fatty acids with reduced levels of n-6 fatty acids in tissues, including the brain. to induce a neuroinflammation, we injected mice intraperitoneally with lipopolysaccharide (lps), components of gram negative bacteria walls. we first studied the microglial phenotype 24 h after lps injection and found that microglia in fat-1 mice presented a significant increase of m2 phenotype markers. we then measured cytokine expression in the hippocampus after a lps challenge and found a significant decrease in il-1b mrna in fat-1mice compared to wildtype littermates. in terms of hippocampal memories abilities, only control mice presented a deficit in the y-maze task whereas fat-1 mice were able to perform the test correctly. our results indicate that 24 h after a lps injection, fat-1 mice presented a decreased of the inflammatory response and normal hippocampal memory abilities compared to wild-type littermates. glia integrity and homeostasis as they have prominent role in blood brain barrier formation and maintenance, as well as in active regulation of an immune response. however, astrocytes are affected in neuroinflammation, when their protective roles might be suppressed and when they might be shifted towards pro-inflammatory phenotype. experimental autoimmune encephalomyelitis (eae) is a widely used model of autoimmune neuroinflammation. chemokine cxcl12 produced by astrocytes and endothelial cells has profound anti-inflammatory and anti-encephalitogenic role in eae. nitric oxide (no) produced by inducible no synthase (inos) within the cns is considered as disease promoting, while interleukin (il)-10 as protective in eae. the aim of this work was to investigate effects of no and il-10 on cxcl12 gene expression in astrocytes. methods: astrocytes were stimulated with supernatant collected from concanavalin a-stimulated splenocytes (conasn) and simultaneously exposed to no donor, sodium-nitroprusside (snp) or anti-il-10 antibody. also, astrocytes were stimulated with ifn-c1il-17 in the absence or presence of il-10. peritoneal macrophages isolated from healthy rats were co-cultivated with astrocytes. after 24h incubation period cxcl12 gene expression in astrocytes was measured by rt-qpcr. phosphorylation of p38 mapk and nf-jb was assessed by immunoblot in astrocytes exposed to snp. results: we found that no released from snp or macrophages significantly reduced cxcl12 gene expression in astrocytes. this reduction was mediated by inhibition of p38 mapk activation, but not of nf-jb signaling. on the contrary, il-10 stimulated and anti-il-10 antibody suppressed cxcl12 gene expression in astrocytes. conclusions: these results imply that expression of cxcl12 in astrocytes is inversely regulated by no and il-10 in the central nervous system affected by inflammation. having in mind, profound regulatory role of cxcl12 in neuroinflammation, these data contribute to our understanding of pro-and anti-inflammatory nature of no and il-10, respectively. in the presence of pathological insults, they acquire reactive phenotypes aimed at re-establishing brain homeostasis and minimizing neuronal damage. however, reactive microglia produce several factors, typical of an inflammatory response, with a potential neurotoxic effect. consequently, the progression and resolution of microglial activation have to be tightly controlled to avoid detrimental secondary effects. signals arising from neuronal cells play an important role in the activation state of microglial cells. among them, inhibitory mechanisms, such as neuronal cd200 and microglial cd200r1 interaction, keep the proinflammatory phenotype of microglia under control. alterations in the expression of cd200 and cd200r1 have been described in pathological conditions, and the modulation of cd200-cd200r signalling could be an interesting target to be considered in therapeutic approaches against neuroinflammation occurring in neurodegenerative diseases. little is known on the molecular mechanisms involved in the regulation of cd200 and cd200r1 expression. the aim of the present work is to study the possible modulation of cd200 and cd200r1 by ppar-c agonists, and the involvement of cd200-cd200r1 in the anti-inflammatory and neuroprotective effects of ppar-c activation. we have used mouse microglial, mixed glial and neuronal cell cultures, as well as neuron-microglia co-cultures. cd200r1 expression was detected in microglial cells, and it was decreased in response to pro-inflammatory stimuli such as lps/ifn-c. the ppar-c agonist 15-deoxy-d 12, 14 -prostaglandin j 2 (15d-pgj 2 ) abrogated the inflammatory response in lps/ ifn-c-treated microglial cells and prevented cd200r1 expression inhibition. cd200 expression was detected in neuronal cultures and, at a lesser extent, in astrocytes in mixed glial cultures. lps/ifn-c-treatment did not modify cd200 expression in neuronal cultures, but it induced an increase in mixed glial cultures. this increase was inhibited by 15d-pgj 2 pre-treatment. 15d-pgj2 also protected against lps/ifnc-induced neurotoxicity in neuron-microglia co-cultures, but this effect was abolished when cd200-cd200r1 interaction was interrupted using an anti-cd200r1 blocking antibody. these results show that ppar-c agonists modulate cd200 and cd200r1 expression in reactive glial cells, and that cd200-cd200r1 interaction is necessary for the neuroprotective effect of ppar-c agonists. to gain insight into the contribution of the cwbc pathway to remyelination in ms, we analyzed the expression pattern of tcf7l2, a downstream target of the cwbc pathway and coactivatior of b-catenin in ms lesions. tcf7l2 was expressed in ol and astrocytes in a subset of early ms lesions, but was also observed in tissue samples from patients with inflammatory, non-demyelinating diseases. in contrast, in chronic lesions, no expression of tcf7l2 was detected. by wb we found slightly increased b-catenin levels in ms lesions compared to normal appearing white matter. aspirin (ass), a non-steroidal anti-inflammatory drug, attenuates bcatenin signaling activity by inhibition of protein phosphatase 2a (pp2a) via increased phosphorylation. therefore, we determined the effect of ass on ol differentiation and myelin gene expression (mge). as a control for the cwbc pathway activation or inhibition the selective cwbc pathway inhibitor (icg-001) was chosen. icg-001 binds specifically to cbp and causes a disruption of the b-catenin/cbp interaction. exposure of ol to 0.3 mm icg-001 for 48 hours increased mge and had a positive effect on ol differentiation. in contrast, ass had no positive effect on process formation and did not promote mge in primary murine ol. our results suggest that icg-001, but not ass increases the expression of myelin genes. further experiments are required to determine the functional role of the cwbc pathway for ol differentiation and remyelination in ol and ms. (braak et al., 2003) . furthermore, neuroinflammation, i.e. activation of microglial cells in the substantia nigra (sn), has been widely implicated in pd progression (mcgeer et al., 1988) . in the present study, we question whether microglial activation occurs in brain regions beside the sn which are affected in pd patients. methods: to this end, we studied microglial activation and protein pathology in the ob and hc of clinically and neuropathologically verified pd patients (braak 4-6) and control subjects (braak 0). human post-mortem formaldehyde-fixed material of pd patients included sn (n 5 13), ob (n 5 9) and hc (n 5 12), and material from age-matched control subjects without neurological deficits included sn (n 5 10),ob(n 5 6), and hc (n 5 8). results: the presence of a-syn pathology concentrated in the anterior olfactory nucleus of the ob and in the ca1-2 region of the hc. furthermore, using cd68 as a microglial marker, we observed a significant increase in the number of immunopositive microglial cells, with an activated, amoeboid morphology, in the sn as well as in the ob and hc of pd patients compared to control subjects. co-localization studies indicated that these activated microglia were found in the proximity of a-syn inclusion bodies and neurites, but did not co-localize suggesting that the microglial cells do not actively phagocytose a-syn. conclusion: we conclude that microglial activation occurs in other brain regions beside the substantia nigra of pd patients. the functional role of the microglial cells in those brain regions and their contribution to pd pathology remains to be established. in the present study, we question whether ccl2 and cx3cl1 and its receptors are present in hippocampal lesions of ms patients. to this end, semi-quantitative rt-pcr was performed on cdna transcribed from rna isolated from hippocampi of ms patients and control subjects. moreover, immunohistochemical analysis of ccl2, cx3cl1 and its receptors ccr2 and cx3cr1 was performed on post-mortem, formalin-fixed hippocampal lesions (plp -) from ms patients and on hippocampal material (plp 1 ) from control subjects. ccl2 and ccr2 mrna was significantly enhanced in demyelinated ms hippocampal homogenates compared to non-demyelinated and control hippocampal homogenates. cx3cr1 mrna was significantly increased in demyelinated ms hippocampal homogenates compared tot non-demyelinated hippocampal homogenates, but cx3cl1 mrna levels showed no difference between groups. ccl2, ccr2, cx3cl1 and cx3cr1 immunoreactivity was present mainly in white matter of control and ms hippocampi and was clearly enhanced in hippocampal grey (cornu amonis) and white (stratum lacunosum, stratum radiatum, alveus) matter lesions. the intensity of the ccl2 and cx3cl1 immunoreactivity was most pronounced when active lesions were present. co-localization studies indicated that ccl2 and cx3cl1 are present in astrocytes, whereas cx3cr1 and ccr2 are present in or on microglial cells. we conclude that the chemokines ccl2 and cx3cl1 and their respective receptors are enhanced in hippocampal ms lesions. as no clear influx of leukocytes is observed in the grey matter lesions, we propose that astrocyte-derived ccl2 and cx3cl1, via interaction with their receptors, affect microglial activation and/or function thereby contributing to neuronal dysfunction in the hippocampus as seen in ms patients. , is a 17 kda cytokine-inducible protein, produced by activated macrophages during chronic transplant rejection and in inflammatory reactions. in central nervous system (cns), iba1 is a sensitive marker associated to activated microglia and is upregulated following neuronal death or brain lesions. iba1-like factors have been described in several metazoan and share a well conserved amino acid primary structure throughout evolution suggesting a common, functional role. the medicinal leech hirudo medicinalis is able to regenerate its cns after injury, leading to a complete functional repair. similarly to vertebrates, leech neuroinflammatory processes are linked to microglia activation and recruitment at the lesion site. we investigated the expression of hirudo iba1 to track the activation state of leech microglial cells involved in nerve repair events. results: we recently identified a gene, named hmiba1, coding a 17.5 kda protein showing high similarity with vertebrate iba1 factor. quantitative rt-pcr analyses showed that hmiba1 is constitutively expressed in cultured nerve chains. a weak down regulation was observed in the days following experimental injury. gene transcripts rise back to basal level one week later. cultured nerve chains stimulated with atp shows a significant increase of hmiba1 transcript 6 hours after treatment. immunoblot analysis, performed with anti-hmiba1 polyclonal antibodies, revealed an immunopositive band at the predicted size. the presence of hmiba1 protein in na€ ıve and experimentally challenged tissues was evaluated by immunohistochemistry. the protein is constitutively present in spread, stellar shaped microglial cells, distributed in connective fibers and in segmental ganglia. a few hours after experimental injury of cns, the amount of immunopositive microglial cells increases at the lesion site and at the cut end of nerve fibers until. the amount of hmiba11 cells in connectives rapidly increases in atp treated nerve chains. this augmentation is visible in ganglia microglia and in connective fibers, where cells located between axon fibers display an elongated and stretched shape. conclusion: hmiba1 is a good marker of activated microglia. like in vertebrates, the atp induces its expression in leech cns. also if the functional role of hmiba1 has to be further elucidated, this molecule appears as a good activation marker of microglia and an interesting tool to study and follow the activity of such cells during nerve repair in leech. hypertension is the single most important risk factor for cardiovascular disease. despite significant advancements, 20-30% of all hypertensives remain resistant to all current available pharmacotherapy. these patients exhibit elevated sympathetic drive, increased norepinephrine spillover, and dampened parasympathetic drive. the dysfunctional autonomic nervous system of these resistant patients indicates a neurogenic origin for their pharmacotherapy resistance. previous studies have proposed that neuroinflammation in the autonomic brain regions plays an important role in neurogenic hypertension. this coupled with the emerging interest in microglia led us to hypothesize that activation of microglial cells in the brain could be a critical event in the initiation and establishment of hypertension pathophysiology. we have previously established that chronic low dose angiotensin ii (ang ii) induced hypertension involves activation of microglia, and increase in proinflammatory cytokines and reactive oxygen species (ros) in cardioregulatory brain areas, such as the paraventricular nucleus of the hypothalamus (pvn). intracerebroventricular (icv) delivery of minocycline, an anti-inflammatory antibiotic that inhibits microglia activation, decreased activated microglia, inhibited increase in cytokines and ros, and attenuated hypertension. in addition, inhibition of brain mitochondrial ros attenuated hypertension, microglial activation, and the overexpression of brain pro-inflammatory cytokines. a time-course experiment demonstrated that there was a significant increase in activated microglia before the increase in blood pressure was detectable by radiotelemetry. furthermore, the origin of microglia in established hypertension appears to be both from resident and bone marrow derivation. these observations indicate that the activation of microglia is a critical player in the development of neurogenic hypertension. they suggest that activation of microglia in cardioregulatory regions of the brain is an early occurrence that may initiates a cascade of signaling events leading to increase sympathetic nerve activity and initiation of hypertension. this research is supported by nih grant (r37 hl 33610 to mkr). our results demonstrate significant inflammatory activation of the neurons and their sgc not only in drg associated but also non-associated with injured nerve. a distinct expression of cytokines and their receptors was identified in sgc surrounding largesized drg neurons. significant inflammatory reactions of sgc were found in all drg of pac-treated rats. moreover, inflammatory activation sgc was also observed in drg of sham-operated rats indicating other kinds of trigger than traumatic nerve injury. the nerve injury and pac-treatment also triggered socs3 expression in sgc to control stat3 activation. inflammatory activation of sgc significantly contributes to ectopic activation of the drg neurons not only associated with injured nerve, and is involved in npp induction. aging is associated with reduced function and degenerative changes of the central nervous system (cns). increasing evidence suggests that changes in microglia cells, i.e. the resident macrophages of the cns, contribute to the age-related deterioration of the cns. the most prominent age-related change of microglia concerns enhanced sensitivity to proinflammatory stimuli of microglia in mice, rats and primates, called priming. we have addressed this issue in ercc1 mutant mice, ercc1 d/mice, a progeroid, dna repair deficient mouse model that displays features of accelerated aging in multiple tissues including the cns. in aged ercc1 d/mice, microglia showed increased proliferation and enhanced immune function including expression of cytokines and antigen presentation molecules, increased phagocytosis and production of reactive oxygen species (ros). this microglial functionality was found to be surprisingly hyper-responsive to immune stimuli indicative of microglia priming. transcriptome analysis revealed an expression pattern that characterizes microglia priming, featuring genes associated with increased antigen pattern recognition and antigen presentation and phagocytic-, adhesion-and chemotactic ability. in mice where the ercc1related dna repair deficit was targeted to forebrain neurons, microglia priming was restricted to forebrain areas, suggesting that the dna damage-induced changes in neurons provided the signals leading to microglial immune priming. acidosis is a clinical consequence of many major diseases. non-infectious diseases are increasing in prevalence and many have been shown to have an inflammatory component, which in the absence of infection, will be sterile. the nlrp3 inflammasome, a multimeric protein complex which induces processing of il-1b into its active form, is a well established mediator of sterile inflammation and is known to drive the worsening of brain injury in numerous experimental disease paradigms. we sought to investigate whether acidic conditions, typical of a disease environment, affected il-1b processing in primary mouse glial cells. mixed glial cultures were grown from wild type and nlrp3 knockout mice. culture media was reduced to ph6.2 and il-1b release was measured following addition of activators of the nlrp3 inflammasome (calcium pyrophosphate dihydrate crystals, monosodium urate crystals, atp) with or without pre-treatment with caspase-1 or cathepsin d inhibitors (yvad-cho or pepstatin a respectively). subsequently, il-1b release was measured following addition of lactic acid with or without pre-treatment with the above inhibitors. at ph6.2, activators of the nlrp3 inflammasome (calcium pyrophosphate dihydrate crystals, monosodium urate crystals, atp) induced the release of il-1b from mixed glial cultures. the il-1b released at this low ph was 20kda in size in addition to the mature 17kda il-1b. this 20kda il-1b release was maintained in nlrp3 deficient cells and was not significantly altered by pre-treatment with the caspase-1 inhibitor yvad-cho. lactic acid, itself released during disease and a common cause of acidosis, also induced the release of 20kda il-1b from mouse glial cultures. as with the nlrp3 activators under acidic conditions, the lactic acid-induced 20kda il-1b release was maintained in nlrp3 knock-out cultures and with pre-treatment with yvad-cho. pre-treatment with the cathepsin d inhibitor pepstatin a, however, significantly reduced the 20kda il-1b released with the nlrp3 activators and lactic acid. here we show that under disease relevant conditions (low ph), 20kda il-1b is released from mouse glial cultures and this 20kda il-1b is independent of the classical nlrp3 inflammasome/caspase-1 pathway and likely mediated by cathepsin d. further investigation of this caspase-1-independent il-1b pathway may in future provide novel targets for the treatment of inflammatory disease. the phoneutria nigriventer (ctenidae-araneaeomorpha) spider venom (pnv) causes reactive gliosis, neuroinflammation and blood-brain barrier (bbb) impairment. nitric oxide(no)-soluble guanylate cyclase(sgc)-cgmp has been implicated in pnv-induced cavernosal relaxation. we investigate whether no-sgc-cgmp signaling acts on astrocytes/microglia activation and neuroinflammation induced by pnv. male wistar rats (6-8-week-old) were pre-treated (i.p.) with sgc inhibitor (odq, 10 mg/kg), nnos inhibitor (7-nitroindazole-7ni, 40 mg/kg), no donor (nitroprussiate-ntp, 3 mg/kg) or pde5 inhibitor (sildenafil, 10 mg/kg). after 30 minutes, the venom (0.85 mg/kg) was injected in the tail vein (i.v.). saline (i.v.), pnv alone or dmso (i.p) followed by pnv were used as controls. one hour after injection, cerebella were processed for immunofluorescence or western blotting. pnv increased gfap, iba-1, ifn-c and sgc, compared to saline control, indicating astrocytes and microglia activation, neuroinflammation and sgc-cgmp involvement. the sgc inhibition by odq intensified the pnv effects, increasing even more gfap, iba-1 and ifn-c levels. this indicates that sgc-cgmp production can be inhibited by venom. in agreement, the cgmp accumulation by sildenafil inhibited the pnv effects, decreasing gfap, iba-1, ifn-c and sgc. the increase of sgc by venom could result from a feedback mechanism, when sgc activity is inhibited and its expression is increased. 7ni and ntp pre-treatment did not show difference compared to venom alone, suggesting that no per se is not involved in the inflammatory effects of venom. however, it is not discarded no and sgs-cgmp coupling in the mediation of pnv effects. we suggest that pnv induces glial reaction and neuroinflammation by sgc-cgmp inhibition. the study gives evidence that sgc-cgmp, coupled or not to no signaling, mediates pnv cerebellar effects including the bbb impairment. pnv can be a useful tool for studies on the mechanisms involved in glial regulation. cnpq/fapesp/faepex support. j. engele, m. puchert, v. € odemis university of leipzig, leipzig, germany it is currently believed that cxcl12 predominantly signals through cxcr4. in addition, it is assumed that the alternate cxcl12 receptor, cxcr7, represents a non-classical g protein-coupled receptor which primarily acts as a modulator of the function of cxcr4. discrepant from this view, we demonstrated recently that in primary rodent astrocytes and human glioma cell lines, sdf-1 exclusively signals through cxcr7 by a g protein-dependent mechanism. we now provide evidence that cxcr4 is essential for cxcl11/i-tac signalling in primary rodent astrocytes and some human glioma cells. treatment of cultured rat astrocytes with cxcl11 for 10 min resulted in the dose-dependent activation (phosphorylation) of erk1/2 and akt with maximum activation in the presence of 100 ng/ml of the chemokine. cxcl-11-dependent activation of both signalling molecules persisted in astrocytes in which expression of the established receptors for cxcl11, cxcr3 and cxcr7, were inhibited by rnai. however, cxcl11-dependent activation of erk and akt was abrogated following sirna-mediated inhibition of cxcr4. likewise, cxcl11 failed to activate erk and akt in cortical astrocytes cultured from a cxcr4-/-transgenic mouse line. moreover, similar to primary astrocytes cxcl11 activated erk and akt in the human glioma cell line, a767. again activation of both signalling molecules was abrogated following rnai-mediated inhibition of cxcr4. cxcl11-dependent activation of erk and akt further remained undetectable in a non cxcr4-expressing subpopulation of a767 cells, previously isolated by flow cytometry. together, these findings unravel a unique processing of cxcl11 and cxcl12 signalling in primary astrocytes which is preserved at least in some malignant astroglial cells. long-lasting activation of gfap-positive astrocytes occurs in brain tissue exposed to injuries that promote epilepsy development. studies in experimental models of epilepsy showed that activated astrocytes release various molecules, such as proinflammatory cytokines and danger signals, that play a role in seizures generation and recurrence. these activated cells also loose their ability to buffer extracellular k 1 and glutamate. this set of evidence suggests, therefore, that astrocytes may contribute to epileptogenesis (i.e. the post-injury phase prodromal to generation of spontaneous seizures), thus representing a putative biomarker of epilepsy. to test this hypothesis, we set up an in vivo longitudinal study using 1 h-magnetic resonance spectroscopy (mrs) to measure the hippocampal levels of metabolites that could reflect the extent and the duration of astrocytes activation after an epileptogenic brain injury. status epilepticus (se), which provokes epilepsy, was induced by pilocarpine in adult male rats. 1 h-mrs measurements were done in the hippocampus every 24 h for 7 d post-se, thus encompassing the epileptogenesis phase, and in chronic epileptic rats using a 7 tesla bruker biospec. spectra were processed and analysed using jmrui and tarquin freeware softwares. we studied changes in myo-inositol (mins) and glutathione (gsh), which reflect astrocytes activation. we found a progressive (2-fold, pwhich was maintained in epileptic rats. immunohistochemical analysis (ihc) of s100ß and gfap confirmed the concomitant activation of astrocytes in separate timematched groups of rats. gsh levels during epileptogenesis showed a negative correlation with the frequency of spontaneous seizures which developed after se. a negative correlation was also found between gsh and mins levels during epileptogenesis and the extent of neurodegeneration in hippocampus of epileptic rats. ihc done in epileptic rats at the end of the mrs experiments, showed that hippocampal s100ß levels positively correlated with spontaneous seizure frequency. since this is a soluble protein, further investigations of its csf and blood levels are warrented. our mrs findings show that gsh levels during epileptogenesis could serve as a predictive biomarker of the ensuing seizure frequency (thus of epilepsy severity) and, together with mins levels, also predict the extent of neuronal cell loss which develops following se in epileptic tissue. notably, ihc-detected s100ß levels in the epileptic tissue also reflect seizure frequency. these findings highlight the potential use of serial 1 h-mrs analysis of astrocyte activation for predicting the severity of epilepsy and the extent of neuropathology in the clinical setting. interleukin-6 (il-6) is a highly plurifunctional cytokine, with many pleitropic actions, considered one of the main cytokines controlling the immune system and coordinating it with the nervous and endocrine systems. il-6 is produced in multiple cell types in the cns, and in turn many cells do respond to it. it is therefore important to ascertain which the contribution of each cell type is in the overall role of il-6 during both physiological and pathological conditions. astrocytes are major responders to il-6 as well as one of the main cns producers of il-6. for this work we used astrocytary il-6 ko (ast-il6 ko) mice, which we already proved to have an important role in physiological conditions (like body weight control and exploratory/ locomotion behavior), in order to test astrocytary il-6 role during a neuroinflammation situation. for this purpose, we induced either an extensively used animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (eae), or a traumatic brain injury (cryolesion) in our mice. regarding eae, results indicate that lack of astrocytary il-6 delays clinical course of eae and ameliorate eae symptomatology in ast-il6 ko respect littermate controls in a gender-dependent way. further immunohistochemistry analyses confirm a decreased number of cellular and lymphocytes infiltrates and lesser demyelination, angiogenesis and gliosis in spinal cord of ast-il6 ko animals. regarding traumatic injury, astrocytary lack of il-6 facilitates microgliosis, lymphocytes infiltration and a faster decrease of the injured area. all these results are likely to bring some answers to astrocytesecreted il-6 involvement in neuroinflammation pathways and eae pathogenesis. interleukin-10 (il-10) is a counterregulatory cytokine that plays an important role in controlling inflammatory and immune reactions. in the central nervous system (cns), production of il-10 has been demonstrated in activated astrocytes and microglia after different types of injuries. the specific role played by il-10 inmodulating glial responses is however still unclear. hence, the objective of this study was to evaluate the effects of local astrocyte-targeted production of il-10 on glial reactivity using the axonal anterograde degeneration paradigm. for this purpose, unilateral perforant pathway transection (ppt) was performed on adult gfap-il10 transgenic (tg) animals and their corresponding wild types (wt) littermates. at 3 and 7 days post-lesion (dpl), animals were intracardially perfused with 4% of paraformaldehyde, brains frozen and parallel free-floating sections processed for glial analysis using immunohistochemistry for iba-1 (microglia) and gfap (astrocytes). our results showed that in comparison with wt animals, microglial cells in the dennervated dentate gyrus molecular layer of gfap-il10tg showed differential reactivity changes. after lesion, astrocytic reactivity presented a higher hypertrophy in gfap-il10tg animals when compared with their corresponding wt littermates. in conclusion, this study demonstrated that local production of il-10 inthe cns can modify the glial response associated with ppt. further studies are warranted to evaluate if these alterations in microglial and astrocytic reactivity have any repercussions for the evolution of the lesion and the associated axonal sprouting. astrocyte and microglia become reactive under many brain pathological conditions, making this process of neuroinflammation a surrogate marker of neuronal dysfunction. several studies have reported that reactive microglia overexpress the translocator protein 18kda (tspo, formerly known as peripheral benzodiazepine receptor). positron emission tomography (pet) using radioligands of tspo is thus considered as a potent technique to detect reactive microglia in situ. however, it is still controversial whether tspo pet imaging is also able to monitor reactive astrocytes in situ. this is an important question as reactive astrocytes and reactive microglia play very different roles in brain physiology and could impact disease progression in opposite ways. to address this question, we used a model of selective astrocyte activation through unilateral lentiviral gene transfer of the cytokine ciliary neurotrophic factor (lenti-cntf) in the rat striatum. cntf induced an extensive activation of astrocytes, which overexpressed gfap and became hypertrophic. microglia, on the contrary, displayed a minimal increase in the expression of markers of reactivity. cntf-activated astrocytes overexpressed tspo at the mrna and protein levels. pet imaging experiments demonstrated a significant and specific binding of two tspo radioligands [ 18 f]dpa-714 and [ 11 c]ssr180575 in the lenti-cntf-injected striatum. we show that reactive astrocytes can be monitored by tspo-pet imaging in the rat brain. this technique is thus well suited to monitor reactive microglia as well as reactive astrocytes, the two cell types involved in neuroinflammation, but would not allow their discrimination in situ. chronicity might establish a neuroinflammatory ganglion profile with inflammatory cells contributing to the hypersensitive phenotype. we first investigated whether, in trigeminal sensory ganglia, cytokines such as tnfa might contribute to a local inflammatory phenotype of a transgenic mouse model of familial hemiplegic migraine type-1 (fhm-1, cacna1a r192q knock-in mice). with respect to wild-type, r192q ki trigeminal ganglia were enriched in activated macrophages and expressed higher mrna levels of il1b, il6, il10 and tnfa cytokines and the mcp-1. functional consequences of crosstalk between macrophages and sensory neurons were studied in primary ganglia cultures, where larger release of soluble factors and larger currents mediated by pain-transducing atp-gated p2x3 receptors were found. consistently, we observed that, following lps injection, tnfa expression and macrophage occurrence were significantly higher in r192q knock-in ganglia with respect to wild-type ganglia. our data suggest that, complex cellular and molecular environment of sensory ganglia could support a new tissue phenotype compatible with a neuroinflammatory profile. we propose that, in selected patients, this condition might contribute to pain pathophysiology through release of soluble mediators, including tnfa and atp that may modulate the crosstalk between sensory neurons and resident glia, underlying the sensitisation process. cultures of astrocyte/microglia are stimulated with ifn-gamma and then infected with tachyzoites of neospora caninum they release nitric oxide (no) that controls parasite proliferation. in order of elucidating the immune response of cns against this parasite, this study investigated the participation of inducible nitric oxide synthase (inos) in the control of its proliferation in co-cultures of neuron-glia obtained from rat brain. the cells were stimulated with ifn-gamma (100 iu/ml-24h) then supplemented with l-ng-nitroarginine methyl ester/l-name, an inhibitor of inos (1.5 mm/ml -60 min) before infection with tachyzoites of n. caninum (1:1 parasite:cell). after 72h, in the cultures supplemented with l-name, it was verified, a reduction of tachyzoites number in 55.7% (control cells) and 59.8% (ifn-y stimulated cells). however, in infected co-cultures, it was not observed any release of no, even when cells where modulated by ifn-g. additionally, in cultures infected and treated with l-name, the levels of il-10 were increased in six fold (non stimulated) and nine fold (ifn-g stimulated). these data suggest that no should not participate as a mediator of n.caninum control in neuron/glia co-culture but, the regulatory pattern of immune response must play a role in its down modulation regulating the inflammatory mediators released during the cns infection, perhaps to protect neurons. methods: adult albino swiss mice were organized in two groups: naive (age-matched control; n 5 6) and lasered (n 5 6). anti-iba1 was used in retinal whole-mounts immunolabelled to analyze the distribution, morphology, density and arbor area of iba11 microglial cells. results: in na€ ıve, contralateral and lasered eyes, iba11 microglias were distributed throughout the retina in the: subretinal space (ss), outer plexiform layer (opl), inner plexiform layer (ipl), nerve-fibre layer (nfl) and ganglion-cell layer (gcl). in na€ ıve eyes, iba11 microglias: i) had rounded bodies with fewer and shorter processes in the ss; ii) exhibited a branched morphology emanating from small cell bodies in the opl and ipl; iii) were less ramified and were related with the retinal vessels in the nfl and in the gcl. by contrast, the morphological features exhibited by iba11 cells in contralateral and oht-eyes differed from na€ ıve: i) somas were more robust; ii) processes were thicker and more branched in contralateral eyes and thicker and retracted in oht-eyes. in oht-eyes and contralateral untreated eyes the iba11 microglial number was increased in comparison with na€ ıve (p < 0.001 and p < 0.05 respectively, t-test). in comparison to na€ ıve retinas the iba-11 cell arbor in the opl and ipl decreased in both oht-eyes (p < 0.001 and p < 0.001 respectively, t-test) and in the contralateral untreated eyes (p < 0.05 and p < 0.001 respectively, t-test). conclusions: two weeks of laser induced-oht triggered microglial retinal changes suggestive of activation in the contralateral untreated and oht-eyes. the microglial activation in contralateral eyes could be related to the immune response. this behaviour in contralateral untreated eyes, lead us to suggest that the use of contralateral eyes as internal controls in experimental unilateral oht, should be reconsidered. interleukin-6 (il-6) is a pleiotropic cytokine involved in inflammatory and non-inflammatory responses. among the latter, it participates in the regulation of body weight and metabolism. il-6-deficient mice develop mature onset obesity and have higher blood glucose, as well as impaired glucose tolerance and elevated blood leptin levels, suggesting that il-6 participates in suppressing adiposity in mice. moreover, transgenic mice with astrocyte-targeted production of il-6 challenged with a high-fat diet are resistant to high-fat diet-induced obesity, highlighting the role of centrally produced il-6 in the regulation of body weight. in this context, we hypothesize that mice lacking il-6 produced by astrocytes (ast-il6ko) will be more prone to develop obesity. we therefore challenged ast-il6ko mice (obtained with the cre-lox technology) with a high fat diet (58% kcal from fat) for 17 weeks and compared their body weight and food intake with those of mice fed a standard diet (18% kcal from fat). on weeks 14 and 15, the metabolic status was evaluated by an insulin tolerance test (itt) and an oral glucose tolerance test (ogtt), respectively. regarding body weight gain, we see a clear increase in ast-il6ko females on a high-fat diet in comparison to their controls with no apparent difference in food intake, which is also already apparent in the control diet. in males a similar tendency is observed. part of this difference can be attributed to the heavier subcutaneous white adipose tissue depots (relative to body weight) in ast-il6ko mice (fig 1) . insulin and oral glucose tolerance are compromised in mice fed a high-fat diet, but no differences between genotypes are observed. taken together, these results indicate that centrally produced il-6 has indeed a major role in the regulation of body weight, affecting subcutaneous white adipose tissue, but without significantly altering peripheral glucose metabolism. we are currently working on assessing hypothalamic neuropeptides with in situ hybridization to study the effects of astrocytic il-6 deficiency at the central level. minocycline is an agent with pleiotropic properties that targets multiple proteins and cellular processes associated with development of neuropathic pain, including inhibition of the injury-induced glia activation. the aim of our study was to examine the effects of minocycline on the injury-induced changes in immune factors and on morphine effectiveness in a rat model of neuropathic pain. chronic constriction injury (cci) to the sciatic nerve in rats was performed according to bennett and xie (1988) and 2 behavioral tests were conducted to measure allodynia (von frey test) and hyperalgesia (cold plate test). minocycline was administered intraperitoneally 16h and 1h before cci and then twice daily for 7 days. the studies were performed using competitive rt-pcr in the spinal cord and drg from control, cci-exposed and minocycline-treated cciexposed rats. morphine was administrated i.t. (chronic catheterization according to yaksh and rudy, 1976) and ip 1h after the last minocycline injection. the experiments were carried out according to iasp rules (zimmermann, 1983) . repeated administration of minocycline attenuated allodynia and hyperalgesia when measured 7 days after cci. minocycline downregulated the spinal and drg level of several immune factors: c1q, mmp-9, mmp-2, il-1beta, il-6, il-18, nos1 and nos2 which were increased in consequence of the injury. moreover, chronic administration of minocycline improved the response to i.t. and i.p. morphine injections. our results demonstrate that minocycline reduces the level of pro-inflammatory factors and increases the effectiveness of morphine, which may have clinical significance for enhancing analgesic effects of opioids in neuropathic pain. to date, there is no standardized simple model available to investigate the biology of human microglia. the aim of this study was to establish a new in vitro microglia model using blood-derived precursor cells. for that purpose, human peripheral blood monocytes were cultured in serum free medium in the presence of a mixture of cytokines and chemokines (m-csf, gm-csf, ngf and ccl2) to generate monocyte-derived microglia (m-mg). monocyte-derived dendritic cells (m-dc) were also generated as a control population using gm-csf and il-4. the human microglia cell line hmc3 was used as control. m-mg were clearly different in morphology, phenotype and function from m-dc, but shared many properties with hmc3 cells. m-mg acquired a ramified morphology with primary and secondary processes, comparable to hmc3. they expressed very low levels of cd45, cd14 and hla-dr, cd11b and cd11c; but a distinct pattern of chemokine receptors, including ccr1, ccr2, ccr3, ccr4, ccr5, cxcr1, cxcr3, cx3cr1. similar to hmc3, under non-activated condition, the m-mg secreted of il-8 and il-6. in comparison with m-dc, m-mg displayed lower t-lymphocyte stimulatory capacity, as well as lower phagocytosis activity. in summary, we have established a new protocol for the generation of human monocyte-derived microglia, which is is feasible, well standardized and reliable, as it uses well defined culture medium and recombinant cytokines, but no serum or conditioned medium. this model will certainly be very helpful for future studies investigating the biology and pathology of human microglia. w. schaafsma university of groningen, neuroscience, section medical physiology, groningen, netherlands background: microglia are the innate immune cells of the cns. like other tissue macrophages, they can activate and commit to distinct reactive phenotypes in response to tissue damage and infections. however, the microglial response to tissue damage may change over age and by past experience. these changes in activation patterns may be caused by epigenetic modifications which in turn can result in changes in gene expression of, for example inflammatory cytokines. a well studied condition with an altered responsiveness of innate immune cells is 'endotoxin tolerance' (et). innate immune cells that are pre-exposed to endotoxins are dampened in their inflammatory response upon re-exposure to these endotoxins. while et is well described for peripheral macrophages/monocytes, very little is known about et in relation to microglia. objective: in our experiments we set out to investigate the presence of et in microglia and if an 'epigenetic' memory is involved. design/methods: in our in vitro experiments, microglia cells were stimulated with lps (100 ng/ml). naive microglia only received one lps stimulation at the end of culture, pre-stimulated microglia received an 24h pre-stimulation of lps (100 ng/ml) and a second stimulation 5 days later. in vivo, male c57bl/6 mice received an i.p. injection with either pbs or lps (1 mg/kg) and 4 weeks later again an i.p. injection with pbs or lps. transcription levels and secretion of il1-b and tnf-a were assessed by use of quantitative pcr and elisa. in order to answer if there is a long lasting epigenetic memory involved in this tolerance we used chromatin immune-precipitation (chip), to investigate changes in covalent histon tail modifications associated with either permissive or repressed chromatin. results : in vitro, we show a 'tolerant' phenotype in microglia which receive a second lps stimulation after 5 days. a reduced expression level of pro-inflammatory genes il1-b and tnf-a and reduced secretion of tnf-a were observed in response to a second lps challenge. furthermore, with in vivo experiments we showed that mice which received a second lps injection after 4 weeks, showed a dampened response in their microglia inflammatory reaction at the level of il1-b and tnf-a gene transcription. in both in vitro and in vivo experiments we showed a corresponding pattern for histon tail modifications in the enrichment of activation marks h3k4me3 and ach3 at the il1-b and tnf-a promotors. we are the first to show a long lasting 'tolerant' phenotype in mouse microglia in vitro and in vivo and the involvement of epigenetic changes at the level of histon modifications. morphological criteria and dual immunofluorescent labelling confirmed expression of er stress protein in neurons, astrocytes, microglia or oligodendrocytes. an intriguing finding was localisation of crt to the rim of oro-positive myelin fragments and the 'patchy' nature of crt staining seen when tissue was dual-labelled with crt and gfap or iba1. these results are the first demonstration of significantly higher levels of crt in rodent eae. chop and p-eif2a data has also not been reported in rat eae. this study highlights the potential importance of er stress in inflammatory demyelination. the authors acknowledge support from the irish research council and from the foundation office of nui, galway. (3). considering the responsible signaling pathways regulating adult neurogenesis (4), we observed differential regulation of wnt members in the hippocampus on transcriptional level, e.g. wnt dependent transcription factors (axin2, tcf4) and ligands (wnt3, 5a, 8b and 11), as well as on translational level represented by the wnt signaling cascade (active-bcatenin, phospo-glycogen synthase kinase 3b) during acute and chronic states of disease. by using in vitro studies in primary hippocampal cultures, we further show the role of transforming growth factorbb1 (tgfb1), a key cytokine early involved during eae (5), in regulation of wnt ligand expression and wnt signaling activity. furthermore we are able to visualize the connection between tgfb1 and wnt signaling by using the axin2-lacz mouse, a wnt reporter mouse, for functional and histological investigation of the hippocampus. taken together our results suggest a cross talk of inflammation and wnt signaling for dysregulated hippocampal neurogenesis. interleukin-6 (il-6) is a cytokine with major regulating effects of the inflammatory response. moreover, il-6 is a neuropoietin that has neurotrophic effects related to neuronal survival and protection. to establish the importance of il-6 produced only in the central nervous system, we have generated mice producing il-6 essentially only in the brain by crossing gfap-il6 mice (transgenic mice with astrocyte-targeted production of il-6) with il-6 ko mice (il-6-deficient). we studied the inflammatory response in a traumatic brain injury model, cryolesion, after 3 and 10 days post-lesion gfap-il6-il6 ko mice, comparing them with appropriate controls. in basal conditions wt and il-6 ko mice showed a similar phenotype. this was also the case for gfap-il6 and gfap-il6-il-6 ko mice, which showed prominent astrogliosis, microgliosis, increased recruitment of t lymphocytes and vascularisation compared with the other two groups. in response to cryolesion of the cortex an increased astrogliosis, microgliosis, recruitment of t lymphocytes and vascularisation was observed. il-6 deficiency produced an altered inflammatory response, in a time-and gender-dependent manner. this study with il-6 ko and gfap-il6 transgenic mice indicates that during an acute neuropathological insult such as traumatic brain injury il-6, from either the brain or the periphery, has an important role on the inflammatory response. increasing evidence suggests that iron accumulation in the brain might contribute to neurodegeneration. iron is a potential source of free radicals as it can catalyze the production of hydroxyl radicals under oxidative conditions. this can lead to amplification of tissue injury caused by oxidative damage. we thus characterized iron storage within the central nervous system (cns) of animal models of different neurodegenerative diseases. we examined animals with acute inflammation mediated by cd8 or cd4 positive t cells and animals suffering from t cell and antibody mediated chronic inflammation due to active immunization. further, we studied lps induced lesions which represent cns disease caused by the innate immune system. similarly, we characterized oxidative damage in the different models for inflammation. we did not find evidence for the presence of oxidized phospholipids or oxidized dna in the experimental lesions, which is in contrast to our results of ms lesions. none of these models showed iron accumulation in glial cells comparable to what is seen in ms tissue. however, some iron positive microglia and perivascular macrophages were observed in acute and chronic models. in preliminary experiments we found evidence that the presence of iron exacerbates h 2 o 2 induced cell death in purified glial cells in vitro. as a next step, we wanted to study a possible amplification of oxidative damage and neurodegeneration by iron in a more elaborate system. for this purpose we treated myelinating spinal cord cultures with iron chloride (fecl3) or ferritin. we observed iron loading in microglia in both experimental setups. moreover we found a selective decrease of microglia in iron chloride treated cultures but not after ferritin application. we did not find iron loaded oligodendrocytes in this in vitro model. iron accumulation is only minimal compared to the human brain in all the tested animal models. these observations necessitate the search for additional animal models mimicking the human situation more closely. objectives: microglia are a brain resident population of immune cells, involved in homeostatic surveillance of the brain parenchyma, with high importance in the regulation of inflammatory processes after injury. there are unresolved questions regarding microglia origin, and their similarities to peripheral macrophage populations. our objective was to compare the capacity of microglia and bone marrow derived macrophages (bmdms) to adopt different phenotypes, and how this influenced cell death after brain injury. methods: we compared the phenotype of bmdms and microglia (both bv2 microglial cell line and primary microglia) after treatment with well known polarising agents. lipopolysaccharide (lps) was used as an inducer of the classical m1 phenotype and il-4 was used to induce an alternative m2 phenotype. microglia or bmdms, of different phenotypes, were added to hippocampal organotypic slices (hosc) subjected to oxygen glucose deprivation (ogd) as an in vitro model of brain injury. results: bmdms and microglia (bv2 and primary microglia) both adopt m1 or m2 phenotypes after treatment with lps or il-4 respectively. however, addition of polarised microglia or bmdms onto hosc resulted in different outcomes. addition of activated bmdms, of either m1 or m2 phenotypes, led to cell death in control hosc and increased death when combined with ogd. on the other hand, addition of bv2-microglia did not induce any cell death in control hosc, and were protective after ogd, except for m1microglia which were toxic. endogenous microglia within the hosc were also able to adopt different phenotypes and exert neuroprotection after il-4 treatment. these ex vivo data correlated with the in vivo situation where we observed increases in microglial activation early after experimental stroke, although the vast majority of these activated cells did not express markers of the m1 phenotype. conclusions: this study highlights functional differences between macrophages and microglia, especially in response to brain injury. although microglia, like peripheral macrophages, can adopt different demyelination, thereby creating an unfavorable environment for remyelination. exploring efficient ways to prevent or overcome tlr-induced fibronectin aggregation by astrocytes might pave the way for new approaches in remyelination-targeted therapeutic strategies in ms. to study the tlr2 response in-vivo, sod1 g93a mice were crossed with the tlr2-luc-gfp reporter mice, a transgenic mouse bearing the dual reporter system luciferase and green fluorescent protein under the transcriptional control of the murine tlr2 promoter. double transgenic mice and littermates controls were monitored longitudinally using in-vivo biophotonic/bioluminescent imaging to measure microglial activation over the course of the disease. surprisingly, this analysis did not reveal an increase of activation in sod1 g93a mice as the disease progressed, with only weak signal coming from olfactory bulb and brain area. however, quantification of this signal suggested a lower level of tlr2 activation in the sod1 g93a mice compared to wild-type littermates. to further study the microglial impairment observed in the sod1 g93a mice, these transgenic mice were given lps with i.p. injections at 5 mg/kg and were followed for 48h by bioluminescence imaging, both at a pre-symptomatic age (60 days) and closer to paralysis (115 days). the level of tlr2 activation was lower in the sod1 g93a mice than the wild-type littermates, both at 60 days and 115 days. immunostaining of olfactory bulbs reveal the same phenomenon, which is less iba1 and less tlr2-positive cells in the sod1 g93a tissue. mrna analysis by in-situ hybridization confirms a similar pattern of microglial response. in a parallel study, primary microglial cell culture, derived from adult mice (60days) were similarly stimulated with lps to further understand the altered sod1 mutant microglia response. here again, sod1 cells appears less responsive to lps stimulation as observed in our mice studies. these combined results suggest that sod1 g93a microglial cells might have a pre-symptomatic suboptimal immune response. the role of reactive glia in the etiology and progress of neurological diseases is still unknown. reactive glia produce several factors, typical of an inflammatory response, with a potential neurotoxic effect, highlighting the relevance of a strict control of the progression and resolution of glial activation. under some circumstances glial activation does not resolve, but it exacerbates or becomes chronic resulting in detrimental secondary effects. it is necessary to develop strategies to halt the negative outcome of glial activation in these situations, by controlling the pro-inflammatory phenotype of reactive glial cells and potentiating their beneficial effects. we studied the temporal pattern of inflammatory reaction in spinal cord and brain regions in the experimental autoimmune encephalomyelitis (eae) model of multiple sclerosis. special attention was paid to the involvement of members of the c/ebp family of transcription factors, which regulate the expression of pro-inflammatory genes in reactive glia, and cd200, cd200r1 and trem-2, membrane-associated inhibitors of the pro-inflammatory response in resting/surveillant microglia. mice were scored for signs of eae for up to 28 days postimmunization (dpi). eae symptoms were present from 10 dpi, reaching score peak at 14 dpi. in the spinal cord, we observed a sustained increase in c/ebpa and a transient increase in c/ebpb and c/ebpd expression peaking at 14 dpi. cd200 expression decreased at 9 dpi, remaining below control values at 28 dpi. cd200r1 and trem-2 expression increased at 14 dpi, thereafter this effect progressively attenuated. no alterations were observed in the brain. an acute increase in the expression of pro-inflammatory genes (il-1b, il-6, tnf-a, inos, cox2) occurred in the spinal cord at 14 dpi. our results suggest that cd200 expression decreases before onset of eae symptoms resulting in downregulation of the microglia inhibitory signal mediated by cd200-cd200r1, which would facilitate the proinflammatory response observed after onset of the eae symptoms. the increase in cd200r1 expression observed after the onset of eae symptoms suggests a concomitant intend of microglia/macrophages to resolve the inflammatory response. the transient increase in c/ebpb and c/ebpd expression could be related to the acute increase in proinflammatory gene expression, while the sustained increases of c/ebpa expression and trem-2 could be involved in the resolution of the inflammatory response. recently, we showed a beneficial effect of myelin reactive t cells on oligodendrocyte precursor cell differentiation in zones of axonal degeneration in the hippocampal dentate gyrus, which mirrors grey matter injury in multiple sclerosis. this effect was associated with a marked expression of t-cell cytokines, such as interferon-c and interleukin-17, enhanced microglial clearance of myelin debris, and an enhanced sprouting of calretinergic axons. to gain better insight in which cytokines and signaling pathways are elevated in response to the t cell enhanced regenerative responses, we performed a mrna microarray study, in which the transcript profile in hippocampi from mice with adoptively transferred proteolipid protein specific t cells combined with an axonal lesion were compared to the transcript profile from mice with axonal lesion. thereby we identified 1446 transcripts, which were differently expressed. we identified the interleukin-1 (il-1) signaling pathway as a potential target by performing pathway analysis using david and amigo databases. we discovered that il-1a, il-1b and il-1 receptor antagonist (ra) mrna as well as the il-1 signaling pathway were highly upregulated in response to myelin reactive t cells. in order to determine if il-1b mrna was translated into protein, we performed immunohistochemical stainings on tissues from mice with 2 days post lesion survival. expression of il-1b was observed in the molecular layer of t cell infiltrated, but not in t cell na€ ıve mice with axonal lesion, suggesting that myelin reactive t cells stimulate il-1b protein expression. ongoing studies will determine the expression of il-1a and il-1ra protein and determine the cellular expression of the il-1 signaling pathway in t-cell infiltrated versus non-t cell infiltrated mice with axonal lesion. understanding the regulation of the expression of il-1a, il-1b and il-1ra in t cell compared to non-t cell infiltrated cns may lead to a better understanding of the functional consequences of inflammatory responses in the cns. when io were challenged with kainate in the presence of conditioned medium (cm) from astrocytes, susceptibility to kainate-induced cell death was reduced, but cm from l-ap4-treated astrocytes reverted kainate toxicity. in contrast, treatment with cm from control or lpsactivated microglia, either untreated or pre-exposed to l-ap4, was not able to affect kainate-induced io cell death. to establish whether mglur4 activation could modulate the antigen presenting activity of microglia, the bv2 microglia cell line was used. treatment with l-ap4 (50 lm/48 h) did not affect changes in morphology induced by activation with lps (0.1 lg/ml/48 h), but significantly reduced the percentage of cells expressing mhc class ii, as detected by flow cytometry. these data suggest that during neuroinflammation, activation of mglur4 directly on io or through astrocytes and microglia may contribute to a protective effect resulting in survival of the oligodendrocyte lineage. mirnas are small non-coding rna molecules that modulate gene expression at a post-transcriptional level and their role in the regulation of biological processes makes them an emerging class of therapeutic targets. dysregulation of mirna networks has been linked to neurodegeneration and immune dysfunctions and has become a research focus in the context of alzheimer's disease (ad). in this work, we demonstrate that mir-155 expression is increased in the brain of 33trg ad transgenic mice, prior to senile plaque formation, and co-localized with intraneuronal app accumulation in the cortex and hippocampus. in view of the pro-inflammatory functions of mir-155 and aiming at clarifying its role in ad, we evaluated the levels of mir-155 in ab-stressed astrocytes and microglia cultures, two brain-related cell types involved in neuroinflammation. we show that mir-155 expression levels are increased in astrocytes and microglia following activation with ab fibrils but not with ab oligomers. these findings correlate with the observed decrease in socs-1 expression, a mir-155 validated target, and with the increase in the production of tnf-a, il-1b and il-6 by these cells. importantly, we show that mir-155 expression is regulated by c-jun, since silencing of this transcription factor led to the reduction of mir-155 levels following astrocyte and microglia exposure to ab fibrils or lps. c-jun was also found to be upregulated in the 33trg ad transgenic model since early ages, which can help to explain the observed early increase in mir-155 expression. overall, our results demonstrate the important role of mir-155 in ad and show that silencing of c-jun in glial cells may constitute an interesting and promising anti-inflammatory therapeutic strategy towards this disease, by targeting mir-155-mediated inflammatory responses. here we demonstrate that tgfb is an important endogenous factor promoting quiescence of microglia in vitro. inhibition of microglial tgfb signalling resulted in a microglia polarisation towards the classical activation state. moreover, tgfb is able to significantly decrease ifnc-induced classical activation of primary microglia and to enhance il4-induced alternative microglia activation. we further provide evidence that il4-mediated alternative microglia activation is dependent on active tgfb signalling. these results demonstrate the essential functions of tgfb as an important regulator of microglia activation states and further suggest that tgfb might be an interesting therapeutical molecule to modulate microglial functions during development and progression of neurodegenerative diseases. we found that young mice lost weight only in the first 24h after infection, whereas aged mice exhibited a biphasic pattern of weight loss, with the second phase of weight loss beginning 1 week after infection and continuing for approximately 2 1 =2 weeks. aged mice also demonstrated a co-ordination and balance deficit in the static rod test 1 week after infection compared to uninfected or 3 week post infection aged mice, whereas young mice were unimpaired at any timepoint. s. typhimurium infection caused a significant, progressive reduction in open field rearing activity at 1 week and 3 weeks after infection in both young and aged mice. a similar but not significant trend was apparent in novel object performance. forced swim test data showed a trend towards depressive behaviour at 1 week after infection, but not at 3 weeks. analysis of pro-inflammatory mediators in the hippocampus did not indicate significantly upregulated il-1b, tnf-a, cox-1 or cox-2 transcript at any timepoint, suggesting that microglia might not play a significant role in mediating these behavioural effects, or that the changes in these molecules were too subtle to reliably detect using qpcr in the hippocampus. the effects of ageing on s. typhimurium induced weight and co-ordination changes may be a result of regional differences in the sensitivity of microglia to peripheral infection within the cns or increased sensitivity to activation of neuronal circuits involved in weight loss or co-ordination with age. we have studied microglial cells with anti-iba1 and anti-cd68 immunohistochemistry in the brains of 18 subjects with pretangle neuropathology ranging from 4 to 51 years of age. regions examined included the locus coeruleus and surrounding brainstem areas, entorhinal cortex, hippocampal formation, and other parts of the mesial temporal lobe. ten of the subjects (median age 34) showed severe neuroinflammation secondary to infectious disease (hiv, sepsis, toxoplasmosis) and cns trauma, while 8 subjects (median age 32.5) died from non-infectious conditions and lacked neuroinflammatory changes (controls). we also examined two 52-year-old subjects with down syndrome both of which showed braak stage vi neurofibrillary degeneration, where one had comorbid sepsis and the other one did not. pretangle neuropathology was staged 1a and 1b in all 18 subjects, i.e. they exhibited primarily subcortical tau pathology in the locus coeruleus and only sporadic lesions in the transentorhinal cortex. our findings show that rampant microglial activation in the ten subjects with infections and trauma was coincident with the same level of tau pathology as seen in the 8 control subjects, demonstrating that neuroinflammation does not initiate or exacerbate neurofibrillary degeneration. in addition, our findings show that the extent of neurofibrillary pathology seen in down syndrome is unrelated to neuroinflammation since the down subject without comorbid sepsis revealed a complete absence of microglial activation. overall we conclude that neuroinflammation is not causally involved in the development of neurofibrillary degeneration which is most characteristically associated with alzheimer's disease dementia. it has been suggested that peripheral infection/inflammation may play a role in the onset or development of not only neurodegenerative diseases but also depression, autism and chronic fatigue syndrome through inflammatory responses in the brain. to investigate the role of microglia in this hypothesis we used a model of neuroinflammation induced by an intraperitoneal (i.p.) injection of the toll-like receptor 3 (tlr3) agonist, polyinosinic:polycytidylic acid (poly i:c) in rats. as we reported previously an injection of poly i:c (i.p) decreased the daily amounts of spontaneous running wheel activity to $60% of the preinjection levels until day 7. microglia were morphologically activated in the prefrontal cortex (pfc) until 72 hrs after the injection of poly i:c. pretreatment with minocycline for the 3 consecutive days (40 mg/kg/ day) blocked the poly i:c-induced decrease in the running wheel activity as well as microglial activation. quantitative analysis of mrna levels demonstrated that interferon-a (ifn-a) increased in the pfc on both days 1 and 7. we found in the present study that poly i:c injection induced increases in mrnas relevant to tlr-signaling such as tlr3, interferon regulatory factor 3 (irf3) and irf7 in the pfc. furthermore, direct application of poly i:c to the primary cultured microglia also induced an enhanced expression of ifn-a, tlr3, irf3 and irf7. it has been reported that permeability of blood-brain barrier is increased after poly i:c injection (wang et al., 2004) . therefore, it is possible that the peripheral poly i:c enters into the brain to induce neuroinflammation by activating microglia. interestingly, intracerebroventricular (i.c.v.) injection of primary cultured microglia activated by poly i:c, but not by lipopolysaccharide, effectively produced a significant decrease in spontaneous activity in rats. taken together, these findings suggest that microglial activation is important for poly i:cinduced neuroinflammation through inducing tlr3-related genes. roles of each gene in the behavioral changes will be further studied. results: microglia produced nanogram levels of pgrn (10 fold higher in microglia than in astrocytes), and pgrn release from microglia was suppressed by the tlr ligands or il-1/ifnc, but increased by il-4 or il-13. unexpectedly, while astrocytes stimulated with proinflammatory factors released large amounts of slpi, none were detected in microglial cultures. we also identified mmp-12 as a pgrn proteolytic enzyme, and slpi as an inhibitor of mmp-12-induced pgrn proteolysis in human microglia. conclusions: our results establish microglia as a significant source of pgrn. mmp-12 and slpi are modulators of microglial pgrn proteolysis. negative and positive regulation of microglial pgrn release by the proinflammatory/th1 and the th2 stimuli, respectively, suggests a fundamentally different aspect of pgrn regulation compared to other known microglial activation products. microglial pgrn appears to function as an endogenous modulator of innate immune responses. at pathological condition such as injury or ischemia to the central nervous system (cns), microglial cells and astrocytes become activate and inflammation occurs like proliferate, migrate and secrete some proinflammatory cytokines, il-1ß, tnf-a or il-6. however, it is unclear that the reason why inflammation is induced in the cns, and whether or not it is necessary for the brain during recovery from pathological situation. to know the molecular mechanisms for inflammation occurs in activated glial cells, we made a stab wound mouse model to the brain. we performed microarray analysis for rna extracted from the brain tissue around stab wound site compared among day after 0, 1, 3 and 7. it revealed that most of the genes from top 20 genes at high expression level around lesion site were concerned in immunological or inflammatory functions. we successfully identified and focused on to osteopontin (opn), which is an inducer of pro-inflammatory cytokine production expressed not only in reactive microglial cells but also in reactive astrocytes around the injured cns. furthermore, we also found receptors against opn functioning in the injured brain. however, functional role of opn in reactive astrocytes is not yet clarified. to analyze the central role of opn in reactive astrocytes, we examined primary culture of astrocytes from opn-deficient (opn/ko) mice and found that the morphology of these cells was unusual. by the stimulation with lipopolysaccharide to the primary culture of astrocytes from opn/ko mice, some of the pro-inflammatory cytokine expression was altered. moreover, reactivity of astrocytes caused by stab wound to the brain was examined using analogue of nucleic acid, bromo-deoxy-uridine (brdu), and clarified that it was decreased in opn/ko mice. from these results, we conclude that opn might play a major role in the activation of astrocytes under inflammation occurred to the cns. here we determined the influence of a hfd on eae. we show that feeding mice with a hfd exacerbates eae severity through the induction of the brain renin angiotensin system (ras), resulting in an increased immune cell infiltration into the cns, oxidative stress and th1 cytokines. moreover, peripheral inflammation was boosted upon a hfd due to a ras independent mechanism, indicating that a hfd exacerbates neuroinflammation in various manners. these data suggest that nutritional modulation may have an important influence on the course of ms and other neuroinflammatory conditions. the brain's immune privilege has been attributed to a lack of dendritic cells (dc) within its parenchyma and the adjacent meninges implying maintenance of antigens rather than their presentation in lymphoid organs (lo). using cd11c-gfp mice, we have recently reported the existence of a cd11c/iba-1/cd11b 1 -population in the juxtavascular parenchyma which extent their processes into the glia limitans, an ideal position for antigen-presentation. therefore, we phenotypically compared them to the cd11c 1 /cd45 1 population (all isolated with the same protocol used for brain cd11c 1 cells) from lung, liver and spleen in healthy mice using 7-color flow cytometry. we found unique, sitespecific expression patterns of f4/80, cd80, cd86, cx3cr1, ccr2, flt3 and mhc-ii. as described before, two different cd45 1 -populations (cd45 high and cd45 int ) in the brain can be separated, whereas liver, lung and spleen exhibit a more homogeneous cd45 high population. a higher percentage of the brain's cd45 1 /cd11c 1 -cells were f4/ 80-positive compared to spleen, liver and lung, but expressed it at lower levels. within the cd45 int /cd11c 1 -population most cells expressed the microglia marker cx3cr1 and ccr2 low (marker for inflammatory, motile cells). most importantly, compared to spleen and liver, cd45 int /cd11c 1 -cells from the brain almost completely lacked mhc-ii expression and cd45 high /cd11c 1 -cells from the brain have a lower percentage of mhc-ii 1 -cells. since the cd45 int cells are widely regarded as the resident, intraparenchymal microglial population and cd45 high as dcs and perivascular macrophages, our data confirm the view that a small subpopulation of microglia (cx3cr1 high , f4/ 80 1 ,ccr2 low ) share established immune phenotypical characteristics of dcs (cd11c 1 ). additionally we showed that intraparenchymal cd11c 1 microglia are unique in their low expression of mhc-ii. their weak expression of cd80 is in line with a tolerogenic phenotype. methods: we performed a series of experiments, where we examined the expression of pro-inflammatory factors in resident microglia and infiltrating macrophages after fluorescence-activated cell sorting (facs) at different eae stages. in order to properly discriminate the two cell types we used a set of markers where resident microglia were defined as cd11b 1 cd45 int ly6c -, and infiltrating macrophages cd11b 1 cd45 hi ly6c 1 . using this protocol we have analyzed the rna expression levels of mhcii (cd74, h2-aa), co-stimulatory molecules (cd80, cd86, cd40, cd274), pro-inflammatory genes (il-1b, tnf-a) and inflammasome-regulated cytokines in these immune cells with quantitative pcr. followed by analysis of mhcii, cd80 and cd40 at the protein level with flow cytometry. results: our results indicate that infiltrating macrophages have a pronounced activated (cd74 and h2-aa) and pro-inflammatory phenotype (il-1b and tnf-a) at rna level. this is supported with an increase of the expression of mhcii and the key co-stimulatory molecules cd80 and cd40 at rna as well as protein level. in contrast, microglia display a decrease in the expression of the co-stimulatory molecules cd80 and cd86 at the rna level and do not up-regulate expression of il-1b and tnf-a during the progression of eae. conclusion: our data suggest that upon eae, infiltrating macrophages display an inflammatory profile whereas microglia are only mildly immune activated. background: age-related hearing loss (presbycusis) affects half of people by the age of 75. it has a large impact on quality of life and is associated with accelerated cognitive decline. however, presbycusis is not an inevitable process of aging, suggesting that its progression could be minimised. hearing relies on sound vibrations from the middle ear to elicit movement of the basilar membrane in the cochlea, resulting in excitation of hair cells. spiral ganglion neurons relay the inner hair cell signals to the central auditory system via the vestibulocochlear nerve. sounds are integrated in the central auditory pathway and then processed by cognitive areas of the brain. degeneration of cochlear structures, including hair cells and spiral ganglion cells occurs in presbycusis. in the central auditory system there are alterations in neuronal plasticity and neurodegeneration may occur. chronic inflammation has previously been correlated with agerelated hearing loss in humans, implicating immune cells in the progression of presbycusis. microglia within the central nervous system are susceptible to priming by neurodegeneration or aging, both factors in presbycusis. it is feasible that microglia in the cochlea, an organ with similar immune privilege as the brain, could also become primed. priming increases the chance of microglia becoming activated by subsequent inflammatory events, such as 'flu, which may cause them to contribute to neurodegeneration in the cochlea and central auditory system. hypothesis: our working hypothesis is that as age-related hearing loss progresses, microglia in the cochlea and central auditory pathway will become primed. we predict that systemic inflammation will cause primed microglia to be activated to a pro-inflammatory damaging phenotype, which will increase the rate of cochlea and auditory pathway degeneration and hence hearing loss progression. methods: c57bl/6j mice are genetically predisposed to develop age-related hearing loss. young and middle-aged mice will be challenged with lps or saline (control) and microglial phenotype assessed in the cochlea and central auditory system. neurodegeneration will be evaluated at corresponding points in the cochlea and auditory pathway using molecular techniques. conclusion: if our hypothesis is proved this would suggest that rigorous control of infection in older individuals would reduce progression of hearing loss by minimising degeneration in the auditory system. this receptor triggers the response via both myd88-and trif-dependent signalling pathways, leading to production of cyto-and chemokines and thereby alarming and attracting the immune cells of the periphery to invade the brain. however, tlr4 is only fully functional in complex with several co/receptors, such as cd14. even though cd14 has been traditionally considered simply as a lps affinity provider, recent data indicate that cd14 is also necessary for the endocytosis of tlr4, which links tlr4 to intracellular signalling via the adapter protein trif. our latest data reveal the critical role of this receptor for the profile as well as the magnitude of microglial cyto/chemokine production in response to diverse lps variants. cd14 increases the sensitivity towards lps in a cell type-specific manner, making microglia far more sensitive to lps than bone marrow and peritoneal macrophages. while striatal applications of low lps doses reveal less incoming neutrophils into a brain of cd14ko mice, as compared to wildtypes, high doses lps lead to an excessive neutrophil infiltration. this phenomenon is well correlating with the observations in vitro, where cd14 absence in microglia stimulated with high doses lps causes an excessive production of selected chemokines, with the highest impact on the neutrophil chemoatractant cxcl1. these regulatory activities of cd14 require its membrane insertion and prolonged functionality, pointing to an involvement of signalling. in order to reveal candidates for such signalling mechanisms, we tested the role of syk and plc. even though these enzymes were described to play a role in cd14-dependent signalling in dendritic cells, they have no contribution in microglia, further pointing to a cell typespecific organization of the cd14/tlr4 complex in microglia. importantly, we identified receptor systems that impose influences on cd14 expression itself, which would thereby determine the extent and impact of a cd14-mediated regulation of tlr4 functions. supported by the dfg (for1336). in neurodegenerative disease misfolded proteins and cues released by dying cells can attract immune cells by chemotaxis and alter their phenotype. to better understand and differentiate between the effect of these cues, we aim to elucidate the spatiotemporal immunological events in the brain in vivo. zebrafish are an excellent model system to study leukocytes in vivo, and we previously showed by live imaging how engulfment occurs in the developing zebrafish brain (van ham et al., curr biol 2012). here we use genetically targeted cell ablation to specifically induce cell death of target brain cells. we subsequently track dying cells and immune cells by single and multiphoton 4d imaging using fluorescent reporter genes. to analyse immunological infiltrate and resident cells in an unbiased fashion, we use large-scale electron microscopy (em) of complete zebrafish cross-sections in parallel as a complementary approach. we find that within a day after onset of cell death multiple types of phagocytic cells, including microglia, accumulate in areas where cell death occurs, engulfing dying cells. granulocytes, do not migrate towards these dying cells and are not found inside the brain at these stages. we also find total numbers of immune cells, microglia in particular, are increased. in addition to microglia, we find phagocytic peripheral cells to be involved in clearance of dying cells. our findings provide an initial in vivo characterization of the immune response to programmed cell death in the brain, indicating involvement of resident and peripheral immune cells. we show spatiotemporal recruitment of these different immune cell types, and proliferation of microglia. the latter together with the recruitment of peripheral leukocytes are likely triggered to increase capacity to dispose of dying cells efficiently. our results indicate zebrafish provides a model to tease apart basic dynamic properties of immune maintenance of the diseased brain in vivo. our current studies focus on identifying the sequence of immunological events in resolving brain injury in vivo and how this is controlled, to ultimately help identify which of these aspects are harmful during brain damage or promote tissue recovery. in humans, lif and osm both signal through the lif receptor (lifr), however osm can also activate its specific receptor, osmr. lifr signaling promotes the survival of glial cells and neurons, and is thought to limit the development of pathogenic t helper 17 cells while promoting differentiation of protective regulatory t cells. osmr signaling is also neuroprotective, however the effects on immune cells are not yet elucidated. in this study, the immunomodulatory effects of lifr and osmr signaling in humans are investigated. we determined which immune cells express the receptors for lif and osm in healthy donors and compared this to the expression levels in ms patients. we found that in blood of healthy donors approximately half of the monocytes express the lifr and osmr, while 5-10% of the t cells and b cells express the receptors. more importantly, in untreated ms patients higher numbers of t cells and b cells express both the lifr and osmr as compared to healthy controls and treated ms patients. available treatments suppress the immune system, resulting in less activated t and b cells, which could explain the lower receptor expression. indeed, we measured the receptor expression on t cells and b cells after activation and demonstrated this strongly induces expression of both receptors. currently, we are investigating the effect of lif and osm on proliferation, cytokine production and antigen presentation of the different immune cell subsets. as blood circulating immune subsets of ms patients have a higher expression of lif and osm receptors, patients show increased susceptibility for modulation by these cytokines. thus, their immunoregulating properties together with the protection of glial cells and neurons indicates that these cytokines are promising candidates for the treatment of ms and other neuroinflammatory diseases. interleukin-6 (il-6) and interleukin-10 (il-10) are key cytokines with an important role in the regulation of the inflammatory and immune responses. in the central nervous system (cns), increased expression of both il-6 and il-10 occurs in a wide range of pathological conditions. meanwhile il-6 is recognized as a cytokine with a dual role acting as a pro-inflammatory or anti-inflammatory signal inducing glial activation, il-10 is well known by its ability to counteract the inflammatory and immune reactions. the objective of the present study was to evaluate the effects of local production of either il-6 or il-10 on the microglial response and the neuronal degeneration induced by facial nerve axotomy, a sterile neuronal injury model. to accomplish that, we used two transgenic mice, gfap-il6tg and gfap-il10tg, which express either il-6 or il-10 under the gfap promoter on astrocytes, i.e. a local production within the cns. unilateral facial nerve 1mm resection was performed, one set of transgenic and wild-type (wt) axotomized animals were sacrificed at 3, 7, 14, 21 and 28 days post injury (dpi) and cryostat free-floating sections were processed for immunohistochemical analysis. another set of animals were sacrificed at 21dpi to study neuronal survival. our results showed that, at 21 days after facial nerve axotomy, in comparison with the usual neuronal death observed in wt, selective cns il-6 production had a detrimental effect on neuronal survival, whereas, il-10 production was able to reduce neuronal death. these effects on neuronal survival correlated with changes in the expression of different molecules associated with microglial activation such as iba1, cd11b, cd16/32, mhc class ii, and some integrins like osteopontin and its receptors (cd44 and a5), along the different time points after injury. remarkably, when compared with their wt littermates, cd11b expression was higher on gfap-il10tg mice, whereas remains lower on gfap-il6tg animals along all the time-points analyzed. similarly, cd44 expression, which increases after axotomy in wt mice, was higher on gfap-il10tg mice at 14 dpi and 28 dpi, whereas no induction of this molecule was detected on the gfap-il6tg axotomized animals. in conclusion, our results indicate that astrocyte-targeted il-6 and il-10 production has a direct impact on neuronal survival, glial activation and integrin mediated signaling, producing a specific outcome of facial nerve axotomy. methods: we used bv-2 cells, a murine microglial cell line. in a first experiment aimed at determining the time course of the induction of expression of the lipoxygenases and receptors, they were incubated with lps. in a second experiment aimed at determining the effects of the resolvins and lipoxin on the production of il-10, il-1b, il-6 and tnfalpha, they were firstly incubated with lxa4, rvd1 and rve1 and then incubated with lps. results: our results indicated that lps only enhanced the expression of alx, the receptor for rve1 and lxa4 ( i2) (p < 0.05). the expression of the 15-lipoxygenase was also affected by lps (p < 0.01) and varied during the time course, reaching a maximum after 6h of incubation (p < 0.05). the production of the proinflammatory cytokines decreased after 18h of incubation: il-1b with rvd1 (100 nm, p < 0.001) and rve1 (10 nm, p < 0.01); il-6 with rve1 (1 and 10 nm, p < 0.05 and p < 0.001); tnfalpha with rve1 (1 and 10 nm, p < 0.001 and p < 0.01). the production of the anti-inflammatory cytokine il-10 increased only when cells were incubated with lxa4 after 6h (1 and 10 nm, p < 0.01). conclusions: these results suggested a potential role of pufa-derivates in the resolution of inflammation. methods: the expression of a panel of m1 and m2 markers on human monocyte derived m1 and m2 macrophages was analyzed using flow cytometry. this revealed that cd40 and mannose receptor (mr) were the most distinctive markers for m1 and m2 macrophages, respectively. we next examined the activation status of macrophages/microglia in ms and control tissue using a panel of m1 and m2 markers. results: our data show that m1 markers, were abundantly expressed by microglia in normal appearing white matter and by activated microglia/macrophages throughout active demyelinating ms lesions. m2 markers, mr and cd163, were expressed by myelin-laden macrophages in active lesions and perivascular macrophages. double stainings using anti-cd40 and anti-mr revealed that 70% of the results and conclusions: the aim of the present study was to evaluate the specific binding of [ 3 h]pk11195, a compound targeting tspo, to human tissue sections from patients with ms, and to correlate these findings with presence of tspo1 microglia in different types of lesions. specific binding of [ 3 h]pk11195 was evaluated using autoradiography, and histological analysis was performed on tissue sections to evaluate lesion type, tspo expression and presence of microglia. there was a clear correlation between level of specific binding of [ 3 h]pk11195 and lesion type, such that there was an increased binding to tissue with active or chronic-active lesions compared to control tissue or tissue with chronic lesions. this was concomitant with an increased tspo1 expression and increased presence of microglia in tissue sections with active or chronic-active lesions. in conclusion, specific binding of [ 3 h]pk11195 is correlated with lesion type, tspo expression and presence of microglia in tissue sections from ms patients. being astrocytes key regulators of microglial cell activation. during inflammatory response, glial cells secrete cytokines such as tnfa, il1b and tgfb, as well as nitric oxide (no), and activate phagocytosis, chemotaxis, increased expression of receptors that bind cytokines and inflammatory ligands. among ligand receptors are induced pattern recognition receptors such as scavenger receptor (srs), including class a receptor (sra), expressed by astrocytes and microglia. we will evaluate if sra has active roles in ab clearance and inflammatory activation of glia. methods: we evaluated the role played by sra in glial cell activation, and the regulatory capacity of astrocytes upon microglia in vitro, assessing production of no griess assay and cytokines by elisa; phagocytosis and activation of activity identity markers of signaling pathways by western blot. results: in primary glial cultures from wild type (wt) and knockout mice (ko) for sra, it was found that lps-stimulated sra ko astrocytes release 50% more no than sra wt astrocytes. these differences were associated with an extended activation of erk signaling pathway. in contrast, evaluation of lps-induced cytokines production, only wt astrocytes released il1b, whereas production of tnfa and tgfb reached similar levels in wt and sra ko animals. the abolition of il1b release by sra ko astrocytes appeared to be associated to the inhibition of activation of jnk and p38 signaling, and to the delayed activation of ijb/nfjb pathway in response to lps. microglia from sra ko mice also showed several differences in response to lps stimulation compared with their wt counterparts. they failed to show activation-associated morphological changes and were unable to secrete il1b, although they showed an increased release of no. in terms of activation pattern, sra ko microglia showed increased expression of mhc-ii, indicating that sra could be involved in a mechanism inhibiting mhc-ii expression that has not been previously documented. conclusions: our results suggest that sra participates in the activation of specific inflammatory responses by astrocytes and microglia, and the activation of signaling pathways involved in the production of soluble molecules, being also needed for astrocytes in order to be able to modulate microglia activation. microglia are the resident macrophages of the brain and the first responders to disturbances of brain homeostasis. they exist in one of two broadly defined states, a rather inaptly named "resting state" in which they exhibit a ramified morphology and, upon tissue disturbance, an "activated state" in which they exhibit an amoeboid morphology. the characterization as "resting" is in stark contrast with the frantic morphological changes that ramified microglia undergo in brain slices and in vivo, constantly extending and retracting processes. while the purinergic receptor p2y12 has been identified as a key receptor via which microglial processes are guided to a source of atp or to the site of an injury, the signals and pathways guiding microglial processes in the healthy tissue remain elusive. here, by imaging microglia in acute hippocampal brain slices, we assessed the baseline and targeted motility of microglial processes in the presence of different pharmacological agents targeting purinergic signaling. we found that while bath application of a p2y12 specific blocker alone (psb-0739 at 500 nm) or of a p2y1 specific blocker alone (mrs2179 at 25 mm) did not affect the baseline motility of microglial processes, bath application of a p2y1/12/13 blocker (mrs2211 at 25 mm) strongly reduced the baseline motility of microglia, leading them to retract most of their processes. moreover, a strong rebound of motility was observed after washing out mrs2211 that did not lead to microglial activation within the time window of the experiment (up to 90 minutes). in experiments measuring the chemotactic response of microglia to a pipette containing 1 mm of atp inserted into the slice, we found that chemotaxis was completely abolished by psb-0739 (bath applied at 2 mm) but not by mrs2211 (20 mm). finally, no chemotaxis was observed in response to a pipette containing udp-glucose (5 mm), an agonist of the p2y14 receptor. these results confirm that p2y12 alone controls the targeted chemotactic response of microglial processes to atp, with no discernable involvement of p2y1, 13 or 14, but suggest that p2y1, 12 and 13 might collectively play a role in controlling the baseline motility of microglial processes in the healthy tissue. how the different p2y receptors interact with each other to control microglial movements remains to be determined. supported by an eu marie curie fellowship, the wellcome trust and the erc. the ubiquitin-proteasome-system (ups) plays a central role in degradation and clearance of short lived regulatory as well as misfolded or damaged proteins. upon stimulation by interferons (ifn) the catalytic subunits b1, b2 and b5 of the 20s proteasomes (s-proteasomes) can be replaced with b1i/lmp2, b2i/mecl-1 and b5i/lmp7 subunits, giving rise to the catalytically active immuno-proteasomes (i-proteasomes), respectively. since i-proteasomes play an important role in clearance of oxidant-damaged proteins that preferentially accumulate upon inflammatory stimulation, and many neurodegenerative diseases, including alzheimer's disease (ad) and parkinson's disease (pd) are associated with a chronic inflammatory reaction, we tested the impact of i-proteasome deficiency on the pathogenesis and progression of ad and pd. while appps1 mice, a model for ad associated extracellular plaque pathology, exhibited increased levels of i-proteasome subunits in the brain, genetic ablation of the b5i/lmp7 subunit, which significantly impairs i-proteasome function, resulted in a reduction of cerebral plaque burden, indicating a disease exacerbating role of iproteasomes in ad. in contrast, transgenic alpha-synuclein (tgasn) mice, a mouse model for intracellular alpha-synuclein pathology associated with pd, crossed to b5i/lmp7 subunit deficient mice, highlight an increase of aggregated alpha-synuclein accompanied by worsening of pathology in tgasn mice lacking functional i-proteasomes, suggesting a protective role of i-proteasomes for intracellular amyloid pathologies. future studies will aim at resolving the mechanistic underpinnings of the seemingly opposing effects of i-proteasome deficiency on the pathogenesis and progression of extracellular and intracellular protein aggregation to aid better understanding and guide novel therapeutic opportunities for neurodegenerative diseases. in the most severe form x-linked adrenoleukodystrophy (x-ald) is a fatal neurodegenerative disorder with inflammatory demyelination of the brain caused by a deficiency of the adrenoleukodystrophy protein (aldp), encoded by the abcd1 gene. aldp, a member of the abc transporter subfamily d, is located in the peroxisomal membrane and transports very long-chain fatty acids (vlcfa) as coa-esters for degradation into the peroxisome. currently, the only curative therapies are allogeneic hematopoietic cell transplantation (hct) or genetically corrected autologous hematopoietic cd34 1 stem cell therapy (hsct). the success probably relies on the settlement of microglia and perivascular macrophages derived from the myelo-monocytic donor cells in the brain of the patient. therefore, we explored the phenotypes of the major immune cell types derived from the cd34 1 stem cell. immune cells from peripheral blood of controls and x-ald patients were isolated by magnetic activated cell sorting (macs). purity of cell isolations was verified by flow cytometry. in purified cells qrt-pcr analysis was used to determine mrna levels of the abcd transporters and gc-ms was used for the quantification of vlcfa levels, a diagnostic marker. the three peroxisomal abc transporters were differentially expressed in cd34 1 derived cell types of healthy controls: abcd1 and abcd2 mrna were inversely expressed in all cell types, whereas abcd3 was equally distributed. abcd2, the closest homolog of abcd1 could be expected to compensate for the loss of abcd1 function. however, the expression pattern of abcd2 was unchanged in x-ald patients; accordingly, cell types lacking abcd2 mrna (e.g. monocytes) displayed the most severe biochemical phenotype concerning vlcfa catabolism, whereas cells with substantial abcd2 expression (e.g. t-lymphocytes) were barely affected. thus, not all investigated immune cells present an intrinsic metabolic defect. therefore, we propose that the beneficial effect of hct and hsct may rely on the replacement of those cells lacking sufficient abcd2 expression in abcd1 deficiency. in addition, these findings support the concept that abcd2 is a target gene for pharmacological induction, as an alternative treatment strategy, to rescue vlcfa metabolism and possibly halt the inflammation in x-ald patients. interleukin-33 (il-33) is a cytokine that has important functions in inflammatory and autoimmune diseases. little is known, however, about il-33 in the brain. we analyzed expression of il-33 in the mouse brain during embryonic and postnatal development. being undetected until late embryogenesis, il-33 was highly expressed during the first two weeks of postnatal life, after which expression gradually decreased and was then absent from the normal adult brain. astrocytes and oligodendrocyte precursors expressed il-33, but not neural progenitors or neurons. the vast majority of il-33 positive cells displayed nuclear staining. apart from its function as a pro-inflammatory cytokine, a role for nuclear il-33, potentially in transcriptional regulation, is also emerging. the important role of neuro-inflammation in traumatic brain injury is being increasingly recognized, and local cytokine production can mediate the inflammatory response. because of its potential for attracting inflammatory cells we analyzed il-33 expression in a cortical contusion infarction model (cci). we found that il-33 expression was induced by injury, with a peak of expression three days after cci, and at six days the levels declined. il-33 positive cells showed an overlap with astrocytic and oligodendrocytic lineage markers, but not with neurons, neural progenitors, endothelial cells or microglia/macrophages. a similar time course for il-33 expression in human brain trauma was found, using cerebral microdialysis, which allows continuous sampling of the parenchymal concentrations of molecules over several days in vivo. the peak of released il-33 for the patient analyzed was 4 days post trauma. il-33 binds to a heterodimeric receptor composed of st2 and il-1racp. following injury of st2 knockout mice we found a general reduction of inflammatory cells at the site of injury, compared to wild type animals, and there were fewer microglia at the trauma site of mice lacking il-33 receptors. our data show that il-33 expression is under tight regulation in the normal brain, but is induced by traumatic injury where is important for attracting inflammatory cells. the small heat shock protein alpha b-crystallin (cryab) is expressed by oligodendrocytes (ol) and astrocytes at several stages of multiple sclerosis (ms) lesions. cryab has been shown to be protective and therapeutic in the eae model of ms, possibly due to its anti-apoptotic functions and regulation of inflammatory pathways. in this study, we further investigated the role of cryab in de-and remyelination in vivo, applying the cuprizone model of demyelination to cryab -/mice. surprisingly, we found that cuprizone-induced lesions are significantly smaller, less severe and less inflammatory in cryab -/mice, compared to wild type (wt) controls. staining for microglia and astrocytes revealed that astrocytes are the main inflammatory cell type that is affected by cryab expression: lesions in cryab -/mice display less reactive astrogliosis than wt controls. conversely, although less severe, lesions in cryab -/mice also contain fewer oligodendrocyte progenitor cells (opc) than in wt mice. in addition, our data suggest that remyelination is less efficient in cryab -/mice than in wt controls and that remyelinated areas in cryab -/mice contain fewer ol than remyelinated areas in wt mice. together, we hypothesize that cryab, due to its anti-apoptotic actions, mediates protection in both astrocytes and ol, enabling on one hand reactive astrogliosis, which contributes to demyelination, and on the other hand ol survival, facilitating remyelination. additional in vitro experiments support this hypothesis, revealing that cryab -/astrocytes are less reactive than wt astrocytes and that sirna-mediated knockdown of cryab affects ol survival. interestingly, analyzing phosphorylation patterns of cryab in cuprizone lesions revealed that cryab is differentially phosphorylated in astrocytes and ol. furthermore, we found that cryab is differentially phosphorylated in astrocytes in active demyelinating ms lesions, indicating that phosphorylation of the protein might underlie its (pathogenic) role in active demyelination. these paradoxical findings not only suggest a role for cryab as a potential target in a regenerative approach for ms treatment, but also point to a possible pivotal role for reactive astrogliosis in cuprizoneinduced demyelination and, more importantly, in early ms pathology. in a comprehensive immunohistochemical survey, we compared cortical ms lesions to those of inflammatory (tuberculous meningitis, rasmussen's encephalitis, b-cell lymphoma, and meningitis) and neurodegenerative (alzheimer's disease) diseases. although complex and fulminant immune responses were seen in many disease cases, primary demyelination was only detected in ms. in search of ms-specific molecular mechanisms, we performed whole-genome microarrays on micro-dissected archival formalin-fixed paraffin-embedded material. apart from cortical ms lesions, we also included brain tissue from patients suffering from tuberculous meningitis (inflammatory control) and alzheimer's disease (neurodegenerative control). additionally, control cases without brain pathology were included. using a restrictive cut-off, we identified 301 genes differentially expressed in ms lesions. more than 80% of these candidate genes could be assigned to t-cell mediated inflammation, microglia activation, oxidative stress, tissue injury, dna damage/repair as well as remyelination and regenerative processes. subsequent neuropathological analysis confirmed that significantly more oxidatively damaged neurons (stained positive for oxidized phospholipids) were present in cortical ms lesions than in any other examined disease. tunel stainings for the detection of dna strand breaks showed that neurons and oligodendrocytes with tunel-positive nuclei were most abundant in ms lesions containing zones of active demyelination and tissue injury. interestingly, immunohistochemical stainings for radical producing enzymes revealed that oxidative stress-mediated tissue damage in cortical ms lesions seems to be driven by nadph oxidases rather than by nitric oxide synthases. taken together, we were able to show that ms-specific primary demyelination is driven by mechanisms of tissue injury that differ from any other investigated inflammatory and neurodegenerative disease. among these mechanisms, oxidative tissue injury seems to play a major role. f. labombarda 1 , s. gonzalez 1 , i. jure 2 , a. de nicola 1 1 ibyme/conicet/uba, buenos aires, argentina 2 ibyme/coni-cet, buenos aires, argentina reactive gliosis and inflammatory mediators are implicated in demyelination and secondary damage after spinal cord injury (sci). we have previously reported that after sci, short-term progesterone treatment (3 days) stimulates oligodendrocyte precursor cells proliferation and decreases reactive gliosis, whereas chronic treatment (21 days) differenciates oligodendrocytes precursor cells into mature oligodendrocytes and enhances remyelination. presently, we further studied whether progesterone was able to modulate the inflammatory reaction and cytokine production by astrocytes and microglial cells. thus, the timecourse mrna expression of pro-inflammatory cytokines (il1b, tnfa and il-6) and pro-inflammatory enzymes (cox-2 and inos) was studied by pcr in real time. results showed that the highest increase in cytokine and pro-inflammatory enzymes mrna production occurred in rat spinal cord 6 h after sci. progesterone treatment significantly decreased the early rise of proinflammatory mediators mrnas at 6h. as progesterone action in spinal cord involves multiple mechanisms, the role played by the classical progesterone receptor (pr) on the progesterone inhibitory effects on cytokines, was assessed in prko mice. in agreement with data obtained in the rat model, sci strongly stimulated tnfa, il1b and il-6 mrna levels in spinal cord of both wild type and prko mice. however, whereas progesterone treatment inhibited the mrnas of cytokines in wild type mice 6h after sci, it was ineffective in prko mice, involving pr in the inhibition of cytokines production. finally, we measured astrocyte and microglial cells density by immunohistochemsitry after 6 h of progesterone treatment. the steroid administration significantly decreases gfap1 and ox-421 cells, which correlated with cytokine and pro-inflammatory enzymes inhibition. we conclude that progesterone attenuates reactive gliosis and inflammatory reaction, probably reducing the secondary spinal cord damage and favouring remyelination. in the steady state they are rarely observed in the cns parenchyma. however, during cns inflammation they cross the blood-brain barrier where together with microglia (cns-resident apc) they can present antigen to infiltrating t cells. the goal of this study was to compare two subpopulations of microglia: cd11c-and cd11c1 with brain dc in terms of promoting different t cell responses. two subpopulations of microglia: (cd11c-cd45dim cd11b1) and (cd11c1 cd45dim cd11b1) as well as (cd11c1 cd45hi) bdc have been sorted from the cns of c57bl/6 mice subjected to eae. sorted cells were used either for ex vivo proliferation and cytokine assays or for qrt-pcr. our results show that bdc and cd11c1 microglia are similar with regard to ability to induce proliferative t cell response from both primed and na€ ıve t cells. nevertheless, they differ in their cytokine profile and ability to promote cytokine release by t cells. our findings suggest that different subtypes of apc in the cns promote different immune responses. this work has been supported by lundbeckfonden. melanocortin 4 receptor (mc4r) is predominantly expressed in the brain and it is the only mcr expressed in astrocytes. our previous results showed that a-melanocyte-stimulating hormone (a-msh) the anti-inflammatory action is mediated by mc4r in astrocytes and in the hypothalamus of male rats and that these effects may lead to neuroprotection. we have already demonstrated that ndp-msh (an a-msh analogue) increased brain-derived neurotrophic factor (bdnf) expression through the camp-pka-creb pathway in astrocytes. in the present study we examined the participation of mitogen activated protein kinases (mapk) and phosphatidylinosotol-3 kinase (pi3k)-akt pathways in mc4r signaling in astrocytes. we also investigated the effect of a-msh on bdnf expression in vivo in brains of male rats.we preincubated cultured rat primary astrocytes with mapk (p38, jnk and erk) and pi3k-pdk1-akt inhibitors 15 min before addition of 1 mm ndp-msh for 1 h. we found that ndp-msh-stimulating effect on bdnf expression assayed by qrt-pcr was abolished only in the presence of erk and pi3k inhibitors. accordingly, levels of phospho-erk1/2 determined by western blot were increased by ndp-msh whereas phospho-akt levels were not modified at 30 min. ndp-mshinduced erk1/2 activation was decreased by adenylate cyclase and pi3k inhibitors but not by a pka inhibitor, suggesting a camp and pi3k involvement in this effect. we also investigated if a-msh was able to induced bdnf expression in vivo. for that purpose we injected male rats with a-msh (0.5 mg/kg, ip) or vehicle (saline) once and sacrificed them after 3 h. rats were also injected twice daily and for two days and killed 48 h after the first injection. we observed that bdnf mrna levels increased after a-msh treatment at 3h in the hypothalamus whereas in the cortex bdnf expression was not modified. at 48 h bdnf expression was not modified by a-msh. we showed by immunohistochemistry that mc4r and bdnf co-localizes with neurons and astrocytes in the brain. since melanocortins have anti-inflammatory and neuroprotective effects in the brain, the mechanisms described in astroglia help understand mc4r action. our results indicate that melanocortins induce bdnf expression in astrocytes through erk and pi3k, suggesting that this effect could be involved in the neuroprotective actions of melanocortins. the fact that bdnf expression also increases in vivo in the hypothalamus reinforce this idea. methods: we analyzed microglia activation and the role of nmda receptors in this process using the microglia cell line bv2 and cortical slice cultures. the in vivo analysis was performed in two models: first, in odtr mice that express the diphtheria toxin receptor specifically in oligodendrocytes (odcs) which allows induced killing of odcs (locatelli et al., 2012) and second in mice that were immunized with mog/ cfa to induce eae. results: the proinflammatory cytokine il-17a plays a pivotal role in the pathogenesis of ms and eae. we showed il-17a activates microglia in vitro. therefore, we activated the microglia cell line bv2 and cortical slice cultures with il-17a to and subsequently treated the cultures with the nmda receptor antagonists mk801 and ap5 to block nmda receptor signaling. this treatment inhibited il-17a-mediated activation of microglia and in particular ros production, proliferation and migration. additionally, we detected increased secretion of il-6 and g-csf. furthermore, il-17a enhanced activation of the nmda receptor through phosphorylation leading to higher influx of calcium upon ligand binding. to further analyze nmda receptor-depended microglia activation we used two different mouse models that provoke different mechanisms of microglia activation. whereas in eae a strong inflammatory process activates microglia, in odtr mice, activation occurs due to induced odc death. interestingly, in the context of eae microglia activation was clearly diminished when mice were treated with mk801 whereas inhibition of the nmda receptor had no effect on microglia activation in the odtr model. in line with the in vivo data we show that inhibition of microglia activation takes place only when the cells were stimulated with il-17a and not upon stimulation with lps. conclusions: our findings show that nmda receptors are not involved in general microglia activation but rather play a role in microglia activation in an inflammatory context with specific cytokines such as il-17a. aims of the study: to investigate whether cx3cl1-cx3cr1 signaling contributes to microglia migratio00ctivation and subsequent astrogliosis following msc transplantation in the cns. methods: first, in order to allow localization of grafted msc in vivo, wt c57bl/6 msc were transduced with a lentiviral vector encoding the blue fluorescent protein (bfp). next, bfp1 msc were injected into the cns (striatum) of cx3cr1 1/-(n 5 10) and cx3cr1 -/-(n 5 7) transgenic mice. these mice have respectively one or both copies of the cx3cr1 gene replaced by the egfp reporter gene. at 10 days posttransplantation, histological analyses were performed to determine iba11 microglia and s100b1 astrocytes within and surrounding the bfp1 msc graft site. astrogliosis was determined based on staining for gfap. cx3cr1 expression was evaluated based on egfp expression. all histological evaluations were quantified using tissuequest and/or imagej cell analysis software. results: (i) bfp1 msc graft survival is observed in both cx3cr1 -/and cx3cr1 1/mice; (ii) microglia migration towards grafted msc occurs independent of functional cx3cr1-cx3cl1 signaling; (iii) down-regulation of egfp gene-expression (and thus also cx3cr1 gene expression) is observed in both cx3cr1 -/and cx3cr1 1/mice on msc graft-invading microglia, but not on msc graft-surrounding microglia; (iv) the absence of functional cx3cr1-cx3cl1 signaling in cx3cr1 -/mice results in significantly less astrogliosis around the msc graft site, as compared to the msc graft site in cx3cr1 1/mice. conclusions: here we identified cx3cr1-cx3cl1 signaling as a potential target to modulate and/or control astroglial scarring following (stem) cell grafting in the cns. the latter is of significant importance as grafted cells can only functionally interact with (injured) brain tissue in the absence of a graft-surrounding astroglial scar. this accumulation is known to be essential for the usual sprouting of injured axons and leads to a functional nerve repair. because of the insignificant infiltration of macrophages in the injured leech cns, we investigate the chemotactic mechanisms of the recruitment of only resident microglial cells in order to explore in fine the crosstalk between damaged neurons and activated microglia leading to the leech cns repair. methods: in vitro and ex vivo chemotactic assays were performed by using recombinant form of hmc1q on microglial cells which were pre-incubated or not with anti-cc1qr or anti-gc1qr antibodies. then, affinity purification analyses using biotinylated c1q were performed from leech microglial cell protein extracts. finally, immunohistochemistry analyses were located the production of newly characterized c1q receptors in microglial cells. results: we demonstrated that rhmc1q contributes to the recruitment of some leech microglial cells. chemotaxis assays showed that the blocking of respective receptors, homologous to human gc1qr and cc1qr, inhibits the hmc1q-dependent recruitment. importantly, affinity purification analyses demonstrated the interaction between both receptors and hmc1q. finally, those receptors were located on distinct microglial subsets. conclusion: this work is used to specify the activation processes of only resident microglial cells. the results show the importance of hmc1q in the microglial accumulation leading to the nerve repair. in h. medicinalis, hmc1q activity is driven through two different receptors which are not present on the same cells suggesting that hmc1q activates two distinct microglial cell subpopulations. the question whether blood-borne immune cells are infiltrating brain areas afflicted with neurodegeneration in ad and other tauopathies yielded contradictory results. some authors showed that the chronic neuroinflammation in ad was provided almost exclusively by resident cns cells without any apparent influx of leukocytes from the blood while others reported that hematopoietic cells can enter the brain in alzheimer's disease and may contribute to an increased inflammatory burden. in order to address this issue we have used two independent transgenic lines expressing human misfolded truncated tau in two different genetic background (wistar and shr). we have shown that tau neurodegeneration can induce inflammatory responses mediated by reactive microglia in both transgenic lines, however the microglial responses showed a striking difference between the lines. we have identified two significantly different inflammatory responses to the same inducer. moreover, we found that the genetic background significantly modified the molecular cascade influencing leukocyte influx into the brain. in both transgenic lines, the majority of the blood cells entering the brain belonged to antigen presenting cell family -monocytes and dendritic cells. at the proteomic level we found striking differences between the lines. while in the wistar transgenic line we observed significant increase of cytokine-induced neutrophil chemoattractant-1, in shr transgenic line, tissue metalloproteinase 3 was significantly reduced. these findings suggest that blood cell infiltration of the brain affected by tau neurodegeneration is modified by genetic background. this inter-individual variability should be taken into the consideration in the development of novel drugs with anti-inflammatory properties. acknowledgement: this work was supported by axon neuroscience and research grants vega 2/0161/11, 2/0205/11, 2/0193/11, apvv 0200-11, apvv 0206-11 and structural fund 26240220046. charit e -universit€ atsmedizin, berlin, germany toll-like receptors (tlrs) are key molecules of the innate and adaptive immune response in vertebrates. the original protein toll in drosophila melanogaster regulates both host defense and morphogenesis during development. tlrs recognize host-and pathogen-derived stimuli. single-stranded rna is sensed by tlr7 localized to the endolysosomal compartment of immune cells. we found that both extracellular viral rna and host-derived micro-rnas induce cell-autonomous and microglia-mediated neuronal cell death through the endosomal receptor tlr7 in vitro and in vivo. however, the exact intracellular signalling cascade and the cellular mechanisms through which tlr7 leads to tissue injury in this context remained unclear. unc93b1 is a molecule specifically involved in trafficking of nucleotide-sensing tlrs, such as tlr7, in immune cells of both humans and mice. unc93b1 physically interacts with this receptor in the endoplasmic reticulum (er), and the function of the membrane protein unc93b1 is to deliver the nucleotidesensing receptors from the er to endolysosomes. making use of real-time pcr, immunocytochemistry, and histochemistry we systematically examined the expression of unc93b1 in the murine cns. whereas a distinct expression for this molecule was observed in microglia and astrocytes, expression of unc93b1 in cultured neurons was negligible. however, expression of this molecule was detected in cortical neurons of brain sections. moreover, unc93b1 was strongly regulated during different embryonic, postnatal, and adult stages of the developing mouse brain. neurons of various brain regions were identified as the main cell type expressing unc93b1 in the developing brain. taken together, our data reveal a specific expression pattern of unc93b1 in the cns, in particular in a developmental context, and lay foundation for further investigation of the pathophysiological significance of this molecule for both injurious and developmental processes in the central nervous system of vertebrates. has benefited from a number of studies that precisely depicted different types of plaques in white or grey matter areas. also, both inflammation and diffuse axonal loss in the normal appearing white matter (nawm) were extensively described. however, little attention was given to the so-called periplaque, which is usually defined as a partially demyelinated ribbon of tissue surrounding the plaque. whether periplaques correspond to expanding lesions, sites of ongoing remyelination or areas of tract degeneration remains uncertain. methods: in this context, our study aimed to bring quantitative insights to the neuropathology of ms periplaques in the spinal cord of primary or secondary progressive ms patients. a neuropathological quantitative assessment of inflammation, axonal loss and myelin loss was performed concurrently in the periplaques, plaques and normal-appearing white matter (nawm) of cervical spinal cord samples from 16 patients with progressive ms. results: periplaques formed large areas of incomplete myelin loss that extended distant away from the border of plaques. axonal loss was quantitatively similar in plaques and periplaques but signs of axonal dystrophy were predominantly observed in plaques. surprisingly, axons that remained myelinated in periplaques presented a thicker myelin sheath than myelinated axons of the nawm. inflammation in the periplaque was mainly characterized by an accumulation of macrophages/microglia that were closely apposed to myelin sheaths but exerted poor phagocytic activity. finally, we found that neuropathological features of periplaques were overall disconnected from that of plaques with regard to size, shape, inflammation and axonal integrity. conclusions: our work indicates that in ms spinal cords, periplaques correspond to demyelinating rather than remyelinating lesions. it further suggests that in progressive forms of ms, periplaques are likely to impact significantly on neurological disability. finally, we propose that tract degeneration might not be the only cause of periplaque extension and that slowly-expanding demyelination, temporally remote from plaque formation, might occur in periplaques. molecular results will be presented that support these hypotheses. cell-adhesion between endothelial cells at the blood-brain barrier (bbb) makes the endothelium impermeable to blood-derivatives and immune cells. to establish and maintain this barrier during development, adulthood, and during disease, brain endo-thelial cells must develop and sustain these strong adhesive contacts, through expression of tight junction molecules. however, we do not know whether netrin supports inter-endothelial cell adhesion at the blood-brain barrier. given this, we hypothesize that netrin tightens the bbb during development, adulthood, and protects it during disease. methods: to test this, we used both human adult primary brainderived endothelial cells and newborn netrin-1 knockout mice and evaluated netrin's effect on inter-endothelial cell adhesion and barrier permeability. we also assessed netrins' therapeutic potential to maintain the barrier and limit immune cell infiltration into the central nervous system (cns) during experimental autoimmune encephalomyelitis (eae). results: our results demonstrate that brain endothelial cells express netrins. they help to form a tighter bbb during development. they also maintain and protect the adult barrier by increasing the expression of endothelial junction molecules, thus promoting inter-endothelial adhesion and reducing protein leakage across the barrier. netrins also reduce bbb breakdown and diminish initial myeloid cell infiltration into the brain and spinal cord during eae, which delays disease onset and ameliorates disease severity. discussion: we conclude that netrins enhance bbb stability, and protects the bbb during neuroinflammatory disease. microglial cells were shown to play an important role in many diseases of the central nervous system, not least due to their capability to become activated upon signs of brain injury. they migrate to lesion sites, proliferate, release cytokines and chemokines, and phagocytose cells or cellular debris. therefore, the microglial cytoskeleton has to be highly rearrangeable. one major element of the contractile system in nonmuscle cells is nonmuscle myosin ii (nm ii). as the role of nm ii in glial cells is poorly characterized, it is an aim of this project to study nm ii-dependent functions in microglia. we found that inhibition of nm ii by blebbistatin, which is a highly selective inhibitor of nm ii adenosine phosphatase, prevents any morphological shaping in freshly plated microglia and leads to functional deficits during migration and phagocytosis of fluorescence-labeled beads. using confocal microscopy, we could find diffuse expression of nm ii in resting microglia in vitro, whereas activation with bacterial lipopolysaccharide (lps) led to perinuclear accumulation of nm ii protein. in an activated state, microglial cells release pro-inflammatory cytokines and reactive oxygen species; interestingly, in the presence of blebbistatin the release of nitric oxide (no) as well as rearrangement of nm ii was prevented. taken together, these results illustrate a key role for nm ii in microglial shaping and functioning. ongoing studies will improve our knowledge on spatial and functional aspects of the actomyosin complex during demyelinating processes and open targets for development of therapeutic strategies towards remyelination in the cns. prolonged activation of the nucleus basalis of meynert (nbm), the primary source of cholinergic projection to the cerebral cortex has been reported to cause significant increases in cerebral cortical blood flow (cbf) in rodents. while the nbm also gives rise to gabaergic and glutamatergic projections to the cerebral cortex, the nbm-driven increase of cbf has been described to be dependent in part on muscarinic acetylcholine receptors. lately, several groups reported that astrocytes modulate local cbf via intracellular ca 21 signaling. considering that cortical astrocytes express machrs and in vivo activation of the nbm leads to machr-dependent ca 21 surges in astrocytes, cholinergic modulation of cbf via astrocytic ca 21 surges is conceivable. we report here that a single train stimulation of the nbm (stnbm; 100 hz, 0.5 s, 200 ma) induces a biphasic (a rapid increase, followed by an overshooting slower decrease that goes back to baseline within a minute) laser doppler flowmetry (ldf) response in the somatosensory cortex. moreover, the stnbm-induced ldf response was sensitive to the machr antagonist atropine. stnbm with a weaker stimulation intensity (50 ma) induces a transient decrease. surprisingly, we find that ip3r2 knockout mice, which lack cytosolic ca 21 surges in astrocytes, show similar ldf responses to stnbm. a.-c. boulay, c. giaume, m. cohen-salmon cirb, paris, france astrocytic endfeet are specialized processes, which enwrap blood vessels and express a large molecular repertoire dedicated to the physiology of the vascular system. one of the most striking properties of astrocyte endfeet is their enrichment in the gap junction proteins connexin (cx) 43 and cx30 allowing for direct intercellular trafficking of ions and small signaling molecules through perivascular astroglial networks. however, the role of these proteins at the gliovascular interface is not fully understood. we have recently demonstrated that astroglial perivascular cxs expression starts after birth during bloodbrain barrier maturation and controls its integrity (ezan et al. cx30 d/d purified vessels showed essentially a strong upregulation (fold change > 12) of sgcg. this gene encodes gamma-sarcoglycan, a membrane glycoprotein associated with the dystrophin-dystroglycan complex which is essential for muscle integrity and is impaired in duchenne muscular dystrophy (petrof et al., 1993) . at the gliovascular interface, the dystrophin-dystroglycan complex anchors astrocyte endfeet to the basal lamina (nico et al., 2010) , and is crucial to the bbb integrity. however, expression of sgcg and its role in the brain has until now only been poorly documented. moreover, we found an upregulation of s100a8 and s100a9 (fold change 6,3 and 5,5, respectively) encoding the calgranulin a and b, two proteins present in neutrophils and monocytes, and involved in their migration to inflammatory sites, attachment to endothelial cells and extravasation (ryckman et al., 2003) . altogether, our results suggest that cx30 might modify the dystrophin-dystroglycan complex thereby influencing blood-brain barrier properties, and control the attachement as well as the transmigration of leukocytes through the endothelium, thus contributing to the regulation of inflammatory processes in the brain. the blood-brain barrier is supported by the perivascular glia. a crucial event during the gliogenesis is the replacement of radial glia by astrocytes. immunohistochemical staining of nestin visualized the first gliovascular connections after e16, and by e20 the radial glial endfeet cover the vessels completely. by e20 numerous nestin immunoreactive astrocyte-like cells participated in it. in parallel, nestin immunoreactivity also appeared in the wall of the vessels. by about p10 both radial glia and nestin immunopositive astrocytes disappeared and gave place to astrocytes immunoreactive to s100 protein and glutamine synthetase, markers of mature astrocytes. in the radial glia these markers were not found, and they colocalized with nestin only scarcely. the s100 and glutamine synthetase immunoreactive cells appeared at first in the middle zone of the pallium about p2, and their population gradually extended to both the pial surface and the deep cortical zones, colonizing the whole thickness by p8, in parallel with the disappearance of nestin immunoreactive glial elements, either radial or astrocytic ones. at p6 gfap immunoreactive cells were still confined to the most superficial and deepest cortical zones, so showed no colocalization with s100 and glutamine synthetase, unlike that found after p8. whether this glial 'takeover' results in a transitory increase of leakage of the blood-brain barrier, it remains to be answered. it seems however, to underly that cerebrovascular fibronectin and laminin immunoreactivities disappear at first from the middle zone of the developing cortex and only later from the superficial and deep ones, between p2 and p10. according to krum and rosenstein (1991) disappearance of these immunoreactivities refers to the fusion of the glial and vascular basal laminae, the maturation of the definite gliovascular connections. surrounded by astroglial scars that inhibit such growth. little is known about how astrocytes assemble to form this scar, and a deeper understanding of the cellular and molecular mechanisms that control this process is needed if this goal is to be achieved. in vitro data suggest that certain bioactive molecules can alter astrocyte morphology into a more growth supporting state. other molecules have been shown to promote axon growth. clinical application of many of these molecules would require neurosurgical delivery directly into the spinal cord. injectable biomaterials have the potential to serve as depots and scaffolds for in vivo delivery of bioactive molecules. we are developing di-block co-polypeptide hydrogels (dch) as fully synthetic biomaterials that could safely and easily be injected into specific sites after sci to deliver molecules in order (i) to understand better, and (ii) to manipulate, the mechanisms that control glial scar formation. we have previously shown that dch are biocompatible after injection into brain or spinal cord and selfassemble into structures with finely controllable properties. our current work shows that dch depots can deliver diffusible bioactive growth factors that influence local neurons or astrocytes in predictable ways and form gradients that are effective up to several mm away from depots. we are now testing the ability of dch depots to deliver specific growth factors that will manipulate scar-forming cells when dch are injected at clinically realistic sub-acute times after sci. to investigate the molecular phenotype of esdm, we performed a whole transcriptome analysis of esdm in comparison to primary cultured microglia, flow cytometry-sorted microglia, different myeloid cells, t cells, astrocyte-and neuron-enriched cultures. while being distinct from t cells, neurons and astrocytes, esdm were closest related to primary cultured microglia followed by flow cytometry-sorted microglia and other myeloid cells. esdm and primary cultured microglia showed a strong overlap in their transcriptome by only having 143 gene transcripts differentially expressed. most of these differentially expressed genes were immune-related genes that were up-regulated in primary cultured microglia. this indicates that esdm were in a less activated state rather reflecting the in vivo biology of these cells. flow cytometry analysis of cell surface markers selected from the microarray confirmed the close relationship between esdm and primary cultured microglia. esdm resemble primary microglia not only in respect to surface marker expression and functional properties, but also display a molecular phenotype similar to primary microglia. our data suggest that esdm are a reliable tool for the replacement of primary microglia. spinal cord injury (sci) is a devastating condition which usually leads to paralysis. this outcome of injury in most cases is permanent due to the cascade of immunological factors that create an anti-repair environment around the site of injury and due to the limited capacity of central nervous system (cns) axons to regenerate, creates a daunting challenge to establish an effective therapy. from many years of research it is apparent that a combination of therapies would be the best course of action for treatment of sci; this however greatly increase the animals cohorts and time to establish the most effective course of treatment. for this reason we have developed an in vitro model of spinal cord injury as a moderate throughput screen, which is in accordance with the 3r's. this culture consist of dissociated mixed rat embryonic spinal cord cells plated onto a monolayer of astrocytes this is essential for the spinal cord cells, obtained from rats of embryonic day 15, which then develop into a carpet of neurites that over time become myelinated forming internodes of myelin interspaced with nodes of ranvier. this mixed cns culture was utilized using a scalpel blade to axotomise the axons and a persistently neurite-free area is created which is accompanied by many features of sci, including demyelination and reduced neurite density adjacent to the lesion and the presence of and reactive astrocytes. these finding are congruent with changes seen in vivo after spinal injury. data will be presented regarding the effects from proven and novel pharmacological agents on neurite outgrowth and myelination looking to understand the mechanism in which they act. as well as modifications of the culture system to incorporate methods to align axons and isolate cell bodies from their processes for a more detailed analysis of regeneration. the role of increased activity of astrocyte networks in structural synaptic plasticity following remote axonal injury is unexplored. we set out to investigate the efficiency of synaptic rearrangements in models where astrocyte reactivity is selectively diminished by the inhibition of the main cytokine pathway in transgenic mice (tg) in comparison to that observed in wild type mice (wt). first, synapse densities were compared in the facial nuclei two weeks after unilateral facial nerve transection by synaptotagmin-1/psd-95 immunolabelling and ultrastructural analysis. we found that the recovery of synapse density was decreased by 40% in tg mice compared to wt mice, and that the reduction in synaptic density correlates with a relative loss of neuronal coverage by reactive astrocyte end-feet in the same conditions. specifically, we observed a 30% reduction in excitatory synapse density in the tg mice. second, we developed an organotypic entorinho-hippocampal slice culture system in which astrocyte activation and synaptic plasticity could be more closely monitored in real time. this was assisted by two-photon microscopy, using glial ca 21 -imaging and dendritic spine motility analysis after perforant pathway transection. in summary, we demonstrated that full astrocyte activation is necessary for partial structural recovery of synapses after axonal injury. our work, using simplified models, may help experimental approaches aimed at optimizing adaptive plasticity in cns injuries. pathological loss of myelin in diseases like multiple sclerosis (ms) is usually followed by a phenomenon of remyelination, in which oligodendrocytes synthesize new myelin sheaths to envelope exposed around axons in the adult central nervous system (cns). the importance of remyelination arises from the fact that this process not only restores saltatory conduction, but also protects axons from different insults and thus limits clinical disability in demyelinating diseases. in this scenario, the mammalian subventricular zone (svz) has garnered attention as a potential source of replacement cells after injury. this zone harbours stem cells and supports long-distance migration, and is activated in ms patients to promote gliogenesis. although ng2 1 precursor cells are the first to react to demyelination and show the highest proliferation rate in comparison with other cells, the relative contribution of the svz with respect to the inflammatory demyelination induced by the theiler's virus infection and oligodendrogenesis has never been addressed. in the present study, we have investigated the behavior of the svz in theiler's murine encephalomyelitis virus -induced demyelinating disease (tmev-idd). we report that in this viral model for ms, there is a preclinical phase of the disease with demyelination in the corpus callosum that is followed later by an attempt of remyelination. this phase is accompanied by an activation of the svz with no apparent activation of ng2 1 precursors, and a strong and clear staingalectins (gals) are a family of soluble lectins that, when binding to b-galactosides, form multivalent complexes with glycoconjugates of the cellular surface and induce a modulation of intracellular signaling pathways for differentiation and survival. recently, galectin-1 has been described as a microglial deactivator which prevents neurodegeneration and promotes neuroprotection in an eae model. however, its action at neuronal level is poorly understood. spinal cord injury (sci) also remains a major challenge to neurological research, as several factors such as extracellular matrix molecules inhibit axonal regeneration. among them, semaphorine 3a (sema3a) contributes to the inhibition of axonal regeneration by acting on microtubules and actin cytoskeleton. neuropilin-1 (nrp-1) is a neuronal receptor whose binding of sema3a generates repulsion of axonal growth. in addition, nrp-1 has been described as a target of gal-1 in non-neuronal systems. the aim of this work was to study the role of gal-1 intraumatic sci, considering that sema3a expression is "turned on" in sci and prevents axonal regeneration via nrp-1. gal-1 knockout mice (lgals1 -/-) were submitted to a full traumatic sci at thoracic level (t9-t10) and treated with different concentrations of recombinant gal-1. we demonstrated that lgals1 -/mice treated with gal-1 have a dose-dependent functional recovery of motility as early as 6 days post-treatment, which is structurally associated to neuronal repopulation and high axonal regeneration. these effects were only observed in animals treated with gal-1 with dimerizing capacity, but not in mice treated with a mutant gal-1, only present in a monomeric form. in addition, we observed that microglial response was "turned off" in gal-1-treated mice. moreover, exogenous dimeric gal-1 bound to the surface of injured motoneurons and promoted the spread of nrp-1. 3d confocal microscopy reconstruction showed gal-1 and nrp-1 spatial proximity. to confirm this result, we carried out immunoprecipitation assays, which allowed us to determine that only the dimeric form of gal-1 interacted with nrp-1 and induced a decrease in the levels of sema-3a bound to nrp-1. our results show that gal-1-based treatments combine its neuronal effect on nrp-1 with its deactivating effect on microglia, which leads to a better restorative process after medullar lesion. the findings presented here support the potential of dimeric gal-1 as a therapeutic agent for human sci patients. theophylline and caffeine. it depresses activation of microglial cells and astrocytes which is associated with neuronal damage during inflammation and hipoxia. it is largely known that ethidium bromide (eb) injection into the cns induces local oligodendroglial and astrocytic death, resulting in primary demyelination, blood-brain barrier disruption and schwann cell invasion, and thus serving as an experimental demyelinating model in animals. the aim of this study was to evaluate if prop had the capacity of affecting glial cell behaviour during the process of demyelination and remyelination following gliotoxic injury with eb. wistar rats were divided into 2 groups: i-rats injected with 10 microlitres of 0.1% eb into the cisterna pontina and treated with prop; ii-rats injected with eb and not treated with the xanthine. prop (agener) treatment was done using 12.5 mg/kg/ day by intraperitonial route during all experimental period. the rats were euthanized from 7 to 31 days after eb injection and brainstem sections were collected and processed for light and transmission electron microscopy studies. results from both groups were compared by using a semi-quantitative method developed for documenting in semithin sections the extent and nature of remyelination in gliotoxic lesions. in general terms, by 7-11 days following gliotoxic lesion, the center of the lesion was expanded and filled with huge amounts of myelin debris among foamy macrophages and demyelinated axons. no astrocytic processes were found in this site and some lymphocytes were seen in the neuropil and around blood vessels. the most prominent feature of the 15 th day was the initial association at peripheral locations between naked axons and remyelinating cells. schwann cells were associated with one or multiple demyelinated axons or already forming thin myelin lamellae around single axons in astrocytic-free areas. oligodendrocytes began to form thin myelin sheaths in areas completely or partially filled with astrocytic prolongations. by 21-31 days, it became evident that rats treated with prop presented a small improve in the repair process when compared to untreated animals, the latter presenting greater amounts of myelin-derived membranes in the central area and a lesser extension of oligodendrocyte remyelination, but without any apparent difference regarding to schwann cells. by 31 days results showed that prop administration following eb injection seemed to stimulate oligodendroglial remyelination (mean remyelination scores of 3. peripheral axotomy results in a disconnection of the neuronal cell body from the target organ. a complex rearrangement of the nerve microenvironment, the so called wallerian degeneration, takes place distally to the lesion and involves schwann cell proliferation as well as macrophage infiltration. the process results in the formation of the bands of b€ ungner which guide the axonal sprouts throughout the nerve distal stump. although this process has been extensively studied, a thorough understanding of the three-dimensional nerve tissue reorganization is not completely known. such knowledge may in turn lead to more effective strategies aiming at the repair of peripheral nerves following transection or crushing. in this sense, the tetrahydrofuran (thf)-based tissue-clearing technique has been adapted for sciatic nerves of mice and used for subsequent observation to 2p-lsm. transgenic mice that express fluorescent proteins in neurons, astrocytes, schwann cells and microglia/macrophages as well as double and triple hybrids were used. additionally, by immunohistochemical analysis, it was possible to observe the structure of the nerve after injury by the expression of neurofilament and s100b, in non fluorescent mice. also, increased non neural cell responsiveness after injury was studied with anti-p75 ntr immunostaining. transmission electron microscopy allowed the evaluation of the ultrastructure of the nerve microenvironment, as well as the regenerative process by 3, 7 and 14 days after injury. microscopic examination of cleared tissues of transgenic mice through 2p-lsm consisted in comparing the different cell type interaction before and after injury, with particular interest on the schwann cells, macrophages and growing axons. question: mass spectrometry (ms) has become nowadays an essential tool for the detection and identification of biomolecules in various contexts of biological problematic. ms can be performed from all types of biological materials ranging from biological fluids to tissues. end of 90's it was shown that with certain instrumentation such as those equipped with a maldi ion source it was possible to record ms spectra directly in situ from tissue sections. more, point to point automated acquisition of maldi ms spectra from a whole tissue section was shown to allow for reconstruction of the 2d images of biomolecules contained in the section distribution through ion density maps (caprioli rm et al., anal. chem. 1997). methods: maldi ms imaging (maldi msi) is a molecular imaging modality that presents several advantages including possible imaging of hundreds of biomolecules in one step acquisition and the untargeted character of the method allowing to study molecules without any prerequisite knowledge or use of probes. results: over the past decade maldi msi has demonstrated its potentiality in terms of applications for biology and clinics (franck j et al., mol. cell. proteomics. 2009). maldi msi can be use to study both endogenous molecules namely metabolites, lipids, peptides and proteins or exogenous ones such as drugs or xeniobiotics and was applied to various problematic. understanding of physiological or physiopathological processes requires knowledge on the identity and localization of molecules within a tissue. maldi msi reseals a clear potential to answer this objective and give a unique contribution. indeed, more recently maldi msi has gained new strategies for identification of molecules in situ on tissues. in particular, many efforts were given to develop confident identification of proteins from tissue sections. conclusion: here we will review this novel technology and discuss some of its current and potential contributions for neurosciences application (wisztorski m et al., dev. neurobiol. 2008). this will be illustrated by presentation of the investigation of invertebrate neuroimmune response as well as vertebrate neuropathological disorders using either fresh frozen or formalin fixed paraffin embedded tissues. the sphingosine 1-phosphate (s1p) signaling pathway is known to influence astroglial reactivity in experimental autoimmune encephalomyelitis and the synthetic s1p analog fty720 has been shown to provide neuroprotection in experimental models of acute stroke. however, the effects of a manipulation of s1p-s1p1 signalling at later time points after experimental stroke have not yet been investigated. we examined whether a relatively late initiation of a fty720 treatment has a positive effect on long-term neurological outcome with a focus on reactive astrogliosis, synapses and neurotrophic factors. we induced photothrombotic stroke (pt) in adult c57bl/6j mice and allowed them to recover for three days. starting on post-stroke day 3, mice were treated with fty720 (1 mg/kg b.i.d.) for 5 days. behavioral outcome was observed until day 31 after photothrombosis and periinfarct cortical tissue was analyzed using tandem mass-spectrometry, taqman v r analysis and immunofluorescence with especial emphasis on markers of astroglial reactivity. fty720 treatment results in a significantly better functional outcome persisting up to day 31 after pt. this is accompanied by a significant decrease in gfap-immunoreactivity (-ir) and an increase in psdsize. however no change in gfap-mrna, gs-ir or cs56-ir was observed. while fty720-treatment leads to a decrease in s1p concentration, it increases vegf-mrna in the periinfarct cortex. the initiation of fty720 treatment in the convalescence period has a positive impact on long-term functional outcome, probably mediated through reduced astrogliosis, a modulation in synaptic morphology and an increased expression of neurotrophic factors. due to the restrictions in regenerative capacity of the brain the tissue most severely damaged after traumatic brain injury (tbi) will end up as a fluid filled cavity. presently, it is an impossibility to effectively restore lost brain tissue, but ongoing research on the stimulation of endogenous neural stem cells has increased the hope to viably and functionally repopulate the injured parenchyma. however, it is crucial that possible therapies can show a long-term effect on both regeneration and functional recovery to be of clinical interest. in this study the enhanced induction of neurogenesis in rats after experimental tbi was evaluated three or six weeks after injury. severe focal tbi was performed using the controlled cortical impact model after which a combination therapy of intracerebroventricular administration of epidermal growth factor (egf) for seven days followed by deposition of extracellular matrix scaffold, containing vascular endothelial growth factor, into the cortical cavity. the treatment was devised to accomplish an optimal effect on the stem cell regeneration. the animals with six weeks survival time were functionally evaluated using the morris water maze (mwm). before sacrifice the animals were injected with bromodeoxyuridine (brdu) to identify newly generate cells. sections were made and immunohistochemically double stained for brdu and cell type markers for neurons, astrocytes and blood vessels. the sections were micrographed and analyzed for hemispheric tissue loss as well as number of new astrocytes, neurons and blood vessels. three weeks after injury there was a significant treatment effect as shown by an increase in neuronal and astrocytic regeneration. however, after six weeks there was no difference in the number of newly generated neurons and astrocytes. evaluation of tissue loss and spatial learning in the mwm corroborated that the treatment had no effect at the later time point. the results clearly show the importance of longterm studies in rodents to ensure that a promising effect on tissue regeneration and functional outcome is not merely temporary. astrocyte activation to extracellular matrix (ecm) -producing cells is inhibitory to regeneration in cns injury or disease. here we show that the cleaved neurotrophin receptor p75 ntr controls transforming growth factor (tgf)-beta-mediated astrocyte activation via regulation of nucleocytoplasmic shuttling of smad2. loss of p75 ntr completely rescues tgf-beta-induced hydrocephaly, astrocyte activation, and ecm production in gfap-tgf-beta transgenic mice. nuclear fractionation of tgfbeta-treated p75 ntr knock out astrocytes revealed reduced nuclear phosphorylated smad2 than their wt counterparts. in accordance to p75 ntr depletion, inhibition of tgf-beta-induced gamma-secretasemediated p75 ntr cleavage also reduces nuclear accumulation of smad2 and the secretion of proteoglycans that inhibit neurite outgrowth. taken together, our results identify regulated intramembrane cleavage of p75 ntr as a mechanism for astrocyte activation resulting in the regulation of tgf-beta signaling and scar formation in the diseased brain. the reaction of glial cells toward brain injury comprises a proliferative response with some reactive astrocytes even reacquiring stem cell potential as indicated by forming long-term self-renewing and mutlipotent neurospheres (for review: robel et al., 2011). here we addressed first the question to which extent the type of injury selectively affects the proliferative response of ng2 glia and astrocytes and to which extent the proliferative reaction of astrocytes may be linked to the stem cell response. interestingly, while both astrocytes and ng2 glia mounted a profound proliferative response after acute invasive injury, such as stab wound and mcao, non-invasive injury models, such as amyloid plaque deposition or widespread neuronal death could not activate the proliferation of these macroglial cells, while microglia proliferated actively in all these lesion models. intriguingly, the proliferative response of astrocytes correlated to the activation of their potential to form self-renewing, multipotent neurospheres with a steep decline with age. we then set-out to determine the signals regulation reactive astrocyte proliferation and neurosphere formation after invasive injury and demonstrate that invasive lesions result in entrance of sonic hedgehog (shh) into the brain from extraneural sources, such as the cerebro-spinal fluid, and activate astrocyte proliferation and neurosphere formation. this response can be blocked by systemic injection of the shh-signaling antagonist cyclopamine as well as inducible astrocyte-specific deletion of the receptor smoothened by glast creert2 . interestingly, shh-agonists can boost the proliferative response of astrocytes in vivo and their subsequent neurosphere-forming capacity, thereby providing a new approach how to activate this response in conditions where it is limited, as e.g. in age or noninvasive injury conditions. taken together, our work highlighted for the first time differences in the proliferative and stem cell response of reactive astrocytes in different injury paradigms and identified the shh signaling pathway as a key signal in this response. tumor necrosis factor (tnf) is a pleotrophic cytokine involved in normal brain function and inflammation, apoptosis, and other responses that occur following injury and disease. there is a considerable amount of confusion as to whether or not tnf activation is beneficial or detrimental following injury to the cns. genetic studies in mice have suggested that inflammation in disease models involves soluble tnf (soltnf) and that maintenance of innate immune function involves transmembrane tnf (tmtnf). these findings suggest that the outcome of selective pharmacologic inhibition of tnf may depend on whether the pharmacologic intervention is targeted towards soltnf or tmtnf. therefore, we took advantage of a dominant-negative inhibitor of soltnf, xpro195, that selectively inhibits soltnf and compared the effect of this inhibitor to a more widely used igg1 fc-tnf receptor (tnfr) 2 fusion protein etanercept, an inhibitor of both soltnf and tmtnf, that has been a successful treatment strategy for several autoimmune diseases. however, etanercept has been shown to display severe side effects by increasing the risk of infections. female c57bl/6 mice were subjected to a spinal cord injury at t9 level. mice were divided into three groups, receiving etanercept, xpro1595, or saline. the drug was delivered directly onto the lesion site using alzet mini osmotic pumps for 3 days starting immediately after introduction of the lesion. mice were allowed to survive 35 days. functional evaluation was performed using the basso mouse scale, rung walk, open field test and hargreaves test. in contrast to etanercept, xpro1595 significantly ameliorated recovery of hind limb function (evaluated by motor recovery score) compared to saline when administered continuously to the lesioned cord, whereas s.c. injections every 3 days for 8 weeks following sci had no effect. our study suggests that selective inhibition of soltnf is beneficial following sci when administered directly to the contused spinal cord and raises the possibility that selective inhibition of soltnf may represent a new therapeutic strategy for the treatment of sci without compromising the innate immune response. here, we show that expression of oncostatin m (osm) is upregulated after spinal cord injury (sci). to reveal the relevance of increased osm signaling in the pathophysiology of sci, osm was applied locally after hemisection. acute osm-treatment significantly improved locomotor recovery after mild and severe sci. delayed osm-treatment in the chronic phase was ineffective. improved recovery in osm-treated mice was associated with a reduced lesion size, less astrogliosis and diminished neuroinflammation. the underlying mechanism involves neuroprotective effects of osm. furthermore, osm promoted nerve fiber regeneration both in vitro and in vivo. thus, local production of osm after sci is an endogenous response that limits cns lesion propagation and promotes recovery. the university of western australia, crawley, australia following neurotrauma, cells beyond the initial trauma site undergo secondary degeneration, with excess ca 21 a likely trigger for loss of neurons, compact myelin and function. treatment of secondary degeneration by limiting excess ca 21 entry into cells using inhibitors of specific ca 21 channels showed promise in preclinical studies, but clinical trials were disappointing and combinatorial approaches are acknowledged as necessary. we assessed efficacy of every possible combination of three ca 21 channel inhibitors at reducing secondary degeneration 3 months after partial optic nerve (on) transection in rat. we used lomerizine to inhibit voltage gated ca 21 channels (for 3 months); oxidised adenosine-triphosphate (oxatp) to inhibit purinergic p2x 7 receptors and/ or 2-[7-(1h-imidazol-1-yl)26-nitro-2,3-dioxo-1,2,3,4-tetrahydro quinoxalin-1-yl]acetic acid (inq) to inhibit ca 21 permeable a-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid (ampa) receptors (both for the first 2 weeks after injury). only the three ca 21 channel inhibitors delivered in combination completely preserved visual function to levels not different from normal animals. in order to determine the mechanism/s by which the three inhibitors delivered in combination preserved function, we assessed neuroprotection, nerve swelling, myelin compaction and node/paranode complexes. preservation of retinal ganglion cell (rgc) somata and axons is unlikely to have accounted for the full recovery of function as only partial neuroprotection of rgcs vulnerable to secondary degeneration was achieved. a range of the ca 21 channel inhibitor combinations prevented swelling of optic nerve vulnerable to secondary degeneration, with no one agent selectively effective. each of the treatments involving lomerizine significantly increased the proportion of axons with normal compact myelin and decreased the proportion of axons with partially decompacted myelin, indicating the importance of sustained control of ca 21 flux via voltage gated ca 21 channels for prevention of chronic myelin decompaction. nevertheless, limiting decompaction of myelin or formation of atypical node/ paranode complexes was not sufficient for preservation of function in our model. it is likely that the additional prevention of the lengthening of the paranodal gap that was only achieved by treatment with the three ca 21 channel inhibitors in combination was an important effect that contributed to the associated preservation of visual function by this combinatorial treatment strategy. to generate myelinating oligodendrocytes opcs undergo a distinct series of morphological and molecular changes. these are regulated by a combination of extrinsic and intrinsic factors, which have only been partially been revealed so far. in the present study we identify a novel signaling mechanism as potent negative regulator of opc maturation. ephrin b3, a member of the b-class family of ephrins, inhibits opc process formation and blocks the formation of mature oligodendrocytes in vitro. the presence of ephrinb3 results in activation rhoa and pkca signalling and deactivation of fak kinase in opcs in vitro. moreover, epha4 rtk, a receptor responsive to ephrinb3 in opcs, is also expressed by opcs in demyelinating lesions. infusion of recombinant ephrin b3 into demyelinating lesions results in a failure of remyelination caused by an inhibition of opc maturation. in contrast, antibody-mediated masking of ephrin b3 epitopes in vitro as well as in demyelinating lesions of aged rats promotes opc differentiation and accelerates myelin regeneration. our results demonstrate an important role of ephrin b3 for the process of remyelination. after peripheral nerve injury, axons rapidly degenerate and start regrowing only after a few days. full functional recovery, however, is rare. a progressive loss of intact axons also determines the clinical phenotype of chronic demyelinating peripheral neuropathies, such as charcot marie tooth disease type 1a (cmt1a). cmt1a is the commonest inherited neuropathy, caused by a duplication of the peripheral myelin protein 22kda (pmp22) gene. we demonstrate that the transgenic neuronal overexpression of the growth factor neuregulin-1 type i enhances axonal regeneration and functional recovery after experimental acute peripheral nerve injury. similarly, neuregulin-1 type i overexpression preserves axonal loss and improves nerve function in a mouse model for cmt1a. importantly, neuregulin-1 type i induced axonal preservation in cmt1a mice was independent of myelination, as myelin sheath thickness was not altered. we conclude that axonal recovery and preservation relies on common supportive factors and that paracrine neuregulin-1 type i constitutes a beneficial signal in peripheral nerve disorders. g. szab o university of tuebingen, tuebingen, germany question: the enormous capacity for repair of the cns after lesion is one of the most amazing property of lower vertebrates. in the mammalian central nervous system (cns) the astrocytes reveal the so-called orthogonal arrays of particles (oaps); the clusterization of the aqp4 molecules at the astrocytic endfeet. in lower vertebrates these structures are lacking. in the case of the amphibia -anolis carolinensis-the caudal spinal cord has the capability for regeneration after loss of the tail, an ability which might be in relation with the presence of tight junctions (tj) between the glial cells like the olfactory ensheating cells act in mammalian olfactory system through the lifespan. the growing axons of the fish optic nerve are embraced by astrocytes which are also interconnected by tjs. tj connected glial cells could contribute to a growth-supportive microenvironment which might reinforce our hypothesis namely the mutual exclusion of aqp4 occurrence in astrocytes and regenerative growth of nerve fiber. methods: we performed immunocyto-and immunohystochemical staining, western blot, freeze-fracture electron microscopy and pcr on cultured aqp4 knockout and wildtype astrocytes and also on formalin fixed paraffin embedded tissue sections regarding junctional proteins, such as occludin, claudin 1,3,5. result: in the light of our experiments, there is no mutual exclusiveness between the presence of aqp4 and tight junctions in astrocytes, at least in mammals. further investigations need to be completed. glutamate and electrical activity influence opc differentiation and myelination in normal development. opcs receive synaptic input from unmyelinated axons, possibly to initiate myelination. both opcs and mature oligodendrocytes respond to glutamate via ampa and nmda receptors. activation of glutamate receptors in vitro increase opc migration but decreases proliferation. here we examine the role of glutamate signalling in remyelination following experimental demyelination in the adult rodent cns. we voltage-clamped opcs in brain-slices of adult rat cerebellar peduncle containing focal areas of primary demyelination and post-identified these by ng2-immunolabelling. recruited opcs mainly expressed ampa receptors at the peak of the opcs proliferation (5-10 days postlesion), as over 90% of the glutamate evoked current was blocked with nbqx (ampa/kainate receptor antagonist) and unaffected by ap5 (nmda receptor antagonist). the demyelinated axons continued to propagate action potentials with latencies similar to those in unmyelinated axons during development. critically, the demyelinated axons established synapses with opcs expressing voltage-gated sodium currents. these synaptic inputs had identical decay times to those recorded during developmental myelination. pharmacological inhibition of neuronal activity or glutamate signalling decreased remyelination efficiency by impairing differentiation. these results indicate that 1) glutamate currents are mainly mediated via ampa and kainate receptors during the opc recruitment 2) opcs establish synaptic contact with demyelinated axons and 3) the neuronal activity is essential for remyelination. together, these data suggest that demyelinated axons remain active and communicate with opcs through glutamate release during the regenerative process to guide their remyelination. there has been much recent interest in the use of intraocular stem cell transplantation to slow or reverse visual loss in glaucoma. however, migration and integration of stem cells into the retina is limited by reactive gliosis, a major barrier to retinal engraftment. our aim was to identify glial modulators of low toxicity and measure their potential to facilitate stem cell entry into the host retina. an explant organotypic culture system was used as a stem cell transplantation model and screening tool for candidate drugs. gfp1 mouse mesenchymal stem cells (mmscs) were co-cultured on the surface of the explants for 4 days.the microglial contribution to mscinduced reactive gliosis was investigated by assessing microglial activation and proliferation using immunohistochemistry for f4/80 and edu. distribution of chondroitin sulphate proteoglycans (cspgs) and their role in the barrier was also explored by immunostaining for chondroitin sulphate (cs-56) and by chondroitinase abc(chabc) treatment. stat3 inhibitor vi and jak1 inhibitor were used to suppress glial fibrillary acidic protein (gfap) expression. the efficacy of each drug to overcome the barrier was evaluated by immunohistochemistry for the glial markers gfap. mmsc integration was assessed by simultaneous immunostaining for gfp. cell viability after drug treatment was investigated by measuring the preservation of retinal thickness and by immunostaining for the neuronal marker neun and for caspase3. co-culture of retinal explants with mmscs caused a 1.5 6 0.4 fold increase in gfap expression. microglial activation and proliferation was not markedly affected by the presence of grafted mmscs on retinal surface. cspgs, highly expressed in the outer retina but only moderately in the inner retina, do not contribute substantially to the barrierto inner retinal engraftment. in contrast, interfering with the jak/stat pathway successfully reduced gfap expression by 60% 6 10% compared to control and facilitated mmscs integration into the host tissue. no toxic effect on cell viability was detected. on the contrary, over the 4 days ex-vivo culture, stat3 inhibitor vi treatment resulted in a neuroprotective effect on the outer nuclear layer, whose thickness (50.84 6 4.4 mm) is better preserved compared to control (38.4 6 9.0 mm) the results show that microglia and cspgs do not play a dominant role in the barrier to retinal engraftment, but the jak/stat pathway represents a promising pharmacological target.targeting modulators of glia reactivity to promote stem cell integration in the host tissue may facilitate stem cell therapy in glaucoma and other neurodegenerative diseases. oecs possess many characteristics favorable for the repair of sci including the secretion of growth and survival factors, the expression of cell adhesion molecules and the ability to integrate with astrocytes. oecs have also been reported to promote exceptional outgrowth of severed axons across the inhibitory environment of the lesion scar. the unique combination of regenerative properties suggests oecs would be beneficial for the repair of ventral spinal root avulsion injuries. after ventral root avulsion anterior horn neurons often fail to regenerate axons back into the surgically re-implanted nerve root resulting in poor functional recovery. to evaluate the efficacy of oec transplants for the repair of ventral root avulsions, the ability of oecs to promote neurite outgrowth from spinal cord neurons was investigated in a spinal cord organotypic co-culture. the integration of oecs with the spinal cord tissue was also examined by confocal fluorescence microscopy. oecs were harvested from adult sprauge dawley (sd) rats, cultured for 14 days and transfected with gfp. slices of cervical spinal cord were prepared from p8 sd pups and cultured on collagen coated membrane inserts. each slice was surrounded by a collagen gel with or without the addition of gfp oecs and glial derived neurotrophic factor (gdnf). our results indicate that oecs alone and in combination with gdnf have a positive effect on neurite outgrowth and morphology. the aim of this study was to obtain an hydrogel for the controlled delivery of vascular endothelial growth factor (vegf-a165). hydrogels are a class of liquid-gel biomaterials with high water content, typically composed of cross-linked, three-dimensional hydrophilic water-soluble polymer networks. the advantages of injectable hydrogel systems are: biocompatibility, biodegradability, adjustable shape, low cost and similarity to the extracellular matrix; moreover, they can be used as growth factor and cell delivery systems for tissue engineering applications. agarose/gelatin (a/gl) hydrogel was cross-linked with genepin. rheological measurements show that a/gl hydrogel can be injected through a syringe into the inner cavity of a nerve guide. in vitro assays performed to evaluate adhesion, proliferation, viability and migrationusing a fibroblast cell line (nih3t3), neonatal olfactory bulb ensheathing cells (nobec) and a schwann cell line (rt4-d6p2t)show that hydrogel allows cell adhesion, proliferation, viability and migration. vegf-a165 is a potent angiogenic factor and angiogenesis has long been recognized as an important and necessary step during tissue repair. vegf is known to have a positive effect on schwann cell proliferation and migration, neuron survival and outgrowth of regenerating nerve fibers. the hydrogel preparation protocol was optimized to be functionally efficient at physiological conditions, allowing the loading of vegf without the risk of its denaturation. different amounts of vegf (50-100-200 ng/ml) were incorporated in the a/gl hydrogel and the releasing rate in vitro up to 65 days was quantified by elisa . released vegf bioactivity was validated in human umbilical vein endothelial cells (huvec) and in nobec by evaluating phosphorylation of vegf-receptor 2 (vegfr2), akt and erk1-2. moreover, dorsal root ganglia explants were cultured on hydrogel containing different amounts of vegf, and an increased neurite outgrowth was quantitatively assessed. these results demonstrate that vegf can be successfully incorporated and bioactive released from a/gl hydrogel inducing vegfr2 activation and neurite outgrowth. in vivo test are ongoing on adult rats: one centimetre lesions on medial nerve were performed, followed by implantation of porous polye-caprolactone tubes filled with a/gl hydrogel containing vegf-a165. functional tests and histological analysis will be carried out to evaluate peripheral nerve regeneration. .spinal cord injury (sci) often leads to death of oligodendrocytes and considerable demyelination. demyelination of axons compromises signal conduction, and contributes to the functional deficits following sci. enhancing remyelination may serve to restore conduction and likely prevents axonal degeneration. here we focussed on a potential therapeutic intervention to promote remyelination, the transplantation of human oligodendrocytes precursor cells (opc)s capable of differentiating into mature oligodendrocytes and remyelinating denuded axons. human platelet derived growth factor (pdgf)-responsive neural precursor (prp) cells were isolated from the fetal forebrain and had been previously shown to differentiate into mature oligodendrocytes with a higher efficiency in vitro than traditional egf/fgf responsive neural stem cells (chojnacki and weiss 2004; deleyrolle et al. 2006 ). therefore, we tested the potential of human prps to differentiate into new oligodendrocytes and remyelinate axons in a rodent thoracic contusion spinal cord injury model. one week following injury, human prps were transplanted rostral and caudal to the lesion site. subsequent histological examination at two, seventeen and forty-two days post-transplantation demonstrated that human prps survived and integrated well into host tissue, particularly when nk cells were depleted. transplanted human prp's labelled with the mature oligodendrocyte marker apc, and 45% of the cells colabelled with the oligodendroglial marker olig2. astrocyte differentiation was also observed, suggesting human prps function as a multipotent glial progenitor following sci. human-specific immunoreactive processes were seen in close association with axons expressing the myelin protein mbp, providing evidence of remyelination by the transplanted cells. overall, these findings indicate that human prps are a source of new oligodendrocytes capable of producing myelin in response to spinal cord injury. remyelination is a regenerative process during which demyelinated axons are enwrapped by newly formed myelin sheaths. in the central nervous system (cns), this process takes place in two stages: first, during the recruitment stage, oligodendrocyte precursor cells (opcs) divide, migrate and engage the demyelinated axons. then, during the differentiation stage, opcs differentiate to myelin-forming oligodendrocytes. under pathological conditions such as multiple sclerosis (ms), remyelination often fails and as a consequence chronic demyelinated areas accumulate in ms. post mortem evidence suggests that opc differentiation is often impaired in ms patients. however, the reason why opc differentiation fails in ms is not completely understood. it has been proposed that ms lesions contain factors that act as specific inhibitors of opc differentiation. amongst them, myelin associated inhibitors play an important role. we have demonstrated that the inhibitory effect of myelin protein extracts (mpe) is mediated by activation of signalling cascades including pkca-marcks in vitro. furthermore, modulation of pkca has been shown to rescue opc differentiation in the presence of inhibitory mpe. here we demonstrate that tamoxifen, a drug that is used in various clinical settings because of its pkc-inhibitory activity, is able to promote opc differentiation in vitro. in a series of in vivo experiments, we tested the hypothesis that inhibition of pkca by tamoxifen promotes cns remyelination. demyelination was induced in the caudal cerebellar peduncle (ccp) of young-adult sprague-dawley rats by the administration of ethidium bromide. animals received daily doses of tamoxifen. although tamoxifen treatment did not induce changes in the number of opcs expressing immature markers (ng2 and pdgfr-a), a significant increase in the number of cells expressing mature marker of oligodendrocytes (cc1/olig2 and plp) was detected. furthermore, analysis of resin-embedded tissue demonstrated that tamoxifen significantly promotes remyelination in the cns when compared to controls (mann-whitney's test, p < 0.05). although further investigations are required to elucidate the mechanisms by which tamoxifen exerts cns remyelination, our results demonstrate that administration of tamoxifen represents a potent and clinically accessible strategy to promote endogenous myelin regeneration. induced pluripotent stem (ips) cell technology now allows development of autologous oligodendrocyte precursor cells for putative remyelination cell therapy for multiple sclerosis (ms). we have successfully developed mouse and human ips cells and converted these into oligodendrocyte precursor cells (opcs) that can differentiate into fully mature oligodendrocytes. furthermore, we have shown that (intracerebral) implantation of ips-derived opcs in mouse ms models enhances remyelination.before implantation-induced remyelination therapy with autologous ips-derived opc can proceed to a clinical trial for remyelination cell therapy in ms patients, we have to provide solid evidence for long term efficacy and to establish the safety thus ultimately excluding contamination of teratogenic (pluripotent) stem cells. these issues are best examined in a nonhuman primate model. the biomedical primate research center (bprc), rijswijk has developed a welldocumented experimental autoimmune encephalomyelitis (eae) model in marmoset monkeys that most closely approximates the pathology of ms in humans. in this animal model we will verify that intrathecally administered ips-derived opcs migrate to and remyelinate eae lesions and determine their long-term fate.we have developed an efficient protocol to induce marmoset ips cells from skin fibroblasts. for that we have used a lentiviral polycistronic construct, containing the four (human) reprogramming genes oct4, klf4, sox2 and cmyc, that is excisable using cre-recombinase. the pluripotent nature of the marmoset ips cells was established based on the endogenous expression of pluripotency transcription factors and the ability to differentiate in-vitro (via embryoid bodies) and in-vivo (subcutaneous injection for teratoma formation) into all the different tissues of the three germ layers. presently we are developing protocols for the differentiation of these marmoset ips cells into an oligodendrocyte cell lineage. published protocols have been modified for production of opcs for research purposes, and produced cells are used for characterization of opcs. ultimately the aim is to define an opc population with optimal myelination capacity. results and discussion: currently, human oligodendrocytes and opcs can be produced from pluripotent stem cells. these cells are routinely characterized in protein expression level, and methods for more comprehensive characterization are under development. produced opcs can be purified with fluorescence-activated cell sorting based on ng2 protein expression in order to study if this cell population is heterogeneous in terms of myelination capacity. conclusions: oligodendrocytes and opcs can be produced from human pluripotent stem cells. studies are ongoing to determine the molecular characteristics and myelination capacity of the differentiated and purified opc-population. remyelination, the regenerative process in which myelin is restored to demyelinated axons, is carried by oligodendrocyte precursor cells (opcs). in a previous study we have shown that oligodendrocyte precursor cells are multipotent and they can differentiate into oligodendrocytes as well as to schwann cells and occasionally astrocytes in the remyelinating lesions. we observed that schwann cells derived from opcs in the central nervous system occupied almost exclusively tissue around blood vessels in astrocyte deficient areas. therefore, we postulated the occurrence of specific niches or microenvironments that modulate opc fate. the aim of present study was to identify the factors and their downstream effectors that significantly discriminate vascular and nonvascular niche and determine the fate of oligodendrocyte precursor cells. we microdissected tissue from vascular and non-vascular regions of lesion areas at 6, 10 and 14 days after bilateral stereotaxic injection of ethidium bromide into the brain white matter of adult rats and performed microarrays to profile the whole transcriptome of both niches separately. this approach allowed us to distinguish between mrnas that are enriched within two separate environmental niches within the same lesion area. the study identified 138 differentially expressed transcripts. comparative bioinformatic analysis of global gene expression combined with signalling transduction pathway structure and genes function analysis revealed specific candidate signaling pathways differentially expressed in local peri-vascular niche compared to nonvascular one. results of rt-pcr-based validation of microarray data have proven that expression gradient of bmp4, bmp7, their antagonist sostdc1, as well as wnt2b, wnt6, dhh and scube1 act as the major regulators of opcs fate in the vascular niche. moreover, we observed that blood vessels undergo substantial regeneration in the course of remyelination and postulated the important role of blood vessels reconstruction in creating the specific microenvironment of neurovascular niche during tissue regeneration. our analysis shown that unique properties of tissue around blood vessels differ from the rest of the lesion which can be one of the factors favoring opcs alternative differentiation in this area of lesion. . however, key aspects of dynamic behavior, such as cell migration or process orientation and motility, can only be monitored by live imaging. to elucidate these key aspects of opc behavior after injury, we used repetitive in vivo two-photon laser scanning microscopy (2plsm) to follow ng2 1 -cells after smaller and larger stab wound injuries in the mouse somatosensory cortex. we therefore used the bac transgenic mouse line sox10 icreert2 (simon et al., 2012) crossed to the inducible reporter line cag-gfp (nakamura et al., 2006) selectively labeling cells of the oligodendrocyte lineage. gfp 1 opcs were imaged through a cranial window at the day of injury and at different time points thereafter. identification of the same cells was possible based on blood vessels labeled by rhodamine dextran as stable landmarks. live imaging revealed that the majority of opcs reacts within 2 days after injury with hypertrophy (enlarged soma and processes), polarization (elongation and concentration of processes towards the injury site), directed migration towards the injury site (defined as movement of the cell body for at least 10 mm) and proliferation (defined by a single cell being replaced by 2 or even more daughter cells). only a small proportion of cells within $500 mm of the injury did not react in any detectable manner. we noted that polarization and hypertrophy occur rather fast after inflicting the injury, while proliferation peaks 4 days after injury. taken together, the live observation of opcs reacting to stab wound injury supports the concept of opc heterogeneity and reveals new insights into the functional role of these cells after injury: the fast process orientation towards the injury site implies a contribution to wound closure and their substantial proliferation sometimes for several rounds amplifies the number of ng2 1 -cells surrounding the injury site with implications for scar formation. the aim of this study was to create a non-invasive tool for cell visualization and cell population tracking that can be used in long term studies. methods: this study was performed using human pluripotent stem cell lines derived in institute of biomedical technology (former regea), university of tampere, finland. cells were differentiated to neural cells using neural differentiation protocol described earlier by lappalainen and colleagues. fluorescent probes used in this study were 1) 5chloromethylfluorescein diacetate (celltracker green cmfda, ct) and 2) 1,1 0 -dioctadecyl-3,3,3 0 ,3 0 -tetramethylindodicarbocyanine perchlorate (did). cell viability and proliferation of the labeled populations was briefly studied. also, the labeled cells were characterized using immunocytochemistry and neuronal functionality was studied using micro electrode array (mea). results: during labeling optimization a wide range of different labeling parameters was tested for both probes to obtain long term labeling. most suitable parameters for human neural cells were 10 mm ct with 72 hours incubation time and 10 mm did with 2 hours incubation time. with these concentrations the labeling was visible up to 4 weeks and had no statistical effect on cell viability. ct labeling was not affecting to cell proliferation but with did slight decrease in proliferation was seen in long term culture. labeled populations contained both map-2 positive neurons and gfap-positive astrocytes. also, the ct or did labeled population and their mixed-cultures expressed normal spontaneous network activity when cultured on mea-platform. conclusions: this study indicated that fluorescent probes ct and did are suitable for long-term labeling of human derived neural cultures in vitro. the labeling had no negative effects on cell behavior with the exception of did labeled cells having slight decrease on cell proliferation. importantly, the labeled neural networks were electrically functional. axonal regeneration after spinal cord injury (sci) is limited mainly due to the presence of an inhibitory microenvironment, especially reactive astrogliosis and glial scar formation. overcoming this inhibitory barrier of reactive astrocytes and inhibitory substances making the microenvironment of the damaged neurons permissive for axonal regrowth might be crucial for cns repair. single-dose irradiation has been proved to be neuroprotective in cns trauma through elimination of reactive astrocytes, however, the irradiation conditions with singledose protocols are not in clinical use. in the present study, we aimed to investigate whether low-dose-fractionated irradiation, which is currently being used widely for clinical treatment of several tumor types, could regulate cell cycle elements of reactive astrocytes just like its use in cancers, inhibit glial scar formation, attenuate astrogliosis-mediated inhibition of axonal regeneration and facilitate recovery of motor function after spinal cord hemi-section in beagle dogs. as expected, our results demonstrated that low-dose-fractionated irradiation reduced reactive astrogliosis, attenuated astrogliosis-associated neural injuries, ameliorated microenvironment of axonal regeneration, and benefited recovery of the animals subjected to spinal cord trauma. accordingly, irradiation might be a promising therapeutic strategy targeting at excessive astrogliosis for sci in the near future. mesenchymal stem cell transplantation has been proven to have beneficial effects in various degenerative diseases, including demyelinating models. both in our lab as well as others have shown that they express a number of trophic factors that are capable of inducing remyelination, mainly by activating nearby oligodendrocyte progenitors towards mature oligodendrocytes that in turn remyelinate the damaged area. however, this effect was only observed locally, in the area surrounding the graft, thus in order to achieve general remyelination in various brain structures simultaneously, we decided to perform bone marrow-derived mesenchymal stem cell injections into the lateral ventricles. in this manner, the cells may attach to various areas such as the hippocampus, corpus callosum and fornix, among others, all of which are demyelinated. previous to the graft, the cells were incubated with iron nanoparticles. this way, it is possible to track in vivo the grafted cells by magnetic resonance imaging (mri). as a result, the cells were observed at different time points (0-15-30-60-90 days) by mri. also, the demyelinated areas can also be visualized and myelin quantified using image analysis software. this allows the quantification myelin density in the same individual mice at different moments before and after transplantation. j. muenzel 1,2 , c. becker 2 , t. becker 2 , a. williams 1 1 university of edinburgh, centre for regenerative medicine, edinburgh, united kingdom 2 university of edinburgh, centre for neuroregeneration, edinburgh, united kingdom central nervous system (cns) remyelination is important for the restoration and protection of proper nervous system function in demyelinating diseases such as multiple sclerosis but is poorly understood, and inefficient in humans. in contrast, zebrafish are able to regenerate many tissues very efficiently. as zebrafish are being increasingly used to study myelination, we aimed to develop and characterise an adult zebrafish model of cns de-and remyelination, to answer the question if remyelination is also very efficient, to describe the biology and to identify similarities and differences with mammalian remyelination. this then may allow us to better understand why zebrafish have a high regenerative capacity compared to rodents and humans. previous studies in teleost fishes have used optic nerve crush as a paradigm of myelin injury to investigate myelin repair. this, however, involves the de novo myelination of regenerated axons, rather than deand remyelination of the same axons. lysophosphatidylcholine (lpc) is a detergent-like toxin, which is widely used in rodent models to demyelinate axons. we administer lpc to the optic nerve of adult zebrafish and observe less myelin by both immunoreactivity and electron microscopy at the lesion site at 8 days post lesion (dpl) and microglia activation along the optic pathway. immunoreactivity and electron microscopy suggest complete regeneration of myelin at 28 dpl. we cannot identify any axonal injury in this model. in young zebrafish (aged 4-6 months), the myelin thickness of remyelinated fibres shows no difference to the pre-lesion state, which is different to mammals, where the myelin thickness is reduced. however, in old fish (aged 18 1 months), although the axon diameter is the same as pre-lesion and in young animals, , remyelinated fibres have thinner myelin, suggesting that the regenerative capacity of zebrafish declines with age. we believe that this new zebrafish model of cns remyelination can be added to the suite of current models to better understand the remyelination process, why it fails, and to test for compounds to improve it, with the added benefit that zebrafish are rapid breeders, transgenesis is easy and there is a high potential for live imaging. although often described as a hard-wired component of the vertebrate body, the nervous system is a plastic and considerably fluid organ system that reacts to external stimuli in a consistent, stereotyped manner, while maintaining incredible flexibility and plasticity of its core components. unlike the cns, the pns is capable of significant repair, but we have only just begun to understand the cellular and molecular mechanisms that underlie this phenomenon. peripheral nerves are composed of axons surrounded by layers of glia and connective tissue. they are ensheathed by myelinating or non-myelintaing schwann cell glia, which are in turn wrapped into a fascicle by a cellular sheath called the perineurium. this structure forms from centrally-derived glial cells and serves as a protective barrier that is essential for nerve function. following an injury, adult peripheral nerves have the remarkable capacity to remove damaged axonal debris, regenerate and re-innervate targets. schwann cells have been shown to play an important role in this process by trans-differentiating, proliferating, clearing debris, and guiding re-growing axons, but less is known about the potential role of perineurial glia. to investigate the role of perineurial cells in pns regeneration, we have developed an injury response assay that uses a micropoint laser to create injuries along the motor nerves in live transgenic zebrafish. time-lapse imaging of injured nerves reveals that perineurial glia rapidly respond to nerve injury and extend processes toward the injury zone. this is in contrast to schwann cells, which we observe orienting towards the distal stump where they engulf and clear axonal debris. these data demonstrate that perineurial glia respond immediately to motor nerve injuries in a manner distinct from schwann cells, and future work is aimed at defining the molecular mechanisms that mediate the cellular responses of perineurial glia and schwann cells, as well as determining if developmental paradigms are recapitulated in these glial populations during the regenerative process. we have previously shown that in schwann cells the transcription factor c-jun acts as a master regulator of wallerian degeneration and is required for successful repair following nerve injury (arthur-farraj et al., methods. we here investigate on the release of the gliotransmitter glutamate from superfused isolated purified glial processes (gliosomes) obtained from adult rat cerebellum. intracellular ca 21 levels were measured by a microfluorimetric technique. immunocytochemical confocal and western blot analysis were also carried on cerebellar gliosomes. results. confocal and western blot analysis confirmed that cerebellar gfap-positive gliosomes are a purified preparation of glia processes. more then 80% of gliosomes were positive for brain-specific lipid binding protein (blbp), indicating that they derived from bergmann cells. by measuring the release of glutamate we obtained evidence for the presence of glutamate release-facilitatory ampa receptors on the bergmann glial processes. in fact, ampa evoked [ 3 h]d-aspartate or endogenous glutamate release which was abolished in ca 21 -free medium; effectiveness of the selective inhibitor naphthylacetyl spermine indicated the involvement of ca 21 -permeable, glua2-lacking ampa receptors. ca 21 imaging confirmed the presence of ca 21 -permeable, glua2lacking ampa receptors on bergmann glia processes. activation of the glua2-lacking ampa receptors triggered vesicular exocytotic glutamate release: inhibition of vesicular loading by the vesicular glutamate transporter (vglut) inhibitors rose bengal or trypan blue, or by the h 1 -atpase inhibitor bafilomycin a1 prevented the ampa-evoked glutamate release (see figure) . confocal analysis confirmed that blbpand gfap-positive processes expressed vglut1 and vglut2. conclusions. we here report evidence that: 1. functional processes of bergmann cells are recovered in purified preparation of adult rat cerebellar gliosomes; 2. ampa receptors located on bergmann glia processes show features of ca 21 permeable, glua2-lacking ampa receptors; 3. activation of the ampa receptors evokes ca 21 entry in isolated bergmann glia processes and ca 21 -dependent vesicular glutamate release from the processes. activation of ca 21 -permeable, glua2lacking ampa receptors coupled to vesicular glutamate release might represent a mechanism by which bergmann glia processes modulate synaptic transmission at parallel fibers/purkinje cell synapses. the formation of brain edema, which accompanies ischemic or traumatic brain injuries, originates from a disruption of ionic/neurotransmitter homeostasis that leads to extracellular k 1 elevation and neurotransmitter accumulation in the extracellular space. an increased uptake of these osmotically active substances, predominantly provided by astrocytes, is accompanied by intracellular water accumulation via aquaporin-4 (aqp4). previously, it has been shown that the removal of perivascular aqp4 via the deletion of alpha-syntrophin, a protein responsible for anchoring aqp4 on the astrocytic membrane, delays edema formation and k 1 clearance (amiry-moghaddam et al.,2003, pnas 11;100(23):13615-20). therefore, we aimed to elucidate the impact of alpha-syntrophin deletion on astrocyte volume changes in the cortex during pathological states, such as hypoosmotic stress or oxygen-glucose deprivation (ogd), using three-dimensional (3d) confocal morphometry in situ. in addition, single cell rt-qpcr profiling was carried out to reveal possible differences in the expression profiles of ion channels/transporters that participate in maintaining ionic/neurotransmitter homeostasis. in order to visualize individual astrocytes that lack alpha-syntrophin, double transgenic mice were generated by crossbreeding gfap/egfp mice ( . 3d-confocal morphometry revealed that alpha-syntrophin deletion results in significantly smaller/slower astrocyte swelling when induced by 20 min hypoosmotic stress (210 mosm), 30 min ogd or by high extracellular k 1 (50 mm), while alphasyntrophin deletion had no effect on the astrocytic shrinkage evoked by hyperosmotic stress (350 mosm). the volume recovery of cortical astrocytes from gfap/egfp/alpha-syntrophin knockout mice was significantly slower following their exposure to hypo-or hyperosmotic stress, whereas no differences were found in astrocyte volume recovery following ogd or after their exposure to high extracellular k 1 . compared to the cortical astrocytes of gfap/egfp mice, single cell rt-qpcr analyses revealed that astrocytes from gfap/egfp/alpha-syntrophin knockout mice express higher mrna levels for two-pore domain k 1 channels (twik-1, task-2, task-3), inwardly or outwardly rectifying k 1 channels (kir3.1, kir5.1, kv1.3, kv1.6) and chloride channels (clc1,clc4), while mrna expression for the glutamate transporter glt-1 is lower. in summary, the deletion of alpha-syntrophin slowed down astrocyte swelling during hypoosmotic stress, ogd or high k 1 ; however, it also resulted in alterations in astrocytic gene expression profiles. supported by grants ga cr 13-02154s and p304/12/g069 from the gacr. microglia undergo a process of activation in any type of pathology which is controlled by many factors such as cytokines, chemokines or growth factors, but also by neurotransmitters. we found that a subpopulation (16%) of freshly isolated microglial cells from the adult brain respond to the muscarinic acetylcholine receptor agonist carbachol with a ca 21 response, indicating the expression of functional receptors. the carbachol-sensitive population increased in microglia/ brain macrophages isolated from the tissue of mouse models for stroke (57%) and alzheimer's disease (26%), but not for glioma and multiple sclerosis. microglia cultured from adult and neonatal brain contained a similar carbachol-sensitive sub-population (17% and 10%) which was increased by treatment with interferon-c to 60% within 12 hours, and was sensitive to blockers of protein synthesis. carbachol was a chemoattractant for microglia and decreased phagocytosis activity, indicating that microglia are a functionally heterogeneous population. t13-05a astrocytes and s1p receptors the family of sphingosine 1-phosphate receptors (s1prs) are g protein-coupled comprising five subtypes (s1p1r-s1p5r). these receptors are expressed in cells of the immune, cardiovascular, and central nervous systems (cns), in addition to others. s1prs play important roles in celular proliferation, differentiation, survival and migration. recently, the immunomodulatory drug, gilenya v r has been approved as the first oral therapy for multiple sclerosis (ms), after proving efficacious in clinical trials. the active ingredient of gilenya v r is the phosphorylated compound fty720 (pfty720), which is a potent agonist on all s1prs, except s1p2rs. pfty720 has been suggested to work as a 'functional antagonist' causing s1p1r internalisation in lymphocytes, thus limiting t cell auto-immunity. in addition to regulating the immune system, the lipophilic nature of the pro-drug fty720 allows it to readily cross the blood-brain-barrier (bbb) where it may also activate s1prs expressed on both neurons and glia. here, the role of s1prs in the cns was investigated, by specifically investigating their role in astrocyte function. a range of methods were used to examine the effects of s1pr activation and their roles in astrocytes, including: (i) s1p1r subtype trafficking, (ii) transient and continued intracelular signalling, (iii) astrocyte cell migration, (iv) release of pro-inflammatory cytokines, (v) oligodendrocyte cell differentiation and survival and (vi) myelination state in brain slice cultures. the data showed that activation of the s1p1r subtype leads to (i) its internalisation and (ii) continued signalling in astrocytes, and that s1p1rs were found to (iii) promote astrocyte migration, (iv) limit release of pro-inflammatory cytokines, (v) promote oligodendrocyte survival and (vi) limit demyelination. these studies demonstrate a role for s1p1rs in regulating astrocyte function and suggest their use a drug targets for neuroinflammtory and neurodegenerative disorders. in particular, both astrocytes and oligodendrocytes express kir4.1, which are essential for setting their strongly negative rmp, as well as the maintenance of [k 1 ] o following neuronal activity. notably, strong expression of the kir7.1 subtype was determined during a whole genome microarray analysis of the mouse optic nerve, a typical cns white matter that contains mainly astrocytes and oligodendrocytes. the kir7.1 subunit is known to be involved in potassium transport in epithelial cells and has been identified in cerebellar purkinje neurons, but has not been reported in glia. here, we examined functional expression of kir7.1 in mouse brain and optic nerve. rt-pcr and western blot confirmed expression of kir7.1 mrna and protein expression in the brain and optic nerve. positive kir7.1 immunostaining was observed in astrocytes and oligodendrocytes in brain sections and in optic nerve explant cultures and the results indicated a developmental increase in kir7.1 expression. in astrocytes, kir7.1 was localised to perivascular end-feet, supporting a potential role in k 1 regulation. in oligodendrocytes, kir7.1 were localised to cell somata, suggesting a developmental role in setting their rmp. in addition, oxygen and glucose deprivation (ogd) experiments were performed on isolated intact optic nerves from mice aged p10 and examined for the effects of the kir7.1 channel blocker vu590. after 60 min ogd, blockade of kir7.1 resulted in increased cell death of optic nerve glia, measured using propidium iodide (pi) labelling, from 80 6 30 in controls to 189 6 49 in vu590 (n 5 4 nerves per group, 6 fov per nerve; p < 0.001, anova). our results demonstrate expression of kir7.1 in glial cells, and indicate they are important in protecting glial cells from ischemic damage. is an eight transmembrane domain protein highly expressed in brain. human mutations in mlc1 lead to the rare genetic disorder "megalencephalic leukoencephalopathy with subcortical cyst" (mlc). characteristic for the disease are the onset of macrocephaly within the first year of life, and a slowly progressive loss of motor skills with epilepsy and cognitive decline. at the cellular level, countless fluid-filled vacuoles occur within myelin sheaths surrounding axons and in astrocytic endfeet. in cell culture models, mlc1 mutations are associated with defects in chloride currents and cell volume regulation. thus far, the expression and function of mlc1 in intact murine and human brain are still controversial. to understand the role and developmental expression of mcl1 in the brain, we have developed a mlc1 mutant null mouse model carrying an egfp reporter gene under the mlc1 promotor. we now show the exclusive expression of mlc1 in astrocytes throughout the brain with specifically higher expression around blood vessels, at the sub-ventricular zone and at the glia limitans. the functional loss of mlc1 in mutant mice recapitulates in part the human mlc disease. ko mice show macrocephaly with high brain wet weight. water filled vacuoles develop in the white matter of the cerebrum and in large fiber tracts of the brainstem. by contrast, the heterozygous loss of mlc1 has no consequences on myelin structural integrity. both heterozygous and homozygous mlc1 ko mice have dysmorphic peri-vascular and peri-ventricular astrocytes. in contrast to humans, ko mice have no motor deficits, but are hyperactive and show an anxiety-like behavior. in the mlc1 ko mouse, a dysfunction of an astrocytic protein causes loss of myelin structural integrity leading to vacuolating myelinopathy. therefore, the mlc mutant mouse could be a key model to study the astrocytic involvement in brain water and ion homeostasis. gaba activated slowly desensitizing responses in ng2 cells which were mimicked by muscimol and inhibited by bicuculline. to elucidate the subunit composition of the receptors we tested its pharmacological properties. co-application of pentobarbital, benzodiazipines and zolpidem all significantly increased the gaba responses. the presence of small tonic currents indicated the presence of extrasynaptic gaba a receptors. to further analyze the subunit expression, single cell transcript analysis was performed subsequent to functional characterization of ng2 cells. the subunits a1, a2, b3, c1 and c2 were most abundantly expressed, matching properties resulting from pharmacological characterization. importantly, lack of the c2 subunit conferred a high zn 21 sensitivity to the gaba a receptors of ng2 cells. to determine the effect of gaba a receptor activation on membrane potential, perforated patch recordings were performed. in the current-clamp mode, gaba depolarized the cells to about 230 mv, indicating a higher intracellular clconcentration (about 50 mm) than previously reported. gaba-induced depolarization in ng2 cells might trigger ca 21 influx through voltage-activated ca 21 channels. schwann cells (sc) play important roles in the development and regeneration of the peripheral nervous system (pns) following injury. several molecules such as neurosteroids and neurotransmitters have been suggested as potential pharmacological targets in regulating sc physiology and regenerative potential. nevertheless, the slow growth rate and difficulties in harvesting limit sc applications in regenerative medicine. adipose-derived stem cells (asc) can be differentiated into a sc-like phenotype (dasc) sharing morphological and functional properties in the present study, we analysed changes in glutamate transporter expression and function following a mechanical lesion in organotypic slice cultures of the mouse hippocampus using immunohistochemistry, western blots and dynamic imaging. the lesion was positioned perpendicular to the stratum pyramidale in the ca1 area and comprised the entire hippocampus proper. after three-six days, a glial scar had formed along the lesion site. activated astrocytes in close proximity (100-150 mm) to the lesion ("scar cells") directed long, palisading gfap-positive processes towards the lesion, had significantly swollen somata and lost their ability to take up sr101. furthermore, some exhibited distinct clustering of glast and glt-1 immunoreactivity. scar cells showed greatly diminished increases in intracellular sodium in response to application of d-aspartate, an agonist for glutamate transporters. astrocytes in the periphery to the lesion, in contrast, maintained their ability to take up sr101 and showed only slight upregulation of gfap, as well as less swollen cell bodies. cells in the periphery displayed only marginal changes in glutamate transporter immunoreactivity and unaltered amplitudes of sodium changes in response to d-aspartate. taken together, our data show that mild astrogliosis in the periphery of a mechanical lesion is not accompanied by a significant change in glial glutamate uptake capacity. at the scar itself, a strong clustering of glutamate transporters is observed that apparently goes along with a severe functional reduction in glutamate uptake. transport of base equivalent (hco 3 -) across the membrane is a crucial process to maintain the intracellular and extracellular proton concentrations within a narrow physiological range. the electrogenic sodium-bicarbonate cotransporter (nbce1, slc4 a4) is a major base transporter in eukaryotes and expressed in many tissues, especially in epithelial cells. nbce1 may transport significant amounts of bicarbonate into and out of the cell. in the brain, nbce1 is predominantly expressed in glial cells and operates with a transport stoichiometry of 1na:2hco 3 -. we have studied the bicarbonate sensitivity of nbce1, in primary cultured mouse cortical astrocytes (c57bl6/n) using live cell fluorescence imaging with confocal microscope and bcecf-am as a proton sensitive fluorescence probe. we have also used the primary cortical astrocyte culture from nbce1-ko mouse to compare the nbce1-mediated processes in wt astrocyte culture. bicarbonate dose response protocols suggest that nbce1 has a very high affinity for bicarbonate (k m <1 mm). due to this high affinity for bicarbonate, nbce1 can sensitize even residual bicarbonate concentrations of 150-300 lm present in nominally bicarbonate-free extracellular solutions (buffered with hepes). the removal of residual bicarbonate by saturating extracellular buffer (hepes) with 100% o 2 reduced these nbce1-mediated intracellular proton changes significantly. in astrocytes of nbce1-ko mice, cytosolic h 1 changes could not be detected at hco 3 concentrations lower than 3 mm. it has been reported that astrocytic nbce1 activity mediates the activation of astrocytic glycolysis by a mechanism dependent of intracellular bicarbonate and ph (ruminot et al 2011 j neurosci 40: 64-71). using a genetically encoded fret-based glucose nanosensor, we show that glucose metabolism can be activated at low millimolar bicarbonate concentration, which was largely absent in astrocytes from nbce1-ko. our results demonstrate the highest bicarbonate sensitivity yet described in animal cells, mediated by nbce1 in cortical astrocytes. nbce1 may function as a bicarbonate sensor in these cells, e.g. for modulating energy metabolism. supported by the deutsche forschungsgemeinschaft de 231/24-1. inward currents which were significantly blocked by the l-type calcium channel antagonist nimodipine. to better investigate the role of l-type calcium channels in ng2 glia at different developmental stages of the cns, we take advantage of the inducible cre-lox system by breeding homozygous cav1.2 floxed mice and cav1.3 flexed mice with ng2-creert2 knock-in mice. we are currently investigating how the removal of cav1.2/1.3 alters proliferation, migration and survival of ng2 glia. a particular focus lies on the analysis of behavioral abnormalities generated by cav1.2/1.3-deficient ng2 glia. chronic neuropathic pain is a frequent consequence of spinal cord injury (sci). yet despite recent advances, upstream releasing mechanisms and effective therapeutic options remain elusive. previous studies have demonstrated that sci results in excessive atp release to the peritraumatic regions and that purinergic signaling, among glial cells, likely plays an essential role in facilitating inflammatory responses and nociceptive sensitization. we sought to assess the role of connexin 43 (cx43) as a mediator of cns inflammation and chronic pain. to determine the extent of cx43 involvement in chronic pain, a weight-drop sci was performed on transgenic mice with cx43/cx30 deletions. sci induced robust and persistent neuropathic pain including heat hyperalgesia and mechanical allodynia in wild-type control mice, which developed after 4 weeks and was maintained after 8 weeks. notably, sciinduced heat hyperalgesia and mechanical allodynia were prevented in transgenic mice with cx43/cx30 deletions, but fully developed in transgenic mice with only cx30 deletion. sci-induced gliosis, detected as upregulation of glial fibrillary acidic protein in the spinal cord astrocytes at different stages of the injury, was also reduced in the knockout mice with cx43/cx30 deletions, when compared with littermate controls. in comparison, a standard regimen of post-sci treatment of minocycline attenuated neuropathic pain to a significantly lesser degree than cx43 deletion. these findings suggest cx43 is critically linked to the development of central neuropathic pain following acute sci. since cx43/cx30 is expressed by astrocytes, these findings also support an important role of astrocytes in the development of chronic pain. mast cells (mcs) are immune cells that reside in normal brain tissue. they store in granules an array of pro-inflammatory mediators, which are rapidly released to the extracellular milieu upon activation through an extracellular ca 21 -dependent mechanism. neurotoxic amyloid peptides are known to induce mcs degranulation but the membrane transduction mechanism remains unknown. here, the possible role of pannexin 1 hemichannel (panx1 hc) known to be permeable to ca 21 was studied. objective: since ca 21 influx is essential for mcs degranulation, we evaluated if the neurotoxic amyloid fragment ab 25-35 peptide promotes mcs degranulation via activation of panx1 hcs. methods: primary na€ ıve mcs were differentiated from bone marrow precursors of wild type (wt) and panx1 knock out (panx1 -/-) c57bl/6 mice using il-3 present in wehi3 conditioned medium. the presence of panxs 1, 2 and 3 mrnas presence was evaluated by rt-pcr analyses. the hc activity was assessed using the 4',6-diamidino-2-phenylindole (dapi) uptake assays and measurements of membrane current using whole cell patch clamp while intracellular ca 21 signal was evaluated using fura-2am. degranulation was assessed by quantification of extracellular histamine as well as release of toluidine blue (tb) from tb preloaded mcs from wt and panx1 -/mice. results: only panx1 mrna was detected in wt mcs. stimulation with ab 25-35 but not ab 35-25 peptide induced histamine and tb release. it also enhanced dapi uptake and total membrane current in wt mcs. these responses were drastically reduced in wt mcs pretreated with 10 mm carbenoxolone and 200 mm 10 panx1, blockers of panx hcs and were absent in panx1 -/-mcs. in wt mcs treated with ab 25-35 increase the intracellular ca 21 signal and was drastically prevented by 10 panx1, absence of extracellular divalent cations and in panx1 -/-mcs. conclusion: these findings indicate that mcs express functional panx1 hcs, which are essential for ca 21 influx required for the degranulation response induced by ab 25-35 . thus, inhibition of panx1 hcs might avoid spreading of mc-dependent inflammatory mediators released during alzheimer's disease progression. gliomas are the most frequent primitive cns tumors and are thought to derive from astrocytes or from neural progenitors/stem cells. however, the precise identity of the cells at the origin of gliomas remains a matter of debate because no pre-neoplastic state has been yet identified. tgf alpha is frequently over-expressed in the early stages of glioma progression. sharif & al (oncogene. 2007 (19):2695-706) previously demonstrated that prolonged exposure of normal astrocytes to tgf alpha is sufficient to trigger their reversion to a neural progenitor-like state. when astrocytes de-differentiated with tgf alpha were submitted to oncogenic stress using gamma irradiation, they acquired cancerous properties: they were immortalized, showed cytogenomic abnormalities, and formed high-grade glioma-like tumors after brain grafting. our study aims to identify and characterize the protein signature of those in vitro transformed cells in an attempt to understand their neoplastic behavior and the effect of transformation on metabolic processes. this involves a global proteomic analysis using the 2d-dige methodology and a study of the metabolism of these cells (carbon source, lactate, glucose and glutamine use, ros metabolism…) by 1hand 13c-nmr nmr quantification of metabolites. such approaches are expected to provide information allowing understanding the metabolic reprogrammation that occurred during transformation. the comparative 2d-dige proteomic analysis of normal and transformed astrocytes shows that during transformation, the cells increase their expression of glycolytic enzymes, thus acquiring the ability to use aerobic glycolysis (warburg effect). moreover, the transformed cells reduce their capacity for tricarboxylic acid oxidation and for neurotransmitters (glutamate and gaba) metabolism. ingenuity pathway analysis indicates major effects on carbohydrates, amino acids and nucleotides metabolic components. using enzymatic activity measurements and the detection of protein isoforms by 2d-western blot and zymography, we document a change in expression and activity of various isoenzymes that may be responsible for those metabolic reprogrammations. r.e. k€ alin, a. jarczewski, f. apel, r. monk, s. kraft, j. radke, f.l. heppner charit e -universit€ atsmedizin berlin, neuropathology, berlin, germany question: glioma are the most frequent malignant primary tumors of the brain. glioma survival and growth is determined by the interaction with brain parenchyma, which includes intense tumor angiogenesis. this process is supported by glioma-invading myeloid (gim) cells, namely brain resident microglia or brain-invading macrophages. depletion of gim cells leads to a significant decrease in glioma volume. in a previous study, we established the novel neuroendocrine hormone apelin as an angiogenic factor and described its upregulation in human glioblastoma multiforme. our aim is thus to study the role of apelin signaling in tumor angiogenesis but also its possible effect on brain monocytes for the regulation of glioma growth. methods: to investigate apelin signaling during gliomagenesis we took advantage of an orthotopic mouse model for tumor formation. intracerebral xenotransplantation of the human glioma cell line u87mg, expressing lentiviral control or apelin shrna, allows us to study the specific contribution of glioma-derived apelin to gliomagenesis. to further dissect a putative immune function of apelin, we used the isogenic mouse glioma cell line gl261 for intracerebral implantation into immunocompetent mice lacking (apelin-ko) or overexpressing apelin (apelin-tg). results: loss-of-apelin expression in u87mg cells resulted in a reduced glioma volume and attenuated the formation of the xenograft vasculature. gl261 implants in apelin-ko mice produced a similar phenotype. in addition, invasion of glm cells was significantly reduced. interestingly, expression analysis showed an upregulation of the apelin receptor apj in brain myeloid cells making them competent to respond to glioma-derived apelin. conclusions: our findings demonstrate that apelin plays an important role in tumor angiogenesis and glioma growth. we show here that both, glioma cell-derived apelin and apelin expressed by the tumor neovasculature are contributing to tumor angiogenesis, gim invasion and glioma growth. we anticipate that our study will provide insights whether apelin signaling may serve as a future novel target for antiangiogenic tumor therapy. tumor infiltrating microglia/macrophages (tims) constitute the largest population of infiltrating cells in glioblastoma (gbm), the most aggressive brain tumor. data from the clinic and from experimental work performed in murine and human models indicate that tims play a significant role in gbm biology as they support proliferation, migration and invasion of tumor cells. evidence is amounting that tumor cells actually polarize tims towards this m2-like, tumor-supportive phenotype. we showed that toll-like receptor 3 ligand reverses this m2-into a m1-like phenotype. pre-activated, m1-like polarized tims incubated with spheroids of gbm cells reduced migration, killed and phagocytosed tumor cells over a 15 day-period, indicating a sustained m1 activation of tims in absence of exogenously added stimuli. in order to analyse in more detail tims-gbm cells interactions, we have undertaken two approaches. we use spheroids of cells (tumor with/without tims) embedded in collagen matrix, in which tims can be implanted, as an experimental model. this three-dimensional in-vitro system is well suited for a qualitative and quantitative monitoring of cell proliferation, death, migration and invasion. data experimentally generated are then implemented in a mathematical model that is, to our knowledge, the first one proposed to take into account tumor cells and tims in order to simulate gbm progressive behaviour. in a first step, the capacity of spheroids made of murine glioma cells to invade collagen matrices was evaluated in absence and presence of microglia. invasion was monitored by photography of the spheroids and images were processed with photoshop cs5 software. in order to achieve a precise determination of invasion, we chose to measure the diameter of the core plus the invasive rim as a read out of expansion rate. untreated microglia promoted growth and invasion of tumor cells. data generated through the proposed in-vitro system were comparable to and reproduced the insilico simulations obtained with the mathematical model, hence validating our mathematical and experimental approaches. we currently evaluate the rate of proliferation and death of tumor cells in various settings that modulate tims polarization, using flow cytometry and confocal (live) imaging. data contributed by these two approaches should facilitate the delineation of a predictive model for tumor progression in a tims-enriched microenvironment with possible therapeutic fallouts. sialic-acid-binding immunoglobulin-like lectins (siglecs) are type 1 membrane proteins displaying an amino-terminal immunoglobulin-like variable (v-set ig-like) domain that binds sialic acid and variable numbers of immunoglobulin-like constant region type 2 (c2-set ig-like) domains. siglec-h is a recently identified mouse-specific cd33-related siglec that signals via the itam linked adaptor protein dap12. so far the function of siglec-h and its ligand are unknown. in this project, we demonstrate gene transcription and protein expression of siglec-h by mouse microglia after interferon-c (ifn-c) treatment or polarization into a m1-subtype. targeting of beads to siglec-h by specific antibodies triggered the phagocytic uptake of the beads by the microglia. a siglec-h fc fusion protein generated by our laboratory selectively bound to cells with an altered glycocalyx, particularly glioma cells, but not to normal control cells. m1-polarized microglia phagocytosed glioma cells and also limited their proliferation in a co-culture system. interestingly, no signs of apoptosis were observed before phagocytosis of the glioma cells by siglec-h expressing microglia. phagocytosis of glioma cells was reduced after shrna mediated siglec-h knock down in microglia. furthermore, microglial clearance of glioma in vitro was dependent on the interaction of siglec-h with its corresponding adaptor protein dap12. our data show that m1-polarized microglial cells can engulf glioma cells via a dap12-mediated and siglec-h dependent uptake. glioblastomas (gbm) are the most common brain tumors in humans. although advances in the chemotherapeutic management of glioblastoma have been made, almost 80% of the patients die within the first 2 years after diagnosis. on the basis of these observations, the identification of new molecules that can counteract the gbm growth and invasiveness appears relevant. previously, we have demostrated that m2 muscarinic receptor agonist arecaidine inhibited cell proliferation in a time and dose-dependent manner and induced a severe apoptosis both in two glioma stable cell lines (u251 and u87) and in primary cultures derived from human biopsies. in order to clarify the mechanisms causing the decreased cell proliferation, we have evaluated the ability of m2 receptor activation to counteract the notch-1 and egfr pathways. the analysis by real time pcr and western blot have demonstrated that, in both cell lines, m2 receptor caused a decreased expression of egfr and notch-1 and its ligands (e.g. delta-1, jagged-1 and 22), suggesting that the decreased cell proliferation may dependent on altered expression and activity of these pathways. although facs analysis have showed that arecaidine induced an arrest of cell cycle progression, we observed, in both cell lines, a significant increase of dividing cells already after 24 hours of treatment. in particular, the number of metaphases increased significantly, while the percentage of anaphases diminished. furthermore we observed in both cell lines, a dis-regulation of mitotic spindle assembly, misalignment of chromosomes and the presence of multipolar spindles. moreover, due to prolonged activation of the promethaphase/methaphase mitotic checkpoint, chromosomes appeared more condensed in cells treated with arecaidine. facs analysis and immunocitochemistry using anti-histone c-h2ax, a marker of double strand breaks, have also demonstrated that arecaidine treatment induced dna damage. on the other hand, m2 agonist induced also oxidative stress, followed by an increased expression of the enzyme superoxide dismutase and sirtuins (sirt1 and sirt2). in conclusion, the data obtained suggest that the activation of m2 receptor induces cytostatic and cytotoxic effects in glioblastoma cell lines, opening new therapeutic perspectives for this receptor in glioblastoma therapy. univdersidade federal do rio de janeiro, rio de janeiro, brazil glioblastoma (gbm) is one of the most aggressive human cancers. despite current advances in multimodality therapies, such as surgery, radiotherapy and chemotherapy, the outcome for patients with high grade glioma remains fatal. there is now a growing awareness that the main limitations in understanding and successfully treating gbm might be bypassed by the identification of a distinct cell type that has defining properties of somatic stem cells, as well as cancer-initiating capacitybrain tumor stem cells, which could represent a therapeutic target. in addition, experimental studies have demonstrated that the combination of antiangiogenic therapy, based on the disruption of tumor blood vessels, with conventional chemotherapy generates encouraging results. emerging reports have also shown that microglial cells can be used as therapeutic vectors to transport genes and/or substances to the tumor site, which opens up new perspectives for the development of gbm therapies targeting microglial cells. finally, recent studies have shown that natural toxins can be conjugated to drugs that bind to overexpressed receptors in cancer cells, generating targeted-toxins to selectively kill cancer cells. further, extensive effort is being dedicated to characterize the molecular basis of gbm resistance to chemotherapy and to explore novel therapeutic procedures that may improve overall survival. we show that a non-cytotoxic concentration of equinatoxin ii (eqtx-ii), a pore-forming toxin from the sea anemone actinia equina, potentiates the cytotoxicity induced by temozolomide (tmz), a first-line gbm treatment, and to etoposide (vp-16), a second-or third-line gbm treatment. we also suggest that this effect is selective to gbm cells and occurs via pi3k/akt pathway inhibition. finally, magnetic resonance imaging (mri) revealed that a non-cytotoxic concentration of eqtx-ii potentiates the vp-16-induced inhibition of gbm growth in vivo. these combination therapies constitute a new and potentially valuable tool for gbm treatment, leading to the requirement of lower concentrations of chemotherapeutic drugs and possibly reducing, therefore, the adverse effects of chemotherapy. a growing body of evidence indicates that glioblastoma stem-like cells (gscs) play a central role in glioblastoma development and resistance to current therapies. we recently described a cluster of micro-rnas, the mir-302-367, which induces the exit of gsc from their stem state and suppresses their tumorigenic properties in an irreversible manner (fareh et al, 2012). we postulated that such a drastic change in the cell phenotype should be accompanied with metabolic alterations that could be instrumental in the loss of functional properties of gscs induced by the micro-rna cluster. metabolome profiling by mass-spectrometry of gscs and gsc-mir-302-367 pointed to changes in the gaba synthesis pathway (see abstract el-habr et al). remarkably, exposure of na€ ıve-gsc to metabolites of the gaba synthesis pathway found to be overproduced in gsc-mir-302-367 reproduced at least in part the effects of mir-302-367, including inhibition of clonality, down-regulated expression of selfrenewal markers, and loss of self-renewal properties. the metabolites studied acted by interfering with progression of the cell cycle and the nuclear localization of transcription factors crucial for maintenance of gsc stem-like properties. studies assaying the effects of the metabolites on gsc tumor-initiating properties are under way. these results demonstrate that metabolic regulations can participate in the control of gsc properties, and opens novel paths in therapeutic targeting of glioblastoma. *lgd and eae contributed equally. glioblastomas are the most frequent and aggressive form of adult primary brain tumors. glioblastoma stem cells (gscs) are thought to play key roles in the development and resistance of the tumor to existing radio-and chemotherapies. our previous results showed that the cluster of micrornas, mirna-302-367, induces loss of gsc stem-like and tumor-inducing properties (fareh et al. 2012). to further understand the molecular pathways controlling the peculiar properties of gscs, we sought for metabolic changes likely to accompany the drastic change in cell phenotype induced by mir-302-367 expression. we measured the intracellular and secreted levels of 271 metabolites by mass spectrometry. reconstitution of the metabolomes revealed significant and coordinated changes in components of the krebs cycle, and the glutamine/glutamate neuropeptide metabolic pathway that suggested altered turnover of the gaba synthesis pathway. exon array hybridization and western blot analysis, revealed changes in expression of several enzymes of the gaba synthesis pathway in mir-302-367-gscs. of note, none of the analyzed enzyme transcripts appeared as a direct target of mir-302-367. our results could be integrated in a complex multistep schema compatible with enhanced turnover of the gaba synthesis pathways, resulting in decreased cell gaba levels and increased levels of gaba by-products, as observed in mir-302-367 gscs. further studies showed that these metabolic changes participate in the induced loss of gsc properties (see abstract by dubois et al). * eae and lgd contributed equally colonization of the brain by carcinoma cells is barely understood, in particular when considering interactions with the host tissue. we recently demonstrated that microglia assist carcinoma cell invasion. current findings indicate that this is part of a danger response of the entire brain tissue. in the brain slice coculture model, contact with both benign and malignant epithelial cells induced a response by microglia as well as astrocytes comparable to that seen at the interface of human cerebral carcinoma metastases. this response tries to protect the brain from intrusion of benign epithelial cells by inducing apoptosis. however, this is ineffective against malignant cells which did not undergo apoptosis and actually exploited this reaction to invade instead. gene expression and functional analyses revealed that c-x-c chemokine receptor type 4 (cxcr4) and wnt signaling are involved in this process. furthermore, cxcr4 regulates the recruitment of microglia towards brain injury in a zebrafish model and was expressed in human stroke patients, suggesting a conserved role of cxcr4 in various danger reactions. we propose that glia-assisted malignant invasion takes advantage of the physiological two-stage glial defense reaction. carcinoma cells escape the second step of cell killing and misuse the damage response to colonize the brain. university of tuebingen, tuebingen, germany aquaporin-4 (aqp4), the main water channel of the brain, is highly expressed in animal glioma and human glioblastoma in situ. in contrast, most cultivated glioma cell lines do not express aqp4, and primary cell cultures of human glioblastoma lose it during the first passages. accordingly, in two glioma cell lines of the rat (c6 cells and rg2 cells) and in sma mouse glioma cell lines we found no aqp4 expression. this let us consider the possibility that aqp4 expression depends on brain microenvironment. aqp4 negative rat glioma cells were implanted into rat brain. within two weeks, a tumor developed. aqp4 staining of the tumor cells was positive. however, if the identical cells were implanted into the rat's flank, they did not express aqp4. in contrast to the normal brain, where aqp4 staining is polarized in the astrocytic endfoot membranes, the aqp4 staining in c6 and rg2 tumors was distributed over the whole glioma cell as in human glioblastoma. we conclude that the micro-environment is crucial for aqp4expression in brain and brain-tumor. glioblastomas (gbm) are infiltrative and highly vascularised brain tumors containing a subpopulation of multipotent stem-like cells. here we questioned whether notch pathway activation in these cells influenced the extent of tumoral vascularisation and dissemination. we used two cd133 1 sox2 1 gbm stem-like cultures generating highly infiltrative but poorly vascularised tumors upon intracranial grafts. the use of a hes5 promoter reporter construct indicated that the notch pathway was only active in a small fraction of cells. sustained notch activation in these cultures, using an activated form of the notch-1 receptor (nicd), induced a profound alteration of their morphology, phenotype and properties. a severe decline of their migration and proliferation rates was observed in vitro and in vivo. intracranial and subcutaneous transplantation revealed that nicd triggered an extensive vascularisation of tumoral cells indicated by the presence of wellformed vessels with large lumens. close interactions between gbm cells and host endothelial cells were readily observed but no evidence for endothelial transdifferentiation was found either in vitro or in vivo. microarray and proteomic analyses showed that nicd induced the expression of vascular adhesion proteins (icam-1, vcam-1, itga9), proangiogenic cytokines (plgf, il-8, hb-egf), metalloproteinase (mmp-9), a-smooth muscle actin and dll4, a notch ligand essential for vascularisation. this was associated with a remarkable transcription factor switch whereby cells became klf9 1 snai2 1 while ascl1, olig2, and sox2 were reduced or lost. thus in addition to its well-described role in vascular development and remodelling, the notch pathway is also central in controlling proliferation, migration and vascularisation of gbm-stem like cells. t04-04b extracellular space diffusion parameters in the mouse thalamus in bral2 knock-out mice retinal wholemounts were immunostained with antibodies against gfap, iba-1 and mhc-ii. results: in the na€ ıve retinas, weak constitutive mhc-ii expression was scarcely found in some iba-11 microglial cells and rarely in gfap1 astrocytes. only a small dendritiform subpopulation of iba-11 cells, located in the juxtapapillary area and in the marginal region of the retina, had a strong mhc-ii immunoreaction. in comparison with na€ ıve both, in contralateral and in oht-eyes: i) gfap was upregulated in m€ uller cells and microglia was activated; ii) mhc-ii was upregulated on macroglia and microglia. in microglia, it was similarly expressed in contralateral and ohteyes. by contrast, in macroglia, mhc-ii upregulation was observed mainly in astrocytes in contralateral eyes and in m€ uller cells in oht-eyes conclusions: both, contralateral and oht-eyes had macro-and microglial retinal changes in mhc-ii expression after two weeks of laser-induced oht approximately 70% of the nmo patients harbor antibodies against the water-channel aquaporin-4, aqp4-igg (seropositive nmo), expressed mainly by perivascular astrocytes. however, some patients diagnosed with nmo don't have aqp4-igg (seronegative nmo). the aim of this work is to compare in vitro the neuroinflammatory profile of igg from seropositive (igg-nmo1) and seronegative (igg-nmo-) nmo patients. pool of purified igg from igg-nmo1 and igg-nmo-patients fulfilling diagnostic criteria of 2006, and from multiple sclerosis (igg-ms) patients and healthy controls (igg-hc) were include in the study. mixed glial cell cultures of astrocytes (40%), oligodendrocytes (40%) and microglia (15%) were obtained from spinal cord of postnatal c57bl6/j mice. after 21 days in vitro, cells were treated with the igg pools for 12 hours for rt-pcr, and 24 hours for western blot and immunocytofluorescence (if) igg-nmo1 caused a stronger upregulation of c/ebpb mrna and nos2 protein than igg-ms and igg-nmo-. all these data show that igg-nmo1 and igg-nmo-induce a different proinflammatory glial activation. the proinflammatory pattern of igg-nmo-is quite similar to that induced by igg-ms patients. these findings raise the question if igg-nmo-could be a different disease. such as gdnf, artemin, bdnf and sonic hedgehog, adhesion molecules including p75ntr, n-cadherin and n-cam and the transcription factor olig1. c-jun also controls the structure of the regeneration tracks. in schwann cell c-jun null mice, the initial rate of axonal regeneration after injury is impeded and long term recovery of function measured by footprint analysis, toe pinching , von frey hairs, and the hargreaves test is not seen 10 weeks after injury. there is widespread death of both large and small drg neurons and although spinal cord motor neurons survive there is widespread failure of both sensory and motor neurons to reinnervate target muscles united states mutations in the neurofibromatosis 2 (nf2) gene cause neurofibromatosis type 2 (nf2) characterized by formation of multiple schwannomas and meningiomas. nf2 encodes a tumor suppressor protein called merlin. loss of function of merlin is associated with an increased level of active rac and p21-activated kinases (pak). the lim domain kinases (limk1 and 2) are rac-pak substrates and modulate actin dynamics and cytoskeletal organization by phosphorylating cofilin, an actin severing and depolymerizing agent acknowledgements: this work was supported by grant ncn 2011/03/b/ nz4/00042 and statutory funds. phenotypes, their functions are fundamentally different in a model of brain injury. these data provide new insights into the protective role of microglia after brain injury. . although the pathogenesis is still not clear, activation of the immune system at some point of the disease process plays a crucial role. it is known that myelin-specific autoreactive t-cells and monocytes cross the blood-brain barrier (bbb) and migrate into the cns. within the cns, these cells secrete proinflammatory cytokines which stimulate local glial cells to produce inflammatory factors, including reactive oxygen species that contribute to demyelination and ultimately axonal loss. the destruction of the bbb and the influx of monocytes and t-cells from the circulation are key events in the progression of ms pathology. tissue transglutaminase (tg2) is a multifunctional enzyme that has been shown to play a role in monocyte/macrophage adhesion and migration onto extra cellular matrix proteins in vitro. this binding occurs via interaction with bintegrins. previously, we observed the appearance of tg2 in monocytes in active human post-mortem ms lesions in the cns.in the present study we question whether tg2 expression is regulated in primary human monocytes by inflammatory mediators and whether it is modulated in ms patients. methods: to this end, tg2 in monocytes was detected by semiquantitative rt-pcr.results and conclusions: our preliminary results show that activation of primary human monocytes with lps or lps1ifnc results in a time-dependent increase in tg2 mrna whereas the expression of factor xiiia (another member of the transglutaminase family) remains stable over time. moreover, the tg2 mrna level is higher in unstimulated monocytes derived from ms patients compared to control subjects while factor xiiia mrna level remains unaffected.these initial data are promising with respect to a possible role for monocyte-derived tg2 being involved in adhesion to and migration across the bbb under inflammatory conditions (e.g. multiple sclerosis). this hypothesis has not been proven yet. in addition, by increasing the number of patients, a consistent difference in tg2 expression in monocytes between ms patients and control subjects may point to tg2 as a novel biomarker for disease. an important aspect of chronic neurodegenerative diseases, such as alzheimer's, parkinson's, huntington's and prion disease, is the generation of an innate inflammatory response within the central nervous system (cns). microglial and astroglial cells play a key role in the development and maintenance of this inflammatory response, showing enhanced proliferation and morphological activation. using a laboratory model of chronic neurodegeneration (me7 murine model of prion disease), we studied the time-course and regulation of microglial proliferation. our results show that resident microglial cells have an increased proliferation rate during the development of the disease, leading to a significant increase in the population, without a contribution from circulating cells. microglial proliferation is differentially regulated in diverse regions of the cns, pointing to a heterogeneous development of the pathology. we have identified novel molecular regulators of the proliferative response, and addressed the significance of the contribution of microglial cells to the pathological course of the disease by modifying their proliferation. we also found a correlation of our results with the scenario present in chronic human neurodegenerative conditions variant creutzfeldt-jakob disease (vcjd) and alzheimer's disease. our results demonstrate that microglial proliferation is an important feature of the evolution of chronic neurodegenerative disease, with direct implications for understanding the contribution of the cns innate immune response to disease progression. here, we examined the underlying mechanism of fibronectin aggregation and address whether inflammatory mediators, such as toll like receptor (tlr) agonists or pro-inflammatory cytokines induce fibronectin aggregation by astrocytes. our findings reveal that only tlr3 and 24 agonists, including the endogenous tlr3 agonist stathmin, increased stable fibronectin aggregation. interestingly, only white matter-derived astrocytes displayed fibronectin aggregation whereas in cortical astrocyte cultures fibronectin remained unaffected, suggesting regional differences in the functional response of astrocytes. furthermore, in rat cerebellar slice cultures, the tlr3 agonists only induce aggregation of fibronectin after lysolecitin-induced demyelination. in this manner, efficient remyelination is impeded and, consequentially, progressive neuronal decline occurs.taken together, tlr3 and 24 agonists promote fibronectin aggregation by primary rat astrocytes. since tlr3 on astrocytes and stathmin are both upregulated in ms lesions, stathmin, as an endogenous tlr3 agonist, might play a role in inducing fibronectin aggregation after c-jun reprograms schwann cells of injured nerves to generate a repair cell essential for regeneration. neuron. 75(4): 633-47). in this study we identify a novel role for c-jun in the activation of notch signalling in the denervated schwann cell. we find that c-jun is required to activate notch signalling, leading to upregulation of the bhlh protein hes1. hes1 then plays two functions in the denervated cell, promoting myelin breakdown and acting as part of a negative feedback loop to reduce c-jun levels. as a result of this, ablating notch signalling specifically in schwann cells acts to increase c-jun levels. we show that this upregulation of schwann cell c-jun accelerates axon outgrowth, target re-innervation and remyelination by generating a cell, which promotes faster than normal functional recovery. these results identify novel functional links between the c-jun and notch signalling pathways. they also show that not only is schwann cell c-jun necessary for successful nerve regeneration, but that nerve repair can be improved by enhancing normal c-jun signalling. it is well known that cell surface immune receptors play a critical role in regulating immune and inflammatory processes in the central nervous system (cns). after injury of the peripheral nervous system (pns), schwann cells and macrophages phagocyte myelin debris in wallerian degeneration in order to regenerate axons to distal targets. the immunoreceptor cd300f is normally expressed in the myeloid line and in nervous system in microglia, oligodendrocytes and neurons under certain conditions. however, little is known about the cd300f ligands. by using a cd300f-fc fusion protein we have analyzed the expression of the cd300f ligands in the pns. moreover, we have also analyzed the possible role of the cd300f immunoreceptor in peripheral nerve regeneration by blocking the interaction between cd300f and its ligands with the same fusion protein. thy1-yfp-h mice sciatic nerves were injected with cd300f-fc, control migg2a or pbs immediately before a crush injury. the results show that cd300f ligand is expressed in schwann cells. moreover, after a sciatic nerve injury, animals injected with the cd300f-fc protein show a lower number of yfp-positive fibers growing into the tibial nerve after 10 days post-lesion (dpl) than control groups. moreover, the cd300f-fc group shows a higher number of macrophages and cd206-positive cells at 10 dpl when compared to control groups. we do not see these differences in axon regeneration and macrophage infiltration after 28 dpl. we have also evaluated the mrna expression of the pro-inflammatory cytokine il-1b at 24 hours after crush and injection of the fusion protein. q-pcr shows an up-regulation of the mrna in control groups and a lower mrna expression level in the cd300f-fc protein group. together these results show that the pair cd300f receptor and ligand is implicated in some aspects of wallerian degeneration and nerve regeneration such as the modulation of both the influx and phenotype of macrophages. in response to the central nervous system (cns) injury, hematopoetic cells migrate to the lesion where they can potentially contribute to tissue regeneration. following cns injury, these cells can secrete a plethora of immunomodulating cytokines and/or pro-regenerative growth factors as well as phagocyte proinflammatory tissue debris. nevertheless, the role of primitive hematopoetic stem/progenitor cells (hspc) -derived from bone marrow -to support cns regeneration has not been studied in detail. therefore, the aim of the present study was to examine characteristics of hspc in vitro as well as to test proregenerative potential of these cells to support cns repair in vivo.highly pure population of primitive hematopoetic stem/progenitor cells was isolated from the bone marrow and the cns with fluorescence activated cell sorting (facs). the number of hspc in the cns lesion was significantly increased compared to intact cns tissue. sorted-bone marrow hspc were co-cultured with primary astrocytes to examine their phenotype according to the presence of specific antigens and gene expression as well as to test their phagocytosis potential. in the presence of astrocytes, bone marrow-derived hspc differentiated in vitro into microglia-like cells expressing specific myeloid/microglia markers and showing high ability to ingest fluorescent microbeads. the in vivo potential of hpsc for supporting regeneration was examined by their transplantation into the cns lesion.results of our study demonstrated that primitive hematopoetic stem/progenitor cells are the source of microglia-like cells which can support regeneration in the central nervous system. when the brain or spinal cord is injured, glial cells in the damaged area undergo complex changes resulting in the formation of the glial scar. whether the scar is beneficial and detrimental to recovery remains controversial. the signals that initiate the formation of the glial scar are unknown. because both canonical and non-canonical wnts are increased after spinal cord injury (sci), we examined the role of canonical wnt signaling in the glial reactions to cns injury. to disrupt b-catenin-dependent wnt signaling specifically in opcs, we created transgenic mice carrying an opc-specific conditionally deleted bcatenin gene. after moderate contusion injuries to the thoracic spinal cord of control mice, opcs proliferate and accumulate in the penumbra region surrounding the injury epicenter. in the b-catenin-depleted mice, there is reduced proliferation and accumulation of opcs after sci, reduced accumulation of activated microglia/macrophages and reduced astrocyte hypertrophy. using a crushed optic nerve model, we show that these reduced glial reactions create an environment that is permissive for axonal regeneration. these results suggest that canonical wnt signaling in glia after cns injury is necessary for the formation of the glial scar and they identify wnt signaling as a new therapeutic target for promoting axon regeneration. the crucial role of central nervous system (cns) glial cells in the integrity and physiology of neuronal networks, is well known. however, little is known about glial cells in the peripheral nervous system (pns). one of the main glial cell types in the pns is the perineuronal satellite glial cells (sgcs), that surround drg neurons in an envelope-like fashion. despite their abundance in peripheral nerves, having stem cells properties and playing a role in neuropathic pain, there is limited information on their physiology. although sgcs have similarities to astrocytes in terms of purinergic-and gap junction-mediated signaling, their membrane properties and ionotrophic glutamate receptor expression are more or less unknown. our aim was therefore to characterize the membrane properties and glutamatergic ion channel expression of sgcs. we performed patchclamp experiments on sgcs wrapped around sensory neurons in the rat dorsal root ganglion (drg) in vitro. whole-cell recordings revealed that sgcs are slightly hyperpolarized in comparison to the neurons they ensheath, with a resting membrane potential of approximately $80mv and a high input resistance (&gt;1gx). moreover, upon membrane depolarization no detectable voltage-gated na 1 , ca 21 or k 1 currents were detected. extracellular application of glutamate agonist, kainic acid, n-methyl-d-aspartic acid (nmda) and 2-amino-3-(3hydroxy-5-methyl-isoxazol-4-yl) propanoic acid (ampa) during the recording, did not evoke any response from the cells, indicating their lack of ionotropic glutamatergic receptor (iglur) expression. double immunocytochemistry on lucifer yellow (ly) filled scgs revealed that they express the scip/oct6 transcription factor, which is also expressed by promyelinating schwann cells.drg ganglion sgcs differ from cns perineuronal cells as they lack glutamatergic receptors, but are similar to astrocytes as they have no voltage-gated ion channels, are gap-junctionally coupled and express glutamine synthetase. thus, we are currently addressing if sgcs respond to neuronal activity in a similar manner to astrocytes in the cns with the use of calcium imaging. these studies will provide further insights into the physiological role of sgcs in the pns. multiple sclerosis (ms), a chronic neuroinflammatory disease with presumed autoimmune etiology, is the most common demyelinating disorder of the central nervous system among young adults. progressive neurological impairment of the patients are associated with accumulation of characteristic brain lesions characterized by inflammation, demyelination, gliosis and axonal damage. demyelination and loss of oligodendrocytes contributes to axonal loss and permanent neurological deficits. remyelination occurs but is limited; one contributing factor is the incapability of oligodendrocyte precursor cells (opc) to differentiate and form new myelin sheaths. k 1 channels are not only known to predominantly set and maintain the membrane potential but also to be important regulators of various cell functions like proliferation and maturation. specific regulation of these cellular functions is based on high channel diversity and their ability to form heteromers as well as alternate mrna splicing and posttranslational modifications. so far the expression and function of potassium channels in cells of the oligodendroglial lineage has not been clearly identified. previous studies indicate that an unspecific blockade of k 1 channels in opc suppresses maturation as well as proliferation. moreover, for one distinct channel (k ir 4.1) a functional relevance has been shown, as deficiency of kir4.1 leads to altered opc maturation and hypomyelination in mice. here, we elucidate the expression and functional relevance of k 1 channels in oligodendrocytes. our first electrophysiological recordings from primary murine opcs revealed the presence of different k 1 channels with both inward and outward rectification. in future experiments, we aim at identifying k 1 channel subtypes that specifically regulate opc cell functions and might influence cell maturation under neuroinflammatory conditions. thereby, we want to offer new insights in the functional role of k 1 channels in opcs/oligodendrocytes and contribute to novel therapeutic strategies for the treatment of ms. aqp4) is the predominant water channel in the mammalian brain and is mainly expressed in the perivascular glial endfeet at the brain-blood interface. aqp4 has been described as an important entry and exit site for water during formation of brain edema and regulation of aqp4 is therefore of broad interest. phosphorylation of some aquaporins has been proposed to regulate their water permeability via gating of the channel itself. protein kinase (pk)-dependent phosphorylation of ser 111 has been reported to increase the water permeability of aqp4 expressed in an astrocytic cell line. this possibility was, however, questioned based on the crystal structure of the human aqp4. our study aimed to resolve if ser 111 was indeed a site involved in phosphorylation-mediated gating of aqp4. the water permeability of aqp4-expressing xenopus oocytes was not altered by a range of activators and inhibitors of pkg and pka. mutation of ser 111 to alanine or aspartate (to prevent or mimic phosphorylation) did not change the water permeability of aqp4. pkg activation had no effect on the water permeability of aqp4 in primary cultures of rat astrocytes. molecular dynamics simulations of a phosphorylation of aqp4.ser 111 recorded no phosphorylation-induced change in water permeability. a phospho-specific antibody, exclusively recognizing aqp4 when phosphorylated on ser 111 , failed to detect phosphorylation in cell lysate of rat brain stimulated by conditions proposed to induce phosphorylation of this residue. thus, our data indicate a lack of phosphorylation of ser 111 and of phosphorylation-dependent gating of aqp4. undocked connexons may form open hemichannels in the plasma membrane when exposed to specific stimuli, e.g. reduced extracellular concentration of divalent cations, and allow passage of fluorescent molecules with masses lower than 1 kda. a range of physiologically relevant molecules, smaller than the assumed molecular cut-off of 1 kda, have therefore been proposed to permeate connexin 43 (cx43) in its hemichannel configuration. however, the permeability profile of cx43 hemichannels remains unresolved as does the molecular substrate for hemichannel activity in astrocytes. exposure of cx43-expressing xenopus laevis oocytes to divalent cation free solution induced a gadolinium-sensitive uptake of the fluorescent dye ethidium. in spite thereof, a range of smaller biological molecules, such as water, glutamate, lactate, glucose, and atp, did not gain detectable access through the pore. in contrast, permeability of glutamate, glucose and atp was observed in oocytes expressing the other major astrocytic connexin, cx30. exposure to low divalent cation solutions also induced a robust membrane conductance in cx30-expressing oocytes but none in cx43-expressing oocytes. expression of cx43 in c6 glioma cells failed to induce hemichannel activity. our results thus call for caution when assigning molecular identity to astrocytic channel activity and when interpreting hemichannel-mediated dye-uptake as equal to permeability of physiologically relevant molecules. atp-gated p2x4 receptor channels expressed in spinal microglia actively participate in central sensitization, making their functional regulation a key process in chronic pain pathologies. p2y6 metabotropic g q -coupled receptors, also expressed in microglia, are involved in the initial response to nerve injury, triggering phagocytosis upon activation by udp. it has been reported recently that expression of both p2x4 and p2y6 is upregulated in activated microglia following nerve injury. we show here, in resting as well as lps-activated primary microglia, that p2y6 decreases p2x4-mediated calcium entry and inhibits the dilation of p2x4 channels into a large-conductance pore measured with a yo-pro-1 uptake assay. furthermore, p2y6 activation modulates the atp-dependent migration of microglia, a process likely involved in their shift from migratory to phagocytic phenotype.reconstituting the p2x4-p2y6 interaction in recombinant systems shows that p2y6 activation decreases p2x4 current amplitude, activation and desensitization rates, and reduces p2x4 channel permeability to the large cation nmdg 1 . phospholipase c-mediated hydrolysis of the phosphoinositide pi(4,5)p 2 , a necessary cofactor for p2x4 channel function, underlies this inhibitory crosstalk. as extracellular levels of both atp and udp are increased in the spinal cord following nerve injury, the control of p2x4 activity by p2y6 might play a critical role in regulating neuropathic pain-inducing microglial responses. with sc, thus representing a valid sc alternative. we have previously shown that dasc express c-aminobutyric-acid (gaba) receptors (i.e. gaba-a and gaba-b receptors), which modulate their proliferation and neurotrophic potential, although little is known about the role of other neurotransmitter systems in asc.in this study we investigated the expression of purinergic receptors in dasc. using rt-pcr, western blot analyses and immunohistochemistry we have demonstrated that asc express p2x 3 , p2x 4 and p2x 7 purinoceptors. interestingly, differentiation of asc towards glial phenotype was accompanied by up-regulation of p2x 4 and p2x 7 receptors. using ca 21imaging techniques, we have shown that stimulation of purinoceptors with adenosine-5 0 -triphosphate (atp) results in intracellular ca 21 signals, indication functional activity of these receptors.. moreover, we have shown that the increase of intracellular ca 21 leads to sc death, an effect that can be prevented using specific p2x 4 or p2x 7 antagonists.altogether, these results show, for the first time, the presence of functional purinergic receptors in sc-like derived from asc and their link with critical physiological processes such as cellular death and survival. the presence of these novel pharmacological targets in dasc might open new opportunities for the management of cell survival and neurotrophic potential in tissue engineering approaches using dasc for peripheral nerve repair. university of freiburg, freiburg, germany intracellular ph homeostasis is a vital function shared by all cells and is mainly regulated by the coordinated action of several acid-base transporters. in the brain, ph homeostasis is of particular importance since changes of extracellular ph (pho) are events associated with both physiological conditions and pathological states in brain function. transient pho changes usually accompany physiological processes, including neuronal activity, and astrocytes respond to a rise in extracellular k 1 with plasma membrane depolarization and intracellular alkalinization. on the other hand, loss of brain ph homeostasis can lead to severe pathological conditions and significant changes of pho have been observed during seizures and spreading depression.in the present study, we sought to investigate regulation mechanisms of the electrogenic sodium/bicarbonate cotranspoter 1, nbce1, and of the vacuolar h 1 -atpase (v-atpase) in primary hippocampal astrocytes following extracellular acid-base changes, following neuronal activity, and using the 4-aminopyridine (4-ap) model of epilepsy in vitro.we show that extracellular acidosis, but not extracellular alkalosis, increased the number of activated astrocytes. our data also show differential regulation of acid-base transporters in the hippocampal glial cells during extracellular acid-base changes. intracellular ph measurements revealed a crucial role for v-atpase in regulation of intracellular ph, a process accompanied by increased membrane expression of the transporter, as assessed by immunofluorescence and surface biotinylation. nbce1-b is the nh2-terminal variant of nbce1 predominantly expressed in glial cells and is transcriptionally regulated followed neuronal activity or treatment of the cells with 4-ap.these data propose transcriptional and post-translational modification of acid-base transporters as putative regulatory mechanisms in astrocytes to cope with changes of extracellular ph serving the maintenance of intracellular ph homeostasis. these astroglial networks fulfil a variety of functions in the brain, e.g. potassium buffering and metabolite transport. in this study we compared glial networks in different brain regions to investigate site specific effects on network formation. we combined electrophysiology and immunohistochemstry with semi-quantitative rt-pcr and western blot analysis to investigate connexin expression, gap junction coupling and antigen profiles. experiments were performed in wild type and transgenic mice with glia specific fluorescence labelling as well as in cx30ko mice. astrocytes were investigated between postnatal days 12-60. gap junction networks in the ca1 region of the hippocampus and the ventrobasal thalamus show abundant coupling in hgfap-egfp mice. intriguingly, we found significant coupling between oligodendrocytes and astrocytes in the thalamus, while in the hippocampus panglial coupling was less abundant. other glial cells did not participate in the networks. we also found that a fluorescent glucose analog, 2-nbdg, propagates through the thalamic panglial network. the function of these panglial networks remains largely unclear.in heterozygous cx43-ecfpki mice, deletion of one allele of the major hippocampal cx43 significantly reduced the number of coupled astrocytes only in the hippocampus, while the thalamic networks remained unchanged. sr101 labelling of astrocytes and subsequent 2p microscopy identified a significant subset of thalamic sr1011 cells lacking cx43-ecfp expression. sr101 did not label oligodendrocytes as analysed in plp-gfp mice. semi-quantitative rt-pcr and western blot analysis revealed stronger expression of cx30 in thalamic nuclei while cx43 levels were higher in the hippocampus. this indicates a minor role for cx43 in gap junction coupling of astrocytes in the thalamus. consistent with these findings, the thalamus of cx30ko mice displayed a strong decrease in astrocytic cell coupling compared to wild type littermates.together, these results indicate that thalamic astrocytes differ in various aspects from their counterpart in other brain regions and support the emerging concept of astrocyte heterogeneity. the mechanism of secondary damage spread after brain trauma remains unresolved. in this work, we redirected the attention to astrocytic communication pathways. using an in vitro trauma model that consists of a scratch injury applied to a rat astrocyte monolayer, we found a significant induction of connexin43 hemichannel activity, demonstrated by ethidium uptake, in regions distal from the injury ($17 mm away and maximal $1 h after injury). this response was abolished by two connexin hemichannel blockers, la 31 and the peptide gap26. in addition, the trauma-induced increase in hemichannel activity was prevented by inhibition of purinergic p2 receptors. the scratch-induced increase in hemichannel activity was absent in astrocytes from cx43 knockout mice. this activity took place with a singular spatial distribution, since cells located at $17 mm away from the scratch gained connexin hemichannel activity. however, the functional state of gap junction channels (dye coupling) was not significantly affected in the same locations. the connexin43 hemichannel activity was also enhanced by the acute extracellular application of 60 mm k 1 . the increase in hemichannel activity was correlated with an increment in apoptotic cells, measured by immunofluorescence to annexin v at 24 h post-trauma, which was totally prevented by gap26 peptide. these findings could open a new approach to prevent or reduce the secondary cell damage due to brain trauma. l. schlosser, a. scheller, f. kirchhoff institute of physiology, saarland university, homburg, germany current research suggests that astrocytes represent heterogeneous neuroglial populations in different regions of the brain. this is particularly evident when the expression pattern of various transmitter receptors is analyzed. hippocampal astrocytes, for example, do not express ampa receptors, while cerebellar bergmann glia is loaded. functional analysis of glial receptors requires dedicated efforts for each distinct brain region and developmental age. unfortunately, the molecular identification using antibody approaches is often hampered by either poor antibody quality or abundant receptor expression on adjacent neuronal membranes. therefore, we are using cre/lox-mediated gene ablation for proper functional analysis.here, we are focusing on the characterization of astrocyte-specific gene deletion of the gaba b1 receptor. metabotropic gaba b receptors consist of two subunits gaba b1 and gaba b2 forming a heteromeric receptor complex of which gaba b1 is essential. in contrast to neurons, activation of astroglial gaba b receptors leads to transient rises of intracellular ca 21 . the functional impact of these glial gaba b -receptors is largely unknown.to address the gaba b receptor function in astrocytes we took advantage of the cre/lox system and crossbred glast-creert2 knockin mice with floxed gaba b1 receptor mice. first immunohistochemical analysis reveals a selective deletion of gaba b1 on a majority of astroglial processes. functional studies addressing the role of these receptors in astroglial ca 21 signaling are in progress. purinergic signaling is the most diverse system in astrocytes to communicate with other glial cells and neurons. astrocytes express a variety of p2x (ionotropic) and p2y (metabotropic) receptors. especially in case of brain injury, atp levels and receptor expression on astrocytes are increased. atp is immediately degraded to adp, amp and adenosine. these nucleotides/nucleosides act back on the preferred purinoreceptor expressed on glial and neuronal cells. in the last decade researchers tried to evaluate the functional impact of astrocytic purinoreceptors on the complex information flux at the tripartite synapse. a prominent role has been suggested for the p2y1 receptor subtype. we generated conditional mouse mutants to investigate the function of p2y1 receptors in astrocytes in vivo and crossbred mice carrying the floxed p2y1 gene with mice that express the inducible dna recombinase (creert2) under the control of the glast (l-glutamate/ l-aspartate transporter) locus. ablation of the p2y1 receptor is induced in development and adulthood by intraperitoneal tamoxifen injections. successful recombination of the targeted p2y1 receptor is evaluated by qrt-pcr on genomic dna and mrna usually 21 days after tamoxifen application. at the genomic level we find $25% and $50% recombination in astrocytes of the cerebellum and hippocampus, respectively. similar recombination frequencies are observed in young (p11) or adult mice (6-10 weeks). when looking at the transcript level, the mrna is reduced to 57% in the cerebellum of adult mice and to 76% in the hippocampus. in young mice we determined a reduction to 40% and 42% mrna expression in the cerebellum and hippocampus, respectively. given the high level of p2y1 expression on other cells of the brain these findings indicate a successful astrocyte-specific knockout. further fluorescence-activated cell sorting (facs) of recombined cells expressing the red fluorescent protein td-tomato will be used to verify the knockout. the potential function of these astroglial receptors in neuronal networks will be as well investigated using histological (em and light microscopy), twophoton ca 21 imaging in situ and in vivo and behavioral approaches. we recently showed that they invade the cortex at 11.5 days of embryonic age (e11.5). they first accumulate at the pial surface and within the lateral ventricles, after which they spread throughout the cortical wall, avoiding the cortical plate region in later embryonic ages. the absence of the expression of mac-2 and mhc ii suggest these cells have a na€ ıve/quiscent phenotype during embryonic development of the cortex. besides immunohistochemical markers is the presence of different k 1 channels on microglia also an indication of their activation stage. however most studies have been conducted on postnatal and adult microglial cells. therefore we aimed at determining the electrophysiological activation phenotype of these embryonic microglia.at the age of e13.5 and e15.5, microglial cells display a small inward rectifying k 1 current and this independent of their location in the embryonic cerebral cortex and their cell morphology. these cells also express functional p2x receptors, which based on the profile of the response are most probably p2x7 receptors. time-lapse analysis showed that embryonic microglia are highly dynamic cells. suggesting that these cells are already very active during fetal development. rapid extracellular removal of glutamate, the major excitatory neurotransmitter in the cns, is essential for normal brain function. this task is primarily accomplished by the action of the sodium-dependent, high-affinity transporters glast and glt-1 (rodent analogous of eaat1 and eaat2), which are mainly expressed by astrocytes. impairment or failure of glast and glt-1 plays an important role in many pathological conditions. question: pelizaeus-merzbacher-like disease (pmld) is a hypomyelinating leukodystrophy caused by mutations in the gjc2 gene encoding the gap junction (gj) protein connexin47 (cx47). cx47 is expressed in oligodendrocytes and forms most gj channels to astrocytes and to other oligodendrocytes. since pmld-associated gjc2 mutations cause loss of cx47 function, gene replacement strategies may be promising for developing future treatments. a mouse model for plmd, the cx32/ cx47 double knockout (ko), is characterized by early onset of severe leukodystrophy at 4 weeks of age leading to death by 6 weeks, offering the possibility to test therapies. the aim of the present study was to generate a lentiviral vector to allow gene delivery specifically to oligodendrocytes, and to examine the transduction efficacy, distribution, duration and levels of egfp reporter gene and cx47 expression throughout the cns, in order to establish a method for oligodendrocyte-targeted gene therapy.methods: the expression cassette with the cx47 gene along with the ires-egfp as a reporter gene under the control of oligodendrocyte-specific cnp promoter was cloned into the lentiviral vector pcclsin.ppt.hpgk.gfp.pre. mock vectors lacking the cx47 gene were also generated as controls. viral particles were produced to high titers and purified, before injection. lentiviral vectors were delivered into the brain of wild type (wt) and cx47ko mice at postnatal day 1 (p1), intraventricularly and in the stratum radiatum of the dorsal hippocampus. egfp and cx47 expression was assessed using immunochemistry and immunoblot analysis at different time points post injection.results: we found widespread expression of virally delivered egfp in different brain regions colocalizing with oligodendrocyte markers (cc1 and olig2). the expression of egfp was detected from p15 until 3 months post-injection. on average 20.3 6 2.56% of oligodendrocytes were egfp positive, with highest rates in the subventricular zone (29.3 6 6.31%, n 5 6) and olfactory bulb (19.9 6 4.36%, n 5 8) and lower rates in the cortex (16.1 6 2.60%, n 5 6) and corpus callosum (12.3 6 3.40%, n 5 3). in cx47ko brain expression of virally delivered cx47 was also found after p21 with formation of gj plaques in a subpopulation of oligodendrocytes.conclusions: our results show that neonatal lentiviral gene delivery may result in stable and widespread cns expression targeted to oligodendrocytes by using cell specific promoters. thus, gene therapy approaches using lentiviral vectors may be feasible for future treatment of leukodystrophy and should be further studied. recent reports identified mrna coding for different cav subtypes like 1.2 and 1.3 in hippocampal ng2 glia. using acute brain slices prepared from developing and mature ng2-eyfp mice, we also found that all ng2 glia upon depolarization in different brain regions elicited a. grimaldi, g. d'alessandro, c. lauro, m. catalano, c. limatola sapienza university of rome, rome, italy glioblastoma (gbm) is one of the most aggressive tumor of the central nervous system, being characterized by a great invasiveness and a very low survival rate of patients. this work wants to better define the role of tumor microenvironment in the modulation of tumor invasiveness. it is known that tumor migration and invasiveness can be modulated by the chemokine cxcl12 and its receptor cxcr4. the expression of both these proteins has been demonstrated in various human gbm cell lines and it has been shown that, blocking this pathway, tumor cells greatly reduce their invasive ability. we have recently demonstrated that an important molecule directly implicated in tumor migration is the intermediate conductance calcium-activated potassium channel (kca3.1). given the expression of these channels also in other cell types infiltrating the tumor mass, like microglia, we investigated the effect of kca3.1 inhibition on microglia-glioblastoma interaction. preliminary data indicate that kca3.1 activity on microglia is involved in the movement of these cells toward the tumor mass. a growing body of evidence indicates that glioblastoma stem-like cells (gscs) play a central role in human glioblastoma development and resistance to current therapies. we recently described a cluster of micro-rnas, the mir-302-367, which induces the exit of gscs from their stem state and suppresses their tumorigenic properties in an irreversible manner (fareh et al, 2012) . to elucidate the gene networks that govern gsc exit from their stem state, we used chip-seq (chromatin immunoprecipitation followed by next generation dna sequencing), to identify gene loci showing enrichment of the active (h3k4me3) and repressive (h3k27me3) epigenetic marks in gsc-mir-302-367 as compared to na€ ıve gscs. functional significance of the chip-seq results was evaluated with confrontation of the chip data with the transcriptomes of the corresponding cells derived from exon array hybridization. western blot analysis showed that global h3k4me3 and h3k27me3 expression levels were similar in gsc and gsc-mir-302-367. chip-seq analysis revealed similar gross number of gene loci associated with h3k4me3 or h3k27me3 (13000 and 5000 genes, respectively) in either cell type. interestingly, 16% (2381 genes) of the analyzed genes exhibited a change in either h3k4me3 or h3k27me3, or both in mir-302-367-gscs as compared to na€ ıve gscs. these changes resulted into a transcript variation in 14% of the cases (332/2381) considering as significant an increase or a decrease of at least 2-fold and the 77% of these genes translated into congruent alterations in the corresponding transcript levels.analysis of the functional significance of the changes observed using functional annotation databases identified novel gene networks, likely to participate in the regulation of gsc properties. studies under way focus on members of these networks specifically activated in mir-302-367 gscs that might alter gsc dialog with their microenvironment. malignant gliomas are the most frequent primary tumors of the brain with poor clinical prognosis. infiltrating peripheral macrophages and resident microglia contribute significantly to the tumor mass. we have previously shown that microglia as the intrinsic immune competent brain cells promote glioma expansion by up-regulating metalloprotease mt1-mmp through toll-like receptor (tlr) and its adaptor protein myd88. in this study we identified tlr2, as the main tlr controlling mt1-mmp expression and pro-tumorigenic signaling in microglia. glioma-derived soluble factors and synthetic tlr specific ligands induced mt1-mmp expression in microglia from wt mice but not tlr2-/-mice. by using the organotypic brain slice model, we found that tumor expansion depended on both parenchymal tlr2 expression and the presence of microglia. tlr2 is also highly expressed in human gliomas and inversely correlates with patient survival. in search for an endogenous tlr2 ligand released from glioma cells, we screened glioma conditioned medium (gcm) by mass spectrometry for endogenous tlr2 agonists produced by glioma cells and found versican, an extracellular matrix proteoglycan and a reported ligand of tlr2. to examine if versican is the factor mediating the glioma-microglia interaction, we silenced versican expression in gl261 cells. primary microglia were then stimulated with gcm from versican sirna and non-target sirna transfected gl261 cells and microglial mt1-mmp expression was analyzed by real-time pcr. an almost 3-fold up-regulation in microglial mt1-mmp expression was observed using gcm from non-target transfectants while the expression was reduced with gcm from versican knock-down gl261 transfectants. our results show that tlr2 activation is an essential part of the signaling cascade employed by glioma cells to convert microglia into a pro-tumorigenic phenotype and tlr2 thus may be a novel target for glioma therapies. two ipsdms were used to determine their responses in the presence of glioblastoma cells. using lc-ms/ms method, proteomic profiles were generated and compared between ipsdms exposed to human glioblastoma cells and ipsdms exposed to normal human astrocytes as control.results: immunolabeling of the two ipsdm cell lines showed specific expression of microglial markers such as iba-1, cd11b, and cd68. the comparative proteomic analyses indicated that microglia exposed to glioblastoma cells showed differential protein expressions relevant for cytoskeletal activity, energy production, and cell survival compared to control.conclusion: this is the first study showing the proteomes of human induced pluripotent stem cell-derived microglia exposed to glioblastoma cells, and the findings could provide further insight of the interaction between microglia and glioblastoma. key: cord-022888-dnsdg04n authors: nan title: poster sessions date: 2009-08-19 journal: eur j immunol doi: 10.1002/eji.200990224 sha: doc_id: 22888 cord_uid: dnsdg04n no abtract the humoral pattern recognition receptors of innate immunity include collectins, ficolins and pentraxins. ptx3, the prototype of long pentraxins, plays a nonredundant role in resistance against a. fumigatus lung infection. the model proposed suggests that upon binding, ptx3 facilitates recognition, phagocitosis and killing of a. fumigatus conidia by alveolar macrophages, dendritic cells and neutrophils and the subsequent development of a properly th1-oriented adaptive response. actually, ptx3-deficient mice are highly susceptible to aspergillosis and develop th2 skewed responses; moreover, ptx3-deficient resident macrophages and neutrophils show defective conidia phagocytosis. both in vitro and in vivo defects can be rescued by the administration of recombinant ptx3, which does not show direct activity on fungal cells. finally, ptx3 alone or in combination with antifungal agents, induces a curative response in mice with aspergillosis, even when given prophylactically. in the present study, we investigated the mechanisms underlying the ptx3-mediated opsonic activity and the involvement of complement, complement receptors and fcg receptors, by in vitro studies and genetic approaches in vivo. in vitro ptx3 amplified the complement-dependent effects on a. fumigatus conidia phagocytosis by human neutrophils, activated through the alternative pathway. accordingly, in the presence of ptx3-opsonised conidia, cd11b activation, internalization, recruitment to the phagocytic cup and cd11b-dependent phagocytosis were increased. as pentraxins interact with fcgreceptors, which in turn can control cd11b activation, the phagocytic assay was performed in the presence of fcgr blocking abs. data obtained strongly suggest that upon conidia opsonisation with ptx3, fcgriia/cd32 mediates inside-out activation of cd11b and consequently phagocytosis of c3b-opsonised conidia. in vivo phagocytosis experiments performed with c1q-and fc common gamma chain-deficient mice and complement inhibitors support in vitro data. these data confirm and extend the paradigm of cooperation among innate receptors, in particular among the humoral arm of innate immunity (complement, ptx3) and the cellular arm (fcgrs, cr3). moreover, they confirm previous studies on the interaction between pentraxins and fcgrs and support the idea that pentraxins behave as predecessors of antibodies. innate immunity is the first line of defence against pathogens and plays a key role in the initiation, activation and orientation of adaptive immunity. the humoral arm of the innate immunity includes soluble pattern-recognition receptors (prrs) such as collectins, ficolins, complement components and pentraxins. the prototypic long pentraxin ptx3 is rapidly produced and released by diverse cell types in response to proinflammatory signals. ptx3 binds selected microorganisms such as aspergillus fumigatus and restores protective immunity against this pathogen in ptx3-/-mice. neonates have an immature innate immune system and are more susceptible to bacterial infection than older children or adult. a beneficial effect of breast feeding on newborn health is highly demonstrated. this protective effect is mediated by nutrients, immunomodulatory mediators (ifn-g, tnfa, or tgf-b), innate immunity factors (soluble cd14, immunoglobulins, lactoferrin), and leukocytes contained in milk that can penetrate the newborn circulation. we thus hypothesized that milk may contain ptx3. we found high concentration of ptx3 in human colostrum (47.62 ± 13.5 ng/ml at day 1 post-delivery) compare to the one found in human serum ( x 2 ng/ml). the presence of ptx3 in human colostrum seems to be due to the secretion of ptx3 by human mammary gland since we report the production of ptx3 by these cells. this prr is also found in human milk cells (hmc), mainly in leukocytes, and penetrate into newborn tissus after suckling. furthermore, human colostrum upregulated the ptx3 production by adult and neonate immunocompetent cells and we demonstrate that neonate mice present a deficit in their ptx3 production after lps injection. collectively, these data demonstrate that newborn have three distinct ways of ptx3 supplying by breast feeding: (i) soluble ptx3 in colostrum (ii) hmc that can secrete ptx3 upon stimulation in the specific tissue, (iii) an increase of ptx3 production by immune cells in the presence of colostrum. thus, soluble or cell-derived ptx3 may participate to the beneficial role of breast feeding on the newborn health. a. m. baru 1 , j. stephani 2 , h. wagner 2 , t. sparwasser 1 1 twincore, institute for infection immunology, hannover, germany, 2 technical university of munich, institute for medical microbiology, immunology and hygiene, munich, germany toll-like receptors (tlrs) represent the best characterized pattern recognition receptor family in mammalian species. the family currently comprise of 10 receptors in humans (tlr 1-10) and 12 in mice (tlr 1-9, 11-13). as transmembrane receptors, tlrs are expressed on the cell surface (tlr 1, 2, 4, 5, 6, (10) (11) (12) (13) and at endosomal membranes (tlr 3, 7, 8 and 9) . toll-like receptors recognize specific patterns of microbial components and regulate the activation of both innate and adaptive immunity. bacterial dna has been shown to possess immunomodulatory activity about a decade prior to the identification of cpg motifs. about 5 years later to this, toll-like receptor 9 (tlr9) was identified and shown to be the receptor for unmethylated cpg dna which is present mainly in non-vertebrate genome. studies have defined potential role of tlr9 as adjuvant enhancing protective immune responses against tumours and infectious diseases in murine models. although promising results are obtained from a few human clinical trials, overall efficacy and safety could not yet be translated entirely from murine studies to human trials. one explanation for these discrepancies could be the fact that expression of human-tlr9 (hutlr9) is restricted to b-cells and plasmacytoid dendritic cells (pdcs) whereas murine-tlr9 (mutlr9) is also expressed on conventional dendritic cells (cdcs). consequently, tlr9 ligands induce distinct cytokine profiles in mice and human thereby probably regulating immune responses in a different manner. by employing bacterial artificial chromosome (bac) technology, we generated transgenic mice with hutlr9 (henceforth called as hut9 mouse) integration in their genome under human epigenetic control. to avoid effects seen due to overlapping ligand specificities, we crossed this mouse onto mutlr9 knock-out background. we expect that hut9-mutlr -/mice mimic the human specific expression pattern of tlr9, i. e. exclusively in b-cells and pdcs, allowing us to investigate detailed in vivo functions of hutlr9. by studying infection and tumour models as well as models for autoimmunity, allergy and transplantation we could then define appropriate and safe implications for employment of tlr9 ligands in human immunotherapy. the fractal analysis provides unique physical insights into the interactions between c1q and the prp protein. if one may take the liberty to extend this to cellular surfaces, where presumably these reactions are taking place, then one has access to a possible avenue by which one may control these reactions in desired directions. if this is true, then surely, this is worth exploring further. any effort, no matter how small that assists in help providing better insights into these debilitating and neurodegenrative disorders such as alzheimers is defintely worth the effort. interleukin-12 is a heterodimeric cytokine consisting of the two subunits p35 and p40. the main inducers of il-12p40 are microbial components activating toll-like receptors with the magnitude of il-12p40 induction depending on the specific tlr engaged. differential induction of il-12p40 upon tlr stimulation correlated with striking differences in the kinetics of nfkb activation. cpg-dna strongly induces il-12p40 due to its outstanding capacity (i) to induce nucleosomal remodelling in proximal il-12p40 promoter region and (ii) to stimulate prolonged rela activity. here we were interested in further changes in chromatin structure of the il-12p40 promoter upon tlr triggering. we did not observe a change in dna methylation, but using chormatin immunoprecipitation (chip) we were able to detect a strong increase in histone 3 and 4 acetylation in specific regions of the proximal promoter region. acetylation of h4 showed a specific distribution pattern and occured mainly in regulatory elements within the il-12p40 promoter, whereas acetylation of h3 took place over all regions analyzed. tlr tolerance has been reported to be associated with specific chromatin alterations. methylation status of lysine residue 4 on h3 turned out to be important for the inhibition of gene expression upon repeated stimulation. modifying the chromatin structure of gene promoter regions therefore seems to be a sensitive mechanism to modify cytokine expression to exogeneous stimuli in innate immune cells thereby allowing adaption of innate immune responses. a. d. koepruelue 1 , w. ellmeier 1 1 medical university of vienna, institute of immunology/division of immunobiology, vienna, austria macrophages are important in innate and acquired immunity. failures are associated with inflammatory and autoimmune diseases. understanding their stimulation is the basis for therapeutic targeting. members of the tec kinase family (bmx, btk, itk, rlk and tec), expressed in the haematopoietic system, constitute the second largest family of non-receptor tyrosine kinases. mutations in btk represent the source of human x-linked agammaglobulinemia (xla). a mutation in the murine btk gene accounts for a similar syndrome, x-linked immunodeficiency (xid). although the tec family members tec, btk and bmx are expressed in monocytes/macrophages, little is known about their function there. tec kinases become activated upon signaling via divers receptors including antigen receptors, receptor tyrosine kinases or tlrs. several studies in xla or xid macrophages and in monocyte/macrophage cell lines implicated roles for tec kinases in tlr signaling and as well as other macrophage effector functions like phagocytosis. inspired by these findings, we aim to determine the role of tec kinases in bone marrowderived macrophages (bmm), during macrophage activation and in other macrophage functions such as recruitment or phagocytosis. in a comprehensive functional analysis of tlr-mediated bmm activation from mice deficient for one or more of the tec family members in vitro, we reveal which of the kinases play a role in which tlr pathway. based on the results of this analysis, we set the goal to further study how tec kinases regulate the respective signaling cascades. our study will contribute insights into the role of tec kinases in this important cell population of the innate immune system. g. lunazzi 1 , m. buxadé 1 , j. minguillón 1 , r. berga 1 , j. aramburu 1 , c. lópez-rodríguez 1 1 universitat pompeu fabra, department of experimental and health sciences (dcexs), barcelona, spain nfat5 is a transcription factor that regulates the expression of cytokines such as tnfa and lymphotoxin b in response to osmotic stress. in addition, nfat5 participates in multiple processes not linked to the response to hypertonicity. in this regards, it has been recently reported that nfat5 is required as a novel host factor that supports hiv replication in macrophages. given the established connections between nfat5, the expression of certain inflammatory cytokines, and its role in the response to specific pathogens in macrophages, we aimed at studying whether nfat5 could be activated by receptors for pathogens expressed in macrophages. the activation of toll-like receptors (tlrs) is central to innate immunity. upon stimulation of tlrs, cells of the immune system induce signalling pathways that lead to the activation of different transcription factors. as a result of that, cells such as macrophages and dendritic cells induce the expression of genes that participate in the response to pathogens such as those encoding proinflammatory cytokines, antimicrobial products, survival factors or mediators of cellular migration. we have analyzed whether nfat5 is expressed in primary macrophages through the activation of different toll-like receptors. likewise, we have explored whether the activity of nfat5 is induced during the response to tlrs. in addition, we have studied whether the specific inhibition of different signalling pathways positioned downstream of tlrs could interfere with the expression of nfat5. our work indicates that nfat5 is a novel transcriptional regulator acting in response to the activation of tlrs. our work extends the knowledge about mechanisms that participate during the innate immune response to pathogens and offers a new regulatory pathway as a possible target to modulate this response. objectives: compelling evidence support a link between inflammation, cell survival, and cancer, with a central role played by nf-xb, a master switch of inflammation. recent studies implicate some tlrs in tumor development or regression, and immune escape. however, mechanisms leading to tumor growth or apoptosis induced by tlr stimulation are not fully understood. several studies strongly suggest that chronic inflammation in lungs induced by chronic bronchitis, chronic obstructive diseases, emphysema, asbestos or tobacco smoke, increases the risk of carcinogenesis. we hypothesized that some tlrs can contribute to lung inflammation and tumor development in vitro and in vivo. methods: tlr expression in lung cancer was assayed by immunohistochemistry or flow cytometry. nfxb activation was determined by western blot and nuclear translocation assay after tlr stimulation. clonogenicity of stimulated cells was analyzed by colony assay. transcriptomic analysis were performed by taqman lda technology. tumor growth in vivo was analyzed in nod/scid mice after subcutaneously engraftment of human lung tumor cell lines. we have observed that primary human lung tumors express tlr3, tlr4, tlr7 and tlr8 and that stimulation of these receptors in lung tumor cell lines by poly i:c, lps, loxoribine or poly u induces nfxb activation through atypical signaling pathway, with phosphorylation of ixba without its degradation and nuclear translocation of p50 and p65 nfxb subunits. interestingly, we observed that tlr3 stimulation induces apoptosis depending of the histological type of the tumor. on the contrary tlr4, tlr7 and tlr8 stimulation induces cell survival and increases clonogenicity. this is correlated with an up-regulation of bcl-2 expression. moreover, despite a common atypical activation of nfxb, our transcriptomic analysis revealed major differences in gene modulation after triggering of tlr3, tlr4, tlr7 and tlr8. finally, in vivo tlr7 stimulation of human lung tumor cells dramatically increases tumor size and metastasis. conclusions: altogether, these data emphasize that tlr4, tlr7 or tlr8 triggering can directly favor tumor development whereas tlr3 signaling can induce tumor cell death. these data suggest that anticancer immunotherapy using tlr adjuvants should take into account the expression of these tlrs in lung tumor cells. objective: dasatinib (bms-354825) is a small molecule src/abl tyrosine kinase inhibitor approved for the treatment of chronic myeloid leukaemia and philadelphia chromosome-positive acute lymphoblastic leukaemia. members of the src family of kinases are involved in normal physiological processes, and play a significant role in the induction and regulation of innate and adaptive immunity. the purpose of this study was to evaluate the inhibitory action of dasatinib on toll like receptor (tlr) signalling, natural killer (nk) cell cytotoxicity as well as antigen-specific cd8 + and cd4 + t cell function. methods: to analyse tlr signalling in vitro murine bone marrow derived (bmd) macrophages were stimulated with the tlr 4 ligand lipopolysaccaride (lps) in the presence of dasatinib and tumour necrosis factor a (tnf-a) in the culture medium was measured. the response to tlr stimulation was also tested in vivo, dasatinib-treated mice were challenged with lps and tnf-a in the serum was quantified. in addition, the clearance of the rma-s cells, a mhc class i deficient thymoma sensitive to nk cell lysis, was analysed in mice undergoing dasatinib treatment. to investigate the inhibitory effects of dasatinib on adaptive immune responses, transgenic cd4 + and cd8 + t cells specific for ovalbumin were utilised to measure antigen specific t cell proliferation. endogenenous cd4 + and cd8 + t cell responses were determined following immunisation of dasatinib-treated mice with a nonreplicating recombinant virus. results: we show that dasatinib impairs: 1. innate immune response; dasatinib treatment reduced the (a) production of tnf-a following tlr 4 stimulation of bmd macrophages in vitro, (b) production of tnf-a in vivo in response to lps and (c) ability of nk cells to eliminate mhc class i deficient cells in vivo . 2. adaptive immune response; dasatinib treatment inhibited (a) proliferation of antigen-specific murine transgenic t cells, (b) endogenous antigen-specific helper t cell recall-responses and (c) t cell-mediated cytotoxic effector function. conclusions: these findings suggest that dasatinib has the potential to modulate the host immune response and highlights scope for off target applications, for example therapeutic immunosuppression in the context of autoimmune pathogenesis, or in combination with other interventions for the treatment of endotoxic shock. i. zanoni 1 , r. oatuni 1 , m. collini 2 , m. caccia 2 , p. castagnoli 3 , g. chirico 2 , f. granucci 1 1 university of milano-bicocca, biotechnology and bioscience, milan, italy, 2 university of milano-bicocca, physics, milan, italy, 3 singapore immunology network (sign), biomedical sciences institutes, immunos, singapore, singapore the recognition of mamps by tlrs expressed on dendritic cells (dcs) plays an essential role for the regulation of the immune responses. by recruiting different combinations of adapter proteins, individual tlrs turn on signal transduction pathways leading to the activation of different transcription factors. interleukin-2 (il-2) is one of the molecules produced by dcs shortly after stimulation with different tlr agonists. based on this observation and by analogy with the events following t-cell receptor (tcr) engagement leading to il-2 production, we hypothesized that the stimulation of tlrs on dcs might lead to activation of the ca2+/ calcineurin and nfat pathway. we found that dc stimulation with lps induces extracellular ca2+ influxes, leading to calcineurin-dependent nfat activation. the activation of this pathway was independent of tlr4 engagement, depending instead exclusively on cd14. we also found that lps-induced nfat activation in dcs was necessary for the efficient synthesis of cyclooxygenase-2 (cox-2) that, by generating prostaglandins (pgs), such as pge2, regulates different dc functions including migration and polarization of t cell responses. our findings reveal novel aspects of the molecular signaling triggered by lps in dcs and define a new role for cd14. given the essential involvement of cd14 in many diseases, including sepsis and chronic heart failure, the discovery of signal transduction pathways activated exclusively via cd14 represents a major step towards the development of potential treatments with modes of action involving interference with cd14 functions. we have examined the interaction of cd55, a 80-kda glycosyl-phosphatidylinositol (gpi)-anchored membrane protein, with the monocyte signalling receptor, cd14. human monocytes were isolated from healthy adult donor's peripheral blood. this involved labelling molecules at saturation with different coloured fluorophores and determining their positions separately by dual wavelength imaging. the cells were labelled at saturation with anti-cd14 antibody coupled to biotin visualised by qd-525-streptavidin and anti-cd55 antibody coupled to allophycocyanin. the images are analysed to quantify the overlap of the particle images and hence determine the extent of co-localization of the labelled molecules. single particle fluorescence imaging (spfi) uses the high sensitivity of fluorescence to visualize individual molecules that have been selectively labelled with small fluorescent particles. the images of particles are diffraction-limited spots that are analysed by fitting with a two-dimensional gaussian function providing the basis for determining the dynamic and associated behaviour of receptors on living cells. changes in the numbers of receptors, and in the proportion of receptors showing colocalisation, indicated that lps promotes the interaction of cd55 and cd14, suggesting a new functional role of cd55 as a member of a multimeric lps receptor complex. l. lundvall 1 , r.r. schumann 1 1 charité -universitätsmedizin berlin campus mitte, institute for microbiology and hygiene, berlin, germany meningitis is a life-threatening disease mainly caused by bacteria and viruses. bacterial components such as lipopolysaccharide (lps), lipoproteins or peptidoglycan breakdown products (i. e. mdp, mesodap) stimulate pattern recognition receptors (prrs), such as toll-like receptors (tlrs) and the intracellular nod-like receptors (nlrs) for an inflammatory response. we hypothesize that a synergistic effect of tlr-induced nf-xb activation and nlr-mediated caspase-1 induction leads to an increased release of mature il-1b during bacterial meningitis in brain-derived cells. a mouse meningitis model with s. pneumoniae (d39) was established for assessing il-1b induction during this disease. the murine raw 264.7 cell line, the human astroglial u-87 mg and the murine microglial cell-line bv-2 were stimulated with the tlr4 ligand lps, the tlr2 ligand pam 2 cys, the nod2 ligand mdp, or the nod1 ligand c12-ie-dap, and, as control, atp alone or in combination. we assessed il-1b by elisa and caspase-1 and pro-il-1b expression by western blot. furthermore, primary mouse astrocytes isolated from the cortices of mouse puppies were used for stimulation followed by sirna suppression of elements of the il-1b induction pathway. s. pneumoniae (d39) infected mice showed a significant increase in il-1b release after 24 hours. in vitro, an increase in il-1b levels after costimulation with lps or pam 2 cys, and mdp or c12-ie-dap was observed in a dose-dependent manner. a synergistic enhancement of il-1b by tlr-and nlr-ligands was observed in raw cells, bv-2 cells, u-87 cells and primary astrocytes. active caspase-1 (p10) was induced by mdp or c12-ie-dap, corresponding with high il-1b responses when lps or pam 2 cys was added. sirna experiments show that a knock-down of nod2 leads to a diminished il-1b release after lps-and mdp-stimulation. the precursor forms of il-1b and caspase-1 seem to be constitutively expressed in astrocytes and microglia. a synergistic enhancement between tlrs and nlrs in il-1b release in brain-derived cells was observed. so a two-step stimulation seems necessary for the release of high levels of mature il-1b by astrocytes and microglia. bacteria containing both, tlr-and nlr-ligands thus have the potential to induce high levels of il-1b which may contribute to disease pathology and may point to novel intervention strategies. j. rosenberg 1 1 toll-like receptors (tlrs), nod-like receptors (nlrs), and rig-i-like (rlrs ) are more well-characterized in their identity and expression as signaling markers which effect the ealry innate immune response and elicit adaptive immunity , . in the case of tlrs most sutides to date have delineated tlr expression and function on antigen presenting cells like dendritic cellof this research. extension of the profiling and presence of tlrs on cell characterized as adaptive immune cells such as t cells is the subject of this line of research. using a cd3 and cd28 activation model system -tlr4 presence on cd4+ cells is found in mouse t cells, human t cells and jurkat cell lines. following cd3/cd28 activation for 48 hours we have identified a small but distinct populationof tlr4+ cells. further characterization indicates these cells to be cd4+cd25+ cells. further characterization of the expression and functional acitvity of the tlr4+ t cells indicates co-expression of tlr4 with md-2 indicating a functional tlr4 receptor. in addition lps activiation did not lead to upregulation of tlr4 expression in t-cells. the data indicate that tcr activation leads to tlr4 expression. the expression appears to be associated with cd4+cd25+ cells and refelecting an activated t cell phenotype which will be further characterized as perhaps related to tregs or other tcell subsets. s. m. lehmann 1 , d. kaul 1 , c. krüger 1 , f. zipp 1 , r. nitsch 2 , s. lehnardt 1 1 charité-universitätsmedizin berlin, cecilie-vogt-clinic for neurology, berlin, germany, 2 charité-universitätsmedizin berlin, institute for cell biology and neurobiology, berlin, germany the innate immune system is the first line of defense against various pathogens and requires the expression of toll-like receptors (tlrs). in macrophages, tlr7 plays a crucial role in immune responses elicited by gu-rich ssrna (i. e. ssrna40) as well as synthetic antiviral chemicals, including imidazoquinoline components (i. e. imiquimod) and some guanine nucleotide analogs (i. e. loxoribine). these compounds were initially described to activate mouse tlr7 (and human tlr8) and are potent immune response modifiers leading to important antiviral and antitumor activities. microglia serve as the major innate immune cells in the central nervous system (cns). employing various techniques including pcr, in situ hybridization, and immunocytochemistry, we demonstrate that tlr7 is expressed in these cells. incubation of microglia with all three of the above mentioned tlr7 ligands leads to activation of these cells displaying an ameboid shape and releasing inflammatory cytokines such as tnf-a and il1-b in a dose-and time-dependent fashion. analysis of wild type (wt) and tlr7 knock out (ko) microglia by realbecause neutrophil apoptosis plays a key role in resolving inflammation, identification of proteins regulating neutrophil survival should provide new strategies to modulate inflammation. using a proteomic approach, coronin-1 was identified as a cytosolic protein cleaved during neutrophil apoptosis. coronin-1 is an actinbinding protein that can associate with phagosomes and nadph oxidase but its involvement in apoptosis was currently unknown. in coronin-1-transfected plb985 cells, coronin-1 overexpression did not modify the kinetics of granulocyte differentiation as assessed by cd11b labeling. concerning apoptosis, increased coronin-1 expression in dmf-differentiated plb985 significantly decreased gliotoxin-induced mitochondrial depolarization as compared with controls. likewise, coronin-1 significantly decreased trail-induced apoptosis with less mitochondrial depolarization, caspase-3 and caspase-9 activities, but not caspase-8 or bid truncation suggesting that coronin-1 interfered with mitochondria-related events. to validate the prosurvival role of coronin-1 in a pathophysiological condition involving neutrophil-dominated inflammation, neutrophils from cystic fibrosis (cf) patients were studied. circulating neutrophils from cf patients had more coronin-1 expression assessed by immunoblotting or proteomic analysis of cytosolic proteins. this was associated with a lower apoptosis rate than those from controls evidenced by delayed phosphatidylserine externalization and mitochondria depolarization. in addition, inflammatory neutrophils from cf patients lungs showed an intense coronin-1 immunolabeling. we concluded that coronin-1 could constitute a potential target in resolving inflammation. p.-n. hsu 1 1 national taiwan university, graduate institute of immunology, taipei, taiwan, republic of china human osteoclast formation from mononuclear phagocyte precursors involves interactions between tumor necrosis factor (tnf) ligand superfamily members and their receptors. many of the proinflammatory cytokines and growth factors implicated in inflammatory processes have also been demonstrated to impact osteoclast differentiation and function. recent evidence indicates that the tnf-related apoptosis-inducing ligand (trail) of the tnf ligand superfamily, which was initially thought to induce apoptosis in many transformed cell lines, can serve as an effector molecule in activated t cells. we show in this work that trail can induce osteoclast formation from human monocytes and murine raw264.7 macrophages. we demonstrated that both cell models differentiate into osteoclast-like cells in the presence of trail in a dose-dependent manner, as evaluated in terms of tartrate-resistant acid phosphatase (trap)-positive multinucleated cells and bone resorption activity. the trail-induced osteoclast differentiation is independent of caspase activation and apoptosis induction activity. however, trail-induced osteoclastogenesis is dependent on activation of nf-xb, erk, and p38 map kinase. the trail-induced osteoclastogenesis was significantly inhibited by treatment with traf-6 sirna and traf-6 decoy peptide, indicating this pathway is traf-6 dependent. thus, our data demonstrate that trail induces osteoclast differentiation via direct engagement with the trail death receptor through a signaling pathway distinct from apoptosis. our results indicate that in addition to triggering apoptosis, trail induces osteoclast differentiation. it provides a novel role for trail in regulating osteoclast differentiation and in osteoimmunology. microglia are considered to be the local antigen presenting cells (apcs) of the central nervous system (cns) which are thought to play a crucial role in local reactivation of autoreactive t cells during cns autoimmunity e. g. in multiple sclerosis (ms) and its animal model experimental autoimmune encephalomyelitis (eae) . in this study we investigated if the anti-inflammatory nuclear transcription factor peroxisome proliferator-activated receptor gamma (pparg) that has been described to negatively regulate macrophage activation has an influence on microglia immunogenicity. sustained activation of pparg both reduced microglial signalling via mhc molecules and costimulatory molecules and concomitantly increased signalling via the coinhibitory molecules b7-h1 and b7-dc. moreover, also production of pro-inflammatory cytokines like tnf-a and il-6 was profoundly reduced if microglia were pre-treated with the pparg-agonist pioglitazone (pio). in contrast to this, the lack of pparg in microglia resulted in increased expression of pro-inflammatory cytokines not only following an inflammatory stimulus but also in the steady-state indicating that pparg might play a cell-intrinsic role in controlling microglia immunogenicity. importantly, if pparg was activated in microglia, the capacity to prime ovalbumin-specific t cells was impaired. t cells primed by pio-treated microglia produced reduced amounts of il-2 and ifn-g which could not be overcome by restimulation with acd3. this indicates that t cells primed by pio-treated microglia did not undergo functional differentiation but were impaired in exhibiting effector functions. furthermore, microglia were able to induce antigen-specific differentiation of naive cd4 t cells into t helper 17 (th17) cells, which have been associated with autoimmune pathogenicity during eae. however, if pparg was activated, microglia were no longer able to induce th17 differentiation. in conclusion, activation of pparg impairs microglial apc function leading to reduced activation of antigen-specific t cells and, in addition, inhibits the induction of th17 cells. therefore, activation of pparg in microglia is a promising approach to limit local activation of autoreactive t cells in the cns in cns-autoimmune deseases. bacterial lipopolysaccharide (lps) triggers monocytes and macrophages to produce several inflammatory cytokines and mediators. however, once exposed to lps, they become hyporesponsive to a subsequent endotoxin challenge. this phenomenon is defined as lps desensitization or tolerance. previous studies have identified some components of the biochemical pathways involved in negative modulation of lps responses. in particular, it has been shown that the il-1 receptor-related protein st2 could be implicated in lps tolerance. the natural ligand of st2 was recently identified as interleukin-33 (il-33), a new member of the il-1 family. in this study, we investigated whether il-33 triggering of st2 was able to induce lps desensitization of mouse macrophages. we found that il-33 actually enhances the lps response of macrophages and does not induce lps desensitization. we demonstrate that this il-33 enhancing effect of lps response is mediated by the st2 receptor since it is not found in st2 ko mice. the biochemical consequences of il-33 pretreatment of mouse macrophages were investigated. our results show that il-33 increases the expression of the lps receptor components myeloid differentiation protein-2 (md2), cd14 and tlr-4 and the myeloid differentiation factor 88 (myd88) adaptor molecule. in addition, il-33 pretreatment of macrophages enhances the cytokine response to tlr-2 but not to tlr-3 ligands. thus, il-33 treatment preferentially affects the myd88-dependent pathway activated by the tlr. c. klotz 1 , b. lenz 1 , r. lucius 1 , s. hartmann 1 1 humboldt-university berlin, molecular parasitology, berlin, germany chronic helminth infections are shown to be negatively associated with allergic disorders in humans and animal models and parasite cysteine protease inhibitors (cystatins) have been identified as a major class of modulators from filarial parasites. recently we showed that recombinant parasite cystatin (avcystatin), derived from the model parasite acanthocheilanema viteae, effectively abolished ova-induced allergic airway responsiveness in a mouse model of asthma (schnoeller et al., 2008) . the cystatin effect was blocked by the application of anti-il-10 receptor antibodies and by depletion of macrophages using clodronate liposomes. we hypothesize that parasite cystatin induced regulatory macrophages characterized by secretion of immune suppressive interleukin-10 (il-10). the aim of the present study was to elucidate the molecular mechanisms by which avcystatin induces il-10 in primary macrophages. in vitro experiments with peritoneal macrophages from balb/c mice confirmed specific and concentration dependent il-10 production after avcystatin stimulation. application of specific inhibitors revealed that the il-10 induction was p38 and erk dependent, and inhibitor titration indicated a higher sensitivity towards p38. western blotting experiments confirmed the phosphorylation of p38 and erk in macrophages after avcystatin stimulation. in addition, by using specific inhibitor and western blotting, we showed that avcystatin induced il-10 is also regulated by the phosphatidylinositol-3-kinase (pi3k) -proteine kinase b (akt) pathway. further analysis indicated a hierarchical signalling pattern and cross regulation of the identified pathways. hence, we conclude that avcystatin renders macrophages into a regulatory state by addressing a broad range of signalling cascades that ultimately lead to the expression of il-10 and possibly other regulatory markers. in general, revealing fundamental knowledge about induction of regulatory macrophages by helminth immunomodulators will help to design new strategies for the treatment of inflammatory disorders. we screened approximately half the (putative) human kinome to identify novel candidates interfering with macrophage activation in response to endotoxin. this screen revealed the impact of several novel kinases as well as kinases with previously established function. one of the top candidates identified to block endotoxininduced tnf-a secretion was carkl, a gene with no previously described function. subsequent biochemical analyses unequivocally revealed that carkl is a phosphotransferase protein using sedoheptulose as a phosphate acceptor and atp as a donor. sedoheptulose is a monosaccharide consisting of seven carbon atoms and a functional ketone group. the product sedoheptulose-7-phosphate (s-7p) is also an intermediate metabolite of the pentose phosphate pathway (ppp) and so far was only known to be produced by condensation of ribose-5p and xylulose-5p via a transketolase reaction. to identify the molecular mechanism by which carkl modulates the immune response, we investigated its endogenous regulation and function in the course of macrophage activation. so far, our data favor a model where post-stimulatory downregulation, i. e. loss of carkl is essential for the activation of macrophages by various pro-inflammatory stimuli. disentangling the signaling pathways responsible for the rapid regulation of carkl unearthed nf-kb and p38/jnk but not erk as driving forces. counterbalancing endotoxin induced loss of carkl by over-expression led to an impaired cytokine response and a concomitant block of free radical production. comparison of wild type and catalyticinactive forms of carkl unveiled that most of the effects of carkl on the inflammatory response were due to its phosphotransferase activity. expression profiling using gene chip analysis further supported the concept that carkl may represent a new key modulator of inflammatory processes. taken together, detailed analyses to study the molecular function of carkl should ultimately lead to a more profound understanding of cellular metabolism and especially clarify new mechanisms involved in the regulation of inflammation. in addition, connecting the ppp and its impact on the cellular redox state with inflammatory disease models might reveal new therapeutic targets. in this context, the sedoheptulose kinase carkl and its product s-7p may provide a novel basis for interfering with adverse immune responses. t. bosschaerts 1 , y. morias 1 , p. de baetselier 1 , a. beschin 1 1 vib, cmim, vub brussels, brussels, belgium the development of classically activated monocytic cells (m1) is a prerequisite for effective elimination of parasites, including african trypanosomes. however, persistent m1 activation causes pathogenic damage including liver injury during infection, resulting in death of the host. we aim to identify mechanisms involved in regulation of m1 activity in order to dampen their pathogenicity and increase the resistance of the host to parasitic diseases. methods: we have scrutinized the phenotype and cellular origin of liver m1 in trypanosoma brucei infected by facs analysis and bone marrow transfer experiments. the contribution of different signaling pathways, including myd88, ifng, il-10, ccr2 and nf-kb to the development and/or recruitment of pathogenic m1 in the liver was investigated using knock-out mice or by delivering il-10 in infected mice. results: we established that cd11b+ly6c+cd11c+ tnf and inos producing dcs (tip-dcs) represent the major m1 liver subpopulation. tip-dcs differentiated in an ifng/myd88-dependent manner from cd11b+ly6c+ inflammatory monocytes in the liver of infected mice. ccr2 promoted the egression of inflammatory monocytes from bone marrow to blood but not their entry, differentiation and maturation to tip-dcs in the inflamed liver. as a consequence, ccr2 ko mice experienced reduced pathogenic symptoms. on the other hand, the absence of il-10 enhanced the recruitment of inflammatory monocytes as well as their differentiation and maturation to tip-dcs, resulting in exacerbated pathogenicity and early death of the host. in addition, the therapeutic liver-specific delivery of il-10 in t.brucei infected mice efficiently limited the differentiation and maturation to tip-dcs, hereby limiting disease-associated pathogenicity. finally, the absence of the nf-kb member p50 was associated with increased tissue injury associated with increased production of pathogenic tnf and no by inflammatory monocytes, but not by tip-dcs. conclusion: our data demonstrate that nf-kb p50 and il-10 play a role in preventing infection-associated pathogenicity in hosts confronted with a chronic inflammatory situation by limiting the activity of pathogenic m1, in particular tip-dcs. the inflammatory activity of liver m1 is controlled by il-10 and/or p50 nf-kb at different levels, including recruitment of inflammatory monocytes to the liver, their differentiation to pathogenic tip-dcs, or their production of tnf and no. a. popov 1 , j. driesen 1 , z. abdullah 2 , a. niño castro 1 , t. chakraborty 3 , m. krönke 4 , o. utermöhlen 4 , c. wickenhauser 5 , j.l. schultze 1 1 limes institute, laboratory for genomics and immunoregulation, university of bonn, bonn, germany, 2 institute of molecular medicine and experimental immunology, bonn, germany, 3 institute of medical microbiology, university of giessen, giessen, germany, 4 institute for medical microbiology, immunology and hygiene, university of cologne, cologne, germany, 5 institute for pathology, university clinic leipzig, leipzig, germany dendritic cells (dc) and macrophages play an important role in pathogen sensing and antimicrobial defense. here we report on a new role for the myeloid antigen presenting cells (apc) in granulomatous infections. infection of myeloid dc and macrophages with listeria monocytogenes results in a distinct regulatory phenotype characterized by expression of multiple inhibitory molecules, including indoleamine 2,3-dioxygenase, cyclooxygenase-2 and cd25 and production of prostaglandin e2 (pge 2 ) and interleukin 10. all these molecules are strictly dependent on autocrine tnf, released during infection, and are in concert suppressing t-cell responses; cd25, expressed by regulatory myeloid cells, acts as an il-2 scavenger. importantly, myeloid cells with regulatory phenotype are characterized by increased resistance to infection and demonstrate significantly improved bactericidal activity against intracellular bacteria. furthermore, infected cells can transfer the regulatory phenotype to the uninfected ones in a cell-cell contact independent manner, thereby extending the pool of infection-resistant myeloid cells. induction of regulatory and protective phenotype in macrophages and dc require at least two signals provided by tnf and either pge 2 or tlr ligands. transcriptional changes in human macrophages, infected by mycobacterium tuberculosis, resemble the ones induced in dc during infection with l.monocytogenes. in fact, granuloma in patients with tuberculosis and listeriosis are enriched for cd25 + ido + cox-2 + regulatory myeloid cells, whereas most effector cell populations, such as t cells, b cells and nk cells, are expelled from the granuloma. of note, in tuberculosis granuloma consist mostly of macrophages, whereas in listeriosis dendritic cells predominate. altogether, our studies provide strong evidence that intracellular pathogens such as m.tuberculosis and l.monocytogenes induce a specific polarization of myeloid dc and macrophages characterized by a functional preponderance of inhibitory mechanisms. we postulate that these regulatory myeloid cells play a dual role during life-threatening granulomatous infections. on one hand, they promote pathogen containment by efficiently killing intracellular bacteria; on the other hand, these myeloid cells inhibit granuloma-associated t cells and thereby might be involved in the retention of granuloma integrity protecting the host from granuloma break-down and pathogen dissemination. the interferon-gamma (ifn-g) component of the immune response plays an important and essential role in infectious and non-infectious diseases. induction of ifn-g secretion by human t and nk cells through synergistic co-stimulation with interleukin 12 (il-12) and il-18 in the adaptive immune responses against pathogens is well known, whereas a similar activity by macrophages is still controversial, largely due to criticisms based on the contamination of macrophages with nk or t cells in the relevant experiments. the possible contribution of macrophages to the interferon response is, however, an important factor relevant to the pathogenesis of many diseases. to resolve this issue, we have determined the production of ifn-g at a single cell level by inmunohistochemistry and by enzyme-linked immunosorbent spot (elispot) analysis and have unequivocally demonstrated that human macrophages derived from monocytes in vitro through the combined stimulation of il-12 and il-18 or with macrophage-colony stimulating factor (m-csf) were able to produce ifn-g when further stimulated with a combination of il-12 and il-18. in addition, naturally activated alveolar macrophages immediately secreted ifn-g upon treatment with il-12 and il-18. therefore, human macrophages in addition to lymphoid cells contribute to the ifn-g response, providing another link between the innate and acquired immune response. a. j. denzel 1 , m. rodriguez gomez 2 , m. niedermeier 2 , y. talke 2 , n. göbel 2 , k. schmidbauer 2 , m. mack 2 1 unversity hospital regensburg, internal medicine ii, regensburg, germany, 2 university hospital regensburg, regensburg, germany we have shown previously that basophils recognize and react to free antigen during a memory immune response in vivo and release large amounts of il-4 and il-6. activation of basophils is dependent on the presence of free antigen, antigen specific immunoglobulins and expression of immunoglobulin fc-receptors. we now have analysed in more detail the binding of antigen to basophils, the recruitment of basophils to lymphoid organs and the basophil dependent migration of other leukocytes during the first days of a memory immune response. following restimulation with soluble antigen only antigen specific basophils but not basophils from naïve mice migrate from bone marrow and spleen to the site of restimulation (e.g. the peritoneum) and the draining lymph nodes. peripheral blood basophils are markedly reduced during the first hours after restimulation. in the blood, spleen lymph nodes and bone marrow basophils can bind intact antigen on their surface for up to 24h, with basophils in the bone marrow binding the lowest amount of antigen. depletion of basophils also affects the recruitment of various other leukocyte subsets in immunized mice. our datas show that basophils are recruited to draining lymph nodes during a memory response. tnf-a is a pro-inflammatory cytokine that mediates inflammation in response to various pathogens, including mycobacterium tuberculosis. it is also a key factor in the pathogenesis of autoimmune diseases like rheumatoid arthritis. three tnf-a-blocking drugs have been approved to treat selected autoimmune diseases; two are monoclonal antibodies against tnf-a (adalimumab and infliximab); the other is a soluble tnf receptor/fc fusion protein (etanercept) . tnf-a-blockers have been shown to increase the risk of reactivation of latent tuberculosis and this risk appears to be higher in patients treated with the monoclonal antibodies. we studied the effects of tnf-a blockers on the maturation of mycobacteria-containing phagosomes in human macrophages. all three drugs had an inhibitory effect on ifn-g-induced phagosome maturation in pma-differentiated human thp-1 cells infected with m. bovis bcg, the avirulent m. tuberculosis h37ra strain and the virulent m. tuberculosis h37rv strain. adalimumab and infliximab, but not etanercept, suppressed phagosome maturation in primary human peripheral blood monocyte-derived macrophages (mdm) in the presence or absence of ifn-g. macrophages secreted tnf-a in response to infection with mycobacteria and this response was enhanced by activation of the cells with ifn-g. treatment of infected macrophages with tnf-a increased maturation of mycobacteria-containing phagosomes. these results suggest a role for tnf-a in activating phagosome maturation and highlight a novel mechanism through which tnf-a blockade can affect the host innate immune response to mycobacteria. z. g. dobreva 1 , l.d. miteva 1 , s.a. stanilova 1 1 trakia university, faculty of medicine, molecular biology, immunology and genetics, stara zagora, bulgaria il-23 is a heterodimeric cytokine composed of a p19 subunit associated with the il-12/23p40 subunit. like il-12, il-23 is expressed by the activated antigenpresenting cells and both cytokines induce ifn-gamma secretion by different t cell subsets. the proper balance between il-12p40-related cytokines controls the appearance of normal th 1 and pathological th 17 mediated immune responses. in this study, we examined the dynamics of inducible il-12p40 and il-23p19 mrna expression and protein production in purified human monocytes and how jnk and p38 mapks inhibitors influenced il-12p40 and il-23 production. the cytokines' quantity determination was performed by elisa. quantitative real-time polymerase chain reaction (qrt-pcr) was performed for mrna transcripts detection. results were calculated in fold increase compared with gene expression in nonstimulated monocytes. il-23p19 gene expression was higher than those observed for il-12p40 gene at all time-points. the level of il-23p19 mrna increased after 6 th h and reaching a maximum level at 9 th h (43.4 fold for c3bgp and 22.7 fold for lps). c3bgp and lps triggered il-12p40 gene transcription were almost equal at the 3 rd h (4.4 and 4.1 fold) and at 9 th h (7.8 and 7.9 fold, respectively) after stimulation. the higher level of il-12p40 gene expression was detected at 6 th h in lps compared to c3bgp stimulated monocytes (8.1 vs. 4.9 fold). however, il-12p40 and il-23 protein production was increased in the highest level after c3bgp stimulation. the inhibition of p38 led to the statistical significant augmentation of c3bgp stimulated il-12p40 production. the inhibition of the same map kinase enhanced lps stimulated il-12p40 production without significant difference. the inhibition of jnk and p38 mapks significantly decreased c3bgp stimulated il-23 production from human monocytic cells.in summary, the present study demonstrates the different time-course and ability of c3bgp and lps to induce the expression of il-12p40 and il-23p19 mrnas in purified human monocytes. we showed that inhibition of p38 mapk down regulated il-23 and upregulated il-12p40 production in stimulated monocytes. we concluded that in human monocytes p38 map kinase activation has an opposite effect on the il-12p40 and il-23p19 expression. neutrophils represent key components of the innate immune system with the ability not only to phagocytose and killing invading pathogens, but also to produce a variety of proteins, including cytokines and chemokines, with important consequences on the recruitment and activation of other immune cells, such as monocytes, dendritic cells, t and b cells. for instance, it has been shown that neutrophils can directly interact with, and induce functional maturation of, immature monocyte-derived dendritic cells (modc). indeed, upon interaction with neutrophils, modc up-regulate the expression of costimulatory molecules, such as cd83, cd86 and cd40, and secrete il-12, thus acquiring the ability to induce proliferation and th1 polarization of naï ve t cells. in order to extend these findings, the present study was designed to address whether human neutrophils interact with peripheral blood-derived dendritic cells and the pathological consequences that such interaction could eventually produce. in human peripheral blood, dendritic cells can be divided in plasmacytoid dendritic cells (pdc) and myeloid dendritic cells (mdc), the latter further divided in three different subsets based on the expression of cd1c, bdca-3, and cd16. by analyzing different chronic inflammatory pathologies, such as crohn's disease, psoriasis and sweet's syndrome, we found that neutrophils co-localize with a subtype of myeloid dendritic cells (mdc) with characteristics resembling the cd16+ subset of mdc. in order to characterize the interaction between the two cell types, autologous neutrophils, highly purified by an in-house built immunonegative selection protocol, and cd16+ dc were isolated from healthy donors and analyzed in a co-culture system under different stimulatory conditions. here we show that neutrophils modulate different effector functions of cd16+ dc, including their survival and their ability to produce il-12p70. besides providing the basis for a better understanding of the cellular interactions that occur in pathological conditions, our results further emphasize the importance of neutrophils in the modulation of the inflammatory response. chitin is a linear polymer of n-acetyl-d-glucosamine (glcnac) residues present in human pathogenic fungi or nematodes. chitotriosidases (cht) and acetic mammalian chitinase (amcase) have been identified as the only functional chitinases in mammalians. the expression of both chitinases appears to be strongly species dependent, indicating distinct physiological functions. amcase is considered as predominant chitinases in mice while cht is regarded as major chitinases in humans. interestingly, cht is constitutively expressed by human phagocytes at high levels while it is absent in mice phagocytes. although, amcase received increased attention as modulator of the innate immune response against chitin in mice, the physiological function of cht in humans is virtually unknown. to evaluate the physiological function of cht we have characterised the substrate specificity of human cht and the mode of substrate cleavage by analysing chtproduced fragments of chitosan, a close but water soluble derivate of chitin. degradation products of chitosan have been investigated by gel electrophoresis and maldi-tof mass spectroscopy. moreover, the application of a computer-based model of cht activity revealed the mode of substrate cleavage. we found that cht is a processive endo-cleaving chitinase resulting in the production of only small diffusible chitin/chitosan fragments. in further studies we could show that those cht-produced small chitin/chitosan fragments exhibit a strong ability to induce a pro-inflammatory response in human blood derived monocytes/macrophages as indicated by an increased release of the pro-inflammatory cytokines tnf-a, il-6, il-8 and mcp-1 involving the transcription factor nfxb. moreover, these stimulated monocytes/macrophages revealed an increase of cht expression indicating an autocrine positive feed-back regulation. our data suggest that human cht is involved in the early recognition of chitin/chitosan containing human pathogens due to the generation of immuno-stimulatory chitin/chitosan fragments. m. hasenberg 1 , s. wolke 2 , a. brakhage 2 , m. gunzer 1 1 otto-von-guericke universität, institut für molekulare und klinische immunologie, magdeburg, germany, 2 hans-knöll-institut, abteilung für molekulare und angewandte mikrobiologie, jena, germany since their discovery in 2004 nucleic extracellular traps (nets) released by certain cell types including neutrophil and eosinophil granulocytes were shown to play a crucial role in mediating innate immune responses towards different bacterial und fungal pathogens. recently it was found by us and others that neutrophil granulocytes release nets also upon contact to the filamentous fungus aspergillus fumigatus. in the present study we aimed to characterize this process in more detail focusing on the kinetics of net-formation as well as clarifying the responsible cell-biological mechanisms. by the use of several microscopic techniques (scanning electron microscopy, fluorescence widefield microscopy, confocal-and 2-photon microscopy) we initially demonstrated the generation of net like structures after coincubation of a. fumigatus germlings and freshly isolated murine or human pmn. the analysis of our time lapse video microscopy data allowed us to examine the exact time course from initial contact to the fungal surface to explosive release of nets up to 3 hours later. moreover, we investigated the dependency of this phenomenon on the induction of an oxidative burst. therefore we added the nadph-oxidase inhibitor dpi to the cell coincubation and found clearly reduced net formation. by fluorescence staining of reactive oxygen species we could demonstrate that ros are released prior to net detection. interestingly, our data currently suggest that in contrast to other pathogens investigated so far, nets are not directly toxic to fungal elements. whether and how nets control growth of a. fumigatus currently remains open. to summarize our data, we found rapid net formation as a commonly observed immune response of neutrophil granulocytes contacting a. fumigatus. consistent with studies on different pathogens this mechanism seems to be ros-dependent, however not toxic for the fungus. thus, in the future we will have to clarify whether net-formation really occurs in vivo and how nets can control the outgrowth of a. fumigatus at sites of infection. production of type i interferons (ifn-i, mainly ifn-a and ifn-b) is a hallmark of innate immune responses to all classes of pathogens. when viral infection spreads to lymphoid organs, the majority of systemic ifn-i is produced by a specialized 'interferon-producing cell' (ipc) that has been shown to belong to the lineage of plasmacytoid dendritic cells (pdc). it is unclear whether production of systemic ifn-i is generally attributable to pdc irrespective of the nature of the infecting pathogen. we have addressed this question by studying infections of mice with the intracellular bacterium listeria monocytogenes. protective innate immunity against this pathogen is weakened by ifn-i activity. in mice infected with l. monocytogenes systemic ifn-i was amplified via ifn-b, the ifn-i receptor (ifnar) and transcription factor interferon regulatory factor 7 (irf7), a molecular circuitry usually characterisitic of non-pdc producers. synthesis of serum ifn-i did not require tlr9. in contrast, in vitro differentiated pdc infected with l. monocytogenes needed tlr9 to transcribe ifn-i mrna. consistent with the assumption that pdc are not the producers of systemic ifn-i, conditional ablation of the ifn-i receptor in mice showed that most systemic ifn-i is produced by myeloid cells. furthermore, results obtained with facs-purified splenic cell populations from infected mice confirmed the assumption that a cell type with surface antigens characteristic of macrophages and not of pdc is responsible for bulk ifn-i synthesis. the amount of ifn-i produced in the investigated mouse lines was inversely correlated to the resistance to lethal infection. based on these data we propose that the engagement of pdc, the mode of ifn-i mobilization, as well as the shaping of the antimicrobial innate immune response by ifn-i differ between intracellular pathogens. t. naessens 1 , s. vander beken 1 , p. bogaert 1 , j. grooten 1 1 ghent university, biomedical molecular biology, zwijnaarde (ghent), belgium introduction: although the effector and modulator functions of activated macrophages in innate and adaptive immunity are well documented, their exact role in the initiation and propagation of immune pathologies is still not fully understood. recent insights in monocyte and macrophage heterogeneity render the picture even more complex. in addition, it is unclear to what extend resident and elicited macrophages differ functionally and hereby differentially contribute to immune pathologies. in this study we focused on the dynamics and function of resident alveolar macrophages (ram) during and after allergic bronchial inflammation. strategy: we used an ovalbumin (ova)-alum based mouse model of allergic asthma and an ova-complete freund's adjuvant (cfa) based mouse model of hypersensitivity pneumonitis, constituting a th1-driven immunological counterpart of the th2-driven experimental asthma. ram were distinguished by prior in situ labelling with fluorescent polystyrene microspheres. as complementary approach, ram and elicited alveolar macrophages (eam) were distinguished using cd45 bone marrow chimeric mice. combined with flow cytometry and fluorescence activated cell sorting, both approaches allowed us to trace resident and elicited am populations in the course of th1-and th2-driven allergic airway inflammation. results: during the acute phase of the allergic response, isolated ram and eam showed distinct gene expression signatures, reflecting a possible functional heterogeneity between these two macrophage subsets. in both types of allergic inflammation, microsphere-tagged cd45.1 + ram remained constant in cell number for the first 2 days of chronic ova-exposure and then dropped sharply, having nearly completely disappeared from the alveoli by day 4 of ova-exposure. as a consequence, following the clearance of inflammation, inflammation-experienced ram replaced the initial ram population. strikingly, in both types of allergic inflammation, this secondary ram population showed a markedly altered responsiveness to lps stimulation. this involved macrophage activation markers and nf-kb inducible inflammatory genes. however, especially genes induced by ifn-beta showed strongly increased expression in secondary ram as opposed to their near lack of induction in primary ram. this switch from an ifn-beta deficient to an ifn-beta adequate phenotype may increase the inflammatory sensitivity of allergic inflammationexperienced lungs as also observed in asthmatic patients, showing an increased sensitivity to bacterial infection. e. schlecker 1 , a. stojanovic 1 , a. cerwenka 1 1 german cancer research center, innate immunity, heidelberg, germany myeloid-derived suppressor cells (mdsc) are a heterogeneous population of cells that expand during cancer, inflammation and infection. these cells play a critical role in suppressing t cell responses. the exact nature and function of mdsc remain unclear. here we show that a subpopulation of mdsc (gr-1 + cd11b + f4/80 + ) isolated from rma-s tumor-bearing mice did not suppress but rather activated nk cells to produce ifn-g. additionally, nk cells eliminated this subpopulation both in vitro and in vivo. in order to identify molecules and pathways that might be involved in mdsc accumulation in tumor bearing mice and their suppressive/activatory function, gene expression profiling of mdsc subpopulations was performed using whole genome microarrays. understanding the reciprocal interaction of mdsc with nk cell could improve the efficiency of cancer immunotherapy. g. solinas 1 , f. marchesi 1 , m. fabbri 1 , s. schiarea 2 , c. chiabrando 2 , a. mantovani 1,3 , p. allavena 1 1 istituto clinico humanitas, rozzano, italy, 2 istituto mario negri, milano, italy, 3 università di milano, milano, italy experimental and clinical evidence has highlighted that tumor-associated macrophages (tam) represent the principal component of the leukocyte infiltrate and are usually associated with tumour growth, progression and metastasis. macrophage population is generally divided into two distinct subsets: m1 and m2. m1 macrophages act as a first line of defence against pathogens whereas m2 cells participate in wound repair and maintenance of tissue integrity. in the tumour micro-environment tam interactions with the extracellular matrix, neighboring cells, and soluble stimuli largely influence their gene expression and behavior. to investigate the role of the tumor micro-environment on macrophage differentiation, we cultured freshly isolated human monocytes with pancreatic cancer cell line supernatants, in the absence of exogenous cytokine addition.. in selected cultures, about 50 % of the monocytes differentiated after 5 days into macrophages. the phenotype analysis of tumor-conditioned macrophages (tc-macro) demonstrated high expression of the mannose receptor, cd16, cd68 and low levels of mhc class ii. tc-macro produced il-10, il-6, tnf but not il-12, even after lps stimulation. moreover, tc-macro produced a panel of chemokines including ccl2, cxcl8, ccl17 and cxcl10. the transcriptional profile of tc-macro revealed that several genes in line with an m2 polarization are highly expressed. the nature of the tumor-derived factors inducing macrophage differentiation is currently under investigation; biochemical analysis indicated that the biological activity is excluded from exosomes and have a high molecular weight ( g 100.000 kda). il-3 and il-6 were not detectable in tumor supernatants whereas m-csf was present at low levels. by mass spectrometric techniques, we surprisingly found that the tumor-derived m-csf had peculiar migration patterns which were different from those expected for the common human homodimeric glycosilated protein, suggesting an interesting structural differences for the tumor-secreted isoforms of this primary regulator of mononuclear phagocyte. the characterization of tumor-derived factors inducing macrophage differentiation could better clarify the intricate cross-talk between tumor cells and macrophages and thus might aid in the process of devising novel anti-tumor treatments. genomic effects of glucocorticoid hormone (gc) are exerted by glucocorticoid receptor (gr)-mediated changes of gene expression. this is relatively timeconsuming process, needing hours to develop. in contrast, non-genomic effects may occur within minutes. gcs are used for a long time for the therapy of anaphylactic reactions, where mast cells play crucial role. moreover, many cells and cell lines of haemopoetic origin are sensitive to gc-induced apoptosis. recent findings indicate, that non-genomic gc effects mediated by mitochondrial gr may have important function in generating pro-apoptotic signals. we aimed the investigation of non-genomic gc effects on in vitro cultured rbl-2h3 rat mast cell line. we demonstrate that gr nuclear translocation begins within 5 minutes and completes after 30 minutes in dx treated rbl-2h3 cells. since genomic effects occur in the nucleus through gene expression changes, we considered gc effects within 5 minutes as non-genomic. studying gc-caused apoptosis, rbl-2h3 cells proved to be gc-resistant and no mitochondrial gr translocation neither impaired mitochondrial function could be observed upon gc treatment. in further experiments we used rbl-2h3 cells sensitized with anti-dnp (dinitrophenyl) ige and dnp-conjugated bovine serum albumin was used for stimulation. 5 minutes of dx treatment inhibited ca 2+ -signaling in antigen stimulated rbl-2h3 cells in the concentration range of 100nm -10mm. moreover, 5 minutes of dx treatment altered the tyrosine phosphorylation pattern of rbl-2h3 cells. dx treatment alone caused slight increase in tyrosine phosphorylation, while dx treatment of activated cells caused also an increase in tyrosine phosphorylation compared to the solvent-treated controls. the tyrosine kinase syk plays indispensable role in regulating mast cell activation through the fc[epsilon] receptor i. our immunoprecipitation studies show, that dx treatment results in decreased syk phosphorylation in both resting and activated cells. this finding raises the possibility, that syk phosphorylation thus kinase activity may be directly or indirectly regulated by gcs via non-genomic pathway. taken together, our experiments along with the clinical experiences suggest that gcs rapidly influence mast cell activation via a non-genomic pathway, too. the elucidation of the exact signal transduction mechanisms behind rapid gc effects need further experiments. high mobility group box 1 (hmgb1) is a non-histone nuclear protein that binds chromatin and has transcriptional and architectural functions. notably, hmgb1 is highly mobile in the nucleus and is passively released by necrotic cells, while it is bound firmly to apoptotic chromatin (1) . extracellular hmgb1 can act as a cytokine and a chemoattractant, mediating inflammatory responses. interestingly, hmgb1 exerts antibacterial functions in human adenoid and testis (2) . recent investigations have revealed that neutrophils eliminate microbes not only by intracellular phagocytosis but also by trapping them in three-dimensional structures called neutrophil extracelluar traps (nets), made of dna fibers, nuclear proteins (histones) and granule proteins. it has been shown that histones on nets have an anti-microbial activity (3). we asked whether hmgb1 from neutrophils is a component of nets and whether it has a function in nets. we purified human primary neutrophils from peripheral blood of healthy volunteers on ficoll gradients. to induce net formation, we stimulated cells for 40 or 120 minutes with 25 nm phorbol ester (pma), 100 ng/ml interleukin 8 (il-8), or 100 ng/ml lps. the presence of nets was assessed by immunofluorescence using antibodies directed against the granule protein myeloperoxidase (mpo) and against a dna-histone h2a-histone h2b complex. dna was stained with hoechst. using a polyclonal antibody we found hmgb1 in the euchromatin of polylobulated nuclei of resting neutrophils and on the filamentous structure of nets induced by all stimuli. elisa assays revealed that hmgb1 is not present in the supernatants of activated neutrophils, confirming its binding to nets. in conclusion, we found that hmgb1 localizes on nets. we hypothesize that net-bound hmgb1 might exert a direct antimicrobial function, or that nets might concentrate hmgb1 locally to recruit macrophages to the site of infection. these receptors were present on the mast cell surface. incubation (37°c, 3 h) of hlmc with vegf-a, vegf-b, vegf-c, vegf-d and placental growth factor-1 induced concentration-dependent chemotaxis that was blocked by a combination of anti-vegfr-1 and anti-vegfr-2 antibodies. these data indicate that human mast cells represent both a source and a target of vegfs and therefore may play a role in inflammatory and neoplastic angiogenesis through the expression of proangiogenic factors and their receptors. macrophages are important effector cells in immunity to intracellular pathogens and at the same time are exploited as host cells by a number of microorganisms such as mycobacterium tuberculosis. a very important mechanism of intracellular killing is delivery of invading microbes to phagolysosomes. whilst mycobacteria can block phagosome maturation in resting macrophages, and hence survive and replicate inside the host cell, the ifn-g activated macrophage utilizes a diversity of defense mechanisms to eliminate the invader. these include putative killing by antibacterial peptides/proteins and overcoming phagosome maturation block, possibly by induction of autophagy, production of reactive nitrogen or oxygen intermediates and deprivation from nutrients such as iron. mycobacteria are not eliminated even upon onset of protective immunity rather leading to persistence. we hypothesize that the very early steps of pulmonary infection directs the outcome of disease. therefore, we investigate initially infected lung cells and their role in infection in the lung with respect to their anti-microbial mechanisms against mycobacteria in vitro as well as in vivo. preliminary data show that m. tuberculosis is able to persist in the alveolar space for several weeks and bacterial numbers do barely drop even after very low dose infection, indication that bacterial killing is inefficient from the very beginning. cells harboring mycobacteria are found during early and late stages of infection. both, autophagy and nitric oxide production seems to contribute to growth restriction of mycobacteria by macrophages. neutrophils, although recruited in vast numbers to infected lungs, are not able to reduce bacterial numbers in the absence of il-18. altogether, the initial response in the barrier organ lung executed by resident and immigrating cells restricted by the local environment can determine the outcome of infection. human cd1 molecules are dedicated to lipid presentation to t cells and are implicated in inflammatory and auto-immune responses. the cd1a protein is almost exclusively expressed at the cell surface of dendritic cells and is dedicated in surveying extracellular environment. our previous studies have demonstrated that ii associated with cd1a and cholesterol-dependent lipid rafts impact on cd1a surface expression and cd1a-restricted t cell response. bacterial infections can induce an increase in self glycolipid synthesis in dendritic cells and such activated dcs acquire the ability to stimulate cd1-restricted autoreactive t cells. this mechanism of self recognition induced by bacterial infection is believed to be involved in the development of auto-immune disorders. sulfatide, which is a major component of the myelin sheath, is also the only known self-antigen presented by cd1 group i molecules. the functional role of these molecules has not been investigated in auto-immune diseases and we propose that regulation of glycolipid presentation by cd1a molecules could impact in such pathologies. we have thus conducted a preliminary study to understand the implication of cd1 molecules in multiple sclerosis. we first analyzed cd1 expression on monocytes from 16 ms patients and the influence of sera and plasma from these patients on dendritic cell differentiation from healthy donors. results obtained in this preliminary study demonstrate that cd1a was not expressed on ms patient monocytes, while the other members of the cd1 family were expressed. moreover ms sera and plasma induced an earlier and more rapid dendritic cell differentiation than ab sera. these preliminary results confirm our hypothesis that cd1 molecule expression is modified in ms and also reveal that serum from patients with ms modifies lipid-antigen presenting cells. further studies should contribute to define precise mechanisms involved in lipid presentation by cd1 molecules in this context. c. ohnmacht 1 , d. voehringer 1 1 ludwig-maximilians-universität munich, institute for immunology, munich, germany basophils are effector cells of the innate immune system which are associated with allergic inflammation and infections with helminth parasites. however, their development and in vivo functions are largely unknown. here, we characterize basophil turnover, tissue localization and effector functions during infection with the gastrointestinal helminth nippostrongylus brasiliensis. for this purpose, brdu incorporation experiments and in situ fluorescence microscopy of il-4 reporter (4get) mice as well as in vivo depletion of basophils are used to uncover their role during type 2 immune responses. our results demonstrate that under homeostatic conditions basophils have a lifespan of about 60h. n. brasiliensis induced basophilia is caused by increased de novo production of basophils in the bone marrow. basophils are found near the marginal zone in the red pulp of the spleen, in the lamina propria of the small intestine and in the lung parenchyma. activated basophils promote systemic eosinophilia, were associated with differentiation of alternatively activated macrophages in the lung and contributed to efficient worm expulsion of n. brasiliensis in the absence of th2 cells. these results demonstrate that basophils play a crucial role as effector cells in type 2 immune responses which might hold great potential for the treatment of helminth infections and allergic diseases. during acute bacterial infections such as meningitis, neutrophils enter the tissue where they combat the infection before they undergo apoptosis and are taken up by macrophages. neutrophils show pro-inflammatory activity and may contribute to tissue damage. in pneumococcal meningitis, neuronal damage despite adequate chemotherapy is a frequent clinical finding. this damage may be due to excessive neutrophil activity. we here show that transgenic expression of bcl-2 in haematopoietic cells blocks the resolution of inflammation following antibiotic therapy in a mouse model of pneumococcal meningitis. the persistence of neutrophil brain infiltrates was accompanied by high levels of il-1beta and g-csf as well as reduced levels of anti-inflammatory tgf-beta. significantly, bcl-2-transgenic mice developed more severe disease that was dependent on neutrophils, characterized by pronounced vasogenic edema, vasculitis, brain haemorrhages and higher clinical scores. in vitro analysis of neutrophils demonstrated that apoptosis inhibition completely preserves neutrophil effector function and prevents internalization by macrophages. the inhibitor of cyclin-dependent kinases, roscovitine induced apoptosis in neutrophils in vitro and in vivo. in wild type mice treated with antibiotics, roscovitine significantly improved the resolution of the inflammation after pneumococcal infection and accelerated recovery. these results indicate that apoptosis is essential to turn off activated neutrophils and show that inflammatory activity and disease severity in a pyogenic infection can be modulated by targeting the apoptotic pathway in neutrophils. objectives: to investigate the existence of systemic inflammatory response to subchronic oral warfarin (wf) consumation in rats. methods: dark agouti (da) rats were treated with warfarin in drinking water (10 mg and 100 mg daily) for 30 days. oxidative activity (cytochemical nbt reduction) and myeloperoxidase (mpo) intracellular content of peripheral blood neutrophils, plasma levels of il-6 and tnf-a (elisa) and superoxide dismutase (sod) activity (red blood cell lysates) were analyzed as inflammatory parameters in rats following warfarin consumation. changes in prothrombin time (pt), as basic biological warfarin activity was determined as well. results: significantly increased pt was noted at the lower wf dose, with tremendous rise after the higher dose. increase of pma-stimulated neutrophil nbt reduction capacity (neutrophil priming) was noted at both wf doses, while increase in mpo intracellular content was noted at the higher wf dose solely. warfarin consumation resulted in no changes in plasma levels of il-6 and tnf-a. significant decrease in the sod activity was detected in red blood cell lysates at both wf doses, suggesting systemic oxidative activity. conclusion: increased neutrophil priming as well as prooxidant activity in peripheral blood of rats following subchronic warfarin consumation imply proinflammatory effects of oral warfarin administration. absence of the rise in inflammatory cytokines in circulation, suggest low-grade inflammation in these rats. this work is funded by serbian ministry of science and technological development (grant 143038). objectives: although many different macrophage receptors and serum proteins have been shown to play a role in phagocytosis of apoptotic cells, the unique microenvironment of an inflammatory site will have considerable influence upon the molecular pathways which are utilized in apoptotic cell removal. we have recently reported that immune complexes (ic) are able to specifically bind to the surface of human apoptotic neutrophils which may have profound implications for their physiological clearance. in disease situations where immune complexes are present neutrophils undergoing apoptosis would be predicted to become coated with ic. here we address the consequences of ic opsonisation of apoptotic cells upon phagocytosis and cytokine response by macrophages that would be expected to be present at the earliest stages of inflammatory responses (type-1 macrophages, mph1), and during resolution of inflammation (type-2 macrophages, mph2). methods: mph1 / 2 were generated by culturing cd14 + human monocytes for 6 days in the presence of gm-csf or m-csf, respectively. phagocytosis by mph1 / 2 of ic opsonised and unopsonised neutrophils was assessed by flow cytometry. after phagocytosis mph1 / 2 were stimulated with lps and secreted il-6, il-8, il-10, il-12p40 and tnf were quantified by elisa. results: mph2 are relatively efficient phagocytes for apoptotic neutrophils whereas mph1 are only poorly phagocytic. opsonisation with ic leads to enhanced neutrophil uptake by both mph1 and mph2 which is specifically inhibited in the presence of a blocking mab for macrophage fcyrii. uptake of ic opsonised neutrophils causes a shift towards an anti-inflammatory cytokine profile. in both macrophage subsets il-6, il-12 and tnf production is suppressed while il-10 secretion is increased. in contrast, engagement of macrophage fcyr with ic alone induces the release of pro-inflammatory cytokines. conclusion: our data demonstrate that ic opsonisation of apoptotic neutrophils increases the proportion of macrophages capable of phagocytosis and that apoptotic cell recognition interactions provide a dominant anti-inflammatory signal, suppressing macrophage responses, even in the presence of ic opsonisation. we suggest that ic present in the inflammatory milieu would opsonise apoptotic neutrophils, enhance macrophage phagocytosis and thereby facilitate the process of resolution of inflammation. excessive production of reactive oxygen species (ros) produced by neutrophils is known to be a factor accelerating ageing because of damaging effect on cells. on the other hand, intracellular heat shock proteins (hsp) are involved in protecting cells from the damaging effects, and provide cell resistance to stress. in this work, correlation analysis was applied to analyze relationship between ros production and intracellular hsp70 in neutrophils of elderly people. neutrophils were isolated from peripheral blood of donors of 90 years old and older (long-livers). intracellular ros and hsp70 levels were registered by flow cytofluorimetry upon labeling with 2',7'-dichlorofluorescin diacetate (invitrogen) and anti-hsp70 antibody (brm-22, sigma), respectively. intracellular level of hsp70 was also estimated in neutrophils after heat shock (hs) performed at 43°c for 10 min. extracellular ros production from zymosan-activated neutrophils was detected by luminol-dependent chemiluminescence. a positive correlation was determined for intracellular ros level and zymosan-mediated extracellular ros release although the dynamics of ros release at 1-15 min time range varied within the group. the correlation was unaffected by hs of neutrophils performed for 1 min at 42°c, although this short heat treatment decreased significantly ros release. there was no correlation between basal intracellular hsp70 (hsp70 basal ) and ros level, both intracellular and extracellular. at the same time increased hsp70 level immediately after hs (hsp70 (0 min)) correlated negatively with intracellular ros (initial and after hs). the hsp70 increase value (hsp70 (0 min) -hsp70 basal ) correlated negatively also with intracellular ros and extracellular ros release in response to zymosan; and the correlation with ros level became lower when hsp70 increase was registered in 60 min after hs (hsp70 (60 min) -hsp70 basal ). thus it was found that within this age group the alteration in hsp70 induced by hs in neutrophils but not basal hsp70 itself is the parameter associated negatively with both spontaneous ros level and ros production in response to activating action of zymosan. this work is supported by istc grant #3303. d. goyeneche-patiño 1 , z. orinska 1 , f. mirghomizadeh 1 , s. bulfone-paus 1 1 forschungszentrum borstel, borstel, germany several studies have shown different roles of mast cells (mc) in innate and adaptative immune responses. in fact, crosstalk between cd8 + t cells and mc has shown to induce multiple genes implicated in the signaling of specific programs such as type 1 ifn. two novel genes, receptor transporter protein (rtp4) and virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible (viperin) are ifn inducible and were found to be over-expressed in chip analysis. the aim of this study is to characterize the expression and protein production of rtp4 and viperin in mast cells after tlr ligand stimulation in mice lacking of ifnra and the adapter proteins myd88 and trif. bone marrow derived mast cells (bmmc) from wt, ifnra -/-, mydd8 -/and trif -/mice, were exposed to tlr ligands (lps, pic, cpg, p(da-dt) and new castle disease virus (ndv)) during 8 and 48 h. mrna and protein extraction were performed for further qrt-pcr and sds page analysis for rtp4 and viperin. intracellular stimulation of tlr was performed transfecting cells with nucleic acids using lipofectamine 2000. stimulation of wt cells with pic, pda-dt and ndv showed an increased expression of viperin and rtp4 in comparison to control cells (untreated). the same trend was observed for mc from the trif and myd88 knockout mice. in contrast, in the ifnra deficient mice, expression of genes and protein production was abrogated to the same levels of wt untreated cells. lipofectamine stimulation does not increase the expression/production of the genes. direct stimulation of the well recognized viral sensors tlr 3 and 9, as well as, infection of mast cells with ndv (rna virus) induce the expression of rtp4 and viperin. the findings suggest that activation of mc with the ulterior expression of genes is type i ifn dependent. in contrast, the adaptor proteins myd88 and trif and the pathways that they represent are not relevant in the expression of rtp4 and viperin. these findings provide bases for performing further studies focused to elucidate the functions of these proteins and show an alternative role of mc in innate immune responses. in recent years, it has been suggested that the phenomenon of "myc-dependent cell competition" described in drosophila melanogaster, could be a critical step when a cell initiates nascent tumour field. we have taken a step forward and applied the phenomenon of cellular competition to the human macrophage system: inflammatory macrophages theoretically have the ability to eradicate cancer due to their tumoricidal capability and, at the same time, acting as antigen-presenting cells (apcs) to activate lymphocytes; but inflammatory macrophages do not express c-myc and, within the tumour, they encounter two powerful rivals: tumoral cells and alternative tumour-associated macrophages which express c-myc. we studied some phenomenons suggested to be myc-dependent such as the ability to feed, the ability to survive in a competitive medium, the ability to proliferate and the ability to eliminate competitors and we observed that alternative macrophages have more resources to survive in a tumoral microenvironment and could be involved in tumour growth by collaborating with tumour cells in transforming inflammatory macrophages into anergic cells which enter into apoptosis and are then phagocyted. finally, using lentiviral vectors, we over-expressed exogenous c-myc in inflammatory macrophages in an attempt to increase their chances of survival in the tumour microenvironment, in vitro and in vivo, and to determine whether it can be utilized as a potential anti-tumoral cell therapy. g. germano 1 , e. erba 2 , r. frapolli 2 , m. d'incalci 2 , a. anselmo 1 , s. pesce 1 , p. allavena 1 , a. mantovani 1, 3 1 humanitas clinical institute, rozzano, italy, 2 mario negri institute, milan, italy, 3 institute of general pathology, university of milan, milan, italy several lines of evidence suggest a strong association between chronic inflammation and tumor progression; therefore the use of anti-inflammatory drugs may be beneficial in anti-tumor therapies. inflammatory mediators (e. g. cytokines, chemokines) are produced at the tumor site both by tumor-associated macrophages (tam) as well as tumor cells, and are attractive target of novel anti-tumor therapies. trabectedin (et-743) is a natural product derived from the marine tunicate ectenascidia turbinate, it binds the minor groove of dna, affects transcriptional factor activity and blocks cell cycle. this novel anti-tumor agent is currently used in phase ii studies in patients with sarcoma, ovarian and breast cancer, with clinical regressions. we previously demonstrated that trabectedin is selectively cytotoxic, in vitro, to monocytes/macrophages, being active at concentrations that spared lymphocytes suggesting a possible alternative target for the anti-tumoral role of this drug. we tested the effect of trabectedin on primary cultures and liposarcoma cell lines showing that at sub-cytotoxic concentrations the production of some inflammatory mediators were down-modulated. trabectedin significantly reduces ccl2, cxcl8 and the inflammatory protein pentraxin3 (ptx3) either at transcriptional and protein level, especially after tnfa/il1b stimulation. down-regulation of ccl2, cxcl8, ptx3 and also of il6 and vegf were confirmed in primary cultures of liposarcoma. according to the previous in vitro data we now show in a mouse model , using the fibrosarcoma mnmcai , that trabectedin treatment selectively affects monocytes in the blood and bone marrow. moreover trabectedin treatment strongly reduces the number of macrophages and of cd31+ vessels in the tumor microenvironment, in line with its selective activity on monocytes/macrophages. overall these results suggest a possible triple role of trabectedin. besides its direct cytotoxic effect on tumor cells, trabectedin also affects tumor associated macrophages and at low dose the transcriptional activity of inflammatory genes involved in the tumor-microenvironment cross-talk. as the local expression of inflammatory mediators may play a role in tumor progression, this newly recognized effect of trabectedin makes it an attractive candidate in inflammation-associated tumors. interleukin-4 (il-4) is a key cytokine of the t helper 2 cell response. il-4 has been found to be a major regulator of immunoglobulin class switching to ige and has important functions in the regulation of allergic diseases. here, the onset of the il-4 production after birth was investigated in equine neonates. the form of equine placentation does not support the transfer of cytokines or immunoglobulins in utero and maternal immunity is exclusively transferred to the neonate with the colostrum after birth. il-4 producing cells were measured in peripheral blood mononuclear cells (pbmc) of neonates and foals by flow cytometric analysis. at day 3-6 after birth, a small population of il-4 producing cells was observed in the absence of any stimuli. the il-4 + population was not detectable at 6 or 12 weeks of age. other cytokine producing cells (ifn-g, il-10) were not detected using these conditions. the stimulation of neonatal pbmc with pma and ionomycin did not alter the il-4 + cell population. phenotyping of the neonatal il-4 + cells showed that they were ige + /mhcii -/cd4cells. the occurrence of cd4 + il-4 producing cells after pma stimulation increased slowly with age and did not reach adult levels by 12 weeks after birth. magnetic cell sorting of the ige + /mhciicells identified them as basophils. previous work has shown that foals do not produce endogenous ige for at least six months of life. ige bound to the surface of neonatal basophils was found to be of maternal origin and transferred with the colostrum after birth. here, the stimulation of neonatal pbmc with anti-ige induced the secretion of il-4 at day 5 after birth. neonatal pbmc collected before colostrum uptake did not produce il-4 in response to anti-ige. in summary, equine neonates provide a model to investigate ige mediated il-4 responses after birth. the transfer of maternal ige from allergic individuals could potentially provide a direct mechanism for the early induction of an allergen-specific neonatal il-4 response mediated by the mare's accumulated acquired immunity to allergens. s. schmechel 1 , d. voehringer 1 1 ludwig-maximilians-university munich, institute for immunology, munich, germany macrophages display broad phenotypic heterogeneity depending on their microenvironment. the initial inflammatory response to th1 cytokines is predominantly mediated by classically activated macrophages whereas macrophages undergo alternative activation in a stat6-dependent manner when stimulated with the th2 cytokines il-4 or il-13. alternatively activated macrophages (aam) are implicated in diverse disease pathologies such as host response to parasitic infection and asthma. furthermore, it has been shown that aam suppress the proliferation of t cells by a yet to be determined mechanism. currently there is still very limited information about the phenotype, migration and function of aam. we began to elucidate whether macrophage turnover and recruitment to inflammatory sites is regulated in a stat6-dependent manner. to this end we generated mixed bone marrow chimeras with bone marrow from congenic wild-type and stat6-deficient mice and infected these chimeras with the helminth nippostrongylus brasiliensis to determine whether lack of stat6 in macrophages affects their turnover and recruitment to the lung side-by-side in the same animal. the highest turnover of macrophages was found in the peritoneum, irrespective of stat6 expression. no major differences in tissue distribution and turnover were observed between both populations suggesting that macrophage proliferation and recruitment during parasite infection is not dependent on stat6 expression in macrophages. we could further confirm that in vitro generated aam from wild-type but not from stat6-deficient mice have a strong inhibitory effect on t cell proliferation. we are now trying to identify the mechanism(s) by which t cell proliferation is inhibited. furthermore, we work on the cellular cross-talk between eosinophils and macrophages and try to determine the plasticity of macrophage differentiation. we have previously shown that aam can recruit eosinophils to inflammatory sites and we now try to clarify which chemotactic factor are involved in this process. to identify potential aam-derived eosinophil chemotactic factors we currently compare the gene expression profile of il-4 exposed macrophages from wild-type and stat6-deficient mice. candidate genes will be expressed using retroviral transfections of stat6-deficient macrophages and supernatants from these cells will then be used to induce eosinophil recruitment in transwell assays. macrophages are an essential component of leukocytes infiltration in the tumor. they are identified as tumor associated macrophages (tams). these cells are also present in pleural effusions which appear as a consequence of spreading of neoplasm in the pleural cavity. the aim of the study was to assess the influence of the pleural macrophages on cells from human malignant cell lines. we tested the dynamics of growth of the malignant cells, their apoptosis and expression of proteins regulating this process under the influence of conditioned media from cultures of macrophages isolated from pleural effusion. we have also attempted to interpret our results by assessing the expression of a variety of immune modulating factors, their receptors on the malignant cells surface as well as the transcription factors. in the study we used macrophages isolated from a total of 38 pleural effusions, including 15 malignant and 23 nonmalignant tumors. the following human malignant cell lines were tested: a549, ht29, hct116, sw60, mcf7, mda-mb231, jurkat and hl60. results: our results suggest that the conditioned media isolated from the cultures of pleural macrophages can up-regulate the proliferative activity of the human malignant cell lines. macrophages from pleural effusions can act as a factor promoting or inhibiting apoptosis of malignant cells. down-regulation of apoptosis may depend on modulation of expression and activity of proteins regulating this process. macrophages can affect the apoptosis regulatory proteins and their activity through the immune-modulatory molecules, e. g. cytokines, chemokines, and growth factors. the up-or down-regulation of transcription factors expression may control the expression of pro-and anti-apoptotic proteins. the results indicate that macrophages from malignant and non-malignant pleural effusions differ from each other insignificantly; however the macrophages isolated from the non-malignant tumors show a pattern comparable to m1, and the tams isolated from the malignant effusions similar to m2. among the alternative stimuli, glucocorticoids are the most effective stimulus up-regulating ms4a4a and ms4a6a: highest trascriptional level after 18h of stimulation with 10-6m dexamethasone. ms4a murine genes are differently expressed respect to the human counterpart and only the homologs of ms4a6a (ms4a6b, 6c and 6d) have a similar regulation. finally, egfp-tagged ms4a4a, ms4a6a, and ms4a7 expressed in cho cells showed that all molecules traffic to the cell membrane. though the biological functions of these ms4a proteins has not jet been defined, their membrane localization and the structural relationship with other better characterized ms4a members suggest a potential involvement in signal transduction, either as components of multimeric receptor complexes or as components of ligand-gated ion channels. during inflammatory reactions endogenously produced cytokines and chemokines act in a network and interact with hormones and neurotransmittors to regulate host immune responses. these signaling circuitries are even more interfaced during infections in which microbial agonists activate toll-like (tlr), rig-like (rlr) and nod-like (nlr) receptors. on the basis of the discovery of synergy between chemokines for neutrophil attraction, we here extended this phenomenon between the chemokine monocyte chemotactic protein-1 (mcp-1)/ccl2 and the gpcr ligand fmlp or the tlr4 agonist lipopolysaccharide (lps) on monocytes. in fact, the bacterial tripeptide fmlp, but not the cytokines il-1b or ifn-g, significantly and dose-dependently synergized with ccl2 in monocyte chemotaxis. furthermore, lps rapidly induced the expression of interleukin-8/cxcl8, but not of the ccl2 receptor ccr2 in monocytic cells. in turn, the induced cxcl8 synergized with ccl2 for mononuclear cell chemotaxis and the chemotactic effect was mediated by cxcr1/cxcr2, because cxcl8 receptor antagonists or antibodies were capable of blocking the synergy, while keeping the responsiveness to ccl2 intact. these data recapitulate in vitro the complexity of innate immune regulation, provide a novel mechanism of enhancing monocyte chemotaxis during bacterial infections with gram-negative bacteria and demonstrate the importance of local contexts in inflammatory and infectious insults. objectives: in recent years the existence and effects of cell-derived vesicles (e. g. exosomes, microparticles) have been revealed in several physiological functions, such as antigen presentation, hemostasis or receptor transfer to innocent cells. most data were collected on endothelial cells and on thrombocytes. however, there are only few data on vesicles derived of neutrophilic granulocytes (pmn), and most of these investigations applied only pharmacological agents. our aim is to investigate pmn-derived cell-free particles and their possible role in bacterial killing methods: preparation of pmn and investigation of bacterial killing by our semi-automatic method was described by rada et al. (blood, 2004) . cell-free vesicles were prepared after co-incubation of human pmns with different activating agents for 20 min at 37°c with gentle shaking, followed by spinning down of pmns for 5 min, at 4°c and 500g. the supernatant was sedimented at 15000g for 10 min, 4°c, and we used the sedimented fraction for our investigations. formation of particles was followed by fluorescent and electron microscopic assays. the amount of particles was estimated with flow cytometer and by their protein content. we observed that upon co-incubation of pmns with s. aureus, opsonized by mixed human serum, pmns produce a well detectable amount of vesicles. omitting opsonization or opsonizing with heat inactivated serum caused a minimal amount of particles. production of particles could be inhibited with diphenyl-iodonium (dpi), cytochalasin b (cb) or with azide treatment. treating pmns with dnase or withdrawing glucose during co-incubation had no effect on vesicle formation. in killing assays we detected remarkable antibacterial effect, which correlated well with the protein content of the used fraction. this antibacterial activity could be inhibited by dpi, cb, azide treatment or by withdrawing glucose from the medium during the killing assay. however, treatment of the microvesicles with dnase had no effect on their antibacterial capacity. for long, cd56 has been used for the detection and identification of natural killer (nk) cells. recently, the presence of a minor subset of cd56 low cd33+ blood monocytes (mo) in healthy individuals and the increase in cd16+cd56+ blood mo in patients with inflammation has been reported. the functional activity of human cd56+ blood mo has been studied in vitro but not tested ex vivo so far. healthy people living permanently in malaria endemic areas are exposed to plasmodium infection, and we hypothesized that blood mo of these individuals could be activated and display increased cd56 expression. we tested if this phenotypic expression was associated with detectable changes in the mo anti-parasitic activity. the mo phenotype of healthy malaria naï ve and malaria exposed individuals was determined by three-color flow cytometry. myeloid cell markers included cd33 and activation markers such as hla-dr and trem-1. percentages of blood mo involved in phagocytosis activity either with or without immune sera were then identified by flowcytometry, and the potential association between a given mo phenotype and phagocytosis activity was then looked for, using spss ® and statview ® softwares. our results showed that, compared with malaria naï ve individuals, there was a 12.3 fold increase (p x 0.0001) in the total number of circulating cd56 low mo present in the blood samples of healthy malaria exposed asian individuals living in thailand. according to the density of surface antigen determined by fluorescence intensity (fi), the decrease in cd33 and the concomitant increase in hla-dr expressions indicated that in this malaria endemic area, blood mo were mature and highly activated by comparison with surface markers of mo from malaria naï ve donors. the relative levels of cd56+ blood mo were associated with the percentages of membrane-bound ifn-g present at the mo surface. in conclusion, (i)-a subset of cd33+ blood mo expressed increased levels of cd56 on mo of healthy malaria exposed individuals; (ii)-blood mo with activated (hla-dr+) and mature (trem-1+) phenotypes were present in these healthy individuals; (iii)-increased expression of hla-dr and cd56 on cd14 high mo was associated with a high phagocytosis activity. introduction: adipokines, initially described for their function within metabolism, have been characterized to exert a regulatory role on the immune response. for instance the appetite-regulating hormone leptin has been identified to modulate the response of the innate as well as the acquired immune system. the present work focuses on the effects of leptin on the reactivity of m1-and m2-polarized human macrophages. methods: monocytes were isolated from the peripheral blood by magnetic cell sorting. polarization to m1 and m2 macrophages was induced by culture in the presence of mcsf or gmcsf respectively. polarized cells were characterized by flow cytometry, stimulated with lps and response assessed by characterization of cytokine profiles via cytometric bead array (cba). results: culture of monocytes in the presence of mcsf or gmcsf induced two different phenotypes. cells cultured in the presence of gmcsf represented the m1 type and were cd14 negative but cd80 and mhcii positive and produced high levels of il-8, tnfalpha and il-6 following lps stimulation. culture in the presence of mcsf resulted in induction of the m2 phenotype. these cells were cd14 positive with intermediate expression of cd80 and mhcii expression and produced high levels of il-10, il-6 and il-8 following lps stimulation. interestingly, already baseline il-8 production was high in these cells. stimulation with leptin alone increased cytokine production in both cell types as compared to cells cultured in medium alone. however, if leptin was present in cultures stimulated with lps, the induction of cytokine production was significantly reduced in both, m1-and m2-polarized cells as compared to cells stimulated with lps alone. summary: whereas presence of leptin enhances baseline cytokine production in polarized macrophages, it reduces the cytokine production in response to stimulation with the tlr4 ligand lps. thus, abundant leptin levels like present in obesity or in the hypertrophied fat as present in crohn's disease patients might exert modulating effects on macrophage response to bacterial antigens. methods: hl60 cell line was used as a model of leukemic myeloid cell differentiation cultured in suspension or on fibronectin matrix prior to pma (50 ng/ml) treatment for 48h. morphological evaluation was performed with conventional microscopy and electron microscopy. immunephenotype and phagocytic activity of the cells were determined by flow cytometry and immunocytochemistry. a colorimetric nitro-blue-tetrazolium reduction assay was performed to assess the production of reactive oxygen species (ros). results : besides their distinctive macrophage morphology and ultrastructure with spindle cell-like features and high granularity, the pma-treated fibronectinadherent hl60 cells expressed antigen receptors cd14, tlr2, tlr4 and cd68 , and displayed enhanced phagocytic activity and production of ros. expression of cd13, cd33 and cd15 was also maintained however the cells were hla-dr and cd1a negative. conclusion: we describe the enhanced ability of fibronectin-adherent hl60 cells to differentiate into macrophages in response to pma. hl60 may provide a functional model for macrophage differentiation. above all, this finding may stimulate further research on myeloid leukemia biology and potential adjuvant therapies. a. aporta 1 , n. ferrer 2 , a. gómez 2 , j. gonzalo 2 , a. arbués 2 , a. anel 1 , c. martín 2 , j. pardo 1 , apoptosis, immunity and cancer 1 university of zaragoza, molecular and cellular biochemistry and biology, zaragoza, spain, 2 university of zaragoza, mycobacterium genetics, zaragoza, spain mycobacterium tuberculosis is an intracellular pathogen that uses alveolar macrophages as its preferred habitat, being capable of produce both a progressive disease and an asymptomatic latent infection. it has been postulated that infected macrophage apoptosis may contribute to host defence against this intracellular infection by, firstly, eliminating supportive environment for bacterial growth and, secondly, by leading to the formation and release of apoptotic vesicles containing mycobacterial antigens. it has been proposed that m. tuberculosis inhibits host cell apoptosis thus interfering with the immune system response. however the biological relevance of this process is not clear. our group has generated so2, a m. tuberculosis phop mutant strain that was shown (perez et al 2001) to be more attenuated than the present attenuated vaccine strain bcg and conferred protective immunity against m. tuberculosis infection in mice and guinea pigs (martin et al 2006) . in the present study, we compare the time course and phenotype of cell death induced by so2, bcg and wild type m. tuberculosis on the murine macrophage cell line j774 and on bone marrow derived mouse macrophages. our results indicate that wild type m. tuberculosis induces macrophage cell death analysed by a clonogeneic assay much faster than the attenuated bacteria. of note cell death presented apoptotic features like caspase-3 activity and nuclear condensation. in order to analyse the consequences of this apoptotis-like cell death, it has been invetigated whether dead cells translocate phosphatydilserine to the outer part of the plasma membrane and if this traslocation is enough to promote phagocytosis by fresh macrophages. experiments are ongoing with macrophages derived from trl2x4 deficient mice and wt animals in order to study the role and implication of those receptor on the susceptibility to infection and death induced by the virulent and attenuated phop m. tuberculosis strain. objectives: vip is a potent anti-inflammatory peptide, mainly acting as endogenous macrophage deactivating factor. type 1 receptor for vip (vipr1) gene is highly conserved through species and, in humans, is highly polymorphic. vipr1 has been reported to be down-modulated in cells of the immune system after activation. an association of some snps with some autoimmune diseases has also been reported. in this study we have investigated the correlation between these snps and gene expression in monocytes exposed to lps. methods: monocytes from 53 blood donors were separated from pbmc and stimulated with lps. total rna was reverse transcribed and the level of vipr1 in untreated or lps-stimulated monocytes was measured by real-time rt-pcr and protein expression. protein level was measured by western blot and densitometric analysis. the kinetic of expression of vipr1 after 3, 6, 9 and 12 h of exposure to lps was firstly analysed in monocytes from five individuals. there were two kinetics: one in which a reasonable high levels ( g 50 %) of mrna was maintained trough time and a second one in which the decrease of mrna was pronounced. the experiments were repeated using monocytes from 53 donors that had been typed for the relevant vipr1 snps. the down-regulation of vipr1 correlates with the presence of a t at rs896 mapping in the 3'-end of the gene (p= 0.004). the vipr1 protein level was decreased about 40 % in monocytes of 3 subjects typed as t/t at rs896 whereas 3 subjects typed as c/c at rs896 maintained a high level of expression after 9h of lps treatment. the data show that different haplotypes of the vipr1 gene correlate with a different kinetics of gene expression in activated monocytes. a possible consequence of these data is that the anti-inflammatory properties of vip governed by the vipr1 vary in different individuals and can eventually contribute to the genetic predisposition to some autoimmune diseases. j. oujezdska 1 , t. vavrochova 1 , d. filipp 1 , immunobiology 1 institute of molecular genetics as cr, immunobiology, prague, czech republic phagocytes which appear in early mouse development (e7.5-13.5) represent a unique embryonic macrophage lineage that differs from adult macrophages phenotypically, biochemically and by their origin. recent studies suggested that there are at least three waves of macrophages populating an early embryo: a maternallyderived one and two waves of extraembryonal, ys-derived phagocytes. in addition, the occurence of early embryonic phagocytes of undetermined origin in the anterior head mesoderm in several invertebrate and vertebrate species is well documented. this origin-related heterogeneity among early embryonic phagocyte subpopulations coupled with the lack of their specific surface markers makes it difficult to distinguish them phenotypically and study their potentially distinct physiological roles in early development. the aim of this study is to identify a set of surface markers expressed on embryonal phagocytes suitable for phenotypic distinction among embryonic phagocyte subpopulations. here we report the temporal and spatial expression of toll-like receptors (tlrs) and cd14 in the early mouse embryo (me). facs analysis of cell suspension prepared from 10.5 day me showed that about 0.7-1 % of cells were positive for cd11b. these cells exclusively were also positive for cd14, tlr2, and cd45 antigens. using qpcr and flow cytometry we show that tlrs and other tir domain-containing signaling molecules are expressed in the embryo through embryonic days 7,5-13,5. reciprocal matings between wild type and mhcii-egfp knock-in mice revealed that while maternallyderived mhcii + macrophages are present in the embryo from early developmental stages (e7,5), embryo-derived mhcii + macrophages start to appear in the embryo around day 13. multicolor facs analysis of cd11b, cd45, cd14, f4/80, tlr2, tlr4, c-kit and mhcii surface markers revealed differential expression of tlr2 and c-kit on embryonal phagocyte subpopulations. moreover, the microarray analysis of cd11b + tlr2 + cells isolated from the e10,5 embryos has revealed significantly upregulated expression of several novel genes in comparison to their expression in murine peritoneal macrophages. these molecules are currently being tested for their use as embryonic phagocyte specific-lineage markers. these results are first to characterize the regulated expression of tlrs on early embryonal phagocytes and demonstate their potential to serve as novel markers for their detection and isolation. humans may be exposed to a variety of mycobacteria ranging from environmental or bcg vaccine to more pathogenic mycobacteria. only a minority of individuals exposed develop disease, this susceptibility may result in part from variability of host immune responses genes through simple (mendelien disease) and complex (polymorphisms with milder effect) inheritance mechanisms. interestingly, key elements of inflammatory pathways are particularly involved in this susceptibility to mycobacteria. il12/il23-dependent ifng pathway of macrophage activation plays a central role in inflammation and cell-mediated immune responses to mycobacteria. due to the high rate of consanguineous marriages in the north african countries, recessive genetic disorders including primary immunodeficiencies occur with a relatively high prevalence. in tunisia, among patients affected with primary immunodeficiencies 16 presented with disseminated bcg infection (bcg-osis). among them, five have an underlying well-defined primary immunodeficiency either a severe combined immunodeficiency or a chronic granulomatous disease and 11 have a mendelien susceptibility to mycobacterial disease. using a candidate gene strategy, we have identified in 9 out of these 11 patients mutations in several ifng pathway genes, other candidate genes are being investigated for the 2 other patients. in the general population, common polymorphisms with milder effect on the risk of tuberculosis have been identified including mhc and nramp1. we did focus on the study of 2 genes which are considered as important pathogen recognition receptors of the innate immune system: tlr2 is the principal mediator of macrophage activation in response to mycobacteria through nfkb pro-inflammatory signaling pathway and dc-sign is the major receptor of m. tuberculosis on human dendritic cells and in contrast induces anti-inflammatory il-10 cytokine. using a case/household-contact cohort we did investigate polymorphisms of these 2 genes in tunisian patients affected with active pulmonary tuberculosis and have shown specific patterns of snp and microsatellite polymorphisms associated with susceptibility/resistance to tuberculosis. host inflammatory responses play a major role in granuloma formation and control of the infection. unraveling these pathways might be crucial in order to identify new therapeutic targets and strategies including immunotherapy e. g. ifng therapy for tuberculosis, particularly in this era of emergence of multi-drug and extensively-drug resistant m. tuberculosis strains. francisella tularensis is a gram negative bacterium that is the causative agent of tularemia. research into francisella has expanded over recent years due to its designation as a potential biological warfare agent. several species of francisella exist and have varying degrees of pathogenicity. f. tularensis live vaccine strain (lvs) is an attenuated strain of the holarctica subspecies and has been shown to be an effective vaccine in humans. however, it is pathogenic in mice which can, therefore, act as a useful model of human tularemia. f. tularensis is an intracellular pathogen and is able to invade several different cell types, in particular macrophages, most commonly through phagocytosis. therefore, if phagocytosis could be disrupted via the addition of inhibitors, uptake of f. tularensis would decrease and antibiotic treatment may be more effective. a flow cytometric assay was developed to measure bacterial uptake. this method used a fitc labelled anti-f. tularensis antibody in conjunction with antibodies to cell surface markers to determine specific cell phenotypes that were positive for bacteria. a series of phagocytic inhibitors have been tested in vitro on an alveolar macrophage derived cell line (mhs) and on ex-vivo mouse lung tissue to determine whether uptake of f. tularensis lvs could be altered. the presented data shows that several inhibitors work efficiently to reduce lvs uptake by up to 70-80 % in both the in vitro and ex vivo assays. however, cytotoxicity of some of the inhibitors was high and, therefore, it was essential to concentrate on inhibitors with low cytotoxicity for further assessment. in addition, bacteriological data suggests that the combination of inhibitors with antibiotics may be a useful therapeutic against f. tularensis. it may also work against other intracellular pathogens that use phagocytic mechanisms to enter their optimal niche.ã crown copyright. dstl, 2009. hsp70 are intracellular proteins but it is known that these proteins can be expressed on cell surface and contained in extracellular medium, in particular in peripheral blood serum. it is also known that extracellular hsp70 have pronounced immunomodulatory properties. to study the pathways of the protein modulating action on immune system we investigated effect of exogenous and cell surface hsp70 on reactive oxygen species (ros) release from phagocytes, namely human neutrophils, during process of phagocytosis (respiratory burst). neutrophils were isolated from human peripheral blood by using a standard protocol. respiratory burst induced by opsonized zymosan was measured by method of luminol dependent chemiluminescence. for the experiments human recombinant hsp70 (low endotoxin) and paraformaldehyde fixed mouse thymocytes exposed surface hsp70 were used. exogenous hsp70 was used in concentration 1-10 ug/ml, fixed thymocytes were added to neutrophil samples in quantitative ratio 1:1 and 2:1 directly before the measuring. as the control we registered amplitude of oxidative burst in samples supplemented with pbs or live mouse thymocytes having no hsp70 on their surface. results demonstrating effect of exogenous hsp70 on phagocytosis-induced ros release from human peripheral blood neutrophils have been obtained. it was demonstrated marked dose-dependent inhibiting action of exogenous hsp70 on amplitude of respiratory burst. the cells expressing surface hsp70 impacted on ros production in this model similarly. the results of chemiluminescence analysis demonstrated that zymosan induced ros production was essentially decreased under action of fixed thymocytes, and was decreased slightly in presence of live thymocytes in the neutrophil samples. the effect was more pronounced for increased amount of thymocytes added to the samples. thus, immunomodulatory effects of exogenous hsp70 might be caused by influence of the protein on ros release from phagocytes. we suppose that the registered effects are connected with ability of hsp70 to inhibit activity of nadp-oxidase -the key enzyme for ros production during respiratory burst. results: we recruited 28 pts, with so far five complete pathological remission, five partial responses and five no responses. no substantial changes were detectable in the number of circulating monocytes. in contrast we observed a clear expansion of cd14/cd86 and cd14/cd163 double positive subsets. this event was transient; it abated at the later time point suggesting a causal relationship to the treatment. it correlated with sensitivity to the treatment. in fact we observed that in the responder patients the expansion of the cd14/86 subset was clear in the first weeks of treatment and decreased there after. in contrast in non-responder patients it was already expanded before the neo-adjuvant therapy. all the patients had an initial expansion of the cd14/163 subset. in the responder patients this population was still present at the time of surgery. the immunohistochemical study revealed a massive tumoral infiltration by macrophages that displayed clear features of alternative m2 polarization. conclusion: these data suggest that neo-adjuvant therapy modulates the cellular components of innate immune responses that could represent valuable predictive factors. m. dimitrijević 1 , i. pilipović 1 , s. stanojević 1 , k. mitić 1 , k. radojević 1 , v. pešić 2 , g. leposavić 1,2 1 institute of virology, vaccines and sera "torlak", immunology research centre "branislav janković", belgrade, serbia, 2 faculty of pharmacy, university of belgrade, department of physiology, belgrade, serbia the primary aim of our current study was to ascertain whether rat resident peritoneal macrophages synthesized catecholamines and to unmask putative effects of catecholamines on nitric oxide (no) and hydrogen peroxide (h 2 o 2 ) production and phagocytic activity of these cells. in addition, given that chronic administration of b-adrenoceptor antagonist increases the density of b-adrenoceptors on both non-immune and immune cells and thereby affects their sensitivity to catecholamine action, we hypothesized that such treatment could also affect macrophage responsiveness. to address our proposition, we determined adrenoceptor expression on peritoneal macrophages from rats subjected to 14-day-long propranolol treatment and measured both no and h 2 o 2 production and phagocytic activity of these cells. using both immunocytochemical and flow cytometric analyses of rat peritoneal exudate cells constitutive expression of tyrosine hydroxylase and both b 2 -and a 1 -adrenoceptors on macrophages was revealed. furthermore, according to the characteristic assemblage of tyrosine hydroxylase and adrenoceptor subtype expression different macrophage subsets were identified. in vitro treatment of macrophages with the non-selective a,b-adrenoceptor agonist arterenol and/or the b-adrenoceptor antagonist propranolol indicated that b-adrenoceptors potentiated no production and suggested a-adrenoceptor-mediated suppression of hydrogen peroxide h 2 o 2 production. an increase in h 2 o 2 production in the presence of the a 1 -adrenoceptor antagonist ebrantil provided support for this. chronic propranolol treatment in vivo led to increased no and h 2 o 2 production by peritoneal macrophages. furthermore, this treatment resulted in opposing effects on the expression of b 2 -and a 1 -adrenoceptors on peritoneal macrophages (a stimulatory effect on b 2 -adrenoceptors and a suppressive effect on a 1 -adrenoceptors). in conclusion, a subset of resident peritoneal macrophages synthesizes catecholamines, which may exert differential effects on h objectives: monocytes display great phenotypical and functional heterogeneity and are divided into two major subsets: cd14 ++ cd16 -('classical') and cd14 + cd16 + ('pro-inflammatory') monocytes. a central monocyte function is cytokine production in response to toll-like receptor (tlr) ligation. the cd14 + cd16 + monocytes display higher tlr2 and -4 expression, produce higher levels of pro-inflammatory cytokines and have increased potency for antigen presentation than the cd14 ++ cd16monocytes, suggesting that the two subsets could play different roles in antimicrobial responses. newborns are vulnerable to infections and an immaturity of both adaptive and innate immunity has been described. studies of neonatal monocyte antimicrobial responses show contrasting results and much remains to be learned, especially regarding monocyte subpopulations. thus we aimed to compare monocytes from newborns and adults, focusing on monocyte subpopulations and responses following tlr2 stimulation. methods: cord blood (n=8) and peripheral-blood (n=8) mononuclear cells were stimulated in vitro for 24hrs with peptidoglycan and subsequently analysed for cd14 and cd16 and intracellular il-12p70 and tnf expression. the mann-whitney u-test was used to evaluate differences between groups. results: a significantly higher percentage of neonatal monocytes were positive for il-12p70, both unstimulated and after peptidoglycan stimulation, as compared to adults. geomfi of il-12p70 was low and similar between groups, although significantly higher in newborns after stimulation. in both newborns and adults, il-12p70 (% positive cells and geomfi) was significantly higher for cd14 + cd16 + cells than for cd14 ++ cd16cells, unstimulated and stimulated. regarding tnf, neonatal and adult monocytes did not differ in unstimulated cultures, however geomfi of tnf was significantly higher in neonatal monocytes after stimulation. whereas the tnf response following stimulation was similar between the adult monocyte subsets, in newborns the cd14 ++ cd16cells were positive for tnf to a significantly higher extent than the cd14 + cd16 + cells. in particular the tnf response to tlr2 stimulation differed between newborns and adults, with neonatal monocytes having a higher per cell production of the cytokine. notably, in newborns the cd14 ++ cd16monocytes were positive to a higher extent for tnf following stimulation pointing towards a functional immaturity of neonatal monocyte subset responses. objective: chronic granulomatous disease (cgd) is an uncommon congenital phagocyte disorder characterized by recurrent life-threatening infections. cgd generally present with recurrent suppurative infections; however, intracranial fungal abscess complicating cgd may cause a diagnostic problem to anyone who is unfamiliar with its clinical and radiological features. we report a 16-year-old boy who admitted with complaints of seizures during the previous 2 months. there was a history of axillary and perianal suppurative skin infections and cavitary pneumonia. the family history was unremarkable, and the parents were unconsanguineous. physical examination was only remarkable for oral moniliasis and skin scars at axillary and perianal region. a large frontol mass with diffuse peripheral vasogenic edema was discovered on mri. subfalcine herniation was noted secondary to mass effect. cgd was suspected and the analysis with flow cytometric dihydrorhodamine assay (dhr assay), for functional analysis of neutrophils was compatible with the diagnosis of cgd and no bimodal histogram pattern spesific for x-cgd was found in the mother and sister. after the diagnosis of cgd, neurosurgical removal of the abscess cavity was performed due to peri-lesional edema and herniation risk. aspergillus fumigates grew from the culture; liposomal amphotericin b and voriconazole were started; which were found to be sensitive to the cultured species. in addition, interferon-g (50 mgr/m2/day, subcutaneously every other day) was started. after 2 months, control mri showed regression of the lesion, and the anti-fungal treatment was continued for 3 months. the screening of the other family members with dhr assay demonstrated that one of his sisters had also cgd and phenotype was autosomal recessive. mutaton analysis in "hot spot" in ncf1 gene concerns the well-known gt deletion in the second exon of ncf1 gene both at the patient and his sister. results: this was an atypical clinical presentation of cgd in an adolescent boy with cerebral aspergillosis, mimicking intra-cranial tumor. we documented a good response to the combination of ifn-g, liposomal amphotericin b and voriconazole after surgery. conclusion: cgd should be considered in the differential diagnosis for all children presenting with invasive fungal infections particularly, those involving the central nervous system. recent data suggest that ubiquitin has anti-inflammatory properties and therapeutic potential after severe trauma and brain injuries. therefore, we hypothesized that ubiquitin treatment can modulate the local inflammatory response triggered after brain injury. to test this hypothesis, a focal cortical contusion was induced using a controlled cortical impact (cci) model in sprague-dawley rats. animals (n = 45) subjected to moderate brain injury were randomized, and received either 1.5 mg/kg ubiquitin or vehicle (placebo) intravenously within 5 min after cci. levels of tnf-a, il-1b, il-6, il-10 and il-1 receptor antagonist were analyzed in brain tissue using real time rt-pcr at 4 and 72 hours after treatment. immune cell infiltration was studied by immunostaining for neutrophils and macrophages/ microglia at 24h and 7 days. data were analyzed with the mann-whitney u test and a two-tailed p x 0 .05 was considered significant. all cytokines were highly up-regulated 4 hours after cci but no differences between the groups were observed at this time point. three days after trauma the levels of il-10 were significantly lower in the ubiquitin treated animals, whereas the levels of il-6 and tnf-a were higher when compared to the placebo group. interestingly, macrophages/ activated microglia were significantly increased in the pericontusional cortex after ubiquitin treatment at day 7. the infiltration of neutropils was not affected by ubiquitin treatment. here, we could demonstrate for the first time that a single injection of ubiquitin immediately after brain trauma is able to modulate the inflammatory response triggered after brain injury at the cellular as well as at the cytokine level. macrophage activation and oxidative metabolic changes are commonly implicated in pulmonary tuberculosis (ptb) patients. efficient plasma antioxidant activities are needed to neutralize high free radical load in pulmonary tuberculosis (ptb) patients. there is limited information about the plasma levels of neopterin (a marker of macrophage activation) and oxidative stress indices such as total plasma peroxide (tpp), total antioxidant activity (taa), malondialdehyde (mda), and oxidative stress index (osi) in ptb patients during chemotherapy with or without micronutrient supplementation. the present study was designed to assess the levels of neopterin, tpp, taa, mda, and osi during chemotherapy with (c+m) or without (c-m) micronutrient supplementation using elisa and spectrophotometric methods. thirty-eight (38) newly diagnosed ptb patients and forty non-ptb apparently healthy subjects volunteered to participate in this study. twenty of the ptb patients were on anti-tuberculosis drugs supplemented with micronutrients (c+m) while 18 were treated with anti-tuberculosis drug alone (c-m) for a period of four weeks. the levels of neopterin (p=0.02), tpp (p=0.00), osi (p = 0.00), mda (p = 0.00) were significantly raised but taa (p = 0.01) was significantly reduced in ptb patients compared with controls. the levels of mda (p = 0.04), neopterin (p=0.00) and tpp (p=0.00) were significantly reduced in c+m after two weeks of treatment compared with baseline values before commencement of treatment. the levels of tpp (p=0.00), mda (p=0.00), neopterin (p=0.02), osi (p=0.00) were significantly reduced while taa (p=0.01) was significantly raised in c+m after 4 weeks of treatment compared with the baseline concentrations. in c-m, only mda showed significant decreased after 4 weeks of treatment when compared with the baseline values. plasma level of neopterin, tpp, osi and mda declined faster in c+m than c-m. therefore, micronutrient supplementation of ptb drugs with synthetic antioxidants or naturally occurring ones (fruits and vegetables) should be attempted. this will improve deranged macrophage activation and reduce oxidative stress indices in ptb patients. a. p. aguas 1 , e.m. cunha 1 , m.j. oliveira 1 1 icbas, university of porto, anatomy, porto, portugal the acute in vivo intake of mercury (hg) microparticles (20 nm in diameter) by neutrophils and macrophages was studied with the use of in situ detection of hg by scanning electron microscopy coupled with x-ray elemental microanalysis (sem-xem). the intracellular distribution of hg particles was compared, at high resolution, between macrophages and neutrophils, and between activated and non-activated phagocytes. balb/c mice were injected intraperitoneally (ip) or in a subcutaneous air-pouch with mercury chloride, and the animals were sacrificed up to 5 minutes after the injection. in some mice, before the hg injection, peritoneal phagocytes were activacted by ip injection of bsa. pre-injections with a selenium (se) salt were also performed in order to study the putative modulatory role of se on hg intake by phagocytes. peritoneal cells were collected by washing of the peritoneal or subcutaneous cavities with pbs, they were cytospinned, fixed with formaldehyde, and processed for observation by sem-xem. five min after the hg injection more than half of the mouse phagocytes were positive for hg. a higher percentage (70 %) of macrophages contained the metal particles than neutrophils (55 %). phagocyte activation enhanced the number of hg particles seen inside the phagocytes. pre-injection of the peritoneal cavity of mice with se resulted in finding that more than half of the hg intracellular particles were coupled with se. subcellular topography of hg particles showed that they were presented in individual small cytoplasmic vesicles. we conclude that hg microparticles are rapidly ingested by macrophages and neutrophils, a processed that is enhanced by cell activation. hg particles are ingested by pinocytosis and sorted in the cytoplasm of macrophages and neutrophils inside individual small vesicles. this study was supported by a grant from fct, portugal. mast cells play central roles in allergic inflammatory reactions and innate immunity. swap-70 is a rac-interacting protein expressed in several cells types of the hematopoietic system including mast cells. in b cells and mast cells swap-70 regulates f-actin cytoskeletal rearrangements, cell polarisation and cell migration. (pearce et al., 2006; sivalenka and jessberger, 2004) . swap-70-/-bone marrow derived mast cells (bmmc) are specifically impaired in fceri-mediated activation and degranulation and in c-kit-induced activation, migration and cell adhesion (gross et al., 2002; sivalenka and jessberger, 2004; sivalenka et al., 2008) . crucial regulators of these processes are members of the rho family of small gtpases such as rac1 and rhoa. swap-70 interacts with rac1 in vitro and preferentially binds the active gtp-bound rac1. swap-70 supports the increase of active rac1 in vitro by a yet to be defined mechanism (shinohara et al., 2002) . in this study, in vitro pull-down assays with purified recombinant proteins were employed to characterize the interaction between swap-70 and rac1. it was found that fulllength swap-70 preferentially binds to constitutively active rac1 (rac1q61l) but not to its dominant negative form (rac1t17n). binding assays with swap-70 truncated mutants showed interaction of swap-70's n-terminus with gtpgs rac1 or rac1 depleted of guanine nucleotide, whereas swap-70 central or c-terminal regions do not bind to any form of rac1. preliminary competitive-binding assays with overlapping 18mer peptides, spanning the entire swap-70 sequence, mapped the rac1 binding site near the n-terminus of swap-70. full-length swap-70 site-specific mutants will be generated to test the relevance of these interactions in mast cells in terms of adhesion, migration and activation of rho gtpases. elucidating the molecular interactions of swap-70 with rho gtpases and the relevance of these will shed light on the biology and biochemistry of mast cells and possibly other hematopoietic cells, which express swap-70. v. c. barbosa 1 , c. d. polli 1 , m.c. roque-barreira 1 , m.c. jamur 1 , c. oliver 1 , g. pereira-da-silva mast cells are essential cells in ige-associated immune responses. fceri crosslinking induces mast cell degranulation and release of proinflammatory mediators. we have previously shown that the lectin artinm induces mast cell activation but the mechanisms involved in this activity remain unknown. objective: the present study was undertaken to further characterize the ability of artinm to activate mast cells. methods: rbl-2h3 cells were sensitized with ige anti-tnp and stimulated with dnp 48 -hsa or artinm. artinm binding to rbl-2h3 cells was assessed by flow cytometry. mast cell degranulation was determined by measurements of released b-hexosaminidase activity. microplate binding assays were utilized to assess artinm binding to ige. to investigate fceri recognition by the lectin, western blots of cell lysates were stained with biotinylated artinm and be's antibodies specific for fceri b-subunit. intracellular protein phosphorylation was detected by specific antibodies and analyzed by confocal microscopy. mcp-1 and tgf-b levels released by mast cells were measured by elisa. results: artinm binding to the cell surface was dependent on sugar recognition and resulted in mast cell degranulation in the presence or absence of ige. the release of b-hexosaminidase doubled when cells were sensitized by the immunoglobulin and was abrogated in the presence of d-mannose, suggesting that mast cell degranulation induced by artinm might be the result of interactions between the lectin crds and glycosylated components on the cell surface, like fceri or ige. indeed, it was observed that the lectin bound to ige in a dose-dependent manner and recognized the fceri b subunit in western blot analysis. exposure to artinm resulted also in phosphorylation of intracellular proteins, mcp-1 release and tgf-b production. significant increases in these activities were observed upon sensitization with ige. conclusions: these results suggest that artinm may bind to glycans of the high affinity ige receptor and/or of the ige (bound to fceri) and that such interactions would be implicated in its ability to activate and degranulate mast cells. in view of the well-established significance of mast cells in allergic inflammation, the participation of sugars as binding receptors on mast cell surface opens new ways of controlling allergic disorders. the adhesion receptor l-selectin is a key player of the innate immune response in the process of leukocyte migration from the blood stream to inflamed tissue. it is expressed on leukocytes and promotes the initial contact to the endothelium resulting in steady rolling and eventually diapedesis. a distinct feature is the exclusive presentation of l-selectin on the tip of finger-like cell membrane protrusions called microvilli which cover the entire leukocyte surface. this topography was shown to facilitate the first transient interactions of the free flowing cell to the static counterreceptor particularly in the context of high dynamic shear. other adhesion molecules such as p-selectin glycoprotein ligand 1 (psgl-1), b1 and b7-integrins also share this special phenotype. taken together, prominent adhesion receptor positioning reflects a widespread biological principle contributing to inflammation as well as hematogenic tumor metastasis. despite the functional relevance and frequent occurrence, however, molecular mechanisms of cell surface receptor compartmentalization remain largely unknown. in this study we identified the highly conserved transmembrane domain of l-selectin to regulate microvillus receptor positioning and adhesion under flow. taking advantage of the inverse surface expression pattern of cd44 (cell body) compared to l-selectin (microvilli) in a myeloid cell line, we investigated domain swapped chimeric receptors regarding their substructural surface localization and their ability to initiate rolling under flow. transmission electron microscopy showed a crucial impact of the transmembrane domain to target the chimeric receptors to a certain cell surface compartment independent of the intracellular anchorage. in turn, the receptor shift from microvilli to the cell body goes along with a substantial decrease of rolling cells in an in vitro parallel flow chamber assay. thus, contrary to the common view of single membrane spanning domains to simply act as a mechanical anchor, our results attach an important functional component as well and might point out a new general principle for targeting receptors to specific membrane compartments. objectives: macrophages are one of the principal effector cells involved in the innate immunity response. they kill microbes through phagocytosis and upon activation, secrete pro-inflammatory cytokines such as il-1b, il-18 and tnf-a. herpes simplex virus 1 (hsv-1) is an enveloped dna virus that infects mostly oral mucosa and sensory neurons. innate immunity responses activated by hsv1 infection consist of: activation of macrophages; activation of the complement cascade, and production and secretion of a variety of cytokines and chemokines. il-18 and tnf-a are cytokines produced by macrophages that contain known anti-hsv properties. the objective of this study was to characterise the secretome of human primary macrophages infected with hsv1. methods: human monocytes were purified from the peripheral blood mononuclear cells of healthy blood donors and differentiated in vitro into macrophages. macrophages were left untreated or primed with poly(i:c) (10ug/ml), a mimetic of double-stranded rna, after which cells were left uninfected or infected with hsv-1 for 18 h. after this, cell culture supernatants were collected, concentrated and proteins purified. the secreted proteins were digested into peptides, identified and quantified using itraq (isotope tagged relative and absolute quantitation) -labelling of the peptides followed by peptide fractionation by cation exchange chromatography and analysis by nanolc-ms/ms. the raw ms/ms data was analysed using proteinpilot 2.0 software. results: in the first itraq experiment over 300 human proteins were identified in the hsv1 infected cell supernatants. from these proteins 119 had at least 3 fold increase after poly(i:c) + hsv1 infection compared to the uninfected cells. hsv1 infected cells had clearly more proteins in their cell supernatants after infection compared to the uninfected cells: itraq labelling showed a total of 2.7 fold increase in the protein amount in the poly(i:c) + hsv1 infected cell supernatant and a 2.6 fold increase in the hsv1 infected cell supernatant when compared to the uninfected cell supernatant. amongst the upregulated proteins there were known inflammatory proteins: chemokine (c-x-c motif) ligand 10, il-6, tnf-a induced protein 6, complement factor b, galectin-1 and mxa. at present, further experiments are on-going for more detailed analysis of the hsv1 infected macrophage secretome. h. p. prakash 1,2 1 german cancer research centre, translational immunology, heidelberg, germany, 2 max planck institute for infection biology, molecular biology, berlin, germany chlamydophila pneumoniae are the major etiological factors for worldwide pneumonia, chd and copd. chlamydia lives and multiplies inside their host epithelial cells where they confer resistance for apoptosis by inducing expression and stability of anti-apoptotic proteins called inhibitor of apoptosis proteins (iaps). the significance of cellular inhibitor of apoptosis protein-1 (ciap-1) and x-linked inhibitor of apoptosis proteins ( xiap) in chlamydia pneumoniae pulmonary infection and innate immune response of macrophages was investigated in ciap-1 and xiap knockout (ko) mice using a novel non-invasive intra-tracheal infection method. in contrast to wildtype, iap knockout mice failed to clear the infection from their lung. wildtype mice responded to infection with a strong inflammatory response in the lung. in contrast, the recruitment of monocytes and macrophages was reduced in iap ko mice compared to wildtype mice. the concentration of interferon gamma (ifn-g) was increased whereastumor necrosis factor (tnfa) was dysregulated in the lungs of infected iap ko mice compared to infected wildtype mice. ex vivo experiments on mouse peritoneal macrophages and splenocytes revealed that iaps are required for innate immune responses of these cells. our findings thus suggest a new immunoregulatory role of iaps in c.pneumonaie pulmaonry infections. methods: human monocytes were purified from venous blood of normal volunteers by ficoll density gradient centrifugation. hrgal-3 (25 mg/ml) binding to monocytes, in the presence or absence of 10mm lactose or sacarose, was assessed by flow cytometry and confocal microscopy. in transwell systems, assays were performed using hrgal3, laminin or fibronectin immobilized or not on the filters. these were added to wells containing soluble hrgal3 or rpmi and monocytes (1x10 5 ) were added into each insert. when necessary, hrgal3 was pre-incubated with 10mm lactose or sacarose. mcp-1 (100ng/ml) was used as positive control. we observed that hrgal-3 binds to the surface of human monocytes through its crd, since this interaction can be inhibited by lactose. we corroborated some data of literature that hrgal-3 is able to induce monocyte migration in a dose-dependent manner, resulting in a bell-shaped curve as seem with other known attractants. when we evaluated the participation of the ecm laminin and fibronectin in monocyte migration induced by hrgal-3, we observed that the association between these glycoproteins and hrgal-3 resulted in a 60 % increase in the number of migrating cells. both n-and c-terminal domains of hrgal-3 are involved in the association between laminin or fibronectin and hrgal-3, since the presence of lactose resulted in 50 % and 20 % inhibition of monocyte migration induced by the lectin, respectively conclusions: our results showed that hrgal-3 induces monocyte migration by haptotaxis, through the interactions established between both n-and c-terminal domains of the lectin and ecm glycoproteins, laminin and fibronectin. in a vertebrate embryo, macrophages develop in two sites (yolk sac and liver) and constitute the primary mechanism of host defense. their phagocytic function may be required during the earliest stages of development both for survival and for organogenesis. recent studies have shown that monocyte heterogeneity is conserved in humans and mice. the different monocyte subsets seem to reflect developmental stages with distinct physiological roles but nothing is known whether the macrophage diversity arises in early ontogeny. in order to study the ontogeny of the monocyte-macrophage lineage, we developed a new culture technique using human embryonic stem cells (hesc).culturing of embryoid bodies for 3 weeks in the presence of bmp4,vegf and a mixture of hematopoietic cytokines resulted in a generation of a significant cell population of cd14+cd45+ cells. the sorted cd14+cd45+ cells were further cultured for 7 -10 days in the presence of m-csf and gave rise to a homogenous population of adherent mature macrophages. embryonic stem cells derived macrophages were identified by several criteria including morphology and ultrastructural features observed by microscopy and by expression of nonspecific esterase and myeloperoxidase by histochemical staining. while virtually all embryonic-derived macrophages expressed the lps-receptor cd14, m-csf receptor cd115 and the scavenger-receptor cd36, we characterized two distinct subpopulations of macrophage based on their difference in size and density and the expression of the cd14 and cd16 (fcgammariii) : the cd14lowcd16-and cd14+ cd16+. trancscriptional, phenotypic and functional assays suggest the alternative (m2) polarization of cd14+cd16+ embryonic stem cell-derived macrophages.(anti-inflammatory cytokines secretion, active phagocytosis, m2 -related gene expression).the exact chemokine receptor expression pattern, phenotype and transcriptional activity of their foetal counterparts are currently under investigation. collectively, our data provide insight into alternative macrophage polarization in humans and and adds further data to the growing body of evidence that establishment of macrophage heterogeneity is related to early ontogeny. b.-s. choi 1 , p. kropf 1 1 imperial college london, immunology department, london, united kingdom the balance between t helper (th) 1 and th2 cell responses is a major determinant of the outcome of experimental leishmaniasis, but polarized th1 or th2 responses are not sufficient to account for healing or nonhealing. we have recently shown that arginase-induced l-arginine depletion results in local suppression of antigen-specific t cell responses in nonhealing leishmaniasis. healing, induced by chemotherapy, resulted in control of arginase activity and reversal of local immunosuppression. moreover, supplementation with l-arginine restored t cell effector functions and resulted in reduced lesions size and parasite load. however, despite the efficient production of ifn-g by cd4 + t cells at the site of infection and despite the reduced pathology, the mice did not heal. we hypothesised that arginase-expressing macrophages contribute to persistent disease and become refractory to ifn-g mediated signals. to test this hypothesis, we used a well-defined model of bone marrow derived macrophages and determined whether the differentiation state of parasitized arginase-expressing macrophages could be altered. in addition, we also tested whether alternatively activated macrophages can be induced to switch off arginase and upregulate inducible nitric oxide synthase (inos) to kill the intracellular parasites. vg9vd2 t lymphocyte are activated following recognition of non-peptidic phosphorylated metabolites. the phosphoantigen isopentenyl pyrophosphate (ipp) is overproduced by tumors following hyperactivation of the mevalonate pathway of isoprenoid synthesis. previous work has shown that a molecular complex homologous to mitochondrial atp synthase (ecto-f1-atpase) is expressed on many cell types and is a possible specific ligand for the vg9vd2 tcr. the present study aims at understanding the role of f1-atpase in antigen regognition. using video microscopy calcium imaging in single vg9vd2 t lymphocytes, we can now show that the t cell response to ipp requires contact with bystander cells of variable tissue origin but that this requirement is not fulfilled by a cell line deprived of surface f1-atpase. purified f1-atpase immobilized on polystyrene beads can partly replace the need for cell-cell contact. ipp in soluble form is highly sensitive to terminal phosphatases and addition of these enzymes in t cell activation assays clearly shows that it is not recognized as such on tumors. however, we could detect nucleotide derivatives of phosphoantigens which are resistant to terminal phosphatases in the cell lysates of stimulatory tumors. one of these, a derivative of ipp, is barely able to stimulate vg9vd2 cells in the absence of apcs, as opposed to the non-nucleotidic antigen ipp. however it can bind stably to f1-atpase. thus the f1-atpase complex acts as a presenting structure for nucleotide phosphoantigens. altogether, our data suggest that vg9vd2 t cells are dedicated to the recognition of phosphoantigens in the form of nucleotide derivatives, on the surface of tissue cells and that antigen recognition involves multiple antigen modification steps, in including final cleavage by a nucleotide pyrophosphatase activity. surface plasmon resonance was used to analyse the molecular interaction between tcr and f1-atpase. by using purified f1-atpase and peptides derived from vg9vd2 tcr sequences, interaction sites between f1-atpase and tcr were identified on both ligands. based on these findings a generalized model for vg9vd2 t cell activation is proposed. ligands for the cytotoxic lymphocyte activating receptor nkg2d are highly expressed on cells stressed by numerous agents including genotoxic damage, thereby contributing to the elimination of transformed cells by nkg2d(+) lymphocytes. a key question is whether this represents a primary inductive means of immune surveillance, or merely enhances responses initiated by dendritic cells and antigen-specific t cells. a second key issue is the scope and scale of events that follow nkg2d activation in vivo. by transiently overexpressing the nkg2d ligand rae-1-beta in the skin of transgenic mice, we showed that this alone provoked rapid, coincident and reversible changes in the organization, morphology and activation state of tissue-resident vgamma5vdelta1 gamma-delta t cells and langerhans cells (lc), that were swiftly followed by epithelial infiltration of unconventional alpha-beta t cells. these data indicate a novel primary immune surveillance pathway whereby epithelial upregulation of nkg2d ligands is sufficient to provoke a series of multicomponent immunological changes. the effects on lc, which lack nkg2d and presumably respond to changes initiated by local gamma-delta t cells, are particularly interesting. ongoing microarray and co-culture experiments are now providing a molecular definition of the immume surveillance response to nkg2d ligands in vivo. to assess the scope of this response, ovalbumin was applied to the skin concomitant with rae1 induction. the primary systemic th2 response is increased by concomitant responses to a stress antigen. we will now resolve whether this increased response contributes to the adaptive memory pool, or whether it is a primary, regulatory response that may limit adaptive responses to auto-antigens exposed during stress. in addition, the many ligands available to the nkg2d receptor suggest that different ones may play unique roles. a novel nkg2d-ligand, h60c, is uniquely expressed in mouse skin. when the expression of this was further increased in a novel transgenic system, there was again an overt alteration in the local immune compartment, but with features that are seemingly distinct from the action of rae-1 induction. such studies may help resolve a long-standing puzzle over the pleiotropy of nkg2d ligands, and dissect immune surveillance of changes in gene expression levels rather than absolute levels. a.-s. invariant natural killer t (inkt) cells are a distinct lineage of t lymphocytes that co-express a highly conserved ab t cell receptor (tcr) along with typical surface receptors for natural killer (nk) cells. these lymphocytes recognize glycolipid antigens presented by the non-classical class i molecule cd1d. inkt cells are characterized by their capacity to produce rapidly large amounts of both th1 (ifn-g, tnf) and th2 (il-4, il-13) cytokines, which enables them to play a role in the regulation of many different types of immune responses, ranging from self-tolerance to responses against pathogens and tumors. converging studies in mouse models suggest that inkt cells can prevent the development of type 1 diabetes. the frequency of inkt cells is lower in non-obese diabetic mice (nod mice). manipulation of inkt cells, either by increasing their frequency or by stimulating them with agonists such as a-galcer, inhibits diabetes onset in nod mice. recently, a new population of cd4 -nk1.1 -inkt cells producing high levels of the pro-inflammatory cytokine il-17 has been identified (inkt17 cells). given that this cytokine has been implicated in several pathologies including autoimmune diseases, we investigated the role of inkt17 cells in type 1 diabetes. interestingly, nod mice exhibit a higher frequency of inkt cells producing il-17 as compared to c57bl/6 mice. this increased frequency was observed in the thymus as well as in peripheral lymphoid tissues. as previously described in normal mice, inkt17 cells present in nod mice were mainly cd4 -nk1.1 -, express the ror-g transcription factor and il-23 receptor, both molecules being usually associated with th17 commitment. we are currently analyzing, using co-transfer experiments, whether these inkt17 cells play a beneficial, a deleterious, or any role in the development of type 1 diabetes in nod mice. j. s. dodd 1 , r. muir 1 , s.s. affendi 1 , p.j. openshaw 1 1 imperial college london, respiratory medicine, london, united kingdom natural killer t (nkt) cells are a heterogeneous population of innate t cells that have attracted interest because of their potential to regulate immune responses to a variety of pathogens. upon activation with their cognate glycolipid antigen presented by cd1d molecules, activated nkt cells produce copious and numerous cytokines which endow these cells with potent immunoregulatory properties. consequently, nkt cells have become the focus for the development of vaccine adjuvants, cancer immunotherapeutics and modulators for autoimmune and inflammatory conditions. respiratory syncytial virus (rsv) is a common cold virus of the family paramyxoviridae. it is the most frequent viral cause of serious lower respiratory tract infection in infants and children worldwide and a significant contributor to winter deaths in the elderly. despite its global impact, there is still no safe and effective vaccine and our understanding of the immunological mechanisms that regulate protection and pathology is incomplete. it is known that cd1d-deficient mice with poor nkt cell responses have inefficient induction of cd8 t cells and reduced clearance of rsv, perhaps because of ifn-g release by activated nkt cells. we now show that activation of lung nkt cells with intranasal agalcer during rsv infection of mice boosts th2 immunity (increasing il-5 and il-10), promoting pulmonary eosinophilia and ablating cd8 t cell recruitment. by contrast, intraperitonal injection of agalcer enhances nk cell recruitment and boosts pulmonary cd8 t cell activity (as measured by cd25 expression), increasing ifn-g production in the airway and lung and inhibiting viral replication. effects on illness (as measured by weight loss) were similarly distinct: intranasal agalcer induced early (d4) weight loss independent of conventional t cells, whereas intraperitonal agalcer enhanced late (d7) weight loss by a cd8 t cell dependent mechanism. therefore, nkt cells stimulated by agalcer administered via different routes induce distinct types of immune response to viral infection in the lung with the intraperitonal route leading to optimal viral clearance. in general, neonatal conventional t cells, especially cd4 + ab t cells, are regarded as immature or t h 2 biased. vg9 + vd2 + t cells are unconventional lymphocytes: they are mhc-unrestricted and can react rapidly upon activation with pyrophosphates (e. g. (e)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (hmb-pp)) or aminobisphosphonates (e. g. zoledronate) in adults. until now, little is known on the functional reactivity of neonatal vg9 + vd2 + t cells towards these activators. because il-23 is preferentially secreted by neonatal dendritic cells (dc) upon tlr stimulation, we investigated the potential costimulatory effect of this cytokine on hmb-pp and zoledronate-treated neonatal vg9 + vd2 + t cells. herein, we observed that zoledronate induced neonatal vg9 + vd2 + t cell proliferation and ifn-g production in cord blood mononuclear cells (cbmc) cultures. other t h 1-like cytokines like tnf-a and gm-csf were also produced upon this stimulation, but less than ifn-g, while t h 2-like cytokines such as il-4 and il-5 were not induced. addition of il-23 to zoledronate selectively costimulated ifn-g production from neonatal vg9 + vd2 + t cells. furthermore, zoledronate/il-23 treatment resulted in neonatal vg9 + vd2 + t cells expressing high levels of the cytotoxic mediators perforin and granzyme a. zoledronate induced the expression of the receptor for il-23 (il-23r) and the transcription factor t-bet, which is known to be important for the production of ifn-g in gd t cells. in addition, costimulation with il-23 resulted in a further increase of t-bet expression in neonatal vg9 + vd2 + t cells. these changes in the expression of il-23r and t-bet likely contribute to the observed selective ifn-g response towards zoledronate/il-23 treatment. of note, in contrast to adult peripheral blood vg9 + vd2 + t cells, hmb-pp had no or only a minor effect on the functional reactivity of neonatal vg9 + vd2 + t cells. altogether, these observations show that neonatal vg9 + vd2 + t cells are functionally active and that this t cell population might play a role in protective immune responses to infections with intracellular pathogens in early life, in particular when dc-derived il-23 is produced in response to microbial stimuli. the evasion of antigen presentation is a feature common to herpesviruses. one of the strategies employed to inhibit antigen presenting molecules is ubiquitination, internalisation and lysosomal breakdown by viral e3 ligases such as hhv8 encoded k3, k5 or mhv68 encoded mk3. these viral genes represent homologues of the march family of cellular genes whose function is the regulation of cell-surface antigen presentation and reduction of the lifetime of loaded antigen complexes. ubiquitination targets surface molecules to the lysosome via the multivesicular body (mvb), a structure which also has an important role in the budding of many viruses. we investigated the existence of alternative fates for antigen presenting molecules post-ubiquitination, and how viral e3 ligases manipulate them. we discovered that both the cellular march and viral e3 ligases ubiquitinate cd1 molecules. however, whereas viral molecules inhibit cd1-antigen presentation, the march molecules are essential for the recirculation and function of the long-lived and lysosome-resistant cd1 molecules. in contrast mhc class ii was only targeted by cellular and not by viral e3 ligases. furthermore cd1 molecules could be found in viral particles as a result of ubiquitination, presumably via the mvb. thus, virally expressed and cellular e3 ligases have opposite effects, despite their homology. how this is achieved is a matter of active investigation. gamma delta (gd) t cells recognize stress-induced auto-antigens and contribute to immunity against infections and cancer. our previous study revealed that vd2 negative ( neg ) gd t lymphocytes isolated from transplant recipients infected by cytomegalovirus (cmv) killed both cmv-infected cells and ht29 colon cancer cells in vitro. in order to investigate the anti-tumor effects of vd2 neg clones in vivo, we generated hypodermal ht29 tumors in immunodeficient mice. concomitant injections of vd2 neg clones, in contrast to vd2 + cells, prevented the development of ht29 tumors. vd2 neg clones expressed chemokine c-c motif receptor 3 (ccr3) and migrated in vitro in response to chemokines secreted by ht29 cells, among which were the ccr3 ligands macrophage inflammatory protein (mip)-1d and monocyte chemoattractant protein (mcp)-4. more importantly, a systemic intraperitoneal (i. p.) treatment with vd2 neg clones delayed the growth of ht29 subcutaneous (s. c.) tumors. the effect of in vivo gd t cell passive immunotherapy on tumor growth could be reverted by addition of a blocking anti-ccr3 antibody. gd t cell passive immunotherapy was dependent upon the cytotoxic activity of the gd effectors towards their targets since vd2 neg clones were not able to inhibit the growth of a431 hypodermal tumors. our findings suggest that cmv-specific vd2 neg cells could target in vivo cancer cells, making them an attractive candidate for anti-tumor immunotherapy. more recently, we generated ht29 cells expressing the luciferase and realized orthotopic injection of ht29-luc cells. progressive tumor development and regression following « gd treatment » will be observed in vivo using bioluminescent imaging. intraepithelial lymphocytes (iel) compose large, oligoclonal, tissue-associated repertoires of non-mhc-restricted t cells that play key roles in immunosurveillance. it is commonly considered that the characteristic iel repertoires are positively selected by thymic epithelial molecules that are also stress-induced in specific tissues, thereby activating iel function. however, no such molecules have been identified. here we characterise skint1, currently the only known determinant of a canonical iel compartment, that is selectively required for vg5vd1 + dendritic epidermal t cell (detc) development. we show that both peripheral and thymic skint1 expression is essential for full detc development. its effects are highly specific since even substantial and ubiquitous over-expression neither negatively selects detc, nor affects any other t cells. unexpectedly, however, skint genes are not expressed by cell lines and are downregulated rather than activated by carcinogenesis. mouse genetic models allow powerful insight into skint1 function; for example, we demonstrate that the constitutive expression of wild-type skint1 fully restores detc development in a skint1 mutant mouse, but does not rescue normal detc function. thus, skint1 provides a novel perspective into how epithelia regulate the development and function of specific tissue-associated t cell compartments, and how normal versus dysregulated tissues may be demarcated. marginal zone (mz) b cells are strategically localized in the mz of the spleen. since most of the blood reaching the spleen is passing through this region such localization favors contact with blood born antigens and pathogens. besides being able to rapidly secrete antibodies, mz b cells may also act as professional antigen presenting cells (apcs). they are known to express high levels of cd1d which is the presenting molecule for nkt cells which are also located in the mz. therefore we hypothesised that mz b cells may be efficient activators of nkt cells. to test this hypothesis, we used freshly sorted splenic mz b cells (cd19 + cd21 hi cd23 lo cd11c -) and splenic conventional dendritic cells (cdcs) (cd11c hi cd8a +/-cd11b +/-b220 -) from wt and cd1d -/mice as apcs for nkt cells from va14-ja18 transgenic or wt mice. the apcs were treated with agalactosylceramide (agalcer) or heat killed (hk) listeria monocytogenes or salmonella typhimurium. both mz b cells and cdcs proved to be highly efficient apcs for priming of nkt cells and induced robust proliferation. in contrast, other populations of b cells failed to activate nkt cells. we showed, using cd1d -/mice as well as blocking antibodies to icosl, that proliferation of nkt cells depends on tcr/cd1d and in case of mz b cells, also on icos/icosl interactions. importantly, apcs primed with hk bacteria were not able to induce nkt cell proliferation. interestingly, mz b cells exclusively induced production of il-4 by nkt cells. in contrast, cdcs mostly induced production of ifn-g and il-4 producing cells were scarce under these conditions. cytokine production by nkt cells proved to be independent of tcr signalling, but dependent on icos/icosl interactions when mz b cells were used as apcs, and gitr-dependent when cdcs were used. taken together, our data suggest that both mz b cells as well as cdc act as professional apcs for nkt cells. notably, the nature of apcs appears to be critical for polarization of the immune response: mz b-cell-primed nkt cells induce cytokine milieu fostering a t h 2 response, whereas cdc-primed nkt cells rather favor a t h 1 response. objectives: il-18 is an innate cytokine present in elevated levels in sera from patients suffering from autoimmunity (eg. sle and ra) and the allergic disease atopic eczema. in mice, injections of il-18 give rise to an early polyclonal isotype switched antibody response which is absent in inkt cell deficient (cd1d -/-) mice. we set out to investigate the activated b cells in il-18 injected mice and how these are regulated by inkt cells. methods: mice received daily i. p. injections of il-18 (2 mg) for 10 days and the antibody response in serum was monitored using elisa. the b cell activation in the spleen at day 14 was evaluated by flow cytometry and immunohistology. results: mice injected with il-18 developed self reactive (anti-pc and anti-dna) antibodies in the serum, in line with the autoreactive antibodies in patients with e. g. sle and atopic eczema. the antibody producing cells formed cd138 + cell clusters in the red pulp of the spleen, a typical feature of extrafollicular activation frequently associated with autoreactive responses. surprisingly, the antibody response induced by il-18 was increased in inkt cell deficient (cd1d -/-) mice, in contrast to published data. an increased response to il-18 was also observed in ja281 -/mice, which lack the a-chain of the tcr used by inkt cells, and thus our data suggest that inkt cells inhibit antibody producing cells in il-18 induced antibody responses. further characterization of the recruitment of b cells in il-18 injected mice revealed a marked expansion of the marginal zone b cell (mzb) population in the spleen, suggesting an important role for mzbs in the il-18 induced autoreactive antibody response. mzbs are innate-type b cells that express high levels of cd1d, are prone to autoantibody production and often involved in early immune responses. the il-18 induced antibody response in mzb deficient (cd19 -/-) mice was either decreased (igg) or delayed (ige), supporting the importance of mzbs in il-18 induced antibody responses. we conclude that the role for inkt cells in il-18 induced antibody responses is to inhibit the production of autoreactive antibodes from mzbs in extrafollicular foci. objectives: amoebiasis is a widespread human parasitic disease caused by the intestinal protozoan entamoeba histolytica. there are two major clinical manifestations of the disease, amoebic colitis and amoebic liver abscess (ala). interestingly, only a small proportion of e. histolytica-infected individuals develop invasive disease, whereas the majority harbors the parasite within the gut without clinical symptoms. so far, cells of the innate immune system have been described to constitute the main host defense mechanism for the control of amoebiasis, relying largely on the early production of interferon-g (ifn-g). however, information is lacking about the sources of early ifn-g production as well as the amoeba antigens involved in this activation process. methods: using a recently developed c57bl/6 mouse model for ala, the contribution of natural killer t (nkt) cells for protection against amoebic disease was investigated. applying nkt cells and dendritic cells as antigen-presenting cells from various ko-mice, the signaling pathways implicated in recognition of amoebic antigens and activation of cytokine-secretion by nkt cells was analysed. results: nkt cells were found to play a key role in the defense against ala. specific activation of nkt cells by a-galactosylceramide (a-galcer) induced significant protection, whereas jalpha 18-/-and cd1d-/-mice lacking inkt as well as dnkt cells suffered from more severe abscess formation. a lipopeptidophosphoglycan, which is present in large quantities on the surfcae of e. histolytica trophozoites (ehlppg), was identified as a major amoeba antigen that activates nkt cells resulting in the production of ifn-g, but not of il-4. moreover, ifn-g production required the presentation of ehlppg by cd1d and signaling through the tlr receptor cascade in combination with a simultaneous secretion of il-12. similar to a-galcer application, treatment of mice with purified ehlppg significantly reduced the severity of ala in amoeba-infected mice. our study provides a mechanism for the innate control of amoeba invasion that might explain why the majority of e. histolytica-infected individuals do not develop amoebic disease. a few years ago, we have observed a significant expansion of circulating effector gamma delta t cells following cytomegalovirus (cmv) infection in kidney transplant recipients (ktr). these unconventional t cells display tcr dependent cytotoxicity against both cmv-infected cells and carcinoma cells. in the present study, an extensive phenotyping of gamma-delta t cells allowed us to demonstrate an over-expression of cd16 in cmv-infected individuals. cd16 is the fcgammariiia, a natural killer cell marker usually absent on conventional t cells. we found that 71.9 ± 15.9 % of gamma-delta t cells from 21 cmv-infected ktr expressed cd16, when compared with only 19.8 ± 16.7% in 11 non cmv-infected ktr (p x 0.0005). similarly, 51.9 ± 25.7 % of gamma-delta t cells from 13 cmv-seropositive blood donors expressed cd16 compared to 27.1 ± 25.1 % in 15 cmv-seronegative donors (p x 0.01). cd16+ gamma-delta t cell lines generated from cmv-infected individuals were able to produce ifn-g (a potent anti-viral cytokine) in a cd16-dependent manner when activated by cmv/igg immune complexes. this production greatly increased in the presence of il-12 and ifn-alpha, two cytokines highly produced during cmv-infection. the supernatants of gamma-delta t cells activated with agonist anti-cd16 mab inhibited cmv replication in vitro and this effect was abrogated in the presence of a blocking anti-ifn-g antibody. cmv/igg immune complexes were also able to induce the expression of the cytotoxicity marker cd107a on cd16+ gamma-delta t cell lines. cd16 is well-known to mediate antibody-dependant cellular cytotoxicity (adcc), especially in natural killer cells. accordingly, we demonstrated that cd16+ gamma delta t cell lines could make adcc against the daudi lymphoma cell line and the a431 skin carcinoma cell line pre-incubated either with rituximab (anti-cd20) or cetuximab (anti-egfr), respectively. in contrast, no addc could be observed against cmv-infected fibroblasts pre-incubated with polyclonal anti-cmv igg (cytogam), probably because cytogam weakly stained infected cells. these data reveal a new cd16-dependent anti-cmv function of gamma-delta t cells through recognition of immune complexes and secretion of ifng. moreover, they demonstrate that these cells are able to kill through adcc lymphoma and skin carcinoma cells, two tumour types frequently encountered in ktr. dendritic epidermal t cells are a prototypic population of intraepithelial gd t cells in the mouse skin. found in the basal layer of epidermis and in close contact with langerhan's cells and keratinocytes detc facilitate vital immunological and physiological processes e. g. wound healing, homeostasis, tumor surveillance and regulation of inflammation. gd t cells respond rapidly to non-peptidic microbial and stress induced self antigens in a non-mhc restricted manner and are therefore proposed to bridge the gap between innate and adaptive immunity. by using gd t cell knock-out mice tcrd-/-, ovalbumin transgenic k5mova mice and a skin grafting model we aimed to elucidate the role of gd-detc in adaptive immune responses associated with elimination of foreign antigen presented in the skin.we show that in the absence of gd t cells in the skin there is a decrease in rejection of ovalbumin expressing skin grafts compared to wildtype mice. we show that optimal regimens of antigen delivered subcutaneously in conjunction with adjuvant elicits comparable responses in wildtype and knockout mice. however frequency of primed host animals is reduced in tcrd-/-mice when antigen is delivered epidermally via skin grafting; suggesting detc enhance cross presentation of classical mhc bound antigens in the skin. considering the incapability of gd t cells to recognize peptide antigens in the context of mhc we plan to dissect the relationship between detc and professional antigen presenting cells in the skin. understanding the underlying mechanisms of this relationship will expand our knowledge of enhancing professional apc function in skin by detc and potentially other epithelia by intraepithelial gd t cells and can be useful in designing therapies to epithelial infections and malignancies. we demonstrate a rapid and hmb-pp-dependent crosstalk between gd t cells and autologous monocytes that resulted in the production of inflammatory mediators including il-6, ifn-g, tnf-a, osm, ccl2, cxcl8, cxcl10, and trail. moreover, under these co-culture conditions monocytes showed enhanced survival and differentiated overnight into inflammatory dcs with antigen-presenting functions. these cells expressed cd40, cd86, hla-dr, and dc-sign, and lost cd14, ccr2, ccr5, and cxcr4. addition of further microbial stimuli (lps, peptidoglycan) induced ccr7 and enabled these inflammatory dcs to trigger antigenspecific cd4 + effector ab t cells expressing ifn-g and/or il-17. importantly, our in vitro model replicated the responsiveness to microbes of effluent cells from pd patients and translated directly to episodes of acute pd-associated bacterial peritonitis, where vg9/vd2 t cell numbers and soluble inflammatory mediators were elevated in patients infected with hmb-pp-producing pathogens. conclusion: our findings suggest a direct link between invading pathogens, microbe-responsive gd t cells, and monocytes in the inflammatory infiltrate, which plays a crucial role in the early response and the generation of microbe-specific immunity. the mechanism(s) responsible for their dichotomous behaviour are poorly understood, and the outcome of nkt cell manipulation remains unpredictable. there is growing evidence that the nkt cell pool is composed of functionally distinct subsets, but such a possibility has not yet been investigated in a model of nkt cellmediated immunosuppression. we examined the differential ability of nkt cell subsets from the thymus and liver to prevent type i diabetes when transferred into prediabetic nod mice. the transfer of abtcr+dn thymocytes (a population enriched for nkt cells) has previously provided robust protection against tid development; however it has not been formally shown that nkt cells are solely responsible for the protection. our study found that while the transfer of thymic dn nkt cells can prevent tid and severe insulitis in nod mice, not all nkt cell subsets show the same tolerogenic capabilities. these findings both formally demonstrate the disease-preventing effects of nkt cell transfer in nod mice and provide further evidence that nkt cells are a functionally heterogeneous population. objective: vg9/vd2 t cells constitute a minor t cell population in human blood that expands specifically and rapidly in response to the microbial metabolite hmb-pp. our previous microarray studies showed that vg9/vd2 t cells stimulated with hmb-pp in the presence of il-21 express markers associated with a possible follicular b cell helper function. we therefore investigated in more detail whether and how hmb-pp and il-21 regulate expression of the b cell attracting chemokine cxcl13/bca-1, its receptor cxcr5, and co-stimulatory molecules involved in b cell help. purified peripheral vg9/vd2 t cells were co-cultured with autologous monocytes or b cells (as feeder cells) for up to 4 days with and without hmb-pp, in the absence or presence of il-2 or il-21, or in medium alone. cells were analysed by flow cytometry and immunofluorescence microscopy. results: high levels of cxcl13 protein were detected in co-culture supernatants only when both il-21 and hmb-pp were provided, implying an il-21-dependent and tcr-dependent expression. vg9/vd2 t cells were confirmed as producers of cxcl13 by flow cytometry and immunofluorescence. under the same conditions, activated vg9/vd2 t cells expressed cd27, cd28, cd40l, cd70, icos and ox40. in contrast, neither cxcr5 nor ccr7 changed markedly by il-21 stimulation of peripheral vg9/vd2 t cells. conclusion: our findings confirm on the protein level that stimulation of vg9/vd2 t cells with hmb-pp and il-21 induces markers typically associated with follicular b helper t (t fh ) cells. these data suggest that gd t cells contribute to humoral immune responses and play a role in germinal centre formation and production of high-affinity antibodies in microbial infection. ongoing analyses of gd t cells in inflamed and non-inflamed lymphoid tissues (tonsils, appendices) aim at demonstrating the physiological relevance of our findings. y. emoto 1 , m. emoto 1 1 gunma university school of health sciences, department of laboratory sciences, maebashi, japan invariant (i) natural killer (nk)t cells become undetectable after stimulation with a-galactosylceramide (a-galcer) or interleukin (il)-12. although downmodulation of surface t cell receptor (tcr)/nkr-p1c (nk1.1) expression has been shown convincingly after a-galcer stimulation, it is unclear whether this holds true for il-12 stimulation. to determine whether failure to detect inkt cells after il-12 stimulation is caused by dissociation/internalization of tcr and/or nkr-p1c or by block of de-novo synthesis of these molecules, and to examine the role of il-12 in disappearance of inkt cells after a-galcer stimulation, surface (s)/ cytoplasmic (c) protein expression as well as mrna expression of tcr/nkr-p1c by inkt cells after stimulation with a-galcer or il-12, and influence of il-12 neutralization on down-modulation of stcr/snkr-p1c expression by inkt cells after a-galcer stimulation were examined. the s/ctcr + s/cnkr-p1c + inkt cells became undetectable after in-vivo administration of a-galcer, which was partially prevented by il-12 neutralization. whereas s/cnkr-p1c + inkt cells became undetectable after in-vivo administration of il-12, s/ctcr + inkt cells were only marginally affected. mrna expression of tcr/nkr-p1c remained unaffected by a-galcer or il-12 treatment, despite the down-modulation of ctcr and/or cnkr-p1c protein expression. in contrast, ctcr + cnkr-p1c + stcr -snkr-p1c -inkt cells and cnkr-p1c + snkr-p1c -inkt cells were detectable after in-vitro stimulation with a-galcer and il-12, respectively. our results indicate that tcr and nkr-p1c expression by inkt cells is differentially regulated by signaling through tcr and il-12r. they also suggest that il-12 participates, in part, in the disappearance of inkt cells after a-galcer stimulation by down-modulating not only snkr-p1c but also stcr. the fetus and infant are highly susceptible to viral infections. a number of viruses, including human cytomegalovirus (cmv), cause more severe disease in early life compared to later life. it is generally accepted that this higher susceptibility to viral infections is due to the immaturity of the immune system. gd t cells are unconventional t cells that can react rapidly upon activation and show mhc-unrestricted activity. herein, we show that upon cmv infection in utero, fetal gd t cells expand and become differentiated. the response was restricted to vg9-gd t cells, irrespective of their vd chain expression. differentiated gd t cells expressed high levels of ifn-g, transcription factors t-bet and eomes, natural killer receptors and cytotoxic mediators including perforin and granzymes. in addition, congenital cmv-infection induced a highly restricted complementary-determining region 3 d1 (cdr3d1) and cdr3d2 repertoire, with a striking enrichment in a specific germline-encoded cdr3d1 sequence. differentiated gd t cells and the enriched cdr3d1 sequence were detected as early as after 21 weeks of gestation. our results indicate that functional fetal gd t cell responses can be generated during development in utero and suggest that this t cell subset could participate in anti-viral defense in early life. results: spectratyping showed only in-frame selection for vd1-jd1 and vgi-jg1.3/2.3 rearrangements in tcrgd thymocytes and to a lesser extent in tcrgd cb cells. in contrast, clear in-frame vd2-jd1 and vg9-jg1.2 selection was seen in pb tcrgd cells. detailed analysis of the cdr3 motifs revealed selection determinants in both vg9-jg1.2 (canonical length and cdr3 motif) and vd2-jd1 (minimal cdr3 length in combination with an invariant t nucleotide) rearrangements. upon evaluation of the replication history we found a clear increase in the number of cell divisions from naïve tcrgd thymocytes (˚4) and tcrgd cb cells (6-7) to tcrgd pb cells (˚10 or more). no increase was seen between cb and pb tcrgd t cells within the first year of life, suggesting that peripheral proliferation occurs later in life. our results indicate that the human peripheral tcrgd repertoire is shaped by (antigenic) selection and proliferation processes. moreover, the ontogenetic changes in the gd repertoire between the central and peripheral immune systems are clearly influenced by proliferation. background: natural killer (nk) t cells have been implied in the regulation of disease in the non obese diabetic (nod) mouse model of type 1 diabetes (t1d). we have previously shown that transgenic expression of a cd1d-restricted, va3.2-vb9 tcr in nod mice lead to an increase in cd1d-restricted type ii nkt cells (24abnkt cells), and prevention of the development of t1d in the transgenic mice. in this study we have investigated the requirements and underlying mechanism of disease protection by type ii nkt cells in a disease transfer model. to investigate the mode of regulation by 24abnkt cells, we explored a disease transfer model into nod.scid mice using transgenic diabetogenic bdc2.5 cd4+ t cells, in the presence or absence of selected cells from 24abnkt cell transgenic mice. results: in 24ab transgenic mice a high frequency of activated transgenic nkt cells was found in the pancreas of the protected mice. in this organ, 24abnkt cells expressed a high level of cxcr3 and a low level of ccr7 and cd62l, a pattern similar to that observed in t cells homing to inflammatory tissues. adoptive transfer of cd4+ bdc2.5 t cells into nod.scid recipients rapidly induced onset of diabetes. using this model, we found that co-transfer of spleen cells from 24ab transgenic mice with bdc2.5 cd4+ cells resulted in the prevention of diabetes development. the protection from disease required a minor cd4+ subset of 24ab+ nkt cells, but was independent of cd25+ t regulatory cells. analogs of alpha galactosylceramide (a-galcer) that may modulate the strong activation of inkt and at the same time prolong their effect upon in vivo administration are a long standing goal of research in this area due to their putative immunotherapeutical applications. a new class of non glycosidic analogues bearing an aminocyclitol ring as galactose surrogate have been synthesized and assayed in their capacity to be presented by cd1d and recognized by inkt. the structural novelty of these compounds resides in the presence of a cyclohexane that substitutes the sugar moeity and the substitution of the o glycosidic linkeage with the ceramide by a n. in this basic structure, substitutions in the cyclohexane ring with oh in different conformations mimicking different sugars, differences in the length of the sphingosine lipid and differences in the orientation of the n linkeage conform a series of analogs that have been analyzed in their capacity to stimulate inkt cells. proliferation assays in bulk splenocyte cultures and cytokine secretion determinations show that inkt cells are specifically stimulated by some of the analogs tested. in particular, the active compound hs44, induces in vitro inkt cell expansion and ifng and il-4 secretion in a similar fashion but less potently than a-galcer. dose response assays show a bias towards a th2 profile response after recognition by nkt cells, more similar to the response induced by och. the degree of structural similarity of the cyclitol ceramides with a-galcer parallels their cellular activities. these data open the way towards the development of a new class of a-galcer lipid analogues having charged amino substituted polar heads resistant to glycosidase degradation, thus enhancing their in vivo biodisponibility, and expands the range of potential inkt cell sphingolipid agonists that can modulate the immune response due nkt cell activation. objectives: invariant natural killer (ink) t cells represent an innate lymphocyte subset with important modulatory functions. in the presence of pathogens or tumors, inkt cells play an adjuvant function that boosts t cell immunity through cytokine secretion and dc maturation. in steady-state conditions, i.e. in the absence of pathogens, inkt cells acquire a regulatory function that promotes t cell tolerance and prevents autoimmune disease. our aim was to assess the mechanism of action of inkt cells in the steady state and, specifically, to test the hypothesis that inkt cells promote immune tolerance through modulation of dcs. methods: to assess the direct influence of regulatory inkt cells on dc maturation in resting conditions, we derived murine inkt cell lines in vitro and, after staining with agalcer-loaded cd1d tetramers and magnetic purification, we tested their capacity to modulate bone marrow-derived myeloid dcs in the absence of any other maturation signals. we analyze the transcriptional profile (microarray analysis) as well as maturation, cytokine expression profile and pro-tolerogenic antigen-presenting function of inkt cell-modulated dcs (inkt-dcs). the cell-cell interaction with inkt cells provoked dramatic phenotypical changes on immature dcs that acquired the cardinal features of tolerogenic dcs such as intermediate levels of mhc class ii and co-stimulatory molecules expression and high secretion of il-10 with no release of pro-inflammatory cytokines. most importantly, inkt-dcs acquired tolerogenic antigen-presenting function inducing the differentiation of regulatory tr1 cells and immune tolerance in vivo. dcs, simultaneously stimulated with inkt cells and through toll-like receptor (lps) completely lost the pro-tolerogenic phenotype and acquired a proinflammatory cytokine profile. conclusion: it is still mysterious how inkt cells can play a dual role and either boost t cell immunity or promote immune tolerance. our results suggest that the same mechanism could underlie both inkt cell functions. in the presence of pathogen-driven maturation signals, the inkt cell-modulation of dcs favors their acquisition of a pro-inflammatory phenotype and function. on the contrary, if inkt cells are activated in the absence of pathogens, e. g. during autoimmune conditions, their interaction with immature dcs promotes their tolerogenic maturation to maintain peripheral tolerance and counter-regulate autoimmune diseases. th17-type immune responses have been reported to fight extracellular bacterial infection, but as well to cause autoimmune diseases and allergy. the th17 immune response is characterized by the secretion of il-17a and il-17f. the il-17 locus encodes the highly conserved il-17a and il-17f cytokines that are syntenic in 44kb distance to each other. besides cd4 + th17 and nkt cells, approximately 50 % of the il17a producers are gd t-cells. like cd4 + th17 cells, il-17 producing gd tcells have recently been implicated to play a major role in the immune response to infections with extra-and intracellular bacteria. our findings show a difference between the il-17 production of gd t cells in the peripheral system and mucosal epithelia. mucosal gd t-cells generally do not produce th17 cytokines. in the periphery, we define novel subsets of gd t-cells that can produce either il-17 or ifn-g. combined with the well known classification of il-17 producing gd t-cells along the markers cd27 and cd122, our data point at specialized functions of the different gd t cell subsets depending on their location and origin. functional studies are currently carried out in order to address the role of the different gd t-cell subsets for th17-type immune responses in vivo. in this context, the potential redundancy of il-17a and il-17f may complicate the analysis. so far, most studies were carried out with il-17a single-deficient or il-17f single-deficient mice. to further clarify these issues, we will have to address the above mentioned findings in il-17a and il-17f double-deficient mice. several subsets of gd tregs have been described and intensively studied, but the potential regulatory role of innate t cells in controlling immune responses remains unclear. lymphocytes expressing gd tcr are involved in both innate and adaptive immune responses. vg9vgd2 t cells, which represent a major peripheral blood gd t-lymphocyte subpopulation in humans, display a broad reactivity against microbial agents and tumors.here we report that tgf-b1 and il-15 differentiate in vitro a subset of gd t lymphocytes with regulatory functions (vd2 tregs) in the presence of specific antigen stimulation. these cells express the forkhead/winged helix transcription factor (foxp3) and, similarly to ab tregs, suppress the proliferation of anti-cd3/anti-cd28 stimulated-pbmc. detailed knowledge about the phenotype and functionality of vd2 tregs will improve our understanding of the role of gd t cells in the pathogenesis and regulation of autoimmune, infectious and cancer diseases. a-galactosylceramide (a-galcer) has the potential to activate invariant (i) nkt cells, which in turn release a wide variety of cytokines that stimulate immunocompetent cells. although this rapid and vigorous cytokine release appears critical for regulation of various immune responses, it remains elusive whether protection against intracellular bacteria can be induced by a-galcer. here we show that treatment with a-galcer ameliorates murine listeriosis, and inhibits inflammation in the liver and spleen following listeria monocytogenes infection. liver infiltration of granulocytes and g/d t cells was accelerated by a-galcer treatment. granulocyte and g/d t cell depletion exacerbated listeriosis in a-galcer-treated mice, and this effect was more pronounced in granulocyte than in g/d t cell depletion. although secretion of gm-csf and il-17 was detected among the nkt cell population in the liver and bone marrow immediately after a-galcer treatment, infiltration of granulocytes into the liver was not prevented by neutralizing mab. yet, in parallel to the numerical increase of granulocytes expressing cd11b in the liver following a-galcer treatment, numbers of cells lacking cd11b diminished in the bone marrow. in addition, respiratory burst in granulocytes was enhanced by a-galcer treatment. our results indicate that a-galcer-induced antibacterial immunity is caused, in part, by accelerated infiltration of inflammatory cells, in particular granulocytes and to a lesser degree g/d t cells, into the liver. we also suggest that the infiltration of granulocytes is caused by an accelerated supply of granulocytes from the bone marrow, rather than by accelerated granulopoiesis. objectives: the aim of this work is to evaluate whether phenotypic and functional features of vgamma9/vdelta2 t cells are influenced by the activity of mevalonate pathway in tumor cells and contribute to determine disease aggressiveness in cll. methods: eighty seven previously untreated cll patients were evaluated for in vitro vgamma9/vdelta2 t cells expansion upon stimulation with zoledronic acid (za) and interleukin-2 (il-2). gammadelta t cells subset distribution and natural killer receptors profile were evaluated by multicolor flowcytometry. the mutational status of the tumor immunoglobulin heavy chain variable region (igvh) was analyzed by dna sequencing. the activity of the mev pathway was determined by 1) the bioinformatic analysis of gene expression profiling data 2) the quantification of mev pathway metabolites. results: proliferation of gammadelta t cells was observed in 43 patients (49 %) (responders, r), whereas 44 patients (51 %) were non-responders (nr). vgamma9/vdelta2 t-cell subset distribution was well balanced in r patients, whereas effectors subsets [i. e., effector memory (tem), and terminally differentiated effector memory (temra)] were largely predominant in nr patients. temra of nr patients mainly expressed the inhibitory receptor ilt2, whereas temra of r patients had an higher expression of the costimulatory molecule nkg2d. the proliferative response of vgamma9/vdelta2 t cells was significantly associated with igvh mutational status, which is a well known prognostic factor in cll. indeed, 82 % of r patients were m, whereas 77 % of um patients were nr (p x 0.001). given this association, we evaluated the activity of the mev pathway in tumor cells of m and um patients. the pathway was more active in tumor cells of um than m patients, suggesting that the former can more easily engage gammadelta t cells and drive their differentiation into functionally exhausted t emra . given the association between the r/nr status and the igvh mutational status we also analyzed the independent prognostic impact of r/nr status in multivariate cox analysis. nr patients had a significantly shorter time to first treatment thus pointing to r/nr status as an independent prognostic factor. conclusion: these data define a novel mechanism of immune escape which can contribute to determine disease aggressiveness in cll patients. the studies reported here were undertaken to ascertain and delineate the ability of kupffer cells to regulate the response of inkt cells to biliary obstruction. methods: c57bl/6 mice were not treated or rendered kupffer cell-depleted by intravenous inoculation of liposome-encapsulated dichloromethylene diphosphonate. to clarify the factors that elicit inkt cell activity, additional mice were administered anti-il-12 p40 (clone r2-10f6; atcc) or anti-cd-1d (clone 1b1) monoclonal antibody (mab) prior to surgery. midline laparotomies were performed; the common bile duct was ligated twice and divided. sham-operated animals served as controls. blood and liver samples were collected at periodic intervals post-surgery. the hepatic lymphoid population was purified and characterized by flow cytometry. the nkt cell population was increased significantly in the livers of control, but not kupffer cell-depleted, mice at 18 hours post-bdl. the response of inkt cells was diminished in mice pretreated with mab specific for il-12p40, a component of both il-12 and il-23; pretreatment with anti-cd1d mab had no effect. il-12rb-deficient mice also exhibited a marked increase in hepatic inkt cells following bdl suggesting that il-12 was not a critical factor. this suggestion is supported by the increased expression of il-23p19 and il-12p40 (but not il-12p35) mrnas by kupffer cells purified from the livers of bdl animals. these findings imply that il-23 production by kupffer cells promotes the response of hepatic inkt cells to biliary obstruction. objectives: p-glycoprotein (pgp or abcb1) is a member of the abc family of transporter proteins which are characterized by their ability to pump molecules across membranes in an atp-dependent manner. although pgp was first identified for its ability to confer resistance to chemotherapeutic agents in tumor cells, it has now also been described in cells of the immune system. our work primarily focuses on gd t cells that complement and regulate the activities of ab t cells, particularly in tissues. we have recently described functional subsets of gd cells based on cd27 expression. gd 27+ cells secrete interferon-g, while gd 27cells are capable of producing il-17. this study investigates the role of pgp in gd cells with specific reference to these recently-identified cd27-defined subsets. methods: pgp activity was measured based on the expulsion of rhodamine 123. cells were incubated with rho followed by a period in the presence or absence of the pgp inhibitor cyclosporine-a. cell populations were identified using monoclonal antibodies and flow cytometry. percentages of subpopulations were compared by anova, statistical results are shown as p values that were calculated using a newman-keuls multiple comparison post-hoc test. results: up to 40 % of intraepithelial lymphocytes (iels) from the small intestine are tcrgd + . of these, virtually all displayed pgp activity. indeed, pgp activity was generally higher in tcrgd + than tcrab + iels. in the thymus, pgp activity was observed in only˚2% of gd 27+ cells but not at all in gd 27cells. by contrast, in peripheral lymph nodes, mesenteric lymph nodes and peyer's patches, 40-60 % of gd 27+ cells were positive for pgp activity, although their gd 27counterparts remained largely negative (p x 0.01). conclusion: this study demonstrates that subsets of gd cells display different levels of pgp activity depending on their location in the body and their expression of the newly identified functional marker cd27. as pgp activity may play a role in cytokine release, cytotoxicity and protection from harmful toxins, it confirms our hypothesis that gd 27+ and gd 27cells have very different roles in immune responses and provides insight into the mechanism by which gd cells cope with diverse body locations. objectives: an effective immune response orchestrates different cellular activities of both innate and adaptive immune compartments. in this context, the vgamma9vdelta2 t cell biology presents some critical features for their ability to display a broad antimicrobial activity by directly killing infected cells and by inducing an effective adaptive immune response. the activation of vgamma9vdelta2 t cells by aminobisphosphonate drugs such as zoledronic acid (zol) results in a massive release of cytokines and chemokines that may induce a bystander activation of other immune cells such as dendritic cells (dcs) and b lymphocytes. the aim of this work was to evaluate the ability of activated vgamma9vdelta2 t lymphocytes to orchestrate granulocytes functions in terms of migration capability, phagocytic activity and alpha defensin release. methods: peripheral mononuclear cells (pbmc) and purified vgamma9vdelta2 t cells from healthy donors were stimulated with different compounds (zol, ipp) for 24 hours and supernatants from these cultures were tested for their ability to induce granulocytes activation. briefly, we analysed the migration activity, the phagocytic activity and the degranulation process by perforimg migration assays, flow cytometry and elisa tests. we showed that soluble factors released by zol-stimulated vgamma9vdelta2 t cells activate granulocytes by inducing their chemotaxis, phagocytosis, and alpha-defensins release. proteomic analysis allowed us to identify a number of cytokines and chemokines specifically released by activated vgamma9vdelta2 t cells. moreover, mcp-2 depletion by neutralizing ab revealed a critical role of this chemokine in induction of granulocyte alpha-defensins release. altogether, these data show a vgamma9vdelta2-mediated activation of granulocytes through a bystander mechanism, and confirm the wide ability of vgamma9vdelta2 tlymphocytes in orchestrating the immune response. conclusion: an immune modulating strategy targeting vgamma9vdelta2 t cells may represent a key switch to induce an effective and well-coordinated immune response, and can be proposed as a way to strengthen the immune competence during infectious diseases. objectives: the aim of this study was to analyse the activity of vg9vd2 t lymphocytes against glioma cells and to verify the possibility to target these innate cells in new immunotherapeutic approaches. human vg9vd2 t cells recognize and kill several cancer cells presenting a disregulation in mevalonate pathway. interestingly, drugs already in clinical use, such as zoledronic acid, are able to promptly activate vg9vd2t cells through an indirect mechanism involving the block of farnesyl pyrophosphate synthase of the mevalonate cycle. the vg9vd2 t cell activation by zoledronic acid results cytokines and chemokines synthesis and cytotoxic activity. glioma are tumors arising from glia in the central nervous system. unfortunately, the majority of glioma patients die in less then of a year from diagnosis and new treatment strategies are therefore hardly needed. methods: in order to analyse the activation of vg9vd2 t cells and their effects on the viability of glioma cells, we expanded in vitro vg9vd2t cells from pbmcs of healthy donors by using phosphoantigen stimulation and tested the ability of vg9vd2t cell lines to kill three different glioma cell lines (t70, u251, u373) by cytokinic/cytotoxic mechanism by flow cytometry. results: our results demonstrated that vg9vd2t cells lines are able to recognize glioma cells, to differentiate in effector memory cells, and to kill glioma cells by releasing perforin. moreover, we analysed whether zoledronic acid treatment could improve the susceptibility of glioma cells to vg9vd2 t lines. we showed that zoledronic acid is able to directly induce cell death on glioma cells and to strongly enhance the cytotoxic activity of vg9vd2 t lines. conclusions: altogether, our results suggest that the induction of a strong antitumor response in vitro of vg9vd2 t cells by using aminobisphosphonates could represent a new interesting immunotherapeutic approach for glioma treatment. viral-induced cancers, such as cervical cancer and liver cancer, contribute to approximately 15 % of all cancers and represent a failure of host immunity to control chronic viral infection. natural killer t (nkt) cells are a population of regulatory t lymphocytes that are pivotal to the outcome of host protection to a range of viral infections and cancers, but their role in controlling host defenses to oncogenic viruses in epithelial and cutaneous tissue is virtually unexplored. using a mouse model of chronic viral infection in the skin, in which human papillomavirus (hpv) oncoproteins are expressed as a transgene in epithelial cells, we investigated the role for nkt cells in abrogating protective immunity to viral antigens in cutaneous tissue. we show that local hpv-e7 protein expression in the skin attracts a large lymphocytic infiltrate, including a population of cd1d-restricted nkt cells. this nkt infiltrate is required to maintain local hpv-e7-induced immune suppression and results in graft survival when transplanted onto a naive, immunocompetent host. the local suppressive environment evident in e7-expressing transplanted skin is dependent on interactions between populations of cd1d-expressing cd11c+/f480+ myeloid cells and nkt cells. removal of either donor-resident or host-infiltrating nkt cells is sufficient to break immune suppression and allow e7 graft rejection. dissecting the suppressive properties of nkt cells in this novel model of chronic viral antigen presentation in the skin will provide valuable new insight into the potential for clinical manipulation of nkt cell populations to restore chronic anti-viral and anti-tumour immunity in epithelial tissues. nkt cells were expanded from total pbmcs from healthy donors by treatment with il-2 and a-galcer. expression of cd1a, cd1d and the costimulatory molecules cd86 and hla-dr, was established by flow cytometry. rna was quantified by real time-pcr. functional assays were performed by analysis of nkts cytokine production (ifn-g, il-4) and cytotoxicity against treated-idcs. results: idcs stimulated with olive pollen lipids up-regulated cd1d expression on the cell surface in comparison with control cells. in contrast cd1a expression was decreased. cd86 and hla-dr slightly increased, indicating certain grade of maturation. the amount of cd1d mrna was higher in treated cells than in control cells. by contrast, there was less transcription of cd1a, cd1b and cd1c genes than in control cells. nkt cells efficiently killed treated idcs as "in vitro" cytotoxic killing assays showed. ifn-g producing cells increased slightly in response to treated idcs compared to unstimulated cells, but the number of il-4 producing cells was not modified. similar results were obtained using monocytes as antigen-presenting cells. conclusions: idcs treated with lipidic extracts from olive pollen up-regulate the expression of cd1d on the cell surface. in addition, nkt cells are able to recognize idcs and monocytes treated with lipids from pollen, producing ifn-g and cytotoxicity. all these data suggest that nkt cells may play a role in the control of the immune response to allergens, such as the lipids present in pollen grains. outline: in humans, 0.5-10 % of circulating lymphocytes express a vg9vd2 t cell receptor, yet strikingly little is known about the function and properties of such unconventional t cells. we performed cdna microarrays to find vg9-enriched genes compared to conventional mhc-restricted cd8 + ab t cells, and found reciprocal enrichment of nectin-like adhesion molecules igsf4 & crtam in gd and ab t cells respectively. because igsf4 binds to crtam, the data fuel a hypothesis that this may be a novel axis of communication between the two cell types. interestingly, previous studies show that activated nk, nkt and cd8 + ab t cells express crtam, and that engagement of igsf4 on epithelial cells renders the latter targets for enhanced cytolytic and cytokine responses. our data extends this to the prospect of cytolytic immunoregulatory interactions between t cells mediated by igsf4/crtam. we therefore sought to answer: 1. what is the function of igsf4/crtam on gd t cells? 2. how is the igsf4-crtam axis regulated in t cells? results and conclusions: flow cytometry showed igsf4 enrichment on resting gd t cells, with expression also detected on˚10 % of ab t cells. the properties of those cells are being examined. however, igsf4 generally correlates with markers of activation/antigen experience such as cd45ro. thus, igsf4 cells may comprise activated-yet-resting/pseudo-memory unconventional t cells and memory-effector conventional t cells. stimulating vg9 + t cells in vitro led to rapid crtam induction, resulting in the majority of cells co-expressing both igsf4 and crtam within 48 hours. however, engagement of igsf4 by crtam or vice versa is not sufficient to induce cytotoxicity, as stable cho cell transfectants expressing either molecules were not specifically lysed by pbmc in vitro, compared to efficient and parallel targeting of mica + cells. instead, our current experiments address the possibility that crtam-igsf4 may regulate cytotoxic interactions promoted by other receptor-ligand interactions, such as mica-nkg2d. this may explain why cells can tolerate co-expression of both molecules, and would refute the hypothesis that crtam-igsf4 interactions are sufficient for cd8 t cells to kill gd t cells and/or vice versa. instead, crtam-igsf4 interactions may set the threshold for cytotoxic immune-surveillance responses. t cell receptor (tcr) is a multisubunit complex in which the invariant subunit cd3z is a 16 kda transmembrane protein indispensable for coupling antigen recognition by tcr to diverse signal transduction pathways. approximately 3-6 % of human peripheral blood lymphocytes express the gd tcr and the majority of these cells express the vd2 tcr variable segment associated with the vg9 segment, and recognize phosphorylated non-peptidic metabolites from microbial or self origin. these compounds trigger vg9vd2 t cells without antigen presentation. in vitro stimulated vg9vd2 t cells with antigens are able to produce ifn-g and tnf-a and exert a powerful cytotoxic activity against infected cells as hiv-infected cells. however, during hiv infection a marked decrease of vg9vd2 t cells was observed and the remaining cells are unable to respond to their non-peptidic ligands. aim of the present work was to study the mechanisms of vg9vd2 t cell anergy observed in hiv+ patients. to this aim, cd3z expression and ifn-g production by vg9vd2 t cells from hiv+ and hiv-subjects were analyzed. we show that vg9vd2 t cells from hiv-infected patients expressed lower level of cd3z compared with healthy donors. a direct correlation between cd3z expression and ifn-g production capability by vg9vd2 t cell was found. however, pkc activation by pma is able to restore cd3z expression and ifn-g production. our findings may contribute to clarify the molecular mechanisms of vg9vd2 t cell anergy found in hiv+ patients and have implication in the design of effective immune-based therapies. l. abeler-dörner 1 , m. swamy 1 , s.l. clarke 1 , a. hayday 1 1 king's college london, immunobiology, london, united kingdom gut intraepithelial lymphocytes (iel) constitute one of the largest t cell compartments in mice and in man. their functions and their interactions with surrounding epithelium are likely to be crucial to the fine-tuned balance between tolerance to harmless food antigens, immunity to gut-associated pathogens, and overall intestinal immune surveillance. intestinal iel comprise many unconventional t cells including tcrgd cells and tcrab cd8aa or cd8 -cd4cells, which have been assigned innate-like immune functions and key roles in surveillance of stressed tissue. unlike conventional t cells, iel might initiate an immune response rather than simply being late effector cells. it is therefore important to elucidate the "immunological information flow" in the gut. to this end, this project characterizes different subsets of iel and their interactions with epithelium in steady state and under immunostimulatory conditions in vitro and in vivo. in the past, it has been notoriously difficult to study iel ex vivo. to solve this problem, we developed a novel culture system that allows us to expand the cells ex vivo and study their responses for up to 15 days. the cells are initially activated by plate-coated acd3 antibody and a cytokine cocktail and maintained further in medium containing low levels of il-2. after a resting period, the cells can be restimulated in vitro. in this new system, we studied responses of different iel subsets to stimulation via tcr, nkg2d and cytokine receptors, either alone or in coculture with epithelial cells. as readouts we monitored proliferation, cytokine secretion (ifng, il-6) and expression of activating and costimulatory molecules. reactivation in response to various stimuli could already be observed after 6 hours. the in vitro data set forms the basis for analysing iel responses in vivo to stimulatory molecules ectopically expressed as transgenes in the gut. the characterization of iel responses opens new insights into the nature of gut immune responses and should provide a better understanding of the immunology of inflammatory bowel diseases which still remain a major problem in the clinic today. objective: behcet's disease (bd) is a multisystemic disorder with a possible underlying pathology of immune-mediated vasculitis. increased expression of cd94 in bd patients suggested that nk receptors may play a pathogenic or regulatory role in the pathogenesis. considering the regulatory functions of nkg2 molecules in heterodimer with cd94, we screened the presence of these receptors on t cell subsets in bd. the expression of nkg2 a/c/d molecules on gd and cd8+ t cells were analyzed in 17 active and 9 inactive patients with bd and 21 healthy controls. expression of nkg2 molecules was evaluated on cd8+, gd t and cd56+ nk cells by using flow-cytometry. results: gd t cells were increased in patients with bd compared to controls (4.4 vs. 2.8 %, p=0.001). in addition to the increase of gd t cells, increased expression of activating nkg2c molecules was also observed on gd t cells (20 % vs. 11 %, p= 0.01). nkg2a expression on gd t cells was found to be higher than nkg2c expression in patients and controls; but nkg2a expression on the t cells was not statistically different in both groups (33.5 vs. 40 %). nkg2d receptors were present on most of the gd t cells in both groups. however these activating molecules on cd8+ cells were decreased in patients with bd compared to controls ( revlimid is a therapeutic agent used to treat myelodysplastic syndrome (mds), a group of haematological disorders characterised by ineffective haematopoiesis. the mechanism of action for revlimid is poorly understood, but there has been increasing interest in the strong association reported between mds and defects within the immunoregulatory nkt cell compartment. indeed, some studies now suggest an important outcome of revlimid treatment is the restoration of normal cytokine production by nkt cell levels and an increase in their overall numbers. we have conducted the most thorough study to date of the nkt cell compartment of mds patients treated with revlimid/ancestim and can report that mds patients had normal nkt cell levels prior to treatment, and no significant increase as a result of revlimid/ancestim treatment. furthermore, nkt cells from mds patients produced high levels of th1 and th2 cytokines when stimulated with pma/ ionomycin and the proportion of nkt cells capable of cytokine production did not increase significantly after revlimid/ancestim treatment. these are highly significant findings given the recent emphasis on nkt cells as a potential therapeutic target for mds. our study provides an extensive analysis of the impact of revlimid/ ancestim treatment on the nkt cell compartment and sheds new light on the role of nkt cells in mds and the mechanism of revlimid immunomodulation. objectives: human gd t cells are potent killers of a variety of tumour cell lines, and mice lacking gd t cells suffer from high incidence of experimentally-induced tumours. however, the molecular mechanisms mediating tumour cell recognition by gd t lymphocytes remain largely unknown. we aim at identifying potential tumour antigens and co-stimulation molecules expressed in ex vivo tumours and in tumour cell lines that activate human gd t cells for tumour cytolysis. as immune evasion mechanisms that down-regulate tumour antigens may operate in vivo, we have identified candidates from human tumour cell lines of hematopoietic origin that constitute in vitro cytolysis targets for vg9/vd2+ lymphocytes. we have screened a panel of 26 lymphoma and leukaemia cell lines using a conventional in vitro killing assay using vg9/vd2+ cells, and selected two susceptible ("target") cell lines (over 60 % death in the assay) and two vg9/vd2+ resistant ("non-target") cell lines (under 20 % death) for cdna microarray analysis. we compared the differential expression in pairs of tumour cell lines of identical origin: the burkitt's lymphoma cell lines daudi (target) vs raji (non-target), and the pre-b cell leukemia cell lines rch-acv (target) vs 697 (non-target), and validated the results by rt-qpcr quantification. results: we identified 37 commonly up-regulated and 50 commonly down-regulated genes that encode cell membrane-associated proteins in susceptible tumours. ulbp1, ifitm1 and prame, for example, are up-regulated, whereas cd274 and clec2d are down-regulated in target cell lines. as these encode membrane-bound proteins with relevant functions in tumour immunity, they constitute potential ligands for gd lymphocyte recognition of tumour cells. the expression of these candidate genes was studied by rt-qpcr in a broader panel of cell lines and primary biopsies. we are currently testing, in functional assays based on rna interference and overexpression, these and other candidate genes in order to determine whether they provide activating or inhibitory signals to gd t cells. the comparison between the transcriptomes of vg9/vd2+ target versus non-target cell lines allowed the identification of candidate genes, whose individual function we are currently dissecting, that may be involved in tumour cell recognition by human gd t cells. mice and humans are the only species in which phenotype and function of inkt cells have been properly described. our aims are to directly identify this cell population and to investigate cd1d, in the rat. mice and rats have very similar cd1d and inkt tcr genes, with the exception of the va14 gene segment, which is a multimember gene family in the rat. novel monoclonal antibodies with nearly identical binding capacities to mouse and rat cd1d revealed a very similar pattern of cd1d distribution, and could inhibit cytokine production after agalcer stimulation of primary cells in both species. response to agalcer was studied in five different rat strains, showing big inter strain differences. notably, ifn-g and il-4 production was 10-100 fold lower in the best responder rat strain (f344) compared to mouse (c57/bl6). since nkrp1a (rat homologue of mouse nk1.1) and tcr are not appropriate markers for rat inkt, cd1d oligomers where tested for binding to inkt-tcr transduced cells. newly generated agalcer loaded rat cd1d dimers, recognized rat inkt tcr and, although less efficiently, bound to mouse inkt tcr. however, mouse cd1d agalcer dimers did not bind to rat inkt tcr. agalcer loaded rat cd1d dimers were then used to stain primary intrahepatic lymphocytes. but, although mouse inkt cells were stained to some extent, the identification of a discrete population in the rat was not possible. the reasons behind could be: that the avidity of the dimers for the tcr is not high enough to stain primary cells and/or that the frequencies are so low that the detection by facs analysis is difficult. in order to clarify these issues we currently produce and test rat cd1d tetramers. burkholderia pseudomallei is a highly virulent bacterium which causes the potentially fatal disease melioidosis in humans. this disease is endemic in tropical regions, especially thailand and northern australia, and has a serious outcome for many infected individuals. b. pseudomallei is an intracellular bacterium and many b. pseudomallei strains are resistant to antibiotics so antibiotic treatment is aggressive and relapse of the disease is frequent. in addition to this, no vaccine is currently available to prevent the disease. human g9d2 t cells are involved in the immune response to infection with a number of intracellular pathogens including brucella suis and mycobacterium tuberculosis. g9d2 t cells respond to non-peptidic phosphorylated molecules known as 'phosphoantigens' which are byproducts of essential metabolic pathways in both bacteria and mammals. phosphoantigens cause expansion and activation of g9d2 t cells during infection with intracellular pathogens including fransicella tularensis and m. tuberculosis. analogues of natural phosphoantigens have been developed to manipulate g9d2 t cell responses as a cancer therapeutic and are currently in clinical trials for the treatment of hepatitis c virus. we aimed to determine in vitro whether enhancing gd t cell responses in human blood using the synthetic phosphoantigen picostim could reduce growth of intracellular b. pseudomallei in the human monocytic cell line thp-1. a significant (p x 0.01) reduction in intracellular bacterial numbers was observed (n=8) in the presence of pbmcs cultured with picostim+il-2 in comparison with pbmcs cultured with il-2 or media alone. picostim+il-2 caused significant expansion and activation of gd t cells following culture of pbmcs for 10-14 days. purified gd t cells stimulated with picostim were able to reduce intracellular b. pseudomallei numbers 100-fold. this data demonstrates that pbmcs, stimulated with the synthetic phosphoantigen picostim+il-2, reduced growth of intracellular b. pseudomallei in a gd t cell-dependent manner. objectives: vgamma9/vdelta2 (gd) t cells play a major role in innate immunity against microbes, stressed and tumor cells. they represent less than 5 % of peripheral blood lymphocytes (pbl), but can be expanded in vitro by zoledronic acid (za)-treated monocytes or dendritic cells (dc).the purposes of this study are: 1) to determine whether dc generated from multiple myeloma (mm) patients are as effective as their normal counterparts in the ability to activate gd t cells; 2) to evaluate whether gd t cells can exert immunoadjuvant activity on dc generated from mm patients and primed with tumor-specific antigens (survivin-sv); 3) to establish whether the same issues could be solved using a simplified protocol of dc generation. 1) dc were generated from cd14 + cells of healthy donors/mm patients; immaturedc on day 6 were induced to fully mature by incubation for 18 hours with tnfa + il-1b + pge2 in the presence or absence of 5 mm za. after 7 days of co-culture dc:pbl, percentages and total counts of gd t cells were determined by flow cytometry; 2) idc generated from cd14 + cells of hla-a*0201 + healthy donors/patients were pulsed with sv-peptide and stimulated for 18 hours with tnfa + il-1b + pge2 in the presence or absence of 5 mm za; after 2 rounds of autologous t cells stimulation by dc, the frequency of sv-specific cd8 + t cells was determined by svpentamers staining; 3) the same experiments were performed both with dc generated following a standard protocol and a 48h protocol (dc fast objective: depletion of or deficiency in gd t cells aggravate colitis in different animal models. additionally, reconstitution of mice with syngeneic gd t cells ameliorated chemically-induced colitis indicating a suppressive or regulatory role for murine gd t cells in intestinal inflammation. therefore, we asked whether human gd t cells possess also suppressive or regulatory potential, which could be of therapeutical use in chronical inflammatory diseases such as ulcerative colitis or crohn's disease. hence, the proliferation, suppressive activity, and cytokine profile of human peripheral gd t cells were determined in vitro. methods: human gd t cells were isolated from whole blood of healthy donors by macs technology. the proliferation was determined by [6-3 h]-thymidine incorporation, while suppression of responder cell proliferation was measured by flow cytometry via cfse fluorescence intensity. the cytokine profile was determined by elisa from culture supernatants as well as by flow cytometry intracellularly. finally, the in vitro characteristics of gd t cells were compared to those of cd4 + cd25 + regulatory t cells (treg). human peripheral gd t cells show suppressive activity against responder cell proliferation, though being themselve anergic, that is, they produce negligible amounts of interleukin-2 on stimulation and proliferate poorly. while the proliferation of gd t cells and treg cells is comparable, the suppression of gd t cells on responder cell proliferation is even stronger than the suppression by treg cells though gd t cells being foxp3 negative. additionally, gd t cells are strong producers for tgf-b, particularly by the vd1 subset. conclusion: human peripheral gd t cells possess regulatory potential and could be of therapeutical use in treatment of chronical inflammatory diseases as they are anergic and act suppressive. their suppressive activity is even superior to treg cells and might be due to strong tgf-b secretion. for application of human gd t cells in therapy their expansion under maintenance of their regulatory properties should be elucidated. there are previous descriptions of gamma-delta t lymphocytes (gd) from behçet's disease patients (bd) but, in most of cases, they are incomplete or contradictory. it has been suggested that nkg2d on gd is involved in bd lesions through interaction with mica molecules. furthermore gdcd8+ have been recently proposed as a new regulatory t subset (treg). objectives: to study gd phenotype in bd active (bda) (n=8) and inactive (bdna) (n=20), versus healthy controls (hc) (n=30) and patients with recurrent oral ulcerations (ru) (n=14). to determine gd cytokine profile and surface markers treg-related in bd (n=9) and hc (n=11). methods: we obtained mononuclear cells from peripheral blood (pbmc). we determined by flow cytometry: -surface expression of: gd tcr, vdelta1, vdelta2, cd8alpha, cd8beta, nkg2d, nkg2a and cd103. -intracellular expression of ctla-4, and foxp3. -intracellular expression of il-2, il-4, ifngamma, il-10 and tgfbeta after pbmc polyclonal stimulation. we used two tailed test for means comparison (mann-whitney u or student's t test). -vdelta2+ cells were significantly increased in ru. vdelta1+ and gdcd8+ lymphocytes were significantly increased in bd versus ru and hc. -the mean fluorescence intensity of nkg2d was slightly increased in gd from bda. -nkg2a expression by gdcd8+ was not different in bd versus hc. -most of gdcd8+ presented cd8alpha-alpha homodimers in bd and hc and were negative for cd103, foxp3 and ctla-4. gdcd8+ and gdcd8-subsets were (in bd and hc): -high ifngamma-producers without differences. -low il-2-producers: il2+ cells were lower in gdcd8+ than in gdcd8-. -low il-10-producers: il10+ cells were lower in gdcd8+ than in gdcd8-. -low tgfbeta-producers: tgfbeta+ cells were lower in gdcd8+ than in gdcd8--very low producers of il4 in most of cases. the hallmark in bd was the increase of gdcd8/vdelta1+. this subpopulation has recently been described as immunosuppressive in infiltrates of human tumours and its function related to nkg2a in intraepithelial intestinal lymphocytes from celiac patients. we did not find a cytokine profile or a phenotype t-reg-related for gdcd8+, except a lower percentage of il-2+ cells than in the gdcd8-subset. gdcd8+ from bd did not show significant differences versus hc. natural killer t (nkt) cells comprise a highly heterogeneous subset of t lymphocytes that co-express a t cell receptor (tcr) and nk cells markers such as cd56 in humans. a subgroup, the invariant nkt cells (inkt), expresses the va24vb11 tcr rearrangement representing a minority subset in peripheral blood and virtually absent in the newborn. objectives: to establish a method to growth cord blood-derived nkt cells (cd3 + cd56 + ), in order to evaluate their phenotypic characteristics and the tcrvb repertoire. methods: mononuclear cells were isolated from 10 healthy umbilical cord blood samples and stimulated with ifn-g (50 ng/ml), anti-cd3 (25 ng/ml) and il-2 (500 ui/ml). these cells were cultured for 21 days and the expanded cd3 + cd56 + cells were isolated by immunomagnetic methods. surface markers were determined by flow cytometry. total rna was extracted from the purified cd3 + c56 + cell suspension using trizol ® reagent and mrna expression of twenty tcrvb gene families was measured by semiquantitative rt-pcr. statistical analyses were performed using mann-whitney u test and one-way anova, a p value of x 0,05 was considered significant. results: we could significantly expand cord blood cd3 + cd56 + nkt cells from 0,87±0,57 % to achieve an enrichment of 46,89±13,31 % (p=0,0002). table 1 shows the percentage (mean±sd,n=10) of phenotypic markers in cd3 + cd56 + cells at baseline (day 0) and after 21 days of culture. expression of mrna for the vb families studied was confirmed in each individual cell culture with a significant high expression of vb4 and vb8 families (p x 0,001). conclusion: our results show that cord blood-derived nkt cells are mainly cd8 + and cd94 + subsets, similar to peripheral blood nkt cell with a low percent of inkt cells. additionally, we confirm a diverse tcr vb repertoire with a significant expression of the vb4 and vb8 families in these cells. l. marischen 1 , d. wesch 1 , p. rosenstiel 2 , a. till 2 , d. kabelitz 1 1 institute of immunology, kiel, germany, 2 institute of clinical molecular biology, kiel, germany gd t cells account for a minority of t cells in human blood, but represent the majority of intraepithelial t cells in the intestinal tract. due to their ability to respond rapidly and in an mhc-independent fashion to particular antigens by cytokine production, gd t cells are considered as a link between innate and adaptive immunity. in addition, the expression of distinct pattern recognition receptors such as toll-like (tlr) and nod-like receptors (nlr) are characteristic for cells of the innate immune response. recent reports have demonstrated the tlr expression in human and murine gd t cells. here we provide evidence also for a gd t cell responsiveness to muramyl dipeptide (mdp), the putative ligand of the nlr family member nod2. peripheral blood mononuclear cells (pbmcs) containing gd t cells as well as freshly isolated gd t cells were stimulated via the gd t cell receptor in the absence or presence of mdp and analyzed for proliferation and ifng-production. while the proliferation of gd t cells within pbmcs was decreased, ifng-production was increased after costimulation with mdp compared to the stimulation with a non-activating dd-stereoisomer of the ligand (mdpi). the enhanced ifng production of pbmcs after costimulation was mediated mainly by gd t cells as shown by intracellular flow cytometric staining. with regard to the ifng-production after co-stimulation with mdp vs. mdpi, freshly isolated gd t cells from different healthy blood donors can be divided into responder and non-responder. responder gd t cells showed a significant increase of the ifng-production due to mdp-stimulation, whereas ifng-production was not influenced in non-responder gd t cells. in further experiments, as first approach to explain the different reactivity patterns of gd t cells, it is planned to analyze the polymorphisms of the nod2 gene in various donors. taken together, our preliminary data indicate that gd t cells are a major source of ifng-producing cells among pbmcs when challenged with specific antigens plus mdp, and support the role of gd t-cells as an important team player in the early immune response against bacteria. objectives & methods: an increasing of gamma-delta t cells during acute p. vivax infection and convalescent period has been reported. moreover, the activation of gamma-delta t cells leads to the inhibition of blood stage p. falciparum parasites in vitro. to determine the killing mechanisms of p. vivax parasites by gammadelta t cells comparing with what has been found in p. falciparum, the gamma-delta t cells were enriched by isopentenylpyrophosphate (ipp) from naïve pbmc. different number of gamma-delta t cells and normal pbmc were incubated with intact of p. vivax parasites and protein extract of p. vivax parasites, recombinant pvmsp1 19 and pvama1 proteins. gamma-delta t cells was daily determined the cytokine and granzyme intracellular releasing by flow cytometry until day 5 culturing. results: among the enriched gamma-delta t cells, the percentage of cells expressing cd69 + and cd25 + was elevated after co-culturing with intact and the proteins of p. vivax parasites. the overall gamma-delta t cells showed proliferation at day 3 after the co-cultivation. moreover, the gamma-delta t cells expressing ifngamma + and cd107a + (lysosomal associated membrane proteins: lamp-1) elevated from the first day of pbmc collection after co-culturing with the intact and p. vivax antigens. this level was correlated with the significantly decreasing number of parasites and the increasing percentage of parasite growth inhibition. our results showed the activation of gamma-delta t cells during p. vivax infection in vitro. this suggests that gamma-delta t cells could be stimulated by p. vivax parasites and these actively activated gamma-delta t cells could kill the parasites via mechanism of granzyme and cytokines at the early stage of cell activation. this study provides more understanding in activation of the innate immunity during acute malaria infection which may lead to the selection of appropriate malaria proteins as vaccine candidates in the future. objectives: several evidence suggest that invariant nkt cells (inkt) connect innate and acquired immune system. they are able to produce both th1 and th2 cytokines after stimulation. atopic dermatitis (ad) is a chronic inflammatory skin disease. th1-like and th2-like cytokines have been implicated in the pathogenesis of ad, but there are controversial data on their role in ad. the frequency and absolute number of inkt cells in mononuclear cells (pbmcs) of peripheral blood of patients with atopic dermatitis (ad) (n=43) and healthy controls (n=13) were determined by flow cytometry using anti-cd3 and monoclonal antibody specific for the cdr3 loop of the invariant tcr a chain of inkt cells (clone:6b11). furthermore, after pma/ionomycin stimulation for 4 hours, intracellular ifng and il-4 cytokines were detected in cd4+cd8-, cd4-cd8-(dn), cd4-cd8+ and cd4+cd8+ subsets of inkt cells by five colour flow cytometry in patients with ad (n=10) and healthy controls (n=10). results: both frequency and absolute number of inkt cells were significantly lower in patients with ad (p x 0.01) compared to healthy controls. the frequency of dn subpopulation was significantly lower in ad patient (p x 0.01). there was a positive correlation between the frequency of dn cells and inkt cells both in ad patients (r=0.726 and p x 0.001) and healthy controls (r=0.693 and p x 0.001). in the intracellular ifng level there were no significant difference in any of the inkt subsets of ad patients, however the intracellular il-4 level was significantly higher in dn subpopulation of inkt cells of ad patients compared to healthy controls (p x 0.05). the frequency, the number of inkt cells and the cytokine producing capacity of the cd4/cd8 inkt subsets are different in peripheral blood obtained from ad patients compared to healthy controls. our result suggest that the dn inkt cell subset can serve as a source of il-4 that promotes the th2 differentiation in ad patients and might play a role in the pathogenesis of this disease. introduction: intrahepatic immune cells (ihic) are known to play central roles in immunological responses mediated by the liver, and isolation and phenotypic characterization of these cells is therefore of considerable importance. aims: in the present investigation, we developed a simple procedure for the mechanical disruption of mouse liver that allows efficient isolation and phenotypic characterization of ihic. these cells are compared with the corresponding cells purified from the liver after enzymatic digestion with different concentrations of collagenase and dnase. results: the mechanical disruption yielded viable ihic in considerably greater numbers than those obtained following enzymatic digestion. the ihic isolated employing the mechanical disruption were heterogeneous in composition, consisting of both innate and adaptive immune cells, of which b, t, natural killer (nk), nk t cells, granulocytes and macrophages were the major populations (constituting 37.5%, 16.5%, 12.1%, 7.9%, 7.9% and 7.5% of the total number of cells recovered respectively). the ihic obtained following enzymatic digestion contained markedly lower numbers of nk t cells (1.8 %) . the b, t and nk t cells among ihic isolated employing mechanical disruption were found to be immunocompetent, i. e. they proliferated in vitro in response to their specific stimuli (lipopolysaccharide, concanavalin a and alpha-galactosylceramide respectively) and produced immunoglobulin m and interferon-gamma. conclusions: thus, the simple procedure for the mechanical disruption of mouse liver described here results in more efficient isolation of functionally competent ihic for various types of investigation. nature killer t cells (nkt) are a special t cell population with co-expresses nk and t cell surface markers. murine nkt cells include cd4 + nkt and cd4 -cd8 -nkt cells. nk1.1 + nkt cells may release large amounts of il-2, il-4, ifn-g and il-10 after they are activated. it has been reported that a-galactorsykeramide (a-galcer), a glycolipid, may induce proliferation of nkt cells with the role of immune regulation by stimulating mouse spleen cells. this study demonstrated that superantigen staphylococcal enterotoxin b (seb) , a kind of peptide, can activate the nkt cells with the function of immune tolerance. the response ability of seb-activating effect cells to cona, lps and il-2 had significantly decreased compared with that of normal lymphocytes. the effect cells exerted an inhibitory effect for the response of normal lymphocytes to cona and il-2. there was a significantly increase in the percent of cd8 + nk1.1 + and tcrvb8 + nk1.1 + nkt cells identified from the seb-activated cells. based on the cell distribution detected in the upper part of the facs picture, expression of cd69 molecule existed in 68.95 % of the cells from large-scale selection. the percent of cd8 + nk1.1 + and tcrvb8 + nk1.1 + nkt cell subsets in the giant lymphocytes were enhanced to 84.0 and 38.9 folds, respectively. under a light microscope at x400 magnification, the seb-activating lymphocytes in size were larger than not only the cona-activated cells but also the adherent macrophages with an increase of 5 fold observed under a microscope. there were a few granules seen in cytoplasm. the value of cytoplasm vs nuclei was less than 1.0 and they are non-adherent cells. the differentiation pathway of the seb-activating cd8 + and tcrvb8 + nkt cells was not relative to a nk source. they were produced directly from t cell population and were considered as a subsets of t lymphocytes. our results suggest that the superantigen seb can act on the cd8 + nkt cell and tcrvb8 + nkt cells. and the two nkt cell subsets may play a critical role in seb mediated tolerance. gd t cells in the intestinal intraepithelial compartment (gd iiel) show an intrinsic activated phenotype. we hypothesised that their t cell receptor gd (tcrgd) is implicated in the activation of gd iiel. because the tcr gd ligands in mice are not well described, monoclonal antibodies (mab) directed against the gd tcr, like the clone gl3 which binds the d subunit of tcr gd, are important tools to specifically activate gd t cells. using cytometric indo-1am measurement, we could detect calcium flux of intestinal and peripheral gd t cells from tcrd-h2begfp reporter mice. stimulation with anti-gd clone gl3 or anti-cd3 clone 2c11 elicited activation of gd t cells suggesting that tcr gd and cd3 molecules in gd t cells are functional and signalling competent. next, using elisa and cytometric bead array, we found that iiel stimulated with plate bound gl3 in vitro produced ccl4, ifng and tnfa. therefore, we were interested whether the ccl4 production of gd iiel influenced the homing of ccr5 cells such as lamina propria (lp) cd4 + foxp3 + cells (tregs). to test this, wt mice were i. p. treated with gl3 mab and lp tregs were analysed by cytometry at various time points post inoculation. we found similar frequencies of lp tregs population but a slight decrease in ccr5 + tregs. however, when we compared wt and tcrd -/mice, we found both lower percentages of total lp tregs and of lp ccr5 + tregs in tcrd -/mice compared to wt mice. in conclusion, our data suggest that intraepithelial activation of gd t cell may directly or indirectly induce changes in the iiel and lamina propria (lp) lymphocyte compartment and influence the ccr5 expression and the homeostasis of lp treg. the ability of nkt cells to serve a variety of different immunoregulatory functions in vivo may reflect a diversity in function of different nkt cell subsets. diversity in cytokine production by nkt cell subsets has been observed in murine and human studies, although this analysis has largely been following in vitro restimulation. here, we investigated cytokine production by murine nkt cell subsets in vivo under conditions where minimal manipulation of the cells was required. to this end, we examined il-4 production in g4 reporter strains in which dna encoding green fluorescent protein (gfp) was inserted into the first exon of the il-4 gene. in the absence of any manipulation gfp was expressed from the il-4 locus in populations of immature thymic nkt cells (predominantly cd4+cd44lotcrhi cells on a balb/c background, and cd4+cd44lonk1.1-on a c57bl/6 background) and some splenic nkt cells, with overall numbers of gfp+ cells in both tissues decreasing with age. after i. v. administration of the nkt cell ligand a-galactosylceramide, il-4 production was induced predominantly in cd4+ nkt cell subsets of the liver and spleen, and after i. n. administration, in cd4+ nkt cells of the airways. spontaneous and a-galcer-induced expression from the il-4 locus occurred in the absence of stat6 signalling, and did not require initial exposure to il-4 protein from other sources in the host. diversification in cytokine expression by nkt cells subsets therefore occurs early in ontogeny, and is also a significant feature of responses to exogenous activating stimuli. interleukin-17 (il-17) plays an important role in neutrophil recruitment. herein, we investigated the role of il-17 receptor signaling in polymicrobial sepsis induced by cecal ligation and puncture (clp). methods: adult c57bl/6 (wt) and il-17 receptor gene-deficient (il-17r ko) mice were subjected to non severe (ns-clp) sepsis. intraperitoneal neutrophil migration, bacteremia, cytokine, chemokines and liver injury were evaluated 6 hours after surgery. the ability of il-17 mediate the neutrophil microbiocidal activity in vitro, as well the neutrophil migration in vivo and in vitro were also evaluated. the means of different treatments were compared by analysis of variance (anova), followed by bonferroni's t test and the survival rate by the mantel-cox log rank test. results: it was observed that il-17r ko mice, subjected to ns-clp sepsis, show reduced neutrophil recruitment into peritoneal cavity, spread of infection, and increased systemic inflammatory response as compared to wt. as a consequence, the mice showed an increased mortality rate. moreover, il-17 induced neutrophil migration in vivo and in vitro. besides, we demonstrated that neutrophils harvested from il-17r ko mice already show reduced microbiocidal activity, compared with wt, suggesting a physiological role of il-17receptor signaling in the microbiocidal activity of neutrophils. furthermore, wt neutrophils treated with il-17 showed strongly enhancement of microbiocidal activity by a mechanism dependent of nitric oxide. conclusion: during ns-clp besides the importance in recruit neutrophils to focus of infection, il-17 also enhances the microbiocidal activity of neutrophils. therefore, our results demonstrated that il-17 receptor signalization plays a critical role on host protection during polymicrobial sepsis. objectives: members of the toll-interleukin-1 receptor (tir) family are important for host defense, inflammation, and immune regulation. their canonical signaling pathway involves adaptor proteins and il-1r associated kinases to activate nfxb and p38 mitogen-activated protein kinase. the il-33-induced signal transduction in mast cells is poorly understood. in this work we studied the signal transduction of il-33 in different mast cell subsets. methods: different mast cells subsets (hmc-1, human cbmcs and murine bmmcs) were stimulated with il-33. the resulting signal transduction was investigated by immunoblot for activated signaling molecules (pc-kit, perk1/2, pakt, pnfxb, p38 and pjnk). additionally, we studied the signal transduction of il-33 in il-33r transfected hek293t cells. results: we found, that a tir family member, il-33r, transactivates the receptor tyrosine kinase c-kit in mast cells and that il-33-induced cytokine production depends on c-kit transactivation. il-33r and il-1r accessory protein (il-1racp) form a physical complex with c-kit. thereby the complexation is dependent on the activity of c-kit. conclusion: these results show for the first time that the biological function of an il-1r family member is dependent on the presence of an activated receptor tyrosine kinase. furthermore, these results reveal that certain il-33-induced signaling pathways and effector functions are dependent on activated c-kit and could therefore explain the effects of il-33 in mast cells in absence of iger activation. (1) . we now provide a molecular mechanism underlying this pathogenic effect by which free heme sensitizes hepatocytes to undergo tnfmediated programmed cell death. independently of newly gene transcription and/or protein synthesis, free heme cytotoxicity is mediated by the unfettered generation of free radicals in response to tnf, presumably due to the participation in the fenton reaction of the fe atom present in the protoporphyrin ix ring. once exposed in vitro to free heme, a sustained c-jun n-terminal kinase (jnk) activation was observed in hepatocytes in response to tnf, an effect that promotes further free radicals production. pharmacologic or genetic (shrna) inhibition of jnk in hepatocytes avoids free radicals accumulation and caspase-3 activation, also mimicked by the anti-oxidants n-acetylcystein (nac) or butylated hydroxyanisole (bha). expression of the heme catabolyzing enzyme heme oxygnease-1 (ho-1) in hepatocytes affords protection against heme sensitization to tnf cytotoxicity. recombinant adenovirus mediated ho-1 expression in the liver suppresses tnfmediated hepatocyte apoptosis and prevents the lethal outcome of plasmodium infection in mice. in conclusion our data reveals a novel signal transduction pathway via which heme sensitizes hepatocytes to undergo tnf-mediated cytotoxic effect, critically involved in the outcome plasmodium infection. the multi-step leukocyte extravasation process is governed by adhesion molecules and chemotactic factors dynamically interplaying in the presence of shear forces. responsiveness to chemotactic ligands is mediated by g protein-coupled receptors (gpcrs) which are finely regulated by a family of cytosolic proteins, betaarrestin 1 and 2. recent evidence indicates that, in addition to playing a regulatory role in gpcr desensitization and internalization, beta-arrestins may contribute to gpcr signaling by functioning as scaffolds for the recruitment of signaling proteins into complexes with agonist-occupied receptors. on this basis, we investigated the physiological role of beta-arrestin 2 in chemokine-driven dynamics associated with leukocyte extravasation, with special interest to the activation of the rap1 small gtpase, recently emerged as pivotal regulator of integrin function. the analysis of kc the (keratinocyte-derived chemokine) rap1 activation profile in rbl (rat basophilic leukemia) cells expressing mcxcr2 shows a bimodal kinetic, with the first peak at 30''/1' and the second at 5' after stimulation. rna interference-mediated depletion of beta-arrestin 2 specifically inhibits the occurence of the second wave of rap1 activation, whilst it has no effect on the early pick, thereby suggesting that beta-arrrestin 2 is involved in rap1 activation and that the oscillations in the formation of rap1-gtp are regulated by different molecular mechanisms. in order to elucidate the gefs and gaps involved in the gtpase regulation we are at present down-regulating the expression of c3g (rap1gef) and spa1 (rap1gap): preliminary results suggest that spa-1 has probably a role in the early activation peak. since this oscillatory chemokine-induced rap1 activation is present on other myeloid cell lines (hl60, 32d) and fresh pmn's we are also translating our research to these more appropriated cells. interestingly betaarrestins amino acid sequence and three-dimensional structure reveal a unique and evolutionary conserved proline-rich sequence in beta-arrestin 2, localized in a solvent exposed loop which may serve as a docking site for migration-associated transducers/adaptors. in order to find sh3 containing proteins that interact with beta-arrestins, we have performed an overlay screening assay of 153 different sh3 domains that revealed over 20 putative beta-arrestins putative interactors, some of which isoform specific. granulocyte-macrophage colony-stimulating factor (gm-csf), interleukin (il)-3 and il-5 stimulate proliferation, differentiation, survival and functional activation of myeloid cells. the cell surface receptors for these cytokines consist of cytokine-specific a subunits and a common b-receptor (bc), required for the activation of intracellular signaling following cytokine engagement. aberrant signalling, stimulated by these cytokines, has been implicated in the pathogenesis of many diseases, including arthritis, asthma and leukemia. as a result, we have sought to define key molecular determinants of these receptor-cytokine interactions in order to gain a greater understanding of receptor activation. here we present novel insights into the role of the ig-like domain of the gm-csfra in gm-csf binding. deletion of the ig-like domain abolished direct gm-csf binding and we identified specific residues directly involved in ligand binding by site directed mutagenesis and binding studies. the results indicate a previously unrecognized role for the ig-like domain of gm-csfra. furthermore, we address a longstanding controversy in the field of gm-csf, il-3 and il-5 receptor biology, by performing a systematic study of the role of n-glycosylation upon on the bc, and related murine b il-3 , in ligand-binding and receptor activation. these data demonstrate definitively that n-glycosylation does not play a role in mediating ligand-binding or receptor activation. these findings clearly establish that the determined human bc structures lacking glycosylation at asn 328 are biologically relevant conformers of the human bc ectodomain. our results appear to suggest that the potency of receptor signalling can be influenced by the biophysical and structural properties of the extracellular receptorligand interactions and it also addresses important, poorly-understood aspects of mechanisms underlying ligand recognition and activation of the gm-csf: gm-csfra: hbc receptor complex. reference: (1) micrornas (mirnas) are endogenous small non-coding rna molecules acting as key regulators of immune cell differentiation and innate immune responses. mirna-146 expression is induced by activation of the toll-like/interleukin-1 receptor pathway (tirpathway), where it targets essential adaptor and signaling molecules, thus serving as a regulator preventing the cells from an exacerbated pro-inflammatory response. since tnfa also up-regulates the expression of mirna-146a, we decided to explore whether this mirna is involved in the regulation of apoptosis. to this end, we used the hela human epithelial cell line as a model system for tnfa signaling. following tnfa and cycloheximide (chx) treatment mirna-146a transfected cells showed significantly reduced levels of the active proapoptotic caspases 8 and 3 (casp8/3). in line with this, mirna-146a conferred enhanced protection against tnfa-induced dna fragmentation and mitochondrial potential drop-down. our results demonstrate that mirna-146a is a regulator of receptor-mediated apoptosis. similar to the tir-pathway, mirna-146a seems to be part of a negative feedback mechanism of the tnfa signaling cascade. ongoing research focuses on the identification of the specific pro-apoptotic molecules targeted by mirna-146a. furthermore, we are exploring the relevance of our observations for the mycobacterial infection of human macrophages, where the regulation of apoptosis is critical. objectives: the aim of this study was to evaluate the role of single nucleotide polymorphisms (snps) located in il-15, il-7r a-chain and il-15r a -chain genes in hiv disease progression. methods: we studied 70 antiretroviral treated patients (progressors) and 71 long term non progressors (ltnp). we analyzed 2 snps in the il-15 gene, 5 snps in the il-7r gene and 3 snps in the il-15r gene. in univariate analysis, we found an association between the presence of at least one mutated a allele in il-15r aa 182 and a higher possibility of being ltnp ( our study suggests that genetic polymorphisms located in il-7r and il-15r genes can influence the rate of disease progression in hiv+ patients, especially when a combination of aplotypes is present. mutations in the coding regions might compromise the binding of the cytokines or the intracellular signal transduction pathways, therefore leading to the alteration of cd4 and cd8 t cells homeostasis. aims: mono-adp-ribosyltransferases (arts) are gpi-anchored ectoenzymes that covalently modify cell-surface or soluble target proteins by transferring an adpribose moiety from extracellular nad+ to specific arginine residues of target proteins. in this study, we report that human tumor necrosis factor (tnf) is adpribosylated by art1, and that adp-ribosylation affects both the release of tnf from cells and its cytolytic action. methods: transcription of art1 in human leukocytes was analyzed by rt-pcr. adp-ribosylation of tnf was detected by monitoring the incorporation of adpribose from labeled nad. release of tnf from transfected hek cells was monitored by elisa. binding of tnf to tnf receptors was analyzed by biacore. tnf cytotoxicity was monitored by flow cytometry. the adp-ribosylation site on tnf was analyzed by lc/ms mass spectrometry. results: we identified art1 transcripts by rt-pcr analysis in human blood leukocytes. soluble art1, released from the surface of transfected cells by phosphatidylinositol-specific phospholipase c (pi-plc), adp-ribosylated recombinant human tnf in vitro. co-transfection of hek293 cells with art1 and tnf resulted in modification of tnf at at least 2 distinct sites, i. e. one within the tnf ectodomain, and one on the stalk that remains connected with the cell membrane after cleavage by tnfa converting enzyme (tace). analysis of modified recombinant tnf by mass spectrometry provided evidence that the tnf ectodomain is adp-ribosylated at r32, a site that has previously been implicated in binding to tnfr2. binding assays indicated that adp-ribosylation inhibited binding of tnf to its receptors. importantly, modified tnf was less potent at inducing cell death in the human t cell lymphoma line kit225 than wildtype tnf. furthermore, cell surface adp-ribosylation of hek cells co-transfected with tnf and art1 resulted in reduced release of tnf into the supernatant. conclusions: adp-ribosylation of tnf or other cell surface proteins interferes with the biology of tnf signals by at least two distinct mechanisms. adpribosylation of tnf blocks binding to its receptors, thereby inhibiting tnf-mediated cytotoxicity. additionally, adp-ribosylation of tnf or another protein on the surface of tnf-producing cells inhibits the proteolytic release of tnf. noninflammatory chronic pelvic pain syndrome : immunological study in ejaculate g. n. drannik 1 , t.v.poroshina 1 1 institut of urology amsci of ukraine, laboratory immunology, kyiv, ukraine chronic prostatitis (cp) is a disease which likely is associated with abnormalities in local immune responses. secretions of the urinary and reproductive tract mucosa contain various protective effector molecules, produced by mucosal cells, lymphocytes, macrophages and neutrophiles. the aim of this prospective study was to observe local immunophenotypic patterns in patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome for further description and as possible surrogate markers for diagnosis and treatment. methods: 23 patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome (cp/cpps) and 11 control men were assessed for slpi, tnf-alpha, il-8 and free tgf-b1 in ejaculate by elisas using stat-fax 303 plus. a 3 to 5 day sexual abstinence period was required from the subjects before semen collection. after liquefaction and centrifugation, seminal plasma samples were kept at -20 degrees c°until assayed. the materials were processed after the standard programmes for statistical analysis.the study was approved by the local ethics committee. the slpi concentration was elevated in all patients (5127.571 ± 971.731 pg/ml, p x 0.001) in seminal fluid, in comparison with the healthy control subjects. the tnf-alpha concentration was elevated in all patients in seminal fluid (125.49 ± 17.9 pg/ml; p x 0.05). the il-8 concentration was elevated in all patients (366.7± 49.5 pg/ml; p x 0.05) in seminal fluid. free tgf-b1 was present in normal seminal plasma in high concentrations (346.4 ± 29.2 pg/ml), while in ejaculates of patients with noninflammatory cp/cpps tgf-b1 concentrations were 36.2 ± 6.2 pg/ml. conclusion: ejaculate's slpi, tnf-alpha, il-8 and free tgf-b1 are possible surrogate markers for the diagnosis and treatment of patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome. m. r. marrakchi 1 , e. a. elgaaeid 1 1 faculté des sciences de tunis, biology, tunisie, tunisia ulcerative colitis (uc) and crohn's disease (cd), collectively referred to the inflammatory bowel disease (ibd), represent a group of multifactorial autoimmune disorders of the gastrointestinal tract sharing many clinical and pathological characteristics, however, differing in histological features and cytokine profiles. the excessive production of either th1 or th2 cytokines due to perturbed regulation of immune system activation results in chronic inflammatory processes and loss of immune homeostasis that may be implicated in the genesis of ibd. studies have identified a gene that encodes the nod2/card15 protein, which is involved in the immune system's response to bacterial infection and confirmed to influence susceptibility to cd. indead, it has been suggested that high rates of asca (saccharomyces cerevisiae ) in absence of panca (perinuclear anca: anti-neutrophil cytoplasmic) antibodies were associated with aggressive forms of cd and that the important rise of panca was more frequent at uc . in a sample of tunisian patients, we examined the contribution of nod2/card15 gene in cd. we performed a cases /controls study upon 75 cd patients and 90 healthy controls. this study suggests that in northen tunisian population, 3020insc mutation in nod2/card15 gene is a prevalent mutation leading to the typical crohn's disease including ileal location, stricturing and penetrating clinical types and asca expression. since conflicting results were obtained on il-10 polymorphisms as risk factor for ibd, the aim of our study was also to explore anti-inflammatory il-10 cytokine genetic profile in patients with ibd. we examined the contribution of il-10 gene promoter polymorphisms (-627 and -1117) to crohn's disease (cd) phenotype, and the possible genetic epistasis between these polymorphisms and card15/nod2 gene mutations in cd presentation and location. in tunisian population, the 3020insc insertion in nod2/card15 gene is a marker of susceptibility to cd, while the a allele at position -627 in the il-10 promoter increases the risk of cd ileal location and severe disease presentation. a genetic epistasis between il-10 gene polymorphisms and card15/nod2 gene mutation was suggested. in conclusion, genetic and serologic markers might be useful in defining patien gangliosides were shown to inhibit the il-2-dependent proliferation of t-cells, implying that gangliosides interfere with one or more of the il-2-driven events. it is known that the major mechanism of inhibition is the direct interaction between ganglioside and the cytokine and, as a result, the capture of il-2 molecule by ganglioside. but gangliosides apparently can also form complexes with il-2r; such complexes influence on the signal transduction through il-2r. this effect of gangliosides may lead to the failure of this pathway. unfortunately, the biological and structural aspects of this problem are poorly understood. in this study we propose possible modes of interactions between exogenous gangliosides and il-2r subunits. in our work we use il-2-dependent cytotoxic t-cell murine line ctll-2. two different approaches for study of possible interaction types between exogenous gangliosides and il-2r subunits were applied: antibody staining of il-2r subunits followed by flow cytometry analysis, and photoaffinity labeling of living cells with 125 i-dcp-gm1 followed by immunoprecipitation of il-2r subunits. the fluorescence intensity of the antibody-labeled il-2r a-subunit substantially decreases after the treatment of cells with gangliosides. it has been shown that the fluorescent labeled cell fraction decreases by 29.5 % after cells incubation with ganglioside gm1, and by 10.7 % after incubation with gm2. labeling of the cells with antibodies to the il-2r b-subunit results in a less significant fluorescence decrease after cells incubation either with gm1 (2.8 %) or with gm2 (5.9 %). to determine the mode of this impressive masking influence of ganglioside gm1, photoaffinity cross-linking has been used. in our modification this method could identify is interaction of gm1 with subunits of il-2r occur with or without incorporation exogenous ganglioside into plasma membrane. electrophoresis followed by immunoprecipitation with appropriate antibodies resulted in appearance of the radioactive band only for b-subunit of il-2r, but not for il-2r a-subunit. these results demonstrate that exogenous ganglioside gm1 can interact with a-and bsubunits of il-2r in different modes. interaction of il-2r b-subunit with ganglioside gm1 requires incorporation of the ganglioside into plasma membrane, but it is not the case for interaction with a-subunit. a. respa 1 , j. bukur 1 , s. purpose: loss of interferon (ifn)-g inducibility of hla class i antigens has been found in some tumor cell lines, but the underlying molecular mechanisms of such deficiencies have not yet been elucidated in detail. this kind of tumor escape mechanism might lead to an inefficient recognition of tumor cells by the immune system. methods: phospho-specific western blot analyses were performed to verify the functionality of the different ifn-g pathway components, intra-and extracellular flow cytometry experiments were employed to determine the expression of antigen processing components and hla class i cell surface antigens, quantitative real time-pcr experiments to confirm the absence of jak2 and presence of pathway relevant molecules as well as, genomic pcr and chromosome typing technique to prove the deletion of jak2. results: different ifn-responsive phenotypes were defined in human melanoma cell lines varying from loss to low, delayed as well as strong ifn-g inducibility. resistance to ifn-g treatment was associated in one melanoma cell line with the lack of jak2 expression due to a gene deletion on chromosome 9, whereas the expression and functionality of other ifn-g signal transduction components like stat1 and jak1 were not affected. jak2 blockade by two jak2-specific inhibitors resulted in reduced levels of hla class i surface antigens. conclusion: structural abnormalities of jak2 leading to a lack of jak2 expression are associated with loss of ifn-g inducibility as well as reduced constitutive hla class i surface expression. in addition the jak2 inhibition modulates the expression of the hla class i antigen processing components. of renal cell carcinomas (rccs) are associated with the size, grade, t, n, m, stage and death of rccs patients. the genotypes were compared with those of a random sample of 506 controls of the spanish population. methods: two il18 polymorphisms located on the il-18 promoter region, snps -607 a/c (rs1946518) and -137 g/c (rs187238) were genotyped using taqman snp genotyping assays. the functional il-18 gene polymorphisms studied do not influence the susceptibility to rccs in the spanish populations (il-18 -607 p=0,318. il18 -137 p=0,740) but may contribute to disease onset and aggressiveness: the genotype il-18 -607 cc genotype, which is associated with higher il-18 production, was significantly associated with higher tumor size (p=0,001), grade (p=0,030), t (p=0,001), m (p=0,012) and stage (p=0,002). the influence of the il-18 -103 gg genotype was less relevant, however was correlated with higher tumor size (p=0,036), grade (p=0,017), t (p=0,026) and stage (p=0,011). the multivariate analysis with cox proportional hazard model revealed that, in this serie, nuclear grade and stage grouping were independent prognostic factors, whereas il-18 polimorphism can not be considered as independent prognosis factors. our results suggest that the polymorphism variants from the il-18 gene (il-18 -607 and -137) may be associated with an worse prognosis of rcc. high levels of il-18 production can play an important role in grow, invasion and metastasis of renal cancer. interleukin-18 (il-18) is a pleiotropic cytokine that is involved in regulation of both the innate and acquired immune response. the most prominent biologic property of il-18 is its ability to induce the production of ifn-gamma in presence of il-12. moreover, it stimulates the expression of tnf-a and il-l, enhances the differentiation of t cells to the thl and impairs the synthesis of the anti-inflammatory cytokine il-l0. then it seems that il-18 has a crucial role in immunity against brucella infection. since the expression of il-18 can be affected by polymorphisms in its gene, we decided to investigate any probable relation between six different il-18 gene polymorph isms and brucellosis. methods: a total of 188 patients with brucellosis and 77 healthy farmer who consumed contaminated row milk and dairy products from animals with brucellosis, were included in this study. all individuals were genotyped for six il-18 polymorphisms at positions -656, -607, 137, +113, +127 and codon 35/3 using polymerase chain reaction-restriction fragment length polymorphism (pcr-rflp). the distribution of alleles for il-18 polymorphisms at position -137 g/+113t/+127c (correlated with high production of il-18), codon 35/3c and -656g/-607c (correlated with higher production of il-18) were significantly higher in the healthy controls than the patients (p=0.000022, p=0.00185 and p=0.0441, respectively). discussion: as data revealed genotypes that have correlation with higher production of il-18 are more frequent in the controls than the patients. then it might be deduced that individuals who inherited these genotypes/alleles are able to produce higher level of il-18 at the onset of infection and it leads to more ifngamma production and control brucella infection before appearing brucellosis. abstract withdrawn by author objectives: a dsyregulated cytokine response has been shown to play a role in inflammatory bowel disease (ibd) pathogenesis. to dissect the influence of these cytokines, specifically interferon gamma (ifng), on the inflammatory response and colitis we have created a cell specific ifng receptor 2 (ifngr2) mouse mutant. we have generated a mouse line carrying a conditional ifngr2 allele using the 2 loxp 2-flp recognition target (frt) approach. a targeting vector with 2 loxp sites flanking exons 4-6 and 2 frt sites flanking the neomycin resistance cassette was generated. after confirmation of homologous recombination the neomycin resistance cassette was deleted by crossing with flp deleter mice. cell specific deletion is being performed by crossing conditional 'flox/flox' mice with specific cre expressing mouse lines. functional inactivation of the receptor has been demonstrated by performing western blots to detect phosphorylated and total stat1 following ifng stimulation. results: successful deletion of the gene and conditional mutants without the presence of the neomycin resistant cassette have been generated. functional inactivation of the receptor with no stat1 activation following ifng stimulation has been demonstrated in the complete knock out mice. furthermore, flox/flox mice retain full responsiveness to ifng. breeding with lysm cre and cd4 cre mice will been completed to create a cell specific deletion of the ifng receptor in macrophages/granulocytes and t cells respectively. the specificity of this deletion will be confirmed through cell sorting and functional assays. in order to determine the influence of ifng on a specific cell type a conditional gene targeting approach has been utilised. this has allowed the generation of conditional mice mutants with deletion of ifngr2 on macrophages and t cells. this has generated a very powerful tool for dissecting the role of this cytokine in numerous disease models. moreover, the ability to cross the conditional mice with additional cre lines will enable the analysis of more cell types in the future. a. gonzalez 1 , r. carretero 2 , p. saenz-lopez 2 , j. cantón 2 , j. carretero 2 , f. ruiz-cabello 2 , l.m. torres 1 , cts-143 1 hospital universitario virgen de las nieves, departamento de ginecología, granada, spain, 2 hospital universitario virgen de las nieves, departamento de análisis clí nicos, granada, spain objetives: cervical cancer is almost associated with infection by human papillomavirus (hpv). however, only a subset of infected women will ever develop the malignancy. ifng dinucleotide (ca) repeat polymorphism is responsible for genetic differences in interferon-gamma production. our objective was to investigated the relationship between ifgn polymorphism and cervical intraepithelial neoplasia (cin) methods: we have studied nineteen women with cin and 130 normal women control. dna was extracted from blood samples, and was genotyped for functional microsatellite (ca) repeats in the first intron of ifn-gamma gene. results: heterozygosis in (ca)12 allele of ifgn is significant more frequent in healthy control than in cin patient (p=0,030) in contrast homozygosis for (ca)12 allele did not show significant differences between both population. analysis of relation between this polymorphism and the cin stage showed that both heterozygosis and homozygosis is correlated with advanced stage (p=0,030 and p=0,045 respectively). conclusions: our study suggest that ifng (ca)12 allele may influence in cin risk and progression. ifng is associated with hpv clearance, but it also plays a role as inflammatory cytokine, promoting cervical malignant progression. further studies of the role of ifng and other cytokines may contribute to the understanding of cin promotions and progression. introduction: macrophages play a fundamental role in controlling of brucella infection, mainly through the secretion of cytokines such ifn-y and tnf-a. interleukin-15 (il-15), a th1-related cytokine, triggers inflammatory cell recruitment and increases the expression of ifn-y. as the cytokine production is under the genetic control, we decided to investigate the association between il-15 single-nucleotide polymorphisms (snps) and susceptibility to brucellosis in iranian patients. methods: 190 patients with brucellosis and 78 healthy animal husband men who kept animals with brucellosis, were included in this study. all individuals were genotyped for il-15 (c267t, g367a, c13687a and a14035t) polymorph isms using pcr-rflp. results: at position 267, the distribution of c allele and cc genotype were significantly lower in the patients than the controls (p=0.025 and p=0.042, respectively). at position 13687, the distribution of c allele and aa genotype were significantly higher and lower in the patients than the controls, respectively (p=0.050, p=0.015). discussion: as shown the frequency of cc genotype and c allele at position 267 were higher in healthy controls than the patients. hence, our study provides evidence that presences of cc genotype and/or c allele are significantly associated with susceptibility to brucellosis. the frequency of aa genotype at position 13687 is lower in patients than the healthy controls and the frequency of c allele at position 13687 is higher in patients than the healthy controls. hence, our study provides evidence that presences of c allele and lack of aa genotype are significantly associated with susceptibility to brucellosis. objectives: increasing evidence is emerging regarding the ability of mammary epithelial cells to respond to various cytokines. our aim was to investigate the cytokine receptor phenotypes of two distinctive human mammary cell lines, mcf-7 and skbr-3, as well as their ability to respond to several cytokines and to the g-1 gpr30 agonist. the mcf-7 and skbr-3 human adenocarcinoma cell lines were investigated by immunocytochemistry and flow cytometry for the expression of the following cytokine receptors: il-2ra, il-2rg, il-3/il-5/gm-csfr, il-4/il-13r, il-5ra, il-6ra, il-6r (gp130), il-7ra, il-10r, tnfr i, tnfr ii, ifngra, cxcr1, cxcr2, cxcr3, cxcr4, cxcr5. cells were incubated with il-10, ifn-g, tnf-a and tnf-b (for which both cell lines displayed receptors) alone and with the gpr30 agonist. cytosolic ca 2+ concentrations [ca 2+ ] i were measured by the microfluorimetric technique. results: different cytokine receptors phenotypes emerged for the two cell lines. the less well differentiated skbr-3 cells were found positive for a larger number of receptors than the mcf-7 cells. both cell lines displayed an important heterogeneity for each of the investigated molecules, in terms of number of positive cells and expression intensity, with chemokine receptors percentages constantly higher than for the other receptors. pretreatment of mcf-7 cells with il-10 reduced the calcium response to g-1, while pretreatment with ifn-g and tnf-a potentiated the calcium response to g-1. tnf-b had no effect on mcf-7 g-1 stimulation. no direct effect on basal [ca 2+ ] i stimulation could be noticed when administering the cytokines alone. in skbr-3 cells, pretreatment with il-10 or tnf-b had no effect on basal [ca 2+ ] i and did not significantly alter the calcium response to g-1, while pretreatment with ifn-g induced calcium oscillations and potentiated the calcium response to g-1. pretreatment with tnf-a produced calcium oscillations and reduced the response to g-1. conclusions: mcf-7 and skbr-3 cell lines express distinctive cytokine receptor phenotypes. furthermore, their ability to respond to cytokines in terms of modulating the gpr30 stimulation proved to be different. the susceptibility towards various soluble factors of these cell lines can offer us some insights on the complexity and individuality of each primary tumor and thus the distinctive evolution of each particular patient. results: in initial analyses of the early infection phase we identified splenic pdcs expressing ifnb/yfp. furthermore, we show for the first time in vivo that these ifnb producing pdcs are not directly infected with mcmv and that not only cdcs but also cd8a -pdcs expressed gfp as a marker for mcmv infection. we observed that at early time points equal frequencies of cd8a -pdcs and cdcs were infected with mcmv, whereas after 24h p. i. the frequency of infected cdcs wins over that of the pdcs. conclusions: with this experimental system we are able to visualize the ifnb response vs. the infection status of mcmv in vivo on a single-cell level. from our initial results we can conclude that infected cells in the spleen induce ifnb production in noninfected pdcs which initiate the antiviral immune response in this organ. recently, we have developed live-attenuated arenavirus vaccine candidates based on lymphocytic choriomeningitis viruses (lcmv) carrying the vesicular stomatitis virus (vsv) envelope gene (rlcmv/vsv-g). since interferon (ifn) signaling is known to be crucial for adaptive immune responses against wildtype (wt) lcmv and control thereof, we wanted to assess the innate and adaptive immune requirements for containing rlcmv/vsv-g infection. mice lacking both, ifn type i and ii receptors generated potent virus-specific cd8 t cells and neutralizing antibody responses against rlcmv/vsv-g, even exceeding the respective responses in wild type animals. in further contrast to wt lcmv infection, ifn type i and ii signaling was dispensable for rlcmv/vsv-g control. rlcmv/vsv-g infection of rag1-deficient mice (lacking mature t and b cells) resulted in persistent levels of circulating viral rna that was solely detectable by qrt-pcr but not by classical measures of infectivity. overt viremia was only found in mice lacking both rag1 and ifn type i receptor (ar). thus, viral attenuation for vaccine use was found to considerably relax the ifn-dependence of adaptive immune responses and virus control. this redundancy of ifn and adaptive immune responses in rlcmv/vsv-g control provides a better understanding of the attenuation of this vector. it furthermore suggests that the virulence of a particular virus may influence the interferon-dependence of cd8 t cell and antibody responses, which may have implications for vaccine development. objectives: the class of type i interferons (ifns) consist of multiple ifnas and a single ifnb subtype. although being important for anti-viral defence they have been shown to be detrimental for the host during bacterial infections. while in general the first ifn to be produced is ifnb, the cell types responsible for its initial production remain unclear. to assess the cellular sources of ifnb and its role for the outcome in bacterial infections we use an ifnb reporter-knockin mouse model, in which yellow fluorescent protein (yfp) is expressed from a bicistronic mrna linked by an internal ribosomal entry site (ires) to the endogenous ifnb mrna. methods: to induce a type i interferon response we intravenously injected the tlr agonists poly(i:c) and cpg 1668, respectively, or infected reporter mice or bone marrow derived macrophages (bmdms) or dendritic cells (bmdcs) with the facultative intracellular pathogen listeria monocytogenes. the spatiotemporal tracking of the ifnb response was done using facs analysis and immunofluorescent microscopy. results: after poly(i:c) injection in vivo a small subpopulation of ifnb + cd8a + dendritic cells relocalize from the red pulp via the marginal zone to the t cell areas of the spleen whereas ifnb + activated pdcs were localized in the splenic white pulp at the t/b-cell interface after cpg administration. in vitro infection of bmdms, bmdcs and flt3-l derived dcs with listeria monocytogenes resulted in a low frequency of ifnb + cells depending on the listeria strain used and multiplicity of infection with bmdms being the most potent producers of ifnb. in vivo mostly activated f4/80 + macrophages were accountable for the ifnb expression. simultanous visualization of the cellular state of infection shows that ifnb + bmdms carry a higher bacterial load then ifnbcells. the cellular detection of ifnb expression reveals a remarkably low frequency of ifnb-producing cells in response to distinct pamps or the infection with intracellular bacteria. this hints at a superior role of few specialised cells for mounting a significant response against distinct stimuli or bacterial disease. additional data will be presented further resolving the timecourse of the ifnb response vs. the state of infection after bacterial challenge. the spleen is a secondary lymphoid organ that is characterized by highly specialized structures and plays a crucial role in the defense of blood-borne pathogens. we investigated the role of the spleen in the generation of antigen-specific cytotoxic cd8 t cell (ctl) responses in a systemic infection with recombinant adenovirus expressing ovalbumin (adova). although adenovirus mainly infects the liver, the ctl response requires the spleen, as splenectomized mice were incapable to mount an antigen-specific ctl response upon systemic challenge with adova. additionally, dendritic cells (dc), macrophages and an intact splenic structure were mandatory for the induction of an efficient ctl response. we detected adova specific ctl responses only in splenectomized mice that received splenic autotransplants but not after adoptive transfer of single cell splenocytes. furthermore, we asked how toll-like receptor (tlr) ligands influence adova-specific ctl responses. tlr ligands as "danger signals" are generally known to exert immune stimulatory effects. importantly, we observed that systemic administration of single-stranded rna (ssrna) prior to adova infection inhibited the generation of antigen-specific ctl responses in a tlr7 and type i interferon (ifn) dependent manner. ssrna injection induced the production of type i ifns as detected in sera and supernatants of splenocytes isolated from wild-type mice. experiments performed in ifnbeta reporter mice revealed that splenic plasmacytoid dcs represented the cellular source of type i ifns. additionally, splenic cd11c+ dcs purified from ssrna pretreated mice showed a reduced capacity to cross-prime ova-specific cd8 t cells upon adova challenge. in vivo pre-activation of endogenous ova-specific cd4 t cells as well as an adoptive transfer of in vitro activated transgenic ova-specific cd4 t cells prevented the ssrna mediated suppression of the ctl activity. we assume that dcs preactivated by systemic ssrna were impaired in their ability to activate naive cd8 t cells in response to an adova infection due to an impaired cd4 t cell help. taken together, we show that type i ifns cannot only stimulate, but also inhibit the induction of ctl responses in the spleen. within hours after infection many viruses induce early type i interferon (ifn) responses that can confer protection against lethal infection. modified vaccinia virus ankara (mva) is a highly attenuated vaccinia virus (vacv) strain generated by more than 500 passages in chicken embryo fibroblasts. mva stimulates systemic ifn responses, whereas vacv-induced cytokine milieus are dominated by il-12. to study the impact of virus-triggered ifn on the induction of t cell responses, we used recombinant mva-gp33 and vacv-gp33 expressing the gp33 epitope of lymphocytic choriomeningitis virus. for adoptive transfer experiments transgenic mice expressing the p14 t cell receptor recognizing the gp33 epitope were intercrossed with mice devoid of the ifn receptor (ifnar -/-) to obtain p14ifnar -/mice. upon adoptive co-transfer of p14ifnar -/and p14ifnar wt/wt t cells and subsequent challenge with mva-gp33, a massive expansion of p14ifnar wt/wt t cells was observed, whereas p14ifnar -/-t cells expanded less efficiently. in contrast, challenge with vacv-gp33 induced a rather similar expansion of ifnar competent and ifnar deficient p14 t cells. in the absence of ifnar-triggering t cell expansion was associated with reduced proliferation capacity and increased apoptosis. to study the impact of ifnar-signaling on the expansion of endogenous t cells, conditional mice with a t cell-specific ifnar-ablation were infected with mva. interestingly, in those mice the virus-specific t cells showed a reduced expansion compared to t cells of wt mice. additionally, we found that ifnar-triggering of dendritic cells but not of macrophages further supported specific t cell expansion. thus, upon virus infection virus-associated properties affected the overall cytokine milieu that influenced the quality and the quantity of expansion of virus-specific t cells. to delineate h5n1-specific signaling patterns we used a genome-wide comparative systems biology approach analyzing gene expression in endothelial cells infected with three different human and avian influenza strains of high and low pathogenicity. blocking of specific signaling pathways revealed that h5n1 induced an exceptionally nf-kb dependent antiviral response. irf3 is essential part of this interferon-response of human endothelia. furthermore, we identified hmga1 as a novel transcription factor specifically responsible for the overwhelming proinflammatory but not anti-viral response induced by h5n1. finally, nfatc4 was found to be a transcriptional regulator for specifically h5n1-induced genes. we therefore describe for the first time defined signaling patterns specifically activated by h5n1 which, in contrast to low pathogenic influenza viruses, are responsible for an imbalance of overwhelming proinflammatory and impaired anti-viral gene programs. objectives: to study the early events of immune antagonism by influenza virus in vivo and how this process impacts the timing at which adaptive immunity is generated. methods: to dissect the contribution of the unique viral antagonist ns1, delta-ns1 influenza virus (a recombinant influenza virus lacking the ns1 gene) was compared side by side with wild type influenza virus in vivo. mice infected with influenza virus were sacrificed at different time points after infection. to study the onset of inflammation during infection lung and blood samples were isolated with a selected panel of inflammatory and antiviral proteins that were measured by multiplex elisa and quantitative pcr (qpcr). to determine the bridging between innate lung inflammation and adaptive immunity, the kinetics of lung antigenbearing dendritic cell (dc) migration to the draining lymph nodes was quantitated in infected mice. further, functional in vitro and in vivo assays in infected animals with influenza-ova viruses were used to determine whether antigen-bearing migratory dcs were capable of priming transgenic t cells. to investigate the effect of ns1 during the onset of immunity in vivo, we systematically studied the early events occurring in the lungs and draining lymph nodes upon infection with influenza virus. strikingly, no sign of innate immunity was detected in the lungs for almost two days after infection when a sudden inflammatory burst including ifns, cytokines, and chemokines occurred. this burst preceded the robust dc migration and t cell activation in the lymph nodes. virus-loaded dcs appear in the lymph node starting 2 days post-infection, reached its maximum at day 4, and triggered t cell priming in vivo. a direct comparison of delta-ns1 virus with wild type virus infection demonstrates that virus can only trigger rapid inflammation in vivo when it lacks the ns1 protein. we demonstrate that the delay in the generation of immediate lung inflammation is mediated by the influenza ns1 protein. thus, we propose that the virally encoded ns1 protein establishes a time limited "stealth phase" where replicating influenza virus remains undetected thus preventing the immediate initiation of innate and adaptive immunity. keratinocytes represent the major cell population of human epidermis which provides a first line of defense barrier for the host. in addition, keratinocytes actively participate in immune response. viral double-stranded rna (dsrna) is the most important viral structure involved in activation of innate immune response. intracellular detection of dsrna triggers secretion of soluble signaling molecules, interferons, and activates pro-inflammatory response and programmed cell death, apoptosis. here we have used subcellular proteomics to characterise dsrna-induced human keratinocytes. cells were transfected with a mimetic of dsrna, poly(i:c), after which the cells were fractionated into cytoplasmic and mitochondrial fractions. these proteomes were analysed using two-dimensional electrophoresis in combination with mass spectrometry, immunoblotting and confocal microscopy. we show that several proteins involved in apoptosis, cytoskeleton reorganization and intracellular transport are up-regulated upon dsrna stimulation. in mitochondrial proteomes the expression of structural proteins, especially fragments of cytokeratins, is highly up-regulated. we show that cytokeratin 18 is cleaved during poly(i:c) stimulation and fragments are solely localized in mitochondria. similar degradation of cytokeratin 18 is seen in emcv-and vsv-infected keratinocytes. cytokeratin fragmentation after dsrna stimulation is dependent on caspase activation, which indicates a role for cytokeratins in the regulation of apoptosis during viral infection. in addition, we show that 14-3-3 proteins are upregulated in both mitochondrial cell fraction and cytoplasm after dsrna-stimulation and during viral infection. viral dsrna also induced transient phosphorylation of 14-3-3 target proteins. thus, these results suggest that 14-3-3 proteins have a regulatory role in host defence against viral infections. i. wessels 1 , d. fleischer 1 , l. rink 1 , p. uciechowski 1 1 institute of immunology, rwth aachen university hospital, aachen, germany objectives: the proinflammatory cytokine interleukin (il)-1b mediates the expression of a variety of proteins responsible for acute inflammation and chronic inflammatory diseases. however, the molecular regulation of il-1b expression is not elucidated, yet. it is known that the il-1b promoter is packaged into a nontranscribed but poised architecture in monocytes, rapidly producing il-1b when stimulated. b-cells which are il-1b non-producers reveal an inaccessible promoter structure. in this study the chromatin structure of the il-1b promoter and the impact of methylation on il-1b expression were examined in a cellular monocytic differentiation model. methods: promyeloid hl-60 cells were differentiated into monocytic cells after dihydroxyvitamine d 3 treatment. the monocytic phenotype was confirmed by flow cytometry. the il-1b promoter was analyzed using the chromatin accessibility by real time pcr (chart) assay. to test the influence of methylation, cells were treated with 5-aza-2-deoxycytodine (aza) and changes in il-1b expression were measured by pcr, elisa and western blot analyses. results: in contrast to undifferentiated hl-60 cells, differentiated cells displayed upregulation of cd14 antigen and acquired the ability to express il-1b. by comparing the accessibilities of il-1b promoter we detected that the il-1b promoter was not accessible in undifferentiated hl-60 cells but highly accessible in differentiated monocytic cells. the accessibilities of differentiated cells were comparable to that observed in primary monocytes. lps stimulation did not affect promoter accessibility in promyeloic and monocytic hl-60 cells, demonstrating that the chromatin remodeling of the il-1b promoter depends on differentiation but is independent of the transcriptional status of the cell. demethylation via aza led to the induction of il-1b expression in both undifferentiated and differentiated cells which could be increased after lps stimulation. conclusion: two independent mechanisms involved in the regulation of il-1b expression were found. our data indicate that the il-1b promoter is reorganized into an open conformation during monopoiesis and that the established poised structure is a privilege of mature monocytes but not of the entire myeloid lineage. as a second mechanism, il-1b expression is regulated by methylation acting independent of the developmental stage of myeloid cells. a. holweg 1 , g. wetzel 1 , h. arnold 1 , a. gessner 1 1 microbiological institute-institute for clinical microbiology, immunology and hygiene, university hospital erlangen, erlangen, germany the p65 family members of interferon (ifn) inducible gtpases also known as guanylate binding proteins (gbps) are among the most abundantly induced transcripts after stimulation with ifn-g. although the stimulatory capacities of the toll like receptor ligands lps and cpg, cytokines like ifn-b and il-1b and the intracellular pathogens listeria monocytogenes and toxoplasma gondii have been described to induce their expression, the function of gbps during bacterial infections is still ill defined. here we report for the first time the massive induction of murine gbps in two independent in vivo models of pneumonia (infection with streptococcus pneumoniae and pseudomonas aeruginosa). a strong and rapid induction of mgbp2, 3, 4, 5 and 6 mrna after intratracheal infection was detected by realtime pcr analysis of bronchoalveolar lavage (bal) cells. using newly generated antibodies in western blots and fluorescence microscopy, we identified macrophages as the main producers of mgbps in bal samples. although the signaling cascade involved in the upregulation of mgbps after ifn-g stimulation has been extensively studied, the mechanisms responsible for mgbp induction upon bacterial stimuli are unclear. in this study we could show that the induction of mgbps upon infection is abrogated in ifn type i/ type ii receptor double-deficient mice and thus absolutely dependent on endogenous interferon production. in contrast, the tlr adaptor molecule myd88 was found to be dispensable arguing for a trif-mediated interferon production subsequentially resulting in enhanced gbp expression. based on these findings our future experiments will address the functional role of gbps in innate and adaptive immune responses against extracellular bacteria. (2)). activation of resident microglia cells and infiltrating brain macrophages appeared to play a role in modulating virus replication shortly after infection but also appeared to be responsible for the inflammation in brains of infected mice. clearance of replicating virus from the cns required mcmv specific cd8 + t lymphocytes whose effectors functions still remain incompletely defined (j. immunol. 2008 aug 1;181 (3)). humoral immunity appeared to also play a role in the control of mcmv infection in developing brain. infected newborn mice treated with mcmv-specific antibodies had lower viral titers in the cns, significantly less cns inflammation and improved neuronal migration and increased cerebellar area as compared to control mice (j. virol. 2008 dec; 82 (24) ). conclusion: peripheral inoculation of the virus induces focal infection and inflammation in the developing mouse cns followed by strong innate and specific immune response that could also alter developmental programs required for normal development of mouse brain. passive immunization of infected newborn mice reduces mcmv-related pathology in infected brain suggesting that antiviral antibodies are an important component of immunological responses during cmv infection of developing cns. chronic inflammation is associated with the promotion and enhancement of malignancy and tumor growth. tumors enhance the accumulation of myeloid derived suppressor cells (mdsc), which contribute to tumor escape, immune tolerance and immune suppression. previously, we have shown that tumor-derived inflammatory cytokines, such as il-1b in the tumor microenvironment can induce a massive accumulation of mdsc in the spleen of tumor bearing mice and induce t cell suppression. in this work, we describe a novel polymorphnuclear mdsc subpopulation -inflammatory mdsc (infmdsc) which accumulates in the bm and spleen of mice bearing inflammatory 4t1 breast cancer cells over expressing il-1b (4t1/il-1b) tumor cells, but rarely in untransfected 4t1 cells. secretion of il-1b from tumor cells is crucial for infmdsc generation and accumulation. infmdsc have the ability to suppress nk cell activity via reduction of the nk activating receptor nkg2d in vivo, and in a cell-cell contact dependent manner in vitro. inflammation up-regulates il-4ra expression on the cell surface, which correlates with tumor growth and induction of suppression on nk cells. our data suggest that tumor derived inflammation enhances a specific mdsc subset which has the ability induce suppression of nk cells, and perhaps can serve as a new chemotherapy target. objectives: lps constitutes a main target of innate immune recognition of gram-negative bacteria and other lps carrying pathogens. cytokine production after in vitro stimulation of whole blood with lps is used as an expression of individual lps reactivity. we assess genome-wide data to analysed the association to lps induced cytokine response, and replicated the top findings in two independent cohorts. materials and methods: we used 130 healthy caucasian blood donors as discovery samples, and replicated snps from affymetrix -genome-wide human snp array 5.0 having p x 10 -4 in two independent cohorts each consisting of 200 blood donors using a customized chip from illumina and real-time pcr respectively. in all the three cohorts whole blood samples was stimulated for 24h and we measured the levels of il-6, il-8, il-10, tnf-alfa and il1-ra with r&d ® assays on luminex platform.. the association analysis was performed with wald statistical test assuming an additive model. the discovery sample statistical analysis revealed 150 snps with p x 1,0*10 -4 . to identify/replicate the association of cytokine production for these 150 we reanalysed these on a 200 cohort. a combined analysis revealed 10 snps with p x 9,1*10 -5 .these results are not genome wide significant. the 10 snps showing nominal association to lps cytokine response are being analysed in a replication cohort conclusion: we find 10 nominal associated snps between lps stimulated cytokines in blood samples of healthy caucasian blood donors. the importance of these snps are to be determined in a replication cohort. adipocytes, so far known for their lipid-storage capacity came into the focus of interest because of their immunoregulatory properties resembling those of innate immune cells. adipocyte-derived pro-inflammatory mediators contribute to the development of chronic inflammation, thereby promoting the progression of insulin-resistance/metabolic-syndrome and diabetes. the physiological signals inducing the secretion of inflammatory mediators by adipocytes are unknown. heat shock proteins, such as hsp60 have been identified as potent regulators of inflammatory innate immune cell-activities, whereas their influence on adipocyteactivities remained elusive. here, we investigated the regulatory effects of hsp60 on adipocytes. for the first time we could show a hsp60-stimulated release of the proinflammatory cytokines il-6, cxcl1 and mcp-1 in a time-and concentration-dependent manner from murine 3t3-l1 adipocytes. analyses of hsp60-signalling in these adipocytes revealed that members of the mapk-family (erk1/2, p38) and the transcription factor nfkb are involved in hsp60-mediated induction of the mediators il-6, cxcl1 and mcp-1. binding-studies with fluorescence-labelled hsp60 demonstrated that the interaction of hsp60 with adipocytes exhibits basic features of a receptor-mediated binding. hsp60-binding to adipocytes was saturable and reached its maximum at 3.5 mm. binding was inhibitable only by the unlabelled ligand (52 %), but not by unrelated proteins, thereby proving the specificity of hsp60-binding. further analyses to characterize hsp60-receptor structures on adipocytes revealed the presence of toll-like receptor (tlr)4 on adipocytes. tlr4 has been found to be expressed on macrophages and to interact with hsp60, therefore suggesting tlr4 as a potential receptor candidate for hsp60 on adipocytes. in order to identify the responsible binding-epitope of hsp60 we investigated the effect of specific antibodies directed against different epitopes of the hsp60-molecule. incubation with antibodies directed against the n-terminus of hsp60 (aa1-200; 5-25 mg/ml) were capable of inhibiting the hsp60-binding to adipocytes (47-80 %) indicating that the n-terminal region of hsp60 is involved in receptor binding. our experiments demonstrate that hsp60 stimulates the release of proinflammatory adipocyte mediators. the findings implicate that the hsp60-mediated induction of adipocyte mediators contributes to inflammatory processes observed in obesity-associated disorders and could serve as a target for the development of therapeutic strategies for patients suffering from diabetes or diabetes-related disorders. legionella pneumophila, a gram-negative facultative intracellular bacterium, is the causative agent of a severe pneumonia known as legionnaires' disease. classically, type i ifns (ifna/b) have been associated with antiviral immunity. ifna/b signal through the ifna/b receptor (ifnar) leading to the induction of hundreds of ifn-stimulated genes (isgs), many of which have anti-microbial activities. recently, it was demonstrated that ifna/b are also produced in host cells infected with (intracellular) bacteria or stimulated with cytosolic dna. here we show by rnai that l. pneumophila infected host cells produced ifnb dependent on irf3. we observed enhanced l. pneumophila replication in mouse macrophages lacking ifnar and human cells after irf3 knock-down, suggesting that endogenously produced ifnb activates a cell-autonomous defence against legionella. moreover, ifnb treatment restricts legionella replication in human and murine host cells. ifna/b impacts formation of large replication vacuoles, but appears not to influence the recruitment of er markers nor fusion of the legionella-containing vacuole with the lysosome. moreover, ifna/b-mediated cellautonomous defence was independent of autophagy and pyroptosis. we thus hypothesize the crucial involvement of antibacterially acting isgs. ongoing studies focus on the role of ifn-induced immunity-related gtpases (irgs). mesenchymal stem cells (mscs) are identified by their capacity to differentiate into connective tissue cell types. mesenchymal stem cells also show a high plasticity and account for a potential therapeutic efficacy. several authors have reported the expression of alfa-smooth muscle actin (a-sm actin) by msc. this protein has been considered a marker for the myofibroblast, has the capacity to polymerize into the cytoplasm and contribute to the cell contractility. this activity may be crucial for the changes of the cell shape, for cell-cell interactions and may therefore be relevant for the physiology of msc. in this work, we study the presence of alfa-sm actin in human msc by flow cytometry and immunoflurescence. we also study the contractility of these cells under the effect of different cytokines. human bone marrow samples were obtained from bone marrow aspirates. bone marrow mononuclear cells were isolated by density gradient centrifugation and cultured in opti-mem culture medium with 3 % of fetal calf serum at 37°c and 5 % co2. bone marrow nonadherent cells were removed after 24 h, and culture medium was refreshed twice per week thereafter. cells grew adherent, with a fibroblastic morphology and expressed cd10, cd29, cd73, cd21 and stro-1 and were negative for cd45. intracellular staining was performed for alfa-sm actin. cell contractility was measured with the collagen gel contraction assay. alfa-smooth muscle actin was detected in almost 100 % of msc. interleukin-2, ifn-gamma and tnf (th1 cytokines) increased msc contractility, whereas il-10 (a th2 cytokine) decreased msc contractility. by immunofluorescence, we observed that il-2, ifn-gamma and tnf increased the incorporation of alfa-sm actin into the stress fibers of msc, whereas il-10 decreased that incorporation. our results suggest that th1 and th2 cytokines regulate msc physiology by acting on their contractility. aims: thapsigargin (tg) is a sesquiterpene lactone (sl) of guaianolide type isolated from the mediterranean plant thapsia garganica l. it is widely recognized as an inhibitor of sarco-endoplasmic reticulum ca 2+ -atpase leading to elevation of intracellular calcium. this activity is shared by trilobolide (tb), a sl from laser trilobum (l.) borkh. tg has been shown to possess prospective immunotherapeutic properties. it kills slowly proliferating and non-proliferating cells, and inhibits replication of viruses. the aim of our work was to investigate possible immunostimulatory potential of tg and tb. methods: the effects of the agents were analyzed under conditions in vitro using rat and mouse resident peritoneal cells (pecs), and human peripheral blood mononuclear cells (hpbmcs). they were cultured in density of 2 × 10 6 /ml in complete rpmi-1640 medium. supernatant levels of ifn-g were determined by elisa. production of no by animal pecs was assayed using griess reagent. possible contamination of the samples with lps was excluded using the chromogenic limulus amoebocyte assay. results: we have found that both tg and tb at as low doses as 40 nm and 400 nm induce ifn-g secretion in rat pecs and hpbmcs, respectively. the concentration of ifn-g produced by the highest dose of the agents (10 mm) at the 24-h culture interval reached the values of approximately 3 ng/ml and 2 ng/ml in rat pecs and hpbmcs, respectively. the increase was apparent within the interval of 2-6 h in rat pecs. the 24-h interval was optimal for accumulation of ifn-g in cultures of hpbmcs. only modestly enhanced secretion of ifn-g was observed in mouse pecs. production of ifn-g was found to depend on activation of nf-xb and map kinases p38 and erk1/2. it was not suppressed by the calcium chelating agents bapta-am and tmb-8. the tg-and tb-induced ifn-g production was associated with activation of the high-output production of no. conclusions: sesquiterpene lactones tg and tb are potent inducers of ifn-g in animal and human cells. the effect is independent of their serca inhibitory potential. underlying mechanism(s) of the immunostimulatory effects remain to be elucidated. acknowledgements: the work was supported by the grant gacr 305/07/0061. pin1 is a peptidil-prolil-cis-trans isomerase that specifically binds phosphorylated ser/thr-pro protein motifs and catalyzes the cis/trans isomerization of peptides. mitotic proteins, cytoskeleton, transcription factors and apoptotic proteins are pin1 substrates and targeting sites. recent data show that pin1 interacts with apo-bec3g (a3g). the pin1/a3g interaction results in a reduced a3g expression and a diminished a3g-mediated restriction of hiv. two single nucleotide polymorphisms (snps) in the promoter region of the pin1 gene (-842 g/c and -667 t/c) modulate pin1 expression; in particular, the -842 gc genotype or cc haplotype are associated with reduced protein levels (neurobiol. aging, 28; 69-74, 2007) . the -842 c/g and -667 t/c polymorphisms in the promoter of pin1 gene as well as pin1 protein levels were analyzed in 30 exposed seronegative individuals (esn), heterosexual partners of hiv-infected patients; 40 hiv-infected patients (hiv) and 40 healthy controls (hc). the genotype and allele distributions of the -842 snp was skewed in esn (genotype: p= 0.008; allele: p= 0.013). in particular esn showed a significantly lower frequency of the -842 gg genotype compared to hiv and hc (p=0.017 and p=0.019, respectively) and consequently a lower g allele frequency (p=0.026 and p=0.028, respectively). no significant differences were found for the -667 snp. these snps are in linkage disequilibrium and combine to form haplotypes. conclusions: our findings support the role of hiv viral replication as the most important promoter of immune activation and prove the importance of art in reducing immune activation and viral replication even in a sub-saharan african environment, where patients are exposed to an abundance of other infectious agents. our data further indicates that hiv replication rather than host genetics is the key determinator of circulating cytokine levels among the studied hiv infected participants. aims: acyclic nucleoside phosphonates (anps) are synthetic analogues of natural nucleoside monophosphates. they have proved to be outstanding antivirals inhibiting replication of both dna-viruses and retroviruses. tenofovir, 9-(r)[2-(phosphonomethoxy) propyl]adenine [(r)-pmpa] is broadly used for therapy of aids, adefovir, 9-[2-(phosphonomethoxy)ethyl]adenine (pmea) has been approved for the treatment of hepatitis b. the aim of our work was to investigate possible interactions of anps with production of cytokines and nitric oxide (no) implicated in antiviral defence mechanisms. the immunobiological effects of anps were screened in vitro using mouse resident peritoneal cells. they were cultured in density of 2 × 10 6 /ml in complete rpmi-1640 medium. secretion of cytokines was determined after the 5-h culture by elisa. production of no was assayed at the interval of 24 h using griess reagent. approximately 300 compounds belonging to several distinct anp groups were included in the study: a) pmea derivatives, b) pmedap i. e. 9-[2-(phosphonomethoxy)ethlyl]-2,6-diaminopurine derivatives, c) 9-[2-hydroxy-3-(phosphonomethoxy)propyl]-adenine (hpmpa), and hpmpdap derivatives, d) guanidino analogues of pmpdap, e) 9-heteroalkyl substituted 2-amino-6-guanidinopurines, and f) 2-amino-3-(purin-9-yl)propanoic acid derivatives. possible presence of lps in the stock solutions of the samples was checked and excluded using the chromogenic limulus amoebocyte assay. results: approximately 30 compounds were found to activate the secretion of anti-hiv effective chemokines rantes and mip-1a and cytokines tnf-a and il-10. although these anps did not stimulate biosynthesis of no on their own, they substantially augmented production of no triggered by ifn-g. no clear-cut relationship between the chemical structure and biological effects of anps was observed. however, the most effective proved to be the n 6 -cycloalkyl derivatives of pme-dap. the effects were produced in a dose-dependent fashion, exhibiting the immunostimulatory effects at as low concentrations as 2 to 5 mm. the remarkably enhanced secretion of chemokines was reached within 2-4 h of the cell culture. the effects were found to depend on activation of nf-kb. conclusions: it may be suggested that anps represent a new generation of antivirotics with combined antimetabolic and therapeutically prospective immunostimulatory properties. acknowledgements. the work was supported by the grant 1m0508. host related immune factors in childhood chronic hepatitis b and change of initial profile with ifn-a treatment needs to be clarified. sixteen patients were included, and 10 million units of ifn-a treatment three times a week for 6 months was initiated. before and after treatment: percentages of the il-2 and ifn-g in cd4+ t cells were assessed to determine intracellular t helper cell 1 (th1) type cytokine expression. similarly, percentages of intracellular il-2 and ifn-g were detected to verify cytotoxic t cell 1 (tc1) type cytokine expression in cd8+ t cells. percentage of th2 and tc2 type cytokine expression, (il-4 and il-13) were determined in cd4+ and cd8+ t cells, respectively. six (50 %) of these were evaluated as having no response and the other half with partial/complete response. all patients had higher percentages of th2 cells with respect to healthy controls before treatment. tc percentages, both tc1 and tc2, were significantly different between these groups, being higher in the patient group. when values of no responder group were compared with healthy controls, il-4 expression was higher and the percentages of th1 type cells were significantly low. il-13 expression in th and tc cells decreased after treatment in the unresponsive group. intracellular cytokine profiles of treatment responders and normal controls were not different. this has been the first study in children comparing baseline and post treatment intracellular cytokine profiles with healthy controls. the frequency (29,8 %) of high concentration of igg anti-oxldl antibodies in patients with hcv infection were significantly elevated in comparison to healthy subjects (6,6 % according to bibliographic data). the immune response was significant but it was not assosiated with the viral load. it is probable that humoral immunity plays a critical role and contributes in an immunoinflammatory reaction of hcv-infection. abstract withdrawn by author t. schwandt 1 , f. juengerkes 1 , b. schumak 1 , g. gielen 1 , j. kalff 2 , p. knolle 1 , b. holzmann 3 , a. limmer 1 1 university hospital bonn, institute of molecular medicine and experimental immunology, bonn, germany, 2 university hospital bonn, department of surgery, bonn, germany, 3 department of surgery, tu munich, munich, germany bacterial translocation is a possible risk of abdominal surgery and could be the cause of life-threatening consequences such as organ failure and septic shock. patients surviving septic shock often suffer from opportunistic infections as well as defects in adaptive immunity. here we investigated the influence of bacteremia and bacterial translocation on systemic adaptive immune responses using murine models. to mimic abdominal surgery, mice were subjected to intestinal manipulation (im). to study septic conditions, mice underwent colon ascendens stent peritonitis (casp) or received e.coli intravenously. we monitored the distribution of gut-derived bacteria by in vivo imaging (xenogen) and additional microbiological assays and determined antigen-specific ctl responses towards subsequent infection with recombinant adenoviruses (av). we detected comparable amounts of bacteria in lung, liver and spleen of mice that underwent casp or were injected i. v. with e.coli. in addition, mice infected systemically with av lacked an antigen-specific ctl response, whereas the ctl responses of locally av infected mice were not affected. in contrast, bacteria were detected in lung and liver but not in spleen of mice that were subjected to im or received e.coli by injection into the hepatic portal vein. here, the ctl response was not impaired. depletion experiments imply that kupffer cells as well as soluble mediators such as tumor necrosis factor alpha play an important role in trapping and clearance of translocated bacteria in liver and lung. our experiments demonstrated that translocation of bacteria did not cause immune suppression as long as they did not reach the spleen in high numbers. we suggest that liver and lung fulfill a filter function to prevent systemic distribution of gut-derived bacteria. failure of or bypassing these barriers might enable bacteria to access the spleen and thus cause systemic suppression of adaptive immunity, whereas induction of local immunity is not affected. objectives: varicella-zoster virus (vzv) is one of the most frequent pathogens for which a vaccine is available. tropism of vzv for skin is the most obvious manifestation of vzv infection, producing vesicular cutaneous lesions that are associated with varicella and herpes zoster. the striking difference in the clinical outcome of infection by rush inducing circulating virulent vzv strains and asymptomatic infection by the vaccine leads to the assumption that the virus interferes with cutaneous immunity. therefore, we comparatively investigated the impact of vzv clinical isolates and the vaccine on the reciprocal crosstalk of immature dendritic cells (idcs) and epithelial gd t cells. methods: skin punch biopsies of herpes zoster patients were analyzed by dual immunofluorescence microscopy. phenotypic changes of cutaneous dcs and monocyte-derived dcs after vzv infection were investigated by flow cytometry. the cytokine profile of vzv-infected dcs and epithelial gd t cells was determined through elisa. results: we observed that innate lymphocytes recognize dcs, which infiltrate herpes zoster lesions, after infection with vzv. strikingly, only the vaccine but not vzv clinical isolates could license the bidirectional crosstalk between idcs and gd t cells resulting in release of ifn-g and il-12, the signature cytokines of antiviral immune responses. this is the first demonstration that virulent virus strains disrupt dendritic cell instruction whereas the corresponding vaccine does the opposite. we describe a novel immune evasion strategy in the skin, which provides the opportunity for efficient and symptomatic virus replication. this result is also of practical importance: future vaccine design has to ensure that candidate vaccines do not impair dc instruction in order to allow stimulation of powerful antiviral immune responses. a. jafarzadeh 1 1 rafsanjan university of medical sciences, rafsanjan, iran, islamic republic of objective: it has been reported that the caga + h. pylori strains induce more severe gastric mucosal inflammation. the aim of this study was to investigate the association of the h. pylori virulence factor caga with serum levels of il-17 and il-23 in h. pylori-infected duodenal (du) patients and h. pylori-infected asymptomatic (as) carriers. methods: totally, 45 h. pylori-infected du patients (23 patients were positive for anti-caga antibody and 22 patients were negative for anti-caga antibody), 30 h. pylori-infected as carriers (15 subjects were positive for anti-caga antibody and 15 subjects were negative for anti-caga antibody) and 15 healthy uninfected subjects (as a control group) were enrolled to study. a blood sample was obtained from all participants and the serum levels of il-17 and il-23 was measured by elisa method. the mean serum levels of il-17 in total du patients (9.28 pg/ml ± 5.48) was significantly higher than those observed in total as subjects (5.19 pg/ml ± 3.75, p x 0.001) and healthy control group (3.55 pg/ml ± 3.76, p x 0.0001). in du group, it was found that the mean serum levels of il-17 in subjects with positive test for anti-caga (10.84 pg/ml ± 5.79) was significantly higher than those observed in subjects with negative test for anti-caga (7.65 pg/ml ± 4.74; p x 0.05). the mean serum levels of il-23 in du (8.66 pg/ml ± 8.41) and as groups (7.25 pg/ml ± 5.66) was significantly higher than those found in uninfected control group (3.64 pg/ml ± 3.36, p x 0.02 and p x 0.03, respectively). however, no significant difference was observed for mean serum levels of il-23 between du and as groups. moreover, in both du and as groups the mean serum levels of il-23 was not significantly differ in subjects with positive test for anti-caga and those were negative for anti-caga antibody. the results of the present study showed higher serum concentrations of il-17 and il-23 in h. pylori-infected subjects as compared with control group. in du group the expression of il-17 influenced by the bacterial caga factor. a. aral 1,2 , a. atak 1 1 gazi university faculty of medicine, department of immunology, ankara, turkey, 2 gazi university institution of health sciences, department of immunology, ankara, turkey objective: ebv is a human herpesvirus, which infects human b lymphocytes latently and immortalizes the cells due to transformation. ebv infection is asymptomatic in childhood while it may cause a self-limiting lymphoproliferative disorder called infectious mononucleosis (im) in adolescence. in immunodefective patients, the virus may lead to malignancies like burkitt's lymphoma, nasopharyngeal carcinoma and immunoblastoma. the virus can also cause latent infections. cytokine production in response to ebv infection differs according to the phase of the infection. neopterin, ifn-g and il-6 levels are elevated in acute ebv infection in vitro; but in chronic phase, particularly, il-6 elevation could not be detected. tnf-a enhances the effects of ifn-g on neopterin synthesis while neopterin enhances the secretion of tnf-a via various stimuli. ifn-g levels are elevated particularly in the acute phase of im. since the elevation of cytokine levels changes according to the phases of disease, it's thought that the association between host defense and viral replication depends on different parameters. ifn-g is the major stimulus for neopterin synthesis, which stimulates monocytes and macrophages primarily. increased production of neopterin in body fluids can be used to monitor the activation of cell mediated immunity. method: in order to analyze the effects of neopterin release and other cytokines, mononuclear cells have been isolated from peripheral blood samples of healthy donors and transformed via ebv. neopterin, ifn-g, tnf-a and il-6 levels have been measured with eia kits in culture supernatants. results: neopterin levels increased dependent on time and independent of ebv transformation. in ebv-transformated cell culture tnf-a levels increased beginning from the 48th hour and reached to maximum levels at the 1st week and decreased again at the 3rd week; however there were no significant differences between the levels among three weeks. likely tnf, ifn-g levels also increased at the 1st week and started to decrease at the 3rd week. il-6 reached to its peak at the 3rd week. conclusion: according to these results, neopterin levels, which increased dependent on time but independent of ebv transformation, may be a helpful marker for evaluating the acute response to viral infection but not for transformation. v. jurisic 1 , m. jurisic 2 1 university of kragujevac, school of medicine, kragujevac, serbia, 2 university of belgrade, school of dentistry, belgrade, serbia tnf-alpha is a pleiotropic cytokine that is considered as a primary modifier of inflammatory and immune reaction in response to various inflammatory diseases and tumour. we investigated tnf concentration in 43 radicular inflamed cysts and 15 odontogenic tumours obtained from patients undergoing surgery, under local anaesthesia, and after aspiration of cystic fluid from non-ruptured cysts. further, we estimated the role of cyst wall on production of tnf-alpha in respect of presence of inflammatory cells, degree of epithelial proliferation and degree of vascularization. the concentrations of tnf-alpha in the cystic fluids were measured by elisa, while proteins analyzed after separation by two-dimensional gel electrophoresis. the presence of pericystic inflammed cells were analyzed in thick section for routine histological examinations and by immunohistochemisty for cd3, cd20 and cd68. tnf-alpha is elevated in both cysts fluid, but higher values were found in radicular inflamed cysts in comparison to odontogenic tumours. higher concentration of tnf-a were associated with higher protein concentration, higher presence of inflammatory cells in peri cystic tissues, cysts wall thickness and higher degree of vascularisation (mann-whitney u-test, p x 0.05) in radicular cysts. no correlation was found, based on these parameters in odontogenic tumours, but all sumple have detectable concentrations of tnf-alpha. objectives: interactions between the neuroendocrine and immune system play an important role in maintaining and restoring homeostasis. in susceptible individuals a dysfunction of the neuroendocrine system may be one of the risk factors involved in the pathogenesis of septicemia and bacterial infection at all. we will study prolactin role in defensive reaction of immune system to bacterial infection. as a type of this bacterial infection we have chosen septic status, where we are expecting the most significant alterations of immune reaction, and specially septic statuses in blood malignancies, where we are expecting deficiency of immune system. our idea is that prolactin takes part in this defensive reaction by its cytokine effects, and it has contraregulative role against activation of adrenocortical system. the aims of this study are to extend our knowledge about the activation of peripheral prolactin expression and by this way to contribute elucidation of its role in periphery. 1) drawings blood from 20 patients and 20 healthy donors. blood of patients and healthy persons were sampled together with past histories after getting their acquainted approval. status of patient had to meet these conditions: a) the presence of systematic inflammatory response syndrome (sirs) according to standard clinical and laboratory criteria. b) positive hemoculture or determination of septic focus with demonstration of microbiologic source. 2) detection of the gene expression of prolactin and tlr-2 in cd14+ peripheral blood monocytes from patients with septic status and from healthy controls has been performed by rt-pcr at the level of mrna and flow cytometry at the level of intracellular protein results: we have found statistical significant differences (p p 0.05) in expression profile between patient and control groups. these differences were at both levels of expression, mrna and protein. conclusion: septic statuses change tlr-2 and peripheral prolactin expression in cd14+ monocytes. the function of interferon (ifn)-induced immunoproteasomes (i-proteasomes) is at present almost exclusively acknowledged in connection with improved processing of mhc-class i antigens. the experiments performed here challenge this existing paradigm of i-proteasome function. we demonstrate in vivo and in cellulo that the key role of i-proteasomes resides in the protection of cells against the formation of protein aggregates, which is ultimately crucial for preservation of cell viability under ifn-induced oxidative stress. ifns up-regulate the ubiquitylation machinery and enhance the formation of oxidant-damaged, nascent, poly-ubiquitylated proteins, which essentially require i-proteasomes for their efficient degradation. i-proteasome deficiency results in the formation of aggresome-like induced structures and increased sensitivity towards apoptosis. enhanced degradation of poly-ubiquitin conjugates designed to protect cells, will therefore also increase the peptide supply for antigen presentation. thus, only by executing their physiological function in the maintenance of protein homeostasis i-proteasome induction also provides a mechanism for cellular immune-adaptation. background: revived by the description of new functions, b cells are considered to be essential in the genesis of autoimmune diseases. in strong support of this interpretation, baff would play a key role in their physiology. however, the correlation between circulating baff levels and disease activity is not clearly established. conflicting results concerning levels of baff and b-cell repopulation after rituximab treatment have been reported. in fact, basal serum levels of baff reported in literature vary according to the antibodies (ab) used in the elisa and the mode of calculation. the aim of this study was to understand these variations. material and methods: different anti-baff abs were tested to verify whether they recognize glycosylated baff purified from u937 culture supernatant. sera from patients with autoimmune diseases were also tested. a western-blot analysis of sera was performed to evaluate anti-baff abs specificity and the best combination of anti-baff abs validated for elisa. then, different commercial baff elisa-quantification kits were compared to our "in-house" elisa. results: unexpectedly, the binding of some anti-baff ab was restricted to glycosylated baff. however, both glycosylated and non-glycosylated forms of baff were found in sera from patients with autoimmune diseases. most of the kits commercially designed to quantify baff suffer from some limitations. some sera are indeed positive with a kit and negative with another and vice versa. furthermore, there seems that rheumatoid factors (rf) interfere and correlate with the optical density of the anti-baff elisa. conflicting results concerning levels of baff and disease activity or auto-abs titers should be reconsidered in light of the glycosylation status of baff. (table-2 ). when tb household contacts and healthy controls were compared, cfp10 and esat6 seemed to be more useful than tst in tb contacts for displaying ltb (table-2) . although cfp10 spot numbers were much more than esat6 spots at the beginning and follow up period, statistically there was no difference in terms of median values(p: 0,069)( table-3 ). both esat6 and cfp10 spots were prone to decline during the follow up period. [ is some evidence to suggest that aspects of innate immunity (e. g. triggering of cytokine production by dendritic cells) may be impaired by hcv. to gain insight into some features of the innate immune response activated in vivo in the context of acute hcv replication, we analysed cytokine and chemokine levels in serum samples from chronic hcv patients undergoing liver transplantation. luminex multiplex assays and elisas were used to quantitate serum levels of g 20 analytes immediately prior to liver transplantation and at sequential time points up to 90 days post-transplantation. the increase in serum hcv rna levels associated with acute viral infection of the transplanted liver was found to be associated in most patients with elevations in serum levels of cytokines and chemokines including ifn-gamma, tnf-alpha, ip-10, il-6 and il-10. notably, the pattern and magnitude of systemic analyte elevations were very similar to those accompanying the acute burst of viral replication in primary hcv infection. high-magnitude elevations in systemic type 1 ifn levels were not observed in either setting, which may reflect an in vivo impairment of plasmacytoid dendritic cell functions by hcv similar to that observed in in vitro studies. we suggest that the liver transplant setting can be used as a model to study aspects of the innate immune response activated in acute hcv infection. to test the hypothesis that virus interactions with sialic acid receptors may play a role in innate antiviral immunity, we used recombinant viruses and differentiated cultures of human airway epithelial cells (hae). the hemagglutinin of the pandemic virus a/hong kong/1/68 (h3n2) (hk) differs from its putative avian precursor by 7 amino acid substitutions. we generated the complete recombinant virus rhk and its ha variants with amino acid reversions back to the ancestral avian sequence (rhk-5aa-i62r, n81d, k92n, s193n, g144a, human (2-6); rhk-r2-l226q, s228g and rhk-7aa-i62r, n81d, k92n, s193n, g144a, l226q, s228g, avian (2-3)). among these variants, the double mutant rhk-r2 and the seven mutant (rhk-7aa) had a typical avian-virus-like receptor-binding specificity owing to substitutions l226q and s228g.we infected hae cultures with the viruses and collected samples from the apical and basolateral sides of the cultures at different times post infection. virus titers were determined in mdck cells, and concentrations of about 50 pro-and anti-inflammatory mediators and chemokines were measured using a multiplex bead assay.concentrations of most cytokines progressively increased at the apical side of the cultures in the course of the infection. many cytokines, including t-cell-attracting chemokines such as ip-10 and rantes, were induced to similar levels by different viruses. however, some mediators were induced significantly stronger by avian-like viruses rhk-r2 and rhk-7aa as compared to rhk and rhk-5aa. in particular, avian-like viruses stimulated a higher release of potent chemo-attractants of innate immune cells, such as g-csf and il-8, shedded adhesion molecules (cd25, vcam-1, icam-1), and pro-apoptotic factors (trail). remarkably, the patterns of secreted cytokines differed between the apical and basolateral sides of the cultures. whereas avian-like viruses typically induced similar or higher levels of cytokines at the apical side than did rhk and rhk-5aa, the human-like viruses were stronger inducers of basolaterally secreted mediators. these data provide the first direct experimental evidence that receptor specificity of influenza viruses can significantly affect patterns of innate immune responses in human airway epithelium. further studies are warranted to determine the role of the observed effects in the host range and pathogenicity of influenza viruses in humans. a total of 98 newborn infants were enrolled in the study. forty-nine newborn infants (group i), who met the criteria of sepsis, had a routine sepsis evaluation as well as measurement of pct, il-10, and neutrophilic cd64 levels, procalcitonin and il-10 were measured by elisa technique while, neutrophilic cd64 by single colour flowcytometric technique. of these 49 "infected" infants, 16 had positive blood culture (subgroup ia: culture-positive sepsis), and 33 infants were diagnosed to have clinical sepsis with negative blood cultures (subgroup ib: culture-negative sepsis). another 49 newborn infants classified as control group (group ii) . results: sensitivity, specificity, positive predictive value, and negative predictive value for diagnosis of sepsis were analyzed for pct, il-10, cd64, and crp. il-10 had the highest sensitivity and specificity, 92 % and 84 %, respectively, using cutoff n 17.3 pg/ml. for pct, the highest sensitivity and specificity, 65 % and 60 %, respectively, were at a cutoff value of n 36.4 pg/ml. neutrophilic cd64 had maximal sensitivity and specificity of 92 % and 71 %, respectively, at cutoff value of 2.6 %. combinations of different markers may improve the sensitivity and specificity of biomarkers such as the tests used in this study. we found that the best combination was il-10 and neutrophilic cd64, which together provided sensitivity and specificity of 95 % and 83 %, respectively, and npv 86 %. the combination of il-10 and crp had high sensitivity and moderate specificity, 93 % and 79 %, respectively. conclusions: il-10 and neutrophilic cd64 levels determination can be used as good tests for early detection of neonatal sepsis. procalcitonin measurement might be used as an additive parameter to improve the diagnostic efficacy of the other markers in neonatal sepsis, but it is not helpful as a single test. objectives: the assembly and activation of inflammasomes are essential processes in the immune response to many inflammatory stimuli. inflammasomes are typically formed by at least one member of the cytosolic innate immune sensor family, the nod-like receptors (nlr). the nlr family members nalp3, naip5 or ipaf and the adaptor asc are involved in caspase-1 activation in response to bacterial infection, triggering the processing and secretion of il-1b and il-18. recent studies have demonstrated that tlr-dependent inflammasome activation in macrophages is modulated by autophagy, a homeostatic mechanism for the catabolism of cytosolic constituents. autophagosome biogenesis and maturation requires activation of the class iii pi3k, vps34 and can be inhibited with the pi3k inhibitors wortmannin and 3 methyladenine (3ma). in contrast, activation of akt, via class i pi3k, results in inhibition of autophagy. the aim of this study was to determine the nature of this inflammasome and whether autophagy influences il-1b secretion in dendritic cells. methods: murine bone marrow-derived dendritic cells (bmdm) were treated with tlr ligands in combination with 3ma, wortmannin or an akt inhibitor. supernatants were analysed for il-1b by elisa. results: 3ma enhanced il-1b secretion by bmdc treated with the tlr3 and tlr4 ligands poly(i:c) and lps, but not with any other tlr ligands tested. similar results were obtained using wortmannin. this increase in il-1b secretion was greatly reduced in bmdc from nalp3 -/mice compared to wild type c57/bl6 controls. treatment with the akt inhibitor had no effect on lps-induced il-1b secretion by bmdc. tlr-dependent secretion of il-1a was also enhanced by treatment with 3ma. conclusions: these data demonstrate that il-1b secretion by bmdc in response to treatment with pi3k inhibitors, in combination with lps or poly(i:c), is dependent on the nalp3 inflammasome. this response is limited to tlr3 and tlr4 agonists. inhibition of akt had no effect on lps-induced il-1b production, suggesting that the effect of wortmannin and 3ma on inflammasome activation is not dependent on the inhibition of akt activation by class i pi3k. objectives: in the last few years, several evidences have shown the modulation of toll-like receptors (tlrs) by g-protein coupled receptors, i. e. our group has recently demonstrated the attenuation of tlr2 signaling by the inflammatory lipid mediator sphingosine 1-phosphate (s1p) through receptors 1 and 2 in human monocytes-macrophages, which could explain some of the s1p anti-atherogenic effects. since adhesion of monocytes to endothelial cells is considered an important event in atherogenesis, our goal was to investigate the putative implication of tlr-s1p receptors crosstalk on the expression of adhesion molecules in endothelial cells. methods: for the study, in vitro cultured endothelial cells from arterial and venous origin were challenged with a combination of tlr ligands and s1p, and later analyzed by flow cytometry. a pharmacological analysis of the s1p receptor subtype involved in the response was also performed. results: data from flow cytometry experiments revealed that icam-1 expression was increased following lps and s1p concomitant stimulation in both venous and arterial cells, suggesting that tlr4 and s1p receptors cooperate in the expression of icam-1. conversely, no cooperation was observed when tlr2 ligands were used. in order to elucidate which s1p receptor subtype was involved in the increase of icam-1 expression, we used a pharmacological approach with s1p receptor inhibitors and pertussis toxin. results showed differences between arterial and venous cells. while in arterial cells elevated icam-1 after lps and s1p challenge was significantly reduced by blocking s1p receptor 3 and the effect was pertussis toxin-insensitive, in venous cells the response was pertussis toxinsensitive and not blocked with inhibitors of s1p receptors 2 and 3, which suggest that s1p receptor 1 might be involved in the effect. conclusions: altogether these data demonstrate that tlr4 and s1p receptors can interact to increase adhesion molecules such as icam-1 in human endothelial cells, and the s1p receptor subtype involved in the effect differs between arterial and venous cells. 15 with ssc without pah) and a pool of 12 sera of healthy controls (hc) were tested. results: in 1 dimension immunoblot, serum igg from ssc patients, patients with ipah and hc tested individually reacted with 7-10, 4-8 and 2-5 protein bands in a human vsmc protein extract with qualitative and quantitative differences between groups, respectively. in 2 dimension immunoblot, igg of pools of patients with ipah, igg of pools of patients with ssc with or without pah, and igg of a pool of hc recognized 145±48, 127±26, 130±25 and 150 protein spots respectively. twenty one protein spots were recognized by more than 80 % of igg of pools of sera in each group of patients and not by igg of hc. twenty seven protein spots were recognized by the great majority of igg of pools of patients with a higher intensity than igg of pools of hc. identified proteins were constituents of cytoskeleton, proteins involved in oxidative stress as stress-induced phosphoprotein 1 and peroxyredoxin 6 and proteins involved in regulation of cell energy metabolism as triosephosphate isomerise. we have identified anti-vsmc abs in the serum of patients with idiopathic and ssc-associated pah. these abs bind to cytoskeleton, oxidative stress and cell cycle antigens. objectives: this study aimed to verify that subcutaneous lymph node transplantation inducing lymphatic regeneration is possible in healthy adult rats, as obtained in other species. methods: this rat model was used to determine the effects of lymph node fragmentation as well as sheep erythrocytes and platelet-rich plasma injection on the regeneration of the transplanted lymph nodes. results: this rat model is adequate to study the regeneration of transplanted lymph nodes. lymph node fragmentation seems to affect transplant regeneration negatively. an immune challenge by injection of sheep erythrocytes in the drainage area of the transplanted lymph nodes does not improve fragment regeneration. however, injection of syngeneic platelet-rich plasma containing several growth factors resulted in an improvement in regeneration. conclusion: lymph node fragment regeneration, although still experimental, could be relevant for lymphedema prevention. acquired lymphedema has a high prevalence in developed countries as a consequence of the removal and/or radiotherapy of tumor-draining lymph nodes in cancer patients. this disease causes lifelong disability due to chronic swelling and increased risk of infections. it currently lacks an effective treatment. methods: 27 patients suffering from different diseases were enrolled in our study. 16 patients were suffering from bone diseases (osteomyelitis, necrosis, tumour) whereas 11 of them were suffering from inflammatoty diseases (soft tissue inflammation, diabetic ulceration). blood specimens were collected before hyperbaric oxygen treatment and the serum levels of icam-1 and vcam-1 were assesed by an enzyme immunoassay (elisa). results: 11 out of the 27 patients (40,7 %) had elevated levels of the intercellular adhesion molecule. 6 out of the 16 patients suffering from bone diseases (37,5%) had raised values (mean value 400 ng/ml) whereas 5 patients out of the 11 suffering from soft tissue diseases and diabetes (45,5 %) had raised values (mean value 735,5 ng/ml). reference value for icam-1 was 130-299 ng/ml. vascular cell adhesion molecule's assesment revealed no elevated levels in our patients. conclusions: our study revealed a high rate of patients (40 %) having increasd levels of icam-1. high icam-1 levels were more prevalent in patients suffering from soft tissue inflammatory diseases and diabetes (45,5 %) than in patients with bone diseases (37.5 %). mean values were found 735,5 ng/ml and 400 ng/ml accordingly. those findings verify the positive correlation between icam-1 and inflammatory diseases and tissue damage but not for vcam-1. colorectal cancer (crc) was the first solid tumour to be successfully targeted with anti-angiogenic therapy in the clinic. tumour angiogenesis is critical for cancer progression in that it permits expansion of the tumour mass and fosters malignant dissemination. angiogenesis is a multistep process involving endothelial cells as well as numerous stromal components within the tumour microenvironment that also represent potential therapeutic targets. inflammation dependent-angiogenesis is increasingly recognised as a central force in tumour growth and progression, while use of anti-inflammatory drugs has been found to reduce incidence of crc carcinoma potentially through repression of tumour angiogenesis. we investigated the link between inflammatory angiogenesis and colorectal cancer in archival tissues across a range of pathologies that represent diverse steps in the progression of crc: 16 cases of ulcerative colitis (urc), 16 adenocarcinomas developed from preexisting tubular or tubulo-villous adenomas, 33 tubular or tubulo-villous adenomas with low grade dysplasia, and 33 infiltrating adenocarcinomas. immunohistoobjectives: to determine the effect from the administration of preoperative pravastatina to therapeutic dose in the expression of cd18 in the leucocitaria adhesion to endotelio vascular in the isquémico-reperfundido miocardico weaveal endotelio vascular en el tejido miocardico isquémico-reperfundido by the circulation extracorpórea (cec). methods: they were included in way randomizada double blind 20 patients with intervened controlled hiperlipidemia of surgery coronary low circulation extracorpórea (cec). 40 mg of pravastatina oral 2 hours they were administered before the procedure (group study, n=10) or placebo (group placebo, n=10), and control (group control, n=10). samples of outlying veined blood were extracted to the 24 hours. the separation of leukocytes was made in peripheral blood, to determine the expression of cd18 in such. in all the samples one quantified the intensity of the expression pattern and the percentage of leucocitarias cells. results: 3 types of patterns were distinguished: cytoplasmic, of membrane and compound. the intensity of the expression was classified in 3 degrees: degree 0. without expression. degree 1. weak; degree 2. moderate; degree 3. intense. in the group control: most of the samples they presented/displayed a mixed pattern (cytoplasmic and of membrane) with an intensity degree 0-1. the placebo group: mixed pattern, degree 1-2. group study (40 mg. oral pravastatina): most of the cells they presented/displayed a predominance of membrane pattern: degree 2-3. the percentage of cells that expressed cd 18 was greater in the group study (40 mg. oral pravastatina). the preoperative oral pravastatina to therapeutic unique dose ours study produces a greater expression cd18 answer induced by the cec; it seems that these molecules located in the leukocytes participate in the adhesion to the activated endoteliales cells, necessary for the extrusion of the lymphocytes through endotelio towards the inflammatory center and in quimiotaxis of the leukocytes towards the inflammation sites. several surface molecules on endothelial and epithelial cells undergo regulated cleavage by the disintegrin and metalloproteinases adam10 and adam17. we recently identified transmembrane chemokines, junctional adhesion molecule-a (jam-a), and members of the proteoglycan family as novel substrates for these proteases. here we demonstrate that cell lines and primary cells of human endothelial or epithelial origin release considerable amounts of soluble jam-a and proteoglycan ectodomains. this release is enhanced by treatment with the proinflammatory cytokines ifng and tnfa. the enhanced release was not caused by an increased gene induction but rather associated with a reduction of the surface expressed molecules. both, constitutive and induced release required the presence of adam17 as demonstrated by specific inhibitors, lentiviral silencing experiments as well as treatment with the recombinant catalytic domain of adam17. these data suggest that the proinflammatory cytokines ifng and tnfa induce enhanced proteolytic shedding of cell surface molecules on endothelial and epithelial cells. to investigate the physiological relevance of this induced shedding, mice were treated systemically with ifng/tnfa leading to increased presence of soluble jam-a in the blood serum. both cytokines also stimulated jam-a release from excised murine aortas with was associated with enhanced adam17 activity in the tissue. in the presence of the adam17 inhibitor induction of jam-a release was suppressed. in cultured epithelial cell lines enhanced shedding of jam-a or proteoglycans was not associated with increased mrna or surface expression of adams but rather with increased activity of cellular adam17 as shown by means of a synthetic substrate assay. our study demonstrates that the proinflammatory cytokines ifng/ tnfa upregulate adam17-mediated shedding activity rendering the protease an important modulator of endothelial and epithelial surface molecules in inflammatory settings. rium, and the haplotype vegf-460/ vegf+405 is associated with rcc risk ( p= 0,017), metastases ( p=0,043), nuclear grade ( p=0,05), tumor stage ( p=0,029), and tumor size (p=0,04). on the other hand, the polymorphism vegf -2578 a/c is not associated with rcc risk and clinical parameters. our results shed a new light to the knowledge on the association between vegf polymorphism and rcc risk and development. these data could help to improve our understanding of the rcc pathogenesis and disease progression. pten is a lipid phosphatase, whose substrate is phosphatidylinositol 3,4,5-trisphosphate. therefore, pten is one of the main antagonists of the pi3-kinase, which plays a major role in many important cellular functions, such as proliferation, migration or response to inflammatory stimuli. here we investigated the role of pten in collagen induced arthritis. we show that conditional deletion of pten under the lysm promoter (lysmcrepten flox/-) leads to a significant reduction in clinical severity of collagen induced arthritis (cia). histological analysis of cia, lysmcrepten flox/mice displayed significantly reduced joint inflammation as well as erosive bone destruction. total anti-collagen antibodies, however, as well as anti-collagen iggs were identical in both groups. upon analysis of inflammatory cytokines in serum after immunisation we found a significant reduction of il-6 as well as il-8 levels. furthermore, pten deficient macrophages and dendritic cells showed reduced induction of il-6 as well as il-12 and il-23 mrna upon stimulation with various tlr-ligands. since these cytokines play an important role in the induction of pathogenic th-17 t cells, we measured th-17 cytokines in lymph nodes after immunisation with collagen. although dendritic cell and macrophage recruitment to the draining lymph node was comparable in both groups, there was a slight reduction of il-17 and a strong reduction of il-22 mrna in the draining lymph node of immunized lysmcrepten flox/compared to wild-type mice. one of the mechanisms through which il-10 exerts its anti-inflammatory effects consists in promoting the release of anti-inflammatory molecules. in this context, particularly important is the production of il-1ra, whose expression is induced by lps in human neutrophils and monocytes and significantly potentiated by the presence of il-10. based on our previous observation that support a direct role of il-10-activated stat3 in the enhancement of il-1ra transcription induced by lps, we plan to characterize the transcriptional activators recruited to the il-1ra promoter in vivo and responsible of the increased rate of transcriptional initiation upon exposure of lps-treated cells to il-10. quantitative chromatin immunoprecipitation (chip) studies were employed to examine the in vivo binding of transcriptional activators to the il-1ra promoter. crosslinked nuclear lysates were immunoprecipitated 30 and 60 min after il-10 addition with different antibodies and immunoprecipitated dna was analyzed by quantitative real-time pcr for the presence of target sequence located in the il-1ra promoter. chip assays showed that the pol ii recruitment to the il-1ra promoter induced by lps is significantly increased by il-10, further strengthening the concept that the rapid enhancement of lpsinduced il-1ra gene expression by il-10 initially occurs by targeting transcriptional events. as expected, real-time pcr of anti-stat3 immunoprecipitated dna showed statistically significant levels of stat3 binding to the il-1ra promoter only in cells stimulated with lps in the presence of il-10. surprisingly, anti-p65 and anti-p50 chip assays revealed enrichment of both p65 and p50 recruitment to the il-1ra promoter when il-10 was added to lps-stimulated cells, suggesting that il-10 enhances the recruitment of nf-kb to the il-1ra promoter. interestingly, when nf-kb is recruited to this promoter in lps + il-10-treated cells, the overall nf-kb nuclear translocation (analyzed by western blot) and dna binding activity (detected by emsa analysis) were not modified with respect to cells stimulated with lps alone. the enrichment of nf-kb at the il-1ra promoter site is dependent on il-10-activated stat3, since it is greatly reduced when stat3 activation by il-10 is impaired. the molecular mechanism through which il-10-activated stat3 promotes the recruitment of nf-kb to the il-1ra promoter is currently under investigation. major components of mast cell secretory granules are proteases. we could recently report that intracellular stored mast cell-produced cytokines regulate mc protease activities and provided evidence that il-15 acts as a specific negative transcriptional regulator of mouse mast cell protease-2 (mmcp-2). we examined the mechanisms underlying the repression of mmcp-2 gene expression. our data show that the "repressor" effects of il-15 on mmcp-2 promoter activity are still operating on the mmcp-2 591 bp long minimal promoter. moreover, il-15 deficiency in mast cells causes a specific dysregulated expression of the transcription factors c/ebpb and yy1. furthermore, chromatin immunoprecipitation revealed that il-15 promoted specific reciprocal recruitment of c/ebpb but not yy1 to the mmcp-2 promoter. finally, il-15 deficient mast cells display a predominantly non-cpg methylated pattern of the mmcp-2. thus, we proposed that the expression of mmcp-2 and possibly other immunoregulatory genes may be regulated by il-15 through epigenetic modification and by balancing the content and binding of c/ ebpb and yy1 in mast cells. i. nagy 1 , k. filkor 1 , a. vörös 1 , l. kemény 2 , a. szász 1 1 bzaka, baygen, szeged, hungary, 2 university of szeged, department of dermatology and allergology, szeged, hungary micrornas (mirnas) are evolutionary conserved small non-coding rnas that act as key regulators of gene expression at post-transcriptional level by targeting mrnas for translational repression and/or degradation. mirnas have been shown to have unique tissue-, developmental stage-and diseases-specific expression patterns. during the last years several studies highlighted that mirnas play critical role in the differentiation and function of the adaptive as well as innate immunity. little is known however, about the differential regulation of mirnas following the activation of pattern recognition receptors. in order to tackle this issue, we treated hacat keratinocytes with staphylococcus aureus-derived peptidoglycan (pgn) once or repeatedly, the latter mimicking persistent infection. after appropriate treatments we first analyzed the expression profile of mirnas mir-203, mir-146a and mir-155, which are known to participate in immune processes of the skin. repeated pgn-treatment significantly decreased mir-203 expression; in contrast, pgn re-stimulation had no further effect on mir-146a and mir-155 expression. next, we investigated the correlation between the expression of mir-203 and its two known direct targets: regulatory protein p63 and suppressor of cytokine signalling-3 (socs-3). although the gene-expression profile of neither p63 nor socs-3 changed, we found that the expression of mir-203 reversibly correlates with both p63 and socs-3 protein expression, a phenotype that we verified by two independent protein analysis methods (western blotting and immunofluorescent labeling). importantly, transfection of hacat cells with anti-mir-203 prior to pgn-treatment completely abolished both p63 and socs-3 down-regulation, revealing the involvement of mir-203 in pgn-induced transcriptional regulation. finally, methylation-specific pcr experiments unravelled the role of dnamethylation in regulating mir-203 expression upon pgn-treatment. taken together, our results strongly suggest that sets of mirnas may be differentially regulated during persistent infection. results: tgfb1+/-had a lower incidence and burden of benign papillomas when compared to tgfb1+/+ animals. however, more scc developed in the tgfb1+/-mice. after acute and chronic promotion, tgfb1+/-skin showed a reduced proliferative response with no increase in epidermal tgfb1 or nuclear p-smad2 compared to tgfb1+/+ mice. tpa-induced pkca activity as well as phosphorylation of specific pkc substrates in keratinocytes correlated with tgfb1 gene dosage. further, pharmacological inhibition of alk5 suppressed tpa-mediated pkca activation suggesting that physiological levels of tgfb1 are required for maximal activation of pkc-dependent mitogenic responses. even though the tpa-induced inflammatory response was greater in tgfb1+/-skin, tgfb1+/+ papillomas had more tumor infiltrating neutrophils. tpa-induced proinflammatory gene expression was sustained in tgfb1+/-skin and primary keratinocytes but it was elevated in v-ras ha -transduced tgfb1+/+ but not tgfb1+/-keratinocytes, indicating that tgfb1 switches from an anti-inflammatory cytokine in the skin to a proinflammatory factor in tumors dependending on an activated ras. despite this differential proliferative and inflammatory response to tpa and enhanced papilloma formation in the tgfb1 +/+ mice, there was no increase in conversion to scc in this genotype. conclusions: tgfb1 acts to promote benign tumors enhancing cell proliferation and inflammation through its ability to regulate pkc activation in skin, yet retains a suppressive function for malignant conversion. background: proto-oncogene survivin has been recently shown as a prognostic marker distinguishing patients with destructive rheumatoid arthritis (ra). in the present study we studied the relationship between survivin and urokinase (upa), a fibrinolytic serine protease being over expressed in the inflamed joints and exhibiting arthritogenic properties. material and methods: levels of survivin and upa were measured in the paired blood and synovial fluid samples of 132 patients with ra, using elisa and compared to controls with non-inflammatory joint diseases. the ability of upa to induce survivin and requirement of upa receptor (upar) was studied in primary synovial fibroblasts and pbmc of ra patients, human monocytic (thp-1) and fibroblast (mrc-5) cell lines employing antibodies against upar, sirna technique, and synthetic inhibitors of intracellular pathways. the ability to prevent urokinase-induced arthritis by interruption of survivin expression was evaluated in mouse model of arthritis. results: in the present material of 132 ra patients and 82 controls the levels of survivin correlated to urokinase (upa) (r=0.46), a plasminogen activator over expressed in inflamed joints and known to exhibit potent arthritogenic properties. we found that 30/132 ra patients had high circulating levels of survivin. these patients had erosive arthritis and were characterized by high levels of upa. in vitro studies showed that upa induced survivin in leukocytes and this process was dependent on signaling through upa receptor. in turn, survivin was required for expression of upar. additionally, survivin was essential for upa production in mrc-5 and synovial fibroblasts. down-regulation of survivin with sirna was followed by significantly reducion of upa synthesis. finally, treatment with downregulation of survivin by sirna in vivo efficiently abrogated upa-induced arthritis in mice model. these findings indicate that survivin is an essential mediator of arthritogenic properties of upa regulating its synthesis in synovial fibroblasts and upar expression in leukocytes. close correlation between survivin and upa in patients with ra supports the improtance of these interaction for the pathogenesis of arthritis. upon cell activation, ubiquitously expressed inositol 1,4,5-trisphosphate 3-kinase type b (itpkb) phosphorylates inositol (1, 4, 5) trisphosphate (ins(1,4,5)p 3 ), a calcium-mobilizing second messenger with pleiotropic effects. itpkb inactivation leads to severe t cell deficiency, altered thymo-independent b cell responses and neutrophil hyperactivation. we here report that itpkb-deficient (itpkb -/-) mice also display profound alterations in mast cell development and function. indeed, while mast cell number, c-kit and fcepsilonri expression were comparable in itpkb-deficient and proficient mice, itpkb -/mast cells were almost completely devoid of granules. this phenotype could be partially reversed upon treatment with sodium cromoglycate. nevertheless, fcepsilonri or c-kit activation on mast cells led to increased ca 2+ responses and to stronger erk phosphorylations. however, itpkb -/mice displayed an attenuated sensitivity to ige-mediated passive systemic anaphylaxis, correlated to the absence of fcepsilonri-dependant histamine release and to downregulation of h1 and h2 receptor expression due to high basal histamine concentrations. production of neosynthesized mediators remained normal. finally, itpkb deficiency also severely impaired scf-induced mast cell differentiation in vitro. taken together these results demonstrate that itpkb is a key regulator of mast cell activation. itpkb antagonists might thus be of therapeutic interest for programmed and progressive depletion of histamine stores. the large percentage of immune relevant genes that are alternatively spliced and the connections between splicing and disease, strongly indicate that alternative splicing plays a central role in the regulation and fine-tuning of physiological immune responses. il-1b is an important proinflammatory cytokine produced by activated macrophages and monocytes. il-1b is produced as an inactive cytoplasmic precursor that is proteolytic processed by the inflammatory caspase-1 to generate the mature secreted active form. caspase-1 is also synthesized as an inactive form that requires processing by the inflammasome to become active. we have used a subset of the trc lentiviral human library to generate loss-of-function phenotypes for most of the splicing factors and splicing regulators. we were able to silence the expression of 425 genes involved in splicing with an average 5-fold coverage. after the primary screen and several rounds of phenotypic validation, we have identified 30 genes that significantly affect the production of il-1b by thp-1 cells after a 24h challenge with pfa-fixed e. coli, as measured by elisa. knockdown levels were analyzed by qrt-pcr for the most significant candidates to validate the phenotypes observed. exon array analysis are being performed to identify possible targets of the most significant splicing factor candidates obtained by the shrna screening in order to dissect their mechanism of action in the regulation of the inflammasome and il-1b secretion. tissue transglutaminase (tg2) has a critical role in the pathogenesis of chronic inflammatory diseases such as celiac or neurodegenerative diseases. we have previously described the key role of tg2 in cystic fibrosis (cf), a genetic disease characterised by chronic lung infections and inflammation. in cf, mutation on the cftr gene results in an increased tg2 expression and activity leading to functional sequestration of the anti-inflammatory pparg and increase of the classic parameters of inflammation. here we tested whether in vivo inhibition of tg2 can reverse inflammation in chronic inflammatory diseases. to assess the importance of tg2 not just in cf but in chronic inflammatory diseases in general, we injected cystamine, a potent tg2 inhibitor, in a transgenic mouse model cf and in the taz10 transgenic mice that spontaneously develop autoimmune thyroiditis. intraperitoneal administration of cystamine had a significant impact on the lung epithelium in the cf model, where it decreased tg expression and activity. the treatment was also able to dampen all the classic inflammatory parameters as well as restoring normal cellular levels of functional pparg. interestingly, cystamine injections could also block inflammation in the taz10 tcr transgenic mouse model with chronic thyroiditis, highlighting the pivotal role of tg2 in generating inflammation in two very different pathologies. this work underlines the critical role of tg2 in inflammation and provides new opportunities to develop therapeutic strategies for sufferers of chronic inflammatory diseases. angiogenesis, the growth of new blood vessels, is a process that is essential during tissue repair, foetal development, and female reproductive cycle. angiogenesis is also a relevant process associated to many pathologic conditions including autoimmune diseases and tumors. we have shown that dendritic cells activated in the simultaneous presence of pro-and anti-inflammatory signals (alternatively activated dc, a-dc) display potent angiogenic activity in vivo which is mediated by the release of biologically active vegf-a. here, we investigates the molecular mechanisms leading to vegf-a secretion in lps+pge 2 stimulated a-dc. preliminary results indicate no accumulation of hif-1alpha in a-dc, therefore suggesting that vegf-a is induced by a non-classical, hif-1alpha independent pathway. in addition, we found that vegf-a secretion depends on the activation of mapk p38 but not erk1/2 or jnk. inhibitor studies, nascent transcript analysis and polimerase ii recruitment on the promoter show that the induction of vegf-a is largely due to new transcription and not to changes in mrna stability. chromatin immunoprecipitation studies aimed at the characterization of the modifications of vegf-a regulatory regions in a-dc and at the identification of transcription factors bound to vegf-a promoters are being performed. this will possibly allow the description of novel transcription factors involved in vegf-a activation in a-dc. wnt proteins are secreted palmitoylated glycoproteins with multiple functions in cell proliferation, migration as well as tissue organization. they are best known for their role in embryonic development and tissue homeostasis. deregulation of wnt signaling has been shown to promote carcinogenesis. recently we identified wnt signaling to be involved in the regulation of inflammatory processes: wnt5a is induced in human macrophages in response to mycobacteria and conserved bacterial structures and contributes to the regulation of the proinflammatory cytokines il-12 and ifn-gamma. to gain deeper insights into wnt mediated modulation of inflammatory processes we now used murine bone marrow derived macrophages and analyzed the effects of the addition of exogenous wnt homologs. we monitored wnt-mediated activation of primary macrophages by measuring the activation of signaling pathways and transcription factors, analyzed the expression of target genes by real-time pcr and measured the secretion of inflammatory cytokines by elisa. exogenous wnt5a -but not wnt3a -was able to induce cytokine expression in primary macrophages. in infection experiments wnt5a promoted the mycobacteria-induced macrophage activation and enhanced the expression of inflammatory mediators in murine macrophages. in contrast, addition of wnt3a reduced the expression of inflammatory mediators upon mycobacterial infection. these data corroborate our previous findings and further support the notion that tlr/nf-kappab and wnt signaling, both being evolutionary highly conserved pathways, are functionally interconnected infection of immunocompetent mammals with t. gondii induces a chronic infection of the brain. t. gondii cysts persist in neurons and escape elimination by the immune system. in immunodeficient individuals, the infection can be reactivated resulting in a lethal toxoplasma encephalitis (te). in te, the parasite is primarily controlled by infiltrating t and b cells. also brain resident cells may contribute to control of the disease and however, the mechanisms of brain resident cells leading to the protection of the vulnerable brain in chronic te are largely unknown. in a previous study, we could show that expression of gp130 on astrocytes in mice is critical for survival of te. in the present study, we analyzed the function of neuronal gp130 in te. after infection with t. gondii, mice lacking neuronal gp130 (synapsin-cre gp130 fl/fl ) died significantly earlier in the chronic phase of infection than control gp130 fl/fl mice. death of synapsin-cre cre gp130 fl/fl was due to a severe encephalitis with larger inflammatory lesions and higher numbers of inflammatory leukocytes. additionally, te of synapsin-cre gp130 fl/fl mice resulted in a substantial apoptosis of neurons both in the vicinity of inflammatory lesions and also in brain areas without inflammation. in vitro, apoptosis of gp130-deficient neurons was also significantly increased upon infection with t. gondii or stimulation with tnf as compared to gp130 expressing neurons. interestingly, the intracerebral parasitic burden was not increased in synapsin-cre gp130 fl/fl mice indicating that the immunoregulatory role of neurons is more important than their anti-parasitic function. t. objectives: persistent production of tnfa in many autoimmune diseases, including intraocular inflammation (uveitis), can lead to significant tissue damage. targeting tnfa with neutralising antibodies or tnf receptor fusion proteins is often, but not always, an effective therapy. high serum concentrations of tnfa, il-1b, il-6 and il-8 have been detected in a spectrum of autoimmune diseases; while in contrast, the levels of il-4, il-10 and tgfb are reduced. this suggests, indirectly, that failure to regulate an appropriate balance of inhibitory factors contributes to the pathogenesis or propagation of tissue inflammation in autoimmunity. thus, understanding the homeostatic control of tnfa by tgfb1 further may generate more effective therapies. as tnfa mrna 3' untranslated region (utr) contains an au-rich element (are), which targets mrnas for degradation, we wished to test whether tgfb1 suppresses tnfa protein production by upregulating the rnabinding protein fxr1, which can bind to tnfa mrna and inhibit translation. methods: using raw 264.7 cells and mouse bone-marrow derived macrophages stimulated with lps and tgfb1, we assessed mrna expression by q-pcr and tnfa protein expression by flow cytometry. the 3'utr of tnfa mrna was isolated and inserted into a luciferase reporter vector on a constitutive promoter. transfected raw cells were treated with lps and tgfb1 and luciferase expression was quantified. cells treated with lps and tgfb1 were also examined for fxr1 expression using pcr and western blot. following fxr1 knockdown using sirna, the influence of tgfb1 and lps on tnfa protein production was examined by flow cytometry. results: we find that while tnfa mrna expression remains constant, lps induced tnfa protein expression is suppressed by tgfb1. using the luciferase-tnf-3'utr vector we show that tgfb1 targets the 3'utr of tnfa. furthermore, tgfb1 and il-10 both upregulate fxr1 mrna and protein; and treatment with tgfb1 and lps can synergistically upregulate mrna expression, more than tgfb1 alone. following sirna inhibition of fxr1, tgfb1 can no longer inhibit lps-induced tnfa production. comtb up-regulated mmp-1 and mmp-3 secretion from saecs, nhbes and fibroblasts to a peak of 2.5 +/-0.5 ng/ml, at 72 hours. interleukin-17 augmented comtb-stimulated up-regulation of mmp-1 and mmp-3 secretion from saecs and fibroblasts in a synergistic manner. in contrast, interleukin-17 down-regulated mmp-9 secretion from saecs by 50 %. interleukin-22 up-regulated mmp-1 and mmp-3 secretion from fibroblasts but not from saecs. timp1 secretion from saecs was enhanced by interleukin-17 but there was no effect of interleukin-22. mmp up-regulation by interleukin-17 and comtb was inhibited by the pi3kinase inhibitor ly294002 and on western analysis akt (protein kinase b) was phosphorylated at 30 minutes. chemical inhibition of the p110d isoform of pi3kinase with ic87114 abrogated the il-17 and comtb driven secretion of mmp-3 from the small airway epithelial cells. chemical inhibition of the tumour suppressor phosphatase, pten (phosphatase and tensin analogue on chromosome 10) accentuated mmp-3 secretion. these inhibitory effects were confirmed with sirna. mmp-3 up-regulation was secondary to increased gene expression with promoter activity peaking 24h after stimulation. in summary, interleukin-17 and interleukin-22 drive transcription dependent mmp-1 and mmp-3 secretion from airway epithelial cells and fibroblasts. interleukin-17 also increases timp but down-regulates mmp-9 gene expression and secretion. this may contribute to the matrix degrading phenotype in tuberculosis. the pi3kinase pathway is central in interleukin-17 driven tissue destruction in the context of m. tuberculosis infection. v. delgado-maroto 1 , l.s. moreira 2 , e. gonzalez-rey 2 , m. delgado 1 1 institute of parasitology and biomedicine lopez-neyra , csic, granada, spain, 2 university of seville, sevilla, spain objectives: atherosclerosis is an inflammatory chronic disease characterized by the formation in the arteries of lesions that involve inflammation, lipid accumulation, cell death and fibrosis. over time, the rupture of these atherosclerotic plaques releases prothrombotic material to the blood and causes thrombotic occlusion at the site of disruption. atherosclerosis will probably become the most common cause of death within 15 years. one of the initial hallmarks of the disease is the uptake of oxldl particles by macrophages, which leads to intracellular cholesterol accumulation and the formation of foam cells. t cells undergo activation after interacting with foam cells, which process and present local antigens including oxldl, generating a t helper 1 response. cholesterol metabolism is regulated by factors such as pparg1 (proliferator activated receptor g), srb1 (a class b scavenger receptor), cd36 or abca1, that can induce cholesterol exit from the macrophage which may help to solve the lesions. expression of these factors depends on intracellular camp. adrenomedullin (am), urocortin (ucn) and vasoactive intestinal peptide (vip) are novel neuropeptides synthesized by immune cells that have various characteristics to be considered as possible therapeutic agents for atherosclerosis. they are potent anti-inflammatory agents, which downregulate a broad spectrum of pro-inflammatory mediators, and inhibit th1 immune response. their mechanism of action involves binding to gpcr and adenylate cyclase activation with subsequent increase of camp in the cell, which is recognized as an anti-inflammatory response. methods: we investigate am/ucn/vip effect on bone marrow-derived macrophages stimulated with oxldl. we determine the levels of pparg1, srb1, cd36, and abca1. we also analyze the cholesterol metabolism of oxldl-stimulated macrophages after neuropeptides incubation using oil red o staining of lipids drops and tritium labelled cholesterol. objective: endovascular aortic repair (evar) is considered a minimally invasive procedure, and the patients are expected to be discharged after a day or two. however up to 60 % develop a systemic inflammatory response syndrome (sirs), resulting in prolonged convalescence. as yet there is no satisfactory explanation to this severe response. previous studies have shown a high level of il-6 in the mural thrombus lining the aneurysm. the thrombus is manipulated during the procedure, but whether or not it is the source of circulating il-6 and/or other cytokines during and after the operation is unknown. methods: quantitative analysis of the pro-inflammatory cytokines il-6, tnf-a, il-1b, il-8 and il-12, and the anti-inflammatory cytokine il-10, in plasma from five patients, as of yet, was carried out by means of cytometric bead array, while analysis of plasma il-23 was performed using the luminex platform. the cytokine levels were compared to the clinical response, in terms of sirs. results: evar induced the production of il-6 and il-10, and in some cases, of il-23. the maximal plasma levels of il-6 and il-10 were found at 24 hours and of il-23 between 48-72 hours. modest plasma levels of il-8 were also observed, with maximal production at various time points (4-24 hours). by contrast, production of tnf-a, il-1b and il-12 did not occur to a significant extent, while production of il-17 occurred sporadically. although our preliminary data indicate that sirs is associated with enhanced cytokine responses, the production of il-6, il-8, il-23 and il-10 also took place in patients who did not develop sirs. conclusion: evar is associated with the sequential production of il-6, il-10, il-8 and il-23, i. e. a mixed pro-and anti-inflammatory response, even in the absence of sirs; but the production seems to be exaggerated in patients developing sirs. further studies involving 165 patients are in progress, and will clarify this. we hypothesize that sirs is elicited by il-6, activated by manipulation of the mural thrombus. to reveal whether this is the case, studies involving immunohistochemistry of the thrombus, will be performed. il-33 is a novel il-1 cytokine family member that is expressed as an intracellular precursor (pro-il-33) and is thought to be cleaved by caspase-1 to yield a mature bioactive form of the molecule (mat-il-33). to date however, evidence of cell-associated proteolytic processing and caspase-1 dependent secretion of mat-il-33 has not been reported. here we show that pro-il-33 but not mat-il-33 is released from uvb-irradiated keratinocytes. we demonstrate binding of pro-il-33 to the il-33r and also il-33r-dependent bioactivity of pro-il-33 on mast cells. we propose a previously unrecognized role for pro-il-33 as a pro-inflammatory mediator and suggest a direct link between uvb-mediated epithelial cell damage and cutaneous mast cell activation. we have previously shown that induction of er stress and tlr signalling synergistically enhance il-23 p19 mrna expression in myeloid cells, and markedly increase secretion of il-23, but not il-12, by dendritic cells. the aim of this study is to investigate the mechanism of this synergy. we examined the il-23 promoter for potential binding sites for er stress induced transcription factors and identified a putative site for chop10. chromatin immunoprecipitation (chip) assays using anti-chop10 and isotype control mab were performed using nuclear lysates from u937 cells and il-23 promoter dna measured by qpcr. chop10 binding on the il-23 promoter was detected following stimulation of u937 cells with lps or tp alone, but this was significantly enhanced when er stress and tlr stimuli were combined. il-23 promoter dna was not detectable following chip with the isotype control antibody. to confirm the role of chop10 in il-23 gene transcription, u937 cells expressing shrna's specific for chop10 or non-specific gene target were tested for their ability to express il-23 following tlr and er stress stimulation. u937 expressing three independent shrna targets for chop10 exhibited significant reductions in il-23p19 mrna (up to 87 % reduction of the response to lps+tp) compared to u937 expressing a control shrna. chop10 shrna expression did not affect the expression of other lpsresponsive genes, including il-1, il-8, ccl3 and sod2. to identify if er stress induction of il-23 mediated by chop10 expression plays a role in a more physiological setting, we examined the role of chop10 in the induction of il-23p19 gene expression following chlamydia trachomatis (ct) infection. infection of u937 cells with live but not g-irradiated ct induced expression of er stress response genes, including chop10. u937 infected with live ct exhibited increased il-23p19 mrna expression compared to u937 infected with nonviable bacteria. chop10 silencing significantly reduced the ability of live ct to induce il-23p19mrna, confirming the important role of chop10 in this response. these data suggest that er stress induction of chop10 could contribute significantly to the pathogenesis of diseases in which il-23 plays an major role, through induction of il-17 and il-22 producing cells. the clonal deletion of thymocytes by negative selection is an important process to ensure immunologic tolerance, even though the underlying molecular mechanisms are poorly understood. here, we show that gadd45b, a regulator of mitogen-activated protein kinases, is critically involved in selection processes in the thymus. gadd45b expression was inducible in different in vitro and in vivo models of negative selection. strikingly, only tcr-ligating peptides resulting in negative selection induced gadd45b expression, while positively selecting ligands or dexamethasone, a tcr-independent apoptosis agonist, failed to do so. expression of gadd45b maintained a sustained activation of p38 kinase and thereby promoted tcr-mediated apoptosis. in contrast, thymocytes from gadd45b-deficient mice showed only transient p38 activation and reduced caspase activation. interestingly, we observed a switch to positive selection in gadd45b-deficient mice since a higher percentage of single positive thymocytes was found. moreover, markers of positive selection as cd5 and cd69 were elevated on gadd45b-deficient thymocytes. thus, we provide evidence that gadd45b and a resulting persistent activation of p38 constitute a novel apoptotic pathway involved in negative selection. these results also provide evidence for the novel concept that not only the on-off switch of a signaling module but also its spatiotemporal regulation may crucially determine cell fate decisions. di santo 1 1 institut pasteur, paris, france, 2 monash university, victoria, australia > the thymus represents the ''cradle'' for t cell development, with distinct thymic stroma components providing multiple soluble and cellular membrane cues that foster in a step-wise fashion developing thymocytes. although il-7 is recognized as an essential factor for thymopoiesis, the nature of the thymic il-7 niche remains poorly characterized in vivo. > using a novel bacterial artificial chromosome transgenic mice in which yellow fluorescent protein (yfp) is under control of il-7 promoter, we identify a subset of thymic epithelial cells (tecs) that co-express yfp and high levels of il7 transcripts (il-7 hi cells). il-7 hi tecs arise during early fetal thymic development, persist throughout life, and co-express homeostatic chemokines (ccl19, ccl25, cxcl12) and cytokines (il15) that are critical for normal thymopoiesis. in the adult thymus, il-7 hi cells are found in cortico-medullary regions and display traits of both cortical or medullary immature tecs. interestingly, the frequency of il-7 hi cells decreases with age, suggesting a mechanism for the age-related thymic involution that is associated with declining il-7 levels. conversely, the frequency of il-7 hi cells is markedly increased under severe lymphopenia imposed by genetic mutations that cause an early and profound block in t cell development. this augment indicates that thymocyte-tec crosstalk may condition il-7-expression by tecs. > together, our temporal-spatial analysis of il-7-expressing cells in the thymus suggests that thymic il-7 levels are dynamically regulated under distinct physiological conditions. this novel il-7 reporter mouse provides a valuable tool to further dissect the molecular and cellular mechanisms that govern thymic il-7 expression in vivo. two lines of evidence have recently demonstrated that the pre-b cell receptor (pre-bcr) is associated with autoimmunity, through its surrogate-light-chain (slc) components l5 and vpreb. it has been shown that pre-bcrs are polyreactive for several self-antigens. the polyreactivity of the pre-bcr induces pre-bcr signaling and activation. furthermore, in human a self-reactive b cell subset was identified that co-expresses immunoglobulin light chain (ig lc) and the slc components. these vpreb + lc + b cells, found in healthy individuals, are potentially harmful as they express autoreactive antibodies associated with autoimmune diseases, like sle and ra. to elucidate the contribution of pre-bcr components to the development and activation of autoreactive b cells, we have recently generated a slc transgenic (slc-tg) mouse model in which all b cells express slc proteins. slc-tg mice exhibit spontaneous igm + plasma cell development. moreover, aging slc-tg mice have elevated anti-nucleosome igm levels, accompanied by immune complex deposition in the kidney, but do not display auto-immune pathology. nevertheless, vpreb + lc + b cells may induce pathology when self-tolerance mechanisms fail. to test this hypothesis, slc-tg mice were crossed on two autoimmune-prone genetic backgrounds: (i) em-bcl2-tg mice with b cell-specific overexpression of the anti-apoptotic protein bcl2 and (ii) fcgriib-deficient mice. both in young slc-tg;em-bcl2-tg double tg mice and in slc-tg;fcgriib -/mice spontaneous germinal center (gc) formation -which is associated with autoimmunity -was significantly enhanced, when compared with control em-bcl2-tg and fcgriib -/mice. in slc-tg;fcgriib -/mice, numbers of splenic igg2 plasma cells and serum igg2 levels were˚5-10 fold increased. importantly, serum from young slc-tg;em-bcl2-tg and slc-tg;fcgriib -/mice contained high titers of igg auto-antibodies in 2/3 and 6/6 cases, respectively. these values were increased when compared with control groups: 1/6 in fcgriib -/and 0/6 in bcl2-tg. finally, we found that the collagen induced arthritis (cia) was significantly enhanced in slc-tg;fcgriib -/mice, compared to fcgriib -/mice. taken together, these findings demonstrate the slc components have the capacity to induce auto-antibody formation in the mouse and and to enhance autoimmune pathology in ra. t cells are generated from progenitor cells that enter the thymus from bone marrow via blood. these progenitor cells once within the thymus have little selfrenewal capacity. differentiation from hematopoietic stem cells to early t lineage cells proceeds through a series of intermediate precursor populations. however, it is largely unknown to what extent these cell populations contribute to t cell development in the presence of other precursor populations and how the earliest intrathymic t cell progenitors are generated from extrathymic precursors. to assess the relative contribution of potential precursors to t lineage differentiation we developed a strategy based on the depletion of well-characterized precursor populations rather than their enrichment and subsequent adoptive transfer together with an equal amount of congenic non-depleted bone marrow. thus, the physiological ratio of extrathymic precursors remained largely intact and we were able to address the question whether there is only one physiological t cell precursor or many. we showed that, under such competitive conditions, t lineage progenitors are confined to the cd27 + cd135 + fraction of bone marrow cells. notably, t lineage reconstitution was not restricted to either cd117 hi cells, representing multipotent progenitors, or cd127 + cells, representing common lymphoid progenitors, both of which contributed to t lineage differentiation with different kinetics. in conclusion, our data suggest that multiple physiological extrathymic t cell precursors exist, which are able to compensate for the loss of depleted populations. thus, our findings may have implications for devising strategies for improved t lineage reconstitution after hematopoietic stem cell transplantation. background: previous results from our group have demonstrated ephb2 and ephb3 expression on both thymocytes and thymic epithelial cells (tecs). we used chimeric models to determine that those molecules govern in an autonomous and non autonomous manner thymocyte and tec development, and how they regulate interactions between both cell types. objectives: in order to better define the importance in thymus of eph-ephrin b interactions we have analyzed the effects of the lack of ephrin b1 and/or ephrin b2, the ligands of ephb2 and ephb3 receptors. this approach is specially interesting taking into account that eph-ephrin signaling is transmitted to both the two cells participating in the interaction and that the cell responses depend on the type of signals (reverse, forward or both), their direction and intensity. methods: for this purpose we have used cre-loxp recombination systems for deleting ephrinb1 or ephrinb2 genes specifically on either thymocytes or tecs. results: animals with ephrin b deficient thymocytes showed thymic hypocellularity and alterations on t-cell development whose severity depended on the background of the analyzed mice. in these mice only a few changes occurred in the cortical tec network. on the contrary, mice with conditioned deletions in tec, especially ephrinb1/b2 double mutants, showed a more severe phenotype that began early in the ontogeny and resulted in very small thymi exhibiting an extremely compact cortical and medullary network, decreased numbers of cd45+ cells in the cortex, increased proportion of k5+k8+ cells and high presence of cysts. in addition, t-cell development was partially blocked at the dn cell stage. conclusion: these data reveal an autonomous and non-autonomous role for ephrinb1 and ephrinb2 in the development of both t cells and tecs, confirming the importance of these molecules in the establishment of a crosstalk between the main two cell types of thymus. we discuss how eph-ephrin contacts modulate cellular homotypic and heterotypic interactions that take place during thymus organogenesis and in t cell differentiation. a. rolink 1 , d. vanhecke 1 1 university of basel, developmental and molecular immunology, basel, switzerland the importance of normal t lymphocyte development in the immune system is exemplified by the occurrence of inherited and acquired human immunodeficiencies where the development or functional maturation of t cells is defective. in order to identify molecules/genes and elucidate developmental processes that are essential for human t cell development we use a novel in vitro tool, the op9-dl1 cell culture system (1) . using this in vitro assay we obtain large numbers of human cycd3 + and cd4 + cd8 + double positive thymocytes starting from umbilical cord blood (ucb) derived cd34 + hsc. signals and molecules that are involved in t cell development are being addressed by using blocking antibodies and/or chemical inhibitors. similar as in mice we found an essential role for il-7 and notch mediated signaling in the development and survival of particular developmental stages of human thymocytes. among the molecules that are rapidly induced in cd34 + cells upon notch signaling is cd7 followed by cd127. t cell specification is accompanied by the induction of cd1a and loss of cd34 on cd7 + cd127 + cells. these cd34 -cd1a + cd7 + cells become dependent on continuous il7 and notch signaling for sustained survival and further differentiation into cd4 + cd8ab + dp thymocytes. we found that flt3l is not essential for the differentiation of cd4 + cd8 + human thymocytes but that addition of exogenous flt3l in the co-cultures increases the number of cd34 + precursors and consequently result in higher yields of developing cd4 + cd8ab + dp thymocytes. finally few mature tcrab + t lymphocytes develop from the cycd3 + cd4 + cd8ab + dp subset in this in vitro assay suggesting that op9 stromal cells lack the required selecting mhc-antigen complexes and/or costimulating molecules to induce and sustain positive selection of human thymocytes. this in vitro assay will allow us now, by using rna interference, to test additional genes for their role during human lymphoid development. from a clinical standpoint, a better understanding of the mechanisms controlling human t-cell development is a fundamental step towards the development of specific therapies for the treatment of primary and acquired immunodeficiencies as well as for the treatment of malignant t-cell disorders. brain derived neurotrophic factor (bdnf) promotes various neuronal functions such as survival, regeneration and synaptic plasticity. emerging evidence also indicates an essential role for bdnf in the immune system, e.g. in the b and t cell lineages. we therefore investigated the impact of bdnf on thymocyte development using bdnf knockout (ko) mice and conditional ko mice lacking bdnf specifically in t cells. in both models, we found reduced thymocyte numbers and a significant increase in double negative thymocytes. in contrast, the percentage of naturally occurring regulatory t cells and the expression of activation markers were unaltered. moreover, the lack of bdnf did not result in enhanced thymocyte apoptosis. the increase in double negative thymocytes was due to a partial block in the transition from the dn3 to dn4 stage, where bdnf and its receptor p75 are expressed as revealed by real-time pcr. the observed partial block in thymic maturation results in mild peripheral lymphopenia without affecting the activation status of peripheral t cells, their homeostatic proliferation and without compromising peripheral immune responses in general. in summary, our findings point to a critical role of t cell lineage derived bdnf in thymocyte development acting in an autocrine and/or paracrine manner. r. berga 1 , c. lópez-rodríguez 1 1 pompeu fabra university, barcelona, spain nfat5 is a transcription factor that belongs to the rel family (nf-kb and nfatc proteins). its expression in primary cells and organs is restricted to certain proliferative tissues, like activated t lymphocytes and thymocytes, where levels of nfat5 are relatively high and its subcellular distribution is predominantly nuclear. recent mouse models suggest that nfat5 participates in thymocyte development and also indicate its involvement in t cell proliferation and survival. nfat5 deficient mice present a t cell immunodeficiency consistent on lymphopenia, which is more accused for cd8 + lymphocytes. these observations are of substantial relevance as we and others have described that, in vivo, nfat5-null mice are unable to mount cd4+-and cd8 + -immune responses. data from our laboratory indicate that nfat5-null mice suffer from hyperosmolarity in plasma (hypernatremia) as a result of the incapacity to induce an osmoprotective gene expression program at a systemic level. to selectively analyze the t-cell autonomous effects derived from the lack of nfat5 during the development of t lymphocytes, we developed mouse models that delete nfat5 at early (lck-cre + /nfat5 flox/flox ) or late (cd4-cre + /nfat5 flox/flox ) stages of thymocyte maturation and that present isotonic plasma. our work indicates that nfat5 is expressed at all stages of t cell development. in addition, analysis of mouse models that lack nfat5 at different points of t cell development indicate that it participates at early stages of the ontogeny of t cells. objectives: apoptosis mediated by the tumor suppressor molecule p53, is regarded as a major player in tumor prevention but this may not be its only role. we have investigated this by creating a mouse (m ¿ pro) lacking residues 58-88 of the proline-rich domain of p53. methods: we compared the ability of various hemaptopoietic tissues from m ¿ pro mice and wild type mice to undergo apoptosis following irradiation or treatment with pro-apoptotic drugs. apoptosis was measured by staining with annexin v in vitro and by detection of caspase activation in vitro and in vivo. we also compared their ability to undergo cell cycle arrest using brdu staining. tumor development was monitored in cohorts of m ¿ pro, p53 null (p53-/-) and wild type (p53+/+) mice, with or without prior irradiation. results: apoptotic function was lacking in m ¿ pro mice, but they were able to arrest cell-cycle progression in hematopoietic tissues. m ¿ pro developed late-onset b-cell lymphoma, but not the thymic t-cell tumors found in p53-/-mice. interestingly, m ¿ pro lymphomas were comprised of incorrectly differentiated b-cells. bcell irregularities were also detected in m ¿ pro prior to tumor onset, in which aged mice showed an increased population of inappropriately differentiated b-cells in the bone marrow and spleen. we propose that the apoptotic function of p53 has an important role in b-cell homeostasis, which, in turn, is important for prevention of b-cell lymphomas moreover, our data suggest that the apoptotic function of p53 is not important for preventing thymic t-cell tumors. s. myrczek 1 , r. pardi 2 , a. gessner 1 1 microbiological institute-institute for clinical microbiology, immunology and hygiene, university hospital erlangen, erlangen, germany, 2 vita-salute san raffaele university school of medicine, milano, italy jab1 is the catalytic subunit of the highly conserved cop9 signalosome. this complex plays a central role in various cellular processes as proliferation and cell cycle control. jab1 regulates the neddylation of ubiquitin ligases and thus contributes to degradation of many proteins. furthermore jab1 regulates the activity of ap1 transciption factors. to date jab1 is thought to be essential for every cell type as jab1 knock out mice are embryonic lethal and t cell development is blocked by t cell selective absence of jab1. to investigate the function of jab1 in b cells we established a mouse strain deficient for jab1 selectively in b cells. mice with floxed alleles of jab1 kindly provided by r. pardi were crossed with a mouse strain expressing the cre recombinase under control of the mb1-locus (m. reth, freiburg). ablation of jab1 expression resulted in an almost complete block of b cell development at the pro b cell stage. the absence of peripheral mature b1 and b2 cells and serum immunoglobulins resulted in chronic arthritis with high pathogen burden after experimental infection with borrelia burgdorferi. the observed block in b cell development is rescued by over expression of the anti apoptotic protein bcl2 under the control of the m enhancer. facs analyses revealed that all b cell subtypes analyzed in the jab1-deficient, bcl2-transgenic mice are present albeit at reduced numbers compared to wild type animals. serum immunoglobulin titers are detectable and after borrelia infection specific antibodies are produced. we confirmed the absence of jab1 in sorted spleen b cells by immunoblot analysis. in summary, we show for the first time that cells are viable and functional without jab1 when apoptosis is prevented. t. nitta 1 , s. murata 2 , k. tanaka 3 , y. takahama 1 1 university of tokushima, tokushima, japan, 2 university of tokyo, tokyo, japan, 3 rinshoken, tokyo, japan how self-peptides are generated and displayed in the thymus to select a useful and self-protective repertoire of t cells is largely unknown, whereas the role of thymic self-peptides in eliminating self-reactive t cells and thereby preventing autoimmunity is well established. a recently identified form of proteasome, termed thymoproteasome, is specifically expressed by thymic cortical epithelial cells (ctec) and is required for the optimum generation of cd8 t cells. here we show that ctec display a thymoproteasome-specific spectrum of class i mhc-associated self-peptides, which is essential for positive selection of major and diverse repertoires of class i mhc-restricted t cells. indeed, cd8 t cells generated in the absence of thymoproteasomes display a markedly altered tcr repertoire that is defective in both allogeneic and antiviral responses. these results demonstrate that thymoproteasome-dependent self-peptides are required for positive selection of a diverse repertoire of immunocompetent cd8 t cells. defects in helper t cell number or function causes susceptibility to infections and in some cases autoimmunity or allergy. our understanding of the genetic control of helper t cell differentiation into specific functional subsets is still far from complete. here we present the results to date from a genome-wide enu mutation screen for mice with inherited deficits in specific helper cell subsets. these deficits were detected by multi-colour facs analysis of peripheral blood samples, and by antibody production following immunization with heat-killed b.pertussis and cgg coupled with the hapten arsonate (aba) in alum, which induce internally polarized th1 and th2 antibody responses, respectively. using this screen, a number of new mutant strains have been isolated with complete or partial loss of cd4+ t cells or functional deficits that selectively interfere with th1 or germinal centre responses. in this talk i will present data from some of the first 12 strains that have been identified including the first 5 strains where we have been able to identify the causative mutation. systematic genetic analysis of helper t cell differentiation in the resulting strains will illuminate how t cell help is correctly polarized for immunity and to avoid immunopathology. intrathymic t-cell development provides a unique model system to study cell fate determination because of the well-defined cellular stages and the confined microenvironment of this process. in order to highlight the differences and similarities between fetal and adult t-cell development at the molecular level we performed a microarray study. labelled rna from facs purified fetal and adult dn1 c-kit high (etp), dn2 and dn3 thymocyte populations was hybridised to affymetrix mouse 430a-2.0 genechips. the resulting data were grouped into four distinct gene clusters: cluster i contained genes over-expressed throughout adult development and included a large proportion of transcription factors (85 out of 623 genes), illustrating a significantly different transcriptional program acting during adult differentiation. conversely, cluster ii consisted of genes that were over-expressed in fetal progenitors and included 64 signal transducers (out of 590 genes) such as acvr1, bmpr1, fzd7, chemokine receptors cx3cr1, cxcr6 and integrins a2, a4, a9, ae and av, pointing to a difference in microenvironments. genes that showed uniform down-regulation during consecutive stages of fetal and adult development were restricted to cluster iii. amongst these were transcripts governing alternative developmental choices, therefore emphasising a common mechanism of lineage restriction during thymopoiesis. on the other hand, cluster iv was limited to genes that were homogeneously up-regulated during development. these included gata-3, tcf-1, notch-1, rag-1, rag-2 and pre-ta, which are indispensable for t cell development. interestingly, levels of expression of these genes were elevated in fetal progenitors, especially at the etp and dn2 stages, suggesting that the molecular program of t-cell development is more advanced in the early stages of fetal differentiation. discriminant analysis with the use of the support vector machine arrived at the same conclusion that demonstrated a nearby clustering of all fetal stages with the adult dn3 population, therefore implying a more committed state of fetal progenitors. finally, transcriptional signatures of each developmental stage were defined by "recursive feature elimination" with support vector machines. this approach can now be used to classify characterised and aberrant hematopoietic progenitors and thus construct an ontological scheme of hematopoietic development based upon transcriptional signatures of populations under normal and pathological conditions. tcrgd+ cells and tcrab+cd8aa+ intraepithelial lymphocytes (iels) of the gut are unconventional t cells that reside in tissues and provide innate-like immune responses to "stressed-self". as these cells share common functional properties in the periphery, we have hypothesised a common mechanism of development in the thymus; their progenitors diverging from the conventional t cell developmental pathway based on tcr signal strength at the dn stage. the pre-t-alpha chain (pta) that pairs with tcrb to generate the pre-tcr, has two isoforms; pta a and pta b . both can form a functional pre-tcr with tcrb. ligand-independent signalling by the pre-tcr is a result of spontaneous oligomerisation (followed by internalisation), that is mediated through charged residues on the pta chain. pta b lacks 3 out of 4 of these essential residues and therefore, we speculate results in higher surface expression and different signalling capabilities. we have hypothesised that pta a and pta b permit differential signal strength through the pre-tcr at the dn stage, facilitating the divergence of the conventional and unconventional lineages of tcrab+ t cells. preliminary semi-quantitative pcr data suggest that pta a and pta b are differentially expressed in wt thymocytes at different stages of ontogeny. retroviral transduction of pta -/-e14 thymocytes with either pta a or pta b alone, followed by fetal thymic organ culture, confirmed the rescue of abt cell development by both isoforms. however the two isoforms appear to differentially regulate the kinetics of thymocyte development by 7-10 days of culture; pta b expression generates a greater percentage of tcrab+ cells while pta a expression results in the accumulation of isps. these results suggest different roles for the two isoforms of pta in the thymic development of abt cells. in order to determine the mechanism by which pta a and pta b may generate qualitatively different signals, site directed mutagenesis was used to produce mutant chains of pta a and pta b that lack the "dimerisation residues" necessary for internalisation of the receptors. in addition, bac transgenic mice that express singly either pta a or pta b under the pta promoter are being generated to fully characterise their role in conventional vs. unconventional lineage commitment. erythroid, myeloid and lymphoid cells are initiated in parallel in appropriate cytokine environments so that specific number of erythrocytes, myeloid cells, natural killer cells, thymocytes and t cells, and precursor of b cells can be detected and counted at day 18 of culture. if needed for further functional analyses, long-term proliferating lines and clones of progenitor t and b cells can be established at this point of "in vitro" development. hence the "in vitro" differentiation of es cells to different hematopoietic cell lineages and their progenitors can be quantified. it allows for testing the efficiencies for hematopoietic development of genetically or epigenetically different es or ips cells. aim: increasing evidence includes wnt proteins inside the group of master-signalling pathways which govern immune and non immune differentiation systems. although their precise functions in bone marrow and thymus are still controversial, numerous studies show that wnt signalling is able to control the proliferation of hsc and thymic progenitors and might also affect both their cell-fate decisions and subsequent maturation. in the present work we analyse the effect of transient stimulation of canonical wnt pathway in the differentiation potential of lin -cd34 + cd1ahuman thymic progenitors, a multipotent and heterogeneous cell population which has the capacity to develop into t cells, nk cells, monocytes, conventional dendritic cells (cdc) and plasmacytoid dcs (pdcs). methods: human thymus samples from patients aged 1 month to 3 years undergoing corrective cardiac surgery were obtained and used according to the declaration of helsinki. transient b-catenin stabilization was triggered culturing purified thymic lin -cd34 + cd1precursors with recombinant wnt3a (100 ng/ml) or with licl (10 mm) for 12hr. active b-catenin, was detected by flow cytometry using anti-human active b-catenin mab (8e7) under conditions of phosphatase activity inhibition. wnt3a or licl pre-treated precursor were assayed in chimeric human-mouse ftoc, in il-15 and scf-supported cultures for generation of nk cells and in co-cultures with murine bone marrow stromal st2 cells suplemented with il-7 and flt3l. phenotype of recovered cells, apoptosis and cytokine receptors were analysed by flow cytometry. expression profile of transcription factors was analysed by real-time quantitative rt-pcr . our results demonstrate that giving a boost to canonical wnt signalling triggered by transient exposure of thymic progenitors to wnt3a or licl, change their differentiation capacity enhancing nk cell production. on the contrary, wnt3a or licl pre-treated thymic progenitors generate a significant lower number of myeloid lineage cells, monocytes and cdc, as well as reduce their capacity to differentiate into pdc lineage. as a possible mechanism for this effect we show that wnt pre-treated progenitors change their expresssion of receptors for cytokines pivotal for their expansion and differentiation, such are il-7 and flt3l and modify the transcription factor profiles of cd34 + cd1thymocytes mainly increasing hes-1 and id3 expression levels. human th17 clones and circulating th17 cells showed lower susceptibility to the anti-proliferative effect of tgf-beta than th1 and th2 clones or circulating th1oriented t cells, respectively. accordingly, human th17 cells exhibited lower expression of clusterin, and higher bcl-2 expression and reduced apoptosis in the presence of tgf-beta, in comparison with th1 cells. umbilical cord blood naï ve cd161(+)cd4(+) t cells, which contain the precursors of human th17 cells, differentiated into il-17a-producing cells only in response to il-1beta plus il-23, even in serum-free cultures. tgf-beta had no effect on constitutive rorgamma t expression by umbilical cord blood cd161(+) t cells but it increased the relative proportions of cd161(+) t cells differentiating into th17 cells in response to il-1beta plus il-23, whereas under the same conditions it inhibited both t-bet expression and th1 development. these data suggest that tgf-beta is not critical for the differentiation of human th17 cells, but indirectly favors their expansion because th17 cells are poorly susceptible to its suppressive effects. m. irla 1 , w. reith 1 1 university of medecine, pathology and immunology, geneva, switzerland objectives: medullary thymic epithelial cells (mtecs) are specialized for inducing central immunological tolerance to self-antigens. to accomplish this, mtecs must adopt a mature phenotype characterized by expression of the autoimmune regulator aire, which activates the transcription of numerous genes encoding tissue-restricted self-antigens. the mechanisms that control mature aire(+) mtec development in the postnatal thymus remain poorly understood. however, the generation of mutant mice exhibiting blocks in thymocyte differentiation at different stages, together with studies on embryonic development of the thymus, have demonstrated that reciprocal interactions between developing thymocytes and tec control not only t cell development but also the differentiation and organization of tec, a phenomenon designated as 'crosstalk'. the aim of the project outlined here is to elucidate the cellular and molecular mechanisms by which thymocytes control the numbers of mature mtec, key mediators of central tolerance. we have demonstrated by generating different transgenic mouse models, that although either cd4(+) or cd8(+) thymocytes are sufficient to sustain formation of a well-defined medulla, expansion of the mature mtec population requires autoantigen-specific interactions between positively selected cd4(+) thymocytes bearing autoreactive t cell receptor (tcr) and mtecs displaying cognate self-peptide-mhc class ii complexes. these interactions also involve the engagement of cd40 on mtecs by cd40l induced on the positively selected cd4(+) thymocytes. conclusion: this antigen-specific tcr-mhc class ii-mediated crosstalk between cd4(+) thymocytes and mtecs defines a unique checkpoint in thymic stromal development that is pivotal for generating a mature mtec population competent for ensuring central t cell tolerance. q. qiu 1 , i. ravens 1 , g. bernhardt 1 1 hannover medical school, institute of immunology, hannover, germany cd155 is originally identified as human poliovirus receptor (pvr) and as rodent tage4, which is overexpressed in rodent colon carcinoma. cd155 is also known as necl-5, a particular notable nectin-like molecule belonging to immunoglobulin superfamily, owning its unique expressing frofiles. cd155 expression is very low in most adult organs, but is abundant in the developing or regenerating liver. in addition, cd155 is overexpressed in transformed cells and promotes the cell cycle. thus, cd155 seems to be an oncofetal protein that functions in embryonic development and cancer progression. t-cell development is characterized by the progression through several phenotypically distinct stages, defined as double negative (dn), double positive (dp) and single positive (sp) based on expression of the co-receptors cd4 and cd8; the dn subset is further subdivided into four stages (dn1-4) by differential expression of cd44 and cd25. thymocytes at different stages of development occupy distinct spatially restricted domains in the adult thymus, indicating that differentiation occurs concomitantly with a highly ordered migration. during their final maturation in the medulla, semi-mature sp thymocytes down-regulate activation markers and subsequently exit into periphery. while semimature cd4+ sp are sensitive to negative selection, it remains elusive when negative selection occurs in the cd8 lineage. here we show that the frequency of terminally matured cd8+ sp cells but not that of cd4+ sp present in thymus varies depending on age. in mice lacking expression of the adhesion receptor cd155, a selective deficiency of mature cd8+ sp thymocytes was observed emerging first in adolescent animals at the age these cells start to accumulate in wild type thymus. evidence is provided that the mature cells emigrate prematurely when cd155 is absent thus cutting short their retention time in the medulla. moreover, in unmanipulated wild type mice semi-mature cd8+ sp thymocytes are subjected to negative selection as reflected by the diverging t cell receptor repertoires present on semi-mature and mature cd8+ t cells. in cd155 deficient animals, a shift in the tcr repertoire displayed by the pool of cd8+ sp cells was found demonstrating that cd155 is involved in negative selection. in the adult, steady-state, homeostatic conditions, lymphohematopoietic cell lineages display high rates of cell turnover. yet, the frequencies of simultaneously cycling cells are small, except in intermediate cellular stages of transit-amplifying precursor cell stages. the analysis of the molecular targets controlling these proliferation rates may provide relevant information to understand differentiation pathways along the ontogeny as well as mechanisms of leukemic transformation (passegué et al. j. exp. med. 2005 , 202, 1599 . during development, hematopoietic stem cells and their derived cell lineages need to expand to cope with continuously-increasing somatic demands. by using complementary, quantitative analyses (brdu labelings, hoescht 33342, propidium iodide), we are dissecting the proliferation rates of hematopoietic cell lineages and their differentiation stages along the whole mouse gestation from e9 (e, gestational day) on, in yolk sac, splanchnopleura/agm, blood, liver, spleen and bone marrow. we have observed that around half of cd71 + ter119 + erythroid and cd45 + cd11b + myeloid cells are simultaneously cycling (s/g2/m) in the post-gastrulation mouse embryo (e10-12). the peak of lymphohematopoietic cell proliferation occurs at e13 in a sort of wave-like pattern. these high-proliferation frequencies are present not only in immature, but also in mature cells, the latter thus displaying a different behaviour from the one present in the adult. later on, the proliferating cell subsets are restricted to fetal liver, whereas the equivalent cells become arrested in the periphery. interestingly, nucleated erythroid cells suddenly go into quiescence 24-48 hours before they enucleate, suggesting that this cell arrest is required for the enucleation process. we are also analysing the proliferation state of the first b and t lymphoid progenitors emerging at e11-12 that give rise to perinatal lymphocytes and, in some cases, to innate-like lymphocytes displaying self-renewal in the adult. we attempt to dissect the mechanisms regulating proliferation and death in the embryo versus those of adult lymphohematopoietic precursors, which may influence the functional activities of the mature cells. objectives: the role of cd40-cd154 interactions in t cell activation of antigen presenting cells and b cells is known, but a role for this receptor-ligand pair in hematopoiesis control has not been described. following an initial discovery that b lineage cells in the bone marrow (bm) as early as pro-b cells express cd40, we hypothesised a role for cd40-cd154 interactions in the control of b cell haematopoiesis. the objectives of this study were to investigate this hypothesis further. methods: flow cytometry was used to investigate cd40 expression by precursor b cells using b cell specific markers. reverse transcription of bm stromal cell rna and pcr were used to assess the presence of cd154 message and cell lineage specific mrna. irradiation and bone marrow transplantation (bmt) in both directions between cd154-/-and wt mice was used to assess potential functional contributions of stromal or haematopoietic cd154 on reconstitution of b cell numbers following depletion. we show that cd40 is expressed by pro-b cells, and these cells proliferate in response to cd40 signalling in vitro. pcr identified a source of cd154, negative for cd3eta, in the bm of wt mice showing this cd154 is not provided by activated re-circulating t cells. we have shown that when cd154 -/-mice are recipients, but not donors of bmt, b cell recovery after irradiation is significantly delayed regardless of the donor cell source. in the in vitro experiments we found that the pta gene is expressed from the dn1 (cd4 -/cd8 -/cd44 + /cd25 -) to the dp (cd4 + /cd8 + ) stage, whereas no yfp expression could be observed in the b lineage. the in vivo analysis of thymocytes confirms the appearance of yfp positive cells during t cell development from the dn1 stage on. in the bone marrow we found yfp + /b220 + and yfp + /b220populations. thus these pta expression analyses show closely similar pattern to those observed with hucd25 preta-reporter transgenic mice (gounari f. et al. 2002 , martin et al. 2003 . the bac pta reporter system can be used together with specific markers of other hematopoietic lineages and their progenitors to trace lymphopoiesis. gounari f et al., nat. immunol. 3, 489-496 (2002 ) martin c. h. et al., nat. immunol. 4, 866-873 (2003 . the individual functions and the reason for the tightly regulated expression of igm and igd during b cell development are poorly understood. our data show, that igd requires stronger stimuli than igm to induce b cell activation and that this silences autoreactive vdj recombination products when expressed as igd. in agreement with this, mhc and dhc, the respective heavy chains of igm and igd, differ dramatically in pre-bcr signaling, which represents the prototype of an autoreactive receptor. together with published data, our results reveal a novel role for igd and suggest that the differential expression of igm and igd is important to raise the activation threshold of mature b cells, thereby avoiding hypersensitivity and ensuring tolerance towards self-antigens. p. d. rymkiewicz 1 , g. klein 1 1 zmf (center for medical research), section for transplantation immunology and immunohematology, tübingen, germany thymic conduits which are exclusively found in the medullary region of the thymic lobules have been recently identified. the core of the conduits consist of fibrillar collagen bundles and is surrounded by a basement-membrane-like structure which contains the typical basement membrane components such as laminins, collagen type iv, nidogens and perlecan. a marker molecule for the conduits in the human thymus is the laminin isoform lm-332 which is synthesized by the medullary thymic epithelial cells (tecs) which tightly surround the conduits. functionally the conduits are too small to transport cells but they are able to transport small molecules x 70kda.mmp-19, a secreted member of the matrix metalloproteinase superfamily, is a protease capable of digesting lm-332. in the human thymus medullary, but not cortical thymic epithelial cells strongly express mmp-19. by western blotting the zymogen and the activated form of mmp-19 can be detected in whole thymus lysates, whereas in lysates from isolated tecs mainly the activated form is present. an in situ zymographic analysis revealed an increased proteolytic activity in the medullary region of the thymus. using confocal laser scanning microscopy double immunofluorescence staining showed that lm-332 and mmp-19 can be found in close neighbourhood, but they do not exactly co-localize. why activated mmp-19 which can be secreted by medullary tecs does obviously not destroy the surrounding basement membrane of the conduits has not been solved so far. two natural inhibitors of mmp-19, timp-2 and timp-3, are found in the thymic medulla, but they are not expressed and secreted by tecs. whether mmp-19 plays a role in processing medullary chemokines which are produced by the thymic epithelial cells is presently under investigation. to study the process of t cells differentiation in more detail, we intend to establish an inducible gene expression system (tet-on system) in primary t cells. the tet-on system comprises two retroviral vectors. the response vector contains an inducible modified minimal cmv promoter which per se is unable to induce expression of the gene of interest (goi). the second vector encodes a transactivator which is constitutively expressed and undergoes conformational changes upon binding of doxycycline. in this state, the transactivator enables the minimal cmv promoter to transcribe the gene of interest. therefore, co-transduction of both vectors is required to achieve transcription of the gene of interest. to date we have tested two tet-on systems (revtet system and retro-x tet-on advanced inducible expression system) that differ in the sequence of their inducible promoters. to monitor successful transfection in retrovirus-generating phoenix cells and transduction in t cells, respectively, we have cloned the reporter gene gfp under the control of a constitutive cmv promoter, into the response vector of the revtet system. this allowed identification of transduced gfp-positive cells via facs. however, when we used a red fluorescent protein, tomato, as a surrogate goi, we detected considerable leakiness of the promoter irrespective of the presence of the transactivator or doxycycline. in contrast, we found comparably low leakiness when using the retro-x tet-on advanced inducible expression system. here, co-transfection of phoenix cells with the transactivator and supplementation of doxycycline yielded an induction of 15-20 % compared to only 5 % basal rate. therefore, the retro-x tet-on advanced inducible expression system appears suitable for our studies. future experiments will aim at establishing this system in primary t cells. although a number of different experimental approaches has been used to elucidate impact of basal levels of adrenal gland-derived glucocorticoids (gcs) on t-cell development, and thereby t-cell-mediated immune response, their relevance for these processes is still far from being understood. the study was undertaken to explore relevance of basal levels of gcs for t-cell differentiation/maturation. eight days post-adrenalectomy in adult male rats thymocyte yield, apoptotic and proliferative rate and relationship among major thymocyte subsets defined by tcrab/cd4/cd8 expression were examined using flow cytometry analysis. it was found that adrenal gc deprivation affects: i) thymocyte apoptosis, producing thymic hypercellularity and ii) kinetics of t-cell differentiation/maturation leading to an overrepresentation of the cd4+cd8+ double positive (dp) tcrab low cells entering selection, and their cd4+cd8+ dp tcrab-immediate precedents followed by underrepresentation of the selected cd4+cd8+ dp tcrab high and the most mature cd4-cd8+ and, particularly, cd4+cd8-single positive (sp) tcrab high cells. the study suggests that withdrawal of adrenal gcs produces alteration in thymocyte selection processes that may affect diversity of functional t-cell repertoire and generation of potentially self-reactive cells as indicated by the reduced proportion and number of cd4-cd8-double negative tcrab high cells. in addition, it indicates that gcs influencing post-selection maturation of thymocytes play a regulatory role in controlling mature cd4+cd8-/cd4-cd8+ sp tcrab high cell ratio. in the thymus a specific subset of thymic stromal cells -medullary thymic epithelial cells (mtecs) -express a highly diverse set of tissue-restricted antigens (tras) representing essentially all tissues of the body, which is known as promiscuous gene expression (pge). this allows self-antigens, which otherwise are expressed in a spatially or temporally restricted manner to become continuously accessible to developing t cells. the scope of central tolerance is to a large extent dictated by the pool of promiscuously expressed genes. thus, even lack of a single tra can result in spontaneous organ-specific autoimmunity. promiscuously expressed gene which have no structural or functional commonality display two prominent features, they are highly clustered in the genome and show a preference for tras. for better understanding these features, we set out to precisely define the genomic organization of this gene pool. in particular, we probed to what extent and according to which rules predefined genomic clusters of tras are transcribed in mtecs. our analysis proceeded from the bioinformatic definition of tra clusters via gene expression analysis in mtecs using whole genome arrays to the in depth analysis of selected tra clusters by rt-pcr at the population and single cell level. patterns emerging from these studies will hopefully yield insight into evolutionary mechanisms responsible for selecting this gene pool. conceivably, positional cues in the genome and/or particular properties of self-antigens (e. g. immunogenicity) could have been driving forces during the co-evolution of pge and adaptive immunity. although catecholamines have been shown to influence thymocyte proliferation and differentiation, long-lasting b-adrenoceptor (ar) blockade failed to show any significant effects on thymic cellularity. bearing that in mind, the present study was undertaken to explore: i) a 1 -ar expression on thymic lymphoid and nonlymphoid cells and ii) putative role of a 1 -ar-mediated mechanisms in modulation of thymic cellularity and t-cell development. for this purpose a 1 -ar expression on thymic cells was assessed using both immunocytochemistry and flow cytometric analyses, while their putative modulatory role in thymopoiesis was estimated by analyses of thymocyte proliferation and apoptosis, as well as expression of major thymocyte differentiation antigens (cd4/ cd8/tcrab), in adult wistar rats subjected to 14-day-long treatment with a 1 -ar blocker urapidil (0.20mg/kg body weight/day s. c.). the a 1 -ar immunoreactivity was found in both thymocytes (mainly less mature cd3and cd3 low cells) and thymic nonlymphoid cells (thymic epithelial cells located mainly at cortico-medullary junction and cortical ed1-postive cells, which comprise macrophages and dendritic cells). chronic treatment with urapidil increased thymic weight and caused the organ hypercellularity. the thymic hypercellularity reflected, at least partly, increased frequency of proliferating thymocytes, which was followed by diminished thymocyte apoptosis. in addition, in these rats changes in distribution of major thymocyte subsets delineated by cd4/ cd8/tcrab expression were observed. these changes comprised of an increase in the percentage of cd4+8+ tcrabthymocytes, which was accompanied by the reduction in that of cd4+8+ tcrab low cells in urapidil-administered rats, and divergent changes in the percentage of the most mature single positive tcrab high thymocytes. compared with saline-administered controls, the percentage of cd4+8-tcrab high thymocytes in urapidil-administered rats was increased, while that of the cd4-8+ tcrab high was reduced. in addition, the percentage of cd4+ t regulatory and cd161+tcrab+ nkt cells was increased. collectively, this study clearly showed the expression of a 1 -ar on both lymphoid and nonlymphoid thymic cells, and indicated that a 1 -ar-mediated mechanisms may be implicated in modulation of multiple steps of t-cell development. we have recently shown that the thymus is a common target for mycobacterial infections. of notice, while bacterial growth is arrested in the spleen around 4 weeks post infection with mycobacterium avium, it takes several weeks longer within the thymus to reach a bacterial plateau. this observation suggests that a specific immune response occurs in the thymus, although this seems to be distinct from that occurring in the spleen. since t cell differentiation occurs, to a large extent, in the thymus, and depends, among other factors, on the antigens encountered within the thymus and on the cytokine milieu of the organ, we decided to characterize the pattern of the thymic immune response against mycobacteria and investigate possible consequences on the normal function of this organ. methods: c57bl/6 mice infected with m. avium (10 6 cfus, iv) were sacrificed at different time points after infection (5, 16 and 25 weeks). non-infected animals were used as controls. bacterial load was assessed in the spleen and thymus and cytokines (such as ifn-g, tnf, il-10) were quantified by rt-pcr in tissues (normalized for hprt expression) or by elisa in the supernatants of cultured thymocytes and splenocytes. statistical significances were determined by anova. we observe increased levels of ifn-g in infected thymi at 16 weeks post infection. this increased expression of ifn-g is concordant with the late bacterial growth arrest within the thymus. at 24 weeks post infection this difference is still present. throughout the course of infection no significant differences are found in the expression of tnf and il-10 in this organ. in the spleen, ifn-g reaches a peak of expression earlier (5 weeks post infection) and this is accompanied by increased tnf expression. conclusion: our cytokine analysis of the thymus and spleen confirm that an immune response against mycobacteria is mounted within the thymus, although different in timing and pattern from the one in the spleen. since the cytokine milieu influences t cell differentiation within the thymus, our observations raise the question on the consequences of such response on the normal function of this organ. such implications should next be investigated. precise regulation of eukaryotic gene expression requires interactions between distal cis-acting regulatory sequences with the looping out of the intervening dna, but how trans-acting regulatory proteins work to establish and maintain dna loops during gene activation remains largely unexplored. lps-induced transcription of the mouse igx gene in b lymphocytes utilizes three distal enhancers and requires the transcription factor nf-xb, whose family members include rela and c-rel. using chromosome conformation capture technology in combination with chromatin immunoprecipitation, here we demonstrate that lps-induced igx gene activation creates chromosomal loops by bridging together all three pair-wise interactions between the distal enhancers and rna polymerase ii, the apparent molecular tie for the bases of these loops. rela and actin polymerization are essential for triggering these processes, which do not require new transcription, protein synthesis or c-rel. we have thus identified both essential and non-essential events that establish higher-order chromatin reorganization during igx gene activation. this investigation was supported by grants gm29935 and ai067906 from nih and grant i-823 from the robert a. welch foundation to wtg, and by grants hl067256 and hl61897 from nih to lst. allelic exclusion of immunoglobulin (ig) genes supports burnet's clonal selection theory. the recognition that m-chain expression is sufficient for the maintenance of the silenced allele status by a process of feedback inhibition is yet not enough to explain the earlier monoallelic activation by the rag complex. attempts to prove the probabilistic or epigenetic nature of monoallelic v(d)j recombination were insightful and favor the epigenetic hypothesis, mainly by the observation that, like autosomal imprinted or x-chromosome inactive genes, ig genes are differentially marked at the chromatin level, and replicate asynchronously in virtually any cell of the mouse organism ever since the embryonic life (mostoslavsky, singh et al. 2001) . we are testing this hypothesis, i. e., that an epigenetic event has previously marked, on each b cell progenitor, which of the ig alleles is going to be activated for rearrangement and expression. for this, we are generating b cell clones from b cell progenitors of (c57bl/6 x balb/c)f1 mice, because the original strains have ig heavy chain (igh) alotypes distinguishable by monoclonal antibodies. we are analyzing if individual heterozygous clones show biased expression of a particular igh allele, and we expect to map the cell stage at which the (epigenetic) allelic marks are fixed. we have already shown, for the igh gene, a clear segregation of monoallelic expressors among b cell clonal lines that were generated from the common lymphoid precursor, but no allele bias was observed among multi-potent progenitor or hematopoietic stem cell b cell clones. this result, although suggesting epigenetic silencing starting at the common lymphoid precursor stage, does not favor the prevailing epigenetic hypothesis in its original formulation. we are currently exploring this result by the analysis of the igh silenced allele rearrangement status in sorted fractions of igm+ b cell clones. we are also testing the same epigenetic hypothesis for the ig kappa light chain gene (igk), using f1 mice in which the igk constant region from both alleles can be distinguished by antibodies. a. giniewski 1 , s. lang 1 , m. stein 1 , t. winkler 1 1 friedrich-alexander university, erlangen, germany vdj recombination is considered to be regulated by lineage and stage specific changes in accessibility of the loci to rag recombinase. accessibility is expected to correlate with certain histone modifications such as acetylation (e. g. h3ac) or methylation of h3 on lysine 4 (h3k4me2/3). previous studies in our lab revealed three regions in the intergenic part of the distal v h cluster (ivars), which are associated with high levels of active chromatin marks (h3ac and h3k4me2/3) only in pro b cells but not in pro t cells. it is also known that vdj h recombination is accompanied by sense and antisense transcription, however, little is known about the function and origin of the antisense transcripts. since one ivar (ivar #3) shows promoter activity in antisense orientation, it was analysed in more detail. we found three transcription start sites by 5'rlm race at the ivar #3 element. background: t-all is a malignancy of the lymphoblast committed to the t-cell lineage with translocations between tcr genes and oncogenes as a genetic hallmark. these translocations are thought to be driven by v(d)j-recombination mechanisms. we believe that these mechanisms only partly facilitate the occurrence of tcr translocations and that the accessibility of involved genes plays an intrinsic role in promoting these events. the lmo2 locus, is thought to be accessible only during the double-negative (dn) 1 and 2 thymocyte stages based on mrna expression, implying that translocations between lmo2 and tcr genes can only occur within these stages. gene expression as a readout for accessibility can not elucidate the involvement of other oncogenes such as tlx1 (hox11), which are not expressed in thymocytes, as being accessible for translocations to occur. objectives: we aimed 1) to evaluate lmo2 and tlx1 breakpoint-site accessibility during thymocyte development; 2) to determine in which stage of development there is an increased chance for lmo2 or tlx1 translocation to occur based on this accessibility. methods: dna of immunologically "healthy" sorted thymocytes was isolated using faire (formaldehyde-assisted isolation of regulatory-elements). this dna was used to quantitatively assess lmo2 accessibility during thymocyte development at both the transcription start site (tss) and negative regulatory element (nre), and within different in t-all documented breakpoint-sites of both the lmo2 and tlx1 loci. results: quantitative analysis on the tss showed a correlation with mrna expression, with the dn1 and dn2 development stages showing the highest accessibility. the nre, showed an inverse pattern of accessibility to the tss region. analysis of breakpoint-sites revealed the highest accessibility levels within the earliest stages of development, dn1, dn2, dn3 and immature single positive (isp) stages for both lmo2 and tlx1. conclusion: our findings show that both the lmo2 and tlx1 loci are accessible during thymic development irrespective of gene expression and that this accessibility is not restricted to dn1 and dn2 stages, suggesting that these loci are much more active than assumed, thus increasing the opportunity for translocations to occur. we addressed this issue, by building a model able to account for of v-ja gene rearrangements observed experimentally during thymus development of mice. we developed, based on experimental data, a numerical model on the whole tra/trd locus to estimate va and ja genes accessibility to rearrangements. the progressive opening of locus to v-j gene recombinations is modeled through windows of accessibility of different sizes and with different speeds of progression. furthermore, the possibility of successive secondary v-j rearrangements was introduced in the modeling. the model points out some unbalanced v-j associations resulting from a preferential access to gene rearrangements and from a non-uniform partition of the accessibility of the j genes, depending on their location in the locus. the model shows that 1 to 3 successive rearrangements are sufficient to explain the use of all the v and the j genes of the locus. finally, the model provides information on the kinetics of rearrangements and on the frequency of each v-j association. the model accounts for the essential features of the observed rearrangements on the tra/trd locus and may provide a reference for the repertoire of the v-j combinatorial diversity. the genetic programs of b-cell differentiation and the first dj h gene rearrangements appear in the post-gastrulation mouse embryo (e10-12), shortly after the first multipotential hematopoietic progenitors do emerge. these dj h joints represent the unselected baseline of the ig repertoires. we have undergone a systematic sequencing of embryo dj h joints obtained from normal balb/c embryos and heterozygous embryos obtained from rag2 -/mothers mated to balb/c males (to discard any mother-derived contribution), as well as newborn and adult control groups. the embryo dj h s displayed unexpected mechanisms of diversity, including short stretches of non-templated n nucleotides in one-third of the studied sequences (in the absence of tdt expression) and frequent dj h s with large nucleotide deletions, as a consequence of ligation to joint-distal microhomology sites. because the dna polymerase m (polm), a highly-homologous tdt member of the x dna polymerase family, showed an increased expression in the embryo, we analysed the dj h s of polm -/mouse embryos. we observed that polm was mainly responsible for introducing n nucleotides at the mouse embryo dj h joints. also, and based on its dna-dependent polymerization ability, polm filled-in small sequence gaps at the coding ends, and ligated highly-processed ends by pairing to internal microhomology sites, although at the cost of germline sequence losses and the generation of "useless" gene products. we think that, more than attempting to increase diversity, polm acts as a "connector" in the embryo, subsequently participating in the repair of rag-induced double-strand breaks, to preserve genomic stability and cellular homeostasis in cells with high proliferation rates. along the end of gestation, further selective pressures acting over these first v-dj h products will contribute to establish the differential neonatal ig repertoires. although mortality from infectious diseases peaks during infancy, many vaccines are ineffective in early life. most children with infantile bronchiolitis are under 6 months of age, and most cases are due to respiratory syncytial virus (rsv) infection, for which vaccines continue to be elusive. we now show that, compared to adults, the antibody response to rsv infection is very poor in neonatal mice and is unaffected by cd4 cell depletion. however, cd8 depletion in infancy led to a remarkable boosting of antibody responses during adult re-challenge. to test the possibility that poor antibody boosting is caused by rsv-specific cd8 t cells killing rsv-infected b cells, we sorted cells from the lungs of infected neonates. viral copy number was high in neonatal b cells, but viral load in surviving b cells was unaffected by cd8 cell depletion. in addition, fas ligand (fasl) deficient gld mice responded to rsv infection in the same way as normal mice, indicating that fasl is not required for the inhibition of antibody responses. this new mechanism of regulation of b cell responses by cd8 t cells has important implications for vaccine development against neonatal infections. (2008) showed that irf8 knockout mice had significantly reduced numbers of pre-pro-b cells in marrow and a phenotype similar to agammaglobulinemia. these results prompted us to consider icsbp/irf8 as a candidate gene in the pathogenesis of defective early b cell development. therefore we decide to undertake direct sequencing of the gene encoding icsbp/irf8 in a small cohort of patients with autosomal recessive agammaglobulinemia. methods: eight patients affected by agammaglobulinemia were included in this study. all patients were under regular ig replacement therapy. informed consent was obtained from all patients. genomic dna was extracted from whole blood and amplified with specific primers designed on the flanking regions of every exon. direct gene sequencing of the eight exons of icsbp/irf8 were obtained using abi prism sequencer. results: seven of the eight patients result wild type while only one patients present a synonymous snp in exon v, yet documented as rs17444416. conclusions: although recent findings indicated that irf8 function is essential for early b cell development, our data in a small cohort of patients affected with autosomal recessive agammaglobulinemia did not evidence any mutations in icsbp/irf8 that may be responsible for this disorder. the hh/ptch signaling system is known to control the development and neoplastic transformation of several cell types. however, the role of hh/ptch for the differentiation of b and t lymphocytes from hematopoietic stem cells (hsc) has not been assessed so far. to analyze the function of hh/ptch for lymphopoiesis in vivo, we have employed a genetically engineered mouse mutant in which the ptch gene can be conditionally inactivated by virtue of the cre/loxp recombination system. we show that targeted disruption of ptch in the adult organism results in a dramatic specification and differentiation defect of the lymphoid lineage leading to rapid disappearance of newly generated b and t lymphocytes from peripheral lymphoid organs. the developmental block occurs at the level of the common lymphoid progenitor cell (clp), which defines an early branching point of hsc differentiation and lineage commitment. in contrast to the lymphoid lineage, development of cell types of the myeloid lineage from common myeloid progenitors (cmp) appears normal. our data identify hh/ptch-induced signaling as a key regulator for proper development of immunocompetent lymphocytes. hence, the progression of tumors, which are initiated upon oncogenic hh/ptch mutations, may be further promoted due to impaired tumor surveillance by a compromised immune system. l. calderon dominguez 1 , t. boehm 1 1 max-planck institute for immunbiology, developmental immunology, freiburg, germany in many organ systems of animals and plants, specialized niche microenvironments maintain and specify stem and progenitor cells. the ability to modify or artificially create such niches in vivo and in vitro has many implications for stem cell research and therapy. by analysis of several mutant mouse strains and subsequent transgenesis in the mouse, we disentangle and individually modulate niche functions responsible for collection, maintenance and specification of multipotent thymocyte progenitors. we demonstrate how an epithelial niche, rendered functionally inactive by disruption of the foxn1 transcription factor, can be specifically rebuilt in a modular and combinatorial fashion to only attract, or attract and maintain, or attract, maintain and specify progenitor cells into the b and t cell lineages, respectively. the strategy of engineering niche functions in a modular fashion might be applicable to other progenitor cell systems. silencing of dc-sign using lentiviral rna interference revealed its critical function for pd-l1 expression on dcs after m. tuberculosis infection. as a counterpart to expression of its ligand, we showed that cd4 and cd8 t cells from tuberculosis patients highly express pd-1 when compared to healthy uninfected individuals. in addition, analysis of pd-1 expression in lung biopsies from tuberculosis patients revealed that pd-1 is expressed on cd4 and cd8 t cells confined to lung granulomatous lesions. finally, blocking of the pd-1/pd-l1 axis using monoclonal antibodies abrogated the down-modulation of t cell proliferation and ifn-g production induced by manlam, a mycobacterial cell wall glycolipid and ligand for dc-sign. taken together, our results suggest that the pd-1/pd-l1 pathway is involved in the exhaustion of t cell responses to m. tuberculosis. the inflammatory canonical nfkb pathway is critically involved in virtually all aspects of inflammation in general. yet, the role of the alternative, non-canonical nfkb pathway in inflammation and adaptive immunity remains largely elusive. the alternative pathway is primarily mediated through the nfkappa-b inducing kinase (nik) which in turn leads to the phosphorylation and the cleavage of p100 to p52. among the receptors engaging nik is the ltbr, which is also required to form the anlage for secondary lympoid tissues (slts). due to a point mutation within nik, alymphoplasia (aly) mice do not develop slts and are highly immunodeficient. however, while the immunodeficiency of aly mice is widely held to stem from their developmental malformation, it has been overlooked, that the mutation of nik itself could potentially lesion the development of immune responses. to verify this notion, we generated a series of bone marrow chimeric mice (bmc) in which the absence of slts was disconnected from the hematopoietic loss of nik function. we generated mice, which lack all slts, but are equipped with a normal systemic immune system (wt 1 aly), and conversely, mice with normal slts, but lacking nik in all leukocytes (aly 1 wt). surprisingly, we discovered that nik is vital for the development of autoimmune disease, while slts (ie. lns, spleen etc.) are essentially dispensable for cell-mediated immunity. we found that nik is required for the polarization of effector t cells and that th17 and th1 cells cannot be generated in the absence of nik. preliminary data implicate the involvement of nik in a discrete and novel pathway required for the formation of cell-mediated immune responses. the family of nfat (nuclear factor of activated t-cells) transcription factors is indispensable for t cells, for example playing an important role in cytokine gene regulation. in peripheral cd4 + t cells, nfatc1 and c2 are predominantly expressed. nfatc1 is synthesized in six isoforms which have partly opposing functions regarding activation and apoptosis. here we address the functional difference of the short isoform nfatc1/a, which is highly induced upon t cell activation, and the long constitutively expressed isoform nfatc1/c. as demonstrated by y2h screen and co-ips, nfatc1/c-specific c-terminus can be highly sumoylated. confocal microscopy studies revealed that upon sumoylation nfatc1/c -but not the unsumoylated nfatc1/a -translocates to promyelocytic leukemia-nuclear bodies (pml-nbs). this leads to interaction with hdacs followed by deacetylation of histones (co-ips), which in turn induces transcriptionally inactive chromatin (chip and confocal microscopy). as a consequence, multiple expression studies revealed sumoylation dependent suppression of the nfatc1 target gene interleukin-2. other lymphokines like ifng and il13 are reversely regulated. interestingly, ntreg cells which do not express il2 exerted only nfatc1/c, but no nfatc1/a expression (qrt-pcr). these findings demonstrate that the modification by sumo converts nfatc1 from an activator to a site-specific transcriptional repressor, revealing a novel regulatory mechanism for nfatc1 function. therefore, especially ntreg cells and anergized cd4 + t cells might be regulated by the long sumoylatable isoform nfatc1/c. lnk/sh2b3 and aps/sh2b2, two members of the lnk/sh2b family of adaptor proteins, play an important role as negative regulators in b cell lymphopoiesis. they possess several protein-protein interaction domains and motifs that allow their interaction with different signalling effectors. mice deficient for these proteins demonstrated that lnk inhibits expansion of pro/pre-b cells while aps controls mature b-1 cell population, suggesting specific roles for these adaptors during b cell development. however, the molecular mechanisms underlying their regulatory function in these cells, have not been identified. to address this question, we used primary and b cell lines at different stages of differentiation as our cellular system. analysis of lnk/aps expression pattern showed that lnk is expressed at all developmental stages, while aps is only detected in immature and mature cells. we then first examined the role of lnk in il-7 signalling in pre-b cells overexpression of lnk dramatically inhibits il-7-dependent growth demostrating that lnk negatively regulates il-7 pathways. furthermore, we showed that il-7 stimulation induces lnk phosphorylation and its subsequent association with important signalling effectors, notably the e3 ubiquitin ligase cbl. we next analyzed the role of aps in mature b cells by imaging and biochemical techniques. our results showed that aps colocalizes with the bcr complex after bcr triggering. interestingly, lnk is not recruted to the bcr signalosome in these cells, suggesting that interaction of the adaptors with the receptor complex regulates their function at different development stages. moreover, we showed, for the first time, that aps can associate, upon bcr stimulation, with the signalling molecules cbl and vav1. to address the functional implications of these interactions, we examined specific b cell responses, notably bcr trafficking and cytoskeleton remodelling. we demonstrated that overexpression of aps enhances ligand-induced endocytosis of bcr, possibly through interaction with cbl and affects the kinetics of bcr-induced cell spreading. our results therefore suggest a regulatory function of aps in bcr internalization and cytoskeleton dynamics. altogether, our findings demonstrate that lnk and aps display sequential specific regulatory roles during b cell development that are important for maintaining b cell homeostasis. signaling through the t-cell receptor (tcr)-cd3 complex is a critical event in adaptive immunity. it is still not clear how ligand binding to the tcr is communicated across the plasma membrane and leads to phosphorylation of the cytoplasmic domains of the cd3 complex. it is widely accepted that dimerization or multimerization of tcr is required for tcr triggering. in our model t-cell activation is initiated by recognition of monomeric mhc/peptide complexes on the surface of antigen presenting cells (apc). critical to tcr triggering is the movement of the t-cell across the apc. engagement of a mhc/peptide complex on the surface of the apc will change the mobility of the tcr leading to partitioning with lipids of lower mobility that are enriched in signaling molecules critical for t-cell activation. furthermore, the change in mobility will lead to dislocation of the itams from the plasma membrane so that they become accessible to tyrosine kinases. to test the hypothesis we established a new approach where we created a soluble bifunctional complex composed of a pmhc and a fab that recognizes an epitope tag that we express on the t-cell surface. binding of the fab to the expressed epitope tag will constrain the lateral mobility of the tcr that is engaged by the pmhc arm of the same complex. the bifunctional complexes induced activation and proliferation as well as ca influx and cytokine production in human cd4 + t-cell clones that displayed the epitope, but not in t-cells that did not display the epitope. activation required interaction of the fab with its epitope on the t-cell surface because no activation was observed when soluble epitope peptide, which acts as a competitor for the fab binding site, was added. these results demonstrate that a monomeric copy of a pmhc is sufficient to trigger tcr and that formation of a tcr dimer is not an obligatory step in t-cell activation. the bifunctional complex we generated may also have a great immunotherapeutic impact. exchanging the fab with a fab or cytokine directed to a surface molecule may allow an antigen specific stimulatory or inhibitory modulation of t-cell responses. adaptor proteins are crucial in signal transduction, cell cycle regulation, apoptosis and stress response. adaptor proteins containing characteristic sh2 or sh3 domains known to mediate protein-protein interactions are key players in these processes. sly2 (sh3 domain protein expressed in lymphocytes 2) was identified as a putative adaptor protein containing a sh3 and a sam domain as well as a bipartite nls. sly2 belongs to a family of three molecules: sly1, sly2 and sash1.in humans, the sly2 gene is located on chromosome 21, in mice on chromosome 16. sly2 is widely expressed for example in immune tissue as well as in hematopoietic cells, brain, lung and pancreas. subcellular fractionation showed that the sly2 protein is located in the cytoplasm and the nucleus and to a lesser extend in the plasma membrane.to elucidate the function of sly2 we searched for possible interaction partners by yeast two hybrid screening with a mouse t cell lymphoma library. this approach identified sin3-associated polypeptide p30 (sap30) as a putative interaction partner of sly2. sap30 is a conserved member of the sin3a-hdac corepressor complex that contains histone deacetylase 1 (hdac1) and histone deacetylase 2 (hdac2) and acts as a transcriptional repressor for a variety of genes. we confirmed this interaction by implementing coimmunoprecipitations with lysates from transiently transfected 293t cells. in addition, we could show a direct interaction between sly2 and hdac1. to investigate the functional impact of this molecular interaction, we performed hdac enzymatic activity assays. we were able to show that sly2 increases the activity of hdac1 in whole cell lysates and, more precisely, in nuclear extracts of 293t cells. the interaction of sly2 with sap30 and hdac1 indicates a transcriptional function of this protein. within the sin3a-hdac corepressor complex sly2 might act as a switch for the activity of hdac1. cd46-cyt1 and cyt2 are co-expressed in human t cells and undistinguishable from the cell surface. in order to determine their specific role in t cell activation, we have expressed chimeric proteins consisting of the extracellular domains of cd19 (b cell marker) fused to the transmembrane and intracellular domain of cd46-cyt1 or cyt2 in primary t cells. we show that these two isoforms differently control human t cell function. specific cyt1 coengagement controlled il-10 secretion, while cyt2 coligation inhibited ifng production. moreover, our preliminary data suggest that cd46-cyt2 inhibits the phosphorylation of several molecules known to be activated by cd46 stimulation. these data suggest that these two isoforms act as molecular switches for t cell activation, either promoting or turning off t cells. they demonstrate for the first time the distinct roles of cd46 cytoplasmic isoforms in primary human t cell activation. this also suggests that the modulation of their expression and/or activation might provide new therapeutic avenues. nck is a ubiquitously expressed adapter protein that is almost exclusively built of one sh2 domain and three sh3 domains. nck connects receptor and non-receptor tyrosine kinases to the machinery of actin reorganisation. in t cells, nck participates in different and interdependent signalling pathways linking t cell activation and effector function with actin remodelling proteins that in turn initiate changes in cell polarity and morphology. we previously showed that nck directs the death factor fasl to the cytotoxic immunological synapse when t cells encounter putative target cells. we now performed a systematic screening for interaction partners of the four individual interaction modules of nck in primary and leukemic t cells. we precipitated putative binding partners from untreated or pervanadate-treated pha blasts, jurkat and hut78 cells with gst fusion proteins containing full length nck, the three sh3 domains or the individual sh3 and sh2 domains. binding proteins were excised from gels after staining with coomassie, silver or flamingo pink and processed by tryptic in gel digestion for mass spectrometrical analysis. as expected, we observed major differences in nck binding proteins precipitated from resting versus activated t cells. we not only verified established interactions (e. g. with the tcr signalling components slp76 and cd3epsilon, the actin-regulatory proteins wasp and wip and the nuclear protein sam68) but also identified novel nck-interacting proteins. the interaction with the actin-binding protein hip55 once more underscores the fundamental role of nck in tcr-mediated actinreorganization. the identification of the nuclear proteins sfpq/nono points to novel, yet unknown functions of nck that might be associated with the recently reported nuclear translocation/localization of nck. accordingly, employing laser scanning microscopy, we clearly detected nck within the nucleus also in human t cells. the present data highlight that nck serves versatile functions in t cells, which include the different interdependent pathways of tcr-induced actin reorganization but also novel, yet poorly defined protein networks that are associated with a nuclear translocation of nck. cytotoxic t lymphocytes (ctl) mediated killing is tightly regulated according to the strength of t cell receptor signal. killing is regulated by the delivery of perforin-containing lytic granules moving along microtubules towards the centrosome, which polarizes and docks at the central supramolecular activation complex (csmac) within the immunological synapse. although much has been learnt about the mechanisms controlling the strength of tcr signal and the mechanisms required for release of the lytic granules, little is known about how the strength of the tcr is able to control the degree of ctl-mediated killing so finely. here we examine how the strength of tcr signal controls polarization of the secretory apparatus leading to ctl-mediated killing using tcr transgenic ot-i ctl. decreasing the tcr signal by reducing the concentration of ova peptide or using the weak agonist peptide, g4, results in a slight reduction in the number of ctl target cell conjugates formed, and the number of conjugates in which a csmac (visualized by a patch of lck-staining at the immunological synapse) was formed. tcr signals result in reduced or absent (in the case of g4) staining with psrc and perk antibodies in the immunological synapse and reduced or absent (g4) degranulation as measured by cd107a assays. the centrosome docks at the csmac of the immunological synapse even with relatively weak tcr signals, but the lytic granules require a certain threshold of signaling to successfully polarize to the immunological synapse. inhibitors support a role for pi3k in granule polarization. together these data demonstrate that the strength of tcr signal controls the level of ctl mediated killing at the single cell level by controlling, the number of conjugates formed, the formation of the csmac and the accumulation of psrc and perk at the synapse. the centrosome polarizes to the csmac even with relatively weak tcr signals, but granule recruitment requires a higher threshold of signaling. these findings reveal how ctl can fine tune the degree of killing in response to tcr signals at the single cell level. cytotoxic t cells play an essential role in the immune system, particularly in the elimination of tumor and virus-infected cells. cytolytic t-cell activity is mediated through the pore-forming molecule perforin allowing granzymes to enter the target cell and to initiate apoptosis. perforin and granzymes are stored in specialized secretory granules, called secretory lysosomes. they are capable of undergoing regulated secretion in response to a t cell receptor engagement which involves binding to a cognate mhc class i-peptide complex. the intracellular transport of lysosomal proteins from the golgi to the lysosomes is mediated by the cationindependent mannose-6-phosphate receptor which exhibits structural and functional similarity to the vps10p-receptor sortilin. sortilin was characterized predominantly in neuronal cells where its function in protein sorting was identified. in the secretory pathway, sortilin is putatively involved in trafficking of proteins in the constitutive and regulated pathway. to explore whether sortilin has a broader functional relevance, we asked if sortilin might act as an alternative receptor for the cation-independent mannose-6-phosphate receptor in cytotoxic t cells. first, we demonstrate that sortilin is expressed in t cells. to examine its function during an adaptive immune response, we analysed sortilin-deficient cytotoxic t cells derived from a knockout mouse strain. in strong contrast to the results reported from neuroendocrine cells, we obtained a reverse phenotype in sortilin-deficient cytotoxic t cells. whereas the regulated release of secretory lysosomes was enhanced, the constitutive release of interferon-g was found to be decreased. the enhanced release of cytotoxic molecules from sortilin-deficient cytotoxic t cells translated into an increased cytotoxicity in vitro. thus, the deletion of sortilin imposed a specific phenotype in cytotoxic t cells which could not be compensated for by other sorting receptors. our localisation studies of sortilin in t cells were consistent with the results previously described in neuronal cells which indicated that sortilin acts as a sorting receptor during the anterograde transport of lysosomal hydrolases from the trans-golgi-network to endosomes and lysosomes. taken together, we suggest that sortilin might play a modulatory role in the regulation of the adaptive immune response through the control of the constitutive and regulated secretory pathway. there is growing interest in the soluble splice variant of ctla-4 (sctla-4) as an immune inhibitor secreted by t cells, because genetically determined variation in its production is associated with susceptibility to autoimmune disease. however, little is known of the biology of sctla-4 in immune responses. using a specific anti-human soluble ctla-4 monoclonal antibody, jmw-3b3 that selectively binds the soluble isoform but not membrane bound ctla-4, or cleaved fragments of it, we demonstrate that sctla-4 plays a vital role in regulating antigen-specific immune responses. we used antibody blockade to show that antigen-specific t cell responses are strongly enhanced upon blockade of sctla-4, secreting increased amounts of cytokines including interferon-g, il-17 and tnf-a, but lower amounts of il-10. soluble ctla-4 was also prepared from sera for use in experiments by antibody based affinity purification techniques. addition of sctla-4 induced secretion of the immunoregulatory cytokine il-10 by human pbmcs both in an antigen-selective and dose-dependent manner, while antibody blockade abrogated that effect. the immunosuppressive indoleamine 2,3 dioxygenase enzyme cascade was also initiated by sctla-4. it is clear that the importance of this natural soluble molecule has been overlooked and like membrane-bound ctla-4 it is crucial to t cell inhibition. membrane-bound ctla-4 exists as a homo-dimer on t cells but sctla-4 is usually considered to be monomeric in form, implying its functional capacity is diminished because of an inability to cross-link b7 ligands on antigen presenting cells. a third important observation from this study is that sctla-4 exists both in serum and culture supernatants as a natural 46kda homo-dimer, and not as a monomer. this goes some way to explaining why this molecule has such potent immunoregulatory effects on antigen-specific immune responses. together, these results lead us to reappraise sctla-4, concluding it to be a mediator of negative feedback, secreted as a recall regulatory t cell response to antigenic stimulus, rather than a product of resting t cells. this work also raises the possibility that where il-10 dependent regulation is most critical, boosting sctla-4 secretion by regulatory t cells could be a novel therapy for immune mediated diseases. recently, we identified a new adaptor protein, swiprosin-1/efhd-2, in lipid rafts of b cell lines that undergo apoptosis after b cell receptor (bcr) stimulation. swiprosin-1/efhd2 is expressed in immature b cells of the bone marrow, in resting and activated splenic b cells, in t cells, macrophages, mast cells and some nonlymphoid tissues. ectopic expression of swiprosin-1/efhd-2 in the immature murine b cell line wehi231 enhanced spontaneous and bcr-induced apoptosis. in contrast, shrna-mediated down-regulation of swiprosin-1/efhd-2 impaired spontaneous and bcr-elicited apoptosis, but not bcr-induced g1 cell cycle arrest. to understand how swiprosin-1/efhd2 enhances pro-apoptotic bcr signals, we analyzed whether swiprosin-1/efhd2 is involved in proximal bcr signalling. in fact, ectopic expression of swiprosin-1/efhd2 enhanced bcr-induced calcium flux in wehi231 cells, whereas shrna-mediated down-regulation of swiprosin-1/ efhd2 impaired bcr-elicited calcium signals. concomitantly, gst-pulldown experiments revealed that swiprosin-1/efhd2 interacts with phospholipase cg2 (plcg2) and with the tyrosine kinase syk (splenic tyrosine kinase), both of which are important for bcr-induced calcium flux. the interaction of plcg2 and swiprosin-1/efhd2 was further established by co-immunoprecipitation. reconstitution of bcr-elicited calcium signals through complementation of swiprosin-1/efhd2 silenced wehi231 cells with swiprosin-1/efhd-2 was inhibited by the syk inhibitor bay 61-3606. in analogy, swiprosin-1/efhd2 regulated syk activity positively. moreover, swiprosin-1/efhd2 re-expression accelerated tyrosine phosphorylation of several proteins, specifically tyrosine phosphorylation of plcg2 and of syk tyrosine residue 352, which is involved in syk activation. finally, reconstitution of swiprosin-1/efhd2 knock-down cells with swiprosin-1/efhd2 mutants revealed that the n-terminal putative sh3-binding site, the first ef-hand, and to a lesser extent, the second ef-hand and the c-terminal coiled-coil domain, are important for bcr-induced calcium flux in wehi231 cells. interestingly, swiprosin-1/efhd2 re-expression in swiprosin-1/efhd2-silenced cells induced already in unstimulated cells raft partitioning of syk, plcg2 and the bcr, which was reversed after 2 min of bcr stimulation. in summary, swiprosin-1/efhd2 is an accelerator of proximal bcr signalling and acts through syk and plcg2 by assembling a syk-dependent calcium initiation complex in lipid rafts. this might be relevant for memory b cell signalling or central b cell tolerance. to test the biological relevance of cbl-b e3 ligase activity, these mice were analyzed for t cell proliferation, susceptibility to autoimmunity, in vivo t cell tolerance responses, and tc1 tumor rejection. results: when stimulated, t cells from rf mutant mice hyperproliferate compared to wild type t cells, even in the absence of cd28 co-stimulation. preliminary data also suggest that rf mutant mice are more susceptible to autoimmunity. in addition, rf/p14 mice die within hours after a second challenge with p33 peptide, indicating a severe defect in t cell tolerance induction. more importantly, cbl-b e3 ligase dead mice can spontaneously reject tc1 tumors. conclusion: cbl-b e3 ligase dead mutant mice phenocopy total body cbl-b knock out mice, thus indicating that cbl-b e3 ligase activity is indispensable for its regulatory in vivo functions. intriguingly, our data suggest that its inactivation could be sufficient to confer anti-tumor activity. to further elucidate the cellular mechanism of cbl-b mediated tumor rejection we have now generated the conditional cbl-b e3 ligase dead mutant mice to for the first time study the cbl-b ubiquitination function in a tissue specific and temporal fashion. our research is also currently focused on identifying the relevant in vivo cbl-b ubiquitination substrates. interferon alpha (ifn-a) has been broadly used in the treatment of specific malignancies and chronic viral diseases. for a long time it was thought that the direct inhibitory effects on malignant or virus infected cells were the major mechanisms involved in the response to ifn-a therapy. however, recent studies in mice have revealed that ifn-a/b also exerts effects on several host immune cells. ifn-a has been shown to enhance cd8 t cells (ctls) responses against soluble antigens in mice. this immunostimulatory activity of ifn-a results at least partly from its direct ability to induce maturation of dendritic cells. several studies have recently demonstrated that ifn-a/b also acts directly on murine ctls, inducing clonal expansion and differentiation into effector and memory cells. to date, little is known about the effects of ifn-a on human ctls. to approach this issue, magnetically sorted untouched human cd8 + cd45ro -t cells (mainly naï ve cells) were unstimulated or stimulated with human ifn-a and gene expression profiles were compared using an affymetrix human array. interestingly, ifn-a stimulation of highly purified human ctls without any other concomitant signals remarkably enhanced the expression of several molecules involved in death receptor signalling (trail) and chemotaxis (ip10 and itac). in a second genome-wide array analysis, we analyzed the effects of ifn-a on human ctls responding to antigen (signal 1) and co-stimulatory signals (signal 2), provided by beads coated with anti-cd3/cd28 antibodies. gene expression patterns were compared for cells stimulated with anti-cd3/cd28 beads alone or along with ifn-a. ifn-a regulates the expression of a number of genes that promote proliferation, activation and survival of ctls, tcr stabilization, chromatin remodelation, and, importantly, enhances the expression of genes involved in ctls effector functions (granzyme-b, ifn-g, trail, fasl) and chemotaxis (ip10, itac). the enhanced expression of granzyme-b, ifn-g, trail and ip10 were further confirmed at the protein levels by flow cytometry analysis and/or elisa. enhancement of granzyme-b-and trail-mediated cytolitic functions was also found by functional assays using anti-cd3-coated p815 cells and trail-sensitive caki-i cells as targets. our results show that ifn-a provides a strong signal-3 to human ctls leading to their differentiation into effector ctls. t cell activation is an important process of the adaptive immune system, which requires recognition of mhc-associated antigens by antigen presenting cells (apcs) via the t cell receptor (tcr). to induce a productive t cell response the interaction of t cells with apcs needs to be stabilized by adhesion molecules. junction adhesion molecules (jams) are a recently discovered group of immunoglobulin (ig) superfamily proteins, which are involved in the regulation of various inflammatory and vascular events. the third member of the jam protein family, jam-c, is highly expressed in platelets and endothelial cells, whereas expression in t cells is largely unknown. to investigate the regulation of jam-c in t lymphocytes, we determined jam-c gene expression in quiescent and activated human t cells. treatment with the polyclonal t cell activator phytohemagglutinin (pha) increased surface and total jam-c expression in t cells time-and dose-dependently, as determined by flow cytometry and immunoblot analysis. by contrast, no up-regulation of jam-a in activated t cells was detectable. the highest level of jam-c up-regulation by pha was observed in cd3 + foxp3 + and cd4 + cd25 high t cells. moreover, t cell receptor activation with combined anti-cd3 and anti-cd28 stimulation induced jam-c expression in t cells. jam-c induction occurred at the mrna level suggesting a transcriptional regulatory mechanism of jam-c expression. accordingly, we studied the regulation of the human jam-c gene promoter in transiently transfected t cells. luciferase activity of a jam-c promoter gene construct with three potential consensus sites for the transcription factor nfat was markedly induced in activated t cells. finally, pretreatment with two pharmacological inhibitors of calcineurin, cyclosporin a and fk-506, but not with mapk inhibitors, blocked jam-c induction in activated t cells. in summary, the present data indicate that jam-c is induced in activated human t lymphocytes via a transcriptional mechanism and suggests a major regulatory function of jam-c for the t cell response. hiv-1 infection leads to immune dysfunction owing to a successive loss of the cd4 + t cell compartment. the molecular mechanisms underlying this depletion are not well-understood but may involve the viral nef protein. nef is a multifunctional accessory protein that is required for full hiv-1 virulence and the maintenance of high viral loads. nef enhances viral infectivity and replication by downregulating cell surface receptors, e. g. cd4 and mhc class i, and modulating signal transduction pathways. the latter is thought to raise the cellular activation level and in this way may increase the infected cell's susceptibility to apoptosis. in this study we identify a signaling complex assembling at the n-terminus of nef, which contains the kinases lck and pkcv. formation of this complex, termed nakc for nef-associated kinase complex, led to activation of lck, as assessed by in-vitro kinase assay, and recruitment of pkcv to membrane rafts, as detected by discontinuous sucrose density gradient ultracentrifugation. recruitment of pkcv to membrane rafts is a hallmark of t cell activation and has been associated with activation of the nfxb transcription factor. however, contrary to our expectations, nef-mediated nakc formation did not activate nfxb. instead, it led to a strong induction of erk1/2. this correlated with a nakc-mediated increase in hiv transcription that was demonstrated by luciferase reporter assays suggesting that erk1/2 directly targets hiv transcription, possibly via induction of transcription factors. to our surprise, however, the effect of nakc on hiv transcription was found to be independent of ap-1, nfat and nfxb suggesting an alternative mechanism of nakc-mediated enhancement of hiv transcription. on the basis of our previous results we propose that nef enhances hiv transcription via removal of inhibitory factors and thus derepression of the hiv promoter. how erk1/2 is involved in this mechanism and whether nakc targets other cellular promoters, which may enhance the cellular activation level and thus sensitize the cell to apoptosis, remains to be determined. p. otahal 1 , t. brdicka 1 , v. horejsi 1 1 institute of molecular genetics as cr, praha, czech republic aims: c-terminal src kinase (csk) and cd45 are key regulators of src-family kinases in leukocytes. while cd45 is a transmembrane phosphatase, csk is localized mostly in cytosol. however, a fraction of csk is found at the cell membrane and in lipid rafts where it inhibits signaling by phosphorylating inhibitory tyrosine of src-family kinases. currently, it is accepted that sh2 domain of csk binds phosphotyrosine 317 of transmembrane adaptor protein pag and via this interaction is recruited to the cell membrane and lipid rafts. however, pag knock-out mice still have cell membrane-associated csk and do not show any apparent dysregulation of signaling which would be expected due to the low levels of membrane csk. thus, the mechanisms of membrane targeting of csk remain unclear. to analyze the role of membrane and lipid raft targeting of csk on lymphocyte signaling we targeted csk to different membrane compartments by fusing csk with transmembrane domains of lat, lax, cd25 and n-terminal part of src kinase. methods: csk chimeras containing n-terminal membrane targeting motif and c-terminal orange fluorescent protein were cloned into retroviral vector pmxs. jurkat t cells expressing individual constructs were subsequently prepared and analyzed for the inhibitory effect of these csk chimeras on t-cell receptor (tcr) signaling by measuring calcium flux and cd69 upregulation. the efficiency of inhibition depended on the membrane targeting motif, while lat-csk chimera completely inhibited tcr signaling and src-csk chimera inhibited the signaling only partially; lax-csk and cd25-csk chimeras showed almost no inhibition of tcr signaling despite efficient presence at the plasma membrane. conclusions: our data demonstrate that the function of csk strongly depends on its targeting to the specific areas of plasma membrane. it also strongly supports the idea that membrane compartmentalization is critical for regulation of t-cell signaling. peripheral cd8 t cell tolerance can be generated outside lymphatic tissue in the liver. however, the course of events leading to tolerogenic interaction of hepatic antigen presenting cells with circulating t cells is unclear. here, we demonstrate that systemically circulating antigen was preferentially taken-up by liver sinusoidal endothelial cells (lsec) and not by other antigen presenting cells in the liver or spleen. uptake and cross-presentation of circulating antigen was followed by rapid antigen-specific naï ve cd8 t cell-retention in the liver but again not in other organs. using bone-marrow chimeras and tie-2kb mice, we could show that antigen cross-presentation by lsec was both essential and sufficient to cause antigen-specific t cell-retention under non-inflammatory conditions, which was followed by cd8 t cell proliferation and expansion, but ultimately led to the development of t cell tolerance. our results show that cd8 t cell tolerance towards circulating systemic antigens is predominantly generated in the liver by lsec, which preferentially take-up and cross-present circulating proteins to cd8 t cells, leading to their rapid local antigen-specific retention and subsequent tolerisation. these insights broaden our understanding not only of physiological immune regulation towards circulating antigens but also of therapeutic manipulation of cd8 t cell responses. alphapix is a rho gtpase guanine nucleotide exchange factor domain-containing signaling protein that associates with other proteins involved in cytoskeletalmembrane complexes. it has been shown that pix proteins play roles in some immune cells, including neutrophils and t cells. in this study, we report the immune system phenotype of alphapix knockout mice. we extended alphapix expression experiments and found that whereas alphapix was specific to immune cells, its homolog betapix was expressed in a wider range of cells. mice lacking alphapix had reduced numbers of mature lymphocytes and defective immune responses. antigen receptor-directed proliferation of alphapix deficient t and b cells was also reduced, but basal migration was enhanced. accompanying these defects, formation of t-cell-b-cell conjugates and recruitment of pak and lfa-1 integrin to the immune synapse were impaired in the absence of alphapix. proximal antigen receptor signaling was largely unaffected, with the exception of reduced phosphorylation of pak and expression of git2 in both t cells and b cells. these results reveal specific roles for alphapix in the immune system and suggest that redundancy with betapix precludes a more severe immune phenotype. s. merluzzi 1 , s. parusso 1 , b. frossi 1 , g. gri 1 , c. pucillo 1 1 university of udine, dstb, udine, italy in this study, we investigated whether primary mcs could modulate the activation and proliferation of primary b cells. we performed co-culture assays using mouse splenic b cells and bone marrow-derived mcs. naï ve and activated b cells proliferation could be induced by nonsensitized mcs while an increase in b cell proliferation was observed when mcs are activated. moreover, b cell proliferation was partially abolished when mcs and b cells were separated by the transwell membranes suggesting that cell-cell contact is important in this event. using both il-6 -/-mcs and anti-il-6 receptor antibody, we demonstrated that in co-culture of primary b cells and mcs, il-6 derived from activated mcs is a key cytokine implicated in the b cell proliferation. moreover, we showed that activated mcs can influence the surface expression of costimulatory molecules as cd40 on naï ve b cells and the interaction of cd40 on b cell surface and cd40l on mcs is important for the further differentiation of b cells to plasmacells. indeed, we presented for the first time evidence that cytokines produced by activated mcs and interaction between cd40l e cd40 on mc and b cells respectively can contribute to differentiate mature b cells to iga secreting cells. in conclusion, in the present report, we showed a novel role of mcs as promoter of both the survival and activation of naive b cells and of the proliferation and further differentiation of activated b cells through soluble factors production and cell-cell contact, suggesting that mcs can contribute to the regulation of specific immune response. e. fourmentraux-neves 1 , n. bercovici 1 , a. caignard 1 1 inserm u567, paris, france inhibitory killer ig-like receptors (kir2dl1-2/3) which bind to hla-c molecules are expressed by human natural killer cells and effector memory cd8 + t cell subsets. these receptors suppress cd8+ t cell activation through recruitment of the src homology 2 domain-containing protein tyrosine phosphatase 1 (shp-1). to further analyse the yet largely unclear role of inhibitory kir receptors on cd4+t cells, kir2dl1 transfectants were obtained from a cd4 + t cell line and primary cells. the transfection of cd4 + t cells with kir2dl1 dramatically increased the t cell receptor (tcr)-induced production of il-2 independently of ligand binding, but inhibited tcr-induced activation after ligation. kir-mediated tcr activation requires intact itim motifs, involves kir2dl1-itim phosphorylation, shp-2 recruitment, zap-70 and pkc-v phosphorylation. synapses leading to activation were characterized by an increase in the recruitment of p-tyr, shp-2 and p-pkc-v but not of shp-1. in contrast, the kir2dl1/hla-cw4 interaction led to a strong synaptic accumulation of kir2dl1 and the recruitment of shp-1/2, inhibiting tcr-induced il-2 production. kir2dl1 may induce two opposite signaling outputs in cd4 + t cells, depending on whether the kir receptor is bound to its ligand. these data highlight unexpected aspects of the regulation of t cells by kir2dl1 receptors. b cell receptor (bcr) binding by antigen initiates activating signaling cascades and facilitates the exposure of specific b cells to powerful co-stimulatory signals, such as t cell help or toll-like receptor ligands. the role of bcr binding in modulating the access to these second signals is complex and varies between stimulatory conditions. by quantitative tracking of b cell responses in vitro we can measure which signals affect b cell proliferation or differentiation, or both, and thereby establish a novel understanding of how b cells respond appropriately to different combinations of stimuli. we utilised hel-specific bcr transgenic sw hel mice to assess the effect of a specific antigen signal on b cell responses to the t-independent mitogen lipopolysaccharide (lps). the presence of antigen renders a greater proportion of cells responsive to lps stimulation and profoundly influences effector cell differentiation. antibody secreting cell formation is dramatically inhibited by hel, but we found that isotype switching to igg1 is strongly upregulated. both of these alterations to differentiation outcomes occur independently to the proliferative effects induced by antigen. when b cells are exposed to antigen for a limited period of time, switching to igg1 still occurs but some capacity to differentiate to antibody secreting cells is recovered, leading to effective secretion of igg1 antibody during these conditions. the observed igg1 switching behaviour mimics that of b cells responding to lps and il-4, but is mediated by a different, stat6-independent pathway. these data are indicative of the important role specific antigen signals play in regulating b cell responses in stimulatory environments. a. quintana 1 , c. schwindling 1 , m. pasche 2 , c. junker 1 , c. kummerow 1 , u. becherer 2 , e.c. schwarz 1 , j. rettig 2 , m. hoth 1 1 saarland university, biophysics, homburg, germany, 2 saarland university, physiology, homburg, germany the adaptive immune response requires the interaction between antigen-presenting cells and t cells. this cell-cell interaction, called the immunological synapse (is), facilitates the activation of t cell receptor-mediated signalling cascades including a rise of cytosolic calcium through the activation of crac/orai1 channels. to allow sustained activity of crac/orai channels, the calcium-dependent inactivation of the channels through local calcium microdomains has to be prevented. objectives: the purpose of the study was to analyze local and global calcium signals in t cells and to test the hypothesis that the is controls these signals through mitochondrial positioning. methods: we used different microscopy techniques including very fast wide-field microscopy with subsequent deconvolution, total-internal reflection microscopy, and confocal microscopy in combination with electrophysiological techniques in primary human t helper cells and cell lines. to test the statistical significance of our data, we used two-sided student t-tests or non-parameterized tests. results: following is formation, we found that mitochondria translocated to the is in a calcium-dependent way. the distance between mitochondria and the plasma membrane at the is was lower than 150 nm. following accumulation at the is, mitochondria limited calcium entry to the orai channels localized right at the is by preventing their calcium-dependent inactivation. in contrast, no calcium influx was observed at sites where no mitochondria were accumulated as orai channels were inactivated at these sites. mitochondrial positioning at the is thus induced local calcium influx at the is without the necessity to enrich orai channels at the is. mitochondria took up calcium at the is distributing it further into the cytosol by releasing it at different sites, which kept the local domain at the is low enough to prevent calcium dependent orai inactivation and to prevent excessive calcium clearance by the calcium aptases in the plasma membrane, which could inhibit an efficient t cell activation. conclusion: mitochondria positioning at the is controls local calcium entry through orai channels. mitochondria prevent orai inactivation and excessive calcium clearance at the is to facilitate calcium-dependent t lymphocyte calcium signalling. we aimed to determine the functional correlates of cd4 + t cell tolerance and immunity in vivo. ovalbumin (ova)-specific transgenic cd4 + t cells were adoptively transferred into syngeneic mice immunized with soluble ova protein ± lipopolysaccharide (lps) by the i. v. route, and analyzed for a variety of immunological parameters over a period of 21 days. under tolerogenic conditions (ova alone), cd4 + t cells showed substantial early activation, but their activation profile differed markedly, both in magnitude and quality (icos, 4-1bb), from t cells activated by ova+lps. this difference in activation also translated into differing cd4 + t cell expansion and contraction kinetics in the early phase of the t cell response (days 1-6). in the late phase of the primary response (days 7-21), under immunizing conditions, the large majority of transgenic cd4 + t cells in the spleen developed into mature effectors with a prominent capacity to secrete il-2, ifn-g, and il-17a, and only few ova-specific foxp3 + regulatory t cells ( x 10 %) were observed. germinal centers were prominent and ova-specific ig of all isotypes were generated. in contrast, under tolerizing conditions, antigen-specific cd4 + t cells failed to migrate into the b cell follicles, but production of ova-specific igm was nevertheless observed. in these animals, the proportion of splenic ova-specific regulatory t cells (30 %) was substantially increased. on day 14, both groups of mice were re-challenged via the airways with ova+lps to functionally assess their immune status. in tolerized animals, the transgenic t cell population in the lung infiltrate was composed of ova-specific regulatory t cells (50 %) or t cells with a reduced capacity to secrete effector cytokines. in contrast, in immunized animals, this population almost exclusively consisted of cd4 + effector t cells with a pronounced inflammatory cytokine profile (ifn-g, il-17a). with this model we provide a comprehensive analysis of the many functional correlates of "immunity" versus "tolerance" to soluble protein antigen in vivo. we identify and characterize a number of the key players (cell surface molecules, cytokines, cell subsets) representing the decision between immunity and tolerance in the immune system. mast cells (mcs) are well-recognized as key effector cells in immunoglobulin e (ige) -associated immune responses and as prototypic regulators of innate immunity. the characteristics, importance, and molecular requirements for interactions between mast cells (mc) and cd8 t cells (tc) remain to be elucidated. using myelin/oligodendrocyte glycoprotein (mog), we demonstrated that mcs induce antigen-specific cd8 tc activation and proliferation. the antigen crosspresentation by mcs induces the secretion of interleukin-2, interferon-g and macrophage inflammatory protein-1 by cd8 tc. in vivo evidence that mcs modulate t cell responses has been obtained so far in the murine experimental autoimmune encephalomyelitis (eae), the standard animal model for multiple sclerosis, in which both cd8 tc and mcs are now recognized as key players. one of the main central nervous system (cns) antigens recognized by autoreactive tc in eae is the myelin oligodendrocyte glycoprotein (mog). to investigate the in vivo-relevance of the identified mc-cd8 tc interactions, we have employed the eae as a model of organ specific autoimmune disease in wild type mice and mc-deficient w/w sh mice. wt and w/w sh mice were immunized with the mog 35-55 protein. our results provide direct evidence that mc contribute to cd8-specific priming in eae and show that the tc proliferation failure is specific for cd8 tc from mog 35-55 -immunized w/w sh mice. the role of mc-cd8 tc interaction in induction of autoimmunity will be further investigated in eae. in summary, we provide the first evidence that mcs regulate antigen-specific responses of primary cd8 tc in vitro and in vivo. our study further supports the emerging concept that mcs, protagonists of innate immunity are also important regulators of adaptive immune responses and corresponding cd8 tc responses. this newly uncovered mc function might be of great biological relevance in situations where effector cd8 tc are critically involved, e. g. viral infections or infections with intracellular pathogens and/or autoimmune diseases such as multiple sclerosis. activation of resting t cells in vitro is triggered by combined t cell receptor (tcr) and cd28 engagement and can be modulated by simultaneous ligation of various other surface receptors. although the fasl is best known for its capacity to initiate cell death in fas-bearing cells, it has recently been implicated in the regulation of t cell activation. thus, a crosstalk between the tcr and fasl is likely, but far from being biochemically elucidated. we report that fasl engagement by immobilized but not soluble fasfc fusion protein and anti-fasl antibodies blocks the activation of primary human peripheral t cells even in the presence of cd28 costimulation at the level of an early signal initiation. inhibition is thus associated with a reduction of tyrosine phosphorylation of a number of key elements in tcr signal transduction and also with a lowered calcium response. the data presented stress the importance of the fas/fasl-system for signal initiation via the tcr/cd3complex and provide further arguments for a retrograde signaling capacity of fasl or a crucial role of fas as a costimulatory molecule. golgi network (tgn). moreover, trim specifically associates with the cytoplasmic tail of ctla-4, but not via any conventional motifs in this region. overexpression of trim augments ctla-4 surface expression, whereas down-regulation of trim expression by shrna results in disturbed ctla-4 localisation, mainly restricted to the tgn. ctla-4 vesicles and surface expression were significantly reduced but not abolished, suggesting that other factors are involved in ctla-4 trafficking. here, we identify additional transmembrane adapter protein (trap) family members as novel binding partners and regulators of ctla-4 expression. although there is some redundancy amongst traps, our results highlight the importance of this family of proteins in ctla-4 transport to the cell surface. it is imperative to reveal the mechanisms by which ctla-4 is transported to the cell surface, given that minor changes in expression can have major effects on t-cell function and in the development of autoimmunity. natural killer t (nkt) cells are found within the liver and are known to exhibit immune regulatory function. upon recognition of glycolipids presented on cd1 molecules, nkt cells are activated and release cytokines, including ifn-g, il-2 and il-4. nkt cells are efficiently recruited to the liver via cxcr6-dependent chemotaxis toward cxcl16 and constitute a large proportion of the liver-resident lymphocytes. we have previously shown, that liver sinusoidal endothelial cells (lsec) can scavenge circulating soluble antigens, and can cross-present these antigens to naive cd8 t cells. cross-presentation leads to initial t cell activation and expansion, but ultimately these cd8 t cells are rendered tolerant. as both naive t cells and nkt cells come into close contact with lsec in the hepatic sinusoids, we investigated whether nkt cells can modulate cd8 t cell tolerisation via interaction with lsec. to this end we analysed cd1d expression on lsec and their ability to activate nkt cells by presentation of the cd1d-binding glycolipid a-galactosylceramide (agalcer). we found that lsec express functional cd1d, as agalcerpresenting-lsec were capable to induce tnf-a, il-2, il-4 and ifn-g production in nkt cells in vitro. the interaction of agalcer-presenting-lsec with nkt cells led to the upregulation of cd54 and b7-h1 on lsec. as naï ve cd8 t cell tolerisation by lsec critically depends on b7-h1, we hypothesise that hepatic nkt cell activity may contribute to the immunological capabilities of the liver by regulating the tolerogenic function of lsec. improved antibody responses by class-switched memory b cells require enhanced signaling from their antigen receptor (bcr). however all bcr classes on naïve and antigen-experienced b cells utilize the canonical iga/igb subunit for signaling. we identified the signal amplification mechanism of the igg-and ige-bcr. for these isotypes tyrosine-based signaling is not confined to iga/igb but extends to a conserved tyrosine residue in the cytoplasmic segments of immunoglobulin heavy chains. the phosphorylated immunoglobulin tail tyrosine recruits the adaptor grb2 in order to sustain protein kinase activation and generation of second messengers causing robust cellular proliferation. hence membrane-bound igg and ige not only recognize antigen but also exert bcr-intrinsic costimulation to render memory b cells less dependent on t cell help for activation. objectives: the majority of circulating human gd t cells harbor tcr containing vg9, jg1.2, and vd2 gene products. they recognize nonpeptide antigens like (e)-4hydroxy-3-methylbut-2-enyl pyrophosphate derived from pathogenic microbes and isopentenyl pyrophosphate (ipp) in malignant cells. recently, we and others found out that gd t cells express a variety of costimulatory molecules including icos, and pd-1. one of the inhibitory receptors, pd-1, is a member of cd28/ctla-4 family and contains a single ig v-like domain in its extracellular region. pd-1 can bind to two b7 homologue molecules, pd-l1 and pd-l2. it has been reported that interaction of pd-1 with its ligands resulted in peripheral immune regulation and tolerance in ab t cells. in this study, we show that pd-1 is expressed on activated human gd t cells and regulates the effctor functions of gd t cells. methods: peripheral blood mononuclear cells were resuspended in yssel's medium and stimulated with 2-methyl-3-butenyl-1-pyrophosphate plus il-2 to obtain gd t cells. pd-l1+ and pd-l1-human tumor cell lines were established from cancer patients. in order to prepare anti-pd-l1 mabs, the pd-l1 extracellular domain was expressed in e.coli as inclusion bodies and refolded in the standard arginine-based buffer. mice were immunized with the refolded protein and mabs were established. to determine the function of pd-1 in gd t cells, we determined cytokine production and cell mediated tumor lysis by activated gd t cells in the presence of inhibitors of pd-1/pd-l1 interaction. results: gd t cells expressed pd-1 upon simulation with nonpeptide antigens and many tumor cell lines expressed pd-l1. we first examined whether or not the engagement of pd-1 receptor could modulate the cytotoxic activity of gd t cells. pd-l1-expressing tumor cells tempered cytotoxic activity of pd-1+ gd t cells, and cytokine production such as tnf-a was down-regulated by pd-1 engagement. in addition, inclusion of anti-pd-l1 mab reversed cytotoxic activity and cytokine production when pd-l1-expressing tumor cells were challenged by pd-1-expressing gd t cells. conclusion: pd-1 delivers inhibitory signals in gd t cells upon engagement with pd-l1. peripheral tolerance plays an important role in preventing t lymphocyte responses to self or harmless antigens. one of the mechanisms that contribute to this form of tolerance is anergy, which is characterized by a lack of proliferation and il-2 production by t cells in response to antigenic challenge. the acquisition of the anergic phenotype is an active process, with negative regulators of t cell signalling being induced. among these are the e3 ubiquitin-protein ligases which recognize target proteins for ubiquitination and catalyse the transfer of ubiquitin to them, directing them to the proteasome or to the endosome-lysosomal pathway, and hence downregulating their activity. the e3 ubiquitin-protein ligases cbl-b, itch and grail have been shown to be upregulated in anergy and to ubiquitinate and downregulate tcr signalling elements. our objectives were to determine the expression of cbl-b, itch and grail in antigen-specific cd4 + t cells in both the induction and maintenance phases of anergy, in vitro and in vivo, and to investigate their functional signalling role(s) in the maintenance of the tolerance phenotype. in order to accomplish these objectives we induced priming or tolerance of ovalbumin (ova 323-339 peptide)-specific t cells from do11.10 tcr transgenic mice in vitro or, following adoptive transfer of near physiologically relevant numbers of such cells into recipients, in vivo and correlated functional outcome (via proliferation and cytokine readout assays or antibody production) with e3 ubiquitin-protein ligases expression and the ubiquitination status of the tcr signalling machinery. cbl-b, itch, grail and target ubiquitination status, in terms of tissue, cellular and subcellular protein expression, modification and localisation, were assessed by a combination of immunoprecipitation and western blotting studies. moreover, we have performed quantitative analysis at the single cell level by tracking such antigen-specific cells in vitro and in vivo by using laser scanning cytometry. our current work focuses on the functional consequences of adenoviral transfection of such antigen-specific t cells by mutant e3 ligase-, signal target-and ubiquitin-constructs. collectively, these approaches have facilitated the dissection of the potential differential roles of ubiquitin signalling in priming and tolerance of antigen-specific t cells. s. j. keppler 1 , p. aichele 1 1 immh, university freiburg, immunology, freiburg, germany interleukin 12 (il-12) is produced by cells of the innate immune system during infection and plays an important role in controlling various pathogens. it was postulated recently that il-12 has a direct influence on cd 8 + t cells in vitro, enhancing expansion and the development of effector functions as a third signal, additionally to tcr engagement (signal 1) and costimulation (signal 2). we analysed direct il-12 signaling to cd8 t -12 signaling exhibited normal degranulation activity, cytolytic functions, ifn-g and tnf-a production. however, cd8 t cells lacking il-12 signaling failed to up-regulate klrg1 and to down-regulate cd127 in the context of listeria but not viral infections. thus direct il-12 signaling to cd8 t cells determines the cell fate decision between short-lived effector cells (slecs) and memory precursor effector cells (mpecs), dependent on the pathogen-determined local cytokine milieu. cd8 + t lymphocytes are required for effective host defense against pathogens but also for mediating effector responses against uncontrolled proliferating self tissues. we could now reveal that individual cd8 + t cells are tightly controlled in their effector functions by cd152 (ctla-4). we demonstrate that signals induced by cd152 reduce the frequency of interferon-gamma (ifn-g) and granzymeb expressing cd8 + t cells. for this novel function cd152 specifically represses the transcription factor eomes, but not t-bet. a cd152 mediated induction of the inhibitory transcription factor ckrox has been ruled out. ectopic expression of eomes reversed cd152-mediated inhibition of effector molecule production. additionally, enhanced cytotoxicity of individual cd8 + t cells differentiated in the absence of cd152 signaling could be demonstrated in vivo. the novel insights that cd152-mediated signal transduction in vivo indeed alters cd8 + t cell cytotoxicity qualitatively at the single cell level and not only quantitatively by enhancing expansion extend the understanding how to selectively modulate immune responses of cd8 + t cells. objectives: atp constitutes a damage associated molecular pattern (damp) and contributes together with pathogen associated molecular patterns (pamp) to the efficient priming of the innate immune system. atp is a ubiquitous extracellular messenger, which activates plasma membrane receptors for extracellular nucleotides termed p2 receptors. p2x 1-7 receptors open to non-selective ion channels, whereas p2y1, 2, 4, 6, 11-14 are g-protein coupled receptors, which bind preferentially adp, udp, utp or udp-glucose. as the role of p2 receptors in the control of b cell activation has been poorly investigated, aim of the present study is to understand better the mechanisms of intracellular atp production and release by human b cell subsets. methods: intracellular atp measurement has been performed using a bioluminescence assay while extracellular atp has been measured by hplc. storage and release of atp by b cells have been elucidated using confocal and tirf microscopy, to study vesicles distribution and dynamics near the plasma membrane. results: in both human naive and memory b cell we observed a prominent increase of atp synthesis upon tlr9 but not bcr stimulation. glycolytic pathway rather than oxidative phosphorilation was involved in atp synthesis. p2x7 antagonists inhibited both proliferation and differentiation to plasma cells of human b cells thus suggesting that atp is released in the pericellular space. labelling of resting and activated human memory b cells with quinacrine, a nucleotide binding component, revealed a typical vesicular pattern of atp, confirmed with subcellular fractionation on sucrose equilibrium gradients. tirf imaging showed a fluorescently labelled vescicle underwent fusion with the plasma membrane after stimulation with anti-ig and this event was ca(2+)-dependent. conclusion: these data provide evidence that atp is produced by b cell preferentially by glycolytic pathway and vesicular exocytosis is a key mediator of atp release in human b cells. atp released in the pericellular space might act as an autocrine and paracrine signalling molecule that regulates the functions of b cells. o. ballek 1 , a. brouckova 1 , d. filipp 1 1 institute of molecular genetics as cr, laboratory of immunobiology, prague, czech republic two src family tyrosine kinases lck and fyn are critical for the proximal t-cell signaling. we have previously demonstrated that induced lck activation outside lipid rafts (lr) results in lck translocation to lr. central in this sequence of events is the rapid translocation of kinase active lck to lr, yet the mechanism underpinning this process is unknown. the main aim of this study is the characterization of molecular mechanisms and its functional elements regulating the early recruitment of signaling molecules to lr and forming immunological synapse. we have recently characterized the c-terminal yqpqp sequence as a novel cis-acting component essential for partitioning of lck to lr. here we report that the expression of the c-terminal truncate of constitutively active lck ( ¿ fqpqp) in nih3t3 cells failed to phosphorylate several proteins detected in the presence of untruncated kinase active y505flck. comparative 2-d gel analyses followed by ms/maldi identified rack1 as a candidate protein for interaction with the c-terminal tail of lck. co-expression in nih3t3 cells of ha-tagged rack1 with either a wild type lck or constitutively active y505flck revealed a significantly enhanced complex formation between y505flck and rack1 compared to that of wtlck. ectopic expression of y505flck with its domain-inactivating mutations showed that lck-rack1 interaction depends on functional sh2, sh3 and the c-terminal tail sequence of lck. lck-rack1 interaction is readily detectable also in primary cd4 + lymph node t cells. upon their activation, only the pool of lck molecules associated with high molecular weight complexes can translocate to lipid rafts. co-purification of rack1 with these fractions further suggests that it plays a role in the translocation of lck to lr. in addition, lck and rack1 co-redistribute to both forming immunological synapse and to antibody-mediated capping clusters. moreover, the importance of interaction between activated lck and rack1 in the context of lck translocation to lr is further strengthen by the observation that rack1 is associated with elements of cytoskeleton. these results are the first to characterize rack1 as a candidate molecule involved in the regulation of lck translocation to lr through linking the c-terminal sequence of lck to cytoskeletal network. human b cells are currently not known to produce the pro-apoptotic protease granzyme b (grb) in physiological settings. we have discovered that b cell receptor stimulation with either viral antigens or activating antibodies in the context of the acute phase cytokine interleukin 21 (il-21) can induce secretion of substantial amounts of grb by human b cells. grb response to viral antigens was significantly stronger in b cells from subjects recently vaccinated against the corresponding virus as compared to unvaccinated subjects. both, naï ve and memory b cells differentiated into grb-secreting cells, which featured a homogeneous cd19+cd20+cd27-cd38-igd-phenotype, improved survival and enhanced expression of co-stimulatory, antigen-presenting and cell-adhesion molecules. b cellderived grb was enzymatically active and its induction required activation of similar signaling pathways as in cytotoxic t cells. our findings suggest grb-secreting b cells play a role in early anti-viral immune responses, thereby contributing to the elevated serum grb levels found in various viral diseases. further studies will elucidate whether b cell-derived grb induces cytotoxicity towards virus-infected cells or exhibits other functions. results: transfer of otii cells augments the wild type th2 response to alumova inducing large early germinal centres and massive plasma cell formation with more than 75 % of these switching to igg1. the plasma cells up-regulate cxcr4, but not cxcr3, a chemokine receptor that attracts plasma cells to inflammatory sites. oti cells respond to alumova by producing ifng, a th1-associated cytokine. when both oti and otii cells are transferred switching is diversified with plasma cells being igm (˚5%), igg2a (˚30 %), igg2b (˚30 %) or igg1 (˚30 %). in addition to cxcr4, some 70 % of these plasma cells strongly express cxcr3. the induction of cxcr3 in these plasma cells correlates with their increased expression of the transcription factor t-bet, which has been linked with igg2a switching during th1 responses. this is functionally significant for oti-dependent cxcr3 expression, as well as induction of switching to igg2a, are dependent on t-bet expression by the responding b cells, although t-bet-deficient b cells still switch to igg2b. t-bet is known to be induced in b cells exposed to ifng or tlr9 stimulation. these two hypothetical mechanisms are currently being tested in mice injected with blocking anti-ifng antibodies or mice deficient in myd88. objective: a successful immune response against malaria has to be tightly controlled. the early production of pro-inflammatory cytokines is required to control the growth of intraerythrocytic parasites but the same cytokines are also involved in the induction of severe malaria in both humans and mice. activation of t lymphocytes through tcr signalling takes place within the context of numerous other cell surface protein interactions. to prevent unnecessary activation of t cells the immune system has developed an intricate balance between positive and negative costimulatory signals. costimulatory signals determine whether antigen recognition by t lymphocytes leads to full activation or to anergy. in contrast negative costimulators expressed by t cells seem to mediate the regulation of immune responses and thus play a pivotal role in the maintenance of peripheral tolerance. recently, btla (b and t lymphocyte attenuator, cd272) was described as a novel negative costimulatory receptor. btla is predominantly expressed on t and b cells and dampens t cell activation. in this study, we analyzed the function of btla during experimental malaria infection. to study the function of btla we employed a mouse model of blood-stage malaria using p. yoelii nl infection of btla-deficient and hvem-deficient mice. p. yoelii provokes a high parasitemia in infected mice that is cleared within three weeks from time of infection. immunity in this model depends on cd4+ t cells and hence the role of negative costimulators that modulate t cell function can be studied using this model. results: peak parasitemia of p.yoelii-infected btla-deficient and hvem-deficient mice was much lower compared to wild type mice. the increased immunity of btla-deficient mice depends largely on cd4+ t cells. we found that btla::hvem interaction regulates the size and the cytokine-production of the responding t cell pool. however, in contrast to the ctla-4 pathway, the manipulation of btla::hvem interaction triggers no pathology during infection. the hvem::btla interaction dampens the protective immune response during experimental malaria and thus manipulation of this pathway is an attractive target for therapeutic interventions. . so far, the contribution of actin regulatory proteins to this process remained largely unknown. here we demonstrate that the actin-bundling protein l-plastin is indispensable for segregation of lfa-1 and cd2 in the psmac of untransformed human t-cells. in marked contrast, tcr/cd3 accumulation in the csmac is not dependent on l-plastin. the relocalization of l-plastin in the immune synapse occurs within seconds of t-cell/apc contact formation and relies on actin polymerization. importantly, binding of calmodulin to l-plastin is required for the maintenance of l-plastin in the immune synapse and inhibition of calmodulin prevents psmac formation. thus, receptor segregation in the immune synapse is a consequence of the combined activities of the actin-bundling protein l-plastin and calmodulin. protective t cell responses are based on expansion and persistence of clones with optimal affinity for antigen. presently, it is unknown which mechanisms guard the selection and expansion of the highest affinity clones from the very diverse naï ve pool. rapid cell division creates shifts in selective pressure, which is a basic biological prerequisite for elimination. therefore we hypothesized that apoptosis might play an important role during this phase of t-cell biology. here we show that the balance between the pro-apoptotic protein noxa and its antagonist mcl-1 regulates interclonal t cell competition during acute and chronic immune activation. we found p53-independent noxa gene induction and mcl-1 downregulation upon t cell activation. concomitant we observed the release of death-inducing factor bim from mcl-1, which was delayed in noxa -/cells. using ot-1 cells and altered peptide ligands we observed that the level of mcl-1 downregulation in activated t-cells depended on the antigen affinity of the t-cell receptor. since mcl-1 -/mice are embryonic lethal, noxa -/mice were used to study the functional implications of this mechanism in vivo. at a young age noxa -/mice have a normal lymphoid compartment, but accumulate effector t cells over time. upon acute influenza infection, normal levels of effector cells were generated. however, the quality of the antiviral (np366) response was impaired in these mice as many subdominant clones persisted in the effector t-cell population of noxa -/mice at the peak of infection. this increased diversity correlated with exacerbated pathology and a reduced rate of viral clearance. in a model of chronic immune activation, effector t cells rapidly accumulated in the noxa -/mice and infiltrated the peripheral organs, culminating in severe multi-organ pathology and premature death. these results establish a novel role for the noxa/mcl-1 axis during immune responses and suggest that the formation of a high-affinity effector population of restricted clonal diversity depends on a darwinian selection of t-cells during the expansion phase based on antigen affinity, with survival of only the fittest clones. cytotoxic t lymphocytes (ctls) are essential for immunosurveillance, a process that requires the presentation of virus-or tumor derived antigenic peptides in context of antigen presenting cells. insight into intracellular mechanisms facilitating lytic granule release and formation of the immunological synapse as a prerequisite for target cell destruction was primarily obtained from loss-of-function mutations in hereditary human diseases and gene-mutated mice. here, we refer to estrogen receptor-binding fragment-associated antigen 9 (ebag9) as a negative regulator of secretory lysosome release. in gene deleted mice we show that loss of ebag9 confers ctls with enhanced cytolytic capacity, in vitro and in vivo. here, we show that ebag9, which was previously identified as a snapin-interacting protein in neuronal cells, interacted with the adaptor molecule g2-adaptin in t cells. both interactions suggested an involvement of ebag9 in endosome-lysosome related organelle biogenesis and membrane fusion. efficiency of granzyme b sorting towards secretory lysosomes was improved, which was consistent with the enhanced kinetics of cathepsin d proteolytic processing. while the formation of the immunological synapse remained unaffected in ebag9-/-ctl, relative size distribution of lytic granules revealed a shift towards smaller granule diameters and volumes in ebag9-deficient ctls. these data imply a role for ebag9 in regulating the formation of mature ctl granules and identify ebag9 as a tunable inhibitor of ctl-mediated adaptive immune response functions. finally, ebag9 defines a novel negative regulator of secretory lysosome release in ctls. thus, the elucidation of the ebag9-related pathway might provide a fresh impuls on therapeutic approaches in the treatment of autoimmune disorders. the liver is known to induce tolerance, rather than immunity, through tolerogenic antigen presentation or elimination of effector t cells. recently, we could show that liver sinusoidal endothelial cells (lsec) inhibit activation of naive cd8 t cells by antigen-presenting dc. this regulatory effect of lsec on dc function was mhc-independent and not limited to soluble mediators, but required physical contact. interestingly, interaction with lsec led to reduced dc expression levels of cd80/86 and il-12. in addition to indirect inhibition of t cell activation by de-licensing of dc, we now detected another influence of lsec in form of direct inhibition of t cell priming. in the presence of lsec, stimulation with acd3/acd28 or pma/ionomycin could not significantly activate cd4 and cd8 t cells. thus, lsec did not only inhibit t cell priming triggered by tcr activation but also after elicitation of ca 2+ influx into the cytoplasm. furthermore, we found that ifn-g secreted by t cells in the early phase of activation is crucial for licensing the inhibitory function of lsec. taken together, these data indicate that the inhibitory effect of lsec is mediated by a machinery induced at the early phase of t cell activation, however, interferes with late events in the t cell activation cascade. we propose a model of "inducible inhibition", where on the one hand naive t cell priming is directly inhibited by lsec, and on the other hand tolerogenic priming by antigen-presenting lsec is still allowed. taken together, these results reveal a novel principle, operative in hepatic tolerance induction, in which lsec not only tolerize t cells themselves, but also inhibit the responsiveness to local activation stimuli. m. almena 1 , s. carrasco 1 , i. merida 1 1 centro nacional de biotecnología csic, inmunología y oncología, madrid, spain self-tolerance acquisition is essential for the immune system to control its own response. t cells achieve self-tolerance trough thymic selection and anergy, two processes where rasgrp1-ras-erk signal intensity is critical to determine the final cell outcome. rasgrp1 is a gef for ras that is activated in a diacylglycerol (dag)dependent manner. dag is generated by plcg after tcr stimulation and is consumed by diacylglycerol kinases (dgk). dag generation, as a result of the concerted regulation of these two enzymes, activates ras, providing a mechanism to translate the strength of the stimulus into a quantitative cell response. 1. analyze the impact that dag metabolism plays in t cell tolerance in vivo, using transgenic mice where dag generation is impaired. 2. develop a method to sense dag production and localization, in both thymocytes and peripheral t cells, and its correlation with the strength of the stimulus used. methods: we generated transgenic mice expressing a constitutively active dgk in t cell lineage. this protein was anchored to the plasma membrane, thus diminishing the lipid levels in this specific location after tcr stimulation. ot-i cd8 cells expressing gfp-c1 domains were used with peptide-pulsed apcs to study dag generation and dynamics by confocal microscopy. results: transgene expression was obtained in thymic and peripheral t cells. no major defects were observed in t cell subsets but analysis of peripheral t cells demonstrated important defects in t cell activation. we are currently studying thymic selection breeding our transgenic with h-y mice, in order to check if t cell populations are being properly selected. using t cell-apc conjugates with peptides with different tcr binding affinities we found a clear correlation between the strength of the stimulus, dag production and ras-mapk activation. conclusion: our data demonstrate that dag generation not only activates c1 domain containing proteins but regulates a mechanism by which t cells sense the magnitude of the stimulus received, translating it into the intensity of the generated response, a process essential in t tolerance. future experiments will help to define the exact contribution of the lipid to tcr signaling pathways and to t cell homeostasis. the inducible costimulator (icos, h4, cd278), a cd28-like costimulatory molecule, has an important role in the development of efficient t cell responses. early data showed that icos costimulation produced th2 biased responses and high production of the anti-inflammatory cytokine il-10, and was essential to the development of germinal centres. however, icos can also help in the il-21-dependent differentiation of inflammatory th17 cells. these different functions could be due to differences in the apcs bound by icos-expressing t cells and/or because of the intervention of distinct molecules binding the cytoplasmic domain of icos. icos shares with cd28 a yxxm cytoplasmic motif that can bind, upon tyr phosphorylation, the regulatory p85 subunit of class ia pi-3 kinases. these can complex with one of the three 110 kda catalytic subunits (p110a, p110b, and p110d) expressed by leukocytes that generate pip 3 affecting cell growth, cell cycle progression, survival, intracellular traffic, cytoskeletal changes and migration. there is also evidence that the regulatory and catalytic class ia pi3k isoforms fulfill specific functions in macrophages and lymphocytes. we have used proteomic and immunochemical approaches to identify molecules binding the phosphorylated or unphosphorylated cytoplasmic domain of icos, and particularly the presence of distinct pi3 kinase isoforms. then, the functional importance of these molecules has been analyzed by using pharmacological inhibitors specific for downstream mediators of icos activation. pull down of t cell lysates using phosphorylated or unphosphorylated synthetic peptides covering the cytoplasmic domain of icos was carried out. proteomic and immunoblot analysis of bound proteins showed that phosphorylated icos bound the different pi3-k regulatory (p85a, p85b, p53a) and catalytic (p110a, p110b, and p110d) pi3-kinase subunits expressed by leukocytes. these data were confirmed in icos immunoprecipitates from pervanadate-activated cells. icos bound regulatory and catalytic subunits in the order p85a g p53a g g p85b and p110a g p110d g g p110b, in agreement with quantitative rt-pcr and immunochemical estimation of subunit abundance in the t cells and t cell lines used. the use of specific pi3-kinase inhibitors has confirmed the relative importance of the catalytic isoforms in icos function, including reorganization of actin cytoskeleton induced by icos ligands, or costimulation of tcr/cd3-induced secretion of il-10 and il-4. during the process of antigen recognition between t-cell and antigen-presenting cell (apc), structural and spatial changes take place at the cell-cell contact, where the molecules involved in the formation of the immune synapse (is) reorganize, displaying a segregated localization. in this context, the translocation of the microtubule-organizing center (mtoc) is an early event that occurs during the formation of the is, bringing with it the golgi apparatus, thus providing the basis for a polarized secretion. however, the molecular mechanisms involve in the localization of the mtoc at the contact area between the t cell and the apc are not completely understood yet. we have studied the possible role of scaffolding protein akap450, a member of the a-kinase anchoring protein (akap) family that localizes at the centrosome and interacts with pka regulatory subunit and other signalling molecules, in mtoc polarization and immune synapse formation. either the overexpression of gfptagged c-terminal cg-nap/akap450 construct that acts as a dominant negative, or sirna knockdown of endogenous akap450 expression in t cells prevents the correct organization of cd3z and pkcv to the is and mtoc reorientation towards t cell-apc contact area in antigen and superantigen-dependent human models, resulting in a disorganized is; lfa-1 localization was also analyzed to assess p-smac architecture and, interestingly, confocal 3d reconstruction revealed that lfa-1 ring was not clear in the akap450-disrupted cells. moreover, akap450 was required for tcr signalling since the knock down with specific sirna and overexpression of c-terminal of akap450 decrease the phosphorylation of molecules such as lat, plcg1 and pkcv. these defective activation events as reflected in a reduction of il-2 production. together, our results underscore a key role for akap450 in the organization of the immune synapse and in the antigen-specific reorientation of the mtoc. the tcrbeta/ptalpha pre-tcr complex signals the expansion and differentiation of developing thymocytes. functional properties of the pre-tcr rely on its unique ptalpha chain, which suggests the participation of specific intracellular adaptors. in fact, we have recently identified cms, a member of the cin85/cms family of adaptors, as a ptalpha-binding protein that specifically interacted with the human ptalpha cytoplasmic domain via its sh3 domains, and to the actin cytoskeleton via its c-terminal region. we found that cms co-localized with polymerized actin in pre-tcr clusters at the pre-tcr activation site, and also in the ptalpha endocytic compartment. since actin polymerization plays a critical role in regulating signalling through the alpha/beta tcr in mature t cells, we decided to investigate the potential function of cms as a regulator of actin polymerization and pre-tcr signalling in pre-t cells. using pre-t cells expressing a mutant pre-tcr lacking the cms-binding motif in the ptalpha tail and short hairpin irna-based gene silencing, we demonstrate that binding of cms to ptalpha contributes to cytoskeleton dynamics and pre-tcr-mediated signalling in human pre-t cells. cms-deficient cells specifically showed defects in pre-tcr-induced ca2+ mobilization and cell activation involving the pi3k, nfat, plcg and erk signalling pathways, together with defects in actin polymerization and cell motility. cms therefore links cytoskeleton dynamics with the function of discrete pre-tcr signalling components, suggesting the functional implication of cms in human t-cell development in vivo. abstract withdrawn by author j objectives: most signaling pathways engaged after bcr activation have been described. however, several negative regulators of these pathways are unknown. the characterization of these regulators is important to understand the control of transduction pathways in adaptative immunity. carabin (tbc1d10c) has been recently described as a negative regulator of tcr signaling. it interacts with calcineurin and inhibits the formation of calcineurin/calmodulin complex, blocking nfat nuclear transport. moreover, carabin maintains ras protein under an inactive form, thus inhibiting ras-mapk cascade. expression of carabin is finely regulated following tcr signaling, and its knockdown (kd) enhances t cell activation. considering the important molecular similarities of antigen receptor signaling pathways in t and b cells, we studied the role of carabin in b cell. could carabin play a role of negative regulator of b cell function? methods: we studied by quantitative rt-pcr 1) the expression of carabin in different purified subsets of bone marrow and splenic mouse b cells, as well as 2) the kinetic of expression of carabin in bcr stimulated murine splenic mature b cells. 3) we then studied the phenotype of carabin kd (shrna expressing) a20 b cells after bcr stimulation. 1) the expression of carabin is significant in murine b cells, with an increase during b cell development, from bone marrow pro/preb to immature, to splenic t1 tot2 b cells and to follicular mature b cells. 2) the kinetic of expression of carabin in bcr stimulated murine mature b cells suggests a fine regulation of carabin expression. 3) bcr simulation, but not lps stimulation, of carabin kd a20 b cells shows an acceleration of ras target erk1/2 phosphorylation, without any for the phosphorylation of mapk jnk, which is not targeted by ras. conclusion: carabin is expressed in murine b cells in a developmental regulated manner, with the highest expression in mature compartment. bcr stimulation leads to a fine regulation of carabin expression in wild-type mature b cells, and to a faster activation of erk1/2 pathway in carabin kd b cells. altogether, these results strongly suggest a role of carabin as a negative regulator of b cell function toll like receptors are pattern recognition receptors, which recognize invariant pathogen associated molecular patterns. toll like receptor 3 (tlr3) binds doublestranded rna, a nucleic acid frequently associated with viral replication. we observed that freshly isolated human cd4 + t cells express tlr3 and respond to the well characterized synthetic tlr3 ligand polyinosinic-polycytidylic acid [poly(i:c)]. the expression of activation markers and cytokine production by cd4 + t cells upon t cell receptor (tcr) stimulation is enhanced in response to co-stimulation via tlr3. tlr3 stimulation on its own had no effect on expression of activation markers and cytokine production. to elicit the molecular basis of a potential cross-talk between tcr and poly(i:c) induced signaling, we used jurkat cells to perform luciferase assays. we observed that costimulation with poly(i:c) in comparison to tcr stimulation alone enhanced nf-kb but not nfat activation in jurkat cells. similarly to jurkat cells, tcr stimulation activated nf-kb in primary cd4 + t cells. this effect was further enhanced by additional poly(i:c) stimulation as shown by real-time-pcr and western blot analysis. on the other hand, we observed that poly(i:c) stimulation on its own activated the transcription factor interferon regulatory factor 3 (irf3) as revealed by realtime-rcr analysis of ifn b and irf7, whose transcription depends on the activity of irf3. combined tcr and poly(i:c) stimulation further enhanced the transcription of these two genes. these results indicate that tlr3 signaling modulates tcr-driven responses and vice versa both in jurkat cells and in freshly isolated cd4 + t cells. this study was supported by dfg spp 1110 "innate immunity" (ka 502/8-3). the initiation of protective t cell responses requires the recognition of mhc-bound peptides from pathogen or tumor antigens by the t cell receptor (tcr). how this signal is transmitted across the t cell membrane to the cytoplasmic signaling motifs is still unknown, and is the focus of this project. in textbooks, the cytoplasmic domains are depicted as flexible chains in the cytoplasm, but biochemical studies show that the cd3e cytoplasmic domain (cd3e cd ) binds to synthetic lipid vesicles that contain acidic phospholipids. this binding is predominantly due to electrostatic interactions between basic residues of cd3e cd and acidic phospholipids. in the cell, acidic phospholipids are enriched in the inner leaflet of the plasma membrane. phosphatidylserine in particular is concentrated on the inner leaflet of the plasma membrane due to active transport mechanisms, explaining how such charge-charge interactions are generated. to study the interaction of the cd3e cd with the membrane in live cells, we have developed a fluorescence resonance transfer (fret) assay which measures the proximity between a fluorescent protein (tfp) attached to the c-terminus of cd3e cd and a fluorescent membrane dye (r18). with this assay, we show that the cd3e cd is membrane-bound in resting cells and that binding is abrogated by introduction of mutations that disrupt lipid binding in the biochemical assay. additionally, in vitro analysis confirm functional domains for cd3e cd lipid binding and conformational change. finally, nmr spectroscopy analysis reveals key features in membrane binding dynamics of cd3e cd to lipid bicells. membrane binding by the cd3e cd could thus be subject to dynamic regulation during the engagement of the tcr and further activation of the t cell. m. xydia 1 , y. ge 1 , u. quitsch 1 , p. beckhove 1 1 german cancer research center (dkfz), heidelberg, germany in peripheral tissues and the factors affecting their proliferation. cd4+ t cell help is believed to contribute to optimal cd8+ memory expansion via cd40l on cd4+ t cells binding cd40 on dendritic cells. however, a few reports suggest that cd40l-cd40 engagement may mediate direct cell-cell contacts between cd4+ and cd8+ t cells. in this study, we investigated the importance of cd4-cd8 co-operation and cd40l-cd40 interactions for t em proliferation. methods: we isolated human cd4+ and cd8+ t em cells from peripheral blood of healthy donors by facs or macs sorting. separated or mixed cd4+ and cd8+ populations were activated in vitro using anti-cd3/cd28 beads. proliferation was measured by [ 3 h]-thymidine incorporation, in some experiments after irradiation of one t em subset and/or incubation with blocking mabs against cd40 or cd40l. furthermore, facs staining was used to assess cell-surface markers. statistical comparison was performed by student's t test. results: upon activation mixed t em populations showed a highly better proliferative response than separated cd4+ or cd8+ t em cells, demonstrating that optimal t em expansion requires direct cd4-cd8 interactions. surprisingly, not only cd8+ but also cd4+ t em cells proliferated much more in mixed populations compared to the separated ones, indicating that optimal cd4+ t em proliferation depends on signals from cd8+ t em cells. activation induced the expression of cd40 on both populations and cd40l on subsets of cd4+ and cd8+ t cells. blocking of cd40l on cd8+ t em cells impaired significantly cd4+ t em proliferation, which confirms that the improved expansive potential of cd4+ t em cells in mixed populations depends on cd40l co-stimulation by the cd8 t em subset. conclusions: our data demonstrate for the first time that activated cd8+ t em cells deliver help to the cd4+ t em subset via cd40l-cd40 signalling and may play an important role for cd4+ t em expansion upon stimulation. the t cell surface glycoprotein cd5, a member of the scavenger receptor cysteine-rich (srcr) family of proteins, targets to the immunological synapse upon t cell binding to antigen presenting cells (apc). however, it has not been established whether this translocation is due to the binding of a ligand expressed in the apc, or to intracellular interactions with signaling molecules or components of the cytoskeleton, that may control cd5 localization upon t cell:apc conjugation. we have questioned which domains of cd5 mediate the localization within the is, and for this we have expressed cd5 mutants as gfp fusion proteins in human t lymphocytes. we have also used jurkat cell lines expressing different cd5 mutants. t cells were incubated with superantigen-loaded raji b cells, and following the establishment of stable interactions between the cells, we analyzed the localization of cd5 by immunofluorescence and confocal microscopy. interestingly, our results show that the translocation of cd5 depends on sequences within the cytoplasmic domain, as a cd5 deletion mutant lacking most of the cytoplasmic tail, cd5.k384 stop , is randomly distributed through the whole cellular surface, even in sustained t-apc interactions. the cytoplasmic domain relevant to cd5 translocation was mapped within amino acids glu 418 and his 449 since the cd5.h449 stop mutant, just short of 22 aa is still able to translocate to the is, whereas cd5.e418 stop , that lacks 2 important tyrosine residues, is no longer transported to the is upon t cell: apc interactions. although these studies do not exclude a role for the extracellular domain binding to an elusive apc-expressed ligand, they suggest that a major mechanism of regulation of cd5 translocation is dependent on molecular association of a short stretch of its cytoplasmic region (glu 418 -his 449 ) to intracellular signaling effectors. however, in hodgkin's lymphoma (hl) ebna-2 is missing, but lmp-1 is still expressed. using a hl derived cell line, we have shown that the cytokine il-4 can induce lmp-1 expression in vitro and can replace ebna-2. we have investigated the molecular events for this mechanism. stat proteins bind to the palindromic ttc(n) x gaa sequence, where × is 2, 3 or 4. a high affinity stat6 binding site is spaced by 4 nucleotides. we found three potential stat binding sites in the lmp-1 promoter, which we named lrs, tr and edl1. they were spaced by 4, 3 and 2 nucleotides, respectively. electrophoretic mobility shift (emsa) experiments were performed with nuclear extracts prepared from il-4-treated or non-treated kmh2-ebv cells. dna binding activity was analyzed using a double stranded oligonucleotide corresponding to the germline (gl) epsilon promoter, which is known to contain a high affinity stat6 binding site, or lrs-stat6. a stat6 complex binding to the gl-epsilon promoter and lrs-stat6 was induced by il-4. the specificity of the stat6 complex was shown by supershift experiments with anti-stat6, but not anti-stat5 antibodies. when gl-epsilon or lrs-stat6 was used as cold competitors in a 100-fold excess, both unlabelled probes could compete out the labeled probe, providing evidence that the lrs-stat6 contains a functional stat6 binding site. oligonucleotides, corresponding to lrs in which the stat6 site had been mutated, could not compete for stat6 binding. interestingly, the unlabeled lrs-tr with 3 nucleotides as spacer could also function as competitor. however, when ttc/gaa palindrom was spaced by 2 nucleotides (lrs-edl1), it could not compete. thus, expression the transforming protein lmp-1 can be induced directly by the t cell derived cytokine il-4 in a stat6 dependent manner. it is likely that this mechanism operates in vivo as well and determines expression of the ebv encoded protein lmp-1 and thus the pathogenesis of ebv carrying hls. established knockout/ knockin mice with a fasl deletion mutant that lacks the intracellular portion (fasl ¿ intra). co-culture experiments confirmed that the truncated fasl protein is still capable of inducing apoptosis in fas-sensitive cells. preliminary immune histochemistry data suggest that, in contrast to published data, the absence of the intracellular fasl domain does not alter the intracellular fasl localization in activated t cells. we are currently investigating signalling and proliferative capacity of b-and t-cells derived from homozygous fasl ¿ intra mice. our data point to a rather inhibitory role of fasl reverse signaling during immune responses. during an immune response numerous receptor-mediated signals delivered to t cells direct their proliferation, survival and differentiation. we are using a quantitative model and in vitro methods to assess the "calculus", or decision-making algorithms t cells use to process these multiple signals. previous experiments with ot-i cd8 t cells revealed that tcr affinity regulated both the frequency of cells responding and the average time taken for cells to reach their first division (ji 2007 (ji . 179:2250 . furthermore, affinity was the sole regulator of the rate of cell death in subsequent divisions. here we examine the same question for cd4 t cells. again we find that lower affinity peptides stimulate t cells to divide rapidly, however, a high proportion of cells die within each division round, revealing an important potential mechanism for affinity maturation and selection of dominant clones over time. in contrast varying the number of dendritic cells used to stimulate cd4+ t cells primarily affect the proportion of cd4+ t cells going into division rather than affecting division time or cell death in subsequent divisions. currently we are using these quantitative methods to measure the effect of cytokines and co-stimulatory molecules cd40, cd80 and cd86 on parameters of cd4+ t cell proliferation to inform quantitative models of the immune response under different conditions. our goal is to develop quantitative models of t cell behaviour that can accommodate information at the molecular, cellular and population level. interaction between cd40, a member of the tumor necrosis factor receptor superfamily constitutively expressed on antigen-presenting cell as b cells, and cd40l, a member of the tumor necrosis factor family transiently expressed on activated t cells, are essential for the development of humoral adaptative immune response. various studies have shown that dual stimulation of b cell through antigen binding on bcr and cd40 leads to an enhancement of ig and cytokine production. the current dogma postulates that these 2 signals are necessary and sufficient to drive naive b cell proliferation and differentiation to ig secreting plasma cells. however, recent evidence suggests that the innate immune responses could regulate humoral adaptive immune response. indeed, b cells can be activated through engagement of a variety of innate immune receptors, including toll-like receptors (tlrs). soluble cd40l is unable to induce murine b cell proliferation. however, we and others have shown that recombinant mouse cd40l (rmcd40l) can increase proliferation induced by tlr3 (poly ic) and tlr4 (lps) agonists. by contrast, we never observed any synergy between rmcd40l and tlr1/2 (pam3csk4) or tlr2/6 (pam2csk4) agonists. to go further in the study of cd40l/tlr agonist synergetic effect, we have developed trimeric synthetic molecule to mimick cd40l, named mini-cd40ls, based on a c 3 -symmetry core holding cd40-binding motif lys-gly-tyr-tyr. in surface plasmon resonance experiments, mini-cd40ls bind to immobilized human cd40 and compete with the binding of cd40l homotrimers and diplayed effector functions that matched those of the much larger recombinant cd40l homotrimers as maturation of mouse dendritic cells and activation of in vivo immune response in a mouse model of trypanosoma cruzi infection. as soluble cd40l, mini-cd40ls synergize tlr4 (lps), tlr3 (poly ic) and tlr7/8 (r848) agonist-induced murine b cell proliferation but no synergy was observed between mini-cd40ls and tlr 1/2 (pam3csk4), tlr 2/6 (pam2csk4) and tlr9 (odn 2395) agonists. synergy between cd40l and tlr agonist provide the ground to use such a combination as adjuvant in vaccination strategy. however, to reach this goal, evaluation of cd40l/tlr combinations on murine and human b cell activation and differentiation in antibody producing cells are under investigation. interaction of naïve cd8 + t cells with immature dendritic cells (idc) expressing self-peptides can result in their abortive activation (aa), which leads to the induction of cd8 + t cell tolerance. we have defined a phenotypic profile for cd8 + t cells undergoing such aa. these cells undergo limited proliferation which is associated with lack of ifn-g production, low cell surface expression of cd25 and cd69, and high levels of expression of cd62l and ly6c. whereas, cd8 + t cells undergoing productive activation (pa), following encounter with mature dc, form effector ctl which is evidenced by extensive t cell proliferation, high levels of ifn-g, cd25 and cd69, and loss of cd62l and ly6c expression. ly6c is a gpi-anchored cell surface glycoprotein expressed on cells of hemopoietic origin: however, its role in peripheral tolerance induction is not understood. in this study, we show that mab-blocking of ly6c in vivo and in vitro results in pa rather than aa. we hypothesize that the interaction of ly6c, expressed on naïve cd8 + t cells, with its ligand on idcs, may be vital in controlling the induction of peripheral tolerance amongst self-reactive cd8 + t cells. objectives: organophosphorus compounds (opcs) are commonly used in the manufacture of insecticides and pesticides. exposure to opcs is associated with neurological toxicity but the effect on the immune system remains ill-defined. in this study, we used a subchronic exposure model to investigate the effect of the organophosphorus compound, paraoxon, on the murine immune system. methods: balb/c mice were injected i. p. daily with saline (control group) or paraoxon (experimental group) for 3 weeks. during the treatment, animals were weighed and blood was collected weekly for determination of acetylcholinesterase activity in red blood cells. at the end of treatment, mice were sacrificed and spleen cells analyzed by flow cytometry. spleen cells were also cultured in the presence or absence of mitogens and supernatants were analyzed for cytokine content by elisa. for in vivo survival studies, mice were treated as described above and then orally infected with a virulent strain of s. typhimurium. animal survival was followed for up to 60 days after infection. results: daily injection of paraoxon induced g 50 % reduction in acetylcholinesterase activity by the end of the first week of treatment, a level which was thereafter maintained during the remaining 2 weeks of treatment. mice exposed to paraoxon exhibited g 80 % reduction in the rate of body weight gain over the treatment period in comparison with control group. at the end of treatment, ex vivo analysis of spleen cellularity and function revealed no significant differences between control and experimental groups. to analyze the status of the immune system in vivo, mice were infected with a lethal dose of a pathogenic strain of s. typhimurium and followed for survival. unexpectedly, paraoxon-treated mice exhibited a significant degree of resistance with 80 % of mice surviving the infection compared to 20 % in control group. protection in paraoxon-treated group was dependent on the reduced acetylcholinesterase activity as it was abrogated by coadministration of a reactivator of cholinesterase. conclusion: our data demonstrate that a reduction in the level of acetyl cholinesterase rendered mice more resistant to a virulent infection. this suggests a hitherto novel function of the neurotransmitter acetycholine in modulating the immune response to infection. t cell-dependent (td) and t cell-independent (ti) igg autoantibodies have been described in the context of the autoimmune disease systemic lupus erythematosus (sle). however, their different roles in autoimmunity are unknown. here we show that ti antigens induce anti-inflammatory igg antibodies and protect from antigen-specific immune pathology. administration of antigen-specific anti-inflammatory igg antibodies was sufficient to mediate this effect independent of the igg inhibitory receptor fcgammariib. ti but not td igg autoantibodies were further associated with inhibition of pro-inflammatory th1 and th17 cells and disease in mice deficient for fcgammariib, a spontaneous model for sle. the data suggest a novel immune regulatory function for ti immune responses through the generation of anti-inflammatory igg antibodies. objective: class i phosphoinositide 3-kinases (pi3k) constitute a family of enzymes that generate 3-phosphorylated polyphosphoinositides at the cell membrane after stimulation of protein tyrosine (tyr) kinase-associated receptors or g protein-coupled receptors (gpcr). the class i pi3k are divided into two types: class ia p85/p110 heterodimers, which are activated by tyr kinases, and the class ib p110g (p110gamma) isoform, which is activated by gpcr. although the t cell receptor (tcr) is a tyr kinase-associated receptor, previous studies showed that p110g deletion affects tcr-induced t cell stimulation. mice lacking p110g show a partial defect in t cell differentiation, activation and survival. p110g participates in signaling pathways that regulate pre-tcr dependent differentiation and cd4+/cd8+ t cell lineage commitment. in the mrl/lpr mouse model of systemic lupus erythematosus, administration of a pi3kg-specific inhibitor causes a reduction in the number of cd4+ memory t cells that mediate renal injury. similarly, pi3kg deletion in p65 pi3k transgenic mice also reduces the numbers of cd4+ memory t cells. there is therefore evidence that pi3kg has an important function in tcr-mediated t cell activation, although the mechanism by which pi3kg regulates this process is not well understood. we studied the specific role of p110g in t cell activation. methods: we studied whether the tcr activates p110g and the consequences of interfering with p110g expression or function on t cell activation. results: we found that after tcr engagement, p110g interacts with and forms a complex with ga q/11 , lck and zap70. tcr stimulation activates p110g, which affects 3-phosphorylated polyphosphoinositide levels at the immunological synapse. we show that tcr-stimulated p110g controls rac1 activity, f-actin polarization, and the interaction between t cells and antigen-presenting cells (apc). we show that p110g deletion affects the activation of many pathways downstream of tcr crosslinking, as well as the interaction between t cells and apc; these findings could explain the defective activation of p110g-/-t cells. our observations clarify the activation mechanism and mode of action of p110g in the control of t cell activation, confirming a crucial role for p110g in tcr-induced t cell activation. we investigated mechanisms controlling central location of lytic granules and kinetics of their release within immune synapses formed by cytotoxic t lymphocytes (ctl). we show that cytolytic granules in ctl can be delivered to the secretory domain via two different pathways -"short" and "long". the choice between these pathways is regulated by the kinetics of early tcr signaling which depends on the strength of tcr/pmhc/co-receptor interactions. meanwhile, the molecular hardware used to deliver the granules remains the same. we conclude that the difference in temporal and spatial coordination of the two principal events, i. e., granule movement toward microtubule organizing center (mtoc) and the mtoc polarization, accounts for two different pathways of granule delivery to the secretory domain that influence efficiency of ctl cytolytic response. our findings reveal a mechanism of well-documented flexibility in t cell responsiveness that is derived from differential use of the similar set of immune receptors, signaling proteins and intracellular effector molecules. objectives: in addition to specific immune cytokines, lymphocyte activation and immune response are modulated by universal mediators like acetylcholine. nicotine was shown to suppress both cellular and humoral immune responses. previously we found that two nicotinic acetylcholine receptor (nachr) subtypes, a4b2 and a7, expressed in mouse b lymphocytes regulate their development within the bone marrow. the aim of the present study was to evaluate the roles of these two nachrs in b lymphocyte activation. methods: b lymphocytes were magnetically separated from the spleens of c57bl/6j mice. they were stained with fluorescently labeled igm-, cd40-or cd23specific antibodies in the presence/absence of unlabeled nachr subunit-specific antibodies to be examined by flow cytometry. b lymphocyte activation was studied by 3 h-thymidine incorporation upon stimulation with anti-cd40 and nachr-specific agonists or antagonists. the antibody response of mice immunized with cytochrome c with or without a7 nachr antagonist methyllicaconitine (mla) was studied by elisa. results: antibodies against a4 or b2 nachr subunits inhibited binding of igm-and cd23-specific antibodies but facilitated that of cd40-specific antibody. in contrast, antibody against a7 subunit prevented binding of anti-cd40 but not of anti-igm or anti-cd23 suggesting that a7 nachrs are located close to cd40, while a4b2 ones are close to bcr/cd23. consequently, anti-cd40-induced b lymphocyte proliferation was increased by mla much stronger than by a4b2-specific antagonist dihydro-b-erythroidine. it was also increased when cells were incubated with the inhibitor of acetylcholine synthesis hemicholine-3. in contrast, proliferation of b lymphocytes from mice consuming nicotine was significantly weaker than that of control mice. mice co-injected with cytochrome c and mla responded with igm antibodies faster than those injected with cytochrome c alone, while the secondary / igg responses were similar. the cd40-mediated b lymphocyte proliferation, but not the igm-igg switch or memory b cell activation, is negatively controlled by either endogenous acetylcholine or consumed nicotine through a7 nachrs. therefore, acetylcholine may be regarded as an auto/paracrine regulator of lymphocyte activation.this work was supported by philip morris usa inc. and philip morris international. binding of cd4 + t h -lymphocytes to antigen presenting cells or of cd8 + cytotoxic t-lymphocytes (ctl) to their target cells lead to a tight contact between these two cells, called immunological synapse (is). formation of the is induces calcium signaling, rearrangement of the actin cytoskeleton, and the recruitment of various molecules to the is, all of which are crucial for t-cell functions such as cytokine release or target cell killing. objectives: using primary human t-lymphocytes, none of the proteins involved in either calcium influx, cytokine release, actin cytoskeleton rearrangement nor in killing of target cells can be analyzed by knock-out strategies. for testing protein functions, down-regulation by rnai technology is thus an important tool. we used short interfering rnas (sirnas) to analyze the role of proteins involved in calcium influx and proliferation (stim1 and trpc3), and to analyze snare proteins which were shown to accumulate at the is and are good candidates to play a role in cytotoxic granule fusion and exocytosis to kill target cells. methods: to validate down-regulation of different mrnas quantitative rt-pcr was used. down-regulation of proteins was confirmed by immunocytochemistry, western blotting and various functional assays depending on the potential role of the protein of interest (calcium imaging, proliferation, cytokine release, killing assay). results: transfection efficiency of sirnas in t-lymphocytes was about 96 %. down-regulation of stim1 was confirmed by qrt-pcr and by calcium imaging, but only for early time points following activation of cd4 + t h -lymphocytes, probably because of stability problems. to increase stability of sirnas within t-lymphocytes we used modified sirnas published by mantei et al. (eji, 2008) . we show that these sirnas down-regulate various snare proteins in ctls more efficiently than non-modified sirnas. the optimal sirna concentrations for transfection in primary human t-lymphocytes was found to be 300-600 pmol, which is lower than the concentrations reported in other cell types. conclusions: following optimization, down-regulation of mrnas by sirna is a powerful tool to investigate the role of different proteins involved in the activation of t-lymphocytes in primary human cells. chemical modifications increase the lifetime and efficiency of the sirnas in primary human t-lymphocytes. stress-inducible heat shock protein 70 (hsp70) has gained plenty of attention because of its potent adjuvant capability to induce antigen-specific cd8 + cytotoxic t-lymphocyte (ctl) and cd4 + t-helper cell (th1) responses. in this study, we investigated the behavior of t-cell subsets stimulated with endotoxin-free recombinant hsp70 with respect to proliferation, cytokine expression, cytotoxicity against allogeneic b-lymphoblastoid cell line (b-lcl) and k562 cells as well as targetindependent cytotoxicity. cd4 + cells exhibited a strong increase in proliferation after stimulation with hsp70, with rates of up to 29 %. in the presence of target cells, a 35-fold up-regulation of granzyme b mrna was observed after stimulation of cd4 + t-helper cells with hsp70 in combination with il-7, -12 and -15. the target cell-independent secretion of granzyme b by cd4 + cells was greatly augmented after stimulation with hsp70 plus il-2 or il-7, -12 and -15. in this study, we have shown that hsp70 is capable of inducing a cytotoxic response of t-helper cells in the absence of lps or any other pamps. the granzyme b secretion and the cytolytic activity of cd4 + t cells is induced in a target-independent way, whereas the cytotoxic activity of cd3 + and cd8 + t cells can be further enhanced in the presence of the target cells. our data provide novel insights into the role of extracellular hsp70 on t-cell immune response concerning the induction of target-independent t-helper cell cytotoxicity. jun n-terminal kinases (jnk) have been shown to play controversial role in regulation of cell fate. cd40, which is responsible for germinal centre formation in lymph nodes, trigger jnk activation. the role of other b cell co-receptor molecules that may be involved in antigen-driven differentiation were not clarified. the aim of this study was to find out whether cd150 receptor contributes to jnk activation in mature human b cells. protein expression and phosphorylation were studied by western blot analysis. protein associations were evaluated by immunoprecipitation and gst-pull down assays. hpk1 overexpression in a model system was achieved by transfection. pjnk1/2 expression in primary hrs cells was assessed by immunohistochemistry. ligation of cd150 on resting (dense) and activated (buoyant) human tonsillar b cells lead to jnk2, but not jnk1 activation. cd40 ligation on primary tonsillar b cells also resulted in jnk2 activation. however, bcr crosslinking did not affect the level of jnk1/2 phosphorylation. cd150-mediated jnk2 activation was independent from sh2d1a/sap adaptor protein expression, and was demonstrated for all studied b-lymphoblastoid, burkitt's lymphoma and hodgkin's lymphoma (hl) cell lines of b cell origin. we were searching for serine/threonine kinase that could coprecipitate with cd150 and link this receptor with jnk pathway. using immunoprecipitation and gst-pull down assays we found that hematopoietic progenitor kinase 1 (hpk1) was associated with cd150 in primary b cells as well as in b cell lines. cd150-hpk1 association was independent from cd150 tyrosine phosphorylation and sh2d1a expression. overexpression of hpk1 in a model system significantly enhanced cd150mediated jnk2 phosphorylation. it is known that tnf family receptors such as cd30, cd40, rank trigger survival signals in hrs cells. we observed the expression of pjnk1/2 in hrs cells of primary classical hl. cd150 could be involved in sustained jnk2 activation in primary hrs cells, and this may reflect the role of cd150 receptor as well as other receptors in the regulation of hrs survival. overall, it was shown that jnk2 is activated via cd150 in primary b cells and in all studied cell lines of b cell origin. serine-threonine kinase hpk1 is involved in cd150-mediated jnk2 activation. objectives: cd5 has been shown to act as a negative regulator of tcr signaling during thymocyte development. however, the molecular mechanisms involved in this process remain elusive. one potential key molecule involved in the downmodulation of tcr signaling is c-cbl, a ubiquitin ligase that physically associates with cd5 upon tcr crosslinking in thymocytes. the objective of this study was to determine which sequences within the cytoplasmic tail of cd5 are involved in c-cbl phosphorylation and association. methods: el4 thymoma cell line was stably transfected with wild-type human cd5 or hcd5 cytoplasmic tail mutants: cd5.k384stop (maintaining only a pseudo itim); cd5.h449stop (lacking the distal s and y in the carboxy-terminal region); cd5. ¿ e418-l444stop (lacking the pseudo-itam, putative site for c-cbl association). phosphorylaton of y700 in c-cbl was analyzed, which is required for vav recruitment and c-cbl dependent degradation by the proteasome. stable clones were stimulated with anti-murine cd3 in combination or not with anti-human cd5 biotinylated antibodies and phosphorylation of c-cbl was detected by flow cytometry after intracellular staining anti-phospho c-cbl (py700) antibody. murine thymocytes were used as positive control. data was analyzed using flowjo software. unpaired two-tailed student t test was used to calculate statistical significance (p x 0.05). in murine thymocytes, co-crosslinking of cd3 with cd5 induces an increase in c-cbl phosphorylation compared to cd3 alone. analysis of the el-4 transfectants showed that mutants cd5.k384stop and cd5.h449stop lost the ability to costimulate cd3-mediated phosphorylation of c-cbl. in contrast, cd5. ¿ e418-l444stop mutant, was able to efficiently costimulate cd3-mediated c-cbl phosphorylation, similarly to the hcd5wt. our results indicate that the absence of the pseudo itam in cd5 does not interfere with c-cbl phosphorylation in response to cd3 plus cd5 crosslinkiing on the other hand, sequences present in the carboxy-terminal region of cd5 appear to be important for c-cbl phosphorylation. therefore, c-cbl phosphorylation might not require physical association with the cd5 cytosplasmic tail, but rather, may indirectly associate with cd5 through the interaction with other sh2-sh3 domain-containing molecules, that may be recruited to cd5 through its carboxy-terminal region. l. kolly 1 , s. narayan 1 , j. tschopp 2 , a. so 1 , n. busso 1 1 chuv, rheumatology, lausanne, switzerland, 2 unil, biochemistry, epalinges, switzerland apoptosis-associated speck-like protein containing a caspase recruitment domain (asc) is an adaptor protein that is essential for the recruitment of pro-capase-1 into inflammasomes and thus plays a key role in regulating capase-1-dependent il-1b and il-18 production. despite recent evidence implicating asc in adaptive immunity against infections, hyperresponsiveness and vaccination, the cellular and molecular basis for asc involvement in adaptive immune responses remains largely unexplored. to investigate the impact of asc on t cell activation and subsequent effector function. asc +/+ and asc -/-t cells or purified cd4 + and cd8 + t cells were activated in vitro through anti-cd3 stimulation and their proliferative potential and cytokine profiles characterized. proliferative responses by asc -/-t cells were significantly inhibited two-fold following tcr-cd3 ligation when compared to asc +/+ t cells. furthermore, cytokine analysis revealed that anti-cd3 activated asc -/-t cells predominantly displayed a more th 2 phenotype, producing more il-10 (199 vs. 692 pg/ml; asc +/+ vs. asc -/-t cells respectively; p=0.0074) and less ifn-g (15,831 vs. 6 ,921 pg/ml; asc +/+ vs. asc -/-t cells respectively; p = 0.0021). when asc +/+ and asc -/-t cells were purified into cd4 + and cd8 + t cell fractions and activated individually using anti-cd3, no inhibition in proliferation was observed amongst activated asc -/-cd4 + and cd8 + t cells. interestingly, the activated asc -/-cd4 + t cell fraction produced significantly more il-10 when compared to activated asc -/-cd8 + t cells and asc +/+ cd4 + and cd8 + t cells (asc -/-cd4 + t cells = 380 pg/ml il-10; asc -/-cd8 + t cells = undetectable il-10; asc +/+ cd4 + t cells = 11 pg/ml il-10; asc +/+ cd8 + t cells = undetectable il-10). cd4 + and cd8 + t cell mixing experiments revealed that asc -/-cd4 + t cells are able to inhibit the proliferative ability of asc -/-cd8 + t cells, asc +/+ cd4 + and cd8 + t cells in vitro and that this suppression appears to be mediated by a soluble factor secreted by activated asc -/-cd4 + t cells. collectively, these results demonstrate that the absence of asc drives cd4 + t cells towards a suppressor cell phenotype, suggesting that asc might play an important role in determining the fate of cd4 + t cells. various members of the eicosanoid family derived from arachidonic acid participate in inflammatory reactions and may act as potent regulators of the immune response. in particular, e-series prostaglandins, pge 1 and pge 2 suppress some t-cell functions including proliferation, activation and cytokine production. pge 2 signals through four types of gpcrs called the ep receptors. at low concentrations, pge 2 is believed to be necessary for t cell function, whereas at higher concentrations, pge 2 inhibits t cell proliferation. these effects are largely governed by various cell specific stimuli and tissue microenvironment. objectives: to delineate, compare and contrast the effects of pge 2 and ep receptor antagonists on t cell activation. methods: flow cytometry, proliferation assays, migration assays. we have observed that pge 2 diminishes expression of early, intermediate and late t cell activation markers. in contrast, pre-treatment of cd4 + t cells with ep receptor antagonists was found to impair cell surface expression of cd71, cd69, cd25 and ox40 but not cd44. suppression of t cell proliferation by pge 2 has already been widely studied. however, blocking ep receptors in cd4 + t cells by the use of ep antagonists prior to activation surprisingly caused a defect in t cell proliferation. migration of cd4 + t cells to the chemokine sdf-1b was also found to be reduced due to pre-treatment with ep antagonists. in order to study the physiological relevance of these findings we studied the trafficking of basal and activated t cells to regional lymph nodes during inflammation in the presence and absence of ep receptor antagonists. this model revealed that the use of ep antagonists causes a reduction in the amount of cd44 + cd4 + adoptively transferred t cells in the regional lymph node following the induction of a local inflammatory response. conclusions: in our study we show for the first time that ep receptors are required for expression of activation markers and activating proliferation in murine cd4 + t cells. our results also suggest that considering pge2-mediated camp signaling in cd4 + t cells, it will be absolutely necessary to distinguish between transient increases, which have potentiating effects, and sustained increases, which have inhibitory effects in t cell activation. objective: our objective was to investigate how ros affect the different stages of t cell activation. because activation is initiated by changes in intracellular calcium concentration, we addressed whether and how ros affect calcium signalling. the experimental results were obtained using a combination of fluorescence microscopy, patch-clamp, t-cell activation assays and molecular biology. results: we show by direct measurement of ros that t-cells are exposed to high concentrations of oxidants when they are in close vicinity of activated phagocytes. the effect of ros on calcium signalling in jurkat t-cells as well as in primary naï ve and effector cd4 + human t-cells was examined. oxidation affects several ca 2+ signalling pathways by altering the activity of ip 3 receptors, trp channels and store operated ca 2+ channels in a concentration dependent manner. interestingly, calcium signalling is differentially affected in naï ve and effector t cells. thiol reducing agents were able to significantly reduce the effects of oxidation implicating thiol oxidation as a major player in the regulation of ca 2+ signalling in t-lymphocytes. cysteins are the main carrier of thiol groups in proteins and we show that orai ion channels contain reactive cysteine groups that mediate ros effects on the calcium influx pathway. conclusion: ros regulate the calcium dependent t-cell activation in a complex way, affecting all three major calcium signalling pathways. by mutational analyses of the orai proteins, we are able to pinpoint molecular targets of regulation. the activation of t cells during an immune response is a crucial but tightly regulated event. to make the grade, the t cells upregulate costimulatory but also inhibitory receptors upon antigen recognition. this enables the t cell to be stimulated for proliferation to keep pace with pathogens infection, but also to become dampened upon successful defense against the pathogens via negative feedback mechanisms. in this study we present data of the signaling mechanisms underlying the potent t cell inhibitory receptor cd147 (emmprin, basigin) , a member of the ig-family. previous studies reported that lymphocytes from cd147 knockout mouse possess enhanced mixed lymphocyte reactions and cd147 monoclonal antibodies can interfere with t cell activation. these observations already pointed to a negative crosstalk of cd147 signals with the t cell antigen receptor or co-stimulatory signals. consistent with these studies, we found that rna interference (rnai) with cd147 in jurkat t cells augments the secretion of the t cell growth-factor interleukin-2 (il-2) upon t cell activation. this up-regulation is at least partially due to an increased activity of the nuclear factor of activated t cells (nfat), which resulted in an enhanced il-2 promoter activity. by reconstituting the rnai-mediated knockdown with various truncated rnai-resistant forms of cd147, we identified the immunomodulatory sub-domain of cd147. supported by the gen-au program of the austrian federal ministry of science and research. mirnas play a critical role in the control of hematopoiesis. the goal of this project is to determine whether mirnas function also during the antigen-induced activation of mature b lymphocytes. therefore, we determined mirna profiles in primary splenic b cells before and after polyclonal activation with either lps (simulates t cell-independent activation) or a combination of anti-igm, anti-cd40 and il4 (simulates t cell-dependent activation). microarray assays identified about 104 mirnas in unstimulated b cells. 35 of these were downregulated and one was upregulated upon stimulation. in silico analyses with various mirna target prediction programs revealed an interesting and promising set of transcripts whose translation/stability could be controlled by mirnas during the antigen-induced activation phase of mature b cells. among these targets are bcr signalling molecules and transcription factors that control proliferation, igh class switch as well as differentiation in antibody-secreting plasma cells. one of these transcripts codes for the interferon regulatory factor-4 (irf-4). the graded expression of this important transcription factor has been shown to coordinate isotype switching with plasma cell differentiation. first results indicate that the expression kinetic of irf-4 transcripts differs from that observed for irf-4 protein abundance after b cell stimulation. further analysis identified the irf-4 transcript as a target whose expression is obviously fine-tuned by a mirna upon antigen stimulation. we are in the process to biochemically verify potential targets for each of the differentially regulated mirnas and determine the effect of ectopic and retrovirally mediated expression of mirnas on b cell differentiation. the work was in part supported by the izkf erlangen, the dfg graduiertenkolleg gk592 and the dfg forschergruppe for832. objective: transforming growth factor-b (tgf-b) signals through type i (tgfbri) and type ii (tgfbrii) tgf-b receptors and receptor regulated smad proteins. tgf-b exerts predominantly anti-proliferative and pro-apoptotic effects which are frequently lost in cancer. the mechanisms of resistance against tgf-b have not been fully elucidated. our aim is to describe how b cell lymphoma cells respond to tgf-b compared to normal peripheral b cells, to create an overview of the different signaling pathways involved, and to characterize the mechanisms behind the loss of sensitivity to tgf-b. methods: proliferation assays were performed on 11 different b-cell lymphoma cell lines and normal peripheral b cells to screen for tgf-b-induced effects. western immunoblotting analysis was conducted to characterize protein expression and phosphorylation related to tgf-b signaling pathways. facs analysis was used to measure tgf-b receptor surface levels. cells were treated with demethylating agents to examine changes in gene expression levels. s. manthey 1 , f. hauck 2 , i. berberich 1 , f. berberich-siebelt 2 , gk 520 -immunomodulation 1 institute for virology and immunobiology, university of wuerzburg, würzburg, germany, 2 university of wuerzburg, department of molecular pathology, würzburg, germany the transcription factor ccaat/enhancer-binding protein b (c/ebpb) can not act only as a transcriptional activator but also as a transcriptional repressor. in murine cd4+ t lymphocytes, the transcription factor is predominantly expressed in t helper 2 (th2) compared to t helper 1 (th1) cells. in contrast, by binding to the c-myc promoter(s), c/ebpb represses c-myc expression thereby arresting t cells in the g1 phase of the cell cycle. both, transactivation and repression depend on the n-terminal transactivation domain of c/ebpb. blimp-1 encoded by prdm1 is a transcription factor necessary for terminal differentiation of b cells to plasma cells. furthermore, blimp-1 is expressed in differentiated effector t cells where it is higher in th2 than th1 cells. the regulation of the blimp1 expression is not fully understood. interestingly, we found that c/ebpb can bind to the prdm1 promoter and activates blimp-1 expression in t cells. as c/ebpb is also expressed in b cells, we hypothesize that this transcription factor might as well influence the expression of blimp-1 in b cells. so far, we were able to show a similar expression profile of c/ebpb and blimp-1 in b cells using cre recombinase. moreover we found a new putative blimp-1 isoform lacking exon 2. currently, we analyze the expression of c/ebpb and blimp-1 in primary b cells and b cell lines after various stimulations. to get more insights into the function of c/ebpb in b and t cells, we are generating mice carrying a b as well as a t cell-specific deletion of c/ebpb. engagement of antigen receptors on lymphocytes leads to rapid increases in intracellular free calcium concentrations via phosphorylation of phospholipase c gamma (plcy) and plays an important role in activation of cells. by screening 53 cvid patients with a flow cytometric assay we demonstrate that calcium flux is significantly reduced in b and t cells isolated from the peripheral blood of patients in the group ia of the freiburg classification as compared to non-ia patients and healthy donors (hd). ia patients are characterized by the expansion of an unusual cd21low b cell population in which calcium mobilization is strikingly lower than in other b cell subsets. common subpopulations like naï ve and mz-like b cells as well as cd4 + t cells but not transitional b cells or cd8 + t cells also revealed significantly decreased calcium peaks. the cytometric data correspond to a semiquantitative rt-pcr assay and functional data showing reduced induction of the calcium dependent macrophage inflammatory protein-1a (mip-1a), and abrogated activation and proliferation, respectively. preliminary data on b cell receptor (bcr) mediated phosphorylation of plcy2 revealed constitutively high background levels in cd21low b cells of ia patients. since phosphorylation in the other b cell populations as well as calcium flux upon ionomycin were the same for patients and healthy donors, we postulate an abrogated amplification or altered inhibitory pathway targeting the signalling events downstream of plcy and upstream of internal store release, thus resulting in defective calcium signalling. the underlying mechanism yet remains to be elucidated and is part of our work in the future. c. balas 1 , v. courtois 1 , k. de luca 1 , r. sodoyer 1 1 sanofi pasteur, marcy l'etoile, france the presence and relative abundance of cytokines at different stages of infection is relatively well documented, but their involvement in immune status, pathogenesis or disease progression is still unclear. a potential explanation to the difficult interpretation of the results obtained might be related to the intrinsic weakness of the analytical techniques. for instance monitoring of the expression level of cytokines, such as il-2, il-4 or il-6 could lead to misinterpretation if molecular isoforms are not detected by antibodies currently used to measure them. the analysis of the human transcriptome is a way to access the subset of genes involved in the immune response upon infection by various pathogens. such an analysis might be completed and enriched by the analysis of the relative expression of some cytokine splice variants. methods: genetic tools (primers and qpcr probes) capable of discriminating and quantifying alternatively spliced messenger rnas from il-2, il-4 and il-6. furthermore, the recognition by several commercial antibodies of the different cytokine isoforms (expressed as recombinant proteins) has been investigated. the genetic tools have been validated on in vitro models as well as on biological samples (please refer to the abstract no a-136-0033-01835). conclusion: implication of such kind of analysis in diagnostic application and disease progression survey will be discussed. in a different context, the same kind of analysis could be applied to the monitoring of the immune response upon vaccination or more generally for new antigens or adjuvant screening. parasitic helminths affect about one third of the world population. therefore the mechanisms, which are involved in the persistence or the expulsion of the parasite, are of special interest. from other parasitic infections it is known, that the regulatory receptor cytotoxic t lymphocyte antigen-4 (ctla-4) plays a crucial role during infections. here, we use the strongyloides ratti infection of mice as an experimental system to investigate the role of ctla-4 during nematode infections. we employed a quantitative real-time pcr (qtpcr) analysis to quantify the migrating larvae (il3) in the tissue and the released eggs and first stage larvae (l1) in the feaces. the cytokine response of lymphocytes, prepared from the spleen and the mesenteric lymphnodes (mln) upon stimulation with polyclonal a-cd3 and s. ratti antigen was determined. additionally the humoral response was analysed in the primary and the secondary infection. to investigate the role of ctla-4 during the infection, a neutralysing antibody (a-ctla-4; 4f10) was administered intraperitoneally (300 mg) two hours before subcutaneous infection with s. ratti il3. the in vivo neutralisation of ctla-4-signalling by applying a-ctla-4 during s. ratti infection led to an altered cytokine response, compared to infected mice treated with a control antibody. we detected an increase in th2 cytokines, such as il-4 and il-5 and a reduction of the proinflammatory cytokines ifn-g and il-17. the investigation of the humoral response showed a remarked increase of the igg1-titer in the serum during secondary infection in mice that had been treated with a-ctla-4 during primary infection. furthermore, the blockade of ctla-4 resulted in a diminished worm burden as indicated by reduced release of l1 in the faeces. these results suggest that the blockade of ctla-4 during s. ratti infection induces an activation of the appropriate effectors of the immune system that are beneficial for the host defence. in particular the transition of the t cell cytokine profile towards a th2 response supports this hypothesis and might be the reason for the reduced worm output in the primary infection. the strong increase of igg1 during secondary infection also reflects the induction of a potent th2 response. objectives: cd36 is a class b scavenger receptor, which has been shown to be involved in the pathogenesis of atherosclerosis as well as in the clearance of apoptotic cells by macrophages. this clearance is important in regulating the immune system to avoid autoimmune reactions, as seen in systemic lupus erythematosus (sle). it was recently described that cd36 is highly expressed also on the marginal zone b cell subtype. we therefore set out to investigate the role of cd36 on the regulation of b cell in the setting of apoptotic cell clearance and autoimmune activation. we used a mouse model for sle where apoptotic cells were injected repeatedly in order to study the auto-reactive antibody response that follows. elisa was used to measure antibody levels and flow cytometry to study cell activation as well as cd36 expression. cd36 knock-out (ko) and wild type mice were used. results: preliminary in vivo data show a tendency for a higher antibody response towards ds-dna and the common self-antigen pc in cd36 ko compared to heterozygous mice. since reduced levels of cd36 are expressed in heterozygous mice we are currently repeating this experiment using wild type mice as controls for comparison. in support of the in vivo findings, the immunosuppressive effect of injected apoptotic cells seen in wild type mice after in vitro stimulation of splenocytes with lps is gone in cd36 ko mice. after one injection of apoptotic cells, cd36 ko b cells are activated while wild type b cells are not. after four injections a break of tolerance is seen and apoptotic cells do no longer have an immunosuppressive effect and we show that cd36 on b-cells are involved in setting this threshold. conclusion: our data suggest that cd36 is involved in the early regulation of b cell response towards apoptotic cells and production of autoreactive antibodies. it does so by being involved in regulation of the tolerance effect exerted by apoptotic cells. successful t cell immunity requires lymphocytes to be at the right time at the right place. the co-receptor cd152 acts as a major check-point of immune responses, but the mechanism by which cd152 controls peripheral t cell responses is unknown. the consequences of cd152 signaling on murine th cell migration were analyzed using chemotaxis assays in vitro and radioactive cell tracking in vivo. the genetic and serological inactivation of cd152 in th1 cells reduced migration towards ccl4, cxcl12 and ccl19. crosslinking of cd152 together with cd3 and cd28 stimulation on activated th1 cells increased expression of the chemokine receptors ccr5 and ccr7, which in turn enhanced cell migration. sensitive liposome technology reveals that mature dendritic cells but not activated b cells were potent at inducing surface cd152 expression and cd152-mediated migration-enhancing signals. importantly, migration of cd152 positive th1 lymphocytes in in vivo experiments increased, as compared to cd152 negative counterparts, showing that indeed cd152 orchestrates specific migration of selected th1 cells to sites of inflammation and antigenic challenge in vivo. these data show that cd152 signaling does not just silence cells, but selects individual ones for migration. this novel activity of cd152 adds to the already significant role of cd152 in controlling peripheral immune responses by allowing t cells to localize correctly during infection. it also suggests that interference with cd152 signaling provides a tool for altering the cellular composition at sites of inflammation and antigenic challenge. here we analyzed the role of cd152 signaling on the longevity of cd28 null t cells. using a sensitive staining method for cd152, we show that human cd4 + cd28 null and cd8 + cd28 null t cells rapidly express surface cd152. serological inactivation of cd152 using specific fab fragments or blockade of cd152 ligands using ctla4ig in cd4 + cd28 null and cd8 + cd28 null t cells reduces the number of non-apoptotic cells in a fas/fasl-dependent manner. cd152 crosslinking on activated cd28 null cells prevents activation-induced cell death (aicd) as a result of reduced caspase activity. apoptosis protection conferred by cd152 is mediated by pi3'k dependent activation of the kinase akt resulting in enhanced phosphorylation and thereby inhibition of the pro-apoptotic molecule bad. we show that signals triggered by cd152 act directly on activated cd28 null t lymphocytes and, due to its exclusive expression as a receptor for cd80/cd86 on cd28 null t cells, prevention of cd152 mediated signaling is likely a major target mechanism taking place during therapy with ctla4ig. objectives: cd45 is a transmembrane protein tyrosine phosphatase (ptp) expressed in all nucleated leukocytes. it activates src family kinases (sfks) by dephosphorylating inhibitory tyrosines in their c-terminal tails. in cd45 -/mouse t cell signaling and development is severely impaired, while other leukocyte populations seem much less affected. at least in part, it is due to the activity of another transmembrane tyrosine phosphatase cd148 (ptprj, dep-1) which acts as a positive regulator of sfk in cd45 -/-b cells and macrophages and can compensate for cd45 deficiency in these cells. indeed, combined deficiency of cd45 and cd148 in mice results in defective macrophage and b cell signaling and development, a phenotype much more severe than the loss of either protein alone. naïve murine t cells do not express cd148 and its expression is increased only after activation. accordingly, no defects in t cell development and signaling in cd148 -/mice were reported so far. however, in human t cells the role of cd148 may be different since naive human t cells express cd148 at a level comparable to b cells. using cd45 -/-/cd148 -/human t cell line (jurkat-derived js-7 cells) we tested the ability of cd148 to complement cd45 deficiency in t cells. we used retroviral transduction to express human cd45 or cd148 in js-7 cells and tested their ability to reconstitute major signaling pathways. we also employed substrate trapping mutant of cd148 to identify direct substrates of this phosphatase. in agreement with previously published data, defective t cell receptor (tcr) signaling was observed in js-7 cells. expression of wild type cd45 or cd148 in js-7 cells resulted in more rapid calcium mobilization, enhanced tyrosine phosphorylation, and increased cd69 upregulation after tcr cross-linking. moreover, the carboxy-terminal tyrosine of lck, major t cell sfk, was hypophosphorylated in js-7 cells expressing cd148 when compared to control cells. finally, cd148 substrate trapping mutant expressed in js-7 cells interacted with lck in vivo suggesting that lck is a direct substrate of cd148 in js-7 cells. the results suggest a level of redundancy between cd45 and cd148 in human t cells not appreciated so far. during the past decades, great efforts have been made to get insights into the complex process of antigen-induced t cell activation and the underlying signal transduction pathways. the t cell antigen receptor signaling cascade is initiated by phosphorylation of itam-tyrosine residues through the t-cell specific src protein tyrosine kinase family member lck. during t cell activation, lck is supposed to undergo structural changes from a closed inactive to an open active conformation followed by phosphorylation of the itam-motifs. in order to resolve conformational changes of lck in living cells with high spatio-temporal resolution, we designed biochemically active conformational-sensitive förster resonance energy transfer (fret) biosensors using cyan and yellow fluorescent proteins inserted at special positions of the complete kinase backbone. for the live-fret imaging and biochemical assays we complemented lck-deficient jurkat t cells (jcam1.6) with the biosensors. by introducing point mutations affecting the two major regulatory tyrosines tyr 505 and tyr 394 we found a dramatic decrease and increase, respectively, of intramolecular fret efficiency compared to the wild type biosensor. these results correspond to unfolding of the biosensor to its active conformation on the one hand and condensation of the kinase structure to its inactive form on the other hand. thus, our biosensor is able to detect phosphorylation modifications of key residues. however, we could not detect any overall change in fret and thus conformation of membrane-associated lck molecules during t cell activation indicating that other mechanisms, presumably reorganization of localization, underlie lck regulation. furthermore, we observed a contribution of intermolecular fret, which indicated homophilic interaction of lck. indeed, by performing single molecule analysis and native 2d immunoblotting we found lck dimers and higher order oligomers. together, these advanced imaging studies in the live cell context provide a novel picture of the function and regulation of this key kinase in signaling via the t cell antigen receptor. it has been reported that mitochondria accumulate under the immunological synapse (is) in response to tcr (t cell receptor) stimulation. this process seems to be required to allow proper tcr-induced calcium influx in t cells in contact with antigen presenting cells (apcs), because mitochondria can sequester calcium and thus keep crac (ca2+ release-activated ca2+) channels open. however, antigen-induced calcium signaling is very fast, and clearly much faster than mitochondrial translocation toward the is. thus, we speculated that other signals are involved in recruiting the organelles to the contact region between t cells and apcs. we found that the adhesion molecule lfa-1 (leukocyte function-associated antigen 1) induces localization of mitochondria at the is. this process is antigenindependent and is enhanced by the presence of chemokines in the t cell environment. however, tcr triggering stabilizes mitochondria at the synapse and it is important to sustain their recruitment in time. our data suggest that, by recruiting mitochondria to the cell-cell contact region, lfa-1 prepares and facilitates tcr signaling. we are performing experiments to understand the signalling pathways involved in mitochondria translocation at the is. burkitt lymphoma (bl) is a high grade b cell malignancy (non-hodgkin lymphoma (nhl)) derived from germinal center b cells, that harbours a chromosomal translocation juxtaposing the protooncogene myc next to the regulatory elements of one of the immunoglobulin loci. however, the precise contribution of myc to the pathogenesis of this tumour is poorly understood. based on the definition of a distinguishing gene expression signature for the molecular burkitt lymphoma (mbl) with myc as one hallmarking signature gene (hummel et al.2006) we describe a non-viral vector based approach (vockerodt et al. 2008) to express myc in primary human gcb cells from pediatric tonsils. comparative whole genome gene expression profiling was performed in 9 independent preparations. our data reveal a global change in gene expression in lymphoma precursor cells by myc giving new insight into potential changes of the gene expression program of gcb cells on the accidental way to bl in addition as a first step the function of selected signature genes in bl is accomplished. in a representative cell line with a mbl signature and with a non-mbl signature rnai directed inhibition of elements of the cd40 signaling cascade was conducted. after activating this particular signaling cascade (cd40) we analysed respective gene expression profiles of ikks, trafs and mapk deficient cells. based on these different rnai-mediated ge-profiles a comparison between both lymphoma types is performed. first attempts are made to reconstruct the topology of the respective signaling pathway by using the nested effects bioinformatic model, which has been described recently (markowetz et al. 2005 ). a rat thymic epithelial cell (tec) line (r-tnc.1) was established from a long-term tec culture. this line was characterized as a type of rat cortical tec with nursing activity (tnc). very little is known about molecular mechanism of the tnc/thymocyes interaction. in our previous studies we investigated molecular mechanisms involved in the binding and emperiopolesis of resting thymocytes by r-tnc.1 cell line in vitro. it was found that a number of adhesion molecules, such as cd2, cd4, cd8, cd11a, cd18, cd54, cd90 was involved in these processes. objectives: a main goal of this study was to define the adhesion molecules involved in the interaction between r-tnc.1 line and activated thymocytes. methods: experiments was performed on inbred ao rats. monoclonal antibodies (mabs)-mediated modulation of thymocyte binding and emperiopolesis was tested by adhesion and engulfment assay, respectively, using a coculture of cona and il-2 activated syngeneic thymocytes and unstimulated or ifn-g stimulated r-tnc.1 cells. we found that both the adhesion (30 min and 3h) of activated thymoytes were partially blocked by mab to cd2 and cd8 molecules (ifn-g unstimulated and ifn-g stimulated r-tnc.1 cells). early adhesion was inhibited by mab to cd90, abtcr, mhc class i molecule (ifn-g stimulated r-tnc.1 cells) and cd4 molecule (ifn-g unstimulated r-tnc.1 cells). after prolonged incubation, significant inhibition was obtained using anti-mhc class i mab (ifn-g unstimulated r-tnc.1 cells). almost all mabs which were inhibitory in the binding assay were inhibitory in the engulfment assay (6h), namely mab to cd2, cd4, cd8, cd90 molecule (ifn-g unstimulated and ifn-g stimulated r-tnc.1 cells) and mhc calss i and mhc class ii molecule (ifn-g unstimulated r-tnc.1 cells). our results also suggest the involvement of cd11a/cd18 dependent -cd54 independent pathway in adhesion and cd11a/cd18 dependent -cd54 dependent pathway in emperiopolesis. the obtained results imply that adhesion, deadhesion and emperiopolesis of activated thymocytes by r-tnc.1 cell line are tightly regulated processes in which multiple adhesion molecules are involved. the crucial roles of cytokines in shaping t cell responses have been documented in both healthy and disease conditions. interleukin-27 (il-27), a recently described cytokine, has been shown to exhibit both pro-and anti-inflammatory properties. il-27 favours naï ve cd4 t cell differentiation into th1 cells to the detriment of th17 or th2 differentiation. the il-27 receptor (il-27r) is a heterodimer composed of tccr, which confers ligand specificity, and gp130, a signal transducing chain that is utilized by several other cytokines. il-27 has been demonstrated to promote cytotoxic lymphocyte functions of mouse cd8 t cells, but the potential impact of il-27 on human cd8 t cells has not been elucidated. our goal is to investigate the impact of il-27 on human cd8 t cell functions. we used peripheral blood mononuclear cells (pbmc) from healthy donors, either exvivo or after short term in vitro activation to perform our analyses. we first assessed whether the il-27r is detectable on ex-vivo t cells using flow cytometry. we observed a greater proportion of cd8 than cd4 t cells expressing the complete surface il-27r (gp130+tccr). however, we detected high amounts of intracellular tccr in both, cd4 and cd8 t cells, but only polyclonal activation (anti-cd3) of cd8 t cells led to an actual increase of il-27r surface expression. purified cd8 t cells from healthy donors were shortly stimulated in vitro and then analyzed using flow cytometry-based functional assays. il-27 activated stat1 and stat3 signalling with rapid kinetics in both cd8 and cd4 t cells, indicating the capacity of il-27 to signal through these molecules. addition of il-27 to anti-cd3 activated cd8 t cells led to a significant dose dependent increase of proliferation (as measured by cfse-based assay) and ifn-gamma and granzyme b production (determined by intracellular staining). these results demonstrate a pro-inflammatory impact of il-27 on human cd8 t cells. defects in immune regulation could result in the breakdown of immune tolerance leading to development of multiple sclerosis (ms). the pd-1/pd-l1 pathway is associated with production of the immunoregulatory cytokine il-10, the suppression of t lymphocytes proliferation by inhibition of akt phosphorylation (pakt), and the elicitation of apoptosis of antigen-specific cells; an impairment in this pathway could play a pathogenetic role in ms. we analysed by flow-cytometry the surface expression of pd-l1 and pd1, as well as myelin basic protein (mbp)-stimulated il-10 production, pakt inhibition, and apoptosis (annexin v), in 50 ms patients with relapsing-remitting disease. twenty-six patients were diagnosed as being affected by acute disease (ams); 24 had a diagnosis of stable disease (sms). results showed that: 1) pd-l1 -expressing cd14+ and cd19+ cells are reduced in ams compared to sms individuals (p=0.04); and 2) pd1 expression is increased in cd4+ t cells of sms individuals and is comparable on cd8+ t cells of ams and sms patients. this is associated with a significant decrease in il-10 production by mbp-stimulated cd14+ and cd19+ cells of ams patients (p=0.03). additionally, cd8+ anexin v+ (av+) cells were diminished and cd8+ pakt+ cells were higher in ams compared to sms patients, while similar percentages of cd4+av+ and cd4+ pakt+ were observed in both groups of individuals. data herein show that the impairments of the pd-l1/pd-1 pathway seen in ams patients result in a reduced mbp-specific il-10 production by cd14+ and cd19+ cells as well as in a reduced apoptosis (annexin v) and an augmented proliferation (pakt) of mbp-specific cd8+ t. the pd1/pdl1 pathway plays an important role in the pathogenesis of multiple sclerosis. monitoring of the expression of these proteins could be a novel diagnostic tool. anti-4-1bb in cd8 cells. this difference could be due to down regulation of cd28 by activated lymphocytes and possible preferential response of cd8 cells to anti-4-1bb costimulation. moreover, increase in ifn-g concentration in costimulated cultures also may enhance the suppressive function of mscs which again could explain the inability of costimulation in proliferation recovery. likewise, reducing tgf-ß by costimulation is not sufficient to abolish suppressive effect of mscs. in overall, these results suggest that lack of costimulation expression by mscs is not the mechanism of msc suppression and other mechanisms are involved. cytotoxic t lymphocytes (ctls) kill target cells by secretion of cytotoxic components contained in lytic granules at the contact zone between the target cell and the ctl, the immunological synapse (is). t cell receptor (tcr) enrichment at the is is one of the early and key events of is formation. objectives: soluble nsf attachment receptor (snare) proteins are required in almost all fusion events in cells. in the present study we tested if the snare protein syntaxin7 (stx7) is part of the is and whether it serves as a key player of is formation and/or the fusion process itself. methods: pcr-techniques, cell transfection, immunocytochemistry and different microscopic techniques like confocal microscopy and total internal reflection microscopy (tirf) were used on primary human ctls to test the function of stx7. rna interference technique was also used to down regulate stx7 expression in primary human ctls. results: we identified stx7 in ctls by pcr and immunocytochemistry. stx7 accumulates at the is after ctl/target cell contact. when stx7 function was blocked by overexpression of a dominant negative stx7 mutant (deletion of the transmembrane region), functional studies with tirf showed a reduced accumulation and fusion of lytic granules at the is. furthermore, confocal studies showed a loss of tcr accumulation at the ctl/target contact side. conclusion: these results imply that the snare protein stx7 is present at the is and moreover is required for is formation in ctls. the observed block of lytic granule release is probably caused by disturbing an upstream process such as vesicle transport, recycling or sorting. objectives: despite the 20 years history of mouse t h 1 and t h 2 subpopulations, relatively little is known about the differences in their signaling mechanisms and the membrane organization of critical receptors and signal transducing molecules. we have developed mouse t h hybridomas to study these differences between polarized t h cells. the in vitro established hybridomas were first characterized as t h 0, t h 1 or t h 2 phenotypes, based on their cytokine production (il-2, ifng or il-4). a comparative analysis of t-bet, ifng and il-4 mrna levels was also done on quiescent and activated t h hybridomas. in the present study, the ca 2+response, membrane raft expression/organization, k + -and ca 2+ -ion channel expression/function and sensitivity to apoptosis (aicd) were compared in these hybridomas. expressions and molecular localizations were investigated by flow cytometry and confocal microscopy, respectively. ion channnels were functionally analyzed by patch-clamp technique. apoptosis was analyzed using three markers (mitochondrial membrane potential, caspase activation, dna fragmentation) and flow cytometry. results: expression level of plasma membrane rafts/gangliosides (assessed by cholera toxin b-staining) showed the following rank: t h 1 g t h 0 g t h 2, although the membrane cholesterol level (detected with anti-cholesterol ab, ac8) was similar in the three cells. in connection, tcr displayed stronger colocalization with rafts and appeared more polarized in t h 1 cells upon activation than in t h 2 cells. t h 1 cells produced a more sustained calcium response with higher amplitude than t h 2 cells to the same tcr-mediated triggering signal. interestingly, this does not coincide with the expression of cav1.2 and kv1.3 ion channels, major functional determinants of the sustained calcium influx. t h 2 cells expressed the highest levels of these two ion channels. there were also marked differences in their sensitivity to activation induced apoptosis (aicd) as assessed by three different markers of apoptosis. the results suggest that a different membrane compartmentation/organization rather than the differential expressions of certain receptors, ion channels and/or other upstream signaling molecules of these t h hybridomas may be responsible for the observed differences in their functional characteristics. objectives: bone morphogenetic proteins (bmps) belong to the tgf-b superfamily, which plays a central role in controlling cellular processes like proliferation, differentiation, apoptosis and migration. whereas tgf-b is well established as one of the most potent negative regulators of hematopoietic cells, the role of bmps in b lymphoid cells remains more elusive. in this study we investigated the effects of bmps on mature human b-cells. methods: b cells were isolated from peripheral blood of healthy donors using cd19-dynabeads. cd19 + isolated cells were facs sorted into cd19 + cd27naïve b or cd19 + cd27 + memory b cells. dna synthesis was measured by 3h-thymidine incorporation, immunoglobulin (ig) levels in cell supernatants were measured by elisa and phospho-protein levels were measured by western immunoblotting analysis. results: all bmps significantly suppressed anti-igm-induced proliferation of cd19 + cd27naï ve b cells, of which bmp-6 and -7 were most efficient (40 % suppression). similarly, all bmps suppressed cpg-induced proliferation of cd19 + cd27 + memory b cells by 40 -50 %. to induce differentiation, both naï ve and memory b cells were stimulated with cd40l and il-21. this increased the production of igm, iga and igg 10 -100-fold compared to medium control, whereas addition of bmps inhibited the production of all ig classes. all bmps highly induced phosphorylation of smad1/5/8 in cd19 + b cells. the mechanisms for how bmps mediate their inhibitory effects are currently being explored in more detail. conclusion: bmps have prominent inhibitory effects on anti-igm-and cpg-induced proliferation of naive and memory human b cells, respectively. they also suppress cd40l/il-21-induced production of igs in mature human b cells. s. gutenberger 1 , k. warnatz 1 1 university medical centre freiburg, freiburg, germany background: signals through the b cell receptor (bcr) and co-receptors are essential for the survival, differentiation and effector function of b cells. the stimulation of the bcr initiates several independent but interrelated signaling pathways. one important pathway leads to the activation of mitogen activated protein kinases (mapk) and especially the phosphorylation of extracellular signal-regulated kinases 1 and 2 (erk 1/2). in a subgroup of patients with common variable immunodeficiency (cvid) we have previously demonstrated intrinsic defects in the activation of b cells revealed by the insufficient cd86 upregulation and proliferation after b cell receptor (bcr) stimulation. therefore we assessed signaling pathways downstream of the bcr in order to identify defects in the activation of b cells. methods: pbmc of 20 hd and 25 cvid patients were stimulated by anti-igm. different igm expressing b cell subsets were analyzed separately for erk1/2 phosphorylation by intracellular flow-cytometry using phospho-specific antibodies to erk1/2. to increase the signal intracellular phosphatases were inhibited by h2o2. as markers of activation and initiation of proliferation, cd86 and ki67 expression were measured after 2 days of in vitro stimulation. k. theil 1 , p. aichele 1 1 immh university freiburg, immunology, freiburg, germany type i interferons are homone-like molecules that are produced early after viral and bacterial infections. they signal via the type i interferon receptor (ifnar) and have pleiotropic effects on different cells of the immune system. their best known function is the antiviral activity. to test the direct effect of type i interferons on cd8 t cells in vivo we adoptively transferred lcmv glycoprotein specific tcr transgenic p14 cd8 t cells that are deficient in type i interferon receptor (ifnar-/-) into wild-type b6-recipient mice and compared their expansion with wild-type (wt) p14 t cells after viral infection. we could demonstrate a severe impairment in the capacity of p14 t cells lacking type i ifnr (ifnar-/-) to expand after lcmv infection. following infection of recipient mice with recombinant vaccinia virus, recombinant vsv (vesicular stomatitis virus) or recombinant listeria monocytogenes expressing lcmv glycoprotein, p14 t cells expansion was considerably less dependent on type i ifnr expression. therefore direct type i ifn signalling is essential for cd8 t cell expansion and survival only after lcmv infection. our experiments showed that the lcmv generated cytokine milieu is responsible for the failure of expansion of ifnar-/-t cells during lcmv infection. a suitable model for elucidating the impact of the lcmv generated cytokine milieu is the transfer of p14 t cells into h8 mice. h8 mice ubiquitously express the lcmv immunodominant glycoprotein-epitope gp33-41. therefore the antigen-presentation can be uncoupled from the lcmv induced cytokine milieu when the h8 mice are infected with lcmv8.7. this is a lcmv variant that has got a point mutation in gp33-41 and consequently cannot be recognized by the p14 t cells. s. frischbutter 1 , r. baumgrass 1 1 deutsches rheuma-forschungszentrum, signal transduction, berlin, germany antigen-specific stimulation of t helper cells induces activation of the main transcription factors nfat, nf-kb and ap1 which are important for expression of cytokines such as il-2, ifng and il17. it is known that the immunosuppressive drug cyclosporin a (csa) blocks the activity of the ser/thr phosphatase calcineurin and thereby the activation of the transcription factor nfat. however, we and others observed that this drug also inhibits the activation of nf-kb. to detect targets of calcineurin within the nf-kb pathway we analyzed phosphorylation and degradation levels of different nf-kb signaling proteins in the presence of csa and other calcineurin inhibitors. we found that phosphorylation of the signaling protein bcl-10 was prolonged in cells treated with inhibitors. our data do not indicate an enhanced bcl-10 phosphorylation but rather an inhibition of bcl-10 dephosphorylation. furthermore, calcineurin and bcl-10 co-precipitated with each other. interestingly, this interaction was observed only in t cell receptor-but not in tnfa-stimulated cells. in our proposed model, we hypothesize that calcineurin interacts with the carma/bcl-10/malt1 signaling complex and dephosphorylates bcl-10 and, thus, promotes nf-kb activation. therefore, calcineurin is not only a hub for nfat but also for nf-kb activation. a. t. fulop 1 , j. lamoureux 1 , c. fortin 1 1 université de sherbrooke, medicine, sherbrooke, canada objectives: aging is accompanied by a decrease in immune functions, called immunosenescence. the exact cause is still not known. changes in t cell subpopulations, thymic involution were invoked. we have demonstrated that the signal transduction is altered with aging. in the present work we studied the negative regulatory molecules in the t cell signaling to explain the altered activation of t cells with aging leading to decreased clonal expansion. methods: 25 healthy young and elderly subjects were studied. lymphocytes were separated by fycoll-hypaque. the molecules participating in the negative control loop of lck were studied by western blot and confocal microscopy. the surface expression of ctla-4 has been studied by facscan. the translocation of the molecules in the membrane lipid rafts (mlr) was also studied by western blot. the activity of phosphatases was also determined. results: we found that the phosphorylation of pag was altered with aging explaining the decreased release of csk from mlr and the decreased lck activation. the activation of fynt was also altered. the phosphatase activity studies showed an increase in their activities with aging. the ctla-4 expression was higher after stimulation in t cells of elderly. there was differences between cd4 and cd8 t cells with aging. conclusion: these results suggest that the negative regulation is preponderant in t cells with aging on the positive activation and as such explaining the defect in t cell functions with aging. this opens new therapeutical avenues in the future. in contrast to other members of the tumour necrosis factor superfamily, fas ligand (cd95l) contains a cytosolic proline-rich domain (prd) that enables interactions with sh3 and ww domain proteins. since fasl surface expression is regulated by adam10-mediated ectodomain shedding and fasl might be subsequently released into the cytosol by regulated intramembrane proteolysis (riping) through the secretase-like enzyme sppl2a, we are interested in defining interactions involving the generated intracellular fragment of fasl. employing a monoclonal antibody directed against the intracellular domain of fasl, we observed that previously described fasl-interacting proteins of the pch family selectively bind to the full length molecule but not to n-terminal fragments (ntfs). in order to identify other sh3 domain proteins that potentially interact with the riped fasl prd, we used a sh3 domain phage display library containing all 288 sh3 domains expressed in humans. the screen confirmed several previously identified interactions but also revealed numerous new and interesting candidate binding proteins includig non-receptor tyrosine kinases and adaptor proteins or enzymes implicated in membrane, organelle, and actin cytoskeleton dynamics. selected interactions were verified biochemically and by laserscanning microscopy in transfected cells. it could be demonstrated that tec kinases known to be involved in immune receptor-associated signal transduction as well as members of the snx9 family, which are crucial regulators of endocytic and endosomal dynamics and trafficking, join the list of known fasl-interacting proteins. of note, in contrast to pch proteins, the snxs bound both ntfs and unprocessed fasl, indicating that individual interactors might influence different facets of fasl biology. in conclusion, the present data provide substantial evidence for a selective binding of individual interaction partners of fasl to the full length protein or ntfs. this more detailed glance at the fasl interactome will facilitate focussed strategies to clarify unanswered questions regarding reverse signalling and functional conseoptimal t cell activation requires the engagement of the t cell receptor (tcr) by the specific mhc/antigen complex and costimulatory signals as the interaction of b7 family members on antigen-presenting cells with cd28 on t cells. remarkably, whereas classical glucocorticoids (gcs) effectively suppress solely tcrtriggered t cell activation in vitro, additional cd28 co-stimulation leads to gc-resistance. in this study, we compared the non-steroidal selective glucocorticoid receptor agonist (segra), compound12, with classical gcs regarding their suppressive effect on cd28-costimulated t cells. human primary t cell subpopulations and jurkat cells were stimulated in vitro with plate-bound anti-cd3 and anti-cd28, and proliferation, cytokine secretion as well as phenotypic activation parameters were determined. remarkably, a clearly improved inhibition of ifn-gamma secretion was observed in cd28-costimulated human memory/effector cd4+ t cells by compound12 than by classical gcs. interestingly, apoptosis and activation antigen expression were similarly regulated. improved inhibition of lymphokine secretion by compound12 was also seen after pma / ionomycin stimulation of human primary t cells and jurkat cells. when investigating the in vivo effects of compound12 and prednisolone in acute and subacute dnfb-induced contact hypersensitivity models in mice, we observed comparable efficacy for inhibition of t cell-dependent skin inflammation when treating before hapten challenge. in contrast, however, when treating around hapten sensitization markedly stronger effects were demonstrated for compound12 than prednisolone. when evaluating possible mechanism for the increased activity of compound12 in inhibition of t cell activation we got hints for a specific inhibition of the calcineurin pathway by compound12 which was not prevented by the partial gc receptor antagonist, ru-486, in vitro. moreover, in vivo we observed less induction of il-1beta and tnf-alpha by pre-treatment with compound12 than with prednisolone. our data indicate that the non-steroidal segra, compound12, may represent a promising drug candidate for the treatment of t cell-dependent inflammatory diseases where therapy with classical gcs is hampered by t cell resistance. influenza a infection of b6 mice elicits robust cd8+ t cell responses, with virus-specific cells showing a distinct pattern of cytokine production: tnfa+ cells always express ifng; and il-2+ cells are contained entirely in the ifng+tnfa+ subset. interestingly, the co-expression of ifng and tnfa varies for different epitope specificities. almost all ifng+ pa 224 -specific cells also express tnfa, but only about half of the ifng+ np 366 -specific cells co-express tnfa. this was originally linked to the avidity of the responding population for the specific peptide/mhc complex, with the ifng+tnfa+ phenotype representing cd8+ t cells with higher avidity and a more differentiated phenotype. however, the same cytokine pattern is seen in adoptively transferred cd8+ t cells expressing a clonal tcr, implying avidity alone cannot control development of cytokine profiles. co-expression of ifng and tnfa by adoptively transferred cfse-labelled ot-i cells following infection with influenza a virus expressing ova 257-264 peptide shows a close correlation with division in vivo. early after antigen encounter (0-2 divisions) the vast majority of cells express only tnfa. after 3-4 divisions cells begin to co-express ifng and tnfa. the emergence of an ifng+tnfa-phenotype increases with subsequent divisions (4-6 divisions), indicating cytokine profile is closely linked to cell cycling, as described previously for both b cells and cd4+ t cells. titration of adoptively transferred ot-i cells, which controls the level of expansion in vivo, reveals that more cd8+ t cells develop an ifng+tnfa-phenotype with increased expansion. thus we conclude that while tcr avidity and co-stimulation can impact the differentiation of cd8+ t cells, expansion plays a very important role in the regulation of cd8+ t cell effector function. in addition to its chemo-attractant function, sdf-1a (stromal-cell derived factor-1a, cxcl12) has been described to costimulate cd4 + t cell during tcr triggering. our objective is to clarify the mechanism regulating this costimulatory activity. tcr-driven proliferation of human cd4 + t cells was increased by immobilized sdf-1a to a level similar to that obtained with the costimulatory molecule cd28. as visualized by real time confocal microscopy, t cells entering in contact with sdf-1a formed a tether and displayed an active scanning activity. since sdf-1a induced a similar activity in t cells stimulated with a sub-optimal dose of anti-cd3 mabs, it is conceivable that the sdf-1a-driven scanning may favour productive tcr engagement. to test this hypothesis, we are studying the effect of sdf-1a on tcr internalization, calcium mobilization, mapk activation and actin cytoskeleton reorganization. we are also studying the role of sdf-1a in the context of cd4 + t activation by antigen-presenting cells secreting sdf-1a. this study should help us to better define how sdf-1a modulates cd4 + t cell activation beyond its chemo-attractant function. background: propolis, an ancient herbal medicine, is well known for the management of respiratory diseases. caffeic acid phenethyl ester (cape), an active component in propolis, is known to have anti-tumor, anti-inflammatory, and antioxidant properties. in this study, the effect of cape on the functions of t cells, which play the major role in chronic airway inflammation of asthma, was evaluated. method: cd4 + t cells isolated from human peripheral mononuclear cells by automacs were stimulated with anti-cd3 and anti-cd28 antibodies and cape for 2 days. cytokine levels were dertermined by elisa and lymphoproliferation was analyzed by 3h-thymidine incorporation method. signaling pathway of t cells was studied by western blot. result: it was found that cape significantly inhibited ifn-g and il-5 production and lymphoproliferation in cd4 + t cells stimulated by anti-cd3/cd28. cape could inhibit nuclear factor-kb (nf-kb) activation, but not mitogen-activated protein kinase (mapk) family phosphorylation in t cells. cape could also inhibit akt phosphorylation. conclusion: these results indicated that cape inhibits cytokine production and lymphoproliferation of t cells which might be related to the nf-kb and akt signaling pathway. this study also provided a new insight into the mechanism of cape in immunology and the rationale for propolis in the treatment of allergic disorders. objective: upon activation, cd4 t cells express a variety of molecules on their surface, such as mhc-class ii, cd80, cd86, cd70, whose ligands are constitutively expressed on resting t cells. whereas these molecules are physiologically expressed on antigen presenting cells, their function on t cells is not understood. we tested the hypothesis that activated cd4 t cells might induce t cell proliferation and differentiation from cd4 resting t cells through interaction of activationinduced surface molecules and their constitutively expressed ligands. methods: cd4 t cells from the peripheral blood of healthy donors were co-cultured with fixed activated t cells from the same donor. after 5 days of co-culture, the phenotype of the resulting cells was analyzed by assessing their surface molecules and production of cytokines. results: cd4 memory t cells but not naive t cells proliferated in response to contact with activated t cells. these cells showed a mild activated phenotype assessed by the expression of cd25, cd30, and cd69. analysis of the cytokine profile of these cells revealed the differentiation of il-10-and ifn-g-double-producing cells in response to contact with th1 effector cells, and il-4-producing cells in response to contact with th2 effector cells. the levels of produced cytokines were, however, significantly lower than those produced by activated cells in response to anti-cd3/cd28 stimulation. whereas neutralization of ifn-g or il-4 during culture did not diminish the frequency of the arising cytokine-producing cells, separation of the responder cell population from effector cells by a transwell system led to a significant decrease of cytokine secretion. blocking particular receptor/ligand interactions by neutralizing antibodies against hla-dr, cd70, cd80, and cd86 could not prevent cytokine production induced by t-t cell interaction. however, simultaneous addition of all antibodies significantly inhibited cytokine production to 64-85 %. conclusion: interaction of cd4 memory t cells with activated t cells resulted in the production of the cytokines il-4, il-10, and ifn-g. given the immunomodulatory capacity of il-4 and il-10, these findings might indicate a novel potential negative feedback mechanism to control t cell-driven immunity. a. nasir 1 , s. thompson 1 , j.j. murphy 1 1 king's college london, division of immunology infection and inflammatory disease, london, united kingdom the murine bcl1 leukaemia cell line can be induced to undergo plasmacytoid differentiation in vitro with cytokines il-2 and il-5 and this is characterised by a marked reduction in proliferation and production of large amounts of secreted igm. these cells were observed to express significant levels of the zinc fingercontaining protein zfp36l1 by western blot analysis. this protein is reported to act in post-transcriptional regulation of gene expression by binding to au rich elements (ares) of mrnas of certain genes and consequently promoting mrna degradation. at a cell functional level, zfp36l1 has been described to have roles in apoptosis, proliferation and differentiation in different cellular contexts. cytokine-induced bcl1 differentiation was observed to be associated with downregulation of zfp36l1 protein. in an attempt to determine whether zfp36l1 downregulation was directly linked to bcl1 differentiation, a zfp36l1 shrna expressing lentivirus (psicor) was employed to knockdown zfp36l1 expression. this reagent downregulated zfp36l1 expression very effectively . shrna infected cells proliferated less well than either control virus infected cells or wild-type cells with or without cytokines. zfp36l1 shrna infected cells also produced more secreted igm per cell than either control virus infected cells or wild-type cells in the presence or absence of cytokines. these results are consistent with a role for zfp36l1 downregulation in promoting bcl1 plasmacytoid differentiation. vidual lysates of peripheral blood lymphocytes (pbl) of 32 patients with igg multiple myeloma and healthy controls were investigated for the expression of sialic acid (sa), galactose (gal) and n-acetylglucosamine (glcnac), the sugars known to specify the glycoforms of human serum igg. the degree of glycosylation and signaling status of all 32 isolated myeloma igg bcrs were correlated and compared with the glycosylation of the igg paraproteins isolated from sera of the same patients. it was shown that bcr igg in myeloma is more heavily sialylated when compared with normal controls, that the increased sialylation of igg bcr is associated with higher levels of tyrosine phosphorylation (signaling activity) of the igg bcr supramolecular complex and that bcr igg and serum igg paraprotein from the same patient differed in all cases in the levels of terminal sugar expression. the results suggest that the development of the malignant clone in mm from postswitch b cells expressing igg bcr at their surfaces to plasma cells secreting igg paraprotein may be followed by permanent glycosylation changes in the igg molecules. caused by thapsigargin-induced release of calcium from the endoplasmic reticulum was insensitive to tpen. conclusion: the signal with fluorescent probes for the detection of calcium ions in response to thimerosal is entirely due to zinc release, and no indication for a calcium signal was detected. in light of these observations, zinc may also contribute to calcium signals caused by mercury containing compounds other than tms, and a potential involvement of zinc release in the immunomodulatory effects of these substances should be considered. although best known for its pro-apoptotic function, it seems clear now that cd95 (fas, apo-1) also exerts anti-apoptotic effects associated with costimulation and the induction of proliferation. we investigated effects of fas co-ligation during tcr/cd3/cd28-triggered activation of freshly isolated human t-lymphocytes. to this end, tcr-triggered cells were incubated in presence or absence of different ligand concentrations of anti-apo1 mab, faslfc or faslstrepfc fusion proteins, or leucin zipper (lz-)cd95l. interestingly for all ligands tested, we could clearly demonstrate a correlation between ligand concentration and t cell response: low doses drastically augmented proliferation in the sense of costimulation, whereas high doses completely blocked tcr-induced cell proliferation without inducing cell death. the positive costimulatory effect of fas at low concentrations is associated with elevated il-2 and ifng production, upregulation of activation markers, adhesion molecules and cell-cycle regulating cdks and cyclins. in addition, we observed an increased activation of important signalling molecules including mapk and caspases. using pharmacological inhibitors, we demonstrate that fas is internalized upon ligation. we also observed an increased tcr internalisation following fas co-incubation potentially resulting in the generation of larger signalling platforms that allow optimal t cell activation. in stark contrast, most fas ligands at high concentrations almost completely inhibited cell proliferation of tcr-triggered lymphocytes. in this context, crucial events associated with t cell activation, i. e. tyrosine and erk1/2 phosphorylation, the expression of various activation markers, the il-2 production and caspase activation were almost completely abrogated. these findings highlight that fas-triggering accelerates or blocks t cell activation, depending on the strength of the stimulus. in addition, we provide further evidence for an anti-apoptotic function of fasl during signal initiation in human t lymphocytes. sponsored by the dfg (sfb415) and the medical faculty kiel (to oj) it has been shown that glycosylation of cell surface proteins controls critical t cell processes, including homing, thymocytes maturation, activation, and cell death. plant lectins have been long used to study changes in cell surface carbohydrate structures, to identify leukocyte cell subsets, and as surrogates for authentic t cell activation stimulus. the galb1,3galnac-specific lectin from amaranthus leucocarpus (all) shows a differential binding pattern to murine thymocytes and peripheral cd4+ and cd8+ t cells. in addition, mitogenic stimulus increase 3-fold the all binding to cd4+ t cells. previous studies in human pbmc showed that all binds to human cd4+ t cells and all-binding increased after a mitogenic stimulus using total cell cultures as murine studies. these data suggest that all detects selectively activation-related changes in cd4+ t cell surface carbohydrate but none study has been performed to examine the all effect on human t cell activation. to examine the effect of all on human t cell activation, we analyzed the anti-cd3-dependent activation of purified cd4+ t cells from pbmc in presence or absent of all by measuring proliferation using cfda-se staining, expression of the surface activation marker cd25 and calcium influx by flow cytometry. results showed that all did not induce significantly t cell proliferation or cd25 expression, but enhanced the anti-cd3-dependent proliferation and cd25 expression of purified cd4+ t cells. analisis of calcium influx showed that all enhanced anti-cd3 dependent calcium influx. our findings indicated that all alone does not affect t cell activation but suggested that all induces a costimulatory effect on human cd4+ t cells by up-regulating t cell activation mediated by anti-cd3 stimulus, as further studies have to be performed to elucidate all-induced costimulatory effect. financed in part by papiit-unam (in214609) a. the adaptor protein lat (linker for activation of t cells) has a prominent role in the transduction of intracellular signals elicited by the tcr/cd3 complex. upon tcr engagement, lat becomes tyrosine-phosphorylated and thereby recruits to the membrane several proteins implicated in the activation of downstream signaling pathways, leading to tightly equilibrated programs of activation and survival or induced cell death. the balance between cell survival and cell death is critical for normal t cell development and activation, and is maintained by signals through lymphocyte antigen receptors and death receptors such as cd95 receptor. it has been previously demonstrated that cd95 ligation in t cells induces the proteolytic cleavage of several adaptor proteins, including gads, slp-76, slap-130 and lat. given the dual role of lat as a transducer of activation and negative signals in t cells, we have analyzed the role of the lat cleavage in t cell functions and studied the proteases responsible for this cleavage. objective: the study is designed to explore preliminarily the need of t cells for cytokines during the culture in vitro, which are associated with the activation, proliferation and apoptosis of t cells, and by detecting the expressions of il-rs, co-stimulatory molecules and apoptotic receptors/ligands onto human peripheral blood lymphocytes (hpbls). the results may lay a theoretic and experimental basis for developing the condition media qualified especially to t cell culture. methods: pbls were isolated , and cultured in different media. both immunocytochemistry staining and cell enzyme linked immunosorbent assay (celisa) were used to detect the expressions of il-1r, il-2r, il-3r, il-7r, il-12r, il-18r, cd27, cd28, cdw137( 4-1bb), cd 95(fas) and cd178 (fasl) on hpbls in different cultured time, i. e. 0d, 1d, 3d, 5d, 7d, 9d, 11d and 14d. using typan blue staining, the living cells, dead cells and total cells of each cultured group were counted, then their cell growth curves were drawn out. to evaluate the cellular activity, growth situation and cell cycle of t cells, both mtt and fcm analysis were also performed separately. 1. the expressions of several membrane immune molecules on the lymphocytes in different cultured conditions. 1) the expressions of membrane immune molecules before cultured. 2) expressions of the membrane molecules on hpbls during culturing. 5% fbs rpmi 1640 group (1640 group), il-2 group, pha group... (1) mtt assay. (2) proliferative times and growth curves of hpbls... 1. during cultured in vitro, there are expression changes of the il-1rs (il-1ra, il-2ra, il-2rg, il-3r, il-7r, il-12r, il-18r), co-stimulatory molecules (cd27, cd28, 4-1bb) and apoptosis associated molecules (fas/fasl) on hpbls in different time and cultured media. the expression patterns of the most molecules checked are similar in 1640 group, il-2 group and pha group, but the rests are different. 2. our data also suggest that the hpbls cultured in cd3mcab+cd28mcab+il2+il1a group has a great proliferative potential compared with the other groups. using this condition medium, may have a practical prospect to tumor therapy. 3. celisa will become probably an effective test to detect the expressions of membrane receptors or molecules quantitatively on a large scale. f. beceren-braun 1 , r. tauber 1 1 zentralinstitut für laboratoriumsmedizin und pathobiochemie, berlin, germany l-selectin is a leukocyte cell surface glycoprotein involved in carbohydrate-specific ligand binding which mediates tethering of leukocytes to the endothelial surface during inflammation. apart from its role in adhesion, l-selectin functions as a signal transduction molecule. crosslinking of l-selectin with antibodies or ligand binding to the receptor have been shown to elicit a wide range of cellular responses. in addition to process signals coming from outside of the cell, the intracellular part of l-selectin (lscyto) is also able to conduct intracellular signals, e. g. activates tyrosine kinase p56lck and the ras/rac2 signalling pathway (1) followed by mitogen-activated protein kinases (2) and c-jun n-terminal kinase (1), which leads to an enhanced binding of l-selectin to soluble ligands (3). in our previous work we described an association of lscyto with isozymes of the pkc family which phosphorylate the receptor on serine residues (4). here we show that the protein phosphatase 2a inhibitor phapii is a novel direct interacting partner of the lscyto. we propose a model in which the l-selectin mediated signalling is regulated by the interaction of pkc, pp2a and phapii: phapii binds to the unphosphorylated lscyto. upon l-selectin crosslinking lscyto is phosphorylated, pha-pii dissociates and inhibits the phosphatase pp2a. in addition we have started structural analysis to investigate ligand binding induced conformational changes of the cytoplasmatic domain of l-selectin. v. heissmeyer 1 , e. glasmacher 1 1 helmholtz center munich, molecular immunology, munich, germany during self-antigen recognition, roquin dependent posttranscriptional downregulation of icos prevents t cell help to b cells and autoantibody production. the molecular mechanism by which roquin interferes with icos translation remained unclear. we have identified two critical regions in roquin. the amino-terminus is required for rna binding and can be functionally replaced by conserved sequences from its paralog mnab. the carboxy-terminus mediates p body localization and has specialized in roquin for efficient repression of icos in t cells. using knockout cells of dicer or ago1-4 genes, we prove that roquin mediated repression of icos occurs in the absence of mirisc formation. instead, roquin function required intact p bodies, and was impaired after knockdown of lsm1 and rck or expression of dominant-negative gw182. interestingly, roquin activity is blocked through induced mirisc formation implicating the mutual regulation of different mechanisms of posttranscriptional gene silencing in immune responses. s objectives: upon encountering their antigens, naï ve t cells are activated and driven to clonal expansion and differentiation into armed effector cells. according to the two-signal hypothesis, the induction of an optimal cd4 + t-cell immune response requires both antigen-specific and co-stimulatory signals. in contrast, stimulating naïve cd8 + t cells with specific antigens and costimulatory signals is insufficient to induce optimal clonal expansion and effector functions. thus, cd8 + t cells require additional signals for full activation and further differentiation into effector cells. methods: in this study, we adopted an in vitro approach to dissect the cellular and molecular requirements for cd8 + t-cell activation and differentiation. naïve cd62l hi cd44 lo cd8 + t cells were sorted and stimulated by anti-cd3 and anti-cd28 antibodies. results: firstly, we show that the activation and differentiation of cd8 + t cells require il-2 provided by activated cd4 + t cells at the initial priming stage after stimulation. secondly, this critical il-2 signal is delivered through il2rbg of cd8 + cells and is independent of il-2ra. besides promoting cell proliferation, il-2 stimulation increases the amount of ifng and granzyme b produced by cd8 + t cells. conclusion: therefore, our studies demonstrate that a full cd8 + t-cell response is elicited by a critical temporal function of il-2 released from cd4 + t cells, providing mechanistic insights into the regulation of cd8 + t cell activation and differentiation. most antigenic peptides recognized by cd8 t lymphocytes are produced through degradation of intracellular proteins by the proteasome. however, some antigenic peptides are produced by a proteasome-independent pathway, which is poorly characterized. mage-a3168-176 is a tumor antigenic peptide presented by hla-a1 and widely used for vaccination of melanoma patients. we observed that proteasome and tppii inhibitors failed to block presentation of the antigen by tumor cells. however, processing of this peptide occurred in the cytosol because tap inhibition prevented its presentation. to characterize the cytosolic peptidase producing mage-a3168-176 we setup an in vitro digestion assay using a 20-mer precursor peptide encompassing the sequence of the antigenic peptide. we observed that only the cytosolic fraction was able to produce the antigenic peptide from this precursor. this production was abolished by treating the cytosolic fraction with o-phenanthroline, a broad-spectrum inhibitor of metallopeptidases. this inhibitor also blocked the presentation of mage-a3168-176 by tumor cells. by electroporating hla-a1 cells with a precursor peptide blocked at the c-and the n-terminus, we could exclude the involvement of exopeptidases in the processing of this peptide, and conclude to a major role of a cytosolic metalloendopeptidase. one such enzyme is insulin-degrading enzyme (ide). we observed that depletion of ide abolished the capacity of a cytosolic fraction to produce the antigenic peptide. furthermore, recombinant ide was able to produce the peptide in vitro from the precursor peptide. lastly, silencing of ide with sirna reduced presentation of the peptide by tumor cells. with tppii, ide is the second example of a proteasomealternative pathway in the production of class-i restricted peptides. antigen-specific t cell based tumor immunotherapy, though extensively studied, has only been of limited clinical success so far. immune escape, due to impairment of hla dependent tumor epitope presentation is believed to be one major reason for this failure. to identify novel mechanisms by which tumors can become refractory to immune elimination, human melanoma cells of different donors expressing the transmembrane mart-1/melan-a tumor antigen were exposed to two or three rounds of brief co-culture with mart-1/melan-a 26-35 specific cytotoxic t lymphocytes (ctls). immune selected melanoma cell clones, being resistant to lysis by mart-1/melan-a 26-35 ctls due to impaired epitope processing were further investigated. our results show that in addition to previously described immune evasion mechanisms like down regulation of mhc class i and mart-1 expression, the ifn-gamma independent endoplasmic reticulum associated degradation (erad) pathway is crucial for mart-1/melan-a 26-35 epitope generation. moreover, deregulation of several erad components is essentially responsible for the observed immune escape of the immune selected melanoma cells. in support, re-expression of down-regulated erad components in ctl-resistant melanoma cells completely restored immune recognition by mart-1/melan-a 26-35 ctls. thus, our studies demonstrate for the first time that erad not only plays a central role in the production of cd8 + t cell epitopes from membrane proteins but also contributes to tumor escape mechanisms by cancer immunoediting. studies of t cell responses to hen egg lysozyme suggest that several conformers of peptide-mhc class ii complexes can be generated for a single peptide epitope and that distinct cd4 t cell repertoires known as type a and type b recognise these different conformers (lovitch and unanue, immunol rev 207: 293-313, 2005) . type a t cells recognise peptide-mhc complexes generated from intact proteins after intracellular antigen processing under h2-m (dm) control, where as type b t cells respond to synthetic peptides in the absence of dm editing, but fail to respond to processed intact protein. type b t cells escape thymic deletion in mice (petersen et al., immunity 11: 453-462, 1999) , with implications for autoimmunity. so we studied whether type a and type b recognition patterns occur in t cell responses to autoantigens such as the rheumatoid arthritis (ra)-associated proteoglycan aggrecan, and whether naturally occurring extracellular ligands that activate type b t cells are found in inflamed joints. lymph node cells from aggrecan-immunised balb/c mice proliferated in response to intact aggrecan and to the immunodominant peptide 84-103, whereas peptide-immunised mice responded to peptide, with low or absent responses to intact aggrecan. t cell hybridomas generated from 84-103 peptide-immunised mice either recognised peptide only (the majority) or peptide and intact aggrecan (the minority), a pattern consistent with type a and type b t cell recognition. responses to staggered and alanine-substituted peptide sets showed that type a and b t cell hybridomas recognized the 84-103 epitope in the same register, consistent with this peptide epitope binding to mhc in distinct conformers. type b t cell hybridomas recognised aggrecan fragments in supernatants from cartilage degraded by stimulation with proinflammatory cytokines that induce raassociated aggrecanases. our data suggest that inflammation generates extracellular peptides that activate type b t cells. we are also characterising human type b t cell responses as well as searching for type b t cell ligands in synovial fluid from ra patients. we propose that extracellular cartilage degradation generates ligands that induce autoreactive type b t cell responses which participate in the pathogenesis of autoimmune arthritis. a to cope with mhc i antigen presentation hcmv encodes for several post-translational strategies which have been extensively studied in transfected cells. in this study we analysed the plc in naturally hcmv-infected cells and monitored the composition of the plc throughout hcmv replication. metabolic labeling experiments revealed the absence of tapasin incorporation into the plc. in contrast, western blot analysis demonstrated only a slow decline of tapasin steady state levels in infected cells, suggesting a blocked synthesis rather than degradation. tapasin mrna levels were found to be continuously downregulated during infection, however, the tapasin transcripts were stable and long-lived. taking advantage of a novel method, in which newly transcribed rna is selectively labeled and analysed (dölken et al, 2008), we found, after an initial induction at 8 hrs p. i., a strong inhibition of tapasin transcription at 24 hrs p. i. furthermore, also reduction of tap1 and tap2 transcription was observed contrasting to the elevated levels of erp57 and mhc i transcripts. importantly, ectopic expression of tapasin restored the incorporation of tapasin into the plc in hcmv-infected cells. the data indicate that hcmv controls mhc i antigen presentation also on a transcriptional level and show for the first time the regulation of tapasin transcription as a viral immune evasive function. most peptides presented by mhc class i molecules are produced by the proteasome during degradation of intracellullar proteins. two main proteasome types have been described, differing in their content of catalytic subunits. the standard proteasome comprises catalytic subunits ß1, ß2 and ß5, which are replaced by their ifng-inducible counterparts ß1i, ß2i and ß5i in the immunoproteasome. the thymoproteasome represents a third proteasome type, where catalytic subunit ß5i is replaced by a thymus-specific subunit ß5t. the standard proteasome is present in most tissues, the immunoproteasome in found in cells exposed to ifng and in dendritic cells, while the thymoproteasome is found exclusively in the thymus. we produced a panel of novel antibodies that recognize subunits ß1i, ß2i, ß5i and ß5 in their native form. using these antibodies for successive immuodepletions performed on tumor lysates, we identified two new proteasome types that are intermediate between the standard proteasome and the immunoproteasome, i. e. they contain only one or two the three catalytic subunits of the immunoproteasome. one comprises ß1,ß2 and ß5i (single intermediate proteasome), and the other comprises ß1i, ß2 and ß5i (double intermediate proteasome). we quantified these intermediate proteasomes in a series a tumor lines of various origins, and found that they represent 10-20 % of the total proteasome content of those tumor cells. they are also present in dendritic cells, where they represent about 50% of the proteasome content. we characterized the activity of these intermediate proteasomes, not only on fluorogenic substrates but also on actual antigenic peptides recognized by anti-tumor ctl. with respect to antigens known to be processed differently by the standard and the immunoproteasome, the intermediate proteasomes often behaved like the immunoproteasome. importantly, we identified two tumor antigens that are processed exclusively by either the single intermediate proteasome ( tapasin is a multi-functional protein dedicated to mhc-i biosynthesis; it serves as a structural component in the so called mhc-i peptide loading complex (plc), as a chaperone putatively acting as an active peptide editor and mhc-i quality control mechanism, as an er retention signal for immature mhc-i, and as a chaperone stabilizing tap expression and increasing tap-performance. furthermore, tapasin has been found outside the er, where it has been suggested to regulate retrograde transport of escaped immature mhc-i back to the er from the trans-golgi compartment. the role of tapasin as an active peptide-editor has been debated and we here set out to study the effect of tapasin on binding of peptides of both high-and low-affinity to a human mhc-i allele (hla-a*0201) using protein interaction-and peptide-competition assays. specifically we wanted to in detail compare the binding of two peptides of the same affinity. at high concentrations all of the tested hla-a*0201 binding peptides (tap-transported high-affinity peptide (ttp-ha), signal-peptide of high affinity (sp-ha), tap-transported mediumaffinity peptide (ttp-ma)) induced dissociation of hla-a*0201 from tapasin, but only ttp-ha dissociated hla-a*0201 from tapasin at lower concentrations. using peptide-competition assays against ttp-ma, a peptide of lower affinity, we could show that ttp-ha, one of the two peptides of equally high affinity was a significantly more efficient competitor than peptide sp-ha. however, analysis of mhc-i peptide loading in the tapasin-negative cell line lcl-721.220-a2 showed no competitive advantage of ttp-ha compared to sp-ha supporting a role for tapasin as a selective facilitator of mhc-i peptide binding. in conclusion, we here show that peptides of different affinities dissociate hla-a*0201 from tapasin in a dose-dependent manner, and that tapasin facilitates ttp-ha, but not sp-ha replacement of a lower-affinity peptide (ttp-ma). together these data strongly suggest a role for tapasin as a selective facilitator of peptide binding to mhc-i. importantly, this study implies that criteria in addition to peptide-affinity determines whether tapasin will promote peptide binding to hla-a*0201. m. basler 1,2 , c. lauer 2 , m. groettrup 1,2 1 biotechnology institute thurgau, kreuzlingen, switzerland, 2 university of constance, division of immunology, department of biology, konstanz, germany two lmp7-dependent antigens have been described that relied on the 'structural presence' of lmp7 in the proteasome but not on the activity of lmp7. here we have investigated processing of the h-2d b -restricted uty 246-254 epitope of the male minor antigen uty reported to be lmp7-dependent. using splenocytes from lmp7 -/-, lmp2 -/and mecl-1 -/mice we found that the uty 246-254 epitope requires lmp7 and lmp2 but not mecl-1. curiously, a selective lmp2 inhibitor did not interfere with uty 246-254 presentation. objective: we investigated why the deletion but not the inhibition of lmp2 interferes with uty 246-254 presentation. we hypothesized that the 'structural' requirement for lmp2 is based on replacement of the caspase-like activity of b1 in the proteasome. methods: it was determined if t1a mutants of lmp2 and/or b1 can rescue the uty 246-254 epitope. we used a b1-selective inhibitor to determine if the inhibition of the caspase-like activity of b1 preserves the epitope. finally we determined by mass spectrometry if the uty 246-254 epitope embedded within a 25mer precursor peptide is differentially cleaved by lmp2-deficient and proficient immunoproteasomes in vitro. results: we found that t1a mutants of lmp2 and b1 rescue presentation of uty [246] [247] [248] [249] [250] [251] [252] [253] [254] . also inhibition of cells with a b1-selective inhibitor preserves uty [246] [247] [248] [249] [250] [251] [252] [253] [254] presentation. an aspartate in position 7 of the uty 246-254 sequence wmhhnmdli is preferentially used as a cleavage site by lmp2-deficient but not half as frequently by lmp2-proficient immunoproteasomes. the generation of the uty 246-254 epitope relies on the replacement of the caspase-like activity of b1 by lmp2 because the b1 activity destroys the uty 246-254 epitope. this is the first example for the 'structural' requirement of lmp2 for generation of an epitope. eliminating the activity of their constitutively expressed homologous subunits may explain the requirement for immuno-subunits of the proteasome also for the generation of other antigens. thus we have discovered a so far unrecognized mechanism how lmp2 and perhaps also lmp7 and mecl-1 exert their function in antigen processing. a. linnemann 1 , a. musiol 2 , r. lindner 1 1 hannover medical school, cell biology, hannover, germany, 2 hannover veterinary school, graduate school for biomedical sciences, hannover, germany objectives: mhc i molecules are constitutively endocytosed and recycled to the cell surface. this process is required for the turnover of aged molecules and for some forms of cross-presentation of exogenous peptides on mhc i. in fibroblasts, mhc i is known to internalize via a clathrin-independent, arf6-regulated pathway that is highly sensitive towards the cholesterol-sequestering drug filipin. although this observation suggests that membrane rafts are involved in the internalization of mhc i, no evidence for an association of mhc i with membrane rafts has been found in this cell type. methods: a novel detergent extraction protocol was used to investigate the association of mhc i with membranes rafts. endocytosis of mhc i was measured with a biotinylation-based biochemical assay and with a cell biological assay employing confocal laser scanning fluorescence microscopy. for characterization of mhc i internalization pathways, dominant negative mutants of gtpases (dynamin and arf6) were overexpressed in 3t3 fibroblasts. we show that antibody-mediated oligomerization of mhc i in 3t3 fibroblasts shifted this molecule from soluble fractions to detergent-resistant membranes. this change in detergent resistance coincided with a switch to a novel internalization pathway: oligomerized mhc i internalized faster and more completely and arrived at different endocytic organelles. the two mhc i internalization pathways differed in their sensitivity towards dominant negative arf6: endocytosis of oligomerized mhc i was not affected, whereas non-oligomerized mhc i endocytosed more slowly and changed its subcellular distribution. unlike transferrin receptor internalization, none of the mhc i endocytosis pathways was affected by overexpression of dominant negative dynamin suggesting internalization mechanisms independent of clathrin, caveolin and rhoa. conclusion: we propose that mhc i switches from an arf6-regulated to a novel, arf6-independent internalization pathway in response to a change in membrane environment induced by oligomerization of mhc i. since mhc i is one of the cellular receptors for sv40 virus and since sv40 binding triggers mhc i oligomerization, this novel pathway may be involved in sv40 uptake. antigen cross-presentation in dendritic cells is a complex intracellular membrane transport process, but the underlying molecular mechanisms remain to be thoroughly investigated. in this study, we tested the effect of sirna-mediated knockdown of 57 rab gtpases, the key regulators of membrane trafficking, on antigen cross-presentation. twelve rab gtpases were identified to be associated with antigen cross-presentation, and among which rab3b, 3c were found to be colocalized with mhc class i molecules at perinuclear tubular structure. tracing with fluorescence protein tagged beta2-microglobulin demonstrated that the mhc class i molecules were internalized from plasma membrane to rab3b and rab3c postitive compartment. moreover, the recycling ligand transferrin was enriched in the rab3b or 3c positive vesicles. furthermore, the rab3b, 3c positive compartment were colocalizd with a fraction of rab27a at a juxtaposition of phagosomes. together these data demonstrate that rab3b and rab3c positive vesicles is involved in and may constitute the recycling compartment of exogenous antigen crosspresentation. introduction: while the proteasome is thought to generate most of the hla peptidome, other proteases were also proposed to be significant for this process. both t cell based assays and proteasome inhibitors were used in the past to follow presentation of specific model hla peptides. the hla-peptidomes presented at the cell surface depend on the rate of peptide generation within the cells, their transport from the cytoplasm and loading in the er, binding stability at the cell surface and retrograde uptake of the hla molecules back into the cytoplasm. objectives: the role of the proteasome in hla peptide presentation was evaluated using proteasome inhibition, while following the turnover rates of the entire hla peptidome. the peptidomes of both the authentic membranal and a recombinant soluble form of the hla molecules were collected for analysis at different time points after the inhibition of the proteasomes. the turnover rates of the hla-peptides were followed using pulse-chase analysis with stable-isotope labeled amino acids concurrently with epoxomicin treatment. the hla molecules were immunoaffinity purified and the peptides were analyzed by capillary chromatography and orbitrap tandem mass spectrometry. both the endogenous membranal and soluble mhc molecules were studied in parallel from the same cells. peptides were identified by their ms/ms fingerprints and the turnover rates were determined by the shift from the 'light' to the 'heavy' leucine of each peptide. results: a few thousands hla-peptides were identified, and for a large portion of them, the turnover rates could be defined. proteasome inhibition did not affect the complexity of the hla peptidomes or reduced significantly the amounts of membranal hla molecules. many peptides were labeled relatively rapidly with heavy leucine, indicating that the hla peptidome contains also the products of newly synthesized and rapidly degrading proteins. the source proteins of the hla peptides seemed to have similar biological functions and cellular origins in both the inhibited and untreated cells. the centrality of proteasomal degradation in hla-peptide presentation is put into doubt and the role of the proteasome in the generation of each peptide and each cleavage site can be defined. epstein-barr virus (ebv) is a ubiquitous y-herpesvirus, infecting over 90 % of adults worldwide. it can cause mononucleosis and several lymphomas and carcinomas, reflecting the tropism of the virus for b-lymphocytes and epithelial cells. ebv persists for life despite the presence of virus-specific adaptive immunity, indicating that it has evolved strategies to counter the host immune response. one such strategy is the persistence of the virus in the latent phase of its life cycle, where expression of viral proteins is minimized. however, for ebv replication and dissemination to occur, it must enter the lytic phase. here, over 80 viral proteins are expressed, creating many potential antigens for presentation to cytotoxic t -lymphocytes. ebv can circumvent possible eradication by cd8+ t lymphocytes during the lytic phase by interference with antigen processing and presentation through hla class i in the infected cell. the viral proteins bnlf2a and bglf5 have been shown to achieve this by impairing peptide-loading of hla class i and inducing the degradation of mrnas encoding hla molecules, respectively. a third ebv lytic phase protein, the g-protein coupled receptor (gpcr) bilf1, has now been found to down-regulate cell surface hla class i expression (zuo et al, plos pathogens 2009). this represents a novel function for a virally-encoded gpcr. bilf1 is expressed early in the ebv lytic cycle and is localized predominantly at the cell surface. there it can interact with hla class i molecules, resulting in their internalization and lysosomal degradation. this has a profound effect on the ability of cytotoxic t-lymphocytes to recognize cells displaying antigens derived from ebv proteins. interestingly, bilf1 displays a differential effect on distinct hla class i haplotypes. furthermore, we have shown that the intracellular c-terminal tail of bilf1 is required for its effect on hla class i expression. however, the ability of the gpcr to activate intracellular signaling pathways is dispensable in this regard. thus, by reducing the cell surface expression of hla class i molecules, ebv bilf1 can hinder the recognition of virally-infected cells by cytotoxic cd8+ t lymphocytes, thereby facilitating the evasion of adaptive immune mechanisms. t lymphocytes mature in the thymus, generating a non-dangerous t cell repertoire. for the adquisition of tolerance, thymocytes suffer positive and negative selection processes. during t cell maturation, tcrs contact with different mhc-peptide complexes on the surface of pressenting cells, allowing tolerization against self proteins. to obtain a non-self-reactive t cell repertoire, it is of most importance that pressenting cells in the thymus express a repertoire of mhc-peptides complexes representative of the proteins that t cells will found in periphery, including tissue restricted antigens (tras)-derived peptides. in the last decade, transcription of tras in thymus has been well-reported. furthermore, the expression of many genes codifying for tras are dependent.on the expression of the autoimmune regulator (aire). aire is mainly expressed in medullar thymic epithelial eells (mtecs), which are involved in negative selection. so far no systematic study have been made to describe the peptide repertoires associated to hla molecules in the thymus. in addition, although many data of tras transcription in thymus have been reported, much less work has been performed at biochemical level, and to our knowledge, no hla ligand arising from any tra have been reported in thymus. in this report we present the results of analyzing the hla-dr-associated peptide repertoire from whole tissue samples of different human thymi by mass spectrometry. we describe 131 natural ligands, including two peptides derived from semenogelin-1, a tissue restricted antigen expressed mainly in the prostate, and present in semen. using qpcr we demonstrate that semg1 is transcribed in thymus from both male and female individuals. finally, we detected the semg1 mrna expression in a fraction enriched in stromal cells, but not in the thymocyte fraction of the thymi. the proteasome is the major protease complex for non-lysosomal protein degradation in eukaryotic cells, which generates most peptides for mhc class i antigen presentation. vertebrates express two sets of catalytic subunits, constitutive (beta1, beta2, beta5) and immuno-subunits (beta1i, beta2i, beta5i). deficiency in beta5i results in profound reduction of mhc class i expression, demonstrating the significance of this subunit for efficient antigen presentation. currently, this is attributed to the specific proteolytic activity of the beta5i subunit, its role in the maturation of immunoproteasomes or both. however, re-expression of catalytically inactive beta5i subunits is capable to rescue antigen presentation suggesting that the proteolytic activity of this subunit is not limiting in this process. here, we show that following infection with listeria monocytogenes induction of beta5i expression increases the cellular proteasome content in the infected organs. our results indicate that this is due to the high chaperone activity of its propeptide which drives proteasome neosynthesis and thus enhances the overall proteasome quantity. further, mhc class i antigen presentation on beta5i-deficient cells could be restored by treatment with d3t, which increases the amount of proteasomes independent of beta5i via induction of mixed proteasomes containing beta1i, beta2i and beta5. consequently, not the lack of the specific proteolytic activity of beta5i or immunoproteasomes, but the reduced proteasome quantity in beta5i deficient cells is the major limiting factor for mhc class i cell surface expression. . we have previously shown that lc, in contrast to ddc, do not express cell surface tlr2, 4 and 5, which results in their inability to respond to both gram-positive and gram-negative extracellular bacteria in terms of maturation into immuno-stimulatory cells and production of inflammatory cytokines. therefore, the question remained what the role is of lc in class ii mhc-mediated activation of anti-bacterial t cells. we determined the capacity of ddc and lc to internalize and process whole bacteria and present bacterial antigens to cd4 + t cells. in vitro generated lcs and ddcs were cocultured with gfp-expressing bacteria and subsequently analysed by clsm and facs for their uptake capacities. furthermore we investigated their capacity to stimulate autologous bacteria-specific t cell lines as a measure for antigen presentation. results: we found that lc are principally able to internalize bacteria, but far less efficient than ddc. moreover, visualisation of bacterial uptake by em revealed different uptake mechanisms by lc and ddc. both in lc and ddc internalized bacteria were detected in the endosomal and lysosomal compartments of the mhcii processing route. nevertheless, presentation of bacterial antigens by lc on mhcii was inefficient compared to that of ddc, as indicated by a low capacity to activate autologous bacteria-specific cd4 + t cells. the presence of exogenous tlr3 and tlr8 ligands did not overcome the differences between lc and ddc, indicating that the impaired capacity to internalize and process bacteria and activate bacteria-specific t cells is not due to the lack of tlr signalling or insufficient expression of co-stimulatory molecules, but could be an intrinsic characteristic of lc. conclusion: we propose that the epidermis of the skin is an immune-privileged site where lc play a minor role in anti-bacterial immunity and may play a role in inducing tolerance to the bacterial skin flora by steady-state presentation of antigens from commensal skin bacteria. e. james 1 , i. bailey 1 , t. elliott 1 1 university of southampton, cancer sciences division, southampton, united kingdom regulatory t cells (tregs) play a pivotal role in the suppression of tumour specific t cell responses. depletion of tregs in balb/c mice results in a robust immunity to the normally poorly immunogenic ct26 colon carcinoma. this response is long lasting and mediated by both cd4 and cd8 t cells. importantly, the treg depleted ct26 specific immunity is cross-protective; capable of mediating rejection of tumour lines of different histological origins (a20, c26, bcl1, renca) implying a broader repertoire of response. we have characterised one of these cross-protective antigens, gsw11, which is h2-d d restricted. analysis of the generation of gsw11 in ct26 revealed that the peptide is susceptible to over-processing by the er-resident aminopeptidase eraap. inhibition of eraap in ct26 cells substantially increased the amount of gsw11 present, observed by increased t cell responses to the tumour in vitro and hplc analysis. this increase was in spite of an overall reduction of mhc class i molecules at the cell surface. to investigate whether the increase in immunogenicity following knockdown of eraap would protect mice, we generated stable eraap knockdown (kd) ct26 and immunised balb/c mice. greater than 80 % of mice injected with eraap kd ct26 were found to reject the tumour. analysis of t cell responses revealed the presence of gsw11-specific t cells, however, these responses were small (0.5-1 %). this compared to a much larger response to ct26 (˚5 %). preliminary results indicate that the majority of the t cell responses (non-gsw11-specific) in these mice are directed toward unstable peptide/mhc complexes, possibly indicating presentation of n-terminally extended peptide antigens. this highlights manipulation of the peptide repertoire as a potent tool for the generation of t cell responses in vivo. minor histocompatibility antigens play important roles in the outcome of stem cell and organ transplantation as they are involved in the development of graftversus-host-disease and in the graft-versus-tumor reactivity in hla-identical stem cell transplantation [1] . the di-allelic hla-a2 restricted minor histocompatibility antigen ha-1 locus codes for the highly immunogenic ha-1 his and the non-immunogenic ha-1 arg nonapeptides, differing in one amino acid. the only difference that could explain the absence of the ha-1 arg immunogenicity was the estimated numbers of cell surface presented copies i. e. 80/cell for ha-1 his and less than 5/ cell for ha-1 arg [2] . as ha-1 his/arg is hematopoietic system specific and shows additional expression on epithelial cancer cells while absent on the normal epithelial cell counterpart, the ha-1 his allele is currently used for boosting the graft-versus-tumor responses after hla matched ha-1 mismatched stem cell transplantation. to elucidate the mechanisms underlying the differential cell surface presentation of the ha-1 allelic peptides, we investigated the impact of the ha-1 his/arg polymorphism on molecular and cellular processes involved in the intracellular generation and stable cell surface presentation of hla class i-bound peptides. therefore, proteasome-mediated digestion experiments, tap translocation analyses, and hla-dissociation assays with ha-1 his and ha-1 arg peptides were performed. moreover, the crystal structures of hla-a2 in complex with either ha-1 his , ha-1 arg or a ha-1 variant with a citrulline residue at position 3 were determined in order to obtain atomic level insights into the conformation of the hla-a2/ha-1 peptide complexes. our results exclude a role for antigen processing in preventing ha-1 arg to be presented at the cell surface and both the structural and hla-dissociation data clearly show that the lack of cell surface expression essentially results from an increased instability of the ha-1 arg allele in the hla-a2 peptide binding groove [3] . they provide a rationale for the lack of ha-1 arg peptide immunogenicity essential for the choice of tumor peptides for stem cell based immunotherapeutical application. proteasomes play an important role in mhc class i antigen processing. exposure of cells to proinflammatory cytokines such as tnfa or ifng leads to the expression of three facultative catalytic proteasome subunits (i. e. immunosubunits) that replace the constitutively expressed subunits in the cellular proteasome population. immunoproteasomes generate many pathogen-derived cd8 t cell epitopes with high efficiency and thereby shape the specificity of the pathogen-specific cd8 t cell response. on the other hand, immunosubunit expression is not essential for development of cd8 t cell-mediated protective immunity, thus the physiological relevance of these cytokine-induced proteasome subunits remains unclear. we observed that mice that lack the immunosubunits lmp7 (ib5) and mecl-1 (ib2) develop a variety of autoimmune responses, including a latent form of t1d (or insulin dependent diabetes mellitus, iddm), following irradiation and bone marrow reconstitution. iddm development in these mice is characterized by inflammation of the islets of langerhans, glucose intolerance and increased water consumption, and is dependent on the presence of cd8 but not cd4 t cells. a cd8 t cell epitope, encoded by the islet beta cell-expressed "islet-specific glucose-6-phosphatase catalytic-subunit-related protein" (igrp) mrna, was identified as an important target of the cd8 t cell response. this epitope, like many other known diabetes-associated epitopes, binds its presenting mhc class i molecule with low affinity. as t cells specific for low affinity binders most likely can escape central and peripheral tolerance while t cells specific for high affine binders do not, we postulate that inflammation-induced immunoproteasome expression primarily functions to replace self-peptides that are derived from tissue-associated antigens and bind mhc class i molecules with low affinity, by a higher affine peptide species towards which t cell tolerance exists. thus, the inducible proteasome subunits may play an important role in immune regulation, by removing the targets of potential auto-immune cd8 t cells that enter inflamed tissues. endocrine epithelial cells, targets of the autoimmune response in thyroid and other organ-specific autoimmune diseases, express hla-ii molecules with compact conformation and are therefore expected to stably bind autologous peptides. the role of these molecules is not known but they could be involved in the maintenance and regulation of the in situ autoimmune response. to study in situ t cell responses without characterizing self-reactive t cells, we have identified natural hla-dr-associated peptides from autoimmune organs that will help finding peptide-specific t cells in situ. here we report the first analysis of hla-dr natural ligands from ex-vivo graves' disease-affected thyroid tissue. using mass spectrometry, 162 autologous peptides were identified from hla-dr-expressing cells, including thyroid follicular cells, some corresponding to predominant molecules of the thyroid colloid. most interestingly, eight of the peptides derived from a major thyroid autoantigen, thyroglobulin. cell-free in vitro binding assays were performed with the thyroglobulin peptides and some other thyroid-eluted peptides as controls, to identify to which hla alleles were these peptides associated in vivo. all but two of the thyroglobulin peptides showed low binding with the corresponding alleles. the two peptides with relatively high binding affinity were presented in the context of dr3 and dr4. analyzing the digestion patterns used for the generation of the thyroid peptides, a preferentially cleavage after a lys and arg was observed for all of them, independent of the restricting allele. our data demonstrate that although the hla-dr-associated peptide pool in autoimmune tissue mostly belong to abundant ubiquitous proteins, peptides from autoantigens are also associated to hla-dr in vivo and therefore may well be involved in the maintenance and the regulation of the autoimmune response. the t cell response generated following herpes simplex virus type 1 (hsv-1) infection is known to be crucial in the clearance of replicating virus and in limiting the severity of infection. despite this, the relative contributions of cd4 + and cd8 + t cells in hsv-1 immunity have yet to be clearly elucidated. to better understand the role of hsv-1-specific cd4 + t cells in immune control we have identified a 13 amino acid epitope derived from glycoprotein d of hsv-1. following flank infection, gd-specific cd4 + t cells were first detected in the draining brachial and axillary lymph nodes (ln) 5-days post-infection (pi), peaking at day 7 and declining thereafter. gd-specific cd4 + t cells were first recovered from the spleen, skin and dorsal root ganglia (drg) at day 6 pi and peaked at day 9. while hsv-specific t cells were first observed in the draining ln at day 5 pi, hybridoma assays showed ex vivo presentation of the gd epitope by brachial ln cells as early as 2 days pi, with peak activity 4 days pi before declining to background by day 7. however presentation of the gd epitope was much more prolonged in vivo as proliferation of transgenic gdspecific cd4 + t cells was observed up to 23 days post-infection in the brachial ln. ex vivo analyses suggest that only cd11c + cells were involved in gd antigen presentation at days 2, 5 and 15 post-infection. subdivision of dendritic cells (dcs) populations indicated that both skin-derived dcs and cd8a + dcs can present the gd antigen to cd4 + t cells at day 2 pi, whereas by day 5 pi the skin-derived dcs were the predominant population presenting the gd epitope. together these data show that following hsv-1 infection, antigen presentation is initiated rapidly and persists well after clearance of replicating virus. furthermore, we present evidence that different dc populations have distinct roles in the presentation of viral antigens and that they may vary during the course of infection. complementary zippers induced complete dimer formation, whereas identical zippers impaired stable interactions of the tagged peptidases. we also verified that the zippers did not influence the substrate "preferences" of the respective erap. our results from in vitro digestions suggest that the stabilised heterodimer is significantly more efficient in the production of a model epitope than the mix of monomeric erap1 and erap2 unable to form dimers. this observation is not due to mere thermodynamic stabilisation but involves positive cooperative effects in the heterodimers. conclusion: allosteric interaction of erap1/erap2 in heterodimeric complexes enhances the global efficiency of precursor peptide trimming in the human er. during the biogenesis of class i molecules, newly synthesized heavy chains fold and acquire disulfide bonds while interacting with the lectin-chaperone calnexin (cnx) and its associated thiol oxidoreductase erp57. upon assembly of the heavy chain with b 2 m, the class i molecule enters a peptide loading complex (plc) that consists of the tap transporter, tapasin, the calnexin homologue calreticulin (crt) plus associated erp57. both crt and erp57 are required for efficient assembly of peptide-loaded class i molecules and their subsequent expression at the cell surface. we examined functional sites on crt and erp57 to gain insights into their mechanisms of action in class i biogenesis. for crt, its lectin function is thought to be crucial for its association with class i molecules. however, when crt mutants lacking lectin function were expressed in crt-deficient cells, they completely complemented all class i biosynthetic defects. thus polypeptide-based contacts either mediated through erp57 or directly between crt and the heavy chain are sufficient to effect the chaperone and quality control functions of crt in class i biogenesis. we also tested the notion that erp57 must be recruited by cnx or crt to function on class i molecules. we found that the rates of heavy chain disulfide formation were normal in cells lacking cnx, crt or both chaperones. furthermore, an erp57 point mutant that fails to bind to cnx or crt was just as effective as wild type erp57 in normalizing rates of disulfide formation. we conclude that erp57 does not require recruitment by cnx or crt and likely acts directly on class i heavy chains to promote disulfide formation. furthermore, in cells expressing the erp57 point mutant, class i heavy chains, crt and the tapasin-erp57 disulfide conjugate were present at normal levels in the plc, indicating that the interaction between erp57 and crt is not required for plc assembly. finally, we show that mutations that destroy the enzymatic function of erp57 have no effect on plc stability or class i surface expression, suggesting that erp57 plays a structural as opposed to catalytic role in plc function. autoimmune pancreatitis (aip) underlies 5-11 % of cases of chronic pancreatitis and is characterized by prominent lymphocytic infiltration. a strong association of aip with the hla-drb1*0405/dqb1*0401 haplotype has been reported, but identification of the predisposing hla gene(s) has been precluded by strong linkage disequilibrium. here, we show that hla-dr*0405 transgenic ab0 nod mice suffer from aip and additional pneumonitis after sublethal irradiation and adoptive t cell transfer from syngenic donors, leading to complete pancreatic atrophy. pancreas histology is characterized by destructive infiltration of the exocrine tissue with cd4+ and cd8+ t cells, b cells and macrophages. mice with complete pancreatic atrophy have reduced serum lipase activity, develop fat stools and loose weight on regular chow. hla-dr*0405 transgenic mice (cd4+ t cell competent) develop aip even unprovoked, similar to ab0 nod mice (cd4+ t cell deficient), while hla-dr*0401, hla-dq8 or hla-dr*0405/dq8 (double-) transgenic controls all remain normal after same treatment. we conclude that hla-dr*0405 fails to protect from aip, likely due to defects in the induction of cd4+ regulatory t cells. our results identify hla-dr*0405 as a prominent risk factor for aip on the hla-drb1*0405/dqb1*0401 haplotype. this humanized mouse model should be useful to study mechanisms that underlie the hla association of autoimmune diseases, but also immunopathogenesis, diagnostic markers and therapy of human aip. s. khan 1 , c. britten 1 , h. overkleeft 2 , g. van der marel 2 , k. melief 1 , d. filippov 2 , f. ossendorp 1 1 leiden university medical center, section tumorimmunology, leiden, netherlands, 2 leiden university, biosynthesis group, leiden, netherlands objective: we have targeted peptide antigens to dendritic cells by the use of synthetic peptides chemically coupled to synthetic tlr ligands to study the impact on mhc class i and class ii antigen presentation. the potency of the vaccine was addressed by monitoring antigen presentation, priming of t-cells and tumor protection. results: our data show that this type of targeting of peptides greatly improves antigen presentation and t-cell priming compared to free peptide. vaccination of mice with the tlr-ligand peptide conjugates induced high numbers of functional cd8 and cd4 t-cells that could protect mice for aggressive melanoma. this potency relies on tlr signaling since peptide coupled to a non-functional tlr ligand was unable to support induction of specific t-cells. these data indicate that simultaneous encounter of antigen and a maturation signal are crucial for optimal t-cell activation by dendritic cells, and show the potency of tlr-l peptide conjugates as a vaccine modality. y. shi 1,2 , x. hu 1,2 , a. kawanatachikawa objectives: nef protein of human immunodeficiency virus (hiv) holds some important immunodominant ctl epitopes. two overlapping 8-mer and 10-mer epitopes (rypltfgwcf (nef138-10) and rypltfgw (nef138-8)) were found to be presented by hla-a*2402 and some immune escape mutants of these two epitopes have also been found in some patients, e. g. y2f, y2w, t5c, f6l, w8r, f10r, f10y etc. or their combinations. it's important to study the molecular basis of the peptide being displayed on the cell surface, through which we can analyze the mechanism of immune escape of hiv. methods: refolding method was used to attain the soluble protein pmhc. crystals are grown using hanging drop vapor diffusion method and x-ray diffraction technology is used to determine the structure. we have determined six peptide-mhc(pmhc) structures containing nef138-10 (wild type) and its four mostly common immune escape mutants (y2f, t5c, y2f&t5c, f6l), and also nef138-8 (wild type). we found that there was little difference between the nef138-10 (wild type) and nef138-10 (y2f) when they were displayed in the peptide-binding groove of mhc molecule, except water molecule distribution near the anchor residue y2 or f2. interestingly the central bulge region of the peptide was becoming very flexible for the nef138-10 (t5c) and nef138-10 (y2f&t5c), which may affect the binding of peptide and the recognition of t cell receptor. for nef138-10 (f6l), the side chain of l6 was more flexible compared to the nef138-10 (wild type). alignment of the nef138-10 and nef138-8 showed that the nef138-8 became flat and the side chain of f6 was not solvent-exposed due to shortening of the length of the peptide. conclusion: as the peptide nef138-10 was featured, while the peptide nef138-8 was featureless, so the different topology of these two epitopes indicates that they have different tcr repertoire diversity in hiv-specific responses. different immune escape mutants of nef138-10 was using different strategies to avoid the killing of host ctls, which indicates that the therapy strategy based on the cellular immune response should be diversity. for the in vivo or ex vivo activation of antigen-specific t cell responses long synthetic peptides are used to activate both cd8+ and cd4+ t cells. in this study we investigated the efficiency and mechanism of cross-presentation of these long synthetic peptides in mhc class i. we observed a large variation in the effectiveness of activation of specific t cells by the extended peptides corresponding to different epitopes, indicating a difference in the efficiency of processing and presentation of these peptides. for the hla-a2 restricted cmvpp65 derived nlv epitope specific t cells were most efficiently activated by n-terminally extended variants of the minimal epitope, while the use of c-terminally extended variants resulted in a 2-3 log reduction of activation efficiency. this pattern was seen for 5/9 epitopes tested in different hla restrictions. furthermore, for all epitopes tested, extending both the c-terminus and n-terminus led to 2-5 log less efficient activation of the specific t cells, compared to the minimal peptide. exchange of the c-terminal sequence of the c-terminal extended hla-b7 restricted cmvpp65 rph peptide with the c-terminal extended nlv peptide led to the enhancement of t cell activation by the exchanged nlv peptide, indicating a role of the extended peptide sequence in the efficacy of processing and presentation of the peptide. tap-deficient t2 cells loaded with extended nlv peptides efficiently activated nlv-specific t cells, indicating that the route of presentation was tap-independent. addition of lactacystin did not affect activation of specific t cells, illustrating that crosspre-sentation was proteasome-independent. primaquine reduced the activation of specific t cells by extended nlv peptides, but not by the minimal nlv 9-mer peptide, suggesting that cross-presentation was dependent on endosomal recycling. these data suggest that long synthetic peptides can be processed by peptidases in endocytic compartments and presented by recycling mhc class i molecules. not all immunogenic epitopes that have been selected in vivo for efficient processing and presentation by the classical pathway may be presented efficiently by cross-presentation. therefore, a rational design of peptides is crucial for efficient activation of cd8+ t cells in approaches of vaccination, adoptive transfer and immune monitoring. antigenic peptides presented by mhc class i molecules are fragments that are usually excised from intracellular proteins while these are degraded by the proteasome. recently, three antigenic peptides were found to result from the splicing of segments that are not contiguous in the parental protein. for two of these peptides, splicing was found to occur in the proteasome by a mechanism of transpeptidation resulting from the nucleophilic attack of an acyl-enzyme intermediate by a free peptide fragment. one of them is derived from melanocytic protein gp100 and requires excision of a four-amino acid intervening segment. the other peptide is derived from protein sp110, and requires splicing in the reverse order of two segments initially separated by six amino acids. the first spliced antigenic peptide described was derived from fibroblast growth factor-5 (fgf-5) and was recognized by human cytotoxic t lymphocytes directed against kidney cancer cells. it is made of two spliced fragments, which are initially separated by a long segment of 40 amino acids. the splicing mechanism of this peptide has not been worked out. the length of the intervening segment made the transpeptidation model more difficult to account for the splicing of this peptide. we therefore evaluated the role of the proteasome in the splicing of this peptide. we observed that the spliced fgf-5 peptide was produced in vitro after incubation of proteasomes with a 49-amino acid long precursor peptide. we evaluated the mechanism of the catalytic reaction by incubating proteasomes with several peptide precursors in a pair wise manner. the results confirmed the transpeptidation model of splicing. we further compared the production of the fgf-5 spliced peptide by cells transfected with mutant constructs encoding fgf-5 proteins where the intervening segment was shortened from 40 amino acids to 30, 20 or 8 residues. we observed an increase in the production of the spliced peptide that was proportional to the reduction in length of the intervening segment, as predicted by the transpeptidation model. finally, using the spliced gp100 peptide model, we observed that splicing did not occur at a significant level between fragments of two distinct proteins in the cell. the polymorphic residues within the peptide binding cleft of hla class i molecules not only diversify the range of peptides presented to cytotoxic t lymphocytes but also influence the pathway of antigen presentation. in order to acquire high affinity peptides, some class i allotypes, such as hla-b*4402, are heavily dependent upon tapasin and other molecules comprising the peptide loading complex (plc). other class i molecules, like hla-b*4405, appear to largely bypass this complex but are consequently loaded suboptimally with peptide. hla-b*4402 and b*4405 are naturally occurring allotypes that differ by only a single amino acid, making this difference in behaviour all the more remarkable. we have previously speculated that such tapasin-independent class i molecules may have been selected in response to viral inhibitors that target the plc, such as the human cytomegalovirus us3 protein. to address this hypothesis, us3 was stably coexpressed in b lymphoblastoid cell lines expressing hla-b*4402 or hla-b*4405. in the presence of us3, the surface expression of hla-b*4402 was substantially reduced whereas hla-b*4405 expression was relatively unaffected. although us3 was able to form complexes with both hla class i allotypes, only hla-b*4402 was retained intracellularly in an immature form whereas hla-b*4405 was transported to the cell surface. accordingly, in the presence of us3, hla-b*4405, but not hla-b*4402, constitutively presented a hla-b44 restricted alloantigen to reporter t cells, suggesting that us3 binds hla-b*4405 without interfering with peptide loading. us3 has been reported by others to bind the plc but surprisingly we have not detected such us3-plc complexes in our system. rather, in the presence of us3 we identified a pool of class i molecules distinct from the plc and only present in us3 expressing cells, implying that us3 may act independently of the plc. these findings demonstrate how hla class i polymorphism not only impacts upon the t cell repertoire and diversifies determinant selection, but also serves to evade the impact of viral inhibitors on antigen presentation. c. massa 1 , b. seliger 1 1 martin-luther-university halle-wittenberg, institute of medical immunology, halle, germany in the attempt to optimize vaccine dc, modifications have been proposed both in the antigen loading and in the maturation protocols. for dc loading "whole antigens" are now preferred to peptides. therefore, it is important to consider not only the costimulatory properties of the vaccine dc, but also their antigen processing abilities. this is even more important since there is the trend to stimulate dc with tlr ligands combined with ifn-y in order to induce dc not only able to correctly migrate, but also secreting the bioactive il12p70. since ifn-g is known to influence the expression of multiple proteases involved in antigen processing, aim of this study was to compare the various maturation cocktails for the consequences on the antigen processing capabilities of the dc in parallel to their costimulatory potential. for this purpose monocyte-derived dc were stimulated for 24h with the gold standard of maturation (tnfa, il1b, il6 and pge2) or a combination of ifn-g and different tlr ligands. the dc obtained exhibit a similar expression of costimulatory and adhesion molecules together with the ability to induce proliferation of allogeneic pbmc, but differ for the pattern of proteases expression as evaluated by real time pcr. with the exception of the downregulation of the tripeptidyl peptidase ii (tppii), no dramatic differences were observed for endo-and aminopeptidase between immature and "gold standard" mature dc. in response to the "ifn-gcontaining" cocktails there was a similar tppii downregulation, but also the induction of many other enzymes. the cytosolic leucine aminopeptidase-3 (lap3) had a more than 20-fold increase in transcription levels, whereas the mrna expression of the aminopeptidases of the endoplasmic reticulum erap1 and erap2 and of the immunoproteasome subunits lmp2 and lmp10 was enhanced between 2 and 5-fold under these culture conditions. with regard to the different tlr ligands used in combination with ifn-g, there was a reproducible higher mrna induction in the presence of the tlr4 ligand mpla in comparison to the tlr-3 and 7/8 ligand polyi:c and r848. these data suggest that the maturation cocktail of dc may alter the peptide repertoire presented by hla class i surface antigens. it has been suggested that mast cells might serve, under certain circumstances, as antigen presenting cells for t cells. however, whether cognate interactions between mast cells and class ii restricted cd4 + t cells actually occur, is still an open question. we addressed this question using peritoneal cell-derived mast cells (pcmc) as an antigen presenting cell model. our results show that in vitro treatment of pcmc with ifn-g and il-4 induced surface expression of mature mhc class ii molecules and cd86. when ifn-g/il-4 primed pcmc were used as antigen presenting cells for cd4 + t cells they induced activation of effector t cells but not of their naive counterparts as evidenced by cd69 up-regulation, induction of proliferation and cytokine production. confocal laser scanning microscopy showed that helper ot-ii t lymphocytes form with pcmc functional immunological synapses, characterized by pkcq enrichment and ifn-g polarized secretion towards the antigen-presenting mast cells. finally, upon cognate interaction with ot-ii t cells, mast cells lowered their threshold of activation via fceri. our results show that mast cells can establish cognate interactions with class ii restricted helper t cells, implying that they can actually serve as resident apc in inflamed tissues. h the vast majority of peptide ligands presented by mhc class i molecules is thought to be produced by cytosolic degradation of source proteins by the proteasome. although, next to cytosolic and nuclear proteins, proteins targeted to the endoplasmic reticulum (er) can also be degraded through this pathway following retrograde transport into the cytosol, antigen processing of er proteins remains little characterized. studying processing and presentation of er-targeted and cytosolic forms of proinsulin (pi), an autoantigen playing a pivotal role in triggering of cellular autoimmune responses in type 1-diabetes, we found that er-targeting of this model antigen has profound effects not only on how pi is degraded, but also on regulation of its synthesis. as expected, proteasome inhibition inhibited degradation of cytosolic pi as well as presentation of the epitope insulin b15-23 to specific cd8+ t cells. in contrast, prior exposure of cells to proteasome inhibitors strongly reduced production of er-targeted pi (pre-pi) through induction of er stress, both in cells infected with a recombinant vaccinia virus and in cells transfected with a tetracycline-regulated expression system. experiments using conditions permissive for pre-pi expression showed that er-targeting modified proteolytic processing of pi for mhc class i presentation. these experiments suggested that two proteolytic pathways contribute to degradation of er-targeted pi, with their relative contribution depending on the stability of the protein. while degradation of unmodified pre-pi was partially dependent on the proteasome, removal of one or several disulfide bridges increased the role of the proteasome in processing of pre-pi for presentation, while introduction of a site for n-glycosylation had the opposite effect. these findings imply that er-targeting together with structural features can have profound effects both on antigen production and on the pathway of proteolytic antigen degradation and presentation. cd8 + t cell immune response to exogenous antigens relies on cross presentation by dendritic cells (dcs) in secondary lymphoid organs. recently, in several infectious murine models, it has been shown that in addition to dc located in tissues, de novo differentiating dc participate in the protective th1 immune response. the role of de novo differentiating dc in cross presentation is however poorly documented, and difficulties of human immunology prevent the accurate identification of the apc subsets patrolling for exogenous ag. a prerequisite for cross presentation is a moderate ag degradation rate in the endocytic pathway, allowing the generation of antigenic epitopes and their binding to mhc molecules. this prerequisite is of special importance considering dc precursors (such as monocytes), which are not yet dcs and may take up antigen before differentiating into dcs. the objective of our in vitro study is to evaluate whether ex vivo purified human blood monocytes are able to cross present long antigenic peptides to cd8+ t cells and whether they are able to sustain this cross presentation while differentiating into dcs. we have previously shown the unique property of dendritic cells to maintain for several days the capacity to stimulate cd8+ tumor-specific t cell clones when pulsed with long antigenic peptides (that need to be processed before presentation to cd8+ t cell clones, faure, 2009, eur j immunol 39 (2): 380-90). in the present study, we address the question of the mechanisms of long peptide cross-presentation by blood monocytes along the course of their in vitro differentiation into dcs. we have shown that despite their high degradative capacity, ex vivo purified monocytes pulsed with long peptides are able to stimulate cd8+ t cells after their in vitro differentiation into dc, 6 days following their antigenic pulse. the delineation of apc subsets able to sustain ag cross-presentation and t cell stimulating potential might be of clinical relevance in immunotherapy using synthetic long peptides. viral genomes contain alternative reading frames (arfs) encoding for mhc-i restricted epitopes (arf-epitope). in the siv/macaque model, ctl responses directed against arf-epitopes participate in controlling viral replication. we previously described that hiv-1 genome contains arfs within gag, pol and env genes encoding for a panel of hla-b*07 restricted epitopes. qprsdthvf (q9vf/5d) is one such epitope but its parental epitope qprsnthvf (q9vf/5n) has a significant higher frequency among hiv-1 isolates. strikingly, q9vf/5d-or q9vf/5n-specific ctls recognize apcs infected with hiv strains encoding for q9vf/5d (e. g. hiv lai ). in contrast, hiv strains (e. g. hiv nl-ad8 ) encoding for q9vf/5n do not activate ctl responses raising the possibility that q9vf/5n epitope is not presented by infected cells. we asked whether introducing mutations within q9vf might be a mean for the virus to escape ctl responses directed against this arf-encoded epitopes. we dissected the mechanism responsible for the lack of q9vf/5n mhc-i presentation. we modified hiv lai to introduce a d to n mutation in q9vf. introducing this single amino-acid mutation abrogated ctl recognition indicating that this asparagine (n) alters q9vf mhc-i presentation. we performed in vitro proteasomal digestions of 28mer peptides encompassing q9vf/5d or q9vf/5n and cleaved polypeptides were analyzed by mass spectrometry. the asparagine (n) in q9vf/5n is a preferential proteasomal cleavage site. thus suggesting that proteasome cleavages within q9vf/5n might be responsible for its lack of mhc-i presentation. we then sought in hiv-infected patients for the presence of proviruses encoding for q9vf/5d or q9vf/5n, and ctls responses directed against these epitopes. far thus, two out of three donors tested recognized the q9vf/5d peptide. we cloned and sequenced hiv-1 genomes from the three donors. surprisingly, out of 20 hiv proviral genomes isolated from pbmcs of q9vf/5d reactive donors, we could not find any virus bearing the q9vf/5d sequence. the isolated hiv sequences either encoded for q9vf/5n or had a stop codon within the epitope. in contrast, viruses encoding for q9vf/5d were isolated from pbmcs of the q9vf/ 5d nonreactive patient. altogether, our data suggest that ctls exert a selection pressure on viral arfs. hiv-1 seems to escape immune surveillance by introducing mutations altering processing of arf-derived epitopes. i. e. flesch 1 , y. wang 1 , d.c. tscharke 1 1 the australian national university, biochemistry and molecular biology, canberra, australia vaccinia virus (vacv) was the live vaccine used to eradicate smallpox and some strains are now being used as vectors for recombinant vaccines. cd8 + t cells recognizing viral peptides in association with mhc class i molecules on infected cells play a crucial role in the defence of viruses. despite the large number of possible mhc class i-peptide combinations, cd8 + t cells only recognize a small number of epitopes, a phenomenon called immunodominance. using recently defined cd8 + t cell epitopes for vacv in mice, we have investigated how heterozygosity of mhc class i molecules influences immunodominance patterns in h-2 bxd f 1 mice compared with their inbred parent strains. we find that the immunogenicity of vacv peptides defined using inbred mice is variable in f 1 progeny, with some peptides being almost equally immunogenic in f 1 and inbred mice, while others elicit responses that are reduced by more than 90 % in f 1 mice. during acute infection as well as memory responses, the dominance hierarchy in inbred mice did not predict the epitopes that would be poorly immunogenic in f 1 mice. in line with these findings, a multiepitope construct expressed by a recombinant vacv was less immunogenic in f 1 mice than would be predicted from its performance in parent strains. in terms of mechanism, we find evidence of altered tcr repertoires including in the case of one epitope, the loss of many diverse tcr vb clones and outgrowth of cd8 + t cells with a restricted vb usage in f 1 mice. these data have implications for our interpretation of experimental vaccine work done in inbred mice and for our understanding of how mhc diversity can alter the range of epitopes that are immunogenic in outbred populations. objective: tlr ligands are being exploited as potential adjuvants, and have impact on the antigen processing and presentation by dendritic cells (dc). therefore we aimed to study the efficacy of a tlr2 agonist, s-[2,3-bispalmitoyiloxy-(2r)-propyl]-r-cysteinyl-amido-monomethoxyl polyethylene glycol (bppcysmpeg), a synthetic derivative of the mycoplasma macrophage activating lipopeptide (malp-2), as an adjuvant for cross-priming against cellular and soluble antigens. malp-2 has been characterized as an effective mucosal adjuvant and synthesis of bppcysmpeg further improved solubility and pharmacokinetic features of the adjuvant. methods: dc isolation, in vitro and in vivo t cell stimulation, intracellular cytokine staining, in vivo cytotoxicity assays. results: systemic administration of bppcysmpeg induced maturation of cd8 + and cd8 -dc in the spleen resulting in enhanced cross-presentation of intravenously co-administered soluble antigen in mice. in addition, administration of bppcysmpeg and cell-associated ova resulted in generation of an effective ctl response against ova in vivo in a t-helper cell-dependent manner, but independent of ifna. delivering antigenic peptides directly linked to bppcysmpeg led to superior ctl immunity as compared to giving antigens and adjuvants admixed. in contrast to other tlr ligands such as cpg, systemic activation of dc with bppcysmpeg did not result in shutdown of antigen presentation by splenic dc subsets, although cross-priming against subsequently encountered antigens was reduced. we provide evidence that bppcysmpeg stimulation of dc via tlr2/6 results in the generation of an effective ctl response and that delivering antigenic peptides linked to bppcysmpeg is a promising strategy for vaccination. while bppcysmpeg-matured dc retain their antigen uptake and presentation capabilities, cross-priming against subsequently encountered antigens is inhibited, indicating that mechanisms beyond down-regulation of macropinocytosis and phagocytosis contribute to shut-down of cross-priming after tlr-mediated dc maturation. altogether our study promotes synthetic lipopeptides as potential adjuvant for specific applications (e. g. viral infections, cancer) for the reason that they can be chemically engineered to carry specific antigenic peptides which allows targeting of antigens and simultaneous activation. tumor immunevasion. to verify whether the loss of erap1 expression could confer a survival advantage on tumor cells and enhance tumor progression, we stably knocked down expression of eraap (murine erap1) in a murine t lymphoma cell line, rma. we used a method that allows an efficient and continuous expression of mirnas that directly silence eraap and obtained several eraap-deficient rma clones with different levels of eraap expression (up to 90 % of reduction at the protein level). microsomal aminopeptidase activity and mhc class i surface expression were decreased in all clones proportionally to eraap expression. moreover, low expression of eraap affected the stability of mhc class i molecules as evaluated after acid and brefeldin a treatment. de-regulated er peptide trimming also drastically affected the tumor formation of rma cells and host survival. eraap-deficient rma clones with different levels of eraap, 50 and 10 % as compared to control rma cells, were injected s. c. in the flank of c57bl/6 syngenic mice, and analysed tumor growth. all mice injected with control rma cells developed a tumor but survived up to 45 days after injection. all mice injected with rma clone with a 50 % level of eraap expression developed a tumor and died within 23 days after injection. surprisingly, any animal injected with rma clone with a 10 % level of eraap died or showed a visible tumor. thus, knockdown of eraap expression appears differently to affect the immunogenicity of rma cells, depending on the eraap silencing level. hemophilia a is an x-chromosome-linked bleeding disorder caused by the absence or dysfunction of clotting factor viii (fviii). treatment consists of regular administration of fviii, but is complicated by the formation of inhibiting antibodies against fviii. both genetic and treatment-related factors play a role in the etiology of inhibitor development in patients with hemophilia a. the development of inhibitory antibodies in hemophilia a patients has been shown to be a cd4 + t-cell driven process. therefore, in order to better understand the process of inhibitor formation, we aim to identify the epitope specificity and phenotype of t cells against fviii in hemophilia a patients using mhc class ii tetramers. cd4+ t-cell responses of two monozygotic twins with severe hemophilia a were analyzed. one of these subjects developed a high titer inhibitor (207 bu/ml) following intensive factor viii (fviii) treatment. high dose immune tolerance therapy together with anti-cd20 therapy resulted in eradication of the inhibitor. in contrast, his twin brother developed a low titer inhibitor (2.5 bu/ml) which declined rapidly after tolerance induction. fundamental differences in the twins' antibody responses were further suggested by elevated and persistent igg4 levels in the subject with the high titer inhibitor. in order to gain a better understanding of processes leading to inhibitor formation versus tolerance, we investigated drb1*0701-restricted t-cell responses of the high titer inhibitor subject, using fluorescent mhc class ii tetramers loaded with 20-mer synthetic fviii peptides to stain epitope-specific cd4+ cells.cd4+ t-cells from the high-titre inhibitor subject recognized three peptides corresponding to the fviii a2 domain: fviii 405-424 , fviii 421-440 and fviii 653-672 , as well as the c1 domain peptide fviii 2093-2112 , but not any c2 domain peptides. the c1 domain peptide contains a sequence that was reported as a promiscuous t-cell epitope (jones td et al., j thromb haemost.85:123-33, 2005 ). analysis of t cells from the lower titer inhibitor subject is expected to reveal differences in the epitope specificity and phenotypes of t cells that may underlie the discordant immune responses of these twins to infused fviii. m. forloni 1 , s. albini 1 , m.z. limongi 1 , l. cifaldi 1 , d. fruci 1 1 ospedale pediatrico bambin gesù, rome, italy neuroblastoma (nb) is a pediatric tumor that derives from neural crest. the most aggressive forms are characterized by amplification of the mycn oncogene and severe reduction of hla class i expression. mycn has been claimed to hinder hla class i expression through affecting the expression of the transcription factor p50 nf-kb subunit. since in many human tumors the expression of hla class i molecules is positively co-ordinated with that of er aminopeptidases, erap1 and erap2, we wondered whether in nb cell lines mycn may impair expression of these aminopeptidases. to explore this possibility, nb cell lines that differ in mycn expression were quantified for expression of mycn, erap1, erap2 and hla class i heavy chains by western blotting and for surface hla class i expression by flow cytometry. we found that mycn negatively correlates with expression of hla class i, erap1 and erap2. this negative correlation was confirmed in a nb cell line expressing a tetracycline repressible mycn transgene. then, by the use of tnfa (a nf-kb nuclear translocation stimulator), sulfasazine and ikba mutant (two nf-kb nuclear translocation inhibitors) and knockdown of p65 nf-kb subunit, we demonstrated that nf-kb is involved in erap1 and erap2 expression in nb cell lines and that mycn does not affect nf-kb expression. furthermore, we showed that mycn and nf-kb are recruited to the promoter regions of erap1 and erap2 and that mycn affects the recruitment of nf-kb binding to these promoter regions. in conclusion, the present results indicate that an enhanced mycn level, linked or not to mycn amplification, represses erap1, erap2 and hla class i expression in nb cell lines by affecting the recruitments of nf-kb binding to their promoters. s. brosch 1 , s. tenzer 1 , h. schild 1 , e. von stebut-borschitz 1 1 uniklinik mainz, mainz, germany infection of inbred mouse strains with the intracellular protozoan parasite leishmania major either leads to self-healing cutaneous disease (resistant phenotype; e. g. c57bl/6 mice) or systemic disease (susceptible phenotype; balb/c mice) depending on the genetic background of an individual. healing of leishmania infections is based on th1 immunity, whereas ifng secretion of both cd4 + th1 and cd8 + tc1 cells is critically important for protection by inducing oxidative radicals in macrophages, which enables them to kill the parasite. stimulation of antigen-specific effector t cells is driven by l. major-infected dendritic cells (dc) in an il-12-dependent manner. proteasome/immunoproteasome-dependent antigen processing is necessary for clearance of viral or intracellular parasitic diseases to induce effective cd8 + t-cell responses via the mhc class i. here, we analysed the role of the ifng inducible immunoproteasome for the priming of cd8 + t cells in l. major infections. using an in vivo model, we show that the functional knock-out mouse in the chymotrypsin-like catalytic domain of the immunoproteasome lpm7 (lmp7 -/-) does not exhibit an altered course of infection (lesion development, parasite loads, cytokine profiles) in intradermal, low dose infections with l. major mimiking natural transmission of the parasite as compared to wild type c57bl/6 mice. in addition, ex vivo co-cultures with infected dc from either lmp7 -/or wild type mice together with antigen-specific t cells from infected wild types showed no differences in tc1 cell ifng secretion and the dc restimulatory capacity of cd8 + t cells. furthermore, significant differences in the proliferation of antigen-specifically restimulated (with soluble leishmania antigen; sla) cd8 + t cells, isolated from low dose infected c57bl/6 wildtype or lmp7 -/mice, were not detected. in summary, our data indicate that despite the fact that cd8 responses in l. major infections are important for disease outcome, processing of antigen and thus priming of cd8 + t cells against l. major is independent of the lmp7 subunit of the immunoproteasome. studies have defined an essential requirement for autoantigen-specific b cells as antigen presenting cells in rheumatoid arthritis. however, the cellular mechanisms involved in antigen processing and presentation of joint-derived autoantigens by b cells are unknown. in this study we have developed a system to investigate how antigen-specific b cells recognise and present the proteoglycan aggrecan, a major component and candidate autoantigen of joint cartilage. we have utilised these cells to characterise the mechanisms by which aggrecan-specific b cells could induce autoimmunity. we have constructed plasmids encoding an aggrecan-specific b cell receptor and have transfected them into the b cell line a20, generating b cell lines that specifically recognise and target aggrecan for presentation to t cells. in addition, we have established conditions for a panel of aggrecan-specific t cell hybridomas to recognise aggrecan pulsed b cells following fixation, to allow the kinetics and mechanisms of aggrecan processing to be studied. we used inhibitors of mhc class ii transport, endosomal ph and enzymes involved in aggrecan degradation. we found that aggrecan-specific b cell lines presented the major arthritogenic cd4 + t cell epitope (84-103) from the g1 domain of aggrecan 10,000 times more efficiently than non-specific b cells and over 100 times more efficiently than the macrophage line j774. however, despite this highly efficient aggrecan capture, processing and presentation of the 84-103 epitope took at least 5 hours, comparable to the time required for presentation of aggrecan by j774. treatment of aggrecan-specific b cells with ammonium chloride to raise endosomal ph or brefeldin-a to disrupt golgi transport inhibited presentation of the 84-103 epitope, suggesting a requirement for low endosomal ph and presentation by newly synthesised mhc class ii. interestingly, aggrecan presentation by antigen-specific b cells was also reduced by phenanthroline, an inhibitor of the aggrecan-degrading metallo-proteinases that are found in abundance in the arthritic synovium understanding the mechanisms of antigen processing and presentation by autoantigen-specific b cells may explain their role in the pathogenesis of diseases such as rheumatoid arthritis. tapasin is an mhc-dedicated chaperone that facilitates peptide loading and optimization of the peptide cargo of mhc class i molecules within the peptide loading complex (plc). class i molecules differ in their dependence on tapasin for efficient cell surface expression, dependence that is determined by the nature of amino acids at positions 114, 115 and 116 at the peptide binding groove. position 116 also determines the strength of tapasin binding and influences peptide specificity, but its precise effect is probably context dependent. the mhc class i antigen b27 is strongly associated to ankylosing spondylitis (as) and other spondyloarthropathies. hla-b27 subtypes differ in their dependence of tapasin for cell surface expression and incorporation into the plc. tapasin also modulates b27 folding but not maturation and although tapasin optimizes the constitutive peptide repertoire of b*2705, peptide loading is relatively independent of this chaperone. we analyzed the effect of b27 subtype polymorphism on tapasin binding and the correlation of this feature with the affinity of the peptide repertoires, the maturation kinetics and the folding efficiency of b27 subtypes. the association of b27 heavy chain with tapasin was analyzed in c1r cells transfected with hla-b27 subtypes and mutants by pulse-chase analysis and co-immunoprecipition with the monoclonal antibody pasta-1, which recognizes human tapasin. we also analyzed the global thermostability, as a measure of the stability of the peptide cargoes, and the optimization of the b27 peptide repertoire with thermostability assays, by pulse-chase analysis and immunoprecipitation with the me1 monoclonal antibody that recognizes b27properly folded b27/peptide complexes. the formation of fully assembled b27 molecules was analyzed by pulse-chase analysis and immunoprecipitation either with the monoclonal antibody hc10, which recognizes mhc class i free heavy chains (hc), or with me1. maturation was analyzed by pulse-chase analysis, immunoprecipitation with me1 and treatment with endoglycosidase h (endo h). hla-b27 polymorphic positions other than 116, both at the a and c/f pockets modulate tapasin binding and the optimization of the peptide cargo. the stability of the peptide repertoires critically influences the folding efficiency of b27 subtypes. from as early as the initial phases of infection, hiv is coated with complement (c) fragments and following seroconversion, the circulating virus forms immunecomplexes with igg and complement. recent in vitro experiments revealed differences with respect to productive infection of immature dendritic cells (idcs) with differentially opsonized hiv. the opsonization pattern of hiv may additionally have profound consequences for the outcomes of the antigen-presenting capacity of dcs and their ability to mount an adequate immune response. in this context, we compared the impact of differential hiv-opsonization on the antigen-presenting capacity of dcs and found that c-opsonized hiv triggered ctl responses, while igg-coated virus did not. these in vitro generated ctls showed an enhanced ifn-g secretion and recognized the help independent ctl epitope slyntvatl. c-generated ctls also degranulated upon stimulation with specific hiv peptides and were able to elicit antiviral activity against hiv-infected cd4 + t cells. our results indicate that c-opsonization of hiv drives the virus towards the mhc class i pathway in dcs, thereby promoting a more efficient stimulation of naïve cd8 + t cells. this ctl-stimulating property of c could be exploited when searching for a novel approach against hiv. igg isolated from patients with high titers of anti-ccp antibodies showed a cross-reactivity with hcit peptides. vaccination experiments supported a triggering role of hcit for the development of arthritis in mice model. conclusions: diamination process is significantly increased in patients with ra while carbamylation is suppressed. production of specific antibodies against diaminated residues in ra patients may have a modulating role for the development of autoimmune arthritis. the classical pathway of mhc class i antigen presentation involves cytosolic degradation of viral proteins by the proteasome. peptides generated entry the endoplasmic reticulum through the transporter associated with antigen processing (tap). previous reports have shown that viral epitopes are presented to ctl independently of tap in smaller viruses. we hypothesized that presentation of vacv by mhc class i might proceed by alternative pathways. the aim of this study was to characterize these alternative pathways in tap-deficient mice. our results show that ctl derived from c57bl/6 mice immunized with vacv, recognized tapdeficient dendritic cells infected with the virus. approximately 15 % of vacv global presentation in the context of h-2 b was independent of tap. in addition, vacv infection induced a virus-specific ctl response in mice deficient in tap. dendritic cells (dc) initiate robust ctl immunity via the presentation of antigen-derived peptides by surface major histocompatibility complex class i molecules (pmhc). two major dc subtypes have been described, cd8+ and cd8-dc, which differ in their mhci antigen presentation capacities. cd8+ dc are the major dc subset responsible for cross presentation (presentation of exogenous antigen by mhci), while cd8-dc display little cross presenting capacity. here, we examined the mhci antigen presentation pathway of cd8+ and cd8-dc in more detail. first, turnover (half-life) of total mhci at the cell surface of cd8+ and cd8-dc was determined. surprisingly, cd8+ dc exhibit rapid surface mhci turnover compared to cd8-dc (following culture in the presence of brefeldin a). following activation of dc with cpg, mhci levels at the surface of both cd8+ and cd8-dc were stabilized and no longer underwent rapid turnover. this suggests that cd8+ and cd8-dc differ in their regulation of surface mhci turnover and that this is subject to regulation by antigen-associated signals. second, we examined the ability of cd8+ and cd8-dc to generate pmhci complexes containing cross presented antigen. we utilized the model antigen ovalbumin (ova) and an antibody that can detect h-2kb loaded with the ova-derived peptide, siinfekl. cd8+ and cd8-dc isolated from ova-expressing mice (actin-ova transgenics) displayed abundant kb-siinfekl complexes at their cell surface. in contrast, in response to exogenous soluble ova protein, only the cd8+ dc, but not the cd8-dc, displayed kb-siinfekl complexes at the cell surface. similarly, when dc were pulsed with ova-coated splenocytes, kb-siinfekl complexes were only detected on the surface of cd8+, and not cd8-dc. this data further validates the role for cd8+ dc as the major cell type responsible for cross presentation and provides insight into the mechanisms that prevent other dc subsets from accessing the important cross presentation pathway. objectives: the action radius of matrix metalloproteinases or mmps is not restricted to massive extracellular matrix (ecm) degradation but extends to the proteolysis of secreted cytokines and membrane-bound receptors and adhesion molecules. although many instances exist in which cells disintegrate, often in conjunction with induction of mmps, the intracellular mmp substrate repertoire or degradome remains relatively unexplored. the aims of the present study were to identify novel intracellular mmp targets and to answer the question whether the proteolytic modification of intracellular proteins alters the immunogenicity of released intracellular contents. methods: multidimensional degradomics technology was developed by the integration of broadly available biotechniques and applied to thp-1 cytosol using gelatinase b/mmp-9 as a model enzyme. in the first dimension, ion exchange chromatography separated the thp-1 proteins by their net charge and/or isoelectric point (pi) followed by cleavage of the proteins by mmp-9. in the second dimension, potential substrates were separated by molecular weight on sds-page. to evaluate the effect of proteolysis by mmp-9 on the immunogenicity of the intracellular protein pool, mice were immunized twice with thp-1 cytosol in complete freund's adjuvant. lymph node t cells were isolated and stimulated with mmp-9-cleaved or intact thp-1 cytosol. proliferation was assessed by measuring incorporated 3 hthymidine. results: 100-200 mmp-9 candidate substrates were isolated, of which 69 were identified, revealing many novel mmp-9 (candidate) substrates from the intracellular matrix (icm), such as actin, tubulin, stathmin,... about 2/3 of the identified substrates were described as systemic autoantigens in one or multiple autoimmune conditions. remarkably, a significantly lower t cell proliferation was observed in the presence of cleaved vs. intact cytosol. conclusion: multidimensional degradomics technology is a valuable tool for high-throughput identification of novel mmp substrates. proteolysis by mmp-9 decreased the immunogenicity of the intracellular contents, suggesting that mmps may contribute to the dampening of inflammation by the clearance of toxic and immunogenic burdens of intracellular (matrix) proteins released after extensive necrosis and tissue injury. a preference for hla-a versus -b molecules; e3-19k did not detectably associate with hla-c molecules under identical conditions. this locus specificity may provide a functional advantage to ads by inactivating t-cell receptors, while avoiding activation of nk receptors. finally, we showed that residue 56 in hla-a11 and residue 93 in e3-19k are highly critical for association of both proteins. this defines a putative interaction surface between e3-19k and class i molecules. conclusions: our studies provide novel insights into the functional relationship between e3-19k and the class i antigen presentation pathway. moreover, because soluble e3-19k can differentiate between polymorphic gene products encoded in the mhc, our results may contribute to define paradigms for how class i substrate specificity is established for er retention. overall, our studies represent an important step towards a molecular understanding of the strategy evolved by ads to establish life-long persistence in host cells. objectives: the transporter associated with antigen processing (tap) belongs to the abc transporter superfamily and is a heterodimer consisting of the two subunits tap1 and tap2. tap transports peptides yielded by proteasomal degradation from the cytosol into the endoplasmic reticulum (er) and is thus a key element of the mhc class i antigen processing machinery (apm). methods: target-specific tap knock downs were generated by shrna technology. the resulting transfectants were subsequently analysed regarding mhc class i surface expression using flow cytometry, whereas mrna and protein expression levels of tap1 and tap2 were analysed by rt-pcr and western blots, respectively. furthermore, the protein stabilizing effect of tap1 on tap2 was investigated in the presence of two distinct proteasome inhibitors. results: previous findings obtained with rare tap1 mutants suggested that the lack of tap1 protein expression is associated with a strong reduction of tap2 protein levels, which could be restored by tap1 gene transfer, whereas no such regulation is found vice versa. to investigate this stabilizing effect of tap1 on tap2 different shrna plasmids specifically targeting tap1 or tap2, respectively, were stably transfected into constitutively tap1 and tap2 expressing hacat keratinocytes and colo794 melanoma cells. in both cell types the shrna-mediated tap1 and tap2 inhibition resulted in a significant downregulation of the respective transcript and protein expression levels. the knock down of tap1 caused not only an almost complete loss of tap1, but also a strong decrease of tap2 protein expression. in contrast, the tap2 knock down exhibited no influence on the tap1 expression. specific inhibition of the proteasome prevented the degradation of tap2 in the tap1 knock down variants. the results of our study emphasize that an unidirectional stabilisation of tap1 on tap2 protein expression is not restricted to rare tap1 mutants, but rather suggest a common regulatory mechanism for the tap complex. uv injury profoundly affects the skin immune homeostasis by promoting strong inflammation and cellular immuno-modulation. in this study, we characterized the inflammatory cell subsets that emigrate in the epidermis the days following uv exposure. therefore, the buttock skin of 27 healthy volunteers was exposed twice to 1.5 minimal erythema dose of uv. blister roofs were then collected before and 1, 4 and 10 days after uv-exposure from un-exposed and exposed skin and the resulting epidermal cells were analysed by flow cytometry. we demonstrated that, along with the rapid activation and migration of langerhans cells (lc), uv skin radiation exposure promotes the infiltration into the epidermis of a monocytic cd14 + cd36 + and of a macrophagic cd14 -cd36 + cell subsets that emerge 1 and 4 days post exposition, respectively. more importantly whereas classical cd1a hi cd207 + lc are the unique dendritic cell (dc) subset found in the epidermis of unexposed skin, we detected two new subsets of epidermal dc namely cd1a low cd207and cd1a low cd207 + that emerged 1 and 4 days post irradiation, respectively. these two distinct populations of epidermal dc (edc) differ from classical epidermal lc by their activation/maturation profile as assessed by the strong expression of cd86 and hladr. finally, 10 days post-exposure, we observed that lc represented almost the only haematopoietic cell population in the epidermis. these results suggesting that the uv-recruited edc and monocytic/macrophagic subsets participate to the progressive recovery of the epidermal immune cell network homeostasis. i. bailey 1 , e. reeves 1 , t. elliott 1 , e. james 1 1 university of southampton, school of medicine, cancer sciences division, southampton, united kingdom there is accumulating evidence that cd4+ cd25+ regulatory t cells (treg) play an important role in anti-tumour immunity by preventing effective t cell responses to tumour antigens. tregs have also been shown to inhibit development of organ-specific autoimmune diseases suggesting they inhibit immune responses to tissue-specific self-antigens. the depletion of tregs prior to challenge with the murine colorectal tumour, ct26, stimulates a robust, protective t cell response which is also protective to challenge with other tumours of different histological origins, such as b cell lymphomas and a renal cell carcinoma. this cross protection has not been seen with other tumour cell lines. we have identified a ct26-derived cross-protective antigen, gsw11, which was found to be encoded within the ectotropic murine leukaemia virus (emv-1) envelope protein, gp70. this protein has previously been shown to encode ct26-specific cd8 and cd4 antigens, implicating it as a 'hot-spot' for ct26 tumour antigens. interestingly, we have identified a truncated version of gp70 which may be responsible for generation of gsw11. expression studies have revealed increased gp70 expression in ct26 compared to other tumour cell lines, indicating the ability to cross-protect is related to the quantity of antigen (gsw11) generated. the current knowledge of hla class ii antigen presentation and peptide binding is mainly based on studies of hla-dr molecules. they contain a large hydrophobic p1 pocket, which can accommodate large hydrophobic amino acid residues and is the most important pocket in selecting and binding peptides, while p4, p6 and p9 tune the peptide repertoire that can be presented by individual hla-dr alleles. the same rules and requirements do not necessarily exists for peptide binding to hla-dp molecules, however. the present study adresses this issue. we have expressed and affinity purified soluble recombinant hla-dp2 molecules from drosophila melanogaster cells and studied its binding of a number of peptides known to bind hla-dr, -dq or dp molecules. unexpectedly, the immunodominant epitope in multiple sclerosis (ms), the myelin basic protein derived peptide, mbp85-99, bound to hla-dp2 with high affinity (10-30 nm). binding studies of mbp85-99 derived peptides containing single alanine substitution at each position revealed that only three of the peptides (f91a, f92a and k93a) were affected, and only by a 20-50 fold reduction in affinity for hla-dp2. the observation that none of the substitutions resulted in a complete loss of binding to hla-dp2 indicates that 1) hla-dp2 binding to peptides does not depend on a large hydrophobic residue accomodated in p1, or 2) mbp85-99 can bind in more than one register. we will present data addressing this issue. the hla-dp peptide binding capacity was increased at neutral as compared to acidic ph, and by the presence of n-butanol, a small organic mhc loading enhancer (mle). in summary, the hla-dp2 molecule binds the immunodominant epitope in ms, mbp85-99, possibly in more than one register. additional studies are required to resolve the hla-dp2 peptide binding properties, and to determine whether expression of hla-dp2 affects the disease course in ms patients. results: depletion of mncs for cd14+ cells abrogated the tg-induced cytokine production and proliferation of cd4+ t cells, indicating a primary role for monocytes as apcs. however, the encounter of t cell with antigens presumably occurs in b cell-rich compartments such as lymph nodes or lymphoid tissue in inflamed organs. to mimic the conditions prevailing there, we depleted pbmcs for g 98 % of the monocytes (without significant loss of t-cells), and compared the tgelicited t-helper cell responses in the presence and absence of b cells. the tg-induced cd4+ t cell proliferation was significantly reduced in cd14/cd19-depleted mnc cultures, as compared to cultures depleted for cd14+ monocytes alone. the same applied to the production of il-2, il-6 and tnf-a. production of ifn-g and il-10 was generally not observed. our data indicate that normal b cells are capable of inducing a pro-inflammatory cytokine response in mnc-cultures, where monocytes and monocyte-derived cells are not preponderant. studies addressing the relative contributions to this cytokine production, by b cells themselves and by t cells (following antigen-presentation by b cells), are in progress. j. kyosiimire-lugemwa 1,2 , p. pala 1 , g. miiro 3 , j. todd 3 , p. kaleebu 1,2 , n. imami 2 , f. gotch 2,3 1 mrc uganda, basic science, entebbe, uganda, 2 imperial college, london, united kingdom, 3 mrc uganda, entebbe, uganda background: hiv-1-specific t-cell responses are preserved in hiv-1 infected individuals with non-progressing hiv-1 disease. "long term non progressors" (ltnps) were defined as art naï ve individuals infected with hiv-1 for g 8 years, maintaining cd4+ t-cell counts g 500, and with minimal cd4+ decline over time. we tested the hypothesis that gag-specific t-cell responses are inversely correlated to disease progression whereas nef-specific t-cell responses are not. methods: 17 art naï ve hiv-1 infected patients from the entebbe cohort in uganda were recruited and stratified by cd4+ t-cell count, cd4+ decline slopes, and time of enrolment, into 2 groups -10 ltnp and 7 rapid progressors (rp). all patients were women reflecting the patient base at the entebbe cohort. we measured plasma viral load, current cd4 t-cell count, and ifn-g, il-2 and il-4 elispot responses to pools of 22 to 34 peptides (18-mers overlapping by 10aa). peptides were based on consensus sequences of gag and nef from hiv-1 clades a1, a2 and d. medians and inter-quartile ranges were calculated and comparisons between groups were performed using the mann-whitney u test. correlations were presented using spearmann's linear correlation coefficients. results: some gag-specific ifn-g and il-4 responses were significantly higher in the ltnp than in rp (p=0.02, ifn-g responses to gaga2 pool 1; p=0.04, il-4 responses to gaga1 pool 2). il-2 responses were low and not significantly different between ltnp and rp. there was a positive correlation between il-4 responses to gaga1 pool 2 and cd4 t-cell counts (r 2 =0.502, p=0.04), but no correlation between either il-4 or ifn-g responses and viral load. cytokine responses to nef peptides were not significantly different between the ltnp and rp. conclusion: overall, gag hiv-1 specific responses were higher in ltnps than in rps confirming previous results. non-specific il-4 responses were high possibly reflecting baseline th2 responses to helminths a common environmental exposure in the study population. objectives: in human and murine tumors and in in vitro oncogene-transformed cells defects in the expression of components of the hla class i antigen processing machinery (apm) have been described, which were associated with a reduced antigenicity of these cells. so far, the molecular mechanisms of such defects have not been elucidated in detail. to investigate whether impaired apm component expression was due to altered transcription and associated with cell growth properties murine her2/neuand her2/neu + fibroblasts were employed. methods: using tapasin as a model molecule its cell cycle-dependent expression was analysed in a time kinetics upon serum starvation followed by stimulation with complete culture medium over time. cells were harvested at different cell cycle phases and expression of tapasin was analyzed by qrt-pcr and western blot. flow cytometry was employed for determination of the distinct phases using 7-aad for dna analysis and specific antibodies directed against the proliferation marker ki-67, the m-phase specific phistone h3 as well as for the h-2l d surface antigens. in addition, chromatin immunoprecipitation (chip) experiments with an antibody directed against rna polymerase ii were performed to investigate the transcriptional levels of tapasin in her2/neuversus her2/neu + cells. results: serum starvation and subsequent stimulation with complete culture medium led to the enrichment of cells at the g 0 /g 1 -, s-and g 2 /m-phases of the cell cycle, which was associated with an altered tapasin transcription during the cell cycle. tapasin mrna level decreased during cell cycle progression, whereas an inverse protein expression was observed with low expression levels at the g 0 /g 1 -phase, which continuously raised and peaked within the s-phase. however, h-2l d surface antigen expression was not altered in her2/neucells during cell cycle progression. in contrast to her2/neufibroblasts the her2/neu + transfectants exhibit a decreased tapasin transcription, which was accompanied by an altered h-2l d surface expression. this was confirmed by a reduced promoter activity and decreased accessibility of the rna polymerase ii to the tapasin promoter. conclusion: these findings lead to an improved understanding of immune escape mechanisms demonstrating a cell cycle dependent and oncogene-mediated tapasin regulation that may provide novel targets for therapeutic intervention. recent studies suggest that dendritic cells (dcs) are key players in shaping the respiratory syncytial virus (rsv) specific immune response. before, dcs within the airway epithelium were characterized as langerhans cells. in this study, in vitro counterparts of langerhans cells expressing langerin (cd207) and ccr6 were cultured from cd34+ stem cells under the influence of tgf-b (tgf-b-dcs) and compared to cd34+ derived dcs, which passed through a monocytic stage . after infection with rsv, both types of dcs generated viral rna and viral-proteins. although tgf-b-dcs expressed higher levels of viral proteins as revealed by flow cytometry and fluorescence microscopy, more than hundredfold more viral particles were released by il-4-dcs. the increased expression of viral proteins is most likely responsible for the pronounced inhibition of t-cell functions by tgf-b-dcs. since there is evidence that langerhans cells are expressed in airway epithelium not before the age of one year, the results may indicate, that an inhibition of rsv replication is characteristic of a more mature answer against rsv. the occurrence of inhibitory antibodies against exogenous factor viii (fviii) remains the major concern of fviii replacement therapy in patients with hemophilia a. initiation of the immune response implies the endocytosis of fviii by professional antigen presenting cells (apcs): b lymphocytes, dendritic cells (dcs) and macrophages (mø). the organ where the anti-fviii immune response is initiated and the type of apcs involved in this process have not been investigated. we hypothesized that the spleen, which is the principal filter for blood-born antigens, is the principal organ where apcs interact with fviii to initiate the anti-fviii immune response. we first administered radiolabeled fviii at therapeutic doses to fviii-deficient mice. fviii was found to preferentially accumulate in the spleen and liver of the mice. levels of fviii in the spleen remained stable for up to 45 min following fviii administration, while they rapidly decreased in the liver. unlabelled fviii was then administered to fviii-deficient mice that had been splenectomized or sham operated and the anti-fviii humoral responses were compared. removal of the spleen resulted in significantly reduced levels of anti-fviii igg. using flow cytometry, fviii was found to preferentially accumulate with splenic mø than dcs and b cells. elimination of apcs by treatment of the mice with clodronate-containing liposomes prior to fviii administration resulted in a drastic reduction of the anti-fviii igg response, as compared to control mice treated with pbs-containing liposomes. taken together, our results suggest that the spleen is the principal organ in the initiation of the anti-fviii immune response and that splenic mø have an important part in this process. the interactions between antigen presenting cells (apc) and t-lymphocytes are a relevant current issue. the area of contact between an antigen presenting cell and a t-lymphocyte is termed immunological synapse (underhill et al, 1999) . the present work started, in experiments with leukocyte of healthy individuals from the finding that under certain experimental conditions, cell-cell association with closely contact between monocyte-derived macrophages and human autologous lymphocytes are produced when the cells are harvested from total leukocyte cell cultures. in this way, such cells selective forming rosettes with a central macrophage and adherent lymphocytes. objectives: as central hypothesis it was postulated that the phenomenon would be due to antigen presentation made (performed) by macrophages to lymphocytes and that would be t cells. methods: autologous total human leukocyte cultures, from samples of 30 healthy blood donors were harvested at various times and centrifuged and performed as previously reported (cabral and novak, 1992, 1999) . cytopreparations of each experiment were performed. statistical analysis: regression model. results: experimentally, it was found a) phagocytosis of autologous antigens by macrophages, stimulates the formation of rosettes, b) a linear relation between rosettes formation and culture-time occurs, (p x 0,0001), anova for regression, c) the cell-cell approximation is very important and was performed by centrifugation of the cells to form pellets, d) the forming rosettes lymphocytes are t-cells, cd4+, e) purified macrophages and lymphocytes produced few rosettes, however if antigens were added, the phenomenon was stimulated, f) if inhibitors of the antigen processing and antigen presentation, such as chloroquine or brefeldin a, were added, rosettes were not formed, g) monoclonal antibodies anti-human mhc ii precluded the formation of rosettes, h) gangliosides diminishes rosettes formation. conclusion: taken together, the findings suggest that the model of rosettes formation might be useful to study cell-cell interactions during antigen presentation in immunological synapses and other immunologic aspects on the cells involved, in short time assays. objective: to investigate if patients with multiple sclerosis (ms), without the typical increase of antibodies in csf, are less likely to develop neutralizing antibodies (nabs) against ifnb-treatment, and whether the absence of such an immunological response might reflect a difference in their antigen presenting ability due to a distinct genetic background. methods: overall, 2252 patients were obtained from the swedish multiple sclerosis registry and the swedish nab registry, and treatment information was available for 2207 of them. for 538 of these patients hla-drb1 data was available. results: a significant correlation between lack of antibodies in csf and nab-negativity was found (p=0.02). patients without csf antibodies were to a lesser extent nab-positive when treated with the ifnb-1a preparations, whereas no differences were shown for ifnb-1b. an association between hla-drb1*11 and nab-negativity was detected (p=0.028). the known associations between hla-drb1*15 and csf-positive ms and hla-drb1*04 and csf-negative ms were confirmed. conclusion: we show for the first time that patients without antibodies in csf have a different propensity to induce nabs compared to csf-positive patients, indicating an extended immunological difference between the two ms sub-groups. hla-drb1 potentially contributes to this, which indicates that it might have something to do with differences in antigen presentation. in csf-negative patients the reaction against ifnb-1a molecules, possibly through a t-cell dependent pathway, is lower than for csf-positive patients. however, reaction against ifnb-1b, which might also be activated through a t-cell independent pathway, shows no difference in seroprevalence between the groups. abstract withdrawn by author tyrosinase-derived epitope was confirmed by five independent assays: flow cytometry on multiple melanoma lines generated from patients, confocal microscopy immuno-staining of melanoma lines, frozen sections staining of authentic melanoma tissue from patients, cytotoxicity assays using tyrosinase-specific ctls, and finally mass spectrometry analysis of peptides isolated from a melanoma cell line. there was no correlation between the level of antigen presentation and mrna expression levels for the three antigens; however, our data suggest that tyrosinase protein stability may play a major role in the high level presentation of this antigen. measurement of the half lives of these proteins revealed a hierarchy in protein stability, with mart-1 and gp100 more stable than tyrosinase. by the use of the cofactor dopa, which stabilizes the tyrosinase protein, significant decrease of hla-tyr complexes presentation was achieved. in addition to the study of antigen presentation, these tcr-like antibodies can also actively participate in immunotherapy as targeting molecules, considering their high affinity and specificity. by generating a whole igg antibody, tumor cell lysis was achieved by antibody-dependent cell-mediated cytotoxicity (adcc). with the addition of point mutations in the fc fragment, which increased the affinity of the fc to the fc receptor, enhanced tumor cell lysis was achieved. g. schiavoni 1 , s. lorenzi 1 , f. mattei 1 , f. spadaro 1 , l. gabriele 1 1 istitituto superiore di sanità, cell biology and neurosciences, rome, italy cross-presentation is a crucial mechanism for generating cd8 t cell responses against exogenous antigens (ag), such as dead cell-derived ag, and is mainly fulfilled by dendritic cells (dc), particularly cd8a + dc. however, apoptotic cell death occurring in steady-state conditions is largely tolerogenic, thus hampering the onset of effector cd8 t cell responses. type i ifn are a family of cytokines induced upon infection and acting as danger signals by stimulating multiple arms of the immune response. in particular, type i ifn have been shown to promote the cross-priming of cd8 t cells against soluble or viral antigens, partly through the stimulation of dc. in this study we evaluate the role of type i ifn to affect dc capacity to capture and cross-present apoptotic cell-derived ag. by using uv-irradiated ova-expressing eg7 thymoma line, we show that type i ifn promote the ability of cd8a + dc to capture apoptotic eg7 cells and to undergo phenotypic activation, both in vitro and in vivo. remarkably, ifn-treatment prolongs the survival of ag-bearing cd8a + dc and the persistence of apoptotic eg7-cell ag within the phagosomal dc compartment, a process that is known to facilitate the recruitment of ag into the mhc-i presentation pathway. accordingly, type i ifn-treatment increases cross-presentation of apoptotic eg7-derived ova ag by dc, as revealed by higher expression levels of siinfekl peptide in association to mhc-i molecules on cell surface of phagocytic cd8a + dc. as a result, eg7-loaded dc become competent at inducing ot-i cd8 t cell proliferation and activation both in vitro and in vivo. our data indicate that type i ifn promote the cross-presentation of apoptotic cell-derived ag by cd8a + dc and suggest that these cytokines may act as a switch signal for cross-presenting dc, thus skewing the immune response from tolerogenic to immunogenic. (2). we have investigated the mechanisms of cross-presentation of soluble antigen in freshly purified splenic dc subsets. using biochemical methods, we show that only cd8+ dc efficiently transfer soluble antigen to their cytosol. the amount of antigen detected in the cytosol increased up to ten-fold after a short exposure to tlr ligands cpg, poly i:c or pam3csk4, and this correlated with enhanced cross-presentation. the increase in antigen accumulation within the cytosol was not due to increased uptake of antigen. measurement of the proteasome activity at different times after exposure to tlr ligands revealed that tlr signalling induced transient inhibition (maximum at two hours) of the proteasome in cd8+ dc but not cd8-dc, thus promoting accumulation of exogenous antigen in the cytosol of cross-presenting dc. this correlated with formation of aggresome-like structures only in cross-presenting dc exposed to tlr ligand. by limiting the degradation of transferred proteins during early activation, when endogenous proteins are being stored in aggresome-like structures, this mechanism could favour the loading of exogenous antigen peptides over endogenous peptides, promoting cross-presentation. to our knowledge this is the first report of a direct, immediate effect of tlr activation on proteasome activity. exosomes are nano-sized membrane vesicles of endosomal origin which can exert both immune stimulatory and tolerance inducing effects depending on their cellular origin. they are currently being investigated for use in vaccination and immune therapy strategies, but their physiological role has not been elucidated. here we explore whether exosomes of different origin can selectively target different immune cells. we compare the binding of exosomes from human breast milk, monocyte derived dendritic cells and b cells to peripheral blood mononuclear cells. flow cytometry, confocal laser scanning microscopy and multispectral imaging flow cytometry (imagestream) reveal that exosomes derived from human dendritic cells and human breast milk preferably associate with monocytes, whereas exosomes from an epstein-barr virus (ebv) transformed b cell line selectively target b cells. our data suggest a highly selective association between cells and exosomes which can be a way to direct their functional effects. one of the hallmarks of cancer cells is the resistance to cell death. it has been suggested that cancer cells also have the capacity to evade the surveillance by the immune system. the proteasome and macroautophagocytosis are attractive effector mechanisms for the immune system, because they can be used to degrade foreign substances, including pathogenic protein, within cells. here, we investigated that dm1, which is a saponin derivative isolated the stem bark of dracaena mannii induced autophagocytosis on a549 human lung cancer cell line. methods: dm1 induced cell cytotoxicity was measured by mtt assay and propidium iodide staining on a549 cells. we examined the morphological change using optical microscope. and gfp-lc3 punctation was measured using confocal. the protein expression was measured by western blot analysis and the mrna expression was measured using reverse transcription poly chain reaction (rt-pcr). results: dm1 was showed high cytotoxicity on various cancer cell line, especially a549 cells. dm1 treated a549 cells did not show regular dna fragmentation and caspases activation. we also analyzed protein expression of apoptotic marker, but protein level didn't change. as a result, we hypothesized that this non-apoptotic cell death is mediated autophagocytosis. we checked lc3 and beclin-1 protein and mrna expression and inhibitory effect of autophagocytosis inhibitor. the expression level of lc3 was increased concentration and time-dependently. beclin-1 also was increased. and then, we examined gfp-lc3 punctation on a549/ gfp-lc3 stable cells using confocal. a549 cells were formed gfp punctuation after dm 1 treatment. to confirm these data, we measured cell death ratio using 3-methyladenine (3-ma), an autophagocytosis inhibitor. after 3-ma treatment, dm1 induced cell death was decreased comparing with dm1 treatment. conclusion: dm1 did not induce apoptotic cell death. but dm1 showed several characteristics of autophagic cell death. taken together, dm1 induced autophagocytosis on a549 cells. these finding indicated that therapeutic potential of dm1 by triggering autophagic cell death. s. b. rasmussen 1 , s. r. paludan 1 1 university of aarhus, department of medical microbiology and immunology, aarhus, denmark during viral infections, different pattern recognition receptors detect specific pathogen associated molecular patterns (pamp)s. in the case of herpes simplex virus (hsv), detection is, among others, conducted by toll like receptor (tlr)9, which is a transmembrane receptor located in the endosomes where it detects unmethylated cpg rich dna of extracellular origin, e. g. viruses. upon binding to dna, tlr9 initiates downstream signalling cascades resulting in induction of antiviral cytokines, interferon (ifn)a and ifnb being some of the most essential ones. the exact route of hsv to the endosomes and thus tlr9 recognition is not clear-cut. the endocytosis pathway is believed to be a central mechanism in which viruses translocate to the endosome, but recently the role of autophagy in tlr mediated viral recognition has been drawn in to focus. we have found indications of an autophagy dependent ifn expression during hsv-1 infection. by use of an entry defect glycoprotein l and glycoprotein h deficient hsv-1, we found that tlr9 dependent ifn regulatory factor 3 activation was abrogated. in addition, inhibition by 3-methyladenine of phosphoinositol 3-kinase class iii, which is crucial in autophagosome formation, abrogates ifnb induction. these findings points to a role for autophagy in tlr9 dependent recognition. in the ongoing project we are examining how hsv-1 triggers formation of autophagosomes. especially the role of endoplasmatic reticulum stress and doublestranded rna-dependent protein kinase will receive attention. also the newly found involvement of ubiquitin and acetylation in autophagy execution and regulation will be investigated. j. zovko 1 , i. berberich 1 , gk 520 -immunomodulation 1 universität würzburg institut für virologie und immunbiologie, würzburg, germany members of the bcl-2 family control the integrity of mitochondria and thereby influence survival and death of cells. most bcl-2 family members can localize to intracellular membranes via hydrophobic sequences within their c-terminal portion. murine a1 and its human homologue bfl-1 are anti-apoptotic members of the bcl-2 family. a1 is expressed in small amounts in the bone marrow and immature b cells, but in high amounts in mature b cells. thus the protein seems to be important for b cell maturation. we analyzed the function of the c-terminus of a1. unless the c-terminal ends of other bcl-2 proteins the tail of a1 does not function as a strong membrane anchor. nevertheless, the last amino acids of a1 are important for the protein. in fact, the c-terminus of a1 serves a dual function by being required for the instability and the anti-apoptotic potential of the protein. we show that a1 undergoes proteosomal degradation controlled by its c-terminus. interestingly, binding to the proapoptotic bcl-2 factor bimel results in increased stability of a1. this is due to reduced ubiquitination of a1 after binding of bimel. we conclude that the cterminus of a1/bfl-1 serves as a docking site for e3 ubiquitin ligase(s) that control the stability of a1 by targeting the protein to the proteasomal pathway. very recently, we have identified a putative e3 ubiquitin ligase that interacted with a1 in a yeast two-hybrid screen. currently, we are trying to confirm this interaction in mammalian cells. suppressors of cytokine signaling (socs), initially identified as negative regulators of cytokine signal transduction, have recently emerged as multi-functional proteins regulating inflammation, survival, differentiation, and apoptosis of immune cells. here we describe novel function of socs-1 in the suppression of rosmediated apoptosis and associated mechanisms using tnf-alpha induced t cell apoptosis as a model system. both in jurkat t cells and primary splenocytes, socs-1 is induced under tnf alpha-induced apoptosis conditions. the tnf-induced apoptosis was mediated by generation of ros, and the over-expression of socs-1 significantly suppressed tnf-induced ros levels and the subsequent apoptosis. such anti-apoptotic function of socs-1 was manifest not only by the suppression of jaks acting upstream of p38 mapk, but also protection of ptps which down-regulate the tnf alpha -induced jak 1/2 activities. we further show that tnf-alphainduced ptp inactivation can be prevented by socs1 which up-regulates thioredoxin levels. finally we present data that there is a molecular interaction between thioredoxin and ptp which is formed in response to ros-generating stimuli and sustained in socs-1 overexpressing cells. the results strongly suggest that socs-1 acts as an anti-oxidant and anti-apoptotic factor to sustain t cell homeostasis and survival under oxidative stress imposed upon inflammatory cytokines during infection or other immune scenarios. aim: inflammation is a cardinal host response to injury, tissue ischemia, autoimmune responses or infectious agents. the effects of inflammatory mediators on the glial function are of particular interest since astrocytes contribute to the local inflammatory responses in the cns by producing cytokines, chemokines, and maintain local homeostasis clearing released neurotransmitters. cxcl12/sdf-1a not only regulates cell growth and migration of hematopoietic stem cells but may also play a central role in brain development. moreover, it has been described that tnf-a mediates in cxcl12 effects on primary astrocytes, and it is clear that tnf-a participates in the pathogenesis of many neurological conditions. methods: we used the astrocytoma cell line u-87, and sk-n-mc as neuroblastoma cells. detection of tnf-a mrna expression was carried out by rt-pcr. transcriptional activity was measured using luciferase reporter gene assays in transiently lipofectin transfected-cells. we performed cotransfection experiments of nfat promoter construct with a dominant negative version of nfat (dn-nfat). neuronal death was performed by mtt and tunel assays. nfat translocation was confirmed by western-blot. p53 and fas-l expression was carried out by western-blot. results: cxcl12 induced mrna-tnf-a and transcriptional activity of tnf-a promoter in human astrocytes. this cytokine by itself was not toxic when added directly to astrocytes, but when we investigated its effect on nb cultures, neuronal death increased in a direct and indirect way. surprisingly, tnf-a did not induce nf-kb activation in nb cells but it induced nfat activity. nfat translocation was confirmed by western-blot. neurotoxicity was absolutely reverted in the presence of ciclosporin. we discard p53 pathway as responsible for tnf-mediated toxicity since we did not find any alteration in p53-ser46 levels in stimulated cells. in addition, we found increased fasl expression in neuroblastoma cells after 24h of tnf-a treatment. conclusions: cxcl12 induced-tnf-a promotes nfat activation in neuroblastoma cells and this event leads to increased apoptosis through an increase of fasl expression without alter p53function/levels. s. y. demiroglu 1 , r. dressel 1 1 universitätsmedizin göttingen, zelluläre und molekulare immunologie, göttingen, germany objectives: intracellular hsp70 is part of the cellular stress response system and can inhibit specific apoptotic pathways. extracellular hsp70 on the other hand, is an immunological danger signal that can induce the antigen-specific activity of cytotoxic t lymphocytes (ctl). interestingly, hsp70 does not protect against cell death mediated by ctl. acute overexpression of hsp70 can even increase the susceptibility of target cells to ctl, which use the granule-exocytosis pathway to induce apoptosis (dressel et al. cancer res 63: 8212) . granzyme b is one of the main effector proteases of cytotoxic granules. therefore, we analyzed the effect of acute overexpression of hsp70 on granzyme b-induced apoptosis. methods: hsp70 was expressed in a human melanoma cell line under the control of a tetracycline-inducible promoter (ge-tet). the effect of hsp70 induction on granzyme b-mediated apoptosis was now analyzed after delivery of granzyme b into the cytosol of the target cells by the endosomolytic activity of adenovirus type 5. results: hsp70 did not protect the melanoma cells against granzyme b-mediated apoptosis when annexin v binding, loss of mitochondrial membrane potential, release of mitochondrial cytochrome c, and activation of caspase-3 were evaluated. instead, we observed a moderate but significant pro-apoptotic effect of hsp70 in ge-tet cells when late apoptosis was analyzed at the nuclear level by sub g1 peak measurements (p=0.01). in contrast, a partial protection of ge-tet cells was observed after acute hsp70 overexpression when apoptosis was induced by staurosporine. conclusion: acute overexpression of hsp70 does not seem to protect tumor cells from granzyme b-induced apoptosis; it appears even to accelerate the progression of apoptosis to dna fragmentation and nuclear condensation. it is conceivable that this hsp70 effect is mediated by chaperoning pro-apoptotic molecules and improving their function as it has been reported for the caspase-activated dnase (liu et al., blood 102:1788) . thus, granzyme b might be able to kill even those tumor cells that undergo an otherwise protective stress response. the work has been supported by the grants grk1034 and dr394/2-3. the authors thank prof. c. j. froelich (evanston, usa) for the granzyme b and dr. t. seidler (göttingen) for the adenoviruses. tumor necrosis factor (tnf) elicits its biological activities by stimulation of tnfr1 and tnfr2, both belonging to the tnf receptor superfamily. tnfr1-mediated signal transduction has been intensively studied and is well understood, especially with respect to activation of the classical nfkb pathway, cell death induction and map kinase signaling. in contrast tnfr2-associated signal transduction is poorly defined. earlier findings demonstrated that only membrane tnf, but not soluble tnf, properly activates tnfr2, resulting in depletion of traf2 from the cytoplasm. here we confirm that tnfr2 induced depletion of cytosolic traf2 by the use of tnfr1-and tnfr2-specific mutants of soluble and membrane tnf. corresponding with the known inhibitory role of traf2 in the alternative nfkb pathway, we show that tnfr2 induced activation of the alternative nfkb pathway. thus, we identified activation of the alternative nfkb pathway as a tnf signaling effect that can be specifically assigned to tnfr2 and membrane tnf. j. c. morales 1 , d. ruiz-magaña 1 , d. carranza 1 , c. ruiz-ruiz 1 1 universidad de granada, instituto de biopatologí a y medicina regenerativa. centro de investigación biomédica, armilla, spain different molecular mechanisms have been involved in the resistance of tumor cells to tumor necrosis factor-related apoptosis-inducing ligand (trail)-mediated apoptosis. epigenetic modifications commonly associated with tumor development, such as histone deacetylation, may influence the resistance of some tumor cells to trail by regulating gene transcription of trail receptors and other trail pathway related-genes. in the present study we have analyzed the effect of several histone deacetylase inhibitors (hdaci), belonging to different structural families, on trail-induced apoptosis in leukemic t cell lines. moreover, we have analyzed the activity of hdaci in normal t lymphocytes. we have found that, to a greater or lesser extent, all hdaci potentiate the induction of apoptosis in leukemic t cells by enhancing the signals triggered upon trail ligation, from the activation of the most apical caspase-8. in contrast, hdaci do not sensitize primary resting or activated t lymphocytes to trail-mediated apoptosis. the analysis of the expression of several pro-and anti-apoptotic proteins involved in the trail-signalling pathway indicates that most hdaci regulate the expression of trail-r2 and c-flip in leukemic t cells, but not in normal cells, which may explain their selective pro-apoptotic effect on leukemic cells. zfp36l1 is a zinc finger containing protein that is involved in post-transcriptional gene regulation. it can bind to mrnas containing adenine uridine rich (are) regions and subsequently mediate their degradation. we have previously reported a role for this protein in promotion of b cell apoptosis. one mechanism whereby zfp36l1 may mediate cell apoptosis could be by degradation of cell survival gene mrnas. the bcl-2 protein is an important cell survival protein at different stages of b cell development. bcl-2 mrna also contains are regions that could possibly be targeted by zfp36l1 protein. in the present study, we have tested the ability of zfp36l1 protein to bind to a bcl-2 mrna are probe and to degrade bcl-2 mrna. recombinant bacterially expressed zfp36l1 protein was shown to bind specifically to a bcl-2 are probe by rna electrophoretic shift assays (remsas). furthermore, remsas using cell lysates of ramos burkitt lymphoma b cells stimulated to express high levels of endogenous zfp36l1 also provided evidence that endogenous zfp36l1 in b cells could bind to the bcl-2 are. in order to examine whether zfp36l1 binding to bcl-2 are resulted in bcl-2 mrna degradation, actinomycin d rna degradation assays were carried out on murine embryonic fibroblast (mefs) cells from zfp36l1 knockout mice and wild-type mice using quantitative real-time pcr analysis. bcl-2 mrna was expressed in both wild-type and knockout mefs. the half-life of bcl-2 mrna was found to be extended in knockout mefs compared to wild-type mefs suggesting that zfp36l1 does play a role in degradation of bcl-2 mrna. overall, our data are consistent with a role for zfp36l1 in degradation of bcl-2 mrna which could be a mechanism for the reported role of this protein in induction of b cell apoptosis. the epstein-barr virus (ebv) is a common human herpes virus, which can predominantly infect two types of human cells: lymphoid cells and epithelial cells. its infection is associated with several human malignancies (hodgkin's lymphoma, burkitt's lymphoma, nasopharyngeal carcinoma), where it expresses limited subsets of latent proteins among which the latent membrane protein lmp1. since lmp1 is able to transform numerous cell types, it is considered as the main oncogenic protein of ebv. the principal mechanism of lmp1 function is based on mimicry of activated member of the tnf receptor super family (tnfr), by its ability to bind a similar sets of adapters and to activate overlapping signalling pathways like nfkb, c-fos-jnk, pi3-kinase...involved in the regulation of cellular processes. we previously generated two unique model, a monocytic (te1) and lymphocytic (nc5) immortalized by ebv and which expressing type ii latency program. here we developed original dominant negative (dn), by generating a fusion between gfp and tes1 or tes2 (transforming effectors site) derived from the c-terminal intracellular part of lmp1, in inducible vectors. then, we generated cell lines conditionally expressing these dns. we showed these dns not only inhibit survival processes resulting to the impairment of nfkb and akt pathway but increase apoptosis in this cell lines. we demonstrated that this pro-apoptotic effect is due to i) the depletion of lmp1's specific adapters and ii) the recruitment of theses adapters by dns interestingly allowed generation of apoptotic complex involved tradd, fadd and caspase-8. using this nc5 tumorigenic model in scid mice, we showed that induction of the dn lmp1-tes2 prevent development of tumours and mouse death. these dominant negative derived from lmp1 could be used to develop therapeutic approaches in malignant diseases associated with epstein-barr virus, but also in inflammatory pathologies. recent studies indicate that suppressors of cytokine signaling (socs) proteins play, in addition to their action as cytokine signaling inhibitors in the immuneinflammatory response, multiple roles in cell survival, differentiation, and apoptosis in diverse cell systems. since tumor cells often exhibit aberrant expression of socs genes, which may be involved in determining resistance to anti-tumor therapies, we have investigated the role of socs isoforms during dna damageinduced apoptotic response and cell cycle changes in various tumor cell types. by using tumor cell lines transduced for over-expression or knock-down of distinct socs isoforms, it is found that socs1 and socs3 differentially affect apoptosis and cell cycle changes induced by dna damaging agents in a cell type-specific manner. in t lymphocytic leukemia cell line jurkat, socs1 exhibited anti-apoptotic effect in response to ionizing radiation, hydrogen peroxide, and etoposide by inducing suppression of p38mapk activities, while socs3 promoted apoptosis with an increase in p38 activities. in contrast, both socs1 and socs3 display proapoptotic effect in rko colon cancer cell lines upon exposure to gamma radiation or ros-generating agents. notably, effects of socs proteins on cell cycle changes induced by dna damaging agents were rather similar in that over-expression of either socs1 or socs3 induced a slight decrease in g1 or s phase cells and a prominent increase in g2/m cells, regardless of their distinct effects on apoptosis. the analysis of cell cycle regulator proteins, however, revealed that different mechanisms are operating to regulate cell cycle via distinct cyclins and cdk inhibitors affecting g1/s transition and g2/m arrest induced by socs1 or socs3. socs1 promoted dna damage-induced p21 induction and g2/m cyclin b expression, while socs3 induced decrease in g1 cyclin e expression. the results suggest that socs isoforms potentially modulate growth of tumor cells exposed to dna damage via complex network involving apoptotic response and cell cycle regulation in a cell type-specific manner. the heat shock protein 90 (hsp90) is a highly conserved a widely expressed molecular chaperone. it is known to regulate the activity of several protein kinases or the proper folding of client proteins. hsp90 has also been identified as an important regulator of cellular survival. besides these intracellular functions, extracellular hsp90 can initiate cross presentation or immune responses. apoptotic cell death occurs permanently in multicellular organisms, without initiation of an immune response. however the mechanisms which prevent an inflammatory response to apoptotic cells are not understood to date. hsp90 is released during necrotic cell death and proinflammatory effects of extracellular hsp90 have been observed. thus, we asked whether apoptozing cells cleave hsp90 during apoptosis or how hsp90 is disposed by these cells. we induced apoptosis either in activated or resting primary human cells and analyzed the hsp90 protein content. we observed that hsp90 is degraded during apoptosis resulting in the formation of a fragment of about 50 kda. this fragment was to be observed exclusively in activated cells, while it was not detected if resting cells were induced to undergo apoptosis. analyzing the isoforms of hsp90 (hsp90 alpha and hsp90 beta) we could show that the 55 kda fragment is formed after degradation of the alpha isoform of hsp90. further, we were able to show, that hsp90 cleavage is dependent on caspase activity and most probably mediated by calpain. analyzing the cytokine response of monocyte derived phagocytes to apoptotic cells in presence or absence of exogenous hsp90 and caspase inhibitors. we observed a rather proinflammatory cytokine profile, if cleavage of hsp90 was inhibited or if exogenous hsp90 was added. these results demonstrate that cleavage of hsp90 represents a mechanism preventing the release of proinflammatory molecules from apoptozing cells. activity. mifc integrates the advantages of flow cytometry and fluorescence microscopy in one system, the imagestream. upon induction of autophagy, cytosolic lc3-i is processed to lc3-ii, which then remains associated with the autophagosome until its degradation upon fusion with the lysosome. an increase in steadystate levels of autophagosomes can be due to enhanced autophagy or decreased lysosomal activity. mcf-7 gfp-lc3 cells were therefore incubated in starvation medium for 3 hours, +/-bafilomycin, which potently inhibits lysosomal activity. classical gating strategies allowed the detection of cell populations of interest, which were further analyzed on single cell levels. we conclude that imagestream-based analysis provides an improved method in terms of objectivity, sensitivity and significance, to quantify autophagic activity. our results clearly show the need for discrimination between "steady-state" levels of autophagosomes and "current flux" of fully functional autophagy, i. e. quantification of autophagic flux. jnk seems to mediate the bcl-2/beclin-1 control of autophagy. recently, jnk was shown to be necessary for beclin-1 upregulation, and jnk-mediated phosphorylation of bcl-2 is associated with both, starvation-and ceramide-induced autophagy. the nfkappab pathway mediates critical survival signals during starvation, which have been linked to the inhibition of autophagy. we report here the novel findings that under conditions of starvation, pharmacological inhibition of nfkappab decreased the autophagic flux in mcf-7 cells, while jnk inhibition shows an enhancing effect on autophagy induction. ingenol 3-angelate (pep005), a novel activator of protein kinase c (pkc), has been shown to induce apoptosis in acute myeloid leukemia cells. we show here, that in contrast to leukemic cells, pep005 provides a strong survival signal to resting and activated t cells. this anti-apoptotic effect was dependent upon the activation of pkcv, a pkc isoform restricted to t cells and myocytes. expression of pkcv in the acute myeloid leukemia cell line nb4 turned their response to pep005 from an increased to decreased rate of apoptosis. furthermore, our data show that pep005 inhibited t cell apoptosis through the activation of nfxb downstream of pkcv, leading to increased expression of the anti-apoptotic proteins mcl-1 and bcl-xl. we conclude that pkcv expression determines whether pkc activation leads to an anti-or pro-apoptotic outcome in the cell types analyzed. this finding may be of considerable importance for the development of pkc -targeting antileukemic therapies. the neuronal growth factors, neurotrophins, and their receptors are widely expressed in a variety of non-neuronal tissues including the immune system. several reports indicate that survival and activation of normal b lymphocytes are regulated by nerve growth factor (ngf) and brain-derived neurotrophic factor (bdnf) autocrine circuits. however, the production and the role of neurotrophins were not evaluated in b lymphoma cells. diffuse large b-cell lymphoma (dlbcl) is a common and often fatal malignancy. despite major advance in the treatment (r-chop protocol) which improves the clinical outcome of patients, a subset of patients does not respond or relapses after the initial treatment; the exact mechanism of such resistance is not entirely clear. we hypothesized that autocrine neurotrophin survival circuits could contribute to the chemoresistance of dlbcl tumor cells. this hypothesis was investigated with dlbcl cell lines (su-dhl). thus, we evaluated the ability of su-dhl cells to produce neurotrophins (ngf, bdnf) and to express their receptors (p75, trka and trkb) in different cell culture conditions. our preliminary data show for the first time the production of neurotrophins by dlbcl tumoral cells whose level decreased in apoptotic conditions, in association with bad dephosphorylation suggesting its pro-apoptotic role. furthermore our results suggest that up-regulation of autocrine circuits (expression of trka known to be involved in survival signaling pathways) may contribute to cell survival and thus drug resistances of tumoral b cells. objectives: ataxia telangiectasia (a-t) is a rare disorder caused by mutations in the ataxia telangiectasia mutated (atm) gene. this gene encodes atm, a protein kinase which has a major role in dna double strand break response. a-t patients suffer from a variety of immune system defects including lymphopenia, immunoglobulin deficiencies and impaired class switch recombination, they also have an increased incidence of cancer especially leukaemia and lymphoma. the susceptibility to lymphoid tumours and immunodeficiency could be partly due to failure of extrinsic apoptotic processes involved in regulation of the immune system. although atm is known to have a central role in the induction of apoptosis in response to unrepaired dna double strand breaks its role in extrinsic apoptotic pathways is unclear. this study aimed to investigate if atm has a role in fas induced apoptosis. a bank of lymphoblastoid cell lines (lcls) derived from a-t and normal individuals and tumour samples with wildtype or mutant atm were used in the study. apoptosis was induced by incubating cells with the fas activating antibody ch11 and analysed by flow cytometry. expression of the caspase 8 inhibitor cflip which inhibits fas induced apoptosis was detected by western blot. results: there was no significant difference in the susceptibility to fas induced apoptosis or cflip protein expression between atm mutant and control groups. however cells expressing high levels of cflip protein do show greater resistance to fas induced apoptosis than those with lower expression. whilst the lcls expressed both long and short forms of cflip, the tumour cells expressed only the long form. conclusion: atm mutations do not affect susceptibility to fas induced apoptosis or alter cflip protein expression in lcls or tumour cells. cflip protein levels and fas susceptibility vary greatly between individuals but this is independent of atm status. high expression of cflip protein correlates with reduced apoptosis in response to ch11 treatment but there is no clear difference in cflip expression between atm wildtype and mutant cells. labdane diterpenoids have a broad spectrum of biological activities including antibacterial, antiviral, and anti-inflammatory properties. however, little is known about their possible role in the apoptotic cell death machinery. we report that labdane diterpenoids induce apoptosis in different tumor cell lines by activating caspase 8 in the extrinsic death receptor pathway, with subsequent participation of mitochondrial signaling. activation of caspase 8 by diterpenoids was followed by a decrease in mitochondrial membrane potential, the release of apoptotic factors from mitochondria to the cytosol, and subsequent activation of caspases 9 and 3. diterpenoids also led to time-dependent cleavage of bid. inhibition of caspase-8 abrogated these processes, suggesting that the death receptor pathway plays a critical role in the apoptotic events induced by labdane diterpenoids. in addition, pretreating cells with neutralizing antibodies to fas ligand, tumor necrosis factor receptor 1 (tnf-r1), and tumor necrosis factor (tnf)-a receptor 2 (trail) inhibited diterpenoid-induced apoptosis, revealing it to be dependent on these death receptors. diterpenoid treatment also induced a significant increase in the generation of reactive oxygen species (ros). however, increased ros production was not directly involved in diterpenoid-triggered apoptosis. these results demonstrate that labdane diterpenoids induce apoptosis through activation of the death receptor pathway. conclusion: cell proliferation and differentiation are tightly regulated networks and it is believed that in cell differentiation, even in cancer form, cells precluded from proliferation. whether these changes affect the level of differentiation or the change of survivin expression can affect the proliferation and differentiation pathways are the hypotheses that need further investigation. synthetic alkyl-lysophospholipids (alps) are a group of unnatural lipids with promising anticancer capability. a prototypic member is the ether lipid 1-o-octadecyl-2-o-methyl-rac-glycero-3-phosphocholine (et-18-och 3 ; edelfosine), which induces selective apoptosis in tumor cells through activation of fas/cd95 independent of its ligand fasl/cd95l. fas/cd95 is activated by edelfosine via its translocation in lipid rafts. in this study we showed that edelfosine promotes cell death in multiple myeloma and various solid tumor cell lines in a death receptor-independent manner. edelfosine-treated cells could not be protected against cell death after inhibition of caspases by zvad-fmk while fasl-stimulated cells stayed mostly alive. furthermore cells could not be rescued by addition of zvad-fmk in combination with necrostatin-1, an inhibitor of death receptor-induced necrosis. fas resistant solid tumor cells overexpressing members of the anti-apoptotic bcl2-family as well as cells overexpressing the cellular regulatory protein flip went in contrast to fas stimulation to apoptosis after treatment with edelfosine. therefore we suggest that edelfosine induces a death receptor-independent cell death pathway in a wide range of tumor cells. apoptosis represents a cellular "suicide" mechanism which allows the control of cell number from tissues and elimination of cells that present dna mutations or having an abberant cell cycle, those cells being predisposed to malignant transformation. thus, elucidating the mechanisms of programmed cell death process seems to be of great importance for malignant transformation, tumour evasion and therefore for anti-cancer therapy. many anti-cancer drugs act during physiological pathways of apoptosis, leading to tumour cell destruction. the present study focused on the potential influence of oncolitical treatment (5-fluorouracyl) associated with natural compounds (curcumin, genistein, quercitin) on the dynamics of the cell cycle and levels of apoptosis in colon cancer cell lines (e. g. colo 201, sw1116, lovo, caco-2, ht-29) . in addition, expression of antigens involved in tumour proliferation and apoptosis (ki-67, pcna, p53, bcl-2) was compared with gene expression in the presence or absence of stimuli treatment. percentages of apoptotic cells were detected by using annexin v/fitc and propidium iodide double staining, while progression through cell cycle phases was evaluated by using pi staining. correlation analyses between the individual profile of the stimuli modulated gene expression with the coded protein expression were performed by using data from rt-pcr with specific primers, and indirect immunofluorescence followed by flow cytometry, respectively. stimuli treatment of colon cancer cell lines differentially induced higher levels of apoptosis as compared to untreated tumour cells, while cell cycle distribution of dna changed. data obtained showed a various expression and functional behaviour of the markers under study associated to colon cancer cells, suggesting their possible involvement in regulating the interactions between tumour cells and host immune system. the results obtained might further lead to the establishment of an experimental pattern for the corroboration of cell and molecular mechanisms involved in the tumour progression and the treatment resistance of colon tumors using cell lines. the effect of modulatoy agents on proliferation and apoptosis could be used in clinical departments in order to elaborate new therapeutical approaches and act as useful instruments in elaboration of individualized treatment schemes. extensive tissue trauma and malnutrition results in disorders of programmed cell death influencing the patients susceptibility to infections. the purpose of our study was to assess the effect of pancreatic cancer surgery and immunonutrition on the apoptotic signaling pathways. the randomized studies were performed in 88 patients after pancreatic cancer resection with preoperative standard or enteral immunonutrition. lymphocytes expressions of bcl-2, bax, caspase 3, 6, 9, nfkb, parp1/89kda, tnfr1/cd120a and cd95/fas were assessed by western-blot and flow cytometry. results: before and after surgery the expression of bcl-2, bax, nfkb, parp1 was significantly lower and expression of caspases, tnfr1 as well as percentage of cd95 cells significantly higher as compared with control group. caspase 3 expression was significantly higher as compared with nfkb, parp1 and tnfr1. in comparison to the standard nutrition preoperative immunonutrition increased bcl-2 and nfkb expressions and decreased caspases and parp1 expressions. in addition, we found a significant down-regulation of bcl-2 expression after surgery, but insignificant in patients with preoperative immunonutrition. conclusion: preoperative enteral immunonutrition has an modulative effect on apoptotic signaling pathways after pancreas resection and possesses antiapoptotic properties. this modulatory effect of glutamine and omega-3 fatty acids has no influence on patients outcome. the capacity of medicinal herbs to modulate cellular and humoral immune response could have useful applications in some immune-mediated disorders, infections and cancers. in this study the immunomodulatory effects of salvia mirzayanii a native plant that is widely distributed to iran was investigated. s. mirzayanii is used for the treatment of infectious and inflammatory diseases and as a tonic in folk medicine. study of the effect of this plant on the activated human peripheral blood lymphocytes showed stimulatory effects at lower concentrations and inhibitory effects at higher ones (p x 0.01). in flow cytometry analysis, accumulation of apoptotic cells in the sub-g1 phase of cell cycle of the mitogen-treated lymphocytes exposed to the inhibitory doses of the extract was observed. dna fragmentation analysis of these cells showed a typical dna laddering. immunization of the extract-treated mice with the antigen decreased delayed hypersensitivity skin reaction as well as the antibody titer at higher concentrations (p x 0.007). these results indicated the presence of immunomodulatory compounds in the extract of s. mirzayanii and suggest that the induction of apoptosis in lymphocytes might be the mechanism responsible for the inhibitory effect of the extract observed at higher concentrations. a new randomized, double-blind, placebo-controlled clinical trial was conducted with 54 healthy volunteers receiving either la1 (10 9 cfu/day) or placebo, during 57 days prior to uv (2 × 1.5 med). blister roofs, liquid and skin biopsies were collected 1, 4 and 10 days after uv exposure from non-irradiated and irradiated skin areas and used for identification of cells involved in uv-induced immune response, quantification of inflammatory cytokines, a dna damage marker (p53). while a similar decrease of lc for both groups was observed on day 1 after uv exposure compared to placebo, la-1 group presented a faster increase of a new subset of epidermal dendritic cells (dc), namely early lc precursors (cd1a low cd207 -) associated with a minor recruitment of monocytes. concomitantly, inhibition of il-10 and a tendency to inhibit il-6 was observed in la-1 group compared to placebo. on day 4, la-1 group presented a greater recruitment of early lc precursors and a trend to increase cd1a low cd207 + lc precursors compared to placebo. additionally, a faster reduction of inflammatory and immunosuppressive cytokines (il-6, tnfa, il-8, and il-10) was observed in la1 group compared to placebo. we show that la1 limits uv-induced immune-suppression and skin inflammation. this contributes to the recovery of the skin immune homeostasis, confirming the previously observed benefits of la1 supplementation for photoprotection at a lower dose (1 log). the thymus is one of the primary lymphoid organs and plays a central role in the immune system. it provides the essential microenvironment for proper t cell development. in the thymus, the maturation of t cells depends on many interactions between t cells and different stromal cell types, mainly composed of epithelial cells (tecs). foxn1 is a winged-helix/forkhead transcription factor, which is crucially required for proper epithelial cell differentiation in the thymus. foxn1 appears to be expressed in all epithelial cells of the early thymic rudiment starting around e11.5. previously, we have used a lineage-tracing system to confirm the existence of a bi-potent epithelial progenitor cell. using the cre-loxp system, we showed that a single epithelial cell, when reverted to express foxn1 in a nude (foxn1-deficient) background, can give rise to a functionally competent thymic microenvironment. hence, we hypothesize that the epithelial progenitor cell expresses foxn1. if true, it should be possible to target this cell type by use of foxn1-promoter driven transgenes. conditional targeted cell ablation is a powerful method to elucidate the physiological function of cell populations and their regenerative capabilities. currently, we are using three different strategies of conditional targeted cell ablation in order to examine functional characteristics of epithelial bi-potent progenitor cells within the thymus. intracellular accumulation of poly glutamines is known to cause neurodegenerative disorders, such as huntington's disease. considering this, we are expressing transgenic egfp variants containing either 19 or 82 glutamine residues under the control of foxn1 promoter, leading to different degrees of tec degeneration. furthermore, also under the foxn1 promoter, we are using the transgenic expression of human diphtheria toxin receptor and the transgenic expression of the bacterial nitroreductase enzyme that converts the pro-drug metronidatole (mtz) into a cytotoxic cross-linking agent for conditional cell ablation. preliminary results describing the phenotypes of these mice will be presented. t. kamei 1,2 , y. toriumi 3 , k. kimura 4 1 european university viadrina, frankfurt (oder), germany, 2 shimane institute of health science, izumo, japan, 3 shimane university faculty of medicine, department of pediatrics, izumo, japan, 4 japan yoga niketan, yonago, japan as a method in relieving stress, yoga is popular today. many reports of physical changes describing how yoga improves respiratory, circulatory, endocrine, and metabolic functions by yogic practice have been reported until now. we examined changes of electroencephalograph (eeg) and cellular immunity before, during, and after yoga exercises, in an endeavor to detect the correlation between them. the subjects consisted of eight yoga instructors who had been practicing yoga for several years. a 10-minute-rest period, followed by a 15-minute yoga exercise called asana, a 15-minute respiratory exercise called pranayama (various specialized respiration methods continuously performed with the eyes closed), and a 20-minute meditation were performed. throughout rest and yoga, brain rhythms were continuously recorded via two disc electrodes placed on each forehead (fp2). blood samples were drawn before and after each exercise. nk activity and percentages of t-cell and b-cell subsets were measured. during the pranayama period, both a positive correlation between the change in abundance of the activated alpha waves and the ratio of changes in nk activity (r=0.83, p x 0.02), and a positive correlation between the change in abundance of the activated alpha waves and the ratio of changes in the number of t lymphocytes (r=0.77, p x 0.05) were observed. furthermore, a positive correlation was also observed between the change in amplitude of the activated alpha waves and the ratio of change in the number of cd4 (r=0.96, p=0.0001). these findings suggest that yoga creates a stress-free and mentally concentrative state which activates the functions of nk cells and t lymphocytes, mainly of cd4, within a short period of time. we conclude from these results that yogic respiratory exercise may be able to activate cellular immunity and to help recover the mental and physical harmony of human. yoga is considered to have an effect of some re-activation of a latent ability of harmonization in which humans naturally possess. t regulatory cells play a central role in the suppression of immune responses thus serving to induce tolerance and to control persistent immune responses that can lead to autoimmunity. several recent studies suggest also that diverse populations of regulatory t cell play an important role in regulating t-helper 2 response to allergens, maintaining functional tolerance and preventing allergy. here we demonstrate that cd4 + cd25 + t regulatory (t reg ) cells are critical in controlling the immediate hypersensitivity response of bone marrow mast cells (mcs) without affecting cytokine release. this effect is shown to require a cell-cell contact and depends on interaction between ox40l expressed on mcs and the constitutive expression of ox40 (members of the tumor necrosis factor [tnf] and tnf receptor family, respectively) on t reg cells. this interaction does not alter the activation of plc-g, syk and lat in ige/ag stimulated mcs upon co-incubation with t reg cells, whereas it induces a decrease in the phosphorylation levels of akt. moreover, we find that upon co-incubation with t reg cells, mcs show increased levels of camp, which is known to inhibit mcs function, as a result of ox40l signal. antagonism of camp in mcs reverses the inhibitory effects of t reg cells restoring normal ca 2+ responses and degranulation. the cross-talk between t reg cells and mcs through ox40-ox40l interaction defines a previously unrecognized mechanism controlling mcs degranulation. loss of this interaction may contribute to the severity of allergic responses or inflammatory disease. active regulation has emerged as a very essential mechanism for both inducing and maintaining peripheral tolerance to non-pathogenic environmental antigens. a healthy immune system responds to antigens with a combination of polarized th1 or th2 effector cells and the induction of antigen specific foxp3+ regulatory t cells (treg). it is believed that the dominant subset determines the quality of the eventual immune response. in allergic asthma there is a clear dominance of th2 cell responses to non-pathogenic environmental antigens. recently it was shown that the specific transcription factors that characterize the th2 and treg subset, gata3 and foxp3 respectively, counter regulate each other (mantel y et al., 2008) . we hypothesize that children with allergic asthma will respond to allergens with low induction of foxp3+ tregs and high gata3+ th2 cells. in order to prove this hypothesis pbmc of children with allergic asthma and non-sensitized healthy controls are stimulated with allergens, tetanus toxoid, and lps. almost 100 million allergic patients are sensitized to the major birch pollen allergen bet v 1, which cross-reacts with major allergens of fagales (e.g., alder, hazel, hornbeam, oak) pollen and plant food allergens. the epitopes of bet v 1 recognized by allergic patients' ige antibodies belong to the conformational type and therefore have not been characterized in detail. here we used antibodies raised against peptides spanning the bet v 1 molecule in ige competition experiments to search for sequences which are involved in ige recognition. the strongest inhibition (i.e., g 90 %) of patients' ige binding to bet v 1 was obtained with polyclonal and monoclonal antibodies specific for peptides comprising aa 30-59 (p2) and aa 74-104 (p6) of bet v 1. cross-reactive ige epitopes between bet v 1 and related pollen allergens and plant food allergens involved primarily p2. p2 and p6 are not adjacent peptides in the bet v 1 sequence but define a surface-exposed patch on the three-dimensional structure of bet v 1. as determined by surface plasmon resonance, monoclonal antibody mab2 specific for p2 and mab12 specific for p6 showed high affinity, i. e., dissociation constants, k(d) = 8.35e-11 m and k(d) = 1.05e-9 m, respectively. interestingly, peptide-specific mabs inhibited allergic patients' ige antibodies equally well as peptide-specific polyclonal rabbit antibodies but only the latter inhibited strongly allergen-induced basophil degranulation. this finding indicates that the surface patch defined with anti-p2 and anti-p6 antibodies contains several ige epitopes. in summary, we have defined a surface-exposed patch on the bet v 1 allergen which seems to harbor the majority of the ige epitopes and may be used for the rational design of active and passive immunotherapy strategies against birch pollen and related allergies. background: antigen-specific th1 cells as well as tc1 cells, induced by biolistic gene transfer using plasmid dna encoding the model allergen b-galactosidase (bgal) under control of the fascin promoter (pfascin-bgal), inhibited the elicitation of systemic th2 immune responses and suppressed ige production in an experimental mouse model. moreover, protective biolistic dna vaccination with pfascin-bgal prevented th2-mediated lung pathology (eosinophilia) in sensitized mice locally challenged with bgal protein, but led to the recruitment of th1/tc1 cells into the lung, associated with substantial neutrophilic infiltration and the induction of airway hyperresponsiveness (ahr). objective: to analyze the modalities of ahr induction in mice biolistically vaccinated with pfascin-bgal. methods: balb/c mice were immunized with pfascin-bgal using the gene gun. subsequently, mice were challenged by consecutive intranasal application of bgal protein. cd4 + and cd8 + t cells, respectively, were depleted before and during the provocation phase by intraperitoneal injection of anti-cd4 (gk1.5) or anti-cd8 (53.6.72) monoclonal antibodies. neutrophilic granulocytes were depleted by treatment of animals with either anti-gr-1 monoclonal antibody rb6-8c5 or monoclonal antibody nimp-r14. one day after the last challenge airway reactivity was assessed by whole body plethysmography, bronchoalveolar lavage (bal) was performed and the frequency of ifn-g-producing cd8 + effector t cells in the lung was determined. results: whereas neutrophilia in the lung of immunized and challenged mice was considerably alleviated by depletion of cd4 + t cells, ahr was not significantly affected, implicating that the elicitation of ahr by cd8 + t cells is dissociated from the activity of neutrophils. this notion was verified by elimination of neutrophils during the provocation phase, likewise leading to unaltered ahr. in contrast to cd8 + t cells, cd4 + t cells induced strong neutrophilic infiltration and ahr. transfer experiments with cd4 + or cd8 + t cell, separated from the airways of vaccinated and challenged mice, will probably reveal details of the effector mechanisms of th1 and tc1 cells operative in the elicitation of airway inflammation. conclusions: robust type 1 immune responses, although highly effective in the counter-regulation of local th2-mediated pathology, might as well trigger inflammatory reactions in the lung and provoke the induction of ahr. respiratory epithelial cells function as physical barrier and have shown to be active participants within the process of defense against pathogens and recognition of allergens. upon activation they release inflammatory mediators thereby creating a micro environment in which recruited immunocompetent cells induce a local immune response. house dust mite (hdm) extract as a source of allergens has been shown to induce a broad panel of genes upon stimulation of epithelial cell line nci-h292. the proteolytic activity of these hdm allergens has been proposed to be involved in the activation process. the aim of this study was to compare the influence of hdm extract on respiratory epithelial cells with grass pollen allergen-induced activation of these cells with regard to the mechanism of activation, gene expression level, and the level of induced cytokine and chemokine release. in contrast to the hdm major allergen der p 1, we were able to show that the major allergen of phleum pratense, phl p 1, although sharing molecular similarities with der p 1, does not display any enzymatic activity under physiological conditions. therefore, in this study respiratory epithelial cells were stimulated with grass pollen extract and purified phl p 1. chemokine and cytokine release was determined by multiplex enzyme-linked immunosorbent assay and mrna was used for cdna-microarray analysis. first data show that both, hdm extract and grass pollen allergens, induce the release of il-6 and il-8 from nci-h292 cells. furthermore, stimulation with hdm extract leads to the release of tnf-a, gm-csf and ifn-g. interestingly none of these mediators was induced after stimulation with grass pollen extract or purified phl p 1. in contrast to hdm extract grass pollen allergens induce the release of mcp-1 from respiratory epithelial cells, as well as moderate levels of il-12. detailed characterization of the response on gene expression level might give new insights into the pathophysiology of grass pollen allergy and a comparison with hdm induced expression profiles will be helpful towards understanding the allergic response in general. (supported by dfg sfb tr22) results: collectively, responses to blg, but not to bsa, were observed in all groups analyzed, included healthy controls. nevertheless, 3 distinct profiles of response were obtained: children with ige mediated cma had a significant increased level of proliferation (mean±sd of stimulation index(si): 7.2±5.7) and of il-13 (mean±sd: 1157±909 pg/ml), and reduced il-10 (mean±sd of il-10-spot forming units/2x10 5 cells (sfu): 912±510), compared to healthy subjects (3.4±2.7 si, p x 0.05; 355±396 pg/ml, p x 0.05; 1272±623 sfu, for proliferation, il-13 and il-10, respectively); children with non-ige mediated cma had a significant reduction of il-13 (192±362 pg/ml), compared to ige patients (p x 0.0004) and an increased, although not statistically significant, production of ifn-g (33.7±54.6 sfu) compared to control (9.5±9.5 sfu) and to ige-allergic patients (0.6±0.8 sfu). finally, tolerant patients showed reduced il-13 (636±1048 pg/ ml, p x 0.05) and proliferation (3.9±3.5 si), compared to acute ige-cma children. interestingly, the high level of il-10 observed in all groups might have a counter-regulatory effect, since its neutralization resulted in an increase of proliferation to blg; by contrast, il-4 was undetectable in all patients even blocking the il4-receptor. conclusion: blg-specific, immune responses can be recalled in peripheral blood of cma patients, as well as of normal and tolerant children. a th2-like response with il-13 and proliferation is dominant in ige-mediated cma patients; by contrast a th1-skewed response with ifn-g is present in non-ige-mediated allergic and in those children who outgrew ige-allergy. y. f. tang 1 , b. chua 1 , f.c. lew 1 , a. ho 1 , k. wong 1 , k.l. wong 1 , d. m. kemeny 1 1 national university of singapore, immunology programme, yong loo lin school of medicine, singapore, singapore allergic inflammation of the airways causes changes in the lung wall that can lead to chronic inflammatory disease such as asthma. using a mouse model, this response can be divided into an induction phase, in which cd4 th2 t cells specific for airborne allergens are produced, and an effector phase, during which they are recruited to the lung. in the lung, recruited th2 cells orchestrate the inflammatory response marked by eosinophilia, mucus hyper secretion and increased airway hyperresponsiveness (ahr). previously we, and others, have shown that transfer of cd8 t cells inhibits the induction of the th2 response. here we have investigated the effect of cd8 t cells on the effector phase of the inflammatory lung response. in vitro activated ot-i cd8 t cells were transferred to ovalbumin (ova)/ alum immunized mice one day before the first of 3 airway challenges with ova. eosinophil infiltration was inhibited by transfer of cd8 + t cells (36.7 %±4.1 % to 17.6 %±2.7 %). when ifn-gamma -/-ot-i cd8 t cells were transferred, we found that the inhibitory effect on eosinophilia was reduced (39.6 %±5.1 %), suggesting an important role for cd8 t cell ifn-gamma. cd11c + cd103 + cd11blung dcs from cd8 transferred mice secreted higher levels (500pg/ml) of il-12p70 following ex-vivo stimulation as compared with animals that were not given cd8 t cells. these data show that, in addition to regulating the induction of the allergic immune response, cd8 t cells can subsequently divert the local lung environment to one that favors th1 immunity. the chain terminator drug abacavir triggers a serious hypersensitivity reaction in 8 % of patients with hiv infection. this reaction is strongly associated with hla-b*5701 and appears to be mediated by cd8+ t cells producing inflammatory cytokines. we show that cd8+t cell responses can be primed in vitro, in normal blood donors who are hla-b*5701+, but not in non-b*5701+ donors. cd8 t cells, but not cd4 t cells, are expanded by abacavir pulsed autologous apc over a13-day culture, producing ifn-gamma and tnf-alpha upon re-stimulation with apc expressing hla-b*5701. similar responses were detected in abacavirhypersensitive hlab*5701+ patients. responses were not detected using mutant apc deficient in tap or tapasin, or when normal apc were aldehyde fixed before loading with abacavir, indicating a reliance on the conventional mhc-i ag presentation. responses were exquisitely restricted to hla-b*5701 since they were undetectable using apc expressing closely related hla-b57 or b58 allotypes. responses to apc expressing mutants of hla-b*5701 demonstrated a crucial role for residue 116. isolation of peptide fractions from abacavir-loaded cells has led to the identification of specific fractions recognised by an abacavir-specific t cell line. our data suggests that abacavir forms a conjugate with an endogenous peptide that is presented by hla-b*5701 triggering cd8 t cells. we speculate that this form of altered self is highly immunogenic, behaving like a form of allogeneic mhc-i, contributing to the responses observed in abacavir naï ve individuals. the molecular mechanisms underlying altered hla-b*5701 may be relevant to the role of other disease-associated class i allotypes such as hla-b27 and b51. a. jenckel 1 , s. bulfone-paus 1 , n. föger 1 1 research center borstel, immunobiology, borstel, germany mast cells play a crucial role in acute inflammatory and allergic reactions. upon activation, mast cells secrete a vast array of preformed and newly synthesized inflammatory mediators. recent work has begun to appreciate an important role of the actin cytoskeleton in mast cell activation. the actin-associated protein coro-nin1a (coro1a), a coronin family protein preferentially expressed in hematopoietic cells, is critically involved in various actin-mediated cellular functions of leukocytes. recent data of our group also indicate a regulatory role of coro1a in mast cell function. coronin proteins have been described to be differentially phosphorylated in vivo. however the molecular mechanisms by which coro1a is regulated in response to physiological stimuli are still poorly characterized. here we investigated the modalities of coro1a phosphorylation during the activation of mast cells. immunoprecipitation studies combined with phospho-specific western blotting techniques revealed a transient phosphorylation of coro1a on serine residues upon antigen-specific engagement of fc-epsilon-receptors. as the phosphorylation status of coro1a can influence its association with the actin cytoskeleton, we analyzed the subcellular localization of coro1a during mast cell activation. cell fractionation experiments demonstrated that the association of coro1a with the actin cytoskeleton significantly decreases in response to mast cell stimulation, concomitant with the increase in coro1a phosphorylation. a functional correlation between coro1a phosphorylation and its association with the actin cytoskeleton in mast cells was further indicated by structure function experiments employing specific phosphorylation mutants of coro1a. thus, coro1a is a downstream effector molecule of fc-epsilon-receptor signaling and likely is involved in the dynamic reorganization of the actin cytoskeleton during mast cell activation. allergen-specific t and b lymphocytes play an important role for the pathogenesis of asthma. t cells orchestrate the infiltration of the lung tissue with eosinophils and neutrophils and provide help for allergen-specific immunoglobulin production. recently, we have shown in a mouse model for allergic airway inflammation that b cells directly interact with t cells in the inflamed tissue and locally produce ige. to analyse t/b-interaction in the inflamed tissue in more detail, we developed a novel adoptive transfer system using ovalbumin-specific t cells and nitrophenolspecific b cells. recipient mice are then challenged intranasally with an np-ova conjugate. this system allows to track single allergen-specific t and b cells in all stages of the immune reaction using flow cytometry and immunohistology. in addition, cells can be re-isolated by flow-sorting for in-depth analysis. using this system we could define several phases of the inflammatory reaction. t and b cells first become activated in the lung-associated lymph nodes. granulocytes can be found very early in the lung tissue and also activated t cells very rapidly emigrate to the site of inflammation. however, clusters of allergen-specific t and b cells can only be found in later stages of the reaction. as another focus, we used our in vivo system to define the role of t cell costimulatory molecules for airway inflammation. costimulatory receptors are key regulators of t cell activation and differentiation and therefore promising targets for therapeutic intervention. of special interest is the t cell-specific icos molecule which is important for t/b cooperation as well as the regulation of chronic inflammatory reactions. using icos knock-out mice we were able to delineate the specific role of icos for the different stages of airway inflammation. in particular, we analysed the impact on t cell subset differentiation, cytokine production and allergen-specific immunoglobulin production. the integrity of the actin cytoskeletal network is critcal for a large variety of cellular functions. coronins constitute a family of evolutionary highly conserved wdrepeat containing proteins that have been implicated in the regulation of actin cytoskeletal dynamics. in mammalians seven coronin family members have been described. the high degree of sequence conservation amongst coronin family proteins suggests common features and functions. however, individual family members may also have developed additional selective and specific functions. our recent studies on coronin1a (coro1a) deficient mice have demonstrated that coro1a exhibits an inhibitory function on the cellular steady-state f-actin content, is required for chemokine-mediated functions in t cells and is involved in the maintenance of t cell homeostasis. coronin1b (coro1b) is a closely related homolog of coro1a and the two genes are co-expressed in hematopoietic cells. to address the question of functional redundancy in vivo, we have generated coro1b deficient mice and crossed them with coro1a deficient mice to obtain coro1a/coro1b double deficient mice. analysis of t lymphocytes from coro1a/coro1b double deficient mice revealed defective chemotactic responses and a severe peripheral t cell lymphopenia in double-deficient mice, which was significantly exacerbated as compared to the respective single knock-outs. an analysis of coronin deficient mast cells also revealed an involvement of coro1a/coro1b in the regulation of actin cytoskeletal dynamics and the function of mast cells. however, in contrast to the inhibitory effects of coro1a/1b deficiency on t cell function, mast cell degranulation and migration was enhanced in coro1a/1b double deficient mast cells. thus, depending on cell type specific requirements, coronin proteins can either exhibit positive or negative regulatory functions. additional studies will investigate molecular and regulatory mechanisms by which coronin proteins control actin cytoskeletal organization and function of immune cells. together, our studies here reinforce and expand our appreciation of the importance of actin-cytoskeleton regulatory proteins for immune cell function. initially found by serial analysis of gene expression, murine samsn1 (also known as hacs1 or sly2) is a putative adaptor and scaffold protein with a sterile-alphamotif (sam), a src homology 3 (sh3) domain and a predicted bipartite nuclear localization signal. the samsn1 gene is located on mouse chromosome 16 and encodes a well conserved protein with 364 amino acids, which is predominantly expressed in hematopoietic tissues. initial overexpression studies suggest a contribution of samsn1 in b cell activation and differentiation, however its physiological function is yet unknown. to investigate samsn1 expression in lymphatic and myeloid cell types in greater detail we employed the sensitive method of quantitative real-time pcr. our data revealed an expression of samsn1 in all tested hematopoietic cell types. the highest expression level of samsn1 mrna was seen in mast cells compared to lower levels in macrophages, dendritic cells, cd4+ and cd8+ t cells and b cells. the other two members of the sly family of adaptor proteins -namely sly1 (hacs2) and sly3 (sash1) -were expressed only at a very low level in mast cells. the high level of samsn1 mrna expression in mast cells, together with minimal expression of other sly family proteins in these cells, implicates an important role of this adaptor protein for mast cells. to address the potential role of samsn1 in mast cell differentiation and function we are analyzing bone marrow derived mast cells from samsn1 deficient mice. initial in vitro experiments indicate normal proliferation and differentiation of samsn1 deficient mast cells. in additional studies we are now investigating the effects of samsn1 deficiency on mast cell activation processes, such as degranulation, cytokine production and the signal transduction cascade. analyzing the role of samsn1 in mast cells will help to define the biological function of this novel class of adaptor proteins. introduction: we showed previously that the ability of murine igg1 antibodies to mediate anaphylactic reaction is directly dependent on the amount of sialic acid residues attached to the carbohydrate chain n-linked to the antibody fc region (silva et al; j.immunol., 2008). then, we hypothesize that differences in the glycan composition mainly the sialylation grade observed between the anaphylactic and non-anaphylactic igg1 abs may be resultant of the differential expression of the glycosyltransferase, essentially sialyltransferase, coding genes during its synthesis in b cells. objective and methods: to prove this hypothesis it was analyzed the expression of st8siai-v; st6galnac i-iv, st3gal ii -v genes quantitatively by real time-pcr in the hybridomas producer of these two types of igg1 abs. results: we observed that the expression of st3gal i, iii and v coding genes was similar in both hybridomas, while the st3gal ii and iv genes were less expressed in the hybridoma producer of non-anaphylactic igg1. in addiction, the expression levels of st8sia and st6galnac genes in the hybridoma producer of anaphylactic igg1 were significantly higher when compared to those observed in the hybridoma producing of non-anaphylactic igg1. conclusion: these data suggest a direct correlation between the sialylation grade observed in the carbohydrate chain attached to the igg1 abs and the expression of sialyltransferase enzymes in the hybridomas producer of these molecules. financial support: cnpq, capes, fapesp. basophils are innate immune cells endowed with important effector functions during allergic inflammation and parasite infection. their activation in terms of histamine and cytokine production is mediated through immunoglobulin-dependent and -independent mechanisms, raising the question whether stimulation of tolllike receptors (tlrs), which have been described in basophils, has a similar effect. we found that, in contrast to other tlr agonists tested, only the doublestranded rna poly(a:u) induced the typical t h 2 cytokine and histamine production in vitro. this compound was also fully active when administered in vivo since it activated basophils and promoted their recruitment to the periphery. we took advantage of a murine model of allergic asthma to establish the pathophysiological relevance of this finding. using both adoptive transfer and depletion of basophils, we established not only that these cells contribute directly to the severity of asthma symptoms, but also that a mimic of viral infection can aggravate the disease through their activation. this is the first evidence for a mechanism of exacerbation of allergic asthma induced by a mimic of viral infections, mediated through basophils. ishes the airway hyperresponsiveness and airway inflammation in experiment murine asthma models. to investigate the effect of activation of nkt cells at different allergic asthma progression, we administered balb/c mice with a-galactosylceramide (a-galcer), a stimulator for nkt cell activation, before or after ova immunization and measured the airway inflammation of that mice after 2 times of intranasal ova challenge. in our results, the total numbers of bronchoalveolar lavage (bal) cells were higher in mice administered with a-galcer before ova immunization compared to that of mice administered with a-galcer after ova immunization. moreover, significant increased percentage and cell numbers of eosinophils in bal of mice administered with a-galcer before ova immunization was noted. il-5 and eotaxin are the most potent cytokine/chemokines for the recruitment of eosinophils. il-5 and eotaxin levels in the bal fluid were higher in mice administered with a-galcer before ova immunization compared to that of mice administered with a-galcer after ova immunization. these data demonstrate that activation of nkt cells at different allergic asthma progression dictates the different outcome of asthma. in addition, the activation of nkt cells in naïve mice induces airway inflammatory responses. the potential risks of treatment with nkt cell activation on human diseases should be considered. objective: bronchial asthma is a complex disease of the lung and is characterized by a variety of symptoms such as airway hyperresponsiveness, reversible airway obstruction, high serum levels of ige and inflammation. histologically, there are infiltrations of eosinophils, degranulated mast cells and hyperplasia of airway globlet cells in addition to lymphocytes. the transcription factor irf1 mediates the differentiation into th1 cells by activating multiple genes which are independently crucial for the development of naive t cells into th1 cells. because irf1 is expressed in many different tissues, it can be considered as a master switch factor for th1 cell differentiation. methods: here, we tested mice deficient in irf1 in the murine acute asthma model to evaluate its importance in this th2 cell-mediated disease. the protocol setup was the following: 3 sensitizations s. c. with ova, followed by 3 challenges via ova inhalation and adoptively transferred wildtype cd8+ t cells prior to initial sensitization. in our experiments, we could demonstrate that only after priming of irf1 deficient mice with the help of adoptively transferred cd8+ t cells, asthma symptoms in these mice were more severe than in wildtype controls. as an example, eosinophil infiltration into the lung was increased by 3.5 fold. likewise, ovaspecific antibodies and numbers of goblet cells (fig. 1) were also significantly higher in irf1 deficient mice. conclusion: interferon regulatory factor 1 plays a role in the severity of the development of asthma. in its absence, proinflammatory parameters in the lung are increased significantly. this effect is only visible in the presence of wildtype cd8+ t cells. mechanistically, a potential counterregulation of asthma by th1 cells is not available in irf1 knockout mice. together with our previous report that irf1 represents a susceptibility gene for allergy in the human, our data highlight irf1 as key in regulating the severity of asthma. the sensory neuropeptide substance p (sp) acts as an important stress mediator with its own stress axis in the skin modulating mast cell as well as antigenpresenting cell (apc) activity. here we postulate that stress-dependent communication between nerve fibers and immune-competent cells can also occur in spleen and affects the course of inflammatory disease. to address this question, atopic dermatitis-like allergic dermatitis (ad) was induced in c57bl/6 mice by intraperitoneal sensitization and intradermal challenge using chicken egg ovalbumin. animals were additionally exposed to noise stress for 24hrs prior to challenge. in this model, stress lead to a relative hyperinnervation of the immune-competent areas of the spleen. at the same time, an increased number of apc could be observed in these areas and contacts between nerve fibers and apc were found. under the same conditions, we were able to show increased nk1-receptor and ppt1 mrna levels. accordingly, sp had the capacity to raise the number of antigen presenting cells in spleen and altered the profile of cd11c expressing apc substets characterized by cd4 and cd8 expression in vitro. in vivo we found a stress dependent shift of cytokine mrna levels towards a th-1 cytokine profile and increased levels of il-2 mrna. further the number of cd25 + t-regulatory cells was increased in vitro. additional analysis of the quality and function of neuro-immune interactions in the spleen will reveal the role of the observed stress-induced alterations in allergic inflammation. proton pump inhibitors (ppis) that are the cornerstone of gastroesophageal reflux disease therapy have been reported to improve asthma and eosinophilic esophagitis (ee) in patients with associated symptoms. the most accepted explanation for these findings is based on the belief that pathologic acidic reflux can act as a triggering factor for these diseases through proximal extent and laryngopharyngeal reflux in asthma and impairment of the epithelial barrier in ee. under these considerations, acid suppression is believed that could prevent these pathogenic mechanisms. nonetheless, a number of evidences suggested the possibility that ppis could have a direct effect in molecular pathways involved in asthma and ee: 1) the inhibitory mechanism of ppis implies alkylation of cysteine residues in gastric atpase3, 2) asthma and ee are prototypic th2 diseases in which the cytokines il-4 and il-13 play a principal role through the activation of the transcription factor stat6, and 3) we have recently demonstrated that some chloromethyl ketones can downregulate stat6 by mechanisms involving cysteine alkylation. on the theoretical basis that cysteine reactivity of ppis may affect the regulation of stat6, we analyzed its effect in the activation of stat6 by il-4 and il-13. we found that treatment of cells with ppis inhibited the ability of il-4 and il-13 to signal stat6 activation in a dose-dependent manner in multiple cell types from different origin. given the important role of these mechanisms in asthma and related diseases, our findings show a novel mechanism to understand the effect of omeprazole in these diseases. in argentina more than three million people suffer from asthma, and the number is rising. asthma is defined as a disorder characterized by chronic airways inflammation that results in high mucus production and airways hyperresponsiveness. a th2 mediated immune response prevails in these patients. in the asthmatic exacerbation period, crisis (cr), triggered by viral infections or other factors, there is a high prevalence of bacterial overinfection. our objective is to compare immunological parameters and in vitro response of lymphocytes to bacterial antigens in the same patient, at that moment and at a time of stability between episodes (i). we studied 12 asthmatic patients both at cr and i. we evaluated eosinophils, basophils and ige expressing b lymphocytes; as well as t regulatory cells (by expression of cd4 and cd25 high (treg)), that might inhibit the development of a th2 response, together with gdt cells, which function in asthma is not completely understood, but could have a role in the increased airway responsiveness. to evaluate the t cell response, mononuclear cells were cultured for 48 hours in the presence of m. tuberculosis (m) or s. pneumoniae (spn), or absence of them (c). then the percentage of activated cells was determined (expression of cd69 or cd25 at 48 hours). all the parameters were evaluated in peripheral blood by flow cytometry. discussion: even though the pathophysiological characteristics of asthmatic patients in periods of cr and i are different, no significant differences were observed in the parameters (cell populations and cell response to bacterial antigens) evaluated when compared for the same patient at cr and i. we might be able to detect differences if we studied cells from the lungs, the target organ. we demonstrate that murine and human lc expressed the h4r. the level of intracellular ccl2 production in human lc was reduced after stimulation with h4r agonists and basal production could be restored when h4r was blocked with the specific antagonist jnj7777120. moreover histamine and a h4r specific agonist augmented the migration of lc from the epidermis as shown in ex-vivo migration experiments using human skin and in-vivo migration experiments in mice. in conclusion, the h4r is expressed on murine and human lc and influences the immunomodulatory function and migration of these cells. these findings underline the relevance of the h4r in allergic skin diseases and encourage further exploration of the h4r as a therapeutic target in allergic skin diseases. expression data of the non-coding rna gene, prins (psoriasis susceptibility-related rna gene induced by stress) identified and characterized by our workgroup, suggests a role for prins in psoriasis susceptibility and in cellular stress response. in order to asses the function of prins, we aimed to identify genes regulated by prins and intracellular molecules interacting with this stress-induced non-coding rna. to identify prins regulated genes, we carried out a cdna microarray chip experiment on hela cells where the expression of prins was silenced. this experiment identified g1p3, an interferon-inducible anti-apoptotic gene that was down-regulated by prins silencing. g1p3 was strongly expressed in proliferating keratinocytes and markedly upregulated in involved psoriatic epidermis compared to healthy epidermis. to detect prins interacting proteins we applied ribonucleoprotein (rnp) purification in hacat cells. with the help of matrix-assisted laser-desorption ionization time-of-flight (maldi-tof) method we identified nucleophosmin, a protein that physically interacts with prins rna. nucleophosmin is a ubiquitously expressed nucleolar phosphoprotein which shuttles continuously between the nucleus and the cytoplasm. immunohistochemical experiments revealed that the expression of nucleophosmin was significantly elevated in psoriatic involved epidermis, localized to the dividing cells of the basal layer. our data indicate that the non-coding prins rna forms a molecular complex with nucleophosmin that regulates stress-induced cellular processes. we suppose that the abnormal functioning of this complex may result in the altered regulation of genes among them the anti-apoptotic g1p3 which can contribute to the pathogenesis of psoriasis. atopic dermatitis (ad) is a chronic inflammatory skin disorder based on a genetic predisposition and triggered by environmental factors characterized by eczematous skin lesions, pruritus, and typical histopathological features. rituximab is a monoclonal anti-cd20 antibody therapy that targets pre-b cells and mature b cells, but not plasma cells. ad is generally considered as a biphasic, with switch to initial th2 to chronic th1-predominant disease, in which rituximab may have multiple effects. objectives: to report three patients with severe ad refractory to conventional treatments and to anti-ige monoclonal treatment (omalizumab). materials and methods: three patients with severe refractory ad with high levels of serum ige that received 4 weekly intravenous infusions of rituximab at a dose 375 mg/m 2 body surface each. subsets of lymphocytes were analyzed with multiparametric-flow cytometry (facscalibur, bd) at baseline and at specific intervals after treatment. serum immunoglobulins levels were quantified by nephelometry. results: at baseline, all patients had highly elevated levels of total ige ( g 5,000; g 6,000; g 19,000 mg/dl, respectively). all patients underwent prior treatment with omalizumab for 6 months, with only partial response. then, we started rituximab therapy, resulting in a clear and complete improvement of ad eczema area and severity of skin lesions in all patients. remission of pruritus was observed from the 2 nd week after initiation of rituximab therapy up to 1 year. whereas allergen-specific ige levels were not altered, we observed a large reduction in total serum ige concentrations after initiation of therapy with rituximab. in the first treated patient (follow-up 1 year), ige levels decreased from 5,512 to 1,500 mg/dl. the other two patients are in the 3 and 1-months of the follow-up period. importantly, during follow-up no other therapies were required for ad control. conclusions: treatment with an anti-cd20 antibody led to a dramatical improvement in our series of patients with severe refractory ad. this study support further evidence on the efficacy and safety of rituximab in severe ad. we have previously demonstrated that chronic topical exposure of mice to the contact allergen dncb or to the respiratory sensitiser trimellitic anhydride (tma) preferentially activates t helper (h) 1 and th2 cells, respectively. in addition, a single application results in divergent cutaneous cytokine production and the migration of langerhans' cells (lc) with different tempos. to explore events occurring after allergen application, balb/c strain mice were exposed to a single topical dose of either 1 %dncb, 25 %tma or to vehicle alone for 0.5-6h. measurement of cytokine production from skin exposed to the allergens was performed by cytokine bead array. exposure to dncb provoked rapid production of il-2 (mean=131pg/ml, n=12, p x 0.001), il-17 (mean=69pg/ml, n=12, p x 0.001) and il-1a (683pg/ml, n=12, p x 0.001) in skin compared with tma-or aoo-treated mice. in subsequent experiments, mice received an intradermal injection of 50ng/ear of murine recombinant il-2 or of the known regulator of lc migration; il-1b. interleukin-1b induced a significant loss of epidermal lc numbers, measured as a function of reduced frequency of mhc class ii positive cells within epidermal sheets, after 4 (32 %) and 16h (31 %) (n=6, p x 0.001). in contrast, il-2 or control injections were without effect. however, il-2 administration caused an increase in cutaneous il-17 production (347pg/ml, n=12, p x 0.001) compared with control injection and naï ve tissue (19 and 63 pg/ml, n=12, ns). in addition, systemic treatment with anti-il-2 antibody failed to impact on lc migration provoked by dncb (33 % reduction; n=6, p x 0.001). in parallel experiments dncb-induced lc migration was blocked by treatment with anti-tumour necrosis factor (tnf)-a antibody, another cytokine known to regulate lc migration. however, dncb-induced cutaneous il-1a (987pg/ml, n=10, p x 0.001) and il-17 (248pg/ml, n=6, p x 0.01) expression was reduced to baseline levels by anti-il-2 treatment. these data demonstrate that il-2 is not involved in the regulation of lc migration, unlike il-1b and tnf-a. however, il-2 is involved in the regulation of the production of other cutaneous cytokines provoked by dncb. therefore it is hypothesised that il-2 may influence lc and dermal dc maturation, via the expression of il-1a and il-17. allergic contact dermatitis (acd) caused by nickel ions (ni) represents the most common form of human contact hypersensitivity. along with other allergies its incidence is increasing in the us and worldwide (nhanes iii survey 2005), but the majority of molecular events underlying this kind of t-cell mediated disease are still widely unknown. to elucidate initial molecular mechanisms (sensitization phase) taking place at the primary allergen contact site in human skin a differential proteomic approach was chosen. by applying dige technology (differential gel electrophoresis), software analysis and mass spectrometric protein identification to cell lysates of allergen stimulated human keratinocytes, seventeen proteins were identified that are specifically regulated by metal allergen ni. in the attempt to further characterize the role of a certain down regulated p38-mapk-pathway related protein (p38prp) in acd, we analysed its regulation, differential distribution of phosphorylated isoforms as well as its subcellular localization. our results strongly support an involvement of p38 mapk pathway in allergenspecific signaling responses. it is expected that identification of differentially allergen-regulated proteins and detailed analysis of acd-associated signaling events in primary keratinocytes will lead to a better biomolecular understanding of the initiation of human contact hypersensitivity. (work supported by eu-project novel testing strategies for in-vitro-assessment of allergens, lshb-ct-2005 -018681, www.sens-it-iv.eu.) objectives: schnitzler's syndrome is a rare disease characterised by chronic urticaria, monoclonal gammopathy, fever, and arthralgia/arthritis with marked elevation of acute phase reactants. in the long term, 15 % of patients develop a lymphoproliferative disorder. schnitzler's pathogenesis is unclear; immunosuppressive treatment is ineffective and high dose steroids are usually required. the recent finding that treatment with il-1 receptor antagonist (il-1ra; kineret) is extremely effective has raised the issue of the role of the inflammatory cytokine il-1b and of il-1-like cytokines in the pathogenesis of the disease. methods: two patients with recently diagnosed schnitzler's syndrome were treated with kineret, obtaining rapid disappearance of fever and urticaria and normalisation of acute phase reactants in one month. blood samples were collected before and after initiation of therapy. serum cytokine levels were measured by elisa, and expression of il-1-related genes by real-time pcr on mrna from blood cd14+ monocytes. results: compared to normal controls, schniztler's monocytes had similar expression of il-18, il-18bp, and caspase-1, both before and after therapy with kineret. il-1b expression was similar to controls before therapy, and was decreased five-fold after therapy. at the serum level, neither inflammatory (il-1b, tnfa, il-12) nor anti-inflammatory cytokines (il-10, tgfb) were detected. as expected, il-1ra was only detectable after therapy. il-18 was detectable in schnitzler's sera at higher levels than in controls (23.0 vs. 10.3 pm) and decreased after therapy (13.2 pm). the circulating il-18 inhibitor il-18bp was lower than in controls and not affected by therapy. thus, free il-18 levels were increased in schnitzler's patients as compared to controls (17.6 pm vs. 7.2 pm in controls) and decreased after therapy (9.9 pm). conclusions: schnitzler's syndrome is not associated to enhanced expression of il-1-related cytokines (il-1b, il-18), nor of the il-1/il-18-converting enzyme caspase-1 in blood monocytes. however, the high circulating levels of il-18 suggest an increased activity of caspase-1, as in the case of autoinflammatory diseases. experiments are in progress to test this possibility. atopic dermatitis (ad) is a chronic relapsing allergic skin disease with a high and growing prevalence. currently around 20 % of the children in industrialized countries are affected. in most cases patients exhibit increased systemic ige-levels (so-called extrinsic form) accompanied by sensitization to allergens. while ad is frequently cleared until adulthood, many patients develop allergic rhinitis and asthma. most ad-patients show topical colonization with staphylococcus aureus indicating a defective innate immune response. as a class of pattern recognition receptors, toll-like receptors (tlrs) are essential for pathogen recognition and critical for the induction of an effective adaptive immunity. all known tlrs except tlr3 signal via myd88 to induce nfxb-dependent gene transcription. tlrs are also known to be involved in the pathogenesis of autoimmune diseases. as chronic ad also has an autoimmune component, the study of myd88 signaling in ad might provide new insights into the function of tlrs. to investigate the role of tlrs in the immunopathology of allergic reactions and skin infections, we induced ad-like symptoms in c57bl/6 myd88-deficient mice by repeatedly sensitizing the mice to ovalbumin (ova) after mechanical disruption of the skin barrier by tape stripping. first results show that myd88-/-mice display reduced inflammation of the treated skin area compared to wildtype mice. immunostainings of skin biopsies reveal reduced acanthosis and infiltration of inflammatory cells into the dermis compared to wildtype. skin-draining lymph nodes are less enlarged in the ova-treated knockout mice compared to wildtype and differ in cellular composition. serum antibody levels determined by elisa show reduced systemic total and ova-specific igg1-titers in ova-treated myd88-/-mice compared to wildtype mice, although the nacl-treated myd88-/-control group has higher total antibody titers than the wildtype nacl control group. total ige levels are increased in the knockout mice compared to wildtype mice under both conditions. to further investigate the role of staphylococcus aureus during ad development, we will include topical application of the superantigen staphylococcal enterotoxin b (seb) or living bacteria into our analyses. following this approach, we anticipate to obtain new insights into the role of the innate immune system in allergic reactions of the skin. introdution: the skin of vertebrates is the target for over 15,000 species of hematophagous arthropods. among these are ticks, which are long-term feeders and interact with host defenses for days to weeks. little is known about specialized strategies for eliminating ectoparasites, but ticks can induce immune responses in hosts. bovines present variable and heritable levels of resistance to the tick rhipicephalus microplus and are the only model in which distinct outcomes of infestation can be examined in the same species of host. in order to obtain some of the immune correlates of these outcomes, we examined expression of candidate genes and quantified populations of leukocytes and subpopulations of lymphocytes present in the inflammatory infiltrates elicited by tick bites in skin of genetically resistant and susceptible bovine breeds, respectively, nelore (bos taurus indicus) and holstein (b. t. taurus). methods: skin biopsies (6 mm punch) were taken at the feeding sites of ticks from susceptible and resistant cattle (each phenotype n = 4) or from non-infested contra-lateral sites. expression of mip-1a, igf-1, mcp-1 and ip-10 genes was quantified with realtime rt-pcr. sections of paraffin-embedded skin were stained with may grünwald-giemsa for differential cell counts. lymphocytes in sections of frozen skin were phenotyped with specific antibodies using immunoperoxidase technique; in infested skin, histological sections were limited to the area of the tick's cement cone. results: as expected, hosts recruit cutaneous inflammatory infiltrates around the tick's mouthparts. however the composition of infiltrates presented with significant differences that varied according to the phenotype of infestation. inflammation of nelores contained significantly more basophils, eosinophils and mononuclear cells expressing cd3, cd4, cd8, cd21, mhc class ii, and p46 than that of holsteins. lymphocytes expressing wc1 and cd21 antigens were significantly diminished in infested skin of holsteins when compared with control skin (p x 0.05). infested skin of nelores contained significantly more message for mip-1a, igf-1, mcp-1 and ip-10 than that of holsteins. conclusions: although ticks secrete molecules that inhibit cell adhesion and chemokines, resistance correlates with the capacity to recruit and maintain populations of leukocytes that generate effector immune responses. supported by cnpq, capes, fapesp, and icttd. in the last decade it has become clear that keratinocytes play an important role in the skin immune system. upon stimulation, keratinocytes produce high amounts of proinflammatory chemokines and cytokines and express receptors which are involved in immunoregulation. in a number of inflammatory skin diseases such as eczema or psoriasis infiltrating lymphocytes are found in close vicinity to keratinocytes, enabling interaction of these two cell types. it has been proposed (goodman et al.) that keratinocytes rather support a th2 response by interacting lymphocytes. we examined this hypothesis with autologous cultures of keratinocytes derived from the outer root sheet of the hair follicle co-cultured with cd3+ t cells from the same donor. during the coculture either seb or antigen were added. in all experimental approaches the addition of keratinocytes resulted in higher production of ifng by t cells. furthermore, we set up an experimental approach were autologous antigen-pulsed monocytes were also added. again, the induction of ifng by the presence of keratinocytes resulted in a marked and significant increase of ifng production by t cells. we were able to show that il-1 plays a crucial role in the induction of ifng in t cells keratinocyte interaction. in addition blocking of lfa-1 in the co-cultures resulted in significantly reduced ifng production by t-cells underlining that icam-1/lfa-1 binding is also crucially important for ifng induction. we conclude from our study that keratinocytes rather support a th1 than a th2 local response pattern by virtue of il-1 secretion and icam-1/lfa-1 interaction. this property of keratinocytes may account for the observed cytokine switch in allergic eczematous skin from a th2 like micromilieu in acute towards a th1 dominated milieu in chronic lesions. the genotyping of ccl4l gene in patients with psoriasis could allow describing subcategories of patients based in clinical parameters and disease severity. therefore, it could be also used as a clinical diagnostic tool, potentially modulating the efficacy of new treatments, or even to be used as a therapeutical target of psoriasis. this work was supported by project grants of merck-serono and instituto de salud carlos iii (pi07/0329). psoriasis is an inflammatory dermatosis with 2 % prevalence among caucasians. hla-cw6 allele is the gene that confers susceptibility to psoriasis and it is placed near to tnf loci with several snp in promoter region. the most common polymorphisms are two g to a transitions in -308 and -238 positions. strong association was found between polymorphisms in the -238 region with psoriasis. in several diseases, the association with hla and clinical manifestation is different between genders, for example in spondyloarthropathy and hla-b27, and this is a question of increasing interest. the objective of this study was to identify clinical and molecular differences between male and female in brazilian psoriatic patients. sixty-nine individuals assisted at the dermatology outpatient clinic of the teaching hospital, university of campinas, with diagnosis of psoriasis of early-onset (up to 40 years of age) were selected. hla-a -b -c -dr -dq alleles and tnf-238 and -308 snp were differentiated by pcr/ssp. analyzing the total group, 21 patients (30.5 %) were male, 48 (69.5 %) were female. in the male group, the mean age at disease onset was 16,3 years. severe forms were seen in this group (psoriatic arthritis in 4 cases and erythroderma in 2). seven patients (33,3 %) had a favorable evolution of the disease, but 14 (66,4 %) developed extensive psoriasis, covering over 30 % of body surface requiring systemic treatment. the main molecular risk factor for the disease, cw*06 allele was positive in 10 cases (47,62 %), tnf 238 g/a genotype was found in 5 (23,8 %) and tnf 308 g/a in 4 (19,1 %). in the female group, the mean age at disease onset was 14,6 years, one case of psoriatic arthritis and one of erythroderma. twenty-nine (60,4 %) had a favorable evolution of the disease and 19 (39,6 %) an unfavorable evolution. cw*06 allele was positive in 26 cases (54,2 %), tnf 238 g/a genotype was presented in 16 (33,3%) and tnf 308 g/a in 8 (16,6 %). severe disease was seen in male patients. there was no difference in frequency of cw*06 allele between male and female groups, but there was a tendency of significant difference in tnf 238 g/a genotype. we found that c57bl/6 mice were more susceptible than balb/c and dba/2 mice. higher susceptibility was reflected by higher footpad swelling and transient systemic dissemination. analysis of serum cytokine level revealed differences in production of proinflammatory cytokines, such as il-6 and mrp8/14, among different inbred strains of mice. furthermore, we identified the cells which are involved in this cytokine production. as expected, histopathological analysis showed that s. aureus infection induces an influx of monocytes and granulocytes. our study shows that not only bacteria-but also host-specific differences are associated with different courses of s. aureus skin infection. aims: to investigate the cause and to study the clinical symptoms and the laboratory findings of the anaphylactic reactions in the pediatric population of our country, considering that these are very often dangerous situations which demand direct treatment and increased alertness. methods: 136 cases, which were studied retrospectively, included children (84 boys and 52 girls), aged 3-14 years, who had an anaphylactic reaction, out of the 915 that were examined in total. the statistical analysis of the data was held with the spss program. the commonest causes were proved to be food (44 %-particularly sea food and dried fruit), drugs (25 %-usually antibiotics and non-steroidal antiinflammatory drugs), as well as insect bites (9 %-mainly caused by hymenoptera). the symptoms included mainly the presence of pruritic pomphus with erythema (76 %), and gastrointestinal symptoms (37 %), while there were quite many cases with dyspnea, nasal congestion, but also angioedema. total ige g 100 was found in 26 out of the 47 severest cases (55,4 %), in which the adequate control was held, while in their vast majority (45 out of 47) there was no previous anaphylactic reaction. on the other hand, it was proved in total, that in 53,7 % (73 cases) there was a hereditary family history of atopy, while in 52 children (38 %) there was also a personal history of asthma. finally, at a great percentage (69 %) eosinophilia was found, while a statistically significant seasonal distribution during spring and summer was registered. conclusions: it has, therefore, been shown that 1)the anaphylaxis is quite often in the pediatric population, with the commonest causes to be food and drugs, which are often thoughtlessly used. 2) in particular, in many cases it is proved that there is a personal but also a family history of atopy. 3) increased attention should be, thus, given in these cases -especially during spring and summer-for their early diagnosis as well as for their effective treatment (adrenaline, antihistamines and corticosteroids) particularly for the severest cases, where the hospitalization of the patient is also necessary. allergic contact dermatitis (acd) is an adaptive inflammatory response of the skin triggered upon exposures to certain chemicals or metal ions. as classical type iv delayed hypersensitivity reaction this response is mediated by t-cells. since many ingredients in consumer products might exert allergenic potency, there is a need for an appropriate screening and characterization of the chemicals used according to this toxicological endpoint. up to now the identification of potential allergens completely relies on animal testing, like buehler assay or guinea pig maximization test (gpmt). due to economical and ethical reasons, as well as driven by the enforcement of certain governmental regulations (i. e., cosmetics directive), the development of an in vitro test system for identification of potential sensitizers is mandatory. since dendritic cells (dcs) play a pivotal role in the initiation of contact dermatitis we chose dcs to characterize known sensitizers in their ability to activate these cells and subsequently examined the molecular interplay between dcs primed by allergens and t-cells in the test tube. the known allergens nickel, dinitrochlorobenzene (dncb) and cinnamic aldehyde were tested for their ability to alter the expression of several immunomodulating surface molecules on dcs derived from monocytes that display a langerhans cell (lc) type-similar phenotype. we used multicolour flow cytometry to detect differences in expression patterns of surface molecules that have been associated with maturation. in addition to the upregulation of cd86 we could observe dose dependent upregulation of programmed death ligand 1 (pdl-1) and downregulation of the dendritic cell immunoreceptor (dcir). furthermore we observed enhanced t-cell proliferation in mixed leukocyte reactions (mlrs) applying lcs stimulated with allergens ex ante. since changes in the expression of only single cell molecules are unlikely of being sufficient for reliable identification of possible contact allergens, we are aimed at analyzing a wide pattern of various surface molecules by multicolour facs and propose that this might be a reasonable approach to screen for contact sensitizing properties of chemicals. our findings are of particular interest for further development of new in vitro assays, using immune cells, to detect the sensitizing potential and quantify the sensitizing potency of chemicals. we want to present the case of a 60 year old iraqui patient with arabic ancestors who had been suffering from psoriatic arthritis since 35 years. in march 2006 a treatment with fumaric acid esters in combination with ibuprofen was introduced. this led to the complete healing of the skin lesions. for this reason the dose could be reduced to one tablet fumaric acid esters (120 mg) every second day. in april 2008 the patient presented himself in the consult with multiple livid papules with a diameter of 3 mm in the area of the auricle. the histological examination showed an hhv-8 positive kaposi sarcoma. the differential blood cell count demonstrated a lymphocytopenia. the hiv-serology was negative. the staging examinations (chest x-ray, gastroscopy, coloscopy, abdominal and lymph node sonography) showed no signs of visceral involvement. after the diagnosis the treatment with fumaric acid esters was discontinued. over the course the livid papules showed a spontaneous complete regression. a spontaneous regression is known from the iatrogenic ks caused by immunosuppressive therapy when the immunosuppression is terminated. as our patient also showed a spontaneous regression of the kaposi sarcoma after stopping the treatment with fumaric acid esters we propose a causative relation. sarcoidosis is a multisystemic granulomatous disease with unknown etiology. although the immunopathogenesis of sarcoidosis remains unknown there are some supportive evidence for the significant role of th1 type immune response. recently, suppressor of cytokine signaling (socs) proteins have been identified as regulators of cytokine signaling pathways. in this study we aimed to evaluate the roles of socs1, socs3 and foxp3 in the immünopathogenesis of sarcoidosis and their association with responsiveness to treatment. peripheral blood (pb) and broncholaveolar lavage (bal) mononuclear (m) cells from sarcoidosis patients in remission following treatment (responders, n:4), the patients who showed recurrence or progression after treatment (non-responders, n:4) and stage i/ii sarcoidosis cases which were followed up without any treatment (untreated, n:7) were evaluated for socs1, socs3 ve foxp3 mrna expressions by taqman pcr, and also flow cytometric analysis was performed for lymphocyte markers including cd3, cd4, cd25, foxp3, cd4 + cd25 high , cd4 + foxp3 + . expression of socs3 and foxp-3 mrna in pbmcs and balmcs from responders were found to be significantly higher in comparison to other two groups . socs1 was found significantly elevated in pbmcs of responders when compared with other two groups. it was also elevated in balmcs of responders when compared with with those of untreated cases. the proportions of cd25, foxp3, cd4+cd25 high , cd4 + foxp3 + cells in pbmcs and balmcs of responders were found to be increased in comparison to nonresponders and untreated cases. our data demonstrates that socs1, socs3 and t regulatory cells may have potential roles in the control of sarcoidosis. we think that if the roles of socs1 and socs3 molecules and t regulatory cells are well characterized, new therapeutic approaches targetting cytokine signal supressors, which can strenghten the regulatory responses, may be beneficial for the sarcoidosis cases resistant to conventional therapy. the inorganic dust, containing free crystalline silicon dioxide (fcs) is critical for the development of silicosis. several studies supported the view that fibrotic responses mainly depends on the regulation of the immune response to the fcs in affected individuals. the role of fcs in induction of a local and systemic inflammation and pulmonary fibrosis are still debates.we studied the changes of neopterin, as a marker for ifn-g dependent macrophage activation and circulating immune complexes (cic), as a marker of humoral immune response, in patients with silicosis and workers exposed to dust containing fcs.we survey a group of 62 silicosis patients, with mild (21), moderate (23) and severe (18) silicosis, 92 coal workers, exposed to inorganic dust containing fcs (exposed), and 43 healthy workers without exposure to dust aerosol (controls).the serum quantity of neopterin and cic, containing iga(igacic), igg(iggcic) and igm(igmcic) was detected by elisa. differences between investigated groups were detected by student's t-test and a p-value less than 0.05 was considered significant.neopterin level was significantly elevated in exposed (3,2±0,8ng/ml) compared to controls (1,8±1,3ng/ml; p=0,0001). moreover, the neopterin level in exposed was similar to silicosis patients (2,9±1,6ng/ml).the levels of iggcic was significantly elevated in the exposed compared to controls (81,9±22,7au vs 64,3±16,7au p=0,0001) and to silicosis patients (69,6±20,0au p=0,002). in contrast, igmcic was significantly elevated in silicosis than in exposed (70,2±18,8au vs 62,1±24,2au; p=0,03).in comparison with exposed, significantly higher igmcic was found only in mild, but no in moderate and severe silicosis. in contrast, the level of iggcic in mild and moderate silicosis was significantly lower compared to the exposed (p=0,001 and 0,02 respectively).the obtained results showed that activation of alveolar macrophages mainly depends on the presence of fcs in the respirable dust fraction and precedes the clinical data for pulmonary fibrosis. the dynamics of cic suggest the involvement of fc-receptors mediated regulation of the immune response in the progression of pulmonary fibrosis, and could be useful marker for exposure to inorganic dust containing fcs. described pathologic similarities between sarcoidosis (sa) and tuberculosis (tbc) suggest m. tuberculosis antigens as caustaive agentes. it seems that in the genetically different predisposed hosts, the same antigens may cause the development of sarcoid or tuberculous inmune response. so different hla haplotypes have been described as a predisposing factor to develop sarcoidosis (hla a*01, b*08, drb1*03 (these of good prognosis) *12/*13/*14/*15) or tbc (drb1*02/*05/*14/*16 and associations with dqa1*02/*03/*05) we describe two cases of two female patients from the same geographic region with mantoux and zhiel-neelsen negative tests and high levels of tnf diagnosed of tbc and sa respectively. both of them debuted with the same clinical manifestations: fever, abdominal pain, and asthenia and shared similarities in the images from the tc study (pulmonary nodules and mesenteric adenopaties). we found the same results for the flow citometry analysis of the non-caseificant granulomes as well as the same anatomopathologic characteristics. after being treated with anti-tbc drugs, the first one presented a good clinical improvement, so she was diagnosed as tbc. the second one did not improved, so she was treated with corticosteroid, with good results. therefore, she was diagnosed as sarcoidosis. after hla analysis, we noticed that the tbc patient was hla a*01, b*08 and drb1*03 (sarcoidosis good prognosis haplotype) and the patient diagnosed as sarcoidosis was hla a*01, drb1*14. as the results show, could there not be a direct relationship between the hla system and the development of sa or tbc, or in contrast, was the first patient missdiagnosed of tbc being a good prognosis sa? objectives: experimental mouse models for acute asthma are well established, yet models for chronic asthma have several shortcomings. for example, current chronic models show decreased inflammation over time and only marginal effects on airway remodelling. experimental models for chronic asthma are essential for development of new therapeutics and must include changes that closely resemble clinical conditions. ovalbumin (ova), house-dust-mite (hdm) and cockroach (cra) proteins are commonly used to trigger an asthma like response in mice and, for this reason, were used in the present study. the objectives of our work were to compare the most frequently used mouse models of chronic asthma and to develop a mouse model of chronic asthma that clearly displays pivotal features of severe human asthma. methods: for the induction of asthma, mice were initially sensitised by intraperitoneal injection of ova, hdm, cra or a combination of all three, followed by repeated challenge by intratracheal application of ova, hdm or cra. inflammation in lung was measured by analysis of cell influx into the bronchoalveolar lavage (bal) and by determination of chemokine and cytokine levels in bal and lung tissue using elisa and multiplex technology. additionally, serum levels of ige and igg antibodies were measured. airway remodelling was assessed by histological staining for mucus production, immune cell influx, smooth muscle thickening and fibrosis. results: significant differences were measured in cell influx, chemokine/cytokine and total ige levels. compared to hdm and cra, ova induced an higher cell influx in the bal, hdm showed an increase of chemokines in bal and increased ige levels in serum. using a combination of all three proteins resulted in the most severe form of asthma. conclusions: to our knowledge, this is the first study that directly compares the most commonly used mouse models in regard to their potential to display a pathology specific of severe asthma. the most sustained and severe form of asthma was induced by the combination model. this model offers particular advantages for evaluating existing and novel therapeutic agents. furthermore, this model could contribute to understanding of the mechanisms underlying chronic asthma. the present study focused on peri-smi connective tissue capsule formation, the most frequent post-operative local complication in patients receiving smi. we investigated the local immune processes via the phenotypic and functional characterization of lymphocytes within the fibrotic tissue. to this end, intracapsular lymphoid cells and peripheral blood mononuclear cells (pbmcs) from the same patients were isolated and analyzed via facs, concentrating on t-effector cells (teff) and t-regulatory cells (tregs: cd4 + , cd25 ++ , foxp3 + ), cytokine profiles, t-cell receptor (tcr) repertoire and reactivity against human heat shock protein 60 (hhsp60). intracapsular tregs were visualized by immunohistochemistry and functionally tested in suppression assays. the cellular composition of intracapsular mononuclear cells showed a preponderance of cd4 + t-helper cells and a significant subset of tcrg/d + cells, exceeding that observed in peripheral blood. il-17, il-6, il-8, tgf-b and ifn-g production prevailed, pointing to a th17/th1 weighted immune response. furthermore, intracapsular t-cells displayed a restricted tcr a/b repertoire (monoclonal/oligoclonal) as well as a preferential reaction with hhsp60. importantly, numbers of intracapsular tregs were inversely proportional to the degree of fibrosis and showed less suppressive capacity as compared to peripheral tregs. our results suggest that silicone triggers a specific local immune response via activated th17/th1 cells, promoting fibrosis due to the production of profibrotic cytokines. clonal restriction of the tcr repertoire is a further indication for a specific antigen driven immune response preceding capsular fibrosis. in this context, hsp60 might be a prominent candidate. taking into consideration that it is ubiquitously expressed, it might be the "missing link" between local and systemic side effects of smi. the inverse correlation between the degree of capsular fibrosis and the number of intracapsular tregs suggest that tregs may initially be able to inhibit the progress of capsular fibrosis. however, as numbers of tregs, as well as their suppressive capacity decreases over time, fibrosis develops. supported by the competence center medicine tyrol (kmt) and the lore-and-udo-saldow donation. objectives: recent findings have proven that silicone induces a local inflammatory response with subsequent fibrotic reactions. the present project deals with the standardization and further development of a modified elisa test system (silisa ® ) for the identification of patients with a risk for fibrotic side effects to silicone mammary implants (smis) based on the protein signature adhering on the surfaces of such devices (1) . the current silisa ® is a test system for the simple detection of the adhesion pattern of proteins from patients' sera to silicone. the optimization of the silisa ® comprised inter-and intra-assay standardization, robustness, specificity and sensitivity. the essay was further developed with antibodies against annotated proteins that were not yet tested in the past. all experiments were carried out in a 384 well plate format for high-throughput analysis. statistical analysis has been performed using spss. the extended essay has been successfully established in the system with antibodies against seven already tested proteins, including c-reactive protein, collagen-i, collagen-iii, fibronectin, igg, c3-complement, myeloid related protein 14 and two new proteins, integrin-ß4 and fibrinogen. data from more than 100 patients have been obtained and exploited so far. the intra-and inter-assay variability of the test was reduced to less than 10 % and 16 %, respectively. patients with fibrotic reactions to silicone were successfully identified using a pattern of protein deposition to silicone. conclusion: applying the silisa ® , sera from five different groups were tested: silicone patients with and without fibrotic reactions, female and male individuals without any contact to silicone and hospital's medical staff with potential silicone contact. the distribution pattern of eight proteins showed differences in patients developing strong fibrotic reactions to silicone compared to controls. muscular lesion is a frequent matter in sportive medicine and myodegenerative diseases. necrosis of the damaged tissue and activation of inflammatory response characterize the initial phase of muscle repair. this work aimed to analyze the tissue repair after induction of lesion in skeletal muscle from mouse lineages with distinct cytokine secretion patterns. it was included at least 3 mice per group with distinct cytokine pattern: th1 (c57bl/10, c57bl/6) and th2 (balb/c). muscular injury was performed by injection of bupivacaine. both th1-dominant strains presented more areas with regenerating myofibers and macrophages at 4 dpi. regional lymph nodes showed significant increase of cellularity and relative numbers of cd3 bupivacaine-inoculated balb/c mice compared to non-inoculated matched mice at 4 dpi. balb/c mice showed increased collagen expression and decrease of mmp-9 activity associated with more mrna for tgf-b1. this study shows that the immune background of the mouse may affect the remodelling processes in skeletal muscles that occur in response to bupivacaine injection promoting muscle regeneration (th1 cytokines) or myonecrosis and collagen deposition (th2 cytokines). the severe, life-threatening heart failure in some of the patients with dilated cardiomyopathy (dcm) is imputed to the stimulatory autoantibodies against the second extracellular loop (ec ii ) of the ß1-adrenergic receptor (anti-ß1ec ii ). to analyze their pathogenic impact as a single causal factor we used a human-analogous lewis rat model of dcm, where monthly subcutaneous immunization of the rats with the ß1ec ii peptide as a glutathione-s-transferase (gst) fusion protein induced production of anti-ß1ec ii and eventually dilated cardiomyopathy. in this model we isolated a ß1ec ii -specific rat monoclonal antibody (clone 13f6), and showed by elisa that it binds to the linearized ß1ec ii peptide. additionally, we confirmed with flow cytometry that 13f6 also binds the ß1ec ii in its native conformation, i. e. directly labeled circular ß1ec ii (dyl649-ß1ec ii ) peptide. moreover, we demonstrated activation of the ß1-adrenoreceptor by 13f6 using a fluorescence resonance energy transfer (fret) assay system in vitro. these data further corroborate the pathogenic role of anti-ß1ec ii antibodies in mediating dcm in this animal model, thus rendering them a potential therapeutic target. therefore, we investigated a novel anti-ß1ec ii -specific peptide-based therapy, by intravenously applying a circular ß1ec ii peptide in the dcm lewis rat model to neutralize the anti-ß1ec ii antibodies. while the peptide therapy strongly reduced the anti-ß1ec ii titers in the serum by up to 80 % and consecutively lead to clinical remission, elispot assays for the detection of ß1ec ii -specific antibody-secreting cells (asc) indicated no difference in the number of long-lived plasma cells in treated animals. in contrast, elispot and flow cytometrical analyses revealed a decrease in the number of ß1ec ii -specific memory b cells in the treated animals, indicating that this cellular compartment is most likely also targeted by the peptide therapy. our newly developed anti-ß1ec ii -specific therapy, thus, not only neutralized the pathogenic autoantibodies, but also depleted antigen-specific memory b cells involved in the generation of these autoantibodies. these results provide the rationale for further development of this therapeutic strategy for eventual application in patients with autoimmune dilated cardiomyopathy. cardiovascular diseases like myocarditis and subsequent dilated cardiomyopathy (dcm), are a frequent cause of mortality in humans with dcm being the most common reason for heart failure in young adults. infections with coxsackievirus b3 or cytomegalovirus can lead to an acute inflammation of the heart muscle that is followed by an autoimmune response directed autoantigens in the heart, such as the alpha isoform of cardiac myosin (myhca). immunization with the well-characterized myhca 614-629 epitope elicits autoreactive cd4 + t cell responses that have been shown to be the major mediators of autoimmune myocarditis in balb/c mice. it is known that professional antigen presenting cells (apcs) such as dendritic cells are crucial for initiating and maintaining t helper (th) cell responses affecting the heart muscle. however, the detailed analysis of the interaction between these cells in the context of autoimmune myocarditis has been hampered by the lack of appropriate analytical tools. we therefore generated a tcr transgenic mouse harboring t cells that specifically recognize the myhca 614-629 peptide. in a first step, hybridoma cells were generated by fusing bw5147 tcra -cd8lymphoma cells with myhca 614-629 -specific th cells. tcr expression and antigen specificity was assessed by facs analysis and elispot assay. following subcloning, the variable regions of the expressed tcr were characterized by pcr-sequencing. the rearranged v(d)j regions were subcloned into tcr cassette vectors and linearized constructs were injected into the pronuclei of fertilized oocytes. using this novel tcr tg mouse we plan to investigate in detail the activation of myhca 614-629 -specific t cells during the process of autoimmune myocarditis. furthermore, this new tool will help to generate a high resolution analysis of the contribution of different apcs in the activation and differentiation of autoreactive th cells during inflammatory heart disease. m. relle 1 , a. schwarting 1 , p.r. galle 1 1 university medical center of the johannes gutenberg university mainz, medical clinic i, mainz, germany several mouse or rat models have been established to explore the role of proteinase 3 (pr3), in anca-associated glomerulonephritis, vasculitis or pulmonary inflammation but these studies have demonstrated that anca alone are not sufficient to induce these diseases directly. therefore, we assessed the expression, mobilization and enzymatic activity of pr3 in mouse bone marrow, kidney, spleen and peripheral blood by immunohistochemistry and immunoblots, as well as the proportion of pr3-positive neutrophils in the peripheral blood of frequently used mouse strains. neutrophils were mobilized from the bone marrow by an intraperitoneal injection with human il-8. pr3-mrna from the murine cancer cell line wehi-274 was amplified by race-pcr and subsequently sequenced. sequence comparisons were done with dnasis software package and the blast tool of the ncbi. promoter analyses were performed with the genomatix software matinspector. we could demonstrate, that mouse bone marrow is a reservoir for functional neutrophils, which are rapidly mobilized after injection of furthermore, we identified an alternative pr3-promoter in the second intron of the mouse pr3 gene. this promoter is active in the bone marrow, in embyros and in cancer cell lines, indicating that its expression is not restricted to myeloid cells. fine structural analyses of this alternative promoter revealed differences not only between the rat and the mouse promoter but also between different mouse inbred strains. taken together, we have shown that the maturation processes of mouse neutrophils differ from those of human granulocytes. the identification of an alternative pr3 transcript and its promoter indicates that the murine pr3 may have additional, as yet not described, functions in hematopoiesis and cancerogenesis. objective: recent studies show that in vivo administration of och, a synthetic lipid that specifically activates natural killer t (nkt) cells, results in suppression of th1 mediated immune responses in autoimmune diseases. nkt cell activation depends on lipid presentation via the mhc-i like molecule cd1d on antigen presenting cells such as mature dendritic cells (mdcs) and upon activation by och nkt cells rapidly produce large amounts of th2 cytokines. the goal of this study was to investigate the effect of och and och-primed dendritic cells on atherogenesis. methods & results: ldl receptor deficient (ldlr -/-) mice were fed a western type diet and atherosclerosis was induced via collar placement around both carotid arteries. subsequently the mice were treated i. p. with och (n=13) or pbs (n=11) twice a week for seven weeks. the injections with och did not affect atherosclerotic lesion size. to improve the presentation of och to nkt cells in vivo, bone marrow-dendritic cells were maturated via tlr4 activation, in the presence/ absence of och. subsequently we transferred 1.5x10 6 mdcs (n=11) or och-primed mdcs (n=11) (3 times) to ldlr -/mice. afterwards the mice were put on a western type diet to induce atherosclerosis. vaccination with och-primed dcs resulted in a 70.6 % reduction in plaque size compared to mice treated with mdcs (p x 0.05). during the experiment no effect on serum cholesterol levels was observed, but at the end of the experiment there was a significant 23.7 % (p x 0.05) reduction in cholesterol levels in the mice treated with och-primed dcs. the number of nkt cells in blood and liver was monitored and a 2 to 3-fold increase in these cells was detected 3 days after the last treatment with och-primed dcs (p x 0.05). additionally, the nkt cells in the liver of mice treated with och-primed dcs produced more il-10. discussion: we conclude that immunotherapy using och-primed dendritic cells efficiently activates nkt cells, resulting in a th2 phenotype of the nkt cells and this leads to an efficient protection against atherosclerosis. these data indicate that immunotherapy based on ligand specific primed dcs may be a novel way to treat atherosclerosis. systemic lupus erythematosus (sle) is characterized by high serum titers of igg anti-nuclear antibodies secreted by plasma cells. however, the characteristics of the igg+ plasma cell antibody repertoire in sle has never been determined on a single cell level and little is known about the role of germinal center (gc) reactions for the development of sle autoantibodies. the igg inhibitory fcgriib knock-out mouse on the c57bl/6 background is a strain specific lupus autoimmune model that is characterized by the spontaneous development of autoantibodies to nuclear antigens such as dsdna and chromatin. to characterize the igg+ plasma cell compartment under normal circumstances and in autoimmunity we have cloned and expressed 350 igg antibodies from single isolated gc b cells and plasma cells derived from spleen, bone marrow and lymph nodes of wild-type c57bl/6 and fcgriib deficient mice. igh and igl chain gene sequence analyses revealed no major differences in the ig gene usage between wild-type and autoimmune mice, but gc b cells of fcgriib were enriched for antibodies with positively charged igh cdr3 regions and anti-nuclear specificity. the overall frequency of autoantibodies was similiar between wild-type and fcgriib deficient mice. however, strongly autoreactive antibodies to dsdna and murine igg2c were isolated only from fcgriib deficient mice, but not from c57bl/6 control mice and somatic mutations contributed to their generation. in summary, our data suggest that the gc reaction plays an important role for the development of self-reactive antibodies in fcgriib deficient mice. the finding that the frequency of autoreactive antibodies is higher in gc b cells than in spleen or bone marrow plasma cells may indicate that autoreactive gc b cells are partly regulated even in the absence of fcgriib. autoantibodies against double-stranded dna (dsdna) and nucleosomes (ncs) represent a hallmark of systemic lupus erythematosus (sle). however, the factors leading to the autoimmune response against these nuclear autoantigens are not fully identified. high mobility group box 1 protein (hmgb1), a nuclear dnabinding protein and an extracellular proinflammatory mediator gets tightly bound to modified chromatin during apoptosis. it is not released, since apoptotic cells are immediately engulfed by phagocytes. conversely, in conditions of clearance deficiency, which is observed in a subset of patients with sle, non-ingested apoptotic cells, may undergo secondary necrosis, thereby releasing ncs containing the "endogenous adjuvant" hmgb1. we investigated if hmgb1-containing ncs contribute to the breakdown of immunological tolerance against dsdna and ncs. we found that hmgb1 remains associated with ncs released from late apoptotic cells in vitro. hmgb1-ncs complexes were detected also in the blood of patients with sle. hmgb1 containing ncs from apoptotic cells induced secretion of il-b, il-6, il-10, and tnfa as well as expression of co-stimulatory molecules on human and murine macrophages and dendritic cells (dc), respectively. cytokine release from murine macrophages was dependent on myd88 and toll-like receptor 2. neither hmgb1-free ncs from living cells nor from apoptotic hmgb1-or hmgb1/2-deficient cells induced marked cytokine production or dc activation. specific inhibition of hmgb1 activity by the antagonistic a box domain significantly reduced capacity of "apoptotic "ncs to induce tnfa and il-10 release by macrophages. immunizations with hmgb1-containing ncs from apoptotic cells induced anti-dsdna and anti-histone igg responses in non-autoimmune mice in tlr2-dependent manner. in conclusion, hmgb1 in complex with ncs activate antigen presenting cells thereby contributing to the loss of immunological tolerance against ncs/dsdna and, hence, to the immunopathogenesis of sle. objective: apoptotic cells are considered to be a major source for autoantigens in autoimmune diseases such as systemic lupus erythematosus (sle). in agreement with this, defective clearance of apoptotic cells has been shown to increase disease susceptibility. still, little is known about how apoptotic cell-derived self-antigens activate autoreactive b cells and where this takes place. methods: injections of fluorescently labelled syngeneic apoptotic cells were traced using immunofluorescence microscopy. binding studies were performed using apoptotic cells and cho cells transfected with class a scavenger receptors (sr). repeated injections of syngeneic apoptotic cells in sr deficient and wild type mice were conducted and antibody production by autoreactive b cells was measured. autoreactivity against sr was followed in two sle prone mice strains over the development of disease and in a cohort of sle patients. an antibody against the sr was injected together with several antigens to directly evaluate the possible role of autoantibodies against the receptors. results: in this study, we find that apoptotic cells are taken up by specific scavenger receptors expressed on macrophages in the splenic marginal zone and that mice deficient in these receptors have a lower threshold for autoantibody responses. autoantibodies against sr are found before the onset of clinical symptoms in sle-prone mice, and they are also found in diagnosed sle patients. furthermore, injections of an antibody binding sr enhance the antibody production by b cells when co injected with either apoptotic cells or tnp-ficoll. conclusion: our findings describe a novel mechanism where autoantibodies toward scavenger receptors can alter the response to apoptotic cells, affect tolerance, and thus promote disease progression. because the autoantibodies can be detected before onset of disease in mice, they could have predictive value as early indicators of sle. e. glasmacher 1 , k.p. hoefig 1 , e. kremmer 1 , v. heissmeyer 1 stretches and helps in the selection of the correct splicing borders. a allele of (r61h) creates a strong binding site for a splicing enhancer protein srp40 according to bioinformatics. our findings indicate that, the putative branch point, r61h snp and the t stretch located downstream of exon two, plays a role in the alternative splicing of bank1. finally, we believe that bank1 delta 2 protein work as a dominant negative isoform in b cell activation and antobodies production, and may antagonize the effect of the full-length protein. these properties of the delta 2 protein may contribute to the observed reduction in sle susceptibility. s. beermann 1 , r. seifert 1 , d. neumann 1 1 hannover medical school, pharmacology, hannover, germany the biological function of histamine is mediated by four different receptors, namely histamine h 1 receptor (h 1 r), h 2 r, h 3 r, and h 4 r. during an immune reaction histamine acts as a local proinflammatory mediator and contributes to the polarisation of the adaptive immune reaction by modulating the activity of dendritic cells and t cells. in these cells, histamine may modulate the synthesis of characteristic t cell cytokines such as ifng, which plays a central role in a number of autoimmune diseases. the present study was initiated to analyze the involvement of histamine on the induced production of ifng by immune cells. mouse spleen cells were stimulated in vitro by either immobilized a-cd3 antibodies or cpg-oligonucleotides (cpg-odn) in the presence or absence of histamine or 4-methylhistamine, a h 4 r-selective agonist. ifng production was evaluated by analysis of cell culture supernatants by elisa. both, histamine and 4-methylhistamine concentration-dependently reduced ifng production in splenocytes obtained from control c57bl/6 mice induced by either a-cd3 antibodies or cpg-odn. this histamine effect was completely inhibited by the h 2 r-specific antagonist famotidine, while h 4 r-, h 1 r-, and h 3 /h 4 r-selective antagonists had no or only moderate effects. interestingly, the h 4 r-selective reagent jnj7777120, which serves as an antagonist on human cells, did not inhibit the histamine-mediated reduction of a-cd3 induced ifng synthesis, but in contrast it slightly enhanced the histamine effect. thus, at the murine h 4 r, jnj7777120 may be a partial agonist. we conclude that histamine modulates the induced production of ifng by t cells via mainly the h 2 r and, to a much lesser extend, the h 4 r. using this assay system, cells obtained from control c57bl/6 mice will be compared to those from sle-prone mrl lpr/lpr mice and the respective wild type strain mrl +/+ . i objectives: resolvins are products of omega-3 fatty acids and they exert potent anti-inflammatory properties. in this study we examined their effects on cytokine release in healthy subjects and autoimmune patients. to test the in vitro effects of 20 ng/ml resolvin e1(rve1) on the release of tgfb, il-6 and il-17 in the culture of peripheral mononuclear cells (5x10 6 /ml) stimulated by phorbol ester (pma) (1nm), and the combination of pma and ionomycine (3 mg/ml) for 72 hours. methods: mononuclear cells were prepared by ficoll-uromiro gradient centrifugation from 7 healthy subjects and from 10 patients each with sle and sjögren's syndrome (ss). level of cytokines was measured by elisa method. results: in the patients with sle (p = 0.010) and sjögren's (p=0.017) mononuclear cell stimulation by pma resulted in a reduced release of tgf b compared with controls. rve1 significantly reduced tgfb release from control mononuclear cells stimulated by either pma (p=0.041) or pma+ionomycin (p=0.021), however rve1 was ineffective at reducing tgfb release in the sle and ss patients. rve1 caused a non-significant decrease in il-6 release from control mononuclear cells, but was again ineffective in sle and ss patients. the production of il-17 was not significantly modified by rve1 in any of the groups tested. the release of tgfb by 20 ng/ml of rve1 can be significantly reduced in healthy control subjects but not in subjects with sle or ss. at the single dose of rve1 tested, il-6 and il-17 release were not significantly affected in healthy or autoimmune patients. omega-3 fatty acid derived rve1 may affect inflammation in healthy patients by reducing tgfb production but its effects on inflammation in sle and ss patients may be expected to be smaller or non-existent. in addition, the tgfb release in the pma activated mononuclear cells of sle and sjögren's patients is less than that of healthy subjects. g. e. fragoulis 1 , a.k. tsirogianni 1 , m. herrmann 2 , h. m. moutsopoulos 1 , m.n. manoussakis 1 1 university of athens, dpt pathophysiology, athens, greece, 2 university of erlangen-numberg, institute for clinical immunology, erlangen-numberg, germany objectives: altered phagocytic capacity has been shown to characterize systemic lupus erythematosus (sle) that is thought to lead to impaired clearance of apoptotic remnants. herein, we assessed comparatively the phagocytic capacity in the peripheral blood of ss and sle patients and investigated the phagocytosis of apoptotic/necrotic cells in the salivary glands of ss patients. methods: patients studied included 29 with primary ss (american-european criteria 2002) and 14 with sle (acr criteria 1997). age-and sex-matched healthy blood donors to the ss and sle groups (13 donors each) were also studied in all assays. the phagocytosis capacity (phagocytosis index) was assessed by flow cytometry, as previously (gaipl et al, j autoimmunity, 2007) using heparinized whole blood from individuals studied mixed with a commercially available preparation of fluorescent microbeads (mb-phagocytosis) or a preparation of propidium iodide-stained necrotic cell-derived material obtained from heat-treated normal pbmc (snec-phagocytosis). salivary gland biopsies of patients with ss with and without malt lymphoma (5 patients each) were also assessed by confocal microscopy for the presence of apoptotic/necrotic material (tunel assay) and the presence of macrophages (cd68-staining). results: in agreement to previous studies, mb-phagocytosis was found significantly decreased in granulocytes and monocytes of sle patients (both for p=0.0001). in ss patients, defective mb-phagocytosis involved only monocytes (p x 0.0001) and significantly correlated with the presence of extraglandular manifestations (p=0.02). compared to controls, snec-phagocytosis was significantly increased in the granulocytes of sle (p x 0.0001) and of ss (p=0.001). in the salivary gland biopsies of ss patients, the lymphoepithelial lesions and germinal center-like structures manifested significantly increased infiltrations by macrophages. these lesions were also characterized by notable accumulation of apoptotic/necrotic material that resided both inside and outside the phagocytes. these phenomena were significantly more intense in the salivary gland lesions that manifested malignant in-situ b-cell lymphoma. conclusion: in a manner similar to sle, ss patients appear to manifest altered phagocytic capacity. this may be associated with the observed accumulation of apoptotic/necrotic cells in the salivary glands that in turn, may participate in the chronic autoimmune reactions and/or the lymphoma-generating processes that characterize the disorder. the autoantibodies to various enzymes are often found out in sera of systemic lupus erythematosus (sle) patients, but clinical value of such antibodies often is not understood. the purpose of work was to study the of antibodies generation to the basic enzyme of purine metabolism -adenozine deaminase (ada) in sle and to reveal the relationship of studied antibodies with clinical and laboratory features of pathological process. methods: 30 healthy persons have been included in our study and 71 sle patients (66 women and 5 men) with various clinical signs (44 persons had 1 st degree of disease activity, 27 persons -2 nd degree of pathological process activity). 18 women had habitual noncarrying of pregnancy (hnp) in anamnesis. antibodies of igg class to ada (anti-ada) determined by technique of indirect elisa developed by us with the use of immobilized form of ada as an antigenic matrix. b 2glicoprotein-i-dependent antiphospholipids (aphl) of igg classes were determined using commercial "anti-phospholipid screen igg/igm" test set (orgentec diagnostica). results: at admission an anti-ada was revealed in 36,6 %, aphl of igg class -in 45,1 % sle patients. it has been noted that igg-aphl were found out in anti-adapositive patients more often and in higher antibody titer, than in anti-ada-negative sle patients (x 2 =6,4; p x 0,02). development of cytopenic syndrome was noted reliable more often in sle patients with associated presence of igg-aphl and an anti-ada in comparison with patients who has not the combinations of these antibodies in blood (x 2 =3,9; p x 0,05). the increased levels of anti-ada were revealed in 11 of 18 women with hnp, and the combination of anti-ada and aphl (9/18) was found out more often than isolated anti-ada (2/18, x 2 =6,5; p x 0,02) or isolated aphl (3/18, x 2 =4,5; p x 0,05). conclusion: taking into account the imbalance of immunoregulatory functions in sle, the further studying of autoantibodies to ada generation seems to be very promising. presence of hnp in anamnesis is the evidence of necessity of careful biochemical monitoring of aphl and anti-ada in women for the prevention of abortus fetus and administration of adequate therapy. objectives: sjögren's syndrome (ss) is a chronic inflammatory and lymphoproliferative autoimmune disease, characterized by dryness of the mouth (xerostomia) and the eyes (keratoconjunctivitis sicca). dendritic cells (dc) are the most potent antigen-presenting cells that play a crucial role in initiating and maintaining primary immune responses. two main subsets of dc have so far been identified in human peripheral blood: myeloid dc (mdc), which can be further divided into mdc1 and mdc2, and plasmacytoid dc (pdc), also known as ifn-a/b producing cells. the pivotal role of dc in inducing and maintaining tolerance could be critical in ss as alterations among dc populations might contribute to autoimmunity. purpose of this study was to quantify mdc1, mdc2 and pdc in peripheral blood from primary ss patients by flow cytometry and compare the results with gender-and age-matched healthy controls. methods: blood samples from 31 pss patients fulfilling the american european consensus group criteria (aecc) and 28 gender-and age-matched healthy controls were collected in heparin tubes. dc populations were stained with the blood dc enumeration kit, miltenyi, according to the manufacturer's manual. cells were analyzed on a facs canto ii, bd, and data analysis was performed with flowjo software, tree star. for the statistical analysis, a two-tailed mann-whitney u test was performed using prism, graphpad. results: pss patients have significantly reduced amounts of pdc (p=0,0002) and mdc2 (p x 0,0001) in peripheral blood. conclusion: alterations in dc populations have been considered to play a role in autoimmune diseases such as systemic lupus erythematosus (sle) or diabetes. in ss patients, up-regulation of interferon-regulated genes has been shown previously. therefore, decreased pdc numbers in peripheral blood from pss patients might explain the fact that an increased ifn signature is found in salivary glands of pss patients, but no elevated levels of ifn-a are measured in serum. recently we reported that malignant cd5+ b cells from patients with b chronic lymphocytic leukemia (b-cll) produce granzyme b (grb) and are rapidly undergoing apoptosis in a granzyme b-dependent manner following interleukin 21 (il-21) stimulation. several autoimmune diseases have been linked to both elevated frequencies of cd5+ b cells and increased il-21 levels. we therefore hypothesized that il-21 may have similar biological effects on cd5+ b cells in autoimmune diseases. here we demonstrate that the amount of il-21 in the serum of systemic lupus erythematosus (sle) patients but not of healthy subjects highly correlated with serum levels of grb. in contrast to b cells from healthy individuals, where no baseline grb expression was found, we demonstrate that up to 14 % of cd5+ b cells in sle individuals expressed grb. in-vitro experiments revealed that il-21 was able to induce expression of grb in b cells from individuals with sle and other autoimmune diseases including psoriasis and rheumatoid arthritis. this effect was direct and was strongly enhanced by engagement of the b cell receptor or toll-like receptor 9. importantly, il-21 significantly decreased the cd5+/cd5-b cell ratio in both sle peripheral blood and healthy cord blood samples, suggesting a preferential induction of cd5+ b cell death. these results suggest that il-21-induced grb may play a regulatory role for cd5+ b cells similar to what we described earlier in b-cll cells. this is the first report uncovering an interrelation between il-21 and grb levels in sle and showing that il-21 reduces the cd5+/ cd5-b cell ratio in b cells from sle peripheral blood and healthy cord blood. endogenous il-21 may therefore play a disease-modifying role and may explain elevated grb serum levels in autoimmune diseases. further studies should evaluate the therapeutic potential of il-21 in sle and other autoimmune diseases. r. de palma 1 , e. d'aiuto 1 , s. vettori 1 , g. abbate 1 , g. valentini 1 1 second university of naples, clinical & experimental medicine, napoli, italy ssc is considered an autoimmune puzzling disease whose pathogenesis is unknown. in the last years, there have been increasing evidences that an interplay between activated t cells and fibroblasts could play a pivotal role in promoting matrix accumulation in systemic sclerosis (ssc). we have previously shown that peripheral t cells from ssc patients with early diffuse disease co-cultured with autologous fibroblasts expand the same t cell clonotypes found in the affected skin. here, using the same experimental approach, we found that the t cell clonotypes expanded in co-cultures are ab positive, hla-dr positive, and promote apoptosis of autologous ssc fibroblasts. we also found that, in these co-cultures, ssc fibroblasts up-regulated fas and underwent apoptosis that paired with the expression of fas ligand (fasl) on cd4+ t cells. finally, when we added a blocking anti-fas antibody to the co-cultures, we observed a marked reduction of fibroblast apoptosis, suggesting that engagement of fas/fasl had a critical role in mediating apoptosis in co-cultured fibroblasts. it has to be reminded that the absence of fasmediated apoptosis in vivo could be due to several reasons, as the increase of soluble fas in sera of patients affected by ssc. moreover, in the co-culture supernatants we found tgf-beta, il-1beta, il-6 and il-8, cytokines known to have a role in promoting fibrosis in systemic sclerosis. taken together, these data suggest that t cell response in ssc may represent an attempt of the immune system to kill fibroblasts, cells that are likely to be altered and expressing (auto)antigens. indeed, fibroblasts of ssc patients have been shown to display a persistently activated phenotype characterized by excessive production of collagen and other extracellular matrix proteins. however, the overall outcome of the t cell response triggered by fibroblasts in ssc, while unable to control the activity and the growth of fibroblasts, contribute to sustain inflammatory loops leading to fibrosis. these findings may lead to change our view about the pathogenesis of this disease and other autoimmune diseases. systemic lupus erythematosus (sle) is a chronic inflammatory autoimmune disease that is associated with a major breakdown in b cell self-tolerance as reflected by elevated serum igg levels of predominantly antinuclear antibodies (anas). serum antibody titers are maintained by antibody-secreting plasmablasts and longlived plasma cells, which reside in survival niches of the bone marrow. however, the antibody repertoire of bone marrow plasma cells, which may include cells expressing autoreactive and potentially pathogenic antibodies, has not been characterized in sle. to determine the frequency, specificity and immunoglobulin gene characteristics of autoantibodies in the long-lived plasma cell compartment in sle, we cloned and expressed 169 igg antibodies from single facs purified cd19+cd27+cd38+cd138+ bone marrow plasma cells of 3 patients with sle and tested the recombinant monoclonal antibodies for self-reactivity. our preliminary data on the ig gene repertoire and reactivity profile of human igg+ sle bone marrow plasma cells in comparison to healthy controls will be discussed. z. amirghofran 1 , e. moazemi godarzi 1 , e. kamali sarvestani 1 , e. aflaki 1 1 shiraz university of medical sciences, shiraz, iran, islamic republic of interleukin 6 (il-6) has been shown to be related to the pathogenesis of systemic lupus erythematosus (sle). two polymorphisms in the promoter region of il-6 gene at positions -572 g/c and -174 g/c have been described that are key regulators of il-6 gene. in the present study the relationship between these two polymorphic sites and disease susceptibility in a group of iranian patients with sle was investigated using polymerase chain reaction-restriction fragment length polymorphism method. the genotype distribution and allele frequencies of il-6 gene polymorphism at -174 position showed no significant difference between sle patients and controls. at this position the frequency of gg genotype as well as g allele was higher than c allele in both patients and control groups. in contrary, both allelic and genotypic frequencies at the -572 position significantly differed in sle patients and controls. at this position gg genotype was observed in 77.9 % of patients compared to 68.9 % in the control group (p x 0.014). the frequency of -572 g allele in patients (87.3 %) was also higher than in controls (83.2 %) (p=0.034). the haplotype study showed no significant difference between patients and healthy subjects. the relationship between these polymorphisms and clinical manifestations and laboratory parameters were investigated. -174 polymorphism was associated with the presence of antinuclear antibodies in all patients and rash and hematuria in male patients (p x 0.04). at -572 polymorphism, a significant difference with regard to photosensitivity in male patients (p=0.04) was found. in conclusion, results of this study showed that -572 polymorphism plays an important role in susceptibility to sle and that -174 polymorphism could influence the presence of antinuclear antibodies in the patients. the eukaryotic constitutive proteasome is the main protease expressed in most tissues. recently we have elucidated a functional importance of the second proteasome form, inducible immunoproteasomes, in regulating nf-kb activity during the intestinal inflammation. in comparison with healthy controls and patients with ulcerative colitis (uc), there was increased expression of immunoproteasomes in the inflamed mucosa of patients with crohn's disease (cd) at both mrna and protein levels. in our very recent work we have shown that the proteasome subunit pattern might be suitable for diagnostic differentiation between cd and uc patients. since ifn-g has been shown to be the main inducer of immunoproteasomes in various murine and human cell lines and the ifn-g levels are highly elevated in inflamed intestine of cd patients, induction of immunoproteasomes in cd might be mediated by this cytokine. our data with human leukemic t cell lines and primary macrophages show a significant increase in the nf-kb controlled production of proinflammatory cytokines after the ifn-g-mediated induction of immunoproteasomes in these cells. in the dss-induced colitis model we have observed a diminished colonic inflammation in the absence of the proteasomal immunosubunits. therefore we here suppose that immunoprteasome are involved in the complex inflammatory response during the chronic intestinal inflammation by increasing nf-kb activity in the epithelial and immune cells. however, it remains to be determined whether these results have an important implication for the treatment of chronic gut inflamation in humans. objectives: inflammatory bowel diseases (ibd) including crohn's disease (cd) and ulcerative colitis (uc) are characterized by unknown etiology and chronic intestinal inflammation. noninvasive serological tests to differentiate cd from uc have been searched for a long time. testing for panca together with ascas has good predictive values to identify patients with ibd.the aim of this study was to find evidences for diversity of ascas and anti-mycobacterium paratuberculosis antibodies (anti-mpt) by elisa method. in addition, to examine whether combination of these elisas is useful for distinguishing cd from uc. methods: the study population contained 161 patients with ibd (89 with cd, 41 with uc, 31 with gluten sensitive enteropathy, gse) and 33 healthy control subjects. serum asca igg, asca iga and anti-mpt antibody levels were measured by solid phase enzyme immunoassay. adsorption of 7 asca positive sera was performed by baker's yeast suspension. results: elevated level of asca igg, iga and anti-mpt was shown in cd and gse but not in uc compared to healthy controls. serum levels of asca igg, iga showed a significant positive correlation with anti-mpt antibody levels in cd. repeated adsorptions with yeast removed asca igg and iga from sera of patients, but did not change levels of anti-mpt. these results indicate the diversity of asca igg, iga and anti-mpt (accordingly their antigens) and suggest that combination of these elisa can have a role in the differential diagnostics of ibd. it is now well recognised that the majority of lymphocytes may be located within tissues, not in blood, and yet these tissue-resident lymphocytes are relatively understudied, especially in humans. we have extracted cells from human gut biopsies (both normal and inflamed gut) in order to characterise the immune cell populations that exist therein and which molecules may be of paramount importance to their function. we show that distinct populations of t cells exist within the gut and that the ratio of these populations changes down the length of the gut, with the so-called 'unconventional' double negative t cell population (ie tcrab+ve, cd4-ve cd8b-ve) predominating in the healthy colon whereas these cells are overwhelmed by infiltrating cd4(+) cells in inflammatory bowel disease (ibd). having previously shown in mice that gut-resident t cells express high levels of the regulator of g protein signalling-1 (rgs-1) protein, we have now found substantial over-expression (10-100 fold) of rgs-1 in human gut-derived t cells, particularly in this unconventional t cell population. furthermore, levels appear even higher in t cells derived from inflamed gut. transfection of rgs-1 decreases primary t cell responses to cxcl12 and ccl19, strongly implying that it may regulate t cell localisation. thus, rgs-1 may be a novel target for modulating t cells in ibd, consistent with which snps in rgs-1 have been associated with both coeliac disease and type 1 diabetes. mechanisms involved in the induction of oral tolerance (ot) or systemic immunization through the oral rout are still poorly understood. in our previous studies we have shown that when normal mice eat peanuts they become tolerant, with no gut alterations. conversely, if they are immunized with peanut proteins prior to a challenge diet (cd) containing peanuts they develop chronic inflammation of the gut. our aim is to evaluate the consequences of the introduction of a novel protein in the diet of animals presenting antigen specific gut inflammation. adult, female c57bl/6j mice were divided in control (c) and experimental (e) groups. c1-c3 received peanut protein immunization, animals of the control groups c4 were sham immunized, and control group c5 received ovalbumin (ova) immunization. the experimental group was immunized with peanut protein extract. before initial exposure to a 30 day peanut containing cd, the experimental group was divided into 5 groups (e1-e5). ova feeding began 7 days prior cd (e1) on day 0 (e2), 7 (e3), 14 (e4) and 21 (e5) during cd. our results show that oral exposure to a novel protein (ova) in the absence of gut inflammation (e1) leads to low levels of systemic antibody titers, comparable to tolerant animals. conversely, as off initial induction of inflammation, groups submitted to ova (ot) protocol develop increasingly higher systemic antibody (ab) titers similar to animals of the immune control group. in conclusion our protocol indicates that timing is more important than the antigenicity when a novel protein is offered, in the diet. nanoparticles of various types are increasingly used as constituents of food supplements and so called nanofood. since nanoparticles induce inflammatory reactions in the lung, there is an urgent need to also study the toxicological potential of nanoparticles in the intestine. therefore, we assessed the effect of particles on dendritic cells (dc) as key players in the manifestation of intestinal immunity.in in vitro studies we could show that ultrafine tio 2 as well as ultrafine silica led to a mature phenotype of the cells when particles were added to cultures of immature bone-marrow-derived dc. this effect appears to result from enhanced cell death in immature dc but also from direct stimulation of the cells.to analyse the mechanisms underlying this effect we looked for apoptosis as well as for induction of the inflammasome since it has been shown that crystalline silica leads to activation of caspase 1 and secretion of bioactive il1-b.in our hands certain nanoparticles induced apoptosis of immature dc, as well as enhanced secretion of active il1-b. we therefore hypothesize that particles can induce the inflammasome which leads to the activation of dc.to study the impact of nanoparticles on intestinal inflammatory processes in vivo, we induced colitis by applying 5 % destrane sulphate sodium (dss) in the drinking water for 8 days to wildtype mice. when ultrafine nanoparticles were administered on day 6 and 7 by gavage feeding, we observed an amelioration of disease symptoms when scoring the degree of epithelial disruption and inflammation. in future experiments we will also analyse the effect of different particles in the il10 -/model of colitis to assess the contribution of particles to the induction and pathogenesis of disease. m. schmohl 1 , n. schneiderhan-marra 1 , m. blum 2 , g. stein 2 , m. schmolz 2 , t. joos 1 1 nmi-natural and medical sciences institute at the university of tübingen, biochemistry, reutlingen, germany, 2 edi gmbh, reutlingen, germany the human immune system represents a highly complex system that protects the organism against diseases. there is an impressive network of immunoregulatory signals within the immune system as well as between the different healthy and diseased organs. epithelial layers function as a barrier against pathogens. as the gastrointestinal tract, which is occupied with a large variety of microorganisms, represents the outside of the body, the immune system has to establish and maintain a strong presence at the mucosal boundary. the ability to discriminate between pathogens while remaining relatively unresponsive to food antigens and the commensal microflora is achieved by a plethora of largely unknown regulatory mechanisms. this ability appears to be breaking down with chronic inflammatory bowel diseases (ibd) like crohn's disease and ulcerative colitis [1] . to date treatment options are restricted to controlling symptoms, putting and keeping the dis-eases in remission and preventing relapse. therefore, there is an urgent need for a more detailed understanding of the inflammatory events taking place during the disease. for this purpose a human organo-typic (hot) co-culture model is used, which allows analyzing the collaborative regulation between the immune system and the gut epithelial cells. the human caco-2 cell line, as a model for the gut-epithelium cells, are cultivated on the top side of special culture vessels, fitting as inserts into carrier wells of 24-well culture plates, containing whole-blood. this co-culture set up mimics the physiological barrier to perorally applied biologicals/drugs and allows measuring their effect on the immune system. as a read out miniaturized and parallelized sandwich immunoassays will be used to detect alterations in the intracellular mapk and rtk-signalling of the epithelial cells as well as in the extracellular communication via cytokines and chemokines at the interface of the two organs. this approach will provide new insight into the inter-and intracellular signalling of gut epithelium and the immune system, which will finally result in a better understanding of the etiology of inflammatory bowel diseases. inflammatory bowel disease (ibd), including crohn's disease (cd) and ulcerative colitis (uc), is characterized by an upregulation of pro-inflammatory cytokines that play an important role in pathogenesis. osteopontin (opn) is a cytokine implicated in several immunological diseases and, although expressed constitutively in normal intestine, is upregulated in intestinal mucosa and in the plasma of ibd patients. opn has been shown to be either pro-inflammatory or anti-inflammatory for experimental uc, indicating a controversy in this field, while its role in experimental cd remains unknown. in our study we investigated the role of opn in experimental colitis using two mouse models: trinitrobenzene sulphonic acid (tnbs) colitis, a t h 1-associated model that resembles cd, and dextran sulphate sodium (dss) colitis, a t h 2-like-associated model for uc. deficiency of opn (either by antibody-mediated neutralization or use of opn -/mice) resulted in suppression of disease phenotype in both colitis models, revealing that opn, and especially, the secreted isoform of opn (opn-s) is important for the initiation of acute intestinal inflammation. importantly, we discovered that opn drives il-17 production and t h 17 polarization and decreases recruitment of cd4 + cd25 + foxp3 + t regulatory (treg) cells in mesenteric lymph nodes (mlns) of mice with colitis. also, there was an effect of opn on recruitment of cd11c + dcs, which were significantly elevated in mlns of opn -/or anti-opn-treated, as compared to opn +/+ or ig-treated control mice. this finding implies that opn deficiency results in enhanced recruitment of regulatory cd11c + dcs which may mediate treg induction and protect from colitis. overall, our findings indicate that opn is proinflammatory in both types of colitis, by promoting pathogenic t h 17 and attenuating treg cell recruitment, implying also common mechanisms in the pathogenesis of cd and uc. c. shen 1,2 , g. van assche 3 , p. rutgeerts 3 , a. liston 1 , j. l. ceuppens 2 1 k.u. leuven, autoimmune & genetics lab, vib, leuven, belgium, 2 k. u. leuven, experimental immunology lab, leuven, belgium, 3 k. u. leuven, department of pathophysiology, gastroenterology section, leuven, belgium background: haptoglobin (hp) is one of the acute phase proteins synthesized during inflammation. hp-1 allele is associated with the disease behavior in crohn's disease but not in ulcerative colitis. however its role in inflammatory bowel disease has not been defined. aim: to determine whether hp modulates the immune responses in experimental colitis. methods: we induced 3 types of colitis dss (th1/th17), tnbs (th1) and oxazolone (th2) in hp ko mice. neutralizing anti-il-17 mab was injected into dss and tnbs hp ko mice. severity of colitis was evaluated by body weight, colon length and histology. th17/th1 cells were analyzed by flow cytometry. cytokines were measured by elisa or rt-pcr. 1) compared to the wt mice, hp ko mice developed much severer dss and oxa induced colitis. dss induced lethal colitis in hp ko but not in wt mice; 2) in dss but not in oxa colitis mice, il-17, ifn-g, tgf-b and il-6 were significantly increased (p x 0.01, dss vs control) in lamina propria and mesenteric lymph nodes (mln), and this is much evident in hp ko mice compared to those in the wt (p x 0.05, ko vs wt). in tnbs colitis, we found elevated il-12 and ifn-g (p x 0.01, tnbs vs control). although not significant, il-17 was also somewhat upregulated; 3) in dss colitis we observed that il-23 enhanced differentiated th17 cells in vitro, this effect could be abrogated by coculture with serum from wt but not hpko mice. furthermore, in vitro in the presence of tgf-b, il-6 and il-21, more mln-t cells from hpko mice differentiated into th17 cells; 4) anti-il-17 mab improved dss and tnbs colitis, and partially rescued hp ko mice from lethal dss colitis. in line with this, mice treated with anti-il-17 showed reduced il-6, il-17 and ifn-g in both mln and lp (p x 0.05, anti-il-17 vs control). our results reveal that hp has a protective role in the development of mucosal inflammation. in dss and tnbs colitis hp may exert its beneficial effect partially through inhibiting production of il-17, supporting further pre-clinical and clinical application of hp for treatment of crohn's disease. p. engelmann 1 , g. talabér 1 , g. süt" o 2 , p. németh 1 , t. berki 1 1 university of pécs, clinical center, department of immunology and biotechnology, pécs, hungary, 2 university of pécs, clinical center, department of immunology and rheumatology, pécs, hungary objectives: inflammatory bowel disease (ibd) resembles as an autoimmune-like disease. ibd is most common in developed countries: it is calculated that 2.2 million people in europe suffer from ibd. several hypotheses are raised in the pathogenesis of inflammatory bowel disease. one of the most favored is the dysregulation of the immune response due to failure of regulatory t cells. the most well known regulatory t cells are the cd4+cd25hi+ t (treg) cells. furthermore, other immune-regulatory cells are known such as invariant natural killer t (inkt) cells producing both th1 and th2 cytokines rapidly upon antigen (lipid) stimulation. methods: based on this hypothesis we aimed to investigate the role of various immune-regulatory t cells in human ibd. we attempt to measure the proportions of inkt cells, treg cells in peripheral blood of patients with crohn's disease (cd) and ulcerative colitis (uc) compared to normal controls. blood samples were collected from normal controls and ibd patients; then lymphocytes were labeled for inkt and treg markers with specific monoclonal antibodies and measured with flow cytometry. results: according to our results a decline in the total inkt cells of ibd patients was observed, interestingly the proportions of cd4+ and double negative (dn) inkt subgroups showed a characteristic shift among the study groups. percentages of dn and cd4+ inkt subpopulations were assessed after gating of total inkt populations. in controls we observed high percentage of dn inkt cells (74.5 ± 3.5 %, mean ± sem), while cd4+ inkt cells ratio was moderate (25.4 ± 3.5 %). in uc and cd patients we found a reduced proportion of dn inkt cells (uc: 38.0 ± 7.1 %; cd: 34.0 ± 5.2 %, mean ± sem), while the percentage of cd4+ inkt cells was elevated (uc: 62.0 ± 7.1 %; cd: 65.9 ± 5.2 %, mean ± sem) in both disease groups. proportions of foxp3+ treg cells also showed a decline in ibd patients comparing to normal controls. conclusion: this study can provide useful data about the pathogenesis of ibd and can lead to identify and characterize new cellular and molecular targets with possible therapeutic use in human autoimmune disorders. objectives: the aim of this project is to explore whether exosomes from tgf-b1 gene modified bone marrow-derived immature dendritic cells (md-imdc) have the function of systemic immune inhibition and protective effect on the development of inflammatory bowel disease (ibd) in mice, the underlying mechanism was also investigated. methods: exosomes were isolated from supernatant of md-imdc transfected with tgf-b1 adenovirus (tgf-b1-exo). the t cell inhibitory function of tgf-b1-exo was determined by mixed lymphocyte reaction (mlr) in vitro. to evaluate the protective effect of tgf-b1-exo in the development of ibd, dextran sulfate sodium(dss) induced murine ibd was established and mice were treated with tgf-b1-exo. the main symptoms of ibd were observed. the inflammatory degree of colon was also evaluated by histological examination. the relative cd4 + foxp3 + treg cell numbers from spleens and mesentery lymph nodes (mlns) were analyzed by facs. results: it was demonstrated that tgf-b1-exo could inhibit the proliferation of t cells in mlr in vitro. in murine ibd model, after treated with tgf-b1-exo, the main symptoms of ibd such as weight loss, diarrhea and grume sanguinopurulent stool were all alleviated and the inflammatory degree of colon was also reduced. analysis of cd4 + foxp3 + regulatory t cells (treg) revealed that the relative numbers of cd4 + foxp3 + treg increased in lymphocytes from mesentery lymph nodes (mlns) of inflammatory site but not from spleens. conclusions: these results demonstrate that immunosuppressive exosomes obtained from tgf-b1 gene modified md-imdc can delay the development of ibd. this protective effect is mediated by the induction of cd4 + foxp3 + treg. tgf-b1-exo might provide a novel strategy for the therapy of ibd. results: hcv-specific cytokine expression by cd8+ t-cells was similar in the four vaccinees as observed by ifng, il-2 production-profiles. however, the killing capacity of expanded cd8+ t-cells was distinct as observed by the competence to kill ns3-peptide presenting transfectants in vitro. as depicted in figure 1 , cd8+ t-cells cells from both vac1 (cleared ) and vac2 (chronic) produced il-2 and ifng after stimulation with ns3-peptide59. however, specific killing of the peptide loaded transfectants was only observed in vac 1, who was able to clear its hcv infection, and this was not observed not in any of the other chimpanzees, who became chronic carriers. [ figure 1 ] killing of ns3 peptide presenting cells was restricted to the vaccinee that was able to clear hcv infection. these results suggest that controlling hcv replication as initiated by this dna-prime mva-boost vaccine-protocol was partly mediated by antigen specific cd8+ t-cells. hence, the effector mechanisms induced were distinct between the animals and clearance of the infection was correlated with induction of killing competent cd8 t-cells. objectives: infection by hepatitis c virus (hcv) is characterized by its high tendency to chronicity, which is usually associated with a low or absent t-cell response against viral antigens. immune response specific for non-structural protein ns3 from hcv was associated with viral clearance. we have demonstrated that fusion of an antigen to the extra domain a from fibronectin (eda) targets the antigen to tlr4-expressing dendritic cells and improves its immunogenicity. thus, we tested if covalent linkage between eda and ns3 might constitute an alternative for vaccination against hcv infection. methods: recombinant plasmids expressing a secretable version of ns3 or eda-ns3 under the control of cmv promoter were prepared. recombinant ns3 and the fusion protein eda-ns3 were produced in e. coli. the recombinant proteins were tested in vitro on their capacity to activate maturation of bone marrow derived dendritic cells and to favour antigen presentation. hhd transgenic mice (expressing the human hla-a2 molecule) were immunized with the recombinant plasmids or with the recombinant proteins, in the absence or presence of poly(i:c) and anti-cd40 agonistic antibodies. elispot and chromium release assays were carried out to measure the immunogenicity of the different vaccination strategies. intrahepatic expression of hcv-ns3 rna was measured after a hydrodynamic injection with a plasmid encoding hcv ns3. results: immunization of mice with the plasmids expressing eda-ns3, but not ns3 alone, induced strong t cell responses against the main hla-a2 restricted cytotoxic t cell determinants from ns3. the recombinant eda-ns3 fusion protein, but not ns3, was able to activate in vitro maturation of bone marrow derived dendritic cells as well as the production of tnf-a by the thp-1 monocyte cell line. immunization of hhd mice with eda-ns3 fusion protein induced both cd4+ and cd8+ t cell responses against ns3 and, when immunized with poly(i:c) and anti-cd40 antibodies, was able to down-regulate the intrahepatic expression of hcv-ns3 rna. the recombinant eda-ns3 fusion protein may be considered for the development of prophylactic or therapeutic vaccines against hcv infection. vaccination is the most efficient strategy to prevent from microbial infections and to control epidemics but are still not available in the case of hiv infection even 25 years after virus detection. therein we propose the intra-dermal inoculation of dna vaccine that present a plasmid vector exploiting the binding capacity of the bovine papillomavirus e2 protein encoding an artificial multi-component hiv antigen. this inoculation is followed by electroporation in order to increase dna uptake. we used skin as site for vaccination because, being the first line in host defence, it is populated with various cells of immune system. among them, langerhans cells (cd207+cd1a+), located in the epidermis, are dendritic cell subset capable to elucidate specific cd8+ responses. the present work emphasizes molecular and cellular biodistribution of the dna vaccine in the skin after intra-dermal vaccination in macaques, as one of the most relevant animal models in hiv studies. technical approach considers an intra-dermal injection of dna followed by topical electroporation of the injection sites. skin and draining ln biopsies were collected at different time points. these biopsies were used for ihc fluorescent staining in order to establish biodistribution dna-encoded antigens and co-localisation with different cell types. kinetic of antigen expression was studied by bioluminescence in vivo imaging. t cell responses were measured by ifn-g elispot assays up to 3 years after dna vaccination. we show that a dna vaccine delivery method combining intra-dermal injection and electroporation dramatically increased the expression of the vaccine antigen selectively in the epidermis, increased the frequency of cd1a+ cells in the draining ln in association with the antigen expression, and increased the cellular response persistence, at high levels, for more than two years after the last vaccine boost. our data suggest that electroporation after intradermal injection of dna vaccine involves langerhans cells from the epidermis that elucidate qualitative anti-hiv immune responses. this new approach that comprise new dna vaccine followed by non-invasive electroporation, induce long-lasting cellular response that could be crucial in prophylactic / therapeutic vaccine design. presenting cells was developed. murine coronavirus-based virus-like particles encoding epitopes from the lymphocytic choriomeningitis virus glycoprotein or human melan-a, in combination with the immunostimulatory cytokine gm-csf, selective targeted dcs in vitro and in vivo resulting in vector-mediated antigen expression, and efficient maturation of dcs. in mice, a single application of only low doses elicited strong and long-lasting cytotoxic t-cell responses which provided protective antiviral and antitumor immunity. furthermore, the efficient activation of human tumor-specific cd8+ t cells by mature dcs transduced with melan-a-recombinant human coronavirus 229e indicates that this novel vaccine platform mediates the delivery of antigens and immunostimulatory cytokines to those cellular components of the immune system that initiate and maintain protective immunity. as the application of gm-csf already enhanced immunogenicity, we are now trying to further modulate the coronavirus vector-induced immune response with the reverse genetic setup of recombinant coronavirus-based vectors expressing different immunostimulatory cytokines. thereby cytokines will be acting on t cell and dc level. to enhance t cell response interleukin 2 (il2) and interleukin 15 (il15) will be involved, and fms-like tyrosin kinase 3 ligand (flt3l) will be expressed to modulate dendritic cells. il2 is known to enhance early t cell expansion and limits t cell overshoot, whereaes il15 guarantees survival of high affinity t cells during memory phase. on the other hand flt3l enhances dc proliferation and accumulation. with these approaches modulation of the immune response generated by this novel vaccine platform will be examined in viral and tumour models to get insight on the antigen specific ctl response, synergistic effects of the cytokines and protective as well as prophylactic vaccination approaches. f. demircik 1 , ag waisman 1 uniklinik mainz, 1. med, mainz, germany in murine cytomegalovirus (mcmv) infection, cytotoxic cd8 t cells and nk cells play a critical role. previously it was shown that mice deficient for b cells are more susceptible to mcmv-related disease, caused by virus reactivation. to better understand the role of b cells and antibodies in the response to mcmv, we made use of different mouse strains that lack b cells, secreted antibodies or il-10 production by the b cells. we found that for the initial t cell response to the virus b cells are important, but antibodies do not play an important role. this implicates b cells as potential important antigen presenting cells (apcs) in the activation of the virus-specific t cells. the reduced t cell response to the virus was observed whether the mice were b cell deficient from birth or if they were depleted later in life. six month after infection mice were tested for the memory cd8 t cell response. interestingly, we found that in mice that lack antibodies (mice that lack b cells all together and mice that have b cells but no secreted antibodies) maintain a rather high t cell response to viral peptides, in a level similar to the acute response 7 days after infection. we conclude that antibodies probably remove residual viruses from the body and therefore prevent the continuous activation of t cells. finally, we tested the role of il-10 produced by b cells by conditional deletion of the il-10 gene in these cells. we found that b cell secreted il-10 has a suppressive effect on the t cell response to mcmv, as this response is elevated in these mice. we conclude that b cells are important for an efficient acute response to mcmv and that antibodies play a role in eliminating residual viral particles, thus implicating a dual role for b cells in the efficient acute and memory response to mcmv. this work is supported by the deutsche forschungsgemeinschaft grant sfb490 to aw. objectives: ebv infection leads to life-long viral persistence. although ebv infection can result in chronic disease and malignant transformation most carriers remain disease-free due to an effective control of the virus by t cells. ebv-specific ifng-producing t cells could be demonstrated in acute and chronic infection by many researchers. recent studies in hiv and leishmania provide, however, evidence that assessing ifng alone is insufficient to assess the quantity and quality of a memory t cell response and support the crucial role of multifunctional t cells in disease control. in this study we therefore analyzed ebv-specific t cell responses in peripheral blood (pb) and bone marrow (bm). methods: paired pb and bm samples were obtained from 8 healthy virus carriers who underwent total hip arthroplasty. t cells were expanded for 10 days in the presence of il-2 and il-7 with exposure to overlapping peptide pools of latent ebna-1 and lytic bzlf-1 antigens. ebv-specific immune responses were assessed exvivo and after expansion by multiparameter flow cytometry staining for live/dead discrimination marker, cd3, cd4, cd8, ccr7, cd45ra, il-2, tnfa, ifng and cd107a. the majority of ex vivo ebv-reactive cd4+ t cells as well as ebna-1-reactive cd8+ t cells were il-2 and tnfa-producing memory cells, the later being more frequent in bone marrow (cd4+, median, ebna-1: bm 0.69 %;pb 0.12 %; bzlf-1: bm 0.37 %;pb 0.01 %, p=0.039). after in vitro expansion a major subset of ebv-specific cd4+ and cd8+t cells displayed a differentiated effector ifng/tnfa phenotype. a comparable number of ebv-specific cd4+ and cd8+ t cells retained, however, a tnfa single, tnfa/il-2 or triple producer phenotype resembling early differentiated or multifunctional memory t cells, respectively. interestingly, both cd4+ and cd8+ t cells generated from bm revealed significantly higher cytotoxic potential. sorting of ccr7/cd45ra differentiation subsets, revealed that ebv-specific t cells were predominantly expandable from the central memory compartment. conclusion: our data shows that multicolor assessment of ifng, tnfa and il-2 delineates various subsets of ebv-specific memory t cells, which reflect the profile of a protective immune response. human adenovirus (hadv) can cause serious morbidity and mortality in immunocompromised patients after allogeneic stem cell transplantation (allosct). reconstitution of hadv-specific t cells has been reported to be associated with sustained protection from hadv disease, but epitope specificity of these responses has not been further characterized. furthermore, the relative contribution of hadv-specific cd4 + and cd8 + t cells in the protection from hadv disease after allosct remains to be elucidated. in this study, we demonstrate, by sensitive measurement using intracellular cytokine staining combined with cd154 or peptide-mhc tetramer staining, that clearance of hadv was associated with a combined hadv hexon specific cd4 + and cd8 + t cell response in both pediatric and adult allosct recipients. based on this observation, we developed a clinical grade method for the rapid generation of t cell lines with high and defined specificity for hadv hexon epitopes for adoptive immunotherapy. activation of hadv hexon-specific cd8 + and cd4 + t cells in peripheral blood with a hexon protein-spanning pool of synthetic 15-mer peptides followed by ifng-based isolation allowed rapid expansion of highly specific t cell lines from healthy adults, including donors without detectable frequencies of hadv hexon-specific t cells. the frequency of hadv-specific t cells was increased to 29-90 % in the t cell lines and the absolute numbers of both hexon-specific cd4+ and cd8+ t cells were 2 to 3 log increased compared to the starting material. detailed analysis showed that hadv-specific t cell lines recognized multiple mhc class i and ii restricted epitopes, including known and novel epitopes, and showed specific and efficient lysis of hadv infected target cells. this strategy may be used for adoptive transfer of donor-derived hadv hexon-specific cd8 + and cd4 + t cells for treatment of disseminated hadv infection after allosct. several studies showed that hbv persistance correlates with a failure of an efficient virus-specific t-cell response. induction of hbv-specific t cells by vaccination may be an innovative approach to overcome virus persistance. dna prime-recombinant adenovirus serotype 5 (ad5) boost strategy proved to be effective in stimulating t cell responses and control of viral infections. woodchuck hepatitis virus (whv) and its host the woodchuck are a useful peclinical model for investigating the new therapeutic approaches. the efficacy of plasmid dna and ad5 vaccine vectors expressing whv core protein was first examinated in c57bl6 mice. groups of mice were immunized with a dna prime-ad5 boost regimen or with dna and ad5 alone. ad5 was injected i. m. or s. c. t cell response was evaluated by intracellular ifng staining of splenocytes stimulated in vitro with whc-derived peptide pools. anti-whc antibodies were detected by elisa. we detected cd8+ t cell responses against peptide pools 1 and 3 in spleens of dna and dna-ad5 immunized mice. however, in prime-boost group the percentage of of detected ifng+ cd8+ t cells was lower in comparison to dna group. in splenocytes of animals vaccinated with ad5 very weak cd8+ t cell response was observed. in dna vaccinated animals we determined high level of anti-whc already after second immunization. after boosting with ad5 level of antibodies did not change. those antibodies were only igg2a subclass what indicates th1 t helper type of response. ad5-immunized mice had over 3-fold lower level of anti-whc: both igg2a and igg1 subtypes were detected. the weak response induced by ad5 may be due to the low expression of whcag. in ongoing expreriments we improved the protein expression level by insertion of an intron. we currenly investigate the new construct in mice. the new peptide construct containing four m2e-peptide sequences coupled to t helper epitopes from the plasmodium falciparum cs protein and the hepatitis b virus antigen was administered together with adjuvants intranasally and subcutaneously as described (mozdzanowska et al., virology journal 2007) into various mouse strains. in contrast to its predecessor peptide, we found that vaccination induced much higher anti-m2e serum ab titers against peptide and native m2e. this correlated with a large number of m2-specific ab-secreting cells in lungs and bone marrow. moreover, the serum of vaccinated mice was also crossreactive against the influenza virus subtype a/fm (h1n1), which contains a variant m2e-sequence different in 3 amino acid positions. importantly, this new peptide vaccine regimen showed significant protection against viral challenge with influenza a strains x31 (h3n2) and the highly pathogenic pr/8 (h1n1) with remarkably reduced viral titers in lungs and noses of mice. in conclusion, our studies show promising results towards the further development of vaccination with m2e as a potential "universal" influenza vaccine. this research is supported by a nih t32 fellowship ca09171-32, the nih grant ai 46457 and a grant from the commonwealth of pa. l. yu 1 1 zhejiang university, zhangzhou, china interleukin-18 (il-18) is a cytokine produced by stimulated mononuclear macrophage system. in this report, 18-day-old chicken embryos were vaccined with the plasmid dna (pci-chil-18) encoding chicken interleukin-18 and the copy numbers of chil-18 in peripheral blood, spleen and bursa of fabricius at different time points post-embryonic-vaccination were detected by real-time fluorescent quantitative pcr. the polyprotein of infectious bursal disease virus (ibdv) was prepared into dna vaccine, and the dna vaccine was co-administrated with pci-chil-18 in 18-day-old chicken embryos, then boosted after two weeks, and challenged with virulent ibdv four weeks later. the results indicated that allantoic cavity vaccinated with pci-chil-18 could accelerate high concentrations of chil-18 in nonage peripheral blood, accelerate high expression of chil-18 in nonage spleen and bursa of fabricius and promote the body early immune response capacity. embryo vaccination with chil-18 could significantly enhance the nonage proliferation responses of t lymphocytes from spleen and b lymphocytes from bursa. meanwhile, it could raise the nonage neutralization antibody level and inhance the protection against virulent ibdv induced by dna vaccine. the results indicated that the nonage immune responsing to ibdv dna vaccine was highly enhanced by embryonic coadministration with chil-18 (p x 0.05). due to the unique role of the hair follicle in percutaneous penetration, drug delivery systems, which target active compounds to the hair follicle, may result in a better penetration and a higher efficiency of hair and skin therapy ("follicular targeting"). applications in immunotherapy, e. g. transcutaneous vaccination, are of particular interest, because skin antigen-presenting cells (apcs) can be found at particularly high densities in hair follicle-bearing skin, where they are concentrated around the upper portion of the hair follicles. in in vitro studies on human skin explants, we demonstrated that nanoparticles, due to their ability to aggregate in the hair follicle openings and to penetrate along the follicular duct, are promising carrier systems for transfollicular drug delivery. transcutaneously applied nanoparticles in the size range of 40nm, were capable of penetrating the epithelium and entered into human epidermal lcs, suggesting that such particles may be used to transcutaneously deliver active vaccine compounds, via the hair follicle. the use of the skin as target organ for vaccine has been spurred by recent implication of epithelial dendritic cells (dc) in cd8 cell cross-priming and suggests that vaccination via the transcutaneous (tc) route may be relevant in the induction of cellular immune responses. advanced studies in vivo using functional vaccines are, however, essential to further assess the potential of particle-based vaccines in transcutaneous vaccination. for this purpose, we developed a standard operating procedure (sop) for transcutaneous vaccine delivery on human skin based on our current knowledge on follicular penetration. in a pilot study on 12 volunteers and a phase i study on 24 volunteers vaccinated with an influenza vaccine, we found that this newly developed sop is safe and efficient at inducing a significant increase in cellular immune responses mostly composed of antigen-specific cd8 cells. induction of t cell responses has become one of the major goals in therapeutic vaccination against viral diseases and cancer. this study proposes new perspectives for the development of vaccination strategies that triggers t cell immune responses in humans. objectives: all anti-hiv-1 neutralizing antibodies are directed toward the viral envelope glycoproteins (gp) 120 and the transmembrane protein gp41. two sites on gp120 and gp41 are attractive targets for vaccine design: the epitope in the third hypervariable region (v3) is recognized by the human monoclonal antibody 447-52d and the epitopes in membrane proximal external region (mper) were recognized by the human monoclonal antibodies 4e10 and 2f5. in order to elicit anti-hiv-1 neutralizing antibodies we have designed virus like particles (vlps) displaying either the gp120-v3 region or the gp41-mper. the vlps are based on the acyltransferase component (e2 chain) of the pyruvate dehydrogenase complex of geobacillus stearothermophilus. the e2 chain self-assembles into a 24 nm protein scaffold resembling a vlp and that contains 60 copies of e2. efficient display and refolding of the v3 and mper regions in e2 vlps are obtained by using engineered plasmid which allows insertion of exogenous oligonucleotides at the 5' of the gene coding for e2. the priming and boosting with a combination of vlps and specific hiv-1 envelope dna were used to immunize mice and rabbits. results: the v3-e2 and mper-e2 vlps were purified as stable 60mers from e. coli cells after refolding in vitro from inclusion bodies followed by gel filtration chromatography. binding of 447-52d, 4e10 and 2f5 antibodies to hiv-e2 monomers was confirmed by western blot. we obtained high titers of hiv-1 gp140-specific antibodies in mice immunized with a combination of vlps plus dna (hiv-1 sf162 gp160). these antibodies generated a low (20 %-27 %) level of neutralization. moreover immunizations were also performed in rabbits, a better model for induction of neutralizing antibodies. three doses of e2 vlps plus dna elicited a low titer of hiv-1 gp140 specific antibodies. additional rounds of immunizations in rabbits will be performed, in combination with gp160 plasmid dna, to enahance the responses to envelope and to induce neutralizing activity against these key epitopes. our results demonstrate that e2 vlps are able to display antigenic determinants of hiv and to induce high titers of hiv-1-specific antibodies. the e2 vlps represent a promising tool for a vaccine design. now a day we paid for vaccination of previous generations. as a result morbidity sharply increases, but we haven't well-tried scheme of immunity renewal yet. every clinic, every center do it in there own way, while vaccination is continued, even when it's not necessary, for example, grip, nobody know strain exactly. the most unpleasantly think is that most of physicians don't know what immunity mean specifically, general they think about vitamins, that isn't fit for forming immunity because of many reasons. we offer a way of immunity according to the world scientific theory and practice. the method is based on biochemical, electrophysiology, and biology way of correction physical status. at first we normalize and activate current settings that are going to the diseased organ, vascular system, gastrointestinal tract, spleen. all of it attends indemnity necessary microelements that were extracted from wild officinal herbals. we don't concentrate only on the one or two types of immunity, fist of all we take into account structure and dynamic of immunogeneration system. in our clinic we use this method; immunity is restored very quickly and kept during long time even if organism gets any complications, which can worsen the situation. that's why when we secure new physical statement in the cns program we forming new nearest and distant men health. we tell local state mechanism of disturbances from disturbances, that develop in blood, lymphatic system, tissues and hypothalamus, when pathological process exist long time. it's completely different disturbances of physician state, which should have different therapeutic approach. the threat of an influenza pandemic has become evident in recent years, emphasizing the requirement for influenza vaccines that are broadly cross-reactive against different subtypes with pandemic potential. we have previously shown that baxter's vero cell-derived h5n1 whole virus candidate vaccines are highly immunogenic both in animal models and in human clinical studies, and cross-protective in mice and ferrets. more recently, it was reported that cross-reactive heterosubtype immune responses against highly pathogenic h5n1 influenza virus could also be achieved by immunizing subjects with a trivalent seasonal influenza vaccine; however the induction of cross-subtype protection could not be addressed in this study with human subjects [1] . the study reported here evaluated whether the seasonal influenza vaccine, when used either as a monotherapy or in combination with a h5n1 whole virus wild-type vaccine, could induce an immune response and protect mice against h5n1 influenza virus infection. a trivalent seasonal influenza vaccine was shown to elicit anti-h5n1 antibody and t cell responses and partially protected mice against a lethal challenge with wild-type h5n1 virus. the protective efficacy of the trivalent vaccine derived mainly from the h1n1 component. moreover, passively transferred serum of mice immunised with seasonal influenza vaccine protected naïve mice from infection with h5n1 virus, suggesting that antibodies are the main contributor to protection. h1n1 specific serum did not inhibit neuraminidase activity of h5n1 virus suggesting that protection was not mediated by neuraminidase n1-specific antibodies. next, we investigated the combination of the trivalent seasonal influenza vaccine and the h5n1 whole virus wild-type vaccine. a prime with the seasonal influenza vaccine followed by immunisation with the h5n1 vaccine enhanced anti-h5n1 antibody response, cellular immunity and protection compared to a single immunization with an equivalent sub-optimal dose of the h5n1 vaccine. hence, hetero-subtype immunity can be achieved by immunization with a trivalent seasonal influenza vaccine, which can be further boosted with a h5n1 candidate vaccine. [1] gioia c et al. aims: to register the compliance of the population to the old and new vaccines of the national vaccination program for the children up to 6 years old, and to investigate the possible causes of the potential shortages, in order to approach even more successfully the further goal of this whole attempt, which undoubtedly is the future control of important generalized infections. methods: in the study we checked the vaccination history of 335 children in the first grade of primary school in the area of central and west macedonia. there were 234 greek and 101 foreign children. as fully vaccinated were considered those who had already undergone at least one dose of hib, meningococcus and pneumococcus, two doses of hav, as well as four doses of dtp-sabin, while in the cases of a lack of vaccination, the causes were investigated and the adequate recommendations and information were given. in all the cases, except for the nationality, the sex, and the educational and social level of the parents were registered. results: the percentages of the compliance found, are presented in the following 1) it should be underlined that, as shown in the table, the percentages of the obligatory-free of charge vaccines were close to 100%. 2) high percentages were noted also for meningococcus, either because it is an old vaccine (it has been available for seven years), or because the bacteria is considered quite dangerous (it has been emphasized through the media). 3) on the contrary, as far as the hepatitis a and the pneumococcus vaccines are concerned, low percentages were found, either because of the lack of adequate information-fact that was also shown in our study-or even because of their cost. 4) finally, a statistically significant difference was found relating the response to the vaccination coverage, between greeks and foreigners, but also between the greeks themselves, in relation to their educational and socioeconomic level. objective: over the past three decades, the incidence of type 1 diabetes has dramatically increased in europe and north america, inversely correlated to the decrease of infections. according to the hygiene hypothesis, pathogens may prevent the onset of the disease. om-85, a bacterial extract of both gram positive and gram negative bacteria already used as an immunomodulatory treatment in children, has been shown to protect non obese diabetic (nod) mice from diabetes development. we aimed here at understanding the mechanism underlying this protection. methods: nod mice and nod-cd28 -/mice, which are devoid of natural regulatory t cells (tregs), were treated with om-85. cytokine secretion, activation and proliferation of b cells and foxp3+ tregs were monitored. as toll-like receptors (tlr) recognise microbial molecules and trigger innate and adaptive immunological response, cells from mice deficient for tlr2, tlr4 or the myd88 adaptor protein were used to further address the mechanisms driving the immunomodulatory activity of om-85. two synthetic tlr4 agonists used as adjuvant in human (om-174-dp and om-197-mp-ac) were also tested for their capacity to protect nod mice from diabetes. the om-85-induced protection of diabetes required natural tregs, as nod-cd28 -/mice were not protected. remarkably, om-85 activated b cells and not t cells, promoting their proliferation and il-10 secretion, two phenomena that were tlr4-and myd88-dependent. om-174-dp and om-197-mp-ac two synthetic murine tlr4 agonists effectively prevented diabetes onset in nod mice, promoted the expansion of cd4 + cd25 + foxp3 + t cells and the proliferation of il-10 secreting b cells in a dose-dependent manner. conclusion: our results argue for the involvement of tlr4 signaling in the protective effect of om-85 on development of diabetes and show that two other tlr4 agonists induce proliferation of b cells and their secretion of il-10 as well as stimulation of regulatory cd4 + cd25 + foxp3 + t cells. activation of the innate immunity by tlr-stimulation using those products already used in clinics, may prevent the onset of diabetes in those at risk of developing the disease. d. de wit 1 , a. legat 1 , s. thomas 1 , m. van mechelen 2 , p. hermand 2 , m. goldman 1 1 institute for medical immunology/université libre de bruxelles, gosselies, belgium, 2 glaxosmithkline biologicals, rixensart, belgium aminoalkyl glucosaminide 4-phosphates (agp) are lipid a mimetics which are considered as interesting candidates for the development of synthetic vaccine adjuvants targeting toll-like receptor 4 (tlr4). since natural lipid a from bacterial lipopolysaccharide (lps) depends on membrane-bound or soluble cd14 (scd14) for its tlr4 ligand activity, we investigated the involvement of both forms of cd14 in the responses elicited by crx-527, a prototypical agp. first, we found that crx-527 efficiently induces nf-kb and irf3 activation in hek cells transfected with tlr4 and md-2 genes, whereas the responses to lps required co-transfection of the gene encoding membrane-bound cd14. likewise, crx-527 efficiently induces the synthesis of nf-kb and irf-3 dependent cytokines in whole blood of a patient with paroxysmal nocturnal hemoglobinuria, a disease in which a defect in membrane-bound cd14 prevents lps responses. we then observed that monocyte-derived dendritic cells (dc) which are devoid of membrane-bound cd14 respond to crx-527 but not to lps in serum-free medium. the addition of the soluble form of cd14 did not modify the levels of il12 and tnf produced by crx-527 stimulated dc but increased the levels of interferon-b (ifn-b). when scd14 was added to hek cells expressing tlr4/md-2, nf-kb activity was not modified but irf3 activity was increased in a dose-dependent manner in response to crx-527. we will further compare the responses induced by crx-527 in wild-type and cd14 deficient mice. we previously showed that the transcriptional transactivator (tat) of human immunodeficiency virus possesses the unusual ability to raise a humoral immune response in the absence of adjuvant. these observations prompted us to examine whether such a property can be used to boost the immune response raised against poorly immunogenic peptides. as we previously observed that the autoadjuvant property is controlled by a determinant located within the core-and cysteine-rich regions of the protein, we decided to investigate whether the grafting or the co-injection of a peptide partially containing this determinant (ptat) can raise a humoral immune response against two model peptides. these two peptides, which originate from diphtheria toxin (pdt) and from toxin alpha (pt), both contain an i-ad restricted t-cell epitope but are nonetheless non-immunogenic in balb/c mice of the h-2d haplotype when injected with alum. the ptat, pdt, pt, ptatpt and ptatpdt constructs were prepared by chemical synthesis, purified by reverse phase hplc and characterized by mass-spectrometry. pdt+ptat, pt+ptat, ptatpt and ptatpdt were respectively injected twice at two weeks interval in balb/c mice and animals were bled 14 and 28 days after the second immunisation. the sera were subsequently incubated in microtiter elisa plates previously coated with pt and pdt peptides respectively in order to assess the humoral immune response. we observed a lack of antibody response for the immunizations made with the mixture of peptides (pdt+ptat and pt+ptat) but an anti-pdt and anti-pt response for the immunizations made with the two hybrid constructs (ptatpt and ptatpdt). our results indicate that a humoral immune response can be raised towards non-immunogenic peptides using a determinant involved in the autoadjuvant property of tat, that the phenomenon requires the covalent coupling to the peptide antigen and that it is therefore not related to a bystander effect. interleukin-15 gene polymorph isms (c267t, g367a, c13687a and a14035t) and susceptibility to brucellosis in iranian patients russian federation some epidemiological and observational data suggest that farm and pets exposure [1] in early childhood may be conducive to reduced atopy. currently, there is a lack of consensus regarding underlying immunological mechanisms, especially in prenatal period. as we previously reported the decreasing of intracellular ifn-g production by cbmc statistical analysis was performed using the kruskal-wallis and mann-whitney tests. results: we revealed that newborns from rural mothers (n=14) have higher amount of both nonactivated (subtype infg+/cd69-, p=0.02) and activated (subtype infg+/cd69+, p=0.028) cbmc, producing ifn-g, as compared with newborns from urban mothers (n=79) exposure to pets and the risk of allergic symptoms during the first 2 years of life intracellular interferon-g production by cord blood mononuclear cells as predictor of atopic dermatitis forming in infants: a one-year prospective birth cohort study pc09/16 to what extent t-spot.tb could be used in the diagnosis of tuberculosis in children exposed to tb infection? s. a tb) in children, especially in bcg-vaccinated is difficult for diagnosis because of the low percentage of smear positivity (12-14 %) and clinical futures only in severe forms of disease. the purpose of the present study was to evaluate the diagnostic value of t-spot.tb (oxford immunotec, oxford, uk) compared to tuberculin skin test (tst) in children exposed to tb contact in the family. forty three children with a history for bcg vaccination/revaccination, treated in the university clinic for lung diseases in children sofia, bulgaria were enrolled in the study. the patients were divided according to age in the following groups: 5 months -3 years (n=22), 4 -7 years (n=15) and 8 -12 (n=6) tb has the highest diagnostic value in children n 4 years of age in early childhood the diagnostic value of t-spot.tb and tst does not differ cfp-10 antigen is more sensitive for detection of tb-specific t cells compared to esat-6 antigen. 4. in children with tst 5-14 mm t-spot.tb has a high diagnostic value objectives: the goal of this study is to determine the role of tlr2 and tlr4 in the development of spontaneous lupus disease by creating tlr2 or tlr4 deficient c57bl/6 lpr/lpr mice. methods: tlr2 and tlr4 deficient lupus prone mice have been generated by crossing c57/bl6-tlr2 -/-or c57/bl6-tlr4 -/-mice with c57/bl6 lpr/lpr mice which develop a moderate type of lupus related to fas deficiency. we analysed the phenotype of the disease, autoantibody production and renal injury. statistical comparisons were performed using the mann-whitney u-test. results: these mice developed a less severe disease and few immunological alterations. indeed, in tlr2 or tlr4 deficient lpr mice, glomerular igg deposits and mesangial cell proliferation were dramatically decreased and anti-nuclear, anti-dsdna and anti-cardiolipin autoantibody titers were significantly reduced. however, the response against nucleosome remained unaffected, indicating a role of tlr2 or tlr4 in the production of autoantibodies directed against certain slerelated autoantigens. analysis of b cell phenotype showed a significant reduction of mz b cells, particularly in tlr4 deficient mice suggesting an important role of tlr4 in the sustained activation of these cells likely involved in autoantibody production. interestingly, the lack of tlr4 also affected the production of cytokines involved in the development of lupus disease. conclusion: our data show that deficiency in tlr4 pc14/13 expression of full length mcl-1 and its splice variant in juvenile systemic lupus erythematosus (jsle) neutrophils: differential modulation by gm-csf granulocytemacrophage colony-stimulating factor (gm-csf) can prolong neutrophil survival by increasing mcl-1, an anti-apoptotic protein. a splice variant of mcl-1 arises by removal of exon 2 and induces cell death rather than preventing it. here we investigate the expression of both the full length mcl-1 (mcl-1l) and its splice variant (mcl-1s) in jsle neutrophils compared to controls and investigate whether the addition of gm-csf changes the expression of both isoforms of mcl-1. method: neutrophils were isolated from children (diagnosed x 17 years) with jsle (n=14) and non-inflammatory conditions (control, n=14) and incubated with control serum, jsle serum alone or with jsle serum plus 20pg/ml gm-csf. quantitative real time pcr was used to assess mcl-1l and mcl-1s mrna expression (mean ± sem) following incubation in the above conditions and immediately following neutrophil isolation the ratio of mcl-1s to mcl-1l was also higher in jsle patients compared to controls (p x 0.05). the addition of gm-csf to jsle serum was associated with an increase in mcl-1l (1.66 ± 0.31) and a decrease in mcl-1s (2.56 ± 1.1) mrna expression the addition of gm-csf to jsle serum can abrogate the increased neutrophil apoptosis. alternative splicing is recognised to play a significant role in the regulation of proteins involved in cell death. our results suggest that jsle neutrophils may be more apoptotic due to differential expression of mcl-1 compared to controls, with jsle neutrophils having greater expression of the pro-apoptotic isoform mcl-1s, and less anti-apoptotic full length mcl-1 cyld is a tumor suppressor gene known to play an important role in the nf-kb pathway. to analyze the function of cyld in vivo we used the cyld ex7/8 mouse strain, which is characterized by loss of the full-length transcript and overexpression of a short splice variant of the cyld gene (scyld) to further investigate the connection between scyld overexpression in t cells and colonic inflammation, we used an adoptive transfer model of colitis. therefore naive cd4 + cyld ex7/8 t cells were transferred into rag1 -/-mice which were analysed by mini-endoscopy weekly after cell transfer. here we could demonstrate that cyld ex7/8 cd4 + t cells exhibit less capacity to induce colitis compared to control cells. consequently we investigated if regulatory t cells (t regs ) of cyld ex7/8 mice are capable to control inflammatory responses. for this purpose cd4 + cd25 + cells were co-transferred with naïve wt cd4 + t cells into rag1 -/-recipients. interestingly, rag1 -/-recipients of cyld ex7/8 t regs displayed strong features of colitis compared to control recipients showing that these cells were unable to inhibit inflammatory responses. our findings demonstrate that overexpression of scyld leads to a hyperresponsive t cell phenotype and higher production of inflammatory cytokines by t cells pc17/16 the role of hla complex in inflammatory bowel disease: crohn's disease and ulcerative colitis de investigación biomédica en red de enfermedades hepáticas y digestivas (ciberehd). university hospital virgen arrixaca the allele frequencies of hla class i in cd and uc patients were not different to those observed in controls, although we found an increased frequency of a*03 in cd vs uc. haplotype frequencies of hla class i and ii in cd and uc were also not different to those observed in controls. however, we found increased frequencies of drb1*13, *01 and *0103 alleles, and a decreased allele frequency of drb1*15 in cd vs uc patients and controls. these data are in concordance with other previous studies suggesting that, in patients with isolated colonic cd, drb1*0103 is associated with the development of severe disease and positive association of cd with drb3*0301 and drb1*13. indeed, drb1*15 was negatively associated with cd. this allele appears to confer protection against all subgroups of cd, in all ethnic groups including japanese. however, hla-drb1*07 frequency allele, associated in unselected patients with cd in other studies, was not different in our cd and uc patients, and controls. additionally, an increased frequency in hla-drb1*04 in cd was not found in our patients in a different manner to other reported studies. on the other hand, in our uc patients, allele frequencies of drb1*15 were strongly increased with respect to cd and controls. however, the frequency of drb1*04 was decreased in uc with respect to cd and controls. in this sense, our data are agreed with other reports showing that hla-drb1*15 is associated with uc in european, north american, japanese and korean populations methods: a total of 176 children were studied, (92 boys and 84 girls), up to 17 years of age, with symptoms suspicious for epstein-barr virus infection. the elisa method was used to look for specific antibodies against the capsid of the virus vcaigg and against the nuclear antigen ebv-igm, while taking into consideration the possible increase of the vcaigg title between two serum samples. results: totally, 51 positive cases of children were found (29 %) with active infection : 28 boys (14 x 5 years of age, 12 5-10 years of age and 2 g 10 years of age) and 23 girls (10 x 5 years of age, 6 5-10 years of age and 7 g 10 years of age). pharyngitis was present in 47 children (92,2%), 39 had fever (76,5%) and 48 had lymphadenitis (94 %). the lab tests revealed leukocytosis up to 20.000 leukocytes in 29 cases (56,9 %) and leukocytosis g 20.000 in 9 cases (17,6 %). the most frequent complication documented was streptococcal superinfection in 13 children (25,5 %) and thrombocytopenia in 8 children (15,7 %). a past infection (negative ebv-igm values and positive vcaigg values) was virus infection is common among children and teenagers serum negative are mainly the children of little age and 3) there is no statistically important difference between the two sexes, while on the contrary there is a seasonal distribution of the infection, with winter and summer outbreaks general hospital of rethymno, rethymno, greece shows that the il-13ra2, previously believed to be a decoy for il-13 only, is able to transmit a signal via il-13. our results support this and may suggest that il-13/ il-13ra2 signalling causes disease in oxazolone-induced colitis. currently we are dissecting the role of single cell populations expressing il-4ra to establish which cells play a role in regulating the immune response to oxazolone-induced colitis. together this data can define a role for il-13 or il-13ra2 and identify specific cell populations methods: splenic apcs exposed to enteroantigen (eag) +/-probiotics were used to stimulate cultured cd4 + cd25 -t cells to which titrated numbers of tregs were added. neutralizing antibodies against il-6 and il-1b and elisa-based cytokine analyses were used to monitor the effect of cytokines secreted in the t cell cultures. results: exposure of apcs to eag and probiotics did not influence eag-specific cd4 + cd25 -t cell proliferation. however, exposure to three of the six probiotics tested (b. bifidum bi-98, l. acidophilus ncfm tm and b. bifidum bi-504) consistently reduced regulatory activity of tregs in a cell-dose dependent manner. the tregreducing activity of probiotics was analyzed using fractionated components of the b. bifidum bi-98 strain. data indicated that bacterial cell-wall components were responsible for reducing treg activity and not components of nucleus or cytoplasm. the probiotic-induced down-regulation of treg activity was not mediated by increased intra-culture secretion of inflammatory cytokines such as il-6 or il-1b. conclusion: we conclude that certain probiotic strains can modify apcs to cause reduced treg activity in an eag-specific t cell proliferation assay. this effect apparently depends on a direct apc-to-treg cell contact and not secreted cytokines. the apc/probiotics-mediated inhibitory effect on tregs may oppose antiinflammatory activities desired from probiotic therapy palmieri 1 1 'la sapienza dysregulated innate and adaptive immune responses against commensal flora lead to crohn disease (cd) and ulcerative colitis (uc), two different forms of inflammatory bowel disease (ibd), a lifelong inflammatory condition of the gastrointestinal tract methods: we analyzed 21 pediatric cd patients (13 active, 8 remission), 24 pediatric uc patients (17 active, 7 remission), and 37 age-matched non-ibd controls. nkg2d/ligand expression was evaluated by immunostaining and multiparametric facs analysis (on pbmc subsets), and by immunohistochemistry and twocolour immunofluorescence (on intestinal biopsies). differences between groups were analyzed with non-parametric and parametric tests; a level of p x 0.05 was considered significant. results: nkg2d expression is selectively upregulated on circulating "innate-like" t cell populations (g/d and cd3+cd56+ nkt cells), in active, but not in quiescent ibd patients; receptor upregulation correlates with disease type (observed in uc, but not in cd patients). in the same patient groups, the appearance of nkg2d ligands on circulating monocytes is also observed. the dramatic increase of nkg2d+ lymphocytes, and the strong upregulation of nkg2d ligands on both epithelial and immune components, are observed in active ibd lesions. conclusions: our observations document the dysregulated expression pattern of nkg2d/ligands on selected innate immunity populations in pediatric ibd patients, both at mucosal and systemic level pc17/26 peripheral and intestinal regulatory t cell dynamics in pediatric ibd patients is a chronic inflammatory condition of the gastrointestinal tract characterized by dysregulated innate and adaptive responses against commensal flora. regulatory t cells (t reg) represent an important mechanism to suppress uncontrolled immune responses to bacterial flora. aims: to evaluate the frequency of regulatory t cells in the peripheral blood, and in inflamed and non inflamed mucosae of pediatric ibd and non mucosal regulatory t cells were identified by immunohistochemistry; circulating regulatory t cells were analysed by immunofluorescence and facs analysis. differences were analyzed with parametric and non-parametric testsconsidered significant. results: foxp3+ t reg were significantly increased in the intestinal lesions of active ibd patients (cd or uc), and returned to normal levels in post-therapy remission phase. at variance, circulating cd4+ t reg frequency was elevated in patients affected by both forms of ibd, independently of disease activity, as it persisted in the remission phase. a selective imbalance in the frequency of t and nk subsets characterized the abundant inflammatory infiltrate present in active intestinal lesions, and the normal immunological profile was only partially restored in mucosal samples of quiescent ibd patients. conclusions: regulatory t cells dynamics are differently regulated in mucosal tissues and at the systemic level, during the distinct phases of disease; t reg dynamics in pediatric ibd patients only partially matches previous data obtained in the adults; quiescent ibd is characterized by the imbalance of selected lymphocyte subsets, both in the mucosa and systemically the increased expression of immunoproteasomes in the inflamed mucosa of ibd patients was shown to contribute to this pathology by enhancing nf-kb activation. due to the relation between nf-kb and the immunoproteasome we have investigated whether specific inhibition of immunoproteasomes is suitable for therapeutic intervention in ibd. lmp7 knock-out mice are deficient in the essential catalytic immunoproteasome-subunit ß5i and therefore are devoid of immunoproteasomes. to test our hypothesis, we employed the dss colitis model. in contrast to wild-type mice, colitis was attenuated in lmp7 knock-out mice characterized by reduced weight loss and less infiltration of lymphocytes in the mucosa confirmed by histology. in addition, lmp7 knock-out mice had lower levels of proinflammatory cytokines and chemokines compared to wild-type mice validated by rt-pcr and elisa. especially nf-kb regulated genes show enhanced induction in wild-type mice unlike lmp7 knock-out mice synaptic systems gmbh, braunschweig, germany objectives: although more than 200 million people worldwide are chronically infected with hepatitis c virus (hcv) no prophylactic or therapeutic vaccines do exist to prevent or cure hcv infections. our major objective is to develop a dendritic cell (dc)-based immunotherapy enhancing virus-specific cellular immune response for treatment of hcv infections based on this approach we aim at generating adec-205 antibodies conjugated with immunodominant hcv proteins to induce hcv-specific protective immunity. methods: recombinant hcv proteins are expressed using "expression-ready-clones" containing n-terminally his-tagged hcv-core (aa 1-191) or hcv-ns3 (aa 1027-1218) sequences. protein purification is performed by metal-affinity chromatography on ni-nta-agarose hcv-specific t cell responses are monitored at different time points after immunization by facs and in vitro t cell proliferation assays. results: to obtain high amounts of recombinant ns3 and core we successfully optimized culture and protein purification conditions. briefly, ns3 was purified natively using pbs-based buffers with ph-gradient. in contrast, purification of core was performed under denaturing conditions in presence of guhcl and urea and a ph-gradient elution. moreover, optimized conditions allowing conjugation of adec-205 to recombinant hcv proteins were established with respect to duration of conjugation and buffer requirements needed to avoid protein precipitation. efficient conjugation was verified by western blot analysis. after successful generation of adec-205/ hcv-protein conjugates we are currently establishing optimized vaccination conditions to induce hcv-specific immune responses pd01/2 mva-nef vaccination induces polyfunctional cd4 t-cells and increases the proliferative capacity of cd8 t-cells in hiv-1 infected individuals under haart several vaccination trials have made use of the modified vaccinia virus ankara (mva) as delivery vector. in a therapeutic vaccination trial, we demonstrated that mva expressing the hiv-1 protein nef (mva-nef) was safe in hiv-1 infected individuals under haart and immunogenic in regard to the elicitation of ifn-g mediated cd4 t-cell responses. recent advancements in polychromatic flow-cytometry technology revealed that the sole evaluation of ifn-g provides limited information on the quality of antigen-specific t-cell responses. the evaluation of several functions is essential, as simultaneous production of multiple cytokines by t-cells is associated with superior control of viral replication. methods: in a retrospective setting, we simultaneously assessed the production of ifn-g, il-2 and mip-1b, the expression of the activation marker cd154 and the differentiation marker cd45ra in nef-specific cd4 and cd8 t-cell populations during the course of the vaccination trial. furthermore we applied a multi-colour cfse based proliferation assay investigating the proliferative capacity and the simultaneous expression of ifn-g, il-2 and mip-1b. results: following mva-nef vaccination, we observed a significant increase of the total nef-specific cd4 t-cell response and a significant increase of polyfunctional nef-specific cd4 t-cells, simultaneously expressing ifn-g, il-2 and cd154. using the standard ics no increase of nef-specific cd8 t-cell responses was observed. however, by the cfse based proliferation assay, we could show a clear expansion and a generally enhanced proliferative capacity of nef-specific cd8 t-cells following mva-nef vaccination. notebly, we observed a correlation between the increase of ifn-g, il-2 and cd154 expressing cd4 t-cells and the increase of proliferating cd8 t-cells suggesting the possibility of a causal link between the two functions. conclusions: the mva-nef vaccine is able to change the quality and quantity of the nef-specific cd4 t-cell immune response and has the potential to increase the proliferative capacity of nef-specific cd8 t-cells in hiv-1 infected subjects under haart this preferential binding to the complex was evident in classical immunochemistry assays, as well as in surface plasmon resonance (spr) tests. this ab inhibited hiv-1 mediated membrane fusion and p24-detected replication. db81 was found to nicely recapitulate the characteristics of the unconventional, protective immune response, which is taking place in naturally resistant esn individuals. further characterization of the antibody and of its binding epitope is ongoing following intradermal vaccination with 25mg dna and electroporation of balb/c mice, splenocytes have been incubated with peptides representing class i and ii epitopes, and specific t cell-responses were examined by elispot-assays. the specific antibody responses have been measured by sandwich eli-sas, and neutralizing antibodies have been investigated by hi-assays. results: the vaccibody constructs have been found to be expressed and correctly folded in vitro. the in vivo experiments further demonstrate the presence of neutralizing antibodies as well as the strong induction of antigen specific cd4 + and cd8 + t cells. conclusion: antibody and cellular immune responses against influenza hemagglutinin are enhanced when targeted to apcs. methods: the hcv recombinant proteins rns4 (1677-1756 aa) and rns5a (2061-2302 aa) were conjugated with immunomax using the heterobifunctional reagent sulfo-smcc. balb/c and dba/2j mice were immunized intraperitoneally 2 times at a month interval with different doses (0.1 -2 mg/mouse) of the proteins without adjuvants, as conjugates with immunomax, or with complete freund's adjuvant (cfa) the other combinations were not immunogenic at given doses. it should be noted that only conjugates stimulated production of antibodies that bound not only to recombinant protein but also to peptides imitating epitopes of ns4 protein. immunization with rns5a-immunomax conjugate and rns5 in cfa (1.4 mg/mouse) induced a similar antibody activity, but a different t-cell responses. the conjugate induced splenic accumulation of t cells specifically reacting in vitro with ns5a recombinant proteins of various genotypes, with peptides and with phages by cell proliferation and/or cytokine secretion. immunization with rns5a in cfa induced cells proliferating in vitro after stimulation only with peptides; none of the antigens stimulated cytokine secretion. conclusion: covalent conjugates of hcv nonstructural proteins with immunomax effectively induce humoral and cell immune responses pd01/21 degree of cross-genotype reactivity of hcv-specific cd8 t cells directed against ns3 the existence of multiple hcv genotypes characterized by marked sequence differences is a challenge for immune control. the aim of this study was to compare the antiviral cd8 t cell response targeting hcv genotype 1 (gt1) and genotype 3 (gt3) as the most predominant genotypes in germany and to determine the extent of cross-genotype reactivity of specific t cells. we analyzed a cohort of patients with past or ongoing intravenous drug use (ivdu) hypothesizing that multiple exposures to different genotypes may occur. methods: 53 subjects (17 with gt1, 22 with gt3 and 14 anti-hcv-pos/hcv-rna-neg) were analyzed. hcv-specific t cells were expanded from pbmc in the presence of peptide pools covering ns3 from gt1 or gt3. individual reactive peptides and the degree of cross-reactivity between the gt1 and gt3 variants were determined by ics. complete ns3 is sequenced from all viremic patients pd01/22 anti-retroviral effects of type i interferon subtypes in vivo ifna subtypes 1, 4, 6 or 9 suppressed fvreplication in vitro, but differed greatly in their antiviral efficacy in vivo. treatment of fv-infected mice with the ifna subtypes 1, 4 or 9, but not 6 led to a significant reduction in viral loads. decreased splenic viral load after ifna1 treatment correlated with an expansion of activated fv-specific cd8 + t cells and nk cells in the spleen, whereas in ifna4-and ifna9-treated mice it exclusively correlated with the activation of nk cells. other ifna subtypes like ifna2, 5 and 11 are under investigation pd01/23 elimination of immunodominant epitopes from multispecific dna-based vaccines allows induction of cd8 t cells that have a striking anti-viral potential immunodominance limits the tcr diversity of specific, anti-viral cd8 t cell responses elicited by vaccination or infection. to prime multispecific t cell responses, we constructed dna vaccines that coexpress chimeric, multidomain antigens (with cd8 t cell-defined epitopes of the hepatitis b virus (hbv) surface (s), core (c) and polymerase (pol) proteins, and/or the ovalbumin (ova) antigen as stress protein-capturing fusion proteins. priming of mono-or multispecific, hla-a*0201-or k b -restricted cd8 t cell responses by these dna vaccines differed. k b /ova 257-264 -and k b /s 190-197 -specific cd8 t cell responses did although chronic infections remain asymptomatic in most cases, immunocompromised patients can suffer from severe and life-threatening ebv-associated diseases, such as posttransplant lymphoproliferative disorders (ptld). thus, immunotherapeutic strategies using adoptively transferred ebv-specific t cells are promising. one option is the generation and expansion of cd4 + and cd8 + t lymphocytes by using ebv-specific synthetic peptides for the stimulation of pre-existing memory t cells. aim of our study was to identify a set of mhc class-ii peptides for each antigen promiscuitive peptides with high syfpeithi scores were tested for immunogenicity using an ifn-g-elispot. pbmcs of at least 16 healthy, randomly chosen blood donors were cultured for12 days in the presence of each candidate peptide. functional and phenotypic analysis of t cells of several donors was performed by multicolor flow cytometry. 48 out of 72 tested peptides could be identified as t-cell epitopes. two of them were defined as immunodominant, as more than 50 % of tested blood donors showed peptide-specific t cell responses. so far, eight of the tested peptides could be identified as mhc class-ii epitopes. furthermore, a highly immunodominant class ii peptide mix consisting of 5 peptides was selected. in conclusion, we could identify several new ebv-specific mhc class-ii epitopes which can be used for united kingdom, 3 hospital de clínicas during persistent hbv infections, patients usually develop poor or no protective immune responses against viral antigens, which not only leads to the chronicity but also the unresponsiveness to conventional treatments.in order to overcome the unresponsiveness and to generate an effective therapeutic strategy for treatment for chronic hbv infections a chimeric tcr against hbsag, which aims to increase the percentage and quality of antigen-specific cd8 + t cells, was developed moreover, we pre-conditioned the liver microenvironment by injection of cpg oligodeoxynucleotides (odn) to optimize the recruitment of transferred cd8 + t cells to the liver and to overcome the tolerogenic microenvironment of the liver. we found that the il-12-exposed cd8 + t cells showed at least five-fold increase of survival rate in vivo than il-2-exposed cd8 + t cells did treatment of the recipients with cpg-odn could increase the percentage and also the total amount of transferred cd8 + t cells mainly in the liver. by in vivo brdu incorporation, we demonstrated that the higher in vivo survival rate of il-12-exposed cd8 + t cells and the effect of cpg-odn were due to the up-regulation of the proliferation of those cells. to sum up, the cocktail therapeutic strategy could not only increase the survival rate of transferred cells but also direct the antigen-specific cd8 + t cells to the liver to exhibit their effector functions. the detailed mechanisms responsible for the il-12 and cpg-odn effects on the regulation hyper igm (him) and wiskott-aldrich syndrome (was) than those of corresponding controls (p x 0.01) . there was a significant elevation of t ada and ada1 activities in iga deficient patients as compared to healthy individuals (p x 0.01) . our results hypothesized that altered ada activity may be associated with altered immunity. therefore, serum ada level could be used as an indicator along with other parameters pd01/61 hiv-1 sequence evolution after dendritic cell-based immune therapy in a phase i/ii clinical trial hiv rna was extracted from plasma samples collected before the startof haart and early after vaccination when haart was terminated. rna was amplified by rt-pcr and sequenced using standard protocols. sequences of the vaccine genes tat, rev and nef as well as control genes vif, vpr, vpu and parts of env were analyzed for variation between pre-and post vaccination time points. hiv sequences spanning known and predicted epitopes of the relevant hla alleles from each participant were analyzed in detail. results and conclusion: immune therapy was well-tolerated and no severe adverse effects occurred. after haart termination, plasma viral load became detectable in all patients after 2-6 weeks. follwing the viral rebound a set point was reached, that was lower than the viral load before start of haart. using various methods we evidenced newly induced or enhanced immunity after immune therapy (see abstract b. de keersmaecker et al.). for studying sequence evolution, complete sets of both pre-haart and post-vaccination sequences were obtained in 12 out of 17 patients. with one exception, variation in sequences of vaccine and control genes of pre-haart samples compared to samples taken early after vaccination was limited. this indicates that there was no significant impact of the immune response on virus evolution at this stage. more focussed analysis on viral sequences spanning specific hla islamic republic of newcastle disease (nd) is regarded throughout the world as one of the most important diseases of poultry, not only due to the serious and high flock mortality, but also through the economic impacts. the purpose of this study was to be informed from the possible influence of infectious bronchitis virus on immune response of chickens to nd live vaccine. one hundred and twenty, 10-day-old ross 308 broiler chickens divided randomly into 3 groups, 2 experimental and a group as control one. the first experimental group vaccinated by a monovalent nd live vaccine with cl/79 strain, and the second experimental group vaccinated by a bivalent newcastle disease and infectious bronchitis live vaccine with cl/79 and h120 strains, via the drinking water at 10 days of age at the same time, and the control group received no nd vaccine. the antibody response to vaccination was assessed using the hemagglutination inhibition (hi) test by taking blood samples three times, first the day before and the next, 7&14 days post vaccination. results indicated that, although the strain of studied nd live vaccines were the same united kingdom t cell-based ifng release assays from blood are an important advance for diagnosing tuberculosis infection but do not permit reliable treatment monitoring or distinction of active tb from successfully treated disease or latent infection. t-cell cytokine profiles vary with in vivo antigen load in viral infections cd4 t cells from 25 patients with active tb and 28 patients with successfully treated tb were analysed for simultaneous expression of ifng and il2 at the single cell level using multi-colour flow-cytometry after 6 hours stimulation with ppd. moreover, cells were stimulated with esat-6 and cfp-10 receiver operator characteristics analysis revealed that a percentage of ifng /il2 dual positive cells x 56 % served as an accurate marker for active tb patients (specificity 100 %, sensitivity 65 %), while frequencies g 56 % were observed in treated as well as active tb patients. in conclusion, quantitation of antigen-specific t cells based on the analysis of ifng only does not allow distinction of patients with active and successfully treated disease pd03/7 necessity of postpone bcg vaccination -lesson from primary immunodeficiencies v. thon methods and results: the czech national database of primary immunodeficiencies (pid) was established in 1993 and is connected with the european database of primary immunodeficiencies (esid). the prevalence of pid in the czech republic (approximately 10 100 000 inhabitants) is 5.33 to 100 000. among these patients there are children diagnosed with severe combined immunodeficiency (scid) and chronic granulomatous disease (cgd) too. according to the czech national database of pid, 12 out of 14 children with later proved scid were immunised with bcg vaccine in the first days of life. nine of them developed disseminated and generalized bcg infections. five children with scid died. moreover, reactivation of bcg was also seen in healthy children after admission of combined vaccines with hepatitis b given at the age of twelve weeks. on the other hand, this was not the case in thousands of children of hbsag positive mothers who were vaccinated against hepatitis b after delivery in the first place and later immunized with bcg vaccine. systematic vigilance against tuberculosis (tb) and vaccination significantly lower the prevalence and risks of tb. in the czech republic, the prevalence of tb is currently 6.12 to 100 000 inhabitants. unfortunately, temporary interruption of bcg vaccination in three large districts in the period of 1986 to 1993 led into higher incidence of tb and appearance of new cases of aviary mycobacteriosis. these complications were not observed in vaccinated children. conclusion: we recommend a change of current practice of bcg vaccination considering new immunization schedule with hexavalent vaccine pd03/8 novel analogues of thalidomide inhibit cd80 expression and production of tnf-a, il-12, ifn-g, cxcl-9 this work describes the synthesis and characterization novel thalidomide analogues, prepared in good yields using simple methodology. our results suggest that anti-inflammatory and immunomodulatory activity of these diamine compounds is potentially applicable in treating enl and other diseases. supported by: cnpq, fapemig and capes, brazil. of the b cell follicle. cta1-dd augmented gc-formations, specific antibody responses and cell-mediated immunity to the t cell-dependent antigen np-cgg, but failed to do so when used together with t cell independent antigens, such as np-ficoll or np-dextran. this effect required adp-ribosyltransferase activity, as mutant cta1r7k-dd failed to exert an adjuvant effect. the adjuvant function appeared to correlate with the fdc-localization and turned out to require complement and/or complement receptors (cr) chitosan formulations varying in molecular weight, counterion and structure (i. e. soluble v/s particulate) were used in assays to examine expression of maturation markers via flow cytometry and cytokine production by elisa. we found that, in contrast to alum, plg and ps particles, chitosan induced bmdc maturation on its own, as determined by the expression of cd80 and cd86. these effects were most prevalent with soluble chitosan chloride formulations but were also notable with soluble chitosan glutamate chitosan. the effect of chitosan on cytokine production was investigated using a panel of different tlr agonists in combination with chitosan particles. results show an increase in the secretion levels of il-1a and il-1b, while il-6 levels were not affected. finally we studied the role of inflammasome activation in the enhancement of il-1b production. using bmdc from nlrp3 -/-mice we examined il-1b production in response to different tlr and chitosan combinations. results show that the ability of chitosan to enhance il-1b production is dependent on nlrp3. collectively our data indicate that upregulation of maturation markers and enhancement in proinflammatory cytokine secretion mediated by chitosan severe sepsis, induced in mice by cecal ligation and puncture (clp), led to ho-1 expression in infiltrating peritoneal leukocytes, kidney and liver. mortality rate of clp increased from 20 % in wild type (hmox1 +/+ ) mice to 87 % in ho 1 deficient (hmox1 -/-) mice. hmox1 -/-but not hmox1 +/+ mice developed end-stage multiorgan failure. mortality of hmox1 -/-mice was associated with increased peritoneal leukocyte infiltration, but not with increased pro-inflammatory cytokine secretion or bacterial load in peritoneum, blood or organs. clp induced a significant increase in cell-free hemoglobin free heme was found to sensitize primary hepatocytes to tnf, anti-fas antibody, h 2 o 2 or peroxynitrite mediated apoptosis. this cell death was associated with outward nuclear translocation and extra-cellular accumulation of the late-stage pro-inflammatory cytokine hmgb1. similarly, circulating and cytoplasmic hmgb1 was increased in hmox1 -/-relative to hmox1 +/+ mice following clp. in conclusion, these data suggest that free hemoglobin and heme, released during severe sepsis, are important factors in the organ failure and death associated with severe and b-1,2-linked mannose residues elicit inhibition effect. it was found that inhibition activity of oligosaccharides increases with chain length. immunization with mannan-hsa conjugate allowed for the maturation of immune response generating specific antibodies with high avidity/affinity, whereas immunization with mannan alone elicited only low-affinity antibodies. in the future, an effective antifungal subcellular vaccine would be constructed using selected mannooligosaccharidic epitope and the appropriate carrier protein as inductor of immunological memory. acknowledgements: this work was supported by the grant agency of slovak academy of sciences all subjects received dtwp vaccine at 4-6 years of age (booster vaccination), following the national vaccination schedule of iran. blood samples were collected before and 2-4 weeks after the vaccination. immunogenicity of the vaccines was assessed by elisa using commercial kits. results: the geometric mean titers (gmt) of the antibodies induced against diphtheria and tetanus by dtwp-local were 7.7 and 9.4 iu/ml and those of dtwp-pasteur were 8.2 and 8.6 iu/ml, respectively. there was no significant difference between the immunogenicity of the two vaccines against diphtheria and tetanus. the gmts of antibodies produced against pertussis were 30.2 eu/ml for dtwp-local and 47.9 eu/ml for dtwp-pasteur vaccines, respectively (p x 0.001). no significant differences were observed in the antibody titers against diphtheria, tetanus and pertussis between the two vaccines before vaccination. conclusion: immunogenicity against diphtheria and tetanus was similar for the two vaccines pd05/18 united kingdom haemorrhagic septicaemia (hs) is an acute disease of cattle and buffaloes in tropical countries, caused by pasteurella multocida serotype b:2 , a gram-negative coccobacillus. jrmt12, an aroa mutant of pasteurella multocida, constructed previously in our laboratory, attenuated for virulence in the mouse and protects mice from challenge with the virulent strain. in this work, the immune response of calves was tested after intramuscular vaccination with single dose of 10 8 cfu of jrmt12. a possible contributory role of cellular immunity against hs was investigated in vaccinated and in control calves after challenge. a lymphocyte stimulation assay was used to assess the effects of a cell-free extract (cfe) of p. multocida on peripheral blood mononuclear cells (pbmcs) isolated from calves at different times after challenge. the results were indicative of a possible immunosuppressive effect of challenge with p. multocida b:2 on calf pbmcs. the suppressive effect was further investigated by in vitro experiments. calf pbmcs obtained from normal calves were treated with cfe for 1 h before adding concanavalin-a (cona) pd06 -vaccination and immunotherapy against parasitic diseases pd06/1 evaluation of simian adenoviral vector adch63 expressing msp-1 as a candidate blood-stage malaria vaccine this successful regime incorporated a human adenovirus serotype 5 (adhu5) prime, boosted eight weeks later with a modified vaccinia virus ankara (mva) vector. adenoviral vectors have generated great scientific interest in recent years and appear to be superior viral vectors with great potential in vaccine regimes. their potential use in humans, however, is limited by natural anti-vector immunity to human adenoviruses, but this problem could be largely circumvented by the use of simian adenoviral vaccine vectors. recent clinical trials have suggested that the simian adenoviral vector adch63 is a promising clinical candidate. we have developed vectors (of human and simian origin) and mva encoding a novel construct based on p. falciparum msp-1 and have undertaken comparative immunogenicity studies in mice. the antigen, termed 'pfm128 while asymptomatic per se, the heterozygous sickle cell trait confers a survival advantage against malaria, the disease caused by plasmointo carbon monoxide (co), iron and biliverdin. when infected by plasmodium hb sad mice are protected against experimental cerebral malaria (ecm), a lethal neuroinflammatory syndrome that in many aspects recapitulates human cerebral malaria. ho-1 expression and activity are strictly required to suppress ecm in hb sad mice, as demonstrated by functional deletion of the hmox1 locus or pharmacologic inhibition of its enzymatic activity. the protective effect of ho-1 is mediated by co, which inhibits the accumulation of protein-free heme in plasma following plasmodium infection conclusion: topical treatment of cutaneous leishmaniasis with gsno accelerated healing and reduced local parasitism in the mouse suggesting that it may be ben gp63 expression was confirmed by sds-page and elisa using monoclonal antibody against gp63. discussion: today researchers attempt to find a suitable vaccine for leishmaniasis. although some researchers have reported proper vaccines of interest, a5-180recp is recognized only by sera collected from resistant bovines infested with all stages of r. microplus, but not by sera from similarly infested, susceptible hosts. furthermore, this recognition was specific since sera from resistant non-infested bovines (naï ve animals) did not react with a5-180recp. our results show that reverse immunogenomics can be useful for discovery of new antigens for development of an anti-tick vaccine. supported by cnpq and fapesp. for the maintenance of ab-mediated vaccine-induced protection after re-challenge with the pathogen or the vaccine antigen. memory b-cell elispot together with ab titres might therefore prove useful as independent marker for duration of protection. objective: this study focused on establishing experimental conditions and optimizing the performance of the memory b-cell elispot assay by detection of specific memory b-cells against anti-tetanus vaccine and naturally acquired toxoplasma gondii infection as a model. methodology: twelve healthy subjects who had received the tetanus vaccine at least 6 month previously were enrolled. peripheral blood mononuclear cells (pbmcs) were isolated from each donor using cell preparation tubes (cpt). plasma was obtained after centrifugation of cpt and stored at -20°c until used for elisa. specific igg-secreting b-cells were determined by elispot assay, using tetanus toxoid (tt) and t. gondii surface antigen as model antigens. results: to optimize our assay, conditions were changed and compared to the previously established protocol. we detected low frequencies of total igg memory b-cells and tt-specific memory b-cells in all donors four seropositive and 2 seronegative donors had positive responses in elispot. no correlations were found with serum antibody titers and frequencies of memory b cells (r=0.216, p=0.641) or with t. gondii-specific b-cells conclusions: following optimization of several assay parameters, we demonstrated that the memory b cell elispot could be reliably used to determine low numbers of antigen-specific memory b-cells in individuals naturally exposed to infection or following vaccination our previous work demonstrated that il-17 also affects the cells of erythroid lineage, by stimulating development of early erythroid progenitors, bfu-e, but inhibiting the growth of late stage erythroid progenitors, cfu-e, from normal murine bone marrow. we also provided in vitro evidence that at least part of its effect on cfu-e is mediated by nitric oxide (no) generation. in the present study we demonstrated that the in vivo reducing effect of il-17 on bone marrow cfu-e was prevented by co-treatment with the no synthase (nos) inhibitor, l-name, implying that this effect is mediated through nos activation. the data obtained in cultured bone marrow cells showed the ability of il-17 to upregulate the expression of mrna for both the inducible (i)nos and the constitutive, endothelial (e)nos isoform. both the nos-inducing effect of il-17 and il-17-related inhibition of cfu-e growth were dependent on p38 mapk activity, since the p38 mapk inhibitor, sb203580, markedly downregulated il-17-induced activation of nos and reversed the growth inhibitory effects of il-17 on cfu-e. the in vivo stimulating effect of il-17 on bfu-e colony growth in the bone marrow was not affected by co-treatment with the nos inhibitor, pointing to different mechanisms for il-17 effects on bfu-e and cfu-e. however, the in vivo exposure of the mice to l-name, increased the number of various hematopoietic progenitor cells in the bone marrow, indicating that no itself is important regulator of hematopoietic progenitor cell activity. overall, the data presented gave an insight into the mechanisms by which il-17 acts on bone marrow cells and also revealed a link between the il-17, no and hematopoiesis. further studies on il-17-mediated induction of both inos and enos methods: a total of 1785 blood donor samples were tested for hbsag and anti-hbc with the immunoenzymic method elisa, while simultaneously, molecular blood test (nat) was applied. the positive samples for anti-hbc were also tested for anti-hbs and anti-hbc igm. results: a total of 68 samples (3,8 %) were found anti-hbc positive conclusions: it is proven, therefore, that in some cases the levels of hbsag, following an infection from the hepatitis b virus, are probable to remain low, so that it is not possible to detect them using elisa method. in these cases anti-hbc can be the only serological marker of the infection. consequently, patients with positive anti-hbc and levels of anti-hbs x 100 iu/l are possibly not immune enough, so that they can become blood donors. that was the reason why some blood donation centers in our country, until recent years when there was no capability for nat testing of blood donors, had 100 iu/l as a limit for anti-hbs levels. however, in present days that nat testing of blood donors is used in our country, it has offered great safety and it is possible that anti-hbc testing will not be necessary, despite the fact that many blood donor centers have preserved the safety limit of 10 iu/l anti-hbs in all the blood units, which also goes for our study pd14/20 neonatal allo immune thrombocytopenia and igg glycosylation patterns michaelsen 1,2 1 national institute of public health in milder cases it can cause petechia and in more severe cases it can cause intracranial hemorrhage and death. the reason behind the variation in clinical symptoms is not fully understood, but is probably not due to differences in immunoglobulin isotypes or antibody affinity. recently influence of glycosylation patterns of igg on the biological activity has been realized. variation in carbohydrate structures attached to asparagine 297 can cause differences in the interaction with fc-receptors, and hence a difference in thrombocyte elimination capacity of the igg molecule. patient sera from norway and the netherlands with different levels of antibody titres and severity of symptoms have been used to affinity isolate igg antibodies against the hpa-1a alloantigen and analyze the glycopeptides using mass spectrometry. the glycosylation patterns have been analyzed for a possible link between severity of symptoms and variation in the glycosylation patterns. so far patients with serious symptoms seem to have increased galactosylation and sialylation and a high level of non core-fucosylated n-glycans on their anti-hpa-1a iggs we monitored 12 children (9 boys and 3 girls) in ages from 3.2 to 17.2 years with average age of 8.9 years. in 11 of them all was diagnosed for the first time. 1 subject had the second relapse of all. one patient received maintenance chemotherapy, all the rest (11 subjects) induction chemotherapy. methods: leukocyte count and hemiluminescent analysis of whole blood were performed for all the patients during infectious complications twice: on neutropenia background and after the recovery of neutrophil number. hemiluminescent analysis for whole blood allows to estimate the functional activity of phagocytes, namely their bactericidal power and phagocytosis completeness. we valuated spontaneous and zymosan induced hemiluminescence. we used onsonised zymosan as the inductor of "respiratory paroxysm mice with a homozygous mutation in the rc3h1 gene, that encodes the zinc finger and ring finger containing protein roquin, develop severe autoimmune disease. the observed lupus-like phenotype involves follicular helper t cells, which express higher levels of icos. these cells provide inappropriate t cell help to b cells, leading to the production of autoantibodies (vinuesa et al. 2005, nature 435, 452-8). it has been shown that the half-life of icos mrna is shortened when roquin is over-expressed. such repression requires the 3'utr of icos, in which a 47bp sequence, containing a possible mir-101 binding site, was sufficient (yu et al. 2007, nature, 450, 299-303). mnab is the paralogue of roquin, and has been shown to bind to nucleic acids (siess et al. 2000, j biol chem 275, 33655-62). we demonstrate that in primary mouse t cells and embryonic fibroblasts roquin, but not mnab, inhibits translation of icos. we map critical domains in the roquin protein to icos repression using deletion-and point-mutants of roquin, as well as chimaeras that swap sequences from roquin to mnab and vice versa. addressing the mechanism of roquin mediated icos repression; we demonstrate binding of roquin to icos mrna in primary mouse t cells and in cotransfection experiments. our current work dissects the requirement of cellular rnai, the stress response pathway or p-body function by testing roquin repression of icos mrna in dicer-, tia-1-and ago2-deficient mef cells and in knockdown approaches. acknowledments: j m m-v is a recipient of a harvard real colegio complutense (rcch) grant. work in dr tsokos' lab is supported by grant phs nih r01 ai 42269. we have recently reported that 6-hydroxyl-1-methylindole-3-acetonitrile (6-hma) isolated from brassica rappa inhibit nuclear factor-kappa b (nf-xb) activity in raw 264.7 macrophages. in this report, we investigated the effect of 6-hma on dextran sulfate sodium (dss)-induced colitis model in mice. methods: we induced colitis with dss in mice and evaluated disease activity index (dai), including body weight, stool consistency and gross bleeding, and tissue myeloperoxidase (mpo) accumulation. through h&e staining, histological change was observed. the expression of inducible nitric oxide synthase (inos), inhibitory kappa b-a (ixba) and nf-xb were detected by western blot and immunohistochemical staining. in-vitro system, the expressions of interleukin-8 (il-8), monocyte chemotactic protein-1 (mcp-1) in ht-29 human colon epithelial cells were measured by rt-pcr. results: in dss colitis model, the dai score and detection of mpo accumulation brevealed 6-hma significantly inhibited loss of body weight, suppression of diarrhea and bleeding, and infiltration of macrophages, leukocytes. moreover, h&e staining also indicated 6-hma suppressed the thickness of muscle layer, edema, mucosal damages by dss. these results were related to the regulation of nf-xb activation. 6-hma attenuated the dss-induced phosphorylation and translocation of nf-xb subunit p65. in addition, this effect was accompanied with parallel blocking degradation of ixba. moreover, pretreatment of 6-hma significantly reduced the mrna levels of il-8 and mcp-1 stimulated by tumor necrosis factor-a (tnf-a) in the ht-29 cells. pretreatment of 6-hma also significantly blocked the ixba degradation and nf-xb p65 nuclear translocation stimulated by tnf-a in the ht-29 cells. these results were concurred with the effect on nf-xb activation in dssinduced colitis model. conclusions: these results for the first time demonstrated that alleviation of 6-hma mediated by regulation of nf-xb activation and suppression of chemokines in vitro and in vivo. therefore, 6-hma could be new potential therapeutic agent for inflammatory bowel disease.cd4 serves as receptor of hiv and is a self-antigen. we have previously characterized the anti-cd4 igg immune response in hiv-1-exposed, seronegative (esn) subjects and we know that there is a peculiar specificity of these antibodies for epitopes induced by gp120-binding and that there is an epitope specificity distinct from that seen in hiv-infected patients (second cd4 domain preferred). to generate antibodies able to inhibit the infection of hiv virus trying to learn from what happen in nature in esn we used a particular immunization procedure. we immunized mice with autologous cells expressing gp120, reacted with the 2 external domains of soluble human cd4, in the absence of the target cells expressing the co-receptor ccr5. the latter is the membrane molecule, which allows the complete reshuffling of the epitopic make-up of the cd4-gp120 complex and trigger the membrane fusion between effector (gp120 expressing) cells and target (ccr5 expressing) cells. thus, in the absence of ccr5 we specifically enriched our immunogens with "frozen" conformational intermediates, that are presumably transiently exposed on the cell membrane during hiv-1 infections. a conventional protocol for the generation of monoclonal antibodies was used. db-81 (igg1, x), one of the anti-cd4 antibodies obtained, recognized preferentially cd4 complexed to gp120, as compared to cd4 alone, not competed for the gp120 binding site on cd4 and was specific for the second extracellular domain of cd4. g. röder 1 , l. geironson 2 , a. darabi 3 , m. harndahl 1 , c. schafer-nielsen 4 , k. skjödt 5 , s. buus 1 , k. paulsson 2 1 copenhagen university, institute of international health, immunology and microbiology, department of experimental immunology, copenhagen, denmark, 2 lund university, immunology bmc d14, lund, sweden, 3 lund university, rausing laboratory, division of neurosurgery, department of clinical sciences, lund, sweden, 4 schafer-n, copenhagen, denmark, 5 department of immunology & microbiology, university of southern denmark, odense, denmarkcytotoxic t-lymphocytes become activated by binding to mhc-i molecules presenting antigenic peptides. the loading of peptides onto mhc-i takes place in the er and involves different chaperones and enzymes. tapasin binds mhc-i molecules, integrates them into peptide-loading complexes, and assures that only 'optimal peptides' are bound to surface exported mhc-i molecules. how tapasin exerts this quality control, and the criteria for being an optimal peptide, are still unknown. here, we have generated the first 87 n-terminal amino acids of human tapasin, tpn , and shown that this fragment of tapasin facilitates peptide dependent folding of hla-a*0201. to further investigate the properties of tpn and tapasin, we generated multiple mouse monoclonal antibodies towards tpn and wildtype human tapasin. one clone, atpn 1-87 /80 , was found to be specific for natural human tapasin and stained cellular er localized tapasin. using peptide chip technology, the epitope of atpn /80 was demonstrated to be located on tapasin [40] [41] [42] [43] [44] , which recently was shown to be a surface-exposed loop of the tapasin structure. together, these results demonstrate that, the first 87 n-terminal amino acids of tapasin are able to facilitate peptide-binding to mhc-i, and as well, this fragment can be recombinantly expressed in e.coli and fold into a structure, which at least partially, resembles that of wild-type human tapasin. we speculate that this region of tapasin might support empty, open and receptive mhc-i peptide-binding clefts effectively allowing an otherwise inherently unstable molecule to exchange peptide; i. e. this tapasin region might be essential for enabling peptide editing. a objectives: antigen processing and presentation through hla class i molecules is critical for an effective destruction of infected or transformed cells by cd8+ t lymphocytes. different intracellular routes governing the processing of endogenous and exogenous antigens have been described. we show here a strategy to introduce epitopes inside the cells for a productive cross-presentation to ctls. methods: to produce genetic in-frame tat fusion proteins, dna sequence encoding for the amino acid region 301-498 of the influenza a virus nucleoprotein (np) was inserted into the expression vector ptat-ha. starting from tatnpflu recombinant protein we produce hybrid proteins, in which the hla-b*2705-restricted np-flu epitope (aa 383-391) was replaced by hla-b27 or hla-a2-restricted epitopes of ebv and hcv, respectively. cross-presentation was evaluated according to the standard 51 cr release assay and through the ifn-g production. results: using hla-b27 or hla-a2 restricted viral epitopes we show that the two molecules cross-present the epitopes following two different pathways of processing: the hla-b27 molecules follow a proteasome-independent pathway which is active in different cell types, whereas the hla-a2 molecules present the epitopes in a classical proteasome-dependent pathway performed by dcs. furthermore, different hla-a2 restricted epitopes can be inserted in tandem and presented to the specific ctls without interfering each other. the data reported here offer new insights on how a same construct containing multiple epitopes from different viral or oncogenic proteins could be designed for vaccinal strategies. these findings also enlighten hla-b27 as a remarkable hla-class i molecule that, differently from hla-a2, can present peptides through additional, unconventional antigen presenting routes. this could concur to an imbalance of the immunological properties of the hla-b27 molecules leading to a more effective response towards viral as well as self -antigens. objectives: although cytotoxic t cells (ctl) in human immunodeficiency virus (hiv-1)-infected individuals can potentially target multiple virus epitopes, the same few are repeatedly recognized. ctl play a key role in limiting viral replication in infections caused by e. g. epstein-barr virus, cytomegalovirus, hepatitis c virus and hiv1. consistent patterns of immunodominant and subdominant ctl-responses have been found between individuals with the same hla-alleles in both acute and chronic infection. as the ctl-response frequency in a population closely correlates with its relative magnitude in an infected individual, the terms immunodominance/subdominance have been used in both contexts. however, the factors determining these ctl-response hierarchies are largely unknown. while structural differences between peptide-hla class i complexes may be important for tcr-repertoire selection and clonal expansion, it is less obvious how they impact ctl-response hierarchy formation and timing. other factors may also contribute, e. g. epitope abundance at the cell surface. methods: antigen processing efficiency of ctl epitopes from the p17-gag and p24 region was determined in vitro. 25mer peptides were digested with i20s and c20 proteasomes and the fragments identified by mass spectrometry. for epitope precursor peptides generated by the proteasome, we then determined tap affinity, trimming by eraap and hla-binding affinities and analyzed patient responses by elispot. results: we show that ctl-immunodominance in regions of hiv-1 p17-and p24-gag correlates with epitope abundance, which is influenced strongly by proteasomal digestion profiles, transporter-associated-with-antigen (tap) affinity and endoplasmatic reticulum aminopeptidase (eraap)-mediated trimming, and moderately by hla affinity. proteasomal cleavage-preferences were affected by flanking and intra-epitope ctl-escape mutations and could modulate the number and length of peptide-epitopes, thereby affecting t cell response avidity and clonality. conclusion: our analyses reveal that antigen processing plays a pivotal role in determining ctl-response hierarchies, that viral evolution may modify cleavage patterns, and suggest strategies for in vitro optimization of ctl-epitope-based vaccines. t. f. gregers 1 , g. koster 1 , o. landsverk 1 , f. skjeldal 1 , o. bakke 1 1 university of oslo, molecular biosciences, oslo, norway mhc ii is synthesized and assembles in the er together with invariant chain (ii). ii facilitates mhc ii assembly followed by transport to the mhc ii loading compartment (miic) where peptide loading occurs. miic is multivesicular late endosomal compartments resembling conventional multivesicular bodies (mvbs) found in all cells. it is not known whether the biogenesis of miics is regulated by the same mechanisms as formation of mvbs. expression of ii induces the formation of enlarged endosomes and we have previously shown that ii modulates antigen processing and presentation. we have suggested that ii itself can act as a tethering factor involved in fusion of ii containing endosomes, and our main question is whether ii can regulate the formation of an endosomal pathway dedicated for antigen processing and mhc ii loading.in order to investigate this we use cell lines expressing ii controlled by an inducible promoter, thus being able to control the ii expression level and thereby the endosomal size. live imaging and high through put microscopy of ii expressing cells treated with inhibitors and/or specific sirnas have revealed that ii induced endosomal fusion is independent on type iii pi3 kinases and thus ptdins(3)p. this is in contrast to conventional endosomal fusion and mvb formation. thus other factors might be important for miic biogenesis. by using small rnai libraries targeting proteins known to be involved in endosomal pathways and microscope based screening we aim to identify factors that are able to knock out the formation of enlarged endosomes in ii expressing cells, and thus potentially identify molecules defining an antigen presenting cell. m. bouvier 1 , l. visvabharathy 1 , j. fu 1 1 university of illinois at chicago, microbiology and immunology, chicago, united statesobjectives: adenoviruses (ads) cause persistent infections. the e3-19k protein from ad targets class i mhc molecules for retention in the endoplasmic reticulum (er), thereby preventing the cell-surface presentation of viral peptides. this escape from immune surveillance allows ads to freely replicate in host cells. the molecular mechanism of e3-19k-mediated class i retention is mostly undefined. it is clear that further characterization of this mechanism is important to understand the susceptibility of the class i antigen presentation pathway to immunomodulatory proteins and to elucidate the molecular basis of ad pathogenicity. we used biophysical and cell-based approaches to examine interaction between ad type 2 e3-19k and class i molecules.results: we showed that e3-19k associates with immature (peptide-deficient) and mature (peptide-filled) hla-a11 molecules, with the mature form being more tightly associated. we also provided evidence that e3-19k does not compete with the class i assembly proteins for binding onto class i molecules. importantly, immature class i molecules sequestered by e3-19k can still bind peptides. together, these results suggest that ads have evolved to interfere with the early and late stages of the class i antigen presentation pathway. evidence was also provided that e3-19k displays an allele-and locus-specificity towards class i molecules with high-density lipoprotein (hdl) reduces the risk for atherosclerotic cardiovascular disease by promotion of cholesterol efflux from macrophage foam cells and by antioxidative as well as anti-inflammatory properties. recent data indicate that qualitative changes of hdl including oxidative modifications and alterations of the protein cargo of hdl may alter its biological activity. here we analyzed the anti-inflammatory potential of hdl and compared it with hdl obtained from patients with end-stage renal disease (esrd), which are characterized by a proinflammatory state and an associated significantly increased cardiovascular mortality. we demonstrate that freshly isolated, but not oxidized hdl from healthy individuals exerts profound anti-inflammatory properties on professional antigenpresenting cells (apc) such as monocytes and dendritic cells, which are regarded as the most potent apc. production of typical proinflammatory cytokines (il-12, il-6, tnf-a) were significantly suppressed by hdl after stimulation of monocytes or dendritic cells with toll-receptor ligands 2 and 4, but also with the t-celldependent stimulus cd40l (cd154) indicating an immunomodulatory effect independent of agonist neutralization by hdl. moreover, surface expression of crucial activation and costimulatory molecules like cd40, cd83, and cd86 was inhibited by freshly isolated, but not oxidized hdl. the negative regulatory effect of hdl on cytokines and surface receptors occurred at the transcription level, while hdl did not modulate the activity of the major inflammatory transcription factor nf-kb or the map kinases p38 and erk-1/2. strikingly, hdl from esrd patients not only failed to block, but rather promoted proinflammatory cytokine production and apc activation. these data identify hdl as a novel potent anti-inflammatory regulator of professional apc, which may help to dampen excessive inflammatory responses of the innate immune system. conversely, qualitative changes of hdl leading to a loss of its anti-inflammatory function might contribute to a proinflammatory state that is linked with excessive cardiovascular mortality in esrd patients. objectives: cd4+ t cell abnormalities may play a role in the autoimmune pathogenesis of churg strauss syndrome (css). on one side, th2 (il-4+) cells may sustain autoantibody formation and eosinophilia, which are hallmarks of css. on the other, th1 (ifn-g+) cells could participate in vessel wall damage and granuloma formation. in order to define this th1 / th2 balance and to identify potential t cell target antigens (ags), we analyzed circulating cd4+ t cell responses to polyclonal stimuli and to myeloperoxidase (mpo) in css and healthy subjects. methods: ifn-g and il-4 expression in peripheral blood cd3+cd4+ lymphocytes were measured in 9 ccs patients and 7 healthy subjects (hs) upon polyclonal stimulation, both by intracellular staining and by elisa. mpo-driven il-4/ifn-g production was assessed by elispot on t cells co-coltured with autologous dendritic cells, stimulated either with heat-inactivated mpo, negative control protein or hexavalent vaccine (positive control recall ags). results: upon polyclonal stimulation, higher il-4 and lower ifn-g intracellular expression were detected in cd4+ t cells from css patients, as compared to hs (il-4: 1.3±0.3 % vs. 0.50±0.21 %, p x 0.05; ifn-g: 14.2±4.5 % vs. 27.0±4.8 %, p x 0.025). similar results were obtained by elisa (il-4: 0.39±0.16 vs. 0.30±0.07 pg/ml, p x 0.05; ifn-g: 31.0±14.3 vs. 79.0±15.0 pg/ml, p x 0.05). elispot counts of hexavalent vaccine-stimulated cd4+ cells were positive for il-4 in 4/5 (80 %) css patients and in 5/5 (100 %) hs, and for ifn-g in 2/9 (22 %) css patients and 7/7 (100 %) hs. mpo stimulation determined significant ifn-g release in 5/8 (62 %) css patients, but not in hs (0/5) no il-4 response to mpo in both groups was observed. conclusion: polyclonally or recall ag-stimulated cd4+ cells from css patients show a th2-polarized cytokine profile. mpo is here first identified as a css-related ag targeted by cd4+ t cells, and responses towards it are instead th1-polarized. these data unfold one molecular target and possible pathogenic mechanisms of cd4+ t cells in css. a. voigt 1 , e. opitz 1 , k. savvatis 2 , k. klingel 3 , k. stangl 1 , u. kuckelkorn 1 , p.-m. kloetzel 1 1 charité -universitätsmedizin berlin campus mitte, berlin, germany, 2 charite -campus benjamin franklin, berlin, germany, 3 universität tübingen, tübingen, germanymurine models of coxsackievirus b3 (cvb3)-induced myocarditis mimic the divergent human disease course of cardiotropic viral infection. immunoproteasomes (ip) are crucial in the modulation of adaptive immune responses, in the maintenance of protein homeostasis and in the preservation of cell viability under stress conditions. our previous work has established that ip expression in the infected myocardium is linked to a strong enhancement of viral epitope generation.here, we investigated the impact of ip function in enterovirus myocarditis. mice, which are deficient in immunosubunit lmp2 of the stress-induced ip, were infected with 1x10e5 pfu cvb3 nancy strain. in concurrence to wt littermates, we observed a pronounced up-regulation of cardiac ip subunit lmp7 as early as day 4 p. i. in lmp2-deficient mice. however, lmp2-deficiency was linked to less severe myocarditis at day 8 p. i. (he stain of cardiac tissue sections: wt 1.95 ± 0.20 vs. lmp2-deficiency 0.71 ± 0.06 (grade of myocarditis; scale 0-4; p x 0.001). whereas the cardiac output (co) was reduced in wt littermates in enterovirusmyocarditis (p x 0.05), there was no difference in lmp2-deficient mice in comparison to sham-treated mice. maximal left ventricular pressure and dpdt max were impaired in acute myocarditis in wt littermates. in contrast, systolic function was not affected by cvb3 infection in lmp2-deficient mice. likewise, diastolic function was preserved in lmp2-deficient mice upon enterovirus infection. our findings of less severe myocarditis in lmp2-deficient mice were associated with tremendously reduced viral load in the myocardium of this strain.in conclusion, this study suggests an impact of lmp2-immunosubunit function in regulatory processes of viral replication. absence of lmp2 confers host protection in enterovirus myocarditis. h. w. liao 1 , j. xu 1 , j.q. huang 1 1 sun yat-sen university, guangzhou, chinathe characteristic of the dengue hemorrhagic fever/dengue shock syndrome (dhf/dss) is hematologic abnormality, which results from multiple factors including thrombocytopenia, coagulopathy and vasculopathy. the pathogenesis of endothelial dysfunction associted with vascular leakage syndrome however remains unknown. in this work, we showed that dengue virus serotype 2 (den-2) strain induced apoptosis in human umbilical vein endothelial cells (huvecs). additionally, fas expression was increased on infected huvecs. trailr1 and tnfr1-2 were constantly very low whereas trailr2-4 decreased after den-2 infection. fasl was expressed at similar levels on huvecs throughout den-2 infection. the apoptotic rates in huvecs were decreased upon addition of caspase family inhibitors and activated caspase 8, caspase 3 were also observed by western blot after by den-2 infection. there were no significant changes of no in our study. we thus proposed that the fas/fasl pathway might be involved in apoptosis induced by dengue virus in vascular endothelial cells in vitro. dermatolymphangioadenitis is a common complication of interruption of afferent lymphatics by cancer surgery combined with partial lymphadenectomy. it seems that skin microbes normally penetrating epidermis during hand work or walking are retained in the skin and subcutis because of lack of lymph drainage and evoke host reaction. aim. to study lymph node cellular reaction to bacterial antigens before and after ligation of afferent lymphatics. materials & methods. group i. s. epidermidis was injected daily for 7 days into wis rat paw web tissue in saline containing 7.5x10 7 cells. group ii. s.epidermis was injected as in group 1 after ligation of lymphatics below the popliteal lymph node. nodes were isolated on day 8.they were weighed, the cell number was counted and cells were stained with mabs for immunohistochemical analysis. immunohistochemical pictures were analyzed by microimage program. results. group i. skin contained some mhcii cells. the popliteal lymph nodes became enlarged on the bacteria injected side. there was an increase in lymph node weight and cell concentration per g of tissue, compared to controls by factors 2,23 and 3,91 respectively (p x 0.05). immunohistochemical pictures showed increase in percentage of ox62 (migrating dendritic cell), mhc ii, his48 (granulocytes), ox7 (stem cells) and cd54 (icam i) subsets in the subcapsular , follicle, paracortex and medullary areas. group ii. after ligation of afferent lymphatics the weight of nodes was not significantly increased. skin showed presence of multiple mhc ii, ed1 (macrophages) and ox62 cells. popliteal lymph nodes contained evidently less of ox62, his48 and mhcii cells than in group i (p x 0.05). summary & conclusions. afferent lymphatics transport microbial cells and/or microbes phagocytized by dendritic cells and macrophages to the regional node. local skin reaction is limited, whereas lymph nodes reveal acute reaction with mobilization of granulocytes from blood perfusing nodes. interruption of lymphatics saves nodes but skin reaction is strong and long-lasting. these observations seem to explain why damage to lymphatics during mastectomy or groin dissection is followed by recurrent attacks of skin inflammation. omega-3 fatty acids, and in particular docosapentaenoic acid (dha) and eicosapentaenoic acid (epa) from fish origin, have recently emerged as nutrients capable of modulating the expression of genes involved in inflammation and atherosclerosis and thus reduce the risk for cardiovascular events. our presentation focuses on the role of omega-3 fatty acids in the prevention and treatment of cardiovascular disease. it is based on reviewing and processing data obtained by search of scientific and medical databases. search terms used were: atheroma, atherosclerosis, cardiovascular disease (cad) ,coronary disease, antiinflammatory drugs, omega-3 fatty acids, epa, dha. we also searched epidemiological research web sites and screened the results of numerous controlled clinical trials which monitored the effects of omega -3 fatty acids consumption. the results indicate that omega-3 fatty acids supplementation is associated with a significant cardioprotection effect on both healthy individuals and patients with an established cardiovascular disease. omega-3 fatty acids appear to work by decreasing endothelial responsiveness to pro-inflammatory and pro-atherogenic stimuli, affecting molecular events not targeted by other drugs thus allowing their use as complementary treatments for the already implemented pharmacological treatments in inflammatory diseases. combined therapy with omega-3 fatty acids and statins shows a synergistic effect. methods: on ultracentrifugation of serum at density 1.24 and 202,000g, top 20 % layer contained lipoproteins only and 20-50 % layer contained lipoproteins as well as immunoglobulins. the bottom layer was shown to contain immune complexes (ic) by binding to coated anti apo(a) and detection with peroxidase labelled anti human immunoglobulins.both these forms of lp(a) were western blotted and probed with jacalin-hrp, anti-gal-hrp and anti apo(a)-hrp. anti-gal was prepared by affinity chromatography on guar galactomannan and complexed with lp(a) in vitro. ic formation by lp(a) was measured in terms of reduction in response in a new elisa for lp(a) involving addition of lp(a) sample to plate-coated jacalin, followed by anti-apo(a)-hrp detection. ic formation was also shown by migration of lp(a) from free lipid layer to ic layer below in ultracentrifugation. results: anti-gal and lp(a) could be liberated from precipitated ic using specific sugar. immune complexed lp(a) in serum was found to be more o-glycosylated, larger in size and binding more anti-gal than lp(a) in free form in western blots. while ic formations within homologous free anti-gal-free lp(a) pairs were few, those within heterologous pairs were more rampant. conclusions: lp(a) is a risk factor in vascular disorders including atherosclerosis, aneurysm, stroke and peripheral vascular diseases and is a component of atherosclerotic plaques, though mechanism of its uptake remains unclear. anti-gal comprising 1 % of serum igg is rich in igg capable of complement fixation and macrophage mobilisation. present results offer a viable mechanism of lp(a)-mediated immune injury to vessel walls leading to vascular damage. even though no receptors have been detected for lp(a), unlike for ldl, the present results may explain the internalization of lp(a) in the form of lp(a) immune complexes by macrophages since the latter can phagocytose ic. extended specificity of the a-galactoside-specific anti-gal for t-antigen in lp(a) is akin to that observed in jacalin, pea nut agglutinin and galectin-1. objective: the aim of this study was to determine if the combination of two genetic alterations, one affecting cell cycle regulation, such as the e2f2 mutation, and other affecting b cell apoptosis control, such as bcl-2 over-expression, can induce the development of ais. methods: mice: mice with both genetic abnormalities were generated in a non-susceptible c57bl/6 (b6) genetic background. e2f2-/-bcl-2tg were obtained backcrossing e2f2-/-and e2f2+/+hbcl-2tg mice. e2f2-/-bcl-2tg, e2f2-/-, e2f2+/+hbcl-2tg and control mice (e2f2+/+) were followed up to 18mo-old. serologic studies: serum samples obtained at 3, 9 and 15 month of age were test for igg and iga ana and anti-dsdna by elisa. histopathologic studies: kidney paraffin sections of 3, 9 and 15 mo-old mice were stained with hematoxylin-eosin (h&e) and masson's trichrome to identify histological changes. immunecomplex deposits were studied by direct immunofluorescence on kidney using fluoresceinated goat anti-mouse igg, igm and iga. to evaluate b cell homeostasis, absolute number of b cell in blood, primary and secondary lymph organs were assessed by flow cytometry. in vitro proliferation was measured with [h3]-thymidine and brdu was used to assess in vivo proliferation capacity of immature b cells. results: overexpression of hbcl-2tg in b lymphocytes of e2f2-/-mice induced the production of high titres of igg and iga ana and anti-dsdna, together with the development of a glomerulonephritis characterized by a moderated mesangial proliferation, mesangial immunecomplex deposits, mainly of the iga isotype, and the presence of tubular casts and lymphoid infiltrates with the presence of glomerular deposits. e2f2-/-bcl-2tg mice showed an altered b cell homeostasis as demonstrated in proliferation and apoptosis studies. e2f2-/-mice showed neither autoantibodies nor nephropathy. this study demonstrates that the isolated deficiency of e2f2 or the overexpression of a bcl-2 tg in the b6 genetic background do not induce an ais. when combined both genetic alterations, involving deregulation of cellular proliferation and survival affect lymphocyte homeostasis, induce a mild ais with overproduction of iga autoantibodies. an alteration in the b cell compartment, but not in the t cell compartment, seems to be underlying the syndrome described in the present work. in different mouse models of the autoimmune disease systemic lupus erythematosus (sle) loss of toll-like receptor 9 (tlr9) abolishes the generation of antinucleosome igg2a and igg2b autoantibodies but exacerbates lupus disease. however, the tlr9-dependent tolerance mechanism is unknown. here we show that loss of tlr9 in b cells of lupus prone mice prevents the generation of protective t cell-dependent self-reactive igm and thereby enhances the development of th1 and th17 t cells. transfer of a synthesized monoclonal polyreactive igm to tlr9 deficient lupus prone mice inhibits t cell activation and abolishes development of lupus disease. thus, these results document a protective tlr9-dependent tolerance mechanism in b cells that induces the generation of self-reactive igm to prevent autoimmunity. cloning and production of polyreactive or antigen-specific igm might therefore be a powerful tool to treat autoimmunity. objectives: to investigate cytokine and autoantibody levels in serum from patients with primary sjögren's syndrome (pss), and to determine possible associations with focal mononuclear cell infiltrates, lymphoid organization, and age at the time of biopsy. methods: minor salivary gland tissue was obtained from a group of patients fulfilling the revised eu-us criteria for pss (n=115) (vitali et al. 2002) . ninety-seven of 115 (84 %) patients had focal mononuclear cell infiltrates corresponding to focus score (fs) g 1 (fs+), while biopsies from 18/115 (16 %) patients lacked characteristic focal mononuclear cell infiltrates (fs-). germinal center (gc)-like lesions were determined in 27/115 (23 %) minor salivary gland biopsies. serum samples were used for cytokine and autoantibody evaluations. the mean level of unstimulated whole saliva was significantly lower in the fs+ patients compared with the fs-patients, and in the gc+ patients compared with the gc-patients (p x 0.05). interleukin (il) 17, il-1ra, il-1beta, il-12p40, il-15, macrophage inflammatory protein (mip) 1alpha, mip-1beta, eotaxin, interferon (ifn) alpha, and il-4 levels were significantly increased in the gc+ patients (n=27) compared with the gc-patients (n=70). in addition, minor differences in cytokine levels were found when comparing age groups. degenerative changes such as atrophy/fibrosis and fatty cell infiltration observed in the minor salivary glands of patients with pss may represent "burned out" inflammation. no significant differences were found in autoantibody levels in either of the groups, nor when comparing cytokine levels in the fs-and fs+ subgroups. the reduced salivary flow observed in gc+ patients may be influenced by the elevated levels of il-4 found in these patients (gao et al. 2006) . increased titers of th17-associated cytokines, il-17, il-1beta and the il-23 subunit il-12p40, may indicate a higher activity of these cells in gc+ patients (nguyen et al. 2008) . differences in cytokine levels may be utilized when sub-grouping the ss patients into disease phases and may consequently have implications for treatment. objectives: c-reactive protein (crp) is an acute phase protein, produced by hepatocytes in response to the pro-inflammatory cytokine il-6. the rapid increase of crp during inflammation makes it an excellent inflammatory marker, but for unknown reasons, blood levels of crp typically remain low in disease flares of systemic lupus erythematosus (sle), a systemic autoimmune disease. another feature of sle is the so called 'interferon (ifn) signature' which implies high levels of ifn-alpha and/or up-regulation of ifn-alpha related genes. ifn-alpha has a wide spectrum of immunomodulatory functions but is mainly known for its antiviral and anti-tumour effects. since high levels of ifn-alpha coexist with a muted crp response in sle disease flares and in viral infections, we hypothesized that ifnalpha inhibits crp synthesis. methods: crp promoter activity was studied in a crp-promoter and luciferase reporter transfected human hepatoma cell-line, hepg2. production of the acute phase protein serum amyloid a (saa) and the negative acute phase protein transferrin were analysed by elisa as reference. results: the crp-promoter activity was inhibited by all ifn-alpha subtypes. mixes of type i ifns that were induced by sle-like immune complexes or virus also inhibited the crp-promoter activity. virus-induced purified leukocyte ifn-alpha had the most prominent inhibitory effect ( g 50 %) on crp promoter activity. saa synthesis was inhibited by ifn-alpha in a similar fashion as for crp promoter activity, whereas transferrin was unaffected. conclusion: our data indicates that ifn-alpha is an inhibitor of crp-promoter activity. we suggest that this could explain the muted crp response seen in sle disease exacerbations. further, i may contribute to differences in crp response between viral and bacterial infections. background: b cell activating factor of the tnf family (baff) is an essential b cell survival and maturation cytokine. mice overexpressing baff (baff tg mice) develop lupuslike autoimmunity, b cell hyperplasia, and lymphomas. autoimmunity in these mice involves proinflammatory autoantibodies driving nephritis and sialadenitis, and was previously found to be t cell-independent (ti) and myd88-dependent. this suggested the involvement of transmembrane activator and caml interactor (taci), which is a receptor for baff that is essential for ti immune responses and is upregulated by myd88-dependent tlr activation. we assessed the role of taci in baff-driven ti autoimmunity. methods: we tested the importance of taci in ti autoimmunity by generating baff tg bone marrow (bm) chimeras reconstituted with taci -/or taci +/+ bm then comparing their disease severity by flow cytometry, autoantibody elisa, immunofluorescence microscopy for ig deposition. results: as expected, baff tg chimeras reconstituted with taci +/+ bm produced high levels of circulating proinflammatory autoantibody isotypes and rheumatoid factors (rhf), and ig deposition in the kidneys and salivary glands was observed. by contrast, baff tg chimeras reconstituted with taci -/-bm had greatly ameliorated levels of circulating proinflammatory autoantibodies, rhf, and ig deposition. b cell hyperplasia was greater in taci -/-1 baff tg chimeras. defects in the regulation of apoptosis contribute to the pathogenesis of human systemic lupus erythematodes (sle). autoantigens not being properly removed and thus exposed to the immune systeme might lead to the emergence of autoantibodies. physiologically apoptotic cells are removed without initiation of an inflammatory immune response and myeloid dendritic cells are believed to actively tolerize t-cells after phagocytosis of apoptotic material. these processes of silent apoptotic cell clearance seem to be disturbed in sle patients. a characteristic of apoptotic cell death is the shedding of membrane coated vesicles from the cellular surface (apoptotic cell blebbing). these microparticles have been recognized as mediators of intercellular communication. therefore, we were interested whether apoptotic cell derived microparticles can influence the function of monocyte-derived dendritic cells and whether those interactions might play a role in the pathogenesis of human sle. we observed an engulfment of microparticles by monocyte-derived dendritic cells. further, apoptotic cell-derived microparticles stimulated differentiation of immature dendritic towards a mature phenotype. however, microparticles caused a remarkable downregulation of mhc class ii molecules. further, we observed only a minor release of proinflammatory cytokines from monocyte-derived dendritic cells pulsed by membrane microparticles when compared to lps stimulated dendritic cells. finally, these dendritic cells pulsed by membrane microparticles did not cause a significant t-cell expansion. interestingly, dendritic cells obtained from sle patients showed significant variations in phenotype and cytokine secretion compared to normal healthy donor cells with absence of the mhc class ii downregulation and a higher constitutive secretion of il-8. objectives: increased levels of il-18, an innate and inflammatory cytokine of the il-1 family, can be detected in serum and organs of human autoimmune pathologies, as well as in autoimmune animal models. here, expression of il-18 and other genes of the il-1/il-1r families was examined in human systemic lupus erythematosus (sle) and in mrl lpr/lpr mice, which develop a chronic progressive lupus-like syndrome. methods: serum, urine, and monocytes were collected from 48 patients and 32 healthy controls. lymphoid (lymph-nodes, spleen, thymus, peyer's patches) and non-lymphoid organs (kidney, lung, liver, salivary and lacrimal glands) were collected from mrl +/+ and lpr/lpr mice of different ages. il-18 and il-18bp were measured by elisa. gene expression was assessed by real-time pcr and expressed relative to b-actin. results: in sle, serum and urine levels of total and free il-18 are higher than in controls. serum il-18 correlates with disease activity and decreases upon remission. monocyte expression of the receptor il-18rb is increased and correlates with disease severity, while expression of tir8/sigirr (a down-regulatory receptor of the il-1r/il-18r family) is reduced. in mrl lpr/lpr mice, expression of il-18, caspase-1 and il-18rb genes precedes disease onset in lymph-nodes. in other organs, changes in il-1-related genes (il-33 and tigirr-1 up-regulation, tir8/sigirr down-regulation) occur after disease onset. free il-18 levels are abnormally high in lpr/lpr lymph-nodes before disease onset, while in other organs the increase occurs with disease. conclusions: free il-18 levels correlate with autoimmune lupus both in mice and humans. free il-18 may be pathogenic in murine lymphadenopathy, while is a disease correlate in lpr/lpr and a severity correlate in sle. both in human and mouse syndromes, upregulation of il-18rb is a marker of pathology, suggesting increased il-18-dependent activation. both in mouse organs and human monocytes, tir8/sigirr expression decreases with disease, suggesting impaired control of il-18r activation. thus, il-18 may be involved in autoimmune lupus pathology, and il-18-related molecules can be both original diagnostic markers and novel therapeutic targets in autoimmunity. in this study we compared the epitope specificity of anti-topo i autoantibodies present in sera of dcssc, lcssc and sle patients. we have constructed an antigen fragment library displayed on bacteriophage lambda and screened this library with igg purified from patients' sera. regions of topo i selected from the library were expressed as recombinant fusion proteins and were tested with elisa and western blot. we unexpectedly found that antibodies against a fragment of topo i (fragment f4 (amino acid (aa) 451-593) could be detected in sera of healthy individuals and patients with inflammatory rheumatic diseases other than ssc and sle. using sera of dcssc, lcssc and sle patients we showed that the pattern of recognized epitopes is different between these patient groups. fragment f4 was recognized by all patients. fragment f1 (aa 5-30) was recognized by 9 of 34 dcssc patients. fragment f8 (aa 350-400) was recognized by 4 of 8 sle patients. analysis of clinical data revealed a significant difference between the f1 negative and f1 positive groups of ssc patients in age and in the duration of the disease. according to our results the newly identified fragments f1 and f8 could represent characteristic epitopes for dcssc and sle, respectively. background: previous studies demonstrated that depletion of regulatory t cells (tregs) results in autoimmunity in mice while their adoptive transfer prevents autoimmune diseases. studies performed by us and others showed that in human connective-tissue diseases a reduced number of tregs exists and this abnormality seems to be correlated with autoantibodies production and disease activity. objectives: based on these observations and the fact that rapamycin (rapa) has the ability to expand tregs and to induce anergy, we proposed to study the possibility to restore peripheral tolerance of cd4 + t cells isolated from systemic lupus erythemaosus (sle) patients by ex vivo expansion of tregs. methods: pbmcs or peripheral cd4 + t cells from sle patients were cultured in the presence of specific stimulation with or without rapa and ril-2. by facs the initial percent of tregs and after expansion protocol were determined. in order to verify the suppressive capacity of expanded tregs, cd4 + t cells enriched in tregs were co-cultured with activated cd4 + effector t cells (teff) stained with cfse, after one week teff cells proliferation was measured by facs. additionally, cytokine and igg release in cell culture media were analyzed by multiplex and elisa, respectively. expanded cd4 + t cells anergy was also evaluated based on cbl-b, grail and foxp3 mrna by realtime rt-pcr. results: in vitro expansion of tregs was more efficient when the starting cells were cd4 + t cells. the presence of rapa during expansion protocol significantly increased the number of tregs. sle tregs cells expanded in vitro in the presence of rapa had the capacity to suppress proliferation of both sle and hd teff cells. rapa inhibits igg secretion in the pbmcs culture, inhibition dependent on tregs level. rapa during tregs expansion protocol stimulated some type of cytokines while suppressed others. rapa had the capacity to re-establish sle cd4 + t cells anergy by induction of anergy genes, grail and cbl-b. conclusions: our data show that the above described protocol permits ex vivo tregs expansion and that suppressive capacity of the expanded tregs depends on the source of both tregs and teff cells. in this study, we look for a more specific approach to remove b-1 cells through targeting p110d by shrnas strategy. methods: we used the drugs, ly294002 and wortmannin, pan-specific inhibitors against pi3ks. then we designed shrnas carried by the lentiviral system and validated that several segments of them can sufficiently knock down the expression of p110d. we then introduced either pan-specific inhibitors against all pi3ks or p110d-targeting shrnas into an sle-prone animal model, nzb/w f1 mice, for therapeutic purposes. the results suggested that pi3ks are not only important for the development of b-1 cells but also remain essential to maintain their population after birth. shrnas carried on lentiviral systems were designed to knock down the expression of p110d. either pan-specific inhibitors against pi3ks or p110d-targeting shrnas were introduced into the sle-prone animal model, nzb/w f1 mice. one inhibitor, ly294002, and shrnas delivered by low dose of lentivirus exhibited certain potential to retard the rising of anti-dna auto-antibodies and prolonged the life span. conclusions: our findings are promising for developing treatments for sle. moreover, knowing pi3ks are critical for the maintenance of b-1 cell populations might shed light on future treating other diseases associated with b-1 cells, such as certain melanoma, lymphoma, or leukemia. a. m. zaghlool 1 , m. alarcón-riquelme 1 , s. kozyrev 1 1 institution of genetic and pathology, uppsala university, uppsala, swedenrecently, we discovered that the bank1 gene, which plays a role in b cells activation pathway, is associated with systemic lupus erythematosus through a nonsynonymous substitution g/a (rs10516487, r61h). we identified that bank 1 gene expresses two alternatively spliced isoforms, a full-length, and a shorter isoform that lacks exon 2 (delta 2). the two isoforms were detected differently in susceptible lupus patients depending on the presence of a risk haplotype. to address the question of how bank1 is spliced and what are the signals governing the expression of each isoform, minigenes with different genetic variants were constructed and the expression of the bank1 isoforms were tested in vitro. qpcr analysis revealed that, another t/c snp (rs17266594), which is in complete ld with r61h snp and located in the putative branch point, has a strong affect on the isoforms expression levels. deletion of a polypyrimidine (py) stretch downstream of the skipped exon produced a dramatic decrease in the full-length expression levels, probably due to the loss of the binding site for protein tia1, which bind to t objectives: cerebral ischemia is the most common presentation of antiphospholipid syndrome (aps), but several other neuropsychiatric features, including chronic headache, dementia, cognitive dysfunction, psychosis, depression, transverse myelitis, multiple sclerosis-like disease, chorea, and seizures have been associated with the presence of antiphospholipid antibodies (apl). we report the case of a subject with atypical movement disorder related to aps successfully treated with oral anticoagulation agents. case report: a 57-year-old woman with a previous history of recurrent foetal losses was admitted to our hospital due to cognitive dysfunction and headache. she presented involuntary movements that were characterized as mioclonic seizures and tonic spasms lasting from few minutes to several hours, followed by bilateral arrhythmic rapid purposeless jerks of the legs. mild executive dysfunction was observed. her deep tendon reflexes were symmetric and normal. pathological reflexes were absent. biochemical analysis, renal, hepatic and thyroid functions were preserved, prothrombin time and partial thromboplastin time were all normal. the immunoglobulin g (igg) isotope of anticardiolipin antibody (acl) was elevated, whereas igm isotype and anti-2gpi antibodies were undetectable. the lupus anticoagulant (la) was negative such as antinuclear antibodies (ana). no evidence of epilepsy was revealed from electroencephalogram or signs of denervation from electromyographic studies. brain magnetic resonance imaging (mri) showed multifocal encephalomalacia probably linked to previous cerebrovascular accidents. she was diagnosed as having an atypical neurologic manifestation probably linked to aps. she was thus discharged with a low-molecular-weight heparin therapy subsequently changed to mild oral anticoagulation . the therapy leads to a late, gradual improvement of symptoms that persisted at the last 1year follow-up evaluation. conclusions: antiphospholipid syndrome may constitute a rare but treatable cause of atypical neurologic manifestation such as myoclonic movements. due to the possibility of an effective treatment, it is important to rule out this diagnosis, moreover in women with other associated features of aps (foetal losses, livedo reticularis, thrombosis). a.-s. korganow 1 1 cnrs 9021, strasbourg, france b lymphocytes from patients with systemic lupus erythematosus are hyperactive and produce autoantibodies. several b cell phenotypic characteristics have been reported, as the expansion of activated populations, and of a newly investigated memory compartment. a few genes have been suggested to be implicated. one of the thing that makes these results difficult to interpret is the heterogeneity of the lupic disease, and sometimes the analysis all together of quiescent, paucisymptomatic and highly symptomatic patients, treated with immunosuppressors or untreated.we made the postulat that "intrinsic" abnormalities of b cells could be a common point in very quiescent patients. we choosed 18 patients, with minor clinical and/or biological manifestations of their disease, for at least 6 monthes. known of them received immunosuppressive drugs since this period. the mean sledai score was below 2. b cell surface markers expression was determined by flow cytometry. we analysed most of the already described and phenotypically distinctive b cell populations. we confirm the presence of activated b cells even in quiescent patients. we do not confirm the significant increase of a specific memory b cell compartment. above all, we described a decreased expression of the cd19 surface protein for all patients. this cd19 lower expression is associated with cd45 lower levels. it is not associated with an evident gene expression alteration and in vitro stimulation restores a control phenotype. these findings suggest some mechanisms in lupus genesis. objectives: tgf-beta is a pleiotropic cytokine with wide ranging effects in proliferation, differentiation, immune suppression and apoptosis. recent work from our group has shown that tgf-beta signalling in t-cells is protective in a mouse model of colitis associated cancer. smad ubiquitin regulating factors (smurf) are ubiquitin ligases that are involved in the regulation of tgf-beta signalling. the aim of this study was to determine the function of smurf2 expression in t-cells on the pathogenesis of experimental colitis associated colon cancer. methods: we could isolate a known splice variant of smurf2 lacking an exon in the c2-domain. to analyse whether this form has a regulatory role in colon associated cancer we generated a transgenic mouse strain that overexpresses smurf2 in t-cells. smurf2 expression were analysed by qpcr. wild type (wt) and transgenic (tg) mice were treated once with the mutagenic agent azoxymethan (aom) followed by three cycles dextran sodiumsulfate (dss). after each cycle, the inflammation of the gut and the tumor growth and size of every mouse were monitored by colonoscopy. results: smurf2 expression was upregulated by tgf-beta stimulation in t-cells and smurf2 was markedly upregulated in tumor infiltrating cd4+ lymphocytes in aom/dss treated mice. whereas wt mice suffered from severe colitis resulting in colon tumors beginning at day 42, smurf2 transgenic mice had less colitis and were significantly protected from tumor development. interestingly, t-lymphocytes overexpressing smurf2 showed an upregulation of the tgfbrii and an activation of smad2 and 3 as compared to wild-type t-lymphocytes, which were previously described as typical smurf2 targets for degradation. in addition the transfection of smurf2 and a caga-luc plasmid into cos-cells for smad3-promotor studies yielded the same effect as shown by upregulation of the smad3 activity. conclusion: although, wt-smurf has been described as a negative regulator of the tgf-beta signalling pathway, our data show surprisingly that a smurf2 splice variant upregulates the tgf-beta receptor expression and increases tgf-beta signalling effects. due to immunosuppressive effects on t-cells smurf2 has beneficial effects on mucosal inflammation and tumor development. smurf2 thus emerges as an attractive target for modulation of chronic intestinal inflammation and colitis associated carcinogenesis. the transcription factor stat3 has important functions in cytokine signalling in a variety of tissues. however, the role of stat3 in the intestinal epithelium is not well understood. we demonstrate that development of colonic inflammation is associated with the induction of stat3 activity in intestinal epithelial cells (iec) both in humans and in mice. studies in genetically engineered mice showed that epithelial stat3 activation in dss colitis is dependent on il-22 rather than il-6. il-22 was secreted by colonic cd11c+ cells in response to toll-like receptor stimulation. conditional knockout mice with an iec specific deletion of stat3 activity were highly susceptible to experimental colitis, indicating that epithelial stat3 regulates gut homeostasis. stat3 iec-ko mice, upon induction of colitis, showed a striking defect of epithelial restitution. gene chip analysis indicated that stat3 regulates the cellular stress response, apoptosis and pathways associated with wound healing in iec. consistently, il-22 and epithelial stat3 was found to be important in wound-healing experiments both in vivo and in cell culture experiments in vitro. in summary, our data suggest that intestinal epithelial stat3 activation regulates immune homeostasis in the gut by promoting il-22-dependent mucosal wound healing. stat3 seems dispensable for gut homeostasis under steady state conditions, but is activated upon challenge to drive tissue regeneration and protection in situations of increased demand, as during colitis and injury. map and ma infection induced an increase in both cd40 and tlr4 expression at day 3 and day 7 after infection. mycobacterial infection did not result in differential tlr2 expression as compared to uninfected cells. cd40 is involved in stimulating th1 pro-inflammatory responses, although map may interfere with cd40 signalling (1) . tlr4 signalling elicits anti-inflammatory responses, which can contribute to bacterial replication (2) .in conclusion, monocyte-derived macrophages from crohn's disease patients show an increase in cd40 and tlr4 receptor expression in response to both map and ma infection. as ma is a known human pathogen of immunocompromised hosts, this findings further support a role for map in the immunopathology of crohn's disease. objectives: for our understanding of the pathogenesis of human ibd, animal models of intestinal inflammation are indispensable. most of them are based on a compromised intestinal barrier, and a deregulated immune response against components of the flora is considered to be critically involved in the development of ibd. the occurrence of extraintestinal manifestations suggests that cross-reactions against hitherto undefined auto-antigens could be responsible for the activation of the adaptive immune system. to further dissect the pathophysiological mechanisms responsible for initiation and progression of ibd and associated extraintestinal manifestations, we established a new antigen-specific model, in which the local activation of cd8 t cells by exogenous antigen leads to colitis. methods: eight million naïve cd8 + ot-i cells, transgenic for a t-cell receptor specific for an ova-derived peptide (siinfekl) in the context of h2-kb, were transferred i. v. into b6 mice. at day 0 and 4, mice were treated intra-rectally (i. r.) with 50 % ethanol. thirty minutes later, ovalbumin (ova) or bovine serum albumine (bsa) were applicated i. r. proliferation of cfse-labelled cells was measured at day 2 after the injection of ot-i cells. the phenotype of effector cells was evaluated at day 5 by measuring ifng production and by in vivo cytotoxicity assay. based on histology and immunhistochemistry for cd8, the severity of colitis was scored. results: local application of the exogenous antigen ova but not of bsa led to antigen-specific activation and proliferation of adoptively transferred naïve ot-i cd8 + t cells. these cells differentiated into fully activated effector t cells with the capacity to secrete ifng upon re-stimulation ex vivo and possessed in vivo cytotoxicity to siinfekl-loaded target spleen cells. furthermore ova treated mice displayed an inflammatory infiltrate in the colonic lamina propria with strongly elevated numbers of cd8 + t cells. our study demonstrates that the local activation of antigen-specific cd8 t cells by exogenous antigen in the colon leads to fully activated effector t cells with the capability to promote local intestinal inflammation in non-immune-compromised b6 mice. aims: to determine the immune system response of the greek population against helicobacter pylori (hp), given the fact that hp infection is a frequent causal factor of gastroduodenal ulcer and gastritis, and to study the distribution by age and sex, as well as the possible correlation with anemia markers (hematocrit, hemoglobin, iron, ferritin etc). the results of express qualitative detection method for igg and iga antibodies were studied of 535 patients, (248 male and 287 female), with age average 69,7 years of age. patients who received antibiotics and excretory medicine in the last year were excluded. anemia laboratory tests were performed (hematocrit, hemoglobin, iron, ferritin), which were followed by statistical processing, using spss, x 2 and t-test programmes. results: in 372 patients (69,5 %,with age average 62,7 years of age) no antibodies were detected. on the contrary, in the remaining 163, 52 male and 111 female, (30,5 %, with age average 76,4 years of age), antibodies were detected. out of them, in 106 cases the results were strong positive (32 male and 74 female) and weak positive in 57 cases (20 male and 37 female). the statistical analysis that followed, showed no statistically important correlation with any of the anemia markers who were determined (hematocrit, hemoglobin, iron, ferritin, mcv and rdw). conclusions: it is proven, therefore, that: 1) helicobacter pylori infection is relatively common in the general population (30,5%).2) there is a statistically important correlation, as far as age (increased in elderly patients) and gender is concerned (clearly greater in women).3) there seems to be no correlation with anemia. it is evident, that the method is very useful, especially in elderly patients with dyspeptic complaints, (who frequently cannot undergo invasive procedures), and should not be neglected, given the fact that there is a great risk of helicobacter pylori infection in our country. abstract withdrawn by author m. durilova 1 , t. ulmannova 1 , k. stechova 1 , k. tesarova-flajsmanova 1 , v. stavikova 1 , j. nevoral 1 1 charles university, pediatrics, prague, czech republicobjective: was to analyze composition of cytokines in breast milk of mothers whose infants were diagnosed with allergic colitis and compare it to cytokine composition in breast milk of healthy controls. methods: breast milk of 20 mothers whose infants were diagnosed with allergic colitis and 20 mothers of healthy infants and no history of allergic disease was analyzed for presence of cytokines. breast milk samples were collected at the time of diagnosis of allergic colitis (2-27 weeks, average 16.8 weeks of infant's age) or at the age of 12 weeks in control group. concentrations of the following cytokines were analyzed using elisa method: il-4, il-6, il-10, il-17, il-23, ifn-gamma, tgf-beta1, egf and eotaxin. man-whitney u test was used for statistical analysis, p x 0.05 was considered statistically significant.results: il-10 as the only cytokine was not detected in any of the tested samples in both groups. significant difference was seen in concentration of ifn-gamma, which was higher (p x 0.001) in breast milk of mothers whose infants were suffering from allergic colitis (range 0-8.45 pg/ml, mean 2.1 pg/ml) than in control group (range 0-3.41 pg/ml, mean 0.35 pg/ml). higher concentrations of il-4 and lower concentration of tgf-beta1 were observed in breast milk received by infants with allergic colitis but the difference was not statistically significant. conclusion: immunologic factors including cytokines present in breast milk passively and actively influence the developing immune system of the newborn. although their role is not exactly known, they are important in regulation of immunologic reactions and might be responsible for protective effects of breast milk from many diseases. inter-individual differences in cytokine composition of breast milk were previously found in many studies and their presence is influenced by various factors. the results of our study indicate that there might be a risk cytokine pattern in breast milk of mothers whose infants are suffering from allergic colitis. supported by national project no. 8310-5. background: ulcerative colitis is associated with excessive neutrophil infiltration into the lamina propria and intestinal crypts leading to the formation of crypt abscesses. the chemokine il-8 (murine homologs kc and mip-2) and its receptor cxcr2 are involved in neutrophil recruitment, thus blocking this engagement offers a new therapeutic strategy for inflammatory bowel disease. this study aimed to develop and characterize a pre-clinical in vivo model to test potential therapeutics targeting neutrophil migration. methods: peritoneal exudate neutrophils from transgenic b-actin-luciferase mice were isolated 12 h post intraperitoneal injection of thioglycollate and phenotypically (facs analysis) and functionally characterized in an in vitro chemotaxis assay. four million exudate cells were injected intravenously into recipients with dextran sulphate sodium (dss) colitis followed by bioluminescence imaging of whole body and ex vivo organs at 2, 4, 16 and 22 h post-transfer. anti-kc antibody or its isotype control was administered at 20mg/mouse one hour before transfer followed by whole body and organ imaging 4 hours post-transfer. results: facs analysis revealed 80 % neutrophil purity, 35 % of which were cxcr2 + . in vitro, the cells migrated towards kc and this was inhibited by anti-kc. in the bioluminescent imaging model, trafficked neutrophils were evident in whole body and ex vivo organ images of dss recipients at all time points. neutrophil recruitment to the colon was detected only in dss recipients and was inhibited by anti-kc, 4 h post cell transfer. this study describes a novel in vivo model of neutrophil trafficking that can be used for pre-clinical studies to evaluate potential inhibitors of neutrophil recruitment. the human gut contains more than 10 15 bacteria (known as the commensal microbiota) that are essential for normal function of our digestive and intestinal immunologic systems. the barrier function of the mucosal epithelium is reinforced by innate defense mechanisms and by immune exclusion mediated by secretory (s)iga and sigm. sigs are generated via epithelial polymeric ig receptor (pigr)-mediated transfer of iga and igm from the lamina propria to the intestinal lumen. to assess the role of sigs in colitis development, we constructed pigr knockout (ko) mice and tested them in the dextran sodium sulfate (dss) colitis model (1.5 % dss in drinking water for 1 week, followed by pure drinking water for 1 week). pigr ko mice suffered increased morbidity and mortality compared with wild type mice, but colitis was cured by depletion of intestinal commensals suggesting that one role of sigs is to prevent pathology induced by commensal microbiota. in contrast, 2 % dss was lethal to all commensal-depleted mice, but these mice became anemic rather than suffering from bloody diarrhea. as previously documented by medzhitov and co-workers (rakoff-nahoum et al, cell 2004), treatment of commensal-depleted mice with the tlr4 ligand lps in drinking water protected against the lethality of 2 % dss. thus, the commensal microbiota serve two distinct roles in the dss colitis model. at dss concentration of 1.5 % they may become pathogenic and drive an intestinal inflammation. at 2 % dss commensals protect against the toxic effect of the chemical via their tlr ligands. in mice lacking sigs, due to deleted pigr, the severity of colitis induced by 1.5 % dss was greatly enhanced suggesting that one role of sigs is to prevent commensal microbiota from becoming pathogenic. ulcerative colitis (uc) is a human inflammatory bowel disease associated with chronic inflammation of the gastrointestinal tract. although uc is associated with a type 2 immune response, current treatment strategies use broad anti-inflammatory drugs which are aspecific for the disease. in a mouse model resembling uc, oxazolone induces il-13 production which is an important pathological factor. neutralizing il-4 or il-13 prevents or ameliorates disease significantly. as many aspects of the mechanisms involving these th2 cytokines in colitis remain undefined, we used mice deficient in il-4/il-13 or the key receptor through which they signal, il-4ra, to further dissect their role in oxazolone-induced colitis. disease was exacerbated in il-4ra -/mice with increased weight loss, mortality, inflammation and immunopathological symptoms. this was in contrast to il-4/il-13 double deficient mice which were protected from colitis. removing il-13 production from il-4ra -/mice, by using il-4ra/il-13 double deficient mice, reversed the susceptible phenotype to protection. together these data strongly suggest that il-13 mediates susceptibility in an il-4ra independent manor. recent evidence pc17/33 introduction: the activation of cd4 + t-cells in the lamina propria play an major role in the pathogenesis of inflammatory bowel disease (ibd). whereas cd is associated with increased production of th1-like cytokines, the cytokines profile in chronic uc is characterized by the increased production of several th1 cytokines, such as il-5,-6 and il-13. however, the functional role of t cell transcription factors such as nuclear factor of activated t cells (nfat) in ibd is poorly understood. the aim of this study was to further analyze the role of this signal transduction pathway and its pathogenic significance in uc. cryosesctions of uc and cd patients were analysed by immunohistochemically methods. a significantly higher expression of nfatc2 was found in uc and cd colonic tissue compared to control specimen. transmitted to the th2-mediated oxazolone-induced colitis model, nfatc2-production is significantly increased in both diseases, too. nfatc2 deficient mice were analyzed in colitis model and are significantly protected against the development of intestinal inflammation compared to control mice, documented by loss of weight, histological score and miniendoscopy. interestingly, cyrosections of inflamed colonic tissue displayed a higher apoptotic rate in nfatc2 deficient mice compared to control mice, which can be observed by tunel assays, caspase3 and annexin v staining, as well as in lamina propria t cells. contrary, anti-apoptotic proteins, like bcl-2 and bcl-xl were downregulated for induction of apoptosis. this observation was associated with a reduced production of il-6, ifn-gamma, il-13 and il-17 by mucosal t lymphocytes, tested by elisa assays. further studies with the oxazoloneinduced colitis model showed that nfatc2 regulates il-23/il-17 in an indirect way. last, administration of il-6 blocked the protective effects of the nfatc2 deficiency in experimental colitis, suggesting that nfatc through il-6 signal transduction plays a direct pathogenic role in vivo. conclusion: our data define a unique regulatory role of nfatc2 in colitis by controlling mucosal t cell activation in an il-6 dependent manner. the examination of this signal transduction pathway emerges as a potentially new therapeutic target for inflammatory bowel diseases. the pivotal role of micrornas in the regulation of gene expression, in particular genes involved in the immune response, indicates that they may play an important role in the pathogenesis of inflammatory bowel disease (ibd) as well. the study of the expression of micrornas in ibd will unravel their role in this disease. in addition, micrornas by their mechanism of action, are promising new therapeutic agents or targets. a possible therapeutic application of micrornas is the introduction of novel, artificial micrornas or microrna mimics to regulate specific genes. because ibd is a heterogeneous disease in human we decided to define microrna expression in a well defined model of experimental colitis. as a result of this study we found a number of micrornas involved in different phases of experimental colitis. to study the role of mirnas in experimental colitis in mice we have used a well defined colitis model that resembles human ibd. this colitis is mediated by cd4cd45rb high t cells that are injected i. p. in scid mice. in control mice in addition to the cd4cd45rb high t cells also regulatory cd4cd45rb low t cells are transferred and no colitis develops. to study mirna expression we collected colonic tissue from the mice at 3 different time points during colitis progression. after 3 weeks a chronic progressive colitis developed characterized by a progressing wasting disease that was terminated at 9 weeks. microrna was isolated from colons of mice in different stages of colitis progression (3, 6 and 9 weeks) and control mice that do not induce colitis (n=3 for each timepoint). from all mice we also processed a part of the colon for immunohistochemistry to determine disease progression at the various time point after induction of colitis.the rna isolation as well as the microarray analysis has been outsourced to miltenyi biotec gmbh, bergisch galdbach, germany. we used the mirxplore tm microarrays for microrna expression profiling. from 11 micrornas that demonstrated an induction during the development of disease we selected 4 micrornas for in situ hybridization and for a proof of principle of the efficacy in the cd4cd45rb high transfer model. objective: the purpose of this clinical trial (id: nct00287677 of www.clinicaltrials.gov) is to investigate whether the expansion of the thymus in adults can restore specific immune responses by administration of growth hormone (gh). methods: successfully highly active antiretroviral therapy (haart) treated hiv infected patients that failed to elicit a humoral response to tetanus toxoid (tt), or to hepatitis a (hva) or to hepatitis b (hvb) virus have been selected for the trial. growth hormone was given for 6 months with the hope that they will reactivate thymic input and restore their specific responses to these vaccine antigens. patients have been randomized in 3 groups: group a (n=8) receiving haart+ gh (for 6 months) + tt+hva/b vaccines (at month 6 post gh adminsitration); group b (n= 6) receiving haart+gh but not vaccines; and group c (hiv control group, n=7) with haart+vaccines (at month 6) but without gh. all patients are followed up 6 months further. results: preliminary results show that an increase in thymic size was observed in gh recipients and not in controls. furthernore after 24 weeks of administaring hormone the absolute numbers of cd4 incresase from 562 ± 93 to 704 ± 112 cells per mm 3 (mean and sem; p x 0.0025). in contrast, pacients who have not received the hormone but have been vaccinated showed a significant decay of the cd4 absolute numbers from 550±97 to 470±103 cells per mm 3 (p x 0.02). viral load remained undetectable in all patients. despite the increase in cd4 counts the percentage of recent thymic emigrants (as assessed by the expression of cd31) as well as the proportion of naï ve and memory cells remained constant throught the trial in all patients. finally, specific responses to hepatitis a virus seem to be restored in a major proportion of patients treated with gh (group a) than in the other groups. conclusions: although the clinical trial is ongoing, the preliminary results seem to indicate an increase in the thymic size and some immmune restoration in patients treated with growth hormone before vaccination. a major problem of current vaccines is the requirement for cold chains to maintain vaccine potency. in the course of the eradication of small-pox, freeze-dried vaccinia virus which proved to be extremely stable was used to overcome this limitation (dryvax ® ). before usage, dryvax has to be reconstituted before vaccination using a bifurcated needle reflecting another drawback of classical vaccination -transmission of blood-borne pathogens. an alarming report by the who has estimated that as many as one-third of immunization injections are unsafe in four of its six geographical regions. each year, an overwhelming number of infections with hiv (80,000-160,000), hepatitis c virus (hcv; 2.3-4.7 million) and hepatitis b virus (hbv; 8-16 million) are thought to originate from the reuse of needles and syringes by health-care providers. in this report, we took advantage of the stability of freeze-dried vaccinia virus mva and directly applied it into the nostrils of mice without prior reconstitution. this direct mucosal application induced systemic antibody and t cell responses comparable to those achieved by intramuscular administration. importantly, mucosal application of lyophilized mva conferred protective immunity against a lethal vaccinia virus challenge. additionally, recombinant mva expressing the model antigen ovalbumin induced long-term and protective immunity against listeria monocytogenes-ova challenge. the data clearly demonstrate the potency of a simple needle-free vaccination, combining the advantages of mucosal application with the stability and efficiency of lyophilized mva. methods and results: seventeen haart-treated asymptomatic hiv-1 infected patients with g 350 cd4 + t-cell counts and plasma hiv-rna levels of x 1.7 log 10 copies/ml were treated with a dc-based vaccine. the vaccine consisted of autologous mature dc electroporated seperately with either sig-tat-dc-lamp, sig-rev-dc-lamp or sig-nef-dc-lamp mrna and were each administered at a distinct anatomical site. after four monthly vaccinations haart treatment was interrupted. pbmc from 4 timepoints, before during and after vaccination, were analysed for ifn-g production (elispot), proliferation (cfse assay) and lytic capacity (fatt-ctl) in response to the antigens used in the vaccine. pbmc were screened upon ex vivo stimulation with pools of overlapping tat, rev, nef and gag peptides and ifn-g secretion was analysed using elispot ('peptide elispot'). elispot was also performed on re-stimulated t-cells with electroporated dc ('dc elispot') in vitro. responses were considered positive when the number of spot forming units per million t-cells was g 50 and when the responses were g 2 times the standard deviation above the mean of replicate negative controls (mock electroporated dc). 16/17 patients were screened with both peptide and dc elispot. an increase of responses to the vaccine-antigens after vaccination was found in both assays. based on the dc elispot data, we observed immune reactivity against tat in 4/16 patients before and 11/16 patients after vaccination. for rev, 7/16 patients showed a pre-existing rev specific response and 14/16 patients responded to rev after vaccination. nef was the most immunogenic antigen used in this vaccine with already 11/16 patients responding before and 16/16 patients responding after vaccination. for our control antigen gag, we observed an anti-gag response in 13 out of 16 patients before vaccination and 16/16 patients after vaccination. the results of the other assays correspond to the dc elispot results be it less pronounced. conclusion: therapeutic vaccination of hiv-infected patients with dc electroporated with tat, rev and nef induces and/or enhances antigen-specific t-cell responses, especially when monitored with the dc elispot. background: enterovirus 71 (ev71) is an etiologic agent responsible for seasonal epidemics of hand-foot-and-mouth disease and causes significant mortality among young children. no effective vaccine for ev71 is available yet. polysaccharide purified from ganoderma lucidum (ps-g) has been known to be a strong immunopotentiator, therefore, we studied the immune enhancing effect of ps-g as the possible adjuvant with ev71 inactivated virus. methods: mice were immunized intraperitoneally with 10 mg inactivated virus /mouse with one of the following samples: pbs, cfa/ifa, and ps-g. each mouse received the same dose of boosters after 0, 2, and 4 weeks. blood samples were collected at 0, 21, 35, and 45 days. the total serum anti-ev71 igg level was determine by elisa, and the neutralization assay was also done. to evaluate the cellular immune responses, spleens were harvested from all mice for splenocyte proliferation assay. cytokines assay regarding ifn-g and il-5 from splenocytes was also measured. results: immunization with ev71/ps-g showed that the anti-ev71 igg levels were significantly increased compared with ev71 alone or ev71/cfa/ifa in balb/c mice. neutralization assays demonstrated an effective protection of ev71/ps-g group compared to ev71 alone. the splenocyte proliferation test showed that production of ifn-g significantly increased in ev71/ps-g-immunized mice compared to those of ev71 or ev71/cfa/ifa-immunized mice, indicating a th1 cells response elicited by heat-inactivated ev71 vaccine with ps-g adjuvant. conclusions: vaccine design is important for the development of immune response for pathogen, and adjuvants play the very important role to enhance the effect of vaccine. the results here suggested that ps-g can be used as a novel, safe and natural vaccine adjuvant. objectives: the search for a vaccine against hepatitis c virus is hampered due to the lack of an animal model to study vaccine-efficacy other than chimpanzees. here we describe the differential modulation of cd8+ t-cell responses induced by a dna prime mva boost vaccine regimen in four individual chimpanzees and their association with viral clearance. methods: an ex vivo expansion protocol was used to map peptide specific cd8+ t-cell responses against hcv-ns3, studying induction of il-2, ifng and tnfa cytokine production as well as killing capacity. to assess the killing capacity and mhc restriction of the peptides, a non-radioactive killing assay was designed. peptides that induced both il-2 and ifng production by cd8+ t-cells were tested for their competence to induce killing of transfected target cells that expressed chimpanzee mhc class i molecules. introduction: high levels of hiv-1-recognizing cd8 + cytotoxic t lymphocytes (ctl) with a widespread specificity, especially against conserved epitopes, are considered to play an important role in the control of hiv-1 replication, and for the prolonged survival of infected individuals. a potential immunotherapeutic strategy would be the adoptive transfer of t cells, which are reprogrammed by introduction of an hiv-specific t cell receptor (tcr). up to now, such ctl were generated by retroviral transfer of tcr-encoding genes, which harbors several challenges (i. e., activation/inactivation of genes, life-long autoimmunity). methods: therefore, we investigated the transfer of tcr-rna into cd8 + t cells by electroporation, and chose tcrs which were able to recognize the hla-a2 restricted hivpol-peptide iv9, or the hivgag-peptide sl9. results: t cells, reprogrammed with these receptors, released the pro-inflammatory cytokines il-2, tnf, and ifng simultaneously, and showed up-regulation of the activation marker cd25, after stimulation with peptide-loaded target cells or target cells (i. e. ebv-transformed b cell and cd4 + t cells) presenting the naturally processed epitope. furthermore, these cells maintained their ability to proliferate after stimulation. more importantly, killing assays demonstrated that the tcrreprogrammed cd8 + t cells were capable to specifically lyse target cells (for at least three days) loaded with the cognate peptide, or presenting the naturally processed epitope. a comparison of our reprogrammed t cells with the parental ctl showed that the transfected t cells were only one order of magnitude lower in avidity than the parental ctl. also, the parental clone's recognition pattern of mutant peptides was preserved in tcr-rna-transfected t cells. the transfection of tcr-encoding rna into cd8 + t cells, may represent a simple, secure, and more flexible alternative to retroviral transduction, but has the benefit that a better evaluation of the transferred tcrs is possible. background: dendritic cells (dcs) are able to capture, internalize, and process antigens leading to potent activation of antigen-specific cellular immunity. the aim of this study was to investigate the capacity of splenic dcs that ingest antigen coated magnetic beads to induce hcv cellular immune responses. methods: splenocytes of flt3l pretreated balb/c mice were incubated for 3 hrs with hcv ns5-coated magnetic beads. the cells were harvested and cells that contained beads were purified by passage over a magnetic column. the isolated population contained g 80 % dcs and was used for immunization. dc expression of the maturation markers cd40, cd80 and cd86 was determined before and after ingestion of ns5-coated beads, showing a significant increase of all maturation markers induced by phagocytosis. cellular immunity was assessed using a conventional ctl assay, a cfse-t-cell proliferation assay, intracellular cytokine staining and tumor challenge (with stably ns5 expressing sp2/0 cells). results: in immunized animals, the ctl activity was increased 3-fold compared to mock immunized mice. accordingly, tumor challenge with ns5 expressing tumor cells showed a significant reduction in tumor growth. the number of cd4 + ifn-g + cells was increased g 3-fold and the number of cd4 + il-2 + increased g 5fold in the dc-ns5-bead immunization group. these results paralleled the proliferative response of splenocytes to ns5 protein obtained from immunized animals with the most significant response in the cd4+ population of dc-ns5-bead immunized animals. the use of ns5 coated beads combines three important aspects of dendritic cell based immunization in a single step: targeting of the antigen, enrichment and maturation of dendritic cells. the induction of a strong th1 biased t cell response makes this approach a promising new tool in therapeutic immunization in chronically hcv infected patients. the success of anti-dec-205 antibody as a stimulator of strong inflammatory immune responses depends on the coadministration of non-specific dendritic cell maturation factors. in their absence, anti-dec-205 induces antigen-specific tolerance rather than immunity. we hypothesize that regulatory t-cell epitopes contained in anti-dec-205 promote a tolerogenic reaction that is only overcome through the co-administration of non-specific immuno-stimulators. this hypothesis is based on our recent discovery of a set of natural regulatory t-cell epitopes derived from human immunoglobulins that induce tolerance by stimulating regulatory t cells. we have verified experimentally that these epitopes generate an expansion of regulatory t cells and suppress inflammatory immune responses. here, we embarked on a proof-of-principle demonstration that a pro-inflammatory and non-tolerogenic anti-dec-205 antibody can be developed. we screened the anti-dec-205 sequence computationally for putative hla dr4-restricted, regulatory t-cell epitopes as targets for mutations that will reduce epitope binding affinity for hla. amino acid substitutions predicted to interfere with hla binding were identified and are being incorporated into an array of anti-dec-205 antibody variants recombinantly fused to a test antigen, hiv gag. variant antibodies that do not interfere with dendritic-cell targeting will be evaluated for reduced tolerogenicity, as well as for enhanced gag immunogenicity, in terms of cellular and humoral responses. we predict that the modification of regulatory t-cell epitopes will significantly diminish tolerogenicity, enabling the use of modified anti-dec-205 as a hiv antigen-delivery system that obviates the dangers associated with non-specific activation of the immune system. supported by nih 1r21ai078800 a live oral vaccine based on human adenovirus (ad)4 has proved safe and effective in us military recruits for nearly 50 years. in these experiments, we have investigated whether replication-deficient ad4 can be an efficient potential vaccine carrier for oral vaccination. ad5 vectors were used throughout to provide a benchmark for efficacy. we generated novel ad4 and ad5 vector systems based on dna recombineering to facilitate manipulation of the vector backbone and high throughput transgene insertion (http://adz.cf.ac.uk). egfp was inserted with a hcmv ie promoter as a model transgene. preliminary in vitro studies on bloodderived human and murine cells demonstrated that primary lymphocytes are less susceptible to transduction with ad4 than ad5. ad5 routinely infected and provided transgene expression in˚10 % of human cd4+ and cd8+ t cells. stimulation of the hcmv ie promoter post infection increased detection of egfp to 25 -30 % of cd8+ t cells present, showing that ad5 infected a surprisingly large proportion of t cells. in comparison, ad4 provided egfp expression in x 2 % of either cell type, even after t cell activation. in contrast, infection rates and transgene expression in dendritic cells of both human and murine origin with ad4 and ad5 vectors were comparable. preferential infection of dcs is likely to be beneficial in the context of a vaccine. in vivo, we observed that following oral delivery both ad4 and ad5 induced restricted yet strong expression in the intestine. the vectors were rapidly taken up into the peyers patches, with optimal expression detected day 3 after dosing, and transgene expression being reduced below detectable levels by day 8. interestingly, when delivered together ad4 and ad5 vectors targeted the same subset of cells. together, these data show that ad4 is a viable alternative to ad5-based vaccines which may also avoid unwanted infection of activated t cells. aims: monoclonal antibodies (mabs) represent some of the most promising agents for anti-cancer and anti-viral immunotherapies (20 recently commercialized; g 300 in clinical trials). to date, their therapeutic antiviral efficiency has mainly been measured by their direct effects on viral spread. however, their indirect effects on long-term immune control of viral replication through their immunoregulatory properties upon interaction with other components of the immune system has hardly been assessed. as induction of long-term protective antiviral immune responses still remains a paramount challenge for treating chronic viral infections, we asked whether neutralizing mabs, in addition to blunt viral propagation, may also modulate the endogenous immune response. methods: we have developed a preclinical mouse model of fatal leukemia induced by the frcas e murine retrovirus. mice were infected with frcas e and treated, or not, with a neutralizing mab (the 667 mab). viral propagation, survival and development of immune responses were followed up for several months. results: using this model, we have shown that 667-treated/infected mice develop a long-lasting protective humoral immune response as well as a strong and sustained cellular immune response with high cytolytic activity which are not observed in leukemic non-treated/infected mice. these results show that neutralizing mabs act as immunomodulatory agents capable of inducing long-term protective immunity ( g 8 months) with both humoral and cellular contributions, despite the fact that they were administered over a short period of time (gros et al, 2005; gros et al, 2008; michaud et al. submitted) . although the initiation and maintenance of this long-term immunity is multi-factorial, we have demonstrated the crucial importance of the uptake of antibody-coated, infected cells by dendritic cells in the development of enhanced primary and memory antiviral t-cell responses. conclusions: our results show that infected-cells/antibody immune complexes play an important role in the induction and maintenance of protective antiviral immunity through enhancement of primary and memory antiviral t-cell responses. our observation might have important consequences on the design of antiviral mab-based immunotherapies. objectives: we have analyzed the potential of virus-like particles (vlps) from rabbit hemorrhagic disease virus (rhdv) as a delivery system for foreign t-cell epitopes. to accomplish this goal, we generated chimeric rhdv vlps incorporating a cd8 + t-cell epitope (siinfekl) derived from chicken ovalbumin (ova). the ova epitope was inserted in the capsid protein (vp60) of rhdv at two different locations: 1) the n-terminus, predicted to be facing to the inner core of the vlps (rhdv-vlp-2), and 2) a novel insertion site predicted to be located within an exposed loop (rhdv-vlp-306). both constructions correctly assembled into vlps and we analyzed the immunogenic potential of both chimeric rhdv vlps in vitro and in vivo. in vitro, dendritic cells (dcs) were able to process and present siinfekl peptide in the context of mhc class i from chimeric rhdv vlps for cd8 + specific recognition in a dose-and insert position-dependent manner. moreover, chimeric rhdv vlps activated dcs for tnf-alpha secretion.in vivo, mice immunized with the chimeric rhdv vlps without adjuvant were able to induce specific cellular responses mediated by cytotoxic (ctl) and memory t cells. although both chimeric rhdv vlps were able to induce specific ifn-g-producing cell priming, insertion of the siinfekl peptide at the amino-terminal position (rhdv-vlp-2) was more immunogenic than insertion at position 306 for induction of ctls and anti-viral immunity.more importantly, immunization of mice with chimeric rhdv vlps at the highest dose tested was able to control an infection by a recombinant vaccinia virus expressing ova in target organs. in addition, immunization with chimeric rhdv-vlp-2 at the highest dose tested was able to resolve vv-ova infection. conclusion: our data demonstrated that immunization with chimeric rhdv vlps was able to protect mice from a viral challenge, suggesting the potential suitability of these constructions for new vaccine development against animal and human viral infections. objectives: a major issue pertaining to use of dna vaccines is that despite successful proof of principle results in small animal models, low efficacies have been reported in human clinical trials and large doses are required to induce protective immune responses. in this study, we describe the targeting of antigen-encoded dna directly to dendritic cells (dcs) through a pathway that results in internalisation and transfection using a cationic lipopeptide containing arginine residues and the lipid dipalmitoyl-s-glyceryl cysteine, a known tlr-2 ligand. methods: agarose gel electrophoresis was used to confirm the electrostatic binding of lipopeptide to dna encoding for green fluorescent protein (gfp), influenza hemagglutinin (ha) or nucleoprotein (np). transfection efficiencies of dcs using these complexes were determined by flow cytometry using specific antibodies for each encoded protein. stimulation of t cells by np-transfected dcs was also investigated by measuring their ability to induce in vitro cytokine secretion by influenza virus-specific cd8+ t cells. subsequently, vaccine immunogenicity was ascertained by immunisation of mice via the intra-nasal route. results: electrostatic binding of the lipopeptide to dna plasmids was confirmed by gel electrophoresis where the positively charged amino acids of the lipopeptide were able to neutralise the negative charged phosphate groups within the dna backbone and retard its ability to migrate towards the anode. high levels of gfp, ha or np were detected in murine spleen-derived cultured dcs following transfection with these complexes concomitant with the upregulation of surface mhc class ii molecules compared to when dna alone was used. the ability of transfected dcs to stimulate cd8+ t cells from influenza virus-infected mice was also investigated. subsequently, vaccination by lipopeptide-dna complexes resulted in the induction of higher numbers of ifn-g producing np 147-155 specific cd8+ t cells in the spleen and lymph nodes of mice compared to those that received dna alone. conclusion: altogether these results demonstrate that the use of a tlr-2 targeting lipopeptide-based system that can facilitate the delivery of dna by directly targeting and concurrently activating antigen-presenting cells, such as the dc, could prove to be advantageous in enhancing cellular responses induced by dna vaccination. the level of interferon in blood serum of non-infected mice was determined by elisa kit and by the cytopathic test in the l929cell culture after aplication of ridostine and ridostine ointment. the effect of the preparation on phagocytic activity of macrophages was evaluated in the monolayer peritoneal cell culture. the statistical processing of the data was carried out by the student t-criterion. ridostine was administered to patients once or twice in the case of high temperature. the clinical signs were recorded (temperature, rhinitis, headache etc). for prophylactic and treatment purposes the ridostine ointment was intranasally applied twice per day during 7 days. the effectiveness of the preparation was evaluated by clinical sings and the level of cd2+, cd4+, cd8+, cd16+ t -lymphocytes. results: ridostine significantly decreased accumulation of the virus in lungs of infected mice at the initial stage of influenza. ridostine and ridostine ointment stimulated synthesis of interferon and phagocytic activity. ridostine in clinical practice decreased the duration of influenza, attenuated clinical signs and was more effective at an early stage of the infection. prophylactic intranasal application of ridostine ointment by healthy volunteers (nurses and doctors) resulted in a high degree of protection during the whole epidemic season and an activation of t-cell immunity. application of ointment at an early stage of disease markedly activated t-cell immunity, reduced the duration of the disease and the intoxication syndrome by 1,5-2-fold. conclusion: interferon inducer based on natural dsrna stimulates some reactions of innate immunity and resistance to influenza virus. the drugs based on dsrna show promise for treatment of influenza. objectives: the creation of gene engineering vaccines against hepatitis c virus (hcv) based on recombinant proteins is one of relevant approach. since the immunogenicity of these proteins is low as a rule, the choice of adjuvant is very important. the aim of this work was to evaluate immunogenicity of covalent conjugates between nonstructural ns4 and ns5a hcv proteins and immunomax ® , an acid peptidoglycan of plant origin, which displays immunomodulating activity. objectives: ifn-g takes part in the development of an anti-infectious reaction of the organism, which is connected with the peculiarities of its immunomodulating action. a/h5n1 influenza virus inhibits the ifn-g synthesis (mibayashi et al., 2007) and causes a decrease in cd4 + and cd8 + t-lymphocytes content in lung and lymphoid tissues associated with an impairment of this cytokine production (tumpley t. m. et al., 2000) . thus, ifn-g is a promising drug for prophylaxis and treatment of avian influenza under conditions of monotherapy or complex therapy. an essential defect of this cytokine is its fast degradation in blood. the goal of this work was to study immunomodulating properties of an ifn-g structural analog with increased proteolytic resistance when it was used alone or in combination with double-stranded ifn inducers. methods: a recombinant human ifn-g analog deltaferon is distinguished by a 10 amino acid deletion at the c-end of the molecule and substitutions of amino acids in positions 129-131 (tat'kov c. i. et al., 2000) . deltaferon was i. p. administered to male non-inbred mice in doses of 2-40*10 4 iu once or twice at an interval of 48 hours, alone or in combination with double-stranded yeast rna preparation (5 mg/kg). the content of ifn-a and ifn-g in mouse blood serum was determined by the immunoenzyme method, proliferative activity of splenocytes -by mtt-test (mosmann t., 1983) . results: when deltaferon was administered in doses of 2-20*10 4 iu alone it did not influence the content ifn-a in blood, but caused a transient increase in the level of ifn-g. the injection of the preparation in a dose of 2*10 4 iu led to a an increase in both spontaneous and conconavalin a-induced proliferation of splenocytes. the two-fold administration of deltaferon in the maximal dose increased a level of ifn-g and inhibited cell proliferative activity. the combined administration of deltaferon (2*10 4 iu) and dsrna markedly increased the level of ifn-a and enhanced splenocyte proliferation. the recombinant human ifn-g analog is able to enhance ifn-g synthesis, splenocyte proliferation and to modulate the effect of ifn inducer. these data suggest that the studies of the preparation as an antiviral agent during influenza are perspective. by using a combined approach of routes of immunization and vaccine delivery systems such as poly-lactic acid (pla) biodegradable nanoparticles, we have dissected the intensity and quality of both cellular and humoral immune responses in mice. we showed that the amplitude and quality of the immune response (humoral, cellular) at systemic and mucosal sites (blood, vagina) could be largely influenced by the choice of a pertinent cutaneous route of vaccination (intradermal (id), transcutaneous (tc), subcutaneous (sc)). while id and tc route remain mostly efficient for the induction of antigen-specific cd8 responses (tetramer+ hiv-1 gag p24 cells), the quality of humoral responses (igg, iga) remained distinct between the two routes. in addition, sc route is less efficient than id and tc routes for the induction of cd8 responses after pla-p24 immunization. we have also shown that a lower antigenic charge of pla particles was needed when pla-p24 were injected using id and tc routes of immunization. currently, we are dissecting innate cellular mechanisms that gave rise to distinct quality of immune responses. this unique possibility to modulate the quality of the immune response according to the skin route of immunization paves the way for new vaccine design strategies and highlights the capacity of nanoparticles-based vaccine delivery system. b. dí az-freitas 1 , c. prego 2 , s. vicente 2 , m.j. alonso 2 , a. gonzález-fernández 1 1 university of vigo. area of immunology, department of biochemistry, genetics and immunology, vigo, spain, 2 university of santiago de compostela, nanobiofar group, department of pharmacy and pharmaceutical technology, santiago de compostela, spainobjectives: the design of effective vaccine delivery nanovehicles is opening up new possibilities for making immunization more equitable, safe and efficient. in this work, we purpose polysaccharidic-based nanocarriers as delivery structures for virus-like particle antigens, using recombinant hepatitis b surface antigen (rhbsag) as a model. our aim was to evaluate in a murine model if these nanocarriers induce an immune response after intramuscular and intranasal administration. materials and methods: loaded chitosan-based nanocarriers were prepared by cross-linking the polysaccharide chitosan, in the presence of the stabilizer poloxamer 188, with a counter ion, tripolyphosphate, containing the free rhbsag. the immunogenicity of these polysaccharidic-based nanocarriers with 1 or 2 immunizations to balb/c female mice (4 weeks old) was assessed following intranasal or intramuscular immunizations. blood samples from the mouse maxillary vein were collected at different intervals (from day 15 to 260 post-primary immunization). specific igg antibodies levels directed against rhbsag in serum were measured by indirect elisa in miu/ml. results: chitosan-based nanoparticles with particle size in nanometric range, positive zeta potential and an important rhbsag loading were prepared. the results of the specific igg levels achieved following intramuscular administration of the antigen-loaded nanocarriers, and also of the alum-adsorbed vaccine showed the significant adjuvant effect of the nanocarriers. the response elicited was delayed respect to the alum based vaccine, and very interestingly, igg concentration was much higher using antigen-loaded nanocarriers than with the conventional vaccine. after intranasal administration, chitosan-based nanoparticles generated a lower immune response compared with the intramuscular route, but increasing over the time, showing an interesting slow release of the antigen. the igg titers elicited were enough to induce full seroprotection against the disease (100 miu/ml). polysaccharidic-based nanocarriers with interesting properties for improving vaccination with complex antigens were designed and in vivo behavior of these nanocarriers suggests their potential utility as controlled release vehicles for reducing the number of intramuscular doses administered. more studies are currently underway to fully validate the potential of these nanocarrier prototypes and to optimize alternative routes of immunization such as the intranasal route. the success of immunotherapeutical approaches strongly relies on specific antigen targeting to dendritic cells (dcs) in an environment that provides optimal immunostimulatory signals. in our research group a bio-safe coronavirus-based vaccine vector platform that delivers multiple antigens to professional antigenbackground: infection with human immunodeficiency virus type 1 (hiv-1) is characterized by dysfunction of hiv-1-specific t cells. to control the virus, antigenloaded dendritic cells (dcs) might be useful to boost and broaden hiv-specific t-cell responses. poly electrolyte microcapsules are potent protein delivery vehicles which can be tailored with ligands to stimulate maturation of dendritic cells. we investigated the immune stimulatory capacity of dendritic cells loaded with these microcapsules, containing both p24 antigen from hiv-1 and the tlr3 ligand poly i:c as a maturation factor. methods: monocyte-derived dc (mddc) from healthy subjects were cultivated with polyelectrolyte microcapsules containing, poly i:c. potential maturation of dc was evaluated by flow cytometry. mddc from hiv-infected patients under highly active anti-retroviral therapy (haart) were similarly pre-incubated with p24 microcapsules containing p24 and poly i:c. these antigen loaded mddc were used to directly stimulate autologous peripheral blood lymphocytes (pbl) in elis-pot. they were also co-cultivated for 10 days with autologous pbl to evaluate the immunogenic capacity. potential expansion of specific t cells was measured by comparing elispot responses of pbl before and after coculture, using a pool of overlapping p24 peptides. intracellular staining of interferon-gamma (ifn-g), interleukin-2 (il-2) and cd107a after p24 stimulation was also performed. results: mddc from hiv(-) subjects, incubated for 24 hour microcapsules alone did not induce maturation of dc, but when poly i:c was present the dc did mature. mddc from haart treated hiv-infected individuals, cultivated with p24 containing microcapsules with poly i:c, were able to efficiently expand and broaden autologous effector memory t cells which contain and secrete ifn-g and il-2, upon p24 peptide restimulation. objectives: we aimed at investigating whether and how the distance of a cytokine from the vlp surface impacts on its function and whether the relative distance towards a co-presented antigen is critical for its biological activity. methods: we inserted one, two or four ig-like domains of hcd16b between our model cytokine il-2 and the minimal gpi-anchor acceptor sequence. subsequently, we compared particle production by western blotting for p30gag, targeting of cytokines to lipid rafts and and vlp upon isopycnic membrane separation and biological activity in il-2 dependent proliferation assays of il-2 variants. results: murine il-2 attached to either of the four fusion partners was biologically active in vitro as shown by induction of proliferation of the il-2 dependent cell line ht-2. we found that the membrane anchors comprising one and four ig-like domains (il-2::1iggpi and il-2::4iggpi) resulted in severely reduced vlp production by 293 producer cells and despite of an increased targeting of il-2::4iggpi to vlp, a reduced stimulatory capacity of producer cell crude supernatant, when compared to il-2 fused to the minimal gpi-anchor acceptor sequence of hcd16b (il-2::gpi). il-2::2iggpi, however, showed comparable particle production and biological activity in vitro when compared to il-2::gpi. furthermore, il-2 fused to 2ig::gpi showed an increased capacity to co-stimulate primary p14 tcr transgenic t-cells specific for lcmv-gp 33-41 in the context of h2-d b . conclustions: besides the minimal gpi-anchor acceptor sequence we have identified one additional membrane anchor, which displays superior capacity to target cytokines to vlp. 2ig::gpi has a biological activity in vitro, which is comparable to the minimal gpi anchor. moreover, il-2::gpi displays increased co-stimulatory potential in the context of specific mhcp complexes. this work was supported by grants sfb f1816-b13 of the austrian science foundation, the austrian research promotion agency (forschungsförderungsgesellschaft) bridge grant 812079 & biomay ag, and the christian doppler laboratory for immunomodulation. a. roemhild 1 , interdisciplinary transplant laboratory 1 charite berlin, insitute of nephrology and medical immunology, berlin, germany immunosuppressive treatments, e. g. after transplantation are often followed by an impaired or dysfunctional immune system. missing viral immunity, particularly against ebv, is an essential key player in the development of severe infections and posttranplant lymphoproliferative disorders (ptld). ptld affects 2-25 % of solid organ transplant recipients, depending on the organ transplanted. healthy individuals control ebv infection by ebv-specific cytotoxic t lymphocytes (ctls), but some patients under immunosuppression are unable to do so. in these cases, immunotherapy is increasingly used as a new approach for re-establishing a functional immune response by retransferring in-vitro expanded autologous virusspecific t cells into the patient. currently these t cells are generated by repetitive stimulations with ebv-infected autologous lymphoblastoid cells (lcls). due to a generation time of 2-3 months, many patients suffering from missing viral immunity and subsequent severe viral disease are excluded from therapeutic benefit. therefore, shortening the generation time would be an important step to make adoptive immunotherapy available for more patients. t cell lines were generated with two different protocols. in the first protocol t cells are generated by repetitive stimulation with ebv-infected autologous lcls. the second protocol is based on stimulation with 6 different overlapping ebv peptide-pools and immunomagnetic cell isolation. expanded t cells were analysed using multicolour flow cytometry. cells were stained for diverse surface markers and intracellular cytokine production. cytotoxic capacity and specificity was determined by a calcein release assay. our group developed a new protocol for the production of ebv specific t cells, thereby shortening the generation time from 2,5 month to 16 days. t cell lines are composed of cd8 and cd4 cells with a mainly effector memory like phenotyp. after restimulation the cells produce more tnfa than ifng. depending on the generation protocol t cells specifically recognized and lysed autologous lcls alone or loaded with ebv-peptides. the detailed characterization of ebv-specific t cell lines should help to further improve the adoptive immunotherapy and its outcome. the novel, short time generation protocol did not affect phenotyp and cytokine production of the t cells. nevertheless their therapeutic potential in vivo has to be tested in further experiments. s. s. schmucker 1 , m. assenmacher 1 , a. richter 1 1 miltenyi biotec gmbh, r&d cell biology, bergisch gladbach, germanyadoptive transfer of virus-specific t cells provides a promising treatment of infection in immunocompromized patients. as expansion of virus-specific t cells from antigen-experienced donors is feasible, no reliable protocols for generation of antigen-specific t cells from naive hosts exist. in this study we established a cell culture system for priming of highly rare naive cmv pp65-specific cd4 + and cd8 + t cells from cmv-seronegative donors in vitro.magnetically isolated naïve (cd45ro -cd25 -) cd4 + and cd8 + t cells from pbmc of cmv-seronegative donors were co-cultured with autologous mature monocytederived dc loaded with cmv pp65 peptide pool and cd3-depleted autologous pbmc as feeder cells in the presence of il-2, il-7, and il-15. already 9-13 days after primary activation pp65 495-503 /a2-tetramer + cd8 + t cells were detectable for 3 hla-a2 + blood donors. to analyze cd8 + t cells having other specificities than for the peptide pp65 495-503 as well as probably primed cd4 + t cells, we looked for the production of cytokines after a second stimulation. we found ifn-g secretion in up to 3.9% of the cd8 + t cells and up to 3.8% of the cd4 + t cells after restimulation with pp65 peptide pool, but not with either irrelevant ie-1 peptide pool or without antigen, in each of eight donors tested. for generation of t cell lines, we magnetically enriched the primed t cells according to their ifn-g secretion. subsequent cultivation for 20 days led to a 10 -100 fold expansion of pp65-specific t cells, defined by their sustained capability to produce ifn-g. evaluation of the antigen-specificity of the expanded t cells also showed upregulation of the activation markers cd154 and cd137 only if restimulated with the pp65 peptide pool. further cytokine analysis of the cells revealed co-production of ifn-g, tnf-a, and il-2, indicating the functionality of the in vitro primed and expanded t cells.in conclusion, we established a cell culture system, which enables the in vitro priming and expansion of cmv-specific cd4 + and cd8 + t cells derived from the naive compartment. this should extend the application of adoptive t cell therapy to patients for whom immune donors are not available. a. i. wolf 1 , k. mozdzanowska 1 , l. otvos 2 , j. erikson 1 1 the wistar institute, philadelphia, united states, 2 temple university, philadelphia, united statesthe influenza virus a matrix protein 2 ectodomain (m2e) sequence has remained highly conserved among various human influenza a strains and is therefore a promising target for a protective vaccine. based on previous work using a synthetic m2e-based multi-antigenic peptide vaccine (mozdzanowska at al., vaccine 2003; virology journal 2007), we generated a novel peptide and investigated its efficacy in inducing an anti-m2e antibody (ab) response and its ability to confer protection against viral challenge.objectives: cytomegalovirus (cmv) causes significant morbidity and mortality in patients after haematopoietic stem cell transplantation (hsct). due to limitations of current antiviral therapies, alternative approaches, involving transfer of donor-derived cmv specific cd8 + t cells, have been considered. clinical data confirm a crucial role for antiviral cd8 + t cells inversely correlating with the incidence of cmv reactivation and disease. cmv specific cells have to reach protective levels in order to be effective. levels of such cells correlating with protection against cmv infection and disease have only been reported in patients expressing hla-a*0201 and hla-b*0702 previously. considering other frequent hla alleles cmv specific cd8 + t cells were monitored longitudinally in 30 hsct patients in this study to establish the cell number thresholds at which patients are protected from cmv reactivation. methods: we have correlated the pattern of different ex vivo cmv peptide specific cd8 + t cell responses (frequency analysis using tetramer staining and interferon gamma elispot analysis) with the cmv viral load (dnaemia) and clinical status in patients. different response groups were compared using the mann-whitney-u test.results: our results demonstrate that the presence of different cmv specific cd8 + t cells inversely correlates with the ability to detect of cmv reactivation in patients at different cell number thresholds. we show that the cell number thresholds for hla-a*2402/pp65 (341-349) (7.6x10 6 cells/l) and hla-b*3501/pp65 (123-131) (4.4x10 6 cells/l) specific cd8+ t cells are significantly lower than those for hla-a*0101/pp50 (245-253) (17.2x10 6 cells/l) and hla-a*0201/pp65 (495-503) (13.2x10 6 cells/l) specific cd8 + t cells in hsct recipients post transplant. this difference is also evident in healthy cmv seropositive volunteers. conclusion: these findings suggest a differing efficiency of the responses restricted by the two sets of alleles. the data merit further studies using larger patient cohorts and are important for considerations regarding the epitope restriction and quantities of ag specific t cells to be monitored after therapeutic strategies for cmv in hsct patients. (2,5 -50 mcg) . no adverse effects were indicated during trials (up to 25 month of observation).hiv-specific antibodies were induced by dose-dependent manner, the most prominent response was detected after 4th immunization with 50 mcg of vichrepol.no differences were detected in cd4+ and cd8+ t cell counts and cd4+/cd8+ ratio, so there was additional safety issue concerned to the possible sensitivity of vaccinees to hiv infection. the results of phase i clinical trials of vichrepol vaccine were approved by who authorized russian national control institution and transition to phase ii immunogenicity trials was recommended. objectives: to improve the vaccination efficiency of adenoviral vectors for anti-retroviral vaccination, we constructed adenoviral nanoparticles by fusion of the vaccine antigen to the adenovirus capsid protein pix. the adenoviral nanoparticle vaccine was evaluated in the friend virus (fv) mouse model and compared to conventional adenoviral vectors. methods: adenoviral nanoparticle vectors were constructed by deletion of pix from the adenoviral genome and insertion of the fusion protein encoding sequence as transgene. for vaccination against fv, that is a retrovirus complex of friend murine leukemia virus (f-mulv) and spleen focus forming virus, we constructed fusion proteins of pix and the f-mulv surface env protein gp70 or gag. to elucidate underlying mechanisms we produced displaying-only nanoparticles and plasmid dna encoding either pixgp70 or gp70 alone. conventional adenoviral vectors were used that express full-length f-mulv env and gag. the vaccines were tested in cb6f1 hybrid mice that are highly susceptible to fv infection and develop viremia and splenomegaly after fv infection. results: vaccination of cb6f1 mice with adenoviral nanoparticles expressing fusion proteins containing gp70 resulted in protection from viremia and splenic enlargement after fv challenge that was superior to vaccination with conventional vectors. immunological analyses showed that the adenoviral nanoparticle vaccine induced a significantly higher number of f-mulv env-specific cd4 + t cells and higher antibody titers than a conventional adenoviral vaccine expressing the vaccine antigen. we could show that for the beneficial effect it is necessary that the fusion protein is incorporated into the adenoviral particle and it also has to be expressed from the adenoviral vector in vivo. conclusion: adenoviral nanoparticles are a useful tool for the induction of antibody and cd4 + t cell responses that are superior to conventional adenoviral vectors. this new type of adenovirus-based vaccination vector combines genetic and protein vaccination and should make adenoviral vectors even more interesting for vaccination purposes. . antibody levels were monitored by elisa and hemagglutination inhibition assay, viral excretion in nasal washes was assessed by quantitative rt-pcr, and cellular production of ifn-gamma was measured via flow cytometry. results: we found that animals vaccinated with caf01 exhibited higher levels of serum igg and mucosal iga than the ones which received the vaccine alone, and that they excreted 90-99 % less virus. animals that received only vaxigrip were producing ifn-gamma after challenge, a sign of infection by low virulence influenza strains, whereas the animals that received also caf01 did not show any increase in their levels of ifn-gamma. conclusion: caf01 enhances the protection conferred by the commercial inactivated vaccine against strains matched by the vaccine. evaluation of the t-cell specific immune response is very important for global eradication of measles and rubella. peripheral blood lymphocytes (pbl) from 13 children aged 1-2 years old (6 boys and 7 girls) -group 1, and 11 children (6 boys and 5 girls) 6-7 years old -group 2 were isolated on a gradient of density before vaccination ( or revaccination) with priorix, 1 week, and 2 months after and incubated with cfse. then 2 million/ ml pbl were incubated in rpmi-1640 supplemented with 10 % fcs (the negative control), at presence of 5 mcg/ml pha (the positive control) or at presence of the measles or rubella viruses antigens in a humidified atmosphere containing 5 % cî 2 at 37°c within 7 day. intensity of a fluorescence estimated on fl1 by flow cytometr facscalibur (bd biosciences, usa). cytokines production was measured in the same cultures by bioplex technology (biorad, usa). in the negative control 90 % pbl in both groups did not enter mitosis. in the positive control 90 % of cells have passed one and more mitoses. in group 1 measles or rubella antigens did not induced lymphocytes to enter mitosis, like in negative control, before the vaccination and in a week, however in 2 months 15-25 % of lymphocytes demonstrated antigen-specific proliferation. in group 2, on the contrary, before the vaccination the most part of cells (75-80 %) has not entered division, but 20-25% of cells have passed 2 and more mitoses. in a week specific lymphocyte proliferation decreased and in 2 months it was increased up to 40-50 %. production of the interleukin (il) 6, ifn-g, tnf-a, il-4, il-1 was more informative than il-5, il-7, il-8, il-12. measles and rubella antigens induced cytokines production in pbl of immune children and did not influence on pbl of intact children. thus, it was shown, that both methods can be applied to revealing the specific cellular immune response to measles and rubella antigens. objectives: broadly neutralizing human monoclonal antibodies (mab) and patients' sera recognizing functionally conserved epitopes on hiv envelope (env), such as the gp120 cd4-binding site (cd4bs), appear to be uncommon. therefore, new approaches are needed to elicit the humoral response on these conserved epitopes. here we describe the generation of two anti-idiotype (ai) murine antibodies recognizing human anti-hiv-1 neutralizing polyclonal iggs directed against the cd4bs. the mabs were shown to react with an anti-cd4bs human neutralizing mab (b12), to elicit antibodies that recognize the gp120 molecule and an anti-hiv-1 neutralizing response in rabbits, confirming them as cd4bs mimotopes. these mabs were also used as probe to detect the expression of clonally distinct anti-gp120 antibodies in sera of hiv-infected individuals. methods: broadly neutralizing sera were collected from long-term non-progressor patients. anti-cd4bs iggs were purified and used to immunize mice for hybridoma generation. mabs reacting in elisa with the anti-cd4bs igg fraction were used to immunize rabbits. rabbit sera were then tested for anti-gp120 titer and hiv neutralizing activity by pseudovirus-based neutralization assay. sera from hiv-infected individuals at various clinical stage of infection were studied to validate an immunoenzymatic assay able to detect the reactivity to the ais. serial dilution of b12 in sera from healthy hiv-negative donors were used to determine elisa sensitivity. results: two clones (p1 and p2) reacted in elisa only with the cd4bs-directed igg fraction. the clones were also recognized in elisa by b12. p1 and p2-immunized rabbit sera showed a strong anti-gp120 titer. in the pseudovirus assay the ais-immunized rabbits showed a neutralization activity against virions bearing hxb2 strain glycoproteins. in particular, 3/5 rabbits in the p1 group and 1/5 in the p2 group showed an 80 % hiv neutralization at dilutions ranging from 1:20 to 1:150. the immunoenzymatic assay used, allowed to detect a p1 and p2 reactivity in hiv-positive sera and was able to detect a b12 concentration equal to 10 ng/ml. conclusions: these data demonstrate that immunogens designed on the idiotype of broadly neutralizing abs are feasible and could help in the design of effective anti-hiv vaccines or diagnostic assays. yellow fever vaccines (17d and 17dd) are well tolerated, with a very low rate of severe adverse events (yf-sae), such as serious allergic reactions, neurotropic (yf-and) and viscerotropic (yf-avd) diseases. viral and host factors have been postulated to explain the basis of yf-sae, especially those able to modify the host immune response to the yf vaccine. however, the mechanisms underlying the occurrence of yf-sae still remain unknown. in the present investigation, we present a detailed immunological analysis of a 23-year-old us citizen female patient, who developed yf-and characterized by encephalitis associated with pancreatitis and myositis following 17d-204 vaccination. our findings highlighted that yf-and exhibited decreased expression of fc-g-r in monocytes (cd16, cd32 and cd64) along with increased levels of nkt-cells (cd3 + cd16 +/-cd56 +/-/cd3 + ratio) and activated t-cells (cd4 + and cd8 + ) and b-lymphocytes. enhanced levels of plasmatic cytokines (il-6, il-17, il-4, il-5 and il-10) besides exacerbated ex vivo intracytoplasmic cytokine pattern, mainly observed within nk-cells (inf-g + , tnf-a + and il-4 + ), cd8 + t-cells (il-4 + and il-5 + ) and b-lymphocytes (tnf-a + , il-4 + and il-10 + ). the analysis of cd4 + t-cells revealed a complex profile with increased frequency of il-12 + and ifn-g + and decreased percentage of tnf-a + , il-4 + and il-5 + cells. depressed cytokine synthesis was observed in monocytes (tnf-a + ) following in vitro antigenic stimuli. these results support the hypothesis that a robust magnitude of the adaptive response and abnormalities in the innate immune system may be involved in the establishment of yf-sae. this is the first case report of yf-sae investigated by members of the international laboratory network for yellow fever vaccine associated adverse events. g. mester 1 , h.-g. rammensee 1 , s. stevanović 1 1 eberhard-karls-universität tübingen, department of immunology, tübingen, germany adenovirus (adv) is a widespread pathogen in humans and can persist in its hosts after infection. persistent virus is an important cause for severe disease in immunocompromised individuals, e. g. bmt recipients, with high rates of mortality. however, the cellular immune response against adv is poorly characterised, and very few t cell epitopes have been published up to now. thus, our aim was to detect dominantly immunogenic adenoviral cd8 t cell epitopes by analysing the responses of healthy blood donors who have overcome infection. we have predicted possible cd8 t cell epitopes for the frequent mhc class i alleles a*01, a*02, and a*24 from the proteins pii (hexon), pviii, and e1a of adv strains ad2 and ad5 by using the syfpeithi software developed by our group (www.syfpeithi.de). subsequently we performed a 12-day recall stimulation of pbmcs from at least 16 healthy donors with synthetic peptide followed by ifn-g elispot screenings to identify naturally occurring t cell responses and assess their frequency in the population. tetramer and intracellular cytokine stainings were also carried out to confirm the presence of specific cd8 t cells. we could identify 27 new peptides eliciting ifn-g responses, several of which were confirmed as novel cd8 t cell epitopes. amongst others we found at least one immunodominant epitope recognised by more than half of the healthy donors for each examined hla restriction as well as, to our knowledge, the first adenoviral epitope derived from a protein other than hexon. these findings will be helpful to identify frequently immunogenic and thus promising candidate peptides for in vitro t cell priming or expansion preceding adoptive transfer, which has been proven to be a valuable therapeutic approach in the treatment of persistent viruses in immunocompromised patients. methods: sle (5 ara criteria) was diagnosed in a 40-year-old african female patient with hiv-1 (clade c) infection. good initial response occurred on hydroxychloroquine and steroids followed by disease flare and drop of cd4 t-cell count x 200 cells/mm 3 . initiation of 500 mg mmf bid was associated with biological and clinical remission of sle and cd4 t-cell increase. no opportunistic infections or cancers were noted during a 3-year follow-up and the patient remained always naive to art. hiv-1-specific cd4 and cd8 t-cell responses were analyzed after 18 months of mmf by ifn-g elispot assay and polychromatic flow cytometry assessing ifn-g, tnf-a and il-2 production following stimulation with a panel of 192 hiv-1-derived optimal epitopes (9/10-mers) covering various hiv regions and a pool of 105 hiv-1-derived peptides (15-mers overlapping by 11 aminoacids) encompassing the entire gag protein. all peptides are derived from hiv-1 consensus strain iiib. results: highly polyfunctional hiv-1 specific cd4 and cd8 t-cell responses against gag were detected. 11 epitope-specific cd8 t-cell responses were identified: except for one response restricted by hla a*23 and another one by hla cw*07, all the others were restricted by hla-b alleles and mostly by b*58 (n = 5). seven out of 11 responses were strong enough to be further analysed with regard to their functional profile and shown to be highly polyfunctional (i. e. ifn-g+, tnf-a+ and il-2+) regardless of the viral region and hla restriction. conclusion: strong, broad and polyfunctional hiv-1 specific cd4 and cd8 t-cell responses known to be associated with nonprogressive infection were detected during mmf treatment.we therefore suggest that mmf use in the context of sle-hiv is not detrimental to the establishment or preservation of protective hiv-1 t-cell immunity. the rabies virus was propagated in the vero cell line. virus was titrated by focus fluorescent units. virus preparations having a titer of 10 6 dl50/ml were inactivated with b-propiolactone. aluminium hydroxide gel or squalene, at different concentrations were adsorbed to the inactivated rabies virus. male mice of the strain cf-1 of 12-16 g and no less than four weeks age, were distributed in six groups for intraperitoneal immunization, group a was immunized with virussqualene, group b with virus-aluminium hydroxide, group c with the antigen alone, group d with saline buffer-squalene, group e with saline buffer-aluminium hydroxide and group e was inoculated with mock-infected cell culture supernatant. mice were boosted at the 7 th day. all mice were properly bled to prepare preimmune sera and hyper-immune sera. at the end of the immunization protocol the igg raised against the rabies virus was tested by an indirect elisa. results: the highest titers of neutralizing antibodies were obtained with similar concentrations of either squalene-or aluminium hydroxide-based vaccine formultaions. there was a significant difference in the neutralizing antibody titers produced by mice immunized with the antigen (inactivated rabies virus) adsorbed to the adjuvant, as compared to those obtained from mice immunized with the antigen alone, as expected, no neutralizing antibodies were detected on mice inoculated with saline buffer or mock-infected vero cell supernatant. conclusions: the use of either squalene or aluminium hydroxide as adjuvant in the canine antirabic vaccine formulation increases immunogenicity, almost to the same extent. aluminum hydroxide adsorbed to the antigen seems to be a better option, since squalene is more expensive than aluminium hydroxide. supported by: concyt-46767, cofaa and cgpi-20090305. . state of vaccine-induced measles immunity was determined by means of elisa in 1-3, 4-6 and 7-9 years since revaccination with live measles vaccine (lmv) before and after tuberculosis chemoprophylaxis. statistic data were processed with t-, w-and u-criteria. results: during the first three years since lmv revaccination igg level was middle (children with negative and long-term positive mt) and high (children with conversion and hyperergic mt). in 4-6 years since lmv revaccination uninfected and long-term infected children showed a significantly decreased (p p 0.05) measles immunity and antibody level much lower (p p 0.05) than among children with mt conversion. in 7-9 years the comparison group kept decreased (p p 0.05) measles immunity, the majority (92±5.54 %) of persons had minimal protected igg level, but the observation groups were characterized by average immunity level, which was higher (p p 0.05) than in the comparison group. comparing measles immunity level before and after tuberculosis chemoprophylaxis demonstrated the following: measles igg level among long-time infected children on completion of chemoprophylaxis decreased (p p 0.05), the majority (83.3±4.1 %) of persons lost protected antibody level; among children with mt conversion in 1-3 years since lmv revaccination immunity state didn't change, but in further periods antibody level decreased (p p 0.05) to low values; among children with hyperergic mt igg level decreased (p p 0.05) and reached low (in 1-3 years), minimal protected (in 4-6 years) and lower than protected (in 7-9 years) values. -at the early stage of tubercular infection process measles immunity was higher compared to uninfected with mycobacterium tuberculosis persons, which fact is connected with immunomodulatory action of low-molecular peptide of bacterial cell wall -muromildipeptide.-in remote periods since lmv revaccination and on completing preventive tuberculosis treatment decreased measles immunity was observed.-in countries with high tuberculosis morbidity chemoprophylaxis level among children with latent infection is high, which can indirectly influence population measles immunity. objectives: to evaluate the balance of ifn-gamma and il-17 producing cells in lungs during the immunotherapy of tuberculosis with the dna vaccine encoding the heat-shock protein 65 (dnahsp65). methods: balb/c female mice were infected by intra-tracheal route with 10 5 h37rv mycobacterium tuberculosis. immunotherapy with endotoxin free dnahsp65 genetic vaccine was done at days 30, 40, 50 and 60 post-infection. each dose consisted of 100 micrograms of dna vaccine in the quadriceps. intracellular cytokine staining of cd4+, cd8+ and gamma-delta t cells from lungs were determined 10 and 50 days after the end of the therapy. bacilli loads, histopathological and morphometric analysis of lungs were also evaluated. differences of p x 0.05 were considered significant (t test). results: at day 10 after the end of the immunotherapy, dnahsp65 treated mice exhibit increased numbers of absolute cd8+ and gamma-delta t cells when compared to non-treated animals. the percentage of ifn-gamma and il-17 producing gamma-delta t cells were the same between treated and non-treated animals. in contrast, dnahsp65 treated mice showed more ifn-gamma producing cells in both cd8+ and cd4+ cell populations. at day 50 after the end of the therapy, the main observation in mice which received dnahsp65 treatment was the augment of all three populations producing ifn-gamma. although non-treated animals also increased the frequency of cd4+ and gamma-delta t cells positive for ifn-gamma, they did not increase the numbers of ifn-gamma cd8+ cells, together with a more frequency of gamma-delta t cells producing il-17. finally, the immunotherapeutic effects of dnahsp65 vaccination also included the diminution of bacilli loads in lungs, spleen and liver and the reduction of inflammation in lungs as determined by the histopathological and morphometric analysis. the results presented here indicates that cd8+ cells producing ifn-gamma and the reduction of the frequency of gamma-delta t cells secreting il-17, are the main effects of dnahsp65 immunotherapy of murine tuberculosis. furthermore, these results have important implications since they indicate the importance of an appropriate balance of il-17 and ifn-gamma levels for the combat of the bacilli and the reduction of the immunopathologic damage in lungs. the detection of quantitative changes in mrna expression levels are currently being performed using either genome-wide (microarray) or single gene (real-time pcr) screening methods. because these techniques are technically challenging and too costly to be applied on a routine basis in resource poor settings, we have developed a reverse-transcriptase multiplex ligation-dependent probe amplification (rt-mlpa) method. rt-mlpa is a reliable, robust, low cost and user friendly technique permitting rapid mrna expression profiling of as many as 60 loci in a single reaction. genes of interest can be selected on a tailor-made basis. the assay is highly reproducible, has an extensive dynamic range of 3-5 log depending on the genes of interest, and a pcr amplification step within the rt-mlpa ensures assay sensitivity, which is an essential prerequisite for the relative quantification of scarcely expressed genes. since this assay is relatively high throughput (96-well format), requires only 100 ng rna per sample, and allows mrna profiling in direct ex vivo whole blood samples (from e. g. pax-gene tubes), it is an exceptionally suitable technique for performing semi-large scale gene expression analyses in human cohort studies. to illustrate this, we have been able to successfully implement this assay in 5 different laboratories in sub-saharan africa. thus far we have applied rt-mlpa to characterize the human immune response to mycobacterium tuberculosis, with particular emphasis on the expression of genes associated with protective host cellular immunity and human disease susceptibility. a particularly useful application of the rt-mlpa is the identification and monitoring of host-biomarker profiles that predict (protection from) tuberculosis (tb) disease in latently infected household contacts or (in)adequate responsiveness to therapy in active tb patients. initial data sets already probe differences in immune reactivity in populations, yielding new candidate biomarkers associated with tb disease. these biomarkers may provide new and relevant information that can be applied in future tb studies for rapid, easy, semi-quantitative and reliable detection of host immune biomarker profiles. preclinical m. leprae infection is a major source for leprosy transmission. therefore, early detection of individuals infected with m. leprae is crucial. however, to date there are no diagnostic tests available that can identify preclinical leprosy. such tests will contribute to the prevention of leprosy disability and its further transmission by otherwise undiagnosed and untreated index cases.newly developed hla based bio-informatic tools combined with comparative genomics have created novel opportunities to help design improved tests for early detection of m. leprae infection.using this post genomic approach, we were able to identify candidate proteins and peptides unique to m. leprae containing predicted t cell epitopes restricted via several major hla-class i and ii alleles. since the selected genes were of unknown function, their expression in m. leprae bacilli was assessed.evaluation of the immunogenicity of these m. leprae proteins in pbmc from a brazilian population showed that 5 candidate antigens induced significant ifn-g levels in m. leprae infected individuals but not in healthy controls from an endemic area.importantly, among exposed healthy controls 71 % had no detectable igm antibodies to the m. leprae specific pgl-i, but instead responded to one or more m. leprae antigen(s). to further improve the diagnostic potential of these m. leprae sequences, synthetic peptides spanning all 5 m. leprae proteins were analyzed similarly. determination of cumulative t cell responses towards 4 of these peptides that activated pbmc of leprosy patients increased the sensitivity compared to single peptides to 100 % in pb, 75 % in rx and 93 % in hhc, without compromising specificity.since diagnostic tools should be applicable in several populations regardless of the genetic background, these m. leprae antigens are also tested in populations on the african (ethiopia) and asian (nepal) continent.in addition, we have applied these antigens in a new user-friendly ucp-lf assay to detect different cytokines. this assay proved to be more sensitive than elisa for detection of ifn-g and can be easily applied in field sites. tuberculosis, an infectious disease caused by mycobacterium tuberculosis (mtb), affects millions of people. m. bovis bcg is the vaccine against tuberculosis but its efficiency is variable for the pulmonary form of the disease. paratuberculosis, an enzootic bacterial disease in ruminants, due to mycobacterium avium subsp. paratuberculosis (map), has a significant economic impact on livestock production, and moreover, map infection may be one of the microbial triggers of crohn's disease in humans. map vaccines can delay apparition of clinical symptoms, but they do not prevent infection and they have a confounding effect in the skin-test based bovine tuberculosis control programs. cd40l, a co-stimulatory molecule preferentially expressed on activated cd4+ t cells, is the ligand of cd40. cd40-cd40l interaction induces the production of il-12 and the initiation of a th1-type immune response. several studies show that cd40l is required for the activation of macrophages and the maturation of dcs. moreover, cd40l enhances the capacity of cd8+ t cells to produce ifn-g and to lyse mtb-infected monocytes. in this study we attempt to improve existing tb and map vaccines with a recombinant bcg expressing cd40l. we have constructed the recombinant bcg strain expressing cd40l (rbcg2) by electroporation of bcg with pgfm11/signalsequenceag85b-cd40lec and an another recombinant strain with empty vector pgfm11 (rbcg1) as a control. the expression of cd40l has been evaluated by western blotting. balb/c mice were vaccinated with the recombinant bcg vaccines. bcg growth kinetics were compared by counting viable bacteria (cfu) in spleen and lungs. the immune response was evaluated by measurement of th1 type cytokine secretion of splenocytes after in vitro restimulation with immunodominant antigens and selected peptides. two months post vaccination, mice were challenged with mtb and map and protection was evaluated. preliminary results show normal persistence of the two recombinant bcgs. analysis of the immune response shows an effect of cd40l 2 weeks after vaccination but not at 4 and 8 weeks. rbcg2 seems to be more protective against paratuberculosis than rbcg1, but not against tuberculosis. another vaccination experiment is required to confirm these results. the effects of bcg-cd40l on cultured dcs in vitro will further be explored. objectives: tuberculosis is a major health problem globally and it is of critical importance to develop an effective vaccine to prevent further spread of the disease. iron is a key nutrient for both mycobacterial infection and for a successful protective immune response by the host. the regulation of iron availability within the host involves the intracellular iron-binding protein ferritin and it is proposed here that the regulation of ferritin is tightly controlled in the host immune response to tuberculosis. methods: using the guinea pig model of mycobacterium bovis bacillus calmette-guérin (bcg) vaccination, populations of immune cells were isolated and restimulated ex vivo over a time-course study using purified protein derivative (ppd) of mycobacterium tuberculosis or infected with bcg or m. tuberculosis. the expression of ferritin in co-ordination with key immuno-regulatory proteins, tnfa, ifng and il-1a, was examined using real-time pcr. to determine whether immuno-regulatory proteins are involved in the regulation of ferritin, cytokine cascades were inhibited in the ppd re-stimulation studies by the addition of guinea pig specific tnfa and ifng antibodies. results: a typical pro-inflammatory immune response was observed with significant up-regulation (p x 0.05) of tnfa, ifng and il-1a after re-stimulation with ppd and mycobacteria. of interest was a trend in ferritin down-regulation after re-stimulation with ppd and bcg and this was significant (p x 0.05) after restimulation with m. tuberculosis. the down-regulation of ferritin was also affected by the addition of tnfa antibody in the ppd re-stimulation study. conclusions: ferritin is important in the storage and management of intracellular iron and its regulation must be tightly controlled to restrict iron availability from invading mycobacteria from sequestering free iron. the data indicate that the regulation of ferritin is very subtle and is affected by cytokine cascades that involve tnfa. these results contribute to our understanding of the role of iron and intracellular ferritin in developing a protective immune response to mycobacteria in the guinea pig model of tuberculosis. this work is funded by health protection agency phd studentship award. methods: anti-cd40 mab and ag85a were chemically treated with sata and sulfo-smcc respectively, in order to produce a stable crosslinker between both proteins. crosslinking was confirmed by western blotting and cd40 binding on cd40 transfected l929 fibroblasts. the conjugates were tested in vivo in wild type and cd4 + cell-depleted mice for the induction of specific anti-ag85a serum antibodies. splenocytes were challenged ex vivo with ag85a and were examined for their ability to produce th1-related cytokines. elispot assays were performed to determine ifng production and flow cytometry was used to analyse intracellular cytokine staining for tnfa, ifng and il-2. we developed a method to successfully crosslink anti-cd40 mab to ag85a. serum antibodies against ag85a were detected after immunisation with this conjugate vaccine in both wild type and cd4 + cell-depleted mice. t cells derived from mice immunised with conjugate vaccine, and stimulated ex vivo, showed an increase in ifng production (elispot), when compared to mice vaccinated with ag85a alone. production of two other th1-related cytokines, tnfa and il2, was also increased in these t cells as shown by intracellular cytokine staining. conclusion: our results suggest that anti-cd40 conjugate vaccines could provide a new way to increase vaccine efficacy. this new conjugate vaccine may be able to by-pass the need for cd4 + t cell help in the production of specific antibodies, which would be a major benefit of any therapeutic vaccine to be used in immunocompromised patients. h. schäfer 1 , r. burger 2 1 robert koch-institute, cellular immunology, berlin, germany, 2 robert koch-institute, infectious diseases, berlin, germanyobjective: immunity against mycobacterial infections is mediated by both cd4-positive and cd8-positive t-lymphocytes. cd8-positve cells respond to peptides derived from cytosolic proteins and presented on mhc class i molecules of antigen presenting cells (apc) via the endogenous pathway. some apc however, are able to take up extracellular antigens and present peptides thereof on mhc class i molecules. this process has been termed cross-presentation and has been shown to be of importance in the immune response against intracellular bacteria. to define the contribution of cross-presentation to activation of cd8+ t cells in the response against mycobacterial antigens, we analyzed the secondary immune response in the guinea pig. methods: purified t lymphocytes from guinea pigs immunized with bcg or complete feund's adjuvant were labeled with the intracellular fluorescent dye cfse and incubated with ppd and/or apc for 5 days. surface phenotype and proliferation of t-lymphocytes were analyzed by flow cytometry.results: up to 30 % of lymph node t lymphocytes form immunized guinea pigs proliferated after in vitro restimulation with ppd-pulsed macrophages. no difference was observed between bcg-(living mycobacteria) and freund's adjuvant (heat killed mycobacteria)-immunized animals. the responses of both t cell subsets were equally strong, although the killed immunogen should primarily target the exogenous pathway of antigen presentation and therefore preferentially prime cd4+ t-lymphocytes in vivo. similarly, the cd4-positive subpopulation should primarily respond to soluble antigens presented on mhc class ii molecules. proliferation of both the cd4+ and the cd8+ subpopulation depended on the presence of apc. stimulation oft cd8+ cells as a consequence of direct loading of peptides onto mhc class i molecules was ruled out by using mhc-class i-positive fibroblast cells instead of professional apc, which did not lead to proliferation of primed t-lymphocytes. conclusion: cross-presentation of soluble antigens to cd8-positive t cells is a highly effective means to stimulate the response of cytotoxic t cells against mycobacterial antigens even without direct contact to infected cells. therefore cross-priming might represent an important mechanism for the induction of the cellular immune response against intracellular pathogens and should be useful for the rational design of vaccines against mycobacterial diseases. objectives: due to broad antigenic cross reactivity of purified protein derivatives (ppd) with bcg vaccine strains and environmental mycobacteria, results of currently used tuberculin skin test (tst) is not reliable to evaluate the specific anti-tuberculosis immune response. therefore, new tools are required to improve mycobacterim tuberculosis (tb) diagnosis and treatment, including enhanced ability to compare new treatment strategies. among different antigens early secretory antigenic target 6 (esat-6) protein is highly specific for tb complex and elicit strong t-cell response in human. in order to monitor the immune response against the pathogen, 83 iranian and afghan adults (38 patients with sputum smear and culture positive tuberculosis, 24 recovered patients during 6 months after full course of chemical treatment and 21 healthy individuals) were recruited to quantify the frequency of esat-6 and ppd specific t-cells in their peripheral blood by home made elispot ifn-gamma assay. results: considering cut off of 4 spot forming unit ( g 20 spots per million), we found detectable response to esat-6 in almost 80 % of patients with active disease. this frequency among treated patients after disease recovery was not significantly different and 77 % of these individuals had detectable esat-6 specific response even after six months completing treatment. neither of healthy individuals showed such response. t cell response against ppd was identified in 14 %, 57% and 62 % of healthy participants, active patients and healing individuals, respectively. conclusion: elispot ifn-gamma assay showed a sharp induction of th1 immune response, against esat-6, in tb patients which persists after successful treatment and full recovery. these results may show potential application of tuberculosis-specific elispot testing as a proxy measure of tb diagnosis and treatment. bcg is the only available vaccine today to fight tuberculosis, but it has been reported to be variably efficacious in the field. both environmental and genetic host factors as well bcg strain variability and virulence of the intruding m. tuberculosis strain have been suggested to affect the efficacy of bcg vaccination. in mouse and bovine models it has been shown that pre-exposure to mycobacterium spp. negatively affected protective efficacy of bcg. we use non-human primates (nhp) for the evaluation of new tb vaccine candidates and possible identification of immune mechanisms of protection. however, in naive rhesus macaques a variable efficacy of bcg reminiscent of the clinical situation was revealed. by meta-analysis we compared immune response parameters measured after vaccination using bcg strain danish 1331 in the context of protection measured by gross pathology evaluation after experimental infection with a constant m. tuberculsosis strain erdman. although numbers of animals used are relatively low, data suggest that both breeding origin as well as the immune status of monkeys impact on the efficacy of bcg. most remarkably, while bcg induced levels of ifng secretion did not correlate with protection, kinetics of secretion monitored after in vitro stimulation of peripheral blood lymphocytes did correlate. our findings would suggest that, in accordance with mouse and bovine experimental data and epidemiologic observations, possible pre-exposure to mycobacterial antigens beyond the current sensitivity of tb diagnostics for nhp, negatively affects the protective efficacy of bcg. together, these results are relevant for evaluation and interpretation of tb vaccine tests in nhp and support further research into the identification of (mechanisms of) protective immunity in the primate host. p. s. nagpal 1 , p.k. upadhyay 1 1 national institute of immunology, pdc-1, delhi, indiaobjective: study was aimed for the preparation of dry powder formulation containing live mycobacterium indicus pranii for pulmonary immunization against tuberculosis. pulmonary delivery evokes both systemic as well as mucosal immune response against the antigen. secondly, pulmonary delivery is a needle-free delivery system, long desired for vaccine delivery. dry powder aerosol one such method, in which vaccine can be directly delivered to lung without any kind of invasion and it has an edge over liquid formulation being feasibility of storage at room temperature, long term shelf stability and higher drug content per unit mass as compared to liquid one. method: sodium alginate solution with suspended mycobacterium indicus pranii was aerosolized using laboratory modified nebulizer assembly. aerosols so generated were entrapped in cacl 2 solution with poly-vinyl alcohol (pva) as surfactant. particle so formed collected by centrifugation and lyophilized for dry powder formulation. pva and alginate concentration varies the size, surface and shape of the particles. formulation so prepared was delivered to c57bl/6 mice directly into lung by endotracheal intubations of mice. proliferation index (pi) of spleenocytes of immunized mice was measured after in-vitro stimulation with mycobacterium tuberculosis antigen. result: 2.4 % alginate, 0.012 % pva concentration gives particles with size of 2-10 micrometer as confirmed by particle size analyzer and scanning electron microscopy. viability of the mycobacterium indicus pranii was best achieved with 5 % trehelose and 3.5 % pvp (poly vinyl pyrollidone). there was 6 fold increase in proliferation index of spleenocytes and releases 600 pico-gram of interferon gamma after 4 week of immunization. formulation also induces the activation of dendritic cells after their in-vitro incubation as shown by 10 % increase in cd86 and 10.5 % in ccr7 expression as compared to blank alginate formulation. bacterial exopolysaccharides (epss) are heterogeneous polymers containing a wide array of homo-or hetero-carbohydrates as well as organic and inorganic substituents. epss are produced by many bacteria and play a critical role in helping these microorganisms to cope with adverse environmental conditions. some epss contain sulphate groups as inorganic substituents. the presence of these groups contributes to the biological activity of epss, which have been shown to have anticoagulant, insulinotropic, antiviral, antitumoral and immunomodulatory properties, among others. b100 is a constitutively sulphated eps produced by halomonas maura, a recently discovered halophilic bacterium. in preliminary experiments we found that modification of its eps by adding sulphate groups to the native polymer (thus obtaining b100s) resulted in vigorous antiproliferative activity in several haematopoietic tumour cell lines. at the same time we found that other epss produced by closely related strains had only a very limited antiproliferative effect on these same tumour cells. it was therefore of interest to determine whether the antiproliferative activity of b100s was mediated by the induction of apoptosis, and if so, to dissect the pathway triggered by b100s. by cell cycle analysis we determined that b100s is able to induce apoptosis in up to 80 % of jurkat and molt-4 t cell leukemias. the examination of a large panel of haematopoietic and nonhaematopoietic cell lines revealed that apoptosis induced by b100s is restricted to cells of the haematopoietic lineage and that leukemic t cells are particularly sensitive to death induced by b100s, but that untransformed cells are not. a time-course of caspases activation indicated that caspase 9 is the first to be cleaved, followed by caspases 3 and 8, thus suggesting that b100s triggers apoptosis through the mitochondrial pathway. it is noteworthy that b100s also induces vigorous apoptosis in primary leukemic t cells obtained from the peripheral blood of patients. therefore, b100s may well provide a satisfactory therapeutic alternative to patients with acute t cell leukemias, since current antitumoral drugs are very inefficient in the treatment of these types of cancer. particulate antigen delivery tools have been shown to enhance the induction of immune responses by targeting dcs. polyelectrolyte microcapsules form a new class of microcapsules generated by the sequential adsorption of oppositely charged polyelectrolytes onto a sacrificial spherical template which is consequently dissolved, yielding a hollow microcapsule surrounded by a thin shell. this layer-by-layer approach allows an efficient incorporation of macromolecules under nondenaturing conditions. by using the biopolyelectrolytes dextran-sulphate and poly-l-arginine, biodegradable microcapsules can be obtained. in this study, we have chosen the lungs as a non-invasive route for vaccine delivery. as demonstrated by flow cytometry and confocal imaging, dextran-sulphate/poly-l-arginine microcapsules were readily taken up by local pulmonary apcs and transported to the mediastinal lymph nodes, making them excellent tools for antigen targeting towards apcs. microcapsule instillation also affected the pulmonary apc activation status, indicated by the emergence of an apc population expressing increased levels of mhcii, the co-stimulatory ligands cd40, cd80 and cd86, and of the inflammatory cytokines il-6, il-23 and mcp-1. using ovalbumin (ova) as a model antigen, we have analysed the adjuvant properties of these polyelectrolyte microcapsules. analysis of the alveolar infiltrate, cd4 t cell and antibody profiles revealed that polyelectrolyte microcapsules display different adjuvant properties than the standard th2 and th1/th17 skewing adjuvants alum and complete freund's adjuvant (cfa). in response to ova aerosol exposure, microcapsule based vaccination resulted in an alveolar infiltrate dominated by monocytes, while alum and cfa respectively induced typical eosinophilic and neutrophilic inflammations. striking differences were also observed on the level of cd4 t cell responses. microcapsule based vaccination resulted in a marked induction of il-17 secreting th17 cells, without inducing strong th1 (cfa) or th2 (alum) responses. these differences were also reflected on the level of the humoral immune response, with microcapsules being the sole adjuvant producing antibodies of all isotypes tested (igg1, igg2c and ige).in conclusion, polyelectrolyte microcapsules allow an efficient targeting of antigens to lung apcs, and possess immune stimulating activities distinct from alum and cfa. due to their capacity to generate th17 responses, polyelectrolyte microcapsules may become interesting tools to combat fungal and bacterial infections. pneumolysin is an important virulence factor produced by virtually all clinical isolates of streptococcus pneumoniae. the protein binds to cholesterol in cell membranes and creates transmembrane pores, leading to cell lysis. published findings have proposed that, at sublytic concentrations, the toxin causes a range of effects including activation of host complement, activation and chemotaxis of cd4 + t cells and increased production of pro-inflammatory cytokines in immune cells. in this study we investigated the interaction of pneumolysin with murine dendritic cells (dc). we found that pneumolysin induced the activation of dc, reflected in the enhanced expression of the costimulatory molecules cd80, cd86 and cd40 and mhc class ii molecules. the toxin alone was found to be a poor inducer of cytokine production by dc but it did enhance the secretion of tlr agonist-induced cytokines such as il-6 and tnf-a. previous published findings have shown that pneumolysin activates peritoneal macrophages in a tlr4-dependent manner. however, we found that pneumolysin was capable of activating dc from both wildtype and tlr4-defective c3h/hej mice, by inducing cell maturation and synergising with tlr agonists to enhance cytokine secretion. importantly, we also found that pneumolysin is a strong inducer of il-1b secretion by dc, through its effects on caspase-1 processing, which is also tlr4-independent. the results suggest that pneumolysin is a potent stimulus for dendritic cell activation and that this does not require tlr4 signalling. objectives: transmission of immune competence from mothers to newborns during pregnancy and lactation is crucial for education of neonate immune system in order to develop optimal protection against early life infections. the objective of the present study was to assess whether maternal supplementation with probiotics may enhance neonatal responses to measles immunization. methods: pregnant balb/c mice were supplemented with placebo (maltodextrin) or probiotics (lactobacillus paracasei ncc2461 (st11) or lactobacillus rhamnosus ncc4007 (lpr), each at 5x10 8 cfu/day), suspended in the drinking water, throughout the gestation period and up to the weaning of pups. at weaning, pups were immunized with live attenuated measles vaccine (mv-s, aventis-pateur). weight evolution of pups was followed from week 1 to week 7 of life. fresh feces were collected at 3, 5 and 7 weeks of life for determination of iga levels (assessed by elisa). pups were bled 2 and 4 weeks after immunization for determination of measles-specific igg1 and igg2a antibodies. analysis of microbiota composition (plating on semi-selective agar media) was performed on fresh feces collected one week after weaning. results: all newborns grew normally and no significant differences in the weight were observed between the groups all along the trial. fecal iga production increased progressively in all pups from weaning, reflecting a normal development status. nonetheless, feeding mothers during pregnancy and lactation did not significantly affect post-weaning s-iga production in pups. lpr supplementation of the mothers significantly potentiated post-weaning measles-specific antibody responses in pups in comparison to control group. interestingly, no significant effect was observed in the st11-fed group. finally, a modification of the microbiota composition was observed in pups of supplemented mothers. particularly, there was a significant increase in lactobacilli in pups from the lpr group as compared to controls. conclusion: this study supports the benefit of perinatal intervention with probiotics during pregnancy and lactation on immune maturation in the offsprings. moreover, these first results seem indicate that the effects are strain specific. chitosan, (1-4)-2-amino-2-deoxy-beta-d-glucan, is a deacetylated form of chitin, an abundant biodegradable, positively charged natural polysaccharide. chitosan (chi) is used for antigen delivery through mucosal barrier due to its ability to disrupt tight junctions. here we studied the ability of chitosan nanoparticles to form complexes with proteins of different size and charge. nanoparticles (chi-np) were prepared from 200 kda chitosan by ionotropic gel formation. bovine serum albumin (bsa) and myoglobin, human immunoglobulin g and superoxidedismutase (sod), and chicken lysozyme were fitc labeled. chi-np were preincubated with proteins at 3:1 ration and washed 3 times. after washing chi-np containing bound proteins were run by denaturating gel electrophoresis. all proteins were able to form complexes. most effective binding was shown for bsa, sod, and lysozyme. the stability of chi-np complexes with proteins was studied in vitro on macrophage cell line raw264.7 by confocal microscopy. for this chi was labeled with rhodamine and nanoparticles were coincubated with fitc labeled proteins before addition to the cells. we showed co-localization of chi and fitc for all proteins studied. these results demonstrate that chi-np form stable complexes which are internalized by macrophages. the family euphorbiaceae consists of a large group of plants whose compounds have been documented to possess anti-inflammatory activities, however, their effects as modulators of innate or acquired immunity has not been described yet. in the present study, different aspects of the immunomodulatory activity of 24 extracts from 10 euphorbiaceaes on peripheral blood mononuclear cells (pbmc) from healthy individuals were evaluated. the pbmc were exposed to the extracts w/o phytohaemagglutinin a (pha), cycloheximide (chx) or lipopolysaccharide (lps) as agents that induce proliferation, apoptosis and cytokine production in pbmc. the lymphoproliferative activity of pbmc was evaluated by thymidine incorporation and cfse dilution assay using flow cytometry. the mitochondrial membrane depolarization (as an early apoptosis indicator) was measured using dioc6/propidium iodide staining by flow cytometry and tnf-a secretion in the culture supernatans by elisa. we found that 15 up to 24 euphorbiaceae's extracts had the ability to modulate one or more of the immune parameters evaluated in this study. however, only the bark extract of croton spp. insoluble in hexane:diclorometane:methanol (hdm) and the latex extracts of euphorbia cotinifolia and euphorbia tirucalli induced strong proliferation, apoptosis and also tnf-a production in pbmc. these extracts were subfractioned by sephadex column chromatography obtaining three subfractions with enhanced activity in comparison to the crude extracts. additionally, we started with the characterization of the specific immune effects of these subfractions on pbmc. all three subfractions induced proliferation predominantly on cd3+ cells. these effect was also observed in isolated t cells indicating that accessory cells are not necessary for the subfractions'activity. the lymphoproliferative activity of these subfractions was also not inhibited by the carbohydrates d-(+) galactose or a-methyl-mannopyranoside. these results demonstrate the presence of immunomodulatory compounds in plants from the euphorbiaceae family and suggest an antigen-presenting cell-and carbohydrate-independent mechanism of the subfractions to exert their effects. we found significant increase on lymphocytes and eosinophils populations obtained from lps + p. acnes-treated group in relation to control group.on 35 th day, we detected a significant negative correlation between eosinophils absolute number and fec. both il-5 and ige serum levels were increased on animals from group i when compared to control.the enhancement on th2 immune response pattern induced by lps and p. acnes treatment diminished drastically parasitic load. conclusion: our findings support the idea of the use of immunostimulant as a helminthiasis control strategy in sheep, which stimulate non-specific mechanisms of resistance and therefore can act against nematodes infections. vaccines based on partially purify populations from the organism or recombinant subunits proteins have been recently developed and are often not sufficiently immunogenic by themselves due to the lack of innate immune stimuli. indeed, current influenza a vaccines do not generate significant immunity against serological influenza a virus subtypes and would thus be ineffective in the face of a pandemic novel variant. hence adjuvant usually needs to be added to those types of vaccines. here we show that wittycell compounds significantly augment cellular and humoral immune responses to commercial seasonal influenza vaccines. experiments performed in mice showed induction of specific cd8+ ctl cells against conserved proteins that were accompanied by the induction of ifn-g producing cd4+t cells, following single immunisation. in addition increased hi titres and higher levels of specific igg2a and igg2b antibodies were found even long periods after single vaccination with reduced doses of vaccines. consequently, protection from lethality was observed following challenge with homologous or heterogonous influenza viruses in vaccinated animals. this promising finding on the improvement of seasonal influenza vaccines by wittycell compounds in these preclinical studies strongly provides support for the careful evaluation in phase i clinical trials in humans. s. lindgren 1,2 , n. almqvist 2 , a. lönnqvist 1 , s. östman 1 , c. rask 1 , e. telemo 2 , a.e. wold 1 1 university of göteborg, department of clinical bacteriology, göteborg, sweden, 2 university of göteborg, department of rheumatology and inflammation research, göteborg, swedenobjective: dietary antigens normally evoke immunological tolerance. a prerequisite is their processing by intestinal epithelial cells, which leads to the appearance of a tolerogenic form in the serum of fed animals that confers antigen-specific tolerance when transferred to naï ve recipients. the gut microbiota may influence the handling of dietary antigens as atopic diseases have increased in western societies in parallel with reduced complexity of the infantile commensal microflora. we have observed that children neonatally colonized with s. aureus in the gut seem protected against development of food allergy. here we examine whether a s. aureus toxin affects tolerogenic processing by the intestinal epithelium. methods: mice were given s. aureus enterotoxin a (sea; 0.8 mg/ml) in the drinking water for 5 days and, 3 days later, 50 mg ovalbumin per os. one hour postfeeding, serum was transferred to naï ve recipients, whose tolerance to ovalbumin was tested in a model of allergic airway inflammation (sensitization followed by intranasal challenge with ovalbumin). results: recipients of serum from sea pretreated ovalbumin-fed donors exhibited increased tolerance compared to recipients of serum from ovalbumin-fed donors not pretreated with sea. this was demonstrated as reduced ovalbumin-induced airway inflammation with diminished influx of eosinophils into the lungs and reduced antigen-induced production of interleukin-5 and interleukin-13. examination of gut sections from sea treated donor mice revealed increased density of cd8a + intraepithelial lymphocytes. our results show that sea promotes oral tolerance induction, possibly by facilitating tolerogenic processing of soluble antigens by the absorptive intestinal epithelium via activation of intraepithelial lymphocytes. abstract withdrawn by author to develop an efficient vaccine against cp pneumoniae we cloned chlamydial genes encoding proteins of the outer membrane like ompa, omcb, and pmp21, proteins of the inclusion membrane like incc, secreted proteins like cpaf, and the heat shock protein groel. cpg-dna 1826, a highly stimulatory oligonucleotide for apcs, was used as adjuvans. subcutaneous co-injection of ompa, omcb, pmp21, or groel together with cpg-dna 1826 reduced the chlamdial burden in nasally infected mice. however, symptoms like substantial loss of body weight were not influenced. in contrast, a low dose infection with cp. pneumoniae almost completely prevented the loss of body weight upon challenge. to improve the efficacy of the vaccine we used poly-dl-lactide-co-glycolide microspheres loaded with the protein ompa or pmp21 together with cpg-dna 1826. the microsphere based vaccine offers the advantage that antigen and adjuvans are delivered to the same apc. intranasal but not subcutaneous vaccination of mice with ompa or pmp21 microspheres efficiently lowered chlamydial burden upon challenge and prevented loss of body weight. pmp21 microspheres induced protective ifng-secreting cd4 + t-cells and raised pulmonary pmp21-specific iga levels in vivo. also, pmp21 microspheres caused lower il-6 serum levels upon administration than the injection of pmp21 together with cpg-dna 1826, indicating fewer side effects. objectives: staphylococcus (s.) aureus superantigens are highly potent t cell mitogens and the causative agents of toxic shock syndrome (tss) and food poisoning. most s. aureus have superantigens and patterns are highly variable. to date, the role of superantigens in bacteraemia is not well defined.to analyse whether superantigens play a role in bacteraemia, we investigated s. aureus strains and anti-superantigen antibody responses in 44 cases of s. aureus bacteraemia in iv drug users and 44 cases in nonaddicts. a rise in neutralising antibody titers indicates that superantigens are produced during infection. the study comprised 44 iv drug users with positive s. aureus blood culture and an equal number of age-and sex-matched nonaddicts from the original fintrova and finlevo trials (ruotsalainen 2006).all s. aureus isolates were analysed by sequence-based genotyping (spa-typing), and multiplex-pcr was applied to determine the superantigen gene pattern. sera from patients were obtained at diagnosis (day 0) and four weeks thereafter (day 28). neutralising capacity of the sera was tested against the superantigen cocktail produced by the respective infecting strain as well as a panel of representative recombinant superantigens.results: genetic analysis confirmed our previous observation that most strains harboured superantigen genes, which were linked to staphylococcal lineages (holtfreter 2007) . there were no major differences in superantigen gene patterns in isolates from iv drug users and nonaddicts. interestingly, the staphylococcal lineage st59 (spa-type t172, agr 1, and sea, seb, sek and seq) was much more prevalent among bacteraemia strains from iv drug users than from nonaddicts (p=0.01).most iv drug users had neutralising antibodies against enterotoxins already at onset of bacteraemia, likely due to previous encounters with the infecting strain. we frequently observed a rise in antibody titers during infection. surprisingly patients with st59 strains did not show any elevations in neutralising antibody levels. conclusion: s. aureus bacteraemia induces an antibody formation against staphylococcal superantigens. this indicates that superantigens are produced during infection. however, the action of superantigens is frequently modulated by specific neutralising antibodies. this and the special behaviour of s. aureus st59 strains need further investigation. objectives: down syndrome (ds) is associated with recurrent infections, hematological malignancies and auto-immune diseases, suggesting immunological changes. to test for more severely disturbed specific antibody response we investigated the antibody response to the highly immunogenic protein antigen tetanus toxoid (tt), which is part of the dutch immunization program. methods: after booster vaccination at 4 and 9 years of age, quantitative (titer) and qualitative (avidity) tt responses were investigated in 15 and 7 ds children, respectively. samples were taken before and 3-4 weeks after vaccination. tt-specific igg and igg-subclass antibodies were measured in serum by quantitative enzyme linked immunosorbent assay (elisa), avidity of igg 1 -anti-tt by an avidity elisa. the results were compared with reference values from the laboratory. results: at 4 years, post-vaccination anti-tt-titers were decreased (geometric mean total igg, igg 1 , igg 2 and igg 4 ). at 9 years, ds children had lower postvaccination geometric mean igg 4 anti-tt-titers only. post-vaccination igg 1 -anti-tt avidity levels were decreased in 8/15 and 4/7 ds children at four years and nine years of age, respectively. the quantitative and qualitative anti-tt-responses in both ds groups are shifted downwards compared to the reference values. although the anti-ttresponse increases towards normal titers with increasing age, the avidity (qualitative response) is still abnormal at that age, showing that ds children have profound and lasting difficulties with specific anti-tt antibody formation. 2 kda; 30, 6 kda; 23, 9 kda; 19, [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] 6 kda and 18, [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] 7 kda were expressed by a majority of examined strains independently of the associated diseases. we assume that these omps could be conservative proteins of h. pylori. conclusion: considering omps as potential targets in the search for disease-related biomarkers and potential vaccine antigens, the identification of h. pylori omps as well as the elucidation of their role in modifying the host immune responses seems to be very important research subjects. the increasing cases of severe diarrhoea and invasive lethal infections in children caused by salmonella typhimurium are a major public health problem in mexico. the rapid dissemination of multidrug-resistant s. typhimurium, and the lack of a licensed vaccine against non-typhoidal infections reduce the possibilities of an effective treatment. the objective of this study was to evaluate if the high incidence of non-typhoidal multidrug-resistant salmonella infections was associated with a reduced in anti-salmonella immunity. a cohort of 100 families, from a mexican agricultural community with a high incidence of endemic salmonella infections, was followed prospectively for an 8-month period. sera were obtained from healthy subjects from the same community (2 months to 88 years of age). the highest incidence of salmonella-associated diarrhea, 74/1000, occurred in children under 5 years of age. the lowest incidence, 10/1000, was observed in the population aged 10 to 59. whereas serum from individuals ranging 15-70 years of age showed maximum igg1, igg3 and iga anti-s. typhimurium titres, children less than 5 years-old did not show detectable igg1 and igg3 titres and had weak igg2, iga and igm antibody levels; only their igg4 levels were comparable to those detected in adults. moreover, the levels of igg2 and igg3 antibodies were lower in adults with a diarrheal-associated episode. interestingly, s. typhimurium yuhs 07-18, a commonly isolated human strain from this endemic area, resisted the complement-fixing activity of antibodies although it was sensitive to opsonisation and to fc-mediated phagocytosis by human monocytes. these data contributes to define the protective immune response involved in anti-s. typhimurium immunity. diseases caused by the yeast of candida genus are a serious clinical and social problem. despite this fact, there is no effective prevention against these opportunistic pathogens yet. although c. albicans is the major cause of the mycoses (67%), the number of the multiresistant non-albicans isolates increases. c. dubliniensis, which was described only recently as serious human pathogen, belongs to the group of these resistant isolates. the surface mannan of candida cells is component of the cell wall mannoprotein complex and participates in an initial contact with its host and subsequently with the host defense mechanisms. because of complexicity of this homopolymer it is necessary to identify a subunit of the mannan that is most effectively recognized by the immune system and thus influences the specificity of the induced antibody response (immunodominant epitope). in our study we prepared oligosaccharides from acid-stable part of mannan c. dubliniensis by conventional acetolysis. this procedure specifically cleaves the a-1,6linked mannopyranose units of mannan backbone and releases the side oligomannosyl branches. obtained oligosaccharides were used in inhibition elisa and spr (surface plasmon resonance) measurements. the reason of these measurements was to quantify interactions of these oligosaccharides with anti-mannan antibodies present in rabbit serum after 7-fold immunization with mannan-hsa conjugate injections in week intervals as well with inactivated c. dubliniensis whole z. neščáková 1 , s. bystrický 1 1 institute of chemistry, slovak academy of sciences, bratislava, slovakiaour newest approach to sub-cellular vaccine against gram-negative bacterial pathogens exploits detoxified lipopolysacharide (detoxified lps) as the target antigen. this is achieved by conjugation of carbohydrate to a protein carrier which secures the t cell dependent immune response. the goal of the immunization with this conjugate is to generate the effective production of memory b cells. here we prepared subcellular conjugate with the detoxified lps from vibrio cholerae strain o135 using polymer carrier and a protein. the cell immunity induced by the vaccination with the conjugate was evaluated in mice, namely their peripheral blood and the spleen. activation and differentiation of b-cell populations in the time-dependent manner was determined by flow cytometry analysis of these samples. a single-platform approach based on flow cytometry and defined number of fluorospheres was used to count b cells. however in our hands this method, previously used in humans, had to be adapted for mouse blood samples first. the protocol allows quantifying cells simultaneously with cytometric immunophenotyping without cell loss or other cell preparation steps. like pan-b cell marker cd19, expressed almost on all blood and tissue b cells, was used. here we investigate the characteristics and development of antibody (iso)types after secondary immunization with mencc or plain polysaccharide and the possible role of certain antibody responses in maintaining immunity after vaccination. methods: volunteers, age 18-55 years, were immunized with mencc or received a secondary immunization with mencc or plain menc ps. blood samples were obtained before and seven time-points after immunization. igg, iga, igm, igg1, igg2 and avidity were assessed by a multiplex immunoassay. functional antibodies were determined by a serum bactericidal assay. results: high levels of antibodies were still present 5 years after primary mencc immunization. secondary immunization resulted in increased igg and sba titers after 5 to 7 days. in primed individuals, igm was still present, and this only increased following a secondary immunization with plain ps. in addition, immunization with ps induced a higher igg2 response compared to mencc immunization. discussion: secondary immune responses are quiet slow. the composition of the ig (iso)type distribution is different between mencc and plain ps and might be of influence on functional titers. although this study indicates that immunological memory was previously induced by a single mencc vaccination, it highlights the importance to sustain protective antibody levels against a rapid invasive organism such as n. meningitidis. the immunological effectiveness of these two semi-synthetic immunogenic conjugates was established according to antigen-specific titers of igg, iga and igm isotypes and by phagocytic and respiratory metabolic activities of granulocytes. results: prime-boost immunization strategy resulted in enhanced production especially dlps-specific igg and igm isotype antibodies in both experimental groups (peak titers 1:3200). igm-igg isotype switch was more pronounced with o-sp. peak values of dlps specific iga isotype were signicantly lower than igg and igm ones (1:800 vs. 1:3200). flowcytometric simultaneous determination of phagocytosis and stimulated oxidative burst of granulocytes revealed conjugate induced enhancement, more evident with o-specific polysaccharide and ip final boost (stimulation index was 5. 85 fold of normal control). subcutaneous immunization gave a weaker stimulation: 1. 52 fold of normal control. the second de-oac conjugate exerted different pattern of stimulation, sc intervention was more effective. our results are indicative for immunological effectiveness of novel dlps derived glycoconjugates; thus promising further application in cholera subcellular vaccine. this work was supported by apvv 0032-06 and vega 2/7029/27 grants of the slovak grant agencies. background: the yeast candida albicans is an opportunistic pathogen that causes infections in immunocompromised individuals with a high morbidity and mortality levels. a long-acting, effective and safe vaccine that protects against medically important candida species should significantly reduce the incidence of various forms of candidasis by these etiologic agents. mannan, polysaccharide component exposed at the most external layer of the fungal cell wall, contains a backbone consisting of a-1,6-linked d-mannopyranose units and many branches composed of a-1,2 a-1,3 and/or b-1,2-linked mannopyranose units that are connected to the backbone. investigation of oligosaccharides immunomodulatory functions could be considered as an important part of their protective immunity against fungal diseases. objective: in this study, for mice immunization, synthetically prepared oligosaccharide (heptamannoside) conjugated to protein carrier (bovine serum albumin, bsa) was used. methods: in order to study the immunogenicity of heptamannoside -bsa conjugate as inducer of hummoral and cell-mediated responses, balb/c mice were subcutaneously immunized without adjuvant (6mg oligosaccharide per one conjugate dose) two times in 14 days intervals and then intraperitoneally or subcutaneously boosted. cell-mediated and humoral responses were analyzed on day 14 after injections by flow cytometric immunophenotyping of peripheral blood leukocytes and by measuring the levels of mannan specific antibodies presented in serum using elisa. results: prepared conjugate was immunogenic and re-injection elicited increase of mannan specific serum antibodies levels. intraperitoneal boost elicited significantly higher igg and igm levels than subcutaneous boost. immunization also induced changes in proportions of major lymphocyte subpopulations in peripheral blood. introduction: bulgarian immunomodulator respivax enhances the natural resistance of organisms and specific immunity towards the most frequent respiratory pathogens. it is composed of killed bacterial bodies and lysates of six microbial species (streptococcus pneumoniae, branhamella catarrhalis, streptococcus pyogenes, haemophilus influenzae, staphylococcus aureus, klebsiella pneumoniae). the immune response in respiratory tract includes not only systemic immunity in lungs, but also balt as a part of common mucosal immune system. most animals develop balt after antigen stimulation and this tissue plays a central role in antigen uptake and local immune response regulation. therefore, immunostimulation of balt may contribute to more efficient mucosal immunity in respiratory tract. aim: to study balt development in different terms after oral application of respivax in guinea pigs. methods: male guinea pigs (250g-350g) were treated orally with 50 mg respivax five consecutive days. after the last application, on days 3, 7, 10, 14, 28 and 42 six animals on each term were sacrificed and lungs were removed. morphological changes were evaluated on 4mm thick serial sections, stained with hemalauneosin. the populations of cd8, cd4 and b cells were identified on cryosections by using indirect peroxidase immunostaining. zio technique was used to detect intraepithelial dendritic cells (dc). results: balt was not identified in control animals. in the treated group on day 3 subepithelial lymphocyte infiltrates and diffuse lymphocytes in lamina propria were found. the following two terms were presented by hyperplasia of lymph epithelium with massive complexes of intraepithelial lymphocytes. they were composed mainly of cd8 positive cells, which number reached maximum at the end of the second week. on day 14 b cell lymphoid follicles with different size were found. lamina propria was presented by abundant lymphocyte infiltrates, composed of cd4 and cd8 positive cells. on days 28 and 42 the morphological reaction in the airways was reduced, characterized with small size lymphocyte accumulates. numerous intraepithelial dc's were detected in treated animals, comparing to controls in which only a few were identified. conclusion: oral administration of respivax in guinea pigs resulted in significant immunomorphological reaction in the airway mucosa presented by increased number of dc and balt development. g. gupta 1 , s. majumdar 1 1 bose institute, molecular medicine, kolkata, indiavisceral leishmaniasis (vl) caused by the protozoan parasite, leishmania donovani, is characterized by the loss of ability of the host to generate an effective immune response in the form of free radicals and proinflammatory cytokines. chemokines, particularly cc chemokines, have been shown to render protection against leishmania infection. there is no clear understanding about the immunoprotective role of cxc-chemokines in vl.in the present study, the comparative potential of cxc chemokines, interferon gamma inducible protein-10 (ip-10) and interleukin-8 (il-8) in restricting leishmania donovani infection via the release of nitric oxide (no) and proinflammatory cytokines was studied in an in vitro model. no, a crucial mediator for ip-10 mediated leishmanicidal activity, was found to be dependent on inducible nitric oxide synthase2 (inos2) expression and was linked to the mapk signaling pathway via antagonistic regulation of p38mapk and erk1/2. further, ip-10 was also able to abrogate the survival of leishmania in an in vivo model of vl by restoration of th1 cytokines and no. thus this study strongly demonstrates that ip-10, like cc chemokines, is involved in rendering a protective response in vl via upregulation of proinflammatory mediators. african trypanosomiaisis (at), known as sleeping sickness, is an orphan and extremely debilitating disease in human, cattle and domestic animals. at is caused by the protozoan trypanosoma brucei and at the present, there's no safe or efficient pharmacology intervention. the dna vaccines could be the answer for this disease by being able to induce production of igg antibodies and induce of th1/th2 cytokines mediated by cd8+ t cells and activating cd4+ t helper cells. in this study, we shows that balb-c mice immunized intramuscularly with a single dose of plasmids encoding three antigenic candidate genes from trypanosoma brucei, named invariant surface glycoprotein (isg), trans-sialidase (tsa), and fosfolipase c (plc) are able to produce igg antibodies anti-trypanosoma. this immunization process was able to control the mortality level when mice were submitted to challenger assay with trypanosoma brucei brucei parasites. in mice co-infected with s. ratti and l. major (nl) neither clearance of l. major nor strongyloides infection was changed. mice co-infected with s. ratti and p. yoelii (nl) showed the same course of parasitaemia as single infected mice. these results suggest a strictly compartmentalized and successful immune response in both murine co-infection models, s. ratti and l. major or p. yoelii. if this compartmentalization is also observed in the antigen specific cytokine response of ex vivo prepared lymphocytes will be the topic of further investigations. in the present study ,we evaluated tsa -encoded dna vaccine against l.major in balb/c mice. igg and ifn-g values were markedly increased in the immunized group ,which were significantly higher than in the control groups (p x 0.05) following immunization and after challenge with leishmania major. il-4 values were increased in all groups, but there was no statistical difference between the groups(p g 0.05) following immunization and after challenge with leishmania major. the immunized mice with the dna vaccine presented a considerable reduction in diameter of lesion comparing to the control mice and indicated a significant difference was observed between the immunized and the control groups (p x 0.05) in this regard . the survival time of the immunized mice with the vaccine was significantly higher than the control groups (p x 0.05) after the challenge with leishmania major. the immunized mice had significantly lower parasite load comparing to the control mice(p x 0.05). the findings of this study indicated that the tsa -encoded dna vaccine increased the cellular response and induced protection against infection with leishmania in the mice. the tsa -encoded dna vaccine may be an excellent candidate for future vaccine developments against leishmania. there is a lot of evidence showing that bcg vaccination at mucosal site via intranasal, intragastric and intrarectal routes are effective in conferring protection against virulent mycobacterium and several non mycobacterial infectious diseases. in this study the protective effect of autoclaved leishmania major (alm) vaccine in combination of either rectal or subcutaneous bcg on susceptible balb/c mice was evaluated.one month after bcg vaccination, balb/c mice were immunized subcutaneously twice with alm+alum at 3 week intervals. three weeks after booster injection, 5×10 5 stationary phase l. major promastigotes were inoculated subcutaneously in one footpad. immunological evaluation at before and post infectious challenge, showed strong proliferative responses in the spleen cells of the rectal immunized group after stimulating with parasite lysate. high level of interferon gamma was induced in the spleen and significant increase in the serum ratio of igg2a/igg1was observed only in rectal immunized group. rectal immunized mice showed comparable nitric oxide production and inos induction in peritoneal macrophages .the obtained results in rectal bcg vaccinated group showed no mortality but low parasite burden in the liver and spleen and suggested protective efficacy of intrarectal bcg immunization against leishmaniasis might be due to the long-lasting induction of type 1 immunity. methods: two groups of balb/c mice were infected by l. tropica. one group was infected subcutaneously into the left footpad and the other group intradermally into the left ear dermis. mice were challenged by l. major in the right footpad after establishment of l. tropica infection. the immune response was evaluated at two intervals: one week and one month after challenge. single cell suspensions were prepared from draining lymph nodes of mice. cells were stimulated by phorbol myristate acetate (pma). cell surface markers and cytokine production were determined by intracellular cytokine assay using flow cytometry. the following parameters were assayed in the two experimental groups: lesion development, delayed type hypersensitivity (dth) to l. major challenge, production of gamma interferon (ifn-g) and interleukin 10 (il-10), and cellular expression of cd4 and cd25. results: infection through subcutaneous route in comparison to the intradermal route induces significantly higher levels of dth and ifn-g, lower levels of cd4+ lymphocytes, and higher protection against l. major challenges. conclusion: intradermal infection of l. tropica, in comparison to subcutaneous infection, induces significantly more protective immunity in balb/c mice. therefore, we propose the route of infection as an important variable in this experimental model. this factor should be considered for development of an appropriate experimental model for human l. tropica infections. objectives: many mammals exhibit a periparturient relaxation of immunity (ppri) to gastrointestinal nematode parasites culminating in increased worm burdens. it has been suggested that the extent of ppri may have a nutritional basis as this effect on host resistance is considerably augmented when protein supply is scarce. subsequent studies have shown that increased dietary protein intake can ameliorate this phenomenon. however, this effect is often confounded with increased food intake and thus increased energy levels. here, we aim to dissect the effects of protein and energy nutrition on the immune status and resistance to gastrointestinal nematodes in the periparturient host. the nippostrongylus brasiliensis lactating re-infected rat model was utilised as a well established model for mammalian ppri. lactating rats, re-infected with 1,600 infective n. brasiliensis larvae on day 2 post parturition, were offered one of three levels of crude protein at one of two levels of metabolisable energy (me). parasite burdens were assessed by counting worms in the small intestine at day 6 post secondary infection. histological counting of intestinal inflammatory cells, assessment of antibody levels and measurement of cytokine mrna levels in the mesenteric lymph nodes were carried out to assess the host immune status. results: increasing cp supply, but not increased me supply, reduced worm burdens. whilst feeding treatment did not affect eosinophil and goblet cell numbers, increased cp supply increased mucosal mast cell numbers and levels of n. brasiliensis specific antibody (total igg, ige, igg1 and igg2a). this was independent of level of me supply. feeding regime did not affect levels of the type-2 cytokines il-4 and il-13. conclusion: this study effectively demonstrates that increasing protein supply per se can decrease periparturient parasite burdens. this anti-parasitic effect correlates strongly with an upregulation of immune effector mechanisms, namely accumulation of mast cells and production of antibody. this data emphasises the role of immunonutrition in combating infectious disease. protein supplementation of periparturient mammals has considerable potential as a non-chemotherapeutic method of controlling gastrointestinal nematode parasites. background: gp63 is the major surface glycoprotein of leishmania that exhibits protease activity and has an important role in the biology of the parasite. the aim of this study was cloning and expression of gp63 of l.major strain mrho/ir/75/er. methods: l.major promastigotes were grown in rpmi1640 supplemented with 10 % fcs. l.major rna extraction and cdna synthesis were carried out. gp63 gene segment was amplified by specific primers and cloned into ptz57r to construct ptz57r/gp63. the presence of gp63 into ptz57r was confirmed by pcr. then, ptz57r/gp63 was sent to determine the sequence of its nucleotides. after that the gp63 gene segment was sub-cloned into pet32 a (+) expression vector and transformed into e.coli bl21 (de3) plyss and gp63 protein was expressed in presence of 1mm iptg. objectives: the development of a vaccine against malaria caused by plasmodium falciparum is an urgent public health priority. influenza virosomes represent an innovative human-compatible antigen delivery system that has already proven its suitability for subunit vaccine design. at appropriate antigen doses, seroconversion rates of 100 % were achieved against two synthetic malaria peptide-mimetics in malaria naï ve volunteers (genton et al., plos one, 2007) . the aim of this clinical trial is to proof that virosomes are a suitable delivery system for malaria peptide-mimetics in malaria semi-immune subjects. objectives include demonstration of safety and tolerability of virosome formulated malaria peptide-mimetics and determination of the humoral and cellular immune responses against these malaria peptide-mimetics. particularly, boosting of pre-existing naturally acquired anti-malaria immunity will be investigated. the study design was a single centre, randomized, controlled, double-blind, age deescalating trial including 50 volunteers. 10 male volunteers (18 and 45 years) for the adult group, and 40 children of both sexes (5-9 years) were enrolled. subjects received virosomal formulations containing 50 mg of ama 49-c1 (pev301t), an apical membrane antigen-1 derived synthetic phospatidylethanolamine (pe)-peptide conjugate and 10 ug of uk39 (pev302t), a circumsporozoite protein derived synthetic pe-peptide conjugate. comparator groups received the influenza vaccine inflexal v. volunteers received two injections at study days 0, and 90. results: safety and tolerability defined as occurrence of local and systemic adverse events and incidence of clinically significant hematological and biochemical abnormalities are assessed. this vaccine showed a very good safety and tolerability profile in all study participants. curcumin dissolved in dmso when administered orally to p.berghei infected mice has been shown to have antimalarial activity, enabling 29 % of the treated mice to survive till 21 days after infection by which time all of the untreated mice had died. under such condition we found that bioavailability of curcumin was only 0.04 % of the amount fed and it remained in circulation in the blood only for 30 minutes post feeding. we therefore prepared curcumin bound to chitosan nano particle to improve it's delivery and found that oral feeding of such particles not only increased its bioavailability to 0.4 % ( of the amount fed but it's circulation was sustained till 6 hrs post feeding. under such conditions when 200mgm of curcumin bound to 300mgm of chitosan nano particles were fed one time daily for 10 days post infection to plasmodium yoelii infected mice 100 % of mice were cured and survived atleast for 100 days without any infection and were resistant to reinfection with the same parasite. curcumin under such condition accumulated preferentially in infected erythrocytes, the quantity increasing with increase in parasitemia and fluorescence microscopy revealed that it was bound to the parasite. like chloroquine, curcumin inhibited hemozoin formation in vivo and heme polymerization in vitro in a dose dependent manner. we believe that it is one of the ways by which curcumin may be killing the parasite. among immune cells, nk and gamma-delta t cells are suspected to play a critical role in the early control of plasmodium falciparum parasitaemia and to influence malaria adaptive immunity. gamma-delta vgamma9vdelta2 t cells, a non-conventional t cell subset specific of primates, are activated and expanded during primary p.falciparum infections in response to malaria non-peptidic phosphoantigens, and they are an important source of ifn gamma. furthermore these cells inhibit in vitro growth of p.falciparum blood stages by a granule exocytosis-dependent cytotoxic pathway and granulysin -an nk and t cell specific cytotoxic molecule has been incriminated. so far, the precise mechanism of the parasite inhibitory capacity of those cells, as well as the parasite blood stages involved remains unclear. to further investigate the anti-parasitic activity of gamma-delta t cells an rnai strategy based on a lentiviral vector approach was undertaken. we demonstrate that granulysin, but not perforin is essential for the anti-parasitic activity of gamma-delta t cells. concerning parasite blood stages, we show that both mature infected red blood cells and the free invasive form (merozoite) trigger gamma-delta degranulation and granulysin release, but noteworthy merozoites were the only stage affected by gamma-delta t cells. in addition, we also provide evidence that such a mechanism may occur in infected patients. altogether these data highlight a new mechanism by which gamma-delta t cells might directly contribute to malaria immunity opening new perspectives based on gamma-delta t cells to prevent or cure malaria. the immune system has a number of mechanisms to prevent self-destructive responses. amongst these, regulatory t cells (treg) have the ability to actively suppress effector responses. many questions surround the issue of antigen specificity of treg, since selective inhibition of only the pathogenic response, leaving the rest of the immune system intact, is the ideal therapeutic goal. the purpose of the project is to develop a model of robust, highly specific regulation operating in vivo that can be studied to understand the underlying mechanisms. such a model is provided by murine autoimmune hemolytic anemia (aiha) induced by immunisation with cross-reactive rat red blood cells (rbc). mice recover from disease due to the development of regulation with exquisite specificity, which suppresses only responses to self-epitopes whilst selectively allowing those to rat-specific determinants to be boosted. the re-establishment of tolerance is associated with the loss of t-cell proliferative responses, and emergence of il-10 responses, to epitopes on the dominant rbc autoantigen, anion exchanger-1 (ae-1, or band 3) protein, and protection can be transferred by injecting splenocytes from recovered mice into naï ve recipients. here we show that transfer of tolerance to naï ve recipients is dependent on ido mediated immunosuppression as mice receiving previously tolerised splenocytes under the cover of 1 methyl tryptophan, an inhibitor of ido, were refractory to tolerance and developed hemolytic disease. induction of ido is therefore an important process in antigen-specific tolerance, and initiators of ido activity, including ctla-4 + regulatory t cells or soluble forms of ctla-4, may also be crucial components of this regulatory pathway. consequently, this finding has important implications for our understanding of tolerance processes in autoimmune disease. objectives: it was shown alpha-fetoprotein (afp) induced immunosuppression of cell-mediated immunity in vivo. our previous work discovered afp-activated mice bone marrow hematopoietic stem cells (hscs) suppressed effector reactions of cell-mediated immunity in vitro. we investigated relationship existed between afp-induced hscs suppressor activity and immunosuppression of cell-mediated immunity during afp-produced teratocarcinoma development. methods: animal models, experimental oncology methods, immunomagnetic separation, cultural methods, cellular biology methods, flow cytometry, multiplex protein analysis, methods of molecular biology and rna interference (rnai) were used in this work. results: as a result, there was a negative correlation (r medium =-0.81) between dynamics of hscs suppressor activity elevation in spleen and inhibition of nk cells, nkt cells and cd8 + t cells cytotoxic activities ex vivo during tumor growth. besides, the inhibition of spontaneous and induced cytokines productions such as ifng, tnf-a and tnf-b from these types of immunocompetent cells negatively correlated with increasing of suppressor factors expression such as tgf-b 1 (r medium =-0.74) and il-10 (r medium =-0.65) in isolated splenic hscs ex vivo. analysis of effector cd4 + t cells in spleen showed decrease of t h 1 cells quantity and simultaneous t h 2 cells number increase during teratocarcinoma development. moreover, it were found elevated numbers of cd4 + cd25 + ctla-4 + -and cd4 + cd25 -il-10 + il-4regulatory t cells in spleen as well as increasing suppressor activity of isolated regulatory t cells ex vivo. number boost kinetics of t h 1, t h 2 and regulatory t cells were correlated (r th1 =-0.74, r th2 =0.86 and r th3 =0.80 and r tr1 =0.68) with kinetics of hscs suppressor activity level. in addition, dynamics of regulatory t cells activity were linear (r th3 =0.84 and r tr1 =0.76) to hscs suppressor activity level in spleen during tumor growth. quantities of tgf-b1-and il-10-produced hscs in spleen were correlated (in some cases negatively but in other positively) with cell-mediated immunity effector reactions alteration during teratocarcinoma development also. however, inhibition of afp expression by rnai caused to inhibition as immunosuppression activity of hscs and their appearance in spleen as well as normalization of cell-mediated immunity effector reactions. conclusion: thus, hscs suppression activity is correlated with changes in cell-mediated immunity during endogenous afp productions by teratocarcinoma cells and may play a role in afp-mediated dysfunction of normal immunoregulation during afp-produced tumor development. syphacia obvelata, a murine pinworm gastrointestinal nematode, is common even in well-managed animal colonies. although often considered as irrelevant, pinworm infections were shown to alter hosts' immune responses and to interfere with the experimental settings. our studies showed that naturally aquired s.obvelata infection also influences the hosts' hematopoietic responses, inducing the increased production and release of the cells of granulocyte-macrophage, as well as of erythroid lineage from the bone marrow of the infected cba mice. while the enhanced myelopoiesis compensates the increased peripheral demand for a larger supply of tissue neutrophils and macrophages, the cause of stimulated erythropoiesis is less obvious, but as infection consequence clearly underscores the disturbed and altered hematopoiesis. beside cellular changes, we also evaluated the impact of the s.obvelata on mitogen-activated protein kinases (mapk) signaling in bone marrow cells and found that infection upregulated all three mapk families, p38, jnk and erk. additionally, s.obvelata enhanced the expression of mrna for the inducible nitric oxide synthase (inos). to evaluate how this pinworm infection modifies hematopoietic cells' reactivity, we also examined the influence of interleukin-17, t cell-derived cytokine implicated in the regulation of hematopoiesis and inflammation, on the bone marrow cells. bone marrow myeloid and erythroid progenitors from s.obvelata-infected mice displayed altered sensitivity to il-17, as compared to non-infected controls. the infection also altered the effect of il-17 on mapk activation by preventing its stimulating effect on p38 mapk. moreover, in s.obvelata-infected animals il-17 markedly down-regulated the expression of both inos and constitutive, endothelial (e)nos, not affecting the low basal nitrite production, which was opposite to the effect previously observed in noninfected mice, i. e. il-17 induced no production through the activation of both inos and enos. besides highlighting the importance of working under pinwormfree conditions when using experimental murine models for immunohematopoietic investigations, the data obtained pointed to the multiple layers of modulatory ability of this pinworm parasite and confirmed that the overall orchestration of the host response to the parasites is a complex process still being unraveled at both the cellular and molecular level. 2.-validation of the method: in order to determine linearity, analytical range, and reproducibility, three different sera with previously identified mc were serially diluted from 1 ⁄2 to 1/64 with a normal serum pool. 3.-implementation as a standard method for analysis of the mc in patients with paraproteinemia. the method showed good linearity: r 2 g 0.98. the analytical range was from 1 g/l to 70 g/l. the coefficient of variation (cv) was x 10 % for [mc] n 1 g/dl, and x 20 % for [mc] x 1 g/dl. this procedure was successfully implemented to quantify the mc in 1100 serum samples between march 2008 and february, 2009. among these samples, we have quantified light chains, heavy chains, igd and biclonal paraproteinemias. conclusions: 1. we have developed a simple, reproducible and low-cost method to quantify the mc using standard analyses of serum protein electrophoresis (spe), serum albumin, and densitometric quantification of mc and albumin regions. 2. the procedure allows monitorization of the mc in patients at diagnosis, after therapy, and evaluation of complete remission. objectives: direct influence of alpha-fetoprotein (afp) on immunosuppressor factors synthesis as well as immunosuppressor activity of bone marrow hematopoietic stem cells (hscs) was detected in our previous work in vitro. we investigated possible role of endogenous-produced afp in induction hscs immunosuppressor activity at tumor-bearing mice. methods: animal models, experimental oncology methods, immunomagnetic separation, cultural methods, cellular biology methods, flow cytometry, multiplex protein analysis, inhibition assay, colorimetric elisa, methods of molecular biology and rna interference (rnai) were used in this work. results: our results demonstrated afp endogenous synthesis adduced to elevation of immunosuppression activity of hscs in bone marrow. this afp effect becomes developed from 7 day and reached plateau level after 30 day of teratocarcinoma insertion. moreover, cd34 + cd38cells showed in spleen and main lymph nodes from 15 day and achieved plateau level after 60 day of teratocarcinoma growth. however, immunosuppression activities of purified hscs from spleen and lymph nodes discovered at 22 day and had a maximum pick at 60 day of teratocarcinoma inoculation. besides, immunosuppression activity of hscs from spleen and lymph nodes was more than 1,5 times lower than in bone marrow in the same period of tumor development. isolated hscs from bone marrow, spleen or lymph nodes produced similar spectrum of suppressor factors such as tgf-b 1 , il-10, pge 2 and no. inhibition of such suppressor factors lead to levelling of hscs immunosuppression activity ex vivo. kinetic of hscs quantity and activity had significant correlation (r cell number =0.81 and r activity =0.92) with afp level dynamics in blood serum. in addition, inhibition of afp expression by rnai caused to diminishing of hscs immunosuppression activity as well as hscs appearance in spleen and lymph nodes. conclusion: therefore, afp plays role not only specific inducer of hscs immunosuppression activity but also as a factor of activated hscs penetration into spleen and lymph nodes during afp-produced tumor development. cd40-ligand molecules -that are powerful immunomodulators -are strongly expressed by activated platelets; membrane-associated cd40l is cleaved to soluble (s)cd40l. we sought to examine the levels of scd40l in platelet concentrates (pcs) having led to an acute transfusion reactions (atr), and to test for its biological effect on b-lymphocytes. we recorded 5 atr episodes that could lead to investigation of residual platelets in container. two fractions of aliquots from each pc (and controls) were prepared, one for assay of individual supernatant fractions and one of corresponding lysates of platelets; scd40l -along with other products -were assayed by quantitative elisa. levels of il8, cd62p and pdgf-ab in pc supernatants and lysates from pcs associated with atr were similar to that in controls. supernatants of pcs associated with an atr contained higher scd40l levels compared to controls, and -in a inversely correlative manner -corresponding platelet lysates contained lower levels of scd40l . to examine if scd40l was biologically active, we stimulated purified b cells recovered from healthy blood donors and exposed those normal b cells to supernatants and cell lysates of pcs implicated in atr, or control material, and we measured il-6 secretion. the il-6 concentration was consistently below 5-10 pg/ml in pc supernatants and lysates, and unstimulated b cells did not secrete detectable levels of il-6. the addition of supernatant from atr-associated pc samples to purified b cells consistently resulted in sustained il-6 production over control (p x 0.05) at d2 after the onset of the culture, while -in a inversely correlative manner -corresponding platelet lysates contained lower levels of scd40l (p x 0.05). pre-incubation of b cells with cd40-blocking antibodies substantially abrogated il-6 secretion, unlike isotype-matched control. the partial blocking of cd40 binding on cd40 + b cells strongly suggests a potentially synergistic role in b cells for cytokines other than scd40l (under investigation) and indicates a sustained role for pc-derived scd40l. these data prompt us to investigate a larger series of events and controls to delineate on the one hand if certain factors can be responsible for an enhanced production of scd40l by collected/stored cpa. objectives: there is accumulating evidence for a role of natural killer (nk) cells in the antitumor response against hematological malignances. nk cells exert their action by means of a large panel of structurally distinct activating receptors that recognize their ligands on target cells. analysis of activating receptor pathways in nk cells has revealed a dominant role for natural cytotoxic receptors (ncrs) and dnax-accessory molecule-1 (dnam-1) in the lysis of acute myeloid leukemia (aml) blasts. here, we investigate the expression of these activating receptors on nk cells from aml patients stratified by age. methods: we analyses by flow cytometry peripheral blood mononuclear cells (pbmcs) from 30 aml patients before specific anti-leukemia therapy and 47 healthy donors. all results were analyzed statistically using spss version 15. results: aml patients under 65 years showed a significant reduction in the expression of dnam-1, nkp30 and nkp46 compared with age-matched controls. both healthy individuals and aml patients older than 65 years showed a reduction of these receptors compared with young donors. in contrast, we have found that nkp44 expression was increased in some patients of aml. on the other hand, the analysis of ligands for these activating receptors on leukemic cells showed a high variability that was not correlated with age or fab subtype. in addition, an inverse correlation in the expression of dnam-1 on nk cells and its ligand cd112 on aml blasts has been found in aml patients under 65 years. to analyze if leukemia cell were involved in the modulation of these receptors we have performed in vitro cocultures of leukemic blast and healthy nk cells. the initial recognition of aml cells by nk cells may represent a crucial process to prevent tumor development. here we described for the first time, a decrease of dnam-1 expression on aml patients and confirm previous reports showing a significant decrease of nkp30 and nkp46 on aml-nk cells. altogether, these alterations of the major receptors involved in nk cell-mediated cytotoxicity of leukemic cells represent an important mechanism of immunoescape that may correlate with disease progression and patient survival. a. stelmaszczyk-emmel 1 , e. gorska 1 , u. demkow 1 , m. wasik 1 1 medical university of warsaw, department of laboratory diagnostics and clinical immunology of developmental age, warsaw, polandglucocorticosteroids are often used in leukemia treatment. their therapeutic use is limited due to several side effects. one of them is multidrug resistance phenomenon, which causes lack of patients response for treatment. dexamethasone is used in schedule of children's all-b treatment and the response on glucocorticosteroids therapy is very important. the aim of this study was to examine whether dexamethasone changes multidrug resistance of lymphoblasts in all-b and their tendency to begin apoptosis. the study involved 10 children with all-b. bone marrow cells isolated by centrifugation on histopaque at the day of diagnosis were cultured for 2 or 48 hours with or without dexamethasone in concentration 10 -6 m. analysis of: p-gp surface expression, p-gp function (rhodamine 123 test), phi (carboxy-snarf) and apoptosis test (annexin-v and pi test) were performed with the use of flow cytometer coulter epics xl. for statistical analysis nonparametric wilcoxon test was used.the results showed that p-gp expression on lymphoblasts was 14,52 %±11,32. after 48 hours of lymphoblasts incubation with or without dexamethasone any statistically significant changes were observed. average percentage of lymphoblasts with rhodamine 123 efflux, which characterized p-gp activity, was 2,62 %±3,15. after 2 hours of cells incubation with dexamethasone there was seen significantly higher percentage of cells able to eliminate rhodamine 123 (4,31 %±4,03, p=0,015). average phi in lymphoblasts was 7,78±0,19. acidification of cells incubated 2 hours with dexamethasone was seen in 10-25 % percentage of cells 17) . rest of lymphoblasts showed alkalization (phi -8,00).the percentage of lymphoblasts in early stage of apoptosis after 48 hours incubation with dexamethasone (annexin-v test) was higher than in control cells (19,34 % vs 14,13 %; p=0,01). we concluded that dexamethasone does not influence surface p-gp expression on lymphoblasts of patients with all b but significantly increases activation of this protein. functional test should be performed to evaluate multidrug resistantace of leukemic cells, because surface expression of p-gp is not identical with its activity. moreover, dexamethasone alkalizes cytoplasm of lymphoblasts and induces early stage of their apoptosis. those effects may contribute to the treatment outcome. . however, small numbers of clonal pc can also be detected in the peripheral blood (pb) of the majority of patients and, in a minority of cases, mm is transformed into pc leukaemia (pcl). here, we describe that tumoral pc can express cd49d/cd29 integrin with high or, sometimes, low affinity states, which is associated with their retention in or release from the bm, respectively. objectives: to evaluate the activation state of cd29 on malignant pc from bm and pb, as well as its regulation. methods: pc and active integrin expression on these cells were detected with anti-cd38 and anti-cd29 (clon huts21) moab, respectively, by flow cytometry. to study the integrin activation with divalent cations and pc index proliferation (brdu+ cells), we used short-term pc cultures (18 hours). results: cd29 active form was expressed in the majority of normal and tumoral bm pc from healthy subjects (67.3±6.6, n=9) and mm patients in the early stages of the disease (32.6±7.5, n=17). in these cells, huts21 epitope was clearly upregulated by mn 2+ . in contrast, circulating pc were almost all huts21 negative, and levels did not significantly augment when these cells were exposed to mn 2+ . moreover, not only pb but also bm malignant cells from pcl patients were also huts21 negative and divalent cation refractory. it was also observed that pc from pcl patients showed an increased proliferative index (2.35±0.9 brdu+ cells, n=3) in comparison to pc from mm patients in the first stages of disease (1.3±0.2 brdu+ cells, n=5). these results suggest that the active form of cd29 must be expressed on pc to retain these cells within the bm environment. moreover, its downregulation is associated with increased numbers of circulating pc and disease progression. multipotent mesenchymal stem cells derived from (hucb) represent promising candidates for the development of future cellular therapy strategies. they are able to self-renew and they terminally differentiate into multiple lineages, including bone, cartilage, muscle, bone marrow, fat and other diverse connective tissues. in the first part of this study, we compared different protocols for the expansion of human mesenchymal stromal cells (hmsc) starting from diagnostic samples of bone marrow aspirates and the cord blood (cb). the protocols differed in the presence of either 10 % fetal bovine serum (fbs) with and without fgf2,or 10 % human platelet lysate (hpl), 5 %hpl, (10 % fbs + 10 % hpl), (10 % fbs + 5 % hpl). we obtained a significantly better expansion with hpl, compared to cells with a selected batch of fbs and in fewer days.in the second part of this study, we focused on proteins that were differentially expressed during osteogenic, adipogenic and vascular muscular differentiation by western blotting. we compared the quality and the quantity of protein expression before and after differentiation (day 18). two bm and two cb differentially expressed spots were observed between the two groups (before and after differentiation).we noted the low pourcentage of hmsc in cb samples: in ten samples, only two made msc colonies as in bm samples. we were also interested to the different coloration: osteogenic diffentiation was determined by alizarin red s staining, for adipogenic differentiation, the cells were stained with oil red o to visualize lipids droplets. background: inherited bone marrow failure syndromes (ibmfs) comprise a group of genetic disorders characterized by single or multiple cytopenias, as well as distinctive clinical features and varied molecular pathways. activation of p53 tumor suppressor pathway leads to cell cycle arrest and initiates apoptosis. we studied the presence of p53 dna (as a marker of cell cycle dysregulation); in bone marrow of children with fanconi anemia (fa) and those with acquired aplastic anemia (aaa). subjects and methods: this is a cross sectional study that involved: 1) ten cases with fa diagnosed on the basis of dna breakage analysis, 2) ten cases with aaa, and 3) ten normal control cases. the presence of p53 dna was measured in both bone marrow and peripheral blood samples using a real-time quantitative pcr by taqman assay. results: p53 dna was demonstrated in bone marrow of 90 % of children with fa, compared to 10 % in children with aaa (p x 0.001), while, no p53 dna was seen in normal control. a positive correlation between dna breakage and presence of p53 dna was seen in bone marrow from fa (p x 0.02, r0.81). the presence of p53 tumor suppressor gene by real time pcr in bone marrow of fa may represent an early indicator of significant dna genetic alteration in those patients. key: cord-257167-rz4r5sj7 authors: nan title: abstracts for the 29th annual meeting of the japan neuroscience society (neuroscience2006) date: 2006-12-31 journal: neuroscience research doi: 10.1016/j.neures.2006.04.004 sha: doc_id: 257167 cord_uid: rz4r5sj7 nan the brain basis of conscious experience is one of the great unsolved mysteries of science. how can a material object became aware of the world around it and of its very own awareness? many scholars think this question is unanswerable. new approaches to this age-old mind-body problem have recently been encouraged by the development of pet and mri and the powerful tools of modern neuroscience. we are now witnessing the explosive growth of a new field, called cognitive neuroscience which focuses on behavior and uses classical reaction-time paradigms together with new computer-based technology. a parallel approach is to cross-correlate formal aspects of conscious experience as the brain spontaneously pursues its regular trajectory through the objectively defined states of waking nrem and rem sleep. at the level of the brain it is possible to record pet and mri and by using an animal model to analyse cellular and molecular mechanisms. the advantage of this approach, which i call the conscious state paradigm, is that it, is quantitative, integrative, and holistic (in the rigorous sense of that word). the brain basis of activation (a) input-output gaining (i) and modulation (m) can now be described in relation to the changes in consciousness associated with them. the data can be used to create a three-dimensional model (aim) which describes the brain-mind trajectory in its state space. neural circuits in many parts of the developing nervous system exhibit highly rhythmic episodes of electrical activity. such activity has generally been considered to fine tune connections that are initially made by axons responding to a complex array of molecular guidance cues. however, chick and mouse spinal cords exhibit activity at early stages as motoneurons are still migrating and beginning to extend their axons. modest alterations in the frequency of such episodes produced changes in the expression of several guidance/recognition molecules and caused motor axon pathfinding errors. interestingly the type of pathfinding decision, the binary dorsal-ventral choice or the subsequent pool specific fasciculation and projection of axons to specific muscles, was differentially affected by decreasing or increasing the frequency respectively. thus, activity generated by developing circuits interacts at early stages with the molecular signaling pathways that govern the formation of precise circuits and any drugs that alter this activity could adversely affect circuit formation. supported by nih grant ns19640. sl2-1-1 frontal cortex and higher-order motor control: preferential use of multiple premotor and prefrontal areas dependent on behavioral context jun tanji brain science research center, tamagawa university, machida, tokyo, japan in the cerebral cortex of primates, there exist a number of motor areas rostral to the primary motor area and caudal to the prefrontal cortex. although each area has been defined based on anatomical and physiological criteria, functional roles played by each of them have been the matter of much debate. in fact, in a recent trend of research reports, it is popular to stress commonalities rather than specificities in the use of multiple cortical areas in motor control. for instance, the involvement of the primary motor cortex in cognitive aspects of motor behavior has been an eye-catching subject of recent reports. the involvement of the prefrontal cortex in movement planning has also been inferred. up to a certain extent, it is true that the use of different areas have some factors in common. however, it is a mistake to ignore profound differences in the use of each area, depending on specific aspects of motor behavior. in this lecture, i will describe such differences that could be clarified only when neural activities are examined properly, by studies designed to reveal individual aspects of functional significance of each area. sl3-1-1 neuronal functions and molecular motor, kinesin superfamily proteins, kifs: from transport of synaptic proteins and mrnas, to brain wiring, neuronal survival and higher brain function nobutaka hirokawa department of cell biology and anatomy, graduate school of medicine, university of tokyo, japan the intracellular transports are fundamental for neuronal morphogenesis, functioning and survival. to elucidate this mechanism we have identified and characterized kinesin superfamily proteins, kifs, using molecular cell biology, molecular genetics, biophysics, and structural biology. kifs play essential roles on neuronal function and survival by transporting synaptic vesicle precursors (kif1a/kif1bbeta), nmda type(kif17) and ampa type(kif5s) glutamate receptors and mrnas(kif5s) such as camkii ␤ mrna and arc mrna with a large rna-transporting protein complex containing at least 42 rna related proteins such as hn rnp-u, staufen, and fmrps. kif2a is fundamental for correct brain wiring by suppressing elongation of axon collaterals through depolymerizing microtubules in growth cones. kif3 suppresses tumorigenesis by transporting ncadherin/beta catenin containing vesicles from golgi to plasma membrane in the neuroepithelium. kif4 controls the activity-dependent survival of postmitotic neurons by regulating parp-1 activity in brain development. thus, kifs play significant roles not only on various neuronal functions but also on brain wiring, development and higher brain functions such as memory and learning. tsukahara award 2-1 what we can learn from the functional recovery after brain injury tadashi isa national institute for physiological sciences, okazaki, japan it is believed that when a part of a neuronal system is damaged, some of the lost functions can be taken over by residual systems through training. such concept is considered as the basis of neurorehabilitation, however, the mechanism of the "take-over" is not well understood. in this talk, i will present our recent progress in two lines of studies using non-human primate models related to this issue. the first topic is on the recovery of dexterous finger movements after lesion of the lateral corticospinal tract at the cervical spinal cord. after the virtually complete lesion, relatively independent finger movements can be recovered in 1-3 months. we have found that bilateral primary motor and ventral premotor cortices are involved at various stages of the recovery process by combining the pet imaging and reversible local inactivation technique. in the second, i will talk about the visuo-motor processing in monkeys with unilateral lesion of the primary visual cortex (v1). after complete ablation of v1, the monkeys recover performance of visually guided saccades toward the blind field in 2 months. saccades to the blind field have low sensitivity and less accurate. however, the monkeys can perform surprisingly cognitively demanding tasks in the blind field. i will discuss on our hypothesis on the bottom-up and top-down control of learning during recovery. takuji iwasato riken brain science center (bsi), wako-shi, saitama, japan in the rodent primary somatosensory (barrel) cortex, the configuration of the whiskers on the face is topographically represented as "barrels", discrete modules of layer iv neurons and thalamocortical afferent (tca) terminals. while barrel formation is an important model of the establishment of patterned topographic connections between the sensory periphery and the brain, the molecular mechanisms underlying this process are poorly understood. we developed and applied mouse reverse genetic technologies to examine these molecular mechanisms. we have focused on the nmda-type glutamate receptor (nmdar) and calcium-stimulated adenylyl cyclases, as nmdar-and camp-cascades are central to various types of neuronal plasticity, both in adulthood and during development. series of global and region-specific knockout mice have revealed the roles of these molecules in the patterning of barrel cortex and differentiated the specific mechanisms at presynaptic tca terminals compared to those at postsynaptic cortical neurons. research funds: presto (jst), kakenhi 17023055, kakenhi 15029261 sy1-1-01-2 distinct roles of two 7-pass transmembrane cadherins in neurite growth control tadashi uemura 1,6 , yasuyuki shima 1,6 , keisuke sehara 2 , manabu nakayama 3 , shinya kawaguchi 4 , mikio hoshino 5 , yoichi nabeshima 5 , tomoo hirano 4,6 1 graduate school of biostudies, kyoto university, kyoto, japan; 2 school of science, japan; 3 kazusa dna research center at chiba, japan; 4 graduate school of science, japan; 5 graduate school of medicine, japan; 6 crest, jst, japan drosophila flamingo (fmi) and mammalian celsr1-3, which are 7pass transmembrane cadherins, have been considered to mediate the regulation of neuron contact-dependent neurite growth. we show that mammalian 7-pass transmembrane cadherins celsr2 and celsr3 are activated by their homophilic interactions and regulate neurite growth in a distinct manner. both gene-silencing and co-culture assay showed that celsr2 enhanced neurite growth whereas celsr3 suppressed it. our result suggested that celsr2 had a stronger activity as g-protein coupled receptor than celsr3 did, most likely due to a difference of a single amino acid residue in the transmembrane domain, and this functional difference resulted in distinct effects in neurite growth regulation. thus, neuron-neuron interactions modulate neurite growth differentially through this couple of 7-pass transmembrane cadherins. masatoshi takeichi riken center for developmental biology, kobe, japan for synapse formation, axons need to recognize their specific partners, and subsequently stabilize their contacts. while a number of cell surface molecules should be involved in such processes, cadherin adhesion molecules play a role. when cadherin activities are blocked, synaptic contacts become destabilized in cultured neurons. this is also the case in vivo; e.g., in the neural retina whose cadherin activities are impaired without perturbing their overall architecture, a certain class of synaptic contacts does not normally form. another series of our study demonstrate that the cadherins cooperate with nectins, a subfamily of ig-domain molecules, for establishment of axon-dendritic contacts: in early hippocampal pyramidal neurons, nectin-1 is preferentially localized in axons; and nectin-3, in both axons and dendrites. we present evidence that the heterophilic binding between axonal nectin-1 and dendritic nectin-3 is important for facilitating the axon-dendritic attachment; and cadherins seem to be required to stabilize the nectin-initiated contacts. thus, multiple classes of adhesion molecules work together to ensure the correct linking between axons and dendrites. hitoshi sakano department of biophysics and biochemistry, university of tokyo, tokyo, japan we have studied how the olfactory sensory neurons (osns) expressing the same odorant receptor (or) gene converge their axons to a specific set of glomeruli in the olfactory bulb (ob). retrogradestaining of osn axons indicated that the dorsal/ventral (d/v) arrangement of glomeruli in the ob is correlated with the expression areas of corresponding ors along the dorsomedial/ventrolateral axis in the oe. in contrast, the anterior/posterior (a/p) arrangement of glomeruli appears to be independent of the epithelial locations of osns and more dependent on the expressed ors. it was found that g proteinmediated camp signals regulate the positioning of glomeruli along the a/p axis in the ob. we also found that multiple sets of cell adhesion molecules, e.g., ephrin-as and eph-as, are expressed in a complementary manner, whose transcription levels are uniquely correlated with the expressing or species. we propose that differential levels of repulsive/adhesive interactions of axon termini may regulate the sorting of like-axons during the process of osn projection. research funds: crest, jst, kakenhi 14104026 sy1-1-01-5 lamina-restricted guidance of hippocampal mossy fibers hajime fujisawa 1 , fumikazu suto 2 1 nagoya university, nagoya, japan; 2 institute of genetics, mishima, japan axons from different sources terminate at particular dendritic segments of target neurons in a laminal fashion. one important issue to be addressed is how individual axons are instructed to invade and arborize in particular laminae. projection of hippocampal mossy fibers is one of good experimental models to analyze molecular mechanisms that govern lamina-restricted termination of axons, because the fibers project to the proximal dendritic segment of ca3 pyramidal cells. we here report the following three mechanisms that provide lamina-restricted projection of mossy fibers. first, a neural repellent sema6a is expressed in ca3 pyramidal cells and principally suppresses invasion of mossy fibers to ca3. second, the repulsive signal of sema6a is mediated by plexin-a4 expressing in mossy fibers. third, the repulsive activities of sema6a are attenuated by plexin-a2 in the proximal dendritic segments of ca3 pyramidal cells, resulting in the segments permissive for mossy fibers to invade. over the course of development, children become increasingly able to control their thoughts and actions (i.e., cognitive control). the term cognitive control is an umbrella term for a set of putative control processes. these control processes may reach adult levels at different rates, depending on the rate of functional development of the specific brain structures involved. the structure most closely associated with cognitive control is prefrontal cortex (pfc). pfc is composed of what are believed to be functionally distinct subregions, including ventrolateral, dorsolateral, rostrolateral, and medial pfc. i will discuss the control processes associated with each of these regions, and how the functionality of these regions differs between school-aged children, adolescents, and young adults. three fmri studies will be presented, focusing on (1) working memory maintenance and manipulation, (2) rule representation and task-switching, and (3) relational reasoning. based on these data, i will discuss some general points about neurodevelopment changes in cognitive control, and outline the approach that our laboratory has taken in our developmental cognitive neuroscientific research. sy1-1-09-5 towards manipulative neuroscience based on non-invasive brain decoding in atr computational neuroscience laboratories, we proposed several computational models such as cerebellar internal models, mosaic, modular and hierarchical reinforcement-learning model. some of these models can quantitatively reproduce subject behaviors given sensory inputs and reward and action sequences which subjects received and generated. these computational models possess putative information representation such as error signals for internal models, action stimulus dependent reward prediction, and they can be used as explanatory variables in neuroimaging and neurophysiology experiments. we named this approach as computationalmodel-based neuroimaging, as well as computational-model-based neurophysiology. this new approach is very attractive and appealing since this is probably the only method with which we can explore neural representations remote from either sensory or motor interfaces. but, sometimes limitation of mere temporal correlation between the theory and data became so apparent, and we started to develop a new paradigm 'manipulative neuroscience' where physical causality is guaranteed. research funds: nict, karc sy1-1-09-6 neural mechanisms in williams syndrome-insights from neuroimaging andreas meyer-lindenberg nimh, bethesda, md, usa williams syndrome (ws), a rare disorder caused by hemizygous microdeletion of −25 genes on chromosome 7q11.23, has long intrigued neuroscientists with its unique profile of striking behavioural abnormalities, such as hypersociability, combined with a differential impact on cognitive functions, with some types of abilities only mildly affected while others are severely impaired. ws, thus, raises fundamental questions about the neural mechanisms of social behaviour, the modularity of mind, and brain plasticity in development, and provides a privileged setting to understand genetic influences on complex brain function in a bottom-uph way. recent months have seen dramatic advances in uncovering the functional and structural neural substrates of ws and a beginning understanding of how these are related to dissociable genetic contributions characterized both in special participant populations and animal models. we will review neuroimaging work indicating abnormal function and structure in subsystems of visual processing, long term memory, and emotional regulation and social cognition, and discuss advances in relating them to the underlying molecular biology of this unique syndrome. daisuke yamamoto tohoku university graduate school of life sciences, japan the fruitless locus of drosophila was originally recognized by its mutants, the males of which preferentially court males rather than females. the fruitless primary transcript is subject to sexually dimorphic splicing mediated by transformer, and encodes a group of proteins that are putative transcription factors of the btb-zn finger protein family. the male-specific fruitless protein is expressed in small groups of cns neurons of males, but not of females. fruitless masculinizes these neurons thereby establishing the neural substrates for male-typical behavior. by experiments that label individually the neurons that express fruitless, we have identified a subset of brain interneurons that display marked sexual dimorphism in their number and projection pattern. fruitless supports the development of those neurons with male-specific dendritic fields, which are programmed to die during development in females as a result of the absence of fruitless. thus, the fruitless protein expression can produce a male-specific neural circuit likely used for heterosexual courtship by preventing cell death in identifiable neurons. hitoshi okamoto riken brain science institute, wako, saitama, japan the emotional behavior depends on the evolutionarily most conserved neural circuits. especially the fear behaviors involve the basal telencephalic nuclei such as the amygdala and the nucleus accumbens. thanks to the progress in understanding of the telencephalic development among different species, we can determine the correspondence of the parts between the teleost and mammalian telencephalons. with these in mind, we initiated the characterization of the emotional neural circuits in the zebrafish brain which are amenable to various modern technology. we already reported the asymmetric axonal projection from the left and right habenulae which act as the relay station to conduct the emotional information from the telencephalon to the monoaminergic neurons in the midbrain and the hindbrain. to investigate whether such asymmetric neural circuits cause the laterality for emotional behaviors, we are now in the process of establishing the paradigms for combining the behavioral assay with genetic manipulations to control the activity of the emotional neural circuit in zebrafish. research funds: kakenhi (17023501) sy1-1-17-3 a molecular biological approach for songbirds to study learned vocal communication kazuhiro wada, erich jarvis duke university, usa songbirds possess one of the most accessible neural systems for the study of brain mechanisms of behavior, particularly that for learned vocal communication. however, neuroethological studies in songbirds have been limited by the lack of high-throughput molecular resources and gene manipulation tools. to overcome this limitation, we generated a resource of full-length cdnas for gene expression analyses and functional gene manipulation in songbirds. we constructed total 21 full-length cdna libraries from brains in different behavioral and developmental conditions. with these cdnas, we created a novel database and 18 k songbird cdna array. we used the arrays to reveal a set of 33 genes regulated by singing behavior. their molecular functions spanned most cellular and molecular categories, including signal transduction, structural, and synaptically released molecules. with the full-length cdnas, we were able to express proteins of singing-regulated genes in targeted brain area, using a lentiviral system. this resource now opens to more thoroughly study molecular neuroethological mechanisms of behavior. research funds: uehara memorial fellowship to k.w. and nih grant to e.j. -1-17-4 stepping pattern learning using mice: histochemical identification of activated neuronal circuits takashi kitsukawa graduate school of frontier biosciences, osaka university, osaka, japan identifying brain areas and neuronal circuits activated in a behavior is a critical step in understanding how the brain works in that behavior. also, identifying neuronal types involved in a behavior is a key step toward connecting behavioral approaches with molecular and genetical approaches. an efficient method of clarifying neuronal types activated by behavior is histochemical identification of neuronal types combined with c-fos staining. i would like to introduce our work as an example of using this method. in order to understand the neural processing involved in sequential motor skill learning we built a wheel running system in which a mouse learns sequential stepping patterns. we double-stained brain sections from mice which performed this task with c-fos and a neuronal marker such as enkephalin, substance p or nitric oxide synthase, each of which denotes a particular neuronal type. our results indicate that particular types of stiriatal neurons are activated during this learning, suggesting that cortico-striatal circuits are involved. synaptic plasticity that is dependent on precise timing of spikes between pre-and postsynaptic neurons plays important role in development and plasticity of brain functions. such spike-timingdependent plasticity (stdp) has attracted wide attentions because of its high computational power and physiologically plausible induction. we previously demonstrated that long-term potentiation was closely associated with structural plasticity of dendritic spines. however, how stdp is associated with structural changes has not been elucidated. we here report that paired two-photon uncaging of a caged-glutamate compound at a single spine and postsynaptic spike of whole-cell clamped neuron rapidly induced long-lasting bidirectional structural plasticity of spines in hippocampal ca1 pyramidal neurons. our results indicate that stdp is intimately associated with bidirectional structural plasticity at the level of single spines. research funds: kakenhi 17680033 sy1-2-02-4 role of camkii as a structural protein that stabilizes actin cytoskeleton in dendritic spines kenichi okamoto, radhakrishnan narayanan, yasunori hayashi massachusetts institute of technology, usa actin serves as a major cytoskeleton which maintains spine structure and exists in a equilibrium between f-actin and g-actin. tetanic stimulation causes a persistent shift of actin equilibrium towards f-actin which enlarges dendritic spines. but the mechanisms which maintain these changes remain elusive. we propose that camkii ␤ acts as an actin stabilizing molecule to maintain spine structure. camkii ␤ is not only an abundant f-actin binding protein, it can also make oligomers. we found that camkii ␤ oligomer crosslinks f-actin and stabilizes actin depolymerization kinetics. in spines, camkii ␤ oligomer slows down actin dynamics and camkii ␤ is enriched in spines by actin polymerization. the suppression of endogenous camkii ␤ alters spine shape to filopodia-like structures. these experiments suggest that camkii ␤ plays a role as a major stabilizer of the actin cytoskeleton to maintain spine structure. we also found that camkii ␤ detaches from f-actin in an activity dependent manner. we will discuss how camkii ␤ maintains actin equilibrium in activity dependent dendritic plasticity. ryohei yasuda duke university medical center, usa the small gtpase protein ras plays central roles in calcium signaling important for many forms of synaptic plasticity and regulation of neuronal excitability. using 2-photon fluorescence lifetime imaging microscopy in combination with a fret-based ras activity sensor, we visualized the activity of ras signaling with high spatiotemporal resolution. our studies indicate that calcium entry due to action potentials causes ras to activate in a supra-linear manner (yasuda et al., 2006) . furthermore, in response to single spine stimulation using 2-photon glutamate uncaging, ras activation initially occurs at the stimulated spine, subsequently spreading into its parent dendrites and nearby spines. these results suggest that nonlinear filtering by ras regulators as well as the spatial spreading of ras and ras regulators shape spatiotemporal patterns of ras signaling. hiroshi shibasaki takeda general hospital, kyoto, japan involuntary movements are unintended, generalized or focal, movements of abnormal nature, and include tremor, myoclonus, dystonia, chorea/ballism, athetosis and dyskinesia. myoclonus is characterized by abrupt, shock-like movements caused by brief muscle contraction (positive myoclonus) or abrupt cessation of on-going muscle contraction (negative myoclonus), or their combination. depending on the estimated origin, it is classified into cortical, brain stem, and spinal myoclonus. cortical myoclonus is short in duration (50 ms). by back averaging eeg or meg time-locked to spontaneous myoclonus, a cortical activity is demonstrated in the corresponding area of the contralateral primary motor cortex immediately preceding the myoclonus (by 20 ms for hand). it is mediated by fact-conducting corticospinal pathway. cortical myoclonus is often stimulus-sensitive (cortical reflex myoclonus), showing extremely enhanced cortical responses to somatosensory or visual stimulus, and enhanced longloop transcortical reflexes. these findings, together with transcranial magnetic stimulation, suggest increased excitability of sensorimotor cortex in cortical myoclonus. mark hallett ninds, bethesda, md, usa there have been many recent advances in the understanding of the physiology of focal dystonia. three main avenues of research have shown abnormalities in cortical inhibition, sensory processing including sensorimotor integration, and plasticity. this lecture will emphasize the abnormal inhibition. abnormal inhibition appears to be the most direct cause of unwanted muscle contractions that make up both the involuntary spasms and the overflow movements also seen in this condition. a loss of inhibition is seen in spinal and brainstem reflexes, but these changes are likely secondary to cortical abnormalities. cortical inhibition is also diminished as demonstrated most clearly with transcranial magnetic stimulation. gaba content may be decreased as shown with magnetic resonance spectroscopy. a particular type of defective inhibition is surround inhibition, the inhibition that normally operates to sharpen fine skilled movements. studies are now in progress to determine the synaptic mechanisms of surround inhibition and how this becomes abnormal in dystonia. understanding about inhibition in dystonia has led to some new treatments including some non-invasive cortical stimulation methods. research funds: nih intramural program sy1-2-10-3 basal ganglia-cortical systems reinforcing tonic motor activity in health and disease peter brown sobell department, institute of neurology, uk the synchronisation of neuronal activity in the beta frequency (∼20 hz) band has been noted in healthy primates, including humans, at both striatal, putamenal and cortical levels. it is most obvious in the motor cortex during tonic motor activity and is suppressed by voluntary movement. in this talk i will develop the idea that beta band synchronisation in the basal ganglia-cortical loop promotes tonic/postural contraction at the expense of new movements. thus, spontaneous phasic increases in beta activity in healthy subjects can be shown to be associated with a slowing of voluntary movements and a reinforcement of transcortical stretch reflexes. beta synchrony is also greatly exaggerated in untreated parkinson disease, where it may bias against new movement and contribute to bradykinesia and rigidity. excessive dopaminergic stimulation, either during treatment for parkinson disease, or in conditions such as dystonia, may overly suppress beta activity in bg-cortical loops leading to excessive movement. recordings of local field potentials in the basal ganglia of patients with movement disorders will be described that support this schema. research funds: mrc sy1-2-10-4 coding of reward value of actions and valuebased action selection in the basal ganglia a damage of the nigrostriate dopamine system results in severe impairments of voluntary movements as well as involuntary behavioral states like rigidity, akinesia and tremor as typically observed in parkinson disease. recent studies revealed that long-term potentiation of corticostriatal synaptic transmission occurs dopaminedependent manner, and that neuronal firing related to external stimuli and body movements are modulated by whether the stimuli and movements are associated with reward or not. we recorded striatal neurons of monkeys who chose between left-and right-handle-turns based on the estimated reward probabilities of the actions. during a delay period before the choices, activity of more than one-third of striatal projection neurons was selective to the values of one of the two actions. during handle-turns, another subset of neurons was activated. these results suggest representation of action values in the striatum, which can guide action selection in the basal ganglia circuit. roles of the basal ganglia circuit in voluntary and involuntary action selection will be discussed. in vivo reporter gene imaging is expected to be a powerful tool in gene and cell therapy monitoring. we designed a new pet reporter gene system with f-18 fluoroestradiol (fes) and human estrogen receptor ligand binding domain (herl), which would work in various tissues with little physiological effect. we have been evaluating its potential in gene therapy monitoring constructing a plasmid co-expressing thymidine phosphorylase (htp), a factor works for revascularization, as therapeutic gene and herl. cos7 cells transfected with the plasmid expressed the both proteins and, when the plasmid was in vivo electroporated into mouse calf muscle, the electroporated muscle accumulated significantly higher amount of fes that the control side. this system was successfully applied to es cell transplantation monitoring also. inducible herl expression system was stably transfected into mouse es cells and viable es cells could be detected in vivo using fes. these data support the prospect that our in vivo reporter gene system would be useful in gene/cell transplantation therapy monitoring. tetsuya suhara molecular imaging center, national institute of radiological sciences, chiba, japan the molecular imaging using positron emission tomography enables to visualize various brain molecules with radio labeled ligand. neurons and glias express various receptors and transporters and those can be a specific target of the imaging. the functions of those molecules can be examined in various types of pharmacologically or genetically modified animal models. amyloid precursor protein transgenic mice provide the target of in vivo imaging of amyloid protein and glial reaction. because pronounced neuronal death is frequently heralded by microgliosis, in vivo analysis of glial activation in a quantitative manner could be a powerful means for assessing neuroglial degeneration. on the other hand, clinical finding of molecular imaging can also provide important cues for the basic research targets. since there is no ideal animal model for psychiatric disorders, the abnormal dopamine d1 receptor found in clinical research indicates a possible therapeutic target of negative symptoms of schizophrenia. the bidirectional interaction between basic research and clinical research using the molecular imaging technique can expand our knowledge in brain disease. sumiko mochida department of physiology, tokyo medical university, tokyo, japan in presynaptic terminals, packages of neurotransmitters, synaptic vesicles (svs), are localized at specific sites in different stages by regulation of proteins complexes. the recent outstanding studies have revealed molecular mechanisms of presynaptic structure and function. for svs, new proteins were found and the anatomy of vesicle structure was clarified. readiness for transmitter release, svs are docked and primed at the cytomatrix at the active zone where proteins complex formation is regulated by phoshorylation. new kinase sad-1 found at the sv and active zone or pka phosphorylates specific proteins. sv exocytosis is triggered by conformational changes in the fusion proteins complex when ca 2+ sensing protein was activated. synchronies of sv fusion are maintained by a ca 2+ sensing protein, synaptotagmin i. after transmitter release svs are recycled: surprisingly, recycled svs are shared between synaptic boutons regulated by cytoskeletal and motor proteins. these new findings suggest fine mechanisms in presynatic terminals that regulates transmitter release. shigeo takamori 21st century coe program, department of neuorology and neurological science, tokyo medical and dental university, tokyo, japan synaptic vesicles are storage organelles for neurotransmitters that recycle in the presynaptic terminals. to achieve their functions, i.e. neurotransmitter uptake and membrane fusion, they have to be equipped with specified proteins that play essential roles for each process. since decades ago, we have witnessed major advances in our knowledge about the molecular constituents on synaptic vesicles and we've functionally characterized many key players on membrane fusion machinery such as snare proteins and the rab gtpases and on neurotransmitter uptake such as vesicular transporters and vacuolar h + -atpase. however, a detailed picture of a vesicle membrane with all of its constituents is not yet available. in the present study, we have applied a combination of biochemical and biophysical approaches on purified synaptic vesicles from rat brains in order to arrive at a comprehensive and quantitative description of synaptic vesicles. in particular, with a newly developed counting method for synaptic vesicles in solution, we estimated the copy number of each molecule in a single synaptic vesicle. sy1-3-11-3 sad: a novel kinase implicated in phosphoproteome at the presynaptic active zone toshihisa ohtsuka department of clinical and molecular pathology, faculty of medicine/graduate school of medicine, university of toyama, toyama, japan sad is a serine/threonine kianse, which has been shown to regulate various neuronal functions during development, including clustering synaptic vesicles, maturation of synapses, and axon/dendrite polarization: these have recently been revealed by genetic studies in c. elegans and mice. to test if sad is also involved in synaptic functions at mature neurons such as neurotransmitter release, we have recently isolated and characterized human orthologues of sad. interestingly, sad localizes both on synaptic vesicles and at the presynaptic active zones. moreover, sad, together with prominent active zone proteins cast and bassoon, is tightly associated with the active zone cytomatrix. in accord with its unique localization at the nerve terminals, sad appears to be involved in a late step of neurotransmitter release, via direct phosphorylation of another active zone protein rim1. thus, these results suggest that sad regulates not only neural polarization during development but also neurotransmitter release at mature synapses. toshiaki sakisaka 1 , takeshi baba 1 , sumiko mochida 2 , yoshimi takai 1 1 department of molecular biology and biochemistry, osaka university graduate school of medicine, osaka, japan; 2 depatment of physiology, tokyo medical university, tokyo, japan two types of factors are involved in ca 2+ -dependent neurotransmitter release: the snare system and its regulators. the regulators of the snare system include many factors, such as the rab3 system, munc-13, munc-18, doc-2, and tomosyn. we have previously reported that tomosyn, a syntaxin-1-binding protein, works as a molecular clamp that controls free syntaxin-1 availability for the formation of the snare complex and thereby regulates synaptic vesicle exocytosis. here we show that pka-catalyzed phosphorylation of tomosyn decreases its binding to syntaxin-1, resulting in enhanced the formation of the snare complex. conversely, rock phosphorylates syntaxin-1, which increases the affinity of syntaxin-1 for tomosyn and forms a stable complex with tomosyn, resulting in inhibition of the formation of the snare complex. thus, tomosyn is likely to be an upstream regulator of the snare system, whose activity is regulated via well known signal transduction pathways. tei-ichi nishiki department of physiology, okayama university, okayama, japan a synaptic vesicle membrane protein, synaptotagmin i is thought to be a ca 2+ sensor for neurotransmitter release. however, the physiological contributions of its ca 2+ -binding domains (c 2 a and c 2 b) are still unclear. we have studied the roles of aspartate (asp) residues in the c 2 b ca 2+ -binding sites. in synaptotagmin i deficient neurons, although synchronous release was abolished as previously reported (cell 79, p. 717) , asynchronous release was significantly increased. this defect was completely rescued by expressing wild-type synaptotagmin i, indicating the crucial roles of synaptotagmin i for triggering synchronous release and suppressing asynchronous release. synaptotagmin i with mutations in the second or third asp inhibited synchronous release, but still could suppress asynchronous release. thus, we conclude that synaptotagmin i maintains the synchrony of transmitter release in two ways. ca 2+ binding to the c 2 b is essential for synchronizing release. suppressing of asynchronous release seems not to require ca 2+ binding to the c 2 b because mutation in the second asp inhibits ca 2+ binding, yet still allows the protein to suppress asynchronous release. yukiko goda, kevin j. darcy, kevin staras, lucy m. collinson mrc cell biology unit and lmcb, university college london, uk it has been assumed that vesicle replenishment at central synapses operates autonomously at individual presynaptic terminals. we tested the classical model of a compartmentalized synaptic vesicle cycle using fluorescent styryl dyes in combination with methods of fluorescence recovery after photobleaching and correlative light and electron microscopy in cultured hippocampal neurons. we found that endocytosed, recycling synaptic vesicles travel along axons and incorporate into non-native synapses by an actin-dependent mechanism. these newly-incorporated vesicles underwent exocytosis upon stimulation, demonstrating that they form part of the functional recycling pool at their host synapses. our findings indicate that synaptic vesicle recycling is not confined to individual presynaptic terminals but rather a substantial proportion of synaptic vesicles are shared constitutively between synapses. research funds: mrc, nih and narsad hiroshi kunugi department of mental disorder research, national institute of neuroscience, ncnp, japan neurotrophins such as brain-derived neurotrophic factor (bdnf) and neurotrophin-3 (nt-3) have been implicated in the phathogenesis of several neuropsychiatric diseases including schizophrenia, mood disorders, and neurodegenerative diseases. in the past decade, we have systematically screened bdnf, nt-3 and its low-affinity receptor p75 genes for polymorphisms and their possible association with neuropsychiatric diseases. as a result, three polymorphisms of the bdnf gene (c270t in the 5 noncoding region, bdnf-linked polymorphic region, and val66met), three polymorphisms of the nt-3 gene (g-3004a, microsatellite in intron 1, and gly-63glu) and a missene polymorphism of the p75 gene (ser205leu) have been found to be associated with susceptibility to schizophrenia, bipolar disorder, depressive disorder, or alzheimer's disease, although some contradictive negative results have also been reported. here i summarize these findings, review the relevant literature, and discuss future directions of the promising role of the genetic variations of neurotrophins and p75 in neuropsychiatric diseases. recently, a single nucleotide polymorphism (val66met) in the bdnf gene resulting in a prodomain substitution at position 66 from a valine (val) to methionine (met) has been shown to lead in humans to altered hippocampal size and function, and susceptibility to neuropsychiatric disorders. we have recently determined in vitro that bdnf met aberrantly engages a specific vps10 protein, sortilin, that is part of a highly specialized sorting machinery that regulate bdnf trafficking to secretory pathways. in order to determine whether these trafficking defects are responsible for impaired hippocampal functioning, we have developed a transgenic knock-in mice containing the genetic variant bdnf (bdnf met/met ). we have determined that there is a regulated secretion defect for bdnf met , as well as altered hippocampal structure and function in these bdnf met mice, in a manner similar to that reported in humans with this variant bdnf. thus, this bdnf met/met mouse may provide an in vivo model system to inform human studies focused on associations of this variant bdnf with clinical disorders. research funds: nih grant# ns052819 sy1-3-19-6 processing of bdnf and brain disorders masami kojima 1,2 1 aist, osaka, japan; 2 sorst, jst, saitama, japan the fact that pro-and mature neurotrophins elicit opposite effects through p75 neurotrophin receptor (p75ntr) and trks, respectively suggests that proteolytic cleavage of proneurotrophins is an important mechanism that controls the direction of neurotrophin actions. here we examined the effects of two rare single nucleotide polymorphisms (snps); 372 (t/g) and 378 (t/g) of the human bdnf gene, causing amino acid substitution (r125m and r127l) near the cleavage site. western blot analysis and two-side elisa demonstrated that these snps prevented the cleavage, resulting in secretion of probdnf, but not mature bdnf. these snps did not affect intracellular distribution or mode of secretion of the protein. application of the uncleavable probdnf (probdnfml) elicited apoptosis of cerebellar granule neurons, but inhibited dendritic growth of basal forebrain cholinergic neurons. together, these results reveal structural determinants for the cleavage of probdnf, and demonstrate distinct functions of probdnf for different populations of neurons. we have now analyzed the brain functions of the mice expressing this form of bdnf. sy1-4-04-1 pleiotropic effects of gdnf in regulation of enteric nervous system development hideki enomoto laboratory for neuronal differentiation and regeneration, riken center for developmental biology, kobe, japan formation of the enteric nervous system (ens) is governed by multiple extracellular signals at a given time during development. inactivation of the gene encoding gdnf, ret or gfr␣1 leads to nearly complete absence of enteric neurons during early development. although this finding establishes gdnf as an essential extracellular signal acting at the initial stage in ens development, little is known about whether and how enccs continue to depend on gdnf later in development. we have generated mice in which function of gfr␣1, the high affinity receptor for gdnf, is conditionally inactivated in a time-specific fashion. we will show how gdnf signal influences cell migration, differentiation, proliferation and survival of developing enteric neurons, and discuss the biological significance of the findings in development and regeneration of the nervous system in general. bone marrow stromal cells (mscs) including the primitive pluriopotent mesenchymal stem cells and the multipotent adult progenitor cells, are attractive targets for cell and gene therapy for the range of central nervous system disorders. we present using replicationincompetent hsv-1 vector that msc population can be efficiently engineered to secrete a series of various cytokines in the large quantities and in long term in vivo to be able to treat the ischemic stroke of the brain potentially. three kinds of gene-transferred mscs, hgf, il2ss+fgf-2, and vegf were prepared and directly transplanted into the lesioned brain of rat transient middle cerebral artery occlusion model. each growth factor gene-transferred mscs achieved the remarkable amelioration of neurological symptoms and apparent decreasing of infarct volume comparison with native research funds: kakenhi (14657350) sy1-4-04-4 hgf gene therapy for the treatment of spinal cord injury masaya nakamura 1 , akio iwanami 1 , kazuya kitamura 1,2 , yoshiaki toyama 1 , hideyuki okano 2 1 department of ophthalmology surgery, keio university, tokyo, japan; 2 department of physiology, keio university, japan hepatocyte growth factor (hgf) has recently been reported to exhibit neurotrophic activity and to play a role in angiogenesis. in this study, we demonstrated the validity of hgf for treatment of spinal cord injury (sci) in adult rats. first, we analyzed temporal expression of hgf and c-met in the injured spinal cord. hgf-mrna expression was relatively low in the acute phase of sci compared with c-met mrna expression. hypothesizing that hgf is insufficient immediately after sci, we induced sci at th10 level in adult rat 3 days after injecting herpes vector-mediated gene transfer of hgf (hgf group) or lacz (control) into spinal cord. motor function was evaluated by bbb score. 1 or 6 weeks after injury, histological analyses were performed. there were significant decreases in apoptotic cell number and significant enhancements of angiogenesis and gap43+fibers in hgf group compared to the control group. animals of the hgf group showed better functional recovery than the controls. these findings suggest that hgf could have therapeutic effects for sci. hiroshi funakoshi, toshikazu nakamura division of molecular regenerative medicine, osaka university graduate school of medicine, osaka, japan hgf was initially identified and molecularly cloned as a mitogen for primary hepatocytes (nakamura et al., 1989) . recently, hgf is found to be a novel neurotrophic factor for various types of neurons, such as hippocampal, cerebral cortex, cerebral granular, motor, and sensory neurons. mutant c-met/hgf receptor knock-in mouse reveals that hgf decreases neuronal survival and axonal elongation of several types neurons, including motor, sympathetic and cerebral granular neurons, during development (maina et al., 1999) . therefore, it is possible to speculate that hgf could play an important role in the retardation or regeneration of neurons in neurodegenerative diseases. here we show the examples of beneficial effects of hgf on model animals of different neural and neurodegenerative diseases, using several delivery methods for hgf including gene therapy approach. we also present the possible application of hgf in modifying the neurogenesis for the disease. references maina et al., 1999 . nature neuroscience. nakamura et al., 1989 . nature. hiroyuki kato center for clinical medicine and research, international university of health and welfare, nasushiobara, japan we examined stroke patients using fmri at acute/subacute and chronic stages, and visualized areas of brain activation during paretic hand movements. normal hand movement activated the contralateral primary sensorimotor cortex, supplementary motor areas, and ipsilateral cerebellum. at the acute/subacute stages, we observed reductions of these activations and/or addition of activation in ipsilateral cortex or contralateral adjacent cortex during paretic hand movements. at the chronic stages, recovery of activation and/or persistent addition of activation were observed. thus, motor functional recovery was accompanied by restoration of brain activation and/or appearance of additional activation within the motor network of the brain. the findings suggest that cortical motor reorganization as well as recovery from reversible injury plays a role in the restoration of motor function. interestingly, the time period during which reorganization occurred was limited to first 1-2 months after stroke, suggesting the presence of a critical period. in the cat, illert et al. (1977) first demonstrated that disynaptic pyramidal excitation in forelimb motoneurons can be mediated via propriospinal neurons located in the c3-c4 segments. in contrast, recently it has been shown that polysynaptic pyramidal epsps are only rarely observed in forelimb motoneurons of macaque monkeys and humans. we reexamined the indirect corticomotoneuronal inputs in the primates, and obtained the following evidence for the pathway. (1) in the macaque, recordings from forelimb motoneurons showed polysynaptic pyramidal epsps after blockade of glycinergic inhibition by strychnine. moreover, we recently identified c3-c4 propriospinal neurons, which receive pyramidal inputs and project to forelimb motor nuclei. (2) in human arm motor units, magnetic stimulation of the motor cortex produced multiple peaks at short latency in the post-stimulus time histogram, whose total duration was longer than the corresponding value of a finger muscle. stimulation of the pyramidal tract in the medulla could also produce multiple peaks, though in a lower frequency. functions of the pathway both in physiological and pathological conditions will be discussed. bisphenol-a (bpa) has been extensively evaluated for toxicity in a variety of tests as the most common environmental endocrine disruptors. we previously reported that prenatal and neonatal exposure to bpa potentiated central dopaminergic neurotransmission, resulting in supersensitivity to psychostimulant-induced pharmacological actions. many recent findings have supported the idea that astrocytes, which are a subpopulation of glial cells, play a critical role in neuronal transmission in the central nervous system. we found that in vitro treatment with bpa caused the activation of astrocytes, as detected by a stellate morphology and an increase in levels of gfap. a low concentration of bpa significantly enhanced the ca 2+ responses to dopamine in both neurons and astrocytes. these findings provide evidence that bpa induces dopaminergic changes in neurons and astrocytes. this phenomenon may, at least in part, contribute to the enhancement by bpa of the development of psychological dependence on drugs of abuse. mami yamasaki, yonehiro kanemura the department of neurosurgery, clinical research institute, osaka national hospital, national hospital organization, osaka, japan l1cam(l1) is a member of the immunoglobulin superfamily of cell adhesion molecules. x-linked hydrocephalus, masa syndrome and certain forms of x-linked spastic paraplegia are now known to be due to mutations in the gene for l1. therefore, these syndromes have been reclassified as l1 syndrome. we performed a nation-wide l1gene analysis and identified li gene mutations in 35 families with l1 syndrome. all the patients showed developmental delay in various degree. we discussed genotype and phenotype correlations, a striking correlation between the mutation class and the severity of symptoms and molecular basis of severity of developmental delay. the loss of extracellular domain functions like l1-mediated cell adhesion and cell migration is considered to be responsible for molecular genesis of ventricular dilatation and disturbance of the functions of cytoplasmic domain would cause symptoms related axon growth in l1 syndrome. research funds: kakenhi (16390424), (16689025) sy1-5-05-4 rett syndrome and developing brain yoshiko nomura segawa neurological clinic for children, japan rett syndrome (rtt) is a neurodevelopmental disorder with mental retardation, autistic feature, and stereotyped hand movements. hypofunction of the brainstem monoaminergic neurons is suggested. pathology showed no degeneration. methyl-cpg-binding protein 2 gene (mecp2) located at xq28 is the causative gene. types of mutation at different functional domains are correlated to clinical severities. x-inactivation also influences phenotypic variability. mecp2 was thought as a global transcriptional repressor, but finding of bdnf as a target gene suggest its role in the neuronal activity-dependent gene regulation. genetic heterogeneities have been suggested and the mutation of cyclin dependent kinase-like 5 gene (cdkl5) manifest as atypical rtt. the mutations of mecp2 are found in other clinical conditions, such as x-linked mental retardation, angelman syndrome, autism, and severe neonatal encephalopathy. thus, the evaluation of rtt gives the clue to study the clinical, pathophysiological, biological and molecular correlation of not only rtt but also other neurodevelopmental disorders. in our previous studies, we have proposed that ros and/or ros-mediated signal play(s) an essential role in 6-ohda-induced, caspase-dependent apoptosis. in contrast, mpp+-mediated death is not blocked by caspase inhibition and is accompanied by an increase in intracellular free calcium. subsequently, we have demonstrated that mpp+ induces release of cytochrome c but not activation of caspase and proposed that depletion of atp and/or calcium-activated calpain-mediated degradation of procaspase-9 are responsible for the absence of subsequent activation of caspases. furthermore, we have identified that degradation of several important proteins by activated calpain and proteasome system is linked to mpp+-mediated dopaminergic neuronal death. as such, we have found that one of onconeural proteins seems to play a role as a potential survival factor, degradation of which is involved in mpp+-induced cell death. taken together, we reason that distinct set of proteases activation is involved in experimental models of pd. therefore, novel strategies interfering activation of these proteases may contribute to prevention of dopaminergic neuronal death. satoshi ogawa department of neuroanatimy, kanazawa university of medical school, ishikawa, japan we discuss the role of er-stress in neuronal cell death in snpc by introducing two models. upregulation of pael-receptor in the substantia nigra pars (snpc) of mice induces endoplasmic reticulum (er) stress leading to a decrease in tyrosine hydroxylase and death of dopaminergic neurons. the role of er stress in dopaminergic neuronal vulnerability was highlighted by their enhanced death in mice deficient in the ubiquitin-protein ligase parkin and the er chaperone orp150, suggesting parkin dysfunction result in er-stress mediated neuronal cell death. conversely, transgenic rats overexpressing megsin (tg meg), a newly identified serine protease inhibitor (serpin), demonstrated intraneuronal periodic-acid schiff (pas) positive inclusions, which distributed throughout the deeper layers of cerebral cortex, hippocampal ca3, and substantia nigrta. enhanced er stress was observed in dopamine neurons in snpc, accompanied with loss of neuronal viability and motor coordination. in both subregions, pas-positive inclusions were also positive with megsin. these data suggest that enhanced er stress causes selective vulnerability in a set of neuronal populations. noradrenaline (na) transmission modulates synaptic excitability and plasticity through distinct receptor subtypes. accumulating evidence has suggested that the central na system modulates consolidation and reconsolidation of long-term emotional memory. here we show that the na system is particularly important for retrieval of reconsolidated emotional memory. the mutation of the gene encoding tyrosine hydroxylase causes a deficit in conditioned taste memory after its reactivation. this memory deficit is restored by pharmacological stimulation of na activity before the test and is also restored by intra-amygdala na stimulation through ␣1or ␤-adrenergic receptors. moreover, intra-amygdala na stimulation in the wild type animals increases their susceptibility to recall reconsolidated memory. our findings indicate that the amygdalar na system, primarily through ␣1and ␤-adrenergic receptors, acts to improve the retrieval of reconsolidated memory trace. shigeru morinobu, shigeto yamamoto, shigeto yamawaki departmnet of psychiatry and neurosciences, hiroshima university, hiroshima, japan as psychophysiological reactivity on exposure to cues resembling an aspect of the trauma is the major symptom in ptsd, it is hypothesized that impaired extinction may be involved in ptsd. rats subjected to single prolonged stress (sps) exhibit the enhanced negative feedback of the hpa axis, exaggerated startle response, and analgesia. thus, sps is a good model of ptsd. we examined whether extinction of fear memory was impaired in sps rats, using the contextual fear conditioning. sps rats exhibited the significant longer freezing during re-exposure to the context 2-4 days after the conditioning. furthermore, repeated administration of d-cycloserine markedly inhibited the development of enhanced freezing in sps rats. we measured the levels of nmda receptor subunits (nr1, nr2a, 2b, 2c), glycine transporter 1, and eaac1, by real-time pcr. no significant changes were found in the hippocampus. based on these findings, it is speculated that the increase in other types of glutamate transporters or nmda receptor modification may play a role in impaired extinction in sps rats. ichiro masai masai initiative research unit, riken, wako, japan in human, there are hereditary retinal diseases such as retinitis pigmentosa. to understand these molecular mechanisms, we performed a large-scale mutagenesis using zebrafish as an animal model. here we report two zebrafish mutants, twilight (tli) and eclipse (els), both of which show no normal erg and okr response. in the tli mutant, photoreceptors initially differentiate but degenerate later. electron-microscopic analyses revealed that photoreceptive membranes are severely disorganized in the tli mutants, suggesting that tli is required for the formation of photoreceptive membranes. in the els mutant, photoreceptors seem normal in morphology, suggesting that phototransduction is compromised. we found that the els gene encodes cgmp phosphodiesterase 6 ␣ -subunit (pde6c), a component of cone-type pde. since genetic mutations of pde6c have not been reported in human, the els mutant provides a good model for studying roles of cone-pde6 in visual functions. shinichi nakagawa 1 , masatoshi takeichi 2,3 , fumi kubo 1,3 1 nakagawa initiative research unit, riken, wako, japan; 2 riken cdb, kobe, japan; 3 department of biostudies, kyoto university, kyoto, japan the marginal region of the optic vesicle contains retinal stem cells that remain undifferentiated and proliferate for a much longer period compared to other progenitor cells in the central retina. we have previously shown that wnt2b, a signaling molecule expressed in a region neighboring the stem cell area, functions as a putative stem cell factor that endows undifferentiated retinal cells with the characteristics of the stem cells. interestingly, wnt2b inhibits cellular differentiation in the absence of notch activity, a well-known signaling receptor that inhibits neuronal differentiation. wnt2b antagonizes proneural gene functions independent of the notch signaling pathway, presumably through unidentified transcriptional repressors. we have isolated several candidate genes that are upregulated upon an activation of the wnt signaling pathway, and some of them are expressed in the stem cell containing region. physiological roles of those genes will be discussed. research funds: kakenhi (16570184) sy1-6-06-3 identification of cell lineage of retinal progenitor cells by cell surface markers sumiko watanabe, hideto koso, shinya satoh department of molecular and developmental biology, university of tokyo, tokyo, japan i would like to discuss about early cellular developmental stages of retina, which we identified by examination of the expression pattern of cell surface markers. we found c-kit and ssea-1 to be spatiotemporal markers of distinct populations of retinal progenitor cells, and these cells dramatically changed their expression profiles of c-kit and ssea-1 during development. c-kit-positive cells expressed various immature retina specific genes; and later onset of rhodopsin expression and stronger proliferation activities were observed. c-kit/ssea-1 double-positive cells showed stronger proliferation activities than ckit single-positive ones. although the number of ssea-1-positive cells was augmented by beta-catenin signal, c-kit-positive cells were positively regulated by notch signaling, suggesting that c-kit and ssea-1 have intrinsically distinct characters. prolonged expression of c-kit by a retrovirus resulted in promotion of proliferation and the appearance of nestin-positive cells in response to scf, suggesting a role for c-kit in retinal development. the retinal photoreceptor cells play a primal and central role in the phototransduction system. they are susceptible to deterioration in human retinal diseases, which lead to severe visual impairment. we have been demonstrated that transcription factors, otx2 and crx play critical roles in retinal photoreceptor development. while otx2 is a key molecule for retinal photoreceptor cell fate determination, crx is essential for the terminal differentiation and maturation of photoreceptors. meanwhile, the photoreceptor cell is a highly polarized neuron and also has epithelial characteristics such as adherens junctions. our investigation of a role of apkc, which has been proposed to play a critical role in the establishment of epithelial and neuronal polarity, in differentiating photoreceptors has shown that apkc is required for the formation of outer & inner segments and ribbon synapse. in addition, we also found that photoreceptor polarity formation has important roles in proper retinal lamination. we would like to present our recent analysis of photoreceptor development. research funds: kakenhi (16790070, 16027257, 17024061) raj ladher riken center for developmental biology, kobe, japan the inner ear translates mechanical energy into neural signals that the appropriate centers of the brain can decode into balance or sound information. the inner ear forms from bilateral thickened discs of ectoderm located on either side of the hindbrain, early during development. induction of the inner ear is mediated by localized signals emanating from the paraxial mesoderm. in the chick, the inner ear is induced by localized fgf19 found in the mesoderm. we find that although fgf19 can induce the inner ear, it is unable to support differentiation of the inner ear. differentiation, that is the development of the chick inner ear hair cells, is triggered by another family member fgf3 and is actually inhibited by fgf19. for full functionality, the inner ear needs to be integrated into the larger auditory complex, made up of the middle ear, the external ear and the auditory centers in the hindbrain. these components develop from diverse origins but are intimately linked during development. we have been trying to understand how integration occurs and present one model by which this could occur. research funds: center support grant, mext leading projects grant sy1-6-06-6 how is olfactory receptor-dependent axonal wiring conducted? shou serizawa, kazunari miyamichi, haruki takeuchi, yuya yamagishi, tokiko tsubokawa, hitoshi sakano department of biophysics and biochemistry, crest jst, university of tokyo, tokyo, japan in the olfactory system, termini of primary axon segregate depending on the type of olfactory receptor (or) expressed, forming the olfactory sensory map. to study how the or-dependent axonal wiring is conducted, we analyzed the gene expression profile in the olfactory epithelium of the transgenic mouse in which the majority of olfactory sensory neurons (osns) express a particular or gene. we found that the expression of the immunoglobulin superfamily gene kirrel2, encoding homophillic adhesion molecule, is down-regulated in the transgenic mouse compared to the wild type control. the expression level of kirrel2 in each osn is found to be correlated with the type of or species expressed in the osn. moreover, kirrel2 promoted fasciculation of osn axon termini in the mosaic gain-of-function experiment. here, we propose that the information of which type of or is expressed in the osn is converted to the expression level of kirrel2 which determines the adhesiveness of axon termini, contributing to or-dependent segregation of osn axons. in spite of its morphological similarity to the other species in the melanogaster species subgroup, drosophila sechellia has evolved distinctive physiological and behavioral characters adapting to its host plant morinda citrifolia, known as the tahitian noni fruit. the ripe fruit of m. citriforia contains hexianoic acid and octanoic acid, the main components of the odor from the fruit. d. sechellia is attracted to these two fatty acids, while the other species are repelled by them. using inter-species hybrid between d. melanogaster deficiency mutants and d. sechellia, odorant binding protein 57e was identified as the gene responsible for this behavioral difference among the species. obp57e forms a gene cluster with obp57d, and these two genes are expressed in the same cells associated with the chemosensory organ. the history of dynamic obp57d/e-cluster evolution was revealed by comparison of the genomic sequences of the obp57d/e region obtained from 30 species phylogenetically located between d. melanogaster and d. pseudoobscula. sy1-6-14-4 an approach of dissociating complex traits into fine genetic elements using consomic strains of mouse aki takahashi 1,2 , akinori nishi 1,2 , toshihiko shiroishi 1,2 , tsuyoshi koide 1,2 1 sokendai, kanagawa, japan; 2 national institute of genetics, shizuoka, japan much of the genetic variation that underlies most behavioral traits is complex and is regulated by loci that have quantitative effect on the phenotype. we have previously shown that laboratory strain c57bl/6 (b6) and wild-derived strain msm/ms have great differences in many behavioral traits. consomic strains were established by natural mating between b6 and msm, and those strains have the same genetic background as b6 except for one chromosome from msm. by examining bunch of consomic strains on many behavioral trait, such as spontaneous activity, anxiety-like behavior, pain sensitivity, and social behavior, we were able to map which chromosome have a locus or loci affecting those phenotype. one strain b6-17msm, which have msm chromosome 17, showed increased fear responses and riskassessment behavior, and thus it is thought that there is a locus/loci related to the emotionality. to identify the gene in the loci, we have made congenic strains, and successfully narrowed the locus down in the telomeric region. research funds: kakenhi (16.12064) sy1-6-14-5 cloning of the major quantitative trait locus underlying capsaicin resistance in mice capsaicin is the main compound of hot chili peppers, and induces sensations of heat and pain. however, sensitivity to capsaicin differs among individuals. a genetic approach using a mouse model reveals some quantitative trait loci for this sensitivity. capsaicin resistance linked on chromosome 2 (capsq1) is the major locus affecting reduced taste sensation in kjr mice. here we show that intracellular recycling of capsaicin receptor (trpv1) was impaired in kjr neurons in contrast to that of c57bl/6j mice. by searching the candidate genes, eh domain-containing four (ehd4), a trp-binding scaffold protein encoding gene was found. ehd4 binds to c-terminal of trpv1. three mutations were found in ehd4 of kjr, which remarkably diminished the binding, leading to changes in the intracellular distribution of trpv1. this study is the first genetic dissection associated with capsaicin/heat resistance in a nature strain and shows a novel binding protein to trpvs. sy1-6-14-6 comprehensive behavioral analysis of genetically-engineered mice tsuyoshi miyakawa hmro, kyoto university, kyoto, japan one of the major challenges in the life sciences of the post-genome era is to elucidate the functions of the genes at the level of individual animals. final output level of the functions of the genes expressed in the brain is behavior, indicating a need for systematic investigations of the behavioral significance of the genes. in our laboratory, we use a "comprehensive behavioral test battery" for genetically-engineered mice to reveal causal relationships between genes and behaviors. the battery covers broad areas of behaviors, from simple reflexes to highly cognitive functions. so far, we have assessed more than 45 different strains of mutant mice with the battery. surprisingly, more than 90% of the strains showed at least one significant behavioral phenotype, suggesting that a large part of the genes expressed in the brain may have some functions. representative results for a few strains of mutant mice and the meta-analytic results of the combined data will be presented. also, a potential impact of our approach to "large-scale neuroscience" will be discussed. research funds: kakenhi (16680015, 16653065, 17017021, 17025023) , jst bird hiroshi takashima department of neurology and geriatrics, kagoshima university, kagoshima, japan inherited neuropathies are clinically and genetically heterogeneous. at least 28 genes and 12 loci have been associated with charcot-marie-tooth disease (cmt) and related inherited neuropathies. most causes of inherited neuropathy have been discovered by positional cloning technique and in the past two years, the pace of cmt gene discovery has accelerated. these recently discovered cmt causing genes/proteins include those which, although showing unpredictable correlations with the peripheral nervous system, are definitely important for the peripheral nerve. their discovery should pave the way for dramatic progress in the understanding of peripheral nerve biology. on the other hand, genotype-phenotype correlations of these genes are also important in order to understand the pathomechanisms of inherited neuropathy since, based on mutation studies, a large number of genes associated with both the demyelinating and axonal forms of cmt have been identified. to clarify the specific features and molecular mechanisms, we reviewed recent progress in cmt research, especially cmt4f caused by prx, and scan1 caused by tdp1. sy1-6-22-2 gangliosides are important for the maintenance of the nodes of ranvier nobuhiro yuki, keiichiro susuki department of neurology, dokkyo university school of medicine, tochigi, japan gangliosides are abundant in vertebrate nervous system, but the function has yet to be elucidated. some patients with guillain-barre syndrome have autoantibodies to gangliosides such as gm1, who show failure of peripheral motor nerve conduction. sensitization of rabbits with gm1 can produce the disease model. in ventral roots from the paralyzed rabbits, igg and complements deposited on the nodes of ranvier, and sodium channel clusters were disrupted. in ganglioside-deficient mice with disrupted gm2/gd2 synthase gene, motor nerve conduction velocities were reduced in the sciatic nerves. some myelin loops failed to contact the paranodal axolemma, and potassium channels were aberrantly localized at the paranodes. the abnormality became prominent with age. these findings using different models showed that gangliosides are important for the maintenance of the node of ranvier and saltatory conduction along the myelinated nerve fibers. hiroshi ueda division of molecular pharmacology and neuroscience, nagasaki university graduate school of biomedical sciences, nagasaki, japan neuropathic pain caused following nerve injury is one of important issues in neuroscience as well as clinics, since its pain pathway is apparently distinct from that in healthy humans and naive experimental animals. this is clearly evidenced by the finding that the tactile information is converted to noxious one in allodynia characterized in neuropathic pain. in our recent paper (nature medicine, 2004) , we firstly demonstrated that lysophosphatidic acid (lpa) and its receptor (lpa1) activation initiate the neuropathic pain. in this and following studies we proposed that the demyelination of nociceptive fibers reorganizes the nociceptive spinal inputs through sprouting and electrical synapses (ephapses). i will discuss four issues, lpa-induced demyelination of dorsal root fibers using in vivo and ex vivo culture models, the signal transduction of underlying lpa-mediated downregulation of myelin proteins, evidence for sprouting and ephapses following demyelination and the origin of injury-specific lpa production in terms of demyelination and allodynia. research funds: kakenhi (17109015) toshihide yamashita department of neurobiology, graduate school of medicine, chiba university, chiba, japan axons of adult central nervous system are capable of only a limited amount of regrowth after injury, and an unfavorable environment plays major roles in the lack of regeneration. some of the axon growth inhibitory effects are associated with myelin. three myelin-derived proteins have been identified to inhibit neurite outgrowth in vitro. these proteins induce activation of rho in some neruons. inhibition of rho or rho-kinase promotes axon regeneration in vivo. these findings establish rho and rho-kinase as key players in inhibiting regeneration of the central nervous system. i will review recent findings regarding the signaling mechanism of axon growth inhibitors. our experiments suggest that several new candidate proteins may be axon growth inhibitors. these proteins activate not only rho/rhokinase but also other signals to inhibit neurite outgrowth from some neurons in vitro. these findings suggest that agents that block the multiple signals elicited by these axon growth inhibitors may provide efficient tools that produce functional regeneration following injuries to the central nervous system. sha mi biogen idec, usa lingo-1 is a cns-specific protein expressed in both neurons and oligodendrocytes. in neurons, lingo-1 mediates the inhibition of axonal growth as a component of the ngr1/p75/lingo-1 and ngr1/troy/lingo-1 signaling complex. inhibition of endogenous lingo-1 by soluble lingo-1 or dominant negative lingo-1 can reverse the inhibition of neurite outgrowth by myelin components. soluble lingo-1 treatment significantly improves functional recovery of spinal cord injured rats as determined by bbb scores. soluble lingo-1 treatment promotes axonal regeneration and reduced axon dieback in the corticospinal tract, rubrospinal tract, and optic nerve. in oligodendrocytes, lingo-1 mediates the inhibition of differentiation and myelination. loss of lingo-1 function using dominant negative lingo-1, lingo-1 rnai, or soluble lingo-1 or lingo-1 knockout increased oligodendrocyte differentiation and myelination, whereas over-expression of lingo-1 led to inhibition of oligodendrocyte differentiation and myelination, in vitro and in vivo. the discovery of a significant role for lingo-1 in neurons and oligodendrocyte biology are an invaluable step for understanding cns axon regeneration and myelination. alex reyes new york university, usa neurons in the auditory cortex exhibit a wide range of firing patterns. to elucidate the cellular properties and circuitry that give rise to these responses, a 2d sheet of excitatory and inhibitory neurons were reconstructed in vitro using an iteratively-constructed network (icn) modified to contain both feedback and feedforward circuits. a disc of neurons was stimulated and the resultant firing pattern and spread was documented. simultaneous whole-cell recordings were performed from pyramidal and interneurons in a slice preparation of the mouse auditory cortex. a computer simulated the activities of thalamic neurons and calculated the net synaptic conductance that would be generated by their firing. this waveform was converted to current, injected into the recorded neurons via a dynamic clamp circuit, and the resultant firing documented. using the icn method, we reproduced the firing of a realistic network of excitatory and inhibitory neurons. we replicated many of the responses recorded in vivo. morever, the firing patterns of neurons depend substantially on their distance from the stimulus center and on the identity of the local interneurons. research funds: nih dc005787-01a1 sy1-7-15-2 neuronal avalanches reveal neuronal wirings of layer 2/3 cell assemblies jun-nosuke teramae, tomoki fukai brain science institute, riken, japan how cortical neurons process information crucially depends on how their local circuits are organized. spontaneous synchronous neuronal activity propagating through neocortical slices displays highly diverse, yet repeatable, activity patterns called 'neuronal avalanches'. they obey power-law distributions of the event sizes and lifetimes, presumably reflecting the structure of local cortical networks. however, the explicit network structure underlying the power-law statistics remains unclear. here, we present a neuronal network model of pyramidal and inhibitory neurons that enables stable propagation of avalanche-like spiking activity. we demonstrate a neuronal wiring rule that governs the formation of mutually overlapping cell assemblies during the development of this network. the resultant network comprises a mixture of feedforward chains and recurrent circuits, in which neuronal avalanches are stable if the former structure is predominant. we investigate how the resultant power laws depend on the details of the cell-assembly formation as well as on the inhibitory feedback. research funds: kakenhi (17700318) sy1-7-15-3 spike-timing dependent and homeostatic plasticity from an optimality viewpoint taro toyoizumi 1 , jean-pascal pfister 2 , kazuyuki aihara 1,3 , wulfram gerstner 2 1 department of complexity science and engineering, university of tokyo, japan; 2 school of computer and communication science & bmi, epfl, japan; 3 aihara complexity modelling project, erato, jst, japan maximization of information transmission by a spiking neuron model predicts changes of synaptic connections that depend on timing of pre-and postsynaptic spikes as well as on the postsynaptic membrane potential. under the assumption of poisson firing statistics, the synaptic update rule exhibits all the features of the bienenstock-cooper-munro rule, in particular regimes of synaptic potentiation and depression separated by a sliding threshold. the learning rule is found by maximizing the mutual information between presynaptic and postsynaptic spike trains under the constraint that the postsynaptic firing rate stays close to some target firing rate. an interpretation of the synaptic update rule in terms of homeostatic synaptic processes and spike-timing dependent plasticity is discussed. research funds: grant-in-aid for jsps fellows 03j11691 and sci. res. on priority areas 17022012 from mext of japan, and swiss natl. sci. found. 200020-108097/1 sy1-7-15-4 timing computations in the auditory brain stem john rinzel center for neural science and courant institute of mathematical sciences, new york university, usa sound localization involves precise temporal processing by neurons in the auditory brain stem. the first neurons in the auditory pathway to receive input from both ears can distinguish interaural time differences (itds) in the sub-millisecond range. these cells in the mammalian medial superior olive have specialized biophysical features: two dendrites, each receiving input from only one side; very short membrane time constant; specialized ionic channel properties, including a low-voltage activated k+ current, i-klt. this i-klt contributes to phasic firing (one spike in response to a step of current), precise phase-locking, and extremely timing-sensitive coincidence detection. we will describe the temporal feature-selecting properties of mso cells based on biophysical (hh-like) modeling, in vitro electrophysiology and application of concepts from dynamical systems theory and coding theory. neuronal information is often inferred by counting spike numbers over tens to hundreds of milliseconds. however, if relative spike timings at the scale of milliseconds would carry information, neuronal circuits could have large information capacity. in response to various visual inputs, the retina fires spike bursts separated by hundreds of milliseconds of silent periods. onsets and spike numbers of these bursts are highly reproducible. we asked if spike patterns, i.e., combinations of interspike intervals within single bursts, carry information. using the retinas of salamanders and mice, we found that bursts have various spike patterns, which are unique to the preceding inputs. differences in spike patterns at the scale of milliseconds encode differences in the input as long as 200-300 ms. when single bursts contain three or more spikes, the multiple interspike intervals combinatorially encode multiple features of the input. this suggests the spike patters are not determined sorely by slowly modulating instantaneous firing rates. we propose that the retina encodes multiple features in hundreds of milliseconds of input into burst spike patterns at the scale of milliseconds. accumulating evidence reveals that the generalized seizure activity can produce regenerative, in addition to degenerative, structural changes in the hippocampus, including the enhancement of progenitor cell division of dentate granule cells. although the regulatory mechanisms underlying such neurogenesis are unknown, we hypothesized that newly generated granule cells may contribute to the reorganization of the hippocampal formation in the early course of seizures, constituting a possible substrate for epileptogenicity. to address this issue, we examined the division of dentate granule cell progenitors in rats after kainic acid administration, or perforant path kindling. the results indicate that initial limbic seizures trigger the enhancement of dentate progenitor cell division, but progenitor cells may become unreactive to prolonged generalized seizures. the degenerative process is not necessary for triggering the upregulation. it is also suggested that newly generated granule cells may play a role in the network reorganization that occurs during epileptogenesis. the molecular basis underlying such neurogenesis will be discussed. keiichi itoi 1 , ikue otaki 1 , saya suzuki 1 , yasunobu yasoshima 2 , kazuto kobayashi 2 1 laboratory of informational biology, graduate school of informational science, tohoku university, sendai, japan; 2 institute of biomedical science, fukushima medical university, japan in order to examine functional roles of the noradrenergic (na) neurons in the locus coeruleus (lc) we developed a novel method to ablate specifically the na neurons in the lc, and examined the behavioral and stress responses using the animal model. a transgenic mouse line was used in which human interleukin-2 receptor ␣ subunit (hil-2r␣) was expressed under the control of dopamine ␤-hydroxylase gene promoter. anti-hil-2r␣ antibody fused to pseudomonas exotoxin was microinjected into bilateral lc of a transgenic mouse stereotaxically to destroy specifically the na neurons. as behavioral paradigms, elevated plus maze and open field test were used. plasma adrenocorticotropin levels were measured following lipopolysaccharide injection intraperitoneally, as an immune stress. thus, the effect of lc ablation how it affects the behavioral and stress responses will be elucidated. -8-16-5 integrated circuits controlling the stress response james p. herman department of psychiatry, university of cincinnati, oh, usa the hypothalamo-pituitary-adrenocortical (hpa) axis is a primary stress-response system in all vertebrates. the end-product of hpa activation, glucocorticoids, serve the general function of redirecting bodily resources to meet a real or perceived challenge. however, prolonged glucocorticoid secretion has deleterious effects on metabolism, immune function and behavior, making control of hpa activity a priority for the organism. this control is exerted in large part by limbic structures in the brain. our studies indicate that the amygdala, hippocampus and prefrontal cortex play major roles hpa axis regulation. the amygdala is primarily stress excitatory, whereas the hippocampus has an inhibitory influence on hpa activity. the role of the prefrontal cortex is considerably more complex; its prelimbic region is primarily stress inhibitory, whereas the infralimbic region may participate in stress activation. all of these regions exert their influence via subcortical relays to hypothalamic paraventricular nucleus (pvn) neurons controlling the hpa response, allowing convergence of information from multiple limbic sources prior to the pvn. sy1-8-24-1 molecular mechanism for the inverse incidence of parkinson's disease and cancer: synuclein as stimulator of tumour differentiation makoto hashimoto department of chemistry and metabolism, tokyo metropolitan institute for neuroscience, tokyo, japan neurodegenerative disease and cancer are major age-associated disorders. however, the pathogenesis of these diseases may be in sharp contrast, since the former is featured by cell death, whereas, the latter is associated with immortalization. in parkinson's disease (pd) research, smoking, the risk factor for a variety of cancers, had been known to reduce the risk of pd. furthermore, epidemiological studies described that the incidence of cancer was reduced in pd patients. recent study provides evidences of the inverse relationship of pd and some cancers at the molecular level. for example, loss of neuroprotection of dj-1 is causative for familial pd, while increased expression of this molecule stimulates oncogenesis. in this context, we show that proteasomal inhibition by ␣-synuclein, which has been thought as one major pathogenic mechanism for pd, may induce differentiation of cancer cells. thus, unifying approach on the basis of the opposite pathogenic mechanism to neurodegenerative disease and cancer might uncover unexpected findings in both fields. kiyomitsu oyanagi department of neuropathology, tokyo metropolitan institute for neuroscience, tokyo, japan neurodegenerative diseases and malignant tumors develop symptoms usually at middle or old-age in humans. however, it is well known that critical periods of some malignancies are in fetal period, which are (1) leukemia in patients exposed with atomic bomb during the iind world war, and (2) brain tumors in rats with ethylnitrosourea administration. as to neurodegenerative diseases, (3) many genetic/familial diseases show clinical symptoms at the middle or old age. (4) epidemiological study revealed that emigrants from guam to the main land of usa show relatively high incidence of amyotrophic lateral sclerosis, and the critical period of exposure to some environmental noxiousness was considered to be childhood/adolescence. (5) relating to parkinson disease, low magnesium intake over generations induced selective degeneration of the dopaminergic neurons in the substantia nigra in rats [oyanagi et al., in press] . these findings indicate that not only certain malignant tumors but also some sporadic neurodegenerative diseases may be induced originally by the insults in embryonic stage/childhood. to understand the role of synuclein, the major component of pathological inclusions, we examine the expression of synuclein in the embryonic mouse cerebral cortex. we found that a-synuclein and b-synuclein were predominantly detected in the subplate neurons, which are known to enter programmed cell death at a postnatal stage. in another line of inquiry, we are interested in a zinc finger protein containing poz domain, rp58, which functions as a sequence specific transcriptional repressor and involved in cortical layer formation. when the rp58 gene is disrupted, apoptosis is enhanced, and a-synuclein, but not b-synclein, is upregulated in the mutant cortex, suggesting that a-synuclein is involved in the cell death. interestingly, in the mutant cortex the expression of s-phase marker, pcna increased, suggesting that rp58 mutant mice are useful to analyze the relation among neurodegeration, synuclein and cell cycle. minoru saitoe, junjiro horiuchi, daisuke yamazaki tokyo metropolitan institute for neuroscience, tokyo, japan age-related memory impairment (ami) is a striking feature of ageassociated neuronal dysfunction. to identify gene mutations that affect ami, we screened ∼100 drosophila lines and found that heterozygous mutants for the pka catalytic subunit (dc0/+) exhibit robust suppression of ami without affecting memory at young ages. this result suggests a causal relationship between pka and ami. of particular interest, igf/pi3k/akt signaling, which results in decreased gsk3 activity, has also been shown to ameliorate ami. both pka and gsk3 phosphorylate the microtubule-associated protein tau, causing tau aggregation and neurodegeneration. while igf signaling suppresses activity of gsk3 at young ages, declining igf levels during aging may increase gsk3 activity in aged animals. in support of this idea, we found suppression of ami in flies fed gsk3 inhibitors. we hypothesize that similar to the mechanisms occurring in neurodegenerative diseases, tau phosphorylation by pka and gsk3 causes neuronal dysfunction during normal aging. research funds: kakenhi sy1-8-24-5 molecular mechanism of cancer progression by gamma-synuclein koji okamoto radiobiology division, national cancer center research institute, tokyo, japan synucleins, a family of small proteins consisting of three known members, are implicated in both neurodegenerative disorder and tumorigenesis. ␣synuclein is involved in the formation of pathologically insoluble deposits characteristic of neurodegenerative diseases such as alzheimer disease and parkinson disease, whereas overexpression of ␥synuclein is associated with progression of breast and ovarian cancer. however, the normal cellular function of synucleins remains largely unknown. in order to get an insight into biological function of synucleins, we focus on cancer progression induced by ␥synuclein. we introduced ␥synuclein into breast cancer cells in order to recapitulate malignant transformation of breast cancer. using such cells, the attempt to elucidate the biochemical function of ␥synuclein is underway. the impact of synuclein over-expression, especially on known tumor suppressor pathways such as the p53 pathway, will be discussed. research funds: kakenhi ryuichi sakai growth factor division, national cancer center research institute, 5-1-1 tsukiji, chuo-ku, tokyo 104-0045, japan numbers of growth factors and their membrane receptors which possess tyrosine kinase activity are involved in proliferation and differentiation of the neural system. shc family docking molecules conduct signals directly downstream of various growth factor receptors as substrates and binding partners of these tyrosine kinases. in the neural systems, two unique shc family molecules, shcb and shcc, are found to be specifically expressed and analysis of mice lacking these proteins revealed that they have redundant functions during mammalian neural development as mediators of ngf/trka signaling. it was recently found that tyrosine phosphorylation of shcc is frequently detected in majority of neuroblastoma cell lines. we showed that hyperphosphorylated shcc detected in some of neuroblastoma cell lines is associated with constitutively activated anaplastic lymphoma kinase (alk) caused by the gene amplification. identification of binding partners of shcc and expression of mutant shcc in several cancer cell lines revealed novel roles of shcc as a regulator of differentiation and proliferation of neuroblastic tumors. research funds: kakenhi sy1-8-24-7 identification of estrogen receptor target genes and role of their gene products in cancer and nervous system satoshi inoue 1,2 1 department of geriatric medicine, university of tokyo hospital, tokyo, japan; 2 research center for genomic medicine, saitama medical school, saitama, japan estrogen has crucial roles in the cancer growth and in the neural function. here, we have isolated and characterized novel estrogenresponsive genes to clarify the molecular mechanism of the estrogen action in target cells using genomic binding-site cloning (gbsc) method. one of the first identified genes is the estrogen-responsive ring finger protein (efp). efp expression was observed in uterus, mammary gland and certain regions of the brain where er is also expressed and positively regulated by estrogen. we revealed that efp targets proteolysis of 14-3-3 sigma, a negative cell cycle regulator that causes g2 arrest and that efp is an essential oncogenic factor in breast cancer growth. on the other hand, another gene identified by gbsc is nr2d, an nmda receptor. this gene was regulated by estrogen in the hypothalamus, together with er, pr and efp. these estrogen responsive genes could mediate roles of estrogen action in specific organs, utilizing differential mechanisms as well as sharing common mechanisms. keiji tanaka 1 , hossein esteky 2 , kiani roozbeh 2 , tadashi sugihara 1 , gang wang 3 1 riken brain science institute, wako, saitama, japan; 2 institute for studies in theoretical physics and mathematics, tehran, iran; 3 kagoshima university, kagoshima, japan individual cells in the monkey inferotemporal cortex, which is the final unimodal stage along the ventral visual pathway, respond to moderately complex features, but not to objects nor to object categories. then, questions arise where and how view-general objects and object categories are represented. a possibility is the representation by a population of inferotemporal cells. to examine it, we recorded responses of 670 inferotemporal cells to 1100 object images in a fixation task. we also conducted psychophysical experiments with monkeys to determine conditions for view-invariant object recognition. the results suggest that a population of inferotemporal cells represent object categories and their relational structure, and that the representation is common to nearby views of objects with up to 60 • rotation. research funds: kakenhi 17022047 alexander thiele 1 , gene stoner 2 , louise s. delicato 1 , mark roberts 1 1 university of newcastle upon tyne, uk; 2 the salk institute, japan a variety of different roles of synchronized activity for sensation and perception have been proposed, ranging from object binding, through attentional enhancement, to mechanisms of learning. we have employed different paradigms to investigate the role of neural synchrony in visual perception and attentional selection in the awake macaque monkey. using two different tasks and stimulus conditions, well suited to probe the role of feature binding in the motion domain, we found no support for the idea that neuronal synchrony in macaque area mt underlies the binding of an object's component features. recent reports have focused on the role of synchrony in the mediation of attention. we will discuss the role of synchronized activity in primate v1 while attentional load was varied, and how it could be mediated by cholinergic mechanisms. research funds: hfsp, wellcome trust, bbsrc sy2-1-01-3 context-dependent changes in noise correlation in mt william newsome, marlene r. cohen stanford university and howard hughes medical institute, usa changes in the correlated firing of a pair of neurons may provide a metric of changes in functional circuitry within the nervous system during ongoing behavior. we studied dynamic changes in functional circuitry by analyzing the noise correlations of simultaneously recorded mt neurons in two behavioral contexts: one that promotes cooperative interactions between the two neurons and another that promotes competitive interactions. we created cooperative or competitive contexts by changing the axis of motion of the discrimination task from trial to trial. we found that identical visual stimuli indeed give rise to differences in noise correlation in the two behavioral contexts. specifically, noise correlations were higher in the cooperative than in the competitive context. this result suggests that mt neurons receive inputs of central origin whose strength changes with the task structure. the changes in correlation appear to reflect differences in how mt neurons are pooled for the purpose of perceptual discrimination, and may derive from higher-level cognitive processes such as feature-based attention. research funds: howard hughes medical institute sy2-1-01-4 effects of task demands on color processing in area te of the monkey hidehiko komatsu 1,2 , kowa koida 1 1 national institute for physiological sciences, okazaki, japan; 2 sokendai, okazaki, japan color vision has two different functions, namely, categorization and discrimination, and the same color stimulus can be processed according to these two functions depending on task demands. lesion studies suggested that inferior temporal (it) cortex of the monkey plays a key role in color vision, and we have recently found that color selective neurons are concentrated in a small region in area te of it cortex. to study how the color selective responses in this region are affected by the task demands, we trained monkeys a color categorization task and a color discrimination task using the same set of color stimuli, and analyzed how the responses are affected. we found response magnitudes of many neurons changed between two tasks while the color tuning is well reserved. in several extreme cases, large gain change almost completely eliminated the responses in one task. these results suggest that color signals are gated by the top-down signal representing task demands in area te and color channels specific to different tasks are formed at this level of the visual cortex. yoichi sugita neuroscience research institute, aist, tsukuba, japan early visual experience is indispensable to shape the maturation of cortical circuits during development1. monocular deprivation in infancy, for instance, leads to an irreversible reduction of visually driven activity in the visual cortex through the deprived eye and a loss of binocular depth perception2-4. here, i show exposure only to monochromatic illumination in infancy results in the disruption of color perception. infant monkeys were reared for nearly a year in a room where the illumination came from only monochromatic lights. after extensive training, they were able to perform color matching. but, their judgment of color similarity was quite different from that of normal animals. furthermore, they had deficits in color constancy; they could not compensate for the changes in wavelength composition. these results indicate that early visual experience is also indispensable for color perception. research funds: crest sy2-2-02-1 dendritic growth, spinogenesis and synaptogenesis in response to neurosteroids in the developing purkinje cell kazuyoshi tsutsui 1 , hirotaka sakamoto 2 , katsunori sasahara 1 , hanako shikimi 1 , kazuyoshi ukena 1 , mitsuhiro kawata 2 1 laboratory of brain science, faculty of integrated arts and sciences, hiroshima university, higashi-hiroshima, japan; 2 department of anatomy and neurobiology, kyoto prefectural university of medicine, kyoto, japan new findings over the past decade have established that the brain synthesizes steroids de novo from cholesterol. such steroids synthesized de novo in the brain are called neurosteroids. recently we have identified the purkinje cell as a major site for neurosteroid formation in the brain. this is the first demonstration of de novo neuronal neurosteroidogenesis in the brain. in mammals, the purkinje cell actively synthesizes progesterone and estradiol de novo from cholesterol during neonatal life, when cerebellar cortical formation occurs. subsequently, our recent studies on mammals using the purkinje cell have demonstrated organizing actions of neurosteroids. both progesterone and estradiol promote dendritic growth, spinogenesis and synaptogenesis via each cognate nuclear receptor in purkinje cells. research funds: kakenhi (15207007 and 16086206 to kt) sy2-2-02-2 roles of estrogen receptors in the regulation of socio-sexual and emotional behaviors-studies with knockout mice and rnai sonoko ogawa kansei, behavioral and brain sciences, university of tsukuba, tsukuba, japan the gonadal steroid estrogen plays a major role in the regulation not only of female reproductive behavior but also an array of social and emotional behaviors in both sexes, by acting through intracellular estrogen receptors (ers), ligand dependent transcription factors. a series of studies using single and double knockout mice for er-␣ and/or er-␤ genes have revealed that activation of er-␣ and er-␤ differentially regulate a number of behaviors as well as neuroendocrine functions. our studies have suggested a unique role of activation of er-␤ in the hypothalamic and limbic brain areas, dorsal raphe nuclei and locus coeruleus in the regulation of socio-sexual and emotional behaviors. in this talk, our findings from behavioral studies using er-␣ and er-␤ knockout mice along with possible brain mechanisms underlying the behavioral effects will be first overviewed. our most recent studies on brain site-specific manipulation of er gene expression with the use of small interference rna combined with adeno-associated virus will then be presented. research funds: kakenhi (17330151, 17051001) sy2-2-02-3 sex steroid receptor function in sexual behavior shigeaki kato 1,2 , takashi sato 1 , takahiro matsumoto 1,2 1 imcb, university of tokyo, tokyo, japan; 2 erato, jst, saitama, japan androgen actions are believed to mediate nuclear androgen receptor (ar)-mediated gene regulations. ar is a member of nuclear receptor, and acts as a hormone-induced transcription factor to control of target genes through chromatin remodeling/histone modification. we generated the floxed ar mice to avoid testicular feminization mutant (tfm) abnormalities with infertility, and then crossed with female ar(−/+) heterozygoutes expressing cre to generate ar(−/−) female mice. the ar(−/y) ko males grew healthy with typical features of tfm abnormalities, and genital organs were atrophic with a marked decrease in the serum testosterone level, but with normal estrogen level (kawano et al., 2003) . no sexual behaviors and reduced aggressive behaviors were seen in ar(−/y) male mice (sato et al., 2004) . female ar ko mice were normal in sexual behavior but exhibited premature ovarian phenotype (shiina et al., 2006) . together with these results, the ar function will be discussed in terms of ar function as a transcription factor. references kawano, h., et al., 2003 . pnas usa 100, 9416. sato, t., et al., 2004 . pnas, usa 101, 1673 . shiina, h., et al., 2006 research funds: probrain sy2-2-02-4 annexin 1: a mediator of cell-cell communication in the neuroendocrine system julia buckingham 1 , helen christian 2 , john morris 2 1 imperial college london, uk; 2 department of human anatomy and genetics, university of oxford, uk annexin 1 (anxa1) plays an important part in mediating the regulatory effects of glucocorticoids (gcs) on neuroendocrine function, particularly within the hpa axis. it is expressed by folliculostellate (fs) cells in the pituitary gland and by ependymal cells and activated glia in the hypothalamus but not by classical secretory cells. gcs act on cells expressing anxa1 to cause the translocation of the protein to the plasma membrane at points with particular accumulation at points where the cells make contact with endocrine cells. this process is effected via a non-genomic mechanism and is dependent upon phosphorylation, lipidation and a transport protein, possibly abca1. the released protein then acts, via cell surface receptors on the endocrine cells to suppress stimulus-evoked peptide release. the nature of the anxa1 receptor is unclear but, increasing data suggest that members of the formal peptide receptor family may be important in this regard. katsuhiko nishimori 1 , yuki takayanagi 2 , masahide yoshida 1 , yoshiyuki kasahara 1 , masaki kawamata 1 1 graduate school of agricultural science, tohoku university, sendai, japan; 2 department of physiology, jichi medical university, minamikawachi-machi, japan we examined the behaviors of mice lacking oxtr gene and discovered that oxtr null females displayed impaired nurturing behavior, and their pups showed defect in ultrasonic vocalization, instead, increased locomotor activity by social isolation test. those are implying impaired mother-infant relationship. oxtr null males also showed more aggressive and having social amnesia as well as the phenotype of oxt null mice. in addition, oxtr null mice failed to maintain their body temperature after acute cold exposure. their rectal temperature rapidly dropped in comparison of that of wildtype animals at 5 • c ambient temperature. our studies demonstrate that oxtr plays a critical role in regulating several aspects of social behavior and the other physiological function, and may have important implications for developmental psychiatric disorders, such as autism. research funds: grant-in-aid for scientific research (b) (14360046) sy2-2-08-1 cortical mechanisms mediating visuomotor control of primate grasp roger n. lemon ucl institute of neurology, uk primates demonstrate an exquisite ability to precisely shape their hand when grasping an object. a network of parietal and frontal motor areas is thought to play a key role in this behaviour. our work shows that: hand shape can be unambiguously determined from emg activity of hand and digit muscles. information about grasp is represented by neuronal populations in the ventral premotor cortex (area f5); f5 activity shows graspspecific discharge soon after an object becomes visible, well in advance of activity in primary motor cortex (m1). local field potential activity in f5 and m1 is also tuned to grasp, and there is strong beta coherence between f5 and m1, indicating reciprocal transmission of information. this is also seen in synaptic responses of m1 neurones to stimulation of f5 (and vice-versa). single pulse stimulation in f5 strongly modulates corticospinal outputs from m1 through corticocortical pathways between these two areas. paired-pulse tms can probe the excitability of these pathways in humans. facilitation of meps is both object and muscle specific and indicates that activity in these pathways is selectively enhanced during object grasp. research funds: wellcome trust, bbsrc sy2-2-08-2 where tactile signals are ordered in time shigeru kitazawa 1,2 1 department of neurophysiology, juntendo university graduate school of medicine, tokyo, japan; 2 crest, japan science and technology agency, saitama, japan how does the brain order successive events? it is generally accepted that the brain can resolve the order of two stimuli that are separated in time by 30 ms. this applies to temporal order judgment of two tactile stimuli, delivered one to each hand, as long as the arms are uncrossed. however, crossing the arms caused misreporting (that is, inverting) of the temporal order. the reversal was not due to simple confusion of hands, because correct judgment was recovered at longer intervals (e.g., 1.5 s). when the stimuli were delivered to the tips of sticks held in each hand, the judgment was altered by crossing the sticks without changing the spatial locations of the hands. we recently found that temporal order judgments of tactile stimuli are strongly affected by visual distractors and/or eye movements. the results suggest that tactile stimuli are ordered in time only after they are referred to relevant locations in space, where multiple modalities of sensory signals converge. results from functional imaging support this idea. sy2-2-08-3 decision making and underlying neural mechanisms-auditory-visual ambiguity solving and preference shinsuke shimojo 1,2 1 biology/cns, california institute of technology, pasadena, ca, usa; 2 jst.erato shimojo implicit brain function project, atsugi, japan we explore mechanisms underlying crossmodal ambiguity solving (passive decision), and preference (active decision). we've employed the illusory flash effect, where a single flash appears to be doubled when accompanied by two sounds. 122-channel meg was employed, while the observer reported number of flashes. partial directed coherence was applied to see if there was a causal influence by the auditory on the visual cortices. the results indicate a strong causal influence in a-v direction in alpha (8) (9) (10) (11) (12) and ranges only in the illusion-reported trials, while stimulus parameters were identical. no such difference was found in v-a direction. for preference, the observer's gaze shifted towards the to-be-chosen stimulus (face) before conscious decision. our fmri study shows activity specific to preference task in the ventral amygdala and the ventromedial prefrontal. while such results enable the same causality analysis, it also raises a question as to what determines active/passive nature of decision. research funds: jst.erato, hfsp sy2-2-08-4 why look there? insights from spatial neglect and the medial frontal cortex masud husain ucl institute of neurology, uk why do we look where we do? studies in humans show that when we look at a scene, our initial fixation patterns can be predicted to a high degree of accuracy. our eyes go to the most salient locations where local feature contrast is greatest. these findings have led to the concept of a salience map which directs attention and gaze bottomup. in humans, damage to the right posterior parietal cortex often leads to dramatic neglect of the left side of space. recent research has begun to unravel the components of this syndrome, demonstrating several underlying mechanisms. these include a disturbance of the salience representation, a failure to keep track of spatial locations across saccades and difficulty in sustaining attention over time. gaze is directed not only bottom-up by but also top-down by voluntary mechanisms. our recent investigations of human medial frontal regions reveal important roles for the supplementary eye field and the pre-supplementary motor areas in the control of competing eye movement plans and deciding where to look. parietal and medial frontal gaze regions appear to play different, complementary roles in controlling why we look where we do. research funds: wellcome trust (061140) sy2-2-08-5 recognizing self actions through externalized eyes atsushi iriki 1,2 1 symbolic cognitive development, riken brain science institute, saitama, japan; 2 cognitive neurobiology, tokyo medical and dental university, tokyo, japan we can recognize ourselves and our own actions through the mirror or video images. thus, human can use such apparatus as externalized eyes (sensory tools), while non-human animals can normally use tools as extension of their effectors (motor tools) at most. human fmri studies revealed that the right temporo-parietal junction region and the mesial superior frontal gyrus are involved in perceiving and manipulating the representation of the self actions under different third person perspectives. japanese monkeys could be trained to use a hand-held video camera as a manipulable extension of their eyes only when their own vision was gradually transferred to the distant cues via motor-tools to extend their body images. the emergence of novel cortico-cortical projections between temporo-parietal junction and the intra-parietal cortex was described in monkeys that were trained to use motor tools, therefore, integrate the tool in their own body image. thus, presence of a self-objectification mechanism is suggested for acquisition of sensory tools as externalized eyes to recognize self actions. yoshiyuki kubota division of cerebral circuitry, nips, okazaki, japan gabaergic nonpyramidal cells in the neocortex are composed of several different subtypes. we found that most of gabaergic cell types, including fs basket and somatostatin martinotti cells, that innervate dendritic spines in addition to the somata and dendritic shafts. most postsynaptic spines also received an asymmetrical input, called double innervated (di) spines. to better characterize the other asymmetrical input on the di spines, excitatory presynaptic terminals were stained by immunohistochemistry for two types of vesicular glutamate transporters (vgluts): vglut1, existing mostly in cortical pyramidal cells, and vglut2, found in thalamocortical fibers. gabaergic inputs were rarely observed in spines innervated by vglut1-expressing terminals (n = 289), but were found in-10% of spines innervated also by vglut2-expressing terminals (n = 444). symmetrical synapses on di spines were positive for gaba, as shown by postembedding immunohistochemistry. these results indicated that some thalamocortical inputs are likely selectively inhibited at the spine level by gabaergic synapses from cortical nonpyramidal cells. research funds: kakenhi sy2-3-03-2 gabaergic recruitment of excitation by cortical axo-axonic cells gabor tamas, csaba varga, gabor molnar, szabolcs olah, pal barzo, janos szabadics university of szeged, hungary the axon has the lowest threshold for action potential generation and axons in the cerebral cortex receive input only at the axon initial segment exclusively from axo-axonic cells (aacs), which use the dominant inhibitory neurotransmitter, gamma-aminobutyric acid (gaba). thus, aacs are considered as strategically placed inhibitory neurons controlling cortical information flow. we applied multiple patch clamp recordings in slices of rat and human neocortex and found that single spikes in aacs can trigger action potentials in pyramidal cells and initiate stereotyped series of multiple synaptic events in the cortical network. the excitatory action of aacs is based on a depolarized reversal potential for axonal relative to perisomatic gabaergic inputs as determined in paired perforated patch recordings. powerful axo-axonic depolarization from the resting membrane potential is supported by a ∼44-fold decrease in the potassium-chloride co-transporter 2 (kcc2) expression from somatic to axon initial segment membranes detected by quantitative immunogold labeling. in my talk i will describe the integrative and plasticity properties of thin basal dendrites of cortical pyramidal neurons. these dendrites receive the majority of the cells' synaptic inputs, so determining their integrative and plasticity properties is of prime importance. previous studies have most often reported global linear or sublinear summation in these dendrites. using confocal imaging and dual-site focal synaptic stimulation of identified thin dendrites in rat neocortical pyramidal neurons we show that thin dendrites provide a layer of independent computational "subunits" that sigmoidally modulate their inputs prior to global summation. next i will describe the plasticity rules used by these fine basal dendrites putting a special emphasis on the role of nmda-spike in local synaptic plasticity processes. yumiko yoshimura department of visual neuroscience, research institute environmental medicine, nagoya university, nagoya, japan neocortical circuits contain fine-scale networks of excitatory neurons interconnected precisely. we previously showed that layer 2/3 pyramidal cells in visual cortex share common excitatory inputs from layer 4 and from within layer 2/3, when they are directly connected. here, we tested whether inhibitory cells are incorporated into the fine-scale specificity of excitatory connections. we recorded photostimulation-evoked synaptic currents from pairs of layer 2/3 cells, consisting of one inhibitory cell and one pyramidal cell in rat visual cortex slices, and measured the extent of common inputs to the pairs based on cross-correlation analysis. fast spiking inhibitory cells shared extensive common excitatory inputs with neighboring pyramids only when the pairs of cells were reciprocally connected. adapting inhibitory cells shared little or no common input with neighboring pyramids, regardless of their direct connectivity. therefore, fine-scale specificity depends on the type of inhibitory cell and on the direct connectivity between neighboring pyramidal-inhibitory cell pairs. research funds: kakenhi (17023026,17500208) sy2-3-03-5 local circuitry of cortical inhibitory neurons edward callaway, takuma mori, xiangmin xu the salk institute, usa we used laser scanning photostimulation to map local input to inhibitory neurons in layer 1 of rat visual cortex and layer 2/3 of mouse barrel cortex. mouse studies used transgenic animals with gfp expressed in subsets of inhibitory neurons. in layer 1, axondescending cells receive excitatory input predominantly from layer 2/3 while neurogliaform cells receive stronger input from deeper layers. layer 2/3 neurons also receive inputs that vary systematically by cell type. two subtypes of martinotti cells, distinguished by calretinin (cr) expression, also differ in morphology and intrinsic physiology. cr+ martinotti cells receive excitatory input predominantly from layer 2/3, while the cr− martinotti cells also receive strong excitation from layer 4. irregular-spiking basket cells also receive strong excitatory input from layers 2/3 and 4, but they often have a gap at the top of layer 4, with little or no input. fast-spiking basket cells and pyramidal cells in mouse barrel cortex receive input indistinguishable from cells in rat visual cortex, with strong input from layers 2/3 and 4, and only weak input from deeper layers. research funds: nih sy2-3-03-6 physiological genomics of cortical microcircuits sacha b. nelson brandeis university, czech republic cortical microcircuits are comprised of highly diverse neuronal cell types that differ in their morphology, synaptic connectivity and intrinsic electrophysiology. presumably, these phenotypic differences are orchestrated and maintained by unique transcriptional programs. in order to begin to reveal those programs we have recently developed methods for measuring genome-wide gene expression from small numbers (30-50) of fluorescently labeled, hand-sorted neurons. subsets of pyramidal neurons and gabaergic interneurons were labeled genetically with gfp or anatomically with fluorescent microspheres. labeled neurons were characterized electrophysiologically and sorted for expression analysis. the resulting expression profiles revealed highly diverse expression of molecules involved in cell-cell signaling and cell-cell adhesion, as well as transcription factors. based on this diversity of expression we constructed a taxonomic tree using an unsupervised clustering algorithm, that correctly reflects known relationships between cortical cell types. research funds: r03 ey015273 , mcknight neuroscience of brain disorders award sy2-3-09-1 axon guidance mediated by localized ca 2+ signals in the growth cone hiroyuki kamiguchi laboratory for neuronal growth mechanisms, riken brain science institute, wako, japan axonal growth cones migrate along the correct paths, not only directed by guidance cues but also contacted by local environment via cell adhesion molecules (cams). many guidance cues attract or repel the growth cone via asymmetric ca 2+ signals. its turning direction depends on the occurrence of ca 2+ -induced ca 2+ release (cicr) through the ryanodine receptor type 3 (ryr3). the activity of ryr3 is controlled by cams via camp and pka. in this way, axon-guiding and cam-derived signals are integrated by ryr3, that serves as a key regulator of axon guidance. attractive ca 2+ signals facilitate intracellular membrane transport to the leading front and subsequent vamp2-mediated exocytosis on the side with elevated ca 2+ . in contrast, repulsive ca 2+ signals do not trigger such membrane dynamics. growth cone attraction, but not repulsion, is prevented by inhibition of vamp2-mediated exocytosis. the results indicate that growth cone attraction is driven by asymmetric membrane dynamics and that growth cone repulsion is driven by different mechanisms, not simply by changing the left/right polarity of the same molecular machinery. sy2-3-09-2 molecular mechanisms for signaling through plexin-a1 hitoshi kikutani, atsushi kumanogoh, toshihiko toyofuku research institute for microbial diseases, osaka university, suita, japan sema3a acts as a guidance cue for axons through a receptor complex comprising neuropilin-1 as the ligand-binding subunit and plexin-a1 as the signal-transducing subunit. the ferm domain-containing gef, farp2, associates directly with plexin-a1. sema3a induces the dissociation of farp2 from plexin-a1, resulting in activation of farp2's rac gef activity, rnd1 recruitment to plexin-a1 and down regulation of r-ras. simultaneously, the ferm domain of farp2 sequesters pipki␥661 from talin, thereby inhibiting its kinase activity. these activities are necessary for sema3a-mediated repulsion of outgrowing axons. plexin-a1 also functions as a ligand binding receptor of a transmembrane semaphorin, sema6d and contributes to cardiac morphogenesis. sema6d exerts a migration-inhibitory activity on cells from the ventricle and a migration-promoting activity on those from the conotruncal segment. plexin-a1 forms a receptor complex with off-track in the ventricle or with vegf receptor type 2 in the conotruncal segment, which are responsible for the effects of sema6d on the respective regions. research funds: crest sy2-3-09-3 axonal transport elicited by axon guidance molecules yoshio goshima department of molecular pharmacology & neurobiology, yokohama city university graduate school of medicine, yokohama, japan for the wiring of individual neurons together into an orderly network, control by axon guidance molecules of navigation to their targets is a critical event, and molecular components destined for specific subcellular domains of neuron must be targeted correctly. we previously reported that semaphorins3a (sema3a), a repulsive axon guidance cue, increases the rate of bi-directional axonal transport in dorsal root ganglia (drg) . to address if the individual molecules rides on the sema3a-facilitated cargo, we used time-lapse imaging of several egfp-fusion proteins expressed in drg. sema3a stimulated the transport of neuropilin-1::egfp, plexin-a4::egfp, and fyn::egfp, which are the components of sema3a signaling, but not neuropilin-2::egfp. interestingly, sema3a accelerated the anterograde transport of trka::egfp. these facts suggest that sema3a selectively facilitates the transport of its own signaling components and that sema3a may modulate ngf-sensitivity of neurites by accelerating the transport of trka. kozo kaibuchi department of cell pharmacology, nagoya university graduate school of medicine, japan neurons are one of the most highly polarized cells, comprised of two structurally and functionally distinct parts, axon and dendrites. however, it remains largely unknown how neuronal polarity is established. we previously showed that collapsin response mediator protein-2 (crmp-2) is enriched in growing axon, and play a crucial role in axon specification. crmp-2 interacts with tubulin dimers to promote microtubule-assembly, and binds to sra-1, an effector of rac1 to regulate wave-dependent reorganization of actin filaments. crmp-2 links kinesin-1 to tubulin dimmers and sra-1, and participates in the kinesin-1-dependent transport of tubulin dimmers and the sra-1/wave complex to growing axons. we also found that the par-6/par-3 complex and the ras/pi3-kinase/akt/gsk-3b pathway are involved in neuronal polarization. akt appears to phosphorylate gsk-3b and inactivates its kinase activity downstream of ras/pi3-kinase, thereby increasing non-phosphorylated active crmp-2 which promotes axon growth. this time, i summarize and discuss functions of these polarity molecules in regulation of neuronal polarity. research funds: grant-in-aid for creative scientific research sy2-3-09-5 regulation of actin dynamics during neurite initiation and axon guidance frank gertler, adam kwiatkowski, doug rubinson, erik dent, leslie mebane mit, usa nervous system development requires extensive cell migration and elaboration of neurites that become axons and dendrites. axons are guided to their targets by motile growth cones. both whole cell and growth cone migration involve dynamic remodeling of the actin and microtubule cytoskeleton in response to environmental cues. the ena/vasp protein family regulates cell migration and axon guidance. ena/vasp proteins modulate actin remodeling and promote the formation of long, sparsely branched actin networks, such as those found in filopodia. results of recent work on phenotypes arising in mice lacking all three ena/vasp proteins (mena, vasp, evl) will be presented. such animals exhibit a "cobblestone cortex" in which groups of neurons migrate beyond the pial membrane. the mutants also contain little if any cortical axonal fiber tracts. analysis of primary cells from the mutants indicates ena/vasp function is required for neurite initiation. mutant neurons express differentiation markers but form few, if any filopodia and exhibit alterations in actin-microtubule interactions. kimitaka anami department of psychiatry, national center hospital for mental, nervous and muscular disorders, ncnp, tokyo, japan recent years, studies using eeg and fmri in simultaneous manner has become flourished. this is because the feasibility that any eeg events is, in principle, able to be mapped on the mri tomographic view has attracted many researchers. applications of this methodology are to basic eeg researches including event-related potentials and background activities, and as clinical aspect, localization of epileptic foci. and applications of this methodology is not matured yet but still developing. in this presentation, we will introduce our study using this method to epilepsy and to other eeg events. masaya misaki 1,2 , takashi abe 1,2,3 , shigeyuki kan 1,4 , satoru miyauchi 1,5 1 national institute of information and communications technology, kobe, japan; 2 japan society for the promotion of science, tokyo, japan; 3 graduate school of biosphere sciences, hiroshima university, higashi-hiroshim, japan; 4 kyushu institute of technology, kitakyushu, japan; 5 crest, japan science and technology agency, tokyo, japan recording fmri and an eeg simultaneously is effective for studying spontaneous brain activities. we used this method to examine the relationship between an eeg rhythm and a bold signal. some studies have hypothesized that the hemodynamic response for a change in power of certain eeg frequency bands, such as alpha waves, is canonical in shape. however, the appropriate response shape for a change in the rhythmic eeg has not yet been determined. we measured the eeg and fmri simultaneously while subjects were in a resting or sleeping state. we applied nonlinear regression analysis using an artificial neural network to detect correlations between the changes in rhythmic eeg waves and fmri signals without a priori hypothesis of the response shape. research funds: crest of jst and grant-in-aid for jsps fellows norihiro sadato, hiroshi toyoda department of cerebral research, national institute for physiological sciences, okazaki, japan previous studies have demonstrated a nonlinear relationship between blood oxygenation level dependent (bold) response and stimulus parameters. however the origin of this nonlinearity still remains unclear. to investigate the origin for the nonlinearity of bold response, we underwent simultaneous measurement of fmri and near infrared spectroscopy (nirs) . temporal dynamics of the responses in oxy-, deoxy-and total hemoglobin concentrations as well as bold signal were simultaneously measured during a visual stimulation with various durations. to quantify the degree of the nonlinearity of responses, we introduced a model using an impulse response function modified with additional nonlinearity scaling. this model was applied to the nirs measures as well as bold responses. the nonlinearity of the response in oxygen extraction fraction (oef) was also estimated from nirs measures. the non-linearity of bold was almost identical to oef across the wide range of nonlinearity of the neuronal responses. and hence we conclude that the non-linearity of bold responses to the neural activity is mainly due to the nonlinear response of oef. the bold-fmri signal is ambiguous regarding the underlying neurophysiology. in our work we attempt a) to better understand the neurophysiological basis of fmri and b) to improve on the information obtained by functional brain imaging by adding additional information, e.g. obtained by electrophysiological measurements. in one series of experiments, we combined transcranial magnetic stimulation with near-infrared imaging in order to clarify how changes in deoxy-hb concentration (the inverse of bold) is related to neuronal inhibition. in another series of experiments, we combine eeg with fmri in order to identify bold correlates of neuronal background rhythms such as alpha rhythm, mu rhythm, etc. in a third series of experiments, we combine fmri with the measurement of high-frequency oscillations in eeg. the latter is an expression of evoked spike burst in the somatosensory cortex, i.e. this kind of measurements adds the information about action potentials to fmri haruhiko akiyama tokyo institute of psychiatry, japan activation of microglia is a part of host defense mechanisms in the brain. microglia remove invading microorganisms as well as cell debris that contains hazardous substances such as lysosomal proteases. brain is particularly vulnerable to the immune and inflammatory attacks and, therefore, has multiple mechanisms that regulate the immune and inflammatory responses more strictly than other organs. nevertheless, many neurodegenerative lesions are associated with activated microglia and low-grade, but sustained, inflammation. neuroinflammation is a term that refers to such conditions. in alzheimer's disease (ad), microglia play a central role for phagocytic removal of amyloid beta-protein (abeta) from the brain. the process is enhanced by complement activation. however, these cellular and humoral responses to abeta may be toxic to neurons in ad. neuroinflammation could be a double-edged sword in the brain. in patients with neurodegenerative diseases, complication of systemic inflammatory diseases, depletion of some neurotransmitters such as catecholamines, and the presence of brain lesions may adjunctly upregulate neuroinflammation, which further accelerates neuronal degeneration. makoto sawada department of brain life science, riem, nagoya university, japan microglia, macrophage-like cells in the cns, are multi-functional cells; they play an important role in removal of dead cells or their remnants by phagocytosis in the cns degeneration as well as are one of important cells in the cns cytokine network. they are thought to be originated from mesoderm, and to be similar cells to other tissue-resident macrophages. activated microglia have been shown to remove potentially deleterious debris and promote tissue repair by secreting neurotrophic factors at the neuronal injury sites, however, they can release potentially cytotoxic substances in vitro, and at least so-called fully activated form of microglia which are observed at the injury site in aids dementia is completely neurotoxic. these suggest that some factor(s) may contribute to change microglial phenotype from protective to toxic, but the detail is not clear. recently we generated hiv-derived nef protein tranduced microglia. they are found to increase both the potential to produce o2-and mpo-like peroxidase activity resulting in the neurotoxicity. therefore, the target protein(s) of nef might to be involved in the control of microglial neurotoxicity. there is abundant evidence that extracellular atp have an important role in pain signaling. the focus of attention now is on the possibility that atp receptor of microglia might be involved in neuropathic pain. neuropathic pain is often a consequence of nerve injury through surgery, bone compression, diabetes or infection. this type of pain state is generally resistant to currently available treatments. we recently reported that the expression of p2x4 receptors in the spinal cord is enhanced in spinal microglia after peripheral nerve injury, and blocking pharmacologically and suppressing molecularly p2x4 receptors produce a reduction of the neuropathic pain behaviour (2003. nature 424, 778-783) . more recently, we have reported that brain-derived neurotrophic factor (bdnf) released from microglia by the stimulation of p2x4 causes the depolarizing shift in reversal potential of anion in li neurons of rats with nerve injury (2005 ( . nature 438, 1017 ( -1021 . understanding the key roles of these atp receptors may lead to new strategies for the management of neuropathic pain. research funds: kakenhi (15209051) sy2-5-05-4 pet imaging of microglia using peripheral benzodiazepine ligands richard b. banati university of sydney, australia brain disease often results in significant changes in the functional state of microglia, the brain's resident tissue macrophages. the response is thought to be an important step in the pathophysiology of traumatic, inflammatory, neoplastic and degenerative brain disease. part of the structural and functional plasticity of microglia is the de novo expression of the 18 kda transposor protein or "peripheral benzodiazepine binding site" (pbbs). the pbbs is bound by the isoquinoline pk11195, which labeled with carbon-11 can be used for positron emission tomography (pet). using 11c-(r)-pk11195 pet in inflammatory and neurodegenerative brain disease as well as receptor autoradiography, we have shown that distributed regional pbbs up-regulation correlates with clinical deficit and mirrors the histologically described activation of microglia in the penumbra of focal lesions, but importantly also in the distant, anterograde and retrograde projection areas of the lesioned neural pathway. sy2-5-10-1 application of simulation of light propagation in tissue to nirs imaging of brain function eiji okada department of electronics and electrical engineering, keio university, japan in nirs imaging, the functional image is obtained from the variation in intensity of detected light caused by concentration change in haemoglobin in cortical tissue. the serious problem of nirs imaging is ambiguity in light propagation in the head caused by the scattering of tissue. this poses results in poor spatial resolution and contrast in the image. the development of simulation model to calculate light propagation in the head to deduce the path length in the brain and the spatial sensitivity profile is very important to improve the nirs imaging. in this study, simulation of light propagation in the head model for nirs imaging is briefly reviewed. the heterogeneity of the tissues in the head, especially low-scattering cerebrospinal fluid (csf), has a strong effect on the light propagation in the brain. the sensitivity to concentration change in haemoglobin in the cortical tissue is improved by the effect of the csf. the simulation of nirs imaging indicates that the intensity and size of activated region in the functional image depend on the relative position of activated region to fibre pairs. yoko hoshi integrated neuroscience research team, tokyo institute of psychiatry, tokyo, japan quantification of near-infrared spectroscopy (nirs) data has been a central issue in the nirs field. over the past 25 years, many approaches to quantification have been tried, and in the case that hb concentration changes are global within the tissue, the quantitative accuracy of time-resolved spectroscopy (trs) and phase-resolved spectroscopy (prs) has been established. when the changes are localized, however, as with functional brain activation, the difficulty of quantification has not yet been fully overcome because elimination of the effects of hemodynamic changes in the extracerebral tissue is also challenging. the temporal profile of detected light intensity measured by trs carries information about depth-dependent attenuation, because light that penetrated into deeper positions in the head is detected later. thus, several time-domain methods to determine absorption changes with depth resolution have been proposed. diffuse optical tomography (dot) is also a potential technique for quantitative detection of focal changes in cerebral hemodynamics. in this symposium, i will outline these approaches. sy2-5-10-3 brain functional imaging in cerebral ischemic disorders: comparison of nirs and fmri kaoru sakatani department of neurological surgery, nihon university school of medicine, tokyo, japan we compared the evoked cerebral blood oxygenation (cbo) responses measured by nirs and activation maps of bold-fmri in stroke patients with mild and severe (misery perfusion) cerebral ischemia (ci). in the age-matched controls, deoxyhemoglobin concentrations decreased with increases in oxyhemoglobin and total hemoglobin in the primary sensorimotor cortex (psmc) during contralateral motor tasks. the psmc on the non-lesion side exhibits normal cbo response pattern. on the lesion side, the mild ci did not affect the cbo response pattern, but the severe ci caused an increase of deoxyhemoglobin during the task associated with increases of oxyhemoglobin and total hemoglobin. the mild ci caused only slight reduction of the activation volumes of bold imaging on the lesion side; however, the severe ci, caused markedly reduction of the activation volumes on the lesion side. misery perfusion caused marked reductions of activation volumes of bold imaging associated with an increase of deoxyhemoglobin during activation. bold-fmri should be performed, giving consideration to the baseline circulatory conditions. masato fukuda, toru uehara, masahiko mikuni department of psychiatry and human behavior, gunma university graduate school of medicine, gunma, japan near-infrared spectroscopy (nirs) has been increasingly employed in psychiatry researches such as personality (2005 . neuropsychobiology 52, 45), aging (2004 . neuroimage 22, 1715 , and psychiatric disorders ("progress in schizophrenia research", nova science publishers, 2006) . frontal lobe reactivity was investigated using multichannel nirs machines in unipolar depression, bipolar depression, and schizophrenia (2004. biol. psychiatry 55, 501; 2006. neuroimage 29, 172) by monitoring changes of oxy-hemoglobin concentration ([oxy-hb]) every 0.1s during a verbal fluency task. the unipolar depression was characterized by smaller [oxy-hb] increase, the bipolar depression by comparable but delayed [oxy-hb] increase, and the schizophrenia by reduced [oxy-hb] increase during the task period followed by [oxy-hb] re-increase during the post-task period, suggesting reduced, preserved but delayed, and inefficient frontal lobe reactivity, respectively. collaborators: itsuro ida, akihiko oshima, makoto ito, tomohiro suto, masaki kameyama, yutaka yamagishi, toshimasa sato, masashi suda sy2-5-10-5 clinical application of nirs to neurorehabilitation optical imaging using near-infrared spectroscopy (nirs) is suitable for assessing cortical activation during human gait because of its flexibility and portability. in healthy subjects, walking induced increase of oxygenated hemoglobin levels that centered in the medial sensorimotor cortex and supplementary motor area. in hemiparetic patients with stroke, cortical activation was characterized by asymmetrical activation in the sensorimotor cortex and recruitment of the premotor and prefrontal cortex. a longitudinal study revealed that locomotor recovery was associated with improvement of asymmetrical activation in the sensorimotor cortex as well as enhanced activation in the premotor cortex. sensorimotor stimulation by facilitation technique induced enhanced activation in the motor related areas, particularly in the premotor cortex. partial body weight support and speed-dependent exercise decreased sensorimotor activation, suggesting relative shift of locomotor control to the hierarchically lower structures including the spinal cord. thus nirs may contribute to establishing brain-based strategies for neurorehabilitation. research funds: funds for comprehensive research of aging and health sy2-5-10-6 measurement of language related brain activities during recovery from aphasia eiju watanabe 1 , yumiko muroi 2 , chizuru nakajima 2 1 department of neurosurgery, jichi medical university, tochigi, japan; 2 department of neurosurgery, tokyo metropolitan police hospital, tokyo mechanism which supports the recovery of language after aphasia is not well understood. it has long been discussed that language related areas including the regions surrounding the language area and compatible cortex on non-dominant side could be candidates which support the recovery. several studies suggest the compensation by these areas using fmri and pet. we used optical topography (ot) to know the participation of these areas during the recovery from the aphasia. we measured 17 aphasics who showed recovery from the aphasia after apoplexy. word generation task was used. in seven cases ot was measured more that twice. seven cases showed the activity on the non-dominant frontal lobe. they all showed activities on the dominant frontal lobe in the follow-up measurements along with the deactivation of non-dominant side. these findings showed dynamic participation of non-dominant frontal lobe during the recovery phase suggesting that the rehabilitation protocol should be changed according to the area activated in each phase. tamami fukushi research institute of science and technology for society (ristex), japan science and technology agency (jst), tokyo, japan recent development of neuroscience has provided remarkable scientific discoveries, as well many newer philosophical, ethical, legal and social issues. for example, the consequences of the progress of non-invasive neuroimaging technologies, such as functional magnetic resonance imaging (fmri) and near infrared spectroscopy (nirs) show the possibility to read the mind of others, which may lead the criminal and commercial applications. brain-machine interface (bmi) technology and pharmacological manipulation of the human brain can cause the unpredictable enhancement of human ability. in advance to the expansion of "applied neuroscience", neuroscientists should consider "what the ethical problem is in the current neuroscience" and "how we learn and interact with the ethics". in this symposium, the panels will talk about the history of neuroethics then provide the ethical aspects of basic research. we will also discuss the future perspective of the neuroethics in japan and world in terms of sharing the problems across neuroscientists, ethicists, mass media, and public. judy illes stanford university, usa akin to genetic testing in the 1990s, the translation of neuroimaging capabilities from the laboratory to the clinical setting has raised ethical questions about how new diagnostic and predictive information will be managed in the absence of effective treatments for certain diseases, about the timing of technology transfer and handling of technology that falls in the regulatory gray zone between research and clinical use, and what impact increasing opportunities for selfreferral to health care will have on patient-physician relationships, medicine, and society overall. potential off-label uses of advanced neuroimaging outside the health care setting -in law, education, employment and even for national security -are already being tested and debated. we will discuss how these issues converge in 21st century neuroethics, the presence of neuroethics in the international domain, and the critical role of ethics in neuroscience in the future. sy2-6-06-3 neuroethics from primate's eyes naotaka fujii bsi, laboratory for symbolic cognitive development, japan neurophysiologists working on monkeys have been trying to understanding how their brains are working. the aim of the studies was not merely revealing functions of primate's brain but also trying to extrapolate the findings in primates onto human brain functions. this was true but not really true due to technical limitations which prevented us from expanding the findings in primates directly to the human brain function. however, recent advancement of technology has changed the world. findings in primates can be directly applied to human studies with or without researcher's intention. technologies invented in primate physiology are now readily transferred into human without much discussion. brain machine interface is one example. now, monkey's brains are forcing us to think about social impact of our research from ethical view, which we have not discussed before. as an experienced primate neurophysiologist but with little ethical training, i will discuss 'what is ethically correct primate research' and 'how our scientific contribution has to be made' from very practical and ground level of neuroethics. sy2-6-06-4 neuroethics of nurturing the brain takao k. hensch critical period mechanisms group, riken brain science institute, japan at no time in life is the brain so easily shaped by experience than in infancy and in early childhood. it is during these critical periods that neural circuits acquire language and other basic brain functions. unraveling mechanisms that limit such dramatic plasticity to early life would pave the way for novel paradigms or therapeutic agents for rehabilitation, recovery from injury or improved learning in adulthood. conversely, a deeper insight into early postnatal brain development will provide valuable inspiration for effective brain-based education methods for our children-perhaps the greatest potential contribution of neuroscience to society. this raises urgent and important ethical questions for our consideration: is there an "ideal" brain we should be nurturing? to what extent can/should drugs be used not merely to correct developmental disorders but also to enhance performance? how do we determine what is good or bad for the maturing brain? research funds: riken bsi sy2-6-06-5 neuroethics beyond laboratories and across cultures daofen chen national institute of neurological disorders and stroke, usa recent progress in systems and cognitive neuroscience poses new ethical challenges to both investigators and to the funding agencies that support scientific investigations. potential uses of many of these recent advances go beyond their intended medical applications. a growing array of neurotechnologies capable of monitoring or even intervening in human cognition makes it imperative to consider the social, ethical, and legal implications of these scientific advances. while it once might had been possible to conduct research with naive ignorance of its societal implications, this is no longer the case. moreover, we need to be cognizant that modern brain science is profoundly influenced by the distinct cultural and social values held by different sectors of the world population. we will discuss what can be done from the perspective of funding agencies to facilitate intercultural dialogue, foster mutual understanding, and develop a framework and strategies to address emerging neuroethical issues and prioritize our future efforts in neuroscience research. sy2-6-06-6 bridging neuroscience and public: neuroethics in cultural contexts osamu sakura 1,2 1 interfaculty initiative in information studies, university of tokyo, tokyo, japan; 2 research institute of science and technology for society (ristex), jst, japan to bridge between neurosciences and public society-it should be one of the important aims of neuroethics. for this purpose we need to draw the outline of neuroscience within the cultural context. the method and the result of natural sciences are universal, of course, but its social consequences are highly variable among cultures. although the systematic comparative researches are open to the future, we should discuss how the neurosciences could create the healthy relationship between the public society, especially focusing on the method for public participation and on the previous successful cases. mitsuru kawamura 1,2 , akira midorikawa 3 , yoshiki kaneoke 4 , shinichi koyama 1 , masato taira 5 , argye hillis 6 1 showa university school of medicine, japan; 2 crest, jst, saitama, japan; 3 national institute of neuroscience, ncnp, tokyo, japan; 4 national institute for physiological sciences, okazaki, japan; 5 nihon university, tokyo, japan; 6 johns hopkins university, baltimore, usa this symposium aims to provide an opportunity to talk between clinical neuropsychologists and neuroscientists. focusing on the visual system, we will discuss up-to-date studies from neuropsychological and neuroscientific viewpoints. the topics include motion perception in brain-damaged patients, neuroimaging of motion perception, surface and depth perception in brain-damaged patients, and neuroimaging of surface and depth perception, and neuropsychological and neuroimaging studies of visual attention. we will discuss consistency and inconsistency of our findings, and discuss what to do in order to produce synergy between clinical neuropsychology and neuroscience. research funds: kakenhi (17022035), crest sy2-6-11-2 impairment of surface perception in patients with ventral occipital damages shinichi koyama 1 , mitsuru kawamura 1 , akira midorikawa 2 , yoshiki kaneoke 3 , masato taira 4 , argye hillis 5 1 showa university, tokyo, japan; 2 national institute of neuroscience, ncnp, tokyo, japan; 3 national institute for physiological sciences, okazaki, japan; 4 nihon university, tokyo, japan; 5 johns hopkins university, baltimore, usa we examined the perception of faces and objects in two patients with ventral occipital damages, using psychophysical techniques. patient 1 was a 61-year-old woman with bilateral damage in the fusiform and parahippocampal gyri. although she could recognize pictures of famous people, she frequently failed to decide the races of unfamiliar faces and occasionally failed to see the hollow-face illusion (gregory 1970) . in addition, her performance in object identification task became poorer when the objects were presented in inverted (negative) pictures. patient 2 was a 63-year-old men with bilateral damage in the lingual and fusiform gyri. his recognition of faces and objects became poorer when they were presented in inverted pictures. based on the above results, the role of the ventral visual cortex in the perception of faces and objects will be discussed. research funds: grant-in-aid for jsps fellows sy2-6-11-3 how do pictorial cues influence 3d information processing in the parietal association cortex? masato taira 1,2 1 arish, nihon university, tokyo, japan; 2 department of applied system neuroscience, nihon university graduate school of medical science, tokyo, japan pictorial cues are one of the most influenced cues for 3d perception. basically, it is thought that the parietal association cortex processes 3d visual information by binocular disparity cues and the temporal association cortex processes that by pictorial cues in the concept of two visual information processing systems in the brain. however, recent studies have suggested that there are many crosstalk of 3d information between these association areas. our recent studies have shown that a group of neurons in the parietal cortex (area cip) is involved in perception of 3d surface orientation and used both disparity and pictorial cues. functional mri data in human also suggest that pictorial cues, such as attached and cast shadow, are processed in the parietal cortex in 3d perception. furthermore, human psychophysical data gives us some insights of how the pictorial cues influence 3d information processing in the parietal association cortex. research funds: kakenhi 17022038 sy2-6-11-4 case report of a patient with posterior cortial atrophy who relatively preserved perception of moving objects akira midorikawa 1 1 national center of neuroscience, national center of neurology and psychiatry, tokyo, japan it has been presented that severe type of bálint syndrome behaves like a blind person; however, it also reported that there are some cases who behave like a blind person but could walk without collision. up to today several cases have been reported, but the underlying mechanism of the phenomenon has not been mentioned. in this report, i will talk about a patient with bálint syndrome due to degenerative disease known as posterior cortical atrophy (pca), who could not only walk around without collision but also play catch very well, nevertheless having blind like behavior. the crucial visual information underlying these phenomenons was assumed to be motion parallax induced by not only objects movement but also self movement. in addition, discrepancy between the patients who could walk and not walk will be discussed. research funds: crest, japan science and technology agency sy2-6-11-5 neural mechanism underlying visual perception of motion as revealed by non-invasive human study yoshiki kaneoke department of integrative physiology, national institute for physiological sciences magnetoencephalography (meg) measures the neural activity representing the synchronized inputs to the millions of pyramidal neurons in the localized cerebral cortex. thus, it will show us another aspect of the neural activity related to the specific brain function that would not be revealed by the recording of single neuronal activity. our meg studies have revealed several important findings in the human visual motion detection system. first, the response properties for the apparent motions indicate the importance of the human mt/v5+ for the perception and the existence of the parallel processing for the motion and light blinking. second, the existence of the spatiotemporal filtering mechanism for the perception of motion speed is shown by the various motion stimuli. third, we present the evidence that the spatial integration of the speed without direction information occurs in our visual system. based on the results, scalar fields theory for the spatial integration of motion is proposed to explain various complex motion perception. research funds: kakenhi (15500221) sy2-6-11-6 neural correlates of visual attention argye hillis 1 , mitsuru kawamura 2 , akira midorikawa 3 , yoshiki kaneoke 4 , shinichi koyama 2 , masato taira 5 1 johns hopkins university, usa; 2 showa university, tokyo, japan; 3 national institute of neuroscience, ncnp, tokyo, japan; 4 national institute for physiological sciences, okazaki, japan; 5 nihon university, tokyo, japan in this paper i report a series of studies of unilateral spatial neglect (usn) in acute stroke, demonstrating a frequent double dissociation between stimulus-centered neglect and viewer-centered neglect, and showing that these types of neglect can be modality-specific. other data reveal that different types of usn are observed when there is hypoperfusion of temporal cortex versus parietal cortex. still other data provide evidence that severity of neglect depends on the volume of hypoperfused tissue in acute stroke, and that reperfusion results in early recovery of neglect. finally, i will review new evidence that right usn is common after left cortical infarcts or hypoperfusion in acute stroke, but the distribution of types of usn is very different from the distribution of types of usn after right hemisphere stroke. takeo kubota, takae hirasawa, kaoru nagai department of epigenetic medicine, university of yamanashi faculty of medicine, chuo, yamanashi, japan how are brains controlled molecularly? this is one of fundamental questions in neuroscience. several lines of evidences have suggested that genes are more strictly controlled in the brain than in other organs. epigenetics is one of such systems to control expression of the genes not based on dna sequence, but based on 'beyond the dna sequence' (chromatin modifications and small rnas). the failure of this system is known to result in neurodevelopmental diseases, such as an autistic rett syndrome. it has recently been demonstrated that the epigenetic system is affected by an environmental stress after birth, and that the system is associated with neural differentiation and cell fate determination and human brain diversity. these findings imply that epigenetics is an important research field to understand mechanisms of neural development and mental diseases. topics from update epigenetic researches in neuroscience will be discussed in this symposium. as one of epigenetic disorders, we introduce studies of rett syndrome (rtt) and its model mouse. rtt is a neurodevelopmental disorder, characterized by mental retardation and peculiar behavior. mutations of the mecp2 gene, encoding methyl-cpg binding protein 2, cause rtt. mecp2 acts as a transcriptional silencer. abnormal expression of some genes due to mecp2 dysfunction is thought to be the first step of rtt pathophysiology. to understand how mecp2 mutation makes rtt, we have two approaches that are morphological investigation of brain and discovery of mecp2-downstream genes. using mecp2-null mice (mecp2 −/y ), we revealed small number of dendritic spines and premature postsynaptic density at presymptomatic period. premature synaptogenesis may be the initial neuronal changes of rtt. we also found that mecp2 directly regulates expression of insulin-like growth factor binding protein 3 (igfbp3) gene in human and mouse brains. pathological features of mecp2 −/y have the similarity of igfbp3 transgenic mice, which show reduction of early postnatal brain growth. over-expression of igfbp3 due to lack of mecp2 may lead to delayed brain maturation. growing evidence suggests that alterations in the epigenetic status such as dna methylation and histone modifications in brain are involved in the behavioral response to environmental factors and the pathogenesis of psychiatric diseases. however, in contrast to mrna profiling, there are few established methods for profiling the genomewide epigenetic status to date. we developed a method for profiling the genome-wide dna methylation pattern using tiling arrays, and focused our analysis on human brain samples derived from psychiatric patients and control subjects. in this symposium, general picture of the genes that are heavily methylated or non-methylated in human brain, and the relationship between dna methylation and mrna expression patterns will be presented. understanding what produces neuronal diversification has been a longstanding challenge for neuroscientists. the recent finding that long interspersed nucleotide elements-1 or l1 (line-1) retroelements are active in somatic neuronal progenitor cells provided an additional mechanism for neuronal diversification. together with their mutated relatives, retroelements sequences constitute 45% of the mammalian genome with l1 elements alone representing 20%. the fact that l1 can retrotranspose in a defined window of neuronal differentiation, changing the genetic information in single neurons in an arbitrary fashion, allows the brain to develop in distinctly different ways. this characteristic of variety and flexibility may contribute to the uniqueness of an individual brain. however, the extent of the impact of l1 on the neuronal genome is unknown. the characterization of somatic neuronal diversification will not only be relevant for the understanding of brain complexity and neuronal organization in mammals but may also shed light on the differences in cognitive abilities, personality traits and many psychiatric conditions observed in humans. sy2-7-07-6 notch-induced acquisition of astrocyte differentiation potential of neural stem cells kinichi nakashima 1 , jun kohyma 1 , tetsuya taga 2 , masakazu namihira 1 1 grad. sch. biol. sci., naist, ikoma, japan; 2 inst. mol. embryol. genet., kumamoto univ., kumamoto, japan neurons and astrocytes are generated from common neural stem/precursor cells, however, neurogenesis precedes astrocytogenesis during brain development. we have previously reported that gfap-positive astrocyte differentiation is dependent on the transcriptional activity of stat3. a cpg dinucleotide in the stat3 recognition sequence within the gfap gene promoter is highly methylated at midgestation which becomes demethylated as the brain develops, enabling stat3 to induce gfap expression. thus, it is conceivable that the epigenetic modification such as dna methylation of cell type-specific gene promoter controls the switch from neurogenesis to astrocytogenesis in the developing telencephalon. here we report that neurons, which are generated earlier than astrocytes can potentiate neural precursors at midgenstation to differentiate into astrocyte through the activation of notch-signaling. the activated notch-signaling accelerates demethylation of stat3 binding element in the gfap gene promoter. sy2-8-12-1 neurogenesis and stem cell supply as therapeutic approach to overcome ischemic stroke masayasu matsumoto department of clinical neuroscience and therapeutics, hiroshima university graduate school of biomedical sciences, japan in order to overcome the brain damage caused by ischemic stroke, several strategies have been so far applied. in the present symposium, i will address the following points to be considered prior to clinical application of neurogenesis and/or stem cell supply to repair the damaged brain function. (1) which type of brain infarction will be a future target of this therapeutic approach? (2) which phase of brain infarction (i.e., acute or chronic phase) will be selected as a future timing of therapeutic intervention? (3) which will be the best way to be applied in the clinical settings, neurogenesis control, stem cell supply or both? the present research status and future directions will be presented and fully discussed. research funds: kakenhi sy2-8-12-2 gene therapy for cerebral ischemia setsuro ibayashi, hiroaki ooboshi department of medicine and clinical science, graduate school of medical sciences, kyushu university, fukuoka, japan cerebrovascular disease is the leading cause of the disabled people in japan and western countries. gene transfer technique may be applicable to the treatment of serious types of cerebrovascular disease. cerebral blood vessels have been targeted by gene transfer with intravascular or perivascular approaches. several experimental studies have revealed potential usefulness of gene therapy for prevention of vasospasm after subarachnoid hemorrhage. as for cerebral infarction, studies using various brain ischemia models have shown effectiveness of gene transfer in reduction of infarct size and functional recovery. our recent studies of post-ischemic gene transfer have provided promising results in attenuation of ischemic damages by inhibiting apoptosis, inflammation and vascular permeability. approaches to cerebral ischemia using gene transfer for angiogenesis and neurogenesis appear to be novel and promising strategies. thus, gene therapy has a potential for the future therapy against cerebral ischemia. isao date department of neurological surgery, okayama university, okayama, japan cerebral ischemia is one of the neurological disorders that cell transplantation is expected to be applied. in this presentation, the author will summarize our recent basic research and clinical application reported in the literature. it is now possible to make several types of neurotrophic factor secreting cell line by genetic manipulation. in order to prevent immunological reaction and tumor formation, we have been using encapsulated cell grafting technique. we transplanted several types of neurotrophic factor secreting cell line into the middle cerebral artery occlusion model and could confirm the histological and behavioral efficacy. we have also been using adultderived neural stem cells as donor cells because they have merits to make autografitng possible. as donor tissue, neural protection can be expected similar to fetus-derived neural stem cells. the effect of neural protection increases when neurotrophic factor secreting genes such as gdnf were inserted into neural stem cells. cell transplantation is considered a new therapeutic approach for cerebral ischemia and clinical application is expected. we now know that (1) motor function may recover after minor injury to the primary motor cortex, (2) this recovery is, at least in part, associated with reorganization of cortical motor representation, (3) the molecular mechanism for synaptic plasticity and axonal regrowth is being elucidated, and (4) recent clinical experience revealed that the motor function in patients with spinal cord injury is improved after transplantation of her/his own olfactory mucosa. furthermore, recent neuroimaging techniques can display the cortical functions as we as the specific fiber connections in individual brain. virtually any part of the brain can be approached with the accuracy of millimeters by the current image-guided neurosurgery. those theoretical and technical backgrounds suggest we might be ready for the reconstruction of brain function. nobuyuki nukina laboratory for structural neuropathology, riken brain science institute, japan a major hallmark of the polyglutamine (pq) diseases is the formation of pq inclusions. recently, misfolding has come to be considered one of the primary factors for pq protein aggregation, although, the nature of misfolding is not yet well known. the protein misfolding induced by pq expansion was investigated with our molecular model system using mutant myoglobin which is inserted different size of pq. expanded polyglutamine stretches form intramolecular and intermolecular beta sheets and amyloid fibrils. the surface of the mutant myoglobin with expanded pq was partially unfolded and destabilized. we also investigated the early phase of fibrillization by small-angle x-ray scattering and electron microscopic studies, revealing that the expansion of pq to 50 repeats induced the formation of quasi-aggregate in the earliest stage of the protein fibrillization. this structure could be closely involved in recruitment of various functional proteins into aggregates, leading to the cellular dysfunction that causes pq diseases. furthermore using cellular model system we also studied the aggregates interacting proteins (aips) by analyzing the purified polyglutamine inclusions and the lists of aip including chaperones, proteasome subunits, ubiquitin interacting proteins and others suggest the pathological role of aips in the disease cascades. sy3-1-01-3 neuronal dysfunctions in dentatorubralpallidoluysian atrophy (drpla) shoji tsuji 1 , toshiya sato 2 , mitsunori yamada 3 1 department of neurology, the university of tokyo, tokyo, japan; 2 center for bioresource-based researches, japan; 3 department of pathology, brain research institute, niigata university, niigata, japan to investigate molecular mechanisms of neurodegeneration in drpla, a polyglutamine disease caused by expansions of cag repeats of drpla gene, we have established transgenic mice harboring a single copy of the full-length human mutant drpla gene with 129 cag repeats. the q129 mice exhibited neurological phenotypes similar to juvenile type of drpla characterized by ataxia, myoclonus and epilepsy. electrophysiological studies disclosed age-dependent abnormalities in the globus pallidus and cerebellum. neuropathological studies revealed progressive brain atrophy without obvious neuronal loss and an age-dependent increase in neuronal intranuclear accumulation of mutant proteins with the regional distribution vulnerable to drpla. expression profiling analyses revealed down-regulated genes including camp responsive genes. these results suggest that "neuronal dysfunction", but not the "neuronal cell death", is the essential mechanism of neurodegeneration in drpla. huntington's disease (hd) is caused by an expansion of a cag repeat encoding polyglutamine in the huntingtin protein and involves progressive motor, cognitive and psychiatric symptoms. using a transgenic mouse model of hd, we have shown that environmental factors can dramatically modify the disease process and delay the onset and progression of motor and cognitive symptoms. further, we have attempted to correlate these behavioural findings with changes in gene expression, neuronal morphology, neurogenesis, and cortical plasticity, in an attempt to elucidate cellular and molecular mediators in hd, and understand how gene-environment interactions can modulate these pathogenic pathways. our findings indicate that the modulatory effects of environmental manipulations are mediated by amelioration of specific molecular and cellular deficits, and provide experimental paradigms for the identification of novel therapeutic targets for hd and related brain disorders. sy3-1-06-1 control of neural organization in the developing cerebral cortex yasuto tanabe mitsubishi kagaku institute of life sciences, tokyo, japan in the developing cerebral cortex, the generation of neurons with distinct identities and patterns of connectivity is controlled by a hierarchical series of cellular interactions that culminate in the laminar organization of distinct cortical areas. over the past three years we have begun to examine cerebral cortical development by focusing on three distinct major neuronal subtypes, namely, cajal-retzius cells, cortical projection neurons, and cortical interneurons. the analyses of these distinct neuronal subtypes allowed us to identify several candidate molecules and cellular interactions that might contribute to the laminar and areal organization of the cerebral cortex. in the first part of my talk, i would like to deal with the issue of ontogeny of cajal-retzius cells, and present the way cajal-retzius cells are generated, migrate and finally distribute in the developing cerebral cortex. then, i would like focus my talk on the issue of the way the acquisition of radial migration and axonal trajectory patterns of distinct cortical projection neurons is controlled during the development of the cerebral cortex. research funds: kakenhi 17390086, 17023061 sy3-1-06-2 mechanisms of the regulation of neuronal migration and corticogenesis kazunori nakajima 1,2 1 dept. of anat., keio univ. sch. of med., tokyo, japan; 2 inst. of dna med., jikei univ. sch. of med., tokyo, japan mammalian cerebral cortex has a six-layered structure where the neurons are aligned depending on their birth-date. to determine whether the migration from the ventricular zone (vz) to beneath the marginal zone (mz) is essential for neuronal segregation into layers, we investigated whether migrating neurons have different cell aggregation properties in vitro depending on their birth-dates, even before they arrive beneath the mz. we analyzed vz cells and cells from the intermediate zone (imz) mainly composed of migrating cells, and found that the cells had acquired a birth-date-dependent preferential segregation mechanism in a reelin-independent manner. these findings suggest that cortical neurons acquire a birth-date-dependent segregation property (or fate) before their somas reach the mz. in silico experiments of the reaggregation culture supported that this mechanism might indeed contribute to the layer formation in the developing cerebral cortex in concert with other mechanisms such as reelin signaling. kenji shimamura division of morphogenesis, institute of molecular embryology and genetics, kumamoto university, japan neurons of the thalamus originate in restricted regions of the proliferative zone of the diencephalic compartment before settling in their final locations in the nuclei. to investigate cellular and molecular mechanisms underlying nucleus formation, we analyzed the sequence and pattern of expression of specific markers that distinguish the subsets of neuronal precursors during development of the thalamus. we found that a morphogen-like activity of sonic hedgehog (shh) precisely defines positions of neurons with distinct properties, and that some gabaergic interneurons migrate from their birth place to distant nuclei in a highly organized manner. we also provide evidence that shh produced by the zona limitans intrathalamica (zli), which abuts the prethalamus and thalamus, is likely to be a cue for this directed migration. our results suggest that local production of prespecified neurons coupled with distinct migration properties and local guidance cues such as compartment boundaries could be principle elements for the nucleus formation. layers and nuclei are important functional units in the vertebrate cns. neurons in these structures have common physiological and anatomical features. despite their importance, mechanisms for nucleogenesis are poorly understood. we focused on the lower rhombic lip (lrl)-derived precerebellar neurons, and utilized exo utero electroporation with an enhanced yellow fluorescent protein (eyfp) gene, to study the process of nucleogenesis. after the unilateral transfer of eyfp to the lrl of embryonic day 12.5 mice, eyfp-labelled neurons migrate tangentially from the lrl in two distinct streams, one toward the ventral metencephalon and the other toward the ventral myelencephalon. the former formed the pontine grey nucleus and reticulotegmental nucleus and the latter the external cuneate nucleus and lateral reticular nucleus. before forming the clusters, the labelled neurons begin to migrate toward the ventricle along the radial fibres, and aggregate as they detach from the fibres. perturbation experiments such as introduction of dominant negative constructs and sirna suggested involvement of several molecules in the migration of these neurons. the brains of fetal alcohol syndrome patients exhibit impaired neuronal migration, but little is known about the mechanisms underlying this abnormality. here we show that ca 2+ signaling and cyclic nucleotide signaling are the central targets of alcohol action in neuronal cell migration. an acute administration of ethanol reduced the frequency of transient ca 2+ elevations in migrating neurons and cgmp levels, and increased camp levels. experimental manipulations of these second messenger pathways, through stimulating ca 2+ and cgmp signaling or inhibiting camp signaling, completely reversed the action of ethanol on neuronal migration in vitro as well as in vivo. each second-messenger has multiple but distinct downstream targets, including camkii, calcineurin, pp1, rho gtpase, mapk and pi 3 k. these results demonstrate that the aberrant migration of immature neurons in the fetal brain caused by maternal alcohol consumption may be corrected by controlling the activity of these second-messenger pathways. sy3-2-02-1 membranes, water and diffusion denis j. le bihan shfj/cea, france among 1905 einstein papers is one which unexpectedly gave birth to a powerful method to explore the brain. molecular diffusion was explained by einstein on the basis of the thermal random translational motion of molecules. in the mid 1980s it was shown that water diffusion in the brain could be imaged using mri. a dramatic application of diffusion mri has been brain ischemia, following the discovery that water diffusion drops immediately after the onset of an ischemic event, when brain cells undergo swelling through cytotoxic edema. also, water diffusion is anisotropic in white matter, because axon membranes limit molecular movement perpendicularly to the fibers. this feature can be exploited to map out the orientation in space of the white matter tracks and image brain connections. more recently, it was discovered that diffusion mri could detect transient swelling of activated cortical cells. this represents a significant breakthrough, allowing non invasive access to a fast and direct physiological marker of brain activation. this approach will bridge the gap between invasive optical imaging techniques and current functional neuroimaging approaches in humans, which are based on indirect and remote blood flow changes. sy3-2-02-2 diffusion tensor fiber tractography using a 3tesla mr system yukio miki department of diagnostic imaging and nuclear medicine, kyoto university, kyoto, japan diffusion tensor imaging (dti) is an mr imaging technique that is sensitive to orientation of mobility in water molecules. dti reveals two specific characteristics: diffusion anisotropy; and directional distribution of water diffusivity. white matter shows high diffusion anisotropy, because diffusion is faster in parallel to fiber direction than in other directions. dti of the brain can be reconstructed to display 3d macroscopic fiber tract architecture, in a process known as fiber tractography. with recent advances in actively shielded 3-t magnets and parallel imaging techniques, high-field mr imaging has become practical in clinical settings. we have demonstrated that depiction of most fiber tracts was improved on 3-t tractography compared to 1.5 t. we have also established an integration of tractography and intraoperative subcortical motor-evoked potential, and demonstrated that diffusion tensor tractography of the corticospinal tract using 3-t mr was able to provide interactive information on fiber tracts, depicting the course of eloquent fiber tracts during an operation. to test whether mr tractography is reproducible and reliable, we used this technique to assess acute tiny infarcts located in the supratentorial brain. we analyzed the data of 14 patients who presented to our institute with sensorimotor symptoms. there was an excellent correlation between the location of the infarct as assessed by tractography and clinical symptoms. next, we applied the technique to patients with evolving symptoms after admission to hospital. we specifically assessed the change in the tract-infarct relationship over time. the data showed that, in most cases when there was symptomatic progression, the distance between the tract and the infarct border depicted on dwi diminished. finally, we studied whether the use of tractography could help predict a patient's prognosis. to simplify the analysis, we specifically focused on patients with lenticulostriate artery (lsa) infarcts. we analyzed the correlation between the extent of cst involvement within the infarcts and the severity of motor deficits. the data indicated that the tractographic technique could be useful to predict a patient's outcome. sy3-2-02-4 anatomical and functional tractography: a combined approach with diffusion tractography and corticocortical evoked potential riki matsumoto department of neurology, kyoto university graduate school of medicine, japan recent advances in diffusion-weighted imaging have raised the possibility of in vivo investigations of brain circuitry in humans. the probabilistic tractography provides estimates of the likelihood of a pathway between two brain regions without tensor estimation and thus could trace the fiber pathways beyond regions of low diffusion anisotrophy into the grey matter. however, the results depend merely on anisotropic movement of water molecules and need validation. for presurgical evaluation of epilepsy patients, we developed an in vivo tracking method, cortico-cortical evoked potential, to electrically track the cortico-cortical connections by stimulating a part of the brain through epicortical electrodes and recording the cortical evoked potentials that emanate from a distant region of the cortex via projections. combined with preoperative diffusion analysis, this invasive evaluation provides a unique opportunity to study the cortico-cortical connectivity both functionally and anatomically. results of the combined approach will be presented. parkinson disease (pd) is the second commonest neurodegenerative disorder after alzheimer disease characterized by tremor, rigidity, bradykinesia, and postural instability. pathologically, the most outstanding change is the neurodegeneration of the nigral dopaminergic neurons. although familial forms of pd can be encountered up to 15% of the patients, the remaining cases are sporadic. it has been postulated that nigral neurodegeneration in pd is induced by the interaction of genetic risk factors and environmental factors. epidemiological studies revealed numbers of environmental factors that are positively correlated with increased risk of pd; such factors include pesticide, herbicides, rural living, well water drinking, metals such as manganese and iron, fuel oil, industrial chemicals, and hydrocarbon solvents. in addition, certain employments were reported to be associated with increased risk of pd; these include steel/alloy industry, wood/pulp plant, farming, carpentry, cleaning, orchard, mining, and welding. these studies suggest importance of environmental factors in the pathogenesis of pd. recent progress in these areas will be discussed. masami ishido national institute for environmental studies, tsukuba, japan there are getting much public concerns about children health since environmental factors such as industrial chemicals cause deficit in developing brains. it has been suggested that they may be incident of attention deficit hyperactivity disorder or autism. epidemiologic studies also suggested that parkinson's disease was found in the peoples who were exposed to pesticides in their childhood. thus, we examined the effects of industrial chemicals, called endocrine disruptors, on rat neurodevelopment. oral administration of an endocrine disruptor (12-60 mg/kg) into male wistar rats (from 5 days to 3 weeks of age) significantly caused hyperactivity at 4-5 weeks old. immunohistochemical analyses of the brain tissues at 8 weeks of age revealed a large reduction of immunoreactivity for tyrosine hydroxylase, but not for glutamic acid decarboxylase, both of which are localized in the substantia nigra, suggesting the specific degeneration by the chemical of dopaminergic neurons. tunel-positive cells were seen in the substantia nigra. thus, environmental insults in early life may be of particular etiologic importance. sy3-2-07-3 non-thermal effects of mobile phones upon the rat brain leif g. salford, b. persson, j. eberhardt, g. grafstrom, l. malmgren, a. brun dept of neurosurgery and the rausing laboratory, lund university, sweden we have shown that rf electromagnetic fields can cause significant leakage of albumin through the bbb of exposed rats as compared to non-exposed animals. one remarkable observation is that sar values around 1 mw/kg give rise to a more pronounced albumin leakage than higher sar values -all at non-thermal levels. if the reversed situation were at hand, we feel that the risk of cellular telephones, base-stations and other rf emitting sources could be managed by reduction of their emitted energy. the sar value of around 1 mw/kg is exists at a distance of more than one meter away from the mobile phone antenna and at a distance of about 150 -200 meters from a base station. another remarkable observation in our studies is the fact that a significant (p < 0.002) neuronal damage is seen in rat brains 50 days after a 2 hour exposure to gsm at sar values 200, 20 and 2 mw/kg. we have followed up this observation in a study where 96 animals were sacrificed 14 and 28 days respectively after an exposure for 2 hours to gsm mobile phone electromagnetic fields at sar values 200, 20, 2, 0.2 and 0 (controls) mw/kg. significant neuronal damage is seen after 28 days and albumin leakage after 14. our findings may support the hypothesis that albumin leakage into the brain is the cause for the neuronal damage observed after 28 and 50 days. sy3-2-07-4 the mitochondrial toxin 3-nitropropionic acid: an environmental toxin to study striatal degeneration in huntington disease emmanuel brouillet neuronal death laboratory, ura cea-cnrs 2210, france huntington disease is a neurodegenerative disorder caused by a mutation in the gene encoding huntingtin. the mechanisms underlying the preferential degeneration of the striatum, the most striking neuropathological change in huntington disease, are unknown. the behavioral and anatomical similarities found between huntington disease and animal models of striatal degeneration using the environmental toxin 3-nitropropionic acid (3np) support the hypothesis that mitochondrial defects could play a role in huntington disease. we will discuss the mechanisms of 3np toxicity and show that 3np and mutated huntingtin have certain mechanisms of toxicity in common. in particular, we show that mutated huntingtin can alter the expression of mitochondrial complex ii, the respiratory chain enzyme specifically inhibited by 3np. in summary, the 3np story is a good example showing how the study of environmental toxins can greatly help to elucidate the complex mechanisms underlying chronic neurodegenerative disorders. we recently demonstrated that rats received intrecisternal injection of 6-ohda or environmental chemicals, such as bisphenol a, nonylphenol, p-octylphenol, diethylhexylphthalate or dibutylphthalate, at 5 days of age showed behavioral hyperactivity at 4-5 weeks of age. immunohistochemical studies revealed a deficit in the development of dopamine (da) neurons. adult rats received these chemicals showed degeneration in nigro-striatal da neurons similarly to parkinson's disease. in this study, we investigated the mechanism of 6-ohda-induced neurotoxicity, using pc12 cells as an in vitro model system. we observed the generation of reactive oxygen species (ros) and p-quinone via auto-oxidation of 6-ohda. we also characterized the oxidation of cellular proteins by 6-ohda and the protective effect of antioxidants such as catalase, glutathione, and n-acetylcysteine with different manner. we will discuss about apoptotic cell death pathway including cytochrome c release and caspase activation induced by 6-ohda, ros and p-quinone. sy3-2-07-6 environmental factors in the pathogenesis of alzheimer's disease joanna l. jankowsky california institute of technology, usa epidemiological studies indicate that environmental factors significantly influence the risk of developing alzheimer's dementia. foremost among those factors are education, occupation, and leisure activities. although not universal, most studies have found that individuals with greater education, more challenging occupation, or active leisure hobbies show relative protection against dementia. animal models for alzheimer's disease have recently been used to explore the mechanism of this effect. transgenic mice designed to recapitulate alzheimer's amyloid pathology are protected from functional decline by enriched housing designed to provide cognitive stimulation. both enriched housing and exercise modify the level of amyloid-beta in the brains of transgenic mice, demonstrating that environmental factors can significantly influence brain biochemistry. intriguingly, traditional environmental enrichment can improve cognitive behavior while paradoxically elevating amyloid-beta levels in transgenic mice, suggesting that environmental stimulation may alter amyloid metabolism and cognitive function by competing mechanisms. the efficacy of synaptic inhibition depends on the number of gaba a receptors expressed on the neuron surfaces. in the present study, we have elucidated the role of prip (plc-related inactive protein) in trafficking of the receptors by analyzing prip knockout (ko) mice; the sensitivity to diazepam was reduced as assessed by biochemical, electrophysiological and behavioral analyses of ko mice, suggesting the dysfunction of the ␥2 subunit-containing receptors. we then examined the mechanisms by which prip molecule regulates cellsurface expression of ␥2 subunit-containing receptors. disruption of the direct interaction between prip and the ␤ subunit of receptors by prip-binding peptide inhibited cell-surface expression of ␥2 subunit-containing receptors in gh3 and hek293 cells. constitutive internalization of the receptors was also modified by the peptide. collectively, prip molecules are involved in trafficking of ␥2 subunit containing gaba a receptors to/from cell-surface membrane. research funds: kakenhi (16109010) sy3-3-03-2 involvement of bdnf in the induction of ltp at visual cortical inhibitory synapses yukio komatsu dept. visual neurosci., res. inst. environ. med., nagoya univ., nagoya, japan high-frequency stimulation (hfs) induces long-term potentiation (ltp) at inhibitory synapses of layer 5 pyramidal cells in rat visual cortex. this ltp requires a postsynaptic ca 2+ rise for induction and spike firing of presynaptic cells for maintenance, although the necessary frequency is low, suggesting that ltp is expressed presynaptically and some information must be sent backwards from the post-to presynaptic cells during induction. in this study, we investigated whether bdnf could act as such retrograde messengers. ltp did not occur when hfs was applied in the presence of k252a at 200 nm, inhibiting trk receptor tyrosine kinases selectively at that dose. hfs induced ltp when k252a application was started soon after hfs or when k252a was loaded into postsynaptic cells. ltp did not occur in the presence of trkb-igg or anti-bdnf antibodies. in cells loaded with bapta, the addition of bdnf to the medium enabled hfs to induce ltp without affecting basal synaptic transmission. these results suggest that bdnf released from postsynaptic cells activates presynaptic trkb, enabling the induction of ltp. research funds: kakenhi (17300101) sy3-3-03-3 autocrine mglur1 activation in cerebellar purkinje cells regulates gaba-mediated synaptic inhibition trevor smart, ian c. duguid university college london, uk in the cerebellum, retrograde signalling is important for the induction of short-and long-term changes to synaptic inhibition at interneuron-purkinje cell (in-pc) synapses. endocannabinoids, via cb1 receptors, mediate a short-term decrease in synaptic efficacy, while glutamate, via presynaptic nmda receptors, induces a sustained increase in gaba release. we now report that dendritically released glutamate also acts as an autocrine messenger, activating mglur1 on pcs to enhance synaptic inhibition via the release of endocannabinoids. this process was triggered by repetitive pc stimulation and blocked by uncoupling the mglur1-gq/11 transduction pathway as well as being initiated by direct mglur1 activation during pc depolarisation. glutamate uptake by excitatory amino acid transporters controlled the extent of autocrine mglur1 activation, whilst basal glutamate levels were unable to enhance endocannabinoid release. our study suggests that autocrine mglur1 activation provides a powerful homeostatic mechanism to dynamically regulate inhibitory synaptic transmission. sy3-3-03-4 regulatory mechanism of inhibitory synaptic transmission in the cerebellum shin-ya kawaguchi 1,2 , tomoo hirano 1,2 1 dept. biophys., grad. sch. sci., kyoto univ., kyoto, japan; 2 crest, jst, kawaguchi, japan at the gabaergic synapses between inhibitory interneurons and a purkinje neuron in the cerebellum, postsynaptic depolarization induces long-term potentiation of transmission efficacy mediated by gaba a receptors (rebound potentiation: rp). the signaling cascades regulating the induction of rp has been clarified. the balance of activities of protein kinases and phosphatases determines whether rp is induced or not. here we show another molecular mechanism involved in the rp induction. using both electrophysiological experiments and computational kinetic simulation of biochemical reactions, we demonstrate how the long-term potentiation of gaba a receptormediated responses is brought about. rp induction was impaired by inhibition of ca 2+ -activated protease calpain or by disturbance of association of gaba a receptor ␥2 subunit with gabarap (gaba a receptor associated protein). binding of gabarap to microtubule was also involved in the regulation of rp. our results suggest that structural alteration of gabarap caused by calpain activity is critical for establishment of rp. sy3-3-08-1 contribution of hebb's "organization of behavior" to the development of brain science masataka watanabe tokyo metropolitan institute for neuroscience, tokyo metropolitan organization for medical research, tokyo, japan more than 50 years have passed since the publication of "organization of behavior". this book has been one of the most influential books in neuroscience. around the time of the 50th anniversary of this book, special issues and articles concerned with this book appeared in several journals. his idea, which is a general framework for relating behavior to synaptic organization through the dynamics of neural networks, has stimulated variety of neuroscience researches in relation to, for example, environmental effects on development, naturenurture interaction, memory consolidation and sensory deprivation. however, he also made some mistakes, for example he advocated frontal lobotomy. in this symposium, i will briefly review how influential this book has been on basic and practical neurosciences, and will re-consider the importance and limitation of studying mental processes, such as emotion, memory and thought by exploring brain mechanisms, in reference to the idea of cell assembly, phase sequence and hebb synapse. sy3-3-08-2 detection of cell assembly in neuroscience experiments and brain-machine interfaces yoshio sakurai 1,2 , susumu takahashi 2 1 department of psychology, kyoto university, kyoto, japan; 2 crest, japan science & technology agency, japan the reality of cell-assembly coding in the working brain depends on how we could detect specific properties of cell assembly from multi-neuronal activities in behaving animals. first in the present paper, we show experimental results indicating some of the properties, i.e., functional overlapping of individual neurons and connection dynamics among multiple neurons, that depend on tasks and events being processed and on the distance among the neurons. second, we demonstrate a newly developed method, brain-machine interface (bmi), to test the reality of cell assembly as neural information and the plastic formation of cell assemblies during learning processes. we introduce our recent bmi system with independent component analysis (ica) and specific multi-electrodes and show some neuronal and behavioral data obtained by the bmi system. hebb postulated that coincident activities of pre-and postsynaptic neurons trigger input-specific plasticity. how relevant is it in protein synthesis-dependent late-phase plasticity (lp)? synaptic tagging hypothesis explains how new proteins reach the activated synapses to establish input-specific lp. using live-imaging techniques, we measured entry of vesl-1s-egfp into dendritic spines (ve trapping) of rat hippocampal neurons in culture, and found that ve trapping activity serves as synaptic tag in many criteria. ve trapping conforms to the hebb's rule in a sense that it required both presynaptic activity and postsynaptic no-pkg pathway, but their coincident time window was far wider (∼h) than that of early-phase plasticity, suggesting an involvement of persistently synchronized rather than transiently coincident activity. no spreading from the activated synapses may persistently prime the postsynaptic tag components at the surrounding synapses, during which brief inputs to these synapses will establish associative and heterosynaptic tags. thus, tagging one synapse would lead surrounding synapses to multiple metaplastic states. tomoki fukai laboratory for neural circuit theory, riken brain science institute, saitama, japan in the cell-assembly hypothesis, cortical neurons are considered to form functional subnetworks depending on a particular demand of information processing. such cell assemblies may be organized through synchronous firing of the constituent neurons and synapses modifiable by hebbian learning. in this talk, i will overview recent in vivo and in vitro experimental findings that provide new evidence for synchronous or precisely timed neuronal activity. i propose a hardwired structure of local cortical networks, "entangled synfire chains", on the basis of the experimental observations of cortical activity. in this model, multiple cell assemblies can be defined by the pattern of neuronal wiring. however, the same experimental findings can lead us to a different type of cortical network models. in this type of models, cortical networks may self-organize to develop a critical dynamical state, which may be useful for realizing a hypothetical "liquid-state machine". i will discuss the characteristic properties of both types of models and the possible implications in cortical computations. research funds: kakenhi (17022036) sy3-3-08-5 impact of hebbian hypothesis on neuroscience keisuke toyama shimadzu institute of basic technology, seikacho, hikaridai, kyoto 619-0237, japan hebb has seeded two major concepts in the modern neuroscience, i.e., cell assembly hypothesis for perception, and hebbian synapses to construct that cell assembly. the former concept stimulated extensive searches for the response selectivity extending from the primary visual cortex to the inferotemporal cortex and even to the hippocampal cortex, while the later concept triggered neuroscience studies of the learning and memory in the developing and adult brains. recently, these concepts refreshed the impact with new dressing of 'dynamics'. cell assembly that was originally assumed to be static, became dynamic and opened a new possibility for the neural computation, combined with dynamic hebbian synapses conceptualized as the spike-timing dependent plasticity (stdp). i would like to discuss about speaker's talks in this context. sy3-4-04-1 tonic gaba a receptor mediated conductances: properties, functions and plasticity alexey semyanov riken brain science institute (bsi), japan communications mediated by non-synaptic receptors are important for information processing in the brain. high affinity extrasynaptic gaba a receptors mediate a persistent "tonic conductance" which reflects their activation by ambient concentrations of gaba. this phenomenon is found in different brain regions, shows cell-type specific differences in magnitude and pharmacology, and changes during brain development. our findings have revealed functional significance of gaba a receptors mediated tonic conductance in the hippocampus. we have shown that it modulates rate-coded information processing by individual neurons, and acts in a cell-type specific manner to regulate the excitability of the local neuronal circuit. the magnitude of the conductance is regulated by efficiency of gaba uptake and membrane potential. gaba a receptor mediated tonic conductance undergoes adaptive plasticity. it is up-regulated in hippocampal pyramidal cells in a model of pilocarpine status epileptics in rats. in mice lacking gad65 the amount of the tonic inhibition is reduced in ca1 hippocampal interneurons, while unchanged in pyramidal cells. sy3-4-04-2 modulatory effects of peri-interneuronal glial cells on neuronal activities in hippocampal ca1 region yoshihiko yamazaki 1 , yasukazu hozumi 2 , kenya kaneko 1 , satoshi fujii 1 , hiroshi kato 1 1 dept. of neurophysiol., yamagata univ. sch. of med., yamagata, japan; 2 dept. of anat. & cell biol., yamagata univ. sch. of med., yamagata, japan glial cells, in addition to their supportive roles in the nervous system, make up a functional unit with neurons and have been suggested to play novel roles in neuronal activities. we focused on interneuron/peri-interneuronal glial cell (pg) pairs in the hippocampal ca1 region and performed dual whole-cell recordings to investigate the modulatory effect of glial cells on neuronal activities. direct depolarization of pg suppressed the excitatory postsynaptic currents in an adjacent interneuron. this suppression was inhibited by adenosine a1 receptor antagonist. moreover, pg activation modulated the firing pattern of the interneuron. since interneurons in the hippocampus are mainly inhibitory and the terminals of a single interneuron make a large number of synapses on a group of pyramidal cells, direct inhibitory regulation via pg would have marked effects on the information processing of neurons in the ca1 region. research funds: kakenhi (15082201) sy3-4-04-3 carbachol-induced beta oscillations in rat hippocampal slices kiyohisa natsume, jun arai graduate school of life science and systems engineering, kyushu institute of technology, fukuoka, japan rat hippocampus has the cholinergic input from medial septum and diagonal band in vivo. the input involves the generation of hippocampal rhythm, theta, gamma rhythm. to mimic the system, we applied carbachol, a cholinergic agent, to rat hippocampal slices. carbachol can induce beta oscillation as well while carbachol can induce theta, and gamma oscillations in the slices. in the present paper, we introduce the beta oscillations. the application of 30 m carbachol induces beta oscillations which occur intermittently with the interval of 20-30 s. during the intervals, gamma oscillations are induced. the mean frequency of the oscillations is 16.9 ± 0.9 hz (mean ± s.e.m.). the oscillations are induced via muscarinic m1, 3, 4 receptors. the frequencies of them are significantly decreased by the application of bicuculline, a gaba a antagonist. they are sensitive to bicuculline, while theta oscillations are not. it is indicated that the character of beta oscillations are different from those of theta oscillations. the neocortex and the hippocampus are connected by way of the entorhinal cortex and the subiculum. to examine ongoing network interactions among these distinct cortices during neocortical slow oscillations (1-3 hz), we recorded intracellular potentials in single neocortical, entorhinal, subicular, and hippocampal neurons, together with hippocampal field and multi-unit activities in adult anesthetized rats. we have found that (1) most entorhinal and subicular neurons displayed bimodal active (up) and quiet (down) states of membrane potential, in synchrony with neocortical slow oscillations, (2) no bimodal up-down transition was present in hippocampal neurons. hippocampal granule cells were directly driven by entorhinal up-state activity, while ca3 and ca1 neurons discharged during both up and down states, (3) gamma and fast (ripple) oscillations were observed in hippocampal ca1 area irrespective of up-down transition. these observations suggest that entorhinal and subicular regions are "neocortex-like" and hippocampal networks can generate self-organized activity independent of neocortical slow oscillations. the cholinergic neurons in the mesopontine reticular formation (mprf) seem to control sleep-wake cycle and hippocampal activity, because stimulation of the mprf elicits rem sleep and hippocampal theta wave. in this study, we recorded neuronal activity in the mprf and pontine and hippocampal eeg during rem sleep and investigated time-relationship between them. our results are summarized as follows: (1) most of the mprf neurons were active during rem sleep; (2) the mprf activity increased over ten seconds before transition from nrem to rem sleep, i.e. from non-theta to theta period; (3) the theta wave was instantaneously accelerated concomitant with activation of the mprf neurons. these results suggest that cholinergic neuron in the mprf is important in generation and maintenance of rem sleep and theta wave. because hippocampal theta waves are involved in memory consolidation during rem sleep, our findings might help to clarify this mechanism. research funds: kakenhi (17605001) sy3-5-09-1 clock mechanisms of the scn involving in the entrainment to the morning and evening light sato honma, natsuko inagaki, nobuko tokumaru, ken-ichi honma dept. physiology, hokkaido univ. grad. sch. med., sapporo, japan the circadian clock, by entraining to the light-dark cycle of different day length, controls seasonality in biological functions. the mechanism is currently explained by morning and evening oscillators which change their coupling intensity depending on the day-length. by using clock gene expression as a marker of clock functions, we examined the localization and molecular bases of the two oscillators. rats and mice were housed under light-dark (ld) 18:6 h and ld 6:18 h. clock gene expression patterns in the entire scn were examined by in situ hybridization on the first day of constant darkness. the phase relations of per1 and per2 rhythms suggest that light-on resets per1 rhythms in both light conditions, while per2 rhythm also relates to the light-off. in cultured scn of transgenic mice expressing luciferase under the control of per1 promoter, we observed two bioluminescent peaks a day only in the anterior scn from the mice kept in ld 18:6. the finding suggests that two distinct oscillators, which respond to the day-length, reside in the anterior scn. the suprachiasmatic nucleus (scn) is the center of the mammalian circadian clock. tissue transplantation of the scn restores the behavioral circadian rhythm in scn-lesioned mice in spite of the impaired neural connection with the host brain. we have investigated whether grafted scn regulates the circadian oscillator in peripheral organs using the scn-transplanted mice that have a limited time information transmission paths. as a result, the grafted scn restored not only circadian behavior rhythm but also the circadian rhythms of peripheral organs. many of clock genes showed dynamic oscillations with identical phase relationship as shown in intact animals, however, per1 and per2 showed low amplitude of oscillation. the findings suggest that diffusible signal molecules released from the transplanted scn entrain the circadian clock in peripheral organs and that they differentially modulate the expression of clock genes. sy3-5-09-3 genome-wide analysis of adrenal-dependent and independent circadian regulation of mouse hepatic genes norio ishida clock cell biology group, national institute of advanced industrial science and technology (aist), ibaraki, japan recent progress in genome-wide expression analysis has identified hundreds of circadian regulated genes in the suprachiasmatic nucleus as well as in peripheral tissues of mammals. adrenal gland is important for circadian regulation for mammalian peripheral clocks. to identify circadian expressed genes regulated by adrenal glands pathways, we performed dna microarray analysis using hepatic rna from adrenalectomized (adx) and sham-operated mice. we identified 169 genes that fluctuated between day and night in the livers, 100 lost circadian rhythmicity in adx mice. these included the genes for key enzymes of liver metabolic functions such as glucokinase, hmg-coa reductase, and glucose-6-phosphatase. the present study showed that the circadian expression of mouse liver genes is governed by core components of the circadian clock such as clock, and the other 100 genes depend on adrenal glands pathway such as glucocorticoids. hitoshi okamura kobe university graduate school of medicine, japan light is a powerful synchronizer of the circadian rhythms, and bright light therapy is known to improve metabolic and hormonal status of circadian rhythm sleep disorders, although its mechanism is poorly understood. in the present study, we revealed that light induces gene expression in the adrenal gland via the suprachiasmatic nucleus (scn)-sympathetic nervous system. moreover, this gene expression accompanies the surge of plasma and brain corticosterone levels without accompanying activation of the hypothalamoadenohypophysial axis. the abolishment after scn-lesioning, and the day-night difference of light-induced adrenal gene expression and corticosterone release, clearly indicate that this phenomenon is closely linked to the circadian clock. the surge of plasma corticosterone after light exposure indicates that environmental light signals are instantly converted to glucocorticoid signals in the blood and csf. the light-induced clock-dependent secretion of glucocorticoids adjusts cellular metabolisms to the new light-on environment. sy3-5-09-5 neuronal and hormonal control of peripheral clock function through suprachiasmatic nucleus shigenobu shibata, naomi hayasaka, takashi kudo, tsuyoshi yaita department of pharmacology, school of science and engineering, waseda university, tokyo, japan the clock genes are expressed not only in the suprachiasmatic nucleus (scn) of the hypothalamus where the master clock exists, but also in other brain regions and various peripheral tissues. in the liver and lung, clock genes are abundantly expressed and show clear circadian rhythm. although oscillation of clock genes in the liver and lung is controlled under the circadian clock mechanism in the scn, we do not know the resetting signals on peripheral clock function. communication between the scn and peripheral tissues occurs through various systems involving the sympathetic, nicotinic and glucocorticoide functions. this symposium mainly describes both anatomical and physiological experiments to reveal the sympathetic and glucocorticoid control over peripheral clock function. sy3-6-10-1 a to z of gene transfer with adenoviral vector-application to neuronal birth date-specific gene transfer using replication-defective adenoviral vectors, we successfully performed 'pulse gene transfer' into progenitor cells in a neuronal birth date-specific manner. when adenoviral vectors were injected into the midbrain ventricle of mouse embryos between embryonic days (e)10.5 to e14.5, the adenoviral vectors introduced a foreign gene into a specific cohort of birth date-related progenitor cells. this technique allows us to distinguish a cohort of birth date-related progenitor cells from other progenitor cells with different birth dates and to introduce a foreign gene into specific subsets of neurons by performing adenoviral injection at specific times. this adenovirus-meditated gene transfer technique will enable us to examine the properties of each subset of progenitor cells that share the same neuronal birth date. i will explain directions how to use an adenoviral vector and application of an adenoviral vector in my talk. research funds: crest sy3-6-10-2 live imaging in the specific neuronal cells by the combination of transgenic mice and viral vectors kaori kashiwagi, naoaki saito lab. mol. pharmacol. biosig. res. ctr, kobe univ, kobe, japan live imaging analysis has revealed that each protein kinase c (pkc) subtype shows spatio-temporally distinct targeting in response to various stimuli. we demonstrated that the trans-synaptic stimulation induced translocation of ␥pkc-gfp in cerebellar slices from bitransgenic mice (nse-tta/tetop-␥pkc-gfp) which express ␥pkc-gfp in time and region-specific manner. this translocation was not restricted, but propagated from the distal to the proximal dendrites close to the soma of purkinje cells. in order to gain further insight in to the molecular mechanisms of pkc translocation, we introduced viral vectors to primary cultured purkinje cells. the propagative ␥pkc-gfp translocation was also observed in cultured purkinje cells derived from nse-tta mice. the molecular mechanisms of pkc translocation in purkinje cells were analyzed by live imaging with various kinds of viral vectors. the combination of tg mice and viral vectors is useful to understand the physiological role of pkc in the specific neuronal cells. research funds: kakenhi (17024040) sy3-6-10-3 visualization and manipulation of the signaling systems in the cns using sindbis viral vectors sho kakizawa dept. of pharmacol., grad. sch. of med., the university of tokyo, tokyo, japan virus vectors can efficiently deliver genes to neurons and other cells in the nervous systems in vitro and in vivo. because many viral vectors are in common use, it is important to select the best viral vector for each specific application, and a number of factors must be considered when making a decision. sindbis virus is an enveloped plus-strand rna virus belonging to the alphavirus genus of the togaviridae family. the sindbis viral vector is characterized by its effective, rapid, high-level and preferential transduction of neurons. these facts indicate that the vector is a powerful tool for the robust expression of target genes in the specific population of neurons. in this symposium, we will introduce our recent topics on the synaptic functions in the cerebellar systems revealed by visualization and manipulation of signaling molecules, such as nitric oxide and inositol 1,4,5-trisphosphate, in the cerebellar purkinje cells. research funds: grant-in-aid for scientific research on priority areas-molecular brain science-from the ministry of education, culture, sports, science and technology of japan sy3-6-10-4 rescue of phenotypes of null-mutant mice by virus vector-mediated gene transfer kazuhisa kohda, wataru kakegawa, kyoichi emi, michisuke yuzaki dept. of physiol., keio univ. sch. of med., tokyo, japan even after the completion of genome project in major species, functions of many molecules remain uncharacterized. a transgene-based rescue approach is one of the powerful methods to decipher the mechanisms of actions of an orphan receptor; however, it is quite labor intensive and time consuming. here, we have developed a virus vector-based rescue approach and applied to investigate the mechanisms of action of the orphan glutamate receptor ␦2 (glur␦2) in the cerebellum. by introducing a sindbis virus carrying a wild-type glur␦2 into glur␦2-null cerebellum in vivo, we could rescue abnormal phenotypes, such as impaired long-term depression at parallel fiber-purkinje cell synapses. by examining whether a mutant glur␦2 lacking a specific domain could similarly rescue the phenotypes, we could evaluate functional importance of the domain. alternatively, by introducing a partial sequence of the gene of interest into wild-type brain and examining its dominant-negative effect, we will be able to identify the region of the gene product that is functionally important. research funds: kakenhi 16650071 sy3-6-10-5 gene transfer into in vivo cerebellar purkinje cells by hiv-derived lentiviral vectors hirokazu hirai advanced science research center, kanazawa university, ishikawa, japan cerebellar purkinje cells are key elements regulating motor coordination and motor learning. gene transfer into purkinje cells is an effective approach for the study of cerebellar function and treatment against cerebellar disorders. although adenoviral vectors or sindbis vectors are frequently used for gene delivery into neurons, the former has extremely low affinity for purkinje cells, while the latter causes substantial damage to the infected cells. to achieve effective gene transfer into purkinje cells, we used human immunodeficiency virus (hiv)-derived lentiviral vectors. purkinje cells were efficiently transduced without significant influence on the cell viability and synaptic functions. gene expression was also detected, though less efficiently, in other cortical cells, whereas no transduced cells were observed outside of the cerebellar cortex. these results suggest that hiv-derived lentiviral vectors are useful for the study of gene function in purkinje cells as well as for application as a gene therapy tool for the treatment of diseases that affect purkinje cells. research funds: jst/presto, kakenhi (17300100) sy3-7-11-1 physiological basis for stereotaxic surgery in basal ganglia atsushi nambu division of system neurophysiology, national institute for physiological sciences, and school of life science, the graduate university for advanced studies, okazaki, japan stereotaxic surgery in movement disorders such as parkinson's disease dramatically improves the symptoms of such diseases. i assume the physiological basis for the treatment is to disrupt abnormally increased information flow through the basal ganglia. i will discuss the pathophysiology of basal ganglia disorders and the effect of stereotaxic surgery in light of the three major pathways in the cortico-basal ganglia loop, i.e., hyperdirect (cortico-stn-gpi), direct (cortico-striato-gpi) and indirect (cortico-striato-gpe-stn-gpi) pathways, that dynamically control the activity of the thalamus and cortex to perform correct motor programs in correct timing. research funds: kakenhi (17022042) sy3-7-11-2 deep brain stimulation of subthalamic nucleus on parkinson's disease fusako yokochi department of neurology, tokyo metropolitan neurological hospital, tokyo, japan parkinson's disease is a progressive and degenerative disease caused by dopamine deficiency attributed to the degeneration of neurons in the substansia nigra. consequently, various symptoms appear, such as cardinal symptoms, those in the advanced stage and those as the side effects of long-term levodopa therapy. many antiparkinsonian drugs have been developed, but all of the symptoms are not improved by these drugs. stereotaxic surgery was started for treating severe tremor and rigidity in the mid-20th century. stimulation by chronically implanted electrodes in the brain, that is, deep brain stimulation (dbs), has recently been applied and has been shown to have marked effects on the symptoms resisted to conventional treatments. in particular, dbs of the subthalamic nucleus (stn) is an effective treatment for the symptoms. much basic research on the function of stn has been reported, but stn function is still unclear. clinical outcomes including the side effects of stn dbs, the neural activities recorded from stn and the localization of clinical effects are reported in this paper. sy3-7-11-3 firing patterns of basal ganglia neurons and effects of deep brain stimulation in parkinson's disease takao hashimoto center for neurological diseases, aizawa hospital, matsumoto, japan high-frequency electrical stimulation of the subthalamic nucleus (stn), internal segment of the globus pallidus (gpi) and thalamus can improve motor signs in patients with parkinson's disease, however, its mechanism of action remains unclear. a leading hypothesis regarding the development of movement disorders of basal ganglia origin suggests that hyperkinetic and hypokinetic disorders occur as a result of changes in the mean discharge rate in the gpi and substantia nigra, which in turn suppress thalamocortical output. on the other hand, altered firing patterns in the basal ganglia have been reported in mptp-induced parkinsonian animals: increases in bursting activity and periodic oscillatory activity in the gpi and stn, and synchronization of gpi, or gpi and striatal neurons. synchronous oscillation in the basal ganglia may break down independent processing in the motor circuit and disrupt signal processing at the cortical level. kaoru takakusaki department of physiology, asahikawa medical college, asahikawa, japan locomotion is composed of volitional and automatic processes. particular attention is required to perform volitional processes such as avoiding obstacles and accurate limb placements during locomotion. however, we are largely unaware of the automatic control processes of rhythmic limb movements, muscle tone and postural reflexes that accompany locomotion. because each process is seriously impaired in parkinsonian patients, the basal ganglia must play a crucial role in integrating the volitional and automatic processes of locomotion. the basal ganglia contribute to the planning and execution of voluntary movements via basal ganglia thalamocortical loops. on the other hand, recent our findings suggest the importance of the direct basal ganglia outflow to the brainstem where fundamental neuronal networks for controlling postural muscle tone and locomotion are located. in this presentation we discuss the role of the basal ganglia in the integration of volitional and automatic movements during locomotion, which has been less understood aspect of gait control. research funds: grant-in-aid for scientific research (c) and priority area (area no.454) sy3-7-11-5 characteristics of neuronal activity within the globus pallidus interna (gpi) in patients with dystonia yoichi katayama 1,2 1 department of neurological surgery, nihon university school of medicine, tokyo, japan; 2 division of applied system neuroscience, nihon university graduate school of medical science, tokyo, japan dystonia represents disordered muscular tonicity of the trunk and/or extremities, and is often dramatically controlled by chronic gpistimulation in humans, indicating that dystonia is attributable to certain abnormal activity of gpi neurons. little is yet known, however, regarding characteristics of neuronal activity within the gpi underlying dystonia. we analyzed activity of gpi-neurons in patients with dystonia or parkinson's disease, which were recorded during surgery for chronic gpi-stimulation. as compared to gpi neurons in patients with parkinson's disease, gpi neurons in patients with dystonia were distinctive with following three characteristics; firing rate (49.4 ± 31.7 hz, n = 27) was low, firing pattern was often composed of irregular pauses and bursts, and many were responsive to body movement with wide receptive fields. these findings suggest that dystonia may be related to unstable movement-related sensory processing within the gpi. it has been considered that dystonia, which is generally characterized by postural abnormalities and involuntary movements including torsion, is caused by dysfunction of the basal ganglia. the purpose of the present work is to clarify the neural mechanisms underlying the onset of dystonia by analyzing the pathophysiology of a model for torsion dystonia, wriggle mouse sagami (wms). the genomic mutation of wms occurs in the gene encoding plasma membrane ca 2+ -atpase isoform 2 that is located on the 6th chromosome. recent immunohistochemical and electrophysiological investigations on wms have shown that (1) d2-type dopamine receptors are downregulated presynaptically in the striatum, and (2) a large number of purkinje cells in the cerebellum express tyrosine hydroxylase (the synthesizing enzyme for dopamine) and their excitability is greatly reduced. in this symposium, the possible correlation between these data and dystonic phenotypes will be discussed. kei-ichiro maeda reproductive science, graduate school of bioagricultural sciences, nagoya university, nagoya, japan gnrh has been well established to regulate reproduction through two modes of secretion: surge and pulse. the surge mode is female-specific and induces lh surge and then ovulation through positive feedback action of estrogen. the pulse mode tonically activates gonads in both sexes with being negatively regulated by gonadal steroids. environmental cues, such as photoperiod or nutrition, are considered to affect reproductive activity through altering pulse mode of gnrh release. pulse mode seems much more robust than surge, because estrogen can induce a surge under a certain condition when the pulse is suppressed. the following four papers aim to unveil the physiological mechanism underlying two modes of gnrh secretion in various experimental models. sy3-8-05-2 metastin: a neuropeptide playing a central role in the regulation of ovulatory cycle hiroko tsukamura, kei-ichiro maeda graduate school of bioagricultural sciences, nagoya university, japan estrous cyclicity is regulated by a sequence of neuroendocrine events consisting of hypothalamus-pituitary-gonadal axis. gonadotropinreleasing hormone (gnrh)/luteinizing hormone (lh) surge is induced by positive feedback action of estrogen secreted by mature ovarian follicles. the central mechanism of positive feedback action of estrogen on gnrh/lh secretion, however, is not fully understood yet. metastin was first isolated as a natural ligand for a g-proteincoupled receptor, gpr54 (2001. nature 411, 613) . recent studies reported that a genetic alteration leading to homozygous loss of function of gpr54 impairs pubertal development in mice and human. we have first demonstrated that endogenous metastin plays a physiological role in inducing ovulation through stimulating gnrh/lh surges to control estrous cyclicity in the female rat (2005. endocrinology 146, 4431) . the present paper focuses on the role of metastin in regulating gnrh/lh surge based on our recent study in rats and discusses possible mechanism underlying positive feedback action of estrogen. suzanne moenter, catherine christian university of virginia, usa a surge in gonadotropin-releasing hormone (gnrh) secretion is the cns signal that triggers the luteinizing hormone (lh) surge, which causes ovulation. the gnrh surge depends on a switch in estradiol (e) feedback from negative to positive and in rodents on time of day, occurring in the pm. treating ovariectomized (ovx) mice with a constant release e capsule (ovx+e) elicits daily pm lh surges; there is no diurnal change in ovx controls. likewise, extracellular recordings of firing activity of gfp-identified gnrh neurons showed no diurnal changes in cells from ovx mice. in contrast, e increased firing in the pm compared to am. gabaergic neurons form a major input to gnrh neurons, and activation of the gabaa receptor can be excitatory in these cells. whole-cell patch-clamp recordings of synaptic activation of gabaa receptors on gnrh neurons revealed e-dependent decreases in transmission during the am (negative feedback) and increases in transmission near surge onset that persisted for some populations of afferents thru the surge peak. together these data indicate one mechanism by which e induces the gnrh surge is by altering gaba transmission to gnrh neurons. yoshitaka oka grad. sch. sci., univ. of tokyo, tokyo, japan the gonadotropin-releasing hormone (gnrh) peptidergic neuronal systems consist of hypothalamic neuroendocrine and extrahypothalamic neuromodulatory gnrh neurons. here, i introduce our recent studies on the physiological properties of neuroendocrine preoptic (poa) gnrh neurons in comparison with the neuromodulatory terminal nerve (tn) gnrh neurons. to study the both types of gnrh neurons, we use a fish model system in which we can easily identify both of them in intact brain preparations in vitro, which is a great advantage over most other vertebrates. the poa-gnrh neurons had quite different basic electrophysiological membrane properties from those of tn-gnrh neurons and showed alternating active and silent phases of firing activities, in contrast to the regular pacemaker activities of tn-gnrh neurons. now that we have various experimental approaches (electrochemical measurement of gnrh release, ca 2+ imaging after single-cell electroporation, single-cell rt-pcr, double patch clamp recording, etc.) in hand, simultaneous multidisciplinary approaches should be possible to study the physiology of gnrh neurons. research funds: kakenhi 15370032 sy3-8-05-5 metabolic regulation of puberty onset using the prepubertal rat model helen i'anson biology department and neuroscience, washington and lee university, usa we hypothesize that glucose availability determines the timing of puberty onset in rats. abdominal fat stores are low in dieted, prepubertal female rats with delayed puberty, suggesting that such rats may be particularly sensitive to dietary fuel changes. such rats are fed a single daily meal and demonstrate decreased blood glucose levels between meals. we hypothesized that such daily hypoglycemic bouts delay onset of puberty during dieting. when glucose supplement was given to prepubertal dieted rats, and they exhibited first estrus with similar timing to previously dieted re-fed rats. conversely, when dieted rats were re-fed ad libitum and simultaneously glucose-deprived, first estrus was delayed. blood glucose levels during glucose-supplementation which induced first estrus, and during glucoprivation and re-feeding which delayed first estrus, were similarly elevated, suggesting that central, rather than peripheral, glucose levels are monitored in the prepubertal animal. in summary, central glucose availability may be an important signal timing puberty onset. research funds: jeffress memorial trust and washington and lee university sy3-8-12-1 molecular mechanisms of thyroid hormone action in developing brain toshiharu iwasaki, noriyuki koibuchi department of integrative physiology, gunma university graduate school of medicine, maebashi, japan thyroid hormone (th) plays an important role in the developing brain. the action is mainly exerted by controlling gene expression through binding to its specific receptor, th receptors (trs). trs are ligand-regulated transcription factors that are expressed in many organs, including brain. trs bind to target dna sequences known as th-response element (tre). coactivators and corepressors are involved in the tr-mediated gene regulation through histone modification. we characterized the coactivator and the corepressor that were expressed in embryo brain. although precise mechanism of the th action for brain development has not been fully clarified, these cofactors may be involved in these actions. th affects brain development only during a limited period, which is referred as the critical period of th action. recently, it has been speculated that environmental chemicals may cause the abnormal brain development. possible mechanism of action of such chemicals on tr system will be discussed. research funds: kakenhi 17510039, 17390060 sy3-8-12-2 thyroid hormone and organic anion transporters in brain hiroyuki kusuhara, yuichi sugiyama graduate school of pharmaceutical sciences, the university of tokyo, tokyo, japan organic anion transporting polypeptides (oatps/slco) currently consist of 15 isoforms in human and rodents. they are initially identified as part of detoxification system in the body, and involved in the tissue uptake of xenobiotics, especially amphipathic organic anions. some members accept a variety of structurally unrelated compounds as substrate. oatp1c1 is characterized by its unique substrate specificity, highly selective for thyroid hormones, particularly for t4 and reverse t3, but the transport activity of t3 is quite low. it is predominantly expressed in the brain capillaries and choroid plexus, acting as barrier of central nervous system, where oatp1a4, the transport activities of t4 and t3 are similar, is also expressed. the uptake of t4 and t3 by the brain determined using in situ brain perfusion technique was saturable and inhibited by oatps substrates and inhibitors. these two transporters may play a role in regulation of brain levels of t4 and t3. research funds: the advanced and innovational research program in life sciences from the ministry of education, culture, science and technology sy3-8-12-3 alterations of gene expression profiles in the developing brain by chemicals disrupting thyroid hormonedependent signals takayuki negishi 1 , masaki takahashi 1 , yasuhiro yoshikawa 2 , tomoko tashiro 1 1 department of chemistry and biological science, aoyama gakuin university, kanagawa, japan; 2 department of biomedical science, the university of tokyo, tokyo, japan there is increasing concern about the possibility that environmental chemicals such as polychlorinated biphenyl (pcb) and its hydroxylated metabolites interfere with normal brain development through acting as thyroid hormone disrupting agents. in this presentation, based on comprehensive dna microarray analysis, we demonstrate alterations in gene expression in the brain of neonatal rats perinatally exposed to 4-hydroxy-2,2 ,3 ,4 ,5pentachlorobiphenyl (anti-thyroid hormone-like), as well as in primary cultured rat hippocampal neurons exposed to 4-hydroxy-2,2 ,3,4 ,5,5 ,6-heptachlorobiphenyl (thyroid hormone-like). among genes whose mrna expression levels were affected by these compounds, a number of genes essential for the establishment of synaptic networks were detected, suggesting that long-lasting effects on higher brain functions may result from exposure of the developing brain to these compounds. hydroxylated metabolites of pcbs (oh-pcbs) have chemical structures similar to thyroid hormones (ths). we reported that low doses of two types of oh-pcb inhibited th-dependent extension of purkinje cell dendrites (2005. dev. brain res. 154, 259) . koibuchi et al. (2004) clarified that they interfere with th-dependent gene expressions in reporter gene assays. further, takasuga et al. (2004) detected some pcb and oh-pcb congeners in human csf, of which 4oh-cb187 is the highest concentration. to determine its effects on developing neurons, we examined 4oh-cb187 in rodent cerebellar culture cells. interestingly, 4oh-cb187 promoted dendritic development of purkinje cells in the absence of th and increased significantly their survival numbers. these results indicate that oh-pcb congeners may disrupt normal brain development by different mechanisms depending on their chemical structures. we have reported that rat pups exposed to an antithyroid agent, propylthiouraci l (ptu), via maternal milk exhibit hyperactivity, impairment in spatial learning and social interaction, and audiogenic hypersensitivity extending to audiogenic seizures, thus this ptu rat can be a possible candidate of animal model for autism. in ptu rats, the delay in migration of the extragranular cells of cerebellum, and in innervation from inferior olive nuclei to purkinje cells were shown. these neurons transiently expressed serotonin-ir, therefore we treated ssri or 5-htp to examine the relevance of serotoninergic function. the treatment of 5-htp but not ssri recovered the delay of cell migration. these effects of serotonin manipulation in ptu rats on the behavioral impairment and the development of cns will be discussed. it is hoped that embryonic stem (es) cells will be used in transplantation therapy for neurological diseases. however, because grafts of neural stem cells derived from es cells may contain residual undifferentiated cells, there would be a risk for teratomas. to reduce this risk, we applied to es cells herpes simplex virus thymidine kinase (hsv-tk) gene and ganciclovir (gcv) treatment. stable mouse and cynomolgus monkey es cell lines expressing hsv-tk were obtained. gcv sensitivity was higher in undifferentiated es cells than in es cell-derived neural stem cells. es cell-derived neurons were resistant to gcv treatment. nude mice with transplants of undifferentiated es cells expressing hsv-tk formed teratomas, but the tumor growth was suppressed after the gcv treatment. suicide gene delivery might increase the safety of the use of es cells in cell replacement therapy. enzymatic degradation of chondroitin sulfate is known to promote axonal regeneration in the central nervous system. the physiological role of chondroitin sulfate up-regulated after injury was examined in the nigrostriatal dopaminergic system which was unilaterally transected and treated with chondroitinase abc. in transected mice, dopaminergic axons did not extend across the lesion. chondroitin sulfate was up-regulated around the lesion and a fibrotic scar containing type iv collagen deposits were developed in the lesion center. in chondroitinase abc-treated mice, numerous dopaminergic axons were regenerated across the lesion. in these animals, chondroitin sulfate immunoreactivity was remarkably decreased and the formation of a fibrotic scar was unexpectedly prevented. these results support our previous supposition that chondroitin sulfate does not act as an obstacle to regenerating axons, but involved in the repair process of the brain injury including the formation of the fibrotic scar (kawano et al., 2005) . reference kawano et al., 2005. j. neurosci. res. 80, 191-202. research funds: kakenhi 16500234 os2a-8-03 neurotransmitters that maintain and suppress the tonic firing of the serotonergic neurons in the dorsal raphe during sleep waking cycles yoshimasa koyama 1 , kazumi takahashi 2 , yukihiko kayama 2 1 cluster of science and technology, fukushima university, fukushima, japan; 2 department of physiology, fukushima medical university, fukushima, japan the present experiment was done to examine, under unanesthetized natural sleep-waking condition, which neural systems were involved to regulate the firing of the serotonergic (5ht) neurons in the dorsal raphe (dr) during sleep waking cycles. using head restrained, unanesthetized rats, single neuronal activity was recorded and each drug was applied iontophoretically or by pressure close to the recording neurons. spontaneous firing of the 5ht neurons in dr were excited by glutamate and orexin a or b. they were inhibited by noradrenaline. an ␣1 receptor agonist (phenylephrine or methoxamine) increased the firing rate during sws or ps, but had no effect when applied during w. in ps-off type dr neurons, cessation of firing during ps was recovered by bicuculline, however in the dr neurons that did not stop firing during ps, bicuculline had almost no effect. os2a-8-04 correlation between regional grey matter volume and proficiency increase in second language: a vbm study arihito nauchi 1,2 , kazuyoshi hirano 2,3 , yukimasa muraishi 2,3 , kuniyoshi l. sakai 1,2 1 department of basic science, university of tokyo, tokyo, japan; 2 crest, jst, japan; 3 secondary education school, university of tokyo, japan although neuroimaging studies have contributed to clarify the brain function, the neural basis of individual variation in cognitive abilities such as language still remains unknown. in the present study using voxel-based morphometry (vbm), a whole-brain unbiased technique for detecting regional differences in mr images, we examined the relationship between the proficiency increase in second language (l2) and grey matter volume among students aged 13-17, who received special classroom training in the use of english verbs. in specific regions including the left lateral premotor cortex and the left inferior frontal gyrus (the grammar center), we found a positive correlation between the regional grey matter volume and improved performance for the grammaticality of english sentences. these results suggest an anatomical basis for the language faculty, such that the capacity of a specific region is related to proficiency increases in l2. os2a-8-05 grammar center activation in honorification judgment of japanese sentences kanako momo 1,2 , kuniyoshi l. sakai 1,2 1 department of basic science, university of tokyo, tokyo, japan; 2 crest, jst, japan one linguistic theory proposes that japanese honorification is a syntactic feature, because syntactic agreement is required between subject/object and honorific forms. to investigate whether such syntactic processing is actually realized in the brain, we examined cortical activation using fmri under several types of normal/anomalous judgment for japanese sentences including honorification. when activation in a honorification task was contrasted with that in a spelling task, we observed significant increase in the left including grammar center (ifg) as well as the bilateral cerebellum for all tested participants. moreover, the lower performance group showed greater activation in the left f3op/f3t and the bilateral cerebellum. these results suggest that syntactic process is required for japanese honorification and that activation in these regions shows modulation according to performance level, even in native language. research funds: crest, jst os2a-8-06 top-down modulation for melody-related activity in the right auditory areas: an meg study takuya yasui 1,2,3 , kimitaka kaga 2 , kuniyoshi l. sakai 1,3 1 dept. of basic science, univ. of tokyo, tokyo, japan; 2 dept. of otolaryngology, univ. of tokyo school of medicine, japan; 3 crest, jst, japan we previously reported right-hemisphere dominance for melody error-induced fields (m140) (neurosci. res. 52, s61). in a subsequent study, we confirmed that m140 was independent from mismatch negativity usually induced by oddballs. in the present study, we examined whether m140 was induced by deviation from a memorized melody. we used four pairs of unfamiliar songs, each pair consisting of an original song and a modified song in which the third note deviated from that of the original. subjects learned these songs and judged whether there were one or two deviations in notes. there was no significant difference in dipole amplitudes between m140 elicited by the original songs and that by the modified ones. however, while m140 without the deviation showed no significant effect of lateralization, m140 with the deviation resulted in significant enhancement in the right hemisphere. these results suggest the existence of memory-induced, i.e., top-down modulation for melody-related activity in the right auditory areas. research funds: crest, jst os2a-8-07 cortical plasticity in adulthood for learning phonics rules for english orthography and phonology makiko muto 1,2 , kuniyoshi l. sakai 1,2 1 department of basic science, university of tokyo, tokyo, japan; 2 crest, jst, tokyo, japan although matching english orthography with correct pronunciation is difficult for second language learners, learning phonics rules may rapidly improve their performance. in the present fmri study, we tested an english matching task during the course of phonics training in 16 sessions, in which infrequent words were visually shown, while matched/unmatched speech sounds were simultaneously presented. comparing the first half of the sessions with the latter half, the left posterior inferior temporal gyrus (the letter center) and a part of the left lateral premotor cortex (the grammar center) showed activation decreases, when the performance was significantly improved. these results suggest that the plasticity of functional systems involving these critical regions is essential for establishing phonics rules and for forming a new link between orthography and phonology. research funds: crest, jst os2a-8-08 hierarchical syntactic processing in the left frontal region: an meg study kazuki iijima 1,2 , naoki fukui 3 , kuniyoshi l. sakai 1,2 1 dept. of basic science, univ. of tokyo, komaba, japan; 2 crest, jst; 3 dept. of linguistics, sophia univ., yotsuya, japan previous erp studies have shown word-related activation based on semantic association or context. however, it remains unclear how syntactic information of preceding words is integrated into the ongoing sentence processing. in the present meg study, we measured brain activity during each of four tasks: a syntactic task, a semantic task, a memory task, and an evaluation task. sentence stimuli consisted of one noun phrase and one verb, where the noun phrase had either an objective or nominative case particle. the first peak of the activity for a verb presentation was observed at the left frontal region as early as 130 ms after the onset. in the objective-case condition, this activity was enhanced only for the syntactic task, while in the nominative-case condition no such task-selectivity was observed. these results are consistent with the current linguistic theory (the minimalist program), which holds that a noun phrase with an objective case particle is directly merged with a verb, to form a new hierarchical level. research funds: crest, jst os2a-8-09 individual difference of brain activity in medial prefrontal cortex and superior temporal sulcus during social cognition koji jimura, seiki konishi, tomoki asari, junichi chikazoe, yasushi miyashita dept. physiol. univ. tokyo sch. med., japan previous neuroimaging studies have reported brain activity in the medial prefrontal cortex (mpfc) and the superior temporal sulcus (sts) during performance of theory of mind tasks. the present fmri study explored individual difference of the mpfc and sts activity by employing false belief paradigms. the task consists of two sessions, study and test. during the study session, subject studied a brief story in which two characters have false beliefs. then, the subject answered questions about the false belief and the fact that constitutes the false belief during the test session. consistent with previous studies, significant activity was observed in the mpfc and the sts during representing the false belief. the individual differences of the mpfc and the sts activity were correlated with psychodiagnostic indices that represent controlled and automatic idealization, respectively. these results suggest that the two indices represent distinct neural mechanisms participating in social cognition. research funds: grant-in-aid from mext (14002005), jsps research fellowship (1611149) os2a-8-10 brain activity of happy facial recognition in mother-daughter relationship jun shinozaki 1 , nobukatsu sawamoto 1 , toshiya murai 2 , takashi hanakawa 1 , hidenao fukuyama 1 1 hbrc, kyoto univ. grad. sch. of med. kyoto, japan; 2 neuropsychiatry, kyoto univ. grad. sch. of med. kyoto, japan relationship between parents and children is special, and affective facial recognition between them should evoke specific neural activity not shared by other personal relationships. eleven healthy females participated in this fmri experiment. the subjects saw happy and neutral faces of their own mothers, and newly learned other subjects' mothers during the scan. when happy face recognition was compared with neutral face recognition, the mother-daughter combination induced greater activity than the non-familial combination in the following areas; the lateral prefrontal cortex, anterior cingulate cortex, middle temporal cortex, striatum, and anterior insula. it has been shown that the lateral prefrontal and anterior cingulate cortices are associated with familial facial recognition, whereas the middle temporal cortex is related to happy facial recognition. the activity in the striatum and anterior insula might be related to positive affection and empathy, respectively. os2a-8-11 anatomical connections among functionally identified brain regions for sentence processing yukari yamamoto 1,2 , atsushi maki 1,2 , kuniyoshi l. sakai 2,3 1 advanced research laboratory, hitachi, ltd., tokyo, japan; 2 crest, japan science and technology agency, saitama, japan; 3 dept. of basic science, univ. of tokyo, tokyo, japan we have functionally identified the left dorsal inferior frontal gyrus (ifg), the left lateral premotor cortex, and the triangular/orbital part of the left ifg (f3t/f3o) as regions associated with sentence and discourse level processing. in the present study, we examined whether there are direct anatomical connections among these regions by using diffusion tensor tractography. f3t and f3o of the left ifg were chosen as seed areas for fiber tracking. fiber bundles that went through two spherical regions were extracted from the tracking data. the central coordinates of these regions were (−54, 27, 21) , (−39, 3, 42) , and (−51, 27, −6) in the standard brain, which are associated with syntactic processing (the first and second coordinates) or sentence comprehension (the third coordinate). direct connections among these regions were consistently observed among the subjects. this result suggests a critical network among multiple regions that are associated with sentence processing. os2a-8-12 effect of the incongruity controlled by semantic distance on visually evoked magnetic fields nobuyoshi harada 1 , sunao iwaki 1 , mitsuo tonoike 2 1 aist, osaka, japan; 2 chiba university, chiba, japan visual incongruities of heads changed on animal pictures, which were controlled by the semantic distance of the word of the animal, were investigated on visually evoked magnetic fields. the semantic distance was decided by the numbers of links of the semantic network of the taxonomic layer in a japanese thesaurus. the words for mammalians were grouped into five semantic categories in the thesaurus. the heads of the animals were changed with those from another semantic category (deviant, d = 4), and with those from an inner semantic category (middle, d = 2), while others were not changed (normal, d = 0). peak amplitudes of waveforms of the root mean square values on the components of 170 ms (f (2/38) = 4.92, p = 0.013) and 220 ms (f (2/38) = 5.23, p = 0.0098) were significantly decreased with increments of the semantic distance in left occipital sensors. the gradient of the decreasing line of the amplitudes of the 170 and 220 ms components indicated the capability of extracting the structure of a typical prototype of the form of the animal. we call this capability, the structural sensitivity for prototype (ssp). ryohei yasuda duke university medical center, usa calcium signaling in dendritic spines is important for many forms of synaptic plasticity. however, the quantitative mechanisms of how calcium elevations are translated into spatial and temporal patterns of biochemical reactions leading to modifications of synaptic strength are unclear. identifying and following the spatiotemporal activation of molecules necessary for synaptic plasticity is crucial for a better understanding of this complex process. to visualize the activity of signaling pathways in neurons deep in brain tissues, we have combined fluorescence lifetime measurements and two-photon microscopy. this technique allowed us to measure spatiotemporal aspects of the activity of signaling proteins including ras gtpase proteins in response to physiologically relevant stimuli with single spine resolution. research funds: burroughs wellcome fund, dana foundation os2p-2-02 mechanisms of p2y purinoceptor-mediated long-term enhancement of inhibitory transmission examined by multiple-probability fluctuation analysis at cerebellar gabaergic synapses yumie ono 1 , xiaoming zhu 1 , takashi tominaga 2 , fumihito saitow 3 , shiro konishi 1,2 1 waseda-olympus bioscience research institute, singapore, singapore; 2 department of neurophysiology, tokushima bunri university, kagawa, japan; 3 department of pharmacology, nippon medical school, tokyo, japan postsynaptic p2y receptor activation by atp enhances ipscs at cerebellar interneuron-purkinje cell (pc) synapses. to investigate the underlying mechanisms, we here employed the non-stationary fluctuation analysis to estimate the number (n) and single channel conductance (i) of gaba a receptors in pcs using whole-cell recordings of evoked ipscs in pcs of rat cerebellar slices before and 5-15 min after application of atp. the atp-induced enhancement of the ipsc amplitude was associated with a significant increase in the single channel conductance, but not the number, of gaba a receptors in pcs: i and n after atp treatment were 160 ± 9.9% and 94 ± 6.1% of the controls, respectively. pretreatment with the protein kinase a inhibitor h-89, but not the calmodulin kinase ii inhibitor kn-62, completely abolished the atp-induced ipsc enhancement. os2p-2-03 activin induces long-lasting nmda receptor activation via scaffolding pdz protein arip1 isao inoue, akira kurisaki, hiromu sugino institute for enzyme research, tokushima university, tokushima, japan calcium entry into the postsynaptic neuron through nmda type glutamate receptors (nmdars) triggers the induction of long-term potentiation (ltp). the ca 2+ permeability of nmdar is regulated by phosphorylation of its tyrosine residues. we report here that activin, a member of the transforming growth factor-b (tgf-b) superfamily, and one of proteins synthesised after ltp, promotes phosphorylation of nmdars and increases the ca 2+ influx through those receptors in primary cultured rat hippocampal neurons. this signal transduction occurs in a functional complex of activin receptors, nmdars, and src family tyrosine kinase, fyn formed on a multimer of postsynaptic scaffolding pdz protein, activin receptor interacting protein 1 (arip1). activin-induced nmdar activation persists more than 24 h, which is complimentary to the transient activation of nmdars by brain derived neurotropic factor (bdnf). our results show that activin is a long-lasting potentiator involved in synaptic plasticity regulatory mechanisms. os2p-2-04 roles of cam kinase i in the hippocampal longterm potentiation kohji fukunaga, takashi komori, shigeki moriguchi department of pharmacology, graduate school of pharmaceutical sciences, tohoku university, sendai, japan cam kinase i (camki) family members are highly expressed in the adult rat hippocampus and camki-alpha is predominantly localized in the cytosol. camki activation requires phosphorylation of thr177 by camkk as an upstream kinase. we here documented a marked increase in camki-alpha-thr177 phosphorylation following ltp induction in rat hippocampal ca1 region. like camkii activation following ltp (fukunaga et al., 1993) , the increased camki-thr177 phosphorylation remained elevated at least for 60 min after ltp induction. the increased camki-thr177 phosphorylation was closely associated with prolonged increases in phosphorylation of creb and myosin light chains in the ca1 region. this is in contrast with transient increases in camkiv and erk phosphorylation. treatment with camkk inhibitor, sto-609 significantly inhibited both creb and mlc phosphorylation with concomitant reduction of ltp in the ca1 region. taken together, camki likely mediates the late phase of creb phosphorylation and an increased mlc phosphorylation in the hippocampal ltp. to investigate how the excitatory postsynaptic inputs of the proximal dendrite effect the information processing of synaptic inputs at the distal dendrite, stimulation was applied to induce bap and epsp at the alveus and the proximal dendrite, respectively. the resulting coincidence of magnitude of bap and epsp at the distal dendrite was enhanced when the bap was delivered at a timing (5 ms) to induce ltp. furthermore, the magnitude of bap at the distal dendrite was attenuated by the input from the proximal dendrite at a timing (20 ms) to induce ltd. these results suggest that the magnitude of bap delivered to the distal dendrite can be amplified or attenuated depending on the relative timing between proximal input and bap. this may be due to an effect on the coding process at the distal dendrite and could support the basis for a novel learning rule in the brain. research funds: kakenhi (17021037) os2p-2-06 mouse brains deficient in neuronal pdgf receptor-␤ develop normally but are vulnerable to injury yoko ishii, takeshi oya, lianshun zheng, masakiyo sasahara department of pathology, faculty of medicine, university of toyama, japan the platelet-derived growth factors (pdgfs) and pdgf receptors (pdgfrs) are widely expressed in the mammalian cns. here, we developed novel mutant mice in which pdgfr-␤ subunit gene was genetically deleted in the neurons of cns to elucidate the role of pdgfr-␤. our mutant mice reached adulthood without apparent anatomical defects. the cerebral damage after cryogenic injury was severely exacerbated in the mutants compared with the controls. furthermore, this exacerbated lesion formation was suggested to be, at least partly, due to the enhanced excitotoxicity after injury, because nmda-induced lesion formation was also extensively enhanced in the cerebral cortex of the mutants without altered nmda receptor expression. this is the first known report to address the postnatal function of pdgfr-␤ expressed in cns neurons, using genetically engineered mutant. it was clearly demonstrated that pdgfr-␤ expressed in neuron protects cns neurons from cryogenic injury and nmda-induced excitotoxicity. early postnatal days (especially the first three weeks in the rat) are the critical period for newborn hippocampal granule cells (gcs) to dynamically migrate from the dentate hilus and form the gc layer. to investigate the mechanism that regulates newborn gc migration, we developed a new slice coculture system. the hilar parts of entorhino-hippocampal slices prepared from postnatal six-day-old (p6) rats that had received a single brdu injection at p5 were substituted with the corresponding region of entorhino-hippocampal slices from p6 rats. after five days in vitro, newborn gcs, detected by brdu and prox1, migrated out of the hilar graft and reached the host gc layer. chronic application of picrotoxin, a gaba a receptor antagonist, facilitated the migration of newborn gcs into the gc layer. these results indicate that gaba a receptors regulate the migration of newborn gcs in early postnatal days. os2p-3-02 cdk5 is required for neuroblast migration in the adult mouse brain yuki hirota 1,2,6 , toshio ohshima 3 , takuji iwasato 4 , ashok b. kulkarni 5 , hideyuki okano 2,6 , kazunobu sawamoto 1,2,6 1 bridgestone lab. keio univ., tokyo, japan; 2 dept. physiol., keio univ., tokyo, japan; 3 dev. neurobiol. riken, tokyo, japan; 4 behavioral gen. riken, tokyo, japan; 5 nih, bethesda, usa; 6 sorst, jst, saitama, japan neuroblasts generated in the subventricular zone (svz) of the lateral ventricles migrate into the olfactory bulb (ob) through the pathway called rostral migratory stream (rms). molecular mechanisms regulating the directional long-distance migration remain largely unknown. here we studied adult function of cyclin-dependent kinase 5 (cdk5) that has been revealed to play a role in neuronal migration in the embryonic brain. crossing the floxed-cdk5 mice to emx1cre mice resulted in decreased size of ob and abnormal distribution of neuroblasts. svz explants from these mice cultured in matrigel showed decreased migration distance. leading process of neuroblasts infected with cre-encoding retrovirus were found in random orientations and frequently failed to migrate out of the svz compared to control cells. these results indicate that cdk5 has a cell autonomous function in neuroblast migration in the adult brain. os2p-3-03 colocaliztion of neuron markers and glial markers in gabaergic neuron progenitors as revealed by singlecell microarray analysis shigeyuki esumi 1 , wu sheng-xi 2 , yuchio yanagawa 3,4 , kunihiko obata 5 , nobuaki tamamaki 1 1 kumamoto univ., kumamoto, japan; 2 fourth military medical univ., xi'an, people's republic of china; 3 gunma univ., maebashi, japan; 4 sokendai, hayama, japan; 5 riken, wako, japan gabaergic neurons and oligodendroglia share many characters in the murine forebrain. both of the cell types has been reported to originate in the medial ganglionic eminence and migrate to the neocortex. in addition, it is reported that they share several glial markers, such as ng2, plp, and cnp at their prematured stages. in order to investigate its nature, we have established a single-cell microarray analysis method. single gfp-positive gabaergic neuron progenitors were corrected from the subventricular zone of the gad67-gfp knock-in mouse neocortex at e18-p0 by dissociation and picking. complemental dna from the single cells was amplified by universal pcr amplification and converted into biotin-labeled crna using t7 rna polymerase. after these procedures, crna sufficient for a microarray analysis was obtained. as the result we found, mbp and s100-␤ expression in the gabaergic neuron progenitors. os2p-3-04 role of ␤-catenin signaling in regulating proliferation of transit-amplifying cells in the adult mouse subventricular zone kazuhide adachi 1,2,3 , masanori sakaguchi 3 , toru yamashita 2,3,6 , yuko fujita 3 , yukiko gotoh 4 , arturo alvarez-buylla 5 , takeshi kawase 1 , hideyuki okano 3 , kazunobu sawamoto 2,3 1 neurosurgery, keio univ. sch. med., tokyo, japan; 2 bridgestone lab. dev. regenerative neurobiol., keio univ. sch. med., tokyo, japan; 3 physiol., keio univ. sch. med., tokyo, japan; 4 inst. mol. cell biosciences, univ. tokyo, tokyo, japan; 5 neurosurgery, ucsf, san francisco, usa; 6 neurol, okayama univ, med, dentistry and pharmaceutical sci, okayama, japan the subventricular zone (svz) continuously produces olfactory bulb neurons in the adult rodent brain. neural stem cells generate migratory neuroblasts via highly proliferative transit-amplifying cells in this region. here, we studied the role of ␤-catenin signaling in the adult mouse svz. ␤-catenin accumulated in the nucleus of only the transitamplifying cells in the svz. activated ␤-catenin signaling promoted the proliferation of transit-amplifying cells, resulting in an increased number of new neurons in the olfactory bulbs. these results suggest that ␤-catenin signaling plays a role in the proliferation of transitamplifying cells in the adult mouse svz. the ciliary marginal zone (cmz) is a region between the neural retina and ciliary epithelium, and contains retinal progenitor cells that give rise to neuron and glia. wnt2b is expressed in cmz, and has been shown to control the differentiation of the retinal progenitor cells. we have isolated a novel bmp antagonist, chick tsukushi (c-tsk), which belongs to the small leucine-rich proteoglycan family. in the eye, the expression of c-tsk is observed in the cmz which is similar with that of wnt2b. to examine the molecular interactions between c-tsk and wnt2b, we co-electroporated them into the optic vesicle at stage 9-10 chick embryo and observed the proliferation of the retinal progenitor cells. we found that c-tsk inhibited the wnt2b activity that sustains prolonged proliferation of retinal progenitor cells. our result suggests that c-tsk controls the proliferation of retinal progenitor cells interacting with wnt2b. to reveal the role of epigenetic gene regulation in neuronal differentiation, we studied subcellular distributions of histone deacetylase (hdac) 9 in developing cortical neurons. an expression vector of gfp-tagged hdac9 was transfected to dissociated cortical cell cultures as well as cortical neurons in vivo. hdac9 was primarily localized in nuclear until 1 week in vitro, but was translocated to cytoplasm in the later stages. such translocation was found in a similar time course after birth in vivo. to examine a possibility that neural activity is involved in the translocation, firing activity of cultured neurons was examined using multi-electrode dishes. as a result, spontaneous firing activity was prominent in the late stages when cytoplasmic translocation occurred. however, ttx addition to the culture medium produced the inverse translocation. these results suggest that activity-dependent intracellular localization of hdac9 contributes to neuronal differentiation in cortical development. research funds: kakenhi (16700286) dept. of physiology, fujita health univ. sch. of med., japan we described electrical synapses in alpha retinal ganglion cells (␣-gcs). precise temporal synchronization of spikes is generated from ␣-gcs (hidaka et al., 2004) . the fraction of open channels in gap junctions were evaluated with techniques of dual patch-clamp, connexin immunocytochemistry, and high-voltage electron microscopy. junction conductance (maximum 2.45 ns) was measured. in high-voltage electron microscopy (hitachi1250m, nips, 2005 , gap junctions (average size 0.86 m long) were present in contacts. in confocal laser-scanning imaging, connexin36 localization at contacts counted gap junctions (seven sites in a pair on average). assuming that the density of connexons would be 5180/m 2 and a single channel conductance is 15 ps, the conductance of each junction would be 45 ns. the presence of seven junctions between a pair will lead to estimate a total junction of 315 ns. the measured conductance could allow to estimate a fraction of open channels as 0.8%. the open fraction is small, when we consider whether electronic transmission acts to synchronize the spikes in the intercellular network. the visual system separates different types of information into parallel, anatomically distinct processing streams. despite their significance for visual processing, the molecular mechanism underlying the physiological stream formation is largely unknown, partially because these physiological streams have not been reported in mice. to identify molecular correlates of functionally distinct streams, we fabricated a custom cdna microarray of higher mammal ferrets. we successfully identified molecules whose unique distribution and developmental profiles define the lgn itself, its constituent layers, or identify cells comprising one of the physiological streams in the lgn. using these molecules as temporal and spatial markers, we investigated mechanisms of the physiological stream formation in the ferret lgn. research funds: kakenhi (17023014), coe research akira muto, herwig baier department of physiology, university of california san francisco (ucsf), usa the visual system operates over a broad range of luminances. this is accomplished by adjustment of photosensitivity, called light adaptation. to study the molecular mechanisms of light adaptation, we screened for zebrafish mutants that showed compromised optokinetic responses (reflexive eye movements to large field motion) after an abrupt dark-to-light transition. in this experimental paradigm, wildtype fish larvae recover their full optokinetic response within about two minutes after being brought back to light. in a screen of almost 2000 genomes, we identified five mutants all of which showed substantially delayed recovery of the okr. positional cloning of one of the loci revealed a mutation in the dna-binding domain of glucocorticoid receptor (gr). gr is known for its role in the stress response, but its function in the visual system is unexplored. we propose that gr is regulating genes essential for light adaptation in the retina. os2p-4-04 multisite recording of the signal propagation pattern in the visual cortex makoto osanai, yusuke takeno, satoshi tanaka, tetsuya yagi graduate school of engineering, osaka university, suita, japan recently, the visual prosthesis systems with implanted stimulus electrode in the visual cortex are developed. but the signal propagation pattern induced by electrical stimuli in the visual cortex is not fully investigated. therefore, we studied the signal propagation pattern induced by electrical stimuli in the mouse visual cortex slice, using a 60 channel multielectrode array and a calcium imaging system. in the electrophysiological study, the responses conducted vertically against the layer of the cortex with layer 4 stimuli and propagated horizontally in the layer 2/3. in the calcium imaging study, the area of the higher calcium concentration region spread vertically with layer 4 stimuli. signal propagation was restricted within several tens m around the stimulus electrode by ap5 + cnqx administration and was completely blocked by ttx administration. administration of bicuculline increased the area of the signal propagation in a dose-dependent manner. we concluded that these restricted patterns of the signal propagation in the visual cortex were due to the inhibitory system. os2p-4-05 presence of two phases in the sensitive period of orientation plasticity shigeru tanaka 1 , toshiki tani 1 , kazunori o'hashi 1,2 , jerome ribot 1 1 brain science institute, riken, saitama, japan; 2 graduate school of life science and systems engineering, kyushu institute of technology, kita-kyushu, japan recently we have revealed that orientation-restricted visual experience induces drastic reorganization of orientation maps in the cat visual cortex. in this study, we examined the effect of release from single orientation exposure on once reorganized orientation maps during the sensitive period using intrinsic signal optical imaging. when kittens were returned to the normal visual environment by removing the goggles after 2 weeks of goggle rearing starting around the age of 3 weeks, the over-representation of the exposed orientation was preserved. on the contrary, when the goggle rearing started around the age of 5 weeks and then the animals were returned to the normal visual environment, orientation maps rapidly changed to represent orientations equally. these findings indicate that the sensitive period of orientation plasticity consists of two phases: orientation map reorganization is irreversible in an early phase and reversible in a late phase. os2p-4-06 residual visuomotor processing in the animal model of blindsight: comparison with normal, near-threshold vision masatoshi yoshida 1,2,3 , tadashi isa 1,2,3 1 dept. dev. physiol., nat'l inst. physiol. sci., okazaki, japan; 2 sch. life sci., grad. univ. adv. stud., hayama, japan; 3 crest, jst, kawaguchi, japan in two macaque monkeys with unilateral v1 lesion performing a visually guided saccade task, saccadic parameters were compared between the saccades to the affected hemifield and those to the intact hemifield. the luminance contrast of the target presented in the intact hemifield was reduced so that the detectability was comparable to that in the affected hemifield (80-90% correct). in the saccades to the affected hemifield, the curvature of the trajectories was smaller and the deviation of the saccadic end points from the target was larger than those to the intact hemifield. these results suggest that without geniculo-striate pathway, online compensation for the variation of the initial saccadic command is not fully functional, thus leading to inaccurate saccades. we propose that the residual visuomotor processing of monkeys with v1 lesion is unlike normal, near-threshold vision. research funds: kakenhi 13854029, kakenhi 16700343 and crest, jst os2p-4-07 comparison of the angle representation in macaque visual areas v1 and v2 minami ito 1,2 , hidehiko komatsu 1,2 1 national institute for physiological sciences, okazaki, japan; 2 the graduate university for advanced studies, hayama, japan previously, we have reported that fairly large number of area v2 neurons has angle selectivity. here, we studied the angle selectivity of area v1, which is the major source of inputs to area v2. we conducted single-unit recordings from the superficial layer of area v1, while animals performed a fixation task. for comparison, we used a similar stimulus set. the stimuli were much larger than the size of the classical receptive fields. area v1 neurons responded mainly to sharp angles (30 • ), straight lines (180 • ) or right corners (90 • ), but not to intermediate angles (60 • or 120 • angle width). this contrasted with area v2, where neurons showed a variety of the optimal angle width including intermediate angles. we also observed several v1 neurons showed fine orientation tuning to short line segments, while weak or no responses were induced by a set of large angle stimuli. we suggest that area v1 neurons largely contribute to representing line components (lines and line-ends) and to sending such information to area v2. os2p-4-08 firing rates and dynamic spatiotemporal patterns of ganglion cells both contribute to retinal information processing xin jin, ying-ying zhang, xue liu, hai-qing gong, pei-ji liang department of biomedical engineering, shanghai jiao tong university, shanghai, china population activities of retinal ganglion cells (rgcs) were recorded using a multi-electrode recording system. single unit analysis showed that firing rate of individual neuron was strongly dependent on the luminance intensity of stimulation. however, population activity of ganglion cells usually showed particular spatiotemporal pattern, in response to a specific velocity of the moving bar. differing from single direction-selective ganglion cell (dsgc), which responds to its preferred direction of movement by firing at its maximal rate, population activity of non-direction-selective ganglion cells may encode the motion information in a temporally ranked manner, independent to their individual firing rates. these results suggest that an efficient and economical coding mechanism may be employed by the retina, where the firing rate of individual neurons and spatiotemporal pattern of population neuronal responses could act in parallel to encode different aspects of visual information. yasuto tanaka, satoru miyauchi, masaya misaki brain information group, nict, kobe, japan visual long-range interaction was reported to be limited in space. here, we show the evidence of long-range interaction extending to an order magnitude larger using the right-left symmetrical configuration. two horizontally collinear gabor signals, one defined as probe and the other cue, were presented at the left and right side of the visual field at mirror symmetrical regions. detection threshold of gs probe reduced with cue-probe separations up to 10 • . the facilitation was highly tuned to the symmetrical locus. furthermore, the facilitation was substantially longer at upper visual field than the lower visual field. the reduction was specific to orientation, phase, and horizontal direction, the results indicate long-range mirror symmetrical interaction across vertical meridian, suggesting symmetrical neuronal communication between early visual cortices. the anisotropy between left-right hemifield (symmetry) and upper-lower hemifield (upper-field advantage) signifies hemifield inhomogenity in human vision. os2p-4-10 integrity of visuospatial attention in a split brain patient noudoost behrad, seyed reza afraz, maryam vaziri, hossein esteky school of cognitive sciences (scs), iran transfer of visual information between hemispheres is severely impaired following transection of posterior part of the corpus callosum. we investigated whether attentive visual object tracking across vertical meridian of the visual field is possible for a posterior callosotomized patient (md). we asked md to track one bouncing ball among four identical distracters while fixating at the center of the screen. target crossed the vertical midline in half of the trials. her performance in crossed conditions was significantly above chance level. also, we asked her to make decision about horizontal alignment of two balls presented simultaneously in one of three conditions: both in right or left hemifield, or each in one hemifield. in this alignment task md was able to compare location of the two bilaterally presented stimuli well above chance level. our data suggest that inter-hemispheric transfer of position information required for spatial attention is preserved without posterior corpus callosum. pei sun, justin l. gardner, mauro costagli, kenichi ueno, r. allen waggoner, keiji tanaka, kang cheng laboratory for cognitive brain mapping, riken brain science institute, wako-shi, japan although the preference for stimulus orientations in human visual cortex has been inferred indirectly in a few studies using fmri, tuning to particular stimulus orientations has not been directly demonstrated using this technique. in an effort towards revealing orientation selectivity and its spatial arrangement in human v1, we have conducted an fmri study with a novel stimulation paradigm and a differential mapping method. we found that responses of the majority of activated voxels were modulated by the grating orientation and individual voxels were sharply tuned to particular orientations. our results provide the first demonstration that orientation selectivity in humans can be directly studied using fmri. os2p-4-12 probing the spatial scale of classifier performance with high spatial resolution fmri justin l. gardner 1,2 , pei sun 2 , keiji tanaka 2 , david j. heeger 1 , kang cheng 2 1 department of psychology and center for neural science, new york university, usa; 2 laboratory for cognitive brain mapping, riken brain science institute, japan recently, classifier analysis with conventional resolution fmri has been used to decode the orientation of a grating stimulus from the fmri responses of early visual cortex. it has been proposed that classifier analysis exploits small but robust orientation biases in voxels that are created by local inhomogeneities in the columnar organization. we have examined this proposal by using classifier analysis to decode stimulus orientation using high spatial resolution fmri (0.75 mm × 0.75 mm × 3 mm voxels) in human v1. we found that many voxels that are weighted heavily in the classifier analysis and carry similar orientation biases closely follow draining veins that are visible on t2*-weighted venograms. we suggest that large draining veins with orientation specific responses, rather than local inhomogeneities in orientation maps, may provide a basis for classifier performance using large voxels. research funds: nrsa fellowship from the nih (1f32ey016260-01) os2p-4-13 relationship between horizontal connections and functional structure in macaque anterior inferotemporal cortex (area te) hisashi tanigawa, kathleen s. rockland, manabu tanifuji riken brain science institute, wako, japan we have studied the relationship between horizontal connections and functional structure in te using a combination of optical imaging, unit recording, and anatomical tracing. intrinsic signal imaging was performed in exposed te, under anesthesia, during presentations of visual object stimuli. this resulted in multiple optical spots evoked by each stimulus. in some animals, subsequently, unit recording was carried out at multiple sites within the imaged region. then, an anterograde tracer was injected into one of the spots. both optical imaging and unit recording revealed regions with stimulus preference similar to that at the injection site. however, these regions and the injection site were not always connected by horizontal axons. some regions sharing a preference to particular stimuli were connected, even though they showed different preferences to the other stimuli. these results suggest that horizontal axons can connect regions with different stimulus preferences in te, in contrast to like-to-like connectivity, as understood in early visual cortices. we recorded single cell responses from the inferotemporal cortex of a fixating monkey while visual stimuli with various durations (18-350 ms; isi = 1 s) were presented. presentation of visual stimuli at all of the tested durations resulted in prolonged responses. the brief presentations evoked multiple phasic responses while the long presentations evoked sustained activities. there was a significant difference in average firing rate of late phase (350-550 ms) of response to optimal stimulus across presentation durations. but no such differences were found for the first phase (70-270 ms). in addition, the optimal stimulus evoked significantly different response magnitudes in the first and second phase particularly in the short presentation durations. but the suboptimal stimulus (∼50% of max response) evoked similar response magnitudes in the first and second phase. these results suggest that stimulus selectivity of inferotemporal cells depends on the stimulus presentation duration and the time window that is used to measure the firing rate. os2p-4-15 the perceptual learning effect in myopes by the lateral masking procedure keiko mizobe 1 , kazuto terai 2 , osamu hieda 3 , shigeru kinoshita 2 1 dept. of ophthal., kyoto second red cross hospital, kyoto, japan; 2 dept. of ophthal., kyoto pref. univ. of med., kyoto, japan; 3 baptist eye clinic hospital, kyoto, japan purpose: the study of the visual cortex revealed the lateral masking collinear configuration modulated the neuronal responses and psychophysical studies also showed perceptual learning improved the visual detection. we asked whether perceptual learning could improve the myopic blurred vision, using the new instrument of the lateral masking technology, neurovision. method: nine low myopes were studied. non-corrected digital visual acuities (va) ranged from 0.4 to 1.2. the logmar average was 0.31. eight sessions of neurovision treatment were performed to each individual. the estimation was done by comparison of va before and after the treatment. results: four eyes showed more than one octave improvement of va. the logmar average of the four was 0.03, improved from 0.50. the residual five eyes showed less or no improvement. the change of logmar average was from 0.15 to 0.08. conclusion: some myopes showed the perceptual learning effects by new treatment, using the lateral masking technology. os2p-5-01 the hindbrain neuroepithelial cells exclude the migrating facial motor neurons by expression of planar cell polarity (pcp) genes hironori wada 1 , hideomi tanaka 1,2 , satomi nakayama 1 , miki iwasaki 1,2 , hitoshi okamoto 1,2 1 laboratory for developmental gene regulation, bsi, riken, japan; 2 crest, jst, japan many neurons migrate tangentially through one cell layer at a specific depth within the brain. in the developing zebrafish hindbrain, the facial (nvii) motor neurons originate in rhombomere (r) 4 and migrate tangentially to r6 near the pial surface of the hindbrain. in this study, we demonstrate that expression of the planar cell polarity (pcp) genes celsr2 and frizzled3a in neuroepithelial cells maintain the nvii motor neurons near the pial surface during their caudal migration in the zebrafish hindbrain. mosaic analyses show that expression of the frizzled3a gene in the surrounding neuroepithelial cells prevented the entry of the nvii motor neurons in the neuroepithelial layer. the demonstration of a role for neuroepithelial cells in excluding differentiated neurons from the neuroepithelial layer may provide new insights into the general mechanisms underlying formation of the layered structures in the mammalian brain, such as in the cerebral cortex. os2p-5-02 disrupted-in-schizophrenia 1 (disc1) regulates the transport of the nudel/lis1 complex to axons via direct interaction with kinesin-1 shinichiro taya, kozo kaibuchi department of cell pharmacology, nagoya university, nagoya, japan disc1 is a candidate gene for susceptibility to schizophrenia. in a scottish family, the chromosome translocation interrupts the coding sequence of disc1. disc1 is reported to interact with nudel, which forms a complex with lis1. although the functional significance of this complex in axon growth and neuronal development has been reported, the transport mechanism of the complex into axons and the functions of disc1 remain largely unknown. here we report that disc1 interacted with kinesin-1, a motor protein of anterograde axonal transport. kinesin-1 interacted with the nudel/lis1 complex through disc1, and these molecules accumulated at the distal part of axons. the knockdown of disc1 by rnai of disc1 induced the delocalization of nudel and lis1 from the axons and reduced axonal growth. the knockdown of kinesin-1 induced the delocalization of disc1 from the axons. taken together, these results indicate that disc1 links kinesin-1 to the nudel/lis1 complex and regulates its transport as a cargo receptor for axon elongation. research funds: mext os2p-5-03 role of a novel collapsin response mediator protein-2 interacting molecule, synaptotagmin-like protein in hippocampal neuron nariko arimura, saeko kawabata, atsushi hattori, kozo kaibuchi department of cell pharmacology, graduate school of medicine, nagoya university, nagoya, japan during the development, neurons recognize the extracellular signals and extend the axons to proper directions. certain kinds of receptors are transported from the nerve cell body to the axon terminal, and participate in the recognition of extracellular environments. however, the mechanism of controlled recruitment of receptors remains unsolved. here, we report that synaptotagmin-like protein 1 (slp1) can mediate the vesicle transport. slp1 is known to associate with rab27. we found that slp1 associates with collapsin response mediator protein-2 (crmp-2), which is a key regulator of axon formation. slp1 could form the trimeric complex with rab27b and crmp-2, and also associate with kinesin-1 through crmp-2. slp1 is accumulated on microtubules at the axonal growth cones, and is co-localized with a receptor of growth factor. these findings suggest that slp1 functions as a mediator of recruitment of certain receptor depending on crmp-2 and kinesin-1. os2p-5-04 absolute quantification of mdr1a, mrp1, mrp4 and bcrp proteins at the mouse brain blood barrier by lc-ms/ms junichi kamiie 1,2 , yuki katsukura 1,2 , sumio ohtsuki 1,2 , xiao-kun cai 1,2 , tetsuya terasaki 1,2 1 graduate school of pharmaceutical sciences, tohoku university, sendai, japan; 2 sorst, jst, japan the abc transporter proteins are thought to limit permeability across the blood-brain barrier as the efflux transporters. however, contribution of each transporter to the bbb function is not clarified. the purpose of this study was to clarify the protein amounts of mdr1a, mrp1, mrp4, and bcrp in the brain capillaries of mouse by newly developed membrane protein quantification method using lc-ms/ms. by this method, the standard curve showed linearity between 10 and 1000 fmol, and amino acid sequence of the detected fragment was confirmed by ms/ms spectrum. in the brain capillaries, the protein amounts of mdr1a, mrp4, bcrp were 3.9, 2.7 and 4.8 fmol/g, respectively, while it of mrp1 was under detection limit of standard curve. this quantitative profile suggests that mrp4 and bcrp function as the efflux transporter at mouse blood-brain barrier as well as mdr1a. os2p-5-05 dominant expression of claudin-5 in highly purified brain capillary endothelial cells sumio ohtsuki 1,2 , hirofumi yamaguchi 1 , saori sato 1 , tmoko asashima 1,2 , tetsuya terasaki 1,2 1 graduate school of pharmaceutical sciences, tohoku university, sendai, japan; 2 sorst, jst, japan claudins are major constituents of tight junctions (tjs). the purpose of this study was to clarify the expression levels of each claudin subtype in brain capillary endothelial cells (bcecs), which form the blood-brain barrier. mouse bcecs were highly purified using endothelial surface antigen (pecam-1) and magnetic cell sorting. mrna expression of caludin-1-23 was measured by real-time rt-pcr. claudin-5 showed the highest mrna expression in the purified mouse bcecs. mrna levels of claudin-1 and -12 were 0.0027% and 0.20% of that of claudin-5. claudin-5 mrna was concentrated in the purified bcecs, while claudin-1 and -12 mrna in the purified bcecs were lower than that in the whole brain. rat claudin-5 mrna was also concentrated in rat brain capillary fraction, but claudin-12 mrna did not. these results suggest that claudin-5 is a dominant tjs protein in bcecs, and expression of claudin-1 and -12, which was reported as tj protein in bcecs, are not restricted in bcecs. os2p-5-06 effects of hydrogen peroxide towards gap junction communication in astrocytes and permeability of blood brain barrier f. ahmad 1 , a. pauzi m. yusof 1 , p.d. mourad 2 , m. bainbridge 3 , s. ab ghani 1 1 universiti sains malaysia; 2 university of washington, seattle, usa; 3 brody school of medicine, east carolina university, nc, usa h 2 o 2 is the main peroxides produced in mammalian cells that consume o 2 . the main source of h 2 o 2 in the brain, produced in large amount, was from the superoxide dismutase catalyzed reaction in mitochondria. therefore, we look into the effects of h 2 o 2 towards the gap junction communication in astrocytes and permeability of blood brain barrier. in this study, by using a h 2 o 2 microsensor, we investigated the level of h 2 o 2 in the brain that altered the permeability of bbb. the microsensor was implanted in the rat's brain and operated amperometrically. we measured h 2 o 2 level from the current generated by the electron transfer at the electrode. we observed a change in permeability when external h 2 o 2 was injected into the brain. fatality occurs when the injected h 2 o 2 exceeds 250 m. these finding showed that the altered paracellular permeability in the presence of h 2 o 2 is caused by a series of events that happen one after another. research funds: short term grants 304pkimia633134 and 304pfar-masi670008 os2p-6-01 somato-ovarian sympathetic reflex discharges in anesthetized rats sae uchida, fusako kagitani, harumi hotta dept. auton. nerv. syst., tokyo metropol. inst. gerontol., tokyo, japan ovarian sympathetic efferent reflex discharges caused by single electrical shock stimulation of spinal (t9-11) afferent nerves or limb (tibial) afferent nerves were studied in urethane anesthetized rats. in central nervous system (cns) intact rats, stimulation of the t9-11 spinal afferent nerve produced early and late a-reflex discharges, and a late c-reflex discharge. after spinalization at the third thoracic level, stimulation of the same spinal afferent nerve produced an a-reflex with the same latency as the early a-reflex in cns-intact rats and an early c-reflex discharge with the similar latency as the late a-reflex in cns-intact rats. on the other hand, stimulation of the tibial afferent nerve produced late a-reflex and c-reflex discharges in cns intact rats, those were not observed after spinalization. it was concluded that ovarian sympathetic aand c-reflex discharges evoked by stimulation of a segmental spinal afferent nerve in cns-intact rats are of spinal and supraspinal origin, and those evoked by tibial nerve stimulation are of supraspinal origin. os2p-6-02 responses of renal sympathetic nerve activity and sodium excretion to 3 days sodium loading in rats misa yoshimoto, nozomi iinuma, rie itokawa, eri hayashi, kenju miki integrative physiol. grad. sch. humanities and sci. nara-women's univ., nara, japan in the present study, a month recording of renal sympathetic nerve activity (rsna) in freely moving rats was made to explore the long-term regulation of rsna and sodium excretion. wistar male rats were instrumented chronically with electrodes for the measurements of rsna and electrocardiogram. after the 7 days recovery period, rsna, heart rate and sodium balance were measured over three weeks. animals were allowed to drink four different concentration of sodium chloride solutions (0, 50, 154, 308 meq./l nacl) over 3 days. the sodium loading with 308 meq./l nacl suppressed rsna significantly and then it gradually recovered while either 0 meq./l nacl or 154 meq./l nacl loading had no effects on rsna. sodium excretion changed significantly in proportion to the each sodium loading levels. these results indicated that the changes in rsna were not always correlated with the changes in sodium excretion in rats. os2p-6-03 cross correlation analysis of respiratoryrelated optical imaging signals yoshitaka oku 1 , haruko masumiya 1 , yasumasa okada 2 1 dept. physiol., hyogo col. med., nishinomiya, japan; 2 dept. med. keio univ. tsukigase rehab. ctr. shizuoka, japan we aimed to establish an objective method to identify the distribution of respiratory-related regions and the timing when these regions are activated relative to the inspiratory activity from optical imaging signals. optical signals were recorded from the ventral medullary surface of neonatal rats in vitro using a voltage-sensitive dye. cross correlation between integrated c4 ventral root (c4vr) activity and each pixel was calculated after cycle-triggered averaging and detrending. the maximum of cross correlation coefficients and the lag at which the cross correlation became maximal (lagmax) were displayed as 3d pseudocolor maps. in all preparations, two respiratory-related regions were consistently identified: (1) a continuous column extending from the para-facial region to the pre-bötzinger complex, and (2) a region corresponding to the ventral horn. pixels where lagmax were negative (meaning that the activity preceded the c4vr activity) tended to be distributed in the para-facial region, and this tendency was more evident when superfusate ph was lowered. os2p-6-04 slow afterhyperpolarization determines the firing pattern of action potentials in rat gnrh neurons masakatsu kato, yasuo sakuma department of physiology, nippon medical school, tokyo, japan gonadotropin-releasing hormone (gnrh) neurons play a pivotal role in the hypothalamo-pituitary-gonadal axis. gnrh neurons must be able to continuously fire in response to depolarizing stimuli. for this type of firing, gnrh neurons may have a certain intrinsic property. to address this issue, we investigated the ca 2+ -activated voltage-independent k + currents underlying afterhyperpolarization. dispersed gnrh neurons from adult gnrh-egfp transgenic rats were cultured overnight and used for an electrophysiological experiment with perforated patch clamp configuration. the gnrh neurons showed a slow afterhyperpolarization current (i sahp ). in contrast to previous reports, the i sahp observed in rat gnrh neurons was potently blocked by an sk channel blocker apamin. in current clamp condition, gnrh neurons evoked a train of action potentials to depolarizing current pulse. apamin increased the susceptibility to spike failure. the results indicate that rat gnrh neurons exhibit an apaminsensitive i sahp , which regulates the firing pattern. research funds: kakenhi 16590180, 16086210 os2p-6-05 the effect of music to sex hormones of elderly person hajime fukui 1 , kumiko toyoshima 2 , kiyoto kuda 1 , katsuhiko iguchi 3 1 nara univ. of edu., nara, japan; 2 grad. school of human sciences, osaka univ. japan; 3 nara city medical clinic, japan it has been known that testosterone or estrogen protects nervous system and regulates cell death in a brain. also, it is pointed out that the decline of t and est accelerates depression. therefore the treatment such as hormone replacement therapy (hrt) has been tried to cure depression and alzheimer's disease. however, it has been pointed out that hrt has serious side effects. on the other hand, there are reports that music influences on a steroid hormone. in addition, it is known that music has certain therapeutic gain toward ad and dementia. in this study, from a point of view of the prevention of ad and dementia, we examined the effect of music to sex hormones of normal elderly person. four males and 36 females participated music session and t and est were evaluated. as a result, in female, in the high hormone group, the values decreased after the session, and in the low hormone group, the values increased. from above, there might be possibility that through a steroid hormone music participates in protection and improvement of function on brain. tuberculous meningitis (tbm) is the most common form of chronic infection of the central nervous system. despite the magnitude of the problem, the general diagnostic outlook is discouraging. this study identifies a specific protein marker in csf, which will be useful in early diagnosis of tbm. we have demonstrated the presence of a 30-kda protein band in csf of 100% (n = 5) of confirmed and 90% (n = 138) of suspected tbm patients out of 153 tbm patients. the 30-kda protein band was analyzed by lc-ms/ms analysis. in the present study we have identified two mycobacterial proteins rv3804c (ag85a) and rv1886c (ag 85b) and one host derived protein as the components of the tbm specific 30-kda protein. involvement of mitochondrial extrinsic and intrinsic apoptotic pathways in dopaminergic neurodegeneration was tested in rotenone-and mpp + -induced rat models of parkinsonǐs disease (pd). hplc-ec, patch clamp, fluorimetry, immunoblot and rt-pcr were used for measuring neurotransmitters/free radicals, membrane currents, caspases activities, levels of proteins and mrna of mitochondria-linked signaling in brain. we report here a retrograde mode of neuronal death via mitochondrial intrinsic pathway in mpp + -, but an extrinsic mode of cell death in rotenone-induced model. drug screening in these models (l-deprenyl as positive control) indicated that quercetin, coenzyme q 10 , vitamin d 3 and melatonin act via interfering the signaling events in neurons. loss of complex-i and -iv activities and changes in some of the protein subunits in pd postmortem brains were confirmed in pd and control cybrids. results from the present study provide evidences for a direct involvement of mitochondria and are suggestive of existence of both intrinsic and extrinsic apoptotic pathways in dopaminergic neuronal death. os2p-7-02 involvement of thioredoxin on the neuroprotective effect of (−)-deprenyl tsugunobu andoh 1 , boon chock 2 , dennis l. murphy 3 , chuang c. chiueh 4 1 dept. applied pharmacol., univ. toayama, toyama, japan; 2 lab. bioch., nhlbi, nih, md, usa; 3 lab. clin. sci., nimh, nih, md, usa; 4 cent, brain diseases and aging, taipei med. univ., taipei, taiwan the present study investigated whether the induction of thioredoxin (trx) involves in the cytoprotective mechanisms of (−)-deprenyl which is known as the inhibitor of mao-b. after confirming (−)-deprenyl protects against mpp + -induced cytotoxicity in human sh-sy5y cells, we observed further that (−)-deprenyl induced trx for protection against oxidative injury caused by mpp+. the induction of trx was blocked by pka inhibitor through a pka-sensitive phosphoactivation of map kinase erk1/2 and the transcription factor c-myc. (−)-deprenyl-induced trx and associated neuroprotection were concomitantly blocked by the antisense against trx mrna in human sh-sy5y cells. consistently, trx increased the expression of mnsod and bcl-2 supporting cell survival. in conclusion, (−)-deprenyl augments the gene induction of trx leading to elevated expression of antioxidative mnsod and antiapoptotic bcl-2 proteins for protecting against mpp + -induced neurotoxicity. os2p-7-03 pgd 2 induces neuronal apoptosis via 15d-12,14 -pgj 2 tatsurou yagami 1 , noboru okamura 2 , toshiyuki sakaeda 2 1 facul. health care sci., himeji dokkyo univ., himeji, japan; 2 kobe univ. grad. sch. med., japan prostaglandin d 2 (pgd 2 ) is abundant in the brain, but its neuropathologic role has been unclear. here, we found that pgd 2 induced neuronal apoptosis in rat cortical cultures. however, a pgd 2 receptor blocker did not suppress neurotoxicity of pgd 2 . little pgd 2 receptor was detected, suggesting an involvement of pgd 2 metabolites in the apoptosis. among pgd 2 metabolites, 15-deoxy-12,14 -prostaglandin j 2 (15d-12,14 -pgj 2 ) caused neuronal apoptosis most potently and rapidly. although 15d-12,14 -pgj 2 is an endogenous ligand for peroxysome proliferator-activated receptor ␥ (ppar␥), ppar␥ activators did not kill neurons, suggesting that 15d-12,14 -pgj 2 induces apoptosis independently of ppar␥ activation. we found specific binding sites of [ 3 h]15d-12,14 -pgj 2 (jbs) in plasma membranes. there was a close correlation between the neurotoxicity of various eicosanoids and their affinity for jbs. in conclusion, we demonstrated that pgd 2 induced apoptosis via 15d-12,14 -pgj 2 in rat cortical neurons, and suggested that jbs in the plasma membrane was involved in the 15d-12,14 -pgj 2 -induced apoptosis. yoshiki iwamoto, daisuke umetsu, shigeru ozaki, naohito terui department of physiology, university of tsukuba, tsukuba, japan stability of a driver's head is crucial for clear vision and consistent, smooth operation of a vehicle. we reported last year that bilateral sternocleidomastoid muscles (scm) of drivers showed a symmetrical increase in activity during forward acceleration of a vehicle. in the present study, we analyzed the relationship between scm activity and vehicle acceleration. emgs of the right and left scm of drivers were recorded during rapid forward acceleration. the time course of the rectified, smoothed emgs did not match that of vehicle acceleration. for a given acceleration, emg was larger when acceleration was increasing than when it was decreasing. we compared emgs and a linear sum of acceleration and its time derivative, jerk. with optimal weights for the two variables and a proper time lag, the linear sum reproduced the emg profile. the optimal weight and lag varied across subjects and vehicles. we suggest that the jerk-related muscle activity may be necessary to quickly restore proper head position after sudden acceleration. grasping is a highly developed movement in primate including human. in contrast to the well-known involvement of cerebral cortex, role of spinal neurons in controlling this behavior has never been examined. here, we show the first direct evidence suggesting the significant contribution of spinal neurons. we trained japanese monkeys to perform the precision grip task, pinching the two springloaded levers with their index finger and thumb, and recorded neural activities through an oval recording chamber implanted over the cervical spinal cord (c6 to t1). majority of the recorded neurons showed movement-related modulation of firing rate, and the modulation sometimes started before movement onset. spike-triggered averaging of muscle activities revealed some neurons had post-spike effects to hand muscles, suggesting that spinal neurons were capable to generate and modulate muscle force during precision grip. we suggest that primate spinal neurons have a significant role in preparation and execution of grasping movement. research funds: kakenhi 17022043 os2p-7-06 compartmentalization of the cerebellar nuclei: aldolase c expression and the olivonuclear projection pattern izumi sugihara, yoshikazu shinoda dept. systems neurophysiol., tokyo med. & dental univ., tokyo, japan the cerebellar cortex is compartmentalized into more than 20 longitudinal stripes by the aldolase c (=zebrin) expression pattern, which is tightly correlated with the topographic olivocortical projection. however, no equivalent compartmentalization has been known in the cerebellar nuclei. we mapped aldolase c labeling of terminals of purkinje cell axons and anterograde labeling of collaterals of olivocerebellar axons in the rat cerebellar nuclei. the cerebellar nuclei were divided into the caudoventral aldolase c-positive and rostrodorsal negative parts, indicating purkinje cells in the positive and negative stripes in the cortex project to the caudoventral and rostrodorsal parts in the nuclei, respectively. olivonuclear projections showed clear topography within these parts, which was completely congruent with the olivocortical topography. these results clarified the compartmentalization of the cerebellar nuclei and supported that the aldolase c expression is tightly related with the functional organization of the cerebellum. we examined a context dependency of neuronal activity of the pedunculopontine tegmental nucleus (pptn) in monkeys during visually guided saccade tasks. about half of movement-related activities occurred for only the saccades to the saccade target in the task, but they did not occur for the saccades outside the task. on the other hand, for the other half of neurons, movement-related activities occurred for every saccade regardless of the task condition. for visual responses, some neurons responded either the initial fixation point or saccade target, and others responded equally to both stimuli. we further analyzed mutual relationship among modulation timing, preferred direction, effect of reward expectation and this context dependency of the activities, and discussed the visuo-motor processing of pptn. in the reinforcement learning theory, the midbrain dopamine (da) neurons send reward prediction error signal to the striatum. the cholinergic pedunculopontine tegmental nucleus (pptn) is one of the strongest excitatory input sources to da neurons. we hypothesized that pptn may play an important role for relaying necessary components of reward prediction error signals to da neurons. during recording of pptn neurons, we utilized reward predictable visually guided saccade tasks where a shape of fixation point indicated a reward volume. for more than half of the neurons, which showed cue related responses, the cue responses were dependent on association of cue feature and reward size. from another population, we recorded reward related activity. in conclusion, pptn neurons may relay both reward and reward prediction signals, sufficient for computation of reward prediction error. research funds: kakenhi (17022027) os2p-7-09 timing activity in supplementary eye field during a saccadic eye movement task shogo ohmae 1 , xiaofeng lu 1,2 , yusuke uchida 1 , toshimitsu takahashi 1,2 , shigeru kitazawa 1,2 1 dept. of neurophysiol., juntendo univ. grad. sch. of med., tokyo, japan; 2 crest, jst, tokyo, japan to act properly in our daily life, the ability to detect and predict timing of events is always required. how do we deal with timing in the brain? to address this question, we trained two japanese monkeys to perform a visually guided saccadic eye movement task in which the monkeys made saccades to each of 16 targets following a gosignal given at a random timing between 500 and 800 ms after the appearance of the target. we recorded neuronal activity from the supplementary eye field (sef) during the task. we found a group of cells that showed activity related to the length of the delay period from target-on to the go-signal. these cells were classified into two types: (1) those that showed buildup activity during the delay period until the go-signal, and (2) those that displayed changed activity after the go-signal in relation to the length of the delay period. the results suggest that sef is involved in timing the onset of the go-signal during the saccadic eye movement task. in reaching, a spatial visuomotor transformation should occur in our brain. we can make the transformation not only when the relationship between visual and motor coordinates is default, but also when a gain for the relationship is changed, for example, in a microsurgery. we trained monkeys to make reaching movements when visuospatially identical targets were presented on a computer display by aligning a cursor that indicated their hand position, while the gain was systematically changed. we recorded and analyzed movement-related neuronal activity in the ventral premotor cortex (pmv) and the primary motor cortex (mi) during reaction time. it was revealed that a majority of the mi neurons and a part of the pmv neurons showed activity changes depending on executed movement direction, amplitude, and velocity, whereas a number of the pmv neurons exhibited activity consistent to the visual location of the targets, but not to motor parameters such as amplitude and velocity. the results indicate that the pmv contributes to gain control of reaching during visuomotor transformation. local oscillatory changes in the human sensorimotor cortex induced by simple motor tasks were investigated using supragyral and intrasulcal surface electrodes which was temporarily implanted for the treatment of intractable deafferentation pain. time frequency spectrogram and coherence between electrodes revealed that, before and after several hundred milliseconds of the motor execution, the coherence in the premotor cortex increased cooperatively between neighboring electrodes but that the coherence in the intrasulcal primary sensorimotor cortex decreased exclusively. this result reflects that the premotor cortex plays a role in motor planning with diffuse network while the primary motor cortex plays a role in selective motor execution with local motor output unit. the human sensorimotor processing may be hierarchical and similar to an artificial neural computer. we have shown that the trigeminal oral nucleus (vor) neurons with the receptive field in the intraoral structures project bilaterally to either the jaw-closing (jc) or jaw-opening (jo) motor nucleus in the cat. it is known that neurons in the somatosensory cortex project to the trigeminal sensory nuclei in the rat. thus, we conducted this study to reveal whether there are vor neurons that receive cortical projections and project to the jc or jo nucleus in the rat. we injected a retrograde tracer, fluorogold (fg), in the vor, and found many retrogradely labeled neurons in the contralateral rostral primary somatosensory cortex (si). thus, we injected an anterograde tracer, biotinylated dextranamine (bda), in the rostral si, and also fg in the jc or jo nucleus in the same animals. we found a considerable number of fg-labeled vor neurons made contact with bda-labeled axon terminals. these results suggest that si neurons control jawreflexes through vor neurons. tsunehiko kohashi, yoichi oda grad. sch. science, nagoya univ., nagoya, japan the mauthner (m) cells, paired large reticulospinal neurons in teleost hindbrain, are known to initiate fast escape from sudden aversive stimuli. to investigate how the fast escape is established during early developmental stages, we examined motor performance of the escape in zebrafish embryos or larvae, and the contribution of mcell activity on the behavior. the rostral portion of the zebrafish, 30-200 h post fertilization (hpf), was embedded in agar and the tail flip in response to water pulse applied to the head was examined. thirty hpf embryos, in which m-cell has already received trigeminal nerve innervation and is still extending its axon in the spinal cord, showed tail flips contralateral to the stimulated side with longer latency (>9 ms) than larvae (>80 hpf, 3 ms). m-cell activity monitored with confocal ca 2+ imaging during the tail flip (>80 hpf) tightly correlated with the initiation of fast escape, whereas delayed escapes without m-cell firing appeared in some cases (<25%) after 100 hpf. thus, the development of the escape behavior coincided with that of m-cell circuit. junctophilins (jps) expressed in the er/sr interacts with plasma membrane thereby constructing junctional membrane complexes (jmc). we here report that lacking neural jps subtypes exhibit an irregular hindlimb reflex and impaired memory. to define neural mechanism of memory deficit in jp-dko mice, we performed whole-cell patch clamp recording of hippocampal neurons. in wild mice, an obvious afterhyperpolarization (ahp) was observed and its ahp was totally blocked by apamin. by contrast, ahp was absent in the jp-dko mice and was insensitive to apamin treatment. the er ca 2+ release through ryanodine receptors, triggered by glutamate receptor-mediated ca 2+ influx, is essential for the activation of sk channels toward ahp generation in the hippocampal neurons. therefore, jp-mediated jmc formation likely plays an essential role in neural excitability underlying neural plasticity and memory. os2p-8-02 distribution of voltage-gated calcium channel ␣ 2 ␦-4 mrna in mouse central nervous system takeshi houtani, satoru sakuma, masahiko kase, tetsuo sugimoto department of anatomy and brain science, kansai medical university, moriguchi, osaka 570-8506, japan the ␣ 2 ␦ subunits are the auxiliary subunit of voltage-gated calcium channels and modulate the biophysical properties of the pore-forming ␣ 1 subunits. these auxiliary subunits are composed of four genetically different molecules, ␣ 2 ␦-1 to ␣ 2 ␦-4. the distributions of ␣ 2 ␦-1, -2, -3 mrna have been intensively investigated in the rat central nervous system by in situ hybridization, but that of ␣ 2 ␦-4 remains to be determined. we cloned ␣ 2 ␦-4 cdna fragment from mouse brain by rt-pcr and examined the distribution of ␣ 2 ␦-4 mrna-expressing cells in the mouse central nervous system by in situ hybridization using digoxigenin-labeled crna probe. while the ␣ 2 ␦-4 mrna was found to be broadly expressed, some neuronal types or sites such as piriform cortex, hippocampal pyramidal cells, paraventricular hypothalamic nucleus, facial nucleus and motor neurons of the ventral horn had intense mrna expression. our results suggest that ␣ 2 ␦-4 subunit may play an important role in learning and memory, neuroendocrine secretion and somatic motor control. the mushroom bodies of insect brains are essential in associative olfactory learning. here we show that the drosophila larval mushroom body calyx, the dendritic region, is organized in about 34 glomeruli, which we have mapped. individual glomeruli receive specific innervation from second order olfactory neurons. by contrast, they contain dendrites from hundreds of mushroom body neurons (kenyon cells), which show low specificity for individual glomeruli. glomeruli therefore potentially transmit specific sensory inputs to a large fraction of kenyon cells. quantitative analysis of dendritic termini of single larval-born kenyon cells suggests that they arborize in about 6 glomeruli in an apparently random manner. this pattern of connectivity is consistent with a model in which kenyon cell dendrites process olfactory input by a combinatorial mechanism that allows the discrimination of a large number of odors. withdrawn os2p-8-05 hypothalamic defense reaction involves purkinje cells in the flocculus folium p via orexin and gaba in anesthetized rabbits, electric stimulation in the hypothalamic defense area either excited or inhibited "simple spike" discharges in purkinje cells located in folium p of the flocculus. iontophoretic application of an orexin antagonist (sb334867) depressed the excitation, while bicuculline depressed the inhibition. h 1 or h 2 histamine antagonist had no effect. labeling orexin fibers by immunocytochemistry showed that they were most numerous in folium p as compared with other folia of the flocculus. stimulation of the hypothalamic defense area produced little field potentials in the folium p unlike those evoked by mossy fibers. these observations suggest that the excitation and inhibition are mediated by orexin-containing fibers, which contact purkinje cells directly and also indirectly via other gabaergic neurons. os3a-5-01 activity-dependent development of corticispinal synapse in mouse slice co-culture takae ohno, masaki sakurai dept. physiol., teikyo univ. sch. med., tokyo, japan we showed nmda-dependent synapse elimination of corticospinal (cs) tract in vitro in rat. in order to use the genetically modified mice to study the underlying molecular mechanisms of this developmental plasticity, we studied development of cs synapses in c57 bl/6 mice. by recording field epsp (fepsp) along 80 m interval lattice in the spinal gray matter in response to the stimulation of deep cortical layer, we evaluated spatial distribution of synapse formation quantitatively. fepsps were recorded diffusely throughout the spinal gray matter at 7-8 div, then the amplitudes of fepsps in the ventral side began to decrease at 9-10 div, and dominated in the dorsal area at 14 div. cs axon terminals labeled anterogradely with biocytin distributed diffusely throughout the spinal gray matter at 7-9 div but the axons terminals in the ventral area were eliminated until 14 div. this synapse elimination from the ventral side was blocked by apv application from 6 div, indicating that this process is also nmda-dependent. in slice coculture study, we showed that corticospinal (cs) axons grow rapidly and reach the ventral spinal gray until 7 div. the number of those ventral axons is reduced before 14 div. to study the behavior of the cs axons at the single axonal level, we transfected a small number of cortical neurons with eyfp expression vector pcag-eyfp by way of electroporation to visualize them and took the time-lapse images of their axons under the confocal microscope equipped with an on-stage co2 incubator. some axons showed rapid growth, reaching the ventral most part of the spinal gray matter already at 6 div. some axons had collaterals at the dorsal part and retracted the ventral branch while extending the dorsal branch during 7-11 div. some ventral axons showed a fragmented tip during retraction, which was indicative of axonal pruning. these observations provide direct evidence that there are early cs axons that once reach the ventral spinal gray and then retract to stay dorsally. we identified click-iii/camki␥ as a novel brain-enriched isoform of the camk-i family that was lipid-anchored by multiple lipid modifications, prenylation and palmitoylation, resulting in enrichment of click-iii into lipid rafts fractions. in situ hybridization revealed the abundant presence of click-iii transcript throughout the central nervous system in mouse embryos. to test the role of click-iii during early neuritogenesis, a shrna vector specific for click-iii was delivered into dissociated cortical culture. we found that knock-down of click-iii resulted in significant decrease in the number and total length of dendrites. results from introduction of click-iii into a click-iii-null context confirmed this finding. surprisingly, lipid modifications of click-iii seemed to contribute to fully elicit such an effect. we thus uncovered a novel signaling mechanism by which lipid raft insertion and local activation of a camk can be efficiently coupled to actin cytoskeletal signaling during dendritogenesis. os3a-5-06 transcription factor control of dendrite arbor ultrastructure adrian moore, reiko amikura, shiho nakao, andrew liu, emi kinameri riken brain science institute, japan the different functions of neurons in a complex nervous system are reflected in a large diversity of dendrite arbor morphologies. the drosophila larva dendritic arbourization (da) neurons consist of four classes (i-iv) with increasing levels of arbor complexity. these diverse arbor shapes develop due to class specific mechanisms of dendrite branching and outgrowth. here we show that these class specific differences in dendrite arbor morphology are controlled by a combinatorial code of transcription factors. we have developed a system to label individual dendrite arbors then subsequently identify them in electron microscopic sections. using this method we illustrate that the dendrites of class i neurons, with a simple arbor, contain a high density parallel array of microtubules; on the other hand class iv neurons, with a complex arbor, contain a low density meshwork of microtubules. we are presently investigating how these differences in ultrastructure are controlled by the transcription factors making up the combinatorial code. os3a-5-07 segmental and hox related cues are involved in the establishment of the somatotopy yasunori murakami igbmc, strasbourg, france in the rodent, trigeminal sensory inputs are topographically relayed, and mapped in the somatosensory cortex. little is known about the mechanism underlying the development of the somatotopic organization. by fate mapping of specific rhombomeres (r), we found that principal sensory (prv) neurons derived from r3 receive predominantly inputs from the maxillary branch of the trigeminal nerve and uniquely contribute to the whisker map. by conditional inactivation, we found that early expression of hoxa2 in r2 is required for pathfinding and positioning of trigeminal nerve afferents. at later stages, hoxa2 expression in prv neurons provides instructive cues for topographic arborization of maxillary axons. moreover, while prv neurons appeared normally specified, loss of hoxa2 function resulted in selective loss of eph4 expression, and altered axonal projections from prv to the ventral posterior medial (vpm) nucleus of the thalamus, and absence of a postnatal whisker map at any level of the neuraxis. thus, hoxa2 dependent cues are required to determine the territory for whisker representation in r3 and the assembly of a somatosensory circuit. os3a-5-08 the second wave of corticospinal innervation after synapse elimination of the first wave tsutomu kamiyama, masaki sakurai dept. physiol, teikyo univ. sch. med., tokyo, japan in the previous study we showed that the rat corticospinal (cs) terminals and synapses were widely distributed at p7 and those in the venrtolateral (vl) area were eliminated from p8 to p10 and that the number of terminals in the dorsomedial (dm) and vl area began to increase again from p12 and further increased thereafter. in the present study we further studied the subsequent developmental time course of cs terminal distribution. cs axons were anterogradely labeled by injection of biotin dextrane (bda) into the sensorimotor cortex. the number of the terminals began to increase from p12, reaching peak around the third postnatal week. labeling of single or a few by microinjection axons revealed that at p14 some additional cs axon branches appeared within the dorsal column of the target spinal segment and further ramified after entering the gray matter. however, the number of axons did not increase in the brainstem and the upper cervical cord. these suggest that the second wave of innervation is explained mainly by branching of cs axons just before and after entering the spinal gray matter. os3a-5-09 proteomics of the growth cone: i. protein profiling of the growth cone the growth cone is a motile tip formed at the developing neuronal processes, and functions for the accurate determination of the axon pathway and the synaptogenesis. in higher organisms, however, the molecular basis of the growth cone is poorly understood for the present, since the information on the protein localization there is insufficient to explain the growth cone functions. proteomics is a powerful strategy for identifying the protein composition in a given cell or a subcellular compartment, and the application of this method to the growth cone should help us solve the above question. we obtained the whole growth cone (gcp) obtained from neonatal rat forebrain and the membrane subfraction of the gcp (gcm), and then those fractions were analyzed using proteomics. we have identified several hundreds of the distinct proteins of these fractions. here, we show the profiling of gcp and gcm, and will discuss the overview of these protein profiles in relation to the growth cone functions. axonal branching is thought to be regulated by not only genetically specified molecules but also neuronal activity. however, the interplay between these two mechanisms remains largely unknown. to study this issue, we analyzed the role of electrical activity in layer-specific thalamocortical (tc) axon branching by using organotypic cocultures. during the second week in vitro, yellow fluorescent protein-labeled tc axons formed branches primarily in the target layer. spontaneous firing was found to increase when branches were formed abundantly. pharmacological blockade of synaptic transmission diminished layer-specific branching considerably. moreover, time-lapse imaging showed that branching was generated dynamically by elimination as well as addition in the target layer and that blockade of synaptic activity reduced this remodeling. these findings suggest that synaptic activity modifies layer-specific tc axon branching by regulating the remodeling process with molecular cues expressed in the target layer. research funds: kakenhi (17023030), kakenhi (15300107) os3a-6-01 the application of navigation-guided repetitive transcranial magnetic stimulation for intractable deafferentation pain naoki tani, yoichi saitoh, haruhiko k., satoru oshino, masayuki hirata, amami katoh, toshiki yoshimine 1 department of physiology, university of osaka, osaka, japan repetitive transcranial magnetic stimulation (rtms) has been applied to control intractable deafferentation pain (dp). but nobody has investigated which cortical area is the most effective target for pain relief. therefore, we stimulated m1, s1, sma, premotor accurately with a navigation-guided rtms and compared their effects of pain relief. at the same time, rtms (1, 5, 10 hz, 500 stimulations) was compared in 20 dp patients. the pain relief was evaluated with visual analogue scale. high frequency (5, 10 hz) rtms of m1 was the only effective stimulation for treating intractable pain in 10 of 20 patients (50%). the pain relief continued for 3 h significantly. we would like to discuss the mechanism of pain relief with high frequency rtms of m1. os3a-6-02 involvement of atp on nociceptive modulation in rat model of masseter muscle pain yasuo sugiura, noriyuki ozaki, masamichi shinoda department of functional anatomy and neuroscience, nagoya university graduate school of medicine, nagoya, japan we determined the role of p2x 3 r on pressure pain and mechanical hyperalgesia in a newly developed rat model of pain in masseter muscle (mm) . the pain in the mm was assessed by the pressure pain threshold (ppt) defined as the amount of pressure required to induce head flinching. the mm injection of ␣,␤-meatp (p2x 1,3,2/3 rspecific agonist) significantly enhanced the behavioral response to the pressure. this enhanced response was completely blocked by the co-application of ␣,␤-meatp with ppads (p2x 1,2,3,5,1/5,4/5 r-specific antagonist). excessive muscular contraction of mm produced by the electrical stimulation significantly decreased the ppt indicating mechanical hyperalgesia of the mm. administration of ppads to the exerted mm produced a complete recovery of decreased ppt. p2x 3 rpositive neurons innervating the exerted mm increased in trigeminal ganglia. our results suggest that p2x 3 r plays an important role in pressure pain, and mechanical hyperalgesia caused by excessive muscular contraction of mm. the present study was undertaken to investigate the change in the activation of the nociceptive neuronal circuit under a neuropathic pain-like state. here we found sciatic nerve ligation (snl) produced a marked increase in the number of c-fos-positive cells in the periaqueductal gray (pag). using the fluoro-gold (fg) microinjection into the pag, numerous fg-labeled cells were detected in the hypothalamus. in the arcuate nucleus (arc) of the hypothalamus, the immunoreactivity (ir) for an excitatory neuronal maker, fosb was increased, whereas the ␤-endorphin (␤-ep)-ir was decreased 7 days after snl. furthermore, the subpopulations of ␤-ep-positive cells were co-labeled with fosb in the arc. the present data suggest that the hypothalamus can be received by snl-induced concomitant nociceptive signals, leading to continuous activation of neurons projecting to the pag. this phenomenon, in turn, indirectly controls pain transmission in the dorsal horn through the descending antinociceptive pathway. os3a-6-04 the cantor-like patterns in rat hippocampal ca1 pyramidal neurons tsuda and kuroda proposed a mathematical model for the cantor coding in the hippocampal ca1. this prediction includes an attractor dynamics expected in the associative network, which was proposed by many authors, since marr's theory of simple memory in the hippocampus. however, our mathematical model is too abstract to describe physiological feature of neurons. then, we have tried to find cantor-like patterns experimentally from the ca1 pyramidal neurons. temporally associated and non-associated electrical stimulations were delivered to schaffer collaterals, and membrane potentials were recorded by patch-clamp recording method. in our results, cantor-like patterns were observed in hippocampal ca1 pyramidal neurons. young songbirds shape their songs using memorized tutor songs and auditory-vocal feedback. we prevented zebra finches from hearing their own vocalizations by exposure to loud noise after 35 days of age, before which they had been reared with song tutors from birth. when the noise stopped at 102-200 days of age, the birds sang unstable and noisy song syllables that did not resemble the tutor syllables. the similarity to the tutor syllables steadily increased until the time of song crystallization (30 days later). these findings show that the memory of tutor syllables still exists well beyond the normal age of song crystallization (d90 of age) and that zebra finches can develop songs using the memory well after the normal period of song development. the temporal order of syllables resembled the tutor model only in birds released from the noise before 80 days of age. thus, different schedules and processes may govern the learning of syllable phonology and syntax. in addition to well-characterized areas, a novel adult neurogenic region; the temporal germinal layer (tgl) was identified in rats (takemura, 2005) . a tracer study revealed that there is an interconnection between the dorsal part of the tgl and the lateral nucleus of the amygdala, suggesting a functional implementation of tgl neurogenesis in amygdala-dependent emotional memory processing. to investigate this possibility, we performed a tgl region-specific low-dose irradiation, which can selectively kill proliferating cells and hence can reduce neurogenesis, using a gamma knife. the tgl-irradiated rats expressed a significantly increased tone-related long-term fear memory, indicating a functional significance of the tgl neurogenesis for aversive memory reduction. we (tsukada and pan, 2005) systematically examine the functional difference between spatio-temporal learning rule (stlr) proposed by tsukada (1996) and hebbian learning rules in a single-layered neural network, computing their ability to differentiate spatiotemporal sequence. in this paper, we tested physiologically the cooperative plasticity without a postsynaptic spike in the ca1 hippocampal network. tsuda and kuroda proposed a mathematical model for the cantor coding in the hippocampal ca1. they also predicted chaotically transitory dynamic behavior called chaotic itinerancy in the hippocampal ca3. this prediction includes an attractor dynamics expected in the associative network, which was proposed by marr and others. the time series of events, which could be output from ca3, may be encoded in ca1 in an efficient way. the proposed cantor coding is effective, because the topology of time series is naturally measured on the cantor set since each element of cantor set represents a single time series. however, our mathematical model is too abstract to describe physiological feature of neurons. then, we have tried to make more realistic model of ca1, using 2-compartment model of neuron, and we found the cantor coding of information of time series in the model ca1. it is known that neurons can propagate action potentials with high temporal precision. however, it is unclear how precisely closely neighbouring neurons synchronize and whether they can code information. here we show that sub-millisecond synchronization can code information as well as the discharge rate modulation. we found that closely neighbouring pyramidal neurons in the ca1 region of the hippocampus synchronize with sub-millisecond precision. the optimal frequency bands for transmitting these synchronizations matched the beta, gamma and fast-ripple oscillations. moreover, we found that the synchronizations were commonly coupled with rate modulations in relation to both internal (retention and comparison) and external (stimulus and motor) events. the synchronization often occurred in relation to stimulus inputs even when rate modulation was clearly absent. therefore, our results suggest that sub-millisecond synchronization plays an important role in propagating information in the hippocampus. the alterations of cerebral motor function by chronic ischemia are poorly understood, since no motor symptoms are noticeable in most of the cases. we evaluated spatial distribution and intensity of eventrelated desynchronization of beta band (beta-erd) evoked in motor area using synthetic aperture magnetometry in 15 patients with chronic ischemia due to diverse vascular occlusive diseases (n = 12) and moyamoya disease (n = 3). contrary to the normal motor activation, ipsilateral beta-erd was dominant during grasping task of affected hand in 8 patients. this abnormal activation was obscured by self-paced finger tapping requiring more selective hand motor programming. and it was more frequently observed in the atherosclerotic hypoperfusion (with white matter change) than in other pathogenesis. ipsilateral beta-erd may be a new indicator of subclinical functional alteration in motor cortices caused by chronic ischemia. os3a-7-02 hypothermia protects against cerebral ischemia by suppressing ␦pkc activation takayoshi shimohata 1,2 , heng zhao 2 , gary steinberg 2 1 department of neurology, brain research institute, niigata university, niigata, japan; 2 department of neurosurgery, stanford university, stanford, usa hypothermia protects the brain from ischemia, but the underlying mechanisms of this effect are not fully elucidated. ␦pkc is reported to induce apoptosis upon activation. its activity is modulated by phosphorylation, translocation and proteolytic cleavage. we investigated effects of hypothermia on ␦pkc activation using a rat permanent distal mca occlusion model. mild hypothermia (30 • c) reduced infarct size by 84%. western blots indicated that ␦pkc cleavage increased markedly in ischemic core but moderately in penumbra after stroke, which is suppressed by hypothermia (p < 0.05). p-␦pkc (t505) dephosphorylated after stroke; this effect is blocked by hypothermia. full-length and cleaved form ␦pkc as well as p-␦pkc (s643) translocate from the cytoplasm to the mitochondria and nucleus, which is suppressed by hypothermia. ␦pkc activator suppressed the protective effect of hypothermia. taken together, hypothermia blocks ␦pkc activation after focal ischemia. this effect might contribute to hypothermic neuroprotection. calcium responses in situ following ischemia remain unclear. we sought to determine, in rats, the calcium changes following transient forebrain ischemia. in anesthetized adult rats, 4-vessle occlusion was induced. fluo-3/am was microinjected, and the fiber-coupled confocal microscope [imaging fiber bundle coupled to the microlensattached nipkow-disk scanner (csu-21, yokogawa, japan) equipped with 10× objective lens] was inserted into the brain. 4-vessle occlusion induced comparable ischemia in both hippocampus and frontal cortex. fluorescence intensity of fluo-3 increased up to 115%, and persistently increased up to 130% during 20-min reperfusion, indicating the long-lasting ca 2+ increase in the ca1 region. in contrast, in the frontal cortex, 10-min ischemia increased fluorescence intensity during ischemia but not reperfusion. in the ca1 region but not in the frontal cortex, transient forebrain ischemia induces long-lasting increase in ca 2+ in situ. research funds: kakenhi #16390407, #16047212 os3a-7-04 reevalution of classical view on resident microglia: neutrophils may play more critical roles than resident microglia at acute phase of ischemic and traumatic brain insults hiroaki matsumoto 1 , h. watanabe 1 , y. kumon 1 , t. ohnishi 1 , chi ii 2 , y. imai 2 , j. tanaka 2 1 dept. neurosurgery, ehime university, japan; 2 dept. molecular and cellular physiology, ehime university, japan resident quiescent microglia (mg) are thought to respond quickly to a variety of pathologic events in the brain, by proliferating and producing a number of bioactive substances including proinflammatory cytokines and nitric oxide (no). in the present study, however, we found that the majority of resident mg died through apoptosis within 36 h after the onset of ischemic and traumatic brain insults. we further noticed that traditional mg markers isolectin b4 and cd11b recognized with ox42 antibody histochemically stained neutrophils, which were identified by neutrophil-specific elastase, rather than iba1+ mg or macrophages. accumulation of neutrophils was observed at the very early phase of the insults, while they expressed proinflammatory cytokines and inducible no synthase. iba1+ amoeboid-shaped mg started to accumulate 3 days after the insults. the data prompted us to reevaluate the roles and the fate of resident mg in the brain. os3a-7-05 insulin regulates the hepatic clearance of amyloid ␤ peptide tetsuya terasaki 1,2 , chihiro tamaki 1 , sumio ohtsuki 1,2 1 graduate school of pharmaceutical sciences, tohoku university, sendai, japan; 2 sorst, jst, japan the liver is the major organ that eliminates amyloid ␤-peptide (a␤) from the circulation, and we have revealed that low-density lipoprotein receptor-related protein 1 (lrp-1) is a molecule responsible for the hepatic clearance. since epidemiologic investigations suggest the high incidence of alzheimer's disease in diabetes mellitus, the purpose of this study was to clarify the effect of insulin on the hepatic clearance of a␤ . insulin infusion into the rat portal vein increased lrp-1 expression in plasma membrane fraction of liver, but did not affect the expression in whole lysate. insulin treatment also increased the hepatic uptake of a␤(1-40), which reached 1.6-fold greater uptake than non-treated control after 10 min treatment. increase of the hepatic uptake of a␤(1-40) by insulin was concentration dependent (ec 50 = 230 pm), and was completely suppressed by rap (2 m), an lrp inhibitor. these results suggest that insulin induces translocation of lrp-1 to the plasma membrane of hepatocytes, leading to increase of a␤ hepatic clearance from the circulation. research funds: sorst, jst os3a-7-06 mr images of intra-arterially administered microglia surrounding ␤-amyloid deposit in the rat brain the therapeutic use of microglial cells has recently received some attention for the treatment of alzheimer disease (ad), but few noninvasive techniques exist for monitoring cells. here we present a magnetic resonance imaging (mri) technology to track micrgolia cells injected intra-arterially in a rat model of ad. we labeled microglia expressing gfp with resovist using the hvj-e vector. we administered labeled microglia into the carotid artery of the rats. mri revealed clear signal changes attributable to resovist-containing microglia in a␤-injected areas. this study demonstrates the usefulness of mri for non-invasive monitoring of exogenous microglia, and suggests a promising future for microglia as therapeutic tools for ad. extravasation of protease-activated receptor (par) activators, such as thrombin, into brain parenchyma can occur after blood-brain barrier breakdown in a number of cns disorders, which causes pathophysiological changes in neurons and glial cells. to elucidate the mechanism of thrombin-induced activation of astroglial cells, we used 1321n1 astrocytomas that show a characteristic retraction of bipolar protrusions after activation of pars with thrombin. the thrombin-induced morphological change of 1321n1 cells was inhibited by an inhibitor of ip 3 receptors, 2-aminoethoxydiphenyl borate (2-apb) or an endoplasmic reticulum ca 2+ -atpase inhibitor, cyclopiazonic acid (cpa). in parallel, thrombin-induced mobilization of ca 2+ was inhibited by 2-apb and cpa. moreover, removal of external ca 2+ accelerated the reversal of thrombin effects. these results suggest that refilling of ca 2+ store by ca 2+ entry play an important role in the cytoskeletal dynamics of astroglial cells. to clarify the occurrence range of neurofibrillary tangles (nft), we reexamined an autopsied alzheimer patient with the onset at age 38 and a 25-year-clinical course. the brain showed severe atrophy (630 g). microscopic examination disclosed that all telencephalic neocortices had nft of more than 100 and sp of more than 10. all limbic cortices and nuclei had nft of more than 100 and sp of more than 10. although there was no sp, various numbers of nft were observed in the following structures: claustrum 85, caudate 9, globus pallidus 6, hypothalamus 22, meynert's nucleus 4, thalamus 74, substantia nigra 6, central gray 12, locus ceruleus 6, purkinje cells 0, posterior root ganglion 0, adrenal medulla 3. this study revealed that there exist nft-rich neurons and free neurons. the latter includes purkinje cells and posterior root ganglion cells. considering the pathogenesis of nft, it must be valuable to clarify qualitative/quntitative differences between nft-rich neurons and free neurons. os3a-7-09 transcriptional regulation of androgen receptor in aging mouse brain androgen receptor (ar) mediates action of androgen, which is involved in memory, behavior and other brain functions that deteriorate with advancing age. in aging mice brain, ar mrna expression was measured by rt-pcr, ar promoter methylation by southern hybridization, and proteins binding to promoter by emsa. ar mrna level was significantly higher in male than female, and it was downregulated by testosterone, but upregulated by estradiol in adult mice. female mice exhibited higher methylation of ar promoter than males. methylation was increased by testosterone, but decreased by estradiol. furthermore, dnasei accessibility to ar promoter was reduced in males, increased by gonadectomy but reduced by sex steroids in adult male. incubation of brain nuclear extract with 32plabeled ar promoter yielded three specific complexes. the intensity of these complexes varied with age and sex. these findings show that ar mrna expression and promoter methylation are inversely regulated by sex steroids in the adult mice cerebral cortex. such regulation of ar expression might influence androgen action and consequently brain function during aging. reliability of synaptic transmission depends on the efficiency of transmitter removal from the synaptic cleft, as well as on the release machinery and the postsynaptic response mechanism. it has been shown in various synapses that postsynaptic and glial excitatory amino acid transporters (eaats) contribute to glutamate removal. however, the role of presynaptic eaats remains unclear. using mouse retinal slices, we examined the contribution of eaats at the rod to rod bipolar cell (rbc) synapse. the kinetics of the rbc current evoked by electrical stimulation of rods was slowed by pharmacological blockade of eaats. recordings of the evoked rbc currents from eaat subtype-deficient mice and the eaat-coupled anion current revealed that functional eaats are localized to rod terminals but not to postsynaptic or glial cells. model simulations suggest that rod eaats are densely packed near the release site, and that rods are equipped with an almost self-sufficient glutamate recollecting system. trpv4 is a thermosensitive trp channel, and activated by body temperature. we found functional-trpv4 was expressed in soma, dendrites and synapses in the neurons. since trpv4 was firstly cloned as an osmotically activated channel, we hypothesized trpv4 might be involved in volume regulation of the spines. therefore, we quantified the spine volume changes by glutamate stimulation, and confirmed trpv4 expression related to the volume increase of spines. next, we compared the resting membrane potential (rmp) between wild type and trpv4-deficient neurons at 37 • c, and found rmp in wild type was more depolarized by approximately 5 mv than rmp in trpv4-deficient neurons. we also performed current-injection experiments in both neurons, and found that trpv4-deficient neurons required much bigger currents to get their firing. thus, we conclude that trpv4 is involved in regulation of both neural activity and spine motility in hippocampus. os3p-2-04 a system for rapid uncaging in defined patterns and its application hiroshi kojima department of intelligent information systems, tamagawa university, tokyo, japan neurons integrate many sysnaptic signals at dendrite. understanding these information processes is a central topics in experimental and computational neuroscience. the use of focused laser beam for uncaging can provide fine spatial resolution to analysis of neural function. however, most experiments were carried out either at spatial locations or in a very simple scanning patterns. we developed a system for performing uncaging in arbitrary pattern in order to emulate realistic neural activity. our system is capable of patterned photorelease of caged neurotransmitters at 100 locations per 200 ms with submicron resolution. ultraviolet laser light is steered by galvano-mirrors and projected onto the surface of preparations for uncaging the caged chemicals. simultaneously, imaging of neurons are obtained by 2-photon microscopy and electrophysiological experiments can be done. we briefly report the present system for rapid uncaging and its application to neurophysiological research. os3p-2-05 d1-like receptors selectively block p/q-type calcium channels to glutamate release onto cholinergic neurons in the rat basal forebrain a number of molecules have been identified in the sensory ganglia including those involved in the signal transmission to the brain. their functions, however, remain largely unknown. we tried to develop a method enabling to inhibit gene expression in the sensory ganglia in vivo by rnai and to evaluate its effect on the synaptic transmission in the brain slices. for this purpose, we selected the nodose ganglion (ng), in which the neurons sending glutamatergic projections to the nucleus tractus solitarii in the brainstem, are located. in anesthetized young wistar rats, synthetic sirna against the genes coding adenosine a 1 receptors (adora1) was introduced to the ng by electroporation. one to five days after sirna delivery, the expression level of adora1 in the ng decreased by >90% of that in the non-treated ng, being not accompanied by a change in mrna level for a 2a receptors. this technique might be promising in analyzing the function of specific molecules involved in transmitter release regulation at the brain synapses. nmda-receptors are specific constituents of glutamatergic system in brain responsible for molecular mechanisms of recognition and learning. activation of neurons by nmda results in intracellular generation of reactive oxygen species (ros) and reorganization of cell metabolism. exposure of rodent and human lymphocytes with nmda results in ros increase within the cells which is suppressed by nmda antagonists. moreover we have demonstrated by rt-pcr technique and by using anti-nmda-antibodies the expression of nmdareceptors on lymphocyte membranes. in addition, we shown that nmda receptor dependent signal from lymphocyte membrane is transformed into specific intracellular reactions controlling caspase-3 activity and interferon-␥ synthesis. in the presentation, properties of nmda-receptors and their functional role in immunnocompetent system are discussed. small molecule g-protein arf1 in combination with phospholipase d (pld) is essential for intracellular trafficking of the proteins from endoplasmic reticulum to golgi apparatus. however, it is recently reported that it also regulate ionic channel activity at the cytoplasmic membrane. to examine possible involvement of arf and subsequent pld in regulation of receptor-induced responses in neurons, we recorded k + -current response to dopamine (da) in the ganglion cells of aplysia under conventional two-electrode voltage clamp. intracellular application of arf1 blockers such as brefeldin a, exo1, and arf1 n-terminal peptide, markedly suppressed the da-induced response. furthermore, intracellular application of ␣-synuclein, a specific blocker of pld, significantly depressed the k + -current response to da. these results suggest that arf1 and subsequent pld may regulate the k + -current response induced by da. os3p-3-05 p250gap, a brain-enriched rhogap, is involved in the nmdar-mediated signaling takanobu nakazawa 1 , toshihiko kuriu 2 , ayako m. watabe 3 , toshiya manabe 3 , shigeo okabe 2 , tadashi yamamoto 1 1 div. of oncology, inst. med. sci., univ. of tokyo, tokyo, japan; 2 dept. of cell biol., tokyo medical and dental univ., tokyo, japan; 3 div. of neuronal network, inst. med. sci., univ. of tokyo, tokyo, japan nmdar regulates structural plasticity by modulating actin organization within spines. however, the signaling pathways that link nmdar activity to the postsynaptic actin cytoskeleton are poorly understood. we identified a brain-enriched rhogap, p250gap, which interacts with the nr2b subunit of nmdar. within neurons, p250gap was highly concentrated in the postsynaptic density and co-localized with nr2b and an actin-binding protein, cortactin. p250gap promoted gtp hydrolysis of cdc42 and rhoa in vitro and in vivo. nmdar stimulation led to de-phosphorylation and redistribution of p250gap. when over-expressed in dissociated neuron, p250gap suppressed the activities of rho gtpases, which resulted in spine elongation. taken together, the results suggest that p250gap is likely to be involved in nmdar activity-dependent actin re-organization in spines. os3p-3-06 non-static method to directly quantify the transfer of firing correlation from one neural population to another: fokker-planck method hideyuki cateau riken brain science institute, saitama, japan firings of only a few neurons are too weak to be transmitted safely, to activate other neurons to fire, or to contract muscles. therefore, we implicitly assume that brain function is exerted by macroscopic population of neurons. to characterize how a macroscopic neural population behave, the simulation method provide an indirect approach. many single neuron simulation runs need to be performed first before extracting macroscopic features by statistically averaging. unlike this method, the fokker-planck (fp) method directly evaluates the macroscopic features, thereby giving a clearer insight into function achievable with neuronal population. despite the lasting interests in firing correlation in coding and conveying information, theoretical studies on it have been largely confined to complicated simulation studies. here, we provide a first non-static fp analysis to directly calculate how correlation and population rates are transferred from one population to another and elaborate a dynamical interplay between these macroscopic quantities at work in time. os3p-4-01 spatial frequency tuning of disparity-selective neurons in macaque v4 hironori kumano 1 , seiji tanabe 2 , ichiro fujita 2 1 grad. sch. of engineering science, osaka univ., osaka, japan; 2 grad. sch. of frontier biosciences, osaka univ., osaka, japan to examine whether convergence across spatial frequency channels contribute to stereoscopic processing, we recorded single neuron activity from area v4 of awake, fixating monkeys. for each neuron tested, we first measured the spatial frequency tuning with sinusoidal gratings or two-dimensional (2-d) filtered noise images, and then examined the disparity tuning with both correlated and anticorrelated dynamic random-dot stereograms (rdss). neurons with broader spatial frequency tuning had more attenuated disparity tuning for anti-correlated rdss. in a subset of v4 neurons, we analyzed responses to various combinations of binocular disparity and spatial frequency by using 2-d filtered noise stereograms. the disparity tuning of most v4 neurons was consistent across a range of spatial frequencies to which they were sensitive. we suggest that v4 neurons pool disparity signals across spatial frequency channels to create an unambiguous representation of stereoscopic depth. os3p-4-02 predicting the monkey's behavioral choice in a stereoacuity task from neuronal responses in area v4 hiroshi shiozaki, seiji tanabe, ichiro fujita lab. cognitive neurosci., grad. sch. frontier biosciences, osaka univ., japan many neurons in visual area v4 of macaque monkeys are selective for binocular disparity. most disparity-selective neurons in v4 are sensitive to small changes in disparity near zero, suggesting that they might contribute to stereoacuity. however, the role of these neurons in stereoscopic depth discrimination has not been directly addressed. we recorded single unit activity from v4 while a monkey was engaged in a fine stereoscopic depth discrimination or stereoacuity task. the monkey was trained to report by saccadic eye movement whether the center region of a random-dot stereogram was nearer or farther than its immediate surround. trial-to-trial fluctuation of visual responses of v4 neurons was correlated with the monkey's subsequent behavioral choice. given the cell's disparity preference, an ideal observer can predict the monkey's upcoming behavioral response from the visual response of v4 neurons. the results suggest that v4 neurons are involved in mediating stereoacuity. os3p-4-03 the role of disparity energy and binocular matching processes in stereopsis takahiro doi, seiji tanabe, ichiro fujita lab. cognitive neurosci., osaka univ., japan the early visual system computes disparity energy of stereo images. some of the next stages retain this information, while other stages perform further computation to solve the stereo correspondence problem. we addressed how the energy and correspondence computations underlie stereopsis. we asked human subjects to discriminate depth of random-dot stereograms with various amounts of disparity. at each disparity level, we manipulated the proportion of dots with the same luminance contrast between the two eyes by reversing the contrast of some dots in one eye. at small disparities, the proportion of correct choices increased monotonically from chance to perfect as the proportion of the same-contrast dots was increased. at large disparities, the subjects perceived reversed depth when contrastreversed dots dominated, and the proportion of correct choices reached only chance level when the two types of dots were balanced. the results suggest that the correspondence and energy computations underlie fine and coarse stereopsis, respectively. we introduce a novel receptive field (rf) analysis, lsrc, which can reveal various aspects of visual receptive fields that were undetectable previously in a single measurement. the visual stimuli are standard wide-field 2-d ternary dynamic random noise, generally refreshed every 26-40 ms. unlike the conventional reverse correlation which computes a spike-triggered average (sta) of the stimuli themselves, lsrc computes the sta of the spectra of localized regions of the stimuli. both simulations and recordings from cat v1/v2 neurons demonstrate that lsrc is capable of revealing details of complex cell rfs, cross-orientation suppression, variations of orientation tuning within rfs that might lead to shape selectivites. since the stimuli can cover a wide visual field area, and few assumptions are made regarding specific shapes or features in stimuli, lsrc is highly suitable for multi-neuron, multi-area studies spanning retina, v1, and especially areas beyond. research funds: mext(13041033), jsps(13308048), coe21 os3p-4-06 analysis of center-surround organization of v1 neurons as a high-order receptive field hiroki tanaka, izumi ohzawa graduate school of frontier biosciences, osaka, japan responses of area 17 (v1) neurons are influenced by stimuli not only in their classical receptive field (rf) center, but also in its surround. such a center-surround organization may be considered as a unified higher-order rf. we have sought to obtain detailed structures of such a rf by harmonic analyses of responses to drifting contrast-modulated sinusoidal gratings that cover both the center and surround regions. of 52 cells analyzed, 80% showed spatial frequency tuning curves that were well fitted with gaussian. by taking the inverse fourier transform of these curves, spatial center-surround rf was obtained as gabor functions with spatial phases between ±90 degrees. highly asymmetric structures were observed for cells with strong surround suppression. estimated sizes of center and surround were well correlated with those from size tuning curves. moreover, there was no space-time tilt in the center-surround rf. the results suggest that neurons with surround suppression are capable of coding various spatial forms of higher-order features (figure-ground borders), but are insensitive to motion of such stimuli. os3p-4-07 spatial organization of receptive fields of complex cells in the early visual cortex kota sasaki 1 , izumi ohzawa 1,2 1 grad. school of eng. sci., osaka univ., japan; 2 grad. school of frontier biosci., osaka univ., japan little is known about the quantitative internal structure of the receptive fields (rf) of complex cells, although this is crucial for understanding how a complex cell acquires its function by collecting inputs from neurons in the preceding stage. therefore, we have analyzed the relationship between the spatial 2nd-order interaction kernels and the rf envelopes of complex cells. extracellular single unit recordings were performed in anesthetized and paralyzed adult cats. threevalued (i.e. gray, dark, and bright) dynamic white noise stimulus with 51 × 51 dots was presented over an area 2 to 4 times larger than the rf of a complex cell. for each dot location, a 2nd-order kernel and its envelope (by hilbert transform) were calculated. the rf envelope of the neuron was determined by summing the envelopes of 2nd-order kernels at all locations. 2nd-order kernels had roughly comparable extent as the rf, and contained 3.3 subregions on average (n = 53). among 35 complex cells, whose rf envelopes were elongated, 16 cells exhibited the horizontal elongation. research funds: mext(13041033), jsps(13308048), coe21 os3p-4-08 orientation tuning of neuron in cat lateral geniculate nucleus tomoyuki naito 1 , osamu sadakane 1 , masahiro okamoto 2 , hironobu osaki 3 , hiromichi sato 1 1 grad. sch. med., osaka univ., osaka, japan; 2 grad. sch. front. biosci., osaka univ., osaka, japan; 3 med. sch., osaka univ., osaka, japan we examined the orientation selectivity of lgn neurons of anesthetized cats and found that although about 19% lgn neurons showed significantly orientation-biased response to the grating with optimal size and spatial frequency (sf), and that 97% of lgn neurons exhibited significant orientation selectivity to gratings with diameter larger than its classical receptive field (crf) and sf higher than the optimal for crf response. two stimulus-size tuning curves measured for responses to stimulation with the optimally-or null-orientated grating exhibited profile similar to each other under the optimal sf condition. however, high sf grating caused stronger surround suppression for response to the orthogonally oriented stimulus than that to the optimally orientated stimulus. our results suggested that elliptic crf center produces orientation-biased response of lgn neurons. furthermore, surround suppression of lgn neurons tuned to particular stimulus orientations enhances orientation selectivity of lgn neurons. os3p-4-09 temporal dynamics of suppressive receptive field surround in cat v1 satoshi shimegi, hiroyuki kida, ayako ishikawa, hiroshi sakamoto, hiromichi sato graduate school of medicine, osaka university, toyonaka, japan in the primary visual cortex (v1), a neuronal response to stimulation of the classical receptive field (crf) is suppressively modulated by the stimulus presented at the receptive field surround (srf). using stationary flashes (500 ms) of sinusoidal grating with optimal parameters and varying radii as stimuli, we examined the temporal dynamics of the surround suppression in v1 cells of anesthetized cats. stimulus slightly larger than the crf caused suppression in early response (<100 ms) but not in middle (100-200 ms) and late responses (200-500 ms). as stimulus size was further enlarged, the middle and late responses were remarkably suppressed while the early response was only moderately or weakly suppressed. radius of surround suppressive field progressively expanded in temporal sequence from 2.5 deg (early response) to 5 deg (middle response) and 7.5 deg (late response). thus, modulation of early response seems to reflect whether stimulus is larger than crf size or not, and late response to reflect how wide area is stimulated. research funds: kakenhi (15500259) os3p-4-10 spatial-frequency dependent surround suppression in cat v1 ayako ishikawa 1 , satoshi shimegi 2 , hiroyuki kida 3 , hiromichi sato 2 1 grad. sch. front. biosci., osaka univ., osaka, japan; 2 grad. sch. med., osaka univ., japan; 3 grad. sch. eng. sci., osaka univ., japan we examined the temporal dynamics of the surround suppression of visual response in terms of spatial-frequency (sf) tuning of neurons in cat v1. we used a stationary flash (duration, 500 ms) of a circular sinusoidal grating patch with optimal orientation and sf as crf stimulus, and that of an annulus (50 ms) with optimal orientation but varying sf as srf stimulus. first, we stimulated crf and srf simultaneously (stimulus-onset-asynchrony (soa) = 0) and analyzed time course of surround suppression. sf tuning of the surround suppression changed along time course of response, and effective sf of surround suppression shifted from the sf lower than that optimal for crf response (c-sf) to that near c-sf. next, changing soa, we examined surround suppression on different temporal phases of crf response. soa-dependency of surround suppression changed according to the temporal phase of response. these results suggest that multiple mechanisms with different sf-and temporal characteristics are involved in the surround suppression. os3p-4-11 contrast-dependency of spatial summation property in cat v1 and lgn masahiro okamoto 1 , tomoyuki naito 2 , osamu sadakane 2 , hiromichi sato 2 1 grad. sch. front. biosci., osaka univ., japan; 2 grad. sch. med., osaka univ., toyonaka, japan we examined contrast-dependent change in a receptive field (rf) size and strength of surround suppression of neurons in the primary visual cortex (v1) and the lateral geniculate nucleus (lgn) of anesthetized cats. rf structure was modeled by spatial interactions of excitatory and inhibitory gaussians. both in v1 and lgn, ratio of gaussians (rog) model captured size-tuning curves of responses better than difference of gaussians (dog) model. under the high contrast stimulus condition, the peak of size tuning curve shrank by 0.76 and 0.68 times in v1 and lgn, respectively. in lgn, surround suppression was strengthened under high contrast stimulus condition, but in v1, the strength of surround suppression did not affected by stimulus contrast on average. we conclude that 1) rog model describes the surround suppression better than dog model both in v1 and lgn, 2) under high contrast stimulus condition, there is a reduction of rf size with a shrinking of excitatory gaussian, which is confirmed with rog model. hiroyuki kida 1 , satoshi shimegi 2 , ayako ishikawa 3 , hiroshi sakamoto 3 , hiromichi sato 2 1 grad. sch. eng. sci., osaka univ., japan; 2 grad. sch. med. sci., osaka univ., japan; 3 grad. sch. front. biosci., osaka univ., japan in the primary visual cortex (v1), neuronal responses to stimulation of the classical receptive field (crf) were suppressed by the presence of stimuli at surround receptive field (srf). we examined whether the suppression varied according to spatial configuration of srf stimuli in v1 neurons of anesthetized cat. the crf stimulus was a circular patch of sinusoidal grating with optimal stimulus parameter. srf was divided into 8 flanks (45 • step), and stationary stimulated with an annulus, oppositely-faced flanks (2-fk) or a flank (1-fk) stimulus. the durations of stimulus presentation were 500 ms for crf and 50 ms for srf stimulation. localized srf stimulation with either 2-fk or 1-fk exerted significant suppression on crf responses. according to the analysis of spatiotemporal change in srf effects, there was no particularly suppressive srf area for 1-fk stimulation throughout the crf response. however, 2-fk stimulation of end position to crf had strong and long-lasting suppression on responses during 80-200 ms after onset. os3p-4-13 temporal-frequency dependency of receptive field size and surround suppression in lgn and v1 osamu sadakane 1 , tomoyuki naito 1 , hironobu osaki 2 , masahiro okamoto 3 , hiromichi sato 1 1 grad. sch. med., osaka univ., japan; 2 med. sch., osaka univ., japan; 3 grad. sch. front. biosci., osaka univ., osaka, japan spatial summation property of neurons in the primary visual cortex (v1) varies depending on stimulus parameters (e.g., stimulus contrast). in this study, we examined how temporal frequency (tf) of grating stimulus affects size-tuning properties of cat v1 neurons. our results showed that, when the tf was higher than the optimal, the strength of surround suppression became weak and receptive field size became larger, suggesting that v1 neurons change their spatial property according to tf in such a way that neurons integrate wide visual field for fast moving stimulus, whereas localized field for slow stimulus. we also tested the effect of changing stimulus size on tf tuning curve. consistent with above-mentioned results, large grating made the peak and the high cut-off of tf-tuning curve higher than those for small grating. in the lateral geniculate nucleus (lgn), we obtained basically similar results to those of v1 neurons, suggesting that the subcortical tf tuning property contributes to that in v1. ryo sasaki, takanori uka department of physiology (i), juntendo university, tokyo, japan a few studies have shown that basic tuning functions in early visual cortex change during visual perceptual learning (schoups et al. 2001; yang and maunsell 2004) . the change in neuronal sensitivity in these studies, however, is small compared to the improvement in behavioral sensitivity. here we hypothesized that the read out of information from sensitive neurons was modified by learning. to test this hypothesis, we investigated whether learning modifies neuronal sensitivity or read out of middle temporal (mt) neurons during learning of a depth discrimination task. two monkeys were trained to report the depth of moving dots (near or far), and we recorded from isolated mt neurons during the course of training. the monkeys showed improvement in discrimination thresholds across 70 daily sessions. in contrast, the sensitivity of mt neurons did not change, whereas the correlation between neuronal activity and the monkey's behavioral choice increased during the course of training. these results suggest that plasticity due to perceptual learning occurs within the neural pathway following area mt. we developed an in vivo method to localize the fine tip of a glassinsulated tungsten microelectrode for chronic recording using 4.7 t mri. the scan conditions were first optimized by imaging a microelectrode that was sunk into copper sulfate solution. the microelectrode tip was precisely localized up to a resolution of 50 m under particular geometrical scan condition. we then examined the applicability of the method in vivo under this optimized scan condition in the temporal cortices of three monkeys. the microelectrode was penetrated into the dorsal or ventral bank of the superior temporal sulcus and the tip was localized by the high-resolution mri. the accuracy of this method was validated by comparing the localized positions of the microelectrode tips with the corresponding electrolytic lesion marks in histological sections. a transient signal change in diffusion-weighted image of the brain has been detected in human visual cortex. the time course of this signal was ahead of the bold signal and characterized by a steep onset. diffusion-mri thus represents a new exciting mechanism for fmri. in order to increase its efficiency we aimed at defining a diffusion response function (drf) as a counterpart of the hemodynamic response function (hrf). an volume of interest was defined using spm with a boxcar function. gamma-variate functions were used to model the steep onset. the parameters of the drf were estimated by fitting the time-course with the drf convolved with a boxcar. although the magnitude of the signal change (around 1%) was smaller than that of bold (>2%), the temporal profile showed a constant precedence of the diffusion signal by 2.4s. os3p-5-03 new insights on normal and pathological brain function from tomographic analysis of magnetoencephalographic signals laboratory for human brain dynamics, brain science institute (bsi), riken, wako-shi, japan tomographic analysis of magnetoencephalography (meg) data combines exceptional temporal resolution with accurate localization, at least for places a few centimeters away from the center of the head [moradi, et al., neuroimage; ioannides et al., cerebral cortex] . this unique capability of probing brain function across the entire cortex and deep brain structures from milliseconds to minutes in the same experiment has already provided new insights about normal [ioannides et al., cerebral cortex;ioannides et al., neuroimage] and pathological [ioannides et al., j. neurosc.] brain function. novel ways of analyzing meg data provide direct measures of regional brain activity over much longer timescales. these new methods are used in ongoing studies to probe the nature of global brain activity in different states of awareness (e.g. different stages of sleep) and explore the relationship between estimates of electrophysiological activity derived from meg with hemodynamic measures of brain activity. os3p-5-04 spatial registration of stand-alone fnirs data to mni space ippeita dan, archana singh, masako okamoto national food research institute, japan the registration of functional brain data to the common brain space offers great advantages for inter-modal data integration and sharing. however, this is difficult to achieve in functional near-infrared spectroscopy (fnirs) because fnirs data is primary obtained from the head surface and lacks structural information of the measured brain. therefore, we present a method for probabilistic registration of fnirs data to the standard montreal neurological institute (mni) template through international 10-20 system without using the subject's magnetic resonance image (mri). the standard deviation in probabilistic registration thus performed for given head surface points is approximately within 1 cm. this means that if the spatial registration error is within an acceptable tolerance limit, it is possible to perform multisubject fnirs analysis to make inference at the population level and to provide information on positional variability in the population, even when subjects' mris are not available. stochastic perturbation in scale is a basic property of biological systems and generates scale-independent structuration and functional dynamics in spatial and temporal patterns, which can be characterized by fractal dimensionality. it allows a user-independent evaluation and does not rely on subjective evaluation in image assessment. we have used a box-counting algorithm in scale-space segmented images to determine the mass fractal dimension of ventricles in different neurological disorders. three groups of subjects [alzheimer disease (ad), obstructive hydrocephalus (oh) and normal controls] were examined. mass fractal dimension is high for ad (1.33), approaching unity (∼1.02) for oh, and in between for control (1.14). statistical analysis was performed and significant differences were observed for these groups (p < 0.01). the observations are accounted by a flow dynamics heterogeneity model. the implications are that stochastic structuration and fractal dimension may be useful to track temporal progression of disease and assess therapeutic management. thrombin, a serine protease essential for blood coagulation, also plays an important role in injury associated with intracerebral hemorrhage. in this study, we revealed that mitogen-activated protein kinase (mapk) pathways contribute to thrombin-induced brain injury in two experimental models. firstly, we employed organotypic cortico-striatal slice cultures. application of thrombin to slice cultures resulted in cortical neuronal injury and striatal shrinkage. the cortical neuronal injury was ameliorated by inhibition of extracellular-signal regulated kinase (erk) but not p38 mapk, while the striatal shrinkage was prevented by both of them. secondly, thrombin was injected into rat striatum. thrombin-induced brain injury determined by immunoreactivity of neuronal marker was reduced by inhibition of erk and p38 mapk. these results suggest that mapk pathways play important roles in thrombin-induced brain injury and they should be therapeutic targets against neurodegeneration associated with blood-brain barrier destruction. positron emission tomography was used to study brain activations during motor imagery of standing and during performance of standing posture in parkinson's disease (pd). eight pd patients performed mental and motor tasks: (1) resting, (2) staring at a standing human object, (3) thinking of standing, (4) standing with eyes open, (5) standing with eyes closed. regional cbf data analyzed by spm2 were compared with normal counterparts. the cerebellar vermis was more activated during imagination of standing in the pd group than in healthy group. as seen in healthy subjects, standing also activated the primary sensorimotor foot area and cerebellar vermis in pd patients, but the between-group comparison generated greater activations in the vermis and prevuneus in pd. the cerebellar vermis engages in postural balance both in mind and reality, and the precuneus may play a more important role in postural control in pd. os3p-6-03 potentiation of nmda receptor-mediated current by metabolic failures through glycine release facilitation in the hypoglossal motoneurons of the rat yu kono 1,2 , eiji shigetomi 2 , kiyoharu inoue 1 , fusao kato 2 1 dept. neurol., jikei univ., sch. med., tokyo, japan; 2 lab. neurophysiol., jikei univ., sch. med., tokyo, japan to elucidate the mechanism underlying the selective vulnerability of motoneurons (mns) to metabolic failures (mfs), we compared the membrane current responses of mns and non-mns to mfs. experiments were performed on neurons in the hypoglossal nucleus (xii) and dorsal motor nucleus of the vagus nerve (dmx) in the young rat brainstem in the presence of ttx. mfs were induced by nacn or oxygen deprivation. in xii neurons, mfs induced large persistent inward currents accompanied by marked increase in strychnine-sensitive synaptic inputs, indicating facilitation of glycine release onto xii neurons. furthermore, nmda receptor-mediated current evoked by exogenous nmda was increased by nacn. in dmx neurons, mfs evoked outward currents without affecting synaptic inputs. these pre-and postsynaptic responses to mfs in mns might play a role in their selective vulnerability in various neurodegenerative diseases including the amyotrophic lateral sclerosis. os3p-6-04 effects of mdma on serotonergic neurons in rat organotypic mesencephalic slice culture including the raphe nuclei yuichi suzuki, megumi higuchi, takayuki nakagawa, shuji kaneko dept. mol. pharmacol., grad. sch. pharmaceu. sci., kyoto univ., kyoto, japan 3,4-methylenedioxymethamphetamine (mdma) is a recreational drug of abused which has been shown to increase serotonin (5-ht) release and cause degeneration of 5-htergic nerve terminals via 5-ht transporter, although the mechanisms are unclear. in this study, we developed rat organotypic mesencephalic slice culture including the 5-htergic raphe nuclei, and examined the effects of mdma and methamphetamine (meth) on 5-ht release and 5-htergic neurotoxicity. immunohistochemical studies for tryptophan hydroxylase revealed abundant 5-htergic neurons around the raphe nuclei. treatment with a 5-htergic neurotoxin 5,7-dihydroxytryptamine dramatically reduced the tissue contents of 5-ht and its metabolite, which was blocked by a selective 5-ht reuptake inhibitor. mdma and meth (0.1-1000 m) increased 5-ht release, and reduced the tissue contents of 5-ht and its metabolite at higher doses. the mesencephalic slice culture including the 5-htergic raphe nuclei may be useful to examine the mechanisms underlying 5-htergic neurotoxic effect of mdma in vitro. os3p-6-05 studies on drug dependence (rept. 421): involvement of platelet-derived growth factor (pdgf) receptor in the morphine-induced rewarding effect masami suzuki, minoru narita, michiko narita, tomoko takeuchi, yasuyuki nagumo, keiichi niikura, tsutomu suzuki dept. of toxicol., hoshi univ. sch. pharm. pharmaceut. sci., tokyo, japan the present study was undertaken to investigate the involvement of platelet-derived growth factor (pdgf) receptor in the morphineinduced rewarding effect in rodents. extensive coexpression of tyrosine hydroxylase with pdgf receptor was apparently observed in the rat ventral tegmental area (vta). the levels of dopamine and its major metabolites in the nucleus accumbens (n.acc.) were markedly increased by the microinjection of pdgf into the rat vta. the morphine-induced rewarding effect was suppressed by intra-vta microinjection of pdgf receptor fc chimera. the increased level of dialysate dopamine produced by morphine in the rat n.acc. was significantly decreased by intra-vta injection of pdgf receptor fc chimera. these findings suggest that the stimulation of -opioid receptors in the vta by morphine leads to the activation of pdgf receptor, which may be directly responsible for the morphine-induced rewarding effect in rodents. os3p-6-06 prostaglandin d 2 is a strong mediator of neuroinflammation in genetic demyelinating mouse model prostaglandin (pg) d 2 , an inflammatory mediator, mainly produced by hematopoietic pgd synthase (hpgds). microglial activation and gliosis are commonly observed during the neuroinflammation. in twitcher (galct wi/twi ), a genetic demyelinating mouse model, we found that hpgds expression was upregulated in activated microglia accompanied by the dp1 receptor induction in hypertrophic astrocytes. using primary culture of glial cells, we demonstrated that activated microglia produced large amount of pgd 2 by hpgds and that astrocytes expressed both dp1 and dp2 receptors and were activated by pgd 2 . we found that gliosis and demyelination were well suppressed in hpgds-or dp1-null twitcher and twitcher treated with an hpgds-inhibitor. these results suggest that pgd 2 is a key molecule of neuroinflammation involved in the demyelination. research funds: 09670806, 12558078 os3p-7-01 on a sodium channel distribution enabling high frequency signal processing go ashida 1,2 , kousuke abe 2 , kazuo funabiki 1 1 grad. sch. medicine, kyoto univ., kyoto, japan; 2 grad. sch. informatics, kyoto univ., kyoto, japan some auditory neurons, such as the owl's nucleus laminaris (nl) cells, can sense very high frequency signals (up to 8 khz). from the theoretical point of view, it seems exceptionally difficult to handle these high frequency signals because the membrane time constant is far longer. first, we discuss a biophysical mechanism of shifting the membrane time constant by connecting the large cell body (soma) with the small node of ranvier. next, we discuss the effect of sodium channel distribution on the impedance function of the membrane. sodium conductance in the soma amplifies low frequency signal components below 1khz, while that in the node does up to 10 khz. last, as a typical example, we discuss the capability of high frequency signal processing in the owl's nl neuron. some biological evidences indicate that sodium channels in the nl neuron are distributed mainly in the nodes but less in the soma. by using an nl neuron model, we show that a neuron with low somatic sodium conductance and high nodal sodium conductance can achieve fine sensitivity to high frequency signals. interaural time difference (itd) is calulated using axonal delay lines and coincidence detector neurons (nucleus laminaris:nl). however, little is known about the cellular mechanisms of coincidence detection. here, we report the results of in vivo intracellular recordings from the barn owl's nl. we used coaxial glass electrodes in which one (microelectrode) was inserted into a patch-electrode type capillary. the inner sharp electrode was protected by the outer one during penetration of the cerebellum. we isolated 140 nl cells from 16 owls and achieved intracellular recordings in 38 of them, as judged by a sudden dc potential drop and the resting membrane potential (mean rp = 58 ± 17 mv). nl neurons produced small spikes and oscillatory potentials whose waveform closely resembled the superposition of the tones delivered to the two ears (sound analogue psps:sap). the amplitude of saps varied as a function of itd. spike rates changed in linear proportion to the amplitude of sap. we evaluated sound localization ability of vision impaired and sighted persons by using a 'two-sound sources discrimination test' in a semianechoic darkroom. in total, 13 vision impaired (7 blind and 6 low vision) and 15 sighted persons participated. the stimuli were pure tone pulses. for each trial, the same single sound pulse was emitted consecutively from a pair of speakers with the same angle either left or right from the midline of the subject. localization ability was assessed whether the subjects are able to discriminate two sound sources or not in each trial. the discriminability of the blind subjects slightly exceeded that of sighted subjects but the difference was not significant. the discriminability of the low vision subjects, on the other hand, was significantly lower than that of blind or sighted. it was suggested that a peculiar 'object perception' of blind persons is not able to measure by means of 'two-sound sources discrimination test.' os3p-8-01 autocrine bmp signaling in astroglia sensitizes the glial scarring masahisa yamada 1 , runa araya 1 , naoto kitamura 1 , yuji mishinsa 2 1 yamada unit, riken bsi, saitama, japan; 2 nihs, nieh, nc, usa bone morphogenetic proteins (bmps) affect growth of glial cells however, contribution of bmps during glial scar formation is unknown. to study the role of bmp signaling in vivo, we disrupted bmpr1a, one of the type i receptors for bmps, in a telencephalic neuronal stem cell-specific manner. we found that aberrant architecture of microvessels that led to a failure in maintaining the blood-brainbarrier in the mutant mice. although mutant mice showed inflammation around the cortical microvessels, proliferation of hypertrophic reactive-astrocytes in the mutant mice was attenuated. disruption of astroglial bmpr1a expression by cre-adenovirus recapitulates the same phenomena. bmps were upregulated in reactive astrocytes in after brain injury. knocking down of bmpr1a by small interfering rna in primary astrocytic culture negatively affected their astrocytic growth injured by scratch, which reinforced the importance of autocrine bmp signaling in astrocytes. this result opens up the understanding of novel mechanisms underlying the autocrine bmp signaling on glial scarring after cns injury. the present study was undertaken to evaluate the functional role of the glial cells in the induction of stress. here, we found that aging mice promoted anxiety-like behaviors as characterized by both the light-dark and elevated plus-maze tests, and they exhibit an increase in astrocytes in the cingulate cortex. a robust increase in gfap-positive astrocytes was noted in the cingulate cortex of nerve-ligated mice that exhibited the anxiety-like behavior. in contrast, iba1-positive microglial cells were dramatically increased as compared to that in control mice and some of them were co-localized with brdu-like immunoreactivity in the hippocampus of mice exposed to chronic psychological stress. our results indicate that the increase in astrocyte or microglia in the cingulate cortex or hippocampus may lead to emotional disorders including aggravated anxiety under aging, chronic pain-like state or exposure to chronic psychological stress. withdrawn os3p-8-04 fucosylation prevents overshooting of the migration by the vagus motor neuron precursors shigeharu kinoshita 1,2 , hideomi tanaka 1,2 , sachiko tsuruoka 3 , hironori wada 1,2 , hitoshi okamoto 1,2 1 riken bsi, wako, japan; 2 jst crest, kawaguchi, japan; 3 riken rrc, wako, japan the vagus motor nuclei are important as the autonomic center for the maintenance of homeostasis. aberrant positioning of nuclei is implicated in the etiology of the sudden infant death syndrome (sids). therefore, control of precursor cell migration into the right position may be crucially important. the zebrafish embryo has two vagus motor nuclei, the dorso-laterally and medially located nuclei (dmx and mmx). the dmx precursors are born near the floor plate, migrate dorso-laterally and then are accumulated at the defined position. in the towhead mutant embryos, ectopic neurons are distributed between bilateral dmx where precursors aberrantly migrate in dorsal direction and fail to stop at the right position. positional cloning and mrna rescue analysis identified towhead as a gdp-mannose 4,6 dehydratase (gmds), a key enzyme for de novo synthesis of a gdp-fucose. as a result, the mutant embryos showed exclusive reduction of fucosylated glycans. our findings represent that fucosylation is responsible for maturation of these neurons. in development of the drosophila visual center, photoreceptor cells extend their axons (r axons) to the lamina ganglion layer and trigger proliferation and differentiation of synaptic partners (lamina neurons) by delivering the inductive signal, hedgehog (hh). this mechanism helps to establish an orderly arrangement of connections between the r axons and lamina neurons, termed a retinotopic map because it results in positioning the lamina neurons in close vicinity to the corresponding r axons. it is found that the bhlh-pas transcription factor single-minded (sim) is induced by hh in the lamina neurons and is required for the association of lamina neurons with r axons. in sim mutant brains, lamina neurons undergo the first step of differentiation but fail to associate with r axons. as a result, lamina neurons are set aside from r axons. the data reveal a novel mechanism for regulation of the interaction between axons and neuronal cell bodies that establishes precise neuronal networks. research funds: kakenhi (17024013) os3p-8-06 initial molecular steps in synaptogenesis in vivo: trans-synaptic interaction of cell adhesion molecule is involved in postsynaptic assembly of psd95-homolog dlg hiroshi kohsaka, etsuko takasu, akinao nose department of physics, university of tokyo, tokyo, japan trans-synaptic interaction via cell adhesion molecules (cam) is essential in constructing synapse structures. although this notion has been supported by various studies in vitro, evidence in vivo has been lacking. here we used live-imaging and genetic analysis to show that a drosophila cam fasciculin2 (fas2) mediates early interaction between pre-and postsynaptic cells in synaptogenesis in vivo. by visualizing gfp-tagged fas2 genetically expressed on a muscle, we found fas2 accumulated at postsynaptic site just after the contact between growth cones and its target muscle. genetic and deletion analysis implied that trans-synaptic interaction with presynaptic fas2 is crucial for the postsynaptic localization of fas2. in addition, postsynaptic localization of a scaffolding protein dlg, psd95-homolog, and glutamate receptors was impaired in fas2 mutants. these results provide the first in vivo evidence that trans-synaptic cell adhesion molecule has a role in inducing the assembly of synapses. gaudilliere brice harvard medical school, usa postsynaptic differentiation of dendrites is an essential step in synapse formation. we report here a requirement for the transcription factor myocyte enhancer factor 2a (mef2a) in the morphogenesis of postsynaptic granule neuron dendritic claws in the cerebellar cortex. a transcriptional repressor form of mef2a that is sumoylated at lys403 promoted dendritic claw differentiation. activity-dependent calcium signaling induced a calcineurin-mediated dephosphorylation of mef2a at ser408 and thereby promoted a switch from sumoylation to acetylation at lys403, leading to inhibition of dendritic claw differentiation. our findings define a mechanism underlying postsynaptic differentiation that may modulate activity-dependent synapse development and plasticity in the brain. research funds: ns4102, ag11085 ps1a-a002 characterization of mrna species that are associated with postsynaptic density fraction by gene chip microarray analysis we previously reported the partial identification by random sequencing of mrna species that are associated with the postsynaptic density (psd) fraction (tian et al., 1999) . we report here further characterization by gene chip analysis of the psd fraction-associated mrnas, which were prepared in the presence of rnase inhibitor. we confirmed that a large number of mrna species are associated with the psd fraction and found that mrnas encoding various postsynaptic proteins were highly concentrated in the psd fraction. we identified some mrna species that were highly concentrated in the psd fraction. we also constructed a cdna library using the psd fraction-associated mrnas as templates, and identified 1152 randomly selected clones by sequencing. our data suggested that the psd fraction-associated mrnas are a very useful resource, in which as yet uncharacterized genes are concentrated. tian et al., 1999. mol. brain res., 72, 147-157. research funds: kakenhi (17500252) ps1a-a003 the distribution of snap-25 protein is regulated in isofom-specific manner makoto itakura, saori yamamori, kouta takano, masami takahashi department of biochemistry, kitasato university school of medicine, sagamihara, japan two isoforms of snap-25 derived from exon 5 splicing are expressed in brain. we generated two specific antibodies for snap-25a and b, and studied the distribution in rodent brain. there was a sticking difference in expression of snap-25a and b during early postnatal period. snap-25b was low at the birth and increased remarkably thereafter. by the contrast, snap-25a increased transiently and attained a maximum level around seven day after birth. furthermore, there seemed to be a difference in their distributions in plasma membrane, since a substantial amount of snap-25b but not snap-25a was recovered in raft-enriched fractions of triton x-100-treated lp1 membrane after sucrose density gradient centrifugation. immunohistochemistry demonstrated that snap-25b was widely distributed throughout brain, whereas, snap-25a was restricted to some particular regions of brain. these results indicate that expression and distribution of snap-25 protein are regulated differently in isoformspecific manners, and snap-25a and snap-25b play different functional roles in brain. ps1a-a004 erc(elks/rab6ip2/cast) regulates syaptic short-term plasticity by recruiting bmunc13-2 to the active zone camkii in the postsynaptic sites is localized as a psd-anchored or a cytoplasmic form. camkii in the two sites is interchangeable by its translocation. translocation and targeting of this kinase to appropriate subcellular compartments are crucial for its physiological function. we have previously suggested that postsynaptic camkii is also localized in lipid raft microdomain (2001. mol. brain res. 89, 20-28) . in this report, we proved the lipid raft localization of camkii by detergent-treatment and successive sucrose floatation assay of spm or cos7 cells expressing camkii, and by cholesterol depletion from membrane using mbcd. we also investigated the mechanism and properties of camkii targeting to lipid raft. camkii targeted to lipid raft microdomain possibly through protein-protein interaction. our data suggest that lipid raft microdomain is a major site of camkii distribution, as well as postsynaptic density and cytoplasmic region, at the postsynaptic site. glial glutamate transporters, glast and glt-1, are co-localized in processes of bergmann glia wrapping excitatory synapses on purkinje cells (pcs). although glast is expressed six-fold more abundantly than glt-1, the decay kinetics of climbing fiber (cf)mediated excitatory postsynaptic currents (cf-epscs) in pcs in glast(-/-) mice are not significantly different from those in wildtype mice. here we attempted to clarify the roles of glial glutamate transporters in cf-pc synapses using glast(-/-) and glt-1(-/-) mice, and a novel antagonist of glial glutamate transporters, (2s,3s)-3-[3-(4-methoxybenzoylamino)benzyloxy]aspartate. our results indicate that glial glutamate transporters can retain the fast decay kinetics of cf-epscs in the normal range if a small proportion (approximately 20%) of functional transporters, glast and/or glt-1, is preserved. glutamate is well known as an essential neurotransmitter in nervous system. how glutamate-mediated synaptic transmission is controlled in neural circuit of live animal, however, remains to be poorly understood. we found that the loss-of-function mutations in vglut (vesicular glutamate transporter) encoded by eat-4 gene led to abnormal sensory behaviors including thermotaxis in c. elegans. thermotaxis defect of eat-4 mutant was caused by malfunction of both thermosensory neuron afd and its downstream interneuron ria, suggesting that thermal signals from afd or ria to their downstream neurons are transmitted by glutamate through eat-4 vglut. a mutation in avr-14 glutamate receptor also led to abnormal thermotaxis. we are trying to investigate whether avr-14 functions in the downstream neurons of afd or ria, and to identify other glutamate receptors involved in thermotaxis. through the analysis of thermotaxis neural circuit, we are hoping to reveal the mechanisms of glutamate-mediated synaptic transmission at neural circuit level. ps1a-a008 biochemical characterisation of the vesicular glutamate transporter 1 stephan schenck, shigeo takamori department of neurology and neurological science, 21st century coe program, tokyo medical & dental university, tokyo, japan vesicular glutamate transporters (vgluts) load synaptic vesicles with glutamate, the major excitatory transmitter in the brain, thus making these transporters of outstanding importance for the function of the central nervous system. the three known isoforms of these secondary active transporters have been characterised in terms of tissue distribution, developmental expression patterns and some pharmacological features. while the third isoform constitutes only a minor fraction, vglut1 and vglut2 are abundantly expressed in the brain with a complementary distribution pattern and divergences in the expression profile during ontogeny. so far, no clear difference in the function of vglut1 and vglut2 has been found. to further characterise the specific properties of the transporters we make use of the vglut1-ko mouse which gives us the opportunity to investigate a brain devoid of vglutl. we focus on synaptic vesicle fractions from ko-mice to study the vglut-associated transport biochemically. in recent years, three isoforms of vesicular glutamate transporters (vgluts) have been molecularly identified in mammals. histological investigations have revealed that the distribution of three vglut isoforms in the cns is largely complementary with limited overlap, suggesting that differential expression of vglut isoforms may contribute to functional diversity in glutamatergic synapses. however, functional differences among the isoforms remained poorly understood. to get insights into their isoform-specific property, we searched for interacting protein(s) to the c-terminus of vglut1 by yeast two-hybrid screening and found endophilin a1. as expected for the interacting molecule to vglut1, endophilin a1 was typically localized to vglut1-positive synaptic terminals in cultured hippocampal neurons. we are currently investigating physiological significance underlying their direct interaction and co-localization. the aim of this study is to investigate the molecular basis for lactate utilization. hippocampal neuronal culture was continuously superfused with glucose or lactate solution and spontaneous excitatory postsynaptic currents (sepscs) were recorded from a voltage-clamped pyramidal neuron. in lactate solution, amplitude of epscs was decreased in 60∼90 min, followed by spontaneously recovered after120 min, while epsc in glucose medium remained unchanged. application of apv+ni in lactate medium, spontaneous recovery was not observed. in neuron cultures, incorporation of 14c-lactate was gradually increased, which was suppressed by applications of inhibitors for calcium calcium channels or protein kinase c. in glial cell cultures, incorpotation of lactate was initially maintained. increased expression of monocarboxylate transporter (mct) 2 was demonstrated in the lactate medium. results suggested that increased mct2 expression of neurons may lead to utilization of lactate to sustain synaptic function via calcium-dependent manner. yumei wu 1 , kazuhito tomizawa 1 , shuang liang 2 , iori ohmori 1 , teiichi nishiki 1 , kohji takei 2 , hideki matsui 1 1 dept. of physiol., okayama univ., okayama, japan; 2 dept. of neurosci., okayama univ., okayama, japan synaptic vesicle endocytosis is regulated by phosphorylation of endocytotic proteins, such as amphiphysin (amph) i and dynamin i. here, we show a novel type of regulation of vesicle endocytosis by proteolysis. in mouse hippocampal slices, amph i was found to be cleaved by a ca 2+ -activated protease, calpain during prolonged depolarization or stimulus trains. the calpain-cleaved n-terminal amph i fragment lost its ability to bind dynamin and inhibited transferrin uptake as overexpressed in cos-7 cells, indicating that the calpain cleavage of amph i inhibits endocytosis. amph i in hippocampus was also cleaved by calpain in vivo after kainate seizure. although the second administration of kainate caused less severe seizure activity than the first one, this relieved second seizure was not observed in pre-treatment with a calpain inhibitor, allm during the first seizure. thus, the proteolytic activity of calpain could protect neurons from excitotoxicity by inhibiting vesicle recycling. synaptic vesicles (svs) are effectively recycled by endocytosis for continuous synaptic transmission. previously, we have suggested that a high level of synaptic transmission is maintained by recycling of svs through two types of endocytosis operating coordinately (27th this meeting). in the present study, we labeled endocytosed svs at nerve terminals of drosophila with fluorescence dyes, fm1-43 and fm4-64, and also measured quantatively exocytosis and endocytosis of svs, using these dyes. egfp-labeled cacophony ca2+ channels and anti hrp stained the active zone and non-active zone at synapse, respectively. imaging analysis revealed that two distinct types of endocytosis of svs occurred at the active zone and the non-active zone of motor nerve terminals. we have previously shown that baclofen, a gaba b receptor agonist, inhibits exocytosis in synapses of mouse hippocampal neurons. syntaxin 1a is also known to modulate exocytosis. to characterize the molecular mechanisms involved, the inhibitory effects of baclofen in neurons transfected with antisense oligonucleotide to syntaxin 1a were investigated by patch-clamp recording and counting the number of release sites. transfected neurons showed higher frequency of miniature epscs and stronger inhibition by baclofen than controls, but no change in number of sites. increased exocytosis is thus induced by increases in transmitter release per site, rather than by more sites due to neurite sprouting. these results suggest that gaba b receptor shares part of the mechanism involved in modulation of exocytosis with syntaxin 1a in mouse hippocampal neurons. we have previously shown a transient localization of tubulin (tub) during synaptic vesicle (sv) cycling in drosophila nerve terminals. the tub localization is detected during sv recycling, while microtuble (mt)-loop is observed throughout sv cycle. in this study, we characterized the two distinct tub localizations and showed their relation with sv pool formation. axonal mts and mt-loops abounded in acetylated (acetyl) tub. the transient localization was either polymerized or depolymerized, and organized by non-acetyl tub. taxol decreased the non-acetyl tub localization but not mt-loops, and inhibited exo/endo cycling pool (ecp) formation. in boutons containing mt-loops, ecp formation was also inhibited. acetyl mt-loops tend to be stable whereas presynaptic non-acetyl tubs are either free dimers or dynamic mts. these results suggest that presynaptic dynamic tub, especially non-acetyl tub, controls ecp formation. presynaptic tub dynamics may regulate functional presynaptic plasticity by controlling sv pools. research funds: grant-in-aid for jsps fellows the mechanism by which pregnenolone sulfate (pregs) enhances synaptic transmission was studied at the rat calyx of held. pregs increased the amplitude of evoked epscs, without affecting that of spontaneous miniature epscs, indicating that the site of its action is presynaptic. pregs facilitated presynaptic voltage-gated ca 2+ channel (vgcc) currents via accelerating their activation kinetics, but had no effect on k + currents, resting conductance, or action potential waveforms. in simultaneous pre-and postsynaptic recordings pregs did not change the relationship between presynaptic ca 2+ influx and epscs, suggesting that exocytotic machinery downstream of ca 2+ influx was not involved in the pregs effect. neither bapta nor gtp␥s loaded into presynaptic terminals blocked the effect of pregs. we conclude that pregs enhances transmitter release via facilitating vgccs by a novel mechanism, which is independent of intracellular ca 2+ or g-proteins. ps1a-b017 spine targeting of endocannabinoid synthesizing enzyme, diacylglycerol lipase-␣ in the cerebellum and hippocampus endocannabinoids are neuromodulator that is released from postsynaptic neurons, acts retrogradely on presynaptic cb1 cannabinoid receptor, and induce suppression of transmitter release. to understand the retrograde signaling mechanisms, we investigated subcellular localization of a major endocannabinoid biosynthetic enzyme, diacylglycerol lipase-␣ (dagl␣), in the mouse brain. in the cerebellum, dagl␣ was predominantly expressed in somatodendritic membrane of purkinje cells, and highly concentrated at the base of spine neck. however, dagl␣ was excluded from the main body of spine neck and head. in hippocampal pyramidal cells, dagl␣ was selective to spines, but widely distributed within spines. these results indicate that dagl␣ is essentially targeted to postsynaptic spines in cerebellar and hippocampal neurons, but its fine distribution within and around spines is differently regulated between the two cell types. synprint site of voltage-gated ca 2+ channels interacts with synaptotagmin. however, its physiological role is not entirely clear. here we report that ap-2 subunit can directly bind with synprint site. this interaction was ca 2+ -dependent, being weaker at concentrations higher than 200 nm. in contrast, the interaction of synaptotagmin with synprint was optimal at 100 m ca 2+ , being weaker at lower or higher concentrations. the binding domain of synprint for ap-2 and synaptotagmin was indistinguishable, and these proteins competed with each other for the synprint site. to assess physiological role of these interactions, we made a peptide containing synprint site, and loaded it directly into the nerve terminal at the calyx of held. this peptide blocked endocytosis measured with capacitance, and gradually diminished exocytosis upon repetitive presynaptic activations. we conclude that ca2+ channel synprint site makes ca 2+ -dependent interactions with ap-2 and synaptotagmin thereby contributing to vesicular endocytosis. ps1a-b019 acl-4, an evolutionarily conserved acyltransferase like gene is required for normal synaptic transmission in c. elegans naoko hara, takao inoue, yasukazu takanezawa, hiroyuki arai department of health chemistry, graduate school of pharmaceutical sciences, university of tokyo, tokyo, japan it is generally accepted that various phospholipid molecular species are formed by phospholipids acyltransferase reactions. however, the physiological significance and the molecular mechanism of the remodeling are largely unknown. to address these questions, we focused on evolutionarily conserved acyltransferase like genes in c.elegans acl-1˜14, and generated their deletion mutants. the mutants of acl-4 gene, which is predominantly expressed in neurons and muscles, showed no apparent phenotype. however, the mutants exhibited severe movement abnormalities in fat-3 mutant background in which long chain polyunsaturated fatty acids are depleted. pharmacological analysis revealed that these mutants showed presynaptic defects in synaptic transmission. these abnormalities were rescued by neuron specific acl-4 expression, suggesting that certain phospholipid species produced by acl-4 are involved in maintaining normal synaptic transmission and motility of c.elegans. daisaku yokomaku 1 , hussam jourdi 1 , akiyoshi kakita 2 , tadasato nagano 1 , hitoshi takahashi 3 , nobuyuki takei 1 , hiroyuki nawa 1 1 dept. mol. neurobiol., brain res. inst., niigata univ., japan; 2 brain resource center, brain res. inst., niigata univ., japan; 3 dept. pathology, brain res. inst., niigata univ., japan scaffolding proteins containing pdz domains interact with synaptic receptors and cytoskeletal components and are therefore implicated in synaptic development and plasticity. little is known, however, about what regulates the expression of the pdz proteins and how the levels of these proteins influence synaptic development. here, we show that ligands for epidermal growth factor (egf) receptors (erbb1) decrease a particular set of pdz proteins and negatively influence synaptic formation or maturation. in neocortical cultures, egf decreased the expression of grip1 and sap97. moreover, egf treatment resulted in a decrease in the frequency of pan-pdzimmunoreactive aggregates on dendritic processes. these findings revealed a novel negative effects of erbb1 receptor ligands that attenuates the expression of the pdz proteins and inhibits postsynaptic maturation in developing neocortex. takatoshi iijima 1 , eriko miura 2 , keiko matsuda 1 , tetsuro kondo 1,3 , masahiko watanabe 2 , michisuke yuzaki 1 1 dept. physiol., sch. med., keio univ., tokyo, japan; 2 dept. anatomy, hokkaido univ., sch. med., sapporo, japan; 3 mol. neurophysiol., aist, tsukuba, japan cbln1 is a member of the c1q and tumor necrosis factor families predominantly produced in cerebellar granule cells. recently, we have shown that cbln1 is secreted as a glycoprotein and plays crucial roles in synaptic plasticity and synaptic integrity of purkinje cells. although other members of the cbln family, cbln2-4, are known to be expressed in the brain, their precise expression patterns and biochemical properties remained unclear. here, we show that each cbln member is expressed in various regions of developing and mature brains. all cbln family members could form both homomeric and heteromeric complexes each other in heterologous cells. like cbln1, cbln2 and cbln4 were secreted as glycoproteins, whereas cbln3 was retained in the endoplasmic reticulum. these results suggest that each cbln member is potentially involved in synapse development and plasticity in various brain regions. s-scam is a synaptic membrane-associated protein with pdz domains, a guanylate kinase domain and ww domains. it interacts with various synaptic components including nmda receptor subunits, psd-95 and neuroligin. as we previously reported, s-scam is recruited to excitatory synapses by ␤-catenin. s-scam forms a ternary complex with neuroligin and psd-95. more importantly, s-scam is involved in synaptic accumulation of neuroligin and subsequently affects the localization of psd-95 at excitatory synapses. in the course of these studies, we observed signals detected by anti-s-scam antibody at inhibitory synapses. we have here examined whether s-scam is indeed localized at inhibitory synapses in hippocampal neurons. we have raised questions which molecules s-scam interacts with at inhibitory synapses and which role s-scam plays in the assembly of inhibitory synapses. eriko fujita, yuko tanabe, takashi momoi division of differentiation and development, department of inherited metabolic disorder, national institute of neuroscience, ncnp, oawahigashi, tokyo, japan igsf4/ra175 (ra175), which is a member of immunoglobulin superfamily having pdz binding domain at c-terminals, has ca2+independent homophilic trans-cell adhesion activity. ra175 participates in synaptic junction and epithelial junctions in various tissues including testis. homozygous null (ra175-/-) male is infertile and shows the defective elongating spermatids and fails to mature further. ra175 interacted with par-3 being involved in the polarity of epithelial cells via pdz binding domain at c-terminals. par-3 was colocalized in the cell adherent region of p19 embryonal teratocarcinoma cells during ra-induced differentiation into epithelial-like cells and mainly localized in the spermatid of ra175+/+ testis, whereas it was undetectable in the spermatid of the ra175−/− testis. ra175 and jam-c were localized around the head portion of spermatid and ra175 deficiency provided the abnormal polarization of the jam-c, which is necessary for the differentiation of round to elongated spermatid. jam-c inhibited the interaction between ra175 and par-3. research funds: 12324122 izumi kawabata, shigeo okabe department of cell biology, tokyo medical and dental university, tokyo, japan coordinated development of excitatory and inhibitory synapses is critical for both stability and temporal fidelity of neuron network in the hippocampus. however, there have been few analyses on postsynaptic molecular assembly in interneurons during development. to address this question, we examined dynamic properties of psd-95 clusters in cultured hippocampal interneurons. higher density of dendritic psd-95 clusters was observed in interneurons at 11 div. at 18 div, this difference was less prominent, mainly due to >3-fold increase of psds in excitatory neurons. psd-95-gfp imaging revealed lower rate of cluster appearance/disappearance in interneurons at 11 div. the higher rate of cluster turnover in excitatory neurons, together with their higher rate of net cluster increase, may explain the delayed boost of cluster density. photobleaching of psd-95-gfp revealed similar kinetics in two neuron types, suggesting additional determinants of cluster dynamics apart from the steady-state assembly rate. possible involvement of other postsynaptic molecules in interneuron psd dynamics is now being investigated. ps1a-c025 two-photon imaging of immature dendritic protrusions and astroglial processes in hippocampal slice cultures hideko nishida, shigeo okabe department of cell biology, tokyo medical and dental university, tokyo, japan several lines of evidences indicate roles of astroglia in synaptogenesis, possibly mediated by either cell adhesion or diffusible factors. however, structural evidences supporting this claim are virtually lacking, mainly due to technical limitations in simultaneous imaging of neuronal and astroglial structures. here we visualized astroglia and pyramidal neurons in hippocampal slice cultures by combining adenovirus-mediated, cre-dependent expression of gfp with electroporation of rhodamine-dextran. two-photon time-lapse imaging of immature dendritic protrusions and astroglial processes in 3-7div slice cultures revealed longer lifetime of dendritic protrusions having experienced astroglial contacts than those without contacts. dendritic protrusions with astroglial contacts also showed higher tendency to form spines. furthermore, expression of mutant rac1 in astroglial cells induced significantly longer, non-spiny protrusions than control. these findings suggest an involvement of direct astroglia-filopodia contacts in subsequent maturation of dendritic protrusions. taiko imura, fusao kato lab. neurophysiol., jikei univ. sch. med., tokyo, japan application of p2x receptor agonists to the neurons in the nucleus of the solitary tract (nts) results in glutamate release facilitation (kato & shigetomi, 2001; shigetomi & kato, 2003) . recently accumulated evidence indicates that astrocytes affect the neuronal excitability by releasing gliotransmitters such as atp. this study was performed to determine whether such astrocyte-neuron interaction takes place in the nts. first, we analyzed the spatial localization of these cells by immunohistochemistry. a large number of gfap-positive cells with processes in the close apposition to the neun-positive neurons were found. second, we analyzed the effect on synaptic activity of localized application of atp using laser-based photolysis of caged atp in brainstem slices. uncaging of atp at neuronal dendrites (2 s, 4-micrometer diameter) resulted in an immediate rise in mepsc frequency, in a manner sensitive to p2x receptor antagonists. these results provide supports for the possible interaction between astrocytes and neuronal presynaptic terminals. research funds: kakenhi (17300123) ps1a-c027 ealy synapsin i accumulation in a granule cell axon at the filopodial attachment site of developing rodent purkinje cell dendrites in vitro isao nagata, junko kimura-kuroda department of brain structure, tokyo metropolitan institute for neuroscience, tokyo, japan synapse formation between the parallel fibers (pf) and dendrites of purkinje cells (pc) occurs at an early stage in the developing cerebellar cortex of the neonatal rodent. however, the precise spatio-temporal pf-pc interaction has not been elucidated. we have found that growth of pc dendrites was initiated by the attachment of axonal neurite bundles of granule cells orienting at right angles in several types of 2d-and 3d-cerebellar cultures. here, we investigated the expression of a synaptic vesicle marker, synapsin i, in granule cell axons by multiple immunofluorescence labelings in these cultures. synapsin i was first expressed at the filopodial attachment site of a pc dendrite as a cluster of faint punctate deposits in a long axon, then they appeared to gather into a slender and finally into a small round deposit. thus, the filopodial attachment of the juvenile pc dendrites to the axons of granule cells may induce rapid formation of presynaptic terminals via local clustering of synaptic vesicles. ps1a-c028 integrative spike dynamics of rat ca1 neurons: an in situ multineuronal imaging study takuya sasaki, rie kimura, norio matsuki, yuji ikegaya department of pharmacology, university of tokyo, tokyo, japan the brain operates through a coordinated interplay of numerous neurons. our new technique with large-scale optical recordings reveals the diversity of synaptic integration in hundreds of neurons. in hippocampal slices bolus-loaded with calcium fluorophores, we stimulated the schaffer collaterals and monitored the bulk presynaptic activity from the stratum radiatum and individual postsynaptic spikes from the ca1 stratum pyramidale. single neurons responded to varying synaptic inputs with unreliable spikes, but at the population level, the networks output a linear sum of synaptic inputs. the network activity varied from trial to trial, even though given constant stimuli. this variation emerged through time-varying recruitment of different neuron subsets, which were shaped by correlated background noise. our imaging approach enables linking single-cell behaviors to their communal dynamics, and we discovered that, even in a relatively simple ca1 circuit, neurons could collectively be engaged in complex information processing. it is assumed based on previous in vitro experiments by other researchers that mglur6 connects with syntenin at the dendrites and mglur7 with pick1 at the axon terminal in on cone bipolar cells. to prove this possibility, we investigated wild-type mouse retinas immunohistochemically and confirmed their co-localized immunopositive labels at the respective places. next, we examined which scaffold protein would connect with mglur7 that was known to be ectopicly expressed in the dendrites of mglur6-deficient on cone bipolar cells. we observed no pick1 but only syntenin at the mglur6-deficient dendrites, and also the syntenin immunopositivity was co-localized with mglur7 immunopositivity. these findings suggest that mglur7 connects with syntenin in place of mglur6 that was knockout from the on cone bipolar dendrites. noriko trpv family is identified as thermosensitive, ca 2+ -permeable channels. trpv1, expressed in sensory neurons, is activated by noxious heat above 42 • c, whereas trpv 4, expressed in keratinocytes, is sensitive to moderate temperatures (>34 • c). here we examined the role of trpv1 and 4 in regulation of body temperature (bt) by using infrared laser as a heat stimulus. in wild type mouse, though the laser irradiation which caused the increase in skin temperature up to 55 • c did not induce the change in bt, desensitization of trpv1 with capsaicin resulted in the increase in bt. on the other hand, in trpv4-knockout mouse, moderate thermal radiation (>43 • c) caused the increase in the bt. the processing of noxious and moderate thermal radiation stimuli may depend on the trpv1 and 4 respectively. research funds: kakenhi (17657052) ps1a-c032 generation and biochemical analysis of a glur␣2 knockout mouse hirotsugu azechi 1 , manabu abe 1 , rie natsume 1,2 , kenji sakimura 1,2 1 department of cellular neurobiology, brain research institute, niigata university, niigata, japan; 2 sorst/jst, saitama, japan glur␣2(glur2) is a key subunit of ampa receptors, since it is a critical determinant of their calcium permeability. to clarify the molecular function of glur␣2, we generated a conditional glur␣2 knockout mouse using the cre/loxp recombination system. we first established a "floxed" mutant line gra2f using c57bl/6(b6) es cell line renka. the homozygous floxed mutants showed no significant abnormalities, thus our gra2f was used as a target of glur␣2 line. by crossing gra2f and tlcn-cre that expressed cre in germ line cells, glur␣2 null ko mice were produced, but most of them died within 3 days after birth. to overcome the lethality, the glur␣2 mutation was transferred onto b6/129 or b6/cd-1 genetic background. subcellular fractionation and quantitative immunoblot showed changes in the amount of ampa receptor subunits. these results indicated a significant role of glur␣2 in the distribution of functional ampa receptors in vivo. ps1a-c033 gisp: a novel brain specific protein that binds to the gaba b1 subunit and promotes its surface expression here we report the identification and characterisation of a novel brain specific 130 kda protein, gaba b r interacting scaffolding protein (gisp), that interacts directly with the gaba b 1 subunit via a coiledcoil domain. gisp coimmunoprecipitates with gaba b 1 and gaba b 2 from rat brain. in cultured hippocampal neurons gisp displays a punctate dendritic distribution and colocalises with gaba b receptors. when co-expressed with gaba b rs gisp increases the amount of gaba b 1 protein and also promotes gaba b 1 surface expression in the heterologous cells. furthermore, gisp increases surface expression of gaba b 1/gaba b 2 complexes. these results suggest that gisp is involved in the forward trafficking and stabilisation of gaba b rs. thus gisp is an novel gaba b 1-binding protein potentially involved in the cell surface and/or synaptic targeting of the gaba b rs. three distinct isoforms of vesicular glutamate transporters (vglut1-3) have been cloned and shown to exhibit differential distribution patterns in the brain. recent work shows the presence of vgluts in synaptic-like microvesicles (slmvs) of endocrine cells. mammalian pineal melatonin-secreting cells, pinealocytes, contain numerous slmvs which likely accumulate glutamate to inhibit melatonin synthesis. vglut1 and vglut2 seem to participate in this glutamate accumulation. in the present study, we found that vglut3 mrna is also expressed in the adult rat pinealocytes. vglut3 immunoreactivity (ir) was distributed throughout the pineal gland, and was co-localized with vglut1-ir or vglut2-ir in many, but not all, processes of pinealocyte. these data indicate that there are some subpopulations of slmvs which differ in the kind of vglut isoforms contained and/or in their combinations, suggesting vglut isoform-dependent sorting of slmvs to pinealocyte processes. kenzi saito 1,2,3 , kenji nakamura 4 , toshikazu kakizaki 1,3 , satoe ebihara 5 , masakazu uematsu 6 , shigeo takamori 7 , minesuke yokoyama 4 , shiro konishi 4 , masayoshi mishina 3,8 , jun-ichi miyazaki 9 , kunihiko obata 10 , yuchio yanagawa 1,3 1 gunma univ., maebashi, japan; 2 sokendai, hayama, japan; 3 sorst, kawaguchi, japan; 4 mitsubishi kagaku inst. life sci., machida, japan; 5 kumamoto univ., kumamoto, japan; 6 toyohashi univ. tech., toyohashi, japan; 7 tokyo med. den. univ., tokyo, japan; 8 univ. tokyo, tokyo, japan; 9 osaka univ., suita, japan; 10 riken, wako, japan the vesicular gaba transporter (vgat) loads gaba from neuronal cytoplasm into synaptic vesicles and is selectively expressed in inhibitory neurons containing gaba and/or glycine. to assess the functional role of vgat in development, we have disrupted the gene encoding vgat using cre-loxp system. western-blot analysis showed that vgat protein was absent in the homozygous embryos, indicating that the mutation had generated in a null allele. vgat knockout mice died around birth. all vgat knockout mice displayed cleft palate and omphalocele. our results suggest that vgat plays essential roles in both palate formation and ventral body wall development. research funds: kakenhi (17052002) ps1a-c036 postnatal changes in the colocalization of vglut1 and vglut2 immunoreactivities at single axon terminals of the mouse neocortex kouichi nakamura 1,2 , akiya watakabe 3 , hiroyuki hioki 1 , fumino fujiyama 1 , yasuyo tanaka 1 , tetsuo yamamori 3 , takeshi kaneko 1,2 1 dept. morphol brain sci., grad. sch. med., kyoto univ., japan; 2 crest, jst, japan; 3 div. brain biol., nat. inst. basic biol., okazaki, japan vesicular glutamate transporter (vglut)1 and vglut2 accumulate transmitter glutamate into synaptic vesicles. the vgluts show a complementary expression pattern in the brain, but colocalize at single axon terminals in some synapses. here we quantitatively evaluated postnatal changes in the colocalization of vgluts at single axon terminals of the developing mouse neocortex by using a pixel-based correlation coefficient (cc) as an index of the colocalization. the cc was calculated from pixel values for vglut1 and vglut2 in each pixel of confocal micrographs of double immunofluorescence-labeled brain sections. in the barrels, the cc showed a prominent increase transiently around p7. the cc was higher in area s1 than areas m1 and area v1 throughout postnatal development. our results indicate that the colocalization of vgluts in the neocortex is regulated in an age-, area-and layer-specific manner. gaba b receptors mediate slow and prolonged synaptic inhibition in the brain, and are members of the g protein-coupled receptors. here we have investigated the role of 5 amp-activated protein kinase (ampk), as an endogenous regulator of gaba b receptor function. site-specific mutagenesis identified multiple phosphorylation sites for ampk within the cytoplasmic tails of both gaba b r1 and r2. the activation of ampk regulated stability of gaba b receptors coupling with k + channels. together highlights a novel role for ampk in regulating the functional properties of gaba b receptors, by direct phosphorylation. given the role of ampk as a sensor of cellular stress this potential mechanism may be relevant in regulating the efficacy of synaptic inhibition under anoxic conditions and during periods of high synaptic activity. takao hirai, hiroaki nishio department of molecular pharmacology, faculty of pharmacy and pharmaceutical sciences, fukuyama university, hiroshima, japan serotonin (5-hydroxytryptamine, 5-ht) is a central neurotransmitter that is widely implicated in the regulation of mood and cognition, and is a peripheral signaling molecule that affects hemostasis, immune function, intestinal physiology, and other systems. there is increasing evidence for contribution of neuronal system to regulation of bone metabolism. this study was thus aimed at elucidation of possible functional expression of serotonergic system in mouse osteoblasts. rt-pcr analysis revealed constitutive expression of mrna for several 5-ht receptor subtypes, 5-ht transporter (5-htt) and vesicular monoamine transporter 1 (vmat1) in primary cultured mouse osteoblasts and mc3t3-e1 osteoblastic cells. sustained exposure to fluoxetine, a selective 5-ht reuptake inhibitor, significantly prevented increase in alkaline phosphatase activities and mineralization in mc3t3-e1. these results suggest that serotonergic system may be functionally expressed to regulate mechanisms underlying cellular differentiation and maturation in mouse osteoblasts. junko motohashi department of physiology, keio university school of medicine, tokyo, japan hotfoot mice are spontaneous mutants with ataxic phenotype. most hotfoot alleles identified so far have deletions of one or more exons coding for portions of the n-terminal domain of the ␦2 glutamate receptor (glur␦2). however, because only genomic dna was available for most hotfoot mutants, it was unclear whether truncated forms of glur␦2 were actually translated and involved in the ataxic phenotype. here, we report that a newly identified hotfoot mutant, ho15j, was caused by a new type of intragenic deletion of the grid2 gene, which was indeed translated as glur␦2 lacking 52-amino acids in the n-terminus. mutant glur␦2 proteins were retained in the soma of purkinje cells and degraded. as a result, ho15j mice exhibited a severe motor discoordination on rotarod tests. furthermore, these mice exhibited sustained innervation of purkinje cells by multiple climbing fibers, and impaired long term depression, which is thought to underlie motor learning. these results indicate the importance of the n-terminal domain in glur␦2 signaling and cerebellar functions. research funds: kakenhi (16200024) ps1a-d040 role of the dry motif in melanin-concentrating hormone receptor 1 in signaling yumiko saito 1 , yoshimi aizaki 1 , mituse nakano 2 , kei maruyama 1 1 dept. pharamacol., saitama med. sch., saitama, japan; 2 international university of health and welfare, tochigi, japan considerable attention has been focused on the functional importance of the highly conserved dry triplet in class a g protein-coupled receptors (gpcr). here we investigated the role of asp140, arg141 and tyr142 in the dry of rat melanin-concentrating hormone receptor 1 (mch1r). in transfected cells, mutation of asp (d/a) resulted in nonfunctional receptor despite of showing moderate level of cell surface expression and an apparent affinity to mch. d/a mutation occurred with no increase in basal signaling pathway, suggesting no indication for constitutive activity. y/a mutation also yielded a loss of function phenotype that is similar to d/a mutation. mutation of the arg (r/a) showed higher ec50 value in signaling with a decrease in mch binding, while the level of cell surface expression exhibited only moderate decrease. these data suggest that a function for dry motif different from that widely accepted for class a gpcrs in regulating mch1r-mediated signal pathway. in this study we confirmed functional heteromultimerization between a 1 r and p2y 1 r electrophysiologically using xenopus oocyte expression system. when a 1 r and p2y 1 r were coexpressed, application of non-hydrolyzable atp analogue induced g i/o response, showing formation of functional heteromultimers with a unique phenotype. it was also observed that the heteromultimers can activate g q/11 pathway by atp analogue and also g i/o pathway by adenosine analogue, maintaining the features of the original subunits. ps1a-d043 dual signaling via metabotropic glutamate receptor1␣ is regulated by a cytoskeletal protein 4.1g michihiro tateyama 1,2 , yoshihiro kubo 1,2 1 department of biophysics and neurology, nips, aichi, japan; 2 sorst, jst, saitama, japan the g protein-coupled metabotropic glutamate receptor 1␣ (mglur1) is known to functionally couple to different types of g proteins. recently we have reported that the signaling pathways through mglur1 are differentially regulated by different types of ligands, glutamate and gd 3+ . on the other hand, several cytoskeletal proteins have been reported to interact with the c-terminal cytoplasmic tail of mglur1. these proteins, such as homer and 4.1g, are also known to change the membrane expression of and modulate the function of mglur1. here we investigated whether or not these cytoskeletal proteins regulate the multi path signaling of mglur1. interestingly, the functional couplings of mglur1 to gq and gs pathways were altered by co-expression of 4.1g, but not by homer. deletion of the c-terminal tail abolished the effect of 4.1g, indicating that the interaction of 4.1g with the c-terminal tail of mglur1 regulates the multi path signaling. ps1a-d044 modulation of the eaac1-mediated glutamate uptake by the addicsin mutant mitsushi j. ikemoto 1,2 , saori akiduki 1,2 1 age dimension research center, aist, ibaraki, japan; 2 graduate school of science, toho university, chiba, japan addicsin is a murine homologue of rat glutamate-transporterassociated protein 3-18 (gtrap3-18), an inhibitory modulator of neural glutamate-transporter excitatory amino acid carrier 1 (eaac1). it contains two potential pkc phosphorylation motifs at positions 18-20 and 138-140. however, its physiological function remains almost unknown. to clarify a significance of these pkc phosphorylation motifs, we investigated eaac1-mediated glutamate transport activity in c6bu-1 cells provided with a mifepristone-inducible expression of addicsin (wt), its mutants mutated at serine 18 into alanine (s18a) or at serine 138 into alanine (s138a). as compared with wt, s18a had no inhibitory effect on glutamate transport activity under exposure to 100 nm pma, and had increased glutamate transport activity under normal condition. by contrast, s138a had the same glutamate transport activity as that of wt. thus, the eaac1-mediated glutamate transport activity may be regulated by a pkc-dependent phosphorylation at serine 18 in addicsin. kaori akashi 1 , manabu abe 1 , toshikazu kakizaki 1 , rie natsume 1,2 , kenji sakimura 1,2 1 department of cellular neurobiology, brain research institute, niigata university, japan; 2 sorst-jst, saitama, japan kainate type glutamate receptors are composed of various combinations of glur␤1-3(glur5-7) and glur␥1-2(ka1-2) subunits. although their physiological functions and subunit compositions have been inferred from various studies, they are still not clear. to clarify the functions and subunit dynamics of kainate receptors, we generated glur␤2ko mice from c57bl/6 es cell line. the glur␤2ko mice were viable, fertile, and displayed no overt phenotype. on the other hand, the amounts of glur␥1 and glur␥2 proteins were significantly decreased in the crude fraction of ca3 region of glur␤2ko. furthermore, subcellular localizations of both subunits were also changed in glur␤2ko. these results suggested that native kainate receptors might function as heteromeric channels (glur␤/␥) and the glur␤2 subunit might determine subcellular localization of the glur␥ subunits, similar to the roles of nmda receptor glur subunits determining stability and distribution of the glur subunits. ps1a-d046 sema4d/plexin-b1 activates gsk-3␤ via r-ras gap activity, inducing growth cone collapse yuri ito, izumi oinuma, hironori katoh, manabu negishi laboratory of molecular neurobiology, graduate school of biostudies, kyoto university, kyoto, japan plexins are receptors for repulsive axonal guidance molecules semaphorins. we have recently reported that semaphorin 4d (sema4d) receptor plexin-b1 induces growth cone collapse by functioning as an r-ras gap. here we characterized the downstream signaling of plexin-b1-mediated r-ras gap activity, leading to growth cone collapse. sema4d suppressed the endogenous r-ras activity in hippocampal neurons, in parallel with dephosphorylation of akt and activation of gsk-3␤. ectopic expression of the constitutively active mutant of akt, myr-akt, or treatment with gsk-3␤ antagonist suppressed the sema4d-induced growth cone collapse. the r-ras gap activity was necessary for plexin-b1-induced dephosphorylation of akt and gsk-3␤. plexin-a1 also induced dephosphorylation of akt and gsk-3␤ through its r-ras gap activity. thus, we conclude that plexin-b1 dephosphorylates akt and gsk-3␤ through r-rasgap activity, inducing growth cone collapse. to find proteins having relations in receptor trafficking, we searched human genome database and selected hepatocyte odd protein shuttling (hops) as a candidate gene. hops had three transmembrane domains, and expressed abundantly on a brain tissue. hops was detected in membranous regions from subcellular fractionation and immunohistochemistry. hops was recruited to membranous structures when overexpressed in cos7 cells. when expressed in hippocampal cultures, hops enhanced the amplitude of mepsc. from antibody feeding assay, we discovered that hops enhanced the recycling of glur2. hops was co-immunoprecipitated with grip1 (glutamate receptor interacting protein 1) when they were co-transfected to hek cells. thus, it was suggested that hops had roles in synaptic transmission enhancement by stabilization of surface glur2 via grip1 binding. serine must be taken up into neurons for their survival, because neurons lack serine biosynthetic enzyme. we have recently identified a serine transporter asc-1. a neural amino acid transporter snat2/ata2 also transports serine. we investigated their roles as serine transporters by comparing the localization of these serine transporters in the rat brain. the asc-1 immunoreactivity (asc-1-ir) was detected in dendrites and somata of pyramidal neurons. the snat2-ir was widely detected in neurons, whose intracellular localization was similar to that of asc-1-ir. deferent from asc-1-ir, snat2-ir was also located in astrocytes and ependymal cells, especially around capillary blood vessels and ventricles. these results suggest the significant contribution of asc-1 and snat2 to the neuronal uptake of l-serine. snat2 might also accumulate l-serine in astrocytes from the extracellular spaces including blood and csf. ps1a-d049 neuronal glutamate transporter eaat4 controls climbing fiber-mediated presynaptic inhibition of gabaergic transmission at cerebellar interneuron-purkinje cell synapses shin'ichiro satake 1 , si-young song 2 , shiro konishi 3 , keiji imoto 1 1 natl. inst. physiol. sci. (nips), okazaki, japan; 2 mitsubishi kagaku inst life sci, tokyo, japan; 3 tokushima bunri univ., sanuki, japan through extrasynaptic diffusion and activation of presynaptic ampa receptors in bc terminals. we here examined possible roles of glutamate transporters in this cf action. the eaat4/glt-1 blocker threo-3-methylglutamate, but not the glt-1 blocker dihydrokainate, augmented the cf-induced inhibition. cf stimulation obviously inhibited gabaergic transmission onto pcs in the lobule iii, where eaat4 expression was low, whereas the cf-induced inhibition was minimal in the lobule x, where eaat4 was abundant. the results suggest that eaat4 plays a major role in regulating the concentration of cf transmitters, possibly glutamate, in the route of its extrasynaptic diffusion, and determining the degree of cf-induced inhibition of gaba release from bcs depending on the regional difference of eaat4 expression in postsynaptic pcs. chitoshi takayama 1 , yoshiro inoue 1 1 department of molecular neuroanatomy, hokkaido university school of medicine, sapporo, japan gaba mediates inhibitory transmission in the adult central nervous system (cns). in contrast, gaba induces depolarization in the immature cns. this developmental shift from depolarization to hyperpolarization may be caused by decreasing of the intracellular chloride ion concentration regulated by two chloride ion co-transporters, na-k-2cl co-transporter 1 (nkcc1) and k-cl co-transporter 2 (kcc2). in this study, we focused on kcc2, which lowers the intracellular chloride ion concentration, and examined the developmental localization of the kcc2 with special reference to the neuronal development in the cerebellum. kcc2 was negative in the proliferating and migrating neurons. post-migratory neurons, which formed synapses, expressed the kcc2. the kcc2-protein was localized at the membrane of dendrites and cell bodies, whereas growth cones, axons and terminals were negative. these results suggested that formation of synapses might induce kcc2-expression and localization, and gabaergic transmission might shift from excitation to inhibition after synapse formation. akinori nakajima 1 , hisashi mori 1 1 molecular neuroscience, university of toyama, toyama, japan the actions of many neurotransmitters are mediated by the members of a superfamily of receptors coupled to heterotrimeric guanine nucleotide binding proteins (g-proteins). the dopamine receptors are classified into two categories, d1-like and d2-like according to their pharmacological properties. the d1-like receptors consist of d1 and d5 receptor, and are coupled to the adenylyl cyclase activating g proteins (gs). in the present study, we have generated a series of d1 receptor mutants and examined the effect on the gs coupled receptor signaling. we found that the expression of the third intracellular loop (3i loop) domain of d1r fused with egfp effectively reduce camp production mediated by d1 and d5 receptors. interestingly, we also identified that the 3i loop domain of d1r interfere with gs coupled beta2 adrenergic receptor signaling. these results suggest that the third intracellular loop of the d1 receptor is a primary determinant in its coupling to gs signaling. the activation of phosphatidylinositol-linked d1-like dopamine receptor profoundly suppresses the exaitatory transmission in the developing hippocampus yoshinobu noriyama 1 , yoichi ogawa 2 , hiroki yoshino 1 , masayuki yamashita 2 , toshifumi kishimto 1 1 dept. psychiatr.; 2 dept. physiol i, nara med. univ., kashihara, japan we studied the effect of dopamine (da) on gabaergic and glutamatergic transmission in neonatal rat hippocampus from the early period of synapse formation by whole-cell patch-clamp recordings from ca1 pyramidal cells. da (100 m) profoundly decreased gaba a receptor-mediated postsynaptic currents to 32% in the first postnatal week, when gaba provides excitatory drive. da also decreased ampa receptor-mediated excitatory post synaptic currents to 29% in the second postnatal week, when glutamate responses first appear. the da-induced inhibition declined after these periods. the receptor subtype involved in the da-induced inhibition was phosphatidylinositol (pi)-linked d1-like receptor, since skf 83959, a selective agonist for pi-linked d1-like receptor, clearly mimicked the action of da. these results suggest that the activation of pi-linked d1-like receptor profoundly suppresses the excitatory transmission during the early period of synapse formation in the developing hippocampus. ayuka ina 1 , jinko konno 1 , sachine yoshida 1 , hideki ohmomo 1 , hitoshi kawano 2 , fumihiro shutoh 1 , haruo nogami 1 , setsuji hisano 1 1 graduate sch., comprehensive human sci, univ. tsukuba, tsukuba, japan; 2 tokyo metro inst neurosci, tokyo, japan supporting critical neurobiological roles of glutamate in mouse corticogenesis, we recently reported that cortical cells express vglut1 or -2 mrna at early fetal ages. to know roles of fetal vglut in cortical development, we studied expressions of vglut proteins in mouse fetuses by immunohistochemistry. on embryonic day 13 (e13), vglut1 immunoreactivity (ir) was first detected in the marginal zone (mz), subplate (sp) and intermediate zone (imz). on e15, vglut1-ir was seen as puncta close to l1-ir thalamocortical fiber tracts in the sp and also localized to fiber tracts expressing l1or tag1-ir in the imz, whereas vglut2-ir was first observed in the sp and upper imz where l1-ir existed. these results show that vglut1ir corticofugal fibers appose to elongating vglut2-ir thalamocortical fibers, suggesting that vglut may play a crucial role in glutamatemediated axon guidance to determine thalamic innervation patterns in the developing cortex. ps1a-d054 developmental changes in the mechanism underlying activity-dependent swelling of the hippocampal ca1 regions michie kon 1 , yoichi avil 2 , hiroshi tsubokawa 1,2 1 dept. of information engineering, tohoku univ., sendai, japan; 2 grad. schl. of information sciences, tohoku univ. to investigate the mechanisms underlying swelling of brain cells in association with neuronal activity, we analyzed interactions between changes in cell volume and synaptic activities in mouse hippocampal slices. swelling of several areas within the ca1 region were detected as increases in transmittance of near infrared light (irt). field epsps (feps) were recorded simultaneously from the stratum radiatum of ca1 region. in adult mice, repetitive stimulation of afferent fibers induced transient increases in irt at both somatic and dendritic regions in a frequency-dependent manner, which was temporally associated with feps. application of the bicuculline, a gaba-a receptor antagonist, reduced these optical signals. however, in mice under 15 days old, the optical signals did not follow by high-frequency stimulation of inputs, and were not affected by an application of bicuculline. these results suggested that gaba-dependency in the mechanisms of cell volume regulation developmentally changes in the hippocampal ca1 region. in the present study, the effects of bilateral injections of glutamatergic agents into the hippocampal ca1 region on morphine-induced conditioned place preference (cpp) were investigated in rats. subcutaneous administration of different doses of morphine (2.5-10 mg/kg) produced a dose-dependent cpp. using a 3-day schedule of conditioning, it was found that intra-ca1 administration of nmda receptor antagonist, mk-801 (2 and 4 g/rat) significantly attenuated the morphine (7.5 mg/kg)-induced cpp. moreover, nmda receptor agonist, nmda (0.1, 0.2 and 1 g/rat) significantly potentiated the morphine (2.5 mg/kg)-induced cpp. these results suggest that the development of morphine-induced cpp may be related to nmda and mk-801 receptors in that the glutamatergic system can modulate opiate reward. takahiro sonomura 1 , kouichi nakamura 2,3 , hiroyuki hioki 2 , masanori uemura 1 , takeshi kaneko 2,3 1 dept. anatomy for oral sciences, grad. sch. med. and dent., kagoshima univ., kagoshima, japan; 2 dept. morphological brain sciense, grad. sch. med., kyoto univ., kyoto, japan; 3 crest, jst the majority of neostriatal neurons are medium-sized projection neurons with spiny dendrites and have so far been classified into three groups: striatonigral neurons producing ppd, and striatopallidal neurons producing ppe, and striatoinnominatal neurons producing pptb. these projection neurons are regulated in part by dopaminergic input from the substantia nigra pars compacta. it has been assumed that d1 receptor are expressed in striatonigral neurons and d2 receptor are expressed in striatopallidal neurons. in recent years, molecular cloning work has shown that there are at least five dopamine receptor genes (d1, d2, d3, d4, d5). in this study, the double-labeling method combining in situ hybridization and immunocytochemistry revealed how these five dopamine receptor subtypes are distributed among three projection neuron groups. the cerebellar tissue is good model system for the analysis of neuronal development, since dynamic neuronal development such as migration and axonal and dendritic outgrowth after birth. in the present study, we examined the localization of chondroitn sulfate proteoglycans (cspgs) by cspg-specific antibodies and lectins. cspgs are mainly observed at molecular layer in developing cerebellum (p10-15) but they scarcely seen at external granular layer. electron microscopic observation demonstrated that phosphacan, one of cspgs, is localized at axonal membrane of parallel fibers. moreover, phosphacan inhibited adhesion and axonal extension of cerebellar granular neurons, while it promoted axonal fasciculation of their aggregated cultures. thus, cspgs, inhibitory molecules for axonal extension, are participated in axonal guidance cue in developing cerebellum. the vasopressin neurons are well knwon to show structural plasticity during chronic physiological stimulation such as salt loading. in the present study, salt loading significantly diminished the levels of chondroitin sulfate proteoglycans (cspgs) in vasopressin neurons. this downregulation is possibly due to proteolysis by tpa, since (1) tpa immunoreactivity was observed at neurosecretory granules of vasopressin dendrites and terminals, (2) salt loading increased protein and mrna levels of tpa in the somata and dendrites in the supraoptic nucleus but reduced protein levels of it in the terminals of the neurohypophysis, (3) depolarizing agent released tpa from isolated neurosecretosomes, (4) tpa knockout mice revealed lower ability of osmotic homeostasis and vasopressin release. thus, it is probable that tpa is participated in regulating structural plasticity of vasopressin neurons by degrading cspgs. chondroitin sulfate (cs) proteoglycans are essential for neuronal morphogenesis, including neural migration, survival and neurite formation in the developing brain. cs chains are modified by various sulfotransferases generating diverse sulfation patterns, which are assumed to be involved in the selective binding to various proteins such as growth factors. in this study, we analyzed the expression patterns of several cs sulfotransferases in the developing mouse cerebrum. using in situ hybridization analysis, it was revealed that cs sulfotransferase mrnas (u2st, galnac4-6st, d4st) were expressed in various types of cells, especially in the ventricular zone, and the cortical plate neurons just below the marginal zone. immunohistochemical analysis with anti-cs antibodies revealed that cs were highly expressed in the ventricular zone and the marginal zone. these results suggest that the cs structural domains generated by these cs sulfotransferases are involved in the regulation of the proliferation of neural progenitor cells and neuronal migration. nobuaki maeda 1 , maki ishii 1 , isao nagata 2 , yumiko shimazaki 1 1 dept. of dev. neurosci., tokyo metro. inst. for neurosci., tokyo, japan; 2 dept. of brain structure, tokyo metro. inst. for neurosci. chondroitin sulfate (cs) is a long polysaccharide with enormous heterogeneity that binds with various proteins in a structure-dependent manner. previously, we revealed that cs is involved in the morphogenesis of the purkinje cell dendrites. in this study, we analyzed the expression of cs in the postnatally developing cerebellum using monoclonal antibodies that recognize specific structural motifs in cs. among the epitopes recognized by these antibodies, the expression of mo-225 epitopes, glca(2s)␤1-3galnac(6s) (d unit)containing structures, remarkably increased during development. detailed immunohistochemical analysis indicated that d unit-rich cs was deposited between purkinje cell surface and the processes of bergmann glia. furthermore, it was found that pleiotrophin bound to d unit-rich cs on phosphacan distributed around purkinje cells. these observations suggest that d-type structure in cs is important for the signaling of pleiotrophin, which play roles in purkinje cell-bergmann glia interaction. nobuna fukazawa 1 , mineko kengaku 2 , nobuaki maeda 1 1 dept. of dev. neurosci., tokyo metro. inst. for neurosci., tokyo, japan; 2 lab. for neuronal cell polarity, riken bsi, wako, japan ptp is a receptor-type protein tyrosine phosphatase, which is synthesized as a chondroitin sulfate proteoglycan that pleiotrophin-ptp signaling regulates the morphogenesis of purkinje cell (pc) dendrites. we previously revealed that ptp associated with delta/notchlike egf-related receptor (dner), which mediates the pc-bergmann glia (bg) interaction and regulates morphological differentiation of these cells. here, we found that ptp was expressed by both pcs and bgs and the expression by pc occurred at relatively late developmental stage. ptp showed patchy distribution in the dendritic shafts of pcs, which partially overlapped with the localization of dner. furthermore, we revealed that multiple tyrosine residues in the cytoplasmic domain of dner were phosphorylated and that these tyrosine phosphorylated residues were efficiently dephosphorylated by the ptp catalytic domain. these results suggested that ptp participate in the pc-bg interaction by regulating tyrosine phosphorylation level of dner. masahiko tanaka 1 , tohru marunouchi 1 1 division of cell biology, institute for comprehensive medical science, fujita health university, toyoake, aichi, japan cerebellar purkinje cells have the most elaborate dendritic trees among the neurons in the cns. to investigate the cellular and molecular mechanisms of dendrite development of purkinje cells, we cocultured purkinje cells on a coverslip with other cerebellar cells such as granule cells and astrocytes on the cell culture insert of 3 m pore size. when purkinje cells were co-cultured with granule cells, dendrite development of purkinje cells was promoted in comparison with that in control conditions. this co-culture effect was abolished by addition of a glutamate antagonist in the cultures. in contrast, dendrite development of purkinje cells was inhibited when purkinje cells were co-cultured with astrocytes. we propose that (i) glutamate secreted by granule cells and diffused through the porous membrane of the cell culture insert promotes the dendrite development of purkinje cells and (ii) astrocytes inhibit the effect of glutamate through their glutamate transporting activity. heparan sulfate (hs) proteoglycans regulate neural development through the interaction with cell surface proteins and extracellular matrix molecules. an extracellular endosulfatase, sulffp1, has been implicated in the regulation of growth factor/morphogen signaling through hs remodeling in vitro, but its physiological roles remain unknown. here we generated knockout mice lacking the sulffp1 gene, and examined the motor control. homozygotes appeared to be normal, showing no sign of ataxia. performances of the rotarod and beam-walking tests were normal compared with the control mice. both short-term and long-term adaptations in the optokinetic response were normal, while the gains in optokinetic response and vestibulocular reflex were significantly reduced. heparan sulfate (hs) proteoglycans regulate a number of developmental signaling through interactions with cell surface proteins and extracellular matrix molecules. these interactions are mediated by the specific sulfation patterns in hs, but the mechanism generating such modifications has not been fully elucidated. here we show that a new class of hs endosulfatases plays an important role in brain development. the mice deficient in either sulffp1 or sulffp2 appeared to be normal, while most of the double knockout mice died soon after birth. mutant brains had higher content of 6-o-sulfated disaccharide units in hs, suggesting a role of sulffps in heparan sulfate remodeling in vivo. the double mutant brains were smaller than the controls and showed some axon guidance defects. these data demonstrate that specific hs modification generated by sulffps is important for normal brain development. recent studies have suggested monoamine affects neural development, but it is unclear which receptor subtypes mediate actions of monoamine. here, we examined roles of 5-hydroxytryptoamine (5-ht), noradrenaline (na) and dopamine (da) in the formation of dendrites and synapses by dissociation culture. embryonic day 16 or 18 rat cerebral cortex was cultured in the presence of 5-ht, na or da. after 4 days, we analyzed dendrite formation using anti-map2 antibody. after 14-21 days, we analyzed synaptogenesis with anti-psd-95, anti-synaptophysin, and anti-map2 antibodies. the addition of 5-ht (100-10000 nm), na (100-1000 nm) or da (10-1000 nm) increased dendritic length of pyramidal neurons. 5-ht (100-1000 nm) also increased the synaptic density. by using receptor agonists and antagonists, it was suggested that dendritic outgrowth may be promoted by 5-ht 1a receptor, ␣ 2a receptor and d 2 receptor, while inhibited by 5-ht 2a and ␤receptors. in addition, synaptogenesis was promoted by 5-ht 2a and 5-ht 2c receptors, whereas inhibited by 5-ht 1a receptor. tatsuya mori, tomoe wada, takahiro suzuki, naoyuki inagaki department of cell biology, nara institute of science and technology, nara, japan most neurons have polarized shape consisting of a single long axon and multiple dendrites. several proteins have been implicated in the establishment of neuronal polarity; however, the mechanism for neuronal polarization is not well understood. in this study, with proteomic approach, we identified a novel protein, singar, as one of the proteins which are up-regulated during neuronal polarization of rat cultured hippocampal neuron. singar was expressed specifically in brain and developmentally up-regulated during neuronal polarization in vitro and in vivo. in 293t cell, singar associated with p85 and p110, the subunits of pi3kinase which is considered as one of the key molecules in neuronal polarization. moreover, inhibition of singar by rna interference induced the formation of multiple axon-like neurites. these data suggest that singar ensures the formation and maintenance of neuronal polarity by suppressing the formation of surplus axons. ps1a-e068 lrfn2, a neuronal leucine-rich repeatcontaining transmembrane protain can interact with psd-95 naoko morimura, takashi inoue, kei-ichi katayama, jun aruga laboratory for comparative neurogenesis, riken bsi, saitama, japan in a variety of organisms, proteins with leucine-rich repeat domain (lrr) function significantly in neural development. lrfn, a neuronal lrr transmembrane family, was expressed in the brain specifically. expression of lrfn2 was low in embryonic brain, and increased dramatically after birth. in the rat dissociated hippocampal neurons, lrfn2 protein was detected predominantly at mature dendrites, where it was accumulated at spines and colocalized with psd-95, a postsynaptic scaffold protein. we examined the physical interaction between lrfn2 and psd-95 by immunocrecipitation and pull-down assay, since lrfn2 contains class i pdz domain-binding motif at its c-terminal tail. we revealed that lrfn2 associated with psd-95/nmda receptor complex in the brain extracts and lrfn2 directly bound to psd-95 via its pdz domain-binding motif. in this study, we suggest that lrfn2 may play an important role in the regulation of synaptic functions. tsuya taneda, shingo miyata, hiroaki okuda, masaya tohyama department of anatomy and neuroscience, graduate school of medicine, osaka university, osaka, japan protein arginine methylation is a common post-translational modification catalyzed by a family of protein arginine n-methyltransferases (prmt1-8). among the prmt proteins, the prmt3 has some characteristic motifs in the n-terminal tract which follows its active methyltransferase site. although little attention has been paid to protein methylation in the nervous system. first of all, we have examined the distribution of the prmt3 in the rat brain. the prmt3 was expressed in the cell bodies and dendrites in the hippocampal neurons. further, the ontogenetic analysis revealed the prmt3 expression increased from the perinatal stages to the adulthood. these findings suggest that the prmt3 relates to the neural function in the young and adult brain. furthermore in order to study the role of the prmt3 in the brain, we tried to identify novel interacting proteins with the prmt3 in rat hippocampal neurons using tandem affinity purification assay coupled with mass spectrometry. ps1a-e070 proteomics of the growth cone: ii. the systematic immunostaining analysis of the growth cone proteins identified by the proteomic research motohiro nozumi 1,2 , michihiro igarashi 1,2 1 div mol cell biol, grad. sch. med dent sci; 2 trans-diciplinary res program, niigata univ., niigata, japan proteomics is a powerful method to understand the molecular composition of a given cell or a compartment of the cell. in the accompanying paper, we applied this method to the growth cone from the rat forebrain, and we identified more than several hundred proteins there. although the proteins have been determined using the powerful methods, the localization of each protein in the neuron should be confirmed; thus, we checked the immunostaining in the cultured rat cortical neurons. currently, we have already performed the immunocytochemistry concerning more than 150 identified proteins including cytoskeletal components, signaling molecules, receptors, and cell adhesion molecules. by quantitative analyzing the fluorescent intensity using the digital imaging, we classified the growth cone proteins into several groups. we have found more than twenty proteins specifically localized in the growth cone by this analysis. research funds: kakenhi 17023019; project-promoting grant from niigata univ ps1a-e071 netrin-1 is involved in the sensory axonal projection toward the spinal cord as a repulsive guidance cue tomoyuki masuda 1 , keisuke watanabe 2 , kazuhiro ikenaka 2 , katsuhiko ono 2 , hiroyuki yaginuma 1 1 dept. anat., fukushima med univ. sch. of med., fukushima, japan; 2 div neurobiol bioinfo, nat inst physiol sci, aichi, japan in higher vertebrate embryos, the ventral spinal cord exerts chemorepulsion for dorsal root ganglion (drg) axons to orient them toward their targets. netrin-1 is known to be a chemorepellent for a subset of axons, the role of netrin-1 for ventral spinal cord-derived repulsion is, however, unknown. by employing culture assays, we report here the involvement of netrin-1 in this repulsion. in the mouse embryo at e11, netrin-1 is expressed in the floor plate and the dermamyotome, and the netrin-1 receptor unc5c is expressed in drg neurons. we show that hek-cell aggregates secreting netrin-1 repelled chick e5 drg axons. moreover, using function-blocking antibody against netrin-1, we revealed the fact that netrin-1 plays an important role in ventral spinal cord-derived repulsion. together, these findings suggest that the ventral spinal cord repels drg axons by secreting netrin-1 to shape the initial trajectories of drg axons. research funds: grants-in-aid on priority area (c) (mecst 17590166 to t.m.) hitoshi maeda, masaki sakurai department of physiology, teikyo university school of medicine, tokyo, japan in the previous reports, we showed that in the early development, corticospinal synapses (cs) were formed widely in the spinal gray matter but those in the ventral side were eliminated later in an activity dependent manner. however, the property of postsynaptic cells to cs input is poorly understood. in the present study, we investigated the electrophysiological and morphological properties of the neurons that receive cs synapses in the acute spinal cord slices of neonatal rat. the postsynaptic neurons that were confirmed by the stimulation of the posterior funiculus, where the cs tract is located in rodents, were whole cell patch clamped and labeled by neurobiotin tm . responsive neurons are widely distributed in the p6 neonates, but the ventral neurons became unresponsive after p9. the majority of the ventral neurons are of multipolar type with large somata showing "repetitive" or "phasic" firing patterns; on the other hand, most of dorsal neurons have smaller somata and multipolar branches with "single" or "phasic" patterns. keisuke watanabe 1 , hirohide takebayashi 1,2 , kazuhiro ikenaka 1 , katsuhiko ono 1 1 div. neurobiol. bioinfo., natl. inst. physiol. sci., okazaki, japan; 2 dev stem cell biol. program, ucsf, usa netrin-1 is a long-range diffusible factor that exerts chemoattractive or chemorepulsive effects on developing axons growing to or away from the neural midline. however, it is not known whether netrin-1 also exerts chemoattractive effect on ventral-ward migrating dorsal interneurons in the developing spinal cord. to test this hypothesis, we examined dorsal interneuron migration in netrin-1 −/− background, using olig3-lacz knockin allele, which marks most of ventral-ward migrating dorsal interneurons. in the embryonic spinal cord of olig3 +/lacz ;netrin-1 −/− mice, ventral migration of olig3 cells was significantly impaired. furthermore, a netrin receptor, dcc was expressed in olig3-positive cells. these results suggest that netrin-1 exerts chemoattractive effects on ventral-ward migrating dorsal interneurons in vivo. netrin-g1 and netrin-g2 are vertebrate-specific membrane-anchored members of the unc-6/netrin family that have no affinity to classic netrin receptors and their function is unknown. here we show that netrin-g1 and netrin-g2 proteins are selectively distributed on axons of distinct pathways, and each interacts with a specific receptor on target dendrites. netrin-g1 and netrin-g2 differentially bind to lrrcontaining proteins, ngl-1 and a related molecule nag14, in vitro. ngl-1 and nag14 in the mouse brain are concentrated in distinct dendritic segments, corresponding to lamina-specific termination of axons expressing netrin-g1 and netrin-g2, respectively. furthermore, in netrin-g1 and netrin-g2 deficient mice, in which axonal pathfinding is normal, there is selective mislocation of individual receptors within dendrites. together, these results suggest that axonal netrin-g proteins transneuronally regulate the localization of distinct receptors on dendrites, and thereby determine the properties of subdendritic segments. jinhong huang 1 , ryuichi sakai 2 , teiichi furuichi 1 1 lab. molecular neurogenesis, brain science institute of riken, saitama, japan; 2 division of cell growth factor, national cancer center of japan, tokyo, japan cas is a tyrosine-phosphorylated docking protein that is indispensable for the regulation of actin cytoskeletal organization and cell migration in fibroblasts. the neuronal function of cas, however, is poorly understood. here we report that cas is dominantly enriched in the brain, especially the cerebellum, of postnatal mice. during cerebellar development, cas is highly tyrosine phosphorylated and is concentrated in the neurites and growth cones of granule cells. cas coimmunoprecipitates with src family protein tyrosine kinases, crk, and cell adhesion molecules. the axon extension of granule cells is inhibited by either rna interference knockdown of cas or overexpression of the cas mutant lacking the crk binding motifs. these results demonstrate that cas acts as a key scaffold to link the proteins associated with tyrosine phosphorylation signaling pathways to the granule cell axon elongation. research funds: huang was a postdoctoral fellowship recipient of jsps in in vitro cerebellum-pons-medulla block preparations isolated from neonatal rats on p4-p8, stimulation of parallel fibers produces excitation of purkinje cells lasting for 600-800 ms. this unusually prolonged response is observed in the lateral region of the cerebellum (paraflocculus/flocculus), where purkinje cells develop primary dendrites on p5-p7. since -agatoxin iva and -conotoxin mviic abolished the prolonged response, we suggest the involvement of p/q type ca channels. immunohistochemical labeling revealed that p/q type ca channels emerged in paraflocculus/flocculus and uvula/nodulus lobules on p4 and that they then locate in purkinje cells, in cell body on p5 and in primary dendrites on p6-p7. the parallel development of p/q type channels, primary dendrites, and the occurrence of prolonged parallel fiber-purkinje cell transmission suggests their causal relationships. naoya ichikawa, yasuo kitagawa, tatsuhiko kadowaki graduate school of bioagricultural sciences, nagoya university, aichi, japan we have recently identified a novel gene, mahya, which is specifically conserved between hymenoptera and deuterostome. mahya encodes a secretory protein with a follistatin-like domain, two immunoglobulin domains, and a c-terminal novel domain. mouse mahya genes (mmahya-1 and mmahya-2) are expressed in the olfactory bulb, hippocampus, and cerebellum of the adult brain. we have found that mmahya-1 protein is specifically synthesized in the pre-migratory granule cells and localized at the molecular layer of the postnatal cerebellum. these results suggest that mmahya-1 is involved in either the migration of granule cells or the dendritic maturation of purkinje cells. we will further report the analysis of the functions of mmahya-1 for the early cerebellum development. toshitaka morishima 1 , erina fukushi 1 , kazuto kobayashi 2 , naohiro hozumi 1 , sachiko yoshida 1 1 toyohashi university of technology, toyohashi, japan; 2 honda electronics co. ltd., toyohashi, japan in cerebellar development, granule cells migrate with elongation their axon, called parallel fibers, and form neuronal circuit in molecular layer. although density and thickness of parallel fibers are important information for cerebellar development, few were simple and useful methods. we have proposed a new method for two-dimensional acoustic impedance imaging for developing cerebellar slices. an acoustic impedance microscopy was obtained by mechanically scanning the transducer and the reflection intensity was interpreted into local acoustic impedance of no treated acute slices with no invasion. the developing parallel fibers were clearly observed as the contrast in acoustic impedance, whereas they were cloudy in immature egl from neonatal rat. the reflection from molecular layer enlarged and floated to deep layer, so that its spatial pattern was changed during cerebellar development. this imaging method is believed to be a powerful tool for observation of neuronal development, as neither fixation nor staining is required. tatsuro yamamoto 1 , hideyuki dekimoto 1 , tomiyoshi setsu 1 , masahiko watanabe 2 , mikio hoshino 3 , yo-ichi nabeshima 3 , toshio terashima 1 1 dept. of anat., kobe univ. grad. sch. of med., kobe, japan; 2 dept. of anat., hokkaido univ. grad. sch. of med., sapporo, japan; 3 dept. of pathol and tumor biol, grad. sch. of med., kyoto univ., kyoto, japan a mutant mouse, cerebelles (cbll), lacks the entire cerebellar cortex but survives into the adult. the responsible gene for this mutation is ptf1a, whose expression is lost in this mutant. in the present study, we examined cerebellar afferent and efferent systems of this mutant mouse by neural tracing methods with a combination of immunohistochemistry. the injection of fluoro-gold (fg) into the cbll thalamus resulted in retrograde labeling of neurons in the contralateral cerebellar nuclei. these fg-labeled neurons were glutaminase-positive. after the injection of bda into the cbll lumbar cord, spinocerebellar terminals projecting to the deep cerebellar nuclei were anterogradely labeled in spite of absence of the cerebellar cortex. these findings suggest that afferent and efferent systems of the cerebellar nuclei of the cbll are preserved in spite of absence of the cerebellar cortex. kumiko ishida 1 , tomoko nishiyama 1 , hitoshi tatsumi 1 , masahiro sokabe 1,2 1 department of physiology, nagoya university graduate school of medicine, nagoya, japan; 2 icorp, cell mechanosensing project, japan science and technology corporation sprouting and synaptic reorganization of the mossy fiber (mf) are commonly found in the hippocampus of temporal lobe epilepsy patients. as the muscarinic agonist, pilocarpine, can induce similar morphological changes, hippocampal slices treated with this drug have been widely used as a model of epilepsy. we found that pilocarpine induced a transient retraction and subsequent elongation of the neurites of granule cells in the slice cultures; the retraction was peaked approximately 12 h and the elongation started at approximately 24 h after the drug application. tetrodotoxin strongly inhibited both the retraction and elongation, while the bdnf sequestering protein, trkb/fc, retarded only the elongation. this result suggests that na + channel dependent neuronal excitation and following activitydependent bdnf releases are essential in the biphasic morphological changes induced by pilocarpine in hippocampal slices. rieko muramatsu, yuji ikegaya, maki k. yamada, norio matsuki, ryuta koyama laboratory of chemical pharmacology, graduate school of pharmaceutical sciences, the university of tokyo hippocampal granule cells extend their axons, i.e. the mossy fibers (mfs), from the dentate gyrus (dg) to the area ca3. once this oneway projection is disrupted, the mfs retrogradely innervate granule cell dendrites and make excitatory synapses that induce epileptic neural activities in the dg. to clarify the mechanism that regulates normal, anterograde mf projections, we used a co-culture system of hippocampal slices. when a dg slice from a gfp(+) rat was juxtaposed to the ca3 region of a host hippocampal slice from a wild type rat, the gfp(+) mfs ran through the host ca3 toward the host dg but failed to invade it even after ten days in vitro. thus the dg seemed to serve as a barrier that blocks retrograde projections of mfs. however, the mfs extended into the dg when forskolin, an activator of adenylate cyclase, was chronically applied. these results suggest that the dg has a mechanism supporting anterograde mf projections to ca3, which is regulated by the levels of adenylate cyclase activation. calcitonin gene-related peptide (cgrp) is a 37 amino acid neuropeptide that is widely distributed in central and peripheral nervous systems. cgrp is expressed from early developmental stage in rat brain, suggesting that cgrp may be involved in not only neurotransmission but also neural development. but roles of cgrp in neuronal development of cerebral cortex and hippocampus remain unclear. in the present study, we made dissociation culture of cerebral cortex and hippocampus of embryonic day (e) 16 or e18 rat. dendritic outgrowth of pyramidal neurons was analyzed after 4 days using anti-map2 antibody. synapse formation was analyzed after 2-3 weeks, using anti-psd-95 and anti-synaptophysin antibodies. in the presence of cgrp (10-1000 nm), both dendritic length and synaptic density were increased. however, the number of dendritic branching was not affected. these results suggest that cgrp promotes dendritic outgrowth and synapse formation. chisako kanamaru, kazunori suda, kouji senzaki, takashi shiga university of tsukuba, graduate school of comprehensive human sciences, tsukuba, japan recent studies have suggested monoamine affects neural development, but it is unclear which receptor subtypes mediate actions of monoamine. in this study, we examined roles of 5hydroxytryptoamine (5-ht) and noradrenaline (na) in the formation of dendrites and synapses using dissociation culture of rat hippocampus. embryonic day 18 rat hippocampus was cultured in the presence of 5-ht or na. after 5 days, we analyzed formation of dendrites using anti-map2 antibody. after 21 days, we analyzed formation of synapses using anti-psd-95, anti-synaptophysin, and anti-map2 antibodies. the addition of 5-ht (500 nm) or na (500 nm) increased dendritic length and number of branches of pyramidal neurons, whereas decreased number of primary dendrites 5-ht (100-1000 nm) and na (10-1000 nm) also increased the synaptic density. by using receptor agonists and antagonists, it was suggested that ␣ 2a receptor promotes dendritic outgrowth, while ␤ receptor suppress dendritic outgrowth and branching. in addition, 5-ht 2a receptor and ␣ 2a receptor promote synapse formation. kenji amano down syndrome cell adhesion molecule (dscam) knock-out (ko) mouse died within 24 h after the birth. to investigate possible etiology of the neonatal death, we examined the respiratory activity using whole body plethysmography and the c4 inspiratory activity using brainstem-spinal cord preparation. the respiratory activity of dscam-ko mice using plethysmography was irregular frequency and small amplitude accompanied with apnea. furthermore, c4 inspiratory activity also showed irregular frequency and narrow duration of the bursting. we then analyzed spatio-temporal pattern of the respiratory neuronal activity using combination of the voltage-sensitive dye (di-2 anepeq) and the imaging system (micam02). in dscam-ko mice, the optical signal which precedes c4 inspiratory activity was depressed. these results suggest that pre-inspiratory neuronal network, which determines respiratory rhythm, does not develop normally in dscam-ko mice and causes lethal respiratory dysfunction. ps1a-f088 hippocampal cells cultured on 3d collagen substrate secrete a dense extracellular matrix, supporting neuritic outgrowth shantanu sur, thomas launey, masao ito brain sc. inst., riken, japan the brain extracellular matrix (ecm) influences neuronal migration and morphogenesis. we explored how hippocampal cells modify their extracellular environment when seeded onto collagen gel, a major component of the ecm. after 2 weeks in vitro, neurons formed a dense layer, >0.1 mm below the gel surface, with neurite outgrowth toward the surface, within the top gel layer (tgl). initially, we thought that hippocampal cells were penetrating the gel, following partial degradation of the collagen matrix. however, (1) collagenasespecific inhibitor did not affect cell depth, (2) limiting gliosis by antimitotics reduced the thickness of the tgl by 40%, (3), neither glial nor neuronal cell body were found in the tgl by gfap/map2 detection, (4) neurite outgrowth was observed only within this tgl, but not toward the bottom of the gel. to see whether the tgl is the remains of the initial collagen substrate, we embedded fluorescent beads in the collagen gel before cell seeding. the tgl was completely devoid of beads after 2 weeks, suggesting that the tgl is newly formed by ecm material, largely secreted by glial cells. emi kumamaru, tadahiro numakawa, yuki yagasaki, hiroshi kunugi disorder research, national institute of neuroscience, ncnp, tokyo, japan the level of glucocorticoid is regulated through hpa axis, and glucocorticoid itself has a negative feedback effect on hpa axis. however, under the intense stress, the glucocorticoid level is increased, and the high level of it is suggested to induce neuronal damage and to cause the mood disorder. on the other hand, it is possible that the reduction of neuronal function mediated by bdnf is partly related to the cause of the disorder. therefore, in the present study, we investigated the effect of glucocorticoid (dexamethasone, dex) on synaptic maturation and function enhanced by bdnf in early developing hippocampal neurons. we found that bdnf increased the expression of synaptic proteins including glutamate receptor and presynaptic protein, however, pretreatment with dex significantly inhibited the up-regulation of these proteins by bdnf. further, increase in release of glutamate and in intracellular ca2+ by bdnf was suppressed after dex pretreatment, suggesting that dex inhibits the maturation of synaptic function mediated by bdnf. takashi ueyama 1 , kazuto kujira 1 , tetsuya kawabe 2 , takao ito 1 , yoshihiro tsuruo 1 1 department of cell biology and anatomy, wakayama medical university, wakayama, japan; 2 department of cardiovascular medicine, wakayama medical university, wakayama, japan in this study, we investigated the effect of castration on the emotional stress response in the brain by comparing the c-fos expression in response to immobilization stress (imo) between castrated rats (cast) and sham-operated rats (sham). increased c-fos immunoreactive cells in response to imo were observed in septum, thalamus, hypothalamus, midbrain, pons and medulla oblongata in accordance with previous findings. in cast compared with sham, the numbers of c-fos-ir cells were significantly lower in the medial parvocellular part of paraventricular hypothalamic nucleus, while they were significantly higher in the supraoptic nucleus and medial amygdaloid nucleus. these data suggest that neuronal activity in these areas is influenced by systemic androgen level. this may underlie the pathophysiology of partial androgen deficiency in aged men (padam). research funds: grant-in-aid for scientific research (c) (17590600) ps1a-f091 metabolic and glucagon response of a genetically heat-tolerant rat to ambient heat and cold fujiya furuyama 1 , hitoo nishino 1 , takehiro yahata 2 1 nagoya city university graduate school of medical sciences, nagoya, japan; 2 nayoro city college, nayoro, japan the inbred fok rat was developed by us using heat selection and inbreeding for generations. fok rats avoided serious multisystem disorders caused by heat stroke and by extreme dehydration. saliva spreads widely over the whole ventral body surface in fok rats. however, no strain difference was not found in vitro in the salivation rate, suggesting exsisting of a negative feedback loop between the central thermoregulation system and evaporation system. on the other hand, body temperature of the fok rats did not decreased in a extream cold environment as those in control rat strain. thermogenesis induced by cold in fok rats was larger than those in control rat strains. the larger increase in thermogenesis was partly attributable to glucagon-induced thermogenesis in brown adipose tissue. blood levels of triglryceride was lower, but polyunsaturated fatty acids were higher in fok rats than those in control rat strains. these changes can be considered to be results of genetically acquired heat-tolerance. oxidative stress is involved in the degeneration of nigrostriatal dopaminergic system in parkinson s disease (pd). vitamin e is a potent antioxidant, and its retention and secretion are regulated by alpha-tocopherol transfer protein (ttp) in brain. dysfunction of ttp has been shown to result in systemic deficiency of vitamin e in human and mice. in the present study, we using the ttp knockout mice, investigated the effect of vitamin e deficiency in pd development by generating mptp mouse model of pd. we confirmed that vitamin e depleted in the brain of ttp knockout mice completely. while the mptp treatment decreased striatal dopamine in the all three ttp genotypic groups, there were no significant differences among them. our results suggest that vitamin e does not play a major protective role in mptp-induced nigrostriatal dopaminergic neurodegeneration in the brain. priyanka dikshit 1 , anand goswami 1 , nobuyuki nukina 2 , nihar ranjan jana 1 1 national brain research centre, india; 2 laboratory for structural neuropathology, riken brain science institute, 2-1 hirosawa, wakoshi, saitama 351-0198, japan a major pathological hallmark of the polyglutamine diseases is the formation of neuronal intranuclear inclusions (niis) of the disease proteins, often associated with various chaperones and proteasome components. but, how the polyglutamine proteins are ubiquitinated and degraded by the proteasome is not known. here, we demonstrate that the expanded polyglutamine proteins that are misfolded, become ubiquitinated. secondly, we identified chip ubiquitin ligase that is able to target polyglutamine expanded huntingtin and ataxin-3 for the misfolding-dependent ubiquitination and degradation by the proteasome. the over expression of chip reduces the aggregate formation and cell death mediated by expanded polyglutamine proteins and the suppressive effect is more prominent when chip is over expressed along with hsc70. finally, we show that the expression of chip is increased in the expanded polyglutamine protein expressing cells. hypothalamic-pituitary-adrenal axis is central to the regulation of stress response. for the comprehensive detection of genes responsive to stress, we identified and catalogued the entire partial complementary dna sequences (expressed sequence tags (ests)) from rat hypothalamus. we have identified the total of 11,092 ests (5,442 non-redundant sequences). 2858 of them matched known genes of rodents in the genbank databases, but 2584 remained unknown. now we classified a full set of hypothalamic ests on the basis of their functional domains. complete profile of them will be presented in the meeting. these ests will also be applied to a cdna microarray for stress experiments. the present study will provide a refined genomic resource for molecular studies of animal models of stress-related disorder. research funds: grants-in-aid from the ministry of health, labor and welfare shinya yanagita, seiichiro amemiya, satoko suzuki, ichiro kita graduate school of science, tokyo metropolitan university, japan our previous study suggests that acute running is one stressor activating corticotropin-releasing hormone (crh) neurons in the hypothalamic paraventricular nucleus (pvn). many studies have reported that several weeks of voluntary running improved stress tolerance during non-exercise stress. it is, thus, possible that housing in cages attached running wheel can alter activation of stress-related neurons during acute running. in this study, we examine the effects of 0, 2, or 4 weeks prior wheel running (i.e. housing in the cages attached running wheel) on activation of stress-related neurons, such as pvn, central nucleus of amygdala (cea), locus coeruleus, dorsal raphe, ventral tegmental area (vta), and prefrontal cortex during acute running using immunohistological methods in rats. prior wheel running altered activation of various stress-related neurons during acute running, especially markedly decreased activation of cea, and increased that of vta. these results suggest that prior wheel running influences stress-related neuronal activity during acute running. ps1a-f096 transforming growth factor-␤ in the brain regulates fat metabolism during exercise kazuo inoue, toma ishikawa, wataru mizunoya, tetsuro shibakusa, tohru fushiki division of food science and biotechnology, graduate school of agriculture, kyoto university, kyoto, japan we have previously reported that the concentration of transforming growth factor-␤ (tgf-␤) increases in the cerebrospinal fluid of rats during exercise and that an increase in fat oxidation was observed following intracisternal administration of tgf-␤. these results led us to postulate that tgf-␤ in the brain regulates the enhancement of fatty acid oxidation during exercise. to test this hypothesis, we carried out respiratory gas analysis during exercise while inhibiting the effect of tgf-␤ in the brain using intracisternal administration of anti-tgf-␤ antibody or sb-431542, an inhibitor of the type 1 tgf-␤ receptor (t␤r1). we found that each reagent blocked the increase in fatty acid oxidation. these results suggest that brain tgf-␤ has a role in enhancing fatty acid oxidation in peripheral tissues during endurance exercise, and this regulation is executed at partly via the t␤r1 signal transduction system. yoshii takanobu it has been demonstrated that vasopressin (avp) might play a role in anxiety-related behavior. we hypothesized that traumatic stress changes avp activity and avp contribute to the symptom of ptsd. we carried out in situ hybridization (ish) for avp mrna expression and avp immunohistochemistry (ihc) with an experimental paradigm of single prolonged stress (sps) as ptsd model. sd male rats were exposed to sps (2 h restraint; 20 min forced-swimming; ether anesthesia) then they were put in untouchable situation for 7 days. avp mrna expression significantly decreased in the son. ihc showed no significant change in avp-ir, but after additive stress (forced swimming 15 min), avp-ir in the son was significantly diminished. we considered that the stress decrease avp synthesis, but has little effect to the storage of avp. mumeko tsuda, takaaki ozawa, aosa fukushi, sonoko ogawa kansei, behavioral and brain sciences, university of tsukuba, tsukuba, japan neonatal maternal separation (ms) is known to affect anxiety and fear responses in adult whereas its effect on socio-sexual behaviors is not fully understood. in the present study, we examined the effect of ms on an array of emotional and socio-sexual behaviors in both sexes of c57bl/6j mice. pups were separated from mothers daily (3 h) on postnatal days 1 through 14. starting at 13 weeks of age they were tested for (1) emotionality and anxiety levels in open field (oft), light-dark transition (ldt), and elevated plus maze tests; (2) responses to social stimuli in social investigation (sit) and social preference tests; and (3) socio-sexual behaviors in aggressive and sexual behavior tests. overall, there was no apparent effect of ms on behaviors measured in the oft and ldt except for higher levels of exploration in the ms group compared to the non-stressed (ns) group in both sexes. during the sit, social investigation time and general activity in ms females were much lower than those in ns females suggesting ms females may be more fearful to social stimuli. in the present study, we investigated the effect of environmental stress applied during perinatal period on spatial learning activity of mouse evaluated by morris water maze test. mice were exposed to the noise of 90 db (so), or were forced to swim (sw). these manipulations were performed for 15 min once a day at 2 weeks after birth (from postnatal days 14 to 18) or 3 weeks after birth (from postnatal day 21 to 25). normal mice were left undisturbed (no). the spatial learning activity was tested at the age of 6 weeks. it was found that the spatial learning activity of both so and sw mice manipulated 2 weeks after birth was impaired as compared to no mice. so mice manipulated 3 weeks after birth exhibited the same learning behavior as no mice, while that of sw mice manipulated 3 weeks after birth was impaired. present results indicated that the effect of the environmental stress on the learning activity of the adolescent mice might be dependent on the period of the stress manipulation. kin-ya kubo 1 , yukiko yamada 2 , mitsuo iinuma 2 , yasuo tamura 2 , fumihiko iwaku 1 , kazuko watanabe 3 , minoru onozuka 4 1 dept. oral anat., asahi univ. sch. dent., japan; 2 dept. ped. dent., asahi univ. sch. dent.; 3 dept. physiol., gifu univ. sch. med., japan; 4 dept. physiol. and neurosci., kanagawa dent. coll., japan recent studies have suggested that occlusal disharmony is related to temporomandibular arthorosis and braxism, which may come from a hypothalamic-pituitary-adrenal (hpa) axis. in addition, aged mice with masticatory dysfunction show deficits in spatial memory, being due to various pathological changes in the hippocampus, suggesting the link between malocclusion induced by abnormal occlusion and hippocampal pathology. in this study, to prove this hypothesis, we examined the effect of this malocclusion on plasma corticosterone levels, the numbers of hippocampal neurons and spatial performance in water maze in samp8 mice. this treatment age-dependently advanced a decline in spatial memory, an increase in plasma corticosterone levels, and a decrease in neuron density in the hippocampal ca3 region. the results suggest that abnormal occlusion may progress hippocampal neuron loss via stress, thereby leading to senile deficits in memory. yurie nakamoto, go mugishima, mitsuko sato, masako miwa, mitsunobu yoshii division of psychobiology, tokyo institute of psychiatry, tokyo, japan it has been shown that pbr are increased after acute stress and decreased under chronic stressful conditions. in our previous studies, expression of pbr was significantly correlated with trait anxiety in normal human subjects, which might reflect polymorphism of the pbr gene. in addition, males appeared to have higher pbr densities than females in their prime lives. the present study was designed to analyze these sexual differences in rats. blood samples were obtained from adult male and female slc wistar rats immediately after acute random electrical footshock and also from these animals after chronic social isolation (for 12 weeks after weaning). in naïve, male rats expressed higher densities of platelet pbr than females. chronic social isolation caused a marked increase in platelet pbr in male rats compared to female. the results indicate that pbr responses to environmentally induced stress are much less in female, probably under the influence of estrogen. kanako tambara 1 , yayoi kitamura 1 , junichi tanaka 2 , yukio hattori 1 , yasushi hayashi 1 1 department of human nutrition, notre dame seishin university, okayama, japan; 2 department of curriculum, teaching and memory, naruto university of education, tokushima, japan we investigated the effects of exogenous putrescine on stressinduced hyperthermia (sih) in male c57bl/6j mice after systemic injection of putrescine to clarify the role of brain putrescine in stressful conditions. in addition, we examined the effects of spermidine, spermine, and the anxiolytic diazepam on sih. the rectal temperature of singly housed mice was measured twice at a 10-min interval, to measure the basal temperature (t 1 ) and stress-enhanced temperature (t 2 ), respectively. the difference ( t = t 2 − t 1 ) gives the sih. in control mice, t was approximately 1 • c. pretreatment with diazepam caused dose-dependent inhibition of the sih. similarly, putrescine reduced t, although it caused a dose-dependent decrease in t 1 . furthermore, spermidine and spermine also lowered t and t 1 at doses lower than that of putrescine. these results suggest that endogenous brain putrescine and other polyamines have an anxiolytic-like effect in stressful conditions. eriko iguchi, yasuhiro tanaka, toshiyuki matsuoka, shuh narumiya department of pharmacology, kyoto university, kyoto, japan prostaglandins (pgs) are synthesized in many organs including the brain. of their synthesis, the rate limiting step depends on cyclooxygenase (cox), which has two subtypes, cox-1 and cox-2. it has been known that, under some stressful conditions, cox-2 is induced in some neurons and increases pgs production. but the roles of the increased pgs under stress are not fully elucidated. in this study, we restrained mice in small tubes individually for 6 h and subjected them to the elevated plus maze task 24 h later. these mice showed more anxiety. immunohistochemistry showed significant induction of cox-2 by restraint in some parts of the brain, such as cerebral cortices and amygdala. next, we examined the effect of indomethacin on this stress-induced anxiety. indomethacin is expected to reduce pgs production. mice treated with indomethacin stayed on open arms longer than control mice. these data suggest that pgs synthesized during stress may have anxiety-increasing effect. ps1a-g104 imaging brain and immune association accompanying cognitive appraisal of acute stressor to investigate association between brain and immune systems accompanying cognitive appraisal of an acute stressor, we recorded 15o-water positron emission tomography, cardiovascular, neuroendocrine, and immune indices, when 11 male subjects conducted a mental arithmetic task in a high controllability (hc) condition and a low controllability (lc) condition. activation in the orbitofrontal (ofc) and medial prefrontal (mpfc) cortices was observed in the lc compared to the hc. furthermore, significant correlations between brain activation and hr, hrv, bp, and nk cells were found commonly in the ofc in the lc, but not in the hc. thus, the ofc is a pivotal region for top-down regulation over immune activity accompanying cognitive appraisal on a stressor. wei zhang 1 , takesi sakurai 2 , yasuitirou fukuda 3 , tomoyuki kuwaki 1,3 1 dept. molec. integ. physiol., chiba univ., japan; 2 dept. pharmacol., univ. tsukuba, japan; 3 dept. autonom. physiol., chiba univ., japan we have previously proposed that orexin plays as a master switch to elicit multiple efferent pathways of the defense response. it is still open question, however, how information of stressor activates the orexinergic neurons. in this study, we examined possible afferent nuclei to activate orexinergic neurons. in urethane-anesthetized mice, a gaba-a receptor antagonist, bicuculline, was microinjected into the amygdala or the bed nucleus of stria terminalis (bnst), of which electrical stimulation induced simultaneous increases in blood pressure, heart rate, and respiration. bicuculline dose-dependently induced cardiorespiratory excitation in both orexin neuron-ablated and wild-type mice. however, dose-response curve was rightward shifted in the former. we conclude that the amygdala and bnst constitute one of the afferent pathways to the orexinergic neurons that involved in the defense response against stressor. in this study, developmental changes of anxiety behavior as well as myelin formation were investigated in male balb/c mice. the early-weaned mice had lower number of entries to the open arms of elevated plus maze at the age of 3-8 weeks, indicating persistent higher anxiety. high performance thin layer chromatography analysis was conducted for amygdaloid galactosyl ceramide, which is a typical lipid of myelin. the early-weaned mice had higher levels of galactosyl ceramide at the age of 5 weeks, and an electron microscopic study suggested increased number of myelinated axon and reduced diameter of myelinated axon in the basolateral amyglaloid nucleus. these results suggest that the early weaning induces precocious myelin formation in the amygdale between 3 and 5 weeks of age, which would be related to higher anxiety state in the early-weaned mice. research funds: sasakawa sci. res. grant takefumi kikusui, yuji mori veterinary ethology, university of tokyo, tokyo, japan we previously reported that early-weaned mice developed persistent increase in anxiety as well as aggression. in this study, developmental changes of brain derived neurotrophic factor (bdnf) protein levels were investigated in early-weaned icr mice. the early-weaned male and female mice had lower number of entries to the open arms of elevated plus maze at the age of 3 weeks, and this change was persistently observed in males. concurrently, the early-weaned males showed decrease of bdnf in the prefrontal cortex between 3 and 8 weeks of age, and in the hippocampus at the age of 3 weeks. however, there was no difference of bdnf expression in females. in addition, the early-weaned males, but not females, showed reduced brdu immunoreactivity in the dentate gyrus. these results suggest that the deprivation of mother-infant interaction during the late lactating period augments the anxiety in the adulthood by decreasing the level of bdnf in the pre-limbic system, and that these stress responses are sexually dimorphic, i.e., male is more vulnerable to early weaning stress. research funds: kakenhi #14760187 ps1a-g108 a systematic analysis of genetic factors associated with behavioral diversity between msm and c57bl/6 koide tsuyoshi 1,2 , aki takahashi 1,2 , toshihiko shiroishi 2,3 , akinori nishi 1 1 mgrl, national institute of genetics, mishima, japan; 2 sokendai, hayama, japan; 3 mammalian genetics lab, nig, mishima, japan in the previous study conducting a multi-phenotype behavioral tests, we observed a great difference of the behavioral phenotype between mouse strains, msm and c57bl/6. in order to elucidate a genetic factors underlying the behavioral difference, we analyzed a series of consomic strains which are made by replacing one of the chromosomes with that of msm strain. the behavioral data clearly indicated involvement of multiple genetic factors for each behavioral phenotype. one of the consomic strains, b6-6cmsm, which carries chromosome 6 of msm, showed extreme behavioral differences from c57bl/6. the strain showed lower activity in home cage and novel cage, and showed decreased number of transition in the light dark box test. by conducting analyses of composite interval mapping and a series of sub-consomic strains, we successfully identified genetic loci for the behavioral phenotype. tomoko soga 1 , yu kajiyama 2 , shigenobu shibata 2 , hiroshi kunugi 1 1 department of mental disorder research, national institute of neuroscience, center of neurology and psychiatry, tokyo, japan; 2 department of electrical engineering and bioscience, waseda university the hypothalamus-pituitary-adrenal (hpa) axis plays an important role in the pathophysiology of depression. alterations of brain derived neuronal factors (bdnf) have been implicated in depression. we examined the effects of synthetic glucocorticoid (dexamethasone; dex) on emotional behavior and gene expression of hpa-related molecules and bdnf in mice. dex treatment for 4 days after birth showed a significant decrease in locomotor activity and a significant rise in the time of immobility during forced swimming test. dex treatment to mature mice resulted in significant decrease in the number of entries into the open arm during elevated plus maze test. there was no change in gene expression of hpa-related molecules in dex-treated group. bdnf gene expression decreased significantly in dex-treated group, which showed behavioral abnormalities. our results lend further support for the involvement of glucocorticoid and bdnf in depression-related behavior. sachiko chikahisa, hiroyoshi sei, atsuko sano, kazuyoshi kitaoka, yusuke morita department of integrative physiology, the university of tokushima graduate school, tokushima, japan music is known to be able to elicit emotional changes including anxiolytic effect. the gonadal steroid hormone estrogen (e2) has been associated with anxiety levels. in this study, we examine whether the effect of music on anxiety is related with ovarian steroid in female mice. behavioral paradigms measuring anxiety (open field, elevated plus maze, dark-light transition and marble burying test) were tested in gonadally intact (sham-operated) and ovariectomized (ovx) female mice treated with placebo (ovx + placebo) or chronic estradiol (ovx + e2) replacement. in three behavioral tests except for open field, sham-operated mice exposed to music showed less anxiety than those exposed to white-noise and silence, while ovx + placebo mice did not show these effects at all. ovx + e2 mice showed the anxiolytic effect of music only in the marble burying test. these results suggest that exposure to music reduce anxiety levels, and ovarian steroids may be, at least partially, involved in the anxiolytic effects of music observed in female mice. tatsuhiro yasuda free, tokyo, japan strength and periodicity of periodical air pressure ascent around ones' ears induced by others' respiration may impact upon ones' awaken level, i.e. cognition. the air vibration acts upon tympanic membrane and then cochlear receptor stereocilia transforms it to neural signals which are sent via cochlear nucleus to inspiration nucleus in the medulla, and inspiration is induced. simultaneously afferent signals generated by external intercostals contraction are forward to medulla, thalamus and cortical areas. the stimuli with larger strength and periodicity compared to ones body size yields to auditory startle reflex. continuation of this may induce hyper ventilation or tension. if the input may be lasting with smaller strength and periodicity, insufficient diaphragm activity after hypoxia and gasping fade-out may induce afferent signal shortage that shrinks various cortical neural activity. lasting this situation may fall into depression. suitable timing of inspiration inducing may keep good mood, strong motivation and effective cognition. body system is suggested to own inherent observer that detects alerting or safe state so called homunculus. kenichi sasaguri 1 , takero in general, it has been proposed that the mandibular retrusive position resulted from either malocclusion or inadequate occlusal reconstruction is one of the causes of indefinite complaint. we determined whether the malocclusion model influences brain activities by using fmri study. the results indicated that in some of volunteers, significantly bold signals in the hypothalamus and the amygdala, being associated with emotion and/or stress increased during clenching. it is, therefore, suggested that malocclusion influences the whole body through emotional system, thereby causing the indefinite complains. ps1a-g113 synaptic organization between the amygdaloid axon terminals and the parvicellular reticular formationprojecting neurons in the retrorubral field of the rat toshiko tsumori, yi qin, shigefumi yokota, tatsuro oka, yukihiko yasui dept. anat. & morphol. neurosci., shimane univ. sch. med., izumo, japan the retrorubral field (rrf) is known as one of the areas containing numerous dopaminergic neurons in the midbrain. in the present study, we showed that the axon terminals from the central amygdaloid nucleus (ace) made synaptic contacts with non-dopaminergic rrf neurons sending their axons to the parvicellular reticular formation (rfp), where many premotor neurons projecting to the orofacial motor nuclei have been well known to exist. the ace axon terminals, which usually contain small pleomorphic vesicles and occasionally contain both small pleomorphic vesicles and large dense-cored vesicles, formed symmetrical synapses with cell bodies and dendrites of the rfp-projecting rrf neurons. moreover, most of these axon terminals showed glutamic acid decarboxylase immunoreactivity. the present study suggests that the ace exerts inhibitory influences upon the non-dopaminergic rfp-projecting rrf neurons to control orofacial movements closely related to emotional behavior. research funds: kakenhi (15500238) ps1a-g114 involvement of nr2b tyrosine-phosphorylation in emotional responses mediated at the amygdala mina delawary 1 , takanobu nakazawa 1 , yuji kiyama 2 , toshiya manabe 2 , tadashi yamamoto 1 1 div. of oncology, ims, univ. of tokyo, tokyo, japan; 2 div. of neuronal network, ims, univ. of tokyo, tokyo, japan nr2b is tyrosine-phosphorylated, with tyr-1472 being its major phosphorylation site. to investigate the role of tyr-1472 phosphorylation, we generated mice with a tyr1472phe knock-in mutation (yf/yf mice). in the elevated plus-maze test, time spent in open arm was reduced in yf/yf mice as compared to that in wild-type mice. similar phenotype was seen in the corticotropin-releasing factor (crf) overexpressing mice. this phenotype of yf/yf mice was canceled by the administration of crf receptor antagonist. as expected, in yf/yf mice, the expression level of crf in the amygdala was increased compared with that in wild-type mice. in the slice of amygdala from wild-type mice, nmda application induced de-phosphorylation of tyr-1472 and up-regulation of crf mrna level. given that crf is important in emotional responses, these data strongly argue that phosphorylation of nr2b is involved in the control of emotional responses by regulating crf content. ps1a-g115 increase in anxiety in transgenic mice overexpressing camkii in forebrain previous studies have shown that ␣calcium/calmodulin dependent protein kinase ii (␣camkii) plays important roles in aggressive and fear response in mice. to understand roles of alpha camkii in emotional behaviors, we have generated transgenic mice overexpressing ␣camkii in forebrain. because these mutant mice showed increase in anxiety in open field and elevated zero maze tests, we here examined effects of administration of selective serotonin reuptake inhibitor (ssri) on anxiety-related behavior of these mutant mice. treatment with ssri suppressed anxiety-related behavior of camkii mutant mice, suggesting that camkii mutant mouse is a mouse model of anxiety disorder. to investigate the mechanisms for increase in anxiety led by overexpression of camkii, we next compared the expression profiles between wild and mutant mice using dna micro array. these mutant mice showed abnormal changes in expression levels of genes related to ca 2+ signal transduction in hippocampus. yumiko ikeda, katsunori kobayashi, hidenori suzuki department of pharmacology, nippon medical school, tokyo, japan environment is known to influence behavior of animals. however, cellular and synaptic mechanisms underlying behavioral changes by environment remain largely unknown. we examined effects of changes in environment on locomotor activity and mossy fiber (mf) synaptic transmission in hippocampal slices. in mice housed in enriched condition for 5 weeks, locomotor activity and longinterval (200, 1000 and 5000 ms) paired-pulse facilitation (ppf) at mf synapses were reduced. in contrast, in mice housed in isolated condition for 5 weeks, there was no detectable change in either the total ambulation distance or the magnitude of ppf. we compared properties of the mf synaptic transmission with the locomotor activity in individual mice used in all experiments and found that the magnitude of synaptic potentiation induced by dopamine was negatively correlated with the ambulation distance. our results suggest that the modification of the hippocampal mossy fiber synaptic transmission could be involved in the environmental regulation of locomotor activity. yilong cui 1,3 , yosky kataoka 1,2 , yasuhisa tamura 3 , yasuyoshi watanabe 2,3 , hisao yamada 1 1 department of anatomy and cell science, kansai medical university, osaka, japan; 2 department of physiology, osaka city university graduate school of medicine, osaka, japan; 3 molecular imaging research program, riken frontier research system, saitama, japan during long-term intracranial self-stimulation (icss; electrical stimulations to the hemi-lateral medial forebrain bundle of rats by their lever pressing behavior at 50-60 times/min), inhibition periods (less than 10 times/min) were often observed 2 h after start of icss. we have been demonstrated that the inhibition was not induced by thermal effect on the neural function or by muscular fatigue. furthermore, the inhibition period was significantly decreased by pre-treatment with ns-398, a selective cox-2 inhibitor. these observations indicate that the arachidonic acid cascade is involved in inhibition of long-term icss and would be in weariness or fatigue sensation. male bluegill, lepomis macrochirus, is known to display alternative reproductive tactics. "parental" males defend nests and provide parental care, and "satellites" or "sneakers" are non-nesting, attempting to achieve parasitic fertilizations via sperm competition. in teleost and other non-mammals, arginine vasotocin (avt), the homologue of mammalian avp, is known as an important hypothalamic peptide involved in the alteration of reproductive behavior. behavioral evaluation and immunohistochemical study in preoptic area (poa) were conducted in parental and satellite bluegills to clear the role of avt in teleost reproductive tactics. parentals displayed more aggressive and courtship behavior than satellites and satellite males had significantly more cells than parentals, while the size of avt cells showed no difference between the male morphs. these results suggested that hypothalamic avt might play some part in the central control of reproductive behavior in teleost. ps1a-g119 impulsive choice in domestic chicks: context dependence and dissociation between delay and handling cost toshiya matsushima 1 , naoya aoki 2 , andras csillag 3 1 biology, hokkaido univ., sapporo, japan; 2 agriculture, nagoya univ., nagoya, japan; 3 anatomy, semmelweis univ., budapest, hungary choice between small/immediate reward and large/delayed reward has been widely used as a behavioral measure of impulsiveness. to study how ecological factors shaped underlying neural processes, we examined week-old chicks in four different tasks with identical economical consequences. in task 1, chicks chose between small reward (one pellet) delivered immediately and large reward (six pellets) after a delay up to 3 s. in task 2, chicks chose between small reward located at 20 cm and large reward at −140 cm, where cues signaled the distance of invisible food. task 3 was similar to the task 2, except that cues signaled the food quantity. in task 4, total handling time differed due to lowered food accessibility, while the delay was kept identical. lesion experiments revealed that ventral striatum was specifically involved in choices based on anticipated proximity (but not quantity), whereas arcopallium (association cortex analogue) in choices based on anticipated handling cost. research funds: kakenhi (15370033, 17021018) ps1a-g120 analysis of the brain regions associated with the dance language of the honeybees taketoshi kiya, takekazu kunieda, takeo kubo dep. biol. sci., univ. tokyo, tokyo, japan social animals have highly developed communicative abilities. the worker honeybees (apis mellifera l.) can transmit location of food sources by the dance language. in spite of the simple structure of the honeybee brain and the stereotyped dance behavior, its neural mechanisms remain totally unknown. previously, we found active brain regions in the dancing workers (dancers) by using a novel immediate early gene, kakusei, as a marker for neural activities and found its prominent expression in the small-type kenyon cells (skcs) of the mushroom bodies. here, we report that kakusei was similarly expressed in the skcs of the foraging workers (foragers), which do not always show the dance behavior. in contrast, the skcspreferential kakusei expression was not observed in the brains of the orienting workers, which were flying to learn the hive location. these results imply that the activities of the skcs in the dancer brain are neither due to dance presentation itself nor sensory inputs during foraging, but complex information processing accompanying the foraging behavior. c. elegans wild type animals are usually attracted to nacl, but show avoidance behaviors after being conditioned with nacl and starvation (food−/nacl+). this behavioral plasticity is not induced under the food−/nacl− or food+/nacl+ conditions. we isolated learning-defective mutants including pe401, which had a missense mutation in the casy-1 gene. several casy-1 deletion mutants also showed learning defects. casy-1 has an extensive similarity to human calsyntenin/alcadein, which is a single-pass transmembrane protein with cadherin-like repeats localized to the postsynaptic membrane of cns synapses. alcadein forms a stable tripartite complex with app and x11l/mint2. however, after dissociation of x11l, alcadein is susceptible to cleavage by protease(s). we found that casy-1 was expressed mainly in neurons and functioned at the adult stage. we are now investigating the localization pattern of the gfp-tagged protein, and whether casy-1 can also be proteolytically cleaved. ps1a-g122 insulin-like signaling is required for association between temperature and feeding state in c. elegans eiji kodama 1 , atsushi kuhara 1 , akiko mohri 1,2 , kotaro kimura 1,3 , masatoshi okumura 1 , masahiro tomioka 4 , yuichi iino 4 , ikue mori 1,5 1 div. of biol. sci., nagoya univ., japan; 2 present address: univ. of texas, health sci. cent., usa; 3 present address: natl. inst. of genet., japan; 4 mol. genet. res. lab., univ. of tokyo, japan; 5 inst. for advanced res., nagoya univ., japan c. elegans can associate cultivation temperature with feeding state. mutations in ins-1 encoding insulin homologue caused defective associative learning, mutations in daf-2 and age-1 encoding the homologues of insulin receptor and pi 3-kinase, respectively, suppressed the defect of ins-1, and the mutation in daf-16 encoding forkhead transcriptional factor caused the learning defect. this suggests that ins-1 antagonizes daf-2 insulin-like signaling for associative learning. interestingly, age-1 animals associate their cultivation temperature with feeding-state quicker than wild type. this defect was rescued by expressing age-1 in some head interneurons. in addition, the activity of these interneurons were down-regulated by starvation through ins-1. we suggest that insulin-like signaling modulates the neuronal activity of interneurons essential for associative learning. ps1a-g123 analysis of ttx-8: novel thermotaxis gene conserved among various organisms akiko miyara, akane ohta, yoshifumi okochi, masatoshi okumura, ikue mori laboratory of molecular neurobiology and institute for advanced research, nagoya university, nagoya, japan c. elegans can memorize the food condition in relation to the cultivation temperature and migrate to the cultivation temperature when looking for the food. this response to temperature is called thermotaxis. several neurons and genes required for thermotaxis have been identified, but molecular mechanism of thermotaxis is still poorly understood. the ttx-8(nj21) and ttx-8(nj34) mutants are obviously defective in thermotaxis and partially defective in chemotaxis. we revealed that ttx-8 encodes novel protein and is expressed in many neurons and functions in several neurons responsible for the thermotaxis behavior. the predicted protein structure of ttx-8 is similar to ric-3, identified in c. elegans at first and conserved among several species (halevi et al., 2002 (halevi et al., , 2003 . ric-3 is thought to be required for the maturation of acetylcoline receptor (halevi et al., 2002) , so ttx-8 may play a similar role such as folding, assembly, transmission or anchoring of some kind of membrane protein. ps1a-g124 analysis of aho-3 mutant that cannot associate cultivation temperature with feeding state in c. elegans nana nishio 1 , akiko mohri 1,2 , eiji kodama 1 , atsushi kuhara 1 , mizuho koike 1 , kotaro kimura 1,3 , ikue mori 1,4 1 div. of biol. sci., nagoya univ., japan; 2 present address: univ. of texas, health sci. cent., usa; 3 present address: natl. inst. of genet., japan; 4 inst. for advanced res., nagoya univ., japan the nematode c. elegans can associate cultivation temperature with feeding state: well-fed animals migrate to and starved animals avoid from the cultivation temperature on a temperature gradient. to identify genes required for this associative learning, we screened mutants that are defective in starvation-induced cultivation temperature avoidance. we isolated aho-3(nj15) mutants that were normal in thermotactic migration after cultivated well-fed state and normal in response to food in locomotion assay (sawin et al., 2000) , indicating that they are normal in temperature and food recognition and may be defective in the associative learning. aho-3 gene encoded a predicted hydrolase and the molecular properties have not been characterized yet, although aho-3 is a highly conserved protein throughout yeast to human. currently, we are trying to dissect the molecular and cellular analysis of aho-3 gene further. we have demonstrated aversive conditioning in lymnaea using 100 mm sucrose presentation as the appetitive stimulus (cs) and mechanical tactile stimulation to the head as the noxious stimulus (ucs). we measured the feeding response before and after pairing with the aversive stimulus to determine whether learning alters the innate preference for sucrose. we also measured the neuronal activity of b3, located in the buccal ganglion. an associative memory, lasting 24 h, was produced with 20 pairings of cs and ucs. the learning was characterized by a shift in the response to the ucs from a whole body withdrawal response to the cessation of feeding behavior. b3 neuron responded with repetitive impulse discharge regularly as fictive feeding patterns to a sucrose application in naive animals, on the other hand cs application failed to generate regular impulse activity rather it resulted in generation of epsps in the conditioned animal. this can interpret that the conditioning decreased the excitability of b3 neuron activity thus to decrease the fictive feeding behavior. yasutaka nomura 1 , dai hatakeyama 1 , tetsuro horikoshi 1 , etsuro ito 2 , manabu sakakibara 1 1 lab. neurobiol. engr, sch. high-tech, tokai univ., numazu, japan; 2 cris, hokkaido univ., sapporo, japan calexcitin, low molecular weight gtp-binding protein is found to be phosphorylated in the visuo-vestibular conditioned hermissenda at the type b photoreceptor. we found positively stained neurons to anti-calexcitin antibody (gift from dr. kuzirian) at the cerebral and pedal ganglion in the circumesophageal nervous system of conditioned lymnaea with two different ways. one was the same conditioning paradigm as hermissenda and the other was taste aversion conditioning. both of these conditioning response is the whole-body withdrawal. no positive neuron was found in naïve animal. neurons in cb cluster and pea cluster showed both positivity to calexcitin and serotonin. this suggested the functional role in conditioning. ken honjo, katsuo furukubo-tokunaga graduate school of life and environmental sciences, university of tsukuba, ibaraki, japan the fruit fly drosophila melanogaster has been utilized as a successful model to study underlying mechanisms of learning and memory. we have established a novel larval olfactory paradigm and found that appetitive and aversive memories are considerably different in their stability whereas both are localized to the mushroom bodies (mbs). we found that larval memory induced by sucrose lasts six times longer than that induced by quinine although the initial learning performances are comparable. by expressing shi ts1 in larval mbs, we demonstrate that disruption of neural output from mbs abolishes both appetitive and aversive memory indicating that both memories are stored before the mb output synapses. moreover, we show that disruption of either creb or amnesiac functions abolishes appetitive but not aversive memory. thus these data suggest that appetitive and aversive reinforcements stimulate different intracellular and/or intercellular signaling pathways generating distinct memory components in mbs. motomi matsuno 1 , minoru saitoe 1,2 , tim tully 3 1 tokyo metropolitan institute for neuroscience, tokyo, japan; 2 department of biology, tokyo metropolitan university, japan; 3 cold spring harbor laboratory, usa we identified ruslan as a novel memory mutant, and found that it encodes a cell adhesion molecule, klingon (klg). klg belongs to the immunoglobulin superfamily and was originally identified as an essential gene for the development of photoreceptor neurons. we report here that klg is necessary for long-term memory as well as early-phase memory. we show that klg expression is dependent on neural activity and functions as a downstream of both the transcription factor, creb and the cell surface receptor notch, both of which are well known to function in ltm formation. transgenic expression of klg improves memory of a klg mutant. since klg protein localizes along the surface between neuropil and neuropil glia, we propose that klg mediates an interaction between neurons and glia that is required for memory formation. we have investigated the ability of context-dependent olfactory learning in the cockroach, periplaneta americana. we trained one group of cockroaches to associate peppermint odor (conditioned stimulus, cs, p) with sucrose solution (appetitive unconditioned stimulus, us+), and vanilla odor (cs, v) with saline solution (aversive us, us−) under illumination (l), and to associate p with us− and v with us+ in the dark (d). another group received training with opposite stimulus setup (l: v+/p−, d: v−/p+). before training, cockroaches preferred v over p. 1 day after training, the former group significantly preferred p over v under illumination but preferred v over p in the dark, and the latter group displayed the invert odor preference. result of the control experiment excluded the possibilities that conditioning hours of the day or its order was used as cues to disambiguate the meaning of css. thus cockroaches are capable of disambiguating the meaning of cs odors according to the visual context. hidehiro watanabe, makoto mizunami graduate school of life sciences, tohoku university, sendai, japan a century had passed since pavlov reported classical conditioning of salivation in dogs. however, the cellular mechanisms underlying this conditioning remain obscure. in insects, salivation is regulated by salivary neurons of the subesophageal ganglion which innervate the salivary grand. here, we established antennal classical conditioning of salivation and that of activities of salivary neurons in cockroaches, periplaneta americana. in insects, antennae are elaborate sense organ that processes many sensory modalities including odor and taste. we found that responses of salivary neurons to an odor was increased after repetitive pairing of the odor with sucrose or saline solution presented to an antenna, but those to an odor paired with water or tactile stimulus presented to an antenna did not changed. the level of salivation to sucrose-associated odor was significantly greater than that to non-associated odor. these results are the first to suggest the classical conditioning of salivation in non-mammalian species. these results are useful to study neural mechanisms underlying classical conditioning of salivation. research funds: kakenhi 17005050 ke zhang 1 , jian z. guo 1 , ai k. guo 1,2 1 institute of neuroscience, chinese academy of sciences, china; 2 institute of biophysics, chinese academy of sciences, beijing, china the cooperation of dopamine system and other brain cortices is essential for decision-making in mammal. drosophila can make clearout choice in visual flight simulator when facing conflicting visual cues based on the saliency of the cues previously trained to follow. here we show this behaviour is impaired when the transmission of dopaminergic neurons or mushroom bodies (mb), was genetically silenced by gal4/uas-shi ts1 system, suggesting that this behaviour is mediated by dopaminergic system acting through mb, a structure shown to be densely innervated by dopaminergic fibers. however, the dopaminergic and mb synaptic activities were required only during the early choice period (<4 min), but not for the sustenance of the chosen flight path. thus the dopaminergic system and mb are specifically devoted to the cognitive function exemplified by the flyǐs choice behaviour and further studies of the circuit in drosophila may help to understand the neural basis of higher cognitive functions. sae unoki, yukihisa matsumoto, makoto mizunami graduate school of life sciences, tohoku university, sendai, japan in mammals, the dopaminergic reward system plays ubiquitous roles in reward learning. previous studies in insects suggested that octopamine (oa) and dopamine (da) mediate various kinds of reward and punishment signals in olfactory learning. however, whether such roles can be generalized to learning of sensory signals other than odors remained unknown. we pharmacologically studied the roles of oa and da in appetitive and aversive forms of visual pattern learning in crickets. crickets injected with oa receptor antagonists exhibited no significant levels of appetitive visual learning, but aversive one was unaffected. the opposite influences were observed by injection of da receptor antagonists. our finding that oa and da participate in reward and punishment conditioning in visual learning, together with results of previous studies in olfactory learning, suggests ubiquitous roles of the octopaminergic reward system and dopaminergic punishment system in insect learning. this suggests conserved roles of aminergic reinforcing systems among different phyla. aiko watanabe, neal a. hessler laboratory for vocal behavior mechanisms, riken brain science institute, saitama, japan in adult songbirds, neural turnover occurs in hvc, a forebrain motor control nucleus. cells labeled by bromodeoxyuridine (brdu), a cell birth marker, appear in the ventricular zone, migrate into hvc, and some of them mature into projection neuron. to assess the role of neurogenesis in adult song plasticity, we deafened adult bengalese finches, whose songs are disorganized and become plastic within the first month after deafening, and then stabilize. deafened birds had more brdu-labeled cells in hvc than control birds within the first month. more tunel-stained apoptotic cells also tended to be seen in hvc of deafened birds. however, number of the brdu-labeled cells decreased 2 months after deafening, when the songs had stabilized. most of the brdu-labeled cells in hvc of deafened birds were immunoreactive for a neuron-specific marker, hu. additionally, amount of singing in deafened birds, which may affect amount of neurogenesis, did not significantly differ from that in control birds. these results suggest that the amount of neurogenesis is related to adult song plasticity. yasko tobari 1,2 , kazuo okanoya 1,2,3 1 lab. for biolinguistics, riken-bsi, wako, japan; 2 grad. sch. of sci. and tech., chiba university, chiba, japan; 3 presto, jst. kawaguchi, japan a set of brain nuclei controls song production in songbirds. among these nuclei, the robust nucleus of arcopallium (ra) is the telencephalic site of direct projections onto vocal motor neurons and respiratory premotor neurons. the projections of ra to the mudulla included the tracheosyrigeal part of the hypoglossal nucleus (xiits), which innervates the syrinx, the birds , vocal organ, and respiratoryrelated nucleus, retroambigualis (ram) were present in bengalese finches. in this study, we have focused our attention on the descending projections of ra, with a view to the presence of contralateral projections to xiits and ram, using in vivo tract-tracing technique. the results indicated that ipsilateral and contralateral projections of ra to respiratory-vocal nuclei in the brainstem were defined in adult male bengalese finches. birdsong is composed of various song elements that have typical frequency modulation. each element is aligned in own sequential rule. especially in bengalese finches, the sequential rule obeys finite state grammar. it has been focused what neural mechanism enables such a complex sequential rule. in order to learn and maintain their own song, they have auditory neural representation of their own song in the forebrain area hvc. we collectively recorded the activities of hvc neurons driven by all possible element pair stimuli. the results show that most of neurons in hvc respond not only the sequence included in their own song but also the sequence not included. each neuron has typical response distribution toward the whole element sequence. in addition, the distribution property is different among neurons in same individual. taken together, information of the entire song element sequence would be stored in the neural ensemble of these neurons as a population coding. hironobu sakaguchi department of physiology and biological information, dokkyo university, school of medicine, japan avian vocal learning provides a good model for human speech learning. young male songbirds learn to imitate their tutor's song during a specific time in development, which is referred to as a sensitive period. many behavioral studies have shown that vocal learning is affected by a song template and social factors. if a young bird is raised without a tutor's song template (father) and/or social contacts with other birds, including its mother and siblings, it produces an abnormal isolated song, meaning that isolation delays the sensitive period for song learning. here, we investigated for the delayed song learning of socially isolated zebra finches from new tutors. consequently, isolated birds, exposed to new tutors from day 120, developed the zebra finch-typical song (song syntax), similar to song acquisition in young birds during the sensitive period of song learning. however, they were not able to imitate the syllable phonology from new tutors. the differences between two aspects of song organization suggest that the schedules and processes of the learning of phonology may be different from those of song syntax. ps1a-h137 facilitatory effects of oxytocin on synaptic plasticity in the olfactory bulb and olfactory learning in young rats fumino okutani, jing-ji zhang, guang-zhe huang, hideto kaba department of integrative physiology, kochi medical school, nankoku, japan oxytocin (ot) within the olfactory bulb (ob) has been reported to be important for the induction of maternal behavior and recognition of offspring. the activity of mitral cells, olfactory relay neurons in the ob is inhibited by granule cells via reciprocal dendrodendritic synapses. electrophysiological studies have revealed that ot modulates mitral cell activity by acting on mitral and granule cells. in a classical conditioning paradigm, young rats show aversion to the odor that has been paired with foot shock. our studies have shown that plasticity in the ob is critical for this olfactory learning. pups that received ot infusion into the ob in the presence of citral odor developed an aversion to the odor without shock, suggesting that ot infusion has a facilitatory effect on olfactory learning. using ob slices, long-term potentiation (ltp) was induced in field epsps recorded in the granule cell layer. ot administration also facilitated ltp. these results demonstrate that ot is involved in olfactory learning in young rats. research funds: kakenhi 17023034 ps1a-h138 the gaba a receptors in the ventral pallidum are involved in the retrieval of conditioned taste aversion in rats tadashi inui, tsuyoshi shimura, takashi yamamoto div. behav. physiol., dept. behav. sci., grad. sch. human sci., osaka univ., japan we examined the effects of microinjections of gaba a receptors antagonist bicuculline into the ventral pallidum (vp) on the retrieval of conditioned taste aversion (cta). in experiment 1, rats received a pairing of saccharin or quinine hydrochloride (cs) with an i.p. injection of 0.15 m lithium chloride (us). after this conditioning, vehicle or bicuculline was bilaterally infused into the vp just before the re-exposure to the cs. the microinjections of bicuculline significantly increased the intake of saccharin cs, but not quinine hydrochloride. in experiment 2, rats were presented with saccharin as cs via an intraoral cannula. the microinjections of bicuculline significantly increased ingestive responses and decreased aversive responses. these results suggest that the gaba a receptors in the vp play an important role in the expression of ingestive and/or aversive responses to saccharin cs during the retrieval of cta so that the microinjection of bicuculline might increase the intake of saccharin cs. research funds: kakenhi (17730431) ps1a-h139 transient blockade, but not genetic deficiency, of c-fos gene expression impairs long-term memory in taste aversion learning roles of c-fos gene expression and its protein product, fos, in conditioned taste aversion (cta) learning were examined using the antisense oligodeoxynucleotide (odn) method in rats and in mice carrying c-fos gene deficiency. infusion of antisense odn (as-odn) directed against c-fos mrna into the parabrachial nucleus (pbn), but not into the amygdala or insular cortex (ic), impaired the acquisition, while infusion of randomized and inverted control odns had no effect. suppression of fos synthesis in the amygdala or ic impaired the retention. retrieval of an acquired cta was not impaired by as-odn infusion into the pbn or amygdala. in contrast, mice carrying c-fos gene deficiency showed normal acquisition and retention. the present results suggest that the fos-mediated signals in the pbn, amygdala or, ic plays key roles in the acquisition and/or consolidation, but not the retrieval, of long-term cta memory. ps1a-h140 gaba receptors in the deep cerebellar nuclei are essential for mouse eyeblink conditioning classical eyeblink conditioning is a useful experimental system to analyze the neuronal substrate underlying learning and memory. the knowledge on the mouse eyeblink conditioning is far less compared with rabbit's. we examined the role of the deep cerebellar nuclei (dcn) during delay eyeblink conditioning in c57bl/6 mice by using gaba a receptors agonist and antagonist. in the acquisition tests, in which muscimol (msc) or picrotoxin (ptx) was injected from beginning of training, acsf-injected control mice learned this task, but both msc-and ptx-injected mice showed a significant impairment in acquisition of conditioned response (cr). in the retention tests, in which the drug was injected after acquisition of training, cr % in acsf-injected mice were kept over 80%, while those in the mscand ptx-injected mice decreased to 30%. these results revealed that gabaa receptors in the dcn play important roles in acquisition and retention of mouse eyeblink conditioning. various forms of synaptic plasticity are found in cerebellar circuits, but their significance in motor leaning remains unknown. in the cerebellum, delphilin is expressed selectively in purkinje cells (pcs) and localized exclusively at parallel fiber (pf) synapses, where it interacts with glutamate receptor ␦2 that is essential for long-term depression (ltd) and motor learning. here, we showed that ablation of delphilin proteins facilitated ltd induction at pf-pc synapses and enhanced optokinetic response adaptation without affecting histology. this finding suggests that threshold regulation of ltd at pf-pc synapses is a limiting step for motor learning efficiency. ps1a-h143 post-training cerebellar cortical activities are necessary for transfer of memory trace of motor learning from cortex to nuclei soichi nagao 1,2 , takehito okamoto 1 , fumihiro shutoh 1,3 1 lab for motor learning control, riken bsi, saitama, japan; 2 sorst, jst, saitama, japan; 3 dept. anat., grad. univ. tsukuba, ibaraki, japan one-hour optokinetic training induces short-term adaptation of horizontal optokinetic response (hokr) gains in mice. succession of 1 h daily training for 1 week induces long-term adaptation. we recently reported that the memory trace of adaptation of hokr is initially acquired within the cerebellar flocculus through long-term depression (ltd), and later transferred to the vestibular nuclei for consolidation. in order to reveal the neural mechanisms underlying the memory transfer, we reversibly inactivated the neural activities of flocculus bilaterally by local application of muscimol immediately after the end of daily training. mice treated with muscimol showed depressed long-term adaptations, while the short-term adaptations were intact, suggesting that the neural activities of cerebellar cortex in a certain period after training are necessary for the transfer of memory trace from flocculus to vestibular nuclei. research funds: kakenhi (16500204) ps1a-h144 modification of gene expression in the cerebellar cortical neurons related with long-term motor learning yuji t. katagiri 1,2 , takehito okamoto 1 , shin-ichi nishimura 1 , fumihiro shutoh 1,3 , soichi nagao 1,4 1 lab. for motor learning control, riken bsi, saitama, japan; 2 univ of the air, chiba, japan; 3 dept. of anatomy, human comprehensive science, grad. univ. tsukba, japan; 4 sorst, jst, japan we recently reported that the cerebellar ltd plays a crucial role for both acquisition and consolidation of memory trace of long-term motor learning using the adaptation paradigm of mouse horizontal optokinetic eye movements (shutoh et al., 2006) . in order to listup the molecules involved in the motor learning, we sampled total rna from the cerebellar flocculus of short-and long-term adapted mice, and quantified amounts of gene expression by the microarray methods. we found that the number of genes modulated by longterm motor learning much exceeded that modulated by short-term motor learning, and the number of down-regulated genes were larger than that of up-regulated genes. we furthermore examined the gene expression of purkinje cells by the laser micro-dissection and quantitative rt-pcr methods. ps1a-h145 influence of spatial cues on hippocampal neuronal activity in spatial navigation tasks in mice hippocampal neurons were recorded while mice performing spatial tasks of searching for unpredictable and predictable rewards. the influence of spatial cues, including distal and proximal cues, on the response of hippocampal cells that exhibited place-related activity was examined. place cells predominantly shifted their fields accordingly by changes of visual and auditory distal cues, and fewer cells shifted their fields by changes of proximal cues. these results provide evidence that hippocampal neurons of mice can use flexibly information of spatial cues to represent the environment, and this ability is important for spatial learning. son ho 1,2 , t kobayashi 1,2 , e hori 1,2 , k umeno 1,2 , t ono 1 , h nishijo 1,2 1 system emotional science, univ. of toyama, toyama, japan; 2 crest, tokyo, japan we investigated a role of the hippocampal formation (hf) in encoding of a moving object in an open field. rats acquired icss rewards if they moved freely. then, a remote-controlled car was placed inside the open field. the rats could receive icss if it chased and approached the car. of a total of 133 place cells recorded, activity of 40 was significantly modulated by the car speed and/or distance between the car and rat; 21, 12 and 7 cells displayed distance-dependent, car speeddependent, and distance and car speed-dependent firing, respectively. furthermore, six cells, which did not show the place field in reference to rat position, but showed the place fields in reference to car position. in a control experiment, the same car was introduced, but the rats could receive icss rewards without relation to relative distance between the rat and car. so far, 16 place cells were recorded in this experiment. of these, six and three place cells displayed distancedependent and car speed-dependent firing, respectively. the results suggest that hf encodes not only spatial information of own location, but also that of other moving object in an environment. hisae gemba, kazuko nakao, ryuiti matsuzaki, yusaku amaya department of physiology, kansai medical university, moriguchi, japan cortical field potentials were recorded by electrodes implanted on the surface and at a 2.0-3.0 mm depth in the cortex of monkeys in the process of learning somatosensory-initiated hand movements and then analyzed. it was found that an s-n, d-p potential, at about 40 ms latency from stimulus, in the caudal bank of the left arcuate sulcus (homolog of broca's area) was related to recognition learning (association of stimulus with movement), and that an s-n, d-p potential in the motor and somatosensory cortices, and areas 5 and 7, contralateral to the operating hand, was related to skill learning (making movements quicker and more appropriate). in visuo-initiated hand movements, the left prefrontal cortex was related to recognition learning; the motor and somatosensory cortices, and area 5 to skill learning, as previously reported. this indicates that motor programming for somatosensory-initiated and visuo-initiated hand movement differs. computational studies of hippocampal function generally assume that ca1 performs a match-mismatch comparison of memory retrieval with sensory input. here we investigated this comparator model using an ensemble recording during task behaviors in the rat. we employed directional memory-guided alternation and visual cue discrimination tasks for the same animal. after training, the animals tended to predict a next direction according to the alternation paradigm even in the visual cue discrimination task. during this task, we found that some ca1 neurons showed specific bursts when a predicted event did not occur or along the trajectories of their corrective movements from a wrong cite to a correct cued one. these data suggest that ca1 plays an important role in the mismatch detecting and correcting process of behavior. n-methyl-d-aspartate (nmda) receptor has high permeability to ca 2+ but is blocked by mg 2+ in a voltage-dependent manner. this property is a molecular basis of nmda receptor-dependent long-term potentiation, which is thought to play a central role in learning and memory. we have generated the genetically engineered mice in which mutated nmda receptors defecting in mg 2+ binding ability are expressed specifically in the granule cells of the dentate gyrus, the entry point to the hippocampal trisynaptic circuit. the mutant mice showed a variety of behavioral abnormalities including hyperactivity, impaired prepulse inhibition. to elucidate the effect of mutation on the information processing in the hippocampus, we recorded the place-related activity from hippocampal ca1 cells, the output stage of hippocampal circuit. the link between the behavioral anomaly and the hippocampal activity is discussed. mikako sakurai, ko zushida, masayuki sekiguchi, keiji wada department of neurodegenerative diseases, national institute of neuroscience, ncnp, tokyo, japan uch-l1 is a component of the ubiquitin system. uch-l1 is expressed at high levels in the hippocampal neurons. however, the functional role of uch-l1 in synaptic plasticity and behavior is not understood. we examined behavior and synaptic plasticity in gad mouse which is an autosomal recessive spontaneous mutant carrying an intragenic deletion in the gene encoding uchl1. gad mice have significantly impaired performance in the open field and one-trial passive avoidance tests. theta burst stimulation (tbs) of shaffer collateral in hippocampal slices from gad mice elicited decremental long-term potentiation (ltp) in the area ca1. in contrast, non-decremental ltp was induced in control wild-type mice. the maintenance of tbsinduced ltp in the wild-type mice was impaired by actinomycin d, an inhibitor of transcription, whereas tbs-induced ltp in gad mice was insensitive to actinomycin d. these results suggest that uch-l1 is a molecule participating in the synaptic plasticity elicited by tbs and the memory function. ps1a-i151 learning stages in rat operant reversal task and cross-correlation between hippocampal and prefrontal local field potential powers yoshinori izaki, tatsuo akema department of physiology, st. marianna university school of medicine, japan to investigate whether the relationship between hippocampus (hip) and prefrontal cortex (pfc) spontaneous local field potentials changes with leaning stages, we analyzed cross-correlation (cc) of these local field potential powers during operant reversal training sessions. rats were trained with initial discrimination task until a stable discriminative performance was achieved (learning stage 0). then the rats received the reversal training. learning stages examined were as following: the first training session (stage i), leaning stage for s+ (stage ii) and for s− (stage iii). different changes of the cc in some frequency-band powers with learning stages were observed. the cc in higher gamma-band (64-120 hz) was strong at stage 0 and changed with leaning stages. particularly, the cc decreased to almost zero at stage ii. these results suggest that functional connection between hip and pfc is reflected in this frequency-band and changes with learning stages. ps1a-i152 longitudinal fiber systems in the dentate gyrus of the rat norio ishizuka, yoshitomo umitsu department of brain structure, tokyo metropolitan institute for neuroscience, tokyo, japan longitudinal fiber systems in the dentate gyrus of the rat were investigated by anterograde labeling method of pha-l and retrograde labeling method with fluorescent dyes. the flattened hippocampal formation allowed sections to be cut perpendicular to the full septotemporal axis of the dentate gyrus. injection of pha-l into the hilar region elucidated that two longitudinal fiber systems existed in the dentate gyrus. the first fiber system gives rise to projections to the superficial portion of the dentate molecular layer, and the longitudinal axonal trajectory of this system ceased within the range of about 1.5 mm from the injection level. in the second fiber system, axonal terminations began to appear at the level of 1 mm apart from the injection level and were distributed in further full septotemporal extent of the dentate molecular layer. the axonal arborizations of the second system were found in the deepest portion of the dentate molecular layer immediately above the granular cell layer. in the experiment of fluorescent dye injection, several kinds of cells in the hilus were retrogradely labeled. ryoichi moki, ryang kim, hisahiro umeeda, akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan recent studies have shown that when conditioned fear memory is retrieved, fear memory becomes labile and requires gene expressiondependent reconsolidation for the re-storage. in addition, previous our study using conditional creb mutant mice indicated that creb is required for reconsolidation of conditioned fear memory. we also observed protein synthesis-dependent reconsolidation of spatial memory using morris water maze. in this study, to understand the mechanisms of reconsolidation of spatial memory, we examined a role of creb in reconsolidation of spatial memory. using conditional creb mutant mice that enable to induce the inhibition of creb activity in a tamoxifen-dependent manner, we found that inhibition of creb activity leads to disruption of spatial memory after the retrieval. this result indicates that creb is required for reconsolidation of spatial memory. shunsuke hasegawa, hirosi hosoda, satoshi kida department of bioscience, tokyo university of agriculture bhlh-pas transcription factor bmal1 ubiquitously expresses in brain. bmal1 functions by forming a heterodimer with either clock or npas2, which has been known to play important roles in control of circadian rhythm and memory formation, respectively. to understand roles of bmal1 in forebrain function, we generated conditional mutant mice that enable to induce the inhibition of bmal1 function in a forebrain. using a dominant negative mutant of bmal1 (bmal1 r91a) that forms a heterodimer with clock but loses the binding activity with e-box (hosoda et al., 2004), we generated transgenic mice expressing this mutant under the control of tetracycline-dependent promoter. these mutant mice were crossed to transgenic lines expressing tetracycline-dependent transcription factors (tta) under the control of alpha camkii promoter. we observed the expression of bmal1 r91a in several double transgenic lines in a tta-dependent manner. behavioral analyses showed that these mutant mice showed an impairment of memory formation, indicating crucial roles of bmal1 in learning and memory. stress sometimes causes memory deficits. and chewing has been shown to reduce stress. however, the chewing-related mechanism in stress-induced memory deficits is unclear. we thus examined the effects of chewing on spatial memory using morris water maze and fos induction in the hippocampus and amygdala in stressed mice. when mice were exposed to restraint stress, reduction in learning ability and density of fos-positive cells in the dg and the bla was seen, but not in the mice chewing a thin wooden bar during stress exposure. the results suggest involvement of the amygdaloidmechanism by which chewing may prevent the stress-induced impairment of hippocampus-dependent memory. seiichiro amemiya, shinya yanagita, satoko suzuki, ichiro kita graduate school of science, tokyo metropolitan university, tokyo, japan we examined the effect of background noise (bgn) on spatial learning and its neuronal activity related to arousal and stress using maze task and c-fos immunostaining. rats performed maze task under different intensity of bgn (0, 70, or 100 db; intermittent white noise). 70 db bgn induced significant decreases in number of error and time to goal in maze compared with 0 and 100 db. although bgn increased fos positive acetylcholinergic neurons (fos-chat) in mesopontine tegmentum (mt) regardless of the intensity, fos-chat in basal forebrain (bf) increased intensity-dependently. in locus coeruleus (lc) and cortex, fos positive cell increased intensitydependently. furthermore, 100 db bgn remarkably enhanced fos expression in stress-related nuclei, such as paraventricular nucleus and central nucleus of the amygdala. these results suggest that bgn improve spatial performance by enhancing arousal following activation of cholinergic neurons in mt and bf, and lc neurons. however, higher bgn intensity could evoke over-arousal and stress responses, thereby prevent the maze task. siriporn chattipakorn 1,2 , anucha pongpanparadorn 2 , wasana pratchayasakul 2 , anchalee pongchaidacha 2 , nipon chattipakorn 2 1 fac. dent. cmu, chiang mai, thailand; 2 cert, cmu, chiang mai, thailand current pharmacotherapy of ad is the use of ache inhibitors. previous in vitro study showed that tde inhibited ache activity. this is the first study investigating the effects of tde on cortical ache activity and neuronal activity in in vivo. we used fos immunohistochemistry to determine the neuronal activity and the colorimetric method to investigate cortical ache activity following the single injection of various tde doses. mean fos-positive neurons in cortex were 101 ± 14, 124 ± 19 and 108 ± 22 in the groups administered 250, 500 and 1000 mg/kg tde, respectively. cortical fos-positive neurons in all three tde-treated groups were greater than those in the control group. percent inhibition of cortical ache activity was 17.4 ± 6.3, 22.7 ± 6.9 and 16.6 ± 5.0 for 250, 500 and 1000 mg/kg tde, respectively. these ache inhibitory effects were significantly different from the control. these findings suggest that tde could be beneficial as a possible novel therapeutic agent for ad. we showed the effects of a 2-h tde administration in animals on the inhibition of cortical ache activity and the enhancement of cortical neuronal activity. ache activity in circulation following a 2-h tde administration was not different compared to the control. this study investigated that the effects of tde on circulating ache activity (cache) in animal models was time-dependent. we used the colorimetric method to investigate cache activity in rats following the single administration of tde at various doses at different time courses (10, 60 and 120 min). percentage inhibition of cache activity following a single tde injection at doses 500 and 1000 mg/kg significantly decreased at 10 and 60 min after tde injection, but not at 120 min. cache inhibitory effects among two doses of tde administrated groups at various time courses were not significantly different. these findings suggest that tde may be a short-acting ache inhibitor. donepezil, galanthamine and tacrine are acetylcholinesterase (ache) inhibitors used for treatment of alzheimer's disease. we examined the neuroprotective mechanisms of ache inhibitors against apoptotic glutamate neurotoxicity using cortical neurons. we show that they protect neurons through mechanisms other than ache inhibition. the protective effects are mediated through nicotinic receptors (nachrs). donepezil and galanthamine protect neurons through ␣4and ␣7-nachr and kinases involved in pi3k-akt pathway, and increase the levels of phosphorylated akt and bcl-2. these results suggest that these ache inhibitors express their neuroprotective effects against glutamate neurotoxicity through nachrs and that donepezil and galanthamine protect neurons through pi3k-akt pathway via ␣4and ␣7-nachrs. ps1a-i160 arachidonic acid preserves hippocampal neuron membrane fluidity in senescent rats yasuto kashiyae 1 , yoshiyuki ishikura 2 , shigeaki fujikawa 2 , yoshinobu kiso 2 , manabu sakakibara 1 1 lab. neurobiol. engr., tokai univ., numazu, japan; 2 inst. health care sci. suntory, shimamoto, japan previous studies indicate that long-term dietary supplementation with arachidonic acid (aa) in 20-month-old rats (oa) effectively restores performance in a memory task and induction of long-term potentiation in the hippocampus to the level of young control animals (yc). this study examined fluorescent recovery after photobleaching (frap) in yc, old control (oc), and oa neurons in hippocampal slice preparations. three measures: mobile fraction (mf), diffusion constant (d), and time constant (τ), were estimated among yc, oc, and oa. each of these parameters was significantly different between oc and yc, suggesting that membrane fluidity is lower in oc than in yc. in contrast, d and τ were almost comparable in oa and yc, indicating that hippocampal neuronal membranes supplemented with aa were more fluid than those in oc, whereas the fraction of available molecules remained smaller than in yc. long-term administration of aa to senescent rats might help to preserve membrane fluidity and maintain hippocampal plasticity. ps1a-i161 thimet oligopeptidase co-exists in gfap-and cd11b-positive glia in rat pc/rsc treated with mk-801 takeshi kato 1 , mohammad arif 1 , michiyuki yamada 2 , toshiyuki chikuma 3 , md. mahiuddin ahmed 4 1 lab. natural info. sci., grad. sch. integr. sci., yokohama city univ., yokohama, japan; 2 grad. sch. integr. sci., yokohama city univ., yokohama, japan; 3 dept. hygien. chem., showa pharmaceut. univ., machida, japan; 4 dept. r&d, bioelectro. anal. sci., japan thimet oligopeptidase (ep24.15) hydrolyzes not only neuropeptides but also the peptides generated by proteasomes. in the present immunohistochem study we found that mk-801 activated gfapand cd11b-positive glia cells in rat posterior cingulate/retrosplenial cortex (pc/rsc) 3 day after the treatment. mk-801 also increased ep24.15 and prolyl oligopeptidase. immunohistochem data showed that ep24.15 co-localized with gfap and cd11b positive glial cells. since mk-801 causes schizophrenia-like psychosis and produces neurotoxicity in adult rodent brain, we further examined the pretreated effect of neuroleptics. clozapine co-administration suppressed the increased ep24.15 in the pc/rsc. these data suggest that ep24.15 in the astroglia and microglia cells of rodent brain might in part control positive and/or negative schizophrenia symptoms. ps1a-i162 effect of age and sex steroids on the expression of alzheimer's disease presenilin (ps) 1 and 2 in the mouse brain soumi ghosh, m.k. thakur banaras hindu university, india alzheimerǐs disease is a neurodegenerative disorder characterized by the impairment of cognition and memory. these functions are improved by supplementation of sex steroids. the genes causing lateonset of ad, presenilin (ps) 1 and 2, code for highly homologous integral membrane proteins. the proteolytic fragments of these proteins are main biological components. we have analysed the effect of age, sex and gonadal hormone supplementation on ps expression at protein level by western blotting. ps1 shows a significant decrease with aging in both males and females. however, there is no significant variation in expression of ps1 and ps2 with sex. gonadectomy also lowers the level of presenilin proteins in old age. ps2 protein shows increase in expression with gonadal hormone treatment in both ages, but estrogen supplementation to old mice lowers ps1 level. these modulatory effects of age, sex and gonadal hormones on ps proteins may explain the therapeutic interventions of hormone replacement therapy. research funds: ministry of science and technology, india ps1a-i163 effects of the monomeric, oligomeric and fibrillar beta-amyloid peptides on the proliferation and differentiation of adult neural stem cells from svz dept. of pharmacol., seoul natl. univ., south korea the subventricular zone is the largest neurogenic area of the adult brain. in this region, neural stem cells (nsc) serve to produce newly generated neurons and glia cells throughout adulthood. however, the common neurogenesis of nsc cannot replace neuronal loss in alzheimerǐs disease (ad) induced by amyloid deposits composed mainly of amyloid␤proteins. in vitro, we examined the effects of various form of a␤peptide on the proliferation and differentiation of nsc from svz of 10-week-old adult mice. in this study, a␤42 peptide was prepared three forms of aggregating stage, monomeric, oligomeric and fibrillar a␤42 peptide. we found that treatment of nsc with oligomeric form of a␤42 peptides remarkably increased the number of neurospheres during proliferation and neurons during differentiation in-vitro. we also found that these neurogenesis was accompanied by morphological change of neuron. the number of secondary and tertiary neurites increased at submicromolar concentrations of oligomeric a␤42 peptide without shrinkage of axonal length. in alzheimer's disease (ad) brain, the formation of senile plaque with accumulated microglia is observed. although the role of microglia in ad is not clarified, their involvement in a␤ clearance is noted. high mobility group box protein-1 (hmgb1) is a non-histone chromosomal protein. here, hmgb1 was associated with senile plaques and protein level was increased in ad brain. diffuse hmgb1 immunoreactivity was observed around dying neurons in the kainic acid-and a␤1-42 (a␤42)-injected rat hippocampi. hmgb1 was not co-localized with a␤ in transgenic mice which show massive a␤ production without neuronal loss. furthermore, co-injection of hmgb1 delayed the clearance of a␤ and accelerated neurodegeneration in a␤42-injected rats. these results suggest that hmgb1 released from dying neurons may inhibit microglial a␤ clearance and enhance the neurotoxicity of a␤. perineuronal nets consisting of chondroitin sulfate proteoglycan (cspg) and hyaluronic acid (ha) are associated with distinct populations in mammalian brain. in the present study, we observed perineuronal net-like structure by rat cortical neurons in dissociated culture using wisteria floribunda lectin, ha binding proteins, and cspgspecific antibodies. this perineuronal net-like structure was observed often at parvalbumin-positive neurons, indicating gabaergic ones. it is well known that perineuornal nets-containing neurons are survive against alzheimer disease in human. to elucidate significance of perineuronal nets in alzheimer disease, we applied beta-amyloid peptide into cultured cortical neurons. perineuronal nets-containing neurons were resistant against beta-amyloid peptide, while negative neurons were often dead. these results indicate that perineuronal nets are participated in protecting neurons from cytotoxic substances such as beta-amyloid. ps1a-i166 x11-like protein regulates metabolism of app in the mouse brain yoshitake sano 1,2 , tadashi nakaya 2 , shigeyoshi itohara 1 , toshiharu suzuki 2 1 riken bsi, saitama, japan; 2 hokkaido university, neuroscience, sapporo, japan abnormal metabolism of amyloid beta precursor protein (app) results in the accumulation of beta amyloid (a␤) in the brain, and contributes to the pathogenesis of alzheimer's disease. app has a functional sequence in its cytoplasmic domain, the yenpty motif, which is involved in trafficking, internalization, and metabolism of app. x11-like protein (x11l) was identified as a molecule that interacts with the motif and regulates app metabolism in cultured cells (1999 . j. biol. chem. 274, 2243 2003. j. biol. chem. 278, 49448) . there is no evidence, however, that endogenous x11l suppresses app metabolism and a␤ generation in vivo. to examine the physiologic role of x11l in app metabolism in the brain, we generated x11l null mutant mice. the mutant mice developed normally without gross anatomic brain abnormalities. there were increased amounts of cterminal fractions cleaved at the ␤-site and a␤, but the amount of total app was unaltered in the mutant mouse brain. these results suggest that x11l suppresses the production of a␤ by inhibiting ␤secretase-induced proteolysis of app. it is still unknown how human's central nervous system (cns) controls its body system to keep the body balanced. this study aims to analyze the characteristics of spectral response of body sway in eyes open and in eyes closed during static upright stance based on a pid control model. in this model, body sway in medial-lateral direction is considered, and the body is simply modelled as a multi-link inverted pendulum system. spectral response analysis showed the gain varied with input frequency and time lag. peaks of the gain were intensively influenced by controller's parameters (kp, kd and ki). parameters identification showed that kd is decreased in eye-closed. by simulation, the spectral responses of the pid model quite agreed with the experimental data. the results proved that the spectral characteristics of body sway is determined by the dynamics of body system and its controller's parameters, suggest the balance-keeping control in cns can be modelled as a pid controller. nuclear dysfunction is a critical element of the pathology of polyglutamine (polyq) diseases. proteome analysis of soluble nuclear proteins in the nuclear matrix of neurons expressing normal or mutant huntingtin or ataxin-1 protein by 2d-electrophoresis and tof-mass delineate that mutant at1 and htt proteins similarly reduce transcriptional factor x1 and x2. immunoprecipitation and pull-down assays support interaction between polyq and factor x1 and x2. immunohistochemistry of hela cells and primary neurons reveal sequestration of factor x1 and x2 into inclusion bodies and reduction of them in the nuclear matrix. compensatory expression of factor x1 and x2 ameliorates poly-q pathology in htt-/at1-expressing neurons and transgenic drosophila. these results suggest that factor x1 and x2 are critical regulators of polyglutamine disease pathology and could be a target for developing therapeutics. ps1a-j170 ba1-42 was reduced in rat brains fed with coconut juice n. radenahmad, p. subhadhirasakul psu, thailand young coconut juice (ycj), cocos nucifera (arecaceae), believed to contain phytoestrogen and other sex hormone-like substances, was investigated for its possible beneficial effects on halting dementia in ovariectomized (ovx) rats, a model system for the postmenopausal condition. sixty ovx rats were divided into six groups, 10 rats/group (g). group 1 received e2 at 2.5 g/kg per day; groups 2 and 3 received ycj at 20 ml, and 100 ml/kg day, respectively, once everyday. group 4 received ycj 100 ml/kg plus e2 at 2.5 g/kg day twice a week, all for 5 weeks. the other two were ovx and sham-operated controls. using a chemiluminescent immunoassay, circulating e2 in group 3 was insignificantly different from the control groups. after rats were sacrificed, brains were removed, fixed and paraffin embedded for ihc staining. using anti-␤-amyloid 1-42 antibody, this alzheimer pathology was found in cytoplasm and dendrites, but not in nuclei or axons, of pyramidal cells both in hippocampus and in layer 3 and layer 5 of cerebral cortex. it was found that amyloid deposition in frontal, temporal and hippocampus of rat brains in group 3 was lesser than ovx and control groups. amyloid deposition was correlated with e2 serum at r = −0.4. ps1a-j171 correlation between semantic memory and regional gray matter volume of anterior aspect of right temporal lobe in normal elderly subjects. a voxel-based morphometry yasuyuki taki 1 , shigeo kinomura 1 , kazunori sato 1 , shinya uchida 1 , ryoi goto 1 , kentaro inoue 1 , ichiro tsuji 2 , hiroyuki arai 2 , ryuta kawashima 3 , hiroshi fukuda 1 1 department of radiology and nuclear medicine, institute of development, aging and cancer, tohoku university, sendai, japan; 2 tohoku univ. grad. school of med., sendai, japan; 3 niche, tohoku univ., sendai, japan the purpose of this study was to determine whether there is a correlation between semantic memory and regional gray matter volume in community-dwelling normal elderly people by voxel-based morphometry. we collected brain magnetic resonance images of 111 community-dwelling normal elderly subjects. we performed multiple regression analysis of raw score in the wais-r information subtest, gender, and regional gray matter volume. the volumes of the right superior and middle temporal gyri showed significant positive correlations with raw score in the information subtest. our study indicated that normal elderly individuals show a significant correlation between regional gray matter volume and semantic memory. research funds: (h13-kenko-008), (h13-choju-007, h13-choju-023) ps1a-j172 effects of fluoxetine on the cognition of patients with mild cognitive impairments arash mowla, azadeh pani shiraz university of medical sciences, iran recent researches suggest a role for monoaminergic hypofunction in age related cognitive decline. in several studies selective serotonin reuptake inhibitors demonstrated neurogenesis in hippocampus. we studied the effects of fluoxetine on cognition of patients with mild cognitive impairment (mci). fifty-two non-depressed patients with mci were randomly assigned to take fluoxetine or placebo. the patients were administered mini-mental status examination (mmse) and wechsler memory scale iii (wmsiii) pre intervention. twenty-six patients completed the 8 weeks trial. treatment response was defined as a final mmse and wms-iii scores. the patients in the fluoxetine group showed improvement in mmse and immediate and delayed logical memory scores of wms-iii. the placebo group had not significant changes in the cognitive measurements. fluoxetine enhanced memory and cognition in the patients. this was consistent with pervious studies that emphasized on the role of fluoxetine in improving memory and promoting neurogenrsis in the hypocampus. however, this conclusion should be tempered by the small sample size. lisa l. cook 1 , d.g. goodenowe 1 , y. yamazaki 1 , j. flax 2 1 phenomenome discoveries inc., saskatoon, canada; 2 precisionmed inc., san diego, ca, usa dementia affects about 10% of the population over the age of 65 and can result from various neuropathological conditions. currently, there is no way to differentiate specific forms of dementia (alzheimer's disease (ad), vascular dementia, etc.) prior to autopsy. pdi has discovered an 8-metabolite biomarker panel within the serum of patients with ad, non-ad dementia and healthy non-demented controls that can simultaneously differentiate the type of dementia and identify cognitive impairment using a non-targeted metabolomics technology based a fourier transform ion cyclotron resonance mass spectrometry (fticr-ms). the accurate measurement of the metabolite mass is sufficient to elucidate its molecular formula, thereby leading to metabolite identification, explication of biological significance and efficient biomarker validation. the 8-metabolite biomarker panel could provide a non-invasive method to aid in the diagnosis of specific subtypes of dementia. the development of a high throughput assay for these markers will also be presented. neurons become photosensitive by genetically introducing one of green algae-derived protein, channelrhodopsin-2 (chr2). in this study, we quantitatively investigated the rapidness of the light-gated current of chr2 expressed in pc12 cells using blue led light. the light-gated current consists of two components, inactivating and noninactivating. the magnitude of inactivating component was almost linearly related to the light intensity. the non-inactivating component showed the tendency to saturate at high illumination. we also found that the activation rate is about 10-fold faster than the inactivating rate, but both are linearly dependent on the light intensity. since the photoactivated current was very rapid in both onset and offset, the neuronal firings were phase-locked to short light pulses in an acute slice of hippocampus. it is suggested that the genetic expression of chr2 is one of the most ideal photostimulation methods of a genetically identified neuron with defined activity patterns in intact nervous system. yujiro hattori 1 , shigeki ohta 1 , kenji hamada 2 , naofumi yamada-okabe 2 , yonehiro kanemura 3 , hideyuki okano 4 , yutaka kawakami 5 , masahiro toda 1,6 1 neuroimmnology research group, keio univ., tokyo, japan; 2 chugai co. ltd., kanagawa, japan; 3 inst. cli. res., onh, japan; 4 physiology, keio univ., tokyo, japan; 5 cellular signaling, institute for advanced medical research, keio univ., tokyo, japan; 6 neurosurgery, keio univ., tokyo, japan to identify neuron specific genes, we performed two gene profiling techniques, dna microarray and est analysis. in this study, we focused on genes expressed specifically in the normal brain tissues but not in glioma tissues and identified the human kiaa1110 gene which was a homologue of rat synarfgef (po). rt-pcr analysis revealed that the human kiaa1110 homologue was expressed only in adult brain tissue. western blot and immunocytochemical analyses showed the kiaa1110 protein was expressed in adult brain tissues and differentiated neuronal cells but not in fetal brain tissues nor neural stem/progenitor cells. in conclusion, we identified an adult neural-specific gene using the combined gene profiling method and our results suggest the usefulness of this method to identify tissue specific genes. ritsuko the objective of this study was to find the proteins related to sexual differentiation and to elucidate its molecular mechanism. methods: developing hypothalamic and cortical cells from fetuses on embryonic day 15 were dissociated. after the cells were treated with 100 nm estradiol-17beta (e2) or ethanol for 8 days, proteins were extracted and labeled with cydyes. two-dimensional difference gel electrophoresis (2d dige) was then performed. the differential protein spots were analyzed by software analysis, subject to in-gel digestion, and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (maldi-tof-ms). results: more than 4000 spots were detected from 2d dige. compared with ethanol treatment, e2 increased the expression of 501 spots in the hypothalamic cells and 169 spots in the cortical cells (p < 0.2, difference >1.5). proteomics analysis showed different effects of e2 for hypothalamic and cortical cells. in order to relate cellular brain structure to function, it is necessary to manipulate neural circuits at the level of individual cell types. genetic methods for neuronal inactivation combined with cell-typespecific promoters will achieve this goal. here, we have developed a genetic method for quickly and reversibly inactivating in vivo mammalian neurons using allatostatin receptor (alstr), which causes neuronal hyperpolarization when treated with peptide ligand allatostatin (al). in rat barrel cortex neurons expressing alstr, al reversibly inactivated neuronal activity evoked by electrical stimulation of the whisker pad. both inactivation and recovery were seen within several minutes. we also confirmed the effectiveness of the al/alstr system in ferret visual cortex, lateral geniculate nucleus (lgn), and monkey lgn. therefore, the al/alstr system will be a powerful tool to investigate neuronal circuits and function. prospective purification of neural stem cells (nsc) through the specific cell surface marker is crucial for functional recovery of the damaged brain. in the last meeting, we showed that living nsc were enriched from mouse whole brain as positive cells for erythro-phytohemagglutinin (e-pha), which binds to complex type asparagine-linked oligosaccharide (n-glycans). in this study, by using facs system, we found that high selective affinity of e-pha binding to the brain cells; e-pha negative cells were neurons, mid-positive cells were nsc, and highly positive cells were endothelial cells, respectively. ligand blot analysis revealed the existence of the e-pha binding proteins different from the known selective nsc markers in e14 days brain homogenate, suggesting that n-glycosylated proteins could be distinctive markers for nsc. masahiro waza, hiroaki adachi, masahisa katsuno, makoto minamiyama, fumiaki tanaka, manabu doyu, gen sobue department of neurology, nagoya university, nagoya, japan the pathogenic gene product of spinal and bulbar muscular atrophy (sbma) is polyglutamine (polyq)-expanded androgen receptor (ar), which belongs to hsp90 client protein family. 17-allylamino-17-demethoxygeldanamycin (17-aag) is a new derivative of geldanamycin that shares its important biological activities but shows less toxicity. 17-aag is now in phase ii as a potential anti-cancer agent because of its ability to selectively degrade several cancer-related client proteins. we examined the efficacy and safety of 17-aag in a mouse model of sbma. administration of 17-aag significantly ameliorated polyq-mediated motor neuron degeneration by preferential proteasome degradation of mutant ar. the ability of 17-aag to preferentially degrade mutant protein would be directly applicable to sbma and other neurodegenerative diseases. modulation of hsp90 function by 17-aag has emerged as a candidate of molecular targeted therapy for neurodegenerative diseases. the avian embryo has long been a popular and an excellent model for studying vertebrate development because of its classical manipulative advantages. in the present study, we tried to develop regulated gene transfer by using the tet regulatory system in the chick. the reverse tetracycline-controlled transactivator was expressed under the control of the motor neuron (mn) specific hb9 promoter. tetracycline responsive elements were used for inducible gfp expression. after these constructs were introduced into neural tube by in ovo electroporation, gfp expression was induced in spinal mns in the presence of doxycycline. approximately 5-20% of mn express gfp very intensely whereas the remaining mns never express gfp, suggesting that, although the transgene is induced in limited numbers of mns, once activated, cells express a large amount of the protein product of the experimental gene. thus, this strategy can be applicable for a variety of experiments that require specially and temporally regulated gene expression in the chicken embryo. ps1a-k181 selective collection of catecholaminergic (ca) neurons in the brain and its application to gene expression analyses hiroaki nakamura 1 , yoshiyuki ishii 1 , kazuto kobayashi 2 , yasufumi sato 3 , keiichi itoi 1 1 lab. info. biol., grad. sch. info. sci., tohoku univ., sendai, japan; 2 dept. mol. genet., fukushima med. univ., japan; 3 inst. develop., aging, cancer, tohoku univ., japan ca neurons are involved in a wide spectrum of physiological functions in the brain. most ca neurons are localized in the brainstem and hypothalamic regions and typically make clusters of cells, among which the noradrenergic (a1, a2 and a6) and dopaminergic (a9, a10 and a12) neurons predominate. in order to explore functional roles of these neurons, we collected ca neurons selectively using tyrosine-hydroxylase (th)-green fluorescent protein (gfp) transgenic mice in which gfp was expressed under the control of th gene promoter. in fetal mice, most gfp-positive neurons expressed th-immunoreactivity in limited brain regions including the locus coeruleus (lc). therefore, lc-containing region was dissected under fluorescent microscopy, and neurons were dispersed by treating with trypsin, then gfp-positive cells were sorted out by flow-cytometry (facs). rna was extracted from the gfp-positive (th) neurons, reverse-transcribed, and analyzed by pcr. atg4b has been shown to play an important role in the processing of lc3, a mammalian homologue of yeast atg8, but the tissue distribution of atg4b remains unknown. to understand the role of atg4b in rat tissue cells, we prepared an antibody to atg4b and pc12 cells in which atg4b expression was knocked down by rnai. in rnaitreated pc12 cells where atg4b expression was 10% of that in the wild-type pc12 cells, the expression of cytosolic lc3-i was similar to that in wild-type cells. the knockdown cell lysates, however, suppressed cleavage of prolc3 to lc3-i. moreover, the expression of atg4b mrna was high in the cerebellum and olfactory bulb, while its protein was evenly distributed in the brain. immunostaining for atg4b was intense in neurons, especially in the cerebellum. these results suggest that atg4b plays a major role in the processing of lc3, while autophagy is deeply associated with the metabolism in neurons, especially in the cerebellum. ps1a-k183 spatial and time-dependent transneuronal propagation of swine coronavirus (hemagglutinating encephalomyelitis virus, hev) in the rat central nervous system after its hind footpad inoculation transneuronal propagation of hev 67n strain into the central nervous system was examined after its subcutaneous inoculation (10 5 pfu) in the rat hind footpad. on day 3 post-inoculation (p.i.), antigenpositive neurons were detected in cell groups of the ipsilateral spinal cord of lumber segments. on day 4 p.i., they increased in number, and higher-order transneuronally infected neurons were observed in restricted brain areas that project to the spinal cord. on day 5 p.i., the viral infection became more extensive and complex, and neurological signs appeared from this period. in this model the 4th day would be critical for the analysis of the long-distance connections. hev can be used as a novel tracing probe, being equivalent to other reported virus probes. hiroyuki hioki 1 , hiroshi kameda 1 , hisashi nakamura 1 , taro okunomiya 1 , koji ohira 1 , kouichi nakamura 1,2 , takahiro furuta 1 , takeshi kaneko 1,2 1 dept. of morphol. brain sci., grad. sch. of med., kyoto univ., kyoto, japan; 2 crest, japan vesicular stomatitis virus g-protein (vsv-g) pseudotyped lentiviral vectors are useful vectors for gene transfer into the central nervous system. vsv-g achieves a broad transduction spectrum and lentiviral vectors provide an efficient vehicle to integrate transgenes into dividing and non-dividing cells. thus, vsv-g pseudotyped lentiviral vectors with ubiquitous promoters, such as human cytomegalovirus (hcmv) promoter, infect and express transgenes in neuronal and glial cells. the purpose in this study is to explore neuron-specific promoters and to quantitatively examine their characteristics. at first, we used five kinds of well-known neuron-specific promoters; hsyn1, rta1, mcamkii, rnse and hpdgf promoters. then, we developed new hybrid promoters by a combination sequence of hcmv enhancer and neuron-specific promoters listed above. all of the new hybrid promoters dramatically improved expression of reporter gene (gfp), but the specificity deteriorated in the rat striatum, thalamus and neocortex. although green fluorescent protein (gfp) is a useful tool to label living neurons, neuronal processes are not completely labeled with gfp. in the present study, we tried to develop dendritic membrane-targeted gfp using non-prolilferative lentivirus vector with human synapsin i promoter. palmitoylation site of gap43 n-terminal and myristoylation site of fyn n-terminal were first tested for membrane targeting of gfp. myristoylated gfp (myrgfp) was efficiently localized at the plasma membrane of infected neurons, but not palmitoylated gfp. since myrgfp was located at both dendritic and axonal membranes, we further added the putative dendrite-targeting or basolateral targeting signals, such as c-terminals of telencephalin (tlc), fc ␥ ii ␤ receptor (fcr), polymeric immunoglobulin receptor (pigr), and low density lipoprotein receptor (ldlr), to c-terminal of myrgfp, and compared their efficiency on dendrite targeting. recently, we developed recombinant rabies virus vectors which were expected to act as a potential neurotracing tool. the vectors infected neurons specifically from axon terminals and were transported to the downstream neurons trans-synaptically. by using two different recombinant vectors, each of which expresses reporter protein of different kind, we attempted double labeling of a neuron. it was expected that we could detect and visualize the divergence or convergence of a neurocircuit. in the study, we could demonstrate the efficiency of double labeling in vivo. in the present study, we examined the potential of this technique particularly in terms of the quantitative detection of double labeled neurons in complicated neurocircuit. the experiments were performed in the hippocampus and the neighboring cortices of rats. we could show that this technique is also useful for the quantitative analysis of neurons which forms projections to different region of the brain. shuchen lee, lihao ge institute of neurobiology, institute of brain science, fudan university, shanghai, china glycine receptors on bullfrog retinal cone photoreceptors were characterized by immunocytochemical and whole-cell patch clamp techniques. cone terminals were both gly␣1 and gly␤ immunoreactive. in freshly dissociated cones, an inward current could be induced while glycine was focally applied to the terminal. the glycine-induced current was strychnine-sensitive and reversed in polarity at a membrane potential, close to the equilibrium potential of chloride ions. these results suggest that glycine, which may be released by glycinergic inplexiform cells, could modulate functions of cone photoreceptors. ␦-catenin has 10 armadillo motifs and a carboxyl terminal type i pdz ligand. in neurons, ␦-catenin is enriched in the postsynaptic density, where it serves as a link between the adherens junction and the post-synaptic protein complex including the nmda and ampa receptors. electrophysiological recordings from ca1 hippocampal neurons overexpressing ␦-catenin demonstrated that ␦catenin increased the ampa receptor-mediated epsc but had no significant effect on the nmda receptor-medicated epsc. the effect of ␦-catenin on the ampar-epsc was medicated by its pdz ligand. in cos7 cells, co-transfection of ␦-catenin/grip showed that ␦-catenin regulated the membrane localization of grip through its pdz ligand. co-transfection of ␦-catenin/grip/gulr2 increased the surface expression of glur2 in cos7 cells compared with grip/glur2 or ␦-catenin/glur2 transfection. this study points to ␦-catenin as a regulator of glur2 receptor trafficking. inseon song, kunihiko obata, alexey semyanov bsi, riken, japan gaba a receptor mediated tonic conductance is a major component of membrane conductance which determines the way how neuron integrates incoming synaptic signals as well as input-output characteristics of the cell. we measured density of picrotoxin (gaba a receptor antagonist) sensitive holding current (which reflects gaba a receptor mediated conductance) in interneurons of hippocampal ca1 area in wild type (wt) and gad65 knockout (ko) mice. this parameter was twice lower in gad65ko mice (wt: 2.0 ± 0.5 pa/pf, n = 7; gad65ko: 0.9 ± 0.3 pa/pf, n = 8; p = 0.038). the total membrane conductance was similar in both types of animals suggesting adaptive compensation. application of gaba (5 m) increased tonic current in both type of mice by the same amount. no significant difference in amplitude or frequency of spontaneous ipscs was detected, although their decay time was shorter in gad65ko animals (wt: 12.4 ± 0.7 ms, n = 14; gad65ko: 10.8 ± 0.6 ms, n = 12; p = 0.047). the changes in inhibition which we have found may explain previously reported behavioral abnormalities in gad65ko. research funds: bsi, riken hiroki mutoh, thomas knopfel lab. for neuronal circuit dynamics, bsi, riken, wako, japan olfactory glomeruli constitute the first stage of central odor processing. yet, their role in integration of odor information is only partially understood. we previously discovered that 5 hz olfactory nerve (on) stimulation induces long-term depression (ltd) in young (p5 to p8) mice. the present experiments were designed to understand in more detail the molecular mechanisms underlying on ltd. bath application of dhpg, a selective group i mglur agonist, induced on ltd and occluded subsequent 5 hz stimulation-induced ltd. on ltd was not induced by activation of group ii or iii mglur agonists. the dhpg-induced on ltd was mediated by mglur1 but not by mglur5 because it was antagonized by the mglur1 antagonist ly367385 but not by the mglur5 antagonist mpep. expression of dhpg-induced on ltd was accompanied by an increase in paired-pulse ratio suggesting that on ltd is caused by a decrease of release probability. we propose that mglur1 is expressed at the on. on ltd may be important for establishment and maintenance of odor maps in the olfactory bulb but may also involve in the regulation of the sensitivity for specific odorants. ps1p-a004 involvement of dopamine system in long-term potentiation of thalamo-prefrontal cortex pathway masatoshi takita 1 , michiko ohtomi 2 1 cognition and action group, national institute of advanced industrial science and technology (aist), ibaraki, japan; 2 department of biomolecular science, faculty of science, toho university, chiba, japan a mesocortical dopaminergic (da) input to prefrontal cortex (pfc) with the d1 receptor is necessary for long-term potentiation (ltp) to occur at hippocampal-pfc synapses, which is involved by working memory (wm) in rats. here the da system was investigated in another wm-involved pathway from mediodorsal nucleus of the thalamus (md) to pfc. preliminarily, local perfusion of the d1 antagonist sch23390 into pfc by using a microdialysis method impaired md-pfc ltp but the d2 antagonist sulpiride did not. extracellular da levels in the pfc robustly increased after the tetanus of md (by 110-120%). as a result both excitatory synaptic inputs to the pfc involved the wm-related da profile, implying da system enables a contrast-emphasis for cooperative crosstalk among several neuroplasticities in the pfc to selectively store intersectional information of multiple brain areas. neuronal activity is necessary for postnatal maturation of synaptic connections only grossly laid out in the neonatal brain. in sensory cortices, synaptic maturation involves strengthening of sensory-evoked responses and development of receptive field (rf) maps with defined rf size and shape. evoked activity is thought to shape synaptic maturation in sensory cortices by mechanisms of competitive hebbian plasticity. dendritic excitability, mediated by voltage-gated na + channels, is required for active backpropagation of axosomatic action potentials (aps) and initiation of dendritic spikes; backpropagating aps and dendritic spikes enable forms of synaptic hebbian plasticity, such as spike-timing dependent plasticity (stdp). here we examined the role of dendritic excitability in synaptic maturation of layer 2/3 pyramidal neurons in the rat somatosensory barrel cortex. in the present study we compared ltp induction in neocortex of captreated and normal rats in present of gaba antagonist, picrotoxin (ptx). the result of present experiment showed that ptx plays an important facilitatory role in the induction of ltp in both normal and cap-treated group. in cap-treated group, in present of ptx, the ltp responses significantly were higher than normal group. we conclude that the enhancement of ltp by ptx can be explained by product of competition between excitatory and inhibitory pathways or synapses. these results suggest that gabaergic system has an important role in synaptic plasticity. also, these results indicated that gabaergic inhibition has been increased in cap-treated group. tohru kurotani, komatsu yukio department of visual neuroscience, research institute of environmental medicine, nagoya university, nagoya 464-8601, japan we showed in previous study that somatic inhibitory synapses of neocortical layer 5 pyramidal neurons undergo long-lasting depression and potentiation depending on the intrinsic firing pattern of the cell that mimics slow wave sleep (sws) and arousal states. in the present study, using a minimal stimulation method, we recorded somatic ipscs from layer 5 pyramidal cells in visual cortical slices prepared from rats at sws like state under urethane anesthesia and in those prepared from rats at arousal state. the average amplitude of somatic ipscs recorded in slices from the former group was significantly larger than that recorded in slices from the latter group. the mean rise time, decay time constant of ipscs and the mean input resistance of the cells were not significantly different between these two groups. the present results further confirmed that the somatic inhibition in neocortical layer 5 pyramidal neurons is bidirectionally modified in accordance with behavioral state. corticothalamic fibers (ct), originated from cerebral layer 6 pyramidal cells, make excitatory synapses with both thalamic relay neurons and reticular neurons. since these pyramidal cells abundantly express kainate receptors (kars) mrna, we studied the effect of kainate on the presynaptic function of the two ct synapses in mouse thalamic vb nucleus. bath application of kainate (200 nm) depressed ct-epscs and increased the paired pulse ratio in relay neurons. in contrast, kainate at the same concentration facilitated ct-epscs and decreased the paired pulse ratio in reticular neurons. these results suggested that kars differentially regulated release at the two ct synapses. furthermore, high frequency stimulation of ct depressed relay cell synapses but facilitated reticular cell synapses. blocking endogenous kars abolished these effects. because reticular cells are the main source of inhibitory input to relay neurons, we suggested that endogenous kars presynaptically regulate the balance of excitatory and inhibitory inputs to thalamic relay neurons. to examine the involvement of ntr1 in the regulatory mechanisms for ltp in the amygdala, we utilized ntr1-knockout (ko) mice. we performed whole-cell patch-clamp recordings from the pyramidal neurons in the basolateral amygdala (bla), where da-nt neurons project. we found that the bla-ltp, induced by la stimulation, was significantly greater in ntr1-ko mice than in wild-type mice. the bla-ltp in ntr1-ko mice was attenuated by sulpiride, a d2 receptor antagonist. these results suggest that d2-ntr1 interaction regulates the extent of ltp in the mouse la-bla synapses. ps1p-a010 facilitation of axonal plasticity in recovery from traumatic brain injury and the role of tnf␣ in mouse model recent studies suggest axonal plasticity as possible mechanism of recovery from brain injury. apart from that, tnf␣, an inflammatory cytokine, has also been suggested to serve neuroprotective roles. the present study evaluated motor function recovery after controlled cortical impact (cci) brain injury, and also the facilitation of plasticity by biotin dextran amine (bda) axonal tracing in tnf␣ko mice and wild type (wt) mice. mice were subjected to left sided cci or served as sham controls, and were evaluated by composite neuroscore and rotarod over 28-day period. bda was injected in right cerebral cortex to observe new axonal connections. so far, we observed recovery of motor function in wt mice, whereas tnf␣ko mice showed continuous functional deficit. we also observed greater number of new axonal connections in wt mice. our results suggest that tnf␣ is necessary for functional recovery after brain injury, and axonal plasticity may be the mechanism involved. disuse of synaptic activity causes homeostatic adaptation presynaptically and/or postsynaptically. here we show that in hippocampal autaptic cultured neurons tetrodotoxin-induced chronic inactivity increases the fraction of high vesicular release probability pool with the entire readily releasable vesicle pool size remained intact. kinetics of short-term plasticity and unchanged apparent ca 2+ sensitivity indicate that ttx-induced presynaptic modification is unlikely due to an increase in the fusion rate crucial for the ca 2+ at the final fusion step. in addition, analysis of neurons genetically lacked the synaptic vesicle protein synaptotagmin-1, and timing-dependent rescues using two different viruses provide a novel conception, namely, vesicle machinery requires prolonged period so that the fast burst vesicle pool orchestrates presynaptic homeostasis system underlying "vesicle mobilization". ps1p-a012 inhibitory modulation of the hippocampal ca3 transmission and plasticity by glucagon-like peptide-2 jun-ichiro oka, takashi iwai lab. pharmacol., fac. pharm. sci., tokyo univ. sci., japan glucagon-like peptode-2 (glp-2) is a proglucagon-derived peptidehormone in the intestine and brain. we reported that glp-2 (i.c.v.) improved the concussive brain injury-or scopolamine-induced amnesia in mice. however, the mechanisms of glp-2 effects on hippocampal neurons are unclear. in this study, we investigated the effects of glp-2 on the synaptic function of neurons in the acute hippocampal slices. hippocampal slices (400 m) were prepared from 7 to 35 days wistar rats of both sexes. patch-clamp recordings were made from pyramidal cells of the ca3 in the whole-cell mode using glass microelectrodes (resistance: 4-8 m ). in extracellular recordings, field excitatory postsynaptic potentials (fepsp) were evoked with a bipolar tungsten electrode, placed in the mossy fibers. glp-2 (10 nm-1 m) inhibited spontaneous excitatory postsynaptic current. glp-2 (10 nm) did not affect fepsp amplitude or the paired-pulse ratio, but attenuated the long-term potentiation. these results suggest that glp-2 may play an inhibitory role in the dg-ca3 transmission. ps1p-b013 quantitative imaging of exo-endocytosis at mossy fiber presynaptic terminals of hippocampus by genetically expressed fluorescent probe takuya hkima, rikita araki, toru ishizuka, hiromu yawo dept. of dev. biol. and neurosci., tohoku univ. grad. sch. of life sci., japan both presynaptic and postsynaptic mechanisms are proposed for the synaptic plasticity. however, the presynaptic mechanisms have been analyzed indirectly on the postsynaptic responses. it has been difficult to quantify the exocytosis at the presynaptic terminals, particularly those in vivo or in acute slices. to measure exocytosis directly, we applied the synaptophluorin (sph) method to the individual presynaptic terminals in hippocampal slices of a mouse genetically expressing a conjugate protein of vamp-2 and superecliptic phluorin selectively in the mossy fiber terminals. the sph fluorescence at individual mossy fiber terminal was increased by nerve stimulation and was followed by its reduction which is blocked by bafilomycin a1, a vesicular h+-atpase inhibitor. therefore, the rising phase of sph fluorescence corresponds to exocytosis whereas the decreasing phase to endocytosis and subsequent re-acidification of vesicles. this method would enable us to evaluate the presynaptic contribution to synaptic plasticity. jyoti parkash, gurcharan kaur gndu amritsar, india we have earlier reported that gnrh nerve terminals in the me continue to express high levels of polysialylated form of neural cell adhesion molecule (psa-ncam) in a cyclic fashion and psa-ncam covers both the gnrh axon surfaces and the associated glial cells in the proestrous phase rats indicating that psa plays important role in the neurosecretory activity in hypothalamus. to further establish the functional significance of psa-ncam molecule, we have studied the expression of psa-ncam on gnrh axon terminals and glial cells in the proestrous phase of cycling rats as well as gaba and pbz treated proestrous rats by using dual immunohistofluorescent staining. both gnrh and psa-ncam immunostaining was much higher in proestrous phase rats, whereas, gaba and pbz treatments significantly reduced their expression. the expression of pst has been studied within gnrh cell bodies as well as at their terminals by combining in situ hybridization with immunohistofluorescent in poa and me-arc regions of cycling female rats as well as in gaba and pbz treated proestrous rats. cortical plasticity has important roles in the development of neural circuits in sensory cortices. however, the roles and mechanisms for various types of ltp and ltd are not clear. we investigated supragranular ltp and two types of supragranular ltd in the slices obtained from the rat auditory cortex, and compared their properties. frontal cortical slices were prepared from male wister rats. supragranular field potentials elicited by the stimulation applied to layer vi were recorded. ltp was induced by tetanic stimulation (ts, 100 hz for 1 s) applied to layer vi. ltd was induced by low-frequency stimulation (lfs, 1 hz for 900 s) applied to layer vi. ltd was also induced by ts applied to supragranular layers near the recording site. lfs-induced ltd and ts-induced ltd were completely abolished in the presence of 50 m apv, 3 m bicuculline, but not 500 m mcpg. lfs-induced ltd and ts-induced ltd occluded each other, suggesting that that both types of ltd share cellular and molecular mechanisms. kazuyoshi kawa department of neurophysiology, tohoku university, graduate school of medicine, sendai, japan using slice-patch techniques, synaptic transmission in neurons of the area postrema (ap) of the rat was studied. when 20 mm kcl was applied from a "y tube" to ap neurons (whole-cell clamped at −10 mv), massive inhibitory postsynaptic currents (ipscs) were induced. most of the evoked ipscs were blocked by bicuculline confirming gabaergic identity. when nicotine (5-100 m) or capsaicin (0.1-1 m) was applied to ap neurons, robust appearance of ipscs with gabaergic identity was induced. after blocking action potential generation in the slice with tetrodotoxin (1 m), nicotine and capsaicin could still induce gabaergic ipscs. interestingly, responses to capsaicin of the synaptic facilitation showed marked desensitization even after 5 min of rigorous washout. it is concluded that nicotinic receptors, as well as capsaicin receptors (presumably, trpv1), are expressed at gabaergic presynaptic terminals in area postrema neurons and play a distinctive role in controlling autonomic neural functions. research funds: grant from the smoking research foundation (japan) takako morimoto-tanifuji 1 , akira komatu 2 , akinao nose 1 1 dept. phys., univ. tokyo, tokyo, japan; 2 dept. physiol., sch. med., tokyo women's med. univ., tokyo, japan the molecular mechanisms that target neurotransmitter receptors to the postsynaptic membrane and keep them clustered remain unknown. we investigated how the localization of glutamate receptors (glurs) is regulated in neuromuscular junctions (nmjs) of drosophila 3rd instar larvae. there are mainly two classes of glurs, containing either gluriia or iib. gluriia has a sequence predicted as ca 2+ -permeable site. when camkii was inhibited by the expression of inhibitory peptide, ala, the content of gluriia in synapses was dramatically increased and the mean amplitude of extrajunctional potential (ejp) was enhanced. the expression of constitutively active form of camkii (t287d) resulted in decreased gluriia content and enhanced gluriib content. although miniature ejp amplitude was reduced, ejp amplitude was normal in t287d expressing larvae, suggesting the existence of some homeostatic mechanisms. taken together, camkii regulates the localization of glurs in a subunitspecific manner and modulates synaptic function in nmjs. ) . notably, neuronal dnrs from dnr1* flies did not show mg 2+ blockade, and dnr1* flies displayed significant impairment in transcription-dependent long-term memory (ltm) but not in transcription-independent acquisition and short-term memory. we identified salient increases in genes involved in l-ltp formation, e.g. homer, and activin, as well as the increase in genes involved in ltm, e.g. staufen, upon ltm formation. however, such increases were absent in dnr* flies. transcription for ltm is mediated, at least, by transcription factors such as creb, adf-1, and notch. we examined how mg 2+ blockade of dnr links to these transcription factors. research funds: kakenhi ps1p-b020 response properties of wind-sensitive giant interneurons in the 4th-instar nymphs of the cricket tetsuya matsuura 1 , masamichi kanou 2 1 dept. of welfare eng., iwate univ., morioka, japan; 2 dept. of biology, ehime univ., matsuyama, japan the response properties of four wind-sensitive giant interneurons (gis) 8-1, 9-1, 9-2 and 9-3 in the 4th-instar nymphs of the cricket gryllus bimaculatus were investigated. air current was presented to the animal from 12 different directions in the horizontal plane. the intensity-response curves showed that the response magnitudes of gi 8-1 increased with stimulus velocity up to 300 mm/s regardless of the stimulus direction. the response magnitudes of gi 9-1 reached a plateau at a stimulus velocity of 30 mm/s in most stimulus directions. the response magnitudes of gis 9-2 and 9-3 increased with stimulus velocity up to 300 mm/s regardless of the stimulus direction. the directional sensitivity curves revealed that the preferential directions of the gis in nymphs were the ipsilateral-side in gi 8-1, the ipsilateralfront and contralateral-rear in gi 9-1, the ipsilateral-rear in gi 9-2 and the ipsilateral-front in gi 9-3, designated with respect to the side of the ventral nerve cord containing the axons, which were basically the same with those of adults. yasuyuki ishikawa, sadao shiosaka division of structural cellular biology, nara institute of science and technology, nara, japan long-term potentiation (ltp) is an enhancement of synaptic strength that may contribute to information storage in the mammalian brain. ltp expression can be regulated by previous synaptic activity, a process known as "metaplasticity." we report a novel form of cellwide metaplasticity in hippocampal area ca1. serine protease, neuropsin, is involved in the regulation of synaptic plasticity. neuropsin increased the stability of ltp induced later at the same inputs via l-type vdcc. moreover, neuropsin-deficient mice impaired l-ltp induction by l-tbs. our findings have revealed the effects of neuropsin on the conversion of e-ltp to l-ltp. ptp, a form of presynaptic short-term plasticity, is mediated by a transient increase in transmitter release probability caused by tetanic stimulation. although it has been known that ptp is induced by the elevation of presynaptic ca 2+ , the molecular mechanism of ptp is poorly understood. in order to elucidate the specific role of presynaptic trkb receptors in ptp, we analyzed ptp using hippocampal slices from conditionally gene-targeted mice in which the knockout of the trkb gene is restricted to presynaptic sites in the ca1 region. we found that ptp induced by the 100-hz tetanus was reduced in mutant mice, and that ptp in control mice was partially reduced by an n-type ca 2+ channel blocker, while ptp in mutant mice was unaltered by the blocker. thus, these data suggest that the n-type ca 2+ channel-dependent component of ptp requires trkb receptor activation. research funds: jsps and mext of japan kiyoshi ohnuma 1 , kunihiko kaneko 1,2 , makoto asashima 1,2 1 grad. sch. of arts & sci., univ. of tokyo, tokyo, japan; 2 jst, tokyo, japan measuring fluctuations or population distributions of a system can be used to understand the dynamics of the system. we have used this approach to study intercellular interaction between neuronal cells. here we show that the shape of the population distribution of intracellular ca 2+ concentration ([ca 2+ ] i ) may change because of nonsynaptic communication. we loaded pc12 cells with a ca 2+ indicator, indo-1 am, and the [ca 2+ ] i of more than 10,000 cells was measured using flowcytometry. the [ca 2+ ] i distribution of unstimulated single cells had a long right tail, suggesting that [ca 2+ ] i is usually low but sometimes becomes high. on the other hand, the distributions of cell clumps and depolarized single cells were bell shaped, suggesting that many ca 2+ -related mechanisms such as channels and pumps were activated by nonsynaptic communication or by depolarization to change the shape into a normal distribution according to the central limit theorem. our results suggest that measuring the distributions is useful in researching intercellular interaction. na x is a sodium channel involved in sensing the sodium level of the body fluid. our recent studies showed that na x is specifically localized to perineuronal lamellate processes of specialized glial cells in the circumventricular organs, the cns organs involved in the sodium reception. however, molecular and cellular mechanisms underlying the sodium reception of the glial cells has not been elucidated. to address this issue, we developed a functional expression system of the channel protein in cultured glial cells, and found that na x enhances glucose uptake and lactate release in an extracellular sodium-dependent manner. these results suggest that na x alters the state of energy metabolism of the glial cells by sensing a physiological increase of the sodium level. the state of inexcitable glial cells thus play a key role for the control of excitable neural cells in the circumventricular organs. we have isolated spinesin/tmprss5 from human and mouse cns. in mouse cns, four isoforms (types 1-4) were expressed. subcellular localization analysis revealed that type 4 (full length) spinesin was predominantly localized to the er, golgi apparatus and plasma membrane, whereas type1 variant was localized to the cytoplasm. furthermore, we performed expression analysis of m-spinesin in some cell lines. nsc34 and nb2a derived from neuronal cell express only type 4, whereas os3 and kt-5 derived from astrocyte express both type 4 and type 1. interestingly, it was observed that the level of spinesin mrna was increased by a dibutyryl cyclic amp treatment only in os3 and kt-5. we analyzed promoter region of m-spinesin gene, and identified that 5 -flanking region from −224 to −188 bp was essential for m-spinesin gene expression. however, this region did not involve camp-dependent regulation of m-spinesin expression. these results indicate that cell-specific expression and regulation of spinesin gene may play multifunctional roles in cns. it has recently been elucidated that l-serine (l-ser) is one of the glia-derived neurotrophic factors in the brain and its biosynthetic enzyme 3-phosphoglycerate dehydrogenase (phgdh), which is the first committed enzyme of l-ser biosynthesis in the phosphorylation pathway, is selectively expressed in glial cells, but not in neurons. since l-ser seems to be important in retinal functions as well, we investigated in the present study the cellular distribution of phgdh in the mouse retina. phgdh immunoreactivity was detected in müller cell soma in internal nuclear layer, being close to internal plexiform layer. immunopositive profiles were cellular processes surrounding rod spherules and retinal neurons in internal nuclear layer through nerve fiber layer. it was suggested that müller cells contribute in l-ser synthesis and its transportation to neurons in the retina. astrocytes frequently show spontaneous intracellular ca 2+ signals, such as intra-and intercellular ca 2+ waves; however, their physiological roles remain elusive. the overexpression of an ip 3 -hydrolyzing enzyme, ip 3 5-phosphatase, suppressed the spontaneous ca 2+ signals in rat hippocampal astrocytes in culture without noticeable effects on their viability. hippocampal neurons were cultured on a monolayer of astrocytes, and their neurite outgrowth was analyzed. the total neurite length and the number of proximal dendrites and branches decreased significantly when neurons were cultured on the monolayer of ca 2+ -signal-deficient astrocytes. moreover, time-lapse imaging revealed that the extension speed of growing neurites was markedly reduced on ca 2+ -signal-deficient astrocytes. these results indicate that spontaneous ca 2+ signals in astrocytes are essential for glial cells to promote neurite outgrowth. katsuyasu sakurai 1 , noriko osumi 1,2 1 tohoku univ. sch. med., japan; 2 crest, jst, japan astrocytes are the most numerous cells in mammalian brain tissues, although factors regulating their structure and function are still poorly understood. we have previously reported that pax6 transcription factor is expressed in gfap positive cells in the rat hippocampus. in the present study, we first investigated the expression patterns of pax6 in postnatal mouse brain and found that pax6 was expressed in almost all astrocytes in the cerebral cortex. to address the role of pax6 in the astrocytes, we examined the morphology of the astrocytes in the wild type (wt) and pax6 heterozygote mutant (sey/+) mice at 8 weeks. confocal imaging revealed that arborization and extension of the astrocytes were poor in sey/+ mice as compared with the wt. in primary culture, the astrocytes isolated from sey/sey cortex showed no morphological difference. however, 6 and 24 h after dibutyryl-camp treatment, the majority of the wt astrocytes had undergone the conversion from a polygonal to stellate shape, while sey/sey astrocytes rarely showed this response. these results suggest that pax6 regulates the morphology of astrocytes, thereby being involved in astroglial functions. we raised mouse monoclonal antibody (mab) dim21 to study its distribution and function in cell membrane and found not only its preferential reaction with ptdglc on tlc, but also its labeling in rodent cns (yamazaki et al., 2006) . we previously reported a unique expression of dim21 ag in developing mouse brain, especially in cell membranes of embryonic radial glia (kinoshita et al., 2005) . we show here that mab dim21 also recognizes adult neural stem cells and glial cells at postnatal period. dim21, brdu and gfap co-expressed in cells of mouse neurogenic subventricular zone. we discuss a possibility that the dim21 ag may be expressed in the radial glia/astrocyte lineage cells. the bone morphogenetic protein (bmp) receptors are thought to have a role in neural patterning of early neuronal development. the bmp receptor is widely expressed throughout the central nervous system (cns) including cerebellum. however, the physiological roles of bmp signaling in mature brain remains obscure. to understand bmp function in cns, we generated a transgenic mouse line that conditionally overexpresses bmp signaling through the type i receptor alk2 (alternatively known as avcri) in a purkinje cell-specific manner using a cre-loxp system. we bred this mouse line with the cre transgenic mouse line of which expression was driven by l7 promoter. tissue specificity of cre recombination was monitored by a bicistronically expressed egfp following a constitutively active alk2 cdna. increased bmp signaling was confirmed by ectopic phosphorylation of smad1/5/8 (p-smads) in purkinje cells. we will discuss functional changes of the purkinje cells which receive excess amount of bmp signaling through alk2. lipopolysaccharide component of the cell wall of certain bacteria is pyrogenic whose administration to spinal cord injured animals was found to inhibit glial scar formation. glial scar being considered as an impediment for axonal growth, it had been proposed in 1950s and 1960s that sub-febrile doses of pyrogen could be considered for spinal cord injury repair research. we tested this ignored hypothesis in paraplegic bonnet monkeys and found that such sub-febrile doses of bacterial pyrogen derived from salmonella typhi was indeed effective in preventing the glial scar formation in short-term and at least prolong the formation of such scar in long term. therefore, pyrogen therapy may be considered as an adjunct to other strategies such as transplantation approaches to treat spinal cord injury. kavita seth, r.k. chaturvedi, s. shukla, a.k. agrawal dev. tox. div., industrial toxicology reserch center, lucknow, india crosstalk between neurons and glial cells (astrocyte and microglia) in neurodegenerative conditions such as parkinson's disease has gained attention of more than supportive interaction. here contribution of glial cells in 6-ohda induced degeneration of dopaminergic neurons was investigated. glial cultures showed significant loss in cell viability after 48 h (34 and 19%) and 72 h (56 and 33%) exposure to 10 −4 and 10 −5 m 6-ohda respectively. it was accompanied by morphological changes and induction of gfap, s-100 and ox42. 6-ohda (10 −6 m, 72 h) was found to cause a significant impairment in 3 h glutamic acid uptake (31%) and gsh levels (27%). further neurons (in coculture with 6-ohda pre exposed glial cells) on exposure to 6-ohda (10 −6 m), showed loss of th expression and significant neuronal cell death (34%). the results of the present study suggest that 6-ohda may impair glial cell functioning, which eventually affect neuronal fate making them more vulnerable toward toxic insults. nestin is an embryonic intermediate filament component, which is transiently expressed by the immediate precursors to neurons and glia during brain development. we studied nestin distribution in the olfactory system after injection of diethyldithiocarbamate in adult rats to cause reversible lesion of the olfactory epithelium (oe). the oe presented a near-complete destruction at 1 day after injection, then started to repair at 3 days and returned to the normal levels at 6 weeks. nestin was expressed in olfactory ensheathing cells (oecs) of the olfactory mucosa at 3∼7 days, but not in those of the olfactory bulb (ob). simultaneously strong expression of nestin was detected in certain population of astrocytes in glomeruli. the reversion of astrocytes in glomeruli to immature phenotype may reflect their involvement in reinnervation of glomeruli. (ng2) is currently considered a marker of multipotent progenitor cells in the brain. in the present study, most iba1+ cells accumulated in stab wounds and ischemic lesions were found to express ng2, of which molecular weight of its core protein was higher by 10 kda than that of ng2 expressed in contralateral brain region. this was due to the lack of shedding of ng2 in the brain lesions. we found that iba1+ cells accumulated in stab wounds and ischemic core lesion, most of which were ng2+/pdgfra+. furthermore, some of these cells expressed gfap, nestin, cd163 and von willebrand factor. ng2+ mg isolated from stab wounds often formed cell aggregates bearing alkaline phosphatase activity turned into cells with neuroectodermal phenotypes in serumfree culture medium. these variety of antigens expressed by ng2+ mg in brain lesions may be related to their multipotentiality to regenerate damaged brain tissue. saroj sharma, l.k. singh, b. ray, t.s. roy 1 all india institute of medical sciences, india oculomotor nerve (on) supplies most of the extra-and intraocular muscles. it shows changes with normal ageing, metabolic and degenerative diseases. though there are various studies on the on, no definitive data regarding the morphometry and the fine structure is available. so, in the present study, neural and the connective tissue organization of the extradural part of the on from 20 cadavers were studied. light microscopy revealed multi-fascicular nerve with myelinated fibers of various calibers. small sized myelinated fibers were noted at the junction of the central and the paracentral zone of most of the nerves. using unbiased stereology techniques the size of myelinated fiber axonal areas showed a multi-modal distribution and presented range from <1 to 40 m 2 . most of the fibers were myelinated and counts produced a mean of 16,891 (12,000-21,500). ultrastructurally, difference in the compactness of arrangement of connective tissue was observed with advancing age. the cell junctions of the perineurial cells and the endoneurial capillaries were observed. myelin thickness ranged from 0.29 to 3.16 m (from fetal age to 60 years age). during the development of the drosophila visual system, retinal axons project to the optic lobe through the optic stalk. the optic stalk is composed of glial cells and adopts tube-like structure. fak is a non-receptor protein tyrosine kinase involved in many aspects of cell behavior including cell migration through the regulation of actin or microtubule dynamics. in drosophila fak (dfak) mutant animals, the optic stalk was abnormally broadened and retinal axons were defasciculated. cdgapr encodes one of gaps that regulate rho-family gtpases. putative cdgapr mutants showed dfak-like phenotype. since dfak and cdgapr interacted genetically, they are likely to act in the same signaling pathway to regulate cytoskeletal rearrangement via rho-gtpases. tissue specific rescue experiment showed that dfak autonomously acts in the glial cells. our results suggest that dfak and cdgapr regulate glial cell rearrangement to establish precise tubelike structure of the optic stalk and organized retinal axon projection. astrocytes are thought to be active participants in synaptic plasticity in the developing nervous system. spontaneous gabaergic postsynaptic activity is reported to be decreased in small neurons of the caudal nts at the end of the first postnatal week. to investigate whether astrocytes might be involved in this phenomenon, we examined developmental expression of gfap, an astrocytic marker. gfap began to be immunohistochemically expressed in the caudal nts at p5-10. costaining with calbindin, a marker for a certain type of small neurons, showed that gfap positive processes were thereafter closely apposed to soma of small calbindin neurons. electron microscopy showed that some astrocytic processes were interposed between orphan gabaergic varicosities and soma of small neurons at the specific developmental stages. these findings indicate that astrocytes may participate actively in regulating the postnatal differentiation of local neural network of the caudal nts. hitoshi ozawa, naoyuki yamamoto, nobuhiko sawai, hao-gang xue department of anatomy and neurobiology, nippon medical school, tokyo, japan it is well known that the hypothalamo-pituitary-adrenal (hpa) axis is an important system for responding and mediating the stress. in addition, hippocampus is also an important area for the stress response. in the hippocampus, the expression of glucocorticoid receptor (gr) has been reported in the ca1, ca2 pyramidal neurons and the dentate gyrus neurons. on the other hand, while astroglia around the hippocampus also expresses gr, the morphological and functional changes under different corticosteroid condition have not been well elucidated. in the present study, we investigated morphological changes of astroglia around pyramidal neurons. under the lack of corticosteroids, astroglia showed well developed morphology with the spread fibrous processes, however the changes recovered to the control level with corticosterone replacement. these suggested that the astroglia were directly regulated by glucocorticoids as associated with the changes of hippocampal neurons. ps1p-d042 impact of s100b on hippocampal spontaneous activities in anesthetized and epileptic conditions seiichi sakatani 1 , akiko seto-ohshima 2 , shigeyoshi itohara 2 , hajime hirase 1 1 neuronal circuit mechanisms research group, japan; 2 lab. for behavioral genetics, bsi, riken, wako, japan s100b is a calcium binding protein mainly expressed in astrocytes and has a role in synaptic plasticity and learning. in order to assess the physiological roles of s100b, we have recorded hippocampal spontaneous activities from urethane anesthetized s100b ko and wt mice. typical eeg patterns including theta (3-8 hz) and sharp wave associated fast ripple (120-180 hz) oscillations were observed in both populations and these patterns were indistinguishable between the wt and ko. when epileptic activity was induced by kainic acid (i.p.), a difference appeared in ca1 radiatum, where ictal event was characterized by hyper-synchronous gamma band (30-80 hz) activity. while both populations developed ictal event within 40 min, mean power during the development was significantly smaller in ko mice. our results suggest that deficiency of s100b does not have a profound impact on neural activity in normal conditions. however, when neural activity was raised, activation of s100b related pathways could potentially be activated. yoshiko takagishi 1 , erina okabe 2 , xiaoyang sun 1 , sen-ichi oda 2 , yoshiharu murata 1 1 riem, nagoya univ, nagoya, japan; 2 grad sch bio-agricult sci, nagoya univ, nagoya, japan shambling (shm) is a spontaneous mouse mutation that causes neurological and motor deficits, characterized by ataxia and the hind limb paralysis. we have recently identified the shm gene that encodes caspr, which constitutes paranodal junction of myelinated nerves. to determine whether the mutation alters the node of ranvier, we performed morphological analysis of myelinated nerves in shambling mice. by electron microscopy, we found that paranodal loops were disorganized and septate-like transverse bands were absent in mutant mice. immunohistochemistry revealed that caspr was diffusely located at the paranodal region, though the staining was extremely weak in mutant sciatic nerves. contactin, a component of the paranodal junction, was distributed similar pattern to that of capsr. further, k + channels were mislocalized to the paranode, while na + channels were normally restricted to the node. these findings suggest that the mutation disrupts the paranodal structure and may disturb salutatory conduction of myelinated nerves in shambling mice. ps1p-d044 regulation of hippocampal neurocircuit activity by glutamate transporter glt-1 noriko koganezawa, shinsuke muraoka, ken-ichiro tsutsui, toshio iijima div. systems neurosci. tohoku univ. grad. school of life science, japan glial cells are now recognized as an essential functional element in synapses. in order to investigate their function, we focused on the activity of glt-1, the glutamate transporter which is expressed in the astrocytes of hippocampus, in the rat brain slice preparations. response to an electrical stimulation of the schaffer collaterals was recorded using the optical imaging technique. by combining the application of the glutamate receptor blockers (nbqx, ap5) and the glt-1 blocker (dhk) with the signal subtraction, we could visualize the activity of glt-1 as a slow, tonic rise of the optic signal following electrical stimulation. then we evaluated the function of glt-1 by applying its blocker dhk. an obvious reduction of neural activity was observed in the hippocampal neurocircuit after application of dhk. furthermore, the blocking of glt-1 function in the ca3 region was elicited by much lower concentration of dhk than that in the ca1 region. ps1p-d045 monoclonal antibody rip specifically recognizes 2 , 3 -cyclic nucleotide 3 -phosphodiesterase in oligodendrocytes the antigen recognized with monoclonal antibody (mab)-rip has been used as marker for oligodendrocytes and myelin sheaths. however, the rip-antigen has been unknown yet. to identify the rip-antigen, we performed immunopurification with mab-rip using the differentiated cg-4 cells lysate. maldi-qit/tof ms n analyses revealed that one of molecules was 2 ,3 -cyclic nucleotide 3phosphodiesterase (cnp). immunocytochemical and immunohistochemical studies showed that rip-antigen colocalized with cnp in rat cerebellum, cultured rat oligodendrocytes and cg-4 cells. moreover, the same localization was also observed in rat cnp1 transfected hek293t cells. overall we first demonstrated that the antigen labeled with mab-rip is cnp in oligodendrocytes. the expression of bdnf gene is regulated by four promoters (pi-piv), and is under activity-dependent control. until now, it has been established that bdnf pi is activated by ca 2+ signal via cre. on the other hand, neuron-restrictive silencer cis-element (nrse), located in bdnf pii, represses bdnf gene expression through binding nrsf and recruiting hdac in non-neural cells. here, we found that nrse repressed the activity of bdnf pi in neuron. using rt-pcr and chip assay, the bdnf exon i expression and the histone acetylation of bdnf pi were increased by the administration of ca 2+ signals or hdac inhibitor. in addition, nrsf bound to bdnf pii in neurons but was detached by ca 2+ signals. these results suggest that bdnf pi activity is regulated by creb and nrsf through an alteration of chromatin structure. since creb and nrsf are playing an important role in neuronal differentiation, it is considered that the bdnf pi is deeply involved in the regulation of neurogenesis. singo suzuki 1,2 , hisatsugu koshimizu 1 , megumi kashihara 1 , tomoko hara 1 , masami kojima 1,2 1 research institute for cell engineering; 2 sorst, jst, japan brain-derived neurotrophic factor (bdnf) plays a crucial role in synapse development, especially, in the central nervous system (cns) . although this concept is now accepted extensively, the underlying molecular mechanisms are poorly understood. here we show that 3-day treatment with bdnf leads to a significant increase in cholesterol content in primary neuron. this change was in its dose-dependent manner and blocked by co-application of a cholesterol synthesis inhibitors. to understand the molecular relationship between cholesterol content and synapse development, we estimated the amount of cholesterol and sv proteins in lipid raft fractions prepared from cultured cortical neurons. the results indicated that bdnf treatment increased the amount of cholesterol and sv proteins in lipid rafts, but not in non-rafts fraction. these data suggested a possibility that bdnf regulated synapse development by increasing the amount of cholesterol and sv proteins in synaptic rafts. (p38) is known to be expressed in the cells of the central nervous system, and supposed to be involved in the control of cell proliferation, differentiation and survival. in this study, we found that p38 expressed in the neural progenitor cells at embryonic 14 days (e14) mice brain. to ascertain the function of p38 on these cells, we treated the cultured e14 cells with the selective chemical inhibitor for p38 (sb203580) for 7 days, and determined the number of neural progenitor cells. the inhibitor specifically enhanced the number of neural progenitors compared to the control cells. this result suggests the involvement of p38 in the proliferation and/or survival of neural progenitor cells in developing mouse brain. hemragul sabit 1 , takashi yamazaki 1,2 , takeshi oya 1 , yoko ishii 1 , masakiyo sasahara 1 1 pathology ii, university of toyama, toyama, japan; 2 oral and maxillofacial surgery, university of toyama, toyama, japan purpose: we had reported the increase of pdgf-b and active src in rat peripheral nerve regeneration. here examined activation of pdgf receptors (pdgfrs) and signals in the peripheral nerve regeneration. method and result: crushed sciatic nerve was removed on 3 to 28 days after injury, and activation of pdgfrs, mapks, akt and p38 were examined by phosphoprotein purification kit (qiagen) and western blot. expression of pdgfrs increased from 3 to 21 days after injury. p-tyr was highly detected from 3 to 21 days after injury, and activation of pdgfrs also increased during this period. activation of erk and jnk increased up to 7 days after injury and then gradually decreased. activation of akt and p38 continuously increased from 3 to 28 day after injury. conclusion: pdgfrs and their signals were activated in rat peripheral nerve regeneration. autocrine signal of pdgf may contribute to the regenerative processes, such as proliferation and differentiation of schwann cells and axonal extension. cbln1 is a cerebellum-specific protein structurally related to the c1q and tumor necrosis factor families. recently, we have shown that cbln1 is secreted from cerebellar granule cells (gc) and controls synaptic structure and plasticity of gc-purkinje cell (pc) synapses. however, because cbln1 was previously shown to serve as a precursor of a pc-specific peptide cerebellin, it remains unclear whether cbln1 needs to be processed before it trans-synaptically activates signaling pathways in pc. here, we show that purified recombinant cbln1 proteins, which formed a hexamer, preferentially bound to spines on pc dendrites. furthermore, cbln1 mutants that did not form a hexamer lost the binding affinity to pc spines. although cerebellin peptide may also contribute to different aspects of signaling, these results indicate that cbln1 released from gc directly bind to postsynaptic pc as a hexamer and activates signaling pathways in pc. activity-dependent gene expression in neurons contributes to expressing a variety of neuronal functions including a long-lasting neuronal plasticity. recently, we found that specific kinds of mrna can be stabilized in an activity-dependent manner. to elucidate the mechanisms for activity-dependent mrna stabilization, we have focused on bdnf, which is a member of neurotrophin family and plays an important role in exerting neuronal functions. we constructed firefly luciferase gene fused to 3 -untranslated region (utr) of bdnf mrna to investigate the effect of the 3 utr on the calcium signal-mediated mrna stabilization. in cultured neurons, we found that the degradation of firefly luciferase-bdnf3 utr mrna induced by the treatment with actinomycin d was prevented by calcium signals evoked via l-type voltage-dependent calcium channels (l-vdccs) and nmda receptors. we are now investigating to identify the cis-regulatory elements involved in the calcium signal-mediated stabilization of bdnf mrna using a series of mutant bdnf3 utr. recently, it has been established that bdnf and pacap regulate the expression of a group of genes which encode proteins involved in expressing neuronal functions. in this study, we found that the treatment of cultured rat cortical neurons with bdnf or pacap acutely induced the mrna expression of the activityregulated cytoskeleton-associated protein (arc) and homer1a, whose products are necessary for the synaptic plasticity. bdnf induced arc mrna expression through the activation of trkb-erk/mapk pathway, whereas pacap induced it partly through the activation of nmda-receptors. using affymetrix genechips, we are now investigating a comprehensive profile of gene expression controlled by bdnf or pacap in cultured rat cortical neurons. ps1p-e056 bmp4 expression in the adult rat brain bone morphogenetic protein-4 (bmp4) is a member of the transforming growth factor ␤ (tgf-␤) superfamily and plays important roles in multiple biological event. although bmp4 expression has been well described in the early development of central nervous system (cns), little information is available for its expression in the adult cns. we, thus, investigated bmp4 expression in the adult rat cns using immunohistochemistry. bmp4 is intensely expressed in most neurons and their dendrites. in addition, intense bmp4 expression was also observed in the neuropil of the gray matters where high plasticity is reported, such as the molecular layer of the cerebellum and the superficial layer of the superior colliculus. furthermore, we found that astrocytes also express bmp4 protein. these data indicate that bmp4 is more widely expressed throughout the adult cns than previously reported, and its continued abundant expression in the adult brain strongly supports the idea that bmp4 plays pivotal roles also in the adult brain. ps1p-e057 hgf as a target-derived trophic factor for rat nigro-striatal dopaminergic (da) system during post natal development wakana ooya, hiroshi funakoshi, toshikazu nakamura div. molecular regenerative medicine, osaka univ. grad. sch. med., osaka, japan hgf is a novel neurotrophic factor in vitro on da neurons. however, little is known about expression and biological activities of hgf in nigral-striatal system in vivo. here we show that hgf is a targetderived trophic factor for rat da system. real-time rt-pcr revealed that c-met mrna was expressed in substantia nigra (sn) and striatum (str), while hgf mrna was expressed in str but not in sn in programmed cell death period. hgf, c-met, phospho-c-met, th, da transporter immunostaining revealed the presence of concentration gradient of hgf from sn to str and c-met was phosphorylated in da nerve end during early postnatal development. phospho-c-metpositive da neurons decreased at later developmental stage, while it became prominent in oligodendrocytic leanage. hgf application into str increased da neuronal number and neurites and modified oligodendrocyte maturation, while opposite effects were observed by the application of blocking antibody for hgf. therefore, hgf may be a critical trophic factor for nigro-striatal da system development. the neuroprotective effects of g-csf were reported in neurological disease models. in the present study, we examined whether g-csf can protect dopaminergic neurons from mptp-induced cell death in a pd. the mice were intraperitoneal injected with mptp for five consecutive days, g-csf is intraperitoneal administered two days and one day before first mptp injection, and 30 min before each mptp injection. in our results, g-csf significantly prevented mptpinduced loss of th-positive neurons, and increased bcl-2 protein, decreased bax protein expression. these findings suggest that g-csf has therapeutic potentiality to protect mptp-induced cell death through increasing the level of bcl-2 expression, decreasing the level of bax expression in c57bl/6 mice. kazue takahata has been shown to increase the expression of brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor, and the activity of superoxide dismutase 1. here, we evaluated the effects of (−)-bpap on the phosphorylation of mitogen-activated protein kinase (mapk) and akt in slice cultures as well as in an in vivo model. (−)-bpap significantly increased phosphorylation levels of mapk, but not those of akt. (−)-bpap attenuated the decrease in nigrostriatal tyrosine hydroxylase immunoreactivity of 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine-treated mice. (−)-bpap may exert antiparkinsonian activity through neuroprotective effects on dopaminergic cells in addition to catecholaminergic enhancement in parkinsonian substantia nigra. shyuichi maeda 1 , yoko tohyama 1 , shinichi kohsaka 2 , tadashi kurihara 1 , kazuyuki nakajima 1,2 1 soka university, hachioji, tokyo, japan; 2 dept. of neurochem., national institute of neuroscience, kodaira, tokyo, japan astrocytes (ast) are a cell type that supports cns not only nutritionally but also neurotrophically, by supplying neurotrophic factors (ntf) required in the neuronal survival, maturation and protection. however, the ability of ast to produce/secrete ntf has not been accurately known. thus, in the present study, we investigate the capacity of ast to produce/secrete various ntf in vitro. ast were prepared from the mother culture of neonatal rat brain. the ntf in the conditioned medium (cm) were detected by immunoblotting. the analysis of neurotrophins in the cm revealed that ast produce/secrete ngf, bdnf and nt-3, and promote the production of them by stimulation with lipopolysaccharide (lps). furthermore, tgf␤2 among tgf␤ family was detected in the cm of ast, and the production was enhanced by stimulation with lps. these profiles of ast were different from those of microglia, suggesting the differential regulation of ntf by glial cells. ritsuko katoh-semba 1 , chiaki nakagawa 1 , masako tsuzuki 1 , motoko matsuda 1 , satoshi ichisaka 2 , yoshio hata 3 1 inst. dev. res., aichi human ser. ctr., kasugai, japan; 2 div. neurobiol., tottori univ., sch. med., yonago, japan; 3 div. integrative biosci., tottori univ. grad. sch. med., yonago, japan the sleep-awake rhythm is formed during the early period after birth. the rhythm is very important for the functional development of brain. when the rhythm formation is disturbed, autism-like behaviors are often observed. brain-derived neurotrophic factor (bdnf) is known to be one of the factors forming circadian rhythms. we have found increases in levels of bdnf in the entorhinal cortex as well as the visual cortex from adult male rats 12 h after beginning an 8-h phase advance of the light-dark cycle. here we planned to reveal the effects of the phase advance on neurotrophins in juvenile rats. we first examined circadian changes in the concentrations of bdnf and neurotrophin-3 (nt-3) in selected brain regions from 14-day-old male rats and compared to those from adults. the changes in levels of bdnf and nt-3 were observed in the neocortex and hippocampus. objective: this study was aimed to investigate the possible beneficial effects of granulocyte colony stimulating factor (g-csf) compared to methylprednisolone (mp) in experimental spinal cord injury (sci). materials and methods: (in vivo) adult female sprague-dawley rats had moderate sci (200 kdyne, ih injury device) at t8/9 and were assigned to three groups; a (placebo), b (mp treated; 30 mg/kg i.v. immediately after injury), c (g-csf treated; 15 g/kg i.v. for 5 days after injury). animals were assessed with the bbb locomotor rating scale for 6 w post injury and then killed for assessment of tissue sparing around the lesion. result: the behavioural recovery rates of the group c was as good as group b and significantly better than that of group a. morphological assessment showed better tissue sparing in group b and c compared to group a. these results suggest that g-csf is a possible neuroprotective agent in sci. research funds: kakenhi (16390427) ps1p-e064 glutamate signals enhance the expression of rnase-l in primary cultured cortical neurons of mice chie sugiyama, kiyokazu ogita dept. pharmacol., setsunan univ., osaka, japan mitochondrial dysfunction results from a decline in the mitochondrial rna (mtrna) transcripts and mitochondrial enzyme activity, as well as from mitochondrial dna (mtdna) damage. to evaluate involvement of mtdna expression in glutamate-induced neuronal death, in this study, we examined the effects of glutamate exposure on mtrna level in cultured cortical neurons of mice. cultured neurons (12 div) exposed to glutamate for 15 min at 100 m. glutamate exposure led to a decrease in mrna of nd1 and nd6, which are subunits of nadhubiquinone oxidoreductase, before cell death. since mtrnas level is regulated at least in part by rna degradation mediated by rnase-l, we next examined the effect of glutamate on expression of rnase-l. rt-pcr analysis revealed that glutamate was effective in increasing the level of rnase-l mrna at least 2-12 h after treatment. the increase in the expression of rnase-l was abolished by the nmda receptor antagonist mk-801. these results suggest that the activation of nmda receptor by glutamate reduces mtrna level probably through enhanced expression of rnase-l in cultured cortical neurons. yuka gotoh, kiyokazu ogita dept. pharmacol, setsunan univ, osaka, japan expression of dj-1 is enhanced by oxidative stresses. although exact functional significance of dj-1 has still unknown, it is thus proposed that dj-1 is protective against neural damage under oxidative stresses. in this study, we tested expression of dj-1 in the hippocampus damaged by trimethyltin (tmt) treatment in mice. tmt was systemically injected into mice to cause neural damage in the dentate gyrus selectively. immunohistochemical analysis indicated that dj-1 was markedly increased in the molecular layer of the dentate gyrus on days 4 and 14 post tmt injection. on day 14 post tmt injection, enhanced expression of dj-1 was observed in the stratum lucidum of the ca3. in glutathione-depleted mice, tmt was more effective in enhancing expression of dj-1, compared with that in untreated mice. furthermore, double staining of dj-1 and gfap demonstrated that most of cells highly immunoreactive to dj-1 were co-localized with gfap in the dentate gyrus of tmt-treated animals, but not of untreated animals. these results suggest that dj-1 is enhanced in the dentate astrocytes activated by tmt treatment. kei higuchi, kiyokazu ogita dept. pharmacol, setsunan univ., osaka, japan the systemic administration of trimethyltin (tmt) is known to induce granule cell death in the dentate gyrus of mice. we have previously shown that an injection of tmt (2.8 mg/kg, i.p.) led to significantly reduction of granule cells in the dentate gyrus 2 days later, with visually apparent recovery of the granule cell layer 14 days afterward. in this study, we examined the effects of glucocorticoids on tmt-induced damage in the dentate granule neurons of mice. tmtinduced neuronal cell damage was assessed by the immunohistochemical analysis using an antibody against single-stranded dna. the systemic injection of dexamethasone (0.5-5 mg/kg) led to a significant reduction in neuronal damage induced by tmt in the dentate gyrus. the neuronal damage induced by tmt at the dose of 2.0 mg/kg was enhanced by adrenalectomy. dexamethasone was effective in completely preventing this neuronal damage in adrenalectomized animals. taken together, these results suggest that glucocorticoid released from adrenal cortex may be capable of protection against tmt-induced dentate granule cell death in mice. masami ishido national institute for environmental studies, tsukuba, japan melatonin, a secretory product of the pineal gland, has antitumor activities and is involved in the regulation of circadian, seasonal rhythms and in inducing osteoblast differentiation. furthermore, melatonin is reported to be a scavenger of a number of reactive oxygen and reactive nitrogen species both in vitro and in vivo. in this chapter, antioxidant nature of melatonin was demonstrated to prevent the cultured neural cells from apoptosis induced by endocrine disrupting chemicals, maneb. neurotoxicity of maneb (1 g/ml) on the pc12 cells was elicited through apoptotic cell death. activation of caspase-3/7 was associated with this process. a fluorescence rationing technique using mitochondrial dye revealed that maneb altered mitochondrial membrane potential of the neural cells. however, melatonin (1 nm) could largely prevent the neural cells from the neural toxicant by inhibition of both caspase-3/7 activation and disruption of the mitochondrial transmembrane potential. thus, melatonin could be a powerful free radical scavenger against manebcaused mitochondrial dysfunction in pc12 cells. ps1p-e068 kinesin superfamily protein 4 (kif4) regulates activity-dependent neuronal survival by suppressing parp-1 enzymatic activity ryosuke midorikawa, yosuke takei, nobutaka hirokawa department of cell biology and anatomy, university of tokyo, tokyo, japan in brain development, apoptosis is a physiological process that controls the final numbers of neurons. here we report that the activitydependent prevention of apoptosis in juvenile neurons is regulated by kinesin superfamily protein 4 (kif4), a microtubule-based molecular motor. the c-terminal domain of kif4 is a module that suppresses the activity of poly (adp-ribose) polymerase-1 (parp-1), a nuclear enzyme known to maintain cell homeostasis by repairing dna and serving as a transcriptional regulator. when neurons are stimulated by membrane depolarization, calcium signaling mediated by camkii induces dissociation of kif4 from parp-1, resulting in upregulation of parp-1 activity, which supports neuron survival. after dissociation from parp-1, kif4 enters into the cytoplasm from the nucleus, and moves to the distal part of neurites in a microtubule-dependent manner. we suggested that kif4 controls the activity-dependent survival of postmitotic neurons by regulating parp-1 activity in brain development. research funds: 1684304 ps1p-e069 expression of hsp105, apg-1, and apg-2 in the hippocampal neural cells by trimethyltin masanari orita, kiyokazu ogita dept. pharmacol, setsunan univ, osaka., japan we tested changes in expression of high-molecular-weight heat shock proteins (hsps) in the hippocampal dentate gyrus in vivo and in the cultured cortical neurons in vitro after trimethyltin (tmt) treatment, which caused neuronal damage in the dentate gyrus and cultured hippocampal neurons. tmt (2.8 mg/kg) was systemically injected into mice, and then an immunohistchemical analysis was performed to identify cells immunoreactive to antibodies against hsps, neun, and gfap in coronal sections of hippocampus. tmt was effective in enhancing the expression of hsp105, apg-1, and apg-2 in the granule cell layer of the dentate gyrus, but not in ca1-ca3 pyramidal cell layer, 16 h to 2 days later. double staining of neun and these hsps revealed that these hsps expressed by tmt almost co-localized with neun in granule cells of the dentate gyrus. whereas hsp105 highly expressed in survival neurons in the culture, apg-1 and apg-2 highly expressed in damaged neurons with nuclear condensation. taken together, the high-molecular-weight hsps may be involved in neuronal survival and damage caused by tmt. brain irradiation is often performed in patients with brain tumors. however, little has been known about radiosensitivity of neurons, especially in the developmental stages. in this study, we investigated the effect of irradiation on immature neurons with that on mature neurons. primary neuronal cultures were prepared from fetal rat hippocampi at embryonic day 18. thirty gray of x-irradiations were performed on the cultured cells at 7 or 21 days in vitro (div). then the cells were fixed at 24 h after the irradiation with dapi. at 7-div, irradiation significantly increased the number of nuclear pyknosis of neurons. in contrast, radiation did not induce any nuclear pyknosis of neurons at 21-div. this indicates that the radiosensitivity of 7-div immature neurons is higher than that of 21-div mature neurons. glutamate receptors are believed to be involved in various neurological disorders via its excitotoxicity. ataxic mice lurcher (lc) are caused by a mutation in the ␦2 glutamate receptor (glur␦2), which shows constitutive channel activities in purkinje cells and leads to the cell death. thus, lc is the first example of neurodegeneration caused by chronic excitotoxicty. interestingly, glur␦2 is also suggested to regulate autophagy via its association with beclin. however, it is unclear how excitation caused by constitutive channel activities is related to the autophagic pathway and cell death. here, using heterologous cells in vitro, we show that continuous influx of na + , but not ca 2+ , was necessary and sufficient to induce autophagic cell death. in addition, we found that intracellular atp levels and subsequent activation of map kinase are involved in this process. junko taniguchi a major pathological hallmark of the polyglutamine diseases is the formation of neuronal intranuclear inclusions (niis) of the disease proteins that are ubiquitinated and often associated with various transcription factors, chaperones and proteasome components. but, how the expanded polyglutamine proteins or their aggregates elicit a complex pathogenic responses in the neuronal cells are not fully understood. here, we demonstrate that the expression of expanded polyglutamine proteins down-regulates the nf-kb-dependent transcriptional activity. expression of expanded polyglutamine proteins increases the stability and the levels of ikb-a and its phosphorylated form. we have also found that various nf-kb subunits and ikb-a aberrantly interacts with the expanded polyglutamine proteins and associates with their aggregates. finally, we have shown several nf-kb-dependent genes are down-regulated in the expanded polyglutamine protein expressing cells. molecular mechanisms for selective neuronal death in polyglutamine diseases remain to be clarified. by microarray analysis, we compared gene expression profiles in cerebellar granular cells under expression of normal and mutant ataxin-1 and found a novel gene down-regulated in response to mutant ataxin-1 in cerebellar granular neurons. we named the novel gene maxcell (mutant ataxin-affected gene in the cerebellum). nothern blot shows that maxcell mrna in human brain is expressed in cerebellum and cerebral cortex. immunohistochemistry with anti-maxcell antibody shows cytoplasmic stains of neurons but not glial cells in mouse brain. confocal microscopy shows that maxell-egfp is colocalized with ribosomal protein s6 as a ribosomal marker. we are analyzing the function of maxcell protein that might relate to sca1 molecular pathology. ps1p-f080 permeability transition in mitochondria isolated from cold perfused brain and spinal cord-a detailed comparison of calcium sensitivity the purpose of this study was to compare the sensitivity of isolated brain and spinal cord mitochondria to ca 2+ -induced permeability transition (mpt). the spinal cord is more traumatized than the brain during the extraction because it takes more time. in order to minimize confounding factors, we induced severe hypothermia in animals prior to removal of tissue and isolation of mitochondria. sensitivity to ca 2+ -induced mpt was evaluated in brain and spinal mitochondria in energized and de-energized model of swelling with or without mpt inhibitor, cyclosporin a (csa). the present findings imply that the general features of mpt are similar in brain and spinal cord mitochondria and that mpt may be an important pharmacological target in disorders affecting the spinal cord. the role of cyclophosphamide monohydrate (cp), which is known as an immunosuppression drug, in the central nervous system (cns) has not been elucidated. in the present study, we found that treatment with cp prevented the cultured cortical neurons from cell death induced by serum deprivation. furthermore, cp exposure induced the activation of both the map kinase (mapk) and pi3 kinase (pi3k) pathways. interestingly the up-regulation of bcl-2, a survival promoting molecule was observed after cp treatment. these observations suggest that cp protects cns neurons from neuronal damage through intracellular signaling pathways. the research of cell death mechanisms has rapidly progressed. however, cell death is an inherently difficult process to measure. to investigate the roles of cell death in vivo, we introduced scat3 probe. scat3 is an indicator protein for caspase-3 activation that uses fret between two types of fluorescent protein, ecfp and venus, linked by a peptide containing the caspase-3 cleavage sequence. using this probe, we could monitor the activation of caspase-3 at the singleneuron level in culture. we will discuss about neural cell death through the detection of caspase activity. keiichi seko, koichi kawada, chie sugiyama, masanori yoneyama, kiyokazu ogita dept. pharmacol., setsunan univ., osaka, japan trimethyltin chloride (tmt) is a kind of organotin derivates that are known to induce neuronal damage in human and rodent. in this study, we examined tmt-induced neuronal death in mouse primary cultured cortical neurons in vitro and the frontal cortex in vivo of mice. in vivo analysis using mice revealed that injection of tmt (2.8 mg/kg, i.p.) led to an increase in single-stranded dna-positive cells, as well as in dnase ii-positive cells, in the frontal cortex 2 days later. in cortical neurons, tmt exposure for 48 h led to a marked decrease in the cell viability, as well as to an increase in nuclear condensation and ldh released. tmt exposure was effective in activating dnase ii in the nucleus. in addition, caspases 3 and 8, but not caspase 9, were significantly activated by tmt treatment. cytochrome c release was not affected by tmt treatment. the caspase inhibitor zvad-fmk completely prevented tmt-induced neuronal death. these results suggest that tmt-induced neuronal death is involved in caspases and dnase ii activated by mitochondria-independent pathway in cortical neurons. we investigated the pattern of hippocampal damage and the levels of brain polyamines after systemic injections of trimethyltin (tmt) chloride (2.5 mg/kg, i.p.) in 3-(3w) and 7-week-old (7w) icr mice. in addition, we measured the brain tin level following tmt injection. tmt induced marked, localized cell death in granule neurons of the dentate gyrus in 3w mice. by contrast, slight, diffuse neuronal damage was found in the ca1 and ca3 subfields and dentate gyrus of 7w mice. the hippocampal putrescine level was elevated markedly in 3w mice on tmt administration, whereas a minor putrescine increase was detected in 7w mice. there was no difference in the brain tin level between these two age groups. these results revealed the age-dependent vulnerability of mice hippocampal neurons to tmt administration, and suggest that massive activation of polyamine metabolism is associated with tmt-induced neurodegeneration. withdrawn ps1p-f086 establishment of memory guided actions of taking food with tweezers in monkeys naoki hirai 1 , toshinori hongo 2 , kimisato naito 2 , shigeto sasaki 2 1 department of physiology, kyorin university, school of medicine, tokyo, japan; 2 tokyo metropolitan institute for neuroscience, tokyo, japan monkeys learned a task of taking food with tweezers (twz) under the visual guidance. the task consisted of sequential actions of looking at twz, grasping it by hand simultaneously shifting gaze to food, bringing twz to food, and picking it up with twz. brief interruption of vision for 0.3-1.0 s during any actions by a liquid crystal shutter disrupted the ongoing actions, indicating that each action needed visual information as guidance. this contrasted with the task of taking directly with hand, which was done without vision. with repeated practice, they developed a mode of using more memory and somatosensory cue as guidance. they directed their gaze to invisible food in advance, and when vision of 0.1 s became available, they grasped twz, brought the twz to memorized location of food and grasped the food without vision. these results show that they acquired food taking actions using twz based on memory and somatosensory cues, the latter allowing monkeys to use twz as an extension of the hand. masahiko nishimura, yoshihiko yoshii university of ryukyus, okinawa, japan we have experienced that the patients with arm impairments by brain disorder were difficult to manipulate the tools with the paralyzed arm, and healthy arm. lt. parietal lobe and ifg are commonly recognized tools-semantic neuro-system. however, nobody knows a neural network contributed to suitability for a purpose of toolsmanipulation. we examined an fmri to evaluate the brain activation of tools-manipulation in 17 volunteers. experiment was performed by three tasks, control task is a forearm rotation, task1 is simulation of tools-manipulation, task2 is execution of tools-manipulation. we found different brain regions by this experiment. task2 to investigate the role of synchronous firing in the prefrontal cortex (pfc), we performed cross-corelational analysis of the pfc neurons, while monkeys performed a path-planning task, which required multiple steps of actions to reach goals. first, we analyzed synchrony among pfc neurons during the execution period in comparison with that during the preparatory period. we found that neuronal synchrony was enhanced transiently for each step of movement during the execution period. next, we examined relationship between neuronal synchrony and task-related activities. we found that the relationship between neuronal synchrony and response selectivity of pfc neurons was more distinct during the preparatory period than during the execution period. we would discuss dynamical roles in neuronal synchrony in planning multiple steps of actions. the functional significance of primate medial prefrontal cortex in the selection of action has been unclear. we studied neuronal activity in this region while monkeys were performing a variant of conflict solving task in which visual cues instructed them to push either the left or right. the location of the cue was either compatible (congruent) or incompatible (incongruent) with the target's location. we found a focus of reaching-related neurons in the medial prefrontal cortex rostral to the pre-sma. the activity of neurons in this newly identified area was dependent on conflict. intracortical microstimulation in this area did not evoke eye movements, distinguishing this area from the sef. we found that the local field potential in this area, but not in other areas, differed when congruent and incongruent trials were intermixed, and when only the congruent trials were presented repeatedly, suggesting the involvement of this area in the selection of actions is dependent on the task demand. masaki maruyama, peter fenwick, andreas a. ioannides laboratory for human brain dynamics, riken-brain science institute, saitama, japan we used infrared corneal reflection, sampling at 1 khz, to record simultaneously and independently the 12 • horizontal saccades of each eye for 12 subjects. two paradigms were used, in go-only sessions saccade direction with the cue to move, and in go/no go sessions, saccade execution, direction and move. mutual information (mi) analysis showed the two eyes were most consistently yoked for position than for velocity, but both provided adequate signals. mi showed coupling between the start and end of saccades and the importance of velocity signals in their ballistic nature. surprisingly leftward movement latency was longer to peak-velocity and showed more complex mi interactions. comparing go-only to go/no go saccades, significant differences were longer onset latencies and a higher eye velocity before the end of saccades. a recent meg study using this protocol, found just before and during the saccade the additional go/no go difficulty led to more interaction between left and right brainstem and cerebellum. these could be related to eye velocity changes with the higher cognitive loading. wriggle mouse sagami (wms) has been presented as a mouse model for dystonia, as it is characterized by postural and motor impairments, such as sever tremor, sustained muscle contractions of the limbs, and wriggling of the neck and trunk without coordination. by extracellular unit recordings under awake conditions, we analyzed neuronal activity in the basal ganglia and the cerebellum of this mutant mouse. in the basal ganglia (globus pallidus, entopeduncular nucleus, substantia nigra pars reticulata), neither rates nor patterns of spike discharges were significantly different as compared to normal mice. on the other hand, the discharge rate of cerebellar purkinje cells in wms was markedly decreased. these results suggest that the decreased activity of purkinje cells may be responsible for movement disorders in wms. hiromi hirata division of biological science, nagoya university, nagoya, japan wild-type zebrafish respond to mechanosensory stimulation with multiple fast alternating trunk contractions at 1 day, whereas bandoneon (beo) mutants contract trunk muscles on both sides simultaneously. muscle voltage recordings confirmed that muscles on both sides of the trunk in beo are likely to receive simultaneous synaptic input from the cns. recordings from motor neurons revealed that glycinergic synaptic transmission was missing in beo mutants. furthermore, immunostaining with glyr antibody failed to show clusters in beo neurons. these data suggest that clustering defect of glyrs at synapse causes the impairment of glycinergic transmission and abnormal behavior in beo. indeed, mutations in the glycine receptor beta subunit were identified in beo. this is the first direct demonstration that glyr␤ is essential for physiologically relevant clustering of glyrs in vivo. since glycine receptor mutations in humans lead to hyperekplexia, a motor disorder characterized by startle responses, zebrafish bandoneon mutant should be a useful animal model for this condition. medium-sized spiny projection neurons in the striatum receive inputs from gabaergic and cholinergic interneurons as well as from extrinsic sources, including the cerebral cortex. in the present study, the effect of gabaergic modulation on striatal projection neuron activity was investigated by infusion of the gaba a receptor blocker gabazine in the vicinity of the recorded neurons in monkeys who performed a memory-guided reaching task. the gabazine infusion enhanced the activity of striatal projection neurons in response to both cortical stimulation and task events, while the neuronal activity specific to the task events was decreased. these results suggest that local gabaergic input may play an important role in fine tuning of striatal projection neuron activity. research funds: kakenhi (17022042) ps1p-f094 dependence of synchrony in the subthalamic network on temporal characteristics of afferent inputs katsunori kitano, fumito kosuga department of human and computer intelligence, ritsumeikan university, japan the subthalamic nucleus (stn) and the external segment of global pallidus (gpe) constitute the indirect pathway of the basal ganglia and highly modulate the basal ganglia functions. the evidence that the emergence of synchronized oscillatory activity in the network of the two nuclei is relevant to movement disorders such as parkinson's disease shows temporal structures of the neuronal firings play an important role for the functions. among possible underlying mechanisms for the abnormal activity, the characteristic membrane properties of stn neurons is likely to be one of the most crucial origins. to clarify the detailed mechanism, we focus on and investigate the dynamical properties of the neuron theoretically and numerically with the model neuron. we apply the phase reduction method to the dynamics of the neuron to analyze the stability of synchronous activity. in particular, how the stability depends on temporal characteristics of afferent inputs to the neurons as well as the intrinsic membrane properties are investigated. during phasic voluntary movement, electromagnetic oscillatory activities of ∼20 hz around the central sulcus show decrement and increment (erd/ers, respectively), that are assumed to reflect the cortical activation and inhibitory/recovery process respectively. we investigated the correlation between personality and these oscillatory changes. from 35 healthy subjects, high and low scorers (n = 12 each) of novelty seeking dimension on the psychometry were selected. magnetic fields were recorded while they performed selfpaced movements of their right index fingers, and frequency analysis was carried through the beta band (18-24 hz). high ns group showed less amount of erd in the left hemisphere, smaller magnitude, larger latency of ers in the right hemisphere and smaller amount of baseline activity in both hemispheres than low ns group. it was suggested that individuals with high ns trait may have less inhibition after the movement and higher readiness during resting state. despite the emerging methodology of combined fmri and tms, the quantitative relationship between tms intensity and bold signals is poorly understood. eight healthy subjects were scanned on a 3-t scanner, with an mri-compatible figure-of-eight tms coil attached for eliciting right hand movement. bold measurement was performed with the stepping stone sequence (tr = 2.7 s) with online monitoring of meps. the intensity of tms pulses was varied from 30% to 95% at a 5% step (frequency at ∼0.15 hz). bold signal changes were assessed in the primary motor cortex. a sharp increase in bold signals was observed above 80% stimulation. bold signals were weakly but significantly correlated with tms intensity adjusted by the resting motor threshold (r = 0.46, p = 0.001). this finding gives a theoretical background for the application of fmri with tms to cognitive brain regions. ps1p-f097 shift of activation areas induced by hand movement during recovery from post-stroke hemiparesis: an nirs study kotaro takeda 1,2 , yukihiro gomi 1 , itsuki imai 3 , nobuaki shimoda 1,2 , hiroyuki kato 1,2 1 international university of health and welfare, ohtawara, japan; 2 crest, jst, kawaguchi, japan; 3 nasu neurosurgical center, nasushiobara, japan we investigated the cerebral hemoglobin (hb) changes in hemiparetic stroke patients under voluntary hand grasping task from acute to chronic phases by using near infrared spectroscopy (nirs). fortyfour channels (22 channels on each side) were placed on the scalp overlying both sensorimotor cortices, and the cerebral hb changes were observed during four to six cycles of 15 s task and 30 s resting periods while sitting on a chair. the amounts of oxy-hb change were significantly increased in the bilateral sensorimotor areas during hemiparetic hand grasping at the acute phase, though the significant increase was mainly observed in the contralateral sensorimotor area during hemiparetic hand grasping at the chronic phase and during normal hand grasping at all phases. this result suggests that the functional recovery from post-stroke hemiparesis may be attributed to neuronal reorganization of sensorimotor areas via recruiting ipsilateral cortex. research funds: crest, jst todd pataky 1,2 , rieko osu 2 , hiroshi imamizu 2 , mitsuo kawato 1,2 1 computational brain project, icorp, jst, japan; 2 atr cns laboratories, japan movement direction encoding in primate single cortical cells has been widely documented. this study was designed to test whether this directional tuning is observable at the voxel level in human fmri. three subjects performed 1 hz isometric force pulses to seven targets separated by 30 • in the shoulder/elbow flexion-extension plane. while in mri, online force feedback was provided by a 2d strain gauge. twenty repetitions of each condition were performed in 12 s blocks (tr = 2 s). all subjects showed broad activation over the contralateral motor area, and from functional rois an average of 661 voxel time series were extracted. many voxels exhibited continuous cosine-like tuning with movement direction. decoding using linear svm revealed that while correct classification rate was only 31.3% (chance: 14.3%), errors were distributed normally about the target such that 80.4% (chance: 59.2%) of the data was classified correctly to within 60 • . these data demonstrate that non-invasive neuroimaging is sufficiently sensitive to study the problem of coordinate system representation. the behavioral thermoregulation is important for living in various temperature environments. however, the neural mechanism of behavioral thermoregulation is poorly understood. in this study, we aimed to establish a new model to analyze the neural mechanism of behavioral thermoregulation using zebrafish. we investigated whether zebrafish perform behavioral thermoregulation against heating. when water temperature was changed from 28 • c, fish showed repetition of short time swimming in the range 32.5-40 • c. the frequency of the heat induced escape behavior was increased with temperature dependant. these results suggest that the heat induced escape behavior is a part of behavioral thermoregulation. the heat induced escape behavior was observed stably in 3-5 days past fertilization, indicating that the neural mechanism which control behavioral thermoregulation is matured in 3 days. in conclusion, we established an effective new model to analyze behavioral thermoregulation. ps1p-f100 to learn with one limb or two: limited transfer between unimanual and bimanual skills recent studies on neural activity in primary motor cortex of nonhuman primates suggest that unimanual and bimanual movements are controlled by partially overlapping neural processes. here we demonstrate that unimanual and bimanual motor learning also reflect a partially overlapping process. first, motor adaptations to reach with a novel force field applied to a limb could not be fully transferred to the same limb across unimanual to bimanual conditions, and vice versa. second, learning acquired during unimanual reaching could not be fully eliminated by repeated bimanual reaching with no loads, and vice versa. rather, some learning remained intact (but invisible) until the original context was again performed. lastly, two conflicting force fields can be learned simultaneously if they are separately associated with unimanual and bimanual reaching. these results support the view of partially overlapping neuronal processes and illustrate the intimate relationship between neural control and motor learning. research funds: jsps and nserc rieko osu 1 , ken-ich morishige 2 , jun nakanishi 1,3 , hiroyuki miyamoto 2 , mitsuo kawato 1 1 atr computational neuroscience labs, kyoto, japan; 2 kyushu institute of technology, japan; 3 jst-icorp, japan human can execute multiple motor tasks by using the same limbs, which makes human different from industrial robots. recently the optimal feedback control hypothesis has given a significant impact on the motor control community because it produces an optimal behavior for a given task by avoiding offline computation of optimal desired trajectory that would result in suboptimal behavior in the presence of noise. it, however, requires considerable amount of resources and learning to realize multiple tasks on nonlinear system. on the other hand, a desired trajectory enables the brain to share resources with multiple tasks and save learning time by dividing a difficult problem into easier sub-problems of plan and execution. considering the modularity of the brain and viability for nonlinear system, the hierarchical implementation is a better solution for global optimality as a versatile creature. here, we experimentally demonstrate that the hand variance modulation during multiple via-point tasks supports the existence of a desired trajectory. the purpose of this study is to computationally predict arm-reaching movements and posture controls from neuronal activity of premotor (pm) and primary motor area (mi). the activity was collected with single-unit recording method during the animal performing a visually guided arm-reaching task. electromyograms (emgs) and kinematics were also measured. we reconstructed the emgs from the neuronal data using a linear regression model, and then we estimated the kinematics from the reconstructed emgs with an artificial neural network model and proportional derivative controller. as a result, these serial processes allowed us to accurately predict the kinematics during both moving and maintaining her posture from the activity. the advantage of our bmi system is to estimate not only the kinematics but also the muscle tension from the neuronal activity. we have recently reported essential role of the tongue in breastfeeding in the hypoglossal (xii) nerve-injured newborn rats. of particular interest were the findings that the rates of the amounts of milk intake in the unilateral xii nerve-injured p1 pups of the surviving cases increased greatly between p4 (30% of the control value) and p7 (52%), suggesting adaptive tongue movement during development. this study was undertaken to reveal underlying basic mechanisms for such adaptation focusing on neural plasticity allowing effective suckling. after resection of the ipsilateral xii nerve on p1, dii, a postmortem neuronal tracer, was applied to the contralateral uninjured xii nerve of p4 and p7 pups. dii-labeled neuronal fibers were successfully traced within the tongue and were found to extend over the xii nerve-injured side with gradual increase from p4 to p7. we show evidence for functional neural plasticity that allows effective suckling in the xii nerve-injured newborns with suckling disturbance. previously we reported that decorticated rats showed abnormal righting movements in the air when dropped from the supine position, while the air righting reflex (arr) could be evoked purposefully (180 • turn around the body axis) in decerebrated ones. thus, the basal ganglia might send interference signals to the arr center via the midbrain tegmentum. to clarify its functional roles in arr control, we examined arr movements in rats with the midbrain lesioned. wistar, male rats were prepared; after the posterior cerebral cortex was removed by sucking, the superior colliculus and surrounding structures were ablated in various degrees. arr movements were examined post-operative 1, 4, and 7 days. in rats with the superior colliculus lesioned extensively on both sides, arr onset were delayed and body turn around the longitudinal axis was weakened, so that either insufficient or no rotation occurred in the air. furthermore, coordination between the body and tail rotations was lost in many cases. the ablated region may relay cortical signals that give a top priority to the arr center. ps1p-g110 role of plateau potentials in feeding system of aplysia kurodai aiko kinugawa 1 , tatsumi nagahama 2 1 dept. of life sci., grad. sch. of sci. & technol., kobe univ., kobe, japan; 2 fac. phar. sci., toho univ., funabashi, japan rhythmic motor activities seen in the animal behaviors can be generated by specific neural circuits termed the central pattern generator (cpg). in the feeding system of aplysia kurodai, the le neuron we identified produces the long-lasting plateau potentials and may be a cpg element. during the feeding-like responses duration of the depolarization of the follower neurons was shortened by hyperpolarization of the le. in this study we found that the le plateau potentials had refractory periods and they were turned to activation periods by application of large depolarizing currents. and various depolarizing pulses tended to produce the stable plateau potentials with almost constant depolarizing size and duration, suggesting that the le can supply the constant long-lasting depolarizing outputs to the follower neurons even when it receives various length and intensity of excitatory inputs from the presynaptic neurons. the le may be an important cpg element to determine the size and duration of the basic depolarization of many buccal neurons. withdrawn ps1p-g112 neural organizations for vocal control in a social rodent, the deguneural organizations for vocal control in a social rodent, the degu naoko tokimoto 1 , sayaka hihara 1 , kazuo okanoya 2 , atsushi iriki 1 1 lab for symbolic cognitive development, bsi, riken, japan; 2 lab for biolinguisutics, bsi, riken, japan vocalizations of most animals are innate, the region for the direct control of such sound is known to be localized in the pag. on the other hand, a few animals with the cortico-medullary projection path can learn a new sound. in this research, we investigated about vocal control in pag of social rodent, the degu (octodon degu). it is known that degus have fifteen kinds of vocal repertoires, and that their courtship song has a complex structure. we verified the hypothesis by electrical stimulation of the pag that the neural mechanism of degus that enables complex vocalization differs from that of guinea pigs with simple vocalization. guinea pig is near relation with degu. as a result, in guinea pigs, each sound is controlled in different area of the pag. in degus, however, multiple sounds are controlled by the same area, and the different sound was occasionally evoked by the different kind of stimulation. the sound with the time-series specific to the spontaneous vocalization of degu was not emitted. the effects of a heat-and steam-generating sheet (hsg sheet) on autonomic nerve activity and bowel movement were examined in women suffering irregular defecation, the hsg sheet was applied to the lumbar or abdominal regions, causing the temperature between the sheet and skin to increase to about 38.5 • c. application of the hsg sheet to either the lumbar or abdominal region significantly increased the rate of miosis in the pupillary light reflex. as for changes in r-r, the hf increased after application, suggesting that the parasympathetic nerve system had become dominant. bowel movement assessed by electrogastrography increased in amplitude. based on the above findings, we concluded that the application of an hsg sheet to the lumbar or abdominal region may lead to dominant parasympathetic nerve activity and improve gastrointestinal motility. ps1p-g115 prostaglandin e 2 -induced thermogenesis involves a gaba-receptive mechanism in the preoptic area toshimasa osaka national institute of health and nutrition, 1-23-1 toyama, shinjuku,162-8636, japan unilateral microinjection of pge 2 into the region around the rostroventral wall of the third ventricle (av3v) elicited thermogenic, tachycardic, vasoconstrictive, and hyperthermic responses simultaneously in urethane-chloralose anesthetized rats. the magnitude of these responses increased dose-dependently in a range of 20-1000 pg, except for the vasoconstrictive response. next, the effects of pretreatment with a gaba a receptor antagonist, bicuculline methiodide (0.5 mm, 100 nl), microinjected into the preoptic area (poa) ipsilateral or contralateral to the pge 2 injection site was examined. this treatment alone had no effect on the o 2 consumption rate and temperatures of colon and skin but elicited a bradycardic response. however, all pge 2 -induced responses were blocked 10 min after the pretreatment with bicuculline, and recovered at ∼70 min. pretreatment with vehicle saline had no effect on the pge 2 -induced responses. these results suggest that the gaba-receptive mechanism in the poa is required for the pge 2 -induced thermogenesis. tetsufumi ito 1 , hiroyuki hioki 2 , kouichi nakamura 2 , takeshi kaneko 2 , yoshiaki nojyo 1 1 dept. anat., univ. of fukui, fukui, japan; 2 dept. of morphological brain sci., kyoto univ., kyoto, japan although gaba-immunoreactive (ir) fibers in the rat superior cervical ganglion (scg) were thought to originate in small cells located in the cervical sympathetic trunk (cst), almost all gaba-ir axon terminals showed markers for sympathetic preganglionic neurons (spns) in our recent report. in this study, we performed series of experiments to confirm the origin of gaba-containing fibers. gad67-ir fibers were not found in dorsal roots (drs), but in ventral roots (vrs), stellate ganglion, cst, and scg. gad67-positive somata were not found in dr ganglia and cst, but in intermediolateral (iml) nucleus of thoracic spinal cord (tsc). after intraperitoneal injection of fluorogold (fg), to label the entire spns, some fg-ir neurons were also positive for gad67. we injected sindbis virus, an anterograde tracer, in iml, and some labeled terminals in scg showed gad67-ir. after cutting t1-t4 vrs and drs, almost all gad67-ir fibers were abolished in scg. these results indicate that gaba-containing fibers in scg originate from spns in iml of tsc. masato nagahama 1 , ning ma 1 , reiji semba 2 , satoru naruse 3 1 dept. of anat. ii, mie univ. school of med., tsu, japan; 2 inst. for developmental research, kasugai, japan; 3 dept. of int. med., nagoya univ. graduate school of med., nagoya, japan aquaporin 1 (aqp1) is first found as a water-transporting protein and has been demonstrated in various organs and tissues. in the present study, we have demonstrated the presence of aqp1 immunoreactivity in a particular neuronal subtype in the enteric nervous system (ens) of the rat ileum. aqp1-immunoreactive (ir) neurons simultaneously expressed a neuronal marker huc/d. moderate numbers of aqp1-ir neuronal somata were found in the myenteric plexus, and a very few were found in the submucosal plexus. aqp1-ir neurons can be classified as dogiel type i cells, which have several short processes and a single long process. many aqp1-ir fibers were found both in the myenteric and submucosal plexi. many aqp1-ir varicose fibers were closely associated with neuronal somata in the ganglia, whereas other aqp1-ir fibers penetrated into the muscle layers. these results suggest that aqp1-ir neurons probably play a significant role within the ens to control gut functions. research funds: kakenhi (13137204) ps1p-g118 abdominal expiratory nerve activity in the decerebrate neonatal rat center for medical sciences, ibaraki prefectural university of health sciences, ibaraki, japan the abdominal expiratory activity was recorded from the iliohypogastric nerve in the decerebrate, vagotomized, paralyzed, ventilated neonatal rat at postnatal days 1-4. the increase in the volume and frequency setting of the artificial ventilator (fio2 = 50%, fico2 = 0%) failed to make the rat apnea. under this condition, the phrenic nerve showed unstable rhythmic inspiratory bursts, and the tail pinch increased the respiratory frequency. although the iliohypogastric nerve showed expiratory discharges, their amplitudes and shapes were not consistent. when fico2 was increased, the cycle period was prolonged and the abdominal expiratory activity was enhanced. in many rats, the iliohypogastric nerve showed biphasic discharges that consisted from the pre-and post-inspiratory discharges. the preinspiratory discharge has larger amplitude and shorter duration than the post-inspiratory discharge. since the post-inspiratory discharge was usually small or indistinguishable in the adult rat, the present results suggest that the pattern of abdominal expiratory activity will change during the postnatal development. we investigated the role of gabaergic neurons in the rostral ventrolateral medulla (rvm) in central respiratory control. we used gad67-gfp knock-in mice in which we could identify gabaergic (i.e., gfp-positive) neurons in a living condition. we recorded gabaergic neuron activities (n = 40) in medullary transverse slices. about 20% of gabaergic neurons were inspiratory, and all of the remaining neurons were non-respiratory. about 50% of gabaergic neurons recorded in the superficial rvm were co 2 inhibitory, and all of the remaining neurons were co 2 insensitive. we suggest that gabaergic inhibition in the rvm respiratory neuron network is mediated mainly by inspiratory neurons. gabaergic neurons are also involved in central chemosensitivity. we investigated two groups of people with a different initial level of an emotional tension before intellectual loading (il). first group had the initially increased emotional level, stressed group (sg), and the second group had not, calm group (cg). reaction time (rt) of simple visual sensorymotor reaction and asymmetry of skin potential level (spla) in two facial zones: forehead and nasal were measured. from research groups, subgroups with 0 and 10 mv spla were extracted. thus the distinction in the greater rt gain after il in subgroups with 10 mv forehead spla, in comparison to subgroups 0 mv forehead spla. such law was common for both for sg and for cg. in two subgroups of 0 and 5 mv nasal zone spla in sg the greater rt gain after il was in 0 mv nasal spla subgroup, and for cg in 5 mv subgroup. it was shown, that individual features of il performance are related to spl lateralization. but the low of the relationships of spl asymmetry in nasal zones depends on the level of emotional tension of investigated groups. mitsuko kanamaru, ikuo homma department of physiology, showa university school of medicine, tokyo, japan we have reported that serotonin (5ht) in the dorsomedial medulla oblongata in mice increases tidal volume and minute ventilation via 5ht2 receptors. peripheral administration of p-chlorophenylalanine (pcpa) reduces the whole brain 5ht level. the present study examined whether peripheral pcpa pretreatment affects hypercapnic ventilatory responses in mice. adult male mice (c57bl/6n) were pretreated with pcpa (0.1 g/10 ml/kg body weight) or saline intraperitoneally for consecutive 4 days. on the next day, each mouse was placed into a double chamber plethysmograph to obtain respiratory flow curves. one hundred percent o 2 inhalation was changed to stepwise 5, 7 and 9% co 2 in o 2 inhalation every 5 min. hypercapniainduced increases in tidal volume and minute ventilation during 9% co 2 inhalation were reduced by pcpa pretreatment; these results suggest that 5ht may increase tidal volume in hypercapnic ventilation. saori nishijima, kimio sugaya, minoru miyazato division of urology, department of organ-oriented medicine, faculty of medicine, university of the ryukyus, okinawa, japan we investigated the effect of gosha-jinki-gan on bladder activity and the autonomic nervous system in rats. forty-two female rats were divided into a control diet group and a 1.08% gosha-jinki-gan diet group. after 4 weeks, continuous cystometry with physiological saline or 0.1% acetic acid solution and biochemical analysis were done. the amplitude of bladder contraction with physiological saline was lower in the gosha-jinki-gan diet group than in the control diet group, and plasma dopamine and serotonin levels were also lower in the gosha-jinki-gan diet group. when cystometry was done with 0.1% acetic acid, the interval between bladder contractions was shortened in the both groups. however, the interval and duration of bladder contractions were longer in the gosha-jinki-gan diet group than in the control diet group. therefore, it is suggested that gosha-jinki-gan inhibits bladder activity by maintaining the balance of the sympathetic nervous system and the parasympathetic nervous system at a low level. satoko suzuki, shinya yanagita, seiichiro amemiya, ichiro kita graduate school of science, tokyo metropolitan university, japan we examined the effects of negative air ions (nai) on physiological responses and neuronal activity with c-fos immunohistochemistry. in addition, we investigated the effect of vagotomy to reveal afferent pathways of nai stimulation. we analyzed neuronal activity of the paraventricular nucleus of hypothalamus (pvn), the locus ceruleus (lc), the nucleus ambiggus (na), and the nucleus of solitary tract (nts). nai significantly decreased blood pressure, heart rate, and respiratory rate, and increased hf component which is an index of parasympathetic nervous activity. nai decreased c-fos expression in the pvn and lc, and enhanced in na significantly. after vagotomy, the physiological responses and changes of c-fos expression in pvn, lc, and na was disappeared. furthermore, increase of c-fos expression in nts induced by nai was also disappeared. these results suggest that effect of nai on sympathetic and parasympathetic nervous activity was induced by reducing the activity of the pvn and lc, whereas enhancing the na activity, and that these effects of nai was caused through vagus nerve. yusuf o. cakmak, umit suleyman sehirli school of medicine, university of marmara, turkey previous assessments of the autonomic nerve supply of testis from vagus and brainstem nuclei were conflicting in the literature. we challenged this consensus by using neuronal tracer fluorogold in rats. fluorogold dye solutions was injected unilaterally under the capsule of rat testis. rats were sacrificed by transcardiac perfusion-fixation in the fifth and seventh days after injection. brainstem of the control group, subdiaphragmatic vagotomy group and main group rats were dissected. in the main group the fluoroscent-labelling were dense in area postrema. dorsal vagal nucleus, nucleus of solitary tract and nucleus ambiguous were also labelled. these preliminary data provide an evidence of testicular innervation by vagus nerve. taking into account that brainstem structures could be labelled from the testis, it can be assumed that the areas detected might be involved in the neural control of testicular functions. the results of this study cautioned that innervation of the testis may not be fully explained by innervation from pelvic and paraaortic ganglia. research funds: scientific researches comittee of medical school of marmara university ps1p-g127 differential control of renal and lumbar sympathetic nerve activity during freezing behaviour in conscious rats yoshimi tahara, misa yoshimoto, keiko nagata, kenju miki integrative physiol. grad. sch. humanities and sci. nara-women's univ., nara, japan the present study was designed to examine sympathetic and hemodynamic responses to loud noise exposure, which induced freezing behaviour, in chronically instrumented rats. wistar male rats were instrumented with electrodes for measurements of renal (rsna) and lumbar (lsna) sympathetic nerve activity and catheters for measurements of systemic arterial and central venous pressure. rats were exposed to 90 db white-noise for 10 min. 90 db noise exposure resulted in an immediate and significant increase in rsna while lsna did not change significantly during the exposure in sham-operated (so) rats. there was a significant difference in the response between rsna and lsna during the 90 db noise exposure in so rats. sinoaortic denervation attenuated the magnitude of the increase in rsna while it had no influence on the changes in lsna observed in so rats. these data suggest that arterial baroreceptor significantly contribute to the differential control of rsna and lsna during freezing behaviour in conscious rats. here, we first demonstrated that in both the kolliker-fuse nucleus (kf) and the rostral ventral respiratory group (rvrg) region, phrenic nucleus (phn)-projecting neurons were embedded in the plexus of axons originating from the ventrolateral subnucleus of the nucleus of the solitary tract (vlnst) and that the vlnst axon terminals made synaptic contacts with somata and dendrites of the phnprojecting neurons, using a combined anterograde and retrograde tracing technique. secondly, we indicated that some of the vlnst neurons innervate both the kf and the rvrg by way of axon collaterals, using the double-labeling method. using retrograde tracing combined with in situ hybridization for mrna encoding glutamic acid decarboxylase 67 (gad67), we finally showed that most of the kf/rvrg-projecting vlnst neurons expressed gad67 mrna. these results suggest that vlnst neurons may exert inhibitory influences upon the phn-projecting kf/rvrg neurons for inspiratory control. we have examined whether the neurons of the dmv have direct synaptic contacts on the myenteric ganglia using wga-hrp. the myenteric ganglia of the stomach were composed of four types of neurons. the average numbers of axosomatic terminals per profile were 2.0 on the small neurons, 3.1 on the medium-sized neurons, 1.2 on the large neurons, and 4.2 on the elongated neuron. most of the terminals contained round vesicles and formed asymmetric synaptic contacts on the small, medium-sized and large neurons. about 80% of the axosomatic terminals on the elongated neurons contained pleomorphic vesicles and formed asymmetric synaptic contacts. when wga-hrp was injected into the dmv, many anterogradely labeled terminals were found around the myenteric neurons. the labeled terminals were large (3.16 m), and contacted exclusively the somata. most of them contained round vesicles and formed asymmetric synaptic contacts. serial ultrathin sections revealed that almost all neurons in a ganglion received projections from the dmv. ps1p-h131 neuronal mechanisms of respiratory rhythm modulation induced by external k + concentration change in the newborn rat brainstem-spinal cord preparation hiroshi onimaru, ikuo homma dept. physiol., showa univ. school of med., tokyo, japan it has been suggested that two distinct rhythm generators (pfrg-pre-i and pre-bötzinger insp) for respiration in the medulla possess different sensitivity to various neuromodulators. we hypothesize that the dominancy of these rhythm generators to determine basic respiratory rhythm depends on the back ground stimulation level. to verify this hypothesis, we studied neuronal mechanisms of respiratory rhythm modulation induced by external [k + ] change. we recorded membrane potential of pre-i neurons, c4 nerve and facial nerve activities. addition of 4 mm k + to the standard superfusate decreased burst rate of c4 activity. addition of 6 or 8 mm k + caused initial inhibition of c4 burst and subsequent high frequency c4 burst. the facial nerve burst was depressed. pre-i neuron was depolarized strongly by application of high k + , and the burst activity was disturbed and action potentials were inactivated. results suggest that pfrg-pre-i or pre-bötzinger insp rhythm generator is dominant in low or high back ground stimulation level, respectively. research funds: kakenhi (16500208) ps1p-h132 regulation of synaptic transmission in the reticular formation of medulla oblongata by substance p we have examined the response of neurons in the reticular formation near the nucleus ambiguous (na) to the administration of substance p (sp). whole-cell recording was applied to the postsynaptic neurons in coronal slice preparations of medulla oblongata isolated from infant rat. bath application of sp (1 m) increased or decreased the frequency of spontaneous activities. several neurons were clamped at −60 mv and recorded epscs evoked by electrical stimulation to dorsoventral adjacent area from recording neurons. in several neurons, evoked inward epscs were augmented by sp perfusion. i-v curve suggested that voltage dependent current was both augmented and not changed by sp. our previous studies have shown that administration of neurokinin 1 receptor (nk1r) antagonist near the na inhibited gastric and respiratory movement in anesthetized rat. these results indicated that sp affect to both post and presynaptic nk1r and regulate the transmittance efficiency to generate the output signal of certain autonomic reactions. ps1p-h133 effects of local warming in the back or abdominal region by means of a heat-and steam-generating sheets on physiological response in the low temperature room to investigate effects of local warming of the body on physiological functions as well as subjective feeling, eegs, ecg, respirometer, bis (bispectrum) index, blood pressure (bp), and local skin temperature of the body were monitored while a steaming heat pack was put on the lower lumber or abdominal region of the subjects for 1 h in the cold room. in the control experiment without the heat pack, lf/hf of hr variability (lf/hf-hr) and lf of bp variability (lf-bp) increased, while hf of hr variability (hf-hr) and skin temperature decreased, suggesting elevation of sympathetic nervous activity. in the warming experiment with the heat pack, an increase in lf/hf-hr and/or lf-bp was suppressed and hf-hr increased. we will discuss these autonomic data in relation to subjective unpleasant or pleasant feeling, eeg and bis data. junichi arai, yasuhisa endo, ryouichi yoshimura, huan wang kyoto institute of technology, japan in the ventromedial hypothalamic-lesioned animals, the abnormal cell proliferation in liver and pancreas are thought to be due to the vagus hyperactivity and/or the sympathetic repression. we conducted the co-culture system of several cell lines and demonstrated that the proliferation of hepatocytes and min-6 cells (a cell line of pancreatic b cells) were stimulated by the administration of carbachol, when they were co-cultured with cell lines of endothelial cells or smooth muscle cells. these effects were also found in the filter-insert coculture system, but never seen in the culture using single cell line. we discuss the possible mechanism of their intracellular signal transduction. research funds: kakenhi to study the correlation between the trans-cranial oxy-and deoxyhemoglobin (hb) dynamics and sbp, we measured hb dynamics (f-nirs ® , omm-3000, shimadzu corp. japan) over the frontal area and sbp (finapres ® , bp monitor, ohmeda 2300, usa) at the right middle finger from 12 volunteers (22.8 ± 2.1 years). mild thermal stimuli (17 ± 1 • or 40 ± 1 • ) were administered every 2 min alternatively to the left hand. some area showed positive correlation between the oxy-hb and sbp, the other showed negative correlation between them. hb dynamics over the frontal area have any correlation to sbp to some extent. so, trans-cranial nirs should be discussed carefully for neural activation. we thank shimadzu corp. for the use of omm-3000. we examined the effects of color environments on cognitive function in 12 healthy subjects and 12 patients after traumatic head injury using p300 components and loreta analyses. the examination was performed in color environments of red, green, or black using visual oddball tasks with photographs of a crying baby face as the target stimuli. the p300 latency in the red environment was significantly shorter in controls than in patients. the p300 amplitude in the red environment was significantly larger in controls than in patients. loreta analysis demonstrated that the neurological activities in the occipital lobes, left tonsillar nucleus, anterior cingulated gyrus, and brodmann area 10 in the red environment were significantly higher in controls than in patients. hironori nakatani, cees van leeuwen riken brain science institute, saitama, japan some figures, such as rubin's vase/face and the necker cube, have two or more distinct interpretations and are, therefore, called 'ambiguous'. when an ambiguous figure is presented continuously for a period of time, we experience spontaneous switching between the alternative interpretations. as this occurs without any changes in the figures themselves, perceptual switching phenomena are eminently suitable to study how perceptual processes are influenced by the intrinsic dynamics of neural activity. we analyzed eye-movement and eeg during perceptual switching in the necker cube. blink probability showed a peak about 800 ms before the button press responses. we found that only blinks that appeared around the peak time led to a characteristic spatiotemporal pattern of eeg. our results indicate that some, but not all, blinks play an active role in perceptual switching processes. ps1p-h139 neural basis of social cognition investigated by functional near infrared spectroscopy and electroencephalograms recorded from the whole brain tsuneyuki kobayashi 1,2 , mikinobu takeuchi 1,2 , takahiro omote 3 , naoyuki yosimura 3 , etsuro hori 1,2 , kazuo sasaki 3 , taketoshi ono 2,4 , hisao nishijo 1,2 1 system emotional science, univ. toyama, toyama, japan; 2 crest, japan science and technology agency, japan; 3 bio-information engineering, univ. toyama, toyama, japan; 4 molcul. & integ. emotional neurosci., univ. toyama, toyama, japan neural basis of social cognition was investigated by functional near infrared spectroscopy (fnirs) and electroencephalograms (eegs). a head cap for recording fnirs and eegs was set on heads of subjects. the probes of the fnirs imaging systems (103 channels) and/or electrodes of the eeg system were attached on the heads of the subjects. the subjects were required to perform social cognition tasks to discriminate (1) human facial stimuli with different gaze directions and (2) simple animation videos representing social interaction. whole brain hemodynamic images were superimposed on the 3d reconstructed mri images of the brains. now we are analyzing hemodynamic images and eeg data related to social cognition, and the results indicated some heterogeneity of the cortex in social cognition. hiroshige takeichi 1 , sachiko koyama 2 , ayumu matani 3 , andrzej cichocki 1 1 riken, wako, japan; 2 hokkaido university, sapporo, japan; 3 university of tokyo, kashiwa, japan to evaluate the level of spoken sentence comprehension objectively and quickly, electroencephalograms (eeg) were recorded from five japanese adults, while they were listening to fifty-second spoken sentences. natural japanese (native) and spanish (foreign) sentences were modulated in amplitude by an eleventh-order m-sequence at 40 hz, and played twice: forward and backward. evoked responses to the modulation were analyzed as follows: (1) circular cross correlation functions were calculated between the eeg data and the m-sequence for each subject. (2) the functions were averaged across subjects. (3) independent component analysis (ica) was applied to the averaged functions and independent eeg components were estimated for each stimulus for each subject. (4) phase-locked component responses to the modulation were inspected. as a result, two components showed differential responses to the comprehensible forward japanese and the other incomprehensible stimuli. research funds: jst and kakenhi (17022004) perceptual rivalry, such as ambiguous figure perception and binocular rivalry, reflects the flexibility of our brain, because it produces fluctuating perception though an unchanging stimulus. in this study, we carried on meg recordings of healthy subjects while they reported perceptual alternation of bistable apparent motion. we investigated power and phase synchronization analyses of meg signals and compared the spatiotemporal patterns during spontaneous perceptual alternation (rivalry condition) with the externally-triggered alternation (replay condition) to extract the inherent dynamics of perceptual alternation. as results, we detected transient anterior-posterior synchronizations in advance of subjects' reports of perceptual alternation in the rivalry condition. these results suggest that these synchronized activities are involved in a higher-order process inducing spontaneous alternations in perceptual rivalry. ps1p-h142 the reflection of category perception of sound in the auditory evoked n1m magnetic responses to periodic complex sounds with equivalent acoustic parameters except for 2 different fundamental frequencies (f0) and 12 different spectral envelopes of vocal, instrumental and linear shapes were recorded to clarify the cortical representation of timbre categorization. responses to vocal and instrumental (nonlinear) sounds were localized significantly anterior to linear sound responses. n1m source strength for nonlinear sounds was significantly larger than that for linear sounds. n1m peak latency only for vocal sounds was not affected by f0. these results suggest that perceptual categorization was reflected in n1m source strength and location (linear or nonlinear), and in n1m latency (vocal or nonvocal). sunao iwaki 1 , hiroko kou 1 , kouichi sutani 1 , mitsuo tonoike 2 1 national institute of advanced industrial science and technology, osaka, japan; 2 chiba univ., chiba, japan interactions between neural activities detected at multiple brain regions involved in the visual target detection processing were assessed using meg and the causal modeling. meg signals were measured during subjects performing a visual infrequent target detection task. distributed source model was used to infer the dynamic neural activities at the multiple regions and the structural equation modeling (sem) was then used to compare two possible causal models underlying the generation of major event-related components, namely p3, related to the target detection. we used akaike information criteria (aic) and goodness-of-fit index (gfi) as measures of the goodness of the models. the results of the comparison of two possible sem models, whose major difference was on the contribution of the activities in the parieto-temporal region to the generation of p3 components, suggested the involvement of frontal and anterior cingulate cortex in the early p3 component (p3a) and the contribution of the parietal and temporal regions to the later component (p3b in our study, we investigate whether or not bilinguals use distinct neural substrates to recognize words in their first and second languages (l1 and l2, respectively). we compared the brain activity of 11 chinese learners of japanese as l2 with that of 28 japanese natives studied in our previous study. we obtained written informed consent from each subjects. in data analysis, we used spm2. while natives showed specifically greater activation in the left middle temporal gyrus than learners, learners showed specifically greater activation in the bilateral parieto-occipital and left occipito-temporal junction than natives. these results indicate that there are distinct neural substrates for word recognition of l1 and l2. neural activations for lexical processes were measured using noun, vowel, and pseudo-character decision tasks with magnetoencephalography (meg) and functional magnetic resonance imaging (fmri) on ten right-handed subjects, and their time courses were analyzed with an fmri-constrained meg-multi-dipole method. the average activations rose at latencies around 100 ms in the occipital gyrus or cuneus (og/cuneus) and ventral occipito-temporal areas (vot), and at latencies around 200 ms in the posterior superior temporal and inferior parietal areas (pst/ipl), anterior temporal area (at), and posterior inferior frontal gyrus (pifg). the differences in activation between tasks are considered to reflect visual-form process in the og/cuneus and r.vot, phonological process in the l.pst/ipl and l.pifg, and semantic process in the l.at. the decay of activation for these areas was found to be well fitted to exponential functions with time constants around 500 ms. the effectiveness of a habituation/dishabituation paradigm for determining the cerebral dominance for language was examined using a 1.5 t fmri. healthy right-handed adult volunteers with prior written informed consent were instructed to listen to analysis-synthesized words. after habituated to a single word presented repeatedly, the subject was presented with contrastive words which comprised comparison and habituation words in a pseudo-random order. the two blocks were repeated alternately for 6 times. comparison words were phonemic or intonational derivative of the habituation word, and presented in respective sessions. the results showed that the left auditory cortex responded more to the phonemic contrast, and the right to the intonational contrast, which is in line with other paradigms/techniques for determining cerebral dominance, while the present paradigm demands little effort on the subject. the issue that whether meaning of kanji words is accessed from orthography, or from both orthography and phonology representations is still debated. the present fmri study investigated brain areas underlying the use of orthography and/or phonology in kanji reading by engaging subjects in semantic categorization task with homophone and orthographic similarity effects. fifteen native japanese volunteers participated. stimuli were pairs of definitions and their target words, including correct words and foils. the subjects were asked to decide the correct target words of definitions. the results showed that homophone versus non-homophone foils increased activation of the left fusiform and middle frontal gyri. orthographically similar versus dissimilar foils increased activation of the left middle and inferior frontal gyri. these findings reflected the roles of both orthography and phonology in kanji reading. moreover, homophone versus non-homophone minus orthographically similar versus dissimilar foils revealed activation of the left fusiform gyrus. this might suggest the role of this area in character-to-sound conversion of kanji words. chieko takamiya 1 , mie matsui 2,3 , tsuneyuki kobayashi 2,4 , hisao nishijo 2,4 , michio suzuki 2,5 , yasuhiro kawasaki 2,5 , masayoshi kurachi 2,5 , jun nakazawa 6 , kyo noguchi 7 , hikaru seto 7 1 neuropsychiatry, univ. toyama, toyama, japan; 2 crest, japan science and technology corporation, japan; 3 psychology, univ. toyama, toyama, japan; 4 system emotional science, univ. toyama, toyama, japan; 5 neuropsychiatry, univ. toyama, toyama, japan; 6 developmental psychology, univ. chiba, chiba, japan; 7 radiology, univ. toyama, toyama, japan an individual has a theory of mind (tom) if he imputes mental states to himself and others. this ability is necessary for our well-rounded social communication. we used functional magnetic resonance imaging (fmri) in ten healthy subjects to study the neural mechanisms underlying tom. we adopted the picture sequencing tasks which demanded inferring mental states to self and others as tom task. as a result, there were significant brain activations in the medial frontal cortex and middle frontal gyrus. these activations coverged with a part of results in previous neuro-imaging studies on tom and social cognitive functions. objective: the purpose of this study was to investigate the neural bases of evaluation of ambiguous facial expression using whole brain functional magnetic resonance imaging (fmri). methods: participants underwent fmri scanning during which they performed a task evaluating facial expression of human (happy or sad). the task consisted of three conditions: ambiguous, middle, and high intensity of facial expression. pictures were chosen from atr facial expression image database. results: subtraction between ambiguous and other conditions revealed the activation of anterior cingulate cortex and prefrontal cortex in evaluation of ambiguous expression. the present results suggest that these area may be involved in evaluation of ambiguously expressed emotions. motoaki sugiura 1 , atsushi sekiguchi 2 , keisuke wakusawa 2,3 , yuko sassa 2,4 , hyeonjeong jeong 2,5 , kaoru horie 5,6 , shigeru sato 5,6 , ryuta kawashima 2,6 1 miyagi university of education, sendai, japan; 2 niche, tohoku univ., japan; 3 dep. pediatrics, tohoku univ. school of medicine, japan; 4 ristex, jst, japan; 5 gsics, tohoku univ., japan; 6 lbcrc, tohoku univ., japan using an fmri, we examined the cortical mechanisms for risk perception during observation of risky tool usage. normal subjects were presented with a picture of a naturalistic situation involving two actors, in which risks related to a tool and the direction of action were modulated in a two-factorial design. after the fmri, each subject self-evaluated the degree of risk in each picture. main effects of object-and direction-related risks were observed in the left ventromedial prefrontal cortex, and dorsolateral parieto-frontal network, respectively, suggesting that the object-and direction-related risk signals are separately processed in these networks. significant positive correlation between self-evaluated risk and cortical activation was observed in the anterior part of the left superior frontal sulcus, suggesting an involvement of this region in phenomenal risk-perception. in this fmri study, we identified cortical areas where activation during experience of risky situation is correlated with the harm avoidance (ha) scores, subscale of temperament and character inventory (tci). forty-six healthy subjects performed a rule speculation task in risky, normal, and safe situations in fmri. each situation was arranged for subjects to gain 10, 50, 100 points or lose 100, 50, 10 points, respectively. cortical activation induced by experience of risky situation was estimated. a significant positive correlation with the ha scores, was observed in activated areas in the right anterior insula in risky versus safe comparison. the results suggested that activation in this region predicts the individual difference in behavioral response to risky situation. this finding indicates that the right insula underlies individual difference in response to risky situation. ps1p-h152 brain activation related to the evaluation of absolute and relative value of outcome juri fujiwara 1 , masato taira 2,3 , toshio iijima 1 , ken-ichiro tsutsui 1 1 div. sys. neurosci., tohoku univ. grad. sch. life sci., sendai, japan; 2 arish, nihon university, tokyo, japan; 3 appl. sys. neurosci., nihon univ. grad. sch. med. sci., tokyo, japan one way to evaluate the behavioral outcome is in terms of absolute gain or loss (absolute value), but the evaluation can also be achieved by comparing the outcome with the possible outcomes of unchosen options (relative value). here we attempted to disentangle the brain processes involved in the absolute and relative value evaluation by using event-related fmri. subjects were instructed to compete with a computer to maximize the income in a task, in which they had to choose one option out of two, each of which were associated with either 0 yen or a gain or loss of 200, 400, or 800 yen. in each trial, a choice period was followed by a serial presentation of the outcomes of the chosen and unchosen options. we analyzed the brain activity during the presentation of each outcome. the activation changes related to the evaluation of absolute and relative value were observed mainly in the basal ganglia and in the cerebral cortex, respectively. ps1p-i153 neural activation during experience-based reasoning chisato suzuki 1,2 , takashi tsukiura 1 , hiroko mochizuki-kawai 1 , yayoi shigemune 1,2 , toshio iijima 2 1 neurosci. res. inst., aist, japan; 2 div. systems neurosci., tohoku univ., japan the aim of this study is to investigate neural activations when reasoning future events based on experienced events. before fmri, subjects encoded two kinds of four-scene comics; the complete version with four scenes and incomplete one without the last scene. after encoding, subjects performed three tasks during fmri. in the first task, subjects chose a last scene associated with the first scene encoded in the incomplete version (memory-based reasoning: mr), whereas in the second task, subjects recognized a last scene encoded in the complete version (memory: m). in the third task, subjects chose a last scene appropriate to the first scene in the new comics (reasoning: r). activations specific to mr was found in a relatively anterior part of the left pfc and right pfc. the common activations between mr and m were identified in the right mtl, whereas a relatively posterior part of the left pfc was activated commonly between mr and r. the findings suggest that the network including bilateral pfc and right mtl may contribute to the experience-based reasoning of future events. to assess neural responses to reciprocal mindreading in socially strained human relationships, we performed an fmri study in 16 healthy subjects who participated in the chicken game. statistical parametric mapping showed that the counterpart effect (human versus computer) activated the anterior paracingulate cortex (pcc) and the posterior superior temporal sulcus (sts). when we analyzed the data to evaluate whether the subjects made aggressive or reconciliatory choices, the posterior sts showed that the counterpart had a reliable effect regardless of risky or safe decisions. in contrast, a significant opponent x selection interaction was revealed in the anterior pcc. it could be inferred that the posterior sts and the anterior pcc play differential roles in mentalizing; the former serves as a general mechanism for mentalizing, while the latter is exclusively involved in socially risky decisions. creativity is the ability to generate new and original ideas. the most of studies of creativity used linguistic tasks which involve multiple aspects oflinguistic information processing in addition to creativity. we used new artistic creativity task such as designing new tools, in which we could quantitatively evaluated the creativity by the originality (os: originality score) of the products. using fmri, we observed bold signal change during designing task in art students (trained) and non-art students (untrained). we observed clear difference between two groups; in the trained highly creative group, the os is correlated with the interhemispheric difference of neural activities of the prefrontal cortex with right hemisphere dominance. in the untrained group we saw no such correlation. thus, our result supports the notion that both right prefrontal dominance and the increase of interhemispheric cooperativity could be the source of the artistic creativity. ps1p-i156 the difference of brain activity elicited by different styles of art hiromi yamamura 1 , yasuyuki kowatari 1,2 , shigeru yamane 2 , miyuki yamamoto 1,2 1 comprehensive human sciences, university of tsukuba, tsukuba, japan; 2 system brain science division, aist, tsukuba, japan artworks are categorized according to time and place where they were produced (cultural effects). surrealistic art is one of those categories and it gives uneasy impression to our mind. we investigated brain activity during viewing pictures of different art styles using functional magnetic resonance imaging (fmri). works of several artists who are well-known as representatives of renaissance, impressionism and surrealism were used as stimuli and results were analyzed by spm2. while renaissance arts or impressionism arts elicited a similar activation pattern in the occipital and inferior temporal areas, surrealisms showed deactivation in parietal with the activation in the right dorsal prefrontal cortex (ba9, ba46). these results suggest that a particular style of artwork may have commonly activated brain regions. research funds: coe(j-3) ps1p-i157 effects of chewing on the activity of the prefrontal cortex in working memory processing: an fmri study in general, it has been proposed that chewing produces holding or enhancing effect on attention. furthermore, recent studies have shown that chewing causes activation of various brain regions, including prefrontal cortex. we therefore examined the influence of chewing on brain activities using fmri. the subjects used were 20-30 aged healthy adults, being conducted continuously to two-back task with intermittent gum-chewing. gum without odors and taste component was used to remove effects other than chewing. the results indicated that chewing tended to increase the bold signals in the prefrontal area including the dorsolateral prefrontal cortex during two-back task. this suggests the possibility that chewing may accelerate the process of working memory. research funds: kakenhi 17,6577 ps1p-i158 the tip-of-the-tongue with an emotional reaction caused by recall of celebrities' names hirohito m. kondo 1 , michio nomura 2 , jun kawaguchi 3 1 ntt commun. sci. labs., ntt corp., atsugi, japan; 2 dept. psychol., tokai women's univ., kakamigahara, japan; 3 dept. psychol., grad. sch. environ. studies, nagoya univ., nagoya, japan the tip-of-the-tongue (tot) phenomenon is a mental state where you cannot recall something though you have every confidence that you know it. the tot state generates emotional reactions, but it is not clear what neural mechanisms are involved in the awareness of frustration. participants were instructed to recall the full names of celebrities when their faces were presented. event-related fmri analysis demonstrated that the anterior cingulate cortex (acc), anterior insular cortex (aic), inferior frontal cortex, intraparietal sulcus, and fusiform gyrus were activated during the tot state with frustration. activity of the acc and right aic was positively correlated with the degree of frustration in unsuccessful retrieval. roi analysis indicated that the acc and right aic were sensitive to retrieval demands and awareness of frustration, respectively. we suggest that the cinguloinsular circuit regulates the self-monitoring processes during the tot state. noriko kudo 1,2,3 , yulri nonaka 1 , katsumi mizuno 4 , kazuo okanoya 1,5 1 riken, bsi, biolinguistics, saitama, japan; 2 chiba university, chiba, japan; 3 jsps, japan; 4 department of pediatrics, showa university, tokyo, japan; 5 presto, jst, japan segmentation of speech stream is a prerequisite for language acquisition. language learners use the transitional probability between vocal tokens to segment continuous auditory stream into distinctive words. we consider that the ability for statistical learning is not specific to language, but more general cognitive competence. and we ask whether this ability could be considered as innate. in this study, we measured erps for 18 neonates within 3 days, in order to examine whether neonates can learn transitional probabilities and statistically segment words. four three-tonal-words were presented in random order without intervals during recording of the eeg. as a result, only the first tone of each word evoked a significant positive component in the frontal area. since this potential is not evident during the first session, this is likely to be due to statistical learning. these results suggest that the ability to distinguish words based on statistical information is innately prepared in humans. using near-infrared spectroscopy (nirs), changes in concentration of oxygenated hemoglobin (oxy-hb) in the prefrontal cortex were evaluated while eleven human subjects performed the paintings appreciation task. in this task, subjects were required to appreciate abstract and representational paintings that appear one after another on a computer monitor. subjects were then required to judge the degrees of interest, beauty, and desirability immediately after the appreciation. it was shown that the peak of averaged oxy-hb change was higher while subjects appreciated abstract paintings. average differentiation for each oxy-hb change revealed that the changes while the appreciation of representational paintings were more accelerated than that while the appreciation of representational paintings. these results suggest the different cortical activity dependent on appreciation of abstract and representational paintings. we used meg to investigate the spatiotemporal cortical activities during mental calculations and their modulation by arithmetic complexity. eleven healthy subjects have participated in the study. three conditions were considered: easy: add three (3) to a two-digits number without carry-over; difficult: stimuli were the same as easy, but with carry-over; nocalc: add zero to the two digits number. probe stimuli were presented 1 s after the presentation of task stimuli (a pair of two-digit and one-digit number), and the subjects were required to respond by lifting the right index or middle finger. root-mean-square values for 12 different meg sensor groups covering entire cortical area were calculated to evaluate local signal power in each condition. increased neural activities in the bilateral frontal/prefrontal and the parietal regions during both calculation conditions were observed in the latencies around 700-900 ms. the activities in the bilateral prefrontal and the left parietal areas in the same latencies were found to be complexity-dependent, i.e., increased activities in these regions were observed in difficult condition compared to easy condition. we investigated an effect of auditory feedback on self-produced speech in children with and without autism by measuring the lombard effect. ten children with autism (9:4-14:11) and 18 agematched typically developing children (9:5-15:2) were instructed to name 50 pictures of objects aloud in control and masking conditions. in masking, weighted-white noise was continuously delivered through a headset. the subjects' speech responses were recorded from a microphone. in typically developed children, the enhancement (masking/control) in masking was significantly greater (duration = 1.2 ± 0.2, loudness = 1.4 ± 0.3) than in the children with autism (duration = 1.1 ± 0.2, loudness = 1.2 ± 0.3) (p < 0.001). the present findings suggest that deficits in speech audio feedback in autistic children and this could be one of the reasons for their delay in speech development. since the mechanism underlying the effect of low power laser irradiation on the soft tissue is still unknown, we examined whether it can influence the muscle contraction as well as its fatigue in the frog (xenopus laevis) gastrocnemius or not. muscle tension continuously induced by a supramaximal stimulus to the sciatic nerve at 0.5/s chronologically attenuated and showed a simple fatigue curve. direct irradiation of laser (532 nm, 180 mw) to the muscle surface (28.3 mm 2 ) significantly delayed its attenuation (p < 0.05). when the rest period was set between stimulating sessions and the laser irradiation was applied during the rest period, averaged muscle tension during stimulating period for 2 min decreased according to the session sequence. however, comparing with no or cooling application during the rest periods, such laser irradiation case significantly delayed the muscle fatigue (p < 0.05). it is suggested that laser irradiation has a potential to more activate atp synthesis during as well as after muscle contraction. ps1p-i165 nedl1, a novel e3 ubiquitin ligase for dishevelled-1, targets mutant superoxide dismutase-1 and interacts with p53 yuanyuan li 1,2,3 , kou miyazaki 1 , toshinori ozaki 1 , akira nakagawara 1 1 division of biochemistry, chiba cancer center research institute, chiba, japan; 2 production technology development center, the furukawa electric co., ltd., ichihara, japan; 3 hisamitsu pharmaceutical co., ltd., tokyo, japan we have cloned a novel hect-type e3 ubiquitin ligase gene termed nedl1. previous study has shown that nedl1 is exclusively expressed in neuronal tissues and its expression level is high in favorable neuroblastomas and undetectable in unfavorable ones. dishevelled-1, a regulatory molecule in the wnt signaling pathway, was identified as the physiological target of nedl1 for uniquitination and proteasome-mediated degradation. on the other hand, nedl1 bound and ubiquitinated mutant (but not wild-type) sod1 in a mutant sod1 type-dependent manner, which is proportionally related with the fals severity. in the present study, we show that nedl1 physically bound p53, and induced apoptosis in a p53-dpendent manner. taken together, our results suggest that nedl1 may play a critical role in neuronal cell death occurring in fals through interacting with mutant sod1 and p53. spinal and bulbar muscular atrophy (sbma) is an inherited motor neuron disease caused by the expansion of polyglutamine tract within the androgen receptor (ar). chip (carboxyl terminus of hsc70interacting protein), u-box type e3 ubiquitin ligase, has been shown to interact with hsp90 or hsp70 and ubiquitylates unfolded proteins trapped by molecular chaperones and degrade them. we demonstrated in a neuronal cell model that transient over-expression of chip reduced the monomeric mutant ar more than the wild-type, suggesting that the mutant ar is more sensitive to chip than is the wild-type. we also demonstrated high expression of chip ameliorated motor impairments in the sbma transgenic mouse model. these findings suggest that chip over-expression ameliorates sbma phenotypes in mice by reducing nuclear-localized mutant ar, which probably due to enhanced mutant ar degradation. we performed an electrophysiological study demonstrating inhibition of spontaneous muscle action potentials within a co-culture of rat muscle and spinal cord by exposure to patients with guillain-barré syndrome (gbs) serum, as well as purified igg, from selected patients with gbs. using a whole-cell recording technique, we then investigated the effects of serum and purified igg from patients with gbs on voltage-dependent calcium currents (vdcc) in ngf-differentiated pc12 cells and cerebellar purkinje cells. serum from selected patients with gbs and purified igg from some serum of patients with gbs inhibited ca 2+ current in both cells. these results suggest that muscle weakness in some patients with gbs might be induced by changes in p/q-type calcium channel function within motor nerve terminals. the aim of the present study was to explore the possible role of cox-2 inhibitor, rofecoxib in pentylenetetrazol (ptz, 40 mg/kg, i.p.)induced kindling. rofecoxib was administered orally daily 45 min before either ptz or vehicle. seizure severity was measured according to a prevalidated scoring scale. biochemical estimations were performed on the 16th day of ptz treatment. chronic treatment with rofecoxib (2.0 and 5.0 mg/kg, p.o.) for 15 days showed significant decrease in ptz-induced kindling score. chronic treatment with ptz significantly increased lipid peroxidation, nitrite levels (no levels), and myeloperoxidase levels and decreased the reduced glutathione (gsh) levels in brain homogenate, which was reversed with rofecoxib treatment. research funds: university supportted study ashish dhir, shrinivas kulkarni uips, panjab university, chandigarh, india the objective of the present study was to elucidate the effect of cyclooxygenase inhibitors on pentylenetetrazol (ptz)-induced (80 mg/kg) convulsions in mice with possible mechanism of action. various cox-inhibitors were administered 45 min prior to the ptz administration. onset, duration of clonic convulsions and percentage mortality/recovery were recorded. pretreatment with cox-inhibitors aspirin (10 and 20 mg/kg, p.o.), naproxen (7 and 14 mg/kg, p.o.), nimesulide (1-5 mg/kg, p.o.) or rofecoxib (1-4 mg/kg, p.o.) dose dependently showed protection against ptz-induced convulsions. rofecoxib (1 mg/kg) or nimesulide (1 mg/kg) also enhanced the subprotective effect of diazepam or muscimol showing gabaergic modulation of cox-2 inhibitors. cox-2 inhibitors also antagonized the effect of flumazenil (4 mg/kg) against ptz-induced convulsions further confirming the gabaergic mechanism. ps1p-j171 cell proliferation after domoic acid-induced neuronal damage in adult rats domoic acid (da) is structurally related to kainic acid, which is a rigid analogue of the putative neurotransmitter l-glutamate that causes neuronal excitation. da-induced convulsions affects limbic structures such as hippocampus and entorhinal cortex. in this study we examined the neuronal damage after intraperitoneal da administration and cell proliferation in the adult rat brain. the most extensive neuronal cell damage was observed in ca3 subfield as evaluated by he staining, while tunel positive cells were mainly observed in the granular cells of cerebellum and dentate gyrus (dg) of the hippocampus. to elucidate the relations between damage and cell proliferation, we examined bromodeoxyuridine (brdu) labeled cells. brdu labeled cells were detected in dg and the granular cells of cerebellum. the cell proliferation was not associated with damage. ps1p-j172 a-type potassium channel truncation mutation in temporal lobe epilepsy the role of voltage dependent calcium channels on the pentylenetetrazol (ptz) kindling induced learning deficits was investigated in rats. in this study animals were divided into three groups. in the test group verapamile were injected in the hippocampus (4 mg/4 min). after 20 min kindling was established in rats with ptz. the control animals were the same age and undergone the same treatment in term of acsf injections and post-kindling waiting time as the kindled animals. and in sham group the animals received saline. one month after induction of kindling spatial learning and memory was tested by morris water maze. results showed that intra-hippocampal injection of verapamil significantly decreased spatial learning, suggesting that only working memory impaired but reference memory remain intact. the results with this study suggest that intera-hippcampal injection of verapamil significantly impaired spatial learning in rats. we showed that 8-oxoguanine (8-oxog) in mitochondrial (mt) dna and cellular rna increased significantly in the ca3 subregion of the mouse hippocampus after kainate administration. laser scanning confocal microscopy revealed that 8-oxog accumulated greatly in mtdna of the ca3 microglia. wild-type and mth1-null mice, the latter lacking an ability to hydrolyze 8-oxo-dgtp and 8-oxo-gtp to the monophosphates to avoid their misincorporation into dna or rna, exhibited similar degree of the ca3 neuron loss after kainate administration, however, levels of 8-oxog accumulated in mtdna and cellular rna in the ca3 microglia were significantly increased in mth1null mice in comparison to wild-type mice. we thus demonstrated that mth1 efficiently suppresses the accumulation of 8-oxog in both cellular dna and rna in the hippocampus, especially in microglia, caused by excitotoxicity. ps1p-j176 transcription factor nrf2 regulates brain response to kainate-induced excitotoxicity yukihiko dan 1 , kosuke kajitani 1 , noriko yutsudo 1 , ken itoh 2 , masayuki yamamoto 2 , yusaku nakabeppu 1 1 kyushu univ., med. inst. bioreg., div. neurofunc. genomics, japan; 2 univ. tsukuba, grad. sch. comp. hum. sci., japan nf-e2 related factor 2 (nrf2) is the key transcription factor that serves to transmit the inducer signal to an antioxidant response element (are), a cis-acting element required for gene expression of a battery of proteins acting on anti-oxidative stress and detoxification of electrophiles. since loss of nrf2 has been reported to increase neuronal death under increased oxidative stress, nrf2 seems to play a role for neuroprotection. administration of kainite, a potent agonist of an excitatory neurotransmitter glutamate, to rodents produces epileptiform seizures followed by a delayed loss of pyramidal cells in the ca3 subregion of hippocampus. to unveil the functional significance of nrf2 in the brain, we compared seizure responses between wild-type and nrf2-null mice after systemic kainate administration. we found that nrf2-null mice exhibited an increased susceptibility to the kainate-induced seizure, and their loss of the pyramidal cells and gene expression profiles are now under investigation. ps1p-j177 synaptic plasticity and 4-aminopyridineinduced epileptic discharges in rat hippocampal slices makoto otani, tetsuo furukawa, kiyohisa natsume department of brain science and engineering, kyushu institute of technology, kitakyushu, japan four-aminopyridine (4-ap) at the concentration below 0.1 mm suppresses k d channel and induces the epileptic discharges in rat hippocampal slices. in the present study, the involvement of the activation of nmda receptor on the ictogenesis of the 4-ap induced discharges in ca3 region was studied. ten m 4-ap induced the epileptic discharges with the frequency of 0.33 ± 0.04 hz (mean ± s.e.m.; n = 3) and the amplitude of 1.23 ± 0.86 mv. when ap-5, an nmda receptor blocker, was applied to the pre-established epileptic discharges, the frequency and the amplitude of the discharges did not change significantly. on the other hand, when ap-5 was applied from the ictogenesis period of the discharges, the discharges did not appear. these results suggest that the nmda receptor-dependent synaptic plasticity involves in the ictogenesis of 4-ap-induced epileptic discharges. chronic exposure of cultured astrocytes to morphine is reported to induce differentiation of the cells. using primary astrocyte cultures, we observed that under thyroid hormone (th) deficient conditions, morphine significantly decreased cell viability. further studies showed that the loss of cell viability was due to apoptosis of the cells. the effect is attenuated by th supplementation to the culture medium. the observed effect of morphine appears to be mediated through the opioid receptor since the opioid antagonist, naloxone, inhibited the decline in cell viability. 7ni, a specific inhibitor of nnos, completely blocked loss of cell viability suggesting that morphine induced intracellular no production, leads to cell death. studies suggested that no acts through a cgmp independent pathway. the involvement of no induced cgmp independent pathway in morphineinduced apoptosis during th deficiency has been investigated. collectively, the present study demonstrates that morphine mediated cytotoxicity of astrocyte is critically influence by the level of thyroid hormone in cultured medium. ps2a-a001 influence of conductance-input signal and prior activation history on spike generation in rat somatosensory cortical neurons takashi tateno 1 , hugh p.c. robinson 2 1 engineering science, osaka university, osaka, japan; 2 university of cambridge, uk in the cortex, a profusion of electrophysiological cell types, which form specific synaptic connections, is becoming apparent. a quantitative understanding of the dynamics of different cell types when responding to complex, natural inputs, is an important prerequisite for understanding the cortical network. neurons compute by transforming excitatory and inhibitory synaptic conductance inputs into a spike train output. we have examined the properties of synaptic conductance inputs which are most effective in evoking spikes, by injecting broad-band excitatory and inhibitory conductance inputs, and using spike-triggered reverse correlation and wiener-kernel estimation to calculate the average conductance input trajectory (acit) preceding spikes. the time course of the acit provides a general description of a neuron's response to dynamic conductance stimuli. our analysis showed that the acit reflects both previous stimulus history and previous discharge history, and that the relative influences of these two factors depend on the cell type. amyotrophic lateral sclerosis (als) is a rapidly progressive neuromuscular disease caused by the destruction of motor neurons. our study has investigated the effects of als-csf on voltage-gated calcium p/q-type channel (␣1a) expression in pre synaptic terminals of rat spinal motor neurons. csf from als and non-als (neurological patients) was injected into the 3-day-old rat pup spinal subarachnoid space at the rate of 1 l/2.5 min. the rats were sacrificed 48 h after csf injection and spinal cord sections were processed for immunocytochemistry with p/q-type channel ␣1a antibody and also for cytochrome oxidase labeling. als-csf significantly increased p/qtype channel expression compared to csf from non als patients. als-csf significantly decreased cytochrome oxidase activity in the rat spinal motor neurons, which may be a sign of degeneration. it is probable that, toxic factors present in the als patients csf might induce the expression of p/q-type channel observed in pre synaptic terminals synapsing on the spinal motor neurons. ps2a-a003 on the membrane potential profile of ca1 pyramidal cells recorded with voltage sensitive dye imaging in rat hippocampal slices takashi tominaga 1,2 , yoko tominaga 1 1 dept. neurophysiol., kagawa sch. pharmaceutical sci., tokushima-bunri univ., kagawa, japan; 2 lab. for dynamics of emergent intelligence, riken bsi, hirosawa 2-1, wako, saitama, japan integration of membrane potential response in a single neuron is a basis of neuronal calculation. we have been aiming to visualize this with voltage sensitive dye (vsd). hippocampal slices, with its unique laminar structure, allow us to assign optical signals to particular membrane fractions. but, it has not been clear whether the profile of optical signal could be a measure of membrane potential profile. to solve this, we visualized rather steady membrane potential change caused by perfusion of high potassium medium. a steep peak in optical signal was seen along stratum pyramidale. an application of ttx diminished this peak, and made the optical signal profile flat along the cell. thus, we concluded that the specificity of the vsd is small. with "neuron", by assuming a population nature to the optical signal, the membrane potential profile in a response to stimulation was successfully simulated. ps2a-a004 overexpression of inwardly rectifying k + channel 2.1 in hippocampal slice culture masayoshi okada, hiroko matsuda department of physiology, kansai medical university, japan the expressions of mrnas for the inwardly rectifying k + channel (kir) 2.1 have been reported in mammalian central nervous system, but regulation of expression or its role in synaptic transmission remains unknown. in our rat hippocampal slice cultures, the endogenous kir current was hardly detected with whole cell recordings in the ca1 pyramidal neurons. then, egfp and kir2.1 expressing virus vectors were constructed, and infected to the neurons in the slices. the vectors succeed to express the kir current, and the translocation of the fusion protein to the plasma membrane was also observed. furthermore, the overexpression significantly reduced the raise in whole-cell membrane potential evoked by depolarizing current injection, suggesting that kir plays a role of noise-filter for synaptic input in central neurons. takeshi otsuka, mieko morishima, yasuo kawaguchi div. cerebral circuitry & structure, nips, okazaki, japan layer 5 pyramidal cells are heterogeneous in morphological and physiological properties, and project to multiple subcortical areas. although recent studies have addressed anatomical features of pyramidal cells identified projection regions, little is known about intrinsic membrane properties of these subtypes. here, we obtained whole cell recordings from rat frontal layer 5 pyramidal cells that project to the striatum (ccs) or pontine nucleus (cpn), identified by injection of fluorescent retrograde tracer to these regions. firing properties of pyramidal cells had similarity depending on the projection regions. ccs cells showed strong adaptation of successive spike intervals in response to the depolarizing current injection. however, cpn cells exhibited very little spike frequency adaptation during current injection. we also examined synaptic inputs from layer 2/3 neurons to these subtypes by single cell stimulation, and detected excitatory inputs in both subtypes. our results suggest that physiological properties of layer 5 pyramidal cells are correlated with their subcortical target. this study aimed to clarify expressional changes in types 1 and 2 of ryanodine receptors (ryr1 and ryr2) in the cerebellum of a ca 2+ channel ␣ 1a subunit mutant, rolling mouse nagoya. semi-quantitative rt-pcr revealed altered mrna signal levels of ryr1 but not ryr2 in the rmn cerebellum: a less ryr1 mrna signals than in the control cerebellum. well consistent with the semi-quantitative rt-pcr results, ryr1 immunostaining in soma and primary dendrites of purkinje cells was less intense in rmn than in control mice. in contrast, ryr2 immunostaining was detected in cerebellar glomeruli but the staining intensity was not different between rmn and controls. the present study suggests that somatodendritic ryr1 expression in purkinje cells was decreased in the cerebellum of rmn. this may suffer ryr1-mediated ca 2+ release, contributing altered ca 2+ homeostasis in the rmn purkinje cells. ps2a-a007 dopamine-based modulation of lateral amygdala neuron excitability: a possible involvement of potassium current ryo yamamoto, yoshifumi ueta, noubuo kato integrative brain sci. med., kyoto univ., kyoto, japan the amygdala and dopaminergic innervation thereonto are considered to cooperatively regulate emotional states and behaviors. in the present slice experiments, we investigated the effects of dopamine (da) on lateral amygdala (la) neurons by whole cell recordings. application of da depolarized la neurons, reduced the action potential threshold, and induced slow afterdepolarization (sadp). this sadp was induced voltage dependently, and lasted for more than 5 s. d1 receptor agonists induced the same sadp. previous reports have repeatedly suggested that sadp is triggered by calcium influx. consistently, calcium channel blockers or chelating intracellular calcium inhibited the present da-induced sadp. a membrane conductance decreased at the peak of sadp current (i sadp ). also, i sadp was suppressed by including cesium in the pipette solution. these results suggest that the present da-induced modulation of la neuron excitability may depend on a potassium current that can be masked by calcium influx. toru aonishi 1,2 , hiroyoshi miyakawa 3 , masashi inoue 3 , masato okada 2,4 1 tokyo institute of technology, japan; 2 brain science institute, riken, japan; 3 tokyo university of pharmacy and life science, japan; 4 the university of tokyo, japan it has been reported that amplification of ap paired with epsp boosts the induction of ltp. there are two alternative hypotheses of such amplification mechanisms; one is activation of the na channel and other is inactivation of the a-type k channel. which is essential? in this talk, by mathematical analyses and the neuron simulator, we demonstrate that the balance of inward and outward currents, which can be controlled by down/up-regulation of the a-type k channel induces a divergence of the membrane input resistance, i.e. a singularity, and such super-sensitivity is the fundamental mechanism for boosting amplification of ap paired with epsp. the balance of na and a currents is essential for controlling dendritic integration manners. we also show that the down-regulation of the a-type k channel, which modifies the ratio between the inward and outward currents, leads to a drastic change from amplifying ap mode to shunting epsp mode. miharu komai 1 , maya yamazaki 1,2 , mika tsujita 1 , manabu abe 1 , rie natsume 1,2 , kenji sakimura 1,2 1 department of cellular neurobiology, brain research institute, niigata university, niigata, japan; 2 sorst/jst, saitama, japan we previously reported that stargazin family (␥2, ␥3, ␥4, and ␥8) not only promoted ampa receptor surface expression but also modulated receptor activity and channel property (yamazaki et al., 2004) . therefore, we assumed these family proteins were auxiliary subunits of ampa receptors. to prove this hypothesis, we generated ␥4 subunit knockout (ko) mice using the cre/loxp recombination system and analyzed their phenotypes. the ␥4 subunit ko mice were viable, fertile, and displayed no overt phenotype. on the other hand, on western blot analysis, protein expression levels of ampa receptor subunits were reduced in ko mice compared with those in wild-type at postnatal day 11, while the reduction was not so significant in adult brain. these results suggested that ␥4 might regulate dynamics of ampa receptor subunits during early development. in the cns, neural damages, such as hypoxia, ischemia and degenerating diseases, are often accompanied by disturbances in the ph environments. ambient ph plays as a significant signal for neural functions. microglia (brain phagocytes) express abundant voltagegated proton (hv) channels which have extremely high selectivity for h + and potent h + efflux ability. exposure to na-lactate (ph 6.8) induced cell acidosis and activation of the hv channels. the channel activation was characterized by increased conductance, facilitation of activation kinetics, prolongation of deactivation kinetics and a shift of the activation voltages to negative potentials. consequently, the hv channel could open more easily over a wide range of the membrane potential during lactic acidosis, and may contribute to a quick relief of the cell acidosis. mari sasaki, masahiro takagi, yasushi okamura okazaki institute for integrative bioscience, aichi, japan here we report a novel four transmembrane protein similar to the voltage sensor domain (vsd) of the voltage-gated channels that exhibits activities of a voltage-gated proton channel. voltage-gated proton channel currents have classically been described in snail neurons and recently in mammalian blood cells. however, the molecular basis underlying this channel has been elusive. here we identify a novel cdna clone named as mouse voltage-sensor domain only protein (mvsop1). cells overexpressing this protein showed depolarization-induced outward currents accompanied by tail currents during repolarization, which reversed at equilibrium potentials for protons. imaging analysis demonstrated that phin recovers rapidly after an acid load in mvsop1-transfected cells. mvsop1induced currents exhibited two key features of native voltage-gated proton channels: ph-dependent gating and zn 2+ sensitivity. neutralization of a positive charge in the s4-like segment caused shift of the voltage-conductance relationship, suggesting that it plays important role in gating. oscillatory extracellular electric fields have been observed in mammalian brains. the electric fields modulate neuronal excitability and synaptic events. to investigate the effect of the oscillatory electric fields on the ca1 pyramidal neuron, we applied sinusoidal electric fields to the rat hippocampal slice and recorded voltage responses with a voltage sensitive dye (rh414). application of sinusoidal electric fields induced transmembrane voltage oscillations in all the layers of the ca1 region. in the pyramidal layer, the amplitudes of the responses to the 1-hz field were the largest. the amplitudes were decreased monotonically when the frequency of the fields became higher. however, in the stratum radiatum, the amplitudes of the responses to the 3-10-hz fields were larger than those to the other frequencies. the frequency preference in the dendritic region may be an underlying mechanism for the synchronization of the membrane potentials among large population of neurons within the theta frequency range. acid sensing ion channels 2 (asic2) have proposed to constitute mechanoreceptors and nociceptors. we examined the localization and characterization of asic2-expressing cells in rat central nervous system (e17-p14) using immunohistochemical techniques. asic2positive fiber first appeared in brain stem and spinal cord at e17-18 stage. asic2-expresseing cells appeared in white matter of brain stem and spinal cord at e20 stage. in early postnatal stages asic2expressing cells appeared in corpus callosum, cerebellar medulla and dorsal horn of spinal cord at p4 stage. these cells were identified as an oligodendroglia by oligodendrocyte specific antibody and immunoelectron microscopy. these results are suggested the hypothesis that the function of asic2 mediate the myelin formation in the developmental stages of central and peripheral nervous system. masato shino, seiji ozawa, yasuhiko saito department of neurophysiology, gunma university graduate school of medicine, maebashi, gunma, japan nucleus prepositus hypoglossi (nph) is involved in horizontal eye movement. previously, we found nph neurons exhibiting a characteristic firing pattern in response to depolarizing current pulses (fil neurons). fil neurons exhibited a spike train with a long first interspike interval (1st isi) that is attributed to a large, slow hyperpolarization (ahp) after the first spike. in this study, we investigated ionic conductances underlying the long 1st isi by whole-cell recordings in rat slices. application of 100 m apamin, an sk-type ca 2+ -activated k + (kca) channel blocker, shortened the 1st isi and decreased the amplitude of the slow ahp. the shortening of the 1st isi was observed when membrane potentials were depolarized. moreover, application of 3 m mibefradil, a t-type ca 2+ channel blocker, shortened the 1st isi. these suggest that the firing pattern of fil neurons arises from activation of sk-type kca channels induced by ca 2+ influx through t-type ca 2+ channels. research funds: kakenhi (c) (17500270) jafar vatanparast 1,2 , mahyar janahmadi 1 , houri sepehri 2 , ali haeri-rohani 2 , ali reza asgari 1 1 neuroscience research center, shaheed beheshti medical sciences university, tehran, iran; 2 dept. of biology, university of tehran, tehran, iran the roles of the ionic channels and muscarinic receptors in paraoxon (px) induced burst firing in snail neurons were studied using current clamp method. px (0.6 m, within 5 min) increased the frequency of spikes and shortened ahp. slow waves of depolarization with superimposed bursts were recorded within 20 min. atropine blocked the depolarization shift but not the other effects of px. px was able to reversibly decrease the duration of calcium spikes elicited in a na + free ringer. this effect observed in the presence of atropine and was along with a decrease in the duration of ca 2+ spike ahp and an increase in the spike frequency. the px blockade of ca + channels may decrease the activation of ca 2+ dependent k + channels that underlies ahp. blockade of these channels possibly makes the neurons susceptible for burst induction, while activation of metabotropic muscarinic receptor by px underlies the depolarization shift with associated bursts. dendritic membrane properties are reported to be non-uniformly distributed in a single neuron and the non-uniformity could be important for synaptic integration. however their distribution is still unclear. we estimated distribution of membrane resistance by fitting a compartment model to voltage imaging data of membrane response in hippocampal ca1 slices to perturbation, such as propagating epsp induced by synaptic inputs and biphasic response to extracellular electric field. by numerical simulations, we found that these imaging data were consistently reproduced if we assume a step function as distribution of membrane resistance. this implies that a steep decrease of membrane resistance exists in distal dendrite of hippocampal ca1 pyramidal neuron. it is known that cooling-induced desensitization of cold receptors, however, its intracellular mechanism has remained unresolved. in this study, we analyzed molecular mechanism of desensitization of cold/menthol receptors (trpm8). repeated menthol application induced trpm8 desensitization. this desensitization was depended on extracellular ca 2+ , indicating that involvement with ca 2+ -dependent kinase. pkc activator (pma) desensitized trpm8 and go6976 (pkc inhibitor) abolished pma-induced trpm8 desensitization. pma similarly desensitized wild type trpm8 and mutant trpm8, in which serine or threonine residues in some putative pkc phosphorylation sites were replaced by alanine. pma treatment did not induce internalization of trpm8. as the basis of cooling-induced desensitization of cold receptors, we conclude that cooling-activated trpm8 causes pkc to desensitize trpm8 itself. yosuke sawada 1 , hiroshi hosokawa 1 , kiyoshi matsumura 2 , shigeo kobayashi 1 1 dept. of int. sci. and tech., grad. sch. of info., kyoto univ., kyoto, japan; 2 dept. of info. sci. and tech., osaka institute of technology, osaka, japan cooling below 17 • c evokes cold pain sensation. however, the molecular basis of the cold pain sensation is still unknown. trpa1 is activated by pungent compounds stimuli. if trpa1 responded to cooling to noxious cold range, it could be candidate for evoking cold pain sensation. however, whether trpa1 is activated by cooling or not is still controversial. here, we investigated that trpa1-expressing hek293 cells responded to noxious cold stimuli. whole-cell recording demonstrated that cooling below threshold evoked inward current. threshold temperature was 17.5 ± 2.7 • c. in inside-out singlechannel recording, cooling activated trpa1 directly. single channel conductance was 74.1 ± 18.8 ps. single channel currents showed inword rectification. in conclusion, trpa1 is the cooling activated cation channel. yoshiki matsuda, foong yen ang, jinsun yoon, noriko ebisu, satoshi takahashi, shinichi kogure dept. bioengin., soka university, tokyo, japan hyperplarization-activated and cyclic-nucleotide-gated nonselective cation channels (hcn1-4) have been demonstrated in the cns. since they contribute to various physiological functions including neuronal pacemaking activity, setting of resting membrane potential and generation of paroxysmal discharge, we examined their expressions as well as functions in the pns using the frog (xenopus laevis) sciatic nerves. western blot analyses for hcn1-4 demonstrated that samples from the nerve and the heart showed an hcn2 band whereas those from the dorsal part of skin and the gastrocnemius did not, and that immunoreactivities for hcn1, hcn3 and hcn4 could not be found in those samples. when an hcn channel blocker, zd7288 was applied on the stimulus portion of sciatic nerve and the nerve was elicited at 0.5/s by a duration of 10 ms pulse with supramaximal intensity, the generation of anode-break-excitation rather than cathode-makeexcitation was significantly blocked (p < 0.01). it is suggested that hcn2 channels exist in the pns and they contribute to the burst or recurrent discharges. ifenprodil, a clinically used cerebral vasodilator, interacts with several receptors, such as ␣ 1 adrenergic, n-methyl-d-aspartate, serotonin and receptors. however, the molecular mechanisms underlying the various effects of ifenprodil remain to be clarified. here, we show that ifenprodil inhibited g protein-activated inwardly rectifying k + (girk; kir3) channels, which play an important role in the inhibitory regulation of neuronal excitability in most brain regions and the heart rate, expressed in xenopus oocytes. in contrast, kir1.1 and kir2.1 channels in other kir channel subfamilies were insensitive to ifenprodil. the girk currents induced by -opioid receptors or ethanol were also attenuated in the presence of ifenprodil. the inhibitory effects of ifenprodil were not observed when ifenprodil was applied intracellularly. our results suggest that inhibition of girk channels by ifenprodil, at submicromolar concentrations or more, may contribute to some of its therapeutic effects and adverse side effects. ps2a-b022 proliferation of rat c6 glioma cells is controlled by the concentration-sensitive na + channel (na c ) shigeru yoshida, hiroyuki yamaguchi, takashi takeuchi, hokuto tanaka, yoshiyuki morimoto, teruki hagiwara department of life science, kinki university, higashi-osaka, japan the role of na + as a regulator of cell growth was studied using the tumor cell line (c6), which has a large quantity of concentrationsensitive na + channels (na c ; c = concentration). cell proliferation was suppressed when [na + ] o was raised from control (140 mm) to 190 or 240 mm. an increase in [na + ] i was revealed by an image processor in c6 cells loaded with a na + indicator (sbfi), under high [na + ] o conditions. [na + ] i elevation was augmented by ouabain or by bumetanide (na + /k + /cl − cotransporter blocker), while it was decreased when na c expression was inhibited by rnai techniques. the real-time pcr method revealed that the expression level of the immediate early gene egr-1, which is involved in cell growth, was concomitantly reduced. it is to be noted that similar alterations in cell growth, egr-1 level and [na + ] i were induced by a na + ionophore (monensin) without raising [na + ] o . these data indicate that na + enters through na c upon [na + ] o increase, and [na + ] i elevation itself is responsible for these phenomena. hiroshi kuba, takahiro ishii, harunori ohmori dept. physiol., univ. kyoto, kyoto, japan na + channels are concentrated in the axon to generate action potentials. however, little is known about how distribution of na + channels contributes to the activity and function of single neurons. in avian nucleus laminaris (nl), neurons act as coincidence detectors for sound source localization, and are tuned to both characteristic frequency (cf) and interaural time difference (itd) of sounds. we show here in the chick that nl neurons have distinct distribution of na + channels along the axon and optimize the itd sensitivity depending on their cf. neurons of high and middle cf (higher than 1 khz) had small action potentials, and had no na + currents in the somatic membrane, but clustered only in the axon at some distance from the soma (20-50 m). while, neurons of low cf generated large overshooting spikes, and na + channels were clustered closer to the soma (5 m) in the axon. thus, nl neurons had a spike generator on the axon, at a greater distance from the soma with the increase of cf. by computer simulation, these unique distributions of na + channels were found essential to enhance the coincidence detection. research funds: kakenhi (17021021) il-sung jang 1 , in-sun choi 1 , eun-ju park 1 , jin-wha cho 1 , man-gee lee 2 , byung-ju choi 1 1 kyungpook national university, school of dentistry, south korea; 2 kyungpook national university, school of medicine, south korea bisphenol a (bpa), an endocrine disrupter, is contained in cans, polycarbonate bottles and some dental sealants. here we report the effect of bpa on gaba a receptors using a conventional whole-cell patch clamp technique from acutely isolated rat ca3 pyramidal neurons. bpa itself elicited a postsynaptic current, which is highly sensitive to bicuculline, in a dose-dependent manner. bpa increased postsynaptic currents induced by gaba at lower concentrations (<10 m), but decreased those induced by gaba at higher concentrations (>100 m). in addition, bpa decreased both the current amplitude and decay time constant of gabaergic mipscs. finally, mechanisms underlying bpa-induced modulation of gaba a receptors will be discussed. we recently generated nav1.1-deficient mice and showed that these mutant mice developed epileptic seizures and died prematurely. we have now used these nav1.1-deficient mice as negative controls to examine nav1.1 distribution in the mouse brain using rna in situ hybridization histochemistry and immunohistochemistry. at low magnification, nav1.1 expression was higher in the thalamus, superior colliculus, inferior colliculus, pons, medulla and cerebellar nuclei relative to other brain regions. contrary to previous studies indicating a somato-dendritic nav1.1 distribution, in the present study, higher magnification analysis revealed that nav1.1 is predominantly distributed to axons in some brain parts. this apparent discrepancy may reflect the lack of specificity of anti-nav1.1 antibodies used in these previous studies, none of which utilized nav1.1-deficient mice. based on our findings, we propose that nav1.1 might be involved in propagating action potential to presynapses. keiji miura 1,2 , masato okada 2,3,4 , shun-ichi amari 4 1 department of physics, kyoto university, kyoto, japan; 2 "intelligent cooperation and control", presto, jst, japan; 3 department of complexity science and engineering, university of tokyo, chiba, japan; 4 brain science institute, riken, saitama, japan we considered a gamma distribution of inter-spike intervals as a statistical model for neuronal spike generation. a gamma distribution is a natural extension of poisson process and it can generate spike trains with various irregularities. the model parameters consist of a time-dependent firing rate and a time-independent spiking irregularity. because the environment changes over time, the firing rate varies for each interspike interval. we used a novel method of information geometry to estimate the spiking irregularity whatever the functional form of the firing rate is. our estimator is simple and easily applicable to experimental data. the estimator is useful for characterizing spiking irregularity which varies among neuron types. it may be possible to classify neurons into functional groups according to their spiking irregularities. research funds: grant-in-aid for scientific research (nos. 14084212 and 16500093) mitsuyo watanabe, yuko ishimaru, taketo nakadai, tomoyuki kanamatsu graduate school of bioengineering, soka university, tokyo, japan we examined the effect of colchicine, inhibitor of axonal flow, on cerebral amino acid metabolism in the rat. the rats were injected with [1-13 c] glucose intravenously (1 g/kg) 24 or 48 h after the intraventricular injection of colchicine (75 g/10 l) and the amino acid fractions were extracted from the brains at 10, 20 or 40 min after the glucose injection. the amount of [1-13 c] glucose in the cerebra was increased, however, the 13 c incorporation into glutamine, glutamate, gaba and aspartate from [1-13 c] glucose were decreased. only glutamine concentration in all amino acids was increased in the cerebra of the colchicine group, compared to those values in the control group. the microdialysis analysis showed that the amount of gln in the dialysate was increased by three times in the colchicine group compared with the control group. these data may suggest that the glycolysis of glucose is decreased and that the influx of glutamine from blood to brain occurs with neuronal dysfunction induced by colchicine. these results indicate that a ␤ alters the bhlh gene expression in neural stem cells toward cell death. ken kojima, akiko nishida, shinji takebayashi, jyuichi ito department of otolaryngology-head and neck surgery, graduate school of medicine, kyoto university, japan basic helix-loop-helix (bhlh) transcription factors play crucial roles in development of the central and peripheral nervous systems. to visualize expression of hes1 or hes5 gene, phes1-and phes5-egfp transgenic (tg) mice were generated (ohtsuka et al., 2006) . in each transgenic mouse, a promoter of hes1 or hes5 gene drives enhanced green fluorescent protein (egfp) gene. in the inner ear, it is suggested that hes1 or hes5 regulate cell division and differentiation of sensory and supporting cell progenitors via notch signaling pathway. by use of immunohistochemical technique, we examined distribution of gfp expressing cells in the inner ear of the transgenic mice from embryonic day 10 (e10) to postnatal day 60 (p60). in the phes1-egfp tg mouse inner ear, gfp immunoreactive (gfp-ir) cells were detected from e10 to p60. in the phes5-egfp tg mouse inner ear, gfp-ir cells were observed from e16.5 to p15. gfp-ir cells in phes1-gfp tg mouse are candidates of sensory cell progenitors in mature mammalian inner ear. ohtsuka et al., 2006. mol. cell neurosci. ps2a-c033 expression of zfh-5 in the developing mouse brain: mrna, antisense rna and protein expression yuriko komine 1 , kenji nakamura 2 , motoya katsuki 1 , tetsuo yamamori 1 1 national institute for basic biology, okazaki, japan; 2 mitsubishi kagaku institute of life science, machida, japan zfh-5 is a transcription factor containing three homeodomains and 18 zn fingers and expressed in differentiating neurons. we have reported that the level of zfh-5 mrna is negatively regulated by antisense transcripts of the zfh-5 gene. in several types of neurons, including pyramidal cells in the hippocampus and granule cells in the cerebellum, the zfh-5 antisense rna is expressed prior to the mrna; as the level of the antisense rna gradually decreases, zfh-5 mrna starts to be expressed. recently, we have raised an antibody against mouse zfh-5 and examined the expression profile of the zfh-5 protein. in the most regions of the brain, the protein expression pattern consisted with that of mrna. however, in the several types neurons mentioned above, zfh-5 protein was not detected even when the zfh-5 mrna was already expressed. this observation together with other data suggested that the zfh-5 protein level is regulated by several mechanisms including suppression by the antisense rna and translational control. takashi inoue 1 , maya ota 1 , katsuhiko mikoshiba 2 , jun aruga 1 1 laboratory for comparative neurogenesis, riken bsi, saitama, japan; 2 laboratory for developmental neurobiology, riken bsi, saitama, japan zic family zinc-finger proteins play various roles in animal development. in mice, five zic genes (zic1-5) have been reported. despite their partially overlapping expression profiles, mouse mutants for each zic gene show distinct phenotypes, suggesting the functional redundancy of zic proteins. it is expected that the common and specific roles of mouse zic proteins can be clarified by studying compound mutant mice. in the present study, we characterized zic1/zic3 compound mutant mice. mice carrying homozygous zic1 mutant allele together with zic3 null allele showed defects in midline structures, including abnormalities in forebrain and thalamus. especially, the compound mutants showed severe anatomical abnormalities in the dorsal and ventral telencephalon and olfactory system, which are not obvious in either zic1-or zic3-single mutant. these observations indicate that zic1, in cooperation with zic3, have an essential role in controlling proliferation and differentiation of the neuronal projenitors in the medial telencephalon. chiaki maruyama, haruo okado department of molecular physiology, tokyo metropolitan institute for neuroscience, japan rp58, a novel zinc finger protein containing a poz domain, functions as a sequence specific transcriptional repressor. rp58 gene disrupted mice show severe abnormalities in brain cortical layer formation, suggesting that rp58 has a crucial role in cerebral development. to understand the role of this protein in brain development, we examined rp58 gene expression in mouse embryo and adult brain by in situ hybridization. as a result, we found that rp58 transcripts are first detected at embryonic day 10 in the neuroepithelium of the spinal cord and telencephalic vesicle. in the day 12-13 embryos, rp58 transcripts are predominantly observed in the preplate region but not in outside the nervous system. at e15, rp58 transcripts were detected throughout the neocortex and hippocampus, but not in the thalamus and striatum. in the cortex, the transcripts were detected primarily in cortical neurons, but not in the marginal zones and ventricular zone. in adult mice, rp58 is expressed in neocortical and hippocampal neurons and granule cells in the cerebellar cortex toshiki kameyama, fumio matsushita, yuzo kadokawa, tohru marunouchi division of cell biology, fujita health university, toyoake, japan neural zinc finger (nzf) proteins are transcription factors with dnabinding domains of c2hc-type zinc finger motifs. using p19 cells, we demonstrated that nzfs were expressed transiently during neuronal differentiation, and forced expression of nzf cdnas resulted in neuronal differentiation. these results suggest that nzf family have a function regulate neuronal differentiation. to elucidate in vivo functions of nzf family in detail, we generate knockout mice of nzf-2 and nzf-3 respectively. nzf-2 null mice are born alive, but die within 10 min after birth with cyanosis. on the other hand, nzf-3 null mice are viable, fertile and appear normal. these mice look normal morphologically. then we generate double knockout mice of nzf-2 and nzf-3 by intercrossing. double knockout mice have a forelimb posture abnormalities like arthrogryposis multiplex congenita. and we find out that the spinal nerves projecting forelimb and trunk are decrease dramatically in the double knockout mice embryo. , 2006) . in the present study, to examine the role of runx3 in the development of drg in more detail, we examined the development of drg neurons in runx3-deficient mice from the early embryonic stages to birth, using various markers for subpopulation of drg neurons. in newborn runx3−/− mice, parvalbumin-positive drg neurons were greatly reduced in number, whereas calretinin-positive neurons were slightly decreased. similar decreases were observed in embryonic days 14.5 and 15.5. shin hisahara 1,2 , susumu chiba 2 , hiroyuki matsumoto 2 , yoshiyuki horio 1 1 department of pharmacology, sapporo medical university, sapporo, japan; 2 department of neurology, sapporo medical university, sapporo, japan in mammalian cns, the function of histone deacetylase sirt1 is still unclear. recent studies indicated that sirt1 interacts with nuclear receptor co-repressor (n-cor) and n-cor represses intracellular domain of notch-icd activation of the hes1 promoter. we performed overexpression of sirt1 and n-cor in neurosphere by nucleofection, then induced differentiation. we found remarkable promotion for neural differentiation by overexpression of sirt1 and n-cor in the sirt1 with n-cor. sirt1 and n-cor suppressed hes1 transcription by notch-icd in the luciferase assay. hes1 transcription was suppressed in overexpression of sirt1 and n-cor, suggesting that interaction between sirt1 and n-cor represses hes1 transcription. consistent with this, chip assays revealed that not only n-cor but also sirt1 bind to the promoter of hes1 gene. taken together, these results indicate that sirt1 and n-cor accelerate neural differentiation of the undifferentiated cells via binding hes1 promoter site and repressing hes1 transcription. yasushi maruyama 1 , mitsuhiko kurusu 2 , masataka okabe 3 , katsuo furukubo-tokunaga 1 1 grad. school life and envir. sci., univ. tsukuba, japan; 2 natl. inst. genetics, mishima, japan; 3 inst. dna medicine, jikei univ. school of medicine, japan during brain development, a large number of neurons are generated by proliferation of neural stem cells. with a characteristic proliferation mode that persists through development, the neuroblasts of drosophila mushroom bodies (mb) provide an attractive model system to study mechanisms of neural stem cell proliferation. here we show that tailless (tll), a member of the orphan nuclear receptor super family, has a crucial function in maintaining cell cycle progression of the mb neuroblasts. mosaic analysis demonstrates that cell autonomous activity of tll is crucial for maintenance of the mb neuroblast cell cycles. moreover, gain-of-function analyses confirm instructive functions of tll in maintaining neuroblast activity. we propose that tll plays pivotal roles in proliferation of the mb neuroblasts and suggest a conserved mechanism of neural stem cell control with the tll/tlx homologs in both drosophila and vertebrate brains. kouji senzaki, masaaki yoshikawa, shigeru ozaki, takashi shiga graduate school of comprehensive human science, university of tsukuba, ibaraki, japan runx family transcription factor is an important component of tgf-␤ and bmp signaling. we reported previously that runx3 mrna is expressed in the dorsal root ganglion (drg) from the early developmental stages, and that runx3 regulates axonal projection of trkcexpressing proprioceptive drg neurons (inoue et al., 2002) . furthermore, we announced previously that runx3 mrna is expressed in cranial ganglia of v, vii, viii, ix and x in mouse developmental stages. the expression was restricted to subset of neurons in each ganglion. to examine the influence of runx3 on the differentiation of trigeminal ganglion neurons, we investigated the expression of neurotrophin receptors, calcium binding proteins and neuropeptides in trigeminal ganglia of runx3 knockout mice using immunohisitochemical staining. we found the decrease of trkc-expressing neurons in trigeminal ganglia of neonatal runx3 knockout mice, however, we observe little change in the proportions of nuen-expressing neurons. kouko tatsumi 1 , hirohide takebayashi 2 , takayuki manabe 1 , kazuhiro ikenaka 2 , akio wanaka 1 1 dept. anatomy, nara med. univ., kashihara, nara, japan; 2 division of neurobiology and bioinformatics, nips, nins, okazaki, aichi, japan our previous study with double labeling of brdu and cell lineage markers suggested that a number of astrocytes were differentiated from resident oligodendrocyte progenitor (opcs)-like cells in the injured adult brain. and we found out that these opcs expressed ng2proteoglycan and olig2 at early phase after injury. to directly trace the lineages of these opcs, we employed double transgenic mice that express tamoxifen-sensitive creer under the control of the olig2 promoter together with rosa-egfp reporter. the gfp positive cells were detected around the injured region, and the almost all of these cells co-expressed gfap at late phase after injury. furthermore, we confirmed that the morphological characteristics of these cells were those of the astrocyte by immunoelectron microscopy. our results clarified that dormant opcs in vivo differentiate into astrocytes in adult injured brain, and suggested that these cells participate in glia scar formation after brain injury. olig2 is a bhlh transcription factor, essential for oligodendrocytes (ols) and motoneurons differentiation in the spinal cord. however, differentiation of olig2 lineage cells in the forebrain is largely unknown. here we examined fates of olig2 expressing cells in the fetal forebrain by tamoxifen (tm)-inducible cre-loxp system. olig2-creer knockin mice were mated with reporter mice, and tm was injected at embryonic day (e) 12.5 or 14.5, when most of olig2+ cells are observed in the basal forebrain. the olig2+ cells at e12.5 gave rise to more neuron than glia that included both ols and astrocytes. majority of neuronal olig2 lineage cells differentiated into gabaergic neurons, and a lesser number, into cholinergic neurons. the olig2+ cells at e14.5 generated more glial cells than neurons. these results indicate that olig2 lineage cells generate three major types of neural cells with a stage dependent manner, and may have multiple functional roles on neural differentiation in the fetal forebrain. mana igarashi 1,2 , masato yano 1,2 , satoru hayashi 1,2 , hirotaka j. okano 1,2 , hideyuki okano 1,2 1 dept. physiol., keio univ. sch. med, tokyo, japan; 2 sorst jst, japan the mammalian neuronal hu rna binding protein family is homolog of drosophila elav protein which is essential for differentiation and maintenance of the nervous system. in mammals, neuronal hu expresses in both early postmitotic and mature neurons and has ability to induce neuronal differentiation by binding to the utrs of specific target mrnas. to understand the molecular mechanism of hu induced neuronal differentiation, we purified hub associated complexes. among them, nf90 family, a double strand rna binding protein which is one of hu associated proteins, is known to bind to utrs of p21, p27 and tau mrna known as hu targets. we generated rabbit polyclonal antibodies against nf90 and nf45, binding partner of nf90, respectively. in mouse embryonic brains, we found that nf45/90 expressed highly in postmitotic neurons where neuronal hu proteins are highly distributed. moreover, we found that hu and nf45/90 formed mrnp complexes in mouse brain extracts. we will discuss the role of hu binding partners in neuronal differentiation through post-transcriptional regulation. sachiko the pallium is specified as a homologous field in the vertebrate telencephalon. however, little is known about how species-specific pallial structures are generated during embryogenesis. to address this issue, we compared several neuronal subtypes and their migration patterns in the developing pallium of the mouse and quail. cell tracing analysis revealed that neurons born at the dorsal pallium tangentially migrated in the developing quail telencephalon, as in the mammalian cortex. next we investigated distribution of later-born neurons in the quail telencephalon using laminar specific genes (er81 and brn2) in the cerebral cortex. in situ hybridization and immunohisitochemical studies indicated that these neuronal markers were expressed in discrete regions of the developing quail telencephalon. our data suggest that early stages of cortex/pallium development are comparable between the mammalian and avian embryos, whereas neuronal specification in later stages is regulated by distinct mechanisms in each species. research funds: kakenhi (16027202) ps2a-d047 protein expression in hippocampal cells dissociated and re-cultured from organotypic slice cultures we established a re-cultivation technique of hippocampal cells dissociated from long-term cultured organotypic slices. protein phenotype of the cells was analyzed using immunocytochemical technique. antinestin immunoreactivity was observed in cells with short processes 2 days in the re-cultivation. the anti-nestin immunoreactivity was progressively declined, whereas number of cells expressing anti-␤iii tubulin immunoreactivity increased through the re-cultivation for 1-4 weeks. presence of neurons, astrocytes and oligodenderocytes was examined using anti-␤iii tubulin, anti-glial acidic fibrillary protein and rip antibody, respectively. apart from the cells expressing one of the markers, the cells marked with multiple sets of antibodies were observed. these results suggest that protein expression was changed backward in normal differentiation course in hippocampal cells once matured in organotypic slices. we have shown that perineuronal ng2 + cells are major populations of proliferating cells in the cerebral cortex of rats. in the adult cortex, ng2 is known as a marker for oligodendrocyte progenitor cells (opcs) that retain ability to proliferate and differentiate into new oligodendrocytes. however, it is still unclear whether all ng2 + cells in the neocortex are the opcs. we investigated about subtypes of ng2 + cells found in the perineuronal regions of the cerebral cortex using cell markers. two subtypes of perineuronal ng2 + cells could be distinguished by the subcellular localization of gst-protein. one is nuclear type, the other is cytoplasmic type. only the nuclear gst-+ cells have the proliferative activity. these data suggest that the nuclear gst-+ /ng2 + cells in the perineuronal territory are progenitor cells engaging in reproduction of cortical cells. muguruma keiko, su hong-lin, matsuo-takasaki mami, watanabe kiichi, sasai yoshiki neurogenesis and organogenesis group, riken center for developmental biology, kobe, japan in this study, we report in vitro generation of math1 + cerebellar granule cell precursors and purkinje cells from es cells by using soluble patterning signals. when neural progenitors induced from es cells in a serum-free suspension culture are subsequently treated with bmp4 and wnt3a, a significant proportion of these neural cells become math1 + . the induced math1 + cells mitotically active and express markers characteristic of granule cells precursors (pax6, zic1, and zipro 1). after purification by facs and coculture with postnatal cerebellar neurons, es cell-derived math1 + cells exhibit typical features of neurons of the external granule cells layer, including extensive motility and a t-shaped morphology. interestingly, differentiation of l7 + /calbindin-d28k + neurons (characteristic of purkinje cells) is induced under similar culture conditions but exhibits a higher degree of enhancement by fgf8 rather than by wnt3a. this is the first report of in vitro recapitulation of cerebellar neurons by using the es cell system. sachiyo misumi, kim hye-jung, hideki hida, hitoo nishino department of neurophysiology and brain science, nagoya city university graduate school of medical science, nagoya, japan regulation of the cell cycle plays an important role in cell proliferation, differentiation, and apoptosis. we have shown that pretreatment with cell cycle blocker increase the number of neurons from neural stem or progenitor cells (npcs) without influencing apoptosis after differentiation. in this study, we investigate the molecular mechanism of neuronal differentiation by cell cycle arrest. in rt-pcr, the expression of p21 cip1 , p27 kip1 and p57 kip2 mrnas were elevated during differentiation to neuron from npcs. especially, prolonged enhancement of p27 kip1 mrna was shown. transfection of p27 kip1 into npcs induced activation of neurod promoter and increase of number of ␤tublin iii-positive cells. treatment with deferoxamine to npcs from e12.5 rat midbrain and hb1.f3 cell line did not activate erk and akt phosphorylation during the treatment. date suggest that prolonged p27 kip1 elevation is related to enhanced production of neuron from npcs, and that cell cycle regulation in g1/s phase did not activate mapk and pi3-k signaling. yuichi tanaka 1 , yusuke tozuka 1 , dai muramatsu 1 , kin-ichi nakashima 2 , tatsuhiro hisatsune 1 1 departement of integrated biosciences, graduate school of frontier sciences, university of tokyo, kashiwa, japan; 2 graduate school of biological sciences, nara institute of science and technology, ikoma, japan we previously reported no definite evidence for in vivo neurogenesis in adult neocortex. however, we also confirmed dividing cells in this area. in this study, we analyzed the characteristics of adult cortical nestin+ cells. in vivo, they belonged to ng2+ and olig2+ cells, showed slowly proliferating ability compared to those in adult dentate gyrus. for in vitro analysis, we precisely isolated progenitor cells by percoll gradient procedure. they differentiated into tuj-1+ or map-2+ neuronal cells by adding retinoic acid or bdnf. more than 90% of newborn neurons expressed gabaergic neuronal markers, gaba, gad or calretinin. we also purified nestin-gfp+ cells from nestin-gfp transgenic mice using the facs system, and confirmed their neuronal potential. moreover, integration of a neural bhlh transcription factor neurod1 significantly promoted this neurogenesis. we demonstrated neurogenic potential of adult cortical nestin+ cells. mie gangi 1 , michiko imanishi 2 , teiko kuroda 2 , masao tachibana 1 , masahiko takada 2 1 department of psychology, graduate school of humanities & sociology, university of tokyo, tokyo, japan; 2 tokyo metropolitan institute for neuroscience, tokyo metropolitan organization for medical research, tokyo, japan a kv3 subfamily of voltage-gated k + channels is thought to play an important role in high-frequency repetitive firings. it is unknown which subtype of kv3 channels is expressed in the frog retina where ␥-range oscillatory spikes are evoked presynaptically by light stimulation. we found immunohistochemically that kv3.1b and kv3.3 were expressed both in the mouse and frog retinas. however, a laminar pattern with two bands in the inner plexiform layer was displayed by kv3.3 in the frog retina and by kv3.1b in the mouse retina. it has been shown that mammalian cholinergic amacrine cells express kv3.1b. thus, the differential expression of kv3 channels may reflect their functional diversity between the frog and mouse retinas. hiroshi jouhou 1,2 , kazunori yamamoto 1 , masayuki hara 1 , akinori homma 1 , akimichi kaneko 3 , masahiro yamada 1 1 tokyo metropolitan univ., hino, tokyo, japan; 2 astellas pharma. inc., osaka, japan; 3 sch. rehabili., seijoh univ., aichi, japan in order to interpret the formation of receptive field surrounds in the retinal neurons, hirasawa and kaneko (2003) proposed a phmediated mechanism to substitute for the gaba-mediated feedback hypothesis from horizontal cells (hcs) to cone photoreceptors. to verify the idea that the depolarized hcs release protons we measured, by a fluorescent ratio imaging technique, the ph of the immediate external surface (ph o ) of hcs isolated from carp or goldfish retina. when hcs stained by 5-hexadecanoylaminofluorescein, a phsensitive lipophilic dye, were depolarized by application of kainate or by high extracellular k + , ph o acidified. the amount of ph o acidification was monotonically dependent on the amount of depolarization, as much as 0.21 ± 0.05 ph unit by 100 mm k + . acidification of pho was suppressed by 0.4 m bafilomycin a1, a specific inhibitor of v-atpase, suggesting that the hc depolarization enhanced an outward proton movement by the outward electrogenic h + pump. ps2a-e055 analysis of spread of activity in the local circuit of superior colliculus by using multi-channel field potential recording system penphimon phongphanphanee, katsuyuki kaneda, tadashi isa national institute for physiological sciences, japan to study how the visual signal is processed in the local circuit of superior colliculus (sc) from the superficial layers (ssc) to the deeper layers (dsc), we analyzed the propagation of excitation following the electrical stimulation of the ssc by using a planar 64-channel field potential recording system in slice preparations obtained from 16 to 24 days old mice. stimulation at ssc induced negative field potential with short latency and short duration (100-200 ms) at the recording site in ssc adjacent to the stimulating site. after application of bath containing 10 m bicuculline, the same stimulus induced a large negative field response with long duration (200-400 ms) that spreads laterally in ssc and ventrally to dsc. these responses disappeared after application of 50 m apv, when only short latency response remained. the results suggest that when gaba a receptormediated inhibition is reduced, visual signal in the ssc propagates to the dsc as large response with long duration and nmda receptors contribute to propagation of the response. osamu hosoya 1 , ken tsutsui 2 , kimiko tsutsui 1 1 dept. of neurobiol. and neuroanat., okayama univ. grad. sch. of med., dent., and pharm. sci., japan; 2 dept. of genomics and proteomics, okayama univ. grad. sch. of med., dent., and pharm. sci., japan amphiphysin ir (amph ir) is alternatively spliced variants of amphiphysin i which is specifically expressed in retina. amph ir is composed of conserved domains including the n-terminal bar, the central clathrin/ap-2 binding, and the c-terminal sh3 domains and the variant specific two novel insertions (a and b). insert a may be a determinant for the retina-specific expression. insert b has no significant homology to known proteins and two shorter transcripts with 3 -truncations in the insert were also expressed. recently, we found that a human retinal pigment epithelia cell line, arpe-19, also expresses amph ir. arpe-19 thus can be a useful tool for investigating the cellular function of amph ir in retina. immunofluorescence analyses with arpe-19 cells revealed that amph ir occasionally colocalized with mitochondria, raising the possibility that amph ir may participate in structural or functional organization of mitochondria. further characterization of the variant is under investigation. hironori takamura 1 , satoshi ichisaka 2 , chihiro hayashi 2 , hirotoshi maki 2 , yoshio hata 1 1 div. integrative biosci., tottori univ. grad. sch. med. sci., yonago, japan; 2 div. neurobiol., sch. life sci., fac. med., tottori univ., yonago, japan monocular deprivation (md) induces significant plasticity in the primary visual cortex (v1) during critical period. it was reported that inhibition of extracellular signal-regulated kinase (erk) activity in the visual cortex suppressed the ocular dominance plasticity. if erk is involved in the mechanism of this plasticity, visual deprivation would change the activity of erk in v1 and such change might be induced only in the critical period. to test this possibility, we examined effects of md on the amount of phosphorylated (activated) erk (perk) in the rat visual cortex. by md, we found a significant decrease in the amount of perk in v1 receiving deprived eye inputs in both young and adult rats. as to the subcellular localization of erk, we found a significant increase of the nuclear perk only after md in young rats. these results suggest that erk signaling might be regulated by different mechanism between young and adult rats. research funds: kakenhi (176959) ps2a-e058 rapid pre-synaptic weakening by experiencedependent competition in mouse visual cortex nobuko mataga, yoko mizuguchi, takao hensch neuronal circuit development, riken brain science institute, saitama, japan in the binocular zone (bz) of mouse visual cortex, critical period (cp) plasticity is accompanied by a transient loss of spines on pyramidal cell dendrites. to explore a correspondingly rapid and local pre-synaptic refinement by sensory deprivation, excitatory intracortical or thalamocortical axon terminals were visualized in the bz by vesicular glutamate transporters (vglut)1 and vglut2, respectively. a complementary distribution of vglut1 and vglut2 was established by postnatal day (p)18 and both signal intensities increased further by p29-30 (peak cp). the immunoreactivity for vglut2 decreased around layer iv after brief monocular deprivation (4dmd) during the cp. interestingly, both signals in all layers were lower in the bz contralateral to an eye injected with ttx than in the ipsilateral bz, consistent with the stronger functional plasticity and rapid dendritic refinement as compared to 4dmd. these results suggest that rapid and local weakening of excitatory inputs corresponds to dendritic spine pruning during experience-dependent competition. reiko meguro, masao norita department of sensory and integrative medicine, niigata university, graduate school of medical and dental sciences, niigata, japan we investigated how the geniculate and the extra-geniculate visual systems reorganize by monocular deprivation at birth. using anterograde/retrograde tracer, biotinylated dextran amine (bda), we made a small injection into the dorsal lateral geniculate nucleus (dlgn) or the lateral posterior nucleus (lp) of the degenerated side of the monocular deprived rat. the geniculate projection terminated mainly in layer iv of area 17, with a small projection to layer vi of areas 17 and 18a. cells in layer vi of area 17 projected to dlgn. in addition, cells in layer v of area 17 projected to dlgn, which is not observed in normal rats. in area 18a, cells in layers v and vi projected to dlgn. the projection from lp terminated mainly in layer iv of 18a. cells in layers v and vi of area 18a projected to lp. smaller number of cells in layer v of area 17 also projected to lp. these findings suggest that major parts of visual system developed normally, but some developed cross talk between geniculate and extra-geniculate systems. ps2a-e060 activity dependent plasticity of feedback projection from the primary visual cortex to the dorsal lateral geniculate nucleus miho yoshida 1 , takemasa satoh 2 , yoshio hata 1 1 div. integrative biosci., tottori univ. grad. sch. med. sci., yonago, japan; 2 div. neurobiol., sch. life sci., fac. med., tottori univ., yonago, japan the projection from the lateral geniculate nucleus (lgn) to the primary visual cortex (v1) shows significant morphological plasticity responding to visual experiences in early life. such experiencedependent plasticity enables the geniculocortical projection to form functionally specific connections. it is not clear whether similar plasticity operates in other neural connections in the visual system. therefore, we tried to investigate the plasticity of feedback projection from v1 to the lgn. we focused on the density of type 1 metabotropic glutamate receptor ␣ (mglur1␣) in the lgn which locate postsynaptically at synapses of feedback projection. immunohistochemical signal for mglur1␣ in the lgn decreased after elimination of v1, showing that this signal reflects density of functional feedback synapses. to explore the activity dependent plasticity, we examined the effect of cortical activity blockade on the mglur1␣ signal in the lgn of young rats. yu morishima 1 , hiroshi sakamoto 2 , takahumi akasaki 1 , yoshio hata 1 1 div. integrative biosci., tottori univ. grad. sch. med. sci., japan; 2 div. neurobiol., sch. life sci., fac. med., tottori univ., japan monocular deprivation (md) during postnatal development reduces cortical response to the deprived eye and input axons serving the deprived eye retract. however, when md is combined with continuous inactivation of the visual cortex by muscimol, cortical neurons lose their response to the open eye and the open eye axons retract. to clarify mechanisms underlying the two forms of ocular dominance (od) plasticity in different direction, we examined other characteristics of them, (1) how rapidly the reverse od shift proceeds and (2) whether the shift is induced only in young animals. we infused the cortex with muscimol in 4-week-old kittens and in adults. the reverse od shift was observed after 6 days md, but not significant after 3 days md. in adults, od distribution remained unchanged. morphological change of individual input axons was also examined after 6 days md. the reverse od shift might reflect a mechanism of developmental plasticity that has a slower time course than the normal od shift. ps2a-e062 experience-dependent plasticity in the absence of ampa receptor subunits in mouse visual cortex youichi iwai 1 , nafiseh atapour 1 , john renger 1 , john roder 2 , peter seeburg 3 , takao hensch 1 1 neuronal circuit dev., riken, bsi, wako, japan; 2 mount sinai hospital, toronto, canada; 3 max planck inst. med. res., heidelberg, germany two ampa receptor subunits (glur1, 2) play prominent roles in hippocampal models of homosynaptic plasticity (ltp/ltd). brief monocular deprivation (1d md) rapidly alters both the phosphorylation state and surface expression of glur1 in visual cortex. here, we addressed whether these coincidental events are essential for subsequent ocular dominance (od) plasticity. mice lacking glur1 (ko) displayed little ltd in visual cortical slices, while baseline transmission was normal. they also exhibited normal visual receptive field properties in vivo and shifted responsiveness toward the open eye after 4d md during a typical critical period. the rate of plasticity appeared somewhat slowed, as 20d md eventually led to full od shifts. in glur2 ko mice, even 4d md robustly activated od plasticity. thus, experience-dependent modification of ampa receptors is not essential for plasticity in vivo, although glur1 may contribute to the very earliest stages. shigeyoshi higo, nobuaki tamamaki department of morphological neural science, kumamoto university, kumamoto, japan virus-assisted transduction with reporter genes is a useful technique to investigate morphology of neurons in the central nervous system. however, the mechanisms to induce reporter expression in vivo often depend on gene-manipulated mice. since mice are not the best experimental animal for the study of mental disorder, we developed an adenovirus in which gfp expression is driven by dlx2 promotor and dlx5/6 enhancer. this virus labels gabaergic neurons and oligodendrocyte in the wild-type mouse neocortex and allows us to trace gfp-labeled axons of gabaergic neurons in serial brain sections. we used this virus to investigate gabaergic neurons with long projecting axon branches beyond a functional area. the virus was injected into the stratum oriens of the mouse hippocampal field ca1 and revealed a nonpyramidal neuron projecting to ca3 and fimbria. further we shall introduce this virus to the cat brain and investigate axon branches of gabaergic projection neurons in the neocortex. akiko yamashita 1 , takao oishi 2 , motoharu hayashi 2 1 div. appl. system neurosci., nihon univ. grad. sch. med. sci., tokyo, japan; 2 dept. cell. and mol. biol., primate res. inst., kyoto univ., inuyama, japan gabaergic cells in the cerebral cortex are divided into subgroups: parvalbumin (pv)-, somatostatin (som)-, calretinin (cr)-, and calbindin-containing types. to clarify inhibitory system in primates, we determined coexistence of these molecules and proportions of these subtypes within gabaergic cells in the various cortical areas. pv, som or cr did not coexist with each other in primates as observed in rodents. more than 60% of gabaergic cells contained pv; showing that pv cells are more abundant in primates than in rodents. proportion of som cells in gabaergic cells was smaller in the primary visual area (5.3%) than in other areas, such as the prefrontal (10.8%), primary motor (9.8%), somatosensory (10.0%) and secondary visual areas (8.5%), indicating cortical differentiation in gabaergic system of the primate cerebrum. our recent retrograde labeling studies in mice and cats showed that the neocortical areas are connected not only by excitatory neurons but also by gabaergic projection neurons. in order to address the importance of the gabaergic projection neurons in the neocortical information processing, we need to know the branching pattern and postsynaptic elements of the gabaergic projection axons. since more than 80% of the gabaergic projection neurons showed npy immunoreactivity, we used npy-cre transgenic mouse that express cre in npy neurons and adenovirus that encodes gfp in the downstream of floxed stop to label the gabeergic projection axons. after injection of the adenovirus into deep layers of the npy-cre mouse neocortex and immunoperoxidase staining of gfp in the brain section, we could reveal gabaergic neurons in a golgi-like image with their axons. also this method seemed to allow us to label gabaergic projection neurons retrogradely. koji ikezoe 1 , guy n. elston 2,3 , tomofumi oga 1 , hiroshi tamura 1,3 , ichiro fujita 1,3 1 osaka univ., japan; 2 univ. queensland, australia; 3 crest, jst, japan layer iii pyramidal cells in adult monkeys exhibit systematic differences in their dendritic morphology among cortical areas. basal dendrites of cells in visual association cortex such as inferior temporal area te spread more extensively and are more branched than those in the primary visual cortex (v1). pyramidal cells in prefrontal cortex, such as area 12, have even more dendritic branches than those in area te. here, we investigated whether a similar regional difference in the dendritic morphology was present in infant monkeys. we stained individual layer iii pyramidal cells in v1 (n = 52), area te (n = 46), and area 12 (n = 43) of a 3-week old monkey (macaca fascicularis) using intracellular dye-injection techniques in lightly fixed tissues. the number of branches and the tangential extent of dendrites was greatest in area 12, followed by area te, and v1. thus, considerable heterogeneity in pyramidal cell structure already exists 3-weeks after birth. hiroaki matsushita 1 , mahito ohkuma 1 , masami watanabe 2 , ei-ichi miyachi 1 1 department of physiology, fujita health university, aichi, japan; 2 department of perinatology, institute developmental research, aichi, japan acetylcholine (ach) receptors are believed to be expressed in developmental and regenerative process of retinal neurons. we performed the patch-clamp recording and fura-2 based calcium imaging in cat retinal ganglion cells (rgcs). under whole cell clamp conditions, transient sodium currents and action potentials were observed in all of normal or axonal regenerated rgcs. however, these currents and spikes were not observed in the 50% of axotomized rgcs. bath application of 100 m carbachol, an ach receptor agonist, rose [ca 2+ ] i in 22% of normal rgcs. although the 85% of rgcs responded to carbachol at 3 days after axotomy, no responsive rgcs appeared during 7-15 days. ach responsiveness recovered in axonal regenerated rgcs (17%). since pycnotic cells were observed few days after axotomy, ach may modulate neurotrophic effect in survived rgcs. these results suggest that ach is an important marker for neuronal degeneration and regeneration in cat rgcs. research funds: kakenhi (17500217 to em, 16591780 to mw) kenichiro miura, masakatsu taki, hiromitsu tabata, kenji kawano dept. integrative brain sci., grad. schl. of med., kyoto univ., kyoto, japan the initiation of smooth pursuit eye movements is facilitated by the bottom-up attention to the target (hashimoto et al., 2004) . to study the effects of the attention on the processing of second-order motion stimuli, we recorded smooth pursuits in three humans with a dualpurkinje-image eye tracker. the pursuit target, presented on a crt monitor (75 hz), was a gaussian patch of texture displayed on a neutral gray background. the gaussian envelope moved at 30 deg/s, while the texture consisting of black and white random-noise pixel blocks remained stationary (drift-balanced stimulus). the number of the frames displaying the target before the motion onset was selected to manipulate the attention to the target, either eight frames (107 ms) or only one frame (13 ms). the initial tracking responses were larger when the target became visible eight frames before the motion onset. the result suggests that the second motion processing underlying the smooth pursuit initiation is facilitated by the attention to the target. ps2a-e069 motion picture effects on eye movements and blood flow in the frontal area atsuhiko iijima 1,3 , tohru kiryu 2 , kazuhiko ukai 3 , takeshiko bando 1 1 div. integrative physiol., grad. sch. med., niigata univ., niigata, japan; 2 div. inform. sci., grad. sch. & tech., niigata univ., niigata, japan; 3 dept. appl. phys., sch. sci. & tech., waseda univ., tokyo, japan motion pictures taken by rider's view of motocross bike elicited horizontal eye movements coherent to the motion vectors in some subjects, and not coherent in the other subjects, while those taken by passenger's view of roller coaster evoked similar eye movements in all of the subjects. 20 subjects watched the two-dimensional motion pictures in random order. eye movements were measured by a binocular video oculography (newopto), and head movements were measured by a magnetic motion sensor (polhemus). blood flow in the frontal area was simultaneously monitored with a near infrared spectroscopy (hamamatsu). the patterns of eye movements and the blood flow variation during movie presentation changed in relation to motion components of the movie. possible mechanisms of the differences will be discussed. kiyoto matsuura 1 , kenichiro miura 1 , masakatsu taki 1 , hiromitsu tabata 1 , naoko inaba 1 , kenji kawano 1 , frederick a. miles 2 1 grad. schl. med., kyoto univ., kyoto, japan; 2 lab. sensorimotor res., nei, nih, bethesda, md, usa human ofrs show winner-take-all behavior when elicited by moving grating patterns composed of two sinusoids (sheliga et al., sfn 2005) . we recorded the ofrs to the motion of vertical grating patterns composed of two sinusoids of spatial frequency 3f and 5f, which created a repeating pattern with beat frequency, f, in two monkeys. motion consisted of successive steps (100 hz), each one-fourth of the wavelength of the beat, so that with each step the two components shifted one-fourth of their wavelengths and had opposite directions, the 5f forwards and the 3f backwards. the contrast of the 5f was fixed at 4, 8, or 16%, while the contrast of the 3f was varied from one-fourth to four times the contrast of the 5f. when the contrast of the 3f (5f) was less than about half that of the 5f (3f), the 5f (3f) dominated initial ofr: winner-take-all. thus, the motion processing underlying the ofr in monkeys, like that in humans, includes nonlinear interactions. masazumi katayama, takahiro fujita department of human and artificial intelligence systems, faculty of engineering, university of fukui, fukuki, japan when executing prehension movement to grasp an object such as a tool, we plans the hand shape and grasping position to grasp a target object. while, goodale proposes the hypothesis that the roles of two visual streams (dorsal and ventral streams) are "vision for action" and "vision for perception", respectively. from the above points of view, we investigated independence of visual estimation and motor execution for grasping position of a target object. in this experiment, grasping positions were measured under the following four conditions: visual estimation without grasping, grasping without lift-up movement, grasping and lift-up movement and visual estimation without grasping. as a result, we found that grasping positions of visual estimation are significantly different from grasping positions of motor execution in the second and third conditions (p < 0.05). we concluded that grasping positions of visual estimation and motor execution are independent and these results support the goodale's hypothesis. ryuichi hishida, masaharu kudoh, katsuei shibuki dept. neurophysiol., brain res. inst. niigata univ., niigata, japan cortical sensory areas are divided into modality-specific domains such as the visual and auditory cortices, in which sensory neurons are driven by modality-specific inputs. recently, wallace et al. found that multimodal neurons clustered in deep layers are present near the borders between sensory cortices. multimodal properties of these neurons may be explained by three types of inputs: overlapped projections from the thalamus, projections from multi-modal sites, or overlapped horizontal projections from the modality-specific sensory cortices. in this study, we tested the third possibility. we prepared the mouse cortical slices including the visual and auditory cortices. the horizontal activity propagation elicited by local electrical stimulation were visualized using flavoprotein fluorescence imaging. these results indicate that cortical areas between the visual and auditory cortices receive horizontal projections originated in the visual and auditory cortices, suggesting that multimodal horizontal connections are important for the multimodal properties of sensory neurons. jumpei naito 1 , yaoxing chen 2 , yukiko tanada 1 1 dept. animal sci., teikyo univ. sci. & tech., uenohara, japan; 2 china agricul. univ., beijing, china twenty white-leghorn chicks (p0-8) were perfused with 1% paraformaldehyde through the heart under deep anesthesia of nembutal (32 mg/kg bw). two to three small crystals of dii were implanted into the optic tectum, thalamus, or hypothalamus under a dissecting microscope. a total of 225 rgcs were classified into six groups according to the somal area and dendritic field (naito and chen, 2004). group ic projected dominantly to the tectum. group is and iiis showed high hypothalamic-and thalamic-dominance, respectively. group iic was non-specific in the central projections. group ivc was tectal-dominant. patterns of the dendritic stratification were counted to 32 in 121 tec-rgcs, 28 in 77 tha-rgcs, and 16 in 38 hyo-rgcs. of these stratification patterns, many patterns were common among tec-, tha-, and hyp-rgcs. in contrast, the rgcs that showed a same dendritic pattern were consisted of a single rgc in most of the non-common rgcs, and their dendrites extended mainly to the superficial inner plexiform layer (sublayers 1-3). yasuro atoji, shouichiro saito laboratory of veterinary anatomy, gifu university, gifu, japan the present study was examined afferent and efferent fiber connections of the intermediate part of the caudal nidopallium (nci) in the pigeon by a tract-tracing method. in the present study we define nci an area which is located lateral to the field l complex and ventral to ncl. following a ctb injection into nci, a large number of neurons was labeled in nci, the mesopallium, and intermediate arcopallium (ai) and in the thalamic posterior dorsointermediate and posterior dorsolateral nuclei. contralateral ai contained a small number of labeled neurons. a few labeled neurons were found in lst. few labeled cells were found in ncl, field l, piriform cortex, or hippocampal formation. following a bda injection into nci, a large number of labeled fibers extended in nci, mesopallium, and ai. lst contained a small number of labeled fibers. few labeled fibers were located in ncv and limbic regions. the diencephalon contained very few labeled fibers. in summary, nci has strong reciprocal connections within nci itself and with the mesopallium and ai, and little connections with the limbic system. hidenori horie 1 , kenji yuda 3 , eiichi okawa 4 , katsuyoshi maruyama 4 , hiroshi uozato 5 , hiroko horie 1 , satomi nakajima 1 , kenkichi tanioka 6 , yuji ohkawa 6 , tomoki matsubara 6 , wolfram tetzlaff 7 1 advanced res. centr. biological sci., waseda univ., tokyo, japan; 2 technomaster co. ltd., yokohama, japan; 3 kikuna yuda eye clinic, yokohama, japan; 4 healthcare business co., matsushita electric ind. co. ltd., yokohama, japan; 5 dept. ophthalmol. & visual sci., kitasato univ., kanagawa, japan; 6 nhk sci. technical res. labo., tokyo, japan; 7 icord, univ. british columbia, vancouver, canada we describe here a highly effective method to improve visual acuity of children with myopia and adult with presbyopia by repeatedly offering a visual object at variable distances while keeping the apparent retinal projection size of the object constant using a novel electronic device. in our experiments on human subjects, we used an lcd screen that was rhythmically moved between 25 and 70 cm toward and away in a high speed (top speed: 1000 mm/s) from the subjects. the device significantly improved visual performances in over 60% of the school-aged children with myopia and 80% of adults with presbyopia. hiroyuki miyamoto 1 , toral s. surti 2 , takao k hensch 1 1 laboratory for neuronal circuit development, riken brain science institute, wako, japan; 2 san francisco, usa competitive plasticity of binocular response following monocular deprivation (md) is prominent in the primary visual cortex (v1) during an early critical period. recently, md has been shown to enhance head-tracking behavior induced by slow rotation of grating stimuli in adult mice and is critically dependent upon the integrity of v1. here, we addressed to what extent these two types of plasticity induced by the same md share common mechanisms. adult mice lacking a gaba-synthetic enzyme (gad65 ko), which do not exhibit ocular dominance (od) plasticity by brief md during the critical period, showed normal optomotor acuity and enhancement with 4 day md. od shifts did not correlate with optomotor enhancement in these mice. finally, early md spanning the entire critical period had no effect on optomotor acuity through the deprived-eye. these observations support the view that adult perceptual learning and classical od plasticity are independent. junya hirokawa 1 , miquel bosch 2 , shuzo sakata 3 , yoshio sakurai 4 , tetsuo yamamori 1 1 division of brain biology, national institute for basic biology, okazaki, japan; 2 mit, ma, usa; 3 rutgers university, nj, usa; 4 department of psychology, kyoto university, japan the brain is able to integrate information from different sensory sources to enhance behavioral responses. to identify the neuronal populations responsible for multisensory enhancement in rats, we have mapped the activation of neurons during an audiovisual integration paradigm (sakata, et al., 2004) by the expression of c-fos. a pronounced c-fos upregulation was found in superior colliculus and in layer iv and deep layer of latero-medial secondary visual area (v2lm). local injection of gaba agonist muscimol into this region selectively suppressed the behavioral enhancement related to multisensory integration, while no suppression was found by the injection into primary auditory and visual areas. these results suggest a key role of v2lm in integration of auditory and visual information to facilitate the behavioral reaction for bimodal stimuli. takashi shinozaki 1 , youichi miyawaki 2 , tsunehiro takeda 1 1 department of complexity science and engineering, university of tokyo, chiba, japan; 2 mathematical neuroscience, riken bsi, saitama, japan drifting grating patterns with different motion directions independently presented to the two eyes induce two sets of perceptual rivalries: interocular rivalry (left or right eye's image) and motiontype rivalry (pattern or component motion). we studied this double rivalry process based on psychophysical and magnetoencephalography (meg) measurements. pattern-motion percept exclusively arose and persisted for a long duration whereas component-motion percept was soon followed by percept of either of left or right eye's single motion direction. reaction time (rt) measurement showed that the pattern-motion was perceived faster than left or right eye's motion direction. we then compared meg signals among those perceptual conditions and found a meg response of interocular rivalry in the latency range expected from the result of rt measurement. these results suggest that the double rivalry process has a hierarchical structure in which motion-type rivalry is resolved before interocular rivalry. visual stimuli evoke several brain potentials with relatively precise time courses. the role of these brain potentials in visual object categorization is not clear. in this study we recorded event related brain potentials (erp) while subjects participated in a face/non-face categorization task. gray face and non-face natural object images were presented briefly (10 ms) followed by a noise mask with pseudo randomly selected stimulus onset asynchrony (soa = 0-990 ms). subjects reported presentation of face or non-face images by pushing one of the two assigned keys. we found that the face category discrimination performance significantly declined only in short soa (10 and 20 ms) with a larger impact of masking on non-face discrimination. in erp, the peak amplitude and latency of p1, n170 and area under curve of a late positive potential expanding from 300 to 500 ms were correlated with the subjects behavioral performance. the effect of backward masking on early erp components may be due to altering sensory processing of visual stimuli while the effect on late erp potential could be related to its impact on decision making processes. yasushi naruse 1 , ayumu matani 1,2 , tomoe hayakawa 2,3 , norio fujimaki 2 1 university of tokyo, kashiwa, japan; 2 nict, kobe, japan; 3 teikyo university, tokyo, japan to study the process of alpha rhythm resetting, we investigated the relationship between visual evoked potential and the seamlessness: how much the phase angle of prestimulus alpha rhythm and the backward-extrapolated phase angle from poststimulus alpha ringing synchronize. alpha ringing is an evoked potential in alpha frequency band around 500 ms in latency. eight clinically normal adult volunteers participated in the experiment, in which the subjects passively viewed a series of 1000 flash stimuli with their eyelids closed throughout the experiment. eeg was simultaneously recorded during the experiment. we classified the trials into four subsets owing to the seamlessness, and then averaged the trials in each subset. the result showed that the larger the amount of the alpha rhythm resetting is, the larger the p100 amplitude becomes. this suggested that a factor of the variability of the p100 amplitude is the amount of the prestimulus alpha rhythm resetting. research funds: a grant-in-aid from the ministry of education, culture, sports, science and technology (no. 16300083) hitoshi sasaki 1 , takuya ishida 1 , masayoshi todorokihara 2 , junichiro miyachi 1 , tahei kitamura 3 , ryozo aoki 1 1 dept. physiol. & biosignal., osaka univ. grad. sch. med., suita, japan; 2 dept. phys. & elec., osaka pref. univ. grad. sch. eng., sakai, japan; 3 dept. elec. eng. & elec., col. industri. tech., amagasaki, japan recently it has been shown that noise can improve detection of sensory stimuli in several modalities. here we investigated whether visual contrast detection sensitivity can be improved by adding a certain amount of noise. contrast detection thresholds of a light changing brightness periodically were measured with or without overlapping noise in five normal participants. the contrast detection threshold, measured by using the psychophysical method (up-anddown method), decreased at around the threshold level of the noise intensity. these findings are consistent with our previous findings obtained by using another psychophysical method and confirm that noise can improve signal detection in human visual perception. narumi katsuyama 1 , nobuo usui 1 , izuru nose 1,2 , masato taira 1,3 1 department of applied system neuroscience, nihon university school of medical science, tokyo; 2 faculty of human science, bunkyo university, saitama, japan; 3 arish, nihon university, tokyo, japan when an object is moving, perception of its 3d trajectory in depth can be strongly influenced by the trajectory of its cast shadow. for example, a ball moving in a diagonal trajectory can appear to rise in a frontal plane when the shadow moves along the horizontal trajectory (rising configuration) or to roll in depth when the shadow follows the same trajectory as the ball, while the trajectory of ball is identical. using fmri, we found that several visual areas, including human mt and the posterior sts and the posterior parietal cortex, are activated in the comparison between rising configuration and ball only condition. additional correlation analysis by modifying the slope of the shadow' s trajectory also showed activation in the posterior part of sts and the posterior parietal cortex, including precuneus. these results suggest that cortical areas in the temporal and parietal cortex might be involved in the processing of apparent motion of ball induced by the moving cast shadow. ps2a-f083 local area network in the gerbil's auditory cortex: reversible focal inactivation with infrared laser irradiation akira yamamoto, hiroshi riquimaroux gratuate school of engineering, doshisha university, scnrl, japan this study investigated local area networks in the primary auditory cortex (a1) and the anterior auditory field (aaf) by blocking neural activities with the near-infrared laser irradiation (wave length = 830 nm). in previous in vivo studies, the laser irradiation could focally inactivate neural activities in a few minutes after the irradiation started, while the activities recovered in a few minutes after its cessation. by using this technique, the present study examined corticocotical relationships in the auditory cortex of the mongolian gerbils (meriones unguiculatus). cf (constant frequency) and fm (frequency modulated) tones were presented to anesthetized animals, and neural responses were extracellularly recorded contralaterally to the ear of stimulation. when irradiated aaf area and recorded neural responses from ai, the irradiation changed phasic responses into tonic responses, and vice versa. these results indicate that there are functional connections within ai or aaf, and between ai and aaf. takashi doi, hiroshi riquimaroux department of knowledge and engineering and computer sciences, doshisha university, kyoto, japan in a previous behavioral study, ablation of right auditory cortex (ac) made the discrimination between ascending and descending frequency modulated (fm) tones by mongolian gerbil (meriones unguiculatus) difficult (wetzel et al., 1998) . this result indicates that some neurons in gerbil's right ac represent the directions of fm sweeps. actually, we could find direction-dependent neurons and these neurons were mainly in anterior auditory field (aaf). in aaf, bfs are gradually shifted along the rostrocaudal direction, and the same bfs are arranged in dorsoventral direction (thomas et al., 1993) . moreover, aaf has dense synaptic connections within the area (budinger et al., 2000) . we made network models based on this structure of aaf and could gain similar responses to the actual responses of directiondependent neurons. this result suggests that aaf in gerbil's ac has good structure to process fm tones. research funds: a grant to rcast at doshisha univ. from mext, innovative cluster creation project by mext it has been demonstrated that the auditory space, namely the direction of a sound source, is represented topographically in the mammalian superior colliculus (sc). however, it is unclear as to how this auditory space map of the mammalian sc is formed in the auditory pathway. the present study investigated the topographical representation of auditory space in the external nucleus of the inferior colliculus (icx) of anesthetized gerbils. the icx is the major auditory nucleus that has projections to the sc. the stimuli were 50-ms noise bursts whose azimuths varied on the horizontal plane in the virtual acoustic space. single-unit responses were recorded from the icx. the majority of units exhibited some degree of spatial selectivity and preference for the azimuth contralateral to the recorded side. for supra-threshold stimulus only, there were topographical gradients of preferred azimuths in the icx. however, the spatial tuning width and preferred azimuth of the units depended markedly on stimulus level. the results indicate that in mammals, the formation of a rigorous auditory space map is incomplete at the icx level. manabu toyoshima 1 , yasuo takeda 2 , yasushi shimoda 1 , kazutada watanabe 1 1 department of bioengineering, nagaoka university of technology, nagaoka, japan; 2 department of clinical pharmacy and pharmacology, kagoshima university, kagoshima, japan nb-2 that we isolated and identified is a neural cell recognition molecule belonging to contactin subgroup. we reported previously that nb-2 expression is prominent in the auditory system. nb-2 knockout mice exhibit impaired neural function in the auditory system. these findings indicate that nb-2 is indispensable for the function of auditory system. here we report the detailed analysis of the nb-2 localization using anti-nb-2 monoclonal antibody that we produced recently. immunohistochemical analysis of the rat brain showed that nb-2 was detected not only in all brain regions of the auditory pathway, but also in substantia nigra (sn), caudate putamen (cpu) and fibers projecting from sn to cpu. the nb-2 immunopositive cells in sn are restricted to gabaergic neurons. since gabaergic neurons play essential roles in the development and function of the auditory system, it is highly likely that nb-2 regulates the development and/or function of gabaergic neuron in the auditory pathway. reiko nagashima, kiyokazu ogita department of pharmacology, setsunan university faculty of pharmaceutical sciences, osaka, japan sensorineural hearing loss can be caused by a variety of insults, including acoustic trauma. there is compelling evidence that reactive oxygen species (ros) are formed in the cochlea during acoustic stimulation. glutathione (gsh) protects against the hearing loss through scavenging ros generated by noise. in this study, we investigated the changes in expression of gamma-glutamylcysteine synthetase (gcs) gene, which is the rate-limiting enzyme in de novo gsh synthesis, in the cochlea following acoustic stimulation. nuclear extracts were prepared from the cochlea at various time points after acoustic stimulation (4 khz octave band, 125 db, 5 h), and then subjected to electrophoretic mobility shift assay to determine activator protein-1 (ap-1) dna binding. ap-1 binding was increased 2-12 h after the exposure. rt-pcr and immunostaining revealed that noise exposure was effective in elevating the expression of gcs in the cochlea 2 h later. taken together, ap-1 may participate in the expression of gcs gene in the cochlea after acoustic stimulation. masaharu kudoh, ryuichi hishida, katsuei shibuki 1 department of neurophysiology, brain research institute, niigata university, niigata, japan multiple formants compose vowels. we have previously reported that bilateral lesions including in the auditory cortex (ac) of rats impaired discrimination learning between synthesized vowel-like sounds with multiple formants, while discrimination between stimuli of a single formant or pure tones was not significantly impaired. in the present study, we determined the responsible auditory fields, which were required for the discrimination leaning between vowel-like sounds. water-deprived rats were trained to discriminate between two sounds including four different formants. licking a spout during presentation of one sound was rewarded with water while the other was not. surprisingly, local lesions in the primary ac or the ventral association cortex had no clear effect on the discrimination learning. in contrast, the dorsal association areas impaired the discrimination learning. these findings indicate that the dorsal auditory association cortex plays a critical role in discrimination learning of vowel-like sounds with multiple formants. hiroaki tsukano, yamato kubota, manavu tohmi, masaharu kudoh, katsuei shibuki department of neurophysiol, brain research institute, niigata university, niigata, japan we used transcranial flavoprotein fluorescence imaging for visualizing cortical responses to missing fundamentals in mice. c57bl/6 mice were anesthetized with urethane. the skull on the auditory cortex was exposed and covered with liquid paraffin to keep the skull transparent. cortical images of green fluorescence in blue light were recorded by a cooled ccd camera. responses in the auditory cortex elicited by sound stimuli (5-26 khz for 500 ms) exhibited mirrorsymmetrical tonotopic maps in the primary auditory cortex (ai) and anterior auditory field. the activity patterns in ai elicited by 5 khz were different from those elicited by 20 or 25 khz. however, the areas activated by 5 khz were also activated by the mixture of 20 plus 25 khz but not by that of 19 plus 26 khz, suggesting that cortical responses to missing fundamentals in ai were visualized using flavoprotein fluorescence imaging. hiroko kosaki national priting bureau, tokyo, japan we constructed a functional scheme of macaque auditory by distribution of calcium binding protein, parvalbumin (pv). auditory cortex is consisted of one core (primary cortex), and five surrounding rings, which correspond with secondary, tertiary, quaric, and quintic cortices. parvabumin showed a graduation, that is, inner core is most pv-rich, and outer rings showed the decrease of pv concentration. comparing with pv staining in visual cortex, these six-levels suggested similar hierarchic and reciprocal structure, which are proposed by deyoe and vanessen by analysis of feed-forward and feedback connections. akihisa kimura, tomohiro donishi, keiichiro okamoto, yasuhiko tamai department of physiology, wakayama medical university, wakayama, japan tonotopically comparable subfields of the primary and non-primary auditory areas in the rat cortex have similar topographies in the projection to the medial geniculate body but reverse topographies in the projection to the thalamic reticular nucleus (trn). in the present study, we determined how cortical and thalamic afferents intersect in the trn with regard to tonotopic organization. in light of the fact that a subset of auditory cells in the trn responds to visual or somatosensory stimulus, we also explored the potential sources of cortical and thalamic afferents that would set up polymodal sensory interaction in the trn. small injections of biocytin into the trn, which were made with guidance of electrophysiological recording of auditory response, resulted in retrograde labeling. retrogradely labeled cortical and thalamic cells exhibited distinctive patterns of distribution depending on the injection sites. the results indicate anatomical nodes in the auditory trn that would implement selective relay of auditory and/or polymodal sensory inputs. ps2a-f092 functional connections between the core and belt fields of the guinea-pig auditory cortex observed by optical recording and partial cortical inhibition using muscimol junsei horikawa, daisuke uchiyama, tatsunori matsui, shunji sugimoto department of knowledge-based information engineering, toyohashi university of technology, toyohashi, japan guinea pigs were anesthetized with ketamine and responses to puretones in the auditory cortex stained with a voltage-sensitive dye rh795 were recorded with a photodiode array. after determining the core (ai and dc) and belt fields, ai or dc was inhibited by putting a muscimol-containing agar piece on each field. the inhibition of ai resulted in reduction of responses in the belt fields by 55-90%, whereas the inhibition of dc resulted in reduction only by 20-50%. the reduction by ai inhibition was larger in the anterior and ventral belt fields and that by dc inhibition was larger in the posterior belt fields than in the other fields. further inhibition of dc after the ai inhibition or vice versa resulted in suppression of the responses in all the fields. these results suggest that the responses of the belt fields are elicited mostly via connections from the core to the belt fields and the belt fields receive differential connections from ai and dc. masataka nishimura, hiroyuki kaizo, wen-jie song department of electrical, electronic, and information engineering, graduate school of engineering, osaka university, suita, japan the auditory cortex of many mammals has a core area and surrounding belt regions. in guinea pigs, the primary auditory area, the secondary auditory area, and many belt regions have been reported. however, the activity of the belt regions has not been fully examined. using a high-resolution optical imaging system, we examined cortical responses to tone stimulations in anesthetized guinea pigs. the auditory cortex of six guinea pigs was exposed to the ventral end and stained with the voltage-sensitive dye rh-795. a novel field in the ventro-anterior region was identified based on its isolated responses to a pure tone stimulation and the relatively long latency of the responses. the field was located ventro-caudal to the primary auditory area, and was close to the ventral edge of the auditory cortex. we thus named the field as ventro-caudal field (vc). smooth frequency gradient was observed in vc in rostro-caudal direction, with the frequency axis in opposite direction to that of the primary auditory area. yoko kato 1,2 , kazuo okanoya 1 1 laboratory for biolinguistics, riken bsi, wako, saitama; 2 graduate school of humanities, university of chiba, chiba, japan bengalese finches sing complex courtship songs. to sing complex sequences, they require auditory feedback during singing. song nucleus nif has a projection from primary auditory area field l and then it projects to sensory/motor nucleus hvc. moreover, bilateral lesion of nif cause song deterioration on complex sequences (hosino and okanoya, 2000) . we recorded auditory responses by multiunit activity from nif and field l. auditory responses of nif showed selectivity to bird's own song (bos) than its reversed song (rev). comparing selectivity of nif and field l, nif showed stronger selectivity than field l. however, nif did not show sequence dependent selectivity. these results suggest that nif relays auditory information and enhances bos selectivity. however, we did not observe a direct evidence that nif related to generation of complex sequences. we started to record responses extracellularly from ac neurons of guinea pigs. in general, animals show a stereotyped pattern of behaviors; they have a quiet, almost-motionless period, usually for tens of min. during this period, animals do not appear to sleep but be sensitive to the environmental disturbance. thereafter they usually fall asleep with their eyes closed. during this presleeping period, the best frequency tone was repeatedly presented 100-500 times at a fixed interval, through a speaker at 50-70 db spl. responses to such repetitive tones are apparently irregular, with the occurrence of spikes in most trials but no spikes in some trials. however, if all the trials are accumulated, there was global phase alternation every a few to tens of seconds. one phase constitutes relatively high rates of spike occurrence, while the other very low rates of spike occurrence. we suppose that, unlike a machine, the brain has a unique mechanism that automatically turns on and off the cortical processing of the redundant sensory stimulus. masashi sakai, sohei chimoto, ling qin, yu sato department of physiology, university of yamanashi, japan a periodic click train produces a continuum of several perceptual qualities: (i) at low repetition rate (<10 hz), the individual clicks are clearly heard as discrete events so that the entire train produces "rhythm" percept, (ii) at high repetition rate (>100 hz), the entire train is heard as a single continuous event leading to a strong "pitch" percept, and (iii) in the transition range, the periodicity can still be detected as "roughness". we physiologically explored how those perceptual qualities are represented in the primary auditory cortex in awake cats. we found that distinct population of cells conducted two coding modes: (i) representing low-rate stimuli through stimuluslocking activity (i.e., temporal code) and high-rate stimuli as only onset responses or (ii) exhibiting sustained responses with generating larger amount of discharges at higher repetition rate (i.e., rate code). in addition, pure-tone stimuli elicited onset responses or sustained responses in each of these cell populations, respectively. we will discuss functional consequences and spike evocation mechanisms of each population. atsuhito toyomaki 1,2,3,4,5,6 1 hokkaido university, sapporo, japan; 2 hokkaido university, sapporo, japan; 3 sakushin gakuin university, utsunomiya, japan; 4 kobe shoin women's university, kobe, japan; 5 riken, wako, japan; 6 sakushin gakuin university, utsunomiya, japan gaps in a continuous sound play important roles for perception of voiceless consonants (i.e./k/,/p/,/t/) and japanese special mora (sokuon). we recorded auditory evoked responses to short gaps and tones from children (8-12 years old, n = 8) and adults (19-23 years old, n = 12). there were six gap conditions with durations of 8, 16, 32, 64, 128 and 256 ms embedded in a continuous tone and six tone conditions with the same durations. the frequency of all the tone was 500 hz. the responses elicited by the onset of gaps differed between the children and the adults: the responses in children were significantly larger and more sustained than those in adults for all the durations. in contrast, an n1 and p2 complex followed the onset of all the tones in all the subjects. thus development time course of neural process is conceivably different between gaps and tones. ps2a-f098 an fmri study on pitch control of voice using transformed auditory feedback method akira toyomrua 1 , tamaki miyamoto 2 , atsushi terao 2 , sachiko koyama 2 , takashi omori 2 , harumitsu murohashi 2 , shinya kuriki 2 1 jst, saitama, japan; 2 hokkaido university, sapporo, japan auditory feedback plays an important role in natural speech production. in the present study, we conducted an fmri experiment while subjects performed a transformed auditory feedback (taf) task to delineate the neural mechanism for control of pitch. the subjects were required to vocalize a and to hold the pitch of a feedback voice constant. in taf condition, the pitch was altered suddenly two or three times, whereas in non-taf condition the pitch was not modulated. under the taf condition, auditory feedback control is selectively expected to work more strongly than the non-taf condition. thus, a comparison between these conditions could neatly extract brain regions involved in auditory feedback control of pitch. as a result, right supramarginal gyrus, right frontal lobe (ba9), right anterior insula, left premotor area and right superior temporal gyrus showed greater activation (12 subjects, p < 0.05 corrected). this result suggests that auditory feedback of pitch is mainly controlled by the right hemisphere. sachiko koyama-takeichi 1 , yuko toyosawa 2 , fumiya takeuchi 3 , michinao matsui 4 , shinya kuriki 1 1 research institute for elecronic science, hokkaido university, sappro, japan; 2 jst, saitama, japan; 3 hokkaido university, school of medicine, japan; 4 kobe shoin institute for linguistic science, kobe, japan sounds with relatively long duration elicit a sustained component (slow field, sf). in the present study, we recorded cortical magnetic responses elicited by vowels and examined whether sf differs between native and non-native vowels (n = 11). four synthesized vowels were used as stimuli (stimulus duration 600 ms). two of the vowels (a, o) are native for japanese and the other vowels (ae, schwa) are not. two inter-stimulus intervals were used (1200/4800 ms). for the native vowels, an early sf (250-300 ms) was larger for the long than for the short interval session in both hemispheres. for the non-native vowels, the early sf was larger for the long than the short interval session only in the right hemisphere. neither an effect of interval nor hemisphere was significant for a late part of sf (400-600 ms) regardless of stimulus types. research funds: japan science and technology agent (brain sciences and education), kakenhi (16500166) ps2a-f100 spatio-temporal representation of frequencymodulated sounds in the auditory cortex revealed by optical imaging shunji sugimoto 1,2 , junsei horikawa 1 1 department of knowledge-based information engineering, toyohashi university of technology, toyohashi, japan; 2 riken brain science institute, wako, japan optical imaging (voltage-sensitive dye, rh 795) showed spatiotemporal response patterns for frequency-modulated (fm) sounds in the multiple fields of the guinea pig auditory cortex. an fm sound evoked a strong onset response spreading widely over the cortex, which was followed by a later response moving across the iso-frequency contours in the core fields. the location of the later response was corresponding roughly to the instantaneous frequency input of each fm sweep. on the other hand, a pure tone evoked a wide-spreading onset response followed by strong and long-lasting inhibitory effects. the later response to an fm sound appeared clearly when the frequency of the fm sweep was modulated over a wider range of frequencies, while it was diminished when the sound frequency was less modulated. these results imply that the cortical representation of such a later response contributes to a detection of frequency modulations in sounds. yamato kubota, kuniyuki takahashi, ryuichi hishida, masaharu kudoh, katsuei shibuki department of neurophysiology, brain research institute, niigata university, niigata, japan mitochondrial flavoprotein fluorescence is intimately coupled with energy metabolism. if the flavoprotein fluorescence is photobleached, energy metabolisms and neural activities can be inactivated. we applied this photo-inactivation technique to demonstrate auditory signal transmission from the anterior auditory field (aaf) to the primary auditory cortex (ai). cortical responses in aaf and ai after sound stimuli (5-20 khz) were visualized using transcranial flavoprotein fluorescence imaging in mice anesthetized with urethane. after determination of tonotopic maps, the auditory cortex was irradiated with strong blue light derived from a xenon lamp for 40 min, while the surface either aaf or ai was covered with a piece of carbon paper for preventing photo-inactivation. although photoinactivation of ai had almost no effect on the responses in aaf, photo-inactivation of aaf significantly reduced the responses in ai. these results suggest the presence of auditory signal transmission from aaf to ai. kousuke abe 1 , go ashida 1,2 , kazuo funabiki 2 1 graduate school of informatics, kyoto university, kyoto, japan; 2 hmro, faculty of medicine, kyoto university, kyoto, japan sound signals are translated to dispersed sporadic firing of the auditory nerves, and are converged to the third auditory station called the nucleus laminaris (nl) in birds. in vivo intracellular recording from owl's nl cells revealed that sound waveforms are observed in the postsynaptic membrane potentials (sound analogue potential; sap). we simulated synaptic inputs to the owl's nl neurons by recruiting the convergence of phase-locked excitatory inputs. several parameters such as the degree of phase-locking, the number of convergence and the time course of a unitary synaptic input affected the amplitude of sap, the amplitude of dc depolarization and the spectral features of synaptic noise in a complex manner. biophysical mechanisms for recreating sound waveforms by synaptic potentials will be discussed. takashi nihashi 1 , shigenori takebayashi 2 , masahiko bundo 2 , masazumi fujii 3 , toshihiko wakabayashi 3 , jun yoshida 3 , hiroyuki fujisawa 1 , kazunori ando 1 , kazumasa hayasaka 1 1 department of radiology, national hospital for geriatric medicine, obu, japan; 2 department of neurosurgery, national hospital for geriatric medicine, obu, japan; 3 department of neurosurgery, nagoya university school of medicine, nagoya, japan we identify si, using fmri in a routine scan for the patient who need a surgical approach. the activation of the brain with a tumor is complicated. we considered the pattern of the response. twelve patients were participated in this study. using 1.5 tesla mr imager, tactile stimulation was applied to bilateral palm, respectively. the statistical threshold was set for individual. contralateral activation on si was found in 11 out of 12 patients in the affected hemisphere. when region is near central sulcus, the multiple sites were activated. on the other hand, when the tumor is from central sulcus, the activation is simple: contralateral si. this method is useful to decide si in affected hemisphere in a short time. however, there are great inter-individual differences due to the locations of the tumor. takayuki iwano, shinya yamamoto national institute of advanced industrial science and technology (aist), neuroscience research institute, tsukuba, japan to examine how body surface with low spatial resolution is represented in the brain, we conducted a tactile identification task on toes. subjects (n = 8) lay on their backs with their eyes closed, and one of their toes was touched with a toothpick. the subjects were required to identify the toe by verbal response. the subjects responded correctly when the great or fifth toe was touched (cf. fein, 1987) . surprisingly, subjects tended to misidentify the second toe as the third (47.2%), and the third toe as the fourth (37.2%), while the reverse misidentification rarely occurred (third as second, 4.5%; fourth as third, 7.1%). this unidirectionality suggests that misidentification arises not only from large overlapping receptive fields associated with the toes, but from some additional factors such as a lack of experience with visuotactile integration, which could be used to reshape the toe receptive fields. ps2a-g105 effects of saccades on subjective temporal order of somatosensory signals toshimitsu takahashi 1,2 , shunjiro moizumi 1 , ayami okuzumi 1 , humine saito 1 , shigeru kitazawa 1,2 1 department of neurophysiology, juntendo university graduate school of medicine, tokyo, japan; 2 crest, jst, saitama, japan morrone et al. (2005) recently reported that subjective temporal order of two successive visual stimuli was reversed when the stimuli were delivered just prior to the onset of a saccade. in this study, we examined whether saccades affect temporal order judgments of tactile stimuli. right-handed subjects were required to make a visually guided rightward saccade (24 • ), and to judge the order of successive tactile stimuli that were delivered one to each hand at various timing relative to the onset of the saccade. with a stimulation interval of 100 ms, subjects generally judged the order correctly as long as the stimuli were delivered after the saccade. however, they often misreported (i.e., inverted) the order when the stimuli were delivered just prior to the onset of the saccade (within 300 ms). the results show that the reversal effect of saccades is multimodal and further suggest that multimodal brain areas are involved in ordering sensory events in time. ps2a-g106 function-directed organization of the postcentral somatosensory cortex representing oral structures takashi toda 1,3 , miki taoka 2,3 1 department neuroscience oral physiology, osaka university graduate school of dental sciences, suita, japan; 2 secondrary cognitives neurobiology, tokyo medical & dental university, tokyo, japan; 3 department physiology, toho university school of medicine, tokyo, japan the representation of oral structures in areas 3b and 1 of four conscious macaque monkeys was studied by recording single-neuron activities. a total of 115 electrode penetrations were made in areas 3b and 1. in 44 penetrations, pairs of adjacent neurons along the track had receptive fields (rfs) on continuous oral portions with or without overlapping, or otherwise on the same portion. in the remaining 71 penetrations, however, 14% of adjacent pairs (311/2153) had rfs on discrete but functionally-related sets of oral portions, e.g., the lip and tongue tip, the cheek mucosa and lateral margin of the tongue, the corresponding portions of the upper and lower lips, the corresponding portions of the palate and tongue, etc. we speculated that such an organization in areas 3b and 1 might be responsible for forming composite rfs of area 2 neurons. those composite rfs often covered discrete but functionally-related oral portions as reported earlier. research funds: kakenhi (12771111, 14771029) miki taoka 1 , michio tanaka 1 , hisayuki ojima 1 , atsushi iriki 2 1 secondrary cognitives neurobiology, tokyo medical and dental university, tokyo, japan; 2 laboratory of symbolic cognitive development, riken brain science institute, wako, japan we previously reported neuronal projections from the hand region of the second somatosensroy cortex (sii) to higher motor cortices (vetral premotor cortex etc.) suggesting that sii may be related to motor control of the hand movement. in the present study, we investigated the activities of sii hand neurons during voluntary movements. we recorded 168 neurons from two animals that were active when animals took small pieces of food by hands and put them into the mouth. among them (44% contra-, 55% bi-and 1% ipsilateral hand movements), we could determine receptive fields for only 43 neurons (25%). most of activities (138 neurons) were related to a certain phase of movements such as reaching, pinching a food piece, and putting it into the mouth. we found neurons showing phasic activities just before/after a certain phase, for example, just before pinching the object, or just after putting it into the mouth. those results suggest that sii hand neurons code the start or end of a certain act of hand. takahiro furuta 1,2 , kouichi nakamura 1 , takeshi kaneko 1 1 department of morphological brain science, graduate school of medicine, kyoto university, kyoto, japan; 2 crulrg, laval university, canada we investigated response properties of whisker-responsive neurons in the nucleus interpolaris (spvi) combining juxtacellular recording and a piezo-stimurator. the spvi is one of the first relay stations in the vibrissal system. rostral part of the spvi sends axons to the posterior thalamic nuclear group, whereas the caudal part of the spvi projects to the ventral lateral part of the ventral posterior medial nucleus. in the rostral part of the spvi, the vast majority of recorded neurons were multi-whisker responsive neurons, which are considered as projection neurons. in the caudal part of the spvi, about a half of neurons were mono-whisker-responsive neurons, which are thought as local circuit neurons. almost all neurons had angular tunings to upward deflection of whiskers in the rostral part of the spvi, while neurons in the caudal part of the spvi exhibited various angular tunings to all directions. these results indicate that the spvi could be divided into two sectors by response properties. seiji komagata 1,2 , shanlin chen 2 , hiroki kitaura 1 , masaharu kudoh 1 , minoru shibata 2 , katsuei shibuki 1 1 department of neurophysiology, brain research institute, niigata university; 2 department plastic surgery, school of medicine, niigata university, japan we visualized neural responses in the mouse somatosensory cortex using transcranial flavoprotein fluorescence imaging. mice were anaesthetized with urethane (1.7 g/kg, i.p.), and somatosensory responses were elicited by vibratory brush stimulation (50 hz for 1 s) applied to the left plantar forepaw. changes in green fluorescence in blue light were observed in the right somatosensory cortex. immediately after cutting the left median and ulnar nerves, the somatosensory responses were almost completely abolished. however, the responses appeared again within a few hours after the partial denervation, and almost complete recovery was observed a few weeks after the partial denervation. the recovered responses were eliminated by cutting the remaining radial and muscle cutaneous nerves. the rapid recovery of the responses observed in the present study may explain the mechanisms for allodynia and cortical plasticity in the somatotopic maps. shin-ya nakamura, takaaki narumi, ken-ichiro tsutsui, toshio iijima division system neuroscience, tohoku university graduate school of life science, sendai, japan in the rat somatosensory pathway, information received with a whisker is conveyed to the barrel cortex via trigeminal and thalamic nucleus mainly by two parallel pathways, the lemniscal and paralemniscal. the former includes the nucleus of trigeminal prv and thalamic vpm, which are known to contain neurons selective to the direction of whisker stimulation, and the latter includes trigeminal spvi and thalamic pom. in this study, we examined the specific involvement of the lemniscal pathway to the discriminative perception of whisker stimulus direction. rats were trained to perform a single-whisker directional discrimination task, and the task performance was evaluated before and after the selective electrolytic lesion or muscimol inactivation of each trigeminal and thalamic nucleus. the lesion or inactivation of prv or vpm significantly impaired the task performance, whereas those of spvi or pom did not. this result suggests the specific involvement of the lemniscal pathway in the single-whisker directional discrimination. kumiko yokouchi 1 , nanae fukushima 1 , tetsuhiro fukuyama 2 , akira kakegawa 1 , tetsuji moriizumi 1 1 department of anatomy, shinshu university school of medicine, matsumoto, japan; 2 department of pediatrics, shinshu university school of medicine, matsumoto, japan to know sensory cues for suckling behavior, rat pups of the suckling period received unilateral or bilateral resection of the infraorbital (io), mental (m) or lingual (l) nerves responsible for sensation of the upper and lower lips, and the tongue. for comparison, unilateral or bilateral removal of the olfactory bulb was done in these pups. the control, unilaterally io or m nerve-injured, and unilaterally bulbectomized pups showed successful suckling by their access to the mother's nipple, oral contact to it and long-lasting sucking. the bilaterally bulbectomized pups could not have access to the nipple. the bilaterally io nerve-injured pups could have access to the nipple with no oral contact, while the bilaterally m nerve-injured pups showed successful suckling. suckling behavior of the bilaterally l nerve-injured pups was characterized by frequent oral contacts for a very short duration, resulting in ineffective milk-intake. the results show fundamental roles of olfaction and sensation of the upper lip and the tongue in suckling. takahiro kawashima 1 , takeshi kawano 1 , hidekuni takao 1,2,4 , kazuaki sawada 1,2,4 , hidekazu kaneko 3,4 , makoto ishida 1,2,4 1 department electrical & electronic engineering, toyohashi university of technology, aichi, japan; 2 issrc, toyohashi university of technology, aichi, japan; 3 aist, hsbe, ibaraki, japan; 4 jst, crest, japan a si microprobe array (probe: au-coated recording site at the tip, 2 m in diameter, 60 m in length; array: 40-m pitch) to record neuronal activities has been developed by using selective si probe growth technique. to examine electrical properties of the array, single motor unit action potentials evoked by the electrical stimulation of the sciatic nerve of a rat were recorded in the left tibialis anterior muscle. signal-to-noise ratio of observed signals decreased with probe impedance, suggesting that lower impedance is better for recording small action potentials. however, lower impedance makes more difficult to record local signals, because signals observed at probes with low impedance were highly correlated (r = 0.91). to record local signals, it is necessary to decrease the area of the recording site of each probe at the expense of an increase in the impedance. research funds: kakenhi (17206038), the 21st century coe program "intelligent human sensing" ps2a-g113 a microelectrode positioning system for longterm extracellular recording of multiple neuronal activities hidekazu kaneko 1 , hiroshi tamura 2 , shinya s. suzuki 1 , takahiro kawashima 3 1 aist, hsbe, ibaraki, japan; 2 laboratory of cognitive neuroscience, graduate school of frontal bioscience, osaka university, osaka, japan; 3 department electrical & electronic engineering, toyohashi university of technology, aichi, japan a novel microelectrode-positioning system was devised that tracks a target neuron by countermoving a microelectrode against uncontrollable movements of brain tissue. the system automatically adjusted the position of a seven-core microelectrode such that the amplitude of each spike of a target neuron at the tip was the same as that at a lateral recording site (differential mode). the differential mode was compared with a conventional (peak-search) mode in which the spike amplitude of a target neuron at the tip was continually maximized. the differential mode was more stable to forced electrode movements and more sensitive to small displacements than the peak-search mode. furthermore, the differential mode enabled stable recording of not only spikes of a target neuron but also those of non-target neurons for over 2 h. seiji matsuda 1 , takehiro terashita 1 , tetsuya shimokawa 1 , kyoujy miyawaki 1 , yuji miguchi 1 , takuya doihara 1 , jie chen 1 , shuang-yan gao 1 , chun-yu li 1 , min wang 1 , zhong wang 1 , bing xue 1 , naoto kobayashi 1,2 , kazuhiro shigemoto 3 1 department anatomy, ehime university of medicine, ehime, japan; 2 medical education c, ehime university of medicine, ehime, japan; 3 department of hygiene, ehime university of medicine, ehime, japan this study shows the phylogenetic development of cajal's initial glomeruli (ig) and dogiel's pericellular nests (pcns) in the dorsal root ganglion of the healthy adult frog, chick, rat, and rabbit. the three-dimensional architecture of the neurons was observed in ganglia by scanning electron microscopy, after removal of the connective tissue. the proportion of neurons having ig or pcns increased with increasing phylogenetic complexity in the species examined here. in the chicks, the stem processes were longer and sometimes tortuous. typical ig were observed not in frogs or chicks, but in rats and rabbits. dogiel's pcns also have been observed in the drg of rats and rabbits. the nerve fibers in the pcns were less than 1.2 m in diameter and had some varicosities. some pcns contain tyrosine hydroxylase-positive nerve fibers and varicosities. masayo okumura, eiji kondo matsumoto dental university, institute for oral science, shiojiri, japan we established a rat nerve injury model using axotomy of the inferior alveolar nerve (ian), and investigated its effect on gene expression in the trigeminal ganglion. microarray analysis three days after surgery showed the up-regulation of some genes which are regulated by transcription factor stat3, whereas other genes known to be regulated by stat3 were not detected. stat3 is expressed in many tissues and plays various roles. however, there have been few reports about the role of stat3 in the peripheral nervous system, despite its welldocumented activation in the central nervous system after injury or stress. the aim of this study is to elucidate the role of stat3 in gene expression in the trigeminal ganglion after ian injury. at various time points, we analyzed and investigated changes of gene expression which are known to be influenced by stat3 and stat3phosphorylation, which indicates transcriptional activity, as well as cell types in which the genes and stat3 are expressed. these results should help us understand injury-induced change mechanisms of the peripheral nerve. hirofumi hashimoto 1 , susumu hyodo 2 , makoto kawasaki 1 , minori shibata 1 , takeshi saito 1 , hiroaki fujihara 1 , takashi higuchi 3 , yoshio takei 2 , yoichi ueta 1 1 department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; 2 laboratory of physiology, department of marine bioscience, ocean research institute, university of tokyo, japan; 3 department of integrative physiology, university of fukui, japan adrenomedullin 2 (am2) (identical to intermedin) belongs to the super family of am. centrally administered am and am2 activated oxytocin (oxt)-secreting neurons and increased plasma oxt level in rats. in the present study, we examined the effects of central administration of am2 on oxt-secreting neurons and sympathetic outflow in comparison with that of am in conscious rats. effects of central administration of am2 was stronger than those of am and the effects of am2 on oxt secreting neurons could not be blocked completely by pretreatment with cgrp or/and am receptor antagonists. these data suggested that am2 would have unknown receptor except cgrp and am receptor. arata oh-nishi 1 , makoto saji 1 , taku uchida 1 , sen-ichi furudate 2 , nobuyuki suzuki 1 1 division of brain science, kitasato university, graduate school of medical science, kanagawa, japan; 2 division of reproduction and fetal development, kitasato university, graduate school of medical science, kanagawa, japan the mechanism whereby neonatal hypothyroidism impairs cognitive function has not been well studied. in this respect, nmda receptors are thought to be crucially involved in cognitive and memory function. we have examined the effect of neonatal hypothyroidism and hyperthyroidism on the nmda receptor function, using rats treated with methylmercaptoimidazole (mmi), which specifically blocks the biosynthesis of thyroid hormone and mmi-treated rats injected with thyroxine, respectively. dose-response curves indicated that the sensitivity to nmda of the nmda receptors was significantly reduced in the hippocampus of the hyperthyroid rats, compared to that of normal and the hypothyroid rats. concomitant with this observation, western blot analysis showed that the nmda receptor subunit nr1 expression significantly decreased in the hippocampus of the hyperthyroid rats, compared to that of normal and the hypothyroid rats. our lab demonstrated that estradiol is endogenously synthesized within hippocampal neurons in the adult male rat (pnas, 2004) . here we report that the density and morphology of spines of pyramidal neurons in ca1 region are rapidly altered by treatments with nm estradiol and bisphenol a (xenoestrogen). hippocampal slices are incubated with estradiol or bisphenol a for 2 h, and then neurons were injected iontophoretically with lucifer yellow. three-dimensional imaging of neurons is performed by confocal laser microscopy, and the analysis of individual spines is performed by neurolucida software. the results showed that in ca1, both estradiol and bisphenol a induce a significant increase in the total spine density, especially the density of thin spine. synaptic plasticity of hippocampal neurons is demonstrated to be rapidly modulated by estrogen and xenoestrogen. we investigated the effects of stress on enhanced green fluorescent protein (egfp) expression in the arginine vasopressin (avp)-egfp transgenic (tg) rats. after bilateral adrenalectomy and intraperitoneal administration of lipopolysaccharide egfp fluorescences were increased in the parvocellular division of the paraventricular nucleus and the external layer of the median eminence. this tg rat is a convenient tool to study dynamic changes of avp expression in the hypothalamus under stressful condition. chitose orikasa, yasuhiko kondo, yasuo sakuma department of physiology, nippon medical school, tokyo, japan we report here a sex difference expression of somatostain mrna within the sexually dimorphic nucleus of the preoptic area (sdn-poa), the volume of which depends on gonadal hormones during the ontogeny. in infant rats aged day 8-15, the volume of somatostain mrna-positive region within the poa was significantly larger in males than in females and overlapped the sdn-poa in both sexes. the sdn-poa visualized by nissl staining in adjacent sections agreed precisely with the extent of somatostain mrna-positive cellular distribution. orchidectomy of males neonates and estrogen treatment of female pups reverse brain phenotypes when examines on day 15. the staining of somatostain mrna in individual neurons was diminished when examined on day 35 or 70, albeit that the sex difference of the volume of somatostain mrna-positive region persisted thoughtout the observed period. somatostatin may play a role in the establishment of the sdn-poa, which lacks classic nuclear receptor for estrogen. ps2a-g121 short chain sugar acid, 2-buten-4-olide, activates oxytocin-secreting neurons in the hypothalamus of rats makoto kawasaki 1 , tatsushi onaka 2 , hirofumi hashimoto 1 , hiroaki fujihara 1 , yoichi ueta 1 1 department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; 2 department of physiology, jichi medical school, tochigi, japan 2-buten-4-olide (2-b4o), an endogenous sugar acid, which may be involved in the regulation of feeding. we examined the effects of 2-b4o on the hypothalamo-neurohypophyseal system in rats. the plasma oxytocin (oxt) levels were significantly increased at 15-60 min after intraperitoneal (i.p.) administration of 2-b4o (100 mg/kg), whereas plasma arginine vasopressin (avp) levels did not change. dual immunostaining revealed that fos-like immunoreactivity (li) was predominantly observed in oxt-secreting neurons in the paraventricular and the supraoptic nuclei 120 min after i.p. administration of 2-b4o. in addition, many fos-li neurons were also observed in the nucleus of the tractus solitarius (nts) after i.p. administration of 2-b4o. these results suggest that peripherally administered high dose of 2-b4o activates oxt-secreting neurons in the hypothalamus through the activation of the nts neurons. ps2a-g122 estrogen receptor ␣ gene promoter activity is a marker for the sexually dimorphic nucleus of the preoptic area tomohiro hamada, yasuo sakuma department of physiology, nippon medical school, tokyo, japan the volume of the sexually dimorphic nucleus in the preoptic area (sdn-poa) is two to four times larger in male rat than in female, however function of this nucleus has not well known. in contrast, estrogen causes the sexually dimorphism by acting in perinatal periods. recently, transgenic rats expressing enhanced green fluorescent protein (egfp) under the control of an estrogen receptor (er) ␣ promoter were generated to tag er␣-positive neurons in the brain. in the present study, we examined gfp expression could be used a marker for the sdn-poa. gfp labeled cells were distributed in the core of sdn-poa of male and female transgenic rats and in the majority of these cells included er␣, immunohistochemically. both area and number of gfp expressed cells in the sdn-poa were larger in male than in female, however, female gfp cells in the sdn-poa showed concentrated distribution than male. these results suggest that gfp labeled cells in sdn-poa could be useful marker to make clear the function of the sdn-poa. recent studies on gonadal steroids imply that testosterone and estradiol are involved in learning and memory with modification of excitatory synapses in the hippocampus. although previous in vivo studies have demonstrated that these steroids increase the number of dendritic spines in neurons, it is still unclear whether each steroid has a direct effect on the modulation of the spatio-temporal patterns of dendritic morphogenesis. in the present study, we investigated steroid-induced morphological changes using cultured hippocampal neurons derived from neonatal or embryonic mice. the neurons were transfected with venus-actin. time-lapse images were taken by laser scanning confocal microscope during steroid treatment. testosterone but not estradiol increased the number of spines/filopodia of the dendrites within 4 h. these results obtained from in vitro studies suggested that testosterone affects dendritic morphogenesis of hippocampal neurons in short term. tetsuya kimoto 1,2 , shinpei higo 1,2 , yasushi hojo 1,2 , kouhei nakajima 1,2 , hironori nakanishi 1,2 , hirotaka ishii 1,2 , suguru kawato 1,2 1 department of biophysics & life science, university of tokyo, tokyo, japan; 2 crest, jst, japan hippocampus is one of the main target of sex steroids (androgen and estrogen) and stress steroids (corticosteroids). neuronal signal transmission in the hippocampus is modulated acutely by these steroids, and we recently demonstrated that the hippocampus of the adult male rat contained enzymes required for the synthesis of these steroids. however, the full diagram of hippocampal neurosteroid synthesis has not been obtained yet. in the present study, we therefore investigated the synthesis of sex steroids and corticosteroids in the hippocampus of adult male rats, by monitoring the metabolism of tritiated steroids with hplc system. ps2a-g125 gaba depolarizes gnrh neurons isolated from adult gnrh-egfp transgenic rats chengzhu yin, nobuyuki tanaka, masakatsu kato, yasuo sakuma nippon medical school, department of physiology, tokyo, japan gnrh neurons are essential in the reproductive neuroendocrine system. in regulation of gnrh neurons, gaba may be one of the major players, especially in relation to gnrh/lh surge. we, therefore, performed a cell-physiological analysis of gaba action on rat gnrh neurons. cells were dispersed from adult gnrh-egfp transgenic rats and cultured overnight. gnrh neurons, were applied to the perforated patch-clamp configuration with gramicidin d. gaba evoked cl − conductance, which was almost completely blocked by either picrotoxin or biccuculin. the reversal potential of the response was ranged from −40 to -10 mv in identified gnrh neurons in both sexes. there was no difference in the reversal potential among the stages of estrous cycle. in unidentified neurons, however, the reversal potential was more negative than -50 mv and most of them were ∼−80 mv. in conclusion, gnrh neurons isolated from adult rats express gabaa receptor and its reversal potential is more positive than the resting potential. although the neural activation in the subfornical organ (sfo) by angiotensin ii (angii) is widely regarded for the increments of angii-induced water intake and vasopressin release, galanin (gal) have been reported to inhibit them. therefore, gal may inhibit neural activity of angii-sensitive sfo neurons. rt-pcr analysis demonstrated existences of all mrnas of gal receptor subtypes, galr1, galr2 and galr3, in the sfo. in extracellular recording on sfo slice preparation, gal dose-dependently led to inhibition of neural activity. all gal sensitive neurons showed excitatory response by angii. galr1 selective agonist m617 induced inhibitory responses, as well as gal. in patch-clamp recordings, gal induced outward current in some neurons. these results suggest that gal inhibits neural activity in sfo neurons through, at least partially, outward current following activation of galr1. hiroaki fujihara 1 , tomoki fujio 1 , david murphy 2 , yoichi ueta 1 1 department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; 2 molecular neuroendocrinology research group, the henry wellcome laboratories for integrative neuroscience and endocrinology, university of bristol, bristol, uk we have generated transgenic (tg) rats expressing an arginine vasopressin (avp)-enhanced green fluorescent protein (egfp) fusion gene. in this study, we investigated the amount of drinking and food intake, the urinary output, the urine osmotic pressure, the urine sodium concentration and body weight after drinking 2% saline for 5 days in 3, 6, 12 and 24 months old tg rats. in 3 and 6 months, there were no difference between tg rats and tg(−) rats about the amount of drinking and food intake, the urinary output, the urine osmotic pressure, the urine sodium concentration and body weight under normal condition and salt loading. in aged tg rats (12 and 24 months old), there were no obvious changes in water balance. these results suggest that the expression of avp-egfp transgene does not disturb body fluid homeostasis in tg rats. ps2a-h128 prolactin-releasing peptide is a potent mediator of stress response in the brain through the hypothalamic paraventricular nucleus takashi mera 1 , hiroaki fujihara 2 , hirofumi hashimoto 2 , makoto kawasaki 2 , tatsushi onaka 3 , takakazu oka 1 , sadatoshi tsuji 1 , yoichi ueta 2 1 department of neurology (division of psychosomatic medicine), school of medicine, university of occupational and environmental health, japan; 2 department of physiology, school of medicine, university of occupational and environmental health, japan; 3 department of physiology, jichi medical school we examined the effects of restraint stress (rts), nociceptive stimulus and acute inflammatory stress on the prolactin-releasing peptide (prrp) gene expression in the hypothalamus and brainstem. moreover, we examined the effects of pretreatment with an anti-prrp antibody on nociceptive stimulus-induced c-fos gene expression in the hypothalamic paraventricular nucleus (pvn). rts, nociceptive stimulus and acute inflammatory stress upregulated the prrp gene expression in the brainstem. pretreatment with anti-prrp antibody significantly attenuated nociceptive stimulus-induced c-fos gene expression in the pvn. these results suggested that prrp is a potent and important mediator of stress response in the brain through the hypothalamic pvn. taieb bousejin 1 , afsaneh eliassi 1 , nasser naghdi 2 , ali ghanbari 1 1 ghsemi; 2 pastor institute, tehran, iran the purpose of this study was to consider the role of the ventromedial hypothalamus (vmh) d1 receptors on histamine-induced gastric acid secretion (gas). the animals were anasthetized and guide cannulas were implanted unilaterally above (0.5 mm) vmh. animals were anasthetized and two polyethylene tubes were introduced into the stomach through esophagus and pylorododenal junction. iv infusion of histamine in sham grup induced marked increase in gas with a peak response that started from 30 min up to the end of experiments (90 min). at the peak acid response, the vmh microinjection skf38393 (0.1,1) significantly reduced the amount of gas (p < 0.001). there was no any effect by microinjected sch23390 (0.1) into the vmh. injecting skf38393 into the vmh, 5 min after sch23390, had no effect on gas in compare with control. but, the acid suppressant effect of skf38393 was completely removed by peripheral injection of sch23390 (p < 0.01). our results show that the vmh d1 dopamine receptors have regulatory mechanisms of gas by interaction with h2 receptors through an inhibitory neural pathways. zhilin song, celia d. sladek department of physiology, uchsc, aurora, co, usa although prior studies demonstrated expression of p 2x purinoceptors in supraoptic neurons (son) and indicated their importance in atp stimulated vasopressin release, in studies monitoring the effect of atp on intracellular ca ++ ([ca ++ ] i ), we have obtained evidence that p 2y purinoceptors (p 2y r) are important in the response to atp. atp stimulated [ca ++ ] i increase was maintained in ca ++ -free medium and reduced by pretreatment with thapsigargin to deplete [ca ++ ] i stores. p 2y r agonists increased [ca ++ ] i in son, with p 2y1 r agonist being the most effective. the possibility that p 2y1 r mediates atp induced [ca ++ ] i increase in son was further evaluated using a p 2y1 r antagonist, mrs2179. atp stimulated increase in [ca ++ ] i was greatly attenuated by mrs2179 (100 m) in 2 mm ca ++ medium. in ca ++ -free medium, there was no significant response to atp in the presence of mrs2179. furthermore, combined treatment with mrs2179 and ppads (10 m, a p 2x r antagonist) also abolished the [ca ++ ] i response to atp. these results demonstrated that p 2y1 r mediates a large portion of the [ca ++ ] i response to atp challenge in son. ps2a-h131 analysis of the ontogenic expression of enzymes for brain neurosteroids in the male rat hippocampus hirotaka ishii 1,2 , yasuhiro sonoki 1,2 , aizo furukawa 2,3 , yasushi hojo 2 , tetsuya kimoto 1,2 , suguru kawato 1,2 1 department of biophysics and life science, university of tokyo, tokyo, japan; 2 crest, jst, japan; 3 kurihama national hospital, japan brain neurosteroids are steroids synthesized endogenously in the brain. our recent studies have demonstrated that the adult male rat hippocampus is equipped with a complete machinery for the synthesis of androgen and estrogen. to define the physiological role of brain neurosteroids in the hippocampal development and function, detailed information about the expression profiles of enzymes for brain neurosteroids in the hippocampus is essential. this study have comprehensively investigated the temporal patterns of enzymes for brain neurosteroids in the male rat hippocampus from postnatal day 1(pd1) to the adult stage using rt-pcr/southern blotting. enzymes required for the synthesis of estradiol from cholesterol were expressed form pd1 to pd14 with a higher level than in the adulthood. these results indicate that the rat hippocampus synthesizes estradiol more vigorously during the postnatal stage than in the adulthood, which may play an important role in the hippocampal development and function. hideo mukai 1,2 , gen murakami 1 , shirou kominami 3 , john h morrison 4 , william g.m janssen 4 , tetsuya kimoto 1,2 , suguru kawato 1,2 1 department of biophysics and life sciences, graduate school of arts and sciences, the university of tokyo, meguro, tokyo 153-8902, japan; 2 crest project, jst, japan; 3 faculty of integrated arts and sciences, hiroshima university, higashi-hiroshima 739, japan; 4 kastor neurobiology of aging laboratories, fishberg research center for neurobiology, usa estrogens elicit rapid non-genomic effects on the synaptic transmission, and spinogenesis in the hippocampus. however, the existence of estrogen receptor alpha (er␣) still remains elusive. with highly purified antibody rc-19, mass spectrometric analysis identified er␣ in the hippocampus and immunohistochemistry showed er␣ localization in principal neurons of ca1, ca3, and granule cells in dentate gyrus. further, western blot revealed that er␣ is contained in psd fraction, confirming the observation with immunoelectron microscopy. these results imply that the synaptic er␣ mediates the effects of estrogen in hippocampal neurons. ken takumi gnrh neuron is the key modulator of reproductive systems, directly regulating the synthesis and secretion of gonadotropins from anterior pituitary gland. gnrh neurons have been reported to be contacted by various neuronal systems, suggesting that the biosynthesis and release of gnrh is controlled by a complex of excitatory and inhibitory inputs. however, anatomical studies which quantified the direct input on gnrh neuron are few. in this study, we quantitatively analysed glutamatergic and gabaergic input onto gnrh neurons of the rhesus monkey by immunofluorescence method and confocal laser scanning microscopy; the close appositions between gnrh neuron and axon terminals immunoreactive for either vgluts or vgat were counted and the densities of the appositions on the dendrites and soma were calculated. sabine gouraud 1 , song t. yao 1 , jing qiu 1 , julian fr paton 2 , david murphy 1 1 university of bristol, hw-line, uk; 2 department of physiology, bristol heart institute, university of bristol, united kingdom the neuropeptide hormone vasopressin (vp) is produced in the magnocellular neurons of the hypothalamic supraoptic (son) and paraventricular (pvn) nuclei and stored in the posterior pituitary (pp). dehydration evokes an increased expression of the vp gene in magnocellular neurons and a massive release of vp from the pp in the circulation to promote the water conservation at the kidney level. in parallel, a functional remodelling of the hypothalamo-neurohypophyseal system (hns) is observed but poorly understood. we investigated this activity dependent plasticity of the hns using proteomic (2d fluorescence difference gel electrophoresis (dige)) combined with maldi mass spectrometry approaches to identify proteins that change in abundance in the son and the pp from 3 days dehydrated rats. a truncated form of prosaas, a granin-like neuroendocrine peptide precursor known as a potent inhibitor of the prohormone convertase 1, has been found decreased in the pp and increased in the son. ichiro nishimura, masakatsu kato, yasuo sakuma department of physiology, nippon medical school, tokyo, japan function of gonadotropin-releasing hormone (gnrh) neurons is regulated by gonadal steroid estrogen. however, the precise mechanism of estrogen action upon these cells has not been clarified. we investigated a direct action of estrogen on the regulation of potassium current in gnrh neuronal cell line gt1-7. delayed rectifier potassium current (i k ) and large-conductance calcium-activated potassium (bk) current were recorded by patch clamp configuration in gt1-7 cells cultured in dmem supplemented with 10% fbs for 3 days. bk current was increased by addition of 17␤-estradiol (e2) in culture medium in a physiological concentration range. this action of e2 was blocked by ici-182,780, a potent estrogen receptor (er) antagonist. we further examined whether e2 acted through er ␣ or er ␤ by using selective agonists ppt and dpn, respectively. the dpn augmented the bk currents similar to the effect of e2 but ppt had no effect. e2 had no effect on the i k . these results indicate that e2 increases the bk current by activating er␤ without affecting the i k . research funds: kakenhi 16590180, 16086210 ps2a-h136 myelin protein zero is one of the components of the detergent-resistant membrane microdomain fraction derived from rat pituitary katsutoshi taguchi 1 , haruko kumanogoh 2 , shun nakamura 2 , seiji miyata 3 , shohei maekawa 1 1 department of biosystems science, kobe-university, kobe, japan; 2 division of biochemistry and cellular biology, national institute of neuroscience, ncnp, tokyo, japan; 3 department of applied biology, kyoto institute of technology, kyoto, japan a membrane microdomain enriched in cholesterol and glycosphingolipids was found to contain many signal transducing and cell adhesion molecules. here, we studied the components of the membrane microdomain fraction derived from rat pituitary, and found specific enrichment of several proteins in this fraction. one of them, 25 kda protein, was identified as myelin protein zero (p0) from mass analysis and this result was confirmed by western blotting that a specific antibody to 25 kda band reacted to an authentic p0 prepared from rat sciatic nerve myelin. p0 is a type i transmembrane glycoprotein and a member of the immunoglobulin superfamily. the expression of p0 has been believed to be restricted to the peripheral myelin in mammals. our result, however, indicates that p0 expresses more widely and participates in cell communications. mari ogiue-ikeda, norio takata, suguru kawato department of biophysics and life sciences, graduate school of arts and sciences, university of tokyo, tokyo, japan 17␤-estradiol (e2) has a rapid effect on synaptic transmission. recently, we found that hippocampal neurons synthesize e2 (hojo et al., 2004) , and express estrogen receptor ␣ (er␣) at synapses. endocrine disrupters are representative estrogenic industrial compounds. while their disrupting effects on reproductive organs are well documented, their effects in the central nervous system are almost unknown. in this study, we investigated the effects of e2 and endocrine disrupters (des, bpa, np, op and tbt) on nmda-induced ltd in the rat hippocampal ca1, ca3 and dg with a custom made multi-electrode measuring system (med64). ltd was enhanced by e2 dose-dependently in ca1, ca3 and dg. des, bpa, np, op and tbt had similar or different effects on ltd dose-dependently. our results suggest that estradiol and endocrine disrupters rapidly modulate synaptic plasticity in the hippocampus and that the action of endocrine disrupters can be quantitatively analyzed by measuring the modulation of ltd of the hippocampal neurons. we examined the effects of chronic salt loading on the hypothalamic expressions of the green fluorescent protein (gfp), arginine vasopressin (avp) and oxytocin (oxt) genes and body fluid balance in avp-enhanced (e) gfp transgenic rats. chronic salt loading caused marked increase of the egfp fluorescence in the hypothalamoneurohypophyseal system in transgenic rats. there were no differences of the avp and oxt gene expressions in the hypothalamus, plasma avp and oxt levels and water balance between nontransgenic and transgenic rats under normal condition and after salt loading. humoral responses to chronic salt loading were maintained in avp-egfp transgenic rats. takeshi saito 1 , takushi x. watanabe 2 , tomoko urabe 2 , hirofumi hashimoto 1 , hiroaki fujihara 1 , yukio hirata 3 , yoichi ueta 1 1 department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; 2 peptide institute, inc., osaka, japan; 3 department of clinical and molecular endocrinology, tokyo medical and dental university, tokyo, japan salusin-␤ was newly discovered as a bioactive endogenous peptide. low concentration of salusin-␤ stimulates the secretion of arginine vasopressin (avp) from perifused rat hypophysis. salusin-␤ coexists with avp, but not oxytocin, in the rat magnocellular supraoptic (son) and paraventricular nuclei (pvn). to further investigate the physiological role of salusin-␤ in body fluid homeostasis, we examined the effects of salt loding for 5 days on salusin-␤-like immunoreactivity (li) in the son and pvn of rats by immunohistochemistry. the marked increase of salusin-␤-li in the son and pvn were observed in the salt loaded rats. the result suggests that salusin-␤ may play a role of body fluid balance by regulating avp release. naoyuki yamamoto 1 , hao-gang xue 1 , yuji ishikawa 2 , yoshitaka oka 3 , hitoshi ozawa 1 1 department of anatomy and neurobiology, nippon medical school, tokyo, japan; 2 national institute of radiological sciences, chiba, japan; 3 department of biological sciences, graduate school of science, the university of tokyo, tokyo, japan the terminal nerve gnrh (gonadotropin-releasing hormone) system, an extrahypothalamic peptidergic system, is thought to modulate neural circuitries involved in the control of motivational status for certain behaviors in teleosts. the major afferent source to the gnrh neurons is a midbrain nucleus, the nucleus tegmento-terminalis in percomorph teleosts, while a comparable nucleus appears to be missing in cyprinids teleosts. here, we examined the presence of such an afferent pathway in medaka oryzias latipes. injections of a dii crystal into the cluster of gnrh neurons resulted in labeled cells in the midbrain tegmentum, and injections to the midbrain tegmentum resulted in labeled terminals close to the gnrh neurons. these results suggest that the afferent pathway to the gnrh neurons is a character shared by "advanced" teleosts like medaka and percomorphs. takeshi yamazaki 1 , eiji munetsuna 1 , asuka kamogawa 1 , suguru kawato 2 , shiro kominami 1 1 graduate school of integrated arts science, hiroshima university of higashihiroshima, japan; 2 graduate school of arts and science, tokyou university of tokyo, japan tributyltin, tbt, an endocrine disruptor, induced increases in estradiol content in rat hippocampal slice culture. to analyze molecular mechanism of stimulation of estrogen synthesis, we determined mrna contents of estrogen biosynthetic enzymes and activity of p450arom in the hippocampus. method: the cultured hippocampus slices from 10 days male rat were treated with 100-1000 nm of tbt for 48 h. after the treatment, total rna was extracted and the levels of mrna of estrogen synthetic enzymes were quantified by real-time rt-pcr. activity of p450arom was determined by quantification of [3h]estradiol from [3h]testosterone. result: forty-eight hours treatment of hippocampal slices with 100 nm tbt induced increases in mrna contents of p450arom, and with 1000 nm tbt induced that of 3␤-hsd. estradiol content was increased by the treatment with 100 nm tbt, but not affected by 1000 nm tbt. tbt may modulate estradiol synthesis by alteration of expression of p450arom. the medial preoptic area (mpoa) is an important neural site for regulation and maintenance of sleep. studies have indicated that gabaergic neurons and terminals at the mpoa are active during sleep. present study was carried out to elucidate the contribution of gaba-a receptor at the mpoa in sleep-wakefulness (sw) in male wistar rats. the sw was assessed by chronically implanted electrodes for eeg, eog, and emg. a bilateral guide cannula was also implanted for drug injection into the mpoa. after recovery, three baseline sleep recordings were taken for 4 h on different days. bicuculline methoiodide (gaba-a receptor antagonist) at a dose of 30, 60, 90 and 120 ng in 200 nl was injected bilaterally into the mpoa in different groups of rats and their sw was studied for subsequent 4 h. the 30 ng dose of bicuculline methoiodide had minimum effect whereas 60 and 90 ng produced arousal. maximal wakefulness was observed at dose of 90 ng with no further increase in wakefulness at higher dose of 120 ng. the results suggest the involvement of gaba-a receptors at the mpoa in sw. yoshiaki isobe 1 , hiroyuki tsuda 2 1 department of neuro-physiology and brain science, nagoya city university, graduate school of medical sciences, nagoya, japan; 2 department of molecular toxicology, nagoya city university graduate school of medical sciences, japan locomotor activity in rodents shows free-running circadian rhythms even under the constant light. constant light exerts a promoting effect on hepatic carcinogenesis. after the partial hepatectomy, hepatic cell proliferation is regulated by circadian rhythm information (via wee1). to know the relation of proliferating factor (cell cycle) with circadian rhythmicity, locomotor activity against a diethylnitrosamine (den), widely used to initiate the hepatic neoplastic foci, is analyzed in preliminary. den was injected (i.p., 200 mg/kg) on rats during the free-running condition under the constant dim light (dd) and constant light (ll). the effects of den were gentle under the dd. however, under the ll, phase delay accompanying the elongation of circadian period () was observed. decrement of an amount of activity in 24 h after the den administration was obvious under the ll compared with that under the dd. this study was designed to investigate the central regulating system of hypothermia during maintenance phase of hibernation. although intracerebroventricular (icv) injection of naloxone (non-selective opioid receptor antagonist) and naloxonazine, (1 antagonist) were effective, naltrindole (␦ antagonist) and nor-bni ( antagonist) did not interrupt the hibernation. the increment of c-fos expression was observed in arcuate nucleus (arc) at 1 h after from hibernation onset compared with before hibernation. in addition, a localized ␤-endorphin-like immunoreactivity (␤-end ir) was observed in neuronal perikarya in arc at 1 h after from hibernation onset. although ␤-end ir in arc got weak, the ␤-end ir of nerve fibers in preoptic nucleus (pon) got strong with progression of hibernation. these results suggest that the ␤-endorphin was transported to pon from arc by axonal flow and then played an important role in maintenance of hypothermia via 1-opioid receptors in hibernation. wei-min qu, zhi-li huang, naomi eguchi, yoshihiro urade, osamu hayaishi department of molecular and behavioural biology, osaka bioscience institute, osaka, japan prostaglandin (pg) d 2 is a potent somnogenic substance, and isomerized from pgh 2 through the action of pgd synthase (pgds). pgds has two distinct types, the lipocalin-type pgds (l-pgds) and hematopoietic pgds (h-pgds). selenium compounds have been reported to decrease sleep by inhibiting pgds in rats. to clarify what type of pgds inhibition is involved in sleep reduction by selenium or whether selenium intoxication decreases sleep, we intraperitoneally injected secl 4 into l-pgds and h-pgds knockout (ko), and their wild-type (wt) mice. in wt mice, secl 4 decreased rapid eye movement (rem) and non-rem sleep for 5 h after injection and, concomitantly, increased wakefulness. similar results were observed in h-pgds ko mice. in contrast, l-pgds ko mice did not exhibit any significant changes in sleep-wake profiles after secl 4 administrations. these findings indicate that pgd 2 plays an essential role in the maintenance of the sleep state under physiological conditions, and l-pgds is a key enzyme for the production of pgd 2 involved in sleep-wake regulation. under baseline conditions, h 1 r ko mice showed essentially identical sleep-wakefulness cycles to those of wild-type (wt) mice but with fewer incidents of brief awakening (<16 s epoch), prolonged duration of non-rapid eye movement (nrem) sleep episodes, a decrease in the number of state transitions between nrem sleep and wakefulness, and a shorter latency for initiating nrem sleep after an intraperitoneal injection of saline. the h 1 r antagonist pyrilamine mimicked these effects in wt mice. these results indicate that h 1 r is involved in the regulation of behavioral state transitions from nrem sleep to wakefulness (huang et al., 2006) . ps2a-h147 dissociation of responsibility in firing activity to dim light between the optic nerve and the suprachiasmatic nucleus neuron of mice koichi fujimura, ai fukushima, takahiro nakamura, toshihiro jogamoto, kazuyuki shinohara division of neurobiology & behaviour, nagasaki university, graduate school of biomedical science, nagasaki, japan the involvement of light response in the optic nerve to the firing activity of suprachiasmatic nucleus (scn) neuron was investigated by extracellular single unit recordings from the optic chiasma and the scn in mice. recordings were carried out during the early night in a light:dark cycle, and the illuminations were applied to a contralateral retina with a high-power led (λ = 500 nm). the scn neurons responded to the light in intensities above 10 11 photons/cm 2 /s and were activated maximally at around 10 15 photons/cm 2 /s, they were about 1.6 log units less sensitive than optic fibers with high sensitivity. a sustained illumination in the intensity range between suprathreshold for the optic fibers and subthreshold for the scn neuron did not suppress the subsequent light response in the scn neurons, except in a few neurons. these results suggest that the most of the light responsive scn neurons are driven by any inputs independent of the high sensitive optic fibers. masayuki ikeda, tomoyoshi kojiya department of biology, faculty of science, toyama university, japan the hypothalamic suprachiasmatic nucleus (scn) has a pivotal role in the mammalian circadian clock. scn neurons generate circadian rhythms in action potential firings and neurotransmitter releases, and the core oscillation is thought to be driven by clock gene transcription-translation feedback loops. we have found robust circadian rhythms in the cytoplasmic concentration of ca 2+ in scn neurons. since cytosolic ca 2+ regulates diverse cellular systems, we have hypothesized that the cytosolic ca 2+ rhythms may mediate the cellular output from the clock gene oscillations. here, to address the clock gene functions on the ca 2+ rhythms, mouse bmal1 and its dominant negative sequence (mbmf1r5) are transfected into the organotypic culture of scn with a yellow cameleon ca 2+ sensor by the gene gun. the results demonstrated that over-expression of bmal1 or mbmf1r5 significantly inhibited the circadian ca 2+ rhythms and thus we concluded that the native bmal1 rhythm is essential for cellular output processes of the murine clock system. ps2a-h149 the activation of ␣2 adrenergic receptor increases the frequency of carbachol-induced ␤ oscillation in rat hippocampal slices masafumi nakano, jun arai, kiyohisa natsume kyushu institute of technology, kyushu, japan recently it is found that locus ceruleus (lc) activation suppresses ␤ rhythm in hippocampus in vivo. noradrenergic fibers derived from lc project to hippocampus. carbachol, a cholinergic agent, can induce ␤ oscillation in rat hippocampal slices like ␤ rhythm in vivo. in the present study, the effect of epinephrine on the generation of carbachol-induced ␤ oscillation in ca3 region of rat hippocampal slices. carbachol (30 m) induced ␤ oscillation with the frequency and the amplitude of 14.8 ± 0.1 hz, and 0.7 ± 0.1 mv, respectively (mean ± s.e.m.; n = 3). epinephrine (50 m) significantly increased the frequency of 16.2 ± 0.1 hz (**p < 0.01), not change the amplitude. clonidine (50 m), an ␣2 receptor agonist, alone significantly increased the frequency at the concentration of 50 m (*p < 0.05). yohimbine, an ␣2 receptor antagonist, suppressed the oscillation. these results suggest that the application of adrenaline will increase the frequency of hippocampal ␤ rhythm via ␣2 receptor. attractor dynamics of recurrent neural network are believed to play an important role in information processing in the brain. we recorded transient activities of two neuron groups by two tetrodes apart 0.4 mm from each other in the hippocampal ca3 region in vitro and applied micro-iontophoresis of glutamic-acid near the tetrodes to activate the neurons selectively. it was found that number of spikes during twosite (pairing) stimuli is fewer than the total number of spikes during the single-site stimuli, suggesting synaptic interaction in the network. peri-stimulus time histogram (psth) of ensemble as well as individual neuronal activity in response to the single-site stimuli applied far from the recording site composed of transient (latency 100-300 ms), oscillatory (2-4 hz) and sustained responses. following the pairing stimuli, the psth showed change in transient response properties (8/9 slices). these results suggest the pairing stimuli would change attractor dynamics of the neural network in the ca3 region. ryozo aoki 1 , hiroshi wake 2 , hitoshi sasaki 1 , kiyokazu agata 3 1 dept. physiol. & biosignal. osaka univ. grad. sch. med., suita, japan; 2 dept. elec. eng. & elec. col. industri. tech., amagasaki, japan; 3 dept. biophys., kyoto univ. grad. sch. sci., kyoto, japan by insertion of a stainless-steel monopolar electrode to the head of planarian, continuous waveform of electrical potential could be first observed in microvolts. the frequency spectrum showed an almost monotonously decreasing distribution likely as 1/f, ranging from 10 − 1 to 10 + 2 hz. during the eeg recordings the planarian was kept still by cooling in several degrees. when it was cooled down to lower temperatures the amplitude of eeg was suppressed, and by warming again restored with spikes provably due to motions. this eeg active state continued beyond 40 min after the electrode insertion but the amplitude gradually decreased, and became natural noise at the time up to 60 min. by observing the sample it turns out the sticked head was degraded. strong photo stimulation suppressed this eeg signals and recovered after over 30 min. however little response to light pulse stimuli was observed on the eeg spectrum. mariko uchida 1 , hiroki sato 1,2 , naoki tanaka 2 , atsushi maki 1,2 1 japan science and technology agency, crest, saitama, japan; 2 advanced research laboratory, hitachi, ltd., saitama, japan previous studies about electroencephalography (eeg) described that alpha-wave power (the frequency band from 8 to 12 hz) decreases and the sleep spindle power (from 12 to 16 hz) increases in falling asleep. the purpose of this study is to analyze the crosscorrelation between the eeg power changes (eegpc) of each band and the cortical hemoglobin concentration changes (hbcc) during sleep. we measured optical topography (ot) and eeg simultaneously. the hbcc was measured at eighty-eight positions covering whole head of subject by ot probes. five females and eight males participated in this measurement. the results showed the high correlation between eegpc and hbcc at the location of dorsolateral prefrontal area, both in the period of (i) dominance of alpha-wave and (ii) dominance of sleep spindle. the time lag from eegpc to hbcc was from 1 to 8 s in (i), and from 8 to 15 s in (ii). we examine these differences between (i) and (ii) in detail. carnitine deficiency disturbs fatty acid oxidation under the fasting condition (fc). we show herein that nocturnal locomotor activity (la) was reduced under fc and recovered to normal by carnitine injection in jvs −/− mice, a model of systemic carnitine deficiency. as judged from eeg/emg profiles, jvs +/+ mice showed prolonged wakefulness under fc, but jvs −/− mice revealed disruption of the prolonged wakefulness with a high frequency of non-rem sleep. as the orexinergic arousal system plays an important role in la, we determined orexin neuronal activity in the fasted mice. fasted jvs −/− mice had fewer c-fos + orexin neurons in their lateral hypothalamus and a reduced orexin-a content in their csf, suggesting that the fasted jvs −/− mice exhibited reduced la and fragmented of wakefulness due to suppressed orexin neuronal activity. juhyon kim 1 , kazuki nakajima 1 , yutaka oomura 2 , kazuo sasaki 1 1 div. of bio-information eng., univ. of toyama, toyama, japan; 2 dept. of integrat. physiol., kyushu univ., fukuoka, japan novel peptide, orexin, identified in the lateral hypothalamus (lh) participates in the regulation of sleep-wakefulness. orexin-containing neurons in the lh project to the pedunculopontine tegmental nucleus (ppt). the ppt is one of brain sites which control sleepwakefulness. thus, we examined effects of orexin on ppt neurons electrophysiologically using brain slice preparations in rats. applications of orexin depolarized the membrane potential of ppt neurons dose-dependently, and the depolarization was associated with the increase in membrane resistance. when extracellular k + concentration was increased, the magnitude of the depolarization significantly decreased. when extracellular na + was replaced by n-methyl-dglucamine, the magnitude of the depolarization also decreased significantly. these results suggest that the ionic mechanism for orexininduced depolarization includes k + channel, non-selective cation channel and/or na + /ca 2+ exchanger, and that orexin participates in the regulation of sleep-wakefulness via the excitatory effect on ppt neurons. ben-shiang deng 1 , wei zhang 1 , akira nakamura 2 , masashi yanagisawa 3 , yasuichiro fukuda 2 , tomoyuki kuwaki 1,2 1 dept. molec. integ. physiol., chiba univ., japan; 2 dept. autonom. physiol., chiba univ., japan; 3 dept. molec. genet., univ. texas, usa we examined whether the respiratory chemoreceptor reflex in prepro-orexin knockout mice (ko) was blunted or not, and if so, whether supplementation of orexin restored the abnormality. we also studied whether pharmacological blockade of orexin in the wildtype mice (wt) resulted in a similar abnormality. ventilation was recorded by whole body plethysmography before and after intracerebroventricular injection of orexin-a, -b, sb-334867 (an orexin receptor antagonist), or vehicle. data were examined for only awake periods because sleeping distorts the chemoreflex. hypercapnic ventilatory responses but not hypoxic responses were attenuated in ko. similar abnormality was reproduced in wt treated with sb-334867. icv injection of orexin partially restored the hypercapnic chemoreflex in ko. our findings suggest that orexin plays a crucial role for co2-sensitivity at least during waking periods. research funds: kakenhi 16590162,17590183 junko hara 1 , taizo matsuki 2 , katsutoshi goto 1 , masashi yanagisawa 3 , takeshi sakurai 1,2 1 department of pharmacology, basic medical science (coe), university of tsukuba, ibaraki, japan; 2 yanagisawa orphan receptor project, erato, jst, tokyo, japan; 3 howard hughes medical institute and department of molecular genetics, university of texas, dallas, texas, usa when the production of inflammatory cytokines is stimulated by acute inflammatory, the nonrem-sleep amount of animals increases. this is possibly due to changes in the biological activity of the tnfalpha system. besides their important function in sleep regulation during acute immune response, cytokines also seem to be involved in physiological sleep regulation. orexins (hypocretins) are recently identified neuropeptides that are derived from a common precursor peptide. recent studies suggest that specific degeneration of orexincontaining neurons occurs in brains of human narcolepsy patients, suggesting critical roles of these neurons in the regulation of vigilance states. here, we examined the effects of inflammatory cytokines on the activity of orexin neurons, by means of patch-clamp recording. these effects might also possibly be involved in the pathophysiology of narcolepsy. ps2a-i157 prenatal exposure to bisphenol a enhances avoidance response to predator odor and impairs sexual differentiation of olfactory response of medial amygdala neurons tetsuya fujimoto 1 , kazuhiko kubo 2 , shuji aou 1 1 dept. brain sci. eng., kyushu inst. technol., kitakyushu, japan; 2 dept. otorhinolaryngol., chidoribashi hospital, fukuoka, japan prenatal exposure to bisphenol a (bpa) impairs the sexual differentiation of exploratory behavior and enhances depressive behavior (fujimoto et al. 2006) . in this study, the effects of bpa on general motor activity and avoidance response to predator odor and olfactory responses in medial amygdala neurons were examined. the smell of fox predominantly suppressed locomotor activity and enhanced avoidance response by bpa. in the electrophysiological study, male medial amygdala neurons showed selective excitatory responses to predator odors. this type of neurons did not respond to plant odors. in contrast female amygdala neurons did not show such selectivity. the sex difference in this neuronal response pattern was attenuated by bpa exposure. these findings suggest that bpa impairs sexual differentiation of medial amygdala neurons which affect emotional responses to the olfactory cues of predators. research funds: grants-in-aid for scientific research (no. 16209006, s.a.) shuji aou 1 , tetsuya fujimoto 1 , yumi ichihara 1 , kimiya narikiyo 1 , toru ishidao 2 , hajime hori 2 , yukiko fueta 2 1 dept. brain sci. eng., kyushu inst. technol., kitakyushu, japan; 2 dept. environm. manage., sch. health sci., univ. occup. environm. health, kitakyushu, japan 1-bromopropane (1-bp), an ozone-depleting substance replacement, has neurotoxicity and exhibited reproductive toxicity in adult animals. in this study, we investigated the effects of prenatal exposure to 1-bp on sexual differentiation of reproductive and non-reproductive behaviors. pregnant rats were exposed to 700 ppm of 1-bp during prenatal period. the open-field test, lashley iii maze test and sexual behavior were evaluated at adult age. 1-bp significantly reduced the locomotor activity and the number of entries into the center area in female rats but not in males in the open-field test. in sexual behavior, the number of ear wiggles, an index of proceptive behavior, was decreased and the rejection score was increased in female rats. these results suggest that 1-bp is the potential candidate of endocrine disruptors which affect brain development. (16651027) ps2a-i159 changes in hippocampal excitability of rats prenatally exposed to 1-bromopropane yukiko fueta 1 , toru ishidao 1 , susumu ueno 2 , yasuhiro yoshida 3 , hajime hori 1 1 department of environmental management, school of health sciences; 2 department of pharmacology; 3 department of immunology, school of medicine, university of occupational and environmental health, kitakyushu, japan inhalation exposure to 1-bromopropane (1-bp), a substitute for ozone depleting compounds, alters the function of gabaergic system in the hippocampus of adult male rats. but the neurotoxcitiy induced by prenatal exposure has not been well investigated. in this study pregnant rats were exposed to 1-bp (700 ppm) during gestational day 1-20 (6 h/day), and the hippocampal excitability in pregnant rats and their offspring was examined. basic excitability was enhanced and disinhibition was observed in the hippocampus of pregnant rats. offspring, however, exhibited an enhancement of averaged s/r curve of ps in the ca1 at the pnd 12-15. conversely, s/r curves of fepsp as well as ps in the ca1 were inhibited at the age of 6-8 weeks. our results suggest that 1-bp causes hyperexcitability in pregnant rats, and disrupts basic excitability in the ca1 of the offspring during development. research funds: grant-in-aid for exploratory research (16651027) ps2a-i160 effects of endocrine disrupting chemical bisphenol a on the development of mouse cerebral cortex keiko nakamura 1,2 , kyoko itoh 1 , takeshi yaoi 1 , tohru sugimoto 2 , shinji fushiki 1 1 dept. pathol. appl. neurobiol., kyoto pref. univ. med, kyoto, japan; 2 dept. pediatr., kyoto pref. univ. med, kyoto, japan bisphenol a (bpa), a widely distributed xenoestrogen, has been shown to disrupt thyroid hormone function. we have thus studied whether prenatal exposure to low-doses of bpa affects morphology and the expression of thyroid hormone-dependent genes in murine fetal neocortex. pregnant mice were injected subcutaneously 20 g/kg of bpa daily from embryonic day 0 (e0). control animals were injected vehicle alone. for evaluating cell proliferation, neuronal differentiation and migration, bromodeoxyuridine (brdu) was given to pregnant mice and processed for immunohistochemistry. the total rna was extracted from embryonic telencephalons at different embryonic period. brdu-labeled cells were decreased in the ventricular zone at e14.5 and e16.5, whereas those cells increased in the cortical plate at e14.5, as compared with control mice. some of the genes associated with neurogenesis and thyroid hormone function were upregulated in bpa-treated group. research funds: jsps grant 15390334 keiko ikemoto 1 , teruko uwano 2 , hisao nishijo 2 , taketoshi ono 2 , masayuki ito 3 , ikuko nagatsu 4 , katsuji nishi 5 , shin-ichi niwa 1 1 dept. neuropsychiat., fukushima med. univ. sch. med.; 2 toyama med. pharm. univ.; 3 faculty med., mie univ., mie, japan; 4 fujita health univ. sch. med., toyoake, japan; 5 dept. leg med., shiga univ. med. sci., japan we examined the effect of maternal repeated cold stress (rcs) on development of catecholamine neurons of offsprings using by tyrosine hydroxylase (th) immunohistochemistry. rcs was loaded to pregnant rats between day 9 and 19 after fertilization. pups were perfused at postnatal day 8. in the frontal cortex, the number of largesized (more than 7 m in diameter) th-immunoreactive (-ir) varicosities was significantly smaller in prenatally rcs rats than controls. in the locus coeruleus of prenatally rcs rats, th immunoreactivity was less than that of controls. in the medullary c1/a1 catecholaminergic field, the size of th-ir neurons was smaller and the quantity of thir fibers were less in prenatally rcs rats, although there were not significant differences. it was suggested that prenatal rcs impaired development of catecholaminergic neurons, especially noradrenergic neurons of neonates. ps2a-i162 developmental exposure to pentachlorophenol affects thyroid hormone responsive gene in the brain but not stress response maiko kawaguchi 1,2,3 , kaori morohoshi 3,4 , rie yanagisawa 3 , erina saita 5 , gen watanabe 6,7 , masatoshi morita 3 , kazuyoshi taya 6,7 , hirohisa takano 3,4 , toshiyuki himi 1,2 , hideki imai 3,8 1 dept. toxicol and pharmacol., facul. pharmacy, musashino univ., tokyo, japan; 2 res. inst. pharmaceut. sci., musashino univ., tokyo, japan; 3 nation. inst. for environ. stud., ibaraki, japan; 4 grad. sch. environ. sci., univ. tsukuba, ibaraki, japan; 5 wildlife rescue veterinarian associ., tokyo, japan; 6 facul. agriculture, tokyo univ. agriculture & technol., tokyo, japan; 7 the united grad. sch. veterinary sci., gifu univ., gifu, japan; 8 div. environ. health sci., dep. social med., facul. med., miyazaki univ., miyazaki, japan antiseptic pentachlorophenol (pcp) treatment to rats affects thyroid hormone (th) system, which is essential for normal development of central nervous system. in this study, we show the exposure to pcp during gestation and lactation suppressed plasma th level, and induces gene expression of neurogranin and th receptor ␤, which play a role in neural formation. the present data suggest that pcp may affect central nervous system development, though stress response was not affected by pcp exposure. ps2a-i163 the effect of psychological stress during pregnancy on the open-filed behavior, the forced swim test, the fos expression in the brain, and the level of plasma corticosterone in offspring rat hiroshi abe, noriko hidaka, kei odagiri, yuko watanabe, yasushi ishida dept. of psychiatry, miyazaki med. coll., univ. of miyazaki, japan one group (psy) was born from the dams which observed, during their pregnancy, that another rat was exposed to the foot-shock stress in a communication box. the other group (c) was born from the dams not exposed to such stress. psy, comparing to c, showed decreased activities in the open-field test and prolonged immobility time in the forced swim test. on the other hand, there were no significant differences between the number of fos immunopositive cells in various regions of the brain in two groups before and after the foot-shock. however, plasma corticosterone was elevated in psy compared with c. these results suggest that the prenatal psychological stress might enhance reactivity to novel environment and depressive behavior induced by forced swim, and chronically elevated level of corticosterone might be involved in this neurobiological substrate. akane nakasato, yasushi nakatani, yoshinari seki, hideho arita department of physiology, toho university school of medicine, tokyo, japan to evaluate roles of da and serotonergic (5-ht) systems in stressinduced anxiety, we measured brain da and 5-ht levels before, during and after a forced swimming test (fst) in autistic model of the rat. the model rat was made by exposing a pregnant rat to valproic acid (vpa). our previous study demonstrated that the autistic model exhibited abnormality of 5-ht system and behavioral impairments related to autism. in the present experiment, we gave a prolonged fst for 60 min in the model rat, which frequently experienced to be drowned after the immobility time during fst. brain da and 5-ht levels were measured from samples collected from the prefrontal cortex (pfc). we found a gradual and steady increase in pfc da level during fst, although 5-ht level showed only transient augmentation. behavioral alteration after fst was characterized by an increased appetite during light phase (sleep) of circadian cycle. we suggest that the feeding abnormality may be caused by the stress-induced anxiety mediated by mesocortical da system. shigeo masaki, eiko aoki, satoshi yonezawa, atsuo nakayama dept. embryology, inst. developmental res., kasugai, aichi, japan neuroligin (nl1-4) is a family of neuronal cell-surface proteins to be involved in intercellular junctional formation and signalling. recently, several studies have implicated nl3 and nl4 in autistic disorders. nls have a relative identical structure (∼70%); nl1 and nl2 localize in the glutamatergic excitatory, and inhibitory synapses, respectively, while nl3 seems express in the olfactory glia, but nl4 distribution is unknown. here we have generated antibody against human nl4, and explored its distribution in the post mortem human tissues. in the central and peripheral nervous system, nl4 was expressed exclusively in the neurons, and was especially abundant in particular subsets of neurons, including neurons producing nonapeptides. nl4 was observed in paraneurons and some endocrine cells outside the cns. these results suggested that nl4 is important for neuroendocrine function. nl4 cdna was transfected to in neuroblastoma. formed spine-like structures on the cells expressing nl4 were rough and thicker than those of nl1 or nl3 transformants. it suggested the unique activity of nl4 for synapse formation. motopsin is a serine protease secreted from pyramidal neurons of cerebral cortex and hippocampus. recently, the truncation of motopsin gene has been reported to cause non-syndromic mental retardation. however, the underlying mechanisms are yet to be elucidated. we report here that the knockout (ko) mice deficient in the expression of motopsin exhibit morphological abnormality. golgi-cox staining revealed that spine density on both apical and basal dendrites of hippocampal ca1 pyramidal neurons in the ko mice significantly decreased than in the wild type mice. similarly, spine density tended to decrease at cingulate cortex of the ko mice than wild type. our results suggest that motopsin affects dendritic spine formation and/or stabilization. mental retardation is a frequent disorder affecting 1-3% of the population. recently the truncation of motopsin/neurotrypsin gene has been identified in algerian family in which four out of eight children affected by a severe impairment of cognitive functions with an iq below 50. here we report that knockout (ko) mice lacking motopsin gene mildly impaired water maze performance and social behavior. the ko mice significantly delayed the latency to the platform area on a probe test of hidden version of morris water maze although they showed the similar performance to wild-type mice during training session. in a social memory test, the ko mice showed significant elongation of sniffing time to an intruder, despite of normal performance of social memory. our results suggest that the ko mice provide insights into the molecular mechanisms important for development of cognitive functions. natsue yoshimura 1 , daisuke horiuchi 1 , tomoyuki miyashita 2 , minoru saitoe 2 , hitoshi okazawa 1 1 department of neuropathology, medical research institute, tokyo medical and dental university, tokyo, japan; 2 tokyo metropolitan institute for neuroscience, tokyo, japan polyglutamine tract binding protein-1 (pqbp-1) was originally isolated as one of the candidates for polyglutamine disease related protein. recently, several groups has reported about pqbp-1 disease that pqbp-1 mutant causes x-linked mental retardation (xlmr). to investigate the function of pqbp-1 in xlmr pathology, we produced two kinds of flies, human pqbp-1 overexpression flies (hpqbp1 flies) and drosophila pqbp-1 knock-down flies (dpqi flies), and examined olfactory learning and memory to analyze their memory consolidation process from short-term memory (stm) to long-term memory (ltm). the hpqbp1 flies showed memory impairment in ltm. in current study, we analyze memory abilities of the dpqi flies to observe detailed function of pqbp-1 in memory formation. seiji hayashizaki, masahiko takada tokyo metropolitan institute for neuroscience, usa when two alternatives are available in instrumental behavior, animalǐs behavior is biased toward responding on one lever with which each behavioral response results in delayed large reward delivery, and against responding on the other lever with which each response results in immediate but small reward delivery. this has been used as an index of impulsive behavior and is known to be susceptible to lesions of brain structures such as the basolateral amygdala (bla) and the nucleus accumbens (na). it has been shown that the bla and na are involved in maintaining reward seeking behavior with a secondary reward when a secondary reinforcer is available. thus, a question arises as to how the behavioral response on the delayed lever is maintained through functions exerted by these structures when no secondary reinforcer is available. to this end, we implanted cannulae bilaterally and electrodes into the bla and na to identify neuronal substances and activities involved in the mediation of 'putative secondary reward' without secondary reinforcer. xue-zhi sun 1 , sentaro takahashi 1 , yoshihisa kubota 1 , rui zhang 2 , chun cui 2 , yoshihiro fukui 2 1 natl. inst. radiol. sci., chiba, japan; 2 sch. med. tokushima univ., tokushima, japan heavy ion irradiation has the feature to administer a large radiation dose in the vicinity of the endpoint in the beam range, and its irradiation system and biophysical characteristics are different from ordinary irradiation instruments like x-or gamma-rays. using this special feature, heavy ion irradiation has been applied for cancer treatment. the safety and efficacy of heavy ion irradiator have been demonstrated to a great extent. for instance, brain tumors treated by heavy-ion beams became smaller or disappearance. however, fundamental research related to such clinical phenotypes and their underlying mechanisms are little known. in order to clarify characteristic effects of heavy ion irradiation on the brain, we developed an experimental system for irradiating a restricted region of the rat brain using heavy ion beams. the characteristics of the heavy ion beams, histological, behavioral and elemental changes were studied in the rat following heavy ion irradiation. yukio imamura department of psychiatry, university of ottawa, on, canada nmdars contain two nr1 subunits paired with two nr2 subunits. nr1 and nr2 (a-d) subunits harbor the glycine and glutamate binding sites, respectively. nmdars are localized in both synaptic and extra-synaptic areas, but they are found at higher density within the synapse. after the peak of synaptogenesis, the nr1/nr2a complex, characterized by rapid offset kinetics, dominates at the synapse, while the nr1/nr2b complex, characterized by slow kinetics, predominates in the extra-synaptic area. the activation of extrasynaptic nmdars by glutamate escaping from the synaptic cleft during episodes of high synaptic activity suggests that they may have a different role. using whole-cell voltage-clamp recordings from ca1 pyramidal neurons from mice (at 12 weeks of age), we found that following induction of ischemia, ifenprodil, a selective nmdar-nr2b antagonist, reduced the inward current of the isolated nmdar at extra-synaptic site while it had less effect at the synaptic nmdar. the molecular mechanisms involved are currently under investigation and these new data will be also presented at the meeting. in the present study, we observed expression and changes of mineralocorticoid receptor (mr) and glucocorticoid receptor (gr) in the gerbil hippocampal ca1 region after ischemia. in blood, corticosterone levels increased biphasically at 30 min and 12 h after ischemia, and thereafter its levels decreased. in the sham group, mr and gr immunoreactivities were weakly detected in the ca1 region. by 3 days after ischemia, mr and gr were not significantly altered in the ca1 region. from 4 days after ischemia, mr and gr immunoreactivities were detected in astrocytes and microglia in the ca1 region, and at 7 days after ischemia. the specific distribution of corticosteroid receptors in glia may be associated with the differences of mr and gr functions against ischemic damage. the present study was investigated the effects of early treadmill training after cerebral infarction in rats. we determined whether treadmill exercise changes cellular expression of caspase-3 and midkine in the mca area. stroke was induced by a 90-min mca occlusion using an intraluminal filament. rats were exercised for 20 min each every day on a treadmill. brain damage in ischemic rats was evaluated by infarct volume. exercised and non-exercised rat brains were processed for immunocytochemistry to quantify the areas of caspase-and mkimmunoreactive calls. no significant differences in infarct volume were found between rats trained with treadmill and non-exercised controls. cellular expressions of mk were significantly increased in striatum (glia) of the exercised rats. treadmill exercise was shown to suppress the decrease in caspase-3 expression in the penumbra. the present study showed the exercise after cerebral infarction might have important implication for post-ischemic recovery. ps2a-j174 reversed astrocytic glutamate transporter glt-1 crucial to the ca 2+ paradox-like insult-induced neuronal death in neuron/astrocyte co-cultures tatsuro kosugi, koichi kawahara, takeshi yamada, motoki tanaka lab. of cellular cybernetics, graduate school of information science and technology, hokkaido univ., sapporo, japan "ca 2+ paradox" is the phenomenon whereby the intracellular concentration of ca 2+ paradoxically increases during reperfusion with normal ca 2+ -containing media after brief exposure to a low ca 2+ solution. the present study aims to characterize the ca 2+ paradoxinduced cell injury in neuron/astrocyte co-cultures. prior exposure of the cultures to a low ca 2+ solution for 60 min significantly injured only neurons after reperfusion with a normal ca 2+ medium for 24 h, but astrocytes remained intact. after the onset of reperfusion, the intracellular concentration of na + in astrocytes increased significantly during the reperfusion episode, resulting in a reversal of the operation of the astrocytic glt-1. the present findings suggested that ca 2+ paradox-induced accumulation of na + in astrocytes was involved in the reperfusion-induced excitotoxic neuronal injury resulting from the reversed operation of astrocytic glt-1 during the reperfusion episode. common genetic mutation in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (cadasil) has been associated with missense mutations of notch3 concerning cysteine residues within the extracellular amino-terminal region. we report new mutations of two japanese cadasil families, which did not directly involve a cysteine residue. exons of the notch3 were amplified by pcr and subsequently analysed for dhplc and direct sequence. the first patient carried the missense mutation c577t, which results in pro167ser. the second patient carried the missense mutation c302g, which results in arg75pro. new mutations had not changed the number of cysteine residues, but coding the extracellular amino-terminal region of the notch3 receptor which may involve an alteration in the ligand binding or putative dimerisation properties. ps2a-j177 mci -186, a radical scavenger, protected cortical neurons from cell death through the activation of mitogen-activated protein kinase and phosphatidylinositol 3kinase madinyet niyaz 1 , tadahiro numakawa 2 , yoshinori matsuki 1 , emi kumamaru 2 , yuki yagazaki 2 , harumi kitazawa 1 , hiroshi kunugi 2 , motoshige kudo 1 1 pathology department of tokyo medical university, tokyo, japan; 2 department of mental disorder research, national institute of neuroscience, ncnp, tokyo, japan the role of mci -186, a radical scavenger, in the central nervous system (cns) has not been fully elucidated. in the present study, we found that treatment with mci -186 prevented the cultured cortical neurons from cell death induced by serum deprivation. furthermore, we found that mci -186 exposure induced the activation of both the map kinase (mapk) and pi3 kinase (pi3k) pathways and that the mci -186-dependent survival effect was blocked by the inhibitors, u0126 (an mapk pathway inhibitor) or ly294002 (a pi3k pathway inhibitor). these results suggested that mci -186 exerts a protective effect on cns neurons via enhancing survival-signaling pathways in addition to a role such as a radical scavenger. osamu tokumaru 1 , noriko yoshimura 1 , tetsuro sakamoto 1 , takaaki kitano 2 , naoko nisimaru 1 , isao yokoi 1 1 dept. physiol., sch. med., oita univ., japan; 2 med. edu. ctr., sch. med., oita. univ., japan protective effects of ethyl pyruvate (ep) on energy metabolism of rat brain exposed to ischemia were investigated by 31 p-nuclear magnetic resonance (25 • c). brain slices were incubated in standard artificial cerebrospinal fluid (acsf) with 2 mm ep (ep-1), acsf replaced by acsf with 2 mm ep after ischemia (ep-2), or acsf only (control). the brain slices were exposed to ischemia by stopping the perfusion for 1 h. high-energy phosphate, creatine phosphate (pcr) and ␥-atp, levels were measured. decrease in pcr level was not different among the three groups when exposed to ischemia. but increase in pcr level after the reperfusion was significantly larger in ep-1 than in control (p < 0.01). these results indicate that ep is effective in the reperfusion period and is more protective when administered before ischemic exposure. the importance of timing of administration of ep in clinical use was suggested. research funds: grant-in-aid for scientific research (c) #17591639 from mext to t.k. hideaki tamai 1 , kuniko shimazaki 2 , norimasa seo 1 1 department of anesthesiology and critical care medicine, jichi medical university, graduate school, tochigi, japan; 2 department of physiology, jichi medical university, tochigi, japan we investigated the effects of acupuncture on cell proliferation in the dentate gyrus (dg) and the lateral ventricle (lv) of adult rats. in this study, acupuncture was performed at the acupoints neiguan (pc6), yintang (ex-hn3) and sanyinjiao (sp6), which have been used for the enhancement of conscious and functional recovery in stroke patients. eight weeks old male wistar rats were used in the experiment. through 5-bromo-2,-deoxyuridine (brdu) immunohistochemistry, a significant increase in cell proliferation in the dg of the acupunctured group was observed. however, the cell proliferation in the lv was not affected with the acupoints pc6, ex-hn3 and sp6. the present findings indicate that the sensitivity on cell proliferation in the dg by acupuncture stimulation is higher than in the lv. yukio ago 1 , keiko takahashi 1 , shigeo nakamura 1 , akemichi baba 2 , toshio matsuda 1 1 laboratory of medicinal pharmacology, graduate school of pharmaceutical sciences, osaka university, osaka, japan; 2 laboratory of molecular neuropharmacology, graduate school of pharmaceutical sciences, osaka university, osaka, japan this study examined the effect of isolation rearing on anxiety-related behavior of mice in the staircase test, an animal model of anxiety. the staircase test consisted of placing an experimentally naive mouse in an enclosed staircase with five steps. in group-reared mice, an anxiolytic diazepam increased the number of steps climbed to the top step of the staircase, but did not affect the frequency of rearing behavior. the anxiogenic drug ␤-cca increased the number of rearing, but did not affect the number of steps climbed. on the other hand, methamphetamine increased the number of steps climbed to the second step. in these circumstances, isolation-reared mice showed an increase in the numbers of steps climbed to the top step and rearing in the staircase. these findings suggest that isolation rearing increases in exploratory and anxiety-like behaviors in mice. tomonori fujiwara 1 , tatsuya mishima 1 , takefumi kofuji 2 , kimio akagawa 1 1 department of cell physiology, kyorin university school of medicine, mitaka, tokyo, japan; 2 radio isotope laboratory, kyorin university school of medincine, mitaka, tokyo, japan hpc-1/syntaxin 1a is believed to regulate the exocytosis of synaptic vesicles. in order to examine the neurophysiological function in vivo, we have produced hpc-1/syntaxin1a knock-out mice. surprisingly, the null mutant mice revealed normal development and basal synaptic transmission in cultured hippocampal neurons appeared to be normal. however, in conditioned fear memory test, consolidation of the memory was impaired in homozygous mutant mice but not in heterozygote. however, once memory consolidation was acquired, the extinction process was disturbed in homozygote. we further examined latent inhibition of cued fear memory (li) to access behavioral property. interestingly, li was suppressed both in heterozygous and homozygous mutant mice unlike the case of conventional conditioned fear memory test. implication of these behavioral abnormalities in hpc-1/syntaxin1a knock-out mouse will be discussed. research funds: kakenhi (15700292) ps2a-k182 effects of local administration of the gaba agonists into the hippocampus ca1 area on active avoidance learning and serotonergic systems in the administration area in rats satoko hatakenaka 1 , hiroko miyakubo 1 , junichi tanaka 1 , yasushi hayashi 2 , yukio hattori 2 , masahiko nomura 3 1 department of curriculum, teaching and memory, naruto university of education, tokushima, japan; 2 department of human nutrition, notre dame seishin university, okayama, japan; 3 department of physiology, saitama medical school, saitama, japan in fischer 344 male rats, bilateral injections of the ␥-aminobutyric acid (gaba) a agonist muscimol into the ca1 area slightly decreased the avoidance rate in an active avoidance task. similar injections of the gaba b agonist baclofen enhanced the avoidance rate. there are significant differences between the muscimol-and baclofen-treated groups in the avoidance rate, implying that gaba a and gaba b receptors have the opposite action on the performance of avoidance learning. perfusion with muscimol through the microdialysis probe decreased the serotonin metabolite 5-hydroxytryptamine (5-hiaa) concentration in the ca1 area, whereas baclofen perfusion had no effect, suggesting that the gabaergic system may exert to inhibit the serotonin release in the ca1 area through gaba a receptors. sawako arai, taku nagai, kenji takahashi, hiroyuki kamei, kazuhiro takuma, kiyofumi yamada lab. neuropsychopharmacol, kanazawa univ., kanazawa, japan we performed immunohistochemical c-fos mapping after a prepulse inhibition (ppi) test of the startle reflex in mice. startle stimulus increased the number of c-fos-positive cells in the somatosensory cortex, nucleus accumbens shell and the caudal pontine reticular nucleus (pnc), while prepulse trials without startle stimulus increased c-fos expression in the lateral globus pallidus (lgp). in mice subjected to startle stimulus with prepulses, most of the startle stimulus-induced c-fos expression was diminished but c-fos expression remained in the lgp. prepulse-induced c-fos expression in the lgp was colocalized with gad-67. fluoro-gold infusion into the pnc and the pedunculopontine tegmental nucleus (pptg) retrogradely labeled neurons in the pptg and lgp, respectively. microinjections of phaclofen, but not picrotoxin, into the pptg impaired ppi of the startle reflex. these results suggest that gabaergic neurons in the lgp which project to the pptg play a crucial role through the activation of gaba b receptors in the ppi of the startle reflex. shiho kitaoka 1 , sho koyasu 1 , akinori nishi 2 , tomoyuki furuyashiki 1 , toshiyuki matsuoka 1 , shuh narumiya 1 1 department of pharmacology, university of kyoto, kyoto, japan; 2 department of physiology, university of kurume, kurume, japan prostaglandins e2 (pge 2 ) exert their actions in various organs through specific receptor, ep1 to 4. the previous study suggests that ep1 modulates da system. to investigate the roles of ep1 in da system, we examined ep1ko mice with behavioral sensitization induced by cocaine. the administration of cocaine elevated da concentration in the nucleus accumbens up to ∼300% in both wild-type and ep1ko mice. however, increase of locomotor activity in ep1ko mice was significantly lower than that in wild-type mice. because locomotor activity is closely related to dopamine d1 receptor (d1r) signaling, we tested the density of d1r and d1r signaling with phosphorylation of darpp-32. there were no differences in d1r binding. d1r signaling was significantly attenuated in the striatal slices from ep1ko mice. the effect of d1r agonist on locomotor activity was also attenuated in ep1ko mice. these results indicate that pge 2 has enhancing effects on locomotor activity via ep1 by potentiating the d1r signaling. central serotonin (5-ht) function has been implicated in impulsivity. the present study examined rats with 5-ht depletion by parachloroamphetamine (pca) in simple and reversal go/no-go visual discrimination tasks, and analyzed the relationships between learning performance and focal concentrations of 5-ht and its metabolites (5-hiaa) in the brain. for both tasks, significant negative correlations between learning performance and 5-ht and 5-hiaa concentrations were observed in the medial prefrontal cortex and nucleus accumbens. in contrast, for reversal task only, significant correlations between learning performance and 5-ht and 5-hiaa concentrations were observed in the orbitofrontal cortex and amygdala. these data suggest the regional difference of 5-ht roles on selective indices of impulsivity. yuki sato 1,2,3 , tatsushi onaka 2 , norimasa seo 1 , eiji kobayashi 3 1 dept. anesthesiol., jichi med. univ., tochigi, japan; 2 dept. physiol., jichi med. univ., tochigi, japan; 3 div. organ replacement research, center for mol. med., jichi med. univ., tochigi, japan cyclosporine is widely used for preventing allograft rejection. however, in a considerable number of transplant recipients, cyclosporine causes neuropsychological side effects such as confusion, depression, and anxiety. cyclosporine inhibits calcineurin activity and forebrain-specific calcineurin knockout mice exhibit deficits in social behaviour. it is thus possible that cyclosporine causes psychological side effects via disturbing social interactions. here, we examined effects of cyclosporine upon anxiety and social behaviour in mice. calcineurin did not significantly change percent entries into open arms and time spent on open arms in the elevated plus maze test. on the other hand, in the social interaction test in home cage, cyclosporine increased the number of particles in home cage, an index of social activity. all these data suggest that impaired social interaction is a cause of psychological side effects of cyclosporine. to investigate the distribution of functionally activated vestibularrelated brainstem neurons during postnatal development, ombined immuno-/hybridization histochemistry of c-fos expression was performed in sprague-dawley rats (p1-21; adult). conscious animals were subjected to rotational or translational stimulus which activates hair cells of the horizontal semicircular canals or utricle, respectively. neuronal activation within brainstem nuclei was defined by the expression of c-fos. labyrinthectomized controls and normal stationary controls showed only a few sporadically scattered fos-expressing neurons. with rotational stimulation that comprised cycles of constant angular acceleration and deceleration, fos-labeled neurons were observed by p4 in the vestibular nucleus and downstream relay stations of vestibular pathways, such as the prepositus hypoglossal nucleus and inferior olive (subnuclei dmcc, ioa, ioc, iok). a later maturation time was evidenced for the utricular system. fos-labeled neurons were only identifiable in the vestibular nucleus by p7; in the prepositus hypoglossal nucleus and inferior olive (subnuclei dmcc and io␤) by p11. within the vestibular nucleus of p7-9 rats, neurons activated by canal or utricular inputs were intermingled throughout its rostro-caudal length. in p21 and adult rats, neurons activated by canal or utricular inputs were intermingled in localized regions of the medial and spinal vestibular nuclei. however, neurons in the rostral half of spinal vestibular nucleus were activated only by utricular inputs. taken together, we have demonstrated that canal-and otolithrelated brainstem neurons that encode rotational and translational movements in the horizontal plane are histologically segregated and exhibit different developmental time frame. to determine whether perineuronal nets (pn) within the vestibular nuclei contribute to plasticity of central connectivity, we studied the presentation of pn within the vestibular nuclei during development (rats, p1 to adult) and after unilateral labyrinthectomy (ul) in the adult. histochemistry with the lectin wisteria floribunda agglutinin was used to map pn about neun-immunopositive neurons within the vestibular nuclei. in normal postnatal rats, pn was detectable by p3 in the vestibular nucleus as fuzziness about neuronal cell bodies. from p9 onwards, the fuzzy pn progressively consolidated into a network organization. the fuzziness was no longer observable after p12. during postnatal development, the number of neurons showing pn increased with age, reaching the adult level by p21. with ul, the pn network on the lesioned side remained compact until 5 days post-lesion when the fuzziness reminiscent of that in early postnatal rats became evident. by 11 days after ul, the pn of some neurons resumed the network pattern as was observed in normal adult rats. this phenomenon was found in the pn of the remaining neurons by 14 days after ul. the pn on the labyrinth-intact side showed the compact network of uninjured age-matched rats. taken together, our findings indicate pn changes that suggest possible correlation with vestibular nuclear neuronal function both during postnatal development of normal rats as well as in adult rats following destruction of the ipsilateral inner ear. minori ueda, takayuki suzuki, hiroyoshi miyakawa laboratory of cellular neurobiology, tokyo university of pharmacy and life science, tokyo, japan dynamics of transmitters in the synaptic cleft depends on many processes such as transmitter release, uptake and diffusion. to better understand these processes, we analyzed ampar-and nmdarmediated epscs and synaptically induced transporter currents (stcs) elicited with high-frequency stimuli. recordings were made from pyramidal cells and astrocytes in the ca1 region of rat hippocampal slices, 100 hz/20 pulse tetanic stimulations were delivered to schaffer-collaterals, and the evoked currents during the course of tetanic stimulation were isolated. the decay time course of the last isolated stc during the tetanic stimulation was not significantly different from that of the first. while the amplitude of the ampar-mediated epscs showed significant decay in the presence of cyclothiazide, there was no marked decay of the amplitude of the nmdar-mediated epscs. these findings imply that synaptic fatigue and saturation of glutamate transporters do not take place during the course of high-frequency stimulation at 100 hz. ikuko yao 1 , hiroshi takagi 1 , hiroshi ageta 1 , tomoaki kahyo 1 , ken hatanaka 1 , kaoru inokuchi 1 , mitsutoshi setou 1,2 1 mitsubishi kagaku institute of life sciences, tokyo, japan; 2 university of tokyo, tokyo, japan; 3 okazaki institute for integrative bioscience, national institute for physiological sciences, okazaki, japan we identified and characterized a novel ubiquitin ligase named scrapper. scrapper is an f-box protein which has leucine rich repeat and c-terminal membrane localization sequence, highly expressed in neurons throughout the brain. to investigate the physiological role of scrapper in the neuron, we recorded mepscs from the neuron over-expressed the egfp-tagged full-length scrapper construct or truncated form of scrapper constructs. they exhibited a strong suppression or enhancement in the frequency of mepscs while showing a non-significant change in mepsc amplitude, rise, and decay time compared with neurons expressing egfp. the passive membrane properties of neurons such as membrane resistance (rm), series resistance (rs), and membrane capacitance (cm) were not statistically different from those of control. these data suggests a presynaptic effect of scrapper protein. ps2p-a003 presynaptic membrane potential-dependent regulatory mechanism of transmitter release tetsuya hori, tomoyuki takahashi department of neurophysiology, university of tokyo graduate school of medicine, tokyo, japan in simultaneous pre-and postsynaptic recordings at the calyx of held, we addressed the mechanism underlying presynaptic membrane potential-dependent changes of transmitter release. a weak sustained depolarization (e.g., 10 mv, 1 s) of calyceal nerve terminal potentiated epscs despite that it diminished presynaptic action potential (a.p.) amplitude. as we further depolarized the terminal epscs became eventually depressed concomitantly with a marked reduction in the a.p. amplitude. when presynaptic ca 2+ currents (i pca ), induced by an a.p.-waveform command pulse, were used to evoke epscs, a weak sustained depolarization enhanced i pca and epscs in parallel. this epsc facilitation was robust at the calyx of held both in rats and mice, but was almost absent in p/q-type ca 2+ channel knockout mice. we conclude that the p/q-type specific ca 2+ channel facilitation plays an essential role in the facilitation of transmitter release following presynaptic depolarization. hiroshi takagi 1 , koji ikegami 1 , ken hatanaka 1,2,3 , yoko fujiwara-tsukamoto 1 , mineo matsumoto 1 , ikuko yao 1 , mitsutoshi setou 1,2,4 1 mitsubishi kagaku institute of life sciences, japan; 2 presto, japan; 3 school of pharmaceutical sciences, the university of tokyo, japan; 4 okazaki institute for integrative bioscience, japan a variety of post-translational modifications to the exposed cterminal tails of tubulin, such as detyrosination/tyrosination, polyglycylation and polyglutamylation would play a crucial role in the neuron. however, evidence for the implication of these modifications in regulating the translocation of channels and receptors is currently unavailable. of the modifications, polyglutamylation is highly abundant in the mammalian brain, thus, this modification might account for the translocation of channels and receptors in the mammalian brain. in the rosa22(−/−) mouse, which shows a gross loss of polyglutamylated ␣-tubulin, transient a-type currents were largely suppressed in hippocampal pyramidal neurons in vitro. we provide herein, using rosa22 mice, the evidence for the implication of ␣tubulin polyglutamylation in the regulation mediated a-type k current. satoshi kawasaki 1 , shingo kimura 1 , reiko fujita 2 , shuji watanabe 1 , kazuhiko sasaki 1 1 dept. of physiol., sch. of med., iwate medical univ., morioka, japan; 2 dept. of chem., sch. of lib. arts & sci., iwate medical univ., morioka, japan application of dopamine (da) induces a slow na + -current response in the identified neurons of aplysia ganglia under voltage clamp. this type of response is produced by the activation of trimeric g-protein sensitive to cholera toxin (ctx) as previously reported. the na +current response to da was gradually and irreversibly depressed after intracellular injection of clostridium difficile toxin b, which is known to inactivate all types of rho family g-proteins. intracellular application of clostridium botulinum exoenzyme c3, a specific toxin to rhoa-c, also depressed the da-induced response irreversibly. furthermore, the da-induced current response was significantly depressed by gap domain of p50rhogap applied intracellulary. in contrast, gef domain of rhogef dbs had a tendency to increase the response. these results suggest that the da-induced na + -current response may be regulated by the activation of rho family g-protein. the ␦2 glutamate receptor (␦2r) plays a crucial role in cerebellar functions. although ␦2r has a putative channel pore domain, and ␦2r displayed ca 2+ -permeable channel activities in lurcher mutant mice, it has been unclear whether wild-type ␦2r functions as a channel. here we introduced a ␦2r transgene, which had a mutation (gln618arg) in the putative channel pore conserved in ca 2+ -permeable glutamate receptors, into ␦2 −/− mice. surprisingly, a mutant ␦2r transgene, as well as a wild-type transgene, rescued all abnormal phenotypes of ␦2 −/− mice, such as ataxia and loss of long-term depression. these results indicate that ca 2+ influx through ␦2r is not required for its function in the cerebellum in vivo, and that wild-type ␦2r may not function as a ca 2+ -permeable ion channel. research funds: kakenhi (17700316) and takeda science foundation ps2p-a007 distribution of tarp -8 on hippocampal neurons and its key role in synaptic and extrasynaptic expression for ampa receptors masahiro fukaya 1 , mika tsujita 2 , maya yamazaki 2 , etsuko kushiya 2 , manabu abe 2 , kaori akashi 2 , masanobu kano 3 , haruyuki kamiya 4 , kenji sakimura 2 , masahiko watanabe 1 1 department of anatomy, hokkaido university school of medicine, sapporo, japan; 2 department of cellular neurobiology, brain research institute, niigata, japan; 3 department of cellular neuroscience, graduate school of medical science, osaka university, suita, japan; 4 department of molecular anatomy, hokkaido university school of medicine, sapporo, japan the -8 is one of four transmembrane ampar regulatory proteins (tarps). pre-and post-embeding immunogold visualized -8 on excitatory synaptic and extrasynaptic membrane. in -8-ko mice, ampars were reduced in hippocampal homogenates (46% of control) and psd fraction (35%). immunogold labeling also exhibited reduction of extrasynpatic (47%) and synaptic (35%) ampars in ca1 pyramidal cells. the reduction of extrasynaptic receptors was particularly severe on dendrites (36%) and spines (38%). ampar-mediated responses were reduced at ca1 synapses (52%). therefore, -8 is the major auxiliary subunit of hippocampal ampars. etsuko tarusawa 1 , yugo fukazawa 1 , elek molnar 2 , masahiko watanabe 3 , ryuichi shigemoto 1,4 1 div. cerebral structure, nips, okazaki, japan; 2 mrc, univ. of bristol, bristol, uk; 3 hokkaido univ., sapporo, japan; 4 sorst, jst, kawaguchi, japan relay cells in the dorsal lateral geniculate nucleus receive two types of glutamatergic inputs; retinogeniculate (rg) and corticogeniculate (cg) synapses. it has been shown that the synaptic transmission at both rg and cg synapses is mediated via ampa and nmda receptors. however, how ampa and nmda receptors are expressed in these two types of synapses have not been elucidated. we examined the expression pattern of ampa and nmda receptors in rg and cg synapses using sds-digested freeze-fracture replica labeling (sds-frl). the sds-frl revealed that synaptic size of individual rg synapses was significantly smaller than that of cg synapses. rg synapses expressed 1.7 to 3 times higher density of ampa receptors than cg synapses. on the other hand, cg synapses expressed 1.6 to 3 times more nmda receptors than rg synapses. these results indicate differential effects on the relay cell by the retino-and cortico-geniculate inputs through ampa and nmda receptors. katsuyuki kaneda 1,2,3 , hitoshi kita 1 1 dept. of anat. & neurobiol., univ. of tennessee, memphis, tn, usa; 2 japan society for promotion of science, tokyo, japan; 3 dept. of developmental physiology, nips, okazaki, japan to investigate the properties of synaptically induced slow responses in globus pallidus (gp) neurons, whole-cell recordings were performed using rat brain slice preparations. repetitive stimulation of the gp and internal capsule induced mixed fast epsps/ipsps followed by a slow ipsp (sipsp), and a long-lasting slow depolarization (sdepo). bath application of nbqx, cpp, and gabazine blocked the mixed epsps/ipsps. the gaba b receptor antagonist cgp55845 abolished the sipsp. an mglur1 antagonist, but not an mglur5 antagonist, partially blocked the sdepo. in addition, cgp55845 enlarged the amplitude of fast ipscs, but not of epscs, that were evoked during the repetitive stimulation, suggesting an involvement of presynaptic gaba b receptors in gaba release. these results indicate that synaptically released gaba and glutamate can evoke gaba b receptor-and mglur1-mediated responses in the gp. contribution of these responses to the control of gp activity will be discussed. research funds: nih and the jsps ps2p-a010 essential contribution of glutamate to gaba depolarization involved in hippocampal seizure-like activity yoko tsukamoto 1 , yoshikazu isomura 1,2 , michiko imanishi 1 , tomoki fukai 2 , masahiko takada 1 1 system neurosci., tokyo met. inst. neurosci., tokyo, japan; 2 neural circuit theory, riken bsi, saitama, japan we have previously shown that neuronal synchronization is achieved by excitatory gabaergic and glutamatergic inputs during a hippocampal seizure-like afterdischarge. however, it still remains unclear how the gaba response is converted from inhibitory to excitatory in the process of afterdischarge induction. here we traced the time-course of amplitude and reversal potential of gabaergic transmission in pyramidal cells and interneurons entraining the afterdischarge, and examined influence of glutamate on the conversion of gaba response. the gaba reversal potential in pyramidal cells rose to spike-threshold levels for >20 s after the induction. gaba amediated cl-influx lasted for 1.5 s, and then glutamate enhanced the conversion effectively in a gaba a -independent manner, which was dependent on an extracellular k increase. coapplication of gaba and glutamate caused a similar oscillatory activity. the results show gaba and glutamate may cooperatively induce as well as maintain seizure-like activity. michiko nakamura 1 , yuko sekino 1,2 , toshiya manabe 1,2 1 division of neuronal network, department of basic medical sciences, institute of medical science, university of tokyo, tokyo, japan; 2 crest, jst, japan profound activity-dependent facilitation of synaptic transmission at hippocampal mossy fiber synapses is a unique and functionally important property. in the present study, we found that this synaptic strengthening was partially mediated by presynaptic gaba a receptor activation during the developmental period (p < 30), using electrophysiological methods and optical imaging. in immature animals (p10), fiber volley amplitudes were activity-dependently increased during short-train stimulation of mossy fibers. this fiber volley facilitation was significantly decreased by either inhibition of gaba a receptors or suppression of gaba release from interneurons. these results suggest that gaba released from inhibitory interneurons and gaba a receptors on mossy fibers contribute to activity-dependent facilitation of the excitatory synaptic transmission during development. takuya nishimaki, il-sung jang, jyunichi nabekura dep. dev. physiol., nips, sokendai, okazaki, crest, jst, japan lateral superior olive (lso) is the first auditory center. during the early postnatal period, the inhibitory synaptic inputs to lso neurons from medial nucleus of the trapezoid body (mntb) change from predominantly gabaergic to glycinergic. we focused on metabotropic gaba b receptor (gaba b r) as the key molecule of difference between gaba and glycine. in immature lso neurons postsynaptic gaba b r could activate k + channels, but this effect ceased by the third postnatal week. baclofen, a gaba b r agonist, reduced ipsc amplitude at mntb-lso synapses in neonate (<p5) with a significant change in the paired-pulse ratio. the presynaptic effect of baclofen was gradually decreased with development (p2-18). in situ hybridization and immunohistochemistry experiment revealed gaba b r expression in mntb neurons was also gradually decreased. the property of mntb-lso synapses in gaba b r knock out mouse remained immature at p14-16 compared with control mouse. thus, gaba b receptor seems to play an important role in development of synaptic property. pyramidal cells in the hippocampal ca1 area receive gabaergic input from interneurons, which make synapses on the soma, dendrites and the axon initial segment (ais). here, we used the sdsdigested freeze-fracture replica labeling method to visualize the cell surface distribution of the ␣1, ␣2, and ␤2/3 subunits quantitatively on distinct subcellular compartments of pyramidal cells. labelings for these subunits were accumulated over clusters of intramembrane particles (imp) on the protoplasmic face (p-face) of the plasma membrane, indicating that many imp clusters of a certain size represent gabaergic synapses. the synaptic labelling densities for gaba-a receptor subunits were not significantly different on the tested subcellular compartments. double labelling experiments showed that the majority of synapses contains ␣1, ␣2, and ␤2/3 subunits. in the cerebellar cortex, the effects of ␣-adrenoceptor activation on inhibitory synaptic transmissions have not yet been fully understood. therefore, we investigated the effects of the ␣ 1 -or ␣ 2 -adrenoceptor agonist on firing rates of presynaptic interneurons (ins). presynaptic cell-attached recordings showed that spontaneous activity of gabaergic ins was enhanced by the ␣ 1 -adrenoceptor agonist phenylephrine, while reduced by the ␣ 2 -adrenoceptor agonist clonidine. immunohistochemical studies showed that ␣ 1 -adrenoceptors were expressed in the molecular layer and the purkinje cell (pc) layer, while ␣ 2 -adrenoceptors were observed in cell bodies of pcs and ins. these results suggest that noradrenaline enhances inhibitory neurotransmitter release via ␣ 1 -adrenoceptors, which were expressed in presynaptic terminals and somatodendritic domains, whereas suppresses the excitability of ins via ␣ 2 -adrenoceptors, which were located in the presynaptic in soma. thus, cerebellar ␣-adrenoceptors play roles in a presynaptic dual modulation of gabaergic inputs from ins to pcs. research funds: kakenhi (16700344) ps2p-b015 involvement of basal pkc and erk 1/2 activities in constitutiveinternalization of ampa receptors in cerebellar purkinje cells tetsuya tatsukawa 1 , takahiko chimura 2 , azumi matsumoto 2 , kazuhiko yamaguchi 2 1 lab. for motor learning control, riken, bsi, japan; 2 lab. for memory & learning, japan ampa receptor (ampar) internalization provides a mechanism for cerebellar ltd, which is the underlying basis of motor learning and motor coordination. however, the relationship between cerebellar ltd and constitutive ampar internalization in parallel fiber (pf)-purkinje cell (pc) synapses remains unclarified. in the present study, we demonstrated that tetanus toxin (tetx, a blocker of exocytosis mediated by vamp) infusion into pcs caused the rundown of pf-epsc amplitude through the constitutive ampar internalization, and then addressed question whether intracellular signals involved in cerebellar ltd induction modify the constitutive ampar internalization at pf-pc synapses using electrophysiological and immunocytochemical techniques. our results suggest that the regulation of actin polymerization is involved in the surface expression of ampars and the surface expressing ampars are constitutively internalized depending on both basal pkc and mek-erk1/2 activities at pf-pc synapses. taisuke miyazaki 1 , kohichi tanaka 2 , masahiko watanabe 1 1 department of anatomy, school of medicine, hokkaido university, sapporo, japan; 2 department of molecular neuroscience, tokyo medical and dental university, tokyo, japan glutamate released to the synaptic cleft has to be removed by glutamate transporter. in the cerebellum, glutamate transporter glast is richly expressed in bergmann glial cells (bg) that enwrap synapses, dendrites and somata of purkinje cells (pcs). in this study, anatomical analysis was applied to glast-deficient mice. in the mutant mice, lamellate processes from bg fibers became atrophic, resulting in incomplete sealing of synaptic cleft. furthermore, the distribution of climbing fiber (cf) terminals was decreased proximally. by anterogradely labeling, multiple cf innervation was demonstrated to be formed by ectopic innervation to and from nearby proximal dendrites, particularly at their crossing point. these results suggest that glast is essential for complete ensheathment of pc synapses and for the establishment of cf mono-innervation by suppressing local ectopic innervation between nearby pc dendrites. miwako yamasaki 1,2 , kouichi hashimoto 2 , taisuke miyazaki 1 , masahiko watanabe 1 , masanobu kano 2 1 dept. of anatomy, grad. sch. of med., hokkaido univ., sapporo, japan; 2 dept. of cellular neuroscience, grad. sch. of med., osaka univ., suita, japan the ␥ isoform of protein kinase c (pkc␥) is highly expressed in cerebellar purkinje cells (pcs), and its deficiency impairs the late phase of climbing fiber (cf) synapse elimination. in the present study, we analyzed multiple cf innervation in pkc␥-deficient mouse by immunohistochemistry combined with anterograde tracer labeling of cfs. in general, cf innervation regressed proximally in the pkc␥-deficient mouse. in some mutant pcs, a considerable part of the proximal dendrites lost association with cf terminals. in the majority of multiply innervated pcs, single main cf innervated broad region of proximal dendrites, whereas surplus cfs innervated the somata and basal portion of the proximal dendrites. moreover, immunoelectron microscopy revealed synaptic contact of cf terminals to somatic spines. these results suggest that pkc␥ is crucial for strengthening innervation by a main cf and elimination of surplus cfs from proximal somatodendritc compartments of pcs. keiji imoto 1,2 , takashi kodama 1,2 , ryuichi shigemoto 2,3,4 , yugo fukazawa 2,3 , yasuo mori 5 1 division of neural signaling, nips, okazaki, japan; 2 school of life science, graduate university for advanced studies (sokendai), okazaki, japan; 3 division of cerebral structure, nips, okazaki, japan; 4 sorst, japan science and technology agency, kawaguchi, japan; 5 department of synthetic chemistry and biological chemistry, kyoto university, kyoto, japan a murine ataxic mutant, rocker, has a point mutation in the ca v 2.1 (p/q-type) ␣ subunit gene and shows the mildest phenotype among the series of ca v 2.1 mutants. although functional defects of the mutant channel were relatively mild, we found that synaptic transmission in parallel fiber-purkinje cell synapses was considerably impaired. we demonstrated that this impairment was mainly attributable to postsynaptic changes, which included reduction of the number of ampa receptors in psd, whereas the presynaptic function remained normal. we also found the dendrites of purkinje cells showed poor arborization in rocker mice. these lines of evidence indicate that ca v 2.1 exerts its strong influence on postsynaptic maturation and dendritic structure. bai-chuang shyu, chia-ming lee, wei-chih chang institute of biomedical sciences, academia sinica, taipei, taiwan roc the aim of the present study was to investigate synaptic transmission and organization of thalamus evoked activities in the anterior cingulate cortex (acc) using a novel mouse brain slice preparation protocol. the mono-and poly synaptic responses in the acc were excited by direct thalamic activation. the synaptic transmission involves both ampa and nmda glutamate receptors. several thalamus-evoked responses were identified: the early negative component (n1) emerged in layer vi near cingulum. subsequently a positive component (p) appeared in layer vi. the second negative component (n2) became apparent in layers ii/iii and v. then the activities spread downward to layer vi and developed as n3 component in a medial-to-lateral direction. we confirmed that functional thalamocingular activities were preserved in our newly developed brain slice preparation. the thalamus-evoked responses were activated and progressed along a lateral-medial-lateral trajectory loop in the acc and this process was modulated extensively by glutamatergic synapses. ps2p-b020 target-dependent extracellular ca-dependence of the short-term plasticity of the solitary complex synapses in the rat lab. neurophysiol., dept. neurosci., jikei univ. sch. med., tokyo, japan the discharge frequency-dependence of the transmission efficiency at the synapses from the vagal primary afferents to the second-order neurons in the solitary complex (sc) forms the primary filter of the visceral information. such frequency filtering of the synaptic transmissions from the solitary tract (ts) to neurons in the sc depends on their short-term plasticity as evidenced by strong correlation between frequency-dependence and the paired-pulse ratio (ppr). here we analyzed the mechanism underlying distinct pprs among distinct types of synapses. in synapses from ts to the neurons in nucleus of the solitary tract, which show prominent low-pass filtering, ppr significantly increased from 0.4 ± 0.2 to 1.1 ± 0.2 (n = 6; mean ± s.d.) by reducing [ca 2+ ] o from 2 to 0.5 mm, whereas that in low-pass synapses from ts to the neurons in dorsal motor nucleus of the vagus was affected only slightly. these results suggest that the synaptic frequency filtering in the sc is under distinct control depending on the function of the postsynaptic target systems. ps2p-b021 response of spinal monosynaptic reflex to high frequency stimulation in newborn rat the susceptibility of synaptic transmission to the stimulation frequency is a characteristic feature of immature animals, and has been attributed to the incompleteness of transmitter release mechanisms. in an isolated spinal cord preparation of newborn rat, monosynaptic reflexes (msrs) evoked by dorsal root stimulation, were mediated by both nmda and non-nmda glutamate receptors. msrs were constant in amplitude at 1/15 s, and were depressed completely by cnqx. when stimulus rate was increased to 1/s, msr amplitudes were greatly reduced initially, and recovered later. this recovery of msr was eliminated by apv. non-nmda component of msrs was eliminated completely at 1/s, but appeared in constant amplitudes in the presence of cyclothiazide. from these results, non-nmda glutamate receptor mediated almost most of msrs at 1/15 s, but not at 1/s due to desensitization. in contrast, nmda glutamate receptor does not mediate msrs, but activated at 1/sec, in place of non-nmda receptor. in conclusion, incompleteness of transmitter release mechanisms could not be solely attributed to the feature of immature synaptic transmission. kenichi ono 1 , keisuke shiba 2 , ken nakazawa 3 , ichiro shimoyama 4 1 department of otolaryngology, chiba university, chiba, japan; 2 department of otolaryngology, kimitsu central hospital, chiba, japan; 3 department of integrative neurophysiology, chiba university, chiba, japan; 4 section for human neurophysiology, research center for frontier medical engineering, chiba university, chiba, japan we determined synaptic source of the respiratory-related activity of laryngeal motoneurons (lms) using spike-triggered averaging of lm membrane potentials triggered by spikes of medullary respiratory neurons in decerebrate, paralyzed cats. in inspiratory (i) lms, monosynaptic epsps were evoked by spikes of i neurons with augmenting firing patterns, and monosynaptic ipsps were evoked by spikes of expiratory (e) neurons with decrementing firing patterns and of i neurons with decrementing firing patterns. in elms, monosynaptic ipsps were evoked by spikes of i neurons with decrementing firing patterns and of e neurons with augmenting firing patterns. we conclude that various synaptic inputs from respiratory neurons contribute to shaping the respiratory-related trajectory of lm membrane potentials. ps2p-b023 in-vivo gene transfer of exogenous protein to the primary afferent neurons chiaki yamada 1,2 , eiji shigetomi 1,3 , fusao kato 1 1 lab. neurophysiol., jikei univ. sch. med., tokyo, japan; 2 dept. basic biol. sci., kyoritsu univ. of pharm., tokyo, japan; 3 jsps, japan in order to understand the mechanism how sensory information is transmitted to the brain, it is indispensable to identify the functional roles of already-identified molecules related to synaptic transmission between the afferent fibers and the central second-order neurons. here we developed a method for efficient in-vivo gene transfer into the neurons in the sensory ganglia and evaluated expression of their gene products. in the anesthetized young wistar rats, we injected pcaggs-egfp plasmid vector (through courtesy of drs. j. miyazaki and k. nakajima) into the nodose ganglion (ng) at the neck and transferred it by electroporation. two days after transfection, the ng was extirpated and fixed to observe egfp signals. a large portion of somata in the ng as well as the centrally and peripherally projecting afferent fibers expressed high levels of egfp with few damaged cells. this technique might enable to analyze the function of specific molecules in the transmitter release from the sensory afferent termini in the brain. research funds: kakenhi (17650116, 176865) ps2p-b024 laminar sources of synaptic input to layer 1 neurons in rat visual cortex takuma mori, edward m. callaway salk institute, usa layer 1 of the mammalian neocortex consists of interneurons, including late-spiking neurogliaform cells and non-late-spiking axondescending cells. very little is known about connectivity of these cells with cells in other cortical layers. we examined the laminar sources of local excitatory and inhibitory functional synaptic inputs onto individual layer 1 neurons of rat visual cortex, using scanning laser photostimulation. we find that axon descending cells get excitatory synaptic inputs primarily from layer 2/3. neurogliaform cells receive more excitatory inputs from layers 4 and 5. the dominant inhibitory synaptic inputs onto both types of cells originate from layer 2/3. these neurons also get inhibitory inputs from layers 1 and 4-6. the presence of convergent excitatory and inhibitory synaptic inputs from multiple cortical layers suggests that the computations in layer 1 are highly integrative. takako nishi 1 , tsukasa gotow 2 , shiro nakagawa 2 1 ins. nat. sci., senshu univ., kawasaki, japan; 2 dept. neurol., kagoshima univ. grad. sch. med.-den. sci., kagoshima, japan four extra-ocular photoreceptors, the photoresponsive neurons, a-p-1, es-1, ip-1 and ip-2 which respond directly to light, exist in the abdominal ganglion of the mollusc onchidium. a-p-1/es-1 of these neurons respond to light with a depolarizing receptor potential, whereas light hyperpolarizes the other ip-1/ip-2. the present study was conducted to examine the functional significance of the above ip-1/ip-2, having a peak sensitivity at 510 nm light. the body wall of the animal transmitted enough 510 nm to hyperpolarize ip-1/ip-2 in the ganglion covered by the body wall. ip-1/ip-2 were coupled with weak electrical synapses and involved in a spontaneous beating or bursting spike activity. ip-1/ip-2 were not coupled to a-p-1/es-1 one another. finally, ip-1/ip-2, the first order photosensory cells were also the second order interneurons or motoneurons relaying various sensory inputs and innervating the pneumostome and viscera. ps2p-c026 cellular mechanism of interaural level difference calculation in the auditory brainstem of the chicken tatsuo sato, iwao fukui, harunori ohmori department of physiology, faculty of medicine, kyoto university interaural level difference (ild) and timing difference (itd) are the major two cues to localize the sound source, and are processed separately. although the details of itd processing are well investigated, not much is known about ild. in the barn owl, it is proposed that ild is calculated in lldp (posterior part of the dorsal lateral lemniscal nucleus). we confirmed already that in vivo lldp calculates ild in the chicken as well. in this work using the patch clamp recording with the brainstem slice preparation, we found that lldp neurons fire tonically under the regulation of two k + channels activated at low and high voltage, and receive both excitatory and inhibitory inputs representing the contralateral and the ipsilateral sound intensity, respectively. we are intending to clarify the cellular mechanism of ild calculation as the interaction of both excitatory and inhibitory synaptic inputs. to understand the functional organization of the cns, it is essential to know how sensory information is processed within the cns. we have been approaching this topic by following the ontogenetic patterning of neural circuit formation related to the cranial and spinal sensory inputs using optical imaging. in this study, we surveyed developmental organization of neural networks related to the olfactory nerve in the embryonic chick forebrain. stimulation applied to the olfactory nerve elicited epsp-related optical signals in the olfactory bulb from the 7-day old embryonic stage. the epsp was mediated by glutamate, and nmda-and non-nmda-receptor components were identified. in more developed stages, in addition to the responses in the olfactory bulb, another response area was discriminated within the cerebrum, which seemed to correspond to the higher-ordered nucleus of the olfactory pathway. the results suggest that the olfactory pathway is functionally generated at early stages of development when neural networks related to other visceral and general somatic sensory inputs are also in the process of developing. yoko momose-sato, katsushige sato dept. physiol., tokyo med. dent. univ. sch. med., tokyo, japan in an accompanying study, using a voltage-sensitive dye recording technique, we examined the developmental organization of the olfactory pathway in the embryonic chick forebrain, and showed that functional synaptic transmission in the olfactory bulb was expressed at around e7. it is known that odor stimuli elicit oscillatory events in the olfactory bulb in various species. we found that oscillatory activity was also generated in the chick olfactory bulb during embryogenesis. at early stages of development (e7-e8), postsynaptic responserelated optical signals evoked by olfactory nerve stimulation exhibited a simple monophasic waveform that lasted a few seconds. as development proceeded, the pattern of the optical signal became complicated, and oscillatory activity was observed in a later phase of the postsynaptic response. the oscillation was restricted to the olfactory bulb, and this spatial pattern was different from that of the propagating wave activity termed the depolarization wave. we examined spatio-temporal patterns of the oscillatory activity in different stages, and studied its developmental dynamics. akiyoshi shimada 1 , nyberg tobias 1 , nahoko kasai 1 , yuriko furukawa 2 , keiichi torimitsu 1,2 , kunihiko obata 3 , yuchio yanagawa 2,4 , tadaharu tsumoto 2,3 1 ntt basic research laboratories, ntt corporation, kanagawa, japan; 2 sorst, jst, saitama, japan; 3 neuronal circuit mechanisms research group, brain science institute, riken, saitama, japan; 4 department of genetic and behavioral neuroscience, graduate school of medicine, gunma university, gunma, japan in the immature stage of cerebral cortex, gaba a receptor activation depolarizes rather than hyperpolarizes the membrane potential of neurons. cortical neurons of glutamic acid decarboxylase 67-green fluorescence protein (gad67-gfp) knock-in mice at postnatal day 0-1 were cultured on a microelectrode array. spontaneous firing of cortical neurons appeared within the first 5 days in vitro (div). when a gaba a receptor antagonist, bicuculline, was added, four types of modulation of firing pattern were observed until at least 14 div: increase, decrease, disappearance and no change. these results and an additional analysis suggest that a portion of the spontaneous activity emerged through an activation of gaba a receptors. ps2p-c030 pathway specific signal modulation by purinergic receptors in the parallel processing system makoto kaneda 1 , toshiyuki ishii 2,4 , yasuhide shigematsu 3 , toshihiko hosoya 2 , yukio shimoda 3 1 dept. physiol., keio univ. sch. med., tokyo, japan; 2 bsi, riken, wako, japan; 3 mri, tokyo womens' medical univ., tokyo, japan; 4 dept. biomolecular science, univ. toho, funabashi, japan the retina divides visual information into on-and off signals, and processes them with independent subsets of neurons. so far no difference between these pathways has been found for neurotransmitter functions. we have previously found that p2x2-purinoceptors are selectively present on the off-type, but not on the on-type cholinergic amacrine cells of the mouse retina. we therefore asked if atp has different functions in the two pathways. as expected, only the off-type, but not the on-type, cholinergic amacrine cells responded to atp. this response was mediated by the p2x2-purinoceptors. furthermore, a p2x-purinergic receptor antagonist activated the tonic phase firing of the off ganglion cells, but not of the on cells. thus atp has off-pathway specific modulatory functions mediated by p2x-purinergic receptors. bhupesh mehta, gulnaz begum, preeti g. joshi department of biophysics, national institute of mental health and neuro science, bangalore, india cultured hippocampal neurons in network exhibit synchronized oscillations of cytosolic ca 2+ . these oscillations are an inherent property of various cell types and are essential for the maintenance and development of neuronal plasticity, and for normal brain functioning. the synchronized ca 2+ oscillations are primarily controlled by glutamate receptor activation, but may be modulated by a variety of other factors. these spontaneous oscillations are inhibited by activation of purinergic receptors. we observed a dose dependent inhibition of the oscillatory activity by purinergic receptor agonists atp and utp, but an enhanced oscillatory pattern was observed when the action of 2mesatp was observed. the inhibition of 2mesatp response by p2y1 receptor specific antagonist mrs 2179 indicates that different subtypes of purinergic receptors play different role in the regulation of synchronized ca 2+ oscillations. data will be presented on the p2 receptor mediated regulation of synchronized ca 2+ oscillations in cultured hippocampal neurons. mahomi kuroiwa, akinori nishi department of pharmacology, kurume university school of medicine, kurume, japan presence of additional d1-like dopamine receptors that selectively couple to gq/phospholipase c (plc) has been proposed. in recent studies, skf83959 was shown to selectively stimulate plc-linked d1-like dopamine receptors. darpp-32 is a phosphoprotein that regulates the efficacy of d1 receptor signaling. activation of d1 receptors that couple to golf/pka signaling leads to darpp-32 phosphorylation at thr34. we investigated the effect of skf83959 on darpp-32 phosphorylation at thr34 by using mouse neostriatal slices. treatment of neostriatal slices with skf83959 increased thr34 phosphorylation. the effect of skf83959 was enhanced by a plc inhibitor, u73122. in contrast, the effect of skf83959 was partially blocked by a d1 receptor antagonist, sch23390, and the sch23390-insensitive increase in thr34 phosphorylation was completely abolished by an adenosine a 2a receptor antagonist, zm241385. thus, analysis of darpp-32 phosphorylation revealed that skf83959 activates at least three signaling cascades in the neostriatum: (1) plc, (2) d1 receptor/golf/pka, (3) adenosine a 2a receptors. ps2p-c033 thiamylal may more effectively reduce neuronal excitability in cerebral cortex than that in hippocampus naoshi fujiwara division of medical technology, niigata university school of health sciences, japan effects of thiamylal (a barbital derivative anesthetic) on excitation propagation in the cerebral cortex and hippocampus were analyzed using membrane potential imaging. cortical and hippocampal slices of the c57bl6 mouse were dyed with a voltage-sensitive dye rh414. in cortical slices, electrical single-pulse stimulation to the layer v evoked transient depolarization in the vicinity of stimulated site, and then this excitatory response propagated to the layers ii-iii and widely expanded in these layers. the excitation propagation in the layers ii-iii was significantly inhibited by thiamylal (200 mol/l). in hippocampal slices, excitation propagation elicited by the same stimulation to the ca1 stratum radiatum remained without significant changes in the presence of thiamylal at the same dose. on the other hand, an ampa/kainate receptor antagonist cnqx (20 mol/l) inhibited excitation propagation in both cortical and hippocampal slices. the results suggest that thiamylal more effectively reduce neuronal excitation in cortex than that in hippocampus. research funds: grant-in-aid from jsps no.15390471 ps2p-c034 brain-derived neurotrophic factor regulating ampa receptor translocation to postsynaptic density through ip3r and trpc calcium signaling hiroko nakata, shun nakamura national institute of neuroscience, tokyo, japan the change in the number of postsynaptic ampa-type glutamatergic receptors (ampars) by neuronal activity is implicated in the excitatory synaptic plasticity. here, we showed that bdnf induced ampar translocation to postsynaptic site of the dissociated cortical pyramidal neurons. using calcium imaging, we located the spines that were activated by bdnf. with thus identified spines, we analyzed the ampar subunit glur1 localization to postsynaptic density (psd) by immunostaining. we found that bdnf increased the ratio of surface glur1 at psd within 20 min. bdnf selectively translocated ampar, but not nmdar. the essential signaling was ca 2+ transients evoked by the activation of trkb/plc␥ pathway. both the intracellular ca 2+ rise, that is, ca 2+ release from ip3r and ca 2+ influx through storeoperated cation channel trpc contributed to the increase of the surface expression of glur1 at psd. these results suggest an important role of bdnf in the regulation of ampar trafficking by spatial and temporal ca 2+ signaling regulation. research funds: health sciences research grant of nano-1 hiroyuki konishi 1 , kazuhiko namikawa 1,2 , keiji shikata 3 , yuji kobatake 1 , taro tachibana 3 , hiroshi kiyama 1 1 dept. anat. & neurobiol., osaka city univ., grad. sch. med., osaka, japan; 2 dept. anat., asahikawa med. col., hokkaido, japan; 3 dept. bioeng., osaka city univ., grad. sch. eng., osaka, japan activation of akt-mediated signaling pathways is crucial for injured neurons to survive and regenerate. in this study, we attempted to identify novel substrates for akt by using an antibody, which recognized phosphorylated consensus motif by akt. using pc12 cells, which overexpressed constitutively active akt, we screened protein spots that exhibited increased immunostaining by the antibody, and consequently identified the neuronal intermediate filament protein, peripherin. using some mutant forms of the recombinant proteins, the phosphorylation site was determined in vitro. we then generated an antibody against phosphorylated peripherin, and demonstrated that peripherin was phosphorylated not only in akt-activated cultured cells but also in nerve-injured hypoglossal motor neurons. these results suggest that peripherin is a novel substrate for akt in vivo and that its phosphorylation may play a role in motor nerve regeneration. the truncated trkb receptor, t1, is involved in the control of cell morphology via the regulation of rho proteins via rho gdi1. however, it is unclear whether t1 regulates the downstream of rho signaling and the actin cytoskeleton. in this study, we investigated this question using c6 rat glioma cells, which express t1 endogenously. rho gdi1 was dissociated from t1 in a bdnf-dependent manner, which also causes decreases in the activities of rho-signaling molecules such as rhoa, rock, pak and erk. moreover, bdnf resulted in the disappearance of stress fibers in the cells treated with lpa, an activator of rhoa, and in morphological changes in cells. furthermore, a competitive assay with cfp fusion proteins of t1-specific sequences reduced the effects of bdnf. these results suggest that t1 regulates the rho signaling pathways and the actin cytoskeleton. anjana kalita eukaryotic gene expression laboratory, national institute of immunology, new delhi, india jak and stat are activated in response to many cytokines and growth factors. this pathway has been primarily studied in non-neuronal origin cells. it has been shown that stat activated cellular transformation, occurs through transcription regulation of specific genes like c-myc, cyclin d1, bcl2. the present study was undertaken to identify target genes of jak/stat pathway in response to tnf-alpha & igf-1 in neuronal origin cells. we also looked at the mechanism of activation of these target genes. we saw that jak/stat pathway was involved in upregulation of cyclind1 in response to igf-1 both at protein & rna levels but no change was observed in c-myc, bcl2 levels. interestingly, mapk-erk, which is also known to activate cd1 in other systems, had no effect on the cd1 level in neuronal cells. on the other hand, pi3kinase not only activated jak/stat pathway but also affected on cyclind1 level. thus it appears that jak/stat is activated by pi3k, which in turn upregulates cyclind1 level. pi3k also activated erk without changing cd1 level, thus we show that jak/stat pathway is important for survival of neurons, activated by pi3k. cd1 is a one of the major downstream targets of jak/stat. hiroko inoue 1 , haruhisa kawasaki 2 , ritsuko fujii 2 , tohru yoshioka 2 , kazunori sango 3 , toshihiko kadoya 4 , hidenori horie 2 1 sch. of sci. and eng., waseda univ., tokyo, japan; 2 advanced res. center for biol. sci., waseda univ., tokyo, japan; 3 tokyo metropolitan inst. for neurosci., tokyo, japan; 4 pharmaceut. res. lab. kirin brewery co., ltd., gumma, japan oxidized galectin-1 (gal-1/ox) stimulates macrophages to promote axonal regeneration in peripheral nerves after nerve injury. but, the mechanism how gal-1/ox stimulates macrophages remains to be clarified. in the present study with rt-pcr and western blotting, we searched for the molecules contributing to the mechanism. rt-pcr analysis with cultured rat peritoneal macrophages revealed that the application of gal-1/ox enhanced mrna expression of nerve regeneration-related factors such as il-6, il-1 ␤. however, those of gdnf, ngf, bdnf, nt-3, nt-4, igf-i, and igf-ii scarcely detected in control condition were not changed by the application of gal-1/ox. since il -6, il-1, and lif can promote axonal regeneration in the in vitro axonal regeneration model, the up-regulation of these cytokines by the treatment with gal-1/ox may enhance nerve regeneration after nerve injury. kennichi katou, taku iwamoto, satoshi kida dep. of bioscience, tokyo univ. of agriculture, japan ␣camkii has been known to play essential roles in synaptic plasticity. in response to increase in ca 2+ concentration, ␣camkii is activated by the interaction with ca 2+ /cam. prolonged increase in ca 2+ level leads to further activation of ␣camkii through autophosphorylation at t286. to understand the molecular dynamics of ␣camkii activation in vivo, we have tried to visualize and monitor the ␣camkii activity in various types of cells using fret. in hela cells, increase in ca 2+ level induces continuous interaction between ␣camkii and cam and conformational change of ␣camkii, indicating that the conformational change of ␣camkii mainly reflects the interaction with cam. in sh-sy5y cells, the increase in ca 2+ level induces only transient interaction between ␣camkii and cam but the conformational change of ␣camkii is maintained following the termination of the interaction with cam, suggesting that conformational change of ␣camkii is induced by the interaction with cam and then maintained by autophosphorylation. thus, mechanisms for regulation of ␣camkii activation is cell-type dependent. akifumi kamata, yusuke takeuchi, kohji fukunaga dept. pharmacol., grad. sch. pharmaceu. sci., tohoku univ., sendai, japan ca 2+ /calmodulin-dependent protein kinase ii (camkii) is highly expressed in brain including dopaminergic neurons, however, the role of camkii in the dopaminergic neurons remains to be unclear. camkii consist of four isoforms, ␣, ␤, ␥ and ␦, and differential isoforms are implicated in the different neural functions. we have examined the isoforms of camkii expressed in rat substantia nigra (sn). rt-pcr and immunoblot analyses revealed that the ␥ and ␦ isoform mrnas including ␦3 isoform, a nuclear isoform of camkii, were predominantly expressed in sn. the immunohistochemical study also confirmed the preferential localization of the ␥ and ␦ isoforms in the sn dopaminergic neurons, and immunoreactivity against anti-camkii␦1-4 antibody was detected in both nucleus and cytoplasm. furthermore, we defined expression of bdnf mrnas having the exon ii and iv in sn. taken together with our previous observation, the camkii␦3 isoform was involved in the expression of bdnf in sn dopaminergic neurons. ps2p-d041 the role of ca 2+ /calmodulin-dependent protein kinase ii in neuronal functions revealed by inactivated camkii␣ knock-in mouse yoko yamagata 1,2 , hiroyuki sakagami 3 , keiji imoto 1,2 , kunihiko obata 4 , yuchio yanagawa 5 1 okazaki, japan; 2 sokendai, okazaki, japan; 3 tohoku univ., sendai, japan; 4 riken, wako, japan; 5 gunma univ., maebashi, japan ca 2+ /calmodulin-dependent protein kinase ii (camkii) is one of the major protein kinase in the central nervous system and proposed to play important roles in various neuronal functions. we engineered knock-in mice with the inactivated ␣ subunit of camkii by replacing k42 with r42 to study functional significance of camkii activity in intact animals. as expected, camkii activity was selectively reduced, while camkii subunit protein levels were comparable to those of wild type controls in homozygous mutants. in situ hybridization revealed normal expression patterns of camkii subunit mrnas, including dendritic localization of camkii␣ mrna. further analyses of these mice will be presented in the meeting. research funds: a grant-in-aid for scientific research from japan society for the promotion of science (kakenhi , #17500218) ps2p-d042 characterization of ca 2+ -dependent membrane binding proteins of rat brain shohei maekawa 1 , katsutoshi taguchi 1 , haruko kumanogoh 2 , shun nakamura 2 1 division of biosystems science, grad. sch. of sci/tech, kobe-u kobe 657-8501, japan; 2 division of biochemistry and cellular biology, national inst. neurosci. kodaira, tokyo 187-0031, japan ca 2+ -dependent membrane binding proteins were purified through a ca 2+ -dependent membrane binding and egta-induced membrane dissociation protocol from a crude triton solubilized membrane fraction. molecular characterization of these ca 2+ -dependent membrane binding proteins through lc-ms/ms identified annexin vii, annexin xi, and copin isoforms, in addition to annexin vi and neurocalcin␣. since the membrane fraction contains phosphatidylethanolamine (pe), cholesterol, and phosphatidylcholine (pc), the membrane binding activity of annexin vi, a major protein in this fraction, was studied using artificial liposomes. annexin vi showed a pe-dependent, but not cholesterol-dependent liposome binding activity. immunostaining of annexin vi in cultured neurons showed a punctuate staining of neuronal cell membrane and this spots increased during maturation. these results suggest the participation of annexin vi in the membrane cycling at the presynaptic region. ps2p-d043 generation of dominant negative mutants of transcription factor crest takuzou simo, kenichi kato, hiroshi hosoda, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan transcription factor calcium-responsive transactivator (crest) has been shown to be required for dendritic growth of neuron. previous studies have shown that crest contains two functional domains; multifunctional domain (mfd; aa251∼322) required for transactivation, nuclear localization and homodimerization and c-terminal transactivation domain (ctd; aa238∼402). to further understand the function of crest on brain function, we tried to generate the dominant negative mutant of crest to use mouse genetics study. indeed, we generated fusion proteins of gal4-dbd with mutants of crest; crest c ( 238∼402) lacking c-terminal transactivation domain, mfd and mfd-nls that is a fusion protein of mfd with nuclear localization signal (nls). from the experiments using gal4 one hybrid system, we found that mfd-nls shows the strong dominant negative effects on transcriptional activation by crest. ps2p-d044 characterization of iq-arfgef, a novel exchange factor for arf6, in mouse brain hiroyuki sakagami, hisatake kondo department of cell biology, tohoku university graduate school of medicine, sendai, japan adp-ribosylation factor 6 (arf6) regulates cytoskeletal dynamics and membrane trafficking that are critical processes for synaptic plasticity. to understand its neuronal functions, we have focused on guanine nucleotide exchange factors (gef) for arf6. in this study, we determined the entire sequence of a partially identified cdna (mkiaa0522) encoding a novel sec 7 domain. the deduced protein contained coiled coil, iq, sec7, plekstrin homology domains. interestingly, it contained a type i pdz domain-binding motif at its cterminal tail. arf pull-down assay demonstrated its ability to active arf6 more selectively than arf1. thus, we named this protein as iq-arfgef. in situ hybridization analysis demonstrated the preferential expression of iq-arfgef in the forebrain and cerebellar granule cells. furthermore, iq-arfgef mrna was localized not only in the hippocampal neuronal layers but also in their dendritic fields. our present findings suggested that iq-arfgef may play important roles in dendrites through activation of arf6 and interaction with various pdz proteins. eriko miura 1 , masahiro fukaya 1 , tokiharu sato 2 , kazushi sugihara 3 , masahide asano 3 , katsuji yoshioka 2 , masahiko watanabe 1 1 dept. anat., hokkaido univ. sch. med., sapporo, japan; 2 dept. mol. cell. biol., cancer res. inst., kanazawa univ., kanazawa, japan; 3 adv. sci. res. center, kanazawa univ., kanazawa, japan the c-jun n-terminal kinase (jnk) is one of the major mitogenactivated protein kinases (mapks), and jsap1 (or jip3) is a jnkassociated scaffold that controls the specificity and efficiency of jnk signaling cascades. here we studied its expression and distribution in mouse brains. jsap1 mrna was expressed in developing and adult brains, showing spatial patterns similar to jnk1∼3 mrnas. in embryos, jsap1 immunolabeling was intense for progenitor cells in the ventricular zone and external granular layer, and for neurons and glial cells differentiating in the mantle zone. in adults, jsap1 was distributed in spines, dendrites, perikarya, and axons of various neurons, and often associated with ser and cell membrane. this wide spatiotemporal expression suggests its fundamental role in mediating external stimuli to jnk mapk pathway and other intracellular machineries. yasukazu hozumi, kaoru goto department of anatomy and cell biology, yamagata university school of medicine, japan diacylglycerol kinase (dgk) is involved in intracellular signal transduction as a regulator of a second messenger, diacylglycerol, and consists of several isozymes. to address the functional implications of dgk isozymes in the brain, we have raised specific antibodies against the isozymes. by immunoblot analysis, we found that the immunoreactive bands for dgk␤, -␥ and -were detected in the light membrane/microsome enriched fraction of rat brains, suggesting that these isozymes localize to the internal membrane system. immunohistochemical examination on rat brain revealed that dgk␤ was detected as dot-like structure in the cell membrane, and dgk␥ as round-shaped structure in the juxtanuclear region of neurons. dgk was detected in the perinuclear region and in the dendrites of purkinje cells. these data show that dgk isozymes localize differentially to the internal membrane system in neurons, suggesting that dgk isozymes play unique roles in distinct internal membrane system. kotaro hattori 1 , shigeo uchino 2 , tomoko isosaka 1,3 , shinichi kohsaka 2 , takeshi yagi 3 , shigeki yuasa 1 1 dept. ultrastractural res., nat. inst. neurosci., ncnp, kodaira, japan; 2 dep. neurochem., nat. inst. neurosci., ncnp, kodaira, japan; 3 kokoro biology group, fbs, osaka univ., suita, japan recently, we have found that fyn-mediated tyrosine-phosphorylation of nmda-r subunits is required for haloperidol-induced catalepsy. haloperidol induced catalepsy in the control mice, but such response was significantly reduced in fyn-deficient mice. fyn activation and enhanced tyrosine-phosphorylation of the nmda-r nr2b subunit were induced after haloperidol injection to the control mice, but both responses were significantly reduced in fyn-deficient mice. both nr2b phosphorylation and the nmda-induced calcium responses increased after dopamine d 2 -r blockade in cultured striatal neurons of the control, but not in fyn-deficient neurons. accordingly, d 2 -r antagonist activates striatal fyn and the subsequent tyrosine phosphorylation of nr2b alters striatal neuronal activity, thereby inducing catalepsy. we also report further proteomics analysis on this molecular pathway. hiroaki okuda, shingo miyata, tsuya taneda, atsushi yamaguchi, shinsuke matsuzaki, natsuko kumamoto, masaya tohyama department of anatomy & neuroscience, graduate school of medicine, osaka university, osaka, japan the schizophrenia is a well-known debilitating psychiatric disorder. recent studies focused on genetic contributions to schizophrenia and have revealed several susceptibility genes, including dysbindin (dtnbp1: dystrobrevin binding protein 1) at 6p22.3. however it is difficult to explain the relationship between the onset of the schizophrenia and only single genetic factor. since, there is an important another factor, an environmental factor, for the schizophrenia. although little attention has been paid to the importance of both factors for the schizophrenia in the nervous system. therefore, to investigate the mechanism of the onset of the schizophrenia, firstly we performed a yeast two-hybrid screening, using the n-terminal region of dys-bindin as a bait. as a result, we detected several transcriptional factors. thus, we further examine that these interactions regulate the expression level of particular genes in the neurons. ryota adachi 1 , naoko oda 2 , ryuzo shingai 1,2 1 21st coe program, iwate university, morioka, japan; 2 department of welfare engineering, faculty of engineering, iwate university, morioka, japan the response to environmental stimuli is important to live for organisms. the soil nematode caenorhabditis elegans responds well to chemical and thermal cues known as chemotaxis and thermotaxis behavior. c. elegans is attracted to some ions, amino acids and alcohol that are byproducts of bacteria, and migrates to the temperature of cultivation with food on a temperature gradient. the sensory neurons detected these stimuli are in the head region. the neural network and genes required in each neuron are well known. however, it is under investigation about the integration of chemical and thermal stimuli. to understand the mechanism of this integration, we investigated the chemotaxis behavior under each temperature using the worms cultivated under each temperature. wild type worms cultivated at 10 • c showed decreased chemotaxis response to water-soluble chemicals and alcohol. alteration of assay temperature did not affect response to cl − but that to na + . to discuss more detail, we used thermotaxis abnormal mutants for chemotaxis assay. takashi takekawa 1 , masaki nomura 2,3 , toshio aoyagi 2 , tomoki fukai 1 1 riken brain science institute, saitama, japan; 2 graduate school of informatics, kyoto university, kyoto, japan; 3 crest, japan science and technology agency, kawaguchi, japan the neuron model proposed by izhikevich can exhibit a variety of firing patterns of cortical neurons and gives an efficient mean to conduct large-scale network simulations. however, simplified models may lose essential properties of real neurons, and hence their properties must be compared with those of more realistic models. in fact, our previous studies have shown that two neuron models with almost identical membrane potential profiles sometimes have quite different phase responses and synchronization properties. here, we study the phase responses of the izhikevich model showing fast rhythmic bursting and demonstrate that it exhibits synchronization properties similar to those shown by complex conductance based models. furthermore, its synchronization properties are consistent with those of realistic models in other firing patterns. therefore, we may conclude that the model with tuned parameters is suitable for simulating the dynamic behavior of large-scale networks. honghai cui, hirotaka sakamoto, mitsuhiro kawata department of anatomy and neurobiology, kyoto prefectural university of medicine, kyoto, japan the amygdala plays a critical role in stress responses, especially for modulation of anxiety and fear. in the present study, we investigated the effects on the neuronal morphology for severe stress in the amygdala by using single prolonged stress (sps) paradigm as ptsd (post-traumatic stress disorder) model. rats were perfused 7 days after sps, and intracellular injections of lucifer yellow were carried out in neurons of central (cea) and basolateral amygdala (bla). at the cellular level, we observed a significant increase of dendritic arborization in bla pyramidal neurons but no effect on the cea neurons. these results suggest that sps-induced structural plasticity of the bla provides a cellular substrate for affective disorders that are triggered in ptsd. takahiko kawasaki 1,2 , tatsumi hirata 1 1 division of brain function, national institute of genetics, sok-endai, mishima, japan; 2 prest, kawaguchi, japan in macrosmatic mammals such as mice, olfactory information is detected by two distinct sensory organs, the olfactory epithelium and the vomeronasal organ, and their signals are integrated into the main olfactory bulb (mob) and the accessory olfactory bulb (aob), respectively. the secondary projections from the mob and aob first run parallel in the lateral olfactory tract and eventually diverge into the discrete targets in the telencephalon. although recent studies have revealed general principles in the primary projections, it has been largely unknown how the secondary projections from the olfactory bulbs are constructed. therefore, we investigated the secondary olfactory projections in mutant mouse lines for several axon guidance molecules and their receptors using a specific dye labeling technique, especially focusing on the distinctive pathways of mob and aob neurons. ps2p-d053 an essential role for click-iii/camki␥ in regulation of neurite extension natsumi ishihara, sayaka takemoto-kimura, hiroyuki okuno, haruhiko bito dept. of neurochem., univ. of tokyo, tokyo, japan click-iii/camki␥, a membrane-anchored camk, is a novel member of the camki family that acts downstream of camkk. in order to uncover the biological function of this kinase, we first examined its subcellular distribution and found that tagged click-iii was concentrated at the tips of filopodia-like processes in cultured hippocampal neurons, in close vicinity of actin cytoskeleton. phenotypic studies in pc-12 cells expressing various mutants of camkk and click-iii revealed that depolarization-induced activation of camkk-click-iii signaling is sufficient to robustly induce neuritogenesis. interestingly, click-iii localization in membrane fractions was regulated via sequential lipidification in an activity-dependent manner. furthermore, lipidification mutants of click-iii were impaired in their ability to facilitate activity-dependent neuritogenesis. our results thus indicate a novel role of click-iii in activity-dependent neurite formation and extension. takuro maruyama 1 , masahiro matsuura 2 , nobuhiko yamamoto 1 1 grad. sch. of frontier biosci., osaka univ., suita japan; 2 riso kagaku corp., tsukuba japan during development, thalamocortical (tc) axons invade into the cortex and stop growing in layer 4. to study tc axonal targeting, we examined the effect of the membrane proteins that are expressed in the developing cortex. the dorsal thalamus dissected from embryonic rat brain was dissociated and plated on the dishes that were coated with preclustered fc-tagged extracellular domains of several candidate molecules. after four days in vitro, axonal growth was enhanced on the substrata with low concentrations of ephrin-a5, sema7a or kit ligand, while a growth-inhibitory effect was found in higher concentrations. a tendency for tc axons to prefer or avoid the region containing these proteins was observed in a new culture method, in which purified proteins were printed on filter membranes in a real laminar size. these results suggest that ephrin-a5, sema7a and kit ligand may contribute to regulating tc axonal growth in the developing cortex. research funds: kakenhi 17023030, kakenhi 15300107 ps2p-e055 expression pattern of the guidance molecules and their receptors during gnrh neuron migration from the nose to the ventral forebrain shizuko murakami 1 , katsuhiko ono 2 1 dept. anat., juntendo univ. sch. of med., tokyo, japan; 2 div. of neurobiol. and bioinfo., nat. ins. for physiol. sci., okazaki, japan using in situ hybridization, we examined the expression pattern of attractive and repulsive guidance molecules and their receptors in the migration pathway of gnrh neurons of chick embryos. netrin-1 expression was seen in the ventral hypothalamus, whereas was absent in the nasal region and the rostral forebrain. a few gnrh neurons expressed netrin-1 receptor neogenin. slit-1 was expressed in the forebrain, but its receptor robo-1 was not expressed in gnrh neurons. semaphorin (sema) 3a expression was detected in the olfactory-forebrain region. within the forebrain, sema3a was absent in the restricted region of the medial forebrain where gnrh neurons migrated in association with a subset of olfactory fibers. at the end of this pathway, sema3a expression was observed in the caudal forebrain, and then gnrh neurons changed their course ventrally. many gnrh neurons expressed sema3a receptor neuropilin-1, suggesting that sema3a may act as a repellent during the migration of gnrh neurons. kazuto fujishima 1,2 , qing-hua jin 1 , junko kurisu 1 , tomoo hirano 2 , mineko kengaku 1 1 riken, bsi, lab. for neural cell polarity, saitama, japan; 2 department of biophysics, graduate school of science, kyoto university, kyoto, japan during development, cortical pyramidal neurons elaborate their dendrites in correct pattern and orientation in response to various environmental factors such as neurotrophins or guidance molecules. dendrite morphology is also regulated by cell-cell contacts with neighboring cells. recent studies have implicated notch signal as a candidate for such a contact-dependent regulator of dendrite patterning in postmitotic cortical neurons. we previously showed that delta/notch-like egf-related receptor (dner), neuron-specific transmembrane protein, acts as a notch ligand and mediates neuron-glia interaction during cerebellar development (eiraku et al., 2005) . dner is expressed in the dendrite of developing cortical pyramidal neurons. here, we analyzed the role of dner in the development of pyramidal neuron dendrites. we quantitatively compared the dendritic morphology of cortical pyramidal neurons in wild-type and dner knock out mice. reelin signal controls neuronal migration in the developing brain. because the binding of reelin to apoer2/vldlr induces dab1 phosphorylation, dab1 is a critical component of reelin signal. recent studies suggested that reelin signal is involved not only in neuronal migration but in other aspects of neural development. we investigated a possible function of the reelin signal in the filopodia and neurite formation. administration of reelin protein increased number of filopodia in a lesser extent than bdnf treatment. pp2 suppressed effects of reelin and bdnf on filopodia formation, suggesting that both signal pathways involve src kinase function. in spite of these similarities of reelin and bdnf functions in the filopodia formation, bdnf did not phosphorylate dab1. reelin treatment of the neurons derived from dab1 deficient mice did not induce significant increase in the number of filopodia. these results suggest that the filopodia and neurite formation involves the reelin-dab1 signal pathways. hidenobu mizuno 1,2 , tomoo hirano 1,2 , yoshiaki tagawa 1,2 1 dept. biophys., kyoto univ. grad. sch. sci., kyoto, japan; 2 crest, jst, kawaguchi, japan the left and right hemispheres are interconnected via the corpus callosum. in mouse visual cortex, callosal axons from one hemisphere make dense projections to a restricted region at the area 17/18 border of the other hemisphere, where they terminate in layers 2/3 and 5. we used in utero electroporation-mediated gene transfer of gfp to assess distribution of callosal projections during development. gfp-labeled callosal axons arrived at the 17/18 border and started to invade the cortical plate around p5. they generated exuberant axonal arborizations by p13 then underwent remodeling to form the adult pattern by p15. neonatal enucleation did not affect the development, suggesting no apparent role of retinal activity. exogenous expression of a potassium channel kir2.1 in callosal projection neurons, a manipulation known to reduce neuronal firing, attenuated axonal arborizations in layers 2/3. area-specific targeting to the 17/18 border was not affected. results suggest that certain stages of callosal axon development require neuronal activity in cortex. ps2p-e060 identification of a novel tetraspan membrane protein, nplp, that is up-regulated after the facial nerve axotomy chihiro akazawa, yoh sasaki, masato hoshi, yasuko nakamura, shinichi kohsaka department of neurochemistry, national institute of neuroscience, ncnp, japan facial nerve axotomy of adult rats, with its extensive ability to regenerate, provides us of a useful model in which to study the molecular mechanisms of nerve regeneration. to identify genes up-regulated during regeneration processes, we constructed a subtractive library enriched for cdnas expressed in the injured facial nucleus. we identified a novel putative tetraspan protein designated as neuronal pmp22like protein (nplp) that has a distant homology to pmp22/claudin family proteins. the mrna expression of nplp was detected on the cell bodies of neurons meanwhile mrna of pmp22 resided on the cell bodies of schwann cells. the nplp mrna level significantly increased in motor neurons of axotomized facial nerve, hypoglossal nerve or sciatic nerve. nplp has a large extracellular domain that may interact with other membrane molecules. overexpression of nplp showed the microspike formation in pc12 cells. our results suggest that nplp plays an important role in the regeneration of injured motor neurons. shinsuke shibata 1 , shin-ichi sakakibara 2 , hirotaka j okano 1 , hideyuki okano 1,3 1 dept. physiol., keio univ., sch. med., japan; 2 dept. histol. neurobiol., dokkyo med. univ., japan ; 3 crest-jst, japan musashi (msi) is an evolutionarily conserved rna-binding protein. drosophila-msi regulates asymmetric cell division in neural development. in mammalian cns we identified two members, msi1 and msi2, which are co-expressed in neural precursor cells including neural stem cells, suggesting msi proteins regulate the immaturity of cns stem cells. to reveal the roles of msi family in vivo, we generated msi1 and msi2 deficient mice. msi1−/− mice frequently died of obstructive hydrocephalus with aberrant cell proliferation of cerebral aqueduct. msi2−/− mice survive to adult show poor weight gain, low spontaneous movement and temperature insensitivity, with the malformation of pns. msi1−/− msi2−/− mice die in a few hours after birth with severe defects in the brain, including malformation of hippocampal dentate gyrus. in vitro and in vivo analysis show msi family proteins control the expression of several downstream target molecules post-transcriptionally, and play important roles cooperatively in mammalian cns and pns development. youhei koshimizu 1 , satoshi yamagoe 2 , kazuo suzuki 2 , michiko ohtomi 1 1 department of biomolecular science, graduate school of science, toho university, chiba, japan; 2 department of bioactive molecules, national institute of infections diseases, tokyo, japan we have previously suggested that lect2 (leukocyte cell-derived chemotaxin 2) is expressed in neurons and may play a role in the regulation of both dendritic extension and morphology of astrocytes in the mouse brain. however, the mechanisms of these regulations have not yet been clarified. in this study, we carried out multi-immunostaining against lect2 and tau or map2 in neuronal cell cultures and in tissues of adult mouse brain to examine the functional sites of lect2 in neurons. at the early stages in cultured neurons, lect2 was remarkably localized in the vicinity of membrane of soma, and was detected around root and end of neurites. in tissues of adult mouse brain, lect2 was localized to soma, axons and dendrites in neurons. these results suggest the possibility that lect2 influences the extension of dendrites and axons from the early stage of neuritic development. furthermore, it is thought that lect2 may regulate the dendritic and axonal morphology in mature neurons. mitsuru noda 1,2 , takahiro moriya 1 , hideyuki terazono 1 , suguru kudo 5 , masahiro yamaguchi 3 , kenji yasuda 4 , izumi nagata 2 , kazuyuki shinohara 1 1 div. neurobiol. & behav., nagasaki univ. grad. sch. biomed. sci., nagasaki, japan; 2 dpt. neurosurg. radiol. nagasaki univ. nagasaki, japan; 3 dpt. life sci., grad. sch. arts & sci., univ. tokyo, tokyo, japan; 4 dpt. physiol., grad. sch. med., univ. tokyo, tokyo, japan; 5 special division for human life technology, national institute of advanced industrial science and technology (aist), japan neural stem cells (nscs) are defined as self-renewing, multipotent progenitor cells that give rise to neurons, astrocytes and oligodendrocytes. using single-cell-based on-chip cell-cultivation system, we analyzed the process of neural differentiation and examined the effects of bdnf. the individual nscs from e12.5 nestin-promoter gfp transgenic mice were placed into 32 pairs of agar microchambers connected by microchannels and were differentiated in the presence or absence of bdnf. we observed that a part of nscs exhibited neuron-like morphology and extended some neurites. the application of bdnf increased the rate of neurite outgrowth. thus we addressed the process of neural differentiation in a single-cell-based level and demonstrated the effect of bdnf. ayumi kyuka, ryoichi yoshimura, yasuhisa endo department of applied biology, kyoto institute of technology, kyoto, japan in order to investigate the relationship between neurite outgrowth and target cells, ng108 cells were co-cultured with vascular smooth muscle cells (sm-3). sm-3 cells promoted the neurite outgrowth, but repelled the contact of their growth cones. the dna microarray analysis indicated that neuropilin-1 mrna was specifically up regulated in ng108 cells. but the western blotting analysis did not show the significant increase of neuropilin-1. sema3a, a candidate of ligands for neuropilin-1, was found in ng108 cells rather than in sm-3 cells. the immunocytochemical analysis revealed that neuropilin-1 was increased in the surface of ng108 cells co-cultured with sm-3 cells or cultured with the conditioned medium of sm-3 cells. these results suggest that neuropilin-1 may be transferred from cytoplasmic pool to surface of ng108 cells by some soluble factors released by sm-3 cells. we will discuss about kinetics of neuropilin and chemorepellents. yoshikuni edagawa 1 , j. nakanishi 2 , h. hamada 1 , y. yokomachi 1 , k. yamaguchi 3 , n. takeda 1 1 inst. biomed. eng., waseda univ., tokyo, japan; 2 riken, japan; 3 kanagawa univ., japan we show a novel method for controlling neurite outgrowth with a photochemical micropatterning technology, which is based on the photoreactive molecule switching its character by irradiation of uv light. this molecule formed a self-assembled monolayer on a glass coverslip, which was used for cell-culture surface. for the neurite outgrowth, pc12 cell was recruited as a model of neuron. by irradiating uv light at small spots on the cell-culture surface, single cells were specifically attached to the corresponding spots with intermediated ecm. to draw narrow paths for neurite outgrowth, new regions were irradiated in adjacent to the cell bodies attached on the spots. ngf stimulated each pc12 cell to extend a neurite along the narrow path: the elongation required both photo-activated path and ngf. the successive irradiation in front of developing neurite achieved bending or branching of the neurite. these results indicate that the timing and direction of neurite outgrowth can be controlled with this functional culture system. tetsuhiro kakimoto, hironori katoh, manabu negishi lab. of molecular neurobiology, grad. sch. of biostudies, kyoto univ., kyoto, japan n-wasp is important for actin polymerization and is strongly expressed in brain. toca-1 was shown to be required for cdc42 to activate n-wasp in vitro, although its physiological role is unknown. here we studied neural function of toca-1. toca-1 is strongly expressed in developing brain. knockdown of toca-1 in pc12 cells significantly enhances neurite elongation. consistently, overexpression of toca-1 suppresses neurite elongation through its amino terminus including the f-bar/efc domain, which induces plasma membrane invagination. in addition, knockdown of n-wasp also enhances neurite elongation in pc12 cells, in clear contrast to the previous report using dominant negative mutants. on the other hand, knockdown of toca-1 in rat hippocampal neurons enhances axon branching a little but not axon elongation, while knockdown of n-wasp enhances both axon elongation and branching. these results suggest that a vesicle trafficking regulating protein toca-1 regulates different aspects of neuronal morphology from n-wasp. research funds: kakenhi (17079003) ps2p-e067 pragmin, a novel effector of rnd2 gtpase, stimulates rhoa activity and regulates neurite outgrowth hironori katoh, hiroko tanaka, manabu negishi kyoto university, japan the rho family small gtpase rnd2 is specifically expressed in brain. although rnd1 and rnd3 display an antagonistic action for rhoa, signaling pathways of rnd2 are not well known. here we have performed a yeast two-hybrid screen using rnd2 as bait and identified a novel rnd2-effector protein, pragmin (pragma of rnd2). pragmin is predominantly expressed in neurons, including cortical and hippocampal neurons. in in vitro and in vivo binding assays, pragmin specifically bound to rnd2 among the rho family gtpases in a gtp-dependent manner. rnd2-bound pragmin significantly stimulated rhoa activity and induced cell contraction through rhoa-and rho-kinase-dependent pathway in hela cells. in pc12 cells, expression of pragmin inhibited the ngf-induced neurite outgrowth in response to rnd2, and knockdown of pragmin by a pragmin-specific small interfering rna enhanced the neurite elongation. therefore, these results suggest that rnd2 controls neurite outgrowth by regulating rhoa activity through pragmin. ps2p-e068 regulation of growth cone motility by collapsin response mediator protein-2 (crmp-2) masumi iketani 1 , yanai shigeki 1 , fumio nakamura 1 , yoshio goshima 1,2 , kohtaro takei 1,2 1 dept. of mol. pharmacol. & neurobiol., yokohama city univ. grad. sch. of med., yokohama, japan; 2 crest, japan sci. & tech. agency, kawaguchi, japan nerve growth cone located at a distal tip of a neurite is known to be the site governing axonal growth and guidance. collapsin response mediator protein-2 (crmp-2) is enriched in the distal part of axon and participated in axonal formation and growth as well as growth cone collapse by semaphorin signaling. the local functions of crmp-2 within growth cones, however, remains unclear. chromophoreassisted laser inactivation (cali) that can inactivate selectively a target protein with high spatial and temporal resolutions. acute localized loss of function of crmp-2 in the entire growth cone region resulted in temporal growth arrest of neurite in chick dorsal ganglion neurons. asymmetric inactivation of crmp-2 in a half region of growth cone caused growth cones to turn temporally toward the inactivated region. these findings suggest that a spatial distribution of crmp-2 activity within the growth cone can regulate growth cone motility and direct neurite outgrowth. research funds: crest, jst ps2p-e069 eph receptors are negatively controlled by protein tyrosine phosphatase receptor type o takafumi shintani 1,2 , masaru ihara 1,2 , hiraki sakuta 1,2 , hiroo takahashi 1,2 , ikuko watakabe 1 , masaharu noda 1,2 1 division of molecular neurobiology, national institute for basic biology, okazaki, japan; 2 crest, jst, kawaguchi, japan eph receptors are involved in numerous events such as cell movements, axonal guidance, the formation of synapses, and the maintenance of synaptic plasticity. eph receptors are activated by autophosphorylation of tyrosine residues upon the binding of their ligands, ephrins, however, the protein tyrosine phosphatases (ptps) responsible for the negative regulation of eph receptors have not been elucidated. here, we identified protein tyrosine phosphatase receptor type o (ptpro) as a specific ptp that efficiently dephosphorylates both epha and ephb receptors as substrates. using the retinotectal projection system, we show that ptpro controls the sensitivity of retinal axons to ephrins, and thereby plays a crucial role in the establishment of topographic projections. ps2p-e070 neogenin acts as a displacement factor that releases rho from rho-gdi katsuhiko hata, toshihide yamashita department of neurobiology, graduate school of medicine, chiba university, chiba, japan repulsive gudiance molecule (rgma) is a potent inhibitor of axon regeneration in the adult central nervous system. rgma inhibits mammalian cns neurite outgrowth by a mechanism dependent on activation of the rho/rho-kinase pathway. here we show that direct interaction of the rho gdp dissociation inhibitor (rho-gdi) with neogenin which is a receptor of rgma initiates the activation of rhoa, and this interaction between neogenin and rho-gdi is strengthened by rgma. we also found that neogenin facilitates the release of prenylated rhoa from rho-gdi. thus, understanding of the molecular mechanisms of rgma may be useful for developing methods to overcome the inhibitory signals. nobuyuki fukushima, masumi nakashima, ryou tokunaga, ryutaro moriyama department of life science, kinki university, higashiosaka, japan lysophosphatidic acid is an extracellular signaling lipid that regulates neurite morphology in neuronal cells through g protein-coupled lpa receptors, including lpa 1 and lpa 2 . here we examine how lpa 3 , third lpa receptor subtype, is involved in neuronal development by using pheochromocytoma 12 (pc12) cells. pc12 cells expressed low levels of endogenous lpa 3 mrna. when pc12 cells were infected with retrovirus expressing lpa 3 and treated with nerve growth factor under serum-free conditions for 3 days, neurite formation was observed in 55% of total infected cells, which was similar to that in control virus-infected pc12 cells. however, the mean length of neurites was increased in pc12 cells expressing lpa3, compared with that in control pc12 cells. interestingly, multiple neurite sprouts were frequently observed in lpa-treated lpa 3 -expressing cells. indeed, the numbers of neurites and neurite branching points per cell were increased in these cells. these results indicated that lpa 3 was involved in neuritogenesis and branch formation. masao sakurai 1 , tomoko aoki 1 , kyoko ishikawa 1 , shingo yoshikawa 2 , john b. thomas 2 , chihiro hama 1 1 cdb, riken, hyogo, japan; 2 salk institute, usa in drosophila, odor information is represented as a combination of activated glomeruli in the antennal lobe (al). each glomerulus is formed by specific synaptic connections between olfactory receptor neurons (orns) and projection neurons (pns). in an attempt to address the question of how these glomeruli are stereotypically organized during development, we identified wnt5 as a regulatory factor for glomerular patterning. in the wnt5 mutant, several glomeruli were shifted to ectopic positions, and this phenotype was partially rescued by wnt5 expression in orns. previous studies in embryos have shown that wnt5 repels the axons expressing derailed (drl). in the als of drl mutants, however, some glomeruli were located at incorrect positions, with the pattern different from that in the wnt5 mutant. this and other genetic data suggest that drl is an antagonist for wnt5 signaling. we propose that wnt5 signal transmitted from orns to pns provide one of mechanisms for glomerular patterning in drosophila. ps2p-e073 a homeodomain transcription factor, homothorax, controls precise dendritic and axonal targeting of the antennal lobe projection neurons in the drosophila brain mai ando, yoko totani, katsuo furukubo-tokunaga grad. sch. life envir. sci., univ. tsukuba, japan the precise neuronal connectivity in the nervous system depends on specific axonal and dendritic targeting of individual neurons. temporal and spatial regulation of gene expression by transcription factors is though to play seminal functions in determining wiring specificity. we are interested in the identification of the genes that control specification and connectivity of antennal lobe projection neurons, which convey olfactory information from the primary olfactory center to the higher order structures such as mushroom bodies and lateral horns. here, we show that homothorax (hth), a homeodomain protein, is expressed in most of the projection neurons. marcm mosaic analyses showed profound targeting defects in both dendritic and axonal projection patterns. currently, we are looking for genes that are either downstream or work cooperatively with hth. based on these data, we will discuss the functional roles of hth in the specification of antennal lobe projection neurons. ps2p-e074 differential microarray analysis of mushroom body transcripts using chemical ablation ayako ino 1 , masatomo kobayasi 1 , taiki nakajima 1 , lydia michaut 2 , indrayani ghangrekar 1 , kuniaki takahashi 3 , ryu ueda 3 , kaoru saigo 4 , walter j. gehring 2 , katsuo furukubo-tokunaga 1 1 grad. sch. life envir. sci., univ. tsukuba, japan; 2 biozentrum, univ. of basel, switzerland; 3 genet. strains res. ctr., natl. inst. genet.; 4 dept. biophys. biochem. grad. sch. sci., univ. tokyo, japan mushroom bodies (mbs) are the centers for olfactory associative learning in the drosophila brain. we have surveyed mb transcripts using a drosophila whole-genome microarray and identified 1,465 genes that exhibited reduced expression levels with pharmacological mb ablation. among the identified genes, we have further selected 102 genes that exhibited a most consistent reduction of expression levels in the ablated brains. in situ hybridization analyses confirmed preferential mb expression patterns for the majority of the identified genes. moreover, we also demonstrate that rnai of many of the identified genes causes mild to strong mb defects. these results provide novel and genome-wide insights into the repertoire of the genes that control differentiation and function of the mb neurons in brain development and plasticity. mikiko inaki 1 , yoshie suzuki 1 , hiroyuki aburatani 2 , akinao nose 1 1 dept. phys., univ. tokyo, tokyo, japan; 2 rcast, univ. tokyo, tokyo, japan in drosophila neuromuscular junction, the axons of individual motoneurons precisely recognize and project to specific muscle cells. to understand the molecular logic of target specificity, we tried to identify systematically target recognition molecules expressed on individual muscles. we focused on two neighboring muscles, 12 and 13, which are innervated by distinct motoneurons, and searched for genes that are differentially expressed between them by using wholegenome microarrays. by comparing gene expression data from the two muscles, we identified ∼170 genes with more than two-fold difference in expression level. for 26 candidate genes out of 33 (78%), the differential expression was confirmed by in situ hybridization and/or quantitative pcr. we focused our functional analyses on those encoding transmembrane or secreted proteins, with confirmed differential expression pattern. ectopic expression and/or knockdown of some of them altered the target specificity of muscles 12 or 13, implicating them in neuronal target recognition. makoto aoki, hiroshi segawa, mayumi naito, hitoshi okamoto laboratory for developmental gene regulation, brain science institute, riken, saitama, japan the sensory neurons have two axons, the central and peripheral axon. previously, we have demonstrated that peripheral axons of sensory neurons are completely abolished in the zebrafish embryos overexpressing the lim domains of islet-2. to find the molecules which regulate the formation of peripheral axons of sensory neurons in the islet-2 signaling cascade, we have created cdna library from trigeminal sensory neurons of zebrafish larvae. most of clones were known genes which are implicated in signal transduction, transcription and cytoskeltal formation, but several clones show no similarity with any known genes. most of them show cns or sensory neuron-specific expression patterns and the expression level of these genes were affected by the overexpression of the lim domains of islet-2. loss of function and gain of function studies of these clones, whose function are unknown, showed that these clones might be involved in the development of peripheral axons or the formation of neural circuitry. hiroshi yamamoto, kiyokazu agata development of biophysics, graduate school of science, kyoto university, kyoto, japan highly conserved netrins are known as bi-functional guidance molecules, attracting some axons and repelling others. they act through receptors of the deleted in colorecal cancer (dcc) and unc5 receptor families. freshwater planarians can proliferate by fission and decapitation, the anterior piece regenerates a tail, and the posterior piece regenerates a head. the planarian central nerve system (cns) can be used as a model for studying neural regeneration and understanding how we can rebuild nervous system after injury. we have identified eight netrin related genes from planarians, dugesiajaponica, including four different netrin homologues and three unc5 homologuesand one dcc homologue. we analyzed expression patterns of eight genes in both intact and regenerating planarians, and then conducted rnai experiments to investigating their functions in the process of regeneration of visual neurons. here we will summarize these results and speculate their molecular actions during eye regeneration in planarians. ps2p-e078 analysis of the significance and mechanism of neurite elimination using c. elegans as a model yu hayashi 1 , takaaki hirotsu 2 , eriko kage 1 , hideaki takeuchi 1 , hirofumi kunitomo 3 , yuichi iino 3 , takeo kubo 1 1 dept. biol. sci., grad. sch. sci., univ. tokyo, tokyo, japan; 2 dept. biol., fac. sci., kyushu univ. grad. sch., japan; 3 mol. genet. res. lab., univ. tokyo, japan during brain development, a large amount of neural connections once formed undergo elimination, the mechanism and physiological significance of which are poorly understood. we previously showed that elimination of neurites occurs in the nematode c. elegans and that a novel transcription factor mbr-1 is involved in this process. in the present study, we attempted to clarify the significance of this phenomenon. the mbr-1 mutant is defective in odor adaptation, and we therefore hypothesized that neurite elimination is critical for maturation of the olfactory circuit. by executing cell-specific rescue experiments, we suggest that mbr-1 functions in a set of interneurons involved in olfactory processing. we are now examining whether neurite elimination occurs in these neurons using gfp reporters. we expect that our research provides a useful model for genetic dissection of neurite elimination. taro asakura 1 , ken-ichi ogura 1 , yoshio goshima 1,2 1 dept. of mol. pharmacol. and neurobiol., yokohama city univ., grad. sch. of med., yokohama, japan; 2 crest, japan sci. and tech. agency, kawaguchi, japan netrin is an evolutionary conserved axon guidance molecule. in c. elegans, netrin/unc-6 is secreted from ventral cells, and attracts some axons ventrally and repels dorsally. one of the neurons guided by netrin/unc-6 is the hsn neuron. the hsn neuron of c. elegans has interesting axon guidance. netrin/unc-6 is required for the first ventral growth of the hsn neurons. however, what molecules participate in the axon guidance is largely unknown. in this study, we found that the vulval precursor cells strongly expressed netrin/unc-6 during the axonal guidance of the hsn neurons. silencing of netrin/unc-6 only at the vulval precursor cells resulted in abnormal axon guidance of hsn. in addition, expression of netrin/unc-6 only at the vulval precursor cells in unc-6 null mutants partially restored the hsn guidance defects. these results suggest that netrin/unc-6 expressed in vulval precursor cells plays essential roles on axon guidance of the hsn neuron. tomomi sato, takanori hamaoka, hidenori aizawa, hitoshi okamoto brain science institute, riken, saitama, japan the optic tectum is a visual center in vertebrates. it receives topographically ordered visual input from the retina in the superficial layers and then sends motor outputs from the deeper layers to the premotor reticulospinal system in the hindbrain. although it is well known how the retinal axons project to the tectum, it has not yet been well understood how the tectal axons project to the hindbrain. here, we established a stable transgenic line tg(brn3a-hsp70:gfp) in zebrafish to visualize the retinotectal and the tectobulbar projections. to explore the question, we developed an efficient single-neuron labeling system in combination with cre/loxp and gal4/uas systems. using the system, we found that the tectum projected to each hindbrain segment with a two-dimensionally ordered pattern. in addition, we showed that ephrinb2a was involved in the patterned projection from the posterior tectum to the hindbrain segment rhombomere 2. these results suggest a neuroanatomical and a molecular basis for the motor map in the tectum. research funds: grant-in-aid for young scientists (b) 16700296 masahiko taniguchi 1 , yoshinori mikami 2 , tomoyuki masuda 3 , tomoyuki yoshida 2 , naoto matsuda 2 , masayoshi mishina 2 , takao shimizu 4 1 department of biochemistry, cancer research institute, sapporo medical university, sapporo, japan; 2 department of molecular neurobiology and pharmacology, graduate school of medicine, university of tokyo, japan; 3 department of anatomy, fukushima medical university, japan; 4 dapartment of biochemistry and molecular biology, faculty of medicine, university of tokyo, japan semaphorin gene family contains a large number of secreted or transmembrane proteins, and some of them function as the repulsive and attractive cues of axon guidance during development. here we report the identification of zebrafish semaphorin 6d (sema6d). the orf of sema6d is 3054 bp (1018 aa). zebrafish sema6d protein showed a 70.8% identity with mouse sema6d protein. to clarify the expression pattern of sema6d, in situ hybridization was performed. in the embryos, sema6d is expressed in the lens and hindbrain. we also found that sema6d has the repulsive activity. these results indicate that sema6d repels specific types of the axons and functions in the nervous system development. research funds: kakenhi on priority areas from the mext (17023013) mayumi yamada 1 , shohei maekawa 2 , seiji miyata 1 1 department of applied biology, kyoto institute of technology, kyoto, japan; 2 division of bioscience, graduate school of science and technology, kobe university, kobe, japan obcam belongs to the immunoglobulin superfamily cell adhesion molecule (cam) and was synapse-specific cam in cortical and hippocampal neurons in vivo. however, it is uncertain how obcam is involved in synaptic functions. in this study, we showed that obcam was highly concentrated on spines of cultured mature neurons. the inhibition of obcam function with its specific antibody resulted in a decrease in the number of presynaptic terminals and postsynaptic spines. the suppression of obcam mrna expression with its specific antisense oligonucleotide also resulted in impairment of synapse formation. in contrast, overexpression of obcam augmented the formation of synapses. moreover, obcam was internalized via a raft-dependent pathway and the internalization was promoted by increased neuronal activity. these results suggest that obcam, a spine-specific cam, is a critical modulator of synaptogenesis and its surface expression is dynamically regulated in response to neuronal activity. ps2p-f083 semaphorin 4d inhibits ␤1 integrin activity through the r-rasgap activity of plexin-b1 izumi oinuma, hironori katoh, manabu negishi lab. mol. neurobiol., grad. sch. biost., kyoto univ., kyoto, japan plexins are cell surface receptors for semaphorins and regulate cell migration in many cell types. we have recently reported that the semaphorin 4d (sema4d) receptor, plexin-b1 functions as a gap for r-ras, a member of ras family gtpases implicated in regulation of integrin activity and cell migration. here we characterized the role of r-ras downstream of sema4d/plexin-b1 in cell migration. activation of plexin-b1 by sema4d suppressed the ecm-dependent r-ras activation, r-ras-mediated pi3-k activation, and ␤1 integrin activation through its r-ras gap domain, leading to inhibition of cell migration. in addition, inactivation of r-ras by overexpression of the r-ras-specific gap or knockdown of r-ras by rna interference was sufficient for suppressing ␤1 integrin activation and cell migration in response to the ecm stimulation. thus, we conclude that r-ras activity is critical for ecm-mediated ␤1 integrin activation and cell migration, and that inactivation of r-ras by sema4d/plexin-b1-mediated r-ras gap activity control cell migration by modulating the activity of ␤1 integrins. shun hamada 1 , emi fukuda 2,3 , shota katori 2,3 , takeshi yagi 2,3 1 dept. nutrition health sci., fukuoka women's univ., fukuoka, japan; 2 grad. sch. frontier biosci., osaka univ., osaka japan, 3 crest, jst, japan cnr/protocadherin (pcdh)␣ is a subfamily of the clustered pcdhs that comprise >50 cadherin-related molecules and are encoded by three gene clusters (␣, ␤ and ␥). the molecular features and synaptic localization of the clustered pcdhs have raised the possibility that they are synaptic recognition molecules. we have demonstrated that overexpressed pcdh␣ family proteins alone in several cell lines are rarely transported into the plasma membrane. furthermore, we found that a stretch of about fifty amino acids located at the c-terminus of pcdh␣s interfered the trafficking to the cell surface. in the present study, we compared the transport properties of a series of the cytoplasmic region truncation mutants and found that truncation mutants lacking 34 or more c-terminal residues were detectable at the cellular surface suggesting a role for lysine-rich motif in the c-terminus of pcdh␣s in the intracellular retention. mdga1 is a novel cell surface glycoprotein similar to ig-containing cell adhesion molecules (igcams) with functions in migration and process outgrowth. mdga1 is expressed by layer 2/3 neurons throughout the neocortex at p7 mice, but is absent in adults. between e15.5 and late p0, stages that span the generation and radial migration of layer 2/3 neurons, mdga is expressed in patterns consistent with its expression by migrating layer 2/3 neurons, suggesting a role for mdga1 in controlling their migration and settling in the superficial cortical plate. we performed loss-of-function studies using rna interference (rnai) with in utero electroporation into the lateral ventricle at e15.5 to transfect progenitors of superficial layer neurons. we found that an rnai suppressing mdga1 protein blocks proper migration of superficial layer neurons to the superficial cortical layer. we conclude that mdga1 acts cell autonomously to control the migration of superficial layer cortical neurons. in various pathological conditions, activated microglia mediate immune responses to injured cns neurons. however, it is not clear whether and how activated microglia affect neurons via direct contacts. this study aimed at examining whether direct contacts between microglia and hippocampal neurons increase following cns injury and whether telencephalin (tlcn), a dendrite specific adhesion molecule, which potentially binds to immune cells, mediates the direct contact. hippocampal neurons were damaged by local injection of excitotoxin, kainic acid (ka). compared to control animals, ka-injected mice showed higher density of contacts between activated microglia and dendrites of ca1 pyramidal neurons. contacts with longer interface appeared in ka-injected mice. these results suggest the importance of direct contacts for the immune response of microglia to injured neurons. similar contact formation was also observed in tlcn-deficient mice, indicating that the direct contacts are mediated by other molecules than tlcn. kilon is belonging to immunoglobulin superfamily of cell adhesion molecules and contains three igg-like domains. western analysis revealed that the expression levels of kilon is low at early neuronal culture and increased with progress of culture days. immunocytochemical observation showed that kilon was localized at elongating axon and growth cones but not at dendrites on 7 days in vitro (div), while kilon was observed at synapses, mainly at presynaptic terminals on 14 div. similar tendency was observed in kilon immunohistochemistry of brain sections in vivo. kilon was observed at axonal fibers of the cerebral cortex on postnatal day 2, but it was seen at synapses in adult brains. these results suggest that kilon is axonal cell adhesion molecule to control axonal guidance and/or extension. ps2p-f088 analysis of mice that show abnormal expression of neuroglycan c, a central nervous system-specific transmembrane proteoglycan sachiko aono 1 , yoshiyuki kuroda 1 , fumiko matsui 1 , yoshihito tokita 1 , keiko nakanishi 1 , michiru ida 1 , masahito ikawa 2 , masaru okabe 2 , katsuhiko ono 3 , atsuhiko oohira 1 1 institute for developmental research, aichi human service ctr., kasugai, japan; 2 research institute for microbial diseases, osaka university, suita, japan; 3 national institute for physiological sciences, okazaki, japan neuroglycan c (ngc) is a membrane-spanning chondroitin sulfate proteoglycan that is exclusively expressed in the central nervous system. to study the role of ngc in the brain, we produced two strains of ngc-mutant mouse by gene-targeting; a mouse strain with no ngcexpression and a strain with low expression (knockdown mice). both mice were viable and fertile. they did not show obvious abnormalities in gross brain anatomy. to examine their behavioral phenotype precisely, the ngc-knockdown mice were subjected to several kinds of behavioral tests sequentially. they displayed obvious abnormalities in morris water maze and passive avoidance tests, suggesting that ngc is involved in learning and memory. we are now carrying out the same experiments using the ngc-knockout mice. research funds: kakenhi (17500246) ps2p-f089 phosphorylation of extracellular signal-regulated kinase in aged rats with acute face inflammation koichi iwata 1 , tatsuhisa watanabe 2 , ikuko suzuki 1 , junichi kitagawa 1 , akiko ogawa 3 , kenro kanda 4 , kazunao kuramoto 5 1 dept. of physiol., sch. of dent., nihon univ., tokyo, japan; 2 dept. of oral and maxillofacial surgery, sch. of dent., nihon univ., tokyo, japan; 3 dept. of oral diagnosis, sch. of dent, nihon univ., tokyo, japan; 4 shinjuku vocational school of acupuncture, moxibustion and judo therapy, tokyo, japan; 5 division of research animal center, tokyo metropolitan institute of gerontology, tokyo, japan the capsaicin-induced perk expression was studied in the aged rats (30-34 months) following noxious face stimulation. a large number of perk-li cells were expressed in the superficial laminae of the trigminal spinal nucleus in adult and aged rats following subcutaneous capsainsin injection into the whisker pad region. the larger number of perk-li cells was expressed in adult rats than aged rats following intravenous administration of naloxone before capsaicin treatment. the present results suggest that the descending modulation system was impaired in the aged rats, resulting in the abnormal pain sensation advancing age. hirokazu katsura 1 , koichi obata 1 , masafumi sakagami 2 , koichi noguchi 1 1 department of anatomy and neuroscience, hyogo college of medicine, hyogo, japan; 2 department of otorhinolaryngology, hyogo college of medicine, hyogo, japan recent studies demonstrated that the activation of extracellular signal-regulated protein kinase (erk) 1/2 and p38 mitogen-activated protein kinase (mapk) in dorsal root ganglion (drg) neurons contributes to the development of inflammatory and neuropathic pain. in the present study, we examined whether the newest member of the mapk family of proteins, erk5 (also known as big mapk 1 or bmk1) is activated in the drg and participate in pain-related behaviors in the complete freund's adjuvant (cfa) model. peripheral inflammation induced an increase in the phosphorylation of erk5, mainly in tyrosine kinase a-containing small-to-medium-diameter drg neurons at days 1 and 3 after cfa injection. furthermore, time course of phosphorylated-erk5 level in the drg matched the emergence of cfa-induced pain hypersensitivity. our data suggest that activation of erk5 in drg neurons may contribute to the development of inflammatory pain. ps2p-f091 activation of erk5 in drg neurons contributes to acute pain toshiyuki mizushima 1,2 , koichi obata 1 , takashi mashimo 2 , koichi noguchi 1 1 department of anatomy and neuroscience, hyogo college of medicine, hyogo, japan; 2 department of anesthesiology, osaka univ. recently, we have reported that phosphorylation of extracellular signal-regulated protein kinase (erk) 1/2 and p38 mitogen-activated protein kinase (mapk) occurred in primary sensory neurons in response to natural noxious stimulation of the peripheral tissue, i.e., activity-dependent activation of erk and p38 in dorsal root ganglion (drg) neurons. however, there has been no study examining erk5 (also known as big mapk 1 or bmk1) activation in drg neurons after noxious stimulation of normal tissue. here, we report intensity-dependent erk5 phosphorylation in drg neurons by painful stimulation. noxious stimulation induced phosphorylated-erk5 in small-to-medium diameter sensory neurons with a peak at 2 min after stimulation. furthermore, we found a stimulus intensitydependent increase in the number of activated neurons. our data suggest that activation of erk5 in drg neurons may contribute to acute pain induced by noxious stimulation. koichi obata, koichi noguchi department of anatomy and neuroscience, hyogo college of medicine, japan there is compelling evidence indicating that the activation of extracellular signal-regulated protein kinase (erk) 1/2 and p38 mitogenactivated protein kinase (mapk) in the dorsal root ganglion (drg) and spinal cord contributes to the development of inflammatory and neuropathic pain. in the present study, we examined whether the newest member of the mapk family of proteins, erk5 (also known as big mapk 1 or bmk1) is activated in the drg and spinal cord and participate in pain-related behaviors in the l5 spinal nerve ligation (snl) model. l5 snl induced an increase in the phosphorylation of erk5 not only in the injured l5 drg, but also in the spared l4 drg at day 7 after surgery. furthermore, l5 snl induced a striking increase in erk5 phosphorylation in glial cells in the ipsilateral dorsal horn. our data suggest that activation of erk5 in the drg and spinal cord may contribute to the development of neuropathic pain. atsushi sakai 1 , minoru asada 1 , naoki seno 2 , hidenori suzuki 1 1 department of pharmacology, nippon medical school, tokyo, japan; 2 pharmaceutical research center, kyowa hakko kogyo co., shizuoka, japan glial cell line-derived neurotrophic factor (gdnf) has been known to alleviate the neuropathic pain. however, the mechanisms of gdnfinduced analgesia remain almost unclear. gdnf binds to gfr␣-1, which forms receptor complex and signals intracellularly through ret. recently, neural cell adhesion molecule (ncam) has been found to be an alternative signal-transducing receptor for gdnf. here, we report that ncam is involved in gdnf-induced analgesia in a rat model of the neuropathic pain. ncam mrna expression was decreased in the ipsilateral dorsal horn of the spinal cord after the nerve injury, but gdnf treatment returned its expression to the normal level. treatment with ncam antisense oligodeoxynucleotide blocked the analgesic effect of gdnf without affecting ret phosphorylation. these results suggest that activation of ncam signaling may provide a new strategy to relieve intractable chronic pain. ps2p-f094 interleukin-1␤ enhanced the excitability of trigeminal root ganglion neurons via activation of satellite glia following inflammation mamoru takeda 1 , jun kadoi 1 , msanori nasu 2 , masayuki takahashi 1 , shigeji matsumoto 1 1 dep. physiol and 2 res. cent. for odont. nippon dent. univ., japan the present study was investigated whether activation of satellite glial cells modulates the excitability of trigeminal root ganglion (trg) neuronal activity via the il-1␤ paracrine mechanism following inflammation. two days after cfa into the whisker pad area, the mean number of trg neurons that were encircled by glial fibrillary acidic protein and il-1␤-immunoreative satellite cells were significantly increased compared with those in the control. fg labeling was used to identify the trg neurons innervating the site of inflammation. in the fg-labeled small trg neurons, the occurrence of il-1␤ induced depolarization in inflamed rats was larger than that in control rats. il-1␤ application significantly increased the firing rate evoked by depolarizing pulses in the inflamed neurons compared with the control neurons. these results suggest that activation of trg satellite glial cells modulates the excitability of trg neuronal activity via the il-1␤ paracrine mechanism following peripheral inflammation. junichi kitagawa 1 , mamoru takeda 2 , jun kadoi 2 , yoshiyuki tsuboi 1 , shigeji matsumoto 2 , koichi iwata 1 1 dept. of physiol., sch. of dent., nihon univ., tokyo, japan; 2 dept. of physiol, sch. of dent. at tokyo, japan, nippon dental univ., tokyo, japan the present study was designed to elucidate an involvement of the primary afferent neurons in the trigeminal neuropathic pain using the rats model with chronic constriction nerve injury of the infraorbital nerve (ion-cci). the mechanical escape threshold was significantly lower in ion-cci rats at day 3 after ion treatment and the threshold decrement was lasting more than day 14. single unit activities of ion were recorded from the ion-cci rats. the firing frequency was significantly higher in a␦ fibers in ion-cci rats as compared with naive at day 3-14 after ion-cci. whole cell patch clamp recording was performed from the middle trg neurons. ik and ia currents were significantly smaller and ih current was larger in ion-cci rats than that of naive rats. the present results suggest that ik, ia and ih currents are involved in abnormal firing of trg neurons in the rats with ion-cci, resulting in neuropathic pain in trigeminal region following peripheral nerve injury. hisako urai, munehiro uda, katsuya kami graduate schools of sport and exercise science, osaka university of health and sport sciences, osaka, japan mechanical hyperalgesia of skeletal muscles has been known to occur following intense eccentric contraction such as downhill running (dhr). the present study examined the number of c-fos-positive neurons in spinal dorsal horn to determine peak of mechanical hyperalgesia following dhr in rats. furthermore, we investigated whether glial cells are activated in dorsal horn with excitation of secondary afferent neurons (san). rats performed an intermittent bout of dhr for 90 min. at 6, 12, 24, 48 and 120 h post-dhr, the rats were applied a weight on the right triceps surae muscle. immunohistochemical staining for c-fos and gfap on spinal cords was performed by freefloating abc method. the number of c-fos-positive neurons detected in superficial dorsal horn were increased at 6 h, peaked at 12 h and then decreased. intense gfap immunoreactivities were also detected at 6 and 12 h post-dhr. these results suggest that dhr generates mechanical hyperalgesia by increasing responsiveness of san, and moreover astrocytes may regulate excitability of san. katsuya kami, hisako urai, munehiro uda department of health science, osaka university of health and sport sciences, osaka, japan a production of inflammatory cytokines is increased in injured skeletal muscles. the present study examined relationship between production of inflammatory cytokines in skeletal muscles and fospositive neurons in spinal dorsal horn following downhill running in rats. the rats performed the downhill treadmill running for 90 min at 25 m/min. after the running, rats were applied the weight on the gastrocnemius muscles for 30 min, and then spinal cord and soleus muscles were removed from the rats. productions of il-1beta, il-6 and tnf-alpha in soleus muscles and expression of fos protein in dorsal horn were examined using immunohistochemical approach. at 6 h post-running, number of fos-positive neurons was increased, peaked at 12 h and then decreased to control level at 24 h post-running. vigorous inflammatory reactions with necrotic myofibers in soleus muscles were observed at 2 days post-running. these results indicated that increased numbers of fos-positive neurons in dorsal horn are induced prior to vigorous inflammation of skeletal muscles. shinichi sugiyo 1 , yusuke sakai 2 , aya masawaki 3 , takashi shimoda 2 , masayuki moritani 1 , motohide takemura 1 1 dept. oral anatomy and neurobiology, osaka university grad. sch. of dentistry, osaka, japan; 2 dept. of fixed prosthodontics, osaka university grad. sch. of dentistry, osaka, japan; 3 dept. of dental anesthesiology, osaka university grad. sch. of dentistry, osaka, japan diabetes mellitus is among the most common causes of painful peripheral neuropathy, worldwide. we examined if there exist the diabetic rat (dm)-specific difference in nociceptive behavioral and c-fos immunoreactivity (ir) by formalin test. injection of formalin into the upper lip 2 weeks before streptozotocin injection induced biphasic specific pain related behavior (prb) for 90 min. first phase was greater in dm than in the control rat (ctrl). in dm, second phase was much greater than ctrl. c-fos ir in the trigeminal caudal nucleus was also greater in dm than in ctrl. these results indicate that dm induced greater prb and c-fos expression following formalin injection into the rat upper lip. yasuko kozaki, satoshi hurune, fukushi kambe, hisao seo, kazue mizumura res. inst. environ. med., nagoya university, nagoya, japan we have reported that prostaglandin ep3 receptor (ep3r) activation attenuates the desensitization of bradykinin (bk)-induced increase of intracellular calcium ([ca 2+ ] i ) in a ptx-sensitive manner in cho cells expressing canine ep3r and mouse bk b2 receptor (b2r). in this study, we examined the involvement of protein kinase a (pka) in the desensitization of the bk response. when bk (1 nm) was applied twice with a 6-min interval to the cells expressing b2r, the second [ca 2+ ] i increase by bk was markedly attenuated. however, the pretreatment with a specific inhibitor of pka, h-89 (1 m) restored the second response. to further confirm camp increase by bk, the expression of a camp responsive reporter gene was examined. bk (10 pm) treatment for 1 h significantly increased the reporter gene expression. it is likely that bk increases the level of intracellular camp, and thus activates pka, resulting in the desensitization of the bk response. these results suggest that the desensitization of bkinduced increase in [ca 2+ ] i was at least in part mediated by pka. ps2p-f100 contribution of peripheral 5ht2a or 5ht3 receptors to fos expression in the trigeminal spinal nucleus (vsp) produced by the masseter muscle injury of rats keiichiro okamoto, akihisa kimura, tomohiro donishi, yasuhiko tamai dept. physiology, wakayama med univ., japan we have recently reported that orofacial nocifensive behavior evoked by the masseter muscle (mm) injury is attenuated by blocking peripheral 5ht2a or 5ht3/r in male rats with tmj inflammation. here we tested if these two 5ht/r subtypes contribute to fos responses in vsp after mm injury. formalin injection into mm produced fos-like immunoreactivity (li) in several areas of vsp and c2. fos-li was distributed mainly in the ventrolateral trigeminal subnucleus interpolaris/caudalis transition (vl-vi/vc) and vc/c2 transition regions. the number of fos-li induced by mm injury was increased in these areas in cfa-evoked tmj-inflamed rats for 7 days compared to naive rats. we tested if local 5ht2a or 5ht3/r antagonist affects fos expression in both groups. the number of fos-li in the vc/c2 but not vl-vi/vc region was reduced when drugs were injected locally prior to formalin injection in tmj-inflamed rats. these data suggest that peripheral 5ht2a and 5ht3/rs play critical roles in mediating mm nociception during tmj inflammation. keiko abe, hidemasa furue, kohei kga, go kato, toshiharu yasaka, akihiro tamae, toshihiko katafuchi, megumu yoshimura department of integrative physiology, graduate school of medical sciences, kyushu university, japan we examined the postsynaptic effects of 5-ht on substantia gelatinosa (sg) neurons in slice preparations of rat spinal cord and their relationship to the morphological features. in ∼60% of sg neurons examined, 5-ht induced an outward current. the outward current was mimicked and suppressed by a 5-ht 1a agonist and 5-ht 1a antagonist, respectively. in ∼10% of sg neurons, 5-ht evoked an inward current which was mimicked by a 5-ht 3 agonist. the outward current was observed mostly in excitatory neurons such as vertical cell, while the inward current was induced in an inhibitory neuron, islet cell. these findings suggest that 5-ht inhibits excitatory neurons and excites inhibitory neurons in the sg through activation of 5-ht 1a and 5-ht 3 receptors, respectively. the reciprocal postsynaptic actions of 5-ht on sg neurons in addition to presynaptic inhibitory effects on primary afferents might play an important role in descending control of nociceptive transmission by 5-ht. we examined effects of levobupivacaine, ropivacaine, bupivacaine and r-bupivacaine on epscs in substantia gelatinosa (sg) neurons of the spinal dorsal horn evoked by dorsal root stimulation, and on action potentials in dorsal root ganglion neurons generated by the dorsal root stimulation. in sg neurons, levobupivacaine reversibly suppressed the amplitude of monosynaptic a␦ and c fiber-evoked epscs. however, a␤ fiber-evoked epscs were slightly inhibited in amplitude. on the other hand, bupivacaine equally suppressed those three fiber-evoked epscs. in drg neurons, ic 50 of bupivacaine and r-bupivacaine were almost equal on a␤, a␦ and c neurons. however, ic 50 of levobupivacaine and ropivacaine on a␦ and c neurons were lower than that on a␤ neurons. the present results suggest that pure s (−) enantiomers especially levobupivacaine effectively inhibits noxious transmission to the spinal dorsal horn by the blockade of ap conduction through a␦ and c fibers. ps2p-g103 nitric oxide-dependent long-term potentiation revealed by real time imaging of nitric oxide production and neuronal excitation in spinal dorsal horn hiroshi ikeda, kei kusudo, kazuyuki murase dept. human & artificial intelligence systems, univ. fukui, fukui, japan no plays an important role in the induction of long-term potentiation (ltp) in spinal dorsal horn, which is believed to underlie hyperalgesia and allodynia. in this study, to elucidate the relationship of no to ltp, we measured the spatiotemporal distribution of no signal with the no-sensitive dye, and neuronal excitation with the voltagesensitive dye, in rat spinal cord slices. in superficial dorsal horn, neuronal excitation evoked by dorsal root stimulation was potentiated for more than 2 h after low-frequency conditioning stimulation (lfs). in the same slices that exhibited ltp, no was produced and distributed in the superficial dorsal horn during lfs. ltp and production of no were inhibited in the presence of no synthase inhibitors and an inhibitor of heme oxygenase, the synthetic enzyme for carbon monoxide (co). research funds: kakenhi to hi (17700306) and km (16500262) and grants from novartis foundation and promotion of science and sumitomo foundation to hi tao liu, tsugumi fujita, akiko koga, masafumi kosugi, terumasa nakatsuka, eiichi kumamoto dept. physiol., facult. med., saga univ., saga, japan in order to know the effect of a pla 2 activator melittin on inhibitory transmission in the substantia gelatinosa (sg; lamina ii of rexed), we applied the blind whole-cell patch-clamp technique to sg neurons in adult rat spinal cord slices. in about 90% of neurons examined, melittin (1 m) superfused for 3 min gradually increased the frequency and amplitude of spontaneous glycinergic inhibitory postsynaptic currents which were recorded at 0 mv in the presence of bicuculline. this action was visible about 2 min after the beginning of its superfusion and subsided within 8 min after washout. these melittin actions were reduced in extent by a pla 2 inhibitor 4-bromophenacryl bromide, while being unaffected by tetrodotoxin, and also by inhibitors of cyclooxygenase (cox) and lipooxygenase (lox). it is concluded that pla 2 activation pre-and postsynaptically enhances glycinergic transmission in sg neurons, possibly not through metabolites of cox and lox; this action would contribute to a modulation of nociceptive transmission. research funds: kakenhi (17500275) ps2p-g105 presynaptic p2y 1 receptor-mediated enhancement of inhibitory synaptic transmission in the rat spinal dorsal horn terumasa nakatsuka, shugo koga, tsugumi fujita, tao liu, masafumi kosugi, eiichi kumamoto department of physiology, faculty of medicine, saga university, saga, japan using whole-cell patch-clamp recordings, we examined whether the activation of p2y receptors can modulate synaptic transmission in dorsal horn (dh) neurons of adult rat spinal cord slices. bath applied 2-methylthio adp (2mesadp, 30 m), a p2y receptor agonist, did not change excitatory transmission, but clearly increased the frequency and amplitude of spontaneous inhibitory postsynaptic currents (ipscs) in about 20% of dh neurons recorded. miniature ipsc in the presence of ttx was increased in frequency by 2mesadp with no change in the amplitude. the 2mesadp-induced increase in miniature ipsc frequency was attenuated in extent by mrs2179 (30 m), a selective p2y 1 receptor antagonist. these results indicate that the activation of presynaptic p2y 1 receptors enhances inhibitory but not excitatory synaptic transmission in a subpopulation of dh neurons. thus, spinal p2y 1 receptors can be involved in an inhibitory effect on pain transmission. research funds: kakenhi (17700370), the japanese health sciences foundation (kh21006) ps2p-g106 presynaptic enhancement by proteinaseactivated receptor-1 agonist peptide of glutamatergic excitatory transmission in rat substantia gelatinosa neurons tsugumi fujita, terumasa nakatsuka, akiko koga, tao liu, masafumi kosugi, eiichi kumamoto dept. physiol., facult. med., saga univ., saga, japan we have previously reported that proteinase-activated receptor (par)-1 but not par-2 agonist (each 1 m) enhances glutamatergic excitatory transmission in substantia gelatinosa (sg) neurons. the present study examined a detail of the par-1 mediated enhancement by applying the whole-cell patch-clamp technique to sg neurons in adult rat spinal cord slices. par-1 agonist (sfllrn, 1 m) reversibly increased the frequency of spontaneous epsc without a change in the amplitude and also in holding current at -70 mv. this facilitatory action was resistant to tetrodotoxin, and was not seen in the presence of par-1 antagonist (yfllrnp, 1 m). these results indicate that the activation of par-1s existing in nerve terminals in the sg results in an increase in the spontaneous release of l-glutamate from there. it is suggested that par-1 activation in glutamatergic neuron terminals in the sg may be involved in the modulation of nociceptive transmission from the periphery. research funds: kakenhi (16700340) ps2p-g107 effect of tramadol metabolite m1 on glutamatergic excitatory transmission in rat spinal dorsal horn neurons akiko koga, tsugumi fujita, tao liu, terumasa nakatsuka, eiichi kumamoto dept. physiol., facult. med., saga univ., saga, japan in order to know the antinociceptive effect of tramadol, we examined the effect of m1, which is one of its metabolites, at 1 mm on glutamatergic excitatory transmission in substantia gelatinosa (sg) neurons of an adult rat spinal cord slice by using the whole-cell patchclamp technique. bath-applied m1 reduced the frequency but not amplitude of spontaneous excitatory postsynaptic currents (epscs) at −70 mv. this action was not seen in the presence of a -opioid receptor antagonist ctap (1 m). m1 also reduced the peak amplitudes of epscs which were monosynaptically evoked at −70 mv by stimulating primary-afferent a␦-and/or c-fibers in a spinal cord slice with an attached dorsal root. we conclude that m1 inhibits the quantal release of l-glutamate from nerve terminals in the sg through the activation of -opioid receptors; this action is not distinct in extent between primary-afferent a␦-fiber and c-fiber transmission. this effect of m1 would give a cellular basis for the antinociceptive effect of systemically-administered tramadol. narihito iwashita, natsu koyama department of physiology, shiga university of medical science, otsu, japan in our previous study, subcutaneous injection of glutamate into the human forearm evoked pain and produced skin temperature increase around the injection site. these results suggest peripheral glutamate receptors contribute to nociceptive signaling and neurogenic inflammation. in order to further investigate which subtype of glutamate receptors is involved in neurogenic inflammation, effect of nmda receptor antagonist mk-801 or non-nmda receptor antagonist cnqx was evaluated in hindpaws of pentobarbital-anesthetized rats. attenuation of skin temperature increase induced by simultaneous mk-801 injection with glutamate was larger than that of skin temperature increase induced by simultaneous cnqx injection with glutamate at the same concentration. on the other hand, inhibition of paw edema formation by cnqx was stronger than by mk-801. these data demonstrate that peripheral nmda receptor predominantly contributes to vasodilatation, while peripheral ampa/ka receptor predominantly contributes to increase of vascular permeability in glutamate-induced neurogenic inflammation. ps2p-g109 studies on pain control system (rept. 57): changes in phosphorylation of nr2b-contained nmda receptor in the spinal cord obtained from rats with painful neuropathy following chronic ethanol consumption kan miyoshi, minoru narita, michiko narita, tsutomu suzuki dept. toxicol., hoshi univ. sch. pharm. pharmaceut. sci., tokyo, japan chronic ethanol consumption produces a painful peripheral neuropathy. mechanical hyperalgesia was clearly observed during ethanol consumption and even after ethanol withdrawal in rats, and it lasted for 14 weeks. under these conditions, the immunoreactivities of phosphorylated-ser-1303 nr2b (p-ser-1303 nr2b) subunit and phosphorylated-conventional protein kinase c (p-cpkc) were significantly increased in the spinal cord following chronic ethanol consumption, whereas p-tyr-1472 nr2b subunit immunoreactivity was not changed in this region. the hyperalgesia induced by chronic ethanol consumption was significantly attenuated by repeated i.p. injection of ifenprodil, a selective nr2b-containing nmda receptor antagonist. these findings provide evidence for a substantial role of the phosphorylation of cpkc-dependent nr2b-contained nmda receptor in the development or/and maintenance of ethanoldependent neuropathic pain-like state in rats. ps2p-g110 prolonged depression of nociceptive response in the prefrontal cortex with high frequency stimulation of the amygdala yumi izawa, yoriko kawakami dept. physiol. tokyo women's medical university, tokyo, japan high frequency stimuli (hfs, 100 hz, 20 a, 30 s) delivered to the basolateral nucleus of the amygdala (bl) induced prolonged depression of the nociceptive specific response in the prefrontal cortex (pfc). we examined the receptor mechanism underlying this depression of pfc neuron activity. extracellular neural activities, induced by nociceptive stimulation applied peripherally, were recorded in the rat pfc. inhibitory effects of hfs delivered to the bl on nociceptive responses were blocked by specific antagonists of a metabotropic glutamate receptor (mglur) or nmda receptor microinjected locally into the pfc. dopamine depletion, produced by 6-ohda injected into the substantia nigra, also reduced the inhibitory effects of hfs. the mglur and dopamine receptor mediated prolonged depressions of nociceptive responses were induced by hfs of the amygdala. our results suggest that emotional condition modulates pain sensation. ps2p-g111 the nav1.3 sodium channel pathologically reconfigures the thalamic pain amplifier-generator after spinal cord injury bryan c. hains, stephen g. waxman yale university school of medicine, usa spinal cord injury (sci) induces pain-related phenomena associated with the aberrant expression of nav1.3, a rapidly repriming voltage-gated sodium channel. in this study we hypothesized that, following sci, neurons in the thalamus undergo similar electrophysiological changes linked to nav1.3. four weeks post-sci, nav1.3 protein was upregulated within thalamic neurons, where unit recordings revealed increased spontaneous discharge, afterdischarge, hyperresponsiveness to innocuous and noxious peripheral stimuli, expansion of peripheral rfs, and bursting. these properties persisted after interruption of ascending spinal barrage. lumbar intrathecal administration of specific antisense oligodeoxynucleotides against nav1.3 caused a significant reduction in nav1.3 expression and reversed electrophysiological alterations. these results show, for the first time, a change in sodium channel expression within neurons in the thalamus after injury to the spinal cord, and suggest that these changes contribute to altered processing of somatosensory information after sci. tomoki fukuda 1 , hiroyuki ichikawa 2 , ryuji terayama 2 , tomosada sugimoto 2 1 department of oral maxillofacial rehabilitation, okayama university, okayama, japan; 2 department of oral function and anatomy, okayama university, okayama, japan ib4-sap is a neurotoxin designed for targeting primary nociceptors with ib4 binding sites on the cell surface. however, the exact cell spectrum that is affected by the toxin has not been thoroughly investigated. we, therefore, unilaterally injected ib4-sap (2.5 l of 0.067% solution for each ganglion) directly into the 4th and 5th lumbar (l4 and 5) dorsal root ganglia (drgs). three weeks later, the rats were killed and drg sections were stained for ib4-binding. after counterstain, the cell body size of neurons were measured. ib4-sap reduced the total number of drg neurons in l4 and 5 ganglia combined by 21% (11652 ± 2213 on untreated side versus 9208 ± 1609 on treated side). small neurons (<1200 m 2 ) were reduced by 28% whereas large ones (≥1200 m 2 ) were not affected. ib4-binding neurons were mostly small (≥96%) and were reduced by 46%. the number of small neurons, that were not stained for ib4-binding, increased by 35% (1994 ± 286 versus 2700 ± 600). schuichi koizumi 1 , kaoru nasu-tada 1 , makoto tsuda 2 , emiko kunifusa 1,2 , kazuhide inoue 2 1 div. pharmacol., natl. inst. hlth. sci., tokyo, japan; 2 dept. mol. system pharmacol., grad. sch. pharmaceut. sci., kyushu univ., fukuoka, japan although microglial p2x4 receptor, a key molecule for the mechanical allodynia, is increased after peripheral nerve injury, the molecular mechanisms underlying its upregulation remain unknown. here, we describe the influence of fibronectin on p2x4 receptor expression in microglia. microglia that were cultured on fibronectin-coated dishes showed a marked increase in p2x4 receptor expression. western blot examination of the spinal cord from rat with spinal nerve injury indicated that fibronectin was upregulated on the ipsilateral side. interestingly, intrathecal injection of atp-stimulated microglia revealed that microglia cultured on fibronectin-coated dishes was more effective in the induction of allodynia than microglia cultured on control dishes. taken together, our results suggest that spinal fibronectin is elevated after the peripheral nerve injury and it may be involved in the upregulation of the p2x4 receptor in microglia, leading to neuropathic pain. research funds: mf16, kakenhi (170230570) ryousuke fujita, hiroshi ueda div. mol. pharmacol. & neurosci., nagasaki univ. grad. sch. of biomed. sci., nagasaki, japan we have reported that intrathecally administered lpa or endogenous lpa generated upon sciatic nerve injury causes demyelination of dorsal root (dr), which is supposed to be one of key molecular mechanisms underlying neuropathic pain (nat. med. 2004). however it remained whether lpa has direct actions on myelinated schwann cells (sc). in the present study we examined the direct effects of lpa on dr fibers in ex vivo culture system. scanning electron microscopy (sem) study revealed that lpa caused a swelling and disruption of myelinated fibers at 24 h. in transmission em analysis, the addition of lpa caused a disruption of myelin sheath of a␦-and a␤-fibers. on the other hand, it was found that c-fibers were separated to each other by scs in naive fibers. following the addition of lpa, c-fibers showed direct contacts and some of them were uncovered. all these effects were also observed either with or without dr ganglion. thus, it is suggested that lpa has direct actions on myelinated and unmyelinated scs to cause demyelination of a-fibers and to uncover c-fibers. research funds: kakenhi 17109015 ps2p-g115 lysophosphatidic acid (lpa) down-regulates myelin associated proteins in cultured dorsal root fibers norikazu kiguchi, ryousuke fujita, hiroshi ueda div. mol. pharmacol. & neurosci., nagasaki univ. grad. sch. biomed. sci. nagasaki, japan we have reported that intrathecally administered lpa or endogenous lpa generated upon sciatic nerve injury causes demyelination of dorsal root, which is supposed to be one of key molecular mechanisms underlying neuropathic pain (nat. med. 2004). these treatments also caused a decrease in myelin protein and their gene expression levels. here we report the biochemical evidence underlying this demyelination in cultured fibers. the addition of lpa at 1 mm decreased the protein levels of myelin basic protein (mbp) at 24 h. this action was significantly inhibited by botulinum neurotoxin/c3 (bont/c3). on the other hand, lpa also caused a decrease in gene expression of various myelin proteins, such as mbp, pmp22, mag, p0 in cultured fibers. the maximal decrease was observed all at as early as 3 h after the addition of lpa. bont/c3 and y27632 abolished the lpainduced down-regulation of mbp gene. all these findings suggest that the down-regulation of gene expression of myelin proteins is through rhoa-rock pathway underlying lpa-induced demyelination. neuropathic pain arise from peripheral never injury. the purpose of this study was to explore behavioral characteristics and investigate the involvement of nmda receptors and opioid receptors in the behavioural responses following spared nerve injury (sni). the hind paw withdrawal threshold to cold-and mechano allodynia and heatyperalgesia were tested at 0 and 3, 7, 14, 21, 28 days after operation. pre-emptive co-administration of mk-801 and morphine were tested. sni produces mechanical and cold allodynia and heat hyperalgesia. co-injection of morphine and mk-801 decreased cold-and mechanoallodynia, but had slightly effect on heat-hyperalgesia. the present data demonstrate that the sni procedure result in severe changes in behavioral responses in whether hyperalgesia or allodynia. coadministration of both drugs seems to be more effective to alleviate induced neuropathic pain. satoshi deyama 1,2 , naomi akiyama 1 , mikie hirata 1 , takayuki nakagawa 2 , shuji kaneko 2 , masabumi minami 1 1 dept. of pharmacol., grad. sch. of pharm. sci., hokkaido univ., sapporo, japan; 2 dept. of mol. pharmacol., grad. sch. of pharm. sci., kyoto univ., kyoto, japan the bed nucleus of the stria terminalis (bst) is involved in the regulation of negative affective states such as anxiety and fear. in this study, we examined the role of the noradrenergic (na) transmission within the bst in the negative affective component of pain in rats. we found that excitotoxic lesion of the bst attenuated intraperitoneal acetic acid-or intraplantar formalin-induced conditioned place aversion (cpa) without reducing nociceptive behaviors. we showed that na release within the bst was significantly elevated by these noxious stimuli. intra-bst injection of a ␤-adrenoceptor antagonist timolol significantly suppressed these noxious stimuli-induced cpa without affecting nociceptive behaviors. these results suggest that visceral and somatic noxious stimuli-induced na release within the bst contributes to the negative affective, but not sensory, component of pain. noriyuki ozaki, mariko kawai, yasuo sugiura department of functional anatomy and neuroscience, nagoya university, graduate school of medicine, nagoya, japan neonatal maternal separation induces visceral hyperalgesia in colon. this study compares the effects of maternal separation on response sensitivity to gastric and colorectal distension in long-evans rats. maternal separation was performed for 3 h per day between postnatal day 1 and 14. visceral sensitivities were assessed in stomach and colon at 10 weeks of age by visceromotor responses induced by either gastric or colorectal distension. somatic pain sensitivities were also assessed by von frey filaments and radiant heat. in contrast to the response to colorectal distension, maternal separation induced decreased response to gastric distension, especially in male rats. no difference was found between control and separated rats in somatic pain sensitivities. these results indicate that maternal separation differentially modulates visceral pain sensation in stomach and colon. research funds: grant-in-aid for scientific research 16500216 ps2p-g119 change by aging in muscular mechanical hyperalgesia after lenghtening contraction k. mizumura, t. taguchi, t. matsuda, t. nasu res. inst. environ. med., nagoya univ., nagoya, japan our previous experiments have shown that the mechanical threshold of the edl muscle underwent lengthening contraction (lec) lowered 1 to 3 days after exercise in rats (8 w old). c-fos expression in the superficial dorsal horn increased in l4 spinal segment when the edl muscle was compressed 2 days after exercise. from these results we have concluded that the muscle became hyperalgesic after lec. in the present experiment, we examined whether this hyperalgesia after lec changes along aging. male sd rats 8, 80 (81-84) and 130 (131-133) w old were used. the basal mechanical threshold (randall-selitto method) of edl muscle tended to be higher in 80 w old rats, but not significant. after lec, the threshold started to decrease 1 day after lec in all three age groups. it returned to the pre-lec level 4 days after lec in 8 and 80 w old rats only. recovery of 130 w old rats delayed up to 7 days after lec. increased c-fos expression in the superficial dorsal horn was observed in l4 as well as in l5 in 130 w old rats. these results suggest that hyperalgesia occurs in larger areas and lasts longer in aged animals. tong liu, hong p. wei, chun y. yuan, ai k. guo institute of neuroscience, chinese academy of sciences, china drosophila can display complex courtship behavior. male-male courtship behavior shown in some fly mutants, but here we report for the first time that the male-male courtship behavior can be induced by disturbance of dopamine level. to up-regulate dopamine level, uas-th/th-gal4 males were used, which showed high level of dopamine and performed active male-male courtship behavior. this behavior was attenuated by decreasing dopamine level either through drug breeding or genetic method. the increased courtship behavior in uas-th/th-gal4 males is specific to male partners, because the males courted females normally. to down-regulate dopamine level, pale ts , th temperature sensitive mutant was used. when raised at restrictive temperature, pale ts showed obvious attraction to wild type males. our study shows that the high level or low level of dopamine can induce male-male courtship behavior in active or passive manner. athushi yokoyama 1 , masaharu akita 2 1 kanagawa life-science res., japan; 2 kamkura, kanagawa, japan we have developed the screening system for drug and chemical compounds of food by the used of ratwhole embryo culture. the advantages of whole embryo culture are to examine the direct effects of l-calnitin (lcal) on embryo and also to find the non-teratogenic agent (d-calnitin:dcal). as the testing agent, lcal was examined in this study using the rat embryo cultured from day 11 to 13 of gestation. in treated embryos of lcal, the embryonic heart beat, the crown-rump length, the embryo weight and the total embryonic somites were not decreased. on the other hand, the malformation (the defects of neural tube) and the short size of head length were observed in the embryos cultured with lcal. in treated embryos of d-calnitin (dcal), there parameter was not decreased. the observed malformation of lcal was not observed in the embryos cultured with dcal. these results might be due to the differences between lcal and dcal in the embryo toxicity. yoshihisa uenoyama, kenji takase, junya hirata, hiroko tsukamura, kei-ichiro maeda laboratory of reproductive science, nagoya university, nagoya, japan the mechanisms underlying the pubertal increase in gonadotropinreleasing hormone (gnrh) secretion are poorly understood. recently, metastin was found to stimulate gnrh secretion and mutations of its receptor are associated with lack of puberty. effect of immunoneutralization of endogenous metastin in the brain on the onset of puberty was examined to clarify the physiological significance of metastin in timing the puberty. when wistar-imamichi strain female rats received an infusion of anti-metastin antibody into the third ventricle during days 25-39 of age, they did not show the first estrus before 52 days of age with mean age of 54.7 ± 2.0 day. in contrast, most of normal mouse igg-treated controls showed the first estrus by 43 days of age with mean age of 42.8 ± 2.2 day. the age of vaginal opening was also delayed in the anti-metastin-treated rats. thus, the present study demonstrates that the puberty onset was delayed by immunoneutralizing central metastin. central metastin may be involved in timing the onset of puberty in female rats. kenji takase, yoshihisa uenoyama, shunji yamada, hiroko tsukamura, kei-ichiro maeda lab. of reproductive science, nagoya university, aichi, japan metastin has been considered to be involved in triggering pulsatile gonadotropin-releasing hormone (gnrh)/luteinizimg hormone (lh) secretion to time the onset of puberty. the present study aimed to determine expression of metastin, a novel kiss-1 gene product, in the rat brain during peripubertal period. wistar-imamichi strain female rat shows vaginal opening on around days 29 of age (d29), and first estrus on around d34. brain tissues were obtained on d21, 26, 31, 36 and 41. kiss-1 mrna expression in the arcuate nucleus-median eminence region (arc-me) and anteroventral periventricular nucleus (avpv) increased significantly from d21 to 26 and was kept at a high level thereafter. gpr54 mrna expression in the medial preoptic area increased significantly from d21 to 31. metastin-immunoreactive cells were not found on d21 but were apparent in the arc-me on d26 onward. these results indicate that metastin expression increases in the arc-me and avpv before vaginal opening, suggesting that metastin triggers the onset of gnrh/lh secretion in female rats. toshiyuki saito 1 , sei-etsu fujiwara 1 , kenjiro konno 2 , takashi yamaguchi 3 , tetsu nemoto 4 , etsuko kasuya 1 , ryosuke sakumoto 1 1 anim. neurophysiol. lab., natl. inst. agrobiol. sci., tsukuba, japan; 2 inst. exp. anim. res., fac. med., gunma univ., maebashi, japan; 3 grad. sch. sci. & eng., yamagata univ., yonezawa, japan; 4 sch. health sci., fac. med., kanazawa univ., kanazawa, japan in the present study, we recorded and examined local field potentials (lfps) in the hippocampus of piglets performing an operant task by a radio-telemetry system. under halothane anesthesia, a pair of tungsten electrodes was implanted into the hippocampus and fixed on the surface of the skull with a transmitter using dental cement. after recovery from surgical procedures, the piglets were moved to a training cage. in the lfps, spike-shaped waves were frequently found just before the piglets pushed a switch with their noses. these waves may represent some of the hippocampal neural activities associated with switch manipulation for getting a food reward. yasuo osawa department of bioscience, tokyo university of agriculture, tokyo, japan memory extinction is an inhibitory learning rather than forgetting or erase of conditioned memory. from the view of treatment of phobia and post traumatic stress disease (ptsd) caused by fear memory, it is important to find the drugs to facilitate extinction of fear memory. importantly, previous studies using pavlovian fear conditioning have shown that d-cycloserine, a nmda receptor agonist, facilitates memory extinction. in this study, to examine whether d-cycloserine is applicable for the treatment with another type of fear memory, we investigated effects of d-cycloserine on extinction of aversive memory in mice. indeed, we performed conditioned taste aversion (cta) task, where the ingestion of a novel taste is paired with transient sickness. our results indicated the injection of d-cycloserine before but not after the re-exposure to cs facilitates extinction of cta. ps2p-g126 hippocampal neural responses during a conditional delayed stimulus-response task in awake mice nobuhide kitabayashi 1 , teruko uwano 1,3 , anh tran 2,3 , eturou hori 1,3 , taketoshi ono 2,3 , hisao nishijo 1 1 system emotional science, univ. of toyama, japan; 2 integrative neurosci, molecular and integrative emotional neurosci., univ. of toyama, japan; 3 crest, japan to investigate a hippocampal (hf) involvement in the representation of temporal sequence in mice, neural responses were recorded during performance of a conditional delayed stimulus-response association task. a trial was initiated by one of two different conditioned tones. after a 2 s delay, two serial reinforcements with an intervening delay was presented; aversive air puff-delay-tube protrusion to evoke licking sucrose solution and the opposite order of the same reinforcements. of 85 hf neurons, 26 responded to the tones, the reinforcements, and during the delay. some neurons responded to a presentation of a sensory stimulus, and other responded differentially during the delay depending on the reinforcement sequence. the results suggest a crucial role of the hf in representation of serial events in episodic memory in mice as well as in rats and primates. further studies will be conducted using genetic modified-mice to clarify the neural substrate in episodic memory. naoko inoue 1 , atsu aiba 2 , kaoru inokuchi 1 1 mitsubishi kagaku inst. life sci. (mitils), tokyo, japan; 2 grad. sch. med., kobe univ., kobe, japan vesl-1s/homer-1a and vesl-1l/homer-1c are splicing isoforms encoded by the vesl-1 gene. vesl proteins bind and regulate mglur1/5, ip3 receptor, ryanodine receptor, and trp channel at the postsynapse. the expression of vesl-1s is upregulated by tetanic stimulation that elicits l-ltp. vesl-1s is thought to play a critical role in the conversion from short-term to long-term memory (ltm). in this study we generated vesl-1s gene-targeting mice (ko) and examined whether vesl-1s plays a role in the ltm formation. analysis with the contextual fear conditioning revealed a defective in consolidation process, reconsolidation process, and remote memory formation in ko. ko further showed an enhanced freezing decrement within a test session, indicating faster within-session extinction. in contrast, consolidation process of the extinction was normal in ko. these results demonstrate that the vesl-1s protein plays critical roles in various processes of the ltm formation. we now examine the signaling pathways important for ltm formation that are altered in ko. hiroshi ageta 1 , r. migishima 1 , s. kida 2 , k. inokuchi 1,3 1 mitsubishi kagaku inst. life sci (mitils), japan, 2 tokyo univ. agricul., japan; 3 crest, jst, japan memory process consists of at least four distinct phases, acquisition, consolidation, maintenance, and retrieval. activin ␤a mrna increases following l-ltp induction in the hippocampus, suggesting that activin plays a role in the memory formation. here, we generated activin and follistatin (antagonizes activin function) transgenic mice in which the transgene expression was tightly regulated by dox in a forebrain-specific manner (tet off system). transgene expression was turned off or on within 3 d by (+/−) dox. contextual fear conditioning with these mice revealed that activin function is required during maintenance phase of fear memory for one week retention. furthermore, activin plays a role in the re-consolidation process. thus, fear memory that was once acquired and consolidated tightly could be "erased" by inhibiting the activin function during maintenance phase. these mice are useful for the study of ptsd. ps2p-h129 sex differences in the effects of chronic estrogen treatment on fear conditioning in c57bl/6j mice takaaki ozawa, mumeko tsuda, sonoko ogawa kansei, behavioral and brain sciences, university of tsukuba, tsukuba, japan it has been suggested that estrogen may play a role in the regulation of learning and cognitive functions. although most of previous studies have focused on elucidating facilitatory effects of estrogen on learning in females, estrogen is also known to affect various behaviors in males. in the present study, we investigated the effects of different doses of estrogen on fear conditioning (fc) learning in both sexes of mice. gonadectomized c57bl/6j mice were implanted with a silastic capsule containing 0, 5, 10 or 20 g of estradiol benzoate. since it is possible that estrogen may indirectly modify learning by affecting general activity, emotionality and anxiety levels, we tested the mice in open-field and light dark transition paradigms prior to fc. mice were then conditioned for fear responses (freezing) to tone stimulus and tested for both contextual and cued fc responses. we found that estrogen facilitated both types of fc learning in females, whereas it inhibited them in males especially at a higher dose, with a small effect on emotional behaviors. ps2p-h130 analysis of brain regions activated during memory consolidation in passive avoidance task zhang yue department of bioscience, tokyo university of agriculture, tokyo, japan short-term memory (stm) is labile. to generate long-term memory (ltm), stm is stabilized through a process known as memory consolidation. importantly, previous studies have shown that memory consolidation requires the function of transcription factor creb whose activation induces c-fos expression. in this study, we tried to understand molecular mechanisms of consolidation of passive avoidance memory that has been known to be amygdala and hipocampusdependent. indeed, we investigated brain regions that are activated following the learning by analyzing the expression level of c-fos using immunocytochemistry. consistent with previous study, we observed increase in c-fos expression in amygdala and hippocampus. more interestingly, we also found this increase in prefrontal cortex, indicating that prefrontal cortex plays critical roles in memory consolidation in light-dark passive avoidance task. hiroshi nomura, norio matsuki laboratory of chemical pharmacology, graduate school of pharmaceutical sciences, university of tokyo, tokyo, japan we have demonstrated the effect of ethanol on reactivated fear memory for the first time, using contextual fear conditioning. rats were conditioned with mild footshock, reexposed to the training context, immediately injected with ethanol or saline, and finally tested 48 h after reexposure. ethanol-treated groups expressed longer freezing and the effect lasted for 2 weeks. reactivation was necessary for the effect. the injection of ethanol itself did not induce a fearful response. as memory retrieval triggers memory extinction and reconsolidation, we investigated whether extinction process is involved in this ethanol effect. increasing retrieval time did not enhance freezing by ethanol, suggesting that ethanol had no effect on memory extinction. post-reactivation injections of anisomycin revealed that retrieval triggered reconsolidation. moreover, picrotoxin inhibited the memory enhancement by ethanol. these studies demonstrate that ethanol enhances reactivated contextual fear memories via activation of gaba a receptors. ps2p-h132 analyses of brain regions activated in reconsolidation and extinction phases of contextual fear memory nori mamiya, akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan the retrieval of conditioned fear memory by conditioned stimulus (cs) initiates two processes; reconsolidation or extinction. we previously found that the change in memory stability after retrieval (reconsolidation) associates with memory extinction. to understand the regulatory mechanisms of memory stability after the retrieval at the anatomical level, we here investigated the brain regions that are activated in reconsolidation and extinction phases. we measured the levels of phospho-creb inducing changes in neural plasticity following the re-exposure to cs. short re-exposure to cs inducing reconsolidation increased in phospho-creb in amygdala and hippocampus. in contrast, longer re-exposure inducing extinction increased in phospho-creb in amygdala and prefrontal cortex. these results indicate that distinct brain areas are activated in response to short or long re-exposure to cs and suggests that amygdala plays crucial roles in the interaction between reconsolidation and extinction. ps2p-h133 analysis of molecular mechanism for the destabilization of retrieved contextual fear memory akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan reconsolidation acts to stabilize, whereas extinction tends to weaken the expression of the original memory. to understand the mechanisms for the regulation of memory stability after the retrieval, we have investigated the relationship between reconsolidation and extinction using contextual fear conditioning. we previously found that memory extinction is associated with regulation of fear memory stability, indicating the interaction between memory reconsolidation and extinction phases. in this study, we compared molecular signatures of reconoslidation and extinction using mice. pharmacological experiments using antagonists for cannabinoid receptor 1 (cb1) and l-type voltage-gated calcium channels (lvgccs) indicated that both cb1 and lvgccs are required for memory extinction but not consolidation and reconsolidation. more interestingly, blockade of either cb1 or lvgccs function prevents the disruption of the original memory by protein synthesis inhibition. these results suggest that cb1 and lvgccs are required for not only memory extinction but also the destabilization of reactivated memory. hotaka fukushima, akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan previous our studies using contextual fear conditioning revealed three distinct time-dependent phases following memory retrieval: stable, reconsolidation, extinction phases. to understand the nature of memory processing following retrieval, we examined the effects of reexposure on memory reconsolidation and extinction using light-dark passive avoidance task. this task is thought to allow us to discriminate between reconsolidation and extinction phases at the time point when mice enter dark box from light box. brief re-exposure to light box did not affect the stability of fear memory (stable phase). further extending re-exposure to light box triggered the requirement of protein synthesis for re-storage of fear memory (reconsolidation phase). in contrast, entry from light into dark box initiated extinction of fear memory (extinction phase). additionally, using pharmacological blockade of cb1 and lvgccs, we also found that cb1 is required for only memory extinction but that lvgccs are required for memory extinction and reconsolidation. wakoto matsuda 1 , takahiro furuta 1 , kouichi nakamura 1,2 , takeshi kaneko 1,2 ps2p-h136 difference in organization of corticostriatal and thalamostriatal synapses between patch and matrix compartments of rat neostriatum fumino fujiyama 1 , tomo unzai 1 , kouichi nakamura 1,3 , sakashi nomura 2 , takeshi kaneko 1,3 1 department of morphological brain science, kyoto university, kyoto, japan; 2 department of physical therapy, kyoto university, kyoto, japan; 3 crest, japan the striatum, which has patch/matrix compartments, receives glutamatergic inputs from cortex and thalamus. in the present study, the differences in synaptology of these inputs between both compartments were examined. axon terminals positive for vesicular glutamate transporter (vglut)2, thalamostriatal inputs, were less dense in patch region, whereas vglut1-positive corticostriatal inputs were evenly distributed. quantitative analysis revealed 84% of vglut2positive synapses in patch region were formed with spines, whereas 70% in matrix region were made with dendritic shafts. in contrast, the targets of vglut1-positive inputs were mainly spines in both regions. moreover, vglut2-positive axospinous synapses in patch region were larger than vglut1-positive ones. the present observation suggests that thalamostriatal connection is more plastic in patch region. research funds: kakenhi (16500217, 17022024,16200025, 17022020 (0131),17650100) ps2p-h137 single cell tracing of thalamostriatal projection neurons with reference to patch and matrix compartments of rat striatum tomo unzai 1 , fumino fujiyama 1 , takeshi kaneko 1,2 1 department of morphological brain science, university of kyoto, kyoto, japan; 2 crest, japan the striatum consists of patch and matrix compartments, and receives glutamatergic inputs mainly from the cerebral cortex and thalamus. thalamic intralaminar nuclei are known to project exclusively to matrix compartment. on the other hand, it has not been clarified which thalamic nuclei project to patch compartment. in the present study, we combined single cell tracing with immunohistochemistry for mu opioid receptor, which is specifically expressed by patch neurons, to reveal the distribution of thalamostriatal axon terminals in relation to striatal compartments. recombinant sindbis virus expressing membrane-targeted green fluorescent protein (palgfp) was injected into the rat thalamus. a single neuron in the thalamic paraventricular nucleus extensively projected to the striatum and preferentially to patch compartment compared with matrix compartment. the axons were also distributed in the thalamic reticular nucleus, accumbens nucleus, amygdala, and cerebral cortex. research funds: kakenhi (16500217, 17022024, 16200025, 17022020(0131) , 17650100) ps2p-h138 lesion of the nucleus accumbens dopamine system shortens the lever pressing interresponse time and delays the response initiation in mice yuji tsutsui 1 , kayo nishizawa 1 , nobuyuki kai 2 , kazuto kobayashi 2,3 1 dept. of psychology, fukushima univ., japan; 2 dept. mol. genet., fukushima medi. univ., japan; 3 crest, jst, kawaguchi, japan dopamine transmission is thought to be important for rodents to perform operant behaviors such as lever pressing. the lever pressing experiment was conducted to examine the effects of 6-ohda injections into the nucleus accumbens (acb) in c57bl/6j mice. all mice were trained to press the lever for a food pellet using a fixed ratio 5 (fr5) schedule. the mice were injected with ascorbate vehicle or 6-ohda into the acb, and then tested post-surgically using the fr5 schedule again. the 6-ohda-injected mice showed the acceleration of response speed, which was revealed by the shortening of interresponse time between each of the five lever pressings, and the suppression of the initiation of the response to the next step. this suppression of initiation was revealed by the increase of time from the last presentation of food to the next initiation. these results suggest that the acb dopamine system is important for the initiation and control of the operant behaviors in rodents. hideshi shibata laboratory of veterinary anatomy, tokyo university of agriculture and technology, fuchu, tokyo, japan retrosplenial area 29 is one of the important structures for spatial memory and behavior in the rat. to understand more fully the functional roles played by area 29, it is essential to clarify the neural circuitry subserving these functions. in the present study, we analyzed the organization of frontal cortical projections to area 29 in the rat, using retrograde transport of cholera toxin b subunit (ctb). ctb injections into area 29d retrogradely labeled cells in the orbital cortex and the caudal parts of the anterior cingulate and primary and secondary motor cortices. ctb injections into area 29c labeled cells in similar cortical regions, except for the orbital cortex. ctb injections involving areas 29a and b labeled cells in the caudal part of the anterior cingulate cortex. the results show that the orbital, anterior cingulate, and primary and secondary motor cortices have a different pattern of projections to each subdivision of area 29, suggesting different functional roles played by each subdivision of area 29 in spatial memory and behavior. eiichi jodo 1 , yoshiaki suzuki 2 , tadahiro katayama 1 , ken-yo hoshino 2 , yukihiko kayama 1 1 dep. of physiol., fukushima med. univ., fukushima, japan; 2 dep. of neuropsy., fukushima med. univ., japan it has been shown previously that the dopaminergic neurons in the ventral tegmental area (vta) selectively respond to a stimulus repeatedly paired with reward stimuli in a classical conditioning paradigm. since the vta receives dense projection from the medial prefrontal cortex (mpfc), such response selectivity of vta neurons may in part be produced by inputs from the mpfc. however, few studies have compared the firing pattern between these two regions. our present experiment was designed to make such a comparison in freely moving rats. two different tones were sequentially presented, one of which (target, 30%) was paired with intracranial simulation of the reward area. the unit activity was recorded from the mpfc and/or the vta. pfc and vta neurons exhibited phasic excitation with the peak latency of about 0.1 s to both tones, while only the target tone induced sustained activation of firing activity lasting until presentation of the reward. masato inoue, akichika mikami primate research institute, kyoto university, inuyama, aichi, japan to investigate the neuronal mechanism in the ventrolateral prefrontal cortex (vlpfc) and inferotemporal cortex (it) for holding information for object and their order of presentation, we examined single neuronal activities in the vlpfc and it while monkeys were performing a serial probe reproduction task. in the task, two sequentially presented objects were memorized and then a target object was selected from memorized objects based on a color stimulus. in 19% out of 438 vlpfc neurons, the delay-period activity showed objectselectivity and order-selectivity. in only 6% out of 254 it neurons, the delay-period activity showed object-selectivity and order-selectivity. the starting time of the order-selective activity was earlier in the vlpfc. these results suggest that the vlpfc plays a role in holding information for object and their order of presentation and the it receives information for object and their order of presentation from the vlpfc. masao yukie, yasutaka oosawa department of behavioral physiology, tokyo metropolitan institute for neuroscience, fuchu, japan relational memory theory (eichenbaum et al., 1994) has been proposed from evidence that the hippocampal damage in rats impairs learning of transverse patterning task (a+ versus b−; b+ versus c−; c+ versus a−). very recent monkey study (alvarado et al., 2005) demonstrated that lesion of the hippocampus produced a significant impairment in that task and supported such a theory. in our study, however, ischemic damage of the hippocampus has not impaired learning of such a transverse patterning task (yukie et al., 2005) . in the present study, we examined effects of lesion of the monkey perirhinal cortex on transverse patterning task using two sets of 2d visual stimuli presented in a wgta. our three monkeys with perirhinal lesions failed to attain a learning criterion within a training limit of 75 sessions in phase 3, although they learned easily the four problems in phases 1 and 2. our results suggest that the perirhinal cortex, but not the hippocampus, is important for learning of transverse patterning task, that is, for formation of relational memory. yasuko sugase-miyamoto 1 , noriyuki higo 1 , munetaka shidara 2 1 neuroscience research inst., aist, tsukuba, japan; 2 grad. sch. of tsukuba univ., tsukuba, japan a recent dopamine d2 receptor study using antisense cdna showed that d2 receptor in rhinal cortex is crucial for learning associations between visual stimuli and reward schedules. neuronal responses in the perirhinal cortex differentiate the visual cues only when the cues are associated with the schedule states, while those in area te are related to physical attributes of the cue independently of the schedule states. to investigate the cellular substrate for d2mediated associative learning, we examined monkey temporal lobe immunohistochemically with a d2 receptor antipeptide antiserum. d2 receptor immunoreactivity was observed in the pyramidal cells in layers ii-vi of the rhinal cortex and area te. the signal was mainly observed in cell bodies, and also in both apical and basal dendrites for some cells. the signal in layers v-vi was stronger in area 36 of the perirhinal cortex than in area teav. the differential localization between area 36 and te suggests the differential roles of the two areas in associative learning process. by using axonal transport of fast blue, diamidino yellow and tritiated amino acids, we determined the afferent and efferent connections of the retrosplenial cortex (rsp) in the macaque monkey. the rsp receives heavy projections from the subiculum, presubiculum and the caudal entorhinal areas (ec-ecl), and projects back to the presubiculum and the ec-ecl. the supracallosal portion of the rsp has connections primarily with the caudal half of the subiculum and presubiculum, as well as the lateral zone of the ec-ecl. the caudoventral portion of the rsp is, in contrast, mainly connected with the rostral half of the subiculum and presubiculum as well as the medial zone of the ec-ecl. the two portions of the rsp, thus, have access to different portions of the medial temporal lobe. these results indicate that there are two distinct neural systems in the retrosplenial-medial temporal network. hideko nakano 1 , natsuko yoshida 2 , kiyohisa natsume 3 1 kyushu kyoritsu university, fukuoka, japan; 2 kyushu institute of technology, fukuoka, japan; 3 kyushu institute of technology, fukuoka, japan eeg activity was examined in english rhythm acquisition of japanese students who learn english as a foreign language (efl). we measured theta, alpha and beta rhythms of five subjects while they were reading aloud the materials and listening to the audio-recording, using eight electrodes attached to their skulls. the result shows that the increase of theta power at f3 and f4 was the highest and suggests that the theta rhythm at f3 and f4 may have a relationship to the process of english rhythm acquisition. moreover we found the highest increase in theta power when the subjects began to orally reproduce every line of the rhythm materials. this finding was observed in three right handlers except a left handler and a right handler who had just returned after 6-month english study experience in australia. these results suggest that the change of theta power at frontal areas may be more closely related to the japanese efl learners' english rhythm acquisition. research funds: kakenhi (1565081) ps2p-h146 neural correlates of music retrieval: an eventrelated fmri study using sparse temporal sampling takamitsu watanabe 1 , sho yagishita 1 , hideyuki kikyo 1,2 1 department of physiology, the university of tokyo school of medicine, tokyo, japan; 2 department of molecular neuroimaging, national institute of radiological sciences, chiba, japan we investigated neural correlates of music memory using eventrelated functional magnetic resonance imaging and sparse temporal sampling technique with originally composed musical materials. written informed consent was obtained from all the subjects in accordance with the declaration of helsinki, and the experimental procedure was approved by the institutional review board of the university of tokyo school of medicine. a 1.5 t scanner system was used (te = 50 ms; tr = 14 s; acquisition time = 3.0 s). we demonstrated that the right hippocampus, bilateral lateral temporal cortices, left prefrontal cortex and left precuneus are involved in music retrieval. in addition, performance-based analysis suggested that the right hippocampus is associated with the accuracy of music memory. in this fmri study, we determined the neural correlates of the intellectual excitement. sentences describing facts in natural and human science were visually presented, and subjects judged whether they know the fact or not. after the fmri, each subject self-evaluates subjective "intellectual excitement" of each sentence. positive correlation with the self-evaluated intellectual excitement for known facts and novel facts were analyzed. significant correlation between cortical activation and self-evaluated intellectual excitement for novel facts was observed in the left and the right parahippocampal gyrus and for known facts was in the left orbital part of inferior frontal gyrus. it suggests the cortical areas related to self-evaluated intellectual excitement are different between getting of novel knowledge and recognition of existing knowledge. hyeonjeong jeong 1 , motoaki sugiura 3 , yuko sassa 2 , keisuke wakusawa 2,4 , kaoru horie 1,5 , shigeru sato 1,5 , ryuta kawashima 2,5 1 gsics, tohoku university, sendai, japan; 2 niche, tohoku university, japan; 3 miyagi university of education, sendai, japan; 4 department of pediatrics, school of medicine, tohoku university, japan; 5 the lbc research center, tohoku university, japan a foreign language word is learned and retrieved either in daily situations (situation) or written text (text), and memory transfer is required when the learning and retrieval modes are different. in this experiment, normal japanese subjects learned korean words in the situation and text modes in video clips. during a subsequent fmri session, subjects were presented with the learned words in different movie clips; half of the learned words was presented in the same mode as in the learning session (match), and the rest was presented in a different mode (mismatch). comparison of the mismatch with match condition revealed significant activation in the orbital part of the left inferior frontal gyrus. the results suggest that this area plays a role in the memory transfer of foreign language words when the learning and retrieval modes are different. georgina e. cruz 1 , christie l. sahley 2 , kenneth j. muller 1 1 physio. & biophys., univ. of miami, miami, fl, usa; 2 biol. sci., purdue univ., west lafayette, in, usa in some animals much is known at the level of single synapses about mechanisms underlying behavioral sensitization, but in no system is the involvement of interactions at the network level well understood. the s-cell network of the medicinal leech is a chain of electrically coupled interneurons spanning the nerve cord with distributed sensory input and motor output and is crucial for sensitization of reflex shortening. its firing increases with sensitization although few additional s-cells initiate impulses during the reflex. we tested the hypothesis that the initial burst of impulses from the s-cell in the stimulated segment suppresses initiations in adjacent segments. hyperpolarizing the central s-cell to reduce its firing during skin stimulation markedly increased the number of initiations in adjacent s-cells, which corroborated the limited expansion of initiation sites seen in the behaving animal. a computational model of s-cell refractoriness further supported the idea of interaction among s-cells during sensitization. research funds: nih, u.s.a. ps2p-h150 sensory/motor modules regulating the development of peer social relationship mamiko koshiba 1,2 , shun nakamura 2 1 jst, crest, japan; 2 ncnp social intelligence is indispensable for animal's survival and could have evolved to language capability. further, as a recent problem in japan, 'fewer children' supposedly causes the more tight interaction of child-parent, reciprocally the less between siblings or friends. in order to study the genetic and epigenetic development of peer social relationship after birth, we controlled peer interaction through limiting a particular sensory/motor modality as social deprivation and examined the effect on the active attachment behavior of domestic chick to conspecific mates. the chick has a merit of being precocial and unique in higher animal with no need of parent-care. comparing to the chicks reared as a group, the isolated chicks didn't develop their active attachment to peers. meanwhile, the behavior study with the chicks deprived not sensory/motor function itself, but only social interaction in auditory, visual, olfactory or tactile system, suggested that vocal communication at least must play a key role for the development. dna-chip study along the different social context brought candidates of social genes. shogo sakata 1 , minoru hattori 2 1 department of behavioral sciences, hiroshima university, higashi-hiroshima, japan; 2 graduate school of biosphere science, hiroshima university, higashi-hiroshima, japan peak interval (pi) 30-s procedure is a very good method to investigate for timing. six male wistar rats were trained for five days a week in pi 30-s procedure over 30 days. the 3-s bin of lever press responses on probe trials showed a clear peak point. the temporal distributions had the peak time of regression curve fitting with the gaussian function. the peak time corresponded to near the 30-s with reinforcement durations. then nicotine was administrated to the rat by intraperitoneal injection before daily pi 30-s session. results showed that the peak time in the nicotine administration was slightly leftward shift compared to the saline injection. however the pattern of temporal distribution of responses was not changed by the nicotine treatment as well as control condition. it suggests that the nicotine administration affects on the time perception that was reflected by the peak durations of responses. ps2p-i152 the effect of random practice schedule on arbitrary stimulus-response association learning satoshi tanaka 1,2 , ritz oshio 1,3 , norihiro sadato 1,4 , manabu honda 5,6 1 nips, okazaki, japan; 2 jsps, tokyo, japan; 3 nagoya univ., nagoya, japan; 4 ristex, jst, tokyo, japan; 5 sorst, jst, tokyo, japan; 6 ncnp, tokyo, japan previous studies suggest that randomly ordered practice facilitates retention and transfer of motor skills compared to blocked or regularly ordered practices. it remains unclear, however, whether the advantageous effects of random practice can be expended to cognitive skill learning in humans. we examined the simultaneous learning of multiple arbitrary stimulus-response (s-r) associations under three different practice schedules: blocked, random and regularly ordered. behavioral data indicate that subjects performing the random practice showed better performance of the retention and transfer of learning compared to those performing the blocked or regularly ordered practice. the present result indicates that random practice schedule is effective also for s-r association learning, which are considered as a bridge between motor control and cognitive control. ps2p-i153 sports rats show increased level of bdnf in the cerebellum, possibly learning and memorizing well masaki morishima, sayuri hara, yutaka nakaya dept. nutrition and metabolism, univ. of tokushima, japan previously, we reported that the activation of hippocampal norepinephrine neurotransmission following a decrease in monoamine oxidase a was observed in sports, a novel hyper-running rat on wheel. this study assessed whether sports show increased bdnf levels and better learning and memory. compared to control, both protein and mrna levels of bdnf in cerebellum were significantly elevated in sports even without wheel running, and slightly increased in hippocampus. in the cerebellum of sports, trkb/pi3k pathway was activated, whereas mapk pathway was activated in the hippocampus. locomotor activity assessed by the open field test showed that the sports were significantly more active in center coat than control. in the passive avoidance test, sports did not enter a dark area at next time indicating that sports showed better passive avoidance learning. these results suggest that bdnf signaling of sports were activated from trkb to mapk and pi3k in the hippocampus and cerebellum, respectively, and that these signaling pathways might play an important role in learning and memory. research funds: kakenhi (17500429) ps2p-i154 selective manipulation of working memory through d1 and d2 receptors: computer simulation shoji tanaka, hiroki yata dept. of electrical & electronics eng., tokyo, japan though a number of experimental results suggest that working memory processes are controlled by the dopaminergic system, its mechanism is still unclear. to elucidate the mechanism, we have constructed a model of the prefrontal cortical neural circuit for working memory. the neurons in the model are leaky integrate-and-fire model with ampa, nmda, gaba, and leak conductances and have dopamine d1 and d2 receptors. the computer simulation with this model shows that d1 receptor activation mainly affect working memory activity itself, while d2 receptor activation affect the termination of working memory, being consistent with the experimental result. the simulation also mimics the hyper-and hypo-dopaminergic states. under such conditions, like schizophrenia, simulated pharmacological treatments using agonists and antagonists of d1 and d2 receptors indicate efficacies of some these treatments for the restoration of working memory. in conclusion, this kind of simulation shows how dopamine controls working memory by using the synergism of the actions of dopamine, glutamate, and gaba. kozo sugioka, tomiyoshi setsu, tatsuro yamamoto, toshio terashima div. anat. & dev. neurobiol., dept. neurosci., kobe univ. grad. sch. med., kobe, japan we examined activity and habituation in rats with experimentallyinduced abnormal morphogenesis of the hippocampus. pregnant rat (jcl:wistar) was injected with saline or 25 mg/kg mam on the 15th day of gestation. the activity of male and female offspring was measured for each 1 h light and dark period, and the habituation to the visual stimulation was observed by measuring the activity with every 1 min interval for 1 h under 5 min dark/light alternative schedule during weaning and adult periods. activity was measured using infra-red sensor in a home-cage placed in the experimental room. the mam-treated rat showed hyperactivity for dark-period during both weaning and adult periods, and showed retarded habituation during weaning period. sex difference of behavioral alteration was evident during adult period in both groups. these behavioral disorders were discussed in relation to the mam-treated rat showing abnormal hippocampus (disruption of the ca1 pyramidal layer and ectopic neuron mass). ps2p-i156 long-lasting tagging of functionally activated neurons in the mouse brain naoki matsuo, leon reijmers, mark mayford the scripps research institute, la jolla, ca, usa immediate-early genes (iegs) have been widely used as activity markers for mapping neurons involved in specific animal behaviors including learning and memory. however, conventional ieg approaches that use immunohistochemistry or in situ hybridization allow to detect neurons only shortly after their activation and does not enable genetic manipulations. here we have developed transgenic mice that allow selective and long-lasting tagging of neurons that were activated in a given brain region at a given time point. the mice consist of two components; c-fos promoter driven tetracycline-controlled transactivator (tta) and teto promoter regulated feedback loop. when strong neuronal activity occurs in the absence of tetracycline analogs such as doxycycline (dox), c-fos promoter driven tta initiates the teto-linked expression of mutant tta (tta*) that is not inhibited by dox. this teto-linked gene expression is then maintained indefinitely by feedback activation via the tta* even in the presence of dox. using this system, we have examined the expression of bicistronic teto promoter driven tau-lacz and egfp-glur1. hamid gholamipour 1 , shirin babri 2 , khameneh saied 3 1 department of physiology, university of tabriz, tabriz, iran; 2 university of tabriz, iran; 3 university of tabriz, iran diabetes mellitus is one of the most prevalent diseases in the world. because hippocampus is an important area for memory formation, the present study is scheduled to investigate the effect of insulin injection in ca1region of hippocampus on memory formation. fifty male rats were divided into five groups. (1) control (2) sham operation (3) test (4) diabetic/saline (5) diabetic/insulin. groups 4 and 5 were made diabetic by treatment with stz (50 mg/kg, i.p.). in all but the control group, two canula were stereotaxically implanted in ca1 region of hippocampus. learning was tested and compared between groups through passive avoidance test. results showed that in the test group the latency increased as compared to control and sham groups (p < 0.05). compared to sham group diabetic/insulin group showed increased latency (p < 0.05) but no significant difference was found between diabetic/saline and diabetic/insulin groups. in conclusion, according to the results obtained in this study, insulin facilitates memory in intact rats but not in diabetic sex differences in hippocampus-dependent memory formation are well documented, but the mechanisms are poorly understood. the ca 2+ /calmodulin (cam) kinase cascade regulates gene transcription in the hippocampus, which is required for long-term memory (ltm) formation. we hypothesized that sex differences in transcriptional regulation may account for the sexual dimorphisms in memory formation. we tested this idea by studying the role of cam kinase kinases (camkks). using mouse molecular genetics we found that camkk␤ is required for spatial, but not contextual ltm. consistent with the impaired spatial memory formation, camkk␤ null mutants lacked spatial training-induced creb activation and had impaired late ltp. in contrast to camkk␤, camkk␣ is required for contextual, but not for spatial ltm. furthermore, female camkk mutants had normal spatial and contextual ltm. thus, we show that there are malespecific mechanisms to regulate gene transcription that may explain sex differences in hippocampus-dependent memory formation. akshay anand, sudesh prabhakar, monika bhatia, c.p. das department of neurology, post graduate institute of medical education and research, chandigarh, india background: parkinson's disease has a prevalence rate of 19 per 100,000 in india. we studied the park 4 polymorphism in north indian population and parkin expression in early parkinson's disease (n = 30) and sporadic parkinson's disease (n = 30). methods: pcr, sscp, rflp and direct sequencing analysis were used to screen mutations. results: our results revealed homozygous exonic mutations in exon-1, 3 and 6 in early pd and exon-1 and 12 in sporadic pd, heterozygous mutations in exon 4 and 9 in five early pd and one sporadic pd patient. frequency of s/n polymorphism was significantly high suggesting that exchange of serine to asparagine at position 167 of protein affects the secondary structure or hydrophobicity of the protein resulting in pathogenicity. our facs analysis of these samples indicates reduced parkin expression correlating with severity of mutations. conclusions: we conclude that high frequency of parkin 4 mutations in pd population in india affect parkin expression resulting in pd. wanida tripanichkul 1 , kittisak sripanichkulchai 2 , david finkelstein 3 1 faculty of medicine, srinakharinwirot university, thailand; 2 faculty of medicine, khon kaen university, thailand; 3 university of melbourne, australia emerging data suggests beneficial effect of estrogen for parkinson's disease (pd), yet the exact mechanisms implicated remain obscured. activated glia observed in mptp mouse model and in pd may participate in the cascade of deleterious events that ultimately leads to dopaminergic nigral neuronal death. estrogen can modify glial expression of inflammatory mediator, such as cytokines and chemokines implicated in neurodegeneration. to determine whether estrogen-elicited neuroprotection in pd is mediated through glia, adult male c57bl/6 mice were pretreated with 17beta-estradiol (e2), injected with mptp on the day 6 and brains were collected on day 11. e2 pretreatment decreased nigral neuronal loss and diminished striatal fibers deficit induced by mptp. the neuroprotective effect of e2 was coincident with an attenuation of a glial response within the snpc and striatum. these findings propose that e2 neuroprotection in mptp mouse model may mediate through reactive glia inhibition. ps2p-i163 effect of angiotensin-converting enzyme inhibitor perindopril in mptp-treated mice; immunohistochemistry and in vivo electron spin resonance (esr) study rumiko kurosaki 1 , fumihiko yoshino 2 , masaichi chang-il lee 2 1 dept. of food sci. and nutrition, showa women's univ., tokyo, japan; 2 clin. care and med. div. of pharmaco., kanagawa dental college, japan we investigated the effects of perindopril on the dopaminergic system and the oxidative stress in mice after mptp treatment. administration of perindopril showed dose-dependent neuroprotective effects against striatal dopamine and its metabolites depletion after mptp treatment. we have reported that th, gfap, pv, nnos and cu, zn sod positive cells in the substantia nigra was changed after mptp treatment in our immunohistochemical study. the administration of perindopril significantly attenuated mptp induced changes of these immunopositive nigral cells. we could measure increased oxidative stress in the brain of mptp and perindopril treatment mice using by in vivo esr technique. our results provide further evidence that the ace inhibitor perindopril may offer a novel therapeutic strategy for parkinsonǐs disease. research funds: kakenhi (17700577) ps2p-i164 recruitment of calbindin into substantia nigra dopamine neurons suppresses the onset of parkinsonian motor signs shigehiro miyachi 1 , kaori sawada 1 , haruo okado 1 , atsushi nambu 2 , masahiko takada 1 1 tokyo met. inst. neurosci., fuchu, tokyo, japan; 2 natl. inst. for physiological sci., okazaki, japan there is a consensus that dopaminergic neurons in the substantia nigra that express calbindin, a calcium-binding protein, are selectively invulnerable to parkinsonian insults. based on this notion, an attempt was made to test the hypothesis that parkinsonism may be suppressed by recruitment of calbindin into a subpopulation of nigral dopamine neurons that does not normally contain calbindin. an adenoviral vector expressing calbindin was injected unilaterally into the striatum of macaque monkeys, to let calbindin express in the dopaminergic neurons via retrograde transport. two to three weeks later, the parkinsonism-inducing drug mptp was systemically administered several times. parkinsonian motor signs, such as muscular rigidity and flexed posture, appeared only on the side ipsilateral to the calbindin recruitment when cumulative doses of mptp exceeded threshold for their bilateral onset. toru yasuda, hideki mochizuki, yoshikuni mizuno department of neurology, juntendo university school of medicine, tokyo, japan using the serotype-2 raav vectors, we have recently reported the protective effect of parkin on the ␣-synuclein (␣s)-induced nigral dopaminergic neurodegeneration in a rat model. here we investigated the neuronal specificity of ␣s toxicity and the effect of parkin co-expression in a primate model. another serotype (type-1) of raav (raav1) carrying αs cdna (raav1-␣s), and a cocktail of raav1-␣s and raav1 carrying parkin cdna were unilaterally injected into the striatum of macaque monkeys, resulting in protein expression in striatonigral gabaergic and nigrostriatal dopaminergic neurons. the injection of raav1-␣s alone caused a decrease of th-immunoreactivity in the striatum, while there was no effect on gabaergic neurons. in the presence of overexpressed parkin, ␣s seemed to be less accumulated and/or phosphorylated at ser129 residue in gabaergic neurons. these suggest that the ␣s toxicity is not expressed in non-dopaminergic neurons but the ␣s-ablating effect of parkin is exerted in all neurons introduced in primates. tomokazu oshima, yohsuke narabayashi narabayashi memorial laboratory of neurology, neurological clinic, tokyo, japan rigidity in aged parkinsonians is often intractable against surgeries. we investigated how their rigidity scored by updrs was related with ␤-band local field potentials (␤-waves) of the surgical targets in thalamic ventrolateral nucleus (vl) and posteroventral pallidal internal segment (pvp). forty patients aged 70-80s gave informed consent for thalamotomy and/or pallidotomy. we divided the patients into groups with rigidity of 0.5-1 (i), 1.5-2 (ii), 2.5-3 (iii), and 3.5-4 (iv). the ␤waves were rated with total periods (%) of 13-27 hz wavelets in 3-s sample records. rigidity was re-scored after the surgeries. the vl ␤-waves were rated 65-70% in groups i-iii with a slightly increasing tendency for increasing rigidity, but declined to about 55% in group iv. the pvp ␤-waves were 60-80%, but with a decreasing tendency for increasing rigidity. the surgeries alleviated rigidity in all the groups, but were least effective in group iv with least vl and pvp ␤-waves. the results suggest that the pathology of aged parkinsonian rigidity develops beyond the pallido-thalamic pathway. yoshihisa tachibana 1,2 , hirokazu iwamuro 1,3 , masahiko takada 4 , atsushi nambu 1,2 1 div. syst. neurophysiol., natl. inst. physiol. sci., okazaki, japan; 2 sokendai; 3 dept. neurosurg., univ. tokyo, tokyo, japan; 4 dept. syst. neurosci., tokyo met. inst. neurosci., fuchu, tokyo, japan to approach a new therapy for parkinson's disease, extracellular unit recordings combined with microinjections of glutamate-related drugs were performed in the external and internal segments of the globus pallidus (gpe/gpi) of mptp-treated parkinsonian monkeys (macaca cyclopus). compared with the normal state, spontaneous oscillatory discharges were so often observed in the gpe/gpi and the subthalamic nucleus (stn) of the parkinsonian monkeys. microinjections of ionotropic glutamate receptor antagonists into the vicinity of recorded gpe/gpi neurons reduced their abnormal oscillations. these results suggest that glutamatergic excitatory input from the stn contributes to the oscillatory activity of gpe/gpi neurons, and that intrapallidal injections of ionotropic glutamate receptor antagonists may ameliorate some of parkinsonian symptoms. one of the pathological features of parkinsonǐs disease (pd) is loss of dopaminergic neurons in the substantia nigra pars comapacta (snpc). and it has been known that ␣-synuclein is involved in the neuronal loss. during the dopaminergic neuronal loss, activated microglia were centered in snpc. we hypothesize that ␣-synuclein may play a role in microglial activation to migrate to the pathological regions and to perform the neuronal cytotoxicity. we demonstrated that ␣-synuclein induced the cd44 expression on microglia and also enhanced the mt1-mmp expression to shed off cd44 at the cell surface and degrade surrounding ecm to open the migratory way. a53t mutant ␣-synuclein showed greater level of cd44 shedding and cell migration. extracellular treated ␣-synuclein also increased cd44 and mt1-mmp expressions dose-dependently. among the multiple signaling pathways, erk pathway was involved in ␣-synuclein induced cell migration. these induced cell migration were also confirmed in human pd patients. research funds: national creative research initiative grant (2004 grant ( -2006 ps2p-j169 serotonergic fibers are involved in the conversion of l-dopa to dopamine in the striatum and the substantia nigra pars reticulata of parkinsonian model rats ryohachi arai 1 , hiromasa yamada 1 , yoshinari aimi 1 , ikuko nagatsu 2 1 department of anatomy, shiga university of medical science, otsu, japan; 2 fujita health university school of medicine, japan dopaminergic neurons in the substantia nigra pars compacta (snc) project their axons to the striatum (st) and their dendrites to the substantia nigra pars reticulata (snr). dopamine released from these axons and dendrites is important in the regulation of motor activity. in parkinson's disease, dopaminergic neurons in the snc degenerate. l-dopa is the most effective drug for this disease. we hypothesize that, in parkinson's disease, a part of administered l-dopa is converted to dopamine in serotonergic fibers of the st and snr. here we produced parkinsonian model rats by the unilateral injection of 6hydroxydopamine into the snc, and found that serotonergic fibers in the st and snr were immunohistochemically positive for dopamine after l-dopa administration in the rats. therefore, it is possible that serotonergic neurons may be involved in the therapeutic effects of l-dopa for parkinson's disease. in mptp-induced pd monkey, reactive microglia are observed around neurons in nigra several years after mptp treatment and may be related to the progression of pd. to evaluate if reactive microglia in striatum and/or nigra of mptp-induced pd mice are present for a long time after mptp administration, like pd monkey. iba 1-and tb4distribution in microglia were immunohistochemically investigated at 0 h and 7 days after twice mptp-treatments (one treatment comprised of 4 intraperitoneal injections of 20 mg/kg mptp at 2 h interval) to c57bl/6 and balb/c at 6 months (mo) interval. the recognizable change of iba 1-and tb4-distibution in microglia of both mice strains was observed even 6 mo after the first treatment. the twice mptp treatments tended to aggravate the symptoms in both mice strains, compared with once treatment. these results suggest that reactive microglia are present for a long time after the treatment by mptp and must play a role in the chronic progression of pd. ps2p-j171 activated microglia affect the nigro-striatal dopamine neurons differently in neonatal and aged mice treated with mptp hirohide sawada 1 , ryohei hishida 2 , yoko hirata 3 , kenji ono 4 , hiromi suzuki 4 , shin-ichi muramatsu 2 , imaharu nakano 2 , kunihiro tsuchida 1 , toshiharu nagatsu 1,4 , makoto sawada 4 1 school of medicine, fujita health university, aichi, japan; 2 division of neurology, jichi medical university, japan; 3 department of biomolecular science, gifu university, japan; 4 research institute of environmental medicine, nagoya university, japan microglia play an important role in inflammatory process of parkinson's disease. we examined the effects between neonatal and aged microglia activated with lps on the nigro-striatal dopamine (da) neurons in mice treated with mptp. by mptp administration to neonatal mice, the number of da neurons in the substantia nigra was significantly decreased, whereas that in mice treated with lps and mptp was recovered. on the contrary, the number of da neurons of the 60 week-old mice treated with mptp was significantly decreased with lps treatment. these results suggest that activated microglia in neonatal mice have neurotrophic potential, in contrast to the neurotoxic effect in aged mice. hyposmia is one of the most characteristic symptoms of parkinson's disease (pd). it may occur even before the motor symptoms start. in the olfactory bulb (ob), dopaminergic cells were present at glomerular layer. furthermore, it has been reported that ob contains neural stem cells. thus, ob has attracted attention because of its unique regenerative potential. in the present study, we established isolation of neurosphere forming cells (nsfcs) derived from adult mice ob, and examined proliferation potential in ob after dopaminergic neuronal loss induced by mptp, a selective toxin for dopaminergic neurons, utilized frequently as pd model. the number of neurospheres derived from adult ob was not decreased with mptp administration, rather significantly increased. we also evaluated nsfcs differentiation into neural subtypes. the isolation of neural stem cells has helped to establish the cellular basis of neurogenesis and the exciting potential for transplant-mediated treatment of degenerative cns disease like pd. ps2p-j173 phosphorylation of erp57 in adult rat brain with neonatal 6-ohda treatment qinghua li, yasuyoshi watanabe department of physiology, osaka city university graduate school of medicine, osaka, japan dopaminergic neuron degeneration occurs in sporadic parkinson's disease (pd), but the mechanism of sporadic pd is not clarified. we prepared neonatal dopamine depleted rats, by i.c.v. injection of 6-ohda at 3(p3) and 6 days after birth, to investigate the mechanism of dopaminergic neuron degeneration. at p56, tyrosine hydroxylase (th) immunostaining cells were significantly reduced in the substantia nigra, and th immunostaining fibers were significantly reduced in the striatum, thus this model mimics the selective dopaminergic neuron degeneraion in sporadic pd. by two-dimensional electrophoresis we found that a certain protein was phosphorylated in the 6-ohda lesioned rats at p56, and it was identified as disulfide-isomerase a3 precursor (erp57) a kind of molecular chaperone of the endoplasmic reticulum (er) by maldi-tof ms. the result suggests that the phosphorylation of erp57 may have the key function to induce dopaminergic neuron degeneration and somehow relates to the pathogenesis of sporadic pd. katsunori nishi department of neurology, tokyo metropolitan institute for neuroscience, tokyo, japan regrowth of survived dopaminergic (da) neurons after the administration of psi, a potent proteasome inhibitor, was examined in vitro. dissociated cell co-culture was prepared from embryonic rat mesencephalon and striatum. psi (20 or 25 nm, 48 h) was applied to cultures at 7 days in vitro and succeeding changes of da neurons were investigated up to 35 days. more than 95% of da neurons reduced in number after the administration of psi, and a few truncated da neurons, devoid of neurites, being observed. non-da neurons were less severely affected at these concentrations of psi. regrowth of da neurites was observed approximately 2 weeks after the administration of psi and continued during the observation period. in most of the regrowing da neurons, one of the processes extended far longer than the rest, suggesting that severely injured neurons retain the capacity to reextend axons. regrowth was less remarkable in mesencephalic culture lacking striatum indicating that target cells are necessary for this effect. in conclusion, psi-damaged da neuron has strong regrowth potential in vitro. ps2p-j175 specific expression of proapoptotic factor pag608 on motor neurons in spinal cords of l-dopatreated parkinsonian models ikuko miyazaki, masako shimizu, francisco j. diaz-corrales, maria f. esraba-alba, masato asanuma dept. of brain sci., okayama univ. grad. sch. of med., dent. and pharmaceut. sci., japan we previously identified a proapoptotic gene, p53-activated gene 608 (pag608), as a dopa-induced gene in the striatum of l-dopa-treated parkinsonian models, which increased p53 expression to promote apoptosis by its nuclear translocation. last year, we also reported specific induction of pag608 in the internal capsule of l-dopatreated and constitutive expression in the smi-32-immunopositive motor neurons in the pontine nucleus and motor nuclei of trigeminal nerve and facial nerve. in the present study, we examined distribution of pag608 in the spinal cords of l-dopa-injected parkinsonian rats by immunohistochemistry. l-dopa treatment showed inducing tendency of pag608 expression on the motor neurons in the anterior corn and lateral corticospinal tract of spinal cords. the expression of pag608 in the motor nuclei of cranial nerves and its induction in the spinal cords suggests its possible involvement in motor dysfunction such as dyskinesia. ps2p-j176 an approach to the generation of ar-jp mouse model: crossbreeding of pael-r transgenic mice with parkin knockout mice hua-qin wang 1,2 , yuzuru imai 2 , haruhisa inoue 1,2 , ayane kataoka 2 , sachiko iita 2 , nobuyuki nukina 2 , ryosuke takahashi 1,2 1 neurology, university of kyoto, kyoto, japan; 2 bsi, riken, saitama, japan since loss of parkin e3 activity appears to be causal of ar-jp, accumulation of potentially toxic parkin substrates should result in degeneration of da neurons. however, parkin knockout mice show no different da neuronal loss even at old ages, presumably due to relative short lifespan of mice. pael-r is one of the best characterized parkin substrates. we generated pael-r transgenic mice and crossbred it with parkin knockout mice. pael-r transgenic mice showed modest alterations in dopamine metabolism and behavioral deficits without displaying obvious dopaminergic neuronal loss at the age of one year. however, when pael-r transgenic mice were crossbred with parkin knockout mice, the da neuronal loss was induced in a pael-r gene dosage-dependant manner. these results strongly support that pael-r accumulation substantially contributes to dopaminergic neurodegeneration in ar-jp. parkinson's disease, a common motor disorder, is caused by a degeneration of dopaminergic neurons in the substantia nigra. after dopamine denervation, an over-activity of glutamatergic pathways has been found and that is implicated in the neuropathology of parkinson's disease. previous study (lai et al., 2004) have found that application of an antisense oligodeoxynucleotide specific for nr1 have successfully knockdown the expression of nr1 gene expression in the striatum of 6-hydroxydopamine-lesioned rats. in the present study, modulation of gene expression of nr1 was re-addressed using a small interfering rna (sirna) specific for nr1. in pc12 cells, reductions of nr1 proteins after a single application of nr1 sirna were found by western blot experiments. and after one single application of nr1 sirna in the striatum of the lesioned rats, a significant reduction in apomorphine-induced rotation was found. slight reductions in the levels of nr1 immunofluorescence were found in the striatum after the sirna treatments. lai et al., 2004. neurochem. int. 45, 11-22. research funds: faculty research grant, frg/04-05/ii-27, hong kong baptist university ps2p-j178 homocysteine and parkinson's disease: effects of acute intranigral administration on dopaminergic system g. chandra, k.p. mohanakumar indian institute of chemical biology, kolkata, india homocysteine (hcy) is implicated in a number of geriatric multisystem disorders and patients with hyperhomocysteinemia exhibit profound neuropsychological abnormalities. parkinsonǐs disease (pd) patients receiving long-term l-dopa therapy are reported to have elevated plasma hcy levels. we studied whether hcy is neurotoxic to the nigrostriatal dopamine (da)-ergic system in sd rats. animals infused unilaterally in substantia nigra pers compacta (snpc) with hcy (0.25-1 mol in 1 l) showed dose dependent loss of da and its metabolites, in the ipsilateral striatum on 19th day. animals with 1 mol hcy exhibited significant motor disabilities and spontaneous and da-ergic drug-induced turning behaviors. in these animals a clear loss of neurons was visible in snpc, which were shown to be daergic by tyrosine hydroxylase immunoreactivity. intra-raphe infusion of hcy did not alter the neurotransmitter levels in the serotonergic perikarya or terminals. these results indicate the toxic potential of hcy to the da-ergic system and suggest that chronic l-dopa therapy in pd patients may further deteriorate the disease. ps2p-j179 ubiquitin proteasome system was impaired by the aggregate formation of mutant ␥pkc found in sca14 takahiro seki 1 , takayuki shimahara 1 , naoko adachi 2 , naoaki saito 2 , norio sakai 1 1 dept. mol. pharmacol. neurosci., grad. sch. biomed. sci., hiroshima univ., hiroshima, japan; 2 lab. mol. pharmacol., biosig. res. ctr., kobe univ., kobe, japan we have previously demonstrated that several mutant protein kinase c gamma (␥pkc), found in several families of spinocerebellar ataxia type 14 (sca14), are susceptible to cytoplasmic aggregation and cause cell death in cho cells, indicating that this property is involved in the etiology of sca14. however, the relationship between the aggregate formation of mutant ␥pkc and cell death remains unclear. accumulating evidences indicate that the impairment of the ubiquitin proteasome system (ups) is related to the pathogenesis of many neurodegenerative disorders. therefore, we examined whether the aggregate formation of mutant ␥pkc affects ups function. the immunoreactivities for ubiquitin and proteasome were intensely accumulated in the aggregates of mutant ␥pkc. decreased proteasome activities were also observed in cells having aggregated mutant ␥pkc. these results indicate that the aggregation of mutant ␥pkc exert cytotoxic effect via the impairment of ups. it is well known that oxidant stress is involved in many pathologic conditions including brain ischemia and neurodegenerative diseases. recently, however, another type of stress, endoplasmic reticulum (er) stress has also been reported to be associated with such diseases. er stress is characterized by accumulation of unfolded proteins in the er that is caused by inhibition of protein modification, disturbance of ca 2+ homeostasis or oxygen deprivation. we recently reported that targeting disruption of herp, a novel er stress-related gene, caused f9 cells vulnerable to er stress. using these cells, we developed a screening system for molecules that suppress er stress. approximately 300 compounds have been screened, and we found some molecules that protect human neuroblastoma cells against er stress and oxidative stress. we speculate that this system could provide novel therapeutic targets to the er stress and oxidative stressrelated diseases. toshiyuki araki 1 , yo sasaki 2 1 department of peripheral nervous system research, national institute of neuroscience, ncnp, tokyo, japan; 2 washington university school of medicine, st. louis, missouri, usa axonal degeneration which is observed in a variety of neuropathological conditions or physical damage to axons is a self-destructive program that is independent from programmed cell death. we previously reported that increased nicotinamide adenine dinucleotide (nad) production by the overexpression of nicotinamide mononucleotide adenylyltransferase1 (nmnat1) or exogenously applied nad can protect neurites from degeneration caused by mechanical or neurotoxic injury of neuronal cells. the mammalian nad biosynthesis is mediated by at least 6 different kinds of enzymes and each enzyme converts different substrate to nad or its precursors. here we investigated whether overexpression of these enzymes or exogenous application of nad precursors protects neurites from degeneration through increased supply of nad. cocaine is considered to affect spine morphology and the composition of postsynaptic density (psd) of medium spiny neurons in nucleus accumbens (nac). we examined the accumulation of several proteins altered by cocaine challenge after withdrawal of repeated cocaine administration in psd fraction of rat nac at different time points. total psd protein yield was decreased at 10 min, but next increased at 2 h and returned to basal at 6 h after cocaine challenge. actin showed a similar pattern but was maintained at high level at 6 h. both psd-95 and glur1 were increased between 2 h and 6 h like actin. by contrast, some proteins such as drebrin were decreased after the peak at 2 h. interestingly, the 20 s proteasome subunit demonstrated a dramatic upregulation at 2 h. these data suggest that the composition of psd proteins is regulated by proteasome activity as well as actin cycling. it is possible that some proteins may be removed from psd by proteasome following transient requirement for organizing psd in the nac of chronic cocaine-administrated animals. ps2p-k184 effects of mdma and 5-meo-dipt on serotonin transporter and dopamine transporter yosuke yamauchi, takaya izumi, takayuki nakagawa, shuji kaneko dept. mol. pharmacol., grad. sch. pharm. sci., kyoto univ., kyoto, japan by two electrode voltage-clamp recordings from xenopus oocytes heterologously expressing serotonin transporter (sert) or dopamine transporter (dat), the effects of two addictive agents, 3,4methylenedioxymethamphetamine (mdma) and 5-methoxy-n,ndiisopropyltryptamine (5-meo-dipt), on sert and dat were examined. as previously reported, mdma (0.3-10 m) dose-dependently induced transport-associated, inward current response in the sertexpressing cells. interestingly, mdma-induced current response was also observed in dat-expressing cells. on the other hand, 5-meo-dipt (0.3-300 nm) evoked an outward current response in sertexpressing cells similarly to that of selective 5-ht reuptake inhibitors. no current response was observed when 5-meo-dipt was applied to dat-expressing cells. these results suggest that mdma is transported not only by sert but also by dat, and that 5-meo-dipt suppresses the spontaneous transport activity of sert. junichi kitanaka 1 , nobue kitanaka 1 , tomohiro tatsuta 1,2 , yoshio morita 2 , motohiko takemura 1 1 department of pharmacology, hyogo college of medicine, nishinomiya, japan; 2 department of neuropsychiatry, hyogo college of medicine, nishinomiya, japan we examined the effects of pretreatment with clorgyline on morphine-induced behavioral changes and antinociception. a single administration of morphine (30 mg/kg, i.p.) to male icr mice induced a hyperlocomotion. the anova analysis revealed statistical significance of a morphine effect (hyperlocomotion) and of a clorgyline pretreatment x morphine interaction effect (inhibition), but not of an effect of clorgyline pretreatment. clorgyline pretreatment itself did not affect the spontaneous locomotion. clorgyline at a dose of 0.1 mg/kg but not other doses tested significantly potentiated morphine-induced antinociception evaluated by tail flick but not hot plate test. clorgyline at the doses of 1 and 10 mg/kg significantly inhibited dopamine and serotonin metabolism. these results suggest that clorgyline showed its inhibitory effect on morphine-induced hyperlocomotion, but not antinociception, through mao inhibition. recent studies in our laboratory have shown that methamphetamine (meth)-induced hyperlocomotion and behavioral sensitization in mice were inhibited by clorgyline, an irreversible monoamine oxidase inhibitor. in this presentation, the effect of clorgyline pretreatment on meth reward was assessed by conditioned place preference (cpp) paradigm, using an apparatus developed with supermex ® sensors. although intact male icr mice showed a significant cpp for meth (0.5 mg/kg, i.p.), pretreatment with subchronic clorgyline (0.1-10 mg/kg, s.c.) did not affect the magnitude of cpp. pretreatment with clorgyline significantly decreased apparent dopamine and serotonin turnovers in the striatum in a dose-dependent manner. these results indicated that clorgyline pretreatment did not influence meth reward in mice. of lobeline pretreatment on methamphetamine-induced stereotypy and monoamine metabolism in mice motohiko takemura 1 , nobue kitanaka 1 , tomohiro tatsuta 1,2 , yoshio morita 2 , junichi kitanaka 1 1 department of pharmacology, hyogo college of medicine, nishinomiya, japan; 2 department of neuropsychiatry, hyogo college of medicine, nishinomiya, japan the effects of lobeline, an alkaloid constituent of indian tobacco, on methamphetamine (meth)-induced stereotypy and monoamine metabolism were investigated in male icr mice. pretreatment with lobeline (3.0-30 mg/kg, i.p.) 15 min prior to drug challenge significantly decreased an intensity of stereotypies and increased its latency to onset in a dose-dependent manner. in saline challenge groups, doses of lobeline examined did not affect the spontaneous locomotion nor induce any stereotyped behaviors. the range of lobeline doses examined except 30 mg/kg did not affect apparent monoamine turnovers in the brain regions including striatum 20 min after drug challenge. these results suggested that the inhibitory effect of lobeline (3.0-10 mg/kg) on meth-induced stereotypy did not attribute to the change in the brain monoamine metabolism. kazuto sakoori, niall murphy riken bsi, wako-shi, japan previously we showed that endogenous nociceptin suppresses drug reward. here, we examined the effect of blockade of nop receptors on methamphetamine (meth) induced behavioral sensitization in order to understand the role of endogenous nociceptin in the chronic response to addictive drugs. first, nop receptor ko and wt mice were treated with 1 mg/kg meth and locomotor activity measured daily for 14 days. wt mice showed gradually increasing sensitivity to meth with repeated treatment of meth, whereas nop receptor knockout mice did not. next, 5 nmol ufp-101 (a nop receptor antagonist) and 1 mg/kg meth were co-administrated to mice and locomotor activity measured daily for eight days. ufp-101 strongly suppressed locomotor activity. thus, it was unclear if ufp-101 suppressed behavioral sensitization to meth during chronic drug treatment. however, when challenged with meth after four or more days without treatment, ufp-101 co-administrated mice showed a lower locomotor response. these results suggest that endogenous nociceptin facilitates the plastic changes induced by chronic treatment with addictive drugs. the influence of olanzapine (a d2dopamine receptor antagonist) on the morphine-induced conditioned place preference (cpp) in male and female mice was investigated in the present study. subcutaneous (s.c.) injection of morphine (1-10 mg/kg, three drug sessions) induced place preference both in male and female mice. intraperitoneal (i.p.) administration of olanzapine (0.5-5 mg/kg) induced place aversion (cpa) in female mice but not in male mice. administration of olanzapine (1, 2.5 and 5 mg/kg, i.p.) reduced both the acquisition and expression of morphine-induced cpp in male and female mice. however, olanzapine (5 mg/kg, i.p.) caused more than 80% mortality in female but not male mice. the effects of olanzapine were reversed by l-arginine (20 mg/kg, i.p.) pre-administration. in conclusion, it seems that olanzapine reduced morphine effects in part via a nitric oxide (no) mechanism. feed-forward associative learning (ffal) theory of cerebellar motor learning proposed by the author presumes that higher motor centers have place-coding systems and the same systems are shared by the cerebellum. when a new motor learning proceeds with respect to a certain movement, previous learning results of the movement will turn out to be modified or erased. ffal theory presumes that transferred memory from the cerebellar cortex to nucleus will serve as the maintenance of the previous learning. from this line, many aspects of saccadic adaptation are successfully demonstrated by computer simulation based on the theory. another theoretical issue is the credit assignment problem of motor error. a motor error is generally an integrated result of maladjusted multiple learning elements, and is to be decomposed to each element credit. this problem naively leads to an idea of a dual redundant system for movement, one for execution and the other for error decomposition. ffal theory naturally and simply resolves the credit assignment problem and demonstrates a computer simulation of motor learning of multi joint movement system, using the place-coding hypothesis. ps2p-d191 regulation of camp responsive element binding protein to stress in rat amygdala and hippocampal formation the department of anatomy and histology, shanghai medical school, fudan university, china amygdala (am) and hippocampal formation (hf) are important structures relating with emotional learning and memory. transcription factor, camp-responsive element binding protein (creb) in am and hf plays important roles in memory modulating processes. creb is a nuclear protein and is wldely accepted as prototypical stimulusinducible transcription factor. creb is activated in response to a vast array of physiological stimuli and then becames phosphorylated creb (pcreb). neurophysiological and neuropharmacological studies said that creb may regulate gene transcription and protein synthesis to maintain the long term and sustaining changing of synaptic efficiency during the long-term process of synaptic plasticity. but we cannot tell exactly via what kind of neurons in am and hf creb regulate these processes. we used the animal model, forced swimming (fs) as emotional stimuli and the experiment methods such as, immunocytochemistry, western-blotting with anti-pcreb antibody. the distributing profiles and changing rules of pcreb immunoreactive nuclei in amygdala and hippocampal formation of both control and experiment groups were investigated. the neuronal types of pcreb immunoreactive nuclei were analyzed by double-labelling immunocytochemistry with anti-pcreb, anti-glu and anti-pv antibodies. the results were: (1) the number of pcreb immunoreactive neuclei and total amount of pcreb in the subnuclei of rat amygdala, dentate gyrus (dg) and cornu ammonis 3(ca3) were increased after fs. the rule of this kind of changing was of region-and time-specific. (2) pcreb immunoreactive neuclei were expressed in glutamate immunoreactive neurons and were devoid in interneurons. these results suggested that pcreb in limbic system regulated the fs process and the regulation was finished via exciting neurons, glutamate neurons. hideto takahashi 1,2 , tomoaki shirao 1 1 dept of neurobiol & behav.; 2 ercgsm, gunma univ. grad. sch. of med., maebashi, japan dendritic spines are developmentally-regulated and activitydependent polymorphic structures based on actin cytoskeleton. drebrin is a spine-rich actin-binding protein regulating spine morphogenesis during development. here we find that chronic blockade of ampa receptors (ampar) inhibits synaptic drebrin clustering during development of hippocampal neurons, but not that of nmdar. further, the analysis of fluorescence recovery after photobleaching for egfp-drebrin a reveals that only 22.7 ± 3.0% of drebrin in the spine is stable, with a turnover time of 5.8 ± 0.4 min. blockade of ampar by 20 m cnqx reduces the population of stable drebrin (8.9 ± 3.6%), and has no effect on a turnover time. on the other hand, blockade of nmdar by 100 m ap5 has no effect on the population of stable drebrin, whereas shortens a turnover time (4.0 ± 0.3 min). these data suggest that ampar activities increase the binding capacity of drebrin in spines, and therefore promote drebrin clustering at spine synapses. instead, nmdar activities regulate spine-shaft shuttling of drebrin. itsuko nihonmatsu 1 , yoshito saitoh 1 , kaoru inokuchi 1,2 1 mitsubishi kagaku inst. life sci. (mitils), tokyo, japan; 2 crest, jst, tokyo, japan dendritic protein synthesis requires dendritic localization of mrnas in neurons. however, ultrastructural localization of these mrnas have not been well described. here we employed in situ electronmicroscopic technique to examine the precise localization of ␣camkii mrna in dendrites. ␣camkii mrna was located at the specific sites of dendritic shafts of pyramidal neurons, close to the spines, rather than in a diffused manner. we observed an increase in the ␣camkii mrna signals at the synaptic layer undergone l-ltp in the hippocampal dentate gyrus in unanesthetized freely moving rats. the increase was transient and returned to the basal level at 1 h. the alteration in the ␣ camkii mrna localization in dendrites may reflect a functional change in the translational apparatus along with synaptic plasticity. reiko okubo-suzuki 1,2 , daisuke okada 1 , kaoru inokuchi 1,2,3 1 mitsubishi kagaku inst. life sci. (mitils), tokyo, japan; 2 yokohama natl. univ. environment information sci., kanagawa, japan; 3 crest, jst, japan late-phase long-term potentiation (l-ltp) depends on de novo protein synthesis. synaptopodin (synpo), an f-actin-associated protein, increases in the activated synapses following l-ltp induction. spine volume and f-actin content in the spines also increase during l-ltp. to reveal the roles synpo plays in the regulation of spine volume and f-actin content, we examined synpo-egfp (se) localization and spine volume in the hippocampal neurons using time-lapse confocal imaging techniques. se-overexpression did not alter spine volume, but the amount of se in spines positively correlated with the spine volume. pharmacological activation of the nmda receptors increased both spine volume and synpo content in spines. furthermore, experiments with ptk2 cells indicated that synpo stabilizes f-actin. these results suggest that synpo synthesized in soma and transported into the activated spines following l-ltp induction stabilizes spine f-actin that may lead to the maintenance of increased spine volume. mineo matsumoto 1 , mitsutoshi setou 1,2 , kaoru inokuchi 1 1 laboratory for molecular gerontology, mitils, japan; 2 laboratory for nano-structure physiology, nips, japan subcellular localization of rna is an efficient way to localize proteins to a specific region of a cell. a requirement for dendritic rna localization and subsequent local translation has been demonstrated in several forms of experience-dependent synaptic plasticity. in spite of several attempts to identify these rnas, the population of rna species present in dendrites as a whole has not been well described. here we show the results of microarray analyses with rnas isolated from rna granule or synaptosome fractions prepared from the rat brain. these analyses revealed the complex nature of the dendritic rna population, which included rnas that were not expected to be in the dendrites. neural activity caused by an electroconvulsive shock triggered a redistribution of the dendritic transcriptome towards the synaptosome, a translationally active region. our results suggest that the redistribution of dendritic rnas is one of the mechanisms regulating local translation in response to synaptic inputs. ps3a-a005 an activity that traps vesl-1s protein into spines serves as synaptic tag synaptic tagging hypothesis explains how new proteins reach the activated synapses to establish input-specific late-phase plasticity, but it has not yet been substantiated. original idea of synaptic tagging is supposed to regulate protein entry into synaptic region including spines. using live-imaging techniques, we measured entry of vesl-1s-egfp into spines (ve trapping) of rat hippocampal neurons in culture, and found that ve trapping activity serves as the synaptic tag in many criteria. ve trapping required synergistic activation of postsynaptic no-pkg pathway and an activity abolished by ttx at 1 m, but not 50 nm. because 50 nm ttx is supposed to suppress na channels only postsynaptically, we concluded that ve trapping is a hebbian-like process that requires both pre-and postsynaptic activities. however, their coincidence time window was far wider (hrs) than that of early-phase plasticity, suggesting a requirement of persistently synchronized, rather than transiently coincident, activities, and a possibility of metaplastic states for late-phase plasticity. ps3a-a006 acute effects of dehydroepiandrosterone sulfate (dheas) on the synaptic transmission and plasticity in rat hippocampal slices yuxia xu 1 , ling chen 2 , masahiro sokabe 1,3,4 1 dept. physiol., nagoya univ., grad. sch. med., nagoya, japan; 2 dept. physiol., nanjing med. univ., nanjing, china; 3 sorst cell mechanosening, jst, nagoya, japan; 4 dept. mol. physiol., nips, okazaki, japan the neurosteroid dehydroepiandrosterone sulfate (dheas) is known to improve memory and learning in mammals. recently we report that chronic administration of dheas facilitates the induction of ltp in the rat hippocampus. to elucidate the underlying synaptic mechanism of the dheas effects, we examined in this study the acute effects of dheas on the synaptic transmission and plasticity at the ca1 region in rat hippocampal slices. an application of 0.1 dheas for 10 min to the slice augmented instantly the epsp, which was terminated within 30 min. however, even 1 h after the drug application, a subthreshold tetanus could induce ltp without alteration of ppf. this facilitating effect of dheas on ltp induction was blocked by a coapplication of a nmda receptor antagonist with dheas for 10 min, suggesting that the dheas effect involves a sustained modulation of the postsynaptic signaling mediated by nmda receptor. xiaoniu dai 1 , ling chen 2 , masahiro sokabe 1,3,4 1 dept. physiol., nagoya univ., grad. sch. med., nagoya, japan; 2 dept. physiol., nanjing med. univ., nanjing, china; 3 sorst cell mechanosensing, jst, nagoya, japan; 4 dept. mol. physiol., nips, okazaki, japan to know whether 17␤-estradiol (e2) can protect ca1 neurons from functional deficit due to ischemia, adult male wistar rats were subjected four-vessel occlusion (4vo) for 10 min, and the effect of e2 against this ischemic injury was examined. the electrophysiological properties of ca3-ca1 synapses were examined by a real-time optical recording method 7 days after ischemia. the ischemic brain showed a decreased synaptic transmission and an impairment of ltp induction but no alteration in paired-pulse facilitation. administration of e2 (1 mg/kg) 3 h before 4vo was able to protect ca1 neurons from these ischemic synaptic dysfunctions. the estrogen receptor-␣ selective agonist ppt (2 mg/kg) produced a similar protective effect, but the estrogen receptor-␤ agonist dpn (8 mg/kg) did not. above results suggest that e2 can protect neurons not only from cell death but also from functional damages caused by cerebral ischemia. ps3a-a008 non-genomic rapid effects of estradiol on hippocampal synapses: multi-electrode dish analysis kohei nakajima 1 , mari ogiue-ikeda 1 , yuki oishi 2 , suguru kawato 1,2 1 department of biophysics and life sciences, graduate school of arts and sciences, university of tokyo at komaba, tokyo, japan; 2 department of physics, university of tokyo, tokyo, japan estradiol has a non-genomic, rapid effect on synaptic transmission, which is manifested within seconds to minutes. recently, hippocampal neurons were shown to synthesize estradiol de novo, and to express estrogen receptor ␣ (er␣) at synapses. although these results imply that estradiol rapidly modulates synaptic plasticity through synaptic er␣, there are few electrophysiological evidence about it. here we investigated effects of estradiol on ltd by using wild type, er␣ hetero and er␤ hetero mouse hippocampal slices with a multi-electrode dish (med, panasonic). med enabled us to measure epsps in ca1, ca3, and dentate gyrus simultaneously. hippocampal slices were perfused with estradiol before nmda-induced ltd. we found that estradiol enhanced ltd both in wild type and er␤ hetero mouse, but not in er␣ hetero mouse. our data suggested non-genomic rapid action of estradiol through synaptic er␣. withdrawn ps3a-a010 morphological changes of dendritic spines mediated by glucocorticoid receptor (gr) in rat hippocampus yoshimasa komatsuzaki 1 , gen murakami 2,3 , tetsuya kimoto 2,3 , suguru kawato 2,3 1 college of humanities and sciences, nihon university, tokyo, japan; 2 department of biophysics and life sciences, university of tokyo, tokyo, japan; 3 crest, jst, japan modulation of hippocampal synaptic plasticity by glucocorticoids has been attracting much attention, due to its importance in stress responses. dendritic spines are essential for memory storage processes. here we investigated the effect of dexamethasone (dex), a specific agonist of glucocorticoid receptor (gr), on density and morphology of dendritic spines in adult male rat hippocampus by imaging of lucifer yellow-injected spines in slices. the application of 100 nm dex induced rapid modulation of the density and morphology of dendritic spines in ca1 pyramidal neurons within 1 h. the total spine density increased from 0.88 spines/m to 1.36 spines/m. dex significantly increased the density of thin and mushroom type spines, however only a slight increase was observed for stubby and filopodium type spines. because the presence of 10 m cycloheximide, an inhibitor of protein synthesis, did not suppress the dex effect, these responses are probably non-genomic. hideki tamura 1 , yuji ikegaya 2 , sadao shiosaka 1 1 division of structural cell biology, naist, nara, japan; 2 laboratory of chemical pharmacology, university of tokyo, tokyo, japan the capacity of activity-dependent synaptic modification is essential in processing and storing information, yet little is known about how synaptic plasticity alters the input-output (i-o) conversion efficiency at the synapses. in the adult mouse hippocampus in vivo, we carefully compared the i-o relationship, in terms of presynaptic activity levels versus postsynaptic potentials, before and after the induction of synaptic plasticity and found that synaptic plasticity led synapses to respond more robustly to inputs, that is, synaptic gain was increased as a function of synaptic activity with an expansive, power-law nonlinearity, i.e., conforming to the so-called gamma curve. in extreme cases, long-term potentiation (ltp) and depression (ltd) coexist in the same synaptic pathway with ltp dominating over ltd at higher levels of presynaptic activity. these findings predict a novel function of synaptic plasticity, i.e., a contrast-enhancing filtering of neural information through a gamma correction-like process. research funds: 21st century coe research ps3a-a012 actin organizations within single dendritic spines in ca1 pyramidal neurons studies with two-photon photoactivation naoki honkura, masanori matsuzaki, haruo kasai center for disease biology and integrative medicine, faculty of medicine, the university of tokyo, japan the major cytoskeleton of dendritic spines is filamentous actin (factin). we have here investigated sub-spine actin organizations using two-photon photoactivation of pa-gfp fused with ␤-actin in rat ca1 pyramidal neurons. we found segregated and discontinuous organizations of two pools of f-actin, dynamic and stable pools, which turned over with time constants of 1.2 min and 17 min, respectively. fractions of the stable f-actin pool were greater in larger spines, therefore, the entire f-actin pool was more stable in larger spines. we succeeded in visualizing a retrograde flow of f-actin in the dynamic pool from the apex to the base of spine, and found that both the speeds (0.2-1.2 um/min) and lengths (0.2-0.7 um) of the f-actin flow were greater in spines with larger head volumes. moreover, spine heads rapidly shrank when actin polymerization was blocked by latrunculin a, suggesting that the rate of actin polymerization in each spine actively and continuously determines the volume of spine head via the length of f-actin. tomoharu nakamori 1 , katsushige sato 2 , kohichi tanaka 1 , hiroko hamazaki 1 1 mol. neurosci., tmdu, tokyo, japan; 2 physiology, tmdu, tokyo, japan the visual wulst (vw) in the thalamofugal pathway in chicks is known to have a critical role in the visual learning. to understand the function of the vw in the learning process of imprinting, we investigated the neuronal activity of vw region in chick brain. the slice stained with a voltage-sensitive dye was prepared for a multiple-site optical recording. when chicks were reared in quasi-dark condition, the extent and amplitude of response induced by electrical stimulation were different between at 1 or 4 days post-hatching (p1 or p4), and at p7. this corresponds to behavioral data showing that chicks have high ability of visual learning in imprinting behavior until p4, but they lose this ability at p7. in addition, the light-exposed chick showed larger optical response than the dark-rearing one. the optical response in the vw was partly inhibited by the glutamate-and gaba-receptor antagonists. these results suggest that the glutamatergic as well as gabaergic neurons are active in the area including vw and that the neuronal activity of vw affects the learning ability for imprinting. withdrawn ps3a-b015 effect of estrogen on hippocampus in male and female mice takanori sugawara 1 , shinji hayashi 1 , victoria luine 2 1 graduated school of integrated sience, yokohama city university, yokohama, japan; 2 department of psychology, hunter college, city university of new york, new york, usa we examined structural difference in the hippocampal neurons with golgi stain among the male, the female and the female treated with estrogen neonatally. the mice were gonadectomized and received 17 ␤-estradiol (e2) or oil-vehicle injections at adult before golgi impregnation. spine densities 10 m of apical dendrites of the pyramidal neurons in the hippocampus ca1 region were calculated with categorization into three shapes, i.e., mushroom type with large head, thin type and filopodia-like type. as a result, only in the female not estrogen treated neonatally, the mushroom type and total spine densities were increased but the thin type spine density was decreased by e2 treatment in adult. the present results indicate that estrogen given at adult induces an enlargement of spine to mushroom type and generates new spines only in the female mice not treated with estrogen neonatally. thus, dendritic spine formation seems sexually dimorphic and depends on the sex steroid environment during the neonatal period. jun-ichi goto 1,2 , takafumi inoue 2,3 , akinori kuruma 1 , katsuhiko mikoshiba 1,2,3 1 lab. developmental neurobiology, brain science inst., riken, saitama, japan; 2 div. molecular neurobiology, inst. medical science, univ. tokyo, tokyo, japan; 3 calcium oscillation project, icorp-sorst, jst, tokyo, japan changes in synaptic efficacy at the parallel fiber (pf)-purkinje cell (pc) synapse are postulated to be a cellular basis for motor learning. although long-term efficacy changes lasting more than an hour at this synapse, i.e., long-term potentiation and depression, have been extensively studied, relatively short lasting synaptic efficacy changes, namely short-term potentiation (stp) lasting for tens of minutes, have not been discussed to date. here we report that this synapse shows an apparent stp reliably by a periodic burst pattern of homo synaptic stimulation. this stp is presynaptically expressed, since it accompanies with a reduced paired-pulse facilitation and is resistant to postsynaptic ca 2+ reduction by bapta injection or in p/q-type ca channel knockout cerebella. this novel type of synaptic plasticity at the pf-pc synapse would be a clue for understanding the presynaptic mechanisms of plasticity at this synapse. aya ishida, wataru kakegawa, michisuke yuzaki department of physiology, keio university, tokyo, japan mitogen-activated protein kinase (mapk) cascade is thought to be essential for the synaptic plasticity and learning. in the hippocampus, three different mapk subfamilies, including extracellular signalregulated kinase (erk), p38 mapk and c-jun nh2-terminal protein kinase (jnk), have been shown to selectively regulate different forms of synaptic plasticity -long-term potentiation (ltp), longterm depression (ltd), and depotentiation after ltp, respectively. although erk was previously shown to play a role in cerebellar ltd in cultured purkinje cells, the role of mapks has not been systemically studied. here, we examined the effect of specific inhibitors of three different mapks on ltd by patch-clamp recordings from cerebellar slices. we found that u0126, a specific inhibitor for erk activation, significantly inhibited ltd induction, whereas sb203580 and sp600125, antagonists for p38 mapk and jnk, respectively, had no effect. therefore, unlike hippocampal ltd, cerebellar ltd was dependent on erk, suggesting involvement of different intracellular downstream pathways. ps3a-b018 regulation of ampa receptor trafficking by aaa atpases in cerebellar purkinje cells: are nsf and vcp playing complementary or antagonistic roles? thomas launey 1 , chou-chi li 2 , yumiko motoyama 1 , junko yamaoka 1 , masao ito 1 1 riken brain sci. inst., japan; 2 national cancer institute, nih, ma, usa the number of postsynaptic ampa receptors (ampar) is regulated by interactions with multiple protein complexes, throughout its synthesis, maturation, transport, synaptic insertion and degradation. aaa atpases influence several of these stages, the most extensively studied being nsf's contribution to ampar trafficking. in cerebellar purkinje cell (pc), we show that valosin containing protein (vcp), an atpase with high homology to nsf, is bound to ampa receptors in pc's dendritic compartment. following glur2 co-ip from molecular layer, vcp was detected by ms/ms and by monoclonal anti-vcp. pull-down assay showed a direct interaction between vcp and glur2 c-term domain, requiring vcp n-term domain and both the nsf and pdz binding domains of glur2. glur2 phospho-ser880 promotes vcp complex dissociation, suggesting a relation with synaptic plasticity. further, pep2m-related peptides, thought to interfere specifically with nsf-regulated ampar trafficking, also blocked the glur2-vcp interaction. yuichi kitagawa 1,2 , shin-ya kawaguchi 1,2 , tomoo hirano 1,2 1 dept. biophys., grad. sch. sci., kyoto univ., kyoto, japan; 2 crest, jst, kawaguchi, japan at inhibitory synapses on a cerebellar purkinje neuron (pn), postsynaptic depolarization induces long-lasting potentiation of the gaba a receptor (gaba a r) responsiveness (rebound potentiation: rp). previous studies have clarified the molecular mechanism regulating rp induction. whether rp is induced or not is determined by the balance of activities of protein kinases (camkii and pka) and phosphatases (pp-1 and calcineurin). to understand the complex behavior of biochemical reactions systematically, a kinetic simulation model to analyze the behaviors of signaling network was developed. computer simulation reproduced the bistable states of gaba a r phospholyration according to stimulation patterns, which apparently corresponded to whether rp was induced or not. we further studied the systematic property of the molecular network, and obtained several experimental predictions. these possibilities were evaluated by experiments such as immunocytochemistry using cultured pns. ps3a-b020 long-term depression of synaptic transmission in a songbird motor nucleus essential for song learning yuki haruta, yachun huang, neal hessler vocal behavior mechanisms riken brain science institute, japan in order to fully understand the neural basis of song learning, it is critical to characterize forms of synaptic plasticity that could be involved in this process. we previously reported that, in synapses of the song motor nucleus ra, participation of postsynaptic nmda receptor nr2b subunits and presynaptic transmitter release both decrease from young birds to adults. here, we tested whether synaptic function could be modified in a similar way by acute stimulation. after pairing slight postsynaptic depolarization with presynaptic stimulation, ltd was reliably induced at both hvc and lman inputs in juvenile birds from 35 to 42 days old. this depression required activation of postsynaptic nmda receptors, and was expressed by decreased transmitter release, which required activation of cannabinoid receptors. no ltd could be induced in normal birds over 60 days old, when song learning is nearly complete, but ltd remained possible in birds over 60 days old who had been isolated from song tutors, and thus retained the capacity for learning. ps3a-b021 involvement of ca 2+ -permeable ampar in the repetitive-ltp induced synaptic enhancement (rise) yukiko ueno, keiko tominaga-yoshino, akihiko ogura graduate school of frontier biosciences, university of osaka, osaka, japan we showed previously that 3 exposures to glu of cultured rat hippocampal slices at 24 h intervals produced a long-lasting enhancement in synaptic strength accompanied by synaptogenesis (rise). we examined here whether the conversion of ampar subunits occurred during the development of rise. immunochemical staining for ampar subunits, glur1 and glur2, showed that the number of glur1-positive puncta increased transiently after the repeated glu exposures, whereas the number of glur2-positive puncta increased gradually and persistently. jstx (a ca 2+ -permeable ampar blocker) suppressed fepsp amplitude recorded at ca3-ca1 synapses by 20-40% in the period corresponding to the transient increase of glur1-positive puncta. this transient increase should represent the delivery of ca 2+ -permeable (glur2-lacking/glur1-including) ampar to synaptic sites. furthermore, jstx application at that period blocked the rise production. these results suggest that the transient delivery of ca 2+ -permeable ampar to synaptic sites is involved in the rise production. yoshihiro egashira, tsunehiro tanaka, yuji kamikubo, yo shinoda, keiko tominaga-yoshino, akihiko ogura osaka univ. grad. sch. frontier biosciences, toyonaka 560-0043, japan long-lasting synaptic plasticity, the cellular basis of long-term memory, is assumed to be associated with protein synthesis. using cultured rat hippocampal slices, we previously found that a long-lasting synaptic enhancement coupled with an increase in the number of synaptic structures was established after 3 inductions of ltp, not after its single induction. this synaptic enhancement required protein synthesis for its establishment. we recently found an apparently mirror-image phenomenon; 3 inductions of ltd led to a long-lasting synaptic decrement coupled with a decrease in the numbers of synaptic structures. to know whether this synaptic decrement also requires protein synthesis, we induced ltd 3 times (24 h intervals) by applications of dhpg (a type i mglur agonist), during or after which anisomycin (a protein translation blocker) was applied. we found that anisomycin did not block the induction of ltd but blocked the establishment of the long-lasting synaptic decrement. haruo mizutani, tetsuya hori, tomoyuki takahashi department of neurophysiology, graduate school of medicine university of tokyo, tokyo, japan bath-application of 5-ht (10 m) attenuated the amplitude of evoked epscs and facilitated paired-pulse ratio without affecting the miniature epsc amplitude, suggesting that its site of action is presynaptic. the 5-ht 1b receptor agonist cp93129 mimicked the presynaptic inhibitory effect of 5-ht. 5-ht 1b receptor antagonist nas-181 reversed the 5-ht inhibitory effect, indicating that the 5-ht induced inhibitory effect occurs by mediating 5-ht 1b receptors. the presynaptic inhibitory effect of 5-ht became weaker as animals matured. in whole-cell recordings from calyceal presynaptic terminals, 5-ht attenuated voltage-dependent calcium currents, but had no effect on potassium currents. this 5-ht effect was characterized with a marked desensitization, but sustained under the fast calcium chelating agents, bapta. these results suggest that 5-ht, upon activating 5-ht 1b receptors, inhibits presynaptic calcium channels thereby inhibiting transmitter release and induces receptor desensitization by calcium influx at the immature calyceal synapse. takako ohno-shosaku 1 , masato ano 1 , yuki hashimotodani 2 , tadasato nagano 3 , masanobu kano 3 1 dept. impair. study, grad. sch. med. sci., kanazawa univ., kanazawa, japan; 2 dept. neurophysiol., grad. sch. med., osaka univ., osaka, japan; 3 dept. cell. neurosci., grad. sch. med., osaka univ., osaka, japan retrograde endocannabinoid signal contributes to activitydependent modulation of synaptic transmissions in various brain regions. endocannabinoid release is triggered by depolarizationinduced elevation of intracellular calcium level or activation of gq-coupled receptors. here we report that nmda receptors can also contribute to generation of endocannabinoid signal. inhibitory postsynaptic currents (ipscs) were recorded in cultured hippocampal neurons prepared from newborn rats. application of nmda induced a transient suppression of cannabinoid-sensitive ipscs but not cannabinoid-insensitive ipscs. the nmda-induced suppression of ipsc was blocked by a cannabinoid receptor antagonist. these results indicate that activation of nmda receptors induces the endocannabinoid release, and suppresses the inhibitory synaptic transmission through activation of presynaptic cannabinoid receptors. the most caudal region of the rat spinal cord, the conus medullaris has a simple anatomical feature, which lacks ventral as well as dorsal root fibers and somatic motor neurons in the ventral horn. a small number of neurons distribute around the central canal, and some of them are nitric oxide synthase (nos) positive. a dense distribution of nerve fibers immunoreactive to cgrp, sp, and npy was found in dorsal part of the conus medullaris similarly to that of other spinal cord levels. in addition, enk-, 5-ht-, and th-immunoreactive varicose fibers were richly distributed throughout the sectional plane. to analyze this unique structure may provide valuable information on the basic neural cytoarchitecture and fiber connections of the spinal cord, particularly for the intraspinal circuitry. for this purpose, we made an electron microscopic study using nadph-diaphorase histochemistry combined with immunohistochemistry for neuronal markers. adenosine has been known to be a neuro-modulator in the nervous systems and four types of adenosine receptor are identified (a1, a2a, a2b and a3). adenosine a1 and a3 receptors have been reported to inhibit high-threshold ca channel currents in neurons. to investigate the interaction between adenosine a1 and a3 receptors in rat striatum neurons in culture, l-type ca channel currents were recorded by whole-cell clamp method before and after administration of a1 agonist (cpa) and a3 agonist (2-cl-ib-meca). ca currents were decreased after administration of low concentration of cpa and 2-cl-ib-meca as reported previously. although ca currents were decreased by 2-cl-ib-meca in the presence of cpa, ca currents applied with cpa were not decreased on cells in the presence of 2-cl-ib-meca. at administration of cpa and 2-cl-ib-meca on cells simultaneously, ca currents were not decreased. these results suggested that adenosine a3 receptor may inhibit adenosine a1 receptor throughout a intracellular pathway in neurons. ps3a-c028 influence of extracellular gaba and taurine to gaba a receptor-mediated actions in radially migrating cortical plate cells with identified by in utero electroporation t. furukawa 1 , j. yamada 2 , k. inoue 1 , y. yanagawa 3 , a. fukuda 1, 2 1 dept. physiol., hamamatsu univ. sch. med., japan; 2 dept. biol. info. process, grad. sch. elec. sci. & tech., shizuoka univ., hamamatsu, japan; 3 dept. developmental and integrative neurosci., gunma univ. sch. med., gunma, japan it is well known that role of gaba a -r mediated actions is important for early cns development. the radially migrating cells may affected by the actions. gaba content in the brain of gad67-gfp knock-in mouse decrease compared with the wild type mice. therefore, we investigate the influence of the circumferential gaba concentration to radially migrating cells. furthermore, as it was known that gaba a -r is affected by taurine, the influence of taurine to radially migrating cells was also investigated. there was no significant difference in distribution of radially migrating cells that was labeled by means of electroporation. evoked gaba a -r mediated currents of labeled cells had dose-dependent manner and had no differences among genotypes. therefore, we have examined the influence of circumferential taurine to gaba a -r mediate actions. takashi hayakawa 1 , hiroyuki hioki 1 , kouichi nakamura 1,2 , hisashi nakamura 1 , takeshi kaneko 1,2 1 dept. morphol. brain sci., grad. sch. med., kyoto univ., kyoto, japan; 2 crest, jst, kawaguchi, japan we previously reported that almost all vesicular glutamate transporter 3(vglut3)-immunoreactive (ir) cells were also gabair in neocortex and choline acetyl transferase (chat)-ir in caudate-putamen in rat. although, in dorsal and median raphe nuclei, many vglut3-positive cells showed immunoreactivity for 5-hydroxytryptamine (5ht), a significant proportion (12.3%) of vglut3-postive cells was 5ht-negative. in this study, triple immunofluorescence staining was performed for vglut3, 5ht and one of the following proteins: neuronal nuclear antigen (neun), glial fibrillary acidic protein (gfap), glutamic acid decarboxylase (gad67) and tyrosine hydroxylase (th). our results showed that all of the vglut3-positive/5ht-negative cells were immunoreactive for neun but not for gfap. furthermore, we found that these vglut3positive/5ht-negative neurons didn't show any immunoreactivities for gad67 nor th, and thus it is indicated that there is a group of exclusively glutamatergic vglut3-positive neurons in these nuclei. research funds: kakenhi 16200025, 17022020, 17650100 ps3a-c030 cortico-striatal and fast-spiking cell activity in the rat frontal cortex during cortical oscillations in vivo: modulation by serotonin m victoria puig 1 , mika ushimaru 1 , yoshiyuki kubota 1 , akiya watakabe 2 , tetsuo yamamori 2 , yuchio yanagawa 3 , yasuo kawaguchi 3 1 div. cerebral circuitry, nips, okazaki, japan; 2 div. brain biology, nibb, okazaki, japan; 3 dept. genetic and behavioral neurosci., gunma univ. graduate school of med., japan we studied how cortico-striatal (cs) and fast-spiking (fs) cells are modulated by slow-wave-sleep (sws) oscillations and by serotonin (5-ht). cs and fs cells were recorded simultaneously with the electrocorticogram in the secondary motor area of anesthetized rats that expressed a gfp in gabaergic interneurons. fs displayed a highsuccess excitation to striatal stimulation, suggesting a control of cs over fs. during sws, both cs and fs fired during the up-states though with different patterns. the stimulation of the dorsal raphe promoted longer up-states. moreover, 60% of the cs were inhibited by 5-ht through 5-ht 1a r and 6% were excited through 5-ht 2a r. however, 44% of the fs cells were inhibited and 28% excited. these results show that cs cells are more inhibited by 5-ht than fs. the expression of 5-htr was confirmed by in situ hybridization. research funds: jsps pe04061 and 15300110 ryohei tomioka, kathleen rockland laboratory for cortical organization and systematics, riken brain science institute, saitama, japan in small mammals, gabaergic neurons have been shown to contribute to ipsi-and contralateral cortical projections. here, we report in monkey as well that some gabaergic neurons send long-distance projections. identification was partly based on golgi-like labeling of the dendritic tree, achieved by injecting adenovirus as a retrograde tracer in areas v4, teo, or tep. aspiny or sparsely spinous nonpyramidal neurons were clearly visualized in the white matter or, less frequently, in cortical gray matter, in mainly layer 3 but also in layer 5 and/or 6. in each of the 3 cases, about 50-100 gabaergiclike neurons were scored, with a preferential location anterior to the injection sites. in addition to their characteristic dendritic morphology, the neurons were identified as positive for gabaergic neuronal markers; namely, gad67, somatostatin, or nos. thus, we conclude that gabaergic projection neurons are phylogenetically conserved; but more work is needed to determine (1) their other features, (2) possible species variability, (3) their functional significance. supported by riken bsi. withdrawn ps3a-c033 regional, cell type, and layer-specific differences in cholinergic modulation of neocortical neurons allan gulledge 1,2 , susanna b. park 2 , greg j. stuart 2 , yasuo kawaguchi 1 1 national institute for physiological sciences, japan; 2 div. neurosci., jcsmr, australian national university, canberra, australia we examined cholinergic modulation of pyramidal and nonpyramidal neurons in 3 neocortical areas (prefrontal, somatosensory, and visual cortex). transient ach exposure (100 m) inhibited layer 5 pyramidal neurons in all areas via activation of an sk-type potassium conductance. pyramidal neurons in layers 2/3 were generally less responsive to ach, but ach inhibited layer 3 cells in visual cortex. prefrontal layer 5 pyramidal neurons were more responsive to ach than were layer 5 cells in other areas of cortex. fast spiking (fs) nonpyramidal neurons were completely non-responsive to ach, even at very high concentrations (5 mm). on the contrary, ach generated fast, nicotinic receptor-mediated responses in 37% of non-fs interneurons (24 of 65 cells). laminar or regional differences in ach responses were not observed in nonpyramidal neurons. these data suggest that ach may act to inhibit the output of cortical projection neurons while preserving information processing in superficial neurons. toshikazu kakizaki 1,2 , kenzi saito 1,3 , yuchio yanagawa 1,2 1 department of genetic and behavioral neuroscience, gunma university graduate school of medicine, maebashi, japan; 2 sorst, jst, kawaguchi, japan; 3 sokendai, hayama, japan a major inhibitory neurotransmitter gaba is synthesized by glutamate decarboxylase (gad), and is accumulated into synaptic vesicles by vesicular gaba transporter (vgat). another inhibitory neurotransmitter glycine could be transported into synaptic vesicles by vgat, and be co-released with gaba. several molecules related to gabaergic or glycinergic neurotransmission are expressed in nonneural tissues, suggesting that gabaergic and glycinergic systems exert their activities outside the cns. vgat-deficient mice die in the perinatal period, and display omphalocele, defect in ventral body wall closure, suggesting that gaba and/or glycine are involved in body wall formation. to further investigate whether gaba is essential for the ventral body wall formation or not, we have been examining how the body wall developed in the gad67-deficient mouse fetus. ps3a-c035 gaba mediated glutamate release from developing cerebellar cortex and ca sensitivity sachiko yoshida, miyuki ohshita, masakazu uematsu, shoichiro hirano, shinya tanaka, naohiro hozumi toyohashi university of technology, toyohashi, japan gaba (␥-amino butyric acid) and glutamate are known to play important roles as modulators in the survival and development of cerebellar neurons. during cerebellar development, gaba-mediated responses, gaba excitations, become depolarized inducing an increase in intracellular calcium concentrations, and are thought to have important trophic effects. many observations of gaba excitations using cultured cells have been reported, whereas few using acute slices. we recently reported the spatial nature of glutamate and gaba releases from acute slice with an enzyme-linked assay system and ccd imaging technology. in the present study, we evolved this measurement system to allow observations of spontaneous or gaba-mediated glutamate release from developing postnatal acute cerebellar slices. glutamate was released spontaneously, but gaba-mediated glutamate release appeared from postnatal 4 to 6 day in egl. its release, especially from premigratory zone, was inhibited by ni 2+ , but cd 2+ couldn't. we suggest that gaba excitation induces granule cell migration. ps3a-c036 gabaergic fiber in the rat trigeminal motor nucleus reorganized following masseter nerve transection hiroyuki hayashi 1 , hiroaki wake 2 , junichi nabekura 2 , osamu takahashi 1 1 department of histology, kanagawa dental college, yokosuka, japan; 2 national institute of physiological science, okazaki, japan it has been reported that gabaergic nerve terminals are seen in the trigeminal motor nucleus (vm) of the rat, and that there are primary afferent inputs from the muscle spindle of masticatory muscles to the vm cell bodies. we recently found that the number of these gabaergic fibers projecting to vm is markedly reduced in postnatal development. in this study, to elucidate the possibility that the re-arrangement of gabaergic circuits could be reproduced after neuronal injury, we examined the effect of axonal injury of the masseter axon on the gabaergic circuits in the vm. two to eight weeks after unilateral surgical transection of the masseter nerve of rats, gabalike immunoreactive (gaba-ir) varicosities were examined using immunofruorescence technique. the significant increase in number of gaba-ir varicosities were seen after eight weeks of the operation. this result suggest that gabaergic inputs may play one of important role for reorganization of afferent inputs in the vm. akiko arata 1 , kunihiko obata 2 , jonathan davies 3 , mark bellingham 3 , peter g. noakes 3 1 lab. for memory & learning, riken-bsi, wako, japan; 2 obata res. unit, riken-bsi, wako, japan; 3 sch. biomed. sci., univ. queensland, queensland, 4072, australia during embryonic development, approximately half of the motoneurons (mns) undergo programmed cell death. this process depends also on glycinergic and/or gabaergic synaptic activity, as suggested by increased mn number in gephyrin-deficient mice (banks et al., 2005) . we investigated the involvement of gaba alone in the mn death using gad67-deficient mice, in which cerebral gaba is reduced to less than 10% of the wild-type. mn numbers at embryonic day (e) 18 were counted by the method of banks et al. brainstemupper spinal cord blocks were prepared from e18 embryos and subjected to electrical recording from the c4 and c8 ventral roots and also gaba measurement. in gad67-deficient embryos, increase in number of brachial mns (139%) and decrease in both spontaneous discharges in the c4, c8 roots and gaba content (less than 20%) were observed, compared with those of the wild-type littermates. gaba might control cell death in developing network. abolghasem esmaeili, joe lynch, pankaj sah queensland brain institute, the university of queensland, australia the amygdala has key role in processing emotional information. distribution of gaba a receptor subunits is crucial for understanding physiology and pharmacology properties of these receptors in the amygdala. we examined the pharmacology of gaba a receptors by expressing different subunit combinations in hek293 cells and comparing the pharmacology with specific gabaergic inputs in the amygdala. dmcm blocked the actions of gaba at expressed ␣1␤1␥2 and ␣2␤1␥2 combinations (75% reduction) but had no effect at ␣1␤1␥1 or ␣2␤1␥1. in slice recordings dmcm blocked ipscs by 70% in the lateral amygdala and had variable effects in the central amygdala. diazepam and zolpidem enhanced ipscs in the lateral whereas the response in the central amygdala was either reduction or enhancement. real time pcr and western blotting revealed differences in the distribution of gaba a receptor subunits between the lateral and central amygdala. we conclude that in the lateral amygdala all inputs have ␥2 subunits whereas in the central amygdala some inputs contain ␥1 while others contain ␥2 subunits. masayuki kobayashi department of pharmacology, nihon university school of dentistry, tokyo, japan noradrenergic agonists have different effects on the excitatory neural transmission according to their subtypes in rat cerebral cortex. the present study aimed to explore what kind of second messengers and the precise site of synaptic membrane, pre-or postsynaptic, is involved in these noradrenergic modulation. the suppressive effect by activation of ␣ 1 -adrenoceptors was mediated by protein kinase c, and excitatory effect by activation of ␤-adrenoceptors was mediated by camp/protein kinase a cascade. phenylephrine suppressed inward currents evoked by puff application of glutamate, and it decreased mepsc amplitude and increased mipsc frequency. isoproterenol increased mepsc frequency and decreased mipsc amplitude. gaba-induced postsynaptic currents were suppressed by isoproterenol. these results suggest that phenylephrine may decrease postsynaptic currents through glutamate receptors and increase the release probability of gaba from presynaptic terminals. on the other hand, isoproterenol may facilitate glutamate release and suppress gaba a receptor-mediated postsynaptic currents. ps3a-d040 hydrogen sulfide modulates synaptic transmission in rat hippocampal neurons mamiko tsugane 1 , takashi iwai 2 , yasuo nagai 1 , junichiro oka 2 , hideo kimura 1 1 dept. mol. genetics, nat'l. inst. neurosci., ncnp, tokyo, japan; 2 lab. pharmacol., fac. pharm. sci., tokyo univ. sci., chiba, japan hydrogen sulfide (h 2 s), which is a well-known toxic gas and facilitates the induction of hippocampal long-term potentiation, has been proposed as a neuromodulator in the brain. the aim of this study is to understand the mechanism of regulation on synaptic transmission by h 2 s. we examined the effect of h 2 s on spontaneous excitatory postsynaptic currents (sepsc) as well as paired-pulse facilitations using both whole-cell and field potential recordings from rat hippocampal slices. sodium sulfide (na 2 s), a donor of h 2 s, reduced the amplitude of field excitatory postsynaptic potentials and increased the ratio of paired-pulse facilitation. the frequency and the amplitude of sepsc were initially reduced by na 2 s then gradually increased, while the inward currents elicited by glutamate were not significantly suppressed by na 2 s. these observations suggest that h 2 s may modulate glutamatergic synaptic transmission by suppressing the release of a transmitter. several studies show that activation of locus coeruleus (lc) play an important role in the symptoms of opiate withdrawal. in this study the effects of lc inactivation on self-administration of morphine and on morphine withdrawal syndrome in rats has been investigated. male rats were anaesthetized and implanted with silastic catheters inserted in to the right jugular vein. after 5 days animals were fitted and the external end of the catheter was connected with a syringedriven pump, then were placed in the self-administration apparatus. lc was inactivated by (1 l) lidocaein (2%) 5 min before training. animals were allowed to self administer morphine (1 mg/kg per inf.) ten consecutive daily 2-h session. during all morphine self administration session lever pressing was measured. our results show that: (1) lc inactivation produced a significant decrease in the initiation of morphine self administration during all session. after the last test session morphine withdrawal symptom signs (mws) precipitated by naloxone were measured. (2) most of mws were decreased by lc inactivation in comparison with morphine group. these results suggest that extracellular atp plays a dual role in astrocytic ca 2+ wave propagation with activation of distinct purinergic receptors in the hippocampus of the rats. the electrophysiological analysis of the rescue effect of 17␤ estradiol from glucocorticoid activity yuki oishi 1 , suguru kawato 2 1 department of physics, graduate school of science, university of tokyo, tokyo, japan; 2 graduate school of arts and sciences, university of tokyo, tokyo, japan it is well known that stress reduces several activity of brain. especially, hippocampus is the largest target of stress. these phenomena are caused by glucocorticoids which are synthesized at adrenal when suffering stress. on the other hand, 17␤ estradiol is one of the neuro protective factors and rescues neural death caused by several neurotoxins, such as ␤-amyloid, glutamate, glucocorticoids. in this study, we focused attention on the acute effects of steroid hormones and researched the effects of glucocorticoids and estradiol on rat hippocampal long term potentiation (ltp), which is the index of learning and memory. the results was that corticosterone (glucocorticoid of rat) acutely reduced ltp via glucocorticoid receptor. 17␤ estradiol rescued this reduction via estrogen receptor ␣ and ␤. so we found that 17␤ estradiol affected not only neuro protection but synaptic protection from stress-induced suppression of synaptic transmission acutely. ps3a-d044 the hypothalamic neuropeptide y neuron system of rats after long-term, high-dose dexamethasone treatment jinko konno, ayuka ina, sachine yoshida, hideki ohmomo, fumihiro shutoh, setsuji hisano lab. neuroendocrinol., graduate sch. comprehensive human sci., univ. tsukuba, ibaraki, japan effects of dexamethasone (dex) on hypothalamic neuropeptide y (npy) expression were evaluated with semi-quantitative in situ hybridization and immunohistochemistry. adult male wistar rats received an injection of dex (0.2 mg/100 g b.w., sc) or sesame oil (vehicle control) everyday for 9-10 days. the two and intact rats (intact control) were decapitated, and the hypothalamus was dissected out, fixed and cut into paraffin sections. npy-immunoreactive axonal varicosities in the external zone of the median eminence were apparently more frequent in the dex-treated rat than in controls. npy hybridization signals in the arcuate nucleus were significantly higher in the treated-rat than in controls. no difference was found between both control animals. these results indicate stimulatory effects of dex on hypothalamic npy production and suggest enhanced npy influences on pituitary function. akiko shingo, idumi yamashita, shozo kito lab. of neuroscience, hyogo university, hyogo, japan we examined estrogen-like actions of isoflavones in the cerebral cortex and hippocampus on the basis of our previous data that estradiol induces igf-1 mrna expression, upregulates estrogen receptors and facilitates ere binding in these brain areas. materials are ovxed and non-ovxed rats. each group of rats were divided into the following groups. a: rats fed with phytoestrogen-free control diet, b: rats fed with diet with soy bean-derived estrogen and c: rats fed with control diet combined with chronic intraperitoneal injections of minimum dose of ␤-estradiol. after feeding, rats were sacrificed to remove the cerebral cortex and hippocampus. expressions of mrnas of igf-1, estrogen receptors ␣ and ␤, and ere binding were analysed. as the results, it was revealed that isoflavones induced increased expression of mrnas of igf-1 and estrogen receptors in both ovxed and non-ovxed rats. difference between estrogen receptor ␣ and ␤ in responses to isoflavones were analysed. isoflavones feeding increased ere binding as much as chronic injections of estrogen did in the ovxed rats. research funds: kampo science foundation, japan ps3a-d046 mechanism of central metabolic control by tgf-beta in the rat brain: using the rat with depletion of hypothalamic noradrenaline teppei fujikawa, kazuo inoue, tohru fushiki division of food science and biotechnology, graduate school of agriculture, university of kyoto, kyoto, japan we have previously reported that activated transforming growth factor-beta (tgf-beta) increase in the rat brain during exercise. intracranial administration of tgf-beta induced an increase in fat oxidation, free fatty acid and keton body in the blood. these results suggest that activated tgf-beta in the rat brain participates in metabolic control of peripheral tissue by cns. it is, however, not known how tgf-beta increases in specifically fat oxidation. many investigations suggest that hypothalamus is essential for central metabolic control. in addition, some reports suggest that noradrenergic system in the hypothalamus may play important role for fat oxidation. in this study we measured concentration of extracellular noradrenaline (na) in the hypothalamus by using microdialysis after injection of tgf-beta. then, we measured respiratory exchange ratio and serum samples, after administration of tgf-beta in the rat with depletion of hypothalamic na by injection of 6-hydroxydopamine. ps3a-d047 the effect of brain-derived neurotrophic factor (bdnf) on neuropeptide y (npy) neurons in the mouse corpus callosum: an examination using organotypic brain slice culture ryoichi yoshimura, kazuto ito, yasuhisa endo department of applied biology, faculty of textile science, kyoto institute of technology, japan the morphology of neuropeptide y (npy) neurons existing in the corpus callosum (cc) and the effects of brain-derived neurotrophic factor (bdnf) on the npy neurons were examined by using organotypic slice culture system. bdnf treatment significantly increased the number of the npy-immunopositive cell bodies and fibers in cc assessed with immunocytochemistry. electron microscopy demonstrated that the npy immunoreactivities were mainly localized in the regions associated with accumulating synaptic or cored vesicles in cc nerve fibers. the sectional area of npy-positive fibers was larger in the bdnf-treated culture than in the control culture. the number of nerve fibers adjacent to the npy-positive fibers was also larger in the bdnf-treated culture than the control. these results suggest that npy may play a key role in the neuronal regeneration, and bdnf takes part in the development of npy neuron fibers as well as the increase of the number of npy neurons in cc. reiji semba 1 , kimi watanabe 1 , munekazu komada 2 1 institute for developmental research, aichi human service center, aichi, japan; 2 graduate school of medicine, kyoto university, kyoto, japan d-serine is hypothesized to be a glia-derived neurotransmitter activating the nmda receptor because d-serine was reported to be formed and localized exclusively in astrocytes. however, we reported strong immunoreactivity of d-serine in some axons. to reveal which cells are producing d-serine in the brain, an in situ hybridization study of serine racemase, the enzyme producing d-serine from l-serine, was performed. using antibodies against neun, a neuronal marker, gfap, an astrocyte marker, and cnpase, an oligodendrocyte marker, type of the cells containing the mrna was examined. coincidentally with our immunohistochemical study of d-serine, strong signals for serine racemase mrna were found in some neurons while weak signals were found in astrocytes. present results suggest that d-serine will be a neurotransmitter activating the nmda receptors produced in a specific type of neurons. takatoshi hikida 1,2 , asif k mustafa 2 , kenji hashimoto 3 , kumiko fujii 2,4 , kazuhisa maeda 2,5 , hiroshi ujike 6 , richard l. huganir 2 , solomon h. snyder 2 , akira sawa 2 1 dept. of systems biology, obi, suita, japan; 2 depts of neurosci. & psychiat, johns hopkins univ. med., baltimore, maryland, usa; 3 chiba univ. forensic mental health, chiba, japan; 4 dept. of psychiat, shiga univ. med. sci., shiga, japan; 5 div. of neuropsychiat, tottori univ., yonago, japan; 6 dept. of neuropsychiat, okayama univ., okayama, japan accumulating evidence from both genetic and clinical studies suggests a critical role of d-serine in schizophrenia (sz). we identified and characterized pick1 as a protein interactor of the d-serine synthesizing enzyme, serine racemase (sr). d-serine levels in the hippocampus and frontal cortex of pick1 knockout mice were significantly lower than those of their wildtype littermates at age of p7, but not in adults, suggesting regulation of pick1 on sr at developing stage. in case-control association study, we observed an association of the pick1 gene with sz, which is more prominent in disorganized sz. our findings suggest that pick1 contributes to sr activity, d-serine production, and nmda neurotransmission in the pathophysiology of sz. ps3a-d050 epileptiform activity is inhibited by taurine which can activate glycine and gaba a receptors in immature rat hippocampus akihito okabe 1,2 , werner kilb 2 , ileana l. hanganu 2 , taizhe qian 2 , daiichiro nakahara 3 , atsuo fukuda 1 , heiko j. luhmann 2 1 dept. of physiol., hamamatsu, japan; 2 inst. of physiol., mainz, germany; 3 dept. of psychol., hamamatsu, japan many studies indicate that the underlying mechanism of epileptic seizures differ between children and adults. the depolarizing gabaergic responses in immature neurons may contribute to higher epilepsy susceptibility. to investigate whether taurine, a neurotransmitter found in high concentrations in the immature cns, modulates epileptiform activity in immature hippocampus, we performed field-potential recordings in neonatal rat hippocampal ca3 region of an intact preparation. 5 mm taurine blocked epileptiform activity induced by mg 2+ free acsf and 20 m 4-ap. this taurine effect was prevented by the glycinergic antagonist strychnine and the gaba a antagonist gabazine. inhibition of taurine uptake by ges also suppressed epileptiform activity in strychnine and gabazine sensitive manner. these results suggest that taurine mediates an inhibition in immature hippocampus via glycine and gaba a receptors that suppresses epileptiform activity. ps3a-d051 responses of pge 2 in undifferentiated and differentiated ng108-15 cells kayoko matsushima 1 , takashi imanishi 1 , akinori kawaguchi 1 , tetsuyuki wada 1 , shigeru yoshida 2 , seiji ichida 1 1 school of pharm. sci., kinki univ., osaka, japan; 2 school of pharm. sci., kinki univ., osaka, japan; 3 school of pharm. sci., kinki univ., osaka, japan; 4 school of pharm. sci., kinki univ., osaka, japan; 5 school of sci. & eng., kinki univ., osaka, japan; 6 school of pharm. sci., kinki univ., osaka, japan our previous findings showed that 5-ht-and bk-induced [ca 2+ ] i increases were enlarged in differentiated ng108-15 cells. for the next stage, we investigated the effect of pge 2 , an inflammatory mediator for 5-ht and bk, on the cells. ng108-15 cells were loaded with fura-2/am, and the change in [ca 2+ ] i was monitored by an image processor. the results showed: (1) pge 2 -induced response was decreased when ng108-15 cells were differentiated by bt 2 camp, (2) 10 −5 m ah6809 and sc19220 irreversibly inhibited pge 2 -induced response by about 50% and 26%, respectively, while 10 −5 m ah23848 and sulprostone had no effect, and (3) pge 2 -induced response was abolished under ca 2+free conditions in about 70% of both ng108-15 cells. these results indicate that the response to pge 2 , via ep1 and ep2 receptors, significantly decreased during differentiation. mitsumasa murano, fumihito saitow, hidenori suzuki department of pharmacology, nippon medical school, tokyo, japan the most of cerebellar outputs are generated as a result of synaptic interaction in the deep cerebellar nuclei (dcn) and by the electrical membrane properties of dcn neurons themselves. this study aimed at examining mechanisms underlying the serotonergic modulations of both the gabaergic transmission at the purkinje-to-nuclear cell synapses and the membrane properties of dcn neurons using cerebellar slices prepared from 11-to 21-day-old rats. bath application of serotonin (5-ht) decreased the amplitude of stimulation-evoked ipscs in dcn neurons in a dose-dependent manner. furthermore, slow inward currents ware observed in dcn neurons during 5-ht application. under the current-clamp recording, 5-ht markedly depolarized and increased action potential discharges of dcn neurons. taken together, these results suggest that 5-ht facilitates the voluntary activity in dcn neurons by both pre-and post-synaptic mechanisms. ps3a-d053 searching for endogenous ligands of trace amine receptors in mammals (1) akira komatsu 1 , airi yamaguchi 2 , noriko makikusa 2 , osamu koizumi 2 1 dept. physiol., tokyo women's med. univ., sch. med., tokyo, japan; 2 neurosci. lab., fukuoka women's univ. fukuoka, japan trace amine receptors were discovered in mammals, but their endogenous ligands have not yet been found. to search for them, we developed a new method to make antibodies against monoamines for immunohistochemistry (ihc). monoamines, phenylethylamine (pea), tyramine (ta) and histamine (ha), were conjugated to a hemocyanine, klh, using an imidoester cross-linker, dimethyl suberimidate (dms). rabbits were immunized by the conjugated macromolecule. the obtained antibodies were assayed by elisa and competitive elisa technique to check their antibody titer and specificity respectively. the antibodies recognized specifically the monoamine-dms part within the complex. for ihc, the rat brain was perfused by 2% dms, post-fixed by 4% formaldehyde and then frozen-sectioned. the antibody against ha revealed the immunoreactive neurons in the hypothalamus, showing that this method is effective to demonstrate the presence and localization of monoamines. the antibodies against pea and ta failed to reveal immunoreactive neurons in the rat brain. ps3a-d054 effects of mg 2+ on neural activity of cultured cortical neurons of the rat and mouse yuriko furukawa 1,2 , nahoko kasai 1 , akiyoshi shimada 1 , keiichi torimitsu 1,2 , kunihiko obata 3 , yuchio yanagawa 2,4 , tadaharu tsumoto 2,3 1 ntt basic research laboratories, kanagawa, japan; 2 sorst/jst, saitama, japan; 3 neuronal circuit mechanisms research group, brain science institute, riken, saitama, japan; 4 dept. of genetic and behavioral neurosci., grad. sch. of med., gunma university, gunma, japan it is well known that mg 2+ plays an important role not only in energy metabolism, but also in neural information processing. however, the mechanism of such a role in cns is not well understood. previously we reported that neural activity and the intracellular ca 2+ concentration are largely affected by mg 2+ removal in cultured cortical neurons of the rat. transient glutamate release was also detected. in the present study, we investigated effects of the mg 2+ removal on neural activity in cultured cortical and hippocampal neurons. in particular, we measured the intra-and extracellular mg 2+ concentration and their actions on neural activity using a mg 2+ indicator, kmg-20-am together with fluo4-am. we observed different effects of the mg 2+ removal on gabaergic and non-gabaergic neurons by using gad67-gfp knock-in mice. research funds: jst/sorst ps3a-e055 transient zinc-positive terminations in the developing rat somatosensory cortical system noritaka ichinohe, daniel potapov, kathleen s. rockland lab. for cortical organization and systematics, bsi, riken, usa synaptic zinc (zn) is a neuromodulator used by a subset of nonthalamic glutamatergic connections, and associated with both experiencedependent and developmental plasticity. during development, transiently high levels of synaptic zn occur in both sensory and nonsensory cortical areas. by injecting the retrograde tracer sodium selenite into barrel cortex, we demonstrated a transient subset of zn + thalamocortical neurons from p7-p13. zn + cortical neurons were also labeled, intrinsic and extrinsic, from p5. unlike in the adult, these were in layer 5, instead of layers 2, 3, and 6. at p9, neurons occurred in layers 2, 3, and 5 and, in some areas, layer 4. at p15, zn + neurons first appeared in layer 6; and at p22, there is the adult lamination. as whisking and exploratory behavior commences in the second postnatal week, these transient zn + terminations may play a role in experience-dependent adjustments in cortical circuitry. research funds: bsi, riken and kakenhi no. 17024064 ps3a-e056 systematic comparison of the structure of the serotonin immunoreactive neurons between insect species masaaki iwano 1,3 , ryohei kanzaki 2 , kei ito 1,3 1 center for bioinform., imcb, univ. of tokyo, tokyo, japan; 2 dept. of mechano-inform., grad. sch. of inform, sci. and tech., univ. of tokyo, tokyo, japan; 3 bird, jst, saitama, japan in the vertebrate central nervous system, the distribution of the serotonin immunoreactive neurons (sirns) is known to be preserved remarkably during evolution. systematic comparison of the invertebrate sirns has not been performed, on the other hand. in the current study we analyzed the morphology of the sirns in the brains of holoand hemi-metabolous insects including flies, bees, moths, beetles, crickets, dragonflies and cicadas. in spite of the large variation in the size and cell numbers of the brain, the number and distribution of the sirns were highly consistent between species. for example, we observed either one or two pairs of bilateral sirns with similar morphology that connect specific subregions of the lateral accessory lobe, a candidate pattern generator of the zigzag locomotion of the insect. variation was greater in the antennal lobe, the insect primary olfactory center, where sirns project either ipsil-or contra-laterally depending on the species. maki kagohashi 1,2 , taizo nakazato 2 , shigeru kitazawa 2 1 neurol, juntendo univ., tokyo, japan; 2 physiol, juntendo univ., tokyo, japan in vivo voltammetry has been used for measuring neurotransmitter releases in the brain of behaving rats (e.g. nakazato, 2005) . however, task freedom was restricted by cables connecting the head and the measurement system. to overcome the difficulty we developed a wireless voltammetry system and examined its sensitivity in vitro (kagohashi et al., jns2005). the system consisted of a wireless transmitter with a potentiostat and a signal receiver. in the present study, we reduced the size and weight and measured dopamine (da) currents in vivo with the wireless system mounted on the back of the rat. a single-step voltage pulse (100 to 250 mv for da; 300 to 450 mv for 5ht) was applied at 4 hz through a carbon electrode that was chronically implanted in the striatum. after administration of l-dopa, da currents showed a gradual increase in good agreement with the data measured with conventional systems. the present wireless system would be applicable to measurement of neurotransmitters in various situations (e.g. social interaction). research funds: scientific research on priority areas (mobiligence) hiroyuki yamazaki, tomoaki shirao department of neurobiology and behavior, gunma university graduate school of medicine, maebashi, japan dendritic spines are multiple functional units that receive most of excitatory inputs in central nervous system. in the purpose of finding a novel molecule that is involved in regulation of dendritic spines, we have done a screening of a novel drebrin binding protein. yeast twohybrid system was conducted with drebrin as bait, and a novel drebrin binding protein was isolated. in neurons, this protein was localized primarily in nucleus and dendritic spines. hence, we named it spikar for its unique intracellular localization in spine and karyoplasm. we studied the role of spikar in spine formation. hippocampal neurons were transfected with shrna expression vector for spikar at several developmental stages. in early stage, spikar knock down (kd) did not affect the density of dendritic protrusions that were mostly filopodia. in contrast, spikar kd reduced spine density at the stage of synapse formation. these results suggest that spikar plays a role in the formation of dendritic spines, without affecting the filopodia formation. ps3a-e059 time-lapse analysis of the translocation of drebrin-actin complex from dendritic spines to dendritic shafts by glutamate stimulation toshiyuki mizui 1,4 , yuko sekino 2,3 , tomoaki sirao 1 1 dept. of neurobiol. & behav., gunma univ. grad. sch. of med., maebashi, japan; 2 div. of neural network, inst. med. sci. univ. of tokyo, tokyo; 3 crest, jst, kawaguchi, japan; 4 jsps, japan we have shown that nmda receptor activation induced translocation of drebrin, with retaining its binding to f-actin, from dendritic spines to their parent dendrites. in the present study, we analyzed the time course of gfp-tagged drebrin a (gfp-da) dynamics after glutamate receptor activation. we prepared primary hippocampal cultured neurons, transfected them with gfp-drebrin a expression vector using microinjection methods at 14 days in vitro (div), and analyzed the dynamic localization of gfp-da at 16 div. glutamate stimulation started gfp-da translocating within 10 s and completed in 2 min. after washout of glutamate, gfp-da gradually re-accumulated in the spine, and the fluorescence intensity of gfp-da is fully recovered in 10 min. these data suggest that translocation mechanism of drebrin from spines to shafts is different from that from shafts to spines. research funds: grant-in-aid for jsps fellows ps3a-e060 distribution of the srf co-activator mal in developing mouse brain mitsuru ishikawa 1 , jun shiota 1 , hiroyuki tsutsumishita 1 , hiroyuki sakagami 2 , masaaki tsuda 1 , akiko tabuchi 1 1 dept. biol. chem., fac. pharm. sci., univ. toyama, toyama, japan; 2 dept. cell biol., tohoku univ., grad. sch. medicine, sendai, japan the srf co-activator mal (megakaryocytic acute leukemia) plays an important role in controlling srf-dependent gene, whose expression is regulated by rearrangement of actin cytoskeleton. recent studies with conditional deletion of srf gene demonstrated that srf was required for inducing genes such as egr-1, c-fos,␤-actin but also for neuronal migration and plasticity. in this study, we investigated the expression of mal in developing mouse brain and the role of mal for dendritic morphology. the in situ hybridization analysis revealed that mal mrna was highly and developmentally expressed in hippocampus and broadly expressed in cortex, olfactory bulb. staining of mal displayed cytoplasmic localization at cell bodies and apical dendrites. furthermore, dominant negative mal mutants and rnai led to a reduction of dendritic number, as well as a decrease of srf transcription. these findings indicate that mal is involved in the formation or the stability of dendrites. research funds: kakenhi (17790055) to a.t. shoko shimizu 1 , shinsuke matsuzaki 1 , tsuyoshi hattori 1 , ko miyoshi 2 , masaya tohyama 1 1 department of anatomy and neuroscience, graduate school of medicine, osaka university, japan; 2 department of brain science, graduate school of medicine and dentistry, okayama university, japan disrupted-in-schizophrenia 1 (disc1) was identified as a novel gene disrupted by a (1;11) (q42.1;q14.3) translocation segregating with schizophrenia and affective disorders in a scottish family. kendrin was identified as a protein which interacts with disc1 at centrosome and residues 446-533 of disc1 (kendrin-binding region: kbr) were essential for the interaction with kendrin. in this study, we show that c-terminal of disc1 downstream of kbr is indispensable structure for kbr to interact with kendrin and also essential for disc1 to target to the centrosome. furthermore, we have shown that inhibition of the disc1-kendrin interaction perturbs the tubulin network formation. these results suggest that the c-terminal region of the disc1 is important to the disc1-kendrin interaction and that a truncated form of disc1 lacking the c-terminal downstream of the translocation breakpoint might affect the microtubule organization. tatsuro kumada, yasuhiko nakanishi, atsushi fukuda department of physiology, hamamatsu university school of medicine migratory cells exhibit dynamic morphological changes in the cell soma and process in both normal developmental program and tumor growth. the morphological changes in the cells are correlated with the rate of cell migration and ion transfer such as ca 2+ or cl − . although the highly invasive migration of glioblastoma in the brain is known to be influenced by a variety of ion channels, there were a little evidence about the relationships among the morphological changes and ion homeostasis. to clarify it, we have developed a glioma cell culture system for the simultaneous observation of the cell movement and ca 2+ and cl − imaging. we found that the relatively low density a172 glioma cells actively moved on the substrate. the movement has the correlation with intracellular ca 2+ oscillation in the cells. the relationship between cell movement and intracellular ion levels is further studied. ps3a-e063 involvement of ca 2+ influx in the unpolarized non-vesicular release of fgf-1 hayato matsunaga, hiroshi ueda division of molecular pharmacology and neuroscience, nagasaki university graduate school of biomedical sciences, nagasaki, japan little is known of molecular basis mechanisms for the er-golgiindependent or non-vesicular release of fgf-1 lacking a conventional signal peptide sequence. we found that fgf-1 is co-released with s100a13, a ca 2+ binding protein from cultured rat astrocytes upon the serum-deprivation stress. here, we report that fgf-1 is co-released with s100a13 from the axon and dendrites in cultured rat hippocampal neurons upon depolarization stimulation, but serum-deprivation stress leads to release, which is seen in neurites as well as in soma. the interaction between fgf-1 and s100a13 required ca 2+ . the overexpression of s100a13 88-98 mutant lacking an ability of interaction with fgf-1 inhibited the their release, suggesting that s100a13 is a cargo molecule. the release of fgf-1 upon either stimulation was abolished by voltage-dependent n-type ca 2+ channel blocker. these findings suggest that ca 2+ influx may be involved in the unpolarized non-vesicular release of fgf-1. in neuron, intracellular calcium involves a large number of physiological phenomena, including cell migration, differentiation, and neurite outgrowth. pc12 cell is a useful model of neural differentiation and neurite outgrowth, and recently we demonstrated that 5-ht has an effect on neurite outgrowth via the increase in intracellular calcium concentration ([ca 2+ ] i ) in pc12 cells. however, it is unclear how [ca 2+ ] i regulates neurite outgrowth via actin cytoskeleton. in this study, we investigated effects of [ca 2+ ] i on actin dynamics in pc12 cells transfected with yfp-actin. filopodial growth speed and actin retrograde flow were increased by treatment with calcium ionophore, a23187. treatment with calcineurin inhibitors decreased the filopodial growth speed, while treatment with camk inhibitor did not. these effects could contribute to 5-ht induced enhancement of neurite elongation. the actin cytoskeleton is a complex protein network that not only provides cellular structure but is fundamental for cellular dynamics. on stimulation of pc12 cells by ngf, proteins that directly interact with f-actin such as actinin rapidly translocate to the f-actin-rich cytoskeleton. clp36 is a pdz-lim protein which was originally identified as an actinin-interacting protein in skeletal muscles. here, we show that clp36 is endogenously expressed in pc12 cells and plays an important role in actin dynamics during ngf-induced neurite outgrowth. immunofluorescent studies showed that clp36 is accumulated in irregular cell surface and membrane extrusion soon after ngf-stimulation, where colocalized with actin filaments. we next performed rnai experiments to explore the role of clp36 in actin dynamics in growth cones and found that knockdown of clp36 expression lead to the suppression of ngf-mediated neurite outgrowth. in addition, we revealed using clp36 deletion mutants that both of pdz and lim domains are necessary for the proper function of clp36. ps3a-e066 screening of genes expressed preferentially in migrating gabaergic neurons of developing cerebral cortex toshiya kimura 1 , tsuyoshi kobayashi 1 , yuchio yanagawa 2 , kunihiko obata 3 , fujio murakami 1,4 1 grad. sch. of frontier biosci., osaka univ., osaka, japan; 2 grad. sch. of medicine, gunma univ., maebashi, japan; 3 bsi, riken, wako, japan; 4 sorst, jst, japan neuronal migration plays a critical role in constructing brain architecture organization. however, molecular mechanisms underlying this process still remain elusive. in an attempt to identify molecules that regulate the motility of migrating neurons, we focused on migrating cortical interneurons, and performed subtractive hybridization, differential screening and in situ hybridization. subtraction was done between the embryonic and postnatal interneurons, because they robustly migrate prenatally but not postnatally. among the 2208 clones tested, two genes, neuronatin and seizure related gene 6 (sez-6) attracted our attention. they were expressed in the subventricular zone of the embryonic cortex, implicating that these molecules are expressed in interneuron subpopulations. postnatally, mrna signals were hardly detectable. these results raise the possibility that they are expressed preferentially in subpopulations migrating cortical interneurons. yan zhu 1,2 , tomoko matsumoto 1,2 , sakae mikami 3 , takashi nagasawa 3 , fujio murakami 1,2 1 grad. sch. of frontier biosci., osaka univ., japan; 2 sorst, jst, japan; 3 inst for frontier med. sci., kyoto univ., japan long distance neuronal migration takes place typically along the tangential plane of the developing neural tube. the migratory behaviour and the underlying molecular mechanisms of tangential migration are poorly understood. we address these issues using the hindbrain precerebellar system as model system. precerebellar neurons, born dorsally in the lower rhombic lip, migrate in close association with the pial membrane (except inferior olive neurons) ventrally or rostroventrally. we therefore studied the role of pia-secreted chemokine sdf-1 and its receptor cxcr4 in the precerebellar migration. we show that cxcr4 is expressed in the migrating precerebellar neurons, and its expression is down-regulated towards the end of migration. in cxcr4 and sdf-1 knock out mice, migrating precerebellar neurons are less confined to the pial surface. more strikingly, the rostrally-directed migration of pontine precerebellar neurons is severely disrupted, leading to a caudalized ectopic pontine-like cluster. ps3a-e068 involvement of an immunoglobulin superfamily molecule, neph2/mkirre in the migration of precerebellar neurons kazuhiko nishida 1,2 , kazuhide nakayama 1 , saori yoshimura 1,2 , fujio murakami 1,2 1 grad. sch. of frontier biosci., osaka univ., osaka, japan; 2 sorst, jst, saitama, japan neural cell migration plays a crucial role in central nervous system development. in this study, we analyze the involvement of neph family transmembrane proteins of the immunoglobulin superfamily in the migration of precerebellar neurons (pcns). postmitotic pcns derived from the rhombic lip in the hindbrain first migrate tangentially along the pial surface, followed by radial migration to settle at their final positions (kawauchi, d., taniguchi, h., watanabe, h., saito, t., and murakami, f., development, in press ). in situ hybridization analysis showed that among neph family members including neph1, neph2/mkirre, and neph3, only neph2/mkirre was strongly expressed in pcns. expression of neph2/mkirre was detected from e13.5 when pcns migrate tangentially. the expression level became weaker at p1, when pcns stop the radial migration, raising the possibility that neph2/mkirre might be involved in the migration of pcns. we are currently analyzing the function of neph2/mkirre in the migration of pcns. hiroki umeshima 1,2 , toshio ohshima 3 , tomoo hirano 2 , mineko kengaku 1 1 lab. for neural cell polarity, riken bsi, wako, japan; 2 department of biophysics, kyoto university, kyoto, japan; 3 lab. for developmental neurobiology, riken, bsi, wako, japan during lamination of the cerebellar cortex, granule cells exit their final mitiosis at the external granular layer and migrate to the internal granular layer. we analyzed the molecular mechanisms regulating migration of granule cells. using an in vivo electroporation system followed by time-lapse confocal microscopy of a slice culture, we found a dominant negative form of cdk5 (cdk5-dn) disrupted the morphology of granule cells during radial migration. recently, centrosome positioning is thought to be one of the important factors for neuronal migration. double-labeling of the centrosome and the whole-cell images by transfecting centrin2-gfp and rfp enabled us to record dynamic movement of the centrosome during radial migration. we found that the motion kinetics of the centrosome was disrupted by cdk5-dn. based on these results, we will discuss the role of centrosome during neuronal migration. keisuke ito 1,2 , takahiko kawasaki 1,2 , tatsumi hirata 1,2 1 division of brain function, national institute of genetics, mishima, shizuoka, japan; 2 department of genetics, school of bioscience, sokendai newly generated neurons migrate through proper pathways toward their own targets, where they are integrated into specific neuronal circuits. we have analyzed a unique tangential migratory stream of early-generated cortical neurons designated as lot cells, and performed pharmacological perturbations to characterize the intracellular mechanism of the migration. among various drugs, we found that a protein kinase inhibitor, k252a has the most interesting effect on the lot cell migration. during the normal migration, leading processes and cell bodies of lot cells move forward in a coordinated manner, but k252a blocks the migratory movement of cell bodies without inhibiting the extension of leading processes. we also found that k252a has a similar effect on cerebellar granule cells. these phenomena are quite intriguing because the drug seemed to switch the neurons from "whole cell migration" to "neurite extension" mode. we are now analyzing possible targets of k252a, aiming for dissection of these phenomena. the conserved ser/thr kinase unc51 functions with unc-76 to regulate axonal transport in drosophila hiroaki mochizuki 1 , hirofumi toda 1,3 , emiko suzuki 2 , joseph gindhart 4 , toshifumi tomoda 3 , katsuo furukubo-tokunaga 1 1 grad. school life and envir. sci., univ. tsukuba, tsukuba, japan; 2 gene net. lab., natl. inst. genet. mishima; 3 beckman res. inst., city of hope, ca, usa; 4 dep. biol., univ. richmond, va, usa neural network develops through regulated guidance of axons and interconnection among them. despite intensive researches in the past years, genetic mechanisms of axonal development still remain unclear. we have identified the drosophila homolog of unc51, which encodes a ser/thr kinase and is required for axonal formation in c. elegans and mouse. we found that unc51 is essential for neural development in drosophila. loss of function of drosophila unc51 results in reduced locomotion and axonal transport defects reminiscent of the phenotypes observed in kinesin mutants. we also found that unc51 genetically interacts with unc-76, an evolutionarily conserved cytoplasmic protein that binds to kinesin heavy chain. in unc51 mutants, unc-76 was separated from synaptotagmin vesicles. these results suggest that unc51 coordinates kinesin-cargo interaction via unc-76 to regulate dynamic axonal transport. ps3a-e072 change in microtubule polarity during the conversion of dendrites into axons kensuke hayashi 1 , daisuke takahashi 2 1 life science inst. sophia university, tokyo, japan; 2 waseda university, tokyo, japan axons and dendrites of neurons differ in the polarity of their microtubules. the mechanism for the difference, however, is not well understood. we found previously that dendrites convert into axons in cultured neurons isolated from rat cerebral cortex. in this study, we examined whether microtubule polarity changes during the conversion. in dendrites of neurons before culture, microtubule polarity was nonuniform. after 24 h of culture, we found that most of microtubules in the original dendrites had their plus ends oriented distal. this indicates that microtubules with their minus-ends distal disappeared during the culture. microtubule movement along actin filaments is a candidate for this mechanism among several types of microtubule movement reported in neuronal processes so far. however, the change of microtubule polarity within dendrites was observed even in the presence of actin polymerization inhibitors. our results suggest a rearrangement of microtubules by a yet-unreported movement in neuronal processes. research funds: kakenhi (16500193) and kakenhi on priority areas (16027207) ps3a-e073 generation and analysis of region-specific rac1-deficient mice hidetoshi kassai 1 , masahiro fukaya 2 , eriko miura 2 , mizuho sakahara 1 , masahiko watanabe 1,2 , atsu aiba 1 1 div. cell biol., kobe univ. grad. sch. med., kobe, japan; 2 div. physiol. sci., hokkaido univ. grad. sch. med., japan rac1 is a member of the rho family of small gtpases, and assumed to be involved in regulation of neuronal development through actin cytoskeletal reorganization. nevertheless, physiological role of rac1 in the cns is poorly understood because of the embryonic lethality of rac1 knockout mice. in this study, we generated and analyzed region-specific rac1-deficient mice (emx1-rac1 ko mice) by the cre-loxp system, in which a promoter for emx1 homeobox gene induces expression of cre recombinase exclusively in the dorsal telencephalon, including cerebral cortex, hippocampus and olfactory bulb. emx1-rac1 ko mice showed partially abnormal layering of cerebral cortex, indicating impaired migration of neuronal cells during cortical development. furthermore, emx1-rac1 ko mice lacked corpus callosum and anterior commissure, both of which connect the left and right cerebral hemispheres. these results suggest that rac1 regulates neuronal cell migration and axonal growth in cerebral cortex. previously we reported overexpression of map1b containing nterminal 126 amino acids promoted neuronal death. to reveal the mechanism of map1b n-terminal induced neuronal death, we searched for the proteins that interact with n-terminal of map1b by two-hybrid system. alpha-tubulin was found to interact with map1b n-terminal and their in vitro interaction was proved with pull-down assay. the interaction of tubulin and map1b n-terminal has not yet been reported. beta-tubulin was also found to interact with map1b n-terminal. when ␤ tubulin was divided in 2 fragment at between amino acid 213 and 214, there was no interaction between ␤ tubulin fragments and map1b n-terminal. interaction needs the continuous region over aa 213 and 214. there were much proportion of round formed cos7 cells in n-terminal containing map1b transfected cells than in n-terminal lacking map1b transfected cells. there might be some interference in interaction between map1b and tubulin in cells express map1b containing n-terminal. otone endo 1,2 , masaaki mizuno 3 , yasukazu kajita 2 , jun yoshida 2 1 department of neurosurgery, ja kainan hospital, aichi, japan; 2 department of neurosurgery, nagoya university, nagoya, japan; 3 department of molecular neurosurgery, nagoya university, nagoya, japan primate es cells have rather different character from rodent ones, but it is inevitable to elucidate mechanism for stable culture, purification and induction into object-oriented differentiation, because human es cells might show wide similarity to cynomolgus ones. we refined the way of large scale culture maintaining totipotency without contacting feeder cells indispensable for primate es cells. our super selective induction method for dopaminergic neurons is also refined, and induced neurons transplanted in vivo which survive without forming tumor such as teratoma for long period, are evaluated not only immunohistologically but eletrophysiologically and ethologically suggesting its enough stability, activity, ability to make neural network system and potentiality to improve clinical symptom of parkinsonism. differentiation of other types of neurons and development of fully functional neural network must be established. shigeki ohta 1 , masae yaguchi 1 , yumi matsuzaki 2 , yoshiaki toyama 3 , yutaka kawakami 4 , hideyuki okano 2 , masahiro toda 1,5 1 neuroimmunology research group, keio univ., tokyo, japan; 2 physiology, keio univ., tokyo, japan; 3 orthopaedic surgery, keio univ., tokyo, japan; 4 institute for advanced medical research, keio univ., tokyo, japan; 5 neurosurgery, keio univ., tokyo, japan we have shown that mouse dendritic cells (dcs) have the ability to induce the proliferation and survival of neural stem cells/progenitor cells (nspcs) in vitro. implantation of dcs into injured mouse spinal cord could improve the motor function through activation of endogenous nspcs in vivo. in this study, to identify an effective dc subtype for the treatment of spinal cord injury (sci), we analyzed the effects of different mouse dc subtypes on the proliferation of nspcs in vitro. among mouse splenic cd11c + dcs, cd8␣ + dcs increased the number of neurospheres most effectively in vitro. furthermore, a significant functional recovery after mouse sci was induced by implantation of cd8␣ + dcs compared to cd11c + dcs. these results suggest that cd8␣ + dcs can be an effective subtype of mouse dcs for the treatment of sci. ps3a-e077 von hippel-lindau protein regulate the neurogenesis in skin-derived precursor cells atsuhiko kubo 1 , hiroshi kanno 1 , takaakira yokoyama 1 , shuichi nakano 2 , naoki sugimoto 2 , nahoko kobayashi 3 , tetsuhiko yoshida 3 , isao yamamoto 1 1 dept. of neurosurgery, yokohama city university graduate school of medicine, yokohama, japan; 2 fiber and dept. of chemistry, konan university, kobe, japan; 3 toagosei co., ltd. corporate research laboratory, nagoya, japan skin-derived precursors (skps), multipotent somatic stem cells, are preferred cell source for autologus cns cell replacement therapy. they are proliferated by the mitogens of egf and bfgf. to investigate the effects of von hippel-lidau (vhl) protein in the neural cell fate commitment, skps were inoculated with hsv vector expressing vhl protein. skps showed promotion of neurogenesis and inhibition of gliogenesis. to detect the intrinsic factors that control lineage commitment, vhl peptides fused with the protein transduction domain (ptd) were synthesized. the ptd-vhl peptides showed rapid cell internalization in nearly 100%, and peptide with the elongin c binding site (residues 157-171) showed a high ability of inducing neuronal differentiation by interacting with jak/stat pathway. these findings are important in its application to the cns cell grafting. ps3a-e078 the effect of pueraria mirifica on erk1/2 and s-100 following sciatic nerve injury in rats pornpen chaiworakul, supin chompoopong department of anatomy, mahidol university, bangkok, thailand to investigate the effects of pueraria mirifica (pm) compared with genistein (g) and estrogen (e2) on the expression of erk1/2 and s-100 following sciatic nerve crush and transection in rats. protein levels of perk1/2 and s-100 in distal segments of nerve at day 7 were determined by western blot analysis. it was demonstrated that pm and g treatments, similar to e2, caused a significant decrease in the expression of perk1/2 levels in both nerve crush and transection injuries. however, transected nerves showed high and sustained levels of erk1/2 phosphorylation. following treatments, levels of s-100 were significantly decreased both in crushed and transected nerves with respect to control group at p < 0.05. this estrogenic effect was blocked by ici182,780. because of their structural similarity to e2, pm may have therapeutic potential in nerve injuries which as previously reported to enhance sfi following sciatic nerve crush in rats after day 7. this study suggested that pm as well as g could enhance nerve regeneration like e2 by interfering with the injury-induced erk signaling pathway. yasuhiro kato 1 , takafumi suzuki 2 , kunihiko mabuchi 2 1 department of advanced interdisciplinary studies, graduate school of engineering, the university of tokyo, japan; 2 department of information physics and computing, graduate school of information science and technology, the university of tokyo, japan mems technologies have been established to fabricate a multichannel neural probe for interfacing with the nervous system. there is, however, no suitable probe for long-term neural recording and stimulation. one main reason is the death of brain tissues damaged by the probe insertion and implantation. thus, a new skeleton-like multichannel flexible neural probe coated with hybrid biodegradable polymer was fabricated. the skeleton-like probe was designed to minimize the volume of the flexible probe and buffer injurious micromotion between the probe and the tissues in a post-implantation. the probe was coated with mixed polyethylene glycol and microspheres with nerve growth factor (ngf) to improve the stiffness for the probe insertion, and deliver ngf for an optimal period to promote regrowth of damaged neural tissues around the probe. damage-induced neuronal endopeptidase (dine) is a newly identified nerve regeneration-associated molecule. it encodes neuronspecific membrane-spanning metalloprotease and belongs to nep/ece family which degrades/processes neuropeptides. although the precise mechanism of dine including substrate is still unclear, dine seems to play a protective role in damaged neurons. the most marked property of dine is a striking response to various kinds of nerve injury in both central nervous system and peripheral nervous system. to clarify the transcriptional regulation of dine after nerve injury, we analyzed 5 untranslated region of dine gene. previously, we found that lif treatment and ngf deprivation additively increased dine mrna. in this study, promoter analysis showed that dine promoter activity was cooperatively up-regulated by atf-3 and stat3, which were induced after nerve injury and activated at the downstream of lif treatment and ngf deprivation. this combination of transcription factors may be pivotal to promote gene expression, which is responsible for nerve regeneration. tomohiro miyashita, takekazu kubo, masashi fujitani, katsuhiko hata, toshihide yamashita department of neurobiology, graduate school of medicine, chiba university, chiba, japan wnt proteins are known as those concerning with formation of central nervous system. we tested whether they play a role in inhibition of axon regeneration after spinal cord injury. cerebral granule neurons from p6-10 wistar rats were cultured. wnt proteins were added into the culture medium. twenty-four hours after culture, neurite length of each neuron was measured. immunohistochemistry was done employing anti-wnts antibody and anti-ryk (wnt receptor) antibody. anti-ryk antibody was injected continuously for two weeks into the subarachnoid space of contused rat spinal cord. locomotor behaviour was evaluated up to six weeks after injury. immunohistochemistry showed that several wnt proteins and ryk were upregulated after spinal cord injury. wnt proteins inhibited neurite outgrowth of cultured cerebral granule neurons. and this effect was abolished by y27632, a rho-kinase inhibitor, and anti-ryk antibody. suppression of wnt proteins may promote axon regeneration and improve locomotor behaviour after spinal cord injury. akihito takeda, richard goris, kengo funakoshi department of neuroanatomy, yokohama city university graduate school of medicine, yokohama, japan in contrast to mammals, spontaneous nerve regeneration after lesion of the spinal cord occurs in fishes. we examined tissue remodeling and axon regeneration after spinal hemisection in the goldfish. in the lesioned spinal cord, neurogenesis reached the maximum level 7 days after the hemisection. glial cells positive for glial fibrillary acid protein (gfap) temporarily increased at the lesion site one day after. many gfap positive cells expressed somatostatin. serotonin (5ht) positive cells increased in number progressively from 1 day to 6 weeks after. six weeks after, the regenerated axons with glial fibers invaded fibrotic scar centered about the lesion site, and 5ht cells surrounded the axons and glia. thus, 5ht may promote these neural elements to invade the fibrotic scar. six weeks after the hemisection, projections from locomotion center in midbrain to spinal motoneurons were restored, and swimming ability was also recovered. these results suggest that the goldfish have ability to reestablish correct projections after the spinal injury. masao koda 1 , yukio someya 2 , ryo kadota 2 , chikato mannoji 2 , tomohiro miyashita 2 , atsushi murata 3 , masashi yamazaki 2 1 department of orthopaedic surgery, togane hospital, 2 department of ortopaedic surgery, graduate school of medicine, chiba university, japan; 3 division of rehabilitation medicine, chiba university hospital, japan objective: anoikis is a type of apoptosis due to the detatchment from the extracellular matrix. preparation of graft cells for cell therapy includes dissociation of cultured cells, which may cause anoikis. here we tested the effect of bdnf for anoikis of schwann cell. methods: (in vitro) schwann cells were cultured from sciatic nerves of neonatal rats. schwann cells were transferred to suspension culture. bdnf was added into the culture medium. cell death was detected 24 h after suspension culture. (in vivo) schwann cells were transplanted with or without bdnf treatment into contused rat spinal cord. immunohistochemistry was performed to detect survival of grafted cells. the olfactory bulb and its caudal extension are unique forebrain regions with the residence of neural stem cells and the ability of persistent neurogenesis. however, evidence for active functional involvement of neural stem cells is still very limited. this study was undertaken to know whether or not newly generated neurons are integrated in olfactory neuronal circuits in the neonatally bulbectomized rats that had been proved to show olfactory discriminative abilities. for this purpose, retroviral vector, a very useful tool to trace neural stem cells, was applied to the anterior part of the subventricular zone of the rats of which olfactory bulbs had been unilaterally ablated at the neonatal stage. we will show cell dynamics of newly generated neurons in the neonatally bulbectomized olfactory nervous system, with special reference to their neuronal circuits. koichi kawada, masanori yonayama, kiyokazu ogita dept. pharmacol., setsunan univ., osaka the subventricular zone (svz) contains undifferentiated cells, which proliferate and generate the olfactory bulb (ob) interneurons. throughout life, these cells leave the svz and migrate to the ob via the rostal migratory stream, where they differentiate. we have shown that trimethyltin (tmt) causes neuronal damage in the hippocampal dentate gyrus. in this study, we examined neuronal degeneration and regeneration in the ob after tmt treatment in mice. ddy mice were given tmt (2.8 mg/kg) to prepare slices for an immunohistochemical analysis using antibodies against single-stranded dna (ssdna), 5-bromo-2 -deoxyuridine-5 -monophosphate (brdu), neuronal nuclei (neun) and nestin. positive cells immunoreactive to ssdna markedly increased in the ob on days 1 after tmt treatment. positive cells immunoreactive to brdu markedly increased in the ob on days 2 after tmt treatment. double staining of brdu and neun in the ob revealed that almost brdu was not incorporated into mature neurons on day 2 after the treatment. these results suggest possible enhancement of neurogenesis in the ob following tmt treatment. ps3a-f087 early migration of human umbilical cord blood neural stem cells transplanted into rat brain miroslaw janowski 1 , hanna kozlowska 1 , marcin jurga 1 , aleksandra habich 1 , elzbieta wanacka 1 , barbara lukomska, krystyna domanska-janik department of neurorepair, medical research center, warsaw, poland many neurological disorders result from progressive cell loss or rapid cell damage. as stem cell technology appeared there is an arising hope for cell replacement therapy and definitive cure. recently, in our laboratory human umbilical cord blood neural stem cell line (hucb nsc) was established. the aim of the study was to analyze the migratory potential of hucb nsc transplanted into intact rat brain. hucb nsc transfected with gfp gene was stereotactically transplanted (tx) into intact brain of csa immunosuppressed adult wistar rats. cell detection was performed 24 h, 48 h, 72 h and 7 days after transplantation using abs anti gfp, hla class i and numa. analysis of rat brains revealed viable gfp positive hucb nsc cells migrating from tx site and dispersed through the host brain tissue 1, 2, 3 and 7 days after grafting. immunohistochemical studies confirmed that these cells were of human origin: hla class i or numa. in future we plan to study their lesion directed migratory potential. heparan sulfate proteoglycans (hspgs) are considered to play roles in cns development, such as axonal guidance. however, little is known about the function of hspgs during nerve regeneration. in this study, we examined the expression of ext2, one of the enzymes for heparan sulfate biosynthesis, after hypoglossal nerve injury. the upregulation of ext2 mrna was detected using in situ hybridization in injured hypoglossal motoneurons, and heparan sulfate glycosaminoglycan was also upregulated in the injured hypoglossal nucleus. we also examined the expression of mrna for hspg core protein. the mrnas for glypican-1 and syndecan-1 were upregulated in injured motoneurons. these results indicate that the synthesis of hspg is upregulated in injured motoneuron and hspg might be involved in nerve regeneration. masami watanabe 1 , hiroe sagawa 2 , masahiro ichikawa 3 , yoshihito tokita 1 1 dept. perinatol., int. dev. res., kasugai, japan; 2 dept. ophthalmol., nagoya univ. sch. med., nagoya, japan; 3 dept. neurosurg., nagoya univ. sch. med., nagoya, japan we examined whether rho/rock inhibitor, y39983, can make injured rgc axons regenerate into the crushed optic nerve (opn) of cats. methods: culture; retinal pieces were cultured in dmem for 14d. after fixation, the neurites were stained with anti-tuj1 antibody to obtain number and length of tuj1 neurites. crush; after an intravitreal injection of drug, the left opn was crushed with thread. on day 12, wga-hrp was injected into the vitreous. sections of opn were reacted for hrp with tmb reaction. masanori yoneyama, kiyokazu ogita dept. pharmacol, setsunan univ., osaka, japan in this study, we evaluated the effects of glutathione depletion on proliferative activity in neural progenitor cells of 15-days-old embryonic mice. neural progenitor cells were prepared from the hippocampus of 15-days-old embryonic mice by culturing in dmem/f12 medium for 9 days in vitro (div). marked round spheres were formed from cells adhered to each other under the culture conditions in the presence of bfgf and egf, and then subsequently proliferated to form large neurospheres in proportion to the duration of cultivation. to evaluate the effects of glutathione depletion on proliferation in the neural progenitor cells, buthionine sulfoximine (bso) were exposed into cultured neural progenitor cells for a period of 0-9 div. treatment with bso resulted in a marked reduction in endogenous glutathione in the cells. mtt assay revealed that the deletion of glutathione led to a marked decrease in surviving neurospheres cultured for 3-9 div. these results suggest that glutathione would positively regulate proliferative activity and/or survival in neural progenitor cells of murine hippocampus. michio hashimoto, eisuke kawakita, masanori katakura, osamu shido dept. of environ. physiol., sch. of med., shimane univ., japan docosahexaenoic acid (dha), one of the main lipids in brain, plays crucial roles in the development and function of brain neurons. we examined the effect of dha on neuronal differentiation of neural stem cells (nscs) in vitro and in vivo. nscs obtained from rat embryos were propagated as neurospheres and cultured with or without dha for 7 days. dha increased the number of tuj1(+) neurons compared with the control, and the newborn neurons in the dha group were morphologically more mature than in the control. dha decreased the incorporation ratio of brdu, the mitotic division marker, during the first 24 h period. thus, dha promotes the differentiation of nscs into neurons by promoting cell cycle exit. furthermore, dietary administration of dha significantly increased the number of brdu(+)/neun(+) newborn neurons in the granule cell layer of the dentate gyrus in adult rats. these results demonstrate that dha effectively promotes neurogenesis both in vitro and in vivo, suggesting that it has the new property of modulating hippocampal function regulated by neurogenesis. research funds: kakenhi (17590205) ps3a-f092 interaction among cues for visual depth motion perception tomokazu shimizu, akitoshi hanazawa kyushu institute of technology, fukuoka, japan when an object surface approaches or leaves us, we perceive visual depth motion. cues for this motion are change in binocular disparity, change in spatial frequency and optical flow. we investigated interactions among these cues by using visual stimuli in which the cues provides opposite depth motion direction. for the stimulus without optical flow component, spatial frequency was changed continuously by presenting uncorrelated random dot patterns filtered by different band-pass filters. when binocular disparity and spatial frequency was oppositely changed, subjects perceived depth motion corresponding to the change in binocular disparity or special frequency. for the stimulus with optical flow component, a random dot pattern filtered by a band-pass filter was expanded or contracted. when binocular disparity and the other two cues were oppositely changed, subjects perceived depth motion corresponding to the change in the other two cues. when depth motion was perceived from binocular disparity, stimulus image was perceived as changing its size. when from the other cues, depth perception from binocular disparity was suppressed. research funds: coe-j19 kazuyuki takahashi, akitoshi hanazawa kyushu institute of technology, japan in phenomena such as biological motion and structure from motion, a global structure is perceived by an integration of local motion signals. to clarify the fundamental mechanism of this motion integration process, we psychophysically examined the influence of directional motion coherency on motion grouping. moving dots were presented in three apertures that were aligned horizontally. before presenting these stimuli, subjects were instructed to detect a dot moving in a direction among noise dots presented in the central aperture. the dots moving in the same as or different from the instructed direction were presented in the side apertures. the performance of the subjects was the best when the dots in the side apertures moved in the same direction as the instructed one. the performance kept high when the directional difference was up to ± 10 • and declined as the difference increased. the high performance would be due to the grouping of the dots presented in the central and side apertures that have the same or similar motion direction. the underlying motion grouping mechanism was suggested to integrate motion signals that have a certain directional variation. ps3a-f094 effect of spatial context on structure-frommotion perception koshi makino, akitoshi hanazawa kyushu institute of technology, japan when viewing an orthographic projection of dots on the surface of a rotating cylinder, one perceives a transparent rotating 3d cylinder. this phenomenon is called structure-from-motion (sfm). the direction of the rotation is ambiguous. we investigated the influence of spatial context on the perceived direction of the rotation. three spatially separated stimuli were horizontally aligned. subjects reported in which direction the central random-dot sfm cylinder rotated. they perceived the same direction of rotation as the side stimuli when the side stimuli were corotating cylinders whose direction of rotation was disambiguated by binocular disparity. this effect was strong when the stimuli consisted of a small number of dots, and was attenuated as the number of dots increased. the perception was also influenced by translational motion stimuli that had front and back planes comprising oppositely moving random-dots whose depth was specified by binocular disparity. these results suggest that the neural mechanism determining the rotation direction of bistable sfm is strongly influenced by the 3d structure of surrounding stimuli defined by binocular disparity. ps3a-f095 rotational motion aftereffect in positive direction for 3-dimensional random-dot pattern masako ono, akitoshi hanazawa kyushu institute of technology, kitakyushu, japan when viewing a unidirectionally moving pattern followed by a stationary pattern, we will see the stationary pattern moving in the direction opposite to the preceding movement. this phenomenon is well known as motion aftereffect (mae). this mae can be perceived for 3-dimensional motion such as rotating cylinders. we found that adaptation to the rotation of a stereoscopic random-dot cylinder generate mae like phenomenon in the same positive direction as the rotation of the adaptation stimulus (positive mae). this positive mae was strong when cylindrical random-dot was used as a stationary test stimulus. this aftereffect could not be perceived for uniformly distributed non-cylindrical random-dot. although ordinary mae declined in a few seconds, this positive mae remained for a few minutes. this is a new phenomenon that is different from known 3dimensional mae. this finding suggests that the visual system has a mechanism that detect 3-dimensional rotation direction specifically, and this mechanism has a property that gives a bias to the perception of stereoscopic rotation direction in an adapted direction. research funds: coe-j19 takanori uka, ryo sasaki department of physiology 1, juntendo university school of medicine, tokyo, japan crowding refers to a subjectǐs difficulty in identifying a target in the presence of distracters. as a first attempt towards identifying the neural mechanism of crowding, we investigated perceptual crowding using a random-dot kinematogram. human subjects were required to report the direction of moving dots within a center patch (3 deg) of a center/surround display presented 10 degrees to the left of fixation, and to ignore the dots in the surround. motion coherence of the dots in the center patch, as well as surround size varied randomly across trials. motion coherence of the surround was always 0 percent. for each of 11 subjects, we calculated direction discrimination thresholds (at 82% correct) at each surround size. consistent with crowding, thresholds increased when surround size was 4.5 and 6 degrees, compared to those with no surround. surprisingly, however, thresholds decreased when surround size was 9 and 12 degrees, relative to 6 degrees. our results show that the spatial resolution of motion direction discrimination improves when the area we have to ignore exceeds a defined size. ps3a-f097 generation of receptive fields in higher visual areas based on v1 columnar structure: a model study yoshitaka toyoda, yoshiyuki shimizu, izumi ohzawa graduate school of frontier biosciences, osaka university, osaka, japan neurons in higher cortical areas of the visual pathway, such as v2 and v4, respond to stimuli with complex shapes. how do these neurons integrate signals from v1? in particular, does the well-known columnar organization of v1 play a role in determining the shape selectivity of higher-order neurons? to explore these questions, we devised a feed-forward hierarchical model. in our model, higherorder neurons sum the activities of v1 neurons linearly according to a neural receptive field (nrf), a weighting function defined over the cortical surface. the manner a nrf sums over multiple columns determines its shape selectivity. since there is no physiological data regarding possible forms for nrf, we have tested simple functional prototypes, gaussians and gabor functions. reponses of these model neurons are examined using non-cartesian gratings and other stimuli, and compared to published physiological data. about 15% of model neurons exhibit responses similar to those of v2 and v4 neurons. odd-symmetric gabor nrfs tend to generate more of these neurons. taihei ninomiya, takahisa m. sanada, izumi ohzawa graduate school of frontier biosciences, osaka university, osaka, japan when images with different spatial frequencies (sfs) are projected onto the two retinae, a 3-d surface slant is perceived (blakemore, 1980) . the relationship between the binocular receptive fields (brfs) and sf tuning properties indicate that the early cortical neurons can signal slant-in-depth (sanada and ohzawa 2006) . however, their measurements of brf were conducted in the spatial domain, and the sf tunings were tested monocularly. in this study, interactions are examined directly in the sf domain between grating stimuli presented to the left and right eyes. frequency-domain brfs were measured by a reverse correlation technique. both binocular and monocular sf profiles were obtained by this method. we predicted binocular sf (bsf) maps from monocular sf profiles, and compared the prediction and the actual bsf maps to assess the binocular interactions. with this method, neural response properties which previous studies couldn't access were revealed. research funds: mext(13041033), jsps(13308048), coe21 ps3a-f099 consistency of simple cell receptive fields: space and spatial frequency domain measurements yuka tabuchi 1 , kota sasaki 2 , izumi ohzawa 1,2 1 grad. school of frontier biosci., osaka univ., japan; 2 grad. school of eng. sci., osaka univ., japan frequency-domain subspace reverse correlation and 2-d spacedomain dynamic dense noise have become increasingly popular for mapping receptive fields (rf) of early visual cortical neurons. however, it is not known whether results from these methods are mutually consistent. to examine this issue, we compared an rf in the space domain measured by 2-d noise stimuli and an rf reconstructed from the response in the spatial frequency (sf) domain measured by flash grating stimuli of various orientation (or), sf and spatial phase presented in rapid succession. we fitted these two rfs by gabor functions, and examined the consistency of their parameters. all parameters including sf, or, spatial phase, and size of the rf agreed well when an expansive nonlinearity is considered for each cell. the optimal sf obtained in the space domain increased over time to the same extent as that obtained in the sf domain. therefore, responses of a simple cell can be encapsulated in a concise framework of a linear filter followed by expansive nonlinearity. research funds: mext(13041033), jsps(13308048), coe21 ps3a-f100 firing statistics and stimulus selectivity of inferior temporal cortical neurons in the monkey shunta tate 1,2 , hiroshi tamura 1,3 , ichiro fujita 1,3 1 graduate school of frontier biosciences, osaka university, osaka, japan; 2 jsps, japan; 3 crest, jst, japan inferior temporal (it) cortical cells are selective for visual shape, and vary in their spontaneous firing pattern among them. cluster analysis indicated that it cells were classified into five groups based on inter-spike interval (isi) histograms of their spontaneous firing. the first two groups showed a single peak at a long or a short isi in isi histograms. the other three had multiple peaks, whose positions and relative heights varied among the groups. principal component analysis and other analyses of visual responses showed that the five groups differed in their stimulus selectivity for a predetermined set of visual stimuli. stimulus selectivity was sharper in the single-peak groups than in the multiple-peak groups. one of the single-peak groups was modulated by natural images more strongly than the other groups. the results suggest that cells with different firing patterns carry different aspects of visual information, and may perform different functions in the coding of visual object images. supported by jsps and crest. research funds: kakenhi 16-07757 ps3a-f101 spatial-frequency dependency of receptive field size and surround suppression in lgn and v1 hironobu osaki 1 , tomoyuki naito 2 , osamu sadakane 2 , masahiro okamoto 3 , hiromichi sato 2,3 1 med. sch., osaka univ.; 2 grad. sch. med., osaka univ.; 3 grad. sch. front. biosci., osaka univ., osaka, japan in the primary visual cortex (v1), neurons change their responses depending on stimulus parameters such as orientation, size, spatial frequency (sf). we investigated how sf of stimulus affects on stimulus-size tuning property of responses of neurons in v1 (n = 93) and lateral geniculate nucleus (lgn) (n = 63) in anesthetized cats. first, we found that v1 neurons exhibited shifts of their sf tuning from high to low according to a change in stimulus size from small to large. second, we measured stimulus-area summation curve of responses and found that a higher sf stimulus caused a reduction of the receptive field (rf) size and an increase of the surround suppression. similar results were obtained for lgn neurons implying that the relationship between sf and area summation properties observed in v1 has its origin in lgn. these results suggest that the sf tuning of rf surround is broader than that of rf center and this center-surround mechanism reduces redundancy in visual information processing. hiroyuki nakamura 1 , akichika mikami 2 , kazuo itoh 1 1 department of morphological neuroscience, gifu university graduate school of medicine, gifu, japan; 2 department of behavioral and brain sciences, section of neurophysiology, primate research institute, kyoto university, inuyama, japan an extrastriate visual area v3a is considered to be involved in the dorsal stream visual areas, however, its connections are not understood. to demonstrate the cortico-cortical connections of v3a, we injected a bi-directional tracer biotinylated dextran amine into the v3a. our results indicated that the v3a has connections with the occipital, parietal and temporal cortices. the v3a may thus be involved in the visual information processing of both the dorsal and the ventral stream visual areas. in addition to these connections, we found that v3a has commissural connections with the v3, the v3a, the parieto-occipital area, the dorsal parietal area, and the ventral intraparietal area, and receives commissural projections from the dorsal and ventral aspect of secondary visual area v2. these commissural connections may convey ipsilateral visual information near the vertical meridian representations. ps3a-g103 activity of neurons in the isthmo-optic nucleus and its relationship with head movements hiroshi ohno, hiroyuki uchiyama department of information and computer science, faculty of engineering, kagoshima university, kagoshima, japan retinopetal neurons in the isthmo-optic nucleus (ion) send their axons to the contralateral retina in birds. the centrifugal visual projection is thought to be involved in attentional modulation of retinal output. we recorded activity of neurons in the ion in awake, headunrestrained japanese quails using an implanted electrode assembly. head movements were videotaped with a high-speed video camera (100 fps), and were also monitored with a 2d or 3d accelerometer. we found two distinct types of activity pattern: phasic and tonic. the majority of neurons in the ion discharge in a phasic manner. phasic and tonic cells are also different one from another in relation to head movements. phasic cells show phasic elevation of activity 10-40 ms after end of head movements, while tonic cells show tonic suppression during head movements. we will discuss the activity profiles of neurons in the ion in terms of their possible role in visually guided behaviors. ps3a-g104 timing of face specificity in fusiform gyrus responses to stimuli in different parts of the visual field yuka okazaki 1,2 , arman abrahamyan 3 , catherine stevens 3 , andreas a. ioannides 1,2 1 brain science institute, riken, saitama, japan; 2 graduate school of life science and systems engineering, kyushu institute of technology, fukuoka, japan; 3 school of psychology, university of western sydney, sydney, australia neuroimaging techniques have demonstrated the preferential responses to faces in the fusiform gyrus (fug). event related potential (erp) and magnetoencephalography (meg) studies have shown that such the responses specificity to faces occurs approximately 170 ms (n170) after stimulus onset by comparing with the other objects. in the present study, we examined whether these and earlier fug activities, which have been already identified by our team (within 100 ms), were selective for face. we achieved this by analyzing meg data elicited by static human faces, hands and shoes stimuli placed in fovea and four quadrants. we found robust statistically significant activities for faces in fug about 100 ms after stimulus onset which depended on the stimulus location in the visual field. narihisa matsumoto 1 , shoutaro akaho 1 , kenji fujikumi 2 , yasuko sugase-miyamoto 1 , masato okada 3 1 aist, ibaraki, japan; 2 ism, tokyo, japan; 3 university of tokyo, chiba, japan to understand the temporal aspects of information encoded at a population level in the inferior-temporal (it) cortex, we applied a cluster analysis method to the responses of 45 neurons. each response was recorded while one of the visual stimuli that consisted of geometric shapes and faces of humans and monkeys was presented. population activity vectors of 45 neurons for 38 visual stimuli were clustered by a mixture of gaussian model. we estimated the number of clusters by using variational bayes algorithm. we assumed that the probability of the number of clusters depended on the one at one time step before. in the early period, the population vectors formed three clusters corresponding to global categories (human versus monkey versus shape). in the subsequent period, each cluster expanded to form sub-clusters corresponding to detailed categories. moreover, the number of clusters changed smoothly over time. these results suggest that the responses of it neurons represent different levels of categorical signals separated along the time axis. ps3a-g106 relationship between color and shape selectivity in area teo of the monkey masaharu yasuda 1,2 , hidehiko komatsu 1,2 1 national institute for physiological science, okazaki, japan; 2 sokendai, okazkaki, japan visual objects typically consist of multiple features such as color, shape, texture etc. it is reported that neurons selective for these object features exist in the inferior temporal (it) cortex of the monkey and some of them are selective for more than one of these features. however, little is known about the relationship between the selectivity for different features. last year, we have reported that there exist many neurons in the posterior part of it cortex (area teo) that are selective for both color and shape. to study the relationship between the color selectivity and shape selectivity, we tested the responses of each neuron using all combinations of the sets of colors and shapes, and conducted svd (singular value decomposition) analysis. we found that some teo neurons exhibited selectivities for color and shape that were independent (separable) each other, whereas in some other neurons they were not independent (nonseparable). these results suggest a possibility that color and shape informations interact at cellular level in this area. ps3a-g107 neural correlates of stimulus shape detection in monkey inferior temporal cortex taijiro doi lab. cogn., neurosci., osaka univ., japan we searched for a neural "correlate" of conscious perception of shape by recording neuronal activities from inferior temporal (it) cortex while a monkey performed a 3-choice shape detection task. the monkey was required to judge whether or not a sample stimulus was presented immediately after a forward masking stimulus. when there was, the monkey was required to select the stimulus identical to the sample from three targets, two shapes and one small dot. trial-totrial variation of firing rates of many it neurons correlated with the monkey's seen versus not-seen choices. the mean choice probability (cp) of it neurons was 0.58, a value significantly larger than the chance level. neurons with stronger visual responses exhibited larger cps. we also searched for temporal firing patterns within the spike train from a single neuron or across 2-3 simultaneously recorded neurons, but failed to find any temporal structure related to the monkey's behavioral choice. the results indicate a link between the firing rates of it neurons with conscious perception of stimulus shape. research funds: mext grant (17022025) ps3a-g108 behavioral visual performance of the zebrafish mutant, eclipse yuko nishiwaki 1 , atsuko komori 1 , tomonori manabe 2 , toshihiko hosoya 2 , hiroshi sagara 3 , emiko suzuki 3 , hitoshi okamoto 2 , ichiro masai 1 1 masai initiative research unit, riken, wako, japan; 2 riken bsi, wako, japan; 3 ims, university of tokyo, minato-ku, japan eclipse was identified as a visual zebrafish mutant that does not show both electroretinogram and optokinetic response. in the last meeting, we reported that the els gene encodes the ␣ subunit of cgmp phosphodiesterase (pde6c), which functions in phototransduction in cone photoreceptors. since genetic mutations of pde6c have not been reported in human patients of hereditary eye diseases, the els mutant is a good model for studying physiological roles of pde6c. here we investigated whether the structural integrity of photoreceptors and visual sensitivity are affected in the els mutants. our electron-microscopic analyses revealed that photoreceptors do not undergo degeneration and are maintained in the els mutant until 9 day-post-fertilization. however, we found that visual response to the contrast is slightly affected in larvae heterozygous for the els mutation. these data suggest that the level of pde6c activity is important for the sensitivity of vision. ps3a-g109 localisation of two markers of oxidative phosphorylation in the ageing human retina: an immunohistochemical study tapas nag, shashi wadhwa aiims, india the enzymes of oxidative phosphorylation are known to be affected by reactive oxygen species, which cause mutations in them, leading to reduced energy production. we examined the distribution of two markers of oxidative phosphorylation (nadh-ubiquinol oxidoreductase and cytochrome c oxidase) in the human retina at different ages. eyeballs of donors (age: 50-94 years) were fixed in paraformaldehyde, frozen retinal sections from macular to midperipheral regions cut and immunolabelled for nadh-ubiquinol oxidoreductase (complex i) and cytochrome c oxidase (complex iv; molecular probe, usa). complex i-immunoreactivity (ir) was moderately present in photoreceptors, outer plexiform layer and few ganglion cells from 50 to 80 years of age, and showed a decline and lack of ir in older retinas (85-94 years). complex iv-ir was intensely present in most ganglion cells, outer plexiform layer and photoreceptors from 50 to 91 years of age, and absent at 94 years of age. thus, complex i and iv-ir decline with age, with the former showing an earlier reduction in its ir. the data signify a reduced mitochondrial activity in the retina with ageing. research funds: aiims ps3a-g110 temporal characteristics of neural activity related to target detection during visual search tomoe hayakawa 1 , norio fujimaki 1 , toshihide imaruoka 2 1 nict, kobe, japan; 2 kit, kanazawa, japan meg and fmri experiments were conducted during the orientation singleton search task, and moment magnitudes of dipoles were estimated with an fmri-constrained meg-multi-dipole method to obtain differences between target-present and -absent conditions in each brain region for the whole time course. activity around the cas consisted of a prominent and a subsequent smaller but still obvious peak (117, 215 ms); the first peak showed no difference between conditions while the second peak was significantly larger in the target-present. activity around the pfug had a prominent peak and subsequent small activity (125, 237 ms), whereas the target's presence or not had no influence on either activity. the activity of the right intraparietal sulcus (ips) was significantly larger than that for the left ips at latencies around 196 ms irrespective of the target's presence or not. the results demonstrate that neural activities of multiple regions had different temporal characteristics and the later activity around the cas was related to the target segregation from its surroundings. kaoru amano 1,2 , derek arnold 3 , alan johnston 4 , tsunehiro takeda 1 1 univ. tokyo, chiba, japan; 2 ntt cs lab., kanagawa, japan; 3 univ. sydney, sydney, australia; 4 ucl, london, uk when a moving border defined by small luminance changes (or by color changes) is shown in close proximity to moving borders defined by large changes in luminance, the low contrast border can appear to jitter at a characteristic frequency -a phenomenon we refer to as misc (arnold & johnston, 2003) . in order to reveal the neurophysiological substrates of this illusion, brain activities measured using magnetoenceohalography (meg) were compared with the perceived rate of illusory jitter measured psychophysically. the result showed that the perceived rate was around 10 hz and matched with the alpha frequency of meg. as 10 hz meg responses were enhanced in the presence of illusory jitter relative to the presence of isoluminant motion and physical 10 hz jitter, we believe that the activity is related to illusory jitter generation rather than to jitter perception or to isoluminant motion per se. these results support our hypothesis that misc is generated within cortex by the dynamic characteristics of a cortical feedback circuit rather than by any physical stimulus properties. ps3a-g112 the internal structure and the visual neuron projection patterns of the ventrolateral protocerebrum (vlpr) in the drosophila central brain kazunori shinomiya 1,2 , kei ito 1,2,3 1 center for bioinform., imcb, univ. of tokyo, tokyo, japan; 2 dept. comput. biol., grad. sch. frontier sci., univ. of tokyo, kashiwa, japan; 3 bird, jst visual information processing in the insect brain has so far been analyzed mainly within the optic lobe. many visual pathways are known to project from the optic lobe to a central brain area called the ventrolateral protocerebrum (vlpr). the vlpr is therefore expected to be one of the major higher-order visual centers. the neural circuits in this area, however, remain essentially unknown. our study is to reveal the detailed internal structure of the vlpr, for the first time, using the drosophila brain as a model system. we have identified 12 discrete glomerulus-like structures (gls) in the vlpr, among which at least five are innervated by the visual projection neurons from the optic lobe. we analyzed the detailed internal structure of these gls by visualizing single cells in each visual pathway using the combination of the gal4 enhancer-trap and the flp-out systems, and revealed the directionality of each pathway by specifically labeling the pre-and post-synaptic terminals. ps3a-g113 dynamic reorganization of orientation maps in a late phase of the sensitive period kazunori o'hashi 1,2 , toshiki tani 2 , shigeru tanaka 1,2 1 graduate school of life science & systems engineering, kyushu institute of technology, japan; 2 laboratory for visual neurocomputing, brain science institute, riken, japan we have found that there are two phases in the sensitive period of orientation plasticity: an early irreversible phase and a late reversible phase. in this study, we attempted to elucidate how orientation maps are reorganized in the late reversible phase, performing intrinsic signal optical imaging several times from the same kittens. we observed the over-representation of the exposed orientation even one day after the onset of goggle rearing around the age of 5 weeks. we also found that when the goggles were removed after 1 or 2 weeks of goggle rearing, drastically reorganized orientation maps returned to regular orientation maps that had been established before goggle rearing. these results suggest that once established orientation maps in an early phase serve as template maps to which later rapidly reorganized orientation maps are restored by the release of single orientation exposure. manavu tohmi, seij komagata, yamato kubota, masaharu kudoh, katsuei shibuki department of neurophysiology, brain research institute, niigata university, niigata, japan fourier analysis of intrinsic signals produced by periodic visual stimuli has been applied for constructing retinotopic maps (kalatsky and stryker, 2003) . in the present study, we used fourier analysis of flavoprotein fluorescence signals for constructing retinotopic maps in the mouse visual cortex. periodic bar stimuli that moved across the visual fields produced periodic fluorescence signals in the visual cortex of anesthetized mice. the fourier components of the signals locked with the periodic stimuli were calculated in each pixel regarding the magnitude and phase. retinotopic maps were constructed based on these components. vascular artifacts could be removed when the stimulus frequency was higher than 0.3 hz, since fluorescence signals but not vascular responses could follow up to these frequencies. combination of flavoprotein fluorescence imaging and fourier analysis is a powerful tool for investigating high-resolution retinotopic maps with short acquisition time in the mouse visual cortex. yoshitake kohei, manavu tohmi, masaharu kudoh, katsuei shibuki dept. neurophysiol., brain res. inst, niigata univ., nigata, japan we have reported that ocular dominance plasticity induced by monocular deprivation can be visualized in mice using transcranial flavoprotein fluorescence imaging. another condition for producing ocular dominance plasticity is strabismus, which causes an increase in the proportion of monocular cells in the visual cortex. however, this possibility has not been tested in mice, mainly because surgical operations for producing large and stable shifts in eye position are difficult in mice. in the present study, we designed a new prism goggle for mice. this goggle was attached on the skull of mice during the critical period. the neural responses in the visual cortex of these mice were investigated using transcranial flavoprotein fluorescence imaging. preliminary experiments suggested that the responses in the monocular zone of the visual cortex were not affected in the strabismic mice. however, binocular interaction, which was additive in the binocular zone of normal mice, turned to be more repulsive in the strabismic mice. ps3a-g116 retinotopy-based morphing of brain activity hiroshi ban, hiroki yamamoto, jun saiki graduate school of human & environmental studies, kyoto university, kyoto, japan the topographic visual field map is a fundamental property of the primate early visual cortex. we propose a new method to represent and sample topographic activities in the space of visual field by extending our previous study (maeda et al., 2003. neurosci. res.) . the procedure was as follows. first, eccentricity and visual angle representations were measured for each subject using standard phase-encoding stimuli. second, individual cortical surfaces were reconstructed. third, the transformation between the position in the visual field and that on the cortical surface was established. finally, by using this transformation, brain activities were sampled and then displayed as an image spanning visual field dimensions, each pixel of which represents the activity of neurons representing a given position in the visual field. this retinotopy-based morphing is useful to analyze brain activity related to spatial and form vision and is more reasonable to integrate individual data than normalizing methods based on stereotaxic coordinates and anatomical structures. masahiro yamada 1 , yasuhiro enami 1 , hiroshi jouhou 2 , takehiko saito 3 , kaj djupsund 4 1 tokyo metropol. univ., hino, tokyo; 2 astellas pharma. inc., osaka, japan; 3 suny upstate med. univ., center for vision and ophthal., ny, usa; 4 univ. kuopio, dept. neurobiol., kuopio, finland on-off type amacrine cells are intensely connected with each other by gap junctions (gjs), forming a syncytium with a wide receptive field. we studied effects of external ph (ph 0 ) on the control of cell functions. photoresponses of the cells were recorded intracellularly. slits of light stimuli simplified the estimation of the current flow in the cellular network into a one-dimensional problem. by lowering ph 0 only 0.2 units from the baseline of 7.60, we found a remarkable reduction of the conduction velocity by 20-30%, an increase of the length constant and a hyperpolarisation of the resting potential. based on our theoretical model, combined with measurements of conduction velocity and length constants of the receptive field, we could estimate both gj and plasmamembrane conductances of the cell. thus, we suggest that protons could contribute to the reduction of conductances, especially at the plasmamembrane but also at gjs. ps3a-g118 analysis of the band-pass filtering of the retinal rod by the ionic current model it is known that the rod network behaves like a band-pass filter. it was found that the time to peak of the response was shorter in rods further away from a slit of light. the band-pass filtering behavior has been attributed to an inductance element, i h , or i k(ca) . however, biophysical mechanism underlying the band-pass filter is not fully understood. to analyze the functional roles of ionic currents in the band-pass properties of rods, a model of the rod network was developed. the model incorporates much of the known parameters in rods, i.e., the phototransduction cascade, ionic currents (i ca , i kv , i k(ca) , i h , i cl(ca) ), calcium system and gap junctions between rods. in simulation, the band-pass properties of the rod was analyzed. it was found that single rod itself behaves as a band-pass filter. the mechanism underlying the band-pass filter was examined by changing model parameters. the result suggests that i k(ca) , i cl(ca) and i h are responsible for the bandpass filtering. research funds: kakenhi (17500195) ps3a-g119 stimulus selectivity and correlated spontaneous activity of distant neurons in monkey inferior temporal cortex go uchida, mitsuhiro fukuda, manabu tanifuji bsi, riken, wako, japan in inferior temporal (it) cortices of anesthetized macaque monkeys, we have previously shown that spontaneous spike activities (sas) of 30% (17 of 57) of neuron pairs (inter-neuronal distance >500 m) are significantly correlated. in the present study, to investigate how the correlated sas relate to functional structure in it cortex, we measured stimulus selectivity for each neuron of the 57 pairs and explored similarity of stimulus selectivity by calculating correlation coefficients of responses to 28 visual stimuli. this analysis revealed that the pairs with correlated sas tended to show more similar selectivity than the pairs lacking correlated sas. in addition, model analysis showed that in 71% (12/17) of the pairs the correlation of sas reflect synchronous transition between two activity states: periods with high and low mean firing rates. these results suggest that a network underlying the synchronous state transition provides circuitry that functionally connects distant it neurons showing similar stimulus selectivity. toshiyuki ishii 1,2 , toshihiko hosoya 1 1 bsi, riken, japan; 2 dept. biomolecular science, toho univ., japan understanding the significance of single spikes can be of critical importance in the analysis of neuronal information coding. it is often assumed that the firing rate is the sole carrier of information. however, if fine temporal patterns of spikes would carry information, the system could have large encoding efficiency. the vertebrate retinal ganglion cells fire burst spikes, separated by hundreds of milliseconds of silent periods. here we show that temporal patterns of spikes within these bursts carry visual information. when three or more spikes are fired, the multiple interspike intervals encode the input in a cooperative, non-redundant manner. this suggests that the spike patterns are not sorely determined by slowly modulating instantaneous firing rates. we also found that millisecond-scale structures in the spike patterns encode light intensity waveforms over 200 ms. we propose that the retina compresses hundreds of milliseconds of light sequences into spike patterns at the scale of milliseconds. kazuhiro shimonomura, takayuki kushima, tetsuya yagi osaka university, osaka, japan purpose of this study is to design a neuromorphic hardware model that emulates fundamental architecture and function in the primary visual cortex (v1). we have constructed a binocular vision system consisting of two silicon retinas and simple cell chips and fpga circuits. the silicon retina has a concentric center-surround laplacian-gaussian-like receptive field. the output image of the silicon retina is transferred to the simple cell chips. the simple cell chip aggregates analog pixel outputs of the silicon retina to generate an orientationselective response similar to the simple cell response in v1. this architecture mimics the feed-forward model proposed by hubel and wiesel, and computes physically a two-dimensional gabor-like receptive field. the fpga circuits compute complex cell responses based on the disparity energy model. the system can emulate the neural image of the binocular complex cells responding to natural scene in real-time and is useful to verify computational models of v1 neurons. masayoshi tsuruoka 1 , masako maeda 1 , bunsho hayashi 1 , ikuko nagasawa 2 , tomio inoue 1 1 dept. physiol. showa univ. sch. dent. tokyo, japan; 2 dept. anestesiol. showa, univ. sch. dent. tokyo, japan the present study investigated the involvement of ventral root looping afferent fibers in visceromotor function. under halothane anesthesia, the t13-l2 dorsal roots were cut bilaterally to eliminate thoracolumbar influences. an electromyogram (emg) of the external abdominal oblique muscle evoked by colorectal distention was measured. colorectal distention (80 mmhg) was produced by inflating a balloon inside the descending colon and rectum. emg activity evoked by colorectal distention significantly increased when the colon was inflamed with mustard oil (5%, 1 ml). the increased emg activity significantly reduced following bilateral l6-s3 ventral rhizotomies. a baseline emg did not significantly alter when the l6-s3 ventral roots were cut bilaterally prior to inflammation. following the development of inflammation, there was less of an increase in emg activities. these results suggest that looping afferent fibers in the ventral root are involved in visceromotor function during colon inflammation. ps3a-g123 hypnotic modulation of the cerebral processing of human visceral sensation using positron emission tomography using positron emission tomography (pet), we examined cerebral processing to visceral perception during neutral, hyperalgesic or analgesic suggestion with standard hypnosis. activation within right dorsolateral prefrontal cortex (dlpfc) and right inferior parietal cortex (ba40) was significantly greater (p < 0.001, uncorrected) during rectal distention with analgesic suggestion than with neutral suggestion. on the other hand, activation within right medial frontal cortex (mpfc) was significantly greater (p < 0.001, uncorrected) during rectal distention with hyperalgesic suggestion than with neutral suggestion. this is the first evidence with pet for a modulation of cerebral processing during visceral stimulation by hypnotic suggestion. these results suggest a role of dlpfc and mpfc in the cognitive control of the interoception. the participation of bladder receptors sensitive to cold temperature has been proposed in overactive bladder for decades. bladder cooling reflex (bcr) which consists of immediate sense of urgency and detrusor contraction in response to ice water infusion may be a neuropathic cause of detrusor overactivity (do). recently, urothelial cells display a number of properties similar to sensory neurons and have many sensors including gene for transient receptor potential (trp). we detected cold sensitive receptor trpm8 in the urothelial cell by immunofluorescence in an animal model for boo. intravesical administration of trpm8 agonist (l-menthol: 0.06-6 mm) in freely moving rats, increased the micturition pressure (mp) in either normal (n = 7) or boo rat (n = 8). the micturition interval (mi) did not change in normal rat, but decreased in boo that have do. the results suggest that bcr is enhanced in boo by increasing trpm8 on the urothelium cell of the urinary bladder. ps3a-g125 caudate projection from the vagal responsive site in the thalamic parafascicular nucleus in monkeys shin-ichi ito 1 , a.d. craig 2 1 dept. physiol, shimane univ. sch. med., izumo, japan; 2 atkinson res. lab., barrow neurol inst, phoenix, usa we investigated efferent projections to the forebrain, from the vagal afferent activation focus in the thalamic lateral parafascicular nucleus (pf) (ito & craig, j neurophysiol 2005) . evoked potentials were mapped in the right thalamus from stimulation of the left cervical vagus nerve, and fluorescent dextrans were iontophoretically injected at the response focus. the injection sites were all located in the ventrolateral part of caudal pf, lateral to the habenulointerpeduncular tract, medial to the basal ventromedial nucleus, and ventromedial to the centre median. labeled terminals were found in the caudate nucleus (cd) in all cases. terminal patches extended longitudinally in the head of cd, concentrated in its ventral aspect. dense terminal patches also occurred throughout the tail of cd. these results suggest that visceral information modulates the portion of the striatum that has been implicated in cognitive function, and they implicate the caudate nucleus in the control of heart rate and respiration. research funds: nih grant ns40413 ps3a-g126 ascending general visceral sensory pathways to the telencephalon via the medial inferior lobe in a percomorph teleost, tilapia masami yoshimoto, naoyuki yamamoto, chun-ying yang, hironobu ito, hitoshi ozawa department of anatomy and neurobiology, nippon medical school, tokyo, japan general visceral sense is relayed to the telencephalon via thalamic and hypothalamic centers in mammals and birds. in teleosts, an ascending connection that corresponds to the thalamo-telencephalic pathway is present. however, it remained unclear whether or not a hypothalamo-telencephalic pathway exists in teleosts. the medial inferior lobe (mil), which corresponds to part of the hypothalamus of other vertebrates, is known to receive general visceral sensory inputs from the rhombencephalon in a percomorph teleost tilapia. hence, telencephalic connections of the mil were studied in this study. tracer injection experiments into the mil revealed that this hypothalamic zone projects to the preoptic area, the ventral telencephalon (i.e., vs, vd, and vv), and the dorsal telencephalon (i.e., dm, rdc, and dl). these findings suggest that the mil corresponds to hypothalamic relay zones in mammals (e.g. ventromedial hypothalamic nucleus). tatsushi onaka, yuki takayanagi department of physiology, jichi medical university, tochigi, japan administration of prolactin releasing peptide (prrp) decreases food intake. we have previously shown that an icv injection of anti-prrp antibodies increases food intake. neurones producing prrp are activated after peripheral administration of cholecystokinin octapeptide, a satiety factor. it is thus possible that prrp may mediate satiety signals in the brain. here we examined effects of anti-prrp antibodies upon total amounts of food intake and meal patterns. an icv injection of anti-prrp antibodies increased the total amounts of food intake and amounts of food intake during a meal but did not significantly change meal frequency. these data suggest that prrp may play an important role in the short-term control of food intake and are consistent with a hypothesis that prrp is a satiety signal within the brain. research funds: grant-in-aid for scientific research (c) ps3a-h128 fasting induced long-chain fatty acid receptor gpr120 expression in the anterior pituitary of mouse ryutaro moriyama, shingo imoto, shinya shano, nobuyuki fukushima department of life science, kinki university, higashiosaka, japan g-protein-coupled receptor 120 (gpr120) is known as a receptor for unsaturated long-chain fatty acids. the present study investigated the effect of 48 h fasting on gpr120 expression in several regions of male mouse by real-time quantitative pcr, in situ hybridization and immunohistochemical method. gpr120 mrna expression was highly observed in the anterior pituitary, lung, colon, rectum, skeletal muscle, adipose tissue and testis in normal fed animals. 48 h fasting induced gpr120 mrna expression increase in the anterior pituitary, lung and rectum. in the anterior pituitary, gpr120-like immunoreactive cells were only observed in fasting animals. these results suggest that long-chin fatty acid regulates endocrine function in the anterior pituitary via gpr120 at least fasting period. ps3a-h129 ketone body sensing cells in the lower brain stem to regulate food intake and reproductive functions kinuyo iwata, mika kinoshita, hiroaki sato, hiroko tsukamura, keiichiro maeda laboratory of reproductive science, graduate school of bioagricultural sciences, nagoya university, nagoya, japan ketone bodies are used for energy in the brain under malnutrition, such as prolonged fasting. we have previously revealed that 3hydroxybutylate (3hb), one of ketone bodies, sensed by the ependymocytes lining the fourth ventricular walls (4v) in the rat brain to regulate reproductive functions and feeding behavior. the present study was aims to determine if the ependymocytes located on the wall of 4v respond to the change in 3hb. change in the intracellular calcium concentration ([ca 2+ ] i ) in vitro was measured in dispersed ependymocytes taken from the 4v in rats. the present results showed that the [ca 2+ ] i increased in response to 3hb, but the increase was blocked by ␣-cyano-4-hydroxycinnamic acid, which is a monocarboxylate transporter 1 (mct1) inhibitor. immunohistochemistry showed that mct1-immunoreactivities were located on the 4v ependymocytes. these results indicate that the ependymocytes may sense 3hb through a mct1-dependent mechanism. research funds: kakenhi 14360177 ps3a-h130 comparison of hypothalamic histamine release by leptin in normal mice and high fat diet-induced obese mice tomoko ishizuka, kouta hatano, atsushi yamatodani dept. med. sci. and technol, grad. sch. allied hlth sci., fac med., osaka univ., osaka, japan leptin is a satiety factor which is produced by the white adipose tissue. peripheral administration of leptin decreases body weight and food intake acting on the hypothalamus. circulating concentration of leptin is in proportion to body fat mass, however, in obese humans, elevated concentrations of endogenous leptin cannot prevent the accumulation of the adipose tissue. we previously reported that leptin decreases food intake via the activation of the histaminergic system. in the present study, the effect of leptin on hypothalamic histamine release was compared in normal and high fat diet-induced obese (dio) mice. leptin (1.3 mg/kg, ip) reduced food intake in normal mice but not in dio mice, suggesting that dio mice have resistance for exogenous leptin like obese humans. the same dose of leptin increased hypothalamic histamine release in normal mice, while it had no effect in dio mice. these results suggest that the lack of the activation of the histaminergic system partly contributes to obesity in leptin-resistant dio mice. tomoya kitayama, yuri onitsuka, katsuya morita, toshihiro dohi department of dental pharmacology, hiroshima university, hiroshima, japan parkinson disease (pd) is neurodegenerative disorder of the substantia nigra accompanied by depletion of dopamine levels. symptoms of pd include disorder of aspiration and mastication, and dysphagia. in this study, rats injected with 6-hydroxydopamine (6-ohda) resulted in an extension of feeding time and a marked increase in the amount of feed powder on cage floor after clump feeding at 4 weeks after 6-ohda without affect on number of neuron in solitary tract. these rats were transplanted with neural progenitor cells at 0 mm; anteroposterior, +3 mm; lateral and −5 and −7 mm; dorsoventral from bregma at 2 weeks after 6-ohda injection. the treatment shortened feeding time and decreased the leavings on the cage floor, as well as achieving decrease of neuronal death in substantia nigra. however, neural progenitor cells were not detected in substantia nigra. these results suggest that transplantation of neural progenitor cells may better 6-ohda-induced eating disorders via protection of neurons. research funds: grant-in-aid for young scientists b 17791323 most tools used by nonhuman animals are extension of their effectors (motor-tools), while humans can use a kind of tools as substitute for their sensory organs (sensory-tools). to understand biological bases of using such tools, we trained japanese monkeys to use a tool as an extension of the eyes, and analyzed its learning processes to proceed as follows: (1) retrieving the food with a rake (a motortool), (2) retrieving the hidden food with a mirror-attached rake, (3) using the reflected image of the food on a mirror separated from the rake, placed stationally beyond hidden food, (4) moving a mirror hung along the rail by hand to find the food, (5) using a rake with a small camera mounted inside, with which the monkeys searched for the food using the live video image captured by the camera on the monitor. finally, they could use a hand-held camera (a sensory-tool) as a manipulable extension of their eyes. thus, acquisition of using the externalized eyes can be achieved by gradual transfer of their own vision to the distant visual cues via motor-tools to extend their body image. kaori sawada 1,2 , shigehiro miyachi 2 , michiko imanishi 2 , masato taira 1 , masahiko takada 2 1 div. applied system neurosci., nihon univ. sch. med., tokyo, japan; 2 dept. system neurosci., tokyo metropol. inst. neurosci., tokyo, japan to investigate the outflow of information from the temporal lobe to the prefrontal cortex, we injected rabies virus into three prefrontal regions: medial area 9 (9m), dorsal area 46 (46d), and ventral area 46 (46v). the retrograde transsynaptic labeling was examined in the temporal lobe cortex 3 days after prefrontal injections when the second-order neurons were labeled. the labeled neurons were observed in the lateral and medial aspects of the temporal lobe. in the lateral temporal lobe, neuronal labeling from 9m, 46d, and 46v was arranged topographically in and around the superior temporal sulcus. the labeing in the medial temporal cortex was also topographically arranged, such that 9m, 46v, and 46d receive multisynaptic projections from the entorhinal cortex, area 36, and both, respectively. these results suggest that there are parallel streams of information flow from the temporal lobe to the prefrontal cortex. research funds: crest, japan science and technology agency ps3a-h137 new neural activities of reward anticipation and task errors h. ogawa 1 , h. ifuku 2 , t. nakamura 3 , s. hirata 4 1 kumamoto kinoh hosp, kumamoto, japan; 2 fac educ, kumamoto univ., kumamoto, japan; 3 nat kikuchi hosp, kumamoto, japan; 4 dept. psych, kumamoto univ. hosp, kumamoto, japan neural activities at reward phase were recorded from the primary (pgc: areas g, 3 & 1-2) and higher-order (hgc: prco & ofc) gustatory cortices of a monkey engaged in a taste discrimination go/nogo task. a lever had to be pressed after led onset when nacl was delivered, but not to water delivery. reward was given ca 2 s after led offset at correct trials. relations between cues and responses were reversed. of 169 reward-related neurons found, 88.7% showed on type responses and the rest usual expectation responses. three types of on responses were noticed; c-type (n = 96) only at correct trial, i-type (n = 5) at around possible reward onset only at incorrect trials, and c-i type (n = 49) at both. two classes of the c-i type were found; class i increased discharges at correct trials but decreased them at incorrect, but class ii increased them at both. all 3 types were found in both cortices, but most class i were found in pgc and most class ii in hgc. i-type and class ii c-i type may represent error signals and reward anticipation. hiroaki ishida, masahiko inase, akira murata department of physiology, school of medicine, kinki university, japan in the macaque monkey, the ventral intraparietal area (area vip) integrated visual-tactile information in the body centered reference frame. the receptive fields of these neurons mapped on the same body parts in each sensory modality, so this area contributes to own body representation often referred to as body image. recent psychological studies implied that shared body representation of self and other might be required in the brain for social interaction. this means other¸s body image is mapped on own body image in the same neuron. in our experiments, we studied visual-tactile receptive field of the bimodal neuron in vip, then recorded activity during observing the experimenter being touched. some of neurons that had receptive fields anchored on the monkey¸s body showed visual response while the experimenter was being touched on corresponding body parts. the results suggested that bimodal neurons in vip may be related to matching mechanism between own body image and others, then we discussed that this area may contribute to the human social ability such as imitation. daichi hirai 1 , takayuki hosokawa 2 , masato inoue 1 , akichika mikami 1 1 section of brain sciences, primate research institute, kyoto university, inuyama, japan; 2 department of psychology, tokyo metropolitan institute for neuroscience, tokyo, japan amygdala is involved in stimulus-reinforcement association learning, and have neural responses related to prediction of rewarding and aversive outcomes. however, it remains unclear whether representation of reinforcement value in the amygdala depends on other available outcomes in a given trial block. to elucidate how rewarding and aversive infomation are coded in the amygdala, we recorded single neuronal activity in monkey amygdala during delayed color matching task. we compared the neural responses to cue that rewarding outcome in two different stimulus-outcome conditions; one included electrical stimulus as aversive outcome, and the other included only rewarding outcomes. we found amygdala neurons to code the relative preference of available outcomes in a given trial block. ps3a-h140 neuronal correlates of expectation-evaluation based on previous and ongoing contextual memories in the monkey prefrontal cortex kyoko matsuda, toshiyuki sawaguchi lab. cogn neurobiol, hokkaido univ. grad. sch. med., sapporo, japan to expect future events based on the ongoing context and to evaluate it are important for flexible control of goal-directed behavior. to examine a possible involvement of the lateral prefrontal cortex (lpfc) in such functions, we recorded neuronal activity from the lpfc of monkeys that performed an oculomotor task. in this task, the target of a saccade was indicated by combinations of successively presented two cues; symmetrically allocated two objects (cue1), and centrally allocated one of the objects presented in cue1 (cue2). the frequency of which object was presented as cue2, i.e., task context, was manipulated across blocks. we focused on cue2 period and found that a subset of neurons showed object preference depending on current task context (cc type) or previous task context (pc type). cc type and pc type activities may be neuronal correlates of expectation-evaluation based on current and previous contexts, respectively. thus, neuronal processes for expectation-evaluation based on previous and ongoing "contextual memories" may progress in the lpfc. ps3a-h141 anterior insular cortex neurons in monkey are activated when reward might be delivered, such as occurs in gambling takashi mizuhiki 1 , barry j. richmond 3 , munetaka shidara 1,2 1 grad. sch. of tsukuba univ., ibaraki, japan; 2 neurosci. ri., aist, tsukuba, japan; 3 lab. neuropsychol., nimh, bethesda, usa the human insular cortex has attracted interest because it is activated during risk-taking or decision-making tasks in fmri studies. to identify related neuronal signals, we recorded single insular neurons while two monkeys worked in a reward schedule task in 2 conditions: (1) a cue is picked at random so it is uncertain whether a correctly performed trial will be rewarded [uncertain condition], (2) a cue indicates whether the current trial will be rewarded or not [certain condition]. in the uncertain condition 84/120 neurons responded in all trials. in the certain condition 50/84 neurons responded in the rewarded trials only. 48 of these 50 showed significant differences in firing rate between in the first trials after reward and other trials. these insular neuron responses seem related to reward expectancy and recent reward delivery. these neuronal responses might underlie the activation identified in imaging studies during gambling and decision-making tasks. research funds: kakenhi (priority areas17022052), aist masamichi sakagami 1,3 , kosuke sawa 2 , xiaochuan pan 1,3 1 bsrc, tamagawa university, tokyo, japan; 2 senshu university, kanagawa, japan; 3 presto, jst, japan reward prediction behavior based on integration of associative information was investigated. monkeys were trained to perform a sequential association task with symmetric reward by symbolic delayed matching-to-sample procedure. at first, they learned two sequences of stimuli: a1-b1-c1 and a2-b2-c2. after monkeys could acquire the sequences, new pairs of stimuli (i.e., d1 and d2, e1 and e2, etc) were introduced to associated with b1 or b2 (d1-b1, d2-b2, etc). the asymmetric reward rule was instructed by pairing c (c1 or c2) with the reward. after this instruction, reward predictive behavior was tested by using trained sequences and new stimuli. monkeys could show reward predictive behavior for not only a1 and a2, which were associated with c1 and c2 in trained sequences, but also new pairs of stimuli, which were not directly associated either with c or reward. these results suggested that monkeys could use reward predicting information by integration of association among trained sequences, c-reward association, and new stimuli. research funds: kakenhi (17022037), hsfp, presto, jst ps3a-h143 reward predicting activity of prefrontal neuron based on group of stimuli xiaochuan pan 1,2 , kosuke sawa 1,3 , masamichi sakagami 1,2 1 bsrc, research institute, tamagawa university, japan; 2 presto, jst, japan; 3 department of psychology, senshu university, japan ability to anticipate a reward based on grouped events is important for guiding appropriate behavior. the main purpose of this study is to examine the pfc neuronal mechanism involved in predicting reward using learned associations among groups of stimuli. monkeys performed a sequential association task with symmetric reward. at first, they learned two sequences of stimuli: a1-b1-c1 and a2-b2-c2. the asymmetric reward rule was instructed by pairing c (c1 or c2) with the reward block by block. monkeys were also trained with two different orders of stimuli (b-c-a and c-a-b). out of 202 neurons from the lateral pfc, 31% showed reward-related activity in the first cue period. and one third of them (sr type) predicted reward only when a preferred stimulus was presented as a first cue. interestingly, the preference was not based on visual properties of stimulus, but on stimulus-group. the results suggest that about 10% of lateral prefrontal neurons predict reward based on stimulus-groups that were formed through the associative learning. attention evoked by novel stimuli is important for behavioral adaptation to new environment. however, it remains unknown whether the novelty is processed in a specific region of the prefrontal cortex. we trained two monkeys on a pavlovian conditioning task interleaved with an instrumental conditioning task and recorded cell activity from the lateral and medial prefrontal cortex (lpfc and mpfc). in a block of the pavlovian task (pv block), a visual stimulus (cs) was paired with a liquid reward and the trial repeated 3 times. in a following block of the instrumental task, the monkey searched a correct action to obtain the cs as positive feedback. the cs was alternated every 4 pv blocks. in many lpfc cells, responses to the cs were enhanced immediately after the change of cs, while such enhancement was less popular in mpfc. this result suggests that lpfc more contributes to coding of stimulus-novelty than does mpfc. when an outcome of action is uncertain, a top-down attention is directed to the coming outcome. to clarify the neural mechanisms, we trained two monkeys on a task with secondary reinforcers and recorded single cell activity of the medial and lateral prefrontal cortex (mpfc and lpfc). in a pavlovian block (pv block), a visual stimulus was paired with a liquid reward. in a following instrumental block (inst block), the monkey searched a correct action based on the visual feedback. the same visual stimulus as the one presented in the preceding pv block followed a correct action, whereas another visual stimulus followed a wrong action. when the monkey made more than 3 consecutive correct trials, a new pv block started. both mpfc and lpfc cells gradually increased their firing toward the visual feedback when the outcome was uncertain, while the onset of the activity was significantly earlier in mpfc than in lpfc. these results suggest that the top-down attention first occurs in mpfc and propagates to lpfc in individual trials. ps3a-h146 neuronal activity in the presupplementary motor area during a bimanual sequential motor task toshi nakajima 1 , hajime mushiake 1 , jun tanji 2 1 department of physiology, tohoku university school of medicine, sendai, japan; 2 brain science research center, tamagawa university, machida, japan to investigate the involvement of the pre-supplementary motor area (pre-sma) in organizing bimanual sequential movements, we recorded neuronal activity while a monkey was performing a motor task consisting of pronation or supination of either arm, with an intervening delay. in this report, we focus on neuronal activity during a period when the monkey was preparing to start the 2-sequence movements in a memorized order. we made regression analysis of neuronal activity in this period. we found that neuronal activity in the pre-sma rarely reflected muscle activity. instead, we found neuronal activity representing forthcoming actions such as supination, regardless of the arm to be used. we also found neuronal activity that reflected the second movement in a preparatory period before the execution of the first movement. we would demonstrate typical examples of pre-sma neurons and discuss their functional implications. ps3a-h147 neuronal activity in the putamen and cm thalamus during response bias and its complementary process yukiko hori, takafumi minamimoto, minoru kimura dept. of physiol., kyoto prefect univ. med., japan we showed previously that cm thalamus participates specifically in complementary process to response bias (minamimoto et al. 2005) . to study the roles of the putamen and cm in response bias and it complementary processes, we recorded activity of cm and putamen projection neurons from two macaque monkeys performing asymmetrically rewarded go-nogo button press task. instruction of go or nogo activated cm neurons (n = 73) preferentially when the instruction was associated with small reward. the instructions activated 3 groups of putamen neurons preferring small reward-(n = 19), large reward-action (n = 3) and both types of action (n = 26). onset latencies of these putamen neurons and rts in large-reward-go trials were shorter than those in small-reward-go trials by 30-50 and 100-150 ms, respectively. putamen neuron activation lead that of cm neurons by 50-70 ms. these results suggested that the putamen plays a major roles in both response bias and its complementary process while cm participates in the complementary process in concert with the putamen. research funds: kakenhi (17022032) ps3a-h148 encoding expected total rewards and their errors through a series of action choices by dopamine neurons naoyuki matsumoto, kazuki enomoto, minoru kimura dept. physiol. kyoto pref univ. med., japan to examine how dopamine (da) neurons represent reward expectation and its error through a series of action choices, we recorded activity of da neurons in two japanese monkeys making trial-anderror and repetition choices to find a correct, rewarding target among three alternatives. there are trials of first (t1), second (t2) and third (t3) choices with reward probabilities of about 30, 50 and 80%, respectively. monkeys got reward after they hit a correct target, and got one more time by choosing the same target in the next trial (r1, 95%). most da neurons (66/85) responded to the start cue of each trial and reinforcer beep after the choices. magnitude of the start cue responses progressively increased from t1 to t2 and to t3 trials, then decreased in r1 trial. in another task with two repetition trials (r1 and r2, 96%), magnitude of start-cue responses decreased gradually from t3 to r1 and to r2 trials. thus, the start cue responses may reflect expected total rewards through a series of action choices for a goal, while reinforcer beep responses may reflect their errors. research funds: kakenhi (17022032) ps3a-h149 striatal neuron activity during decisions and action selections for probabilistic, scheduled rewards hiroshi yamada 1,2 , hitoshi inokawa 1 , minoru kimura 1 1 dept. of physiol. kyoto prefect univ. med., kyoto, japan; 2 jsps, japan to study roles of the striatum in decision and selection of actions for probabilistic, multiple rewards, we recorded 146 striatal projection neurons from a monkey. after depressing a start button, the monkey chose 1 of 3 target buttons with correct rates at 1st, 2nd, 3rd and repetition trials of 33, 50, 89 and 96%, respectively. correct choices were followed by reward water. neuronal firing rates at starting each trial were related either to expected reward probability or to schedule states to obtain reward twice (11/55) rather than to upcoming choice of target (2/55). during the target choice, another subset of neurons showed firings selective to choosing particular target (29/57) rather than to expected reward probability (12/57). after the target choices, another group of neurons fired related to expected reward (41/56) rather than to chosen action (19/56). our results suggested that striatal neurons encode expected reward probability, schedule states to obtain multiple rewards and choice of actions during decision and action choices for a goal. research funds: kakenhi (17022032), jsps fellows ps3a-h150 encoding of reinforcement after rewardbased action selection by tonically active neurons in the striatum hitoshi inokawa, hiroshi yamada, minoru kimura department of physiology, kyoto pref. univ. of med., japan to study the signals encoded by tonically active neurons (tans) in the striatum, presumed cholinergic interneurons, reward-based decision and action selection, activity of 169 tans was recorded from the putamen and caudate nucleus of a japanese monkey. after depressing a start button, the monkey chose 1 of 3 target buttons at average correct rates of 33 (first), 50 (second), 89 (third) and 96% (repetition choices). correct and incorrect choices were followed by high-tone beep, reward water and low-tone beep, respectively. about a half of tans (80/169) responded differentially to the high and low tone beep respectively. number of responsive tans and magnitudes of the responses to high-tone beep was highest at the first choices, then, decreased gradually at second, third and repetition choices. these results suggested that the tans may encode reinforcement after reward-based action choices which is modified by reward expectation errors and motivation. research funds: kakenhi (17022032) ps3a-i151 representation of value of action, action and its outcome in sub-populations of striate neurons y. ueda 1 , k. samejima 2 , k. doya 3 , m. kimura 1 1 dept. physiol., kyoto pref. univ. med.; 2 brain sci. res. center, tamagawa univ.; 3 irp, oist to know the mechanisms of reward-based action selection in the basal ganglia, we recorded activity of 236 striatal projection neurons of two macaque monkeys performing a free choice task with probabilistic reward. after a 1 s delay, monkeys chose between left-and right-handle turn, followed by water reward at probability of 10, 50 or 90%. a linear regression of neuronal discharge rates showed: 39 neurons encoded reward values of either action during delay period before go signal, with most (64%) of them not having the action value signal in other task epochs. another subset of neurons encoded action signal selectively during action selection after go signal (n = 33), while other 37 neurons encoded presence or absence of reward at reinforcer epoch after the action selection. neurons encoding action values were in more anterior part of putamen than the neurons encoding actions. these findings suggested that sub-populations of striate neurons process action values and selection of actions during rewardbased decision and action selection. research funds: kakenhi (17022032) ps3a-i152 delay period activity of the monkey striatum in duration discrimination task atsushi chiba, ken-ichi oshio, masahiko inase dept. physiol., kinki univ. sch. med., osaka sayama, japan neuronal activity was recorded from the striatum of a monkey during a duration discrimination task. two visual cues (a blue or red square) were presented consecutively followed by delay periods, and the subject then chose the cue presented for the longer duration. durations of both cues, order of cue duration (long-short or short-long), and order of cue color (blue-red or red-blue) were randomized on a trial-by-trial basis. striatal neurons phasically responded during the first cue (c1), first delay (d1), second cue (c2), second delay (d2), and response periods. activity during the d1 and d2 periods was analyzed in this study. firing rates during the d1 period linearly depended on c1 durations. on the other hand, d2 period activity depended on trial types (ls and sl), but not on the variety of c2 durations in each trial type. our results suggest that striatal neurons encode, in the delay periods, not only temporal information with monotonic dependence on cue durations to prepare a comparison to a forthcoming cue duration, but also encode discrimination results between two cue durations. research funds: kakenhi (17021039) ps3a-i153 neuronal activities in the anterior inferior temporal cortex of monkeys during an asymmetrical pair association task based on facial identity satoshi eifuku 1 , ryoi tamura 1 , teruko uwano 1 , taketoshi ono 2 1 dept. integrative neurosci., univ. toyama, toyama, japan; 2 dept. molecular integrative emotional neuroscience, univ. toyama, toyama, japan to elucidate neuronal basis of face memory, neuronal activities in the area teav of monkeys were recorded during a pair association paradigm that involves recognition of facial identity (i-apa task). in the i-apa task, monkeys were required to memorize 4 paired associates of patterns and facial identity. each association has a particular direction, either the 'face to pattern' direction in which a cue stimulus which is a face is associated with a test stimulus which is a pattern, or the 'pattern to face' direction in which a cue stimulus which is a pattern is associated with a test stimulus which is a face. during the i-apa task, neuronal responses to a particular paired associate were identified. many of these neurons showed asymmetrical activities during the delay periods which were dominant in the 'face to pattern' trials. this asymmetrical delay activity are indicative of the crucial role of the teav area in face memory. research funds: kakenhi (16500260 17021016) ps3a-i154 reflexive social attention elicited by biological motion in monkeys and humans yoshiya mori 1 , mikio inagaki 1 , wu lisa 2 , taijiro doi 1 , eishi hirasaki 1 , hiroo kumakura 1 , ichiro fujita 1 1 osaka univ., japan; 2 massachusetts institute of technology, usa determining where another individual is attending and preparing for his/her upcoming action is crucial for members of a social group. here we report that the walking direction of another individual elicits a reflexive shift of visuospatial attention in monkeys and humans. we examined how the reaction time to peripheral visual targets was affected by a prior, brief presentation of a walking biological motion (bm) stimulus. during the task, subjects responded to a target point after the disappearance of the bm stimulus and fixation point. the walking direction of the bm stimulus was not predictive of the target direction, and was irrelevant for performing the task. we found that the reaction times in congruent trials, where the walking direction of the bm stimulus and the direction of the target appearance were the same, were significantly shorter than those of incongruent trials. we believe the attention mechanisms driven by bm may be part of the intentionality inference system. research funds: grants from 17022025 and takeda science foundation ps3a-i155 response properties of posterior parietal neurons during a multidimensional visual search task tadashi ogawa, hidehiko komatsu natl. inst. physiol. sci., aichi, japan the posterior parietal cortex (ppc) is thought to be one of crucial areas to direct spatial attention toward the target in visual search. visual sensory information (e.g. stimulus features) might be integrated in ppc to form a saliency map that controls spatial attention. to examine this hypothesis, we recorded the neural activity from the lateral intraparietal (lip) and 7a areas of monkeys performing a multidimensional visual search task. the monkeys had to make a saccade to either shape or color singletons in a stimulus array depending on the instructed search dimension. ppc neurons increased their activity when the receptive field stimulus became the target. some neurons showed target enhancement depending on the stimulus condition (singleton type and stimulus features), whereas others exhibited it irrespective of the stimulus condition. the mixed existence of these two distinct types of activities suggests that ppc is one of critical stages that integrate feature-dependent signals to produce featureindependent signals identifying the target location toward which spatial attention should be directed. monkeys utilize visual information in social communication. to elucidate visual function to categorize sexes, (1) performance of visually guided sex discrimination task and (2) neuronal activity during the task in orbitofrontal cortex (obf), the region could be related to sex recognition and vision processes, were investigated. monkeys were trained to discriminate the sex of a monkey shown in a picture that was presented on the display. the monkeys pressed the right bar for pictures of males and the left for females to get water reward. as a result, the monkeys were able to discriminate the sexes of monkeys shown in pictures. extracellular recordings of neurons in obf during the task showed that some cells responded to the pictures in a sexspecific manner. the present results suggest that visual information alone sufficiently contribute to discriminate sex in monkeys. obf could be involved in visual categorization of sex. research funds: kakenhi (a) (16209006) (sa) and coe program in kit from the mext ps3a-i157 activities of bursting neurons during color discrimination task in the monkey prefrontal cortex naoki ishikawa 1 , satoshi katai 2 , masanori saruwatari 1 , masato inoue 1 , akichika mikami 1 1 section of brain sciences, primate research institute, kyoto university, inuyama, japan; 2 third department of internal medicine, shinshu university, school of medicine, matsumoto, japan the neurons in the prefrontal cortex of monkeys are involved in the behavioral control of saccadic eye movements. on the other hand, cerebral cortex consists of different types of neurons. in this study, we trained macaque monkeys to perform a delayed matching to sample task with saccadic eye movement. and we classified neurons whether they had burst episode or not, and then classified bursting neurons into fast spiking (fs), fast rhythmic bursting (frb), and intrinsic bursting (ib) neurons (katai et al. neuro 2004) . most of bursting neurons activated during the target presentation or during the saccade period were selective to the target location or saccade direction. these results suggest that the bursting neurons have the significant role in the target selection and decision-making of the eye movement toward the specific direction. atsushi matsumoto 1 , tetsuya iidaka 2 1 department of psychology, nagoya university, nagoya, japan; 2 department of psychiatry, nagoya university, nagoya, japan several studies indicated that gamma band activity (gba: 30-80 hz) reflects the process to form mental representation of objects or information. we investigated whether the gba is observed during subliminal visual word processing as well as supraliminal word processing. gba were observed both in masked and unmasked condition. at the 400-600 ms time window, gba was significantly higher in the word condition compared to the nonword condition in the unmasked condition. similarly, in the masked condition, gba of the word condition was significantly higher than that of the nonword condition at that time window. these results indicate that the unconscious lexical processing was reflected in the gba at that time window. furthermore, at the 600-700 ms time window, gba induced by word was significantly higher than that induced by nonword. this effect was not observed in the masked condition. in addition we found the significant semantic priming effect, indicating that the information of briefly presented words was processed unconsciously. wakayo yamashita, junichi hayashi, tomoki murakami, gang wang department of bioengineering, kagoshima university, kagoshima, japan the purpose of this study was to investigate the dependency of view association learning on the separation of the views. each stimulus set included 16 images (4 objects × 4 views). 4 novel objects were generated by deforming a prototype in four directions. for 30 deg-interval object sets, 4 views were obtained by rotating each object with the interval of 30 deg, 60 deg-interval set and 90 deg-interval set were with 60 deg and 90 deg interval respectively. task performances were evaluated while the subjects performed an object matching task, in which the subjects had to recognize one object from others regardless of the viewpoint. the performance across 60 deg separated views was significantly higher in the trials with 30 deg-interval sets than those with 60 deg-interval sets. similarly, the difference was also found in the performances across 90 deg separated views between those with 30 deg-interval sets and 90 deg-interval sets. the results suggest that the exposure of interpolated views significantly improved the association learning of the views. ps3a-i160 brain regional activity during attention task ( the kana pick-out test, treated as inspecting higher brain function, has been proposed to be suitable for screening dementia, which is widely used among public health nurses in japan. however, few fmri studies while demonstrating the test have reported. we therefore assessed the effect of brain regional activity with computerized kana pick-out test projected on the screen with clicking a mouse button to pick kana out under fmri running. executing the test resulted in significant increases in bold signals in right prefrontal area, bilateral hippocampus and broca's area. the results indicate the existence of the attention pathway from and/or to prefrontal area as association mechanisms for execution of kana pick-out test, suggesting that this test is useful in screening dementia. ps3a-i161 obsessive compulsive symptoms in middle school students and its association with tic disorder, body dysmorphic disorder and trichotilomnia in shiraz, iran, 2005 ashkan mowla, arash mowla shiraz university of medical sciences, iran aim: the aim of this study is to evaluate ocd symptoms, tic disorder, body dismorphic disorder (bdd) and trichotilomnia (ttm) among middle school students of shiraz, iran. methods: 1682 middle school students were selected in a cluster random sampling from the four educational regions of shiraz, iran.persion standardized moci was used to assess obsessional symptoms. for evaluating bdd, tic disorder and ttm symptoms, a semi-structured interview was done according to dsm-iv-tr criteria. results: students with more obsessional symptoms were more girls and demonstrated more positive family history.they were more likely to be from lower socioeconomic class and with lower school average. they also showed more association with body dysmorphic disorder and tic disorder. conclusion: girls especially those from lower socioeconomic class demonstrated more obsessional symptoms. this study, like pervious ones, confirmed bdd symptoms and tics to be more in individuals with ocd symptoms. it was seen that ocd symptoms would affect school performance. ps3a-i162 sirna-induced nr1 knockdown causes hypofunction of nmda-r and cognitive deficit m. saji 1,2 , t. utida 2 , a. ohnishi 2 , k. noda 1 , m. ogata 1 , h. akita 1 , n. suzuki 1,2 1 physiol, health sci. sch. kitasato univ., sagamihara, japan; 2 brain sci., graduate sch. kitasato univ., sagamihara, japan blockade of nmda-r by antagonists causes psychomimetic effects, suggesting involvement of nmda-r dysfunction in mental disorders like schizophrenia. however, the relationship between mental disorders and molecular abnormality has not been cleared. to identify the role of nmda-r in brain function, we performed sirnainduced knockdown of nmda-nr1 using hvj-envelope vectors. we confirmed that marked down-regulation (50%) of nr1 expression occurred only in the hippocampus among various brain regions 4-8 days after intra-ventricular injection of sirna-vector complex. in the hippocampal slice from rats with the nr1 knockdown, the nr1 down-regulation prevented depressive effects of nmda on fepsps, while the treatment did not affect ltp or ltd. in rats with the nr1 knockdown, the nr1 down-regulation caused disruption of prepulse inhibition, while the same treatment did not affect locomotor activity. these results suggest that hypofunction of hippocampal nmda-r by sirna-treatment causes a deficit of cognition. ken hatanaka 1,2 , hiroshi ageta 2 , ikuko yao 2 , kaoru inokuchi 2 , yutaka kirino 1 , mitsutoshi setou 2,3 1 graduate school of pharmaceutical sciences, the university of tokyo, tokyo, japan; 2 mitsubishi kagaku institute for life sciences, tokyo, japan; 3 okazaki institute for integrative bioscience, national institute for physiological sciences, okazaki, japan schizophrenia is a severe psychiatric disorder that characterized by psychotic symptoms in particular delusions and hallucinations, reduced interest and drive, altered emotional reactivity and disorganized behavior. to know the molecular mechanisms of the disease, we screened altered gene expression on the brain of schizophrenic patients by using microarray analysis, and found that the expression of ubl3 mrna was significantly decreased in the enthorinal cortex, whose size is known to be reduced in some schizophrenic patients. ubl3 is a highly conserved protein, which has a ubiquitin-like domain (ubl domain) and caax motif which is a membrane localization signal. we found that ubl3 mrna was expressed in the hippocampus, and purkingie cells of the cerebellum. the putative molecular function of ubl3 wil be discussed. research funds: grant-in-aid for young scientists (b), presto ps3a-i164 decreased interneurons in the pax6 mutant mouse limbic system hasumi haba 1 , tadashi nomura 1 , yoshinobu hara 1,2 , noriko osumi 1,2 1 div. dev. neurosci., ctaar, tohoku univ. sch. med., sendai, japan; 2 crest, jst, japan core features of schizophrenia are impairments in certain cognitive functions such as working memory, in which a number of brain regions in the corticolimbic system are involved. recent studies have revealed abnormality in distribution of interneurons in these regions. we have previously found that pax6 heterozygous mutant rats show behavioral abnormalities including impairment in fearconditioned memory and sensorimotor gating. in the present study, we thus analyzed distribution of interneurons in several regions of pax6 heterozygous mutant mouse (sey/+) brain. we focused on three subpopulations of interneurons: parvalbumin (pv)-, calretinin-, and somatostatin-posive interneurons. immunohistochemical studies indicated marked decrease in pv-positive interneurons in two brain regions of sey/+ mice, i.e., the olfactory bulb and the amygdala. reduced number of pv-positive interneurons was observed in the sey/+ amygdala at 8 weeks, but not at 4 weeks. our results suggest that age-dependent decrease of pv-positive interneurons might underlie behavioral abnormalities in sey/+ mice. schizophrenia is a complex genetic disorder, characterized by multiple susceptibility genes. dysbindin (dtnbp1) is a susceptibility gene for schizophrenia. genetic evidence for the association between the disorder and the dysbindin gene has repeatedly been reported in various populations world wide. recently, decreased expression levels of dysbindin mrna and protein have been reported in postmortem brain in patients with schizophrenia. thus, we performed behavioral analysis in sandy mouse, which has a deletion in dysbindin gene and expresses no protein. sandy mouse showed decreased locomotor activity and time in the center in the open field test. and an acute treatment of atypical antipsychotic, olanzapine (0.2 mg/kg, i.p.), improved the decrease in time in the center. moreover, subtle behavioral abnormality was observed in elevated plus maze test and social interaction test in sandy mouse. our results suggest that dysbindin might be involved in anxiety-related behavior in novel environment. research funds: 16790712, 17025055 ps3a-i166 gene expression analysis of dysbindin mrna in peripheral blood in schizophrenia sachie chiba 1,2 , satoko hattori 1 , hiroaki hori 1 , tetsuo nakabayashi 3 , hiroshi kunugi 1 , ryota hashimoto 1 1 department of mental disorder research, national institute of neuroscience; 2 tokyo university of agriculture and technology department of biotechnology and life science, koganei, japan; 3 musashi hospital, ncnp, kodaira, japan although many efforts have been spent to discover a biological marker of schizophrenia, no biological marker has been established. as genetic evidence suggested that dysbindin (dtnbp1) is a susceptibility gene for schizophrenia, we measured dysbindin mrna expression level in peripheral blood samples of 38 patients with schizophrenia and 38 age-sex matched healthy controls by a quantitative real time rt-pcr method. we quantified the expression levels of two major dysbindin transcripts among several known splicing variants. no significant difference in the expression levels of examined dysbindin transcripts was observed between control and schizophrenia. further examination measuring other dysbindin transcripts should be warranted to find a biological marker for schizophrenia. research funds: 16790712, 17025055 ps3a-i167 genetic variation in dysbindin influences memory and general cognitive ability ryota hashimoto 1 , hiroko noguchi 1 , hiroaki hori 1 , tetsuo nakabayashi 2 , satoko hattori 1 , sachie chiba 1 , seiichi harada 2 , osamu saito 2 , hiroshi kunugi 1 1 department of mental disorder research, national institute of neuroscience, national center of neurology and psychiatry, kodaira, japan; 2 musashi hospital, ncnp, kodaira, japan; 3 tokyo university of agriculture and technology department of biotechnology and life science, koganei, japan dysbindin (dtnbp1) is a susceptibility gene for schizophrenia, a neuropsychiatric disorder characterized by cognitive dysfunction. we examined the possible association between genetic variants in the dysbindin gene and memory and iq in 165 healthy volunteers and 72 patients with schizophrenia. individuals who did not carry a protective haplotype had lower performance in several memory domains wms-r, although this haplotype did not affect iq measured by wais-r. a risk independent polymorphism for schizophrenia influences both memory and iq in the opposite direction. these data suggest that dysbindin gene may have impact on the cognitive function such as memory and iq and that memory might be an intermediate phenotype of dysbindin on risk for schizophrenia. research funds: 16790712, 17025055 ps3a-i168 detection of 18f-dopa signal in brainstem monoaminergic nuclei in schizophrenia yuri kitamura 1 , nicola bright 3 , toshio yanagida 1 , masatoshi takeda 2 , paul grasby 3 1 department of physiology, osaka university, japan; 2 department of psychiatry, osaka university, japan; 3 cyclotron unit, imperial college, hammersmith hospital, uk we used 18 f-dopa pet to investigate presynaptic dopamine dysfunction in schizophrenic patients. the object of this study was to test that a schizophrenic cohort would show elevated aadc activity in the substantia nigra, midbrain raphe and locus coeruleus compared to normal controls. all subjects and 18 f-dopa scans were obtained from a database of scans published in mcgowan et al. 2004 , archives general psychiatry. the 14 schizophrenic patients all met dsm-iv criteria on medication and 10 healthy volunteers were compared. we attempted to improve the quality of the 18 f-dopa signal by implementing a fbf-realignment movement correction method. significant increases in 18 f-dopa uptake were found in the striatum, substantia nigra and raphe nuclei of schizophrenic patients (p > 0.02). our result suggests that an elevated presynaptic dopamine function is present in dopaminergic neurons that innervate striatal areas associated with enhanced dopamine activity in schizophrenia. in this study, we analyzed the p300 component of the visual eventrelated potential in 14 patients with schizophrenia and 14 healthy controls, and also performed loreta analysis. the ethics committee of kurume university approved this study. the p300 amplitude for the crying face was significantly smaller in patients than in controls. in controls, the p300 amplitude was significantly larger for the crying face than for the laughing face, while in patients, there was no significant difference in the p300 amplitude between the 2 faces. loreta analysis demonstrated that there were significant differences in the activity in brodmann area 10 between the 2 faces in controls, while in patients, there was no significant activity difference between the 2 faces. stimulation with crying face induced higher activities in the 10 and right 21 areas in controls than in the patients. these results indicated that the cognitive function was influenced by affective stimulus. ps3a-j170 inappropriate input produces schizophrenialike working memory deficits in a simulated neural circuit kensuke nomura 1 , shoji tanaka 2 , koki yamashita 2 , motoichiro kato 1 , haruo kashima 1 1 department of neuropsychiatry, school of medicine, keio university; 2 department of electrical and electronics engineering, sophia university a number of studies indicate that the prefrontal cortex (pfc) is intrinsically linked to working memory (wm) and that dopamine critically modulates wm activity. according to the hypothesis proposed by goldman-rakic and her colleagues, we constructed an electrophysiological circuit model for wm which represents eight directions. the computer simulation with this model shows that the working memory activity is dampened by cue-irrelevant inputs and greater noise inputs lose the directional selectivity of the representation. a lot of studies suggested that increase of noise was related to schizophrenia, especially in wm disturbance. our study indicates that noise inputs cause wm impairment in patients with schizophrenia and that working memory performance is not always positively correlated with the neuronal activity of the pfc. ps3a-j171 pericentrin is localized to the base of neuronal primary cilia in the developing cerebral cortex ko miyoshi, ikuko miyazaki, masato asanuma department of brain science, okayama university, okayama, japan we previously identified pericentrin, a mammalian centrosomal protein, as a binding partner of the product of disc1, a candidate gene for schizophrenia. in this study, we analyzed in vivo expression of pericentrin in the mouse embryo. in the developing cerebral cortex, pericentrin mrna was highly expressed in migrating cells of the intermediate zone, though proliferating neuroepithelial cells and mature neurons revealed a low expression level of pericentrin. the pericentrin protein was shown to be localized to the base of primary cilia in the pre-plate of the developing cerebral cortex, in agreement with a recent study demonstrating the involvement of pericentrin in primary cilia formation. specific subtypes of receptors such as 5-ht6 are known to be localized to the plasma membrane of neuronal primary cilia in certain regions of the brain, and then our results raise the possibility that pericentrin dysfunction may result in perturbed chemosensory function of neuronal primary cilia and increased vulnerability to psychiatric disorders. dysregulation of gr has been thought to play an important role in the pathophysiology of mood disorders. two isoforms of human gr-alpha and -beta arise from alternative splicing of the pre-mrna primary transcripts. previously, we evaluated these two isoforms mrna level in the peripheral white blood cells of the patients with mood disorders. we found that the reduced gr-alpha mrna level in the patients with both bipolar and major depressive disorders, while gr-beta mrna level was not altered. these results suggest that dysregulation of alternative splicing play an important role in the pathophysiology of mood disorders. to test this, we evaluated mrna level of alternative splicing-related sr protein family, which regulate alternative splicing in several genes including gr, in the peripheral white blood cells of the patients with mood disorders. we did not find any differences in 7 of the 10 sr protein mrnas level in the patients compared to healthy controls and now, we are examining other sr family mrnas level. ps3a-j173 alteration of neocortical long-term depression following electroconvulsive shock yoshifumi ueta 1 , ryo yamamoto 1 , shigeki sugiura 2 , kaoru inokuchi 3 , nobuo kato 1 1 dept. integrat. brain sci., grad. sch. med., kyoto univ.; 2 nara med. univ.; 3 mitsubishi kagaku inst. life sci. electroconvulsive therapy is useful in treating drug-resistant depressive disorders, though its mechanism remains unclear. there have been a few reports that studied effects of electroconvulsive shock (ecs) on long-term potentiation. however, its effects on long-term depression (ltd) have not been investigated to date. the present experiments examined roles of ecs in inducing ltd at a variety of corticocortical synapses in rat cortex slices by using whole-cell patch clamp. following ecs, ltd magnitude at layer ii/iii-to-vi pyramidal cell synapses was significantly reduced in comparison to no-ecs subjects. as described in recent microarray studies, homer1a/vesl-1s was identified as one of the most up-regulated molecules after ecs. we therefore injected homer 1a protein by diffusion from patch pipettes. homer 1a injection, as well as with ecs treatments, reduced ltd magnitude only at layer ii/iii-to-vi pyramidal cell synapses, implicating that homer 1a may be a biological mediator of ecs effects. masanori kasai 1 , nozomi miyagi 1 , norio kawashiro 2 , daisuke torizuka 2 1 dept. of chem. & biosci., faculty of sci., kagoshima univ., kagoshima, japan; 2 sanko shokuhin co., ltd., tokyo, japan it is well known that zinc is an essential mineral necessary for a multitude of body functions, including acuity of taste. to know a change of serum level in adjuvant-induced inflammation, we measured a zinc level in serum from male lewis rats received a suspension of complete freund's adjuvant (1.0 mg), injected intradermally into the tail. body weight, food intake and water intake were also measured. all rats showed signs of systemic inflammation (weight loss, hind paw swelling, nodules around eyes and penis) after the 11th day. the rats were sacrificed to measure the serum mineral contents (zn, na, cl, p, ca, k, mg) on the 2nd, 7th, 14th, 21st, 28th and 35th days. the serum zinc level was decreased on all of the measurement and the average of serum zinc (75.1 ± 12.1 g/dl, n = 7) on the 35the day was significantly lower than that in intact rats (139.4 ± 11.9 g/dl, n = 7). this decrease of zinc was correlated with weight loss but not hind paw swelling. other minerals did not show any significant changes throughout the measurement period. ps3a-j175 molecular cloning of a novel candidate for ethanol-responsive genes, yy1ap-related protein (yarp), in rat brain in order to elucidate the molecular mechanisms of etoh action on the cns, we investigated changes in gene expression in the adult rat brain after chronic etoh treatment. by means of cdna subtraction, we identified a candidate for etoh-responsive genes in the hippocampus. cdna cloning and sequence analysis revealed that this gene encodes a novel homolog of yy1ap (yy1-associated protein) and is well conserved in rats and humans. homology search for functional domains predicted that the yarp polypeptide contains nlss', a dnabinding motif, and a chromatin decondensation domain, as well as yy1-binding and transactivation domains previously demonstrated in yy1ap. in the brain, neurons such as hippocampal pyramidal cells were stained by in situ hybridization, and co-expression of yarp and yy1 genes was demonstrated in the same neurons. analogous to yy1ap as a co-activator of transcription factor yy1, it is postulated that yarp can regulate cerebral gene expression in response to etoh treatment. ps3a-j176 excitotoxic degeneration of hypothalamic orexin neurons: involvement of nr2b-containing nmda receptors and rescue by gaba a receptor stimulation hiroshi katsuki, shinsuke kurosu, toshiaki kume, akinori akaike department of pharmacology, graduate school of pharmaceutical sciences, kyoto university, kyoto, japan selective degeneration of orexin neurons, a pathological hallmark of narcolepy, is in part reproduced in hypothalamic slice cultures by application of quinolinic acid (qa), an endogenous nmda receptor agonist. we report here that nr2b-selective nmda antagonists ifenprodil (3 and 10 m) and ro25-6981 (0.1 and 1 m) markedly inhibited degeneration of orexin neurons induced by 24 h application of nmda (45 m) or qa (1.5 mm). we also show that stimulation of gaba a receptors by muscimol (10 and 30 m) or isoguvacine (30 and 100 m) potently inhibited qa cytotoxicity. in addition, the protective effect of gaba (100 m) plus a gaba uptake blocker nipecotic acid (1 mm) was abolished by a gaba a antagonist picrotoxin (100 m). norepinephrine and serotonin did not provide a neuroprotective effect. thus, gabaergic inhibition may be decisive on survival of orexin neurons under excitotoxic stimuli mediated by nr2b-containing nmda receptors. yoshika kurokawa, shinji tsukahara, hidekazu fujimaki national institute for environmental studies, tsukuba, japan to evaluate neurotoxicological influence of volatile organic chemicals (vocs), such as toluene, on hippocampal function, we attempted to develop an in vivo optical imaging technique for the hippocampus of mice with or without receiving voc inhalation. we dissected out the cerebral cortex in mice anesthetized with pentobarbital in order to prepare an optical window for monitoring the dorsal surface of the hippocampus, and stained the hippocampus with voltage-sensitive dye (rh795). we then monitored optical signals responding to electrical single-pulse stimulation to the parahippocampal region or hippocampal formation with a time resolution of 1 ms. we also examined optical signals in the hippocampus during toluene inhalation. as a result, neural excitation of the superficial layer was observed in the hippocampal formation after electrical stimulation. on the other hand, acute perinasal exposure of toluene gas did not alter any signal pattern in the hippocampal formation. we will discuss the usefulness of this technique for examination of the neurotoxicological influence of vocs. ps3a-j178 a simple method for fabricating electrodes array for multichannel neural recording -investigation of the alignment of the array and the measurement system-noriyuki taniguchi 1 , osamu fukayama 2 , takashi sato 2 , takafumi suzuki 2 , kunihiko mabuchi 1,2 1 dept. biomed. eng., univ. tokyo, tokyo, japan; 2 dept. info. physi. comp., univ. tokyo, tokyo, japan various types of electrodes have been developed for use as brain-machine interface (bmi) to record signals from neurons. electrode arrays can be purchased from vendors. however, economic considerations and the adjustment of the array alignment for experimental design still make it worthwhile to develop fabrication methods inhouse. thus we developed a low-cost multichannel microwire array electrodes for recording from the cerebral cortex of conscious rats. the electrodes were able to align for the experimental paradigms. the effectiveness of the arrangement of the array as a bmi device was investigated. the electrodes were implanted in the primary motor cortex of wistar rats. we used a wheel-formed rat exercising kit to measure the walking speed of a rat. the neural signal of the rat and the rotating speed of the wheels were simultaneously recorded. and we evaluated the estimation of the walking speed by multiple electrodes with different alignments. ps3a-j179 on-chip electrophysiological measurement of artificially constructed single-cell based neuronal networks ikurou suzuki 1 , yasuhiko jimbo 2 , kenji yasuda 1 1 department of life sciences, graduate school of arts and sciences, university of tokyo, tokyo, japan; 2 department of precision engineering, graduate school of engineering, university of tokyo, tokyo, japan we have developed a single-cell-based on-chip 8 um-diameter multielectrode arrays with an agarose microchambers (amc) for topographical control of the network patterns of living neurons. this system enables flexible and precise control of the cell positions and the pattern of connections through photo-thermal etching. and sampling rates of measurement are 100 khz in 64ch electrodes simultaneously. using this system, we formed a single-cell-based neural network pattern of rat hippocampal cells within the amc array and controlled the growth direction of axon/dendrite selectively using photo-thermal etching methods during cultivation, and recorded the spontaneous firings and evoked responses. moreover, we identified propagation along patterned neural network and found the effects of tetanic stimulation within this neural network. in the meeting we will present the results in detail and will discuss the potential of our method. yuichi yamashita 1 , tetsu okumura 1 , kazuo okanoya 2 , jun tani 1 1 lab. for behavior & dynamic cognition, riken-bsi, japan; 2 lab. for biolinguistics, riken-bsi, japan how the brain generates and learns temporal sequences is a fundamental issue in neuroscience. the production of birdsongs, a process which involves complex learned sequences, provides researchers with a good biological model to study this phenomenon. bengalese finches (bf) learn highly complex songs that have grammatical structure. the underlying neural mechanisms that allow the birds to learn these songs are however not fully understood. to address this issue, we developed a neural network model of bf's songs that might explain how different regions of the brain work together. to test the model, we also conducted empirical experiments on the brains of bf. the model shows that complex grammatical songs can be replicated by simple interactions between deterministic dynamics of a recurrent neural network and random noise. moreover, comparison between the model and the empirical data on real birds shows similar trends. this work is a part of an integrated research project combining model simulations and empirical study. please see also the empirical component of this project as reported by okumura. ps3a-k181 local administrations of muscimol into the nif alter song grammar of the bengalese finches (bf) tetsu okumura 1 , yuichi yamashita 1 , kazuo okanoya 2 , jun tani 1 1 behav & dynamic cognition, riken-bsi, saitama, japan; 2 biolinguistics, riken-bsi, japan songs of passerines are learned behavior which used by males to attract females. their songs consist of several song notes, and these notes are produced in a fixed temporal order. among the passerines, however, bfs sing complex song which follows finite state syntax. the song control system of bf consists of a set of discrete nuclei including the hvc and nif. previous study showed that nif lesioned bfs sung simpler songs, with less phrases to phrases branching. therefore, nif-hvc connection may play important role in generating song grammar. in this study, we perfused nif with muscimol via microdialysis probes as a perturbation on nif-hvc system. following a local perfusion, song grammar was modified. some of chunks in their grammar were disappeared and introductorily notesǐ duration was elongated. nif is also known as one of auditory relay nucleus to hvc. part of the effects is possibly caused by disruption of auditory feedback. we also developed a neural network model of nif-hvc system. please refer yamashitaǐs poster for details of this model. the reason for the emergence of reward expectancy neurons suggested by a model using reinforcement learning and an artificial neural network katsunari shibata 1 , shinya ishii 1 , munetaka shidara 2 1 dept. of e&e engineering, oita univ., oita, japan; 2 grad. sch. of comprehensive human sci., univ. of tsukuba, tsukuba, japan in the experiment of multi-trial schedule task to obtain a reward, reward expectancy neurons, which respond only in the non-reward trials prior to the reward trial, have been observed in the anterior cingulate cortex of monkeys. it is difficult to explain directly by reinforcement learning why they do not respond in the reward trial. here, we interprets that such neurons emerge as an intermediate representation to generate appropriate value and actions in reinforcement learning by simulation analysis using a model that consists of an artificial recurrent neural network trained by reinforcement learning. the simulation result suggests that the reward expectancy neurons emerge to realize smooth temporal increase of the state value by complementing the neurons that respond only in the reward trial. [1] s. ishii, et al., "a model to explain the emergence of reward expectancy neurons using reinforcement learning and neural network", neurocomputing, 2006 behavior is adjusted by outcomes of actions. to examine the neural mechanisms of the behavioral adjustment, we recorded single cell activity of the medial prefrontal cortex (mpfc) of two monkeys performing a behavioral adjustment task. the monkey searched a correct action (left or right lever press) on the basis of the two kinds of visual feedback, one (cs+) paired with a liquid reward and the other (cs−) that did not appear in a preceding pavlovian conditioning. cs+ followed a correct action and cs− followed a wrong action. when the monkey made more than 3 consecutive correct trials, a new block of pavlovian conditioning started. we calculated the prediction errors provided by cs+ and cs− on the basis of a reinforcement learning model of action selection. we found that the neuronal activity corresponds to the prediction error of value of the selected action. this result suggests that mpfc contributes to behavioral adjustment by providing prediction errors of action values. makoto miyazaki 1 , shinya yamamoto 2 , sunao uchida 3 , shigeru kitazawa 4,5 1 faculty of hum sci., waseda univ., tokorozawa, japan; 2 neurosci. res. inst, aist, tsukuba, japan; 3 faculty of sport sci., waseda univ., tokorozawa, japan; 4 dept. of neurophysiol, juntendo univ. grad. sch. med., tokyo, japan; 5 crest, jst, saitama, japan our judgment of temporal order of two sensory signals is not always fixed but subject to changes due to prior experiences, such as repeated exposure to a constant stimulus sequence. to date, such perceptual changes occurred so that signals in the order of the most frequent sequence are judged as simultaneous. in this study, we examined temporal order judgment of two tactile stimuli, delivered one to each hand, using stimulation intervals sampled from biased gaussian distributions (mean = ±80 ms, s.d. = 80 ms). previous studies predict that the point of simultaneity would be shifted toward the peak of the gaussian, i.e. toward the most frequent interval. however, the point of simultaneity was shifted away from the peak by about 50 ms. our results disagree with the previous studies, but conforms to a contrasting prediction from a bayesian integration theory. research funds: kakenhi (15200031) ps3a-k185 single measurement of oxy-and deoxyhemoglobin for a functional near infra-red spectroscopy ichiro shimoyama 1 , fumiko sato 2 , ken nakazawa 3 , kenichi ono 3 1 chiba university, japan; 2 field of home economics, faculty of education, chiba univ., japan; 3 department of integrative neurophysiology, graduate school of med. chiba univ., japan to study single dynamics for oxy-and deoxy-hemoglobin to a single task, we measured near infra-red spectroscopy (omm-3000, shi-madzu) over the frontal area (45 channels) for 7 volunteers (19-23 y). thirty tasks were presented visually every 30 s, the subjects were asked to think about the question immediately following the sentences and asked not to think moreover if the question was difficult (e.g., how to cook curried rice? or how to fold paper into a turtle? etc). a comprehension-test was done just after the record. easy/difficult serial tasks were selected, and the oxy-and deoxy-hemoglobin differences between 2 tasks were calculated to obtain correlation coefficients between the oxy-and deoxy-hemoglobin. grand averaged correlation coefficient was −0.4+/−0.45 between the dynamics of the oxy-and deoxy-hemoglobin. the correlation should be considered in discussing neural activation for nirs. we thank shimadzu corp. for providing the nir station. kazuya ishibashi 1,2 , kosuke hamaguchi 3 , masato okada 1,2,3 1 department of complexity science and engineering, graduate school of frontier sciences, university of tokyo, kashiwa, japan; 2 jst, japan; 3 riken bsi, wako, japan a synfire chain is one of the networks which generate stable synchronous pulse packets. although the networks with a single stable synfire state is intensively analyzed by using several neuron models, the networks with several stable synfire states have not yet been investigated so thoroughly. by using leaky integrate-and-fire neuron model we construct a layered associative feedforward network embedded with several memory patterns. we analyse the network dynamics with the fokker-planck equation. first, we analyze the activity of the network when we activated one memory pattern of the first layer. we show that the layered associative network has stable synfire state. second, we investigate the activity when we activated 2 different memory patterns. then we observe several characteristic phenomena, which are not observed in the conventional homogenous synfire chain. we will report the details of those phenomena. research funds: kakenhi (14084212) and (16500093) ps3a-k187 auditory erps can be identified as corresponding stimuli by classifier with naive bayes method akitoshi ogawa 1,2 , sachiko koyama 1,2 , takashi omori 3 , takashi morotomi 4 1 research institue for electronic science, hokkaido university, sapporo, japan; 2 japan science and technology agency, saitama, japan; 3 graduate school of information science and technology, hokkaido university, sapporo, japan; 4 sakushin gakuin university, utsunomiya, japan in an attempt to reversely estimate the input stimulus from measured erps, we developed computational classifier using naive bayes method. correct classification rates could be index values of the erp characteristic. in this study, we applied the classifier to identify auditory erps (n = 13). the erps were elicited by tones (500 hz) with different durations (8, 16, 32, 64, 128, 256 ms) and gaps (8, 16, 32, 64, 128 , 256 ms) embedded in a continuous pure tone (500 hz). to confirm the generality of the method, we used leave-one-out cross validation. erps of each subject were identified by the classifier which was constructed from the others' erps. as a result, the correct rates for 32 and 64 ms were high both for the tones (32 ms, 38%; 64 ms, 61%) and the gaps (32 ms, 46%; 64 ms, 46%). ps3a-k188 determination of channel parameters for construction of a neural model of caenorhabditis elegans kazumi sakaa, akane andoh, taro ogurusu laboratory of bioscience, faculty of engineering, iwate university, iwate, morioka, japan caenorhabditis elegans (c. elegans) is one of the most suitable model animal for investigation of the relationship between the connection and the function of the neural network because its connection was revealed with the electronmicroscopy. on the other hand, it has been difficult to build a precise model neuron because the neuronal electrophysiological data of c. elegans has not been sufficient. we have been developing a precise neural model by extracting parameters required for model of voltage dependent channels from the electrophysiological data by the genetic algorithm with a neural simulator genesis and parallel genesis. using these simulation softwares, not only the optimum parameter set was determined for each channel but also the ratio of the conductances of several channles were determined. we report validity of obtained parameters and the possibility of the existence of unknown channel. supported by grant from jsps. ps3a-k189 theoretical consideration about nmda current change and its effect on synaptic plasticity shigeru kubota, tatsuo kitajima department of bio-system engineering, yamagata university, yonezawa, japan it is well known that nmdar plays an important role in learning and memory. several experiments have shown that the property of nmdar epsc can change within a few weeks after birth, leading to the shortening of its decay time course. since the calcium current through nmdar is involved in ltp and ltd induction, it is possible that such change can work as the modulation of the plasticity rule or higher-order plasticity. here we show by the biophysical compartmental model that the alteration of nmdar property can modulate the calcium influx into the spine, which finally switches plasticity rule. we also show that this type of plasticity switch can promote synaptic competition and separate postnatal synapses rapidly into two groups of either strong or week ones. our results suggest that changing nmdar time course is very useful for the developing animals in order to promote fast and stable formation of the polysynaptic circuit. manish kumar jain department of psychiatry, r.d. gardi medical college, india introduction: i want to inform you regarding the some of challenges coming across my practice with the person with the psychiatric disorder in social rehabilitation like education and training, work and employment, family, groups, social, sexual, environmental and regional, coordination with the other health group and care giver, insurance problems, medical, physical, occipital vocational, languages problems mostly how to give oppurtinies with in the society and many more to be come in future. method: i keep the records with me since i join the medical college and my during practice but this is really challenging to calm down for question with their relatives and care givers. results: it is always to see the experience of the other people including self help groups in this regards and most challenging with near by perfect action and required more interaction with the rehabilitation groups because some are social problems in psychiatric disorder. conclusions: there is big challenge in the for social rehabilitation for the persons with psychiatric disorder as multifactor involvement s are there in this groups with early intervention and long term rehabilitation so that we can produced many working induals with in the society among the person with psychiatric disorder the more interaction among the society and care giver working in this field as well as neuroseiencents working in this field so that we will able to achieve almost complete social rehabilitation as till today we are not able to achieve social rehabilitation up to 50% till now. hepatic encephalopathy (he) refers to acute neuropsychiatric changes accompanying fulminant hepatic failure (fhf). in the present study we investigated changes in lipid composition of membranes isolated from cerebral cortex of rats treated with thioacetamide (taa), a hepatotoxin which induces fhf and thereon he. estimation of phospholipid fatty acid content in cerebral cortex membranes from taa treated rats revealed a decrease in monounsaturated fatty acid namely oleic acid and the poly unsaturated fatty acids ␥-linolenic acid, decosa hexanoic acid and arachidonic acid compared to controls. assesment of membrane fluidity with pyrene, 1,6-diphenyl-1,3,5-hexatriene, and 1-[4 (trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene revealed a decrease in annular membrane fluidity while the global fluidity was unaffected. the level of thiobarbituric acid reactive species-marker for lipid peroxidation also increased in membranes from taa treated rats indicating prevalence of oxidative stress. results from the present study demonstrate gross alterations in cerebral cortical membrane fatty acid composition and fluidity during taa induced he and their possible implications in the pathogenesis of this condition are also discussed. nagatoki kinoshita, shigenobu yonemura cellular morphogenesis, cdb, riken, kobe, japan rho-gtpases are well known as regulators of cytoskeletal reorganization and many cellular morphogenetic movements. however, little is known about their distributions and their physiological functions in vertebrates. immunohistology of chick embryos revealed apical accumulation of rho, rac and cdc42 in neural plate cells, especially in bending hinge points. after neural tube closure, the apical accumulation decreased. coordinately, activities of rho-gtpases and myosin ii in neural plate cells were higher during neurulation than after neural tube closure. inhibitions of actin filament formation, myosin ii-mediated contraction or rho-associated kinase activity affected neural tube formation. inhibition of rho activity induced the disruption of its apical accumulation and the defects of neural tube formation. these results suggest that rho-gtpases in an active form accumulate in the apical surface of neural plate cells and play important roles in neurulation. furthermore, we are screening regulators and effecters of rho-gtpases transiently expressed in neural plate cells during neurulation. setsuko sahara, dennis dm o'leary mnl-o, the salk institute, usa gradients of morphogens are postulated to establish the initial patterning of the mammalian forebrain, but little is known about their downstream targets and the mechanisms of patterning. here we report mouse buttonhead homogoues, the sp gene family, as candidates of downstream of those morphogens: sp5 expression correlates with wnts/bmps in the cortical hem, sp8 with fgfs in the cop, and sp9 with shh in the ventral midline and mge. by using in utero electroporation, we show that sp8 regulates anterior-posterior patterning of the cortex into areas by controlling distinct fgfs that having opposing effects. sp8 and fgf8 exhibit reciprocal induction, indicating that sp8 is a positive feedback regulator of fgf8. surprisingly, though, ectopic expression of both sp8 and its dominant active form shift cortical areas in the opposite manner to fgf8, suggesting that sp8 activates additional targets that overcome fgf8 function. our results indicate that fgf10 is an additional target of sp8, showing effect on patterning similar to sp8. these findings indicate that sp8 balance the proper cortical arealization through fgf8 and fgf10. research funds: nihr37ns31558 ps3p-c003 fyn-fak signal transduction is involved in the radial migration of late-generated neocortical neurons eiko nakahira 1 , kotaro hattori 1 , takeshi yagi 2 , shigeki yuasa 1 1 dept. ultrastructural res., nat. inst. neurosci., ncnp, tokyo, japan; 2 kokoro biology group, fbs, osaka univ., suita, japan fyn tyrosine kinase posphorylates focal adhesion kinase (fak) that is involved in cell migration. taking into account the defective formation of neocortical layers ii-iii in fyn-deficient mice, fyn-fak signal transduction might be involved in the control of the migration of neocortical neurons. accordingly, we analyzed the neuronal migration in the mutant neocortex and compared the phenotypes to the changes induced by fak gene-knock down by foreign gene transfer by means of in utero electroporation. late-generated neocortical neurons exhibited defective radial migration in the mutant and this defect was rescued by the transfer of fyn-expression vector to the neocortical primordium. fyn and fak were colocalized in the migratory neurons, and fak sirna transfer into neocortical primordium induced migration defect similar to that in fyn deficiency. these findings strongly suggest that the coordination of fyn and fak is essential for the radial migration of late-generated neocortical neurons. noriyo ishibashi 1 , kazuko keino-masu 1 , tatsuyuki ohto 1 , satoshi kunita 2 , satoru takahashi 2 , masayuki masu 1 1 dept. of mol. neurobiol., grad. sch. of comprehensive human sci., univ. of tsukuba, tsukuba, japan; 2 laboratory animal resource center, univ. of tsukuba, tsukuba, japan heparan sulfate (hs) proteoglycans regulate developmental patterning through the interactions with cell surface proteins and extracellular matrix molecules. these interactions are mediated by the specific hs structures generated by sulfation and epimerization. a recently identified extracellular sulfatase, sulffp1, has been implicated in the regulation of growth factor/morphogen signaling through hs remodeling in vitro, but its physiological roles remain unknown. here we generated knockout mice lacking the sulffp1 gene, and examined the brain development. a previous study showed that the brain-specific disruption of the ext1 gene, which encode a hs synthesizing enzyme, led to severe brain defects including hypoplasia of the cerebral cortex and cerebellum. in this study, we thus examined the morphological changes of the cerebellum in the neonatal and adult sulffp1-deficient mice. heparan sulfate (hs) proteoglycans play a crucial role in mediating important signaling by wnt, hedgehog and fgf. recently, novel sulfatases, sulffp1/sulfatase-1 and sulffp2/sulfatase-2, which have hs 6-o-endosulfatase activity have been isolated. since these sulffps are detected in the extracellular space, sulffps are thought to regulate cell surface signaling through hs remodeling. in order to examine the function of sulffp genes in zebrafish, we isolated zebrafish sulffp1 and sulffp2. here we report the isolation and the characterization of the third homologue, sulffp3. sulffp3 has about 56% and 71% overall amino acid homology with sulffp1 and sulffp2, respectively. at 24 h postfertilization, sulffp3 is expressed in the ventral region of spinal cord, whereas sulffp1 is expressed only in the floor plate and sulffp2 is expressed in the lateral floor plate and ventral regions of spinal cord. detailed expression patterns of sulffp3 will be presented. masahiko ajiro, kenichi arai, mika maeda-sato, masuo obinata, wataru shoji dept. of cell biology, idac tohoku univ., japan collapsin response mediator proteins (crmps) are cytosolic proteins involved in neuronal differentiation and axonal guidance. a member of this family, crmp2 was shown to mediate the repulsive effect of sema3a on axons. crmps appear to play more complex roles in axonal differentiation, elongation and branching during development. since less is known about their in vivo function, we studied their roles during development using transparent zebrafish embryos. at early axogenesis stage, zebrafish crmps are expressed in specific patterns. in trigeminal sensory ganglia, crmp2, 3, 4, and 5 are highly expressed. knocking down of these gene results in disorganization of the ganglia, separating into several clusters. however, their axonal patterns including direction, extension, and branching appears normal. same defects were observed in the knockdown of neuropilins, receptor component for class 3 semaphorins. these results suggest that crmps may functionin keeping trigeminal neurons as a ganglia by mediating semaphorin-neuropilin signals. ps3p-c007 developmental origin of diencephalic sensory relay nucley in teleosts y. ishikawa 1 , n. yamamoto 2 , m. yoshimoto 2 , t. yasuda 1 , k. maruyama 1 , t. kage 1 , h. takeda 3 , h. ito 2 1 nat. inst. rad. sci., chiba, japan; 2 nippon med. sch., japan; 3 tokyo univ., japan we propose a novel interpretation of the embryonic origin of cells of diencephalic sensory relay nuclei in teleosts, based on our studies in the medaka embryonic brain. it has been proposed that the relay system in teleosts is unique among vertebrates. teleost relay nuclei, the preglomerular complex (pg), have been assumed to originate from the basal plate (posterior tuberculum, pt) of the diencephalon, whereas relay nuclei in mammals are derived from the alar plate. our results show, however, that many pax6-or dlx2-positive cells migrate laterally and ventrocaudally from the diencephalic alar plate to the basal plate during development. massive clusters of the migrated alar cells become localize in the mantle layer lateral to the pt neuroepithelium, from which the pg appear to differentiate. we therefore consider most neurons in the pg are be of alar, not basal origin. thus, the teleost pg can be regarded as migrated alar nuclei. the organization of the diencephalic sensory relay system may have been conserved across vertebrates. hideyuki dekimoto, yoshihiro oomiya, satoshi kikkawa, toshio terashima, yu katsuyama department anatomy and developental neurobiology, kobe university graduate school of medicine laminaiton is one of features unique to the brain of vertebrates. to understand the evolution of layer formation in the vertebrate brains, we are studying genes which exhibit layer-specific expression. since one of ets family transcription factors, er81 is expressed specifically in the layer v of the mouse neocortex, we selected this gene for the purpose of our study. here we cloned zebrafish er81 homologue (zfer81), and found that the amino acid seuqence of the putative protein is highly conserved throughout the entire length. expression of zfer81 was observed in multiple sites of developing brain. the expression disappears sequentially in some sites, whereas it persisted in other sites until adult stage. er81 expressing sites in the brain was basically conserved between mouse and zebrafish, whereas expression pattern in each site (i.e. telencephalon, tectum) was different. based on these observations, evolution of the gene expression in the brain lamination will be discussed. hiroyuki koizumi, teruyuki tanaka, joseph g. gleeson university of california, san diego, usa doublecortin (dcx), encoding a microtubule-associated protein, is critical for neuronal migration, as mutations result in x-linked lissencephaly in hemizygous males and subcortical band heterotopia in heterozygous females, whereas in mouse, rnai-mediated knockdown but not germline knockout shows abnormal positioning of cortical neurons. dclk (doublecortin-like kinase) is one of the homologous genes of dcx, encodes for protein with an n-terminus that is 70% identical to dcx, but also additional c-terminal protein kinase domain. here, we report that the dclk functions in a partially redundant pathway with dcx in the formation of axonal projections across the midline and migration of cortical neurons in mouse. dosagedependent genetic effects were observed in both interhemispheric connectivity and migration of cortically and subcortically derived neurons. rnai-mediated knockdown of either gene results in similar migration defects. these results indicate the dcx microtubuleassociated protein family is required for proper neuronal migration and axonal wiring. hiraki sakuta 1,2 , hiroo takahashi 1,2 , takafumi shintani 1,2 , kazuma etani 1 , masaharu noda 1,2 1 div. of mol. neurobiol., nibb, okazaki, japan; 2 crest, jst, japan in the developing chick retina, the expression of bmp4 is relieved by that of bmp2 at around e5 with a change from a dorsal high to dorsotemporal high pattern, complementary to that of ventroptin, a bmp antagonist. we previously demonstrated that misexpression of ventroptin altered the retinotectal projection along both the dv and ap axes. here, we show that topographic molecules along the dv axis, together with ephrina2, are expressed in a double-gradient fashion from e6 on like ventroptin and bmp2. when bmp2 expression is manipulated by using the gene-specific knockdown and the reagent-inducible gene expression techniques, the expression patterns of these double-gradient molecules are all changed. moreover, in the bmp2 knockdown and ephrina2-misexpressing embryos, the retinotectal projection is altered along the two axes. the expressional switching from bmp4 to bmp2 thus appears to play a key role in retinal patterning and consequently in topographic retinotectal projection, by changing the direction of the dv axis toward the posterior side during retinal development. noriyuki morita, teiichi furuichi lab. for molecular neurogenesis, riken-bsi, wako, japan the mammalian cerebellum is anteroposteriorly and mediolaterally compartmentalized at the level of neuroanatomy and also at the level of gene expression. to elucidate the molecular mechanisms underlying the establishment and the maintenance of functional cerebellar compartment, genes responsible for mouse cereballar development transcriptome were examined for patterned expression in cerebellum by whole-mount in situ hybridization. not a few known and novel genes were found to be expressed in parasagittal band pattern in the embryonic mouse cerebellum, which could be categorized as "early-onset-genes". parasagittally expressed genes were classified in comparison with the band pattern of en2, wnt7b and pcp2/l7 gene expression in declival vermal lobule, to investigate the correlation between spatial expression profiles and transcriptional regulatory elements. our accumulating data suggest that not only patterning genes like engrailed and wnts, also genes related in later events in neural development such as synaptogenesis are expressed as earlyonset-genes. yasufumi tanaka, tomiyoshi setsu, hideyuki dekimoto, yu katsuyama, toshio terashima kobe university graduate school of medicine, japan the nissl staining of the brains of the adult reeler and normal mice showed that the size of the pontine nuclei (pn) was reduced in the reeler compared with the normal counterpart. the injections of dii and 4di-10asp into the left and right hemicerebellum, respectively, resulted in that only a few pn neurons were doubly labeled in the control, but in the reeler most of pn neurons were doubly labeled. the placements of solutions of dii and 4di-10asp into the left and right cerebellar peduncles of paraformaldehyde-fixed brains resulted in that dii-labeled or 4di-10asp-labeled pontocerebellar fibers made a fascicular formation in the cerebellum of the normal mouse, but such a fascicular formation was not recognized in the reeler and labeled terminals of mossy fibers were randomly arranged along the course of the pontocerebellar projection. reelin mrna and reelin were both expressed in the pn of the normal mice. these data elucidate that the reelin may play a key role in fasciculuation and collateral formation of pontocerebellar projections in addition to cell positioning or migration of pn neurons. kudoh suguru 1,2 , takahisa taguchi 1 1 aist, ikeda, japan; 2 presto, jst the spatiotemporal patterns of spontaneous action potential were analyzed, using the multi-site recording system for extracellular potentials of neurons and the living neuronal network cultured on a 2-dimensional electrode array. the map of functional connections between neurons revealed that each culture contained some hublike neurons and the distribution of the number of functionalconnections approximated a power-law distribution. we confirmed that the spatiotemporal pattern of spontaneous action potentials became more complex pattern along with developmental stage, and the constant pattern of stimulation promote this developmental change. in addition, the spatiotemporal pattern and the functional connections between neurons were drastically re-organized by real-time feedback stimulation. these results strongly suggest that the network structure of the cultured hippocampal neurons is neither stable nor random, but is functionally dynamic and is suitable for certain types of information processing. research funds: presto, jst ps3p-d014 laterality of the human cerebral hemisphere taiko kitamura, jinzo yamada department of anatomy, tokyo medical university, tokyo, japan it has been reported that some functional predominance is located in the right or left hemisphere of the human brain. especially, the speech center and the center related to thought and emotion are located in the left and in the right hemisphere, respectively. in this study, the laterality between the right and the left human hemisphere was investigated macro-anatomically. we measured the weight, the medial-lateral width (m-l), the anterior-posterior lenght (a-p), and the width of the medial surface in the right and the left human hemisphere using in anatomical practice for medical students. the weight of each hemisphere was roughly equal. the m-l was wider in the right side than the left side. the a-p was longer and the width of the medial surface was larger in the left side than in the right side. because of the longer a-p and the larger width of the medial surface in the left hemisphere, it appeared that the left hemisphere overspreads the medial-dorsal marginal surface of the right hemisphere by the naked eye. such overspreading suspects that the left hemisphere develops earlier and faster than the right hemisphere. ps3p-d015 synchrony-induced transition behaviors organized under spike-timing dependent plasticity for retrieving the memorized patterns takaaki aoki 1 , toshio aoyagi 2 1 department of physics, kyoto university, kyoto, japan; 2 graduate school of informatics, kyoto university, kyoto, japan temporally correlated spikes, such as spike synchrony, have been observed in relation to behaviors or cognitions. however, it is unclear how the neurons read out the incoming spike synchronization in the dynamical behavior of network. in this modeling study, considering a network of excitatory and inhibitory neurons organized under spiketiming dependent plasticity, we present a type of network model in which incoming spike synchrony causes a transition between learned activity patterns in the order they were experienced in the learning process. furthermore, using appropriate training patterns, this network exhibits a context-dependent transition, in which the network switches to multiple patterns from a single pattern depending on the temporal structure of neuronal activity at the onset of incoming spike synchrony. this ability of the network may provide one of mechanisms by which a neuronal system can be trained to carry out tasks in a context-dependent manner. shozo kito, maiko kitagawa, akiko shingo lab. of neurosci., hyogo univ., kakogawa japan in our previous studies, we showed that a part of nicotine's beneficial effects on hippocampal and cortical neurons were due to increased igf-1 mrna expressions. nevertheless, the situation may be somewhat different as far as nicotine's effects on the neuronal progenitor cell, which is still on the way of differentiation are concerned. to clarify this problem, nicotine was intraperitoneally injected into 4 weekold wistar strain rats in several doses followed by successive injections of brdu for the next 4 days. then rats were sacrificed and vertical sections of the hippocampus formation were offered for double immunohistochemical staining of brdu/psa-ncam, brdu/neun or brdu/gfap. as the results, numbers of both brdu(+)/psa-ncam(+) cells and brdu(+)/neun(+) cells were much decreased nicotine-dose dependently. on the other hand, as much as 1 mg/kg was needed for nicotine to exert its effect on the number of brdu(+)/gfap(+) cells. these results reveal that nicotine inhibits neurogenesis and plasticity in the hippocampus of adult rats. ps3p-d017 the establishment of the organotypic slice culture of postnatal rat forebrain involving egfp-labeled neural progenitors kaoru sato 1 , james e. goldman 2 1 division of pharmacology, national institute of health sciences, tokyo, japan; 2 department of pathology, columbia university, new york, usa after injecting egfp-encoding retrovirus into p0 rat svz, sagital sections of forebrain were made at p3 and cultured for 6 days. the migration pattern of the egfp-labeled neural progenitors in the cultured slices is almost same as that at the corresponding age. the expression patterns of the glial differentiation-markers were also in accordance with those at the corresponding age. when slices were cultured with anti-␣3 integrin antibody, the migration of the neural progenitors inside svz was significantly enhanced along the rostrocaudal extent. these results suggest that the organotypic slice culture of postnatal rat forebrain is an efficient experimental system for pharmacological studies about migration and differentiation of neural progenitors. radial glia is involved in the contact guidance of neuronal migration and also the neuronal and astroglial precursors. to make clearer the role of radial glia, we developed a method for the selective ablation of a subset of radial glia. it has been reported that tenascin-c (tn-c) is one of the markers for radial glia. accordingly, diphtheria toxin (dt)gene and enhanced green fluorescence protein (egfp)-gene both driven by tn-c gene promoter were co-transferred into the ventricular zone cells of the mouse neocortical primordium by means of in utero electroporation. the numbers of egfp-labeled cells in that tn-c gene promoter and subsequently dt gene are activated selectively decreased by this approach. using this method, the examination of radial glial morphology and neuronal migration following selective ablation is in progress. takayuki manabe, kouko tatsumi, eri makinodan, manabu makinodan, takahira yamauchi department of 2nd anatomy, nara medical university, kashihara, nara, japan it has been well documented that neurogenesis persists at the subventricular zone and the subgranular layer of the dentate gyrus in the adult mammalian brain. in the adult mice, we demonstrated that cells around a cryo-injured cortical lesion had a proliferative activity (labeled with brdu in vivo) and formed neurosphere-like aggregates in the sphere-forming culture condition. significantly lager number of spheres was observed in the culture from the injured hemisphere, which excluded the neurogenic regions (i.e. the svz and hippocampus), than those cultured from the control (contralateral and intact) hemisphere. furthermore, the sphere-forming cells differentiated to neuronal-and glial-marker positive cells in vitro. these results suggest that the cells forming sphere-like aggregates in vitro may function as a kind of progenitor cells in the injured brain. if this is a case, it would be tempting to transplant these sphere-forming cells to cure brain injury or disease. further characterization of the cells is underway. ps3p-d020 localization of neurotrophin receptors trka in pc12 cells: 3d reconstruction analysis of membrane proteins tomoki nishida 1 , hiroshi jinnai 2 , tatuo arii 3 , akio takaoka 4 , ryoichi yoshimura 1 , yasuhisa endo 1 1 department of applied biology, kyoto institute of technology, kyoto, japan; 2 department of polymer science and engineering, kyoto institute of technology, kyoto, japan; 3 national institute for physiological sciences, myodaiji, okazaki, japan; 4 osaka university, mihogaoka, ibaraki, osaka, japan it was previously reported that trka (ngf receptor) was associated with caveolae, small invaginations on the cell membrane, but its subcellular localization is not clarified in detail. we performed immunocytochemistry of trka and caveolin-1 in pc12 cells, analyzed by high-voltage electron microscopy, and reconstructed 3d structure of their subcellular distribution by imod. our results indicated that localization of caveolin-1, known as an integral membrane protein of caveolae, was never found in the invagination structure in pc12 cells, but trka and caveolin-1 immunoreactivities were mainly found as a mesh-like structure in the cytoplasmic matrix. kensuke shiomi, kazuko keino-masu, masayuki masu department of molecular neurobiology, graduate school of comprehensive human sciences, university of tsukuba, tsukuba, japan the wnt signaling plays important roles in cell growth, differentiation, polarity formation, and neural development. previously we identified ccd1, a third-type of the dix domain-possessing protein, as a positive regulator of the wnt/␤-catenin pathway. ccd1 mrna was mainly detected in the neural crest derivatives and differentiated neurons in mouse embryos, suggesting the importance of ccd1 in the wnt-mediated neuronal development. there are three subtypes of mouse ccd1 gene products, ccd1a, ccd1b and ccd1c, which are generated by different promotor usage. mouse ccd1a as well as zebrafish ccd2a has a calponin homology domain which can mediate the interaction with the actin cytoskelton. we found that in the ccdtransfected hela cells, only the type a ccd proteins co-localized with the actin filament. in order to examine the function of the type a ccd proteins, we are now doing overexpression and functional blocking experiments using zebrafish embryos and cell culture. research funds: kakenhi (15770137, 17300098) ps3p-d022 analysis of a role of r-spondin2 on proliferation of the cortical neuroepithelium yumiko hatanaka 1 , masahiro yamaguchi 2 , fujio murakami 3 , masayuki masu 1 1 grad. school of comprehensive human sci., univ. of tsukuba, japan; 2 grad. school of med., univ. of tokyo; 3 grad. school of frontier biosci., osaka univ r-spondin2 (rspo2) is a secreted activator of wnt/␤-catenin signaling (kazanskaya et al. 2004) . rspo2 is expressed in the developing medial cerebral wall and transgenic mice expressing rspo2 in the entire neuroepithelium show enlarged lateral ventricle with a slight increase of brain size (hatanaka et al. 2005) . since wnt3a has a role for expansion of caudomedial cortical progenitor cells (lee et al. 2000) , these findings lead us to the idea that rspo2 may synergistically promote proliferation of cortical neuroepithlial cells together with wnt3a. to clarify their role on proliferation of cortical neuroepithelial cells, we first introduced a ␤-catenin/tcf reporter gene into these cells of embryonic day 11.5 mouse. an application of wnt3a on these cells increased level of the reporter expression, and an addition of rspo2 further increased its level. we are now monitoring incorporation of brdu in neuroepithelial cells to know whether wnt3a and rspo2 directly promote their proliferation. tae sun kim, hideki hida, tomoko narita, sachiyo misumi, hitoo nisino department of neurophysiology & brain science, nagoya city university graduate school medical sciences, nagoya, japan to investigate whether physiological low oxygen during development and cytokines expressed in the dopamine (da)-depleted striatum increase the number of da neurons from es-derived neural progenitor cells (npcs), npcs were treated with cytokine cocktail (il-1␤, il-11, lif, gdnf) or lowered o2 (3.5%), followed by tyrosine hydroxylase (th) immunostaining. low oxygen increased total number of th (+) cells (1.86-fold) as compared to normal o2. cytokine cocktail significantly increased th (+) cells (2.11-fold) compared to nontreated control. treatment of lif and il-1␤ to npcs exhibited major contribution in the effect of cytokine cocktail. data suggest that physiologically relevant low oxygen in development and cytokines and trophic factors that were enhanced in da-depleted striatum cause in the increase of daergic neurons from es-derived npcs. ps3p-d024 structural basis for reelin signaling: determination of receptor-binding site and its three-dimensional structure norihisa yasui 1 , terukazu nogi 1 , mitsuharu hattori 2 , kenji iwasaki 1,3 , junichi takagi 1 1 research center for structural and functional proteomics, inst for protein res., osaka univ., suita, japan; 2 dept. of biomed. sci., grad. sch. of pharm. sci., nagoya city univ., nagoya, japan; 3 core research for evolution and technology (crest) a large secreted glycoprotein reelin acts on target neurons through its receptors (apoer2 and vldlr), resulting in tyrosine phosphorylation of dab1. in the present study, we have carried out structural and functional studies on the reelin signaling. first, we determined the structure of a single reelin repeat by x-ray crystallography. it had a horseshoe-like globular structure with some similarities to carbohydrate binding modules from many enzymes. moreover, electron micrographic 3d reconstruction of four-domain reelin fragment (i.e. r3-6) revealed an elongated rod-like structure. next we determined minimum active unit within reelin. a fragment containing both the fifth and sixth reelin repeats (r5-6) was capable of binding to the receptor (apoer2), and was also able to induce tyrosine phospholylation of dab1 in primary neuronal culture. ps3p-d025 effects of astrocyte-derived factor and cell-cell communication on uni-directional differentiation from mouse embryonic stem cells into neural cells embryonic stem (es) cells uni-directly differentiate into neurons via neuroectoderm and neural stem cells by neural stem sphere (nss) method. cultured with astrocyte-derived factor, colonies of es cells give rise to nsss. we analyzed structure and gene expression of cell spheres formed under various culture conditions, in order to elucidate mechanisms of the uni-directional differentiation into neurons. quantitative real-time rt-pcr analysis demonstrated that the neuronal differentiation did not occur in the cell spheres. these results suggest that astrocyte-derived factor and cell-cell communication are necessary for the differentiation. we have previously established es cell differentiation system, by which we can derive neurospheres containing neural stem/progenitor cells (ns/pcs) with the identity of early caudal neural tube. taking advantage of this culture system, we have recently found conditioned medium of a stromal cell line (cmsc) has the activity to support the formation of neurospheres. this activity was more prominent when cultured at low cell density than when cultured at high cell density, suggesting that it supports the survival of ns/pcs. moreover, rt-pcr analysis of regional identities of the cmsc treated neurospheres revealed elevated expression of pax3 and pax7 compared with those of untreated neurospheres, indicating that cmsc promotes dorsalization of ns/pcs or selective proliferation of dorsal ns/pcs. elucidation of underlying mechanisms may provide important tools to derive early ns/pcs which can generate variety of projection neurons and be applicable to regenerative medicine. research funds: sorst jst ps3p-d027 neudesin, a secreted factor, promotes neural cell proliferation and neuronal differentiation in mouse neural precursor cells neudesin expressed in adult mouse brain encodes a secreted signal with neurotrophic activity in neurons (j neurosci res 79:287, 2005) . most neurotrophic factors are involved in neural cell proliferation and/or differentiation. however, the role of neudesin in neural development remains to be elucidated. neudesin mrna was expressed in the neural precursor cells before the appearance of neurons. therefore, roles of neudesin in neural development were examined using the neural precursor cells. neudesin significantly promoted neuronal differentiation. in addition, neudesin transiently promoted neural cell proliferation early in the developmental process. the differentiation was mediated though activation of the pka and pi-3k pathways. in contrast, the proliferation was mediated through the mapk and pka pathways. the expression profile and activity indicate that neudesin plays unique roles in neural development. ps3p-d028 fabp7 is required for maintenance of neural stem/progenitor cells in the postnatal hippocampus motoko maekawa 1 , miho matsumata 2,3 , yuji owada 2 , shigeki yuasa 1 , noriko osumi 2,3 1 natl. inst. of neurosci., ncnp, tokyo, japan; 2 tohoku univ. sch. of med., sendai, japan; 3 crest, jst pax6 transcription factor is a key player for brain patterning and embryonic neurogenesis, and also expressed in the postnatal brain. we have previously shown that pax6 is necessary for keeping neural stem/progenitor cells in the hippocampus. in this study we have focused on a fatty-acid binding protein fabp7, a downstream of pax6, regulating maintenance of embryonic neural stem/progenitor cells (arai et al., 2005) . fabp7 was expressed in neural stem/progenitor cells in the hippocampal dentate gyrus (dg). 56% of fabp7-expressing cells co-expressed gfap (a marker for early progenitors), and 33% of them co-expressed psa-ncam (a marker for late progenitors). fabp7 expression was also overlapped with pax6, and expression of fabp7 was down-regulated in the dg of pax6 deficient rats and mice. finally, brdu-labeling analysis revealed decreased cell proliferation in the dg of fabp7 knockout mice. taking all together, it is concluded that fabp7 is required for maintenance of neural stem/progenitor cells in dg. ps3p-d029 involvement of the psa-ncam expressing cells in early development of the vascular system of the forebrain momoko miyakawa, tatsunori seki department of anatomy, juntendo university school of medicine, tokyo, japan early development of the vascular system of the forebrain were studied in the chick embryo. staining of vascular endothelial cells by fitctomato lectin and immunohistochemical staining of the surrounding cells were performed on the same cryostat sections of embryos of embryonic day 4-7. sections were examined under a confocal laser scanning microscope. capillaries were found in the lateral pallium and seemed to grow from psa-ncam-positive outer zone to negative inner zone of the pallium. psa-ncam is thought to be expressed in the immature neurons. the rims of capillaries were immunoreactive with psa-ncam in both zones. immunoreaction of doublecortin (neuronal marker) and punctate immunostaining of laminin also were observed on rims of capillaries. by immuno-electron-microscopy it appeared that the endothelium were covered with very thin processes of cells of which outer surface was immunoreactive with psa-ncam. psa-ncam expressing cells may be involved in the development of the vascular system of the forebrain by supporting or guiding the growing capillaries. masaharu kotani 1,6 , shiki okamoto 2 , masato imada 3 , kouichi itoh 4 , atsushi irie 5 , hitoshi sakuraba 6 , hideo kubo 7 1 department of molecular biologu, ohu univ., koriyama, japan; 2 dept. deve. physiol., natl. inst. physiol. sci., okazaki, japan; 3 dept. anatomy, nihon univ. shl. med., tokyo, japan; 4 dept. mol. pharma., univ. tokushima bunri, sanuki, japan; 5 dept. biochem. cell res., tokyo metro. inst. med. sci., tokyo, japan; 6 department of clin. genet, tokyo metro. inst. med. sci., tokyo, japan; 7 dept. med. biol, tokyo metro. inst. med. sci., tokyo, japan as randam-2 shows the highest expression level with the proliferating stage of neural stem cells (nscs), it is thought that the isolation of nscs based on the expression level of randam-2 is possible. in the present, we show that the isolated randam-2 high+ cells enrich nscs. the randam-2 high+ cells had the characteristics as the highly self-renewal capability and potential for multilineage differentiation into neural cells. in contrast, almost all of the randam-2 low+/− cells exhibited not only the extremely low self-renewability but the differentiation capability restricted to neurons. the results demonstrate that randam-2 is a usefule marker for the isolation of nscs by facs. yasuharu takamori 1 , yasuhisa tamura 1,3 , yosky kataoka 1,2 , yilong cui 1,3 , hisao yamada 1 1 department of anatomy and cell science, kansai medical university, osaka, japan; 2 department of physiology, osaka city university graduate school of medicine, osaka, japan; 3 morecular imaging reserch program, riken frs, saitama, japan lamins are major structural proteins of nuclear envelope. three lamin subtypes, a/c, b1 and b2 are mainly present in mammalian somatic cells. to investigate the pattern of lamin expression during neuronal differentiation, we immunohistochemically analyzed the existence of lamins in two neurogenic regions of rat brain; subgranular zone of dentate gyrus and subventricular zone, with confocal microscopy. gfap-positive primary progenitor cells possess lamin a/c (++), b1 (++), b2 (++), psa-ncam-positive subsequent progenitor cells possess lamin a/c (−), b1 (+++), b2 (+), and mature neurons possess lamin a/c (++), b1 (+), b2 (+++), in both neurogenic regions. these observation showed that the composition of lamin subtypes was distinct in particular differentiation stages during adult neurogenesis. yusuke tozuka 1 , yuichi tanaka 1 , tatsuhiro hisatsune 1 1 department of integrated biosciences, university of tokyo, chiba, japan recent work has shown that nestin + neural progenitor cells exist in the adult brain, and suggested that neural activity itself could act directly on these progenitor cells. it has been unclear, however, how do adult progenitor cells sense activity signals from surrounding neural circuit. in the hippocampus where new neurons are continuously produced throughout life, nestin + adult progenitor cells received gabaergic inputs. the gabaergic activity depolarized these progenitor cells, and then promoted their neuronal differentiations. although neuronal production does not readily occur in the adult neocortex, nestin + neural progenitor cells exist in this area too. interestingly, these progenitor cells also received excitatory gabaergic inputs. this gabaergic inputs inhibited their cell proliferations. from these results, we here propose that adult progenitor cells are a direct target of gabaergic neuronal networks, and that this networkto-progenitor cell interaction influences progenitors development by regulating their cell proliferations and/or neuronal differentiations. ps3p-e033 new migration pattern in the postnatal neurogenesis of the dentate gyrus takashi namba 1,2 , hideo namiki 2 , tatsunori seki 1 1 dept. of anat, juntendo univ. sch. of med., tokyo, japan; 2 integrative biosci. and biomed. eng, sch. of sci. and eng, waseda univ., tokyo, japan in the hippocampus, granule cells continue to be generated from embryonic to adult stages. the early postnatal neurogenesis is a transitional state between the embryonic and adult neurogenesis. previously, we have suggested that the postnatal hilus contains astrocytic neural progenitors that divide and differentiate into neuroblasts, and that finally the neuroblasts settle in the granule cell layer (gcl). however, the questions remain how astrocytic progenitors divide and differentiate into neurons, and how the neuroblasts migrate to the gcl. to observe them, we developed a time-lapse imaging system. retrovirus-gfp was injected into the rat hippocampus at p5. three days after the injection, the hippocampal slices were prepared for the time-lapse imaging. the present data show that neuroblasts migrate from the hilus to the gcl, changing the direction of their movement. this is inconsistent with the previous report suggesting simple radial migration (rickmann, et al., 1987) . the dividing pattern is currently under investigation. akiya watakabe 1 , noritaka ichinohe 2 , sonoko ohsawa 1 , tsutomu hashikawa 3 , kathleen s. rockland 2 , tetsuo yamamori 1 1 div. of brain biol, nibb, okazaki, japan; 2 lab. for cortical organization and systematics, bsi, riken, wako, japan, 3 lab. for neural architecture, bsi, riken, wako, japan by using gene expression profiles, we have tried to classify layer 6 neurons in several areas of monkey neocortex. we previously reported that nurr1, ctgf and sema3e mrnas are specifically expressed in subsets of layer 6 neurons. we further show here that cholecystokinin (cck) mrna is expressed in a subset of excitatory neurons in layer 6. by double ish, layer 6 neurons in monkeys are roughly divisible into cck(+) and sema3e(+) subgroups. each subgroup was further subdivided by other markers. tracer experiments showed that cck and sema3e mrna expression correlate well with corticocortical and corticothalamic connectivity, respectively, but the correlation was only partial. from this, we infer that subtypes defined by gene expression may not directly correspond to classical neuronal types. the implication of our findings will be discussed in terms of constancy of laminar structure across areas and species. research funds: kakenhi 17024055 ps3p-e035 rbp-j regulates the cortical laminar formation kenji tanigaki 1 , kazue muraki 1 , norio yamamoto 2 , tasuku honjo 2 1 shiga medical center, research institute, shiga, japan; 2 department of medical chemistry, kyoto university, kyoto, japan precise patterns of cell cycle exit and migration of neural progenitors are crucial for the formation of cortical layer structure. to examine involvement of notch-rbp-j signaling in the cortex laminar formation, we deleted rbp-j from neural progenitors in anatomically restricted areas by in vivo electroporation of cre-expressing plasmids. such studies revealed that rbp-j deficiency caused transformation of glutamatergic pyramidal neurons in layer ii/iii to layer iv neurons with concomitant loss of astrocytes. the loss of rbp-j accelerated neuronal differentiation and changed their laminar fates. in addition, time-lapse studies indicated the migration defect of rbp-j-deficient neurons. the results showed that notch-rbp-j signaling regulates migration of differentiated neurons as well as the timing of the cell cycle exit of neuronal progenitors to determine the laminar and cellular fates of neural progenitors. ps3p-e036 search for the genes that define mammalian cortical progenitor cells using single-cell gene expression profiles ayano kawaguchi 1 , tomoko ikawa 1 , yuya kasukawa 2 , hironori ueda 2,3 , kazuki kurimoto 4 , michinori saitou 4 , fumio matsuzaki 1,5 1 lab. for asymmetric cell division, cdb, riken, kobe, japan; 2 functional genomics subunit, cdb, riken, kobe, japan; 3 lab. for systems biology, cdb, riken, kobe, japan; 4 lab. for mammalian germ cell biology, cdb, riken, kobe, japan; 5 crest, jst, japan in the mammalian brain, cellular heterogeneity of the progenitor cells has largely hindered the molecular analysis of neuronal diversity. to overcome this problem, we randomly picked individual vz/svz cells of mouse embryos, and constructed cdnas from each of them by global pcr amplification method. we could classify these "single cell derived cdnas" into several groups retrospectively based on the expression of marker genes, including cell cycle related genes, transcription factors, and regional marker genes. 30 samples that showed typical marker gene expression pattern of the groups were applied for genechip analysis. the obtained data were confirmed by quantitative pcr and in situ hybridization. by this strategy, we identified nine genes that were specifically expressed in the svz progenitor cells. research funds: kakenhi (15700264) ryosuke tatsuno 1 , tomoaki sai 2,3 , masahiro otsu 2 , kuniko akama 1 , takashi nakayama 4 , tosifusa toda 5 1 grad. sch. of sci. and tech., chiba univ., chiba, japan; 2 lab. regener neurosci., tokyo metropol. univ. fac. health sci., tokyo, japan; 3 dept. orthop. surg., jikei univ. sch. med., tokyo. japan; 4 dept. biochem., yokohama city univ. sch. med., yokohama, japan; 5 proteomics collab. res., tokyo metropol. inst. of gerontol., tokyo, japan embryonic stem (es) cells possess pluripotency and self-renewal. however, the proteomic analysis of neural stem cells and neurons differentiated in vitro from es cells has not so proceeded yet. we investigated the expression levels of proteins during in vitro differentiation of mouse es cells into neurons via neural stem cells by neural stem sphere (nss) method, using 2-d gel electrophoresis and maldi-tof ms. we identified vimentin, creatine kinase, atp synthase beta subunit, and some proteins with no annotation in murine brain the database, which were up-regulated in neural stem cells, and down-regulated in es cells and neurons. these results suggest that the neural stem cells have characteristic protein expression profile. ps3p-e038 identification of se90, a novel gene expressed in the nural progenitor cells shin-ichi sakakibara, kazuhiko nakadate, shiichi ueda department of histology and neurobiology, dokkyo university school of medicine, tochigi, japan identification of the genes regulating neural progenitor or neural stem cell functions is critical to understand the mechanisms of the adult neurogenesis and neurodegenerative disease. we compared the gene expression profile of proliferating neural stem cell cultures with those of differentiated cells. a subtractive library was constructed by using the suppression subtractive hybridization and the differential screening was performed. among two thousand of the differentially expressed subtracted clones, we identified 150 genes that significantly upregulated in neural stem cell culture. these included several novel genes, in addition to the known genes involving in the cell cycle and signal transduction. in situ hybridization and the developmental northern analysis demonstrated that these mrnas were enriched in the germinal neuroepithelium, embryonic ventricular zone and the postnatal subventricular zone surrounding the lateral ventricles. we further analyzed the expression pattern of the novel gene se90 in developing and matured cns. teiichi furuichi 1 , akira sto 1,2 , yukiko sekine 1 , noriyuki morita 1 , tetsushi sadakata 1 , satoshi shoji 1 , jin-hong huang 1 , toshio kojima 2 1 laboratory for molecular neurogenesis, riken brain science institute, japan; 2 comparative systems biology team, riken genome sciences center, yokohama 230-0045, japan mouse cerebellum develops through a series of cytogenetic and morphogenetic events that are genetically coded within the first three weeks of life. we have extensively investigated the spatio-temporal gene expression profiles during the postnatal development of mouse cerebellum by differential display, rt-pcr, genechip, cdna microarray, and in situ hybridization. we have informatively systematized all the profiles in an online neuroinformatics database cdt-db (http://www.cdtdb.brain.riken.jp) with various search functions. we have demonstrated that the postnatal development of mouse cerebellum is genetically programmed by thousands of genes that exhibit differential expression patterns in time and space. further studies on a scale that includes the underlying expression of all genes and more detailed studies on their transcriptional regulation will shed light on the genetic basis for cerebellar development. miwako ozaki 1 , makoto mizuno 2 , kazuhisa sakai 4 , yoshimoto kiyohara 4 , kazuhiko yamaguchi 3 , tsutomu hashikawa 4 , hiroyuki nawa 2 1 institute of biomedical engineering, waseda university, tokyo, japan; 2 department of molecular neurobiology, brain research institute, niigata university, niigata, japan; 3 laboratory for memory and learning, bsi, riken, saitama, japan; 4 laboratory for neural architecture, bsi, riken, saitama, japan neuregulin (nrg), a neurotrophic factor, involved in the development, differentiation and repair of the nervous system, regulates the activation of ion channels and neurotransmitter receptors. in order to examine the molecular mechanism on the relationships between network, synapse formations and higher orders functions, we prepared ig-nrg1 knock out mice (nrg1 type i and iv were disrupted). the mutant mice showed motor disco-ordination and abnormality of synaptic structure in related areas in cerebellar nuclei and cortex. in addition, the number of vesicles in presynaptic neurons decreased in their synapses. the study on cerebellum that is very clear in the network input information would give some suggestions to the relationship between synaptic functions and behaviors. ps3p-e041 psd-95 protein expression in rat oromaxillofacial motoneurons during postnatal development kohji ishihama 1,2 , satoshi wakisaka 1 , shiho honma 1 , akira ito 1,2 , kei azuma 1,2 , mikihiko kogo 1 1 department of oral anatomy and developmental biology, osaka university graduate school of dentistry, osaka, japan; 2 first department of oral and maxillofacial surgery, osaka university graduate school of dentistry, osaka, japan postsynaptic density (psd), which is composed of diverse proteins, involved in synaptic structure, neurotransmission and signal transduction. psd-95 implicates in formation and maturation of excitatory synapses. psd-95 regulates the localization of the nmda receptor by means of binding with nr2. rhythmical oro-maxillofacial activities, such as suckling and chewing, are generated in the brainstem, and we showed that nmda receptors played critical role for the rhythm and pattern generation and signal transmission around the trigeminal motor nucleus during prenatal and early postnatal development. here we examined the temporal distributions of psd-95 protein using with immunohistochemical study, in developing rat brainstem from suckling to mature chewing stage. there was early emergence of psd-95 expression in the interneurons located at medial of the trigeminal motor nucleus. masami miura, masao masuda, toshihiko aosaki neural circuits dynamics research group, tokyo metropolitan institute of gerontology, japan the striatum, an input stage of the basal ganglia, contributes to habit formation as well as motor functions. recent studies suggest that striatal interneurons play an important role in processing of cortical input. we investigated the synaptic connections between interneurons using paired whole-cell recordings and immunohistochemical techniques. we found that fast-spiking (fs) interneurons sent gabaergic inhibitory input to cholinergic interneurons, which were gaba a receptor-mediated and suppressed by gaba b receptor agonist skf97541. in turn, cholinergic interneurons sent cholinergic excitatory input to fs interneurons. because the excitatory postsypnatic potentials (psps) were blocked by hexamethonium and dihydro-␤-erythroidine, the psps were nicotinic acetylcholine receptor-mediated. these results suggest that gabaergic interneurons and cholinergic interneurons mutually influence their excitability and might modulate the activity of striatal local circuits. ps3p-e043 ocular following responses (ofrs) to a brief background motin are modulated in relation to preparation for upcoming pursuit hiromitsu tabata, kenichiro miura, kenji kawano dept. integ brain sci., grad. schl of med., kyoto univ., kyoto, japan recently, our group reported that the ocular responses to a brief perturbation of a small target during fixation increased when subjects (humans, monkeys) were preparing for upcoming smooth pursuit eye movements (spems) rather than preparing for saccades or stationary fixation. here, we report that the increase in ocular responses based on the anticipation of spems was also observed in monkeys when a large-field visual stimulus (background) was moved briefly prior to pursuit. the result indicates that the visual region where the gain of the visuomotor transmission increased is not limited to a small region near the target but spreads to a larger field. in other words, the anticipation of upcoming spems could affect the generation of ofrs. furthermore, directionally biased ocular responses to the brief background motion were observed when the animals repeatedly performed spems toward one direction, implying that the prediction of the upcoming spem direction might cause the directional asymmetry of the visuomotor transmission gain. ps3p-e044 comprehensive characterization of motor neurons related with locomotory central pattern generator in the earthworm by imaging toshinobu shimoi 1 , kenji mizutani 2 , hiroto ogawa 3 , kohji hotta 1 , kotaro oka 1 1 ctr. for biosci. and info, keio univ., yokohama, japan; 2 neuro, karolinska inst, stockholm, sweden; 3 bio, saitama med. sch., saitama, japan in this study, we comprehensively identified and characterized motor neurons concerning with locomotory central pattern generator (cpg) in the earthworm by calcium imaging as multiple recording. the candidates of motor neurons were stained with dextran conjugated calcium indicators using retrograde labeling from projection nerves. we obtained the responses of up to 50 cell bodies of motor neurons and sensory neurons on the ventral surface of the segmental ganglion (25% or less for all neurons on the ventral surface). we analyzed the activity patterns of the candidates of motor neurons using pattern matching method comparing between calcium responses or between calcium responses and locomotory motor pattern. as a result, we detected motor neurons as pairs of neurons having strong synchrony to each other neuron or to motor pattern. these results were great progress to identify motor neurons related with locomotory cpg in the earthworm. ps3p-e045 three dimensional (3d) pursuit eye movement signals in cerebellar dorsal vermis takuya nitta, teppei akao, sergei kurkin, kikuro fukushima department of physiology, hokkaido university school of medicine, sapporo, japan for pursuit of a target moving in 3d space, signals for frontal and vergence-pursuit must be synthesized. studies in our laboratory have demonstrated that 3d pursuit signals are generated in the frontal eye fields, and also present in cerebellar floccular region. however, the majority of floccular purkinje (p-) cells discharged after onset of vergence-pursuit. cerebellar dorsal vermis is another cerebellar area for frontal pursuit. to examine whether 3d pursuit signals are present in this area, we examined simple-spike discharge of vermal pursuit p-cells in 3 monkeys. of a total of 77 p-cells that were examined during both frontal and vergence-pursuit, 46% discharged for both, 40% only for vergence, and 14% only for frontal pursuit. these results indicate that most of vermal pursuit p-cells discharged for vergence and that about half of them had 3d pursuit signals. majority (70%) of these p-cells discharged before onset of vergence eye movements with the typical lead time of 50 ms, suggesting their involvement in the initiation of vergence-pursuit. research funds: kakenhi (17022001) ps3p-e046 information processing in fef-rnrtp pathway for smooth pursuit seiji ono, michael j. mustari division of sensory-motor systems, yerkes national primate research center, emory university, atlanta ga, usa the frontal eye field (fef) cortex is known to play a role in smooth pursuit (sp). this role is supported by fef projections to the rostral nucleus reticularis tegmenti pontis (rnrtp) which projects heavily to the vermis. using multiple linear-regression modeling, we have shown that sp neurons in rnrtp were biased towards eye acceleration. however, the functional characteristics of sp related fef neurons that project to rnrtp have never been described. therefore, we used micro-electrical stimulation to deliver single pulses in rnrtp to antidromically activate fef neurons. the majority of sp related fef neurons that we identified as projecting to rnrtp were most sensitive to eye acceleration and much less sensitive to eye velocity. the neurons in fef-rnrtp pathway carry signals that could play a primary role in sp initiation. our antidromic studies may help address a fundamental question regarding whether basilar pontine nuclei integrate signals from multiple cortical areas or mostly relay signals with little transformation to cerebellum. research funds: nih grants ey13308, rr00165 aya takemura 1 , yumi murata 1,3 , kenji kawano 1,2 1 neurosci. res. insti, aist, tsukuba, japan; 2 dept. integ brain sci., grad. sch. med., kyoto univ., japan; 3 grad. sch. compreh hum sci., univ. tsukuba, japan previous studies in monkeys suggest that the medial superior temporal (mst) area is involved in visual motion processing. to understand the role of the mst in optokinetic nystagmus (okn) and afternystagmus (okan), we examined the effects of bilateral chemical lesions in the mst in two monkeys. when each monkey was injected with ibotenic acid (15 mg/ml, 36-37 l total), the initial rapid rise in okn was reduced. consequently, it took longer for the eye velocity to reach a steady state (i.e., an eye velocity close to the stimulus velocity). by contrast, the steady state okn was not affected and the okan persisted. the initial amplitude and falling time constant of the okan increased. the results suggest that the mst is part of the direct pathway for the initial rapid rise in the okn, but is not involved in the velocity storage mechanism for the steady state okn and okan. smooth pursuit is performed by coordination of eye and head movements. we have reported that the majority of fef pursuit neurons in monkeys with their head free to rotate about a vertical axis were modulated not only during eye-and gaze-pursuit but also head-pursuit to a moving reward feeder while the monkeys fixated an earth-stationary spot without gaze movement. to examine the origin of head-pursuit modulation, we moved the reward feeder in a ramp trajectory at 20 • /s with random intervals. the majority of pursuit neurons discharged before the onset of head movements with the mean lead time of 58 ms. discharge modulation during head-pursuit and passive whole body rotation was not correlated in most neurons. these results suggest that proprioceptive neck inputs or vestibular inputs are not the main origin of head-pursuit modulation. rather, our results suggest that the main origin reflects pursuit commands. ps3p-e049 the local feedback loop of the saccadic system: an analysis of the eye movements induced by pdb stimulation rikako kato department of developmental physiology, national institute for physiological sciences, okazaki, japan saccadic amplitude are controlled by a comparator that calculates dynamic motor error. some models place the comparator in the superior colliculus while others assign this role to the reticular formation. to decide between the two hypotheses one would need to stimulate pathways in between their putative comparators. we stimulated collicular axons descending in the pdb. our data demonstrate that electrical stimulation of the pdb evokes saccades and they always terminate before the end of the stimulus train. the characteristics of evoked saccades are comparable to those spontaneously generated by the cat. our data clearly demonstrate that the feedback path of the local loop of the saccadic system closes downstream of the superior colliculus. katsuo fujiwara 1 , kenji kunita 2 , kaoru maeda 1 , takeo kiyota 1 1 department of human movement and health, graduate school of medical science, kanazawa university, kanazawa, japan; 2 institute for health and sport sciences, osaka city university, osaka, japan we investigated changes in visual evoked potential (vep) during postural adaptation process while subjects maintaining standing posture on an oscillation floor with periodic vision shut. the subjects were 17 undergraduate students. a shutter goggle was used as a vep stimulator which was opened periodically for 30 ms with 800-ms intervals. the oscillation trial (0.5-hz frequency and 2.5-cm amplitude) (65-75 s) was repeated 5 times. postural steadiness was evaluated by mean fluctuation speed of the center of foot pressure. the mean speed decreased as trial was repeated, and reached a plateau before the 5th trial. a significant correlation was shown between 5th-1st trial differences in mean speed and vep amplitude (r = 0.79). this indicates that the role of visual information is different among subjects with various adaptation processes of postural control. ps3p-e051 primary motor cortex contributes to generating manual following response toshitaka kimura 1 , naoki saijo 1 , hiroaki gomi 1,2 1 ntt cs labs, kanagawa, japan; 2 erato shimojo implicit brain function proj, jst, saitama, japan a large-field visual motion during arm movements induces a shortlatency, involuntary arm response called as manual following response (mfr). the mfr exhibits similar features to the ocular following response (ofr) elicited by the similar visual stimulus, with respect to the stimulus-response directional characteristics and the spatiotemporal frequency tuning property. this suggests that computational mechanism is shared for both responses. however, the neural basis of the mfr motor command generation remains unclear, while ofr is known to be generated subcortically. here we show, by using transcranial magnetic (tms) and electrical (tes) stimulation over the primary motor cortex (m1), that (1) an emg response evoked by tms was facilitated during mfr, while that by tes was not, and (2) intracortical inhibition within m1 assessed by paired-pulses tms was reduced during mfr. these results suggest that mfr is generated through activity of interneuronal networks within m1. such cortical mechanisms for mfr generation are distinct from the subcortical processes for ofr generation. naoki saijo 1 , hiroaki gomi 1,2 1 ntt cs labs., kanagawa, japan; 2 erato shimojo implicit brain function proj, jst, saitama, japan when a visual target is suddenly shifted during a reaching movement, we can quickly adjust the arm movement. however, the computational mechanism to generate quick adjustment is still unclear. here we investigated this mechanism from the viewpoint of visuomotor coordinate transformation. we observed the hand responses to the target shifts in 8 radial directions applied during reaching. the data show that the direction of the initial phase (150-200 ms) of hand response acceleration was slightly biased from the corresponding target shift direction, whereas the direction of the late phase (250-300 ms) was little biased. additionally, when we use a target shift having less-motion energy, the response latency greatly increased and the directional bias significantly decreased. these results suggest that the on-line reaching adjustment would be generated by two different mechanisms: a reflexive controller which is induced by visual motion with short latency and generates spatially inaccurate response, and voluntary controller which generates spatially accurate response with long latency. ps3p-e053 spatial relationship between gaze and reaching-target modulates manual following response naotoshi abekawa 1 , hiroaki gomi 1,2 1 ntt cs labs., kanagawa, japan; 2 erato shimojo implicit brain function proj, jst, saitama, japan to explore the functional mechanism of the manual following response (mfr) induced by a large-field visual motion during arm movement, we examine its modulation caused by the spatial relationships between gaze, target, and background. on a large vertical screen placed in front of the subject, full field checker pattern, two markers (upper and lower), and a gray mask around one of the markers, were displayed. in the first condition, subjects kept watching the upper marker, and pointed the upper (congruent) or lower (incongruent) marker instructed before every reaching. the checker pattern suddenly moved either rightward or leftward brief after reaching start. in the second condition, subjects did the same task with watching the lower marker. in both conditions, the mfr amplitude was significantly grater in the congruent condition than in the incongruent condition, whereas the mask location did not significantly affect the mfr amplitude. this suggests that the spatial relationship between gaze and target is important in modulating mfr. misako komatsu, eizo miyashita dept. compu. intelligence & systems sci., tokyo tech., yokohama, japan when a subject performed pointing to a remembered target under eyes fixated, we have reported that endpoints tended to sift closer to the fixation point. moreover, we have noted that the greater the distance between a target and the fixation point, the larger the errors. the result was consistent even when the position of the fixation point was changed. the above tendency was considered to occur in eye-or gaze-centered coordinates. it is open question, however, if the brain correctly compensates the difference of the relative position of eyes to the head? to answer this question, we investigated the dependency of the endpoint errors on the positions of a monitor and the fixation point. the subjects, sitting in front of the monitor, were asked to point a remembered target as accurately as possible using a computer mouse. all the results were consistent with the previous ones regardless of the position of monitor or the fixation point. these results suggest either the eye-position doesn't affect how we recognize the target position, or the brain correctly compensates the eye-position with a fixed head position. ps3p-f055 influence of the coupling of muscle activity on rhythmic movements of ipsilateral hand and foot tetsuro muraoka 1 , takashi obu 2 , kazuyuki kanosue 3 1 asmew, waseda university, saitama, japan; 2 graduate school of human sciences, waseda university, saitama, japan; 3 faculty of sports sciences, waseda university, saitama, japan the aim of this study was to investigate the influence of the coupling of muscle activity on rhythmic movements of ipsilateral hand and foot. the subjects (n = 6) were supine, and their hand was prone. they performed cyclical flexion-extension coordinations of the hand and foot in the iso-(iso) or opposite-(oppo) directions, and those with an elastic load against wrist flexion (el-iso and el-oppo) at 1.5, 1.75, and 2.0 hz. over 99% success rate was observed in all tasks except oppo (78-97%). the in-phase muscle activity of wrist and foot muscles was obserbed in all tasks except oppo. it was suggested that the in-phase muscle activity might be an important factor in a coordinated movement of ipsilateral hand and foot. research funds: the special coordination funds for promoting science and technology, mext, japan ps3p-f056 simultaneous muscle activity stabilizes the coordinated movement of ipsilateral hand and foot takashi obu 1 , tetsuro muraoka 3 , kazuyuki kanosue 1,2,3 1 faculty of human sciences, waseda university, saitama, japan; 2 faculty of sport sciences, waseda university, saitama, japan; 3 asmew, waseda university, saitama, japan in human, voluntary opposite-directional movement (antiphase) of ipsilateral hand and foot is more difficult than iso-directional movement (inphase). the purpose of the present study was to investigate the influence of the coupling of muscle activity on these movements. eight normal subjects lay in supine position with hand prone and their foot was forcedly moved by a dynamometer cyclically at 1, 1.33, and 1.66 hz. they were asked to perform 4 tasks, concentric/eccentric contraction of ankle dorsiflexors with in-phase/antiphase wrist extension/flexion. all tasks were performed successfully. muscle activity of hand flexors was observed in concentric-antiphase and eccentric-inphase tasks, indicating simultaneous muscle activity of hand and foot. it may be suggested that simultaneous muscle activity would make the movement easier regardless of the direction of movement. ps3p-f057 activities of erector spinae muscles during jaw clenching in man kayoko yasunaga 1,2 , tadachika yabushita 1 , kazuo toda 2 , kunimichi soma 1 1 orthodontic science, tokyo med. & dent. univ., tokyo, japan; 2 div. integrative sensory physiology, nagasaki univ., nagasaki, japan recent studies focused the functional relationships between the masticatory and the posture system. the hypothesis of our present study is an existence of functional connections between the masticatory system and the spinal muscles which maintain the posture. therefore, we investigated the effect of the maximum jaw clenching on the spinal muscle activities. bipolar needle electrodes were inserted into erector spinae muscles to record the motor unit activities when the sitting subjects relaxed and performed maximal jaw clenching. as a result, the instantaneous frequencies of the spinal muscles decreased with clenching, compared with relaxed jaw position. our results suggested that there were some relationship between spinal muscle activities and jaw clenching. the effects of bipedal walking on the central nervous systems-influence of bipedal walking on the spinal reflex-naomi wada 1 , sachiko motoyama 1 , futoshi mori 1 , shigemi mori 2 1 department of veterinary physiology, yamaguchi university, yamaguchi, japan; 2 national institute for physiological science, okazaki, japan the one of the biggest questions in the vertebrate evolution is how human got the highly developed brain. many investigators suggest that upright posture and bipedal walking caused remarkable development of brain and produced the human being. the purpose of our experiments is to show the influences of bipedal habits on central nervous systems. we have established the bipedal walking model using rats (rbm) by amputation of forelimbs and training of upright posture and bipedal walking. after training of upright posture and bipedal walking for 12-20 weeks, rats got abilities of the stable upright posture and bipedal walking with symmetrical hindlimb movements between left and right side. in the present experiments, we studied about the effects of bipedal habits on the lumbar spinal reflex. the results of out experiments showed that bipedal habits inhibit the spinal reflex pathways. ps3p-f059 neuronal activity in primary motor cortex during quadrupedal locomotion of the japanese monkey katsumi nakajima 1 , futoshi mori 2 , akira murata 1 , masahiko inase 1 1 dept. of physiol., kinki univ. schl. of med., osakasayama, japan; 2 dept. of vet. physiol., facult. of agr., yamaguchi univ., yamaguchi, japan to elucidate cortical mechanisms related to the control of primate locomotion, we recorded neuronal activity in m1 of the monkey walking quadrupedally on the treadmill. tungsten microelectrodes were inserted into m1 hindlimb region using a custom-made micromanipulator. we found that all neurons recorded in m1 modulated their discharge phasically time-locked to the step cycle or increased their discharge frequency tonically during simple locomotion. the neuron exhibiting phasic modulation peaked once or twice per step. the peak activity occurred at widely different times during the step cycle in different recorded neurons. as the treadmill speed increased, most of recorded neurons increased their discharge frequency. all these results suggest that m1 output in monkeys directly and/or indirectly acts on spinal circuitries generating a basic pattern of rhythmic activity during simple locomotion in a manner different from that in subprimates. research funds: kakenhi (16500271) ps3p-f060 activity of putaminal neurons receiving inputs from motor cortical areas in behaving monkeys sayuki takara 1,2 , nobuhiko hatanaka 1,2 , masahiko takada 3 , atsushi nambu 1,2 1 school of life science, the graduate university for advanced studies, japan; 2 division of system neurophysiology, national institute for physiological sciences, japan; 3 tokyo metropolitan institute for neuroscience, japan the putaminal (put) neurons receive motor cortical inputs and change their activity in relation to movements. to investigate how these inputs contribute to put neuron activity in behaving monkeys, extracellular unit activity was recorded from identified put neurons during the performance of a memory-guided reaching task. based on orthodromic spikes evoked by cortical stimulation, individual put neurons were defined in terms of whether they receive input from the primary motor cortex (mi), the supplementary motor area (sma), or both. the results showed that mi-recipient neuron activity was responsive to the movement, while sma-recipient neuron activity was responsive to the cue stimuli and/or the delay period. the activity of neurons receiving convergent inputs was related to both the movement and the delay period. we previously reported that electrical stimulation of cerebrofugal fibers induced short latency facilitation and succeeding suppression on phrenic activities, while train pulse stimulation of caudal raphe nuclei (raphe magnus, rm, and raphe pallidus, rp) induced suppression or facilitation on respiratory neural activities in cats and rats. in this study, in order to analyze the cerebral and raphe projections to the respiratory neuron network, we examined the effects of stimulation of cerebrofugal fibers and caudal raphe nuclei on activities of ventral respiratory group neurons (vrgs) in the medulla and upper cervical inspiratory neurons (ucins). animals were anesthetized, immobilized and artificially ventilated. stimulation of cerebral peduncle (cp) induced short latency facilitation and succeeding suppression on activities of ucins. stimulation of rm or cp evoked inhibitory postsynaptic potentials in the caudal vrgs. these results suggest that rm and cerebral cortex directly inhibit main respiratory output neurons in vrg. ken muramatsu 1 , sei-ichi sasaki 2 , yuichiro cho 1 , kenji sato 1 1 anatomy and physiological science, tokyo medical and dental university, tokyo, japan; 2 department of physiology, ibaraki prefectural university of health sciences, ibaraki, japan distribution of average diameters of external anal sphincter (eas) motoneurons and peripheral motor fibers were examined in cats. to identify eas motoneurons, horseradish peroxidase was applied to the central cut end of the anal branches of the pudendal nerve. eas motoneurons were found in the onuf's nucleus of s1 and s2 spinal levels. to examine size of peripheral motor fibers, ganglionectomy was performed onl7 -s3 spinal segments which contain afferent fibers of eas muscles. after 3 weeks survival period, anal branches of the pudendal nerve was examined. histograms of the distribution of average diameters of cell body and motor fiber shows unimodal distri bution. also, distribution of muscle spindles of eas muscle were examined by serially sectioning the distal colon and staining with mayer's haemotoxylin and eosin. no muscle spindles were found. these results suggest that eas muscle is controlled without gamma loop. mariko miura, yoshiki iwamoto, kaoru yoshida neurophysiol., univ. tsukuba, tsukuba, japan saccade accuracy is ensured by an adaptation mechanism. the speed and magnitude of adaptation vary greatly across experiments even for the same subject. one factor that might cause this variability is adaptation history. the present study aims to clarify whether preceding adaptation influences subsequent adaptation over several days. gain decrease adaptation was induced in a monkey by stepping the target backward during saccades. adaptation experiments were repeated for 4 consecutive days. we compared adaptation in day1 and that in day4. the gain decrease for the first 700 saccades in day 4 (0.140 ± 0.036) was larger than that in day 1 (0.080 ± 0.021) (p = 0.026, n = 5, paired-t test). the rate of adaptation in day 4 (1.739 ± 0.393 × 10 −4 /sac) was higher than that in day 1 (1.120 ± 0.240 × 10 −4 /sac) (p = 0.011). the overall gain change (1800 saccades) in day 4 (0.219 ± 0.030) was larger than that in day 1 (0.112 ± 0.022) (p = 0.002). thus, both the speed and magnitude of adaptation were increased by preceding adaptation. the present study suggests that the memory of saccadic adaptation is retained for days and facilitates following adaptation. research funds: kakenhi (16300129) ps3p-f064 asymmetry of the anticipatory convergence eye movement haruo toda, takehiko bando div. integr. physiol., grad. sch. med. sci., niigata univ., niigata, japan typically, convergence eye movement is known as symmetric adduction of the both eyes. but asymmetrical convergence also found in the natural condition. these asymmetrical convergence may reflect asymmetries of central control of convergence eye movement. the lateral suprasylvian (ls) areas are extrastriate cortices which receive visual information from v1. the ls has contralateral dominant receptive fields and convergence eye movements evoked from the long latency regions were asymmetrical. cats (n = 7) were trained to start convergence by an alarm signal (buzzer sound or combination of buzz and blinking of led), preceding target movement by 4 s. after training, ocular convergence was elicited by the alarm signal before target movement (predictive open-loop convergence) in 60% of trials. in three cats, we used training with obliquely approaching target. after training, asymmetrical anticipatory eye movements were observed. based on these findings, related ls neuronal activities and results from lesion study, we will discuss the role of ls in asymmetry of anticipatory and visually-evoked convergence eye movement. yusuke uchida 1 , xiaofeng lu 1,2 , shogo ohmae 1 , toshimitsu takahashi 1,2 , shigeru kitazawa 1,2 1 dept. of neurophysiol., juntendo univ. grad. sch. of med., tokyo, japan; 2 crest, jst, tokyo, japan we examined reward related neural activity in the supplementary eye field (sef). for this purpose, two monkeys were rewarded after each visually guided saccade from a central fixation point to one of 16 targets that were arranged in a radial pattern. a target appeared while the monkeys were fixating on the central point, and the monkeys made a saccade to the target when the fixation point disappeared and held on the target until the target turned off. reward was delivered during or after target-hold period. we found that many sef cells became active during the period of reward delivery (r-cell). more than half of r-cells showed enhancement of the neural discharge in the specific target directions but not other directions in which the same amount of reward was given (rd-cell). interestingly, most of rd-cells displayed activity with the clear directional tuning. these results demonstrate reward dependent activity specific to spatial direction in the sef, and further suggest that sef cells provide reinforcement mechanism. research funds: kakenhi 17022033 ps3p-f066 frontal pursuit area is involved in the retinalslip dependent adaptation of monkey post-saccadic pursuit eye velocity hiromasa kitazawa 1 , soichi nagao 1,2 1 lab. for motor learning control, riken bsi, saitama, japan; 2 sorst, jst, saitama, japan smooth pursuit is under learning control by several brain areas including cerebrum and cerebellum. smooth pursuit velocity is modifiable by repetition of target velocity for a brief period at its onsets. role of cerebellar vermis and hemisphere in the adaptive control of smooth pursuit is suggested by lesion experiments, but the role of frontal pursuit area (fpa) is not known. to reveal possible involvement of fpa in the adaptation of smooth pursuit, we identifying fpa by unit recording and microstimulation, and reversibly inactivated it by local injection of muscimol. we found that inactivation of fpa not only reduced of the velocities of pursuit in the ipsi-and contra-versive directions to the inactivated fpa, but also appreciably depressed its adaptation, suggesting that fpa is involved in the adaptation of smooth pursuit. shinji matsutani department of functional morphology, kitasato university school of nursing, kanagawa, japan distribution of terminals on individual centrifugal axons in the main olfactory bulb was studied using an anterograde tracer to elucidate function of the centrifugal system. the tracer was injected into olfactory cortical areas, and individual labeled axons were traced from serial sections. as already reported in the last meeting, the centrifugal axons had multiple terminals with discrete locations. distribution of these terminals was examined in reconstructed maps in which localization of the terminals was projected onto a sagittal plain. in most axons, the terminals were clustered to form a patch that was stretched in a rostrocaudal direction. it was also common that patches belonging to the same axon were found in distant locations and in both sides of the single bulb. while most of the terminals were seen in the granule cell layer, those located in the glomerular layer and in the external plexiform layer were found following injections into the anterior olfactory nucleus. the centrifugal fibers may couple the activity of discrete and distant subsets of bulbar neurons. ps3p-f068 projection targets of the drosophila taste receptor neurons in the primary gustatory center of the brain takaaki miyazaki 1,2 , kei ito 1,2,3 1 dept. of comput. biol., grad. sch. of frontier sci., univ. of tokyo, japan; 2 center for bioinform., imcb, univ. of tokyo, japan; 3 bird, jst, japan in order to figure out the way of information processing linking gustatory stimulus and taste-associated behavior, systematic knowledge about the underlying neural networks is required. drosophila melanogaster is an attractive model organism for this task, thanks to its relatively simple brain structure and a wide variety of molecular and genetic tools available. gustatory sensory neurons in the labellum of the mouth project their axons via the labial nerve to the suboesophageal ganglion (sog) of the brain. to understand the entire neural circuits of these first-order neurons in the primary gustatory center, we searched for the gal4 enhancer-trap strains that visualize specific neural fibers in the sog and the labial nerve. screening 4,000 strains, we identified about 180 candidate lines. the projection targets of the labeled neurons were classified into seven areas. the terminals of the already identified sensory neurons appear to fall into specific subsets of these areas. research funds: bird, jst ps3p-f069 immunoreactivity and voltage-gated channels of mouse taste bud cells kennji kimura 1 , yoshitaka ohtubo 1 , takashi kumazawa 2 , kiyonori yoshii 1 1 graduate school of life science and systems engineering, kyushu institute of technology, kitakyushu, japan; 2 department of applied chemistry, saitama institute of technology, fukaya, japan mammalian taste buds comprise four heterogeneous cell types, type i to iv, and their collaboration seems to generate taste sensation. we investigated the electrophysiological properties of these cell types except type iv with taste buds preserved in mouse lingual epithelia. type i cells elicited smaller ttx-sensitive, tea-sensitive, and teainsensitive currents in magnitude than other cell types. type ii cells elicited a smaller tea-sensitive current and a larger tea-insensitive current than type iii cells. these results suggest that type ii and iii cells elicit action potentials with different ionic mechanisms, and that the difference results from the functional differences of these cell types. research funds: kakenhi (16300094) and the 21st coe program (center #19) granted by mext of japan ps3p-f070 inositol monophosphatase maintains synapse localization and regulates behavior in the mature nervous system of c. elegans yoshinori tanizawa 1 , atsushi kuhara 1 , hitoshi inada 1 , eiji kodama 1 , takafumi mizuno 1 , ikue mori 1,2 1 lab. of mol. neurobiol., nagoya univ., japan; 2 institute for advanced research, nagoya univ., japan inositol monophosphatase (impase) is suggested to be relevant to bipolar disorder. although lithium is believed to exert therapeutic effect by inhibiting impase in patients, the mechanism underlying lithium therapy is largely unknown. here we show that the loss of impase causes defects in behavior and localization of synapses in c. elegans. mutations in ttx-7 gene encoding impase exhibit defective thermotaxis behavior, which is attributable to the loss of impase activity in the most essential integrative interneuron ria in the nervous system. the ttx-7 mutations also cause mislocalization of synaptic proteins in ria. both behavioral and synaptic defects in ttx-7 mutants were rescued by expression of impase at adult stage and inositol application, and were mimicked by lithium application in wild type animals. these results suggest that impase is required in the mature nervous system for maintaining synapses of the central interneurons in order for animals to behave properly. research funds: kakenhi ps3p-f071 postnatal alterations in expression of vesicular glutamate transporters in the main olfactory bulb (ob) of rats h ohmomo, f shutoh, a. ina, s. yoshida, h. nogami, s. hisano lab. neuroendocr., graduate sch., univ. tsukuba, tsukuba, japan olfactory information is conveyed to the brain by transmission from primary olfactory neurons to mitral or tufted cells. however, little is known about development of these ob glutamatergic neurons in early postnatal life. vesicular glutamate transporters (vglut) have been used as the best histological markers to identify glutamatergic neurons. we here studied expressions of two vglut isoforms (vglut1 and -2) during rat ob development from postnatal day 1 (p1) to p10 by in situ hybridization and immunohistochemistry. at p1 vglut1 immunoreactivity (ir) was detected in all layers except the olfactory nerve layer, and thereafter its localization expanded and intensity increased. vglut1 mrna signals were detectable in the mitral cell layer from p1 to p10. in contrast, vglut2 ir was prominent in the glomerulus at all days examined, and only at p1 and p3 in mitral cells. despite mitral vglut2 ir disappeared at p10, the mrna signals were still detectable. these results suggest that glutametergic neurons in the rat ob continue to develop even after birth. ps3p-f072 v1r genes multiplied in amphibian and expressed in the main olfactory system atsuko date-ito 1,2 , masumi ichikawa 3 , yuji mori 2 , kimiko hagino-yamagishi 1 1 tokyo metrop. inst. med. sci., tokyo, japan; 2 the univ. of tokyo, tokyo, japan, 3 tokyo metrop. inst. neurosci., tokyo, japan in rodent, v1r gene family is expressed specifically in the vomeronasal organ (vno) and is thought to be responsible for pheromone reception. however, teleost fishes lacking for the vno have a single v1r gene, which is expressed in the olfactory epithelium (oe). to examine when the v1rs function as pheromone receptors in the course of evolution, we analyzed the amphibian xenopus tropicalis genome, and identified 23 v1r sequences. these v1rs were not expressed in the vno, but most of them were expressed in the oe of the middle cavity, which is considered for reception of water-soluble odorants. from these results, we speculate that the amphibian v1rs get a chance to receive diverse odorants such as pheromones by gene multiplication and sequence diversification. our results raise the possibility that pheromonal information is transmitted via the main olfactory system. ps3p-f073 analyses of ligand binding sites and snps on sweet taste receptor system in human noriatsu shigemura, a.a. islam, yuki nakamura, shinya shirosaki, yuzo ninomiya sect. oral neurosci., grad. sch. dent science, kyushu univ., japan recent studies have shown that t1r2/t1r3 heterodimer plays a role as a sweet taste receptor. but, mice lacking t1r3 showed diminished but not abolished behavioral and nerve responses to sugars, suggesting t1r3-independent sweetener binding site also exist in mice. in this study, to predict binding sites on t1r2/t1r3 and/or other sweet receptor in human, we measured sensitivity thresholds to various sweet compounds and examined the qualitative similarities. we also used gymnemic acid and ␥-cyclodextrin, which selectively inhibits sweet responses and reduces the inhibitory action of it. the ten sweet compounds were classified into five groups [(1) sucrose, glcose, fructose, (2) saccharin, aspartame, acesulfame-k, glycine, (3) d-phenylalanine, (4) d-tryptophan, (5) l-proline]. in sequencing analysis, four and two snps with amino acid substitution were revealed in t1r2 and t1r3, respectively. these results suggest that there may be at least five binding sites in human sweet receptor system. the individual differences in sweet sensitivities may be due to these snps. keiko yasumatsu 1 , sachiko saito 2 , yuko murata 3 , ding ming 4 , tatsu kobayakawa 5 , robert f. margolskee 4 , yuzo ninomiya 1 1 sect. oral neurosci., grad. sch. dent. sci., kyushu univ., fukuoka, japan; 2 saito sachiko taste and smell research institution, ibaraki, japan; 3 national res. institute of fisheries sci., kanagawa, japan; 4 dept. of physiol. & biophys., mount sinai sch. med., new york, usa; 5 national institute of advanced industrial science and technology, ibaraka, japan the effect of unsaturated fatty acids on taste responses was examined by measuring perceived taste intensity in human, behavioral short-term lick responses and electrophysiological taste responses recorded from the chorda tympani and glossopharyngeal nerves in mice. the results showed that dha and other polyunsaturated fatty acids inhibit responses to bitter taste compounds without affecting other taste stimuli. we also found fatty-acid inhibition on bitter responses in an in vitro g-protein activation assay using bovine taste membrane, but lack of the bitter taste inhibition in ggustducin ko mice. these results suggest that fatty acids specifically inhibit responses to bitter stimuli by suppression of activation of t2r receptors which coupled with ggustducin. ps3p-f075 newborn infant body odor attenuates their mother's postpartum moods shota nishitani 1 , mayumi kokuryo 1 , tsunetake miyamura 2 , kazuyuki shinohara 1 1 div. neurobiol. & behav., nagasaki university, japan; 2 obstet. & gynecol. of miyamura hospital, japan mothers are attracted to the body odor of newborn infants, but little is known about its reason. in the present study, we examined whether the body odor of newborn infants exert effects on moods in postpartum mothers. the body odors of newborn infants were collected from their undershirts. postpartum mothers were exposed to odors of a part of the undershirt with control odors, their own infant body odors or other infant body odors. we used the poms to assess the effects of infant body odors on postpartum moods. this study was approved by the ethics committee of nagasaki university. the infant body odors significantly increased hedonics and friendliness scores, and significantly decreased anxiety, depression and fatigue scores, whether infant odors may be originated from their own infants or other infants. these results suggest that body odors of newborn infants attract their mothers because they have calming effects on postpartum mothers. research funds: japan science and technology agency (jst), research institute of science and technology for society (ristex) ps3p-f076 human prefrontal activity in taste encoding: an fnirs study masako okamoto 1 , mari matsunami 2 , haruka dan 1 , tomoko kohata 2 , kaoru kohyama 1 , ippeita dan 1 1 national food research institute, tsukuba, japan; 2 nippon suisan kaisha, ltd., japan taste remains one of the least-explored human senses. using multichannel functional near-infrared spectroscopy (fnirs), we examined the lateral prefrontal cortex (lpfc) of healthy volunteers (n = 10) while they tasted and encoded the quaternary taste mixtures. the contrast between the cortical activation under encoding conditions and that under control conditions without memory requirement revealed activation in the bilateral ventro-lpfc and the right posterior portion of the lpfc. the activation pattern, which was in line with those that have been associated with intentional encoding of non-verbal materials of other senses, supported an amodal role of lpfc in intentional encoding, at least at a macro structural level. this study also demonstrates that, by using fnirs, lpfc functions on taste can be examined with experimental paradigms comparable to those used for other senses. recently, we performed simultaneous respiration and electroencephalographic recordings during odor stimulation. we sought to identify changes in respiratory pattern, inspiratory phase-locked alpha oscillation (i-␣) and location of dipoles estimated from the potentials. electroencephalographic dipole tracing identified the location of dipoles from the i-␣ in the limbic area and the cortex; the entorhinal cortex, hippocampus, amygdala, premotor area and orbitofrontal cortex. in this study, we compared the respiratory pattern during odor stimulations, i-␣, dipole localizations without habituation with those with habituation of odors. onset of inspiration was used as a trigger for averaging, and potentials were averaged before and after the habituation period. habituation of odor caused to return to the normal respiratory pattern, decrease of amplitudes of ␣, and entorhinal cortex, hippocampus, amygdala were less active. akio tsuboi, takaaki miyazaki, takeshi imai dept. of biophys. & biochem., univ. of tokyo, tokyo, japan vertebrate odorant receptor (or) genes are divided phylogenetically into two distinct classes, the fish-like class i and the terrestrialspecific class ii. in the present study, we systematically analyzed mouse class i or genes (42 subfamilies) to elucidate the expression profiles in the olfactory epithelium (oe) and the projection sites of their olfactory sensory neurons (osns) in the olfactory bulb (ob). in situ hybridization (ish) revealed that most class i or genes (36 subfamilies) were expressed in the dorso-medial zone (zone 1) of the oe. furthermore, there appeared to be no significant differences in the distributions of osns expressing class i genes within zone 1. these results indicate that there is a clear boundary between zone 1 and non-zone 1 areas in the oe. some class i ors are known to possess ligand specificity for aliphatic acids, aldehydes and alcohols. our ish analysis has revealed that osns expressing the class i ors in zone 1 tend to converge their axons on a cluster of glomeruli in an antero-dorsal domain that is assumed to be involved in responses to the aliphatic compounds on the ob. research funds: kakenhi (16500196) ps3p-g079 taste response characteristics of putative interneurons in the rat gustatory cortex tatsuko yokota, kunihiro eguchi, katsunari hiraba department of physiology, school of dentistry, aichi-gakuin university, nagoya, japan previous studies have indicated that the extracellular spike waveforms and discharge rate properties of cortical neurons differed between pyramidal cells and interneurons, the latter tending to have narrower spike-widths and higher discharge rates. taste-sensitive neurons in the rat gustatory cortex were classified according to (1) best-taste profiles and (2) spike-widths which were found to form a bimodal distribution (narrow and broad). narrow-spike neurons had a significantly larger response to nacl than broad-spike neurons, but no differences were found to other tastants. the proportion of narrow-spike neurons in the n-best neurons was higher than that in the h or nh-best neurons. these results indicate that putative interneurons may play an important role in the coding of salt taste information. research funds: kakenhi (11671860) of japan to t.y. yuki sato, nobuhiko miyasaka, yoshihiro yoshihara laboratory for neurobiology of synapse, riken bsi, wako, japan in the fish olfactory system, individual olfactory sensory neurons (osns) are thought to express only one or at most a few different odorant receptors (ors) from the large or family consisting of ∼100 members. here, we investigated the mechanisms underlying or gene choice by using transgenic zebrafish that carried a modified bac containing a zebrafish or gene cluster. replacement of the or coding regions in the bac transgene with reporter genes allowed the reporters to be expressed in a small population of osns in the transgenic fish. in situ hybridization analysis using or-specific probes revealed that or genes expressed in reporter-positive cells were mostly restricted within the same or subfamilies to which the replaced ors belonged. additionally, the reporter-expressing osns projected their axons to a topographically fixed cluster of glomeruli in the olfactory bulb. these findings suggest the hierarchical regulation of or gene choice, whereby an individual osn may express one or gene from a limited subpopulation that is chosen from the entire repertoire in advance. research funds: kakenhi (16300105) ps3p-g081 identification of perisomatic-targeting granule cells in the mouse olfactory bulb hiromi naritsuka 1 , kazuhisa sakai 2 , tsutomu hashikawa 2 , kensaku mori 1 , masahiro yamaguchi 1 1 dep. physiol. grad. sch. med., univ. of tokyo, tokyo, japan; 2 laboratory for neural architecture, bsi, riken, saitama, japan in the olfactory bulb (ob), odor information is processed by the local circuit that includes inhibitory interneurons. granule cells (gcs) are major interneurons in the ob, but their diversity is not well understood. in the ob of adult transgenic mice expressing gfp under the control of nestin gene regulatory regions, we observed gcs with strong gfp expression (referred to as type s cells). their dendrites branched and formed spines within the granule cell layer, internal plexiform layer and mitral cell layer but did not reach the external plexiform layer, where typical gcs make synapses with dendrites of mitral and tufted cells. type s cells had huge protrusions at their dendritic ends, which formed contact with mitral cell somata. electron microscopic analysis revealed the existence of reciprocal synapses between type s cell protrusions and mitral cell somata. characteristic morphology of perisomatic-targeting gcs indicates that they have functions distinct from typical gcs in the ob. keiko moriya-ito, kentaroh endoh, yuuki ishimatsu, masumi ichikawa department of neuroscience basic technology, tokyo metropolitan institute for neuroscience, fuchu, tokyo, japan a coculture system of accessory olfactory bulb (aob) neurons and vomeronasal neurons was established for studying the functional roles of aob neurons in pheromonal signal processing. in this study, the effect of vomeronasal neurons on the development of aob neurons was examined in a coculture system. the densities of dendritic spines were lower in the coculture than in single culture. the ratio of the density of synaptophysin-immunopositive spine/total spine density was larger in the coculture than in the single culture. the volume of spine head was larger in the coculture than in single culture. by electron microscopic observation, the synapses on dendritic shafts were decreased and the synapses on dendritic spines were increased in the coculture. the synapses between aob neurons and vomeronasal neurons were recognized in the coculture. these observations suggest that synapse formation of aob neurons is modified by synaptic contact with vomeronasal neurons. ps3p-g083 nacl induced responses of mouse fungiform taste cells: existence of amiloride sensitive and insensitive taste cells ryusuke yoshida, tadahiro ohkuri, keiko yasumatsu, noriatsu shigemura, yuzo ninomiya sect. of oral neurosci., grad. sch. of dental sci., kyushu univ., fukuoka, japan previous electrophysiological studies showed that the chorda tympani nerve contains two types of nacl-responsive fibers, amiloride sensitive (n-type) and insensitive (e-or h-type) fibers, suggesting the existence of amiloride sensitive and insensitive taste receptor cells in fungiform papillae. in this study, we examined nacl responses of mouse fungiform taste cells in isolated taste bud and amiloride sensitivity of them. some taste cells respond to apical restricted nacl stimulation with increase in firing frequency and their responses were concentration dependent. amiloride mixed with apical nacl solution inhibited nacl responses in some taste cells [amiloride sensitive (as) cells] but not in others [amiloride insensitive (ai) cells]. ai cells responded to other electrolytes such as kcl and hcl. these results suggest the existence of at least two types of nacl sensitive cells, as and ai cells. n-or e-type fiber may selectively innervate as or ai cells respectively. research funds: kakenhi (15209061), kakenhi (17791325) ps3p-g084 integration of olfactory and oral sensory input in the rat insular cortex hideki kashiwadani, kensaku mori department of physiology, university of tokyo, tokyo, japan axonal connections between olfactory cortex and insular cortex suggest that insular cortex integrates olfactory information and information originated from the oral cavity (taste, tactile, temperature). however cellular mechanisms underlying the integration of multimodality are poorly understood yet. in this study, we examined single-unit spike responses of insular cortical neurons to odor stimulation and intraoral water stimulation in urethane-anesthetized rat. we found that more than 25% of recorded neurons in the insular cortex responded to odors. about half of the odor-responsive neurons were activated by intraoral water stimulation, indicating the convergence of olfactory and oral sensory information onto individual neurons in the insular cortex. when odor stimulation and intraoral water stimulation were simultaneously applied, some neurons showed spike responses larger than the responses evoked by each stimulus. the integration of olfactory and oral sensory information in the insular cortex might contribute to form the flavor sensation. research funds: kakenhi (17023012) ps3p-g085 odor combination selectivity of the rat piriform cortex neurons ikue yoshida, kensaku mori dept. physiol. grad. sch. med., univ. of tokyo, tokyo, japan olfactory cortex is thought to integrate signals from different odorant receptors to form the olfactory image of objects. however, the manner of integration at the level of individual cortical neurons is not well understood yet. using single-unit recording method, we examined the response selectivity of individual neurons in a dorsocaudal part of the anterior piriform cortex (apc) to 8 classes of odorous compounds, each class being present in odors from many different vegetables and fruits. individual neurons typically responded to more than 2 classes of odorants. each neuron was uniquely tuned to a specific combination of odorant classes, and different neurons typically showed different odor combination selectivity. single-unit responses to odor mixtures showed mixture facilitation and mixture suppression. these results suggest that individual neurons in the apc can be characterized by the odor combination selectivity and that the apc neurons may integrate signals from different odorant classes. research funds: kakenhi (13gs0007) ps3p-g086 odor-driven activity in the anterior piriform cortex of an in vitro isolated whole brain with the olfactory epithelium takahiro ishikawa 1 , takaaki sato 2 , akira shimizu 1 , ken-ichiro tsutsui 1 , toshio iijima 1 1 div. of systems neuroscience, grad. sch. of life sciences, univ. of tohoku, sendai, japan; 2 res. inst. for cell engineering, aist, amagasaki, japan to examine the neural mechanisms underlying odor-induced response in the anterior piriform cortex (apc), we analyzed odorinduced local field potential (lfp) and multiunit activity in an in vitro preparation, isolated guinea-pig whole brain with the olfactory epithelium. in apc, odor-induced lfps consisted of a phasic initial component followed by a fast oscillatory activity in the beta range (20 hz). by comparison a result of current source-density analysis with unit activity data, we confirmed that the initial component of odor-induced response has a characteristic temporal pattern, generated by a relatively weak direct afferent input, followed by an intracortical associative response, which was associated with a phasic inhibition. the beta oscillation might be generated by the repetition of these network activities. these electrophysiological data were consistent with the results of previous studies that used slice or anesthetized in vivo preparations. ps3p-g087 chemotaxis of c. elegans to concentration gradient of an attractant superimposed on a uniformly distributed attractant lin lin, hiroyuki oikawa, miyako sasaki, tokumitsu wakabayashi, ryuzo shingai department of welfare engineering, iwate university, morioka, japan to investigate the informational interaction between pathways from different sensory inputs to the behavior in the nervous system of c. elegans, chemotaxis toward the concentration gradient of an attractant spotted on a uniformly distributed another attractant was investigated. lysine and chloride ions are water soluble chemoattractants. when 3 m lysine was spotted on ammonium chloride background, 0.003-0.01 m and 0.05 m background did not influence lysine chemotaxis, while 0.03 m background augmented and 0.07-0.1 m background suppressed the chemotaxis. in contrast, when 0.1 m ammonium chloride was spotted on the lysine background, the background did not alter or suppressed the chemotaxis. interaction between informational pathways from different sensory inputs could be seen also in the presentation of an odorant spotted on chemoattractant background, and vice versa. ps3p-g088 glutamate receptors are regulated by the ras-mapk pathway in neural circuit-dependent odor adaptation in c. elegans takaaki hirotsu 1,2,3 , takeshi ishihara 1 , eisuke nishida 3 , yuichi iino 2 1 dept. biol., fac. sci., kyushu univ., japan; 2 mol. genet. res. lab., univ. of tokyo, japan; 3 grad. sch. biostudies., kyoto univ., japan c. elegans shows a decrease in chemotaxis to odorants after exposure to the odorant for 5 min. this plasticity, called early adaptation, requires aiy interneurons, which receive synaptic inputs from olfactory neurons, indicating that early adaptation depends on neural circuit. the ras-mapk pathway is activated by odorant exposure in aiy and plays essential roles for early adaptation. the function of glr-1, a non-nmda type glutamate receptor, in aiy is also important for early adaptation. glr-1 appears to localize at postsynaptic sites in aiy. this localization was changed by odorant exposure in early adaptation. mutation of the ras-mapk pathway impaired localization of glr-1. in vitro kinase analyses revealed the possibility that mapk directly phosphorylates glr-1. these results suggest that the ras-mapk pathway controls odor adaptation by directly regulating glr-1 localization in aiy neurons. kohei ueno 1 , yoshiaki kidokoro 2 1 dept. behav. sci., grad. sch. med., gunma univ., maebashi, japan; 2 inst. mol. cel. reg., gunma univ., maebashi, japan sodium chloride (nacl) is the major substance that induces nacl taste. in rodents, some strains prefer nacl solutions (∼1%), but others do not or even avoid them. although it is reported that the difference is based on the genetic background, the molecular information involved in the difference is not known. in the 26th ns annual meeting, we have shown that nacl preference in several wild-type strains of drosophila melanogaster is variable and p-element insertion in a single gene suppressed nacl preference. here, we carried out the sequencing analysis and found eight single-nucleotide polymorphisms (snps) in the gene. moreover, we found that one of the snps was correlated with nacl preference among wild-type strains. we generated transgenic flies and rescued the low preference phenotype of p-element insertion strain using the gal4/uas system. finally, we examined the expression pattern of the gene and found the gene is expressed in taste organs. taken together, we suggest that the gene is a novel nacl receptor gene. ps3p-g090 spatial and temporal organization of odor representation by moth antennal lobe output neurons shigehiro namiki 1,2 1 graduate school of life and environmental sciences, university of tsukuba, ibaraki, japan; 2 department of mechano-informatics, graduate school of information science and technology, university of tokyo, tokyo, japan the antennal lobe (al) is the first relay station for olfactory information in the insect brain and is the anatomical equivalent of the mammalian olfactory bulb. both systems have common structures called glomeruli, functional units of olfactory processing. odor-evoked spatial and temporal patterns by an array of glomeruli are both important in olfactory coding. but the details of olfactory coding mechanisms are still unclear. we confirmed that projection neurons (pns, al output cells) innervating the same glomerulus had similar olfactory responses in the silkmoth. by pooling data from many pns that innervate identified glomeruli i reconstructed odor representations. i found that olfactory information is encoded by distributed spatiotemporal activity of a pn population and that there are no clear correlation between the similarity of slow temporal patterns of pns and spatial distances of innervating glomeruli. research funds: brain ps3p-g091 medial nucleus amygdala neurons have morphologically and electrophysiologically heterogeneous properties makoto yokosuka 1 , yoshinori sahara 2 , shinichiro horie 2 , masumi ichikawa 3 , shun nakamura 2 1 st. marianna univ. schl. med., kawasaki, japan; 2 ntl. inst. neurosci., ncnp, tokyo, japan; 3 tokyo metropol. inst. neurosci., tokyo, japan we characterize the electrophysiological and morphological properties of the medial nucleus amygdala (mea) neurons using whole-cell recordings in mice slice preparations. most mea neurons showed either tonic-bursting or adapting burst of action potentials to deporalizing currents. biocytin labeling showed that mea neurons possessed bipolar to multipolar cell bodies and dendritic fields covering projection areas from the accessory olfactory bulb. norepinephrine increased the frequency of spontaneous ipscs in some neurons, while serotonin increased spontaneous epscs in others. morphologically and physiologically heterogeneous mea neurons seem likely to produce multiplex outputs of many instinct behaviors. hideyuki matsumoto, kensaku mori department of physiology, graduate school of medicine, university of tokyo, tokyo, japan olfactory sensation sometimes lasts even after odorant stimulation has ceased. neuronal mechanisms for the olfactory afterimage are not well understood yet. single unit recordings from mitral/tufted cells in the mouse olfactory bulb (ob) showed that some neurons continued to discharge for more than 10 s even after the cessation of odorant stimulation. the induction of the sustained spike discharge depended on the intensity of odorant stimulation, and showed an allor-none behavior. spike discharges during the sustained discharge mode phase-locked to the respiration cycle and the phase-locking pattern during the sustained discharge mode differed from that during odor stimulation. these results suggest that neuronal mechanism in the ob may be responsible for the induction of the post-stimulus sustained discharges. the respiratory-phase-locked sustained discharges were recorded from juxta-glomerular cells. this implies that neuronal interactions within the glomeruli are involved in the induction of the sustained spike activity of mitral/tufted cells. ps3p-g093 synaptic transmission shows state-dependent change in the urethane-anesthetized rat olfactory bulb yusuke tsuno, hideki kashiwadani, kensaku mori department of physiology, graduate school of medicine, the university of tokyo, tokyo, japan olfactory cortex (oc) shows a state-dependent sensory gating that is controlled under the modulatory inputs from the basal forebrain and brainstem. since the olfactory bulb (ob) receives the modulatory inputs heavily, neuronal activity in the ob might change in a state-dependent manner. in the present study, we demonstrate a clear state-dependent change in the magnitude of the transmission of granule-to-mitral dendrodendritic inhibitory synapses and olfactory cortex-to-granule excitatory synapses. transmission of granule-tomitral synapses and olfactory cortex-to-granule synapses was facilitated during slow-wave state and suppressed during fast-wave state. in addition, we observed synchronous slow oscillations (about 1 hz) in the granule cell layer of the ob, layer iii of the oc, and the occipital cortex. thus the ob shows state-dependent synaptic modulation and presumably receives top-down periodic signals from the cortex. research funds: kakenhi (17023012) ps3p-g094 rem sleep deprivation decreases na-k atpase phosphorylation gitanjali das, birendra n. mallick school of life sciences, jawaharlal nehru university, new delhi, india it has been hypothesized that "one of the functions of rem sleep is to maintain brain excitability" rem sleep deprivation increases noradrenaline in the brain that increases the na-k atpase activity causing increased brain excitability. however, the molecular mechanism of such increased na-k atpase activity was unknown; although it was known that dephosphorylated state is the active form of na-k atpase. rats were rem sleep deprived by flower-pot method; large platform and recovery from lost rem sleep were carried out as controls. at the end of experiment, brains were quickly removed by cervical dislocation and synaptosomes prepared, which were used for western blotting against phosphoserine and phosphothreonine antibodies as well as for na-k atpase activity. after rem sleep deprivation the activity increased, while the level of phosphorylated form of na-k atpase decreased in the same sample. this confirms our hypothesis that rem sleep deprivation induced increased activity is due to dephosphorylation of na-k atpase. research funds: icmr (govt. of india) and upoe (govt of india) takeshi fujii 1,2 , ken yoshikawa 2 , yuki takatori 1 , koichiro kawashima 2 1 dept. of pharmacol., fac. of pharmaceut sci., doshisha women's coll., japan; 2 dept. of pharmacol., kyoritsu univ. of pharmacy, japan stimulation of muscarinic (machr) and nicotinic (nachr) receptors with respective agonists induces ca 2+ signals in t cells. in the present study, using rna interference approach, we investigated roles of machr and nachr subtypes in ca 2+ signals in ccrf-cem (cem) cells, a human t cell line, as a model of t cells. cem cells express m 1 , m 3 , m 4 and m 5 machr subtypes, and ␣3, ␣5, ␣6, ␣7, ␣9, ␣10 and ␤4 nachr subunits. transfection of anti-m 3 , anti-m 5 and anti-␣7 small interfering rna (sirna) significantly down-regulated respective mrna expression, while no changes were observed in gene expression of other machr subtypes or nachr subunits. ca 2+ signals evoked by oxotremorine-m, a non-selective machr agonist, were reduced by anti-m 3 or anti-m 5 sirna. ca 2+ signals evoked by nicotine were reduced by anti-␣7 sirna. these findings indicate that m 3 , m 5 machr and ␣7 nachr subtypes play major roles in ca 2+ signals to acetylcholine in t cells, and suggest that these receptors are involved in regulation of immune function. research funds: kakenhi (16590060) ps3p-g101 is "seronegative" mg explained by autoantibodies to musk? kazuhiro shigemoto 1 , sachiho kubo 2 , seiji matsuda 3 , naoki maruyama 2 1 dept. of preventive medicine, ehime univ. schl. of med., ehime, japan; 2 dept. of mol. path., tokyo metro inst. for gerontology, tokyo, japan; 3 dept. of integrated basic medical science, ehime univ. schl. of med., ehime, japan muscle-specific kinase (musk) is critical for the synaptic clustering of nicotinic acetylcholine receptors (achr). musk is activated by agrin, which is released from motoneurons, and induces achr clustering at the postsynaptic membrane. although autoantibodies against the ectodomain of musk have been found in a proportion of patients with generalized myasthenia gravis (mg), it is unclear whether musk autoantibodies are the causative agent of generalized mg. in the present study, rabbits immunized with musk ectodomain protein manifested mg-like muscle weakness with a reduction of achr clustering at the nmj. the autoantibodies activated musk and blocked achr clustering induced by agrin or by mediators that do not activate musk. thus, musk autoantibodies rigorously inhibit achr clustering mediated by multiple pathways, an outcome that broadens our general comprehension of the pathogenesis of mg. (shigemoto et al., j. clinical investigation, 2006) research funds: kakenhi (16590831) ps3p-g102 dynamic changes in the thalamo-cortical system associated with thalamic neurodegeneration shin-ichi kyuhou, hisae gemba department of physiology, kansai medical university, japan in purkinje cell degeneration (pcd) mice, degenerating thalamic neurons were found morphologically in the particular thalamic nuclei including the ventral medial geniculate nucleus around postnatal day 50. electrophysiologically, auditory evoked potentials in the primary auditory cortex began to decrease gradually in amplitude from postnatal day 45. analysis of spontaneous cortical field potentials by fast fourier transform, revealed that high frequency oscillation (hfo) of around 25 hz appeared prominently in the auditory cortex. local injection of kynurenic acid, a glutamate receptor blocker, into the thalamus suppressed the hfo in the auditory cortex, indicating that the thalamus is involved in the generation of the hfo. the real time polymerase chain reaction analysis demonstrated the upregulation of the mrna of nmda receptors in the auditory cortex. these results suggested dynamic changes occurred in the thalamo-cortical system after thalamic neurodegeneration in pcd mice. research funds: grant c1 from kansai medical university ps3p-h103 unusually folded sod1 species sequester specific motor molecules and inhibit the axonal transport of their cargos minako tateno 1 , yumiko simazaki 1 , fuminori saitoh 1 , ryosuke takahashi 2 , toshiyuki araki 1 1 national institute of neuroscience (ncnp), tokyo, japan; 2 dept. of neurology, kyoto university, kyoto, japan misfolding of mutant sod1 protein is thought to be responsible for the selective loss of motoneurons in sod1-related familial amyotrophic lateral sclerosis (als), although the molecular mechanisms underlying the toxicity of such unusually folded sod1 species are not yet clarified. since we have detected accumulation of unusual sod1 species in motoneuronal axons from g93a sod1-tg mice, we fractionated the ventral white matter of spinal cords to isolate the unusual sod1 species. immunoprecipitation analyses revealed specific interaction of unusual sod1 species with certain kinds of motor molecules. moreover, the axonal transport of cargos mediated by those molecules was found to be significantly reduced in symptomatic mutant sod1-tg compared with wt sod1-tg mice. these data strongly suggest that the toxic property of unusual sod1 proteins is partially ascribable to the transport inhibition of specific cargos. research funds: grant-in-aid for scientific research c (17590907) ps3p-h104 relationship between the amount of the cathepsin d expression and the symptomatic manifestation of neuronal ceroid-lipofuscinosis in a mouse model masahiro shibata, masato koike, yasuo uchiyama department of cell biology and neuroscience, osaka university graduate school of medicine, japan mice deficient in cathepsin d (cd), a representative lysosomal aspartic proteinase, have been shown to be an excellent model of neuronal ceroid-lipofuscinosis (ncl). here we report that the phenotype of mice in which cd is partially expressed is decided depending on the amount of the protein expression of cd. the proteolytic activity and protein expression of cd in the mutant mice were approximately 30% of those in the wild-type mice, while the growth of the mice appeared intact until postnatal day 24. the mice started to show ncl symptoms on p25, and their life span was prolonged for one to three days, compared to that of the cd-null mice. the protein expression of cd in the heterozygous mice was approximately half of that in the wildtype mice and the mice showed no pathological finding. these results indicate that a threshold of the cd expression required for the manifestation of ncl symptoms in the mice may be present in the range from 30% to 50% of that in the wild-type mice. research funds: kakenhi (10343253) ps3p-h105 neuronal toxicity of expanded polyglutamine depends on intracellular distribution among cells with similar expression levels mamoru satoh, atsuyoshi shimada, noriko kawamura, yoichi chiba, yuko saitoh, hiromi keino, masanori hosokawa dept. pathol., inst. develop. res., aichi human service center, aichi, japan we previously reported that expanded polyglutamine (polyq) tracts induced cellular toxicity of neuro2a cells in the form of massive cytoplasmic aggregates but not of intranuclear inclusion. however, we did not rule out the possibility that such toxicity depends on the level of intracellular expression of polyq. in this report, we compared the toxicity of polyq among cells expressing polyq tracts with a variety of intracellular distribution but at similar expression levels. damages were most remarkable in cells with cytoplasmic massive aggregate in terms of shrunken cellular and nuclear sizes. cells with cytoplasmic homogeneous distribution, cytoplasmic punctate distribution and intranuclear inclusion of polyq tracts were relatively spared. these data suggest that the severity of cell damages depends on the type of intracellular distribution of polyq tracts in cells expressing polyq tracts at similar level. ayumi takamura 1 , katsumi higaki 1 , junichiro matsuda 2 , yoshiyuki suzuki 3 , eiji nanba 1 1 division of functional genomics, research center for bioscience and technology, tottori university, tottori, japan; 2 national institute of biomedical innovation, osaka, japan; 3 clinical research center, international university of health and welfare, tochigi, japan g m1 -gangliodisosis is an autosomal recessive lipid storage neurodegenerative disorder. due to a deficiency of lysosomal ␤-galactosidase, excessive lysosomal accumulation of gm1 is observed in patients and animal model brains. however pathogenesisi of this disease is still unclear. since gm1 is known to be a major sialoglycolipid constituent of plasma membrane (pm) in neuron, we examined the analysis of brain of mouse model. cerebellar granule cells from this mouse showed gm1 accumulation of lysosome and pm and the membrane fluidity was also reduced. gm1-bound phosphorylated trka was markedly decreased in cultured neuron and brain tissues. subsequent plc␥, known as a downstream signal of trka, was also impaired. these results suggest that dysfunction of neurotrophin signaling may cause the onset of neurodegeneration in g m1 -gangliosidosis. katsuya inoue 1,2 , katsuaki endo 1 , takamitsu fujikawa 1 , seijyun fukuda 2 , tatsuo nakamura 2 1 department of physical therapy, university of aino, osaka, japan; 2 institute for frontier medical science, kyoto university, kyoto, japan regeneration of spinal cord injury is an important thema in rehabilitation science as well as basic one. the experiment was designed to reveal the process after spinal cord injury by asphyxia. to establish the animal model of spinal cord injury produced by asphyxia, we used adult cats with aorta occulusion under deep pentabarbital anesthesia. twenty minutes after occulusion electrical reflex activity of spinal cord disappeared. after 40 min occulusion, irreversible functional changes were observed, long term depression of reflex activities and disorders of motorsensory function. we also traced time course of electrical and functional changes after 40 min occulusion. ps3p-h108 development of a rodent behavioral model to study the direct interactions of reward and learning adam weitemier, niall p. murphy riken brain science institute, japan cognitive and reward processes often occur simultaneously, and perhaps interdependently. learning is a necessary condition in many experimental models aimed at assessing the rewarding value of a given stimulus. conversely, reward is often used as an experimental tool to engage mnemonic processes in studies aimed at investigating learning and memory. recent studies have demonstrated shared neurobiology between memory and reward. a direct behavioral interaction between reward and memory has never been studied. cognitive impairments observed in psychiatric conditions of dysregulated reward, such as drug abuse and depression, make this issue important, particularly in light of ongoing efforts to investigate higher brain functions. we are developing a rodent behavioral model with which to directly assess the influence of reward processes on learning and memory. we will introduce our recent progress with this new model, including two variations of the procedure designed to study the influence of reward on memory acquisition and memory recall. tetsuya ando 1 , yuya kawanaka 1 , minoru saito 2 , hiroaki mochizuki 1 , ken honjo 1 , hirofumi toda 1,3 , toshifumi tomoda 3 , akira sawa 4 , katsuo furukubo-tokunaga 1 1 grad. school of life & envir. sci., univ. tsukuba, japan; 2 molecular physiol., tokyo metropolitan inst. neurosci. tokyo, japan; 3 beckman res. inst., city of hope. california, usa; 4 dept. of psych. & neurosci. johns hopkins univ. school of medicine. baltimore, usa the disrupted-in-schizophrenia-1 (disc1) gene, originally identified at the breakpoint of a chromosome (1;11) (q42.1; q14.3) translocation in a scottish schizophrenia pedigree, is a promising candidate gene for schizophrenia and affective disorder. however, cellular and molecular mechanisms underlying cognitive impairments are yet to be elucidated. to address disc1 functions in vivo, we expressed disc1 in drosophila and examined developmental and behavioral phenotypes. overexpression of disc1 resulted in marked suppression of olfactory associative learning in flies whereas it caused no symptoms of neural degeneration even in aged animals. we anticipate that the drosophila system will serve as a novel model system amenable to a variety of genetic manipulations for the study of schizophrenia. ps3p-h110 effect of hypothermia on discrepancy between memory learning ability and anatomical brain damages in rats with neonatal hypoxic ischemic encephalopathy yuji miyatake 1 , ayumi kamo 1 , kenji minato 1 , hitoshi haruna 1 , hiritsugu fukuda 2 , yuji murata 2 , takayoshi hosono 1 1 department of bomedical engineering, osaka electro-communication university, japan; 2 graduate school of medicine, osaka university, japan we investigated the effect of brain hypothermia on neonatal hypoxic ischemic encephalopathy (hie) in hie-model rats using olton t-maze and anatomy. the common carotid artery of 22 of 25 7-day-old rats was ligated and cut under anesthesia. after the operation the rats were put in a box containing 8% oxygen at 40 • c for 15 min. after the insult, 11 of the 22 rats were put in a box at 35 • c for 12 h (hypothermia, h-group). the other 11 rats were returned to their mother without hypothermia (normothermia, n-group). sham operations were performed on three rats (s-group). eight weeks after the operation, their learning and memory ability was assessed by olton t-maze, and no statistical difference was observed in either the working or reference memory in the three groups although the anatomical brain size in the n-group was significantly smaller than in the h-group and s-group. withdrawn ps3p-h112 tau hyperphosphorylation in ts1cje, a partial trisomy 16 mouse model for down syndrome ebrahim abdul 1 , a. shimohata 1 , w. yu 1 , m. yamaguchi 1 , m. murayama 3 , d. chui 3 , t. akagi 2 , t. takeuchi 1 , k. amano 1 , h.s. karthik 1 , t. hashikawa 2 , h. sago 4 , c.j. epstein 5 , a. takashima 3 , k. yamakawa 1 1 research scientist; 2 lab. for neural arch.; 3 lab. for alzheimers disease; 4 div. of fetal med. ncchd; 5 ucsf, usa although down syndrome (ds) or trisomy 21 is the most common genetic cause of mental retardation, its neuropathology remains unclear. ts1cje, a ds mouse model partially trisomic for chromosome 16, shows learning and behavioral abnormalities mimicking ds mental retardation. the trisomic segment, corresponding to parts of human chromosome 21q22, has about 97 genes. importantly, sod1 and app, which may contribute to the ds phenotype, are excluded from the ts1cje trisomic segment. here we report that ts1cje brains show hyperphosphorylation of tau in the absence of nft formation, as well as increased gsk3␤ and jnk/sapk activities without alterations in a␤pp metabolism. our results suggest that genes on the trisomic ts1cje segment other than app and sod1 can cause hyperphosphorylation of tau, which in turn may be critical in the pathogenesis of ds mental retardation. research funds: kakenhi number: 17790737 ps3p-h113 increased oxidative stress and mitochondrial dysfunction in ts1cje, a down syndrome mouse model atsushi shimohata 1 , ebrahim a. s. 1 , m. yamaguchi 1 , w. yu 1 , h. sago 2 , c.j. epstein 3 , k. yamakawa 1 1 lab. for neurogenetics, riken-bsi, japan; 2 div. of fetal med. ncchd, japan; 3 dept. pediatrics, ucsf, usa down's syndrome (ds), caused by chromosome 21(hsa21) trisomy, is the most common genetic cause of mental retardation and affects every major organ in the body. ts1cje is one of a number of segmentally trisomic ds mouse models, and is triplicated for a region of mouse chromosome 16 extending from sod1 to znf295, containing 97 genes syntenic with hsa21. since these mice show learning and behavioral abnormalities mimicking ds mental retardation, ts1cjespecific trisomic segment genes may be involved in the ds phenotype. in the present study, we observed increased levels of reactive oxygen species (ros), mitochondrial function impairment in primary cultured astrocytes and hippocampal neurons, and increased cabonylated proteins in ts1cje brains. collectively, our results implicate dosage imbalanced genes other than sod1 and app in both ros generation and mitochondrial dysfunction, which in turn possibly contribute to the ts1cje ds mental retardation-like phenotype. ps3p-h114 polyinosinic-polycytidylic acid injection in early pregnancy causes the hypomyelination in the hippocampus, but not in the cortex manabu makinodan 1,2 , kouko tatsumi 2 , takayuki manabe 2 , takahira yamauchi 1,2 , eri makinodan 2 , juro shimoda 1 , toshifumi kishimoto 1 , akio wanaka 2 1 department of psychiatry, nara medical university, kashihara, japan; 2 department of 2nd anatomy, nara medical university, kashihara, japan polyinosinic-polycytidylic acid (poly i:c) elicits maternal immune response similar to anti-viral ones. recent studies demonstrated that poly i:c injection into pregnant mice resulted in behavioral changes including deficits in prepulse inhibition in the offspring, rendering this system an animal model of schizophrenia. in the present study, we observed such behavioral abnormalities reproducibly in the experimental group born from poly i:c-injected mice, but not in the control group born from pbs-injected mice. they showed decreased myelination in the hippocampus at juvenile period with unaltered number of oligodendrocytes. on the other hand, myelination in the cerebral cortex did not significantly differ between the experimental and control mice. the hypomyelinaton in the hippocampus at the juvenile period may be a possible cause for the behavioral changes in later periods. joanna doumanis, ritsuko kazama, adrian moore, nobuyuki nukina riken brain science institute, japan the fruitfly drosophila melanogaster is well established as a model system in the study of human neurodegenerative diseases. to model the polyglutamine expansion disease, huntington disease (hd), we have established stable, inducible cell lines expressing n-terminal truncated huntingtin fused to egfp with an expanded (62q) polyglutamine repeat in a drosophila larval central nervous system-derived cell line. induction of expression results in the formation of protein aggregates, characteristic of hd. utilising rnai, we have carried out a high-throughput screen for modifiers of aggregate formation in these cells. 7200 genes, encompassing around 50% of the drosophila genome, were screened, resulting in the identification of 370 candidates that either suppress or enhance aggregation. most candidates identified have mammalian orthologues, validating the use of drosophila to screen for genes relevant to human disease. we established in vivo models of hd by expressing polyq-egfp in the drosophila nervous system and are further characterising selected candidates in our model. the rodent model of harmaline-induced tremor has been used as an animal model of essential tremor. the present study investigated effects of harmaline on olivocerebellar systems of mice and rats. systemic administration of harmaline produced generalized tremors in both types of rodents. immunohistochemical studies revealed significant degeneration of purkinje cells that was associated with activated microgliosis in the cerebellar cortex, following administration of harmaline in rats but not in mice. however, in mice but not rats, microgliosis was induced following administration of harmaline in the inferior olivary nucleus (ion). numbers of neurons in the mouse ion did not decrease, suggesting the possibility that microgliosis in ion might not be a simple neurotoxic effect. presumably, differences in sensitivity of purkinje cells between rats and mice may be related to differences in functional alterations in their respective olivocerebellar systems induced by harmaline. recognition of these species-specific differences is an important consideration for experimental analysis of the rodent model of tremors. ps3p-h117 analysis of ␣-synuclein expression in young mouse model of multiple system atrophy kimiko nakayama, yasuyo suzuki, ikuru yazawa laboratory of research resources, national institute for longevity sciences, aichi, japan multiple system atrophy (msa) is a sporadic neurodegenerative disease that affects oligodendrocytes and neurons in human central nervous system. glial cytoplasmic inclusions (gcis) are diagnostics of msa. gcis are shown to be abnormal accumulation of filamentous ␣-synuclein. yazawa et al. (2005) generated a transgenic (tg) mice overexpressing human wild-type ␣-synuclein in oligodendrocytes under the control of the 2 , 3 ,-cyclic nucleotide 3 -phosphodiesterase (cnp) promoter. tg mouse study demonstrated that formation of gci-like ␣-synuclein inclusions in the oligodendrocyte leads directly to neuronal degeneration, as shown by motor impairment and novel accumulation of mouse ␣-synuclein in neuron. to elucidate the mechanisms of neurodegeneration in tg mice, we prepared primary cultures of neurons and glial cells from tg mice. the cells are examined the effects of ␣-synuclein accumulation. ps3p-h118 dysregulation of sodium channel ␤4 subunit by expanded polyglutamine in huntington disease transgenic mice fumitaka oyama, haruko miyazaki, kazumasa okamura, yoko machida, kurosawa masaru, takashi sakurai, nobuyuki nukina laboratory for structural neuropathology, riken bsi, wako-shi, japan sodium channel ␤4 (␤4) is a very recently identified auxiliary subunit of the voltage gated-sodium channels. we have identified ␤4 as an est that was significantly downregulated in the striatum of hd model mice and found that reduction in ␤4 started at a presymptomatic stage of the hd model mice. in contrast, spinal cord neurons, which generate only negligible levels of expanded polyq aggregates, maintained normal levels of ␤4 expression even at the symptomatic stage. expanded polyq with nls expression suppressed the promoter activity of ␤4 gene in pc12 cells. forskolin, an activator of the camp/pka pathway, did not affect b4 promoter activity, indicating that ␤4 is not camp-responsive gene. these findings strongly suggest that sodium channel ␤4 subunit is a novel molecule, which is an upstream non-camp-responsive gene in hd pathogenesis. ps3p-h119 repeat length-and age-dependent changes in behavioral phenotypes of drpla transgenic mice harboring a single copy of a full-length human drpla gene kazushi suzuki 1 , yuji takahashi 1 , jun goto 1 , mutsuo oyake 2 , toshiya sato 3 , shoji tsuji 1 1 department of neurology, the university of tokyo, tokyo, japan; 2 department of neurology, brain research institute, niigata university, niigata, japan; 3 center for bioresource-based research, brain research institute, niigata university, niigata, japan we carried out detailed analyses of the behavioral phenotypes of drpla transgenic mice carrying an expanded cag repeat of 76 (q76), 96 (q96), 113 (q113), or 129 (q129). in the accelerating rotarod (9w), the latencies of q76, q96, q113 and q129 were 113%, 81%, 34% and 24%, respectively. in the open field, moving distances of q96, q113, and q129 were decreased to 98%, 50%, and 20%, respectively, while that of q76 was increased to 134%. home cage activity was decreased depending on the repeat length. the q113 mice, however, showed increased ratios of the activity during the light time to that during the total day at 8 weeks (115%) and 24 weeks (208%), suggesting that drpla mice display not only impaired motor coordination, but also changes in emotional behavior, and disrupted night and day activity patterns. ps3p-h120 the mice lacking schnurri-2 show multiple behavioral abnormalities related to psychiatric disorders keizo takao 1 , nobuyuki yamasaki 1 , keiko toyama 1 , tsuyoshi takagi 2 , shunsuke ishii 2 , tsuyoshi miyakawa 1 1 hmro, kyoto university graduate school of medicine, kyoto, japan; 2 riken, tsukuba, japan schnurri-2 (shn-2) is a zinc finger transcription factor, a mouse homologue of human hiv-ep2, that binds to nuclear factor kappa b-binding site in the hiv long terminal repeat. shn-2 is known to play important roles in the mammalian immune systems. however, the role of shn-2 in the central nervous system (cns) is still unknown. to investigate the functional significance of shn-2 in mammalian brain, we analyzed the shn-2 knockout (ko) mice using a comprehensive behavioral test battery. shn-2 ko mice were dramatically hyperactive under novel environment and in their home cage. they also showed increased acoustic startle response and impaired prepulse inhibition, indicating their impairment in sensorimotor gating. anxiety-like behavior and depression-like behavior were also significantly reduced in shn-2 mice. our results demonstrate a critical role of shn-2 in cns and suggest that shn-2 ko mice may serve as an animal model of psychiatric disorders. research funds: kakenhi (16680015, 16653065, 17017021, 17025023) , jst bird ps3p-h121 comprehensive brain-behavior phenotyping of camkii␣ heterozygous knockout mice nobuyuki yamasaki, koichi tanda, keiko toyama, yasuyuki fukui, keizo takao, tsuyoshi miyakawa hmro, kyoto university graduate school of medicine, kyoto, japan ca 2+ /calmodulin-dependent protein kinase ii (camkii) is a ubiquitous serine/threonine protein kinase that is abundant in brain as a major constituent of the postsynaptic density and critically involved in synaptic plasticity, learning and memory. several behavioral abnormalities of camkii␣ mutant mice were reported, but systematic assessments of behaviors of camkii␣ mutant mice have not been conducted. to analyze the behavioral effects of camkii␣ deficiency, we subjected camkii␣ heterozygous knockout mice to a comprehensive behavioral test battery. the mutant mice showed hyperactivity, decreased anxiety, decreased depression-related behavior, increased offensiveness, selective spatial working memory deficit, and dramatic periodic change of locomotor activity in home cage. to identify the mechanism underlying these behavioral abnormalities, gene expression analysis was conducted. the potential involvement of camkii␣ in pathogenesis/pathophysiology of psychiatric disorders will be discussed. research funds: kakenhi (16680015, 16653065, 17017021, 17025023) , jst bird ps3p-h122 effects of various factors on the results of a comprehensive behavioral test battery for genetically engineered mice: a factor analytic study hiroshi ougino, nobuyuki yamasaki, koichi tanda, keiko toyama, keizo takao, tsuyoshi miyakawa hmro, kyoto university graduate school of medicine, kyoto, japan we have been using a behavioral test battery to reveal unknown phenotypes of genetically engineered mice. for the adequate experimental design and interpretation of data, it is essential to know experimental variables which may potentially influence results, and various kinds of factors which underlie many indices measured in the tests. in this study, we investigated the effects of background strains (c57bl/6j, c57bl/6n, c57bl/6c, 129svev, balb/c), body weight, age at test, and start time of test on the results of each test, by analyzing data of more than 1200 mice (, including wild type and mutant mice from 25 strains of genetically engineered mice), which had been tested in our laboratory. also, we conducted factor analyses of a large set of data to examine the relationship between behavioral indices. the potential implications of our findings for the improvement of the behavioral test battery will be discussed. calcium-and calmodulin-dependent protein kinase iv (camkiv) is a protein kinase that activates the transcription factor, camp responseelement binding protein (creb). camkiv has been hypothesized to play a significant role in synaptic plasticity and in learning and memory. however, functions of camkiv in a variety of behaviors, e.g., motor function, nociception, fear, anxiety, depression, learning and so on, have not yet been fully elucidated. to gain more insight into behavioral significance of camkiv, we subjected camkiv−/− mice to a battery of behavioral tests. camkiv−/− mice did not display any deficit in spatial reference memory and working memory tests, but had mild performance deficit in fear conditioning tests. these results indicated selective and specific involvement of camkiv in regulating emotional behavior. research funds: kakenhi (16680015, 16653065, 17017021, 17025023) , jst bird ps3p-h124 comprehensive behaivoral analysis of ryanodine receptor type3 knockout mouse suzuko ohsako 1 , koichi tanda 1,2 , nobuyuki yamasaki 1 , keiko toyama 1 , hiroshi takeshima 3 , tsuyoshi miyakawa 1 1 kyoto university graduate school of medicine, kyoto, japan; 2 dep. of pediatrics, kyoto prefectural univ. of medicine, kyoto, japan; 3 dep. of biochem. and mol biol., tohoku univ. graduate school of medicine, miyagi, japan ca 2+ signaling is essential for the regulation of neuronal processes including synaptic transmission and transmitter release. ryanodine receptors (ryrs) are family of intracellular calcium channels and mediate calcium-induced calcium release from the endoplasmic reticulum. ryr3 is highly expressed in the hippocampus, caudate putamen, and thalamus. to investigate the behavioral effects of ryr3 deficiency, we subjected ryr3 knockoout mice to a battery of behavioral tests. ryr3 knockout mice exhibited hyperactivity and abnormal behavior in social interaction test, while they did not show any deficit in motor function, depression, attention, and working memory tests. these results suggest a role of ryr3 in regulating general locomotor activity and social behavior. research funds: kakenhi (16680015, 16653065, 17017021, 17025023) , jst bird ps3p-h125 comprehensive behavioral analysis of neuronal nitric oxide synthase knockout mouse keiko toyama 1 , koichi tanda 1,2 , nobuyuki yamasaki 1 , tsuyoshi miyakawa 1 1 hmro, kyoto university graduate school of medicine, kyoto, japan; 2 dept. of pediatrics, kyoto prefectural univ. of medicine, kyoto, japan nitric oxide (no) plays several important roles in the brain, including in regulation of synaptic signaling and plasticity. no is synthesized from the amino acid l-arginine by the enzyme nitric oxide synthase (nos). in neurons, no is produced by neuronal nitric oxide synthase (nnos), representing one of three nos isoforms expressed in most tissues. to elucidate function of nnos/no in a variety of behaviors, e.g., activity, motor function, nociception, attention, anxiety, depression, social interaction, learning and so on, we subjected nnos knockout mice to a battery of behavioral tests. nnos knockout mice exhibited increased locomotor activity and decreased depressionrelated behavior. furthermore, they displayed increased social contacts in novel environment and homecage. these results indicate that nnos/no is involved in regulation of their behaviors. research funds: kakenhi (16680015, 16653065, 17017021, 17025023) , jst bird ps3p-h126 primate model of attention-deficit/hyperactivity-disorders (adhd) shintaro funahashi 1 , keiko shimizu 2 1 grad. sch. human and environmental std, kyoto univ., kyoto, japan; 2 primate res. inst., kyoto univ., inuyama, japan adhd is one of the prevalent childhood psychiatric disorders. children with adhd show hyperactive behavior and attention problems, suggesting prefrontal (pfc) contribution to adhd. adhd is also known as dopamine (da) related dysfunctions, because methylphenidate is the most effective drug for the treatment of adhd. pfc is the cortical area where the strongest da innervation is observed. injection of da-related drugs to pfc produces behavioral deficits in cognitive tasks. these suggest that da-related dysfunction in pfc could be a candidate of biological causes of adhd. to prove this notion, we injected 6-ohda into bilateral pfc to destroy da innervation in infant monkeys and examined whether these monkeys exhibited hyperactivity. 6-ohda injected monkeys showed significant increase of spontaneous activity in test cages. oral administration of methylphenidate reduced spontaneous activity in 6-ohda injected monkeys. these results suggest that monkeys injected 6-ohda into pfc are good candidates of the primate model of adhd. research funds: kakenhi (17021022) ps3p-i127 training-induced recovery of precision grip after primary motor cortex damage in the adult monkey yumi murata 1,2,3 , noriyuki higo 1,3 , takao oishi 1,3,4 , akiko yamashita 5 , keiji matsuda 1 , motoharu hayashi 4 1 neurosci. res. inst, aist, tsukuba, japan; 2 grad. sch. compreh. hum. sci., univ. of tsukuba, tsukuba, japan; 3 crest, jst, kawaguchi, japan; 4 dept. cell mol. biol., primate res. inst., kyoto univ., inuyama, japan; 5 div. applied sys. neurosci., nihon univ. sch. med., tokyo, japan in the present study, we compared the motor recovery between monkeys that received daily training and that did not receive any training after lesion of the primary motor cortex (mi), in order to investigate the effects of postlesion training on motor recovery. we derived a hand representation map in mi, and ibotenic acid was then injected to destroy the digit region, which resulted in hand paralysis. after one or two months of postlesion training, skilled use of the affected hand including a precision grip was recovered. untrained monkeys also became able to grasp objects with their affected hand, but they couldn't use a precision grip. this suggests that recovery of precision grip requires postlesion training. research funds: a grant-in-aid for scientific research on priority areas from mext (17021055) mouse mutants with behavioral abnormality are indispensable tools to elucidate molecular pathways underlying behavior. in order to develop numbers of novel behavioral mutants, we have been carrying out dominant behavioral screening in potential mouse mutants that was randomly induced point mutations by a chemical mutagen enu (n-ethyl-n-nitrosourea). we screened about 2,500 g1 animals (dba/2j × enu-treated c57bl/6j) for home-cage activity, open-field activity, and passive avoidance response, and obtained 13 lines of dominant behavioral mutants. by linkage analysis, the causative genes were mapped in 5 of 13 mutant lines. hyperactivity was predominant phenotype, and 7 of 13 mutants showed hyperactivity in home-cage and/or open-field. we will report the recent results of initial characterization and the progress of fine mapping in these enuinduced mutants. ps3p-i129 ubiquitin signal in neurons of cathepsin ddeficient mouse brains with special reference to the autophagic process masato koike, masahiro shibata, yasuo uchiyama dept. of cell biol. and neurosci., osaka univ. grad. sch. of med., suita, japan we have shown that autophagy contributes to the accumulation of vacuolar structures in neurons obtained from cd−/− and cb−/−cl−/− mice, murine models for neuronal ceroid lipofuscinoses (ncls) (koike et al., 2005) . until recently, it remains unknown what signaling is essential for autophagosome formation. interestingly, in the conditional atg7-knock-out mice where autophagy is absent specifically in the liver, numerous ubiquitinated aggregates are detected in the cytosol of hepatocytes (komatsu et al., 2005) , suggesting that protein ubiquitination may serve as a signal to the autophagic process. we therefore examined the immunohisto/cytochemical localization of ubiquitin and lc3, and found that in our ncl model mice, positive signals for ubiquitin and lc3 were co-localized on the membranes of granular structures in the neuronal perikarya. these results suggest that protein ubiquitination may be involved in signaling for autophagosome formation in ncls. research funds: grant-in-aid for young scientists (b)(17790141) and creative scientific research (16gs0315) ps3p-i130 activation of medial prefrontal cortex neurons by systemic phencyclidine is primarily mediated via ampa/kainate glutamate receptors tadahiro katayama 1 , eiichi jodo 1 , yoshiaki suzuki 2 , ken-yo hoshino 2 , yukihiko kayama 1 1 dept. of physiology, fukushima medical university, fukushima, japan; 2 dept. of neuropsychiatry, fukushima medical university, fukushima, japan it has been shown that tonic activation of the medial prefrontal cortex (mpfc) plays a pivotal role in development of behavioral abnormalities induced by systemic phencyclidine (pcp). however, receptors mediating such activation are not clearly specified, though several studies indicate the increase of extracellular acetylcholine, dopamine, and glutamate in the mpfc. here, we examined effects of local application of those antagonists on increased firing activity of mpfc neurons by systemic pcp in anesthetized rats. after tonic activation of mpfc neurons by pcp had been established, cnqx, sch23390, mecamylamine or scopolamine was locally applied with iontophoresis or gas pressure on the recorded neuron. cnqx reduced pcp-induced augmentation of firing activity to the baseline level, while others gave little changes. these results suggest that pcpinduced activation of mpfc neurons be mediated primarily via ampa/kainate receptors. ps3p-i131 increased depressiveness and decreased sensitivity to antidepressants in calcium/calmodulin-dependent protein kinase iv (camkiv)-knockout mice jiro kasahara 1 , hiroyuki sakagami 2 , hisatake kondoh 2 , kohji fukunaga 1 1 department of pharmacology, graduate school of pharmaceutical sciences, tohoku university, sendai, japan; 2 department of histology, graduate school of medicine, tohoku university, sendai, japan calcium/calmodulin-dependent protein kinase iv (camkiv) is expressed abundantly in the nuclei of neurons and thought to regulate ca-dependent gene expressions mediated by the transcriptional factors such as creb. recently, we found that chronic treatments of the rats with antidepressants increased camkiv activity and creb phosphorylation in the prefrontal cortex, suggesting the importance of camkiv in the effects of antidepressants. this result led us to perform the behavioral assessments of depressiveness and the sensitivity to antidepressants in camkiv-knockout mice by some experimental paradigms. from the experiments, the increased depressiveness and decreased sensitivity to antidepressants were observed in the mice, suggesting the importance of camkiv for the regulation of depressiveness and the effects of antidepressants. ps3p-i132 severity of audiogenic seizures is influenced by multiple factors in vlgr1-mutated mice hideshi yagi 1,2 , makoto sato 1,2 1 division of cell biology and neuroscience, department of morphological and functional sciences, faculty of medical sciences, university of fukui, fukui, japan; 2 research and education program for life science, university of fukui, fukui, japan epilepsy is a highly prevailed disorder and reports are accumulating that demonstrate that single gene mutation causes such disorders. we made vlgr1-mutated mice and found that they showed high susceptibility to audiogenic seizure, one of the reflex seizures provoked by loud noise. to evaluate whether the genetic backgrounds influence on phenotype of the audiogenic seizure in our mice, we made c57bl/6 backcrossed vlgr1-mutated mice and 129/svs4 backcrossed vlgr1-mutated mice. these two backcrossed lines showed different susceptible periods and severity of audiogenic seizure from the original line. furthermore, phenotype of audiogenic seizure was altered by restraining mice from free moving while being exposed to loud noise. these observations suggest that genetic factors and environmental factors may modify the phenotype of seizures and our vlgr1-mutated mice are good model of reflex epilepsies that are evoked by multifactors. ps3p-i133 reduction in the density of parvalbumin-positive cells in the medial frontal cortex of rats behaviorally sensitized to methamphetamine tomoko kadota 1 , ken kadota 1,2 1 department of bioenvironmental medicine, university of chiba, chiba, japan; 2 chiba institute of psychiatry, chiba, japan our previous study demonstrated that the development of behavioral sensitization of rats to methamphetamine (map) corresponded in time with the progress of neurotoxic changes induced in the medial prefrontal cortex (mfc). the present study further examined morpholological changes of rats that were administered a daily dose of 5 mg/kg of map i.p. for 12 days (d 1 d 12) and then withdrawn from the drug for 7 28 days (wd 7 wd 28). the regimen reduced the densities of parvalbumin positive cells (pac); these were probably gabaergic cells and distributed in the strata covering layers ii, iii and v in the anterior cingulate cortex (cg 1) and mfc. the decrease in the density of pac was first observed in cg 1 and then in mfc. the reduction began on d 6 and advanced to higher levels on d 12 and subsequently wd 7. these findings suggest that the behavioral sensitization regimen leads to the deterioration of inhibitory processes in the neural circuits in cg 1 and mfc, particularly in layers ii and iii. ps3p-i134 up-regulation of ␤ 2 -adrenergic receptor immunoreactivity in astrocytes in the spinal cord after dorsal rhizotomy teruyoshi kondo, yoshihiro ishibashi, kei-ichiro nakamura department of anatomy, division of microscopic and developmental anatomy, kurume university school of medicine, kurume, japan stimulation of ␤ 2 -adrenergic receptor (␤ 2 -ar) induces astroglial proliferation and activation after brain injury, but little is known concerning the potential role of adrenergic receptors in the spinal cord. present study demonstrated that rhizotomy induced a marked and prolonged up-regulation of ␤ 2 -ar-immunoreactivity (ir) in the regions of the dorsal root entry zone and dorsal funiculus containing the central processes of the injured primary sensory neurons. ␤ 2 -arimmunoreactive cells coexpressed gfap-ir and were positive for nestin which is characteristic of reactive astrocytes. a population of ␤ 2 -ar-immunoreactive cells were labeled with ki-67, a marker of cell proliferation, indicating some of them went into cell mitotic state. interestingly, a major population of ␤ 2 -ar-immunoreactive cells also exhibited fgf-2-ir. these findings suggest that ␤ 2 -ar may play important roles in astrocytic activation and neuroprotection associated with induction of synthesis of growth factor such as fgf-2. ps3p-i135 effects of lateral fluid percussion injury (fpi) on the optical signals in dentate gyrus of the rat brain slice preparations shin yamashita 1 , norihiro muraoka 2 , hiroshi hasuo 1 , takashi akasu 1 , minoru shigemori 2 1 dept. of physiology, kurume univ. sch. of med., kurume, japan; 2 dept. of neurosurgery, kurume univ. sch. of med., kurume, japan we investigated the effects of experimental traumatic brain injury on the neuronal function in dentate gyrus (dg) using optical recording techniques with voltage-sensitive dye (rh482). horizontal hippocampal slices were obtained from the control and the fpi rats (one week after the single moderate impact). electrical stimulation of perforant path (pp) produced the optical signal spread in the molecular layer of dg. temporal change in the optical signal, obtained from an area on the propagation pathway, had two peaks (fast and slow peaks). increment of stimulus intensity (10-50 v) increased the amplitude of both fast and slow peaks. the intensity for producing the maximal response was 30-40 v. the amplitude of slow peak in fpi group was about 50% larger than that in control group, while the amplitudes of fast peak were not different in the two groups. these data suggest that the excitatory pp synapse onto granule cells of dg is facilitated after fpi. ps3p-i136 comparative study of neural activities in mouse hippocampal slices by flavoprotein autofluorescence and ca 2+ imaging chikafusa bessho, yasuharu mitsushima, ryo matsumoto department of physics, kyoto sangyo university, kyoto, japan recently k. shibuki et al. have succeeded in flavoprotein autofluorescence imaging of neural activities in the rat brain. we examined neural activities in mouse brain (hippocampal) slices by the modified method and ca 2+ imaging. the slices (300 m) were prepared from the block in an ice cold acsf medium using microslicer and incubated for 1 h in the oxygenated medium at room temperature. a slice was placed on a recording chamber perfused with the medium at a flow rate of 1 ml/min. green autofluorescence (>520 nm) of the slices illuminated by blue light (460-490 nm) was observed by an inverted microscope. images of the autofluorescence were recorded using a calcium imaging system. ca 2+ imaging was also performed in the slices. slices were incubated in acsf medium containing 10 m of fluo3/4 am for 1 h at 37 • c. the ca 2+ image was recorded with an excitation wavelength of 460-490 nmand an emission wave length of >520 nm. the autofluorescence and ca 2+ responses wereobserved in slices perfused with l-glutamate (10 mm). takuya hayashi 1 , hiroshi sato 1 , shinichi abe 2 , takashi hanakawa 2 , hiroshi watabe 1 , hidenao fukuyama 2 , babak aldekani 3 , hidehiro iida 1 1 department of investigative radiology, national cardiovascular center research institute, osaka, japan; 2 human brain research center, kyoto university, kyoto, japan; 3 nathan kline institute for psychiatric research, ny, usa we show connectivity pattern between cortex and striatum in macaque and human by using the non-invasive method of diffusionweighted magnetic resonance imaging (dwi). in macaque, the dwibased striatal connectivity of brodmann's area 9 corresponded to that revealed by the tracer (mncl 2 ) tractography. the dwi-based connectivity pattern also isolated a part of the ventral striatum corresponding to the histochemically-specific 'shell' region in both human and macaque. in addition, we confirmed the species-homology in intra-striatal topography of cortical connection by quantitatively analyzing the connectivity; however, we found that human striatum was more intensively connected to prefrontal cortex and less connected to extra-frontal cortices. these results suggest that human striatum has a dominant and specific role in processing prefrontal information. research funds: h17-kokoro-025 ps3p-i138 optical analysis of synaptic transmission by a fluorescent glutamate probe shigeyuki namiki, hirokazu sakamoto, sho iinuma, kenzo hirose department of cell physiology, nagoya university graduate school of medicine, nagoya, japan glutamate is an essential excitatory neurotransmitter in the central nervous systems. for optical analysis of glutamatergic synaptic transmission, we have developed a fluorescent glutamate probe called eos. by imaging with eos, we successfully detected the synaptically released glutamate following axon firings in cultured hippocampal neurons; the spatial distribution of the glutamate release was non-uniforml along dendrites. we also succeeded in monitoring the phorbol ester-induced potentiation of the glutamate release. furthermore, we found spontaneous and stochastic glutamate release which was confined to small regions. neither application of tetrodotoxin nor removal of extracellular calcium blocked the release. high concentrations of sucrose increased the frequency of the release. these features are reminiscent of those of miniature epsc in electrophysiological recordings and thus suggest that the spontaneous release is quantal vesicular release. in conclusion, our probe directly visualizes the presynaptic release. shingo miyata 1,2 , yasutake mori 1,2 , tsuya taneda 1,2 , hiroaki okuda 1,2 , masaya tohyama 1,2 1 department of anatomy and neuroscience, graduate school of medicine, osaka university, osaka, japan; 2 21st coe program, tokyo, japan local protein synthesis in neuronal dendrites is one of the mechanisms that may mediate a rapid and synapse-specific mobilization of proteins from the resident mrnas. a great deal of effort has been made in analyzing the dynamic state of protein synthesis in the living cells, chiefly by quantifying protein level. however, the protein level cannot mirror the spatio-temporal alteration of translation, because it cannot be affected only by protein synthesis but also by other factors like degradation. therefore, it is problematic to visualize the dynamic state of translation by the present methods. to solve the problem, we applied fret (fluorescence resonance energy transfer) technique to in situ detection of the assembly and disassembly cycle among a pair of translation initiation factors (eifs), thereby showing that bdnf and ephrin could potentiate local protein synthesis in the dendrites of hippocampal neurons. ps3p-i140 a model selection of glm applied to fmri data using aic jobu watanabe 1 , fumikazu miwakeichi 2 , andreas galka 3,4 , ryuta kawashima 4 , tohru ozaki 3 , sunao uchida 1,5 1 institute for biomedical engineering, consolidated research institute for advanced science and medical care, waseda university, japan; 2 department of medical system engineering, faculty of engineering, chiba university, chiba, japan; 3 institute for statistical mathematics, tokyo, japan; 4 institute of experimental and applied physics, university of kiel, keil, germany; 5 new industry creation hatchery center, tohoku university, sendai, japan; 6 faculty of sport sciences, waseda university, tokorozawa, japan in the general linear model (glm) that is widely used in analyses of functional neuroimaging data, several combinations of explanatory variables are possible. the akaike information criterion was applied as a basis of comparison and selection among several glms that analyze block-designed functional magnetic resonance imaging (fmri) data. the glms with/without a resting condition, head motion covariates, time derivatives and dispersion of hemodynamic response function were compared. we demonstrate that a combination of these explanatory variables can effectively improve the model and that aic is a useful tool for model selection in fmri studies. ryuzo shingai, katsunori hoshi, tokumitsu wakabayashi department of welfare engineering, iwateuniversity, morioka, japan to investigate the relationship between the behavior and function of the nervous system of caenorhabditis elegans, quantitative analysis of behavior that indirectly represents the internal states of the worm is necessary. we devised an automated analysis system of c. elegans locomotion. the system is well suited for detecting four locomotion states: forward or backward movement, curl and rest. the system was applied to a phenotype that when a worm is transferred from a seeded plate to a bacteria-free plate, the worm shows frequent backing and short duration of forward movement for 20-30 min and then a gradual increase in the duration of forward movement. accuracy of the state identification for wild type and several mutants was sufficiently high, indicating the system is robust in studies of locomotion. ps3p-j148 flavoprotein fluorescence responses elicited by thalamic stimulation in slices obtained from the mouse barrel cortex daiki kamatani, ryuichi hishida, masaharu kudoh, katsuei shibuki dept. neurophysiol., brain res. inst., niigata univ., niigata 951-8585, japan we have reported that whisker trimming induced activity-dependent changes in the barrel cortex of rat cortical slices using flavoprotein fluorescence imaging. however, contribution of thalamo-cortical afferents in this plasticity was not clear, since specific stimulation of thalamo-cortical afferents was not possible in the coronal cortical slices obtained from rats. in the present study, we used the mouse cortical slices that kept thalamocortical connections to the barrel cortex intact. the cortical activities in layer iv were observed as fluorescence responses after thalamic stimulation. the magnitude of the fluorescence responses was increased as the amplitude of cortical field potentials was increased. these cortical responses were suppressed by antagonists of glutamate receptors such as cnqx and apv, and almost completely abolished in the presence of cnqx plus apv. in preliminary experiments, we confirmed that whisker trimming induced activity dependent changes in the barrel cortex of mice. ps3p-j149 effects of implicit emotional processes on encoding-related activations of episodic memory: an eventrelated fmri study yayoi shigemune 1,2 , takashi tsukiura 1 , hiroko mochizuki-kawai 1 , chisato suzuki 1,2 , toshio iijima 2 1 neurosci. res. inst., aist, tsukuba, japan; 2 div. systems neurosci., tohoku univ. grad. sch. life sci., sendai, japan in this study, we investigated the effects of implicit emotional processes on encoding-related activations of episodic memory using fmri. nineteen healthy right-handed male participated in this study. we prepared emotional pictures with three kinds of emotional valence (negative: nega, neutral: neu and positive: posi) and line drawings for encoding. in the fmri scanning, subjects memorized line drawings, which were presented after the emotional pictures. after the scanning, subjects were presented with the names of line drawings, and were required to judge whether or not line drawings with the names were learned. we found significant activations of the right anterior cingulate gyrus specifically in the nega condition, the right lingual gyrus in the neu condition and the right amygdala and pulvinar in the posi condition. these results suggest that encodingrelated activations of episodic memory may be modulated by the implicit primer with emotional valence. ps3p-j150 different neural correlates of stimulus-actiondependent and stimulus-dependent reward predictions revealed by fmri masahiko haruno 1 , kenji kansaku 2 , yu aramaki 2 , mitsuo kawato 1 1 atr cns, kyoto, japan; 2 institute of physiology, okozoki, japan efficient decision making requires multiple reward predictions in switching different contexts and learning. we conducted a human fmri experiment (n = 14) to examine stimulus-action-dependent and stimulus-dependent reward predictions. in condition a, each of two fractal figures specifies a monetary reward associated with a button push (left or right). if the button is pressed correctly, 50 or 10 yen is provided with a probability of 0.8, but only with 0.2 if pressed wrongly. the key difference in condition b is that a fractal determines the reward but not the action. subjects had learned the two conditions fully before scanning. at the fractal onset, the putamen, lateral ventral and medial dorsal prefrontal cortices showed stronger activity correlated with the predicted reward (p < 0.01) in a, while it was more prominent in the caudate, dorsolateral prefrontal cortex and cerebellum in b. the striatum also showed a similar difference correlated with the reward prediction error at reward feedback, suggesting the different neural substrates for different reward predictions. research funds: nict ps3p-j151 brain networks for communicative speech production: feeling inference and speech content production yuko sassa 1,2 , motoaki sugiura 3 , hyeonjeong jeong 1,4 , keisuke wakusawa 1,5 , kaoru horie 6 , shigeru sato 6 , ryuta kawashima 1 1 niche, tohoku university, sendai, japan; 2 ristex, jst, tokyo, japan; 3 miyagi university of education, sendai, japan; 4 gsics, tohoku university, sendai, japan; 5 department of pediatrics, tohoku university, sendai, japan; 6 the lbc research center, tohoku university, japan communicative speech production often accompany inference of the targetperson's feeling. in this fmri study, we segregated the brain networks forthe feeling inference and speech content production processes incommunicative speech production. during presentation of a picture showingan actor's utterance in a balloon, normal subjects covertly talked to theactor (speech), inferred feeling (feeling), or described the action (des). greater activation in the contrasts speech-feeling was observed in themedial prefrontal cortex, and that in the contrast feeling-des wasobserved in the right superior temporal sulcus extending to the temporalpole. the results suggest that these two regions play roles in the speechcontent production and feeling inference, respectively. research funds: the 21st coe program ps3p-j152 the construction of a brain-computer interface using the brain activity measured by near infrared spectroscopy takafumi miyoshi 1 , yasuhisa fujibayashi 2 , yoshiharu yonekura 2 , tatsuya asai 2 1 department of human and intelligence systems, university of fukui, fukui, japan; 2 biomedical imaging research center, university of fukui, fukui, japan people with severe motor disabilities can increase the quality of life if they can communicate with the external world. a brain-computer interface using brain activity is one of the ways to provide such communication without depending on muscular controls. brain activity was measured non-invasively by multi-channel near infrared spectroscopy (nirs) during various motor tasks from healthy subjects. these spatial brain activities were fed to neural networks, and pattern learning was carried out by matching the tasks and the brain activities. we propose that nirs signals may be used to construct a brain-computer interface. ps3p-j153 imaging of brain activity by near infrared spectroscopy in response to various sounds tatsuya asai 1 , kuniyoshi shinya 1 , tetsuo araki 2 , masahiro kusakabe 2 , yasuhisa fujibayashi 3 , yoshiharu yonekura 3 1 department of nuclear power and energy safety engineering, university of fukui, fukui, japan; 2 department of human and intelligence systems, university of fukui, fukui, japan; 3 biomedical imaging research center, university of fukui, fukui, japan brain activity can be monitored non-invasively by near infrared spectroscopy (nirs). in the present study, we measured changes in cerebral hemoglobin concentrations during a listening task using multi-channel nirs from healthy right-handed subjects, and hemispheric dominance for various sounds including verbal sounds was assessed. we have found asymmetrical brain activity when subjects listened to sounds with their left or right ear. these results suggest that hemispheric sound dominance may exist in addition to language dominance in healthy humans. kazuo kitamura 1,2 , winfried denk 3 , michael hausser 2 1 department of cellular neuroscience, graduate school of medicine, osaka university, osaka, japan; 2 university college london, london, uk; 3 max-plank institute, heidelberg, germany we describe a new approach for making targeted patch-clamp recordings from single neurons in vivo visualized using two-photon microscopy. the method involves using a patch electrode to perfuse the extracellular space surrounding the neuron of interest with a fluorescent dye, thus allowing the neuron to be visualized as a negative image and identified on the basis of its somatodendritic structure. the same electrode can then be placed on the neuron under visual control to allow gigaseal formation. we demonstrate the reliability and versatility of the method using recordings from principal neurons and interneurons in mouse and rat barrel cortex and cerebellum. we also show that the method can be used for in vivo juxtacellular labelling in identified cell types. this approach thus offers the prospect of targeted recording and labelling of single neurons in the intact native mammalian brain without the need to pre-label neuronal populations. research funds: wellcome trust, gatsby foundation, jsps and uehara foundation ps3p-k161 analysis on viability of gabaergic neurons in cerebral cortical slices of adult mice yasuyo tanaka 1 , yasuhiro tanaka 1 , takeshi kaneko 1,2 1 dept. of morphological brain science, kyoto univ., kyoto, japan; 2 crest, jst, kawaguchi, japan whole cell clamp recording and intracellular staining in adult brain slices are technically difficult because of their low viability. we analyzed the effect of slice cutting and incubation conditions on viability of cortical gabaergic neurons, using gad67-gfp knock-in mice. we considered gfp positive cells as having survived. we observed more gfp-positive cells in the slices when nacl in cutting solution was replaced with n-methyl-d-glucamine (nmdg) chloride, choline chloride or sucrose. however, the viability was lower after 3 h incubation in nmdg-based solution than in nacl-based solution. cutting at 0 • c did not reduce the number of gfp-positive cells, but decreased gfp fluorescence in single neurons as compared with cutting at 20 • c. the viability after 3 h incubation was better kept at 20 • c than at 0 or 37 • c. we thus recommend that slices be cut at 20 • c in na-free solution, and incubated at 20 • c in nacl-based solution. we thank dr yanagawa for his generous gift of knock-in mice. research funds: kakenhi (16200025,17022020,17650100) ps3p-k162 contribution of reduced and oxidized glutathione to signals detected by magnetic resonance spectroscopy as indicators of local brain redox state takumi satoh 1 , yoshichika yoshioka 2 1 faculty of engineering, iwate university, morioka, japan; 2 iwate medical university, takizawa, japan we evaluated gsh signals by the mega-press (a frequencyselective refocusing technique) signals assessed by magnetic resonance spectroscopy (mrs). gsh gave a single positive signal (2.95 ppm) by mega-press. in contrast, gssg gave a multiplet of reversed signals (3.03, 3.23, and 3.34 ppm). a phantom solution mimicking the normal condition (gsh:gssg = 100:1) gave a single positive peak. gsh was prominent and gssg signals were minimal. thus, the signals originated from gsh, not from gssg. in the phantom solution (creatine: gsh: aspartate: gaba = 7:3:1:1), the creatine signal overshadowed the other signals. through mega-press, a single peak of gsh stood out over other signals. in vivo, the brains of healthy volunteers gave similar signals as the in vitro phantom solution, indicating that the signal originated from gsh. the estimated concentration of gsh in the human brain was 1.9 mm. in conclusion, mega-press allowed us to assess gsh levels in vivo non-invasionally. hiroshi kadota, hirofumi sekiguchi, yasoichi nakajima, yutaka kohno, makoto miyazaki department of sensory and communicative disorders, research institute, national rehabilitation center for persons with disabilities, tokorozawa, japan we investigated the brain regions related to the inhibition of habitual responses by using functional mri. we used the rock-paper-scissors game as an example of a familiar habitual behavior. it is considered that making positive attempts to lose when presented with the gesture of a rock, paper, or scissors is associated with the inhibition of habitual responses. in this study, the subjects were randomly assigned to one of the following two groups: the "win group" and the "lose group." a comparison between these groups showed that the lose group displayed activation of multiple cortical areas in the brain. with regard to the prefrontal cortex, the comparison revealed a higher activation in the left middle frontal gyrus (brodmann area 9) and the right superior and middle frontal gyri (brodmann area 10) in the lose group. these findings suggest that these regions play a role in the inhibition of habitual responses. ps3p-k164 cortical commissural connection in macaque and human callosum using diffusion mri rishu piao 1 , takuya hayashi 1 , hiroshi sato 1 , shinichi abe 1,2 , takashi hanakawa 2 , hidenao fukuyama 2 , hidehiro iida 1 1 national cardiovascular center, osaka, japan; 2 human brain research center, kyoto university, kyoto, japan we investigated the cortical commissural connection in human and macaque using the non-invasive diffusion-weighted magnetic resonance imaging (dwi). we used the probabilistic algorithm to track connection paths between a pair of the left and right homologous in 9 subcortical areas. in macaque, the classification of callosum based on the highest interhemispheric connections paralleled with the results of tracer studies. however, the territory corresponding to the interfrontal connectivity extended more posteriorly than suggested in the tracer studies. the human interhemispheric connectivity showed similar topography in callosum as in the current macaque study, except that the connectivity territory of the frontal areas extended more posteriorly than in macaque. this study revealed that the commissural connectivity of the two species has a common intra-callosal topography. ps3p-k165 optimal resolution of eeg/meg source imaging by spatial filtering wan xiaohong 1,2 1 niche, department of qutantum science and energy engineering, tohoku university, sendai, japan, 2 niche, tohoku university, sendai, japan nowadays, electro-and magnetoencephalography (eeg/meg) is the sole invasive technique which is able to directly measure the human brain neural cortical dynamics. although we are well aware that it is impossible to accurately estimate the 3-d neural cortical activity using the 2-d eeg/meg surface potential topography, the upper limit of these techniques is not well described. during the past decades, various inverse approaches based on different criteria have been proposed, from the single dipole or multiple dipoles to the distributed current dipoles. however, it is difficult to systematically evaluate their efficiencies due to the different criteria and regularizations adopted in these methods. in this paper, we ask the question whether there exists an optimal approach based on a systematical criterion. this motivation firstly seems to be conflicted with the primary knowledge that there is no unique solution for the bioelectromagnetic inverse problem. essentially, here we are trying to find an optimal inverse solution that is closest to the real current distribution. ps3a-c192 sensitivity of serotonin synthesis to synthesis inhibitor gtp cyclohydrolase i in senescence-accelerated mouse-prone inbred strain (samp8) nobuyuki karasawa 1 , kazuko watanabe 2 , keiki yamada 3 1 seijoh universitry, tokai, japan, 2 dept. physio., sch. med., gifu univ., gifu, japan, 3 dept. anat., sch. health sci., fujita health univ., toyoake, japan to study the relationship between aging and levels of monoaminergic neurons, 2,4-diamino-6-hydroxypyrimidine (dahp), an inhibitor of monoamine synthesis, was intraperitoneally administered to senescence-accelerated mouse-prone (samp8) mice. time course of immunoreactive intensity for serotonergic (5-ht) neurons in the dorsal raphe nucleus, which were stained using laboratory-raised serotonin-specific antibody, was quantitatively evaluated using an image analysis system. results showed that 5-ht neruons are not highly sensitive to a synthesis inhibitor common to both catecholaminergic and 5-ht neurons. katsuya yamada 1,2,5 , yoshihiro matsumura 2 , takashi miki 3 , makoto wakui 1 ␣-smooth muscle actin + arterioles were fewer in kir6.1(−/−) barrel cortex than in wild-type one. in addition, whisker stimulation-induced increase in local cerebral blood flow was much smaller in kir6.1(−/−) barrel cortex than in wild-type one for short (2 s, 5 hz) but not long (30 s) stimulation, suggesting crtical involvement of thin arterioles in a short-time neuro ps3a-h135 learning to use sensory-tools by japanese monkeys yumiko yamazaki 1 , hiromi namba 1 support by public health research foundation (japan). acknowledgement supported by hkrgc. we wish to thank professor miyashita for valuable advice. ps2p-h147 mechanisms for processing of intellectual excitement kazuhiko yanai, hongmei dai dept. pharmacol tohoku grad. univ. sch. med., sendai, japanthe aim of this study was to investigate the role of histamine h1 receptor (h1r) in cognition in physiological and pathological conditions by using h1r mutant (h1−/−) mice. in normal condition, several behavioral studies indicated h1−/− mice show impaired object recognition and spatial memory, improved conditioned fear memory. moreover, hippocampal long-term potentiation was reduced in h1−/− mice. these results indicate h1 receptor is involved in memory process for which the frontal cortex, amygdala and hippocampus interact. in pathological condition, both h1−/− and control mice were subjected to social isolation, an animal model of schizophrenia. social isolation impaired locomotion, prepulse inhibition of startle response and water maze performance in control mice, but not in h1−/− mice. mutation of h1 receptor decreases isolation-induced hyperactivity of cortical dopaminergic neurons. these findings indicate blockage of h1r attenuates social isolation-induced behavioral changes. in conclusion, blockage of h1r impairs cognition in normal conditions, whereas h1r blocking inversely improves cognition in disease models of schizophrenia.research funds: kakenhi (17390156; 14370027) ps3a-h133 5-ht2a receptor gene polymorphism modulates activation in the human ventrolateral frontal lobe during go/no-go task michio nomura 1,2 , hirohito-m. kondo 2 , makio kashino 2,3 1 department of psychology, tokai women's university, kakamigahara, japan; 2 ntt communication science laboratories, ntt corporation, atsugi, japan; 3 shimojo implicit brain function project, erato, jst, kawaguchi, japan impulsive behavior has been suggested to be due to a dysfunction of 5-ht neurotransmission. we examined whether this 5-ht2a receptor gene polymorphism is involved in impulsive aggression by evaluating a reward-punishment go/no-go task using fmri. participants were required to learn to respond to active stimuli and inhibit their response to passive stimuli both under the reward-only (r) condition and the punishment-only (p) condition. the r condition, compared with the p condition caused right prefrontal activation mainly seen in ventrolateral regions. it has been reported that the possible involvement of the 5-ht2a receptor gene polymorphism in impulsive behavior (nomura et al., 2005) , together with the present findings, this observation indicates the involvement of 5-ht2a receptor gene polymorphisms in ventrolateral frontal lobe.research funds: shimojo implicit brain function project, erato, jst ps3a-h134 role of cortical thin arterioles in neurovascular coupling; analyses of kir6.1-containing atpsensitive potassium channel-deficient mice ps3p-g095 rem sleep deprivation increases serum ceruloplasmin level manoj jaiswal 1 , chinmay k. mukhopadhyay 2 , birendra n. mallick 1 1 school of life sciences, jawaharlal nehru university, new delhi, india; 2 special centre of molecular medicine, jawaharlal nehru university, new delhi, india rapid eye movement sleep (rems) is present across higher species and is essential for life. its loss predisposes one to several pathophysiological conditions. continuous loss of rems leads to several diseases and extreme loss may be fatal. rems loss is reported to increase metabolism and food intake though associated with hypothermia. hence, we proposed that the rems deprivation would affect acute phase response protein. in this study rats were rems deprived by platform method. free moving normal, large platform and recovery from rems loss were used as controls. blood was collected from the same rat before and after experimental as well as control periods. level of serum cruloplasmin, a positive acute phase response protein, was detected using western-blot analysis. the results showed that rems deprivation increased the serum ceruloplasmin level suggesting that the rems deprivation triggers an acute phase response at least in rats.research funds: council of scientific and industrial research, india and dst, india the indirect cytopathic effect in hiv-1 and the direct infection of hsv-1 are critical in their pathogenesis. we established murine neurosphere and evaluated with cocultivation of hiv-1 jrfl-infected macrophages or with hsv-1. the generation of primary neurospheres did not suppressed by hiv-1-infection or by hsv-1 infection at no more that moi 10. in the secondary neurospheres, cd133 + neural stem cells were intact in these infections, although beta-3-tubulin + cells were decreased in hiv-1 infection and intact in hsv-1-infection. in the differentiation assay, neun + nfp + neurons in hiv-1-infection and gfap + s100 + astrocytes in hsv-1 infection were significantly decreased. the migration capacity of the neurosphere cells was suppressed in hiv-1 infection and in hsv-1 infection. we conclude that neural stem cells in vitro are resistant to cytopathic effect by hiv-1 and hsv-1 infection and their differentiation capacities are different in these infections. our assay will be one of the significant methods in neurovirological research.research funds: kakenhi 17790579 grant-in-aid for young scientists(b) ps3p-g097 effects of attraction to favorite opposite gender on nervous, endocrine, and immune systems masahiro matsunaga 1,2 , taeko yamauchi 2 , toshihiro konagaya 2 , hideki ohira 1 1 department of psychology, nagoya university, japan; 2 department of internal medicine, aichi medical university school of medicine, japan everybody can "fall in love". thus everybody knows that attraction to favorite opposite gender invokes positive feelings and often makes us energetic. to investigate effects of this positive emotion on the biological systems, we recorded various parameters, namely mood states, heart rate, skin conductance level (scl), serum levels of several hormones, and proportions of t cells and natural killer (nk) cells in the lymphocytes simultaneously when subjects viewed the video films of their favorite opposite genders. when the subjects were evoked their attraction to favorite opposite gender, they became more vigorous and felt better. as for the biological systems, scl and the proportion of nk cells in the lymphocytes significantly increased. these results suggest the possibility that attraction to favorite opposite gender may have a role in activating nk cell-related innate immune system by means of the activation of scl-related sympathetic nervous system. hiroko ikeshima-kataoka 1 , shen jin-song 2 , saburo saito 1 , shigeki yuasa 3 1 dept. mol. immunol., inst. dna med., jikei. univ. sch. med., tokyo., japan; 2 dept. gene ther., inst. dna med., jikei univ. sch. med., tokyo, japan; 3 dept. ultrastruc. res., natl. inst. neurosci., ncnp, tokyo, japanto investigate the role of tenascin (tn)-expressing astrocytes played in the injured brain, we analyzed tn knockout (tn/ko) mouse. we have previously reported that tn is one of the essential molecules for proliferation of the primary culture of astrocytes. from injured mouse brain model with stab wound, gfap expression was down regulated sharply at earlier stages in tn/ko mouse than in the wt mouse. some of the inflammatory cytokines are known to be expressed in injured cns, and also those receptors are expressed in the primary culture of astrocytes. to evaluate immune responses in the cns, some of the inflammatory cytokine production was determined in the lesioned mouse brain compared with tn/ko and wt mouse. from rt-pcr method, tn seemed to have the possible roles for some of the cytokine prodution at the cns lesion sites. we are currently investigating the function of tenascin for the cytokine production around the lesion site. aiko hori 1 , tomoko yamamoto 1 , kiyoshi matsumura 2 , hiroshi hosokawa 1 , shigeo kobayashi 1 1 dept. of intelligence science and technology, grad. sch. of informatics, kyoto university, kyoto, japan; 2 dept. of information science and technology, osaka institute of technology, osaka, japan intracerebroventricular (i.c.v.) injection of arachidonic acid (aa) evokes fever. this response has been thought to occur simply because aa is converted to prostaglandin e 2 (pge 2 ), the final mediator of fever. however, our recent study suggested that aa might not only be the precursor of pge 2 but also induce an enzyme cyclooxygenase-2 (cox-2) that catalyses aa to form prostaglandins. we here examined in rats whether i.c.v. injection of aa induces cox-2, and whether cox-2 is involved in aa-induced fever. two hours after i.c.v. injection of aa, cox-2 was expressed in the perinuclear region of brain endothelial cells. aa-induced fever was partly suppressed with a cox-2 specific inhibitor, ns-398. these results indicate that aa itself or its metabolites induces cox-2 that accelerates the formation of pge 2 from aa, and, hence, enhances fever. mitsunari abe 1 , tatsuya mima 1 , shinichi urayama 1 , toshihiko aso 1 , nobukatsu sawamoto 2 , hidenao fukuyama 1 1 human brain research center, kyoto university graduate school of medicine, japan; 2 nano-medicine merger education unit, kyoto university, japanrepetitive transcranial magnetic stimulation (rtms) can induce lasting changes in the cortical excitability. however, its cellular mechanism remains unknown. diffusion weighted imaging (dwi) is a useful tool for measuring microscopic states of the brain tissue by probing water diffusion.we examined changes of dwi following rtms to further understand its effects. four healthy volunteers received rtms at 0.9 hz (10 min; 90% of the rest motor threshold) applied over the left primary motor area (m1). we scanned 4 sets of dwi (before, and 0 min, 10 min and 20 min after rtms) using 3-t mr scanner, and calculated apparent diffusion coefficient (adc). in 3 out of 4 subjects, the adc decreased (mean 1.72 × 10 −5 /mm 2 s −1 ) in the left m1 just after the rtms, which recovered at 20 min. it is possible that the rtms-induced change of adc might occur as the cellular response. further examination is needed for confirming this point. vahe poghosyan, andreas a. ioannides laboratory for human brain dynamics, brain science institute riken, wako-shi, japanretinotopic areas v6 and v6a in macaques occupy almost the entire extend of the anterior bank of parieto-occipital sulcus (pos). v6a located more dorsal has a larger receptive field size then v6. both areas lack a foveal magnification. we used meg to record brain activity while human subjects were viewing stimuli presented at two different eccentricities in each quadrant of visual fields. to verify the reliability of results, for each subject, the experiment was repeated on three different days. tomographic analysis of meg signal, in each subject, identified highly reproducible activations throughout visual cortex in accord with the known organization. two new areas along the pos with a similar retinotopy to that of macaques v6 and v6a were identified. in the ventral one, activations in response to each stimulus were spatially separated. the foveal magnification was much reduced compared to v1 in both areas and in the more dorsal area activations elicited by stimuli in the same quadrant could not be separated. given the above finding we suggest these areas as possible homologues of macaques v6 and v6a.ps3p-i144 measurement of magnetic evoked field of ratmeasurement of magnetic evoked field of rat using micro squid naohiro tsuyuguchi department of neurosurgery, osaka city university graduate school of medicine, japanthe study of neural activity in rodents would be enhanced by the stimulation of neuronal function in vivo. magnetoencephalography (meg) is used to study brain function in humans, but the limited resolution and sensitivity of conventional instruments have precluded the use of meg to study neuronal function in rodents. we demonstrate that micro meg developed for use with small animals, can be used to detect assess neuronal activity in conscious rodent brain. we used a micro 9-channel magnetometer consisting of a 3 × 3 matrix of superconducting quantum interference device (squid) with its integrated base of 2.5 × 2.5 mm to measure the visual evoked magnetic field (vef) and auditory evoked field (aef) of rats. we obtained the vef wave with 60-80 ms peak by the white led flashing stimulation and the aef wave with 10-20 ms peak by the tone and burst stimulation. this study demonstrate that micro meg can be used for serial assessment of neuronal function of individual, live animals with a minimal degree of invasiveness, has the potential for use in the study of brain function and plasticity. kentaroh takagaki, michael t. lippert, jian-young wu department of physiology and biophysics, georgetown university, washington, dc, usa voltage-sensitive dye (vsd) imaging is used to study visually evoked responses in rat visual cortex, in single trials without averaging. the signal is small, and a diode array with an effective dynamic range of 19 bits was used, along with a "blue dye" (rh 1691) with small heartbeat artifact. a subtraction algorithm was used to further remove heartbeat artifact in the data. with the combination of the array, the blue dye and the algorithm, we were able to visualize sensory evoked wave activity with a high signal-to-noise ratio in single trials. the signals were 0.1-0.05% of the resting fluorescence intensity. spatiotemporally, the evoked response manifested as propagating waves in the visual areas. there were large trial-to-trial variations in the propagating velocity and directions of the waves. the evoked wave apparently interacted with spontaneous waves in the cortex, and varied greatly according to anesthetic regimen. visualizing evoked waves may contribute to the understanding of cortical dynamics underlying sensory processing. masahito nemoto 1 , yoko hoshi 1 , chie sato 1 , susumu terakawa 2 1 tokyo institute of psychiatry, tokyo, japan; 2 photon medical research centre, hamamatsu university school of medicine, hamamatsu, japanwe investigated interhemispheric interactions and neurovascular coupling by simultaneous recordings of neuronal and hemodynamic signals in rats. bilateral somatosensory cortices were activated with a stimulus time lag between test stimuli (electrical pulses to contralateral hindpaw) and conditioning stimuli (to a homologous somatosensory region of the contralateral hemisphere). we measured electrophysiological signals (local field potentials and multiunit activity) and optical intrinsic signals (586 and 610 nm, indicators of cbv and oxygenation), and analyzed the dependence of the signals on the time lag. the results showed that both neuronal and hemodynamic signals were suppressed around 20-ms time lag. average and trial-by-trial correlation analyses suggested that the hemodynamic signals reflected a balance of neuronal excitation and inhibition via callosal connections. we can infer some parts of underlying neural interactions by imaging of the hemodynamic signals. ps3p-j147 multiple-site optical detection of spontaneous activity in the rat sensorimotor cortex akihiko hirota, shin-ichi ito department of physiology, shimane university school of medicine, izumo, japan multiple-site optical recording provides a powerful tool for the cerebral cortical neurophysiology, but its application has largely been restricted to reproducible, stimulus-evoked activation. we have developed the recording system with longer continuous recording capacity and larger signal-to-noise ratio to detect spontaneous activity in a single sweep. we applied this system to the sensorimotor cortex of rats anesthetized with a mixture of urethane and ␣-chloralose. the hindlimb region was exposed and stained with rh414, a voltage sensitive dye. optical records, after compensation for pulsation artifacts, contained deflections time-locked to the high amplitude transient waves, characteristic to ␣-chloralose anesthesia, recorded with a wire electrode placed in the optically sampling area. as the transient in the electrocorticogram fluctuated, the optical signal also varied. this signal was distributed over a broad region, whose latency, amplitude or shape varied systematically within the region, probably reflecting the regional differences in the transient activity. yuko tanaka, r. allen waggoner, kenichi ueno, keiji tanaka, kang cheng bsi, riken, saitama, japanobjective: in this fmri study, we attempted to identify the brain regions involved in the process completing objects with degraded image information.method: fourteen healthy subjects were studied using a 4t mri scanner while performing a matching-to-sample task with three task conditions. the main condition required the subject to judge in a 4-s trial if a trial-unique, degraded animal image matched a contour image. in the comparison condition, we reversed the order presenting intact and degraded images (id epoch).results: comparing images acquired in di epochs with those acquired in id epochs, significant activation was found in the left parietooccipital cortex spanning the cuneus (ba18), superior occipital gyrus around the parieto-occipital sulcus (ba19) and superior parietal lobule (ba7). other activated foci include the left anterior cingulated cortex, left dorsal frontal gyrus and right middle frontal gyrus.conclusion: these results indicate that the parieto-occipital cortex is critically involved in the object completion with degraded images.ps3p-j155 influence of task difficulty during meter inspection: an fmri study naoki miura 1,2 , makoto takahashi 3 , jobu watanabe 2,4 , shinya uchida 2,5 , shigeru sato 6 , kaoru horie 6 , masaharu kitamura 2 , toshio wakabayashi 2 , katsuki nakamura 1,7 , ryuta kawashima 2 1 crest, jst, kawaguchi, japan; 2 niche, tohoku univ. sendai, japan; 3 graduate school of engineering, tohoku univ. sendai, japan; 4 bme institute, waseda univ., tokyo, japan; 5 idac, tohoku univ., sendai, japan; 6 lbc research center, tohoku univ., sendai, japan; 7 department of animal models for human disease, ncnp, tokyo, japanthe purpose of the study was to analyze the cognitive process of a subject facing a human-machine interface (hmi) using fmri. we compared brain activation during meter inspection tasks with different task difficulty. during the meter inspection tasks, the subjects were instructed to inspect the three meters, and to press the button, if the subject found abnormal state. the task difficulty was devised by controlling the rate of change for the value to be displayed. in the right occipitotemporal area and the left cerebellar posterior lobule, activation during analog meter inspection was greater when the task difficulty was higher case. the results suggest that these regions are related to attention and perception of visual appearance of hmi.ps3p-j156 neural connectivity among brain areas related to language function shinya uchida 1,2,3 , naoki miura 2,4 , jobu watanabe 5 , shigeo kinomura 1 , kazunori sato 1 , yasuyuki taki 1 , kentaro inoue 1 , ryoi goto 1 , ai fukushima 2 , kaoru horie 6 , shigeru sato 6 , katsuki nakamura 3 , hiroshi fukuda 1 , ryuta kawashima 2 1 department of nuclear medicine and radiology, idac, tohoku university, sendai, japan; 2 niche, tohoku university, sendai, japan; 3 national institute of neuroscience, ncnp, kodaira, japan; 4 japan science and technology agency, kawaguchi, japan; 5 bme institute, asmew, waseda university, tokyo, japan; 6 graduate school of international cultural studies, tohoku university, sendai, japanthe present study examined the neural connectivity of languagerelated regions using functional mri (fmri) and diffusion tensor imaging tractography (dtt). twenty subjects were participated. functional region of interest (roi) in the left inferior frontal gyrus (lifg) defined by fmri during speech production task was used as a seed point for dtt. in more than 80% of subjects, tracts between the roi and the left thalamus (lth) were estimated. post hoc fmri analysis showed activation in the lth during speech production tasks. therefore, cortical connectivity between the lifg and lth may have certain functional roles in speech production. keisuke wakusawa 1,2 , motoaki sugiura 3 , yuko sassa 1,4 , hyeonjeong jeong 1,5 , kaoru horie 5 , shigeru sato 5 , hiroyuki yokoyama 2 , kazuie inuma 2 , ryuta kawashima 1 1 niche, tohoku univ., sendai, japan; 2 department of pediatrics, tohoku univ. graduate school of medicine, sendai, japan; 3 miyagi univ. of education, sendai, japan; 4 ristex, jst, tohoku univ., sendai, japan; 5 gsics, tohoku univ., sendai, japan; 6 lbc rc, tohoku univ., sendai, japanthis study examines the cortical mechanisms of comprehension of implicit social meanings such as irony and metaphor. healthy subjects judged whether the utterance in a picture such as irony, metaphor, or control expressions was situationally appropriate (s), or literally correct (l). greater activation during s than l task was analyzed to identify the activation for implicit meanings and neural responses to irony or metaphor were analyzed. the left medial prefrontal cortex showed higher activity during the s than l task. the medial orbitofrontal cortex and the right temporal pole showed responses selective to the irony; the responses in the former were observed during s task only, while the latter in both tasks. no selective response to metaphor was observed. keiichi onoda 1 , yasumasa okamoto 1 , kazuhiro shishida 1 , akiko kinoshita 1 , shigeru toki 1 , kazutaka ueda 2 , hidehisa yamashita 1 , shigeto yamawaki 1 1 department of psychiatry and neuroscience, hiroshima university, hiroshima, japan; 2 training and research center for clinical psychology, hiroshima university, higashi-hiroshima, japan anticipation of emotional events may affect perceptual and cognitive processes when the events actually happen. we studied the effects of anticipation of positive and negative affective images on neural processes estimated with meg and event-related fmri. participants were presented emotionally positive or negative images with cue stimuli. the cue stimulus indicated the emotional valence of the image which followed a few seconds later. in meg study, visual evoked field (vef) was smaller for the anticipatable negative image than the anticipatable positive image. this result suggests that when the presentation of a negative image is anticipated before the event, neural processing for the image is depressed compared to when a positive image is anticipated. furthermore, we report the difference of brain activation between anticipation of positive images and that of negative images in event-related fmri study. makoto wada 1,3,4 , kenji yoshimi 1,2 , noriyuki higo 4 , yong-ri ren 2 , hideki mochizuki 2 , yoshikuni mizuno 2 , shigeru kitazawa 1,3,4 1 dept. of physiol, juntendo univ. schl. of medicine, tokyo, japan; 2 dept. of neurol., juntendo univ. schl. of medicine, tokyo, japan; 3 crest, jst, tokyo, japan; 4 neurosci. res. inst., aist, tsukuba, japanwe developed a new method for comparing immunopositive cell densities across groups of animals and creating statistical parametric maps on standardized sections. as an example, we compared iba-1 positive glial cell densities in rats with and without unilateral injection of mpp+. immunopositive cell density map was automatically created in each animal over a coronal section in the midbrain (bregma −5.9 mm). after the map was normalized to a template section, positive cell densities of the two groups were compared in each pixel and a statistical parameter was mapped on each pixel. we were able to detect significant increases of microglias in the side of the injection not only in the substantia nigra pars compacta but also in the white matters. the new method was proven to be useful for detecting significant changes of cell densities over the entire area of immunostained sections.